Programa de Doctorado

“Salud y Desarrollo en los Trópicos”

TESIS DOCTORAL:

“Epidemiología y diagnóstico de la
amphimeriosis en Ecuador”.

William Fernando Cevallos Trujillo

2018
Certificación:

Prof. Dr. Antonio Muro Álvarez, Catedrático de Parasitología y Prof. Dr. Pedro
Fernández Soto, Profesor Contratado Doctor del Departamento de Biología
Animal, Parasitología, Ecología, Edafología y Química Agrícola de la Universidad
de Salamanca, y Prof. Dr. Manuel Calvopiña Hinojosa, Docente-Investigador,
Universidad de las Américas (UDLA), Quito-Ecuador

Certifican:
Que la Tesis Doctoral titulada “Epidemiología y Diagnóstico de la
Amphimeriosis en Ecuador”, que se presenta para optar al grado de Doctor por
la Universidad de Salamanca en la modalidad de Tesis por compendio de
publicaciones, ha sido realizada por William Fernando Cevallos Trujillo, con CC.
No. 170989629-2, Médico por la Universidad Central del Ecuador y Magister en
Medicina Tropical por la Fundación Oswaldo Cruz de Rio de Janeiro-Brasil, bajo
nuestra dirección en el Centro de Biomedicina de la Universidad Central del
Ecuador y en el Centro de Investigación de Enfermedades Tropicales de la
Universidad de Salamanca, dentro del Programa de Doctorado Salud y Desarrollo
en los Trópicos. Consideramos que reúne, a nuestro juicio, originalidad y
contenidos suficientes, por lo que autorizamos su presentación para ser evaluada.

Y para que así conste, a efectos legales, expiden el presente certificado en

Salamanca, a 20 de Febrero de 2018.

Fdo. Dr. Antonio Muro Álvarez Fdo. Dr. Pedro Fernández Soto

Fdo. Dr. Manuel Calvopiña Hinojosa

Dedicatoria

Dedico no esta tesis, sino el camino que recorrí hasta llegar a ella,
a todos aquellos que me acompañaron a recorrer caminos,
y muy especialmente:
A mi esposa, amiga y compañera de eternas jornadas: Nancy Pepita,
mi ejemplo de sencillez y perseverancia.
A mis adorados hijos: Samy Abigail y Andrés Paúl,
mi luz al final del camino.
A mi querida hermana Irene Elsie,
por su calidez y apoyo incondicional.
A mi familia.

“Per aspera, ad astra”

“Por el sendero áspero, a las estrellas”
Séneca, el joven.
 A. Agradecimientos
La realización de este trabajo fue posible gracias a la ayuda de varias personas e
instituciones a las cuales expreso mi sincero agradecimiento, en especial a:

Los pobladores de las comunidades Chachis por recibirme siempre con los brazos
abiertos y en medio de la carestía supieron entregarme lo mejor que se alberga en este
pueblo sencillo y trabajador: su amistad y afecto.

Al profesor y amigo Manuel Calvopíña Hinojosa, por su valiosa orientación

académica y científica, por la amistad sincera de todos estos años y por las enseñanzas
impartidas durante las jornadas de trabajo en los diversos proyectos que hemos
desarrollado.

Al profesor Antonio Muro Álvarez, por la confianza depositada desde los primeros
días del programa de doctorado, por la atención amiga durante la estancia en su
laboratorio y en la fase de escritura de los textos inacabados de esta tesis.

Al profesor Julio López Abán, quien a la distancia supo guiarme en el procesamiento

y conservación de muestras biológicas, sin ellas nada de este trabajo habría sido
posible. Gracias también por su hospitalidad en la bella y querida ciudad de Salamanca.

Al profesor Pedro Fernández Soto, por su orientación en el laboratorio de biología

molecular y por su acompañamiento en las intensas pero felices jornadas de trabajo.

A la profesora Belén Vicente Santiago por su inmensa y generosa ayuda con los
primeros ensayos en la técnica inmunológica, gracias por tu alegría y palabras de
aliento.

A la licenciada Victoria Nipáz, por su excelente colaboración con la preparación y

mantenimiento del antígeno parasitario.

Al profesor Ángel Guevara por su apoyo en la revisión del manuscrito de la técnica

inmunológica.

Al profesor Hiromu Sugiyama por el apoyo financiero para la recolección de

muestras en papel de filtro y por la donación de los parásitos trematodos asiáticos.

A los colegas del programa de doctorado: Juan Hernández y Javier Gandasegui por la
atención amiga durante el trabajo de laboratorio.

A todos los colegas y amigos del Instituto de Biomedicina de la Universidad Central

del Ecuador, por su amistad sincera y por su apoyo en la preparación y mantenimiento
de muestras, y a mi amigo el Dr. Alberto Narváez O. PhD, por el apoyo desinteresado.
Mi eterno agradecimiento a las siguientes instituciones:

A la Universidad Central del Ecuador, en la persona del Señor Rector Dr. Fernando
Sempértegui PhD., por su apoyo decidido para el perfeccionamiento de la planta
docente. A todas las personas que conforman la Dirección General de Investigación y
Posgrado por el apoyo logístico y financiero para la realización de este trabajo y la
Unidad de Gestión por su asesoría en los trámites administrativos.

Al Centro de Investigación de Enfermedades Tropicales de la Universidad de

Salamanca (CIETUS), por todo el apoyo brindado durante el trabajo de laboratorio,
pero, sobre todo, por recibirnos con los brazos abiertos.

Sin instituciones comprometidas en apoyar la investigación científica, todo esfuerzo

personal naufragaría en el mar del olvido.
 B. Índice de figuras
Figura 1. Mapa de intensidad global de la endemicidad por regiones de las
Enfermedades Olvidadas Emergentes y Re-emergentes (EReNTDs)..................................... 5
Figura 2. Clasificación de las principales trematodosis transmitidas por alimentos. 13
Figura 3. Características morfológicas de Amphimerus spp. .............................................. 15
Figura 4. Imágenes de microscopía óptica y electrónica de huevo Amphimerus spp. 16
Figura 5. Ciclo biológico de Amphimerus spp. . ......................................................................... 17
Figura 6. Peces de agua dulce involucrados en la transmisión de Amphimerus spp. en
Ecuador. ....................................................................................................................................................... 18
Figura 7. Distribución geográfica de Amphimerus spp. en las Américas. ........................ 20
Figura 8. Ecografía de paciente ecuatoriano infectado con Amphimerus spp. . ............ 25
Figura 9. Representación esquemática de la reacción LAMP. ............................................. 29
Figura 10. Localización geográfica del área de estudio. ......................................................... 32
Figura 11. Mapa del área de las tres comunidades Chachis estudiadas ........................... 32
Figura 12. Vista panorámica del acceso a las comunidades del Río Cayapas. ............... 33
Figura 13. Forma de preparación y consumo de alimentos en la población Chachi. .. 34
Figura 14. Obtención de parásito Amphimerus spp. adulto. ...............................................117
 C. Índice de tablas

Tabla 1. Trematodos transmitidos por alimentos de importancia médica. ........................ 7

Tabla 2. Principales trematodosis hepáticas transmitidas por alimentos. ......................... 8
Tabla 3. Ictiozoonosis reportadas en humanos y animales a nivel mundial. ..................... 9
Tabla 4. Distribución de Amphimerus spp. y hospedadores definitivos en América….21
Tabla 5. Componentes y condiciones de reacción de la PCR ...............................................120
Tabla 6. Componentes utilizados en la reacción LAMPhimerus ........................................122
 CONTENIDO

Dedicatoria

Agradecimientos

Índice de figuras

Índice de tablas

1. INTRODUCCIÓN .................................................................................................................................. i
1.1 Introducción .............................................................................................................................. 1
1.2 Enfermedades Emergentes y Re-emergentes ............................................................... 1
1.3 Enfermedades Tropicales Desatendidas. ....................................................................... 3
1.4 Trematodosis transmitidas por alimentos .................................................................... 6
1.5 Amphimerus spp. y amphimeriosis ................................................................................ 12
1.5.1 Definición .......................................................................................................................... 12
1.5.2 Breve reseña histórica ................................................................................................. 12
1.5.3 Taxonomía ........................................................................................................................ 12
1.5.4 Morfología ........................................................................................................................ 14
1.5.5 Ciclo biológico ................................................................................................................. 16
1.5.6 Epidemiología ................................................................................................................. 19
1.5.7 Mecanismos patogénicos ............................................................................................ 22
1.5.8 Manifestaciones clínicas ............................................................................................. 24
1.5.9 Diagnóstico....................................................................................................................... 26
1.5.9.1 Métodos parasitológicos .................................................................................... 26
1.5.9.2 Técnicas inmunológicas ..................................................................................... 27
1.5.9.3 Diagnóstico molecular ........................................................................................ 27
1.5.10 Tratamiento ..................................................................................................................... 30
1.5.11 Prevención y control. ................................................................................................... 30
1.6 Zona geográfica del estudio. ............................................................................................. 31
1.7 Bibliografía .............................................................................................................................. 35
2. HIPÓTESIS Y OBJETIVOS ............................................................................................................. 46
2.1 Hipótesis................................................................................................................................... 47
2.2 Objetivo general .................................................................................................................... 47
2.2.1 Objetivos específicos .................................................................................................... 47
3. ARTÍCULOS DE INVESTIGACIÓN.............................................................................................. 48
3.1 ARTÍCULO 1: High prevalence of human liver infection by Amphimerus spp.
Flukes, Ecuador. ................................................................................................................................... 49
3.2 ARTÍCULO 2: High prevalence of the liver fluke Amphimerus spp. in domestic
cats and dogs in an area for human amphimeriasis in Ecuador. ...................................... 54
3.3 ARTÍCULO 3: Enzyme-linked immunosorbent assay for diagnosis of
Amphimerus spp. liver fluke infection in humans................................................................... 64
3.4 ARTÍCULO 4: LAMPhimerus: a novel lamp assay for detecting Amphimerus
spp. DNA in human stool samples. ............................................................................................... 71
3.5 ARTÍCULO 5: Diagnosis of amphimeriasis by LAMPhimerus assay in human
stool samples long term storage onto filter paper. ................................................................ 88
4. CONCLUSIONES ............................................................................................................................100
5. OTROS ARTÍCULOS DE INVESTIGACIÓN ............................................................................102
5.1 Sensibilidad de la técnica de Kato-Katz para la detección de huevos de
Amphimerus en muestras de heces, y prevalencia de infección en Amerindios Chachis.
…………………………………………………………………………………………………………….103
6. ANEXOS ............................................................................................................................................116
Anexo 1. Metodología ......................................................................................................................117
1. Obtención del parásito adulto. ...........................................................................................117
2. Preparación de antígeno somático de vermes adultos de Amphimerus spp.....118
3. Desarrollo de la técnica de ELISA. .....................................................................................118
4. Desarrollo de la TD-PCR. ......................................................................................................119
5. Desarrollo de la técnica LAMPhimerus. ..........................................................................121
5.1 Obtención de ADN de Amphimerus spp. ..................................................................121
5.2 Obtención de ADN de otros parásitos. .....................................................................121
5.4 Diseño del LAMP ...............................................................................................................121
6. Método LAMP para la amplificación de ADN de Amphimerus spp. ......................123
7. Detección de los productos de amplificación. ..............................................................123
7.1 TD-PCR. ................................................................................................................................123
7.2 LAMP. ....................................................................................................................................123
8. Análisis estadísticos................................................................................................................124
Anexo 2. Otras publicaciones con índice de impacto ..........................................................125
Anexo 3. Contribuciones en Congresos ....................................................................................130
1. INTRODUCCIÓN
Introducción

1.1 Introducción

El ser humano se ve constantemente amenazado por antiguos y nuevos agentes

patógenos, entre ellos los parásitos trematodos causantes de un sinnúmero de
enfermedades. Estos parásitos tienen complejos ciclos de vida que incluyen
hospedadores intermediarios, definitivos y reservorios en un intrincado y complejo
mecanismo de adaptación y supervivencia.

Las regiones tropicales y subtropicales de países pobres, son las más propicias para
el desarrollo de ciertas parasitosis humanas, ya que cuentan con un ambiente natural y
social que les son favorables para su cadena reproductiva.

Los adelantos científicos y tecnológicos en el conocimiento de estas enfermedades,

no siempre van de la mano con las mejoras en el ambiente, el saneamiento e higiene
que permitan interrumpir los ciclos de transmisión de las parasitosis humanas.

El desarrollo de nuevos métodos diagnósticos se hace imperioso para conocer la real

carga de infección y enfermedad que los parásitos producen, así como para descubrir
nuevos agentes infecciosos que pudiendo estar en otros hospedadores no humanos
pueden infectar a éstos. Es así como, a partir del descubrimiento de la infección en
humanos por el trematodo de vías biliares denominado Amphimerus spp. en el año
2011, existe la necesidad de desarrollar métodos diagnósticos sensitivos, específicos,
reproducibles y accesibles para establecer su carácter zoonótico y su prevalencia real y
distribución geográfica. El único método diagnóstico disponible hasta el momento es el
análisis coproparasitario de heces, el cual tiene limitada sensibilidad y especificidad.

Desarrollar nuevos métodos diagnósticos inmunológicos y moleculares es un

imperativo para aumentar el conocimiento de esta parasitosis a fin de establecer un
diagnóstico oportuno, un tratamiento adecuado y contar con herramientas para la
vigilancia epidemiológica en zonas endémicas y no endémicas.

1.2 Enfermedades Emergentes y Re-emergentes

Los Centros de Control de Enfermedades de los Estados Unidos de Norteamérica

(CDC; del inglés, Centers for Disease Control and Prevention), define como
enfermedades emergentes a aquellas nuevas infecciones producidas por nuevos
patógenos desconocidos o enfermedades infecciosas conocidas las cuales se descubren
en nuevas áreas geográficas. Enfermedades re-emergentes serían aquellas infecciones
que aumentan o amenazan incrementarse con el tiempo, como consecuencia de la
resistencia a los medicamentos o a una falta de control apropiado derivado de las
políticas sanitarias de cada país (CDC, 2014). Los CDC, reconocen más de cincuenta
Enfermedades Emergentes y Re-emergentes y desde 1990 mediante un “Programa de

1
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Infecciones Emergentes”, se ha expandido por todo el mundo (CDC, 2014 a; CDC 2014b;
Breiman et al., 2013).

De acuerdo con la Organización Mundial de la Salud (OMS), cada año aparecen en el

mundo nuevas enfermedades infecciosas (WHO, 2007). La mayoría se consideran
zoonosis, siendo más de tres cuartas partes originadas en animales silvestres (Jones et
al., 2008). Aproximadamente, el 64% de las enfermedades infecciosas que afectan a las
personas, son causadas por patógenos que se transmiten entre diferentes especies de
animales domésticos y silvestres (Heeney, 2006; Davis, 2008; Taylor et al., 2001).

Esta tendencia, ha dado lugar a un enfoque más holístico e integrado de la salud

humana, animal y ambiental, conocida como “Una Salud”, la misma que viene siendo
impulsada por Naciones Unidas (Pappaioanou et al., 2008). Esta propuesta ha merecido
especial atención en las últimas décadas, buscando una estrategia global para mejorar
el diagnóstico, control y tratamiento de estas enfermedades (Kaplan et al., 2009;
Heymann et al., 2014; Mackey et al., 2012). Este nuevo paradigma, ha sido aplicado, por
ejemplo, mejorando el diagnóstico en las trematodosis zoonóticas desatendidas
(Johansen et al., 2015).

En un mundo cada vez más globalizado e interconectado, nuevos y viejos parásitos

amenazan la salud de las poblaciones humanas de otros continentes a los de su origen.
La globalización no solo implica el intercambio de mercancías y tecnología, sino
también de patógenos causantes de enfermedades. Sucediendo esto también a nivel
regional e incluso dentro de un país con la migración y el turismo interno, influyendo
entre otros el compartir e intercambiar alimentos (Trostle et al., 2008).

Los Institutos Nacionales de Salud de los Estados Unidos (NIH; del inglés, National
Institutes of Health), clasifican la aparición de nuevas enfermedades y el resurgimiento
de otras, en tres grupos: el grupo 1 corresponde a enfermedades emergentes
reconocidas como nuevas en los últimos 20 años, el grupo 2 son las enfermedades re-
emergentes propiamente dichas, y, el grupo 3 son las enfermedades producidas por
agentes que pueden ser utilizados en acciones bioterroristas (NIAID, 2017).

Esta emergencia o re-emergencia de infecciones nuevas o conocidas, se ve afectada

entre otras razones por el acelerado desarrollo humano, incluyendo numerosos
cambios demográficos, poblacionales y ambientales, así como el rápido desarrollo de
nuevas técnicas de diagnóstico. Estas infecciones se producen sobre todo en países
pobres, constituyendo una importante barrera para el desarrollo social y de la salud
humana. Debemos resaltar además que no solo existen discrepancias entre países sino
también dentro de cada país. Así, hay comunidades que son más afectadas y vulnerables
como son las poblaciones indígenas y afrodescendientes, donde las madres y niños,

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presentan alta morbimortalidad (Molyneux et al., 2010; WHO, 2013; Baker et al., 2010;
Conteh et al., 2010).

1.3 Enfermedades Tropicales Desatendidas.

Existen en el mundo lugares más específicos donde la mayoría de las enfermedades

infecciosas emergentes y re-emergentes se desarrollan y atacan, estas son las zonas
tropicales y subtropicales. Históricamente, las enfermedades que se presentan en estas
zonas de países pobres y en vías de desarrollo son endémicas y continúan desatendidas
por los organismos gubernamentales regionales, nacionales e internacionales (WHO,
2010; Liese et al., 2010; Molyneux, 2010). En el año 2007 la OMS reunió a más de 200
expertos para tratar este problema acuñándose el término: “Enfermedades Tropicales
Desatendidas” (Neglected Tropical Diseases – NTDs por sus siglas en inglés) para
denominar estas afecciones (WHO, 2008).

Las NTDs representan la causa más frecuente de enfermedad en 2.7 billones de

personas pobres, es decir, aquellas que viven con menos de 2 dólares diarios (Hotez et
al., 2010). Estas enfermedades no solo dificultan el desarrollo infantil y causan
incapacidad crónica a lo largo de la vida, sino que además impiden el desarrollo
económico y social de las poblaciones humanas (Hotez et al., 2006; Fürst et al., 2012).

Son causadas principalmente por virus, bacterias, protozoos y helmintos. Muchas

son zoonosis y/o transmitidas por un sinnúmero de artrópodos vectores. Las veinte
NTDs que considera la OMS son: dengue, rabia, tracoma, úlcera de Buruli,
treponematosis endémica, lepra, enfermedad de Chagas, tripanosomosis Africana
humana, leishmaniosis, teniosis/cisticercosis, dracunculosis, equinococosis,
trematodosis transmitidas por alimentos, filariosis linfáticas, oncocercosis,
esquistosomosis, helmintosis transmitidas por el suelo, pian, mordedura de serpientes,
escabiosis y otras ectoparasitosis (Hotez, 2013; WHO, 2017).

La mayoría de las comunidades en las que se presentan estas enfermedades se ven

afectadas por limitados recursos gubernamentales tanto humanos como económicos,
baja accesibilidad a servicios de salud, escaso saneamiento e higiene, pobreza extrema,
nutrición deficiente, afectación de su cohesión social debida a proyectos de desarrollo,
prácticas de consumo de alimentos inadecuadas. Muchas de estas enfermedades son
prevenibles y/o tratables a través de intervenciones de bajo costo (Eisenberg et al.,
2009; WHO, 2013; Baker et al., 2010; Conteh, 2010).

El estigma social, los hábitos culturales, la marginación, la extrema pobreza de las

poblaciones afectadas y la baja mortalidad son varios factores que contribuyen en el
“olvido” de estas enfermedades. Su prevalencia en zonas geográficas y ambientales
específicas fuera del mundo desarrollado y su cuota de mercado insignificante para la

3
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industria farmacéutica reduce aún más la importancia de estas enfermedades en el

debate sobre salud global (WHO, 2016). La negligencia u olvido, también es evidente en
términos económicos, ya que estas enfermedades reciben una pobre financiación
estatal. Así, por ejemplo, durante el periodo 2003-2007, la asignación para proyectos
de lucha contra VIH fue del 36,6%, para malaria 3,6% y para tuberculosis 2,2%. Por el
contrario, la proporción media para el control de NTDs fue del 0,6% (Liese & Schubert,
2009). La asignación de recursos para la investigación también muestra una asimetría,
pues el 80% de recursos se destinan únicamente al abordaje de la malaria, tuberculosis
e infección por VIH (Moran et al., 2009).

En América Latina, las NTDs no sólo afectan a la población más pobre, sino que
también se concentran en poblaciones vulnerables, especialmente comunidades
indígenas y con descendencia africana (Hotez et al., 2008). En esta región del mundo,
se estima que el 7% del total de la población y el 40% de la población rural pertenecen
a un solo grupo étnico (PAHO, 2007). La extrema pobreza afecta a poblaciones
indígenas, particularmente en Bolivia, Colombia, Ecuador, Perú, Guatemala y México,
donde reside aproximadamente el 80% de esta población (Holveck et al., 2007).

Las enfermedades zoonóticas desatendidas o Neglected Zoonosis Diseases (NZDs),

son un subconjunto de las NTDs. De acuerdo a la OMS, 7 de las 20 NTDs, son
consideradas enfermedades zoonóticas, a saber: rabia, tripanosomosis africana
humana, leishmaniosis, teniosis/cisticercosis, equinococosis, trematodosis
transmitidas por alimentos y esquistosomosis (Hotez, 2013; WHO, 2013).

En muchos países del mundo coexisten varias NTDs, lo cual demuestra la

complejidad al abordar este tipo de enfermedades (Figura 1).

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Figura 1. Mapa de intensidad global de la endemicidad por regiones de las Enfermedades

Olvidadas Emergentes y Re-emergentes (EReNTDs). Fuente OMS, 2013 (Mackey et al., 2014).

De acuerdo a la clasificación del Banco Mundial, los países que tienen bajos ingresos
económicos y que además están ubicados en la línea ecuatorial, aumentan el riesgo para
la transmisión y dispersión de las enfermedades zoonóticas (Hotez et al., 2010). Los
factores ecológicos y el desarrollo económico, juegan un papel importante en la
dinámica de transmisión (McMichael, 2004; Sachs et al., 2001). Así, las regiones
alrededor de la línea ecuatorial reciben mayor influencia de los rayos solares, lo que
permite una mayor capacidad de supervivencia de las plantas (Thornthwaite, 1948),
dando como resultado regiones tropicales con mayor biodiversidad del planeta (Waide
et al., 1999). Esto tiene como resultado la presencia de nichos ecológicos propicios para
la transmisión de enfermedades infecciosas entre diversos animales (Keesing et al.,
2010).

En el Ecuador, las enfermedades tropicales y zoonóticas desatendidas, son un

verdadero problema de Salud Pública. Así, la enfermedad de Chagas causada por T. cruzi
y transmitida por diferentes especies de chinches vectores persiste en amplias zonas
tropicales y subtropicales (Dumonteil et al., 2016). El dengue, si bien ha sido
considerado una enfermedad urbana, recientemente se ha incrementado en zonas
rurales y estaría reemplazando a la malaria como causa de fiebre (Cifuentes et al.,
2013).

El control de la rabia canina continúa siendo un desafío en zonas rurales del país
donde además se han documentado casos transmitidos por murciélagos (Cartelle
Gestal et al., 2015). Uno de los mayores éxitos del Ecuador, ha sido el control y posterior

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eliminación a través de la distribución masiva de ivermectina, de la transmisión de

Onchocerca volvulus causante de la oncocercosis, que afectaba principalmente a
pobladores afroecuatorianos y Chachis en el Noroeste del país (Lovato et al., 2014;
WHO, 2016). La equinococosis quística causada por Echinococcus granulosus, es un
problema creciente, posiblemente debido a un mejor diagnóstico a través de métodos
más sensibles y a una mejor comprensión de la enfermedad (Cartelle Gestal et al., 2015).

En relación al complejo Teniosis/Cisticercosis, varios estudios en zonas endémicas

andinas, han demostrado una alta exposición a las oncosferas de T. solium pero baja
prevalencia de neurocisticercosis (Rodríguez-Hidalgo et al., 2003; 2006). Otro
importante problema de Salud Pública en Ecuador es la leishmaniosis que afecta a 21
de sus 24 provincias especialmente en zonas subtropicales y tropicales, aunque
también está presente en zonas de los Andes (Hashiguchi et al., 1991; Calvopiña et al.,
2004). Las helmintosis transmitidas por el suelo continúan siendo un importante
problema de salud en sectores pobres y carentes de servicios sanitarios básicos con
altas tasas de prevalencia (Romero-Sandoval et al., 2017; Cepon-Robins et al., 2014).

1.4 Trematodosis transmitidas por alimentos

Es el mayor grupo dentro de las NTDs. Se estima que cerca de 40 millones de

personas están infectadas y más de 750 millones están en riesgo de infectarse (más del
10% de la población mundial), y que habitan principalmente en el sudeste asiático
(Hotez et al., 2008; Keiser & Utzinger, 2005).

Se estima que la carga global de estas enfermedades es de 665.352 años de vida

perdidos, debidos a la discapacidad que producen (Fürst et al., 2012). Además, en 1994,
la Agencia Internacional de Investigación del Cáncer (IARC; del inglés International
Agency for Research on Cancer) clasificó a O. viverrini y C. sinensis como agentes
carcinogénicos (Working Group of the International Agency for Research on Cancer,
1994).

Estas enfermedades causadas por parásitos que pertenecen al Phylum

Platyhelminthes, clase Trematoda, subclase Digenea, son clasificadas de acuerdo a la
localización anatómica de los parásitos adultos en el organismo del hospedador
definitivo. Más de 80 especies han sido reportadas infectando a humanos (Chai, 2007;
Blair et al., 2007; Mas-Coma et al., 2007; Fürst et al., 2012). Se localizan principalmente
en pulmones, vías biliares y tubo digestivo (Keiser & Utzinger, 2007) (Tabla 1), y
mantienen complejos ciclos de vida en la naturaleza, los cuales han sido bien detallados
en la literatura (Keiser et al., 2009).

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Tabla 1. Trematodos transmitidos por alimentos de importancia médica y de Salud Pública.

Órganos Género Especie Fuente Hospedador

afectados infección natural final de la
humana infección

Hígado y Clonorchis C. sinensis Peces Perros y otros

vías carnívoros que
biliares consumen peces.
Opisthorchis O. viverrini Peces Gatos y otros
O. felineus carnívoros que
consumen peces.
Amphimerus* Amphimerus spp. Peces Gatos, perros, aves,
ratones, tortugas,
raposas, delfines.

Pulmones Paragonimus Paragonimus spp. Cangrejos de Gatos, perros y

río carnívoros que
comen crustáceos.
Fasciola F. hepatica Vegetales Ovejas, ganado y
F. gigantica acuáticos otros herbívoros

Intestino Haplorchis H. taichui, Peces Pájaros y otros

H. pumilio mamíferos que
Metagonimus M. yokogawa Peces consumen peces.

* Parásito reportado por nuestro grupo de investigaciones.

Fuente: WHO 2011; Sripa et al., 2010; Calvopiña et al., 2012.

A pesar de que han sido descritos hace cientos de años (Mas-Coma et al., 2005), aún
existen dudas respecto a su taxonomía, ya que nuevas especies continúan siendo
identificadas y descritas (Chai, 2007; Blair et al., 2007).

Más de 18 millones de personas en el mundo se ven afectadas por la infección de

trematodos hepáticos pertenecientes a la familia Opisthorchiidae (Chai et al., 2009;
WHO, 1995) y más de 500 millones están en riesgo de ser infectados (dos Santos &
Howgate, 2011). La tabla 2, muestra las principales especies de trematodos hepáticos
transmitidas por alimentos de importancia en Salud Pública, especificando la fuente de
transmisión y la zona endémica donde se producen.

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Tabla 2. Principales especies de trematodos hepáticos transmitidos por alimentos de

importancia en Salud Pública.

Familia Género Especie Fuente Zona endémica

infección
humana

Opisthorchiidae Clonorchis C. sinensis Pescado Asia Oriental.

Opisthorchis O. viverrini Pescado Asia Oriental.
O. felineus Pescado Asia Central y Este
de Europa.
Amphimerus Amphimerus spp. Pescado Américas y Asia.

Fasciolidae Fasciola F. hepatica Plantas Europa, Asia y

F. gigantica Plantas América.

Las trematodosis zoonóticas transmitidas a través del consumo de peces crudos o

insuficientemente cocinados son denominadas ictiozoonosis (Tabla 3), las cuales han
generado un gran problema de Salud Pública en el mundo (WHO, 1995; dos Santos &
Wowgate, 2011). Entre los trematodos que afectan al hombre, más de 50 son
transmitidos por el consumo de peces (Furst & Uzinger, 2012; Hung et al., 2013). Entre
ellas, encontramos a la amphimeriosis, producida por Amphimerus spp., que es
conocida desde hace más de 100 años como una infección de reptiles, aves y ciertos
mamíferos (Calvopiña et al., 2011). Al parecer, este parásito fue descrito anteriormente
infectando a humanos en Ecuador bajo la denominación de Opisthorchis guayaquilensis
(Rodriguez et al., 1948, Moreira et al., 2008).

Las especies de mayor importancia para la salud humana son las ictiozoonosis que
afectan al hígado, producidas por parásitos de la familia Opisthorchiidae, los cuales
parasitan los conductos biliares pequeños del hospedador humano o animal. Tienen
una amplia distribución a nivel mundial, presentando alta prevalencia especialmente
en países asiáticos, causando elevada morbilidad (Chai et al., 2005; Keiser & Utzinger,
2009; dos Santos et al., 2011; Mas-Coma & Bergues, 1997). Por ejemplo, en Taiwán, C.
sinensis tiene tasas de prevalencia de hasta un 57%. Se estima que una región con una
prevalencia mayor al 20% debe ser considerada como área hiperendémica y donde el
tratamiento masivo con praziquantel estaría justificado (Chen, 1991; Rim, 1986). Estas
infecciones son reconocidas por causar enfermedad grave en ciertas zonas geográficas
del mundo. Colangitis, coledocolitiasis, pancreatitis y colangiocarcinoma se asocian a
un patrón de infección crónica (Chai et al., 2009; Mas-Coma & Bergues, 1997).

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Tabla 3. Ictiozoonosis reportadas en humanos y animales a nivel mundial.

Familia Género Especie Zona endémica

Localización hepática y vías biliares

Opisthorchiidae Clonorchis C. sinensis Asia Oriental.

Opisthorchis O. viverrini Asia Oriental.
O. felineus Asia Central y
Este de Europa.
Opisthorchis Asia y Europa.
tenuicollis
Amphimerus Amphimerus spp. Américas, Asia.
Metorchis albidus Europa, Asia.
Metorchis bilis Europa.
Metorchis conjunctus Norte América
Metorchis orientalis Asia Oriental.
Pseudamphistomum Pseudamphistomum África, Europa y
spp. Norte América.

Localización intestinal

Nanophyetidae Nanophyetus Nanophyetus Rusia, Estados

salmincola Unidos América.
Echinostomatidae Echinochasmus Echinochasmus spp. Asia y África.
Echinoparyphium E. paraulum Asia, Europa,
Echinostoma Echinostoma spp. Nueva Zelandia,
Episthmium E. caninum Brasil

Heterophyidae, Appophalus Appophalus donicus Europa, Canadá.

Ascocotyle Ascocotyle spp. Asia, África,
Centrocestus Centrocestus spp. América, Europa.
Cryptocotyle Cryptocotyle lingua Asia, Europa, EUA
Haplorchis Haplorchis spp. Asia, África,
Heterophyes Heterophyes spp. América.
Heterophyopsis Heterophyopsis Asia.
continua
Isthimiophora Isthimiophora melis Asia, EUA, Europa
Metagonimus Metagonimus spp. Asia
Phagicola Phagicola sp. Las Américas
Procerovum Procerovum spp. Asia, África.
Pygidiopsis Pygidiopsis summa Asia Oriental.
Stellantchasmus Stellantchasmus spp. Asia Oriental.
Stictodora Stictodora spp. Corea y Japón.
Fuente: Hung et al., 2013.

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Varias son las razones para el incremento de estas ictiozoonosis: el desarrollo de

nuevas y mejores técnicas diagnósticas, el incremento de consumo de pescado mal
cocinado en las poblaciones endémicas en forma de sashimi, sushi, ceviche, encurtido;
el incremento en viajes y comercio de pescado de cultivo en criaderos tanto para
consumo local como para exportación, entre éstos la tilapia y truchas, el desarrollo de
la acuacultura y el descubrimiento de estas parasitosis en zonas donde antes no se había
documentado la transmisión (dos Santos & Wowgate, 2011; Calvopiña et al., 2011;
Hung et al., 2013).

Estas enfermedades de origen alimentario se desarrollan en un ambiente eco-

epidemiológico específico en el que confluye un pobre saneamiento e higiene, hábitos
culturales de consumo de alimentos, presencia de reservorios domésticos y silvestres,
variedad de hospedadores intermediarios, pobreza y un diagnóstico tardío, ya que
muchas infecciones pasan desapercibidas. La pobreza como expresión social de la falta
de escolaridad, baja calidad de la vivienda, falta de acceso a agua segura y pobre
saneamiento ambiental es uno de los determinantes sociales claves para el
mantenimiento y el aumento de estas enfermedades que producen discapacidad, una
importante morbi-mortalidad a largo plazo y disminución de la productividad, lo cual
genera un círculo vicioso de pobreza-enfermedad-pobreza (WHO, 2010; Hotez, 2007;
Liese et al., 2010; Hotez et al., 2010).

Otros determinantes sociales de la salud son por ejemplo, el papel social asignado a
las mujeres y niños como agentes de recolección de agua y juego dentro de ríos, lo cual
les expone más a enfermedades vectoriales y consumo de alimentos poco o mal
preparados. Esto representa una dinámica de trasmisión de la enfermedad específica
que requiere intervenciones focalizadas como acceso a agua segura, educación y mejora
en el saneamiento (Guernier et al., 2004; Rathgeber et al., 1993). Las condiciones de
trabajo dedicados a agricultura y pesca de subsistencia determinan un mayor riesgo de
contraer las NTDs (Coutinho et al., 1997). También el bajo nivel de escolarización les
impide conocer las formas de evitar o disminuir el riesgo de exposición, comparado con
poblaciones con mayores niveles de educación (Conteh et al., 2010, Vecchiato, 1997;
Ho, 2004; Boelaert et al., 2010).

El saneamiento inadecuado influye en su transmisión, ya que la falta de una

adecuada eliminación de excretas aumenta el riesgo de infección de los hospedadores
intermediarios. Así, la mejora del abastecimiento de agua y construcción de letrinas
adecuadas, son medidas imprescindibles para mejorar el saneamiento y reducir la
transmisión de estas enfermedades (Relman & Choffnes, 2011; Aagaard-Hansen, 2010;
Fuller et al., 2016).

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El control de la exposición humana a los hospedadores intermediarios, como son los

pescados de agua dulce, resulta ser un componente crítico en la prevención ante la falta
de vacunas y la imposibilidad de su eliminación dada la presencia de otros reservorios
domésticos y silvestres (WHO, 2013; Karesh et al., 2012).

Entre las trematodosis transmitidas por alimentos en el Ecuador, destacan la

paragonimosis pulmonar, comúnmente subdiagnosticada y transmitida por la ingestión
de cangrejos dulceacuícolas insuficientemente cocinados (Calvopiña et al., 2014);
también la fasciolosis, la cual se contrae por la ingesta de plantas acuáticas infectadas
con metacercarias que son consumidas crudas en ensaladas. Esta parasitosis es
endémica en ciertas comunidades alto andinas con presencia de ambientes ecológicos
propicios para su transmisión (Trueba et al., 2000).

Los esfuerzos para la prevención y el control de estas enfermedades deben iniciarse

con la identificación de la carga de infección, los hospedadores intermediarios
involucrados en la transmisión y los factores de riesgo asociados. De esta manera se
podrán llevar a cabo campañas de prevención mediante educación sanitaria a la
población para evitar el consumo de hospedadores intermediarios o promoviendo su
consumo adecuado. Adicionalmente, otros métodos para promover un control
integrado se basan en mejorar el saneamiento e higiene en la población (Relman &
Choffnes, 2011; Rollinson et al., 2013; Mazigo et al., 2012).

La OMS recomienda 5 estrategias para el control de estas enfermedades: (i)

quimioterapia preventiva, (ii) manejo de la enfermedad y acceso a la atención
especializada, (iii) control de hospedadores intermediarios, (iv) atención a la salud
pública veterinaria en su relación con la salud humana y, (v) provisión de agua segura,
saneamiento e higiene (WHO, 2012).

Dentro de la subclase de trematodos digénidos, está la familia Opisthorchiidae. Los

más importantes desde el punto de vista de Salud Pública son Clonorchis sinensis,
Opisthorchis viverrini y O. felineus que infectan al hombre y otros reservorios por comer
pescado de agua dulce en platos típicos como sushi y sashimi y que son endémicos en
el sudeste del continente asiático (Sripa et al., 2007). Dentro de esta familia, en los
últimos años se ha reportado la infección causada por Amphimerus spp., trematodo
hepático descubierto en poblaciones indígenas del noreste de la costa Pacífica
ecuatoriana (Calvopiña et al., 2011) y que es objeto de estudio de la presente tesis
doctoral.

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1.5 Amphimerus spp. y amphimeriosis

1.5.1 Definición

El parásito trematodo hepático Amphimerus spp. debe su nombre a los vocablos

latinos Amphi que significa “en ambos sitios” y merus “posterior”, refiriéndose a la
disposición de sus glándulas vitelinas en el estadio adulto, las cuales están divididas en
dos grupos, un grupo de dos anteriores y un grupo de dos posteriores a nivel del ovario.

La amphimeriosis es la enfermedad zoonótica causada por Amphimerus spp. que se

transmite al ingerir pescados de río crudos o escasamente cocinados.

1.5.2 Breve reseña histórica

Dentro del género Amphimerus, el primer trematodo de vías biliares estudiado fue
Amphimerus pseudofelineus, descrito originalmente por Ward en 1901 en un gato de
Nebraska, Estados Unidos de América. Posteriormente, Barker en 1911 lo clasificó
dentro del género Amphimerus diferenciándolo de aquellos pertenecientes a la familia
Opisthorchiidae como Clonorchis sinensis y Opisthorchis viverrini. A partir de entonces,
A. pseuodofelineus ha sido descrito ampliamente en las Américas infectando también a
gatos en los estados de Illinois y Michigan. Además, se ha estudiado el parásito a través
de infecciones experimentales en Manitoba, Canadá.

En 1948, Rodríguez y colaboradores descubren un trematodo hepático infectando a

humanos en Ecuador, denominándolo Opisthorchis guayaquilensis (Rodriguez et al.,
1948). Yamaguti en 1971, indica que este parásito previamente reportado en Ecuador,
podría tratarse de Amphimerus spp. (Yamaguti, 1971). Así, es referido con este nombre
en otras publicaciones posteriores (de Moraes N et al., 1998). No obstante, son
necesarios estudios moleculares para identificar con exactitud este trematodo de vías
biliares.

1.5.3 Taxonomía

La taxonomía básica de los trematodos transmitidos por alimentos se muestra en la

figura 2, enfatizando a aquellos que infectan al hombre. Hemos incluido a Amphimerus
spp. recientemente descubierto por nuestro grupo de investigación (Keiser & Utzinger,
2009; Calvopiña et al., 2011).

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Familia

Figura 2. Clasificación de los principales trematodos transmitidos por alimentos.

El género Amphimerus (Barker, 1911) pertenece al phylum Platyhelminthes, clase

Trematoda, subclase Digenea, familia Opisthorchiidae (Calvopiña et al., 2011). Está
estrechamente relacionado con los trematodos hepáticos de la familia Opisthorchiidae:
Opisthorchis spp. y Clonorchis sinensis. Infecta a mamíferos del continente Americano
incluyendo Canadá, Estados Unidos, México, Costa Rica, Panamá, Venezuela, Colombia,
Ecuador, Brasil y Perú; también en el sur de Asia, Corea del Sur, India y Filipinas
(Bowman, 2002; Yamaguti, 1971; Miyazaki et al., 1978; Rivillas et al., 2004; de Moraes
Neto et al., 1998; Artigas et al., 1962; Thatcher, 1970; Calvopiña et al., 2011, Eom et al.,
1984). No se ha reportado su presencia en otros continentes como África, Oceanía, Asia
o Europa. Actualmente, han sido descritas las siguientes especies: A. lancea (Diesing,
1850), A. speciosus (Stiles & Hassall, 1896), A. interruptus (Braun, 1901), A.
pseudofelineus (Ward, 1901), A. noverca, en India (Braun, 1902), A. ovalis (Barker, 1911)
que es la especie tipo, A. anatis (Yamaguti, 1933), A. elongatus (Gower, 1938), A. pricei
(Foster, 1939). A. caudalitestis (Caballero, Grocott & Zerecero, 1953), A. neotropicalis
(Caballero, Geis & Caballero, 1963), A. parciovatus (Franco, 1967) y A. bragai N.sp. (de

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Moraes Neto A et al., 1988). De éstos, A. ovalis es un trematodo de reptiles; A. speciosus,

A. interruptus, A. anatis y A. elongatus, son parásitos de aves; las demás son parásitos de
mamíferos (Franco, 1967).

1.5.4 Morfología

Amphimerus spp. es un trematodo hepático hermafrodita. Los parásitos adultos

habitan en los conductos biliares pequeños y medianos. Son de morfología aplanada y
alargados en forma de hoja, presentan una coloración rojo- rosada y miden entre 8-13,6
mm de largo por 0,5-1,1 mm de ancho. Pasados unos minutos en solución salina los
gusanos se enrollan en forma de “S” y se vuelven transparentes o blanquecinos. En su
estructura destacan las glándulas vitelinas distribuidas en cuatro grupos, 2 anteriores
y 2 posteriores; la ventosa ventral es mayor que la oral y los testículos son redondeados
o ligeramente lobulados (Calvopiña et al., 2011). En contraste, las glándulas vitelinas en
Clonorchis y Opisthorchis existen solo en la zona frontal a los testículos (Figura 3).
Adicionalmente, Clonorchis tiene dos testículos grandes altamente ramificados y en
Opisthorchis son siempre lobulados (Yamaguti, 197; Bowman, 2002).

Los huevos presentan un opérculo en su parte anterior y una espina inferoposterior.

Tienen un tamaño de 28-33 µm x 12-15 µm. A la observación por microscopía
electrónica, la superficie de los huevos de Amphimerus spp. muestra diferencias con los
huevos de otros trematodos de la familia Oposthorchiidae. Así, mientras es rugosa e
irregular en forma de parches en Amphimerus spp., es fina, regular y reticulada en C.
sinensis (Figura 4).

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Figura 3. Características morfológicas de Amphimerus spp, Clonorchis sinensis y Opisthorchis

viverrini. A. Amphimerus spp. adulto de Ecuador (corte ventral). Ventosa ventral (A) es mayor
que la Ventosa oral (S). Poro genital (G) se abre delante de la ventosa ventral; Glándulas
vitelinas (V) están divididas en dos partes; los Testículos (T) son redondeados. Estas son las
diferencias comparadas con los parásitos trematodos asiáticos O. viverrini y C. sinensis. R:
Receptáculo seminal, O: Ovario, U: Útero, C: Conducto vitelino, y; F: Faringe. Comparado con B.
Clonorchis sinensis, y; C. Opisthorchis viverrini (Sripa et al., 2007).

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Figura 4. Imágenes de microscopía óptica y electrónica de huevo de Amphimerus spp. y

Clonorchis sinensis. (A) Huevo de Amphimerus spp. ecuatoriano obtenido de persona a
microscopía óptica (X 3) y (B) a microscopía electrónica. (C) Huevo de C. sinensis a microscopía
electrónica. A pesar del tamaño similar, las diferencias en la superficie permiten diferenciar los
dos géneros (Calvopiña et al., 2011).

1.5.5 Ciclo biológico

Conocemos muy poco acerca del ciclo biológico de Amphimerus spp. Sin embargo,
nuestras primeras investigaciones evidencian que este parásito produce infección en
humanos y animales domésticos a través de la ingestión de peces crudos o
insuficientemente cocinados. Al igual que en otros trematodos de la familia
Opisthorchiidae, los peces son portadores de metacercarias activas, las cuales una vez
ingeridas se separan de la carne del pescado en el estómago por la acción del jugo
gástrico, avanzando hasta el duodeno, donde son liberadas por acción combinada de
proteasas como tripsina y cisteína. Posteriormente, los trematodos liberados
atraviesan la ampolla de Vater y alcanzan las vías biliares intrahepáticas, sitio en el cual
se desarrolla el parásito adulto, pudiendo vivir hasta 30 años y produciendo
aproximadamente 4.000 huevos por día. (Attwood, 1978; Kim et al., 2009). La
maduración a parásito adulto se produciría aproximadamente en dos meses, como en
el caso de O. viverrini y C. sinensis. Los humanos, reptiles y ciertos mamíferos como
perros y gatos que ingieren peces de río pueden servir como hospedadores definitivos
(Taylor et al., 2001).

Una vez iniciada la reproducción sexual, el parásito adulto produce huevos

embrionados los cuales pasan a las vías biliares y son eliminados a través de las heces

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en las orillas de los ríos o lagunas. Los huevos son ingeridos por un caracol, que sirve
de primer hospedador intermediario. Cada huevo libera un miracidio, el cual atraviesa
varias etapas de desarrollo intramolusco (esporoquistes, redias y cercarias). Las
cercarias liberadas por el caracol tras un breve período de tiempo en el agua, penetran
en la carne de un pez de agua dulce que actúa como su segundo hospedador
intermediario (Figura 5).

Existen más de 100 especies de caracoles que pueden servir de hospedadores

intermediarios primarios de diferentes trematodos. Hasta el momento, desconocemos
que género y especie de caracol sirve como primer hospedador intermediario de
Amphimerus spp. A través de la técnica de PCR-RFLP, se han logrado identificar
metacercarias de Amphimerus spp. en cuatro especies de peces de agua dulce: Rhoadsia
altipinna (familia Characidae), Bryconamericus bucay, Andinoacara rivulatus (familia
Cichlidae) y Lebiasina aureoguttata (familia Piabucinae; Fowler, 1911) (Figura 6). Las
tasas de infección natural fueron del 80%, 10%, 18% y 1% respectivamente. La
identificación de estas 4 especies de peces transmisores de Amphimerus spp. que
incluyen al menos 3 familias taxonómicas, sugiere que este trematodo tiene varios
peces como hospedadores intermediarios secundarios (Calvopiña M, comunicación
personal).

Figura 5. Ciclo biológico de Amphimerus spp.

(Adaptado de http://www.dpd.cdc.gov/DPDx/HTML/Opisthorchiasis.htm).

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Figura 6. Peces de agua dulce involucrados en la transmisión de Amphimerus spp. en

Ecuador.

(Fotografía cortesía de Dr. Daniel Romero)

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1.5.6 Epidemiología

En la actualidad, la infección en humanos por Amphimerus spp. ha sido reportada

solamente en Ecuador (Calvopiña et al., 2011). Sin embargo, se han identificado
infecciones por el parásito en otros hospedadores vertebrados en diferentes países del
continente americano con el consiguiente riesgo de infección para la población humana
(Thatcher, 1970; Miyasaki et al., 1976; de Moraes et al., 1998). Así, existen informes en
Colombia y Perú donde se han identificado huevos pertenecientes a la familia
Opisthorchiidae en heces humanas semejantes a Amphimerus spp. (Restrepo M, 1962;
Beltran M. & Naquira C, 1999). Estas evidencias demuestran la importancia del estudio
de esta parasitosis en humanos en países vecinos a Ecuador.

Respecto a las especies de Amphimerus spp. identificadas en hospedadores no

humanos, en la figura 7 se muestra un mapa del continente americano con catorce
especies de parásitos descritas hasta la fecha. Esta información se completa con los
datos referidos en la tabla 4 en la que se detalla los hospedadores en los que se aislaron
las diferentes especies del parásito.

Al igual que en otras trematodosis de origen alimentario, la prevalencia e intensidad

de la amphimeriosis está determinada por la cultura profundamente arraigada entre
determinadas poblaciones de ingerir pescado de rio crudo o insuficientemente
cocinado. En zonas endémicas para C. sinensis y Opisthorchis spp. existe la creencia de
que el consumo de pescado crudo es altamente nutritivo (Phan et al. 2011; Qian et al.,
2013).

Entre los determinantes ambientales y ecológicos que afectarían la endemicidad de

esta parasitosis, sería de gran importancia conocer las especies de hospedadores
intermediarios primarios (caracoles terrestres) y secundarios (peces de agua dulce). A
día de hoy no se conocen los caracoles transmisores de Amphimerus spp., aunque en el
caso de trematodos afines como C. sinensis, las especies identificadas han sido
Parafossarulus striatulus, Bithynia fuchsiana, Alocinma longicornis y Melanoides
tuberculata (Lun et al., 2005; WHO, 1995). En cuanto al segundo hospedador
intermediario, estudios preliminares realizados por nuestro grupo dan cuenta de que
la mayoría de especies de peces infectadados con metacercarias de Amphimerus spp.
corresponden al género Rohadsia altipinna, pertenecientes a la familia Characidae
(datos no publicados). Animales domésticos como perros y gatos, sirven también como
hospedadores definitivos de Amphimerus spp., actuando tambien como reservorios,
pudiendo mantener el ciclo de vida del parásito sin la participación del hombre.

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Figura 7. Distribución geográfica de Amphimerus spp. en las Américas.

20
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Tabla 4. Distribución de Amphimerus spp. y sus hospedadores definitivos en las Américas.

Parásito Hospedador País (localidad) Registro

A. pseudofelineus No reportado Canadá (Lago Minatoba) Ward, 1901

Felis felis (Catus) Venezuela (Maracay- Mayaudon, 1969
domesticus Aragua)
Felis felis (Catus) Estados Unidos de
domesticus Norteamérica
A. speciosus No reportado Estados Unidos de Stiles & Hassall,
Norteamérica 1896
A. pseudofelineus Dildelphis marsupialis. Brasil (Amazonía) Artigas & Pérez,
minutus 1964
A. lancea Sotalia tucuxi y Nectomys Brasil (Amazonía, Goiás) Diesing, 1850
squamipes
A. parciovatus Dildelphis marsupialis Brasil (Belém do Pará) Franco, 1967
marsupialis L.
A. bragai Nectomys squamipes Brasil (Minas Gerais) de Moraes Neto A
et al., 1988
A. minutus Dildelphis marsupialis Perú (Huánuco) Artigas & Perez,
marsupialis 1964
A. ruparupa Philander opossum Perú (Huánuco y Loreto) Kifune & Uyema,
1976
O. guayaquilensis Cannis familiaris Ecuador (P.P. Gómez) Rodríguez et al.,
X Philander opossum L. & Colombia (Buenaventura, 1949
A. guayaquilensis Dildelphis marsupialis L. Valle y Antioquia) Thatcher V, 1970
Dildelphis marsupialis &
Felis felis (Catus) Panamá (Colón) Caballero et al.,
domesticus 1953

A. neotropicalis Philander fpossum L. & Colombia (Buga y Villa Thatcher V, 1970

Dildelphis marsupialis L. Carmelo, Valle) Caballero et al.,
Philander opossum Costa Rica 1963
Dildelphis peruguayensis Perú Miyazaki I, 1991

A. minimus Philander opossum L. Colombia (Buga, Valle) Thatcher, 1970

A. pricei Caluromys derbianus Panamá Foster, 1939

A. caudalitestis Chironectes minimus Panamá Caballero et al.,

1953
A. interruptus No reportada México Braun, 1901

A. elongatus Phalacrocorax auritus, Estados Unidos (Louisiana Pense & Childs,

birds. y Michigan) 1972
Ducks, swans Gower W, 1938
Amphimerus spp. Homo sapiens sapiens, Ecuador (Río Cayapas- Calvopiña et al.,
Cannis familiaris y Felis Esmeraldas) 2011
felis (Catus domesticus)

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1.5.7 Mecanismos patogénicos

Los mecanismos de agresión, defensa y evasión inmunológica desencadenados en la

amphimeriosis son desconocidos. Sin embargo, estos mecanismos están bien descritos
en otros trematodos de la familia Opisthorchiidae como C. sinensis y O. viverrini, los
cuales son responsables de producir infecciones de larga duración, e incluso
desencadenar carcinoma de vías biliares o colangiocarcinoma (CCA) (Choi et. al., 2004;
Sripa et al., 2007; Sripa et al., 2012).

Basados en estudios de estos trematodos, mayoritariamente realizados en modelos

experimentales, describiremos a continuación los diferentes mecanismos patogénicos
conocidos hasta el momento.

Mecanismos de agresión

Se pueden agrupar en tres acciones principales: (i) daño mecánico, (ii) efecto tóxico,
y (iii) proceso inflamatorio.

Es evidente que Amphimerus spp. debería ocasionar cambios en los conductos

biliares originando procesos obstructivos y de alteración de la mucosa. Daños
similares se han documentado en la infección por C. sinensis durante el proceso de
alimentación a través de su ventosa oral (Sripa et al., 2010).

Para sobrevivir largos períodos en entornos hostiles como el fluido biliar, los
trematodos hepáticos liberan activamente productos de excreción/secreción (ES) a
través del tegumento y del poro excretor, algunos de los cuales son altamente
inmunogénicos (Sripa et al., 2000; Mulvenna et al., 2010). Además, pueden ser tóxicos
o interactuar con el epitelio biliar para estimular la inflamación, promover la
proliferación y suprimir la apoptosis (Kim et al., 2008; 2009). Varios estudios
demuestran que los productos de ES del parásito adulto C. sinensis provocan cambios
en el perfil de expresión del transcriptoma, proteoma y micro ARN en células humanas
(HuCCT1) con CCA y en hígados de ratones (Pak et al., 2009; 2014). Además, estos
productos pueden producir hiperplasia de células biliares normales a células
adenomatosas con posterior transformación a CCA, debido a la alteración
transcripcional de genes diana carcinogénicos, tales como el gen Mcm7, a través de
modificaciones de histonas (Sripa et al., 2010). Con los recientes avances en la
caracterización del transcriptoma (Laha et al., 2007; Young et al., 2010) y del proteoma
de productos de ES de O. viverrini y C. sinensis (Mulvenna et al., 2010; Pak et al., 2009),
se han identificado varias moléculas del parásito con actividad carcinogénica
(Daorueang et al., 2012; Smout et al., 2009). Entre ellas destaca una proteína con gran
similitud al factor de crecimiento en mamíferos, denominada granulina (Ov-GNR-1).

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Esta proteína es el único factor de crecimiento derivado de helmintos reportado hasta

la fecha que puede causar la proliferación de células de mamíferos (Smout et al., 2009).

Una pro-granulina humana (PGRN), se asocia con muchos cánceres agresivos y se

sobre expresa en muchos tumores humanos (Bateman & Bennett; 1998). Se ha visto
además que la PGRN estimula la angiogénesis, suprime la apoptosis, promueve la
invasión tumoral y apoya la expansión del tumor en un entorno intersticial
desfavorable (Ong et al., 2003; Zanocco-Marani et al., 1999). De hecho, la expresión de
PGRN impulsada por IL-6 da como resultado un mayor crecimiento de CCA (Frampton
et al., 2012). Sería por tanto de gran interés conocer el genoma, transcriptoma y
proteoma de Amphimerus spp. con la finalidad de identificar moléculas clave en la
relación parásito-hospedador y en la posible dilucidación de un efecto carcinogénico.

Los Toll-like receptors (TLRs) son precursores muy potentes de la respuesta

inflamatoria y permiten el reconocimiento de patógenos incluso en la fase inicial de la
infección (Kawai & Akira; 2011). Sin embargo, en la inflamación prolongada existe una
producción excesiva de citocinas inflamatorias mediada por los TLRs, lo cual podría ser
perjudicial por causar toxicidad en el hospedador con el consiguiente daño tisular
(Sripa et al., 2012). En infecciones experimentales con C. sinensis, los TLR2 y TLR4
fueron altamente expresados en ratones, lo que indica una probable participación de
estos TLRs en la estimulación de la respuesta inmune innata (Yan et al., 2015). Además,
para estudiar las primeras etapas de la inmunopatología en el tracto biliar de las
personas infectadas con O. viverrini, se estimuló una línea celular de colangiocitos
humana normal (H69) con productos de ES del parásito, induciendo una mayor
expresión de TLR4 (Ninlawan et al., 2010). Por otro lado, diversos estudios han
demostrado niveles altos de IL-6 específica contra los productos de ES del parásito en
pacientes con fibrosis periductal avanzada (Sripa et al., 2009). Esta citocina también
podría estar implicada en otras anomalías hepatobiliares incluido el CCA (Sripa et al.,
2012). Estos trabajos indican nuevamente la importancia de estudiar la respuesta
inflamatoria desencadenada en la infección por Amphimerus spp. Una primera
limitación se debe a la inexistencia en la actualidad de modelos experimentales de
infección con este trematodo.

Mecanismos de defensa

Es bien conocido que la generación de respuestas inmunológicas Th1-dependientes,

están no solamente asociadas con la eliminación del parásito, sino también, con la
inducción de lesiones graves en el propio hospedador. Por el contrario, la activación de
la vía tipo Th2 conduce al desarrollo de respuestas inmunológicas beneficiosas para el
parásito, facilitando la progresión de la enfermedad (Liu & Ding; 2016). En este sentido,
la infección crónica producida por C. sinensis, se asocia predominantemente a

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respuestas tipo Th2, induciendo la supresión de citocinas involucradas en la respuesta

Th1 y por tanto facilitando la infección (Choi et al., 2003; Kim et al., 2012). En la misma
dirección, estudios realizados en animales de experimentación infectados con C.
sinensis indican que el incremento de las poblaciones linfocitarias Th2 y Treg juega un
papel decisivo en el desarrollo de fibrosis biliar (Zhang et al., 2017).

Aunque se han realizado trabajos para dilucidar el papel de las repuestas Th17 en
estas infecciones, se ha visto un incremento de estas poblaciones celulares en ratones
infectados con C. sinensis. Sin embargo, aún hacen falta más estudios para conocer con
exactitud la función específica de estas poblaciones linfocitarias en la defensa contra
estos trematodos (Yan et al., 2015).

Mecanismos de evasión

Existen escasos datos experimentales sobre los mecanismos de evasión de

trematodos de la familia Opistorchiidae. Sin embargo, estudios del proteoma de C.
sinensis ponen de manifiesto las diferencias moleculares existentes entre las fases
juveniles y los parásitos adultos (Jex et al., 2012). Esta composición y recambio es
crucial para desarrollar estrategias de evasión parasitaria.

Por otro lado, se han identificado proteasas y enzimas antioxidantes, altamente

expresadas en las fases adultas de estos trematodos. Estas moléculas pueden impedir
los ataques inmunológicos del hospedador inactivando los anticuerpos generados por
respuestas inmunológicas tipo Th2 (Yoo et al., 2011; Young et al., 2010).

1.5.8 Manifestaciones clínicas

Hasta la fecha, no existen descripciones exhaustivas de la sintomatología causada

por la infección de Amphimerus spp. A priori, la mayoría de las personas infectadas que
hemos observado en las zonas endémicas son asintomáticas (datos observacionales no
publicados). Si tenemos en cuenta los datos clínicos de las trematodosis afines como
clonorquiosis u opistorquiosis, algunos autores estiman que solo el 5 – 10% de las
personas infectadas presentan sintomatología inespecífica como dolor abdominal,
flatulencia o fatiga (Pungpak et al., 1983; Upatham et al., 1984). Sin embargo, hay que
tener en cuenta que estos síntomas pueden ser ocasionados por otro tipo de parasitosis
intestinal presente en zonas endémicas (Vasco et al., 2014).

En un estudio piloto realizado por nuestro grupo de investigación en nueve pacientes

con elevada carga parasitaria de Amphimerus spp, sólo dos tuvieron un leve aumento
de transaminasas hepáticas y un tinte subictérico de mucosa conjuntival. Se realizó
ecografía hepática, observando zonas hipodensas en vías biliares intrahepáticas
traduciendo una leve dilatación de las mismas (Figura 8). El resto de los pacientes no

24
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presentaron sintomatología o alteración de la función hepática (datos no publicados).

Los datos de laboratorio mostraron eosinofilia en tan solo un paciente con un 14,5% de
porcentaje de eosinófilos.

Figura 8. Ecografía hepática de paciente ecuatoriano infectado con Amphimerus spp. La

ecografía muestra la dilatación leve de vías biliares intrahepáticas en paciente de 47 años de
edad procedente de la zona endémica Chachi.

Las manifestaciones clínicas presentes en las trematodosis afines más estudiadas

indican que en pacientes con alta carga parasitaria (entre 100-1000 parásitos) los
síntomas que pueden presentarse son debilidad generalizada, malestar en epigastrio,
parestesias, pérdida de peso, taquicardia, diarrea, vértigo, y disfunción hepática
(Belding, 1965; Keiser & Utzinger; 2009). En pacientes con una elevadísima carga
parasitaria (>25.000 trematodos) los síntomas incluyen dolor en cuadrante superior
derecho, marcada disfunción gastrointestinal, hepatoesplenomegalia, ictericia, ascitis y
cirrosis portal (Keiser & Utzinger; 2009; Mas-Coma & Bergues; 1997). En este punto es
importante señalar que estudios histopatológicos realizados en gatos y en un cormorán
de doble cresta infectados con Amphimerus spp. se demostró la presencia de cirrosis
hepática y pancreatitis (Rothenbacher & Lindquist; 1963; Pense & Childs; 1972).

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No tenemos datos de complicaciones en las infecciones humanas producidas por

Amphimerus spp. Sin embargo, son bien conocidas las complicaciones de la infección
crónica por C. sinensis, como colecistitis, colangitis por Escherichia coli y más
frecuentemente colelitiasis (Qiao et al., 2014). Adicionalmente, pueden presentarse
casos de abscesos hepáticos y pancreatitis (Hou, 1955; Kim, 1999). En niños se ha
reportado diarrea, inapetencia, malnutrición, anemia y hepatomegalia, lo cual genera
retardo del crecimiento (Quian et al., 2016). Pero, sin lugar a dudas, la mayor
complicación de las infecciones por C. sinensis y O. viverrini es el desarrollo de CCA,
estando estos parásitos clasificados actualmente como agentes carcinogénicos del
grupo 1 por la Agencia Internacional para la Investigación del Cáncer (Bouvard et al.,
2009; Sripa, B et al., 2011).

1.5.9 Diagnóstico

1.5.9.1 Métodos parasitológicos

En la actualidad, el diagnóstico de las infecciones en los seres humanos por

Amphimerus spp. se basa en la observación microscópica directa de huevos del parásito
en las heces. La sensibilidad de este método diagnóstico es hasta diez veces menor
comparada con la técnica de concentración de formalina-éter (Calvopiña et al., 2011;
2015).

En un estudio preliminar realizado por nuestro grupo se comparó la sensibilidad de

cuatro métodos de detección de huevos en heces de Amphimerus spp. Los métodos
incluyeron la técnica de Kato-Katz, la técnica de sedimentación simple, la concentración
con formalina-éter y el examen en fresco mediante microscopía. La sensibilidad de los
métodos utilizados fue de 25,7%, 21%, 18% y 1% respectivamente (Calvopiña et al.,
2017; submitted).

Los huevos de otros parásitos de la familia Opisthorchiidae pueden ser detectados

en las heces, lo cual representa la mejor manera de obtener un diagnóstico definitivo,
aunque este enfoque se vuelve cada vez menos fiable en casos de cargas parasitarias
bajas (Johansen et al., 2010; Kim et al., 2011). Por lo tanto, pueden pasar desapercibidas
infecciones debidas a Amphimerus spp. o tener dificultades para realizar un diagnóstico
en zonas endémicas. El trabajo realizado por Calvopiña y colaboradores (2011) mostró
el 3,4% de positividad mediante el método de observación microscópica directa de
huevos de Amphimerus spp. en heces. Posteriormente y utilizando la técnica de
concentración de Ritchie mejoró la sensibilidad hasta alcanzar el 34%. Sin embargo,
esta técnica debe realizarse preferiblemente 2 o 3 veces en días diferentes, ya que la
eliminación del parásito no se produce de forma continua. Esto justifica el desarrollo de
técnicas en etapas tempranas de la infección posibilitando así su diagnóstico precoz.

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1.5.9.2 Técnicas inmunológicas

Hasta el momento no se dispone de métodos serológicos que permitan el diagnóstico

de la amphimeriosis. Las técnicas inmunológicas, tales como los métodos basados en
detección de anticuerpos que utilizan el ensayo inmunoabsorbente ligado a enzimas
(ELISA), han mostrado una alta sensibilidad y especificidad para diagnosticar diversas
infecciones parasitarias (Elkins et al., 1991; Guevara et al., 1995). De particular
importancia es la técnica de ELISA utilizada para detectar parásitos de la familia
Opisthorchiidae (Meniavtseva et al., 1996). Clonorchis sinensis y Opistorchis spp.
inducen niveles elevados de IgG en animales experimentales, lo cual es similar a las
observaciones en seres humanos (Elkins et al., 1991; Gómez-Morales et al., 2013).

Se han utilizado antígenos de extractos crudos de vermes adultos, aunque con

diferentes niveles de sensibilidad y especificidad (Sawangsoda et al., 2012;
Poogrushpong et al., 1990). Posteriormente se han empleado antígenos recombinantes
derivados de proteínas del tegumento (CsTP20.8) de C. sinensis, incrementando la
especificidad, pero disminuyendo la sensibilidad de la prueba (Quian et al., 2016).
También han sido utilizados antígenos recombinantes derivados de productos de ES de
C. sinensis, denominado GRCsP (proteína rica en glicina), cuyos resultados han sido
altamente específicos y sensibles (Kim et al., 2009).

En un trabajo reciente se han detectado antígenos circulantes de C. sinensis, útiles

para la evaluación de pacientes que han recibido tratamiento (Nie et al., 2014).

1.5.9.3 Diagnóstico molecular

Como se ha referido en apartados anteriores, el diagnóstico de la infección por

Amphimerus spp. generalmente se realiza mediante el examen microscópico de las
heces y la observación directa de los huevos del parásito (Calvopiña et al., 2011;
2015). Sin embargo, es difícil la diferenciación morfológica con otros huevos de
trematodos hepáticos afines como C. sinensis u Opisthorchis spp. y con trematodos
intestinales diminutos (Johansen et al., 2010). Por lo tanto, es necesario el desarrollo de
técnicas de diagnóstico directo e indirecto más sensibles y específicas que eviten
errores de identificación.

El uso de técnicas moleculares se ha convertido en una herramienta importante para

diferenciar especies estrechamente relacionadas. En los últimos años, se han
desarrollado varios métodos de diagnóstico molecular basados en la amplificación de
ADN mediante la reacción en cadena de la polimerasa (PCR) para la detección de
trematodos, incluidas las especies que están estrechamente relacionadas con
Amphimerus spp., como C. sinensis (Parvathi et al., 2007; Kim et al., 2009; Rahman et al.,
2011; Cai et al., 2012; Sanpool et al., 2012; Huang et al., 2012) y O. viverrini

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(Wongratanacheewin et al., 2002; Muller et al., 2007; Lovis et al., 2009; Kaewkong et
al., 2013). Aunque estos estudios han demostrado que los métodos basados en la PCR
son muy sensibles y específicos, aún no se utilizan de manera rutinaria en países de baja
renta ya que se necesita personal capacitado y equipos costosos, lo que los hace
inviables para su aplicación en condiciones de campo.

Por otro lado, hay que tener en cuenta que la sensibilidad obtenida mediante la
técnica PCR depende en la mayoría de los casos de la calidad del proceso de extracción
de los ácidos nucleicos, ya que se conocen diversos inhibidores de la Taq polimerasa
presentes en las heces. Además, algunos compuestos y derivados de los alimentos (p.e.
polifenoles, taninos, terpenos, polisacáridos y resinas) pueden limitar la extracción de
ADN (Arimatsu et al., 2012). Así, al realizar la PCR usando ADN extraído de parásitos
adultos, donde la contaminación es menor a la de otros compuestos, la reacción se
produce con una sensibilidad más elevada, mientras que si la reacción se lleva a cabo
con ADN obtenido a partir de heces, la sensibilidad se reduce considerablemente. Por
tanto, sería extremadamente difícil aplicar una técnica de PCR en condiciones de campo
ya que no se dispone de las condiciones necesarias para la realización de esta técnica
en sitios remotos.

Por estas razones, el desarrollo de una técnica de amplificación de ADN en

condiciones isotérmicas y de fácil implementación en el campo para el diagnóstico de
Amphimerus spp. es de gran importancia. En dicho contexto, en el año 2000, fue
desarrollada la técnica LAMP (Loop mediated isothermal amplification) la cual permite
una amplificación rápida y altamente específica de ADN en condiciones isotérmicas
(Notomi et al., 2000) (Figura 9). Diferentes estudios han demostrado su elevada
sensibilidad y especificidad en diversas enfermedades parasitarias (Aritmasu et al.,
2015), incluyendo varias especies de trematodos (Ai et al., 2010; Cai et al., 2010; Chen
et al., 2011; Fernández-Soto et al., 2014; Le et al., 2012). La detección visual mediante
turbidez o colorimetría y su sencillez en la realización le facilitan su aplicación en
condiciones de campo (Njiru, 2010; Mori et al., 2013).

Se ha desarrollado un método LAMP basado en la amplificación de secuencias

altamente conservadas del ITS-1 para la detección de
O. felineus, F. gigantica y Haplorchis spp. (Aritmasu et al., 2015). No obstante, sería
necesario diferenciar entre trematodos que cohabitan en una misma zona. En este
sentido, se ha desarrollado un LAMP frente a C. sinensis con alta sensibilidad,
amplificando el gen mitocondrial nad1 (Le et al., 2012) o el gen cox1 (Rahman et al.
2017). Además, un estudio usando microsatélites ha permitido diferenciar
entre Opisthorchis viverrini y O. felineus (Aritmasu et al., 2015).

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En Ecuador, las infecciones humanas con Fasciola hepatica y Paragonimus

mexicanus son endémicas en la misma zona donde se ha detectado Amphimerus
spp. Por tanto, sería de gran utilidad el desarrollo de un método LAMP para la detección
específica de la amphimeriosis. Además, disponer de un método molecular de fácil
aplicación en campo permitiría avanzar en el diagnóstico de esta parasitosis.

Figura 9. Representación esquemática de la reacción LAMP.

(Imagen tomada de Notomi et al., 2000).

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1.5.10 Tratamiento

Es conocido que el fármaco praziquantel (PZQ) destruye los ovarios y testículos de

los trematodos inhibiendo la producción de huevos por los parásitos adultos (Lee et al.,
1987), aunque su mecanismo preciso de acción es todavía poco comprendido. Tiene un
amplio espectro de actividad contra varios trematodos con un excelente perfil de
seguridad, lo que le convierte en un fármaco de elección para muchas trematodosis
(Keiser & Utzinger, 2004; Utzinger & Keiser, 2004). El PZQ es la droga de primera
elección en el manejo y tratamiento de las otras opistorquiosis producidas por C.
sinensis y Opisthorchis spp. Sin embargo, ya se han descrito resistencias en infecciones
por Schistosoma mansoni y S. japonicum (Chai, 2013).

En un estudio piloto realizado por nuestro grupo de investigación se utilizó PZQ a

dosis de 25mg/kg de peso, vía oral, tres veces al día durante tres días consecutivos en
pacientes con infección por Amphimuerus spp., obteniendo altas tasas de curación
clínica y parasitológica superiores al 95% (datos no publicados). Son necesarios más
estudios para evaluar la dosis, eficacia y posibles efectos adversos. A pesar de que este
fármaco consta en el del cuadro nacional de medicamentos básicos del Ecuador (CNMB,
2016), no se encuentra disponible en el mercado ecuatoriano.

1.5.11 Prevención y control.

Las intervenciones en Salud Pública para prevenir las infecciones por trematodos
transmitidos por alimentos deben incluir disponibilidad y acceso a la quimioterapia,
adecuado saneamiento e higiene, cambio de prácticas de uso y consumo de alimentos y
campañas de información, comunicación y educación. La implementación de un control
integrado que incluya todas estas medidas comparado con la utilización exclusiva de
tratamiento ha sido más eficaz en el control de O. viverrini en Tailandia, valorada
mediante la tasa de reinfección (Sornmani et al., 1984). En China, la prevalencia de
Paragonimus spp. fue reducida significativamente sobre todo con la implementación de
educación en salud (Blair et al., 2007). Adicionalmente, está bien documentado que la
mejora en el saneamiento e higiene influye en la prevalencia de estas enfermedades. Un
estudio realizado en Laos mostró una significativa asociación entre opistorquiosis y
escaso saneamiento e higiene en poblaciones de bajos recursos económicos (Sayasone
et al., 2007).

Los cambios culturales requieren de tiempo y perseverancia con la finalidad de

evitar las fuentes de contagio. Lo que se busca es un cambio de actitudes y
comportamientos culturales relacionados con la producción, preparación y consumo de
alimentos. Otro elemento que se debe tener en cuenta es lograr una seguridad
alimentaria que permita a las poblaciones en riesgo poder suplantar las fuentes de

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proteína animal en la dieta, siendo una acción necesaria que debe ser considerada por
los gobiernos locales y nacionales.

No hay vacunas contra ninguna de las trematodosis, incluida la amphimeriosis. Para

su desarrollo es imprescindible llegar a conocer los mecanismos patogénicos
involucrados en la infección, así como disponer de la secuenciación del genoma de
Amphimerus spp. y del conocimiento de su transcriptoma y proteoma.

1.6 Zona geográfica del estudio.

El área de estudio está situada en el noroeste de la República del Ecuador, cantón

Eloy Alfaro de la provincia de Esmeraldas (Figura 10). En este Cantón existen alrededor
de 150 comunidades. Los pueblos están situados a lo largo de tres ríos, el Río Cayapas,
Río Santiago y Río Onzole, ríos que desembocan en el estuario de la ciudad de Borbón,
y que reciben un sinnúmero de afluentes. Borbón es la ciudad más grande del área con
cerca de 5.000 habitantes. En total, aproximadamente 25.000 personas residen en la
región.

El estudio se realizó en tres comunidades indígenas Chachis a lo largo del río Cayapas
(Figura 11). Esta área forma parte del bosque lluvioso tropical llamado “Chocó
biogeográfico del Pacífico” que cubre parte de las costas de Ecuador, Colombia y
Panamá. Esta área ha sido catalogada como un “biological hotspot” (Myers et al., 2000),
es decir, una zona considerada de alta biodiversidad pero en peligro de extinción por la
rápida pérdida de sus recursos naturales. El clima es cálido y húmedo, con una
temperatura promedio de entre 24°C y 28°C y una humedad relativa promedio del 85%
(Sierra, 1999).

De acuerdo al censo nacional ecuatoriano del 2010, casi un 55% de la población se

autodefine como afroecuatoriano y un 13% como Chachi, el grupo indígena que
predomina en la región (Instituto Nacional de Estadísticas y Censo, 2001). Los demás
se autodefinen como mestizos, mulatos, blancos, cholos, categorías que refieren a
tendencias migratorias recientes provenientes de otras provincias del país (Minda,
2002). En general, los pueblos lejanos a Borbón tienen mayores porcentajes de
afroecuatorianos, excepto en los altos de los Ríos Cayapas y Onzole donde predominan
los Chachis. El promedio de años de educación varía entre 3,1 y 6 (Trostle et al., 2008).

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Figura 10. Localización geográfica del área de estudio en la provincia de Esmeraldas,

Ecuador, a 320 km desde la capital Quito y a 127 a 132 km del borde del Océano Pacifico.

Figura 11. Mapa del área donde se muestran en círculos rojos las 3 comunidades estudiadas
a lo largo del Río Cayapas y su afluente Río San Miguel.

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Los indígenas Chachis viven en comunidades remotas donde la única forma de llegar
es por canoa a lo largo del río (Figura 12). Mantienen una infraestructura poco
desarrollada con aguas residuales no tratadas que son eliminadas detrás de las casas,
rudimentarios sistemas de eliminación de residuos sólidos. Una de las principales
fuentes alimenticias es el pescado y crustáceos recolectados en los ríos principales y
sus afluentes.

Figura 12. Vista panorámica del acceso a las comunidades del Río Cayapas.

La fuente de su agua es principalmente de ríos y arroyos y la consumen sin

tratamiento, aunque el agua de lluvia se utiliza de manera intermitente; algunas
comunidades tienen pozos o reciben agua corriente de fuentes superficiales. Las
instalaciones de saneamiento son de calidad variable, aunque generalmente se
clasifican como no mejorados mediante criterios de la Organización Mundial de la
Salud; los inodoros son poco frecuentes.

Las personas son cazadoras y comen pescado poco cocinado y capturado en los ríos
vecinos casi todos los días y la comida se acompaña con arroz cocido y plátano (Figura
13). Mayor información de la zona de estudio y sus habitantes se encuentra muy bien
detallada en la literatura (Whitten, 1965; 1974; Sierra, 1998; 1999).

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Figura 13. Forma de preparación y consumo de alimentos en la población Chachi.

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45
Hipótesis y Objetivos

2. HIPÓTESIS Y OBJETIVOS

46
Hipótesis y Objetivos

2.1 Hipótesis

De la revisión bibliográfica podemos extraer las siguientes conclusiones: (i) aunque

la amphimeriosis ha sido descrita en casos aislados, se desconoce actualmente su
situación epidemiológica y, (ii) los métodos de diagnóstico parasitológicos clásicos son
insuficientes para conocer el alcance real de esta infección.

Por lo tanto, la hipótesis de esta tesis doctoral es probar que la amphimeriosis es una
zoonosis prevalente en comunidades con hábitos alimentarios de consumo de peces de
agua dulce crudos o insuficientemente cocinados. Para ello es necesario disponer de
nuevas herramientas más útiles para su diagnóstico.

2.2 Objetivo general

Estudio epidemiológico y desarrollo de técnicas de diagnóstico inmunológico y

molecular para la evaluación de la amphimeriosis en Ecuador.

2.2.1 Objetivos específicos

1. Determinar la prevalencia de la infección por Amphimerus spp., en población

indígena residente en zonas del noroeste de Ecuador.
2. Establecer si la amphimeriosis es una zoonosis, mediante el análisis de muestras
de heces de animales domésticos en la misma zona de estudio.
3. Desarrollar, estandarizar y aplicar una técnica diagnóstica basada en el análisis
inmunoenzimático tipo ELISA que permita la detección de IgG específicas anti-
Amphimerus spp. en sueros humanos.
4. Diseñar, desarrollar y evaluar un método molecular tipo LAMP para la detección
de ADN de Amphimerus spp. en muestras de heces humanas.
5. Definir la viabilidad del método LAMP para la detección de Amphimerus spp. en
muestras de heces utilizando papel de filtro como soporte.

47
Artículos de Investigación

3. ARTÍCULOS DE
INVESTIGACIÓN

48
Artículos de Investigación

3.1 ARTÍCULO 1: High prevalence of human liver infection by

Amphimerus spp. Flukes, Ecuador.
Manuel Calvopiña, William Cevallos, Joseph Eisenberg.

Emerging Infectious Diseases Vol. 17. No. 12. 2011.

RESUMEN

Es conocido que el trematodo Amphimerus spp. infecta a mamíferos, pero las

infecciones en humanos aún no han sido confirmadas. La microscopía de muestras
fecales de 297 personas de Ecuador, reveló la presencia de huevos de la familia
Opisthorchiidae en 71 (24%) personas. La microscopía óptica de parásitos adultos y la
microscopía electrónica de barrido de huevos fueron compatibles con las descripciones
de Amphimerus spp. Este patógeno solo se observó en las comunidades que
consumieron pescado mal cocinado.

49
 High Prevalence high prevalence of human infection with a trematode of the
genus Amphimerus in Ecuador.
of Human Liver
Infection by
The Study
In June 2009, during a routine fecal examination

Amphimerus spp.
for the parent study, 4 samples tested positive for eggs
of the Opisthorchiidae family in 3 indigenous Chachi

Flukes, Ecuador
communities along the Cayapas River in the northern
coastal rainforest of Ecuador. In January 2010, a follow-
up survey was conducted in the same 3 communities (total
Manuel Calvopiña, William Cevallos, population 589); all villagers, whether symptomatic or
Hideo Kumazawa, and Joseph Eisenberg not, were asked to provide a fecal sample. Specifically,
a community meeting was held in each village, study
Amphimerus spp. flukes are known to infect mammals,
objectives were explained, and villagers were asked for
but human infections have not been confirmed. Microscopy
of fecal samples from 297 persons from Ecuador revealed
their voluntary participation. Flasks were distributed to
Opisthorchiidae eggs in 71 (24%) persons. Light microscopy all villagers and collected the next day in the school and
of adult worms and scanning electron microscopy of eggs by going house to house. The Chachis, the predominant
were compatible with descriptions of Amphimerus spp. This group in these 3 communities, represent 13% of the 24,000
pathogen was only observed in communities that consumed inhabitants in the region. Afro-Ecuadorians and mestizos
undercooked fish. also reside in this region (10,11).
A total of 297 (50.4%) community members 3–77
years of age provided samples. To each person providing a
T he genus Amphimerus Barker 1911 infects mammals
from the Americas, including Canada, the United
States, Costa Rica, Panama, Colombia, Brazil, and Peru.
sample, a questionnaire was administered regarding types of
food eaten and cooking practices. Samples were preserved
in 10% formalin, transported to a laboratory in Quito, and
Eleven species are reported (1–7). In Ecuador, a trematode
stored at 4°C until examination by light microscopy. Eggs
resembling Amphimerus spp. but identified as Opisthorchis
were concentrated by using the formalin-ether technique.
guayaquilensis has been reported (8,9).
In addition, 120 fecal samples from Afro-Ecuadorian
Amphimerus spp. are parasitic liver flukes in the bile
and mestizo persons were examined. The villagers were
ducts of mammals, birds, and reptiles (1). Although these
informed of the study in their local Chapalache language
digenetic trematodes of the Opisthorchiidae family are
by community health community workers. The ethical
closely related to the genera Clonorchis and Opisthorchis,
committee of the Central University approved this study.
there are morphologic differences. The vitellaria in adult
Duodendoscopy was performed in 4 patients by a
Amphimerus spp. trematodes are distributed in 4 groups, 2
gastroenterology specialist to examine the biliary liquid;
anterior and 2 posterior; the latter groups extend beyond the
the microscopy of this liquid showed eggs identical to those
posterior testis; the ventral sucker is larger than the oral, and
found in their feces. These patients received praziquantel
the testes are rounded or slightly lobulated. In contrast, the
(75 mg/kg in 3 doses/3 d), and were purged with 10 mg
vitellaria in Clonorchis and Opisthorchis spp. trematodes
of bisacodilo. Fecal samples were collected and examined
exist only in front of the testes. Additionally, Clonorchis
for worms as previously described (12). Recovered
spp. trematodes have 2 large highly branched testes; testes
worms were fixed with 10% formalin, stained with Diff-
in Opisthorchis spp. worms are always lobulated (1,2). The
Quik fixative (Sysmex, Kobe, Japan), and identified by
eggs of the flukes from these genera can be differentiated
comparing their morphologic features to known adult
only by using scanning electron microscopy (SEM).
Clonorchis and Opisthorchis spp. worms. Community
Definitive diagnosis using light microscopy of flukes
health workers collected and examined the livers of 3
of the Opisthorchiidae family, therefore, is not possible
cats and 3 dogs from 1 of the 3 communities. All 6 livers
unless the adult worm is collected and identified. Through
had eggs and high numbers of adult parasites in the bile
isolation of adult worms and SEM of eggs, we found a
ducts. Adult parasites were stained, and microscopic
studies showed them to be identical to those in the human
Author affiliations: Universidad Central del Ecuador Centro de
specimens.
Biomedicina, Quito, Ecuador (M. Calvopina, W. Cevallos); Kochi
A total of 71 (24%) of the 297 fecal samples from
University School of Medicine, Kochi, Japan (H. Kumazawa); and
the indigenous Chachi were positive for Opisthorchiidae
University of Michigan, Ann Arbor, Michigan, USA (J. Eisenberg)
eggs (Table). In contrast all 120 samples from Afro-
DOI: http://dx.doi.org/10.3201/eid1712.110373 Ecuadorian and mestizo persons were negative. Eggs

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 17, No. 12, December 2011 2331
DISPATCHES

were yellow-brown and measured 28–33 μm ×12–15 μm Table. Prevalence of Amphimerus eggs in feces in 3 villages,
(n = 20). The operculum and the shoulders, however, were Ecuador
not prominent as they are in Clonorchis and Opisthorchis No.
eggs. Occasionally, a small knob, but most frequently a Total samples No. (%) Distance to the
Village population examined positive coast, km
curved spine, was seen on the abopercular end. Although, 1 116 82 28 (34.1) 120
by light microscopy, the shape and size of the eggs 2 248 86 23 (26.7) 91
resembled that of the other liver flukes, the patterns of 3 253 129 20 (15.5) 85
the eggshell surface were distinct when viewed with Total 617 297 71 (23.9)
SEM (Figure 1). This observation is corroborated with
published photographs (3). spp. trematodes are believed to be transmitted, as are
After participants were treated with praziquantel, a the other members of the Opisthorchiidae family, by
total of 8 worms were recovered from 4 human participants ingestion of raw or undercooked fish (2). In our survey,
and dozens from cat and dog livers; all were placed in most Chachis reported eating smoked fish caught in the
saline. The worms were delicate, leaf-shaped, elongated, rivers. Food sharing is more common among Chachi than
and red-pink and measured 8–13.6 mm long (average 10.2 Afro-Ecuadorians and mestizo families (13). Notably, the
mm) × 0.5–1.1 mm wide (n = 15). After a few minutes, most remote village (120 km inland from the coast) had the
the worms coiled in an S shape and became transparent highest prevalence. Our results suggest that Amphimerus
or whitish. Once stained, the following features were spp. flukes are zoonotic pathogens of domestic animals
observed: 1) the vitellaria divided into an anterior and living with humans.
posterior group with the posterior group extending the level Amphimeriasis should be considered an endemic liver
of the posterior testis; 2) a ventral sucker larger than oral fluke infection among residents of this Chachi population
sucker; and 3) 2 rounded testes (Figure 2). On the basis in Ecuador. Further studies are needed to determine the
of these morphologic characteristics of the adults and complete epidemiology and geographic distribution of
the SEM findings of the eggs, the parasite was identified infection in this region, as well as in other provinces of
as Amphimerus spp. Ecuador where freshwater fish is eaten undercooked or
where the same tropical ecology is found. For example,
Conclusions the Amazonian region has indigenous groups where other
Our study demonstrates that the liver fluke of the foodborne trematodiasis-like paragonimiasis are endemic
genus Amphimerus can infect humans. We found a high (14). Amphimerus spp. flukes infecting domestic and wild
prevalence (15.5%–34.1%) of infection with Amphimerus animals have been reported from Ecuador’s neighboring
spp. trematodes in the surveyed communities (Table). countries as well as from Central and North America.
Samples from the Afro-Ecuadorian and mestizo population The existence of undiscovered foci of human infections is
were all negative for Opisthorchiidae eggs. Amphimerus possible.
Figure 1. Scanning electron
microscopy images of A)
an egg of the Ecuadorian
Amphimerus spp. trema-
tode (original magnification
×3) obtained from a
human and B) an egg
of the Asian Clonorchis
sinensis trematode (original
magnification ×4). Although
the size is similar, the
pattern of the surface is
different, thus differentiating
the 2 genera.

2332 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 17, No. 12, December 2011
 Human Liver Infection by Amphimerus spp.

This study was supported by a grant from the US National

Institute of Allergy and Infectious Disease, National Institutes of
Health, grant no. RO1-AI050038.
Dr Calvopiña is a professor in the Department of Molecular
Parasitology and Tropical Medicine, Centro de Biomedicina,
Universidad Central del Ecuador, Quito, Ecuador. His major
research interest is parasitic diseases, including leishmaniasis,
paragonimiasis, onchocerciasis, and intestinal parasite infections.

References

1. Yamaguti S. Synopsis of the digenetic trematodes of vertebrates.

Vols. 1 and II. Tokyo: Keigaku Co; 1971. p. 1074.
2. Bowman DD. Amphimerus pseudofelineus (Ward 1901) Barker,
1911. In: Feline clinical parasitology. 1st ed. Ames (IA): Iowa State
University Press; 2002. p. 151–53.
3. Miyazaki I, Kifune T, Habe S, Uyema N. Reports of Fukuoka Uni-
versity scientific expedition to Peru, 1976. Department of Parisi-
tology, School of Medicine Fukuoka University, Fukuoka, Japan.
1978;1:1–28.
4. Rivillas C, Caro F, Carvajal H, Velez I. Algunos trematodos dige-
neos (Rhopaliasidae, Opistorchiidae) de Phillander Opossum (Mar-
supialia, mammalia) de la Costa Pacifica Colombiana, Incluyendo
Rhopalias caucensis N.SP. 2004 [cited 2011 Oct 20]. http://www.
Figure 2 . Amphimerus spp. adult trematode (10.1 mm) recovered accefyn.org.co/revista/Vol_28/109/14_591_600
from a human, Ecuador. 5. de Moraes Neto AHA, Thatcher VE, Lanfredi RM. Amphimerus br-
agai N.sp. (Digenea: Opisthorchiidae), a parasite of the rodent Nec-
tomys squamipes (Cricetidae) from Minas Gerais, Brazil. Mem Inst
Oswaldo Cruz (Rio de Janeiro). 1998;93:181–86.
In 1971, Yamaguti (1) suggested that a parasite 6. Artigas PT, Perez MD. Consideracoes sobre Opisthorchis pricei Fos-
ter 1939, O. guayaquilensis Rodriguez, Gomez e Montalvan 1949 e
previously reported in Ecuador (8) as O. guayaquilensis
O. pseudofelineus Ward 1901. Descricao de Amphimerus pseudofel-
might in fact be Amphimerus spp. Subsequently, publications ineus minumus n. sub. sp. Mem Inst Butantan. 1962;30:157–66.
referred to this parasite as A. guayaquilensis (5,7); however, 7. Thatcher VE. The genus Amphimerus Barker, 1911. (Trematoda:
the accuracy of this reclassification is unclear. Molecular Opisthorchiidae) in Colombia with the description of a new spe-
cies. Proceedings of the Helminthological Society of Washington.
analysis could help clarify the ambiguities in genus/species
1970;37:207–11.
identification of O. guayaquilensis parasites and the 8. Rodriguez JD, Gomez-Lince LF, El Montalvan JA. Opisthorchis
conspecific species of Amphimerus (15). guayaquilensis. Rev Ecuat Hig Med Trop. 1949;6:11–24.
We have much to learn about the pathology and 9. Moreira J, Gobbo M, Robinson F, Caicedo C, Montalvo G, Anselmi
M. Opistorquiasis en Esmeraldas: Hallazgo casual o problema de im-
epidemiology of Amphimerus spp. flukes. For example,
portancia epidemiológica? Boletin Epidemiologico. 2008;5:24–30.
nothing is known about the clinical and pathologic 10. Instituto Nacional Ecuatoriano de Censos. VI Censo de población y
significance of infections with this parasite. Praziquantel de vivienda [Sixth census of population and housing]. Quito, Ecua-
eliminated the parasites in these patients, but whether dor: Instituto Nacional de Estadisticas y Censo; 2001.
11. Eisenberg JN, Cevallos W, Ponce K, Levy K, Bates SJ, Scott JC, et al.
the dose and treatment time were adequate are unknown.
Environmental change and infectious disease: how new roads affect
Additionally, little is known about epidemiologic factors the transmission of diarrheal pathogens in rural Ecuador. Proc Natl
responsible for the differences in the number of infections Acad Sci U S A. 2006;103:19460–5. doi:10.1073/pnas.0609431104
among the different population groups. Future studies 12. Chai JY, Park JH, Han ET, Guk SM, Shin EH, Lin A, et al. Mixed
infections with Opisthorchis viverrini and intestinal flukes in resi-
can help determine the direct and indirect public health
dents of Vientiane Municipality and Saravane Province in Laos. J
implications of this new foodborne zoonosis. Helminthol. 2005;79:283–9. doi:10.1079/JOH2005302
13. Trostle JA, Hubbard A, Scott J, Cevallos W, Bates SJ, Eisenberg JN.
Acknowledgments Raising the level of analysis of food-borne outbreaks: food-sharing
networks in rural coastal Ecuador. Epidemiology. 2008;19:384–90.
We thank the community health workers of Borbon and Rio doi:10.1097/EDE.0b013e31816a9db0
Cayapas, Esmeraldas, for informing study participants, preparing 14. Calvopiña M, Guderian RH, Paredes W, Cooper PJ. Comparision
and obtaining the consent, and translating to the local language of two single-day regimens of triclabendazole for the treatment of
in the communities surveyed. We also thank Jeyson Abarca for human pulmonary paragonimiasis. Trans R Soc Trop Med Hyg.
2003;97:451–4. doi:10.1016/S0035-9203(03)90088-5
performing duodendoscopy and Ronald Guderian for revising the
manuscript.

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 17, No. 12, December 2011 2333
DISPATCHES

15. Park GM. Genetic comparison of liver flukes, Clonorchis sinensis Address for correspondence: Manuel Calvopiña, Department of Molecular
and Opisthorchis viverrini, based on rDNA and mtDNA gene se- Parasitology and Tropical Medicine, Centro de Biomedicina, Universidad
quences. Parasitol Res. 2007;100:351–7. doi:10.1007/s00436-006-
Central del Ecuador, Sodiro N14-121 e Iquique, Quito, Ecuador; email:
0269-x
[email protected]

2334 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 17, No. 12, December 2011
Artículos de Investigación

3.2 ARTÍCULO 2: High prevalence of the liver fluke Amphimerus spp.

in domestic cats and dogs in an area for human amphimeriasis in
Ecuador.
Manuel Calvopiña, William Cevallos, Richard Atherton, Matthew Saunders, Alexander
Small, Hideo Kumazawa, Hiromu Sugiyama.

PLoS Negl Trop Dis. 2015 Feb 3; 9 (2): e0003526.

RESUMEN

Recientemente, hemos demostrado que Amphimerus spp. es un parásito trematodo

de vías biliares, el cual presenta una alta prevalencia de infección entre el grupo
indígena Chachi, residente en la selva tropical, ubicada en el noroeste de la provincia de
Esmeraldas, Ecuador, Sudamérica. Al momento, desconocemos los animales que
pueden actuar como reservorios y/u hospedadores definitivos para Amphimerus spp.
En esta zona endémica, se realizó un estudio para determinar la prevalencia de
infección en gatos domésticos y perros. Esta información es de vital importancia para
entender la epidemiología, el ciclo de vida y el control de este parásito.

En julio de 2012, se realizaron encuestas en tres comunidades Chachis localizadas

en el Río Cayapas, provincia de Esmeraldas, Ecuador. Un total de 89 de los 109 hogares
registrados participaron en el estudio. De los 27 gatos y 43 perros encontrados que
vivían en las comunidades, se recogieron muestras de heces de 14 gatos y 31 perros. Se
examinó microscópicamente la presencia de huevos de Amphimerus spp. La prevalencia
de infección fue de 71,4% en gatos y 38,7% en perros, con índices similares de infección
en las tres comunidades. Significativamente más gatos que perros fueron los infectados
por el parásito (p = 0.042).

Los datos muestran una alta tasa de infección por Amphimerus spp. en gatos
domésticos y perros que residen en las comunidades Chachis. Se puede concluir que
estos animales actúan como hospedadores definitivos y reservorios del parásito y por
tanto que la amphimeriosis es una enfermedad zoonótica. Estos hallazgos proporcionan
importantes datos epidemiológicos que ayudarán en el desarrollo e implementación de
estrategias de control contra la transmisión de Amphimerus.

54
 RESEARCH ARTICLE

High Prevalence of the Liver Fluke

Amphimerus sp. in Domestic Cats and Dogs
in an Area for Human Amphimeriasis in
Ecuador
Manuel Calvopiña1‡*, William Cevallos1‡, Richard Atherton1, Matthew Saunders2,
Alexander Small3, Hideo Kumazawa4, Hiromu Sugiyama5
1 Centro de Biomedicina, Carrera de Medicina, Universidad Central, Quito, Ecuador, 2 Royal Liverpool
University Hospital, Liverpool, United Kingdom, 3 Sandwell and West Birmingham Hospitals NHS Trust,
Birmingham, United Kingdom, 4 Department of Parasitology Kochi Medical School, Kochi, Japan,
5 Department of Parasitology, National Institute of Infectious Diseases, Tokyo, Japan

‡ These authors contributed equally to this work.

* [email protected]

OPEN ACCESS

Citation: Calvopiña M, Cevallos W, Atherton R, Abstract

Saunders M, Small A, Kumazawa H, et al. (2015)
High Prevalence of the Liver Fluke Amphimerus sp. Background
in Domestic Cats and Dogs in an Area for Human
Amphimeriasis in Ecuador. PLoS Negl Trop Dis 9(2):
Amphimerus sp. is a liver fluke which recently has been shown to have a high prevalence of
e0003526. doi:10.1371/journal.pntd.0003526 infection among an indigenous group, Chachi, who reside in a tropical rainforest in the
Editor: Hector H Garcia, Universidad Peruana
northwestern region of Ecuador. Since it is unknown which animals can act as a reservoir
Cayetano Heredia, PERU and/or definitive hosts for Amphimerus sp. in this endemic area, a study was done to deter-
Received: June 18, 2014
mine the prevalence of infection in domestic cats and dogs. This information is important to
understand the epidemiology, life cycle and control of this parasite.
Accepted: January 8, 2015

Published: February 3, 2015 Methodology/Findings

Copyright: © 2015 Calvopiña et al. This is an open In July 2012, three Chachi communities located on Rio Cayapas, province of Esmeraldas,
access article distributed under the terms of the
were surveyed. A total of 89 of the 109 registered households participated in the study. Of
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any the 27 cats and 43 dogs found residing in the communities, stool samples were collected
medium, provided the original author and source are from 14 cats and 31 dogs (total of 45 animals) and examined microscopically for the pres-
credited. ence of Amphimerus eggs. The prevalence of infection was 71.4% in cats and 38.7% in
Data Availability Statement: All sequences of the dogs, with similar rates of infection in all three communities. Significantly more cats were in-
ribosomal DNA ITS2 region of the flukes are available fected than dogs (p = 0.042).
in GenBank (Accession numbers, deposited in the
GenBank/EMBL/DDBJ nucleotide database, are
AB678442, AB926429 and AB926430 for Conclusions/Significance
Amphimerus sp from humans, cats and dogs, The data show a high rate of Amphimerus sp. infection in domestic cats and dogs residing
respectively.)
in Chachi communities. It can be concluded that these animals act as definitive and reser-
Funding: This study was funded by the grants from voir hosts for this liver fluke and that amphimeriasis is a zoonotic disease. These findings
the Universidad Central del Ecuador (CUP
provide important epidemiological data which will aid in the development and implementa-
91750000.0000.374072) and in part by the Japan
Society for the Promotion of Science, JSPS tion of control strategies against the transmission of Amphimerus.
(KAKENHI: Grant No. 25305011) and by grants for

PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0003526 February 3, 2015 1/9

 Amphimerus sp. Fluke in Ecuador

Research on Emerging and Re-emerging Infectious

Diseases (H23-Shinko-ippan-014 and H26-Shinko- Author Summary
ippan-009) from the Ministry of Health, Labor and
Welfare of Japan. The funders had no role in study Amphimerus sp. is a fluke that infects the bile ducts of its definitive hosts. Recently, it has
design, data collection and analysis, decision to been shown that an indigenous Amerindian group, the Chachi, living in a rural and re-
publish, or preparation of the manuscript. mote tropical area of Ecuador, are infected with this parasite. The epidemiology and life
Competing Interests: The authors have declared cycle of this parasite remains elusive, and research is needed to understand the mode of
that no competing interests exist. transmission and zoonotic potential of the parasite. It was hypothesized that the domestic
animals of the Chachi households may act as definitive and reservoir hosts for Amphi-
merus infection. Hence, the presence and prevalence of infection in these animals residing
in communities endemic for human amphimeriasis was investigated. Some 45 animal
stool samples were examined microscopically for the presence of Amphimerus eggs. The
results showed an infection rate of 71.4% in cats and 38.7% in dogs. The data provided evi-
dence that these domestic animals act as both definitive and reservoir hosts for the parasite
and that amphimeriasis is a zoonotic disease. The implementation of a mass treatment/
control program must target both humans and animals in order to minimize the transmis-
sion of this liver fluke.

Introduction
Amphimerus Barker, 1911 is a genus of parasitic liver fluke which are flat helminths (Platyhel-
minthes) of the Trematoda class and belong to the Opisthorchiidae family. The adult flukes re-
side within the bile ducts of a definitive host [1]. Infection by liver flukes of this family, which
include Clonorchis sinensis and Opisthorchis spp. can occur through the consumption of raw or
undercooked, metacercariae infected freshwater fish [1–5]. Liver fluke infection is one of the
more important food-borne diseases worldwide and is considered by the World Health Orga-
nization as a neglected tropical disease [6]. Affected individuals with liver flukes of the
Opisthorchiidae family can suffer from suppurative cholangitis, cholelithiasis and cholangio-
carcinoma [3,5,6].
In the Americas, ten species of Amphimerus which infect mammals have been described:
A. pseudofelineus, A. pseudofelineus minutus, A. caudalitestis, A. price, A. lancea, A. parciovatus,
A. bragai, A. minimus, A. neotropicalis and A. ruparupa [7,8]. In Ecuador, flukes found in the
bile ducts of dogs were previously described as Opisthorchis guayaquilensis [9] but later, this
species, without any further comparative studies was named as being Amphimerus guayaqui-
lensis. However, Artigas and Perez (1964) considered to A. guayaquilensis to be synonym of
A. pseudofelineus. A few years later, A. guayaquilensis was considered to be distinct from
A. pseudofelineus and was instead regarded as a synonym of A. parciovatus [7]. Furthermore,
Thatcher (1970) did not agree with this synonymy and contemplated A. guayaquilensis distinct
from A. pseudofelineus because of the extent of the vitellaria. The validity of some species in
this genus is controversial, since speciation is based only on morphological and morphometric
features present in the adult flukes and the assignment of species names must be regarded
as speculative.
Amphimerus spp. have been demonstrated to infect birds, reptiles and certain mammals in-
cluding cats, dogs, ducks, the double-crested cormorant, Amazonian dolphins, opossums
(Didelphis marsupialis, Philander opossum) and the rodent Nectomys squamipes [1,9–16]. For
other members of the Opisthorchiidae family known to infect humans e.g. C. sinensis and
Opisthorchis spp., cats and dogs are the most important animal reservoirs for human infection
[2,3,17,18]. However, it is currently unknown whether domestic animals may act as a definitive
and/or reservoir host for human transmission in the recently reported focus of infection in

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 Amphimerus sp. Fluke in Ecuador

Ecuador [10]. Given the similarities between C. sinensis, Opisthorchis spp. and Amphimerus
spp., it was hypothesized that these mammals may also act as reservoirs for Amphimerus sp. in-
fection in Ecuador.
The indigenous group, Chachi, who live along the Rio Cayapas and its tributaries in the
north-western coastal rainforest of Ecuador, have been shown to have a high prevalence of in-
fection (15.5% to 34.1%) with Amphimerus sp. [10]. Afro-Ecuadorian and mestizo populations
living in separate communities but along the same rivers were not found to be infected [10].
The solo infection of the Chachi population was postulated to be related to their cultural tradi-
tion of eating smoked fish and food sharing [10,11].
In a previous pilot study, conducted by the authors, in the same endemic communities for
human infection, both cats and dogs were found to be positive for Amphimerus eggs. There
was no evidence of infection in any other animals investigated (e.g. pigs and chickens). The ob-
jective of this study was, therefore, to investigate the prevalence of infection of Amphimerus sp.
in domestic cats and dogs and to determine their role in the transmission in the Ecuadorian vil-
lages endemic for human infection.

Materials and Methods

Study area
The study was conducted in three indigenous Chachi communities along the Rio Cayapas in the
Province of Esmeraldas, located in the northwest coastal rainforest of Ecuador (Fig. 1). The in-
digenous Chachi, living alongside Afro-ecuadorian and mestizo populations, is the predominant
ethnic group in this area, and represent 13% of the inhabitants in this region of Ecuador [19,20].
These communities are the same as those studied previously, showing the resident Chachi hav-
ing a high prevalence of infection with Amphimerus sp. [10]. They live in remote villages where
the only way to reach the communities are by boat along the river; the source of their water is
mainly from rivers and streams and is consumed untreated. Sanitation facilities are lacking, and
flush toilets are uncommon. The people are hunters and eat fish caught in the neighboring rivers
almost every day and the meal is accompanied with cooked rice and plantain.
The province of Esmeraldas, forms part of the tropical rainforest known as “Choco Biogeo-
gráfico del Pacífico” which covers a section of the coast of Ecuador, Colombia and Panamá.
This area has been labelled as a biological hotspot; an area with an extraordinary concentration
of animal species [21]. The climate of this region is warm and humid, with an average tempera-
ture of between 24°C and 28°C and an average relative humidity of 85% [22].

Sampling
This study was based on a previous census conducted in January 2012 where each household
was given an identification number and a total of 109 households were recorded in the three
communities. In July 2012, all house owners were asked to participate in the study by providing
a stool sample from any cats and/or dogs residing in the respective household, simultaneously
a census of dogs and cats residing in the participating communities was conducted. A team of
community health workers informed the villagers of the study in their local Chapalache lan-
guage and residents were free to refuse entry to their household or access to their domestic ani-
mals at any point during data collection.

Sample processing
Stool samples were collected by their owners from each animal directly after the deposit was
made. Plastic flasks containing stool samples were labelled with type of animal, house code and

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 Amphimerus sp. Fluke in Ecuador

Fig 1. Map of the study area. It showed the geographical location of the study area in the Canton Eloy
Alfaro, province of Esmeraldas, 320 km from the capital Quito. In red circles are the 3 communities studied
along the Rio Cayapas and its tributary Rio San Miguel.
doi:10.1371/journal.pntd.0003526.g001

date. The samples were preserved in 10% formalin and transported to the parasitology labora-
tory at Centro de Biomedicina in Quito where they were stored at 4°C until they were exam-
ined. Samples were concentrated using the formalin-ether technique as previously described
[10] and were examined under light microscopy by at least two laboratory technicians for the
presence of Amphimerus eggs. Samples were then verified by an expert in animal stool exami-
nation who was not involved in the data collection. The primary outcome variable was Amphi-
merus infection, defined as positive if eggs of the parasite were visualized by light microscopy.
The yellow-brown eggs measured 20 to 32 um in length and 14 to 16 um in width; they are pyr-
iform with a visible operculum at the narrower anterior end. In the centre of the posterior end
there is a small bud (Fig. 2).
The adult parasites were obtained from the livers of two cats and one dog. Animals were
presented by their owners as sick and showed severe emaciation. They were euthanized with
ether and necropsied. The livers were collected in saline solution, squeezed and sliced in small

PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0003526 February 3, 2015 4/9

 Amphimerus sp. Fluke in Ecuador

Fig 2. An egg of Amphimerus sp. observed in stools from a cat. It is seen to be morphologically similar, using light microscopy, to eggs of Clonorchis
sinensis and Opisthorchis spp. (dimensions 31 μm × 15 μm).
doi:10.1371/journal.pntd.0003526.g002

pieces and maintained for 30 minutes. Flukes were then removed from the saline and were
fixed in both 70% ethanol and 10% formalin.

Molecular characterization
For molecular analysis, genomic DNA samples were extracted from each of the Amphimerus
adult specimens from the cats and dogs using a DNeasy Blood & Tissue Kit (QIAGEN K. K.,
Tokyo, Japan). The ITS2 region of the ribosomal DNA was then amplified by PCR using Ex
Taq DNA polymerase (Takara Bio, Shiga, Japan). The primers used were 3S (forward, 50 -
GGTACCGGTGGATCACTCGGCTCGTG-30 ) [23] and A28 (reverse, 50 -GGGATCCTGGT-
TAGTTTCTTTTCCTCCGC-30 ) [24]. DNA sequencing of amplicons was performed with a
3100-Advant Genetic Analyzer (Life Technologies, Foster City, CA, USA).

Statistical analysis
Data was analysed using SPSS version 19 (Statistical Product and Service Solutions, Chicago, IL,
USA). The data was stratified by village and animal species, and prevalence of Amphimerus in-
fection in the animals calculated for each village. A chi squared test was used to detect any sig-
nificant differences in the prevalence of infection between animal species and between villages.

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 Amphimerus sp. Fluke in Ecuador

Ethics
Ethical approval of the study was given by ethic committee of the Universidad Central del Ec-
uador (licence number LEC IORG 0001932, FWA 2482, IRB 2483.COBI-AMPHI-0064–11).
All villagers were asked for their verbal consent to access their domestic animals and collect
stool samples. Infected animals with any parasite were treated with specific drugs free of charge
in the following months. The study was conducted according to the above institution’s guide-
lines for animal welfare.

Results
Of the 109 houses recorded in the communities, 89 (81.6%) agreed to take part in the survey
(Table 1). From these 89 houses, a total of 27 cats and 43 dogs were counted at the time of data
collection. Stool samples from 45/70 animals (64.2%), 14 from cats and 31 from dogs were col-
lected and examined microscopically for the presence of Amphimerus eggs (Table 1). Samples
of the remaining 25 animals were not able to be collected because they either did not defecate
on the collection day or were not present in the home at the time. In cats the prevalence of
Amphimerus infection was 71.4% (95% confidence interval [CI] = 47.7–95.1), and in dogs,
38.7% (95% CI = 21.6–55.8) (Table 1). The overall prevalence of Amphimerus sp. in the two an-
imal species investigated was 48.9% (95% CI = 34.3–63.5). Cats were approximately four
times more likely to be infected with Amphimerus sp. than dogs, OR = 3.95 (95% CI 1.01–15.6,
p = 0.042). There was no statistically significant difference between the communities with re-
gard to prevalence of Amphimerus infection in the animals studied.
The eggs found in the stools of cats and dogs demonstrated the morphological characteris-
tics consistent with other members of the Opisthorchiidae family (Fig. 2). In order to confirm
that the eggs were from Amphimerus sp., two positive cats and one positive dog were eutha-
nized and adult flukes were obtained from the bile ducts and subjected to morphological and
molecular characterization. The recovered flukes just after extraction from the bile ducts were
flat, leaf-like, reddish, flexible and elongated with active movements in saline, with a thinner
anterior than posterior extremity, measuring from 15 to 28 mm in length by 2 to 4 mm in
width. When fixed in formalin 10%, the flukes became whitish and shorter measuring around
10 to 18 mm long. Flukes were stained with borax carmine and photographed (Fig. 3). Adults
of Amphimerus sp. can be differentiated from Clonorchis sinensis and Opisthorchis spp. by cer-
tain morphological features e.g.: 1) The presence of two rounded testes, which lie one behind
the other in the posterior portion, 2) The vitellaria occupy both lateral sides of the fluke,

Table 1. Demographics of three villages in northwest Ecuador and the prevalence of Amphimerus sp. infection in domestic cats and dogs of
these villages.

Village No. of houses No. of dogs No. of cats

Total Surveyed (%) Total Examined (%) Positive (%) Total Examined (%) Positive (%)
1 22 17 (77.2) 10 8 (80) 3 (37.5) 8 4 (50) 3 (75)
2 44 32 (72.7) 19 13 (69) 6 (46.1) 10 5 (50) 3 (60)
3 43 40 (93) 14 10 (72) 3 (30) 9 5 (56) 4 (80)
Total 109 89 (81.6) 43 31 (72) 12 (38.7) 27 14 (52) 10 (71.4)

List of accession numbers/ID numbers for the sequences of the ribosomal DNA ITS2 region of the flukes obtained from humans, cats and dog
Accession numbers, deposited in the GenBank/EMBL/DDBJ nucleotide database are: AB678442 for Amphimerus sp. from humans
AB926429 for Amphimerus sp. from cats
AB926430 for Amphimerus sp. from a dog.

doi:10.1371/journal.pntd.0003526.t001

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 Amphimerus sp. Fluke in Ecuador

Fig 3. Adult fluke obtained from the biliary ducts of a cat. The major difference of Amphimerus flukes from the others Opisthorchiidae is that the vitelline
glands situated along both lateral sides of the body are divided into anterior and posterior clusters at the level of the ovary, distributed in four groups, 2
anterior and 2 posterior extending nearly to the excretory pore.
doi:10.1371/journal.pntd.0003526.g003

outside of the intestinal branches and are conspicuously distributed in four groups, 2 anterior
and 2 posterior extending backwards nearly to the excretory pore, and 3) the ventral sucker is
larger than the oral [1,10]. Furthermore, sequences of the ribosomal DNA ITS2 region of the
flukes obtained from the cats and dog were identical to that obtained from humans in the pre-
vious study [10]. Accession numbers, deposited in the GenBank/EMBL/DDBJ nucleotide data-
base, are AB678442, AB926429 and AB926430 for Amphimerus sp. from humans, cats and
dogs, respectively.

Discussion
The current study is the first to identify Amphimerus sp. infection in the domestic animals
from Chachi communities in which high rates of infection have been demonstrated [10]. The
overall prevalence of Amphimerus sp. infection in cats and dogs was relatively high and it can
be concluded that these species act as definitive hosts for Amphimerus sp. in the study region.
As the eggs and adult flukes were demonstrated to be morphologically similar and molecularly
identical to those obtained from humans it can be further assumed that they act as reservoir
hosts for human infection. This confirms that amphimeriasis is a zoonosis from domestic ani-
mals living together with humans. Importantly, the prevalence of infection in the cats and dogs
recorded in this study was significantly higher than in the humans of the same communities
(48.9% in animals compared to 24% in humans) [10]. These findings suggest that these animals
play a role in the transmission of Amphimerus to humans. It is of interest to note that the ani-
mals studied have contact with villages inhabited by Afro-Ecuadorians and mestizo groups and
defecate in and contaminate the community streams, thus represent a risk to these populations
which were not found to be infected in the previous human study [10].
The prevalence reported here for Amphimerus sp. infection in cats and dogs is similar to
that of other members of the Opisthorchiidae family. In China and Korea, the average preva-
lence of C. sinensis infection in cats and dogs ranged from 64.1% to 73.2% and from 56.4% to
69.4%, respectively [2]. Furthermore, in a study of O. viverrini infection in four districts of
Thailand, cats had a much higher prevalence (35.5%) than dogs (0.37%) [18].
Amphimerus spp. flukes are thought to be transmitted by ingestion of raw or undercooked
freshwater fish [1–5]. This was previously shown in 2011, when most of the Chachi included in
the study, admitted eating smoked fish caught in nearby rivers [10]. There are two possibilities

PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0003526 February 3, 2015 7/9

 Amphimerus sp. Fluke in Ecuador

how these domestic animals can become infected. Firstly, cats and dogs could ingest the left-
overs of smoked fish containing the metacercariae of Amphimerus sp. Secondly, the animals
themselves may swim in the river and feed, thus acquiring infection directly from live fish. The
prevalence of Amphimerus sp. in cats was significantly higher than in dogs. This is probably be-
cause of a cat’s preference for fish and increased capability to catch them in streams. Both cats
and dogs have been observed fishing and eating leftovers of human food. Overall 230 freshwa-
ter fish from the Cayapas River are currently being examined for metacercariae. A number of
trematode metacercariae have been identified but none were Amphimerus. It is presumed the
intermediate host is only found in the rainy season or the natural infection is very low.
Though the study was successful in identifying a zoonotic link between domestic animals
and humans, there are some important limitations that indicate the need for further research
in order to validate the findings. Firstly, a total sample size of 45 is small and although this did
account for over 64% of all cats and dogs living in the three communities, a larger sample is re-
quired to obtain a more accurate estimate of Amphimerus infection and more reliable conclu-
sions regarding its transmission dynamics. Furthermore, only one sample was obtained from
each animal on one occasion. Although the samples in this study were concentrated to improve
sensitivity, future study could involve taking multiple samples on different occasions with du-
plicate analyses to further improve diagnostic accuracy. On the other hand, surveyed animals
were not examined for clinical symptoms, though some were presented sick and emaciated,
with others looking healthy. Future research will examine infected animals in more depth and
record any visible signs of ill-health.

Conclusions
This study is of importance in showing that the liver fluke Amphimerus sp. can infect and is
common in cats and dogs living in Chachi communities of Ecuador, where human amphimer-
iasis is prevalent. The key finding of the study is that cats and dogs serve as definitive hosts and
represent reservoirs for human infection. It can therefore be concluded that amphimeriasis is a
zoonotic disease. These results provide relevant data that could be used for policy makers for
conducting effective control strategies and measures against Amphimerus infection. As this
study found a high prevalence of infection in cats and dogs, the recommended public health
measure to control transmission would be to treat these domestic animals, as well as humans,
with a specific drug for flukes such as praziquantel.

Acknowledgments
We thank the residents of El Progreso, Loma Linda and Guadual for their kindness in partici-
pating in this study. We are grateful to Margoth Barrionuevo for her assistance with the micro-
scopical examination of animal parasites. The authors gratefully acknowledge Ronald
Guderian for reviewing this manuscript.

Author Contributions
Conceived and designed the experiments: MC WC. Performed the experiments: MC WC RA
MS AS HK HS. Analyzed the data: MC WC MS RA. Wrote the paper: MC WC MS RA.

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Artículos de Investigación

3.3 ARTÍCULO 3: Enzyme-linked immunosorbent assay for diagnosis

of Amphimerus spp. liver fluke infection in humans.
William Cevallos, Manuel Calvopiña, Victoria Nipáz, Belén Vicente-Santiago, Julio
López-Abán, Pedro Fernández-Soto, Ángel Guevara, Antonio Muro.

Mem Inst Oswaldo Cruz. 2017 May;112 (5): 364-369.

RESUMEN

El objetivo principal de este trabajo fue desarrollar, estandarizar y aplicar un ensayo

de inmunoabsorción enzimática (ELISA) utilizando antígeno crudo de Amphimerus
spp., para el diagnóstico serológico de la amphimeriosis. Amphimerus spp. es un
parásito que puede potencialmente provocar enfermedad del hígado infectando a los
seres humanos y animales domésticos. Es muy frecuente en algunas comunidades
ecuatorianas en Sudamérica. Actualmente, el diagnóstico se basa en la observación
microscópica directa de los huevos en las heces, pero con una baja sensibilidad de
detección. Se necesitan métodos de diagnóstico más sensibles.

Los antígenos somáticos se obtuvieron de parásitos adultos de Amphimerus spp. Se

analizaron 219 sueros humanos: 48 de individuos con infección confirmada por
microscopía óptica con Amphimerus spp., 78 de ecuatorianos no infectados y que
residen en zonas endémicas, 60 de personas no infectadas y que viven en zonas no
endémicas (20 ecuatorianos, 20 europeos y 20 africanos) y 33 que tuvieron otras
infecciones parasitarias y no parasitarias. Los resultados se analizaron utilizando el
análisis mediante curvas ROC. El área bajo la curva fue de 0,967, indicando que la
exactitud del ELISA fue alta. La sensibilidad fue del 85,0% [intervalo de confianza del
95% (IC): 80,3-89,7%] y la especificidad del 71,0% (IC del 95%: 65,2-76,8%). Se detectó
reactividad cruzada contra Paragonimus mexicanus, Fasciola hepatica, Schistosoma
mansoni, Taenia solium, Strongyloides stercoralis, Mansonella spp. y Vampirolepis nana.

Hemos desarrollado la primera técnica ELISA que detecta IgG anti-Amphimerus spp.
en sueros humanos con buena sensibilidad, repetitividad y reproducibilidad. Sin
embargo, se necesitan antígenos más específicos para mejorar el rendimiento de este
ensayo. Esta prueba ELISA podría ser útil para el diagnóstico precoz y el tratamiento
oportuno de infecciones por Amphimerus spp.

64
364 Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 112(5): 364-369, May 2017

Enzyme-linked immunosorbent assay for diagnosis

of Amphimerus spp. liver fluke infection in humans
William Cevallos1,3, Manuel Calvopiña2, Victoria Nipáz1, Belén Vicente-Santiago3,
Julio López-Albán3, Pedro Fernández-Soto3, Ángel Guevara1/+, Antonio Muro3
1
Universidad Central del Ecuador, Centro de Biomedicina, Carrera de Medicina, Quito, Ecuador
2
Universidad de Las Américas, Quito, Ecuador
3
Universidad de Salamanca, Faculty of Pharmacy, Tropical Disease Research Centre, Group e-INTRO, Salamanca, Spain

BACKGROUND Amphimerus spp. is a liver fluke that infects humans and domestic animals. It is highly prevalent in some
Ecuadorian communities. Currently, diagnosis is based on the microscopic observation of eggs in faeces, but this has variable
sensitivity. More sensitive methods are needed for diagnostic testing.
OBJECTIVE The main objective of this work was to develop an enzyme-linked immunosorbent assay (ELISA) using crude
antigens from Amphimerus spp. adult worms to detect anti-Amphimerus IgG in human sera.
METHODS Crude somatic antigens were obtained from adult Amphimerus spp. worms. Human sera from 119 patients were
tested: 48 from individuals with a confirmed Amphimerus spp. infection, 78 from non-infected Ecuadorians living in the endemic
region, 60 from persons living in non-endemic areas (20 Ecuadorians, 20 Europeans, and 20 Africans), and 33 who had other
parasitic and non-parasitic infections.
PRINCIPAL FINDINGS Results were analysed using the receiver-operator characteristic (ROC) curve analysis with an area under
curve (AUC) value of 0.967. The accuracy of the ELISA was high. The sensitivity was 85.0% [95% confidence interval (CI): 80.3-
89.7%] and the specificity was 71.0% (95% CI: 65.2-76.8%). Some cross reactivity was detected against Paragonimus mexicanus,
Fasciola hepatica, Schistosomiasis, Taenia solium, Strongyloides stercoralis, Mansonella spp., and Vampirolepis nana.
MAIN CONCLUSIONS We have developed the first ELISA technique that detects anti-Amphimerus IgG in human sera with good
sensitivity, repeatability and reproducibility. However, more specific antigens are needed to further enhance performance of this
assay. Regardless, this ELISA test could be useful for early diagnosis and prompt treatment of human Amphimerus spp. infections.

Key words: Amphimerus spp. - Ecuador - ELISA - diagnosis

Amphimeriasis is a zoonotic disease caused by in- ter fish, an estimated 20,000 people are at risk of acquir-
fection with the liver fluke Amphimerus spp., a member ing this disease (Calvopiña et al. 2011). Furthermore, a
of the Opisthorchiidae family that includes Clonorchis recent study reported a very high prevalence of infection
sinensis, Opisthorchis viverrini and O. felineus. The in domestic cats and dogs living in Chachi communities
genus Amphimerus (Barker, 1911) infects several wild (Calvopiña et al. 2015). Further studies by the authors
and domestic mammals in the Americas, and it has been (MC & WC) found infected people in several other prov-
reported in cats, dogs, marsupials, and rodents from inces of Ecuador (unpublished observations).
Canada, the United States, Costa Rica, Panama, Colom- Adult parasites of the genus Amphimerus spp. grow
bia, Ecuador, Brazil and Peru (Artigas & Perez 1962, and parasitise the host’s intra- and extra-hepatic bile ducts
Thatcher 1970, Miyazaki et al. 1978, de Moraes Neto et (Calvopiña et al. 2015). It is well documented that other
al. 1998, Bowman 2002). These flukes infect humans members of the Opisthorchiidae are responsible for heavy
after ingestion of raw or undercooked freshwater fish and long-lasting infections that lead to hepatobiliary dis-
parasitised with viable metacercariae. Recently, Amphi- eases including hepatomegaly, cholangitis, cholecystitis,
merus spp. infections were found in 34% of an indige- and cholangiocarcinoma (Sripa et al. 2011). Amphimerus
nous Chachi population living in the tropical rain forest spp. infection may also occur in humans. However, it is
of Northwestern Ecuador. Since the Chachi community mostly an asymptomatic disease, occasionally causing
habitually consumes smoked or lightly cooked freshwa- non-specific, generalised symptoms. To date, there are
no comprehensive descriptions of this disease. However,
histopathological studies in cats and a double-crested cor-
morant infected with Amphimerus spp. showed the pres-
ence of liver cirrhosis and pancreatitis (Rothenbacher &
doi: 10.1590/0074-02760160426 Lindquist 1963, Pense & Childs 1972).
Financial support: DGIP-Universidad Central del Ecuador (CUP At present, the diagnosis of Amphimerus spp. infec-
91750000.0000.37 4072). It was also funded in part by IBSAL-CIETUS, tions in humans is achieved by direct microscopic obser-
Spain (DTS16/00207, PI16/01784).
vation of eggs in the patient’s faeces. Observation of eggs
+ Corresponding author: [email protected]
Received 20 September 2016 after formalin-ether concentration has also been utilised.
Accepted 24 January 2017 The formalin-ether method is used on samples from cats

online | memorias.ioc.fiocruz.br
 ELISA for Amphimerus spp. human infection • William Cevallos et al. 365

and dogs, but this technique is not routinely conducted thal dose of ketamine. The adult worms were washed
in local laboratories in Ecuador (Calvopiña et al. 2015). with sterile phosphate-buffered saline (PBS) until the
The sensitivity of direct microscopic observation is up to host’s blood was completely removed. Afterwards, clean
ten times lower than the formalin-ether method (Calvo- liver flukes were suspended in sterile PBS at a concen-
piña et al. 2011). Likewise, it has been demonstrated that tration of 30 worms/mL and homogenised in a buffer [10
the fluke eggs of other Opisthorchiidae parasites can mM Tris base, 1 mM EDTA, 500 µL of a protease inhib-
be detected in the stools. This represents the best way itor cocktail (UltraCruz® Protease Inhibitor Cocktail
to obtain a definitive diagnosis, although this approach Tablet, Santa Cruz Biotechnology, catalogue number sc
becomes increasingly unreliable in cases of low-worm - 29130)]. The suspension was frozen and thawed three
burden (Johansen et al. 2010). Moreover, human amphi- times and sonicated three times at 70 kHz for one min-
meriasis is asymptomatic in most cases and does not ute each cycle using a sonicator (Vibra-Cell, Sonics and
show pathognomonic signs and symptoms. Therefore, Materials Inc., Danbury, CT, USA). The samples were
physicians can easily miss Amphimerus spp. infections then centrifuged at 16000 g for 30 min at 4ºC. The crude
or have difficulties making a differential diagnosis in antigen was lyophilised and stored at 4ºC until used.
endemic areas and, even more so, in non-endemic are- The protein concentration of the antigen was determined
as where infected migrant people may require medical using the commercial micro-BCA™ Protein Assay Kit
attention. A reliable diagnosis test is needed to ensure (Thermo Scientific Pierce, Waltham, MA, USA).
appropriate treatment and prevent chronicity to reduce
Human sera - A total of 219 human serum samples
the risk of developing liver damage.
were used to test the diagnostic value of the crude an-
Immunological techniques, such as antibody-based
tigen by ELISA. Of these, 48 sera samples were from
methods using enzyme-linked immunosorbent assay
patients who had received a definitive diagnosis, which
(ELISA), have shown high sensitivity and specificity
was demonstrated microscopically by the presence of
for diagnosing various parasitic infections (Elkins et
Amphimerus spp. eggs in their stools [true positive (TP)].
al. 1991, Guevara et al. 1995). Of particular note, ELI-
Additionally, 60 sera samples were from people living
SA was used to detect parasites of the Opisthorchiidae
family, and this technique performed the best among all in non-endemic areas, including 20 Ecuadorians, 20 Eu-
serological tests evaluated (Meniavtseva et al. 1996). ropeans, and 20 Africans, who were free of trematode
Clonorchis sinensis and Opisthorchis spp. induce robust infections [true negative (TN)]. A group of 78 serum
immune responses and significantly increase the levels samples were obtained from people living in endemic
of IgG in experimental animals, which is similar to ob- areas, all of whom tested negative for a Amphimerus spp.
servations in humans (Elkins et al. 1991, Gómez-Mo- infection based upon a coprologic test. Finally, 33 serum
rales et al. 2013). The detection of specific antibodies has samples were derived from patients infected by other
been considered a complementary tool to establish a de- parasites or viruses including Paragonimus mexicanus
finitive diagnosis for liver fluke infections (Upatham & (2), Fasciola hepatica (3), Schistosoma mansoni (2),
Viyanant 2003). Antigen-based techniques using crude Schistosoma haematobium (2), Echinococcus granulo-
adult extracts have been used for immunodiagnosis of sus (2), Mansonella spp. (2), hookworms (2), Vampirole-
O. viverrini and C. sinensis infections, albeit with var- pis nana (2), Strongyloides stercoralis (2), Taenia solium
ying levels of sensitivity and specificity (Wongratana- (1), Ascaris lumbricoides (2), Onchocerca volvulus (1),
cheewin et al. 1988, 2003, Poopyruchpong et al. 1990, Leishmania spp. (1), Giardia intestinalis (2), Chilomas-
Sawangsoda et al. 2012). tix mesnili (1), Blastocystis hominis (1), Microsporidium
For Amphimerus infections, there are no immunologi- parvum (1), Plasmodium falciparum (1), Dientamoeba
cal diagnostic methods currently available. In the present fragilis (1), and hepatitis B virus (2). Of all the serum
study, we developed an immunological assay using total samples used in this study, 81% were from adults (14-98
crude somatic extract antigens to detect anti-Amphimerus years old), and approximately 45% were from females.
IgG antibodies in sera from infected people in Ecuador. Serum samples from Europeans and Africans, as
well as individuals with other parasitic and viral infec-
MATERIALS AND METHODS tions, were provided by IBSAL-CIETUS. The diagno-
Ethics statement - The present study was approved by ses of parasitic infections were based on parasitological
the Ethics Committee of the Universidad Central del Ec- and/or serological tests. Sera from Ecuadorians were
uador (License number LEC IORG 0001932, FWA 2482, obtained by venipuncture using disposable needles and
IRB 2483, COBI-AMPHI-0064-11). Oral informed con- 10 mL vacuum tubes (VACUETTE® Bio-one GmbH,
sent was obtained from all individuals participating in Austria). These samples were obtained between January
the study prior to the collection of biological samples for and May 2015. Blood was allowed to clot, and was sub-
parasitological and immunological evaluation. Further- sequently centrifuged for 15 min at 1000 g. Serum was
more, written consent from animal owners was obtained then aliquoted in cryovials and stored at -20ºC until use.
prior to the euthanisation of cats that had been naturally All participants provided oral informed consent before
infected with Amphimerus spp. to obtain adult parasites. blood samples were collected.
Crude somatic extract of Amphimerus spp. and anti- ELISA - A standard ELISA protocol was developed to
gen preparation - Adult Amphimerus spp. were obtained test all human serum samples. Briefly, 96-well microtiter
from cat livers following euthanasia by injection of a le- plates (Sigma, St. Louis, MO, USA) were coated with 100
366 Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 112(5), May 2017

µL/well of 4 µg/mL Amphimerus spp. crude extract anti- significant differences between values for group 1 and
gens from adults. The crude extract had been reconstituted those for the remaining groups (p < 0.0001). The area un-
in carbonate buffer (pH 9.6). The plates were incubated der curve was 0.967 for the mean OD value of the dupli-
overnight at 4ºC. Next, the plates were washed three times cates and 0.970 for the SI, indicating that the two param-
with PBS (pH 7.3) containing 0.05% Tween-20 (PBS-T20) eters provided equally accurate results (Fig. 2). The ROC
(Sigma). Plates were then blocked in 200 µL/well of PBS optimised cut-off was 18% for SI and 0.624 for the OD
with 0.5% BSA and 1% Tween-20 at 37ºC for 1 h. After ad- values; on the basis of these cut-off values, the sensitivity
ditional washing steps, 100 µL of sera diluted 1:50 in PBS- reached 85% (95% CI 78.3% to 91.7%) and the specificity
T-20 was added to each well in duplicate, and plates were was assessed as 94% (95% CI: 89.5% to 98.5%).
incubated at 37ºC for 1 h. After another wash step, 100 µL/
well of 1:1000 diluted goat anti-human IgG peroxidase-la-
belled antibody (Sigma) was added and incubated at 37ºC
for 1 h. Finally, another wash was performed and 100 µL/
well of 5.3 µg/mL O-phenylene-diamine (OPD) and 8 µL of
hydrogen peroxide in citric acid buffer were added. Plates
were incubated at room temperature for 15 min, and the re-
action was stopped by adding 50 µL/well of 1N H2SO4 solu-
tion. Optical density (OD) values were obtained by reading
the plates at 492 nm using an ELISA plate microtitre reader
(Ear400FT ELISA reader Lab. Instruments). All samples
were tested in duplicate, and the OD mean was calculated.
Statistical analysis - A serological index (SI) was
calculated for each OD, and this was used to establish a
cut-off value for the ELISA using the following formula:
SI = [(PS-NC) / (PC-NC)] X 100, where NC and PC are
the negative and positive controls, respectively, and PS is
the problem sample (Hernández-González et al. 2008). Fig. 1: values of optical density determined for the Amphimerus spp.
Receiver-operator characteristic (ROC) curve anal- crude antigen determined by enzyme-linked immunosorbent assay
ysis was used to determine the diagnostic value. The (ELISA) using sera from patients. We included sera from patients di-
ROC curve is a graphical plot of the sensitivity versus agnosed with amphimeriasis based upon the detection of eggs (group
“1 - specificity” for a binary classifier system. Using 1), sera from healthy people living in amphimeriasis-affected areas
various cut-off values, this allows the selection of the (group 2), sera from healthy people living in areas unaffected by am-
phimeriasis (group 3), and sera from patients suffering from unrelated
cut-off value that gives the best balance of sensitivity helminthic, protozoan, or viral infections (group 4). The horizontal
and specificity for the test under consideration (Gómez- line represents the mean values. The asterisk represents significant
Morales et al. 2013). In order to determine which cut-off differences in the mean between group 1 and the other groups.
provided the most accurate result, we used the mean OD
of the duplicates and the SI to calculate the area under
the ROC curve for each cut-off. The specificity and sen-
sitivity were interpreted according to the ROC analysis.
The SI and OD in the different groups were expressed as
the mean and standard error of the mean (SEM). Posi-
tive and negative predictive values were calculated, as
described elsewhere (Fernández & Días 2003).
All statistical calculations were performed using
SPSS (version 22.0), which is available from: https://
www.ibm.com.
RESULTS
ROC curves were built with data from two defined true
positive and true negative reference populations. The ODs
of the 219 sera analysed are presented in Fig. 1. The mean
OD ± standard deviation (SD) for the group of patients
with confirmed amphimeriasis was the highest (0.938
± 0.304) of those calculated. The remaining groups pre-
sented low means: ODs: 0.627 ± 0.144 for healthy patients
Fig. 2: the receiver-operator characteristic (ROC) curve. The ROC curve
from zones endemic for amphimeriasis, 0.412 ± 0.120 for was generated using data from 48 sera samples from people infected with
healthy people from non-endemic areas, and 0.566 ± 0.206 Amphimerus spp. and 60 sera samples from healthy people residing in
for patients with other helminths, protozoan, and viral in- non-endemic areas. The area under the curve (accuracy) for the serologic
fections. Comparative analysis of the mean ODs detected index (SI) was 0.970. For the optical density (OD) was 0.962.
 ELISA for Amphimerus spp. human infection • William Cevallos et al. 367

TABLE chi population of Ecuador (Calvopiña et al. 2011). Our

Diagnostic sensitivity, specificity, and positive and negative recent unpublished findings demonstrate that people in
predictive values [cut-off 18%, 95% confidence interval (CI)] other communities along the Pacific coast of Ecuador are
for serum samples from people diagnosed with Amphimerus also infected with Amphimerus spp. In those studies, di-
spp. infection. The infection was confirmed by detection of rect egg detection via faecal examination by microscopy
Amphimerus eggs in stool samples (infected). Serum samples was performed. However, as reported before, the for-
from people living in endemic areas who were negative for malin-ether concentration technique was more sensitive
the presence of Amphimerus eggs in stool and serum samples than a direct smear (Calvopiña et al. 2011). For Opistor-
from people suffering from other parasitic or non-parasitic chiidae family members, many of these trematodes have
infections (uninfected) were also examined morphologically indistinguishable eggs. This includes
minute intestinal trematodes, such as Haplorchis spp.,
Amphimerus spp. infection Echinostoma spp., Metorchis spp., and Metagonimus spp.
As a result, the low specificity of egg identification dur-
ELISA result Infected n Uninfected n Total ing common low-grade infections is a major problem for
diagnostic methods (Johansen et al. 2010). Several studies
Positive TP 41 FP 48 89
for other Opisthorchiidae flukes such as C. sinensis and
Negative FN 7 TN 123* 130 O. viverrini, have demonstrated that crude antigens work
well for inducing a B-immune response during human in-
Total 48 171 219
fections (Wongratanacheewin et al. 1988, Meniavtseva et
Sensitivity: = 85.0% (95% CI: 80.3 - 89.7%), it was calculated al. 1996). Thus, more sensitive and versatile serodiagnos-
as TP/(TP+FN); specificity: = 71.0% (95% CI: 65.2 - 76.8%), tic tests based on ELISAs have become the predominant
it was calculated as TN/(TN+FP); positive predictive value = assays used in biomedical research to diagnose clonorchi-
46.0% (95% CI: 39.4 - 52.6%), it was calculated as TP/(TP+FP); asis, opistorchiasis, paragonimiasis, and fasciolosis.
negative predictive value = 86.5 (95% CI: 84.2% to 88.8%), it To the best of our knowledge, no serological tests
was calculated as TN/(TN+FN); ELISA: enzyme-linked im- have been developed and validated for amphimeriasis
munosorbent assay; FN: false negative; FP: false positive; TN: using crude antigens. This was our rational for evalu-
true negative; TP: true positive; *: for this value we eliminated ating ELISAs against a large panel of sera from indi-
78 healthy people from endemic amphimeriasis area. viduals confirmed to have a Amphimerus spp. infection;
people living in endemics area who are negative for
eggs; people infected with viruses, protozoa, and other
helminths; and, lastly, healthy people.
The diagnostic sensitivity and specificity of the ELISA Regarding sensitivity, we found that our ELISA de-
in this study, including serum samples from people who tected 85.0% (95% CI: 80.3-89.7%) of the TP samples. Our
lived in endemic areas and people with other parasitic and results are in accordance with those obtained for other par-
non-parasitic infections, are summarised in Table. The sen- asites of the Opisthorchiidae family using crude antigens.
sitivity of the test was 85% (95% CI: 80.3% to 89.7%), but For instance, the sensitivity of a previously reported ELISA
the specificity dropped to 71% (95% CI: 65.2% to 76.8%) for C. sinensis infection was 88.2% (Choi et al. 2003), and
with a positive predictive value of 46% (95% CI: 39.4% for O. viverrini infection, the range was between 83.3% and
to 52.6%) and negative predictive value of 86.5 (95% CI: 100% (Poopyruchpong et al. 1990). This moderate sensi-
84.2% to 88.8%). For this reason, we eliminated 78 healthy tivity could be explained by level of worm burden, as the
people from areas where amphimeriasis is endemic. serum IgG levels against O. viverrini correlated with the
Seven serum samples (17%) yielded false-negative overall egg count (Elkins et al. 1991, Wongratanacheewin
results, and 48 samples (28%) yielded false-positive et al. 2003). We propose that the sensitivity could also be
results. Of these false-positives, 34 were from people affected by free-living parasites in the bile ducts.
who live in endemic areas, and 14 were from persons In our results, 34 (19.8%) serum samples were not
with other parasitic infections (two were infected with identified as positive for an infection by microscopic
P. mexicanus, three with S. mansoni, and one for each examination. However, these samples were correctly
of these parasites: T. solium, S. stercoralis, F. hepática, scored as positive sera by ELISA. This considerable im-
Mansonella spp., and V. nana). provement in the identification of seropositive cases for
Amphimerus spp. in egg-negative individuals is probably
DISCUSSION
due to the presence of a paucisymptomatic disease or a
The present study shows for the first time that the Am- delayed diagnosis, which is common in Opisthorchis
phimerus spp. liver fluke infection induces an immune re- spp. infections (Armignacco et al. 2008). This suggests
sponse in human hosts. This response is characterised by that this ELISA will be able to positively diagnose more
the production of IgG antibodies, which can be detected infected people than faecal examination by microscopy
by ELISA using crude somatic extract antigens. Our data can. In the ELISA, more than 95% of the differences be-
demonstrate that the ELISA has good sensitivity for sera tween OD values of serum duplicates were less than two
from patients confirmed to be infected with this parasite. SDs, indicating the high reproducibility of the ELISA.
Human amphimeriasis is an emerging food-borne On the other hand, the specificity of the ELISA
trematode infection commonly found in tropical areas of dropped to 71% (95% CI: 65.2% to 76.8%) when sera
Ecuador. It has a high prevalence in the indigenous Cha- from people living in endemic areas or from those with
368 Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 112(5), May 2017

other health disorders unrelated to Amphimerus spp. AUTHORS’ CONTRIBUTION

were tested. Several authors have hypothesised that the AG, WC, MC and AM - Designed the study, performed ex-
main drawback of serologically based diagnostic meth- periments, wrote the draft, and reviewed the manuscript; MC,
ods that detect circulating antibodies, especially for in- VN and WC - did the fieldwork to collect Amphimerus spp. adult
fections caused by helminths, is the cross-reactivity that samples and Ecuadorian sera samples; AG and VN - prepared the
can occur when testing crude extracts containing para- crude Amphimerus spp. Antigen; BVS, JLA, PFS and WC - per-
sites (Hong 1988, Sirisinha et al. 1990). This is particu- formed the ELISAs; WC, JLA and PFS - performed the statistical
larly important in developing countries where people analysis and interpreted the results. All authors participated in
could be infected with several species of liver and intes- drafting the article and approved the submitted version.
tinal flukes, in addition to other helminths (Sawangsoda
et al. 2012). The relatively low specificity we observed REFERENCES
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ragan P, Wongratanacheewin S. Opisthorchis viverrini: relation-
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Opisthorchis felineus infection in humans from low trematode
as defined by sensitivity, repeatability, and reproducibil- endemic areas. PLoS ONE. 2013; 8(5): 5-10.
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early diagnosis and prompt treatment. Y. Antibody isotypes, including IgG subclasses, in Ecuadorian
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To Jeff Guderian, for reviewing this paper, and Juan Hernández-González A, Muro A, Barrera I, Ramos G, Orduña A,
Hernández, for his help with the benchwork. We also thank Siles-Lucas M. Usefulness of four different Echinococcus granu-
the Chachi communities in Borbón-Esmeraldas and Jipijapa- losus recombinant antigens for serodiagnosis of unilocular hyda-
tid disease (UHD) and postsurgical follow-up of patients treated
Manabi, for participating in this study.
for UHD. Clin Vaccine Immunol. 2008; 15(1): 147-53.
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Artículos de Investigación

3.4 ARTÍCULO 4: LAMPhimerus: a novel lamp assay for detecting

Amphimerus spp. DNA in human stool samples.
William Cevallos, Pedro Fernández-Soto, Manuel Calvopiña, Cristina Fontecha-Cuenca,
Hiromu Sugiyama, Megumi Sato, Julio López Abán, Belén Vicente, Antonio Muro.

PLoS Negl Trop Dis. 2017 Jun 19; 11(6):e0005672. doi: 10.1371.

RESUMEN

La amphimeriosis es una enfermedad transmitida por el consumo de peces de agua

dulce y causada por el trematodo hepático Amphimerus spp. Recientemente ha sido
reportada como endémica y con alta prevalencia en humanos y animales domésticos
que viven en la costa pacífica de Ecuador. El diagnóstico se basa en el examen de heces
para identificar los huevos del parásito, pero carece de buena sensibilidad. Además, la
morfología de los huevos puede confundirse con otros trematodos hepáticos e
intestinales. Hasta la fecha no se han desarrollado métodos inmunológicos o
moleculares. Se requiere de nuevas técnicas de diagnóstico en muestras clínicas para la
detección sensible y específica de ADN de Amphimerus spp.

Se diseñó el método LAMP dirigido a una secuencia de la región de transcripción

interna (ITS2) de Amphimerus spp. Para optimizar los ensayos moleculares se obtuvo
ADN de parásitos adultos recuperados de animales. La PCR convencional se realizó
usando los cebadores externos F3-B3 para verificar la correcta amplificación de la
secuencia de ADN diana de Amphimerus spp. La técnica LAMP se optimizó utilizando
diferentes mezclas de reacción y temperaturas. Finalmente se estableció la más
apropiada como LAMPhimerus. Se evaluó la especificidad y sensibilidad de la PCR y
LAMP. El límite de detección fue de 1 pg de ADN genómico. Las pruebas de campo se
realizaron utilizando 44 muestras de heces humanas recolectadas en localidades donde
esta trematodosis es endémica. Veinticinco muestras fueron microscópicamente
positivas para la detección de huevos de Amphimerus spp. En las pruebas moleculares,
la PCR (F3-B3) resultó negativa cuando se analizó el ADN de muestras fecales. Cuando
se analizaron todas las muestras de heces humanas incluidas en nuestro estudio, la
sensibilidad y especificidad diagnósticas para nuestro ensayo LAMPhimerus, fueron
76,67% y 80,77%, respectivamente.

En conclusión, hemos desarrollado y evaluado, por primera vez, un ensayo LAMP

específico y sensible para detectar Amphimerus spp. en muestras de heces humanas. El
procedimiento ha sido denominado LAMPhimerus y tiene el potencial de ser adaptado
para el diagnóstico de campo y la vigilancia de la amphimeriosis en áreas endémicas.
Futuros estudios a gran escala evaluarán la aplicabilidad de este novedoso ensayo
LAMP.

71
 RESEARCH ARTICLE

LAMPhimerus: A novel LAMP assay for

detecting Amphimerus sp. DNA in human stool
samples
William Cevallos1,2☯, Pedro Fernández-Soto2☯*, Manuel Calvopiña3, Cristina Fontecha-
Cuenca2, Hiromu Sugiyama4, Megumi Sato5, Julio López Abán2, Belén Vicente2,
Antonio Muro2*
1 Centro de Biomedicina, Carrera de Medicina, Universidad Central del Ecuador, Quito, Ecuador,
2 Infectious and Tropical Diseases Research Group (e-INTRO), Biomedical Research Institute of
a1111111111 Salamanca-Research Centre for Tropical Diseases at the University of Salamanca (IBSAL-CIETUS), Faculty
a1111111111 of Pharmacy, University of Salamanca, Salamanca, Spain, 3 Carrera de Medicina, Universidad De Las
a1111111111 Américas (UDLA), Quito, Ecuador, 4 Department of Parasitology, National Institute of Infectious Diseases,
a1111111111 Tokyo, Japan, 5 Graduate School of Health Sciences, Niigata University, Niigata, Japan
a1111111111
☯ These authors contributed equally to this work.
* [email protected] (PFS); [email protected] (AMA)

OPEN ACCESS Abstract

Citation: Cevallos W, Fernández-Soto P, Calvopiña
M, Fontecha-Cuenca C, Sugiyama H, Sato M, et al.
(2017) LAMPhimerus: A novel LAMP assay for Background
detecting Amphimerus sp. DNA in human stool
samples. PLoS Negl Trop Dis 11(6): e0005672.
Amphimeriasis is a fish-borne disease caused by the liver fluke Amphimerus spp. that has
https://doi.org/10.1371/journal.pntd.0005672 recently been reported as endemic in the tropical Pacific side of Ecuador with a high preva-
Editor: David Blair, James Cook University,
lence in humans and domestic animals. The diagnosis is based on the stool examination to
AUSTRALIA identify parasite eggs, but it lacks sensitivity. Additionally, the morphology of the eggs may
Received: January 24, 2017
be confounded with other liver and intestinal flukes. No immunological or molecular methods
have been developed to date. New diagnostic techniques for specific and sensitive detec-
Accepted: May 30, 2017
tion of Amphimerus spp. DNA in clinical samples are needed.
Published: June 19, 2017

Copyright: © 2017 Cevallos et al. This is an open Methodology/Principal findings

access article distributed under the terms of the
Creative Commons Attribution License, which A LAMP targeting a sequence of the Amphimerus sp. internal transcribed spacer 2 region
permits unrestricted use, distribution, and was designed. Amphimerus sp. DNA was obtained from adult worms recovered from ani-
reproduction in any medium, provided the original
mals and used to optimize the molecular assays. Conventional PCR was performed using
author and source are credited.
outer primers F3-B3 to verify the proper amplification of the Amphimerus sp. DNA target
Data Availability Statement: All relevant data are sequence. LAMP was optimized using different reaction mixtures and temperatures, and it
within the paper and its Supporting Information
files.
was finally set up as LAMPhimerus. The specificity and sensitivity of both PCR and LAMP
were evaluated. The detection limit was 1 pg of genomic DNA. Field testing was done using
Funding: This study was funded by the grants from
the Universidad Central del Ecuador (CUP
44 human stool samples collected from localities where fluke is endemic. Twenty-five sam-
91750000.0000.374072) (WCT) (http://www.uce. ples were microscopy positive for Amphimerus sp. eggs detection. In molecular testing,
edu.ec/) and in part by the Japan Society for the PCR F3-B3 was ineffective when DNA from fecal samples was used. When testing all
Promotion of Science, JSPS (KAKENHI: Grant No.
human stool samples included in our study, the diagnostic parameters for the sensitivity and
25305011) and by grants for Research on
Emerging and Re-emerging Infectious Diseases specificity were calculated for our LAMPhimerus assay, which were 76.67% and 80.77%,
(H23-Shinko-ippan-014 and H26-Shinkoippan- respectively.

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0005672 June 19, 2017 1 / 16

 LAMPhimerus assay for detecting Amphimerus sp.

009) from the Ministry of Health, Labor and Conclusions/Significance

Welfare of Japan (http://www.mhlw.go.jp/english/).
Additional funding was supported by the Health We have developed and evaluated, for the first time, a specific and sensitive LAMP assay
Research Projects: Technological Development for detecting Amphimerus sp. in human stool samples. The procedure has been named
Project in Health, grant number DTS16/00207 LAMPhimerus method and has the potential to be adapted for field diagnosis and disease
(AMA) and Health Research Project, grant number
PI16/01784 (PFS) of funding institution Instituto de
surveillance in amphimeriasis-endemic areas. Future large-scale studies will assess the
Salud Carlos III (http://www.isciii.es/). The funders applicability of this novel LAMP assay.
had no role in study design, data collection and
analysis, decision to publish, or preparation of the
manuscript.

Competing interests: The authors have declared

Author summary
that no competing interests exist.
Amphimeriasis, a fish-borne zoonotic disease caused by the liver fluke Amphimerus spp.,
is a highly prevalent parasitic infection affecting an indigenous Amerindian group, the
Chachi, living in rural and remote tropical areas along the Rı́o Cayapas and its tributaries
in the north-western coastal rainforest of Ecuador. Very little is known about the clinical
course and treatment of this disease, and the only method for diagnosing it is the parasito-
logical microscopic detection of eggs from Amphimerus spp. in patients’ stool samples.
This method lacks sensitivity, and the morphology of the eggs may be confounded with
other liver and intestinal flukes. New diagnostic tools that can improve the sensitivity and
specificity for diagnosing Amphimerus spp. infection would be desirable. At present,
LAMP technology shows all the characteristics required of a real-time assay with simple
operation for potential use in the clinical diagnosis of infectious diseases, particularly in
the field conditions in developing countries for most neglected tropical diseases. In this
study, we developed and successfully evaluated a LAMP assay for detecting Amphimerus
sp. in human stool samples. After further validation, our LAMP assay (LAMPhimerus)
could be readily adapted for effective field diagnosis and disease surveillance in amphi-
meriasis-endemic areas.

Introduction
Amphimerus spp. are digenean parasitic flatworms in the bile ducts of birds, reptiles and
mammals, and they are closely related to the genera Clonorchis and Opisthorchis within the
Opisthorchiidae family [1, 2]. As for other members of the Opisthorchiidae family, the life
cycle of Amphimerus spp. is highly complex, involving both freshwater snails and fish as inter-
mediate hosts and vertebrates, including humans, as definitive hosts [3]. Humans or fish-eat-
ing animals are infected with Amphimerus spp. through the ingestion of raw or undercooked
freshwater fish containing metacercariae [3]. Recently, Amphimerus sp. has been reported, for
the first time, as endemic in rural communities in the tropical Pacific side of Ecuador with a
high prevalence in humans and domestic cats and dogs, causing amphimeriasis [3, 4]. Several
foodborne trematodiases around the world are now considered by the World Health Organi-
zation as neglected tropical diseases (NTDs) [5] with high prevalence, especially in East Asia
[6], and they have serious consequences, such as cholangiocarcinoma [7,8]. Amphimeriasis
has been reported as a new emerging foodborne zoonotic disease [3].
Amphimerus spp. adult stages are located in the bile ducts of the definitive host, and the
eggs are shed in the feces [3]. Diagnosis of human and animal infection can be performed with
the wet mount technique for examining feces, allowing for microscopic visualization of para-
site eggs; the formalin-ether concentration method has been shown to increase the sensitivity

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0005672 June 19, 2017 2 / 16

 LAMPhimerus assay for detecting Amphimerus sp.

ten-fold [3]. Detection of the eggs in bile or duodenal fluid can also be performed. However,
microscopic examination is cumbersome and time consuming, and it could have a low sensi-
tivity in cases of light infections. In addition, the morphological similarity of the Amphimerus
spp. eggs to those of closely related species belonging to genera Clonorchis and Opisthorchis as
well as to minute intestinal flukes, makes diagnosis difficult. It would be necessary to use
scanning electron microscopy to accurately observe the differences between the coatings of the
different species [3]. Therefore, the development of a new method that can improve the sensi-
tivity and specificity for diagnosing Amphimerus spp. infection is urgently required.
To overcome these limitations, the use of molecular approaches has become a powerful tool
for the diagnosis, identification and differentiation of closely related species. In recent years,
several polymerase chain reaction (PCR)-based molecular diagnostic methods have been
developed for detecting many parasitic trematodes, including those species that are closely
related to Amphimerus spp., such as C. sinensis [9–14] and O. viverrini [15–18]. Although these
studies have demonstrated that PCR-based methods are very sensitive and specific, they are
not still widely used in low-income countries because well-trained personnel and expensive
equipment are needed, making them unviable for routine application in field conditions in
endemic areas that are generally undeveloped and have a high disease prevalence. Loop-medi-
ated isothermal amplification (LAMP) could be a good alternative amplification technology
[19] because it has several salient advantages over most PCR-based methods [20, 21]. At pres-
ent, LAMP technology has all the characteristics required of a real-time assay along with sim-
ple operation for potential use in the clinical diagnosis of infectious diseases, particularly
under the field conditions in developing countries [22, 23]. Additionally, several LAMP assays
have already been successfully described for detecting trematode parasites, including a number
of species causing foodborne trematodiases, such as Fasciola spp. [24], Clonorchis sinensis [25,
26], Opisthorchis viverrini [27–29] and Paragonimus westermani [30].
With the aim of developing new, applicable and cost-effective molecular tools for the diag-
nosis of amphimeriasis, we have developed and evaluated, for the first time, a LAMP assay for
the specific detection of Amphimerus sp. liver fluke in human stool samples.

Methods
Ethics statement
The study protocol was approved by the Ethics Committee of Universidad Central del Ecuador
(License number: LEC IORG 0001932, FWA 2482, IRB 2483. COBI-AMPHI-0064-11) and the
Ethics Committee of the University of Salamanca (protocol approval number 48531). Partici-
pants were given detailed explanations about the aims, procedures and possible benefits of the
study. Written informed consent was obtained from all subjects prior to the collection of bio-
logical samples for parasitological and molecular evaluation. Parents or guardians of children
who participated in the study provided written informed consent on the child’s behalf. All
samples were coded and treated anonymously.

Study area and population

The study was conducted during February 2016 in two indigenous Chachi villages alongside
the Cayapas River in the Esmeraldas province, located in the northwest coastal rainforest of
Ecuador [4]. The indigenous Chachi, living together with the Afro-ecuadorian and mestizo
populations, belong to the predominant autochthonous group in this area, representing 13%
of the inhabitants in this region. These communities are the same as those studied previously
and have a high prevalence of infection (15.5% to 34.1%) with Amphimerus sp. Prevalences are
also high in local cats and dogs [3, 4]. They live in remote villages where the only way to reach

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 LAMPhimerus assay for detecting Amphimerus sp.

them is by boat along the river. Sanitation facilities are lacking, and the members are hunters
who habitually eat undercooked freshwater fish (mainly smoked fish) caught in the neighbor-
ing rivers [4]. More details on the region can be accessed elsewhere [31, 32].

Human stool samples and parasitological tests

Human stool samples were obtained from indigenous Chachi communities during February
2016. Each participant who enrolled in the study was given a copro-parasitological flask for
stool collection. Samples were collected within a few hours of stool passing. After collection,
samples were transported to the Parasitology Laboratory (Centro de Biomedicina, Universidad
Central del Ecuador, Quito, Ecuador) for parasitological screening under light microscopy by
direct examination, simple sedimentation, formalin-ether concentration and Kato-Katz tech-
niques. All samples were examined by two qualified laboratory technicians according to the
basic laboratory methods in medical parasitology recommended by the World Health Organi-
zation (WHO) [33]. After parasitological screening, a total of 44 stool samples were selected,
including 25 (56.81%) that were positive for Amphimerus sp. eggs-by one or more parasitologi-
cal methods-and 19 (43.18%) negative samples. Afterwards, the 44 stool samples that were
well-preserved in 80% ethanol were sent to the Research Center for Tropical Diseases (CIE-
TUS) at the University of Salamanca, Spain, for further DNA extraction and molecular analysis
as described below.

DNA extraction for molecular analyses

DNA from human fecal samples. Approximately 250–300 mg from each of 44 stool sam-
ples preserved in 80% ethanol solution was used for DNA extraction. First, excess ethanol was
removed from each vial; subsequently, DNA extraction was performed using the Mini Stool
DNA Extraction kit (Macharey-Nagel) according to the manufacturers’ instructions. Purified
DNA samples were stored at -20˚C until use.
DNA from parasites. Amphimerus sp. genomic DNA was extracted from frozen adult
worms that were previously obtained from the livers of naturally infected cats and dogs of Cha-
chi communities, as described elsewhere [4], using a G-spin Total DNA Extraction Kit (Intron
Biotechnology) according to the manufacturers’ instructions. DNA was measured using a
Nanodrop ND-100 spectrophotometer (Nanodrop Technologies) and then diluted with ultra-
pure distilled water to final concentrations of 5 ng/μL and 0.5 ng/μL. Serial 10-fold dilutions
from adult Amphimerus sp. DNA were prepared with ultrapure water, ranging from 1x10-1 to
1x10-9, and stored at -20˚C until use. DNA thus prepared was used as a positive control in all
PCR and LAMP reactions as well as for assessing the sensitivity of both molecular assays.
To determine the specificity of PCR and LAMP assays to amplify only Amphimerus sp.
DNA, a total of 16 DNA samples from several helminths, including trematodes (Clonorchis
sinensis, Opisthorchis viverrini, Fasciola hepatica, Dicrocoelium dendriticum, Schistosoma man-
soni, S. haematobium, S. japonicum, and S. intercalatum), cestodes (Echinococcus granulosus
and Taenia truncata), nematodes (Onchocerca volvulus, Strongyloides venezuelensis, and Trichi-
nella spiralis) and protozoa (Entamoeba histolytica, Cryptosporidium parvum, and Giardia duo-
denalis) were used. The concentration of all DNA samples was measured by the same method
as described for Amphimerus sp. DNA, which was then diluted with ultrapure water to a final
concentration of 0.5 ng/μL and kept at -20˚C until use in molecular assays.

Designing LAMP primers

An 459 base pair (bp) sequence, corresponding to a linear genomic DNA partial sequence in
the ITS2 region of Amphimerus sp. HS-2011 isolated from human host, was selected and

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 LAMPhimerus assay for detecting Amphimerus sp.

retrieved from GenBank (Accession No. AB678442.1) [4] for the design of the specific primers.
The 459 bp sequence was tested using BLASTN analysis [34] for similarity in the available
online genome databases. A set of LAMP primers complementary to the nucleotide sequence
was designed using the online Primer Explorer V4 software (https://primerexplorer.jp/elamp4.
0.0/; Eiken Chemical Co., Ltd., Tokyo, Japan) according to criteria described by Notomi et al
[19]. A final complete set of four primers-including a forward outer primer (F3), a reverse
outer primer (B3), a forward inner primer (FIP) and a backward inner primer (BIP)-was
selected based on the criteria described in “A guide to LAMP primer designing” (http://
primerexplorer.jp/e/v4_manual/index.html) of LAMP primers; the locations and target
sequence are shown in Fig 1. All the primers were of HPLC grade (Thermo Fisher Scientific
Inc., Madrid, Spain). The lyophilized primers were resuspended in ultrapure water to a final
concentration of 100 pmol/μL and stored at -20˚C until use.

PCR using outer primers F3 and B3

The outer LAMP primer pair (F3 and B3; Fig 1) was initially tested for Amphimerus sp. speci-
ficity by a PCR to verify whether the correct target was amplified. PCR was conducted in 25 μL

Fig 1. Design of LAMP primers for detecting DNA of Amphimerus sp. (A) Schematic representation of the 459 bp selected sequence of
Amphimerus sp. HS-2011 isolated from human host (AB678442.1). (B) Location of the LAMP primers within the selected sequence. Arrows
indicate the direction of extension. (C) Sequences of LAMP primers. F3, forward outer primer; B3, reverse outer primer; FIP, forward inner
primer (F1c and F2 sequences); and BIP, reverse inner primer (B1c and B2 sequences).
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 LAMPhimerus assay for detecting Amphimerus sp.

of a reaction mixture containing 2.5 μL of 10x buffer, 1.5 μL of 25 mmol/L MgCl2, 2.5 μL of 2.5
mmol/L dNTPs, 0.5 μL of 100 pmol/L F3 and B3, 2 U Taq-polymerase and 2 μL (10 ng) of
DNA template. Initial denaturation was conducted at 94˚C for 1 min, which was followed by a
touchdown program for 15 cycles with successive annealing temperature decrements of 1.0˚C
every 2 cycles. For these 2 cycles, the reaction was denatured at 94˚C for 20 s followed by
annealing at 64˚C-58˚C for 20 s and polymerization at 72˚C for 30 s. The subsequent 15 cycles
of amplification were similar, except that the annealing temperature was 57˚C. The final exten-
sion was performed at 72˚C for 10 min. All PCR reactions were performed in a Mastercycler
Gradient-96well (Eppendorf).
The specificity of PCR F3-B3 was tested using heterogeneous DNA samples from other par-
asites included in the study. The sensitivity was also assayed to establish the detection limit of
Amphimerus sp. DNA with 10-fold serial dilutions prepared as mentioned above. All PCR
assays were performed with 2 μL of the DNA template (5 ng/μL) in each case. Negative con-
trols (ultrapure water) and positive controls (genomic DNA from Amphimerus sp.) were
always included. The PCR products (3–5 μL/each) were subjected to 1.5–2% agarose gel elec-
trophoresis stained with ethidium bromide and visualized under UV light.

Establishing the LAMP assay

We evaluated the LAMP primer set designed by using different reaction mixtures to compare
results in Amphimerus sp. DNA amplification. LAMP reactions mixtures (25 μL) contained 40
pmol each of FIP and BIP primers, 5 pmol each of F3 and B3 primers, 1.4 mM each of dNTP
(Intron), 1x Isothermal Amplification Buffer-20 mM Tris-HCl (pH 8.8), 50 mM KCl, 10 mM
(NH4)2SO4, 2 mM MgSO4, 0.1% Tween20 (New England Biolabs, UK)-betaine (0.8, 1, 1.2, 1.4
or 1.6 M) (Sigma, USA), supplementary MgSO4 (2, 4, 6 or 8 mM) (New England Biolabs, UK)
and 8 U of Bst polymerase 2.0 WarmStart (New England Biolabs, UK) with 2 μL (1 ng) of tem-
plate DNA.
LAMP reactions were performed in 0.5-mL micro-centrifuge tubes that were incubated in a
simple heating block at a range of temperatures (61, 63 and 65˚C) for 60 min to optimize the
reaction conditions and then heated at 80˚C for 5–10 min to terminate the reaction. The opti-
mal temperature was determined and used in the following tests. Because of the high sensitiv-
ity of the LAMP reaction, DNA contaminations were prevented using sterile tools at all times,
performing each step of the analysis in separate work areas and minimizing manipulation of
the reaction tubes. Template DNA was replaced by ultrapure water as a negative control in
each LAMP reaction.
The specificity of the LAMP assay to amplify only Amphimerus sp. DNA was tested against
16 DNA samples obtained from other parasites used as heterogeneous controls, as mentioned
above. To determine the lower detection limit of the LAMP assay, genomic DNA from Amphi-
merus sp., 10-fold serial diluted as mentioned above, was subjected to amplification compared
with the PCR F3-B3.

Detection of LAMP products

The LAMP amplification results could be visually inspected by adding 2 μL of 1:10 diluted
10,000X concentration fluorescent dye SYBR Green I (Invitrogen) to the reaction tubes. Green
fluorescence was clearly observed in the successful LAMP reaction, while it remained original
orange in the negative reaction. In addition, the LAMP products (3–5 μL) were monitored
using 1.5–2% agarose gel electrophoresis stained with ethidium bromide, visualized under UV
light and then photographed using an ultraviolet Gel documentation system (UVItec, UK).

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 LAMPhimerus assay for detecting Amphimerus sp.

Statistical analysis
To estimate the accuracy of the LAMP assay method as a diagnostic test, the percentages of the
sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV)
were calculated using the MedCalc statistical program version 16.8.4 (MedCalc Software,
Ostende, Belgium) according to the software instruction manual (www.medcalc.org).

Results
PCR using outer primers F3-B3: Sensitivity and specificity
The expected 229 bp PCR product was successfully obtained with outer primers F3 and B3
from Amphimerus sp. DNA. According to sensitivity, the minimum level of Amphimerus sp.
DNA detectable by PCR was 0.001 ng (1 pg) (Fig 2A). Additionally, when DNA samples from
other parasites included in the study were subjected to this PCR assay, no amplicons were
obtained (Fig 2B).

Examination of human stool samples by PCR F3-B3

We tested the 44 human stool samples by PCR using the outer primers F3 and B3, and very
faint bands of the expected size (229 bp) were only obtained in 3 samples (nos. 31, 34 and 45)
(S1 Fig).

Fig 2. PCR verification, detection limit and specificity using outer primers F3 and B3 for DNA amplification of Amphimerus sp. (A)
Detection limit of PCR F3-B3. Lane Amp, DNA of Amphimerus sp. (10 ng); lanes 10−1–10−9, 10-fold serial dilutions of Amphimerus sp. DNA.
(B) Specificity PCR F3-B3. Lanes Amp, Cs, Ovi, Sm, Sh, Sj, Si, Fh, Dd, Ov, Sv, Ts, Tt, Eg, Cp, Gd, Eh: DNA samples of Amphimerus sp.,
Clonorchis sinensis, Opisthorchis viverrini, Schistosoma mansoni, S. haematobium, S. japonicum, S. intercalatum, Fasciola hepatica,
Dicrocoelium dendriticum, Onchocerca volvulus, Strongyloides venezuelensis, Trichinella spiralis, Taenia truncata, Echinococcus
granulosus, Cryptosporidium parvum, Giardia duodenalis and Entamoeba histolytica, respectively. In all panels: lane M, molecular weight
marker (100 bp Plus Blue DNA Ladder) and lane N, negative controls (ultrapure water, no DNA template).
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 LAMPhimerus assay for detecting Amphimerus sp.

Establishing the LAMP assay: LAMPhimerus

Subsequent to testing different reaction mixtures and temperature conditions, the best amplifi-
cation results (based on the most evident color change by adding the fluorescent dye and the
intensity of the multiple bands on agarose as well as reproducibility of tests) were always
obtained when the LAMP master mixture contained 1 M betaine combined with supplemen-
tary 6 mM MgSO4 (resulting in a final concentration of 8 mM MgSO4 in 1x Isothermal Ampli-
fication Buffer) and was incubated at 63˚C for 60 min in a heating block (Fig 3A). When we
evaluated the sensitivity of the established LAMP assay, the limit of detection in Amphimerus

Fig 3. Establishing the LAMP assay. (A) LAMP amplification results obtained using the established LAMPhimerus assay with the addition
of SYBR Green I (up) or visualization on agarose gel (down). Lane M, molecular weight marker (100 bp Plus Blue DNA Ladder); lane Amp,
Amphimerus sp. DNA (1 ng); and lane N, negative control (ultrapure water and no DNA template). (B) Sensitivity assessment of
LAMPhimerus using serial dilutions of Amphimerus sp. genomic DNA. Lane M, molecular weight marker (100 bp Plus Blue DNA Ladder);
lane Amp, Amphimerus sp. DNA (1 ng); lanes 10−1–10−9, 10-fold serial dilutions; and lane N, negative control (ultrapure water and no DNA
template). (C) Specificity of the LAMPhimerus assay. Lane M, molecular weight marker (100 bp Plus Blue DNA Ladder); lane Amp, Cs, Ovi,
Sm, Sh, Sj, Si, Fh, Dd, Ov, Sv, Ts, Tt, Eg, Cp, Gd, and Eh: DNA samples of Amphimerus spp., Clonorchis sinensis, Opisthorchis viverrini,
Schistosoma mansoni, S. haematobium, S. japonicum, S. intercalatum, Fasciola hepatica, Dicrocoelium dendriticum, Onchocerca volvulus,
Strongyloides venezuelensis, Trichinella spiralis, Taenia truncata, Echinococcus granulosus, Cryptosporidium parvum, Giardia duodenalis
and Entamoeba histolytica, respectively; and lane N, negative control (ultrapure water and no DNA template).
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 LAMPhimerus assay for detecting Amphimerus sp.

sp. genomic DNA amplification was identical to that obtained when using PCR with outer
primers, specifically 0.001 ng (1 pg) (Fig 3B). To determine the specificity of the LAMP assay
for Amphimerus sp., a panel of 16 additional DNA samples from other parasites was tested for
amplification. A positive result was only obtained when Amphimerus sp. DNA was used as
template, while DNA samples from other specimens were not amplified, demonstrating its
high specificity (Fig 3C).
In this way, the best reaction mixture, in addition to the specific primers designed, was
established as the most fitting assay for amplification of Amphimerus sp. DNA and was named
"LAMPhimerus" in all successive LAMP reactions.

Application of LAMP in human stool samples: LAMPhimerus analysis

The 44 human stool samples were tested with LAMPhimerus assay using two incubation times
for reaction, 60 min and 120 min (Fig 4). To prevent potential cross-contamination, amplifica-
tion assays were performed in four batches of 11 samples each for easy handling. When testing
stool samples using an incubation time of 60 min (Fig 4A), we obtained LAMP positive results
in 14/44 (31.81%) samples, including 5 samples (nos. 36, 45, 47, 68 and 99) that were negative
in all parasitological tests applied. When using an incubation time of 120 min (Fig 4B), the
number of positive results was increased to 22/44 (50%), which also included 4/5 negative
parasitological samples as before (nos. 36, 45, 68 and 99). In all LAMP positive reactions, green
fluorescence was clearly visualized under natural light conditions. Positive controls always
worked well and negative controls were never amplified. All positive results obtained when
performing the assay for 60 min were supported at 120 min, except one sample (no. 47). For
120 min, in sample no. 47, a mix between green and orange was observed in the reaction tube;
also a very faint smear was visible on agarose gel, indicating poor DNA amplification. Taking
together the results obtained from the two incubation assays, we finally considered sample no.
47 as positive, resulting in a total of 23/44 (52.27%) positive LAMPhimerus results.
In summary, of the total of 25 parasitologically positive stool samples, we obtained 9/25
(36%) and 18/25 (72%) positive results when we applied LAMPhimerus for 60 min and 120
min, respectively. Additionally, positive results included 5/19 (26.31%) samples (nos. 36, 45,
47, 68 and 99) that were negative in all previously applied parasitological tests. Of the 11 sam-
ples (nos. 6, 27, 30, 32, 33, 42, 54, 60, 79, 84, and 85) that were simultaneously positive on three
parasitological tests (including the formalin-ether concentration technique, FECT; simple sed-
imentation technique, SST; and Kato-Katz technique, KKT), 9 (9/11; 82%) were also positive
on the LAMP assay; only the 2 samples (nos. 42 and 54) with the same very low egg count
(FEC = 1; EPG = 24) were negative on the LAMP assay. Fig 5 shows a comparison of the results
obtained for detecting Amphimerus sp. in human stool samples when using the classical parasi-
tological techniques applied and the 120 min-LAMPhimerus assay.
Considering the results obtained, the following diagnostic parameters for the sensitivity
and specificity were calculated for our LAMPhimerus assay: 76.67% sensitivity (95% CI:
52.72% -90.07%); 80.77% specificity (95% CI: 60.65% -93.45%); 82.14% positive predicted
value (95% CI: 67.13% -91.20%) and 75.00% negative predicted value (95% CI: 60.43%
-85.49%).

Discussion
Human amphimeriasis, caused by the Amphimerus spp. liver fluke, has been recently reported
as an emerging zoonotic food-borne trematodiasis [3, 4]. The conventional diagnosis of liver
fluke infections in humans is based on the demonstration of eggs in different clinical samples,
especially in feces. However, the morphological identification of eggs is troublesome in

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 LAMPhimerus assay for detecting Amphimerus sp.

Fig 4. LAMPhimerus analysis of human stool samples in this study. (A) Incubation time of 60 min. (B)
Incubation time of 120 min. Lane M, molecular weight marker (100 bp Plus Blue DNA Ladder); lane Amp,
Amphimerus sp. DNA (1 ng); lane N, negative controls (ultrapure water and no DNA template); and numbers 5–104,
analyzed human stool samples. The highlighted red numbers correspond to samples that were positive by one or
more applied parasitological methods.
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 LAMPhimerus assay for detecting Amphimerus sp.

Fig 5. Comparison of the results obtained by the LAMPhimerus assay and classical parasitological techniques applied in this
study for detecting Amphimerus sp. in human stool samples. DME, direct microscopy detection; FECT, formalin-ether concentration
technique; SST, simple sedimentation technique; KKT, Kato-Katz technique; FEC, fecal egg count; EPG, eggs per gram of feces; -,
negative for egg detection; and +, positive for egg detection. Values indicated for FEC and EPG correspond to the numbers of detected eggs
and numbers 5–104 correspond to the analyzed human stool samples.
https://doi.org/10.1371/journal.pntd.0005672.g005

endemic areas where co-infection with other zoonotic trematodes usually exists. Additionally,
stool examination lacks analytical sensitivity, particularly for light infections, requiring serial
fecal sampling and an intensive effort in resource-poor settings [35]. To solve these limitations,
many immunological and molecular diagnostic approaches have already been developed and
applied to detect the presence of several human zoonotic trematode infections with varying
accuracy [36, 37].
For detecting Amphimerus spp. infection, conventional coprological techniques are the
only ones available, and no immunological or molecular methods have been developed to
date. Among the possible molecular methods to be developed, LAMP tests are rapidly becom-
ing an attractive diagnostic option for use under field conditions in laboratories with basic
facilities [22, 23]. Hence, in this study, with the aim of improving the diagnostic testing for
amphimeriasis, we have developed and evaluated, for the first time, a specific LAMP assay to
detect Amphimerus sp. DNA in field samples collected from humans.
At present, nucleotide information for Amphimerus spp. DNA is very scarce, and only a few
DNA partial sequences, corresponding to five isolates from hosts (including human, dog, cat,
and two softshell turtles), are available in the Genbank database for potential LAMP primers
design. The 459 bp sequence of the ribosomal DNA ITS2 region of Amphimerus sp. HS-2011
isolated from human hosts [4] was selected as a target of amplification. This sequence matches
those later reported for isolates from a dog (dog-2012) and cat (cat-2012) residing in the same
studied endemic indigenous Chachi communities for human amphimeriasis [4]. Therefore,
the selection of the target region was appropriate because it seems to contain an identical
sequence for all geographical isolates of Amphimerus spp. circulating in the same area, and the

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 LAMPhimerus assay for detecting Amphimerus sp.

assay could be suitable for easily diagnosing both infected animals and humans in endemic
areas of amphimeriasis with limited resources.
First, we established the proper operation, sensitivity and specificity of both conventional
PCR (using the outer primers) and the LAMP assay (using four specific primers: LAMPhi-
merus) in the amplification of the Amphimerus sp. DNA target sequence. Both assays were
shown to be highly specific for Amphimerus sp. because no cross-reactivity could be observed
when DNA from other parasites, including those closely related such as C. sinensis and O.
viverrini, were used as a template in the reactions. Identical sensitivities (1 pg of parasite geno-
mic DNA) were obtained for both PCR and LAMPhimerus although the LAMP technique is
usually 10–100 fold more sensitive than PCR [38]. However, the sensitivity obtained was the
same as that previously reported for O. viverrini detection targeting the ITS1 region in rDNA
(ITS1-LAMP) [27, 29] or the mitochondrial nad1 sequence (mito-OvLAMP) [28]. A higher
sensitivity (10−5 pg) has been reported for detecting C. sinensis targeting the cathepsin B3 gene
[12]. Perhaps, in this study, a greater sensitivity could have been achieved for our LAMPhi-
merus assay if other DNA target sequences for designing LAMP primers had been available to
analyze in databases.
When PCR was specifically tested with the 44 field-collected stool samples, only 3 very faint
PCR-positive results were obtained. Varying sensitivity of PCR detection for O. viverrinii [27]
and C. sinensis [14] has already been noted when analyzing human stool samples because Taq
DNA polymerase inhibitors are frequent in stool specimens. Substances typically present in
human feces and dietary components can also limit DNA extraction success [39]. Therefore,
improvement of DNA preparation before extraction from stool samples could be a key factor
for obtaining better PCR results in Amphimerus sp. DNA detection, as has been previously
described for other similar parasites, such as O. viverrini [40, 27]. In our study, the PCR assay
is not emphasized because of its very low performance and inconvenience of application in
poorly equipped and often short-staffed laboratories in endemic areas.
Better results were obtained when LAMPhimerus method was applied to test human stool
samples. A better performance of LAMP assays over conventional PCR methods when analyz-
ing stool samples has been widely reported in the literature because LAMP is more tolerant to
sample-derived inhibitors than PCR for diagnostic applications [41, 42]. Therefore, using the
initial established reaction time of 60 min, we obtained 14/44 (31.81%) positive results, includ-
ing 9/25 (36%) that tested positive by microscopy. It has been already suggested that a longer
incubation reaction time in the LAMP assay improves the sensitivity and that LAMP negative
samples should be incubated longer to reduce false negatives [43]. According to this, a subse-
quent increase to 120 min of the standard incubation time protocol for the LAMPhimerus
assay allowed us to increase the number of positives results up to 23/44 (52.27%), including
18/25 (72%) microscopy-confirmed Amphimerus sp. infections. It should be noted that 5 stool
samples with no parasite eggs (nos. 36, 45, 47, 68 and 99) were positive on LAMPhimerus test-
ing regardless of the reaction time used for amplification. These samples could be truly Amphi-
merus sp. infections that have been microscopically undetected because of the classically low
sensitivity of the parasitological diagnosis [35]. Moreover, up to 10 samples without egg counts
were also LAMPhimerus positive. This result confirms a greater sensitivity of the LAMPhi-
merus assay over microscopic examination.
By contrast, 7 truly microscopy Amphimerus-positive samples (nos. 34, 42, 46, 54, 62, 70
and 81) were never amplified. For these samples, values of FEC using the Kato-Katz thick
smear method were minimal (between zero and 1–2 eggs), resulting in very low EPG levels.
The absence of amplification in these samples was likely not due to the ineffectiveness of LAM-
Phimerus method because we obtained positive results in other microscopy-positive samples
with low EPG levels too. A possibility for the lack of amplification could have been the small

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 LAMPhimerus assay for detecting Amphimerus sp.

quantity (250–300 mg) of the field-collected stool samples finally used for DNA extraction in
the laboratory for the LAMP assay. Because eggs of parasites are not equally distributed among
the stool specimens [44], it is possible that eggs could have been easily missed in working sam-
ples, compromising the Amphimerus sp. DNA obtained and thus subsequent amplification. It
is also important to note that we established the minimum amount of Amphimerus sp. geno-
mic DNA detectable by LAMP is 0.001 ng (1 pg). It has been reported that a single egg of a
closely related trematode O. viverrini yields 3.72 ng of genomic DNA [45]. Then, theoretically,
our LAMP assay would detect Amphimerus sp. DNA corresponding to less than one single egg
in a stool sample. Another possibility could have been a mistake in the morphological identifi-
cation of parasite eggs when performing the stool microscopic examination. This observation
would further confirm the specificity of LAMPhimerus method in the amplification of Amphi-
merus sp. DNA alone.
However, as noted elsewhere, the need for a decision in case management dictates unequiv-
ocal result interpretation [22] and some of the drawbacks of LAMP assays, such as potential
DNA contamination and carry-over of amplified products when opening the tubes to use the
dye, should be considered because they may compromise the test results.
In summary, we have developed, for the first time, a LAMP assay (namely, LAMPhimerus)
for the sensitive and specific detection of Amphimerus sp. DNA in human stool samples. After
further research for validation, the method could be readily adapted for effective field diagno-
sis and disease surveillance in amphimeriasis-endemic areas. Future work will be aimed at
large-scale studies to further assess the applicability of this novel diagnostic tool.

Supporting information
S1 Fig. Examination of human stool samples by PCR F3-B3. Analysis of human stool sam-
ples included in the study by PCR using outer primers F3 and B3 to detect Amphimerus sp.
DNA. In all panels: lane Amp, DNA of Amphimerus sp. (10 ng); lane M, molecular weight
marker (100 bp Plus Blue DNA Ladder); lane N, negative control (ultrapure water, no DNA
template); and numbers 5–104, stool samples analyzed.
(TIF)

Acknowledgments
The authors would like to thank Dr. Angel Guevara for kindly provided Onchocerca volvulus
DNA used in specificity trials.

Author Contributions
Conceptualization: WCT PFS MCH CFC HS MS JLA BVS AMA.
Funding acquisition: WCT PFS MCH CFC HS MS AMA.
Investigation: WCT PFS MCH CFC HS MS AMA.
Methodology: WCT PFS MCH AMA.
Project administration: WCT PFS MCH AMA.
Resources: WCT PFS MCH HS MS JLA BVS AMA.
Supervision: WCT PFS MCH AMA.
Validation: WCT PFS MCH CFC AMA.

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 LAMPhimerus assay for detecting Amphimerus sp.

Visualization: WCT PFS MCH AMA.

Writing – original draft: WCT PFS MCH AMA.
Writing – review & editing: WCT PFS MCH HS AMA.

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Artículos de Investigación

3.5 ARTÍCULO 5: Diagnosis of amphimeriasis by LAMPhimerus assay

in human stool samples long term storage onto filter paper.
William Cevallos, Pedro Fernández-Soto, Manuel Calvopiña, María Buendía-Sánchez,
Julio López Abán, Belén Vicente, Antonio Muro.

Plos ONE. Accepted.

RESUMEN

La anfimeriosis es una enfermedad zoonótica transmitida por el consumo de peces

crudos e infectados con el trematodo hepático Amphimerus spp. Es una enfermedad
emergente recientemente reportada en un grupo Amerindio, los Chachis, que viven en
Ecuador. El único método existente para diagnosticar la amphimeriosis era hasta la
fecha la detección microscópica de huevos del parásito en muestras de heces de
pacientes con muy baja sensibilidad. Nuestro grupo desarrolló una técnica ELISA para
la detección de IgG anti-Amphimerus en suero humano y un método molecular basado
en la tecnología LAMP (LAMPhimerus) para la detección específica y sensible del ADN
del parásito. El método LAMPhimerus demostró ser mucho más sensible que los
métodos parasitológicos clásicos para el diagnóstico de amphimeriosis utilizando
muestras de heces humanas para el análisis. El objetivo de este trabajo es demostrar la
viabilidad de utilizar muestras de heces secas conservadas en papel de filtro que sirvan
como fuente de ADN en combinación con la eficacia de nuestro ensayo LAMPhimerus
previamente diseñado para la detección de Amphimerus spp. en muestras clínicas de
heces. Un total de 102 muestras de heces no tratadas, sin diluir, tomadas de la población
Chachi, se extendieron como una fina capa sobre papel de filtro común para
transportarlas fácilmente a nuestro laboratorio y almacenarlas a temperatura ambiente
durante un año hasta la extracción del ADN. Cuando se aplicó el método LAMPhimerus
para la detección de ADN de Amphimerus spp., se detectó un mayor número de
resultados positivos (61/102; 59,8%) en comparación con los métodos parasitológicos
(38/102, 37,25%), incluyendo 28/61 (45,9%) infecciones de Amphimerus spp.
confirmadas por microscopía. La sensibilidad y especificidad del diagnóstico para
nuestro ensayo LAMPhimerus, fueron 79.17% y 65.98%, respectivamente.
Demostramos, por primera vez, que el papel de filtro común es útil para la recolección
fácil y el almacenamiento a largo plazo de muestras de heces humanas para la posterior
extracción de ADN y el análisis molecular de huevos de trematodos parásitos de
humanos. Este método simple, económico y, de fácil manejo, combinado con el ensayo
LAMPhimerus específico y sensible, tiene el potencial de ser utilizado como una prueba
de análisis molecular efectiva a gran escala para áreas endémicas de amphimeriosis.

88
 RESEARCH ARTICLE

Diagnosis of amphimeriasis by LAMPhimerus

assay in human stool samples long-term
storage onto filter paper
William Cevallos1,2☯, Pedro Fernández-Soto2☯*, Manuel Calvopiña3, Marı́a Buendı́a-
Sánchez2, Julio López-Abán2, Belén Vicente2, Antonio Muro2*
1 Centro de Biomedicina, Carrera de Medicina, Universidad Central del Ecuador, Quito, Ecuador,
2 Infectious and Tropical Diseases Research Group (e-INTRO), Biomedical Research Institute of
Salamanca-Research Centre for Tropical Diseases at the University of Salamanca (IBSAL-CIETUS), Faculty
a1111111111 of Pharmacy, University of Salamanca, Salamanca, Spain, 3 Carrera de Medicina, Universidad De Las
a1111111111 Américas (UDLA), Quito, Ecuador
a1111111111
a1111111111 ☯ These authors contributed equally to this work.
a1111111111 * [email protected] (PFS); [email protected] (AMA)

Abstract
OPEN ACCESS Amphimeriasis, a fish-borne zoonotic disease caused by the liver fluke Amphimerus spp.,
Citation: Cevallos W, Fernández-Soto P, Calvopiña has recently been reported as an emerging disease affecting an indigenous Ameridian
M, Buendı́a-Sánchez M, López-Abán J, Vicente B, group, the Chachi, living in Ecuador. The only method for diagnosing amphimeriasis was
et al. (2018) Diagnosis of amphimeriasis by
the microscopic detection of eggs from the parasite in patients’ stool samples with very low
LAMPhimerus assay in human stool samples long-
term storage onto filter paper. PLoS ONE 13(2): sensitivity. Our group developed an ELISA technique for detection of anti-Amphimerus IgG
e0192637. https://doi.org/10.1371/journal. in human sera and a molecular method based on LAMP technology (named LAMPhimerus)
pone.0192637 for specific and sensitive parasite DNA detection. The LAMPhimerus method showed to be
Editor: Ana Paula Arez, Universidade Nova de much more sensitive than classical parasitological methods for amphimeriasis diagnosis
Lisboa Instituto de Higiene e Medicina Tropical, using human stool samples for analysis. The objective of this work is to demonstrate the fea-
PORTUGAL
sibility of using dried stool samples on filter paper as source of DNA in combination with the
Received: October 13, 2017 effectiveness of our previously designed LAMPhimerus assay for successfully Amphimerus
Accepted: January 26, 2018 sp. detection in clinical stool samples. A total of 102 untreated and undiluted stool samples
Published: February 14, 2018 collected from Chachi population were spread as thin layer onto common filter paper for eas-
ily transportation to our laboratory and stored at room temperature for one year until DNA
Copyright: © 2018 Cevallos et al. This is an open
access article distributed under the terms of the extraction. When LAMPhimerus method was applied for Amphimerus sp. DNA detection,
Creative Commons Attribution License, which a higher number of positive results was detected (61/102; 59.80%) in comparison to parasi-
permits unrestricted use, distribution, and tological methods (38/102; 37.25%), including 28/61 (45.90%) microscopy-confirmed
reproduction in any medium, provided the original
Amphimerus sp. infections. The diagnostic parameters for the sensitivity and specificity wer-
author and source are credited.
ecalculated for our LAMPhimerus assay, which were 79.17% and 65.98%, respectively. We
Data Availability Statement: All relevant data are
demonstrate, for the first time, that common filter paper is useful for easy collection and
within the paper and its Supporting Information
files. long-term storage of human stool samples for later DNA extraction and molecular analysis
of human-parasitic trematode eggs. This simple, economic and easily handling method
Funding: This study was funded by the grants from
the Universidad Central del Ecuador (CUP combined with the specific and sensible LAMPhimerus assay has the potential to beused as
91750000.0000.374072) (to William F. Cevallos) an effective molecular large-scale screening test for amphimeriasis-endemic areas.
(http://www.uce.edu.ec/) and in part by the Japan
Society for the Promotion of Science, JSPS
(KAKENHI: Grant No.25305011) to Manuel

PLOS ONE | https://doi.org/10.1371/journal.pone.0192637 February 14, 2018 1 / 11

 Diagnosis of amphimeriasis by LAMPhimerus assay using filter paper

Calvopiña and by grants for Research on Emerging Introduction

and Re-emerging Infectious Diseases (H23-
Shinko-ippan-014 and H26-Shinkoippan- 009) Amphimeriasis, a fish-borne zoonotic disease caused by the liver fluke Amphimerus spp.
from the Ministry of Health, Labor and Welfare of (within the family Opisthorchiidae), was recently reported as an endemic disease in the tropi-
Japan (http://www.mhlw.go.jp/english/). Additional cal Pacific side of Ecuador. Data showing high prevalence of infection among an indigenous
funding was supported by the Health Research group, the Chachis, and also domestic cats and dogs residing in the same communities have
Projects: Technological Development Project in
been noted and, actually, human amphimeriasis has been reported as a new emerging food-
Health, grant number DTS16/00207 (to Antonio
Muro) and Health Research Project, grant number borne zoonotic disease. Parasites of the genus Amphimerus infect humans after ingestion of
PI16/01784 (to Pedro Fernández-Soto) of funding raw or undercooked freshwater fish containing viable metacercariae. Human disease is mostly
institution Instituto de Salud Carlos III (http://www. asymptomatic, occasionally causing non-specific, generalised symptoms. However, histopath-
isciii.es/). The funders had no role in study design, ological studies in cats and a double-crested cormorant infected with Amphimerus spp. showed
data collection and analysis, decision to publish, or
the presence of liver cirrhosis and pancreatitis [1,2]. Similarly, as occur in other human infec-
preparation of the manuscript.
tions by parasites of the family Opisthorchiidae, affected individuals with Amphimerus spp.
Competing interests: The authors have declared can suffer from suppurative cholangitis, cholelithiasis and cholangiocarcinoma [3–5]. Since
that no competing interests exist.
the Chachi community habitually consumes smoked freshwater fish, an estimated 13% of the
inhabitants living along the Rio Cayapas in the Province of Esmeraldas are a risk of acquiring
amphimeriasis [6].
Until very recently, the only method for diagnosing the disease was the microscopic detec-
tion of eggs from the parasite in patients’ stool samples, but it lacks in sensitivity [6]. To over-
come this limitation, our investigation group developed, for the first time, an ELISA technique
for detection of anti-Amphimerus IgG in human sera [7] and, afterwards, the first molecular
method based on LAMP technology (named LAMPhimerus) for specific and sensitive parasite
DNA detection. The LAMPhimerus method showed to be much more sensitive than the classi-
cal parasitological methods for amphimeriasis diagnosis using human stool samples for analy-
sis [8]. In that study, a number of human stool samples from Chachi communities were
preserved in 80% ethanol solution for later DNA extraction to test by LAMP assay. It is known
that collection of fresh stool samples for diagnostic purposes can be quite difficult in some
population groups. Besides, the handling, management and storage of a large number of
patients’ stool samples can be very laborious in large-scale field trials in poor settings with min-
imal infrastructures. This fact is especially true for many tropical diseases since they are fre-
quently in populations remote from sophisticated diagnostic facilities. Dried samples spots or
smears collected onto filter paper provide a potentially useful and economic means of over-
coming these drawbacks. The use of dried specimens -especially blood and sera samples- for
the diagnosis and surveillance of infectious diseases has been recently reviewed [9]. In general,
dried specimens perform with sensitivities and specificities very similar to gold standard sam-
ple types when using for DNA extraction and subsequent analysis by PCR-based molecular
methods. However, a standardization methodology is still needed. For collection, preservation
and easy handling of stool samples onto filter paper there are very few cases, and only includ-
ing protozoa studies [10–13].
It should be very interesting to join the advantages of using filter paper for easy collection
and preservation of human stool samples and the easy LAMP technology [14]. Considering a
number of salient advantages of LAMP over most PCR-based molecular methods [15, 16],
LAMP technology shows a potential use in clinical diagnosis and surveillance of infectious dis-
eases, particularly under field conditions in developing countries for most tropical diseases
[17, 18].
As mentioned above, we have recently developed a sensible and specific LAMP assay for
the successful detection of Amphimerus sp. DNA in human stool samples from a Chachi com-
munity. Now, the objective of this work is to demonstrate the feasibility of using dried stool
samples on filter paper as source of DNA in combination with the effectiveness of our

PLOS ONE | https://doi.org/10.1371/journal.pone.0192637 February 14, 2018 2 / 11

 Diagnosis of amphimeriasis by LAMPhimerus assay using filter paper

previously designed LAMPhimerus assay for successfully Amphimerus sp. detection in clinical
stool samples.

Materials and methods

Ethics statement
The study protocol was approved by the Ethics Committee of Universidad Central del Ecuador
(License number: LEC IORG 0001932, FWA 2482, IRB 2483. COBI-AMPHI-0064-11) and the
Ethics Committee of the University of Salamanca (protocol approval number 48531). Partici-
pants were given detailed explanations about the aims, procedures and possible benefits of the
study. Written informed consent was obtained from all subjects prior to the collection of bio-
logical samples for parasitological and molecular evaluation. Parents or guardians of children
who participated in the study provided written informed consent on the child’s behalf. All
samples were coded and treated anonymously. Procedures were performed in accordance with
the ethical standards laid down in the Declaration of Helsinki as revised in 2013.

Study area and population

The study was conducted during February 2016 in two indigenous Chachi villages (El Progreso
and Estero Vicente) in the Canton Eloy Alfaro alongside the Cayapas River in the Esmeraldas
province, located in the northwest coastal rainforest of Ecuador, 320 km from the capital
Quito. The indigenous Chachi -living together with Afro-ecuadorian and mestizo populations-
is the predominant autochthonous group in this area, representing 13% of the inhabitants in
this region. In these Chachi communities high prevalence of human (15.5% to 34.1%) and
local cats and dogs (71.4% and 38.7%, respectively) with Amphimerus spp. have been previ-
ously reported [6, 19]. They live in remote villages where the only way to reach them is by boat
along the river. Sanitation facilities are lacking. The members are hunters who typically eat
undercooked freshwater fish (mainly smoked fish) caught in the neighboring rivers and food
sharing is usually common [6, 19]. The main economic activities are agriculture, fishing and
exploitation of forest resources. The province of Esmeraldas, forms part of the tropical rainfor-
est known as “Choco Biogeográfico del Pacı́fico” which covers a section of the coast of Ecua-
dor, Colombia and Panamá. This area has been labeled as a biological hotspot, an area with an
extraordinary concentration of animal species. More details on the region can be accessed else-
where [20].

Human stool sampling and parasitological tests

A total of 102 participants living in two indigenous Chachi communities were enrolled in the
study, including 56 females (54.90%) and 46 males (45.09%) with a median age of 20.39 (range
1–65 years). Each participant was given a copro-parasitological flask for stool collection. Sam-
ples were collected within a few hours of stool passing. A single stool sample was individually
obtained from each participant. After collection, samples were transported to the laboratory of
parasitology (Centro de Biomedicina, Universidad Central del Ecuador, Quito, Ecuador) for
parasitological screening under light microscopy by simple sedimentation technique (SST),
formalin-ether concentration technique (FECT) and Kato-Katz technique (KKT). All samples
were examined by two qualified laboratory technicians according to the basic laboratory pro-
cedures in Medical Parasitology, recommended by the World Health Organization (WHO)
[21].
In addition, a portion of each sample was spread with a swab onto a filter paper (10 X 2 cm,
approximately), air-dried, numbered, folded in a half, and individually wrapped in foil. In that

PLOS ONE | https://doi.org/10.1371/journal.pone.0192637 February 14, 2018 3 / 11

 Diagnosis of amphimeriasis by LAMPhimerus assay using filter paper

way, samples were stored at room temperature until shipped to the Research Centre for Tropi-
cal Diseases at the University of Salamanca, Spain, for further DNA extraction (during Febru-
ary 2017) and molecular analysis as described below.

DNA extraction for molecular analyses

DNA from parasites. Amphimerus sp. genomic DNA was extracted from frozen adult
worms that were previously obtained from the livers of naturally infected cats and dogs of Cha-
chi communities, as described elsewhere [19], using a G-spin Total DNA Extraction Kit
(Intron Biotechnology) according to the manufacturers’ instructions. DNA was measured
using a Nanodrop ND-100 spectrophotometer (Nanodrop Technologies) and then diluted
with ultrapure distilled water to final concentration of 0.5 ng/μL to use as positive control in all
LAMP reactions.
DNA from human stool samples smeared on filter papers. DNA from human stool
samples smeared on filter papers was extracted 12 months after collection and preparation.
Steps followed for DNA extraction are shown in Fig 1. DNA extraction procedure was per-
formed in batches of 10 samples each for easy handling and also to prevent potential cross-
contamination. DNA extraction was performed using the i-genomic Stool DNA Extraction
Mini Kit (Intron Biotechnology) according to the manufacturers’ instructions with some addi-
tional procedures as follows. The smeared portion of filter papers were cut with scissors into
thin strips. Scissors were always sterilized before cutting the next sample to prevent contami-
nation. Thin strips of each sample were first placed into a 1.5 mL tube immersed in a lysis mix-
ture -TE (400 μL; pH 8.0), lysis buffer (200 μL Buffer SL) and proteinase K (20 μL)-, vortexed
vigorously, and subsequently incubated for 30 min at 65˚C in a thermoblock. During incuba-
tion, to help dissolve feces until complete lysis the tubes were vortexed or inverted at about

Fig 1. Human stool samples processing for DNA extraction. A. Batches organization. B. Smeared stool sample on filter paper. C, D. Filter paper is cut with
scissors into thin strips. E, F Thin strips of each sample are placed into a 1.5 mL tube immersed in a lysis mixture an incubated for 30 min at 65˚C in a
thermoblock G. Mixture is transfer for begining DNA extraction with the commercial kit i-genomic Stool DNA Extraction Mini Kit (iNtRON Biotechnology).
https://doi.org/10.1371/journal.pone.0192637.g001

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 Diagnosis of amphimeriasis by LAMPhimerus assay using filter paper

5–10 min intervals. After incubation, a volume of 500 μL approximately of the mixture was
transferred into a IR Spin Column for proper binding, washing, and elution steps. A final elu-
ate of 100 μL of genomic DNA (gDNA) was obtained from each sample and divided into two
aliquots of 50 μL each. After measuring the concentration using a Nanodrop ND-100 spectro-
photometer (Nanodrop Technologies), DNA samples were stored at -20˚C until use in molec-
ular assays.

LAMPhimerus assay
All the human stool samples were tested using the reaction mixture and specific primer set for
LAMP assay (LAMPhimerus) previously established by our group [8]. The LAMPhimerus
method amplifies a sequence of the Amphimerus sp. internal transcribed spacer 2 region (Gen-
Bank acc. no. AB678442.1). Briefly, the reaction was carried out with a total of 25 μL reaction
mixture containing 40 pmol of each FIP and BIP primers, 5 pmol of each F3 and B3 primers,
1.4 mM of each dNTP (Intron), 1x Isothermal Amplification Buffer -20 mM Tris-HCl (pH
8.8), 50 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Tween20- (New England Biolabs,
UK), 1 M betaine (Sigma, USA), supplementary 6 mM of MgSO4 (New England Biolabs, UK)
and 8 U of Bst 2.0 WarmStart DNA polymerase (New England Biolabs, UK) with 2 μL of tem-
plate DNA. Reaction tubes were placed in an economic heating block (K Dry-Bath) at a con-
stant temperature of 63˚C for 90–120 min and then heated at 80˚C for 5 min to stop the
reaction. In all LAMPhimerus trials positive controls (Amphimerus sp. gDNA) and a negative
controls (water instead DNA) were included.
The LAMP amplification results could be visually inspected by the naked eye by colour
change after adding 2 μL of 1:10 diluted 10,000X concentration SYBR1 Green I (Invitrogen)
to the reaction tubes. To avoid as much as possible, the potential risk of cross-contamination
with amplified products, all tubes were briefly centrifuged and carefully opened before adding
the fluorescent dye. Green fluorescence was clearly observed in successful LAMP reaction,
whereas it remained original orange in the negative reaction. The LAMP products (3–5 μL)
were also monitored using 1.5–2% agarose gel electrophoresis, visualized under UV light and
then photographed using an ultraviolet image system (Gel documentation system, UVItec,
UK).

Statistical analysis
To estimate the accuracy of the LAMP assay method as a diagnostic test, the percentages of the
sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV)
were calculated using the MedCalc statistical program version 16.8.4 (MedCalc Software,
Ostende, Belgium) according to the software instruction manual (http://www.medcalc.org).

Results
Parasitological tests
Of the total of 102 stool samples examined microscopically for the presence of Amphimerus
eggs, 38 (37.25%) resulted positive at least by one of the parasitological techniques applied,
including 27 (26.47%) positive by the simple sedimentation technique (SST), 19 (18.62%) posi-
tive by the formalin-ether concentration technique (FECT), and 27 (26.47%) positive by the
Kato-Katz technique (KKT). Up to 15 (15/102; 14.70%) stool samples resulted simultaneously
positive for the three parasitological tests; only 3 (3/102; 2.94%) stool samples resulted simulta-
neously positive for two parasitological tests, including 1 for SST and KKT and 2 for SST and
FECT.

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 Diagnosis of amphimeriasis by LAMPhimerus assay using filter paper

LAMPhimerus analysis
Amplification assays were performed in batches of 10–11 samples each for easy handling and
to prevent potential cross-contamination. All the samples were analyzed in duplicate with
identical result. We obtained LAMP positive results in 61/102 (59.80%) samples, including 33/
61 (54.09%) samples that were negative in all parasitological tests applied and 28/61 (45.90%)
samples that were positive at least by one of the parasitological technique applied. Of the 15
samples (nos. 6, 27, 28, 30, 31 32, 33, 42, 54, 60, 79, 84, 85, 93, 97) that were simultaneously
positive on three parasitological tests (FECT, SST and KKT), up to 13 (13/15; 86.66%) (nos. 27,
28, 30, 31 32, 33, 42, 54, 60, 79, 84, 85, 93) were also positive by LAMPhimerus assay; only 2
samples (2/15; 13.33%) (nos. 6 and 97) were negative on the LAMPhimerus assay. In all LAMP
positive amplifications, green fluorescence was clearly visualized under natural light condi-
tions and also by electrophoresis in agarose gels (Fig 2). Positive controls always worked well
and negative controls were never amplified.
In Fig 3 a total comparison of the results obtained by LAMPhimerus assay and parasitologi-
cal techniques applied for detecting Amphimerus sp. in human stool samples is showed.
Considering the microscopy findings by parasitological techniques as the reference stan-
dard, the following diagnostic parameters for the sensitivity and specificity were calculated for
our LAMPhimerus assay in this study: 79.17% sensitivity (95% CI: 65.0% -89.53%); 65.98%
specificity (95% CI: 55.66% -75.30%); 53.52% positive predicted value (95% CI: 45.72%
-61.16%) and 86.49% negative predicted value (95% CI: 78.36% -91.88%).

Discussion
The indigenous Chachi communities, who live in remote villages along the Rı́o Cayapas in the
north-western coastal rainforest of Ecuador, have been shown to have a high prevalence of
infection (15.5%-34.1%) with Amphimerus sp. [6]. Infection in domestic cats and dogs residing
in this endemic area has also been reported as high (71.4% and 38.7%, respectively) and these
animals have been proposed to serve as definite hosts and reservoirs for the parasite [19]. The
prevalence data obtained in these studies were assessed according to eggs findings in both
human and animal stool samples by classical parasitological methods.
Recently, in a pilot study using 44 human stool samples preserved in 80% ethanol solution
from that area, a novel LAMP assay (LAMPhimerus) showed to be more sensitive than parasi-
tological techniques for diagnosing human amphimeriasis [8]. Therefore, LAMPhimerus was
proposed as a new molecular tool that could be readily adaptable for effective field diagnosis in
amphimeriasis-endemic areas. However, the handling, management and storage of a large
numbers of patients’ fresh or frozen stool samples for diagnosing amphimeriasis in remote
areas with poor infrastructure can be very difficult in large-scale field trials. Filter paper poten-
tially provides a useful medium to overcome a number of difficulties of fresh sample collection,
preservation and transportation. This method has been widely used as a specimen substrate
when performing diagnostic or epidemiological surveys, especially in remote areas in
resource-poor settings [9]. However, most studies have used filter papers for blood and sera
collection and studies applying this method in human faecal samples for subsequent molecular
detection of parasites are still very limited; a few reported examples are Enterocytozoon bieneusi
[10, 11], Giardia duodenalis [12] and Blastocystis spp. [13]. Thus, the aim of this work is to
demonstrate the feasibility of using filter paper for collection and preservation of human stool
samples as source of DNA in combination with the effectiveness of our previously designed
LAMPhimerus assay for successfully Amphimerus sp. detection in clinical stool samples.
There are several kinds and brands of filter paper available consisting of 100% cellulose and
varying in thickness and pore size that have been used in different studies for PCR-based

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 Diagnosis of amphimeriasis by LAMPhimerus assay using filter paper

Fig 2. LAMPhimerus analysis of human stool samples in this study. Lanes M, molecular weight marker (100 bp Plus
Blue DNA Ladder); lanes C, Amphimerus sp. genomic DNA (1 ng); lanes N, negative controls (ultrapure water and no
DNA template); numbers 1–107, analyzed human stool samples.
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 Diagnosis of amphimeriasis by LAMPhimerus assay using filter paper

Fig 3. Comparison of the results obtained by the LAMPhimerus assay and classical parasitological techniques applied in this study. FECT, formalin-ether
concentration technique; SST, simple sedimentation technique; KKT, Kato-Katz technique; FEC, fecal egg count; EPG, eggs per gram of feces; +, positive for egg
detection. Values indicated for FEC and EPG correspond to the numbers of detected eggs. Numbers 1–107 correspond to the analyzed human stool samples.
https://doi.org/10.1371/journal.pone.0192637.g003

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 Diagnosis of amphimeriasis by LAMPhimerus assay using filter paper

detection of DNA from humans, plants, animals, viruses, bacteria and parasites [9, 13]. In
some cases, filter papers are impregnated with a proprietary mix of chemicals which provide
protection of DNA of samples thus avoiding degradation and subsequent successful extraction.
In addition, filter paper technology, such as FTA (Flinders Technology Associates)-treated
matrix cards, may inactivate highly pathogenic organisms for safety transporting and long-
term storage [13, 22]. However, some disadvantages of FTA paper are the use of a restricted
diluted faecal sample volume of 15 μL for detection of protozoa and the whole procedure to
get DNA template ready for PCR amplification takes approximately 3 hours [11].
In our preservation method, we used an economic common filter paper (100% cellulose
with smooth surface and normal hardness) which is used for routine laboratory procedures
such as basic filtration. Untreated and undiluted stool samples were spread as thin layer onto
the filter papers for easily transportation to our laboratory and stored at room temperature for
one year until DNA extraction. In our case, the whole procedure to get DNA template ready
for LAMPhimerus assay, including the cutting of the strips, pre-incubation with the lysis mix-
ture and DNA extraction with the commercial kit, can be performed in just 45 min. In addi-
tion, when measuring the DNA concentration of samples, the procedure yielded enough
quantity of quality DNA for molecular detection by LAMPhimerus assay. According to this,
long-term storage of dried stool samples onto common filter paper at room temperature
worked very well for subsequent DNA extraction.
Thus, when LAMPhimerus method was applied to test human stool samples for Amphi-
merus sp. DNA detection, a higher number of positive results was detected (61/102; 59.80%) in
comparison to parasitological methods (38/102; 37.25%), including 28/61 (45.90%) micros-
copy-confirmed Amphimerus sp. infection. It is important to note that up to 33/61 (54.09%)
samples that were negative in all parasitological tests applied were LAMPhimerus-positive.
These samples could be truly Amphimerus sp. infections undetected because of the known clas-
sically low sensitivity of the microscopy diagnosis in trematode infections [23]. This data rein-
forces the previous greater sensitivity of the LAMPhimerus assay over microscopic
examination when testing human stool samples preserved in 80% ethanol solution [8]. On the
other hand, only 8 truly parasitological Amphimerus-positive samples (nos. 3, 6, 11, 16, 71, 94,
97 and 107) were never amplified by LAMPhimerus assay. We think that the inoperative
amplification in these samples was not due to the ineffectiveness of LAMPhimerus method
because we obtained positive results in other microscopy-positive samples with lower EPG lev-
els. Besides, the minimum amount of Amphimerus sp. genomic DNA detectable by LAMPhi-
merus assay (1 pg) has been reported to correspond to less than one single egg of the parasite
in a stool sample [8]. An explanation for the inoperative amplification could be that the
amount of the sample onto the filter paper was not enough to obtain Amphimerus sp. DNA for
analysis. Perhaps, an inaccuracy in microscopy identification of parasite eggs occurred since
morphological similarity of the Amphimerus spp. eggs to those of closely related species
belonging to Opisthorchiidae family and to minute intestinal flukes makes diagnosis very diffi-
cult. Sometimes, it is necessary to use scanning electron microscopy to accurately observe the
differences between the coatings on the different species [6]. This observation would further
reinforce the specificity of LAMPhimerus method in the solely amplification of Amphimerus
sp. DNA.

Conclusions
In conclusion, to the best of our knowledge, we demonstrate for the first time that common fil-
ter paper is useful for long-term storage of human stool samples for later quality DNA extrac-
tion of human-parasitic trematode eggs. Additionally, this simple, economic and easily

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 Diagnosis of amphimeriasis by LAMPhimerus assay using filter paper

handling method combined with the specific and sensible LAMPhimerus assay has the poten-
tial to be used as an effective molecular large-scale screening test for amphimeriasis-endemic
areas. The system ’air-dried stool sample on filter paper’-LAMP assay could also be very inter-
esting and useful for molecular diagnosis of other human infectious parasitic diseases in
remote areas with poor settings.

Supporting information
S1 Checklist. STARD checklist.
(DOCX)

Author Contributions
Conceptualization: William Cevallos, Pedro Fernández-Soto, Manuel Calvopiña, Julio López-
Abán, Antonio Muro.
Data curation: Pedro Fernández-Soto, Marı́a Buendı́a-Sánchez, Antonio Muro.
Formal analysis: Pedro Fernández-Soto, Antonio Muro.
Funding acquisition: William Cevallos, Pedro Fernández-Soto, Manuel Calvopiña, Antonio
Muro.
Investigation: William Cevallos, Pedro Fernández-Soto, Manuel Calvopiña, Julio López-
Abán, Antonio Muro.
Methodology: Pedro Fernández-Soto, Marı́a Buendı́a-Sánchez.
Project administration: William Cevallos, Pedro Fernández-Soto, Manuel Calvopiña, Belén
Vicente, Antonio Muro.
Resources: William Cevallos, Belén Vicente.
Supervision: Pedro Fernández-Soto, Manuel Calvopiña, Julio López-Abán, Antonio Muro.
Validation: Pedro Fernández-Soto, Antonio Muro.
Writing – original draft: William Cevallos, Pedro Fernández-Soto, Manuel Calvopiña, Marı́a
Buendı́a-Sánchez, Antonio Muro.
Writing – review & editing: William Cevallos, Pedro Fernández-Soto, Manuel Calvopiña,
Antonio Muro.

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Conclusiones

4. CONCLUSIONES

100
Conclusiones

CONCLUSIONES

1. Se describe por primera vez la amphimeriosis como infección humana

causada por el trematodo hepático Amphimerus spp. en humanos residentes
en la selva tropical húmeda del Ecuador. Anteriormente solo se conocía la
infección en varias especies de animales domésticos y silvestres en varios
países de las Américas y Asia.

2. La prevalencia de Amphimerus spp. en personas residentes en las

comunidades indígenas Chachis del Noroeste de Ecuador estudiadas es
elevada, llegando hasta el 34% de infección.

3. Los animales domésticos -gatos y perros- que habitan en las comunidades

estudiadas también presentan altos niveles de infección por Amphimerus spp.
(71,4% y 38,7%, respectivamente) demostrando que actúan como
hospedadores definitivos y reservorios del parásito. De esta manera, se
evidencia el carácter zoonótico de esta tramatodosis.

4. La técnica ELISA desarrollada por primera vez para la detección de

anticuerpos IgG anti-Amphimerus spp., presenta alta sensibilidad (85%). Se
requieren futuros estudios para mejorar su rendimiento utilizando moléculas
más específicas.

5. Se ha desarrollado y evaluado por primera vez un método molecular basado

en la tecnología LAMP (LAMPhimerus) útil para el diagnóstico de la
amphimeriosis en muestras de heces humanas, presentando una sensibilidad
del 76,7% y una especificidad del 80,8%.

6. El uso de papel de filtro para la recolección y conservación de muestras de

heces humanas durante largos períodos de tiempo a temperatura ambiente
ha resultado ser un método efectivo para la posterior aplicación del método
LAMP.

7. El análisis de muestras de heces almacenadas sobre papel de filtro mediante

LAMPhimerus es de gran utilidad para el diagnóstico de la amphimeriosis en
condiciones de campo y sería de gran utilidad en la vigilancia epidemiológica
de esta parasitosis.

101
Otros Artículos de Investigación

5. OTROS ARTÍCULOS DE
INVESTIGACIÓN

102
Otros Artículos de Investigación

5.1 Sensibilidad de la técnica de Kato-Katz para la detección de

huevos de Amphimerus en muestras de heces, y prevalencia de
infección en Amerindios Chachis.
Manuel Calvopiña, Fernanda Diaz, Daniel Romero, William Cevallos, Hiromu Sugiyama.

Submitted to PLOS Neglected Tropical Diseases.

RESUMEN

El presente estudio compara la sensibilidad de cuatro métodos coproparasitarios

diferentes para detectar huevos del trematodo hepático Amphimerus spp. en heces y
evidencia la prevalencia de infección en residentes indígenas Chachi residentes en un
bosque tropical lluvioso de la costa Noroeste de Ecuador. Se examinaron un total de 105
muestras aplicando las técnicas de Kato-Katz (KK), sedimentación simple en tubo
(SSTT), concentración de formalina-éter (FEC) y microscopía de frotis directa (DM). La
sensibilidad de cada método fue del 25.7% (IC 95%: 17.5-34%), 21% (IC 95%: 13.1-
28.7), 18% (IC 95%: 10.7-25.3) y 1 % (95% CI: 0.9-2.9), respectivamente. Combinando
los cuatro métodos, 38 muestras fueron positivas con una prevalencia de infección de
36,1%. Nuestros resultados indican que el KK solo obtuvo el mejor resultado y detectó
27 (71%) de las 38 muestras positivas. Combinando KK y SSTT se diagnosticó
amphimeriosis en 35 muestras (92.1%) y en 31 muestras (81.6%) mediante KK y FEC.
La técnica de SSTT sola, detectó 22 muestras (57.9%) y sería recomendado realizar esta
técnica en estudios de campo debido a su simplicidad. La menor sensibilidad mostrada
por el método de DM, plantea una seria preocupación. Realizar dos técnicas en una sola
muestra mejora la detección de la infección por Amphimerus spp.

103
 SENSITIVITY OF THE KATO-KATZ TECHNIQUE IN COMPARISION TO
THREE OTHER DIAGNOSTIC METHODS FOR THE DETECTION OF
AMPHIMERUS EGGS AND THE PREVALENCE OF INFECTION IN CHACHI
AMERINDIANS IN ECUADOR

Manuel Calvopina1, Daniel Romero-Alvarez2, Fernanda Díaz3, William Cevallos3 and

Hiromu Sugiyama4

1. Carrera de Medicina, Facultad de Ciencias de la Salud, Universidad De Las Américas

(UDLA), Quito, Ecuador.
2. Department of Biology, Kansas University USA.
3. Carrera de Medicina, Universidad Central del Ecuador, Quito, Ecuador.
4. Department of Parasitology, National Institute of Infectious Diseases, Tokyo, Japan.

Abstract
The present study compares the sensitivity of four different coproparasitological methods
for detecting eggs of Amphimerus spp. liver fluke in stools and evidenced the prevalence
of infection in indigenous Chachi residents in a tropical rain forest in the northwest coast
of Ecuador. A total of 105 samples were examined applying the Kato-Katz technique
(KK), the spontaneous sedimentation technique in tube (SSTT), the formalin-ether
concentration technique (FEC), and direct smear microscopy (DM). The sensitivity of
each method was 25.7% (95% confidence interval (CI): 17.5–34%), 21% (95% CI: 13.1–
28.7), 18% (95% CI: 10.7–25.3), and 1% (95% CI: 0.9–2.9), respectively. Combining the
four methods, 38 samples were positive with a prevalence of infection of 36.1%. Our
results indicated that KK alone performed the best, detecting 27 (71%) of the 38 positive
samples. Combining KK and SSTT, amphimeriasis were diagnosed in 35 samples
(92.1%), and in 31 samples (81.6%) by KK and FEC methods. SSTT alone detected 22
samples (57.9%) and would be recommended for field studies because of its simplicity.
The lowest sensitivity showed by the DM method raises a concern. Performing two
techniques on a single sample enhanced the detection of Amphimerus infection.

INTRODUCTION
A high prevalence of human infection by the Amphimerus spp. liver fluke, was recently
reported in the indigenous population of Chachi located in the province of Esmeraldas, in
the northwest region of Ecuador. The prevalence, which varied from 15.5% to 34.1%,
was closely associated with the distance inland from the coast (Calvopina et al. 2011).
More recently, a higher prevalence of infection in cats and dogs living in the same
communities where humans are infected, was evidenced (Calvopina et al. 2015).
Amphimerus is a trematode belonging to the Opisthorchiidae family, along with the
Clonorchis sinensis and Opisthorchis spp., which can also affect the bile ducts but are
only endemic in Asian countries. Liver fluke infection is one of the more important food-
borne diseases worldwide and is considered by the World Health Organization as a
neglected tropical disease (WHO, 2010). C. sinensis and Opisthorchis spp. are classified
by the International Agency for Research on Cancer (IARC) as carcinogenic group 2A

1
and class 1, respectively considering the high association of their presence and the
development of cholangiocarcinoma (Keiser and Utzinger, 2009).

The genus Amphimerus Barker, 1911 (Trematoda: Opisthorchiidae) had been described
mainly in the American continent including Canada, USA, Costa Rica, Panamá,
Colombia, Venezuela, Ecuador, Brazil and Perú (Calvopiña et al. 2011) as well as from
South Korea, India and the Philippines (Eom et al. 1984, Yamaguti 1958). There are no
known reports of its presence in other continents such as Africa, Oceania, and Europe.
Adult worms are hermaphrodites and parasitize the bile ducts and gall bladder,
eliminating their eggs via stools. Consequently, microscopy examination of stool samples
searching for eggs is the most commonly used technique for diagnosing Amphimerus
infection (Calvopina et al. 2011 & 2015). Other diagnostic immunological and molecular
methods have been developed very recently, ELISA and LAMPhimerus, showing some
sensitivities of 85% and 76.6%, and some specificities of 71% and 80.7%, respectively.
(Cevallos et al. 2017a; Cevallos et al 2017b).

In the previous studies on human and domestic animals in Ecuador, diagnosis was made
by microscopic observation of Amphimerus eggs in stools (Calvopina et al. 2011 & 2015).
The only coproparasitological technique used was the formalin-ether concentration (FEC)
because the direct smear microscopy (DM) showed low sensitivity (Calvopina et al.
2011). DM is the most commonly used method in the public and private laboratories for
parasites examination of stools in Ecuador and neighboring countries.

For the detection of human helminth parasites, where their eggs are eliminated via stools,
WHO currently recommends the use of the Kato-Katz (KK) method (WHO, 2002). Other
commonly used methods include direct smear microscopy, formalin-ether concentration,
formalin-ethyl acetate, McMaster, FLOTAC and mini-FLOTAC (WHO, 2002).
Spontaneous sedimentation technique in tube (SSTT) has been described by Tello (1988)
(This article doesn’t appear in the Reference section) being a coproparasitological
technique with high sensitivity to detect the majority of intestinal parasites, including
eggs, larvae, cysts, and trophozoites and required less costly materials and equipment
(Tello et al. 2012). Sedimentation techniques use solutions of lower specific gravity than
the parasitic organisms, thus concentrating eggs and larvae in the sediment.

Sedimentation techniques are recommended for general diagnostic laboratories because

they are easier to perform and less prone to technical errors (CDC, 2013). All of these
techniques rely on visual examination of prepared stool samples under a light microscopy.
With the exception of SSTT all of the others use a small amount of fecal material.

For Amphimerus liver fluke there is a lack of studies comparing detection techniques to
the KK method. Hence, we conducted a study to determine the best method, or
combination of methods, to detect the eggs of Amphimerus in stools under field conditions
of individuals in a remote amphimeriasis endemic area and to determine the prevalence
of Amphimerus infection in a Chachi community-based sample.

2
MATERIALS AND METHODS
Study design and area. This is a cross-sectional study performed in a remote village
alongside the Cayapas river, located in the northwestern coastal region of Ecuador, in the
province of Esmeraldas, canton Eloy Alfaro, latitude of 0.721283°, longitude of -
78.906783°, at an elevation of 34 meters above sea level. The only means of
transportation to reach this community is by boat and is located approximately 131 Km
inland from the coast. The ecosystem, characteristics of this region, is described
elsewhere in previous studies (Sierra 1999, Calvopina et al. 2011 & 2015; Eisenberg et
al. 2006)

Study population. The study was first socialized and discussed with the local Chachilla
in a general assembly, where they were informed of the objectives of the study. Family
leaders were asked that all family members participate in the study by providing a stool
sample taken in the next three days. People were provided with a plastic stool flask
collector. A community health worker translated the information into the local language
“cha´palaachi”. Chachi indigenous group are Amerindians and represent around 13% of
the 24,000 inhabitants in the region. Fishing and farming are their main activities.

Stool collection and parasitological examination. The study was conducted in August
2015. Within a few hours of stool collection, each stool specimen was processed as
follow: 1) for the direct smear method (DM), a wet mount was prepared with
approximately 20 mg of feces suspended in Lugol’s staining solution and observed
microscopically; 2) a single KK thick smear, 3) five grams of fecal material was
suspended in 10 mL of warm saline solution, and 4) a smear was spotted in a FTA card
filter paper, 3 x 2 cm in size and preserved at room temperature for future studies. All
samples, except for formalin-ether sedimentation (FEC), were processed, observed under
light microscopy at the magnification of 100x and 400x and results were recorded, while
in the community. The remaining amount of fecal material was preserved in 70% ethanol
and 10% formalin and transported to the parasitology laboratory at Centro de
Biomedicina in Quito, Universidad Central del Ecuador, where it was used for FEC.

Kato Katz (KK) technique. A 3% methylene blue-glycerol solution was prepared in

advance. Cellophane strips of the size of a slide were cut and immersed in the solution
for 24 hours before their use. The KK technique was performed following the WHO
protocol. A single thick smear slide was prepared using 41.7 mg punched plastic
templates. Amphimerus egg count were recorded as total count and then converted to
eggs per gram (EPG) of stool as appropriate.

The EPG was obtained by multiplying the number of eggs per slide by 24 (WHO, 2002).
Formalin-ether concentration (FEC). The procedure followed was a modified protocol
described by Ash and Orihel (1991). Briefly, 3 grams of stool was weighed, homogenized,
and diluted to 14 mL with 10% formalin in a 15 mL plastic tube. After manually shaking
the tube to mix the fecal content, the mixture was filtered throughout a double surgical
gauze into a second conical screwed 15 mL plastic tube. The supernatant was manually

3
decanted and the plastic tube with its content allowed to stand for 10 minutes. After the
addition of 3 mL ethyl ether (Fisher Chemical, New Jersey, USA), the tube was filled up
till 10 mL with 10% formalin, capped and vigorously agitated for 30 seconds by hand.
The sample was centrifuged at room temperature at 2500 rpm for 5 minutes. This
procedure assured the formation of three layers within the tube. The upper 2 layers were
decanted and then 50 μl of 10% formalin was added to the sediment. Approximately 50
μl of sediment was placed on a slide and covered with a cover slip. A small drop of
Lugol´s staining solution was placed between the slide and cover slip and then examined
under the light microscope; first at 100x and confirming the observation with 400x
magnification.

Spontaneous Sedimentation Technique in Tube (SSTT). The protocol used was that
described by Tello (1988) with some modifications. Briefly, five grams of fresh feces
were weighed and homogenized by strong manual agitation in a 50-mL plastic tube
containing 25 mL of 0.9% saline (Na Cl) solution. It was then filtered through a double-
layer surgical gauze and poured to another clean 50-mL plastic tube and filled up to 45
mL with warm (40oC) saline and mixed again for around 30 seconds. If the feces
consistency was hard, it was macerated by a wooden tongue depressor and left upright at
room temperature for 2 hours. The supernatant was manually decanted and a sample of
the sediment was removed with a plastic pipet. The sample was placed on a glass slide
with Lugol´s staining solution, covered with a cover slip and observed under a light
microscopy (100x and 400x).

Two laboratory technicians observed the samples for the 4 different methods and one
expert (MC) verified and made an external control of the slides. An individual was
considered positive for Amphimerus infection if one or more eggs were observed by one
or more of the methods employed.

Statistical analyses
Since a “gold” standard test is not available for the detection of an Amphimerus infection,
the operational characteristics (sensitivity and negative predictive values (NPV) were
estimated using the combined results from the four methods employed as the “gold”
standard (Joseph J. et al. 1995; Dendokuri et al. 2004; Goodman et al. 2007). Data were
analyzed using SPSS version 16 and JavaStat software. Sensitivities, NPV, and kappa
were determined for the various tests to evaluate their operational characteristics. 𝑃 values
<0.05 were considered statistically significant. The sensitivity of each method and
combinations of methods were calculated based on comparison with those results
obtained by all methods combined.

The sensitivity and negative predictive value (NPV) were assessed for each diagnostic
technique in comparison to a composite reference standard, which was defined as being
positive if any of the 4 tests were positive.

4
Ethics
Signed consent was obtained by the leaders and school teachers of the community and
informed consent by each participant and from the guardians if children were enrolled.
Individuals were free to refuse their participation and its delivery of stool samples. Ethical
committee of the Universidad Central del Ecuador reviewed and approved this study
(license number LEC IORG 0001932, FWA 2482, IRB 2483. COBI-AMPH-0064-11).
In a second visit to the community, all participants positive for any kind of parasites, were
treated with antiparasitic medication following the guidelines of Ecuadorian Ministry of
Public Health.

RESULTS
The total population of the study village was 135 Chachi inhabitants. Of them, a total of
107 individual samples were collected; two were discarded due to inadequate amount of
fecal matter. Of the participating individuals 59 were females and 46 males, with a mean
age of 21.7 years, ranging from 1 to 65 years; 37% were of the 1-9 age group, 20% of 10-
19, 11% of 20-29, 12% of 30-39, and 19% of those greater than 40 years. Of the 105
samples examined for Amphimerus eggs, 27 (25.7%) (95% confidence interval (CI):
17.5–34%) were positive when examined by KK technique, 22 (21%) (95% CI: 13.1–
28.7) by the spontaneous sedimentation method, 19 (18%) (95% CI: 10.7–25.3) using the
formalin-ether concentration method, and 1 (1%) (95% CI: 0.9–2.9) based on direct smear
microscopy. The combined results of the four techniques used, showed an Amphimerus
infection prevalence rate of 36.2% with 38 of the 105 stool samples positive. There was
no significant difference in the infection rate by sex or age groups. The occurrence and
prevalence of soil transmitted helminths (STH) will be reported elsewhere.

The sensitivity of Amphimerus eggs detection as determined by each method or

combination of methods, is shown in table 1. Comparing each method separately the KK,
SSTT and FEC provided the higher sensitivities, however there was no statistically
significant difference between them (P <0.05). KK technique showed the highest
sensitivity (71%). However, when the 3 methods were compared with DM there was a
significantly higher difference (P >0.01). Combination of KK with either SSTT, or FEC
had a higher sensitivity in detecting eggs than any one method used alone.

The typical characteristics of Amphimerus eggs observed under microscopy utilizing the
four methods are showed in Figure 1. The eggs observed by light microscopic were oval
or piriform-shaped, with a thick yellow-brown shell surrounding it, operculate, measuring
26-33 µm x 13-16 µm, with a small knob seen on the abopercular end, showing the
developed miracidium takes up the interior of the eggs (Figure 1A). However, in the KK
technique, the interior miracidium disappear and the membrane became thin and
transparent, some were difficult to recognize (Figure 1B).

5
TABLE 1. Sensitivity of Amphimerus eggs detection by a single and combination of
methods when the combined results of four methods are considered the “gold”
standard.
Methods n (%) 95%
Confidence Interval
KK 27 (71) 57 – 85
SSTT 22 (57.9) 42.3 – 73.5
FEC 19 (50) 34.2 – 65.8
DM 01 (2.6) -2.4 – 7.6
KK + SSTT 35 (92.1) 83.6 – 100
KK + FEC 31 (81.6) 69.3 – 93.9
KK + DM 28 (73.7) 59.8 – 87.6
SSTT + FEC 27 (71) 57 – 85
SSTT + DM 27 (71) 57 – 85
FEC + DM 20 (52.7) 36.9 – 68.5
All (“gold” standard) 38 (100)
KK= Kato-Katz, SSTT= spontaneous sedimentation, FEC= formalin-ether concentration,
DM= direct smear microscopy.

The sensitivity and negative predictive values of the individual methods compared with
the combined results, which was considered our diagnostic “gold” standard, are shown in
table 2.
TABLE 2. Sensitivity and negative predictive values (NPV) of the methods used
when considering the combined results as the diagnostic “gold” standard.
Technique Sensitivity NPV
Kato Katz 71% (CI 95%: 66 – 75%) 85% (CI 95%: 79 – 91%)
Spontaneous 57.9% (CI 95%: 48.9 – 66.9%) 80% (CI 95%: 73 – 87%)
Sedimentation
Formalin-ether 50% (CI 95%: 41 – 59%) 77% (CI 95%: 69 – 85%)
Direct Microscopy 2.6% (CI 95%: 0.4 – 5.6%) 64% (CI 95%: 55 – 73%)

TABLE 3. Kappa index and its interpretation on the methods used when considering
the combined results as the diagnostic “gold” standard.
Technique Kappa Índex– Interpretation p-value
Kato Katz 0.758 – Good concordance 0.0001
Spontaneous 0.637 - Good concordance 0.0001
Sedimentation
Formalin-ether 0.561 - Moderate concordance 0.0001
Direct Microscopy 0.033 – Very low concordance 0.182

6
DISCUSSION
This is the first study to report the sensitivity on Amphimerus spp. egg detection by the
KK technique in comparison with three other coproparasitological methods. Our results
demonstrated that using a non-duplicated KK method showed the higher sensitivity,
identifying 27 (71%) of the 38 stool samples confirmed as Amphimerus positive by the
four combined methods. For the second and third ranking, the spontaneous sedimentation
technique in tube (SSTT) and the formalin-ether concentration (FEC) found 22 (58%)
and 19 (50%) of the samples to be positive, respectively. However, only 1 (2.6%) sample
was detected positive by the direct smear microscopy method (DM), this presents a great
concern particularly in Ecuador where DM is the only method provided by private and
public laboratories of the Ministry of Public Health (MSP).

At the moment, there is no “gold” standard diagnosis test for Amphimerus liver fluke,
even using a ELISA and LAMP methods where sensitivity reached only to 85% and
76.6%, and specificity to 71% and 80.7%, respectively (Cevallos et al. 2017; Cevallos et
al. 2017b). Here, we used the combined results of the 4 different methods as the diagnostic
“gold” standard for Amphimerus infection as has been used in previous studies for STH
(Santos et al. 2005, Goodman et al, 2007, Steinmann et al. 2008, Utzinger et al. 2008).
Summing together the results of the 4 methods employed, a high prevalence rate of 36.2%
for Amphimerus infection was found in the Chachi community studied. Merging KK and
SSTT results, the sensitivity raised to 92.1% (35 positive samples) and to 81.6% (31
samples) by combining KK and FEC methods. Therefore, performing two techniques on
a single sample enhanced the detection of Amphimerus infection.

In the present study, SSTT detected 9 extra positive samples that were negative with KK,
enhancing the diagnosis by 25% as compared with KK. This observation might be
explained for that with the SSTT, 5 g of fecal matter are used as compared to 41,7 mg for
KK. SSTT has the advantage of not needing expensive reagents; just warm saline and two
50-mL plastic tubes that could be recycled after careful washing. On the contrary, KK
requires 3% malachite-green glycerol or 3% methylene blue-glycerol solution, and FEC
method needs 10% buffered-formalin and centrifuge equipment. SSTT had already been
used in several studies for diagnosis of intestinal parasites in developing countries and
showed to be highly effective and inexpensive (Tello et al. 2012). For this study KK kits
were purchased for $400 USD expecting to do 400 stool examinations.

According to Tello et al (2012) the cost for SSTT is approximately $0.03 USD per sample.
Another important advantage of the SSTT method as compared to KK in field work
setting, is that eggs of Opisthorchiidae members are hard and are not easily broken.
Therefore, a stool sample can be fixed, transported, and preserved in 10% formalin,
merthiolate-iodine-formalin (MIF) or sodium acetate-acetic acid-formalin (SAF)
solutions for several days before SSTT is performed whilst with the KK method,
examination needs to be processed using fresh stool samples and observed the same day.
Hence, based on our field experience for detecting Amphimerus infection, we would

7
recommend SSTT because this method is easy to perform in field conditions, cost-
effective and capable for high volume screening of large populations.

Low sensitivities for DM has been reported elsewhere (WHO 2002, Goodman et al. 2007,
Keiser & Utzinger, 2009, Endris et al. 2013, Tello et al. 2012) as well as in our previous
study for Amphimerus (Calvopina et al. 2011). Unlike KK, SSTT and FEC, very small
amount of fecal material is processed for DM, approximately 2 mg, which could explain,
in part, its low sensitivity. Given the low sensitivity of DM and therefore the high
probability of missing Amphimerus infections, the utilization of this technique should be
discouraged for amphimeriasis diagnosis, especially when used alone.

The observed prevalence of Amphimerus infection based on the KK technique was higher
in comparison with the other 3 methods. However, there was no significant difference
statistically. In contrast, comparing the KK, SSTT and FEC methods against DM, a
significant difference was found. It is also corroborated by the Kappa index where both
KK and SSTT has good concordance whilst moderate and very low concordance by FEC
and DM, respectively. Examination of duplicate 41.7-mg stool sample and/or multiple
KK thick smear from one or 2 stool samples and in different days could enhance the
sensitivity of Amphimerus diagnosis, but it would increase labor, costs, and be more time
consuming (Utzinger et al. 2008, Goodman et al. 2007). Furthermore, processing multiple
samples for KK under field-work conditions, with limited resources such as power
supply, as we did in the present study, would be challenging. It is noteworthy to mention
that using the KK method, Amphimerus eggs were not detected in 11 (29%) stool
specimens that were observed using the other 3 methods, this can be explained because
the Amphimerus eggs can became transparent, thin shelled and could be easily missed as
is showed in figure 1B.

Also they can even disappear due to glycerin when long delays occur between the
preparation and the microscopic observation. A previous study showed that using
duplicate slides for KK, the sensitivity for detecting STH increased from 74 to 95% at
high infection intensity, however, dropped to 53-80% in low intensity settings (Nikolay
2014). In our study, existed a positive correlation between the intensity of infection, based
on fecal egg counts demonstrated by KK technique, with the detection of eggs observed
by SSTT and FEC methods (data not shown). A disadvantage for FEC method is that
reagents used may be hazardous or can be irritant; ether is highly flammable. The
sedimentation technique used at CDC is the formalin-ethyl acetate technique, a diphasic
sedimentation technique that avoids the problems of flammability of ether, however this
technique needs a centrifuge and is time-consuming.

Methods requiring a centrifuge as FLOTAC have a distinct disadvantage in field

laboratory settings, and also involve more procedural steps. The Mini-FLOTAC needs a
closed chamber for flotation and mixing, and a separate reading disc, all resulting in
higher costs and procedures. McMaster method is a flotation technique and therefore does

8
not detect eggs of trematodes as is Amphimerus or Schistosoma mansoni (Assefa et al.
2014).

Additionally, our study confirms the high prevalence of human infection by Amphimerus
in the Chachi group, who reside in the tropical rain forest along the Cayapas river, in the
province of Esmeraldas, Ecuador where the first human infections were described
(Calvopina et al. 2011). The prevalence of infection in this study (36.2%) is much higher
compared to that previously reported of 24% (Calvopina et al. 2011). In the previous
study, the most remote village (120 km inland from the coast) had the highest prevalence.
In the present report the study community was 135 km inland from the coast, suggesting
that the villagers living alongside the upper tributaries of the Cayapas river, in this area,
are more infected.

It is important to note that trematode species-specific diagnosis based on egg morphology

poses a problem for liver fluke of the Opisthorchiidae and for minute intestinal flukes of
Heterophyidae families (Kaewkes 2003). The size and shapes of eggs of the members of
the above mentioned families are quite similar to Amphimerus eggs (Calvopina et al. 2011
and 2015). However, it should not be a concern in South American countries, including
Ecuador, because those liver and intestinal flukes are only found in Asian countries (see
below) and has not been reported in humans of the Americas (Keiser and Utzinger, 2009).
In addition, definitive diagnosis of eggs found was confirmed to be Amphimerus by
scanning electronic microscopy (SEM) and examination of adult worms obtained from
the bile ducts of humans, cats and dogs in the same area where this study was performed
(Calvopina et al. 2011 and 2015).

In conclusion, the routinely used laboratory technique of DM method will underestimate

the prevalence of Amphimerus infection. Therefore, the use of additional diagnostic
methods is mandatory. The KK technique detected the greatest number of positive
samples for eggs of Amphimerus. However, 11 (29%) additional samples were detected
positive by SSTT and FEC, that were missed by KK method. Our results indicate that
SSTT would be the more cost effectiveness method in field conditions and the screening
a stool sample by 2 or 3 methods is likely to detect more Amphimerus infections in
endemic communities.

9
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FIGURE 1. A. Four eggs of Amphimerus spp. in an Lugol´s stained wet mount of
concentrated stool (200x magnification). B. An egg of Amphimerus spp. observed in a
slide by the Kato-Katz method (400x) in comparison with a fertile Ascaris lumbricoides
egg.

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6. ANEXOS

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Anexo 1. Metodología
1. Obtención del parásito adulto.

Los parásitos adultos de Amphimerus spp. fueron obtenidos del hígado de un gato de
la zona endémica del Río Cayapas, previo a una eutanasia del animal y al consentimiento
informado de sus dueños. El hígado fue lavado con agua destilada y posteriormente
depositado en un recipiente con NaCl al 0,9% (Figura 14). Se realizaron varios cortes
transversales al eje horizontal, se recuperaron parásitos adultos durante 2 h y a
continuación fueron colocados en tubos tipo Falcon de 15 mL conteniendo PBS estéril.

Figura 14. Obtención de parásito Amphimerus spp. adulto.

A. Hígado de gato. B. Lavado con agua estéril para posterior conservación del parásito.

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2. Preparación de antígeno somático de vermes adultos de Amphimerus spp.

Se realizó directamente en el campo a partir de parásitos adultos de Amphimerus

spp. que fueron lavados tres veces con PBS estéril (pH 7,4) temperatura de a 4°C en una
cámara pequeña con hielo seco. Se colocaron 90 parásitos en tubos de 15 mL (Falcon
®) añadiendo 5 mL de tampón de lisis que contiene Buffer Tris-base10mM-EDTA1mM

y 500 µL de una mezcla de inhibidores de proteasas (UltraCruz® Protease Inhibitor

Cocktail Tablet). Los parásitos fueron congelados a -20°C hasta el siguiente día.
Posteriormente, fueron homogenizados completamente con la ayuda de un sonicador
(ultrasonic processor). La sonicación se realizó en 4 ciclos con 60% de amplitud cada
ciclo con una duración de 1 min y 3 min de descanso. La solución generada en el
sonicado se centrifugó 2 veces durante 15 min a 3.500 rpm a 4 oC.

El sobrenadante fue dializado utilizando una membrana de diálisis (Spectra/Por®

Dialysis MWCO: 12-14,000). Se utilizó agua destilada estéril (H2Od) y 0.01% de
mertiolate blanco. La diálisis se realizó durante 72 h con cambios de agua cada 24 h a
4°C.

Para concentrar el antígeno dializado se realizó una segunda diálisis de saturación

con sucrosa en polvo, colocando la membrana en un recipiente y cubriéndolo
totalmente con sucrosa durante 5 h. El producto se almacenó a -40°C en alícuotas de 10
µL hasta su utilización como antígeno crudo total soluble de Amphimerus spp.

El antígeno obtenido fue liofilizado y almacenado en tubos Nunc™ a -20 °C hasta la

realización de la prueba ELISA. En el CIETUS (Universidad de Salamanca, Salamanca,
España), una alícuota del antígeno fue reconstituida en 600 µL de PBS estéril. La
cuantificación de la concentración de antígeno se valoró mediante colorimetría,
utilizando Albúmina Sérica Bovina (BSA) como proteína de referencia. Se determinó
una concentración de 0,21 µg/µL.

3. Desarrollo de la técnica de ELISA.

Para el desarrollo de la técnica ELISA, los antígenos fueron conservados en todo

momento en alícuotas de 100 µL a -20 oC. Tras varios ensayos de estandarización, el
protocolo final aprobado fue el siguiente: (1) Tapizado de la placa de poliestireno de
fondo plano con 100 µL de antígeno somático de Amphimerus spp., a razón de 4 µg/mL
diluido en tampón carbonato pH 9,6. Se cubrió y se dejó incubar toda la noche (12-18
h) a 4 oC. (2) Se diluyeron los sueros en PBS-Tween y se incubaron a 4 oC. (3) Al siguiente
día se realizó el bloqueo de la placa con BSA al 2% (PBS-Tween). La primera placa de
96 pocillos se preparó en 4 mL de PBS + 0,08 g de BSA. Se colocó 100 µL por pocillo y
se incubó a 37 oC durante 1 h. (4) Lavado de la placa con PBS-Tween 20 al 0,05%
durante 3 min. Se repite esta operación 3 veces. Se colocó 200 µL por pocillo. (5) Se

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añadió el suero problema a la dilución de 1:50 en PBS-Tween. 100 µL por pocillo. Se

cubrió la placa y se incubó a 37 oC durante 1 h. (6) Lavado de la placa con PBS-Tween
20 al 0,05% durante 3 minutos. Se repite esta operación por 3 ocasiones. Se colocó 200
µL por pocillo. (7) Se añadió el conjugado o anticuerpo secundario (Sigma), marcado
con la enzima peroxidasa a una dilución 1:2000 en PBS-Tween, 100 µL por pocillo. Se
incubó la placa durante 1 h a 37 oC. (8) Lavado de la placa con PBS-Tween 20 al 0,05%
durante 3 min. Se repite esta operación tres veces. Se colocó 200 µL por pocillo. (9)
Posteriormente, se reveló la reacción utilizando solución de revelado (C6H8O7,
PO4HNa2, H2O y OPD), se colocó 100 µL/pocillo. Incubar en oscuridad durante 2-10 min.
Es importante que el agua oxigenada se añada al final de la preparación de la solución
de revelado. Finalmente se paró la reacción con 50 µL/pocillo de H2SO4 3N y se leyó la
densidad óptica (DO) en un lector de ELISA (Ear400FT ELISA reader Lab. Instruments),
utilizando un filtro de longitud de onda de 492 nm.

Materiales y reactivos
1. Péptido sintético o extracto del parásito (antígeno).

2. Sueros humanos (anticuerpo primario).

3. Anticuerpo secundario (IgG anti-humano) acoplados a peroxidasa.

4. Tampón Fosfato Salino al 5%; pH 7,20: Na2HPO4 (1,07g), NaH2PO4 (0,39g),

NaCl (8,5g), H2O (1L), Tween 20 (500 µL).

5. Tampón Carbonato. pH 9,6: Na2CO3 (0,159 g), NaHCO3 (0,293 g), H2O (100
mL).

6. Tampón Citrato. pH 5: C6H8O7 (2,14 g), PO4HNa2 (1,4 g), H2O (400 mL).

7. Ácido Sulfúrico. 3N: H2SO4 (8mL), H2O (92mL).

8. Solución de Revelado: Tampón Citrato (20 mL) y OPD (0,0053 g). Para la placa
de 96 pocillos se utilizaron 0,0028 g en 10 mL H2O2 (8 µL). Éste se añade justo
antes de colocarlo en los pocillos.

4. Desarrollo de la TD-PCR.

Para comprobar si el anillamiento de los cebadores externos F3 y B3 generaba una

amplificación del tamaño esperado in silico según el diseño previo informático (209 pb),
se realizó una touchdown PCR (TD-PCR). En esta variante de la PCR la temperatura de
anillamiento va disminuyendo en cada ciclo de amplificación de manera que garantiza
un abanico de temperaturas de anillamiento de los cebadores idóneo para la correcta
amplificación de la secuencia diana. Se ensayaron diferentes condiciones. En la tabla 5,

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se muestran las concentraciones finalmente utilizadas para la amplificación y los

reactivos usados para la mezcla de la reacción. Todas las reacciones de PCR se llevaron
a cabo en un termociclador Mastercycler Gradient®-96 well (Eppendorf).

Para determinar la sensibilidad de la PCR (F3-B3) se utilizaron diluciones seriadas

desde 1×10-1 a 1×10-9 ng/µL de ADN de Amphimerus spp. las cuales fueron preparadas
con agua ultrapura y almacenadaa a -20 oC. Todos los ensayos de la PCR se realizaron
con 2 μL de ADN diana (5 ng/μL) en cada caso. Siempre se incluyeron controles
negativos (agua ultrapura) y controles positivos (ADN genómico de Amphimerus spp.).
Para valorar la especificidad se usaron muestras de ADN de otros parásitos disponibles
en el CIETUS y también de Clonorchis sinensis y Opisthorchis viverrini (0,5 ng/µL). En
todos los casos se utilizó 2 μL de ADN molde y en cada reacción se incluyeron controles
negativos a (con agua ultrapura en lugar de ADN) y controles positivos (con ADN de
Amphimerus spp.)

Tabla 5. Componentes y condiciones de reacción de la PCR (F3-B3).

Mezcla de reacción y condiciones de amplificación utilizadas en la TD-PCR con los cebadores

externos F3 y B3 para la amplificación de la secuencia nucleotídica de 209 pb de ADN de
Amphimerus spp.

Componentes Volumen Tª (°C) Tiempo Ciclos

H20 15.1 µL
10X Buffer 2.5 µL 94 1 min x1
MgCl2 (25 mM) 1.5 µL 94 20 s x2
dNTPs (2.5 mM) 2.5 µL 64-58 20 s
F3 (5 pmol) 0.5 µL 72 30 s
B3 (5 pmol) 0.5 µL 94 1 min
x 15
Taq-polimerasa (2 U) 0.4 µL 94 1 min

ADN molde 2 µL 57 20 s
TOTAL 25 µL 72 30 s
72 10 min x1

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5. Desarrollo de la técnica LAMPhimerus.

5.1 Obtención de ADN de Amphimerus spp.

Para la obtención del ADN genómico de Amphimerus spp. se utilizó un total de 100
parásitos adultos obtenidos de pacientes de las comunidades de Corriente Seca y Estero
Vicente del Río Cayapas. Los ejemplares adultos fueron recuperados tras el tratamiento
de los pacientes con praziquantel y mantenidos en alcohol hasta la extracción del ADN
en el laboratorio del CIETUS mediante el kit comercial G-spin Total DNA Extraction Kit
(Intron Biotechnology) siguiendo las instrucciones del fabricante. El ADN obtenido se
cuantificó mediante espectrofotometría utilizando un Nanodrop (ND-1000) y se diluyó
en agua ultrapura a concentraciones finales de 5 ng/µL y 0.5 ng/µL; a partir de estas
concentraciones se realizaron diluciones seriadas desde 1×10-1 a 1×10-9 ng/µL. Este
ADN se utilizó como control positivo en todas las reacciones de PCR y LAMP realizadas
y también para evaluar la sensibilidad de las dos técnicas moleculares.

5.2 Obtención de ADN de otros parásitos.

Para evaluar la especificidad de la PCR y de la técnica LAMP se utilizó un total de 16

muestras de ADN de otros parásitos disponibles en el laboratorio del CIETUS,
incluyendo varios helmintos y protozoos. Los ADN utilizados fueron de los siguientes
parásitos: Clonorchis sinensis, Opisthorchis viverrini, Schistosoma mansoni, S.
haematobium, S. japonicum, S. intercalatum, Fasciola hepatica, Dicrocoelium
dendriticum, Onchocerca volvulus, Strongyloides venezuelensis, Trichinella spiralis,
Taenia truncata, Echinococcus granulosus, Cryptosporidium parvum, Giardia duodenalis
y Entamoeba histolytica. La concentración utilizada para cada muestra fue de 0.5 ng/µL.

5.3 Diseño del LAMP

Tras la búsqueda de secuencias disponibles en las bases de datos de GenBank

(http://www.ncbi.nlm.nih.gov) y comparación mediante BLAST (Altschul et al., 1990),
se seleccionó una diana de amplificación correspondiente a una secuencia de 459 pb de
ADN lineal de Amphimerus spp. HS-2011 aislado de hospedador humano (Genbank
accesion no. AB678442.1) (Calvopiña et al., 2015) sobre la que se diseñaron los
cebadores (F3, B3, FIP y BIP) utilizando el software Primer Explorer V4
(https://primerexplorer.jp/e). Inicialmente, el software generó varios juegos de 4
cebadores; se seleccionó el más apropiado en función de los criterios descritos en el
"Manual del software para la generación de cebadores para LAMP"
(http://primerexplorer.jp/e/v4_manual/index.html). Una vez seleccionados, su
síntesis se encargó a la empresa Fisher Scientific y se resuspendieron en agua ultrapura
a una concentración final de 100 pmol/µL. El juego de cebadores se probó con
diferentes mezclas de reacción empleando distintas enzimas (Bst ADN polimerasa

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Large Fragment, Bst ADN polimerasa 2.0 y Bst ADN polimerasa 2.0 Warm Start) y
concentraciones variables de MgSO4 y betaína a distintas temperaturas de incubación
(61oC, 63 oC y 65 oC). Finalmente, se seleccionó la temperatura de 63 oC como más
idónea para la amplificación utilizando la mezcla de reacción que aparece en la tabla 6.

Tabla 6. Componentes utilizados en la reacción LAMPhimerus

Mezcla de reacción utilizada en la reacción LAMPhimerus. La enzima Bst polimerasa 2.0

WarmStart, buffer y MgSO4 fueron suministrados por New England Biolabs; la betaína por
SIGMA y los dNTPs por Intron.

Componentes Volumen (μL)

H2O 7,7
Betaína (1M) 5
MgSO4 (0.8 mM) 1,5
dNTPs (2.5 mM) 3,5
10X Buffer 2,5
FIP (40 pmol/µL) 0,4
BIP (40 pmol/µL) 0,4
F3 (5 pmol/µL) 0,5
B3 (5pmol/µL) 0,5
Bst polimerasa 2.0 Warm Start 1
ADN 2
TOTAL 25

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6. Método LAMP para la amplificación de ADN de Amphimerus spp.

Para comprobar el correcto funcionamiento del juego de 4 cebadores sintetizados,

se realizaron diversas reacciones de amplificación utilizando ADN de Amphimerus spp.
como control positivo de la reacción. Todas las reacciones se incubaron en un
termobloque K Dry-Bath® (No. DB-006) a 63 oC durante 60 min o 120 min; transcurrido
ese tiempo, la temperatura se elevó a 80 oC durante 10 min para desactivar a la enzima
y detener la reacción.

Para valorar la especificidad del LAMPhimerus se utilizaron las muestras de ADN de

los distintos parásitos mencionados anteriormente. El límite de detección de la técnica
se estableció con las diluciones seriadas (10-1 a 10-9 ng/µL) obtenidas a partir de ADN
de gusanos adultos de Amphimerus spp.

7. Detección de los productos de amplificación.

7.1 TD-PCR.

Para la detección de los productos obtenidos mediante PCR se utilizó la

electroforesis en gel de agarosa al 1,5% (100 mL de TBE, 1,5 g de agarosa y 5 μL de
bromuro de etidio) a 60v durante 20 min y posteriormente a 100v durante
aproximadamente 1h. Los geles se visualizaron utilizando un transiluminador con luz
UV (Gel documentation system, UVItec, UK). Por último, los geles se fotografiaron y
almacenaron en un soporte informático para su manejo y edición.

7.2 LAMP.

En el caso de la amplificación mediante la técnica LAMP, la visualización de los

resultados se realizó mediante:

A. Colorimétricamente. Se añadió a los tubos de reacción 2 μL (dilución 1:10;

10.000X) del colorante fluorescente SYBR Green I (InvitrogenTM). En los tubos en los
que hubo amplificación se produjo un viraje del colorante de naranja a verde. En los
tubos negativos se mantuvo el color naranja original propio del colorante.

B. Electroforesis en gel de agarosa. De igual forma que para la detección de los

productos obtenidos mediante TD-PCR.

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8. Análisis estadísticos

Para estimar la exactitud los métodos de ELISA y LAMPhimerus como pruebas

diagnósticas se calcularon los porcentajes de sensibilidad, especificidad, valor
predictivo positivo (VPP) y valor predictivo negativo (VPN); también la curva ROC
(Receiver Operating Characteristic o Característica Operativa del Receptos) para el
ELISA, utilizando los programas estadísticos SPSS v.22 (disponible en
https://www.ibm.com) y MedCalc v. 15.2.2 (MedCalc Software, Ostende, Belgium;
www.medcalc.org).

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Anexo 2. Otras publicaciones con índice de impacto

Determinants of Short-term Movement in a Developing Region and Implications
for Disease Transmission.

Kraay ANM, Trostle J, Brouwer AF, Cevallos W, Eisenberg JNS.

Epidemiology. 2018 Jan;29(1):117-125.

Intralesional Infiltration with Meglumine Antimoniate for the Treatment of

Leishmaniasis Recidiva Cutis in Ecuador.

Calvopiña M, Cevallos W, Paredes Y, Puebla E, Flores J, Loor R, Padilla J.

Am J Trop Med Hyg. 2017 Nov;97(5):1508-1512.

Coinfection of Leishmania guyanensis and Human Immunodeficiency Virus-

Acquired Immune Deficiency Syndrome: Report of a Case of Disseminated
Cutaneous Leishmaniasis in Ecuador.

Calvopina M, Aguirre C, Cevallos W, Castillo A, Abbasi I, Warburg A.

Am J Trop Med Hyg. 2017 May;96(5):1151-1154.

I get height with a little help from my friends: herd protection from sanitation on
child growth in rural Ecuador.

Fuller JA, Villamor E, Cevallos W, Trostle J, Eisenberg JN.

Int J Epidemiol. 2016 Apr;45(2):460-9.

Antibiotic Resistance in Animal and Environmental Samples Associated with

Small-ScalePoultry Farming in Northwestern Ecuador.

Braykov NP, Eisenberg JN, Grossman M, Zhang L, Vasco K, Cevallos W, Muñoz D,

Acevedo A, Moser KA, Marrs CF, Foxman B, Trostle J, Trueba G, Levy K.

mSphere. 2016 Feb 10;1(1). pii: e00021-15.

Distribution of Enteroinvasive and Enterotoxigenic Escherichia coli Across Space

and Time in Northwestern Ecuador.

Bhavnani D, Bayas Rde L, Lopez VK, Zhang L, Trueba G, Foxman B, Marrs C, Cevallos W,
Eisenberg JN.

Am J Trop Med Hyg. 2016 Feb;94(2):276-84.

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Anexos

Association of Household Food Insecurity with the Mental and Physical Health of
Low-Income Urban Ecuadorian Women with Children.

Weigel MM, Armijos RX, Racines M, Cevallos W, Castro NP.

J Environ Public Health. 2016 Sep 26. PubMed PMID: 27752266.

Food Insecurity Is Associated with Undernutrition but Not Overnutrition in

Ecuadorian Women from Low-Income Urban Neighborhoods.

Weigel MM, Armijos RX, Racines M, Cevallos W.

J Environ Public Health. Epub 2016 Mar 23. PubMed PMID: 27110253.

Effects of selection pressure and genetic association on the relationship between

antibiotic resistance and virulence in Escherichia coli.

Zhang L, Levy K, Trueba G, Cevallos W, Trostle J, Foxman B, Marrs CF, Eisenberg JN.

Antimicrob Agents Chemother. 2015 Nov; 59 (11): 6733-40.

Unexpected distribution of the fluoroquinolone-resistance gene qnrB in

Escherichia coli isolates from different human and poultry origins in Ecuador.

Armas-Freire PI, Trueba G, Proaño-Bolaños C, Levy K, Zhang L, Marrs CF, Cevallos W,

Eisenberg JN.

Int Microbiol. 2015 Jun;18 (2): 85-90.

A national survey to determine prevalence of Trypanosoma cruzi infection among

pregnant women in Ecuador.

Costales JA, Sánchez-Gómez A, Silva-Aycaguer LC, Cevallos W, Tamayo S, Yumiseva CA,

Jacobson JO, Martini L, Carrera CA, Grijalva MJ.

Am J Trop Med Hyg. 2015 Apr; 92 (4): 807-10.

Spatial Variability of Escherichia coli in Rivers of Northern Coastal Ecuador.

Rao G, Eisenberg JN, Kleinbaum DG, Cevallos W, Trueba G, Levy K.

Water (Basel). 2015 Feb 13;7 (2): 818-832.

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Anexos

Identifying etiological agents causing diarrhea in low income Ecuadorian

communities.

Vasco G, Trueba G, Atherton R, Calvopiña M, Cevallos W, Andrade T, Eguiguren M,

Eisenberg JN.

Am J Trop Med Hyg. 2014 Sep; 91(3): 563-9.

Household effectiveness vs. laboratory efficacy of point-of-use chlorination.

Levy K, Anderson L, Robb KA, Cevallos W, Trueba G, Eisenberg JN.

Water Res. 2014 May 1; 54: 69-77.

Impact of rainfall on diarrheal disease risk associated with unimproved water

and sanitation.

Bhavnani D, Goldstick JE, Cevallos W, Trueba G, Eisenberg JN.

Am J Trop Med Hyg. 2014 Apr; 90(4): 705-11.

Heavy rainfall events and diarrhea incidence: the role of social and
environmental factors.

Carlton EJ, Eisenberg JN, Goldstick J, Cevallos W, Trostle J, Levy K.

Am J Epidemiol. 2014 Feb 1; 179 (3): 344-52.

HIV and syphilis infection in pregnant women in Ecuador: prevalence and

characteristics of antenatal care.

Sánchez-Gómez A, Grijalva MJ, Silva-Aycaguer LC, Tamayo S, Yumiseva CA, Costales JA,
Jacobson JO, Chiriboga M, Champutiz E, Mosquera C, Larrea M, Cevallos W.

Sex Transm Infect. 2014 Feb; 90(1): 70-5.

Molecular identification of Giardia duodenalis in Ecuador by polymerase chain

reaction-restriction fragment length polymorphism.

Atherton R, Bhavnani D, Calvopiña M, Vicuña Y, Cevallos W, Eisenberg J.

Mem Inst Oswaldo Cruz. 2013 Jun; 108 (4): 512-5.

Social connectedness and disease transmission: social organization, cohesion,

village context, and infection risk in rural Ecuador.

Zelner JL, Trostle J, Goldstick JE, Cevallos W, House JS, Eisenberg JN

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Am J Public Health. 2012 Dec; 102 (12): 2233-9.

Synergistic effects between rotavirus and coinfecting pathogens on diarrheal

disease: evidence from a community-based study in northwestern Ecuador.

Bhavnani D, Goldstick JE, Cevallos W, Trueba G, Eisenberg JN.

Am J Epidemiol. 2012 Sep 1; 176 (5): 387-95.

In-roads to the spread of antibiotic resistance: regional patterns of microbial

transmission in northern coastal Ecuador.

Eisenberg JN, Goldstick J, Cevallos W, Trueba G, Levy K, Scott J, Percha B, Segovia R,

Ponce K, Hubbard A, Marrs C, Foxman B, Smith DL, Trostle J.

J R Soc Interface. 2012 May 7; 9 (70): 1029-39.

Where science meets policy: comparing longitudinal and cross-sectional designs

to address diarrhoeal disease burden in the developing world.

Markovitz AR, Goldstick JE, Levy K, Cevallos W, Mukherjee B, Trostle JA, Eisenberg JN.

Int J Epidemiol. 2012 Apr; 41 (2): 504-13.

Plesiomonas shigelloides infection, Ecuador, 2004-2008.

Escobar JC, Bhavnani D, Trueba G, Ponce K, Cevallos W, Eisenberg J.

Emerg Infect Dis. 2012 Feb;18 (2): 322-4.

Rapid changes in rotaviral genotypes in Ecuador.

Hasing ME, Trueba G, Baquero MI, Ponce K, Cevallos W, Solberg OD, Eisenberg JN.

J Med Virol. 2009 Dec; 81 (12): 2109-13.

Raising the level of analysis of food-borne outbreaks: food-sharing networks in

rural coastal Ecuador.

Trostle JA, Hubbard A, Scott J, Cevallos W, Bates SJ, Eisenberg JN.

Epidemiology. 2008 May; 19 (3): 384-90.

Relating diarrheal disease to social networks and the geographic configuration

of communities in rural Ecuador.

Bates SJ, Trostle J, Cevallos WT, Hubbard A, Eisenberg JN.

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Anexos

Am J Epidemiol. 2007 Nov 1; 166 (9): 1088-95.

Symptomatic and subclinical infection with rotavirus P[8]G9, rural Ecuador.

Endara P, Trueba G, Solberg OD, Bates SJ, Ponce K, Cevallos W, Matthijnssens J,

Eisenberg JN.

Emerg Infect Dis. 2007 Apr; 13(4): 574-80.

High prevalence of enteroinvasive Escherichia coli isolated in a remote region of

northern coastal Ecuador.

Vieira N, Bates SJ, Solberg OD, Ponce K, Howsmon R, Cevallos W, Trueba G, Riley L,
Eisenberg JN.

Am J Trop Med Hyg. 2007 Mar; 76 (3): 528-33.

Environmental change and infectious disease: how new roads affect the
transmission of diarrheal pathogens in rural Ecuador.

Eisenberg JN, Cevallos W, Ponce K, Levy K, Bates SJ, Scott JC, Hubbard A, Vieira N, Endara
P, Espinel M, Trueba G, Riley LW, Trostle J.

Proc Natl Acad Sci U S A. 2006 Dec 19; 103 (51): 19460-5.

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Anexo 3. Contribuciones en Congresos

Divulgación científica de resultados ELISA, PCR y LAMPhimerus.

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131