CHAPTER 2-Parasit
CHAPTER 2-Parasit
CHAPTER 2-Parasit
protozoan Infections
Intestinal Amebae
Pilarita T. Rivera, Windell L. Rivera, Juan Antonio A. Solon
20
Chapter 2: protozoan Infections 21
although natural infection of primates has been Infection with E. histolytica occurs when cysts
reported. The quadrinucleate cyst is resistant to are ingested from fecally-contaminated material
gastric acidity and desiccation, and can survive (Figure 2.1). Other modes of transmission
in a moist environment for several weeks. include venereal transmission through fecal-oral
and cytophagocytosis. In vitro, amebic killing study involving 206 patients with probable
of target cultivated mammalian cells involve ALA as diagnosed by ultrasound, the two
receptor-mediated adherence of ameba to most frequent manifestations were fever in
target cells, amebic cytolysis of target cells, 77% and RUQ pain in 83%. Pain is either
and amebic phagocytosis of killed or viable localized in or referred to the right shoulder.
target cells. E. histolytica trophozoites adhere The liver is tender, especially in acute cases,
to the colonic mucosa through a galactose- and hepatomegaly is present in 50% of cases.
inhibitable adherence lectin (Gal lectin). Then, Chronic disease (>2 weeks duration) is found
the amebae kill mucosal cells by activation of in older patients and it involves wasting with
their caspase-3, leading to their apoptotic death significant weight loss rather than fever. Only
engulfment. 30% of ALA cases have concurrent diarrhea.
Recent studies have shown that susceptibility However, daily stool cultures revealed that 72%
of humans to E. histolytica infection is associated harbored trophozoites even in asymptomatic
with specific alleles of the HLA complex. infections. Mortality in uncomplicated ALA is
Majority of cases present as asymptomatic less than 1%.
infections with cysts being passed out in The onset of amebic colitis may be sudden
the stools (cyst carrier state). The recent after an incubation period of 8 to 10 days, or
differentiation of E. dispar and E. histolytica after a long period of asymptomatic cyst carrier
by PCR has confirmed the high prevalence state. ALA may have all acute presentation of
of non-pathogenic E. dispar compared to the less than 2 weeks duration or a chronic one of
pathogenic E. histolytica. However, studies also more than 2 weeks duration. The recurrence
revealed that most E. histolytica infections in rate was found to be 0.29% in a five-year study
endemic communities are asymptomatic. of ALA in Mexico.
Amebic colitis clinically presents as gradual The most serious complication of amebic
onset of abdominal pain and diarrhea with or colitis is perforation and secondary bacterial
without blood and mucus in the stools. Fever peritonitis. Colonic perforation occurs in 60%
is not common and it occurs only in one third of fulminant colitis cases.
of patients. Although some patients may only In ALA, the most serious complications are
have intermittent diarrhea alternating with rupture into the pericardium with a mortality
constipation, children may develop fulminant rate of 70%, rupture into the pleura with
colitis with severe bloody diarrhea, fever, and mortality of 15 to 30%, and super infection.
abdominal pain. Intraperitoneal rupture, which occurs in 2 to
Ameboma occurs in less than 1% of 7.5% of cases, is the second most common
intestinal infections. It clinically presents as complication. However, it is not as serious as
a mass-like lesion with abdominal pain and colonic perforation because ALA is sterile.
a history of dysentery. It can be mistaken for Secondary amebic meningoencephalitis
carcinoma. Asymptomatic ameboma may also occurs in 1 to 2%, and it should be considered
occur. in cases of amebiasis with abnormal mental
Amebic liver abscess (ALA) is the most status. Renal involvement caused by extension
common extra-intestinal form of amebiasis. of ALA or retroperitoneal colonic perforation is
The cardinal manifestations of ALA are fever rare. Genital involvement is caused by fistulae
and right upper quadrant (RUQ) pain. Several from ALA and colitis or primary infection
studies have shown these two as the most through sexual transmission.
frequent complaints, particularly in acute Natural or innate immunity to E. histolytica
cases (<2 weeks duration). In a Philippine in the intestines involves mucin inhibition of
24 MedICal parasItology In the phIlIppInes
amebic attachment to the underlying mucosal Acute amebic colitis should be differentiated
cells. In the systemic circulation, the mechanism from bacillary dysentery of the following
is that of complement-mediated killing of etiology: Shigella, Salmonella, Campylobacter,
trophozoites. Acquired immunity primarily Yersinia, and enteroinvasive Escherichia coli
involves cell-mediated responses, although (Table 2.1). Although stools may be grossly
humoral responses may also contribute to bloody or heme-positive in both conditions,
anti-amebic immunity. Activated T-cells kill fever and significantly elevated leukocyte count
E. histolytica by: a) directly lysing trophozoites are less common in amebic colitis. Another
in a contact-dependent process; b) producing differential is inflammatory bowel disease.
cytokines which activate macrophages and other Amebic colitis should be ruled out before
effector cells (neutrophils and eosinophils); and steroid therapy for inflammatory bowel disease
c) providing helper effect for B-cell antibody is started because of the risk of developing toxic
production. In vitro studies using activated megacolon.
murine and human T-cells demonstrated The differential diagnoses of ALA include
significant killing of trophozoites in a contact- pyogenic liver abscess, tuberculosis of the liver,
dependent and antibody independent manner. and hepatic carcinoma. On the other hand,
Cytokine studies revealed that interferon (IFN) genital amebiasis should be differentiated
and interleukin (IL-2) may have a role in from carcinoma, tuberculosis, chancroid, and
activating macrophages for amebicidal activity. lymphogranuloma venereum.
More recent studies demonstrated that activated
macrophages produce nitric oxide (NO) which Table 2.1. Comparison of bacillary and amebic
was lethal to trophozoites. Tumor necrosis factor dysentery
(TNF) was shown to stimulate NO production.
Bacillary Dysentery Amebic Dysentery
Although it is known that antibodies are
May be epidemic Seldom epidemic
produced against amebic antigens, there has
Acute onset Gradual onset
been no direct evidence of T-cell help for
Prodromal fever and No prodromal features
B-cells. Studies have revealed that the principal malaise common
antibody-dependent cell cytotoxicity (ADCC) Vomiting common No vomiting
did not work against amebae. Antibodies which Patient prostrate Patient usually ambulant
were detected by seroepidemiologic studies and
Watery, bloody diarrhea Bloody diarrhea
secretory IgA isolated in the gut may merely
Odorless stool Fishy odor stool
be an indicator of current or recent invasive
Stool microscopy:
amebiasis. numerous bacilli, pus
Amebic modulation of host immune cells,
responses exists. For instance, infected human macrophages, red cells, Stool microscopy: few
no Charcot-Leyden bacilli, red cells,
subjects and animals have been shown to be in crystals trophozoites with
a state of immunosuppression during the acute ingested red blood
cells, Charcot-Leyden
stage of amebiasis. This state, characterized crystals
by T-cell hyporesponsiveness, suppressed Abdominal cramps Mild abdominal cramps
proliferation and cytokine production, depressed common and severe
delayed-type hypersensitivity (DTH), and Tenesmus common Tenesmus uncommon
macrophage suppression, is favorable for amebic Natural history: Natural history: lasts for
survival. It is the reversal of these modulatory spontaneous recovery weeks; dysentery
in a few days, weeks or returns after remission;
effects, which is the key in controlling amebiasis. more; no relapse infection persists for
years
Chapter 2: protozoan Infections 25
countries, prevalence depends on the level of cases should be done. Food handlers should be
sanitation, crowding, socio-economic status, screened for cyst carriage, and asymptomatic
cultural habits, and age. In developed countries, cyst carriers should be treated.
infection is usually caused by E. dispar, and Vaccines can be a cost-effective and
is prevalent in certain groups: immigrants, potent strategy for amebiasis prevention
travelers from endemic countries, homosexual and eradication. Unlike in other protozoan
males (men having sex with men), HIV patients, infections, amebic vaccine development has
and institutionalized people. fewer problems. The ameba life cycle is simple,
A microscopic study of diarrheic stools in and no intermediate hosts are involved. Amebae
Australia (n=5,921) revealed 177 (3%) positive are extracellularly located, and do not undergo
samples. PCR detected 5 E. histolytica, 63 E. antigenic variation. All these characteristics are
dispar, and 55 E. moshkovskii infections. The supportive of an achievable amebic vaccine.
latter two species, which are both commensals, Studies have also demonstrated the
are 10 times more prevalent than E. histolytica. acquisition of protective immunity to amebae,
A stool survey done in Iran (n=16,592) showed particularly that of mucosal immune response.
226 positive samples. Only 101 isolates were Trials with recombinant amebic antigens as
successfully cultured in Robinson’s medium. vaccines have proven to be more advantageous
Of these isolates, 93 (92.1%) were E. dispar, than inactivated/attenuated amebae. The
and only 8 (7.9%) were E. histolytica or mixed candidate vaccine molecules which have been
infections by PCR- RFLP. most intensely studied are the serine-rich E.
A field study in Northern Philippines histolytica protein (SREHP), the adherence
(n=1,872) showed 137 (7.3%) E. dispar, and lectin (Gal/GalNAc lectin), and the 29 kDa
18 (0.96%) E. histolytica by PCR. A study in a cysteine-rich amebic antigen. However, most
mental institution (n=113) showed E. histolytica of these studies have utilized animal models
or E. dispar in 43 subjects (38.1%), while PCR and artificial infection during challenge.
detected 74 (65.5%) E. histolytica-positive Testing these candidate vaccines in humans
samples, and 6 (5.3%) E. dispar/E. histolytica and developing them as food-based vaccines
mixed samples. will be in the forefront of future directions of
amebiasis control.
Prevention and Control
References
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depends on integrated and community-based Ali IK, Clark CG, Petri WA Jr.. Molecular
efforts to improve environmental sanitation, epidemiology of amebiasis. Infect Genet
and to provide for sanitary disposal of human Evol. 2008;8(5):698–707.
feces, safe drinking water, and safe food. These Diamond LS, Clark CG. A redescription of
efforts become more sustainable through health Entamoeba histolytica Schaudinn, 1903
education and promotion. The proper use of (Emended Walker, 1911) separating it
latrines and practice of proper hygiene, such from Entamoeba dispar Brumpt, 1925. J
as washing of hands, should be emphasized. Eukaryot Microbiol. 1993;40:340–4.
In communities where potable water is not Farthing M, Cevallos A, Kelly P, Cook G.
available, drinking water should be boiled or Manson’s tropical disease. 20th ed. London:
filtered. Vegetables and fruits which are eaten WB Saunders Co. Ltd.; 1996. p. 1255–69.
raw should be thoroughly washed. The use Fotedar R, Stark D, Beebe N, Marriott D,
of night soil for fertilizer should be avoided. Ellis J, Harkness J. PCR Detection of
Prompt diagnosis and treatment of amebiasis Entamoeba histolytica, Entamoeba dispar,
28 MedICal parasItology In the phIlIppInes
and Entamoeba moshkovskii in stool samples histolytica and Entameba dispar in the
from Sydney, Australia. J Clin Microbiol. Northern Philippines as detected by the
2007;45(3):1035–7. polymerase chain reaction. Am J Trop Med
Garcia E, Tiu W. Immunodiagnosis of some Hyg. 1998;59(6):916–22.
parasitic infections in the Philippines. Rivera WL, Tachibana H, Kanbara H.
Taguig: National Academy of Science and Application of polymerase chain reaction
Technology; 1998. p. 23–8. (PCR) in the epidemiology of Entamoeba
Hira PR, Iqbal J, Al-Ali F, Philip R, Grover histolytica and Entamoeba dispar infections.
S, D’Almeida E, et al. Invasive amebiasis: Tokai J Exp Clin Med. 1999;23:413–5.
challenges in diagnosis in a nonendemic Rivera WL, Tachibana H, Silva-Tahat MR,
country (Kuwait). Am J Trop Med Hyg. Uemura H, Kanbara H. Differentiation of
2001;65(4):341–5. Entamoeba histolytica and E. dispar DNA
Hooshya H, Rezaian M, Kazemi B, Jeddi- from cysts present in stool specimens
Tehrani M, Solaymani-Mohammadi S. by polymerase chain reaction: its field
The distribution of Entamoeba histolytica application in the Philippines. Parasitol
and Entamoeba dispar in Northern, Res. 1996;82:585–9.
Central, and Southern Iran. Parasitol Res. Salazar N, Pasay C, Avenido A, Macapsir S,
2004;94:96–100. Lena M, Maguinsay V, et al. Detection
John DT, Petri WA. Markell and Voge’s medical of Entamoeba histolytica in routine stool
parasitology. 9th ed. St. Louis: Elsevier examination. Phil J Microbiol Infect Dis.
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Petri WA Jr. Recent advances in amebiasis. Crit Stark DJ, Fotedar R, van Hal SJ. Prevalence of
Rev Clin Lab Sci. 1996;33(1):17–23. enteric protozoa in HIV-positive and HIV-
Ravdin JI. Amebiasis: series on tropical negative men who have sex with men from
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Imperial College Press; 2000. p. 65–159. 2007;76:549–52.
Rivera PT, Rivera WL, Escueta AS, Villacorte Van Hal SJ, Stark DJ, Fotedar R, Marriott
EA, Limlingan ET, Lazaro CM, et al. D, Ellis JT, Harkness JL. Amoebiasis:
Prevalence of serologically positive amebic current status in Australia. Med J Aust.
liver abscess cases in the Philippines. Acta 2007;186(8):412–6.
Med Philipp. 2007;41(2):31–6. Walsh JA, Ravdin JI. Prevalence of Entamoeba
Rivera WL, Kanbara H. Detection of histolytica infection. Amebiasis. Human
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by polymerase chain reaction. Parasitol Res. York: Wiley; 1988. p. 93–105.
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Chapter 2: protozoan Infections 29
Commensal Amebae
Pilarita T. Rivera, Vicente Y. Belizario, Jr., Juan Antonio A. Solon
E. histolytica, E. hartmanni does not ingest red by the following features: 1) a more vacuolated
blood cells. or granular endoplasm with bacteria and
debris, but no red blood cells; 2) a narrower,
Entamoeba coli
less-differentiated ectoplasm; 3) broader and
Entamoeba coli is cosmopolitan in blunter pseudopodia used more for feeding
distribution, and is considerably more common than locomotion; 4) more sluggish, undirected
than other human amebae. Trophozoites of E. movements; and 5) thicker, irregular peripheral
coli measure 15 to 50 µm in diameter. It can chromatin with a large, eccentric karyosome in
be differentiated from E. histolytica trophozoite the nucleus (Plate 2.7).
Chapter 2: protozoan Infections 31
Diamond LS, Clark CG. A redescription of Neva FA, Brown HW. Basic Clinical Parasitology.
Entamoeba histolytica Schaudinn, 1903 6th ed. Connecticut: Appleton & Lange;
(Emended Walker, 1911) separating it 1994.
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Eukaryot Microbiol. 1993;40(3):340–4. AM, White MC. Sexual transmission
Esparar DG, Belizario VY, Relos JR. Prevalence of enteric protozoa and helminths in a
of parasitic infection among food-handlers venereal disease clinic population. N Engl
in a dietary service of a tertiary hospital in J Med. 1981; 305(11):603–6.
Manila. Phil J Microbiol Infect Dis. 2004; Roberts LS, Janovy J. Foundations of
33(3):99–103. parasitology. 5th ed. Dubuque: Wm. C.
Imperato PJ. A historical overview of amebiasis. Brown Publishers; 1996.
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John DT, Petri WA. Markell and Voge’s medical SR, Lena MJ, Maguinsay VM, et al.
parasitology. 9th ed. St. Louis: Elsevier Detection of Entamoeba histolytica in
Saunders; 2006. p. 36–48. routine stool examination. Phil J Microbiol
Mahmoud AA. Tropical and geographical Infect Dis. 1990;19(2):57–60.
medicine companion handbook. 2nd ed. Van Hal SJ, Stark DJ, Fotedar R, Marriott
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34 MedICal parasItology In the phIlIppInes
Acanthamoeba spp.
Parasite Biology
increased susceptibility to infection, and may fluorescence microscopy. GAE usually occurs
lead to disseminated disease in the lungs and in immunocompromised hosts including
brain (GAE). the chronically ill and debilitated, and
Symptoms of AK include severe ocular those on immunosuppressive agents such as
pain and blurring of vision. Corneal ulceration chemotherapy and anti-rejection medications.
with progressive corneal infiltration may occur. The acquired immune deficiency syndrome
Primary amebic infection or secondary bacterial (AIDS) epidemic in the 1980’s dramatically
infection may lead to hypopyon formation. increased the numbers of person with GAE,
Progression of infection may cause scleritis and but these numbers have since fallen with the
iritis, and may ultimately lead to vision loss. advent of highly effective antiretroviral therapy.
Major differentials which need to be ruled out Signs and symptoms of GAE are generally
include fungal and herpetic keratitis. related to destruction of brain tissue and the
associated meningeal irritation. Systemic
B. Granulomatous Amebic Encephalitis
manifestations early in the course include fever,
Acanthamoeba was documented as the malaise, and anorexia. Neurologic symptoms
causative agent of human GAE by Stamm in may include increased sleeping time, severe
1972. Amebae were demonstrated in brain headache, mental status changes, epilepsy, and
sections of a GAE patient using indirect coma. Neurologic findings depending on the
36 MedICal parasItology In the phIlIppInes
location of the lesions include hemiparesis, Diagnosis of GAE is usually made post-
blurring of vision, diplopia, cranial nerve mortem in most cases. The rarity of the
deficits, ataxia, and increased intracranial disease and unfamiliarity of most physicians
pressure. with the pathogen contribute to frequently
Entry of Acanthamoeba into the central missed diagnosis. Signs and symptoms of
nervous system is still incompletely understood. disease are usually attributed to more common
From a primary site of infection in the differentials. Moreover, recovery of ameba from
skin or lungs, the likely route of invasion is cerebrospinal fluid is exceedingly rare, and
hematogenous. Direct infection through the imaging results are generally nonspecific.
olfactory valves has also been proposed, but Immunocompromised patients such
not conclusively demonstrated. Recent reviews as those with AIDS are at the highest risk
have focused on blood-borne invasion, with for acquiring GAE. While opportunistic
a combination of host factors, elucidation of infections of the central nervous system such
serine proteases, and parasite adhesion using as Cryptococcus meningitis and toxoplasmosis
a mannose-binding protein all contributing to are much more common than GAE, the lack of
brain endothelial cell damage and subsequent response despite appropriate treatment should
breakdown of the blood-brain barrier. prompt a more thorough evaluation for more
Gross examination of neural tissue post- esoteric organisms.
mortem reveals cerebral hemispheres that are Specific diagnosis depends on demonstrating
edematous and soft, with areas of hemorrhage the trophozoites or cysts in tissues using
and focal abscesses. The most affected areas histopathologic stains and microscopy. The
of the brain are the posterior fossa structures, organisms can rarely be demonstrated in the
thalamus, and the brainstem. In the affected cerebrospinal fluid and can be cultured for
areas, the leptomeninges are opaque and exhibit further studies.
purulent exudates and vascular congestion.
Treatment
The incubation period from initial
inoculation is approximately 10 days, with a Medical treatment of AK has been met
subacute and chronic clinical course of infection with increasing success in recent years. While
that lasts for several weeks to several months. historically, only surgical excision of the infected
The clinical manifestations of disease include cornea with subsequent corneal transplantation
decreased sensorium, altered mental status, was curative, early recognition of AK coupled
meningitis, and neurologic deficits. The natural with aggressive combination anti-amebic
course of the disease eventually results in coma agents can preclude the need for extensive
and death. surgery. D’Aversa and his colleagues have
achieved acceptable results with clotrimazole
Diagnosis
combined with pentamidine, isethionate,
Acanthamoeba keratitis is diagnosed by and neosporin. Other agents that have been
epithelial biopsy or corneal scrapings for used include polyhexamethylene biguanide,
recoverable ameba with characteristic staining propamidine, dibromopropamidine isethionate,
patterns on histologic analysis. Amebae have neomycin, paromomycin, polymyxin B,
also been isolated from the contact lens and lens ketoconazole, miconazole, and itraconazole.
solution of patients. Species-specific identification Topical corticosteroids should be avoided, as
can be made from culture and molecular analysis this retards the immune response. Advanced
through PCR. Known species that have caused AK usually requires debridement, but complete
AK include A. castellani, A. culbertsoni, A. excision of the cornea can be avoided if the
hutchetti, A. polyphaga, and A. rhysoides. infection is confined to more superficial areas.
Chapter 2: protozoan Infections 37
Deep lamellar keratectomy is the procedure of of the risk of infection, and physicians treating
choice. these patients should maintain a high index
Clinically apparent neurologic disease in of suspicion in the presence of compatible
GAE usually heralds a fatal outcome within signs and symptoms of infection which do not
3 to 40 days. A few patients have shown good respond to conventional antimicrobial therapy.
responses to combinations of amphotericin
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38 MedICal parasItology In the phIlIppInes
Naegleria spp.
Plate 2.10. Naegleria fowleri trophozoites in ameboid (left) and ameboflagellate (right) forms
(Accessed from www.dpd.cdc.gov/dpdx)
Naegleria fowleri has three stages, cysts, by penetrating the nasal mucosa and migrating
trophozoites, and flagellated forms, in its life to the brain via the olfactory nerves. N. fowleri
cycle. The trophozoites replicate by promitosis trophozoites are found in cerebrospinal fluid
(nuclear membrane remains intact) and can turn (CSF) and tissue, while flagellated forms are
into temporary non-feeding flagellated forms, occasionally found in CSF. Cysts are not seen
which usually revert back to the trophozoite in brain tissue (Figure 2.4).
stage. Trophozoites infect humans or animals
Chapter 2: protozoan Infections 39
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46 MedICal parasItology In the phIlIppInes
Giardia duodenalis
Juan Antonio A. Solon
body temperature and stable at a pH of 7.8 be asymptomatic. For acute cases, patients
to 8.2. The parasite may also produce a lectin experience abdominal pain, described as
which, when activated by duodenal secretions, cramping, associated with diarrhea. There is
is able to facilitate attachment. Once attached, also excessive flatus with an odor of “rotten
the organism is able to avoid peristalsis by eggs” due to hydrogen sulfide. Other clinical
trapping itself in between the villi or within the features include abdominal bloating, nausea,
intestinal mucus. and anorexia. Diarrhea is the most common
Upon attachment to the intestinal cells, symptom, occurring in 89% of cases. It is
G. duodenalis is able to cause alterations in followed by malaise and flatulence. Spontaneous
the villi such as villous flattening and crypt recovery occurs within 6 weeks in mild to
hypertrophy. These alterations lead to decreased moderate cases. In untreated cases, patients may
electrolyte, glucose, and fluid absorption, and experience diarrhea with varying intensities, for
cause deficiencies in disaccharidases. Studies on weeks or months.
Giardia muris-infected mice showed diffuse loss Chronic infection is characterized by
of microvillous surface area which investigators steatorrhea, or the passage of greasy, frothy
also correlated to decreased maltase and stools. In some cases, periods of diarrhea have
sucrase activities. The physiologic disturbances been observed to alternate with normal or even
subsequently result in malabsorption and constipated bowel periods. There may be weight
maldigestion, which in turn cause the signs and loss, profound malaise, and low-grade fever. In
symptoms experienced by the patient. Bacterial developing countries, it has been described as a
colonization of the area may further worsen the cause of the failure-to-thrive syndrome.
damage already caused by the parasite.
Diagnosis
In other studies, G. duodenalis was shown
to rearrange the cytoskeleton in human colonic Diagnosis is made by demonstration of G.
and duodenal monolayers. Cytoskeleton is duodenalis trophozoites (Plate 2.11) and/or cysts
essential for proper cell attachment to the (Plate 2.12) in stool specimens. Trophozoites
extracellular matrix and the other neighboring in direct fecal smears may be characterized as
cells. Changes observed in apoptotic cells having a floating leaf-like motility. To detect
include disruption of the cytoskeleton that leads
to structural disintegration and detachment
from the substrate. Hence, the parasite has
been suggested to cause enterocyte apoptosis.
This finding was strengthened by another study,
which showed the ability of the parasite not
only to disrupt cellular tight junctions but also
to increase epithelial permeability, thus, leading
to the loss of epithelial barrier function. With
this loss of barrier function, luminal contents
may penetrate the submucosal layers causing
more damage in the intestinal tissue.
From ingestion of the cysts, it takes about Plate 2.11. Giardia duodenalis trophozoite
1 to 4 weeks (average of 9 days) for the disease (Courtesy of the Department of Parasitology,
to manifest. Half of the infected patients may UP-CPH)
Chapter 2: protozoan Infections 49
The first published study on Giardia or by infected food handlers. Normal water
genotypes in the Philippines showed that the chlorination will not affect cysts, but usual
majority (86%) of the isolated genotypes belong water treatment modalities should be adequate.
to assemblage B.
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Baldo ET, Belizario VY, de Leon WU, Kong Esparar DG, Belizario VY, Jr., Relos JR.
HH, Chung DI. Infection status of Prevalence of intestinal parasitic infections
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Med Public Health. 2008;39(6):991–9. duodenalis isolates among residents of slum
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Chapter 2: protozoan Infections 53
Trichomonas vaginalis
Juan Antonio A. Solon
acridine orange stains. Trichomonas can also be detection tests and polymerase chain reaction
cultured using Diamond’s modified medium, (PCR) assays are commercially available, but
and Feinberg and Whittington culture medium. not widely used locally. PCR among females
The Pap smear may also show trichomonads does not seem to offer an added diagnostic
(sensitivity 60%; specificity 95%). Antigen advantage. Among males, however, diagnosis is
Chapter 2: protozoan Infections 55
characterized molecularly showing low genetic Gumbo FZ, Duri K, Kandawasvika GQ,
polymorphism. Kurewa NE, Mapingure MP, Munjoma
It is relevant to discuss trichomoniasis in the MW, et al. Risk factors of HIV vertical
context of HIV. In Zimbabwe and South Africa, transmission in a cohort of women
trial participants diagnosed with trichomoniasis under a PMTCT program at three peri-
were more likely to test positive for HIV in their urban clinics in a resource-poor setting. J
next visit. Perinatal transmission of HIV was Perinatol. 2010;30(11):717–23.
likewise more likely if the mother had vaginal Ju e c o N L , A r a n e t a C A , Ta d i n a E G .
infections. Epidemiology of Trichomonas vaginitis
among selected group of women in Manila.
Prevention and Control
Acta Med Philipp. 1988;24(3):85–6.
Prevention is best achieved by reducing Mavedzenge SN, Pol BV, Cheng H, Montgomery
the risk of exposure. Limiting the number of ET, Blanchard K, de Bruyn G, et al.
sexual partners, and proper use of protective Epidemiological synergy of Trichomonas
devices such as condoms and spermicidal foams vaginalis and HIV in Zimbabwean and
may help prevent infection. To prevent “ping- South African women. Sex Transm Dis.
pong” or recurrent infections, there should 2010;37(7):460–6.
be simultaneous treatment of sexual partners. Monzon OT, Santana RT, Paladin FJ, Bautista
Prompt follow-up of patients and their contacts, A, Fajutagana L, Eugenio S. The Prevalence
as well as health and sex education about of sexually transmitted diseases (STDs) and
venereal disease are also important. human immunodeficiency virus (HIV)
infection among Filipino sex workers. Phil
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Arambulo PV, Cabrera BD, Osteria TS, Baltazar Rivera WL, Ong VA, Masalunga MC. Molecular
JC. A comparative study of Trichomonas characterization of Trichomonas vaginalis
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1977;8:298. Schwebke JR, Burgess D. Trichomoniasis. Clin
Basaca-Sevilla V, Cross JH, Alquiza L, Lacap T. Microbiol Rev. 2004;17(4):794–803.
Prevalence of Trichomonas vaginalis in some Spence MR, Harwell TS, Davies MC, Smith JL.
Filipino women. Southeast Asian J Trop The minimum single oral metronidazole
Med Public Health. 1986;17(2):194–6. for treating trichomoniasis: a randomized,
Beaver PC, Jung RC, Cupp EW. Clinical blinded study. Obstet Gynecol. 1997;89(5):
parasitology. 9th ed. Philadelphia: Lea and 699–703.
Febiger; 1984. Van der Pol B. Trichomonas vaginalis infection:
Belding DL. Textbook of parasitology. New the most prevalent nonviral sexually
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Chapter 2: protozoan Infections 57
Non-Pathogenic Flagellates
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indicated. Preventive and control measures Southeast Asian J Trop Med Public Health.
include improved sanitation and personal 1981;12(1):12–8.
hygiene. Carney WP, de Veyra VU, Cala EM, Cross
JH. Intestinal parasites of man in
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Med Public Health. 1980;11(4):473–9. Hersh SM. Pulmonary trichomoniasis and
Carney WP, Banzon T, de Veyra VU, Papasin Trichomonas tenax. J Med Microbiol.
MC, Cross JH. Intestinal parasites of 1985;20(1):1–10.
man in Oriental Mindoro, Philippines, Lewis KL, Doherty DE, Ribes J, Seabolt JP,
with emphasis on schistosomiasis. Bensadoun ES. Empyema caused by
Trichomonas. Chest. 2003;123(1):291–2.
Chapter 2: protozoan Infections 59
Coccidians
Winifreda U. de Leon
A study done in San Lazaro Hospital attempted gametes. The microgametes fertilize the
to describe Cryptosporidium among diarrheic macrogametes to produce oocysts, which are
patients and reported a prevalence of 8.5%, passed out with feces when the host cells are
while a study done in the Philippine General sloughed off from the intestinal wall. The
Hospital on diarrheic patients had a much lower oocysts undergo complete sporulation within
prevalence at 1.7%. 7 to 12 days in a warm environment.
It is assumed that the oocyst is the infective
Prevention and Control
stage and when ingested, the sporozoites are
Water-borne transmission is the most released and enter intestinal cells to go through
common source of cr yptosporidiosis. schizogony and gametogony. The different
Chlorination does not affect the parasite. The developmental stages of the parasite may be
synergistic effect of multiple disinfectants and found in the intestinal tissue (Figure 2.9).
combined water treatment processes may reduce
Pathogenesis and Clinical Manifestations
C. hominis oocysts in drinking water. Natural
water and swimming pool water should not be Initial symptoms include malaise and low
swallowed. Contamination of drinking water by grade fever, which may occur 12 to 24 hours
human and animal feces should be prevented. after exposure. Chronic and intermittent watery
diarrhea occurs early in the infection and may
Cyclospora cayetanensis alternate with constipation. The diarrhea
may continue for 6 to 7 weeks with six or
When first associated with diarrhea,
more stools per day. Other symptoms such as
this organism was thought to be a
fatigue, anorexia, weight loss, nausea, vomiting,
m ember of cyan o ba ct er i a b e cau s e it
abdominal pain, flatulence, bloating, and
showed photosynthesizing organelles and
dyspnea may develop. D-xylose malabsorption
autofluorescing particles characteristic of the
has been found to develop in some of the
blue green algae.
patients. Infections are usually self-limiting
Parasite Biology and immunity may result with repeated
infections. No death has been associated with
Cyclospora cayetanensis was originally cyclosporidiosis.
called a cyanobacterium-like body (CLB)
but upon careful study, it was found to be Diagnosis
a coccidian parasite. Similar to the other
Direct microscopic examination of fecal
intestinal coccidians, the life cycle begins with
smears under high magnification (400x)
the ingestion of sporulated oocyst, which
is recommended. Various concentration
contains two sporocysts with two sporozoites
techniques and acid-fast staining (Kinyoun’s
each. The released sporozoites invade the
stain) are also useful. Oocysts are auto-
epithelial cells of the small intestines, although
fluorescent and under fluorescent microscopy,
the site of predilection was found to be the
they appear as blue or green circles depending
jejunum. Multiple fissions of these sporozoites
on the filter (365-450 DM). This technique
take place inside the cells to produce meronts,
is useful for screening. Safranin staining
which contain 8 to 12 merozoites during the
and microwave heating are also helpful. A
first generation, and only four merozoites in
polymerase chain reaction (PCR) technique has
the second generation. Some of the merozoites
been developed to differentiate Cyclospora from
develop into male (micro) and female (macro)
closely related Eimeria species.
Chapter 2: protozoan Infections 63
Pathogenesis and Clinical Manifestations Other concentration techniques that can also
be used include zinc sulfate and sugar flotation.
Among the immunocompetent, infection is
Oocysts are thin walled, transparent, and ovoid
generally asymptomatic or may present as a self-
in shape. They appear as translucent, oval
limiting gastroenteritis. However, in more severe
structures measuring 20 to 33 µm by 10 to
infections, severe diarrhea and fat malabsorption
19 µm. Alternatively, oocysts can be seen in a
can occur. Symptoms include low-grade fever,
fecal smear stained by a modified Ziehl-Neelsen
anorexia, vomiting, general body malaise,
method, where they stain granular red color
anorexia, weight loss, and flatulence. Stools
against a green background. Phenol-auramine,
usually contain undigested food, mucus, and
as well as iodine staining of the specimen
Charcot-Leyden crystals.
can help visualize the organism. Acid-fast
Infection in immunocompromised
stain, such as Kinyoun’s stain or an auramine-
individuals ranges from a self-limiting enteritis
rhodamine stain, is also useful. A considerable
to severe diarrheal illness resembling that of
amount of stool may have to be examined
cryptosporidiosis, giardiasis or cyclosporiasis.
because oocysts in the samples are often few in
Mucosal bowel biopsy may reveal flattened
number. Charcot-Leyden crystals may be seen
mucosa and damaged villi. Infiltration of the
in the stool specimen. In blood examination,
lamina propria with lymphocytes, plasma cells,
peripheral eosinophilia is common. String
and eosinophils has been reported. However,
capsule (Enterotest®) and duodenal aspirate
the mechanism by which the parasite produces
examinations may be of value. Molecular based
these lesions is still not clear.
techniques may prove useful as an additional
Diagnosis diagnostic tool.
The oocysts of C. belli may be detected Treatment
in the feces by direct microscopy or formalin-
Asymptomatic infections may be
ether/ethyl acetate concentration (Plate 2.13).
managed with bed rest and a bland diet,
while symptomatic infections, such as those
occurring in AIDS patients, can be treated with
trimethoprim-sulfamethoxazole 160/800 mg
four times per day for 10 days, then two times
per day for 3 weeks. Combination therapy with
pyrimethamine and sulfadiazine for 7 weeks has
also been used successfully.
Epidemiology
of those with AIDS were infected; in South Microsporidia, Isospora and Cyclospora. Ann
America, 10%, and in Haiti and Africa, a range Intern Med. 1996;124:429–441.
of 7 to 20% was observed. The disease has also He y w o r t h M F. Pa r a s i t i c d i s e a s e s i n
been reported among those with lymphoma, immunocompromised hosts,
leukemia, and organ transplants. Considered cr yptosporidiosis, isosporiasis and
endemic are the following: Africa, Australia, strongyloidiasis. Gastroenterol Clin North
the Caribbean Islands, Latin America, and Am. 1996;25:691–707.
Southeast Asia. Cystoisosporiasis has been Hoepelman IM. Human cryptosporidiosis. Int
reported in both adults and children, but severe J STD AIDS. 1996;7(suppl)l:28–33.
diarrhea is common among infants. Both sexes Jueco NL, Belizario VY, Jr., de Leon WU,
were found susceptible to infection. Tan-Liu N, Bravo LC, Gregorio GV.
Cryptosporidiosis among selected patients
Prevention and Control
in the Philippine General Hospital. Acta
Cystoisosporiasis can be prevented by Med Philipp. 1991;27:244–247.
following good sanitary practices, thorough Lindsay DS, Dubey JP, Blagburn BL. Biology
washing and cooking food, and drinking safe of Isospora spp. from humans, non human
water. primates and domestic animals. Clin
Microbiol Rev. 1997;10:19–34.
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Chapter 2: protozoan Infections 69
Toxoplasma gondii
into bradyzoites that are protected by a cyst antibodies against T. gondii. A seroconversion
wall and proliferate at a slower rate. Cysts to a positive titer or a four-fold increase in titers
can be found in the brain, skeletal and heart is indicative of an infection. The Sabin-Feldman
muscles, and retina. Clinical manifestations methylene blue dye test is very sensitive and
become apparent when the immune system specific but it requires the maintenance of
is suppressed as in old age, drug-induced live organisms in the laboratory. High titers
immunosuppression after organ transplantation, (>1,024), although usually indicating an acute
or in the case of AIDS. More often, symptoms infection, may also be seen in chronic cases,
appear when there is relapse of chronic hence the need for IgM antibody detection
infections as a result of a suppressed immune through either the IgM indirect fluorescent
system rather than as a response to an acute antibody technique or through a double
infection. Among the immunocompromised sandwich IgM enzyme immunoassay. Handling
patients, the most common manifestation is of live trophozoites may result in accidental
encephalitis. Myocarditis and focal pneumonia infection of the laboratory personnel. Other tests
have also been reported. It is also possible are the indirect hemagglutination test, indirect
that the immunosuppressed patient acquires fluorescent antibody test, and enzyme-linked
the infection from blood transfusion or immunosorbent assay. Latex agglutination test
organ transplantation. Clinical manifestations is also available. Differentiating pre-existing
include retinochoroiditis, lymphoreticular antibody from passively transferred antibody
hyperplasia with enlargement of the posterior from the mother or antibody related to illness
cervical lymph node, hepatitis, splenomegaly, is important in the assessment of serological
pneumonia, extramedullary hematopoiesis, and test results.
failure to gain weight. Better diagnostic assays are being developed
Stillbirth and abortion may result when because toxoplasmosis has been recognized
mothers acquire the infection during the first as an important disease associated with
trimester of pregnancy. Babies may exhibit AIDS. Polymerase chain reaction (PCR) has
clinical manifestations like chorioretinitis, been successfully used in the diagnosis of
epileptic seizures, jaundice, hydrocephaly, and toxoplasmosis using samples taken from the
microcephaly. Death of the infected newborn patient, which include serum, amniotic fluid,
babies is usually due to anemia with pneumonia. cerebrospinal fluid, and broncheoalveolar
There are cases when clinical manifestations lavage, especially in cases where there is very
may not be apparent during the neonatal little amount of specimen available.
period, but will appear later in childhood. Most
Treatment
babies will harbor the infection and grow up
without any clinical manifestation until such Treatment consists of pyrimethamine
time later in life when their immune system is (25-100 mg daily) and sulfadiazine (1-1.5 g
suppressed and there is reactivation of chronic four times daily) used in combination for one
toxoplasmosis. month. These drugs keep the Toxoplasma under
control but do not kill it. Since pyrimethamine
Diagnosis
can lower blood counts in most people, it
Identification of the parasite can be done should be given together with leucovorin (folic
through examination of tissue imprints stained acid). Sulfadiazine may cause serious allergic
with Giemsa. Tissue sections can be processed reactions like fever and rash, but it can be
and stained with hematoxylin and eosin. substituted with clindamycin. Spiramycin,
Serodiagnostic methods are used to detect azithromycin, clarithromycin, dapsone, and
72 MedICal parasItology In the phIlIppInes
Sarcocystis spp.
Alice Alma C. Bungay, Raezelle Nadine T. Ciro
diagnosis. Currently, 24 wall types have been America, China, India, Tibet, and Southeast
identified in 62 species. S. hominis and S. Asia.
suihominis both have walls of type 10. The wall Of fecal specimens examined from children
of S. hominis is up to 6 µm thick and appears in Poland and Germany, 10.4% and 7.3% were
radially striated from villar protrusions that are found positive, respectively. In Tibet, Sarcocystis
up to 7 µm long. The wall of S. suihominis is was detected in 42.9% of beef specimens
4 to 9 µm thick, with villar protrusions up to examined from the marketplace, and S. hominis
13 µm long. and S. suihominis were found in stool samples
Recently, polymerase chain reaction of 21.8% and 7% of 926 persons, respectively.
(PCR) amplification of the 18S rRNA was Stool examination among Thai laborers showed
demonstrated to be useful in distinguishing S. that Sarcocystis infection had a prevalence of
hominis, S. fusiformis, and S. cruzi sarcocysts about 23%; all cases were asymptomatic which
and oocysts. The technique makes possible probably explained the lack of recognition. A
amplification and identification of species- study of 100 human tongues obtained post
specific gene sequences based on DNA extracted mortem in Malaysia revealed an infection rate
from as few as seven excreted sporocysts (the of 21%. There was no sex difference and the age
equivalent of 3 ½ oocysts) from freshly prepared range was 16 to 57 years (mean 37.7 years). A
material, or as few as 50 sporocysts from fecal seroepidemiological survey in West Malaysia
samples that had been stored in potassium found that 19.7% of 243 persons had antibodies
dichromate (K2Cr2O7) for as long as 6 years. for Sarcocystis.
In the Philippines, studies involving
Treatment
the examination of muscle tissues obtained
Because infection is often asymptomatic, from water buffaloes, cattle, pigs, and goats
treatment is rarely required. There have been revealed the presence of S. cruzi in backyard
no published trials so treatment remains cattle (Bos taurus) possessing a type 7 sarcocyst
empirical. Agents that have been used include wall, S. levinei in water buffaloes (Bubalus
albendazole, metronidazole, and co-trimoxazole bubalis) possessing a type 7 sarcocyst wall
for myositis. Corticosteroids have also been used with similarities to S. cruzi, S. miescheriana in
for symptomatic relief. domestic pigs (Sus scrofa domestica) with a type
10 sarcocyst wall, and S. capracanis in domestic
Epidemiology
goats (Capra hircus) with a type 14 sarcocyst
There are very few large-scale population wall. There is a lack of local studies on human
surveys that have been conducted for Sarcocystis sarcocystosis.
in humans. Prevalence data for Sarcocystis Prevention and Control
infections often come from case reports and
findings of physicians, public health workers, Intestinal sarcocystosis can be prevented
and scientists with specific interests. by thoroughly cooking or freezing meat to kill
Human infection is considered rare with bradyzoites in the sarcocysts. Alternatively,
less than 100 published cases of invasive freezing the meat at –5°C for several days
disease (approximately 46 cases reported by will kill the sporocysts. Where contaminated
1990). These figures may represent a gross drinking water is suspected, boiling should be
underestimate of the human burden of disease. considered to ensure disinfection.
Sarcocystosis has been reported in Africa, The administration of anticoccidial
Europe (Germany, Spain, and Poland), the drugs, amprolium and salinomycin, as
United States (California), Central and South chemoprophylactic agents was effective
Chapter 2: protozoan Infections 77
the vacuolar form and the precystic form, as flatulence, mild to moderate diarrhea without
this stage allows the parasite to ingest bacteria fecal leukocytes or blood, nausea, vomiting, low
in order to enhance encystment. Studies grade fever, and malaise. Symptoms usually last
of Tan and Suresh have revealed that the about 3 to 10 days, but may sometimes persist
ameboid forms predominated in isolates from for weeks or months.
symptomatic cases. It has been found that in subjects suffering
Granular forms are multinucleated and from immunosuppression, Blastocystis showed
are mainly observed from old cultures. The a significant association with gastrointestinal
diameter of the cell varies from 10 to 60 µm. symptoms. Other studies have also provided
The granular contents develop into daughter evidence of changes in the cellular immune
cells of the ameba-form when the cell ruptures. function of infected individuals.
Multiple fission forms arise from vacuolated
Diagnosis
forms. It is believed that these multiple fission
forms produce many vacuolated forms. Specific diagnosis based on clinical
The size of the resistant cystic form is presentation alone may prove difficult, because
about 3 to 10 µm in diameter, and has one or the spectrum of symptoms is seen in other
two nuclei. It has a very prominent and thick, intestinal infections. Laboratory detection of
osmophilic, electron dense wall. It appears the organism from stool is needed to confirm
as a sharply demarcated polymorphic, but the diagnosis. Multiple stool samples should
mostly oval or circular, dense body surrounded be collected from patients showing clinical
by a loose outer membranous layer. This signs and symptoms. Microscopic examination
membranous layer seen in phase contrast using direct fecal smear is useful, but sensitivity
microscopy corresponds to the fibrillar layer is increased when concentration techniques
described around the cyst at the ultrastructural are used. Hematoxylin or trichrome staining
level, and is the easiest diagnostic feature to offers a very convenient and easy method to
identify. differentiate the various stages of Blastocystis.
It is postulated that the thick-walled cyst Leukocytes are usually seen in fecal smears and
may be responsible for external transmission, stool eosinophilia may also be observed. The
while those cysts with thin walls may be the organism can be cultured using the Boeck and
cause of reinfection within a host’s intestinal Drbohlav’s or the Nelson and Jones media.
tract.
Treatment
Pathogenesis and Clinical Manifestations
Blastocystis is difficult to eradicate. It hides
Infection with B. hominis is called in the intestinal mucus, as well as sticks and
blastocystosis. B. hominis as a cause of holds on to intestinal membranes. The drug of
gastrointestinal pathology is controversial. choice is metronidazole given orally, 750 mg
Several studies have shown that the presence three times daily for 10 days (Pediatric dose:
of the parasite in a majority of patients was not 35-50 mg/kg/day in three doses for 5 days)
associated with symptoms; or, it was found or iodoquinol given at 650 mg three times
with other organisms that were more likely to daily for 20 days. However, there have been
be the cause of the symptoms. However, other reported cases of resistance. Trimethroprim-
studies have concluded that the presence of sulfamethoxazole (TMP-SMX) has also been
Blastocystis in large numbers produces a wide found to be highly effective against Blastocystis.
variety of intestinal disorders, such as abdominal Nitazoxanide has been clinically tested on
cramps, irritable bowel syndrome, bloating, patients with blastocystosis, and was found to
80 MedICal parasItology In the phIlIppInes
resolve symptoms in 86% of patients after 3 Blastocystis similar to those found in humans.
days of administration. Evidence has also shown that Blastocystis is
present in house lizards and cockroaches,
Epidemiology
raising the possibility that food and water
Blastocystis hominis has been reported contaminated by fecal droppings of these “home
virtually worldwide, with infections occurring visitors” may transmit Blastocystis.
most commonly in tropical, subtropical, and In the Philippines, studies of 32
developing countries. Studies from developed morphologically similar isolates from different
countries have reported approximately 1.5 to hosts: 12 from humans, 12 from pigs, and 8
17.9% overall prevalence of B. hominis. All from chickens, using the restriction fragment
ages are affected, but symptomatic cases are length polymorphism (RFLP) analysis of small
more often found in children and in those with subunit rDNA (SSUrDNA), have shown
weakened immune systems. A prevalence of up extensive genomic polymorphism.
to 11.6% was reported from Stanford University
Prevention and Control
Hospital. Prevalence rates of 32.6 % and as high
as 52.3% had been reported from China and Available data on B. hominis indicate that
Malaysia, respectively. the disease can be prevented by consuming safe
Occurrence of the parasite in temperate drinking water. While food has not been fully
countries is generally associated with recent implicated, provisions for sanitary preparation
travel to the tropics and consumption of may be of value in efforts to prevent and
untreated drinking water. This indicates that control this infection. The cysts of B. hominis
infection is possibly through the oral route, can survive up to 19 days in water at normal
and it is more likely to occur in crowded and temperature, and have shown resistance to
unsanitary conditions. Outbreaks of B. hominis chlorine at the standard concentrations.
in day-care centers have been reported in Spain
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(13.4%). Avila MS, Garcia MR, Narcelles MV, Serra
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S. Isolate resistance of Blastocystis hominis Hepatol. 2005;3(10):987–91.
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82 MedICal parasItology In the phIlIppInes
Dientamoeba fragilis
Vicente Y. Belizario, Jr., Timothy M. Ting
Plasmodium spp.
Vicente Y. Belizario, Jr., Carlos Miguel P. Perez
positive for P. knowlesi. The life cycle of P. in humans consists of schizogony, which leads to
knowlesi is microscopically indistinguishable the formation of merozoites, and gametogony,
from P. malariae, and differentiation is only which leads to the formation of gametocytes.
achieved through polymerase chain reaction The sexual cycle in the mosquito involves
(PCR) assay and molecular characterization. sporogony, which leads to the formation
These protozoans are pigment producers of sporozoites. The life cycles of all human
and are ameboid in shape, with some being species of malaria are similar. The infected
more ameboid than the others. Their asexual female Anopheles mosquito bites and sucks
cycle occurs in humans, the vertebrate and blood from the human host. In the process,
intermediate host, while the sexual cycle occurs salivary fluids containing sporozoites are also
in the Anopheles mosquito, the invertebrate and injected. These sporozoites, the infective stage
definitive host. of the parasite, are immediately carried to the
liver and enter the parenchymal cells. The
Parasite Biology
parasites then commence exo-erythrocytic
Various processes comprise the life cycle schizogony, which produces the merozoites
(Figure 2.14) of the parasite. The asexual cycle in varying duration and amounts, depending
on the species. Merozoites proceed to the gut as an ookinete, which then develops into
peripheral blood to enter the erythrocytes. Some an oocyst. The oocyst grows and produces
merozoites of P. vivax and P. ovale re-invade the sporozoites, which escape from the oocyst and
liver cells forming hypnozoites, while the other enter the salivary glands of the mosquito. These
species do not. These dormant exo-erythrocytic sporozoites may be injected into another human
forms may remain quiet for years. Within host when the mosquito takes a blood meal.
the red blood cell, the merozoite grows as a The entire developmental cycle in the mosquito
ring form developing into a trophozoite. The takes 8 to 35 days, depending to some extent
trophozoite has an extended cytoplasm and on ambient temperature.
a large chromatin mass which further divides Morphologically, the early trophozoite
to form more merozoites within schizonts. form is ring-shaped with a red chromatin dot
The merozoites of P. falciparum develop in the and a scant amount of blue cytoplasm when
parasitophorous vacuolar membrane (PVM) stained with Giemsa or Wright’s stain. The
within the mature red cells and modify the trophozoite form has a large chromatin mass
structural and antigenic properties of these and a prominent ameboid cytoplasm, which
cells. The parasites feed on the hemoglobin is spread through the erythrocyte. The parasite
resulting in the production of pigment known develops into a schizont when the chromatin has
as hematin. Soon after, the erythrocytes rupture divided into two or more masses of chromatin
and the merozoites are released into the blood, with small amounts of cytoplasm, the so-called
ready to enter new erythrocytes. This asexual merozoites. The number of merozoites is species
cycle is synchronous, periodic, and species- dependent. Clumps of pigment accumulate in
determined. the middle of a mature schizont.
Some merozoites develop into The gametocyte stage fills the entire
microgametocytes (male) or macrogametocytes red blood cell and is characterized by a large
(female) which are picked up by feeding female chromatin mass and a blue cytoplasm with
mosquitoes for completion of the life cycle. In pigment. It is round to banana-shaped. The
the gut of the mosquito, the male gametocytes microgametocyte has a lighter blue cytoplasm,
exflagellate and produce eight sperm-like while the cytoplasm of the macrogametocyte
microgametes which may fertilize the female is a darker blue. Species identification depends
macrogamete to form a zygote. The zygote on various characteristics of these stages of
becomes motile and penetrates the mosquito’s development as described in Table 2.5.
Plasmodium species
Parameter P. falciparum P. vivax P. ovale P. malariae
(malignant tertian) (benign tertian) (benign tertian) (quartan)
Infected red blood Normal: multiple Larger than normal, Somewhat larger than Larger than normal,
cells (RBC) infection of RBC pale, often bizzare; normal, often with pale, often bizzare;
very common Schüffner's dots fringed or irregular Schüffner's dots
are often present; edge, and oval in are often present;
multiple infection shape; Schüffner's multiple infection
of RBC not dots appear even of RBC not
uncommon with younger uncommon
stages; stains more
readily and deeply
than in P. vivax
88 MedICal parasItology In the phIlIppInes
Plasmodium species
Parameter P. falciparum P. vivax P. ovale P. malariae
(malignant tertian) (benign tertian) (benign tertian) (quartan)
Small trophozoite Same as P. vivax Signet-ring form with Small, darker in Same as P. vivax
but with small heavy red dot and color, and but with blue
threadlike blue blue cytoplasmic generally more cytoplasmic circle,
cytoplasmic ring solid than those smaller, thicker and
circle with one of P. falciparum; heavier
or two small red Schüffner's dots
chromatin dots; regularly present
double chromatin in almost 100% of
common; infected cells
marginal forms
common
Growing trophozoite Remains in ring Like small trophozoite, Resembles closely Chromatin rounded
form but grows as above, same stage of or elongated;
resembling small with increased P. malariae but cytoplasm
trophozoite of P. cytoplasm is considerably compact or in
vivax in size; usually and ameboid larger; pigment narrow band
the oldest asexual activity; small- is lighter and less across cell:
stage seen in yellowish brown conspicuous dark brown
peripheral blood pigment granules granules may
in cytoplasm, have peripheral
increasing with arrangement
age of parasite
Large trophozoite Seldom present Large mass of Seldom present Chromatin often
chromatin; elongate,
loose, irregular, or indefinite in outline;
close compact cytoplasm dense,
cytoplasm with compact, in
increasing amount rounded oblong
of fine brown or band forms;
pigment; parasite pigment granules
fills cell in 30 to 40 larger, darker than
hours P. vivax parasite fills
cells frequently
Schizont Not present Chromatin divided; About 25% of Same as P. vivax
(presegmenting) cytoplasm shows infected cells are except parasite
varying degrees definitely oval is smaller, shows
of separation shaped: usual less chromatin
into strands and picture is that of a division, more
particles; pigment round parasite in delayed clumping
collects in parts of the center of an of pigment
the parasite oval cell; many
cells with indefinite
fringed outline;
pigment lighter
and less coarse
than in P. malariae
Schizont Rarely present; 8-24 12-24 merozoites; Usually eight 6-12 (average of
(mature) merozoites; smaller pigment in one merozoites 8-10) merozoites
than other species to two clumps; arranged around in rosette form;
parasite almost fills a central block of parasite almost
enlarged cells pigment fills cell
Pathogenesis and Clinical Manifestations species involved, this may range from 11 days
to 4 weeks. The average pre-patent period for
The interval from sporozoite injection to
P. falciparum is 11 to 14 days, for P. vivax, 11
detection of parasites in the blood is referred
to 15 days, for P. ovale, 14 to 26 days, and 3
to as the pre-patent period. Depending on the
to 4 weeks for P. malariae. The incubation
Chapter 2: protozoan Infections 89
Plasmodium species
Parameter P. falciparum P. vivax P. ovale P. malariae
(malignant tertian) (benign tertian) (benign tertian) (quartan)
Gametocyte Present in peripheral Microgametocyte: Distinguished from P. Same as P. vivax
blood stream, light red to pink malariae by size except smaller; fills
similar to P. vivax; chromatin, of infected cells or almost fills cells
crescent or diffuse, central; and by Schüffner’s
sausage shape gives tint to light dots; less easy to
blue cytoplasm; differentiate from
yellowish P. vivax
brown pigment
throughout
cytoplasm; usually
round and about
the size of normal
RBC
Macrogametocyte:
small, compact,
dark red eccentric
chromatin;
cytoplasm dark
blue, no vacuoles;
abundant dark
brown pigment
scattered
throughout the
cytoplasm
Stages in peripheral Ring forms and All stages present All stages present All stages present
blood gametocytes;
other stages rare
Length of asexual 48 hours or less 48 hours 48 hours 72 hours
cycle
Note: P. knowlesi is microscopically indistinguishable from P. malariae.
period, the time between sporozoite injection with the associated asymptomatic intervals.
and the appearance of clinical symptoms, is Prodromal symptoms may include: a feeling
typically 8 to 40 days, depending again on of weakness and exhaustion, a desire to stretch
the involved species. For P. falciparum, it lasts and yawn, aching bones, limbs, and back, loss
an average of 8 to 15 days, for P. vivax, 12 to of appetite, nausea and vomiting, and a sense of
20 days, for P. ovale, 11 to 16 days, and for P. chilling. At the onset, symptoms may include
malariae, 18 to 40 days. The incubation period malaise, backache, diarrhea, and epigastric
may range from 9 days to 3 years, depending discomfort. The classical malaria paroxysms
on the parasite strain, the dose of sporozoites have three stages: the cold stage, the hot stage,
inoculated, the immune status of the host, and and the sweating stage. The cold stage starts
the host’s malaria chemoprophylaxis history. with a sudden inappropriate feeling of coldness
Partial or incomplete prophylaxis may prolong and apprehension. Mild shivering quickly turns
the incubation period several weeks after to violent teeth chattering and shaking of the
termination of medication. Any person who entire body. Although the core temperature is
has traveled to a malaria-endemic area must be high or may be rising quickly, there is intense
considered at risk of developing malaria up to peripheral vasoconstriction. The patient may
2 years and even longer upon leaving the area. vomit and febrile convulsions may ensue at
There are no absolute clinical features of this stage in young children. These rigors last
malaria except for the regular paroxysms of fever for 15 to 60 minutes after which the shivering
90 MedICal parasItology In the phIlIppInes
ceases, and the hot stage or flush phase begins. distribution. They also depend on the age,
The patient becomes hot and manifests with genetic constitution, state of immunity, general
headache, palpitations, tachypnea, epigastric health and nutritional status of the host, and
discomfort, thirst, nausea, and vomiting. The on any chemoprophylaxis or chemotherapy
temperature may reach a peak of 41°C or even previously used.
more. The patient may become confused or There may be a tendency to recrudesce or
delirious, and the skin may be notably flushed relapse over a period of months to several years.
and hot. This phase lasts from 2 to 6 hours. In Recrudescence is the renewal of parasitemia
the sweating stage, defervescence or diaphoresis or clinical features arising from persistent
ensues with the patient manifesting with undetectable asexual parasitemia in the absence
profuse sweating. The temperature lowers over of an exo-erythrocytic cycle. Relapse is renewed
the next 2 to 4 hours, and symptoms diminish asexual parasitemia following a period in which
accordingly. The total duration of a typical the blood contains no detectable parasites
attack is 8 to 12 hours. The classic periodicity (Figure 2.15). Relapses, which occur with vivax
of attacks develops only if the patient is left and ovale malaria, result from the reactivation
untreated until the time when the life cycle of hypnozoite forms of the parasite in the liver.
phases become synchronized and sufficient Cold, fatigue, trauma, pregnancy, and infections
numbers of red blood cells containing schizonts including intercurrent falciparum malaria may
rupture at about the same time. The interval precipitate reactivation.
between attacks is determined by the length of The pathological processes in malaria
the erythrocytic cycle. For P. falciparum, it is are the result of the erythrocytic cycle. Once
48 hours. For P. vivax and P. ovale, paroxysms the merozoites of P. falciparum invade the
occur on alternate days. For P. malariae, they erythrocytes, the cells reduce their deformability,
occur every 72 hours, causing paroxysms on the degree of which is directly proportional
days 1 and 4, hence the term, quartan malaria. to parasite maturity. This reduction in
Due to the lack of an exoerythrocytic stage in deformability is due to changes in the red
P. knowlesi, fever follows a quotidian pattern, or blood cell cytoskeleton and the increase in
is noted to be non-relapsing. membrane stiffness and cytoplasmic viscosity.
The five species also differ in the age of In the course of invasion, electron-dense sub-
infected erythrocytes. The non-falciparum membranous structures appear and enlarge.
species infect erythrocytes only of a certain These become the so-called “knobs” which are
age: P. vivax and P. ovale infect only young red important in cytoadhesion. They contain several
blood cells, while P. malariae infects only aging proteins such as rosettins, riffins, histidine-
cells. This limits the number of red blood cells rich proteins (HRP), and the Plasmodium
that can be parasitized to less than 3% of all falciparum erythrocyte membrane protein 1
erythrocytes. P. falciparum, as well as P. knowlesi, (PfEMP-1), which is the most adhesive protein
may infect erythrocytes of all ages. As the among the knobs. PfEMP-1 is encoded by a
infected erythrocytes rupture, more falciparum multi-gene family termed var and is clonally
malaria parasites are released to infect more red variant enabling it to evade specific immune
blood cells. The severity of complications and responses. Rosettins and PfEMP-1 are the
mortality increase as the level of parasitemia ligands for rosette formation. They adhere to
increases. The course and severity of the attack parasitized and non-parasitized cells as well
of malaria depend on the species and the strain as blood platelets. In more recent studies, it
of the infecting parasite; therefore, geographical has been suggested that febrile temperatures
origin of infection plays a major role in disease induce the cytoadherence of the ring-staged
Chapter 2: protozoan Infections 91
Figure 2.15. Diagram of the course of malaria infections showing the primary attack, relapses,
and recrudescence (From World Health Organization. Chemotherapy of malaria and resistance to
antimalarials: report of a WHO scientific group. Technical report series no. 529.
Geneva: World Health Organization; 1973.)
P. falciparum erythrocytes, and that the factor cytokines at the time of schizont rupture.
responsible for this heat-induced cytoadherence The combination of altered red cell surface
is PfEMP-1. HRP, on the other hand, localize to membranes and the host’s immunological
the cytoadherence ligands making the adhesion response to the parasite antigens bring about the
more effective. pathologic changes such as alteration in regional
Infected erythrocytes also undergo blood flow in the vascular endothelium, altered
altered membrane transport mechanisms. biochemistry, anemia, and tissue and organ
The hemoglobin is digested forming the hypoxia. Other destructive tissue processes
pigment hematin, and variant strain-specific include increased capillary permeability which
neoantigens are expressed. The soluble antigens allows fluid to leak into surrounding tissues, and
of P. falciparum are potent inducers of pro- congestion in blood vessels resulting in tissue
inflammatory as well as anti-inflammatory infarction and necrosis.
cytokines from monocytes and macrophages. In severe forms of malaria, impairment
Glycosylphosphatidyl inositol (GPI) moieties of consciousness and other signs of cerebral
that are seen covalently linked to the surface dysfunction, such as delirium and generalized
antigens of these protozoans act like the convulsions, are commonly observed. Other
endotoxin of gram-negative bacteria, manifestations are severe hemolytic anemia
lipopolysaccharide (LPS). They stimulate the with a hematocrit less than 20%, hemoglobin
monocytes to release tumor necrosis factor levels less than 7 g/dL and hyperbilirubinemia
(TNF) or cachexin, which is implicated as with levels more than 50 mmol/L (Table 2.6).
the cause of malarial fever. The fever, febrile Cerebral malaria generally manifests with
paroxysms, headache, various aches and pains, diffuse symmetric encephalopathy. Other signs
and prostration, which are the more familiar and symptoms include retinal hemorrhage,
symptoms of an acute malarial attack, are bruxism (fixed jaw closure and teeth grinding),
probably the result of the release of these and mild neck stiffness. Pouting may occur or
92 MedICal parasItology In the phIlIppInes
Table 2.6. Clinical features and laboratory findings in severe malaria infection
Source: World Health Organization. Management of severe malaria: a practical handbook. Geneva: World Health Organization; 2000.
a pout reflex may be elicited by stroking the affected. Malaria ARF is defined as having a
sides of the mouth. Lumbar tap usually reveals serum creatinine of more than 265 mmol/L
a normal to elevated opening pressure, clear (3 mg/dL) and a 24-hour urine output of
cerebrospinal fluid (CSF) with fewer than 10 less than 1 ml/kg/hr, despite rehydration, in
leukocytes/mL, and slightly elevated protein and patients with asexual forms of the parasite
CSF lactic acid concentration. If left untreated, present in their peripheral blood smear. The
symptoms progress to persistent coma and patient may also present with hyperkalemia
death. The neurological complications, once and hyperuricemia earlier in the course.
promptly and adequately treated, are reversible The cytoadherence, rosette formation, and
and a majority of the patients make a complete sequestration of parasitized erythrocytes lead
recovery. to a decrease in tissue perfusion resulting in
Respiratory findings are also a major decreased renal blood flow. The increase of TNF
feature of severe malaria. Altered pulmonary in tubular epithelial cells leads to inflammatory
function is common, and it includes air flow cell infiltration in the interstitium and altered
obstruction, impaired ventilation and gas tubular transport, which result in tubular
transfer, and increased pulmonary phagocytic damage and dysfunction. The presence of GPI
activity. In African children, pneumonitis from and other falciparum malaria antigens lead to
sequestered, parasitized RBC and inflammatory release of cytokines and mediators that decrease
cells are seen in postmortem pulmonary the systemic vascular resistance and increase
vasculature, while in adults, non-cardiogenic renal vascular resistance. All these changes
pulmonary edema and acute pulmonary distress eventually lead to acute tubular necrosis causing
syndrome (ARDS) may be observed. There is a acute renal failure.
high mortality rate (over 80%) when pulmonary Malaria in pregnancy increases the risk of
edema develops in a patient with severe malaria. maternal death, maternal anemia, intrauterine
Factors which predispose to pulmonary edema growth retardation, spontaneous abortion,
include hyperparasitemia, renal failure, and stillbirth, and low birth weight associated with
pregnancy. risk for neonatal death. Non-immune pregnant
The incidence of acute renal failure (ARF) women are susceptible to all complications
reaches up to 60% of patients with severe associated with severe malaria such as cerebral
falciparum malaria, with more males being malaria, hypoglycemia, and pulmonary edema.
Chapter 2: protozoan Infections 93
For partially immune pregnant women, Not everyone infected with the malaria
especially primigravid, severe anemia may parasite becomes seriously ill or dies. In areas
develop but the other complications of severe where endemicity is stable, repeated exposures
malaria are unlikely to occur. Falciparum to the parasite lead to specific immunity.
malaria may induce uterine contractions, thus This restricts occurrence of serious problems
may push the patient to premature labor. In in young children, while older patients have
severe malaria, the prognosis of the fetus is poor. relatively mild febrile illness. In people who are
Falciparum malaria in a young child is exposed to malaria for the first time, possible
considered a medical emergency for it can be outcomes may range from apparent resistance to
rapidly fatal. The initial symptoms may be death. Any resistance, therefore, is nonspecific.
atypical and difficult to recognize, but within It also does not necessarily depend on prior
hours, life-threatening complications may start exposure to malaria and may be either acquired
to occur. The most common complications or innate. Poor prognostic factors in falciparum
of severe malaria in children are cerebral malaria include hyperparasitemia defined as
malaria, severe anemia, respiratory distress, and a peripheral count more than 250,000/µL or
hypoglycemia. Children with severe malaria more than 5% of the RBCs infected, and the
most commonly present with seizures. These presence of mature or immature schizonts in
convulsions are common before or after the a peripheral blood smear. It has been shown
onset of coma and are significantly associated that a peripheral count of 10% or more of
with neurologic sequelae. Opisthotonos may red blood cells infected has a mortality rate
also be observed in some children. As much as of 50%, particularly in non-immune cases,
10% of children who survive cerebral malaria despite treatment. The clinical indicators of
will develop sequelae such as hemiparesis, poor prognosis include deep coma, absence
cerebellar ataxia, speech disorders, generalized of corneal light reflex, respiratory distress
spasticity, or some behavioral disturbances (acidosis), circulatory collapse, decerebrate
(Table 2.7). or decorticate rigidity, opisthotonos, and age
Table 2.7. Comparison of sign and symptoms of sever malaria in adults and children
These tests can be performed in 15 to 30 differentiate between current and past infections
minutes without the use of electricity, special and are therefore most helpful in epidemiologic
equipment, or any training in microscopy, and studies. Current studies are using PCR to
most kits have more than 90% specificity. More significantly enhance microscopic diagnosis of
recent studies have shown that test kits based malaria especially in cases of low parasitemia
on HRP II have a sensitivity and specificity of and in cases of mixed infection.
92.5% and 98.3% respectively, while kits based
Treatment
on the pLDH antigen have a lower sensitivity
(88.5%) albeit a higher specificity at 99.4%. Antimalarial drugs have selected actions
The use of RDTs can be easily taught to village on the different phases of the life cycle of the
health workers and the results can likewise be malaria parasite. These drugs may be classified
easily interpreted. The main disadvantages into causal prophylactic drugs, which prevent
of RDTs compared to microscopy are: the the establishment of the parasite in the liver,
lack of sensitivity at low levels of parasitemia; and blood schizonticidal drugs, which attack
the inability to quantify parasite density; the the parasite in the red blood cell, preventing
inability to distinguish among P. vivax, P. ovale, or terminating the clinical attack. Tissue
and P. malariae, as well as sexual and asexual schizonticides act on pre-erythrocytic forrns
stages; the persistently positive tests (for some in the liver. Gametocytocidal drugs destroy
antigens) despite parasite clearance following the sexual forms of the parasite in the blood.
chemotherapy; and the relatively higher cost Some drugs are hypnozoitocidal or antirelapse
per test. drugs, which kill the dormant forms in the liver.
In recent studies conducted in various areas Sporonticidal drugs inhibit the development
of the Philippines to describe the validity of a of the oocysts on the gut wall of the mosquito,
few specific malaria RDT kits, results showed which has fed on a gametocyte carrier so that
sensitivity and specificity levels below the the mosquito cannot transmit the infection.
WHO recommended ideal of 95% and 90%, The main uses of antimalarial drugs are:
respectively. Reasons for these findings could be (a) protective (prophylactic), (b) curative
manufacturer-related problems, the instability (therapeutic), and (c) preventive. Drugs for
of the substances used in the diagnostic prophylaxis are used before the infection occurs
technique to varying environmental conditions or before it becomes evident, with the aim of
such as extremes of temperature and humidity, preventing either the occurrence of the infection
and user-related problems. Quality assurance of or any of its symptoms. A blood schizonticidal
these malaria RDT kits is, therefore, necessary drug may have minimal effects on parasites
before they are deployed on a larger scale in growing in the liver, but if it is still present in
remote and rural areas. More recent studies are the blood when the merozoites leave the liver
now concentrating on quality assurance of these and invade the blood cells for the first time, it
tests and on identifying the factors which may will effectively prevent symptomatic malaria.
affect RDT performance in the field. Curative or therapeutic use refers to action on
Malaria can also be diagnosed serologically the established infection, which involves the use
but presently available methods are not of blood schizonticidal drugs for the treatment
capable of making a definitive diagnosis of of the acute attack and in the case of relapsing
acute malaria. Available serologic tests like malaria, radical treatment of the dormant liver
indirect hemagglutination (IHA), indirect forms. Prevention of transmission means the
fluorescent antibody test (IFAT), and enzyme- deterrence of infection of mosquitoes with
linked immunosorbent assay (ELISA) cannot the use of gametocytocidal drugs to attack the
96 MedICal parasItology In the phIlIppInes
gametocytes in the blood of the human host. It be given in pregnancy and in children less than
also means the interruption of the development 4 years of age.
of the sporogonic phase in the mosquito when In contrast with falciparum malaria, vivax
it feeds on the blood of an infected person who malaria remains sensitive to chloroquine. Clinical
has been given the appropriate sporonticidal studies and extensive in vitro observations have
compound. shown that P. vivax is still generally sensitive to
Chloroquine was the mainstay of chloroquine, although resistance is prevalent
antimalarial treatment for the last 50 years. and increasing in Indonesia, Peru, and Oceania.
Because of emergence of multidrug-resistant Moreover, vivax malaria is sensitive to all other
(MDR) strains, subsequent chloroquine use antimalarial drugs albeit slightly less sensitive to
has been rendered ineffective against falciparum artesunate plus sulfadoxine-pyrimethamine. The
malaria, and the current DOH Malaria asexual stage of P. vivax remains susceptible to
Control Program (MCP) recommends the use primaquine; therefore, combination treatment
of artemisinin-based combination therapies with chloroquine and primaquine affords blood
(ACTs) for severe and uncomplicated falciparum stage and liver stage treatment, respectively.
malaria, replacing the chloroquine plus Often referred to as radical treatment, the use of
sulfadoxine-pyrimethamine combination. The primaquine, together with chloroquine, allows
following drug combinations are recommended: for the prevention of relapse in vivax malaria. In
artemether plus lumefantrine, artesunate plus comparison with no primaquine treatment, the
amodiaquine, artesunate plus mefloquine, risk of relapse decreases for every additional mg/
and artesunate together with sulfadoxine- kg of primaquine given. Repeated vivax malaria
pyrimethamine. For severe malaria, parenteral relapses are debilitating at any age, hence they
antimalarial treatment should be started must be prevented. At least a 14-day course of
without delay after rapid clinical assessment and primaquine is needed for the radical treatment
confirmation of the diagnosis. The following of P. vivax.
antimalarial drugs are recommended: artesunate Resistance of P. malariae and P. ovale
intravenous (IV) injection or intramuscular to antimalarials is not well characterized,
(IM) injection, quinine IV or IM, or artemether and infections with these species are still
IM. In a placebo-controlled trial, patients with considered sensitive to chloroquine. The
severe malaria who could not be treated orally treatment for relapsing fever caused by P.
and where access to IM and IV treatment was ovale is similar to that of vivax malaria (i.e.,
unavailable, a single artesunate suppository at chloroquine and primaquine). In the case of
the time of referral reduced the risk of death or mixed malarial infections, ACTs remain the
permanent disability. mainstay treatment. Moreover, the use of
Current guidelines also recommend artemisinin-based compounds and a partner
the use of gametocytocidal drugs to reduce drug with a long half-life (i.e., artesunate
transmission. Seen in the context of malaria plus amodiaquine and dihydroartemisinin
elimination, the use of primaquine 0.75 plus piperaquine) has been effective against
mg base/ kg body weight single oral dose in chloroquine-resitant vivax malaria. Radical
demonstrates an added benefit to artemisinins treatment with primaquine should also be
in eliminating gametocytes. The addition of a considered in cases of confirmed P. vivax and
single dose of primaquine to current ACTs is P. ovale infections.
therefore recommended provided that the risk Artemisinin and its derivatives (Qinghaosu
for hemolysis in G6PD deficient patients is derivatives), artesunate, and artemether produce
considered. Moreover, primaquine should not rapid clearance of parasitemia and rapid
Chapter 2: protozoan Infections 97
shows an initial reduction in parasitemia after without previously meeting any of the criteria
treatment but there is failure to clear the blood of ETF; and (b) presence of parasitemia and
of asexual parasites and soon after an increase an axillary temperature of 37.5°C (or history
of parasitemia follows. RIII is the severest form of fever) on any day from Day 4 to Day 28,
of resistance in which parasitemia will either without previously meeting any of the criteria
show no significant change with treatment or for ETF. Late parasitological failure for intense
will eventually increase. transmission areas is defined as presence of
MDR malaria is considered when treatment parasitemia on Day 14 and axillary temperature
failure occurs with three or rnore antimalarial of 37.5°C without previously meeting any of the
agents. In this case, a combination of artesunate criteria for ETF or late clinical failure.
has been combined with mefloquine and is now For low to moderate transmission areas,
the first-line regimen for MDR malaria in some late parasitological failure is defined as presence
Southeast Asian countries. of parasitemia on any day from Day 7 to Day
Classification of response to malaria 28 and axillary temperature of 37.5°C without
treatment can be divided into early treatment previously meeting any of the criteria for ETF
failure, late treatment failure, and adequate or late clinical feature. Adequate clinical and
clinical and parasitological response. Early parasitologic response for intense transmission
treatment failure (ETF) is present when there areas is defined as absence of parasitemia on Day
is: (a) development of danger signs or severe 14 (Day 28 for low to moderate transmission
malaria on Day 1, 2, or 3 in the presence of areas) irrespective of axillary temperature
parasitemia; (b) parasitemia on Day 2 higher without previously meeting any of the criteria of
than the Day 0 count irrespective of axillary ETF, late clinical failure, or late parasitological
temperature; (c) parasitemia on Day 3 with failure.
axillary ternperature of 37.5°C; and (d) In cases of renal failure in severe malaria,
parasitemia on Day 3 which is 25% of count dopamine may be given at 3 to 5 µg/kg/
on Day 0. This classification of ETF holds for minute. If the patient remains unresponsive
both intense transmission and low to moderate despite adequate rehydration and other forms
transmission areas. Late treatment failure (LTF) of therapeutic management, and blood urea
is further divided into late clinical failure and and creatinine are rising progressively, dialysis
late parasitological failure. The definitions for is indicated.
these two would differ depending on whether For control of seizures, diazepam may be
the area is an intense transmission area or a given at 10 mg intravenous (pediatric dose at
low to moderate one. Late clinical failure for 0.3 mg/kg IV up to 10 mg) or in cases of status
intense transmission areas is defined as: (a) epilepticus, phenytoin at a loading dose of 13
development of danger signs or severe malaria to 18 mg/kg and a maintenance dose of 3 to 5
after Day 3 in the presence of parasitemia, mg/kg per day (pediatric dose: loading dose of
without previously meeting any of the criteria 15-20 mg/kg slow IV push and maintenance
of ETF; and (b) presence of parasitemia and dose of 5 mg/kg in two divided doses). The
axillary temperature equal to or greater than following are now not considered useful and are
37.5°C on any day from Day 4 to Day 14, therefore not recommended in the management
without previously meeting any of the criteria of cerebral malaria: corticosteroids, other anti-
for ETF. For low to moderate transmission inflammatory agents, low molecular weight
areas, late clinical failure is defined as: (a) dextran, epinephrine, and heparin.
development of danger signs or severe malaria Proper management of malaria also
after Day 3 in the presence of parasitemia, includes general and supportive measures
Chapter 2: protozoan Infections 99
especially in P. falciparum infections. If fluid malaria have shrunk considerably over the past
replacement or blood transfusion is necessary, 50 years, but control is becoming more difficult,
it must be administered with care to avoid and past gains have been threatened. The spread
pulmonary edema. Antipyretics and sponging of the disease is linked to activities like road
for high fever are important especially in building, mining, logging, and new agricultural
children to prevent convulsions. Blood sugar and irrigation projects, particularly in “frontier”
should be monitored regularly especially in areas (e.g., forest fringe, mountain valleys and
severe malaria. If hypoglycemia develops, 50 reclaimed areas). Elsewhere, disintegration
mL of 50% dextrose (1.0 mL/kg for children) of health services, armed conflict, and mass
diluted in an equal volume of infusion fluid movements of refugees have worsened the
should be infused over a 5-minute period, malaria situation.
followed by a continuous intravenous infusion Malaria remains a public health problem
of 5 to 10% dextrose. today in more than 90 countries inhabited by
a total of some 3.3 billion people (Figures 2.17
Epidemiology
to 2.19). Of these, 2.1 billion are at low risk
Malaria is the world’s most important (<1 case per 1,000 population), 94% of whom
tropical parasitic disease. The disease kills live in areas outside the WHO African region.
more people than any other communicable The 1.2 billion at high risk (>1 case per 1,000
disease except tuberculosis. In many developing population) live in the WHO African (47%)
countries, especially in Africa, malaria has an and Southeast Asian Regions (37%).
enormous toll on lives, medical costs, and days In 2010, the WHO reported an estimated
of labor lost. The geographical areas affected by 216 million cases of malaria, of which 81% or
Figure 2.17. Global distribution of malaria: malaria-free and malaria-endemic countries in phases of
control, elimination and prevention of reintroduction (From World Health Organization. World Malaria
Report 2009. Geneva: World Health Organization; 2009.)
100 MedICal parasItology In the phIlIppInes
Figure 2.18. Distribution of malaria in the WHO Southeast Asia Region: areas in red indicate malaria-
endemic countries (Accessed from http://www.map.ox.ac.uk)
Figure 2.20. Malaria cases per 100,000 population in the Philippines from 2000 to 2009
(From Department of Health-National Center of Disease Prevention and Control. Malaria medium term
development plan 2011-2016. Manila (Philippines): Department of Health; 2011.)
morbidity since 2006. Cases have been notably reported in 2009 (Figure 2.21). Similarly, the
decreasing as reported in 2009 (Figure 2.20). malaria morbidity rate has decreased by 58.3%,
However, disease prevalence, seen in the 2010 with 18,781 malaria cases reported in 2009.
DOH-Malaria Control Program (MCP) Among blood smears examined from 2005 to
report, remains markedly high in Regions 2009, 69.4 to 80% of patients were diagnosed
IV-B, Caraga, III, XII, and II. There remains with P. falciparum, 17.0 to 23.4% with P. vivax,
an estimated 10.8 million people still at risk approximately 1% with P. malariae, and a small
for the disease, consisting mostly of farmers, number of cases (0.5%) were diagnosed to have
indigenous cultural groups, miners, forest mixed malaria infection, falciparum and vivax
product gatherers, and soldiers. Fifty nine out of malaria being the usual mixed infection.
the 80 provinces in country are endemic for the Macrostratification of malaria endemic
disease, with 60.4% of the endemic provinces areas serves to classify the different provinces
located in Luzon, 39.5% in Mindanao, and based on annual incidence of the disease
0.1% in Visayas. As of 2009, the provinces of in each respective province (Table 2.8).
Cagayan, Isabela, Palawan, Sulu, and Tawi-Tawi Macrostratification provides an opportunity
comprise the five provinces having the highest for planning, policy making, and resource
number of malaria cases reported. allocation of the provincial MCP. The number
It appears that in areas of low malaria of provinces in Category A has been reduced
endemicity, there is a clustering of cases, from 26 in 2000, to nine in 2005 and finally to
resulting in pockets of high endemicity. five in 2008. The values reported for Category
Mortality rate for malaria has markedly B provinces have changed from 22 in 2000 to
decreased by 88.2% from 2005 figures to values 31 in 2005, and to 27 in 2008. Malaria-free
102 MedICal parasItology In the phIlIppInes
Figure 2.21. Malaria-related deaths per 100,000 population in the Philippines from 2005 to 2009
(From Department of Health-National Center of Disease Prevention and Control. Malaria medium term
development plan 2011-2016. Manila (Philippines): Department of Health; 2011.)
by contaminated needles and syringes. Blood breeding sites, and estimation of mosquito
from semi-immune donors without clinical density.
symptoms may contain malaria parasites. In
Prevention and Control
congenital malaria, infected mothers transmit
parasites to their child before or during birth. Early diagnosis and prompt treatment
The evaluation of the amount and of malaria are essential for malaria control.
conditions of transmission of malaria in a Breeding sites of Anopheles flavirostris should
given locality is called the malaria survey. be detected early and contained. Personal
Disease control efforts must always take into protection measures against mosquito bites
consideration the findings of the malaria are also helpful and cost-effective. The use
survey. The survey involves looking into of insecticide- (permethrin or deltamethrin)
epidemiologic data regarding the disease, such as treated nets (ITNs) and long lasting insecticide-
malaria mortality and morbidity, investigations treated nets (LLIN) remains the major vector
relating to the human host, and investigations control strategy coupled with indoor residual
relating to the insect vector. Investigations spraying (IRS), with the latter used in epidemic
relating to the human host include blood and situations, areas with stable transmission but
spleen examinations. Investigations relating without reduction of malaria incidence, and
to the vector, on the other hand, may include areas of displaced populations. Wearing of
identification of mosquito vectors and their light colored clothing, which cover most of the
104 MedICal parasItology In the phIlIppInes
body (since dark colors attract mosquitoes), and rice fields and bacterial insecticide (PG-14
using insect repellants containing DEET Bacillus thuringiensis), and chemical control
(N,N-diethyl-m-toluamide, optimally as a 35% such as the use of mosquito repellants and
concentration lotion) on exposed parts of the insecticide treated mosquito nets.
body, using a insect spray containing pyrethrum In the field of molecular entomology,
in living areas, and use of permethrin insecticide stable germline transformation of the Anopheles
as a repellant spray for clothing have known to mosquito is being investigated. This research
be effective personal protection measures as well. involves inserting genes (e.g., immune response
Chemoprophylaxis may be protective to genes) that will inhibit the development of
travelers who have no immunity to malaria, the parasite in the mosquito. With the recent
although no chemoprophylactic regimen sequencing of the genomes of Plasmodium
ensures 100% protection. Because of this, falciparum and of the Anopheles mosquito,
precautions to avoid mosquito bites are new areas of research for malaria treatment and
needed even if antimalarials have been taken. prevention are now being explored.
Prophylactic drugs should be taken with good
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Chapter 2: protozoan Infections 107
Babesia spp.
Florencia G. Claveria
fission, and eventually forms sporokinetes. Once the organs of the larva, nymphs, and adult
the sporokinetes are released, they continue to ticks. With the stage-to-stage (transstadial)
infect and multiply in various organs, including transmission, each of the developmental stages
the ovaries of the replete tick, until death ensues. is generally capable of parasite transmission to
The transovarian route represents one pattern mammals. The complicated phase of Babesia
of parasite transmission in the vector, which life cycle in the tick vector ends with the
terminates with the death of the vector. formation of the infective sporozoites in various
With the passage of sporokinetes to eggs organs or in the salivary glands, for subsequent
(transovarian), similar cycles of multiple fissions transmission to the mammalian hosts during
continue to take place in the embryo and in blood feeding (Figure 2.23).
Pathogenesis and Clinical Manifestations bigemina (4-5 µm by 2-3 µm) and B. caballi (3
µm by 2 µm) are less virulent. Several factors can
Smaller forms like Babesia bovis (2.4 µm
influence the susceptibility of hosts to infection,
by 1.5 µm) and B. equi (2 µm by 1 µm) are
like the age and breed of farm animals, and the
more pathogenic, while larger forms such as B.
health and immune state of humans.
Chapter 2: protozoan Infections 109
blood or NOD/sch-scid mice with the patient’s The drug combination azithromycin and
blood can be useful in parasite detection and atovaquone is as effective as clindamycin-
identification. quinine, with less adverse effects. Both drug
The polymerase chain reaction (PCR) is combinations are ineffective in suppressing
highly specific and is generally considered to disease progression in immunosuppressed
be the gold standard for Babesia detection. It is, patients. Very recently, there have been reports
however, unrealistic for epidemiologic surveys of immunocompromised patients who,
because it is time consuming and expensive. during 28 days of uninterrupted treatment
The current practice is the use of PCR in the with azithromycin-atovaquone, manifested
isolation of the SSU rDNA from asymptomatic recurrence of marked parasitemia, suggesting
patients, followed by gene sequencing and its the development of drug-resistant B. microti.
comparison with known SSU rDNA gene Ar temisinin, pyrimethamine, and
sequences from pathogenic strains. pamaquine can strongly inhibit the growth of
The continued work on the isolation of B. equi and B. caballi in vitro. Pyrimethamine
specific and highly immunogenic antigens can indirectly interrupt the parasite life cycle
of Babesia species and isolates, and their through its inhibitory effect on dihydrofolate
intended utilization in the development reductase, essential in folate metabolism, while
of immunochromatographic test (ICT) is pamaquine can interfere in the recycling of
practicable for epidemiologic surveys in the endosomal proteins into the plasma membrane
field. ICT is simple, quick, reliable, sensitive, by direct interaction with the endosomes.
and inexpensive, and has gained acceptability in
Epidemiology
the diagnosis of both acute and latent infections.
Currently, there are ICT strips or dipsticks Babesiosis is essentially a zoonotic infection,
employed in the detection of infected livestock. regarded of major economic importance
to livestock, particularly in the cattle and
Treatment
horse industry. Its documentation in humans
The standard treatment of human babesiosis worldwide has increased its recognition as
utilizes a drug combination of clindamycin and a disease of public health concern. The first
quinine, or azithromycin and atovaquone. The human case attributed to the cattle form was
clindamycin and oral quinine combination reported in 1956, in Europe, and followed
was first used in 1982 in a newborn infant with reports of more cases in Europe and in
suffering from babesiosis, and since then, this North America, including the discovery of
combination has become the drug of choice. the transmission of a B. microti-like species to
Clindamycin is given 1.2 g intravenously twice humans in 1970. A review of the 136 human
a day or 600 mg orally three times a day, and cases examined in New York (1982-1991)
combined with oral quinine at a dose of 650 revealed almost all cases were among those
mg three times a day. Clindamycin-atovaquone living in Suffolk, Long Island. Of the 103
combination is efficacious in clearing the (76%) who were hospitalized, seven patients
parasites in normal individuals and prevents previously underwent splenectomy, 31 patients
recurrence of infection, but produces adverse had symptoms of babesiosis and Lyme disease,
effects like vertigo, tinnitus, and gastrointestinal and seven died. The Asian cases reported have
symptoms. Supportive and symptomatic been few and sporadic with the first records in
management is important. In severe cases where 1984, in Yunnan, China, and a recent report
there is progressive exacerbation of hemolytic in Japan attributed to B. microti (Table 2.10).
anemia, total blood exchange may be indicated.
Chapter 2: protozoan Infections 111
Table 2.10. Summary of human cases of babesiosis reported in some Asian countries
The Centers for Disease Control and habitation with livestock and wild animals,
Prevention, USA has confirmed more than and where ticks were abundant. The parasites
40 human cases that contracted the disease detected were pyriform-shaped, suggestive of
from transfusion of packed RBC and tested Babesia. One case recorded in the rural area
positive for anti-B. microti antibodies. In Asia, in Southwestern Taiwan was serologically and
the two cases have been associated with renal morphologically diagnosed with a chronic and
transplantation and blood transfusion. Thus, subclinical infection of a geographic isolate
subclinical or asymptomatic cases cannot simply of Babesia named Taiwan isolate (TW1). The
be ignored, considering their potential role in detection of anti-Babesia antibodies in 83%
the spread of human babesiosis. Rattus coxinga endemic in the locality where the
Phylogenetic analyses of the gene sequences Taiwanese patient lived, suggested the rodents
of the SSU rDNA of Babesia spp. obtained as the highly likely source of infection. The SSU
from human cases helped clarify three patterns rDNA isolated from the Japanese patient and
or groupings, worldwide, namely: (a) human from the NOD/sch-scid mice inoculated with
babesiosis attributed to the B. divergens-related the patient’s blood revealed 99.2% sequence
parasites in Europe; (b) human babesiosis caused homology with the US B. microti SSU rDNA
by B. microti principally in the Northeastern (Genbank/EMBL/DDBJ: U09833).
USA; and (c) human babesiosis caused by newly In Japan the wild rodents, Apodemus
emerging species, the WA1-type in the Western speciosus and A. argenteus, are infected with B.
USA, tentatively grouped with B. microti or microti-like forms. In Taiwan, the bandicoot
alternatively with Theileria spp. Recently in rats, Bandicola indica, and the spiny rat, R.
Italy and Austria, parasites obtained from coxinga, carry morphologically similar B.
splenic cases revealed SSU rDNA sequences microti-like forms. The TW1 isolated from
more closely related to B. odocoileus, a species the first human case in Taiwan is serologically
that bears morphological, molecular, and related to the US B. microti SSU rDNA.
immunological similarities with B. divergens. In the Philippines, studies on animal
The B. divergens-related species now has been babesiosis have been limited and mainly
expanded to include B. odocoileus and possibly concentrated on hematological parameters and
B. bovis. In Asia, the etiologic agent of human clinical manifestations in cattle B. bigemina and
babesiosis has been identified as B. microti or B. argentina (syn. B. bovis), and B. canis. Using
B. microti-like isolate or strain. the ICT, 13 (28%) stray dogs in an impounding
Human cases recorded in China were facility in Dasmarinas, Cavite tested positive for
generally among farmers living in close anti-p50 truncated B. gibsoni antigen. The dogs
112 MedICal parasItology In the phIlIppInes
had infestation mainly with Rhipicephalus ticks, In Europe and the USA, the Ixodes ricinus
suggestive of their putative role in the spread of and Ixodes triangulicep, and the Ixodes scapularis
canine babesiosis in the country. and Ixodes pacificus are the principal vectors,
Slaughtered and race horses in the respectively. In Asia, the tick vectors are poorly
Philippines tested seropositive for B. caballi established. The predominance of Ixodes
and/or B. equi infection, using ICT containing a granulatus in Southeast Asian countries makes
recombinant B. caballi 48-kDa rhoptry protein it a favorable vector, though this warrants
(rBc48) and a recombinant truncated B. equi confirmation (Plate 2.17). In Japan, Ixodes
merozoite antigen 2 (rEMA-2t). Serological ovatus has been suggested as a highly likely
data correlated well with the detection of the tick vector of human babesiosis for its human
morphologies of the specific etiologic agent(s) biting activity.
in blood smears.
Plate 2.17. Ixodes sp. A. Non-engorged female. B. Engorged female. C. Mouthparts showing
the hypostome (H), pedipalp (P), and scutum (S). (Courtesy of Ms. Mary Jane Cruz-Flores
and Dr. Florencia Claveria)
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1972;11:81–91. Toxoplasmosis and babesiosis in Asia. Vol
Clarke CR, Burrows GE, MacAllister 4, Asian Parasitology. Chiba (Japan): The
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horses. Am J Vet Res. 1982;53:2292–5. Ristic M, editor. Babesiosis of Domestic
Cruz-Flores MJ, Claveria FG, Verdida R, Xuan Animals and Man. Boca Raton, Florida:
X, Igarashi I. First detection of Babesia CRC Press; 1988. p. 209–28.
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by immunochromatographic test (ICT). H, Slemenda SB, Piccaluga P, Martinelli
Veterinarski Arhiv. 2008;78:149–57. G, et al. Molecular characterization of a
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(Theileria equi Mehlhorn and Schein, Seng, S. Tum, S., et al. Role of CD4+ T
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Chapter 2: protozoan Infections 115
into the body through broken skin, or through result in cardiomegaly, congestive heart failure,
mucous membranes that are rubbed with fingers thromboembolism, and even arrhythmias. Less
contaminated with the bug’s feces. severe signs and symptoms associated with
the chronic phase of the disease include chest
Pathogenesis and Clinical Manifestations
pain, palpitations, dizziness, syncopal episodes,
Chagas disease can be divided into an abnormal electrocardiogram findings, achalasia
acute and a chronic phase. The acute phase is associated with megaesophagus, and chronic
characterized by a focal or diffuse inflammation constipation associated with megacolon. About
mainly affecting the myocardium. Non- one-third of patients in the latent stage develop
specific signs and symptoms, such as fever, some manifestation of chronic Chagas disease
malaise, nausea, vomiting, and generalized after several years or decades. The majority of
lymphadenopathy often accompany the acute symptomatic, chronic patients manifest with
phase. Cutaneous manifestations of the disease the cardiac form, while the rest develop the
can sometimes appear during this phase, gastrointestinal form.
usually associated with a localized inflammatory
Diagnosis
reaction at or near the site of inoculation.
Chagomas are furuncle-like lesions associated A complete patient history is the primary
with induration, central edema, and regional tool for diagnosing Chagas disease. Possible
lymphadenopathy. These lesions represent exposure to T. cruzi should be established, and
the site of entry of the parasite. If the parasite risk factors such as place of residence or work,
penetrates through the conjunctiva, eyelid recent blood transfusion in an endemic area,
swelling called Romaña’s sign may form. This and contact or exposure to T. cruzi intermediate
lesion is characterized by unilateral painless host should be evaluated.
bipalpebral edema and conjunctivitis, and may The definitive diagnosis of Chagas
involve the lacrimal gland and surrounding disease during its acute phase relies on direct
lymph nodes. After 1 or 2 months, symptoms visualization of the parasites in thick and thin
resolve, and the patient goes into a latent or blood smears using Giemsa stain. Cerebrospinal
indeterminate, but usually asymptomatic phase. fluid (CSF), tissue samples, or lymph can also be
During this phase, patients infected with T. used for parasite visualization. However, only in
cruzi are still capable of transmitting it to others the first two months of acute disease can T. cruzi
through insect vectors, blood transfusion, or trypomastigotes be seen by direct examination.
organ transplantation. Other diagnostic techniques include
The pathophysiology of the chronic concentration methods (microhematocrit),
phase of the disease was initially thought to blood culture, and polymerase chain reaction
be autoimmune in nature; however, this is (PCR). Xenodiagnosis, wherein laboratory-
controversial. Newer evidence shows that reared triatomine bugs are allowed to feed on
chronic Chagas disease is multifactorial, suspected patients and are later examined for the
and dependent on the interaction between presence of T. cruzi metacyclic trypomastigotes,
parasite and host. Nonetheless, the chronic may also be utilized as a diagnostic modality.
phase is manifested by fibrotic reactions that During the chronic phase, a variety of
cause injury to the myocardium, cardiac serologic tests may be used, such as enzyme-
conduction network, and enteric nervous linked immunosorbent assay (ELISA), indirect
system (decrease in nerve ganglia leading to hemaglutination, indirect immunofluorescence,
megasyndromes). The heart is the primary and PCR. The WHO recommends using at
organ affected during this phase, which may least two techniques with concurrent positive
Chapter 2: protozoan Infections 119
Chagas disease is estimated to have infected There have been major breakthroughs
more than 10 million people worldwide. Most in the control and prevention of American
cases are reported in the Latin Americas, where trypanosomiasis, particularly by Brazil, Chile,
more than 25 million people are at risk for the and Uruguay. Vector control and blood
disease. Serologic techniques have identified that transfusion regulations have delivered positive
up to 13% of populations in certain endemic outcomes, in terms of disease prevention in
regions are positive for T. cruzi antibodies. An these countries. Spraying of insecticides, use
estimated 10,000 to 12,000 people die of the of insecticide-treated bed nets, and house
disease annually. improvements to prevent vector infestation
120 MedICal parasItology In the phIlIppInes
have been proven cost-effective. International highly fatal disease caused by two subspecies of
organizations such as the WHO and the Trypanosoma brucei: T. brucei gambiense and T.
manufacturers of the antiparasitic drugs are brucei rhodesiense. A third subspecies, T. brucei
working in tandem to ensure the availability of brucei, primarily affects wild and domestic
drugs for the treatment of the disease. animals; collectively, the three subspecies
In Mexico and non-endemic countries represent the Trypanosoma brucei complex. The
near endemic countries, the coverage, quality earliest reports of sleeping sickness in Africa
and safety of blood transfusion screening date back to 1734, while the formal correlations
is being evaluated as avenues for disease between the symptoms, the parasite in blood
prevention. Vaccine development has not and CSF, and the relationship between the
yet been successful, but the advent of newer parasite and its insect vector were established
technologies and characterization of the T. cruzi during the early 1900s.
genome may aid in future vaccine research.
Parasite Biology
Trypanosoma brucei gambiense Members of the T. brucei complex belong to
Trypanosoma brucei rhodesiense the trypanosome family Salivaria. The parasite
is usually transmitted via the bite of the blood-
Human African trypanosomiasis (HAT), sucking tsetse fly (Glossina spp.) feeding from an
also known as African sleeping sickness, is a infected mammalian host (Figure 2.25). Since
the disease relies heavily on the tsetse fly for its through mechanical methods (accidental needle
transmission, HAT cases are localized in regions pricks, other blood sucking insects), as well as
of sub-Saharan Africa, primarily in remote rural vertically, via mother-to-child infection through
areas where tsetse fly habitats are located. the placenta.
T. brucei gambiense is localized mostly in
Pathogenesis and Clinical Manifestations
the western and central regions of sub-Saharan
Africa. It primarily affects humans, but utilizes Human African trypanosomiasis has two
dogs, pigs, and sheep as reservoir hosts. It is types, acute and chronic, depending on the
responsible for the chronic type of sleeping subspecies causing the disease. Trypanosoma
sickness, and accounts for 95% of all HAT cases. brucei gambiense sleeping sickness manifests
T. brucei rhodesiense is found in east Africa months or years after initial infection, while
and is primarily a zoonosis of cattle and wild symptoms of T. brucei rhodesiense sleeping
animals, with humans being accidental hosts. sickness may appear just weeks after infection.
It causes the more acute and rapidly fatal form The initial lesion of African trypanosomiasis
of sleeping sickness, and accounts for the begins as a local, painful, pruritic, erythematous
remaining 5% of HAT cases. chancre located at the bite site, progressing
Only the epimastigote and trypomastigote into a central eschar, and resolving after 2 to
forms are exhibited by the T. brucei complex. 3 weeks. This trypanosomal chancre is more
The trypomastigotes are polymorphic: there common in Gambian sleeping sickness. Several
are typical slender forms, and short, stumpy days after the development of the chancre,
forms. They are flattened and fusiform in usually within 3 to 10 days, the next stages of
shape, 14 to 33 µm in length and 1.5 to 3.5 the disease manifest.
µm in width. The body tapers anteriorly and is Both types of HAT undergo two stages:
blunt posteriorly. The centrally located nucleus early and late. During the early phase of HAT,
contains a large central karyosome. There is an called the hemolymphatic stage, the parasites
undulating membrane, and a single flagellum proliferate in the bloodstream and lymphatics.
that runs along the edge of the undulating The patient may manifest with irregular bouts
membrane and becomes free anteriorly. of fever, headache, joint and muscle pain, and
Once ingested by the intermediate malaise. Anemia, myocardial inflammation,
host, Trypanosoma brucei trypomastigotes disseminated intravascular coagulation, and
undergo several developmental changes from renal insufficiency may occur. Frequently,
trypomastigote into procyclic forms in the in Gambian trypanosomiasis, the posterior
insect’s midgut. After multiplying for 15 to 20 cervical lymph nodes are enlarged, non-tender,
days, the epimastigotes migrate to the foregut and rubbery in consistency (Winterbottom’s
into the insect’s salivary glands, where they sign). Other lymph nodes, such as axillary
mature into metacylic trypomastigotes. When and supraclavicular lymph nodes, may also be
the infected fly bites another mammalian host, involved in both types of sleeping sickness. The
these infective trypomastigotes are injected into signs and symptoms manifested within this
the new host where they multiply and mature phase are due to tissue damage, either from
in blood and connective tissue. In humans, parasitic toxins or immune complex reactions
T. brucei lives in the blood, in the reticular that target organs and RBCs. The early systemic
tissue of lymph and spleen, and the CSF. The phase lasts from 1 to 6 months.
long, slender trypomastigotes multiply by The late phase of the disease, known as
longitudinal binary fission. the meningoencephalitic stage, marks the
Though mostly transmitted through its involvement of the central nervous system.
insect vector, the disease can also be transmitted The brain and meninges become involved
122 MedICal parasItology In the phIlIppInes
as the parasites find their way into the CNS due the relative higher levels of parasitemia.
through the bloodstream. This usually occurs Serial examinations may be necessary due
3 to 10 months after initial infection in to varying levels of parasitemia. Other
Gambian infections, but can manifest after diagnostic techniques include enzyme-linked
just a few weeks in Rhodesian trypanosomiasis. immunosorbent assay, immunofluorescence,
Neurologic symptoms become evident, such indirect hemagglutination test, mini-anion
as apathy, behavioral changes, headache, and exchange centrifugation technique, and PCR.
sleep pattern changes. These may be followed CSF examination is mandatory in patients
by more severe symptoms, such as convulsions, with suspected HAT to detect CNS involvement.
tremors, speech defects, disturbances in speech Abnormal CSF findings include increase in cell
and reflexes, and even paralysis. Kerandel’s sign count, opening pressure, protein concentration,
may manifest as a deep, delayed hyperesthesia and IgM levels. The latter is considered
(delayed bilateral pain out of proportion to pathognomonic for the meningoencaphalitic
the extent of tissue injury). In the later stages, stage of the disease.
somnolence manifests, followed by a deep coma. Card agglutination test for trypanosomiasis
Death eventually follows either from the disease (CATT) detecting circulating antigens in
itself, or from intercurrent infection due to persons infected with T. brucei complex is
immunosuppression. available commercially and can be used in the
Areas of the CNS usually involved in the field setting to screen at-risk populations. This
meningoencaphalitic phase include the frontal technique provides a rapid and highly specific
lobes, pons, medulla, and perivascular areas. method of screening for HAT cases; however,
Parasites may also be seen in the CSF. Autopsy the method has low sensitivity for certain strains
of HAT patients reveals edema and numerous, of T. brucei gambiense in certain areas of West
small, and confluent hemorrhages. Africa.
Trypanosomes are able to evade the immune
Treatment
response of the host through a process called
antigenic variation. This refers to the ability Treatment of African sleeping sickness
of the trypomastigote to continuously change depends on the stage of the disease. For the first
its surface coat, composed of variant surface stage, intravenous suramin sodium for both T.
glycoproteins, so that the host’s antibodies brucei gambiense and T. brucei rhodesiense, and
cannot recognize the parasite in subsequent intramuscular pentamidine for the Gambian
recurrent waves of parasitemia. form can be used. These drugs have side effects,
which include fever, rash, renal insufficiency,
Diagnosis
muscle pain, and paresthesia for suramin; and
Diagnosis of human African trypanosomiasis tachycardia, hypotension, and hypoglycemia
depends upon the demonstration of highly for pentamidine. These drugs do not cross the
motile trypomastigotes in expressed fluid from blood-brain barrier, and so, they cannot be used
a chancre, lymph node aspirate, and CSF. for the CNS stage of the disease.
Thick and thin blood films can be stained Once CNS involvement occurs, intravenous
with Giemsa. Buffy coat concentration method melarsoprol is the drug of choice for both types
is recommended to detect parasites when of sleeping sickness. This arsenic-containing
they occur in low numbers. Examination for drug can cause fatal arsenic encephalopathy
trypomastigotes is usually done during the (usually prevented by co-administration of
hemolymphatic stage of the disease, and is more corticosteroids), and resistance to the drug
useful for the diagnosis of T. brucei rhodesiense has also been observed. A febrile episode
Chapter 2: protozoan Infections 123
arises from the kinetoplast and extends to the incubation period ranges from two weeks to
anterior tip. several months. An erythematous papule or
Promastigotes have a single free flagellum nodule, called an “oriental button,” is produced
arising from the kinetoplast at the anterior end. at the inoculation site. The lesion has raised
They measure 15 to 20 µm in length and 1.5 to edges and a central crater. During the course
3.5 µm in width. The infective promastigotes in of several weeks, the papule forms a violaceous
the proboscis of the sandfly are injected into the ulcer as it enlarges in size. The lesion may heal
host’s skin during feeding (Figure 2.26). They spontaneously after a few months, leading to
then invade the cells of the reticuloendothelial a disfiguring scar; in the case of New World
system, transform into amastogotes, and leishmaniasis, CL may progress to other forms
multiply via binary fission. When the parasitized of leishmaniasis.
cell ruptures, the amastigotes that are released
B. Diffuse Cutaneous Leishmaniasis
either invade new cells, or are taken up by
sandflies during feeding, where they transform The manifestation of DCL, also called
into promastigotes in the gut, multiply by anergic or lepromatous leishmaniasis, is
binary fission, and migrate to the foregut. characterized by a localized, non-ulcerating
Leishmania spp. may also be transmitted papule, eventually developing numerous
congenitally, through blood transfusion, by diffuse satellite lesions that affect the face and
contamination of bite wounds, and by direct extremities. This type of leishmaniasis may be
contact with contaminated specimens. initially diagnosed as lepromatous leprosy.
Pathogenesis and Clinical Manifestations C. Mucocutaneous Leishmaniasis
cost-effective drug formulation for cutaneous mostly poor and malnourished children below
and visceral leishmaniasis. 15 years old.
In India, where sodium pentavalent Leishmaniasis is primarily a disease of
antimony resistance is high, the antineoplastic poverty. It affects people living in squalid
drug miltefosine was introduced in 2002 to treat conditions, and is associated with poor housing,
VL. Miltefosine is the only oral drug currently malnutrition, a weak immune system, and
given to VL patients. lack of resources. Environmental changes such
Pentamidine is another second-line drug as deforestation, new irrigation schemes, and
for cutaneous as well as the visceral form of the urbanization are also linked to changes in the
disease. However, due to side-effects and the epidemiology of the disease. In urban areas
development of drug resistance, pentamidine where leishmaniasis occurs, there is a greater
use has been limited. For the cutaneous form of epidemic threat.
leishmaniasis, topical paromomycin has shown Visceral leishmaniasis is an important
efficacy in certain areas. opportunistic infection in AIDS patients. VL/
Combination therapy using two or more of HIV co-infection is currently a major threat in
the anti-leishmanial drugs is being studied. The the control and prevention of either disease.
presence of drug resistance especially towards Immunosuppression from HIV predisposes
the pentavalent antimonials, poor treatment to VL, while VL infection accelerates HIV
outcomes of complicated cases (such as HIV replication and progression to AIDS. VL/
coinfection), the potential for greater efficacy, HIV co-infection has been documented in
better compliance, and fewer side effects are 35 countries, with most cases coming in from
reasons why combination therapy for VL Ethiopia, southern Europe (Spain, Italy, France,
is the current consensus. Among the drug and Portugal), and Brazil.
combinations currently being used or under In the Philippines, there have been
clinical trials are: sodium stibogluconate plus imported cases of cutaneous lesions referred
paromomycin, and liposomal amphotericin B to the University of the Philippines—College
plus either miltefosine, or sodium stibogluconate. of Public Health, where amastigotes were
identified from the patients.
Epidemiology
Prevention and Control
Leishmaniasis is a global disease distributed
across 88 countries in four continents. It affects Preventive measures against leishmaniasis
more than 12 million people worldwide, and include usage of insect repellants containing
more than 350 million are at risk for the DEET and permethrin, insecticide-treated
disease. New cases of cutaneous leishmaniasis clothing, and fine-mesh bed nets. Use of fine
number between 1 to 1.5 million per year, the mesh screens and spraying of houses and
majority of which occur in Afghanistan, Brazil, buildings are also being done in certain areas.
Iran, Peru, Saudi Arabia, and Syria. American However, interval spraying predisposes to
soldiers deployed in Afghanistan and Iraq have resistance of sandflies to the insecticides, not
also demonstrated cases of CL. Mucocutaneous to mention the impact of insecticides on the
leishmaniasis occurs in Bolivia, Brazil, and environment.
Peru, while half a million new cases annually Regulation of reservoir hosts is another
of visceral leishmaniasis occur primarily in important aspect in the control and prevention
Bangladesh, Brazil, India, Nepal, and Sudan. of leishmaniasis. Insecticide-treated dog collars,
In 2009, there was a noted upsurge in VL cases mass testing of domestic dogs, and even
in Sudan compared to previous years, affecting extermination of infected dogs are current
128 MedICal parasItology In the phIlIppInes
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At present, there is no commercially Saunders Company; 1992.
available form of either active or passive Nantulya VM. TrypTect CIATT a card indirect
chemoprophylaxis against leishmaniasis. agglutination trypanosomiasis test for
However, in immunocompetent individuals, diagnosis of Trypanosoma gambiense and
a form of immunity persists after resolution T. rhodesiense infections. Trans R Soc Trop
of active lesions. Certain countries, such as Med Hyg. 1997;9(1):551–3.
endemic areas in the Middle East, have been Neva FA, Brown HW. Basic clinical parasitology.
using live parasites either from infected insect 6th ed. Connecticut: Appleton & Lange;
vectors, or in recent years, from cultures, to 1994.
inoculate inconspicuous areas (such as the Roberts LS, Janovy J. Foundations of
buttocks) so as to protect themselves from parasitology. 5th ed. Dubuque: Wm. C.
disfiguring facial lesions from future infections. Brown Publishers; 1996.
Commercial vaccines are currently under Wilson WR, Sande MA. Current diagnosis
development. and treatment in infectious diseases. USA:
Lange Medical Books, McGraw-Hill; 2001.
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