Discovered in 1909, Chagas disease, caused by the parasite Trypanosoma cruzi, continues to be an ... more Discovered in 1909, Chagas disease, caused by the parasite Trypanosoma cruzi, continues to be an important tropical disease in Latin America and Caribbean, affecting more than 7 million people. Recently, Chagas disease has also been reported in US and Europe mainly because of unscreened blood donations by immigrants from endemic areas. Current Chagas disease diagnostic relies on search for parasites on blood smears using optical microscopes, screening for serological response, xenodiagnosis, hemoculture and, more recently, polymerase chain reaction (PCR). Each of these tests has its own problems, mainly because of the changes in the immunological profile of the patients and number of circulating parasites in the blood throughout the evolution of the disease. In endemic areas, logistic issues are an additional difficulty for delivering the results to the patients, so a rapid and sensitive point of care test is desirable. In this work, we present results of an on-chip test able to detect equivalent amounts of parasites’ DNA such as those typically present in both acute and chronic phase of the disease. We developed a silicon chip-based PCR using a portable thermocycler with fluorescent detectors that has many advantages over the conventional PCR. When compared to the conventional PCR, our on-chip reaction shows similar sensitivity (between 1 and 0.1 genome equivalents), but a shorter reaction time (35 min versus 90 min). The results presented herein are the first step towards the development of a portable diagnostic test using a fast, sensitive and specific reaction for the detection of Trypanosoma cruzi.
Hepatitis Delta is a disease caused by exposure to hepatitis B (HBV) and hepatitis D (HDV) viruse... more Hepatitis Delta is a disease caused by exposure to hepatitis B (HBV) and hepatitis D (HDV) viruses, usually with a more severe clinical outcome when compared to an HBV monoinfection. To date, the real prevalence of HDV infection is underestimated and detection methods are poorly available, especially in more endemic regions. Therefore, a one-step RT-qPCR method for quantification of HDV-RNA was developed. Biological samples were selected between 2017–2023 from patients at the Ambulatório Especializado em Hepatites Virais of the Centro de Pesquisa em Medicina Tropical de Rondônia and Serviço de Assistência Especializada and underwent the test developed by this study and a second quantitative RT-qPCR assay. The slope of the initial quantitative assay was − 3.321 with an efficiency of 100.04% and amplification factor equal to 2. Analysis of the repeatability data revealed a Limit of Quantification of 5 copies/reaction and Limit of Detection (95%) of 2.83 copies per reaction. In the dia...
In this study, the chromogenic medium CHROMagar Candida™ and semi-nested PCR-based assay (sn- PCR... more In this study, the chromogenic medium CHROMagar Candida™ and semi-nested PCR-based assay (sn- PCR) were compared in relation to their capacity to identify the species of 52 clinical isolates of Candida sp. By using the chromogenic medium, 39 (75%) yeasts isolates were presumptively identified as C. albicans (n = 22), C. glabrata (n = 9), C. tropicalis (n = 5) and C. krusei (n = 3). Thirteen isolates (25%) were not distinguishable to the species level. Through the sn-PCR, that is based on the ribosomal RNA genes (5.85 – 28S) cluster amplifications, 43 (83%) isolates were identified as C. albicans (n = 24), C. glabrata (n = 11), C. tropicalis (n = 5), C. parapsilosis (n = 3) and nine isolates could not be identified to the species level. Among the 52 analyzed isolates, 34 (65.4%) were in accordance with both methods, 12 (23.1%) showed discrepant results, and 6 (11.5%) could not be identified by any of the methodologies. The results indicate that both methods are limited, but the sn-PC...
Anais do IV International Symposium on Immunobiological e VII Seminário Anual Científico e Tecnológico de Bio-Manguinhos, 2019
Introduction: The rapid and continuous emergence of epidemic arboviral diseases (i.e. Zika, Dengu... more Introduction: The rapid and continuous emergence of epidemic arboviral diseases (i.e. Zika, Dengue, Chikungunya, and Yellow Fever) presents a serious challenge to public health. The multiple Aedes-transmitted diseases with similar clinically indistinguishable febrile syndromes, underscores the need for sensitive and specific diagnostic tests that can differentiate between them. Objective: Here we present the development of a multiplex RT-qPCR lab-on-a-chip-based point-of-care-platform (POC) for differential diagnosis of Zika, Dengue 1, 2, 3 and 4 and Chikungunya viruses. Methodology: The overall system consists of a single-use disposable microfluidic chip and an instrument to operate and read out the tests. The overall protocol consists of a fully integrated and automated cartridge, containing all reagents required to carry out the assay, which consists of whole blood sample uptake, lysis, RNA extraction and purification, cDNA synthesis and qPCR amplification. Results: First, the conventional one-step RT-qPCR protocol previously developed at IBMP to detect and differentiate the viruses above mentioned was implemented on-chip using a modular approach for each step of the protocol. The optimized and validated processes and conditions compatible with the performance observed on regular laboratory instruments were used to design the integrated cartridge prototype and the respective instrument to control the complete processing on-chip. The fluidic protocol was initially validated with liquid colored solutions, and after that with buffers and lyophilized reagents. Whole blood artificially contaminated with virus-like particles was used to validate the analytical process. Reagents stability study and performance of the system with clinical samples are under evaluation to validate the platform. Conclusion: The chip can perform simultaneously a wide range of tests based on RT-qPCR distributed on its 16 reaction chambers.
2016 31st Symposium on Microelectronics Technology and Devices (SBMicro), 2016
Whooping cough is caused by the bacteria Bordetella pertussis and, despite massive global vaccina... more Whooping cough is caused by the bacteria Bordetella pertussis and, despite massive global vaccination campaigns, still poses a threat to infants, to elders well as to immunocompromised patients. In this work, we used a silicon lab-on-chip and a portable, lightweight thermocycler to develop a molecular-based detection reaction for two B. pertussis genes (IS48I and ptxS1). Each bacterial target is detected concomitantly to the detection of the human gene 18S. Thus, these duplex reactions have an internal control, which evaluates the performance of the whole system (chip temperature cycling and biological reaction). We found no significant difference between the results obtained in the portable equipment and in the standard benchtop instrument, the ABI7500. The results presented here are a first step towards a point of care test for B. pertussis diagnosis, adapted to primary health centers with low infrastructure as well as small hospitals and private health clinics.
SARS-CoV-2 has spread rapidly around the world, with Brazil currently considered an epicenter of ... more SARS-CoV-2 has spread rapidly around the world, with Brazil currently considered an epicenter of the pandemic. The Northern region has the second highest incidence coefficient, as well as the third highest mortality rate in the country. This study aimed to investigate information about the evolutionary history of epidemic spread and genetic aspects of strains isolated on the Western Amazon, in the State of Rondônia, Brazil. It was possible to detect a total of 22 mutations. Some of these alterations may possibly be related to effects on transmissibility, the fidelity of RNA replication, the ability of cancer patients to respond to infection, beyond a mutation that emerged after the introduction of SARS-CoV-2 in Rondônia. At least two events of introduction were detected, corresponding to the B.1 and B.1.1 European lineages. An introduction was observed possibly through Argentina, where strains originated that circulated in the Minas Gerais and Ceará Brazilian states, prior to Rondôn...
Journal of Clinical Images and Medical Case Reports, 2021
Background: Chronic infection with the Hepatitis Delta Virus (HDV) is often associated with sever... more Background: Chronic infection with the Hepatitis Delta Virus (HDV) is often associated with severe liver decompensation and fulminant hepatitis, but in some cases, it can present a stable clinical presentation. Objectives: This study evaluated the clinical evolution of HDV-3 carriers from an endemic region of the western Brazilian Amazon. Methods: Cross-sectional study was carried out with Anti-HDAg reagent patients, seen at an outpatient clinic specialized in viral hepatitis located in Rondônia, Brazil. Findings: A total of 19 patients, 68.4% male and 31.6% female, aged between 23 and 65 years old, were evaluated; 84.2% were clinically classified as carriers of the decompensated disease and 15.8% as carriers of the inactive disease. The results of the clinical evaluation were related to viral load; 30.8% had detectable viral RNA, and even though it was not possible to establish an association between the stage of the disease and persistent viral replication (p> 0.05), persistent...
Foodborne outbreaks caused by parasites have long been a public health issue. Among the available... more Foodborne outbreaks caused by parasites have long been a public health issue. Among the available contamination detection methods, qPCR is one of the most sensitive and specific. However, it can be cumbersome and error-prone, if used by unexperienced users. Moreover, qPCR reagents usually require freezer temperatures for transportation and storage. We present a gelified reaction format that allows the reagents to be stored at 2-8°C for up to 90 days without losing performance. The gelification process eliminates most operator mistakes during reaction setup, and renders the qPCR plates ready-to-use. The new reaction makeup was evaluated using artificially contaminated samples of distinct food matrices for sensitivity, specificity, repeatability, reproducibility, and stability. Samples consisted of cilantro leaves and raspberry fruits spiked with Cyclospora cayetanensis oocysts, as well as açai pulp and sugarcane juice tainted with Trypanosoma cruzi trypomastigotes. No significant difference between the gelified and the non-gelified qPCR was found. Our results suggest that gelifying the assay may help to achieve more reproducible qPCR data across laboratories, thus supporting surveillance actions.
The purpose of the study was to classify, through phylogenetic analyses, the main arboviruses tha... more The purpose of the study was to classify, through phylogenetic analyses, the main arboviruses that have been isolated in the metropolitan region of Porto Velho, Rondônia, Brazil. Serum samples from patients with symptoms suggesting arboviruses were collected and tested by One Step RT-qPCR for Zika, Dengue (serotypes 1–4), Chikungunya, Mayaro and Oropouche viruses. Positive samples were amplified by conventional PCR and sequenced utilizing the Sanger method. The obtained sequences were aligned, and an evolutionary analysis was carried out using Bayesian inference. A total of 308 samples were tested. Of this total, 20 had a detectable viral load for Dengue, being detected DENV1 (18/20), co-infection DENV1 and DENV2 (1/20) and DENV4 (1/20). For Dengue serotype 3 and for the CHIKV, ZIKV, MAYV and OROV viruses, no individuals with a detectable viral load were found. A total of 9 of these samples were magnified by conventional PCR for sequencing. Of these, 6 were successfully sequenced an...
Arboviruses have been emerging and reemerging worldwide, predominantly in tropical and subtropica... more Arboviruses have been emerging and reemerging worldwide, predominantly in tropical and subtropical areas. As many arbovirus infections, including dengue (DENV), Zika (ZIKV), and chikungunya (CHIKV), have similar signs and symptoms, clinical diagnosis of arbovirus infections is challenging. Therefore, reliable laboratory tests are necessary to improve the clinical management of patients with suspected arbovirus infections. Real-time reverse-transcription PCR (RT-qPCR) is among the more effective methods to distinguish these viruses. The aim of this study was to construct a unique positive external control derived from a unique plasmid using genetic engineering for specific use in RT-qPCR assays to detect Zika, dengue (1–4), and chikungunya. An external control derived from the MS2 bacteriophage was constructed using sequences from arbovirus and human genomes. Laboratories were asked to test the control in the ZDC Biomol kit, a RT-qPCR kit which is able to detect Zika, dengue serotype...
Revista do Instituto de Medicina Tropical de São Paulo
Several arboviruses have emerged and/or re-emerged in North, Central and South-American countries... more Several arboviruses have emerged and/or re-emerged in North, Central and South-American countries. Viruses from some regions of Africa and Asia, such as the Zika and Chikungunya virus have been introduced in new continents causing major public health problems. The aim of this study was to investigate the presence of RNA from Zika, Dengue and Chikungunya viruses in symptomatic patients from Rondonia, where the epidemiological profile is still little known, by one-step real-time RT-PCR. The main clinical signs and symtoms were fever (51.2%), headache (78%), chills (6.1%), pruritus (12.2%), exanthema (20.1%), arthralgia (35.3%), myalgia (26.8%) and retro-orbital pain (19.5%). Serum from 164 symptomatic patients were collected and tested for RNA of Zika, Dengue types 1 to 4 and Chikungunya viruses, in addition to antibodies against Dengue NS1 antigen. Direct microscopy for Malaria was also performed. Only ZIKV RNA was detected in 4.3% of the patients, and in the remaining 95.7% of the patients RNA for Zika, Dengue and Chikungunya viruses were not detected. This finding is intriguing as the region has been endemic for Dengue for a long time and more recently for Chikungunya virus as well. The results indicated that medical and molecular parameters obtained were suitable to describe the first report of symptomatic Zika infections in this region. Furthermore, the low rate of detection, compared to clinical signs and symptoms as the solely diagnosis criteria, suggests that molecular assays for detection of viruses or other pathogens that cause similar symptoms should be used and the corresponding diseases could be included in the compulsory notification list.
BACKGROUND Trypanosoma cruzi is an important protozoan parasite and the causative agent of Chagas... more BACKGROUND Trypanosoma cruzi is an important protozoan parasite and the causative agent of Chagas disease. A critical step in understanding T. cruzi biology is the study of cellular and molecular features exhibited during its growth curve. OBJECTIVES We aimed to acquire a global view of the gene expression profile of T. cruzi during epimastigote growth.
Reports of arboviral transmission via blood transfusion may be a cause of concern among asymptoma... more Reports of arboviral transmission via blood transfusion may be a cause of concern among asymptomatic infected donors. This study evaluated the presence of arboviruses in donated blood products during the 2016 outbreak in Vitória da Conquista (Bahia-Brazil). Serum samples (n = 676) were screened for ZIKV, CHIKV and the four DENV serotypes using a one-step reverse transcriptase-based Real-Time Polymerase Chain Reaction (RT-PCR). No samples tested positive for any of the targets, whereas positive controls performed as expected. The results suggest a low risk of arboviral transmission via transfusion of blood products in the endemic area studied during the 2016 outbreak. This article is protected by copyright. All rights reserved.
Canine visceral leishmaniasis (CVL) is a systemic disease caused by Leishmania infantum. A precis... more Canine visceral leishmaniasis (CVL) is a systemic disease caused by Leishmania infantum. A precise CVL diagnosis would allow for a faster and more specific treatment. Quantitative PCR (qPCR) is a sensitive and specific technique that can diagnose CVL and also monitor parasite load in the animal during the course of the infection or treatment. The aim of this study was to develop a ready-to-use (gelified and freezer-free) duplex qPCR for the identification of infected animals. We combined a new qPCR protocol that detects the canine 18S rRNA gene with an existing protocol for L. infantum kDNA detection, creating a duplex qPCR. This duplex method was then developed into a ready-to-use format. The performance of the duplex and singleplex reactions were compared in the traditional format (liquid and freezer-stored). Furthermore, the duplex qPCR performance was compared between the ready-to-use and traditional formats. The singleplex and new duplex qPCR exhibited the same detection limit in the traditional format (0.1 parasites/reaction). The ready-to-use format showed a detection limit of 1 parasite/reaction without affecting the reaction efficiency. The performance of the new qPCR protocol in the two formats was assessed using canine tissue samples from 82 dogs in an endemic CVL area that were previously characterized by standard serological and parasitological protocols. Splenic aspirates provided a higher rate of positivity (92.9%) followed by skin (50%) and blood (35.7%). The reported detection limits were observed for all tissues studied. Our results show that the amplification of L. infantum kDNA and canine DNA in a single tube, using either the traditional or ready-to-use format, exhibited the same diagnostic performance as amplification of the parasite kDNA alone. The detection of the host gene strengthens the qPCR results by confirming the presence and quality of DNA in the samples and the absence of polymerase inhibitors. The ready-to-use duplex qPCR format has many advantages. By joining two qPCR protocols into one, more results can be obtained in the same amount of time with reduced costs and embedded quality control. Reagents are preloaded and stored on the plate, reducing the operator's hands-on time to set up a reaction, as well as decreasing manipulation steps, which reduces the risk of mistakes or contamination. Thus, the ready-to-use duplex format turns qPCR into a robust, easy-to-use tool, which could help increase the availability of qPCR for CVL diagnosis.
Nucleic acid extraction (NAE) plays a vital role in molecular biology as the primary step for man... more Nucleic acid extraction (NAE) plays a vital role in molecular biology as the primary step for many downstream applications. Many modifications have been introduced to the original 1869 method. Modern processes are categorized into chemical or mechanical, each with peculiarities that influence their use, especially in point-of-care diagnostics (POC-Dx). POC-Dx is a new approach aiming to replace sophisticated analytical machinery with microanalytical systems, able to be used near the patient, at the point of care or point of need. Although notable efforts have been made, a simple and effective extraction method is still a major challenge for widespread use of POC-Dx. In this review, we dissected the working principle of each of the most common NAE methods, overviewing their advantages and disadvantages, as well their potential for integration in POC-Dx systems. At present, it seems difficult, if not impossible, to establish a procedure which can be universally applied to POC-Dx. We a...
Discovered in 1909, Chagas disease, caused by the parasite Trypanosoma cruzi, continues to be an ... more Discovered in 1909, Chagas disease, caused by the parasite Trypanosoma cruzi, continues to be an important tropical disease in Latin America and Caribbean, affecting more than 7 million people. Recently, Chagas disease has also been reported in US and Europe mainly because of unscreened blood donations by immigrants from endemic areas. Current Chagas disease diagnostic relies on search for parasites on blood smears using optical microscopes, screening for serological response, xenodiagnosis, hemoculture and, more recently, polymerase chain reaction (PCR). Each of these tests has its own problems, mainly because of the changes in the immunological profile of the patients and number of circulating parasites in the blood throughout the evolution of the disease. In endemic areas, logistic issues are an additional difficulty for delivering the results to the patients, so a rapid and sensitive point of care test is desirable. In this work, we present results of an on-chip test able to detect equivalent amounts of parasites’ DNA such as those typically present in both acute and chronic phase of the disease. We developed a silicon chip-based PCR using a portable thermocycler with fluorescent detectors that has many advantages over the conventional PCR. When compared to the conventional PCR, our on-chip reaction shows similar sensitivity (between 1 and 0.1 genome equivalents), but a shorter reaction time (35 min versus 90 min). The results presented herein are the first step towards the development of a portable diagnostic test using a fast, sensitive and specific reaction for the detection of Trypanosoma cruzi.
Hepatitis Delta is a disease caused by exposure to hepatitis B (HBV) and hepatitis D (HDV) viruse... more Hepatitis Delta is a disease caused by exposure to hepatitis B (HBV) and hepatitis D (HDV) viruses, usually with a more severe clinical outcome when compared to an HBV monoinfection. To date, the real prevalence of HDV infection is underestimated and detection methods are poorly available, especially in more endemic regions. Therefore, a one-step RT-qPCR method for quantification of HDV-RNA was developed. Biological samples were selected between 2017–2023 from patients at the Ambulatório Especializado em Hepatites Virais of the Centro de Pesquisa em Medicina Tropical de Rondônia and Serviço de Assistência Especializada and underwent the test developed by this study and a second quantitative RT-qPCR assay. The slope of the initial quantitative assay was − 3.321 with an efficiency of 100.04% and amplification factor equal to 2. Analysis of the repeatability data revealed a Limit of Quantification of 5 copies/reaction and Limit of Detection (95%) of 2.83 copies per reaction. In the dia...
In this study, the chromogenic medium CHROMagar Candida™ and semi-nested PCR-based assay (sn- PCR... more In this study, the chromogenic medium CHROMagar Candida™ and semi-nested PCR-based assay (sn- PCR) were compared in relation to their capacity to identify the species of 52 clinical isolates of Candida sp. By using the chromogenic medium, 39 (75%) yeasts isolates were presumptively identified as C. albicans (n = 22), C. glabrata (n = 9), C. tropicalis (n = 5) and C. krusei (n = 3). Thirteen isolates (25%) were not distinguishable to the species level. Through the sn-PCR, that is based on the ribosomal RNA genes (5.85 – 28S) cluster amplifications, 43 (83%) isolates were identified as C. albicans (n = 24), C. glabrata (n = 11), C. tropicalis (n = 5), C. parapsilosis (n = 3) and nine isolates could not be identified to the species level. Among the 52 analyzed isolates, 34 (65.4%) were in accordance with both methods, 12 (23.1%) showed discrepant results, and 6 (11.5%) could not be identified by any of the methodologies. The results indicate that both methods are limited, but the sn-PC...
Anais do IV International Symposium on Immunobiological e VII Seminário Anual Científico e Tecnológico de Bio-Manguinhos, 2019
Introduction: The rapid and continuous emergence of epidemic arboviral diseases (i.e. Zika, Dengu... more Introduction: The rapid and continuous emergence of epidemic arboviral diseases (i.e. Zika, Dengue, Chikungunya, and Yellow Fever) presents a serious challenge to public health. The multiple Aedes-transmitted diseases with similar clinically indistinguishable febrile syndromes, underscores the need for sensitive and specific diagnostic tests that can differentiate between them. Objective: Here we present the development of a multiplex RT-qPCR lab-on-a-chip-based point-of-care-platform (POC) for differential diagnosis of Zika, Dengue 1, 2, 3 and 4 and Chikungunya viruses. Methodology: The overall system consists of a single-use disposable microfluidic chip and an instrument to operate and read out the tests. The overall protocol consists of a fully integrated and automated cartridge, containing all reagents required to carry out the assay, which consists of whole blood sample uptake, lysis, RNA extraction and purification, cDNA synthesis and qPCR amplification. Results: First, the conventional one-step RT-qPCR protocol previously developed at IBMP to detect and differentiate the viruses above mentioned was implemented on-chip using a modular approach for each step of the protocol. The optimized and validated processes and conditions compatible with the performance observed on regular laboratory instruments were used to design the integrated cartridge prototype and the respective instrument to control the complete processing on-chip. The fluidic protocol was initially validated with liquid colored solutions, and after that with buffers and lyophilized reagents. Whole blood artificially contaminated with virus-like particles was used to validate the analytical process. Reagents stability study and performance of the system with clinical samples are under evaluation to validate the platform. Conclusion: The chip can perform simultaneously a wide range of tests based on RT-qPCR distributed on its 16 reaction chambers.
2016 31st Symposium on Microelectronics Technology and Devices (SBMicro), 2016
Whooping cough is caused by the bacteria Bordetella pertussis and, despite massive global vaccina... more Whooping cough is caused by the bacteria Bordetella pertussis and, despite massive global vaccination campaigns, still poses a threat to infants, to elders well as to immunocompromised patients. In this work, we used a silicon lab-on-chip and a portable, lightweight thermocycler to develop a molecular-based detection reaction for two B. pertussis genes (IS48I and ptxS1). Each bacterial target is detected concomitantly to the detection of the human gene 18S. Thus, these duplex reactions have an internal control, which evaluates the performance of the whole system (chip temperature cycling and biological reaction). We found no significant difference between the results obtained in the portable equipment and in the standard benchtop instrument, the ABI7500. The results presented here are a first step towards a point of care test for B. pertussis diagnosis, adapted to primary health centers with low infrastructure as well as small hospitals and private health clinics.
SARS-CoV-2 has spread rapidly around the world, with Brazil currently considered an epicenter of ... more SARS-CoV-2 has spread rapidly around the world, with Brazil currently considered an epicenter of the pandemic. The Northern region has the second highest incidence coefficient, as well as the third highest mortality rate in the country. This study aimed to investigate information about the evolutionary history of epidemic spread and genetic aspects of strains isolated on the Western Amazon, in the State of Rondônia, Brazil. It was possible to detect a total of 22 mutations. Some of these alterations may possibly be related to effects on transmissibility, the fidelity of RNA replication, the ability of cancer patients to respond to infection, beyond a mutation that emerged after the introduction of SARS-CoV-2 in Rondônia. At least two events of introduction were detected, corresponding to the B.1 and B.1.1 European lineages. An introduction was observed possibly through Argentina, where strains originated that circulated in the Minas Gerais and Ceará Brazilian states, prior to Rondôn...
Journal of Clinical Images and Medical Case Reports, 2021
Background: Chronic infection with the Hepatitis Delta Virus (HDV) is often associated with sever... more Background: Chronic infection with the Hepatitis Delta Virus (HDV) is often associated with severe liver decompensation and fulminant hepatitis, but in some cases, it can present a stable clinical presentation. Objectives: This study evaluated the clinical evolution of HDV-3 carriers from an endemic region of the western Brazilian Amazon. Methods: Cross-sectional study was carried out with Anti-HDAg reagent patients, seen at an outpatient clinic specialized in viral hepatitis located in Rondônia, Brazil. Findings: A total of 19 patients, 68.4% male and 31.6% female, aged between 23 and 65 years old, were evaluated; 84.2% were clinically classified as carriers of the decompensated disease and 15.8% as carriers of the inactive disease. The results of the clinical evaluation were related to viral load; 30.8% had detectable viral RNA, and even though it was not possible to establish an association between the stage of the disease and persistent viral replication (p> 0.05), persistent...
Foodborne outbreaks caused by parasites have long been a public health issue. Among the available... more Foodborne outbreaks caused by parasites have long been a public health issue. Among the available contamination detection methods, qPCR is one of the most sensitive and specific. However, it can be cumbersome and error-prone, if used by unexperienced users. Moreover, qPCR reagents usually require freezer temperatures for transportation and storage. We present a gelified reaction format that allows the reagents to be stored at 2-8°C for up to 90 days without losing performance. The gelification process eliminates most operator mistakes during reaction setup, and renders the qPCR plates ready-to-use. The new reaction makeup was evaluated using artificially contaminated samples of distinct food matrices for sensitivity, specificity, repeatability, reproducibility, and stability. Samples consisted of cilantro leaves and raspberry fruits spiked with Cyclospora cayetanensis oocysts, as well as açai pulp and sugarcane juice tainted with Trypanosoma cruzi trypomastigotes. No significant difference between the gelified and the non-gelified qPCR was found. Our results suggest that gelifying the assay may help to achieve more reproducible qPCR data across laboratories, thus supporting surveillance actions.
The purpose of the study was to classify, through phylogenetic analyses, the main arboviruses tha... more The purpose of the study was to classify, through phylogenetic analyses, the main arboviruses that have been isolated in the metropolitan region of Porto Velho, Rondônia, Brazil. Serum samples from patients with symptoms suggesting arboviruses were collected and tested by One Step RT-qPCR for Zika, Dengue (serotypes 1–4), Chikungunya, Mayaro and Oropouche viruses. Positive samples were amplified by conventional PCR and sequenced utilizing the Sanger method. The obtained sequences were aligned, and an evolutionary analysis was carried out using Bayesian inference. A total of 308 samples were tested. Of this total, 20 had a detectable viral load for Dengue, being detected DENV1 (18/20), co-infection DENV1 and DENV2 (1/20) and DENV4 (1/20). For Dengue serotype 3 and for the CHIKV, ZIKV, MAYV and OROV viruses, no individuals with a detectable viral load were found. A total of 9 of these samples were magnified by conventional PCR for sequencing. Of these, 6 were successfully sequenced an...
Arboviruses have been emerging and reemerging worldwide, predominantly in tropical and subtropica... more Arboviruses have been emerging and reemerging worldwide, predominantly in tropical and subtropical areas. As many arbovirus infections, including dengue (DENV), Zika (ZIKV), and chikungunya (CHIKV), have similar signs and symptoms, clinical diagnosis of arbovirus infections is challenging. Therefore, reliable laboratory tests are necessary to improve the clinical management of patients with suspected arbovirus infections. Real-time reverse-transcription PCR (RT-qPCR) is among the more effective methods to distinguish these viruses. The aim of this study was to construct a unique positive external control derived from a unique plasmid using genetic engineering for specific use in RT-qPCR assays to detect Zika, dengue (1–4), and chikungunya. An external control derived from the MS2 bacteriophage was constructed using sequences from arbovirus and human genomes. Laboratories were asked to test the control in the ZDC Biomol kit, a RT-qPCR kit which is able to detect Zika, dengue serotype...
Revista do Instituto de Medicina Tropical de São Paulo
Several arboviruses have emerged and/or re-emerged in North, Central and South-American countries... more Several arboviruses have emerged and/or re-emerged in North, Central and South-American countries. Viruses from some regions of Africa and Asia, such as the Zika and Chikungunya virus have been introduced in new continents causing major public health problems. The aim of this study was to investigate the presence of RNA from Zika, Dengue and Chikungunya viruses in symptomatic patients from Rondonia, where the epidemiological profile is still little known, by one-step real-time RT-PCR. The main clinical signs and symtoms were fever (51.2%), headache (78%), chills (6.1%), pruritus (12.2%), exanthema (20.1%), arthralgia (35.3%), myalgia (26.8%) and retro-orbital pain (19.5%). Serum from 164 symptomatic patients were collected and tested for RNA of Zika, Dengue types 1 to 4 and Chikungunya viruses, in addition to antibodies against Dengue NS1 antigen. Direct microscopy for Malaria was also performed. Only ZIKV RNA was detected in 4.3% of the patients, and in the remaining 95.7% of the patients RNA for Zika, Dengue and Chikungunya viruses were not detected. This finding is intriguing as the region has been endemic for Dengue for a long time and more recently for Chikungunya virus as well. The results indicated that medical and molecular parameters obtained were suitable to describe the first report of symptomatic Zika infections in this region. Furthermore, the low rate of detection, compared to clinical signs and symptoms as the solely diagnosis criteria, suggests that molecular assays for detection of viruses or other pathogens that cause similar symptoms should be used and the corresponding diseases could be included in the compulsory notification list.
BACKGROUND Trypanosoma cruzi is an important protozoan parasite and the causative agent of Chagas... more BACKGROUND Trypanosoma cruzi is an important protozoan parasite and the causative agent of Chagas disease. A critical step in understanding T. cruzi biology is the study of cellular and molecular features exhibited during its growth curve. OBJECTIVES We aimed to acquire a global view of the gene expression profile of T. cruzi during epimastigote growth.
Reports of arboviral transmission via blood transfusion may be a cause of concern among asymptoma... more Reports of arboviral transmission via blood transfusion may be a cause of concern among asymptomatic infected donors. This study evaluated the presence of arboviruses in donated blood products during the 2016 outbreak in Vitória da Conquista (Bahia-Brazil). Serum samples (n = 676) were screened for ZIKV, CHIKV and the four DENV serotypes using a one-step reverse transcriptase-based Real-Time Polymerase Chain Reaction (RT-PCR). No samples tested positive for any of the targets, whereas positive controls performed as expected. The results suggest a low risk of arboviral transmission via transfusion of blood products in the endemic area studied during the 2016 outbreak. This article is protected by copyright. All rights reserved.
Canine visceral leishmaniasis (CVL) is a systemic disease caused by Leishmania infantum. A precis... more Canine visceral leishmaniasis (CVL) is a systemic disease caused by Leishmania infantum. A precise CVL diagnosis would allow for a faster and more specific treatment. Quantitative PCR (qPCR) is a sensitive and specific technique that can diagnose CVL and also monitor parasite load in the animal during the course of the infection or treatment. The aim of this study was to develop a ready-to-use (gelified and freezer-free) duplex qPCR for the identification of infected animals. We combined a new qPCR protocol that detects the canine 18S rRNA gene with an existing protocol for L. infantum kDNA detection, creating a duplex qPCR. This duplex method was then developed into a ready-to-use format. The performance of the duplex and singleplex reactions were compared in the traditional format (liquid and freezer-stored). Furthermore, the duplex qPCR performance was compared between the ready-to-use and traditional formats. The singleplex and new duplex qPCR exhibited the same detection limit in the traditional format (0.1 parasites/reaction). The ready-to-use format showed a detection limit of 1 parasite/reaction without affecting the reaction efficiency. The performance of the new qPCR protocol in the two formats was assessed using canine tissue samples from 82 dogs in an endemic CVL area that were previously characterized by standard serological and parasitological protocols. Splenic aspirates provided a higher rate of positivity (92.9%) followed by skin (50%) and blood (35.7%). The reported detection limits were observed for all tissues studied. Our results show that the amplification of L. infantum kDNA and canine DNA in a single tube, using either the traditional or ready-to-use format, exhibited the same diagnostic performance as amplification of the parasite kDNA alone. The detection of the host gene strengthens the qPCR results by confirming the presence and quality of DNA in the samples and the absence of polymerase inhibitors. The ready-to-use duplex qPCR format has many advantages. By joining two qPCR protocols into one, more results can be obtained in the same amount of time with reduced costs and embedded quality control. Reagents are preloaded and stored on the plate, reducing the operator's hands-on time to set up a reaction, as well as decreasing manipulation steps, which reduces the risk of mistakes or contamination. Thus, the ready-to-use duplex format turns qPCR into a robust, easy-to-use tool, which could help increase the availability of qPCR for CVL diagnosis.
Nucleic acid extraction (NAE) plays a vital role in molecular biology as the primary step for man... more Nucleic acid extraction (NAE) plays a vital role in molecular biology as the primary step for many downstream applications. Many modifications have been introduced to the original 1869 method. Modern processes are categorized into chemical or mechanical, each with peculiarities that influence their use, especially in point-of-care diagnostics (POC-Dx). POC-Dx is a new approach aiming to replace sophisticated analytical machinery with microanalytical systems, able to be used near the patient, at the point of care or point of need. Although notable efforts have been made, a simple and effective extraction method is still a major challenge for widespread use of POC-Dx. In this review, we dissected the working principle of each of the most common NAE methods, overviewing their advantages and disadvantages, as well their potential for integration in POC-Dx systems. At present, it seems difficult, if not impossible, to establish a procedure which can be universally applied to POC-Dx. We a...
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Papers by Rita Rampazzo