Leíla I. A. R. C. Coelho

Comparison between Quantitative Nucleic Acid Sequence-Based Amplification, Real-Time Reverse Transcriptase PCR, and Real-Time PCR for Quantification of Leishmania Parasites

ABSTRACT DNA or RNA amplification methods for detection of Leishmania parasites have advantages regarding sensitivity and potential quantitative characteristics in comparison with conventional diagnostic methods but are often still not routinely applied. However, the use and application of molecular assays are increasing, but comparative studies on the performance of these different assays are lacking. The aim of this study was to compare three molecular assays for detection and quantification of Leishmania parasites in serial dilutions of parasites and in skin biopsies collected from cutaneous leishmaniasis (CL) patients in Manaus, Brazil. A serial dilution of promastigotes spiked in blood was tested in triplicate in three different runs by quantitative nucleic acid sequence-based amplification (QT-NASBA), quantitative real-time reverse transcriptase PCR (qRT-PCR), and quantitative real-time PCR (qPCR). In addition, the costs, durations, and numbers of handling steps were compared, and 84 skin biopsies from patients with suspected CL were tested. Both QT-NASBA and qRT-PCR had a detection limit of 100 parasites/ml of blood, while qPCR detected 1,000 parasites/ml. QT-NASBA had the lowest range of intra-assay variation (coefficients of variation [CV], 0.5% to 3.3%), while qPCR had the lowest range of interassay variation (CV, 0.4% to 5.3%). Furthermore, qRT-PCR had higher r2 values and amplification efficiencies than qPCR, and qPCR and qRT-PCR had faster procedures than QT-NASBA. All assays performed equally well with patient samples, with significant correlations between parasite counts. Overall, qRT-PCR is preferred over QT-NASBA and qPCR as the most optimal diagnostic assay for quantification of Leishmania parasites, since it was highly sensitive and reproducible and the procedure was relatively fast.

Mucosal Leishmaniasis Caused by Leishmania (Viannia) braziliensis and Leishmania (Viannia) guyanensis in the Brazilian Amazon

Background Leishmania (Viannia) braziliensis is a parasite recognized as the most important etiologic agent of mucosal leishmaniasis (ML) in the New World. In Amazonia, seven different species of Leishmania, etiologic agents of human Cutaneous Leishmaniasis, have been described. Isolated cases of ML have been described for several different species of Leishmania: L. (V.) panamensis, L. (V.) guyanensis and L. (L.) amazonensis. Methodology Leishmania species were characterized by polymerase chain reaction (PCR) of tissues taken from mucosal biopsies of Amazonian patients who were diagnosed with ML and treated at the Tropical Medicine Foundation of Amazonas (FMTAM) in Manaus, Amazonas state, Brazil. Samples were obtained retrospectively from the pathology laboratory and prospectively from patients attending the aforementioned tertiary care unit. Results This study reports 46 cases of ML along with their geographical origin, 30 cases caused by L. (V.) braziliensis and 16 cases by L. (V.) guyanensis. This is the first record of ML cases in 16 different municipalities in the state of Amazonas and of simultaneous detection of both species in 4 municipalities of this state. It is also the first record of ML caused by L. (V.) guyanensis in the states of Pará, Acre, and Rondônia and cases of ML caused by L. (V.) braziliensis in the state of Rondônia. Conclusions/Significance L. (V.) braziliensis is the predominant species that causes ML in the Amazon region. However, contrary to previous studies, L. (V.) guyanensis is also a significant causative agent of ML within the region. The clinical and epidemiological expression of ML in the Manaus region is similar to the rest of the country, although the majority of ML cases are found south of the Amazon River.

American Tegumentary Leishmaniasis and HIV-AIDS Association in a Tertiary Care Center in the Brazilian Amazon

American tegumentary leishmaniasis (ATL) and human immunodeficiency virus (HIV) are both common infectious diseases in the Brazilian Amazon with overlapping expansion areas, which leads to the occurrence of Leishmania/HIV coinfection. Most ATL/HIV-acquired immunodeficiency syndrome (AIDS) association cases have been reported from areas where Leishmania (Viannia) braziliensis is the main pathogen; this finding is in contrast with the Amazon region, where L. (V.) guyanensis is the most implicated agent, implying distinct clinical and therapeutic aspects. We describe 15 cases of ATL/HIV coinfection treated in a tertiary care center in the Brazilian Amazon between 1999 and 2008. Thirteen patients presented with diverse clinical manifestations of cutaneous leishmaniasis, and four of them had disseminated forms; two patients presented with mucosal leishmaniasis (ML). Seven patients required more than one course of treatment. The particularities of ATL/HIV-AIDS association in L. (V.) guyanensis-endemic areas require efforts for an increased understanding of its burden and subsequent improvements in case management.

Trypanosoma cruzi strain TcI is associated with chronic Chagas disease in the Brazilian Amazon.

BackgroundChagas disease in the Amazon region is considered an emerging anthropozoonosis with a predominance of the discrete typing units (DTUs) TcI and TcIV. These DTUs are responsible for cases of acute disease associated with oral transmission. Chronic disease cases have been detected through serological surveys. However, the mode of transmission could not be determined, or any association of chronic disease with a specific T. cruzi DTU’s. The aim of this study was to characterize Trypanosoma cruzi in patients with chronic Chagas disease in the State of Amazonas, Brazil.MethodsBlood culture and xenodiagnosis were performed in 36 patients with positive serology for Chagas disease who participated in a serological survey performed in urban and rural areas of Manaus, Amazonas. DNA samples were extracted from the feces of triatomines used for xenodiagnosis, and the nontranscribed spacer of the mini-exon gene and the mitochondrial gene cytochrome oxidase subunit II (COII) were amplified by PCR and sequenced.ResultsBlood culture and xenodiagnosis were negative in 100% of samples; however, molecular techniques revealed that in 13 out of 36 (36%) fecal samples from xenodiagnosis, T. cruzi was characterized as the DTU TcI, and different haplotypes were identified within the same DTU.ConclusionThe DTU TcI, which is mainly associated with acute cases of Chagas disease in the Amazon region, is also responsible for chronic infection in patients from a region in the State of Amazonas.

Serological survey for Chagas disease in the rural areas of Manaus, Coari, and Tefé in the Western Brazilian Amazon

INTRODUCTION Deforestation, uncontrolled forest, human population migration from endemic areas, and the large number of reservoirs and wild vectors naturally infected by Trypanosoma cruzi promote the endemicity of Chagas disease in the Amazon region. METHODS We conducted an initial serological survey (ELISA) in a sample of 1,263 persons; 1,095 (86.7%) were natives of the State of Amazonas, 666 (52.7%) were male, and 948 (75.1%) were over 20 years old. Serum samples that were found to be reactive, indeterminate, or inconclusive by indirect immunofluorescence (IFI) or positive with low titer by IFA were tested by Western blot (WB). Serologically confirmed patients (WB) were evaluated in terms of epidemiological, clinical, ECG, and echocardiography characteristics. RESULTS Fifteen patients had serologically confirmed T. cruzi infection, and 12 of them were autochthonous to the state of Amazonas, for an overall seroprevalence of 1.2% and 0.9% for the state of Amazonas. Five of the 15 cases were males, and the average age was 47 years old; most were farmers with low education. One patient who was not autochthonous, having originated from Alagoas, showed right bundle branch block, bundle branch block, and anterosuperior left ventricular systolic dysfunction with an ejection fraction of 54%. CONCLUSIONS The results of this study ratify the importance of monitoring CD cases in Amazonia, particularly in the state of Amazonas.

Cardiopatia chagásica crônica na Amazônia: uma etiologia a ser lembrada

This study assessed the frequency of chronic chagasic cardiopathy (CCC) in 37 autochthonus patients from Amazon region with left ventricular systolic dysfunction of undefined etiology. Three cases were diagnosed in the studied sample, with an 8.1% frequency.Este estudio evaluo la frecuencia de cardiopatia chagasica cronica (CCC) en 37 pacientes autoctonos de la Amazonia con disfuncion sistolica ventricular izquierda sin etiologia definida. Se diagnosticaron tres casos con frecuencia del 8,1% en el grupo estudiado.

Chronic chagasic cardiopathy in Amazon region: an etiology to remember

This study assessed the frequency of chronic chagasic cardiopathy (CCC) in 37 autochthonus patients from Amazon region with left ventricular systolic dysfunction of undefined etiology. Three cases were diagnosed in the studied sample, with an 8.1% frequency.Este estudio evaluo la frecuencia de cardiopatia chagasica cronica (CCC) en 37 pacientes autoctonos de la Amazonia con disfuncion sistolica ventricular izquierda sin etiologia definida. Se diagnosticaron tres casos con frecuencia del 8,1% en el grupo estudiado.

Mucosal Leishmaniasis - Clinical and Epidemiological Aspects of 234 Cases Treated in a Tertiary Reference Center in the Brazilian Amazon

Presented here is a retrospective study including patients of the Brazilian Amazon region who were diagnosed with mucosal leishmaniasis (ML) and treated in a tertiary referral center between July 1992 to December 2008. In total, 234 patients fulfilled the inclusion criteria. There was a clear predominance of males, comprising 193 (82.5%) cases, and the mean age was 48 years. No fewer than 186 (79.5%) patients had undertaken activities that are considered to place one at risk of acquiring American Tegumentary Leishmaniasis (ATL) – agriculture, animal raising, or mineral extraction. Furthermore, 193 (82.5%) presented with a prior history of cutaneous leishmaniasis (CL); the average time between the onset of CL and the first symptoms of ML in 138 cases was 16.3 years. Finally, 156 cases (66.7%) were located in municipalities that lie on the south bank of the Amazon River, suggesting that Leishmania (Viannia) braziliensis was the likely etiological agent in these cases.

Cross-Sectional Serological Survey of Human Fascioliasis in Canutama Municipality in Western Amazon, Brazil

Background Fascioliasis is an important parasitic disease. In the northern region of Brazil, a human parasite infection has been reported through a coprological survey. Eggs of Fasciola hepatica were found in fecal samples of 11 individuals. Knowledge of the infection in animals or the presence of snails is necessary to address the possibility of the parasite cycle occurrence in that region. The aim of this study was to describe the transmission of human fascioliasis in Canutama, Amazonas, in Western Amazonia, Brazil. Methods Serological (ELISA and Western Blot, WB) and parasitological analyses were carried out in humans. In addition, the presence of the intermediate snail host within the community was examined. Results A total of 434 human samples were included in the study, of which 36 (8.3%) were reactive by ELISA and 8 (1.8%) were reactive by WB. Fasciola hepatica eggs were found in one human sample. The occurrence of the intermediated host was recorded and 31/43 specimens were identified as Lymnaea columella. Conclusion. Canutama constitutes a focus of transmission of human fascioliasis. This study describes the first serological survey for human fascioliasis, as well as its simultaneous occurrence in human hosts and possible intermediates performed in northern Brazil.

Cardiopatía chagásica crónica en la Amazonía: una etiología que recordar

This study assessed the frequency of chronic chagasic cardiopathy (CCC) in 37 autochthonus patients from Amazon region with left ventricular systolic dysfunction of undefined etiology. Three cases were diagnosed in the studied sample, with an 8.1% frequency.Este estudio evaluo la frecuencia de cardiopatia chagasica cronica (CCC) en 37 pacientes autoctonos de la Amazonia con disfuncion sistolica ventricular izquierda sin etiologia definida. Se diagnosticaron tres casos con frecuencia del 8,1% en el grupo estudiado.