RESUMO
BACKGROUND: The role that the genetic diversity of natural Trypanosoma cruzi populations plays in response to trypanocidal treatment of chronic Chagas disease (CD) patients remains to be understood. We analysed the genetic polymorphisms of parasite bloodstream populations infecting chronic CD patients enrolled in the E1224 clinical trial. METHODS: A total of 506 baseline and post-treatment follow-up samples from 188 patients were analysed. T. cruzi satellite DNA (satDNA) was amplified and sequenced using cruzi1/cruzi2 primers, and samples with TcI/III, TcII, TcIV or hybrid satDNA sequences were identified. Minicircle signatures were obtained after kinetoplast DNA amplification using 121/122 primers and restriction enzyme digestion. Genetic distances between baseline and post-treatment minicircle signatures were estimated using the Jaccard coefficient. RESULTS: At baseline, 74.3% TcII, 17.9% hybrid and 7.8% TcI/III satDNA sequences were found, whereas at the end of follow-up the distribution was 55.2% TcII, 35.2% hybrid and 9.5% TcI/III. The placebo arm was the treatment group with the highest variation of satDNA sequences between baseline and post-treatment follow-up. Genetic distances between baseline and post-treatment minicircle signatures were similar among all treatment arms. No association between minicircle signature variability and satDNA type distribution was found. CONCLUSIONS: Genetic variability of T. cruzi bloodstream populations during post-treatment follow-up did not differ from that observed during chronic infection in the absence of treatment, suggesting that there were no selection events of E1224-resistant parasite populations. This is the first report documenting the genetic polymorphism of natural T. cruzi populations in chronic patients in the context of clinical trials with trypanocidal drugs.
Assuntos
Doença de Chagas , Trypanosoma cruzi , Adulto , Doença de Chagas/tratamento farmacológico , Humanos , Polimorfismo Genético , Trypanosoma cruzi/genéticaRESUMO
This work evaluated a serial blood sampling procedure to enhance the sensitivity of duplex real-time quantitative PCR (qPCR) for baseline detection and quantification of parasitic loads and posttreatment identification of failure in the context of clinical trials for treatment of chronic Chagas disease, namely, DNDi-CH-E1224-001 (ClinicalTrials.gov registration no. NCT01489228) and the MSF-DNDi PCR Sampling Optimization Study (NCT01678599). Patients from Cochabamba (n = 294), Tarija (n = 257), and Aiquile (n = 220) were enrolled. Three serial blood samples were collected at each time point, and qPCR triplicates were tested for each sample. The first two samples were collected during the same day and the third one 7 days later. A patient was considered PCR positive if at least one qPCR replicate was detectable. Cumulative results of multiple samples and qPCR replicates enhanced the proportion of pretreatment sample positivity from 54.8% to 76.2%, 59.5% to 77.8%, and 73.5% to 90.2% in Cochabamba, Tarija, and Aiquile cohorts, respectively. This strategy increased the detection of treatment failure from 72.9% to 91.7%, 77.8% to 88.9%, and 42.9% to 69.1% for E1224 low-, short-, and high-dosage regimens, respectively, and from 4.6% to 15.9% and 9.5% to 32.1% for the benznidazole arm in the DNDi-CH-E1224-001 and MSF-DNDi studies, respectively. The addition of the third blood sample and third qPCR replicate in patients with nondetectable PCR results in the first two samples gave a small, non-statistically significant improvement in qPCR positivity. No change in clinical sensitivity was seen with a blood volume increase from 5 to 10 ml. The monitoring of patients treated with placebo in the DNDi-CH-E1224-001 trial revealed fluctuations in parasitic loads and occasionally nondetectable results. In conclusion, a serial sampling strategy enhanced PCR sensitivity to detecting treatment failure during follow-up and has the potential for improving recruitment capacity in Chagas disease trials, which require an initial positive qPCR result for patient admission.
Assuntos
Doença de Chagas/tratamento farmacológico , DNA de Protozoário/sangue , Monitorização Fisiológica/métodos , Carga Parasitária/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Adolescente , Adulto , Humanos , Pessoa de Meia-Idade , Nitroimidazóis/uso terapêutico , Placebos/administração & dosagem , Tiazóis/uso terapêutico , Resultado do Tratamento , Triazóis/uso terapêutico , Tripanossomicidas/uso terapêutico , Trypanosoma cruzi/efeitos dos fármacos , Adulto JovemRESUMO
Data on the distribution and abundance of Lutzomyia spp. (Diptera: Psychodidae) in Bolivia is scarce. Sand flies from an area of Leishmania (Viannia) braziliensis endemicity in the Isiboro-Secure National Park in the Department of Cochabamba were captured and identified to species. In total, 945 sand flies (789 females and 156 males) belonging to 15 species were collected from the four collection points in two study villages in 2007. With 549 (58.1%) specimens, Lutzomyia shawi was the most abundant species, followed by Lutzomyia (Trichophoromyia) sp. (22.2%), Lutzomyia llanosmartinsi (8.3%), Lutzomyia antunesi (4.3%), and Lutzomyia olmeca (2.1%). Abundance and species composition varied between rainy and dry seasons, with 99.3% of all sand flies being collected outdoors. Because of species abundance and confirmed Leishmania infection in previous entomological collections, we believe Lu. shawi is the vector of L. (Viannia) braziliensis in Isiboro-Secure National Park.
Assuntos
Biodiversidade , Leishmaniose Cutânea/transmissão , Psychodidae , Animais , Bolívia , Feminino , Humanos , Leishmania braziliensis , Masculino , Densidade Demográfica , Estações do AnoRESUMO
BACKGROUND: Current treatment for Chagas disease with the only available drugs, benznidazole or nifurtimox, has substantial limitations, including long treatment duration and safety and tolerability concerns. We aimed to evaluate the efficacy and safety of new benznidazole monotherapy regimens and combinations with fosravuconazole, in the treatment of Chagas disease. METHODS: We did a double-blind, double-dummy, phase 2, multicentre, randomised trial in three outpatient units in Bolivia. Adults aged 18-50 years with chronic indeterminate Chagas disease, confirmed by serological testing and positive qualitative PCR results, were randomly assigned (1:1:1:1:1:1:1) to one of seven treatment groups using a balanced block randomisation scheme with an interactive response system. Participants were assigned to benznidazole 300 mg daily for 8 weeks, 4 weeks, or 2 weeks, benznidazole 150 mg daily for 4 weeks, benznidazole 150 mg daily for 4 weeks plus fosravuconazole, benznidazole 300 mg once per week for 8 weeks plus fosravuconazole, or placebo, with a 12-month follow-up period. The primary endpoints were sustained parasitological clearance at 6 months, defined as persistent negative qualitative PCR results from end of treatment, and incidence and severity of treatment-emergent adverse events, serious adverse events, and adverse events leading to treatment discontinuation. Primary efficacy analysis was based on the intention-to-treat and per-protocol populations and secondary efficacy analyses on the per-protocol population. Safety analyses were based on the as-treated population. Recruitment is now closed. This trial is registered with ClinicalTrials.gov, NCT03378661. FINDINGS: Between Nov 30, 2016, and July 27, 2017, we screened 518 patients, and 210 were enrolled and randomised. 30 patients (14%) were assigned to each treatment group. All 210 randomised patients were included in the intention-to-treat population, and 190 (90%) were included in the per-protocol population. In the intention-to-treat analysis, only one (3%) of 30 patients in the placebo group had sustained parasitological clearance at 6 months of follow-up. Sustained parasitological clearance at 6 months was observed in 25 (89%) of 28 patients receiving benznidazole 300 mg daily for 8 weeks (rate difference vs placebo 86% [95% CI 73-99]), 25 (89%) of 28 receiving benznidazole 300 mg daily for 4 weeks (86% [73-99]), 24 (83%) of 29 receiving benznidazole 300 mg daily for 2 weeks (79% [64-95]), 25 (83%) of 30 receiving benznidazole 150 mg daily for 4 weeks (80% [65-95]), 23 (85%) of 28 receiving benznidazole 150 mg daily for 4 weeks plus fosravuconazole (82% [67-97]), and 24 (83%) of 29 receiving benznidazole 300 mg weekly for 8 weeks plus fosravuconazole (79% [64-95]; p<0·0001 for all group comparisons with placebo). Six patients (3%) had ten serious adverse events (leukopenia [n=3], neutropenia [n=2], pyrexia, maculopapular rash, acute cholecystitis, biliary polyp, and breast cancer), eight had 12 severe adverse events (defined as interfering substantially with the patient's usual functions; elevated alanine aminotransferase [n=4], elevated gamma-glutamyltransferase [n=2], elevated aspartate aminotransferase [n=1], neutropenia [n=3], leukopenia [n=1], and breast cancer [n=1]), and 15 (7%) had adverse events that led to treatment discontinuation (most of these were in the groups who received benznidazole 300 mg daily for 8 weeks, benznidazole 300 mg once per week for 8 weeks plus fosravuconazole, and benznidazole 150 mg daily for 4 weeks plus fosravuconazole). No adverse events leading to treatment discontinuation were observed in patients treated with benznidazole 300 mg daily for 2 weeks or placebo. There were no treatment-related deaths. INTERPRETATION: Benznidazole induced effective antiparasitic response, regardless of treatment duration, dose, or combination with fosravuconazole, and was well tolerated in adult patients with chronic Chagas disease. Shorter or reduced regimens of benznidazole could substantially improve treatment tolerability and accessibility, but further studies are needed to confirm these results. FUNDING: Drugs for Neglected Diseases initiative (DNDi). TRANSLATION: For the Spanish translation of the abstract see Supplementary Materials section.
Assuntos
Doença de Chagas/tratamento farmacológico , Nitroimidazóis/administração & dosagem , Triazóis/administração & dosagem , Adulto , Bolívia , Método Duplo-Cego , Esquema de Medicação , Quimioterapia Combinada , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/epidemiologia , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/patologia , Feminino , Humanos , Masculino , Nitroimidazóis/efeitos adversos , Carga Parasitária , Resultado do Tratamento , Triazóis/efeitos adversos , Adulto JovemRESUMO
Major histocompatibility complex (MHC) class I-specific inhibitory natural killer receptors (iNKRs) are expressed by subsets of T cells but the mechanisms inducing their expression are poorly understood, particularly for killer-cell immunoglobulin-like receptors (KIRs). The iNKRs are virtually absent from the surface of cord blood T cells but we found that KIR expression could be induced upon interleukin-2 stimulation in vitro. In addition, KIR expression was enhanced after treatment with 5-aza-2'-deoxycytidine, suggesting a role for DNA methylation. In vivo induction of KIR expression on cord blood T cells was also observed during a human congenital infection with Trypanosoma cruzi which triggers activation of fetal CD8(+) T cells. These KIR(+) T cells had an effector and effector/memory phenotype suggesting that KIR expression was consecutive to the antigenic stimulation; however, KIR was not preferentially found on parasite-specific CD8(+) T cells secreting interferon-gamma upon in vitro restimulation with live T. cruzi. These findings show that KIR expression is likely regulated by epigenetic mechanisms that occur during the maturation process of cord blood T cells. Our data provide a molecular basis for the appearance of KIRs on T cells with age and they have implications for T-cell homeostasis and the regulation of T-cell-mediated immune responses.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Doença de Chagas/congênito , Doença de Chagas/imunologia , Doenças do Recém-Nascido/imunologia , Receptores KIR/metabolismo , Trypanosoma cruzi/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Antígenos CD/metabolismo , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Complexo CD3/imunologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Metilases de Modificação do DNA/antagonistas & inibidores , Decitabina , Inibidores Enzimáticos/farmacologia , Feminino , Sangue Fetal/citologia , Sangue Fetal/imunologia , Humanos , Imunofenotipagem , Recém-Nascido , Interleucina-2/farmacologia , Subfamília C de Receptores Semelhantes a Lectina de Células NK/metabolismo , Gravidez , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Receptores KIR2DL1/metabolismo , Receptores KIR2DL2/metabolismo , Receptores KIR2DL3/metabolismo , Receptores KIR3DL1/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
Species identification is highly relevant for improved prognosis and adequate treatment of American tegumentary leishmaniasis (ATL). PCR-based methods are available for this purpose but should be simplified to improve accessibility. As a first step in this process, this paper describes a simplified protocol for collection of clinical samples. Using samples from 44 Bolivian patients with confirmed ATL, we demonstrated that hsp70 PCR-RFLP on skin scrapings collected with a tooth pick allowed identification of the parasite species with a sensitivity of 95% and specificity of 100%. Our method should greatly facilitate individual patient management and epidemiological surveillance of ATL.
Assuntos
Leishmania/classificação , Leishmaniose Cutânea/parasitologia , Animais , DNA de Protozoário/genética , Proteínas de Choque Térmico HSP70/genética , Humanos , Leishmania/genética , Leishmania/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Fragmento de Restrição , Proteínas de Protozoários/genética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Pele/parasitologia , Manejo de Espécimes/métodosRESUMO
Human-made and environmental changes constitute a major risk factor for the (re-)emergence and spread of leishmaniasis; surveillance of the transmission cycle is essential in this context. This study integrated entomological and molecular parasitological techniques to document the transmission pattern of a peridomestic focus of Leishmania in the Isiboro Secure area of Bolivia. First the spatial distribution and relative density of phlebotomine sand flies, genus Lutzomyia, were established. Lutzomyia shawi was the predominant species in domestic and peridomestic environments (90% from all collections). Second, direct application of the hsp70 PCR to sand fly extracts detected Leishmania infections in Lu. shawi only, and gave an estimated infection rate of 0.21 to 0.38%. The cleavage of the hsp70 amplicon with restriction enzymes (hsp70 PCR-RFLP) allowed identification of Le. (V.) braziliensis and Le. (V.) guyanensis in Lu. shawi captured in the same village. These two parasite species were also found in humans from the study region, supporting the co-existence of two transmission cycles involving the same sand fly species. This study demonstrated the use of PCR-RFLP in the identification of Leishmania in sand fly pools which could lead to the development of methods for screening large sand fly populations in Latin America.
Assuntos
Insetos Vetores/parasitologia , Leishmania braziliensis/isolamento & purificação , Leishmania guyanensis/isolamento & purificação , Leishmaniose Cutânea/transmissão , Psychodidae/parasitologia , Animais , Bolívia , Feminino , Humanos , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de RestriçãoRESUMO
Real-Time PCR (qPCR) testing is recommended as both a diagnostic and outcome measurement of etiological treatment in clinical practice and clinical trials of Chagas disease (CD), but no external quality assurance (EQA) program provides performance assessment of the assays in use. We implemented an EQA system to evaluate the performance of molecular biology laboratories involved in qPCR based follow-up in clinical trials of CD. An EQA program was devised for three clinical trials of CD: the E1224 (NCT01489228), a pro-drug of ravuconazole; the Sampling Study (NCT01678599), that used benznidazole, both conducted in Bolivia; and the CHAGASAZOL (NCT01162967), that tested posaconazole, conducted in Spain. Four proficiency testing panels containing negative controls and seronegative blood samples spiked with 1, 10 and 100 parasite equivalents (par. eq.)/mL of four Trypanosoma cruzi stocks, were sent from the Core Lab in Argentina to the participating laboratories located in Bolivia and Spain. Panels were analyzed simultaneously, blinded to sample allocation, at 4-month intervals. In addition, 302 random blood samples from both trials carried out in Bolivia were sent to Core Lab for retesting analysis. The analysis of proficiency testing panels gave 100% of accordance (within laboratory agreement) and concordance (between laboratory agreement) for all T. cruzi stocks at 100 par. eq./mL; whereas their values ranged from 71 to 100% and from 62 to 100% at 1 and 10 par. eq./mL, respectively, depending on the T. cruzi stock. The results obtained after twelve months of preparation confirmed the stability of blood samples in guanidine-EDTA buffer. No significant differences were found between qPCR results from Bolivian laboratory and Core Lab for retested clinical samples. This EQA program for qPCR analysis of CD patient samples may significantly contribute to ensuring the quality of laboratory data generated in clinical trials and molecular diagnostics laboratories of CD.
Assuntos
Doença de Chagas/tratamento farmacológico , Nitroimidazóis/uso terapêutico , Reação em Cadeia da Polimerase em Tempo Real/métodos , Triazóis/uso terapêutico , Tripanossomicidas/uso terapêutico , Doença de Chagas/sangue , Humanos , Monitorização Fisiológica/métodosRESUMO
Although Trypanosoma cruzi can be transmitted transplacentally and induce congenital infection, no data are available about the presence of this parasite in human amniotic fluid. We examined 8, 19, and 4 amniotic fluid samples (collected at delivery or by aspiration of gastric content of neonates) from control uninfected mothers (M-B-), infected mothers delivering uninfected newborns (M+B-), and mothers of confirmed congenital cases (M+B+), respectively. Polymerase chain reaction (PCR), using nuclear and kinetoplastic DNA primers (Tcz1-Tcz2 and 121-122), were negative for all control M-B- samples, but positive for 5 of 19 M+B- and 2 of 4 M+B+ samples. To determine the number of parasites in the positive samples, real-time PCR using S35/S36 kinetoplastic DNA was performed. Only one M+B+ sample presented a high parasitic DNA amount, whereas the other six PCR-positive samples displayed traces of T. cruzi DNA. In conclusion, the release of parasites in amniotic fluid is probably a rare event that cannot be helpful for the routine diagnosis of congenital Chagas disease.
Assuntos
Líquido Amniótico/parasitologia , Doença de Chagas/diagnóstico , Diagnóstico Pré-Natal , Trypanosoma cruzi/isolamento & purificação , Animais , Primers do DNA , Feminino , Humanos , Recém-Nascido , Gravidez , Reprodutibilidade dos TestesRESUMO
The aim of this study was to validate the method of microhematocrit tube, as a rapid method to estimate the parasitemia in blood and to associate the parasites concentration with the morbidity and mortality of new born children with congenital Chagas diseases. Our results were determined experimentally and shown that the detection limit of the microhematocrit tube method is 40 parasites/ml when at least one of the four observed tubes is positive. Besides, it was also established that when the four examined tubes are positive the parasitemia in blood reaches more than 100 parasites/ml. It is important to highlight the modification made by our laboratory in the microscopic observation of the microhematocrit tubes with respect to the methodology used by previous investigators. A positive association exists between a high number of parasites in blood and the morbi-mortality of the newly born children with congenital chagas. The results of positive association between the parasitic load and the morbility and mortality could constitute an argument to understand the possible role of the parasite in the pathology of the disease.
Assuntos
Doença de Chagas/congênito , Doença de Chagas/parasitologia , Contagem de Ovos de Parasitas/métodos , Parasitemia , Trypanosoma cruzi/isolamento & purificação , Animais , Peso ao Nascer , Bolívia/epidemiologia , Doença de Chagas/diagnóstico , Feminino , Hematócrito/instrumentação , Hematócrito/métodos , Humanos , Recém-Nascido , Masculino , Camundongos , Parasitemia/mortalidade , Sensibilidade e Especificidade , Cordão UmbilicalRESUMO
An international study was performed by 26 experienced PCR laboratories from 14 countries to assess the performance of duplex quantitative real-time PCR (qPCR) strategies on the basis of TaqMan probes for detection and quantification of parasitic loads in peripheral blood samples from Chagas disease patients. Two methods were studied: Satellite DNA (SatDNA) qPCR and kinetoplastid DNA (kDNA) qPCR. Both methods included an internal amplification control. Reportable range, analytical sensitivity, limits of detection and quantification, and precision were estimated according to international guidelines. In addition, inclusivity and exclusivity were estimated with DNA from stocks representing the different Trypanosoma cruzi discrete typing units and Trypanosoma rangeli and Leishmania spp. Both methods were challenged against 156 blood samples provided by the participant laboratories, including samples from acute and chronic patients with varied clinical findings, infected by oral route or vectorial transmission. kDNA qPCR showed better analytical sensitivity than SatDNA qPCR with limits of detection of 0.23 and 0.70 parasite equivalents/mL, respectively. Analyses of clinical samples revealed a high concordance in terms of sensitivity and parasitic loads determined by both SatDNA and kDNA qPCRs. This effort is a major step toward international validation of qPCR methods for the quantification of T. cruzi DNA in human blood samples, aiming to provide an accurate surrogate biomarker for diagnosis and treatment monitoring for patients with Chagas disease.
Assuntos
Doença de Chagas/sangue , DNA de Protozoário/análise , Reação em Cadeia da Polimerase em Tempo Real/métodos , Trypanosoma cruzi/genética , Doença de Chagas/diagnóstico , Doença de Chagas/genética , Doença de Chagas/parasitologia , DNA de Protozoário/isolamento & purificação , Humanos , Cooperação Internacional , Ensaio de Proficiência Laboratorial , Tipagem Molecular , Parasitemia/sangue , Parasitemia/diagnóstico , Parasitemia/genética , Sensibilidade e Especificidade , Trypanosoma cruzi/isolamento & purificaçãoAssuntos
Doenças Transmissíveis Emergentes/epidemiologia , Leishmaniose/epidemiologia , Adolescente , Adulto , Idoso , Bolívia/epidemiologia , Criança , Pré-Escolar , Doenças Transmissíveis Emergentes/transmissão , Métodos Epidemiológicos , Feminino , Humanos , Leishmaniose/transmissão , Masculino , Pessoa de Meia-Idade , Adulto JovemAssuntos
Técnicas Bacteriológicas/métodos , Mycobacterium tuberculosis/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Tuberculose Pulmonar/diagnóstico , Bolívia , Humanos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crescimento & desenvolvimento , Sensibilidade e Especificidade , Escarro/microbiologia , Tuberculose Pulmonar/microbiologiaRESUMO
In the Gran Chaco region the reinfestation by Triatoma infestans remains a major problem for control of Chagas disease. Trypanosoma cruzi the agent of the illness presents a broad genetic intraspecific variability which is poorly documented in the Bolivian Gran Chaco. This work presents the identification of the discrete typing units (DTUs) currently recognized for T. cruzi in T. infestans populations collected before and after residual insecticide spraying in four villages in this region. Before spraying, of 84 samples, the frequencies of the DTUs identified by using the multiplex PCR based on the non transcribed spacer of the mini-exon gene (MMPCR) were 0.21 for TcI, 0.70 for TcII/TcV/TcVI, and 0.17 for TcIII/TcIV and no significant difference was observed after spraying (76 samples). Moreover 13% of the total sample corresponds to T. infestans specimens with mixed infection of DTUs of which three were TcII/TcV/TcVI with TcIII/TcIV. The partial sequences of T. cruzi Gpi gene obtained from 14 PCR products agree the MMPCR DTU identification and allowed to precise the occurrence of TcIII, TcII and hybrid TcV/TcVI stocks which were not discriminated by the MMPCR. Given the high prevalence of hybrid stocks, the authors ask whether the recombination event at the origin of hybrids would have taken place in the Gran Chaco where the putative parents are also present.
Assuntos
Triatoma/parasitologia , Trypanosoma cruzi/genética , Animais , Bolívia/epidemiologia , Doença de Chagas/epidemiologia , Doença de Chagas/parasitologia , DNA de Protozoário , Feminino , Genótipo , Glucose-6-Fosfato Isomerase/genética , Humanos , Controle de Insetos , Masculino , Filogenia , Proteínas de Protozoários/genéticaRESUMO
BACKGROUND: The analytical validation of sensitive, accurate and standardized Real-Time PCR methods for Trypanosoma cruzi quantification is crucial to provide a reliable laboratory tool for diagnosis of recent infections as well as for monitoring treatment efficacy. METHODS/PRINCIPAL FINDINGS: We have standardized and validated a multiplex Real-Time quantitative PCR assay (qPCR) based on TaqMan technology, aiming to quantify T. cruzi satellite DNA as well as an internal amplification control (IAC) in a single-tube reaction. IAC amplification allows rule out false negative PCR results due to inhibitory substances or loss of DNA during sample processing. The assay has a limit of detection (LOD) of 0.70 parasite equivalents/mL and a limit of quantification (LOQ) of 1.53 parasite equivalents/mL starting from non-boiled Guanidine EDTA blood spiked with T. cruzi CL-Brener stock. The method was evaluated with blood samples collected from Chagas disease patients experiencing different clinical stages and epidemiological scenarios: 1- Sixteen Venezuelan patients from an outbreak of oral transmission, 2- Sixty three Bolivian patients suffering chronic Chagas disease, 3- Thirty four Argentinean cases with chronic Chagas disease, 4- Twenty seven newborns to seropositive mothers, 5- A seronegative receptor who got infected after transplantation with a cadaveric kidney explanted from an infected subject. CONCLUSIONS/SIGNIFICANCE: The performing parameters of this assay encourage its application to early assessment of T. cruzi infection in cases in which serological methods are not informative, such as recent infections by oral contamination or congenital transmission or after transplantation with organs from seropositive donors, as well as for monitoring Chagas disease patients under etiological treatment.
Assuntos
Doença de Chagas/parasitologia , DNA Satélite/genética , Reação em Cadeia da Polimerase Multiplex/métodos , Carga Parasitária/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Trypanosoma cruzi/genética , Adulto , Criança , Pré-Escolar , Feminino , Humanos , Recém-Nascido , Masculino , Reação em Cadeia da Polimerase Multiplex/normas , Carga Parasitária/normas , Gravidez , Reação em Cadeia da Polimerase em Tempo Real/normas , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: The Leishmaniases are a group of clinically diverse diseases caused by parasites of the genus Leishmania. To distinguish between species is crucial for correct diagnosis and prognosis as well as for treatment decisions. Recently, sequencing of the HSP70 coding region has been applied in phylogenetic studies and for identifying of Leishmania species with excellent results. METHODS: In the present study, we analyzed the 3'-untranslated region (UTR) of Leishmania HSP70-type I gene from 24 strains representing eleven Leishmania species in the belief that this non-coding region would have a better discriminatory capacity for species typing than coding regions. RESULTS: It was observed that there was a remarkable degree of sequence conservation in this region, even between species of the subgenus Leishmania and Viannia. In addition, the presence of many microsatellites was a common feature of the 3'-UTR of HSP70-I genes in the Leishmania genus. Finally, we constructed dendrograms based on global sequence alignments of the analyzed Leishmania species and strains, the results indicated that this particular region of HSP70 genes might be useful for species (or species complex) typing, improving for particular species the discrimination capacity of phylogenetic trees based on HSP70 coding sequences. Given the large size variation of the analyzed region between the Leishmania and Viannia subgenera, direct visualization of the PCR amplification product would allow discrimination between subgenera, and a HaeIII-PCR-RFLP analysis might be used for differentiating some species within each subgenera. CONCLUSIONS: Sequence and phylogenetic analyses indicated that this region, which is readily amplified using a single pair of primers from both Old and New World Leishmania species, might be useful as a molecular marker for species discrimination.
Assuntos
Regiões 3' não Traduzidas/genética , Proteínas de Choque Térmico HSP70/metabolismo , Leishmania/genética , Leishmania/metabolismo , Regulação da Expressão Gênica/fisiologia , Marcadores Genéticos , Proteínas de Choque Térmico HSP70/genética , Filogenia , Especificidade da EspécieRESUMO
Data on Leishmania spp. infection in dogs in Bolivia is scarce. Dogs from an area where 90% of human cutaneous leishmaniasis (CL) cases are due to Leishmania (Viannia) braziliensis were screened for Leishmania infection using established enzyme-linked immunosorbent antibody test (ELISA) protocols. Although none of the 51 dogs surveyed had clinical lesions indicative of CL, 6 out of 51 (11.8%) sampled dogs tested positive by ELISA.
Assuntos
Doenças do Cão/parasitologia , Leishmaniose/veterinária , Animais , Anticorpos Antiprotozoários/sangue , Bolívia/epidemiologia , Doenças do Cão/epidemiologia , Cães , Ensaio de Imunoadsorção Enzimática/veterinária , Feminino , Leishmaniose/sangue , Leishmaniose/epidemiologia , Masculino , PrevalênciaRESUMO
We describe the first case of Leishmania/HIV co-infection reported in Bolivia. Initially hospitalized with a diagnosis of pneumonia and bronchitis, the patient had numerous cutaneous and mucosal lesions caused by Leishmania (Viannia) braziliensis. The patient was also diagnosed as severely immunocompromised because of HIV infection.
Assuntos
Infecções por HIV/complicações , Leishmania braziliensis , Leishmaniose Mucocutânea/complicações , Leishmaniose Mucocutânea/patologia , Adulto , Animais , Anti-Infecciosos/uso terapêutico , Bolívia/epidemiologia , Infecções por HIV/epidemiologia , Humanos , Leishmaniose Mucocutânea/epidemiologia , MasculinoRESUMO
The leishmaniases are protozoan, zoonotic diseases transmitted to human and other mammal hosts by the bite of phlebotomine sandflies. Bolivia has the highest incidence of cutaneous leishmaniasis (CL) in Latin America (LA), with 33 cases per 100,000 population reported in 2006. CL is endemic in seven of the country's nine administrative departments. Visceral leishmaniasis (VL) is comparatively rare and is restricted to one single focus. Most CL cases are caused by Leishmania (Viannia) braziliensis (85% cases); VL is caused by L. (L.) infantum. Seven sandfly species are incriminated as vectors and Leishmania infections have been detected in several non-human mammal hosts. Transmission is associated with forest-related activities, but recently, cases of autochthonous, urban transmission were reported. Because most cases are caused by L. (V.) braziliensis, Bolivia reports the greatest ratio (i.e., up to 20% of all cases) of mucosal leishmaniasis to localized CL cases in LA. Per national guidelines, both CL and VL cases are microscopically diagnosed and treated with pentavalent antimony.