Sequence analysis of the 3-untranslated region of HSP70 (type I) genes in the genus Leishmania: Its usefulness as a molecular marker for species identification

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, evenbetween 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 hylogenetic 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. © 2012 Requena et al.; licensee BioMed Central Ltd.Ministerio de Ciencia y Tecnología (BFU2009-08986); Fondo de Investigaciones Sanitarias (ISCIII-RETIC RD06/0021/0008-FEDER and ISCIII-RETIC RD06/0021/0009-FEDER); Agencia Española de Cooperación Internacional para el Desarrollo (AECID, A/024740/09); Fundación Ramón ArecesPeer Reviewe

Usefulness of Serial Blood Sampling and PCR Replicates for Treatment Monitoring of Patients with Chronic Chagas Disease

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-CHE1224-001 (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

New regimens of benznidazole monotherapy and in combination with fosravuconazole for treatment of Chagas disease (BENDITA): a phase 2, double-blind, randomised trial

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.Fil: Torrico, Faustino. Fundación Ciencia y Estudios Aplicados para el Desarrollo en Salud y Medio Ambiente; Bolivia. Universidad Mayor de San Simón; BoliviaFil: Gascón, Joaquim. Instituto de Salud Global de Barcelona; España. Universidad de Barcelona; EspañaFil: Barreira, Fabiana. DNDi Latin America; BrasilFil: Blum, Bethania. DNDi Latin America; BrasilFil: Almeida, Igor C. University of Texas at El Paso; Estados UnidosFil: Alonso Vega, Cristina. DNDi Latin America; Brasil. Instituto de Salud Global de Barcelona; EspañaFil: Barboza, Tayná. DNDi Latin America; BrasilFil: Bilbe, Graeme. Drugs For Neglected Diseases Initiative; SuizaFil: Correia, Erika. DNDi Latin America; BrasilFil: Garcia, Wilson. Universidad Mayor de San Simón; Bolivia. Fundación Ciencia y Estudios Aplicados para el Desarrollo en Salud y Medio Ambiente ; BoliviaFil: Ortiz, Lourdes. Universidad Autónoma Juan Misael Saracho; Bolivia. Fundación Ciencia y Estudios Aplicados para el Desarrollo en Salud y Medio Ambiente; BoliviaFil: Parrado, Rudy. Universidad Mayor de San Simón; BoliviaFil: Ramirez Gomez, Juan Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud "Dr. C. G. Malbrán". Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben"; ArgentinaFil: Ribeiro, Isabela. Drugs For Neglected Diseases Initiative; SuizaFil: Strub Wourgaft, Nathalie. Drugs For Neglected Diseases Initiative; SuizaFil: Vaillant, Michel. Luxembourg Institute Of Health; LuxemburgoFil: Sosa-Estani, Sergio Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Epidemiología y Salud Pública. Instituto de Efectividad Clínica y Sanitaria. Centro de Investigaciones en Epidemiología y Salud Pública; Argentina. DNDi Latin America; Brasi

First external quality assurance program for bloodstream Real-Time PCR monitoring of treatment response in clinical trials of Chagas disease

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

Analytical Performance of a Multiplex Real-Time PCR Assay Using TaqMan Probes for Quantification of Trypanosoma cruzi Satellite DNA in Blood Samples

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 CLBrener 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.Fil: Duffy, Tomas. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones En Ingeniería Genética y Biología Molecular; ArgentinaFil: Cura, Carolina Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; ArgentinaFil: Ramírez, Juan C.. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; ArgentinaFil: Abate, Teresa. Universidad Central de Venezuela. Instituto de Medicina Tropical; VenezuelaFil: Cayo, Nelly M.. Universidad Nacional de Jujuy. Instituto de Biologia de la Altura; ArgentinaFil: Parrado, Rudy. Universidad San Simón; BoliviaFil: Diaz Bello, Zoraida. Universidad Central de Venezuela. Instituto de Medicina Tropical; VenezuelaFil: Velazquez, Elsa Beatriz. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud. Instituto Nacional de Parasitología; ArgentinaFil: Muñoz Calderón, Arturo. Universidad Central de Venezuela. Instituto de Medicina Tropical; VenezuelaFil: Juiz, Natalia Anahí. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; ArgentinaFil: Basile, Joaquín. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; ArgentinaFil: Garcia, Lineth. Universidad San Simón; BoliviaFil: Riarte, Adelina. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud. Instituto Nacional de Parasitología; ArgentinaFil: Nasser, Julio Rubén. Universidad Nacional de Salta. Facultad de Ciencias Naturales; ArgentinaFil: Ocampo, Susana B.. Universidad Nacional de Jujuy. Instituto de Biologia de la Altura; ArgentinaFil: Yadon, Zaida E.. Pan-American Health Organization; Estados UnidosFil: Torrico, Faustino. Universidad San Simón; BoliviaFil: Alarcón de Noya, Belkisyole. Universidad Central de Venezuela. Instituto de Medicina Tropical; VenezuelaFil: Ribeiro, Isabela. Drugs and Neglected Diseases Initiative; SuizaFil: Schijman, Alejandro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; Argentin

Leishmaniases in Bolivia: comprehensive review and current status.

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

Prevalence of Leishmania spp. infection in domestic dogs in Chapare, Bolivia.

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

Sequence analysis of the 3'-untranslated region of HSP70 (type I) genes in the genus Leishmania: its usefulness as a molecular marker for species identification

AbstractBackgroundThe 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.MethodsIn 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.ResultsIt 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.ConclusionsSequence 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.This work was supported by grants from the Ministerio de Ciencia y Tecnología (BFU2009-08986), the Fondo de Investigaciones Sanitarias (ISCIII-RETIC RD06/0021/0008-FEDER and ISCIII-RETIC RD06/0021/0009-FEDER), and Agencia Española de Cooperación Internacional para el Desarrollo (AECID, A/024740/09). Also, an institutional grant from Fundación Ramón Areces is acknowledged.Peer Reviewe