Rudy Parrado

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 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.

Killer cell immunoglobulin-like receptor expression induction on neonatal CD8(+) T cells in vitro and following congenital infection with Trypanosoma cruzi.

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‐γ 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.

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

BackgroundThe 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 Hae III-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.

Predominance of hybrid discrete typing units of Trypanosoma cruzi in domestic Triatoma infestans from the Bolivian Gran Chaco region

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.

Leishmaniasis in Chaparé, Bolivia

To the Editor: In Bolivia, most cases of leishmaniasis are caused by Leishmania (Viannia) braziliensis (1). The parasite is transmitted zoonotically by several sandfly species and, when transmitted to humans, may cause cutaneous leishmaniasis (CL), and potentially, mucosal leishmaniasis (ML) (2). Data on the prevalence and effects of CL in Bolivia have been scarce, even though anecdotal and official reports indicate a dramatic increase in the number of human CL cases in Bolivia in the past decade (1,3). Also, although CL was originally a sylvatic disease in Bolivia, some evidence indicates that the transmission cycle has adapted to the peridomestic habitat. However, this evidence is largely based on individual case reports. No information is available on parasite species, vectors, and reservoirs in such a peridomestic transmission cycle. A preliminary study to guide future research focus and assist in immediate leishmaniasis prevention and control policy decision making is underway in Isiboro-Secure National Park, Chapare, Bolivia. Our objectives were to collect data on the prevalence of leishmaniasis in that area and evidence for peridomestic Leishmania transmission. A survey was carried out during April–July 2007 in 2 communities in Isiboro-Secure National Park, San Gabriel (16°40′31′′S and 65°37′38′′W) and San Julian (16°41′59′′S and 65°38′10′′W). These 2 communities were selected because of local knowledge of disease in the community, their moderate degree of urbanization (i.e., ≈50% of the communities’ houses are clustered around the main access road), and the accessibility of the sites to the field team. In this area, CL is transmitted from April through October. Households in both communities were visited by a team of experienced medical staff who interviewed heads of household to collect demographic data (sex, age) and diagnose the clinical condition of all present household members (presence/absence of CL lesions or scars, number of lesions, date of lesion onset) by using a standardized, pretested questionnaire. The study protocol was approved by the Ethical Committee Review Board of the World Health Organization (WHO). All patients with active cases were treated with meglumine antimoniate according to the standard protocol (2). We surveyed 133 and 52 households in San Gabriel and San Julian, which represented 86% and 80% of the total households of the respective communities; 21 and 13 households, respectively, were visited but did not participate because the owners refused or were not present. Of the 965 persons surveyed, 488 (50.6%) were male and 476 (49.3%) were female; 9 (0.9%) had active CL lesions and 62 (6.4%) had CL scars. One person had ML, and 3 had evidence of past ML; all ML patients were male. Of those with CL lesions, all had 1 lesion only. The mean lesion size was 2.3 cm (range 1.5–3 cm), and the mean lesion duration (to survey date) was 5.6 months (range 1–11 months). The clinical CL lesions were parasitologically confirmed by microscopy (n = 4) or PCR (n = 8). Parasite culture was performed on patient isolates (n = 6), and L. (V.) braziliensis was identified and characterized as the etiologic agent of these CL cases. Active lesion and scar prevalence were associated with male sex (lesions: Fisher exact test, odds ratio [OR] = 7.90 [95% confidence interval (CI) 1.01–169.09], p 15 years (lesions: Fisher exact test, OR = 0.19 [95% CI 0.01–1.46], p = 0.094; scars: Yates-corrected χ2 test, OR = 0.09 [95% CI 0.03–0.27], p<0.001) (Figure). Active lesion and scar prevalence were also associated with prolonged migration into the forest before the survey (lesions: Fisher exact test, OR = 28.10 [95% CI 3.49–184.29], p<0.01; scars: Fisher exact test, OR = 35.76 [95% CI 13.49–93.53], p<0.001). Figure Age prevalence curve of persons with lesions (white bars) and scars (black line) from cutaneous leishmaniasis, Bolivia, 2007. Whether the surveyed population is representative of the total population living in the study area is debatable. However, on the basis of current population figures (i.e., 16,000) and observed prevalence of CL, we estimate up to 1,440 CL cases in Isiboro-Secure currently. The low prevalence of active disease and scars indicates that L. (V.) braziliensis was introduced into Isiboro-Secure fairly recently, which is corroborated by the short median time since the cure of persons with CL scars (i.e., 7.5 years, range 0.4–30.5 years). Combined with the association of CL with male sex, age, and migration to the forest, we conclude that in Isiboro-Secure, most L. (V.) braziliensis transmission is sylvatic rather than peridomestic. This transmission pattern implies that prevention and control approaches that focus on the person (e.g., use of repellents, early treatment seeking) will most likely be more effective than approaches that focus on the household (e.g., indoor residual spraying with insecticides, insecticide-treated bednets). Current analyses are underway to establish CL risk factors. Additionally, a prevention and control strategy adapted to the local context is being planned to minimize the population’s exposure to sandflies, prepare health professionals for adequate (per protocol) management of cases, and minimize the likelihood that L. (V.) braziliensis transmission becomes peridomestic.

Sand fly fauna in Chapare, Bolivia: an endemic focus of Leishmania (Viannia) braziliensis.

ABSTBACT 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.

Co-Infection of Leishmania (Viannia) braziliensis and HIV: Report of a Case of Mucosal Leishmaniasis in Cochabamba, Bolivia

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.

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.

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.

Multiprimer PCR System Diagnosis of Pulmonary Tuberculosis in Cochabamba, Bolivia

Bolivia has one of the highest incidence rates of tuberculosis (TB) in the Americas. An estimated 15,000 new cases per year are detected ([1][1]), which corresponds to an incidence rate of 112 cases per 100,000 population; 1,600 deaths due to TB are reported to occur annually ([10][2]). The actual