Browsing by Autor "Rudy Parrado"

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American tegumentary leishmaniasis: direct species identification of Leishmania in non-invasive clinical samples
(Oxford University Press, 2006) Ana Lineth García; Rudy Parrado; Simonne De Doncker; Hernán Bermúdez; Jean‐Claude Dujardin
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.
AMNIOTIC FLUID IS NOT USEFUL FOR DIAGNOSIS OF CONGENITAL TRYPANOSOMA CRUZI INFECTION
(American Society of Tropical Medicine and Hygiene, 2006) Myrna Virreira; Sabrina Sales Martínez; Cristina Alonso‐Vega; Faustino Torrico; Marco Solano; Mary Cruz Torrico; Rudy Parrado; Carine Truyens; Yves Carlier; Michal Svoboda
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.
Analytical Performance of a Multiplex Real-Time PCR Assay Using TaqMan Probes for Quantification of Trypanosoma cruzi Satellite DNA in Blood Samples
(Public Library of Science, 2013) Tomás Duffy; Carolina Cura; Juan Carlos Ramı́rez; Teresa Abate; Nelly M. Cayo; Rudy Parrado; Zoraida Díaz Bello; Elsa Velázquez; Arturo Muñoz-Calderón; Natalia Juiz
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.
Caracterización molecular de virus respiratorios en población pediátrica del Hospital Albina Patiño en Cochabamba, Bolivia
(2024) Rudy Parrado; Nattaly Grecia Torrico Villarroel
Las infecciones respiratorias agudas son enfermedades frecuentes en todo el mundo, la mayoría producidas por virus. La población más afectada son niños menores de cinco años, quienes tienen mayor riesgo de padecer enfermedades respiratorias graves asociadas a mortalidad. En Cochabamba no existía información publicada sobre circulación de virus respiratorios antes de la pandemia por SARS-CoV-2/COVID-19. Objetivos: detectar y caracterizar virus respiratorios en pacientes pediátricos internados con infecciones respiratorias en el Hospital Pediátrico "Albina Patiño". Métodos: el período de estudio fue entre septiembre de 2017 y agosto de 2018, habiéndose incluido una población de 202 pacientes menores de cinco años. Se utilizó la reacción en cadena de la polimerasa en tiempo real (qPCR) multiplex para detectar y caracterizar los virus respiratorios relacionados con infección respiratoria. Resultados: El 61,4% de las muestras analizadas dieron positivo a virus respiratorios. Los resultados incluyeron: Adenovirus 1,5 %, Metapneumovirus 3,5 %, Influenza virus 8 %, Parainfluenza virus 9,9 % y VSR 35,7 %. El virus más común responsable de las infecciones respiratorias fue el VSR. El grupo de niños infectados más afectado son los menores de dos años. Las infecciones virales alcanzaron el rango epidémico completo entre mayo y julio de 2018. Conclusiones: Se logró detectar y caracterizar virus respiratorios en pacientes pediátricos, se evidencia un patrón de circulación de estos virus respiratorios específico para cada población, incluso antes de la circulación de SARS-CoV-2. Es necesario tener en cuenta que los países vecinos con condiciones similares de climatología pueden tener un patrón de circulación diferente.
Co-Infection of Leishmania (Viannia) braziliensis and HIV: Report of a Case of Mucosal Leishmaniasis in Cochabamba, Bolivia
(American Society of Tropical Medicine and Hygiene, 2009) Faustino Torrico; Rudy Parrado; Rosario Castro; Carla Jimena Marquez; Mary Cruz Torrico; Marco Solano; Richard Reithinger; Ana Lineth García
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.
Epidemiological monitoring of American tegumentary leishmaniasis: molecular characterization of a peridomestic transmission cycle in the Amazonian lowlands of Bolivia
(Oxford University Press, 2007) Ana Lineth García; Tatiana Tellez; Rudy Parrado; Ernesto Rojas; Hernán Bermúdez; Jean‐Claude Dujardin
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.
Erratum—Vol. 15, No. 2
(Centers for Disease Control and Prevention, 2009) Ernesto Rojas; Rudy Parrado; Raúl Delgado; Richard Reithinger; Ana Cristina Bicharra García; Editor JAS; A Garca; R Parrado; E Rojas; R Delgado
[Estimation of the parasitemia in Trypanosoma cruzi human infection: high parasitemias are associated with severe and fatal congenital Chagas disease].
(National Institutes of Health, 2005) Mary Cruz Torrico; Marco Solano; José Miguel Guzmán; Rudy Parrado; Eduardo Suárez; Cristina Alonzo-Vega; Carine Truyens; Yves Carlier; Faustino Torrico
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.
First external quality assurance program for bloodstream Real-Time PCR monitoring of treatment response in clinical trials of Chagas disease
(Public Library of Science, 2017) Juan Carlos Ramı́rez; Rudy Parrado; Elena Sulleiro; Anabelle de la Barra; Marcelo Rodríguez; Sandro Villarroel; Lucía Irazú; Cristina Alonso‐Vega; Fabiana Alves; María Á. Curto
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.
Genetic polymorphism of Trypanosoma cruzi bloodstream populations in adult chronic indeterminate Chagas disease patients from the E1224 clinical trial
(Oxford University Press, 2021) Juan Carlos Ramı́rez; Gonzalo R. Acevedo; Carolina Torres; Rudy Parrado; Anabelle de la Barra; Sandro Villarroel; Lineth García; Joaquím Gascón; Lourdes Ortiz; Faustino Torrico
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.
Killer cell immunoglobulin‐like receptor expression induction on neonatal CD8+ T cells in vitro and following congenital infection with Trypanosoma cruzi
(Wiley, 2009) Emmanuel Hermann; Aurélie Berthe; Carine Truyens; Cristina Alonso‐Vega; Rudy Parrado; Faustino Torrico; Yves Carlier; Véronique M. Braud
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.
Leishmaniases in Bolivia: Comprehensive Review and Current Status
(American Society of Tropical Medicine and Hygiene, 2009) Ana Lineth García; Rudy Parrado; Ernesto Rojas; Raúl Delgado; Jean‐Claude Dujardin; Richard Reithinger
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.
Multiplex RT-qPCR strategy for SARS-CoV-2 variants detection in developing countries without ngs: The Bolivian experience
(Cambridge University Press, 2025) Rudy Parrado; Carolina X Cuba-Grandy; Eugenia Fuentes-Luppichini; Nattaly Grecia Torrico Villarroel; Yercin Mamani Ortiz; Javier Méndez; Betty Melgarejo; Irenice Coronado-Arrázola; Nair A. Montaño; Leonardo I. Almonacid
The rapid evolution of SARS-CoV-2 has led to the emergence of variants of concern (VOCs) characterized by increased transmissibility, pathogenicity, and resistance to neutralizing antibodies. Identifying these variants is essential for guiding public health efforts to control COVID-19. Although whole genome sequencing (WGS) is the gold standard for variant identification, its implementation is often limited in developing countries due to resource constraints. In Bolivia, genomic surveillance is a challenge due to its limited technological infrastructure and resources. An RT-qPCR-based strategy was designed to address these limitations and detect the mutations associated with VOCs and variants of interest (VOIs). The multiplex RT-qPCR commercial kits AllplexTM Master and Variants I (Seegene®) and the ValuPanelTM (Biosearch®) were used to target mutations such as HV69/70del, E484K, N501Y, P681H, and K417N/T. They are characteristic of the Alpha (B.1.1.7), Beta (B.1.531), Gamma (P.1), Omicron (B.1.1.529), Mu (B.1.621), and Zeta (P.2) variants. A total of 157 samples collected in Cochabamba from January to November 2021 were evaluated, identifying 44 Gamma, 2 Zeta, 20 Mu, and 10 Omicron were identified. The strategy's effectiveness was validated against WGS data generated with Oxford NanoporeTM technology, showing a concordance rate of 0.96. This highlights the value of the RT-qPCR strategy in guiding the selection of samples for WGS, enabling broader detection of new variants that cannot be identified by RT-qPCR alone.
Multiprimer PCR System Diagnosis of Pulmonary Tuberculosis in Cochabamba, Bolivia
(American Society for Microbiology, 2007) Rudy Parrado; Daniel Lozano; Lineth García; Mary Cruz Torrico; Raúl Delgado; Faustino Torrico; Monica Laserna; Richard Reithinger
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
Predominance of hybrid discrete typing units of Trypanosoma cruzi in domestic Triatoma infestans from the Bolivian Gran Chaco region
(Elsevier BV, 2012) Esdenka Pérez-Cascales; Marcelo Monje; Boris Chang; Rosio Buitrago; Rudy Parrado; Christian Barnabé; François Noireau; Simone Frédérique Brénière
Prevalence of Leishmania spp. infection in domestic dogs in Chapare, Bolivia
(Elsevier BV, 2010) Rudy Parrado; Ernesto Rojas; Raúl Delgado; Mary Cruz Torrico; Richard Reithinger; Ana Lineth García
Real Time PCR for the Evaluation of Treatment Response in Clinical Trials of Adult Chronic Chagas Disease: Usefulness of Serial Blood Sampling and qPCR Replicates
(2018) Rudy Parrado; Ramírez Jc; Anabelle de la Barra; Cristina Alonso‐Vega; Natalia Juiz; Lourdes Ortiz; Daniel Illanes; Faustino Torrico; Joaquím Gascón; Fabiana Alves
Abstract This work evaluated a serial blood sampling procedure to enhance the sensitivity of duplex real time PCR (qPCR) for baseline detection and quantification of parasitic loads and post-treatment identification of failure in the context of clinical trials for treatment of chronic Chagas disease, namely DNDi-CH-E1224-001 ( NCT01489228 ) and 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 per sample. The first two samples were collected during the same day and the third one seven 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 pre-treatment 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 and increased cumulative 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 regimes, respectively; and from 4.6 to 15.9% and 9.5 to 32.1% for the benznidazole (BZN) arm in the DNDi-CH-E1224-001 and MSF-DNDi studies, respectively. The monitoring of patients treated with placebo in the DNDi-CH-E1224-001 trial revealed fluctuations in parasitic loads and occasional non-detectable results. This 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.
Sand Fly Fauna in Chapare, Bolivia: An Endemic Focus ofLeishmania(Viannia)braziliensis: Table 1.
(Oxford University Press, 2012) Marinely Bustamante; Mery Diaz; Jorge Espinoza; Rudy Parrado; Richard Reithinger; Ana Lineth García
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.
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
(BioMed Central, 2012) José M. Requena; Carmen Chicharro; Lineth García; Rudy Parrado; Concepción J. Puerta; Carmen Cañavate
Usefulness of Serial Blood Sampling and PCR Replicates for Treatment Monitoring of Patients with Chronic Chagas Disease
(American Society for Microbiology, 2018) Rudy Parrado; Juan Carlos Ramı́rez; Anabelle de la Barra; Cristina Alonso‐Vega; Natalia Juiz; Lourdes Ortiz; Daniel Illanes; Faustino Torrico; Joaquím Gascón; Fabiana Alves
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.