Molecular detection and parasite load of Trypanosoma cruzi in digestive tract tissue of Chagas disease patients affected by megacolon

Chagas disease, caused by the Trypanosoma cruzi parasite in the Americas affects similar to 7 million people, 30% with cardiac tissue damage and 10-15% with digestive disorders. In this study, we have developed a protocol to detect the presence of the parasite and estimate its load in resected dysfunctional tissue segments of chronically infected patients with digestive megacolon. We have included samples from 43 individuals, 38/5 with positive/negative serology for Chagas disease and digestive syndromes. Samples of 1.5 to 2.0 cm(2) were taken from different points of the dysfunctional digestive tract in specialized centres in Cochabamba, Bolivia. T. cruzi cultures were performed by inoculation with NNN-LIT culture medium, and genomic material was obtained from the samples for multiplex qPCR with TaqMan probes targeting satellite nuclear DNA. Cultures failed to isolate T. cruzi but qPCR reached a sensitivity of 42.1% (16/38) with all three spots and in triplicate. A new quantification methodology using synthetic satellite DNA as quantitation standard revealed parasite loads ranging from 2.2 x 10(2) to 1.0 x 10(6) satellite DNA copies/mu l. Positive samples from the distal end showed a higher parasite load. The results of the present study strengthen and add further evidence to previous findings in an experimental mouse model of chronic T. cruzi infection, providing a valuable tool to improve scientific knowledge on the relevance of the digestive tract in parasite persistence, and underline the need of a better understanding of host-pathogen interaction in digestive tissues, considering pathophysiology, disease immunology and response to treatment

Clinical and immunological characteristics of tegumentary leishmaniasis cases in Bolivia

Background Tegumentary leishmaniasis (TL) is a parasitic disease that can present a cutaneous or mucocutaneous clinical form (CL and MCL, respectively). The disease is caused by different Leishmania species and transmitted by phlebotomine sand flies. Bolivia has one of the highest incidences of the disease in South America and the diagnosis is done by parasitological techniques. Our aim was to describe the clinical and immunological characteristics of CL and MCL patients attending the leishmaniasis reference center in Cochabamba, Bolivia, in order to gain updated clinical and epidemiological information, to evaluate the diagnostic methods used and to identify biomarkers related to clinical disease and its evolution. Methodology/Principal findings The study was conducted from September 2014 to November 2015 and 135 patients with lesions compatible with CL or MCL were included. Epidemiological and clinical data were collected using a semi-structured questionnaire. Two parasitological diagnostic methods were used: Giemsa-stained smears and culture of lesion aspirates. Blood samples obtained from participants were used to measure the concentrations of different cytokines. 59.2% (80/135) were leishmaniasis confirmed cases (CL: 71.3%; MCL: 28.7%). Sixty percent of the confirmed cases were positive by smears and 90.6% were positive by culture. 53.8% were primo-infections. Eotaxin and monokine induced by IFN-γ presented higher serum concentrations in the MCL clinical presentation compared to CL cases and no-cases. None of the cytokines presented different concentrations between primo-infections and secondary infections due to treatment failure. Conclusions/Significance In Bolivia, parasitological diagnosis remains the reference standard in diagnosis of leishmaniasis because of its high specificity, whereas the sensitivity varies over a wide range leading to loss of cases. Until more accurate tools are implemented, all patients should be tested by both smears and culture of lesion aspirates to minimize the risk of false negatives. Our results showed higher concentrations of several cytokines in MCL compared to CL, but no differences were observed between CL and no-cases. In addition, none of the cytokines differed between primary and secondary infections. These results highlight the need of further research to identify biomarkers of susceptibility and disease progression, in addition to looking at the local cellular immune responses in the lesions

Usefulness of Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry in the Characterization of Leishmania Strains Causing Tegumentary Leishmaniasis in Bolivia versus hsp70 Gene Sequencing

Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) is a proteomic technique with proven efficiency in the identification of microorganisms, such as bacteria, fungi, and parasites. The present study aimed to evaluate the usefulness of MALDI-TOF MS for the characterization of Leishmania species circulating in Bolivia using hsp70 gene sequencing as a reference technique. 55 Leishmania strains that were isolated from patients with tegumentary leishmaniasis were analyzed. MALDI-TOF MS identified two species of the L. braziliensis complex (L. braziliensis, n = 26; L. braziliensis outlier, n = 18), one species of the L. guyanensis complex (L. guyanensis, n = 1), one species of the L. lainsoni complex (L. lainsoni, n = 2), and two species of the complex (, n = 5; and L. garnhami, n = 3). All of the strains were correctly identified at the subgenus, genus, and complex level, but 10 of them (18%) were misidentified as other species within the same complex by the hsp70 gene sequencing, with 7 of these corresponding to possible hybrids. Thus, one L. braziliensis corresponded to L. peruviana, two L. braziliensis corresponded to L. braziliensis / L. peruviana possible hybrids, two corresponded to , and three L. garnhami and two corresponded to / possible hybrids. Accordingly, MALDI-TOF MS could be used as an alternative to molecular techniques for the identification of Leishmania spp., as it is low cost, simple to apply, and able to quickly produce results. In Bolivia, its application would allow for the improvement of the management of patient follow-ups, the updating of the epidemiological data of the Leishmania species, and a contribution to the control of tegumentary leishmaniasis. IMPORTANCE The objective of the study was to evaluate the usefulness of MALDI-TOF MS for the characterization of Leishmania species circulating in Bolivia, in comparison with the sequencing of the hsp70 gene. In our study, all of the isolates could be identified, and no misidentifications were observed at the complex level. Although the equipment implies a high initial investment in our context, MALDI-TOF MS can be used in different areas of microbiology and significantly reduces the cost of testing. Once the parasite culture is obtained, the technique quickly yields information by accessing a free database that is available online. This would allow for the improvement of the management of patients and follow-ups, the updating of the epidemiological data of the species, and a contribution to the control of tegumentary leishmaniasis in Bolivia. Likewise, it can be used to determine a specific treatment to be given, according to the causal species of Leishmania, when there are protocols in this regard in the area

Phylogenetic Analysis of Bolivian Bat Trypanosomes of the Subgenus Schizotrypanum Based on Cytochrome b Sequence and Minicircle Analyses

The aim of this study was to establish the phylogenetic relationships of trypanosomes present in blood samples of Bolivian Carollia bats. Eighteen cloned stocks were isolated from 115 bats belonging to Carollia perspicillata (Phyllostomidae) from three Amazonian areas of the Chapare Province of Bolivia and studied by xenodiagnosis using the vectors Rhodnius robustus and Triatoma infestans (Trypanosoma cruzi marenkellei) or haemoculture (Trypanosoma dionisii). The PCR DNA amplified was analyzed by nucleotide sequences of maxicircles encoding cytochrome b and by means of the molecular size of hyper variable regions of minicircles. Ten samples were classified as Trypanosoma cruzi marinkellei and 8 samples as Trypanosoma dionisii. The two species have a different molecular size profile with respect to the amplified regions of minicircles and also with respect to Trypanosoma cruzi and Trypanosoma rangeli used for comparative purpose. We conclude the presence of two species of bat trypanosomes in these samples, which can clearly be identified by the methods used in this study. The presence of these trypanosomes in Amazonian bats is discussed

Evaluación de la contaminación microbiológica de la lechuga (lactuca sativa) en la cadena alimentaria, provincia de Quillacollo, Cochabamba, Bolivia 2015

Las enfermedades transmitidas por alimentos constituyen un problema de salud, diversos estudios demuestran la alta contaminación microbiológica de la lechuga en la cadena alimentaria. El objetivo de la presente investigación fue evaluar la contaminación microbiológica de la lechuga (Lactuca sativa) en la cadena alimentaria, en la provincia Quillacollo-Cochabamba-Bolivia. Muestras de lechugas fueron tomadas en los meses de abril y mayo, con el mismo de número de muestras, en tres puntos de la cadena alimentaria: parcelas de producción (localidad Combuyo), puestos de venta de lechuga y de comida de los Mercados 9 de diciembre y Central de Quillacollo. El procesamiento de las muestras fue realizado en los laboratorios de Microbiología de LABIMED, y Post Grado de Medicina Tropical de la Facultad de Medicina, UMSS. Los resultados demuestran la contaminación de las lechugas con bacterias Aerobias Mesófilas, colifor-mes totales y fecales en concentraciones superiores a los estándares permitidos en los tres puntos de la cadenas alimentaria evaluadas; identificándose Salmonella sp en una de las muestras de lechugas procesada de los puestos de venta de comida de los mercados. La contaminación parasitaria fue evidenciada en los puestos de venta solo en el mes de mayo en 83% (n=10) de las 12 muestras analizadas, detectándose quistes de Entamoeba coli y larvas de Strongyloides sp, contaminación relacionada con la utilización de abono de aves en el cultivo y la manipulación inadecuada de la lechuga en la cadena alimentaria. En conclusión podemos indicar que las lechugas presentan una importante contaminación microbiana en los tres puntos evaluados de la cadena alimentaria, por lo que constituye un riego para la salud pública.Palabras claves: lechuga, contaminación microbiológica, cadena alimentari

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

Efectos de la infección materna por Trypanosoma cruzi en el desarrollo del embarazo y del recien nacido.

In the endemic regions of Bolivia the infection of the feminine population in fertile age by T. cruzi is frequent (20 to 50 % of the women in fertile age) and the rate of fetal maternal transmission is of approximately 5%. A great percentage of infected women do not transmit the infection to the fetus. The intention of the present study carried out at the Maternal-Infantile Hospital Germán Urquidi of Cochabamba (Bolivia) is to contribute to the knowledge regarding the pregnancy and birth of a newborn of Chagas infected women who do not transmit the infection to the fetus. 2124 mothers and 2,155 newborns were studied. The prevalence of infection by T. cruzi among these pregnant women is of 26,3%. Two groups of mothers were studied: 554 that presented infection by T. cruzi (group M+B-) and 1520 not infected (group control M-B-). Both groups of mothers are comparable in their anthropometric and obstetrical antecedents. The mothers (M+B+) are in average older than those not infected (p<0.05), which will probably have an influence on the number of gestations and abortion antecedents, which were of p<0.05 and p=0.01 respectively. Among the different anthropometric and biological parameters studied in newborns of groups M+B- and M-B -, no statistically significant differences between both groups were found. It can be inferred that the chronic maternal infection by T. cruzi seems to have no clinical influence, neither on the course of the pregnancy nor during birth, if a group of T. cruzi infected mothers is compared to a non infected group.English AbstractJournal Articleinfo:eu-repo/semantics/publishe

Estatus inmunológico de las madres infectadas por T. cruzi.

The mechanisms of congenital transmission of Chagas disease remain largely unknown. To better understand the role of maternal immunology during pregnancy in congenital Chagas transmission, we studied the cytokine production and the parasitic load in three groups of mothers: infected mothers who transmitted the disease to their babies (M+B+-), infected mothers who did not transmit the disease to their babies (M+B-) and not infected mothers as a control group (M-B-). M+B+ mothers produced less IFNgamma and more IL-10 than the M+B- mothers, and they are not able to produce IL-2. M+B+ mothers showed a higher parasitic load. These results, indicated that the congenital Chagas transmission is associated with an immunological imbalance and a high parasitic load in the M+B+ mothers.English AbstractJournal Articleinfo:eu-repo/semantics/publishe