Tripanosomátidos aislados de mamíferos silvestres en tres departamentos de Bolivia (Cochabamba, Potosí y Santa Cruz de la Sierra) / Trypanosomatidae isolated from wild mammals in three departments of Bolivia (Cochabamba, Potosi and Santa Cruz de la Sierra)

Objetivos: el objetivo de la investigación fue aislar protozoarios kinetoplástidos a partir de mamíferos silvestres en tres departamentos de Bolivia, con la finalidad de identificar reservorios de tripanosomátidos que podrían causar infección en diferentes reservorios y enfermedades en el humano. Métodos: Los mamíferos silvestres fueron capturados en el Chaco, valles interandinos y la zona tropical de Bolivia, utilizando trampas Sherman, Havahard y Tomahawk. Los animales capturados fueron anestesiados para realizar el xenodiagnóstico y la extracción de sangre por punción cardiaca; el aislamiento de tripanosomátidos se realizó por hemocultivo utilizando medios de cultivo NNN y su respectiva identificación por las técnicas de PCR-RFLP en el laboratorio de Biología molecular IIBISMED. Resultados: fueron capturados 236 mamíferos silvestres pertenecientes a 30 especies, de las cuales 7 especies presentaron infección por hemoflagelados. Trypanosoma cruzi fue aislado de Didelphis marsupialis, D. albiventris, Galea musteloides, Graomys domorum y Andalgalomis pearsoni; T.c marinkellei y T. dionisii fueron aislados de Carolia perspicillata (murciélagos) y otros kinetoplástidos no identificados por herramientas moleculares disponibles fueron aislados de mamíferos del género Graomys y Andalgalomys, capturados en las provincias Campero de Cochabamba y Cordillera del departamento de Santa Cruz. Conclusiones: El T. cruzi, T.c. marinkellei, T. dionisii y otros tripanosomátidos se encuentran infectando a marsupiales (Didelphis), roedores (Graomys y Andalgalomys) y cobayos silvestres (Galea) los cuales se encuentran en su ciclo silvestre en las zonas estudiadas.

Objectives: The aim of this research was isolate kinetoplastid protozoan from wild mammals in three departments of Bolivia, to identify Trypanosomatids reservoirs that could cause infection in different reservoirs and disease in humans. Methods: The wild mammals were caught in the Chaco, valleys and the tropical zone of Bolivia, using Sherman, Havahard and Tomahawk traps. Captured animals were anesthetized and xenodiagnosis and blood cardiac puncture was performed; trypanosomatides isolation using blood culture was done in NNN culture media and the respective identification was performed by PCR-RFLP techniques in the molecular biology laboratory of IIBISMED. Results: 236 wild mammals belonging to 30 species were captured, of which 7 species showed infection by hemoflagellates. Trypanosoma cruzi was isolated from Didelphis marsupialis, D. albiventris, Galea musteloides, Graomys domorum and Andalgalomis pearsoni; T.c. marinkellei and T. dionisii were isolated from Carolia perspicillata (bats) and other kinetoplastid not identified by available molecular tools were also isolated from Andalgalomys and Graomys mammals genus, from Campero and Cordillera provinces of Cochabamba and Santa Cruz. Conclusions: The T. cruzi, T.c. marinkellei, T. dionisii and other trypanosomatids are infecting marsupials (Didelphis), rodents (Graomys and Andalgalomys) and wild guinea pigs (Galea) which are found in a sylvatic cycle in the studied areas.

Estudio de costo/beneficio de un programa de control de Enfermedad de Chagas Congénita en Bolivia / Cost effectiveness study of a control program of congenital Chagas disease in Bolivia

Cost effectiveness analysis of Chagas' vertical transmission control program in Bolivia: Today, Bolivia is the most concerned country in America by Chagas disease: Trypanosoma cruzi infection affects 20% of whole population, around 1800000 inhabitants, and mother-to-child transmission is around 5%, from 1.6 to 9.8%. Direct and indirect costs derived from disease complications and death, from birth to adulthood, add up around US$ 21 millions per year for 2,718 infected new-borns. This cost falls on individual, family and society, when the nation is struggling in a depressed economy. On the other side, an effective control program could detect and treat all cases with an investment of US$ 123 per infected new-born, or US$ 1.2 per new-born in Bolivia. Indirect benefits, apart of suffering relieve and improving of life quality, are related with Chagas vector control program, increasing the demand thanks to increasing risk awareness and also induced demand testing all pregnant women in endemic areas. So the conclusion is that such investment is profitable.

Estatus inmunológico de las madres infectadas por T. cruzi / Immunological status of mothers infected with Trypanosoma 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.

Respuesta inmune en neonatos no infectados nacidos de madres infectadas por Trypanosoma cruzi / Immune responses of non-infected neonates of mothers infected with Trypanosoma cruzi

We have investigated if maternal T. cruzi infection could induce in utero innate and/or adaptive immune responses in uninfected neonates by measuring specific IgM and IgA antibodies in cord blood plasma, and by performing phenotypic and functional studies of umbilical cord blood cells of their newborns (M+B- group). We detected T. cruzi-specific IgM and IgA antibodies in M+B- cord blood, indicating they had mounted in utero a strong B cell response, although they are not infected. On the other hand, circulating T cells of such uninfected neonates displayed a low level of activation, as seen bya slightly increased expression of the activation markers CD45RO on CD4+ T cells and HLA-DR on CD8+ T cells, although the proportion of CD4+ and CD8+ T cells was unmodified as compared to newborns from uninfected mothers (MB- group). This activation did not give rise to a proliferative response upon stimulation by T. cruzi antigens in vitro. However, M+B- cells produced low levels of lymphokines (IFN-gamma and IL-13) upon mitogenic stimulation, which was not the case of M-B- newborn cells. Beside this, M+B- blood cells produced higher levels of inflammatory cytokines (IL-1b, IL-6, TNF-alpha) than M-B- cells when stimulated with the T. cruzi lysate or LPS, suggesting the over-activation of the innate response in M+B- newborns. Monocytes participated in such inflammatory response since M+B- purified cord blood monocytes produced higher levels of TNF- when incubated with LPS or a T. cruzi lysate than M-B- cells. Altogether, these results show that, even in the absence of congenital infection, maternal T. cruzi infection triggers in utero both adaptive and innate immune responses in their babies. This indicates that parasite circulating antigens have been transferred from mothers to their fetuses.

Las lesiones placentarias en la infección humana por Trypanosoma cruzi / Placental lesions in human Trypanosoma cruzi infection

This histopathological study analyzes placentas of babies congenitally infected with T. cruzi (M+B+), or babies not infected but born from infected- (M+B-), or non infected-mothers (M-B-). Placentas M+B+ showed lesions of chorionitis, chorioamnionitis and cord edema with lymphocyte infiltration, whereas such lesions were infiltrated only with polymorphonuclear cells in M+B- and M-B- placentas. Parasites were found in M+B+ placentas, in fibroblasts and macrophages of chorion, membranes, chorionic plate, mainly in the area of membrane insertion, as well as in cells of Wharton jelly and myocytes of umbilical cord vessels. These results suggest that the materno-fetal transmission of parasites occurs mainly through the marginal sinus, spreading into the chorionic plate infecting fibroblasts and macrophages so far as to found a fetal vessel, inducing a fetal infection by hematogenous route.

Detección de la heterogeneidad molecular de Trypanosoma cruzi / Detection of molecular heterogeneity of Trypanosoma cruzi

Congenital transmission of T. cruzi in Cochabamba affects 6% of newborns from infected mothers. Only limited information is available on the type of transmitted parasites. However, it is well established that T. cruzi isolated from various vectors as well from host animals are highly heterogeneous. In our presentation we analyse aspects of molecular heterogeneity of T. cruzi and we review methods used for the molecular typing of T. cruzi lineages. Experimentally, we performed the PCR amplification of [quot ]Sequence-characterised region Markers[quot ] for typing T. cruzi isolated from umbilical blood of newborns in Cochabamba. We compared these results with those we obtained from general infected population. All 16 analysed, congenitally infected samples were of lineage IId. Our data also indicated that this lineage was found in about 80% of samples originated from general infected population in Cochabamba.

Efectos de la infección materna por Trypanosoma cruzi en el desarrollo del embarazo y del recien nacido / Effects of maternal infection with Trypanosoma cruzi in pregnancy development and in the newborn infant

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.

Comparación de métodos de PCR para el diagnóstico de la infección congenita por Trypanosoma cruzi / Comparison of PCR methods for the diagnosis of congenital Trypanosoma cruzi infection

PCR is a potentially interesting diagnostic tool to detect congenital T. cruzi infection. We have compared the sensitivity and capacity of a battery of T. cruzi PCR primers to detect the complete spectrum of known T. cruzi lineages, in order to improve and simplify the detection of infection in neonatal blood. We found that the primers Tcz1/Tcz2, targeting the 195 bp satellite repeat, detected all the parasitic lineages with the same sensitivity For all other tested primers (nDNA primers: BP1/BP2, 01/02, Pon1/ Pon2 and Tca1/Tca2; kDNA primers: S35VS36, 121/122), either, the intensity of amplicons varied according to T. cruzi lineages, or the assess were less sensitive. In order to better assess such PCR protocol, we assayed 311 samples of neonatal blood previously tested with parasitological methods. Reliability of our PCR test was demonstrated since all the 18 blood samples from newborns with congenital T. cruzi infection were positive, whereas the remaining samples (30 from control newborns of uninfected mothers and 262 out of 263 from babies, parasitologically negative, born from infected mothers) were negative. As our PCR method is simple, reliable, robust and cheap, it appears suitable for the detection of T. cruzi infection in neonatal blood.

Dosificación de anticuerpos IgM e IgA anti-trypanosoma cruzi en sangre de recién nacidos de madres con serología positiva para Chagas / Serum levels for IgM and IgA antibodies to anti-trypanosoma cruzi in samples of blood from newborns from mothers with positive serology for Chagas disease

This study compares the levels of specific antibodies IgM and IgA for Chagas in samples of blood from newborns. Three groups of cord blood samples have been analysed: a group of 42 samples from newborns, displaying positive parasitemia, of seropositive mothers (M+B+), 68 samples from newborns with negative parasitemia whose mothers were seropositive (M+B-) and a group of 45 control newborns coming from mothers with negative serology for Chagas. From the 42 M+B+ samples with congenital Chagas disease, 81 and 82.9% displayed detectable levels of IgM and IgA antibodies, respectively In the M+B- group, 70.6 and 33.8% presented antibodies of IgM and IgA classes, respectively, whereas in the control group M-B-, we detected 6% and 11.1% of IgM and IgA antibodies, respectively. The calculated sensitivity of detection of congenital cases using IgM or IgA antibodies was of 82.9% and 80.9% respectively, whereas the specificity of detection was of 29.4% for IgM antibodies and of 66.1% for IgA antibodies.

Estimación de la parasitemia en la infección humana por Trypanosoma cruzi: las altas parasitemias están asociadas con la severa y fatal enfermedad de Chagas congenita / Estimation of the parasitemia in Trypanosoma cruzi human infection: high parasitemias are associated with severe and fatal congenital Chagas disease

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.

Tratamiento integral de la enfermedad de Chagas congenita: la experiencia boliviana / Integral treatment of congenital Chagas disease: the Bolivian experience

We have analyzed the response to the treatment with benznidazol in newborns and nurslings in the Hospital Materno Infantil Germán Urquidi of Cochabamba, Bolivia, between 1999 and 2002. It is important an integral treatment of the nursling with a subsequent information directed to the family. The response was close to 100% when the treatment was correctly administrated. They were not adverse effects and the detected biochemical alterations did not present clinical significance.

Nivel de endemia de la infección por Trypanosoma cruzi en el lugar de residencia de la madre y desarrollo de la enfermedad de Chagas congénita en Bolivia / Endemic level of congenital Trypanosoma cruzi infection in the areas of maternal residence and the development of congenital Chagas disease in Bolivia

In Bolivia, the prevalence of infection by T. cruzi in women in fertile age can vary between 20 and 60%. The present study made in the Maternity Germin Urquidi of Cochabamba - Bolivia, it has demonstrated, that 19.9% of the mothers who go to this hospitable center to be taken care of in the childbirth, they are carrying of the infection and that 4,6% of them, they are going to transmit, by transplacentaria route, the infection to its babies. Of the 71 children born with congenital Chagas, only 47,8 % present/display some type of alteration or of development(Apgar to 1 minute low, BPN, prematuridad, pathological dismadurez) or signs (SDR, hepatomegalia, esplenomegalia, neurological signs, cardiomegalia, anasarca, petequias). When investigating the effect of the differences in the vectorial density (low, medium and high) of the zone of maternal residence, on the transmission of the infection of the mother infected to the fetus, we concluded that the rate of transmission of the congenital infection of T. cruzi is not modified by the level of endemicidad of the zone of maternal residence. By another infected new born sides whose mothers reside in zones of high endemicidad present/display, most frequently and of significant way, Apgar to 1 minute < to 7, low weight when being born and prematuridad or an association of these alterations with respiratory syndrome of distress or anasarca, when one compares them with new born of resident mothers in the zones of loss or medium endemicidad, mortality in this group is greater. These results suggest calls to account it of the mothers, in areas of high endemicidad, she is associate with a serious increase in the risk of Disease of newborn severe and mortal congenital Chagas in.