This work compares the results of two epidemiologic and clinical surveys on the consequences of maternal chronic Trypanosoma cruzi infection. They were conducted in 1992-1994 and 1999-2001 in the same maternity clinic in Bolivia, a country highly endemic for infection with this parasite. In both surveys, the materno-fetal transmission of parasites occurred in 5-6% of the infected mothers. Maternal chronic T. cruzi infection had no effect on pregnancy outcome and health of newborns when there was no materno-fetal transmission of parasites. Comparisons between the older and the more recent surveys highlighted significant reductions in frequencies of symptomatic cases (from 54% to 45%), Apgar scores < 7, and low birth weights and prematurity (from 32-50% to 6-16%) among congenitally infected babies. Neonatal mortality related to congenital Chagas disease also decreased from 13% to 2% in the interval between both studies. These results suggest that the decrease in poverty that has occurred in Bolivia between both surveys might have contributed to reduce the morbidity and mortality, but not the transmission rate of T. cruzi congenital infection, which remains a serious public health problem in this country.

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Abstract. The polymerase chain reaction (PCR) is a potentially interesting diagnostic tool for detecting congenital Trypanosoma cruzi infection at birth. We have compared the sensitivity and capacity of a group of T. cruzi PCR primers in detecting the complete spectrum of known T. cruzi lineages, and to improve and simplify the detection of infection in neonatal blood. We found that the two primers, Tcz1/Tcz2 and Diaz1/Diaz2, which target the 195-basepair satellite repeat, detected all parasitic lineages with the same sensitivity. However, the intensity of the amplicon was somewhat higher with Tcz1/Tcz2. For other tested primers (nuclear DNA primers BP1/BP2, O1/O2, Pon1/Pon2, and Tca1/Tca2 and kinetoplast DNA primers S35Ј/S36Ј and 121/122), either the intensity of amplicons varied according to T. cruzi lineages or the PCR assay was less sensitive. The use of the Tcz1/Tcz2 primers, which target a tandem repetitive sequence, requires a careful determination of the appropriate amount of Taq polymerase to avoid the formation of smears and multiple amplicon bands. The Tcz1/Tcz2 primers resulted in an intense 200-basepair amplicon with DNA extracted from blood equivalent to 0.02 parasites per assay when used with a simple DNA extraction method and of a low amount of Taq polymerase from a standard PCR kit. To better assess such PCR protocol, we assayed 311 samples of neonatal blood previously tested by parasitologic methods. The 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 of 263 from babies born to infected mothers) were negative. Since our PCR method is simple, reliable, robust, and inexpensive, it appears suitable for the detection of T. cruzi infection in neonatal blood, even in laboratories that are not equipped for performing the PCR.

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. MethodsWe 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 perprotocol population. Safety analyses were based on the as-treated population. Recruitment is now closed. This trial is registered with ClinicalTrials.gov, NCT03378661.

The conditions and mechanisms of congenital transmission of Trypanosoma cruzi remain largely unknown. In the present study, we compared the parasitic loads and the immune responses of pregnant T. cruzi-infected women who transmitted parasites to their fetus ("M+B+ mothers") with those of such women who did not transmit parasites to their fetus ("M+B- mothers"). M+B+ mothers had a higher frequency of positive results of hemoculture for T. cruzi than did M+B- mothers, in association with depressed production of parasite-specific interferon- gamma by blood cells that persisted after delivery. In contrast, the production of interleukin (IL)-2, IL-4, and IL-10 and transforming growth factor- beta 1 was similar between both groups of infected mothers, after stimulation with T. cruzi lysate. Flow cytometric analysis showed that T cells and monocytes of M+B+ mothers were less activated than were those of M+B- mothers. Altogether, these results indicate that congenital transmission of T. cruzi is associated with high parasitic loads and peripheral deficient immunological responses in mothers.

Abstract. To better understand the factors involved in maternal-fetal transmission of Trypanosoma cruzi, we compared DNA levels-obtained by use of quantitative real-time PCR and parasitic genotypes determined by PCR amplification followed by hybridization-in Bolivian mothers and their congenitally infected newborns. Mothers and their neonates displayed markedly different parasitic DNA levels, as most maternal estimated parasitemias (> 90%) were < 10 parasites/mL, whereas those of 76% of their newborns were > 1,000 parasites/mL. Comparison of T. cruzi TcII sublineages infecting mothers and newborns showed identity, without evidence of mixed infection in mothers or neonates. Analysis of minor variants of TcIId-genotyped parasites using sequence class probes hybridizing with hypervariable domains of kDNA minicircles showed discrepancies in half of mother/newborn pairs.

This study aims to typify the Trypanosoma cruzi (sub)lineage(s) in umbilical cord blood of congenitally infected Bolivian newborns, using PCR amplifications of "Region Markers", mini-exon or kDNA fragments followed by hybridization or sequencing. New probes were also designed to distinguish three variants within the TcIId sublineage. The IIb, IId, or IIe T. cruzi sublineages, as well as different variants of the IId sublineage, were detected in infected neonates, whereas mixed infections were not found. The frequencies of the IId sublineage were similar in neonates (95.1%) and adults of the same area (94.1%). The IId-infected newborns displayed either asymptomatic, or severe and fatal clinical forms of congenital Chagas disease, as well as low or high parasitemia. Altogether these data show that T. cruzi DNA polymorphism, based on the presently available markers, is not associated with the occurrence of congenital infection or the development of severe clinical forms of congenital Chagas disease.

Bolivia is one of the most endemic countries for Chagas disease. Data of 2005 shows that incidence is around 1.09‰ inhabitants and seroprevalence in children under 15 ranged from 10% in urban areas to 40% in rural areas. In this article, we report results obtained during the implementation of the congenital Chagas program, one of the biggest casuistry in congenital Chagas disease, led by National Program of Chagas and Belgian cooperation from 2004 to 2009. The program strategy was based on serological results during pregnancy and on the follow up of children born from positive mothers until one year old; if positive, treatment was done with Benznidazole, 10 mg/Kg/day/30 days with one post treatment control 6 months later. Throughout the length of the program, a total of 318,479 pregnant women were screened and 23.31% were detected positive. 42,538 children born from positive mothers were analyzed at birth by micromethod, of which 1.43% read positive. 10,120 children returned for their second micromethod control of which 2.29% read positive, 7,650 children returned for the serological control, of which 3.32% turned out positive. From the 1,093 positive children, 70% completed the 30 day-treatment and 122 returned for post treatment control with 96% showing a negative result. It has been seen that maternal-fetal transmission rates vary between 2% and 4%, with an average of 2.6% (about half of previously reported studies that reached 5%). In this work, we show that it is possible to implement, with limited resources, a National Congenital Chagas Program and to integrate it into the Bolivian health system. Keys of success are population awareness, health personnel motivation, and political commitment at all levels.