Analytical Validation of Quantitative Real-Time PCR Methods for Quantification of Trypanosoma cruzi DNA in Blood Samples from Chagas Disease Patients.

An international study was performed by 26 experienced PCR laboratories from 14 countries to assess the performance of duplex quantitative real-time PCR (qPCR) strategies on the basis of TaqMan probes for detection and quantification of parasitic loads in peripheral blood samples from Chagas disease patients. Two methods were studied: Satellite DNA (SatDNA) qPCR and kinetoplastid DNA (kDNA) qPCR. Both methods included an internal amplification control. Reportable range, analytical sensitivity, limits of detection and quantification, and precision were estimated according to international guidelines. In addition, inclusivity and exclusivity were estimated with DNA from stocks representing the different Trypanosoma cruzi discrete typing units and Trypanosoma rangeli and Leishmania spp. Both methods were challenged against 156 blood samples provided by the participant laboratories, including samples from acute and chronic patients with varied clinical findings, infected by oral route or vectorial transmission. kDNA qPCR showed better analytical sensitivity than SatDNA qPCR with limits of detection of 0.23 and 0.70 parasite equivalents/mL, respectively. Analyses of clinical samples revealed a high concordance in terms of sensitivity and parasitic loads determined by both SatDNA and kDNA qPCRs. This effort is a major step toward international validation of qPCR methods for the quantification of T. cruzi DNA in human blood samples, aiming to provide an accurate surrogate biomarker for diagnosis and treatment monitoring for patients with Chagas disease.

Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples.

BACKGROUND: Trypanosoma cruzi has been classified into six Discrete Typing Units (DTUs), designated as TcI-TcVI. In order to effectively use this standardized nomenclature, a reproducible genotyping strategy is imperative. Several typing schemes have been developed with variable levels of complexity, selectivity and analytical sensitivity. Most of them can be only applied to cultured stocks. In this context, we aimed to develop a multiplex Real-Time PCR method to identify the six T. cruzi DTUs using TaqMan probes (MTq-PCR). METHODS/PRINCIPAL FINDINGS: The MTq-PCR has been evaluated in 39 cultured stocks and 307 biological samples from vectors, reservoirs and patients from different geographical regions and transmission cycles in comparison with a multi-locus conventional PCR algorithm. The MTq-PCR was inclusive for laboratory stocks and natural isolates and sensitive for direct typing of different biological samples from vectors, reservoirs and patients with acute, congenital infection or Chagas reactivation. The first round SL-IR MTq-PCR detected 1 fg DNA/reaction tube of TcI, TcII and TcIII and 1 pg DNA/reaction tube of TcIV, TcV and TcVI reference strains. The MTq-PCR was able to characterize DTUs in 83% of triatomine and 96% of reservoir samples that had been typed by conventional PCR methods. Regarding clinical samples, 100% of those derived from acute infected patients, 62.5% from congenitally infected children and 50% from patients with clinical reactivation could be genotyped. Sensitivity for direct typing of blood samples from chronic Chagas disease patients (32.8% from asymptomatic and 22.2% from symptomatic patients) and mixed infections was lower than that of the conventional PCR algorithm. CONCLUSIONS/SIGNIFICANCE: Typing is resolved after a single or a second round of Real-Time PCR, depending on the DTU. This format reduces carryover contamination and is amenable to quantification, automation and kit production.

Plasmodium vivax malaria during pregnancy, Bolivia.

Plasmodium vivax is a major cause of illness in areas with low transmission of malaria in Latin America, Asia, and the Horn of Africa. However, pregnancy-associated malaria remains poorly characterized in such areas. Using a hospital-based survey of women giving birth and an antenatal survey, we assessed the prevalence rates of Plasmodium spp. infections in pregnant women in Bolivia, and evaluated the consequences of malaria during pregnancy on the health of mothers and newborns. P. vivax infection was detected in 7.9% of pregnant women attending antenatal visits, and placental infection occurred in 2.8% of deliveries; these rates did not vary with parity. Forty-two percent of all P. vivax malaria episodes were symptomatic. P. vivax-infected pregnant women were frequently anemic (6.5%) and delivered babies of reduced birthweight. P. vivax infections during pregnancy are clearly associated with serious adverse outcomes and should be considered in prevention strategies of pregnancy-associated malaria.

Evaluation of compliance to congenital Chagas disease treatment: results of a randomised trial in Bolivia.

BACKGROUND: A randomised, unblinded, clinical trial comparing two benznidazole regimens for congenital Chagas disease was carried out to determine whether simplification and reduction in the length of treatment could lead to better treatment compliance. METHODS: This study was conducted in Santa Cruz, Bolivia. Serological screening was carried out in pregnant women, and parasites were sought in the blood of newborns from seropositive mothers. Infected infants were randomly assigned to two treatment groups. Recovery was assessed by parasite seeking at 1 month and 2 months as well as serological tests at 9 months. Assessment of treatment adherence was based on weekly home visits and use of electronic monitors. RESULTS: Benznidazole was given to 63 newborns in group A (5 mg/kg in two daily doses for 60 days) and 61 newborns in group B (7.5 mg/kg in a single daily dose for 30 days). There was no difference in compliance between the two groups. The study confirmed the efficacy and good tolerance of both benznidazole regimens in the treatment of congenital Chagas disease. CONCLUSIONS: The short treatment should be preferred as it allows reducing the dose of benznidazole as well as the cost of treatment.

Influence of pregnancy on Trypanosoma cruzi parasitemia in chronically infected women in a rural Bolivian community.

To determine the role of pregnancy on Trypanosoma cruzi parasitemia, a matched cohort study was carried out in a rural Bolivian community comparing parasite rates in gravidae, puerperae, and non-pregnant infected women. A selection of 67 chronically infected women, who delivered between March 2004 and May 2005, were initially evaluated during the third trimester of pregnancy and again after delivery. They were matched for age, parity, and location with 104 seropositive non-pregnant women, who likewise had submitted blood for microscopic examination for T. cruzi parasites in June 2005. Seroreactive pregnant women had a higher rate of T. cruzi parasitemia (14.9%) than matched non-pregnant infected women (2.9%; P = 0.004). After delivery, parasitemia significantly decreased during puerperium (1.5%) compared with the period of pregnancy (14.9%; P = 0.03). This study showed an increase of parasite loads in maternal peripheral blood, during the third trimester, and a significant decline after delivery.

Door-to-door screening as a strategy for the detection of congenital Chagas disease in rural Bolivia.

OBJECTIVE: To demonstrate the feasibility of a house-to-house screening system used for congenital Chagas disease in rural areas based on an active search for pregnant women and newborns in their homes in addition to passive case detection in health facilities. METHODS: Exploratory phase conducted by the research team followed by an operational period coordinated by municipal health service. A blood sample was taken for serological and parasitological tests of Trypanosoma cruzi from pregnant women who were searching antenatal care or visited at home by field investigators. Infants born to T. cruzi-infected women were examined for infection at birth and again at 1 and 7 months of age. RESULTS: 64.5% of the pregnant women were infected. Congenital infection was diagnosed at birth in 4.0% (12/299) of the children born to seroreactive mothers. Twelve additional cases of infection (4%) were diagnosed in children between 1 and 7 months of age. Finally, 37% of the children were lost to follow-up in the exploratory phase and 53% during the operational phase (P=0.002), significantly fewer than in most passive case detection studies. CONCLUSION: Despite poorer outcomes after door-to-door screening activities have been transferred to the health system, a combined strategy based on active and passive case detection appeared to be efficient for identifying rural cases of congenital Chagas disease.

Detectable Trypanosoma cruzi parasitemia during pregnancy and delivery as a risk factor for congenital Chagas disease.

Vector control has led to a drastic decrease in the prevalence of acquired Chagas disease in Latin America, thus redirecting attention to congenital Chagas disease. We report results of a longitudinal study of 359 pregnant women in Yacuiba in southern Bolivia, of whom 147 (40.9%) were infected with Trypanosoma cruzi, to evaluate the relationship between the patency period of the parasitemia and the risk of congenital infection. Maternal infection was assessed by using T. cruzi-specific serologic tests, and parasitemia in mothers and newborns was diagnosed by using microscopic examination of blood in heparinized microhematocrit tubes. Parasitemia was present in 28.6% of the infected women. Its prevalence increased during the third trimester, then decreased at delivery. The likelihood of congenital infection was significantly correlated with the parasite density in the mother's blood. The risk of transmission increased during the third trimester of pregnancy and could explain premature births or low-weight newborns for infected mothers.

Highly effective serodiagnosis for Chagas' disease.

Many proteins of Trypanosoma cruzi, the causative agent of Chagas' disease, contain characteristic arrays of highly repetitive immunogenic amino acid motifs. Diagnostic tests using these motifs in monomeric or dimeric form have proven to provide markedly improved specificity compared to conventional tests based on crude parasite extracts. However, in many cases the available tests still suffer from limited sensitivity. In this study we produced stable synthetic genes with maximal codon variability for the four diagnostic antigens, B13, CRA, TcD, and TcE, each containing between three and nine identical amino acid repeats. These genes were combined by linker sequences encoding short proline-rich peptides, giving rise to a 24-kDa fusion protein which was used as a novel diagnostic antigen in an enzyme-linked immunosorbent assay setup. Validation of the assay with a large number of well-characterized patient sera from Bolivia and Brazil revealed excellent diagnostic performance. The high sensitivity of the new test may allow future studies to use blood collected by finger prick and dried on filter paper, thus dramatically reducing the costs and effort for the detection of T. cruzi infection.

Antibody drop in newborns congenitally infected by Trypanosoma cruzi treated with benznidazole.

OBJECTIVE: To compare the drop of Chagas antibody titres between non-infected and congenitally infected newborns treated by two doses of benznidazole, aiming at evaluating the recovery time and giving recommendations regarding serological criteria of recovery. METHODS: During a clinical trial, the drop of Trypanosoma cruzi antibody titres measured by ELISA tests was followed during the first year of life in congenitally infected newborns treated with different doses of benznidazole and compared to T. cruzi antibody titres in non-parasitaemic newborns. Confirmation of recovery was given by two negative serological tests: Chagas Stat-Pak (CSP) (immunochromatography) and Chagatest v3.0 (ELISA). RESULTS: In non-parasitaemic infants of infected mothers, antibodies of maternal origin disappeared in <8 months while in infected infants, T. cruzi antibodies decreased more slowly and disappeared in 9-16 months allowing to confirm the recovery. All CSP tests were negative before the ninth month while about 10% of ELISA tests remained positive at the 12th month. CONCLUSIONS: Recovery may be confirmed in most cases at 10 months. The CSP test was compared to Chagatest v3.0 ELISA and appeared to give a reliable response. The decrease rate of antibodies does not depend on treatment modes.

Sensitivity and specificity of Chagas Stat-Pak test in Bolivia.

OBJECTIVE: To compare the results of an immunochromatographic test performed on whole blood, Chagas Stat-Pak, with those of an ELISA test using recombinant antigens. METHOD: We tested 995 subjects of a rural population of all ages in the south of Bolivia, 459 pregnant women of the same population and 1030 urban women giving birth from the east of Bolivia. RESULTS: The sensitivity of the CSP test for the entire studied population (n = 2484) was 94.73% [93.35-96.10]; the specificity was 97.33% [96.50-98.15]. However, the specificity differed significantly between rural pregnant and urban birthing women, which could be attributed either to differences of parasite strain or Chagas prevalence. CONCLUSION: The test is simple of use, reliable, relatively inexpensive (<2 US$ each test) and its performances are compatible with a field use for large-scale screenings.

Epidemiological evaluation of Chagas disease in a rural area of southern Bolivia.

We evaluated the prevalence of Chagas disease using a rapid screening test (Chagas Stat-Pak), confirmed by ELISA, in Caraparí, a village of 9000 inhabitants in southern Bolivian Chaco. The prevalence of Trypanosoma cruzi was estimated in a sample of 995 people. The prevalence adjusted on age was 51.2% and was proportionally related to age. We also observed a very significant cline from the south to the north of the locality, where the prevalence ranged from 40 to 80%. In children younger than 11 years, the prevalence was 21.5%, which confirmed the importance of residual vector transmission despite several years of vector control. Among women of procreation age, the prevalence was 63.9%, resulting in a high risk of congenital transmission. The control of the disease requires an increase in vector control and improvement of dwellings before considering children's treatment with trypanocide.