International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients.

BACKGROUND: A century after its discovery, Chagas disease still represents a major neglected tropical threat. Accurate diagnostics tools as well as surrogate markers of parasitological response to treatment are research priorities in the field. The purpose of this study was to evaluate the performance of PCR methods in detection of Trypanosoma cruzi DNA by an external quality evaluation. METHODOLOGY/FINDINGS: An international collaborative study was launched by expert PCR laboratories from 16 countries. Currently used strategies were challenged against serial dilutions of purified DNA from stocks representing T. cruzi discrete typing units (DTU) I, IV and VI (set A), human blood spiked with parasite cells (set B) and Guanidine Hidrochloride-EDTA blood samples from 32 seropositive and 10 seronegative patients from Southern Cone countries (set C). Forty eight PCR tests were reported for set A and 44 for sets B and C; 28 targeted minicircle DNA (kDNA), 13 satellite DNA (Sat-DNA) and the remainder low copy number sequences. In set A, commercial master mixes and Sat-DNA Real Time PCR showed better specificity, but kDNA-PCR was more sensitive to detect DTU I DNA. In set B, commercial DNA extraction kits presented better specificity than solvent extraction protocols. Sat-DNA PCR tests had higher specificity, with sensitivities of 0.05-0.5 parasites/mL whereas specific kDNA tests detected 5.10(-3) par/mL. Sixteen specific and coherent methods had a Good Performance in both sets A and B (10 fg/µl of DNA from all stocks, 5 par/mL spiked blood). The median values of sensitivities, specificities and accuracies obtained in testing the Set C samples with the 16 tests determined to be good performing by analyzing Sets A and B samples varied considerably. Out of them, four methods depicted the best performing parameters in all three sets of samples, detecting at least 10 fg/µl for each DNA stock, 0.5 par/mL and a sensitivity between 83.3-94.4%, specificity of 85-95%, accuracy of 86.8-89.5% and kappa index of 0.7-0.8 compared to consensus PCR reports of the 16 good performing tests and 63-69%, 100%, 71.4-76.2% and 0.4-0.5, respectively compared to serodiagnosis. Method LbD2 used solvent extraction followed by Sybr-Green based Real time PCR targeted to Sat-DNA; method LbD3 used solvent DNA extraction followed by conventional PCR targeted to Sat-DNA. The third method (LbF1) used glass fiber column based DNA extraction followed by TaqMan Real Time PCR targeted to Sat-DNA (cruzi 1/cruzi 2 and cruzi 3 TaqMan probe) and the fourth method (LbQ) used solvent DNA extraction followed by conventional hot-start PCR targeted to kDNA (primer pairs 121/122). These four methods were further evaluated at the coordinating laboratory in a subset of human blood samples, confirming the performance obtained by the participating laboratories. CONCLUSION/SIGNIFICANCE: This study represents a first crucial step towards international validation of PCR procedures for detection of T. cruzi in human blood samples.

Maternal infection with Trypanosoma cruzi and congenital Chagas disease induce a trend to a type 1 polarization of infant immune responses to vaccines.

BACKGROUND: We previously showed that newborns congenitally infected with Trypanosoma cruzi (M+B+) display a strong type 1 parasite-specific T cell immune response, whereas uninfected newborns from T. cruzi-infected mothers (M+B-) are prone to produce higher levels of proinflammatory cytokines than control neonates (M-B-). The purpose of the present study was to determine if such fetal/neonatal immunological environments could alter the response to standard vaccines administered in early life. METHODOLOGY: Infants (6-7 months old) living in Bolivia, an area highly endemic for T. cruzi infection, and having received Bacillus Calmette Guerin (BCG), hepatitis B virus (HBV), diphtheria and tetanus vaccines, were enrolled into the M+B+, M+B-, M-B- groups mentioned above. The production of IFN-gamma and IL-13, as markers of Th1 and Th2 responses respectively, by peripherical blood mononuclear cells stimulated with tuberculin purified protein derivative of Mycobacterium tuberculosis (PPD) or the vaccinal antigens HBs, diphtheria toxoid (DT) or tetanus toxoid (TT), as well as circulating levels of IgG antibodies against HBsAg, DT and TT were analyzed in infants. Cellular responses to the superantigen SEB were also monitored in M+B+, M+B-, M-B-infants and newborns. PRINCIPAL FINDINGS: M+B+ infants developed a stronger IFN-gamma response to hepatitis B, diphtheria and tetanus vaccines than did M+B- and M-B- groups. They also displayed an enhanced antibody production to HBsAg. This was associated with a type 1-biased immune environment at birth, since cells of M+B+ newborns produced higher IFN-gamma levels in response to SEB. M+B- infants produced more IFN-gamma in response to PPD than the other groups. IL-13 production remained low and similar in all the three groups, whatever the subject's ages or vaccine status. CONCLUSION: These results show that: i) both maternal infection with T. cruzi and congenital Chagas disease do not interfere with responses to BCG, hepatitis B, diphtheria and tetanus vaccines in the neonatal period, and ii) the overcoming of immunological immaturity by T. cruzi infection in early life is not limited to the development of parasite-specific immune responses, but also tends to favour type 1 immune responses to vaccinal antigens.

Epidemiological monitoring of American tegumentary leishmaniasis: molecular characterization of a peridomestic transmission cycle in the Amazonian lowlands of Bolivia.

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.

Are maternal re-infections with Trypanosoma cruzi associated with higher morbidity and mortality of congenital Chagas disease?

BACKGROUND: Comparing two surveys performed in Bolivia in 1992-1994 and 1999-2001, we reported a significant decrease in the proportions of severe and mortal forms of congenital Chagas disease. This might be due to a reduction of vectorial density (VD) in maternal residence area, raising the question of a possible causal relationship between such VD, maternal parasitaemia and prognosis of congenital infection with Trypanosoma cruzi. METHOD: Comparisons of haematological and parasitological data obtained from Bolivian mothers infected with T. cruzi, and of clinical and biological data obtained from their infected and uninfected newborns, stratified according to VD in the area of maternal residence. RESULTS: i) Blood hematocrit rates or hemoglobin amounts were within the normal ranges and similar in all the maternal groups, whatever the VD in their areas of residence; ii) mothers living in high VD areas displayed a higher frequency of hemocultures positive for T. cruzi; iii) newborns congenitally infected with T. cruzi, but not uninfected babies born from infected mothers, displayed higher frequencies of very low Apgar scores, low birth weights, prematurity, respiratory distress syndrome or anasarca, as well as higher mortality rates when their mothers lived in areas of high VD. CONCLUSION: Frequent bites of blood sucking Reduvidae during pregnancy do not induce maternal anaemia, but, likely through multiple maternal re-infections with T. cruzi, increase maternal parasitemia and worsen congenital Chagas disease. Maternal dwelling in areas of high VD is associated with a serious increased risk of severe and mortal congenital Chagas disease.

Genomic changes of Chagas disease vector, South America.

We analyzed the main karyologic changes that have occurred during the dispersion of Triatoma infestans, the main vector of Chagas disease. We identified two allopatric groups, named Andean and non-Andean. The Andean specimens present C-heterochromatic blocks in most of their 22 chromosomes, whereas non-Andean specimens have only 4-7 autosomes with C-banding. These heterochromatin differences are the likely cause of a striking DNA content variation (approximately 30%) between Andean and non-Andean insects. Our study, together with previous historical and genetic data, suggests that T. infestans was originally a sylvatic species, with large quantities of DNA and heterochromatin, inhabiting the Andean region of Bolivia. However, the spread of domestic T. infestans throughout the non-Andean regions only involved insects with an important reduction of heterochromatin and DNA amounts. We propose that heterochromatin and DNA variation mainly reflected adaptive genomic changes that contribute to the ability of T. infestans to survive, reproduce, and disperse in different environments.