Sylvia Ortiz

DNA evidence of Trypanosoma cruzi in the Chilean wild vector Mepraia spinolai (Hemiptera: Reduviidae).

Molecular evidence showed 46.2% of Trypanosoma cruzi infection in Mepraia spinolai insects from North-Central Chile, which is significantly higher than previous reports of up to 26% by microscopic observation. Our results show similar infection levels among nymphal stages, ranging from 38.3 to 54.1%, indicating that younger nymphs could be as important as older ones in parasite transmission. A cautionary note must be stressed to indicate the potential role of M. spinolai in transmitting T. cruzi in country areas due to the high infection level detected by molecular analysis.

Trypanosoma cruzi isolates from Chile are heterogeneous and composed of mixed populations when characterized by schizodeme and Southern analyses.

In total, 61 Chilean isolates of Trypanosoma cruzi, were analysed using schizodeme and Southern analysis, using as probes the highly variable regions of minicircles from cloned parasites. Isolates were collected and amplified from domestic and wild triatomines, and from infected subjects in all the endemic areas of Chile. Three major parasite genotypes could be detected in the domestic transmission cycle, whilst 1 major T. cruzi genotype is circulating in the wild transmission cycle. Schizodeme analysis suggested that T. cruzi isolates are mixed populations, whereas the Southern analyses detected only 3 mixed isolates using 4 selected minicircle segments as probes.

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.

Evaluation of Nifurtimox Treatment of Chronic Chagas Disease by Means of Several Parasitological Methods

ABSTRACT Currently, evaluation of drug efficacy for Chagas disease remains a controversial issue with no consensus. In this work, we evaluated the parasitological efficacy of Nifurtimox treatment in 21 women with chronic Chagas disease from an area of endemicity in Chile who were treated according to current protocols. Under pre- and posttherapy conditions, blood (B) samples and xenodiagnosis (XD) samples from these patients were subjected to analysis by real-time PCR targeting the nuclear satellite DNA of Trypanosoma cruzi (Sat DNA PCR-B, Sat DNA PCR-XD) and by PCR targeting the minicircle of kinetoplast DNA of T. cruzi (kDNA PCR-B, kDNA PCR-XD) and by T. cruzi genotyping using hybridization minicircle tests in blood and fecal samples of Triatoma infestans feed by XD. In pretherapy, kDNA PCR-B and kDNA PCR-XD detected T. cruzi in 12 (57%) and 18 (86%) cases, respectively, whereas Sat DNA quantitative PCR-B (qPCR-B) and Sat DNA qPCR-XD were positive in 18 cases (86%) each. Regarding T. cruzi genotype analysis, it was possible to observe in pretherapy the combination of TcI, TcII, and TcV lineages, including mixtures of T. cruzi strains in most of the cases. At 13 months posttherapy, T. cruzi DNA was detectable in 6 cases (29.6%) and 4 cases (19.1%) by means of Sat DNA PCR-XD and kDNA PCR-XD, respectively, indicating treatment failure with recovery of live parasites refractory to chemotherapy. In 3 cases, it was possible to identify persistence of the baseline genotypes. The remaining 15 baseline PCR-positive cases gave negative results by all molecular and parasitological methods at 13 months posttreatment, suggesting parasite response. Within this follow-up period, kDNA PCR-XD and Sat DNA qPCR-XD proved to be more sensitive tools for the parasitological evaluation of the efficacy of Nifurtimox treatment than the corresponding PCR methods performed directly from blood samples.

Molecular Epidemiology of Chagas Disease in the Wild Transmission Cycle: The Evaluation in the Sylvatic Vector Mepraia spinolai from an Endemic Area of Chile

The sylvatic transmission cycle of Chagas disease in Chile is composed of wild mammals and insects of the genus Mepraia. We determined infection rates and Trypanosoma cruzi genotypes in Mepraia spinolai. We collected 227 insects from two ecologically contrasting areas to assess T. cruzi infection. Polymerase chain reaction (PCR)-amplified minicircle DNAs were characterized by Southern blot and hybridization tests with genotype-specific probes. Infection in insects from the more fertile area was almost 2-fold higher than in the poorer area. The genotype TCI was the most prevalent and other genotypes such as TCIIb, TCIId, and TCIIe were found at lower rates. The areas differed in their genotype distribution but not in their genotype diversity. We suggest that the difference in abundance and richness of mammals between the areas may be producing the detected infection levels in vectors. Our results are compared with those reported for mammals from the same area.

Predominance of Trypanosoma cruzi genotypes in two reservoirs infected by sylvatic Triatoma infestans of an endemic area of Chile

We report results of Trypanosoma cruzi infection and parasite genotypes in the wild Octodon degus and synantropic reservoir Rattus rattus from an endemic area with sylvatic Triatoma infestans as the only detected vector. The infection status was determined by hemi-nested PCR directed to minicircles DNA and genotyping by hybridization tests with a panel of five specific probes, including two probes for TcI subgroups (clones 19 and 20). O. degus was found infected with 13.3% and mainly with sublineage TcIId, and less with TcIIb and TcI. Meantime the synantropic R. rattus was found infected with 27.7% and mainly with TcI and much less with TcIId, TcIIb and TcIIe. The results are discussed to explain the distribution of T. cruzi genotypes between these two reservoirs and the importance of sylvatic foci of T. infestans allowing the permanence of the wild and peridomestic cycle of Chagas disease.

Trypanosoma cruzi Genotypes of Insect Vectors and Patients with Chagas of Chile Studied by Means of Cytochrome b Gene Sequencing, Minicircle Hybridization, and Nuclear Gene Polymorphisms

Fifty-six Trypanosoma cruzi stocks from Chile and neighboring countries and different hosts, humans, and Triatoma infestans and Mepraia sp., vectors of domiciliary and natural environments were characterized by using three molecular markers. These were cytochrome b (Cyt b) gene sequencing, minicircle DNA blotting, and hybridization with five genotype-specific DNA probes and nuclear analysis of 1f8 and gp72 by polymerase chain reaction-restriction fragment length polymorphism. The results with all three molecular markers are concordant, with minor limitations, grouping T. cruzi stocks into four discrete typing units (DTUs) (TcI, TcII, TcV, and TcVI). TcI and TcII stocks were heterogeneous. TcI and TcII stocks were clustered in two main subgroups determined by Cyt b gene sequencing and minicircle hybridization. However, TcV and TcVI stocks were homogeneous and not differentiated by Cyt b gene sequencing or minicircle DNA hybridization. The discriminatory power and limitations of the molecular markers are discussed, as well as the distribution of the four DTUs in the domiciliary and sylvatic transmission cycles of Chile and the limitations of each marker for molecular epidemiological studies performed with T. cruzi stocks rather than the analysis of direct T. cruzi samples from natural hosts.

Presence of Trypanosoma cruzi in pregnant women and typing of lineages in congenital cases.

The objective of this study was to determine the presence of Trypanosoma cruzi in blood samples of mothers with chronic Chagas disease and their newborn by conventional PCR targeted to minicircle kinetoplastidic DNA (kDNA), and to determine the lineages in mother/newborn pairs of the congenital cases by hybridization assays with probes belonging to the TcII, TcI and TcV Discrete Typing Units (DTU). In 63 (57.2%) of the mothers the presence of circulating T. cruzi was demonstrated by PCR immediately before delivery and in three newborn (3%) congenital transmission was confirmed by serial PCR and conventional serology between 1 and 16 months of life, at which point treatment was started. The hybridization signals showed that two of the newborn had the same DTU as their mother (TcI, TcII and TcV), whilst in the third congenital case only TcV was detected in the cord blood, suggesting that in this infant TcI and TcII did not cross the placenta or the parasite was not present at a detectable level. Levels T. cruzi DNA was determined by TaqMan Probe based Real Time PCR assay targeted to nuclear satellite sequences in these three pairs of samples.

Trypanosoma cruzi infection in the sylvatic kissing bug Mepraia gajardoi from the Chilean Southern Pacific Ocean coast.

The Southern Pacific Ocean coast has been traditionally considered a non-active transmission area for Chagas disease. In this report, we show evidence of Trypanosoma cruzi infection in the sylvatic kissing bug Mepraia gajardoi from the northern Chilean coast.

Trypanosoma cruzi Genotypes in Mepraia gajardoi from Wild Ecotopes in Northern Chile

We evaluated Trypanosoma cruzi infection in 397 wild Mepraia gajardoi specimens from five coastal localities in northern Chile by detection of minicircle DNA by polymerase chain reaction. The wild capture sites were classified into two ecotopes: a fully wild ecotope (ecotope 1) and a wild ecotope near human dwellings (ecotope 2). Infection rates varied between 11% and 27%. Minicircle hybridization assays showed that T. cruzi lineages Tc II and Tc VI were commonly detected in ecotope 1 and ecotope 2, respectively. These results are discussed in the context of insect proximity to human dwellings, the alimentary profile of Mepraia sp., T. cruzi lineages detected in the past in the same disease-endemic area circulating in humans, and Triatoma infestans.