The intergenic region of spliced-leader (SL-IR) genes from 105 Trypanosoma cruzi I (Tc I) infected biological samples, culture isolates and stocks from 11 endemic countries, from Argentina to the USA were characterised, allowing identification of 76 genotypes with 54 polymorphic sites from 123 aligned sequences. On the basis of the microsatellite motif proposed by Herrera et al. (2007) to define four haplotypes in Colombia, we could classify these genotypes into four distinct Tc I SL-IR groups, three corresponding to the former haplotypes Ia (11 genotypes), Ib (11 genotypes) and Id (35 genotypes); and one novel group, Ie (19 genotypes). Genotypes harboring the Tc Ic motif were not detected in our study. Tc Ia was associated with domestic cycles in southern and northern South America and sylvatic cycles in Central and North America. Tc Ib was found in all transmission cycles from Colombia. Tc Id was identified in all transmission cycles from Argentina and Colombia, including Chagas cardiomyopathy patients, sylvatic Brazilian samples and human cases from French Guiana, Panama and Venezuela. Tc Ie gathered five samples from domestic Triatoma infestans from northern Argentina, nine samples from wild Mepraia spinolai and Mepraia gajardoi and two chagasic patients from Chile and one from a Bolivian patient with chagasic reactivation. Mixed infections by Tc Ia + Tc Id, Tc Ia + Tc Ie and Tc Id + Tc Ie were detected in vector faeces and isolates from human and vector samples. In addition, Tc Ia and Tc Id were identified in different tissues from a heart transplanted Chagas cardiomyopathy patient with reactivation, denoting histotropism. Trypanosoma cruzi I SL-IR genotypes from parasites infecting Triatoma gerstaeckeri and Didelphis virginiana from USA, T. infestans from Paraguay, Rhodnius nasutus and Rhodnius neglectus from Brazil and M. spinolai and M. gajardoi from Chile are to our knowledge described for the first time.

Abstract. Epidemiologic evidence suggests a preferential association of Trypanosoma cruzi genotypes TCI and TCII with marsupials and placental mammals, respectively. We identify T. cruzi genotypes from 117 infected mammals. Minicircle DNA amplified by polymerase chain reaction and hybridization with a panel of four specific probes showed frequencies for the T. cruzi genotypes TCI, TCIIb, TCIId, and TCIIe of 38%, 41%, 26%, and 9%, respectively, in wild mammals. In peridomestic mammals, frequencies for the same clones were 29%, 33%, 43%, and 14%, respectively. As a whole, mixed infections are found in more than 31% of the cases, which indicates the coexistence of multiclonal strains circulating in nature, and the absence of specific associations between T. cruzi genotypes and reservoir hosts, including marsupials. The direct characterization of parasite genotypes emphasizes the importance of obtaining unbiased epidemiologic information from parasite-endemic areas. Results are discussed in the context of competition or facilitation of T. cruzi genotypes within hosts.

Patients suffering from Chagas' disease, as determined by positive serological results, were tested for further evidence of Trypanosoma cruzi infection by xenodiagnosis and PCR. The patients included 67 children aged from 0 to 10 years and 75 adults. All children were positive by PCR on their pre-therapy sample, while only 69% of the seropositive adults and none of the 78 seronegative control adults were PCR positive. Xenodiagnosis was positive in 79% of the children, but only in 21% of the adults. A group of 66 children was treated with nifurtimox, and followed up every 3 months during the first year and every 6 months during the second and third year post-therapy, by PCR, xenodiagnosis and serology. We concluded that PCR was the most effective test to monitor children for 3 years post-chemotherapy, when all the cases converted from positive to negative. Conventional serology, however, remained positive after that period in most cases. In contrast, conversion to negative xenodiagnosis occurred very early after treatment.

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Eleven years after they had been given itraconazole or allopurinol for the treatment of chronic American trypanosomiasis, 109 adult patients were checked for electrocardiographic abnormalities and evidence of Trypanosoma cruzi infection. The parasitological investigations included xenodiagnosis, in which the faeces of Triatoma infestans that had fed on the patients were checked under the microscope for flagellates. In addition, a PCR-based assay and a hybridization assay were used to test blood samples from the patients, and faeces from the Tri. infestans that had fed on the patients, for Try. cruzi DNA. For the data analysis, the patients were divided into four groups known as normal/normal, abnormal/normal, normal/abnormal and abnormal/abnormal, according to whether the patients had been found to have normal or abnormal electrocardiograms (ECG) shortly before the first treatment and to have normal or abnormal ECG when checked at the 11-year follow-up. The 51 normal/normal and 24 normal/abnormal patients were assumed to have been in the 'indeterminate' phase of the disease when they were treated, whereas the 16 abnormal/normal and 18 abnormal/abnormal patients all had evidence of chagasic cardiopathy at that time. When checked 11 years post-treatment, 40 (78.4%), 17 (70.8%), 14 (87.5%) and 17 (94.4%) of these patients, respectively, were each found positive for Try. cruzi in at least one of the parasitological tests. The hybridization assay, whether applied to human blood or bug faeces, appeared a significantly more sensitive test than the PCR-based assays or microscopically assessed xenodiagnosis (P<0.05). Only the 21 patients who appeared to be negative for Try. cruzi could be considered parasitologically cured (although all still appeared to have anti-Try. cruzi antibodies in their blood). Only 13 of these parasitologically cured patients (seven of those treated with itraconazole and six of those given allopurinol) had normal ECG at the 11-year follow-up. In Chile at least, itraconazole, which caused fewer adverse effects than the allopurinol while being no less effective at preventing cardiopathy, appears to be the drug of choice to treat chronic American trypanosomiasis in adults.

To identify Trypanosoma cruzi clones from chronically infected individuals, they were transferred to triatomines by the xenodiagnosis test (XD) with Triatoma infestans. Polymerase chain reaction (PCR) and hybridization assays were performed to detect minicircle DNA in human blood samples and triatomine feces, using probes to determine the T. cruzi clones present. T. cruzi clone 19 (TcI) resulted the most prevalent in humans, with a frequency of 0.70 compared with a frequency of 0.53 in triatomines. T. cruzi clone 39 (TcIId) was the most prevalent in T. infestans, with a frequency of 0.65 compared with 0.33 in humans. The T. cruzi clone 43 (TcIIe) was not detected in blood samples; nevertheless, it was present at a rate of 0.17 in T. infestans feces. In conclusion, the T. cruzi clones are associated to each host, suggesting that selective amplification of clones occurs in human and triatomines.

MLP analysis represents a new alternative to existing molecular methods for T. cruzi typing. It might offer an invaluable support to clinical and epidemiological studies and to control programs.

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