Abstract Trypanosoma cruzi is the etiological agent of Chagas disease, a public health challenge due to its morbidity and mortality rates, which affects around 6-7 million people worldwide. Symptoms, response to chemotherapy, and the course of Chagas disease are greatly influenced by T. cruzi‘s intra-specific variability. Thus, DNA mutations in this parasite possibly play a key role in the wide range of clinical manifestations and in drug sensitivity. Indeed, the environmental conditions of oxidative stress faced by T. cruzi during its life cycle can generate genetic mutations. However, the lack of an established experimental design to assess mutation rates in T. cruzi precludes the study of conditions and mechanisms that potentially produce genomic variability in this parasite. We developed an assay that employs a reporter gene that, once mutated in specific positions, convert G418-sensitive into G418-insenstitive T. cruzi. We were able to determine the frequency of DNA mutations in T. cruzi exposed and non-exposed to oxidative insults assessing the number of colony-forming units in solid selective media after plating a defined number of cells. We verified that T. cruzi‘s spontaneous mutation frequency was comparable to those found in other eukaryotes, and that exposure to hydrogen peroxide promoted a two-fold increase in T. cruzi‘s mutation frequency. We hypothesize that genetic mutations in T. cruzi can arise from oxidative insults faced by this parasite during its life cycle.

Amostras de displasias ou carcinomas do colo uterino e de condilomas acuminados da região genital foram analisados retrospectivamente por hibridização in situ (HIS) com sondas de DNA biotiniladas de papilomavirus humano (HPV) tipos 6, 11, 16 e 18. Nenhum caso do grupo controle foi positivo para DNA de HPV. Em displasias leve/moderada, 4 casos (14%) foram positivos para HPV 6 ou 11 e 2 casos (7%), para HPV 16. No grupo de displasia acentuada/carcinoma in situ, 9 casos (31%) tinham DNA de HPV tipos 16 ou 18. Seis carcinomas invasores (20%) foram positivos para HPV tipos 16 ou 18. Entre os condilomas acuminados, 22 casos (73%) foram positivos para HPV tipos 6 ou 11. Em todos os casos positivos pela HIS somente um tipo viral foi encontrado. Não foi observada correlação entre a positividade para DNA de HPV e achados histológicos de infecção por HPV. Apesar de menos sensível que algumas outras técnicas de biologia molecular, a hibridização in situ com sondas de DNA biotiniladas mostrou-se simples e adequada para detecção e tipagem de HPV em amostras enviadas rotineiramente para estudo histopatológico.

Specimens from cervical dysplasias or carcinomas and genital condylomata acuminata were retrospectively analysed by in situ hybridization (ISH) with bioti-nylated DNA probes for human papillomavirus (HPV) types 6, 11, 16 and 18. In the control group no case was positive for HPV DNA. In mild/moderate dysplasias, 4 cases (14%) were positive for HPV 6 or 11 and 2 cases (7%), for HPV 16. In the severe dysplasia/in situ carcinoma group, 9 cases (31%) showed presence of DNA of HPV types 16 or 18. Six invasive carcinomas (20%) were positive for HPV type 16 or 18. Among condylomata acuminata, 22 cases (73%) were positive for HPV types 6 or 11. In all ISH-positive cases only one viral type was detected. No correlation between HPV DNA positivity and histological findings of HPV infection was observed. Although less sensitive than some other molecular biology techniques, in situ hybridization with biotinylated DNA probes proved to be simple and useful for detecting and typing HPV in samples routinely received for histopathological analysis.