Relationship of spermatoscopy, prostatic acid phosphatase activity and prostate-specific antigen (p30) assays with further DNA typing in forensic samples from rape cases.

In the forensic laboratory the biological analyses for rape investigation commonly include vaginal swabs as sample material combined to biochemical tests including sperm cytology (SC) and detection of acid phosphatase activity (AP) and prostate-specific antigen (PSA, p30) for the conclusive identification of semen components. Most reports comparing these tests relied on analysis of semen samples or donor swabs taken under controlled conditions; however their individual or combined efficacy under real live sampling conditions in different laboratories is largely unknown. We carried out SC, APA and PSA analyses in vaginal swabs collected from casework rapes submitted to Mexican Forensic Laboratories at Texcoco and Toluca. On the basis of positive and negative results from each assay and sample, data were classified into eight categories (I-VIII) and compared with those obtained in the two only similar studies reported in Toronto, Canada and Hong Kong, China. SC and APA assays had the higher overall positivity in Toluca and Texcoco samples respectively and otherwise PSA had a lower but very similar positivity between these two laboratories. When compared to the previous studies some similarities were found, namely similar frequencies (at a ratio of approximately 1 out of 3) of samples being positive or negative by all techniques (Categories I and VI respectively) and a comparable overall positivity of APA and SC but higher than that of PSA. Indeed the combined results of using SC, APA and PSA tests was considered as conclusive for semen detection from approximately 1 out of 3 cases (Category I) to approximately 1 out of 2 cases in a scenario where at least SC is positive, strongly presumptive in 2 out of 3 cases (with at least one test positive) and the remainder 1 out of 3 cases (Category VI) suggested absence of semen. By determining Y-STR polymorphisms (12-loci) in additional samples obtained at Toluca laboratory, complete DNA profiles were determined from all Category I samples, none marker was detected from all Category VI samples and mostly partial profiles were obtained from samples of other categories. These observations give an overview on the variability in efficacy of each test performed at different laboratories and provide a general notion about the in praxis contribution of SC, APA and PSA tests for further DNA typing in the forensic analysis of rape.

In vitro resistance to 5-nitroimidazoles and benzimidazoles in Giardia duodenalis: variability and variation in gene expression.

The susceptibility of Giardia duodenalis trophozoites exposed in vitro to sublethal concentrations of metronidazole (MTZ) and albendazole (ABZ) may exhibit inter-culture (variability) and intra-culture (variation) differences in drug susceptibility. It was previously reported that MTZ-resistant trophozoites may display changes in pyruvate:ferredoxin oxidoreductase (PFOR) expression while changes at the beta-tubulin molecule are apparently absent in ABZ-resistant cultures. To assess the levels of gene expression of these molecules, we obtained cloned cultures growing at concentrations up to 23 microM MTZ (WBRM23) and up to 8muM ABZ (WBRA8) and gene sequence and expression of pfor and beta-tubulin loci were compared with these of drug-susceptible clone WB1. Neither the pfor nor the beta-tubulin genes showed changes at sequence level but the MTZ-resistant clones WBRM21 and WBRM23 showed up-regulation of the pfor RNA using the gdh gene as reference. By using WB1 and WBRA8 clones in representational difference analyses of gene expression (RDA) an insert referred to as ARR-VSP was selected and sequenced. It showed the highest homology to one VSP molecule in the Giardia Genome Database (orf GL50803_101765). This isogene was up-regulated in five ABZ-resistant clones and the clone WBRA8 exhibited the highest RNA expression level. When successive progenies of clones WB1, WBRM23 and WBRA8 were analyzed in Northern blot assays to detect pfor and ARR-VSP RNAs respectively, the expression patterns showed variation for both genes but it was much lower in the clone WBRA8. These results suggest that G. duodenalis cultures either susceptible or resistant to MTZ and ABZ may display variability and variation at RNA expression levels albeit these were more marked in the MTZ-resistant parasites. These data might have further implications defining major mechanisms involved in drug resistance of Giardia.

Variability and variation in drug susceptibility among Giardia duodenalis isolates and clones exposed to 5-nitroimidazoles and benzimidazoles in vitro.

OBJECTIVES: We analysed, in a cell-by-cell study, the in vitro susceptibility of Giardia duodenalis strains, including Mexican isolates and their clones to 5-nitroimidazoles and benzimidazoles. METHODS: Fluorogenic dye staining (FDA-PI) and cell morphology (CM) assays, two fast and direct techniques, replaced the indirect 'gold standard' method (subculture in liquid medium) in the evaluation of 5-nitroimidazoles and benzimidazoles, respectively. RESULTS: Under these conditions, the activity of several 5-nitroimidazole and benzimidazole derivatives was consistent with their known efficacy, but parasite stocks exhibited a greater variability in response to 5-nitroimidazoles compared with benzimidazoles. Also, consecutive progenies from single stocks maintained in continuous subculture in drug-free media displayed changes (variations) in the proportions of drug resistant (R/T) subpopulations when exposed to sublethal concentrations of 5-nitroimidazoles and benzimidazoles. These were again more variable upon exposure to 5-nitroimidazoles than to benzimidazoles. Variations were not due to drug susceptibility shifts in parent trophozoites since analysis of cytokinetic processes showed a predominant pattern of susceptible/susceptible or resistant/resistant daughters, whereas susceptible/resistant daughters were scarce. CONCLUSIONS: Our observations support the idea that G. duodenalis cultures exhibit variations in their response to 5-nitroimidazoles and benzimidazoles as a result of a drug-independent competition between drug-susceptible and drug-resistant subpopulations when parasites are subcultured.