Marina Muñoz

Human Papillomavirus Detection from Human Immunodeficiency Virus-Infected Colombian Women's Paired Urine and Cervical Samples

Infection, coinfection and type-specific human papillomavirus (HPV) distribution was evaluated in human immunodeficiency virus (HIV)-positive women from paired cervical and urine samples. Paired cervical and urine samples (n = 204) were taken from HIV-positive women for identifying HPV-DNA presence by using polymerase chain reaction (PCR) with three generic primer sets (GP5+/6+, MY09/11 and pU1M/2R). HPV-positive samples were typed for six high-risk HPV (HR-HPV) (HPV-16, -18, -31, -33, -45 and -58) and two low-risk (LR-HPV) (HPV-6/11) types. Agreement between paired sample results and diagnostic performance was evaluated. HPV infection prevalence was 70.6% in cervical and 63.2% in urine samples. HPV-16 was the most prevalent HPV type in both types of sample (66.7% in cervical samples and 62.0% in urine) followed by HPV-31(47.2%) in cervical samples and HPV-58 (35.7%) in urine samples. There was 55.4% coinfection (infection by more than one type of HPV) in cervical samples and 40.2% in urine samples. Abnormal Papanicolau smears were observed in 25.3% of the women, presenting significant association with HPV-DNA being identified in urine samples. There was poor agreement of cervical and urine sample results in generic and type-specific detection of HPV. Urine samples provided the best diagnosis when taking cytological findings as reference. In conclusion including urine samples could be a good strategy for ensuring adherence to screening programs aimed at reducing the impact of cervical cancer, since this sample is easy to obtain and showed good diagnostic performance.

Determining Clostridium difficile intra-taxa diversity by mining multilocus sequence typing databases

BackgroundMultilocus sequence typing (MLST) is a highly discriminatory typing strategy; it is reproducible and scalable. There is a MLST scheme for Clostridium difficile (CD), a gram positive bacillus causing different pathologies of the gastrointestinal tract. This work was aimed at describing the frequency of sequence types (STs) and Clades (C) reported and evalute the intra-taxa diversity in the CD MLST database (CD-MLST-db) using an MLSA approach.ResultsAnalysis of 1778 available isolates showed that clade 1 (C1) was the most frequent worldwide (57.7%), followed by C2 (29.1%). Regarding sequence types (STs), it was found that ST-1, belonging to C2, was the most frequent. The isolates analysed came from 17 countries, mostly from the United Kingdom (UK) (1541 STs, 87.0%). The diversity of the seven housekeeping genes in the MLST scheme was evaluated, and alleles from the profiles (STs), for identifying CD population structure. It was found that adk and atpA are conserved genes allowing a limited amount of clusters to be discriminated; however, different genes such as drx, glyA and particularly sodA showed high diversity indexes and grouped CD populations in many clusters, suggesting that these genes’ contribution to CD typing should be revised. It was identified that CD STs reported to date have a mostly clonal population structure with foreseen events of recombination; however, one group of STs was not assigned to a clade being highly different containing at least nine well-supported clusters, suggesting a greater amount of clades for CD.ConclusionsThis study shows the usefulness of CD-MLST-db as a tool for studying CD distribution and population structure, identifying the need for reviewing the usefulness of sodA as housekeeping gene within the MLST scheme and suggesting the existence of a greater amount of CD clades. The study also shows the plausible exchange of genetic material between STs, contributing towards intra-taxa genetic diversity.

Classical molecular tests using urine samples as a potential screening tool for human papillomavirus detection in human immunodeficiency virus-infected women.

ABSTRACT Human papillomavirus (HPV) is the main risk factor associated with the development of cervical cancer (CC); however, there are other factors, such as immunosuppression caused by the human immunodeficiency virus (HIV), that favor progression of the illness. This study was thus aimed at evaluating the functionality of classical PCR-based molecular tests for the generic identification of HPV DNA (GP5+/GP6+, MY09/MY11, and pU1M/2R primers, individually or in combination) using cervical and urine samples from 194 HIV-positive women. Infected samples were tested with type-specific primers for six high-risk types (HPV-16, -18, -31, -33, -45, and -58) and two low-risk types (HPV-6 and -11). HPV infection prevalence rates were 70.1% for the cervical samples and 63.9% for the urine samples. HPV-16 was the most prevalent viral type in the cervical and urine samples, with higher rates of multiple infections than single infections detected in such samples. HPV DNA detection by PCR (mainly with the pU1M/2R primer set) in urine samples was positively associated with abnormal cytological findings (atypical squamous cells of undetermined significance/squamous intraepithelial lesions [ASCUS/SIL]). It was determined that the operative characteristics for detection of cytological abnormalities were similar for cervical and urine samples. This suggested using PCR for the detection of HPV DNA in urine samples as a potential screening strategy for CC prevention in future prevention and control programs along with currently implemented strategies for reducing the impact of the disease, i.e., urine samples are economical, are easy to collect, have wide acceptability among women, and have operative characteristics similar to those of cervical samples.

Estimating the Intra-taxa Diversity, Population Genetic Structure, and Evolutionary Pathways of Cryptococcus neoformans and Cryptococcus gattii

Members of the Cryptococcus complex, includes Cryptococcus neoformans (most common fungal infection of the brain) and Cryptococcus gattii (high-impact emerging pathogen worldwide). Currently, the fungal multilocus sequence typing database (Fungal MLST Database) constitutes a valuable data repository of the genes used for molecular typing of these pathogens. We analyzed the data available in the Fungal MLST Database for seven housekeeping genes, with the aim to evaluate its contribution in the description of intra-taxa diversity, population genetic structure, and evolutionary patterns. Although the Fungal MLST Database has a greater number of reports for C. neoformans (n = 487) than for C. gattii (n = 344), similar results were obtained for both species in terms of allelic diversity. Phylogenetic reconstructions revealed grouping by molecular type in both species and allowed us to propose differences in evolutionary patterns (gradualism in the case of C. neoformans and punctuated evolution in the case of C. gattii). In addition, C. neoformans showed a population genetic structure consisting of 37 clonal complexes (CCs; CC1 being predominant), high crosslinking [without sequence type (ST) grouping by molecular type], marked divergence events in phylogenetic analysis, and few introgression events (mainly between VNI and VNIV). By contrast, C. gattii showed 50 CCs (with greater homogeneity in ST number by CC) and clustering by molecular type with marked crosslinking events in phylogenetic networks being less evident. Understanding relationships at the molecular level for species of the Cryptococcus complex, based on the sequences of the housekeeping genes, provides information for describing the evolutionary history of these emerging pathogens.

New Insights into Clostridium difficile (CD) Infection in Latin America: Novel Description of Toxigenic Profiles of Diarrhea-Associated to CD in Bogotá, Colombia

Clostridium difficile (CD) produces antibiotic associated diarrhea and leads to a broad range of diseases. The source of CD infection (CDI) acquisition and toxigenic profile are factors determining the impact of CD. This study aimed at detecting healthcare facility onset- (HCFO) and community-onset (CO) CDI and describing their toxigenic profiles in Bogotá, Colombia. A total of 217 fecal samples from patients suffering diarrhea were simultaneously submitted to two CDI detection strategies: (i) in vitro culture using selective chromogenic medium (SCM; chromID, bioMérieux), followed verification by colony screening (VCS), and (ii) molecular detection targeting constitutive genes, using two conventional PCR tests (conv.PCR) (conv.16S y conv.gdh) and a quantitative test (qPCR.16s). The CD toxigenic profile identified by any molecular test was described using 6 tests independently for describing PaLoc and CdtLoc organization. High overall CDI frequencies were found by both SCM (52.1%) and conv.PCR (45.6% for conv.16S and 42.4% for conv.gdh), compared to reductions of up to half the frequency by VCS (27.2%) or qPCR.16S (22.6%). Infection frequencies were higher for SCM and conv.16S regarding HCFO but greater for CO concerning conv.gdh, such differences being statistically significant. Heterogeneous toxigenic profiles were found, including amplification with lok1/3 primers simultaneously with other PaLoc markers (tcdA, tcdB or tcdC). These findings correspond the first report regarding the differential detection of CDI using in vitro culture and molecular detection tests in Colombia, the circulation of CD having heterogeneous toxigenic profiles and molecular arrays which could affect the impact of CDI epidemiology.

Community-acquired infection with hypervirulent Clostridium difficile isolates that carry different toxin and antibiotic resistance loci: a case report

BackgroundClostridium difficile infection (CDI) leads to the onset of antibiotic-associated diarrhea (AAD) and a wide range of gastrointestinal pathologies. Currently, CDI is one of the most important opportunistic infections at the intrahospital level and an exponential increase in community-acquired infections has been reported. Herein, we evaluated the relationships (at phylogenetic and genetic population structure levels), as well as the molecular toxigenic and antibiotic resistance profiles of a set of isolates established from a case of community acquired-CDI.Case presentationA 30-year-old woman with no history of hospitalization who was exposed to antibiotics (ampicillin/sulbactam and metronidazole) after a cat-bite wound was presented. The patient had a continuous episode of diarrhea; a stool sample was then collected and community acquired-CDI was confirmed by molecular tests and in vitro culture. Seven isolates were established and subsequently subjected to: (i) Multilocus sequence typing, all isolates belonging to ST-1 (associated with hypervirulent strain (027/BI/NAP1); (ii) description of their toxigenic profile: two of the isolates (Gcol.49 and Gcol.91) were positive for the genes coding for the major toxins (tcdA and tcdB) and their negative regulator (tcdC). All isolates were positive for the cdtB gene encoding one of the binary toxin subunits, while only two (Gcol.51 and Gcol.52) were positive for cdtA; and (iii) identification of antibiotic resistance molecular markers, where there was no difference in gyrA or gyrB gene polymorphisms (related to quinolone resistance), but rather at loci presence/absence, being just one isolate negative, whereas the others showed a differential presence of the tet, ermB and Tn916 regions. The former was associated with resistance to tetracycline and the other two for erythromycin/clindamycin.ConclusionsThis case represents the first report of community acquired-CDI in Colombia associated with hypervirulent strains and shows that isolates obtained from a single patient can carry different toxin and antibiotic resistance loci.