Assessment of Lactobacillus species colonizing the vagina of apparently healthy Nigerian women, using PCR-DGGE and 16S rRNA gene sequencing

The Cellient™ Automated Cell Block System (Hologic) can be used to analyze cells of HPV-positive cervical scrapes staining positive with the biomarker p16. For this study fourteen cervical scrapes of Tanzanian women infected with HIV testing positive for HPV were selected. The paraffin Cellient sections were stained with the Papanicolaou method, with hematoxylin eosin (HE), and with the biomarker p16. This pilot study was limited to cases classified as atypical squamous lesion of unknown significance (ASCUS) and high-grade squamous lesion (HSIL) as diagnosed in the ThinPrep slide. The Cellient paraffin sections (cut from paraffin blocks prepared from the residual cervical sample) were classified into negative, atypical, CIN 1, CIN 2, and CIN 3. Multiple HPV genotypes were encountered in 79% of the scrapes. HPV16 was found in six scrapes and HPV52 in four. In the Papanicolaou sections, it was easy to detect dyskeratotic cells. Eleven of the 14 cases were p16 positive and five contained p16 positive dyskeratocytes. Of the 10 ASCUS scrapes, two contained p16 positive CIN 1 epithelial fragments. All four HSIL cases contained p16 positive CIN 3 epithelial fragments. In HIV-positive HPV-positive women, the Cellient system resulted in high quality histology sections with perfect p16 images of dyskeratocytes. © 2012 Boon et al.; Licensee Lifescience Global

Biofilms in infectious disease and on medical devices

Objective: The objective was to examine the use of a tailor-made DNA microarray containing probes representing the vaginal microbiota to examine bacterial vaginosis. Study Design: One hundred one women attending a health center for HIV testing in South Africa were enrolled. Stained, liquid-based cytology slides were scored for bacterial vaginosis. An inventory of organisms was obtained using microarray technology, probing genera associated with bacterial vaginosis in more detail, namely Gardnerella, Atopobium, Dialister, Leptotrichia, Megasphaera, Mobiluncus, Peptostreptococcus, Prevotella, and Sneathia. Results: Of 101 women, 34 were diagnosed positive for bacterial vaginosis. This condition was associated with an increased microbial diversity. It is no longer useful to base the diagnosis of bacterial vaginosis on Gardnerella alone. Rather, its presence with Leptotrichia and Prevotella species, and especially Atopobium was more indicative of an aberrant state of the vaginal flora. Conclusion: To understand the vaginal microbiota in more detail, microarray-based identification can be used after microscopic scoring. © 2011 Mosby, Inc. All rights reserved

Aggregation by fragilis and non-fragilis Bacteroides strains in vitro

Vaginal lactobacilli assessed by PCR-based microarray and PCR-based genotyping of HPV in South African women at risk for HIV and BV. Vaginal lactobacilli can be defined by microarray techniques in fixed cervical samples of South African women. Cervical brush samples suspended in the coagulant fixative BoonFix of one hundred women attending a health centre for HIV testing in South Africa were available for this study. In the Ndlovu Medical Centre in Elandsdoorn, South Africa, identification of 18 hr-HPV genotypes was done using the INNO-LiPA method. An inventory of lactobacilli organisms was performed using microarray technology. On the basis of the Lactobacillus and Lactobacillus biofilm scoring, the cases were identified as Leiden bacterial vaginosis (BV) negative (BV-; n = 41), Leiden BV intermediate (BV±; n = 25), and Leiden BV positive (BV+; n = 34). Fifty-one women were HIV positive and 49 HIV negative. Out of the 51 HIV positive women, 35 were HPV infected. These 51 HIV positive women were frequently infected with HPV16 and HPV18. In addition, HPV35, HPV52, HPV33, and HPV66 were often detected in these samples. Lactobacillus salivarius and Lactobacillus iners were the most prevalent lactobacilli as established by the microarray technique. In women with HPV infection, the prevalence of Lactobacillus crispatus was significantly reduced. In both HIV and HPV infection, a similar (but not identical) shift in the composition of the lactobacillus flora was observed. We conclude that there is a shift in the composition of vaginal lactobacilli in HIV-infected women. Because of the prominence of HPV35, HPV52, HPV33, and HPV66, vaccination for exclusively HPV16 and HPV18 might be insufficient in South African HIV+ women. Copyright © 2012 Wiley Periodicals, Inc

Molecular assessment of bacterial vaginosis by Lactobacillus abundance and species diversity

Background: To date, women are most often diagnosed with bacterial vaginosis (BV) using microscopy based Nugent scoring or Amsel criteria. However, the accuracy is less than optimal. The aim of the present study was to confirm the identity of known BV-associated composition profiles and evaluate indicators for BV using three molecular methods. Methods: Evaluation of indicators for BV was carried out by 16S rRNA amplicon sequencing of the V5-V7 region, a tailor-made 16S rRNA oligonucleotide-based microarray, and a PCR-based profiling technique termed IS-profiling, which is based on fragment variability of the 16S-23S rRNA intergenic spacer region. An inventory of vaginal bacterial species was obtained from 40 females attending a Dutch sexually transmitted infection outpatient clinic, of which 20 diagnosed with BV (Nugent score 7–10), and 20 BV negative (Nugent score 0–3). Results: Analysis of the bacterial communities by 16S rRNA amplicon sequencing revealed two clusters in the BV negative women, dominated by either Lactobacillus iners or Lactobacillus crispatus and three distinct clusters in the BV positive women. In the former, there was a virtually complete, negative correlation between L. crispatus and L. iners. BV positive subjects showed cluster profiles that were relatively high in bacterial species diversity and dominated by anaerobic species, including Gardnerella vaginalis, and those belonging to the Families of Lachnospiraceae and Leptotrichiaceae. Accordingly, the Gini-Simpson index of species diversity, and the relative abundance Lactobacillus species appeared consistent indicators for BV. Under the conditions used, only the 16S rRNA amplicon sequencing method was suitable to assess species diversity, while all three molecular composition profiling methods were able to indicate Lactobacillus abundance in the vaginal microbiota. Conclusion: An affordable and simple molecular test showing a depletion of the genus Lactobacillus in combination with an increased species diversity of vaginal microbiota could serve as an alternative and practical diagnostic method for the assessment of BV