Flow cytometric quantification of bacteria in vaginal swab samples self-collected by adolescents attending a gynecology clinic

Schellenberg, John; Ball, T. Blake; Lane, Michael; Cheang, Mary; Plummer, Frank

doi:10.1016/j.mimet.2008.03.004

Cited by 16 publications

(13 citation statements)

References 58 publications

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“…A separate swab was taken and rolled onto a glass slide for evaluation of BV using the criteria of Nugent (24). Swab heads were snipped into freezing buffer and stored at Ϫ80°C until DNA extraction as described previously (27). Sample DNA was extracted by using Instagene Matrix (Bio-Rad, Mississauga, Ontario, Canada).…”

Section: Methodsmentioning

confidence: 99%

Pyrosequencing of the Chaperonin-60 Universal Target as a Tool for Determining Microbial Community Composition

Schellenberg

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Hill

et al. 2009

Appl Environ Microbiol

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We compared dideoxy sequencing of cloned chaperonin-60 universal target (cpn60 UT) amplicons to pyrosequencing of amplicons derived from vaginal microbial communities. In samples pooled from a number of individuals, the pyrosequencing method produced a data set that included virtually all of the sequences that were found within the clone library and revealed an additional level of taxonomic richness. However, the relative abundances of the sequences were different in the two datasets. These observations were expanded and confirmed by the analysis of paired clone library and pyrosequencing datasets from vaginal swabs taken from four individuals. Both for individuals with a normal vaginal microbiota and for those with bacterial vaginosis, the pyrosequencing method revealed a large number of low-abundance taxa that were missed by the clone library approach. In addition, we showed that the pyrosequencing method generates a reproducible profile of microbial community structure in replicate amplifications from the same community. We also compared the taxonomic composition of a vaginal microbial community determined by pyrosequencing of 16S rRNA amplicons to that obtained using cpn60 universal primers. We found that the profiles generated by the two molecular targets were highly similar, with slight differences in the proportional representation of the taxa detected. However, the number of operational taxonomic units was significantly higher in the cpn60 data set, suggesting that the protein-encoding gene provides improved species resolution over the 16S rRNA target. These observations demonstrate that pyrosequencing of cpn60 UT amplicons provides a robust, reliable method for deep sequencing of microbial communities.

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Section: Methodsmentioning

confidence: 99%

Pyrosequencing of the Chaperonin-60 Universal Target as a Tool for Determining Microbial Community Composition

Schellenberg

Links

Hill

et al. 2009

Appl Environ Microbiol

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100

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“…Samples were collected from African women as part of a larger study, ongoing since the 1980s, examining the factors associated with resistance to human immunodeficiency virus infection in a cohort of commercial sex workers in Nairobi, Kenya (22). Other samples were collected from young women from North America within the scope of a research program aimed at developing methodologies for studying the vaginal microbiota (36). The study procedures were reviewed and approved by the Health Research Ethics Boards of the University of Manitoba and the University of Nairobi.…”

Section: Methodsmentioning

confidence: 99%

“…For the samples from Africa, swabs of the midvaginal canal were collected at the time of speculum examination and were rolled onto a glass slide, air dried, stained with Gram stain reagents, and scored by using the criteria of Nugent et al (29) for the laboratory diagnosis of BV by two trained and experienced evaluators. Samples were collected from young women in Canada, and Gram-stained slides were evaluated by using the criteria of Ison and Hay (20), as described previously (36). For both study groups, a duplicate vaginal swab was collected simultaneously.…”

Section: Methodsmentioning

confidence: 99%

Multiplex Detection of Bacteria Associated with Normal Microbiota and with Bacterial Vaginosis in Vaginal Swabs by Use of Oligonucleotide-Coupled Fluorescent Microspheres

et al. 2009

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Bacterial vaginosis (BV) is a recurrent condition that is associated with a range of negative outcomes, including the acquisition of human immunodeficiency virus and other sexually transmitted diseases, preterm births, and pelvic inflammatory disease. In contrast to the Lactobacillus-dominated normal vaginal microbiota, BV is characterized by a lack of lactobacilli and an abundance of anaerobic and gram-negative organisms, including Gardnerella vaginalis and Atopobium vaginae. To date, the laboratory diagnosis of BV has relied upon the fulfillment of criteria determined by microscopic observation of Gram-stained vaginal swabs. We describe a molecular-based method for the easy determination of the species profile within the vaginal microbiota based on the amplification of the chaperonin-60 genes of all bacteria present in the swab and hybridization of the amplicon to species-specific oligonucleotide-coupled fluorescent beads that are identified by flow cytometry with a Luminex instrument. We designed a nineplex Luminex array for characterization of the vaginal microbiota and applied it to the analysis of vaginal swabs from individuals from Africa and North America. Using the presence of A. vaginae or G. vaginalis, or both, as the defining criterion for BV, we found that the method was highly specific and sensitive for the diagnosis of BV using microscopy as a gold standard.Bacterial vaginosis (BV), which is defined by a reduction in the vaginal Lactobacillus populations and an increase in the number of microbial species present, including Gardnerella spp., Atopobium spp., and others, is increasingly recognized as an important risk factor for adverse reproductive health outcomes, such as miscarriage and premature birth, and sexually transmitted diseases, including human immunodeficiency virus infection (25). Despite intense investigation, the etiology and clinical course of BV have not been well defined, probably because the normal variation of the vaginal microbial communities between individuals and their dynamics over time are complex and poorly understood. Consequently, the accurate diagnosis of BV, as well as the elaboration of effective prevention and treatment strategies, remains a major challenge (26, 44).The current "gold standard" for the diagnosis of BV in the laboratory setting relies on microscopic profiling of microbial types in Gram-stained vaginal swab smears by using the criteria of Nugent et al. (29) or Ison and Hay (20). These scoring systems are based on the relative abundance of Lactobacillus morphotypes (large gram-positive rods) compared to the abundance of the bacterial morphotypes suggestive of BV (gramnegative or gram-variable coccobacilli and curved rods and gram-positive cocci). Although this method provides reliable information and is particularly well suited for use in resourcepoor settings, it requires well-trained, highly experienced individuals to interpret the results and is simplistic in its reflection of the variety of organisms present in the microbial communities present in he...

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“…Although this is a uniquely designed EQA program that appears so far suitable for Xpert MTB/RIF verification using different strains from different culture methods, the individual components are not unfamiliar to the field: filter paper has been used for the transportation and molecular testing of M. tuberculosis DNA (7,14), and flow cytometry has been used for the analysis of M. tuberculosis (2,10,16,18,(20)(21)(22)(23). Flow cytometry has the advantage of rapidly and accurately identifying inactivated single whole bacterial cells, which circumvents conventional, time-consuming CFU enumeration methodologies.…”

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confidence: 99%

Dried Culture Spots for Xpert MTB/RIF External Quality Assessment: Results of a Phase 1 Pilot Study in South Africa

et al. 2011

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Implementation of Xpert MTB/RIF requires quality assessment. A pilot program using dried culture spots (DCSs) of inactivated Mycobacterium tuberculosis is described. Of 274 DCS results received, 2.19% generated errors; the remainder yielded 100% correct Mycobacterium tuberculosis detection. The probe A cycle threshold (C T ) variability of three DCS batches was <3.47. The study of longer-term DCS stability is ongoing.The Xpert MTB/RIF assay (Cepheid, Sunnyvale, CA) (1, 3-5, 9, 12, 13, 15, 19, 25) for the diagnosis of Mycobacterium tuberculosis has recently been endorsed by the WHO (28), and recommendations for data collection to quantify the impact of this GeneXpert (GX) technology are provided (26). Guidance, however, with respect to appropriate external quality assessment (EQA) programs is lacking (17). Current international tuberculosis (TB) EQA programs focus on microscopy, culture, and susceptibility testing laboratories (24) and highlight the difficulties in expansion due to labor-intensive preparatory work and the high cost and regulations associated with shipping drug-resistant isolates (27).Criteria for a verification ("fit for purpose") and EQA program suited to the characteristics of the Xpert MTB/RIF assay (3, 8) will require the following elements. (i) The testing material must contain whole M. tuberculosis (8).(ii) Transportation of EQA material needs to be safe. (iii) The testing procedure needs to be safe and compatible with the Xpert MTB/ RIF current testing protocol. (iv) Health care workers who do not have laboratory skills must be able to perform the testing in nonlaboratory settings. (v) Finally, the programs will need to be cost-effective and sustainable. Such a program using whole inactivated M. tuberculosis spotted onto filter paper was developed and piloted in South Africa as part of the National Health Laboratory Service (NHLS) GX rollout. H37Ra]) and well-characterized local clinical strain MYCTU 15, and (iii) the ATCC 25618 (H37Rv) laboratory strain grown for single-cell-organism suspensions (11). The MGIT cultures S-MYCTU-02-P2 and MYCTU 15 and clinical isolates were pooled in their respective batches (with strains kept separate and not mixed), centrifuged (3,000 ϫ g for 15 min at 4°C) to pellet cells, and resuspended in 40 ml phosphate-buffered saline (PBS) followed by addition of 80 ml (2:1 ratio of buffer to culture) of the Xpert sample reagent (SR) buffer. For the H37Rv strain, 200 ml of culture was harvested (by centrifugation at 3,500 ϫ g) at room temperature for 10 min, and cells were resuspended in PBS to 40 ml followed by addition of 80 ml SR buffer (2:1 ratio of buffer to cells). Both MGIT-grown and H37Rv strain cultures were inactivated in SR buffer for 2 h at room temperature, with intermittent mixing. The inactivated material was washed twice with sterile PBS and resuspended in final volumes of 10 ml (S-MYCTU-02-P2 and MYCTU 15) and 40 ml (H37Rv) PBS. For confirmation of inactivation, washed cultures (0.5 ml) were reinoculated into new MGIT tubes in Bactec cabinets fo...

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Flow cytometric quantification of bacteria in vaginal swab samples self-collected by adolescents attending a gynecology clinic

Cited by 16 publications

References 58 publications

Pyrosequencing of the Chaperonin-60 Universal Target as a Tool for Determining Microbial Community Composition

Pyrosequencing of the Chaperonin-60 Universal Target as a Tool for Determining Microbial Community Composition

Multiplex Detection of Bacteria Associated with Normal Microbiota and with Bacterial Vaginosis in Vaginal Swabs by Use of Oligonucleotide-Coupled Fluorescent Microspheres

Dried Culture Spots for Xpert MTB/RIF External Quality Assessment: Results of a Phase 1 Pilot Study in South Africa

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