Development and Validation of a Highly Accurate Quantitative Real-Time PCR Assay for Diagnosis of Bacterial Vaginosis.

Bacterial vaginosis (BV) is the most common gynecological infection in the United States. Diagnosis based on Amsel's criteria can be challenging and can be aided by laboratory-based testing. A standard method for diagnosis in research studies is enumeration of bacterial morphotypes of a Gram-stained vaginal smear (i.e., Nugent scoring). However, this technique is subjective, requires specialized training, and is not widely available. Therefore, a highly accurate molecular assay for the diagnosis of BV would be of great utility. We analyzed 385 vaginal specimens collected prospectively from subjects who were evaluated for BV by clinical signs and Nugent scoring. We analyzed quantitative real-time PCR (qPCR) assays on DNA extracted from these specimens to quantify nine organisms associated with vaginal health or disease:Gardnerella vaginalis,Atopobium vaginae, BV-associated bacteria 2 (BVAB2, an uncultured member of the orderClostridiales),Megasphaeraphylotype 1 or 2,Lactobacillus iners,Lactobacillus crispatus,Lactobacillus gasseri, andLactobacillus jensenii We generated a logistic regression model that identifiedG. vaginalis,A. vaginae, andMegasphaeraphylotypes 1 and 2 as the organisms for which quantification provided the most accurate diagnosis of symptomatic BV, as defined by Amsel's criteria and Nugent scoring, with 92% sensitivity, 95% specificity, 94% positive predictive value, and 94% negative predictive value. The inclusion ofLactobacillusspp. did not contribute sufficiently to the quantitative model for symptomatic BV detection. This molecular assay is a highly accurate laboratory tool to assist in the diagnosis of symptomatic BV.

UPC2A is required for high-level azole antifungal resistance in Candida glabrata.

Candida glabrata, the second most common cause of Candida infections, is associated with high rates of mortality and often exhibits resistance to the azole class of antifungal agents. Upc2 and Ecm22 in Saccharomyces cerevisiae and Upc2 in Candida albicans are the transcriptional regulators of ERG11, the gene encoding the target of azoles in the ergosterol biosynthesis pathway. Recently two homologs for these transcription factors, UPC2A and UPC2B, were identified in C. glabrata. One of these, UPC2A, was shown to influence azole susceptibility. We hypothesized that due to the global role for Upc2 in sterol biosynthesis in S. cerevisiae and C. albicans, disruption of UPC2A would enhance the activity of fluconazole in both azole-susceptible dose-dependent (SDD) and -resistant C. glabrata clinical isolates. To test this hypothesis, we constructed mutants with disruptions in UPC2A and UPC2B alone and in combination in a matched pair of clinical azole-SDD and -resistant isolates. Disruption of UPC2A in both the SDD and resistant isolates resulted in increased susceptibility to sterol biosynthesis inhibitors, including a reduction in fluconazole MIC and minimum fungicidal concentration, enhanced azole activity by time-kill analysis, a decrease in ergosterol content, and downregulation of baseline and inducible expression of several sterol biosynthesis genes. Our results indicate that Upc2A is a key regulator of ergosterol biosynthesis and is essential for resistance to sterol biosynthesis inhibitors in C. glabrata. Therefore, the UPC2A pathway may represent a potential cotherapeutic target for enhancing azole activity against this organism.

Detection of epidemic USA300 community-associated methicillin-resistant Staphylococcus aureus strains by use of a single allele-specific PCR assay targeting a novel polymorphism of Staphylococcus aureus pbp3.

In recent years, the dramatic increase in community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections has become a significant health care challenge. Early detection of CA-MRSA is important because of its increased virulence associated with the arginine catabolic mobile element (ACME), Panton-Valentine leukocidin (PVL), and other toxins that may contribute to disease severity. In particular, the USA300 epidemic clone has emerged and now represents the cause of as much as 98% of CA-MRSA skin and soft tissue infections in the United States. Current diagnostic assays used to identify CA-MRSA strains are based on complex multiplex PCRs targeting the staphylococcal cassette chromosome mec (SCCmec) DNA junction, a multitude of genes, and noncoding DNA fragments or on a number of lengthy sequence-typing methods. Here, two nucleotide polymorphisms, G88A and G2047A, that were found to be in strict linkage disequilibrium in the S. aureus penicillin-binding protein 3 (pbp3) gene were also found to be highly associated with the USA300 clone of CA-MRSA. Clinical isolates that contained this pbp3 allele were also positive for the presence of SCCmec type IV, the ACME, and the PVL toxin gene and matched the t008 or t121 molecular spa types, which are associated specifically with the USA300 CA-MRSA clone. A single allele-specific PCR targeting the G88A polymorphism was developed and was found to be 100% sensitive and specific for the detection of USA300 CA-MRSA and 91.5% sensitive and 100% specific for the detection of all CA-MRSA isolates in this study.

Gain-of-function mutations in UPC2 are a frequent cause of ERG11 upregulation in azole-resistant clinical isolates of Candida albicans.

In Candida albicans, Upc2 is a zinc-cluster transcription factor that targets genes, including those of the ergosterol biosynthesis pathway. To date, three documented UPC2 gain-of-function (GOF) mutations have been recovered from fluconazole-resistant clinical isolates that contribute to an increase in ERG11 expression and decreased fluconazole susceptibility. In a group of 63 isolates with reduced susceptibility to fluconazole, we found that 47 overexpressed ERG11 by at least 2-fold over the average expression levels in 3 unrelated fluconazole-susceptible strains. Of those 47 isolates, 29 contained a mutation in UPC2, whereas the remaining 18 isolates did not. Among the isolates containing mutations in UPC2, we recovered eight distinct mutations resulting in putative single amino acid substitutions: G648D, G648S, A643T, A643V, Y642F, G304R, A646V, and W478C. Seven of these resulted in increased ERG11 expression, increased cellular ergosterol, and decreased susceptibility to fluconazole compared to the results for the wild-type strain. Genome-wide transcriptional analysis was performed for the four strongest Upc2 amino acid substitutions (A643V, G648D, G648S, and Y642F). Genes commonly upregulated by all four mutations included those involved in ergosterol biosynthesis, in oxidoreductase activity, the major facilitator efflux pump encoded by the MDR1 gene, and the uncharacterized ATP binding cassette transporter CDR11. These findings demonstrate that gain-of-function mutations in UPC2 are more prevalent among clinical isolates than previously thought and make a significant contribution to azole antifungal resistance, but the findings do not account for ERG11 overexpression in all such isolates of C. albicans.

Arv1 lipid transporter function is conserved between pathogenic and nonpathogenic fungi.

The lipid transporter Arv1 regulates sterol trafficking, and glycosylphosphatidylinositol and sphingolipid biosyntheses in Saccharomyces cerevisiae. ScArv1 contains an Arv1 homology domain (AHD) that is conserved at the amino acid level in the pathogenic fungal species, Candida albicans and Candida glabrata. Here we show S. cerevisiae cells lacking Arv1 are highly susceptible to antifungal drugs. In the presence of drug, Scarv1 cells are unable to induce ERG gene expression, have an altered pleiotrophic drug response, and are defective in multi-drug resistance efflux pump expression. All phenotypes are remediated by ectopic expression of CaARV1 or CgARV1. The AHDs of these pathogenic fungi are required for specific drug tolerance, demonstrating conservation of function. In order to understand how Arv1 regulates antifungal susceptibility, we examined sterol trafficking. CaARV1/CgARV1 expression suppressed the sterol trafficking defect of Scarv1 cells. Finally, we show that C. albicansarv1/arv1 cells are avirulent using a BALB/c disseminated mouse model. We suggest that overall cell survival in response to antifungal treatment requires the lipid transporter function of Arv1.

Antifungal resistance of Candida glabrata vaginal isolates and development of a quantitative reverse transcription-PCR-based azole susceptibility assay.

A multiplex quantitative reverse transcription-PCR assay was developed to detect azole resistance in Candida glabrata, an important opportunistic pathogen that develops resistance rapidly. Resistance was defined as a >or=3-fold increase in CDR1 expression by this assay, which proved to be 100% sensitive and 95% specific in comparison to the gold standard broth microdilution assay.

Survey of vaginal-flora Candida species isolates from women of different age groups by use of species-specific PCR detection.

A retrospective survey of 93,775 samples testing positive in Candida species-specific PCR tests performed on cervicovaginal swabs over a 4-year period demonstrated consistent yearly distributions of Candida albicans (89%), C. glabrata (7.9%), C. parapsilosis (1.7%), and C. tropicalis (1.4%). However, the species distributions among different age groups revealed increases in the percentages of non-albicans species with increases in age.

Utilization of molecular methods to identify prognostic markers for recurrent bacterial vaginosis.

BACKGROUND: Recurrent bacterial vaginosis (BV) after antimicrobial therapy is a major problem, affecting >50% of patients within 1 year. The objective of this study was to determine if prospective identification of patients at risk for recurrence using molecular methods is feasible. METHODS: Women were evaluated for BV by Amsel criteria and Nugent score. Vaginal specimens were analyzed using a panel of quantitative real-time polymerase chain reactions (qPCRs) at three times: pre-treatment, 7-10days post-treatment and 40-45days post-treatment. The PCRs quantified DNA of the following organisms: Gardnerella vaginalis; Atopobium vaginae; Bacterial Vaginosis-Associated Bacteria-1 (BVAB1), -2 (BVAB2) and -3 (BVAB3); Leptotrichia/Sneathia; Megasphaera Phylotypes 1 and 2; and Lactobacillus spp. (L. crispatus, L. gasseri, L. iners and L. jensenii). RESULTS: Out of 84 women diagnosed with BV (Amsel ≥3 and Nugent ≥4), 77 (91.7%) were successfully treated after 7-10days (asymptomatic and Amsel of either 0 or 1 with elevated vaginal pH and Nugent ≤6). Of these 77 women, 46 (59.7%) remained cured after 40-45days and 31 (40.3%) developed recurrent BV. In univariate analysis, we found that women who would have recurrent BV during the study had greater concentrations of Megasphaera Phylotype 2 (P=0.001) and BVAB2 (P=0.015) at initial diagnosis and greater vaginal pH (P=0.030), higher Nugent score (P=0.043) and a greater concentration of G. vaginalis (P=0.012) post-treatment, when compared to women who were cured during the study. These differences largely remained when cure was defined as Nugent ≤3 or when only women treated with intravaginal metronidazole were evaluated. CONCLUSION: Molecular analysis of BV is a useful adjunct to clinical and microscopic analysis to prospectively identify patients at high risk for recurrent BV.

Draft Genome Sequence of a Metronidazole-Resistant Gardnerella vaginalis Isolate.

We report the draft genome sequence of a Gardnerella vaginalis strain (3549624) isolated from a vaginal specimen. G. vaginalis is associated with bacterial vaginosis, the most common cause of vaginal discharge, which is often treated with metronidazole. This isolate is highly resistant to metronidazole (MIC, 500 µg/ml) and may be useful for comparative genomic studies to determine the molecular basis of metronidazole resistance in this species.

X-Plate Technology: a new method for detecting fluconazole resistance in Candida species.

Candida species are responsible for many opportunistic fungal infections. Fluconazole is a well-tolerated antifungal drug, commonly used in the treatment of candidiasis. However, with fluconazole resistance ever increasing, rapid detection and antifungal susceptibility testing of Candida is imperative for proper patient treatment. This paper reports a cost-effective, simple and rapid chromogenic agar dilution method for simultaneous Candida species identification and fluconazole susceptibility testing. The results obtained by X-Plate Technology were in absolute concordance with standard microbroth dilution assays. Analysis of 1383 clinical patient samples with suspected vulvovaginal candidiasis revealed that this technology was able to detect and speciate the Candida isolate and determine the fluconazole susceptibility. The prevalence and susceptibility profiles of the clinical isolates using this method were highly similar to published reports using the microbroth dilution method.

Staphylococcal Cassette Chromosome mec type and antibiotic susceptibility profiles of vaginal and nonvaginal MRSA clinical isolates.

Methicillin-resistant Staphylococcus aureus (MRSA) causes nosocomial and community-associated infections, representing significant healthcare concerns. Limited studies have investigated cervicovaginal MRSA colonization and antibiotic susceptibility. Upon comparing clinical cervicovaginal MRSA isolates to nonvaginal isolates by Staphylococcal Cassette Chromosome mec type, presence of Panton-Valentine Leukocidin toxin, antibiotic susceptibility, and presence of associated resistance genes, no significant differences were observed between the anatomical sites, but were observed between our hospital- and community-associated MRSA isolates. There was a significant increase in erythromycin resistance in our vaginal MRSA isolates compared to previous vaginal MRSA reports and an increase in clindamycin, doxycycline, and mupirocin resistance in our nonvaginal MRSA isolates compared to previously reported community-based skin and soft tissue MRSA isolates. Additionally, this is the first report of mupirocin resistance in vaginal MRSA isolates.