Bacterial Vaginosis-Associated Bacteria and Uterine Fibroids: A Nested Case-Control Study.

BACKGROUND: Reproductive tract infections are hypothesized to influence uterine fibroid development, yet few studies have investigated the common condition of bacterial vaginosis (BV). The literature is currently limited to data using self-report of BV. METHODS: We conducted a nested case-control study of 200 women (100 cases and 100 controls) from a large study of 23- to 35-year-old African American women, 1310 of whom were fibroid-free and prospectively followed up for 5 years to identify incident fibroids with standardized ultrasound examinations. We used quantitative polymerase chain reaction, an objective molecular method, to assess 9 BV-associated and 4 Lactobacillus species from vaginal swab specimens. We used hierarchical logistic regression to compute odds ratios and 95% confidence intervals to examine associations between bacterial species (both individually and grouped as (1) "optimal" Lactobacillus and (2) BV-associated species) with fibroid incidence and number. We also examined vaginal imbalance (quantitatively more BV-associated bacteria than optimal Lactobacilli). RESULTS: Contrary to our hypothesis, we found no increase in fibroid incidence or number among women with more BV-associated bacteria. High imbalance (only BV-associated bacteria, no optimal Lactobacillus bacteria) was actually inversely associated with fibroid incidence (odds ratio, 0.38; 95% confidence interval, 0.17-0.81). CONCLUSIONS: This is the first study of ultrasound-detected incident fibroids and molecular vaginal bacterial assessment. We found no evidence that BV-associated bacteria increase the risk of fibroid incidence or number.

Mycoplasma genitalium and Bacterial Vaginosis-Associated Bacteria in a Non-Clinic-Based Sample of African American Women.

BACKGROUND: Mycoplasma genitalium is associated with adverse reproductive problems. However, prevalence estimates from studies that screen women not seeking care are rare. Studies have reported co-occurrence of M. genitalium with bacterial vaginosis (BV), but no prior study of specific BV-associated bacteria has been conducted in African Americans whose reproductive tract infection burden is high. METHODS: Using quantitative polymerase chain reaction, we screened vaginal swabs for M. genitalium, 9 BV-associated bacteria, and 4 Lactobacillus species from 200 participants drawn from a cohort of African Americans 23 to 35 years old. Sexual history, herpes serostatus, and Nugent score had been assessed. Prevalence of M. genitalium was computed. The associations of other vaginal bacteria with M. genitalium were examined with binomial regression. RESULTS: M. genitalium prevalence was 18%. Detection and quantity of 2 BV-associated bacteria were significantly associated with a higher prevalence of M. genitalium (Leptotrichia/Sneathia: detection prevalence ratio (PR) of 2.9 [95% confidence interval {CI}, 1.1-7.7] and quantity PR of 1.2 [95% CI, 1.0-1.3]; Megasphaera phylotype 1: detection PR of 2.2 [95% CI, 1.2-4.2] and quantity PR of 1.1 [95% CI, 1.0-1.2]). Increased quantity of L. iners was also positively associated with M. genitalium (PR, 1.3 [95% CI, 1.0-1.8]). Nugent ≥7, herpes serostatus, and lifetime number of sex partners were not associated with M. genitalium. CONCLUSIONS: Specific BV-associated microbes and L. iners were associated with M. genitalium, but Nugent ≥7 was not. Studies are needed to confirm a high prevalence of M. genitalium in African Americans and to understand its interactions with other vaginal bacteria.

Trichomonas vaginalis is most frequently detected in women at the age of peri-/premenopause: an unusual pattern for a sexually transmitted pathogen.

BACKGROUND: Trichomonas vaginalis is the most common nonviral sexually transmitted infection. However, because it is not a reportable disease in the United States, there is limited information on the age of infected individuals and their geographic distribution. OBJECTIVE: The purpose of this study was to evaluate the detection rates of T vaginalis infection compared with Chlamydia trachomatis by age and state in a commercial laboratory setting. STUDY DESIGN: Quantitative real-time polymerase chain reactions were used to detect the presence of T vaginalis and C trachomatis in cervicovaginal samples that were obtained during gynecologic examinations. A total of 1,554,966 and 1,999,077 samples from females 10-79 years old were analyzed retrospectively for the presence of T vaginalis and C trachomatis, respectively. RESULTS: The highest detection rate of an infection with T vaginalis was ages 47-53 years. For C trachomatis, the highest detection rate was ages 14-20 years. T vaginalis detection rate distribution by age shows a bimodal pattern with first peak at ages 21-22 years (4.0-4.1%) and a higher second peak at ages 48-51 years (5.4-5.8%). C trachomatis prevalence distribution by age shows a maximum peak of 8.6% at age 17 years and a rapid decline thereafter. In general, the detection rates of both pathogens were higher in the southeast and in states along the Mississippi River Valley than in other parts of the country. A nucleotide polymorphism associated with T vaginalis metronidazole resistance (ntr6TVK80STOP) was not associated with age and was found most frequently in specimens from New Mexico and Vermont. CONCLUSIONS: The detection rate of T vaginalis does not appear to decrease with age as observed for C trachomatis and reaches maximum rates in women 48-51 years old. The geographic distribution of T vaginalis appears to be broadly similar to that of other sexually transmitted diseases. The ntr6TVK80STOP polymorphism did not have a specific association with age or geography.

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.

Novel antifungal drug discovery based on targeting pathways regulating the fungus-conserved Upc2 transcription factor.

Infections by Candida albicans and related fungal pathogens pose a serious health problem for immunocompromised patients. Azole drugs, the most common agents used to combat infections, target the sterol biosynthetic pathway. Adaptation to azole therapy develops as drug-stressed cells compensate by upregulating several genes in the pathway, a process mediated in part by the Upc2 transcription factor. We have implemented a cell-based high-throughput screen to identify small-molecule inhibitors of Upc2-dependent induction of sterol gene expression in response to azole drug treatment. The assay is designed to identify not only Upc2 DNA binding inhibitors but also compounds impeding the activation of gene expression by Upc2. An AlphaScreen assay was developed to determine whether the compounds identified interact directly with Upc2 and inhibit DNA binding. Three compounds identified by the cell-based assay inhibited Upc2 protein level and UPC2-LacZ gene expression in response to a block in sterol biosynthesis. The compounds were growth inhibitory and attenuated antifungal-induced sterol gene expression in vivo. They did so by reducing the level of Upc2 protein and Upc2 DNA binding in the presence of drug. The mechanism by which the compounds restrict Upc2 DNA binding is not through a direct interaction, as demonstrated by a lack of DNA binding inhibitory activity using the AlphaScreen assay. Rather, they likely inhibit a novel pathway activating Upc2 in response to a block in sterol biosynthesis. We suggest that the compounds identified represent potential precursors for the synthesis of novel antifungal drugs.

Trichomonas vaginalis metronidazole resistance is associated with single nucleotide polymorphisms in the nitroreductase genes ntr4Tv and ntr6Tv.

Metronidazole resistance in the sexually transmitted parasite Trichomonas vaginalis is a problematic public health issue. We have identified single nucleotide polymorphisms (SNPs) in two nitroreductase genes (ntr4Tv and ntr6Tv) associated with resistance. These SNPs were associated with one of two distinct T. vaginalis populations identified by multilocus sequence typing, yet one SNP (ntr6Tv A238T), which results in a premature stop codon, was associated with resistance independent of population structure and may be of diagnostic value.

Cervical and vaginal flora specimens are highly concordant with respect to bacterial vaginosis-associated organisms and commensal Lactobacillus species in women of reproductive age.

Matched vaginal and cervical specimens from 96 subjects were analyzed by quantitative PCR for the presence and concentration of bacterial vaginosis-associated microbes and commensal Lactobacillus spp. Detection of these microbes was 92% concordant, indicating that microbial floras at these body sites are generally similar.

Flucytosine antagonism of azole activity versus Candida glabrata: role of transcription factor Pdr1 and multidrug transporter Cdr1.

Infections with the opportunistic yeast Candida glabrata have increased dramatically in recent years. Antifungal therapy of yeast infections commonly employs azoles, such as fluconazole (FLC), but C. glabrata frequently develops resistance to these inhibitors of ergosterol biosynthesis. The pyrimidine analog flucytosine (5-fluorocytosine [5FC]) is highly active versus C. glabrata but is now rarely used clinically due to similar concerns over resistance and, a related concern, the toxicity associated with high doses used to counter resistance. Azole-5FC combination therapy would potentially address these concerns; however, previous studies suggest that 5FC may antagonize azole activity versus C. glabrata. Here, we report that 5FC at subinhibitory concentrations antagonized the activity of FLC 4- to 16-fold versus 8 of 8 C. glabrata isolates tested. 5FC antagonized the activity of other azoles similarly but had only indifferent effects in combination with unrelated antifungals. Since azole resistance in C. glabrata results from transcription factor Pdr1-dependent upregulation of the multidrug transporter gene CDR1, we reasoned that 5FC antagonism might be similarly mediated. Indeed, 5FC-FLC antagonism was abrogated in pdr1Δ and cdr1Δ strains. In further support of this hypothesis, 5FC exposure induced CDR1 expression 6-fold, and this upregulation was Pdr1 dependent. In contrast to azoles, 5FC is not a Cdr1 substrate and so its activation of Pdr1 was unexpected. We observed, however, that 5FC exposure readily induced petite mutants, which exhibit Pdr1-dependent CDR1 upregulation. Thus, mitochondrial dysfunction resulting in Pdr1 activation is the likely basis for 5FC antagonism of azole activity versus C. glabrata.

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.

Detection of erythromycin and clindamycin resistance genes in Group B Streptococcal clinical isolates and cervicovaginal-rectal swabs.

A multiplex PCR assay was used to detect the erythromycin (EM) and clindamycin (CM) antibiotic resistance genes, ermB, ermTR, and mefA/E, in Group B Streptococcal (GBS) clinical isolates and in DNA extracted from the corresponding cervicovaginal-rectal (CVR) swabs. We compared these results to the standard EM/CM double disk diffusion assay of 46 isolates. Given that these genes are present in other CVR flora and are found on mobile genetic elements, the PCR assay was unable to predict GBS resistance directly from the swabs. Therefore, PCR can only accurately detect resistance genes and predict the resistance phenotype from purified GBS isolates.

SepBCTF4 is required for the formation of DNA-damage-induced UvsCRAD51 foci in Aspergillus nidulans.

SepB is an essential, conserved protein required for chromosomal DNA metabolism in Aspergillus nidulans. Homologs of SepB include yeast Ctf4p and human hAnd-1. Molecular and bioinformatic characterization of these proteins suggests that they act as molecular scaffolds. Furthermore, recent observations implicate the yeast family members in lagging-strand replication and the establishment of sister-chromatid cohesion. Here, we demonstrate that SepB functions in the A. nidulans DNA damage response. In particular, analysis of double mutants reveals that SepB is a member of the UvsC(RAD51) epistasis group. In accord with this prediction, we show that UvsC(RAD51) forms DNA-damage-induced nuclear foci in a manner that requires SepB function. We also provide evidence that implicates SepB in sister-chromatid cohesion, thereby suggesting that cohesion may play a role in regulating the localization and/or assembly of UvsC(RAD51) complexes.

Vaginal Dysbiosis from an Evolutionary Perspective.

Evolutionary approaches are powerful tools for understanding human disorders. The composition of vaginal microbiome is important for reproductive success and has not yet been characterized in the contexts of social structure and vaginal pathology in non-human primates (NHPs). We investigated vaginal size, vulvovaginal pathology and the presence of the main human subtypes of Lactobacillus spp./ BV-related species in the vaginal microflora of baboons (Papio spp.). We performed morphometric measurements of external and internal genitalia (group I, n = 47), analyzed pathology records of animals from 1999-2015 (group II, n = 64 from a total of 12,776), and evaluated vaginal swabs using polymerase chain reaction (PCR) (group III, n = 14). A total of 68 lesions were identified in 64 baboons. Lactobacillus iners, Gardnerella vaginalis, Atopobium vaginae, Megasphaera I, and Megasphaera II were not detected. L. jensenii, L. crispatus, and L. gasseri were detected in 2/14 (14.2%), 1/14 (7.1%), and 1/14 (7.1%) samples, respectively. BVAB2 was detected in 5/14 (35.7%) samples. The differences in the vaginal milieu between NHP and humans might be the factor associated with human-specific pattern of placental development and should be taken in consideration in NHP models of human pharmacology and microbiology.

Identification of intrinsically metronidazole-resistant clades of Gardnerella vaginalis.

Gardnerella vaginalis is associated with bacterial vaginosis (BV), the most common cause of vaginal discharge. Metronidazole is a front-line therapy for BV, and treatment failure and recurrent disease are common problems. Whole-genome sequencing studies have revealed that G. vaginalis has a population structure that consists of 4 clades: clades 1 and 3 are associated with BV, whereas clades 2 and 4 are not. To determine if metronidazole susceptibility is associated with population structure, we analyzed 87 clinical isolates and found that metronidazole resistance (MIC ≥32 µg/mL) was highly associated with clade (P<0.0001), as 14/14 clade 3 isolates (100%) and 22/22 clade 4 isolates (100%) exhibited resistance, compared to only 16/37 clade 1 isolates (35%) and 1/14 clade 2 isolates (7.1%). The identification of intrinsically metronidazole-resistant G. vaginalis clades will facilitate future studies on the relationship between metronidazole resistance and BV treatment failure.