ABSTRACT
Background: Numerous bacteria are involved in the etiology of bacterial vaginosis (BV). Yet, current tests only focus on a select few. We therefore designed a new test targeting 22 BV-relevant species. Methods: Using 946 stored vaginal samples, a new qPCR test that quantitatively identifies 22 bacterial species was designed. The distribution and relative abundance of each species, α- and ß-diversities, correlation, and species co-existence were determined per sample. A diagnostic index was modeled from the data, trained, and tested to classify samples into BV-positive, BV-negative, or transitional BV. Results: The qPCR test identified all 22 targeted species with 95 - 100% sensitivity and specificity within 8 hours (from sample reception). Across most samples, Lactobacillus iners, Lactobacillus crispatus, Lactobacillus jensenii, Gardnerella vaginalis, Fannyhessea (Atopobium) vaginae, Prevotella bivia, and Megasphaera sp. type 1 were relatively abundant. BVAB-1 was more abundant and distributed than BVAB-2 and BVAB-3. No Mycoplasma genitalium was found. The inter-sample similarity was very low, and correlations existed between key species, which were used to model, train, and test a diagnostic index: MDL-BV index. The MDL-BV index, using both species and relative abundance markers, classified samples into three vaginal microbiome states. Testing this index on our samples, 491 were BV-positive, 318 were BV-negative, and 137 were transitional BV. Although important differences in BV status were observed between different age groups, races, and pregnancy status, they were statistically insignificant. Conclusion: Using a diverse and large number of vaginal samples from different races and age groups, including pregnant women, the new qRT-PCR test and MDL-BV index efficiently diagnosed BV within 8 hours (from sample reception), using 22 BV-associated species.
Subject(s)
Gardnerella vaginalis , Lactobacillus , Microbiota , Real-Time Polymerase Chain Reaction , Vagina , Vaginosis, Bacterial , Female , Vaginosis, Bacterial/diagnosis , Vaginosis, Bacterial/microbiology , Humans , Vagina/microbiology , Microbiota/genetics , Lactobacillus/isolation & purification , Lactobacillus/genetics , Real-Time Polymerase Chain Reaction/methods , Adult , Gardnerella vaginalis/isolation & purification , Gardnerella vaginalis/genetics , Young Adult , Sensitivity and Specificity , Prevotella/isolation & purification , Prevotella/genetics , Megasphaera/isolation & purification , Megasphaera/genetics , Actinobacteria/isolation & purification , Actinobacteria/genetics , Actinobacteria/classification , Middle Aged , Lactobacillus crispatus/isolation & purification , Lactobacillus crispatus/genetics , Adolescent , Bacteria/genetics , Bacteria/isolation & purification , Bacteria/classification , Pregnancy , RNA, Ribosomal, 16S/geneticsABSTRACT
Bartonella henselae is a gram-negative rod-shaped bacterium and is the primary causative agent of Cat Scratch Disease (CSD). Although the prevalence of CSD is low in the human population, the possibility of developing multi-organ complications, especially in vulnerable individuals, remains a serious cause for concern. The immunofluorescent assay (IFA) is currently one of the most common laboratory tests for the detection of antibodies to B. henselae in serum, however, it has several disadvantages. The enzyme-linked immunosorbent assay (ELISA) technique offers a more quantitative, sensitive, and cost-effective alternative to conventional IFAs. Here, we report the purification of a novel bioidentical polyclonal antibody from discarded human serum for use as a standard in ELISAs against B. henselae. This novel method of antibody production overcomes the many limitations of animal-derived antibodies while also offering a more robust, reproducible, and scalable antibody production alternative for the diagnosis of CSD.
Subject(s)
Antibodies, Bacterial , Bartonella henselae , Cat-Scratch Disease , Enzyme-Linked Immunosorbent Assay , Bartonella henselae/immunology , Bartonella henselae/isolation & purification , Humans , Antibodies, Bacterial/blood , Antibodies, Bacterial/immunology , Cat-Scratch Disease/diagnosis , Enzyme-Linked Immunosorbent Assay/methods , Animals , Sensitivity and SpecificityABSTRACT
Vaginal dysbiosis in women reduces the abundance of Lactobacillus species and increases that of anaerobic fastidious bacteria. This dysbiotic condition in the vagina, called bacterial vaginosis (BV), can be symptomatic with odorous vaginal discharges or asymptomatic and affects a third of women of reproductive age. Three unclassified bacterial species designated BV-associated bacteria 1, 2, and 3 (BVAB-1, -2, and -3) in 2005 were found to be highly preponderant in the vagina of females with BV. Here, we used sequence homology and phylogenetics analyses to identify the actual species of BVAB-1, -2, and -3 and found BVAB-1 to be Clostridiales genomosp. BVAB-1, BVAB-2 to be Oscillospiraceae bacterium strain CHIC02, and BVAB-3 to be Mageeibacillus indolicus, respectively. These are anaerobic and uncultured species that can be identified only through metagenomics. Long-read sequencing of BV specimens can also enable a genomic reassembly of these species' genomes from metagenomes. Species-specific identification of these pathogens and the availability of their genomes from assembled metagenomes will advance our understanding of their biology, facilitate the design of sensitive diagnostics and drugs, and enhance the treatment of BV. IMPORTANCE For many years since 2005, BVAB, an important pathogen of the female vaginal tract that is associated with BV, has been identified using PCR without knowing its actual species. Without a full genome of these pathogens, a better understanding of their pathogenicity, treatment, resistance, and diagnostics cannot be reached. In this analysis, we use the DNA of BVAB-1, -2, and -3 to determine their actual species to enhance further research into their pathogenicity, resistance, diagnosis, and treatment.
Subject(s)
Vaginosis, Bacterial , Female , Humans , Vaginosis, Bacterial/diagnosis , Vaginosis, Bacterial/genetics , Vaginosis, Bacterial/microbiology , Bacteria/genetics , Vagina/microbiology , Metagenome , Polymerase Chain ReactionABSTRACT
Background: A retrospective study of a single laboratory's results from patients in the United States to investigate high-risk human papillomavirus (HPV) genotype distribution according to cervical cytology and age was performed. Methods: Anonymous results of 23 580 patients' cervical specimens sent to Medical Diagnostic Laboratories, LLC, for cervical cytology and HPV testing between August 2020 and August 2021 were analyzed. Results: Overall, any of the 14 high-risk HPV genotypes identified were detected in 2302 of the 23 580 patients (9.8%), with HPV 52 (1.4%), HPV 39 (1.3%), HPV 51 (1.3%), and HPV 16 (1.2%) being the most frequent in all patients. Multiple high-risk HPV infection was observed in 1.3% of all patients. HPV 16 was most likely to be a single high-risk genotype detected as compared with detection with other high-risk HPV genotypes, in contrast to HPV 33, which is least likely to be a single high-risk genotype detected as compared with detection with other high-risk HPV genotypes. High-risk HPV detection was greatest in patients under 25 years old (<21-year-olds, 24.6%, and 21-25-year-olds, 25.4%). In patients with low-grade squamous intraepithelial lesions, the most frequently detected high-risk HPV genotypes were HPV 51 (10.5%) and HPV 39 (9.1%), and in patients with high-grade squamous intraepithelial lesions, the most frequently detected high-risk HPV genotypes were HPV 16 (25.6%) and HPV 52 (17.1%). Conclusions: HPV genotyping and cervical cytology data analysis may contribute to and inform cervical cancer screening and HPV vaccination programs.
ABSTRACT
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.
Subject(s)
Chlamydia Infections/epidemiology , Chlamydia trachomatis , Trichomonas Infections/epidemiology , Trichomonas vaginalis , Adolescent , Adult , Age Distribution , Aged , Anti-Infective Agents/pharmacology , Child , Chlamydia Infections/microbiology , Drug Resistance, Bacterial/genetics , Female , Humans , Metronidazole/pharmacology , Middle Aged , Perimenopause , Polymorphism, Genetic , Premenopause , Prevalence , Real-Time Polymerase Chain Reaction , Retrospective Studies , Trichomonas Infections/microbiology , Trichomonas vaginalis/genetics , United States/epidemiology , Young AdultABSTRACT
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.
Subject(s)
Bacteria/classification , Bacteria/genetics , Vaginosis, Bacterial/epidemiology , Vaginosis, Bacterial/microbiology , Adolescent , Adult , Anti-Infective Agents/therapeutic use , Bacterial Load , Female , Humans , Longitudinal Studies , Middle Aged , Prognosis , Real-Time Polymerase Chain Reaction , Recurrence , Risk Factors , Vaginosis, Bacterial/drug therapy , Young AdultABSTRACT
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.
Subject(s)
Microbiological Techniques/methods , Molecular Diagnostic Techniques/methods , Real-Time Polymerase Chain Reaction/methods , Vaginosis, Bacterial/diagnosis , Adolescent , Adult , Animals , Female , Humans , Longitudinal Studies , Middle Aged , Sensitivity and Specificity , United States , Young AdultABSTRACT
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.
Subject(s)
Anti-Bacterial Agents/pharmacology , Drug Resistance, Bacterial , Gardnerella vaginalis/drug effects , Gardnerella vaginalis/isolation & purification , Genotype , Metronidazole/pharmacology , Female , Gardnerella vaginalis/classification , Gardnerella vaginalis/genetics , Gram-Positive Bacterial Infections/microbiology , Humans , Microbial Sensitivity Tests , Vaginosis, Bacterial/microbiologyABSTRACT
We report the genome sequence of a metronidazole-resistant derivative of Gardnerella vaginalis ATCC 14019. This strain was obtained after serial selection to increase the MIC from 4 to ≥500 µg/ml. Two coding changes, in genes encoding a response regulator and an NAD(+) synthetase, arose during selection.
ABSTRACT
We report the draft genome sequence of a vaginal isolate of Atopobium vaginae vaginae (strain 44061), an organism linked to bacterial vaginosis (BV), the most common gynecological infection in the United States. This species is often highly resistant to metronidazole, which is a front-line therapy for BV. Strain 44061 is a metronidazole-susceptible isolate (MIC, 16 µg/ml), and its genome sequence will be useful for comparative studies to elucidate the molecular basis of metronidazole resistance in this species.
ABSTRACT
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.
ABSTRACT
Podoplanin (PDPN) is a unique transmembrane receptor that promotes tumor cell motility. Indeed, PDPN may serve as a chemotherapeutic target for primary and metastatic cancer cells, particularly oral squamous cell carcinoma (OSCC) cells that cause most oral cancers. Here, we studied how a monoclonal antibody (NZ-1) and lectin (MASL) that target PDPN affect human OSCC cell motility and viability. Both reagents inhibited the migration of PDPN expressing OSCC cells at nanomolar concentrations before inhibiting cell viability at micromolar concentrations. In addition, both reagents induced mitochondrial membrane permeability transition to kill OSCC cells that express PDPN by caspase independent nonapoptotic necrosis. Furthermore, MASL displayed a surprisingly robust ability to target PDPN on OSCC cells within minutes of exposure, and significantly inhibited human OSCC dissemination in zebrafish embryos. Moreover, we report that human OSCC cells formed tumors that expressed PDPN in mice, and induced PDPN expression in infiltrating host murine cancer associated fibroblasts. Taken together, these data suggest that antibodies and lectins may be utilized to combat OSCC and other cancers that express PDPN.
Subject(s)
Antibodies, Monoclonal/pharmacology , Antineoplastic Agents, Phytogenic/pharmacology , Carcinoma, Squamous Cell/pathology , Membrane Glycoproteins/antagonists & inhibitors , Molecular Targeted Therapy , Mouth Neoplasms/pathology , Neoplasm Proteins/antagonists & inhibitors , Phytohemagglutinins/pharmacology , Administration, Oral , Animals , Animals, Genetically Modified , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/therapeutic use , Antineoplastic Agents, Phytogenic/administration & dosage , Antineoplastic Agents, Phytogenic/therapeutic use , Carcinoma, Squamous Cell/virology , Cell Line, Tumor , Cell Movement/drug effects , Cell Survival/drug effects , Fibroblasts/pathology , Humans , Membrane Glycoproteins/immunology , Membrane Glycoproteins/physiology , Membrane Potential, Mitochondrial/drug effects , Mice , Mouth Neoplasms/virology , Neoplasm Proteins/immunology , Neoplasm Proteins/physiology , Papillomaviridae/isolation & purification , Papillomavirus Infections/pathology , Papillomavirus Infections/virology , Phytohemagglutinins/administration & dosage , Phytohemagglutinins/therapeutic use , Xenograft Model Antitumor Assays , Zebrafish/embryologyABSTRACT
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.
Subject(s)
Bacterial Load , Biota , Cervix Uteri/microbiology , Lactobacillus/isolation & purification , Vagina/microbiology , Vaginosis, Bacterial/microbiology , Adolescent , Adult , Cross-Sectional Studies , Female , Humans , Middle Aged , Real-Time Polymerase Chain Reaction , Young AdultABSTRACT
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.
Subject(s)
Metronidazole/pharmacology , Nitroreductases/genetics , Protozoan Proteins/genetics , Trichomonas vaginalis/drug effects , Trichomonas vaginalis/genetics , Antiprotozoal Agents/pharmacology , Codon, Terminator/genetics , Drug Resistance/genetics , Parasitic Sensitivity Tests , Polymorphism, Single NucleotideABSTRACT
Lactobacilli play a key role in promoting vaginal health. Depletion of these bacteria is associated with bacterial vaginosis (BV), the most common vaginal disorder. Here we describe the development and laboratory validation of a novel single-tube multiplex TaqMan quantitative polymerase chain reaction (qPCR) assay for the identification and quantitative assessment of the four major vaginal Lactobacillus species: L. crispatus, L. jensenii, L. gasseri, and L. iners. The assay utility was evaluated by the analysis of lactobacilli in non-cultured clinical vaginal swab specimens collected from BV patients and healthy individuals. As confirmed by the assay, L. crispatus, L. jensenii, and to a lesser extent L. gasseri, are common in the vagina of healthy women, whereas L. iners dominance is associated with BV. The major assay limitation was preferential detection of dominant Lactobacillus species in samples with mixed lactobacilli resulting in lower sensitivity for minor species. The multiplex qPCR assay described here is an advance in the detection and quantitation of the major vaginal lactobacilli, potentially facilitating the molecular diagnosis of BV and post-therapy restoration of the vaginal microflora.
Subject(s)
Bacterial Load/methods , Lactobacillus/isolation & purification , Multiplex Polymerase Chain Reaction/methods , Real-Time Polymerase Chain Reaction/methods , Vagina/microbiology , Adolescent , Adult , Female , Humans , Lactobacillus/genetics , Young AdultABSTRACT
OBJECTIVE: To measure the performance characteristics of an immunochromatographic rapid antigen test for respiratory syncytial virus (RSV) and determine how its interpretation should be contextualised in patients presenting to the emergency department (ED) with bronchiolitis. DESIGN: Diagnostic accuracy study of a rapid RSV test. SETTING: County hospital ED. INTERVENTION: We took paired nasal samples from consecutively enrolled infants with bronchiolitis and tested them with a rapid immunochromatographic antigen test and reverse transcriptase PCR gold standard. OUTCOME MEASURES: Sensitivity, specificity, the effect of point prevalence, clinical findings and overall context on predictive values. We used these to construct a graphical contextual model to show how the results of RSV antigen tests from infants presenting within 24 h should influence interpretation of subsequent antigen tests. RESULTS: We analysed 607 patients. The sensitivity and specificity for immunochromatographic testing was 79.4% (95% CI 73.9% to 84.2%) and 67.1% (95% CI 61.9% to 72%) respectively. We found little evidence of spectrum bias. In our contextual model the best predictor of a positive RT-PCR test was a positive antigen test OR 5.47 (95% CI 3.65 to 8.18) and the number of other infants having positive tests within 24 h OR 1.48 (95% CI 1.26 to 1.72) per infant. Increasing numbers presenting to the ED with bronchiolitis in a given day increases the probability of RSV infection. CONCLUSIONS: The RSV antigen test we examined had modest performance characteristics. The results of the antigen test should be interpreted in the context of the results of previous tests.
Subject(s)
Antigens, Viral/blood , Bronchiolitis, Viral/diagnosis , Chromatography, Affinity/methods , Respiratory Syncytial Virus Infections/diagnosis , Respiratory Syncytial Viruses/immunology , Chromatography, Affinity/standards , Emergency Service, Hospital , Female , Humans , Infant , Male , Nasopharynx/virology , Reagent Kits, Diagnostic/standards , Reverse Transcriptase Polymerase Chain Reaction , Sensitivity and SpecificityABSTRACT
Gardnerella vaginalis is an important component of the human vaginal microflora. It is proposed to play a key role in the pathogenesis of bacterial vaginosis (BV), the most common vaginal condition. Here we describe the development, validation and comparative analysis of a novel molecular approach capable of G. vaginalis identification, quantification and subtyping in noncultured vaginal specimens. Using two quantitative PCR (qPCR) assays, we analysed G. vaginalis bacterial loads and clade distribution in 60 clinical vaginal-swab samples. A very high pathogen prevalence was revealed by species-specific qPCR not only among BV patients (100â%), but also in healthy women (97â%), although the G. vaginalis concentration was significantly lower in non-BV samples. G. vaginalis clades identified in vaginal specimens by subtyping multiplex qPCR, which targets four clade-specific genetic markers, had frequencies of 53â% for clade 1, 25â% for clade 2, 32â% for clade 3 and 83â% for clade 4. Multiple clades were found in 70â% of samples. Single G. vaginalis clades were represented by clade 1 and clade 4 in 28â% of specimens. A positive association with BV was shown for clade 1 and clade 3, while clade 2 was positively associated with intermediate vaginal microflora, but not with BV. Clade 4 demonstrated no correlation with the disorder. The presence of multiple clades had a high positive association with BV, whereas G. vaginalis identified as a single clade was negatively linked with the condition. Polyclonal G. vaginalis infection may be a risk factor for BV.
Subject(s)
Bacterial Load/methods , Gardnerella vaginalis/classification , Gardnerella vaginalis/isolation & purification , Gram-Positive Bacterial Infections/diagnosis , Molecular Diagnostic Techniques/methods , Real-Time Polymerase Chain Reaction/methods , Vaginosis, Bacterial/diagnosis , Adult , Female , Gardnerella vaginalis/genetics , Genotype , Gram-Positive Bacterial Infections/microbiology , Humans , Middle Aged , Vaginosis, Bacterial/microbiology , Young AdultABSTRACT
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.