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.

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.

Is the interpretation of rapid antigen testing for respiratory syncytial virus as simple as positive or negative?

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.

Development and Diagnostic Potential of a Novel Bartonella henselae-Specific Immunoglobulin.

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.

Molecular characterization of vaginal microbiota using a new 22-species qRT-PCR test to achieve a relative-abundance and species-based diagnosis of bacterial vaginosis.

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.

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.

Detecting DNA methylation of the BCL2, CDKN2A and NID2 genes in urine using a nested methylation specific polymerase chain reaction assay to predict bladder cancer.

PURPOSE: Detection of methylated DNA has been shown to be a good biomarker for bladder cancer. Bladder cancer has the highest recurrence rate of any cancer and, as such, patients are regularly monitored using invasive diagnostic techniques. As urine is easily attainable, bladder cancer is an optimal cancer to detect using DNA methylation. DNA methylation is highly specific in cancer detection. However, it is difficult to detect because of the limited amount of DNA present in the urine of patients with bladder cancer. Therefore, an improved, sensitive and noninvasive diagnostic test is needed. MATERIALS AND METHODS: We developed a highly specific and sensitive nested methylation specific polymerase chain reaction assay to detect the presence of bladder cancer in small volumes of patient urine. The genes assayed for DNA methylation are BCL2, CDKN2A and NID2. The regions surrounding the DNA methylation sites were amplified in a methylation independent first round polymerase chain reaction and the amplification product from the first polymerase chain reaction was used in a real-time methylation specific polymerase chain reaction. Urine samples were collected from patients receiving treatment at Wolfson Medical Center in Holon, Israel. RESULTS: In a pilot clinical study using patient urine samples we were able to differentiate bladder cancer from other urogenital malignancies and nonmalignant conditions with a sensitivity of 80.9% and a specificity of 86.4%. CONCLUSIONS: We developed a novel methylation specific polymerase chain reaction assay for the detection and monitoring of bladder cancer using DNA extracted from patient urine. The assay may also be combined with other diagnostic tests to improve accuracy.

Detection rates of trichomonas vaginalis, in different age groups, using real-time polymerase chain reaction.

OBJECTIVE: The study aimed to compare the overall detection rate of Trichomonas vaginalis to Chlamydia trachomatis and Neiserria gonorrhea and report detection rates by age groups. MATERIALS AND METHODS: Real-time polymerase chain reaction was used to detect the presence of T. vaginalis, C. trachomatis, and N. gonorrhea in cervical samples obtained from patients during gynecological examinations. A total of 78,428, 119,451, and 117,494 samples from women age 12 to 75 years were retrospectively analyzed for the presence of T. vaginalis, C. trachomatis, and N. gonorrhea, respectively. T. vaginalis and C. trachomatis detection rates in Florida, New Jersey, and Texas were calculated in different age groups. RESULTS: The overall detection rate was 4.3% for T. vaginalis, 3.8% for C. trachomatis, and 0.6% for N. gonorrhea. The overall detection rate of T. vaginalis in Florida was 4.7% (n = 22,504), in New Jersey was 3.6% (n = 22,249), and in Texas was 4.5% (n = 33,675). Calculation of infection rates with T. vaginalis revealed differences between selected age groups with the highest detection rates in all 3 states found in age group 46 to 55 years (6.2%), which was higher than the overall detection rates in other age groups (p < .05 for all states). For C. trachomatis, the highest detection rate was found in age group 12 to 25 years (7.3%). CONCLUSIONS: The overall infection rates of T. vaginalis were higher compared with those of C. trachomatis and N. gonorrhea. Detection rates of T. vaginalis were found to be highest among women age 46 to 55 years and may be due to T. vaginalis infiltrating the subepithelial glands and being detected only during hormone-induced or antibiotic-induced changes in the vaginal flora.

High-Risk HPV Genotype Distribution According to Cervical Cytology and Age.

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.

Oncoprotein DEK as a tissue and urinary biomarker for bladder cancer.

BACKGROUND: Bladder cancer is a significant healthcare problem in the United States of America with a high recurrence rate. Early detection of bladder cancer is essential for removing the tumor with preservation of the bladder, avoiding metastasis and hence improving prognosis and long-term survival. The objective of this study was to analyze the presence of DEK protein in voided urine of bladder cancer patients as a urine-based bladder cancer diagnostic test. METHODS: We examined the expression of DEK protein by western blot in 38 paired transitional cell carcinoma (TCC) bladder tumor tissues and adjacent normal tissue. The presence of DEK protein in voided urine was analyzed by western blot in 42 urine samples collected from patients with active TCC, other malignant urogenital disease and healthy individuals. RESULTS: The DEK protein is expressed in 33 of 38 bladder tumor tissues with no expression in adjacent normal tissue. Based on our sample size, DEK protein is expressed in 100% of tumors of low malignant potential, 92% of tumors of low grade and in 71% of tumors of high grade. Next, we analyzed 42 urine samples from patients with active TCC, other malignant urogenital disease, non-malignant urogenital disease and healthy individuals for DEK protein expression by western blot analysis. We are the first to show that the DEK protein is present in the urine of bladder cancer patients. Approximately 84% of TCC patient urine specimens were positive for urine DEK. CONCLUSION: Based on our pilot study of 38 bladder tumor tissue and 42 urine samples from patients with active TCC, other malignant urogenital disease, non-malignant urogenital disease and healthy individuals; DEK protein is expressed in bladder tumor tissue and voided urine of bladder cancer patients. The presence of DEK protein in voided urine is potentially a suitable biomarker for bladder cancer and that the screening for the presence of DEK protein in urine can be explored as a noninvasive diagnostic test for bladder cancer.

Prevalence of Bordetella pertussis and Bordetella parapertussis in infants presenting to the emergency department with bronchiolitis.

BACKGROUND: The clinical presentation of Bordetella pertussis can overlap with that of respiratory syncytial virus (RSV), and coinfection does occur, but management differs. HYPOTHESIS: The prevalence of B. pertussis is < 2% among Emergency Department (ED) patients with bronchiolitis. Our secondary hypothesis was that the prevalence of Bordetella parapertussis is also < 2% among these patients. METHODS: Nasal washings were obtained from children up to 18 months of age (inclusive) who presented to a county hospital ED with a clinical diagnosis of bronchiolitis. These washings were frozen to -70°C before testing for B. pertussis and B. parapertussis using species-specific real-time polymerase chain reaction (PCR) assays. The assays were optimized to target conserved regions within a complement gene and the CarB gene, respectively. A Bordetella spp. genus-specific real-time PCR assay was designed to detect the Bhur gene of B. pertussis, B. parapertussis, and B. bronchiseptica. RSV antigen detection was also performed. RESULTS: There were 227 patients enrolled. After exclusions, 204 remained in the analysis. RSV antigen testing was positive in 109/186 (59%) of the patients in whom it was performed. All samples were tested for B. pertussis. B. parapertussis testing could not be completed on 23 samples. No cases (0/204; 95% confidence interval [CI] 0-1.8%) tested positive for B. pertussis or B. parapertussis (0/181; 95% CI 0-2%). CONCLUSION: The prevalence of B. pertussis in children presenting to the ED with bronchiolitis was < 2%.

Genital herpes simplex virus type 1 in women: detection in cervicovaginal specimens from gynecological practices in the United States.

Herpes simplex virus types 1 and 2 (HSV-1 and -2) are significant human pathogens causing clinically indistinguishable facial and genital lesions. Recently, the number of reported genital herpes cases caused by type 1 virus has increased. Identifying the HSV type is of clinical importance to determine proper treatment, as there is no licensed vaccine or cure. We assessed, by PCR, the frequency of HSV-1 and HSV-2 present in more than 60,000 clinical cervicovaginal specimens derived from samples originating from 43 continental U.S. states. Fourteen percent were positive for HSV-1 and/or HSV-2. This likely represents subclinal shedding. It was not a measurement of the prevalence of HSV infection. While the majority were HSV-2, 32% were HSV-1. The distribution of HSV types varied between the states with the largest number of specimens, New Jersey, Florida, and Texas. Specimens from women under the age of 24 had an HSV-1 positivity rate of 47 percent. Importantly, in New Jersey, an observed age effect was the disproportionately high prevalence of genital HSV-1 in young women. This represents the largest analysis of HSV types reported and has important public health implications, particularly for younger women.

Uropathogenic Escherichia coli dominantly suppress the innate immune response of bladder epithelial cells by a lipopolysaccharide- and Toll-like receptor 4-independent pathway.

Urinary tract infections are a major source of morbidity among women, with the majority caused by uropathogenic Escherichia coli. Our objective was to test if uropathogenic E. coli suppress the innate immune response of bladder epithelial cells. We found that bladder epithelial cells secrete interleukin-6 and interleukin-8 in response to non-pathogenic E. coli, whereas they failed to do so in response to uropathogenic E. coli. Uropathogenic E. coli prevented interleukin-6 secretion in response to non-pathogenic E. coli and a panel of Toll-like receptor agonists, as well as to interleukin-1beta, but not to tumor necrosis factor alpha. These results indicate that receptors with a Toll/interleukin-1 receptor domain are specifically targeted, and that suppression is not a consequence of toxicity. One candidate for mediating immune suppression is bacterial lipopolysaccharide. However, lipopolysaccharide isolated from either uropathogenic or non-pathogenic E. coli stimulated interleukin-6 secretion to similar levels. In addition, uropathogenic E. coli did not stimulate interleukin-6 secretion from cells expressing a dominant negative Toll-like receptor 4, and prevented cells lacking Toll-like receptor 4 from secreting interleukin-6 in response to synthetic lipoprotein. We conclude that uropathogenic E. coli suppress the innate immune response through a pathway partially independent of lipopolysaccharide and Toll-like receptor 4.

Atopobium vaginae triggers an innate immune response in an in vitro model of bacterial vaginosis.

Bacterial vaginosis is the most common vaginal disorder among women of reproductive age. The pathogenesis of bacterial vaginosis is poorly understood, but is defined by a transition in the vaginal flora from the predominant Lactobacillus species to other bacterial species such as Atopobium vaginae and Gardnerella vaginalis. This change is associated with an increase in vaginal cytokine secretion. We hypothesize that vaginal epithelial cells respond to bacterial vaginosis-associated bacteria by triggering an innate immune response. We observed that vaginal epithelial cells secreted interleukin-6 and interleukin-8 in response to Atopobium vaginae and Gardnerella vaginalis, but not to Lactobacillus crispatus. Atopobium vaginae induced increased levels of interleukin-6 and interleukin-8 transcripts, as well as increased transcripts for the antimicrobial peptide beta-defensin 4. This innate immune response required live bacteria capable of protein synthesis in direct contact with vaginal epithelial cells. The response of vaginal epithelial cells was mediated by Toll-like receptor 2, required the adaptor protein MyD88, and involved activation of the NFkappaB signaling pathway. These results suggest that Atopobium vaginae stimulates an innate immune response from vaginal epithelial cells, leading to localized cytokine and defensin production, and possibly contributes to the pathogenesis of bacterial vaginosis.

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.