Melinda B. Nye

Development and Validation of a Semiquantitative, Multitarget PCR Assay for Diagnosis of Bacterial Vaginosis

ABSTRACT Quantitative PCR assays were developed for 4 organisms reported previously to be useful positive indicators for the diagnosis of bacterial vaginosis (BV)—Atopobium vaginae, Bacterial Vaginosis-Associated Bacterium 2 (BVAB-2), Gardnerella vaginalis, and Megasphaera-1—and a single organism (Lactobacillus crispatus) that has been implicated as a negative indicator for BV. Vaginal samples (n = 169), classified as positive (n = 108) or negative (n = 61) for BV based on a combination of the Nugent Gram stain score and Amsel clinical criteria, were analyzed for the presence and quantity of each of the marker organisms, and the results were used to construct a semiquantitative, multiplex PCR assay for BV based on detection of 3 positive indicator organisms (A. vaginae, BVAB-2, and Megasphaera-1) and classification of samples using a combinatorial scoring system. The prototype BV PCR assay was then used to analyze the 169-member developmental sample set and, in a prospective, blinded manner, an additional 227 BV-classified vaginal samples (110 BV-positive samples and 117 BV-negative samples). The BV PCR assay demonstrated a sensitivity of 96.7% (202/209), a specificity of 92.2% (153/166), a positive predictive value of 94.0%, and a negative predictive value of 95.6%, with 21 samples (5.3%) classified as indeterminate for BV. This assay provides a reproducible and objective means of evaluating critical components of the vaginal microflora in women with signs and symptoms of vaginitis and is comparable in diagnostic accuracy to the conventional gold standard for diagnosis of BV.

Sequence Diversity in the Glycoprotein B Gene Complicates Real-Time PCR Assays for Detection and Quantification of Cytomegalovirus

ABSTRACT Real-time quantitative PCR systems (Q-PCR) for the rapid detection and quantification of microorganisms in clinical specimens employ oligodeoxyribonucleotide primers and probes for specificity, which makes them vulnerable to false negatives caused by sequence diversity in the template. Schaade et al. (J. Clin. Microbiol. 39:3809, 2001) reported a sequence variant (C630T) in the cytomegalovirus (CMV) glycoprotein B (gB) gene that, although detectable in their Q-PCR assay, could not be accurately quantified. In an effort to evaluate the impact of CMV sequence variants in our patient population by use of a similar Q-PCR assay, we surveyed 54 isolates of CMV, each from a different patient. We detected evidence for the C630T variant in 4 of 54 (7.4%) patients. Furthermore, isolates from two additional patients were completely negative in the test. Sequencing of these false-negative isolates revealed multiple mutations within the probe hybridization sites. A Q-PCR that targeted the CMV polymerase gene instead of gB detected all 54 isolates. We suggest that Q-PCR assays for viral load be rigorously tested on large panels of viral isolates to assess the impact of sequence diversity on detection as well as quantification.

Detection of Trichomonas vaginalis DNA by Use of Self-Obtained Vaginal Swabs with the BD ProbeTec Qx Assay on the BD Viper System

ABSTRACT Trichomonas vaginalis is the most prevalent nonviral sexually transmitted infection worldwide, and improved diagnostic methods are critical for controlling this pathogen. Diagnostic assays that can be used in conjunction with routine chlamydia/gonorrhea nucleic acid-based screening are likely to have the most impact on disease control. Here we describe the performance of the new BD T. vaginalis Qx (TVQ) amplified DNA assay, which can be performed on the automated BD Viper system. We focus on data from vaginal swab samples, since this is the specimen type routinely used for traditional trichomonas testing and the recommended specimen type for chlamydia/gonorrhea screening. Vaginal swabs were obtained from women attending sexually transmitted disease or family planning clinics at 7 sites. Patient-collected vaginal swabs were tested by the TVQ assay, and the Aptima T. vaginalis (ATV) assay was performed using clinician-collected vaginal swabs. Additional clinician-collected vaginal swabs were used for the wet mount and culture methods. Analyses included comparisons versus the patient infection status (PIS) defined by positive results with the wet mount method or culture, direct comparisons assessed with κ scores, and latent class analysis (LCA) as an unbiased estimator of test accuracy. Data from 838 women, 116 of whom were infected with T. vaginalis, were analyzed. The TVQ assay sensitivity and specificity estimates based on the PIS were 98.3% and 99.0%, respectively. The TVQ assay was similar to the ATV assay (κ = 0.938) in direct analysis. LCA estimated the TVQ sensitivity and specificity as 98.3 and 99.6%, respectively. The TVQ assay performed well using self-collected vaginal swabs, the optimal sample type, as recommended by the CDC for chlamydia/gonorrhea screening among women.

Comparison of Nucleic Acid Amplification Assays with BD Affirm VPIII for Diagnosis of Vaginitis in Symptomatic Women

ABSTRACT A commercially available, nonamplified, nucleic acid probe-based test system (BD Affirm VPIII) was compared with nucleic acid amplification (NAA)-based assays for determining the etiology of vaginitis in a cohort of 323 symptomatic women. First, a semiquantitative, multiplexed PCR assay (BV-PCR) and the Affirm VPIII Gardnerella vaginalis test were compared with a unified bacterial-vaginosis (BV) reference standard incorporating both Nugent Gram stain scores and Amsel clinical criteria. In the evaluable population of 305 patients, BV-PCR was 96.9% (191/197) sensitive and 92.6% specific (100/108) for BV, while Affirm VPIII was 90.1% sensitive (179/197) and 67.6% specific (73/108). Second, a multiplexed PCR assay detecting Candida albicans and Candida glabrata (CAN-PCR) was compared with the Affirm VPIII Candida test using a reference standard for vulvovaginal candidiasis (VVC) of yeast culture plus exclusion of alternate vaginitis etiologies. In the population evaluated (n = 102), CAN-PCR was 97.7% sensitive (42/43) and 93.2% specific (55/59) and Affirm VP III was 58.1% sensitive (25/43) and 100% specific (59/59) for VVC. Finally, the results of a commercial NAA test (GenProbe Aptima Trichomonas vaginalis assay; ATV) for T. vaginalis were compared with the Affirm VPIII Trichomonas vaginalis test. In the absence of an independent reference standard for trichomonal vaginitis (TV), a positive result in either assay was deemed to represent true infection. In the evaluable cohort of 388 patients, the sensitivity of ATV was 98.1% (53/54) versus 46.3% (25/54) for Affirm VPIII. The diagnostic accuracy of the combined NAA-based test construct was approximately 20 to 25% higher than that of the Affirm VPIII when modeled in populations with various prevalences of infectious vaginitis.

Suboptimal Adherence to Repeat Testing Recommendations for Men and Women With Positive Chlamydia Tests in the United States, 2008–2010

BACKGROUND Chlamydia is prevalent among young persons in the United States. Infected persons have a high prevalence of infection several months later, most likely from reinfection. Retesting of all men and women with a positive test is recommended 3 months after treatment. A test-of-cure is recommended for pregnant women 3-4 weeks after treatment. METHODS We analyzed 2008-2010 chlamydia testing data from a large US laboratory to estimate test positivity by patient demographic characteristics and diagnoses, and patterns of repeat testing of men and nonpregnant women with a positive test and tests-of-cures of pregnant women with a positive test. RESULTS During the study period, 7.0% of 0.40 million tests performed in men and 4.0% of 2.92 million tests performed in women were positive. Among young women, positivity rates were highest among those aged 15-19 years, ranging from 8.5% to 10.0%. Retesting rates of persons with a positive test were suboptimal, with 22.3% of men and 38.0% of nonpregnant women retested. Although 60.1% of pregnant women with a positive test were retested, only 22.0% received a test-of-cure within the 4-week recommended time frame. Repeat tests were positive in 15.9% of men, 14.2% of nonpregnant women, and 15.4% of pregnant women. CONCLUSIONS Analyses of laboratory testing data provided important insights into chlamydia testing, retesting, and positivity among a diverse US population of men and women. Too few persons were retested as recommended, and interventions are needed to increase both healthcare provider and patient adherence to recommendations for retesting men and women with positive tests.

Infrequent Testing of Women for Rectal Chlamydia and Gonorrhea in the United States

Background Anal sex is a common sexual behavior among women that increases their risk of acquiring rectal infection with Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (GC). Methods We estimated the frequency and positivity of rectal CT and GC tests for women aged 15-60 years performed by a large US commercial laboratory between November 2012 and September 2015. We also estimated the frequency and positivity of pharyngeal and genital specimens also performed on the same date. Among women with a positive CT or GC result, we estimated the frequency and positivity of recommended repeat testing within 12 months. Results Of 5499 women who had rectal CT and GC tests, positivity was 10.8%. On the same date, approximately 80% also had genital CT tests, genital GC tests, and pharyngeal GC tests, while 40% had pharyngeal CT tests. Rectal CT or GC infection was associated with genital CT or GC infection, but 46.5% of rectal CT and GC infections would not have been identified with genital testing alone. Among women with a rectal CT or GC infection, only 20.0% had a recommended repeat rectal test. Of those who had a repeat test, 17.7% were positive. Conclusions Testing women for rectal CT and GC was infrequent, but positive tests were often found in women with negative genital tests. Most women with positive rectal tests were not retested. Interventions are needed to increase extragenital CT and GC testing of at-risk women.

Age-specific chlamydial infection among pregnant women in the United States: evidence for updated recommendations.

Background In the United States, chlamydia screening has been recommended for all pregnant women by the Centers for Disease Control and Prevention (CDC) but only for pregnant women who are at increased risk by the US Preventive Services Task Force (USPSTF). Very limited evidence, such as age-specific chlamydia positivity in pregnant women, has been used to develop these recommendations. Methods We analyzed data from a large commercial laboratory corporation in the United States in 2013. At the first prenatal visit made by women aged 15 to 44 years for whom a chlamydia test was performed between June 2008 and July 2010, we estimated positivity of chlamydia by age, insurance coverage, geographic region, and test type. Results Of 601,001 pregnant women aged 15 to 44 years who had routine prenatal care, 62.9% had private insurance and 32.9% had Medicaid coverage, 60.3% resided in the South region, and 43.2% were aged 15 to 24 years, 26.8% were aged 25 to 29 years, and 19.1% were aged 30 to 34 years. Chlamydia positivity was 3.6% overall, and significantly decreased as age increased (15–19 years: 9.6 %; 20–24 years: 5.2%; 25–29 years: 1.8%; 30–34 years: 0.9%; and 35–44 years: 0.6%; P < 0.05). Conclusions Our findings of higher positivity among younger pregnant women suggest that the yield is likely to be greater from screening younger pregnant women than from screening older pregnant women to identify chlamydia infection. The benefits of harmonizing CDC and USPSTF recommendations for pregnant women could be explored by reviewing age-specific positivity data and estimating the frequency of prenatal adverse health outcomes caused by chlamydia to develop consensus regarding the age limit for pregnant women who should be screened.

O1-S01.04 Suboptimal repeat testing of women with positive chlamydia tests in the USA, 2008–2010

Background Women treated for chlamydia have a high prevalence of infection several months later, likely caused by reinfection from an untreated or new infected sex partner. To prevent potential adverse outcomes of chlamydia, US guidelines recommend repeat testing 3 months after treatment, regardless of partner treatment. If retesting at 3 months is not possible, women should be retested at their next clinical encounter within 12 months. A chlamydia test-of-cure is also recommended for all infected pregnant women 3–4 weeks after treatment. We assessed adherence to retesting guidelines using data from a US laboratory corporation that has a large share of the US market. Methods Among tests performed from June 2008 to May 2010, we estimated the percentage of women who were retested ≥3 weeks later by test result, age and pregnancy status. We also estimated the positivity rate among repeat chlamydia tests and the mean time between an initial test and the first repeat test. We assumed that for each woman in the database all chlamydia tests during the study period were performed by this laboratory corporation. Results Among 2.90 million chlamydia tests performed in 1.77 million women, 4.0% (114 963) were positive. Among the 1.77 million women with tests, 1.34 million (75.7%) had only a single test and 0.43 million (24.3%) had at least one repeat test. If an initial test was positive, 48.6% were retested compared to 23.5% if the initial test was negative (p<0.01); a repeat test was more likely to be positive in women with an initial positive test (13.3%) than a negative one (3.3%) (p<0.01). The mean time interval between the initial and repeat test was shorter if the initial test was positive (117 days) than negative (149 days). Women aged 15–24 years with a positive test had a lower retesting rate than those aged 25–34 years (46.8% vs 53.3%). The percentage of women with a positive test who were retested differed significantly by pregnancy status (60.0% pregnant vs 44.2% nonpregnant), and pregnant women had a repeat test within 93 days compared to 125 days in nonpregnant women. Conclusions These data from a large laboratory corporation provide insight into chlamydia testing practices among women in the USA, and suggest suboptimal adherence to retesting recommendations for both pregnant and nonpregnant women. These data can be useful to monitor the effectiveness of interventions to improve follow-up testing of women with chlamydia.

Detection of Chlamydia trachomatis and Neisseria gonorrhoeaewith the cobas CT/NG v2.0 test: performance compared with the BD ProbeTec CT Qx and GC Qx amplified DNA and Aptima AC2 assays

Objectives Infections due to Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) are among the most common bacterial sexually transmitted infections worldwide, most of which are asymptomatic. Detection of infection using a variety of specimen types in symptomatic and asymptomatic subjects is important to effectively combat CT/NG infections. The performance of the cobas CT/NG v2.0 test was assessed for urogenital swabs, urine and cervical cytology samples collected in PreservCyt Solution from 5266 symptomatic and asymptomatic women (including 202 who were pregnant), and urine from 738 men. Methods Sensitivity and specificity were estimated compared with a patient infected status determined using two US Food and Drug Administration–cleared nucleic acid amplification tests. Results Among 6004 participants, 487 CT (8.1%) and 159 NG (2.6%) infections were identified. Sensitivity estimates for CT for women ranged from 91.2% to 97.6% depending on specimen type, and the estimate for male urine specimens was 98.4%. Specificity for CT ranged from 99.2% to 99.7%. Sensitivity estimates for NG ranged from 95.6% to 100.0% for women, and the estimate for men was 100.0%. Specificity for NG ranged from 99.3% to 100.0%. Conclusions The cobas CT/NG v2.0 test performs well using urogenital swabs, urine and cervical samples collected in PreservCyt solution.

Multicenter study establishing the clinical validity of a nucleic-acid amplification–based assay for the diagnosis of bacterial vaginosis

The present study sought to validate the clinical performance of a previously described PCR-based assay for the diagnosis of bacterial vaginosis (BV). A total of 1579 patients were enrolled in 5 locations; samples were classified as BV positive (n=538) or negative (n=1,041) based on an algorithm utilizing quantitative Gram-stain analysis of vaginal discharge and clinical evaluation (Amsel criteria); a next-generation sequencing (NGS) approach to determining diversity of vaginal microbiota was used to resolve discordant results between BV-PCR and Nugent/Amsel. BV-PCR demonstrated a sensitivity of 96.0% (483/503) and a specificity of 90.2% (885/981) when measured against the conventional test standard, with 95 samples (6.0%) being classified as indeterminate. After resolution of discordant results by NGS, including elimination of the PCR indeterminate category, the resolved sensitivity, specificity, and positive and negative predictive values of the BV-PCR assay were 98.7%, 95.9%, 92.9%, and 96.9%, respectively. The results of this study conclusively demonstrate that a relatively simple, 3-biomarker, molecular amplification construct can effectively diagnose BV in symptomatic women. Results generated using this assay were congruent with those obtained using conventional and molecular reference methods.