Comparison of Bordetella species identification among differing rt-PCR assays in the United States.

In the United States, the general laboratory method for diagnosing pertussis, caused by Bordetella pertussis, is real-time PCR (rt-PCR) targeting insertion sequence 481 (IS481). Other Bordetella species (parapertussis, holmesii, and bronchiseptica) can also cause a pertussis-like syndrome, and some commercial laboratory assays include the insertion sequence 1001 (pIS1001) that can detect B. parapertussis/B. bronchiseptica (BppBb). Because IS481 exists in B. pertussis and B. holmesii, current commercial assays cannot differentiate these two species. We used a multiplex rt-PCR assay containing species-specific targets to Bordetella to evaluate clinical specimens detected as B. pertussis/B. holmesii (BpBh) or BppBb by commercial laboratories. A sample of 3,984 clinical specimens positive for IS481 or pIS1001 from two commercial laboratories during 2012-2019 were re-tested at CDC. Agreement of Bordetella species between the CDC and commercial laboratory assays, and the proportion of commercial laboratory specimens that were non-B. pertussis by CDC's assay was assessed. Overall agreement in Bordetella species detection and identification between the CDC and commercial lab assays was 85%. Agreement for identifying B. pertussis was 87% for 3,663 BpBh specimens and 98% for identifying B. parapertussis in 310 BppBb specimens. CDC's assay detected B. holmesii in 55/3,984 (1.4%) specimens. Most discrepant results (410/490, 82%) were BpBh specimens interpreted as indeterminate B. pertussis at CDC. We found a small portion of B. holmesii in a sample of IS481-positive clinical specimens originally identified by commercial laboratory rt-PCR assays, suggesting that commercial PCR assays are a reliable diagnostic tool for correctly identifying Bordetella species in most patients with suspected pertussis. IMPORTANCE: When testing specimens collected from patients with suspected pertussis, large-scale commercial laboratories in the United States employ an IS481-based assay that cannot differentiate between Bordetella pertussis and Bordetella holmseii. The level of B. holmesii causing pertussis-like illness in the United States is not well-understood given that only B. pertussis is nationally notifiable. After re-testing with a multiplex, species-specific rt-PCR assay, our data show low levels of B. holmesii identified in a sample of IS481-positive clinical specimens originally identified by commercial laboratory rt-PCR assays. These results reinforce the validity of large-scale commercial rt-PCR testing as a reliable diagnostic tool for pertussis in the United States.

Chlamydia trachomatis Variants Escaping Detection in the Aptima Combo 2 Assay in the United States.

BACKGROUND: The Aptima Combo 2 (AC2) assay manufactured by Hologic, Inc., detects Neisseria gonorrhoeae and/or Chlamydia trachomatis (CT) in urogenital and extragenital specimens by targeting either a 16S rRNA (N. gonorrhoeae) or 23S rRNA (CT) region. In 2019, a mutation (C1515T) in the 23S rRNA region was reported to cause false-negative/equivocal results in specimens collected in Finland. Specimens containing this variant (Fl-nvCT) were also discovered internationally. Working with specimens submitted to a large commercial laboratory, we sought to determine if this variant was also present in the United States. METHODS: A subset (n = 401) of specimens tested with the AC2 assay collected during a 5-week period in late 2019/early 2020 were evaluated using an updated AC2 assay. RESULTS: Although the FI-nvCT variant was not detected within this specimen panel, 2 CT variants containing 23S rRNA mutations (A1518G, G1526A) were identified. The updated AC2 assay targeting an additional region of the 23S rRNA detected both of these variants. A retrospective study of >18 million AC2 results tested between 2018 and 2019 did not display a decrease in CT positivity. CONCLUSIONS: Although we did not detect the Fl-nvCT variant among US specimens, we show evidence that the low occurrence of similar diagnostic-escape mutants can be detected with an updated AC2 assay using multiple 23S rRNA targets.

Trichomonas vaginalis Detection in Urogenital Specimens from Symptomatic and Asymptomatic Men and Women by Use of the cobas TV/MG Test.

Trichomonas vaginalis is a prevalent sexually transmitted infection (STI). Diagnosis has historically relied on either microscopic analysis or culture, the latter being the previous gold standard. However, these tests are not readily available for male diagnosis, generally only perform well for symptomatic women, and are not as sensitive as nucleic acid amplification tests (NAATs). Men are largely asymptomatic but carry the organism and transmit to their sexual partners. This multicenter, prospective study evaluated the performance of the cobas T. vaginalis/Mycoplasma genitalium (TV/MG) assay for detection of T. vaginalis DNA compared with patient infection status (PIS) defined by a combination of commercially available NAATs and culture using urogenital specimens. A total of 2,064 subjects (984 men and 1,080 women, 940 [45.5%] symptomatic, 1,124 [54.5%] asymptomatic) were evaluable. In women, sensitivity ranged from 99.4% (95% confidence interval [CI] 96.8 to 99.9%) using vaginal samples to 94.7% (95% CI 90.2 to 97.2%) in PreservCyt samples. Specificity ranged from 98.9 to 96.8% (95% CI 95.4 to 97.8%). In men, the cobas TV/MG assay was 100% sensitive for the detection of T. vaginalis in both male urine samples and meatal swabs, with specificity of 98.4% in urine samples and 92.5% in meatal swabs. The cobas TV/MG is a suitable diagnostic test for the detection of T. vaginalis, which could support public health efforts toward infection control and complement existing STI programs.

Prevalence of Mycoplasma genitalium infection in women with bacterial vaginosis.

BACKGROUND: Bacterial vaginosis (BV) is a common condition in reproductive-age women and is known to be positively associated with risk of acquisition of sexually transmitted infections (STI) such as chlamydia and gonorrhea. Mycoplasma genitalium is an emerging STI that has been linked to increased risk of pelvic inflammatory disease, adverse pregnancy outcomes and infertility. In the present study we sought to examine whether women diagnosed with symptomatic BV were at increased risk of having concurrent infection with Mycoplasma genitalium. METHODS: We used a novel PCR-based assay (ResistancePlus MG; SpeeDx Pty. Ltd., Sydney, Australia) to determine the prevalence of Mycoplasma genitalium infection and 23S rRNA macrolide-resistance mediating mutations (MRMM) in a cohort of 1532 women presenting with symptoms of vaginitis. RESULTS: M. genitalium was detected in 4.0% (62/1532) of samples with 37.1% (23/62) harboring MRMMs. The prevalence of M. genitalium infection in subjects with BV was significantly higher than in subjects with non-BV vaginitis (7.0% v 3.6%; OR = 1.97 (95% CI: 1.14-3.39). CONCLUSIONS: Prevalence of M. genitalium infection is associated with BV in women with symptomatic vaginitis. Improved management of BV is needed as a component of STI prevention strategies.

Syphilis Testing among Men Who Have Had Rectal Gonorrhea and Chlamydia Tests, United States.

The Centers for Disease Control and Prevention (CDC) recommends syphilis screening at least annually for sexually active men who have sex with men (MSM). The objective of this study is to assess the frequency of MSM testing for syphilis and how syphilis test results compared with results of rectal gonorrhea and chlamydia tests. In collaboration with a large US commercial laboratory, we identified men aged 15-60 years who had rectal chlamydia or gonorrhea tests during 09/01/2013-09/30/2015 as presumed MSM. We classified MSM as having current or past syphilis if during the study period they had (1) either a reactive qualitative non-treponemal test or at least a 1:1 quantitative non-treponemal test, and (2) they had a reactive treponemal test. Of 52,771 MSM, 14.3% had no syphilis testing, 4.8% had only treponemal testing (37.8% were reactive), 63.2% had only non-treponemal testing (2.0% were reactive), and 17.7% had both non-treponemal and treponemal testing (86.6% had current or past syphilis). Of those MSM who had reactive qualitative non-treponemal tests, at least 90% had no quantitative non-treponemal tests. Current or past syphilis was more common among MSM with positive rectal gonorrhea or chlamydia tests (24.1%) than MSM with negative rectal gonorrhea and chlamydia tests (13.0%, p < 0.005). Of MSM with any syphilis testing during 09/01/2013-09/30/2014, 64.8% also had annual repeat testing. Syphilis testing in general and repeat syphilis testing were frequent but suboptimal among MSM. It is important to continually monitor syphilis for MSM, especially for those MSM who had rectal chlamydia or gonorrhea infection.

Evaluation of the Performance of the Cobas CT/NG Test for Use on the Cobas 6800/8800 Systems for Detection of Chlamydia trachomatis and Neisseria gonorrhoeae in Male and Female Urogenital Samples.

The clinical performance of the Cobas CT/NG assay on the Cobas 6800/8800 systems (Cobas) for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae was established in a multisite, prospective collection study using male and female urogenital specimens; supportive data from archived specimens were also included. The results obtained with the Cobas assay were compared with the patient infected status derived from a combination of U.S. Food and Drug Administration-approved nucleic acid amplification tests to determine the sensitivity and specificity of detection from each sample type. The sensitivity of Cobas for the detection of C. trachomatis in female specimens was 95.6% (95% confidence interval [CI], 92.4% to 97.4%) for urine; 98.6% (95% CI, 95.2% to 99.6%) and 99.2% (95% CI, 95.4% to 99.9%) for clinician- and self-collected vaginal swab specimens, respectively; 93.3% (95% CI, 89.6% to 95.7%) for endocervical swabs; and 92.5% (95% CI, 88.7% to 95.1%) for cervical swab samples in PreservCyt. The specificity for the detection of C. trachomatis was ≥98.8% for all female sample types. Sensitivity and specificity estimates of Cobas for the detection of C. trachomatis in male urine samples were 100% (96.8% to 100.0%) and 99.7% (95% CI, 99.2% to 99.9%), respectively. The sensitivity of Cobas for the detection of N. gonorrhoeae in female specimens was 94.8% (95% CI, 89.6% to 97.4%) for urine; 100.0% (95% CI, 87.9% to 100.0%) and 100.0% (95% CI, 87.9% to 100.0%) for clinician- and self-collected vaginal swab specimens, respectively; 97.0% (95% CI, 91.5% to 99.0%) for endocervical swabs; and 96.6% (95% CI, 90.6% to 98.8%) for cervical samples in PreservCyt; the specificity for all female sample types was >99.0%. The sensitivity and specificity of Cobas for detecting N. gonorrhoeae in male urine were 100.0% (95% CI, 95.8% to 100.0%) and 99.5% (95% CI, 98.8% to 99.8%), respectively. Fully automated assays help fill the clinical need for a sensitive, high-throughput screening tool to aid public health efforts to control C. trachomatis and N. gonorrhoeae infections.

Detection of Chlamydia trachomatis and Neisseria gonorrhoeae with 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.

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.

Rectal Infection With Neisseria gonorrhoeae and Chlamydia trachomatis in Men in the United States.

BACKGROUND: Centers for Disease Control and Prevention guidelines recommend at least annual rectal screening of men who have receptive anal intercourse for Neisseria gonorrhoeae (GC) and Chlamydia trachomatis (CT). Only limited national data are available on the prevalence of rectal GC and CT infection among US men. METHODS: In collaboration with a large US commercial laboratory, we estimated positivity of the first rectal GC and CT test ("index" test) in men aged 15-60 years tested between January 2013 and May 2015. We estimated the frequency and positivity of pharyngeal or urine specimens tested for GC and CT on the index date, and the frequency and positivity of repeat rectal testing or any follow-up testing at any anatomic site after the index date. RESULTS: Of 52 063 tested men aged 15-60 years, approximately 6.1% were positive for GC only, 8.3% for CT only, and 2.7% for both GC and CT on their index date. On that date, 86.5% had either urine or pharyngeal specimens collected, and 56.1% had both specimens collected. Pharyngeal GC infection was highly associated with rectal GC infection. Follow-up testing after 12 months ranged from 42.4% among uninfected men to 56.7% among infected men on the index date. Positivity was at least 5.7% in rectal GC, rectal CT, or pharyngeal GC at their last test. CONCLUSIONS: This analysis of a large number of male rectal specimens tested for GC and CT suggest that routine testing and timely repeat rectal GC and CT testing should be prioritized among men who report receptive rectal sex.

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.

Comparison of nucleic acid amplification assays with BD affirm VPIII for diagnosis of vaginitis in symptomatic women.

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 Gardnerellavaginalis 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.

Development and validation of a semiquantitative, multitarget PCR assay for diagnosis of bacterial vaginosis.

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.

H-NS binding and repression of the ctx promoter in Vibrio cholerae.

Expression of the ctx and tcp genes, which encode cholera toxin and the toxin coregulated pilus, the Vibrio cholerae O1 virulence determinants having the largest contribution to cholera disease, is repressed by the nucleoid-associated protein H-NS and activated by the AraC-like transcriptional regulator ToxT. To elucidate the molecular mechanism by which H-NS controls transcription of the ctxAB operon, H-NS repression and binding were characterized by using a promoter truncation series, gel mobility shift assays, and DNase I footprinting. Promoter regions found to be important for H-NS repression correlated with in vitro binding. Four main H-NS binding regions are present at ctx. One region overlaps the high-affinity ToxT binding site and extends upstream, another overlaps the ToxT low-affinity binding site around the -35 element, and the remaining two are located adjacent to one another downstream of the transcriptional start site. Competition for binding to the overlapping H-NS/ToxT binding sites was observed in gel mobility shift assays, where ToxT was found to displace H-NS from the ctx promoter region. In addition, regulatory differences between the ctx and tcpA promoters were examined. H-NS was found to have a higher relative binding affinity for the ctx promoter than for the tcpA promoter in vitro. In contrast to ToxT-dependent activation of the tcpA promoter, ToxT activation of ctx did not require the C-terminal domain of the α-subunit of RNA polymerase. These findings demonstrate that transcriptional regulation of ctx and tcpA by H-NS and ToxT is mechanistically distinct, and this may lead to important differences in the expression of these coregulated genes.

Comparison of APTIMA Trichomonas vaginalis transcription-mediated amplification to wet mount microscopy, culture, and polymerase chain reaction for diagnosis of trichomoniasis in men and women.

OBJECTIVE: We evaluated the performance characteristics of APTIMA Trichomonas vaginalis (ATV) transcription-mediated amplification (TMA) for diagnosis of T vaginalis (TV) infection from female vaginal swab, endocervical swab, and urine specimens and from male urethral swab and urine specimens. Performance of ATV TMA was compared with wet mount microscopy, culture, and polymerase chain reaction (PCR). STUDY DESIGN: In all, 296 female and 298 male subjects who attended the Jefferson County Health Department sexually transmitted diseases clinic were enrolled in the study and provided specimens for each test. Results were analyzed using 3 interpretative algorithms. RESULTS: For women, vaginal swab ATV TMA was significantly more sensitive than wet mount or culture. In male subjects, urethral swab ATV TMA was significantly more sensitive than culture or PCR. CONCLUSION: ATV TMA provides a sensitive, commercially available nucleic acid amplification test for improved diagnosis of TV in male and female patients.

Successful treatment of Pasteurella multocida meningitis with aztreonam.

This is the first reported case of the successful treatment of Pasteurella multocida meningitis with aztreonam in a patient with multiple antibiotic allergies.

Successful treatment of Pasteurella multocida meningitis with aztreonam.

This is the first reported case of the successful treatment of Pasteurella multocida meningitis with aztreonam in a patient with multiple antibiotic allergies.

Sequence diversity in the glycoprotein B gene complicates real-time PCR assays for detection and quantification of cytomegalovirus.

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.

Vibrio cholerae H-NS domain structure and function with respect to transcriptional repression of ToxR regulon genes reveals differences among H-NS family members.

H-NS is an abundant bacterial protein involved in transcriptional silencing of a variety of environmentally responsive genes during growth under non-permissive conditions. We have previously demonstrated a direct role for H-NS in the negative modulation of expression of several genes within the ToxR virulence regulon of Vibrio cholerae. Here we have undertaken extensive mutagenesis of the structural and functional domains of the H-NS protein to determine the contribution of each to the regulation of gene expression. Insertions within, or truncations of, the C-terminal conserved DNA-binding domain prevent repression of toxT and ctx, as expected. Dominant negative experiments demonstrate that V. cholerae H-NS represses gene expression as an oligomeric protein. Hydrophobic coiledcoil interactions have been shown to provide oligomerization capability in other H-NS orthologues. We used site-directed mutagenesis to construct altered V. cholerae H-NS proteins, including an extensive internal deletion within the predicted coiledcoil domain. Remarkably, these proteins were competent to repress gene expression and to form oligomers. Chimeric H-NS proteins, using sequences from both Escherichia coli and V. cholerae H-NS orthologues, revealed that V. cholerae H-NS possesses a second oligomerization domain in the N-terminal 24 amino acids of the protein. Overall, our results suggest DNA binding and protein oligomerization, provided by either the central coiledcoil or N-terminal domain, are required for repression of promoters responsive to H-NS within the ToxR regulon.