Justin Hardick

Mycoplasma genitalium as a Contributor to the Multiple Etiologies of Cervicitis in Women Attending Sexually Transmitted Disease Clinics

Objectives: The purpose of this study was to investigate the prevalence of Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, and Mycoplasma genitalium, in women attending a sexually transmitted disease (STD) clinic, as well as the frequency of coinfections, and relationship of each organism to cervicitis. Methods: In this cross-sectional study of 324 women attending Baltimore City STD Clinics, C. trachomatis, N. gonorrhoeae, T. vaginalis, and M. genitalium were detected using nucleic acid amplification tests. Demographic characteristics and risk factors were ascertained. Results: Overall prevalence of infection with C. trachomatis, N. gonorrhoeae, T. vaginalis, and M. genitalium was found to be 11.1%, 4.6%, 15.3%, and 19.2%, respectively. Prevalence in women with cervicitis was 15.8%, 6%, 18.9%, and 28.6% for C. trachomatis, N. gonorrhoeae, T. vaginalis, and M. genitalium, respectively. Percentages of coinfections were high. C. trachomatis and M. genitalium were significantly associated with cervicitis in univariate analysis, but only M. genitalium was significantly associated with cervicitis (AOR: 2.5) in multiple logistic regression models. Conclusion: Knowledge of the statistical association of M. genitalium with cervicitis in this study increases the need for further confirmation of the etiologic significance of this organism with cervicitis in more diverse populations. The high prevalence merits more study and may have implications for diagnosis and treatment of cervicitis.

Quantitative PCR Assay Using Sputum Samples for Rapid Diagnosis of Pneumococcal Pneumonia in Adult Emergency Department Patients

ABSTRACT Accurate diagnosis of pneumococcal pneumonia in the acute-care setting remains a challenge due to the inadequate sensitivity of conventional diagnostic tests. Sputum cultures, which are likely to have the highest diagnostic yields of all specimen types, have been considered unreliable, due to their inability to differentiate colonization from infection. Our objective was to evaluate the potential clinical utility of a rapid quantitative real-time PCR assay using sputum samples for Streptococcus pneumoniae in adult patients with community-acquired pneumonia (CAP). A prospective clinical observational study of consecutively enrolled emergency department patients with CAP was performed; only those patients with excess good-quality sputum samples were included for evaluation. Sputum samples were tested for the presence of S. pneumoniae by using a quantitative PCR that targets the pneumolysin gene. PCR findings were compared with those of a composite reference standard comprising Gram staining of sputum samples and sputum/blood cultures. The area under the curve (AUC) and a log-transformed threshold, which provides the maximal sensitivity and specificity, were calculated. Of 487 subjects enrolled, 129 were evaluable. Receiver operating characteristic curve analysis demonstrated an AUC of 0.87. Sensitivity and specificity were 90.0 percent and 80.0 percent, respectively; positive and negative predictive values were 58.7 percent and 96.2 percent, respectively. We have demonstrated that a quantitative rapid pneumolysin PCR assay has favorable accuracy for diagnosis of pneumococcal pneumonia in adult patients with CAP; this assay may be a useful diagnostic adjunct for clinicians, particularly those practicing in the acute-care setting, where rapid pathogen identification may assist in selection of the most appropriate antibiotics.

Rapid PCR-based diagnosis of septic arthritis by early Gram-type classification and pathogen identification.

ABSTRACT Septic arthritis (SA) is a rheumatologic emergency associated with significant morbidity and mortality. Delayed or inadequate treatment of SA can lead to irreversible joint destruction and disability. Current methods of diagnosing SA rely on synovial fluid analysis and culture which are known to be imprecise and time-consuming. We report a novel adaptation of a probe-based real-time PCR assay targeting the 16S rRNA gene for early and accurate diagnosis of bacterial SA. The assay algorithm consists of initial broad-range eubacterial detection, followed by Gram typing and species characterization of the pathogen. The platform demonstrated a high analytical sensitivity with a limit of detection of 101 CFU/ml with a panel of SA-related organisms. Gram typing and pathogen-specific probes correctly identified their respective targets in a mock test panel of 36 common clinically relevant pathogens. One hundred twenty-one clinical synovial fluid samples from patients presenting with suspected acute SA were tested. The sensitivity and specificity of the assay were 95% and 97%, respectively, versus synovial fluid culture results. Gram-typing probes correctly identified 100% of eubacterial positive samples as to gram-positive or gram-negative status, and pathogen-specific probes correctly identified the etiologic agent in 16/20 eubacterial positive samples. The total assay time from sample collection to result is 3 h. We have demonstrated that a real-time broad-based PCR assay has high analytical and clinical performance with an improved time to detection versus culture for SA. This assay may be a useful diagnostic adjunct for clinicians, particularly those practicing in the acute care setting where rapid pathogen detection and identification would assist in disposition and treatment decisions.

Comparison between the Gen-Probe Transcription-Mediated Amplification Trichomonas vaginalis Research Assay and Real-Time PCR for Trichomonas vaginalis Detection Using a Roche LightCycler Instrument with Female Self-Obtained Vaginal Swab Samples and Male Urine Samples

ABSTRACT This study compared two assays for Trichomonas vaginalis detection, Gen-Probes transcription-mediated amplification (TMA) assay for Trichomonas vaginalis and BTUB FRET PCR, using self-obtained clinical samples from 611 patients. Infection status was defined as two positive results by two different tests. The initial TMA assay sensitivity was 96.7%; specificity was 97.5%. The TMA assay was comparable to BTUB FRET PCR.

Point of care diagnostics for sexually transmitted infections: perspectives and advances

Accurate and inexpensive point-of-care (POC) tests are urgently needed to control sexually transmitted infection epidemics, so that patients can receive immediate diagnoses and treatment. Current POC assays for Chlamydia trachomatis and Neisseria gonorrhoeae perform inadequately and require better assays. Diagnostics for Trichomonas vaginalis rely on wet preparation, with some notable advances. Serological POC assays for syphilis can impact resource-poor settings, with many assays available, but only one available in the U.S. HIV POC diagnostics demonstrate the best performance, with excellent assays available. There is a rapid assay for HSV lesion detection; but no POC serological assays are available. Despite the inadequacy of POC assays for treatable bacterial infections, application of technological advances offers the promise of advancing POC diagnostics for all sexually transmitted infections.

Performance of the Gen-Probe Transmission-Mediated Amplification Research Assay Compared to That of a Multitarget Real-Time PCR for Mycoplasma genitalium Detection

ABSTRACT Mycoplasma genitalium (MG) can cause nongonococcal urethritis and is potentially associated with urethritis, endometritis, and cervicitis. Several assays have been developed to detect MG. Molecular amplification assays for organism detection can be problematic due to the potential for false-positive and false-negative results. Confirmatory testing is often required in these situations, requiring additional time and resources. Use of multigene targets could integrate both detection and verification at lower cost. Utilizing two targets, the MgPa adhesion gene and the 16S rRNA gene, a multitarget real-time (MTRT) PCR for the detection of MG was developed. Samples from patients attending sexually transmitted disease clinics were collected in duplicate. Urine samples from males (n = 286) and self-collected vaginal swabs from females (n = 321) were analyzed by MTRT PCR for MG and the Gen-Probe transmission-mediated amplification (TMA) assay, which targets MG rRNA for detection (TMA-MG research use only). Utilizing the criteria of any two targets being positively amplified, the MTRT PCR had a sensitivity and specificity of 91.8% (101 positive samples/110 samples tested) and 99.5% (495/497), respectively, with a positive predictive value (PPV) of 98.1% (101/103) and a negative predictive value (NPV) of 98.2% (495/504). The Gen-Probe TMA-MG assay had a sensitivity, specificity, PPV, and NPV of 98.1% (108/110), 98.1% (488/497), 92.3% (108/117), and 99.5% (488/490), respectively. Comparison between the MTRT PCR and TMA-MG assay by kappa statistic analysis indicated that an overall kappa value was 0.941 (95% confidence interval, 0.907 and 0.976). Both assays demonstrated accuracy in the detection of MG from urine samples from male patients and self-collected vaginal swabs from female patients.

Use of the roche lightcycler instrument in a real-time PCR for Trichomonas vaginalis in urine samples from females and males

ABSTRACT Trichomonas vaginalis is the agent of a highly prevalent sexually transmitted infection (STI) that can result in vaginitis, urethritis, and preterm birth. Traditional methods of diagnosis, including wet preparation, can be unreliable. In this study, we describe the adaptation of an existing PCR method for specific detection of T. vaginalis DNA into a rapid real-time PCR assay based on fluorescence resonance energy transfer (FRET) probe chemistry. The FRET-based assay described demonstrated high sensitivity with a detection limit of 1.06 organisms, as well as a high specificity. A total of 253 urine samples collected prospectively from both men and women were tested for T. vaginalis DNA with both the FRET-based assay and a previously validated PCR assay. When the validated PCR assay was used as the “gold standard” and after discrepancies had been resolved, our FRET-based assay demonstrated an analytical sensitivity and specificity of 90.1 and 100%, respectively. Overall results suggest that FRET-based assays can provide rapid, accurate, and high-throughput detection of T. vaginalis and may prove useful in clinical settings and for large-scale screening programs.

Testing and Validation of High Density Resequencing Microarray for Broad Range Biothreat Agents Detection

Rapid and effective detection and identification of emerging microbiological threats and potential biowarfare agents is very challenging when using traditional culture-based methods. Contemporary molecular techniques, relying upon reverse transcription and/or polymerase chain reaction (RT-PCR/PCR) provide a rapid and effective alternative, however, such assays are generally designed and optimized to detect only a limited number of targets, and seldom are capable of differentiation among variants of detected targets. To meet these challenges, we have designed a broad-range resequencing pathogen microarray (RPM) for detection of tropical and emerging infectious agents (TEI) including biothreat agents: RPM-TEI v 1.0 (RPM-TEI). The scope of the RPM-TEI assay enables detection and differential identification of 84 types of pathogens and 13 toxin genes, including most of the class A, B and C select agents as defined by the Centers for Disease Control and Prevention (CDC, Atlanta, GA). Due to the high risks associated with handling these particular target pathogens, the sensitivity validation of the RPM-TEI has been performed using an innovative approach, in which synthetic DNA fragments are used as templates for testing the assays limit of detection (LOD). Assay specificity and sensitivity was subsequently confirmed by testing with full-length genomic nucleic acids of selected agents. The LOD for a majority of the agents detected by RPM-TEI was determined to be at least 104 copies per test. Our results also show that the RPM-TEI assay not only detects and identifies agents, but is also able to differentiate near neighbors of the same agent types, such as closely related strains of filoviruses of the Ebola Zaire group, or the Machupo and Lassa arenaviruses. Furthermore, each RPM-TEI assay results in specimen-specific agent gene sequence information that can be used to assess pathogenicity, mutations, and virulence markers, results that are not generally available from multiplexed RT-PCR/PCR-based detection assays.

Mycoplasma genitalium compared to chlamydia, gonorrhoea and trichomonas as an aetiological agent of urethritis in men attending STD clinics.

Objectives: To investigate prevalence of Mycoplasma genitalium, Chlamydia trachomatis, Neisseria gonorrhoeae and Trichomonas vaginalis in men, frequency of co-infections, and association of organisms with urethritis in men. Methods: This was a cross-sectional study of 290 men (age range 19–34 years) attending Baltimore City STD clinics. M genitalium, C trachomatis, N gonorrhoeae and T vaginalis, during 2004 were detected using nucleic acid amplification tests (NAATs) (153 with urethritis and 137 without urethritis). Demographic characteristics and risk factors were ascertained. Results: The overall prevalences of infection with C trachomatis, N gonorrhoeae, T vaginalis and M genitalium were 20.3%, 12.8%, 3.4% and 15.2%, respectively. Prevalences in men with urethritis were 32.7%, 24.2%, 5.2% and 22.2% for C trachomatis, N gonorrhoeae, T vaginalis and M genitalium, respectively. Percentages of co-infections were high. All men with N gonorrhoeae had urethritis. C trachomatis and M genitalium were found to be significantly associated with urethritis in univariate analysis and in multiple logistic regression analysis. Conclusion: The association of M genitalium with urethritis in this study provides confirmation of the importance of screening men for M genitalium as a cause of non-gonococcal urethritis and supports treatment considerations for urethritis for agents other than gonococci and chlamydia.

Surveillance of Chlamydia trachomatis and Neisseria gonorrhoeae infections in women in detention in Baltimore, Maryland

Background In conjunction with a program to expand syphilis and HIV infection services, women were also offered screening for Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (GC) during intake at the Baltimore Womens Detention Center. Goal The goal was to assess the effectiveness of a routine screening program for CT and GC in women in a detention setting. The association among infection, race, and area of residence was also explored. Study Design CT and GC prevalences were determined and analyzed by demographic data, including zip code, for 1858 women enrolled over a 48-week period. Informed consent was obtained, and infections were detected with use of urine samples tested by ligase chain reaction. Results Overall, the population had prevalence rates of 5.9% (109/1858) and 3.4% (63/1858) for CT and GC respectively. Among whites, CT and GC prevalences were 9.0% (29/323) and 8.7% (28/323), respectively. Among African Americans the prevalence rates were 5.1% (77/1510) and 2.3% (34/1510) for CT and GC, respectively. White women <25 years of age were associated with the highest CT and GC prevalences, at 20.0% (13/65) and 13.9% (9/65), respectively. African American women <25 years of age also were associated with the highest CT and GC prevalences, at 13.9% (24/173) and 5.8% (10/173), respectively. Multivariate analysis of risk factors and demographic data indicated that ages <25 years and 25 to 34 years, white race, and certain zip codes of residence were risk factors for infection. Conclusion This study illustrated that urine-based screening for CT and GC is feasible in detention settings and can be productive in high-prevalence areas. Geographic analysis demonstrated no definitive relationship among race, infection, and area of residence, although it did demonstrate clustering of infected individuals and could be useful in future interventions. These findings demonstrated the need for implementing screening programs for sexually transmitted infections in detention centers.