Jeanne E. Hryciuk

Impact of Trichomonas vaginalis Transcription-Mediated Amplification-Based Analyte-Specific-Reagent Testing in a Metropolitan Setting of High Sexually Transmitted Disease Prevalence

ABSTRACT Trichomoniasis is a significant sexually transmitted disease (STD) in the spectrum of public health and primary care because of its association with agents such as human immunodeficiency virus and Neisseria gonorrhoeae. However, its true significance may be underestimated due to diagnostic modalities that exhibit poor sensitivity. A total of 1,086 genital specimens from two urban emergency departments, a suburban urgent-care facility, and a metropolitan outpatient physician group were subjected to transcription-mediated amplification-based Trichomonas vaginalis analyte-specific-reagent (ASR) testing (Gen-Probe, Inc.). The rate of positive molecular ASR results (14.5%) doubled that of direct saline preparation (7.0%; P < 0.0002). Analogous increases were observed at one emergency department and within the outpatient physician group (P < 0.0002). No significant increase in the rate of positive molecular ASR results was observed from the facilities that encountered a lower frequency of black/African American patients. While positive T. vaginalis findings via direct saline preparation did not have a significant association with concomitant Chlamydia trachomatis or N. gonorrhoeae infection overall, a positive T. vaginalis ASR result was a better predictor of concomitant C. trachomatis or N. gonorrhoeae infection (odds ratios of 2.34 and 4.46, respectively; P < 0.0001). The increased rate of positive T. vaginalis ASR results was observed in both point-of-care (P = 0.02 versus direct saline preparation) and laboratory (P = 0.003) testing. Highly sensitive T. vaginalis molecular ASR not only transcends issues of specimen integrity and microscopic acumen but also has an increased ability to predict the likelihood of additional STDs in defined populations.

Comparison of Carrot Broth- and Selective Todd-Hewitt Broth-Enhanced PCR Protocols for Real-Time Detection of Streptococcus agalactiae in Prenatal Vaginal/Anorectal Specimens

ABSTRACT The reporting of accurate Streptococcus agalactiae screening results in a short time frame is of tremendous clinical benefit. A total of 203 consecutive primary vaginal/anorectal specimens were cultured in selective Todd-Hewitt broth (LIM broth) and with the StrepB carrot broth kit (carrot broth). One-day broth cultures were subjected to both centrifugation and clarification of a 500-μl aliquot prior to sample lysis (protocol A) and direct lysis of a 50-μl aliquot (protocol B). The lysates were subsequently analyzed by the BD GeneOhm StrepB assay. The results were compared to the carrot broth culture results derived from visualization of pigment on day 1 or from a subculture of carrot broth. Thirty-four carrot broth cultures (16.7%) generated diagnostic pigment following overnight incubation; an additional 26 (12.8%) were positive for S. agalactiae upon subculture. Carrot broth-enhanced PCR by the use of either protocol A or protocol B trended toward a higher rate of positive results (33.0%) than the rate observed by either the LIM broth-enhanced PCR (30.5%) or full carrot broth culture analysis (29.6%). In the context of the result on day 1, both carrot broth- and LIM broth-enhanced PCRs generated more true-positive results (P < 0.001) than carrot broth culture visualization. The predictive values for both protocols of carrot broth- or LIM broth-enhanced PCR were ≥95.4%. Whereas protocol A resolved the results for 99.8% of the specimens in the evaluation upon initial testing, a 5.7% initial unresolved rate and a 1.5% final unresolved rate were determined by the use of protocol B. The use of carrot broth within a rapid and highly accurate molecular reflex testing algorithm can limit follow-up testing to cultures without evidence of pigmentation.

Screening of Male Patients for Trichomonas vaginalis with Transcription-Mediated Amplification in a Community with a High Prevalence of Sexually Transmitted Infection

ABSTRACT Trichomonas vaginalis infection in males has been largely uncharacterized. Past reports indicated increased susceptibility to other sexually transmitted infection (STI) agents such as human immunodeficiency virus and Neisseria gonorrhoeae with concurrent T. vaginalis infection. This warrants a more thorough review of male T. vaginalis incidence. A retrospective 3-year investigation of transcription-mediated amplification (TMA)-based urethral swab and first-void urine screening for T. vaginalis within a regional health care system was performed to address T. vaginalis prevalence in males. Of 622 total samples tested, 6.6% were positive for T. vaginalis. Delineation of all specimens by ZIP code of patient residence revealed 11 predominant ZIP codes with respect to testing volume and detection rates. Within these 11 ZIP codes, representing 78.3% of total testing volume, urine was the preferred specimen source compared to urethral swabs. Seven of these 11 ZIP codes contained majority African American populations. The aggregate T. vaginalis detection rate trended higher than that of the remaining four ZIP codes, which were comprised primarily of Caucasian populations (8.9% versus 5.0%, respectively; P = 0.15). The average age of a T. vaginalis-infected male (39.9 years) was significantly greater than those for Chlamydia trachomatis or N. gonorrhoeae (27.6 and 25.9 years, respectively; P < 0.001). Given the significant rate of T. vaginalis detection, with age distribution analogous to that reported in females, TMA-based detection of T. vaginalis can be a routine constituent within a comprehensive STI screening panel for males in high-prevalence STI communities.

Female Epidemiology of Transcription-Mediated Amplification-Based Trichomonas vaginalis Detection in a Metropolitan Setting with a High Prevalence of Sexually Transmitted Infection

ABSTRACT Recent literature has reported increased accuracy of Trichomonas vaginalis transcription-mediated amplification (TMA)-based analyte-specific reagent (ASR) testing in female populations. A retrospective investigation assessed 7,277 female first-void urine, cervical, or vaginal specimens submitted from a high-prevalence sexually transmitted infection (STI) community to characterize prevalence of disease etiologies. The most common STI phenotype reflected detection of solely T. vaginalis (54.2% of all health care encounters that resulted in STI detection). In females with detectable T. vaginalis, codetection of Chlamydia trachomatis and Neisseria gonorrhoeae occurred in 7.8% and 2.7% of health care encounters, respectively. The mean age of women with detectable T. vaginalis (30.6) was significantly higher than those for women with C. trachomatis or N. gonorrhoeae (22.3 and 21.6, respectively; P < 0.0001). T. vaginalis was the predominant sexually transmitted agent in women over the age of 20 (P < 0.0002). C. trachomatis was the most commonly detected agent in females under the age of 21, particularly from cervical specimens. However, first-void urine detection rates for T. vaginalis and C. trachomatis within this age demographic demonstrated no difference (P = 0.92). While overall and cervical specimen-derived detection of T. vaginalis within African American majority geographical locales outweighed that within majority Caucasian geographical regions (P ≤ 0.004), this difference was not noted with first-void urine screening (P = 0.54). Health care professionals can consider TMA-based T. vaginalis screening for a wide age range of patients; incorporation of first-void urine specimens into screening algorithms can potentiate novel insight into the epidemiology of trichomoniasis.

Trichomonas vaginalis transcription-mediated amplification-based analyte-specific reagent and alternative target testing of primary clinical vaginal saline suspensions.

Following wet mount analysis, 255 vaginal saline suspensions were aliquoted to lysis medium for transcription-mediated amplification (TMA)-based Trichomonas vaginalis analyte-specific reagent testing (ASR) (Gen-Probe, San Diego, CA). Specimens with visible T. vaginalis were then refrigerated, with additional aliquoting at later intervals. Twenty-four wet mount-positive specimens (9.4%) yielded a median luminescent value (x1000, relative light unit [RLU]) of 4736. In contrast, RLU ranged from 1 to 21 following ASR of 204 wet mount-negative specimens. Twenty-seven wet mount-negative specimens (10.5%) were positive by ASR and subsequently positive via T. vaginalis alternative target TMA (Gen-Probe). Discrepancies were additionally resolved by demonstration of T. vaginalis nucleic acid from a separate endocervical collection. T. vaginalis nucleic acid was detectable following prolonged storage, following minimal incubation in lysis medium, and from low-volume aliquots of sparsely populated specimens. T. vaginalis ASR adequately detects T. vaginalis from vaginal saline suspension aliquots, providing a simple specimen alternative for a highly sensitive laboratory diagnosis of trichomoniasis.

Evaluation of Gen-Probe APTIMA-Based Neisseria gonorrhoeae and Chlamydia trachomatis Confirmatory Testing in a Metropolitan Setting of High Disease Prevalence

ABSTRACT Prompted by reports challenging the validity of the low-positive Neisseria gonorrhoeae and Chlamydia trachomatis results generated by the APTIMA Combo 2 assay (Gen-Probe, Incorporated) and by a Centers for Disease Control and Prevention recommendation to confirm N. gonorrhoeae- or C. trachomatis-positive screens by using an alternative amplification target, we report on a comparison of this means of confirmation with an in-house algorithm of repeat testing. Primary clinical specimens yielding N. gonorrhoeae- or C. trachomatis-specific luminescent values between 100,000 and 1,000,000 were repeat tested in duplicate. A subset of specimens was forwarded for confirmatory assays (Gen-Probe) individualized for alternative N. gonorrhoeae or C. trachomatis targets. An 18-month audit revealed that 230 of 29,977 C. trachomatis screens (0.8%) and 41 of 29,064 N. gonorrhoeae assays (0.1%) yielded low-positive data. When a subset of 40 low-positive N. gonorrhoeae screens was repeat tested, 20 (50.0%) remained positive; 22 (55.0%) of the screens remained positive following performance of the confirmatory assay. In contrast, repeat testing of 153 low-positive C. trachomatis screens yielded a positive result for fewer specimens (n = 97; 63.4%) than when commercial confirmatory testing was used (n = 124; 81.0%). However, confirmation of the results for additional C. trachomatis screens by use of an alternative target did not translate into significant differences in the calculated overall C. trachomatis-positive screen rates (7.39% by repeat testing versus 7.52% by the confirmatory assay; P = 0.53). Furthermore, use of the confirmatory assay raised the positive predictive value only 1.8% over that of repeat testing. Molecular confirmatory testing did not significantly enhance the reliability of C. trachomatis- or N. gonorrhoeae-specific nucleic acid amplification testing in this metropolitan setting compared to the reliability of repeat testing.

Cost-Effective Modification of a Commercial PCR Assay for Detection of Methicillin-Resistant or -Susceptible Staphylococcus aureus in Positive Blood Cultures

ABSTRACT Real-time detection of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) in cases of clinical bacteremia may promote appropriate antimicrobial therapy and infection control. Expense inherent to molecular diagnostics may prevent laboratories from utilizing real-time PCR for this purpose. BD GeneOhm StaphSR assay master mix was reconstituted and aliquoted into SmartCycler tubes in 25-μl volumes (freshly reconstituted master mix), with a portion being frozen at −70°C (frozen master mix). Incubation of 40 previously analyzed lysates from positive BacT/Alert SA and SN blood culture bottles (identified as 10 MRSA strains, 10 MSSA strains, 12 coagulase-negative Staphylococcus strains, and 8 Micrococcus strains) in freshly reconstituted master mix and master mix frozen between 1 week and 6 months generated the expected results in all PCRs. Similarly, positive- and negative-control reagents stored frozen at −70°C for up to 18 weeks yielded the expected reactions. Prospective analysis of 244 positive blood culture samples utilizing 1-week-frozen master mix and freshly reconstituted master mix yielded a 1.2% discordant rate upon initial testing due to three unresolved results (two unresolved results for freshly reconstituted master mix and one unresolved result for frozen master mix). Repeat testing produced a final 100% concordance rate between the two master mix preparations. Use of master mix that was frozen up to 6 months did not compromise performance of the BD GeneOhm StaphSR assay. This modification, resulting in less reagent waste, may allow a greater number of laboratories to consider real-time PCR methodology for detection of bacteremia caused by MRSA and MSSA.

Retrospective Assessment of Transcription-Mediated Amplification-Based Screening for Trichomonas vaginalis in Male Sexually Transmitted Infection Clinic Patients

ABSTRACT Transcription-mediated amplification (TMA) enhances detection of Neisseria gonorrhoeae and Chlamydia trachomatis from rectal and pharyngeal sources. The utility of TMA for detection of Trichomonas vaginalis has recently been described. We report on the performance of TMA for detection of sexually transmitted infection (STI) agents from extraurogenital sources, with a focus on T. vaginalis. Within a 21-month interval, 1,314 consecutive male patient encounters at an STI clinic resulted in collection of 2,408 specimens for C. trachomatis, N. gonorrhoeae, and T. vaginalis TMA screening. A total of 471 encounters were managed with a single specimen collection (94.9% urine), with 12.7% positive for at least one STI agent. This detection percentage increased to 14.4% with collection of specimens from two sources and to 20.3% with collection from three sources (P = 0.03 versus single-source sampling). A total of 44.4% of encounters were managed by collection of urine and pharyngeal specimens and 19.1% by the addition of a third (rectal) collection. While procurement of urine and rectal specimens resulted in greater detection of C. trachomatis (6.1% and 11.3% rates, respectively) than of other STI agents, 858 pharyngeal specimens yielded a 2.9% T. vaginalis detection rate compared with 2.1% for N. gonorrhoeae and 1.6% for C. trachomatis. All T. vaginalis pharyngeal detections were confirmed by TMA-based alternative target testing. A total of 38.1% of T. vaginalis-positive pharyngeal specimens were derived from symptomatic patient encounters. A total of 85.7% of males with T. vaginalis-positive pharyngeal collections indicated strictly heterosexual preference. Additional specimen source sampling is necessary to make STI screening comprehensive. Incorporation of extraurogenital sources into assessment for T. vaginalis detection may identify additional symptomatic and asymptomatic male STI carriers.

Three-Year History of Transcription-Mediated Amplification-Based Trichomonas vaginalis Analyte-Specific Reagent Testing in a Subacute Care Patient Population

ABSTRACT A total of 7,899 specimens submitted for live clinical Trichomonas vaginalis analyte-specific reagent (ASR) screening from 2008 to 2010 were audited on the basis of patient gender, specimen source, molecular Neisseria gonorrhoeae and Chlamydia trachomatis results, and relative light unit (RLU) data yielded by T. vaginalis ASR. Only 1.4% of the screening was ordered by emergency department clinicians. The screening volume in 2010 was 126% higher than that in 2008. The proportions of annual female and male screening remained consistent throughout the 3-year interval (∼92 and 8%, respectively). Although 71.8 and 9.5% of screening was performed on endocervical and vaginal specimens, respectively, over the 3-year period, no significant difference was noted in the T. vaginalis detection rates (8.9 and 8.6%, P = 0.85). Increased T. vaginalis detection was derived from female urine specimens (12.6%) compared to female genital swabs (P = 0.0004). The proportion of female urine screening increased during the 3-year interval (P < 0.0002). T. vaginalis detection rate in males was 6.6%, with no difference between urethral and urine T. vaginalis detection (P = 0.53). The mean RLU value for 714 positive specimens was 3,971,441; analogous values for each female specimen source and combined male source testing showed no variance (P ≥ 0.29). Combined-gender T. vaginalis detection rate (9.1%) was significantly greater than those of C. trachomatis (5.9%) and N. gonorrhoeae (1.5%; P < 0.0002). Equivocal results presented at a rate of 0.4%. T. vaginalis ASR is an increasingly utilized assay that yields higher detection rates than other sexually transmitted infection etiologies in this community subacute care setting.

Clinical Laboratory Assessment of Mycoplasma genitalium Transcription-Mediated Amplification Using Primary Female Urogenital Specimens

ABSTRACT Following analysis of primary cervix, vagina, and first-void female urine specimens for Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis via commercial transcription-mediated amplification (TMA), residual material was subjected to Mycoplasma genitalium research-use-only TMA. Representation within a 2,478-specimen retrospective study set was established by comparison to a 6-month audit of clinical C. trachomatis TMA (12,999 specimens) on the basis of the C. trachomatis detection rate, specimen source distribution, clinic location, and age. M. genitalium was detected in 282 (11.4%) patients. This rate was higher than those seen with T. vaginalis (9.0%; P = 0.005), C. trachomatis (6.2%), and N. gonorrhoeae (1.4%). Positive M. genitalium results were confirmed by repeat testing or alternative-target TMA at a rate of 98.7%. The mean age of the M. genitalium-infected females (24.7 years) was lower than that of the T. vaginalis-infected females (mean, 30.1 years; P < 0.0001) and higher than that of the C. trachomatis-infected females (mean, 23.8 years; P = 0.003). Of 566 patient encounters positive for at least one sexually transmitted infection (STI), 35.9% exhibited sole detection of M. genitalium (P ≤ 0.0004 versus sole detection of other STI agents) and 26.1% were solely positive for T. vaginalis (P < 0.0002 versus C. trachomatis). The M. genitalium and T. vaginalis detection rates among 755 patients at urban emergency departments were 14.6% and 13.0%, respectively (P = 0.37). A 10.0% M. genitalium detection rate from other facilities exceeded that of T. vaginalis (7.2%; P = 0.004). Incorporation of M. genitalium TMA into comprehensive testing programs would detect M. genitalium in a significant proportion of females, particularly those in outpatient obstetrics and gynecology (OB/GYN) settings.