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

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.

Retrospective assessment of transcription-mediated amplification-based screening for Trichomonas vaginalis in male sexually transmitted infection clinic patients.

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.

Assessment of screening practices in a subacute clinical setting following introduction of Trichomonas vaginalis nucleic acid amplification testing.

OBJECTIVE: Trichomonas vaginalis analyte-specific reagent is a highly sensitive assay for T vaginalis detection. We report how this diagnostic innovation influenced the sexually transmitted infection ordering practice patterns of 20 subacute-care clinicians. METHODS: T vaginalis, Neisseria gonorrhoeae, and/or Chlamydia trachomatis screening data were audited on female swab submissions when only wet mount testing was available for detection of T vaginalis (2004-2007) and when T vaginalis detection options included analyte-specific reagent and wet mount (2008-2010). RESULTS: Analyte-specific reagent availability resulted in more screening and detection of T vaginalis, prompted less utilization of wet mount microscopy, and increased overall RNA-based screening for N gonorrhoeae and C trachomatis (P < 0.0002). CONCLUSION: Clinician familiarity with T vaginalis analyte-specific reagent can benefit both clinical practice and public health.

Cost-effective modification of a commercial PCR assay for detection of methicillin-resistant or -susceptible Staphylococcus aureus in positive blood cultures.

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-mul volumes (freshly reconstituted master mix), with a portion being frozen at -70 degrees 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 degrees 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.

Cost-effective frozen master mix modification of a commercial methicillin-resistant Staphylococcus aureus PCR assay.

The expense inherent to molecular diagnostics may be an overriding concern for a variety of clinical laboratories in the development of PCR-based methicillin-resistant Staphylococcus aureus (MRSA) active surveillance programs. BD GeneOhm MRSA assay master mix was reconstituted, aliquoted into SmartCycler tubes in 25-microl volumes, and frozen at -70 degrees C. One hundred percent of archival nasal swab lysates yielded the expected PCR results when incubated in master mix frozen for 1, 2, 3, and 4 weeks. A 98.8% concordance of the final result was observed upon prospective PCR analysis of 320 clinical lysates utilizing freshly reconstituted master mix and 2-week-frozen master mix. Initial unresolved rates generated by frozen master mix and freshly reconstituted master mix differed by 1.6% (P = 0.16). Of 50 MRSA-positive lysates, the titers of 32 (64%) were determined to the same value upon initial tandem frozen master mix and freshly reconstituted master mix utilization; the titers of an additional 14 were determined to the same value upon repeat testing. Frozen master mix maintains potency for at least 4 weeks, facilitating detection of MRSA from nasal swab lysates, and may decrease the amount of unused reagent up to an average of 33%.