Mycoplasmoides genitalium nucleic acid semi-quantitation and molecular macrolide resistance detection via automated assays: gender and specimen source considerations.

A laboratory-developed test (LDT) using analyte-specific reagents has been optimized on a commercial platform to detect macrolide resistance-associated mutations (MRM) in 23S rRNA from Mycoplasmoides genitalium from primary clinical specimens. In this study, MRM-LDT was applied to a multi-specimen source study set. One thousand four hundred ninety-five primary specimens testing positive for M. genitalium by commercial transcription-mediated amplification (TMA) were initially titered by the TMA assay using serial 10-fold dilutions to semi-quantitate target nucleic acid burden. Primary specimens were then processed for MRM detection using the MRM-LDT. Findings were stratified by gender and specimen source. The mean log10 target nucleic acid titer of a TMA-positive specimen was 3.51 (median 3; range 0-10). Male specimens (n = 1145) demonstrated a mean log10 M. genitalium TMA titer of 3.67; that value observed in 350 female specimens was 2.98 (P < 0.0001). The MRM-LDT detection rate (88.7%) from specimens with log10 M. genitalium TMA titers ≥ 4 was increased over specimens with log10 titers ≤ 1 (4.5%; P < 0.0002). In females, MRM-LDT was positive from 51.3% of vaginal swab and 34.7% of urine specimens (P = 0.01). In males, MRM-LDT was positive from 65.0% of rectal swab and 55.7% of urine specimens (P = 0.002). Differences were also observed in log10 M. genitalium TMA titers as a function of specimen source. M. genitalium macrolide resistance rates among multiple specimen sources, as determined by MRM-LDT, are high in the United States and can be consistent with target nucleic acid burden within the primary specimen. Caveats experienced within subgroupings support MRM reflex testing on primary M. genitalium-positive specimens. IMPORTANCE: First-line macrolide treatment failure is of increasing concern with Mycoplasmoides genitalium in multiple settings. Recent sexually-transmitted infection treatment guidelines from the United States Centers for Disease Control and Prevention have predicated therapeutic approaches on the availability of a macrolide resistance/susceptibility result from a primary clinical specimen. In this report, we investigate potential correlation between macrolide resistance mutation detection rates (identified by a molecular amplified laboratory-developed test) and transcription-mediated amplification-based rRNA target semi-quantitation. Data reveal that rRNA semi-quantitation and laboratory-developed test detection rate differences exist as a function of gender and specimen source. These data can guide providers in proper specimen selection not only for the laboratory diagnosis of M. genitalium but also macrolide resistance mutation determination from primary clinical specimens.

Optimization and Clinical Evaluation of an Automated Commercial Analyte-Specific Reagent Assay for Mycoplasmoides genitalium Macrolide Resistance Detection in Primary Clinical Specimens.

With improvement in laboratory diagnosis of Mycoplasmoides genitalium infection through molecular diagnostics, macrolide resistance determination within M. genitalium-positive patients is necessary. In this study, we report baseline parameters for an analyte-specific reagent (ASR) macrolide resistance real-time reverse transcriptase PCR on an open access analyzer and evaluated detection of macrolide resistance-mediated mutation (MRM) within 23S rRNA in a clinical specimen set. Initial use of 1.2 µM M. genitalium primer and 0.8 µM M. genitalium detection probe concentrations yielded an 80% false-positive detection rate when challenged with 10,000 copies of wild-type RNA. Optimization experiments showed that lowering primer/detection probe and MgCl2 concentrations minimized these false-detections of wild-type 23S rRNA, while higher levels of KCl increased rates of MRM detection with concomitant lower cycle threshold values and higher fluorescence emission. Lower limit of A2058G mutation detection was 5000 copies/mL (180 copies/reaction; 20/20 detections). Utilization of a baseline correction slope limit of 250 units further mitigated false-detection from wild-type 23S rRNA at challenges up to 3.3 billion copies/mL. MRM was detected in 583/866 (67.3%) clinical specimens initially positive for M. genitalium by commercial transcription-mediated amplification. These data included 392/564 detections (69.5%) from M. genitalium-positive swab specimens and 191/302 (63.2%) from M. genitalium-positive-positive first-void urine specimens (P = 0.06). Overall resistance detection rates did not vary by gender (P = 0.76). Specificity of the M. genitalium macrolide resistance ASR was 100% (141 urogenital determinations). MRM detection by the ASR was confirmed at a concordance rate of 90.9% by Sanger sequencing of a clinical specimen subset.

Outcome of Electronic Order Alert Intervention Relative to Toxigenic Clostridium difficile PCR Analysis and Hospital-Onset C difficile Infection in a Multihospital Health Care System.

OBJECTIVES: Concern exists regarding overdiagnosis of Clostridium difficile infection (CDI) via molecular modalities. We determined effects of a preanalytic order intervention on laboratory and CDI prevention measures in a multihospital system. METHODS: Intervals before and following implementation of a CDI electronic order alert (relative to appropriate testing scenario) were assessed for C difficile test volume and positivity rate, hospital-onset CDI frequency, and hospital-onset C difficile standardized infection ratio (SIR). C difficile detection occurred by PCR throughout the study. RESULTS: During the first half of 2015, testing volume was 1,578, with 88 hospital-onset CDIs. Following implementation, 18.9% and 56.8% reductions in volume and hospital-onset CDIs were realized, respectively, in the first half of 2017. Regression analysis revealed decreasing trends in PCR volume, positivity rate, hospital-onset CDI frequency, and SIR in larger facilities. CONCLUSIONS: Preanalytic considerations affect not only the microbiology laboratory but also hospital infection prevention in the context of CDI.

Clinical laboratory assessments for Mycoplasma genitalium in a high-prevalence sexually-transmitted infection community reveal epidemiologic dichotomies with Trichomonas vaginalis.

INTRODUCTION: Mycoplasma genitalium is an emerging agent of sexually-transmitted infection and is responsible for clinically-significant genital tract disease in both females and males. Similar to scenarios recently experienced with the urogenital flagellate Trichomonas vaginalis, an evolving molecular diagnostic reference standard based on transcription-mediated amplification allows for accurate detection of the organism, plus additional insight into disease epidemiology. Areas covered. The basis for this article includes primary peer-reviewed literature plus compilations of data derived from routine clinical laboratory screening of females and males for agents of sexually-transmitted infection. Introductory laboratory and epidemiologic data related to T. vaginalis provides not only a foreshadowing to the dichotomies inherent to M. genitalium prevalence but also advocacy of a common non-invasive specimen source that could be used to screen females for both agents. This review also documents increased prevalence rates of M. genitalium in both females and males by way of transcription-mediated amplification. Expert commentary. Molecular detection of M. genitalium should be a consideration in the development of comprehensive sexually-transmitted infection screening programs for both females and males. Transcription-mediated amplification has additionally identified novel facets of M. genitalium and T. vaginalis epidemiology that warrant further investigation.

Expansion of Comprehensive Screening of Male Sexually Transmitted Infection Clinic Attendees with Mycoplasma genitalium and Trichomonas vaginalis Molecular Assessment: a Retrospective Analysis.

Of 1,493 encounters of males at a sexually transmitted infection (STI) clinic in a community with a high prevalence of STI, Chlamydia trachomatis was detected in 8.7% and Neisseria gonorrhoeae was detected in 6.6%. Additional Trichomonas vaginalis and Mycoplasma genitalium screening found 17.4% and 23.9% of the encounters, respectively, to be positive for STI. STI agents were detected in 13.7% of urine specimens; addition of pharyngeal and rectal collections to the analysis resulted in detection of STI agents in 19.0% and 23.9% of encounters, respectively. A total of 101 (23.8%) encounters of identified STI involved sole detection of M. genitalium Expansion of the STI analyte panel (including M. genitalium) and additional specimen source sampling within a comprehensive STI screening program increase identification of male STI carriers.

Application of Alternative Nucleic Acid Extraction Protocols to ProGastro SSCS Assay for Detection of Bacterial Enteric Pathogens.

As an alternative to automated extraction, fecal specimens were processed by investigational lysis/heating (i.e., manual) and by chromatography/centrifugation (i.e., column) methods. ProGastro SSC and Shiga toxin-producing Escherichia coli (i.e., STEC) indeterminate rates for 101 specimens were 1.0% to 3.0% for automated, 11.9% for manual, and 24.8% to 37.6% for column methods. Following freeze-thaw of 247 specimens, indeterminate rates were 1.6% to 2.4% for manual and 0.8 to 5.3% for column methods. Mean processing times for manual and column methods were 30.5 and 69.2 min, respectively. Concordance of investigational methods with automated extraction was ≥98.8%.

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.

Detection of Mycoplasma genitalium from male primary urine specimens: an epidemiologic dichotomy with Trichomonas vaginalis.

A total of 2750 male urines subjected to a transcription-mediated amplification (TMA)-based Mycoplasma genitalium assay yielded 188 positive results (6.84%). This rate was similar to Chlamydia trachomatis (6.87%; P = 0.96) and greater than Neisseria gonorrhoeae (4.0%) and Trichomonas vaginalis (2.3%; P < 0.0002). Mean age of M. genitalium-infected males (30.8) was similar to N. gonorrhoeae (P = 0.78) but less than T. vaginalis (mean, 41.6; P < 0.0001). A total of 266 STI clinic encounters had at least 1 sexually transmitted infection (STI); 36.5% of these encounters had sole detection of M. genitalium (P ≤ 0.009 versus sole detection of other STI agents). In 209 community encounters with at least 1 STI, 22.0% exhibited sole detection of M. genitalium (P = 0.0007 versus sole M. genitalium detection in STI clinic males), while 18.7% had sole detection of T. vaginalis (P < 0.0002 versus detection in STI clinic males). TMA-based M. genitalium screening identifies additional cases of nongonococcal urethritis.

Comparison of commercial hybridization and automated transcription-mediated amplification modalities for detection of high-risk human papillomavirus nucleic acid.

Assessment of 4,056 cytology collections by Cervista HPV HR and APTIMA HPV yielded 88.7% concordance, with increased detection by Cervista in patients with atypical squamous cells of undetermined significance (ASC-US) and patients negative for intraepithelial lesions and malignancy (NILM) (P ≤ 0.02). Both assays detected ≥91.7% of cervical intraepithelial neoplasia grade 2+ (CIN2+) lesions. A total of 262 specimens demonstrated luminescence within all three Cervista oligonucleotide mixtures (triple positive). APTIMA HPV and PCR-based microarray confirmed triple-positive results at rates of 7.3% and 5.9%, respectively.

Screening of male patients for Trichomonas vaginalis with transcription-mediated amplification in a community with a high prevalence of sexually transmitted infection.

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.

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.

Female epidemiology of transcription-mediated amplification-based Trichomonas vaginalis detection in a metropolitan setting with a high prevalence of sexually transmitted infection.

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.

Three-year history of transcription-mediated amplification-based Trichomonas vaginalis analyte-specific reagent testing in a subacute care patient population.

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.

Temporal characterization of carrot broth-enhanced real-time PCR as an alternative means for rapid detection of Streptococcus agalactiae from prenatal anorectal and vaginal screenings.

Analysis of overnight carrot broth culture using the BD GeneOhm StrepB assay (carrot broth-enhanced PCR) yields increased sensitivity compared to that of carrot broth culture alone for the detection of Streptococcus agalactiae. We investigated the prospect of reducing the carrot broth incubation time prior to PCR performance. In vitro experimentation demonstrated that carrot broth-enhanced PCR nominally detected 10 CFU S. agalactiae after 4 h of carrot broth incubation with competitive flora. Detection rates improved with inocula of 100 and 1,000 CFU S. agalactiae, with the majority of these aliquots demonstrating detection after 2 h of carrot broth incubation. Carrot broth was prospectively inoculated with clinical vaginal/anorectal swabs, with 500-µl aliquots collected. Early aliquots from 227 specimens were subjected to carrot broth-enhanced PCR (early-aliquot carrot broth-enhanced PCR) in instances of subsequent positive carrot broth culture or positive overnight clinical carrot broth-enhanced PCR. The S. agalactiae detection rate by early-aliquot carrot broth-enhanced PCR (66.1%) exceeded that observed for 227 remnant swabs retrospectively tested by direct swab PCR (56.4%; P=0.03). Early-aliquot carrot broth-enhanced PCR detection rate differences were most pronounced in aliquots from 83 carrot broth aliquots collected after 6 h (84.3%) compared to detection rates from either direct swab PCR of these samples (51.8%; P<0.0002) or early-aliquot carrot broth-enhanced PCR of 144 carrot broth aliquots collected after fewer than 6 h of incubation (55.6%; P<0.0002). Enhanced sensitivity of early-aliquot carrot broth-enhanced PCR versus direct swab PCR suggests that this assay could serve as a surrogate rapid detection method facilitating the prevention of group B streptococcal disease.

Enzyme-mediated protein haptenation of dapsone and sulfamethoxazole in human keratinocytes: I. Expression and role of cytochromes P450.

Cutaneous drug reactions (CDRs) are among the most common adverse drug reactions and are responsible for numerous minor to life-threatening complications. Several arylamine drugs, such as sulfamethoxazole (SMX) and dapsone (DDS), undergo bioactivation, resulting in adduction to cellular proteins. These adducted proteins may initiate the immune response that ultimately results in a CDR. Recent studies have demonstrated that normal human epidermal keratinocytes (NHEKs) can bioactivate these drugs, resulting in protein haptenation. We sought to identify the enzyme(s) responsible for this bioactivation in NHEKs. Using immunofluorescence confocal microscopy and an adduct-specific enzyme-linked immunosorbent assay (ELISA), we found that N-acetylation of the primary amine of SMX and DDS markedly reduced the level of protein haptenation in NHEKs. Detection of mRNA and/or protein confirmed the presence of CYP3A4, CYP3A5, and CYP2E1 in NHEKs. In contrast, although a faint band suggestive of CYP2C9 protein was detected in one NHEK sample, a CYP2C9 message was not detectable. We also examined the ability of chemical inhibitors of cytochromes P450 (aminobenzotriazole and 1-dichloroethylene) and cyclooxygenase (indomethacin) to reduce protein haptenation when NHEKs were incubated with SMX or DDS by either confocal microscopy or ELISA. These inhibitors did not significantly attenuate protein adduction with either SMX or DDS, indicating that cytochromes P450 and cyclooxygenase do not play important roles in the bioactivation of these xenobiotics in NHEKs and thus suggesting the importance of other enzymes in these cells.

Metalloproteases from Pseudomonas aeruginosa degrade human RANTES, MCP-1, and ENA-78.

The gram-negative bacterium Pseudomonas aeruginosa is an opportunistic human pathogen associated with both an acute lung disease in patients with hospital-acquired pneumonia and a chronic, progressive lung disease in individuals with cystic fibrosis. A unique characteristic of this bacterium in its natural environment is the secretion of a wide variety of factors designed to ensure its growth and survival. Evidence suggests, however, that when present in the human host, these same factors may contribute to disease. In the course of studying the effect of P. aeruginosa secretory factors on airway epithelial cells, we observed that metalloproteases in bacterial-conditioned medium, as well as purified alkaline protease and elastase, degraded human RANTES, monocyte chemotactic protein-1 (MCP-1), and epithelial neutrophil-activating protein-78 (ENA-78). Under identical conditions, interleukin-8 (IL-8) was significantly more resistant to proteolysis. Degradation was accompanied by a loss of chemotactic activity. These data suggest that metalloproteases from P. aeruginosa could alter the relative amounts of critical immunomodulatory cytokines in the airway and, thus, could contribute to the pathophysiology observed in P. aeruginosa-associated lung disease.