OBJECTIVES. Emerging Laboratory Diagnostic Options for Sexually-transmitted Infections
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1 Emerging Laboratory Diagnostic Options for Sexually-transmitted Infections OBJECTIVES I. Appreciate the changing epidemiology of trichomoniasis and clinician ordering patterns on the basis of improved laboratory diagnostics Erik Munson Wheaton Franciscan Laboratory Marquette University Milwaukee, Wisconsin II. III. Characterize the distribution of Mycoplasma genitalium infection in women and men on the basis of a new diagnostic option Describe the evolution of molecular screening for human papillomavirus and application to cervical cytology Gonorrhea United States, Sexually Transmitted Disease Surveillance Sexually Transmitted Disease Surveillance Chlamydia United States, Sexually Transmitted Disease Surveillance Sexually Transmitted Disease Surveillance
2 In vitro CHALLENGE C. trachomatis Elementary Bodies TMA Result PCR Result Light Units (x) Interpretation detected detected detected detected detected not detected. detected not detected. detected not tested. not detected not tested. not detected not tested. not detected not tested J. Med. Microbiol. : -; EXTRA-UROGENITAL GONOCOCCUS EXTRA-UROGENITAL TMA (since ) Source Modality Sensitivity (%) Specificity (%) Culture.. Pharynx DNA amplification (PCR).. RNA amplification (TMA).. Culture.. Rectum DNA amplification (PCR).. RNA amplification (TMA).. Gender Source n Detection Rate (%) C. trachomatis N. gonorrhoeae Female Pharynx.. Rectum.. Male Pharynx.. Rectum.. Sex. Transm. Dis. : -; Wheaton Franciscan Laboratory In-house Data CHLAMYDIA TRENDING Metropolitan Statistical Area Mean Annual Rank Aggregate Rate/, Memphis.. Milwaukee.. Virginia Beach-Norfolk.. Philadelphia.. Indianapolis.. Jacksonville.. San Antonio.. Richmond.. Saint Louis.. Birmingham.. Trich is just an itching disease; why should I care? United States NA. Expert Rev. Anti Infect. Ther. : -;
3 SOCIAL/PUBLIC HEALTH I HIV transmission facilitated by T. vaginalis infection Sex. Transm. Dis. : -;. relative risk for per-act probability of HIV transmission for T. vaginalis-positive women at start of two-year surveillance (P =.) J. Infect. Dis. : -; Estimated $ million for T. vaginalis-attributable HIV infection Sex. Transm. Dis. : -; SOCIAL/PUBLIC HEALTH II Human papillomavirus (HPV) / cervical neoplasia Difference in median time to clear HPV infection Arch. Pediatr. Adolesc. Med. : -; MOLECULAR DETECTION RATES STI Agent from Females Initial Validation (n = ) J. Clin. Microbiol. : -; J. Clin. Microbiol. : -; Community Audit (n = ) Chlamydia trachomatis.. Neisseria gonorrhoeae.. Trichomonas vaginalis.. OTHER T. vaginalis DETECTION Modality Performance Indices (%) Sensitivity Specificity Reference Wet mount microscopy.... Antigen detection Nucleic acid hybridization.. J. Clin. Microbiol. : -; Diagn. Microbiol. Infect. Dis. : -; Sex. Transm. Infect. : -; J. Clin. Microbiol. PMID J. Clin. Microbiol. : -; Male urogenital Female urogenital Total ORDERING PRACTICES Number of Screens Percentage of Female Testing Modality Genital Swabs - - P value Any wet mount preparation.. <. Point-of-care wet mount preparation.. <. Any assessment for Trichomonas vaginalis.. <. Chlamydia trachomatis/neisseria gonorrhoeae TMA.. <. I II I II I II Biannum J. Clin. Microbiol. : -; Wisc. Med. J. : -;
4 DETECTION RATE Specimen Trichomonas vaginalis Chlamydia trachomatis Neisseria gonorrhoeae Endocervix... Vagina... Female urine... DETECTION RATE Specimen Trichomonas vaginalis Chlamydia trachomatis Neisseria gonorrhoeae Endocervix. P =... Vagina... Female urine... J. Clin. Microbiol. : -; J. Clin. Microbiol. : -; DETECTION RATE Specimen Trichomonas vaginalis Chlamydia trachomatis Neisseria gonorrhoeae Endocervix. P =... Vagina..P <.. Female urine... Vagina.% Male urine.% Urethra.% Female urine.% I Male urine.% Urethra.% Vagina.% Female urine.% II Cervix.% Cervix.% J. Clin. Microbiol. : -; J. Clin. Microbiol. : -; Trichomonas vaginalis Chlamydia trachomatis Neisseria gonorrhoeae Percentage Positive Testing of Males for T. vaginalis Under to to to to Over J. Clin. Microbiol. : -;
5 CLINICAL/PUBLIC HEALTH Non-gonococcal urethritis; persistent, scant, mucoid discharge; prostatitis Sex. Transm. Dis. : -; J. Chemother. : -; Complications include infertility, hypogonadism HIV transmission Clinical condition T. vaginalis urethritis Non-T. vaginalis urethritis J. Clin. Microbiol. : -; Median copies HIV RNA/mL semen. x. x Sex. Transm. Dis. : -; POPULATION STUDIES/PROSTATE % of prostate cancer pts. with T. vaginalis Ab % of age-matched controls with T. vaginalis Ab Cancer Epidemiol. Biomarkers Prev. : -;. odds ratio for prostate cancer in men with T. vaginalis seropositivity Int. J. Cancer. : -;. odds ratio for prostate cancer in physicians with T. vaginalis seropositivity J. Natl. Cancer. Inst. : -; INCREASED CARCINOGENICITY? Increased risk of advanced-stage prostate cancer diagnosis in men with positive T. vaginalis serostatus Cancer Epidemiol. Biomarkers Prev. : -; Odds ratio of. (% CI.-.) for diagnosis of lethal prostate cancer in seropositive patients with documented history of T. vaginalis infection J. Natl. Cancer. Inst. : -; Prostate carcinogenesis mechanism undefined PLoS Pathog. : e; EVOLVING REFERENCE STANDARD Am. J. Obstet. Gynecol. :.e-.e; (adapted) Trichomonas vaginalis Chlamydia trachomatis Neisseria gonorrhoeae Percentage Positive Under to to to to Over J. Clin. Microbiol. : -;
6 MILWAUKEE COUNTY ZIP CODES RACE/ETHNICITY (Males) Parameter Geographical Region Race Majority African American Caucasian P value Percentage urine submissions... Mean screenings per ZIP code... Percentage detection of: African American (): Mean difference between African American and Caucasian populations was.% per ZIP Trichomonas vaginalis Chlamydia trachomatis Neisseria gonorrhoeae Caucasian (): Mean difference between Caucasian and African American populations was.% per ZIP J. Clin. Microbiol. : -; Chlamydia trachomatis Neisseria gonorrhoeae Trichomonas vaginalis Mycoplasma genitalium Urine Rectal Pharyngeal Specimen J. Clin. Microbiol. : -; Int. J. Syst. Bacteriol. : -; CLINICAL SIGNIFICANCE (MALES) Increased M. genitalium molecular detection in males with acute non-gonococcal urethritis (NGU) than in males without NGU J. Eur. Acad. Dermatol. Venerol. : -; M. genitalium-positive males more likely to exhibit urethritis than C. trachomatis-positive males Sex. Transm. Infect. : -; Male infertility; spermatozoa motility Clin. Microbiol. Rev. : -; ASSESSMENT OF MALES Prospective sampling of residual urines via M. genitalium research-use only TMA Previous TMA testing: Source of specimens Chlamydia trachomatis Neisseria gonorrhoeae Trichomonas vaginalis Outpatient clinics (community setting) Milwaukee STI clinic (demographics, history available)
7 DETECTION RATE (MALES) P =. AGE OF POSITIVE MALES Detection Rate (percentage) P <. P =. Agent Median Mean Range Mycoplasma genitalium. - Chlamydia trachomatis. - Neisseria gonorrhoeae. - Trichomonas vaginalis. - All mean detection age comparison P., except N. gonorrhoeae vs. M. genitalium (P =.) Mycoplasma genitalium Chlamydia trachomatis Neisseria gonorrhoeae Trichomonas vaginalis Agent Diagn. Microbiol. Infect Dis. : -; Diagn. Microbiol. Infect Dis. : -; STI CLINIC; M. genitalium ONLY Preferences.% heterosexual.% bisexual.% homosexual Partners median mean. range -.% symptomatic for urogenital disease Discharge.% Dysuria.% Burning/tingling.% Itching.% STI Phenotype Percentage of Patients Delineated by Healthcare Setting M. genitalium Chlamydia Neisseria Trichomonas STI Clinic Outpatient P value <. Any detection of Mycoplasma genitalium... Any detection of Chlamydia trachomatis... Any detection of Neisseria gonorrhoeae... Any detection of Trichomonas vaginalis.. <. Diagn. Microbiol. Infect Dis. : -; Diagn. Microbiol. Infect Dis. : -; CLINICAL SIGNIFICANCE (FEMALES) Females with high-burden M. genitalium more likely to shed HIV- DNA than M. genitalium-negative females J. Infect. Dis. : -; Associated w/ reproductive disease (meta-analysis) Cervicitis pooled OR. PID pooled OR. Pre-term birth pooled OR. Spontaneous abortion pooled OR. Clin. Infect. Dis. : -; ASSESSMENT OF FEMALES Retrospective/random selection of urine, cervical, or vaginal specimens via M. genitalium research-use only TMA Previous TMA testing: Chlamydia trachomatis Neisseria gonorrhoeae Trichomonas vaginalis Assessment of study set via comparison to -month audit of routine C. trachomatis TMA age, location, detection rate, specimen source
8 DETECTION RATE (FEMALES) AGE OF POSITIVE FEMALES Detection Rate (percentage) P =. P =. P <. Mycoplasma genitalium Chlamydia trachomatis Neisseria gonorrhoeae Trichomonas vaginalis Agent J. Clin. Microbiol. PMID Agent Median Mean Range Mycoplasma genitalium. - Chlamydia trachomatis. - Neisseria gonorrhoeae. - Trichomonas vaginalis. - Mean M. genitalium, N. gonorrhoeae ages P =. Mean C. trachomatis, N. gonorrhoeae ages P =. J. Clin. Microbiol. PMID DETECTION BY SOURCE (FEMALES) Detection Rate (percentage) Mg specimen sources P. Ct specimen sources P. Tv urine P =. versus genital Cervix Urine Vagina Specimen Source Ng specimen sources J. Clin. Microbiol. PMID P. EMERGENT VERSUS SUBACUTE Setting n M. genitalium detection (%) T. vaginalis detection (%) P value Emergency Department... Non-Emergency Department... J. Clin. Microbiol. PMID HPV AND CANCER % of cervical cancers caused by HPV Int. J. Cancer. : -; High-risk HPV types:,,,,,,,,,,,,, Courtesy of Charlotte Gaydos, Dr.Ph. (via Fred Jones, Ph.D.) Obstet. Gynecol. : -;
9 EVOLUTION OF HPV DIAGNOSTICS J. Natl. Cancer Inst. : -; Chemistry Product Manufacturer Mechanism Hybrid capture H c Digene (Qiagen) DNA hybridization Invader Cervista Third Wave (Hologic) DNA hybridization PCR Cobas Roche DNA amplification TMA APTIMA HPV Gen-Probe (Hologic) RNA transcription ALTS Use molecular testing to triage ASC-US patients Test Performance H c Participants Mean Age Colposcopies % with no lesion (no biopsy) % with no pathology upon biopsy % CIN % CIN+ Detection of CIN+ (%) via HPV DNA Detection of CIN+ (%) via HPV DNA Negative predictive value for CIN+ (%). Negative predictive value for CIN+ (%). Referral to colposcopy (%) PCR concordance (%). J. Natl. Cancer. Inst. : -; CERVISTA EVALUATION Cytologic Classification P value Cervista H c HSIL ASC-US Total... Diagn. Microbiol. Infect. Dis. : -; Test Performance H c Cervista Participants ~ Mean Age Colposcopies % with no lesion (no biopsy) % with no pathology upon biopsy % CIN % CIN+ Detection of CIN+ (%) via HPV DNA Detection of CIN+ (%) via HPV DNA Negative predictive value for CIN+ (%).. Negative predictive value for CIN+ (%).. Referral to colposcopy (%) PCR concordance (%).. Gynecol. Oncol. : -;
10 CERVISTA TOO POSITIVE?? SUMMARY OF METHODS ThinPrep vials for H c performance at reference laboratory during nine months of - ThinPrep vials for Cervista performance in-house during nine months of - Am. J. Clin. Pathol. : -; FREQUENCY OF DIAGNOSIS RETROSPECTIVE COMPARISON Percentage Diagnosis Cytologic Classification P value Cervista arm H c arm HSIL ASC-US.. <... <. Cytologic Classification P value Cervista H c HSIL ASC-US J. Clin. Microbiol. : -; J. Clin. Microbiol. : -; RETROSPECTIVE COMPARISON TRANSCRIPTION Cytologic Classification P value S subunit Evidence of bioactivity Evidence of persistence Cervista H c ASC-US; age <... ASC-US; age... ; age <... ; age... RIBOSOME P A messenger RNA J. Clin. Microbiol. : -; S subunit
11 A NEW PARADIGM PROSPECTIVE COMPARISON.%.%.% Detection of E/E (RNA) transcripts from high-risk HPV Potential to ascribe (precursory) oncogenic function to HPV nucleic acid detected in ThinPrep specimens.% Cervista positive/gen-probe positive Cervista negative/gen-probe negative Cervista positive/gen-probe negative Cervista negative/gen-probe positive % of underlying CIN+ detected by both assays J. Clin. Microbiol. : -; HPV PCR ADJUDICATION ThinPrep vials subjected to Cervista ; also HPV PCR via Cobas.% concordant positive.% concordant negative.% Cervista negative; Cobas positive.% Cervista positive; Cobas negative Of COBAS negative/ Cervista positive specimens, HPV sequencing revealed low-risk HPV and HPV-negative results th General Meeting American Society for Microbiology th General Meeting American Society for Microbiology RETROSPECTIVE COMPARISON Cytologic Classification P value Cervista Gen-Probe HPV HSIL... ASC-H ASC-US..... <. Cumulative.. <. J. Clin. Microbiol. : -; CERVISTA POSITIVES Pool of high-risk HPV oligonucleotides distributed among three mixes DNA extract Mix Mix Mix Hybridization signal versus background: Positive if one or two mixes exceeds. Positive if all three exceed. (triple-positive; TP)
12 Cervista negative/ Gen-Probe positive Frequency Cervista negative/ Gen-Probe positive Frequency ASC-H ASC-US ASC-US HSIL ASC-H ASC-US ASC-US HSIL Cervista positive/ Gen-Probe negative Frequency Cervista positive/ Gen-Probe negative Frequency J. Clin. Microbiol. : -; ASC-H ASC-US ASC-US HSIL J. Clin. Microbiol. : -; ASC-H ASC-US ASC-US HSIL RELATIVE LUMINESCENT OUTPUT Gen-Probe Result n Mean Cervista Luminescence from: a Mix b Mix b Mix b Genomic DNA positive.... negative. c. c. c. d a From Cervista TP specimens b TP hybridization signal versus background exceeds. for all three mixes c P <. versus mean luminescence from Gen-Probe positive specimens d P =. versus mean luminescence from Gen-Probe positive specimens BASIS FOR GENOTYPING HPV high-risk types,, most associated with cervical cancer % of squamous cell carcinoma % of adenocarcinoma Co-detection of high-risk types, more common in adenocarcinoma (%) than squamous cell carcinoma (%) J. Clin. Microbiol. : -; Lancet Oncol. : -; BASIS FOR GENOTYPING Mean age of patients with cervical cancer Type :. Type :. Type :. Other types:. Lancet Oncol. : -; Integration frequency predictive of quicker progression to invasive cancer J. Med. Virol. : -; J. Pathol. : -; Cancer Res. : -; Type : -% Type % Type : -% Type : % Type : %
13 GEN-PROBE GENOTYPING Cytologic Percentage Positive HPV High-risk a Classification < years years b.. Non- c.. Cytologic Percentage Positive HPV High-risk / a Classification < years years b.. Non- c.. a Dataset derived from HPV high-risk screen-positive specimens b total specimens c total non- specimens Wheaton Franciscan Laboratory In-house Data SUMMARY Changing epidemiology of trichomoniasis on basis of highly-accurate molecular assay M. genitalium dichotomy within females and males on basis of novel molecular assay Cervista and Gen-Probe HPV acceptable triage performance; Gen-Probe HPV specificity Benefits for HPV, / genotyping
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