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Division of STD Prevention

National Center for HIV/AIDS, Viral Hepatitis, STD and TB Prevention

Overview of molecular diagnostics and antimicrobial resistance detection in STD

pathogens

Allan Pillay, Ph.D.

Laboratory Reference & Research Branch, Division of STD Prevention, Centers for Disease Control &

Prevention, Atlanta, USA

Division of STD Prevention

National Center for HIV/AIDS, Viral Hepatitis, STD and TB Prevention

Nucleic acid amplification tests (NAATs) currently in use for sexually transmitted agents

New diagnostic tests

Whats on the horizon?

AMR & detection methods

AMD/WGS

Outline

Division of STD Prevention

National Center for HIV/AIDS, Viral Hepatitis, STD and TB Prevention

Global estimates of curable STIs

Gonorrhea, chlamydia, syphilis, trichomoniasis

Division of STD Prevention

National Center for HIV/AIDS, Viral Hepatitis, STD and TB Prevention

Syndromic management has been a valuable approach to STD control efforts in developing or resource-limited countries

Periodic testing of specimens from patients diagnosed and treated using syndromic management algorithms is necessary to ensure that syndromic diagnosis is effective in identifying the infections targeted for intervention

Both ulcerative and non-ulcerative STIs are a risk factor for the transmission and acquisition of HIV therefore early diagnosis and treatment is important

Background

Division of STD Prevention

National Center for HIV/AIDS, Viral Hepatitis, STD and TB Prevention

Organisms causing genital discharge

Neisseria gonorrhoeae urethritis (men), epididymitis, cervicitis, endometritis, PID

Chlamydia trachomatis D-K (chlamydial infection) urethritis (men), epididymitis, cervicitis, endometritis, PID, proctitis (both sexes)

Trichomonas vaginalis premature rupture of membranes, low birth weight, preterm delivery, vaginitis, urethritis (men)

Mycoplasma genitalium urethritis in men & women, cervicitis, endometritis, ?PID

Organisms causing genital ulcer disease

Treponema pallidum syphilis; multiple-stage (1o, 2o, 3o) with varied clinical manifestations

Herpes simplex virus 1 & 2, genital herpes

Haemophilus ducreyi chancroid, painful lesions; cutaneous lesions in children

Klebsiella granulomatis (Calymmatobacterium) Donovanosis

Chlamydia trachomatis, L1-L3 lymphogranuloma venereum (LGV), lesion, proctitis

Other STDs HIV, HPV, Hepatitis B, Ebola virus (Mate et al. N Engl J Med 2015)

Division of STD Prevention

National Center for HIV/AIDS, Viral Hepatitis, STD and TB Prevention

Commercial molecular diagnostic tests for CT/NG

Panther, Hologic/Gen-Probe M2000 RealTime, Abbott Mol. Viper XTR, BD Cobas 4800, Roche Mol. Diag.

118 93 92 94

Non-batch random access system

Batch System Batch System Batch System

Single unitfor extraction &

detection

Separate units Single unit Separate units

Commercial molecular diagnostic tests for CT/NG

Test APTIMA COMBO 2 CT/GCAPTIMA CTAPTIMA GC

BD ProbeTec ET GC/CT Qx

Cobas 4800 Abbott m2000RealTimeCT/NG

Xpert CT/NG

Manufacturer Hologic/Gen-Probe

Becton Dickinson Roche Abbott Cepheid

Technology TMA SDA Real Time PCR Real Time PCR Real Time PCR

Target - CT 23S rRNA cryptic plasmid cryptic plasmid & genomic target

cryptic plasmid, 2 diff regions

1 Genomic target

nvCT Yes Yes Yes Yes YesTarget - GC 16S rRNA Pilin DR-9, direct

repeat regionOpa genes 2 genomic targets

Specimen Type endocervical swab, clinician-& patient-collected vaginal swab; male urethral swab; urine

endocervical swab, male urethral swab; urine; patient-collected vaginal swab

endocervical swab; clinician-& patient-collected vaginal swab & urine

endocervical swab, clinician-& patient-collected vaginal swab; male urethral swab; urine

endocervical swab, patient-collected vaginal swab; urine

FDA/CE-IVD Approved

Y/Y Y/Y Y/Y Y/Y Y/Y

Sensitivity* (%) Urine: 88SCVS: 96.2

75.590.6

81.184.6

76.998

>95>98

Specificity* 8898.4

75.5100

81.199.6

76.9100

>99>99

*Based on a head-to-head comparison (excluding GeneXpert), Chernesky et al. JCM 2014

Division of STD Prevention

National Center for HIV/AIDS, Viral Hepatitis, STD and TB Prevention

Commercial molecular diagnostic tests

Organism Commercial test Lab developed test

Trichomonas vaginalis Affirm VPIII (BD), APTIMA TV (Hologic/Gen-Probe) ProbeTec Qx TV (BD) Xpert TV (Cepheid)

Yes

Mycoplasmas genitalium See below YesTreponema pallidum None YesHerpes simplex virus Magicplex HSV 1/2 Real-time Test

(Seegene)MultiCode-RTx (EraGen)

Yes

Haemophilus ducreyi None YesKlebsiella granulomatis None YesChlamydia trachomatis (LGV) None Yes CT, NG, TV, MH, MG, UU, UP Anyplex II STI-7 Molecular Kit

(Seegene)Yes, not all agents

Division of STD Prevention

National Center for HIV/AIDS, Viral Hepatitis, STD and TB Prevention

Gene Xpert CT/NG; TV POC/near patient test

Division of STD Prevention

National Center for HIV/AIDS, Viral Hepatitis, STD and TB Prevention

POC/near patient tests for CT/NG

Gaydos & Hardick 2013

Division of STD Prevention

National Center for HIV/AIDS, Viral Hepatitis, STD and TB Prevention

POC Molecular Test for HSV 1/2

HSV1/2 in genital & oral lesions

Isothermal amplification Results within 1.5 hrs

Equipment required- Heat block

IsoAmp HSV Assay, BioHelix Corp

Division of STD Prevention

National Center for HIV/AIDS, Viral Hepatitis, STD and TB Prevention

New Technologies

Melendez et al. 2013

Division of STD Prevention

National Center for HIV/AIDS, Viral Hepatitis, STD and TB Prevention

Direct detection of Treponema pallidum

Radolf et al. MCM10, 2011PCR sensitivity in early syphilitic lesions 75 - 80%

Division of STD Prevention

National Center for HIV/AIDS, Viral Hepatitis, STD and TB Prevention

Antimicrobial resistance

Neisseria gonorrhoeae all classes of antibiotics

Trichomonas vaginalis metronidazole

Mycoplasma genitalium azithromycin, fluoroquinolone

Treponema pallidum azithromycin

Herpes simplex virus acyclovir

Haemophilus ducreyi

Chancroid, -lactams, fluoroquinolones, tetracyclines, trimethoprim, sulfamethoxazole

Cutaneous lesions none reported

Division of STD Prevention

National Center for HIV/AIDS, Viral Hepatitis, STD and TB Prevention

Azithromycin resistance in Treponema pallidum

Azithromycin treatment failure first reported in San Francisco in 2002 and spectinomycin failure was documented in 2009 in the Czech Republic

Macrolide treatment failure is associated with either a A2058G or a A2059G point mutation in both copies of the 23S rRNA gene

Macrolide-resistant T. pallidum with the A2058G mutation are prevalent in the USA , Canada, Europe, and China

Division of STD Prevention

National Center for HIV/AIDS, Viral Hepatitis, STD and TB Prevention

Antimicrobial resistance in H. ducreyi

Chancroid prevalence has declined significantly worldwide

Antimicrobial susceptibility testing is labor intensive and rarely performed

Plasmid-mediated resistance has been described for tetracycline, chloramphenicol, sulfonamides, penicillin and aminoglycosides in H. ducreyi strains

Chromosomally mediated antimicrobial resistance has been described for trimethoprim, penicillin and fluoroquinolones

Division of STD Prevention

National Center for HIV/AIDS, Viral Hepatitis, STD and TB Prevention

Prevalence of cutaneous H. ducreyi

High prevalence of H. ducreyi causing cutaneous lesions in children under 15 years old with clinically suspected yaws

Country Prevalence by PCR (%)

Ghana 27.4

Solomon Islands 32

Vanuatu 38.6

Papua New Guinea 60

Division of STD Prevention

National Center for HIV/AIDS, Viral Hepatitis, STD and TB Prevention

Azithromycin dosing study - Ghana

Division of STD Prevention

National Center for HIV/AIDS, Viral Hepatitis, STD and TB Prevention

4-week follow-up after 30 mg/kg AZM

Division of STD Prevention

National Center for HIV/AIDS, Viral Hepatitis, STD and TB Prevention

Antimicrobial resistance in M. genitalium

Mutations in 23S rRNA gene associated with azithromycin resistance

A2059G, A2058G, A2059C, A2059T, A2062T, C2038T, T2185G

Mutations associated with fluoroquinolone resistance

gyrA - T321A, G285C + 5 additional mutations

parC G248A, G259T, A260T, A290G + 7 additional mutations

Division of STD Prevention

National Center for HIV/AIDS, Viral Hepatitis, STD and TB Prevention

Antimicrobial resistance in HSV

Resistance to acyclovir, penciclovir, & brivudin associated with mutations in the thymidine kinase and DNA pol genes mainly in immunocompromised patients

Division of STD Prevention

National Center for HIV/AIDS, Viral Hepatitis, STD and TB Prevention

Azithromycin resistance in N. gonorrhoeae

Division of STD Prevention

National Center for HIV/A