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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

Overview of molecular diagnostics and antimicrobial ... · Overview of molecular diagnostics and antimicrobial resistance detection in STD pathogens Allan Pillay, Ph.D. Laboratory

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Page 1: Overview of molecular diagnostics and antimicrobial ... · Overview of molecular diagnostics and antimicrobial resistance detection in STD pathogens Allan Pillay, Ph.D. Laboratory

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

Page 2: Overview of molecular diagnostics and antimicrobial ... · Overview of molecular diagnostics and antimicrobial resistance detection in STD pathogens Allan Pillay, Ph.D. Laboratory

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

What’s on the horizon?

AMR & detection methods

AMD/WGS

Outline

Page 3: Overview of molecular diagnostics and antimicrobial ... · Overview of molecular diagnostics and antimicrobial resistance detection in STD pathogens Allan Pillay, Ph.D. Laboratory

Division of STD Prevention

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

Global estimates of curable STIs

Gonorrhea, chlamydia, syphilis, trichomoniasis

Page 4: Overview of molecular diagnostics and antimicrobial ... · Overview of molecular diagnostics and antimicrobial resistance detection in STD pathogens Allan Pillay, Ph.D. Laboratory

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

Page 5: Overview of molecular diagnostics and antimicrobial ... · Overview of molecular diagnostics and antimicrobial resistance detection in STD pathogens Allan Pillay, Ph.D. Laboratory

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)

Page 6: Overview of molecular diagnostics and antimicrobial ... · Overview of molecular diagnostics and antimicrobial resistance detection in STD pathogens Allan Pillay, Ph.D. Laboratory

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

Page 7: Overview of molecular diagnostics and antimicrobial ... · Overview of molecular diagnostics and antimicrobial resistance detection in STD pathogens Allan Pillay, Ph.D. Laboratory

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

Page 8: Overview of molecular diagnostics and antimicrobial ... · Overview of molecular diagnostics and antimicrobial resistance detection in STD pathogens Allan Pillay, Ph.D. Laboratory

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

Page 9: Overview of molecular diagnostics and antimicrobial ... · Overview of molecular diagnostics and antimicrobial resistance detection in STD pathogens Allan Pillay, Ph.D. Laboratory

Division of STD Prevention

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

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

Page 10: Overview of molecular diagnostics and antimicrobial ... · Overview of molecular diagnostics and antimicrobial resistance detection in STD pathogens Allan Pillay, Ph.D. Laboratory

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

Page 11: Overview of molecular diagnostics and antimicrobial ... · Overview of molecular diagnostics and antimicrobial resistance detection in STD pathogens Allan Pillay, Ph.D. Laboratory

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

Page 12: Overview of molecular diagnostics and antimicrobial ... · Overview of molecular diagnostics and antimicrobial resistance detection in STD pathogens Allan Pillay, Ph.D. Laboratory

Division of STD Prevention

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

New Technologies

Melendez et al. 2013

Page 13: Overview of molecular diagnostics and antimicrobial ... · Overview of molecular diagnostics and antimicrobial resistance detection in STD pathogens Allan Pillay, Ph.D. Laboratory

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%

Page 14: Overview of molecular diagnostics and antimicrobial ... · Overview of molecular diagnostics and antimicrobial resistance detection in STD pathogens Allan Pillay, Ph.D. Laboratory

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, β-lactams, fluoroquinolones, tetracyclines, trimethoprim, sulfamethoxazole

Cutaneous lesions – none reported

Page 15: Overview of molecular diagnostics and antimicrobial ... · Overview of molecular diagnostics and antimicrobial resistance detection in STD pathogens Allan Pillay, Ph.D. Laboratory

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

Page 16: Overview of molecular diagnostics and antimicrobial ... · Overview of molecular diagnostics and antimicrobial resistance detection in STD pathogens Allan Pillay, Ph.D. Laboratory

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

Page 17: Overview of molecular diagnostics and antimicrobial ... · Overview of molecular diagnostics and antimicrobial resistance detection in STD pathogens Allan Pillay, Ph.D. Laboratory

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

Page 18: Overview of molecular diagnostics and antimicrobial ... · Overview of molecular diagnostics and antimicrobial resistance detection in STD pathogens Allan Pillay, Ph.D. Laboratory

Division of STD Prevention

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

Azithromycin dosing study - Ghana

Page 19: Overview of molecular diagnostics and antimicrobial ... · Overview of molecular diagnostics and antimicrobial resistance detection in STD pathogens Allan Pillay, Ph.D. Laboratory

Division of STD Prevention

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

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

Page 20: Overview of molecular diagnostics and antimicrobial ... · Overview of molecular diagnostics and antimicrobial resistance detection in STD pathogens Allan Pillay, Ph.D. Laboratory

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

Page 21: Overview of molecular diagnostics and antimicrobial ... · Overview of molecular diagnostics and antimicrobial resistance detection in STD pathogens Allan Pillay, Ph.D. Laboratory

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

Page 22: Overview of molecular diagnostics and antimicrobial ... · Overview of molecular diagnostics and antimicrobial resistance detection in STD pathogens Allan Pillay, Ph.D. Laboratory

Division of STD Prevention

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

Azithromycin resistance in N. gonorrhoeae

Page 23: Overview of molecular diagnostics and antimicrobial ... · Overview of molecular diagnostics and antimicrobial resistance detection in STD pathogens Allan Pillay, Ph.D. Laboratory

Division of STD Prevention

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

Evolution of resistance in Neisseria gonorrhoeae

Unemo & Shaffer CMR 2014

Page 24: Overview of molecular diagnostics and antimicrobial ... · Overview of molecular diagnostics and antimicrobial resistance detection in STD pathogens Allan Pillay, Ph.D. Laboratory

Division of STD Prevention

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

Ceftriaxone is the last remaining option for empirical first-line antimicrobial monotherapy

No new drug classes have been licensed to replace extended spectrum cephalosporins

Page 25: Overview of molecular diagnostics and antimicrobial ... · Overview of molecular diagnostics and antimicrobial resistance detection in STD pathogens Allan Pillay, Ph.D. Laboratory

Division of STD Prevention

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

Dual-antimicrobial therapy, i.e., mainly ceftriaxone and azithromycin, have been introduced in the United States, the United Kingdom, and all of Europe

Dual-antimicrobial therapy may be short-lived as susceptibility of gonococcal isolates to ceftriaxone has been decreasing globally, and resistance to azithromycin is already prevalent in many settings

XDR isolates with high-level cefixime and ceftriaxone MICs have been identified in Japan, France, and Spain

Between 2006 -2011 MICs of cefixime in the US and other countries has been increasing suggesting that the effectiveness of this antibiotic may be waning

Page 26: Overview of molecular diagnostics and antimicrobial ... · Overview of molecular diagnostics and antimicrobial resistance detection in STD pathogens Allan Pillay, Ph.D. Laboratory

Division of STD Prevention

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

Acquired AMR determinants are stably maintained in the gonococcal population many decades after removal of specific antimicrobials from treatment regimens

Continual mutation and internal recombination – rapidly evolving gonococcal populations

Acquisition of all or part of external resistance or virulence genes from other Neisseria spp.

The gonococcus is highly transformable – frequently releases DNA and also efficiently incorporates exogenous DNA acquired from other Neisseria sp. and closely related bacteria

Page 27: Overview of molecular diagnostics and antimicrobial ... · Overview of molecular diagnostics and antimicrobial resistance detection in STD pathogens Allan Pillay, Ph.D. Laboratory

Division of STD Prevention

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

gyrA mutations/SNPs, e.g., S91F, D95N, and D95G, in the QRDR, reduce quinolone binding to DNA gyrase

parC SNPs, e.g., D86N, S88P, and E91K, in the QRDR, reduce quinolone binding to topoisomerase IV

Many additional mutations in the QRDR of gyrA and parC have been described

An overexpressed NorM efflux pump also slightly enhances quinolone MICs

Quinolone resistance (ciprofloxacin and ofloxacin)

Page 28: Overview of molecular diagnostics and antimicrobial ... · Overview of molecular diagnostics and antimicrobial resistance detection in STD pathogens Allan Pillay, Ph.D. Laboratory

Division of STD Prevention

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

23S rRNA SNPs, i.e., C2611T and A2059G (in 1 to 4 alleles), result in a 23S rRNA target (peptidyltransferase loop of domain V) with a reduced affinity for the 50S ribosomal macrolide target.

mtrR mutations in the promoter (mainly an [A] deletion) or coding sequence result in overexpression of and increased efflux from the MtrCDE efflux pump.

erm genes (ermB, ermC, and ermF), encoding rRNA methylases that methylate nucleotides in the 23S rRNA target, block the binding of macrolides.

MacAB efflux pump overexpression increases the MICs of macrolides.

mef-encoded efflux pump exports macrolides out of the bacterial cell and increases the MICs of macrolides.

Macrolide resistance (azithromycin, erythromycin)

Page 29: Overview of molecular diagnostics and antimicrobial ... · Overview of molecular diagnostics and antimicrobial resistance detection in STD pathogens Allan Pillay, Ph.D. Laboratory

Division of STD Prevention

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

Mosaic penA alleles encoding mosaic PBP2s with a decreased PBP2 acylation rate. Derived from horizontal transfer of partial penA genes from mainly commensal Neisseria spp.

9 Mutations in mosaic PBP2s verified to contribute to resistance

penA SNPs, i.e., A501V and A501T, in nonmosaic alleles can also enhance cephalosporin MICs.

mtrR mutations, overexpression of and increased efflux from the MtrCDE efflux pump

penB mutations, reduce influx

“Factor X,” an unknown, nontransformable determinant increases penicillin MICs 3- to 6-fold

Cephalosporin resistance (cefixime, ceftriaxone)

Page 30: Overview of molecular diagnostics and antimicrobial ... · Overview of molecular diagnostics and antimicrobial resistance detection in STD pathogens Allan Pillay, Ph.D. Laboratory

Division of STD Prevention

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

Antibiotic resistance testing

Agar dilution method remains the “gold standard” for antimicrobial susceptibility testing - not routinely done; batch testing is preferred

No commercial molecular test for same day diagnosis and an antimicrobial susceptibility profile for NG

Page 31: Overview of molecular diagnostics and antimicrobial ... · Overview of molecular diagnostics and antimicrobial resistance detection in STD pathogens Allan Pillay, Ph.D. Laboratory

Division of STD Prevention

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

PPNG PCR Assay

PPNG-PCR assay Penicillin is used to treat gonorrhea in some remote Western Australian communities

PPNG-PCR assay that targets a region of DNA on the gonococcal plasmids carrying the penicillinase gene but not the gene

Evaluation of the PPNG-PCR using gonococcal isolates from throughout Australia PPNG resistant strains gave a false negative result

A novel 1,885-bp deletion in the plasmid type (Australian plasmid)

The assay has since been modified to include a stable marker for GC Buckley et al. 2015 JCM

Page 32: Overview of molecular diagnostics and antimicrobial ... · Overview of molecular diagnostics and antimicrobial resistance detection in STD pathogens Allan Pillay, Ph.D. Laboratory

Division of STD Prevention

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

PCR Assay for 23S rRNA AzR mutations

Trembizki et al. 2015 JAC

• 1-4 alleles of 23S rRNA• Mix of WT & mutant alleles

amongst the 4 copies

• Cross reactivity with melt curve analysis but Ct values of commensal strains were higher

Page 33: Overview of molecular diagnostics and antimicrobial ... · Overview of molecular diagnostics and antimicrobial resistance detection in STD pathogens Allan Pillay, Ph.D. Laboratory

Division of STD Prevention

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

Whole genome sequencing AMR data

Metagenomics - Direct sequencing of microbial genomes from clinical specimens

GC culture is still essential for antimicrobial susceptibility testing

Advanced molecular detection

Page 34: Overview of molecular diagnostics and antimicrobial ... · Overview of molecular diagnostics and antimicrobial resistance detection in STD pathogens Allan Pillay, Ph.D. Laboratory

Save the Date!

2016 STD Prevention

Conference

Atlanta, GA │20-23 September 2016www.cdc.gov/stdconference/