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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
What’s 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, β-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/AIDS, Viral Hepatitis, STD and TB Prevention
Evolution of resistance in Neisseria gonorrhoeae
Unemo & Shaffer CMR 2014
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
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
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
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)
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)
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)
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
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
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
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
Save the Date!
2016 STD Prevention
Conference
Atlanta, GA │20-23 September 2016www.cdc.gov/stdconference/