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
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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
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/