Genotypic Detection of Antimicrobial Resistance

Jeanette Teo
25th Sept 2013

Advantages / Limitations Basis Techniques Examples

Microbiology Technical Workshop

ESBL/CPE MRSA VRE

Phenotypic Susceptibility Testing

Incidence

2013

Clinical Relevance

Nucleic acid amplification

PCR: simple, multiplex, real-time, digital Isothermal: LAMP, NASBA

Hybridization-based
Macro, Micro, Liquid-arrays

Sequencing
Sanger, Pyro, Next-generation

Resistance determinant

Complexity Simplicity

Next-Generation Sequencing
CA TT AA

Gene

mecA mecA

MRSA
Methicillin resistant Staphylococcus aureus
MSSA MSSA

MRSA

mecA

orfX

mecA

Culture 25min 80hrs

Xpert 2min 75min

CPE
Carbapenemase producing Enterobacteriaceae

Dubouix-Bourandy et al. 2011. J Clin Microbiol. 49(12):4225-30

Class A kpc Class D OXA-48 like Encode enzymes that degrade carbapenems

In-house
Class B ndm
Class A kpc Class B ndm

Class D OXA-48 like

MDR-TB XDR-TB

Nucleotide changes associated with drug resistance

gyrA WT1 88

gyrA WT2 gyrA WT3 90 91 94

Line-probe Assay

Line-probe Assay for Second-line DST

GenoType MTBDRsl (Hain Lifescience)
Conjugate control.. Amplification control.. MTB control. gyrA locus control.. gyrA WT probe 1.. gyrA WT probe 2.. gyrA WT probe 3.. gyrA mutation probe 1.. gyrA mutation probe 2.. gyrA mutation probe 3A. gyrA mutation probe 3B. gyrA mutation probe 3C. gyrA mutation probe 3D. rrs locus control.. rrs WT probe 1.. rrs WT probe 2.. rrs mutation probe 1. rrs mutation probe 2. embB locus control. embB WT probe 1. embB mutation probe 1A. embB mutation probe 1B. Color marker.

Resistance

None

FQ

FQ + EMB + KAN

FQ + EMB

http://www.medscape.com/viewarticle/710937_2

Limitations of Molecular Tests

100% sensitivity

Restricted test sets Population prevalence

Kiet et al. 2010. J Clin Microbiol. 2010 Aug;48(8):2934-9

High concordance (>99%) between phenotypic and predicted AS WGS - an alternative ?