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Genotypic Detection of Antimicrobial Resistance

Jeanette Teo
25th Sept 2013

Microbiology Technical Workshop

Advantages / Limitations Basis

Techniques Examples

Incidence

ESBL/CPE
MRSA VRE

2013

Phenotypic Susceptibility Testing

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

Complexity Simplicity

Resistance determinant

CA

TT AA

Gene

Next-Generation Sequencing

MRSA
Methicillin resistant Staphylococcus aureus

mecA
mecA

MSSA

MRSA

mecA

MSSA

orfX

mecA

Culture 25min 80hrs

Xpert 2min 75min

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

CPE
Carbapenemase producing Enterobacteriaceae

Class A kpc Class D OXA-48 like

Class B ndm

Encode enzymes that degrade carbapenems

In-house
Class A kpc Class B ndm

Class D OXA-48 like

100% 80% 60% 40% 20% 0% 2011 2012 2013 Others OXA-48 like KPC NDM

MDR-TB XDR-TB

Nucleotide changes

associated with drug resistance

gyrA WT2 gyrA WT1 88 90 91 gyrA WT3 94

Line-probe Assay

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

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

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

Limitations of Molecular Tests


100% sensitivity

Restricted test sets

Population prevalence

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

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