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Genotypic Detection

of AntimicrobialResistance

25th Sept 2013

Jeanette Teo

Microbiology Technical Workshop

Advantages / Limitations

Basis

Techniques

Examples

ESBL/CPE

MRSA

VRE

2013

Incidence Phenotypic

Susceptibility

Testing

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Clinical

Relevance

Nucleic acid amplification

PCR: simple, multiplex, real-time, digital

Isothermal: LAMP, NASBA

Hybridization-basedMacro, Micro, Liquid-arrays

Sequencing

Sanger, Pyro, Next-generation

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Complexity

Simplicity

Resistance determinant

Gene

A AC AT T

Next-Generation Sequencing

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MRSAMethicillin resistant

Staphylococcus aureus

mecA

MSSA

mecA

MRSA

mecA

MSSA

orfX mecA

Culture Xpert

25min 2min80hrs 75min

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

CPECarbapenemase producing

Enterobacteriaceae

Class Akpc

Encode enzymes thatdegrade carbapenems

Class Bndm

Class D

OXA-48 like

Class Akpc

Class Bndm

Class D

OXA-48 like

In-house

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MDR-TBXDR-TB

Nucleotide changes

associated with

drug resistance88 90 91 94

gyrA WT1

gyrA WT2

gyrA WT3

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

Line-probe Assay

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 m utation probe 1.rrs m utation probe 2.

embB locus control.embB WT probe 1.

embB mu tation probe 1A.embB m utation probe 1B.

Color marker.

Resistance None

Line-probe Assay for Second-line DSTGenoType MTBDRsl (Hain Lifescience)

FQ +EMB +

KAN

FQ FQ +EMB

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

Limitations of Molecular Tests

100% sensitivity

Population prevalence

Restricted test sets

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High concordance (>99%)

between phenotypicand predicted AS

WGS - an alternative ?