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New Diagnostic Methods
PROFESSOR PETER M. HAWKEY
Title of
The University of Birmingham
presentation
Edgbaston, B15 2TT
Subtitle
of presentation
Health
Protection Agency West Midlands
Public Health Laboratory, Birmingham
Heartlands and Solihull NHS Trust, B5 9SS
[email protected];[email protected]
Why would you want to use a
Molecular diagnostic method?
• Cheaper?!
• Faster?
• Gives unique information
Culture - free Microbiology –
Molecular diagnostics
Application of Molecular microbial diagnostics
• Identification of pathogen (species)
• Identification of genes
• Antibiotic resistance
• Pathogenicity genes
• Identification of strain type
Advantages of genotypic
sensitivity testing methods
• Can be potentially performed on specimens
• Do not rely on phenotypic expression
• Organisms do not have to grow (or be
viable!)
• Potential for automation
Problems with genotypic
detection of antibiotic
resistance
•
•
•
•
Plethora of mechanisms
Cost
Rapidity, is it necessary?
Expression
– ‘silent’ genes
– different levels of resistance in different hosts
• Resistance genes occurring in commensal flora
when direct testing
• Familiarity with technology
Problems with genotypic
detection of antibiotic
resistance
Cost
• Single PCR reaction aprox 2 euro + licensing
fees
• Automation with fluoro-chrome labelled
primers/multiplex PCR will reduce cost
• Longer term use of micro-array with mass
production
Problems with genotypic
detection of antibiotic resistance
Rapidity, is it necessary?
Only if:
a) growth rate slow
b) selection of empirical antibiotic not
possible without extra information
c) resistant bacteria represent
significant risk of cross-infection
Molecular identification of VRE
in a clinical laboratory
Conventional method (CBT)
• Bile esculin, 6mg/L Vancomycin
• Colonies screened with PYR, Xylose &
motility
• Confirmation on Micro Scan & BHI
Vancomycin 6 mg/L plate
Page etal.Diag.Micro.Infect.Dis
2002, 42:91-97
PCR
• Multiplex PCR (MPCR) to detect van A &
vanB together with identification as
Entercococus
Petrich et al. 1999 Mol.Cell.Probes.
13:275-288
COSTS for 1 year service (1400 specimen)
MPCR
• Capital cost of equipment + depreciation $17,116
• Training of lab staff ($22.K)
• CBT
$24,314
Payback period = 3 years
Emergence of MRSA
S. aureus has acquired a large genetic element known as
Staphylococcal chromosome cassette mec (SCCmec).
Origin of MRSA Clones
• Arise by acquisition of Staphylococcal Cassette
Chromosome mec (SCC mec)
• 21-67 kb DNA fragment integrates at unique site attBscc
orfx (highly conserved) near oriC
• process different to transposition as attBscc 15bp repeats
not all outside mobile elements and no duplication of
SCCmec
• attBscc recognised by recombinase encoded by ccrA/B
genes.
• SCCmec has NO phages, tra genes
transposases or virulence genes
• “Antibiotic resistance island”
• 5 classes of mec gene complex I-IV
• Type I and IV has mecI and 3' region mecR1
deleted. II and III intact mecI/R1.
SCC mec Elements
Size k.b.
Type I SCCmec
Type 1
Class B
Type II SCCmec
Type 2
Class A
Type III SCCmec
Type 3
Class A
Type IV SCCmec
Type 2
Class B
Type V SCCmec
Type 4
Class C*
ccr complex
34
52
67
21-24
21-28
mec complex
* Also S. haemolyticus
Lanes 1-4, MRCoNS; lane 5, MSCoNS; lane 6, MSSA; lanes 7 and
8, MRSA; lane C, negative control
Direct Detection of MecA in
nasal swabs
• Primers to SCCmec/orfX junction and molecular
beacon probe in the amplimer
Huletsky, et al. 2002. CMI, 8, suppl1, 85.
+
Target
Molecular
Beacon
Hybrid
100
Fluorescence
80
60
Unspiked nasal
sample
40
Nasal samples spiked with:
10-4
20
200 MRSA
cfu
0
10
20
30
-20
Cycles
40
Direct Detection of MecA in
nasal swabs
• Primers to SCCmec/orfX junction and molecular
beacon probe in the amplimer
• 205 MRSA
252 MSSA
203
13
+ve
+ve
(1% false negative)
(5.2% false positive)
• Time to result < 1 hour
• Sensitivity 2-10 genome equivalents
Huletsky, et al. 2002. CMI, 8, suppl1, 85.
MREJ type
ccr
mec
complex complex
SCCmec
right extremity
orfx
Product size bp
i
176
ii
278
iii
223
iv
215
v
196
vii
176
Schematic representation of the MecA right extremity junction( MREJs) of MRSA strains MREJ types i - vii
IDI-MRSA evaluation at Washington
Univ. Sch. Med.
288 patients
Inclusion criteria any of:
i. Prior MRSA colonisation
ii. Hospital stay >3 days
iii. Known colonisation with HAP
Warren, et al, JCM, 2004 42:5578-81
Direct plating: MSA then M-H + 6µg/ml
oxacillin
Enrichment:
TSA + 6.5% NaCl then
M-H + ox
IDI-MRSA versus culture
No. samples with IDI
result
Culture
result
Positive
Negative
Direct Plating
Positive
64
1
Negative
16
207
Enrichment
Positive
61
5
Negative
19
203
Either method
Positive
66
6
Negative
14
202
Sensitivity%
Specificity %
PPV%
NPV%
98.5
92.8
80
99.5
92.4
91.4
76.2
97.6
91.7
93.5
82.5
97.1
6 samples +ve by culture PCR negative
• No inhibitors
• 4 isolates available
• 3 grew on oxacillin agar, mecA negative
Possible gene targets for
molecular detection
M. tuberculosis
rifampicin (97%)*
rpoB
isoniazid (78%)
katG/inhA/
aphC
streptomycin (60%)
rpsL
fluoroquinolones (90%) gyrA/B
Molecular typing of S.aureus for
cluster investigation
PFGE – “Gold Standard”
MLST
REP-PCR
Coa and Spa gene repeat polymorphisms
Microarrays
VNTR
Analysed using bionumerics –
allows grouping of like isolates
Molecular typing of S.aureus for
cluster investigation
PFGE – “Gold Standard”
MLST
REP-PCR
Coa and Spa gene repeat
polymorphisms
VNTR
Koreen, et al, J.Clin.Micro.,
2004, 42: 792
Strain
EtrA
EtrB
VNTR No.
I
42
II
21
III
32
1
5
7
13 15 16 21
Hardy, et al, Microbiology,2004, 150: 4045
Table 1: Characteristics of staphylococcal interspersed repeat units (SIRUs).
Locus
Number
Genome repeat
sequence
identified in
Repeat
Length
(bp)
%
Conservation
% GC
Content
1
EMRSA-16
55
98
40
5
EMRSA-16
60
98
36
7
EMRSA-16
56
95
46
13
MW2
64
100
30
15
MW2
131
99
32
16
MW2
159
98
35
21
N315
48
94
45
Hardy, et al, 2004, 150: 4045
SIRU Dendrogram
PFGE profiles
EMRSA Type
EMRSA-8
EMRSA–10
EMRSA-15
EMRSA-5
EMRSA-3
EMRSA-16
EMRSA-6
EMRSA–13
EMRSA–14
EMRSA-2
EMRSA-12
EMRSA-4
EMRSA-7
EMRSA-9
EMRSA-1
EMRSA–11
SIRU Profile
3114572
3113782
15038X2
311X532
1313521
1222532
3113313
3113313
3113313
2113313
3113413
3113322
3113322
3113322
2113322
2113322
MLST Type
ST250
ST254
ST22
ST247
ST5
ST36
ST8
ST8
ST8
ST8
ST8
ST239
ST8
ST240
ST239
ST239
Fig 8: Dendrogram deduced from the clustering analysis of the EMRSA strains, showing the PFGE profiles, the SIRU profile and the corresponding
ST. The box highlights SIRU profiles that only differ from each other by a maximum of two loci.
Hardy, et al, 2004, 150: 4045
CONCLUSIONS
• Genotypic methods for the detection of antibiotic
resistance have revolutionised our understanding
of the epidemiology of resistance genes
• In the routine diagnostic laboratory some specific
applications have emerged with commercial/home
brewed PCR’s
• Widespread usage probably depends on
commercial development of
conventional/microelectronic silicon chips
The Future……
Move away from complex,
equipment/laboratory based assay to ……
Simple, bedside, (cheap!?) disposable single
use tests to give immediate results.