PROFESSOR PETER M. HAWKEY

1
New Diagnostic Methods
PROFESSOR PETER M. HAWKEY
PROFESSOR PETER M. HAWKEY
The University of Birmingham Edgbaston, B15 2TT
Health Protection Agency West Midlands Public
Health Laboratory, Birmingham Heartlands and
Solihull NHS Trust, B5 9SS

p.m.hawkey_at_bham.ac.ukpeter.hawkey_at_heartsol.wmids.
nhs.uk
2
Why would you want to use a Molecular diagnostic
method?

• Cheaper?!
• Faster?
• Gives unique information

3
Culture - free Microbiology Molecular
diagnosticsApplication of Molecular microbial
diagnostics

• Identification of pathogen (species)
• Identification of genes
• Antibiotic resistance
• Pathogenicity genes
• Identification of strain type

4
(No Transcript)
5
(No Transcript)
6
(No Transcript)
7
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

8
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

9
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

10
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

11
Molecular identification of VRE in a clinical
laboratoryConventional 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, 4291-97

12
PCR

• Multiplex PCR (MPCR) to detect van A vanB
  together with identification as Entercococus
• Petrich et al. 1999 Mol.Cell.Probes.
• 13275-288

13
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

14
Emergence of MRSA

• S. aureus has acquired a large genetic element
  known as Staphylococcal chromosome cassette mec
  (SCCmec).

15
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.

16

• 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.

17
(No Transcript)
18
SCC mec Elements
Size k.b.
Type I SCCmec
34
Type 1
Class B
Type II SCCmec
52
Class A
Type 2
67
Type III SCCmec
Type 3
Class A
21-24
Type IV SCCmec
Type 2
Class B
Type V SCCmec
21-28
Class C
Type 4
ccr complex
mec complex
Also S. haemolyticus
19
Lanes 1-4, MRCoNS lane 5, MSCoNS lane 6, MSSA
lanes 7 and 8, MRSA lane C, negative control
20
(No Transcript)
21
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.

22

Target
Hybrid
Molecular Beacon
23
Unspiked nasal sample
Nasal samples spiked with
10-4
200 MRSA cfu
24
Direct Detection of MecA in nasal swabs

• Primers to SCCmec/orfX junction and molecular
  beacon probe in the amplimer
• 205 MRSA 203 ve (1 false negative)
• 252 MSSA 13 ve (5.2 false positive)
• Time to result lt 1 hour
• Sensitivity 2-10 genome equivalents
• Huletsky, et al. 2002. CMI, 8, suppl1, 85.

25
(No Transcript)
26
ccr complex
mec complex
SCCmec right extremity
MREJ type
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
27
(No Transcript)
28
(No Transcript)
29
(No Transcript)
30
(No Transcript)
31
(No Transcript)
32
IDI-MRSA evaluation at Washington Univ. Sch. Med.

• 288 patients
• Inclusion criteria any of
• Prior MRSA colonisation
• Hospital stay gt3 days
• Known colonisation with HAP

Warren, et al, JCM, 2004 425578-81
33

• Direct plating MSA then M-H 6µg/ml
  oxacillin
• Enrichment TSA 6.5 NaCl then
• M-H ox

34
IDI-MRSA versus culture
35

• 6 samples ve by culture PCR negative
• No inhibitors
• 4 isolates available
• 3 grew on oxacillin agar, mecA negative

36
Possible gene targets for molecular detection
37
(No Transcript)
38
(No Transcript)
39
(No Transcript)
40
Molecular typing of S.aureus for cluster
investigation

• PFGE Gold Standard
• MLST
• REP-PCR
• Coa and Spa gene repeat polymorphisms
• Microarrays
• VNTR

41
Analysed using bionumerics allows grouping of
like isolates
42
Molecular typing of S.aureus for cluster
investigation

• PFGE Gold Standard
• MLST
• REP-PCR
• Coa and Spa gene repeat polymorphisms
• VNTR

43
Koreen, et al, J.Clin.Micro., 2004, 42 792
44
(No Transcript)
45
1 5 7 13 15 16 21
Hardy, et al, Microbiology,2004, 150 4045
46
Table 1 Characteristics of staphylococcal
interspersed repeat units (SIRUs).
Hardy, et al, 2004, 150 4045
47
Hardy, et al, 2004, 150 4045
48
(No Transcript)
49
(No Transcript)
50
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 PCRs
• Widespread usage probably depends on commercial
  development of conventional/microelectronic
  silicon chips

51
The Future

• Move away from complex, equipment/laboratory
  based assay to
• Simple, bedside, (cheap!?) disposable single
  use tests to give immediate results.

52
(No Transcript)