TB outbreaks and the usefulness of molecular typing

1
TB outbreaks and the usefulness of molecular
typing

• Dr Deborah Modha
• CCDC Eastmidlands south health protection unit

2
Molecular epidemiology, the way forward for TB
control?

• Key factors in control of TB
• Rapid detection of cases esp smear positive
• early clinical review and establishment of
  adequate therapy
• Thorough review by TB nurse specialists to ensure
  compliance
• Thorough contact tracing to arrest further
  transmission

3
Molecular epidemiology, the way forward for TB
control?

• Traditional epidemiological investigation of
  cases and contacts has been the bedrock of
  control
• However with the number of new cases continuing
  to rise year on year there is a need for new
  approaches
• Developments in DNA technology and molecular
  biology have led to methods for rapid detection
  of mycobacterial DNA in clinical specimens by in
  vitro nucleic acid amplification

4
Molecular epidemiology, the way forward for TB
control?

• Furthermore methods have been developed that
  enable us to trace transmission of tuberculosis
  by differentiation of clinical isolates based on
  polymorphisms in repetitive elements in the
  genomic DNA of M.tuberculosis. The most important
  and widely used examples include
• IS6110 RFLP typing
• Spoligotyping
• VNTR/MIRU typing

5
IS6110 RFLP typing

• Isolate grown up for 2 weeks at least
• DNA harvested and cleaved with a specific
  endonuclease pvu11
• Restriction fragments separated by
  electrophoresis on an agarose gell
• Southern blot hybridisation with IS6110
• Pattern visualised

6
Typing of M.tuberculosis,

• IS6110 RFLP typing
• Historically most widely utilized technique. The
  dutch reference laboratory has used this
  technique to type all isolates since 1993 and the
  information generated used to inform contact
  tracing
• Has proved useful in USA ,the far east and the UK
  to inform TB control
• Has enabled isolates of Mtb to be assigned to
  genetic families and has improved our
  understanding of the epidemiology of TB
• Large amounts of DNA is needed, time consuming
  and labour intensive results cannot be directly
  compared between laboratories.
• Poor discrimination of isolates containing less
  than 5 copies of IS6110

7
(No Transcript)
8
spoligotyping

• Can be used directly on clinical material
• Has been applied to ancient DNA, formaline fixed
  parafin-embedded tissue and ZN stained slides
• Has been used to track strains from outbreaks in
  USA
• Has detected new outbreaks by showing the
  clustering of one particuler spoligotype
• Used to provide a snapshot of moving and
  expanding clones of M.tuberculosis and their
  global distribution
• This study included 13008 patterns from greater
  than 90 countries. It defined 36 major clades
  CAS1 and x of interest to us

9
spoligotyping

• Method based on detection of polymorphisms in the
  Direct Repeat DR region of genome
• DR locus made up of multiple well conserved 36bp
  direct repeats interspersed with non repetitive
  spacer sequences 34-41 bp long
• Strains vary in the number of direct repeats and
  in the presence and absence of particular spacers
  so isolates can be distinguished from one another
• Sequence of spacer nucleotides are known
• 43 spacer oligonucleotides produced bound to a
  membrane
• DR region amplified using primers directed at the
  repeat sequences
• PCR products hybridised with membrane
• Digital output produced

10
Variable number tandem repeat VNTR

• 5 loci ETR-A through ETR-E
• VNTR loci amplified by PCR using primers
  complementary to the flanking regions
• Number of tanden repeat units determined by
  estimating the size of the PCR product on agarose
  gels.
• Results expressed as a 5 digit allele profile in
  which each digit represents the number of copies
  a a particular locus
• Not discriminatory enough to be utilised in an
  outbreak setting but a good screening tool before
  using MIRU

11
Mycobacterial interspersed repetitive unit
genotyping MIRU

• Expands on the original VNTR typing method
• PCR based method based on the number of variable
  repeat units in mycobacterial interspersed
  repetitive units
• 12 MIRU loci have been selected out of 44 present
  that offer the most discrimination as determined
  by a variety of relatedness comparative studies
• Each locus is amplified and the number or
  repetitive units at each locus determined by the
  size of the products

12
(No Transcript)
13
Epidemiological links
Pilgrimage
Placeof worship
School
Case 2
Instruction
Case 3
Case 1
Family
Case 6
Case 5
Case 7
Case 4
Case 8
14
Molecular epidemiological links
Case 1 Case 2 Case 3
Case 4 Case 5
no isolate from Case 3 but very strong
epidemiological link to Case 1
Case 8 - not TB atypical
Case 7
Case 6
conclusion not an outbreak
15
Concluding remarks

• consequences of failing to consider tuberculosis
  as a diagnosis
• children with tuberculosis can be very infectious
• tuberculosis in schools is not always due to
  transmission from adults
• large outbreaks are scrutinised by
• the media
• politicians