Gillespie, S.H.

Lipman, M.

McHugh, T.D.

Shorten, R.J.

Sule, O.

English

UCL Discovery

Sir,There are many reports of transmission of mycobacteria by contaminated bronchoscopes 1-8. We undertook a retrospective investigation after a bronchoscope was found to be faulty and could not be decontaminated effectively. This revealed that Mycobacterium tuberculosis had been isolated from four patients who had had broncho-alveolar lavage specimens collected using this instrument.   A specimen from a fifth patient was culture negative, but positive by a  nucleic  acid  amplification  technique  (Becton  Dickinson  ProbeTec  strand  displacement amplification SDA method). It was possible that the four M. tuberculosis culture positive patients had genuine infections, or that their specimens had been cross contaminated by the faulty bronchoscope during the procedure  or  that  contamination  had  occurred  within  the  laboratory.  To  clarify  this,  the organisms were strain-typed 9.The  current  international  gold  standard  for  strain  typing  M.  tuberculosis  is  restriction fragment length polymorphism (RFLP) using the insertion sequence IS6110 10.  In order to perform this method, 200ng high quality DNA is required, which can only be obtained from cultures incubated for a considerable length of time, often more than 6 weeks. As a rapid alternative  approach,  MIRU-typing  (mycobacterial  interspersed  repeating  units)  has  been proposed. MIRU typing is a polymerase chain reaction (PCR) based tool 11, 12. MIRU typing provides different sized PCR products related to the number of tandem repeats that a strain possesses at 12 different genomic loci and is simple and rapid to perform resulting in a 12 digit strain type that can be compared between isolates. This method also has the advantage of using smaller quantities of poor quality DNA, and can therefore be performed on much younger cultures than RFLP and may also be performed directly on clinical specimens. For these reasons, the four M. tuberculosis isolates, together with the specimen from the SDA-positive patient were investigated by the MIRU methodology. Table I: MIRU patterns for the four isolates. PCR products could not be generated from the specimen from the SDA-positive patient as the SDA  extraction  method  proved  unsuitable  for  use  with  the  MIRU  PCR  and  insufficient sample  remained  to  extract  mycobacterial  DNA  by  a  suitable  protocol.  Products  were generated from the DNA extracted from liquid media culture from the other four patients.The four isolates possess distinct MIRU patterns and therefore, there is no evidence that cross contamination or nosocomial cross infection has occurred (see Table I).  Had  these  specimens  been  typed  using  IS6110  RFLP,  these  results  would  have  taken  a minimum  of  4  weeks  rather  than  the  one  week  turnaround  for  MIRU  at  our  laboratory. Currently there is not an extensive database of MIRU typing as there is for IS6110, so the wider applications of the latter method have obvious benefits. However, in small suspected outbreaks, such as the one described, the MIRU typing is invaluable in providing a definitive, rapid result which can guide clinical practice.MIRU loci and corresponding allelesIsolate 2 4 10 16 20 232 24 262 27 31 39 4003:409 Patient A 2 2 2 2 2 5 1 7 3 5 4303:420 Patient B 2 2 3 4 1 5 1 1 3 3 2203:434 Patient C 2 2 3 1 2 8 1 5 2 3 2103:485 Patient D 2 2 5 1 2 5 1 1 3 3 22Table I: MIRU patterns for the four isolates.References1. W. Leers, Disinfecting endoscopes: how not to transmit Mycobacterium tuberculosis by bronchoscopy. Can Med Assoc J 1980; 123: 275-280.  2. K.E. Nelson, P.A. Larson, D.E. Schraufnagel and J. Jackson, Transmission of tuberculosis by flexible fiberbronchoscopes. Am Rev Respir Dis 1983; 127: 97-100.3. P.W. Wheeler, D. Lancaster and A.B. Kaiser, Bronchopulmonary cross-colonisation and infection related to mycobacterial contamination of suction valves of bronchoscopes. J Infect Dis 1989; 159: 954-958.  4. T. Agerton, S. Valway, C. Gore et al., Transmission of a highly drug-resistant (strain W1) of Mycobacterium tuberculosis. Community outbreak and nosocomial transmission via a contaminated bronchoscope. JAMA 1997; 278: 1073-1077.   5. T.M. Michele, W.A. Cronin, N.M. Graham et al., Transmission of M. tuberculosis by a fiberoptic bronchoscope. Identification by DNA fingerprinting. JAMA 1997; 278: 1093-1095.   6. D.J. Dawson, J.G. Armstrong and Z.M. Blacklock, Mycobacterial cross-contamination of bronchoscopy specimens. Am Rev Respir Dis 1982; 126: 1095-1097.   7. A.C. Steere, J. Corrales and A. Von Graevenitz, A cluster of Mycobacterium gordonaeisolates from bronchoscopy specimens. Am Rev Respir Dis 1979; 120: 214-216.   8. J. Jackson, J.E. Leggett, D. Wilson and D.N. Gilbert, Mycobacterium gordonae in fiberoptic bronchoscopes. Am J Infect Control 1996; 24: 19-23.9. Kanduma E, McHugh TD, Gillespie SH. <http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=12694442> Molecular methods for Mycobacterium tuberculosis strain typing: a users guide. J Appl Microbiol 2003; 94: 781-91. 10. van Embden JD et al. Strain identification of Mycobacterium tuberculosis by DNA fingerprinting: recommendations for a standardised methodology J Clin Microbiol 1993; 31:  406-409.11. Supply et al. Automated high-throughput genotyping for study of global epidemiology of Mycobacterium tuberculosis based on Mycobacterial interspersed repetitive units. J Clin Microbiol 2001; 39: 3563-3571.12. Supply et al. Variable human minisatellite-like regions in the Mycobacterium tuberculosis genome. Molecular Microbiology 2000; 36: 762-771R J Shortena, S H Gillespieb O Sulea, M Lipmanc, T D McHughb,*aDepartment of Medical Microbiology, Royal Free Hampstead NHS Trust, Pond Street, London NW3 2QGbCentre for Medical Microbiology, University College London, Hampstead Campus, Rowland Hill Street, London NW3 2PFcDepartment of Respiratory Medicine, Royal Free Hampstead NHS Trust, Pond Street, London NW3 2QG*Corresponding author. t.mchugh@medsch.ucl.ac.uk <mailto:t.mchugh@medsch.ucl.ac.uk>, Tel. +44 (0)20-7472-6402   

Molecular strain typing of M. tuberculosis isolates

from a suspected outbreak involving a faulty

bronchoscope

Shorten, R. J.

Gillespie, S. H.

McHugh, T. D.

University of St Andrews Research Portal

Molecular strain typing of M. tuberculosis isolates from a suspected outbreak involving a faulty bronchoscope [3]

University of St. Andrews - Pure

Molecular strain typing of M. tuberculosis isolates from a suspected outbreak involving a faulty bronchoscope

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