Optimized molecular resolution of cross-contamination alerts in clinical mycobacteriology laboratories
© Martín et al; licensee BioMed Central Ltd. 2008
Received: 17 July 2007
Accepted: 14 February 2008
Published: 14 February 2008
The phenomenon of misdiagnosing tuberculosis (TB) by laboratory cross-contamination when culturing Mycobacterium tuberculosis (MTB) has been widely reported and it has an obvious clinical, therapeutic and social impact. The final confirmation of a cross-contamination event requires the molecular identification of the same MTB strain cultured from both the potential source of the contamination and from the false-positive candidate. The molecular tool usually applied in this context is IS6110-RFLP which takes a long time to provide an answer, usually longer than is acceptable for microbiologists and clinicians to make decisions. Our purpose in this study is to evaluate a novel PCR-based method, MIRU-VNTR as an alternative to assure a rapid and optimized analysis of cross-contamination alerts.
MIRU-VNTR was prospectively compared with IS6110-RFLP for clarifying 19 alerts of false positivity from other laboratories. MIRU-VNTR highly correlated with IS6110-RFLP, reduced the response time by 27 days and clarified six alerts unresolved by RFLP. Additionally, MIRU-VNTR revealed complex situations such as contamination events involving polyclonal isolates and a false-positive case due to the simultaneous cross-contamination from two independent sources.
Unlike standard RFLP-based genotyping, MIRU-VNTR i) could help reduce the impact of a false positive diagnosis of TB, ii) increased the number of events that could be solved and iii) revealed the complexity of some cross-contamination events that could not be dissected by IS6110-RFLP.
The false diagnosis of tuberculosis (TB) due to laboratory cross-contamination is a well-known phenomenon and has been reported to occur in 0.1–3% of cases [1–5]. It has an obvious epidemiological, clinical and therapeutical impact -each misdiagnosis of tuberculosis due to laboratory cross-contamination has been estimated to cost on average 10,872 dollars . We recently presented data indicating that laboratory cross-contamination events are more frequent than expected  and that these alerts require a faster clarification. False positivity due to laboratory cross-contamination is suspected when i) Mycobacterium tuberculosis (MTB) is cultured from only one of the serial specimens of a patient, ii) the bacterial yield in the culture is low and iii) the suspected sample has been processed together (or in a short period of time apart) with at least one other from a patient with a high bacterial load. The final confirmation of false positivity requires the application of molecular tools to prove that the MTB isolates from the co-processed specimens share identical genotypic patterns (after having ruled out epidemiological links between the cases involved). Unfortunately, the reference MTB genotyping method, IS6110-RFLP, requires well-grown cultures and takes a long time to provide an answer, usually longer than is acceptable for microbiologists and clinicians to make decisions.
A rapid PCR-based MTB genotyping tool, MIRU-VNTR (Mycobacterial-interspersed-repetitive-units-Variable-number-tandem-repeats) , has recently been developed and has proved useful in different epidemiological studies [9, 10]. It could allow quicker resolution of cross-contamination alerts although studies evaluating the efficiency of MIRU-VNTR in this context in a prospective design are lacking, and only a few isolated examples of its potential in identifying false-positive cases have been reported . Our purpose in this study is to evaluate MIRU-VNTR as an alternative to assure a rapid analysis of cross-contamination alerts in reference laboratories.
Results and Discussion
We prospectively evaluated whether MIRU-VNTR could be an alternative to RFLP for the fast resolution of laboratory cross-contamination alerts in reference genotyping centres. Therefore, we applied both IS6110-RFLP and MIRU-VNTR in a pilot study to analyze all the alerts received from laboratories in Almería, Spain. We compared the response time and the correlation between the diagnosis of either true positivity or laboratory cross-contamination using both techniques. The response time was measured from the moment the culture was received until MIRUtype for all the 12 loci assayed, or an RFLPtype were obtained. We decided to accept only those results obtained within a reasonable time frame (below 90 days), because longer times were not considered useful for resolving the alerts.
As indicated above, RFLP could not offer an answer in six alerts, either because some of the cultures (in three alerts) did not lead to the bacterial yield required for RFLP or because the 90-day limit was exceeded. In all the cases that remained unsolved by RFLP, MIRU-VNTR provided a result and, in two of them, it identified a cross-contamination that would have gone undetected if only RFLP analysis had been available. Each one of these two cross-contamination events revealed only by MIRU-VNTR showed interesting features (Figure 2). The first one (alert 15) involved a polyclonal isolate, with two variants in one loci, similar to others previously reported . The second (alert 19) was a complex situation in which two cross-contamination events occurred simultaneously. Each of two different sources (PS1 and PS2) contaminated a specimen from two independent patients (CCA1 and CCA2) and both (PS1 and PS2) were also involved in the simultaneous contamination of an additional specimen from another case (CCA3). This double contaminated case (CCA3) could be detected by MIRU-VNTR because its pattern was the combination of the MIRUtypes of the sources (PS1 and PS2), with two different alleles in three of the loci (Figure 2). In this alert, the 90-day limit was exceeded by RFLP and the profiles are not shown; however, the complex patterns of the isolates involved (14 bands and 8 bands) led to a 22-band pattern in the double-contaminated false-positive case that prevented identification of false-positivity by RFLP but not by MIRU-VNTR.
Our data mean that MIRU-VNTR is more adequate than RFLP for analyzing cross-contamination alerts. It was faster than RFLP, the correlation with RFLP diagnosis was high and it succeded in resolving alerts even under circumstances that were not appropriate for the RFLP analysis requirements. A permanently suspicious attitude on the part of the clinical mycobacteriologist together with access to a fast resolution of cross-contamination alerts could enable more rapid management of suspected false-positive cases, because culture-results would only need to be retained for a short time before clarification. Unlike standard RFLP-based genotyping, MIRU-VNTR could help reduce the impact of a false positive diagnosis of TB.
Clinical specimens were processed according to standard methods and grown in Lowenstein-Jensen slants and in MGIT (Becton Dickinson, Sparks, Md) liquid media.
For IS6110-RFLP we followed the standard procedures , and for MIRU-VNTR we applied the 12-loci set , trying to apply the simplest and fastest MIRU format and also attempting to obtain a result by directly amplifying a crude extract of the culture (after boiling and sonicating for ten and five minutes, respectively). MIRU-VNTR products were separated by electrophoresis at 45 V for 17 h 30 min, using MS8 2% agarose gels (Pronadisa, Madrid, Spain). Fragment sizes were calculated with the ChemiDoc system (BioRad, CA, USA) and the Diversity database (BioRad), using a 100-bp ladder (Invitrogen, CA, USA) as a molecular weight marker. The number of repeats in each locus was calculated by applying the corresponding conversion tables (P. Supply, personal communication)
Molecular patterns were analyzed using Bionumerics 4.6 (Applied Maths, Sint-Martens Laten, Belgium). Results were interpreted as false positivity if both the potential source and the cross-contamination alert had identical MIRU-VNTR and IS6110 RFLP and the converse for true positivity.
We are indebted to Thomas O'Boyle for proofreading and editing the manuscript.
This study was partially financed by Fondo de Investigaciones Sanitarias (FIS030654; FIS030986; FIS060882; FIS061467; 06/90490; 06/90357), Junta de Andalucía (0453/06, 151/05) and by the Instituto de Salud Carlos III (CIBER Enfermedades Respiratorias CB06/06/0058). A.M is receptor of a grant from Comunidad de Madrid cofinanced by the European Social Fund (Order no 5297/2006). INDAL-TB group: MI Sánchez, MC Rogado, T Cabezas, W SánchezYebra, J Martínez, MA Lucerna, P Barroso, I Cabeza-Barrera, LF Díez, M Rodríguez, M Escámez, P Marín, A Lazo, J Gamir, J Vázquez, C Gutiérrez, A Reyes and T Peñafiel.
- Ruddy M, McHugh TD, Dale JW, Banerjee D, Maguire H, Wilson P, Drobniewski F, Butcher P, Gillespie SH: Estimation of the rate of unrecognized cross-contamination with mycobacterium tuberculosis in London microbiology laboratories. J Clin Microbiol. 2002, 40 (11): 4100-4104. 10.1128/JCM.40.11.4100-4104.2002.PubMed CentralView ArticlePubMedGoogle Scholar
- Small PM, McClenny NB, Singh SP, Schoolnik GK, Tompkins LS, Mickelsen PA: Molecular strain typing of Mycobacterium tuberculosis to confirm cross-contamination in the mycobacteriology laboratory and modification of procedures to minimize occurrence of false-positive cultures. J Clin Microbiol. 1993, 31 (7): 1677-1682.PubMed CentralPubMedGoogle Scholar
- Multiple misdiagnoses of tuberculosis resulting from laboratory error--Wisconsin, 1996. MMWR Morb Mortal Wkly Rep. 1997, 46 (34): 797-801.Google Scholar
- de Boer AS, Blommerde B, de Haas PE, Sebek MM, Lambregts-van Weezenbeek KS, Dessens M, van Soolingen D: False-positive mycobacterium tuberculosis cultures in 44 laboratories in The Netherlands (1993 to 2000): incidence, risk factors, and consequences. J Clin Microbiol. 2002, 40 (11): 4004-4009. 10.1128/JCM.40.11.4004-4009.2002.PubMed CentralView ArticlePubMedGoogle Scholar
- de C Ramos M, Soini H, Roscanni GC, Jaques M, Villares MC, Musser JM: Extensive cross-contamination of specimens with Mycobacterium tuberculosis in a reference laboratory. J Clin Microbiol. 1999, 37 (4): 916-919.PubMed CentralPubMedGoogle Scholar
- Northrup JM, Miller AC, Nardell E, Sharnprapai S, Etkind S, Driscoll J, McGarry M, Taber HW, Elvin P, Qualls NL, Braden CR: Estimated costs of false laboratory diagnoses of tuberculosis in three patients. Emerg Infect Dis. 2002, 8 (11): 1264-1270.PubMed CentralView ArticlePubMedGoogle Scholar
- Martinez M, Garcia de Viedma D, Alonso M, Andres S, Bouza E, Cabezas T, Cabeza I, Reyes A, Sanchez-Yebra W, Rodriguez M, Sanchez MI, Rogado MC, Fernandez R, Penafiel T, Martinez J, Barroso P, Lucerna MA, Diez LF, Gutierrez C: Impact of laboratory cross-contamination on molecular epidemiology studies of tuberculosis. J Clin Microbiol. 2006, 44 (8): 2967-2969. 10.1128/JCM.00754-06.PubMed CentralView ArticlePubMedGoogle Scholar
- Supply P, Lesjean S, Savine E, Kremer K, van Soolingen D, Locht C: Automated high-throughput genotyping for study of global epidemiology of Mycobacterium tuberculosis based on mycobacterial interspersed repetitive units. J Clin Microbiol. 2001, 39 (10): 3563-3571. 10.1128/JCM.39.10.3563-3571.2001.PubMed CentralView ArticlePubMedGoogle Scholar
- Blackwood KS, Wolfe JN, Kabani AM: Application of mycobacterial interspersed repetitive unit typing to Manitoba tuberculosis cases: can restriction fragment length polymorphism be forgotten?. J Clin Microbiol. 2004, 42 (11): 5001-5006. 10.1128/JCM.42.11.5001-5006.2004.PubMed CentralView ArticlePubMedGoogle Scholar
- Hawkey PM, Smith EG, Evans JT, Monk P, Bryan G, Mohamed HH, Bardhan M, Pugh RN: Mycobacterial interspersed repetitive unit typing of Mycobacterium tuberculosis compared to IS6110-based restriction fragment length polymorphism analysis for investigation of apparently clustered cases of tuberculosis. J Clin Microbiol. 2003, 41 (8): 3514-3520. 10.1128/JCM.41.8.3514-3520.2003.PubMed CentralView ArticlePubMedGoogle Scholar
- Allix C, Supply P, Fauville-Dufaux M: Utility of fast mycobacterial interspersed repetitive unit-variable number tandem repeat genotyping in clinical mycobacteriological analysis. Clin Infect Dis. 2004, 39 (6): 783-789. 10.1086/423383.View ArticlePubMedGoogle Scholar
- Supply P, Allix C, Lesjean S, Cardoso-Oelemann M, Rusch-Gerdes S, Willery E, Savine E, de Haas P, van Deutekom H, Roring S, Bifani P, Kurepina N, Kreiswirth B, Sola C, Rastogi N, Vatin V, Gutierrez MC, Fauville M, Niemann S, Skuce R, Kremer K, Locht C, van Soolingen D: Proposal for standardization of optimized mycobacterial interspersed repetitive unit-variable-number tandem repeat typing of Mycobacterium tuberculosis. J Clin Microbiol. 2006, 44 (12): 4498-4510. 10.1128/JCM.01392-06.PubMed CentralView ArticlePubMedGoogle Scholar
- Oelemann MC, Diel R, Vatin V, Haas W, Rusch-Gerdes S, Locht C, Niemann S, Supply P: Assessment of an optimized mycobacterial interspersed repetitive- unit-variable-number tandem-repeat typing system combined with spoligotyping for population-based molecular epidemiology studies of tuberculosis. J Clin Microbiol. 2007, 45 (3): 691-697. 10.1128/JCM.01393-06.PubMed CentralView ArticlePubMedGoogle Scholar
- Shamputa IC, Jugheli L, Sadradze N, Willery E, Portaels F, Supply P, Rigouts L: Mixed infection and clonal representativeness of a single sputum sample in tuberculosis patients from a penitentiary hospital in Georgia. Respir Res. 2006, 7: 99-10.1186/1465-9921-7-99.PubMed CentralView ArticlePubMedGoogle Scholar
- van Embden JD, Cave MD, Crawford JT, Dale JW, Eisenach KD, Gicquel B, Hermans P, Martin C, McAdam R, Shinnick TM: Strain identification of Mycobacterium tuberculosis by DNA fingerprinting: recommendations for a standardized methodology. J Clin Microbiol. 1993, 31 (2): 406-409.PubMed CentralPubMedGoogle Scholar
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