- Email: [email protected]
Identification of nontuberculous mycobacteria in clinical samples using molecular methods: a 3-year study
Research Notes
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References 1. Woodford N, Ward ME, Kaufmann ME et al. Community and hospital spread of Escherichia coli producing CTX-M extended-spectrum b-lactamases in the UK. J Antimicrob Chemother 2004; 54: 735–743. 2. Fang H, Lundberg C, Olsson-Liljequist B et al. Molecular epidemiological analysis of Escherichia coli isolates producing extended-spectrum b-lactamases for identification of nosocomial outbreaks in Stockholm, Sweden. J Clin Microbiol 2004; 42: 5917–5920. 3. Mamlouk K, Boutiba-Ben Boubaker I, Gautier V et al. Emergence and outbreaks of CTX-M b-lactamase-producing Escherichia coli and Klebsiella pneumoniae strains in a Tunisian hospital. J Clin Microbiol 2006; 44: 4049–4056. 4. Pitout JDD, Gregson DB, Church DL, Elsayed S, Laupland KB. Community-wide outbreaks of clonally related CTX-M-14 beta-lactamaseproducing Escherichia coli strains in the Calgary health region. J Clin Microbiol 2005; 43: 2844–2849. 5. Clinical and Laboratory Standards Institute. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 6th edn. M7A6. Wayne, PA: CLSI, 2005. 6. Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing; fifteenth informational supplement (M100-S15). Wayne, PA: CLSI, 2005. ¨ sterblad M, Hakanen AJ, Huovinen P, Jalava J, the Finn7. Nyberg SD, O ish Study Group for Antimicrobial Resistance. Detection and molecular genetics of extended-spectrum beta-lactamases among cefuroxime-resistant Escherichia coli and Klebsiella spp. isolates from Finland, 2002–2004. Scand J Infect Dis 2007; 39: 417–424. 8. Haanpera¨ M, Forssten SD, Huovinen P, Jalava J. Typing of SHV extended-spectrum b-lactamases by pyrosequencing in Klebsiella pneumoniae strains with chromosomal SHV b-lactamase. Antimicrob Agents Chemother 2008; 52: 2632–2635. 9. Kaufmann ME. Pulsed-field gel electrophoresis. Totowa: Humana Press, 1998. 10. Manninen R, Auvinen H, Huovinen P, the Finnish Study Group for Antimicrobial Resistance. Resistance to second- and third-generation cephalosporins among Escherichia coli and Klebsiella species is rare in Finland. Clin Microbiol Infect 1997; 3: 408–413. 11. Livermore DM, Canton R, Gniadkowski M et al. CTX-M: changing the face of ESBLs in Europe. J Antimicrob Chemother 2007; 59: 165–174. 12. Brigante G, Luzzaro F, Perilli M et al. Evolution of CTX-M-type b-lactamases in isolates of Escherichia coli infecting hospital and community patients. Int J Antimicrob Agents 2005; 25: 157–162. 13. Fang H, Ataker F, Hedin G, Dornbusch K. Molecular epidemiology of extended-spectrum beta-lactamases among Escherichia coli isolates collected in a Swedish hospital and its associated health care facilities from 2001 to 2006. J Clin Microbiol 2008; 46: 707–712.
1161
Identification of nontuberculous mycobacteria in clinical samples using molecular methods: a 3-year study
I. Couto1,2, D. Machado1, M. Viveiros1, L. Rodrigues1,3 and L. Amaral1,3 1) Unidade de Micobacte´rias, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa (IHMT/UNL), Lisbon, 2) Centro de Recursos Microbiolo´gicos (CREM), Faculdade de Cieˆncias e Tecnologia, UNL, Caparica and 3) UPMM, IHMT/UNL, Lisbon, Portugal
Abstract Nontuberculous mycobacteria (NTM) are being increasingly isolated in clinical laboratories and present technical and therapeutic challenges. In the present study, we report our experience with the identification of NTM received from 12 Lisbon hospitals over a 3-year period using GenoType Mycobacterium (CM/AS) assays (HAIN Lifescience GmbH, Nehren, Germany). Together, the two kits identified 96.6% of all NTM isolates tested. Among the 18 NTM species identified, Mycobacterium avium complex was the most frequent, although it accounted for only 34% of all NTM. Introducing these methods for the rapid identification of NTM highlights the importance of NTM as potential pathogens and assisted the selection of adequate therapy.
Keywords: Atypical, identification, molecular, mycobacteria, nontuberculous Original Submission: 8 April 2009; Revised Submission: 3 July 2009; Accepted: 14 September 2009
Editor: M. Drancourt Article published online: 14 October 2009 Clin Microbiol Infect 2010; 16: 1161–1164 10.1111/j.1469-0691.2009.03076.x Corresponding author and reprint requests: L. Amaral, Unidade de Micobacte´rias, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Rua da Junqueira 96, 1349-008 Lisbon, Portugal E-mail: [email protected]
Although Mycobacterium tuberculosis is the main cause of mycobacteriosis in humans, other species of mycobacteria may also cause infection [1,2]. The increasing importance of ª2009 The Authors Journal Compilation ª2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 16, 1155–1171
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nontuberculous mycobacteria (NTM) in the clinical laboratory is now generally recognized [3–6]. Among the many factors that may contribute to such an increase are the HIV/AIDS pandemics and other immunocompromising diseases, the technical improvements in NTM recovery and identification, the increased interest in NTM identification and the improvement of public health services for tuberculosis [4,5]. Conventional identification of mycobacteria is achieved by standard culture and biochemical methods, all of which are time consuming. The rise in NTM isolation demands faster methods for their identification and for selection of appropriate therapy. In the present study, the GenoType Mycobacterium CM and AS assays (HAIN Lifescience GmbH, Nehren, Germany) were jointly employed for the identification of NTM present in isolates received in our laboratory. These genetically based assays are designed for identifying the clinically most relevant mycobacterial species isolated in culture. From 1 January 2005 to 31 December 2007, our laboratory received a total of 1192 acid-fast bacilli (AFB) isolates from 1174 patients presenting with presumptive active mycobacteriosis at 12 hospitals of the Greater Lisbon Area (Portugal). All isolates were processed for acid-fast staining (Ziehl-Neelsen stain) and inoculated into MGIT tubes of the BACTEC MGIT 960 System (Becton-Dickinson Diagnostic Instrument Systems, Towson, MD, USA) [7]. When necessary, decontamination was carried out by the conventional NaOH-NALC method [8]. M. tuberculosis strains isolated from culture were identified by the Accuprobe system (Gen-Probe Inc., San Diego, CA, USA). Fully-grown AFB cultures, negative for M. tuberculosis, were identified by GenoType Mycobacterium (CM/AS) kits. The experimental procedure involves the isolation of DNA from cultures using the QIAamp DNA mini kit (Qiagen GmbH, Hilden, Germany), PCR amplification of a region of the 23S rRNA gene region [9] with biotinylated primers, and reverse hybridization of the biotin-labelled amplicons with probes immobilized on membrane strips. Identification is achieved by comparing the hybridization patterns obtained with those provided by an interpretation chart. Together, the GenoType Mycobacterium CM (Common Mycobacteria) and the GenoType Mycobacterium AS (Additional Species) kit allow the identification of 30 species, listed in Table 1. Mycobacteria not identifiable by any of these systems were identified by partial 16S rDNA sequencing using the primers 27f and 519r as previously described [10]. When available for a given species, NTM Accuprobe probes were used for comparison with the GenoType CM results. During the 3-year period, out of 1192 specimens received, 1181 were identified as members of the Mycobacterium genus, whereas the 11 non-mycobacterial acid-fast bacilli
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cultures were identified as Nocardia spp. (nine isolates), Tsukamurella (one isolate) and Rhodococcus (one isolate) by 16S rRNA gene sequencing. Among the 1181 mycobacterial cultures, 1032 (87.4%) were positive for the M. tuberculosis complex. The remaining 149 cultures were NTM, corresponding to 12.6% of the total number of cultures from which mycobacteria were isolated. During the study period, NTM prevalence increased steadily, starting with 8.7% in 2005 and rising to 15.2% in 2007. The joint use of the CM and AS kits identified 96.6% of all NTM isolates tested; these results are comparable to those reported elsewhere [9, 11–15]. The GenoType Mycobacterium CM system identified 136 out of 149 NTM cultures tested. Of the remaining 13 isolates, eight were identified with the GenoType AS kit (Table 1). When available, there was complete agreement between the Accuprobe identification result and that provided by the GenoType CM system (data not shown). Sequencing of the 5¢-termini of 16S rDNA identified the remaining five isolates as Mycobacterium arupense/Mycobacterium nonchromogenicum (three isolates) and Mycobacterium terrae (two isolates). A total of 18 NTM species were identified (Table 1). Although Mycobacterium avium was the most frequently found species, the relatively low percentage of M. avium among all of the NTM isolates was one of the important findings of the present study. Even if we consider the M. avium complex (M. avium plus Mycobacterium intracellulare isolates), it would account for only one-third (34%) of all NTM isolates received in our laboratory. The species distribution of the remaining NTM was in agreement with that reported earlier in a multicountry survey on the frequency and distribution of NTM, which included Portugal [4], except for Mycobacterium kansasii, fourth among the NTM species, together with M. intracellulare and Mycobacterium fortuitum, whereas, in the previous study it was not found among the Portuguese isolates [4]. Although the NTM-containing specimens were mainly respiratory (Table 1), over 10% of NTM were isolated from sterile sources, namely blood and cerebral-spinal fluids. The isolation of NTM from these sources is particularly relevant because it illustrates situations where the ability of the laboratory to identify NTM rapidly and precisely plays a major role in the clinical outcome of the infection. In the present study, all NTM identified were considered to be clinically relevant, either as the cause of infection or as confounders of diagnosis and therapeutics. Among the different methods available for NTM identification, the Accuprobe system, based on a hybridization protection assay with DNA probes, is the one most widely used because of its accuracy and rapidity. In our laboratory, the Accuprobe system remains the standard for identification of
ª2009 The Authors Journal Compilation ª2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 16, 1155–1171
Research Notes
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TABLE 1. Nontuberculous mycobacteria species identified with the Genotype CM/AS systems during the 3-year period
Species/complex identifiable
Identified in the present study
Number of isolates (frequency)a
Source (number of isolates)
GenoType Mycobacterium CM Mycobacterium avium spp.
Yes
37 (24.8%)
Mycobacterium gordonae Mycobacterium fortuitum Mycobacterium intracellulare
Yes Yes Yes
23 (15.4%) 14 (9.4%) 14 (9.4%)
Yes Yes Yes Yes Yes Yes Yes No No
14 (9.4%) 10 (6.7%) 9 (6%) 7 (4.7%) 6 (4%) 1 (0.7%) 1 (0.7%) – –
Blood culture (14); sputum (8); bronchial secretions (5); bronchoalveolar lavage (3); articular fluid (1); biopsies (2); myeloculture (1); urine (1); Gastric fluid (1); pus (1) Sputum (20); gastric fluid (2); bronchial secretions (1) Sputum (10); bronchial aspirate (3); bronchial secretions (1) Sputum (8); bronchial secretions (2); bronchoalveolar lavage (1); gastric fluid (1); articular fluid (1); urine (1) Sputum (8); bronchial secretions (4); bronchoalveolar lavage (1); synovial fluid (1) Sputum (7); bronchial secretions (1); biopsies (1); pus (1) Sputum (6); bronchial aspirate (1) bronchial secretions (1); bronchoalveolar lavage (1) Sputum (5); biopsies (1); bronchial secretions (1) Sputum (4); bronchoalveolar lavage (1); gastric fluid (1) Sputum (1) Sputum (1) – –
Not applicableb
–
–
Yes Yes Yes Yes Yes No No No No
1 4 1 1 1 – – – –
No No Not applicablec No No No No
– – – – – – –
Mycobacterium kansasii* Mycobacterium chelonae Mycobacterium xenopi Mycobacterium peregrinum Mycobacterium abscessus Mycobacterium scrofulaceum Mycobacterium malmoense Mycobacterium interjectum Mycobacterium marinum/ Mycobacterium ulcerans Mycobacterium tuberculosis complex Geno type Mycobacterium AS Mycobacterium simiae Mycobacterium mucogenicum Mycobacterium lentiflavum Mycobacterium heckeshornense Mycobacterium celatum Mycobacterium smegmatis Mycobacterium goodii Mycobacterium genavense Mycobacterium szulgai/ Mycobacterium intermedium Mycobacterium phlei Mycobacterium haemophilum Mycobacterium kansasii* Mycobacterium ulcerans Mycobacterium gastri Mycobacterium asiaticum Mycobacterium shimoidei
(0.7%) (2.6%) (0.7%) (0.7%) (0.7%)
Blood culture (1) Cerebral-spinal fluid (2); Bronchoalveolar lavage (1); biopsies (1) Sputum (1) Sputum (1) Bronchial secretions (1) – – – – – – – – – – –
*Both kits are able to identify M. kansasii, for which Genotype AS distinguishes four possible hybridization patterns. The AS kit distinguishes M. ulcerans. Among a total of 149 nontuberculous mycobacteria isolates that included five isolates not identifiable by any of the kits (see text). b All M. tuberculosis complex isolates were identified by Accuprobe. c All M. kansasii isolates were identified with the Genotype CM kit. a
isolates from the M. tuberculosis complex. However, the increasing isolation of NTM by the collaborating hospitals proved challenging to our work algorithm. The Accuprobe system is currently limited to the identification of five species/complexes (M. tuberculosis complex, M. avium, M. intracellulare, or the M. avium complex as a whole, M. kansasii and Mycobacterium gordonae) and, although these correspond to the most frequently isolated mycobacteria, these probes failed to identify 40.2% of all NTM isolated and identified in the present study by the combined use of the CM and AS kits. An important advantage provided by the GenoType kits is the possibility to detect mixed infections. In the present study, such mixed infections were detected in sputa, blood, and urine. The co-existing species isolated from these specimens were M. tuberculosis and M. avium, M. gordonae or Mycobacterium abcessus. The co-isolation of M. tuberculosis and other mycobacteria and their distinction was highly relevant for the selection of therapy.
The GenoType methodology provided identification within 6 h. The major drawbacks were cost (approximately €40 per test) and the need to have a fully-grown culture to obtain reliable results. Despite the advantages provided by molecular methods, conventional methods for the identification of NTM must be retained and judiciously used when necessary. In countries with a high incidence of tuberculosis and, particularly, multidrug-resistant M. tuberculosis (MDR-TB) such as Portugal [16], therapeutic failure with isoniazid and rifampicin is anticipated to be the result of an MDR-TB strain. Because many NTM species are resistant to these drugs [3], the identification of the mycobacteria causing therapeutic failure (MDR-TB vs. NTM) is of major importance [17]. This has been appreciated by the clinicians at the collaborating hospitals and has resulted in an increased awareness of the importance of being able to identify NTM rapidly as potential pathogens and the key role played by the laboratory in assisting the selection of therapeutic modality.
ª2009 The Authors Journal Compilation ª2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 16, 1155–1171
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Acknowledgements 12.
We thank the members of the TB Task Force of Greater Lisbon and its Fast and Faster Track TB Programmes for their kind cooperation. This work was presented in part at the 45th Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, September 2007.
13.
14.
Transparency Declaration
15.
This study was supported by grants SDH.IC.I.01.17-TB and TB Faster Track for Greater Lisbon, from the Fundac¸a˜o Calouste Gulbenkian. D. Machado and L. Rodrigues were supported by grants PTDC/BIA-MIC/BIC-01/2008 and SFRH/ BD/24931/2005 from Fundac¸a˜o para a Cieˆncia e a Tecnologia (FCT, Portugal). The authors declare that they have no conflicts of interest.
16.
References 1. Tortoli E. Impact of genotypic studies on mycobacterial taxonomy: the new mycobacteria of the 1990s. Clin Microbiol Rev 2003; 16: 319– 354. 2. Tortoli E. The new mycobacteria: an update. FEMS Immunol Med Microbiol 2006; 48: 159–178. 3. Griffith DE, Aksamitt T, Brown-Elliott BA et al. An official ATS/IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases. Am J Respir Crit Care Med 2007; 175: 367– 416. 4. Martı´n-Casabona N, Bahrmand AR, Bennedsen J et al. Non-tuberculous mycobacteria: patterns of isolation a multi-country retrospective survey. Int J Tuberc Lung Dis 2004; 8: 1186–1193. 5. Piersimoni C, Scarparo C. Pulmonary infections associated with nontuberculous mycobacteria in immunocompetent patients. Lancet Infect Dis 2008; 8: 323–334. 6. Primm TP, Lucero CA, Falkinham JO III. Health impacts of environmental mycobacteria. Clin Microbiol Rev 2004; 17: 98–106. 7. Siddiqi S, Ru¨sch-Gerdes S. MGIT Procedure Manual for BACTEC MGIT 960 TB System (Also applicable for Manual MGIT). Mycobacteria Growth Indicator Tube (MGIT) Culture and Drug Susceptibility Demonstration Projects. Foundation for Innovative New Diagnostics Ed. 2006. Available at: http://www.finddiagnostics.org/resource-centre/reports_brochures/071130_mait_manual.html (last accessed 3 July 2009). 8. Kent PT, Kubica GP. Public health mycobacteriology: a guide for the level III laboratory. Atlanta, GA: US Department of Health and Human Services, Centers for Disease Control, 1985. 9. Richter E, Ru¨sch-Gerdes S, Hillemann D. Evaluation of the GenoType Mycobacterium assay for identification of mycobacterial species from cultures. J Clin Microbiol 2006; 44: 1769–1775. 10. Lane DJ. 16S/23S rRNA sequencing. In: Stachebrandt E, Goodfellow M, eds. Nucleic acid techniques in bacterial systematics. New York, NY, John Wiley and Sons, 1991; 115–175. 11. Gitti Z, Neonakis I, Fanti G, Kontos F, Maraki S, Tselentis Y. Use of the GenoType Mycobacterium CM and AS assays to analyze 76
17.
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nontuberculous mycobacterial isolates from Greece. J Clin Microbiol 2006; 44: 2244–2246. Ma¨kinen J, Marjama¨ki M, Marttila H, Soini H. Evaluation of a novel strip test, GenoType Mycobacterium CM/AS, for species identification of mycobacterial cultures. Clin Microbiol Infect 2006; 12: 481–483. Roth A, Reischl U, Streubel A et al. Novel diagnostic algorithm for identification of mycobacteria using genus-specific amplification of the 16S-23S rRNA gene spacer and restriction endonucleases. J Clin Microbiol 2000; 38: 1094–1104. Ruiz P, Gutierrez J, Zerolo FJ, Casal M. GenoType Mycobacterium assay for identification of mycobacterial species isolated from human clinical samples by using liquid medium. J Clin Microbiol 2002; 40: 3076–3078. Russo C, Tortoli E, Menichella D. Evaluation of the new GenoType Mycobacterium assay for identification of mycobacterial species. J Clin Microbiol 2006; 44: 334–339. Direcc¸a˜o Geral de Sau´de. Programa Nacional de Luta contra a Tuberculose (PNT). Ponto da situac¸a˜o epidemiolo´gica e de desempenho, ano 2007, Lisboa, Portugal, DGS 2008; 1–17 Tabarsi P, Baghaei P, Farnia P et al. Nontuberculous mycobacteria among patients who are suspected for multidrug-resistant tuberculosis – need for earlier identification of nontuberculosis mycobacteria. Am J Med Sci 2009; 337: 182–184.
Pyrazinamide resistance in multidrugresistant Mycobacterium tuberculosis isolates in Japan
H. Ando1, S. Mitarai2, Y. Kondo3, T. Suetake3, J.-I. Sekiguchi1,2, S. Kato2, T. Mori2 and T. Kirikae1 1) Department of Infectious Diseases, Research Institute, International Medical Centre of Japan, 2) Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association, Tokyo and 3) Third Department, Research and Development Laboratory, Nipro Corporation, Shiga, Japan
Abstract Thirty-six multidrug-resistant (MDR) Mycobacterium tuberculosis isolates collected in Japan were examined for pyrazinamide susceptibility and pyrazinamidase activity, and analysed by pncA sequencing and a hybridization-based line probe assay (LiPA), which was used to detect pncA mutations for the rapid identification of pyrazinamide-resistant isolates. Pyrazinamide resistance was found in 19 (53%) of them. All pyrazinamide-resistant isolates had no pyrazinamidase activity and at least one mutation in pncA. Among the pncA mutations, 11 had not been previously reported. The results of the LiPA were fully consistent with the DNA sequencing results. A majority of MDR M. tuberculosis isolates in Japan were resistant to pyrazinamide.
Keywords: Line probe assay, multidrug resistance, Mycobacterium tuberculosis, pncA, pyrazinamide
ª2009 The Authors Journal Compilation ª2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 16, 1155–1171
CMI
References 1. Woodford N, Ward ME, Kaufmann ME et al. Community and hospital spread of Escherichia coli producing CTX-M extended-spectrum b-lactamases in the UK. J Antimicrob Chemother 2004; 54: 735–743. 2. Fang H, Lundberg C, Olsson-Liljequist B et al. Molecular epidemiological analysis of Escherichia coli isolates producing extended-spectrum b-lactamases for identification of nosocomial outbreaks in Stockholm, Sweden. J Clin Microbiol 2004; 42: 5917–5920. 3. Mamlouk K, Boutiba-Ben Boubaker I, Gautier V et al. Emergence and outbreaks of CTX-M b-lactamase-producing Escherichia coli and Klebsiella pneumoniae strains in a Tunisian hospital. J Clin Microbiol 2006; 44: 4049–4056. 4. Pitout JDD, Gregson DB, Church DL, Elsayed S, Laupland KB. Community-wide outbreaks of clonally related CTX-M-14 beta-lactamaseproducing Escherichia coli strains in the Calgary health region. J Clin Microbiol 2005; 43: 2844–2849. 5. Clinical and Laboratory Standards Institute. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 6th edn. M7A6. Wayne, PA: CLSI, 2005. 6. Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing; fifteenth informational supplement (M100-S15). Wayne, PA: CLSI, 2005. ¨ sterblad M, Hakanen AJ, Huovinen P, Jalava J, the Finn7. Nyberg SD, O ish Study Group for Antimicrobial Resistance. Detection and molecular genetics of extended-spectrum beta-lactamases among cefuroxime-resistant Escherichia coli and Klebsiella spp. isolates from Finland, 2002–2004. Scand J Infect Dis 2007; 39: 417–424. 8. Haanpera¨ M, Forssten SD, Huovinen P, Jalava J. Typing of SHV extended-spectrum b-lactamases by pyrosequencing in Klebsiella pneumoniae strains with chromosomal SHV b-lactamase. Antimicrob Agents Chemother 2008; 52: 2632–2635. 9. Kaufmann ME. Pulsed-field gel electrophoresis. Totowa: Humana Press, 1998. 10. Manninen R, Auvinen H, Huovinen P, the Finnish Study Group for Antimicrobial Resistance. Resistance to second- and third-generation cephalosporins among Escherichia coli and Klebsiella species is rare in Finland. Clin Microbiol Infect 1997; 3: 408–413. 11. Livermore DM, Canton R, Gniadkowski M et al. CTX-M: changing the face of ESBLs in Europe. J Antimicrob Chemother 2007; 59: 165–174. 12. Brigante G, Luzzaro F, Perilli M et al. Evolution of CTX-M-type b-lactamases in isolates of Escherichia coli infecting hospital and community patients. Int J Antimicrob Agents 2005; 25: 157–162. 13. Fang H, Ataker F, Hedin G, Dornbusch K. Molecular epidemiology of extended-spectrum beta-lactamases among Escherichia coli isolates collected in a Swedish hospital and its associated health care facilities from 2001 to 2006. J Clin Microbiol 2008; 46: 707–712.
1161
Identification of nontuberculous mycobacteria in clinical samples using molecular methods: a 3-year study
I. Couto1,2, D. Machado1, M. Viveiros1, L. Rodrigues1,3 and L. Amaral1,3 1) Unidade de Micobacte´rias, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa (IHMT/UNL), Lisbon, 2) Centro de Recursos Microbiolo´gicos (CREM), Faculdade de Cieˆncias e Tecnologia, UNL, Caparica and 3) UPMM, IHMT/UNL, Lisbon, Portugal
Abstract Nontuberculous mycobacteria (NTM) are being increasingly isolated in clinical laboratories and present technical and therapeutic challenges. In the present study, we report our experience with the identification of NTM received from 12 Lisbon hospitals over a 3-year period using GenoType Mycobacterium (CM/AS) assays (HAIN Lifescience GmbH, Nehren, Germany). Together, the two kits identified 96.6% of all NTM isolates tested. Among the 18 NTM species identified, Mycobacterium avium complex was the most frequent, although it accounted for only 34% of all NTM. Introducing these methods for the rapid identification of NTM highlights the importance of NTM as potential pathogens and assisted the selection of adequate therapy.
Keywords: Atypical, identification, molecular, mycobacteria, nontuberculous Original Submission: 8 April 2009; Revised Submission: 3 July 2009; Accepted: 14 September 2009
Editor: M. Drancourt Article published online: 14 October 2009 Clin Microbiol Infect 2010; 16: 1161–1164 10.1111/j.1469-0691.2009.03076.x Corresponding author and reprint requests: L. Amaral, Unidade de Micobacte´rias, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Rua da Junqueira 96, 1349-008 Lisbon, Portugal E-mail: [email protected]
Although Mycobacterium tuberculosis is the main cause of mycobacteriosis in humans, other species of mycobacteria may also cause infection [1,2]. The increasing importance of ª2009 The Authors Journal Compilation ª2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 16, 1155–1171
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nontuberculous mycobacteria (NTM) in the clinical laboratory is now generally recognized [3–6]. Among the many factors that may contribute to such an increase are the HIV/AIDS pandemics and other immunocompromising diseases, the technical improvements in NTM recovery and identification, the increased interest in NTM identification and the improvement of public health services for tuberculosis [4,5]. Conventional identification of mycobacteria is achieved by standard culture and biochemical methods, all of which are time consuming. The rise in NTM isolation demands faster methods for their identification and for selection of appropriate therapy. In the present study, the GenoType Mycobacterium CM and AS assays (HAIN Lifescience GmbH, Nehren, Germany) were jointly employed for the identification of NTM present in isolates received in our laboratory. These genetically based assays are designed for identifying the clinically most relevant mycobacterial species isolated in culture. From 1 January 2005 to 31 December 2007, our laboratory received a total of 1192 acid-fast bacilli (AFB) isolates from 1174 patients presenting with presumptive active mycobacteriosis at 12 hospitals of the Greater Lisbon Area (Portugal). All isolates were processed for acid-fast staining (Ziehl-Neelsen stain) and inoculated into MGIT tubes of the BACTEC MGIT 960 System (Becton-Dickinson Diagnostic Instrument Systems, Towson, MD, USA) [7]. When necessary, decontamination was carried out by the conventional NaOH-NALC method [8]. M. tuberculosis strains isolated from culture were identified by the Accuprobe system (Gen-Probe Inc., San Diego, CA, USA). Fully-grown AFB cultures, negative for M. tuberculosis, were identified by GenoType Mycobacterium (CM/AS) kits. The experimental procedure involves the isolation of DNA from cultures using the QIAamp DNA mini kit (Qiagen GmbH, Hilden, Germany), PCR amplification of a region of the 23S rRNA gene region [9] with biotinylated primers, and reverse hybridization of the biotin-labelled amplicons with probes immobilized on membrane strips. Identification is achieved by comparing the hybridization patterns obtained with those provided by an interpretation chart. Together, the GenoType Mycobacterium CM (Common Mycobacteria) and the GenoType Mycobacterium AS (Additional Species) kit allow the identification of 30 species, listed in Table 1. Mycobacteria not identifiable by any of these systems were identified by partial 16S rDNA sequencing using the primers 27f and 519r as previously described [10]. When available for a given species, NTM Accuprobe probes were used for comparison with the GenoType CM results. During the 3-year period, out of 1192 specimens received, 1181 were identified as members of the Mycobacterium genus, whereas the 11 non-mycobacterial acid-fast bacilli
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cultures were identified as Nocardia spp. (nine isolates), Tsukamurella (one isolate) and Rhodococcus (one isolate) by 16S rRNA gene sequencing. Among the 1181 mycobacterial cultures, 1032 (87.4%) were positive for the M. tuberculosis complex. The remaining 149 cultures were NTM, corresponding to 12.6% of the total number of cultures from which mycobacteria were isolated. During the study period, NTM prevalence increased steadily, starting with 8.7% in 2005 and rising to 15.2% in 2007. The joint use of the CM and AS kits identified 96.6% of all NTM isolates tested; these results are comparable to those reported elsewhere [9, 11–15]. The GenoType Mycobacterium CM system identified 136 out of 149 NTM cultures tested. Of the remaining 13 isolates, eight were identified with the GenoType AS kit (Table 1). When available, there was complete agreement between the Accuprobe identification result and that provided by the GenoType CM system (data not shown). Sequencing of the 5¢-termini of 16S rDNA identified the remaining five isolates as Mycobacterium arupense/Mycobacterium nonchromogenicum (three isolates) and Mycobacterium terrae (two isolates). A total of 18 NTM species were identified (Table 1). Although Mycobacterium avium was the most frequently found species, the relatively low percentage of M. avium among all of the NTM isolates was one of the important findings of the present study. Even if we consider the M. avium complex (M. avium plus Mycobacterium intracellulare isolates), it would account for only one-third (34%) of all NTM isolates received in our laboratory. The species distribution of the remaining NTM was in agreement with that reported earlier in a multicountry survey on the frequency and distribution of NTM, which included Portugal [4], except for Mycobacterium kansasii, fourth among the NTM species, together with M. intracellulare and Mycobacterium fortuitum, whereas, in the previous study it was not found among the Portuguese isolates [4]. Although the NTM-containing specimens were mainly respiratory (Table 1), over 10% of NTM were isolated from sterile sources, namely blood and cerebral-spinal fluids. The isolation of NTM from these sources is particularly relevant because it illustrates situations where the ability of the laboratory to identify NTM rapidly and precisely plays a major role in the clinical outcome of the infection. In the present study, all NTM identified were considered to be clinically relevant, either as the cause of infection or as confounders of diagnosis and therapeutics. Among the different methods available for NTM identification, the Accuprobe system, based on a hybridization protection assay with DNA probes, is the one most widely used because of its accuracy and rapidity. In our laboratory, the Accuprobe system remains the standard for identification of
ª2009 The Authors Journal Compilation ª2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 16, 1155–1171
Research Notes
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TABLE 1. Nontuberculous mycobacteria species identified with the Genotype CM/AS systems during the 3-year period
Species/complex identifiable
Identified in the present study
Number of isolates (frequency)a
Source (number of isolates)
GenoType Mycobacterium CM Mycobacterium avium spp.
Yes
37 (24.8%)
Mycobacterium gordonae Mycobacterium fortuitum Mycobacterium intracellulare
Yes Yes Yes
23 (15.4%) 14 (9.4%) 14 (9.4%)
Yes Yes Yes Yes Yes Yes Yes No No
14 (9.4%) 10 (6.7%) 9 (6%) 7 (4.7%) 6 (4%) 1 (0.7%) 1 (0.7%) – –
Blood culture (14); sputum (8); bronchial secretions (5); bronchoalveolar lavage (3); articular fluid (1); biopsies (2); myeloculture (1); urine (1); Gastric fluid (1); pus (1) Sputum (20); gastric fluid (2); bronchial secretions (1) Sputum (10); bronchial aspirate (3); bronchial secretions (1) Sputum (8); bronchial secretions (2); bronchoalveolar lavage (1); gastric fluid (1); articular fluid (1); urine (1) Sputum (8); bronchial secretions (4); bronchoalveolar lavage (1); synovial fluid (1) Sputum (7); bronchial secretions (1); biopsies (1); pus (1) Sputum (6); bronchial aspirate (1) bronchial secretions (1); bronchoalveolar lavage (1) Sputum (5); biopsies (1); bronchial secretions (1) Sputum (4); bronchoalveolar lavage (1); gastric fluid (1) Sputum (1) Sputum (1) – –
Not applicableb
–
–
Yes Yes Yes Yes Yes No No No No
1 4 1 1 1 – – – –
No No Not applicablec No No No No
– – – – – – –
Mycobacterium kansasii* Mycobacterium chelonae Mycobacterium xenopi Mycobacterium peregrinum Mycobacterium abscessus Mycobacterium scrofulaceum Mycobacterium malmoense Mycobacterium interjectum Mycobacterium marinum/ Mycobacterium ulcerans Mycobacterium tuberculosis complex Geno type Mycobacterium AS Mycobacterium simiae Mycobacterium mucogenicum Mycobacterium lentiflavum Mycobacterium heckeshornense Mycobacterium celatum Mycobacterium smegmatis Mycobacterium goodii Mycobacterium genavense Mycobacterium szulgai/ Mycobacterium intermedium Mycobacterium phlei Mycobacterium haemophilum Mycobacterium kansasii* Mycobacterium ulcerans Mycobacterium gastri Mycobacterium asiaticum Mycobacterium shimoidei
(0.7%) (2.6%) (0.7%) (0.7%) (0.7%)
Blood culture (1) Cerebral-spinal fluid (2); Bronchoalveolar lavage (1); biopsies (1) Sputum (1) Sputum (1) Bronchial secretions (1) – – – – – – – – – – –
*Both kits are able to identify M. kansasii, for which Genotype AS distinguishes four possible hybridization patterns. The AS kit distinguishes M. ulcerans. Among a total of 149 nontuberculous mycobacteria isolates that included five isolates not identifiable by any of the kits (see text). b All M. tuberculosis complex isolates were identified by Accuprobe. c All M. kansasii isolates were identified with the Genotype CM kit. a
isolates from the M. tuberculosis complex. However, the increasing isolation of NTM by the collaborating hospitals proved challenging to our work algorithm. The Accuprobe system is currently limited to the identification of five species/complexes (M. tuberculosis complex, M. avium, M. intracellulare, or the M. avium complex as a whole, M. kansasii and Mycobacterium gordonae) and, although these correspond to the most frequently isolated mycobacteria, these probes failed to identify 40.2% of all NTM isolated and identified in the present study by the combined use of the CM and AS kits. An important advantage provided by the GenoType kits is the possibility to detect mixed infections. In the present study, such mixed infections were detected in sputa, blood, and urine. The co-existing species isolated from these specimens were M. tuberculosis and M. avium, M. gordonae or Mycobacterium abcessus. The co-isolation of M. tuberculosis and other mycobacteria and their distinction was highly relevant for the selection of therapy.
The GenoType methodology provided identification within 6 h. The major drawbacks were cost (approximately €40 per test) and the need to have a fully-grown culture to obtain reliable results. Despite the advantages provided by molecular methods, conventional methods for the identification of NTM must be retained and judiciously used when necessary. In countries with a high incidence of tuberculosis and, particularly, multidrug-resistant M. tuberculosis (MDR-TB) such as Portugal [16], therapeutic failure with isoniazid and rifampicin is anticipated to be the result of an MDR-TB strain. Because many NTM species are resistant to these drugs [3], the identification of the mycobacteria causing therapeutic failure (MDR-TB vs. NTM) is of major importance [17]. This has been appreciated by the clinicians at the collaborating hospitals and has resulted in an increased awareness of the importance of being able to identify NTM rapidly as potential pathogens and the key role played by the laboratory in assisting the selection of therapeutic modality.
ª2009 The Authors Journal Compilation ª2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 16, 1155–1171
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Clinical Microbiology and Infection, Volume 16 Number 8, August 2010
Acknowledgements 12.
We thank the members of the TB Task Force of Greater Lisbon and its Fast and Faster Track TB Programmes for their kind cooperation. This work was presented in part at the 45th Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, September 2007.
13.
14.
Transparency Declaration
15.
This study was supported by grants SDH.IC.I.01.17-TB and TB Faster Track for Greater Lisbon, from the Fundac¸a˜o Calouste Gulbenkian. D. Machado and L. Rodrigues were supported by grants PTDC/BIA-MIC/BIC-01/2008 and SFRH/ BD/24931/2005 from Fundac¸a˜o para a Cieˆncia e a Tecnologia (FCT, Portugal). The authors declare that they have no conflicts of interest.
16.
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Pyrazinamide resistance in multidrugresistant Mycobacterium tuberculosis isolates in Japan
H. Ando1, S. Mitarai2, Y. Kondo3, T. Suetake3, J.-I. Sekiguchi1,2, S. Kato2, T. Mori2 and T. Kirikae1 1) Department of Infectious Diseases, Research Institute, International Medical Centre of Japan, 2) Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association, Tokyo and 3) Third Department, Research and Development Laboratory, Nipro Corporation, Shiga, Japan
Abstract Thirty-six multidrug-resistant (MDR) Mycobacterium tuberculosis isolates collected in Japan were examined for pyrazinamide susceptibility and pyrazinamidase activity, and analysed by pncA sequencing and a hybridization-based line probe assay (LiPA), which was used to detect pncA mutations for the rapid identification of pyrazinamide-resistant isolates. Pyrazinamide resistance was found in 19 (53%) of them. All pyrazinamide-resistant isolates had no pyrazinamidase activity and at least one mutation in pncA. Among the pncA mutations, 11 had not been previously reported. The results of the LiPA were fully consistent with the DNA sequencing results. A majority of MDR M. tuberculosis isolates in Japan were resistant to pyrazinamide.
Keywords: Line probe assay, multidrug resistance, Mycobacterium tuberculosis, pncA, pyrazinamide
ª2009 The Authors Journal Compilation ª2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 16, 1155–1171