RESEARCH ARTICLE
Nontuberculous Mycobacteria Among Patients Who are Suspected for Multidrug-Resistant Tuberculosis—Need for Earlier Identification of Nontuberculosis Mycobacteria Payam Tabarsi, MD, MPH, Parvaneh Baghaei, MD, MPH, Parisa Farnia, PhD, Nahal Mansouri, MD, Ehsan Chitsaz, MD, Fatemeh Sheikholeslam, MSc, Majid Marjani, MD, MPH, Nima Rouhani, MD, MPH, Mehdi Mirsaeidi, MD, MPH, Narges Alipanah, MS, Majid Amiri, MD, MPH, Mohammad R. Masjedi, MD and Davood Mansouri, MD, MPH
Abstract: Background: In this study, we intended to find the prevalence of nontuberculosis mycobacteria (NTM) among patients who are referred as suspected multidrug-resistant tuberculosis (MDR-TB) cases to the only referral center in Iran. Methods: All patients referred to our center in 2002–2006 as MDR-TB with histories of treatment with standard and CAT II World Health Organization regimens were included in the study. Sputum smear and culture for acid-fast bacilli were performed for all patients 3 times. Sputum polymerase chain reaction was also performed for all patients. Mycobacterial identification was performed via polymerase chain reaction and routine identification tests for all culture-positive cases. Results: Of the 105 patients in the study, 12 (11.43%) were identified to have NTM infection. The identified mycobacteria were classified in order of prevalence as Chelonae (8 cases), Simiae (2 cases), Aloei (1 case), and Farcinogen (1 case). Based on radiologic findings, most of the cases demonstrated bilateral nodularity (83.3%) and also multifocal bronchiectasis (75%). Notably, cavitary lesions were present in 41.7% of the cases. Conclusion: Based on the findings of this study, it is essential that such cases be identified before commencing MDR-TB treatment. Key Indexing Terms: Nontuberculosis mycobacterium; CAT II regimen; Multidrug-resistant tuberculosis; PCR. [Am J Med Sci 2009;337(3):182–184.]
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t is now proved that various types of nontuberculosis mycobacteria (NTM) can affect human and may cause either symptomatic or asymptomatic infection.1 Currently, more than 125 different types of NTM exist in the environment, which usually prompt relatively vague and nonspecific symptoms.1–3 To date, there is no evidence of human– human transmission, even in cases with underlying lung diseases, such as bronchiectasis, cystic fibrosis, etc. However, some reports have described the animal to human transmission, eg, through pets and domestic animals or through the litter of infected animals.4,5 Unfortunately, no precise data is available of their prevalence particularly in developing countries.6 –10 Yet in industrial countries, the prevalence has been reported to be 1 to 1.8 per 100,000.11 In the recent decades, NTM infections have experienced an increasing incidence worldwide. This is mainly attributable to 2 major factors: the first factor is the rising incidence of HIV infection in the world, whereas the other is the significant improvement
From the Mycobacteriology Research Center, NRITLD, Masih Daneshvari Hospital, Shahid Beheshti University of Medical Science and Health Services. Submitted May 7, 2008; accepted in revised form July 7, 2008. Correspondence: Payam Tabarsi, MD, M.P.H., Masih Daneshvari Hospital, Darabad, Niavaran Sq, Tehran, Iran (E-mail:
[email protected]).
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in the laboratory techniques and diagnostic methods that makes it possible to distinguish between varieties of mycobacteria species.1 In resource-limited countries, tuberculosis (TB) treatment is mainly based on sputum smear, and culture may not be performed in many regions. Hence, it is not unlikely that a positive sputum smear is in fact due to an NTM that is then erroneously treated with standard anti-TB medications. As many NTM are resistant to first-line anti-TB medications,12 most of these cases, as CAT I treatment failures, are subsequently treated with World Health Organization (WHO) CAT II regimen. On failure of the latter regimen, the patients are referred to the referral center as chronic cases. Currently, no specific information regarding these patients is available in Iran. Furthermore, no definitive data have been reported so far. Therefore, this study seeks to find the number of NTM patients among the chronic cases referred to the TB referral center.
METHODS Setting This study was conducted at National Research Institute of Tuberculosis and Lung Disease at Masih Daneshvari Hospital, the WHO collaborating center, and the sole national referral center for tuberculosis in Iran possessing the National Mycobacteriologic Reference Laboratory. Patient Recruitment Based on the national protocol in Iran, all patients diagnosed with TB are supposed to receive WHO CAT I regimen.12 If the treatment fails, WHO CAT II regimen is then administered.13 In the event of CAT II regimen failure, the patients are referred to our center for evaluation and treatment of multidrug-resistant tuberculosis (MDR-TB). All patients who were admitted to our center in 2002– 2006 suspected for MDR-TB infection are included in the study. For each patient sputum smear and culture for acid-fast bacilli have been performed 3 times. Sputum polymerase chain reaction (PCR) was undertaken for all patients to confirm Mycobacterium tuberculosis (MTB). Mycobacterial identification was performed via PCR and routine identification tests for all culture-positive cases (CAT I regimen: HRZE for 2 months then HR for 4 months – CATII regimen: HRZES for 2 months then HRZE for 1 month then HRE for 5 months).12 Identification Method Specimens obtained from patients were cultured on the Lowenstein–Jensen (LJ) slants so that 100 to 120 colonies grew. Whenever polymerase chain reaction using IS6110 probe and spoligotyping were negative, conventional biochemical
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tests14 were performed for both the patient strains and standard NTM (obtained from the Department of Mycobacteriology, National Institute of Public and the Environment, The Netherlands). The tests included photoinduction test, niacin production, nitrate reduction, catalase (heat stable and semiquantitative), Tween hydrolysis (10 days), and urease (Murphy–Hawkins disk method). In addition, the growth on LJ culture media at 25, 37, 40, and 45°C were determined. On the basis of data reported in Bergey’s Manual of Systematic Bacteriology,14,15 the only species whose results matched the specific pattern seemed to be NTM. PCR Method DNA Extraction Bacterial DNA was prepared as described by van Soolingen et al.16 Briefly, 1 loopful of bacteria cultured on LJ medium was suspended on 400 L of TE buffer (0.01 M Tris–HCl, 0.01 M EDTA [pH 8.0]). The samples were heated for 20 minutes at 80°C to kill the cells, and then cooled at room temperature. Lysozyme was added to a final concentration of 1 mg/mL and the tube was incubated at 37°C overnight. Seventy-five microliters of 10% sodium dodecyl sulfate and proteinase K (at 10 mg/mL) were added and mixture was incubated at 65°C for 10 minutes. Hundred microliters of saturated NaCl and 100 L of N-acetyl-N,N,N,-trimethylammonium bromide were added. The tubes were incubated for 10 minutes at 65°C. An equal volume of chloroform was added and the mixture was centrifuged for 5 minutes. Four hundred fifty microliters of isopropanol was added to supernatant to precipitate the DNA. After 30 minutes at 20°C and centrifuging for 15 minutes at 12,000 rpm at room temperature, the pellet was washed once with 70% ethanol and the air-dried pellet was dissolved in 50 to 150 L sterile distilled water.
TABLE 1. The different types of nontuberculous mycobacteria isolated from our patients’ specimens Chelonae Aloei Farcinogen Simiae
The scientific and ethics committee of National Research Institute of TB and Lung Disease in Iran approved the study protocol.
RESULTS Totally, 105 patients were included in the study, among whom mycobacteriologic studies showed 12 (11.43%) cases to be affected by NTM. Nine (75%) of these cases were women and the average age of the patients was 50.75 ⫾ 12.9 (range: 22–70). Eleven patients (91.7%) were Iranian and 1 was Afghan. All patients were negative for HIV and none had a history of smoking or opium use. All patients had a history of prior treatment with WHO CAT II regimen. The variety of mycobacteria and their radiologic manifestations are shown in Tables 1 and 2. On confirmation of infection with NTM, the appropriate regimen was administered for the patients according to the American Thorasic Society (ATS) 2003 Guidelines.1 Having received the treatment for NTM infection, there was no mortality in the treatment course and the 18-month follow-up period. Furthermore, no cases showed further progress in the disease or emergence of MTB infection.
DISCUSSION
PCR A segment of the 65-kDa heat shock protein gene (hsp65) was amplified by 2 specific primers as described by Telenti et al.17 The amplification was done with a 25-L reaction mix containing 10 mM Tris–HCl (pH 8.0), 50 mM KCl, 2 mM MgCl2, 2% DMSO, 200 mM each deoxynucleoside triphosphate, 20 pmol of the primers, 1 U Taq DNA polymerase (all reagents from Gene Craft, Germany), and 5 L of DNA. The thermal profile involved 45 cycles with the following steps: initial denaturation for 5 minutes at 95°C followed by 20 seconds of denaturation at 95°C, 1 minute of annealing at 55°C, and 40 seconds of extension at 72°C. The presence of amplified products was confirmed by 2% agarose gel electrophoresis. Restriction Digestion and Analysis of Restriction Patterns To digest the PCR product, 2 restriction enzymes (BstE II and Hea III) were used as described by Telenti et al.17 Briefly, 5 L of PCR product was added to a mixture containing 1 L (10 U/ L) of enzyme, 1 L of appropriate restriction buffer (10⫻), and 2 L of sterile distilled water. The mixture was incubated at 37°C for 2 hours. Restriction fragments were electrophoresed on a 10% polyacrylamide gel at 150 V for about 2 hours until the dye front migrated to approximately 1 cm from the end of the gel. Fragment band sizes were estimated on comparison with 100-bp ladder as molecular size standard. The estimated bands were compared with algorithm for differentiation of strains of Mycobacteria. Mycobacterium strains were reference strains from the American Type Culture Collection or from the collection of the Italian Reference Laboratory for Mycobacteria and clinical isolates from the Mycobacteriology Laboratory of Verona Hospital.18 © 2009 Lippincott Williams & Wilkins
8 (66.7%) 1 (8.3%) 1 (8.3%) 2 (16.7%)
In countries with high burden of tuberculosis, the isolated mycobacterium from the suspected individuals is most often MTB. Although the prevalence of NTM varies in different countries, it is proved to be more common among the HIV-positive patients.13 And most of the studies reported on NTM have been performed on HIV-positive patients.19,20 However, all cases in this study were negative for HIV infection. NTM can cause clinical and radiologic manifestations similar to MTB. As it is not feasible to microscopically distinguish between MTB and NTM, in the case that sputum culture and identification tests are not performed, some NTM cases may be misdiagnosed and treated as TB. Fundamentally, NTM treatment is different from TB treatment. Consequently, most
TABLE 2. Radiologic findings in chest x-rays of nontuberculous mycobacterial patients Nodularity Bilateral Unilateral No Cavitary lesions Bilateral Unilateral No Bronchiectasis Focal Multifocal
10 (83.3%) 1 (8.3%) 1 (8.3%) 0 5 (41.7%) 7 (58.3%) 3 (25%) 9 (75%)
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of these patients will not show favorable response to anti-TB therapy. Moreover, as NTM isolates are frequently resistant to first-line anti-TB medications, this may lead to the problem that these cases be identified and treated as MDR-TB cases. Particularly, this is more probable in countries with limited resources, where mycobacteriologic culture and identification tests are not routinely performed. The current study demonstrated that a significant portion (11.43%) of referred patients as suspected MDR-TB were in fact NTM cases. This indicates that distinguishing between MTB and NTM cases is of significant importance as: 1. MDR-TB treatment is costly and time-consuming (24 months). Thus, identifying NTM cases prevents unnecessary expenditure of money and healthcare resources. 2. Considering the adverse effects of second-line anti-TB medications,21 identifying NTM, and administering the proper treatment is quite important. 3. As resources are limited to take isolation measures, correct diagnosis can aid in the beneficial usage of such resources. In this study, the most common isolated mycobacteria belonged to the rapid-growing mycobacteria of Chelonae family. Interestingly, it has been shown in a study that 1 of the risk factors associated with acquiring rapid-growing mycobacterium is previous infection with other mycobacteria.1 Because the patients in our study did not have sputum culture samples at the commencement of treatment, it is impossible to prove that fast-growing mycobacterium is the cause of their initial clinical syndrome. On the other hand, it is still possible that after a primary infection with MTB, the patients have caught superinfection with NTM. Another important finding in this study is the existence of cavitary lesions in 41.7% of the patients. This demonstrates that the presence of lung cavities, even in endemic areas, does not necessarily indicate an MTB infection and NTM must also be considered as a potential differential diagnosis. The current study was conducted at a referral center and among patients suspected of MDR-TB. Consequently, these findings may not accurately reflect the exact prevalence of NTM among patients treated for TB in the community. Nonetheless, the study indicates that in resource-limited settings, before commencing MDR-TB treatment and in addition to the drug susceptibility tests, mycobacterium identification must be performed. Additionally, to prevent unnecessary expenditure of time and resources, this study recommends that culture be obtained from all TB cases and identification tests be performed on all NTM-suspected cases.
CONCLUSION NTM cases encompass 11.43% of the suspected MDR-TB patients referred to our referral center and hence should be contemplated in treatment failures. With regard to radiologic manifestations, existence of lung cavitary lesions in 41.7% of cases was noticeably significant. Moreover, in resource-limited settings, mycobacterium identification before MDR-TB treatment initiation is recommended. REFERENCES 1. Griffith DE, Aksamit 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 2003;175: 367– 416.
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2. McNabb A, Eisler D, Adie K, et al. Assessment of partial sequencing of the 65-kilodalton heat shock protein gene (hsp65) for routine identification of Mycobacterium species isolated from clinical sources. J Clin Microbiol 2004;42:3000 –11. 3. Tortoli E. Impact of genotypic studies on mycobacterial taxonomy: the new Mycobacteria of the 1990s. Clin Microbiol Rev 2003;2:319 –54. 4. Lind A, Larsson LO, Bentzon MW, et al. Sensitivity to sensitins and tuberculin in Swedish children. I. A study of schoolchildren in an urban area. Tubercle 1991;72:29 –36. 5. Reznikov M, Robinson E. Serologically identical Battey mycobacteria from sputa of healthy piggery workers and lesions of pigs. Aust Vet J 1970;46:606 –7. 6. von Reyn CF, Waddell RD, Eaton T, et al. Isolation of Mycobacterium avium complex from water in the United States, Finland, Zaire, and Kenya. J Clin Microbiol 1993;1:3227–30. 7. Meissner G, Anz W. Sources of Mycobacterium avium-complex infection resulting in human disease. Am Rev Respir Dis 1997;116: 1057– 64. 8. Ahrens P, Giese SB, Klausen J, et al. Two markers, IS901-IS902 and p40, identified by PCR and by using monoclonal antibodies in Mycobacterium avium strains. J Clin Microbiol 1995;33:1049 –53. 9. Guerrero C, Bernasconi C, Burki D, et al. A novel insertion element from Mycobacterium avium, IS1245, is a specific target for analysis of strain relatedness. J Clin Microbiol 1995;33:304 –7. 10. Tanaka E, Kimoto T, Matsumoto H, et al. Familial pulmonary Mycobacterium avium complex disease. Am J Respir Crit Care Med 2000;161:1643–7. 11. Horsburgh CR Jr. Epidemiology of Mycobacterium avium complex. In: Korvick JA, Benson CA, editors. Mycobacterium avium complex infection: progress in research and treatment. New York (NY): Marcel Dekker; 1996. p. 1–22. 12. World Health Organization. Treatment of tuberculosis. Guidelines for national TB programmes, 3rd ed. WHO/CDS/TB/2003.313. Geneva, Switzerland: WHO; 2003. 13. World Health Organization. Guidelines for the programmatic management of drug resistance tuberculosis. WHO/HTM/TB/2006.361. Geneva, Switzerland: WHO; 2006. 14. Holt JG, Krieg NR, Sneath PHA, et al, editors. Bergey’s manual of systemic bacteriology. Baltimore: Williams & Wilkins; 1994. p. 597. 15. Kent PT, Kubica GP. Public health Mycobacteriology: a guide for the level III laboratory. Atlanta (GA): Department of Health and Human Services, Centers for Diseases Control; 1985. p. 36 –138. 16. van Soolingen D, Hermans PW, de Haas PE, et al. Occurrence and stability of insertion sequences in Mycobacterium tuberculosis complex strains: evaluation of an insertion sequence-dependent DNA polymorphism as a tool in the epidemiology of tuberculosis. J Clin Microbiol 1991;29:2578 – 86. 17. Telenti A, Marchesi F, Balz M, et al. Rapid identification of mycobacteria to the species level by polymerase chain reaction and restriction enzyme analysis. J Clin Microbiol 1993;31:175– 8. 18. Brunello F, Ligozzi M, Cristelli E, et al. Identification of 54 Mycobacterial species by PCR-restriction fragment length polymorphism analysis of the hsp65 gene. J Clin Microbiol 2001;39:2799 – 806. 19. Matos ED, Santana MA, de Santana MC, et al. Nontuberculosis mycobacteria at a multiresistant tuberculosis reference center in Bahia: clinical epidemiological aspects. Braz J Infect Dis 2004;8:296 –304. 20. Kim SJ. Drug-susceptibility testing in tuberculosis: methods and reliability of results. Eur Respir J 2005;25:564 –9. 21. Iseman MD. Treatment of multidrug-resistant tuberculosis. N Engl J Med 1993;329:784 –91.
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