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Journal of Islamabad Medical & Dental College (JIMDC); 2015:4(1):42-47 Review Article An Update on the Laboratory Diagnosis of Tuberculosis 1 Khurshid Ahmad1, Farhat Khurshid2 Consultant Microbiologist, Islamabad Diagnostic Centre; Scientist Emeritus, National Institute of Health, Islamabad 2 Department of Microbiology, Islamabad Diagnostic Centre, Islamabad fundamental epidemiological attributes, mycobacterial species are divided into two major groups: Mycobacterium tuberculosis complex (MTB Complex). Comprises 3 major species: o Mycobacterium tuberculosis o Mycobacterium bovis (including M. bovis BCG) o Mycobacterium africanum The Non-tuberculous Mycobacteria (NTM) – also called Atypical Mycobacteria : All mycobacterial species other than MTB complex have been included in this group.6 Species belonging to Mycobacterium tuberculosis complex occur in humans (and animals); although all three are capable of causing disease, Mycobacterium tuberculosis is the predominant species responsible for tuberculosis in humans. Differentiation of these three species for routine microbiological diagnosis is not only difficult and complicated but also of little or no medical importance in the diagnosis and clinical management of the disease except when required for epidemiological and public health purposes.7 Tuberculosis primarily affects the lungs. Infection occurs by inhalation of infectious airborne droplet nuclei containing Mycobacterium tuberculosis exhaled during coughing and sneezing (even speaking and singing) by “open” cases of TB lungs. Therefore pulmonary tuberculosis is by far the commonest form of disease produced by these organisms; also it has probably the smallest infective dose of any infectious disease – inhalation of a single viable organism of MTB complex has been shown to lead to infection.7 In a small percentage of infected patients, extra-pulmonary TB occurs which may affect the lymph nodes, gastrointestinal tract, central nervous system (meningitis), bones and joints, peritoneum, genitor-urinary tract, pericardium etc. Outcome of primary infection with MTB complex is variable; a) in the majority the cellular immune system of the host is able to contain the organisms; bulk of the organisms is killed; those that survive are surrounded by T cells and macrophages to form granulomas which limit their multiplication and spread. After a variable period of time 15-20% of these people develop clinical tuberculosis. b) In others the mycobacteria do not produce disease but lie within the granulomas in a dormant state for years and decades. Such people have a latent TB infection (LTBI) Introduction Tuberculosis is one of the most serious diseases afflicting mankind since times immemorial; it has been present in all ages and climates.1 Using the most modern DNA techniques, presence of Mycobacterium tuberculosis has been confirmed in the lesions from the lungs of a 1000-year old mummy in Peru.2 Recent decades have witnessed resurgence in tuberculosis, both in the developing as well as in developed countries – more people are currently dying of TB than at any other time in human history. According to WHO estimates, one third of the entire human population is presently infected with TB, most of it in developing countries; a person is newly infected every second. Up to 8 million individuals develop active TB infection, with 2.9 million deaths, annually. Among the 22 countries that have the highest burden of TB cases in the world Pakistan is listed in the top eight.3 Prevalence of pulmonary tuberculosis in Pakistan among adults over the age of 15 years stands confirmed at 295 per 100,000 populations.4 At least 400,000 new TB cases occur every year including an alarming 15000 cases of multi-drug resistant tuberculosis. These disturbing statistics call for a redoubling of efforts to properly diagnose and treat all those who have been infected with tuberculosis. Mycobacteria Microorganisms belonging to the genus Mycobacterium are thin, non-motile, non-sporing and non-capsulated rods measuring 0.2-0.4 x 2-10um. Three characteristic features differentiate these organisms from other pathogenic bacteria: a) they cannot be stained by the classical Gram stain, b) they are acid-fast – once stained, they resist decolourisation by acid - hence the name Acid Fast Bacilli or AFB; both these characteristics owe their occurrence to the high lipid content of the cell wall, and c) they have extremely slow growth in culture – 2-8 weeks as opposed to 18-24 hours for other common pathogens. Currently the genus Mycobacterium comprises more than 70 recognized species.5 Based on their Corresponding Author: Dr Khurshid Ahmad Email: [email protected] association with human or animal disease and on their 42 Journal of Islamabad Medical & Dental College (JIMDC); 2015:4(1):42-47 but not clinical tuberculosis. c) A variable percentage of primary infections (up to 5%) develop directly into progressive primary tuberculosis. Thus tuberculosis can be active which is symptomatic and highly infectious or it can be latent and inactive without any symptoms. Individuals with latent TB infection are at the risk of developing active disease whenever their immune status changes (e.g. due to immunosuppressive therapy or HIV infection) or clinical circumstances are altered. Each person having a LTBI has about 10% chance of progressing to active disease. successive days are usually recommended for detection of AFB by smear microscopy; at least one, if not all three, must be early morning sample. Other pulmonary specimens may include broncho-alveolar lavage, broncho-alveolar brushings and trans-tracheal aspirate. Extra-pulmonary Tuberculosis Most common specimens from cases of extra-pulmonary tuberculosis consist of body fluids (including pleural, peritoneal, pericardial, synovial and CSF), urine and pus. Others include lymph nodes, bone marrow, gastric aspirate and tissues. From cases of renal tuberculosis, mid-stream urine is collected on three successive days. Specimens are transported to the laboratory immediately, at most within 2 hours of collection. In case of delay, they should be refrigerated. Tuberculosis and HIV/AIDS HIV/AIDS infection has played a crucial role in the resurgence of tuberculosis particularly in Africa and South Asia. TB and HIV/AIDS are fatally synergistic. Immunity to MTB complex is primarily cell-mediated. Because of suppressive effect of HIV on cell mediated immune function, co-infection with HIV greatly increases the risk of developing active tuberculosis with rapidly developing primary disease, instead of latent infection, and markedly accelerates its progress.8 WHO estimates that up to 40% of AIDS patients in Africa and South Asia die of tuberculosis. 3 Diagnostic Modalities Currently Available The clinical microbiology laboratory is faced with the challenge of providing a rapid, reliable and cost effective diagnosis of both active and latent tuberculosis. Techniques presently available include the following: Safety Precautions Organisms of MTB complex carry a very high risk of laboratory acquired infection (Hazard Group 3). Even the simplest procedures like making AFB smears should be undertaken using disposable gloves and a face mask. Extreme care should be taken when sterilizing the inoculating loop after making smear. Any amount of sputum remaining in the loop spurts in the flame and may create aerosols. This maneuver should be performed using a hooded flaming device. It is safer to use a tooth pick instead of wire loop which can be discarded in disinfectant without flaming. Beyond this, all procedures on TB specimens should be carried out in a bio-safety level 2 facility (Class II safety cabinet). WHO further advises that all laboratories handling TB cultures, must have Biosafety level 3 (BSL-3) facilities. Conventional techniques 1 AFB Smear Microscopy 2. Tuberculin Skin Test (TST) – (Mantoux test) 3. AFB Culture – Lowenstein-Jensen (LJ) solid medium New technologies 1. BACTEC MGIT 960 Culture System (Becton Dickinson, USA) 2. Interferon Gamma Release Assays – IGRAs a. Quantiferon TB Gold b. T-SPOT.TB 3. Nucleic acid Amplification Tests (NATs) - PCR 4. Bacteriophage-based Tests – FASTPlaqueTB Serological tests 1. Rapid Immunochromatographic assays (ICTs) 2. Enzyme-linked Immunosorbant Assays (ELISA) Except for the basic procedures of smear microscopy and tuberculin skin test, most of the advanced technologies, including culture, conventional as well as MGIT, described herein belong to the domain of specialized tuberculosis laboratories. Collection and Transport of Clinical Specimens Successful detection and isolation of MTB complex depends on the type and quality of the specimen obtained from the patient which in turn is determined by the clinical presentation of the case. Pulmonary tuberculosis Spontaneously produced early morning sputum from a deep and vigorous cough is the specimen of choice. It should be collected in a clean (not necessarily sterile), dry, widemouthed, leak-proof container and should be free from saliva. Patients should be asked to cover their mouths carefully during sputum collection or they should be asked to collect it in a bath room. Up to three specimens on three AFB Smear Microscopy In Pakistan and other resource poor countries with a high TB prevalence, smear microscopy continues to be the mainstay of TB diagnosis. Because of its simplicity and minimum requirements in terms of equipment and supplies, 43 Journal of Islamabad Medical & Dental College (JIMDC); 2015:4(1):42-47 it is by far the most widely used procedure in TB diagnostics not only in detecting TB cases but also in establishing their infectiousness. AFB smear microscopy may be performed directly or after concentration. If positive, result should be reported only as “Acid Fast Bacilli seen” with quantification/grading as follows (WHO / CDC): > 9 AFB / oil field (++++) 1 – 9 AFB / oil field (+++) 1 – 9 AFB / 10 oil fields (++) 1 – 9 AFB / 100 oil fields (+) 1 – 2 AFB in the entire smear Doubtful. Repeat on fresh specimen Direct smears A loopful from a thick purulent portion of the sputum is spread evenly on a slide to make a smear taking care that it should neither be too thin nor too thick. A slide which has already been used for sputum smear and washed should never be reused for making a fresh smear. The smear is airdried and fixed by gently heating the slide on the flame. Sensitivity and specificity of AFB smear microscopy An AFB stained smear from clinical specimens requires at least 5000-10000 bacilli per ml for detection. In skilled and experience hands, overall sensitivity and specificity of smear microscopy is excellent – up to 80% and 100% respectively.11,12 There have been suggestions that sputum smear microscopy may be more reliable for evidence-based diagnosis of pulmonary tuberculosis than X-ray chest which may often result in over-diagnosis. Use of direct ZN microscopy for spinal, pleural and other body fluids for detection of AFB is an unrewarding exercise; some experts consider it a wasteful use of time and energy as these samples are rarely positive. Many laboratories have stopped reporting direct AFB smears on CSF and other effusions.7 However a highly specialized test, adenosine deaminase level, is now available for the reliable diagnosis of tuberculous effusions particularly the pleural and ascetic effusions.13 Concentration method: AFB can be concentrated by centrifugation after treating the sputum with an equal volume of 5% sodium hypochlorite (commercial bleach) for 10-15 minutes which also renders the specimen nonConcentration technique significantly infectious.9,10 increases the sensitivity of smear microscopy (by up to 50%) for the detection of AFB in sputum . Staining Smears are stained by the standard Zeihl-Neelsen technique or by Auramine-O stain (if facilities for fluorescent microscopy are available). ZN staining is a hot stain procedure as it requires heating the slide for better penetration of stain into the mycobacterial cell wall. AFB Fluorescent microscopy Fluorescent staining using auramine O stain is more sensitive than conventional ZN staining but this advantage is more than offset by the high cost of fluorescent microscope and reagents as well as the requirement of highly trained and experienced staff for interpretation. It has been strongly recommended that all positive results of auramine staining must be confirmed either by a second trained observer or by staining the same smear with ZN stain.7,12,14 Examination, interpretation and reporting of AFB smears ZN stained smears are examined under oil immersion. While applying oil on the smear the slide should never be touched with oil applicator or the nozzle of oil-containing bottle. A small oil droplet should be dropped on the smear without touching it. Examination of smears should be carried out by scanning at least 300 oil immersion fields (roughly equivalent to 3 horizontal sweeps over an area 2 cm x 1 cm) before giving a negative result.7 Oil should be wiped off from the objective before examining the next smear, especially in case of a positive smear; AFBs from a positive smear may come off the slide and float in the oil remaining on the objective and may give a false positive result while examining the next smear. In a well stained smear, typical acid fast bacilli, when present, appear as red or purple, slightly curved rods, 2-8 um long, against a crisp blue background of mucous threads and debris. Bacilli of MTB complex often appear beaded or banded (Fig.1). Tuberculin Skin Test (TST) – Mantoux test 15 Next to smear microscopy, tuberculin skin test, in spite of its limitations, is probably still the most commonly performed test for detection of tuberculosis. Introduced by Robert Koch nearly 100 years ago, it is still used as a standard screening test in the United State for latent TB infection. Reaction is based on the premise that infection with MTB complex elicits delayed type hypersensitivity based on cell mediated immune response to antigenic components of MTB complex. In the standard Mantoux skin test, 0.1 ml of purified protein derivative (PPD) from tubercle bacilli containing 5 tuberculin units is injected intradermally on the forearm. After 72 hours, the site is examined for induration (area of firmness as a result of influx of immune cells). The diameter of induration (not erythema which is usually present with or without induration) is measured. Result is Reporting The following information should be included in the final report: Specimen quality (Thick, mucoid, muco-purulent, muco-salivary, blood stained etc), staining method used (ZN stain), quantification or grading of result. 44 Journal of Islamabad Medical & Dental College (JIMDC); 2015:4(1):42-47 reported according to the interpretative criteria given in table 1. A positive Mantoux skin test establishes exposure to tuberculosis but does not distinguish between active and latent infection. Infection with non-tuberculous mycobacteria and BCG vaccination may yield a false positive tuberculin skin test; BCG effect may last for several years. On the other hand a negative skin test does not rule out the presence of tuberculosis. instrument monitors fluorescence every 60 minutes and gives a signal when detected. Cultural methods Specimen processing: Sputum and other specimens from sites containing resident flora require decontamination and digestion before culture. 4% NaOH or a mixture of NaOH and N-acetyl l-cystein – NALC (a mucolytic agent) not only decontaminates but also digests and liquefies the mucoid material and organic debris which may wall off AFBs from nutrients in the medium.16,17 Specimens from sterile sites do not require decontamination. They should only be concentrated by centrifugation. AFB smear microscopy Table 2: Summary of diagnostic procedures for tuberculosis Test Culture Medium 0 – 4 mm 5 – 9 mm = / > 10 mm All individuals - Healthy individuals - Close contacts of active TB cases - HIV positive individuals - Injecting drug users All individuals 4-8 weeks 11-13 days 48-72 hours Comments Mainstay of TB diagnosis; rapid, simple, cost-effective Gold standard for TB diagnosis Does not distinguish between active & latent TB; mainly used to evaluate a person who has symptoms of TB or for detecting LTBI in asymptomatic persons; false negative & false positives (after BCG and NTM infection) may occur As above but highly IFN-γ release assays 12-24 hours sensitive & specific; (IGRAs) – most reliable test for T-SPOT.TB, LTBI, not affected by Quantiferon TB BCG and most NTMs; but costly & labour intensive Technically demanding, Bacteriophage based 2 days assay -FASTplaque can only be used for TB sputum specimens. Few Highly sensitive and NA amplification hours specific but technically tests – PCR complex and expensive 20 - 30 Inconsistent results – Rapid Serological tests- ICTs minutes not endorsed by WHO Conventional ELISA 2-3 Insufficient evidence of hours diagnostic accuracy A schematic representation of the processing of sputum by smear microscopy and AFB culture is given in Figure 2. Footnote: ICT: Immuno-chromatographic Test, IGRA: Interferon Gamma Release Assay, LJ: Lowenstein-Jensen Medium, LTBI: Latent Tuberculosis Infection, MGIT: Mycobacterial Growth Indicator Tube, NTM:: Nontuberculous Mycobacteria, PCR: Polymerase Chain Reaction, TST: Tuberculin Skin Test. BACTEC MGIT (Mycobacterial Growth Indicator Tube) consists of modified Middlebrook 7H9 broth medium which is known to yield better recovery and faster growth of MTB complex.18 Table 1: Interpretation of Mantoux skin test (5 tuberculin units) Person tested LJ Bactec MGIT Tuberculin Skin Test (TST) – Mantoux test Conventional AFB Culture A definitive diagnosis of tuberculosis requires culture which is still considered to be the gold standard for laboratory diagnosis of tuberculous disease. It is much more sensitive than microscopy, being able to detect as low as 10 viable tubercle bacilli per ml of specimen. However, the conventional culture on egg-based solid LJ medium has a long detection time, taking anywhere from 4 to 8 weeks for the growth to appear. Area of induration Turnaround time <2 hours Result Negative Borderline positive Positive Positive Positive Positive A fluorescent compound embedded in silicon sensor at the base of the tube monitors oxygen consumption by growing AFB if present in the specimen. Growth of AFB and resulting O2 depletion is detected as an increase in fluorescence. Fluorescence can be detected visually in UV light, manually by Mini-MGIT or automatically by the MGIT 960 system (Beckton Dickensen, USA) in which the Mean detection time in the MGIT system is 11 – 13 days. Growth is confirmed by ZN staining of a smear from the 45 Journal of Islamabad Medical & Dental College (JIMDC); 2015:4(1):42-47 culture tube which will show AFBs in groups or cords (cording). Interferon Gamma Release Assays – IGRAs Interferon-gamma release assays (IGRAs) are based on the principle that the T-cells of individuals who have acquired TB infection respond to re-stimulation with M. tuberculosis by secreting interferon gamma (IFN-γ). These assays employ specific antigens of M. tuberculosis (ESAT-6 & CFP-10) which are shared neither by BCG strains nor (with rare exceptions) by non tuberculous mycobacteria, making these assays virtually specific for MTB complex. The QuantiFERON-TB Gold (Cellestis U.K. - [email protected]) measures the IFN-γ produced in response to these M. tuberculosis antigens in whole blood.19,20 In contrast, the TSPOT.TB assay (Oxford Immunotec UK) detects the number of “spot forming effector T cells” in peripheral blood which produce IFN-γ in response to the above antigens (Fig 3). Like Mantoux skin test, IGRAs do not distinguish between active and latent TB infection. However unlike skin test, these assays are not affected by BCG and by most non-tuberculous mycobacteria. 21-24 Nucleic acid Amplification Tests (NATs) - PCR NATs have been successfully used for the direct detection of MTB in sputum and blood.25 In spite of being rapid, highly sensitive and specific, these tests suffer from a number of limitations: they are too expensive to be used in countries where they are most needed, require special expertise and may not be specific for active infection as DNA from a dead organism can be amplified and detected by PCR with equal efficiency. Assays from Roche and GenProbe have been approved by FDA for use on AFB-smearpositive specimens only because of less than optimum sensitivity on smear-negative specimens. Recently WHO has endorsed a NA assay for TB control programmes in developing countries by the name of GeneXpert MTB/RIF.3. It is an automated, cartridge-based NA amplification assay specifically for the simultaneous detection of MTB complex as well as rifampicin resistance directly from sputum samples in less than two hours. Fig. 1: Sputum smear strongly positive for AFB – ZN stain Sputum Direct Concentration Decontaminate & liquify 4% NaOH (or NaOH + NAC) Neutralise (1N-HCl) AFB smear Microscopy (ZN stain) Centrifuged deposit Inoculate Bactec MGIT Incubate 37oC – 42d LJ Slope Incubate 37oC 6-8 wks If there is growth confirm by AFB smear microscopy Fig. 2: Suggested flow diagram for processing of sputum samples for AFB Bacteriophage-based assay – FASTplaque TB FASTPlaque TB (Biotec UK) utilizes the ability of a specific mycobacteriophage (Actiphage) to lyse and destroy viable MTB complex in decontaminated sputum. The test takes 48 hours to complete but is technically demanding and can only be used for sputum specimens.10 Serological tests Antibody-based serological tests for the detection of antibodies to MTB complex, including immunochromatographic assays and ELISA, are simple to use, comparatively inexpensive and easily available. Despite widely being used in routine laboratories, these tests have important limitations as clearly stated in the following WHO recommendations.26 “It is strongly recommended that these commercial tests not be used for the diagnosis of pulmonary and extra-pulmonary diagnosis. Fig. 3: T-SPOT.TB test showing “spot forming effector T cells”. Each spot represents an IFN-γ-producing T cell in response to TB antigens 46 Journal of Islamabad Medical & Dental College (JIMDC); 2015:4(1):42-47 Currently available commercial sero-diagnostic tests (also referred to as serological tests) provide inconsistent and imprecise results. There is no evidence that existing commercial serological assays improve patient outcomes, and high proportions of false positive and false negative results may have an adverse impact on the health of the patients.” A summary of diagnostic procedures for tuberculosis and their clinical correlations is given in table 2. 12. Lipsky BJ, Gates J, Tenover FC. Factors affecting the clinical value of microscopy for acid fast bacilli. Rev Infect dis 1984;6:214 13. Banales JL , Pineda PR, Fizgerald JM. Adenosine deaminase in the diagnosis of tuberculous pleural effusions: a report of 218 patients and review of literature. Chest 1991; 99:355 14. Steingart K, Henry M, Ng V, et al. Fluorescence versus conventional sputum smear microscopy for tuberculosis: A systematic review. Lancet Infectious Diseases 2006; 6(9):570-81. 15. Huebner RE, Scheun ME, Bass JB Jr. The tuberculin skin test. Clin Infect Dis 1993; 17:968 16. Sommers HM, Good RC. Mycobacterium. In Lennette EH, Balows A, Hausler WJ, Shadomy HJ editors: Manual of Clinical Microbiology, 4th ed. 1985. Washington DC; American Society of Microbiology: pp 216 – 248. 17. Yajko DM, Nassos PS, Sanders CA et al. Comparison of four decontamination methods for recovery of Mycobacteria. J Clin Microbiol 1993;31:302 18. Anargyros P, Astill DS, Lim IS. Comparison of improved BACTEC and Lowenstein Jensen media for culture of Mycobacteria from clinical specimens. J Clin Microbiol 1990; 142: 725. 19. Centres for Disease Control and Prevention. Guidelines for using the QuantiFERON-TB test for detecting States. Mycobacterium tuberculosis infection, United MMWR Morb Mortal Wkly Rep 2005; 54 (RR-15):4955. 20. Kobashi Y, Obase Y, Fukuda M at al. Clinical evaluation of the QuantiFERON TB-2G test as a diagnostic method for differentiating active tuberculosis from non-tuberculous mycobacteriosis. Clin Infect Dis 2006; 43: 1540-1546. 21. Lalvani A, Pathan AA, Durken H et al. Enhanced contact tracing and spatial tracking of Mycobacterium tuberculosis infection by enumeration of antigen-specific T-cells. The Lancet 2001; 357 : 2017-21. 22. Ewer K, Decks J, Alvarez L et al. Comparison of a T-cellbased assay with tuberculin skin test for diagnosis of Mycobacterium tuberculosis infection in a school tuberculosis outbreak. The Lancet 2003; 36:11681173. 23. Brodie D, Lederer DJ, Gallardo JS et al. Use of an interferongamma release assay to diagnose latent tuberculosis infection in foreign-born patients. Chest 2008;133: 869874. 24. World Health Organisation. Use of tuberculosis interferongamma release assays (IGRAs) in low- and middle-income countries: policy statement. 2011; ISBN 978 92 4 150267 2 (NLM classification: WF 220): Geneva, World Health Organisation. 25. Forbes BA. Current and future applications of mycobacterial amplification assays. Clin Microbiol Newsletter 1995;17:145 26 World Health Organization. Laboratory Based Evaluation of 19 commercially available rapid diagnostic tests for tuberculosis (Diagnostics Evaluation Series No 2); Policy statement 2011: Geneva; World Health Organisation, Acknowledgments We are grateful to Dr. Aftab Ahmad for the use of his excellent camera microscope for taking picture of AFB smear (Fig. 1) and to Mr. Najam Farooq for providing picture of a positive T-Spot.TB test performed at Islamabad Diagnostic Centre (Fig. 3) References 1. Bates JH, Stead WW. The history of tuberculosis as a global epidemic. Med Clin North Am. 1993; 77:1205. 2. Salo WL, Aufderheide AC, Buikstra J. Identification of Mycobacterium tuberculosis DNA in a pre-Columbian Peruvian mummy.. Proc Natl Acad Sci 1994 USA; 91:2091. 3. World Health Organisation Global Tuberculosis Report 2014. Geneva: World Health Organisation. www.who.int/tb/publications/global_report/en/ 4. National TB prevalence survey – Preliminary Report 2012. National TB Control Programme (NTCP); Ministry of InterProvincial Coordination, Islamabad. Pakistan. 5. Shinnick TM , Good RC. Mycobacterial taxonomy. Eur J Clin Microbiol Infect Dis. 1994; 13:884 6. Debrunner M, Salfinger M, Brandli O et al. Epidemiology and clinical significance of non-tuberculous mycobacteria. Clin Infec Dis 1992;15:330 7. Forbes BA, Sahm DE, Weissfeld AS. Mycobacteria. In Bailey and Scott’s Diagnostic Microbiology, 6th Ed., 2002. St. Louis. Mosby; pp 538 - 571 8. Snider DE, Roper WL. The new tuberculosis. N Eng J Med 1992; 326:703 9. Krasnow I, Wayne LG. Comparison of methods for tuberculosis bacteriology. Appl Microbiol 1969;18:915. 10. Cheesbrough M. Distric Laboratory Practice in Tropical Countries, Part 2. Cambridge University Press; 2000; pp 73 11. Centres for Disease Control and Prevention: In Kent PT and Kubica GP, editors: Public Health Mycobacteriology 1995. Atlanta, US Department Health and Human Services, Public Health Service. 47