Indian Journal of Tuberculosis IMPORTANCE OF BLOOD SAMPLES FOR DIAGNOSIS AND DRUG SENSITIVITY TESTING IN HIV POSITIVE PATIENTS WITH SUSPECTED TUBERCULOSIS Original Article (Original Received on 30.6.2003; Revised version received on 27.10.2003; Accepted on 30.10.2003) Summary: Background: Diagnosis of tuberculosis is difficult in HIV positive patients since they often present with atypical symptoms and are susceptible to pulmonary infections that mimic tuberculosis. Sputum collection may not be possible even in patients with pulmonary involvement since a productive cough is not always present. In such patients, blood smear and culture for AFB apart from serving as a diagnostic tool can be used for testing drug sensitivity. Objectives: This study was undertaken to explore the value of blood culture for diagnosis in patients with suspected TB. In addition, a comparison of drug sensitivity patterns of blood and sputum isolates in 10 of these patients was also carried out. Methods: Blood and sputum samples were processed, cultured and isolates tested for their drug susceptibility and for niacin production, nitrate reduction as well as catalase activity at 68o C. Results: All 24 blood samples were culture positive although only 6 were smear positive. On the basis of the biochemical investigations, 22 strains were identified as Mycobacterium tuberculosis. All the 10 sputum samples were culture positive despite 4 being smear negative. Comparison of drug sensitivity profiles from blood and sputum revealed concordance to five first or second line drugs in 5 of 10 patients. Additionally, 2 patients demonstrated discordance for only one first or second line drug. Conclusion: The study demonstrates the importance of blood culture in confirming diagnosis of tuberculosis and testing for drug sensitivity in HIV positive patients without a productive cough. The level of discordance in drug sensitivity profiles between blood and sputum the same individual is suggestive of infection with multiple strains. Testing for the occurrence of multistrain infections through individual colony examination of a single isolate is necessary since such infections would affect treatment of non-responder patients having HIV-TB dual infections. Key Words: HIV and TB, Diagnosis of, Drug sensitivity Testing 1. Scientific Assistant ‘C’* 2. Senior Research Officer* 3. Clinical Consultant*,** 4. Professor-Director**,*** 5. Director and Trustee* * Foundation for Medical Research, Mumbai ** AIDS Research and Control (ARCON) Centre, Mumbai *** University of Texas, Houston, USA Correspondence: Dr. Tannaz J. Birdi,Senior Research Officer, Foundation for Medical Research, 84-A R.G.Thadani Marg, Worli, Mumbai 400 018. E-mail : frchbom@bom2.vsnl.net.in INTRODUCTION Tuberculosis(TB) is the most common opportunistic infection in persons with HIV infection. It is estimated that around 15% of the TB incidence occurs among such individuals. The most striking clinical feature of TB in HIV positive patients is the high frequency of extrapulmonary involvement, often with concomitant pulmonary tuberculosis. However, recognition of TB can be difficult in these patients since they often present with atypical symptoms and are also susceptible to pulmonary infections that may mimic TB1. Sputum collection may not be possible even in patients with pulmonary involvement since a productive cough is not always present. The World Health Organization has reported a global prevalence of 14% of drug resistant tuberculosis2. Drug resistant TB is a serious concern among HIV positive patients in whom mortality from TB can be as high as 80%3. In such patients blood smear and culture, apart from serving as a useful diagnostic tool, can be used for testing drug sensitivity. A study was undertaken primarily to determine the value of blood culture in diagnosis of [Indian J Tuberc 2004; 51:77-81] Desiree T.B. D’souza1, Tannaz J. Birdi2, Yatin Dholakia3, Subhash Hira4 and Noshir H. Antia5 ,, Indian Journal of Tuberculosis TB in a case series of 24 patients with suspected pulmonary and extra-pulmonary TB. In addition, a comparison of drug sensitivity patterns between blood and sputum isolates in 10 of these patients, from whom both sample types were available, was carried out. MATERIAL & METHODS Patients HIV positive patients attending the AIDS Research and Control (ARCON) Centre’s out-patient department for HIV infected individuals at Sir J. J. Hospital, Mumbai over a period of six months were taken up for the study. These patients were on regular follow-up to assess HIV disease progression. They were considered for evaluation for M. tuberculosis infection on the basis of the presence of one or more of the following symptoms: prolonged fever, marked weight loss, cough for more than 3 weeks not responding to broad spectrum antibiotics, symptoms related to specific organ systems. The diagnosis of pulmonary TB was confirmed on the basis of chest radiographs and/or sputum examination for AFB. Investigations in patients suspected to be suffering from extra- pulmonary TB included complete blood counts, erythrocyte sedimentation rate, Mantoux test, fine needle aspiration cytology (FNAC) / biopsy of accessible peripheral lymph nodes examined by histopathology and Ziehl Neelsen stain, cytochemistry and bacteriological examination of body fluids like pleural, peritoneal or cerebrospinal fluid and ultrasound examination of the abdomen. On the basis of these investigations, 24 patients suspected to be suffering from TB were included in the study. Twelve patients had pulmonary symptoms while the remaining 12 patients did not. Chest radiographs were taken for all 24 patients. Blood was collected from all the 24 TB suspects. Sputum could be collected from only 10 patients since the remaining 14 patients did not have a productive cough. Blood and/ or sputum samples were collected once, on suspicion of TB, before starting therapy. Processing of blood samples Ten ml of heparinized blood was collected from each patient and processed with modifications to the method described by Saltzman et al4. Briefly, an equal volume (i.e. 10 ml) of lysis buffer (50 mM Tris + 75 mM sucrose pH 8) was added to the heparinized blood. Following a 2 min incubation with lysis buffer an equal volume (i.e. 20 ml) of sterile Minimal Essential Medium (MEM) (Gibco) with 10% Fetal Calf Serum(FCS) (Gibco) was added. The sample was centrifuged at 500 rpm for 5 min and the pellet consisting of cell debris discarded. The supernatant was recentrifuged at 2000 rpm for 20 min, the pellet washed and resuspended in 2 ml of MEM containing 10% FCS. Processing of sputum samples Sputum samples collected in sterile SV-60 Laxbro vials were processed by the Modified Petroff’s Method5. Repeat samples were collected if there was a delay in transport of the samples to the laboratory and/or if the sample was unsatisfactory. Smears were stained using the ZN method. The samples were inoculated into Dubos’ medium (Himedia) containing 10% glycerol as well as 10% FCS and on to Lowenstein-Jensen (LJ) (Himedia) slants. The isolates obtained from the blood and sputum samples were tested for their drug susceptibility using a modified Buddemeyer assay6. As part of routine practice, samples were sent to the National Tuberculosis Research Centre, Chennai and the Swedish Institute for Infectious Disease Control, Karolinska, Sweden for quality assurance of the drug sensitivity results. Biochemical tests such as niacin production, nitrate reduction and catalase activity at 680C were also carried out on the bacterial isolates obtained to differentiate between Mycobacterium tuberculosis and Mycobacteria other than tuber- culosis (MOTT)7. DESIREE T.B. D’SOUZA ET AL Indian Journal of Tuberculosis of either laboratory cross contamination or a mini- epidemic due to a common source of exposure11. Since DNA extracted from isolates grown in liquid media were used for spoligotyping, no inference on mixed infections can be drawn at this stage. However, investigations are under way to explore this possibility. Testing for the occurrence of multi-strain infections through individual colony examination of a single isolate8 appears to be vital in circumstances wherein the drug susceptibility profile does not correlate with the clinical status of the patient and /or the profile is discordant for varying sample types such as sputum and blood. Such infections would affect treatment of non-responder patients having HIV-TB dual infections. ACKNOWLEDGEMENTS The financial support from the Industrial Credit and Investment Corporation of India Limited and the Bombay Community Public Trust is acknowledged. We thank Dr. N. F. Mistry for her valuable suggestions and help in the preparation of the manuscript. REFERENCES 1. Hira SK, Dupont HL, Langewar DN, Dholakia YN. Severe weight loss: The predominant clinical presentation of tuberculosis in patients with HIV infection in India. Nat Med J India 1998; 11 : 256-258. 2. WHO Anti-tuberculosis drug resistance in the World (the WHO/IUATLD Global project on anti- tuberculosis drug resistance surveillance 1994-1997) WHO/TB/97-229. 3. Frieden TR, Sterling T, Pablos-Mendez A, Kilburn JO, Cauthen GM, Dooley SW. The emergence of drug resistant tuberculosis in New York city. N Eng J Med 1993; 328 : 521-526. 4. Saltzman BR, Motyl MR, Friedland GH, McKitrick JC, Klein RS. Mycobacterium tuberculosis bacteremia in the Acquired Immunodeficiency Syndrome. JAMA 1986; 256: 390-391. 5. Baker JF, Silverton RE. Routine bacteriological examination of specimens. In : Introduction to Medical Laboratory Technology, 5th ed., Butterworths, London; 1978 p. 528-530. 6. Shah DH, Devdhar MN, Ganatra RD, Kale PN, Virdi SS, M. D. Deshmukh. Modified rapid radiometric method for the detection of Mycobacterium tuberculosis isolates from sputum samples. Int J Nucl Med Biol 1985; 12 : 333-335. 7. Kent PT, Kubica GP. Public Health Mycobacteriology: A guide for the level III laboratory, U.S. Department of Health and Human Services, Centres for Disease Control, Atlanta, GA; 1985. 8. Braden CR, Morlock GP, Woodley CL, Johnson KR, Colombel AC, Cave MD et al. Simultaneous infection with multiple strains of Mycobacterium tuberculosis. Clin Infect Dis 2001; 33(6) : e42-47. 9. Chaves F, Dronda F, Alonso-Sanz M, Noriega AR. Evidence of exogenous reinfection and mixed infection with more than one strain of Mycobacterium tuberculosis among Spanish HIV-infected inmates. AIDS 1999; 13 (5) : 615-620. 10. Theisen A, Reichel C, Rusch-Gerdes S, Haas WH, Rockstroh, Spengler U et al. Mixed strain infection with a drug-sensitive and multi-drug-resistant strain of Mycobacterium tuberculosis. Lancet 1995; 345 : 1512- 1513. 11. Mistry NF, Iyer A, D’souza DT, Taylor GM, Young DB, Antia NH. Spoligotyping of Mycobacterium tuberculosis isolates from multiple drug resistant tuberculosis patients from Bombay, India. J Clin Microbiol 2002; 40 : 2677- 2680. IMPORTANCE OF BLOOD SAMPLES FOR TB DIAGNOSIS 81