Indian Journal of Tuberculosis 21 ISOLATION OF MYCOBACTERIUM AVIUM COMPLEX AND M. SIMIAE FROM BLOOD OF AIDS PATIENTS FROM SEVAGRAM, MAHARASHTRA* Pratibha Narang1, Rahul Narang2, Deepak Kumar Mendiratta3, Devashis Roy4, Vijayshree Deotale5, M. A. Yakrus6, Toney Sean6, and Kale Varsha7 * Presented at the 58th National Conference on Tuberculosis & Chest Diseases held in Mumbai from 1st to 4th January, 2004 1. Professor of Microbiology and Dean, MGIMS 2. Reader 3. Professor and Head 4. Demonstrator 5. Professor Dept. of Microbiology, Mahatma Gandhi Institute of Medical Sciences, Sevagram, Wardha District, (Maharashtra) 6. Tuberculosis/Mycobacteriology Branch, National Centre for HIV, STD and TB Prevention, Centre for Disease Control and Prevention, Atlanta 7. Research Assistant, National AIDS Research Institute, Pune. Correspondence: Dean, Mahatma Gandhi Institute of Medical Sciences, Sevagram-442 102, Wardha District (Maharashtra) Email – deanmgims@rediffmail.com Summary: Background and Methodolgoy: Mycobacterium avium complex (MAC) has not been reported as an opportunistic pathogen among patients with AIDS in the Indian subcontinent. Blood samples were cultured for mycobacteria using BACTEC 460TB system from 71 HIV seropositive and 33 seronegative patients, tested at Mahatma Gandhi Institute of Medical Sciences, Sevagam between August 2001 and December 2002. Results: MAC and M. simiae were isolated from three patients each. All the six patients with mycobacteremia were AIDS cases. Clinically, none of them was diagnosed as a case of mycobacteremia. However, laboratory results confirmed the dissemination of MAC and M. simiae among these patients. Conclusion: These results confirm that disseminated MAC and M. simiae disease exist among AIDS patients in India also. Key Words: HIV, MAC, M. simiae, Mycobacteremia INTRODUCTION Disseminated Mycobacterium avium complex (MAC) infection is a common problem among AIDS patients living in the developed countries, where almost half of these patients are found to have histological evidence of MAC infection at autopsy1. Today, owing to improved treatment regimens, prolonged survival of AIDS patients is becoming common, allowing MAC to infect most AIDS patients, who do not succumb to other illnesses2, 3. In developing countries, the first recognition of the occurrence of Non-tubercular mycobateria (NTM) in AIDS patients was reported in Sao Paulo, Brazil in 1993. The minimum estimated rate of disseminated MAC infection, defined by bone marrow culture, was 0.88% 4. In Africa, the first report of an association of MAC with AIDS patients was published in 1995 and the minimum rate for disseminated MAC disease was estimated to be 1%5. No reports of isolation of MAC from blood of AIDS patients from India are so far available, probably because MAC infection, as such, is a rare entity and, moreover, very few laboratories are testing blood for these organisms. The present study was undertaken to isolate mycobacterial species from the blood of HIV seropositive patients attending a rural hospital. MATERIAL AND METHODS This prospective study was conducted in the Department of Microbiology, Mahatma Gandhi Institute of Medical Sciences, Sevagram between August 2001 and December 2002. During this period, a total of 167 patients were found to be seropositive for HIV 1 and/or 2 by ELISA kits (Micro ELISA, J. Mitra, India) and Rapid test (ImmunoComb® II HIV 1& 2 Bi Spot, Orgenics, Israel) as per National AIDS Control Organization (NACO) guidelines. All results were confirmed by National AIDS Research Institute, Pune. [Indian J Tuberc 2005; 52:21-26] Indian Journal of Tuberculosis 22 Out of 167 patients tested positive for HIV, blood samples from 71 were cultured for mycobacteria and 67 of these were also clinically diagnosed as cases of either pulmonary and/or extra- pulmonary tuberculosis. Detailed history was obtained from each patient and results of physical examination and laboratory tests were recorded. Five ml of blood was inoculated into a BACTEC 13A bottle (Becton Dickinson). The bottles were incubated at 37oC and read by BACTEC 460TB instrument as per the recommended protocol. Whenever the growth index (G.I.) reached >20, the vial was considered positive. Positive BACTEC cultures were confirmed for the presence of acid-fast bacilli by Ziehl Neelsen staining and were subcultured further in the laminar flow inoculation hood on i) Lowenstein Jensen medium for isolation and identification of mycobacterial species; ii) in BACTEC 12B medium (Becton Dickinson) for NAP test and iii) Paraffin Slide Culture for paraffin baiting of NTM using Infectech IdentikitTM, USA6. All necessary precautions as per the protocols were taken in order to detect media contamination and to prevent cross contamination. The isolated mycobacteria were identified by niacin and NAP test along with other basic biochemical and biological tests. Confirmation of the species was done by high performance liquid chromatography (HPLC) of mycolic acids at the Centre for Disease Control and Prevention (CDC) in Atlanta, Georgia, USA. In addition to blood, other specimens processed for mycobacteria were sputum, stool, and peritoneal fluid, depending upon requisition from the treating physician. Blood samples of 33 HIV seronegative TB patients were also processed for mycobacteria using BACTEC 460 TB system. These acted as controls. RESULTS Among the seventy-one HIV seropositive patients, male to female ratio was 3.2:1and the age ranged from 4 to 65 years, of which 85.9% were between 20-49 years. Majority of the patients were from rural area (77.5%) and none was on anti- retroviral therapy or MAC prophylaxis. The profile of these 71 patients is given in Table 1. MAC was isolated from blood of 3 patients (4.23%) and M. simiae from another three (4.23%). No M. tuberculosis was isolated. Five of the six patients were residents of rural area. The clinical profile of the patients with mycobacteremia is given in Table 2. All the patients with mycobacteremia had history of fever and weight loss. Cough was a predominant symptom (83.33%), night sweats and Table 1: Profile of 71 HIV sero-positive patients included in the study PRATIBHA NARANG ET AL No. (%) Gender Male Female Place of residence Rural Urban Clinical Features Fever Weight loss Cough Lymphadenopathy Night sweats Breathlessness Chest pain Haemoptysis Hepatomegaly Splenomegaly Neurological symptoms Diarrhoea Jaundice Asymptomatics Clinical diagnosis TB Pulm. TB TB Lymph node Pulm. TB + TB Lymph node Pleural Effusion TB Meningitis TB Abdomen Pulm. TB + TB Abdomen Sputum culture positivity in 67 TB patients 54 (76.1) 17 (23.9) 55 (77.5) 16 (22.5) 67 (94.4) 58 (81.7) 53 (74.6) 20 (28.2) 15 (21.1) 14 (19.7) 14 (19.7) 6 (8.5) 6 (8.5) 5 (7.0) 5 (7.0) 4 (5.6) 1 (1.4) 4 (5.6) 67 (94.4) 53 (74.6) 6 (8.5) 3 (4.2) 2 (2.8) 1 (1.4) 1 (1.4) 1 (1.4) 19 (28.36) Indian Journal of Tuberculosis 23 lymphadenopathy were noted in 66.67% and breathlessness in 50% of the six patients. One subject with blood culture positive for MAC also had ascitis, pleural effusion and hepatosplenomegaly. The results of investigations in these 6 patients are given in Table 3. Total lymphocyte counts for these six patients ranged between 500-1300/µl. The haemoglobin for these patients ranged between 2.2 and 9.0gm%. In one patient (Case no. 1) with MAC bacteremia, AFB were also demonstrated on cytology in pleural and peritoneal fluid; the specimens were not sent for culture. However, the sputum culture for this patient grew acid-fast bacilli, but it could not be speciated as the isolate was lost. Sputum from another patient (Case no. 3) with MAC bacteremia also grew M. tuberculosis indicating mixed infection. In two cases with M. simiae bacteremia (Cases no. 4 and 5), lymph node aspirates showed non- granulomatous lesions with acid-fast bacilli and in one of the two patients (Case no. 5), aspirate from the parotid swelling also showed the same picture. (Table 3) The time taken for isolation of mycobacteria by BACTEC 13A ranged from 5 days to 26 days. All isolates grew on subculture on LJ, BACTEC 12B and Infectech Identikit with paraffin baiting. Blood cultures in the 33 HIV seronegative tuberculosis patients were negative for both M. tuberculosis and NTM. However, M. tuberculosis was cultured from sputum of 13 of these 33 patients. Table 2: Clinical profile of 6 patients with Mycobacteremia Case 1 Case 2 Case 3 Case 4 Case 5 Case 6 Age (Years) 47 30 40 20 25 49 Body weight 40 Kg 37 Kg 31 Kg 34 Kg 40 Kg 35 Kg Fever + + + + + + Loss of weight + + + + + + Cough + + + + - + Night sweat + + + - - + Lymphadenopathy + + - + + - Breathlessness + - + - - + Ascitis + - - - - - Pleural effusion + - - - - - Haemoptysis - - + - - - Splenomegaly + - - - - - M. AVIUM AND M. SIMIAE IN AIDS PATIENTS Indian Journal of Tuberculosis 24 DISCUSSION A thorough search of the literature revealed that this is the first Indian report on isolation of MAC and M. simiae from blood of AIDS patients. The rate of 4.23% isolation for MAC in the present study from central India is, however, much lower than that reported in the West 2, 3, but is higher than that reported from other developing countries4, 5. Only three reports on isolation of mycobacterial species from blood of HIV positive Table 3: Results of investigations in patients with Mycobacteremia (-): Investigations not done, (): normal values. *18 colonies of AFB grown but could not be speciated. **M. tuberculosis PRATIBHA NARANG ET AL Case 1 Case 2 Case 3 Case 4 Case 5 Case 6 Haematological Investigations: Haemoglobin gm% (13-18 gm%) 8.6 7.1 2.2 4.5 9 8 TLC/µl. (4000-11000) 5200 5900 3100 4500 4200 5400 DLC (N%,L%,M%) (45-75,20-45,2-9) 78,22 66,18,16 78,16,6 80,16,4 80,15,5 80,20 Total lymph. (1200-3400/µl) 1150 1100 500 800 600 1300 ESR (Westergren) (0-15 mm/1st hr) 70 80 170 120 - - Platelets (1.5-4 Lakhs/µl) - - Reduced Adequate Reduced - Reticulocyte (1-2%) - - 1.8% - 0.5% - Cytological/Histopathological Investigations: FNAC Cervical LN Reactive Hyperplasia Cervical LN Reactive Hyperplasia - Axillary LN AFB+ Supra Clav. LN and parotid AFB+ - Cytology- Peritoneal Fluid Tubercular - - - - - Cytology- Pleural fluid Tubercular - - - - - Microbiological Investigations: Sputum AFB S - C+* -ve S+C+** - ve -ve -ve Blood Culture isolate MAC MAC MAC M. simiae M. simiae M. simiae Radiological Investigations: X-Ray Rt.Pleural Effusion NAD Lt. lower lobe lesion Rt. lower lobe lesion NAD Bilateral Calcification, Infiltration Sonography Hepatosple- nomegaly - - Intra- abdominal Lymph node - - Indian Journal of Tuberculosis 25 patients are available from India. The first was a case report of mycobacteremia in 2 AIDS patients from Mumbai where M. tuberculosis was isolated using MYCO/F LYTIC medium and BACTEC 9500 system7. In another study on spoligotyping of 65 multiple-drug resistant M. tuberculosis from Mumbai, 12 isolates of M. tuberculosis from blood were from 22 HIV seropositive cases, recovered by using the lysis-centrifugation methods, Dubo’s liquid medium and Lowenstein-Jensen slants8. In the third study from Chennai, blood samples of 105 patients with tuberculosis (85 HIV seropositives and 20 seronegatives) were cultured for mycobacteria using BACTEC 13A medium and BACTEC 460TB system. M. tuberculosis was isolated from blood of three (3.5%) and M. phlei from one among the 85 HIV seropositive patients only9. The authors have not come across any report from India on MAC mycobacteremia, though the organism has been isolated from other clinical specimens and in at least 3 published studies, it was considered as pathogenic or potentially pathogenic, when isolated from sputum and other pulmonary samples in HIV seronegative patients10-12. MAC has also been isolated from the environmental samples of water and soil in south India13. However, among the HIV seropositive patients, there is no published report of isolation of MAC, from either pulmonary or extra-pulmonary sites, except for the study conducted by some of the authors in the present group14, in which paraffin baiting technique (Infectech IdentikitTM) was used to isolate NTM from stool (80 HIV seropositives and 40 HIV seronegatives) and from sputum samples (42 HIV seropositives and 128 HIV seronegatives) of tuberculosis patients. MAC was isolated from stool in 4 cases and sputum in 2 cases of HIV seropositive patients only. In addition, 2 strains of M. fortuitum were isolated from stool and one unspeciated NTM was isolated from sputum sample, also from HIV seropositive patients. No NTM was isolated from HIV seronegative subjects. In the present study, blood samples were collected from all patients who were recruited, but samples from other sites including pulmonary samples were processed for culture only when the physician requisitioned it. Isolation of mycobacteria from blood is an indicator of the disseminated infection. However, samples from different sites may provide multiple isolates of the same organism, confirming the disseminated form of disease. Specific criteria for selection of other specimens, for example, as given by Kiehn et al15, were not followed in the present study. However, in 2 patients (Cases no. 4 and 5), there was evidence of acid fast bacilli at other sites also, though the culture was not done (Table No. 3). M. simiae has been isolated from tap water, but reports of human infections are relatively few. Clinical disease appears similar to that caused by MAC and includes chronic pulmonary disease, osteomyelitis and disseminated disease with renal involvement. Cases of M. simiae infection in AIDS patients have also been reported16. In India, M. simiae has not been isolated from clinical or environmental samples, except one isolate from sputum in Bangalore12. CONCLUSION The present study concludes that mycobacteremia, both by MAC and M. simiae, is also present in AIDS patients, in India and this issue needs to be addressed. This study, as well as those reported earlier by other authors, further point to the fact that mycobacteremia is a problem mainly of immunocompromised state as none of the HIV seronegative patients in any of the Indian studies was positive either for NTM or M. tuberculosis. Western literature reports that many NTM species, though resistant to conventional anti-mycobacterial agents, are amenable to treatment with other drugs like newer macrolides, fluoroquinolones, aminoglycosides, etc. Therefore, clinicians even in India should suspect this condition and in addition to other samples, must ask for mycobacterial blood culture. It may be the only specimen that would clinch the diagnosis. With increasing incidence of HIV and TB co-infection, the laboratories in India must equip themselves to handle and process M. AVIUM AND M. SIMIAE IN AIDS PATIENTS Indian Journal of Tuberculosis 26 these samples. ACKNOWLEDGEMENTS We are grateful to Dr. A. P. Jain, Professor and Head, Medicine, MGIMS for permitting us to collect samples and to the patients for giving their consent. We are also thankful to Dr. R. A. Ollar, Head, Molecular Biology Unit, Department of Neurology, St. Vincent’s Hospital and Medical Centre, New York, for providing us with the Infectech Identikits and the guidance throughout this study. We are also grateful to National AIDS Research Institute for confirming HIV positive sera and to Mr.P. M. Dhone & Mr. D. U. Ingley for their technical help. REFERENCES 1. Horsburgh CR Jr. Mycobacterium avium complex infection in the acquired immunodeficiency syndrome (AIDS). N Engl J Med 1991; 324:1332-1338. 2. Nightingale SD, Byrd LT, Southern PM, Jockusch JD, Cal SX, Wynne BA. Incidence of Mycobacterium avium intracellulare complex bacteremia in human immunodeficiency virus-positive patients. J Infect Dis 1992; 165:1082-1085. 3. PregoV, Glatt AE, Roy V, Thelmo W, Dincsoy H, Raufman JP. Comparative yield of blood culture for fungi and mycobacteria, liver biopsy, and bone marrow biopsy in the diagnosis of fever of undetermined origin in human immunodeficiency virus-infected patients. Arch Intern Med 1990; 150:333-336. 4. Barreto JA, Palaci M, Ferrazoli L, Martins MC, Suleiman J, Lorenco R, Ferreira OC Jr, Riley LW, Johnson WD Jr, Galvao PAA. Isolation of Mycobacterium avium complex from bone marrow aspirates of AIDS patients in Brazil. J Infect Dis 1993; 168: 777-779. 5. Charles FG, Brindle RJ, Mwachari C, Batchelor B, Bwayo J, Kimari J, Arbeit RD, von Reyn CF Disseminated Mycobacterium avium infection among HIV-infected patients in Kenya. J Acquired Immune Defic Syndr 1995; 8(2): 195-198. 6. Ollar RA, Dale JW, Felder MS, Favate A. The use of Paraffin wax metabolism in the speciation of Mycobacterium avium intracellulare. Tubercle 1990; 71: 23-28. 7. Deodhar L. Mycobacteraemia in AIDS patients-Report of 2 cases. Indian J Med Microbiol 1999; 17(4):196- 197. 8. Mistry NF, Iyer AM, D’souza DTB, Taylor GM, Young DB, Antia NH. Spoligotyping of Mycobacterium tuberculosis isolates from multiple-drug-resistant tuberculosis patients from Mumbai, India. J Clin Microbiol 2002; 40(7):2677-2680. 9. Ramachandran R, Swaminathan S, Somasundram S, Azgar VN, Parmesh P, Paramsivan CN. Mycobacteremia in tuberculosis patients with HIV infection. Indian J Tuberc 2002; 50:29-31. 10. Kumar R, Khurana S. Incidence of atypical mycobacteria in Ludhiana. Indian J Med Microbiol 1990; 8:139-144. 11. Chauhan MM. Non-tuberculous mycobacteria isolated from an epidemiological survey in rural population of Bangalore district. Indian J Tuberc 1993; 40:195- 197. 12. Bose M., Isa M and Dam T. Isolation of pathogenic non-tuberculous mycobacteria from patients with chronic respiratory disease. Indian J Tuberc 1996; 42:11-14. 13. Kamala T, Paramasivan CN, Herbert D, Venkatesan P, Prabhakar R. Evaluation of procedures for isolation of non-tuberculous mycobacteria from soil and water. Appl Environ Microbiol 1994; 60(3):1021-1024. 14. Narang P, Narang Rahul, Bhattacharya S, Mendiratta DK. Paraffin slide culture technique for isolating non tuberculous mycobacteria from clinical specimens of stool and sputum of HIV seropositive patients. Indian J Tuberc 2004; 51:23-26. 15. Kiehn TE, Edwards FF, Brannon P, Tsang AY, Maio M, Gold JWM, Whimbey E, Wong B, McClatchy JK and Armstrong D. Infections caused by Mycobacterium avium complex in immunocompromised patients: diagnosis by blood culture and faecal examination, antimicrobial susceptibility tests, and morphological and seroagglutination characteristics. J Clin Microbiol 1985; 21:168-173. 16. Nolte FS and Metchock B. Mycobacterium. In: Murray PR, Baron EJ, Pfaller MA, Tenover FC and Yolken RH (Eds). Manual of Clinical Microbiology, 6th Edition. ASM Press Washington DC 1995: 404. PRATIBHA NARANG ET AL