A comparative evaluation of mycobacteria growth indicator tube and Lowenstein-Jensen medium for the isolation of mycobacteria from clinical specimens

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1 Original Article Vol. 29 No. 2 Comparative evaluation of mycobacteria culture:-kaur KP, et al. 67 A comparative evaluation of mycobacteria growth indicator tube and Lowenstein-Jensen medium for the isolation of mycobacteria from clinical specimens Kawal Preet Kaur, MBBS, M.D. (Microbiology) 1, Bharti Arora, MBBS, M.D. (Microbiology) 1, Deepinder K Chhina, MBBS, M.D. (Microbiology) 2, Veenu Gupta, MBBS, M.D. (Microbiology) 2 ABSTRACT This study was undertaken to compare sensitivity, time to detection and contamination rates of culture in Mycobacteria Growth Indicator tube (MGIT) and on Lowenstein-Jensen (L-J) media. A total of 300 pulmonary and extra-pulmonary samples were processed. All samples were stained by Ziehl-Neelsen (ZN) and Auramine-O method. The samples were inoculated on L-J media and in MGIT after performing NALC- NaOH (N-Acetyl-L-cysteine NaOH) procedure according to standard protocol. All isolates obtained were identified biochemically using standard procedures. Auramine-O staining was more sensitive (64%) compared to ZN staining (48%), while ZN staining had higher specificity (99.64%) than Auramine-O staining (98.20%). The sensitivity of MGIT and L-J media was 95.35% and 58.14% respectively. The mean detection time of L- J medium (32.88 days) was significantly more than MGIT (20.12 days). The contamination rate of MGIT (10%) was higher than L-J medium (7%). (J Infect Dis Antimicrob Agents 2011;29:67-74.) INTRODUCTION Tuberculosis (TB) is the leading cause of death from a curable infectious disease. TB has affected mankind for over 5,000 years, and it still continues to be a leading cause of morbidity and mortality. The bacilli was discovered more than a century back by Sir Robert Koch in 1882 and effective drugs for treatment were available for more than half a century. Globally more than 1.3 million people die of the disease every year. Nearly one third of the world s population is infected with TB bacilli, approximately 10% of them have a life time risk of developing TB disease. 1 The emergence of resistance to drugs used to treat tuberculosis and particularly MDR-TB (Multi- Drug Resistant Tuberculosis), has become a 1 Maharaja Agrasen Medical College, Agroha, Hisar, Haryana, India. 2 Dayanand Medical College, Ludhiana, Punjab, India. Reprint request: Dr. Kawal Preet Kaur, MBBS, M.D. (Microbiology), Assistant Professor, Maharaja Agrasen Medical College, Agroha, Hisar, Haryana, India. dockawu25@gmail.com Keywords: Tuberculosis, MDR, MGIT, LJ media, NALC-NaOH 67

2 68 J INFECT DIS ANTIMICROB AGENTS May-August 2012 significant public health problem in a number of countries and an obstacle to effective TB control. 2 In response to altered clinical and epidemiologic situations, several changes in laboratory practice have evolved over the past several years. Several new techniques have been introduced that are directed towards the detection of Mycobacterium tuberculosis directly in clinical sample by nucleic acid amplification, the more effective recovery of mycobacteria from clinical specimens, the rapid identification of mycobacteria and the determination of drug susceptibility profiles. 3 Examination by mycobacterial culture provides the only definitive diagnosis of tuberculosis. As M. tuberculosis grows slowly, conventional culture with visual detection of bacterial colony formation usually requires 2-6 weeks. Recently, a number of growth indicators have been used, often involving liquid media and automated systems that shorten the detection period to 1-3 weeks in most cases. 4 Therefore, this study was undertaken to compare the sensitivity, time to detection and contamination rates of culture in MGIT and on L-J media. MATERIAL AND METHODS This prospective study was conducted over a period of one year (1 st August 2009 to 31 st July 2010) in the Department of Microbiology, Dayanand Medical Table 1. Distribution of various samples. Samples (n = 300) Pulmonary Samples (n=217) Positive Samples (n = 43) Negative Samples (n = 257) S putum (n = 143) 22 (15.38) 121 (84.62) B ronchoalveolar Lavage (n = 59) 10 (16.95) 49 (83.05) E ndotracheal Secretions (n = 15) 3 (20.00) 12 (80.00) Extra-pulmonary Samples (n=83) P leural Fluid (n =2 6) 2 (7.69) 24 (92.31) P us / Wound Swab (n = 19) 4 (21.05) 15 (78.95) Endometrial Biopsy (n = 18) 0 18 (100.00) S oft Tissue (n = 6) 1 (16.67) 5 (83.33) Drain Fluid (n = 3) 0 3 (100.00) L ymph Node Aspirates (n = 3) 1 (33.33) 2 (66.67) Ascitic Fluid (n = 2) 0 2 (100.00) Cerebrospinal Fluid (n = 2) 0 2 (100.00) Pericardial Fluid (n = 2) 0 2 (100.00) Semen (n = 1) 0 1 (100.00) Bone Marrow Aspirates (n = 1) 0 1 (100.00)

3 Vol. 29 No. 2 Comparative evaluation of mycobacteria culture:-kaur KP, et al. 69 College and Hospital, Ludhiana. From 269 suspected cases of tuberculosis admitted in various wards and ICUs, 300 pulmonary and extra-pulmonary samples were received. Blood and urine samples were not included since they are not recommended to be processed using MGIT. All the samples were subjected to staining by ZN and Auramine-O method. All the samples were inoculated on L-J media (conventional) and in MGIT for rapid culture method after performing the NALC- NaOH procedure for digestion and decontamination according to standard protocol. 3 All the isolates obtained were identified biochemically using standard procedures. 3 RESULTS Out of 269 suspected cases of tuberculosis, 39 cases were positive for AFB (Acid fast bacilli) smear/ culture. From these cases a total of 43 samples (single sample from 35 cases, 2 sputum samples each from 3 cases and 2 bronchoalveolar lavage samples from 1 case) were obtained. From these 43 samples, 22 samples were positive smear positive (ZN/ Auramine). Forty one isolates of mycobacteria were obtained. Among the pulmonary samples, 15.38% sputum samples were found to be positive, whereas the positivity of bronchoalveolar lavage and endotracheal secretions was 16.95% and 20.00% respectively. Among the extra-pulmonary samples, 33.33% of the lymph node aspirates showed positivity, whereas the positivity of pus/ wound swab and soft tissue samples was 21.05% and 16.67% respectively. The distribution of smear and culture positivity according to various samples is shown in Table 2. Out of 43 positive samples, 13 (30.23%) were found to be positive with ZN staining, while 19 (44.19%) were positive with Auramine staining. Culture by conventional method on L-J media yielded 25 isolates, while culture in MGIT recovered 41 isolates. A total of 20 samples were both smear and Table 2. Distribution of positive samples. Smear Positive Culture Positive Samples ZN Staining Auramine Staining L-J media MGIT (n = 13) (n = 19) (n = 25) (n = 41) Pulmonary Samples (n = 35) Sputum (n = 22) Bronchoalveolar lavage (n = 10) Endotracheal secretions (n = 3) Extra-pulmonary Samples (n = 8) Pleural Fluid (n = 2) Pus/ Wound Swab (n = 4) Soft Tissue (n = 1) Lymph Node Aspirates (n = 1) T otal ( n = 43) 13(30.23) 19 (44.19) 25 (58.14) 41(95.35) 69

4 70 J INFECT DIS ANTIMICROB AGENTS May-August 2012 Table 3. Smear positivity by ZN and Auramine Staining. Staining Method Smear Positivity (n=22) Z N Staining 13 (59.09) A uramine Staining 19 (86.36) Table 4. Correlation of ZN Staining/Auramine-O Staining with growth on L-J media. Results Culture Result on LJ media Positive Negative ZN Staining Positive (n = 13) 12 1 Sensitivity 48% Negative(n = 287) Total (n = 300) Auramine-O staining Specificity 99.64% Positive (n = 19) 16 3 Negative (n = 281) Total (n = 300) Sensitivity 64% Specificity 98.20% p = (for Sensitivity) p = (for Specificity) Table 5. Correlation of smear positivity with culture. Smear Examination Culture Positive (n=41) Culture Negative (n=259) S mear Positive (n=22) 20 (48.78) 2 (0.77) S mear Negative (n=278) 21 (51.22) 257 (99.23)

5 Vol. 29 No. 2 Comparative evaluation of mycobacteria culture:-kaur KP, et al. 71 Table 6. Sensitivity of L-J media and MGIT. Smear Results No. of Positive Cultures (% recovery) L-J media MGIT S mear Negative (n = 21) 7 (33.33) 21 (100) S mear Positive (n = 22) 18 (81.82) 20 (90.91) T otal (n = 43) 25 (58.14) 41 (95.35) p = Table 7. Mean Detection Time of Growth on L-J media & in MGIT for Smear Positive and Smear Negative Samples. Smear Examination Smear Smear Positive (n = 20) Negative (n = 21) L -J medi a ( n = 25) M GIT (n = 41) days days days days p-value culture positive while 21 samples were smear negative and culture positive. Smear positive and culture negative samples were 2 in number. Out of the 300 samples, 278 were smear negative whereas 22 were positive % were positive with ZN staining while 86.36% were positive with Auramine staining. Six samples which were missed by ZN staining were detected by Auramine staining. The sensitivity and specificity of ZN and Auramine-O staining were calculated with growth obtained on L-J media as gold standard. The sensitivity of ZN staining (48%) was found to be lower than that of Auramine staining (64%). The difference between the sensitivity of the two staining methods was found to be statistically non-significant (p= ). The specificity of ZN staining (99.64%) was found to be slightly more than that of Auramine- O staining (98.20%). The difference between the specificity of the two staining methods was found to 71 be statistically non-significant (p= ). Out of the total 300 samples, 41 yielded growth of mycobacteria on culture, while 259 were culture negative. Twenty-two samples were positive by direct AFB smear examination while 278 were smear negative. Hence culture detected 21 cases of tuberculosis which were missed on direct smear examination. Two out of the smear positive samples failed to yield growth of mycobacteria on conventional as well as rapid culture method for mycobacteria. When analysed retrospectively, both the patients were on antitubercular treatment. Out of the 41 isolates obtained, 40 isolates were slow growers taking an average time of ± days for growth. All the isolates were non-chromogens. One isolate was a rapid grower, in which growth was obtained on 4 th day. On the basis of the biochemical identification scheme, all the slow growers were

6 72 J INFECT DIS ANTIMICROB AGENTS May-August 2012 identified as Mycobacterium tuberculosis while the rapid grower was identified as M. cheloneae. Overall MGIT recovered all the 41 isolates hence the sensitivity of MGIT was found to be 95.35% as compared to 58.14% with L-J media which could recover only 25 out of 41 isolates. The sensitivity of MGIT was found to be statistically significant as compared to L-J media (p= ). The rate of contamination on L-J media (7%) was lesser as compared to MGIT (10%). The difference between the contamination rate on both the media was statistically non-significant (p= ). The mean detection time of growth on L-J media was ± days as compared to ± days in MGIT which was statistically significant (p= ). For L-J media, the mean detection time was ± 2.73 days for growth of the AFB smear negative samples while it was ± days for AFB smear positive samples. This difference of mean detection time for growth on L-J media for smear negative samples and smear positive samples was found to be statistically significant (p= ). For MGIT, the mean detection time was ± days for growth of the AFB smear negative samples while it was ± days for AFB smear positive samples. This difference of mean detection time for growth in MGIT for smear negative specimens and smear positive specimens was found to be statistically significant (p = ). DISCUSSION Despite recent advances in mycobacteriology, early laboratory diagnosis of TB still relies on the examination of stained smears. Conventional methods such as ZN staining and fluorescent staining are rapid, simple techniques with low cost. The use of fluorescent microscopy significantly increases the diagnostic value of the smear, particularly where there are low-density bacilli which may escape detection by ZN method. A study conducted in Iran had reported the sensitivity and specificity of 51%, 100%, and 57%, 100% for the ZN and auramine phenol staining methods, respectively. 5 A study conducted by Ulukanligil M et al reported a sensitivity of 61% with ZN staining as compared to 83% with FM staining and specificity of 100% and 99% respectively. 6 In contrast, the sensitivity of ZN staining was 48% and fluorescent staining was 64% in our study. The specificity of ZN staining and fluorescent staining was 99.64% 98.20% respectively. M. tuberculosis culture is still the cornerstone on which definitive diagnosis of TB relies. Egg-based Lowenstein-Jensen media has been used for cultivation of mycobacteria for several decades. Liquid media have been introduced to increase the diagnostic yield of mycobacteria. MGIT was introduced about a decade ago with the aim of increasing the sensitivity of culture. A study conducted in Spain had reported the sensitivity of MGIT to be 93% as compared to only 60% with L-J medium. 7 The findings were concordant with our study. We observed that the sensitivity of MGIT was 95.35% as compared to only 58.14% with L-J medium. However, in literature sensitivity of L-J medium had been reported to be 59.7%, 76.9% and 87% as compared to MGIT which showed the sensitivity of 85.4% 86.5% and 93% Liquid culture media have problems of contamination and the need for subculture on solid media for the confirmation of identity and purity. Various studies have reported a higher contamination rate with liquid media. A study conducted in Zimbawe had reported a contamination rate of 14.7% with MGIT and 5% with L-J media. 11 Katila et al 12 had demonstrated a contamination rate of %, however, another study 9 found the contamination rate of 3-4% with both the media. We found that the contamination rate was 10% in MGIT

7 Vol. 29 No. 2 Comparative evaluation of mycobacteria culture:-kaur KP, et al. 73 and 7% on L-J media. The contamination rate was more due to the higher enrichment of the Middlebrook 7H9 broth base. Liquid cultures generally require a shorter incubation time than solid media for detection of M. tuberculosis. A Malaysian study had shown the mean detection time to be 14 days with MGIT and 33 days with L-J medium. 13 Chew et al had shown the mean detection time to be 22 days with MGIT and 27 days with L-J medium. 9 Our findings were in agreement to other studies. The mean detection time with MGIT (20.12 days) was significantly lower than that with L-J media (32.88 days). Thus liquid media facilitate earlier detection of mycobacteria. We also recorded the mean detection time of growth on L-J media and in MGIT for both AFB smear positive and smear negative samples. The mean detection time of growth on L-J media for AFB smear positive samples was days as compared to days for AFB smear negative samples similar to a study which reported the values to be 31.2 days and 35.3 days respectively. Further study had also reported that the mean detection time of growth in MGIT for AFB smear positive samples was days as compared to days for AFB smear negative samples whereas in our study, the mean detection time of growth in MGIT for AFB smear positive samples was days as compared to days for AFB smear negative samples. 13 The present trend and pattern of MTB infection which include its opportunistic and lethal synergy with HIV, therapeutic complications due to emergence of drug resistant MTB strains and infection by nontuberculous mycobacteria (NTM) species necessitate the scaling up of routine diagnostic procedures at least to include culture and identification of causative mycobacterial species. There is need to identify MTB complex (slow strains) from nontuberculous mycobacteria (fast growers) to enable 73 prompt eradication of the infecting strain from the host. Studies have found a much lower incidence of NTM in India. A study from CMC Vellore found M. tuberculosis to be the predominant isolate (96.1%) as compared to only 3.9% NTM. 14 Even in HIV positive patients, only 1 out of 23 isolates was found to be atypical while the other 22 were all Mycobacterium tuberculosis isolates. 15 In our study, out of the 41 isolates, 40 were Mycobacterium tuberculosis (97.56%) while 1 was M. cheloneae (2.44%). The clinical microbiology laboratory has a critical role in the detection and control of infection caused by clinically significant Mycobacterium species. Rapid, sensitive and accurate detection of these organisms in clinical specimens can hasten the administration of appropriate antimycobacterial therapy and prevent the spread of infection to susceptible contacts. Broth media such as Middlebrook 7H9 have been developed to speed the growth and recovery rate of mycobacteria in the laboratory. However, there is an increase in costs compared to those of conventional cultivation techniques. This must be weighed against the benefits gained by early diagnosis and the higher sensitivity of detection. However, in our centre, we obtained enhanced diagnostic yield in terms of more number of tuberculosis cases detected and with a much less time to detection. Hence, we found out Auramine-O staining to be better than ZN staining and MGIT to be better than L-J media for the detection and isolation of mycobacteria. ACKNOWLEDGEMENTS The present study was carried out over a one year period in Dayanand Medical College, Ludhiana. We are thankful to the late Dr Raj Kumar for his support, invaluable guidance and blessings. The technological support for the study was provided by the institute itself. The various laboratory tests were

8 74 J INFECT DIS ANTIMICROB AGENTS May-August 2012 conducted in the Microbiology Department of the institute and the tests were used for routine diagnostic purposes. The expenses incurred were charged from the patients as per the protocol being followed in the hospital. There were no other sources of financial support. References 1. TB India 2010: RNTCP Status Report [Internet] [cited 2010 Jun 1]. Available from: pdfs/tb%20india% pdf 2. India Ministry of Health and Family Welfare. Revised National Tuberculosis Control Programme DOTS-Plus Guidelines [Internet] [cited 2010 Jun 1]. Available from: Guidelines_Jan2010.pdf 3. Winn W Jr, Allen S, Janda W, et al. Mycobacteria. In: Winn W Jr, Allen S, Janda W, et al., eds. Koneman s Color Atlas and Textbook of Diagnostic Microbiology. 6 th ed. Philadelphia: Lippincot Williams & Wilkins, 2006: World Health Organization. Diagnostics for tuberculosis: global demand and market potential [Internet]. Geneva: WHO, 2010 [cited 2010 Jun 1]. Available from: _eng.pdf 5. Ziaee M, Namaei M, Khazaei M, Azarkar G. Comparison of the value of two different sputum staining for diagnosis of acid-fast bacilli. Iranian J Clin Infect Dis 2008;3: Ulukanligil M, Aslan G, Tasci S. A comparative study on the different staining methods and number of specimens for the detection of acid fast bacilli. Mem Inst Oswaldo Cruz 2000;95: Idigoras P, Beristain X, Iturzaeta A, Vicente D, Perez-Trallero E. Comparison of the automated nonradiometric Bactec MGIT 960 system with Lowenstein-Jensen, Coletsos, and Middlebrook 7H11 solid media for recovery of mycobacteria. Eur J Clin Microbiol Infect Dis 2000;19: Lu D, Heeren B, Dunne WM. Comparison of the Automated Mycobacteria Growth Indicator Tube System (BACTEC 960/MGIT) with Lowenstein-Jensen medium for recovery of mycobacteria from clinical specimens. Am J Clin Pathol 2002;118: Chew WK, Lasaitis RM, Schio FA, Gilbert GL. Clinical evaluation of the Mycobacteria Growth Indicator Tube (MGIT) compared with radiometric (Bactec) and solid media for isolation of Mycobacterium species. J Med Microbiol 1998;47: Badak FZ, Kiska DL, Setterquist S, Hartley C, O Connell MA, Hopfer RL. Comparison of mycobacteria growth indicator tube with BACTEC 460 for detection and recovery of mycobacteria from clinical specimens. J Clin Microbiol 1996;34: Apers L, Mutsvangwa J, Magwenzi J, et al. A comparison of direct microscopy, the concentration method and the Mycobacteria Growth Indicator Tube for the examination of sputum for acid-fast bacilli. Int J Tuberc Lung Dis 2003;7: Katila ML, Katila P, Erkinjuntti-Pekkanen R. Accelerated detection and identification of mycobacteria with MGIT 960 and COBAS AMPLICOR systems. J Clin Microbiol 2000;38: Fadzilah MN, Ng KP, Ngeow YF. The manual MGIT system for the detection of M. tuberculosis in respiratory specimens: an experience in the University Malaya Medical Centre. Malays J Pathol 2009;31: Jesudason MV, Gladstone P. Non tuberculous mycobacteria isolated from clinical specimens at a tertiary care hospital in South India. Indian J Med Microbiol 2005;23: Patwardhan NS, Bansal MP, Pradhan J. Characterization of mycobacterial species in clinically diagnosed cases of pulmonary tuberculosis and their HIV status. Indian J Pathol Microbiol 1997;40:365-7.