BANGALORE, KARNATAKA PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION
Name of the candidate and address (in block letters)
KEMPEGOWDA INSTITUTE OF MEDICAL SCIENCES ,
Name of the Institution
KEMPEGOWDA INSTITUTE OF MEDICAL SCIENCES ,
Course of the study and subject
M. D. MICROBIOLOGY
Date of admission to course
01 JUNE 2013
Title of the topic: “Comparative study of Microscopy and Culture methods in detection of Mycobacterium tuberculosis among smear negative Pulmonary Tuberculosis cases and all Extra Pulmonary Tuberculosis cases in KIMS Hospital and Research centre, Bangalore”
Brief resume of the intended work
Need for the study:
Tuberculosis(TB) is a significant infectious disease in many parts of the world, which is of great concern. Prompt detection, isolation, identification and susceptibility testing of Mycobacterium tuberculosis from clinical specimens is essential for appropriate management of patients with tuberculosis.
According to RNTCP annual report 2011, India accounts for nearly one fifth of tuberculosis cases worldwide and every year around 2 million people develop TB.1 As per WHO Global report, 2013, out of these 2 million cases, 27% are smear negative cases of pulmonary tuberculosis and 20% are extra pulmonary tuberculosis.2 So together they account for nearly 50% of total tuberculosis cases.
Direct microscopy has been the main stay in diagnosis of TB, but has less sensitivity when compared to various culture methods, which is considered gold standard, especially when the bacillary load is less as in smear negative pulmonary TB and extra pulmonary TB.
There is an estimated proportion of Multidrug resistant TB(MDR-TB) in India of 2.1% in new TB cases and 15% in previously treated cases.2 It is therefore necessary to check the drug sensitivity of the culture isolates.
Hence there is a need for early diagnosis of sputum smear negative cases and extra pulmonary cases and prompt treatment after assessing the sensitivity to drugs. This goes a long way in reducing patient morbidity and mortality.
Review of literature:
Tuberculosis, for many centuries, has been the most important of human infections, in its global prevalence, devastating morbidity and massive mortality. It is estimated that a third of the world’s population is infected with the tubercle bacilli. Each year more than eight million new cases and three million deaths due to Tuberculosis is being reported worldwide.3
Detection of Acid Fast Bacilli(AFB) in sputum samples constitutes the mainstay of diagnosis in this disease. However this method has low sensitivity and has little value in patients who do not expectorate significant amount of sputum spontaneously. It has been reported that approximately 25-30% of adult patients with suspected pulmonary Tuberculosis(PTB) do not produce sputum spontaneously or have negative AFB smears. With increase in the prevalence of TB-HIV co-infection, a rise in the proportion of sputum-negative PTB patients is anticipated.4 Though pulmonary form is the commonest presentation, the extra pulmonary Tuberculosis(EPTB) is also an important emerging clinical problem. Extra pulmonary tuberculosis infections are more often smear negative than pulmonary cases which makes its diagnosis difficult to establish.5
Ziehl-Neelsen(ZN) method is a useful special staining procedure that specifically stains all members of the genera Mycobacterium. The limit of detection with this method is that it requires ≥10,000 bacilli per ml of sputum. Whereas the fluorescent staining method using Auramine-O and Rhodamine is associated with a higher rate of detection, since slides can be examined at lower magnifications. It is usually given preference over Ziehl-Neelsen method or its modifications especially when large number of specimens have to be examined.6
Culture for growth of Mycobacterium tuberculosis is more sensitive when compared to microscopy. It has an ability to give positive results on a clinical specimen containing as low as 10-100 bacilli per ml. The conventional culture method using Lowenstein Jensen(LJ) medium takes 6-8 weeks for primary isolation of the organism. While the rapid slide culturing(RSC) method is more rapid, simple and safe method and culture results are available in 7 days.7 However in RSC, drug susceptibility testing cannot be performed.
Few studies have been conducted to compare the sensitivities of LJ method and rapid slide culture method among smear negative pulmonary TB cases. A study conducted by Hemavathi, Pooja et al reported that out of 169 smear negative samples the studied, 35(20.71%) samples were RSC positive and 41(24.26%) were LJ culture positive.8 Another study by Sanjeev.H, Karnaker Vimal K et al reported that among the 50 smear negative , 16 were RSC positive and 15 were positive on LJ medium cultures. Of these 11 were positive by both RSC and LJ medium culture and 30 were negative by both the methods.3
The diagnosis of extrapulmonary TB is not always promising with conventional methods, due to longer time required for the cultivation and the paucibacillary nature of samples. Therefore for a definitive diagnosis an array of manual and automated systems have been developed to trim down the time to detect and identify bacteria in the clinical specimens. In this series the Mycobacterium Growth Indicator Tube(MGIT) system has been launched and extensively evaluated.5
The Mycobacterium growth Indicator Tube(MGIT) system is a non-radiometric method which senses the decrease in oxygen concentration and is considered to have a higher sensitivity. Also the recovery period is shortened, on an average, by 2 weeks.In a study on BAL samples in sputum smear negative pulmonary TB, Jagadish Rawat, Debasis Biswas et al reported a sensitivity of 47.55% for MGIT method when compared to 42.5% in LJ method.4 Another study by Rushi Bunger, Varsha A Singh et al reported that MGIT system was found more sensitive in detecting mycobacteria in smear negative samples. LJ method detected 12/198 (6.06%) and MGIT method detected 18/198 (10.10%).5 Another study by S.Rishi, P.Sinha et al reported that smear negative samples gave a recovery rate of 18.91% in LJ media and 39.18% in MGIT media, whereas the direct smear positive extra pulmonary samples gave 66.66% and 100% sensitivity in LJ and MGIT respectively and direct smear negative extra pulmonary samples gave sensitivity of 3.62% in LJ media and 20.28% in MGIT media.9
Mycobacterial antigen, MPT64, is a highly specific protein for M. tuberculosis. This can be easily used for rapid identification and confirmation of M.tuberculosis isolates. 10
In closing it is worthwhile to mention the emergence of Multi drug resistant TB in India at an alarming rate. Hence it is necessary to use diagnostic tests which have higher sensitivity in smear negative cases of pulmonary TB and extra pulmonary TB cases and perform drug sensitivity on the isolates. This will help to make an early diagnosis and aid in the usage of the appropriate drugs, prevent the emergence of MDR TB and hence reduce the overall morbidity and mortality.
Objectives of the study:
To compare the sensitivity of Microscopy and Culture methods in detection of Mycobacterium tuberculosis among smear negative pulmonary TB and extra pulmonary TB cases.
To detect the drug resistance pattern to 1st line drugs among Mycobacterium tuberculosis isolates
Material and Methods:
7.1 Source of data: The study will comprise of suspected cases of smear negative pulmonary tuberculosis and extra pulmonary tuberculosis in KIMS, Bangalore.
Duration of study:18 months
Study design: comparative study
Sample Size: 100
Sampling design: Purposive sampling
Patients having radiological lesions suggestive of active pulmonary/extra pulmonary Tuberculosis
Patients having strong clinical evidence suggestive of extra pulmonary Tuberculosis
Sputum smear negative for M.tuberculosis among clinically suspected pulmonary TB cases
Patients on anti-tubercular treatment.
7.2 Methodology: Specimen collection:
Specimens like sputum/BAL fluid/pleural fluid/ ascitic fluid/cerebrospinal fluid/synovial fluid/lymph node aspirate are collected under aseptic precautions.
The Specimen for detection of Mycobacterium tuberculosis is subjected to
Direct microscopy by Ziehl-Neelsen staining and fluorescent staining using Auramine-O.11
Specimen is decontaminated by modified Petroff’s method using N-acetyl-L-cysteine-NaOH(NALC-NaOH) digestion decontamination technique.5
0.1ml of specimen is inoculated into Lowenstein-Jensen media and incubated at 370 C for 8 weeks. Slopes are examined for growth twice a week during first 2 weeks, followed by once a week till 8 weeks.5
Two smears are made at lower 1/3rd of slide and are dipped in Human Blood Medium in McCarteny’s bottle and incubated at 370 C for 7 days. On seventh day slides are taken out, washed off excess blood and stained by the Ziehl-Neelsen method and fluorescent method using Auramine-O.3
0.5ml of specimen is inoculated into MGIT tube containing 7ml modified middle brook 7H9 broth with supplement and incubated at 370 for maximum 42 days. Reading is taken daily for first 3 weeks and once daily till end of 42 days in Micro MGIT system.5
Sterile specimens as pleural fluid, ascitic fluid, CSF, pericardial fluid are inoculated directly into MGIT tubes without decontamination.
Appropriate biochemical tests like Niacin test, Nitrate reduction test, Pyrazinamidase test are performed to confirm the positive isolates from Lowenstein-Jensen media and MGIT.
A rapid TB antigen detection is performed on the isolates from Lowenstein-Jensen media and MGIT for MPT64 Ag (SD BIOLINE).
Positive isolates confirmed as Mycobacterium tuberculosis is subjected to drug sensitivity using MGIT method for 1st line drugs - Streptomycin, Rifampicin, Isoniazid and Ethambutol.
Data collected will be analyzed using descriptive statistical methods by computing percentage, mean and standard deviation. Appropriate tests will be employed for categorical, parametric or non parametric data. Wherever necessary the results will be depicted in the form of graphs and percentages.
7.4 Does the study require any investigations or interventions to be conducted
On patients or other humans or animal? If so, please describe briefly.
Samples are collected as a part of routine diagnostic workup and patient management.
No animal experiments are required for the study.
7.5 Has ethical clearance been obtained from your institution in case of 7.3 Yes. The certificate has been enclosed.
1. Revised National TB Control Programme. Annual Status Report,TB INDIA; 2011:8
2. World Health Organization. Global Tuberculosis report; 2013: 122
3. Sanjeev, H., Karnaker, V. K., Rai, R., Pai, A. K. B., Ganesh, H. R., and Krishnaprasad, M. S., Rapid slide culture: Relevance to the Modern Day Diagnosis of Tuberculosis, Journal of Clinical and Diagnostic Research. 2012 May; 6(3):378-381
4. Rawat, J., Biswas, D., Sindhwani, G., Masih, V., and Chauhan, B. S., Diagnostic role of MGIT Culture of BAL Samples in Sputum Smear-Negative Pulmonary Tuberculosis, Indian Journal of Tuberculosis.2013; 60:77-82
5.Bunger, R., Singh, V. A., Avneet, Mehta, S., and Pathania, D., Evaluation of BACTEC Micro MGIT with Lowenstein Jensen Media for Detection of Mycobacteria in Clinically Suspected Patients of Extra Pulmonary Tuberculosis in a tertiary Care Hospital at Mullana (Ambala), Journal of Medical Microbiology & Diagnosis.2013; 2(2)
6.Palomino, J. C., Nonconventional and new methods in the diagnosis of tuberculosis: feasibility and applicability in the field, European Respiratory Journal.2005;26:339-350
7.J.Jena, S.K. Nema, B.N. Panda, K.E. Rajan., Comparative efficacy of Rapid slide culture of M.Tuberculosis and conventional LJ medium culture in diagnosis and management of pulmonary Tuberculosis cases, Indian Journal of Tuberculosis.1995;42:151-154
8. Hemavati, Sarmah, P., and Ramesh, D. H., Comparative Evaluation of the Rapid Slide Culture and Microscopy with the Conventional Culture Method in the Diagnosis of Pulmonary Tuberculosis,Journal of Clinical Diagnostic Research. 2012 April; 6(2):192-194
9. Rishi, S., Sinha, P., Malhotra, B., and Pal, N., A Comparative Study for the Detection of Mycobacteria by BACTEC MGIT 960, Lowenstein Jensen Media and Direct AFB Smear Examination, Indian Journal of Medical Microbiology.2007; 25(4): 383-386
10.Hasegawal,N., Miura, T., Ishii, K., Yamaguchil, K., Lindner, T. H., Merrit, S., Mathews, J.D. and Siddiqi, S. H., New Simple and Rapid Test for culture Confirmation of Mycobacterium Tuberculosis Complex: a Multicenter Study, Journal of clinical Microbiology. 2002 March; 40(3): 908-912
11. Manual for Sputum Smear Fluorescence Microscopy. Revised National Tuberculosis Control Programme. Central TB Division, Directorate General of Health Services, Ministry of Health and Family Welfare, Nirman Bhavan, New Delhi.
Signature of the candidate:
Remarks of the guide:
Name and designation of the guide (in block letters): 11.1 Guide : DR. PRATIBHA MALINI.J