COMPARISON OF ACID FAST BACILLI SMEAR AND CULTURE FOR THE DIAGNOSIS OF EXTRA-PULMONARY TUBERCULOSIS

The study was conducted with the objectives to compare the Acid Fast Bacilli (AFB) staining and Culture for the diagnosis of extra-pulmonary tuberculosis and to find out the burden of extra-pulmonary tuberculosis from the different site of the body in suspected TB patients. The standard Ziehl-Neelsen Technique was done for AFB staining and culture was done in Ogawa Medium. Of the 292 extra-pulmonary samples examined by AFB smear only 2(0.7%) were positive and of the 1058 extra-pulmonary samples cultured 20 (1.9%) were positive. The isolation rate for extra-pulmonary samples was three fold higher in culture in comparison to AFB Smear. The confirmation rate of extra-pulmonary tuberculosis was approximately 1/ 8th of the pulmonary tuberculosis by conventional bacteriological diagnostic methods. Of the extra-pulmonary tuberculosis renal, endometrial or pelvic and cold abscess were common in this study. M.tuberculosis was also isolated from peritoneal fluid, pericardial fluid, Synovial fluid, lymph node and cerebro spinal fluids only by culture. In conclusion, the standard AFB culture has significant role for diagnosis of extra-pulmonary tuberculosis, even though the conventional bacteriological technique could diagnose very low number of extra-pulmonary tuberculosis. The new methods of diagnosis should be considered to confirm more extra- pulmonary  tuberculosis. Key Words : Extra-pulmonary tuberculosis, AFB Smear, Culture, Diagnosis, Nepal.


INTRODUCTION
It is estimated that 1.7 billion individuals are infected with M. tuberculosis.Every year 8 million people get infected.Ninety five percent of the cases are in developing countries and approximately 3 million patient die each year. 1 South Asian Association for Regional cooperation (SAARC) region bears 22% of the global population and 29% of the global burden of tuberculosis.It is estimated that SAARC region has 2.5 million new cases and 0.6 million death per year.Nepal is among the poor developing countries in SAARC region with population of 23 million and it gets 201 tuberculosis cases per 100,000 populations of which 90/100,000 is smear positive tuberculosis. 2agnosis of extra-pulmonary tuberculosis is often difficult to establish using standard methods, 3 although a history of previous exposure to tuberculosis and X-ray changes consistent to old pulmonary diseases were found in many cases. 4urrently, developing countries rely on AFB staining and culture of sputum in conjunction with assessment of clinical symptom and radiographic evidence to diagnose tuberculosis.Detection by AFB staining and culture lack sensitivity particularly in case of sputum negative diseases.Extrapulmonary tuberculosis presents even more problems as the sputum samples are often not available and obtaining specimen from the suspected sites of infection often involve highly and expensive procedures. 5The Study done in University Hospital in Spain revealed that 20% of all bacteriologically confirmed cases were extra-pulmonary and the most frequent site was urinary tract 6 .Extra-pulmonary tuberculosis was generally observed most often in immunosupressed patients but 34% of cases were observed in people without any underlying diseases or risk factors.Delay to diagnosis was especially long in nonimmunosupressed patients 7 .The women, non-Hispanic black and HIV positive persons have been found to be higher risk for extra-pulmonary tuberculosis. 8e present study was done with the objectives to compare the detection rate of the extra-pulmonary tuberculosis in different site of the body by AFB smear and culture and to find out the common extra-pulmonary tuberculosis among the suspects.

MATERIALS AND METHODS
This study was conducted in Tribhuvan University Teaching Hospital (TUTH) Kathmandu Nepal from June 1997 to May 2001.Standard method for Acid Fast Bacilli (AFB) staining and culture was followed. 9For sputum samples modified Petroff methods was used and sputum culture was done only clinically suspected and AFB smear negative samples.All the suspected patients of pulmonary and extra-pulmonary tuberculosis from any site of the body were included in this study.Samples from the sterile site such as endometrium, peritoneal fluid, pleural fluid, synovial fluid, lymph node aspirate and bone marrow were centrifuged at 3000xg for 15 minutes, and then supernatant was discarded and deposit was inoculated in 2 tubes of 3% Ogawa medium.Lymph node, endometrial tissue or aspirate and other surgically resected tissue were cut into small piece with sterile scalpel or scissors.The specimen was homogenized in a sterile mortar using 0.5 to 1 ml sterile saline and a small quantity of sterilized sand.The suspension was mixed with 5ml of sterile saline, mixed well and centrifuged at 3000xg for 15 minutes, then supernatant was discarded and 0.1 ml of sediment was inoculated in each 3% Ogawa medium in duplicate.Whenever doubt existed about the contamination of the specimen, an untreated portion of the specimen was inoculated into a nonselective bacteriological medium e.g.nutrient agar and incubated for 24 hours to check for the presence of fast growing nonmycobacterial organisms.The remaining portion of the specimen was kept untreated in refrigerator until the absence of contamination was confirmed.Those samples having grown contaminants were processed like urine sample.
Urine sample: Patients were instructed to collect 24 hours urine sample in a sterile container.The urine was kept at refrigerator for over night for sedimentation purpose.Then the sediment was centrifuged at 3000x g for 15 minutes.The supernatant was discarded, then equal volume of 4% sulphuric acid was added on sediment, mixed and kept at room temperature for 15 minutes, then 15 ml of sterile saline was added in it and centrifuged at 3000xg for 15 minutes.The supernatant was discarded and sediment was neutralized with 4% NaOH containing a phenol red indicator.Then 0.1 ml each of sediment was inoculated in two tubes of 3% Ogawa medium and incubated at 37 0 C. Cultures were observed every week for 8 weeks.When there was no growth observed until 8 week incubation the culture was discarded.

RESULTS
Of the 1413 samples, 1121 (79.3%) from the pulmonary and 292(20.7%)from the extra-pulmonary were subjected for smear microscopy.Similarly out of 1521 samples cultured for M. tuberculosis, 463(30.4%) of pulmonary and 1058 (69.6%) samples were from the extra-pulmonary sources.Out of 1121 pulmonary samples for AFB smear 182(16.2%)smears were positive.Out of 292 extra-pulmonary smears only 2(0.7%) smears were positive.Of the 463 samples cultured, only 22(4.8%) were culture positive.Similarly, out of 1058 samples cultured from extra-pulmonary sources 20(1.9%)were culture positive.Of the 20 isolates obtained from the culture 7 from urine, 4 from endometrial tissue, 4 from cold abscess, one each from peritoneal, pericardial, Synovial fluid and lymph node aspiration.In comparison to 16.2% and 4.8% of pulmonary tuberculosis isolated from the AFB staining and culture of the pulmonary sources, only 0.7% and 1.9% tuberculosis were confirmed by AFB staining and culture from the extra-pulmonary sources (Table I).

DISCUSSIONS
The percentage of isolation among the suspects of extrapulmonary tuberculosis was very low in comparison to pulmonary suspects in this study.The isolation rate of extrapulmonary tuberculosis was approximately 1/8 th of the pulmonary tuberculosis.Similar to present study extrapulmonary tuberculosis was found 20% of the all the bacteriologically confirmed tuberculosis in Spain. 6Many laboratories of the resource poor countries have to still relay on smear examination and AFB culture, although more sensitive methods are available for diagnosis of extrapulmonary tuberculosis. 5Similar to present study significantly higher number of suspected cervical tuberculosis was confirmed by culture in comparison to AFB smear staining.Polymerase chain reaction (PCR) was highly sensitive than culture but specificity was lower in PCR in comparison to culture. 10t of the investigated samples from the various extrapulmonary sites most of the isolates were obtained from urinary, pelvic and cold abscess in this study.The incidence of urogenital tuberculosis has significantly decreased over the years due to unknown reason and the incidence of other extra-pulmonary tuberculosis remained same. 11In contrast to present study the most commonly observed site was pleura in a region of high prevalence of tuberculosis and low prevalence of HIV. 12 The peripheral lymph node and abdominal tuberculosis were the most commonly observed in Denis clinic. 13The diagnosis of tuberculosis peritonitis was less than 3% of cases by using acid fast staining of asitic fluid and culure of M. tuberculosis could detect up to 20% of cases in several trials. 14The present study was able to isolate M tuberculosis from 4.5% of the investigated samples which were negative in AFB smear.Only 4(0.6%) of the endometrial or pelvic samples were positive for M tuberculosis in present study.The isolation rate of Mycobacterial culture from endometrial aspirates of this study was comparable to Indian studies. 15In contrast to present and Indian studies, high percentage of endometrial tuberculosis was detected by BACTEC culture and histopathological examination in Rawal Pindi, Pakistan 16 .About 10% of patients of pulmonary develop genital tuberculosis although less then 1% of salppingitis can be attributed to Mycobacterium tuberculosis. 17ogenital tuberculosis was the most commonly found exta pulmonary tuberculosis in this study.Urogenital tuberculosis occurs with the haematogeneous spread of tubercle bacilli to the glomeruli.The infection spreads to involve renal pelvis, ureter, bladder, seminal vesicles, epididymis and testes. 18The urogenital tuberculosis comprises about 15 to 20 % of extra-pulmonary tuberculosis in developing countries but it was found 6% of the extra-pulmonary tuberculosis in United States 19 .Similar types of result were found in spain 3 where as lymph node and bone tuberculosis was more common in Shanghai. 20 conclusion, the isolation rate of M.tuberculosis by AFB smear examination and culture was low in present study and it is agreed with the conclusion that there is a urgent need for improving the diagnosis of extra-pulmonary tuberculosis 21 .The new methods of diagnosis should be considered on the basis of sensitivity, specificity, the cost per test, and time taken to perform the test.In addition it should also address the demographic factors, HIV status, acceptability of the new technique, etc. 22