Indian Journal of Tuberculosis PARAFFIN SLIDE CULTURE TECHNIQUE FOR ISOLATING NON-TUBERCULOUS MYCOBACTERIA FROM STOOL AND SPUTUM OF HIV SEROPOSITIVE PATIENTS P. Narang1, Rahul Narang2, S. Bhattacharya3 and D.K. Mendiratta Summary: Objective: Paraffin slide culture method (PSC) was used to isolate Non-tuberculous mycobacteria (NTM) from stool and sputum samples of HIV seropositive and negative patients. Material & Methods: Eighty stool and forty two sputum samples from both symptomatic or asymptomatic HIV sero-positive patients; and 40 stool and 128 sputum samples from symptomatic but HIV seronegative patients were cultured by PSC to assess its utility in isolating NTM from the clinical specimens. The samples were simultaneously processed by culture on Lowenstein Jensen (LJ) medium for comparison with regard to isolation rate, isolation time and contamination rate. Results: The PSC proved to be as good as LJ in isolating NTM from clinical specimens and, in addition, had the advantage of in situ staining for acid fast bacilli and lower contamination rate. The PSC was also used for typing NTM by biochemical tests. Key words: Paraffin slide culture, HIV, Non-tuberculous mycobacteria INTRODUCTION Non-tuberculous mycobacteria (NTM) or the so-called atypical mycobacteria or mycobacteria other than tuberculosis (MOTT) have been recognized since Koch’s time but did not gain as much importance as Mycobacterium tuberculosis. Today, the recovery of NTM from patients’ specimens and from environmental sources is of concern to microbiologists, epidemiologists and physicians. In developed countries, as the incidence of tuberculosis decreased, the occurrence of NTM in pulmonary diseases increased1-3. Haematogenous dissemination of NTM has been reported with higher frequency after the advent of the acquired immunodeficiency syndrome (AIDS). The immunosuppressed individuals infected by human immunodeficiency virus (HIV) infection have become the most significant risk factor for disseminated NTM disease and of these, 95% are due to Mycobacterium avium complex (MAC)4,5. In developing countries, little is known about NTM infections, either in AIDS or non AIDS patients. In India, there is paucity of data relating NTM to disease. They have, however, been isolated from patients’ specimens but are considered only as commensals or contaminants6,7. Infectech IdentikitTM which utilizes capacity of NTM to use paraffin wax as the sole source of carbon, was successfully used by our laboratory at the M.G. Institute of Medical Sciences, Sevagram, to culture known strains of NTM and Nocardia and also to speciate NTM by putting up biochemical reactions8. The present study was undertaken to find out the utility of this kit in isolating NTM from clinical specimens of stool and sputum of HIV seropositive and seronegative subjects. The growth was compared with the gold standard Lowenstein Jensen medium with regard to isolation rate, isolation time and contamination rate, and was also correlated 1. Dean, MGIMS and Professor 2. Lecturer 3. Demonstrator 4. Professor & Head Department of Microbiology, Mahatma Gandhi Institute of Medical Sciences, Sevagram. Correspondence: Dr. P. Narang, Dean, MGIMS & Professor of Microbiology, Mahatma Gandhi Institute of Medical Sciences, Sevagram-442 102 District Wardha, Maharashtra. E-mail: narangpr@rediffmail.com/deanmgims@rediffmail.com Original Article (Received on 30.6.2003; Accepted on 3.9.2003) [Indian J Tuberc 2004;51:23-26 ] Indian Journal of Tuberculosis with signs and symptoms of the patients. MATERIAL AND METHODS The study was conducted between November 1999 and March 2001. Stool and sputum specimens were collected from HIV seropositive patients, with or without symptoms of mycobacterial infection and disease (80 stool and 42 sputum), and HIV seronegative symptomatic patients (40 stool and 128 sputum) attending Kasturba Medical Hospital, Sevagram. Due to unavoidable reasons, only one sample of stool and sputum could be obtained from each patient, hence repeated isolation could not be demonstrated. Processing of faecal samples Fresh faecal suspension was prepared from stool by inoculating a loopful of sample into sterile saline. Aliquots of 500ì l were added to 4.5ml of sterile Czapek broth (3.0g NaNO3, 1.0g K2HPO4, 0.5g MgSO4, 0.5g KC1, 0.01g FeSO4 in 1L of distilled water at pH 7.5) with antibiotic cocktail of PANTA Plus, Becton and Dickinson (1:100) in a sterile test tube containing paraffin wax coated slide, PSC (Infectech IdentikitTM USA). Two such tubes, one for staining and other for subculture, were inoculated. Stool samples were also cultured on two slopes of LJ medium after decontamination with 3% oxalic acid for 60 minutes. Processing of sputum samples For sputum samples, 500ì l of sputum was added to 4.5ml of sterile Czapek broth with PANTA Plus (1:100) in each of 2 tubes containing paraffin wax coated slides (PSC). Before inoculation on 2 slopes of LJ medium, modified Petroff’s method was used to process the sputum samples. The PSC tubes and the LJ bottles were incubated at 370 C in an incubator and were checked daily for growth. If visible growth was observed on PSC, one of the slides was removed from the tube for in situ acid fast staining by Kinyoun’s method. The slide was first fixed with alcohol and was then immersed in a tube containing the staining reagents8. The stained slides were then examined under microscope (10x, 40x, and 100x) for presence of acid fast colonies and acid fast organisms. If acid fast organisms were seen, NTM species were suspected as they, along with Nocardia, are the only acid fast organisms which utilize paraffin as the sole source of carbon and grow on this media. The growth from the other slide was then subcultured on to LJ media and 5 fresh tubes of PSC for further identification of the species. The growth on the freshly inoculated tubes was used for putting up tellurite reduction, nitrate reduction, urea hydrolysis and Tween 80 hydrolysis tests. Separate slides with growth of the isolated NTM were used for each test in the test tube containing Czapek broth and the reagents. Uninoculated media, with paraffin slide without growth were used as reagent control8. Speciation of growth on LJ medium was also performed by conventional methods. RESULTS From stool samples of 6 HIV seropositive patients, NTM grew by PSC, and five of them were suffering from diarrhoea. Out of these 6 isolates, 4 belonged to Mycobacterium avium complex (MAC) while 2 belonged to M. fortuitum complex. LJ could detect 5 NTM isolates, 4 MAC and 1 M. fortuitum (Table 1). None of the HIV seronegative subjects P. NARANG ET AL Table 1: Isolation of NTM from stool samples ** 4 M.avium complex and 2 M. fortuitum Figures in parentheses indicate horizontal percentages Symptoms HIV +ve subjects (n=80) No. tested PSC LJ Diarrhoeal 17 5 (29.41) 4 (23.52) Non-diarrhoeal 63 1 (1.59) 1 (1.59) Total 80 6** (7.5) 5 (6.25) 24 Indian Journal of Tuberculosis had NTM isolated from their stool samples. Mean isolation time for MAC was 19 days by PSC, while on LJ it was 21 days. For M.fortuitum complex isolation time was the same (12 days by both methods). Contamination rate for stool samples by PSC and LJ was 9.16% and 13.33% respectively (Table 3). M.tuberculosis was not isolated in any of the stool samples. In sputum samples also, NTM were isolated from samples of HIV seropositive subjects only. Three isolates of NTM (two MAC and one un- speciated) were isolated from a total of 42 sputum samples of HIV seropositive subjects. Positivity by PSC and LJ was the same. In one subject, MAC was isolated from both stool and sputum sample. The isolation time for MAC was the same (21 days) by PSC as well as LJ. The contamination rate was lower in PSC, 5.29% as compared to LJ, 7.05% (Table 3). As expected, no M.tuberculosis was isolated on PSC. However, on LJ medium, M.tuberculosis was the main isolate from sputum in both the groups but HIV seropositive subjects had more positivity (45.23%) than HIV seronegative (27.34%) (Table 2). DISCUSSION PSC technique proved to be a good method of isolation for NTM from clinical specimens of the patients. Since M.tuberculosis does not grow on PSC, any growth on the paraffin slide is an immediate indication of NTM or Nocardia species. In addition, the in situ acid alcohol fast staining procedure allows differentiation between these organisms. The Nocardia species are not acid alcohol fast using Kinyoun’s method and are filamentous on PSC, and therefore can be differentiated in the first instance. The risk of contamination was found to be low as compared to LJ. The reason could be that very few human pathogens and commensals are able to grow on paraffin wax and the technique is made more selective from the possible contamination by Pseudomonas aeruginosa or Candida tropicalis, the organisms which can contaminate the slides by the addition of antibiotic and antifungal cocktail, PANTA (Becton and Dickinson). This technique was used in our laboratory to speciate the known strains of mycobateria and was found to be easy and useful 8. In the present study, the technique has been used to isolate the NTM from clinical specimens and to speciate them. Six NTM species including 4 M.avium and 2 M.fortuitum from the stool specimens and 3 NTM species including 2 M. avium and one unspeciated from sputum samples were isolated. Isolation of MAC was equally good on PSC when compared to LJ. For stool samples, isolation time for MAC was lesser while it was the same for PARAFFIN SLIDE CULTURE TECHNIQUE FOR NTM ISOLATION Table 2: Isolation of mycobaterial species from sputum samples by PSC and LJ Table 3: Comparison of contamination on PSC and LJ (*) NTM; (**) Nocardia Figures in parentheses indicate horizontal percentages Specimen LJ PSC Stool (n=120) 16 (13.33) 11 (9.16) Sputum (n=170) 12 (7.05) 9 (5.29) Total (n=290) 28 (9.65) 20 (6.89) Subjects Positive for AFB on ZN staining Culture positive on PSC Culture positive on LJ for MTB and NTM HIV +ve (n=42) 16 (38.09) 3* (7.14) 19+3* HIV -ve (n=128) 24 (18.75 1** (0.78) 35+1** ) 25 Indian Journal of Tuberculosis sputum. This method has also been utilized for putting up drug resistance tests in our department (data not given here) and by other workers9. Also the PSC has been utilized for isolation of NTM from blood samples of HIV positive patients in our laboratory and other laboratories10. In this case, the sample is first cultured in the 13 A medium and then subcultured in PSC. PSC growth has also been utilized for DNA extraction and further characterization of NTM at genetic level (Ollar R.A. personal communication). Isolation of NTM using rapid methods and their identification up to species level using genetic probes is being done in the developed countries routinely. The exorbitant cost involved in these techniques is the major inhibitory factor for their incorporation in the laboratories of the developing countries. 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