Frequency of Non Tuberculosis Mycobacteria Among Pulmonary Tuberculosis Patients in Khartoum State

Abstract

This study aimed to detect the frequency of non tuberculous mycobacteria among tuberculous patients in Khartoum state by conventional and molecular methods. This study is a cross-sectional laboratory-based study in which sputum samples were collected from patients attending Abu-Anga Teaching Hospital, El Shaab Teaching Hospital and the Tuberculosis Reference Laboratory at the national Health Laboratory in Khartoum, Sudan, during the period from January to March 2010. Patients were consented and informed. Sputum samples that showed AFB- positive results were included. Two tubes of the Lowenstein-Jensen medium were inoculated with 20μl of the neutralized sputum sample that was obtained from the patients and decontaminated. One of the tube contained pyruvic acid to isolate Mycobacterium bovis if encountered. All sputum samples were inoculated and in LJ media, 40 (23.4%) showed MTC- like colonies, 10 (5.8%) were considered rapidly growing mycobacteria, 2(1.2%) showed contamination and 119 (69.6%) no growth. The recover rapidly growing colonies were identified by conventional methods. Out of the 10 rapidly growing, 4 (2.3%) of the isolates were non tuberculosis mycobacteria organisms which were identified by conventional methods and PCR. The biochemical tests regarding the NTM isolates showed that 4 out of 4 (100%) were sensitive for Para-nitrobenzoic acid (growth was inhibited by PNB); 4 out of 4 (100%) were resistant to Thiophene – 2 – Carboxylic Acid Hydrazide TCH; 2 out of 4(50%) were positive for nitrate reduction, all the 4 isolates were negative for catalase test at 68°C while 3 out of 4 (75%) were catalase positive at room temperature. Three out of the four isolates (75%) showed the standard patterns of mycolic acid components when thin layer chromatographic technique was used. Then the result of the conventional methods was confirmed by PCR. When the one hundred and fourty five Mycobacterial isolates were subjected to PCR. Four isolates showed a band typical in size (136 bp) to the target gene (rpoB) of NTM as indicated by the standard DNA marker. The rest one hundred and fourty one showed a band typical in size (123 bp) to the target gene (IS6110) of MTB as indicated by the standard DNA marker. These results revealed a prevalence of (4,2.3%) isolates having phenotypic properties typical for members of the genus Non Tuberculosis Mycobacteria, and revealed clearly the importance of conventional methods and PCR technique in the diagnosis of pulmonary patients especially if there is other invader of non tuberculosis mycobacteria.