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.
