Polymerase chain reaction targeting insertion sequence IS6110 for the diagnosis of pulmonary tuberculosis among sudanese children and young adults

This study was carried out in Khartoum State during the period from January 2011 to 1 December 2013 to improve the rate of detection of Mycobacterium tuberculosis [MTB] in children with symptoms of tuberculosis [TB] infection using different conventional and ,: advanced diagnostic techniques. One hundred and ninety-seven specimens of gastric f lavage and sputum were collected from different hospitals in Khartoum State, including | Elbolok Hospital, Jafar Ibn Owf Hospital, Elasha'ab Teaching Hospital, Soba University Hospital and Academy Charity Hospital. ; All children participating in the study were subjected to the Mantoux test after obtaining: appropriate consent injected by 5 tuberculin units of tuberculin purified protein derivative, and the results were recorded after three days. Specimens were decontaminated and inoc-i ulated on Lowenstein-Jensen media according to the modified Petroffs method. Two smears were prepared and stained by Ziehl-Neelsen stain and Auramine fluorescent dye; bacterial DNA was extracted from each specimen by using phenol chloroform method, and then the Polymerase Chain Reaction technique was adopted to detect Insertion Sequence IS6110 gene of MTB in these specimens. This study showed that the positive results for TST, ZN, Auramine, Culture and PCR were 86 [43.7%], 16 [8.1%], 22 [11.2%], 32 [16.2%] and 35[17.8%], respectively. The study concluded that the PCR technique is the most sensitive and specific technique for a quick identification of MTB in gastric lavage and sputum from children who are unable to expectorate a good quality sputum sample or who are diagnosed as negative using conventional diagnostic methods

Detection of multidrug-resistant tuberculosis using PCR compared to the conventional proportional method

To evaluate the PCR technique for the rapid defection of Multidrug-Resistant [MDR] Mycobacterium tuberculosis compared to the conventional proportional drug sensitivity testing. Cross sectional laboratory based study. Alshaab Teaching Hospital, Abu-Angah Hospital and the National Health Laboratory, Sudan. One hundred thirty tuberculosis suspected individuals of both sexes and of different ages were included in the study. Sputum samples were cultured on Lowenstein-Jensen [LJ] medium. Resistant strains were tested for the presence of mutations conferring resistance using molecular techniques to amplify 315 base pair [bp] rifampicin [RIF] and 146 bp isoniazid [INH], as markers for MDR among Mycobacterium tuberculosis. One hundred nineteen [91.5%] showed Mycobacterium tuberculosis-like colonies, 65 of which were randomly subjected to PCR and examined for the presence of IS6110 insertion sequences. Fifty-six [86.2%] were confirmed members of the Mycobacterium tuberculosis. The result of antibiotics susceptibility testing revealed that 32/56 [57.1%] of the strains were resistant to RIF, 36/56 [64.3%] to INH and 30/56 [53.6%] were resistant to both drugs [MDR]. The conventional method showed 21/56 [37.5%] were resistant to RIF, 32/56 [57.1%] to INH and 16/56 [28.6%] were resistant to both drugs [MDR]. Not all resistant strains detected by conventional were detected by PCR method; 14 [25%] were missed for RIF, 9 [16.1%] for INH and 4 [7.1%] for both. This represents a significant lack of sensitivity of the PCR technique, which could be due to the presence of other types of mutations that needs other primers and PCR protocol

Molecular detection of mycobacterium other than tuberculosis among patients with pulmonary infection

Isolation of Mycobacterium other than tuberculosis [MOTTs] among patients with pulmonary infection and evaluation of the usefulness of polymerase chain reaction [PCR] as a confirmatory test. Descriptive laboratory based study. National Health Laboratory [Stack], Elasha'ab Teaching Hospital, Abo Anga Hospital, Ebrahem Malek Hospital and Academy Charity Hospital. One hundred and seventy-one sputum samples were collected from suspected pulmonary tuberculosis patients, males and females with different ages. Informed consent was taken. Sputum samples were inoculated on LJ medium and organisms were identified according to their Ziehl-Neelsen stain, cultural characteristics and biochemical proprieties. The rapidly growing isolates were subjected to PCR. In this study, males were found to be more affected than females 125 [73.1%]. The patients between 21-50 years were the most affected with TB and NTM. On LJ medium, 40 [23.4%] of the isolates gave characteristic growth of Mycobacterium tuberculosis and were identified according to their Ziehl-Neelsen stain and cultural characteristic. Ten [5.8%] isolates were identified as rapid growers, 6 out of which were identified as MOTTs according to their indirect Ziehl-Neelsen stain, cultural characteristics and biochemical proprieties. On PCR, six of the ten rapid growers showed a band typical in size [136 bp] to target rpoB gene as indicated by the standard DNA marker. The results revealed clearly the importance of conventional methods including Z.N stain and culture techniques in the diagnosis of TB and NTM. As well as it proved the effectiveness of PCR as a sensitive, specific and rapid diagnostic and confirmatory test