Background: The aim of this study was to evaluate the genotypic diversity of Mycobacterium tuberculosis strains using IS6110 RFLP, spoligotyping and MIRU-VNTR typing. Materials and methods: Between June 2008 and June 2009, all smear positive pulmonary specimens were collected prospectively at the national tuberculosis program (NTP) center of Duhok province. The specimens were processed for culture by modified Petroff's method and were inoculated into two tubes of Löwenstein-Jensen (LJ) media. The isolates were identified as M. tuberculosis by using biochemical tests and growth rate. Molecular fingerprinting of all M. tuberculosis strains was performed by IS6110 RFLP, spoligotyping and MIRU-VNTR. Results: M. tuberculosis strains were isolated from 53 Iraqi patients with pulmonary TB. Spoligotyping of M. tuberculosis isolates showed T family (30%) as the predominant genotype. By using the three molecular techniques, there were four spoligotyping clusters of strains (“3540 & 3516”, “3565 & 3563”, “3605 & 3618” and “865, 877 & 13811”). Complete concordance with RFLP was observed in one cluster of spoligotyping, but no concordance with MIRU-VNTR profile (234426153433 and 236424183433). Conclusions: Molecular fingerprinting methods are vital for differentiating a reactivation of latent infection from a recent transmission; however, it should be coupled with clinical epidemiological investigation. The low clustering rate in this study suggests that either reactivation of latent infections may be the main driving force for the endemic situation of the disease in Duhok, or it may indicate that a big circle of TB transmission is missed in the community, which means effective control measures have not been achieved yet in Duhok.

Background: Conventional biochemical tests are the standard for the identification of Mycobacterium species, but molecular identifications are becoming more prevalent. The rpoB gene encodes the β-subunit of RNA polymerase and is utilized for the identification of Mycobacterium species. In the present study, a stepwise Mycobacterium species identification algorithm using the 16S rRNA encoding gene and rpoB analysis was evaluated for its effectiveness. Methods: A total of 172 clinical Mycobacterium isolates were tested, and concordant results were obtained with 108 strains by using the conventional method and molecular methods (AccuProbe or DDH method). Results: In these 108 strains, 4 strains were identified by 16S rRNA gene analysis, but rpoB indicated no identical Mycobacterium species with more than 99% similarity. The remaining 64 strains were not identified by conventional method and commercial kits. Forty-two showed concordant results with 16S rRNA and rpoB analysis, and 13 strains were identified by 16S rRNA gene analysis although rpoB indicated no identical Mycobacterium species. On the other hand, 4 strains included 2 strains of Gordona and 2 strains of M. celatum type II which were identified by rpoB but not by 16S rRNA gene analysis. Finally, 5 strains could not be identified by analysis of either gene. The rpoB analysis can differentiate M. kansasii from M. gastri; M. malmoense from M. szulgai; M. abscessus from M. chelonae; M. peregrinum from M. septicum; M. porcinum from M. fortuitum; and M. farucinogense from M. senegalense—pairs that are not differentiated by 16S rRNA analysis. Additionally, Nocardia asteroids, Rhodococcus equi, Gordona aichiense, G. aurantiaca, G. bronchialis and G. terrae are able to be analyzed by using rpoB. Conclusions: The 16S rRNA gene identification is a rapid and prevalent method but still has some limitations. Therefore, the stepwise combination of rpoB with 16S rRNA gene analysis is an effective system for the identification of Mycobacterium species.

Background: There is a paucity of published data on burden and pattern of dual infection with Mycobacterium tuberculosis and HIV among migrants from South Asia, South-east Asia and sub-Saharan Africa entering the Middle-East, particularly Kuwait. Therefore, this study assessed the overall prevalence of HIV infection and pulmonary tuberculosis (TB) and evaluated the ecological relationship between them. Methods: Time series cross-correlation analysis was used to determine the ecological time-lagged relationship between the monthly proportions (per 100,000) of HIV seropositive and pulmonary TB cases among migrant workers that entered Kuwait between January 1, 1997 and December 31, 2006. Results: During the study period, overall prevalence (per 100,000) of HIV seropositivity and pulmonary TB among the migrants was 21 (494/23,28,582) (95% CI: 19–23), and 198 (4608/23,28,582) (95% CI: 192–204), respectively. Estimated cross-correlation function revealed a significant positive correlation (0.292±0.093) at lag −3 representing a positive relationship between the proportions of HIV seropositive (per 100,000) migrants tested 3 months earlier and the proportion of pulmonary TB (per 100,000) cases detected among migrants in a given month. Thus, the peak in proportion of pulmonary TB cases preceded the peak in proportion of HIV seropositive migrants indicating a direct time-lagged association between HIV seropositivity and the prevalence of pulmonary TB among migrants. Conclusions: HIV infection seemed to have played a significant role in the re-activation of latent M. tuberculosis infection in this migrant population. While currently less evident, in near future, however, TB and HIV/AIDS control programmes in the countries of origin of migrants may face a crucial challenge. Knowledge of serious consequences of association between HIV infection and pulmonary TB allows the promotion of public heath education to reduce the exposure to these infections. Future studies may focus on evaluating the impact of public health education programs on this dual burden of HIV infection and pulmonary TB in migrants.

Background: The use of molecular techniques is a major improvement for the rapid routine detection and control of multidrug-resistant tuberculosis (MDR-TB). Materials: In this study, the multiplex allele-specific polymerase chain reaction (MAS-PCR) was developed to simultaneously detect the most frequent mutations associated with isoniazid (INH) and rifampin (RIF) resistance in a single assay. Results: The assay was tested with 53 clinical isolates. Among them, 27 were MDR strains, 17 were mono-resistant to INH, one was mono-resistant to RIF, and eight were susceptible. The MAS-PCR assay showed a specificity of 100% in detecting drug resistance. An equivalent sensitivity of 92.6% in detecting MDR and RIF-resistance was found. The sensitivity for the detection of INH-resistance was 88.6%. Conclusions: The MAS-PCR assay was a simple and rapid method for detecting the INH and RIF-resistance in Mycobacterium tuberculosis (MT) clinical strains. It is also easy to perform and to interpret. The assay is inexpensive and a less-demanding technique.

Objective: The aim of this study was to assess the performance of a molecular line probe assay, GenoType^® MTBDRplus, for rapid detection of rifampicin and isoniazid resistance in the Mongolian situation. The sensitivity and specificity of GenoType^® MTBDRplus to detect rifampicin and isoniazid resistance-associated mutations in culture specimens and directly in smear-positive clinical specimens was examined. Method: 218 MDR-TB subjects aged between 14 and 75 years old from eight districts in Ulaanbaatar city (between July 2009 and May 2010) were included in this study .The GenoType Mycobacterium tuberculosis drug resistance first line (MTBDR plus) assay (Hain Life-science, Nehren, Germany) was tested on 109 clinical isolates and directly on 41 sputum specimens for the ability to detect the resistances. Results were compared with conventional culture and drug susceptibility testing on solid medium. Results: The high correlation of the results from GenoType^® MTBDRplus and conventional drug susceptibility testing was obtained from this study. The results clearly showed a high performance of GenoType^® MTBDRplus with almost 100% accuracy for all the important indicators, such as sensitivity, specificity, positive and negative predictive values and detection of rifampicin resistance. Discrepancies were obtained in comparison with DNA sequencing results. Conclusions: The Genotype^174; MTBDRplus assay was demonstrated as a rapid, reliable and highly accurate tool for early detection of MDR-TB through examining smear positive cases.

Objective: To investigate the performance of “tofu-whey liquid medium” for the propagation of Mycobacterium tuberculosis (MTB) strain H37Rv. Method: Two hundred micro liters (200μl) of 1 McFarland standard (1mg/ml-bacillary suspension) were inoculated into different batches of tofu-whey liquid medium. Each series contained three trials of test (tofu-whey liquid medium) and control media (Middlebrook 7H9 medium). Turbidity was measured within three weeks of inoculation using a nephelometer. The combinations of various tofu-whey liquid culture media were as follows; T1 (tofu-whey+ADC+glycerol+Potassium sulfate+Magnesium citrate+Sodium glutamate); T2 (tofu-whey + ADC + glycerol + Potassium sulfate + Magnesium citrate); T3 (tofu-whey + ADC + glycerol + Potassium sulfate); T4 (tofu-whey + ADC + glycerol); T5 (tofu-whey + ADC); and T6 (tofu-whey only). Results: In all test and control liquid culture media, the multiplication of M. tuberculosis was documented under light and fluorescence microscopy. Of various tofu-whey medium used, T1 demonstrated the most potential for MTB propagation. The increased turbidity reading represented by the value in “unit drop of % transmittance” was higher (25 scores) in the T1 tofu-whey medium, compared with the T6 tofu-whey medium (8 scores). The overall growth was significantly better in Middlebrook 7H9 culture media, although by the third day of incubation, the bacillary growth was superior in the T1 tofu-whey culture media. Sub-cultures in Lowenstein–Jensen (L–J) medium yielded between 87% (47 of 54) and 89% (48 of 54) recovery rate with between 7% and 13% contamination rate with coagulase-negative staphylococci. Conclusion: Tofu-whey media can be used as an economical alternative to Middlebrook 7H9 in resource-limited settings.

Disseminated Mycobacterium kansasii infection is a rare infection in non-HIV patients. This research has uncovered a very rare manifestation of disseminated M. kansasii infection in a non-HIV patient with lung and pericardial involvement.

Immobilization of bacterial cells onto surfaces is critical for imaging with an atomic force microscope. In this paper, polyethylenimine (PEI) coated silicon plates are shown to be suitable for immobilizing and imaging Mycobacterium tuberculosis.