Tuberculosis continues to cast a huge impact on humanity with its high incidence and mortality, especially in developing countries. For tuberculosis case detection, microscopy continues to be indispensible, given its low cost, rapidity, simplicity of procedure and high specificity. Modifications have attempted to improve the sensitivity of microscopy which include: concentration methods such as centrifugation, N-acetyl cysteine–sodium hydroxide, bleach, ammonium sulfate or chitin. Furthermore, classical Ziehl–Neelsen (ZN) staining has been subjected to varying carbol fuchsin concentrations or replaced by Kinyoun staining, fluorescent microscopy or immune-fluorescence. Currently, light emitting diode fluorescence is recognizably the most plausible method as an alternative to ZN staining.

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Nontuberculous mycobacteria (NTM) are emergent pathogens whose importance in human health has been gaining relevance after being recognized as etiological agents of opportunist infections in HIV patients. Currently, NTM are recognized as etiological agents of several respiratory and extra-respiratory infections of immune-competent individuals. The environmental nature of NTM together with the ability to assemble biofilms on different surfaces plays a key role on their pathogenesis. In the present work the ability of three fast-growing NTM (Mycobacterium smegmatis, Mycobacterium fortuitum and Mycobacterium chelonae) to persist within a model of human alveolar macrophages was evaluated. Most often human infections with NTM occur by contact with the environment. Biofilms can work as environmental reservoirs. For this reason, it was decided to evaluate the ability of NTM to assemble biofilms on different surfaces. Scanning electron microscopy was used to elucidate the biofilm structure. The ability to assemble biofilms was connected with the ability to spread on solid media known as sliding. Biofilm assembly and intracellular persistence seems to be ruled by different mechanisms.

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Purpose: PCR assay is a highly sensitive, specific and reliable diagnostic tool for the identification of pathogens in many infectious diseases. Genome sequencing Mycobacterium leprae revealed several gene targets that could be used for the detection of DNA from clinical and environmental samples. The PCR sensitivity of particular gene targets for specific clinical and environmental isolates has not yet been established. The present study was conducted to compare the sensitivity of RLEP, rpoT, Sod A and 16S rRNA gene targets in the detection of M. leprae in slit skin smear (SSS), blood, soil samples of leprosy patients and their surroundings. Method: Leprosy patients were classified into Paucibacillary (PB) and Multibacillary (MB) types. Ziehl–Neelsen (ZN) staining method for all the SSS samples and Bacteriological Index (BI) was calculated for all patients. Standard laboratory protocol was used for DNA extraction from SSS, blood and soil samples. PCR technique was performed for the detection of M. leprae DNA from all the above-mentioned samples. Results: RLEP gene target was able to detect the presence of M. leprae in 83% of SSS, 100% of blood samples and in 36% of soil samples and was noted to be the best out of all other gene targets (rpoT, Sod A and 16S rRNA). It was noted that the RLEP gene target was able to detect the highest number (53%) of BI-negative leprosy patients amongst all the gene targets used in this study. Conclusion: Amongst all the gene targets used in this study, PCR positivity using RLEP gene target was the highest in all the clinical and environmental samples. Further, the RLEP gene target was able to detect 53% of blood samples as positive in BI-negative leprosy cases indicating its future standardization and use for diagnostic purposes.

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Representative strains of Mycobacterium avium, Mycobacterium intracellulare and Mycobacterium scrofulaceum (MAIS) grew at equal rates in laboratory medium at 21% (air) and 12% oxygen. Growth in 6% oxygen proceeded at a 1.4–1.8-fold lower rate. Colony formation was the same at 21% (air) and 6% oxygen. The MAIS strains survived rapid shifts from aerobic to anaerobic conditions as measured by two experimental approaches (Falkinham (1996) ^[1]). MAIS cells grown aerobically to log phase in broth were diluted, spread on agar medium, and incubated anaerobically for up to 20 days at 37 °C. Although no colonies formed anaerobically, upon transfer to aerobic conditions, greater than 25% of the colony forming units (CFU) survived after 20 days of anaerobic incubation (Prince et al. (1989) ^[2]). MAIS cells grown in broth aerobically to log phase were sealed and vigorous agitation led to oxygen depletion (Wayne model). After 12 days anaerobic incubation, M. avium and M. scrofulaceum survival were high (>50%), while M. intracellulare survival was lower (22%). M. avium cells shifted to anaerobiosis in broth had increased levels of glycine dehydrogenase and isocitrate lyase. Growth of MAIS strains at low oxygen levels and their survival following a rapid shift to anaerobiosis is consistent with their presence in environments with fluctuating oxygen levels.

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Nontuberculous mycobacteria (NTM) are a diverse group of bacterial species that are distributed in the environment. Many of these environmental bacteria can cause disease in humans. The identification of NTM in environmental sources is important for both clinical and epidemiological purposes. In this study, the distribution of NTM species from environmental and clinical samples in the Middle East was reviewed. In order to provide an overview of NTM, as well as recent epidemiological trends, all studies addressing NTM in the Middle East from 1984 to 2014 were reviewed. A total of 96 articles were found, in which 1751 NTM strains were isolated and 1084 of which were obtained from clinical samples, 619 from environmental samples and 48 were cited by case reports. Mycobacterium fortuitum was the most common rapid growing mycobacteria (RGM) isolated from both clinical (269 out of 447 RGM; 60.1%) and environmental (135 out of 289 RGM; 46.7%) samples. Mycobacterium avium complex (MAC) was the most common slow growing mycobacteria (SGM) isolated from clinical samples (140 out of 637 SGM; 21.9%). An increasing trend in NTM isolation from the Middle East was noted over the last 5 years. This review demonstrates the increasing concern regarding NTM disease in the Middle East, emphasizing the need for regional collaboration and coordination in order to respond appropriately.

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Background: Nigeria ranks 10th among 22 high TB burden countries with low TB case detection that relies on passive case finding. Although there is increasing body of evidence that active case finding (ACF) has improved TB case finding in urban slums in some parts of the world, this strategy had not been implemented in Nigeria despite the pervasiveness of urban slums in the country. Objective: To assess the yield and profile of TB in urban slums in Nigeria through ACF. Methods A prospective, implementation study was conducted in three urban slums of southeastern Nigeria. Individuals with TB symptoms were identified through targeted screening using a standardized questionnaire and investigated further for TB. Descriptive and bivariate analyses were performed using SPSS. Results: Among 16,743 individuals screened for TB, 6361 (38.0%) were identified as TB suspects; 5894 suspects were evaluated for TB. TB was diagnosed in 1079 individuals, representing 6.4% of the screened population and 18.3% of those evaluated for TB. Of the 1079 cases found, 97.1% (n = 1084) had pulmonary TB (PTB), and majority (65%) had new smear-positive TB. Children (<15 years) accounted for 6.7% of the cases. Also, 22.6% (216) of the cases were HIV co-infected, among whom 55.1% (n = 119) were females. The average number of individuals needed to screen to find a case of TB was 16. Conclusions: There is high prevalence of TB in Nigeria slum population. Targeted screening of out-patients, TB contacts, and HIV-infected patients should be optimized for active TB case finding in Nigeria.

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Objective: To determine the utility of polymerase chain reaction (PCR) for diagnosing pediatric pulmonary tuberculosis (PPTB). Method: A prospective cross-sectional study was carried out on 100 children less than 14 years of age, with strong clinical suspicion and radiological evidence suggestive of pulmonary tuberculosis (TB). Sputum samples/gastric lavage were collected. Direct smears and culture on Lowenstein Jensen (LJ) media were performed. DNA extraction and amplification was performed using Genei™ Amplification Reagent set for Mycobacterium tuberculosis (MTB) (by Genei, Bangalore, India). This test is based on the principle of single-tube nested PCR which amplifies the repetitive insertion sequence IS6110. Results: When compared with culture, sensitivity and specificity of PCR was 93.55% and 92.75%, respectively. The PPV was 85.29% and the NPV was 96.97%. When intention to treat (ITT) was used as the standard, sensitivity, specificity, PPV and NPV of PCR was 47.88%, 93.1%, 94.4%, and 42.19%, respectively, and that of culture was 40.85%, 100%, 100% and 40.85%, respectively. Against response to treatment (RTT), PCR demonstrated sensitivity, specificity, PPV and NPV of 50.9%, 93.1%, 93.33% and 50%, respectively, and for culture it was 43.64%, 100%, 100% and 48.33%, respectively. Conclusion/recommendation: The present study reinforces better case detection rate with PCR-based nucleic acid amplification test as compared with microscopy and culture in pediatric pulmonary TB. PCR showed a higher correlation with clinical diagnosis as compared with microscopy and solid culture. Hence, a molecular platform should be the test of choice for detecting PPTB.

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Background: To date, the advancements in polymerase chain reaction (PCR) assures accurate, fast identification and mycobacterial speciation in clinical settings, which promotes a better tuberculosis (TB) treatment regimen. Methods: In this study, a total of 78 clinically suspected cases of TB were processed for the detection of Mycobacterial infections by standard Ziehl Neelsen (ZN) staining, conventional Lowenstein–Jensen (LJ) and BACTEC MGIT-960™ liquid culture. Strain typing was performed by using Double Repetitive Element PCR (DRE-PCR) and Duplex PCR (DPCR) to differentiate Mycobacterium tuberculosis complex (MTB) from non-tuberculous mycobacteria (NTM), respectively. Results: Of 78 clinical isolates, 25 (32%) were drug-susceptible, and 53 (68%) were resistant to at least one drug. The BACTEC MGIT-960™ showed the highest (88.5%) positivity rate, compared with conventional LJ (82%) and ZN smear (61.5%). The mean time detection and drug susceptibility for MTB was 28 and 40 days in LJ culture, and 10 and 13 days in BACTEC MGIT-960™ culture. Using DPCR, Mycobacterium avium infection was identified in HIV-positive (2.56%) and MTB in HIV-negative patients (97.4%), and the DRE-PCR system divulged 15 unique genotype patterns, and an institutional-based epidemiology database was created. Conclusions: The combination of an in-house DRE–DPCR system could possibly identify and differentiate MTB from other mycobacterial species in a single reaction. In addition, restriction polymorphism analysis and DNA sequencing of NTM could assist in species identification directly from clinical isolates.

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Background: This study was conducted in Kassala Teaching Hospital, Kassala State, Sudan (January 2006–June 2008) to determine the rate of Mycobacterium drug resistance to anti-tuberculous treatment and to explore the genotype of Mycobacterium tuberculosis resistant isolates using rpoB gene. Methods: 53 isolates of Mycobacterium isolated from pulmonary tuberculosis (PTB) patients from Kassala State were subjected to drug susceptibility testing (DST) to anti-tuberculous drugs; 10 M. tuberculosis complex (MTBC) resistant isolates were subjected to polymerase chain reaction (PCR), and commercially the amplified DNA was sequenced. Results: DST detected resistance in 23/53 (43.39%) isolates, among which rifampicin had a high number of resistant isolates (13/23), followed by streptomycin (11/23), and multi-drug resistance was detected in 5 isolates. DNA sequence analysis of 10 MTBC-resistant isolates detected variations within and outside the rifampicin resistant determining region (RRDR). Variation within RRDR was detected at positions 512 (AGC/ATC, Ser/Ile), and 528 (CGC/CTC, Arg/Leu). Outside the RRDR region variations were detected at positions 498 (GTG/GGG, Val/gly), 488 (ACA/ACC, Thr/Thr), which is a silent mutation. Insertions were observed at positions 484, 496 (GTG/GTGA, CGG/CAGG, respectively). Deletion was observed at position 487 (ATC/_TC). Discussion and conclusion: This study revealed that high resistance to rifampicin was associated with various point mutations in and out of the RRDR of the rpoB gene. Molecular methods are needed for early detection of TB disease and drug resistance.

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Objective: To determine the utility of light-emitting diode fluorescent microscopy (LED-FM) for the diagnosis of pulmonary tuberculosis (PTB) in HIV-infected patients. Material and methods: A cross-sectional study was performed on 400 HIV-infected, clinically or radiologically suspected PTB patients. Two sputum specimens were collected from each patient. Two direct smears were prepared from each sputum specimen. One was stained by ZN method and another by auramine-O method and reported as per the Revised National Tuberculosis Control Programme (RNTCP) guidelines. LED-FM stained smears were reported by two readers. All specimens were cultured on LJ medium after digestion and decontamination. Address and contact details of all the patients were recorded in case record form. They were contacted for follow-up if required. Results: Of the 800 sputum specimens processed, 130 were positive by LED-FM method and 33 were positive by ZN method; 77 specimens showed growth of MTB on LJ medium. When compared with solid culture as a reference standard, LED-FM has a sensitivity of 67.53%, specificity of 88.71%, PPV of 40% and NPV of 96.08%. Seventy-eight LED-FM positive and culture negative specimens had scanty grading. Of these, 15 were confirmed as having PTB as they responded to anti-TB treatment. The concordance between two readers was 98.75%. Conclusion: LED-FM can be a good screening test for the diagnosis of PTB in HIV-infected patients. However, all scanty grade positive smears need to be confirmed by WHO approved gold standard.

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Mutations in genes involved in drug metabolism have been well-associated with drug resistance. Sequence analysis of known antimycobacterial drug-resistant genes is often used to predict resistance to antibiotics. However, some polymorphisms in such genes may serve a phylogenetic purpose rather than resistance to drugs. The Beijing family of Mycobacterium tuberculosis (MTB) is prevalent worldwide and has been associated with the emergence of multidrug resistance. Sequence type (ST) 10 of the Beijing family is the most predominant in countries like Peru, Taiwan and Thailand. A sequence analysis was performed of 81 previously reported drug-resistant associated genes in multidrug-resistant and pan-susceptible strains of the Beijing family sequence type 10 of MTB. This analysis revealed 10 synonymous and 12 nonsynonymous single nucleotide polymorphisms (SNPs) that are shared by all strains under study. One frameshift mutation was also observed to be common to all. These data might be useful in excluding some observed SNPs in drug-resistant-associated genes of MTB Beijing ST 10 when performing genotypic drug susceptibility assay.

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Background: Mycobacterium tuberculosis (MTB) PE_PGRS genes belong to the PE multigene family. Although the function of PE_PGRS genes is unknown, it is hypothesized that the PE_PGRS genes may be associated with antigenic variability in MTB. Material and methods: Whole genome sequencing analysis was performed on (n = 37) extensively drug-resistant (XDR) MTB strains from Pakistan, which included Lineage 1 (East African Indian, n = 2); Other lineage 1 (n = 3); Lineage 3 (Central Asian, n = 24); Other lineage 3 (n = 4); Lineage 4 (X3, n = 1) and T group (n = 3) MTB strains. Results: There were 107 SNPs identified from the analysis of 42 PE_PGRS genes; of these, 13 were non-synonymous SNPs (nsSNPs). The nsSNPs identified in PE_PGRS genes – 6, 9 and 10 – were common in all EAI, CAS, Other lineages (1 and 3), T1 and X3. Deletions (DELs) in PE_PGRS genes – 3 and 19 – were observed in 17 (80.9%) CAS1 and 6 (85.7%) in Other lineages (1 and 3) XDR MTB strains, while DELs in the PE_PGRS49 were observed in all CAS1, CAS, CAS2 and Other lineages (1 and 3) XDR MTB strains. All CAS, EAI and Other lineages (1 and 3) strains showed insertions (INS) in PE_PGRS6 gene, while INS in the PE_PGRS genes 19 and 33 were observed in 20 (95.2%) CAS1, all CAS, CAS2, EAI and Other lineages (1 and 3) XDR MTB strains. Conclusion: Genetic diversity in PE_PGRS genes contributes to antigenic variability and may result in increased immunogenicity of strains. This is the first study identifying variations in nsSNPs and INDELs in the PE_PGRS genes of XDR-TB strains from Pakistan. It highlights common genetic variations which may contribute to persistence.

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