Background: The tuberculosis and infections caused by nontuberculous mycobacterial (NTM) species are increasing in patients presented with respiratory illness, and it is crucial to document the epidemiology of these infections. Objectives: To study the mycobacterial species and in vitro drug susceptibility trends of Mycobacterium tuberculosis found in the respiratory specimens. Materials and methods: A prospective descriptive study from July 2009 to December 2012. The BACTEC MGIT system tubes with growth were used in the study. GenoType Mycobacterium (Hain Diagnostika, Nehren, Germany) assays were used to identify the mycobacteria. The drug susceptibility testing was performed by the MGIT 960 system. Results: A total of 1745 MGIT 960 system positive tubes were included. M. tuberculosis complex (MTC) constituted 67.45% of the yield isolated, 30.83% were nontuberculous mycobacterial species, 0.17% were Mycobacterium bovis BCG and 1.55% were not interpretable to species levels. Mycobacterium fortuitum (45.71%), Mycobacterium abscessus (26.21%) and Mycobacterium intracellulare (10.41%) were major NTM identified. The drug susceptibility study showed that 6.88% (81/1177) of MTC were drug-resistant TB, 56 isolates were resistant to one of the first-line anti-TB drugs, 25 isolates were found to be resistant to 2 or more first-line anti-TB drugs, of which 19 (20.46%) were MDR-TB and one of the isolates in the year 2011 was confirmed XDR-TB. Conclusion: M. tuberculosis, M. fortuitum, M. abscessus and M. intracellulare were major mycobacterial species detected in the respiratory samples. The drug susceptibility testing showed that the majority of MTC were sensitive to first-line anti-TB drugs.

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Mycobacterium tuberculosis (MTB) Central Asian Strain (CAS) lineage strains are predominant in South Asia. Mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) typing is an effective way of determining genetic diversity of strains. A maximum of 24 loci-based MIRU-VNTR typing can be used, however, it is important to investigate the relevance of specific MIRU loci for regional strains for more cost-effective MIRU typing. MIRU-VNTR typing was performed on MTB strains from Pakistan. Strains were comprised of CAS (n = 113) and non-CAS lineages (n = 87) – both multi-drug resistant (MDR) and drug susceptible. Hunter Gaston Discriminatory Index (HGDI) for each MIRU loci was interpreted as poor, moderate or highly discriminatory. Results were analyzed using Bionumerics software and miru-vntrplus database link. Clustering analysis revealed 185 different MIRU types. Eight clusters of 2 strains each were present amongst MDR (3 clusters) and drug susceptible (5 clusters) isolates. MDR clusters had orphan and Haarlem strains, whereas drug susceptible strain clusters were comprised of CAS and Beijing lineage strains. The HGDI for 15 loci-based MIRU typing of all isolates was 0.620, whereas HGDI for CAS was lower than non-CAS lineage strains (p-value: 0.023). HGDI of 8 MIRU-VNTR loci (Qub 26b, 10, 26, 4156, Mtub 04, 16, 31 and ETR-A) were all highly discriminatory. The average HGDI based on these 8 loci was significantly lower for CAS than non-CAS strains (P value: 0.03). The lower discriminatory index for CAS using both 15 and 8 MIRU loci-based analysis suggests less genetic diversity in these isolates than in other lineages. The eight highly discriminatory MIRU loci for CAS may help in monitoring the transmission of MTB strains in regions with high CAS lineage prevalence.

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Background: Nontuberculous mycobacteria (NTM) are a heterogeneous group of microorganisms with distinct clinical relevance. The treatment of NTM infections depends significantly upon the crucial identification of species at this level. The steady increase of mycobacteria species, the use of time-consuming techniques and the lack of standardized identification methods makes the achievement of this goal a demanding challenge. Additionally, inaccurate diagnosis can lead to therapeutic approaches consistent with Mycobacterium tuberculosis infection that are useless. In the present study, the performance of public databases in the accurate identification of NTM by sequence analysis of 16S rRNA and hsp65 genes were evaluated and compared. An algorithm is proposed to achieve an accurate classification of NTM in the geographic region of Portugal (Western Europe). Methods: Partial sequencing of 16S rRNA and hsp65 genes of 22 reference strains and 54 clinical isolates was performed. The resulting sequences were analysed by public web databases since their performance is evaluated statistically. The phenotypic characteristics of the isolates were also evaluated. Results: The use of commercial kits allowed the accurate identification of 57.4% of the clinical isolates. This result was improved either by the use of 16S rRNA (75.9%) and hsp65 (88.9%) genes analysis alone or combined (96.3%). Conclusions: Analysis of 16S rRNA gene alone is insufficient for the accurate identification of NTM. A stepwise algorithm combining 16S rRNA and hsp65 gene analysis by multiple public databases is proposed to identify NTM at the species' level.

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Background: Tuberculosis (TB) patients co-infected with human immunodeficiency virus (HIV) often lack the classic symptoms of pulmonary tuberculosis, making the diagnosis difficult. Current practices in resource-limited settings often indicate that these co-infected patients are diagnosed when they clinically manifest disease symptoms, resulting in a delayed diagnosis and despite continued transmission. The aim of this study is to determine the prevalence of undiagnosed pulmonary tuberculosis cases through active case finding and including multidrug-resistant TB (MDR-TB) among HIV-infected patients. Materials and methods: A total of 250 HIV-infected patients, aged 18 years and above were evaluated in a cross-sectional design between February 2012 and November 2012. Socio-demographic and clinical data were collected using a structured questionnaire. Sputum samples were collected from all participants for acid fast bacilli (AFB) direct smear microscopy and Mycobacteria culture. A PCR-based RD₉ deletion and genus typing, as well as first-line anti-TB drug susceptibility testing, was performed for all culture-positive isolates. Results: Following active TB case finding, a total of 15/250 (6%) cases were diagnosed as TB cases, of whom 9/250 (3.6%) were detected by both smear microscopy and culture and the remaining 6/250 (2.4%) only by culture. All the 15 isolates were typed through RD₉ typing of which 10 were Mycobacterium tuberculosis species; 1 belonged to Mycobacterium genus and 4 isolates were non-tuberculous mycobacteria. The prevalence of undiagnosed pulmonary TB disease among the study participants was 4.4%, which implies the possibility of identifying even more undiagnosed cases through active case finding. A multivariate logistic regression showed a statistically significant association between the presence of pneumonia infection and the occurrence of TB (OR = 4.81, 95% CI (1.08–21.43), p = 0.04). In addition, all the isolates were sensitive to all first-line anti-TB drugs, except for streptomycin, seen in only one newly diagnosed TB patient, and MDR-TB was not detected. Conclusion: The prevalence of undiagnosed pulmonary TB infection among HIV-infected patients in Gondar was 4.4%. Additionally, the possibility of these undiagnosed TB cases in the community could also pose a risk for the transmission of the disease, particularly among family members. Active screening of known HIV-infected individuals, with at least one TB symptom is recommended, even in persons with opportunistic infections.

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Buruli ulcer is a cutaneous mycobacterial disease caused by Mycobacterium ulcerans, whose incidence is increasing steadily, especially in West Africa. This study reports a first documented case of M. ulcerans infection which can be attributed to a water bug bite at the site of the primary lesion.

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Objective: Bangladesh is a high tuberculosis burden country. It is always challenging to diagnose active pulmonary tuberculosis (PTB) cases in rural areas where the setting up of conventional microscopic and cultural diagnostic tools is difficult. The objective of the present study is to find a feasible, reliable and easily accessible alternative diagnostic approach for PTB in the rural areas of Bangladesh. Methods: A total of 86 sputum samples were collected from clinically suspected PTB patients of Anantapur village, an underdeveloped remote area of Netrokona district, Bangladesh. Sputum samples were screened by Ziehl-Neelsen (Z-N) and fluorescence staining methods and were categorized as smear-positive active PTB cases (n = 50) and smear-negative controls (n = 36); then the performance of three popular immunological tests were evaluated, including ICT, ELISA and Mantoux tests (MT). Results: The sensitivity of ICT, ELISA, and MT (10 mm induration size) was 68%, 84% and 96%, respectively, and the specificity of these tests was 94.4%, 80.6% and 52.8%, respectively. When the cut-off size of induration in MT was changed from 10 to ≥15 mm, the sensitivity and specificity of MT became 92% and 83.3%, respectively. It was also found that the interpretation of MT was not significantly affected by BCG vaccination when ≥15 mm induration was taken as a cut-off value. Conclusion: Considering the resource-constraints of rural and remote areas, the Mantoux test could be an alternative tool for the diagnosis of active PTB.

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Background and objectives: The objective of this study is to synthesize and evaluate 1-(4-chlorophenyl)-4-(4-hydroxy-3-methoxy-5-nitrobenzylidene) pyrazolidine-3,5-dione (CD59) analogues to establish structure–activity relationship and mechanism of action. Methods: Thirty analogues of reported antitubercular CD59 were prepared by two-step synthetic protocols and characterized. The compounds were evaluated for in vitro activities against Mycobacterium tuberculosis (MTB), cytotoxicity against RAW 264.7 cells. The molecules were also evaluated for three mycobacterial enzymes to study the mechanism of action. Results: Among the compounds, 4-(2-bromobenzylidene)-1-(4-chlorophenyl)pyrazolidine-3,5-dione (4k) was found to be the most active compound in vitro with MICs of 4.13 μM against log-phase culture of MTB and also non-toxic up to 50 μM. Conclusions: Amongst all, the compounds 4g, 3i and 3n were most active against the enzymes MTB Pantothenate synthetase, lysine amino transferase and Alanine dehydrogenase, respectively. Further screening of these molecules was required in the in vitro dormant MTB models.

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This study aimed to compare traditional tests (Johnin test, fecal staining and fecal culture) with advanced laboratory tests (ELISA, LCD array and IS900 PCR) for detection of Johne's disease. A total of 365 Holstein–Friesian dairy cattle (40 express profuse diarrhea unresponsive to treatment and 325 contacting them) tested with Johnin test, blood collected for ELISA and fecal samples for fecal staining as well as fecal culture, application of LCD array and PCR using IS900 on DNA extracted from Mycobacterium paratuberculosis bacilli (from feces and culture). Johnin test was 40/40 (100%) and 25/325 (7.69%), fecal staining was 13 (37.1%) and 2 (50%), ELISA was 35/40 (87.5%) and 4/25 (16%) for clinical cattle and apparently healthy contacting them respectively. Isolation was 12/13 (92.3%) of the (Johnin test +ve, ELISA +ve and Acid Fast Bacilli +ve) from the clinically positive cattle and 1/2 (50%) of the (Johnin test +ve, ELISA +ve and Acid Fast Bacilli +ve) from apparently healthy contacting them while LCD array and IS900 gave 100% confirming the isolation results. In conclusion, LCD array depending on 16S RNA and DNA hybridization with specific probes for detection of M. paratuberculosis are fast, sensitive and labor-saving when combined with IS900.

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A 16-year-old female presented with a 6-month history of a gradually increasing swelling of the left side of her face. A panoramic radiographic view of the mandible showed diffuse radiolucency in the ramus of the mandible with a loss of cortication on the superior and anterior portion of the condyle. The computed tomography (CT) scan revealed destruction of the mandibular bone and a large retromandibular and inferior temporal fossa mass with areas of breakdown. The biopsy was consistent with tubercular osteomyelitis. Antitubercular therapy resulted in a marked reduction of the size of the swelling over a 9-month period.

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Background: Tuberculosis (TB) represents a serious public health problem in Mozambique, with an estimated incidence rate of 548 cases per 100,000 population in 2011. Information on the molecular epidemiology of Mycobacterium tuberculosis (MTB) strains circulating in Mozambique is limited. This study provides the first description of the genetic diversity of MTB strains circulating in Beira city, the second largest town in Mozambique. Methods: A total of 67 MTB isolates were tested to determine genetic lineages and diversity. The genetic lineages were determined using real-time PCR while genetic diversity was assessed by obtaining Mycobacterial Interspersed Repetitive Unit-Variable Numbers of Tandem Repeat profiles. Results: Only three of the six major lineages were represented, with 41 (61%) strains belonging to lineage 1, 25 (37%) belonging to lineage 4 and the remaining isolate belonging to lineage 3. No lineage 2 strains (containing the Beijing family) were identified. A high degree of diversity amongst the strains from both lineages 1 and 4 were observed. Comparison of the profiles of representative strains with those of reference strains in the MIRU-VNTRplus database revealed that all lineage 1 isolates clustered with the Eastern African Indian (EAI) 5 sub-family. The lineage 4 strains clustered with a variety of different sub-family strains, including the Latin-American-Mediterranean (LAM) 1 sub-family, the Haarlem, Uganda 1 and Cameroon sub-families and the T2-S sub-family. Conclusions: The TB epidemic in Beira city is caused by a diverse group of MTB strains predominantly belonging to lineages 1 and 4.

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Background: There is a paucity of published data on the effect of TB chemotherapy on nitric oxide (NO) synthesis and metabolism in newly diagnosed and relapsed patients with or without multi-drug resistant tuberculosis (MDRTB). Methods: The pattern of NO response in 140 patients with pulmonary TB, including 74 with MDR-TB and 66 without MDR-TB has been studied and compared to the NO status of 30 healthy donors. Patients comprised those with newly diagnosed TB and recurrent or relapsed TB. The NO status was assessed by measuring inducible NO synthase (iNOS) and nitrites and nitrates levels. This was measured prior to treatment initiation and two months after the prescribed chemotherapy. Results: Increased levels of NO indices were found in patients with tuberculosis when compared to healthy controls. After two months of chemotherapy a significant decrease in NO indicators was observed in the patients with TB, particularly in those without MDR-TB and newly diagnosed TB. The decline in NO activity was less prominent in patients with recurrent TB and MDR-TB, which suggests lower level of immunologic and reparative processes in such patients. Conclusion: Changes in serum levels of nitrites and nitrates as well as iNOS activity in neutrophils may serve as diagnostic criteria to differentiate various clinical forms of TB and help as prognostic tool to predict treatment outcome.

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Background: Early identification of determinants of tuberculosis (TB) treatment failure is urgently needed in resource-limited settings. This study describes the profile and determinants of TB treatment failure in a high-incidence setting where patients were managed at a TB control program with significant resource limitations. Methods: This was a retrospective case-control study carried out in one tertiary and one secondary hospital in Southeastern Nigeria. Cases were adult (≥15 years) TB patients with a positive sputum smear after 5 months of treatment (treatment failure). Controls were adult TB patients whose sputum smear was positive at the beginning of the treatment but who were smear-negative in the last month of treatment and on at least one previous occasion (cured). Cases were compared with controls to assess determinants of treatment failure. Results: Of the 1668 TB patients registered during the study period, 985 (59%) had smear-positive pulmonary TB. Of these, 694 (70.5%) were aged ≤40 years, 602 (61.1%) were males, 707 (71.8%) were rural residents, and 898 (91.2%) received care at the private facility. The prevalence of treatment failure was 2.5%. Significant determinants of treatment failure were: older age (>40 years) (P< 0.001), male gender (P = 0.04), previous treatment for TB (P = 0.045), and positive sputum smears after two month of anti-tuberculosis treatment (0.001). Conclusion: This study showed that the treatment failure rate among smear-positive TB patients is low in Nigeria. Education and improved clinical and laboratory interventions for the identified at-risk groups may reduce TB treatment failure in resource-limited settings.

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Isoniazid (INH) susceptibility testing for 100 Mycobacterium tuberculosis performed by conventional minimum inhibitory concentration (MIC) method was stratified using additional drug concentrations. Introduction of additional drug concentrations did not greatly improve the discriminatory capacity, but can be used in specialized studies pertaining to cross resistance between structural analogues of INH.

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