Occurrence of RD149 and RD152 deletions in Mycobacterium tuberculosis strains from Pakistan

Introduction: Central Asian Strain 1 (CAS1) is the predominant Mycobacterium tuberculosis genotype in Pakistan. The occurrence of deletions in regions of differences (RDs) among CAS1 and other predominant genogroups in the country were investigated. Methodology: Using stratified random sampling, 235 M. tuberculosis (185 pulmonary, 50 extrapulmonary) strains were selected from 926 previously spoligotyped isolates, including 171 CAS strains (133 CAS1 (ST26), 38 CAS subfamily), 8 Beijing isolates, 47 isolates belonging to other previously defined ( Other ) clusters, and 9 previously undefined Unique isolates. Commonly reported RD deletions, RD1, RD750, RD207, RD149, RD152, RD105, RD150, RD142 and RD181, were investigated using a PCR - based method. Results: Deletions in RDs 750, 149 and 152 were identified among CAS strains, and in RDs 207, 149, 152, 105, 150, 142 and 181 in Beijing isolates. CAS1 strains showed more frequent RD149 deletions compared with CAS subfamily strains (p=0.036), and more frequent RD152 deletions compared with Other clusters (p=0.003). RD149 and RD152 deletions were more frequent in Beijing isolates compared with CAS1 strains (p \u3c 0.001). Concurrent RD149 and RD152 deletions were more frequent in CAS1 compared with Other clusters (p \u3c 0.001) and in Beijing strains compared with CAS1 (p \u3c 0.001). No significant difference was detected in RD deletion patterns between pulmonary and extra pulmonary isolates. Conclusion: Higher frequencies of RD149 and RD152 deletions and of concurrent RD149 and RD152 deletions were found in CAS1 and Beijing strains compared with CAS subfamilies, Other clusters and Unique strains. No association between these deletions and disease presentation, pulmonary or extrapulmonary tuberculosis, was observed

Characterization of Mycobacterium tuberculosis Central Asian Strain1 using mycobacterial interspersed repetitive unit genotyping

<p>Abstract</p> <p>Background</p> <p>The Central Asian Strain1 (CAS1) genogroup of <it>Mycobacterium tuberculosis </it>(MTB) is the most prevalent in Pakistan, India and Bangladesh. Mycobacterial interspersed repetitive units variable number tandem repeat (MIRU-VNTR) typing is a reliable and reproducible method for differentiation of MTB isolates. However, information of its utility in determining the diversity of CAS1 strain is limited. We performed standard 12 loci based MIRU-VNTR typing on previously spoligotyped CAS1 strains and 'unique' strains in order to evaluate its discriminatory power for these isolates.</p> <p>Methods</p> <p>Twelve loci based MIRU- VNTR typing was used to type178 CAS1 and 189 'unique' MTB strains. The discriminatory index for each of the loci was calculated using the Hunter Gaston Discriminatory Index (HGDI). A subset of these strains (n = 78) were typed using IS<it>6110 </it>restriction fragment length polymorphism (RFLP). MIRU-VNTR profiles were studied together with their drug susceptibility patterns.</p> <p>Results</p> <p>A total of 349 MIRU patterns were obtained for the 367 strains tested. The CAS1 strains were subdivided into 160 distinct patterns; 15 clusters of 2 strains each, 1 cluster of four strains and 144 unique patterns. Using HGDI, seven MIRU loci, (numbers 26, 31, 27, 16, 10, 39, and 40) were found to be "highly discriminatory" (DI: ≥0.6), four MIRU loci (numbers 20, 24, 23, and 4) were "moderately discriminatory" (DI: 0.3–0.59), and one locus (number 2) was "poorly discriminatory" (DI< 0.3). Loci 26 and 31 were the most discriminatory for the CAS1 isolates. Amongst 'unique' strains in addition to loci 26, 31, 27, 16, 10, 39, and 40, locus 23 was highly discriminatory, while no locus was poorly discriminating. DI values for loci 4, 10 and 26 were significantly lower (P-value < .01) in CAS1 strains than in 'unique' strains. The association between CAS1 strains and MDR was not found to be significant (p value = 0.21).</p> <p>Conclusion</p> <p>We propose that MIRU typing could be used to estimate the phylogenetic relatedness amongst prevalent CAS1 strains, for which MIRU loci 26, 31, 16, 10, 27, 39 and 40 were found to be the most discriminatory.</p

Extensively Drug-Resistant Tuberculosis, Pakistan

Frequency of extensively drug-resistant tuberculosis in Pakistan increased from 1.5% in 2006 to 4.5% in 2009 (p<0.01). To understand the epidemiology, we genotyped selected strains by using spoligotyping, mycobacterial interspersed repetitive units–variable number of tandem repeats, and IS6110 restriction fragment length polymorphism analysis

Genotyping and drug resistance patterns of M. tuberculosis strains in Pakistan

<p>Abstract</p> <p>Background</p> <p>The incidence of tuberculosis in Pakistan is 181/100,000 population. However, information about transmission and geographical prevalence of <it>Mycobacterium tuberculosis </it>strains and their evolutionary genetics as well as drug resistance remains limited. Our objective was to determine the clonal composition, evolutionary genetics and drug resistance of <it>M. tuberculosis </it>isolates from different regions of the country.</p> <p>Methods</p> <p><it>M. tuberculosis </it>strains isolated (2003–2005) from specimens submitted to the laboratory through collection units nationwide were included. Drug susceptibility was performed and strains were spoligotyped.</p> <p>Results</p> <p>Of 926 <it>M. tuberculosis </it>strains studied, 721(78%) were grouped into 59 "shared types", while 205 (22%) were identified as "Orphan" spoligotypes. Amongst the predominant genotypes 61% were Central Asian strains (CAS ; including CAS1, CAS sub-families and Orphan Pak clusters), 4% East African-Indian (EAI), 3% Beijing, 2% poorly defined TB strains (T), 2% Haarlem and LAM (0.2). Also TbD1 analysis (<it>M. tuberculosis </it>specific deletion 1) confirmed that CAS1 was of "modern" origin while EAI isolates belonged to "ancestral" strain types.</p> <p>Prevalence of CAS1 clade was significantly higher in Punjab (P < 0.01, Pearsons Chi-square test) as compared with Sindh, North West Frontier Province and Balochistan provinces. Forty six percent of isolates were sensitive to five first line antibiotics tested, 45% were Rifampicin resistant, 50% isoniazid resistant. MDR was significantly associated with Beijing strains (P = 0.01, Pearsons Chi-square test) and EAI (P = 0.001, Pearsons Chi-square test), but not with CAS family.</p> <p>Conclusion</p> <p>Our results show variation of prevalent <it>M. tuberculosis </it>strain with greater association of CAS1 with the Punjab province. The fact that the prevalent CAS genotype was not associated with drug resistance is encouraging. It further suggests a more effective treatment and control programme should be successful in reducing the tuberculosis burden in Pakistan.</p

Mycobacterium tuberculosis genotypes in patients developing pulmonary tuberculosis related acute respiratory distress syndrome

Background: Acute Respiratory Distress Syndrome is an uncommon but frequently fatal presentation of pulmonary TB. We attempted to identify whether a specific M. tuberculosis genotype occurs more commonly in patients who develop ARDS.Methods: An observational study carried out at the Aga Khan University Hospital, Karachi, Pakistan enrolled all ARDS patients with tuberculosis. MTB isolates were spoligotyped for strain identification.Results: 725 patients were admitted with pulmonary tuberculosis during the study period. Only 2.5% (18 patients) developed ARDS. Inpatient mortality rate was 58% (7 patients). Genotypes of M. tuberculosis isolates were predominantly CAS I (58.3%) and Unique strains (25%). Beijing and CAS subfamilies were less common; with each genotype identified in 8.3% patients respectively. All except one of the isolated strains were sensitive to usual first line anti-­tubercular drugs

M. tuberculosis Central Assian Strain 1 MDR isolates have more mutations in rpoB and katG genes compared with other genotypes

Pakistan ranks eighth globally among TB burden countries, with a MDR rate of 2-5%. The most prevalent MTB genotype is Central Asian Strain1 (CAS1) followed by the Beijing genogroup. We investigated common mutations in multidrug resistance encoding genes rpoB, katG and inhA of CAS1 and Beijing strains using DNA sequencing and fluorescent resonance energy transfer (FRET) probe based real-time PCR methods. 30 CAS1, 12 Beijing and 20 unclustered spoligotypes, and 10 susceptible MTB strains were tested. The most common mutations in the rpoB gene were at codons 531 (60%), 526 (23%) and 516 (5%). CAS1 strains had a higher frequency of mutations at codon 526 (

Reduced TNF-alpha and IFN-gamma responses to Central Asian strain 1 and Beijing isolates of Mycobacterium tuberculosis in comparison with H37Rv strain

Pakistan ranks eighth in terms of tuberculosis burden worldwide, with an incidence of 181/100 000. The predominant genotypes of Mycobacterium tuberculosis are reported to be the Central Asian strain 1 (CAS1) and Beijing families. Mycobacterium tuberculosis down-regulates host pro-inflammatory cytokines, which are essential for protection against infection. There is currently little information regarding the interaction of the CAS1 genotype with host cells. We studied the growth rates of CAS1 and Beijing clinical isolates, and their ability to induce cytokines compared with the laboratory reference strain H37Rv. Host responses were studied using a THP-1 monocytic cell tine model and an ex vivo whole blood assay. Growth rates of CAS1 and Beijing isolates were significantly lower (P = 0.011) compared with H37Rv. All clinical isolates induced significantly tower levels of TNF-alpha secretion (P = 0.003) than H37Rv in THP-1 cells and in the whole blood assay of healthy donors (n = 8). They also induced tower IFN-gamma secretion in the whole blood assay (P \u3c 0.001). A positive correlation was observed between the growth indices (GI) of H37Rv, Beijing and CAS1 strains and the TNF-alpha responses they induced [Pearson\u27s correlation coefficient (R(2)): 0.936, 0.775 and 0.55, respectively], and also between GI and IFN-gamma production (R(2): 0.422, 0.946, 0.674). These findings suggest that reduced growth rate, together with down-modulation of pro-inflammatory cytokines, is a contributory mechanism for the predominance of the CAS genotype

Spoligotyping of Mycobacterium tuberculosis Isolates from Pakistan Reveals Predominance of Central Asian Strain 1 and Beijing Isolates

The estimated incidence of tuberculosis in Pakistan is 181 per 100,000; however, there is limited information on Mycobacterium tuberculosis genotypes circulating in the country. We studied 314 M. tuberculosis clinical isolates; of these, 197 (63%) isolates grouped into 22 different clusters, while 119 (37%) had unique spoligotypes. Eighty-nine percent of the isolates were pulmonary (Pul), and 11% were extrapulmonary (E-Pul). We identified Central Asian Strain (CAS), Beijing, T1, Latin American-Mediterranean, and East African-Indian genogroups. Beijing strains, reportedly the most prevalent spoligotype worldwide, constituted 6% of our strain population. The CAS1 strain comprised 121 (39%) of the study isolates. No difference was observed between clustered isolates from cases of Pul and E-Pul tuberculosis. However, E-Pul isolates included a greater number of unique spoligotypes than Pul isolates (P = 0.005). The overall percentage of drug resistance was 54%, and that of MDR strains was 40%. While CAS1 strains were not associated with drug resistance, the relative risk of MDR was significant in Beijing strains compared to the non-Beijing groups (95% confidence interval, 1.2 to 8.9). The fact that the predominant strain, CAS1, is not associated with drug resistance is encouraging and suggests that an effective tuberculosis control program should be able to limit the high incidence of disease in this region

Prevalence of ST26 among untreated smear-positive tuberculosis patients from Karachi indicating ongoing transmission

Tuberculosis (TB) control is a major healthcare priority for Pakistan. We have studied Mycobacterium tuberculosis strains from the sputa of 100 treatment-naive, smear-positive pulmonary TB cases from Karachi, Pakistan, to identify strains most responsible for active transmission in this population. DNA extracted from M. tuberculosis isolates were subjected to spacer oligotyping (spoligotyping). Sixty-six (66%) clinical isolates were grouped into 9 different clusters. The largest cluster comprised the Central Asian Strain (CAS) 1 or ST26 (n = 40). The remaining isolates (34%) had unique spoligotypes. We conclude that ST26 being the most prevalent strain in smear-positive cases contributes greatly towards ongoing transmission in Karachi. Our data further suggest that ST26 may have a selection advantage not afforded by other genotypes. This conclusion is further supported by DESTUS analysis (Detecting Emerging Strains of Tuberculosis Using Spoligotypes) identifying ST26 as the only emerging spoligotype. Reasons for the spread of ST26 require further study