Research Article: Emergence of mixed infection of Beijing/Non-Beijing strains among multi-drug resistant Mycobacterium tuberculosis in Pakistan

Date Published: April 19, 2016

Publisher: Springer Berlin Heidelberg

Author(s): Samar Mustafa, Hasnain Javed, Jawairia Hashmi, Nazia Jamil, Zarfishan Tahir, Abdul Majeed Akhtar.


Tuberculosis (TB) remains as one of the deadliest diseases after HIV globally with 95 % of deaths confined to low-and-middle income countries. Pakistan is fifth among the 22 high-burden TB countries with the incidence rate of 230/100,000 persons, however, studies related to prevalent Mycobacterium tuberculosis strains and their spread, drug resistance pattern and evolutionary genetics are inadequate. The present study was undertaken to highlight the circulation of M. tuberculosis strains causing drug resistant TB in our community by targeting the molecular marker IS6110 and then characterization of these strains as Beijing and Non-Beijing genotypes. Sputum samples from 102 MDR TB suspects from different cities of Punjab were collected and their record was stored in a database. Sputum samples were evaluated by Ziehl Neelson staining and cultured on Lownstein Jensen medium by Modified Petroff’s method. DST was performed for first-line anti-mycobacterial drugs by indirect proportion method. Mycobacterium tuberculosis isolates were investigated for the presence of IS6110 and further identification as Beijing, Non-Beijing or mixed genotype. Percentage of male and female patients was found to be 58.8 and 41.2 % respectively. DST showed resistance of 93 % of isolates to isoniazid and rifampicin. All of the isolates showed positive results for IS6110 amplification. Based on PCR amplification of Beijing and non-Beijing primer sets 4.9 % of the patients showed infection with pure Beijing isolates, 14.7 % with both Beijing and non-Beijing isolates and 80.3 % with pure non-Beijing isolates. Analysis of IS6110 and Beijing sequences showed the presence of putative transposase conserved domain while non-Beijing sequences were epitomized with RAMP_I_III superfamily domain (CRISPR-associated protein family). TB in Pakistan is predominantly caused by Non-Beijing genotypes, but Beijing strains showed incessant circulation in our community as both single and mixed (co-infecting Non-Beijing and Beijing) strains.

Partial Text

Tuberculosis (TB) continues as one of the deadliest contagious diseases across the globe. Nearly 9 million people developed TB in 2013 and the year ended with the demise of 1.5 million people due to TB. Of the total TB cases and deaths, around 56.25 % are men but the burden of the disease is eminent in women as well. To no surprise, out of the total TB cases occurred in 2014, 58 % belonged to the South-east Asian and Western Pacific regions (WHO 2015).

Acid-fast bacilli (AFB) microscopy and mycobacterial culturing have been practiced for years for the lab diagnosis of M. tuberculosis. But, being labour intensive and time consuming, these methods demand for rapid, more efficient and reliable methods for TB diagnosis. Timely and accurate diagnosis is crucial to the commencement of effective antimycobacterial therapy of TB cases. PCR is greatly used for the detection of M. tuberculosis either using sputum or the cultures obtained from sputum (Huang et al. 2010; Kaul 2001; Soini and Musser 2001).In case of smear positive pulmonary TB, diagnosis is confirmed or dismissed in just 48 h using PCR rather than 2–8 weeks for culturing (Balamurugan et al. 2006). Newer molecular methodologies have identified extensive genetic diversity among M. tuberculosis isolates thereby making it quite difficult to classify different disease causing strains precisely (Van Embden et al. 1993). In the present study, PCR amplification targeting genetic markers specific to distinct evolutionary lineages was utilized to differentiate M. tuberculosis isolates on the basis of presence or absence of these markers. This method provided us with a simplified approach to estimate the prevalence of Beijing and Non-Beijing strains of M. tuberculosis in our community.




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