Spoligotyping analysis of Mycobacterium tuberculosis in Khyber Pakhtunkhwa area, Pakistan

Abstract

Background: Spoligotyping is a reproducible, reverse hybridization approach for genotyping of Mycobacterium tuberculosis complex (MTBC). Molecular typing of MTBC is helpful for understanding and controlling tuberculosis epidemics. Methods: Spoligotyping was performed on 166 clinical isolates of Mycobacterium tuberculosis (MTB) collected from 25 districts of Khyber Pakhtunkhwa, Pakistan. Results were compared to SITVIT2, an online database developed by the Institut Pasteur de la Guadeloupe, France. Results: Spoligotyping results showed that 145 strains (88%) displayed known patterns while 21 (12%) were new. Lineage 3/Central Asian strain (L3/CAS) was the predominant family (73%, χ ²=19.9, P=0.001), followed by L2/Beijing (5.4%) and L4 (4.2%). L3/CAS1-Delhi was the major sublineage (82%) among the L3/CAS family (χ ²=664, P=0.0001). Analysis showed that the majority of the clinical isolates with an unknown pattern had an evolutionary link with the L3/CAS strain, and nine (5.4%) of the unknown strains were epidemiologically linked and were tentatively named L3/CAS-KP (Khyber Pakhtunkhwa). Conclusion: The present study demonstrated that L3/CAS is the predominant lineage of MTB, widely distributed in different areas of the Khyber Pakhtunkhwa province of Pakistan. Spoligotyping patterns of some clinical isolates could not be matched to other reported patterns in an international database. Other tools, such as mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR), will be helpful in future investigations into the epidemiological characteristics of clinical isolates in the Khyber Pakhtunkhwa province.

Keywords: L3/CAS; VNTR; genetic diversity; genotyping; molecular characterization; spoligotyping.

Figure 1

Graphical analysis of 166 Mycobacterium tuberculosis isolates collected from different areas of Pakistan. Note: Lineage 3/Central Asian strain (L3/CAS), shown in black, is the dominant spoligotype family. New unclustered strains are shown in the black dotted segment. Less frequent spoligotypes are grouped in the “Others” category in the gray segment.

Figure 2

Maximum parsimony analysis of new unclustered spoligotypes.