Detection of Insertion Sequence IS60 Deficient Mycobacterium tuberculosis Strains from Myanmar

Mi Mi Htwe, Wah Wah Aung , Phyu Win Ei, Aye Su Mon, Su Mon Win, Lai Lai San, Nan Aye Thida Oo,Wint Wint Nyunt & Jong Seok Lee

Myanmar Health Sciences Research Journal, 2021: 33,(1-3): 68-73

Insertion sequence (IS) 6110 is a widely used target for detection of Mycobacterium tuberculosis (MTB). Molecular diagnosis of tuberculosis (TB) especially extra-pulmonary TB in Myanmar is based on detection of IS 6110 from clinical samples. However, 8-11% of MTB stains in South-East Asia do not contain this target, thus it can lead to false negative results for molecular diagnostic testing using this target. The present study was carried out to determine the occurrence of MTB complex that lacking IS6110 in Myanmar. IS6110 PCR assay was performed to detect IS6110 deficient strains among 232 MTB isolates collected during 2015-2017 from adult TB patients in different regions of Myanmar. These isolates included 125 rifampicin-resistant and 107 rifampicin-sensitive strains detected by Xpert MTB/RIF. 

Approximately 1.7 billion people of the world’s population has been infected with Mycobacterium tuberculosis (MTB), the causal bacterium of tuberculosis (TB) and  5-15% of them were estimated to develop TB disease during their lifetime. Myanmar is one of the high TB and multidrug-resistant TB (MDR-TB) burden countries worldwide with the incidence of 338/100,000 population in 2018.¹ Insertion sequences (IS) IS6110 is a mobile genetic element, which is inserted in a 36-bp array Direct Repeat region (DR region: Rv2813-Rv2820c, RD207) of almost all MTB complex isolates. 

Study design and study period

A cross-sectional descriptive study was carried out from May 2018- April 2019.

Study population

Altogether 232 MTB isolates collected during 2015-2017 from adult TB patients in different regions of Myanmar included in this study. These isolates were 125 rifampicin-resistant and 107 rifampicin-sensitive strains detected by Xpert MTB/RIF in routine diagnosis program by National Tuberculosis Program.

Isolation of M. tuberculosis from stock culture isolates

The colonies from stock culture isolates were taken using a disposable loop and put in
a conical tuber with glass beads and vortexed for 2 minutes to disrupt colony clumps
and was stand 15 min to allow the undisrupted colony clumps to settle. Supernatant was taken and adjusted turbidity to McFarland of M. tuberculosis were cultured on Lowenstein Jenson (LJ) median BSL2+ laboratory, Advanced Molecular Research Centre, Department of Medical Research according to the following standard procedures.¹⁴

Although previous studies reported as 8-11% of MTB strains in South-East Asia do not contain IS6110 sequence^{8, 9}, the present study found low percentage, 1.3% of IS6110 deficient strains among 232 clinical MTB isolates. The detected proportion of IS6110 negative MTB isolates was lower than that of other Asian countries such as India and Vietnam, both of which detected no-copy strains using RFLP method only, and in which the frequency of these strains was estimated at respectively 8% and 11% of tested isolates. In the present study, we used the IS6110 PCR and the chance of having false negative for IS6110 PCR-based detection was controlled by optimizing PCR reactions with positive and negative controls, and repetition of negative tests. 

1.       World Health Organization. Global  Tuberculosis Report 2018 [Internet]. WHO, Geneva, 2019. Available from: https://www. who.int/ tb/publications/global report/en/ [Accessed 10 October 2019].

2.       Genewein ATA, Bernasconi C, et al. Molecular approach to identifiying route of transmission of tuberculosis in the community. Lancet 1993; 342: 841-844.

3.       Ei PW, Aung WW, Nyunt WW, Swe TL, Htwe MM, Win SM, et al. Extensively drug-resistant tuberculosis in Myanmar: burden and mutations causing second-line drug resistance. The International Journal of Tuberculosis and Lung Disease 2018; 22(1): 47-53.

4.       Howard ST, Oughton MT, Haddad A & Johnson WM. Absence of the genetic marker IS6110 from a strain of Mycobacterium tuberculosis isolated in Ontario. The Canadian Journal of Infectious Diseases. 1998 Jan-Feb; 9(1): 48-53.

5.       Van Soolingen D, de Haas PE, Hermans PW, Groenen PM & van Embden JD. Comparison of various repetitive DNA elements as genetic markers for strain differentiation and epidemiology of Myco-bacterium tuberculosis. Journal of Clinical Microbiology. 1993; 31(8): 1987-95.