Association of lineage 4.2.2 of Mycobacterium tuberculosis with the 63-bp deletion variant of the mpt64 gene

Abstract

ABSTRACT Tuberculosis (TB) remains the primary cause of death from an infectious disease worldwide. The MPT64 antigen assay is an expedited diagnostic method that is increasingly adopted in many TB diagnostic laboratories and clinical settings worldwide. However, the diagnostic accuracy of the MPT64 antigen assay is influenced by the presence of 63bp deletion variants within the mpt64 gene, and the potential association of this variant with the lineage or sub-lineage of Mycobacterium tuberculosis complex (MTBC) remains unverified. In this study, we analyzed the MPT64 antigen assay using isolates from the major lineage and sub-lineage of MTBC combined with whole-genome sequencing. Notably, we found a significant association between the 63bp deletion variant in the mpt64 gene and M. tuberculosis Lineage 4.2.2 (L4.2.2) globally. Phylogenetic analysis revealed that L4.2.2 isolates were divided into two clusters (Clades A and B). Importantly, all isolates within Clade A exhibited the 63bp deletion variant in the mpt64 gene. Furthermore, using the Bayesian Skyline population model, we observed that the effective population size of Clade A has remained relatively constant. These findings highlight that MPT64-based testing should be cautiously used, and MPT64 negative results should be validated using an alternative assay in regions where L4.2.2 isolates are prevalent, such as China and Vietnam. These L4.2.2 isolates with the 63bp deletion variant in mpt64 warrant greater consideration during the development of diagnostic tests and study of their immunomodulatory role. Overall, this study provides valuable insights into a better understanding of the virulence and diagnosis of MTBC isolates based on the MPT64 protein. IMPORTANCE To date, rapid diagnostic methods based on the MPT64 antigen assay are increasingly utilized to differentiate between non-tuberculous mycobacteria and TB disease in clinical settings. Furthermore, numerous novel techniques based on the MPT64 release assay are continuously being developed and applied for the identification of both pulmonary and extrapulmonary TB. However, the diagnostic accuracy of the MPT64 antigen assay is influenced by the presence of 63 bp deletion variants within the mpt64 gene. To our knowledge, this is the first report on the association between the 63 bp deletion variant in mpt64 and Mycobacterium tuberculosis L4.2.2 globally, which highlights the need for the cautious utilization of MPT64-based testing in regions where L4.2.2 isolates are prevalent, such as China and Vietnam, and MPT64 negative results should be confirmed with another assay. In addition, further studies on vaccine development and immunology based on MPT64 should consider these isolates with 63 bp deletion variant.