Epidemiology of molecular probes in Xpert MTB/RIF assay in Azad Jammu And Kashmir, Pakistan: a retrospective study

Abstract

In spite of lot of advanced techniques, tuberculosis and multidrug are major cause of mortality in world. If AuNPs (gold nano particles) were attached with probes, it could enhance the efficacy of detection than dye probes for real time pcr such as Genexpert MTB/RIF. A total of 15,000 samples were collected from TB suspects and subjected to Xpert MTB/ RIF assay, where 6800 (45.3%) were detected as MTB positive, 280 (4.3%) were detected to harbor mutations in the RRDR, while invalid /errors were found in 690 (4.6%) cases. The mutations were detected by probe E, 199 (71.1%), while probes B and D, 30 and 26 (10% and 9%), respectively. In the Xpert MTB/RIF Assay were found mutations picked by probes E and B codons 529–533 (71%) and 512–518 (10%), respectively. The fast-rising works of TB nano-diagnostics, of Xpert probes, may improve by the applications of gold nanoparticle probes
Tuberculosis is the major infectious disease found all parts of the world. Pakistan ranks 5th among 30 high burden countries with tuberculosis and 4th among multidrug resistant (MDR) tuberculosis. A molecular diagnostic technique called Xpert MTB/RIF assay is used to detect Mycobacterium tuberculosis (MTB) and rifampicin resistance (RR-TB) at the same time. It is a fast and fully automated cartridge-based real-time PCR that uses three specific primers and five unique molecular beacon probes to target the rpoB gene.This study was designed to detect rifampicin-resistant tuberculosis (RR-TB) cases and the frequency of missing probes in different study populations in the Azad Jammu and Kashmir (AJK) state of Pakistan.These probes target and detect mutations in the rpoB gene.
• Methods: The current study was carried out in SRL TB lab of District Headquarters hospital Mirpur (TB State reference laboratory AJK). A total of 2790 specimens from March 2016-August 2019 were collected. Among the total cases, 94 % are Pulmonary TB (PTB) where as 6 % are extra-pulmonary cases. All respiratory and non-respiratory samples were analysed for fluorescence smear microscopy (AFB) and real time PCR test (Xpert MTB/RIF assay) to detect MTB and RRTB.
Results: Out of total 2790 suspected MTB patients, 734(26%) were detected MTBC (Mycobacterium Tuberculosis Complex) on Xpert MTB/ RIF whereas 564(20%) were positive on fluorescence microscopy. Of these MTB positive patients, 720(98%) had pulmonary TB and 14(2%) were EPTB cases. Resistance to rifampicin (RR) was detected in 66(9%) cases of which 97% were pulmonary and 3% were extra-pulmonary. The frequency of probe E (Codon 529-533) missing was highest (34%) followed by probe D(Codon 523–529) (26%) and lowest (3%) in probe C(Codon 523-529).The frequency of probe B(Codon 512-518) missing was 15.4% whereas probe A (Codon 518-523) was 9.4%.

Conclusion: The rifampicin resistance of Mycobacterium tuberculosis complex (MTBC) is mostly conferred by a single nucleotide change (point mutation) in rpoB gene present in the bacterial genome. Molecular diagnostic techniques such as Xpert MTB/RIF assay can provide fast identification and rifampicin resistance detection (RRD). This investigation supports to provide baseline data for 81 bp mutations in the rpoB gene and emphasizes the need for further evaluation of mutation patterns in this state.