Diagnostic accuracy of mycobacterium tuberculosis DNA as detected by the top assay among the individuals with paucibillary TB

Abstract

Background: One of the major reasons leading to the increased incidence of TB is the lack of a diagnostic with high sensitivity that can be relied on to capture even patients with low burden TB. This had been attempted by increasing the sensitivity of Xpert MTB/RIF assay with the Xpert MTB/RIF Ultra cartridge; however it has led to PCR results that are not diagnostic(Trace-positive) that require further study so that the result could be interpreted correctly, provide the right treatment for patients, and prevent the progression to active stages of TB infection(1). ‘Totally Optimized PCR (TOP)’, is a highly sensitive new nucleic acid amplification test that utilizes a combination of efficient sample processing, novel target selection, modern primer design techniques, and an extended PCR for selective target isolation and amplification. The assay targets a gene (ponA1) involved in the assembly of peptidoglycans in the MTB bacterial wall which is highly specific to Mycobacteria in the MTB complex and therefore it is not affected by background noise (2). Methods: A nested cross-sectional study in Trace Ultra Result iNsight in TB screening (TURN-TB) study was carried out to evaluate the diagnostic accuracy of Mycobacterium tuberculosis DNA as detected by the top assay among individuals with paucibillary TB. A total of 71 samples were tested on TOP assay, GeneXpert ultra tests were already done on the same specimens, 66 samples had valid TOP assay results and 5 had indeterminate results 30/66 had both sputum GeneXpert Ultra and sputum culture done. Results: Relative to sputum Xpert Ultra, the sensitivity of TOP assay was 73.53% (n/N=25/34, 95% CI, 62.89-84.17) and specificity was 84.38% (n/N=27/32, 95% CI, 75.62-93.13), 45.46% (30/66) samples tested with TOP had both sputum Xpert Ultra and culture results which were compared to the microbiological reference standard ( incorporating both sputum Xpert and culture), among these patients, 23.33% (7/30) had both sputum Xpert Ultra and culture positive results, among these cases, 71.43% (5/7) were detected by TOP assay, while 28.57% were not detected. Furthermore, 40% (12/30) of the patients had both sputum Xpert Ultra and culture negative results. Among these negative cases, 66.67% (8/12) were also negative on TOP, however, 33.33% (4/12) were considered false positive compared to the microbiological reference standard. The diagnostic sensitivity of TOP assay consistently increased in patients with higher sputum mycobacterial loads, as indicated by the semi-quantitative results of sputum Xpert ultra. Specifically, among the 22 cases with Xpert Ultar Low results, 90.91% (20/22) were detected by TOP, for the 11 cases with Very Low results, 45.45% (5/11) were detected by the TOP assay,. Finally, one case with a Trace-positive result was not detected by TOP.