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The insertion sequence (IS) 6110 is a repetitive mobile element specific for the Mycobacterium tuberculosis complex (MTBC) used for years to diagnose and genotype this pathogen. It contains the overlapping reading frames orfA and orfB that encode a transposase. Its genetic variability is difficult to study because multiple copies are present in the genome. IS 6110 is randomly located, nevertheless some preferential locations have been reported, which could be related to the behaviour of the strains. The aim of this work was to determine the intra- and inter-strain genetic conservation of this element in the MTBC. For this purpose, we analysed 158 sequences of IS 6110 copies from 55 strains. Eighty-four copies were from 17 strains for which we knew all the locations in their genome. In addition, we studied 74 IS 6110 copies in 38 different MTBC strains in which the location was characteristic of different families including Haarlem, LAM, S, and L6 strains. We observed mutation in 13.3% of the copies studied and we found 10 IS 6110 variants in 21 copies belonging to 16 strains. The high copy number strains showed 6.2% of their IS 6110 copies mutated, in contrast with the 31.1% in the low-copy-number strains. The apparently more ancient copy localised in the DR region was that with more variant copies, probably because this was the most studied location. Notably, all Haarlem and X family strains studied have an IS 6110 in Rv0403c , suggesting a common origin for both families. Nevertheless, we detected a variant specific for the X family that would have occurred in this location after the phylogenetic separation. This variant does not prevent transposition although it may occur at a lower frequency, as X strains remain with low copy number (LCN) of IS 6110. [ABSTRACT FROM AUTHOR]

Abstract Background This study aims to detect Mycobacterium tuberculosis complex (MTBC) DNA in intraocular fluid from clinically suspected tuberculous uveitis patients using multiplex polymerase chain reaction (PCR) and investigate the diagnostic utility of multiplex PCR for tuberculous uveitis. Methods Primers targeting three specific genes (MPB64, CYP141, and IS6110) within the MTBC genome were designed. Multiplex PCR was conducted using DNA from the H37Rv strain as well as DNA extracted from fluids of confirmed tuberculosis patients to assess primer specificity and method feasibility. Intraocular fluid samples were collected during the initial visit for multiplex PCR detection of MTBC DNA. The results of multiplex PCR tests were correlated with intraocular fluid findings and clinical profiles of patients clinically diagnosed with tuberculous uveitis who underwent standard antituberculosis therapy. Results Multiplex PCR was employed to detect MTBC DNA in intraocular fluid samples from 15 patients clinically suspected of having tuberculous uveitis, with no amplification bands observed in the DNA lanes for the three target genes. T-cell spot test (T-SPOT) results were positive in 11 patients (100%), while purified protein derivative (PPD) tests were positive in 5 patients (45.5%). Abnormal chest CT findings were noted in 4 patients (36.4%), including one case of active pulmonary tuberculosis and three cases of inactive pulmonary tuberculosis. Retinal vasculitis was observed in 6 eyes (46.2%), panuveitis in 5 eyes (38.5%), and intermediate uveitis in 2 eyes (15.4%). The average duration of antituberculosis therapy administered to the 11 patients was 7.1 months (range: 6–10 months). The medium LogMAR Best Corrected Visual Acuity (BCVA) significantly improved at the last follow-up (Z=-2.371, P = 0.018). Conclusions Standard antituberculosis therapy demonstrated effectiveness in treating 11 patients clinically suspected of having tuberculous uveitis despite the absence of detectable MTBC DNA in intraocular fluid via multiplex PCR. Further investigation is warranted to elucidate the role of PCR in diagnosing ocular tuberculosis among Chinese individuals.