Browsing by Author "Minnies, Stephanie"

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    Extract from used Xpert MTB/ RIF Ultra cartridges is useful for accurate second-line drug-resistant tuberculosis diagnosis with minimal rpoB-amplicon cross-contamination risk
    (Nature Research (part of Springer Nature), 2020) Venter, Rouxjeane; Minnies, Stephanie; Derendinger, Brigitta; Tshivhula, Happy; De Vos, Margaretha; Dolby, Tania; Ruiters, Ashley; Warren, Robin M.; Theron, Grant
    Xpert MTB/RIF Ultra (Ultra) detects Mycobacterium tuberculosis and rifampicin resistance. Follow-on drug susceptibility testing (DST) requires additional sputum. Extract from the diamond-shaped chamber of the cartridge (dCE) of Ultra’s predecessor, Xpert MTB/RIF (Xpert), is useful for MTBDRsl-based DST but this is unexplored with Ultra. Furthermore, whether CE from non-diamond compartments is useful, the performance of FluoroType MTBDR (FT) on  CE, and rpoB cross-contamination risk associated with the extraction procedure are unknown. We tested MTBDRsl, MTBDRplus, and FT on CEs from chambers from cartridges (Ultra, Xpert) tested on bacilli dilution series. MTBDRsl on Ultra dCE on TB-positive sputa (n = 40) was also evaluated and, separately, rpoB amplicon cross-contamination risk . MTBDRsl on Ultra dCE from dilutions ≥103 CFU/ml (CTmin <25, >“low semi-quantitation”) detected fluoroquinolone (FQ) and second-line injectable (SLID) susceptibility and resistance correctly (some SLIDs-indeterminate). At the same threshold (at which ~85% of Ultra-positives in our setting would be eligible), 35/35 (100%) FQ and 34/35 (97%) SLID results from Ultra dCE were concordant with sputa results. Tests on other chambers were unfeasible. No tubes open during 20 batched extractions had FT-detected rpoB cross-contamination. False-positive Ultra rpoB results was observed when dCE dilutions ≤10−3 were re-tested. MTBDRsl on Ultra dCE is concordant with isolate results. rpoB amplicon cross-contamination is unlikely. These data mitigate additional specimen collection for second-line DST and cross-contamination concerns.
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    Novel and rapid tests for diagnosis of tuberculosis using non-sputum specimens
    (Stellenbosch : Stellenbosch University, 2023-12) Minnies, Stephanie; Theron, Grant; Reeve, Byron; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.
    ENGLISH ABSTRACT: Diagnosis of TB remains a challenge, as in 2021, 60% of those who developed active TB were diagnosed. Xpert Ultra MTB/RIF (Ultra) is endorsed for TB diagnosis on sputum, but at the advent of the study, more data on the usefulness of Ultra on non-sputum specimens, particularly in HIV-endemic settings, were needed. Moreover, the impact of different sample processing such as sample concentration of non-sputum specimens by centrifugation on Ultra has not been previously investigated. Lastly, data on how Ultra on site-of-disease non-sputum specimens directly compares to other tests, either on site-of-disease or non-site-of-disease non-sputum specimens remain limited. Firstly (chapter 2), we showed that in patients with presumptive TB lymphadenitis (TBL), Ultra detects more TBL cases than Xpert MTB/RIF (Xpert), Ultra’s predecessor, and results in more people being placed on treatment. Ultra’s increased sensitivity on fine needle aspirate biopsies (FNABs) does however come with decreased specificity, and this was not significantly associated with HIV status or the use of alternate reference standards. Furthermore, we showed that study Ultra detected more TBL cases than programmatic Ultras when both tests were done, indicating that optimisation of programmatic testing of FNABs would result in improved TBL diagnosis. Moreover, we showed that FNAB Ultra false-negative results are associated with PCR inhibition. Lastly, we showed that in patients with presumptive TBL, urine-Ultra had low sensitivity. Thereafter (chapter 3), we found that in people living with HIV (PLHIV) with presumptive TB pericarditis, Ultra on unconcentrated pericardial fluid had higher sensitivity and lower specificity overall when compared to Xpert. We also found that comparing Ultra to alternate reference standards did not improve sensitivity. Exclusion of Ultra results is the superior recategorization strategy in pericardial fluid (unlike reclassifying trace results as negative). Additionally, we showed that using concentrated pericardial fluid on Ultra resulted in higher Ultra specificity but more non-actionable results. This suggests that laboratories with adequate fluid volume and capacity should concentrate pericardial fluid when possible. Furthermore, we showed that the high sensitivity of uIFN-γ on pericardial fluid is offset by its’ poor specificity, indicating that Ultra is the superior test on pericardial fluid. Lastly, Urinary Ultra and TB-LAM had low sensitivity but could reduce the need for pericardiocentesis for TB pericarditis diagnosis in 4% of patients, highlighting their potential. Thirdly (chapter 4), in patients with presumptive TB pleuritis, we showed that Ultra had similar sensitivity but higher diagnostic yield compared to Xpert, and exclusion or reclassification of trace results to negative does not increase sensitivity. Additionally, alternate reference standards and HIV status did not significantly increase Ultra’s sensitivity. Furthermore, we showed that testing Ultra with concentrated pleural fluid increases Ultra specificity, but this also increases non-actionable results, and this was also observed in pericardial fluid. Moreover, we showed that uIFN-γ on pleural fluid had high sensitivity and moderate specificity, suggesting that laboratories with sufficient funding and infrastructure should use uIFN-γ concentration for TB pleuritis diagnosis. Finally, we showed that Ultra and TB-LAM on urine could reduce the need for thoracentesis in a subset of patients for TB pleuritis diagnosis, particularly in PLHIV. Lastly (chapter 5), we showed that in bronchial fluid (BF), Ultra’s diagnostic accuracy was not significantly different between bronchoalveolar lavage fluid (BALF) and bronchial wash fluid (BWF), and thus they were not stratified in downstream analyses for TB diagnosis. We also showed that Ultra on concentrated BF had higher sensitivity and lower specificity when compared to Xpert (HIV and alternate reference standards did not significantly change Ultra’s sensitivity and specificity). Moreover, 4 in 5 Ultra “false-positives” started empirical treatment, which suggests that Ultra on BF could be detecting TB cases missed by culture. We also showed that programmatic Ultra testing on BF would benefit from optimisation as study Ultras detected more TB cases. Moreover, we showed that uIFN-γ should not be used on BF for TB diagnosis due to its’ poor sensitivity. Lastly, we showed that urinary-Ultra had low sensitivity, but still detected TB missed by tests on site-of-disease fluid, highlighting its’ usefulness. In terms of outputs, this dissertation has resulted in four first author manuscripts. One has been published in a peer reviewed journal (chapter 2) and the others’ (chapters 3, 4 and 5) are submission ready. Additionally, three ancillary publications (one of which was co-first authored) are briefly discussed in chapter 8 and can be found in the appendices. Some of this research was presented by the candidate at an international and national peer-reviewed conference. In summary, this work shows Ultra’s high sensitivity and moderate sensitivity on FNABs, pericardial fluid, pleural fluid, and BF in patients with presumptive TBL, TB pericarditis, TB pleuritis and PTB. We can therefore recommend a positive Ultra, with the inclusion of trace results, for TB diagnosis in these non-sputum specimens.