A pre-clinical validation plan to evaluate analytical sensitivities of molecular diagnostics such as BD MAX MDR-TB, Xpert MTB/Rif Ultra and FluoroType MTB

Abstract

Rapid diagnosis of tuberculosis (TB) and antibiotic resistances are imperative to initiate effective treatment and to stop transmission of the disease. A new generation of more sensitive, automated molecular TB diagnostic tests has been recently launched giving microbiologists more choice between several assays with the potential to detect resistance markers for rifampicin and isoniazid. In this study, we determined analytical sensitivities as 95% limits of detection (LoD95) for Xpert MTB/Rif Ultra (XP-Ultra) and BD-MAX MDR-TB (BD-MAX) as two representatives of the new test generation, in comparison to the conventional FluoroType MTB (FT-MTB). Test matrices used were physiological saline solution, human and a mucin-based artificial sputum (MUCAS) each spiked with Mycobacterium tuberculosis in declining culture- and qPCR-controlled concentrations. With BD-MAX, XP-Ultra, and FT-MTB, we measured LoD95TB values of 2.1 cfu/ml (CI95%: 0.9-23.3), 3.1 cfu/ml (CI95%: 1.2-88.9), and 52.1 cfu/ml (CI95%: 16.7-664.4) in human sputum; of 6.3 cfu/ml (CI95%: 2.9-31.8), 1.5 cfu/ml (CI95%: 0.7-5.0), and 30.4 cfu/ml (CI95%: 17.4-60.7) in MUCAS; and of 2.3 cfu/ml (CI95%: 1.1-12.0), 11.5 cfu/ml (CI95%: 5.6-47.3), and 129.1 cfu/ml (CI95%: 82.8-273.8) in saline solution, respectively. LoD95 of resistance markers were 9 to 48 times higher compared to LoD95TB. BD-MAX and XP-Ultra have an equal and significantly increased analytical sensitivity compared to conventional tests. MUCAS resembled human sputum, while both yielded significantly different results than normal saline. MUCAS proved to be suitable for quality control of PCR assays for TB diagnostics.

Fig 1. Schematic overview of LoD₉₅ experiments.

10x pre-dilutions of H37Rv and an MDR MTBC isolate were prepared and spiked in human sputum, MUCAS or saline solution. In phase 1 three 10-fold dilution series (10⁴ to 0 cfu/ml) were produced in all test matrices. The orienting LoD was determined for each matrix/test combination (in the example, 10⁰ negative & 10¹ positive = > LoD 10 cfu/ml). In phase 2 the LoD₉₅^{TB / HR} were determined using at least six 2-fold dilutions per matrix/test around the respective orienting LoDs. Calculated LoD values were corrected according to colony counts of plates and interpolated using GraphPad Prism 8.0.2 to yield the 95% LoD and the 95%-CIs (GraphPad Software, USA).

Fig 2

LoD₉₅^TB of BD-MAX MDR-TB (BD-MAX), Xpert MTB/Rif Ultra (XP-Ultra) and FluoroType MTB (FT-MTB) in human sputum (A), MUCAS (B) and physiological saline solution (C). Red arrow: calculated LoD₉₅^TB, solid black line: predicted positive assay, blue dashed line: 95% probability of a positive assay, black dashed lines: upper and lower CI₉₅ intervals.

Fig 3. Whiskers Min to Max of IS6110 copies at the limit of detection of BD-MAX, XP Ultra and FluoroType in different test matrices.

Assays were performed in human sputum (Sputum), artificial sputum (MUCAS) and 0.85% saline solution (Saline). The limit of detection (LoD) was determined using two-fold dilutions of H37Rv spiked in test matrix. Number of test series of BD-MAX, XP-Ultra, FT-MTB in human sputum, MUCAS and saline were 8, 10, 6, and 8, 9, 9 and 9, 6, 9, respectively. **** p≤0.0001, *** p≤0.001, ** p≤0.01, * p≤0.05 (one-way ANOVA with Tukey’s multiple comparison test).