Validation of the FluoroType MTBDR Assay for Detection of Rifampin and Isoniazid Resistance in Mycobacterium tuberculosis Complex Isolates

MATERIALS AND METHODS

Mycobacterial cultures were performed at the National Mycobacterial Reference Laboratory in Borstel, Germany, between May 2016 and October 2017 using mycobacterial growth indicator tubes (MGIT) (Becton Dickinson, USA) and Loewenstein-Jensen and Stonebrink culture slants (Artelt-Enclit, Germany). All cultures were identified to species level using the GenoType MTBC version 1.0 (Hain Lifescience, Nehren, Germany) according to the manufacturer's instructions. The MGIT 960 isoniazid-rifampin-ethambutol (IRE) kit (Becton Dickinson) was used according to the manufacturer's instructions. The critical concentrations of rifampin and isoniazid were 1 μg/ml and 0.1 μg/ml, respectively (11).

For DNA extraction, an aliquot of 500 μl was obtained from the bottom of a positive MGIT or two loops (10 μl) of bacterial growth were collected from Loewenstein-Jensen or Stonebrink culture slants and added to 500 μl of 0.9% NaCl solution. Samples were centrifuged at 3,000 × g for 15 min. Supernatants were removed, and DNA was extracted using the Fluorolyse kit (Hain Lifescience, Nehren, Germany) according to the manufacturer's instructions. In brief, 100 μl of lysis buffer and 2 μl of internal control were added to the remaining pellets. The mixed samples were incubated at 95°C for 5 min, centrifuged for 1 min, and resuspended in 100 μl neutralization buffer. Following centrifugation at 10,000 × g for 5 min, supernatants were transferred into a 0.5-ml cryovial. DNA was stored at −20°C. For each batch, one negative control using 100 μl of lysis buffer, 100 μl of neutralization buffer, and 2 μl of internal control was included.

DNA was analyzed with the Genotype MTBDRplus version 2 and FluoroType MTBDR assays (Hain Lifescience, Nehren, Germany) as per the manufacturer's instructions. In brief, for the FluoroType MTBDR, 20 μl of DNA solution was added to 6 μl master mix component A and 14 μl master mix component B and transferred into 96-well plates. The plates were then sealed, centrifuged, and analyzed via FluoroCycler 96. For each plate, one positive and one negative control were included. For the Genotype MTBDRplus version 2, 5 μl of DNA solution was added to 45 μl of master mix containing 10 μl master mix component A and 35 μl master mix component B. Twenty microliters of PCR products was added to 20 μl denaturation reagent in a hybridization well. Hybridization reagents were prepared, preheated, and loaded onto the GT-Blot48 (Hain Lifescience, Nehren, Germany). After an initial hybridization step, strips were added and hybridization performed as per the standard protocol. For each batch, one negative control (45 μl master mix, 5 μl DNA-free water) was tested. Interpretation of the results was performed according to the manufacturer's instructions.

The package insert of the FluoroType MTBDR identifies the following rpoB, katG, and inhA mutations in amino acid codons (e.g., S531L) and regulatory regions (e.g., T-8A): T508A, S509T, E510H, L511P, S512K, Q513L, Q513P, Q513R, D516A, D516F, D516V, D516Y, N518I, S522L, S522Q, H526C, H526D, H526G, H526L, H526N, H526P, H526Q, H526R, H526S, H526Y, R529K, S531F, S531L, S531L, S531Q, S531W, L533E, L533P, S315T1, S315T2, S315N, S315R, G-17T, A-16G, C-15T, G-9A, T-8A, T-8C, and T-8G. In contrast the Genotype MTBDRplus is able to detect and specify the following mutations: D516V, H526Y, H526D, S531L, S315T1, S315T2, C-15T, A-16G, T-8C, and T-8A.

Molecular results were interpreted without knowledge of phenotypic DST results. Sanger sequencing in the key regions of rpoB, katG, inhA, and aphC (12) was performed for isolates with discordant results between either the two molecular methods or the molecular and phenotypic methods. If the Genotype MTBDRplus showed a missing wild-type band but not a mutation band, Sanger sequencing in the respective key region was performed to confirm the FluoroType MTBDR result. Sanger sequencing was performed using an ABI 3130xl genetic analyzer (Applied Biosystems) and an ABI BigDye Terminator cycle sequencing kit (version 3.1) according to the manufacturer's instructions.

Molecular results were coded as wild type, individual mutation, mutations in a region of the gene, or indeterminate. The molecular results were interpreted as susceptible, resistant, or indeterminate. Indeterminate results in the Genotype MTBDRplus were defined as detection of MTBC DNA without the presence of a gene locus control. All samples yielding an indeterminate result were retested using a new aliquot of DNA. A mixed culture (heteroresistance) was defined as both wild-type and mutant DNA being present as identified by wild-type and mutant bands (Genotype MTBDRplus) and/or by respective double peaks in the DNA sequence.

To determine the accuracy of the FluoroType MTBDR assay, the results were compared with phenotypic DST results. Sensitivity and specificity were calculated for RMP and INH separately. Sensitivity was defined as the proportion of isolates correctly determined as resistant by the FluoroType MTBDR compared to the phenotypic DST results. Specificity was defined as the proportion of isolates correctly determined as susceptible by the FluoroType MTBDR compared to the phenotypic DST results. The McNemar test on paired proportions was used to compare the sensitivities of the FluoroType MTBDR and the Genotype MTBDRplus for INH. All statistical analysis were performed using Stata 14.0.

Information was extracted from the laboratory information system anonymously. The ethics committee of the University of Luebeck approved the study. Individual consent was not sought as no additional patient information was obtained.