Comparison of the Genedia MTB/NTM Detection Kit and Anyplex plus MTB/NTM Detection Kit for detection of Mycobacterium tuberculosis complex and nontuberculous mycobacteria in clinical specimens

Abstract Background Real‐time (RT) PCR is a rapid and accurate method that is widely used for the detection of Mycobacterium tuberculosis complex (MTB). The aim of this study was to evaluate and compare the performance of the Genedia MTB/NTM Detection Kit and the Anyplex plus MTB/NTM Detection kit in the detection of MTB and nontuberculous mycobacteria (NTM) in clinical specimens. Methods From October 2017 to February 2018, 236 respiratory specimens and 137 non‐respiratory specimens, from patients with suspected tuberculosis, were examined. AFB smear, culture, and RT‐PCR using the Genedia MTB/NTM Detection kit (Green Cross Medical Science Corp.) and the Anyplex plus MTB/NTM Detection kit (Seegene) were applied. PCR performance in the detection of MTB and NTM was evaluated in relation to culture results and between the two assays. Results Culture was positive for MTB in 30 (8.0%) of the 373 specimens and for NTM in 23 (6.2%). The sensitivity and specificity of MTB detection with the Genedia kit were 76.7% and 99.7%, respectively, whereas the Anyplex kit sensitivity and specificity for MTB detection were 86.7% and 97.5%, respectively. Both kits exhibited the same sensitivity (73.9%) for NTM detection, and the specificity was 100% and 99.4% for the Genedia and Anyplex kits, respectively. Conclusions The Genedia and Anyplex kits demonstrated high sensitivity and specificity for the detection of MTB and NTM. Both kits have a high concordance rate and can be used more widely in clinical laboratories for the early detection of tuberculosis.


| Samples
We selected respiratory and non-respiratory samples that were transferred to the Department of Laboratory Medicine in a tertiary university hospital for MTB/NTM PCR from October 2017 to February 2018.
All clinical samples were examined by AFB smear, conventional culture, and RT-PCR. This study was approved by the institutional review board of Chungnam National University Hospital.

| AFB smear and culture
Respiratory specimens were treated with NALC-NaOH (2% N-acetyl-L-cysteine-sodium hydroxide) after centrifugation at 3000 × g for 20 minutes. The AFB smear procedure involved staining with auramine-rhodamine fluorescent stain, followed by confirmation using Ziehl-Neelsen staining. Smears were graded, and those from 1 to 4 were defined as smear-positive. All specimens were cultured in liquid (MGIT960 system) and solid (2% Ogawa agar) media for a maximum of 6 and 8 weeks, respectively. Identification of MTB and NTM was carried out by MPT 64 antigen detection using immunochromatographic method.

| Real-time PCR kits
RT-PCR was performed using two kits (Genedia MTB/NTM Detection kit and Anyplex plus MTB/NTM Detection kit) according to the F I G U R E 1 Distributions of tests performed for respiratory and nonrespiratory specimens. CSF, cerebrospinal fluid manufacturers' instructions. One hundred microliters of decontaminated sample was mixed with 100 µL DNA extraction solution from each assay kit and heated at 100°C for 10 and 20 minutes, for the Genedia and Anyplex assays, respectively. Each total mixture including PCR mixture, primer mixture, internal control DNA, and template DNA was centrifuged and prepared for PCR reactions according the manufacturers' instructions. Assays were run on a CFX96TM real-time PCR system (Bio-Rad). Positive and negative controls were included in each run for both instruments. Results were automatically interpreted via the instrument software, using pre-defined threshold and cutoff values.

| Statistical analyses
Sensitivity, specificity, positive predictive value, and negative pre-

| RE SULTS
Of the 411 clinical specimens collected, 38 were excluded due to Specimens were examined by AFB smear, culture, and using both RT-PCR tests. Of these, 37 (9.9%) were AFB-positive, including 36 respiratory specimens and 1 synovial fluid specimen; the remaining 337 (90.3%) specimens were AFB-negative (Table 1).
Of the 236 respiratory specimens cultured, 26 (11.0%) were positive for MTB and 22 (9.3%) were positive for NTM. Of the 137 non-respiratory specimens cultured, 4 (2.9%) were positive for MTB and 1 (0.7%) for NTM. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of each assay were calculated using the culture results as the "gold standard" (Table 1) tory specimens, sensitivity and specificity were 71.0% and 94.9%. 8 We found that sensitivity of both kits was lower with AFB smearnegative samples, which is consistent with previous findings. Nine false-positive results were detected in AFB smear-negative specimens using the Anyplex assay, resulting in a lower PPV (76.5%) than that of the Genedia assay (95.8%). In another previous study, the

TAT (Turn Around Time) of Genedia kit was about 140 minutes and
showed high correlation and accuracy with conventional PCR and other real-time PCR methods (AdvanSure kit, Real-Q kit). 9 In this study, the specificity and NPV calculated for AFB smear-positive specimens were significantly lower than those for smear-negative specimens. This may be due to the small number of specimens, which is a limitation of this study.

The United States Centers for Disease Control and Prevention
(CDC) recommends that nucleic acid amplification testing (NAA), using a Food and Drug Administration (FDA) approved test, should be performed on at least one respiratory specimen from each patient with signs and symptoms of pulmonary TB. 3 The Korean guidelines for TB recommend that when an individual is suspected of having pulmonary TB, NAA tests should be performed at least once in combination with AFB smear and culture. 4 RT-PCR kits using NAA techniques have been developed with increasing sensitivity and specificity than the initial product launch and have been used in many clinical laboratories with the advantage of obtaining rapid results. 5 In summary, both the Genedia MTB/NTM Detection kit (Green Cross Medical Science) and the Anyplex plus MTB/NTM Detection kit (Seegene) have the capacity to report high sensitivity and specificity in a short time and both demonstrate a high concordance rate. We therefore propose that these kits can be used more widely in clinical laboratories. It is considered necessary to study the problem of increased false-positive rate when targeting AFB-negative samples. Further research is needed in near future, including identification of non-detected and false-positive samples in larger numbers of specimens.