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1 Research Article imedpub Journals DOI: / Rapid Identification of Mycobacterium Species in Human Formalin-Fixed Paraffin Embedded Tissues by REBA Myco-ID Assay Using an Automated PCR- Reverse Blot Hybridization Assay Instrument, HybREAD 480 Sung Bae Park 1,2, Yoon Sung Choi 1,2, Jin Young Bae 1,2, Sunghyun Kim 1,2*, Jiyoung Lee 3, Ji-Hoi Kim 4, Mi Ran Kang 4, Dongsup Lee 5, Ji Young Park 6 and Hee Kyung Chang 7 1 Department of Clinical Laboratory Science, College of Health Sciences, Catholic University of Pusan, Busan, Republic of Korea 2 Clinical Trial Specialist Program for In Vitro Diagnostics, Brain Busan 21 Plus Program, Graduate School, Catholic University of Pusan, Busan, Republic of Korea 3 Department of Biomedical Laboratory Science, College of Health Sciences, Yonsei University, Wonju, Republic of Korea 4 YD R&D Center, YD Diagnostics, Yongin, Republic of Korea 5 Department of Biomedical Laboratory Science, Hyejeon College, Hongsung, Republic of Korea 6 Department of Internal Medicine, Kosin University Gospel Hospital, Busan, Republic of Korea 7 Department of Pathology, College of Medicine, Kosin University, Busan, Republic of Korea * Corresponding author: Sunghyun Kim, Ph.D, Department of Clinical Laboratory Science, College of Health Sciences, Catholic University of Pusan, Busan 46252, Republic of Korea, Tel: ; shkim0423@cup.ac.kr Received Date: Nov 12, ; Accepted Date: Nov 21, ; Published Date: Nov 23, Citation: Park SB, Choi YS, Bae JY, Kim S, Lee J, et al. () Rapid Identification of Mycobacterium Species in Human Formalin-Fixed Paraffin Embedded Tissues by REBA Myco-ID Assay Using an Automated PCR-Reverse Blot Hybridization Assay Instrument, HybREAD 480. Ann Clin Lab Res. Abstract Mycobacterium tuberculosis (MTB) remains one of the main causative agents of tuberculosis (TB) although infection from nontuberculous mycobacteria (NTM) in human tissue has also significantly increased in recent years. The most widely used method to detect MTB in formalin-fixed paraffin-embedded (FFPE) tissue is the acid-fast bacilli stain; it is simple, rapid and cost-effective. However, the conventional stain method is a little low sensitivity and specificity and cannot identify Mycobacterium species. In patients with infected mycobacterium species, precise and rapid identification of Mycobacterium species is important for determining accurate and effective therapy. To address this, the present study therefore used automated PCR-reverse blot hybridization assay (REBA) instrument for the rapid identification of Mycobacterium species in various types of human tissue. A total of 52 FFPE human tissue samples were collected from the Department of Pathology at Kosin University Gospel Hospital (Busan, Republic of Korea) including lung, lymph node, skin and bronchus. As shown in the results, it was evaluated that MTB and NTM could be detected at the same or superior level compared with conventional nested PCR using the REBA Myco-ID method and positive and negative predictive value of 100%. We investigated the clinical performance through the patterns of mycobacterial infection comparison REBA Myco-ID method with the histopathologically-diagnosed tubercle, chronic inflammation, and necrotic samples. The overall results are as follows that 11 samples were detected MTB positive, and 8 samples of them (72.7%) were tuberculous, 2 samples (18.2%) were chronic inflammation, and 1 sample (9.1%) was necrotic material. In addition, 21 samples were detected NTM-positive, and 10 samples of them (47.6%) were tuberculous and 11 samples (52.4%) were chronic inflammation. Also, we investigated the patterns of mycobacterial infection according to the various type of tissue using a REBA Myco- ID method. The results show that MTB was more highly detected in lung, lymph node, colon and NTM species were more highly detected in colon, lung, and ileum than in the other types of tissue. It was detected 11 MTB positive, 21 NTM positive, 7 MTB and NTM positive in the colon, lungs, lymph nodes, ileum, bronchus, testis, ear, tendon, and soft tissue. In conclusion, the automated PCR-REBA system proved useful to identify Mycobacterium species more rapidly and with higher sensitivity and specificity than the conventional assay; it might therefore be the most suitable tool for identifying Mycobacterium species in various types of human tissue for precise and accurate diagnosis of mycobacterial infection. Keywords: Tuberculosis; Mycobacterial infection; Rapid molecular identification; HybREAD 480; Formalin-fixedparaffin-embedded tissues Introduction Tuberculosis (TB) is a lethal infectious disease caused by Mycobacterium tuberculosis (MTB) which continues to pose Copyright imedpub This article is available from: 1

2 serious problems worldwide. According to a World Health Organization report, approximately 10 million people suffer health problems caused by TB annually [1]. Moreover, the report outlines that TB was the most frequent cause of death from a single infectious agent, ranking higher than HIV/AIDS for the past five years. In addition, the numbers of HIV-infected people with TB and/or multi-drug resistant-tb have also increased, making it difficult to treat and control the TB in these situations [2]. Infection of the lungs and other organs by nontuberculous mycobacterium (NTM) species has also varied and increased significantly in recent years. Early detection and the effective and rapid differentiation between NTM and MTB complexes are therefore very important for the prevention and treatment of TB [3,4]. Globally, the most widely used conventional method for detecting MTB in formalin-fixed paraffin-embedded (FFPE) tissue is an acid-fast bacilli (AFB) stain. This is a rapid and simple method; however, FFPE specimens can contain low bacterial load, and the granuloma formation and necrosis caused by Mycobacteria can be also found in other bacterial and fungal infections. Additionally, AFB staining displays low sensitivity in the detection of MTB and the method cannot differentiate MTB from NTM species [5,6]. Alternative diagnostic tools are therefore required for the rapid identification of mycobacterial species which is very important for treatment of the infections they cause [7]. Recently, molecular diagnostic assays based on nucleic acid amplification tests, such as real-time polymerase chain reaction (PCR), line probe, and reverse blot hybridization assays (REBA), have been shown to effectively identify mycobacterial species in various respiratory specimens and culture samples [8]. A number of previous studies have used FFPE tissue to evaluate these diagnostic assays and report higher specificity (99%) but variable sensitivity according to the kind of commercial kit used [9-12]. To address the apparent disadvantages of conventional PCR assays, a commercial molecular diagnostic machine was designed and built by YD Diagnostics (Yongin, Republic of Korea) to perform PCR-REBA. The original REBA Myco-ID was shown to provide rapid and accurate detection and identification of 22 mycobacterial species, particularly in cultured respiratory samples, including MTB, M. avium, M. intracellulare, M. scrofulaceum, M. abscessus, M. massiliense, M. chelonae, M. fortuitum complex, M. ulcerans/m. marinum, M. kansasii, M. gastri, M. haemophilum, M. genavense/m. simiae, M. terrae/m. nonchromogenicum, M. celatum, M. gordonae, M. szulgai, M. mucogenicum and M. aubagnense [13]. According to the other report, when comparing REBA Myco-ID results using direct respiratory specimens and cultured samples, the concordance rate was 98.6% [14]. Additionally, another report showed that the results of REBA Myco-ID in FFPE tissues, the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were 95%, 97%, 88.3% and 16.7%, respectively [9]. However, the complicated post-amplification process in PCR-REBA means that it would take a long time to obtain results from varied samples [15]. In order to overcome this limitation, YD Diagnostics designed and developed the line-probe assay automatic system, HybREAD 480 ; this system was developed a fully automatic REBA instrument possible for all of the process of denaturation to chromogenic step and scan to interpretation so that has the advantages of not only being sensitive and specific, but also having no difference in the results between the experimenter, and it is not labor-intensive since the manual procedure has been eliminated therefore being less time consuming. In the present study, 52 FFPE human tissue samples including lung, lymph node, skin, and bronchus were analyzed using the automated REBA Myco-ID HybREAD 480 in order to evaluate the diagnostic efficacy of its rapid Mycobacterium identification system. Materials and Methods Patients and clinical specimens A total of 52 FFPE human tissue samples were collected from lung, lymph node, and other various tissue; these clinical samples were obtained between 2011 and 2017 from the Department of Pathology at Kosin University Gospel Hospital (Busan, Republic of Korea). Among these 52 samples, five were positive and 11 were negative for mycobacterial infection according to a Ziehl-Neelsen AFB stain. The status of the remaining 36 samples could not be confirmed by AFB stain. Deparaffination and genomic DNA extraction Tissue sections 4 µm thick were cut from the paraffin blocks and ten paraffin sections were placed in sterile e-tubes. For deparaffination and DNA extraction, a QIAamp DNA FFPE Tissue Kit (Qiagen, Hilden, Germany) was used according to the manufacturer s instructions. In brief, 1 ml of xylene was added to each tube and the samples were then vortexed for 10 sec and centrifuged at 13,000 g for 2 min; the supernatants were discarded. To remove residual xylene, 1 ml of 100% ethanol (EtOH) was added and the samples were vortexed for 10 sec and centrifuged at 13,000 g for 2 min. The supernatants were again discarded and residual pellets were dried at 37 C for 10 min. After drying, 180 μl of ATL buffer and 20 μl of proteinase K solution were added to each tube and the samples were vortexed for 10 sec. Each tube was incubated at 56 C for 1 hr. and then at 90 C for 1 hr. Quick spin down was conducted to remove drops from the inside of the lid and 200 μl of AL buffer was added and the solution mixed thoroughly by vortex. Then, 200 μl of absolute EtOH was added and gently mixed again. After further quick spin down, the entire lysate was carefully transferred to a QIAamp MinElute column (Qiagen) and centrifuged at 7,000 g for 1 min. After flow through was removed, 500 μl of AW1 buffer was added and centrifuged at 7,000 g for 1 min. Flow through was again removed and 500 μl of AW2 buffer was added and the column centrifuged at 7,000 g for a further 1 min. Subsequently, the column was centrifuged at full speed (13,000 g) for 3 min to dry the membrane completely. After flow through removal, 30 μl of ATE buffer was added to the 2 This article is available from:

3 center of the membrane for genomic DNA elution. The column was incubated at room temperature for 1 min and centrifuged at full speed for 1 min. The extracted genomic DNA was measured concentration and purity by Nano Drop 2000 (Thermo Fisher Scientific, Waltham, Massachusetts, USA). Nested PCR test for detection of Mycobacterium tuberculosis A 0.5 μl aliquot of enzymes and a mixture of Taq polymerase and buffer were added to 15.5 μl of a mixture of the primers of MTB, dntps, buffer and dye. Next, 4.0 μl of the extracted DNA were added to the reaction mixture for the first PCR round. Positive and negative controls were used. After the first PCR round, 18.0 μl of the first PCR round product and 0.5 μl of the second PCR round enzyme were mixed with 1.5 μl of the first PCR round product. Both PCR cycles were conducted with an initial 5 min denaturation step at 95 C, three repeating cycles of 30 sec each at 94 C, 68 C, and 72 C, followed by 25 cycles of 30 sec at 94 C, 65 C, and 72 C, and 5 min of the final extension at 72 C for 30 sec. A 5 μl aliquot of the PCR product was resolved on 1.5% agarose gel. The products were visualized by Ultraviolet Transillumination compared to the positive control. The first PCR band was revealed at 256 bp and the second was revealed at 181 bp. PCR-REBA Myco-ID using a HybREAD 480 The REBA Myco-ID was used according to the manufacturer s instructions to rapidly identify Mycobacterium species in various tissue samples. First, PCR was performed using 20 μl of reaction mixture (YD Diagnostics) containing 10 μl of PCR premix, 2 μl of primer mixture, 5 μl of extracted template DNA, and sterile distilled water to give a final volume of 20 μl. The 40 PCR cycles were comprised of initial predenaturation at 95 C for 5 min followed by denaturation at 95 C for 30 sec, then annealing and extension at 65 C for 30 sec. After the final cycle, samples were maintained at 72 C for 10 min to complete the DNA synthesis. The amplified PCR products were loaded into the HybREAD 480 system and denaturation, hybridization, washing, and chemiluminescent reaction were then automatically performed by the instrument. After all processes were complete, the data was interpreted using HybREAD 480. Results ear (n=2, 3.8%), and bronchus, testis, tendon, kidney, appendix, soft tissue, and finger joint (all n=1, 1.9%). Comparison of results from the nested PCR and REBA Myco-ID assays To evaluate the automated diagnostic performance of the REBA Myco-ID assay, its results were compared with those from the conventional nested PCR tests for detecting MTB and NTM infection. In conventional nested PCR tests, among the 52 samples, 25 samples (48%) were MTB positive and 27 samples (52%) were negative. In contrast, when conducted REBA Myco-ID test, among the 52 samples, 11 samples (21%) were MTB positive, 20 (39%) were NTM positive, 8 (15%) were MTB-NTM double positive, and 13 (25%) were negative. The PPVs for MTB, NTM and NPV of REBA Myco-ID test were 63.2%, 55.0% and 69.2%, respectively (Table 2). The overall coincidence rate of the assays was 61.5%. Table 1 Population characteristics. Characteristics No. of samples, n (%) Age (median=51.7 SD ± 16.3 yrs, range=17-87) <30's 11 (21.1) 40-50's 21 (40.4) >60's 20 (38.5) Sex Male 35 (67.3) Female 17 (32.7) Locations Colon 14 (26.9) Lung 13 (25.0) Ileum 7 (13.6) Lymph node 6 (11.6) Skin 3 (5.8) Ear 2 (3.8) Bronchus 1 (1.9) Testis 1 (1.9) Tendon 1 (1.9) Kidney 1 (1.9) Appendix 1 (1.9) Soft tissue (hand) 1 (1.9) Lt. finger joint 1 (1.9) Population characteristics The characteristics of the patients involved in this study are shown in Table 1. The mean age of the enrolled subjects was 51.7 years (17 to 87 years) and the male-to-female ratio was 35:17 (67.3%:32.7%). FFPE tissue was obtained from several types of organ including colon (n=14, 26.9%), lung (n=13, 25%), ileum (n=7, 13.6%), lymph node (n=6, 11.6%), skin (n=3, 5.8%), Total 52 (100) Comparison of results from the nested PCR and REBA Myco-ID assays with histopathological diagnosis in discordant samples To determine the accuracy and reliability of the two molecular diagnostic tests, results from those two assays, the Copyright imedpub 3

4 nested PCR and REBA Myco-ID assays, were compared with histopathological diagnosis in discordant samples. Among the 17 tubercle samples, when conducted the conventional nested PCR test, 10 samples (59%) were MTB positive and seven samples (41%) were negative. In contrast, when conducted REBA Myco-ID test, three samples (17%) were MTB positive, 10 samples (59%) were NTM positive and four samples (24%) were negative. Likewise, among the 13 chronic inflammation samples, when conducted the nested PCR test, three samples (23%) were MTB positive and 10 samples (77%) were negative. On the other hand, when conducted REBA Myco-ID test, two samples (15%) were MTB positive and 11 samples (85%) were NTM positive. Table 2 Comparison of results from the nested PCR and REBA Myco-ID assays with histopathological diagnosis in discordant samples. REBA Myco-ID Nested-PCR Variables MTB, n (%) NTM, n (%) Negative, n (%) Total, n (%) MTB, n (%) 12 (23%) 9 (17%) 4 (8%) 25 (48%) Negative, n (%) 7 (14%) 11 (21%) 9 (17%) 27 (52%) Total, n (%) 19 (37%) 20 (38%) 13 (25%) 52 (100%) PPV, % (n) 63.2% (12/19) 55.0% (11/20) NPV, % (n) % (9/13) -- Concordance, % (n) 61.5% (32/52) PPV: Positive Predictive Value, NPV: Negative Predictive Value Patterns of mycobacterial infection according to histopathological diagnosis The REBA Myco-ID results were analyzed to observe mycobacterial infection patterns according to histopathological diagnosis. Of 31 tubercle samples, eight (25.8%) were positive for MTB, 10 (32.2%) were positive for NTM, seven (22.6%) were MTB-NTM double positive, and six (19.4%) were negative. Of the 18 samples presenting chronic inflammation, two (11.1%) were positive for MTB, 11 (61.1%) were positive for NTM, and five (27.8%) were negative. Among three other samples, one (33.3%) was positive for MTB and two (66.7%) were negative (Figure 1). Of the 11 samples that originally tested positive for MTB, eight (72.7%) were tuberculous, two (18.2%) were chronic inflammation, and one (9.1%) was necrotic material. Of the original 21 NTM-positive samples, 10 (47.6%) were tuberculous and 11 (52.4%) were chronic inflammation. Coinfection of MTB and NTM was only found in originally tuberculous samples. Figure 1 Comparison of patterns of mycobacterial infection according to histopathological diagnosis. More detailed results of NTM species identification are shown in Table 3. Of the 10 NTM cases identified by REBA Myco-ID, five involved Mycobacterium; these were one case each of M. abscessus, M. massilense, M. intracellulare, M. kansasii, and M. avium-m. kansasii co-infection. Of the seven MTB-NTM double positive cases, four were found to be MTB 4 This article is available from:

5 and M. chelonae co-infection, two were MTB and M. kansasii co-infection, and one was MTB, M. chelonae, and M. kansasii co-infection. Within the seven cases of chronic colitis, one was positive for MTB, three were Mycobacterium species, and one was M. intracellulare. Among the five cases of chronic ileitis, three were Mycobacterium species and one was M. intracellulare, and of the two cases of chronic lymphadenitis, one was M. avium and one was M. gordonae. One case of necrotic material and one of chronic inflammation tested positive for MTB, and one chronic bronchitis case was M. intracellulare. Table 3 Patterns of mycobacterial infection according to the histopathological diagnosis. Histopathological diagnosis Nested PCR, n (%) REBA Myco-ID, n (%) TBC (n=17) MTB 10 (59%) MTB 3 (17%) Chronic inflammation (n=13) Negative 7 (41%) NTM 10 (59%) Negative 4 (24%) MTB 3 (23%) MTB 2 (15%) NTM 11 (85%) Negative 10 (77%) Negative 0 (0%) Patterns of mycobacterial infection according to the type of tissue The REBA Myco-ID results were analyzed to investigate the mycobacterial infection patterns according to type of tissue; the data are shown in Table 4. The original 11 MTB-positive samples included three (27.3%) that were from the colon, four (36.5%) from the lungs, two (18.2%) from the lymph nodes, and one (9%) each from ear and soft tissue. Of the 21 NTM-positive cases, six (28.6%) were colon samples, five (23.8%) were lung, two (9.5%) were lymph node, one (4.75%) was kidney and one (4.75%) was appendix. Among the seven MTB-NTM co-infection cases, two (28.5%) were lung samples, and one each (14.3%) was colon, lymph node, bronchus, testis, and tendon. Of the 14 colon samples, three (21.4%) tested positive for MTB, 6 (42.9%) were positive for NTM, one (7.1%) was MTB- NTM double positive, and four (28.6%) tested negative for any Mycobacterium. Four (30.8%) of the 13 lung samples were positive for MTB, five (38.4%) were positive for NTM, two (15.4%) were coinfected, and 2 (15.4%) were negative for any species of Mycobacterium. Among the seven ileum samples, six (85.7%) were positive for NTM and one (14.3%) was negative for any mycobacterial infection. For the six lymph node samples, two (33.3%) were positive for MTB and two (33.3%) for NTM, one (16.7%) was MTB-NTM double positive, and one (16.7%) was completely negative. The single bronchus, testis, and tendon samples were MTB- NTM double positive, the kidney and appendix samples were NTM-positive, and the ear and soft tissue samples tested positive for MTB (Table 5). Table 4 Comparison of Mycobacterium species infection patterns of various regions. Variables REBA Myco-ID results MTB positive, n (%) NTM positive, n (%) Negative, n (%) Tubercle (n=31) 8 (25.8) Mycobacterium spp. 5 (16.2) MTB, M. chelonae 4 (13.0) 6 (19.3) M. abscessus 1 (3.2) M. avium and M. kansasii 1 (3.2) MTB, M. chelonae, M. M. massilense 1 (3.2) kansasii 1 (3.2) M. intracellulare 1 (3.2) MTB, M. kansasii 2 (6.5) M. kansasii 1 (3.2) Chronic colitis (n=7) 1 (14.3) Mycobacterium spp. 3 (42.8) -- 2 (28.6) M. intracellulare 1 (14.3) Chronic ileitis (n=5) Mycobacterium spp. 3 (60.0) -- 1 (20.0) M. intra 1 (20.0) Chronic Lymphadenitis (n=2) M. avium 2 (100.0) -- Chronic dermatis (100.0) Copyright imedpub 5

6 (n=2) Necrotic material Furuncle Chronic inflammation Chronic bronchitis Chlesteatoma 1 (100.0) (100.0) 1 (100.0) M. intracellulare 1 (100.0) (100.0) Total Table 5 Comparison of Mycobacterium species infection patterns of various regions. Specimens REBA Myco-ID results MTB positive, n (%) NTM positive, n (%) MTB & NTM double positive, n (%) Negative, n (%) Colon (n=14) 3 (21.4) Mycobacterium spp. 4 (28.6) MTB, M. kansasii 1 (7.1) 4 (28.6) M. intracellulare 2 (14.3) Mycobacterium spp. 1 (7.7) M. abscessus 1 (7.7) Lung 4 (30.7) M. avium, kansasii 1 (7.7) 2 (15.4) 2 (15.4) (n=13) M. massiliense 1 (7.7) M. intracellulare 1 (7.7) MTB, M. chelonae Mycobacterium spp. 4 (57.1) Ileum (n=7) M. intracellulare 1 (14.3) M. kansasii 1 (14.3) -- 1 (14.3) lymph node (n=6) 2 (33.2) M. avium 1 (16.7) M. gordonae 1 (16.7) MTB, M. kansasii 1 (16.7) 1 (16.7) Skin (n=3) (100.0) Bronchus -- MTB, M. chelonae 1 (100.0) Testis -- TB, M. chelonae, M. kansasii 1 (100.0) Tendon -- TB, M. chelonae 1 (100.0) Kidney Mycobacterium spp. 1 (100.0) -- Appendix Mycobacterium spp. 1 (100.0) -- Ear 1 (100.0) (100.0) 6 This article is available from:

7 Soft tissue (hand) 1 (100.0) Lt. finger joint (100.0) Total Discussion TB remains a serious health problem in the Republic of Korea [16]. Moreover, cases of NTM infection have been misdiagnosed as TB and treated unnecessarily [17]. Accurate diagnosis of mycobacterial infection by MTB and other NTM species is therefore required. In order to evaluate the clinical usefulness of automated PCR-REBA instrument, genomic DNA was extracted from various FFPE human tissue samples and the diagnostic performance of a REBA Myco-ID using a HybREAD 480 system (YD Diagnostics) was compared with conventional nested PCR assays. Of the 25 samples that tested MTB positive in nested PCR, 12 cases (48%) were similarly identified as MTB by the REBA Myco-ID; however, nine cases (36%) were identified as NTM and four (16%) as negative for any mycobacterial infection. This means that where nested PCR only identified MTB, the HybREAD 480 system identified MTB and NTM. In addition, it is thought that existing nested PCR techniques might amplify the nucleotide sequence which can cause crossreaction between NTM and MTB. Among the 27 samples that returned negative results in nested PCR, nine cases (33%) were also negative for mycobacterial infection in REBA Myco-ID; however, seven cases (26%) were identified as MTB and 11 (41%) as NTM. Additional DNA sequence analysis with discordant samples is required in both assays to compare their accuracy as this could not be confirmed because of the limited template DNA samples in this study. The pattern of mycobacterial infection according to histopathological diagnosis was analyzed. Of 31 tubercle samples, eight (25.8%) tested positive for MTB, 10 (32.2%) were positive for NTM, seven (22.7%) displayed MTB-NTM coinfection, and six (19.3%) tested negative. In the 18 cases of chronic inflammation, two (11.1%) tested positively for MTB, 11 (61.1%) for NTM, and five samples (27.8%) were negative. These results suggest that MTB was more frequently found in tubercle lesions than NTM, while NTM were more frequently found in chronic inflammation than MTB. In this way, MTB might play a role in tubercle formation in human tissue and NTM might be responsible for causing chronic inflammation. Furthermore, mycobacterial infection patterns according to tissue type were analyzed. Three (21.4%) of the 14 colon samples were positive for MTB, six (42.9%) were positive for NTM, one (7.1%) demonstrated MTB-NTM co-infection, and four (28.6%) samples were negative for any mycobacteria. Of the 13 lung samples, four (30.7%) returned positive MTB results, five (38.5%) were positive for NTM, two (15.4%) had MTB-NTM co-infection, and the remaining two (15.4%) were negative. Six (85.7%) of the seven ileum samples were positive for NTM and 1 (14.3%) was negative. These results suggest that MTB appears more frequently in lung, colon, and lymph node tissues than at other sites, while NTM species are most highly detected in colon, ileum, and lung. In NTM infections, M. intracellulare and M. avium have been identified more often than other species in previous research [13]. In the present study, these particular species were identified similarly in NTM infections, frequently. In contrast, other studies have identified M. chelonae in fewer than one case [3,18] but this study has revealed a number of multi-infections with TB. As a result, it can be said that the proportion of M. chelonae is relatively high in NTM in the Busan area. Conclusion In conclusion, the REBA Myco-ID system provided a rapid and effective method of simultaneously detecting MTB, NTM species, and MTB-NTM co-infection in various tissue samples. Although the system is more expensive than conventional assays, its overall economy and time consumption are advantageous. The REBA Myco-ID can therefore provide accurate identification of mycobacterial species for the effective antibiotic treatment of infection. Nevertheless, further evaluation should be performed with larger volumes of varied clinical samples. Acknowledgments This study was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2016R1C1B108888) and BB21 plus Project in. References 1. World Health Organization (2017) Global tuberculosis report. 2. Vidyaraj CK, Chitra A, Smita S, Muthuraj M, Govindarajan S, et al. (2017) Prevalence of rifampicin-resistant Mycobacterium tuberculosis among human-immunodeficiency-virusseropositive patients and their treatment outcomes. J Epidemiol Glob Health 7(4): Wang HY, Kim H, Kim S, Kim DK, Cho SN, et al. (2015) Performance of a real-time PCR assay for the rapid identification of Mycobacterium species. J Microbiol 53(1): Park YS, Lee CH, Lee SM, Yang SC, Yoo CG, et al. (2010) Rapid increase of non-tuberculous mycobacterial lung diseases at a tertiary referral hospital in South Korea. Int J Tuberc Lung Dis 14(8): Copyright imedpub 7

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