Comparison of One-Tube Nested-PCR and PCR-Reverse Blot Hybridization Assays for Discrimination of Mycobacterium tuberculosis and Nontuberculous Mycobacterial Infection in FFPE tissues원문보기
Park, Sung-Bae
(Department of Clinical Laboratory Science, College of Health Sciences, Catholic University of Pusan)
,
Park, Heechul
(Department of Clinical Laboratory Science, College of Health Sciences, Catholic University of Pusan)
,
Bae, Jinyoung
(Department of Clinical Laboratory Science, College of Health Sciences, Catholic University of Pusan)
,
Lee, Jiyoung
(Department of Biomedical Laboratory Science, College of Health Sciences, Yonsei University)
,
Kim, Ji-Hoi
(YD R&D Center, YD Diagnostics)
,
Kang, Mi Ran
(YD R&D Center, YD Diagnostics)
,
Lee, Dongsup
(Department of Biomedical Laboratory Science, Hyejeon College)
,
Park, Ji Young
(Department of Internal Medicine, Kosin University Gospel Hospital)
,
Chang, Hee-Kyung
(Department of Pathology, Kosin University College of Medicine)
,
Kim, Sunghyun
(Department of Clinical Laboratory Science, College of Health Sciences, Catholic University of Pusan)
Currently, molecular diagnostic assays based on nucleic acid amplification tests have been shown to effectively detect mycobacterial infections in various types of specimen, however, variable sensitivity was shown in FFPE samples according to the kind of commercial kit used. The present study theref...
Currently, molecular diagnostic assays based on nucleic acid amplification tests have been shown to effectively detect mycobacterial infections in various types of specimen, however, variable sensitivity was shown in FFPE samples according to the kind of commercial kit used. The present study therefore used automated PCR-reverse blot hybridization assay (REBA) system, REBA Myco-ID HybREAD 480®, for the rapid identification of Mycobacterium species in various types of human tissue and compared the conventional one-tube nested-PCR assay for detecting Mycobacterium tuberculosis (MTB). In conventional nested-PCR tests, 25 samples (48%) were MTB positive and 27 samples (52%) were negative. In contrast, when conducted PCR-REBA assay, 11 samples (21%) were MTB positive, 20 samples (39%) were NTM positive, 8 samples (15%) were MTB-NTM double positive, and 13 samples (25%) were negative. To determine the accuracy and reliability of the two molecular diagnostic tests, the one-tube nested-PCR and PCR-REBA assays, were compared with histopathological diagnosis in discordant samples. When conducted nested-PCR assay, 10 samples (59%) were MTB positive and seven samples (41%) were negative. In contrast, when conducted PCR-REBA test, three samples (17%) were MTB positive, 10 samples (59%) were NTM positive and four samples (24%) were negative. In conclusion, the automated PCR-REBA system proved useful to identify Mycobacterium species more rapidly and with higher sensitivity and specificity than the conventional molecular assay, one-tube nested-PCR; 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.
Currently, molecular diagnostic assays based on nucleic acid amplification tests have been shown to effectively detect mycobacterial infections in various types of specimen, however, variable sensitivity was shown in FFPE samples according to the kind of commercial kit used. The present study therefore used automated PCR-reverse blot hybridization assay (REBA) system, REBA Myco-ID HybREAD 480®, for the rapid identification of Mycobacterium species in various types of human tissue and compared the conventional one-tube nested-PCR assay for detecting Mycobacterium tuberculosis (MTB). In conventional nested-PCR tests, 25 samples (48%) were MTB positive and 27 samples (52%) were negative. In contrast, when conducted PCR-REBA assay, 11 samples (21%) were MTB positive, 20 samples (39%) were NTM positive, 8 samples (15%) were MTB-NTM double positive, and 13 samples (25%) were negative. To determine the accuracy and reliability of the two molecular diagnostic tests, the one-tube nested-PCR and PCR-REBA assays, were compared with histopathological diagnosis in discordant samples. When conducted nested-PCR assay, 10 samples (59%) were MTB positive and seven samples (41%) were negative. In contrast, when conducted PCR-REBA test, three samples (17%) were MTB positive, 10 samples (59%) were NTM positive and four samples (24%) were negative. In conclusion, the automated PCR-REBA system proved useful to identify Mycobacterium species more rapidly and with higher sensitivity and specificity than the conventional molecular assay, one-tube nested-PCR; 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.
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제안 방법
To evaluate the automated diagnostic performance of the REBA Myco-ID® assay, its results were compared with those from the conventional one-tube nested-PCR tests for detecting MTB and NTM infection.
성능/효과
To determine the accuracy and reliability of the two molecular diagnostic tests, results from those two assays, the one-tube nested-PCR and PCR-REBA assays, were compared with histopathological diagnosis in discordant samples (Table 3). 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 PCR-REBA assay, three samples (17%) were MTB positive, 10 samples (59%) were NTM positive and four samples (24%) were negative.
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 one-tube nested-PCR, nine cases (33%) were also negative for mycobacterial infection in PCR-REBA; 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 the present study.
In conclusion, the conventional one-tube nested-PCR assay provides a more sensitive in detecting MTB, however, cannot discriminate between MTB and NTM species. However, the automated REBA Myco-ID HybREAD 480® system provided a rapid and effective method of simultaneously detecting MTB, NTM species, and MTB-NTM coinfection in various tissue 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 PCR-REBA assay, 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 one-tube nested-PCR test, three samples (23%) were MTB positive and 10 samples (77%) were negative.
In conventional one-tube nested-PCR tests, among the 52 samples, 25 samples (48%) were MTB positive and 27 samples (52%) were negative. In contrast, when conducted the PCR-REBA assay, among the 52 samples, 11 samples (21%) were MTB positive, 20 samples (39%) were NTM positive, 8 samples (15%) were MTB-NTM double positive, and 13 samples (25%) were negative. The positive predictive values (PPVs) for MTB, NTM and negative predictive value (NPV) of REBA MycoID® test were 63.
Of the 25 samples that tested MTB positive in one-tube nested-PCR assay, 12 cases (48%) were similarly identified as MTB by the PCR-REBA assay; 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 REBA Myco-ID HybREAD 480® system identified MTB and NTM.
The positive predictive values (PPVs) for MTB, NTM and negative predictive value (NPV) of REBA MycoID® test were 63.2%, 55.0% and 69.2%, respectively (Table 2).
To determine the accuracy and reliability of the two molecular diagnostic tests, results from those two assays, the one-tube nested-PCR and PCR-REBA assays, were compared with histopathological diagnosis in discordant samples (Table 3). Among the 17 tubercle samples, when conducted the conventional nested-PCR test, 10 samples (59%) were MTB positive and seven samples (41%) were negative.
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