Hiroko Yoshida1, Kenichi Onohara2, Tomomi Tazawa2, Yuki Tsurinaga3, Masashi Kurokawa3, Yuki Han3, Yoshitaka Tamura3, Takayuki Nagai3, Shoji Hashimoto3, Ichirou Kawase3. 1. Division of Laboratory, Osaka Prefectural Medical Center for Respiratory and Allergic Disease, Osaka Prefectural Hospital Organization, 3-7-1, Habikino, Habikino-shi, Osaka 583- 8588 Japan. yoshidahi@opho.jp 2. Division of Laboratory, Osaka Prefectural Medical Center for Respiratory and Allergic Disease, Osaka Prefectural Hospital Organization, 3-7-1, Habikino, Habikino-shi, Osaka 583- 8588 Japan. 3. Division of Infectious Disease, Osaka Prefectural Medical Center for Respiratory and Allergic Disease, Osaka Prefectural Hospital Organization, 3-7-1, Habikino, Habikino-shi, Osaka 583- 8588 Japan.
Abstract
UNLABELLED: OBJECTIVE; The COBAS TaqMan real-time polymerase chain reaction (PCR) assay (TaqMan assay) is a well-accepted and widely distributed molecular-based diagnostic test for tuberculosis. In the present study, we evaluated the efficacy of the LAMP assay (loopamp MTBC detection kit) as an alternative molecular-based diagnostic kit for tuberculosis, through comparison with the TaqMan assay. STUDY PERIOD AND METHODS: This study was conducted over a period of approximately 2 months, between May and July 2012. We collected 48 samples (43 sputum, 2 gastric fluid, 2 pleural fluid, and 1 pus fluid samples) from patients who had been diagnosed with tuberculosis through the culture method, but had not received any treatment for more than one week. All samples were processed using the CC-E pre-treatment reagent (Japan BCG) prior to performing the TaqMan and LAMP assay. For the TaqMan assay, 100 microL of supernatant was used after centrifugation at 1,000 rpm for 1 minute, whereas 60 microL of the precipitate in the same sample was used for the LAMP assay. RESULTS: In total, 23 out of 48 samples were identified as positive for tuberculosis according to smear microscopy tests, among which 15, 4, and 4 samples had smear test scores or 1+, 2+, and 3+, respectively. All the samples that tested positive in the smear test, regardless of the score, also tested positive in both the TaqMan and TB-LAMP assays (100%). Of the 25 smear-negative samples, we noted that 16 tested positive by the TaqMan assay (64%), whereas 20 tested positive by the LAMP assay (80%). DISCUSSION: Compared with the TaqMan assay, the LAMP assay showed a higher positive rate among the smear-negative samples. We believe that this is because substances in the samples acted as co-precipitating agents, resulting in the presence of a larger number of bacteria in the precipitates than in the supernatants. Thus, the findings indicate that the application of the LAMP method to precipitates obtained following CC-E pre-treatments may lead to prompt diagnosis of tuberculosis, with a level of sensitivity comparable to that of culture tests.
UNLABELLED: OBJECTIVE; The COBAS TaqMan real-time polymerase chain reaction (PCR) assay (TaqMan assay) is a well-accepted and widely distributed molecular-based diagnostic test for tuberculosis. In the present study, we evaluated the efficacy of the LAMP assay (loopamp MTBC detection kit) as an alternative molecular-based diagnostic kit for tuberculosis, through comparison with the TaqMan assay. STUDY PERIOD AND METHODS: This study was conducted over a period of approximately 2 months, between May and July 2012. We collected 48 samples (43 sputum, 2 gastric fluid, 2 pleural fluid, and 1 pus fluid samples) from patients who had been diagnosed with tuberculosis through the culture method, but had not received any treatment for more than one week. All samples were processed using the CC-E pre-treatment reagent (Japan BCG) prior to performing the TaqMan and LAMP assay. For the TaqMan assay, 100 microL of supernatant was used after centrifugation at 1,000 rpm for 1 minute, whereas 60 microL of the precipitate in the same sample was used for the LAMP assay. RESULTS: In total, 23 out of 48 samples were identified as positive for tuberculosis according to smear microscopy tests, among which 15, 4, and 4 samples had smear test scores or 1+, 2+, and 3+, respectively. All the samples that tested positive in the smear test, regardless of the score, also tested positive in both the TaqMan and TB-LAMP assays (100%). Of the 25 smear-negative samples, we noted that 16 tested positive by the TaqMan assay (64%), whereas 20 tested positive by the LAMP assay (80%). DISCUSSION: Compared with the TaqMan assay, the LAMP assay showed a higher positive rate among the smear-negative samples. We believe that this is because substances in the samples acted as co-precipitating agents, resulting in the presence of a larger number of bacteria in the precipitates than in the supernatants. Thus, the findings indicate that the application of the LAMP method to precipitates obtained following CC-E pre-treatments may lead to prompt diagnosis of tuberculosis, with a level of sensitivity comparable to that of culture tests.