Y Hu1, X Wu2, J Luo1, Y Fu1, L Zhao1, Y Ma1, Y Li1, Q Liang1, Y Shang1, H Huang3. 1. National Clinical Laboratory on Tuberculosis, Beijing Key Laboratory for Drug Resistant Tuberculosis Research, Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis and Thoracic Tumor Institute, Beijing, China. 2. Department of Tuberculosis, Beijing Chest Hospital, Capital Medical University, Beijing, China. 3. National Clinical Laboratory on Tuberculosis, Beijing Key Laboratory for Drug Resistant Tuberculosis Research, Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis and Thoracic Tumor Institute, Beijing, China. Electronic address: huanghairong@tb123.org.
Abstract
OBJECTIVES: Despite the importance of pyrazinamide (PZA) in tuberculosis treatment, PZA susceptibility testing is not routinely performed because of its acid pH requirement. We evaluated the Microplate Alamar Blue assay (MABA) to detect resistance to PZA using nicotinamide (NIC) as a surrogate in neutral pH and identify the appropriate cutoff point for the assay. METHODS: The NIC minimal inhibition concentrations (MICs) for 125 Mycobacterium tuberculosis clinical isolates were tested by MABA at nine different concentrations (8-2000 μg/mL). The PZA susceptibility testing by the BACTEC MGIT 960 system was used as a reference method. The pncA gene and its promoter region were sequenced for all the recruited strains. RESULTS: A total of 64 of 125 clinical isolates were identified as resistant by MGIT 960. Using a minimum inhibitory concentration (MIC) of >500 μg/mL as the cutoff concentration to define resistance presented the best fit of the MABA assay with the MGIT 960 outcomes. MABA demonstrated sensitivity of 100% (95% confidence interval, 92.6-100), specificity of 95.2% (95% confidence interval, 86.0-98.8) and an accuracy of 97.6% compared to the MGIT 960 method. Nine PZA susceptible strains defined by MGIT 960 also had pncA mutations; however, three of them were defined as PZA resistant by NIC MABA with MIC ≥2000 μg/mL. CONCLUSIONS: The NIC substitution method for PZA susceptibility test is reliable, cheap, rapid and easy, which makes it promising for use in clinical laboratories.
OBJECTIVES: Despite the importance of pyrazinamide (PZA) in tuberculosis treatment, PZA susceptibility testing is not routinely performed because of its acid pH requirement. We evaluated the Microplate Alamar Blue assay (MABA) to detect resistance to PZA using nicotinamide (NIC) as a surrogate in neutral pH and identify the appropriate cutoff point for the assay. METHODS: The NIC minimal inhibition concentrations (MICs) for 125 Mycobacterium tuberculosis clinical isolates were tested by MABA at nine different concentrations (8-2000 μg/mL). The PZA susceptibility testing by the BACTEC MGIT 960 system was used as a reference method. The pncA gene and its promoter region were sequenced for all the recruited strains. RESULTS: A total of 64 of 125 clinical isolates were identified as resistant by MGIT 960. Using a minimum inhibitory concentration (MIC) of >500 μg/mL as the cutoff concentration to define resistance presented the best fit of the MABA assay with the MGIT 960 outcomes. MABA demonstrated sensitivity of 100% (95% confidence interval, 92.6-100), specificity of 95.2% (95% confidence interval, 86.0-98.8) and an accuracy of 97.6% compared to the MGIT 960 method. Nine PZA susceptible strains defined by MGIT 960 also had pncA mutations; however, three of them were defined as PZA resistant by NIC MABA with MIC ≥2000 μg/mL. CONCLUSIONS: The NIC substitution method for PZA susceptibility test is reliable, cheap, rapid and easy, which makes it promising for use in clinical laboratories.
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