Rapid Detection of Mycobacterium tuberculosis and Pyrazinamide Susceptibility Related topncAMutations in Sputum Specimens through an Integrated Gene-to-Protein Function Approach

Abstract

cTesting the pyrazinamide (PZA) susceptibility of Mycobacterium tuberculosis isolates is challenging. In a previous paper, we described the development of a rapid colorimetric test for the PZA susceptibility of M. tuberculosis by a PCR-based in vitro-synthesized-pyrazinamidase (PZase) assay. Here, we present an integrated approach to detect M. tuberculosis and PZA susceptibility directly from sputum specimens. M. tuberculosis was detected first, using a novel long-fragment quantitative real-time PCR (LF-qPCR), which amplified a fragment containing the whole pncA gene. Then, the positive amplicons were sequenced to find mutations in the pncA gene. For new mutations not found in the Tuberculosis Drug Resistance Mutation Database (www .tbdreamdb.com), the in vitro PZase assay was used to test the PZA resistance. This approach could detect M. tuberculosis within 3 h with a detection limit of 7.8 copies/reaction and report the PZA susceptibility within 2 days. In an initial testing of 213 sputum specimens, the LF-qPCR found 53 positive samples with 92% sensitivity and 97% specificity compared to the culture test for M. tuberculosis detection. DNA sequencing of the LF-qPCR amplicons revealed that 49 samples were PZA susceptible and 1 was PZA resistant. In the remaining 3 samples, with new pncA mutations, the in vitro PZase assay found that 1 was PZA susceptible and 2 were PZA resistant. This integrated approach provides a rapid, efficient, and relatively low-cost solution for detecting M. tuberculosis and PZA susceptibility without culture.

A Global Perspective on Pyrazinamide Resistance: Systematic Review and Meta-Analysis

“…tuberculosis clinical isolates. According to WHO region, five [ 60 , 68 , 69 , 78 , 102 ] articles were from Africa, 14 [ 27 , 70 – 72 , 79 , 81 – 83 , 97 , 100 , 105 , 107 , 114 , 120 ] from the Americas, two [ 80 , 92 ] from the Eastern Mediterranean, 17 [ 61 , 62 , 84 – 86 , 93 , 96 , 103 , 104 , 106 , 108 – 110 , 115 – 117 , 119 ] from Europe, six [ 59 , 63 , 73 , 118 , 121 , 123 ] from South East Asia, and 22 [ 64 – 67 , 74 – 77 , 87 – 91 , 94 , 95 , 98 , 99 , 101 , 111 – 113 , 122 ] from the Western Pacific. A SNP in the ...…”

Assessing mycoplasma contamination of cell cultures by qPCR using a set of universal primer pairs targeting a 1.5 kb fragment of 16S rRNA genes

“…This optimized method was called “m16S_qPCR”. That a Real-Time PCR of long DNA fragments can be performed with non-degenerate primers has been independently reported to detect two bacterial non-ribosomal genes [ 20 , 21 ].…”

Immunological detection of pyrazine-2-carboxylic acid for the detection of pyrazinamide resistance in Mycobacterium tuberculosis

“…However, high rates of mortality have been reported when PZA is administered empirically to patients who are infected with PZA-resistant strains [5]. PZA resistance is associated with resistance to other TB medications: current estimates indicate that 50% of MDR-TB cases are also PZA resistant [6][7][8] and, alarmingly, the emergence of Mycobacterium tuberculosis (Mtb) strains resistant to all known anti-TB drugs has been reported [9]. This is likely to be caused by the mismanagement of pharmacological protocols.…”