ERRATUM: Rapid and Simple Detection of Isoniazid-Resistant Mycobacterium tuberculosis Utilizing a DNA Chromatography-Based Technique.

Volume 74, no.3, p.214-219, 2021. Page 214, affiliation "1TBA Co., LTD, Sendai; 2Hokkaido University Research Center for Zoonosis Control, Sapporo; 3Hokkaido University, GI-CoRE Global Station for Zoonosis Control, Sapporo; 4Zambia National Public Health Institute, Ministry of Health, Lusaka, Zambia; 5Department of Pathology and Microbiology, University Teaching Hospital Ministry of Health, Lusaka, Zambia; and 6Ministry of Health, Ndeke House, Lusaka, Zambia." should read "1TBA Co., LTD, Sendai, Japan; 2Hokkaido University Research Center for Zoonosis Control, Sapporo, Japan; 3Hokkaido University, GI-CoRE Global Station for Zoonosis Control, Sapporo, Japan; 4Zambia National Public Health Institute, Ministry of Health, Lusaka, Zambia; 5Department of Pathology and Microbiology, University Teaching Hospital Ministry of Health, Lusaka, Zambia; and 6Ministry of Health, Ndeke House, Lusaka, Zambia".

Rapid and Simple Detection of Isoniazid-Resistant Mycobacterium tuberculosis Utilizing a DNA Chromatography-Based Technique.

Despite the availability of anti-tuberculosis drugs, the treatment of tuberculosis has been complicated by drug-resistant tuberculosis. The early detection of drug resistance makes early treatment possible. However, the available tools are mainly for rifampicin resistance detection, and the existing isoniazid resistance detection method is expensive, highly technical, and complicated, making it unsustainable for use in developing nations. This study aimed to develop a simple, rapid, and low-cost diagnostic kit for isoniazid-resistant tuberculosis using the single-stranded tag hybridization method to target an isoniazid resistance-conferring mutation. Specificity and sensitivity were assessed using DNA extracted from 49 isoniazid-resistant and 41 isoniazid-susceptible Mycobacterium tuberculosis clinical isolates cultured in mycobacterial growth indicator tubes. Positive signals were observed on mutant and wild-type lines with 100% sensitivity and specificity compared with Sanger sequencing results. In contrast, no positive signal was observed for non-tuberculosis mycobacteria. The detection limit of this method was 103 CFU or less. The STH-PAS system for isoniazid-resistant M. tuberculosis detection developed in this study offers a better alternative to conventional phenotypic isoniazid resistance determination, which will be of both clinical and epidemiological significance in resource-limited nations.

Rapid detection of rifampicin-resistant Mycobacterium tuberculosis, based on isothermal DNA amplification and DNA chromatography.

Rapid and easy detection of nucleotide point mutations in bacterial pathogens associated with drug resistance is essential for the proper use of antimicrobials. Here, we developed a rapid and simple method for the detection of mutations using Loop-mediated isothermal amplification (LAMP) combined with the single-tag hybridization (STH) chromatographic printed array strips (PAS) method. This procedure is able to detect four mutations (C1349 T, A1295C, G1303 T, A1304 T) in Rifampicin Resistance Determining Region (RRDR) of rifampicin-resistant Mycobacterium tuberculosis (RR-TB), simultaneously. LAMP reactions contained a LAMP primer and eight allele-specific primers for each mutation. The allele-specific primers products were detected by nucleic acid chromatography using PAS. Four detection lines were detected there, one of which was detected at different positions depend on the wild type and the mutant type. We carried out the four mutations detection using 31 genomic DNA (2 A1295T, 1 G1303 T, 6 A1304 T, 22 C1349 T) from clinical isolate. The mutations have been confirmed by sequence analysis. The detection results were completely consistent with the sequence analysis. In the present study, four mutations could be detected, but only 60% of RR-TB could be detected with these four. It is expected that the detection rate will increase by adding more mutant primers. The combined LAMP and STH chromatographic PAS method is a simple and rapid method for detecting point mutations in clinical isolates as a point-of-care testing (POCT) technique. In addition, it does not require special equipment and can meet the demand in areas where drug-resistant bacteria are endemic, such as developing countries.

PCR-Dipstick Chromatography for Differential Detection of Carbapenemase Genes Directly in Stool Specimens.

A PCR-dipstick chromatography technique was designed and evaluated for differential identification of blaNDM, blaKPC, blaIMP, and blaOXA-48 carbapenemase genes directly in stool specimens within 2 h. It is a DNA-DNA hybridization-based detection system where PCR products can be easily interpreted by visual observation without electrophoresis. The PCR-dipstick showed high sensitivity (93.3%) and specificity (99.1%) in directly detecting carbapenemase genes in stool specimens compared with multiplex PCR for genomic DNA of the isolates from those stool specimens.

Incipient complex formation between AP endonucleases and DNA containing AP site: a vital role of the tryptophan residue.

To elucidate whether the tryptophan residues in the vicinity of the catalytic site are involved in AP site recognition and are critical for AP endonuclease activity, the AP endonucleases of the four subtypes in the ExoIII AP endonuclease family were characterized and compared the positions of the tryptophan residues. The positions of the catalytic amino acid residues, corresponding to Glu-34, Asp-229, and His-259 of ExoIII, are strictly conserved. On the other hand, the positions of the tryptophan residues, which are critical to the incipient complex formation, do not exist at a fixed position. There are four patterns at the position of the essential tryptophan residue.