Simultaneous detection of omicron and other SARS-CoV-2 variants by multiplex PCR MassARRAY technology.

The rapid emergence of SARS-CoV-2 variants with high severity and transmutability adds further urgency for rapid and multiplex molecular testing to identify the variants. A nucleotide matrix-assisted laser-desorption-ionization time-of-flight mass spectrophotometry (MALDI-TOF MS)-based assay was developed (called point mutation array, PMA) to identify four major SARS-CoV-2 variants of concern (VOCs) including Alpha, Beta, Delta, and Omicron (namely PMA-ABDO) and differentiate Omicron subvariant (namely PMA-Omicron). PMA-ABDO and PMA-Omicron consist of 24 and 28 mutation sites of the spike gene. Both PMA panels specifically identified VOCs with as low as 10 viral copies/µl. The panel has shown a 100% concordant with the Next Generation Sequencing (NGS) results testing on 256 clinical specimens with real-time PCR cycle threshold (Ct) values less than 26. It showed a higher sensitivity over NGS; 25/28 samples were positive by PMA but not NGS in the clinical samples with PCR Ct higher than 26. Due to the mass of nucleotide used to differentiate between wild-type and mutation strains, the co-infection or recombination of multiple variants can be determined by the PMA method. This method is flexible in adding a new primer set to identify a new emerging mutation site among the current circulating VOCs and the turnaround time is less than 8 h. However, the spike gene sequencing or NGS retains the advantage of detecting newly emerged variants.

Simultaneous Detection of Omicron and Other SARS-CoV-2 Variants by Multiplex PCR MassARRAY Technology.

The rapid emergence of SARS-CoV-2 variants with high severity and transmutability adds further urgency for rapid and multiplex molecular testing to identify the variants. A nucleotide matrix-assisted laser-desorption-ionization time-of-flight mass spectrophotometry (MALDI-TOF MS)-based assay was developed (called point mutation array, PMA) to identify four major SARS-CoV-2 variants of concern (VOCs) including Alpha, Beta, Delta, and Omicron (namely PMA-ABDO) and differentiate Omicron subvariant (namely PMA-Omicron). PMA-ABDO and PMA-Omicron consist of 24 and 28 mutation sites of the spike gene. Both PMA panels specifically identified VOCs with as low as 10 viral copies/ µl. The panel has shown a 100% concordant with the Next Generation Sequencing (NGS) results testing on 256 clinical specimens with real-time PCR cycle threshold (Ct) values less than 26. It showed a higher sensitivity over NGS; 25/28 samples were positive by PMA but not NGS in the clinical samples with PCR Ct higher than 26. Due to the mass of nucleotide used to differentiate between wild-type and mutation strains, the co-infection or recombination of multiple variants can be determined by the PMA method. This method is flexible in adding a new primer set to identify a new emerging mutation site among the current circulating VOCs and the turnaround time is less than 8 hours. However, the spike gene sequencing or NGS retains the advantage of detecting newly emerged variants.

Molecular Characterization of Mutations in Isoniazid- and Rifampicin-Resistant Mycobacterium tuberculosis Isolated in Thailand.

The control of drug-resistant tuberculosis (TB) is a major challenge. The frequency and mutation characteristics indicate the efficiency of molecular tests for the rapid detection of TB drug resistance. This study examined the existence of katG and inhA mutations for isoniazid (INH) resistance and rpoB mutations for rifampicin (RFP) resistance. In total, 178 drug-resistant Mycobacterium tuberculosis (MTB) isolates were analyzed. Mutations in katG encoding and inhA regulatory regions were detected in 136/168 (81.0%) and 29/168 (17.3%), respectively, with the most prominent mutation of Ser315Thr substitution in katG in 126/168 (75.0%), and -15 C to T substitution in the regulatory region of the inhA (26/168; 15.5%). Two distinct katG mutations (Tyr337Cys, 1003InsG) were identified. Of 125 RFP-resistant isolates, 118 (94.4%) carried mutations affecting the 81-bp RFP resistance-determining region, with the most commonly affected codons 450, 445, and 435 identified in 74 (59.2%), 26 (20.8%), and 12 (9.6%) isolates, respectively. Genetic mutations were highly associated with phenotypic INH and RFP resistance, and the majority shared similarities with those reported in previous studies in Thailand and other Asian countries. These data are useful for guiding the use and improvement of molecular tests for TB drug resistance.

Drug-resistant Mycobacterium tuberculosis and its genotypes isolated from an outbreak in western Thailand.

BACKGROUND: Multidrug-resistant TB (MDR-TB) outbreaks have occurred in the Thamaka district, Kanchanaburi province in Thailand. METHODS: Seventy-two isolates, which included 7% mono-, 30.6% MDR and extensively drug-resistant TB (XDR-TB), were genotyped by spoligotyping, mycobacterial interspersed repetitive unit-variable-number tandem repeat (MIRU-VNTR) and single nucleotide polymorphism genotyping, and their drug resistance was analysed. RESULTS: The spoligotyping results showed that Beijing spoligo-international type (SIT)1 was predominant (n=38; 52.8%) while the remaining were non-Beijing sublineages (n=34). The MIRU-VNTR analysis showed that Beijing isolates, most of which belonged to the modern type (n=37), formed 5 clusters and 13 individual patterns. In katG, only mutation Ser315Thr was identified. In rpoB, Ser531Leu was predominant, except for His526Arg and Leu533Pro, which were found in two isolates. A cluster of 14 Beijing strains contained these common mutations and shared the MIRU-VNTR genotype with isolates in the Thamaka district that had spread previously. Two U SIT523 isolates contained the mutations A1400G in rrs and Asp94Gly in gyrA genes, indicating a spread of XDR-TB. CONCLUSIONS: Most mutations were associated with drug resistance and the specific MDR Beijing and XDR-TB in U SIT523 isolates remain. This genotyping is a key tool for tracking TB transmission in the Thamaka district of Thailand.

Characterization of katG, inhA, rpoB and pncA in Mycobacterium tuberculosis isolates from MDR-TB risk patients in Thailand.

INTRODUCTION: Multidrug-resistant tuberculosis (MDR-TB) is commonly found in Thailand especially in the public health region 5, the Western region of Thailand. This study's aim was to characterize katG, inhA, rpoB and pncA genes in Mycobacterium tuberculosis. METHODOLOGY: One hundred strains of Mycobacterium tuberculosis (MTB) were isolated from sputum samples of MDR-TB risk patients in the laboratory of the Office of Disease Prevention and Control 5th Ratchaburi province, Thailand from January to December 2015. Drug susceptibility testing (DST) was performed using a BACTEC MGIT 960 system. Furthermore, the genes katG, inhA, rpoB and pncA were characterized by DNA sequencing. RESULTS: Of a total of 100 MTB samples which underwent drug susceptibility testing, 42% showed isoniazid (INH) and rifampicin (RIF) resistance, and a further 25% showed INH mono-resistance (25%). The most common gene mutations found using DNA sequencing were katG_Ser315Thr (70%), rpoB_Ser531leu (81%) and pncA_Ile31Thr (84%). The common mutation of pncA_Ile31Thr substitution was detected in 26 of 91 (29%) pyrazinamide (PZA) susceptible isolates. CONCLUSION: Using DNA sequencing to screen for gene mutations conferring drug resistance may be feasible and use less time than using DST to detect resistance patterns.

An optimized genomic VCF workflow for precise identification of Mycobacterium tuberculosis cluster from cross-platform whole genome sequencing data.

Whole-genome sequencing (WGS) data allow for an inference of Mycobacterium tuberculosis (Mtb) clusters by using a pairwise genetic distance of ≤12 single nucleotide polymorphisms (SNPs) as a threshold. However, a problem of discrepancies in numbers of SNPs and genetic distance measurement is a great concern when combining WGS data from different next generation sequencing (NGS) platforms. We performed SNP variant calling on WGS data of 9 multidrug-resistant (MDR-TB), 3 extensively drug-resistant tuberculosis (XDR-TB) and a standard M. tuberculosis strain H37Rv from an Illumina/NextSeq500 and an Ion Torrent PGM. Variant calls were obtained using four different common variant calling tools, including Genome Analysis Toolkit (GATK) HaplotypeCaller (GATK-VCF workflow), GATK HaplotypeCaller and GenotypeGVCFs (GATK-GVCF workflow), SAMtools, and VarScan 2. Cross-platform pairwise SNP differences, minimum spanning networks and average nucleotide identity (ANI) were analysed to measure performance of the variant calling tools. Minimum pairwise SNP differences ranged from 2 to 14 SNPs when using GVCF workflow while maximum pairwise SNP differences ranged from 7 to 158 SNPs when using VarScan 2. ANI comparison between SNPs data from NextSeq500 and PGM of MDR-TB and XDR-TB showed maximum ANI of 99.7% and 99.0%, respectively, with GVCF workflow while the other SNP calling results showed lower ANI in a range of 98.6% to 95.1%. In this study, we suggest that the GVCF workflow showed the best performing variant caller to avoid cross-platform pairwise SNP differences.

First Complete Genome Sequence of Human Coronavirus HKU1 from a Nonill Bat Guano Miner in Thailand.

Human coronavirus HKU1 (HCoV-HKU1) was first detected in a patient with viral pneumonia from Hong Kong in 2004. Here, we report the first complete genome sequence of HCoV-HKU1 from Thailand, obtained from a nonill person who worked in a bat cave. Phylogenetic tree analysis revealed it as a group B HCoV-HKU1.