Validation and implementation of whole-genome sequencing-based analytical methods for molecular surveillance and relatedness analysis of complex isolates at a national reference laboratory.

Whole genome sequencing-based methodologies have become extremely relevant for the molecular surveillance of human pathogens and are being increasingly introduced into national reference laboratory services. In this study, we describe the validation and implementation of core-genome Multi-Locus Sequence Typing (cgMLST) and whole genome single-nucleotide polymorphism (wgSNP) analysis at the Irish Mycobacteria Reference Laboratory, as a replacement for Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeat (MIRU-VNTR) typing. Concordance of clustering, discriminatory power, and ease-of-use of both WGS analytical methods were evaluated.

Investigation of genomic mutations and their association with phenotypic resistance to new and repurposed drugs in Mycobacterium tuberculosis complex clinical isolates.

Background: WGS has the potential to detect resistance-associated mutations and guide treatment of MDR TB. However, the knowledge base to confidently interpret mutations associated with the new and repurposed drugs is sparse, and phenotypic drug susceptibility testing is required to detect resistance.

Methods: We screened 900 Mycobacterium tuberculosis complex genomes from Ireland, a low TB incidence country, for mutations in 13 candidate genes and assessed their association with phenotypic resistance to bedaquiline, clofazimine, linezolid, delamanid and pretomanid.

Towards clinical breakpoints for non-tuberculous mycobacteria - Determination of epidemiological cut off values for the Mycobacterium avium complex and Mycobacterium abscessus using broth microdilution.

Objective: For non-tuberculous mycobacteria (NTM), minimum inhibitory concentration (MIC) distributions of wild-type isolates have not been systematically evaluated despite their importance for establishing antimicrobial susceptibility testing (AST) breakpoints.

Methods: We gathered MIC distributions for drugs used against the Mycobacterium avium complex (MAC) and Mycobacterium abscessus (MAB) obtained by commercial broth microdilution (SLOMYCOI and RAPMYCOI) from 12 laboratories. Epidemiological cut-off values (ECOFFs) and tentative ECOFFs (TECOFFs) were determined by EUCAST methodology including quality control (QC) strains.

Diagnostic Performance of Xpert MTB/RIF Ultra Compared with Predecessor Test, Xpert MTB/RIF, in a Low TB Incidence Setting: a Retrospective Service Evaluation.

The aim of this study was to evaluate the performance of Xpert MTB/RIF Ultra (Ultra) compared with its predecessor, Xpert MTB/RIF (Xpert), in the diagnosis of tuberculosis (TB) in a low TB incidence country. Retrospective analysis was performed on 689 clinical samples received between 2015 and 2018, on which Xpert was performed, and on 715 samples, received between 2018 and 2020, on which Ultra was performed. Samples were pulmonary ( = 830) and extrapulmonary ( = 574) in nature, and a total of 264 were culture positive for Mycobacterium tuberculosis complex (MTBC).

False detection of rifampicin resistance using Xpert MTB/RIF Ultra assay due to an A451V mutation in .

Background: In a 12 month period, three Irish-born adult cases with pulmonary TB were initially diagnosed by Xpert MTB/RIF Ultra assay, which detected a rifampicin resistance-conferring mutation prompting treatment as potential MDR cases.

Methods: Further laboratory investigations on the cultured isolates included GenoType MTBDR assay, phenotypic drug susceptibility tests using the BD BACTEC MGIT culture system and MIC broth microdilution tests. Sequencing of the gene was performed using Sanger sequencing and WGS.

Draft Genome Sequences of Clinical Respiratory Isolates of Mycobacterium goodii Recovered in Ireland.

Here, we describe the draft genomes of five Mycobacterium goodii isolates that were recovered from respiratory clinical specimens in Ireland. Currently, one complete genome and one draft genome exist publicly for M. goodii.

Overcoming the Challenges of Pyrazinamide Susceptibility Testing in Clinical Mycobacterium tuberculosis Isolates.

Pyrazinamide (PZA) is one of the first-line agents used for the treatment of tuberculosis. However, current phenotypic PZA susceptibility testing in the Bactec MGIT 960 system is unreliable, and false resistance is well documented. Rapid identification of resistance-associated mutations can confirm the phenotypic result.

Genomic Analysis of Mycobacterium abscessus Complex Isolates Collected in Ireland between 2006 and 2017.

Members of the complex (MABC) are multidrug-resistant nontuberculous mycobacteria and cause opportunistic pulmonary infections in individuals with cystic fibrosis (CF). In this study, genomic analysis of MABC isolates was performed to gain greater insights into the epidemiology of circulating strains in Ireland. Whole-genome sequencing (WGS) was performed on 70 MABC isolates that had been referred to the Irish Mycobacteria Reference Laboratory between 2006 and 2017 across nine Irish health care centers.

Antimicrobial Susceptibility of Clinical and Environmental Mycobacterium chimaera Isolates.

is a slow-growing nontuberculous species belonging to the complex (MAC). It has been identified globally as the cause of a large outbreak of cardiovascular infections following open heart surgery, but it can also cause respiratory infections in individuals with underlying structural pulmonary disease. Invasive infections are associated with poor clinical responses, and the optimal antibiotic treatment regimen for these infections is not known.

Investigation of reduced susceptibility to glycopeptides among methicillin-resistant Staphylococcus aureus isolates from patients in Ireland and evaluation of agar screening methods for detection of heterogeneously glycopeptide-intermediate S. aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) isolates (n = 3,189) from 2,990 patients were investigated by agar screening and by the Etest macromethod for reduced susceptibility to glycopeptide. No vancomycin-resistant S. aureus or glycopeptide-intermediate S.

The emergence and importation of diverse genotypes of methicillin-resistant Staphylococcus aureus (MRSA) harboring the Panton-Valentine leukocidin gene (pvl) reveal that pvl is a poor marker for community-acquired MRSA strains in Ireland.

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) carrying pvl is an emerging problem worldwide. CA-MRSA tends to harbor staphylococcal cassette chromosome mec type IV (SCCmec IV), to be non-multiantibiotic resistant, and to have different genotypes from the local hospital-acquired MRSA (HA-MRSA). However, in Ireland, 80% of HA-MRSA isolates have the non-multiantibiotic-resistant genotype ST22-MRSA-IV.