Evaluation of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Molecular Typing of Acinetobacter baumannii in Comparison with Orthogonal Methods.

Colonization and subsequent health care-associated infection (HCAI) with Acinetobacter baumannii are a concern for vulnerable patient groups within the hospital setting. Outbreaks involving multidrug-resistant strains are associated with increased patient morbidity and mortality and poorer overall outcomes. Reliable molecular typing methods can help to trace transmission routes and manage outbreaks. In addition to methods deployed by reference laboratories, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) may assist by making initial in-house judgments on strain relatedness. However, limited studies on method reproducibility exist for this application. We applied MALDI-TOF MS typing to A. baumannii isolates associated with a nosocomial outbreak and evaluated different methods for data analysis. In addition, we compared MALDI-TOF MS with whole-genome sequencing (WGS) and Fourier transform infrared spectroscopy (FTIR) as orthogonal methods to further explore their resolution for bacterial strain typing. A related subgroup of isolates consistently clustered separately from the main outbreak group by all investigated methods. This finding, combined with epidemiological data from the outbreak, indicates that these methods identified a separate transmission event unrelated to the main outbreak. However, the MALDI-TOF MS upstream approach introduced measurement variability impacting method reproducibility and limiting its reliability as a standalone typing method. Availability of in-house typing methods with well-characterized sources of measurement uncertainty could assist with rapid and dependable confirmation (or denial) of suspected transmission events. This work highlights some of the steps to be improved before such tools can be fully integrated into routine diagnostic service workflows for strain typing. IMPORTANCE Managing the transmission of antimicrobial resistance necessitates reliable methods for tracking outbreaks. We compared the performance of MALDI-TOF MS with orthogonal approaches for strain typing, including WGS and FTIR, for Acinetobacter baumannii isolates correlated with a health care-associated infection (HCAI) event. Combined with epidemiological data, all methods investigated identified a group of isolates that were temporally and spatially linked to the outbreak, yet potentially attributed to a separate transmission event. This may have implications for guiding infection control strategies during an outbreak. However, the technical reproducibility of MALDI-TOF MS needs to be improved for it to be employed as a standalone typing method, as different stages of the experimental workflow introduced bias influencing interpretation of biomarker peak data. Availability of in-house methods for strain typing of bacteria could improve infection control practices following increased reports of outbreaks of antimicrobial-resistant organisms during the COVID-19 pandemic, related to sessional usage of personal protective equipment (PPE).

Single base mutations in the nucleocapsid gene of SARS-CoV-2 affects amplification efficiency of sequence variants and may lead to assay failure.

Reverse transcriptase quantitative PCR (RT-qPCR) is the main diagnostic assay used to detect SARS-CoV-2 RNA in respiratory samples. RT-qPCR is performed by specifically targeting the viral genome using complementary oligonucleotides called primers and probes. This approach relies on prior knowledge of the genetic sequence of the target. Viral genetic variants with changes to the primer/probe binding region may reduce the performance of PCR assays and have the potential to cause assay failure. In this work we demonstrate how two single nucleotide variants (SNVs) altered the amplification curve of a diagnostic PCR targeting the Nucleocapsid (N) gene and illustrate how threshold setting can lead to false-negative results even where the variant sequence is amplified. We also describe how in silico analysis of SARS-CoV-2 genome sequences available in the COVID-19 Genomics UK Consortium (COG-UK) and GISAID databases was performed to predict the impact of sequence variation on the performance of 22 published PCR assays. The vast majority of published primer and probe sequences contain sequence mismatches with at least one SARS-CoV-2 lineage. We recommend that visual observation of amplification curves is included as part of laboratory quality procedures, even in high throughput settings where thresholds are set automatically and that in silico analysis is used to monitor the potential impact of new variants on established assays. Ideally comprehensive in silico analysis should be applied to guide selection of highly conserved genomic regions to target with future SARS-CoV-2 PCR assays.