Evaluation of a combined Multi-Locus Sequence Typing and Whole-Genome Sequencing Two-step Algorithm for Routine typing of Clostridioides difficile

Multi locus sequence typing (MLST) is a low resolution but rapid genotyping method for Clostridioides difficile. Whole-genome sequencing (WGS) has emerged as the new gold standard for C. difficile typing but cost and lack of standardization still limits broad utilization. this study, we evaluated the potential to combine the portability of MLST with the increased resolution of WGS for a cost-saving approach to routine C. difficile typing. C. difficile strains from two New York City hospitals (Hospital A and Hospital B) were selected. WGS single nucleotide polymorphism (wgSNP) were performed using established methods. Sequence types (ST) were determined using PubMLST while wgSNP analysis was performed using the Bionumerics software. An additional analysis of a subset of data (Hospital A) was made comparing the Bionumerics software to the CosmosID pipeline. Cost and turn-around time to results were compared for the algorithmic approach of MLST followed by wgSNP versus direct wgSNP. Among the 202 C. difficile isolates typed, 91% (n=185/203) clustered within the representative ST, showing a high agreement between MLST and wgSNP. While clustering was similar between the Bionumerics and CosmosID pipelines, large differences in the overall number of SNPs was noted. Two-step algorithm for routine typing results in significantly lower cost than routine use of WGS. Our results suggest that using MLST as a first step in routine typing of C. difficile followed by WGS for MLST concordant strains is a less technically demanding, cost-saving approach for performing C. difficile typing than WGS alone without loss of discriminatory power.

Interlaboratory Diagnostic Validation of Conformation-Sensitive Capillary Electrophoresis for Mutation Scanning

Background: Indirect alternatives to sequencing as a method for mutation scanning are of interest to diagnostic laboratories because they have the potential for considerable savings in both time and costs. Ideally, such methods should be simple, rapid, and highly sensitive, and they should be validated formally to a very high standard. Currently, most reported methods lack one or more of these characteristics. We describe the optimization and validation of conformation-sensitive capillary electrophoresis (CSCE) for diagnostic mutation scanning. Methods: We initially optimized the performance of CSCE with a systematic panel of plasmid-based controls. We then compared manual analysis by visual inspection with automated analysis by BioNumerics software (Applied Maths) in a blinded interlaboratory validation with 402 BRCA1 (breast cancer 1, early onset) and BRCA2 (breast cancer 1, early onset) variants previously characterized by Sanger sequencing. Results: With automated analysis, we demonstrated a sensitivity of >99% (95% CI), which is indistinguishable from the sensitivity for conventional sequencing by capillary electrophoresis. The 95% CI for specificity was 90%–93%; thus, CSCE greatly reduces the number of fragments that need to be sequenced to fully characterize variants. By manual analysis, the 95% CIs for sensitivity and specificity were 98.3%–99.4% and 93.1%–95.5%, respectively. Conclusions: CSCE is amenable to a high degree of automation, and analyses can be multiplexed to increase both capacity and throughput. We conclude that once it is optimized, CSCE combined with analysis with BioNumerics software is a highly sensitive and cost-effective mutation-scanning technique suitable for routine genetic diagnostic analysis of heterozygous nucleotide substitutions, small insertions, and deletions.