Genomic Surveillance Enables Suitability Assessment of Salmonella Gene Targets Used for Culture-Independent Diagnostic Testing.

Rebecca J Rockett,Vitali Sintchenko,Alicia Arnott,Peter Howard,Qinning Wang,John P Dekker

doi:10.1128/jcm.00038-20

Rebecca J Rockett, Vitali Sintchenko + Show 4 more

Open Access

https://doi.org/10.1128/jcm.00038-20

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Abstract

Salmonella is a highly diverse genus consisting of over 2,600 serovars responsible for high-burden food- and waterborne gastroenteritis worldwide. Sensitivity and specificity of PCR-based culture-independent diagnostic testing (CIDT) systems for Salmonella, which depend on a highly conserved gene target, can be affected by single nucleotide polymorphisms (SNPs), indels, and genomic rearrangements within primer and probe sequences. This report demonstrates the value of prospectively collected genomic data for verifying CIDT targets. We utilized the genomes of 3,165 Salmonella isolates prospectively collected and sequenced in Australia. The sequences of Salmonella CIDT PCR gene targets (ttrA, spaO, and invA) were systematically interrogated to measure nucleotide dissimilarity. Analysis of 52 different serovars and 79 multilocus sequencing types (MLST) demonstrated dissimilarity within and between PCR gene targets ranging between 0 and 81.3 SNP/kbp (0 and 141 SNPs). The lowest average dissimilarity was observed in the ttrA target gene used by the Roche LightMix at 2.0 SNP/kbp (range, 0 to 46.7); however, entropy across the gene demonstrates that it may not be the most stable CIDT target. While debate continues over the benefits and pitfalls of replacing bacterial culture with molecular assays, the growing volumes of genomic surveillance data enable periodic regional reassessment and validation of CIDT targets against both prevalent and emerging serovars. If PCR systems are to become the primary screening and diagnostic tool for laboratory diagnosis of salmonellosis, ongoing monitoring of the genomic diversity in PCR target regions is warranted, as is the potential inclusion of two Salmonella PCR targets in frontline diagnostic systems.

Highlights

Salmonella is a highly diverse genus consisting of over 2,600 serovars responsible for high-burden food- and waterborne gastroenteritis worldwide
We examined the variability in culture-independent diagnostic testing (CIDT) targets and the robustness of current CIDT systems utilizing a comprehensive Salmonella genome collection spanning more than two summer seasons in New South Wales (NSW), Australia
Salmonella isolates included in this study represent all isolates referred to the NSW Enteric Reference Laboratory, Institute of Clinical Pathology and Medical Research (ICPMR), NSW Pathology, that underwent whole-genome sequencing as part of routine public health outbreak investigations in NSW, Australia between October 2015 and December 2018 (n ϭ 3,256)

Summary

Introduction

Salmonella is a highly diverse genus consisting of over 2,600 serovars responsible for high-burden food- and waterborne gastroenteritis worldwide. In 2006, it was reported that a C. trachomatis variant was circulating and that this variant harbored a deletion in the PCR primer target region contained within the cryptic plasmid [11] This highly conserved multicopy plasmid had been thought to be the ideal diagnostic target, as it offered increased sensitivity for detecting C. trachomatis. Emergence of a variant harboring a deletion within the cryptic plasmid caused a complete loss of sensitivity in the majority of commercial CIDTs, resulting in the ongoing transmission of C. trachomatis and an increased number of severe sequalae after unrecognized and prolonged C. trachomatis infections [12] These false-negative results for patients with C. trachomatis variant infections were noted only after a significant decrease in the incidence of C. trachomatis triggered an investigation by public health authorities. The current Salmonella CIDTs are often reliant on a single PCR target region

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