A real-time PCR for the differentiation of typhoidal and non-typhoidal Salmonella

Abstract

22Rapid and accurate differentiation of Salmonella spp. causing enteric fever from non-23 typhoidal Salmonella is essential for clinical management of cases, laboratory risk 24 management and implementation of public health measures. Current methods used 25 for confirmation of identification including biochemistry and serotyping as well as 26 whole genome sequencing analyses, takes several days. Here we report the 27 development and evaluation of a real-time PCR assay that can be performed directly 28 on crude DNA extracts from bacterial colonies, for the rapid identification of typhoidal 29 and non-typhoidal Salmonella. 30 This novel two-hour assay identifies the genus Salmonella by detecting the ttr gene, 31 encoding tetrathionate reductase, and defines typhoidal Salmonella by the detection 32 of S. Typhi and Paratyphi-specific gene combinations. PCR assay performance was 33 determined using 211 clinical cultures of Salmonella (114 non-typhoidal and 97 34 Typhoidal strains) and 7 clinical non-Salmonella cultures. In addition, the specificity 35 of the assay was evaluated in silico using a diverse in-house collection of 1882 36 Salmonella whole genome sequences. The real-time PCR results for 218 isolates 37 and the genomic analysis of the 1882 isolates produced 100% sensitivity and 100% 38 specificity (based on a 7 gene profile) for identifying typhoidal Salmonella compared 39 to the Salmonella whole genome sequening identification methods currently used at 40 Public Health England.41 This paper describes a robust real-time PCR assay for the rapid, accurate 42 identification of typhoidal and non-typhoidal Salmonella which will be invaluable for 43 the urgent screening of isolates from symptomatic individuals, the safe processing of 44 isolates in laboratories and for assisting the management of public health risks. 45 46 Salmonella are a diverse genus of gastrointestinal pathogens that cause a wide 47 spectrum of disease from self-limiting gastroenteritis (non-typhoidal salmonellae, 48 NTS) to systemic enteric fever (typhoidal salmonellae -Salmonella enterica serovar 49 Typhi, Paratyphi A, B and C). Salmonellosis is global but typhoidal Salmonella are 50 found mainly in sub-Saharan Africa and South Asia where enteric fever is endemic 51 (1); although the detailed local surveillance data from endemic regions remains poor 52(2). A current concern is the increase in bacteraemia (and focal infections) 53 associated with multi-drug resistant NTS infection in sub-Saharan Africa. In high 54 income countries such as the UK, invasive NTS infection is mainly confined to 55 immune-compromised hosts and so the major risks are local outbreaks of NTS 56 through poor food hygiene and typhoidal infections associated with travel to endemic 57 regions. 58 Diagnostic hospital microbiology laboratories make only a presumptive identification 59 of Salmonella spp.: they do not usually hold a sufficient range of specific antisera for 60 full identification and rapid identification systems, such as Matrix Assisted Laser 61 Desorption/I...