The Detection Of Carrier State For Salmonella Serovar Typhi

Abstract

Enteric fever is a severe multi-systemic illness that is endemic in most tropical and subtropical countries with limited sanitation. It is propagated by asymptomatic biliary carriers who intermittently shed Salmonella serovar Typhi (Salmonella ser. Typhi) and/or Salmonella serovar Paratyphi A (Salmonella ser. Paratyphi A) in their stool for years. The aim of this study was to establish if the presence of Salmonella in bile as determined by PCR and culture, correlated with the detection by ELISA of immunoglobulin G (IgG) and immunoglobulin A (IgA) to the 50KDa outer membrane protein (OMP) of typhoidal Salmonella, in the sera of gastroenterology patients. Bile was artificially spiked with 1-107cfu/mL of pure Salmonella ser. Typhi and Salmonella ser. Paratyphi A cultures. Various DNA extraction methods were compared for the ability to yield PCR inhibitor-free DNA extract from bile. There was a 5-fold increase in DNA yield for either Salmonella species when commercial kits were used for DNA extraction compared to conventional methods (p<0.01). PCR sensitivity was higher for both Salmonella ser. Typhi (2.46 x 10 ◦cfu/mL) and Salmonella ser. Paratyphi A (2.66 x 10 ◦ cfu/mL) DNA extracted by the commercial kits. To keep costs minimal, a low-cost DNA extraction method utilizing Dowex ion-exchange resin was developed. The Dowex based protocol successfully overcame PCR inhibition. The resin selectively bound to inhibitors not to target DNA (P=0.669). The PCR sensitivity of the DNA extract was comparable to that of commercial DNA extraction kits (2.6 x 10² for Salmonella ser. Typhi and 2.8 x 10◦ for Salmonella ser. Paratyphi A). The method was shown to be repeatable (P=0.73), and adopted in subsequent DNA extractions. About 320 bile and corresponding blood specimens were collected from patients undergoing endoscopic retrograde cholangiopancreotography (ERCP) and biliary surgery at the Asian Institute of Gastroenterology, Hyderabad, India; and at hospital Tuanku Ja’afar (Seremban), Negeri Sembilan, Malaysia. Bile was cultured for Salmonella using standard protocols. Of the 150 isolates recovered, there was only 1 Salmonella ser. Typhi and 1 Salmonella spp. Lactose fermenters (124), Pseudomonas (12), Shigella (2), Enterococcus (1) and Proteus spp. (9) were also isolated. DNA was extracted from bile using the established Dowex method, and amplified in multiplex Salmonella PCR. Culture and PCR were both 100% sensitive and specific for Salmonella.

A sandwich ELISA kit (TyphiLiza, Malaysian Bio-Diagnostics Research Sdn Bhd, Selangor, Malaysia), was standardized to enable detection of serum immunoglobulin IgG (IgG) and immunoglobulin A (IgA) to the 50KDa outer membrane protein (OMP) of Salmonella ser. Typhi in carriers. Cut-off values of OD 0.8 and 0.3 at 205nm for IgG and IgA respectively, were established and adopted in the interpretation of serological data. The 50KDa OMP was non-specific, and performed poorly as a sub-marker for IgG and IgA in carriers. No relationship could be established between isolation and positive Salmonella PCR; with positive IgG and IgA levels to the 50KDa OMP of Typhi. Isolation rates of Salmonella were below expected (<1%). It is possible that the incidence of Salmonella carrier state has declined since the last reports in literature, most probably due to the increase in use of fluoroquinolones in the treatment of acute typhoid cases. Key words: enteric fever; chronic carrier; bile; PCR; Salmonella serovar Typhi; Salmonella serovar Paratyphi A; 50KDa OMP