Development of a PCR-Based Method for the Diagnosis of Salmonella Typhi Infections from Filter Paper Human Blood Blots

Abstract

The most common diagnostic test for typhoid fever in endemic countries is the Widal lest because it is last and easy. Unfortunately, specificity is less than 75% and false positives of about 33% occur. Culture of Salmonella typhi on the other hand, is expensive and takes 7 days to produce significant detectable growth. Also, large volumes of blood used for diagnosis even from acute cases are not suitable for anaemic or distressed patients. A sensitive and specific PCR-based method for the detection of S. typhi from less than 500µl of blood has been applied successfully on field samples. The present study assessed PCR on DNA extracts from filter paper blood blots, which require much smaller volumes of blood from patients. A set of nested primers was designed for typhoid fever diagnosis by PCR. The first primer pair is Salmonella genus specific and amplifies a 872bp fragment while the second pair is .S. typhi specific and amplifies a 477bp fragment. To check for specificity of (he primers, they were used in a PCR reaction on DNA extracts from isolates of Escherichia. Staphylococcus aureus, S treptococcus pyogenes and Bacillus snhlilis. PCR results showed no amplification products from these bacterial cells but gave the expected product sizes from Salmonella typhi DNA extracts. Uninfected blood seeded with S typhi was serially diluted and thereafter, aliquots plated on plate count agar (PCA). After 24 hours of incubation, bacterial colony forming units (CFUs) were counted on each plate. I lie serially diluted blood was blotted on filter paper lo determine (lie threshold of detection of the PCR method. I lie results indicated that a minimum of 1.6CI U of bacteria in 1ml blood could be detected. Blood samples from 185 suspected typhoid patients at University Hospital. Legon, were screened by both the Widal and the PCR methods. Out ('I (his number, 58 (31.4%) samples were Widal positive for 0 and II antigens at a litre of 1/160 and seven (3.8%) were PCR positive. Blood dims of 138 of the samples were screened for the malaria parasite and 12 (6.89%) were positive. A hundred and twenty-two cultured blood samples yielded 9 (7.14%) Staphylococcus aureus positives and I (0.79%) each for Scrralia marccsccns and Yersinia enterolilica. There was no S. typhi culture positive. Restriction digestion of positive PCR products with Oral gave the expected product sizes of 315 and 162 bps. This PCR assay can be used to detect S. typhi in blood blots; however, it needs to be evaluated against bacterial culture of large volumes of blood over a more extensive sample size.