Optimisation of culture diagnostic conditions and evaluation of a molecular assay for the detection of Shigella species from diarrhoeagenic stool specimens

Abstract

Shigella spp. are major diarrhoeal pathogens, affecting all age groups particularly children from low-to-middle income countries (LMIC). The global burden of shigellosis has been underestimated due to the use of insensitive Shigella spp. detection methods. This study aimed to optimise the current culture conditions and evaluated a conventional polymerase chain reaction (PCR) assay for the recovery and detection of Shigella spp. from stool specimens.

A South African diarrhoeal surveillance study showed that molecular assays detected more shigellosis cases compared to culture methods. However, molecular assays require expensive reagents and equipment and current molecular assays target the invasion plasmid antigen H (ipaH) gene, which is present in both Shigella spp. and enteroinvasive Escherichia coli (EIEC).

Culture conditions were optimised using stool specimens spiked with a Shigella flexneri reference strain. The optimised culture method entailed the transport of stool specimens at 4°C in Buffered glycerol saline (BGS) within 24 hours (hrs), growth on MacConkey (MAC) agar and xylose lysine deoxycholate (XLD), enrichment in Gram-negative (GN) broth and subculturing on XLD and Colorex Shigella media. The pentaplex PCR assay was evaluated for detection of the Shigella spp. genus (invasion gene C, invC) - and species specific genes [S. flexneri (O-antigen polymerase gene, rfc), S. sonnei (putative epimerase/dehydratase gene, wbgZ) and S. dysenteriae (O-antigen biosynthesis gene, rfpB] and an outer membrane protein A (ompA) gene as an internal control. However, the pentaplex PCR assay ompA internal control caused non-specific amplification. An alternative conventional multiplex PCR (mPCR) assay, (F-mPCR assay) was evaluated as a direct screening tool for detection of the invC, rfc, wbgZ and rfpB genes of Shigella spp. The routine and optimised culture methods and the F-mPCR assay were compared to each other and to a real-time PCR assay as a reference standard with 100% sensitivity and specificity based on diagnostic accuracy, costs and turnaround times (TAT).

A total of 77 fresh stool specimens were screened of which 12 were Shigella spp. positive from the combined results of the four diagnostic tests. Detection of Shigella spp. was highest with direct screening methods [F-mPCR and real-time PCR assays: 83% (10/12)], followed by the optimised culture [33% (4/12)] and routine culture [8% (1/12)] methods. The F-mPCR assay showed the highest sensitivity (80%), followed by the optimised culture (40%) and routine culture (10%) methods. The TAT for the F-mPCR assay (8 hrs) was comparable to the real-time PCR assay (7 hrs) and shorter compared to both culture methods (80 hrs). The lowest cost was for the routine culture (R 113) and optimised culture (R 195) methods. Direct detection using the F-mPCR assay (R 935) was more affordable compared to the real-time PCR assay (R 1 323).

The F-mPCR assay proved to be a sensitive, rapid and cost-effective screening tool for the direct detection of Shigella spp. in stool specimens. Future studies should design alternative primers for the detection of Shigella spp. serotypes in South Africa and explore stable gene markers for Shigella spp. detection and differentiation from EIEC.