Abstract
BackgroundIn Malaysia, Shigella spp. was reported to be the third commonest bacterial agent responsible for childhood diarrhoea. Currently, isolation of the bacterium and confirmation of the disease by microbiological and biochemical methods remain as the "gold standard". This study aimed to detect the prevalence of four Shigella virulence genes present concurrently, in randomly selected Malaysian strains via a rapid multiplex PCR (mPCR) assay.MethodsA mPCR assay was designed for the simultaneous detection of chromosomal- and plasmid-encoded virulence genes (set1A, set1B, ial and ipaH) in Shigella spp. One hundred and ten Malaysian strains (1997–2000) isolated from patients from various government hospitals were used. Reproducibility and sensitivity of the assay were also evaluated. Applicability of the mPCR in clinical settings was tested with spiked faeces following preincubation in brain heart infusion (BHI) broth.ResultsThe ipaH sequence was present in all the strains, while each of the set1A, set1B and ial gene was present in 40% of the strains tested. Reproducibility of the mPCR assay was 100% and none of the non-Shigella pathogens tested in this study were amplified. The mPCR could detect 100 colony-forming units (cfu) of shigellae per reaction mixture in spiked faeces following preincubation.ConclusionsThe mPCR system is reproducible, sensitive and is able to identify pathogenic strains of shigellae irrespective of the locality of the virulence genes. It can be easily performed with a high throughput to give a presumptive identification of the causal pathogen.
Highlights
In Malaysia, Shigella spp. was reported to be the third commonest bacterial agent responsible for childhood diarrhoea
A recent study by Lee & Puthucheary [2] on bacterial enteropathogens in childhood diarrhoea in a Malaysian urban hospital showed that Shigella spp. was the third most common bacteria isolated
Bacterial strains and growth conditions A total of 110 Shigella strains of S. flexneri (n = 84), S. sonnei (n = 15), S. dysenteriae (n = 10) and S. boydii (n = 1) were used in this study. These strains were isolated from patients with diarrhoea in Peninsular Malaysia from 1997–2000, and were provided by the Institute for Medical Research (IMR), Malaysia
Summary
In Malaysia, Shigella spp. was reported to be the third commonest bacterial agent responsible for childhood diarrhoea. Members of the genus Shigella, namely S. flexneri, S. dysenteriae, S. sonnei and S. boydii have caused and continue to be responsible for mortality and/or morbidity in high risk populations such as children under five years of age, senior citizens, toddlers in day-care centres, patients in custodial institutions, homosexual men and, war- and famine-engulfed people. A recent study by Lee & Puthucheary [2] on bacterial enteropathogens in childhood diarrhoea in a Malaysian urban hospital showed that Shigella spp. was the third most common bacteria isolated. S. flexneri and S. dysenteriae type 1 infections are usually characterized by frequent passage of small amounts of stool and mucus or blood. S. sonnei and S. boydii infections are less severe with watery faeces but little mucus or blood
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