Abstract
M. Tech.
Diarrhoeal disease is an important public health problem worldwide as it is responsible for approximately 4 billion cases of diarrhoea per annum, of which 1.8 billion cases result in death. In most cases the causative agents are bacterial entero-pathogens such as Escherichia coli (E. coli), Salmonella, Shigella and Vibrio species. They enter the human body after consumption of contaminated water and food via the faecal-oral route of transmission. These pathogens are therefore identified from faecal matter with microbiological methods such as culture based techniques. There are however certain factors which negatively impact on the diagnosis. Recent literature has shown that bacterial pathogens may not be detected when they enter into a viable but non-culturable state (VBNC) making it difficult to detect the bacterial pathogens with culture based methods. The aim of the study was to detect entero-pathogenic bacteria from stool specimens using optimised protocols and a novel collection technique called the Bio-wipe kit.
In the past sterile containers were used to collect and transport faecal matter to the WHRU laboratory for analysis. The disadvantage of this collection technique was that individuals were hesitant to provide faecal matter in a transparent container due to their social and moral status. The Bio-wipe kit eliminated some of the problems encountered with the previous collection technique as it is used in the same way as toilet tissue. Factors such as storage time and temperature was investigated for the recovery of faecal matter from the Bio-wipes since it was used in rural villages where the stool samples can not be refrigerated and transported to the lab immediately after the diarrhoeal episode. It was shown that the bacterial DNA can be recovered from the Bio-wipes within 5-10 days after usage when stored at 30°C and within 14 days after usage when stored at ambient temperature.
Comparison of two in-house DNA extraction methods with the commercially available QIAamp® DNA stool mini kit indicated that the Guanidium thiocyanate without alpha casein method (GuSCN non ά-casein) could efficiently recover bacterial DNA from faecal matter free from the presence of inhibitors. This methodology could successfully recover amplifiable bacterial DNA in 92% (181/197) of the clinical Bio-wipes collected from individuals in the rural areas of the Vhembe region of the Limpopo province of South Africa.
Various multiplex PCR’s (m-PCR) were optimised for this study for the detection and classification of diarrhoeagenic E. coli types, Salmonella, Shigella and Vibrio species. These
m-PCR’s were proven to be very sensitive at detecting diarrhoegenic E.coli, Salmonella, Shigella and Vibrio species bacteria from the Bio-wipes. The extracted bacterial DNA from Bio-wipes recovered from clinical samples was amplified with the single genus specific multiplex PCR and 92% (181/197) of the samples tested positive for the E. coli mdh housekeeping gene, 3% (7/197) tested positive for the sodB housekeeping gene for V. cholerae spp, 5% (10/197) tested positive for the IpaH and Ial virulence genes for Shigella spp. and entero-invasive E. coli (EIEC) whereas none of the samples tested positive for the Salmonella virulence gene (IpaB). These results were confirmed with species specific multiplex PCR for each pathogen.
It was concluded from this study that the Bio-wipe kit could be used for the collection of diarrhoeal and non-diarrhoeal faecal matter. The bacterial DNA could effectively be isolated from the recovered faecal matter using the GuSCN non α-casein DNA extraction method. The genus specific m-PCR was able to amplify low levels of bacterial DNA isolated from the Bio-wipes and thus the causative agents for diarrhoeal disease can successfully be diagnosed with the genus specific m-PCR. The Bio-wipe kit can be implemented for routine analysis and during diarrhoeal outbreaks as it is a cost effective, easy to use collection kit. The bacterial pathogens can easily and rapidly be diagnosed using the optimised molecular techniques instead of classical culture-based techniques.