Microbial Water Quality Assessment and Molecular Assay Optimization for Vibrio Cholerae Detection

Abstract

Access to clean water and adequate sanitation is critical for public health, yet microbial contamination of water sources remains a pervasive challenge, particularly in developing regions like Sub-Saharan Africa. One of the most devastating consequences of this contamination is cholera- a severe diarrheal disease that causes rapid dehydration and is considered one of the fastest fatal infections if not treated immediately. This study investigates the microbial water quality and its associated health risks within various water sources in Dar es Salaam, Tanzania. The objective of this study was to 1) Conduct microbial water quality assessment to identify and quantify the presence of total Coliforms and fecal coliform indicator (E. coli), in water samples collected from various district locations to improve public health; 2) assess risks to diarrhea diseases associated with water sources in these districts; and 3) optimize a molecular assay for the detection of Vibrio cholerae (V. cholerae) and testing primer concentration using SimpliAmp and Bio-Rad Polymerase Chain Reaction (PCR) platforms. A total of 160 water samples were assessed for microbial water quality by testing for the presence of Total Coliform (TC) and Escherichia coli (E. coli) using the Colilert method. E. coli results were used to determine the risk for diarrhea diseases using the World Health Organization (WHO) guidelines. For detection of V. cholerae, four biomarkers, ompW, ctxA, O1 and 0139 genes were targeted for optimization using PCR. Overall, 66.9% of samples tested positive for Total coliforms while, 37.4% were positive with E. coli contamination. Among the three districts, Kinondoni recorded significantly higher TC compared to both Ilala (Z = -4.73, p < 0.001) and Temeke (Z = 5.06, p < 0.001) respectively. Similarly, significant higher levels of E. coli were recorded in Kinondoni compared to Ilala (Z = -7.93, p < 0.001) and Temeke (Z = 7.99, p < 0.001). All samples from the stream showed an intermediate risk level for disease, followed by 27.9% of open wells samples and 6.7% of public taps. All water samples from closed wells and vendors were in conformity with WHO guidelines and, therefore, considered safe. Our optimized PCR assay for V. cholerae detection, reduced cycling time for the multiplex assay (ctxA, O1 and O139) from the baseline study by 30 seconds for denaturation and annealing time, while extension time was reduced by 15 seconds. Higher annealing temperature was also achieved for ompW and the multiplex assays from our baseline. Our assay detected clear/strong bands for ompW between 1 µM (baseline concentration) to 0.5 µM and is sensitive enough to detect the presence of V. cholerae with 299 gBlocks copies or bacteria cells. We observed a comparable performance on both thermocycler platforms. Our results show evidence of fecal contamination. While samples purchased from vendors and those from close wells were safe for consumption, other water sources have low to intermediate risk to diarrheal diseases. With fewer reagents and a cost-effective thermocycler, costs will be reduced, and more reagents can be saved to assess other samples, which could enhance more surveillance, outbreak identification, and response to potential cholera outbreaks, thereby potentially preventing widespread transmission, lowering mortality, and lessening the strain on already overburdened health systems. By monitoring the levels of coliforms and E. coli in these water sources, this study has provided valuable insights into the level of fecal contamination and the potential health risk it poses to individuals in these communities that rely on these water sources both for drinking, cooking, and recreation. There is an urgent need for a concerted effort at the household, community, and government levels to improve access to quality water for all.