Determinants of diarrhoeal infections among users of shared sanitation in informal settlements in Durban, South Africa.

Diarrhoeal disease continues to be a major health problem in many parts of the world, especially in developing countries, mainly due to the lack of access to sanitation, water, and hygienic living conditions. Identifying the determinants of diarrhoeal infections continues to be a challenge in developing countries. In this study, we ascertained the factors behind diarrhoea among inhabitants of informal settlements in the city of Durban, South Africa. Prevalence of diarrhoea in the study area varied between 7-year historical clinical records and data collected during the current study (primary data), with the primary data giving the highest monthly prevalence odds ratio (POR) up to 18.1 (±1.6)%. The main factors associated with diarrhoeal infections were open defaecation (POR = 1.8; 95% confidence interval (CI): 0.9-3.12), use of shared sanitation (POR = 1.7; 95%; CI: 1.05-2.26), and exposure to faecal matter around the homes (POR = 1.69; 95% CI: 1.25-3.10). Several other factors were also determined to be associated with diarrhoeal infections, such as hygiene practices in the communities, the non-treatment of water before use, and the presence of solid waste and faecal materials around the households. This study shows that diarrhoeal disease infections in informal settlements could be multifactorial; therefore, a multifactorial approach is needed to reduce these infections. These could include improving education on hygiene practices within the home setting as well as in public places, such as the community ablution blocks.

Development and evaluation of a molecular based protocol for detection and quantification of Cryptosporidium spp. in wastewater.

Infections caused by protozoan parasites are a major public health concern globally. These infections are commonly diagnosed during water-borne outbreaks, necessitating accurate and highly sensitive detection procedures to assure public health protection. Current molecular techniques are challenged by several factors, such as low parasite concentration, inefficient DNA extraction methods, and inhibitors in environmental samples. This study focused on the development and validation of a molecular protocol for DNA extraction, efficient protozoan (oo)cyst recovery and quantification of protozoan parasites from wastewater using droplet digital polymerase chain reaction (ddPCR). Five DNA extraction methods, including commercial kits, custom phenol-chloroform, and in-house modified methods, were evaluated. The efficiency of each method was assessed via spectrophotometric analysis and ddPCR amplification using specific primers. Lastly, the developed protocol was evaluated for the detection and quantification of Cryptosporidium parvum in wastewater from different regions in South Africa. The conventional phenol-chloroform extraction method yielded the highest DNA concentration of 223 (±0.71) ng/µl and detected the highest number of Cryptosporidium parvum (1807 (±0.30) copies/ddPCR reaction) compared to other methods evaluated in this study. Additionally, the phenol-chloroform method demonstrated high sensitivity in extracting DNA from as few as one cyst/L of Cryptosporidium parvum, corresponding to 5.93 copies/ddPCR reaction. It was also observed that analysis of both the filtered supernatant and pellets after centrifugation improves the recovery efficiency of oocysts from wastewater by 10.5%, resulting in a total recovery of 64.1%. This optimized protocol was successfully applied to measure protozoan concentration in wastewater from different regions in South Africa. The improved DNA extraction and quantification method proposed in this study would be effective in monitoring protozoan concentration in the environment, which will help in instituting mitigation measures to reduce water-borne infections.

A review on application of next-generation sequencing methods for profiling of protozoan parasites in water: Current methodologies, challenges, and perspectives.

The advancement in metagenomic techniques has provided novel tools for profiling human parasites in environmental matrices, such as water and wastewater. However, application of metagenomic techniques for the profiling of protozoan parasites in environmental matrices is not commonly reported in the literature. The key factors leading to the less common use of metagenomics are the complexity and large eukaryotic genome, the prevalence of small parasite populations in environmental samples compared to bacteria, difficulties in extracting DNA from (oo)cysts, and limited reference databases for parasites. This calls for further research to develop optimized methods specifically looking at protozoan parasites in the environment. This study reviews the current workflow, methods and provide recommendations for the standardization of techniques. The article identifies and summarizes the key methods, advantages, and limitations associated with metagenomic analysis, like sample pre-processing, DNA extraction, sequencing approaches, and analysis methods. The study enhances the understanding and application of standardized protocols for profiling of protozoan parasite community from highly complexe samples and further creates a resourceful comparison among datasets without any biases.

Microplastics in the environment: Interactions with microbes and chemical contaminants.

Microplastics (MPs) are contaminants of emerging concern that have gained considerable attention during the last few decades due to their adverse impact on living organisms and the environment. Recent studies have shown their ubiquitous presence in the environment including the atmosphere, soil, and water. Though several reviews have focused on the occurrence of microplastics in different habitats, little attention has been paid to their interaction with biological and chemical pollutants in the environment. This review therefore presents the state of knowledge on the interaction of MPs with chemicals and microbes in different environments. The distribution of MPs, the association of toxic chemicals with MPs, microbial association with MPs and the microbial-induced fate of MPs in the environment are discussed. The biodegradation and bioaccumulation of MPs by and in microbes and its potential impact on the food chain are also reviewed. The mechanisms driving these interactions and how these, in turn, affect living organisms however are not yet fully understood and require further attention.

Comparative assessment of DNA extraction procedures for Ascaris spp. eggs.

A central and critical step in the molecular detection of soil-transmitted helminths from environmental sources is the extraction of DNA from the eggs. In this study, we investigated the yield of DNA extracted from known quantities (500, 100, 50, 20, 10 and 5) of Ascaris suum eggs, as well as directly from wastewater and sludge samples containing Ascaris spp. eggs, using six commercial DNA extraction kits. The amount of DNA extracted was quantified with NanoDrop, Qubit and Ct values from quantitative polymerase chain reaction (qPCR) assay using CFX96 Touch™ real-time PCR equipment. The PowerLyzer Ultraclean Microbial DNA isolation kit and PowerSoil DNA isolation kit gave the highest yield of DNA based on the NanoDrop, Qubit and Ct values. However, the qPCR results indicate that in some of the kits, PCR inhibitors may have been carried over to the PCR reaction. DNA extraction kits that incorporate a bead-beating step as well as other mechanical eggshell disruption steps were superior in extracting DNA from Ascaris spp. eggs. Additionally, for the accurate quantification of extracted DNA, the use of Ct values from qPCR and Qubit readings gives better results compared to the NanoDrop readings. For efficient downstream applications, the use of DNA extraction kits with superior inhibitor removal technology is essential, in addition to a high yield of DNA.