The risk factors of soil-transmitted helminth infections : a need for appropriate measurement methods

Abstract

Soil-transmitted helminths are a major health concern, especially in tropical and sub-tropical regions. Poor sanitation and poverty are major pre-disposing factors contributing to increase in infections. Infection with STH is mainly through exposure to water, soil and food contaminated with the eggs of these parasites. Accurate detection and quantification of STH eggs in environmental samples is therefore critical for the determination of infection risks from exposure. Accurate detection of these eggs is also important in the adoption of risk reduction strategies. This thesis presents the development of a revised method for the accurate detection and quantification of STH eggs in different environmental matrices, such as wastewater, sludge etc. It further presents the application of this method in the comparative determination of STH egg reduction efficiencies of centralized wastewater treatment plants and decentralized wastewater treatment (DEWATS) plants in Durban, South Africa and Maseru, Lesotho. The concentration of viable STH eggs in dried sludge from Durban, South Africa and Dakar, Senegal was also determined and compared with both WHO guidelines and South African national standards for sludge reuse. The risks of infection with STHs for different populations exposed (directly and indirectly) to wastewater, wastewater contaminated surface water and sludge were determined using both quantitative microbial risks assessment and epidemiological approaches. Despite the plethora of methods available for the detection and quantification of STH eggs in the environment there is no internationally accepted method, however the most commonly used methods are based on the principles of sedimentation, differential flotation and microscopy. These are mainly adaptations of the WHO and USEPA methods. These methods were found to be similar with a few differences which affected the recovery rates reported. However, the major challenges with the conventional methods are the time needed for sample analysis and the use of reagents that could possibly affect the recovery of viable STH eggs. A new revised method was developed based on review of literature and laboratory experiments. In this method the heterogeneity of environmental samples was accounted for by the development of different pre-processing steps, involving the use of detergents to aid in the separation of eggs from particles in samples such as sludge, UD waste and untreated wastewater. Additionally, the use of sieves of different pore sizes ensured that the number of debris on the microscope slides was reduced considerably. The use of these sieves also reduced the time need for sample analysis, due to the elimination of the spontaneous sedimentation step, which is commonly used. This spontaneous sedimentation step takes between 12-24 hours therefore prolonging the time needed for sample analysis. Reagents such as acetoacetic acid and ethyl acetate were found to result in considerable loss of egg viability after just 5 minutes of exposure. This new method therefore does not involve their usage. The elimination of the use of acetoacetic acid and ethyl acetate step also reduces the number of steps involved in sample analysis. This reduces room for error as well as helping in fast analysis of samples. In addition to a much faster sample analysis the method has recovery percentages of 80.25% to 97.63% in sludge and wastewater samples respectively, with sensitivity of 2-3 eggs per liter in wastewater samples and 5-7 eggs per 20 gram of sludge. Exposure to STH eggs in the environment is mainly through wastewater, either treated or untreated, this exposure could therefore be eliminated through wastewater treatment. Centralized wastewater treatment systems are the most favored treatment options globally. These centralized treatment systems incur high cost of construction, maintenance and operations which may hamper the robustness in developing countries and rural areas. One of the most widely used alternative means of wastewater treatment is the anaerobic baffled reactors (ABRs) and planted gravel filters

(PGFs) (collectively referred to as DEWATS in this thesis), which have been considered as low cost, effective wastewater treatment options. However, there is lack of comparative assessment of the STH egg removal efficiency of these two different wastewater treatment approaches. Eggs of Ascaris spp, hookworm, Trichuris spp, Taenia spp and Toxocara spp were the commonly recorded STH eggs in the untreated wastewater at the inlets of the centralized wastewater treatment plants as well as the DEWATS plants (except for Toxocara spp). There was variation in STH egg concentrations between and within the study areas, indicating difference in STH infections among the populations both in Durban and Maseru. STH egg removal varied between and within the different wastewater treatment plants as well. The DEWATS plants achieved 95-100% STH egg removals as compared to the 67 to 100% in the centralized wastewater treatment plants. This could be attributed to the difference in treatment processes. Among the different STHs, reduction in Ascaris spp eggs was significantly higher, irrespective of the type of treatment, which is attributed to the high relative density of the egg resulting in a higher settling velocity than the other STH eggs. Reduction or elimination of STH eggs through wastewater treatment is achieved by removing the eggs from the wastewater into the sludge. STH egg concentration in sludge is therefore mostly higher than in the wastewater. Sludge from Durban and Dakar after 60 days of drying under ambient environmental conditions contained very high concentration of viable STH eggs. Ascaris spp, hookworm, Trichuris spp, Taenia spp and Toxocara spp were the commonly recorded STH eggs, except for Dakar were Taenia spp and Toxocara spp were not detected in the sludge. STH egg concentrations were higher in Dakar than in Durban, with viable STH egg concentrations exceeding both the USEPA regulatory value (≤0.25 eggs/g TS) and the WHO guideline value (≤1 eggs/g TS). This variation in egg concentration could be attributed to the difference in prevalence and intensity of STH infections in the two study areas. Over a ten-month study period concentration of viable eggs in the sludge from Durban varied considerably, probably influenced by the environmental conditions. A decay rate of 0.0056 per day was calculated for egg die-off during drying. The rate of decay is low therefore drying alone cannot produce sludge meeting both local and international standards and guidelines for sludge reuse.

Determination of STH infection risks due to exposure to wastewater and sludge either directly or indirectly is critical in the prevention of infection. Exposure to the effluents during wastewater irrigation is one major route of infection. STH egg concentrations in the final effluents from the centralized and DEWATS wastewater treatment plants were consistently higher than the WHO recommended guideline for unrestricted agricultural use (≤ 1 helminth egg/L), whereby the direct reuse of the effluents for agriculture was found to pose a higher risk than the WHO tolerable risk of infection (1 ×10-2 pppy) for farmers and consumers. Annually the use of effluents from the DEWATS plants poses the least risk of infection (1.9 ×10-2 (±2.4×10-4)), which is marginally higher than the WHO tolerable risk value. Well maintained DEWATS plants are more efficient in removing or reducing the concentration of STH eggs in wastewater and therefore pose the least risks of infection compared to centralized wastewater treatment plants. Consumers of vegetables from these farms are also at considerable risks of STH infections.

Probabilistic assessment of the STH infection risks showed that farmers applying sludge from Durban and Dakar without adequate protective measures had risks of infections higher than the WHO tolerable risks figure (1×10-2 pppy). Based on the estimated risks of infection after decay, exposure to farm soil after 40-50 days of sludge application may reduce the risks of infection to

levels lower the WHO tolerable risks value. However, this may not be practical due to the need for farmers to attend to their crops frequently. Incorporation of the decay of the eggs into the risks assessment also indicated that, using lettuce as a representative vegetable, harvesting of vegetables in Dakar could be done after 40 days of sludge application to reduce the risks of infection to the WHO tolerable value but in Durban harvesting after 30 days ensures that consumers are protected. Therefore, to protect both the farmers and consumers exposed to STH eggs through wastewater/sludge reuse in agriculture the implementation of the WHO multi-barrier approach to risk reduction is required. Risks of STH infections could be directly estimated using epidemiological approaches. By using this approach, the concept of STH infection risks for farmers using wastewater was assessed through direct measurements of the concentration of STHs both in wastewater used for irrigation and the farm soil, as well as the actual load of STHs ova in the stool of farmers and their family members. In Kumasi, Ghana, wastewater used for irrigation of vegetables and the farm soil contained high concentration of STH eggs. There was positive correlation between STH concentrations in the wastewater/soil and STH eggs load in stool of the exposed farmers. Stool analysis after 3 months, following deworming, showed a fast re-infection rate. Farmers exposed to the wastewater were three times more likely as compared to the control group of non-farmers to be infected with Ascaris spp (OR = 3.9, 95% CI, 1.15-13.86) and hookworm (OR = 3.07, 95% CI, 0.87-10.82). These risks of infection were higher in the rainy season than the dry season. This corresponds to a higher egg concentration in wastewater used for irrigation during this period. This indicates a relationship between STH infection and egg concertation in the environment. This study therefore contributes to the evidence-based conclusion that wastewater irrigation contributes to a higher incidence of STHs infection for farmers. In conclusion, this thesis therefore presents a new revised method that can be used to determine the STH egg concentration in different environmental samples. The development of this method also provides an opportunity to comparatively assess the STH egg reduction/removal efficiency of the more commonly used centralized wastewater treatment plants and DEWATS plants. The accurate quantification of viable STH eggs provide inputs for the probabilistic assessment of STH infection risks for different populations exposed to effluents from these two wastewater treatment approaches. This assessment of risks provides a public health perspective to the wastewater treatment. Additionally, it was concluded with the used of this method that drying of sludge for 60 days in Durban or Dakar does not produce sludge of good quality for agricultural application. This was confirmed by the estimates of STH infection risks determined using quantitative microbial risks assessment. This thesis therefore shows the importance of accurate quantification of STH eggs in the determination of infection risks either though QMRA or epidemiological approache