Isaac Amoah

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• Malla, M., Malambule, N., Amoah, I., Featherston, J., Ismail, A., Bux, F., & Kumari, S. (2025). The plastisphere ecology: Assessing the impact of different pollution sources on microbial community composition, function and assembly in aquatic ecosystems. Environmental Chemistry and Ecotoxicology, 7. doi:10.1016/j.enceco.2024.10.010
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  In aquatic ecosystems microplastics (MPs) provide new habitat for microbes, forming the plastisphere. While, the effect of different pollution sources on microbiome compositions, functions and assembly processes remains largely cryptic, and hence requires further investigation. Thus, in this study microplastic and surrounding water samples were collected from four different locations and performed meta-analysis to evaluate the impact of different pollution sources on microbial community composition, function and assembly in plastisphere and surrounding environment. Results demonstrated that pollution source had a significant effect on microbial diversity (p = 0.0012) and composition (PERMANOVA F = 16.386; R2 = 0.15, p < 0.001) in surface water and plastisphere. Specifically, plastisphere harboured distinct microbial community and recruited unique taxa compared to surface water, suggesting that microplastics serve as new ecological habitats. We observed a clear shift in microbial community composition, with Bacteroidetes being significantly higher in surface water significantly, whereas α- and β-Proteobacteria dominated the plastic surface (p < 0.05). These change in microbial communities were more likely due to unique chemical properties and substrates enrichment on plastic surfaces and different pollution sources. Genes involved in metabolism, signaling, cell motility, vesicular transport energy production and defence were significantly enriched in plastisphere (p = 0.001). The environmental factors such as DO and salinity drive the microbial communities in plastisphere. Niche-based selection process govern assembly in plastisphere microbiome, while as stochastic processes dominated the assembly process in aquatic microbial communities. These finding suggest that trajectory, continued microplastic emission and transport in aquatic ecosystems could pose serious planetary and health issues.
• Amoah, I., Bux, F., Kumari, S., & Ng’etich, A. (2024). Anthelmintic resistance in soil-transmitted helminths: One-Health considerations. Parasitology Research, 123(1). doi:10.1007/s00436-023-08088-8
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  The One-Health approach recognizes the intricate connection between human, animal, and environmental health, and that cooperative effort from various professionals provides comprehensive awareness and potential solutions for issues relating to the health of people, animals, and the environment. This approach has increasingly gained appeal as the standard strategy for tackling emerging infectious diseases, most of which are zoonoses. Treatment with anthelmintics (AHs) without a doubt minimizes the severe consequences of soil-transmitted helminths (STHs); however, evidence of anthelmintic resistance (AR) development to different helminths of practically every animal species and the distinct groups of AHs is overwhelming globally. In this regard, the correlation between the application of anthelmintic drugs in both human and animal populations and the consequent development of anthelmintic resistance in STHs within the context of a One-Health framework is explored. This review provides an overview of the major human and animal STHs, treatment of the STHs, AR development and drug-related factors contributing towards AR, One-Health and STHs, and an outline of some One-Health strategies that may be used in combating AR.
• Huang, Q., Wang, B., Shen, J., Xu, F., Li, N., Jia, P., Jia, Y., An, S., Amoah, I., & Huang, Y. (2024). Shifts in C-degradation genes and microbial metabolic activity with vegetation types affected the surface soil organic carbon pool. Soil Biology and Biochemistry, 192. doi:10.1016/j.soilbio.2024.109371
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  The incorporation of plant-derived biomass by microorganisms into deceased microbial biomass, i.e., a “microbial carbon pump”, is essential for forming a soil carbon (C) pool. Therefore, microbial communities and associated functions could shape the formation of soil organic C (SOC) composition and persistence. However, the mechanism by which microorganisms mediate the degradation of various types of biomass (such as plant or microbial) and its impact on SOC formation under different vegetation types remains unclear. Herein, we analyzed microbial communities, carbohydrate-activated enzymes (CAZymes), absolute quantification of C-degradation genes, and extracellular enzyme activities to track microbial-mediated SOC formation under three vegetation types on the Loess Plateau. Changes in vegetation type influenced microbial community structure and distributions of certain bacterial groups. Specifically, dominant bacterial taxa shifted from oligotrophic Actinobacteria to eutrophic Proteobacteria from grass to forest soil. The proportion of microbial CAZymes responsible for decomposing plant-derived components (86–89%) exceeded that of microbial-derived components (11–14%), suggesting a greater capacity for the degradation of deceased plant biomass by microorganisms. There was a reduction of 14%–17% in the absolute abundance of specific C-degradation genes for hemicellulose and cellulose in forest soil compared to grass, while an increase of 20%–32% was observed in lignin and chitin degradation. This indicated a higher decomposition potential of lignin and chitin by microorganisms in forest soil. The number of CAZymes genes involved in the degradation of bacteria-derived biomass (peptidoglycan) was higher than fungi-derived biomass (chitin and glucans) and had a close correlation with the qCO2, microbial biomass C (MBC), and particulate organic C (POC). The abundance of C-degrading genes increased with the increase in the corresponding enzyme activity, indicating that enzyme activity is regulated by functional genes. In conclusion, shifts in CAZymes genes encoding for the degradation of diverse carbon sources could impact microbial metabolic activity through a microbial “carbon pump” regulation process. This mechanism could facilitate the formation of soil organic carbon and its fractions across different vegetation types.
• Mtetwa, H., Amoah, I., Kumari, S., Bux, F., & Reddy, P. (2024). Optimisation of analytical methods for tuberculosis drug detection in wastewater: A multinational study. Heliyon, 10(10). doi:10.1016/j.heliyon.2024.e30720
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  Wastewater-based epidemiology (WBE) is a robust tool for disease surveillance and monitoring of pharmaceutical consumption. However, monitoring tuberculosis (TB) drug consumption faces challenges due to limited data availability. This study aimed to optimise methods for detecting TB drugs in treated and untreated wastewater from four African countries: South Africa, Nigeria, Kenya, and Cameroon. The limit of detection (LOD) for these drugs ranged from a minimum of 2.20 (±1.02) for rifampicin to a maximum of 2.95 (±0.79) for pyrazinamide. A parallel trend was observed concerning the limit of quantification (LOQ), with rifampicin reporting the lowest average LOQ of 7.33 (±3.44) and pyrazinamide showing the highest average LOQ of 9.81 (±2.64). The variance in LOD and LOQ values could be attributed to factors such as drug polarity. Erythromycin and rifampicin exhibited moderately polar LogP values (2.6 and 2.95), indicating higher lipid affinity and lower water affinity. Conversely, ethambutol, pyrazinamide, and isoniazid displayed polar LogP values (−0.059, −0.6, and −0.7), suggesting lower lipid affinity and greater water affinity. The study revealed that storing wastewater samples for up to 5 days did not result in significant drug concentration loss, with concentration reduction remaining below 1 log throughout the storage period. Application of the optimised method for drug detection and quantification in both treated and untreated wastewater unveiled varied results. Detection frequencies varied among drugs, with ethambutol consistently most detected, while pyrazinamide and isoniazid were least detected in wastewater from only two countries. Most untreated wastewater samples had undetectable drug concentrations, ranging from 1.21 ng/mL for erythromycin to 54.61 ng/mL for isoniazid. This variability may suggest differences in drug consumption within connected communities. In treated wastewater samples, detectable drug concentrations ranged from 1.27 ng/mL for isoniazid to 10.20 ng/mL for ethambutol. Wastewater treatment plants exhibited variable removal efficiencies for different drugs, emphasising the need for further optimisation. Detecting these drugs in treated wastewater suggests potential surface water contamination and subsequent risks of human exposure, underscoring continued research's importance.
• Mthethwa-Hlongwa, N., Amoah, I., Gomez, A., Davison, S., Reddy, P., Bux, F., & Kumari, S. (2024). Profiling pathogenic protozoan and their functional pathways in wastewater using 18S rRNA and shotgun metagenomics. Science of the Total Environment, 912. doi:10.1016/j.scitotenv.2023.169602
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  Despite extensive research, little is known about the composition of eukaryotic protists in environmental samples. This is due to low parasite concentrations, the complexity of parasite diversity, and a lack of suitable reference databases and standardized protocols. To bridge this knowledge gap, this study used 18S rRNA short amplicon and shotgun metagenomic sequencing approaches to profile protozoan microbial communities as well as their functional pathways in treated and untreated wastewater samples collected from different regions of South Africa. Results demonstrated that protozoan diversity (Shannon index P-value = 0.03) and taxonomic composition (PERMANOVA, P-value = 0.02) was mainly driven by the type of wastewater samples (treated & untreated) and geographic location. However, these WWTPs were also found to contain a core community of protozoan parasites. The untreated wastewater samples revealed a predominant presence of free-living, parasitic, and potentially pathogenic protists typically found in humans and animals, ranging from Alveolata (27 %) phylum (Apicomplexa and Ciliophora) to Excavata (3.88 %) (Discoba and Parasalia) and Amoebozoa (2.84 %) (Entamoeba and Acanthamoeba). Shotgun metagenomics analyses in a subset of the untreated wastewater samples confirmed the presence of public health-importance protozoa, including Cryptosporidium species (3.48 %), Entamoeba hystolitica (6.58 %), Blastocystis hominis (2.91 %), Naegleria gruberi (2.37 %), Toxoplasma gondii (1.98 %), Cyclospora cayetanensis (1.30 %), and Giardia intestinalis (0.31 %). Virulent gene families linked to pathogenic protozoa, such as serine/threonine protein phosphatase and mucin-desulfating sulfatase were identified. Additionally, enriched pathways included thiamine diphosphate biosynthesis III, heme biosynthesis, Methylerythritol 4-Phosphate Pathway, methyl erythritol phosphate (MEP), and pentose phosphate pathways. These findings suggest that protozoan pathogens may possess metabolic and growth potential within WWTPs, posing a severe risk of transmission to humans and animals if inadequately disinfected before release. This study provides a baseline for the future investigation of diverse protozoal communities in wastewater, which are of public health importance.
• Rajcoomar, S., Amoah, I., Abunama, T., Mohlomi, N., Bux, F., & Kumari, S. (2024). Biofilm formation on microplastics in wastewater: insights into factors, diversity and inactivation strategies. International Journal of Environmental Science and Technology, 21(4). doi:10.1007/s13762-023-05266-0
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  Microplastic (MP) occurrence in wastewater poses serious threats to aquatic organisms due to their ecological impact. Additionally, these microplastics may provide a unique habitat for microbial biofilms. This study explored the possible factors that facilitate biofilm formation on different MPs in a wastewater environment. Biofilm formation was determined via measurement of optical density (OD) and confirmed using scanning electron microscopy. Furthermore, microbial community profiling was performed via 16S rRNA amplicon sequencing. The highest biofilm formation occurred after 3 weeks of exposure, followed by a decline to its lowest after 5 weeks. Biofilms were predominant on MPs that were exposed to dark conditions, mesophilic temperature (25 °C) and aerobic conditions. Under these conditions, a positive correlation was observed between the OD value and ammonia (NH3) (r = 0.824) and nitrite (NO2) concentrations (r = 0.1). However, a negative correlation (r = −0.673) was found between the OD value and nitrate (NO3) levels in the medium. Furthermore, the highest biofilm formation was observed on polyethylene particles. The most dominant phyla in both the biofilms and wastewater medium were Proteobacteria, Bacteroidetes and Planctomycetes, with Methylotenera being the most abundant genera in the biofilms. In general, the polypropylene particles supported the most diverse bacterial community (H' = 2.51138 and Simpson index = 11.096), while high-density polyethylene supported the least diverse bacterial community (H' = 0.88779 and Simpson index = 1.5324). The study also demonstrated that both UV and chlorine treatments were effective in inactivating these biofilms, within 30 and 10 min, respectively.
• Xu, K., Lv, X., Yue, F., Zhang, L., Wang, P., Amoah, I., Tang, K., Yao, Y., & Li, R. (2024). Effects of phosphate-solubilizing bacteria Aspergillus flavus AF-LRH1 on promoting phosphorus solubilization, wheat growth and soil heavy metal remediation. Journal of Environmental Chemical Engineering, 12(6). doi:10.1016/j.jece.2024.114357
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  Increasing nutrient supply level and eliminating pollution risk are essential to promote farmland soil use. In the soil-plant nutrition cycle, phosphorus is a limiting nutrient, toxic metals such as Cd and Pb are stress threats. Utilizing microorganisms as a bio-amendment could be a viable approach to enhance the cycling of soil phosphorus nutrients and immobilize Cd and Pb in the soil. This study involved the isolation of Aspergillus flavus AF-LRH1, a phosphate solubilizing fungal strain from soil. The strain exhibited the capacity to solubilize calcium phosphate, attaining a phosphorus concentration of 220.73 mg L−1 in the medium. High performance liquid chromatography analysis showed that AF-LRH1 produced a combination of organic acids (tartaric acid 260.7 mg L−1, fumaric acid 0.8 mg L−1, citric acid 911.9 mg L−1) to dissolve Ca3(PO4)2 by providing H+ ions. AF-LRH1 also utilizes NH4+ for the biosynthesis of amino acids and releases protons, which facilitate the dissolution of insoluble Ca3(PO4)2 and calcium phytate. Through these processes, calcium phosphate is partly converted into hydroxyapatite because of biological solubilization. AF-LRH1 strain showed the capability to recover phosphorus from various types of sludge-based solid residue by increasing the solubility of insoluble phosphate compounds for plant nutrient supply. In addition, the combining addition of AF-LRH1 strain and phosphate-containing amendments (calcium phosphate, sludge ash and biochar) benefited the decontamination of Pb and Cd contaminated soil. This finding confirmed the AF-LRH1 could be a promising environmental-friendly biofertilizer suitable for agricultural applications and bioremediation of heavy metal contaminated soil.
• da Silva Santos, L., de Simone Souza, H., Amoah, I., Magri, M., Nobuyoshi Ide, C., & Loureiro Paulo, P. (2024). Treated domestic effluents for non-potable reuse: microbial risk assessment and economic viability. Urban Water Journal, 21(3). doi:10.1080/1573062X.2023.2295309
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  In this study, we applied quantitative microbial risk assessment and net present value methodologies to evaluate the economic viability and safety of wastewater treatment to produce water for reuse. We found payback periods of three to over 15 years for investment in the universalisation of sanitation services in the selected municipalities, wherein the most feasible scenarios were those in which water reuse was carried out at its maximum potential. Microbial risk was assessed in terms of reduced concentrations of Escherichia coli. Results show that reuse of wastewater treated by different plant configurations for crop irrigation, involving only secondary treatment processes, presented high risk of infection for both farmers and consumers (3.50E–02 ± 3.10E–03 and 5.80E 02 ± 3.30E–03, respectively). In the urban and industrial context, it did not pose any risk beyond the tolerable risk recommended by WHO, except for the worker’s droplet ingestion route of exposure (2.40E–03 ± 3.80E–03).
• Mtetwa, H., Amoah, I., Kumari, S., Bux, F., & Reddy, P. (2023). Exploring the role of wastewater-based epidemiology in understanding tuberculosis burdens in Africa. Environmental Research, 231. doi:10.1016/j.envres.2023.115911
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  Tuberculosis (TB) remains a persistent challenge to public health and presents a substantial menace, especially in developing nations of sub-Saharan Africa. It exerts a considerable strain on healthcare systems in these regions. Effective control requires reliable surveillance, which can be improved by incorporating environmental data alongside clinical data. Molecular advances have led to the development of alternative surveillance methods, such as wastewater-based epidemiology. This studyinvestigated the presence, concentration, and diversity of Mycobacterium tuberculosis complex, the cause of TB, in from six African countries: Ghana, Nigeria, Kenya, Uganda, Cameroon, and South Africa. Samples were collected from wastewater treatment plants. All samples were found to contain Mycobacterium species that have been linked to TB in both humans and animals, including Mycobacterium tuberculosis complex, Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum, and Mycobacterium caprae, at varying concentrations. The highest median concentration was found in Ghana, reaching up to 4.7 Log copies/ml for MTBC, 4.6 Log copies/ml for M. bovis, and 3.4 Log copies/ml for M. africanum. The presence of M. africanum outside of West Africa was found in South Africa, Kenya, and Uganda and could indicate the spread of the pathogen. The study underscores the usefulness of wastewater-based epidemiology for tracking TB and shows that even treated wastewater may contain these pathogens, posing potential public health risks.
• Mtetwa, H., Amoah, I., Kumari, S., Bux, F., & Reddy, P. (2023). Surveillance of multidrug-resistant tuberculosis in sub-Saharan Africa through wastewater-based epidemiology. Heliyon, 9(8). doi:10.1016/j.heliyon.2023.e18302
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  The spread of multidrug-resistant tuberculosis (MDR-TB) is a serious public health issue, particularly in developing nations. The current methods of monitoring drug-resistant TB (DR-TB) using clinical diagnoses and hospital records are insufficient due to limited healthcare access and underreporting. This study proposes using Wastewater-Based Epidemiology (WBE) to monitor DR-TB in six African countries (Ghana, Nigeria, Kenya, Uganda, Cameroon, and South Africa) and examines the impact of treated wastewater on the spread of TB drug-resistant genes in the environment. Using droplet-digital polymerase chain reaction (ddPCR), the study evaluated untreated and treated wastewater samples in selected African countries for TB surveillance. There was a statistically significant difference in concentrations of genes conferring resistance to TB drugs in wastewater samples from the selected countries (p-value
• Amoah, I., Abunama, T., Awolusi, O., Pillay, L., Pillay, K., Kumari, S., & Bux, F. (2022). Effect of selected wastewater characteristics on estimation of SARS-CoV-2 viral load in wastewater. Environmental Research, 203. doi:10.1016/j.envres.2021.111877
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  Wastewater-based epidemiology has been used as a tool for surveillance of COVID-19 infections. This approach is dependent on the detection and quantification of SARS-CoV-2 RNA in untreated/raw wastewater. However, the quantification of the viral RNA could be influenced by the physico-chemical properties of the wastewater. This study presents the first use of Adaptive Neuro-Fuzzy Inference System (ANFIS) to determine the potential impact of physico-chemical characteristics of wastewater on the detection and concentration of SARS-CoV-2 RNA in wastewater. Raw wastewater samples from four wastewater treatment plants were investigated over four months. The physico-chemical characteristics of the raw wastewater was recorded, and the SARS-CoV-2 RNA concentration determined via amplification with droplet digital polymerase chain reaction. The wastewater characteristics considered were chemical oxygen demand, flow rate, ammonia, pH, permanganate value, and total solids. The mean SARS-CoV-2 RNA concentrations ranged from 648.1(±514.6) copies/mL to 1441.0(±1977.8) copies/mL. Among the parameters assessed using the ANFIS model, ammonia and pH showed significant association with the concentration of SARS-CoV-2 RNA measured. Increasing ammonia concentration was associated with increasing viral RNA concentration and pH between 7.1 and 7.4 were associated with the highest SARS-CoV-2 concentration. Other parameters, such as total solids, were also observed to influence the viral RNA concentration, however, this observation was not consistent across all the wastewater treatment plants. The results from this study indicate the importance of incorporating wastewater characteristic assessment into wastewater-based epidemiology for a robust and accurate COVID-19 surveillance.
• Amoah, I., Kumari, S., & Bux, F. (2022). A probabilistic assessment of microbial infection risks due to occupational exposure to wastewater in a conventional activated sludge wastewater treatment plant. Science of the Total Environment, 843. doi:10.1016/j.scitotenv.2022.156849
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  Exposure to pathogens during wastewater treatment could result in significant health risks. In this paper, a probabilistic approach for assessing the risks of microbial infection for workers in an activated sludge wastewater treatment plant is presented. A number of exposure routes were modelled, including hand-to-mouth and droplet ingestion of untreated wastewater, droplet ingestion and inhalation of aerosols after secondary treatment, and ingestion of sludge during drying. Almost all workers exposed to untreated wastewater could be infected with the three selected potential pathogens of pathogenic E. coli, Norovirus and Cryptosporidium spp. Hand-to-mouth ingestion is the single most significant route of exposure at the head of works. There is also a risk of infections resulting from ingestion of droplets or inhalation of aerosols at the aeration tanks or contaminated hands at the clarifiers during secondary wastewater treatment. For sludge, the risks of infection with Norovirus was found to be the highest due to accidental ingestion (median risks of 2.2 × 10−2(±3.3 × 10−3)). Regardless of the point and route of exposure, Norovirus and Cryptosporidium spp. presented the highest risks. The study finds that occupational exposure to wastewater at wastewater treatment plants can result in significant viral and protozoan infections. This risk assessment framework can be used to establish and measure the success of risk reduction measures in wastewater treatment plants. These measures could include the use of personal protective equipment and adherence to strict personal hygiene.
• Mtetwa, H., Amoah, I., Kumari, S., Bux, F., & Reddy, P. (2022). Molecular surveillance of tuberculosis-causing mycobacteria in wastewater. Heliyon, 8(2). doi:10.1016/j.heliyon.2022.e08910
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  The surveillance of tuberculosis infections has largely depended on clinical diagnostics and hospitalization data. The advancement in molecular methods creates an opportunity for the adoption of alternative surveillance systems, such as wastewater-based epidemiology. This study presents the use of conventional and advanced polymerase chain reaction techniques (droplet digital PCR) to determine the occurrence and concentration of total mycobacteria and members of the Mycobacterium tuberculosis complex (MTBC) in treated and untreated wastewater. Wastewater samples were taken from three wastewater treatment plants (WWTPs) in the city of Durban, South Africa, known for a high burden of TB/MDR-TB due to HIV infections. All untreated wastewater samples contained total mycobacteria and MTBC at varying percentages per WWTP studied. Other members of the MTBC related to tuberculosis infection in animals, M. bovis and M. caprae were also detected. The highest median concentration detected in untreated wastewater was up to 4.9 (±0.2) Log10 copies/ml for total mycobacteria, 4.0 (±0.85) Log10 copies/ml for MTBC, 3.9 (±0.54) Log10 copies/ml for M. tuberculosis, 2.7 (±0.42) Log10 copies/ml for M. africanum, 4.0 (±0.29) Log10 copies/ml for M. bovis and 4.5 (±0.52) Log10 copies/ml for M. caprae. Lower concentrations were detected in the treated wastewater, with a statistically significant difference (P-value ≤ 0.05) in concentrations observed. The log reduction achieved for these bacteria in the respective WWTPs was not statistically different, indicating that the treatment configuration did not have an impact on their removal. The detection of M. africanum in wastewater from South Africa shows that it is possible that some of the TB infections in the community could be caused by this mycobacterium. This study, therefore, highlights the potential of wastewater-based epidemiology for monitoring tuberculosis infections.
• Mtetwa, H., Amoah, I., Kumari, S., Bux, F., & Reddy, P. (2022). The source and fate of Mycobacterium tuberculosis complex in wastewater and possible routes of transmission. BMC Public Health, 22(1). doi:10.1186/s12889-022-12527-z
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  Background: The Mycobacterium tuberculosis complex (MTBC) consists of causative agents of both human and animal tuberculosis and is responsible for over 10 million annual infections globally. Infections occur mainly through airborne transmission, however, there are possible indirect transmissions through a faecal-oral route which is poorly reported. This faecal-oral transmission could be through the occurrence of the microbe in environments such as wastewater. This manuscript, therefore, reviews the source and fate of MTBC in the wastewater environment, including the current methods in use and the possible risks of infections. Results: The reviewed literature indicates that about 20% of patients with pulmonary TB may have extra-pulmonary manifestations such as GITB, resulting in shedding in feaces and urine. This could potentially be the reason for the detection of MTBC in wastewater. MTBC concentrations of up to 5.5 × 105 (±3.9 × 105) copies/L of untreated wastewater have been reported. Studies have indicated that wastewater may provide these bacteria with the required nutrients for their growth and could potentially result in environmental transmission. However, 98.6 (± 2.7) %, removal during wastewater treatment, through physical-chemical decantation (primary treatment) and biofiltration (secondary treatment) has been reported. Despite these reports, several studies observed the presence of MTBC in treated wastewater via both culture-dependent and molecular techniques. Conclusion: The detection of viable MTBC cells in either treated or untreated wastewater, highlights the potential risks of infection for wastewater workers and communities close to these wastewater treatment plants. The generation of aerosols during wastewater treatment could be the main route of transmission. Additionally, direct exposure to the wastewater containing MTBC could potentially contribute to indirect transmissions which may lead to pulmonary or extra-pulmonary infections. This calls for the implementation of risk reduction measures aimed at protecting the exposed populations.
• Mthethwa, N., Amoah, I., Reddy, P., Bux, F., & Kumari, S. (2022). Development and evaluation of a molecular based protocol for detection and quantification of Cryptosporidium spp. in wastewater. Experimental Parasitology, 234. doi:10.1016/j.exppara.2022.108216
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  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.
• Mthethwa, N., Amoah, I., Reddy, P., Bux, F., & Kumari, S. (2022). Fluorescence and colorimetric LAMP-based real-time detection of human pathogenic Cryptosporidium spp. from environmental samples. Acta Tropica, 235. doi:10.1016/j.actatropica.2022.106606
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  Public health concerns related to pathogenic protozoa are widespread, causing significant morbidity and mortality worldwide. Due to the lack of rapid and cost-effective diagnostic methods, timely treatment and control interventions are hindered. In this study, loop mediated isothermal amplification (LAMP) methods were optimised and evaluated for the rapid detection of human pathogenic Cryptosporidium species in environmental samples. Real-time fluorescence and colorimetric detection were tested simultaneously. As a reference method, the results were compared to the well-established droplet digital polymerase chain reaction (ddPCR) method. Both LAMP-based methods successfully detected the Cryptosporidium parvum (GP60 gene) and the entire Cryptosporidium genus (SAM gene) from environmental samples with 100% specificity and no cross-reactivity. Furthermore, both colorimetric and fluorescent methods demonstrated a high sensitivity, with the same limit of detection (LOD) of 1.1 copies of C. parvum per 25 µl reaction (0.02 ng/µl). For real-time fluorescence monitoring, the LAMP reaction can be completed within 28 min, and for visual detection, within 30 min. In addition, both fluorescent and colorimetric LAMP methods showed substantial agreement with the reference method (ddPCR) used. The developed protocols were able to detect C. parvum and Cryptosporidium spp. in 50–85% (n = 60) of environmental samples (treated and untreated wastewater, sludge, and surface water) compared to 58–98% (n = 60) detected by ddPCR. The results further demonstrate that LAMP is an efficient technique for detecting Cryptosporidium spp. in environmental samples due to its simplicity, low cost, sensitivity, and specificity. Therefore, it has great potential as a useful diagnostic tool for disease control and public health protection.
• Ngubane, Z., Bergion, V., Dzwairo, B., Troell, K., Amoah, I., Sokolova, E., & Stenström, T. (2022). Water quality modelling and quantitative microbial risk assessment for uMsunduzi River in South Africa. Journal of Water and Health, 20(4). doi:10.2166/wh.2022.266
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  South African rivers generally receive waste from inadequate wastewater infrastructure, mines, and farming activities, among others. The uMsunduzi River in KwaZulu-Natal, South Africa, is among these recipients with recorded poor to very poor water quality. To identify parts of the uMsunduzi River that are polluted by Cryptosporidium and Escherichia coli (E. coli), this study mapped out pollutants emanating from point and non-point sources using the Soil and Water Assessment Tool (SWAT). Streamflow calibration in the upper and lower reaches of the catchment showed good performance with R2 of 0.64 and 0.58, respectively. SWAT water quality output data were combined with a Quantitative Microbial Risk Assessment (QMRA) to understand the microbial health implications for people using river water for drinking, recreational swimming, and non-competitive canoeing. QMRA results for Cryptosporidium and pathogenic E. coli showed that the probability of infection for most users exceeds the acceptable level for drinking and recreation as outlined in the South African water quality guidelines, and by the World Health Organization (WHO). The results of this study can be used as a baseline to assess the economic and health implications of different management plans, resulting in better-informed, cost-effective, and impactful decision-making.
• Pillay, L., Amoah, I., Kumari, S., & Bux, F. (2022). Potential and Challenges Encountered in the Application of Wastewater-Based Epidemiology as an Early Warning System for COVID-19 Infections in South Africa. ACS ES and T Water, 2(11). doi:10.1021/acsestwater.2c00049
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  The use of wastewater-based epidemiology (WBE) as a disease surveillance tool during the COVID-19 pandemic has been mainly to achieve two goals: early warning and determining changes in infection numbers over time. This study focused on the utilization of WBE as an early warning tool for COVID-19 infection in the eThekwini municipality of South Africa. Four wastewater treatment plants treating mainly domestic wastewater were chosen for this study. A central wastewater treatment plant was first used to determine the early warning potential of WBE, and thereafter, the tool was applied at three other locations. Viral concentrations in the raw wastewater were determined via the droplet digital polymerase chain reaction (PCR) method. The SARS-CoV-2 concentration varied from 4 to 7 Log10/100 mL of raw wastewater during the study period. The changes in viral concentration corresponded with the active COVID-19 cases within the study area. As an early warning tool, the WBE data was able to detect increasing infections in the community at least 5 weeks prior to increasing clinical cases during the third wave of COVID-19 infections in the country. Similarly, SARS-CoV-2 concentrations began increasing on November 30, 2021 prior to an increase in clinical cases on December 14, 2021, giving an almost a 2 week lead time. However, statistically, a 2-3 week lead time was determined to show the highest relationship with increasing COVID-19 cases. This study therefore identified the lead time as being between 2 and 3 weeks for early warning of COVID-19 infections using WBE data. Application of the early warning system at the three other catchments during the fourth wave of COVID-19 infections gave similar results of a lead time of 2 weeks. Therefore, the findings in this study further support the use of WBE as an early warning system. However, further studies are still required to address the challenges hindering the accurate and efficient use of this tool.
• Ramlal, P., Lin, J., Buckley, C., Amoah, I., & Stenström, T. (2022). An assessment of the health risks associated with shared sanitation: a case study of the community ablution blocks in Durban, South Africa. Environmental Monitoring and Assessment, 194(3). doi:10.1007/s10661-022-09815-x
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  Shared sanitation facilities have been hailed as an innovative approach to solve the challenge with sanitation access. However, these facilities may act as hotspots for disease transmission due to unhygienic conditions. In this study we used quantitative (based on Escherichia coli contamination) techniques to assess the health risks associated with the use of community ablution blocks (CABs). The most contaminated surfaces were the cistern handle (5.7 Log10 cfu/cm2) and internal pull latch (5.8 Log10 cfu/cm2). Based on the E. coli contamination, at least two people out of 100 CAB users might be potentially infected when they touch “hot” surfaces. These risks were modelled assuming transfer of potentially pathogenic E. coli from these surfaces to the mouth. The incorporation of risk-reduction measures, such as wiping of these surfaces or washing of hands, could potentially result in significant reduction of infection risks. The most significant risk-reduction intervention was determined to be wiping of the contact surfaces, especially twice prior to contact. A combination of risk-reduction interventions could further reduce the risks. This study shows that contamination of contact surfaces within shared CABs could lead to increased risks of infections, requiring measures aimed at reducing the associated risks. The risk assessment framework used in this study could therefore be applied in similar settings to estimate associated health risks with the use of such facilities.
• Ramlal, P., Lin, J., Buckley, C., Amoah, I., & Stenström, T. (2022). Determinants of diarrhoeal infections among users of shared sanitation in informal settlements in Durban, South Africa. Journal of Water and Health, 20(10). doi:10.2166/wh.2022.201
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  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 ¼ 1.8; 95% confidence interval (CI): 0.9–3.12), use of shared sanitation (POR ¼ 1.7; 95%; CI: 1.05–2.26), and exposure to faecal matter around the homes (POR ¼ 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.
• Amoah, I., Mthethwa, N., Pillay, L., Deepnarain, N., Pillay, K., Awolusi, O., Kumari, S., & Bux, F. (2021). RT-LAMP: A Cheaper, Simpler and Faster Alternative for the Detection of SARS-CoV-2 in Wastewater. Food and Environmental Virology, 13(4). doi:10.1007/s12560-021-09489-7
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  Reverse transcription loop-mediated isothermal amplification (RT-LAMP) has the potential to become a cheaper and faster option for monitoring COVID-19 infections through wastewater-based epidemiology. However, its application in COVID-19 surveillance has been limited to clinical testing only. We present in this paper two optimized RT-LAMP protocols based on colour change and fluorescence detection and application of these protocols for wastewater monitoring from four wastewater treatment plants over 4 weeks. The optimized RT-LAMP protocols have a limit of detection of 10 copies/25 µl reaction with positive amplification within 35 minutes. Over the 4 weeks of monitoring, the colorimetric protocol detected a prevalence of 12.5%, when 1 µl of extracted RNA with 92.7(± 28.2) ng/µl concentration was analysed. When the RNA template was increased by fivefold, the prevalence increased to 44%. The fluorescent RT-LAMP had a prevalence of 31% and 47% for starting templates of 92.7(± 28.2) ng/µl and 480(± 134.5) ng/µl of the extracted RNA, respectively. All samples were positive for SARS-CoV-2 when analysed with droplet digital PCR, with viral loads ranging from 18.1 to 195.6 gc/ml of wastewater. The RT-ddPCR, therefore, confirms the presence of the viral RNA in the wastewater samples, albeit at low concentrations. Additionally, the RT-LAMP protocols positively detected SARS-CoV-2 in wastewater samples with copies as low as 20.7 gc/ml. The results obtained in our study show the potential application of RT-LAMP for the detection of SARS-CoV-2 in wastewater, which could provide a cheaper and faster alternative to RT-qPCR or RT-ddPCR for wastewater-based epidemiological monitoring of COVID-19 and other viral infections.
• Amoah, I., Pillay, L., Deepnarian, N., Awolusi, O., Pillay, K., Ramlal, P., Kumari, S., & Bux, F. (2021). Detection of SARS-CoV-2 RNA on contact surfaces within shared sanitation facilities. International Journal of Hygiene and Environmental Health, 236. doi:10.1016/j.ijheh.2021.113807
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  Contamination of contact surfaces with SARS-CoV-2 has been reported as a potential route for the transmission of COVID-19. This could be a major issue in developing countries where access to basic sanitation is poor, leading to the sharing of toilet facilities. In this study, we report SARS-CoV-2 contamination of key contact surfaces in shared toilets and the probabilistic risks of COVID-19 infections based on detection and quantification of the nucleic acid on the surfaces. We observed that 54–69% of the contact surfaces were contaminated, with SARS-CoV-2 loads ranging from 28.1 to 132.7 gene copies per cm2. Toilet seats had the highest contamination, which could be attributed to shedding of the virus in feces and urine. We observed a significant reduction in viral loads on the contaminated surfaces after cleaning, showing the potential of effective cleaning on the reduction of contamination. The pattern of contamination indicates that the most contaminated surfaces are those that are either commonly touched by users of the shared toilets or easily contaminated with feces and urine. These surfaces were the toilet seats, cistern handles and tap handles. The likelihood (probability) of infection with COVID-19 on these surfaces was highest on the toilet seat (1.76 × 10−4(1.58 × 10−6)) for one time use of the toilet. These findings highlight the potential risks for COVID-19 infections in the event that intact infectious viral particles are deposited on these contact surfaces. Therefore, this study shows that shared toilet facilities in densely populated areas could lead to an increase in risks of COVID-19 infections. This calls for the implementation of risk reduction measures, such as regular washing of hands with soap, strict adherence to wearing face masks, and effective and regular cleaning of shared facilities.
• Govender, R., Amoah, I., Adegoke, A., Singh, G., Kumari, S., Swalaha, F., Bux, F., & Stenström, T. (2021). Identification, antibiotic resistance, and virulence profiling of Aeromonas and Pseudomonas species from wastewater and surface water. Environmental Monitoring and Assessment, 193(5). doi:10.1007/s10661-021-09046-6
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  Aquatic environments are hotspots for the spread of antibiotic-resistant bacteria and genes due to pollution caused mainly by anthropogenic activities. The aim of this study was to evaluate the impact of wastewater effluents, informal settlements, hospital, and veterinary clinic discharges on the occurrence, antibiotic resistance profile and virulence signatures of Aeromonas spp. and Pseudomonas spp. isolated from surface water and wastewater. High counts of Aeromonas spp. (2.5 (± 0.8) – 3.3 (± 0.4) log10 CFU mL−1) and Pseudomonas spp. (0.6 (± 1.0) – 1.8 (± 1.0) log10 CFU mL−1) were obtained. Polymerase chain reaction (PCR) and MALDI-TOF characterization identified four species of Aeromonas and five of Pseudomonas. The isolates displayed resistance to 3 or more antibiotics (71% of Aeromonas and 94% of Pseudomonas). Aeromonas spp. showed significant association with the antibiotic meropenem (χ2 = 3.993, P < 0.05). The virulence gene aer in Aeromonas was found to be positively associated with the antibiotic resistance gene blaOXA (χ2 = 6.657, P < 0.05) and the antibiotic ceftazidime (χ2 = 7.537, P < 0.05). Aeromonas recovered from both wastewater and surface water displayed high resistance to ampicillin and had higher multiple antibiotic resistance (MAR) indices close to the hospital. Pseudomonas isolates on the other hand exhibited low resistance to carbapenems but very high resistance to the third-generation cephalosporins and cefixime. The results showed that some of the Pseudomonas spp. and Aeromonas spp. isolates were extended-spectrum β-lactamase producing bacteria. In conclusion, the strong association between virulence genes and antibiotic resistance in the isolates shows the potential health risk to communities through direct and indirect exposure to the water.
• Marques, F., Magri, M., Amoah, I., Paulo, P., & Stenström, T. (2021). Development of a semi-quantitative approach for the assessment of microbial health risk associated with wastewater reuse: A case study at the household level. Environmental Challenges, 4. doi:10.1016/j.envc.2021.100182
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  This study proposes a modified methodology for semi-quantitative microbial risk assessment (SQMRA) based on the risk matrix presented in the World Health Organization (WHO) Sanitation Safety Planning manual adding the variable ranking for probability of infection in the estimations of risk scores, which composes the risk matrix. The approach is applied to cases where quantitative microbial risk assessment (QMRA) studies are hindered by the lack of information regarding pathogens in the (waste)water, collected as a set of primary data. The methodology is fully described and its application is demonstrated by comparing the microbial risk to which individuals are exposed within a residence in different scenarios of direct or indirect exposure to wastewater. Three current scenarios and one proposed scenario, with four sub-scenarios, focused on the use of a decentralized treatment system and greywater reuse in a single-family residence. Scenarios were selected for application of the SQMRA to elucidate if solutions considering source separation and reuse would bring more health-related risks, or attenuate the existing ones related to the conventional onsite wastewater treatment and soil disposal. None of the studied alternatives achieved risk score above 160 (very high). Considering the average score of each alternative by exposed group, the current scenarios, representing low-cost, informal solutions, posed the highest risks for children. All proposed reuse types reduced the risks from high to medium (score 43.9) for children and from medium to low (score 22.2) for adults. The adapted approach was demonstrated to be a useful tool, aiding the decision-making process and clarifying uncertainties raised when source separation and household reuse are proposed.
• Mtetwa, H., Amoah, I., Kumari, S., Bux, F., & Reddy, P. (2021). Wastewater-based surveillance of antibiotic resistance genes associated with tuberculosis treatment regimen in kwazulu natal, south africa. Antibiotics, 10(11). doi:10.3390/antibiotics10111362
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  Essential components of public health include strengthening the surveillance of infectious diseases and developing early detection and prevention policies. This is particularly important for drug-resistant tuberculosis (DR-TB), which can be explored by using wastewater-based surveillance. This study aimed to use molecular techniques to determine the occurrence and concentration of antibiotic-resistance genes (ARGs) associated with tuberculosis (TB) resistance in untreated and treated wastewater. Raw/untreated and treated (post-chlorination) wastewater samples were taken from three wastewater treatment plants (WWTPs) in South Africa. The ARGs were selected to target drugs used for first-and second-line TB treatment. Both conventional polymerase chain reaction (PCR) and the more advanced droplet digital PCR (ddPCR) were evaluated as surveillance strategies to determine the distribution and concentration of the selected ARGs. The most abundant ARG in the untreated wastewater was the rrs gene, associated with resistance to the aminoglycosides, specifically streptomycin, with median concentration ranges of 4.69–5.19 log copies/mL. In con-trast, pncA gene, associated with resistance to the TB drug pyrazinamide, was the least detected (1.59 to 2.27 log copies/mL). Resistance genes associated with bedaquiline was detected, which is a significant finding because this is a new drug introduced in South Africa for the treatment of multi-drug resistant TB. This study, therefore, establishes the potential of molecular surveillance of wastewater for monitoring antibiotic resistance to TB treatment in communities.
• Pillay, L., Amoah, I., Deepnarain, N., Pillay, K., Awolusi, O., Kumari, S., & Bux, F. (2021). Monitoring changes in COVID-19 infection using wastewater-based epidemiology: A South African perspective. Science of the Total Environment, 786. doi:10.1016/j.scitotenv.2021.147273
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  Monitoring of COVID-19 infections within communities via wastewater-based epidemiology could provide a cost-effective alternative to clinical testing. This approach, however, still requires improvement for its efficient application. In this paper, we present the use of wastewater-based epidemiology in monitoring COVID-19 infection dynamics in the KwaZulu-Natal province of South Africa, focusing on four wastewater treatment plants for 14 weeks. The SARS-CoV-2 viral load in influent wastewater was determined using droplet digital PCR, and the number of people infected was estimated using published models as well as using a modified model to improve efficiency. On average, viral loads ranged between 0 and 2.73 × 105 copies/100 ml, 0–1.52 × 105 copies/100 ml, 3 × 104–7.32 × 105 copies/100 ml and 1.55 × 104–4.12 × 105 copies/100 ml in the four wastewater treatment plants studied. The peak in viral load corresponded to the reported COVID-19 infections within the districts where these catchments are located. In addition, we also observed that easing of lockdown restrictions by authorities corresponded with an increase in viral load in the untreated wastewater. Estimation of infection numbers based on the viral load showed that a higher number of people could potentially be infected, compared to the number of cases reported based on clinical testing. The findings reported in this paper contribute to the field of wastewater-based epidemiology for COVID-19 surveillance, whilst highlighting some of the challenges associated with this approach, especially in developing countries.
• Amoah, I., Kumari, S., Reddy, P., Bux, F., & Stenström, T. (2020). Impact of informal settlements and wastewater treatment plants on helminth egg contamination of urban rivers and risks associated with exposure. Environmental Monitoring and Assessment, 192(11). doi:10.1007/s10661-020-08660-0
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  The quality of surface water could be influenced by both anthropogenic and natural factors. This study was designed to determine the impact of informal settlement and wastewater treatment plants on helminth egg contamination of urban rivers and the risks associated with everyday use. We also ascertained the accumulation of these eggs in the river sediments. The study was carried out in two rivers in the eThekwini Municipality of South Africa. Grab samples were taken at different points over a 10-month period. Ascaris spp., hookworm, Toxocara spp., Trichuris spp. and Taenia spp. were the helminth eggs detected in both the water column and sediments, with mean Ascaris spp. eggs of 0–6.3 (± 5.1)/L in the water and 0–6.8 (± 5.2)/kg in sediment samples. The helminth egg concentrations showed seasonal variation, probably due to changes in infection levels of the populations or natural factors, such as rainfall. The informal settlements had a greater impact than treated wastewater. For every 10,000 recreational users of the rivers 19 to 58 may be infected under undisturbed conditions, increasing to 29–88 individuals when the riverbed is disturbed. The risk from agricultural use of the rivers was above the tolerable risk values applicable for wastewater reuse, recommended by the World Health Organization. This calls for a re-evaluation of the policies governing surface water quality assessment, where the inclusion of helminth eggs and sediment monitoring will be critical.
• Amoah, I., Singh, G., Troell, K., Reddy, P., Bux, F., & Stenström, T. (2020). Comparative assessment of DNA extraction procedures for Ascaris spp. Eggs. Journal of Helminthology, 94. doi:10.1017/S0022149X19000683
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  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.
• Deepnarain, N., Nasr, M., Amoah, I., Enitan-Folami, A., Reddy, P., Kumari, S., Bux, F., & Stenström, T. (2020). Impact of sludge bulking on receiving environment using quantitative microbial risk assessment (QMRA)-based management for full-scale wastewater treatment plants. Journal of Environmental Management, 267. doi:10.1016/j.jenvman.2020.110660
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  During sludge bulking in wastewater treatment plants (WWTPs), high amounts of potentially pathogenic bacteria would release into the environment, causing various human-health risks. This is the first study attempting to assess the microbial infections associated with the reuse of WWTP effluents under various bulking conditions. Three common waterborne pathogens, viz., E. coli O157:H7, Salmonella, and Mycobacterium, were quantified from full-scale WWTPs using DNA extraction and qPCR at different sludge volume indices (SVIs). The detected pathogens were incorporated into a quantitative microbial risk assessment (QMRA) model to determine the applicability of WWTP discharge for recreational (bathing) activities and agricultural practices. The QMRA exposures were children, women, and men during swimming, and farmers and vegetable consumers during irrigation. Bacterial abundance in the treated wastewater increased in response to SVIs, and the QMRA values at all bulking events exceeded the tolerable risk of one case of infection per 10,000 people per year. Hence, various disinfection scenarios (chlorination, ultraviolet, and ozonation) were hypothetically tested to control the risks associated with pathogenic bacteria, allowing for safe disposal and reuse of the treated effluent. The ultraviolet application provided the highest ability to inactivate the pathogenic bacteria, except for the case of children exposed to Salmonella infection during swimming. The reduction of Mycobacterium infection risks with either chlorination or ozonation showed inefficient results. This study would be helpful for the management of human health risks associated with effluent wastewater containing pathogens, i.e., particularly concerning the case of sludge bulking.
• Govender, R., Amoah, I., Kumari, S., Bux, F., & Stenström, T. (2020). Detection of multidrug resistant environmental isolates of acinetobacter and Stenotrophomonas maltophilia: a possible threat for community acquired infections?. Journal of Environmental Science and Health - Part A Toxic/Hazardous Substances and Environmental Engineering, 56(2). doi:10.1080/10934529.2020.1865747
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  Acinetobacter spp. and Stenotrophomonas maltophilia are bacteria commonly associated with infections at the clinical settings. Reports of infections caused by environmental isolates are rare. Therefore, this study focused on determination of the antibiotic resistance patterns, antibiotic resistance genes, efflux pumps and virulence signatures of Acinetobacter spp. and S. maltophilia recovered from river water, plant rhizosphere and river sediment samples. The isolates were identified and confirmed using biochemical tests and PCR. The antimicrobial resistance profiles of the isolates were determined using Kirby Bauer disk diffusion assay and presence of antibiotic resistance and virulence genes were detected using PCR. S. maltophilia was more frequent in plant rhizosphere and sediment samples than the water samples. Acinetobacter spp. were mostly resistant to trimethoprim-sulfamethoxazole (96% of isolates), followed by polymyxin b (86%), cefixime (54%), colistin (42%), ampicillin (35%) and meropenem (19%). The S. maltophilia isolates displayed total resistance (100%) to trimethoprim- sulfamethoxazole, meropenem, imipenem, ampicillin and cefixime, while 80% of the isolates were resistant to ceftazidime. Acinetobacter spp. contained different antibiotic resistance genes such as sul1 (24% of isolates), sul2 (29%), blaOXA 23/51 (21%) and blaTEM (29%), while S. maltophilia harbored sul1 (8%) and blaTEM (20%). Additionally, efflux pump genes were present in all S. maltophilia isolates. The presence of multidrug resistant Acinetobacter spp. and Stenotrophomonas maltophilia in surface water raises concerns for community-acquired infections as this water is directly been used by the community for various purposes. Therefore, there is the need to institute measures aimed at reducing the risks of these infections and the resulting burden this may have on the health care system within the study area.
• Clack, K., Pietruschka, B., Amoah, I., Muchaonyerwa, P., Odindo, O., Palomo, M., Buckley, C., Ngwane, Z., & Mladenov, N. (2019). Transfer of coliform bacteria to duckweed harvested from anaerobic baffled reactor effluent. Bioresource Technology Reports, 8. doi:10.1016/j.biteb.2019.100314
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  Efforts to recover nutrients from wastewater have drawn interest in effluent polishing with duckweed, a small angiosperm known to grow in high nutrient environments. One important consideration is that pathogens may be transferred to suspended duckweed biomass and ultimately spread into agricultural or other environmental settings. Our objective was to evaluate transfer of fecal indicator bacteria to duckweed (Lemna minor and Wolffia arrhiza) from anaerobic baffled reactor effluent. The presence of duckweed resulted in significant removal (~1.5-log) of Escherichia coli and >65% decrease in turbidity in reactors. There was E. coli transfer of ~7-log CFU/g from effluent to harvested duckweed, and viable E. coli persisted (at 3.6-log CFU/g) even after several days of biomass drying, representing a health risk for agricultural applications.
• Amoah, I., Reddy, P., Seidu, R., & Stenström, T. (2018). Concentration of soil-transmitted helminth eggs in sludge from South Africa and Senegal: A probabilistic estimation of infection risks associated with agricultural application. Journal of Environmental Management, 206. doi:10.1016/j.jenvman.2017.12.003
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  The use of sludge in agriculture has been encouraged as a means of increasing soil nutrient content and improving the water holding capacity. On the negative side, major public health concerns with sludge application prevail, mainly due to the high concentration of pathogenic microorganisms. Soil-transmitted helminths (STHs) are of major health concern in this regard, especially in endemic regions, mainly due to the high environmental resistant of the eggs combined with a low infectious dose. In this study the concentration of STH eggs in two months dried sludge from Durban, South Africa and Dakar, Senegal was determined and compared. Sampling was carried out from January to October 2016 and in September 2016 for Dakar. Ascaris spp, hookworm, Trichuris spp, Taenia spp and Toxocara spp were the commonly recorded STH eggs. STH egg concentrations were higher in Dakar than in Durban, with viable STH egg concentrations exceeding both local and international guidelines. Due to the high concentration of viable STH eggs, risks of Ascaris spp infection was very high for farmers applying this sludge on their farms in both Durban (7.9 × 10−1 (±1.7 × 10−2)) and Dakar (9.9 × 10−1 (±1.3 × 10−5)). Consumption of lettuce grown on sludge amended soil will result in probable infections but harvest after 30 days between sludge application and harvest in Durban gave median probability infection risks with a risk level similar to the WHO tolerable risk value (10−4). This time period need to be prolonged to harvest in Dakar to 40 days to reduce the risks of infection to the tolerable risks values. Further treatment of the sludge either through composting or drying for longer periods of time is thus recommended from a public health perspective.
• Amoah, I., Reddy, P., Seidu, R., & Stenström, T. (2018). Removal of helminth eggs by centralized and decentralized wastewater treatment plants in South Africa and Lesotho: health implications for direct and indirect exposure to the effluents. Environmental Science and Pollution Research, 25(13). doi:10.1007/s11356-018-1503-7
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  Wastewater may contain contaminants harmful to human health; hence, there is the need for treatment before discharge. Centralized wastewater treatment systems are the favored treatment options globally, but these are not necessarily superior in reduction of pathogens as compared to decentralized wastewater treatment systems (collectively called DEWATS). This study was therefore undertaken to assess the soil-transmitted helminth (STH) and Taenia sp. egg reduction efficiency of selected anaerobic baffled reactors and planted gravel filters compared to centralized wastewater treatment plants in South Africa and Lesotho. The risk of ascariasis with exposure to effluents from the centralized wastewater treatment plants was also assessed using the quantitative microbial risk assessment (QMRA) approach. Eggs of Ascaris spp., hookworm, Trichuris spp., Taenia spp., and Toxocara spp. were commonly detected in the untreated wastewater. The DEWATS plants removed between 95 and 100% of the STH and Taenia sp. eggs, with centralized plants removing between 67 and 100%. Helminth egg concentrations in the final effluents from the centralized wastewater treatment plants were consistently higher than those in the WHO recommended guideline (≤ 1 helminth egg/L) for agricultural use resulting in higher risk of ascariasis. Therefore, in conclusion, DEWATS plants may be more efficient in reducing the concentration of helminth eggs in wastewater, resulting in lower risks of STH infections upon exposure.
• Amoah, I., Reddy, P., & Stenström, T. (2017). Effect of reagents used during detection and quantification of Ascaris suum in environmental samples on egg viability. Water Science and Technology, 76(9). doi:10.2166/wst.2017.324
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  Soil-transmitted helminths (STHs) are a major health concern globally. Infection is mostly through contact with contaminated water, food or soil. Therefore to break the cycle of viable transmission STH eggs must be quantitatively detected in the environment. The effect of different reagents on the viability of Ascaris suum eggs during laboratory detection and quantification was assessed and different incubation solutions compared. Sulphuric acid gave a slightly higher recovery percentage of viable eggs (91.2%) than distilled water (90.0%) and 0.5% formalin (87.6%), although the difference was not statistically significant (p > 0.05). Acetoacetic acid, ethyl acetate, ammonium bicarbonate, zinc sulphate, magnesium sulphate and Tween 80, are reagents widely used in test protocols for the detection and quantification of STH eggs. Eggs were exposed to these reagents for different time durations. Acetoacetic acid resulted in the highest loss of viability (3.4 ±0.7% viable), while magnesium sulphate resulted in the least effect (88.5±1.2% viable). In conclusion the use of the selected reagents in the detection of these eggs was found to affect the viability of exposed eggs, especially during prolonged exposures. Therefore we recommended that eggs be exposed for ≤5 minutes, to reduce the risk of viability loss.
• Amoah, I., Abubakari, A., Abaidoo, R., Seidu, R., & Stenström, T. (2016). Contribution of Wastewater Irrigation to Soil Transmitted Helminths Infection among Vegetable Farmers in Kumasi, Ghana. PLoS Neglected Tropical Diseases, 10(12). doi:10.1371/journal.pntd.0005161
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  Wastewater irrigation is associated with several benefits but can also lead to significant health risks. The health risk for contracting infections from Soil Transmitted Helminths (STHs) among farmers has mainly been assessed indirectly through measured quantities in the wastewater or on the crops alone and only on a limited scale through epidemiological assessments. In this study we broadened the concept of infection risks in the exposure assessments by measurements of the concentration of STHs both in wastewater used for irrigation and the soil, as well as the actual load of STHs ova in the stool of farmers and their family members (165 and 127 in the wet and dry seasons respectively) and a control group of non-farmers (100 and 52 in the wet and dry seasons, respectively). Odds ratios were calculated for exposure and non-exposure to wastewater irrigation. The results obtained indicate positive correlation between STH concentrations in irrigation water/soil and STHs ova as measured in the stool of the exposed farmer population. The correlations are based on reinfection during a 3 months period after prior confirmed deworming. Farmers and family members exposed to irrigation water were three times more likely as compared to the control group of non-farmers to be infected with Ascaris (OR = 3.9, 95% CI, 1.15–13.86) and hookworm (OR = 3.07, 95% CI, 0.87–10.82). This study therefore contributes to the evidence-based conclusion that wastewater irrigation contributes to a higher incidence of STHs infection for farmers exposed annually, with higher odds of infection in the wet season.
• Singh, G., Vajpayee, P., Rani, N., Amoah, I., Shanker, R., & Stenström, T. (2016). Exploring the potential reservoirs of non specific TEM beta lactamase (blaTEM) gene in the Indo-Gangetic region: A risk assessment approach to predict health hazards. Journal of Hazardous Materials, 314. doi:10.1016/j.jhazmat.2016.04.036
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  The emergence of antimicrobial resistant bacteria is an important public health and environmental contamination issue. Antimicrobials of β-lactam group accounts for approximately two thirds, by weight, of all antimicrobials administered to humans due to high clinical efficacy and low toxicity. This study explores β-lactam resistance determinant gene (blaTEM) as emerging contaminant in Indo-Gangetic region using qPCR in molecular beacon format. Quantitative Microbial Risk Assessment (QMRA) approach was adopted to predict risk to human health associated with consumption/exposure of surface water, potable water and street foods contaminated with bacteria having blaTEM gene. It was observed that surface water and sediments of the river Ganga and Gomti showed high numbers of blaTEM gene copies and varied significantly (p < 0.05) among the sampling locations. The potable water collected from drinking water facility and clinical settings exhibit significant number of blaTEM gene copies (13 ± 0.44-10200 ± 316 gene copies/100 mL). It was observed that E.crassipes among aquatic flora encountered in both the rivers had high load of blaTEM gene copies. The information on prevalence of environmental reservoirs of blaTEM gene containing bacteria in Indo-Gangetic region and risk associated will be useful for formulating strategies to protect public from menace of clinical risks linked with antimicrobial resistant bacteria.