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.

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.

Concentration and reduction of antibiotic residues in selected wastewater treatment plants and receiving waterbodies in Durban, South Africa.

In the province of KwaZulu-Natal, South Africa the incidence of resistant tuberculosis, upper respiratory tract diseases as well as diarrhoeal and parasitic infections is high. Treatment of these diseases with antibiotics is partly reflected by the excretion of the respective antibiotics and their subsequent occurrence in wastewater. Their quantitative reduction in wastewater treatment reflects their potential environmental as well as human impact, the latter due to the use of the recipient water for domestic purposes and for irrigation. Information of the occurrence and reduction of different classes of antibiotics in wastewater treatment is sparse, especially the particle bound fraction of these. Due to this, analyses of aqueous and particle bound antibiotics in untreated wastewater of four selected wastewater treatment plants (WWTPs) and their receiving water bodies was carried out in Durban, South Africa. The treatment step especially considered was the biological one, represented by activated sludge and trickling filters. The treatment further included secondary clarifiers and final chlorine disinfection. Composite samples were collected during the period February 2017 to May 2017 and analysed with online solid phase extraction - high performance liquid chromatography mass spectrometry (SPE-HPLC-MS). For the 13 assessed antibiotics, the limit of detection (LOD) and the limit of quantification (LOQ) ranged from 0.07 to 0.33 ng L-1 and 0.23 to 1.09 ng L-1 respectively, while the total percentage recovery was in the range of 51 to 111%. The percentage of individual antibiotics bound to the particulate fraction normally lost by sample (influent) filtration, if not analysed in parallel, was in the range of 2.6%-97.3% (n = 32). In this fraction (sludge from centrifuge sample), the concentration of bound antibiotics of all the target antibiotics were detected in the influent of all WWTP in concentration ranges between 1.3 ng L-1 (Azithromycin; AZI) to 81,748 ng L-1 (Ciprofloxacin; CIP). The antibiotics with the highest median concentrations in receiving water bodies of the respective WWTP were; Sulfamethoxazole; SUL (239 ng L-1) WWTP "K", Ciprofloxacin; CIP (708 ng L-1) WWTP "S" and Albendazole; ALB (325 ng L-1 and 683 ng L-1) WWTP "P" and "I" respectively. The overall percentage removal efficiency for the four WWTPs ranged from 21% to 100%. The biological treatment steps, activated sludge and trickling filters, were effective in removing antibiotics especially with the trickling filter and the impact of the sedimentation stage after activated sludge treatment.

Identification of antibiotics in wastewater: current state of extraction protocol and future perspectives.

The release and occurrence of antibiotics in the aquatic environment has generated increased attention in the past few decades. The residual antibiotic in wastewater is important in the selection for antimicrobial resistance among microorganisms and the possibility of forming toxic derivatives. This review presents an assessment of the advancement in methods for extraction of antibiotics with solid phase extraction and liquid-liquid extraction methods applied in different aquatic environmental media. These advanced methods do enhance specificity, and also exhibit high accuracy and recovery. The aim of this review is to assess the pros and cons of the methods of extraction towards identification of quinolones and sulphonamides as examples of relevant antibiotics in wastewater. The challenges associated with the improvements are also examined with a view of providing potential perspectives for better extraction and identification protocols in the near future. From the context of this review, magnetic molecular imprinted polymer is superior over the remaining extraction methods (with the availability of commercial templates and monomers), is based on less cumbersome extraction procedures, uses less solvent and has the advantage of its reusable magnetic phase.

Evaluation of phytotoxicity effect on selected crops using treated and untreated wastewater from different configurative domestic wastewater plants.

This study investigated the phytotoxicity effect of untreated and treated wastewater collected from two different configurations of domestic wastewater treatment plants in South Africa. The phytotoxicity effect on vegetable seed growth was studied in terms of germination index (GI), relative seed germination (RSG) and relative root elongation (RRE) using four commercial crop varieties, viz., tomato (Lycopersicon esculentum), radish (Raphanus sativus), carrot (Daucus carota) and onion (Allium cepa). According to phyototoxicity limits, 80% germination and above is regarded as non-toxic and less than 50% GI is regarded as highly toxic and not suitable for agricultural purposes. In our study, seeds were irrigated with concentrations of 25%, 50%, 75%, 100% of treated effluent (TE) and untreated effluent (UTE). The TE results were best with the highest GI (%) recorded as tomato, 177; carrot, 158.5; onion, 132; and lettuce, 124. The results of this study indicate that TE showed no phytotoxicty effects and recorded above 80% GI. The UTE irrigated crops reached a GI of only 50% and above which is clear evidence of the beneficial effect of waste water treatment. The overall results confirmed that treated wastewater has a beneficial effect on agricultural crops and can be used as a liquid fertilizer.

Quantification of pathogen inactivation efficacy by free chlorine disinfection of drinking water for QMRA.

To support the implementation of quantitative microbial risk assessment (QMRA) for managing infectious risks associated with drinking water systems, a simple modeling approach for quantifying Log10 reduction across a free chlorine disinfection contactor was developed. The study was undertaken in three stages: firstly, review of the laboratory studies published in the literature; secondly, development of a conceptual approach to apply the laboratory studies to full-scale conditions; and finally implementation of the calculations for a hypothetical case study system. The developed model explicitly accounted for variability in residence time and pathogen specific chlorine sensitivity. Survival functions were constructed for a range of pathogens relying on the upper bound of the reported data transformed to a common metric. The application of the model within a hypothetical case study demonstrated the importance of accounting for variable residence time in QMRA. While the overall Log10 reduction may appear high, small parcels of water with short residence time can compromise the overall performance of the barrier. While theoretically simple, the approach presented is of great value for undertaking an initial assessment of a full-scale disinfection contactor based on limited site-specific information.

Virus removal by unsaturated wastewater filtration: effects of biofilm accumulation and hydrophobicity.

Enhanced treatment of septic tank effluent can improve the hydraulic function and performance of infiltration systems and constructed wetlands. By intermittent spray application of septic tank effluent onto a coarse-grained filter media, an unsaturated flow regime beneficial for pathogen removal is created. A column filtration study showed an increase in PRD-1 removal by time of operation with corresponding biofilm accumulation in the filter material. The same increased removal was observed for 1 mum polystyrene beads, irrespective of their hydrophilic/hydrophobic surface properties. A control experiment with sorption of 1 mum hydrophobic and hydrophilic polystyrene beads to different glass surfaces with hydrophobic and hydrophilic properties indicate that mechanisms other than hydrophobic interactions may govern the rate of attachment to the filter media. For a given volumetric flow-rate in the columns, the presence of biofilm altered the hydrodynamic characteristics and this resulted in increased retention time and particle removal.

Energy forest irrigated with wastewater: a comparative microbial risk assessment.

In this study, risks for human infection associated with irrigation of municipal wastewater on short rotation willow coppice (Salix) were evaluated in three countries. The aim was also to determine the reduction of indicator organisms and pathogens in the treatment plants. Two of the field sites were chosen for further evaluation by QMRA (quantitative microbial risk assessment) applied to three scenarios: accidental ingestions of wastewater, exposure to aerosols and ingestion of groundwater. The risks of infection for bacteria (Salmonella), virus (rotavirus) and protozoa (Giardia, Cryptosporidium) were characterised as probability of infections per exposure and number of infections per year.The highest risk for infection was associated with exposure to rotavirus in Culmore (Northern Ireland), by either accidental ingestion of wastewater or ingestion of groundwater (P(inf) 8 x 10(-1)). For Kvidinge (Sweden) the risk for virus infection by ingestion of wastewater were in the same range (P(inf) 7 x 10(-1)). The risk for Giardia infection differed between the two sites due to differences in concentration of this pathogen in the wastewater. The groundwater was found to have suffered faecal contamination due to the wastewater irrigation. Use of partially treated wastewater for irrigation of energy crops could be a sustainable option if site-specific recommendations are developed.

Variability analysis of pathogen and indicator loads from urban sewer systems along a river.

The pathogen loads within surface waters originating from urban wastewater sources needs to be assessed to support drinking water risk estimations and optimal selection of risk reduction measures. Locally reported discharges from sewer systems (>100,000 persons connected) were used to simulate the potential microbial loads into the Göta älv river, Sweden. Using Monte Carlo simulations, the median and 95% percentile (i.e. worst case) of total microbial load from wastewater treatment plants, sewer network overflows and emergency discharges were assessed and presented for dry and wet weather conditions. Wastewater treatment plants with secondary treatment represented a major source of E. coli, norovirus, Giardia and Cryptosporidium. During wet weather, comparably high microbial loads were found for sewer overflows due to heavy rains. Substantial loads were also associated with an incident of the emergency discharge of untreated wastewater. Simulated river water concentrations of faecal indicators (E. coli, sulfite reducing clostridia, somatic coliphages) and pathogens (norovirus, Giardia, Cryptosporidium) were confirmed by river sampling data, suggesting that urban wastewater is the major microbial source for this river.

Identification and management of microbial contaminations in a surface drinking water source.

Microbial contamination of surface waters constitutes a health risk for drinking water consumers which may be lowered by closing the raw water intake. We have evaluated microbial discharge events reported in the river Göta älv, which is used for raw water supply to the city of Göteborg. Elevated levels of faecal indicator bacteria were observed during periods of closed raw water intake. High bacteria levels were, however, also occasionally detected during periods of open intake, probably as a result of microbial discharge far upstream in the river which may be difficult to predict and manage by closing the intake. Accumulated upstream precipitations, resulting in surface runoff and wastewater contaminations in the catchment, correlated positively with the levels of total coliforms, E. coli, intestinal enterococci and sulfite-reducing clostridia. Levels of faecal indicator organisms were negatively correlated to the water temperature due to enhanced survival at lower temperatures. Wastewater discharges from a municipality located just upstream of the water intake resulted in elevated E. coli concentrations downstream at the raw water intake for Göteborg. To improve the prediction of microbial contaminations within the river Göta älv, monitoring data on turbidity and upstream precipitation are of particular importance.

Evaluation of the microbial risk reduction due to selective closure of the raw water intake before drinking water treatment.

Short-term peaks in pathogen concentrations may increase the risks for waterborne diseases considerably. In this study the occurrence of indicator organisms and pathogens in the river Göta älv at the raw water intake to Göteborg was evaluated and related to risk for drinking water consumption. About half of the 24 pathogen samples, taken during event and non-event conditions, were positive for at least one of the following: Cryptosporidium, Giardia, norovirus, enterovirus, Campylobacter and E. coli O157. Positive pathogen detects were often associated with heavy rainfalls and viruses with a sewage emergency discharge. The annualised probability of infection from this type of event was calculated from pathogen concentrations in a QMRA model. Given that the water intake is not closed, the risk given present water treatment seems to be acceptable for Giardia; however, it is at a borderline for Cryptosporidium and insufficient for noro- and enteroviruses. Present results emphasise the need for an appropriate intake regulation with respect to high pathogen loads, as the risk increases with time of exposure to pathogen contaminants. Rather than a threshold level on E. coli, reports on upstream microbial discharges are valuable for quick pathogen indications.

Removal of noro- and enteroviruses, Giardia cysts, Cryptosporidium oocysts, and fecal indicators at four secondary wastewater treatment plants in Sweden.

The aim of this study was to investigate variation in the occurrence and removal of enteroviruses, noroviruses, Giardia cysts, Cryptosporidium oocysts, and the most commonly used fecal indicators in four Swedish secondary wastewater treatment plants (WWTPs). Paired samples were taken from the inlet and outlet of each WWTP. (Oo)cysts and indicators were enumerated with standard methods and viruses with a reverse transcriptase polymerase chain reaction. Giardia cysts and enteroviruses were constantly detected (mean numbers were 10(3.31) cysts and 10(4.44) polymerase chain reaction (PCR) units L(-1), respectively). Oocysts were found in 5 out of 19 samples (mean number was 20 L(-1)). Noroviruses were found between November and February, with an average titer of 10(3.29( L(-1). Mean cyst removal was 2.6 log, while noroviruses and enteroviruses were removed by 0.9 and 1.3 log, respectively. There was no correlation between the removals of pathogens and indicators (p > 0.05). Coliphage removal resembled human viral removal better than did F-specific phage.

Removal of viruses, parasitic protozoa and microbial indicators in conventional and membrane processes in a wastewater pilot plant.

The aim of this study was to investigate variations in the occurrence and removal of enterovirus and norovirus genomes, Giardia cysts, Cryptosporidium oocysts and the most commonly used faecal indicators in a Swedish wastewater pilot plant. Paired samples were taken from the inlet and outlet of each treatment line: tertiary filtration, membrane bioreactor (MBR) and upflow anaerobic sludge blankets (UASB). (Oo)cysts and indicators were enumerated using standard methods and viruses using RT-PCR. Giardia cysts and enteroviruses were constantly detected, mean numbers 10(3.11) cysts and 10(4.0) PCR units L(-1), respectively. Oocysts were found in 5/19 samples, mean number 5 L(-1). Noroviruses were found in 6/7 influent samples, with an average titre of 10(3.28)L(-1), during winter, but only in 2/15 in the rest of the year (mean 200 L(-1)). MBR treatment removed indicators more efficiently than did the other two lines, with 5log removal of E. coli. Human virus genome removal did not differ between the MBR and tertiary treatment line. Microorganism removal in UASB was significantly lower for all the organisms studied. E. coli, enterococci and Cl. perfringens removal was correlated (p<0.05) with enterovirus genome removal, with R-values around 0.4. However, values for removal of indicators were more strongly correlated to each other. Removal of viruses based on enumeration using RT-PCR probably underestimates infectious virion removal.

Biofilms in an urban water distribution system: measurement of biofilm biomass, pathogens and pathogen persistence within the Greater Stockholm Area, Sweden.

Distribution pipe biofilms can provide sites for the concentration of a wide range of microbial pathogens, thereby acting as a potential source of continual microbial exposure and furthermore can affect the aesthetic quality of water. In a joint project between Stockholm Water, the MISTRA "Sustainable Urban Water" program, the Swedish Institute for Infectious Disease Control and the Royal Technical University, Stockholm, the aim of the current study was to investigate biofilms formed in an urban water distribution system, and quantify the impact of such biofilms on potential pathogen accumulation and persistence within the Greater Stockholm Area, Sweden. When used for primary disinfection, ultra-violet (UV) treatment had no measurable influence on biofilm formation within the distribution system when compared to conventional chlorination. Biofilms produced within a model pilot-plant were found to be representative to those that had formed within the larger municipal water distribution system, demonstrating the applicability of the novel pilot-plant for future studies. Polystyrene microspheres (1.0 microm) and Salmonella bacteriophages demonstrated their ability to accumulate and persist within the model pilot-plant system, where the means of primary disinfection (UV-treatment, chlorination) had no influence on such phenomena. With the exception of aeromonads, potential pathogens and faecal indicators could not be detected within biofilms from the Stockholm water distribution system. Results from this investigation may provide information for water treatment and distribution management strategies, and fill key data gaps that presently hinder the refinement of microbial risk models.

QMRA (quantitative microbial risk assessment) and HACCP (hazard analysis and critical control points) for management of pathogens in wastewater and sewage sludge treatment and reuse.

Hazard Analysis and Critical Control Points (HACCP) was applied for identifying and controlling exposure to pathogenic microorganisms encountered during normal sludge and wastewater handling at a 12,500 m3/d treatment plant utilising tertiary wastewater treatment and mesophilic sludge digestion. The hazardous scenarios considered were human exposure during treatment, handling, soil application and crop consumption, and exposure via water at the wetland-area and recreational swimming. A quantitative microbial risk assessment (QMRA), including rotavirus, adenovirus, haemorrhagic E. coli, Salmonella, Giardia and Cryptosporidium, was performed in order to prioritise pathogen hazards for control purposes. Human exposures were treated as individual risks but also related to the endemic situation in the general population. The highest individual health risk from a single exposure was via aerosols for workers at the belt press for sludge dewatering (virus infection risk = 1). The largest impact on the community would arise if children ingested sludge at the unprotected storage site, although in the worst-case situation the largest number of infections would arise through vegetables fertilised with sludge and eaten raw (not allowed in Sweden). Acceptable risk for various hazardous scenarios, treatment and/or reuse strategies could be tested in the model.

Biofilms, thermophilic amoebae and Legionella pneumophila--a quantitative risk assessment for distributed water.

A simplistic quantitative microbial risk assessment (QMRA) based on the maximum risk curve (r = 1) was developed for Legionella within a water distribution system. Both biofilms and a thermophilic isolate of acanthamoebae were shown to increase the resistance of Legionella to conventional thermal disinfection by between one and two logs respectively. The level of risk presented to consumers was shown to exceed the USEPA 10(-4) benchmark in many cases tested. This was caused, in part, by the sensitivity of the risk model but also through a lack of reliable dose-response data for Legionella. Not withstanding this, the current study provided comparative information on the efficacy of conventional disinfection against Legionella. Combined chlorine was shown to reduce the risk of infection by as much as 1-log when compared to free chlorine, although thermal disinfection provided the most effective means of risk reduction. Biofilm detachment and the interaction of Legionella with acanthamoebae were two important ecological factors that significantly increased the risk of legionellosis, and thus should be further considered in the refinement of QMRA models.

The fate of legionellae within distribution pipe biofilms: measurement of their persistence, inactivation and detachment.

Distribution pipe biofilms present a currently unquantified public health risk to consumers receiving water for domestic potable and non-potable use. The aim of this study was to quantify the numbers of legionellae, used here as model bacterial pathogens, that may accumulate, persist within and detach from distribution pipe biofilms. L. pneumophila recovered by standard culture from an 8 week-old biofilm formed within a novel pilot-scale water distribution system represented 1% of those present in the adjacent bulk water. A combined chlorine concentration exceeding 0.2 mg x L(-1) eliminated culturable sessile legionellae altogether, though the reduction in FISH-positive cells represented just 75+/-25% of the original amount, compared to a 5-log reduction in culturable cells during the same period. Where there was < 0.1 mg x L(-1) combined chlorine, an exponential decay/loss of sessile L. pneumophila was observed (k = 0.37 - 0.41) over the course of a 38-day experimental period. The inoculation of the system with 1 microm fluorescent microspheres and legionellae demonstrated that removal of the latter was dominated by chemical disinfection, with erosion and biological grazing playing lesser roles. Under turbulent (Re approximately 5000) conditions, larger clusters of biofilm become detached from substrata, with more than 90% of sessile legionellae mobilised into the bulk water phase. Interaction with both biofilms and a thermophilic Acanthamoeba isolate reduced the susceptibility of legionellae to thermal inactivation by between one and two orders of magnitude, though it increased their sensitivity to chemical (free and combined chlorine) disinfection.

A theoretical approach to assess microbial risks due to failures in drinking water systems.

A failure in treatment or in the distribution network of a surface water-works could have serious consequences due to the variable raw water quality in combination with an extended distribution. The aim of this study was to examine the theoretical impact of incidents in the drinking water system on the annual risk of infection in a population served by a large water treatment plant in Sweden. Reported incidents in the system were examined and a microbial risk assessment that included three pathogens, Cryptosporidium parvum, rotavirus and Campylobacter jejuni, was performed. The main risk incidents in water treatment were associated with sub-optimal particle removal or disinfection malfunction. Incidents in the distribution network included cross-connections and microbial pollution of reservoirs and local networks. The majority of the annual infections were likely to be due to pathogens passing treatment during normal operation and not due to failures, thus adding to the endemic rate. Among the model organisms, rotavirus caused the largest number of infections. Decentralised water treatment with membranes was also considered in which failures upstream fine-pored membranes would have little impact as long as the membranes were kept intact.

Growth and reduction of microorganisms in sediments collected from a greywater treatment system.

AIMS: To study the effects of competitive microbiota, temperature and nutrient availability on Salmonella, Enterococcus, Campylobacter spores of sulphite reducing anaerobes and bacteriophages MS2 and phiX174 in sediments from a greywater treatment system. METHODS AND RESULTS: Standard culture methods were used. Bacteria died off rapidly under normal conditions (20 degrees C, competitive microbiota) but remained stable or grew in the other conditions studied. When the sediments became nutrient depleted after 2 weeks, a log-linear die-off was observed for Salmonella, which was higher at 20 degrees C than at 4 degrees C. Bacteriophage decay was shown to be log-linear from day 0, with T90 values ranging from 9 (phiX174, 20 degrees C) to 55 days (phiX174, 4 degrees C). The MS2 phage had a significantly higher decay rate in tyndallized sediments (T90 = 17 days) than in original sediments (T90 = 47 days) (P < 0.001), with temperature not shown to affect the decay rate. Spores of sulphite-reducing anaerobes were not significantly reduced during the study period (35 days). Campylobacter died-off rapidly or entered a viable but non-culturable state and subsequently results were not provided. CONCLUSIONS: Competition was the most important factor to suppress pathogenic bacterial growth in an eutrophic environment. When nutrient depleted conditions prevailed, temperature was more important and log-linear decay of microorganisms could be observed. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings suggest that the normally occurring microbiota will suppress pathogenic bacterial growth in nutrient rich sediments. With lower nutrient status, temperature is the more important factor in reducing pathogens.

Adequacy of in situ glass slides and direct sand extractions to assess the microbiota within sand columns used for drinking water treatment.

Historically, Cholodny-Rossi buried glass slide techniques have been used to study the microbiota of subsurface environments, yet the bias of such a technique has not been compared against direct sand extraction using modern in situ probing. Over a period of 34 wk, four separate 4-m-deep sand columns receiving raw lake water were examined to compare direct extraction of sand filter biofilm material against in situ glass slide biofilms. Significantly different DAPI direct counts and fluorescent in situ hybridization signals for major phylogenetic groups were observed. Not only were lower proportions (P < 0.001) of EUB338-probed DAPI cells observed on in situ glass slides, but also fewer gamma-Proteobacteria (12%-21%) and more alpha-Proteobacteria (16%-33%) when compared to direct sand extracts. Hence, investigators of the microbial ecology of even simple sand biofilms must consider the inherent biases from "accepted" methods and seek further independent methods to identify those which may be most accurate.