Early Warning Diagnostics for Emerging Infectious Diseases in Developing into Late-Stage Pandemics.

The spread of infectious diseases due to travel and trade can be seen throughout history, whether from early settlers or traveling businessmen. Increased globalization has allowed infectious diseases to quickly spread to different parts of the world and cause widespread infection. Posthoc analysis of more recent outbreaks-SARS, MERS, swine flu, and COVID-19-has demonstrated that the causative viruses were circulating through populations for days or weeks before they were first detected, allowing disease to spread before quarantines, contact tracing, and travel restrictions could be implemented. Earlier detection of future novel pathogens could decrease the time before countermeasures are enacted. In this Account, we examined a variety of novel technologies from the past 10 years that may allow for earlier detection of infectious diseases. We have arranged these technologies chronologically from pre-human predictive technologies to population-level screening tools. The earliest detection methods utilize artificial intelligence to analyze factors such as climate variation and zoonotic spillover as well as specific species and geographies to identify where the infection risk is high. Artificial intelligence can also be used to monitor health records, social media, and various publicly available data to identify disease outbreaks faster than traditional epidemiology. Secondary to predictive measures is monitoring infection in specific sentinel animal species, where domestic animals or wildlife are indicators of potential disease hotspots. These hotspots inform public health officials about geographic areas where infection risk in humans is high. Further along the timeline, once the disease has begun to infect humans, wastewater epidemiology can be used for unbiased sampling of large populations. This method has already been shown to precede spikes in COVID-19 diagnoses by 1 to 2 weeks. As total infections increase in humans, bioaerosol sampling in high-traffic areas can be used for disease monitoring, such as within an airport. Finally, as disease spreads more quickly between humans, rapid diagnostic technologies such as lateral flow assays and nucleic acid amplification become very important. Minimally invasive point-of-care methods can allow for quick adoption and use within a population. These individual diagnostic methods then transfer to higher-throughput methods for more intensive population screening as an infection spreads. There are many promising early warning technologies being developed. However, no single technology listed herein will prevent every future outbreak. A combination of technologies from across our infection timeline would offer the most benefit in preventing future widespread disease outbreaks and pandemics.

High occurrence of viable forms of Cryptosporidium and Giardia in domestic sewage from an agricultural region of Brazil.

Cryptosporidium and Giardia are the main etiologies of waterborne outbreaks caused by protozoa. These parasites are commonly detected in wastewater; however, there is little knowledge about the concentration of viable forms in treated sewage, mainly in small communities. To understand more about the presence of viable oocysts and cysts in domestic sewage, we monitored the affluent and effluent of a wastewater treatment plant (WWTP) in inner-city Brazil. Ten samplings and seven follow-ups were performed in 2020. Samples were concentrated by centrifugation, filtration and purified by fluctuation. Viability was accessed by propidium-monoazide (PMA) associated with nPCR and qPCR. Both viable protozoa were detected in all raw sewage samples (average: 438.5 viable oocysts/L). Regarding treated sewage, Cryptosporidium was detected in all of the samples (average: 92.8 viable oocysts/L) and Giardia was detected in 70% with viable cysts in 30%. Considering the follow-ups, 31.17% of Cryptosporidium viable oocysts remained in the effluent after the treatment. High amounts of Cryptosporidium and a high frequency of Giardia were detected, therefore both arrived at WWTP and were discharged into the river. These alert the presence of agro-industrial effluents into domestic sewage and demonstrated the effectiveness of the concentration technique for monitoring protozoa in wastewater.

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

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

Dead-End Ultrafiltration and DNA-Based Methods for Detection of Cyclospora cayetanensis in Agricultural Water.

Cyclospora cayetanensis is a protozoan parasite that causes foodborne and waterborne diarrheal illness outbreaks worldwide. Most of these outbreaks are associated with the consumption of fresh produce. Sensitive and specific methods to detect C. cayetanensis in agricultural water are needed to identify the parasite in agricultural water used to irrigate crops that have been implicated in outbreaks. In this study, a method to detect C. cayetanensis in water by combining dead-end ultrafiltration (DEUF) with sensitive and specific molecular detection was developed and evaluated. Triplicates of 10-liter agricultural water samples were seeded with 200, 100, 25, 12, and 6 C. cayetanensis oocysts. Surface water samples were also collected in the Mid-Atlantic region. All water samples were processed by DEUF and backflushed from the ultrafilters. DNA was extracted from concentrated samples and analyzed by quantitative PCR (qPCR) targeting the C. cayetanensis 18S rRNA gene. All water samples seeded with 12, 25, 100, and 200 oocysts were positive, and all unseeded samples were negative. Samples seeded with 6 oocysts had a detection rate of 66.6% (8/12). The method was also able to detect C. cayetanensis isolates in surface water samples from different locations of the Chesapeake and Ohio Canal (C&O Canal) in Maryland. This approach could consistently detect C. cayetanensis DNA in 10-liter agricultural water samples contaminated with low levels of oocysts, equivalent to the levels that may be found in naturally incurred environmental water sources. Our data demonstrate the robustness of the method as a useful tool to detect C. cayetanensis from environmental sources.IMPORTANCECyclospora cayetanensis is a protozoan parasite that causes foodborne and waterborne outbreaks of diarrheal illness worldwide. These foodborne outbreaks associated with the consumption of fresh produce and agricultural water could play a role in the contamination process. In this study, a method to detect C. cayetanensis in agricultural water by combining a robust filtration system with sensitive and specific molecular detection was developed and validated by the FDA. The results showed that this approach could consistently detect low levels of C. cayetanensis contamination in 10 liters of agricultural water, corresponding to the levels that may be found in naturally occurring environmental water sources. The method was also able to detect C. cayetanensis in surface water samples from a specific location in the Mid-Atlantic region. Our data demonstrate the robustness of the method to detect C. cayetanensis in agricultural water samples, which could be very useful to identify environmental sources of contamination.

Investigations from Northern Greece on mussels cultivated in areas proximal to wastewaters discharges, as a potential source for human infection with Giardia and Cryptosporidium.

Marine bivalves are usually cultivated in shallow, estuarine waters where there is a high concentration of nutrients. Many micro-pollutants, including the protozoan parasites Giardia duodenalis and Cryptosporidium spp., which also occur in such environments, may be concentrated in shellfish tissues during their feeding process. Shellfish can thus be considered as vehicles for foodborne infections, as they are usually consumed lightly cooked or raw. Therefore, the main objective of this study was to investigate the presence of both parasites in Mediterranean mussels, Mytilus galloprovincialis that are cultivated in Thermaikos Gulf, North Greece, which is fed by four rivers that are contaminated with both protozoa. Moreover, the occurrence of these protozoa was monitored in treated wastewaters from 3 treatment plants that discharge into the gulf. In order to identify potential sources of contamination and to estimate the risk for human infection, an attempt was made to genotype Giardia and Cryptosporidium in positive samples. Immunofluorescence was used for detection and molecular techniques were used for both detection and genotyping of the parasites. In total, 120 mussel samples, coming from 10 farms, were examined for the presence of both protozoa over the 6-month farming period. None of them were found positive by immunofluorescence microscopy for the presence of parasites. Only in 3 mussel samples, PCR targeting the GP60 gene detected Cryptosporidium spp. DNA, but sequencing was not successful. Thirteen out of 18 monthly samples collected from the 3 wastewater treatment plants, revealed the presence of Giardia duodenalis cysts belonging to sub-assemblage AII, at relatively low counts (up to 11.2 cysts/L). Cryptosporidium oocysts (up to 0.9 oocysts/L) were also detected in 4 out of 8 samples, although sequencing was not successful at any of the target genes. At the studied location and under the sampling conditions described, mussels tested were not found to be harboring Giardia cysts and the presence of Cryptosporidium was found only in few cases (by PCR detection only). Our results suggest that the likelihood that mussels from these locations act as vehicles of human infection for Giardia and Cryptosporidium seems low.

Helminth Egg Automatic Detector (HEAD): Improvements in development for digital identification and quantification of helminth eggs and their application online.

Helminths are parasitic worms that constitute a major public health problem. Conventional analytical techniques to evaluate helminth eggs in environmental samples rely on different steps, namely sedimentation, filtration, centrifugation, and flotation, to separate the eggs from a variety of particles and concentrate them in a pellet for direct observation under an optical microscope. To improve this process, a new approach was implemented in which various image processing algorithms were developed and implemented by a Helminth Egg Automatic Detector (HEAD). This allowed identification and quantification of pathogenic helminth eggs of global medical importance and it was found to be useful for relatively clean wastewater samples. After the initial version, two improvements were developed: first, a texture verification process that reduced the number of false positive results; and second, the establishment of the optimal thresholds (morphology and texture) for each helminth egg species. This second implementation, which was found to improve on the results of the former, was developed with the objective of using free software as a platform for the system. This does not require the purchase of a license, unlike the previous version that required a Mathworks® license to run. After an internal statistical verification of the system was carried out, trials in internationally recognized microbiology laboratories were performed with the aim of reinforcing software training and developing a web-based system able to receive images and perform the analysis throughout a web service. Once completed, these improvements represented a useful and cheap tool that could be used by environmental monitoring facilities and laboratories throughout the world; this tool is capable of identifying and quantifying different species of helminth eggs in otherwise difficult environmental samples: wastewater, soil, biosolids, excreta, and sludge, with a sensitivity and specificity for the TensorFlow (TF) model in the web service values of 96.82% and 97.96% respectively. Additionally, in the case of Ascaris, it may even differentiate between fertile and non-fertile eggs.

Contribution of hospitals to the occurrence of enteric protists in urban wastewater.

We assessed the potential contribution of hospitals to contaminations of wastewater by enteric protists, including Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi in raw wastewater. Wastewater samples were collected from storage tanks in two hospitals and one associated wastewater treatment plant in Shanghai, China, from March to November 2009. Enteric pathogens were detected and identified using PCR and DNA sequencing techniques. Among a total of 164 samples analyzed, 31 (18.9%), 45 (27.4%), and 122 (74.4%) were positive for Cryptosporidium spp., G. duodenalis, and E. bieneusi, respectively. Altogether, three Cryptosporidium species, four G. duodenalis assemblages, and 12 E. bieneusi genotypes were detected. Cryptosporidium hominis, G. duodenalis sub-assemblage AII, and E. bieneusi genotype D were the dominant ones in wastewater from both hospitals and the wastewater treatment plant. A similar distribution in genotypes of enteric pathogens was seen between samples from hospitals and the wastewater treatment plant, suggesting that humans are one of the major sources for these pathogens and hospitals are important contributors of enteric parasites in urban wastewater. Data from this study might be useful in the formulation of preventive measures against environmental contamination of waterborne pathogens.

Current Trends in the Application of Nanomaterials for the Removal of Emerging Micropollutants and Pathogens from Water.

Water resources contamination has a worldwide impact and is a cause of global concern. The need for provision of clean water is becoming more and more demanding. Nanotechnology may support effective strategies for the treatment, use and reuse of water and the development of next-generation water supply systems. The excellent properties and effectiveness of nanomaterials make them particularly suitable for water/wastewater treatment. This review provides a comprehensive overview of the main categories of nanomaterials used in catalytic processes (carbon nanotubes/graphitic carbon nitride (CNT/g-C3N4) composites/graphene-based composites, metal oxides and composites, metal-organic framework and commercially available nanomaterials). These materials have found application in the removal of different categories of pollutants, including pharmaceutically active compounds, personal care products, organic micropollutants, as well as for the disinfection of bacterial, viral and protozoa microbial targets, in water and wastewater matrices. Apart from reviewing the characteristics and efficacy of the aforementioned nanoengineered materials for the removal of different pollutants, we have also recorded performance limitations issues (e.g., toxicity, operating conditions and reuse) for their practical application in water and wastewater treatment on large scale. Research efforts and continuous production are expected to support the development of eco-friendly, economic and efficient nanomaterials for real life applications in the near future.

Bacteria and microeukaryotes are differentially segregated in sympatric wastewater microhabitats.

Wastewater purification is mostly performed in activated sludge reactors by bacterial and microeukaryotic communities, populating organic flocs and a watery liquor. While there are numerous molecular community studies of the bacterial fraction, those on microeukaryotes are rare. We performed a year-long parallel 16S rRNA gene and 18S rRNA-gene based analysis of the bacterial and of the microeukaryote communities, respectively, of physically separated flocs and particle-free liquor samples from three WWTPs. This uncovered a hitherto unknown large diversity of microeukaryotes largely composed of potential phagotrophs preferentially feeding on either bacteria or other microeukaryotes. We further explored whether colonization of the microhabitats was selective, showing that for both microbial communities, different but often closely taxonomically and functionally related populations exhibiting different dynamic patterns populated the microhabitats. An analysis of their between plants-shared core populations showed the microeukaryotes to be dispersal limited in comparison to bacteria. Finally, a detailed analysis of a weather-caused operational disruption in one of the plants suggested that the absence of populations common to the floc and liquor habitat may negatively affect resilience and stability.

Precipitation effects on parasite, indicator bacteria, and wastewater micropollutant loads from a water resource recovery facility influent and effluent.

The variability of fecal microorganisms and wastewater micropollutants (WWMPs) loads in relation to influent flow rates was evaluated for a water resource recovery facility (WRRF) in support of a vulnerability assessment of a drinking water source. Incomplete treatment and bypass discharges often occur following intense precipitation events that represent conditions that deviate from normal operation. Parasites, fecal indicator bacteria, and WWMPs concentrations and flow rate were measured at the WRRF influent and effluent during dry and wet weather periods. Influent concentrations were measured to characterize potential bypass concentrations that occur during wet weather. Maximum influent Giardia and C. perfringens loads and maximum effluent Escherichia coli and C. perfringens loads were observed during wet weather. Influent median loads of Cryptosporidium and Giardia were 6.8 log oocysts/day and 7.9 log cysts/day per 1,000 people. Effluent median loads were 3.9 log oocysts/day and 6.3 log cysts/day per 1,000 people. High loads of microbial contaminants can occur during WRRF bypasses following wet weather and increase with increasing flow rates; thus, short-term infrequent events such as bypasses should be considered in vulnerability assessments of drinking water sources in addition to the increased effluent loads during normal operation following wet weather.

Molecular analysis of Blastocystis sp. and its subtypes from treated wastewater routinely used for irrigation of vegetable farmlands in Iran.

Treated wastewater samples were collected, filtered using sterile 47-mm cellulose nitrate membrane and DNA extracted from the filtered materials. The presence of Blastocystis sp. was confirmed via polymerase chain reaction (PCR) targeting the SSU rRNA gene of Blastocystis sp. in 5/12 of samples. Based on the subtype analysis after sequencing, 2, 2 and 1 of ST2, ST6 and ST8 were detected among the isolates, respectively. Furthermore, both ST6s were allele 139, alleles 11 and 138 were identified in ST2 and the only ST8 was allele 95. The phylogenetic tree showed that one of ST2 was clustered together with those ST2 that were already reported from humans and animals. The presence of Blastocystis sp. in treated wastewater can indicate the potential role of this type of water for irrigation in the transmission of pathogenic microorganisms to downstream farmlands.

Evaluation of Occurrence, Concentration, and Removal of Pathogenic Parasites and Fecal Coliforms in Three Waste Stabilization Pond Systems in Tanzania.

In Tanzania, waste stabilization ponds (WSPs) are employed to treat wastewater, and effluents are used for urban agricultural activities. The use of untreated or partially treated wastewater poses risks of disease transmission, including parasitic and bacterial infections, to exposed communities. Little is known about the occurrence, concentration, and removal of parasites and fecal coliform (FC) bacteria in WSPs in Tanzania. This study evaluates the occurrence and concentration of parasites and FCs in wastewater, the efficiency of WSPs in removing parasites and FCs, and the validity of using FCs as an indicator of parasites. This was a cross-sectional study conducted between February and August 2018. Wastewater samples were collected from three WSPs located in the Morogoro, Mwanza, and Iringa regions. APHA methods were used to test physicochemical parameters. The modified Bailenger method and Ziehl-Neelsen stain were used to analyse parasites. Membrane filtration method was used to analyse FCs. Data were analysed using IBM SPSS version 20. Helminth egg removal ranged from 80.8% to 100%. Protozoan (oo)cyst removal ranged from 98.8% to 99.9%. The Mwanza WSP showed the highest FC reduction (3.8 log units (100 mL)-1). Both the parasites and FCs detected in the effluents of assessed WSPs were of higher concentrations than World Health Organization and Tanzania Bureau of Standards limits, except for helminths in the Morogoro WSP and FCs in the Mwanza WSP. FCs were significantly correlated with protozoa (p < 0.01) and predicted protozoa occurrence well (p=0.011). There were correlations between physicochemical parameters, parasites, and FC bacteria in the WSP systems. Inadequate performance of these systems may be due to lack of regular maintenance and/or systems operating beyond their capacity. FC indicators were observed to be a good alternative for protozoa monitoring, but not for helminths. Therefore, during wastewater quality monitoring, helminths should be surveyed independently.

Evaluation of a PCR Method for Detection of Entamoeba polecki, with an Overview of Its Molecular Epidemiology.

Entamoebapolecki is a parasite of human and nonhuman primates, other mammals, and birds. Due to overlapping morphological features, cysts of E. polecki may be confused with those of other Entamoeba species commonly found in human fecal samples, including immature cysts of Entamoeba histolytica Although the presence of E. polecki in human Entamoeba-positive stool samples may be rare, its prevalence is likely underestimated due to such confusion. Here, we give examples of diagnostic approaches applied so far and summarize data on the molecular epidemiology of E. polecki, including host specificity and phylogeography. Moreover, we evaluate a novel diagnostic conventional PCR developed for the screening of fecal samples for E. polecki The assay was highly sensitive and specific when used on genomic DNA extracted directly from stool and Swedish wastewater samples. The PCR enabled the identification of all four subtypes (ST1 to ST4) of E. polecki by PCR product sequencing. Most (23/28) subtyped E. polecki-positive samples detected in patients in Sweden between 2002 and 2015 reflected colonization by ST4 and were seen in travelers/foreigners. Two and three human cases of ST2 and ST3, respectively, were also detected. Subtypes 1, 2, and 3 were detected in 3/21 wastewater samples, suggesting local endemicity of these E. polecki subtypes; interestingly, ST4 was not detected in wastewater. In conclusion, the current PCR assay enables simple and cost-effective screening of fecal and wastewater samples for E. polecki Human cases of E. polecki appear to involve primarily ST4, while E. polecki detected in wastewater may be primarily of animal origin.

Soil-transmitted helminth infections associated with wastewater and sludge reuse: a review of current evidence.

OBJECTIVE: To review current evidence on infections related to the concentration of soil-transmitted helminth (STH) eggs in wastewater, sludge and vegetables irrigated with wastewater or grown on sludge-amended soils. METHOD: Search of Web of Science, Science Direct, PubMed and Google Scholar databases for publications reporting on STH egg concentration in wastewater, sludge and vegetables and for epidemiological studies on wastewater/sludge reuse and STH infections. RESULTS: STH egg concentrations were variable but high in wastewater and sludge especially in developing countries. They ranged from 6 to 16 000 eggs/L in wastewater and from 0 to 23 000 eggs/g in sludge and far exceed limits set in the WHO guideline for wastewater/sludge reuse. Numbers of STH eggs on vegetables ranged from 0 to 100 eggs/g. The concentration of STH eggs in wastewater, sludge and vegetables therefore relates to risks of infection through different exposure routes. CONCLUSION: Epidemiological evidence reveals an increased prevalence of STH infections associated with direct exposure to wastewater or sludge (farmers) and consumption of vegetables grown on soil treated with it. This calls for increased efforts to reduce the adverse health impact of wastewater and sludge reuse in line with the WHO multi-barrier approach.

Temperature-Dependence of Predator-Prey Dynamics in Interactions Between the Predatory Fungus Lecophagus sp. and Its Prey L. inermis Rotifers.

Temperature is considered an important factor that influences the bottom-up and top-down control in water habitats. We examined the influence of temperature on specific predatory-prey dynamics in the following two-level trophic system: the predatory fungus Lecophagus sp. and its prey Lecane inermis rotifers, both of which originated from activated sludge obtained from a wastewater treatment plant (WWTP). The experiments investigating the ability of conidia to trap rotifers and the growth of fungal mycelium were performed in a temperature range that is similar to that in WWTPs in temperate climate. At 20 °C, 80% of the conidia trapped the prey during the first 24 h, whereas at 8 °C, no conidium was successful. The mycelium growth rate was the highest at 20 °C (r = 1.44) during the first 48 h but decreased during the following 24 h (r = 0.98), suggesting the quickest use of resources. At a medium temperature of 15 °C, the tendency was opposite, and the r value was lower during the first 48 h. At 8 °C, the growth rate was very low and remained at the same level even though numerous active rotifers were potentially available for the fungus. The temperature also influences the production of new conidia; on the 7th day, new conidia were observed in 96% of the wells at 20 °C, but no new conidia were observed at 8°C. These results show that the prey (rotifers)-predator (Lecophagus) dynamics in WWTPs is temperature-dependent, and a temperature of 8 °C is a strongly limiting factor for the fungus. Moderate temperatures ensure the most stable coexistence of the fungus and its prey, whereas the highest temperature can promote the prevalence of the predator.

Interaction Between a Bacterivorous Ciliate Aspidisca cicada and a Rotifer Lecane inermis: Doozers and Fraggles in Aquatic Flocs.

Activated sludge is a semi-natural habitat composed of macroaggregates made by flocculating bacteria and inhabited by numerous protozoans and metazoans, creating a complicated interactome. The activated sludge resembles the biological formation of naturally occurring floc habitats, such as "marine snow." So far, these two types of habitat have been analyzed separately, despite their similarities. We examined the effect of a bacterivorous ciliate, Aspidisca cicada, on the quality of the macroaggregate ecosystem by estimating (i) the floc characteristics, (ii) the proliferation of other bacterivores (rotifers), and (iii) the chemical processes. We found that A. cicada (i) positively affected floc quality by creating flocs of larger size; (ii) promoted the population growth of the rotifer Lecane inermis, an important biological agent in activated sludge systems; and (iii) increased the efficiency of ammonia removal while at the same time improving the oxygen conditions. The effect of A. cicada was detectable long after its disappearance from the system. We therefore claim that A. cicada is a very specialized scavenger of flocs with a key role in floc ecosystem functioning. These results may be relevant to the ecology of any natural and engineered aggregates.

The detection of Entamoeba histolytica and Toxoplasma gondii in wastewater.

To increase current knowledge on the epidemiology of protozoan parasites in wastewater treatment plants (WWTP), the occurrence of Entamoeba histolytica and Toxoplasma gondii in raw and treated wastewater was investigated. Samples were collected from WWTP twice a month over a period of 8 months. Determination of protozoa was performed using polymerase chain reaction (PCR) and light microscopy. After concentration and purification of wastewater samples, DNA extraction was conducted followed by PCR amplification of small subunit ribosomal RNA (SSU rRNA) gene sequences of E. histolytica and B1 gene of T. gondii. Loop-Mediated Isothermal Amplification (LAMP) primer set was designed from E. histolytica hemolysin gene HLY6. Amplification of DNA in the LAMP mixture was monitored by naked eye as a blue color solution after addition of, hydroxynaphthol blue (HNB) to the reaction tube. Light microscopy revealed the presence of Entamoeba in all raw wastewater samples and treated water samples. PCR amplification of DNA products revealed that all, (9/9) wastewater samples were positive for Entamoeba. None was positive for Toxoplasma. These findings, which corroborate recent observations, indicate that E. histolytica may pose a public health risk.

Soil-transmitted helminth eggs assessment in wastewater in an urban area in India.

Water quality and sanitation are inextricably linked to prevalence and control of soil-transmitted helminth infections, a public health concern in resource-limited settings. India bears a large burden of disease associated with poor sanitation. Transformative onsite sanitation technologies are being developed that feature elimination of pathogens including helminth eggs in wastewater treatment. We are conducting third-party testing of multiple sanitation technology systems in Coimbatore (Tamil Nadu) India. To ensure stringent testing of the pathogen removal ability of sanitation technologies, the presence of helminth eggs in wastewater across the town of Coimbatore was assessed. Wastewater samples from existing test sites as well as desludging trucks servicing residential and non-residential septic tanks, were collected. The AmBic methodology (based on washing, sieving, sedimenting and floating) was used for helminth egg isolation. We tested 29 different source samples and found a 52% prevalence of potentially infective helminth eggs. Identification and enumeration of helminth species is reported against the septage source (private residential vs. shared toilet facility) and total solids content. Trichuris egg counts were higher than those of hookworm and Ascaris from desludging trucks, whereas hookworm egg counts were higher in fresh wastewater samples. Surprisingly, no correlation between soil transmitted helminth eggs and total solids was observed.

Investigating the bacterial community and amoebae population in rural domestic wastewater reclamation for irrigation.

Reclamation of domestic wastewater for agricultural irrigation is viewed as a sustainable option to create an alternative water source and address water scarcity. Free-living amoebae (FLA), which are amphizoic protozoa, are widely distributed in various environmental sources. The FLA could cause considerable environmental and health risks. However, little information is available on the risk of these protozoa. In this study, we evaluated the feasibility using rural domestic wastewater for agricultural irrigation, and analyzed dynamic changes of the microbial community structure and FLA populations in raw and treated wastewater, as well as the phyllosphere and rhizosphere of lettuce production sites that were irrigated with different water sources. The bacterial community dynamics were analyzed by terminal restriction fragment length polymorphism (T-RFLP). The bacterial community structures in the influent were similar to that in the effluent, while in some cases relative abundances varied significantly. The populations of Acanthamoeba spp. and Hartmannella vermiformis in the anaerobically treated wastewater were significantly higher than in the raw wastewater. The vegetables could harbor diverse amoebae, and the abundances of Acanthamoeba spp. and H. vermiformis in the rhizosphere were significantly higher than in the phyllosphere. Accordingly, our studies show insight into the distribution and dissemination of amoebae in wastewater treatment and irrigation practices.