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Wastewater surveillance (WWS) has been globally recognised to be a useful tool in quantifying SARS-CoV-2 RNA at the community and residential levels without biases associated with case-reporting. The emergence of variants of concern (VOCs) have given rise to an unprecedented number of infections even though populations are increasingly vaccinated. This is because VOCs have been reported to possess higher transmissibility and can evade host immune responses. The B.1.1.529 lineage (Omicron) has severely disrupted global plans to return to normalcy. In this study, we developed an allele-specific (AS) RT-qPCR assay which simultaneously targets the stretch of deletions and mutations in the spike protein from position 24-27 for quantitative detection of Omicron BA.2. Together with previous assays that detect mutations associated with Omicron BA.1 (deletion at position 69 and 70) and all Omicron (mutation at position 493 and 498), we report the validation and time series of these assays from September 2021 to May 2022 using influent samples from two wastewater treatment plants and across four University campus sites in Singapore. Viral RNA concentrations at the treatment plants corroborate with locally reported clinical cases, AS RT-qPCR assays revealed co-incidence of Omicron BA.1 and BA.2 on 12 January 2022, almost two months after initial BA.1 detection in South Africa and Botswana. BA.2 became the dominant variant by the end of January 2022 and completely displaced BA.1 by mid-March 2022. University campus sites were similarly positive for BA.1 and/or BA.2 in the same week as first detection at the treatment plants, where BA.2 became rapidly established as the dominant lineage within three weeks. These results corroborate clinical incidence of the Omicron lineages in Singapore and indicate minimal silent circulation prior to January 2022. The subsequent simultaneous spread of both variant lineages followed strategic relaxation of safe management measures upon meeting nationwide vaccination goals. Ministry of Education (MOE) National Environmental Agency (NEA) National Research Foundation (NRF) Published version This research is supported by the National Research Foundation, Prime Minister's Office, Singapore, under its Campus for Research Excellence and Technological Enterprise (CREATE) program funding to the Singapore Centre for Environmental Life Sciences Engineering (SCELSE) and the IntraCREATE Thematic Grant (Cities) grant NRF2019-THE001-0003a to SW & JT, the National Environmental Agency (NEA) of Singapore, and funding from the Singapore Ministry of Education and National Research Foundation through an RCE award to SCELSE.

Arboviruses are viral pathogens transmitted by blood-borne vectors that impose a great social and economic burden globally. Most clinical surveillance of arbovirus outbreaks underestimates the true prevalence as a large proportion of cases exhibit no or only mild clinical symptoms (i.e., are subclinical). Reports of urinary shedding of several arboviruses such as Dengue (DENV), yellow fever (YFV), and Zika (ZIKV) viruses suggest the possibility of utilizing wastewater surveillance to assess the prevalence of arboviral outbreaks. To determine the feasibility of wastewater surveillance, we investigated the decay of representative arboviruses (including DENV-2, DENV-3, YFV, and ZIKV) along with murine hepatitis virus (MHV) as a surrogate for human coronavirus within a wastewater matrix at 6, 25, or 37 °C using RT-qPCR. DENV-2, DENV-3, YFV, ZIKV, and MHV experienced a one log10reduction within 3.95-6.21 days at 25 °C and within 2.60-5.12 days at 37 °C, while incubation at 6 °C did not indicate substantial decay within 21 days. Our work suggests that these arboviruses or their RNA could persist sufficiently in wastewater over a range of temperatures, supporting the potential for wastewater-based surveillance of arboviral outbreaks. Ministry of Education (MOE) National Research Foundation (NRF) Singapore-MIT Alliance for Research and Technology (SMART) This research was supported by the National Research Foundation, Prime Minister’s Office, Singapore, under its Campus for Research Excellence and Technological Enterprise (CREATE) program, through Intra-CREATE Thematic Grant (Cities) Grant NRF2019-THE001-0003 to J.T. and E.J.A., the Singapore-MIT Alliance for Research and Technology (SMART) Antimicrobial Resistance Interdisciplinary Research Group (AMR IRG), and funding from the Singapore Ministry of Education and National Research Foundation through an RCE award to the Singapore Centre for Environmental Life Sciences Engineering (SCELSE).

On November 26, 2021, the World Health Organisation classified the B.1.1.529 SARS-CoV-2 variant as the Omicron variant of concern (VOC). Reports of higher transmissibility and potential immune evasion triggered flight bans and heightened health control measures across the world to stem its distribution. Wastewater-based surveillance has demonstrated to be a useful complement for community-based tracking of SARS-CoV-2 variants. Using design principles of our previous assays that detect VOCs (Alpha and Delta), here we report three allele-specific RT-qPCR assays that can quantitatively detect and discriminate the Omicron BA.1 and BA.2 variants in wastewater. The first assay targets the nine-nucleotide deletion at the L24-A27S of the spike protein for detection of BA.2. The second targets the six-nucleotide deletion at 69-70 of the spike protein for detection of the Omicron BA.1 variant, and the third targets the stretch of mutations from Q493R to Q498R for simultaneous detection of both Omicron BA.1 and BA.2. This method is open-sourced, can be implemented using commercially available RT-qPCR protocols, and would be an important tool for tracking the introduction and spread of the Omicron variants BA.1 and BA.2 in communities for informed public health responses.

Motivation Sanitation and Water for All (SWA) is a global partnership addressing challenges to universal water, sanitation, and hygiene (WaSH) access. Shortly following adoption of the United Nations' Sustainable Development Goals, the Research and Learning (R&L) constituency of SWA undertook a systematic study to determine global research priorities and learning needs. Purpose We aimed to identify priority topics where improved knowledge would aid achievement of Goal 6, by developing a global WaSH research agenda, and to describe evidence-use challenges among WaSH professionals. Approach and methods We delivered a tailored, semi-structured electronic questionnaire to representatives from countries, R&L institutions, and other SWA partners (external support agencies, civil society, and private sector). The survey gathered views from 76 respondents working in an estimated 36 countries across all world regions. Data were analyzed quantitatively and qualitatively to identify patterns and themes. Findings Most responses indicated lowered confidence on at least one Goal 6 target area, especially managing untreated wastewater and faecal sludge. Both brief and lengthy information formats were valued. WaSH information was perceived as conflicting or unreliable among non-R&L constituencies, suggesting differences in perceptions and information-seeking approaches. While the R&L constituency appeared saturated with learning and training opportunities, others perceived barriers to participating (e.g. not receiving notice or invitation). Research and other WaSH activities were frequently constrained by upward accountability to funders, while stakeholders were inconsistently included in research processes. Policy implications This study offers insight into perceived research and decision challenges related to Goal 6 targets. It develops a unified research agenda focused on high priority topics, and recommends renewed attention to evidence synthesis, learning and implementation support, research engagement, and multisectoral coordination.

Controlled struvite (NH4MgPO4·6H2O) precipitation has become a well-known process for nutrient recovery from wastewater treatment systems to alleviate the pressures of diminishing, finite rock phosphate reservoirs. Nonetheless, coprecipitation of potential microbial and chemical hazards is poorly understood. On the other hand, antimicrobial resistance (AMR) is a major global public health concern and wastewater is thought to disseminate resistance genes within bacteria. Fecal indicator bacteria (FIB) are typically used as measures of treatment quality, and with multiresistant E. coli and Enterococcus spp. rising in concern, the quantification of FIB can be used as a preliminary method to assess the risk of AMR. Focusing on struvite produced from full-scale operations, culture and qPCR methods were utilized to identify FIB, antibiotic resistance genes, and human enteric viruses in the final product. Detection of these hazards occurred in both wet and dry struvite samples indicating that there is a potential risk that needs further consideration. Chemical and biological analyses support the idea that the presence of other wastewater components can impact struvite formation through ion and microbial interference. While heavy metal concentrations met current fertilizer standards, the presence of K, Na, Ca, and Fe ions can impact struvite purity yet provide benefit for agricultural uses. Additionally, the quantified hazards detected varied among struvite samples produced from different methods and sources, thus indicating that production methods could be a large factor in the risk associated with wastewater-recovered struvite. In all, coprecipitation of metals, fecal indicator bacteria, antimicrobial resistance genes, and human enteric viruses with struvite was shown to be likely, and future engineered wastewater systems producing struvite may require additional step(s) to manage these newly identified public health risks.

The Delta (B.1.617.2) variant has caused major devastation in India and other countries around the world. First detected in October 2020, it has now spread to more than 100 countries, prompting WHO to declare it as a global variant of concern (VOC). The Delta (B.1.617.2), Delta plus (B.1.617.2.1) and Kappa (B.1.617.1) variants are all sub-lineages of the original B.1.617 variant. Prior to the inception of B.1.617, vaccine rollout, safe-distancing and timely lockdowns greatly reduced COVID-19 hospitalizations and deaths. However, the Delta variant, allegedly more infectious and for which existing vaccines seemed less effective, has catalyzed the resurgence of cases. Therefore, there is an imperative need for increased surveillance of the B.1.617 variants. While the Beta variant is increasingly outpaced by the Delta variant, the spread of the Beta variant remains of concern due to its vaccine resistance. Efforts have been made to utilize wastewater-based surveillance for community-based tracking of SARS-CoV-2 variants, however wastewater with its low SARS-CoV-2 viral titers and mixtures of viral variants, requires assays to be variant-specific yet accurately quantitative for meaningful interpretation. Following on the design principles of our previous assays for the Alpha variant, here we report allele-specific and multiplex-compatible RT-qPCR assays targeting mutations T19R, D80A, K417N, T478K and E484Q, for quantitative detection and discrimination of the Delta, Delta plus, Kappa and Beta variants in wastewater. This method is open-sourced and can be implemented using commercially available RT-qPCR protocols, and would be an important tool for tracking the spread of B.1.617 and the Beta variants in communities.

In the ongoing Second Decade of Action for Road Safety, road traffic crashes pose a considerable threat especially in low-income countries. Uganda shows a vast burden of non-fatal injuries and resides at the top range of countries with the highest death rates due to unsafe roads. However, little is known about the differences in road traffic associated injuries between urban and rural areas and potential influence factors. Here, we used a cross-sectional study conducted by a retrospective medical record review from trauma cases admitted in 2016 to hospitals in rural and urban areas in Uganda. Injury severity scores were calculated and descriptive analysis was carried out while multivariate logistic regression was applied to assess significant covariates. According to the 1683 medical records reviewed, the mean age of trauma patients in the dataset under investigation was 30.8 years with 74% male. The trauma in-hospital mortality was 4% while prevalence of traumatic injuries is 56.4%. Motorcycle users (49.6%) and pedestrians (33.7%) were identified as the most vulnerable groups in both urban and rural setting while mild injuries of extremities (61.6%) and the head/neck-region (42.0%) were registered most. The frequency of road traffic injuries was homogenous in the urban and rural hospitals investigated in this study, interventions should therefore be intensified ubiquitously. The identification of significant differences in road traffic crash and injury characteristics provides the opportunity for specific programmes to decrease the socio-economic and health burden of unsafe roads. In addition to law enforcement and introduction of a Systems Thinking approach to road safety including infrastructural and educational concepts, the strengthening of trauma care and health resources is recommended.

On November 26, 2021, the B.1.1.529 COVID-19 variant was classified as the Omicron variant of concern (VOC). Reports of higher transmissibility and potential immune evasion triggered flight bans and heightened health control measures across the world to stem its distribution. Wastewater-based surveillance has demonstrated to be a useful complement for clinical community-based tracking of SARS-CoV-2 variants. Using design principles of our previous assays that detect SARS-CoV-2 variants (Alpha and Delta), we developed an allele-specific RT-qPCR assay which simultaneously targets the stretch of mutations from Q493R to Q498R for quantitative detection of the Omicron variant in wastewater. We report their validation against 10-month longitudinal samples from the influent of a wastewater treatment plant in Italy. SARS-CoV-2 RNA concentrations and variant frequencies in wastewater determined using these variant assays agree with clinical cases, revealing rapid displacement of the Delta variant by the Omicron variant within three weeks. These variant trends, when mapped against vaccination rates, support clinical studies that found the rapid emergence of SARS-CoV-2 Omicron variant being associated with an infection advantage over Delta in vaccinated persons. These data reinforce the versatility, utility and accuracy of these open-sourced methods using allele-specific RT-qPCR for tracking the dynamics of variant displacement in communities through wastewater for informed public health responses. Ministry of Education (MOE) National Research Foundation (NRF) Published version This research is supported by the National Research Foundation, Prime Minister’s Office, Singapore, under its Campus for Research Excellence and Technological Enterprise (CREATE) program funding to the Singapore-MIT Alliance for Research and Technology (SMART) Antimicrobial Resistance Interdisciplinary Research Group (AMR IRG) and the Intra-CREATE Thematic Grant (Cities) grant NRF2019-THE001- 0003a to JT and EJA and funding from the Singapore Ministry of Education and National Research Foundation through an RCE award to Singapore Centre for Environmental Life Sciences Engineering (SCELSE). FW is supported by the Faculty Startup funding from the Center of Infectious Diseases at UTHealth and the UT system Rising STARs award.

Wastewater-based surveillance (WBS) has been widely used as a public health tool to monitor the emergence and spread of SARS-CoV-2 infections in populations during the COVID-19 pandemic. Coincident with the global vaccination efforts, the world is also enduring new waves of SARS-CoV-2 variants. Reinfections and vaccine breakthroughs suggest an endemic future where SARS-CoV-2 continues to persist in the general population. In this treatise, we aim to explore the future roles of wastewater surveillance. Practically, WBS serves as a relatively affordable and non-invasive tool for mass surveillance of SARS-CoV-2 infection while minimizing privacy concerns, attributes that make it extremely suited for its long-term usage. In an endemic future, the utility of WBS will include 1) monitoring the trend of viral loads of targets in wastewater for quantitative estimate of changes in disease incidence; 2) sampling upstream for pinpointing infections in neighborhoods and at the building level; 3) integrating wastewater and clinical surveillance for cost-efficient population surveillance; and 4) genome sequencing wastewater samples to track circulating and emerging variants in the population. We further discuss the challenges and future developments of WBS to reduce inconsistencies in wastewater data worldwide, improve its epidemiological inference, and advance viral tracking and discovery as a preparation for the next viral pandemic. Ministry of Education (MOE) National Research Foundation (NRF) This work was supported by the MIT Center for Microbiome Informatics and Therapeutics, funding from the Massachusetts Consortium on Pathogen Readiness (MassCPR) and China Evergrande Group (EJA), the National Research Foundation, Prime Minister’s Office, Singapore, under its Campus for Research Excellence and Technological Enterprise (CREATE) program funding to the Singapore-MIT Alliance for Research and Technology (SMART) Antimicrobial Resistance Interdisciplinary Research Group (AMR IRG), the Intra-CREATE Thematic Grant (Cities) grant NRF2019-THE001–0003a to JT and EJA and funding from the Singapore Ministry of Education and National Research Foundation through an RCE award to Singapore centre for Environmental Life Sciences Engineering (SCELSE) to SW and JT. FW is supported by the Faculty Startup funding from the Center of Infectious Diseases at UTHealth, the UT system Rising STARs award, and the Texas Epidemic Public Health Institute (TEPHI), which is housed within and supported administratively by The University of Texas Health Science Center at Houston (UTHealth).

Wastewater-based epidemiology (WBE) has emerged as a critical public health tool in tracking the SARS-CoV-2 epidemic. Monitoring SARS-CoV-2 variants of concern in wastewater has to-date relied on genomic sequencing, which lacks sensitivity necessary to detect low variant abundances in diluted and mixed wastewater samples. Here, we develop and present an open-source method based on allele specific RT-qPCR (AS RT-qPCR) that detects and quantifies the B.1.1.7 variant, targeting spike protein mutations at three independent genomic loci highly predictive of B.1.1.7 (HV69/70del, Y144del, and A570D). Our assays can reliably detect and quantify low levels of B.1.1.7 with low cross-reactivity, and at variant proportions between 0.1% and 1% in a background of mixed SARS-CoV-2. Applying our method to wastewater samples from the United States, we track B.1.1.7 occurrence over time in 19 communities. AS RT-qPCR results align with clinical trends, and summation of B.1.1.7 and wild-type sequences quantified by our assays strongly correlate with SARS-CoV-2 levels indicated by the US CDC N1/N2 assay. This work paves the path for rapid inexpensive surveillance of B.1.1.7 and other SARS-CoV-2 variants in wastewater.

The presence of biofilms in drinking water distribution systems (DWDS) can affect both water quality and system integrity; yet these systems remain poorly studied due to lack of accessibility. We established two independent full-scale DWDS Testbeds (A and B) on two different campuses situated in a tropical urban environment and equipped them with online sensors. Testbed B experienced higher levels of monochloramine and lower water age than Testbed A within the campus. Based on long amplicon-sequencing of bacterial 16S rRNA genes extracted from the mature biofilms (MPB) growing on pipes and young biofilms (YSB) growing on the sensors, a core community was identified in the two testbeds. The relative abundances of operational taxonomic units at the family level, including Mycobacteriaceae, Methylobacteriaceae, Rhodospirillaceae, Nitrosomonadaceae, and Moraxellaceae, were consistent for MPB and YSB on each campus. The MPB community was found to be influenced by conductivity, sample age, and pipe diameter as determined by both canonical correlation analysis and fuzzy set ordination. MPB displayed higher α-diversity based on Hill numbers than YSB; in general, second order Hill numbers correlated positively with conductivity and sample age, but negatively with ORP and nitrite. Pseudomonas spp. together with Bacillus spp. likely initiated biofilm formation of YSB on Testbed A under conditions of reduced monochloramine and high water age. Significant levels of orthophosphate were detected in YSB samples at two stations and associated with higher levels of stagnation based on long-term differential turbidity measurement (DTM). Orthophosphate and DTM may act as indicators of the biofilm growth potential within DWDS.Highlights- Established two testbeds to study biofilms in full-scale distribution system- Biofilms on pipes and sensors had core community- Temporal effect and higher α-diversity for biofilms on pipes- Water chemistry was related to biofilm community differencesGraphical Abstract

The critical need for surveillance of SARS-CoV-2 variants of concern has prompted the development of methods that can track variants in wastewater. Here, we develop and present an open-source method based on allele-specific RT-qPCR (AS RT-qPCR) that detects and quantifies the B.1.1.7 variant, targeting spike protein mutations at three independent genomic loci that are highly predictive of B.1.1.7 (HV69/70del, Y144del, and A570D). Our assays can reliably detect and quantify low levels of B.1.1.7 with low cross-reactivity, and at variant proportions down to 1% in a background of mixed SARS-CoV-2. Applying our method to wastewater samples from the United States, we track the occurrence of B.1.1.7 over time in 19 communities. AS RT-qPCR results align with clinical trends, and summation of B.1.1.7 and wild-Type sequences quantified by our assays matches SARS-CoV-2 levels indicated by the U.S. CDC N1 and N2 assays. This work paves the way for AS RT-qPCR as a method for rapid inexpensive surveillance of SARS-CoV-2 variants in wastewater. Ministry of Education (MOE) National Research Foundation (NRF) Published version This research is supported by the National Research Foundation, Prime Minister's Office, Singapore, under its Campus for Research Excellence and Technological Enterprise (CREATE) program, Intra-CREATE Thematic Grant (Cities) Grant NRF2019-THE001-0003a to J.T. and E. J.A., and funding from the Singapore Ministry of Education and National Research Foundation through an RCE award to the Singapore Centre for Environmental Life Sciences Engineering (SCELSE) to J.T. This work was also supported by funds from the Massachusetts Consortium on Pathogen Readiness and China Evergrande Group (M.M. and E.J.A.).

Fecal contamination of surface water is commonly evaluated by quantification of bacterial or viral indicators such as Escherichia coli and coliphages, or by direct testing for pathogens such as enteric viruses. Retention of fecally derived organisms in biofilms and sediments is less frequently considered. In this study, we assessed the distribution of E. coli, somatic coliphages, and enteric viruses including human adenovirus (HAdV), enterovirus (EV), norovirus genogroup GII (NoV GII) and group A rotavirus (RoV) in an urban river environment in Germany. 24 samples each of water, epilithic biofilms and sediments were examined. E. coli and somatic coliphages were prevalent not only in the flowing water, but also in epilithic biofilms and sediments, where they were accumulated compared to the overlying water. During enhanced rainfall, E. coli and coliphage concentrations increased by approximately 2.5 and 1 log unit, respectively, in the flowing water, whereas concentrations did not change significantly in epilithic biofilms and sediments. The occurrence of human enteric viruses detected by qPCR was higher in water than in biofilms and sediments. 87.5% of all water samples were positive for HAdV. Enteric viruses found less frequently were EV, RoV and NoV GII in 20.8%, 16.7% and 8.3% of the water samples, respectively. In epilithic biofilms and sediments, HAdV was found in 54.2% and 50.0% of the samples, respectively, and EV was found in 4.2% of both biofilm and sediment samples. RoV and NoV GII were not detected in any of the biofilms and sediments. Overall, the prevalence of enteric viruses was in the order of HAdV > EV > RoV ≥ NoV GII. In conclusion, epilithic biofilms and sediments can be reservoirs for fecal indicators and enteric viruses and thus should be taken into consideration when assessing microbial pollution of surface water environments.

Recreational, reclaimed and drinking source waters worldwide are under increasing anthropogenic pressure, and often contain waterborne enteric bacterial, protozoan, and viral pathogens originating from non-point source fecal contamination. Recently, the capsid integrity (ci)-qPCR, utilizing the azo-dyes propidium monoazide (PMA) or ethidium monoazide (EMA), has been shown to reduce false-positive signals under laboratory conditions as well as in food safety applications, thus improving the qPCR estimation of virions of public health significance. The compatibility of two widely used human adenovirus (HAdV) qPCR protocols was evaluated with the addition of a PMA/EMA pretreatment using a range of spiked and environmental samples. Stock suspensions of HAdV were inactivated using heat, UV, and chlorine before being quantified by cell culture, qPCR, and ci-qPCR. Apparent inactivation of virions was detected for heat and chlorine treated HAdV while there was no significant difference between ci-qPCR and qPCR protocols after disinfection by UV. In a follow-up comparative analysis under more complex matrix conditions, 51 surface and 24 wastewater samples pre/post UV treatment were assessed for enteric waterborne HAdV to evaluate the ability of ci-qPCR to reduce the number of false-positive results when compared to conventional qPCR and cell culture. Azo-dye pretreatment of non-UV inactivated samples was shown to improve the ability of molecular HAdV quantification by reducing signals from virions with an accessible genome, thereby increasing the relevance of qPCR results for public health purposes, particularly suited to resource-limited low and middle-income settings. Published version

Our surrounding environment has been influenced by the COVID-19 pandemic situation. The second wave of COVID-19 in India has proven to be more devastating and aggressive than the first wave of the pandemic, which led to recognizing India as one of the world's topmost worst-hit nations considering >4000 fatalities reported in a single day in May 2021. Such “resurgence and acceleration” of COVID-19 transmission has been fuelled by the MahaKumbh festival and political mass gathering (elections rallies) events, where the COVID-19 protocols have been ignored by millions of pilgrims/followers. The present review discusses only the consequences of this year's MahaKumbh festivals, the largest religious mass gathering on earth, which was held during the COVID-19 pandemic in India, and its impact on both the spread of SARS-CoV-2 among participants and their families and its influence on the quality of the river Ganga. This article tries to give readers outside of India an overview of how much impact of any such single large gathering of any relgion in any part of the world can drive coronavirus infections and effectively commence the second/third wave outbreak with this case study. Furthermore, the religious large scale celebration are widely accepted through out the world that have played a significant role in the spread of the pandemic into remote villages and towns all over the subcontinent/world, thus affecting many areas with insufficient healthcare facilities that have been relatively spared. This review also highlights the potential risk of transmission from infected humans into the aquatic environment of the river Ganga. Besides the obvious relevance of SARS-CoV-2, a large variety of other water-related disease vectors (bacteria, viruses, and protozoa) stemming from visitors to the religious congregation were introduced into the upstream regions of the Ganga river. Their sheer number is assumed to have had a severe influence on its delicate ecosystem, including endangered mammals such as the river Dolphins. The detailed epidemiological and clinical study on transmission routes of SARS-CoV-2 is the need of the hour to understand the pathogenesis of RNA virus infection and prevent the massive spreading of such infectious respiratory diseases. An interdisciplinary approach, rooted in evidence-based efficient learning, contextual strategies, and a streamlined unified approach should be adopted to help in the development of a proactive prevention model during future MahaKumbh festival (and similar religious gatherings) instead of just “picking up the pieces” in a conventional post-event model.

Pathogenic free-living amoebae (FLA), such as Naegleria fowleri, Balamuthia mandrillaris and Acanthamoeba species isolated from aquatic environments have been implicated in central nervous system, eye and skin human infections. They also allow the survival, growth and transmission of bacteria such as Legionella, Mycobacteria and Vibrio species in water systems. The purpose of this study was to investigate the co-occurrence of potentially pathogenic FLA and their associated bacteria in hospital water networks in Johannesburg, South Africa. A total of 178 water (n = 95) and swab (n = 83) samples were collected from two hospital water distribution systems. FLA were isolated using the amoebal enrichment technique and identified using PCR and 18S rDNA sequencing. Amoebae potentially containing intra-amoebal bacteria were lysed and cultured on blood agar plates. Bacterial isolates were characterized using the VITEK®2 compact System. Free-living amoebae were isolated from 77 (43.3 %) of the samples. Using microscopy, PCR and 18S rRNA sequencing, Acanthamoeba spp. (T3 and T20 genotypes), Vermamoeba vermiformis and Naegleria gruberi specie were identified. The Acanthamoeba T3 and T20 genotypes have been implicated in eye and central nervous system infections. The most commonly detected bacterial species were Serratia marcescens, Stenotrophomonas maltophilia, Delftia acidovorans, Sphingomonas paucimobilis and Comamonas testosteroni. These nosocomial pathogenic bacteria are associated with systematic blood, respiratory tract, the urinary tract, surgical wounds and soft tissues infections. The detection of FLA and their associated opportunistic bacteria in the hospital water systems point out to a potential health risk to immune-compromised individuals. Electronic supplementary material The online version of this article (doi:10.1007/s00436-016-5271-3) contains supplementary material, which is available to authorized users.

This study investigated the occurrence of free-living amoebae (FLA) in a public hospital in South Africa. A total of 97 water and biofilm samples from the municipal water inlet of the hospital, theatres, theatre sterilization service unit, central sterilization service unit, endoscopy/gastroscopy unit, intensive care unit and the renal unit were collected and examined for the presence of FLA using an amoebal co-culture and molecular techniques. Of the 97 samples, 77 (79.4%), 40 (52%) water and 37 (48.1%) biofilm, contained FLA. The genera Acanthamoeba, Vermamoeba (formerly Hartmanella) and Naegleria were detected by morphology, 18S rRNA PCR (polymerase chain reaction) and sequence analyses. Further sequence analysis of the five Acanthamoeba-positive isolates revealed a close resemblance with the potentially pathogenic T20 genotype. These results show a potential health risk to immuno-compromised patients and health care workers as some of the species detected are pathogenic and may harbor potential intracellular bacteria responsible for nosocomial infections. To date, this is the first report on the detection of potentially pathogenic amoebae from South African hospital water systems.

Despite the great sensitivity of PCR in monitoring enteric viruses in an aquatic environment, PCR detects viral nucleic acids of both infectious and noninfectious viruses, limiting the conclusions regarding significance for public health. Ethidium monoazide (EMA) and propidium monoazide (PMA) are closely related membrane impermeant dyes that selectively penetrate cells with compromised membranes. Inside the cells, the dye can intercalate into nucleic acids and inhibit PCR amplification. To assess whether EMA and PMA pretreatment is a suitable approach to inhibit DNA amplification from noninfectious viruses upon heat treatment, UV exposure or chlorine treatment, viruses were measured by qPCR, EMA-qPCR, PMA-qPCR and cell culture titration. EMA/PMA-qPCR of UV- and heat-treated viruses did not correlate with the results of the cell culture assay. However, the data from EMA/PMA-qPCR of chlorine-inactivated viruses was consistent with the cell culture infectivity assay. Therefore, a dye treatment approach could be a rapid and inexpensive tool to screen the efficacy of chlorine disinfection, but it is not able to distinguish between infectious and noninfectious viruses inactivated via heat treatment or UV irradiation. Indeed, different viruses may have different trends and mechanisms of inactivation; thus, the assay must be evaluated for each virus separately.