The coagulation process for enveloped and non-enveloped virus removal in turbid water: Removal efficiencies, mechanisms and its application to SARS-CoV-2 Omicron BA.2.

The coagulation process has a high potential as a treatment method that can handle pathogenic viruses including emerging enveloped viruses in drinking water treatment process which can lower infection risk through drinking water consumption. In this study, a surrogate enveloped virus, bacteriophage Փ6, and surrogate non-enveloped viruses, including bacteriophage MS-2, T4, ՓX174, were used to evaluate removal efficiencies and mechanisms by the conventional coagulation process with alum, poly­aluminum chloride, and ferric chloride at pH 5, 7, and 9 in turbid water. Also, treatability of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a recent virus of global concern by coagulation was evaluated as SARS-CoV-2 can presence in drinking water sources. It was observed that an increase in the coagulant dose enhanced the removal efficiency of turbidity and viruses, and the condition that provided the highest removal efficiency of enveloped and non-enveloped viruses was 50 mg/L of coagulants at pH 5. In addition, the coagulation process was more effective for enveloped virus removal than for the non-enveloped viruses, and it demonstrated reduction of SARS-CoV-2 Omicron BA.2 over 0.83-log with alum. According to culture- and molecular-based assays (qPCR and CDDP-qPCR), the virus removal mechanisms were floc adsorption and coagulant inactivation. Through inactivation with coagulants, coagulants caused capsid destruction, followed by genome damage in non-enveloped viruses; however, damage to a lipid envelope is suggested to contribute to a great extend for enveloped virus inactivation. We demonstrated that conventional coagulation is a promising method for controlling emerging and re-emerging viruses in drinking water.

Quantitative microbial risk assessment of the gastrointestinal risks to swimmers at Southeast Asian urban beaches using site-specific and combined autochthonous and fecal bacteria exposure data.

Recreational exposure to microbial pollution at urban beaches poses a health risk to beachgoers. The accurate quantification of such risks is crucial in managing beaches effectively and establishing warning guidelines. In this study, we employed a quantitative microbial risk assessment (QMRA) framework to assess marine water quality and estimate the risks associated with Vibrio parahaemolyticus, an autochthonous pathogen that causes gastrointestinal illnesses, and enterococci, a traditional fecal bacteria indicator. The microbial contamination levels of V. parahaemolyticus and enterococci were determined from 48 water samples collected at two beaches in Thailand during dry and wet seasons. The accidentally ingested water volumes were obtained through a survey involving 438 respondents. The probability of illness (Pill) was estimated using dose-response models and Monte Carlo simulation. The results revealed that enterococci posed a higher risk of illness than V. parahaemolyticus at all seven study sites. The median combined gastrointestinal (GI) risk from both bacteria at all sites met the US EPA risk benchmark of 0.036 and the 0.05 benchmark set by the WHO, but the 95th percentile risk data at all sites exceeded the benchmarks. This emphasizes the need for the continuous monitoring and management of microbial pollution at these sites. The site-specific exposure data showed higher estimated risks with increased variations compared to the WHO-referenced values, which highlights the significance of locally measured microbial concentrations and survey exposure data to avoid underestimation. Estimating the risks from recreational exposure to waterborne bacteria can inform beach management policies aimed at reducing public health risks to swimmers. The study findings improve the understanding of the risks associated with water recreation activities at Southeast Asian beaches and offer valuable insights for the development of water quality guidelines, which are crucial for the sustainable development of the blue economy.

Microbial source tracking using molecular and cultivable methods in a tropical mixed-use drinking water source to support water safety plans.

Microbial contamination deteriorates source water quality, posing a severe problem for drinking water suppliers worldwide and addressed by the Water Safety Plan framework to ensure high-quality and reliable drinking water. Microbial source tracking (MST) is used to examine different microbial pollution sources via host-specific intestinal markers for humans and different types of animals. However, the application of MST in tropical surface water catchments that provide raw water for drinking water supplies is limited. We analyzed a set of MST markers, namely, three cultivable bacteriophages and four molecular PCR and qPCR assays, together with 17 microbial and physicochemical parameters, to identify fecal pollution from general, human-, swine-, and cattle-specific sources. Seventy-two river water samples at six sampling sites were collected over 12 sampling events during wet and dry seasons. We found persistent fecal contamination via the general fecal marker GenBac3 (100 % detection; 2.10-5.42 log10 copies/100 mL), with humans (crAssphage; 74 % detection; 1.62-3.81 log10 copies/100 mL) and swine (Pig-2-Bac; 25 % detection; 1.92-2.91 log10 copies/100 mL). Higher contamination levels were observed during the wet season (p < 0.05). The conventional PCR screening used for the general and human markers showed 94.4 % and 69.8 % agreement with the respective qPCR results. Specifically, in the studied watershed, coliphage could be a screening parameter for the crAssphage marker (90.6 % and 73.7 % positive and negative predictive values; Spearman's rank correlation coefficient = 0.66; p < 0.001). The likelihood of detecting the crAssphage marker significantly increased when total and fecal coliforms exceeded 20,000 and 4000 MPN/100 mL, respectively, as Thailand Surface Water Quality Standards, with odds ratios and 95 % confidence intervals of 15.75 (4.43-55.98) and 5.65 (1.39-23.05). Our study confirms the potential benefits of incorporating MST monitoring into water safety plans, supporting the use of this approach to ensure high-quality drinking water supplies worldwide.

Wastewater monitoring in tourist cities as potential sentinel sites for near real-time dynamics of imported SARS-CoV-2 variants.

Wastewater-based epidemiology (WBE) complements the clinical surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants' distribution in populations. Many developed nations have established national and regional WBE systems; however, governance and budget constraints could be obstacles for low- and middle-income countries. An urgent need thus exists to identify hotspots to serve as sentinel sites for WBE. We hypothesized that representative wastewater treatment plants (WWTPs) in two international gateway cities, Bangkok and Phuket, Thailand, could be sentineled for SARS-CoV-2 and its variants to reflect the clinical distribution patterns at city level and serve as early indicators of new variants entering the country. Municipal wastewater samples (n = 132) were collected from eight representative municipal WWTPs in Bangkok and Phuket during 19 sampling events from October 2021 to March 2022, which were tested by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) using the US CDC N1 and N2 multiplex and variant (Alpha, Delta, and Omicron BA.1 and BA.2) singleplex assays. The variant detection ratios from Bangkok and Phuket followed similar trends to the national clinical testing data, and each variant's viral loads agreed with the daily new cases (3-d moving average). Omicron BA.1 was detected in Phuket wastewater prior to Bangkok, possibly due to Phuket's WWTPs serving tourist communities. We found that the Omicron BA.1 and BA.2 viral loads predominantly drove the SARS-CoV-2 resurgence. We also noted a shifting pattern in the Bangkok WBE from a 22-d early warning in early 2021 to a near real-time pattern in late 2021. The potential application of tourist hotspots for WBE to indicate the arrival of new variants and re-emerging or unprecedented infectious agents could support tourism-dependent economies by complementing the reduced clinical regulations while maintaining public health protection via wastewater surveillance.

SARS-CoV-2 RNA surveillance in large to small centralized wastewater treatment plants preceding the third COVID-19 resurgence in Bangkok, Thailand.

Wastewater surveillance for SARS-CoV-2 RNA has been a successful indicator of COVID-19 outbreaks in populations prior to clinical testing. However, this has been mostly conducted in high-income countries, which means there is a dearth of performance investigations in low- and middle-income countries with different socio-economic settings. This study evaluated the applicability of SARS-CoV-2 RNA monitoring in wastewater (n = 132) to inform COVID-19 infection in the city of Bangkok, Thailand using CDC N1 and N2 RT-qPCR assays. Wastewater influents (n = 112) and effluents (n = 20) were collected from 19 centralized wastewater treatment plants (WWTPs) comprising four large, four medium, and 11 small WWTPs during seven sampling events from January to April 2021 prior to the third COVID-19 resurgence that was officially declared in April 2021. The CDC N1 assay showed higher detection rates and mostly lower Ct values than the CDC N2. SARS-CoV-2 RNA was first detected at the first event when new reported cases were low. Increased positive detection rates preceded an increase in the number of newly reported cases and increased over time with the reported infection incidence. Wastewater surveillance (both positive rates and viral loads) showed strongest correlation with daily new COVID-19 cases at 22-24 days lag (Spearman's Rho = 0.85-1.00). Large WWTPs (serving 432,000-580,000 of the population) exhibited similar trends of viral loads and new cases to those from all 19 WWTPs, emphasizing that routine monitoring of the four large WWTPs could provide sufficient information for the city-scale dynamics. Higher sampling frequency at fewer sites, i.e., at the four representative WWTPs, is therefore suggested especially during the subsiding period of the outbreak to indicate the prevalence of COVID-19 infection, acting as an early warning of COVID-19 resurgence.

Integrated analyses of fecal indicator bacteria, microbial source tracking markers, and pathogens for Southeast Asian beach water quality assessment.

The degradation of coastal water quality from fecal pollution poses a health risk to visitors at recreational beaches. Fecal indicator bacteria (FIB) are a proxy for fecal pollution; however the accuracy of their representation of fecal pollution health risks at recreational beaches impacted by non-point sources is disputed due to non-human derivation. This study aimed to investigate the relationship between FIB and a range of culturable and molecular-based microbial source tracking (MST) markers and pathogenic bacteria, and physicochemical parameters and rainfall. Forty-two marine water samples were collected from seven sampling stations during six events at two tourist beaches in Thailand. Both beaches were contaminated with fecal pollution as evident from the GenBac3 marker at 88%-100% detection and up to 8.71 log10 copies/100 mL. The human-specific MST marker human polyomaviruses JC and BK (HPyVs) at up to 4.33 log10 copies/100 mL with 92%-94% positive detection indicated that human sewage was likely the main contamination source. CrAssphage showed lower frequencies and concentrations; its correlations with the FIB group (i.e., total coliforms, fecal coliforms, and enterococci) and GenBac3 diminished its use as a human-specific MST marker for coastal water. Human-specific culturable AIM06 and SR14 bacteriophages and general fecal indicator coliphages also showed less sensitivity than the human-specific molecular assays. The applicability of the GenBac3 endpoint PCR assay as a lower-cost prescreening step prior to the GenBac3 qPCR assay was supported by its 100% positive predictive value, but its limited negative predictive values required subsequent qPCR confirmation. Human enteric adenovirus and Vibrio cholerae were not found in any of the samples. The HPyVs related to Vibrio parahaemolyticus, Vibrio vulnificus, and 5-d rainfall records, all of which were more prevalent and concentrated during the wet season. More monitoring is therefore recommended during wet periods. Temporal differences but no spatial differences were observed, suggesting the need for a sentinel site at each beach for routine monitoring. The exceedance of FIB water quality standards did not indicate increased prevalence or concentrations of the HPyVs or Vibrio spp. pathogen group, so the utility of FIB as an indicator of health risks at tropical beaches maybe challenged. Accurate assessment of fecal pollution by incorporating MST markers could lead to developing a more effective water quality monitoring plan to better protect human health risks in tropical recreational beaches.

Performance of viral and bacterial genetic markers for sewage pollution tracking in tropical Thailand.

Identifying sewage contamination via microbial source tracking (MST) marker genes has proven useful for effective water quality management worldwide; however, performance evaluations for these marker genes in tropical areas are limited. Therefore, this research evaluated four human-associated MST marker genes (human polyomaviruses (JC and BK viruses [HPyVs]), bacteriophage crAssphage (CPQ_056), Lachnospiraceae Lachno3, and Bacteroides BacV6-21) for tracking sewage pollution in aquatic environments of Thailand. The viral marker genes, HPyV and crAssphage were highly sensitive and specific to sewage from onsite wastewater treatment plants (OWTPs; n = 19), with no cross-detection in 120 composite swine, cattle, chicken, duck, goat, sheep, and buffalo fecal samples. The bacterial marker genes, Lachno3 and BacV6-21, demonstrated high sensitivity but moderate specificity; however, using both markers could improve specificity to >0.80 (max value of 1.00). The most abundant markers in OWTP samples were Lachno3 and BacV6-21 (5.42-8.02 and nondetect-8.05 log10 copies/100 mL), crAssphage (5.28-7.38 log10 copies/100 mL), and HPyVs (3.66-6.53 log10 copies/100 mL), respectively. Due to their increased specificity, the abundance of viral markers were further investigated in environmental waters, in which HPyVs showed greater levels (up to 4.33 log10 copies/100 mL) and greater detection rates (92.7%) in two coastal beaches (n = 41) than crAssphage (up to 3.51 log10 copies/100 mL and 56.1%). HPyVs were also found at slightly lower levels (up to 5.10 log10 copies/100 mL), but at higher detection rates (92.6%), in a freshwater canal (n = 27) than crAssphage (up to 5.21 log10 copies/100 mL and 88.9%). HPyVs and crAssphage marker genes were identified as highly sensitive and specific for tracking sewage pollution in aquatic environments of Thailand. This study underlines the importance of characterizing and validating MST markers in host groups and environmental waters before including them in a water quality management toolbox.

Inactivation kinetics and efficiencies of UV-LEDs against Pseudomonas aeruginosa, Legionella pneumophila, and surrogate microorganisms.

To demonstrate the effectiveness of UV light-emitting diodes (UV-LEDs) to disinfect water, UV-LEDs at peak emission wavelengths of 265, 280, and 300 nm were adopted to inactivate pathogenic species, including Pseudomonas aeruginosa and Legionella pneumophila, and surrogate species, including Escherichia coli, Bacillus subtilis spores, and bacteriophage Qß in water, compared to conventional low-pressure UV lamp emitting at 254 nm. The inactivation profiles of each species showed either a linear or sigmoidal survival curve, which both fit well with the Geeraerd's model. Based on the inactivation rate constant, the 265-nm UV-LED showed most effective fluence, except for with E. coli which showed similar inactivation rates at 265 and 254 nm. Electrical energy consumption required for 3-log10 inactivation (EE,3) was lowest for the 280-nm UV-LED for all microbial species tested. Taken together, the findings of this study determined the inactivation profiles and kinetics of both pathogenic bacteria and surrogate species under UV-LED exposure at different wavelengths. We also demonstrated that not only inactivation rate constants, but also energy efficiency should be considered when selecting an emission wavelength for UV-LEDs.

Analysis of Hydroxyl Radicals and Inactivation Mechanisms of Bacteriophage MS2 in Response to a Simultaneous Application of UV and Chlorine.

The simultaneous application of UV and chlorine (expressed as UV/Cl2) as a water treatment method may be a good disinfection option for UV-resistant microorganisms, such as human adenoviruses (HAdVs). In this study, we developed two approaches using UV/Cl2: one to quantitate the OH• radicals based on the degradation of the probe compound para-chlorobenzoic acid (pCBA) and the other to use bacteriophage MS2 to understand the virus inactivation mechanisms in response to UV, chlorine and UV/Cl2 disinfection using reverse-transcription quantitative polymerase chain reaction (RT-qPCR), attachment and genome penetration assays. The results revealed that OH• radicals were produced at a concentration of 2.70 × 10-14 M in the UV/Cl2 treatment with a practical chlorine dose of 1 mg/L and with a minimum UV254 fluence of approximately 10 mJ/cm2, whereas UV or chlorine alone did not produce OH• radicals. In the UV/Cl2 treatment, synergistic effects on viral genome damage were observed, but were not directly due to OH• radicals. The ability of MS2 to penetrate the genome of the host bacteria was impaired, but its ability to attach to the host was not affected by the treatment. We concluded that the major cause of virus inactivation in response to UV/Cl2 was the damage to the viral genome caused by combination actions of chlorine species and OH• radicals.

Sequential and Simultaneous Applications of UV and Chlorine for Adenovirus Inactivation.

Adenoviruses are water-borne human pathogens with high resistance to UV disinfection. Combination of UV treatment and chlorination could be an effective approach to deal with adenoviruses. In this study, human adenovirus 5 (HAdV-5) was challenged in a bench-scale experiment by separate applications of UV or chlorine and by combined applications of UV and chlorine in either a sequential or simultaneous manner. The treated samples were then propagated in human lung carcinoma epithelial cells to quantify the log inactivation of HAdV-5. When the processes were separate, a fluence of 100 mJ/cm(2) and a CT value of 0.02 mg min/L were required to achieve 2 log inactivation of HAdV-5 by UV disinfection and chlorination, respectively. Interestingly, synergistic effects on the HAdV-5 inactivation rates were found in the sequential process of chlorine followed by UV (Cl2-UV) (p < 0.05, ANCOVA) in comparison to the separate processes or the simultaneous application of UV/Cl2. This implies that a pretreatment with chlorine may increase the sensitivity of the virus to the subsequent UV disinfection. In conclusion, this study suggests that the combined application of UV and chlorine could be an effective measure against adenoviruses as a multi-barrier approach in water disinfection.