Viruses In Sludge Research Articles

Adapted methods for monitoring influenza virus and respiratory syncytial virus in sludge and wastewater

Wastewater-based surveillance constitutes a valuable methodology for the continuous monitoring of viral circulation, with the capacity to function as an early warning system. It holds particular significance in scenarios where respiratory viruses exhibit overlapping clinical presentations, as occurs with SARS-CoV-2, influenza virus (IV), and respiratory syncytial virus (RSV), and allows seasonal virus outbreaks to be distinguished from COVID-19 peaks. Furthermore, sewage sludge, given it harbors concentrated human waste from a large population, serves as a substantial reservoir for pathogen detection.To effectively integrate wastewater-based epidemiology into infectious disease surveillance, the detection methods employed in wastewater samples must be adapted to the distinct characteristics of sludge matrices. In this study, we adapted and applied protocols for the detection of IV and RSV in sewage sludge, comparing their performance with the results obtained in wastewater. To assess the efficiency of these protocols, sludge and wastewater samples were spiked with IV and RSV RNA, either free or incorporated in lentiviral particles. Samples were concentrated using the aluminum hydroxide adsorption-precipitation method before viral RNA extraction. Absolute virus quantification was carried out by RT-qPCR, including an internal control to monitor potential inhibitory factors. Recovery efficiencies for both free IV and RSV RNA were 60 % in sludge, and 75 % and 71 % respectively in wastewater, whereas the values for IV and RSV RNA in lentiviral particles were 16 % and 10 % in sludge and 21 % and 17 % in wastewater respectively. Additionally, the protocol enabled the quantification of naturally occurring IV and RSV in wastewater and sludge samples collected from two wastewater treatment plants during the winter months, thus affirming the efficacy of the employed methodologies.

Diversity and fate of human pathogenic bacteria, fungi, protozoa, and viruses in full-scale sludge treatment plants

Sewage sludge, as the by-product of urban sewage biological treatment, is a potential reservoir and source of pathogens. However, the distribution and fate of different pathogens during sludge treatment and disposal remain unclear. Here, we investigated the diversity and fate of human pathogenic bacteria, fungi, protozoa, and viruses in sludge from four full-scale sludge treatment plants (STPs) using high-throughput sequencing approaches. The results indicated that the diversity of pathogens in different STPs was different, which may be related to sludge characteristics and local epidemics. Sludge treatment steps differentially affected pathogens reduction, and anaerobic digestion (AD) reduced the relative abundance and richness of pathogens. AD combined with thermal hydrolysis pretreatment effectively reduced most pathogenic bacteria except for Clostridium perfringens. Sanitary performance of AD was also varied by substrate characteristics (high-solid or low-solid sludge) and compositions (mono-digestion or co-digestion). Resistance of pathogens to sludge treatment was type-dependent, and pathogenic protozoa and fungi showed greater resistance to AD. Meanwhile, some pathogenic bacteria and viruses (Adenovirus and Herpesvirus) were widespread in digestate, revealing a biological risk in disseminating pathogenicity. These findings reveal the diversity and fate of human pathogens during sludge treatment and disposal, which would be crucial in monitoring and controlling pathogen proliferation.

Interactions between virus surrogates and sewage sludge vary by viral analyte: Recovery, persistence, and sorption

Sewage sludge, as a reservoir of viruses, may pose threats to human health. Understanding how virus particles interact with sludge is the key to controlling virus exposure and transmission. In this study, we investigated the recovery, survivability, and sorption of four typical virus surrogates with different structures (Phi6, MS2, T4, and Phix174) in sewage sludge. The most effective elution method varies by viral analyte, while the ultrafiltration method could significantly reduce the recovery loss for all four viruses. Compared with nonenveloped viruses, the poor recoveries of Phi6 during elution (<15%) limited its efficient detection. The inactivation kinetics of four viruses in solid-containing sludge were significantly faster than those in solid-removed samples at 25 °C, indicating that the solid fraction of sludge played an important role in virus inactivation. Although enveloped Phi6 was more vulnerable in both solid-removed and solid-containing sludge samples, it could remain viable for several hours at 25 °C and several days at 4 °C, which may pose an infection risk during sludge collection, transportation, and treatment process. The adsorption and desorption behavior of viruses in sludge could be affected by virus envelope structure, capsid proteins, and virus particle size. Phi6 adsorption to sludge was great with log KF of 6.51 ± 0.53, followed by Phix174, MS2, and T4. Additionally, more than 95% of Phi6, MS2, and T4 adsorbed to sludge were strongly bound, and a considerable fraction of strongly-bound virus was confirmed to retain viability. These results shed light on the environmental behavior of viruses in sewage sludge and provide a theoretical basis for the risk assessment for sludge treatment and disposal.

Comprehensive investigation of SARS-CoV-2 fate in wastewater and finding the virus transfer and destruction route through conventional activated sludge and sequencing batch reactor

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA transmission route was thoroughly investigated in the hospital wastewater, sewage collection network, and wastewater treatment plants. Samples were taken on four occasions from December 2020 to April 2021. The performance of two different wastewater treatment processes of sequencing batch reactor (SBR) and conventional activated sludge (CAS) was studied for virus destruction. For this purpose, liquid phase, solid phase and bioaerosol samples were taken from different units of the investigated wastewater treatment plants (WWTPs). The results revealed that all untreated hospital wastewater samples were positive for SARS-CoV-2 RNA. The virus detection frequency increased when the number of hospitalized cases increased. Detection of viral RNA in the wastewater collection system exhibited higher load of virus in the generated wastewater in areas with poor socioeconomic conditions. Virus detection in the emitted bioaerosols in WWTPs showed that bioaerosols released from CAS with surface aeration contains SARS-CoV-2 RNA posing a potential threat to the working staff of the WWTPs. However, no viral RNA was detected in the bioaerosols of the SBR with diffused aeration system. Investigation of SARS-CoV-2 RNA in WWTPs showed high affinity of the virus to be accumulated in biosolids rather than transporting via liquid phase. Following the fate of virus in sludge revealed that it is completely destructed in anaerobic sludge treatment process. Therefore, based on the results of the present study, it can be concluded that receiving water resources could not be contaminated with virus, if the wastewater treatment processes work properly.

A comparison of two extraction methods for the detection of Enteroviruses in raw sludge

The aim of this study was to compare two viral extraction methods for the detection of naturally occurring Enteroviruses in raw sludge. The first method (M1) is based on an ultracentrifugation step. In the second one (M2), viral RNA was extracted directly after viral elution from suspended solids. Genomes of enteroviruses were quantified by a quantitative real time PCR (qRT-PCR) in sludge samples. Somatic coliphages and F-specific RNA phages, considered as viral indicators of enteric viruses in sludge, were enumerated by the double layer agar technique. Results showed that direct assay of RNA extraction yielded higher genomic copies of enteric viruses (with an average of 5.07Log10genomiccopies/100mL). After the ultracentrifugation assay in the second method, genomic copies number decreases (with an average of 4.39Log10genomiccopies/100mL). This can be explained by an eventual concentration of inhibitors existing in sludge samples. Phages enumeration results showed their presence in all sludge samples with an average of (5.69Log10PFU/100mL) for somatic coliphages and (4Log10PFU/100mL) for F-specific RNA phages. This emphasizes the use of somatic coliphages as viral indicators for enteroviruses in environmental samples and especially in raw sludge samples in wastewater treatment plants prior to agricultural use.

Enhanced virus recovery from municipal sewage sludge with a combination of enzyme and cation exchange resin

There is a great difficulty in virus enumeration in sewage sludge because viruses in sludge are firmly captured by sludge solids. In order to determine the precise number of viruses in sludge, an enhanced virus recovery method with a combination of an enzyme and a cation exchange resin (CER) was developed. Test viruses were seeded to a sample sludge obtained from a municipal wastewater treatment plant, and the sludge were incubated with various eluents. The quantity of eluted viruses in the liquid phase was then measured by the plaque assay technique. Using the eluent containing only water, CER, and CER with enzyme exhibited 0%, 19% and 39% of virus recovery, respectively. While the conventional USEPA method exhibited a virus recovery of 21%. Furthermore, viruses eluted by the eluent containing the CER and the lysozyme included not only surface-attached viruses but also solids-embedded viruses.

The detection of astrovirus in sludge biosolids using an integrated cell culture nested PCR technique.

The work presented here demonstrates the utility of the integrated cell culture-reverse transcriptase-polymerase chain reaction (ICC-RT-PCR) coupled with nested PCR to detect human astroviruses and enteroviruses in sludge biosolids. Viruses were concentrated by beef extract elution and organic flocculation prior to analysis by a plaque assay and ICC-RT-PCR. Astroviruses were detected in all but one sample and all of the samples were positive for enteroviruses. We have demonstrated the prevalence and frequency ofastrovirus in sludge and validated the ICC-RT-PCR/nested PCR technique as a useful tool to detect viruses in sludge.

Persistence of Viruses in Desert Soils Amended with Anaerobically Digested Sewage Sludge

Pima County, Ariz., is currently investigating the potential benefits of land application of sewage sludge. To assess risks associated with the presence of pathogenic enteric viruses present in the sludge, laboratory studies were conducted to measure the inactivation rate (k = log(10) reduction per day) of poliovirus type 1 and bacteriophages MS2 and PRD-1 in two sludge-amended desert agricultural soils (Brazito Sandy Loam and Pima Clay Loam). Under constant moisture (approximately -0.05 x 10 Pa for both soils) and temperatures of 15, 27, and 40 degrees C, the main factors controlling the inactivation of these viruses were soil temperature and texture. As the temperature increased from 15 to 40 degrees C, the inactivation rate increased significantly for poliovirus and MS2, whereas, for PRD-1, a significant increase in the inactivation rate was observed only at 40 degrees C. Clay loam soils afforded more protection to all three viruses than sandy soils. At 15 degrees C, the inactivation rate for MS2 ranged from 0.366 to 0.394 log(10) reduction per day in clay loam and sandy loam soils, respectively. At 27 degrees C, this rate increased to 0.629 log(10) reduction per day in clay loam soil and to 0.652 in sandy loam soil. A similar trend was observed for poliovirus at 15 degrees C (k = 0.064 log(10) reduction per day, clay loam; k = 0.095 log(10) reduction per day, sandy loam) and 27 degrees C (k = 0.133 log(10) reduction per day, clay loam; k = 0.154 log(10) reduction per day, sandy loam). Neither MS2 nor poliovirus was recovered after 24 h at 40 degrees C. No reduction of PRD-1 was observed after 28 days at 15 degrees C and after 16 days at 27 degrees C. At 40 degrees C, the inactivation rates were 0.208 log(10) reduction per day in amended clay loam soil and 0.282 log(10) reduction per day in sandy loam soil. Evaporation to less than 5% soil moisture completely inactivated all three viruses within 7 days at 15 degrees C, within 3 days at 27 degrees C, and within 2 days at 40 degrees C regardless of soil type. This suggests that a combination of high soil temperature and rapid loss of soil moisture will significantly reduce risks caused by viruses in sludge.

The Minimisation of Microbiological Hazards Associated with Latrine Wastes

Investigations of latrine wastes in Botswana were undertaken to verify that sludge stored in a pit latrine chamber for at least one year does not constitute an unacceptable microbiological hazard on handling or reuse. Levels of faecal bacteria, Ascaris, Taenia, Schistosoma and human enteric viruses in sludges which had been stored for periods of more than one year were compared with levels in improperly stored sludges. In all cases, the importance of long term storage was confirmed. However, it is noted that substantial sociological and educational inputs are required if latrine users are to obtain the maximum health and economic benefits of latrine sanitation and sludge reuse, particularly in cultures where there is traditionally little interest in such practices.

Detection of environmental viruses in sludge: Enhancement of enterovirus plaque assay titers with 5-iodo-2′-deoxyuridine and comparison to adenovirus and coliphage titers

Enteroviruses present in the primary sludge of two wastewater treatment plants were quantitated by plaque assay using a continuous African green monkey kidney cell line (BGM). Incubation of BGM monolayers with 5-iodo-2′-deoxyuridine (50 μg ml −1) for 4 days prior to use enhanced the number of PFU detected in 10 of 10 concentrated sludge samples. Coliphages also present in these samples were detected using as hosts E. coli C and E. coli A-19, an Hfr strain. E. coli C coliphage titers were consistantly higher than E. coli A-19 titers, and were 10 2-10 3 times higher than the enterovirus titers obtained with BGM cells. Adenoviruses present in the sludge samples were detected by immunofluorescence assay. Interestingly, those results suggested that adenoviruses were also more numerous than enteroviruses (as detected by either IDU-treated or untreated BGM plaque assay).

Low-temperature stability of viruses in sludges.

Enteroviruses survived for up to 38 days without diminishing in numbers in extended-aeration sludges maintained at 5 degrees C. In oxidation ditch sludges similarly maintained, enteroviruses survived for up to 17 days without diminishing in numbers. The pHs of the sludges in this study were well inside the pH 6 to 8 corridor in which destruction of enteroviruses by the detergents and ammonia present in sludges reportedly does not occur. Unexplained, however, was the survival of large numbers of enteroviruses in sludges at pH 3.5, a pH at which some anionic detergents commonly present in sewage are rapidly virucidal. The long survival of enteroviruses in these sludges at 5 degrees C indicates that such sludges can probably be stored under refrigeration in the laboratory for extended periods while awaiting processing without suffering significant losses in enterovirus numbers.

Fate of Human Enteric Viruses (Rotaviruses and Entero Viruses) in Sewage after Primary Sedimentation

A wastewater treatment plant, which treats sewage by either mechanical primary sedimentation or lime coagulation, was used for modelling the fate of human enteric viruses after these treatments. The occurrence of human rotaviruses and enteroviruses was monitored in sewage influent, effluent and sludge samples. The level of indigenous enteric viruses in raw sewage was very high (up to 10 000 I.U./l); the values for rotavxrus and enterovxrus were of the same magnitude, although no correlation was found between the levels of both kinds of human enteric viruses. Both rotaviruses and enteroviruses were removed from sewage and transferred to sludge at similar rates after mechanical sedimentation, although rotaviruses were more efficiently recovered from sludge samples than enteroviruses. After chemical coagulation by lime treatment, most human enteric viruses in sludge were inactivated. Lime coagulation should be regarded as a most effective single-step treatment for the removal of viral pathogens such as rotaviruses and enteroviruses from sewage.

Detection and Stability of Enteric Viruses in Sludge, Soil and Ground Water

Digested sludge from a municipal sewage treatment plant was systematically placed on the surface of pure diluvial sand in a forest plantation. In the area examined, sludge application started in September. Viruses could be demonstrated in the sludge deposits up to 21 weeks but not 22 weeks after the application. Polio-, coxsackie- and adenovirus types were detected. The method employed was elution with beef extract at pH 8 followed by hydroextraction. No virus was demonstrated in soil samples 5 weeks after sludge application. Ground water samples from bores established before sludge application were examined employing 5 litre samples, but these were all negative. Four larger samples (30 to 100 1) were examined. One 30 litre sample contained poliovirus type 2 and coxsackievirus type B3. It was collected in the 11th week after sludge application at a depth of 3 m under the surface. The 3 other samples were negative. A soil sample of 30 kg collected at a depth of 35 to 100 cm was brought to the laboratory. Aliquots of 300 g were suspended in ground water from the area in flasks of 500 ml. Antibiotics were added. The pH was 4.5. In the same way a system employing a sandy loam soil was set up. Here the pH was 7.2. In each series 3 flasks were seeded with coxsackievirus B3, adenovirus 1 and echovirus 7 respectively. The flasks were completely filled with water, stoppered airtight and kept dark at 4° to 7°C. During an 8 month period 20 ml samples were taken 7 times after mixing. The samples were centrifuged. The supernatant and eluates of the sediments were examined for virus. Adenovirus was not demonstrated in the supernatants, echovirus only in the one from the sandy soil, while coxsackievirus was demonstrated in both supernatants. Except for adenovirus in the sandy loam soil the viruses were quite stable in the sediments. Ground water samples seeded with the same 3 viruses were set up in parallel but without added soil. The results obtained from these water samples were quite similar to the sediment samples except for echovirus which could not be demonstrated in the ground water from the sandy soil area after 2 months.

Bacteria and Viruses in Aerobically Digested Sludge: Influence of Physical and Chemical Treatments on Survival and Association with Flocs under Laboratory Conditions

The effect of chemical (NaF, NaN3) and physical (heating, autoclaving) treatments of sludge flocs on bacteria and viruses in sludge undergoing aerobic digestion was studied as part of studies on the mechanisms of removal of these agents by aerobic waste treatment. Following either chemical or physical treatments, aerobically digested sludge flocs adsorbed only a small portion of added or indigenous bacteria. These treatments also reduced inactivation of bacteria as compared with untreated sludge flocs. In contrast, these treatments had relatively little effect on either adsorption of enteroviruses to sludge flocs or on inactivation of viruses. The treatments that decreased bacterial association with sludge flocs and decreased the inactivation rate of bacteria also reduced the number of active protozoans and other predators. It is concluded that enteric viruses and bacteria have different affinities for sludge flocs and are inactivated by different mechanisms during aerobic digestion of sludge.

Destruction by Anaerobic Mesophilic and Thermophilic Digestion of Viruses and Indicator Bacteria Indigenous to Domestic Sludges

In raw sludges and in mesophilically and thermophilically digested anaerobic sludges, large variations in numbers of viruses occurred over narrow ranges of numbers of fecal coliforms, total coliforms, and fecal streptococci, demonstrating that the bacteria were poor quantitative reflectors of the numbers of the viruses detected. Mesophilic and thermophilic digestion of anaerobic sludges destroyed all three indicator bacteria more rapidly than such digestion destroyed the viruses. The relative rates for the destruction of viruses, fecal coliforms, and fecal streptococci in the digested sludges were consistent over the 17-month study. Fecal coliforms were 7 to 8 times more sensitive than the viruses to mesophilic digestion and 9 to 10 times more sensitive to thermophilic digestion. Total coliforms were even more sensitive. The rates at which fecal streptococci were destroyed by mesophilic and thermophilic digestion of anaerobic sludges approached those at which the viruses were destroyed by those processes; this suggested that the rates at which fecal streptococci in sludges are destroyed by those processes may serve as useful indicators for the rates at which viruses in sludges are destroyed by those processes.

Development of quantitative methods for the detection of enteroviruses in sewage sludges during activation and following land disposal.

The development and evaluation of methods for the quantitative recovery of enteroviruses from sewage sludge are reported. Activated sewage sludge solids were collected by centrifugation, and elution of the solid-associated virus was accomplished by mechanical agitation in glycine buffer at pH 11.0. Eluted viruses were concentrated either onto an aluminum hydroxide floc or by association with a floc which formed de novo upon adjustment of the glycine eluate to pH 3.5. Viruses which remained in the liquid phase after lowering the pH of glycine eluate were concentrated by adsorption to and elution from membrane filters. The method of choice included high pH glycine elution and subsequent low pH concentration; it yielded an efficiency of recovery from activated sludge of 80% for poliovirus type 1, 68% for echovirus type 7, and 75% for coxsackievirus B3. This method was used to study the survival of naturally occurring virus in sludge at a sewage treatment plant and after subsequent land disposal of the solids after aerobic digestion. Reduction of enterovirus titers per gram (dry weight) of solids were modest during sludge activation but increased to a rate of 2 log 10/week after land disposal.

Inactivation of enteric viruses in wastewater sludge through dewatering by evaporation

The effect of dewatering on the inactivation rates of enteric viruses in sludge was determined. For this study, water was evaporated from seeded raw sludge at 21 degrees C, and the loss of viral plaque-forming units was measured. Initial results with poliovirus showed that recoverable infectivity gradually decreased with the loss of water until the solids content reached about 65%. When the solids content was increased from 65 to 83%, a further, more dramatic decrease in virus titer of greater than three orders of magnitude was observed. This loss of infectivity was due to irreversible inactivation of poliovirus because viral particles were found to have released their RNA molecules which were extensively degraded. Viral inactivation in these experiments may have been at least partially caused by the evaporation process itself because similar effects on poliovirus particles were observed in distilled water after only partial loss of water by evaporation. Coxsackievirus and reovirus were also found to be inactivated in sludge under comparable conditions, which suggests that dewatering by evaporation may be a feasible method of inactivating all enteric viruses in sludge.