Removal of indigenous coliphages and enteric viruses during riverbank filtration from highly polluted river water in Delhi (India).

Emerging countries frequently afflicted by waterborne diseases require safe and cost-efficient production of drinking water, a task that is becoming more challenging as many rivers carry a high degree of pollution. A study was conducted on the banks of the Yamuna River, Delhi, India, to ascertain if riverbank filtration (RBF) can significantly improve the quality of the highly polluted surface water in terms of virus removal (coliphages, enteric viruses). Human adenoviruses and noroviruses, both present in the Yamuna River in the range of 10(5) genomes/100 mL, were undetectable after 50 m infiltration and approximately 119 days of underground passage. Indigenous somatic coliphages, used as surrogates of human pathogenic viruses, underwent approximately 5 log10 removal after only 3.8 m of RBF. The initial removal after 1 m was 3.3 log10, and the removal between 1 and 2.4 m and between 2.4 and 3.8 m was 0.7 log10 each. RBF is therefore an excellent candidate to improve the water situation in emerging countries with respect to virus removal.

Development and application of a one-step low cost procedure to concentrate viruses from seawater samples.

A novel and simple procedure for concentrating adenoviruses from seawater samples is described. The technique entails the adsorption of viruses to pre-flocculated skimmed milk proteins, allowing the flocs to sediment by gravity, and dissolving the separated sediment in phosphate buffer. Concentrated virus may be detected by PCR techniques following nucleic acid extraction. The method requires no specialized equipment other than that usually available in routine public health laboratories, and due to its straightforwardness it allows the processing of a larger number of water samples simultaneously. The usefulness of the method was demonstrated in concentration of virus in multiple seawater samples during a survey of adenoviruses in coastal waters.

Quantitative detection of enteroviruses in activated sludge by cell culture and real-time RT-PCR using paramagnetic capturing.

We have compared in extracts of activated sludge the number of enteroviruses detectable with buffalo green monkey (BGM) cell-cultures versus the number of enteroviral genomes determined by reverse-transcription quantitative real-time PCR (RT-qPCR). In order to find conditions adequate for quantifying enteroviral RNA isolated from (waste)water we have investigated affinity capture of RNA with polystyrene beads (Dynabeads). The capture efficiency strongly depended on the genomic region chosen for the affinity binding. Capture of the RNA by its 3'-tail was most efficient (almost 100%); other regions within the genome yielded variable but lower results. Indirect capture (first hybridization of the RNA to the oligonucleotides, then attachment of the duplex molecules to the beads) was much more efficient than direct capture (attachment of the oligonucleotides to the beads first, then binding of the RNA), and resulted in RNA capture of maximally 60-80%. At least partly, this was due to incomplete hybridization of the RNA to the complementary oligonucleotides. No correlation was found between the number of cytopathic effects (CPE) determined by cell culture and the number of genomes quantified by RT-qPCR; RT-qPCR values were consistently much higher than the number of CPE. This points to overestimation of infectious enteroviruses by RT-qPCR and/or underestimation by the cell culture approach.

[The EU Bathing Water Directive. Risk assessment and standards]. / Die Novelle der EU-Badegewässerrichtlinie. Aspekte der Risikobewertung bei der Grenzwertsetzung.

In an attempt to reduce the risk of infection in natural bathing waters the European Union is in the process of improving the Bathing Water Directive 76/160/EWG, which regulates the safety of such waters. The proposal contains several positive innovations which will improve the protection of the bathers: (1) health-related indicators, (2) harmonized detection methods, (3) requirements for active bathing water management, and (4) stricter standards for coastal waters. One of the most salient features of the current draft is the introduction of bacterial standards that are more stringent for coastal than for fresh waters. This decision on different standards seems unjustified: it was taken solely on the grounds that in two epidemiological studies-one carried out in coastal, the other in fresh waters-the maximum excess rate of gastroenteritis among bathers in coastal waters was higher than among bathers in fresh waters. However, it was not taken into account that the concentrations of bacterial indicators at which the gastroenteritis rate began to increase was nearly identical in both studies. The ratio between the standard concentrations of E. coli and intestinal enterococci in the draft was set at 2.5. This value does not correspond to the ratio found in German surface waters with low pollution levels, with ratios ranging from 2.7 to 4.0, and to the even higher ratios found in raw and treated sewage effluents. As a consequence in a majority of cases the non-compliance of bathing waters in Germany would be caused exclusively by a violation of E. coli standards. In assessing risks of infection it must also be taken into account that the adequacy of E. coli and intestinal enterococci for signaling the presence of viruses in water is far from optimal. The decay of viruses in water-estimated by the decay of bacteriophages-was found to be substantially slower than the die-off of indicator bacteria.

Contribution of the colmation layer to the elimination of coliphages by slow sand filtration.

River bank or slow sand filtration is a major procedure for processing surface water to drinking water in central europe. In order to model the performance of river bank and slow sand filtration plants, we are studying the different mechanisms by which the elimination of pathogens is realized. An important question concerning the mode of action of slow sand filters and river bank filtration units is the role of the colmation layer or "schmutzdecke" on the elimination of human pathogens. The schmutzdecke is an organic layer which develops at the surface of the sand filter short after the onset of operation. We have inoculated a pilot plant for slow sand filtration with coliphages and determined their rate of breakthrough and their final elimination. In the first experiment, with a colmation layer still missing, the breakthrough of the coliphages in the 80 cm mighty sandy bed amounted to ca. 40 %. In contrast, less than 1 % of coliphages escaped from the filter as the same experiment was repeated two months later, when a substantial colmation layer had developed. Our preliminary conclusions are that the colmation layer is extremely efficient in eliminating of viruses.

Persistence of heterologous nucleic acids after uptake by mammalian cells.

Plasmids containing the complete genome of poliovirus-1 are transcribed at random in transfected cells and give rise to infectious RNA molecules. These generate viruses which can be detected easily in a plaque assay. Using this system, we analyzed the persistence of the biologically active portion of transfected poliovirus cDNA by determining its infectious activity in mammalian cells. Transfection and the cultivation of the cells for up to 16 days were performed under the influence of guanidine or other drugs which inhibit plaque formation. Removal of the drug then allowed viral development to start at defined time points. We thus ensured that the reduction of plaques correlated with the decay of the transfected polio cDNA or the viral RNA synthesized exclusively from that DNA. We showed that the intracellular cDNA kept its full capacity to generate viruses for as long as 8 to 10 days post-transfection. After this time, this capacity declined, and no viruses were detected after 14 to 16 days. The plaque-producing activity depended primarily on the stability of the cDNA and its ability to be transcribed and not on the stability of the RNA transcripts, which decayed within hours.

Wastewater treatment and elimination of pathogens: new prospects for an old problem.

Although the development of wastewater treatment technology is more than one hundred years old, most wastewater treatment plants existing today do not eliminate pathogens satisfactorily. Even in highly developed nations, receiving waters, serving in many cases as drinking water resources, are contaminated with pathogens. Surface waters also contain large concentration of phosphate due to long lasting wastewater discharges. Cyanobacteria and algal overgrowth is the consequence. Present drinking water technology only partially overcomes the pollution; it can not be ruled out that drinking water originating from polluted resources contains pathogens. This situation frequently goes on unnoticed because current indicator organisms are not representative for all pathogens. As studies have shown that small concentrations of pathogens also pose a risk for the consumer health, this state of affairs is a matter of concern. Microfiltration technology is able to significantly eliminate bacteria and protists from wastewater. Viruses, although smaller than the pore size of the filters, are reduced too because, in wastewater, they are frequently bound to larger particles. If the microfiltration of wastewater is preceded by the addition of coagulants for the precipitation of phosphate, the precipitate will be retained by the filter. The effluent obtained contains very low concentrations of phosphate. As viruses also adsorb to the precipitate, the amount of viruses eliminated increases and with increasing amounts of coagulant they become undetectable.

[Reduction of bacteria and viruses in purification plant discharges using microfiltration in a model plant]. / Reduzierung von Bakterien und Viren in Klärwerksabläufen durch Mikrofiltration in einer Modellanlage.

Microfiltration through a membrane matrix of a nominal pore size of 0.2 microns has been applied for advanced treatment of mechanically and biologically treated wastewater. Elimination of bacteria and coliphages as well as the decrease in some chemical constituents were studied at a flow rate of 80 l/h. Microfiltration resulted in a reduction of E. coli, coliform bacteria, fecal streptococci and of coliphages by more than 4 logs in the filtrate. Thus, the quality requirements of EC Directives for bathing water (EC, 76/160) could be maintained. As a result of microfiltration, a 43 +/- 13% removing of the total phosphorous compounds (Pt) in the pre-filtered secondary effluent from 11 +/- 2 mmol/m3 (0.34 +/- 0.06 mg/l) to 6 +/- 2 mmol/m3 (0.19 +/- 0.06 mg/l) was measured. The dosage of FeCl3 (between 30 and 150 mmol/m3) as coagulant before microfiltration improved the reduction of Pt to result in an average value of 75 +/- 16%. After adding FeCl3, orthophosphates (PO4-P) could be efficiently reduced by microfiltration. Thus, PO4-P concentrations in effluent samples were, in most cases, below the detection limit (0.01 mmol/m3).

Differentiation of vaccine and wild-type polioviruses using polymerase chain reaction and restriction enzyme analysis.

Genomic amplification followed by selective digestion of restriction enzymes was used to differentiate polioviruses. The method was based on conserved and variable components of the 5'-noncoding region. The differences between Sabin vaccine and wild-type viruses made it possible to identify rapidly an isolated poliovirus as vaccine-related or wild-type virus. A total of 60 isolates and strains were tested and all of them were correctly identified. This method is recommended as a sensitive, specific and rapid way to differentiate polioviruses in clinical isolates and environmental samples.

[Inactivation of bacteria and coliphages in surface water highly polluted by secondary effluent and purified by flocculation and filtration by means of UV irradiation at a pilot plant scale]. / Inaktivierung von Bakterien und Coliphagen in abwasserbelastetem und durch Flockungsfiltration aufbereitetem Oberflächenwasser mittels UV-Bestrahlung in einer halbtechnischen Anlage.

The water of a channel in Berlin which is highly polluted by municipal sewage effluent is treated at the phosphate elimination plant (PEP) Tegel by flocculation and filtration in order to reduce eutrophication in the following Lake Tegel. The elimination of bacteria and coliphages in the effluent of the PEP was investigated in a scale pilot UV irradiation reactor installed at the outlet of the PEP Tegel. The influence of technical parameters such as flow rate and the arrangement of 23 UV lamps in the reactor on the inactivation was tested. The UV irradiation dose was calculated 119 mJ/cm2 and 49 mJ/cm2 at a flow rate of 50 m3/h and 120 m3/h, respectively and for an irradiation zone of 97.5 cm. The colony count of bacteria and concentrations of coliform organisms, E. coli, and feacal streptococci as well as the plaque forming units of coliphages in the influent of the UV reactor were reduced 2-3 lg units by an irradiation dose of 119 mJ/cm2. These elimination was found being only one lg unit at a UV irradiation dose of 49 mJ/cm2. The concentration of E. faecalis and Coliphages f2 seeded into the influent of the UV reactor decreased after UV irradiation by 119 mJ/cm2 by 2-4 lg units and 1-2 power of magnitude, respectively. A UV dose of 49 mJ/cm2 caused only a 90% elimination of E. faecalis and a 75% inactivation of Coliphages f2. Due to heterogenous distribution and the different retention period of the inflowing water in the irradiation zone, the inactivation of E. faecalis and Coliphages f2 was unequal. Both test organisms decreased in the middle of the reactor up to 2 lg units more than at the sides of the reactor. The hygienic-microbiological quality of a secondary effluent from sewage treatment plants can be improved by a combination of flocculation-filtration and UV irradiation due to their additive elimination effect. However, this UV reactor, which was tested under field conditions can only ensure the inactivation of bacteria and coliphages in the pretreated effluent, if more homogenous distribution of the inflowing water can be achieved. Further, the water must be irradiated by a higher UV dose.

Detection of naturally occurring enteroviruses in waters by reverse transcription, polymerase chain reaction, and hybridization.

Comparison in virus-seeded mineral water of three detection methods for enteroviruses, direct hybridization, cell culture, and reverse transcription into cDNA followed by polymerase chain reaction and hybridization, showed that the last procedure was 10 to 1,000 times more sensitive than detection by cell culture and 10(5) to 10(7) times more sensitive than direct hybridization. The presence of naturally occurring enteroviruses was also demonstrated in activated sludge and in concentrated and non-concentrated surface water samples by reverse transcription-polymerase chain reaction-hybridization. However, in activated sludge and in concentrated surface waters, enzymatic amplification was sometimes inhibited by contaminants.

Lack of evidence for strand-specific inhibition of poliovirus RNA synthesis by 3-methylquercetin.

Buffalo Green Monkey cells were infected with poliovirus-1 in the presence of 3-methylquercetin or guanidine, and the formation of positive- and negative-strand viral RNA was monitored using single-stranded RNA probes. Both 3-methylquercetin and guanidine prevented the formation of plus-strand as well as minus-strand viral RNA, although, due to the high multiplicity of infection used, a high number of genomic viral RNA was always present in the cells.

[Adsorption of selected enteroviruses on sand from a groundwater stratum: effect of pH, grain size, water solutes and organic contamination]. / Adsorption ausgewählter Enteroviren an Sand aus einem Grundwasserleiter: Einfluss von pH, Korngrösse, Wasserinhaltsstoffen und organischer Belastung.

Elimination of human pathogenic viruses during the filtration of contaminated water or during groundwater recharge with surface water relies a great deal on the adsorption and permanent binding of the viruses to the soil or to the aquifer material. In the present study we have investigated the influence of organic contamination, of grain size, and of the presence of several ions and other compounds on the adsorption of four enteroviruses to sand. Results showed an impaired adsorption of viruses in organic contaminated soil. Smaller sand grain sizes, specially the colloidally suspended particles, did bind viruses more efficiently than the larger ones. This points to the possibility of designing filters containing sand of reduced grain sizes where elimination of viruses is a main objective. Several cations increased the binding of viruses to sand and caused the viruses to aggregate. This property might be taken advantage of during coagulation-sedimentation processes, in order to optimize viral reduction.

[The ATP concentration of drinking water compared to the colony count]. / Die ATP-Konzentration von Trinkwasser im Vergleich zur Koloniezahl.

During a four months' period we have monitored the drinking water of the city of Berlin for its ATP-content and for its Total Colony Count. After concentrating the drinking water by a factor of 1000 by filtration, we obtained ATP-values which were always significantly above the blanks. The profile of the ATP-values roughly paralleled that of the Colony Counts; however, as we never observed Colony Counts nearly approaching 100 colonies/ml (which is the value not to be exceeded according to current German Law), we are unable to positively assess how the ATP-value would have behaved in samples with Colony Counts above 100 ml. Analysis of increasing dilutions of sewage tested for both Colony Counts and ATP indicate that values above 100 colonies/ml would have yielded ATP-values well above those normally seen in drinking water. Therefore, ATP could probably be taken as a measure for the Total Colony Count.