Comparison of techniques for the detection of helminth ova in drinking water and wastewater.

Many countries use wastewater for irrigation. The World Health Organization established, as reuse guidelines, a maximum value of 1 helminth ovum/L for irrigation. Various techniques for enumerating helminth ova in water have been published. To determine the most adequate method for Mexico, four techniques were compared: the U.S. Environmental Protection Agency (U.S. EPA), membrane-filter, Leeds I, and Faust. Two types of water were used: drinking water and municipal wastewater effluent. Sensitivity, discrimination coefficients, precision, recovery efficiency, and cost were determined. In addition, several unseeded wastewater samples were analyzed. For drinking water, U.S. EPA and the membrane-filter techniques demonstrated comparable results; however, when wastewater was used, the membrane technique showed some deficiencies. Because the U.S. EPA technique may be used for samples with both high and low solids content, allows for the recovery of helminth ova with different specific gravities, and has the lowest total cost, it was selected as the best technique.

Liming as an advanced treatment for sludge sanitisation: helminth eggs elimination--Ascaris eggs as model.

The presence of helminth eggs (Ascaris eggs) in sewage sludge may constitute a sanitary risk when used as agricultural fertiliser. Sanitisation of sewage sludge can be achieved by treatment with quick lime, a process that destroys sludge pathogens in two ways: pH increase and temperature rise. Among the pathogens of epidemiological relevance, Ascaris eggs are the most resistant to liming, and, hence, may serve as indicators of hygienic quality of biosolids. This research aims at defining, between 50 degrees C and 60 degrees C, the time required in the case of limed sludge to obtain a product with a negligible level of viable Ascaris eggs. To achieve this objective, investigations on inactivation kinetics of Ascaris eggs were conducted in the following products: contaminated milk of lime; naturally contaminated sludge treated with slaked lime and heat; naturally contaminated sludge treated with quick lime; and sludge treated at full scale with quick lime. For the inactivation kinetics where a negligible level of Ascaris eggs was reached, the inactivation threshold was determined. Depending on the experimental situation, the inactivation threshold period was found to fluctuate between 5 and 75 min at 55 degrees C and between 1 and 8 min at 60 degrees C.

Parasite contamination of liquid sludge from urban wastewater treatment plants.

This study was performed on sludge samples from 20 wastewater treatment plants located in the north west of France with capacities of 1,000-20,000 inhabitant equivalents. The types of treatment studied were activated sludge low charge with and without denitrification. Respectively, 110 samples of fresh sludge and 84 samples of discharged sludge for spreading were analysed. Globally 78.6% of samples contained helminth eggs belonging to the cestodes (6.1%) and nematodes (93.9%). Most of the nematode eggs detected were viable with 135 positive samples. The distribution, according to genera, indicated a high prevalence of Toxocara eggs (77.4%) followed by Capillaria (13.2%), Trichuris (8.1%) and Ascaris (1.3%). For viable nematode eggs, the concentrations detected ranged from < 1 to 28/4 gDM for fresh sludge and from < 1 to 9.6/4 gDM for discharged sludge.

Optimised immunofluorescence procedure for enumeration of Cryptosporidium parvum oocyst suspensions.

The aim of this study was to evaluate an optimised immunofluorescence assay in terms of the variability of sets of counts for Cryptosporidium parvum oocyst suspensions and data recovery and the reliability of the procedure. A coefficient of variation (CV) of 10% was determined to be the maximum value acceptable for count variability. It was found that the optimised IFA tested provided a high precision for the sets of enumerations for suspensions containing 800-20,000 oocysts/mL. The procedure was found to be robust and providing high recovery level (96.3%). In terms of counting precision, the technique described here approaches the performance of flow cytometry and surpasses other manual techniques with a CV of 10% for a concentration close to 800 oocysts/mL. The procedure described is particularly suitable for the production of seed doses and for other applications requiring the titration of oocyst suspensions with a high degree of precision and accuracy.

Surface properties of Ascaris suum eggs: hydrophobic potential and Lewis acid-base interactions.

The cell surface properties of Ascaris suum eggs were investigated to determine the hydrophobic properties of eggs of different origins. Hydrophobic potential measurements were based on adhesion to hydrocarbons. Freshly laid eggs with or without the external coat and eggs that had been stored for 3 months at -20 degrees C were tested. The hydrophobic potential differed according to the hydrocarbon used (hexadecane or octane) and the eggs tested (stored or fresh). The hydrophobic potential with octane was 95% for fresh eggs and 72% for eggs stored at -20 degrees C. Tests with Ascaris eggs with and without an external coat showed similar hydrophobic characteristics; the presence or absence of this external structure did not appear to influence hydrophobic interactions. Acidic interactions also appeared to play no part in the adhesion of eggs to the surface of octane emulsions.

Experimental investigations and numerical modelling of Cryptosporidium parvum transport behaviour in aquifers.

The purpose of this study was to improve understanding of the potential for transfer of the protozoan pathogen Cryptosporidium parvum through aquifers to drinking water wells. Therefore, the factors characterising this transport were experimentally determined. We have developed a continuously recirculating column assay. The latter allows small amounts of C. parvum oocysts to be manipulated providing as much protection as possible from the risks of contamination. As the analysis of oocyst samples is time consuming, a numerical model, simulating the transport phenomena of oocysts under the experimental conditions of assays, was developed to establish the whole experimental curve of results using a small number of experimental points. The comparisons drawn between analytic solutions, experimental results with tracer (NaCl solution) and numerical simulation were in good agreement. A continuously recirculating column assay was performed using oocysts in suspension (flow rate = 1.43 mL/min). Treated sand was used as previous experiments had shown that no adsorption occurs. We observed almost total filtration (99.85%). To check this result, an assay with an open column was carried out under the same conditions. We observed a filtration value of 97%. Consequently, we may say that the continuously recirculating column assay provides satisfactory results.

Comparison of sludge and clinical isolates of Listeria monocytogenes.

AIMS: Listeria monocytogenes strains isolated in the same geographical area from sewage sludge and from patients presenting with listeriosis were compared. METHODS AND RESULTS: All isolates were typed by serotyping, phage typing and SmaI/ApaI pulsed-field gel electrophoresis (PFGE). Among the sludge isolates (n=32), 22 subtypes could be distinguished by the combination of all typing methods. The human isolates (n=11) were distributed into 10 subtypes which clearly differed from those observed among sludge isolates, except for one cluster formed by two related human isolates which showed high similarity in PFGE patterns (SmaI: 92%; ApaI: 89.5%) with one sludge isolate. CONCLUSION: These results suggest the existence of an epidemiological link between sludge and human isolates, but they may also be reflecting the distribution of L. monocytogenes types within the environment. SIGNIFICANCE AND IMPACT OF THE STUDY: Sludge and human L. monocytogenes may be related but further epidemiological studies are necessary to elucidate this point.

Irradiation of Ascaris ova in sludge using an electron beam accelerator.

The efficiency of sludge disinfection by irradiation was investigated using an electron beam accelerator, with the Ascaris ovum as a model. Ova suspensions prepared by worm dissection, immediately after preparation and after storage at 4 degrees C for 2 months were tested. Suspensions of ova extracted from slaughterhouse sludge were also tested. The ova were irradiated in sludge to determine, by probit analysis, the dose that inactivated 90% of viable ova. The D10 values obtained for irradiation of residual sludge contaminated with ova depended on the source of the ova, the D10 values were 788 +/- 172 Gy for suspensions of ova extracted from slaughterhouse sludge and 1125 +/- 145 Gy for suspensions freshly prepared by dissection. Ova suspensions freshly prepared by dissection were more proof against irradiation. Similarly, the D10 value was affected by storage: 1125 +/- 145 Gy for freshly produced ova suspensions and 661 +/- 45 Gy for suspensions of ova stored for 2 months at 4 degrees C in deionized water. The medium in which the ova were irradiated (deionized water or sludge) also affected D10 values, the indirect effects were smaller in samples of contaminated sludge, which were rich in organic matter, with the action of the radiation being mostly direct.

Monitoring of bacterial and parasitological contamination during various treatment of sludge.

Wastewater treatment results in the production of large quantities of sludge. Agricultural use is an interesting alternative provided that the health risks are taken into account. In France, a decree (1998) defines "sanitized" sludge as sludge that has been treated so that pathogens (Salmonella, enteroviruses, viable nematode eggs) are no longer detectable. The aim of this study was to determine the impact of various sludge treatments on 2 types of pathogens (Salmonella and nematode eggs). We also assessed the levels of micro-organisms that indicate faecal contamination (E. coli, Enterococci, sulfite-reducing anaerobes spores). Various treatment processes were analyzed: 4 biological, 3 chemical, 1 heat, 2 storage and 4 sampling campaigns were carried out. Concerning the parasitological contamination of sludge at the entrance of all sites, concentrations of total nematode eggs and viable nematode eggs were highly heterogeneous (respectively, from 2 to 53 eggs/10 g DM and from 2 to 45 eggs/10 g DM) with viable eggs present in all samples. The total concentration of nematode eggs in the sludge was not greatly affected by treatment or storage, and was in the range < 1-66 eggs/10 g DM. However, the situation was different for mean viable egg counts. For four treatments, no viable eggs were detected. Mesophilic processes are inefficient at eliminating viable nematode eggs. Salmonella were systematically detected in all sludge samples at the entry to the station, except for limed sludge intended for storage. At the end of four treatments Salmonella had been totally eliminated. For the fecal contamination indicators, the largest decrease in E. coli contamination was observed following liming and after thermophilic treatment. The mesophilic treatments resulted in very small decreases in contamination. A similar pattern was observed with Enterococci. For the spores of sulfite-reducing anaerobe bacteria, with the exception of heat treatment, which reduced contamination by 3.2 log units, the maximum decrease in contamination was 1.7 log. For storage of sludge, E. coli and Enterococci levels had decreased at first but then increased between 6 and 8 months of storage.

Determination of Giardia cyst viability in environmental and faecal samples by immunofluorescence, fluorogenic dye staining and differential interference contrast microscopy.

Amongst the techniques suggested for the determination of Giardia cyst viability, the use of the fluorogenic dyes, fluorescein diacetate (FDA) and propidium iodide (PI) is the most often recommended, even though it appears to overestimate the number of viable cysts. In the present study, the replacement of FDA with 4',6-diamidino-2-phenylindole (DAPI) allowed simultaneous direct immunofluorescence with monoclonal antibody labelled with fluorescein isothiocyanate (MAb-FITC). Under these conditions, it was possible both to quantify the cysts according to the immunofluorescence technique, and to appreciate their viability by using fluorogenic dye staining (DAPI and PI) and differential interference contrast (DIC) microscopy. This method proved to be significantly better than the counting methods normally suggested. The technique has been applied to Giardia cysts recovered from faeces and wastewater sludge.

Epidemiological study of intestinal helminthiasis in a Marrakech raw sewage spreading zone.

It has been suspected for some time that raw sewage is a potential source of protozoan and helminthic infections (10). Shuval et al. (16, 17) and W.H.O. (21) have shown that irrigating crops with untreated wastewater causes significant intestinal infections in both consumers and farm workers. This study compared the stools of "sewage farming" children exposed to raw sewage (El Azzouzia area) and those of a control group. Stool specimens from 253 exposed children and 275 controls were analyzed. 73% of the exposed were infected with one or more helminths verses 30% of the control group. The main parasites were Ascaris and Trichuris. The El Azzouzia children were more heavily infected and their polyparasitism index (PPI: 13%) was considerably higher than that of the controls (PPI: 2%). Thus "sewage farming" children are exposed to detectable risk from the parasitic nematodes in raw sewage.

[Treatment of sludge from purification stations with the purpose of ameliorating soils intended for agriculture: parasitic contamination and model development with a view to managing the sanitation risk]. / Valorisation des boues de stations d'épuration en vue de l'amélioration des sols destinés à l'agriculture: contamination parasitaire et modélisation en vue de la gestion du risque sanitaire.

Helminth eggs (Ascaris, Taenia...) present in urban sludge constitute a sanitary risk when used for the enrichment of agricultural soil. These eggs are very resistant in the environment and their survival could reach 6 months to one year in soils. To control the risks for the public health, we have to determine parameters leading to the eggs destruction in soils. Then the aim of the work is to study several conditions (humidity, temperature, texture,...) that could influence the survival of intestinal nematode eggs in various types of soil. Survival experiments were carried out in clayey soil, silty-loam soil and sandy soils with humidity levels corresponding to: field capacities, wilting point and variation between this 2 limit point (storage temperatures 4 degrees C, 19 degrees C and 30 degrees C). The result of this study has shown that the temperature conditioned for a great part the survival of the eggs in all types of soils with survival times superior at two year with a temperature of 4 degrees C. Between survivals at 20 degrees C and 30 degrees C, no difference has been observed. The humidity is the second parameter that condition the egg survival with a good viability preservation at the wilting point. This study also shows better survival in the conditions of deep soil with the protection of the egg from drying. With these data and with the simulation of continental temperate conditions, a contamination rate of 0.35% could be found on the surface soil after one year and a more important rate in the deep soil with 10.6%. The last step is the validation of this model and calculated values seem to be a good evaluation of the results found on fields. All these data must be take into account for the establishment of the regulation about the urban sludge reused for agricultural purpose.

Hydrophobic and electrostatic cell surface properties of Cryptosporidium parvum.

Microbial adhesion to hydrocarbons and microelectrophoresis were investigated in order to characterize the surface properties of Cryptosporidium parvum. Oocysts exhibited low removal rates by octane (only 20% on average), suggesting that the Cryptosporidium sp. does not demonstrate marked hydrophobic properties. A zeta potential close to -25 mV at pH 6 to 6.5 in deionized water was observed for the parasite. Measurements of hydrophobicity and zeta potential were performed as a function of pH and ionic strength or conductivity. Hydrophobicity maxima were observed at extreme pH values, with 40% of adhesion of oocysts to octane. It also appeared that ionic strength (estimated by conductivity) could influence the hydrophobic properties of oocysts. Cryptosporidium oocysts showed a pH-dependent surface charge, with zeta potentials becoming less negative as pH was reduced, starting at -35 mV for alkaline pH and reaching 0 at isoelectric points for pH 2.5. On the other hand, variation of surface charge with respect to conductivity of the suspension tested in this work was quite small. The knowledge of hydrophobic properties and surface charge of the parasite provides information useful in, for example, the choice of various flocculation treatments, membrane filters, and cleaning agents in connection with oocyst recovery.

Irrigation with waste water: parasitological analysis of soil.

In third world countries, the agricultural use of treated wastewater represents an interesting solution. A recent report of W.H.O. shows the importance of strict parasitological criteria for such reuse. The aim of this paper is to study the conditions under which Ascaris eggs have been recovered from artificially contaminated soils (sandy, clay or loamy soil, mould garden). The eggs elution has been carried out from soil particles using various solutions (detergents, distilled water, formaldehyde, sodium hydroxide, sodium hypochlorite). The recovery percentage analysis showed the superiority of the sodium hypochlorite solution titrating 10 chlorometric degrees, whatever the soil type. The eggs concentration in the eluates has been carried out through the flotation technique by testing various reagents with densities ranging from 1.16 to 1.44. The zinc sulphate solution at 50%, 55% and 66% prove to be flotation agents that are well adapted to this sampling type. By carrying out the elution with a sodium hypochlorite solution titrating 10 chlorometric degrees and the concentration by flotation with a zinc sulphate solution at 55% the recovery percentages, which are independent of the parasitic load, vary from 66 to 78%.

Quantification of helminth eggs in waste water.

Agricultural use of waste water has been regulated mostly by its bacteriological quality. Recently the importance of parasitological criteria for waste water standards was recognized by establishing that waste water containing less than 1 intestinal Nematode egg per liter is released for unrestricted use of effluents. At present, most of the techniques borrowed from parasitological coprology are only qualitative. In this report different protocols for helminth egg quantitation were compared. Three flotation techniques (Janeckso-Urbanyi, Faust, Arther) and two sedimentation techniques (Bailenger, Ritchie) and their performances were evaluated. Although all the techniques were based on the same number of on the whole positive samples, significant differences were observed in both the number of species identified and in the type of egg preferentially concentrated. Among the flotation methods tested, the Janeckso-Urbanyi modality offers undeniable advantages in efficiency and in the variety of species concentrated, but it is relatively costly. Among the sedimentation techniques, the one described by Bailenger is relatively inexpensive and concentrates all the types of eggs normally found in waste water samples, except for the relatively rare Enterobius with globally satisfactory yields.

[Study of the behavior of poliomyelitis virus type 1 in diffrerent hydrous media (author's transl)]. / Etude du comportement du virus poliomyélitique de type 1 dans différents milieux hydriques

After having analyzed several surveys on natural inactivation of viruses in water, the authors have attempted to determine the survival life of poliomyelitis virus type 1 in hydrous medium, of known composition, well-buffered and devoid of any bacterial contamination on the one hand, and on the other hand, in a similar medium with addition of organic humate-type substances. The experiment was carried out at 4 degrees C and at laboratory room temperature (18 degrees C-24 degrees C). Results tend to confirm that the presence of humate-type substances seems to have no significant influence on the development of poliomyelitis virus type 1. Moreover, under the experimental conditions used, the survival life of the virus is much longer than the one previously described. It represents an average of 296 days for a storage temperature of 18 degrees C-24 degrees C. It is longer in water kept at 4 degrees C; in fact, according to statistical calculations and to titrations carried out over a period of 550 days, the total disappearance of the virus was detected only after a period of 10 to 15 years.

[Concentration of viruses in water by the 2 phase polymer method: quantitative study]. / Concentration des virus dans l'eau par la méthode des polymères à deux phases: étude quantitative

The authors attempt to determine the sensibility of polymer two phases system applied to the concentration of viruses from water. They show that this technic agrees to the research of poliomyelitic viruses in waters since viral recuperation is always upper than 74.5%. Besides, they notice that average percentages of regained viruses are always 100% where the initial concentrations of viruses are 10(2) or 10(3) per liter; on the other hand if the initial concentration is higher the yield of this method is decreasing.