Enterovirus circulation in wastewater and behavior of some serotypes during sewage treatment in Monastir, Tunisia.

Introduction

Human enteroviruses (HEVs) are a large, clinically important group of human viruses. The genus Enterovirus contains 64 serotypes, which are divided into five groups: poliovirus (PV serotypes 1-3); HEV-A (Coxsackieviruses A2-A8, A10, A12, AH, A16. and Enterovirus 71); HEV-B (Coxsackie viruses A9 and B1-B6; Echoviruses 1-7, 9, 11-21, 24-27, and 29-33; and Enterovirus 69); HEV-C (Coxsackie viruses A1, A11. A13, A15, A17-22, and A24); and HEV-D (Enteroviruses 68 and 70) (King et al., 1999). Nonpolio enteroviruses (NPEVs) circulate in all populations, and infection can be associated with a vast variety of expressions, ranging from no symptoms to acute flaccid paralysis resembling poliomyelitis (Zaoutis & Klein, 1998). NPEVs are the most frequently identified cause of viral meningitis, particularly in infants and young children (Andreoletti et al., 1998; Modlin, 1997). Other illnesses with a chronic course, such as Type 1 diabetes mellitus, have also been associated with enterovirus infections (Hovi, Stenvik, & Rosenlew, 1996).

Numerous studies have documented the presence of enteroviruses in raw and treated wastewater (Payment, Fortin, & Trudel, 1986) and sewage sludge (Craun, 1984). Enteroviruses in the environment represent a public health risk because these viruses can be transmitted via the fecal-oral route through contaminated water (Nicand, Teyssou, & Buisson, 1998). Many studies from other countries provide such information, but few data of this nature are available from Tunisia. Our study monitored enteroviruses in wastewater plants of three types, namely plants using activated-sludge, trickling-filter, and oxidation-pond processes, from October 2000 to September 2001 in the region of Monastir, a tourist zone situated on the central Tunisian coast. This article gives an overview of the seasonal incidence and the prevalence of serotypes during wastewater treatment.

Materials and Methods

Study Area

The Monastir area, situated on the central Tunisian coast, attracts numerous tourists. This coastal area is known for tourist attractions and sport fishing. Wastewaters from the city of Monastir are treated in biological wastewater treatment plants of three types and are discharged directly to the Mediterranean Sea.

Sewage Samples

Each month, a total of 10 samples were collected. The samples were taken from wastewater at different treatment stages in three wastewater treatment plants, each plant using a different type of treatment, as indicated in Table 1. The study period was one year: October 2000 to September 2001.

Virus Extraction, Concentration, and Decontamination of Samples

Viruses were extracted from samples as described by Belguith, Hassen, and Aouni (2006). Samples were added to 1 percent (v/v) of 0.05-moles-per-liter (0.05-M) aluminum chloride and adjusted to pH 3.5 with HCl. The mixture was homogenized and centrifuged at 2500 g with a Hettich centrifuge (Model Rotina 35R, d-78532, Rotor 1718, Germany) for 15 minutes at 4[degrees]C. The pellet was suspended in 100 mL of 10 percent beef extract (LP029, Oxoid LTD, Basingstoke, Hampshire, England), and the pH was adjusted to 7. The mixture was once again homogenized and centrifuged at 10.000 g for 30 minutes at 4[degrees]C. The supernatant was used for virus detection.

The extract, concentrated by precipitation with polyethylene glycol 6000 (PEG) as described by Lewis and Metcalf, was added to 5 percent PEG (w/v) (1988). After an overnight incubation at 4[degrees]C, the extract was centrifuged at 10,000 g for 45 minutes, and the pellet was suspended in 10 mL of phosphate buffer (0.1 M) with pH of 7. This suspension was filtered through a membrane with 0.22-um pore size (Millex-GS, SLGS0250S, Molsheim, France) and collected as a virus concentrate.

Enterovirus Isolation on Cell Culture

Isolation and serotyping was conducted as recommended by the World Health Organization (WHO) with three cell lines: 1) L20B cells, a mouse L-cell line genetically engineered to express the human poliovirus receptor; 2) RD cells, derived from a human rhabdomyosarcoma; and 3) HEp-2 Cincinnati cells, derived from a human epidermoid carcinoma of the larynx (WHO, 2000). When complete characteristic enteroviral cytopathic effect (CPE) had been obtained, the infected cells were harvested and kept frozen (-20[degrees]C) until typing.

Identification of Viral Isolates

Seroneutralization of the CPE on cell cultures and intratypic differentiation of PVs were carried out in the WHO Regional Reference Laboratory of Poliomyelitis and Measles in the Pasteur Institute of Tunis.

Serotype Identification by Seroneutralization of CPE on Cell Cultures

Microneutralization tests with pools of antisera specific for the most common HEV serotypes were used, according to standard protocols (WHO, 2000), for all virus isolates obtained: type-specific PV antisera, enterovirus A-G pools (National Institute of Public Health and the Environment, Bilthoven, Netherlands) and in-house-produced monospecific immune antisera specific for the CBV serotypes. Virus isolates that were not identified by these standard seroneutralizations were qualified as untypable enteroviruses.

Intratypic Differentiation of Polioviruses

All isolated PV were systematically tested for their wild or vaccine origin by two distinct methods, according to WHO-recommended protocols (WHO, 2000): one antigenic method and one genetic method. The antigenic method was the EL1SA test, using cross-adsorbed Sabin-specific or wild-specific rabbit antisera; the genetic method was a hybridization assay using recombinant riboprobes specific for vaccine-related isolates.

Results and Discussion

Poliovirus Isolation

All isolated PV strains were PV vaccine-related. These findings are certainly due to the effectiveness of the extensive supplemental immunization activities that were made in the country up to 1997. They are consistent with previous epidemiological studies carried out in Tunisia (Triki et al., 1999).

Nonpolio Enterovirus Isolation

Seroneutralization of CPE on cell culture allows the identification of seven echovirus serotypes (E-4, E-6, E-13, E-11, E-25, and E-33). Use of this technique enabled the identification of 77 percent of the NPEV isolates. The lack of neutralization by antisera pools may have been due to one of the following two reasons: 1) the isolates belonged to serotypes not covered by the RIVM A-G antisera pools used in the study of seroneutralization, or 2) this particular viral strain had evolved, so that it was no longer neutralized by the antiserum produced against the reference strain of the same serotype.

Among all isolated serotypes, 64 percent were echoviruses; no coxsackieviruses were isolated (Table 2). These results are consistent with those of other reports (Chambon et al., 2001; Nairn & Clements, 1999). In fact, outcomes of epidemiological studies are certainly altered or modified by the type of cell culture used. Peigne-Lafeuille and co-authors (1989) showed that the number of isolated coxsackieviruses decreased significantly after the use of primary monkey kidney cells was abandoned during the mid-1980s. Furthermore, during our study, echoviruses seemed to be easy to isolate with different human cell lines. The frequency of CPEs on three cell lines is shown in Table 3. The maximum positive CPE (58 samples) was detected on the RD cell line. This line seemed to be twice as sensitive as HEp-2 cells (23 samples). For all samples from which enteroviruses were isolated on HEp-2, enteroviruses also were isolated on RD. Positive CPE on line L20B was very weak. This cell line was chosen to exclude PV isolates (WHO, 2000). Sedmak, Bina, and MacDonald (2003) reported that RMK-I, HEp-2, and Buffalo green monkey kidney (BGM) cell lines were sensitive to Coxsackie virus B, whereas cell line RD was more sensitive to Coxsackie virus A than was RMK-I. Thus, using RD and HEp-2 cell lines, we could conclude that echoviruses were the only nonpolio enteroviruses circulating in the wastewater of the Monastir area.

E-6 was found in 50 percent of the serotyped samples (Table 2). These results confirm those of a previous Tunisian epidemiological study of NPEV done with clinical samples (Bahri et al., 2005). This finding demonstrates that there is a correlation between clinical enterovirus serotype and sewage serotype circulating in the country. E-6 has also been reported to be among the serotypes most frequently isolated in other countries (Meqdam, Khalousi, & Al-Shurman, 2002; Trallero et al., 2000). E-11 and E-30, reported to be endemic NPEVs in Tunisia and in other countries (Bahri et al., 2005), were not frequently isolated in our study. Of the two, only E-11 was isolated in the wastewater of the Monastir area.

Seasonal Occurrence of Enteroviruses

Positive CPEs were found during the whole period of investigation, from October 2000 to September 2001 (Table 3). This result agreed with the finding of Riding, Stewart, Clements, and Galbraith (1996), who used RT-PCR and observed a year-round occurrence of enteroviruses associated with aseptic meningitis. In wastewater, the percentage of positive results decreased from February to June (CPE < 45 percent) and increased during the summer, during the autumn, and particularly in the beginning of the winter period, from September to January. This finding confirmed the results of Tani, Dohi, Kurumatani, and Yonemasu (1995), who mentioned an enterovirus peak level in urban river water during summer that persisted to autumn-winter (1995). Many reports, however, have shown that in temperate climates, enteroviruses are encountered far more commonly during summer and autumn than in winter and spring (Melnick, 1996).

E-6 was the most frequently isolated serotype (in 23 of 47 samples), circulating during the whole period of investigation (Table 2), except in the spring (March, April, May). The dominance of E-6 in wastewater can predict, or hint at, the presence of this serotype in clinical manifestations during this period. An epidemiological study of enterovirus serotypes carried out in the Milwaukee area from August 1994 to December 2002 revealed that for many years, the enterovirus serotype most commonly detected in clinical settings was also the serotype most commonly detected in sewage (Semak et al., 2003). In our study, different serotypes occasionally appeared, namely serotypes E-13 (May and September), E-11 (September), E-25 (March), and E-33 (December). Eight enteroviruses that were not typable by RIVM pool appeared from March to December. Sedmak and co-authors reported that sewage enteroviruses detected in late winter or early spring can at times predict some of the enterovirus types that will predominate clinically during the following summer.

Enterovirus Removal During the Wastewater Treatment

To determine if enteroviruses were selectively eliminated during wastewater treatment, we isolated them at different steps during each of the wastewater treatment processes. The results are shown in Table 4. Eight serotypes were isolated. Enterovirus serotypes were found at all steps of the treatment process in all three wastewater treatment plants. E-6 and PV were present there as well as in effluent and sludge samples from the three plants, indicating a high level of resistance to the wastewater treatments. Whereas E-11 was isolated only in sludge samples, E-13 was isolated in primary wastewater and in digested sludge, but only in the trickling-filter plant. Trickling filter and anaerobic digestion were ineffective at removing E-13 and E-6. PV1 was not detected in raw wastewater, perhaps because of the limitations of the detection method. It was, however, detected in stable sludge and in treated effluents. Payment, Fortin, and Trudel (1986) previously observed that PV1 and PV2 better withstand the step of activated-sludge treatment than does PV3. Many studies have shown the efficiency of activated-sludge treatment in the elimination of enteroviruses, especially PV (Clarke, Stevenson, Chang, & Kabler, 1961; Farrah, Goyal, Gerba, Conklin, & Smith, 1978; Malina, Sagik, & Moore, 1975; Safferman & Morris, 1976). Two phenomena can explain this fact, namely adhesion-aggregation of viruses to sludge and viral inactivation due to bacterial activity in activated sludge. In fact, Block, Collin, Jofret, Rolland, and Folliguet demonstrated that PV1 adsorption was instantaneous for 90 percent of viral particles and that 99.9 percent were absorbed after five minutes of contact (1979). Many studies have shown the presence of antiviral components in mixed liquor of activated sludge even in the liquid and solid phases (Arraj, Bohatier, Laveran, & Traore, 2005; Knowlton & Ward, 1987; Ward, 1982). The study of adhesion-aggregation and inactivation of PV1 in groundwater stored in a hydrophobic container has demonstrated that viral inactivation and adhesion-aggregation in water are related phenomena (Gassilloud & Gantzer, 2005). Like PV1, E-6 may be using adhesion-aggregation phenomena to escape viral inactivation in the different wastewater and sewage sludge treatments. The phenomena of adhesion-aggregation and endemicity are certainly related. Endemic strains may use adhesion-aggregation to resist and persist in the environment. Clarifying this relationship offers good prospects for cutting off the enterovirus endemic cycle.

Conclusion

Enteroviruses are present year round in the wastewater of the Monastir, Tunisia, area, but occur with low frequency at the end of the winter and in the spring. In all seasons, E-6 was the most noted serotype. Different serotypes, such as E-11, E-4, E-25 and E-13, occasionally appeared. Resistance to wastewater treatment differed from one serotype to another. PV1 and E-6 were the most resistant serotypes. Studying the circulation of enteroviruses and enterovirus behavior in wastewater treatment can be a good tool for the surveillance of enterovirus outbreaks.

Acknowledgements: We are grateful to Professor Triki Henda, Director of the WHO Regional Reference Laboratory of the Pasteur Institute of Tunis, Tunisia, for her assistance in serotyping enterovirus isolates, and to Professor Hans-W Ackermann, of the Department of Microbiology, Faculty of Medicine, Laval University in Quebec, Canada, for helpful discussion and critical review of the manuscript. We also thank the director and sanitary technicians of the Office National de l'Assainissement of Monastir, Tunisia, for technical assistance.

Corresponding Author: Khaoula Belguith, Assistant Professor, Pharmacy University of Monastir, Transmissible Diseases and Biological Active Substances Laboratory, 5000 Avicen Street, Monastir, Tunisia. E-mail: [e-mail].

REFERENCES

Andreoletti, L, Blassel-Dammam, N., Dewilde, A., Vallee, L., Cremer, R., Hober, D., & Wattre, P. (1998). Comparison of use of cerebrospinal fluid, serum, and throat swab specimens in diagnosis of enteroviral acute neurological infection by a rapid RNA detection PCR assays. Journal of Clinical Microbiology, 36(2), 589-591.

Arraj, A., Bohatier, J., Laveran, H., & Traore, O. (2005). Comparison of bacteriophage and enteric virus removal in pilot scale activated sludge plants. Journal of Applied Microbiology, 98(2), 516-524.

Bahri, O., Rezig, D., Ben Nejma-Oueslati, B., Ben Yahia, A., Ben Sassi, J, Hogga, N., Sadraoui, A., & Triki, H. (2005). Enteroviruses in Tunisia: Virological surveillance over 12 years (1992-2003). Journal of Medical Microbiology, 54(1), 63-69.

Belguith, K., Hassen, A., & Aouni, M. (2006). Comparative study of four extraction methods. Bioressources Technology, 97, 414-419.

Block, J.C., Collin, J.F., Jofret, J.C., Rolland, D., & Folliguet, J.M. (1979). Elimination des enterovirus au cours du traitement par boues activees des eaux usees urbaines [Enterovirus elimination during activated-sludge treatment of urban sewage]. Techniques et Sciences Municipales L'eau, 74, 201-206.

Chambon, M., Archimbaud, C, Bailly, J.L., Henquell, C, Regagnon, C, Charbonne, E, & Peigue-Lafeuille, H. (2001). Circulation of enteroviruses and persistence of meningitis cases in the winter of 1999-2000. Journal of Medical Virology, 65(2), 340-347.

Clarke, N.A., Stevenson, R.E., Chang, S.L., & Kabler P.W. (1961). Removal of enteric viruses from sewage by activated sludge treatment. American Journal of Public Health, 51, 1118-1129.

Craun, G.E (1984). Health aspects of groundwater pollution. In G. Bitton & C.P Gerba (Eds.), Groundwater Pollution Microbiology (pp. 135-179). New York: John Wiley & Sons.

Farrah, S.R., Goyal, S.M., Gerba, C.P, Conklin, R.H., & Smith, E.M. (1978). Comparison between adsorption of poliovirus and rotavirus by aluminum hydroxide and activated sludge flocs. Journal of Applied and Environmental Microbiology, 35(2), 360-363.

Gassilloud B., & Gantzer, C. (2005). Adhesion-aggregation and inactivation of poliovirus 1 in groundwater stored in a hydrophobic container. Journal of Applied and Environmental Microbiology, 71(2), 912-20.

Hovi, T., Stenvik, M., & Rosenlew, M. (1996). Relative abundance of enterovirus serotypes in sewage differs from that in patients: Clinical and epidemiological implications. Epidemiology and Infection, 116, 91-97.

King, A.M.Q., Brown, F., Christian, P., Hovi, T., Hyypia, T., Knowles, N.J., Lemon, S.M., Minor, P.D., Palmenberg, A.C., Skern, T., & Stanway, G. (2000). Virus taxonomy: Seventh report of the International Committee on Taxonomy of Viruses. In M.H.V Van Regenmortel, C.M. Fauquet, D.H.L. Bishop, E.B. Carstes, M.K. Estes, S.M. Lemon, J. Maniloff, M.A. Mayo, D.J. McGeoch, C.R. Pringle, & R.B. Wickner (Eds.), Picornaviridae, (pp. 657-673). New York: Academic Press.

Knowlton, D.R., & Ward, R.L. (1987). Characterization of virucidal agents in activated sludges. Journal of Applied and Environmental Microbiology, 53(4), 621-626.

Lewis, G.D., & Metcalf, T.G. (1988). Polyethylene glycol precipitation for recovery of pathogenic viruses including hepatitis A and human rotavirus from oyster, water, and sediments. Journal of Applied and Environmental Microbiology, 54(8), 1983-1988.

Melnick, J.L. (1996). Enteroviruses: polioviruses, coxsackievirus, echoviruses, and newer enteroviruses. In B.N Fields, D.M. Knipe, P.M. Howley, R.M. Chanock, J. L. Melnick, T.P Monath, B. Roizman, & S.E. Straus (Eds.), Virology (pp. 655-712). Philadelphia: Lippincott-Raven Publishers.

Malina, J.F, Sagik, K.R., & Moore, B.E. (1975). Poliovirus inactivation by activated sludge. Journal of Water Pollution Control Federation, 47(8), 2178-2183.

Meqdam, M., Khalousi, M., & Al-Shurman, A. (2002). Enteroviral meningitis in northern Jordan: Prevalence and association with clinical findings. Journal of Medical Virology, 66(2), 224-228.

Modlin, J.F (1997). Update on enterovirus infections in infants and children. Advances in Pediatric Infectious Disease, 12, 155-180.

Nairn, C, & Clements, G.B. (1999). A study of enterovirus isolations in Glasgow from 1997 to 1999. Journal of Medical Virology, 58, 304-312.

Nicand, E., Teyssou, R., & Buisson, Y (1998). Le risque fecal viral en 1998 [Viral enteric risk in 1998]. Virologie, 2(2), 103-116.

Payment, P., Fortin, S., & Trudel, M. (1986). Elimination of human enteric viruses during conventional wastewater treatment by activated sludge. Canadian Journal of Microbiology, 32(12), 922-925.

Peigne-Lafeuille, H., de Champs, C, Laveran, H., Labbe, A., Chambon, M., Beytout, D., & Cluzel, R. (1989). Survey of viruses of the gut in 10,477 children admitted to general paediatric wards from 1981 to 1986. Journal of Hospital Infection, 14(8), 357-378.

Riding, M.H., Stewart, J., Clements, G.B., & Galbraith, D.N (1996). Enteroviral polymerase chain reaction in the investigation of aseptic meningitis. Journal of Medical Virology, 50, 204-206.

Safferman R.S., & Morris M.E. (1976). Assessment of virus removal by a multistage activated sludge process. Water Research, 10(5), 413-420.

Sedmak, G., Bina, D., & MacDonald, J. (2003). Assessment of an enterovirus sewage surveillance system by comparison of clinical isolates with sewage isolates from Milwaukee, Wisconsin, collected August 1994 to December 2002. Journal of Applied and Environmental Microbiology, 69(12), 7181-7187.

Tani, N., Dohi, Y, Kurumatani, N., & Yonemasu, K. (1995). Seasonal distribution of adenoviruses, enteroviruses and reoviruses in urban river water. Microbiology Immunology, 39(8), 577-580.

Trallero, G., Casas, I., Tenorio, A., Echevarria, J.E., Castellanos, A., Lozano, A., & Brena, P.P. (2000). Enteroviruses in Spain: Virological and epidemiological studies over 10 years (1988-1997). Epidemiology and Infection, 124(3), 497-506.

Triki, H., Bahri, O., Guillot, S., Van der Avoort, H.G., Sassi, J.B., Arrouji, A., Arrouji, Z., Slim, A., Crainic, R., & Dellagi, K. (1999). Molecular epidemiology of poliovirus infection in Tunisia. Journal of Medical Microbiology, 48, 569-576.

Ward, R.L. (1982). Evidence that microorganisms cause inactivation of viruses in activated sludge. Journal of Applied and Environmental Microbiology, 43(8), 1221-1224.

World Health Organization. (2000). Manual for the virological investigation of poliomyelitis. Geneva, Switzerland: Author.

Zaoutis, T, & Klein, J.D. (1998). Enterovirus infections. Pediatrics in Review, 19(6), 183-191.

Although most of the information presented in the Journal refers to situations within the United States, environmental health and protection know no boundaries. The Journal periodically runs International Perspectives to ensure that issues relevant to our international constituency, representing over 60 countries worldwide, are addressed. Our goal is to raise diverse issues of interest to all our readers, irrespective of origin.

Khaoula Belguith, Ph.D., C.I.H.

Abdennaceur Hassen, Ph.D., R.E.H.S.

Lamjed Bouslama

Sdiri Khira

Mahjoub Aouni

TABLE 1 Origin of Wastewater Samples

Process           Treatment Step

Trickling filter
                  Primary wastewater
                  Secondary wastewater
                  Digested sludge
                  Trickling-filter wastewater
Activated sludge
                  Primary wastewater
                  Secondary wastewater
                  Activated sludge
                  Thickened sludge
Oxidation ponds
                  Primary wastewater
                  Secondary wastewater

TABLE 2 Numbers of Enterovirus Serotypes Isolated from Wastewater and
Sewage Sludge

                             Enterovirus Serotypes
Date       Echovirus 4  Echovirus 6  Echovirus 13  Echovirus 11

October    -             2           -             -
November   -             2           -             -
December   -             5           -             -
January    -             6           -             -
February   -             1           -             -
March      -             -           -             -
April      -             -           -             -
May        -             -           1             -
June       1             1           -             -
July       -             3           -             -
August     -             3           -             -
September  -             -           1             2
Total      1            23           2             2

                           Enterovirus Serotypes
Date       Echovirus 25  Echovirus 33  PV*  NTP**  NE ([dagger])  Total

October    -             -             4           1               7
November   -             -             3    -      -               5
December   -             1             -    -      -               6
January    -             -             -    -      -               6
February   -             -             1    -      -               2
March      1             -             -    1      -               2
April      -             -             -    2      -               2
May        -             -             -    -      -               1
June       -             -             -    1      -               3
July       -             -             -    1      -               4
August     -             -             -    1      -               4
September  -             -             -    2      -               5
Total      1             1             8    8      1              47

*PV = vaccinal poliovirus.
**NTP = not typable by RIVM pool.
- = Not detected.
([dagger])NE = Not enterovirus.

TABLE 3 Percentage Positive Cytopathic Effect (CPE), by Cell Line and
Date of Sampling

                   Cell Lines
Date       RD                    HEp-2

October    63.6%                 18.2%
November   45.5%                 18.2%
December   81.8%                 27.3%
January    72.7%                  9.1%
February   27.3%                 27.3%
March      18.2%                  0.0%
April      18.2%                  9.1%
May         9.1%                  0.0%
June       27.3%                  9.1%
July       63.6%                 27.3%
August     45.5%                 27.3%
September  54.5%                 36.4%
Average    43.9% [+ or -] 23.8%  17.4% [+ or -] 11.9%

           Cell Lines
Date       L20B

October    9.1%
November   9.1%
December   0.0%
January    0.0%
February   9.1%
March      0.0%
April      0.0%
May        0.0%
June       0.0%
July       0.0%
August     9.1%
September  0.0%
Average      3% [+ or -] 4.5%

TABLE 4 Serotypes Isolated at Different Treatment Steps

Process           Treatment Step               Serotypes

Trickling filter
                  Primary wastewater           E-6, E-4, E-13, PV2
                  Trickling-filter wastewater  E-6
                  Digested sludge              PV1, E-6, E-11, E-13,
                                                 E-33, NTP
                  Secondary wastewater         E-6, E-25, NTP
Activated sludge
                  Primary wastewater           E-6
                  Activated sludge             E-6, E-11
                  Thickened sludge             PV1
                  Secondary wastewater         NTP
Oxidation ponds
                  Primary wastewater           E-6
                  Secondary wastewater         PV1