Academic journal article Bulletin of the World Health Organization

Assessment of Cell Culture and Polymerase Chain Reaction Procedures for the Detection of Polioviruses in Wastewater

Academic journal article Bulletin of the World Health Organization

Assessment of Cell Culture and Polymerase Chain Reaction Procedures for the Detection of Polioviruses in Wastewater

Article excerpt

Voir page 978 le resume en francais. En la pagina 979 figura un resumen en espanol.


Significant progress has been made in WHO's programme for the eradication of poliomyelitis by the year 2000 (1). However, poliovirus remains endemic in some areas, including parts of sub-Saharan Africa. For example, over 600 cases were reported in Angola during March and April 1999 (ProMED mail, 30 April 1999). The strategy for poliomyelitis eradication includes routine immunization, surveillance for acute flaccid paralysis, and mopping-up activities. The final stage, in which eradication is certified, requires surveillance of clinical cases of acute flaccid paralysis in order to obtain evidence of the absence of wild-type polioviruses. It is considered that environmental monitoring, mainly involving the screening of wastewater, can provide additional evidence of the circulation or absence of wild-type polioviruses (1). The ratio of subclinical infections to cases of acute flaccid paralysis may exceed 1000:1. Poliovirus excretors occur at a frequency of 1 per 2000 persons (1, 2). Furthermore, it has been observed in many parts of the world that the screening of wastewater is a more sensitive tool for the detection of wild-type polioviruses circulating in communities than is surveillance for cases of acute flaccid paralysis. Furthermore, wild-type polioviruses have frequently been isolated from wastewater in the absence of cases of acute flaccid paralysis (1-7). Environmental surveillance may not only confirm the elimination of wild-type polioviruses but may also prove useful for monitoring the success of vaccination and detecting the reintroduction of wild-type strains into communities previously considered free of poliomyelitis (8, 9).

Various techniques are being used for the environmental surveillance of polioviruses, but there is little information on their efficiency and reliability. WHO has therefore recommended that research be carried out to improve methods for the detection of polioviruses in the environment (10) and has established a working group on the formulation of standard procedures for the environmental surveillance of these viruses (1, 2).

The present study deals with an evaluation and optimization of techniques for the detection of polioviruses in wastewater in South Africa. The L20B mouse cell line (11-13), the PLC/PRF/5 human liver cell line (PLC) (14), the buffalo green monkey kidney cell line (BGM), and primary vervet monkey kidney cells (PVK) (15, 16) were compared for the isolation of polioviruses. These cells were selected on the basis of evidence on their susceptibility to enteric viruses (14-19). L20B mouse cells carry the receptor site for polioviruses, and this makes them selectively susceptible to polioviruses in the presence of human enteric viruses (11). Techniques involving the polymerase chain reaction (PCR) were used to detect enteroviruses in water samples and suspensions of cell cultures with or without cytopathogenic effect (CPE). A glass wool adsorption-elution procedure was used to recover viruses from water samples (20). The study was carried out on environmental waters containing a variety of viruses at a range of concentrations. Samples were collected from wastewater in a community of low socioeconomic status before, during, and after two rounds of mass immunization of children with oral poliovirus vaccine (OPV), from wastewater in an abattoir, and from river and dam water polluted with domestic and animal waste.

Materials and methods

PLC (PLC/PRF/5 cell line, ATCC Code CRL 8024, passage 85-100), BGM (BGM cell line, BioWhittaker Catalogue No. 71-176B, passage 80-95), L20B (L20B cell line, passage 50-65, kindly supplied by Dr David J. Wood, National Institute for Biological Standards and Control, UK) and PVK cells were cultured by established procedures (14, 19). These essentially consist of cultivation in Eagle's minimum essential medium (MEM) with Earle's salt solution and antibiotics (penicillin and streptomycin) supplemented with appropriate concentrations of fetal calf serum for the cultivation or maintenance of cell cultures. …

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