Detection of enteroviruses and hepatitis A virus in sludge and sludge amended soil using the polymerase chain reaction.

Abstract

Human enteroviruses and hepatitis A virus may be present in significant concentrations in municipal sewage sludge. Land application of sludge on irrigated crop land may introduce these pathogens into the environment if they are not destroyed by the sludge treatment process. If infectious, these viruses could contaminate ground- and surface water sources used for municipal drinking water sources. Conventional cell culture methods used to detect human enteric viruses are costly and time consuming. The advent of recombinant DNA technology and related fields have made it possible to detect these pathogens in a matter of hours using the polymerase chain reaction (PCR). However, the methods used to recover these viruses from sludge and sludge amended soil are not optimized for PCR analysis. Optimization of these samples for detection of enteroviruses and hepatitis A virus by PCR required treatment employing spun column chromatography. This was accomplished using a combination of size exclusion chromatography (Sephadex G- 50) and ion exchange chromatography (Chelex-lOO). This treatment method was tested using undigested sewage sludge, anaerobically digested sewage sludge and digested sludge amended to 10 different soil samples. These samples were subsequently seeded with 10³ plaque forming units (PFU) of poliovirus type 1 and subjected to reverse transcription and 30 cycles of PCR. Positive amplification was observed in all seeded samples treated using Sephadex G-50 and Chelex-IOO. Based on this success, 4 undigested sewage sludge samples, 4 anaerobically digested sewage sludge samples and 24 field soil samples that had been amended with anaerobically digested sludge were screened for naturally occurring enteroviruses using PCR. Enteroviruses were detected in all 4 undigested sludge samples, all 4 anaerobically digested sludge samples, and 19 of 24 field soil samples. In addition, indigenous hepatitis A virus was detected in 7 of the 8 sewage sludge samples. Subsequent tissue culture analysis resulted in detection of enteroviruses in all of the sewage sludge samples but none of the field soil samples. These results indicate that PCR detection of enteroviruses and hepatitis A virus may serve as a useful presumptive test for their detection in these kinds of samples. Because of the ability of PCR to detect non-culturable viruses, positive results should be confirmed by cell culture techniques.