The efficiency of concentration methods used to detect enteric viruses in anaerobically digested sludge

Abstract

The presence of enteric viruses in biosolids can be underestimated due to the inefficient methods (mainly molecular methods) used to recover the viruses from these matrices. Therefore, the goal of this study was to evaluate the different methods used to recover adenoviruses (AdV), rotavirus species A (RVA), norovirus genogroup II (NoV GII) and the hepatitis A virus (HAV) from biosolid samples at a large urban wastewater treatment plant in Brazil after they had been treated by mesophilic anaerobic digestion. Quantitative polymerase chain reaction (PCR) was used for spiking experiments to compare the detection limits of feasible methods, such as beef extract elution and ultracentrifugation. Tests were performed to detect the inhibition levels and the bacteriophage PP7 was used as an internal control. The results showed that the inhibitors affected the efficiency of the PCR reaction and that beef extract elution is a suitable method for detecting enteric viruses, mainly AdV from biosolid samples. All of the viral groups were detected in the biosolid samples: AdV (90%), RVA, NoV GII (45%) and HAV (18%), indicating the viruses' resistance to the anaerobic treatment process. This is the first study in Brazil to detect the presence of RVA, AdV, NoV GII and HAV in anaerobically digested sludge, highlighting the importance of adequate waste management.

Fig. 1. mean numbers in log10 units of viruses recovered [genome copies (GC) g-1] from biosolid samples. Methods were performed in triplicate. Ten-fold serial dilution of nucleic acids are shown in paren thesis (1:10 and 1:100) on the x axis. Viral titres spiked are described in Materials and Methods. AdV: adenoviruses; bars: minimum and maximum value; HAV: hepatitis A virus; Method 1: ultracentrifuga tion; Method 2: beef extract; NoV GII: norovirus genogroup II; RVA: rotavirus species A; square in black: mean value.

Fig. 2. recovery efficiency (%) from each method performed in trip licate used for detecting enteric viruses. AdV: adenoviruses; HAV: hepatitis A virus; Method 1: ultracentrifugation; Method 2: beef ex tract; NoV GII: norovirus genogroup II; RVA: rotavirus species A.