The analytical performances of the new Abbott RealTime hepatitis C virus (HCV) and human immunodeficiency virus type 1 viral load assays were compared at nine laboratories with different competitor assays. These included the Abbott LcX, Bayer Versant bDNA, Roche COBAS Amplicor, and Roche COBAS TaqMan assays. Two different protocols used during the testing period with and without a pre-m1000 RNA isolation spin were compared. The difference proved to be nonsignificant. A uracil-N-glycosylase (UNG) contamination control option in the HCV test for previous Roche COBAS Amplicor users was evaluated. It proved to decrease amplicon carryover by 100-fold independent of the amplicon input concentration. The protocol including UNG proved to overcome problems with false-positive negative controls. Comparison with other assays revealed only minor differences. The largest difference was observed between the Abbott HCV RealTime assay and the Roche COBAS Amplicor HCV Monitor version 2.0 assay.Hepatitis C virus (HCV) and human immunodeficiency virus type 1 (HIV-1) are major causes of mortality in both developing and developed countries. For both viruses, relatively effective therapies have been available in developed countries for quite some time. HCV and HIV-1 viral loads are important parameters in patient management both before initiating therapy and during therapy. The decline of the HCV viral load during the first 3 months of therapy is, for instance, a strong indicator of the final outcome of therapy (9, 23). Moreover, in many countries, the HCV viral load in infected health care workers determines whether they are allowed to perform surgical procedures. HIV-1 viral load is monitored during therapy, and a viral load above a certain threshold, which may differ per treating physician, requires a switch in medication (6, 13).The first tests that were described for determining the viral load were based on target amplification techniques like reverse transcriptase PCR (RT-PCR) and nucleic acid sequence-based amplification (NASBA). These tests had readout on agarose gel or readout with enzymatic detection of the amplicon after amplification with biotinylated primers (1, 3, 15, 17). Subsequently, signal amplification techniques were developed for the quantitative detection of HCV and HIV-1 (5, 16). Those techniques were improved and commercialized. The suppliers of the most widely used systems at the moment are Roche and Abbott, for RT-PCR-based systems (the COBAS Amplicor and LcX systems, respectively); bioMerieux (formerly Organon Technica), with a NASBA-based technique; and Bayer, with the Versant bDNA system being a signal amplification technique (4,18,19). Each technique has its own advantages and disadvantages. The relatively small dynamic range is a disadvantage that all current assay formats have in common. All assay formats are also rather sensitive to contamination, especially at the lower limit of detection (21). Handling of the sample after target amplification is a major cause of contamination for the NASBA-and RT-PC...

Understanding the abundance and fate of human viral pathogens in wastewater is essential when assessing the public health risks associated with wastewater discharge to the environment. Typically, however, the microbiological monitoring of wastewater is undertaken on an infrequent basis and peak discharge events may be missed leading to the misrepresentation of risk levels. To evaluate diurnal patterns in wastewater viral loading, we undertook 3-day sampling campaigns with bi-hourly sample collection over three seasons at three wastewater treatment plants. Untreated influent was collected at Ganol and secondary-treated effluent was sampled at Llanrwst and Betws-y-Coed (North Wales, UK). Our results confirmed the presence of human adenovirus (AdV), norovirus genotypes I and II (NoVGI and NoVGII) in both influent and effluent samples while sapovirus GI (SaVGI) was only detected in influent water. The AdV titre was high and relatively constant in all samples, whereas the NoVGI, NoVGII and SaVGI showed high concentrations during autumn and winter and low counts during the summer. Diurnal patterns were detected in pH and turbidity for some sampling periods; however, no such changes in viral titres were observed apart from slight fluctuations in the influent samples. Our findings suggest that viral particle number in wastewater is not affected by daily chemical fluctuations. Hence, a grab sample taken at any point during the day may be sufficient to enumerate the viral load of wastewater effluent within an order of magnitude while four samples a day are recommended for testing wastewater influent samples.

Human enteric viruses are responsible for waterborne and shellfish-associated disease outbreaks worldwide. Quantitative reverse transcription PCR (qRT-PCR) is often used to assess the health risks associated with shellfish and environmental water, but viral titres in sediments are less commonly investigated. In this study, we developed and validated two multiplex qRT-PCR assays for aquatic sediment and shellfish samples targeting viruses that are a common cause of gastroenteritis (norovirus GI, GII and hepatitis A virus), two emerging viruses (sapovirus and hepatitis E virus), along with mengovirus (MgV), which is often used as a sample process control for the assessment of RNA extraction efficiency. Singleplex and multiplex assays demonstrated comparable PCR efficiencies and gave reliable results over a wide concentration range. The multiplex assays showed remarkable sensitivity with a limit of detection of 1 RNA copy/µL nucleic acid extract for all target viruses and limits of quantification of 3–18 RNA copies/µL for the targeted human pathogenic viruses and 20–40 RNA copies/µL for MgV. The results demonstrated the veracity of multiplex qRT-PCR for the estimation of viral titres in sediment and shellfish, allowing the rapid assessment of viral infection risks associated with environments exposed to wastewater contamination.