► The Tm of a TaqMan probe employed in a widely used RT-qPCR for HEV was calculated. ► Calculations showed that the melting temperature (Tm) of the probe was suboptimal. ► A Minor Groove Binder ...version of probe was synthesised in order to increase its Tm. ► MGB probe detected HEV in 6 samples that were negative with the unmodified probe. ► False negative results were due to a C→T point mutation in the probe binding region.
Hepatitis E virus (HEV) is a major cause of acute viral hepatitis in many parts of the developing world. It is responsible for both sporadic infections and large scale epidemics and may be associated with significant mortality during pregnancy. Over the past two decades many serological and nucleic acid based diagnostic tests for HEV have been developed, including several reverse transcription real-time polymerase chain reaction assays (RT-qPCR). One of the most widely used of these RT-qPCRs is that developed by Jothikumar and colleagues (Journal of Virological Methods 2006, 131, 65–71). Whilst reviewing this assay we calculated the predicted melting temperature of its TaqMan probe and consequently synthesised a minor groove binder (MGB) version in order to increase its hybridisation stability. In this report the performance of the original unmodified probe is compared with that of the MGB-modified version. We demonstrate that the MGB-modified probe detected HEV RNA in plasma samples from six patients with serologically confirmed hepatitis E in whom the unmodified probe had failed to detect HEV RNA. Sequence analysis of the ORF3 segment targeted by the RT-qPCR was possible in 4 of the 6 patients and revealed an identical C→T single nucleotide mutation in the probe binding region in each case.
The hepatitis B virus (HBV) surface antigen (HBsAg) is a complex protein, and understanding accurately the impact of amino acid changes on the antigenicity of the immunodominant a determinant must ...take this complexity into consideration. Epitope mapping with four mAbs was used to phenotype HBsAg directly from patients' sera to investigate the effect of mutations in their native genetic backbone. The expected mAb reactivity was established initially for samples harbouring 'wild-type' HBsAg sequences across genotypes A-E. The alteration of HBsAg antigenicity, defined by mAb epitope loss, was demonstrated in a number of samples with sequence-inferred amino acid changes. Individual mutations within the mapped epitopes to which the mAbs were directed usually affected their binding. However, the loss of more than one epitope was observed as the number of mutations within a sequence increased. Conversely, not all mutations occurring in the a determinant altered the HBsAg conformation. The genotype backbone, the specific amino acid substitution and amino acid changes occurring outside the major antigenic region appeared to be important in determining expression of the predicted epitope loss. These data clearly demonstrate that sequence-based methods alone may not accurately define HBsAg phenotype. This phenotyping methodology allows for the rapid and accurate identification of antigenically altered viruses and will greatly enhance current HBV surveillance, research and diagnostic activities. The data generated can be used to inform on public health issues relating to prevalence, transmission and impact of HBsAg mutants in HBV-infected populations.
The deficiency of readily biodegradable organic carbon can be a significant limitation to effective nitrogen removal during wastewater denitrification. Waste-activated sludge (WAS) is a source of ...carbon produced directly at wastewater treatment plants (WWTPs). Raw WAS has a large molecular weight and complex chemical structure molecules that are not easily available for microorganisms. In this study, easily biodegradable organic fractions were released using pH control and/or nitrites and nitric acid (NO2/FNA). The obtained results indicated that WAS can be a sufficient carbon source for denitrification in WWTPs that are at risk of minor effluent violations. The implementation of WAS disintegration with the use of pH control and NO2/FNA allowed for the denitrification of an additional 0.5 and 0.8 mgN-NO3/L. WAS disintegration, besides being a source of carbon generation, reduces the volume of sludge and leads to the implementation of a closed-loop system.
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