RNA is an important marker of acute HEV infection, especially during early stages, before the antibody response becomes evident. Serological testing alone may fail to diagnose acute infection, especially in immunocompromised patients, which justifies the use of molecular assays for diagnosis. Several protocols based on Real-time PCR including commercial assays with good sensitivity are now available for HEV diagnosis. A recent study compared five Real-time PCR procedures for HEV RNA detection with detection rates ranging from 83 to 100%
. In another study aiming at investigating the performance of molecular-based assays (conventional and Real Time PCR assay) using a panel of HEV-containing plasma samples, the authors found a marked difference in sensitivity between the assays, with the most sensitive methods being those based upon real-time RT-PCR
. Although real time PCR provides sensitive detection of HEV genome, it is not useful for molecular characterization and typing. Indeed, the length of the fragments amplified by real time PCR is usually less than 100 nucleotides and they are located on conserved parts of the genome to reach higher PCR efficacy and sensitivity
. Genotyping and phylogenetic analysis requires longer fragments within more variable regions. Conventional PCR followed by sequencing of PCR products can identify the genotype and, depending on the region, the subtype, thus helping in defining the origin of infection and tracing the source of contamination. However, the great genetic diversity of HEV viruses, classified into four major genotypes and several subtypes within each genotype, makes it very difficult to design sensitive universal primers. Several RT-PCR assays have been developed capable of detecting different HEV types, including those derived from animals. In this work we compared the performances of 5 widely used broad spectrum methods targeting the ORF1, ORF2, and ORF2/3 regions, using a collection of positive serum samples previously confirmed as HEV-positive by anti-HEV IgM and IgG assays as well as by Real-Time PCR
. In total, only 46% of serum samples tested positive for HEV RNA by nested PCR, combining the results obtained with the 5 assays. The conventional PCR assays were less sensitive than the Real-Time assay, being able to detect only a portion of the positive samples. It should be noted, however, that a comparison of the sensitivity of Real-Time PCR vs. conventional PCR was out of the scope of this study; indeed the quantitative and qualitative analyses were not performed simultaneous, on the same extracted RNAs. Serum samples were collected from 2004 to 2013 in Italy and analyzed immediately after collection, by a diagnostic strategy based on genomic (Real-Time) and serological assays
. They were then stored at -70°C until the genome extraction for this study; the newly extracted RNAs were then aliquoted and thawed only once. Thawing of serum and RNAs are possible causes of HEV RNA degradation and could partly explain the lower sensitivity of PCR assays even if a correlation between time of storage and performances of PCR assays was not observed.
In samples found positive by both quantitative and qualitative PCR, we found, in general, a positive trend between the yield of RT-PCR positivity and the viral load. However, some samples were not detected by the nested assay despite the high viral load; on the other hand, samples with low RNA quantity were detected. In these cases, the sensitivity of the nested PCR assays was not linked to the viral load, therefore likely depending on the genetic variability of the strains. Eleven samples were positive for HEV-RNA by one or more of the above assays: 9 were positive for G1 (imported) and two for G3 (authoctonous). The countries from which HEV cases seem to have been imported are Bangladesh, India, and Thailand, in agreement with previous Italian studies
[6, 7]. Phylogenetic analysis showed a match between sequences derived from patients with travel-related HEV and sequences from the geographical regions in which infection was acquired. The two HEV G3-positive patients had not travelled outside Italy; unfortunately risk factors for these infections have not been investigated. Sequences from patients with autochthonous HEV clustered on the same branch with published swine HEV isolates, which are thought to play a role in the transmission of HEV. It is noteworthy that they also clustered with HEV sequences detected in sewage and river samples in Italy. In industrialized countries HEV has been detected in different water environments
; moreover, infectious particles have been reported to occur in sewage, indicating the existence of a potential public health risk from the contamination of surface water with HEV
Analysis of the relationship between the genotype and the viral load showed that HEV G1 (imported, travellers) infections use to display a higher viral load than HEV G3 (authoctonous, non-travellers) infections. The overall yield of RT-PCR testing found among samples from travellers was twice higher than the yield obtained among the non-travellers. Differences in viral concentration may possibly be linked with the severity of symptoms; indeed, in a previous study, we found that the course and outcome of clinical illness in patients developing travel-related HEV were different from those observed in patients developing autochthonous HEV. Patients from the first group were all hospitalized due to severe symptoms; the second group of patients, on the other hand, showed less severe symptoms and did not require hospitalization
As for the performance of the different assays, Method A which targets the MTase gene displayed a marked higher analytical sensitivity than the other assays, with 8/11 positivities detected; moreover it detected both G1 and G3 HEV strains. Method B, which targets the RdRp detected only 3 out of 11 positive samples. This method was chosen for this study since it was successfully used recently for the identification of HEV in symptom-free migrants and environmental samples in Italy
; in the present study the sensitivity was low, despite having attempted to improve PCR sensitivity using different RNA dilutions or PCR conditions (data not shown). However, it was able to detect two additional G1-positive samples (not detected by Method A). Considering both assays, therefore the ORF1 region seems to be the most suitable for HEV genotype identification, with 10/11 positivities detected. However the ORF1 assays are not useful for subtype characterizations which will require the sequencing of the capsid region. The capsid assays (Methods C and D) were also found to be less sensitive than the ORF2/3 assay; similar results were obtained by Inoue and coworkers who found the ORF2/3 (Method E) to be two to three times more sensitive than ORF2 PCR (Method C)
Although the higher sensitivity of Real-Time assays makes them more promising for diagnostic use, it is important to note that they are not useful for sequencing so the improvement of conventional RT-PCR assays is still needed to obtain information on molecular epidemiology of HEV.