In the present study, a one-step RT-PCR assay followed by pyrosequencing was developed to potentially and simultaneously detect and rapidly type all known lyssaviruses. The 3′ terminus of the viral genome as a target for the amplified product allowed us to properly identify the virus involved in the infection, either by applying pyrosequencing or classical sequencing. We demonstrated that the pyrosequencing protocol could easily differentiate among lyssavirus species, and results were in complete agreement with those obtained using the Sanger sequencing method.
To date, real-time and heminested RT (hnRT)-PCRs have been favored in the diagnosis of rabies, as they provide a better performance if compared to endpoint, not nested, protocols. However, the one-step RT-PCR developed in this study demonstrated high sensitivity and specificity levels and a high value of accuracy here as well as in previously published studies (5
). The method was in fact capable of detecting less than 1 TCID50
/ml and 10 gene copies/μl of the synthetic RNA of RABV.
Several advantages characterize this newly developed method, such as the rapidity in obtaining a final diagnosis, the lower cost, and the possibility to directly identify the lyssavirus species involved in the infection. A maximum time span of about 5 h from the submission to the final typing of a positive field sample is needed to analyze a suspect sample in laboratory conditions. Starting from the amplified products, a pyrosequencing analysis requires only 2 h to obtain the final result in almost half the time of that for classical Sanger sequencing. Pyrosequencing analysis does not need any terminator enzymes or prior purification steps, thus resulting in limited costs per analysis if compared to classical sequencing. In fact, we have estimated the average cost (calculation made according to European standards) for the developed pyrosequencing protocol between 3.87 to 6.03 euros/sample (for 96 or 25 samples analyzed/run, respectively), compared to 9.86 euros/reaction for the classical sequencing method.
We found a total of only 9 discrepant results out of the analyzed 897 samples using multiple diagnostic tests (). This occurrence is a rather expected event when a large set of clinical samples are included in a validation process, and this could have several explanations, such as the different targets and sensitivity levels of the applied protocols. Molecular methods are able to reveal low infection levels, as well as low levels of contamination potentially occurring during the diagnostic process, from the collection of samples to the final step of amplification.
Although the occurrence of detecting failures could not be completely excluded, the use of the one-step RT-PCR in association with pyrosequencing presents the advantage of being a mutation-resistant method, since it is based on primers designed on conserved regions, while the most variable region is pyrosequenced. Any unexpected polymorphisms occurring in the target sequence could be easily identified. This is in contrast with probe-based methods, such as real-time PCR, for which results can be negatively affected by the occurrence of viral mutations since the design of the probe is on the most variable region. In the case of pyrosequencing, the 30- to 40-nt fragment is sequenced more efficiently than that obtained by the Sanger method. The purpose of applying pyrosequencing is to give rapid information on the species responsible for the infection. Although further molecular analyses and phylogeny are beyond the aims of the method, results obtained can be easily integrated by conventional sequencing. Thus, the use of PCR products previously obtained and primers used for the one-step RT-PCR optimize and reduce time of testing. Further details about the lineage of the virus and its phylogeny will be obtained by sequencing the region amplified (603 bp).
The method that we developed has the advantage of being applicable to a variety of samples of human and animal origins and represents the main diagnostic tool when standard protocols are not applicable, i.e., intra vitam
diagnosis. The majority of samples analyzed were brain specimens, and only 70/897 samples were from different matrices (). However, the analytical sensitivity of the method has been tested on saliva, showing the same sensitivity values as those for brain specimens and cell culture supernatants (). In addition, we have analyzed a panel of skin biopsy specimens in parallel, comparing this method to the previously described hnRT-PCR (9
) and obtaining an almost perfect agreement (see Table S2 in the supplemental material). We acknowledge that further evaluation of the method on a larger panel of matrices is advisable before applying it routinely on these types of clinical specimens. Only 33 saliva specimens/oral swabs and 26 skin biopsy samples were tested in this study (). However, it should also be taken into account that these matrices are not regularly submitted to the laboratories, and therefore, they are not easily available for validation purposes.
We analyzed over 300 RABV representatives from 4 different continents, and strains belonging to LBV, MOKV, DUVV, EBLV-1, EBLV-2, and ABLV. Moreover, results from in silico
analyses clearly indicate that the method has the potential to detect all known lyssaviruses, including those belonging to putative new species. A further screening of a wider panel representative of the worldwide diversity of known and emerging lyssaviruses should be applied in order to assess the performances of the method developed in this study. Improvements and subsequent revalidation of molecular diagnostic protocols should always be taken into consideration, not only for rabies but also in the cases of other viral diseases (4
In the last decades, the use of molecular methods has largely been adopted as an alternative to diagnose viral diseases, proving to be a valid aid to a diagnostic virological technique (10
). The lack of standardization and interlaboratory reproducibility and quality issues, such as cross-contamination events or false-negative results, have been recognized as major constraints to the use of molecular methods. International organizations are therefore extremely cautious in suggesting standard molecular protocols for rabies diagnosis. The OIE and WHO recommend applying RT-PCR as a typing method only in specialized laboratories, while following, however, recommended validation guidelines (1
), and not as a routine technique for postmortem diagnosis. From a more comprehensive point of view, a diagnostic approach based on the application of good laboratory practices, interlaboratory standardization, and the complementary use of both classical and molecular protocols will help overcome the intrinsic limitations of diagnostic methods currently available for rabies (10
). The use of an internal amplification control (IAC) is recommended to eventually identify false-negative samples resulting from the presence of PCR inhibitors or the degradation of the nucleic acid (21
). In this regard, endogenous (i.e., a housekeeping gene) or exogenous nucleic acid (i.e., a microorganism which is usually not found in the sample type to be tested) can be used as an internal control. Such internal controls can be applied to many different tests, running independently of the specific virus assay and currently available in molecular diagnostic laboratories. Alternatively, a competitive IAC (i.e., the target and the IAC are amplified with one common set of primers and under the same conditions in the same PCR tube) can be developed, bearing in mind that all these approaches have both advantages and limitations. For example, if a competitive IAC approach is applied, the different amplification assays will be competing for the same reagents in the same tube; thus, target detection sensitivity may be adversely affected (14
). Although at the moment molecular methods cannot completely replace standard techniques, they should be taken into account as confirmatory tests in the case of inconclusive results for intra vitam
diagnosis of human rabies and for lyssavirus characterization. The one-step RT-PCR developed in this study, followed by pyrosequencing, was validated in compliance with international guidelines for diagnostic molecular techniques (1
). The method can be notably used in combination with standard methods as an early-warning detection tool, since it is capable of rapidly revealing the emergence or the introduction of a novel lyssavirus species in a given susceptible population or geographical area.