Improved PCR Methods for Detection of African Rabies and Rabies-Related Lyssaviruses

Abstract

Eleven different lyssavirus species, four of which occur on the African continent, are presently recognized. These viruses cause rabies, the burden of which is highest in the developing world, where routine laboratory diagnosis is often not available. From an epidemiological and control perspective, it is necessary that diagnostic methods detect the diversity of lyssaviruses present in different regions of the world. A published and widely used heminested reverse transcription-PCR (hnRT-PCR) was evaluated for its ability to detect a panel of diverse African lyssaviruses. Due to the limitations experienced for this assay, an alternative hnRT-PCR was developed. The new assay was found to be accurate and sensitive in the detection of African lyssavirus RNA in a variety of clinical specimens. The assay was further adapted to a real-time PCR platform to allow rapid, one-step, quantitative, and single-probe detection, and an internal control for the verification of sample preparation was included. The limit of detection of the real-time PCR assay was 10 RNA copies per reaction, with inter-and intra-assay variability below 4%. Subsequently, in demonstrating utility, both assays were successfully applied to antemortem rabies diagnosis in humans. We believe that the quantitative real-time PCR assay could find application as a routine confirmatory test for rabies diagnosis in the future and that it will serve as a valuable research tool in the biology of African lyssaviruses. Alternatively, the hnRT-PCR assay can be used in laboratories that do not have access to expensive real-time PCR equipment for sensitive diagnosis of lyssaviruses.

Lyssavirus Detection and Typing Using Pyrosequencing

“…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 (5,9,19,20,35). The method was in fact capable of detecting less than 1 TCID 50 /ml and 10 gene copies/l of the synthetic RNA of RABV.…”

“…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 (4,5,20,30,40).…”

“…Molecular protocols dedicated to RNA viral detection demonstrate good performances for human rabies diagnosis and are currently adopted as diagnostic tools. They are based on nested and real-time reverse transcription (RT)-PCR (5,9,19,30) or nucleic acid sequence-based amplification (NASBA) (36,37). Other protocols have also been developed to be used in animal diagnosis (3,20,35,38,39).…”

Antibodies against Duvenhage Virus in Insectivorous Bats in Swaziland

“…After sampling the bats were released. Swabs were tested for lyssavirus RNA with the use of a reverse transcription-real time polymerase chain reaction assay (Coertse et al, 2010) after total RNA extraction with the use of Trizol (Invitrogen, Carlsbad, California, USA) as described by the manufacturer. Virus-neutralizing antibodies (VNA) in serum samples were determined by a modification of rapid fluorescent focus inhibition test (RFFIT; Kuzmin et al, 2008), with DUVV2006 used as a challenge virus.…”

A case of human survival of rabies, South Africa

“…All the specimens repeatedly tested negative by rabies virus real-time PCR. 9 Extensive investigation to determine other possible causes of viral encephalitis and bacterial meningitis did not provide the diagnosis. It was subsequently revealed that the child was also scratched on the forehead by a neighbour's dog on 29 March 2012, for which the child received no medical attention or PEP.…”