The use of a rapid automated NASBA technique was successfully applied to the ante-mortem saliva and cerebrospinal fluid (CSF) of four rabies patients in Thailand and shown to have a ten-fold increase in sensitivity compared to RT-PCR [33] . The assay detected rabies viral RNA as early as two days after onset of symptoms. The NASBA technique involves the use of three enzymes (reverse transcriptase, RNase H and T7 RNA polymerase) to synthesise multiple copies of target RNA under isothermal conditions. Briefly, a large number of RNA copies are generated using a pair of specific primers, one of which contains the T7 RNA polymerase binding site, and the other which has an electrochemiluminescence detection region attached to the 59 end. The Table 3 . Conventional, gel-based PCR-assays for the detection of lyssavirus species other than RABV. www.plosntds.org amplified RNA is detected using an automated reader, enabling rapid throughput testing. It is relatively easy to use and the whole process from extraction to detection can take as little as four hours. This technology has already been applied for point of care testing of bacterial pathogens [34] and viral pathogens [35, 36] . The NASBA technique has also been adapted to investigate rabies virus replication in situ, whereby the relatively lower isothermal temperatures of NASBA compared to in-situ RT-PCR ensure that cell integrity is maintained [37] .