Highlights
- •Rabies remains a neglected disease in most developing countries.
- •Significant progress and efforts towards rabies control have been recorded in some enzootic countries.
- •A stepwise approach to the global elimination of canine-mediated rabies is now being championed by health authorities worldwide and the strategy for rabies diagnosis and surveillance should be adapted to the progress made.
- •Several assays are available for the diagnosis of rabies virus (RABV) infection in animals and humans.
- •The various assays all have strengths and weaknesses, and their performance varies with the type of sample analyzed, their conservation during shipment, the expertise of personnel, and the laboratory environment (equipment, maintenance).
- •The use and the criteria for selecting these assays will vary according to the sample analyzed and the objective of the diagnosis, which will also vary depending on whether the setting in endemic or non-endemic.
Summary
Keywords
1. Introduction
Roux EPP. Des nouvelles acquisitions sur la rage. Paris: A. Parent, Imprimeur de la Faculté de Médecine; 1883, Available at: http://www2.biusante.parisdescartes.fr/livanc/?cote=TPAR1883x398&do=pdf (accessed January 20, 2016).
2. Principles of RABV diagnostic tests
Test | Principle | Advantages | Limitations | Ref. |
---|---|---|---|---|
Detecting viral replication | ||||
Mouse inoculation test (MIT) | Intracerebral inoculation into young mice for virus amplification | Sensitive; amplifies virus for identification; easily performed; possibility of isolating infectious virus | Delayed results (up to 28 days); more expensive than RTCT; not recommended by WHO; requires animal facilities and adequate containment; potential animal ethics issues, as alternative methods exist | 12 |
Rapid tissue culture infection test (RTCT) | Inoculation of sample onto cell cultures (e.g., neuroblastoma cells) | Faster and cheaper than mouse inoculation test; sensitivity comparable to MIT; no mice sacrificed | Requires training and manpower, as well as cell culture systems and fluorescence microscopy facilities; sensitive to toxic and bacterial contamination; amplification of live virus may require adequate biosafety (safety cabinets and BSL-3 laboratory) | 12 , 15 |
Detecting viral RNA | ||||
Reverse-transcriptase PCR (RT-PCR) | Transcribes viral RNA to cDNA and amplifies it using specific primers with further detection of PCR products in agarose gel | Applicable to any sample; highly sensitive and specific; PCR products can be used for further nucleotide sequencing | Time- and resource-intensive; cross-contamination and false-positives are a risk; no commercial diagnostic kits currently available | 2 , 12 , 29 |
Real-time reverse-transcriptase PCR (RT-qPCR) | Transcribes viral RNA to cDNA and amplifies it using specific primers and probes with detection of PCR products in real time | Less cross-contamination; applicable to any sample; sometimes more sensitive than conventional RT-PCR; highly specific probes | Single sequence mismatch between the primer or probe sequence and the target viral sequence may alter the sensitivity of the test and even cause false-negative results; PCR products are too short and unsuitable for nucleotide sequencing; no commercial diagnostic kits currently available | 12 , 29 , 30 |
Detecting viral antigens/proteins | ||||
Direct fluorescent antibody test (DFAT) | Use of polyclonal or monoclonal FITC-conjugated antibodies for detection of rabies virus antigens by means of fluorescence microscopy | Gold standard for fresh or fixed brain samples; high sensitivity and specificity, even on fixed specimen; results obtained quickly; commercial diagnostic kits are available | Interpretation requires well-trained personnel (results highly observer-dependent) and a costly fluorescence microscope; less suitable on degraded samples | 2 , 8 , 12 |
Antigen capture ELISA: rapid rabies enzyme immunodiagnosis (RREID) | Immunohistochemical technique based on the capture of various rabies antigens by specific antibodies labelled with enzyme | Highly specific but less sensitive than DFAT (96% agreement between DFAT and RREID test results); usable even on partly degraded brain samples; qualitatively readable with the naked eye; a large number of samples can be tested at the same time (screening) | Can be used on brain tissues only; requires great care to preserve specificity; no commercial diagnostic kits available | 12 , 29 , 35 , 36 , 37 , 38 , 39 , 40 , 41 |
Rapid immunodiagnostic test (RIDT) | Immunochromatographic assay based on monoclonal antibodies to capture rabies antigens | Highly sensitive and specific but usually less so than DFAT; usable on brain and saliva samples from animals; results obtained rapidly | Dedicated for research purposes only; need for further validation before either OIE or WHO can recommend its use | 11 , 43 , 44 |
Direct rapid immunohistochemical test (dRIT) | Biotinylated monoclonal antibodies for the detection of rabies virus antigens by means of normal light microscopy | High sensitivity and specificity; no need for fluorescence; results obtained quickly | Reagents difficult to obtain: need to identify an uninterrupted supply chain of quality-controlled monoclonal antibodies for sustainability | 12 , 29 , 45 |
2.1 Detecting virus: inoculation tests
2.2 Detecting viral RNA in samples
2.2.1 Reverse-transcriptase PCR (RT-PCR)
2.2.2 Real-time reverse transcriptase PCR (RT-qPCR)
2.3 Detecting viral antigens
2.3.1 Direct fluorescent antibody testing (DFAT)
2.3.2 Rapid rabies enzyme immunodiagnosis (RREID)
2.3.3 Rapid immunodiagnostic test (RIDT)
2.3.4 Direct rapid immunohistochemical test (dRIT)
2.4 Detecting antibodies
3. Why and when should we diagnose rabies?
World Health Organization. Criteria for application withholding of post-exposure treatment. Geneva, Switzerland: WHO. Available at: http://www.who.int/rabies/human/generalconsid/en/ (accessed February 5, 2016).
4. Using diagnostic techniques in the operational setting
4.1 Performance issues

4.2 Operational issues
4.3 Practical and logistical issues of rabies diagnosis in animals
4.4 Practical issues of rabies diagnosis in humans
- Tarantola A.
- Crabol Y.
- Mahendra B.J.
- In S.
- Barennes H.
- Bourhy H.
- Peng Y.
- et al.

4.5 Shipment issues
International Air Transport Association. UN 3373—biological substance, category B. IATA; 2015. Available at: https://www.iata.org/whatwedo/cargo/dgr/Documents/packing-instruction-650-DGR56-en.pdf (accessed January 20, 2016).
5. Conclusions
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