If you don't remember your password, you can reset it by entering your email address and clicking the Reset Password button. You will then receive an email that contains a secure link for resetting your password
If the address matches a valid account an email will be sent to __email__ with instructions for resetting your password
Institut Pasteur, Lyssavirus Dynamics and Host Adaptation Unit, National Reference Centre for Rabies, WHO Collaborating Centre for Reference and Research on Rabies, Paris, France
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
The diagnosis of dog-mediated rabies in humans and animals has greatly benefited from technical advances in the laboratory setting. Approaches to diagnosis now include the detection of rabies virus (RABV), RABV RNA, or RABV antigens. These assays are important tools in the current efforts aimed at the global elimination of dog-mediated rabies. The assays available for use in laboratories are reviewed herein, as well as their strengths and weaknesses, which vary with the types of sample analyzed. Depending on the setting, however, the public health objectives and use of RABV diagnosis in the field will also vary. In non-endemic settings, the detection of all introduced or emergent animal or human cases justifies exhaustive testing. In dog RABV-endemic settings, such as rural areas of developing countries where most cases occur, the availability of or access to testing may be severely constrained. Thus, these issues are also discussed along with a proposed strategy to prioritize testing while access to rabies testing in the resource-poor, highly endemic setting is improved. As the epidemiological situation of rabies in a country evolves, the strategy should shift from that of an endemic setting to one more suitable for a decreased rabies incidence following the implementation of efficient control measures and when nearing the target of dog-mediated rabies elimination.
The development of rabies diagnostic tests began after the routine inoculation of rabbits with rabies virus (RABV)-infected brain and saliva samples in 1880,
Several different assays and diagnostic approaches are now available, and these represent important assets in the renewed global efforts to eliminate dog-mediated human rabies.
But how should these tests be used in the operational setting, especially in countries with a high caseload of dog-mediated rabies? In what sequence? For which expected level of performance?
This review comprises an overview of currently available assays and their strengths and weaknesses. Furthermore, a rabies testing strategy based on the authors’ field experience is proposed. The assays are examined by type and in terms of the type of sample used and their usefulness in the endemic and non-endemic settings. The aim of this review is to guide virologists on the constraints and priorities of surveillance and detection, as well as animal and human rabies programme managers on the diagnostic methods and their performance.
2. Principles of RABV diagnostic tests
The various available reference diagnostic approaches resulting from international and academic initiatives have been described extensively in previous publications.
Evaluation of a rapid immunodiagnostic rabies field surveillance test on samples collected from military operations in Africa, Europe, and the Middle East.
Table 1Summary of RABV laboratory techniques and their advantages and limitations
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
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)
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
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
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
Evaluation of a rapid immunodiagnostic rabies field surveillance test on samples collected from military operations in Africa, Europe, and the Middle East.
Historically and in the research setting, RABV infection is identified by infecting cells and detecting virus. This can be done either through the mouse inoculation test (MIT) or by inoculation of samples onto cultures of murine neuroblastoma or other cells (rapid tissue culture infection test, RTCT).
Following intracerebral inoculation of mice aged 3–4 weeks, MIT test results are available after an incubation period of up to 28 days. Some strains are associated with a longer incubation period. In laboratories with cell culture facilities and an appropriate level of bio-containment, the RTCT provides results within 24–48 h, which is far quicker than intracerebral inoculation.
Comparative field evaluation of the fluorescent-antibody test, virus isolation from tissue culture, and enzyme immunodiagnosis for rapid laboratory diagnosis of rabies.
As RABV does not cause any cytopathic effect, the detection of the virus must therefore be evidenced by direct fluorescent antibody testing (DFAT, see below). In addition, MIT requires animal facilities to produce mice (or a supplier that can quickly provide animals of a suitable age in sufficient numbers), as well as animal facilities with a high level of bio-containment (ASL3) to maintain the inoculated animals. Animal ethics regulations also recommend avoiding the use of animals when an efficient cell culture system exists.
As results are usually required urgently and due to animal protection issues, the World Health Organization (WHO),
now recommend replacing the MIT with the isolation of RABV in cell culture whenever possible.
2.2 Detecting viral RNA in samples
The international health organizations do not currently consider molecular assays to be reference techniques for the post-mortem diagnosis of RABV in humans and animals. However, they are recommended for intra vitam diagnosis in humans. Although they require laboratory technicians to be trained and validated in molecular techniques, a PCR assay has been shown to be more sensitive than DFAT for the detection of lyssaviruses.
Development of a hemi-nested RT-PCR method for the specific determination of European bat lyssavirus 1. Comparison with other rabies diagnostic methods.
RT-PCR is the method most often used to detect RABV RNA for intra vitam diagnosis of rabies in humans. Saliva samples or skin biopsies taken at the nape of the neck (being careful to include hair follicles)
can be tested using this technique. The test reaches 100% sensitivity when at least three successive saliva samples are collected at 3- to 6-h intervals (due to irregular viral shedding) and tested.
Positive results can be obtained as soon as the patient is admitted. Human and animal brain samples collected by various methods can also be tested with this technique, even when they have been kept at relatively high ambient temperatures or when they have become degraded.
Several sets of more or less consensual primers for RABV have been developed. In some cases, primers specific for variant types of RABV have been developed for precise and geographically limited purposes, especially research. Although time- and resource-intensive, the sequencing of PCR amplicons can improve the specificity of the technique. Specificity is very high when the technique is implemented rigorously, but false-negatives and false-positives may occur. RT-PCR is highly susceptible to cross-contamination in the operational setting, unless standardization and procedures are stringent for the PCR itself and for the sample extraction
These assays are based on the transcription of viral RNA to cDNA before amplification (RT). This phase is followed by PCR, which uses specific primers and probes or a dye to provide real-time quantification of DNA. These assays reduce the risk of cross-contamination thanks to closed tubes and show an improved sensitivity compared to conventional RT-PCR protocols.
However, as the probes used are highly specific for known sequences, sequence mismatch between the primer/probe sequences and the target viral sequence may adversely affect the sensitivity of the test, leading to false-negative results. Because the PCR fragments generated are usually very short, the sequencing of the amplicons may provide diagnostic confirmation but cannot be used for in-depth molecular phylogenetic analyses.
DFAT is the main assay used worldwide; it is the WHO and OIE recommended gold standard for the diagnosis of rabies in fresh or frozen brain samples. The latter are important in tropical countries, as preserving fresh samples at 4 °C is often a challenge.
This assay is based on attaching fluorescein isothiocyanate (FITC) to polyclonal antibodies targeting the RABV ribonucleocapsid, or monoclonal antibodies targeting the RABV nucleoprotein (N). If the targeted RABV antigen is present in the sample fixed on a slide, antibodies attach to it, remain attached despite washing, and can be observed using a fluorescence microscope.
Results are available within 1–2 h and are expressed as positive or negative. The sensitivity and specificity of DFAT nears 99% in an experienced laboratory, but is extremely observer-dependent.
Comparative field evaluation of the fluorescent-antibody test, virus isolation from tissue culture, and enzyme immunodiagnosis for rapid laboratory diagnosis of rabies.
At least two observers must spend enough time on each slide once the quality of the sample has been ensured. This test is best performed on fresh brain samples; the reliability of this assay to diagnose rabies in degraded animal brain samples or corneal smears is low.
ELISA techniques have been adapted to detect RABV antigens in samples using monoclonal or polyclonal antibodies. These use microplates coated with purified polyclonal or monoclonal anti-RABV IgG targeting the nucleocapsid. Several versions of these assays have been developed (RREID, WELYSSA, etc.) and some have been commercially available for some time.
Test results can be evaluated qualitatively with the naked eye. However, these techniques are less sensitive than DFAT (96% agreement between DFAT and RREID test results).
For this reason, they should not replace DFAT in laboratories where DFAT is already performed. These are now implemented in a very limited number of countries, using home-made reagents.
2.3.3 Rapid immunodiagnostic test (RIDT)
The Antigen Rapid Rabies Ag Test Kit (BioNote, Inc., Gyeongi-do, Republic of Korea) is a lateral flow device based on a qualitative chromatographic immunoassay developed for the detection of RABV antigen in fresh animal brain tissue (e.g., canine, bovine, raccoon dog). This type of new RIDT appears suited to the field and frontline laboratories. It is performant (specificity and sensitivity) for brain samples from any animal and is highly dependent on the antibodies (polyclonal or monoclonal) used. This test has a sensitivity ranging from 91.7% to 96.9% and a specificity ranging from 98.9% to 100% when compared to a fluorescent antibody test.
Evaluation of a rapid immunodiagnostic rabies field surveillance test on samples collected from military operations in Africa, Europe, and the Middle East.
Furthermore, a result can be obtained within 15–20 min, including the preparation time. This test appears to be very accurate in detecting RABV antigen, but other tests are required if questionable results are obtained. However, the RIDT kit requires further validation before it can be recommended for use by either the OIE or WHO. At this stage, the RIDT should be implemented (1) for research purposes only, or (2) in frontline laboratories to improve the surveillance and control of rabies in remote places from which the shipment of samples to a central laboratory would be difficult or even impossible (or where classical rabies laboratory diagnosis using recommended techniques cannot be established for financial or logistical reasons). Many different products using the same methodology are commercially available and these exhibit highly variable intrinsic properties. Laboratories aiming to use the RIDT should therefore evaluate the kit carefully and assess the specificity and sensitivity – or should rely on available evaluations performed by international reference laboratories – before routine use.
2.3.4 Direct rapid immunohistochemical test (dRIT)
This promising method was developed recently by the US Centers for Disease Control and Prevention (CDC). It is based on the detection of rabies N protein in brain smears fixed in formalin, using highly concentrated monoclonal antibodies in the presence of streptavidin peroxidase and a substrate colouring agent. Test results are available within 1 h. This test can be implemented in the field and no fluorescence microscope is required.
This method can also be used for samples that have been frozen or preserved in glycerol. Cost-effective indirect immunochemistry assays have been developed that can also provide indications on the RABV variant.
A major concern, however, is access to uninterrupted supplies of controlled batches of monoclonal antibodies, which are only available through a few laboratories specialized in rabies diagnosis.
The detection of antibody in serum in the absence of a history of rabies vaccination, or in cerebrospinal fluid (CSF), provides indirect evidence of rabies infection. However, the interpretation of test results may be difficult, since the host immune response may vary among individuals: the sensitivity and negative predictive value of antibody detection methods in rabies patients is very poor,
as patients with suspected rabies overwhelmingly die before they can mount an antibody response. The antibody response is only detectable in the blood (or CSF) after 8–10 days,
These tests are thus better suited to assess the protection of laboratory or veterinary workers or of pets before transboundary travel, or to check for the appropriate immune response in patients receiving post exposure prophylaxis (PEP) as part of research (vaccine evaluation, seroprevalence studies). Considering these constraints and the low sensitivity in the context of rabies diagnosis in humans and animals, the techniques used for serology will not be described here.
3. Why and when should we diagnose rabies?
In non-endemic settings, it is imperative that all suspected human and animal cases be documented. Rabies laboratory diagnosis is essential to detect the importation or emergence of rabies and to guide the public health response or individual case management.
In endemic settings, the early diagnosis of rabies in the head of a suspect animal after euthanasia or natural death (the biting animal) is essential, as it can help inform PEP and the consequent need for rabies immunoglobulin. Assays such as RIDT and dRIT (or ELISA assays, such as RREID, if available) offer the possibility of testing the biting animal's head at frontline bite centres to guide timely and adequate PEP for bite victims in rural settings of endemic countries, where most human rabies deaths occur. However, a diagnosis of ‘probable rabies’ in humans can routinely rely simply on the presence of a bite from a potentially rabid dog or other mammal and clinical signs in the patient, especially those compatible with furious rabies.
Laboratory diagnosis remains essential in the endemic setting to guide public health surveillance rather than individual patient management for an incurable disease. Testing is also helpful in human cases of ‘paralytic’ rabies, as encephalitis may be caused by RABV or several other pathogens co-circulating in many rabies-endemic settings.
Finally, when the biting dog's head is not available for testing, a positive diagnosis in a patient can help guide PEP in other bite victims if they have remained asymptomatic; these victims should receive PEP even months after the bite.
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).
The laboratory diagnosis of rabies in humans is also a priority particularly when PEP with adequate and adequately conserved vaccine has been undertaken and failed, in order to identify the virus.
Finally, the laboratory diagnosis of rabies in endemic settings provides reliable, laboratory-confirmed data to complete the few existing estimations of rabies burden documented in humans as part of research efforts.
Findings from such research projects are crucial to increase awareness about the rabies burden among policy-makers and to prioritize resources towards its control.
4. Using diagnostic techniques in the operational setting
4.1 Performance issues
Assays used will vary, depending on the time elapsed since infection or the objective sought (Figure 1). In practice, direct diagnosis by RABV protein detection using DFAT is the reference technique that should be implemented in national reference laboratories whenever possible. Direct diagnosis by detection of genome fragments using PCR-based assays must be used only by experienced teams, bearing in mind that this technique is based on validated in-house tests and not standardized, commercially available kits. Cell culture isolation of the virus is usually reserved for reference laboratories and research. Isolation of the virus, as well as PCR-based methods, may be used as a second-line test to confirm negative results of other tests such as DFAT in animals, or in the case of isolation to amplify live virus from original samples.
Figure 1Proposed rabies testing algorithm based on the objectives of testing and the methods to be used in a specialized laboratory.
In post-mortem human brain samples, DFAT and PCR-based techniques are highly effective for the detection of RABV antigen and genome, respectively. In such samples, RT-PCR and RT-qPCR are useful alternatives to DFAT (Figure 1). These assays can also be used to test saliva or nuchal skin biopsy samples for intra vitam or post-mortem diagnosis in humans.
4.2 Operational issues
The choice of assay may vary depending on the nature of the sample to be tested and also its state of preservation
(Figure 1). DFAT, for example, remains less expensive than RT-PCR and can be used to test samples locally, however the latter remains more effective when testing degraded samples.
In many endemic settings, the absence of a comprehensive network to transport samples to the laboratory makes it difficult to maintain staff competence in DFAT, and fluorescent conjugates may reach their expiry date before being fully used. Method simplicity, the availability of laboratory equipment, and the cost and ease of maintenance of this equipment are important factors to consider in the choice of laboratory assays before their implementation.
but a level 2 biosafety laboratory (BSL-2) is considered adequate containment for diagnosis and other activities, as long as they are performed by duly immunized personnel and are not associated with a high potential for droplet or aerosol production and do not involve large quantities or high concentrations of infectious material.
Diagnosis and routine activities are undertaken by trained and duly immunized personnel in BSL-2 laboratories in most rabies-endemic developing settings that do not routinely have BSL-3 laboratory capacity.
4.3 Practical and logistical issues of rabies diagnosis in animals
Testing live animals for ongoing suspected rabies is not recommended
(Figure 1). Animals should be quarantined for 10–14 days (depending on local regulations) or, in the case of suspected rabies, be humanely euthanized with no damage to the head. Brain samples can then be analyzed in the laboratory setting.
One set of tools should be used for each specimen. The brain must be extracted from the animal's cranium by trained and immunized personnel in a safe and dedicated laboratory with a clean autopsy table and sterilized equipment. These precautions will prevent false-positive results due to sample contamination. After extracting the brain attached to the cerebellum and medulla oblongata, tissue samples should be taken from more than one site among Ammon's horns (hippocampus) and the medulla oblongata (in the brainstem). Additional recommended samples include the following: cerebral tissue for OIE,
A pipette or a straw can also be used to rapidly collect brain samples during field studies in wild or domestic animals, or when necropsy of all animal heads is impossible due to too many samples coming to the laboratory for testing.
4.4 Practical issues of rabies diagnosis in humans
As in animals, post-mortem diagnosis in humans can easily be performed on brain samples obtained in a pathology laboratory, as above. Autopsies, however, are increasingly refused by next of kin for cultural reasons, or when funeral rites must be performed without delay.
If a post-mortem autopsy cannot be performed for a human subject, minimally invasive methods may be used to sample tissue for rabies diagnosis while respecting the corpse's integrity (Figure 2).
Figure 2Minimally invasive tissue sampling techniques for rabies diagnosis in humans.
Nuchal skin biopsies can be performed post-mortem but also intra vitam in sedated patients. An optimal diagnostic strategy will associate nuchal biopsy with testing of at least three consecutive saliva samples (ideally at 3–6 h intervals), which is highly specific and sensitive, irrespective of the time of collection (i.e., 1 day after the onset of symptoms until just after death).
Two other minimally invasive methods for post-mortem brain sampling without craniotomy in humans have also been described: sub-occipital cisternal puncture and the retro-orbital route using a catheter (Figure 2).
Nuchal samples are best shipped frozen, but can be shipped simply with ice packs. Saliva samples should be shipped frozen. Brain samples can be frozen or preserved in 50% glycerol–saline solution if freezing is not readily available.
Samples should never be preserved in formalin. The reference laboratory must be contacted before the shipment of samples with suspected RABV. They should be sent in a double-enclosed waterproof container, with cooling material and absorbent material. All of these should be placed in a leak-proof outer container, in observance with national and International Air Transport Association (IATA) guidelines as ‘category B’ necessitating UN 3373/650 packaging (only RABV cultures are considered ‘category A’).
Sampling and shipping procedures must be well-established and communicated effectively to laboratory staff before they have to be put into use. Samples may be sent on filter paper at ambient temperature for easier shipment.
Filter paper such as FTA also has the advantage of inactivating RABV, reducing the hazard and facilitating shipment while preserving nucleic acids.
5. Conclusions
The laboratory diagnosis of rabies is imperative to guide public health measures in non-endemic settings, document protection (intra vitam serology on vaccinees’ blood samples), and to conduct research (seroprevalence studies, death despite timely PEP).
Although laboratory testing is desirable, it is extremely limited in resource-poor settings and/or rural areas of endemic countries, where most cases occur worldwide. Public health surveillance needs in the endemic setting with high caseloads may therefore be satisfied by resorting to syndromic case definitions and past history of a dog bite.
Diagnosis based on molecular biology tests and virus isolation, however, may help guide PEP, is especially useful in ‘paralytic’ rabies cases, and may provide information on phylogeny for documenting the introduction/emergence of RABV in a geographic area.
As the epidemiological situation evolves, the strategy should shift from that suited for an endemic setting to one adapted to a non-endemic setting. Many assays available to date are sensitive and specific but must be implemented by an experienced team with uninterrupted supplies. Rapid tests (dRIT and RIDT) have recently been developed, offering the possibility for first-line rabies laboratory diagnosis in less-equipped laboratories or in more remote areas. Should further external validation of the RIDT confirm its initial performance and usability in both saliva and brain samples,
Comparative field evaluation of the fluorescent-antibody test, virus isolation from tissue culture, and enzyme immunodiagnosis for rapid laboratory diagnosis of rabies.
this assay may become a good alternative in the field, for example in provincial hospitals or vaccination centres where reference laboratory methods of diagnosis are not readily available. The same is true for dRIT when reagents become standardized and available on a regular basis, with adequate quality control.
Conflict of interest: The authors have no conflict of interest. Philippe Buchy is an employee of GSK Vaccines.
Evaluation of a rapid immunodiagnostic rabies field surveillance test on samples collected from military operations in Africa, Europe, and the Middle East.
Comparative field evaluation of the fluorescent-antibody test, virus isolation from tissue culture, and enzyme immunodiagnosis for rapid laboratory diagnosis of rabies.
Development of a hemi-nested RT-PCR method for the specific determination of European bat lyssavirus 1. Comparison with other rabies diagnostic methods.
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).
Biosafety: guidelines for working with pathogenic and infectious microorganisms.
in: Coico R. McBride A. Quarles J.M. Stevenson B. Taylor R.K. Current protocols in microbiology. John Wiley and Sons, Inc,
Hoboken, NJ2009 (p. 1A.1.1–1A.1.14)
31003-7&id=gr1.jpg){kind=link}
31003-7&id=gr2.jpg){kind=link}