Markotter_257-262.indd INTRODUCTION Rabies is caused by all members of the lyssavirus genus in the family Rhabdoviridae of the order Mononegavirales, a group of single stranded nega- tive sense RNA viruses currently consisting of seven genotypes (gts). Classical rabies viruses (RABV) are grouped in the lyssavirus genotype 1 (gt 1) and occur worldwide (Tordo, Benmansour, Calisher et al. 2004). Two biotypes of RABV occur in Africa; the mongoose biotype and the canid biotype (Von Teich- man, Thomson, Meredith & Nel 1995; Nel, Sabeta, Von Teichman, Jaftha, Rupprecht & Bingham 2005). In addition, gt 2 (Lagos bat virus (LBV)), gt 3 (Mokola virus (MOKV)) and gt 4 (Duvenhage virus (DUVV)) have been reported exclusively from the African continent (Nel & Markotter 2007). There is little sur- veillance for these gts but all have been reported from South Africa and other African locations in re- cent years (Paweska, Blumberg, Liebenberg, Hew- lett, Grobbelaar, Leman, Croft, Nel, Nutt & Swane- poel 2006; Markotter, Kuzmin, Rupprecht, Randles, Sabeta, Wandeler & Nel 2006a; Markotter, Randles, Rupprecht, Sabeta, Wandeler, Taylor & Nel 2006b; Sabeta, Markotter, Mohale, Shumba, Wandeler & Nel 2007; Kuzmin, Niezgoda, Franka, Agwanda, Markotter, Beagley, Urazova, Breiman & Rupprecht 2008). The gold standard for lyssavirus diagnostics approved by both the World Organization for Animal Health (OIE) and the World Health Organization (WHO) (WHO 2005), the fluorescent antibody test 257 Onderstepoort Journal of Veterinary Research, 76:257–262 (2009) Evaluation of a rapid immunodiagnostic test kit for detection of African lyssaviruses from brain material W. MARKOTTER1*, D. YORK2, C.T. SABETA3, W. SHUMBA3, G. ZULU3, K. LE ROUX4 and L.H. NEL1 ABSTRACT MARKOTTER, W., YORK, D., SABETA, C.T., SHUMBA, W., ZULU, G., LE ROUX, K. & NEL, L.H. 2009. Evaluation of a rapid immunodiagnostic test kit for detection of African lyssaviruses from brain material. Onderstepoort Journal of Veterinary Research, 76:257–262 A rapid immunodiagnostic test kit was evaluated against a selection of isolates of lyssavirus geno- types occurring in Africa. The test was carried out in parallel comparison with the fluorescent antibody test (FAT) and isolates representing previously established phylogenetic groups from each genotype were included. The specificity of the rapid immunodiagnostic test compared favourably with the FAT and was found to detect all representatives of genotypes 1, 2, 3 and 4 in brain samples of either field cases or suckling mouse brain inoculates. Keywords: Africa, diagnostics, lyssavirus, rabies, rapid test * Author to whom correspondence is to be directed. E-mail: wanda.markotter@up.ac.za 1 Department of Microbiology and Plant Pathology, University of Pretoria, Pretoria, 0002 South Africa 2 Molecular Diagnostic Services, Private Bag X20, Westville, Dur ban, Kwazulu Natal, 3630 South Africa 3 OIE Rabies Reference Laboratory, Onderstepoort Veterinary Institute, Private Bag X05, Onderstepoort, 0110 South Africa 4 Allerton Veterinary Laboratory, Private Bag X2, Cascades, Pieter maritzburg, 3202 South Africa Accepted for publication 1 July 2009—Editor 258 Evaluation of immunodiagnostic test kit for detection of African lyssaviruses from brain material (FAT) (Dean, Abelseth & Atanasiu 1996), is per- formed on brain tissue. These preparations are stained with anti-lyssavirus serum (conjugate) la- belled with fluorescein isothiocyanate (FITC) and viewed under a fluorescence microscope. This test therefore cannot be performed under field condi- tions since specialized equipment is needed. It is important that rabies diagnosis be quick and reliable in order to ensure correct post exposure prophylaxis (PEP) decisions and prevent fatal infec- tions in cases of human exposures. Diagnostics in epidemiological studies aimed at prevention and control of the disease in the relevant reservoir spe- cies should also be as practical as possible. Routine diagnostic tests should focus on brain tissues (brain- stem is the optimal region). Diagnosis of lyssa- viruses in humans is fundamentally different from diagnosis in animals due to the fact that the former most often requires ante-mortem diagnosis while in animals it is usually performed post-mortem (Tri- marchi & Smith 2002). Methods involved in ante- mor tem diagnosis depend on a high level of techni- cal expertise and a well equipped laboratory. In Africa and other developing regions of the world, lyssavirus diagnostics and surveillance are seriously hampered due to the lack of facilities and logistical support for reliable execution of the FAT. Fur ther- more, in some instances where a diagnostic facility does exist and is operational, the need to efficiently transport samples to a central facility can often not be met. These obstacles in obtaining a diagnostic result from field specimens have led to serious under- reporting of the disease and have ultimately resulted in a lack of commitment to control the disease. A rapid immunodiagnostic test kit (RIDT) for RABV, that could offer advantages towards overcoming some of the difficulties mentioned above, has been developed (Kang, Oh, Lee, Park, Park, Hong, Lee, Cho & Song 2007). This lateral flow test uses gold conjugated detector antibodies, including a mono- clonal antibody directed against the lyssavirus nu- cleoprotein. According to Kang et al. (2007), the test is rapid and simple and does not require any spe- cialized equipment or technical expertise—but their study included only gt 1 viruses of which none were of African origin. Since gt 1–4 lyssaviruses occur in Africa, the test should be able to identify all these gts in order to be of any potential use in Africa. It was therefore our aim to evaluate this test for its ability to detect the most diverse isolates within those African lyssavirus gts and variants known to us. MATERIALS AND METHODS Samples A total of 25 samples that included 21 diverse rep- resentatives of all the African lyssavirus genotypes and four negative controls (Table 1), were tested. Samples were selected based on previous phyloge- netic studies to be representative of different phylo- genetic groups within each genotype (Nel et al. 2005; Sabeta et al. 2007; Cohen, Sartorius, Sabeta, Zulu, Paweska, Mogoswane, Sutton, Nel, Swane- poel, Leman, Grobbelaar, Dayson & Blumberg 2007; Markotter, Kuzmin, Rupprecht & Nel 2008). Fluorescent antibody test (FAT) Results were compared with those obtained with the standard fluorescent antibody test (FAT) (Dean et al. 1996) using a polyclonal fluorescein isothio- cyanate conjugated immunoglobulin (Onderstepoort Veterinary Institute, OIE Rabies Reference Labor a- tory). Rapid immunodiagnostic test (RIDT) The RIDT test was performed as described by the manufacturer (Animal Genetics, Inc). Briefly, a 20 % brain suspension of each isolate was prepared in PBS. A swab supplied with the test kit was dipped into the brain homogenate after which it was trans- ferred to the assay diluent for extraction. After 1 min, four drops of the sample were added to the sample well, using the supplied dropper. Final re- sults were read 5 min after application of the sam- ple. All samples were tested in duplicate. In each case, the appearance of two lines was considered a positive result (one in the test zone and one in the control zone), while the formation of only one line in the control zone was considered negative. Purified monoclonal antibody against the rabies virus nu- cleoprotein was attached to the test zone and puri- fied goat anti-mouse IgG was attached to the con- trol zone (Kang et al. 2007). RESULTS All 21 samples that tested positive with the FAT also tested positive with the RIDT and all four negative samples tested negative (Table 1 and Fig. 1). Al- though the intensity of the test lines was found to vary between different virus samples, all the tests were clearly readable and there were no cases of doubtful interpretation. Most samples reacted after about 2 min and none of the samples indicated any 259 W. MARKOTTER et al. TABLE 1 List of lyssavirus-positive samples included in the evaluation of the specificity of the rapid immunodiagnostic test (RIDT) against African lyssaviruses* Sample number Genotype Host Sample Laboratory identification number Year of isolation Geographical origin Reference 1 Gt 1 (Canine biotype) Canine Original canine brain 272/06 2006 South Africa, Limpopo Cohen et al. 2007 2 Gt 1 (Canine biotype) Canine Original canine brain 567/04 2004 South Africa, KwaZulu Natal Cohen et al. 2007 3 Gt 1 (Canine biotype) Canine Original canine brain 479/96 1996 South Africa, Limpopo Cohen et al. 2007 4 Gt 1 (Canine biotype) Black-backed jackal Original jackal brain 819/05 2005 South Africa, North West Province Cohen et al. 2007 5 Gt 1 (Canine biotype) Bat-eared fox Original bat-eared fox brain 31/05 2005 South Africa, Eastern Cape Cohen et al. 2007 6 Gt 1 (Mongoose biotype) Galerella sanguinea Original mongoose brain 22107 1994 Zimbabwe Nel et al. 2005 7 Gt 1 (Mongoose biotype) Cynictis penicillata Original mongoose brain 669/90 1990 South Africa, Mpumalanga Nel et al. 2005 8 Gt 1 (Mongoose biotype) Cynictis penicillata Original mongoose brain 767/95 1995 South Africa, Free State Nel et al. 2005 9 Gt 1 (Mongoose biotype) Cynictis penicillata Original mongoose brain 364/96 1996 South Africa, Eastern Cape Nel et al. 2005 10 Gt 1 (Mongoose biotype) Atilax paludinossus Original mongoose brain 113/91 1991 South Africa, Western Cape Nel et al. 2005 11 Gt 2 (Lineage A) Bat (Epomophorus wahlbergi) Original bat brain LBVSA2008 2008 South Africa Unpublished 12 Gt 2 (Lineage A) Bat (Rousettus aegyptiacus) Suckling mouse brain LBVAFR1999 1999 Senegal Markotter et al. 2008 13 Gt 2 (Lineage B) Bat (Eidolon helvum) Suckling mouse brain LBVNIG1956 1956 Nigeria Markotter et al. 2008 14 Gt 2 (Lineage C) Mongoose (Atilax paludinossus) Suckling mouse brain LBVMongoose 2004 2004 South Africa Markotter et al. 2008 15 Gt 3 Feline Suckling mouse brain 12341 1981 Zimbabwe Sabeta et al. 2007 16 Gt 3 Feline Suckling mouse brain 543/95 1995 South Africa, East London Sabeta et al. 2007 260 Evaluation of immunodiagnostic test kit for detection of African lyssaviruses from brain material Sample number Genotype Host Sample Laboratory identification number Year of isolation Geographical origin Reference 17 Gt 3 Feline Suckling mouse brain 97/252 1997 South Africa, Pietermaritzburg Sabeta et al. 2007 18 Gt 3 Canine Suckling mouse brain 173/06 2006 South Africa Sabeta et al. 2007 19 Gt 4 Human Suckling mouse brain DUVVSA2006 2006 South Africa Paweska et al. 2007 20 Gt 4 Bat (unknown) Suckling mouse brain DUVVSA1981 1981 South Africa Paweska et al. 2007 21 Gt 4 Human Suckling mouse brain DUVVSA1970 1970 South Africa Paweska et al. 2007 * As negative controls, phosphate buffered saline (PBS), uninfected mouse, uninfected canine and uninfected bat brain were used FIG. 1 Results of the Rapid immunodi- agnostic test (RIDT) after testing negative control samples as well as representatives of Afri- can lyssaviruses (Table 1). Sam- ples 1–21 indicate a positive re- sult with different representatives of African lyssaviruses. Negative control samples (PBS, canine and bat uninfected brain) indi- cated a neg ative result. C = con- trol line and T= test line 261 W. MARKOTTER et al. non-specific reactions. No difference in results be- tween field samples and isolates that were pas- saged in suckling mouse brains was observed. DISCUSSION Rabies is endemic in most developing countries. For most countries in Africa no rigorous epidemio- logical data for lyssaviruses exist—largely due to the lack of operational rabies diagnostic facilities. The FAT requires a fluorescent microscope, which is not only expensive but needs to be well maintained and is sensitive to power surges and blackouts that are common throughout Africa. Many Afri can gov- ernments simply cannot comply with the require- ments that would ensure fully competent FAT diag- nostics. In other cases, where a diagnostic facility may exist, its usefulness is restricted to specific geo graphic locations and samples from remote ar- eas seldomly or never reach these laboratories. Under these circumstances, the RIDT may be a use- ful tool—in our hands it has demonstrated a high specificity (100 %) against representatives of all the known African lyssavirus genotypes when com- pared to the FAT. This is a very simple test that can be performed in less than 10 min without any spe- cialized equipment, infrastructure, or high level of training. There are no critical points to field use such as cold storage, since the test kit contains every- thing required for the diagnosis and is stable at am- bient temperatures. As indicated in another evalua- tion of the test, it is capable of detecting low amounts of virus – at an excellent level of sensitivity (slightly less sensitive than a well executed FAT) (Kang et al. 2007). In our hands, several field isolates were examined, including a gt 2 infected bat brain, and all field isolates were detected as positive using the RIDT. From these results, the RIDT could be a very useful additional tool in field surveillance for lyssa- virus infections in those many areas where no other diagnostic method is available or where samples simply cannot easily reach a diagnostic laboratory due to logistical and infrastructural limitations. In these latter scenarios, suspect rabies cases are simply not tested at all (personal observations at several locations in Africa). The RIDT could specifi- cally assist in better understanding of the epidemiol- ogy of lyssavirus infections in wildlife if application in an on-site manner is considered. Just one exam- ple applies to kudu (Tragelaphus strepsiceros) an- telope, in which rabies is, in fact, a significant, but still fairly unqualified problem in Namibia and poten- tially elsewhere. Areas of wildlife reserve are often very remote, and it is rarely possible for conserva- tionists, game rangers or farmers to duly collect and send samples to a diagnostic laboratory for testing. Brain material is required for the RIDT and where no adequate necropsy facilities are available, other methods for removal of brain material without the risk of opening the scull, could be applied—includ- ing, for example, a trucut needle biopsy through su- perior orbital fissure (Tong, Leung, Lee & Lam 1999). It must be stressed that a reliable rabies diagnosis can only be made post-mortem using brain material and that saliva must not be recommended. The sim- plicity of the RIDT kit also urges us to point out a further area of caution: All personnel performing the test should be vaccinated against rabies and trained to follow the safety regulations and procedures for working with lyssaviruses, and in cases of clear po- tential human exposures, positive diagnosis should be considered as a matter of course while additional testing by FAT should be sought as a matter of ur- gency. In conclusion, we do not argue that the RIDT should be used as replacement for the well accept- ed gold standard FAT. In fact, the preferential use of the FAT should be encouraged and implemented wherever possible. We do, however, consider that the RIDT could be a useful onsite test under field conditions and in developing countries with limited diagnostic resources. The technique has the poten- tial of enhancing epidemiological surveillance of lys- saviruses under such conditions and in remote ar- eas where lyssaviruses infections otherwise go unnoticed or unquantified. After the diagnosis sam- ples can also be stored in phosphate-buffered 50 % glycerol saline and stored at 4 °C, or can be kept at room temperature for up to 4 months for future char- acterization, if needed. In this regard the RIDT, to- gether with a renewed global focus on rabies as one of the most seriously neglected zoonoses, may as- sist in obtaining at least some rabies epidemiologi- cal information from those many parts of Africa that report rabies haphazardly or not at all. ACKNOWLEDGEMENTS We thank Animal Genetics, Inc who supplied the Rapid Immunodiagnostic kits for this study. REFERENCES COHEN, C., SARTORIUS, B., SABETA, C., ZULU, G., PAW ES- KA, J., MOGOSWANE, M., SUTTON, C., NEL, L., SWANE- POEL, R., LEMAN, P.A., GROBBELAAR, A.A., DYASON, E. & BLUMBERG, L. 2007. 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