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Evaluation of a rapid immunodiagnostic rabies field surveillance test on samples collected from military operations in Africa, Europe, and the Middle East.

Rabies is an acute, progressive, viral encephalomyelitis with the highest case fatality rate of any conventional etiological agent. It is one of the oldest described infectious diseases, having been recognized more than 4,000 years ago. Rabies has a substantial international presence in that it is distributed on all continents but Antarctica. It is the leading viral zoonosis with a significant global burden on veterinary and human public health. Globally, the number of human rabies exposures per year is estimated to be in the tens of millions; the number of human rabies deaths per year was estimated to be over 55,000 in 2005. (1) The rabies virus is a negative strand RNA-virus belonging to the genus Lyssavirus, family Rhabdoviridae of the order Mononegavirales. (2,3)

Different animal species are involved in the maintenance and transmission of rabies around the world. While the predominance of any one reservoir species varies by geographical region, the domestic dog remains the most significant reservoir for human rabies in overall case numbers and with regard to transmission, accounting for more than 90% of rabies exposures worldwide and more than 99% of human rabies deaths. Although rabies control and elimination is possible in dogs, stray and free-roaming, infected dogs present barriers to success. (1,4,5)

In the Eastern Mediterranean region as defined by the World Health Organization (WHO), which includes Afghanistan and Iraq, (6) rabies continues to be a public health problem, predominantly affecting vulnerable, impoverished populations living in remote, rural locations. In 2002, 5,000 human rabies deaths were recorded in the region, mostly from Afghanistan, the Islamic Republic of Iran, and Pakistan, whereas most other countries reported fewer than 10 cases per country. (7) Unlike in developed countries, vaccination coverage of domestic animals in Afghanistan, Iraq, and similar countries is low. Absence of adequate vaccination within these countries makes them high risk for rabies in terrestrial animals and has resulted in a high prevalence of rabies within the dog population. (7,8)

Unfortunately, current reported numbers of animals infected with rabies in Iraq and Afghanistan are variable. Infrastructure is not in place to support testing for rabies in these regions, so reporting of cases is often based on clinical signs and is therefore limited and inconclusive. (9) These countries lack a much needed, effective surveillance network to assess the magnitude of disease and to focus vaccination and control efforts. These countries also lack proper diagnostic facilities. Reliable national, systematic surveillance of rabies-related human deaths and animal rabies prevalence is urgently needed to garner support for effective prevention strategies. (4,10,11)

For animal rabies diagnosis, the direct fluorescent antibody test (DFAT) is the most frequently used and is the gold-standard test approved by the Centers for Disease Control and Prevention (CDC), WHO, and the World Organisation for Animal Health. This test is performed on brain tissue from animals suspected of being rabid and can only be performed postmortem. (12-15) The DFAT is one of the quickest and most reliable testing methods, providing an accurate diagnosis in 98% to 100% of rabies suspect cases. (13,16,17)

In remote locations, the use of DFAT is often not feasible because of transportation issues and lack of adequate cold chain. Laboratories are not able to comply with the strict requirements to perform DFAT accurately. The lack of infrastructure and logistical support hinders DFAT as a realistic expectation. Currently cases go undetected, and surveillance is not actively pursued. The lack of diagnostic and surveillance capability results in a low level of awareness of the actual incidences of rabies in these regions, and the virus remains hidden and endemic with a potential to increase. (18) The US Army did evaluate the use of another test, the direct rapid immunohistochemical test, for field surveillance. While this is an inexpensive test with excellent sensitivity and specificity comparable to DFAT, the need for CDC-certified training, refrigeration, multiple chemicals, and proper microscopic training made this unrealistic as a suitable surveillance test in military environments.

There are many challenges in the implementation of effective rabies diagnosis and surveillance programs in developing countries. Development and international acceptance of a validated test that can be used worldwide are essential to overcome these challenges. Financial and logistical barriers are additional obstacles that prevent use of such a test in developing countries with the greatest need. A rapid immunodiagnostic test (RIDT) for rabies virus has been developed, and this test shows potential in meeting these criteria. This lateral flow test uses gold conjugated detector antibodies, including a monoclonal antibody directed against the lyssavirus nucleoprotein. (18) Although this method has only been used for qualitative analysis, it provides rapid detection of rabies antigen. Advantages over conventional immunoassays include lower cost, inexpensive equipment, simplicity of procedure, rapid operation, and long-term stability over a range of environmental conditions. The test is suited for on-site testing by personnel with limited technical expertise. (18,19)

The objective of this study was to evaluate the use of the Anigen Rapid Immunodiagnostic Test Kit for detection of rabies virus in clinical samples for application as a surveillance test among animal populations in areas with deployed military units. Clinical samples had previously been submitted to the US Army Public Health Command Region Europe Veterinary Laboratory Europe (VLE), in Landstuhl, Germany, for rabies testing with the DFAT.


Clinical Samples, Field Isolates, and Diagnosis

A total of 79 clinical samples collected between 2004 and 2011 were examined for rabies using the Anigen RIDT. An additional clinical sample from 1996 was also tested. Total samples numbered 80. As shown in Table 1, all specimens were brain tissue collected from the following animals: canine (n=46), bovine (n=5), feline (n=18), macaque (n=3), porcine (n=1), mongoose (n=1), equine (n=1), jackal (n=1), rat (n=1), and bat (n=3). The majority of samples tested with RIDT originated from Iraq (n=26) or Afghanistan (n=45). Samples also originated from Turkey (n=1), Bosnia (n=1), Germany (n=3), Kuwait (n=3), and Qatar (n=1). Detailed information about the animals was not available. All samples were collected by US military personnel and submitted to VLE. When testing with the RIDT was first initiated in 2010, all specimens were from banked samples stored at VLE and previously tested with DFAT and, in some cases, with rabies murine neuroblastoma cell culture (MN). Thus, at that time, all results were previously known positives or negatives, and the investigators were not blinded to the results. Since that initial batch run of 39 tests, the RIDT was used concurrently on samples at the time of testing with DFA and MN. The sensitivity and specificity of the RIDT were determined using DFAT as the reference method.

Rapid Immunodiagnostic Test Kit

Test Principles

The Anigen Rapid Rabies Antigen Test Kit (Bionote, Inc, Hwaseong, Korea) is an immunochromatographic assay designed for the qualitative detection of rabies virus antigen in canine, bovine, and raccoon dog salivary secretions and brain homogenates. The test uses gold conjugated detector antibodies to detect rabies virus antigen. (20)

Application of the RIDT

The rapid immunodiagnostic test was performed according to the instructions supplied by the manufacturer. (20) In the study, final results were read 5 to 10 minutes after application of the sample as per the guidelines. In 2 cases, final results were read at 30 minutes after application of the sample to the test well. Examples of positive and negative results are shown in the Figure.


Sensitivity and Specificity of the RIDT Kit

Of the 80 samples used in the study, 45 were negative on DFAT, 32 were positive on DFAT, and 3 had an indeterminate result on DFAT. When the RIDT was run on these samples, there were 49 negative results and 31 positive results. Although the intensity of the test lines was found to vary among the different samples, all tests were clearly readable. Seventy-eight samples reacted within the 10-minute cut-off time for interpreting the test, but 2 samples were negative at 10 minutes and had faint positive results at 30 minutes. The 3 tests that were indeterminate on DFAT were negative on the RIDT.

Thirty-six of the 41 samples tested with both DFAT and RIDT in 2011 had also been tested with rabies murine neuroblastoma cell culture (MN). Of the 36 samples, 31 were negative on all tests, 3 were positive on all tests, and 2 were indeterminate on DFAT and MN but negative on RIDT.

One of the samples that was indeterminate on DFAT and MN but negative on RIDT was from a bat from Afghanistan. The other sample that was indeterminate on DFAT and MN but negative on RIDT was from a canine from Afghanistan. The third sample that was indeterminate on DFAT and negative on RIDT was from a canine from Kuwait. This sample was not subjected to the MN test.

Using DFAT as the reference method for the results of the samples tested, the RIDT kit was 96.9% sensitive and 100% specific. The 3 tests that were indeterminate on DFAT and MN were not used in the calculations.

Results by Species and Geographic Region

Results obtained from using the RIDT were evaluated by species (Table 2) and geographic region (Table 3). Given the concern regarding interactions between military personnel and canines, the results for this species in Afghanistan and Iraq, 2 regions with significant military presence, are further highlighted here.

Canine--Forty-six canine samples were tested. Excluding the samples with indeterminate results, sensitivity and specificity were both 100% for canine samples in the study.

Afghanistan--Excluding the indeterminate results, the RIDT was 100% sensitive and specific for Afghanistan samples.

Iraq--Based on interpretation of the delayed test results as positive, the RIDT was 95% sensitive and 100% specific for Iraq samples. Using the 10-minute recommended cutoff time for RIDT interpretation, the sensitivity was 85%.


In the present study, we describe a simple and rapid surveillance test for rabies virus infection based on the principle of immunochromatography. This lateral flow immunoassay system is widely used and accepted for the diagnosis of many human and animal diseases.

Rabies surveillance is lacking for many areas where troops are deployed, and no rigorous epidemiological data exist largely because of the lack of operational rabies diagnostic capabilities. The RIDT is a practical tool for field application to gather surveillance data of rabies-suspect animals, especially in resource poor regions where fluorescent antibody testing is impractical. (18)

In this study, the RIDT was highly sensitive (96.9%) and highly specific (100%) compared to the DFAT. The sensitivity and specificity of the RIDT, both 100%, compared to DFAT for the canine samples tested demonstrate the utility of the RIDT as a surveillance tool among canines, the rabies reservoir of significant concern for transmission to military troops. The significance of data in species other than canines is limited for this study by the small sample sizes. Sensitivity and specificity were also calculated separately for Afghanistan and Iraq, the 2 regions that had enough samples for meaningful interpretation. Afghanistan samples were 100% sensitive and 100% specific with the RIDT. Iraq samples were 95% sensitive and 100% specific with the RIDT. The data are presumably not reflective of the true extent of rabies in the region since samples were collected only by Army personnel in selected locations and not by local veterinarians throughout the regions. In addition, sensitivity and specificity may be affected by the ability of the RIDT to detect regional rabies virus variants.

The RIDT can fulfill the CDC surveillance objectives for "uniformity, simplicity, and brevity." (21) It is a straightforward test that is simple and quick to perform. There are no critical points to field use such as cold storage since the test kit is self contained and stable when stored at room temperatures or refrigerated. Kang el al (14) demonstrated that the test is capable of detecting low amounts of virus at an excellent sensitivity level that is slightly less than that of a well-executed FAT. The study by Markotter et al (18) found excellent correlation of results when testing samples with both FAT and the RIDT.

As with any new surveillance or diagnostic tool, strict quality control and test validation are essential before the test can be relied upon for meaningful results. Preliminary validation studies performed by Kang et al (14) showed the RIDT to have significant potential as a rabies diagnostic method. In the present study, the sensitivity of samples from Iraq must be considered in light of the 2 DFAT-confirmed positive tests that were initially negative on the RIDT after 10 minutes, but turned positive after 30 minutes. The delayed positive results could have been a result of the samples having a viral load close to the limit of detection, thus delaying the result. However, accurate interpretation is not possible outside of the manufacturer's recommend time frame, 10 minutes for the RIDT. These 2 samples highlight the need for additional test validation.

While further laboratory and field evaluations are needed, these results are promising for the benefit of the RIDT as a surveillance tool in those regions without other immediate capability. Capacity for effective rabies surveillance programs is crucial to determine those geographical areas of operations where soldiers may be at risk for encountering a rabid animal, especially among free-roaming dogs and dog packs, which are a significant burden on military installations.

Even though the RIDT was not evaluated on specimens other than dog, cattle, and raccoon dog in the initial study by Kang et al, (14) the results from the present study suggest that the RIDT may have application as a surveillance tool for multiple species. Further research with greater case numbers in multiple species is necessary to determine if the RIDT is capable of detecting multiple rabies virus variants.

Surveillance is an essential component of infectious disease risk assessment of military members during deployments. (22,23) As a surveillance tool, the RIDT can help guide the most appropriate and cost effective use of animal control procedures and resources where they are most needed and will be beneficial to long-term rabies control. Continued on-site surveillance using the RIDT can also serve as a validation of vaccination control efforts. Military use of the RIDT has the potential to lead to country-wide acceptance and approval of this test with implementation among local veterinary organizations. Because it does not require specialized equipment or training, RIDT kits would be an excellent addition to military veterinary field supplies. This will further allow easy and accessible rabies surveillance capabilities throughout the regions. According to the manufacturer (BioNote, November 6, 2013), a single test costs approximately $9.60 USD, which is significantly less than the costs associated with establishing a laboratory equipped with fluorescent microscopy.


We thank the staff and soldiers of the Veterinary Laboratory Europe who processed samples and participated in evaluation of the RIDT. Special thanks to Leslie Fuhrmann for providing quality assurance in the rabies department and for her expertise in the testing process, and to LTC Jerry Cowart, MS, USA, Veterinary Pathology Division, Laboratory Science, US Army Public Health Command Region-Europe, for his review of the manuscript.


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(8.) Animal & Insect-Borne Diseases FAQ [internet]. Aberdeen Proving Ground, MD:US Army Public Health Command. Available at: ics/discond/aid/Pages/FAQ.aspx. Accessed November 30, 2011.

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(12.) Centers for Disease Control and Prevention. Rabies Diagnosis: Accuracy of the Tests [internet]. April 22, 2011. Available at: http:// Accessed January 11, 2012.

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(19.) Wang H, Feng N, Yang S, et al. A rapid immunochromatographic test strip for detecting rabies virus antibody. J Virol Methods. 2010;170(1-2):80-85.

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(22.) Murray CK, Horvath LL. An approach to prevention of infectious diseases during military deployments. Clin Infect Dis. 2007;44(3):424-430.

(23.) United States Air Force Guide to Operational Surveillance of Medically Important Vectors and Pests: Operational Entomology. Version 2.1. Washington, DC: US Dept of the Air Force; August 15, 2006. Available at: files/pubs/guides/operational_surveillance_guide.pdf. Accessed August 1, 2012.

Kristen M. Voehl, DVM, MPH, DACVPM

LTC Greg A. Saturday, MS, USA

Dr Voehl is a staff veterinarian at the Army Veterinary Treatment Facility, Kaiserslautern, Germany.

When the study described in this article was conducted, LTC Saturday was Chief, Veterinary Pathology Division, Army Veterinary Laboratory Europe, Landstuhl, Germany. He is currently with the Comparative Pathology Branch, Research Support Division, US Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, Maryland.

Table 1. Species and number of brain
tissue samples tested with RIDT in
this study from 1996 to 2011

Year    Species      Number of
                   Samples Tested

1996     Bovine           1
2004     Canine           1
2005     Canine           2
2005    Porcine           1
2006     Canine           3
2007     Canine           3
2007    Mongoose          1
2008     Canine           5
2008     Bovine           4
2008     Jackal           1
2009     Canine          11
2009     Feline           2
2009    Macaque           2
2009     Equine           1
2010     Canine           1
2011     Canine          20
2011     Feline          16
2011      Rat             1
2011      Bat             3
2011     Monkey           1


Bovine--Bos primigenius
Canine--Canis familiaris
Porcine--Sus domesticus
Jackal--Canis sp.
Feline--Felis catus
Macaque--Macaca sp.
Equine--Equus ferus
Rat--Rattus sp.
Bat--Chiroptera sp.

Table 2. Comparison of the rapid
immunodiagnostic test (RIDT) to the
direct fluorescent antibody test (DFAT,
reference method) and murine neuroblastoma
cell culture (MN) by species tested.

Species    Results             Number of

                           RIDT   DFAT   MN

Canine     Positive        23     23     3
           Negative        23     21     14
           Indeterminate   2      1
Feline     Positive
           Negative        18     18     15
Bovine     Positive        4      5
           Negative        1
Jackal     Positive        1      1
Macaque    Positive
           Negative        3      3      1
Bat        Positive
           Negative        3      2
           Indeterminate   1      1
Rat        Positive
           Negative        1      1      1
Mongoose   Positive        1      1
Porcine    Positive        1      1
Equine     Positive        1      1
Total      Positive        31     32     3
           Negative        49     45     31
           Indeterminate   3      2

NOTE: Canine--Canis familiaris
Feline--Felis catus
Bovine--Bos primigenius
Jackal--Canis sp.
Macaque--Macaca sp.
Bat--Chiroptera sp.
Rat--Rattus sp.
Porcine--Sus domesticus
Equine--Equus ferus

Table 3. Comparison of the rapid immuno-diagnostic test (RIDT)
to the direct fluorescent antibody test (DFAT, reference method)
and murine neuroblastoma cell culture (MN) by geographic region.

Region        Results         Number of Samples

                                 RIDT         DFAT      MN

Afghanistan   Positive            11           11       3
              Negative            34           32       25
              Indeterminate                    2        2
Iraq          Positive           19 *          20
              Negative            7            6        2
              Indeterminate   ([dagger])
Bosnia        Positive            1            1
Turkey        Positive
              Negative            1            1
Germany       Positive
              Negative            3            3        1
Kuwait        Positive
              Negative            3            2        2
              Indeterminate                    1
Qatar         Positive
              Negative            1            1        1
Total         Positive            31           32       3
              Negative            49           45       31
              Indeterminate       3            2

* Includes the 2 samples that did not show a positive result
for 30 minutes

([dagger]) One sample that was positive for DFAT and negative
on RIDT was also positive on the DRIT.
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Author:Voehl, Kristen M.; Saturday, Greg A.
Publication:U.S. Army Medical Department Journal
Article Type:Report
Geographic Code:60AFR
Date:Jul 1, 2014
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