Comparative evaluation of Direct Rapid Immuno-Histochemical Test (DRIT) with Direct Fluorescent-Antibody Test (DFAT) for laboratory diagnosis of animal Rabies in Ethiopia

Introduction

Rabies is a lethal zoonotic disease with a worldwide distribution and is transmitted frequently by carnivores to humans and livestock. It is presented to cause a large number of deaths in animals and humans each year. Human rabies is present in 150 countries and territories and on all continents (Barecha et al., 2017). Of these countries, Ethiopia is one of the worst affected (Hampson et al., 2015). According to Tariku Jibat et al. (2018) research suggestions in most rabies endemic countries, reliable documents of occurrence data on rabies and rabies exposure are missing. However, estimates of burden have always been uncertain due to the absence of reliable data (Taylor and Nel, 2015). Official record usually underestimate the actual number of human rabies cases and hence the actual burden. For example, in sub-Sahara African countries such as Tanzania, the occurrence of human rabies predicted based on active surveillance information on bite occurrences was up to 100 times larger than the officially reported number of deaths. In Ethiopia, the national annual assesses from official records present 12 exposure cases per 100,000 population and 1.6 rabies deaths per 100,000 populations. However, the true numbers are expected to be higher as many cases are not reported (Beyene et al., 2018). Recently, rabies is a considerable disease that has been detected for many years in Ethiopia. The first rabies epidemic in Ethiopia reported in Addis Ababa in August 1903. It is believed that communication between the public and animal health sector were not enough to inexistent considering reporting and control of rabies cases (Pieracci et al., 2016). At present time, direct rapid immuno-histochemical test (DRIT) is mentioned as the gold-standard test by the World Organization for Animal Health (OIE) and World Health Organization (WHO)(Tekki et al., 2016) for rabies detection; however, the limitation of use of DRIT  in developing countries is that the method is technically demanding and needs the use of a fluorescence microscope, which is expensive and difficult to maintain, and it also needs a specific rabies conjugate as well as technical expertise. Thus, there is a necessity for a rapid diagnostic test that has comparable sensitivity and specificity as fluorescent antibody (AB) test (FAT), which is economical and can be adapted to field as well as laboratory conditions in resource-constraint countries (Singh et al., 2017). The DRIT is an experimental assay, which is used in limited field trials in Africa, such as Nigeria and South Africa (Coetzer et al., 2014). It has been demonstrated to be as specific and sensitive as the gold standard direct fluorescent antibody test (DFAT). In Ethiopia, the surveillance system is very week due to lack of rabies laboratory and also the burden of rabies is under expected or under-reported. DFAT is currently available only in three regions of Ethiopia, because establishing DFAT in all regions requires high-level expertise, expensive laboratory equipment, reagents, and sophisticated laboratory setups. As in all other endemic countries, an establishment of a diagnostic laboratory set-up as the priority for the improvement of a national rabies control program is needed. Any technical advances that make diagnosis of rabies more rapid, accurate, and cost-effective, will consequently facilitate recruiting such programs in resource-limited countries. According to Kevin Middel et al. (2017), DRIT was presented to have a sensitivity and specificity of 100% and 98.2%, respectively. Moreover, positive and negative test agreement was reported to be 98.3% and 99.1%, respectively, with an overall test agreementof98.8%. The average cost to test a sample was $3.13 CAD for materials, and hands-on technical time to complete the test was estimated at 0.55 h. Also, DRIT procedure was proposed to be accurate, inexpensive, fast, easy to perform, and a great tool for monitoring the progression of rabies incursion. DRIT not only has a diagnostic efficacy equal to that of the gold standard DFAT, but is also faster, cheaper and easier to elucidate by an inexperienced reader. These findings highlight the versatility of the DRIT as a potential WHO and OIE accredited rabies diagnostic assay (DRIT SOP).The authors attempted to demonstrate which of the current laboratory techniques, being applied for rabies diagnosis worldwide, has unique advantages over the other by comparing the gold standard laboratory technique DFAT and DRIT so that the result of the study would serve to trigger government officials both in the ministry of health and agriculture to take further action in scaling up the suitable laboratory diagnostic techniques, provided the limited laboratory set up. For internal quality purpose, the researchers agreed to perform mice inoculation test (MIT) by selecting 12 randomly selected brain samples and inoculated into mice brain tissue. The rationale behind performing MIT was that there are indeterminate conditions, usually 1%, while performing both DRIT and DFAT. In situations where skills and facilities for other tests [e.g., cell culture, polymerase chain reaction, (PCR)] are not available, MIT could be served as an optional confirmatory technique. In a country like Ethiopia, where rabies is highly prevalent, besides with a very limited access for laboratory diagnosis of rabies, the result of this research would have several implications like enhancing the rabies laboratory surveillance in Ethiopia and its suitability regarding the time taken, technical ease, user acceptability, and the stability of the DRIT to perform the test makes the present study crucial.

Materials and methods

Addis Ababa, the capital city of Ethiopia, covers an area of 530 km² and is divided into 10 sub-cities. Ethiopian public health institute is located at Gulele sub-city. Currently, the institute is focusing on priority disease research and strengthening the national public health laboratory services in the country. It is also the technical hand of the Federal Ministry of Health. So, all dogs inflicting bite including laboratory diagnosis of rabies in and around Addis Ababa is managed through the national diagnostic center; which is Ethiopia Public Health Institute (EPHI) (Figure 1).

Sample Size

A total of 100 brain specimens were collected during the study period (December, 2020 – June, 2021) from various regions and different species of animals and brought to the Ethiopian public health institute rabies diagnostic laboratory.

Protocol for DFAT

In the present study, DFAT, monoclonal antibody-conjugate, were applied. The working (reagent) dilution after titration was prepared at 1:40 in accordance with the manufacturer’s recommendations (Standard Operating Procedure for DRIT, center for disease control (CDC); Reagent for the study was manufactured and supplied by CDC, Atlanta). A small piece of the brain tissue specimens was smeared using a wire loop on one part of a slide, which was subsequently air-dried and fixed in cold acetone at -20°C for one hour. Then, the slides were air-dried and the rabies conjugate was used at 1:40 and incubated at 37°C for 30 minutes in a humid chamber after which excess conjugate was removed from the slides by rinsing it with 7.4 pH PBS solution about 3-5 minutes and was conducted to air-dry. The coverslips were mounted with buffered glycerol mounting medium and the slides were evaluated by a fluorescence microscope within two hours after staining. When brilliant apple-green fluorescence color or greenish-yellow objects a represented against a black background, the test slide is positive. If no specific apple green fluorescence was presented, the test slide is negative (Figure 3B).

Protocol for DRIT

1. Routine touch impressions of suspect CNS tissues were made on labeled glass microscope slides                       (Standard positive and negative controls were included).
2. Slides were air-dried for 5 minutes at room temperature. 
3. Slides were immersed in 10% buffered formalin at room temperature for 10 minutes (Dish I).
4. Slides were removed and dip-rinsed several times to wash off any excess fixative in wash buffer TPBS (PBS with 1% tween 80; Dish II). 
5. Slides were immersed in 3% hydrogen peroxide for 10 minutes (Dish III).
6.  Excess hydrogen peroxide was removed by dip-rinsing slides in TPBS (Dish IV). Then slides were transferred to the next rinse, Dish V (after dipping, excess buffer was shaken off, and blotted from the slide edges surrounding the impression). One slide at a time was done leaving the remaining slides immersed within TPBS rinse.
7. Slides were incubated in a humidity chamber (Plastic top to a 96-well plate was used), at room temperature with primary antibody - biotinylated anti-rabies mAb for 10 minutes (Enough of this primary antibody were added by dropping to cover the impression). 
8. After incubation, excess conjugate was shaken off. Slides were Dip-rinsed with TPBS, Dish V (The same wash buffer was used through step 10. Slides were incubated with streptavidin-peroxidase complex (enough of this complex was added to the slide by dropping to cover the impression) in a humidity chamber at room temperature for 10 minutes. After incubation, excess of the complex was shaken off.
9. Slides were dip-rinsed with TPBS, Dish V (excess buffer was shaken off and also blotted from slide edges surrounding the impression).
10. Slides were incubated with peroxidase substrate, amino-ethyl carbizole (AEC) –The working dilution was prepared just prior to use. Enough of this substrate was added to the slide by dropping to cover the impression in a humidity chamber at room temperature for 10 minutes. Excess substrate was shaken off after incubation.
11. Slides were dip-rinsed in deionized/distilled water (Dish VI).
12. Slides were counterstained with Gills Hematoxylin (diluted 1:2 with deionized/distilled water) for 2 minutes (Dish VII).
13. Immediately, the stain was dip-rinsed with deionized/distilled water (Dish VIII). A second dip-rinsing was made of slides with fresh deionized/distilled water (Dish IX) to ensure removal of excess stain.
14. Slides were transferred to fresh distilled water (Dish X). Slides were mounted with water-soluble mounting medium and cover-slipped. (One slide at a time was made; Excess deionized/distilled water was shaken off and blotted from slide edges surrounding the impression). Slides were allowed to air-dry prior to cover slipping.  
15. Slides were viewed by light microscopy, using a 20x objective to scan the field, and a 40x objective for higher power inspection (rabies virus antigen appeared as red inclusions against the blue neuronal background).
16. Finally, results were recorded.