Validation of a Rapid Rabies Diagnostic Tool for Field Surveillance in Developing Countries

One root cause of the neglect of rabies is the lack of adequate diagnostic tests in the context of low income countries. A rapid, performance friendly and low cost method to detect rabies virus (RABV) in brain samples will contribute positively to surveillance and consequently to accurate data reporting, which is presently missing in the majority of rabies endemic countries.; We evaluated a rapid immunodiagnostic test (RIDT) in comparison with the standard fluorescent antibody test (FAT) and confirmed the detection of the viral RNA by real time reverse transcription polymerase chain reaction (RT-qPCR). Our analysis is a multicentre approach to validate the performance of the RIDT in both a field laboratory (N'Djamena, Chad) and an international reference laboratory (Institut Pasteur, Paris, France). In the field laboratory, 48 samples from dogs were tested and in the reference laboratory setting, a total of 73 samples was tested, representing a wide diversity of RABV in terms of animal species tested (13 different species), geographical origin of isolates with special emphasis on Africa, and different phylogenetic clades. Under reference laboratory conditions, specificity was 93.3% and sensitivity was 95.3% compared to the gold standard FAT test. Under field laboratory conditions, the RIDT yielded a higher reliability than the FAT test particularly on fresh and decomposed samples. Viral RNA was later extracted directly from the test filter paper and further used successfully for sequencing and genotyping.; The RIDT shows excellent performance qualities both in regard to user friendliness and reliability of the result. In addition, the test cassettes can be used as a vehicle to ship viral RNA to reference laboratories for further laboratory confirmation of the diagnosis and for epidemiological investigations using nucleotide sequencing. The potential for satisfactory use in remote locations is therefore very high to improve the global knowledge of rabies epidemiology. However, we suggest some changes to the protocol, as well as careful further validation, before promotion and wider use

Rabies knowledge and practices among human and veterinary health workers in Chad

To prevent human rabies deaths close communication between the veterinary and human health sector is needed for timely and adequate treatment after a bite exposure. Good practice treatment and efficient One Health communication depends heavily on the knowledge and practices (KP) of both human health and veterinary workers. We have evaluated the level of KP of both these sectors during a one-day joint training program to kick start a large scale rabies burden and vaccine demand study in selected regions of Chad. Participants were evaluated through a questionnaire before and after training to get insight into the basic knowledge of rabies and the improvement of this knowledge after the training session. In addition to 20 questions on rabies derived from the educational platform of the Global Alliance for Rabies Control, the questionnaires included a pre-training knowledge self-evaluation and a post-training evaluation of the course. Overall 247 workshop participants with varied level of educational background responded to at least one questionnaire. Around 75% of respondents were from the human health sector and 20% from the veterinary sector. Knowledge level did not differ significantly between the two sectors nor between rural or urban working backgrounds. Respondents with a university degree scored significantly higher in pre-and post-training questionnaire compared to respondents with high school level degree or lower. Knowledge was also dependent on study region and sex of the respondent. In general, the importance of a One Health approaches, such as vaccination of dogs to prevent human rabies, is well understood in both sectors. Regarding treatment, many participants did not know the adequate number of doses required for a full course of PEP, but through the training, this knowledge improved. Detailed knowledge of atypical transmission routes and pathophysiology (neurotropism of the virus) was generally lacking and did not significantly improve through the training. The study revealed considerable deficiencies and challenges in the knowledge level of both veterinary and human health workers in Chad. Rabies control programs need to anticipate these challenges to implementation and provide sufficient time and funds for training workshops and follow-up

Evaluation of the RIDT Anigen test in NRC-R in an inter-laboratory trial.

<p>Evaluation of the RIDT Anigen test in NRC-R in an inter-laboratory trial.</p

Examples of RIDT results.

<p>(A) Results from NRC-R, with six positive results including the two middle cassettes (samples 93101 and 87006) demonstrating only a weak test line in the results window. (B) Results from Chad, with one negative result (sample 18/12) and four positive results. The upper line visible in the results window is the control (C) and the lower line represents the test (T). The presence of two lines depicts a positive result, whereas the presence of only the upper line indicates a negative result. The test is considered as invalid when the upper line is not visible (not shown).</p

Description of samples tested in Chad and results obtained after FAT, RIDT and RT-qPCR on FTA papers.

<p>All specimens correspond to original brain samples collected from dogs.</p

Detection of viral RNA with RT-qPCR on Anigen test strip used in field conditions (IRED), in laboratory conditions (NRC-R) or combined.

<p>Detection of viral RNA with RT-qPCR on Anigen test strip used in field conditions (IRED), in laboratory conditions (NRC-R) or combined.</p

Comparison results of the detection of viral RNA with RT-qPCR performed on FTA Whatman cards and Anigen test strips.

<p>Comparison results of the detection of viral RNA with RT-qPCR performed on FTA Whatman cards and Anigen test strips.</p

Limit of detection of the RIDT using 8 different titrated rabies virus suspensions.

<p>Limit of detection of the RIDT using 8 different titrated rabies virus suspensions.</p

The importance of dog population contact network structures in rabies transmission

<div><p>Canine rabies transmission was interrupted in N’Djaména, Chad, following two mass vaccination campaigns. However, after nine months cases resurged with re-establishment of endemic rabies transmission to pre-intervention levels. Previous analyses investigated district level spatial heterogeneity of vaccination coverage, and dog density; and importation, identifying the latter as the primary factor for rabies resurgence. Here we assess the impact of individual level heterogeneity on outbreak probability, effectiveness of vaccination campaigns and likely time to resurgence after a campaign. Geo-located contact sensors recorded the location and contacts of 237 domestic dogs in N’Djaména over a period of 3.5 days. The contact network data showed that urban dogs are socially related to larger communities and constrained by the urban architecture. We developed a network generation algorithm that extrapolates this empirical contact network to networks of large dog populations and applied it to simulate rabies transmission in N’Djaména. The model predictions aligned well with the rabies incidence data. Using the model we demonstrated, that major outbreaks are prevented when at least 70% of dogs are vaccinated. The probability of a minor outbreak also decreased with increasing vaccination coverage, but reached zero only when coverage was near total. Our results suggest that endemic rabies in N’Djaména may be explained by a series of importations with subsequent minor outbreaks. We show that highly connected dogs hold a critical role in transmission and that targeted vaccination of such dogs would lead to more efficient vaccination campaigns.</p></div

Outbreak probabilities and rabies cases north of the river Chari from 2012 and 2016.

<p>For each week the vaccination coverage was calculated using a deterministic transmission model. Vaccination coverages were then translated to outbreak probabilities using the contact network model.</p