Comparison of five different laboratory techniques for the rabies diagnosis in clinically suspected cattle in Brazil

https://doi.org/10.1016/j.jviromet.2020.113918Get rights and content

Highlights

  • If dFAT results are negative or inconclusive, rabies diagnosis may require auxiliary tools.

  • RABV non-homogenous distribution in CNS may interfere in the diagnosis.

  • Molecular techniques can detect RABV in low viral load samples.

Abstract

The direct-fluorescent antibody test (dFAT) is considered the “gold standard” assay to diagnose rabies. However, it is crucial to develop molecular techniques, such as RT-PCR and RT-qPCR, since many laboratories lack the needed supplies for performing complementary methods (viral isolation, for example). For this purpose, diagnostic techniques must be specific and sensitive to guarantee accuracy. This present investigation aimed to detect rabies virus (RABV) in 126 clinically suspected cattle in Brazil using different diagnostic tests [dFAT, mouse inoculation test (MIT), immunohistochemistry (IHC), RT-PCR and RT-qPCR] and to compare those results obtained under routine laboratory conditions. The results of the present investigation demonstrate that the molecular techniques are more sensitive and may detect low viral load, even though the non-homogeneous viral distribution caused a false-negative result in dFAT. We also observed a usual alteration in antigens distribution among regions of the central nervous system (CNS). By both dFAT and IHC assays, the most reliable CNS structures were thalamus and midbrain. Although this investigation demonstrated diagnostic sensitivity and specificity close to 100 % in all laboratory techniques employed, a dFAT auxiliary test is required for bovine specimens, such as molecular techniques, when there are poor sampling conditions (low viral load combined with unavailability of brainstem structures).

Introduction

Rabies is enzootic and epizootic disease in Brazil, affecting mainly livestock and bats. This disease has shown a change in its epidemiological pattern: domestic dogs became less relevant as rabies reservoirs and wild animals have gained relevance (Castilho et al., 2018). Vampire bats, as important rabies reservoirs, have caused substantial financial losses in livestock farming (Kobayashi et al., 2006).

Cattle are very susceptible to rabies and may contribute as sentinel species to the rabies virus (RABV) circulation, particularly in regions with inadequate vampire bats surveillance (MAPA, 2009). The Brazilian Ministry of Agriculture, Livestock and Food Supply (MAPA) reported the occurrence of over 38,000 bovine rabies cases between 1999 and 2019 (MAPA, 2020). However, misdiagnosis of cattle rabies, along with an under-reporting, may underrate the real number of cases.

The Pasteur Institute (São Paulo, Brazil) tests approximately 8000 animal samples per year for rabies diagnosis. During warmer seasons, the specimen submission amount can surpass 80 samples per day. As a result, detect RABV by a high-throughput method is essential. Rabies diagnosis must be fast and reliable to assess the infection risk of an exposed person. Laboratory diagnosis, obtained by testing brains of animals, plays an important role in the fight against rabies, because it is the basis for making decisions regarding human post-exposure prophylaxis and for managing an outbreak (Bourhy et al., 1989; Léchenne et al., 2016).

The direct-fluorescent antibody test (dFAT) is considered the “gold standard” assay to diagnose rabies: it is a rapid method (∼2−3 h) and has high sensitivity and specificity (∼100 %) to detect viral antigens on postmortem nervous tissue imprints (Dean et al., 1996). However, dFAT accuracy depends on skilled technicians, appropriate fluorescence microscope, high-quality anti-rabies conjugate, and sample conservation (Dean et al., 1996; Dupuis et al., 2015). Brain tissue degradation and denaturation by heat or repeated freeze-thaw cycles may reduce sensitivity (Hanlon and Nadin-Davis, 2013).

Because RABV does not infect all central nervous system (CNS) structures uniformly, the election of CNS fragment to make imprints for dFAT may also affect the test results (Bingham and Van Der Merwe, 2002). The RABV antigens spread throughout the CNS in most animals positive for rabies, usually on those collected in the final stages of the disease, when the levels of RABV antigens are higher. However, it is difficult to confirm a rabies-positive result and the viral distribution may be limited to the brainstem on those samples with only a small amount of viral antigen, notably when the animal is euthanized before or during the early stage of the disease (Dean et al., 1996; Hanlon and Nadin-Davis, 2013).

World Health Organization (WHO) recommends an alternative test when dFAT results are negative or inconclusive, and when an animal had contact with a human to confirm the rabies diagnosis (WHO Expert Consultation on rabies: Third report, 2018). For some time, the mouse inoculation test (MIT) has been the most common confirmatory test of dFAT results. This test allows the propagation and amplification of the etiologic agent, as well as its preservation for further studies (Bourhy et al., 1989; Koprowski, 1996).

Immunohistochemistry (IHC) on formalin-fixed paraffin-embedded (FFPE) sections has specificity and sensitivity equivalent to dFAT, with the advantage that tissue fixation with formalin preserves the tissue and allows inactivation of RABV (Jogai et al., 2000). Another advantage of IHC on FFPE tissue is the observation of pathological changes and RABV antigen localization in the CNS (Last et al., 1994).

Many molecular assays have been evaluated as confirmatory tests for the dFAT results, including conventional gel-based reverse-transcription PCR (RT-PCR) assays with nested/semi-nested protocols to detect the RABV RNA in clinical samples, particularly in decomposed samples or archival specimens (Heaton et al., 1997; Lopes et al., 2010). Molecular techniques detect the RABV RNA in poor quality brain samples, even when dFAT is negative (Araújo et al., 2008). One advantage of molecular techniques is their speed to obtain results when compared to the MIT (Heaton et al., 1997).

Real-time RT-PCR (RT-qPCR) technique, which does not involve post-amplification handling steps, substantially curtailing the frequency of cross-contamination due to the “closed-tube” character of the analysis. This method also has high-throughput and provides quantification of the viral load (Silva et al., 2013). Analytical specificity is ensured by a hybridization reaction within the nucleoprotein gene with the hydrolysis fluorogenic probes, which have the potential for RABV genetic lineages typing (Hoffmann et al., 2010).

As the fatality rate of rabies remains higher than 99 %, a false-negative result could have a catastrophic effect on human mortality. Therefore, it is essential to have accurate and rapid laboratory techniques to diagnose positive rabies cases from suspected animals. Also, early diagnosis with highly sensitive and specific tests will reduce unnecessary prophylaxis and treatment (Dupuis et al., 2015).

Additionally, the commercial availability of standardized and validated kits of molecular tools for the efficient detection of street rabies viruses are becoming progressively indispensable to improve accuracy. This present study aimed to diagnose rabies in 126 clinically suspected cattle in Brazil by using different diagnostic tests (dFAT, MIT, IHC, RT-PCR and RT-qPCR) and to compare those results performed under routine conditions.

Section snippets

Samples

Between March 2015 and March 2016, the Pasteur Institute of Sao Paulo (Brazil) received 126 CNS samples from cattle for routine rabies diagnosis. Most of the specimens were submitted refrigerated or frozen, either as whole CNS (cortex, hippocampus, thalamus, brainstem [medulla oblongata, pons, and midbrain], cerebellum, and spinal cord) or as pieces of CNS removed from the cranium by private and government veterinarians. Due to severe autolysis, it was impossible to identify the CNS structures

Results

Of the 126 routine rabies diagnostic cattle specimens tested, 40 (31.7 %) were positive for RABV by dFAT, MIT, and IHC procedures. In the RT-PCR assay, 33.3 % of samples were positive (42/126) and 66.6 % were negative (84/126). On the other hand, RT-qPCR assay detected 42 positives (33.3 %), 82 negatives (65.1 %) and two inconclusive samples (1.6 %) (Table 1).

After 10.5 ± 2.4 days of intracerebral inoculation, the animals showed the onset of the disease. The shortest incubation period in our

Discussion

Although there were no significant differences among the analyses, the rabies diagnosis should not only rely on dFAT results. It should be supported at least by one confirmatory test, especially with dFAT negative or inconclusive results when an animal had contact with a human (WHO Expert Consultation on rabies: Third report, 2018). The sensitivity of dFAT necessarily depends on the quality of the investigated specimen (Dean et al., 1996; Dedkov et al., 2018).

MIT can require a 30-day waiting

CRediT authorship contribution statement

N.H.F. Centoamore: Investigation, Validation, Formal analysis, Writing - original draft, Writing - review & editing. M.E.R. Chierato: Investigation, Validation, Formal analysis, Writing - original draft, Writing - review & editing. V.B.V. Silveira: Investigation, Writing - original draft, Writing - review & editing. K.M. Asano: Investigation, Methodology, Writing - original draft, Writing - review & editing. K. Iamamoto: Investigation, Writing - original draft, Writing - review & editing. W.O.

Declaration of Competing Interest

We declare that we have no conflict of interest.

Acknowledgements

This study was supported by the Sao Paulo Research Foundation (FAPESP), grant #2015/17807-0. We would like to acknowledge the National Council for Scientific and Technological Development (CNPq) for providing a Master´s fellowship to N.H.F. Centoamore, and Coordination for the Improvement of Higher Education Personnel (CAPES) - Finance Code 001. We would also like to thank Dr. Mary S. Varaschin for providing the anti-RABV antibody.

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