Diagnosis and molecular typing of rabies virus in samples stored in inadequate conditions

Abstract

Introduction: The exposure of nervous tissue samples to high temperatures affects the sensitivity of rabies virus diagnostic tests, causing degradation of the viral structure. This study evaluated reverse transcriptase polymerase chain reaction (RT-PCR) for the diagnosis and molecular characterization of brain tissue samples in an advanced state of decomposition and poorly conserved viral isolates by comparing it with routine diagnostic tests. Methodology: A panel of three canine brain samples exposed to controlled decomposition for 7, 15, 30, and 120 days were evaluated using fluorescence antibody test (FAT), mouse inoculation test (MIT), and RT-PCR. In addition, 14 isolates of rabies variants, representing the largest circulation in Argentina, preserved in inadequate cooling for six to eight years were analyzed. Molecular typing of strains was performed using a 159-nucleotide region corresponding to the nucleoprotein gene. Results: The three samples analyzed were positive by RT-PCR at all the decomposition times evaluated, in contrast to results observed with FAT and MIT, which rapidly became negative. In addition, 100% of the inadequately preserved samples were characterized molecularly. The limit of detection of RT-PCR was 0.5 MICDL 50 /0.03 mL. Conclusion: RT-PCR can be useful for rabies diagnosis and typing of putrefying samples or rabies isolates stored in inadequate conditions.

Application and Comparative Evaluation of Fluorescent Antibody, Immunohistochemistry and Reverse Transcription Polymerase Chain Reaction Tests for the Detection of Rabies Virus Antigen or Nucleic Acid in Brain Samples of Animals Suspected of Rabies in India

“…In addition, 35 of the 36 samples, which were deemed to be unfit for either DFA or dRIT due to decomposition of the tissue, also yielded the expected amplicons by RT-PCR. Indeed, RT-PCR can detect the presence of nucleic acid in samples in decomposing conditions and collected several days earlier, transported at ambient temperatures, archived in frozen or fixed condition for several years, exhumed bodies, or in some cases, as an intravitam diagnostic assay [ 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 ]. This is not surprising since nucleic acids, especially fragments, are likely to be more resistant to tissue decomposition than proteins.…”

Clarifying Indeterminate Results on the Rabies Direct Fluorescent Antibody Test Using Real-Time Reverse Transcriptase Polymerase Chain Reaction

“…2 However, the sensitivity and specificity of the test can be affected by the integrity of the sample. 3,4 The DFAT relies on rabies protein structures (epitopes) to which fluorescently labeled anti-rabies antibodies bind. Decomposition, desiccation, or exposure to chemicals (eg, bleach, formalin) can cause structural changes in epitopes that result in less efficient binding of the diagnostic antibodies.…”

Effect of Postmortem Degradation on the Preservation of Viral Particles and Rabies Antigens in Mice Brains. Light and Electron Microscopic Study

“…A molecular test has been recently developed that can be suitable for the postmortem diagnosis of rabies [ 34 ]. The results have demonstrated the robustness of viral RNA, compared to viable virus or antigen in a decomposing animal, at least 70 days after death, even when the integrity of the brain tissue was compromised [ 6 , 35 ], which supports the use of a RT-PCR as a more sensitive and specific diagnostic test, since it can detect the rabies virus genome in samples highly decomposed [ 35 , 36 , 37 ], even when the Direct Fluorescent Antibody Test (DFA) and the Mouse Inoculation Test (MIT) present negative results [ 35 ]. However, there are financial and logistical barriers that prevent the routine use of molecular trials for the rabies diagnosis in many parts of the world [ 4 ].…”