Pathlogical investigation of naturally infected canine rabies cases from…
Pathlogical investigation of naturally infected canine rabies cases from Sri Lanka
Rabies diagnostic tools
IHC methods for rabies detection provide
sensitive and specific means to detect rabies in formalin-fixed tissue
Negri bodies is pathognomonic for rabies, so it confirms rabies, but the lack of rabies does means that diagnosis remains inconclusive.
no longer recommended for primary diagnosis as it has reduced sensitivity compared with immunological and molecular tests.
challenges for testing for rabies
Artefactual change can prevent accurate rabies diagnosis
fresh brain tissues
screening of rabies involves 'fresh brain tissue'.
of handling tissues
limited in resources
in 3rd world countries: require specialised equipment, ie. no cold chain + hotter climates--> rapid autolysis/ artefacts
: Can fixing brain tissue with formalin paraffin be effectively used in rabies diagnosis instead of fresh tissue samples? Even if it is less reliable for FAT ? Could use for IHC as an additional test? or for PCRs? Will artefact present a problem?
unequal distribution of viral antigen throughout brain tissues
need for reliability and user friendly tests that are relatively inexpensive and do not require special storage
lack of adequate diagnostic tests in the context of low income countries- "under field laboratory conditions"
lack of rabies laboratories
Alternative diagnostic tools that cannot be applied to rabies
seroconversion cannot be used for rabies. is the time period during which a specific antibody develops and becomes detectable in the blood. This cannot be used for rabies as it occurs very late in the disease. Rabies is present in nervous tissue (and not blood like many other viruses),
the ideal tissue to test for rabies antigen is brain.
Clinical presentation is not enough, general symptoms: pyrexia (ie fever), ataxia, paralysis prior to death
you must euthanise animal in order to confirm whether it had rabies. Shedding via saliva may be intermittent and so therefore not a reliable test.
Fluorescent Antibody Test (FAT)
The rabies antibody used for the dFA test is primarily directed against the nucleoprotein (antigen) of the virus
requires a florescent microscope
unsuitability for highly decomposed samples
requires trained technician
can be used on formalin/ glycerol preserved tissues but this is
than using fresh sample
has 99% sensitivity and specificity
uses an anti-rabies Ig with a fluorescein tag. detected after 30min incubation at 37celsius. When labeled antibody is incubated with rabies-suspect brain tissue, it will bind to rabies antigen. Unbound antibody can be washed away and areas where antigen is present can be visualized as fluorescent-apple-green areas using a fluorescence microscope. If rabies virus is absent there will be no staining
seem much more reliable, reported to give 100% specificity and sensitivity. important
can identify specific
. ie. novel lyssaviruses
not recommended by WHO/ OIE
as it requires fulfillment of quality control.
PCRs usually undertaken on fresh tissues. One study using formalin fixed tissues reported HnRT-PCR not to work (however it was not paraffin embedded)
heminested assay a second amplification of the template increases the sensitivity being capable of detecting minimum quantities of virus .
Rapid IHC> FAT
The FAT depends heavily on the quality of the immunofluorescent conjugate, the maintenance of the fluorescent microscope and also on an experienced technician reading the microscope slides. In contrast, RIDT is a very easy-to-use kit, which does not require a high level of expertise. This technique is simple to perform and to interpret.
histochemical test using
low-cost light microscopy
, the direct rapid immunohistochemical test (dRIT).
Brain tissues were all FFPE
Variations in brain region between case studies
on average: 2,3 anatomic areas per case
Only 13/ 57 cases had fresh brain tissue
57 canid rabies suspect cases based on neurological changes.
Histopathology and IHC- immuno staining
Histopathology (HE) staining
Purpose: - 1) Assess the distribution of rabies viral antigen and inflammatory changes within the brain. 2) Suitability of FFPE for PCRs. 3) the effect of artefact
negri bodies presence correlated with low scores of autolysis and freeze thaw artefact.
Not an accurate enough test to use for rabies diagnosis.
No use in autolysed specimens
Negri bodies present in only 60% of positive cases. Negri bodies, most often located in the hippocampus.
A perivascular cuffing: accumulation of various leukocytes seen in infectious, inflammatory, or autoimmune diseases.
observed mostly in the brain stem, least often in the hippocampus
inflammation may be higher in the brainstem than hippocampus as it was infected before hyppocampus. *due to retrograde transport of the virus antigen"
anatomical regional differences
more negri bodies
perivascular cuffing least observed here
more perivascular cuffing
least negri bodies observed, this may be because inflammation may obscure negri body observation. Also it is easier to see negri bodies in large purkinje neurones that are in higher numbers in the dentate gyrus.
satellitosis and neuronophagia significantly higher in brainstem and cerebrum...so everywhere??
IHC labelling score for viral antigen highest in brain stem
A condition marked by an accumulation of neuroglia cells around neurons or blood vessels of the central nervous system.
phagocytosis of neurons
the effect of artefact
artefact degeneration associated wth decrecreased immunoreactivity but does not affect overall positive or negative diagnosis.
FFPE is an advantage
autolysis and bacterial contamination reduce sensitivity
formalin fixation may make a sample unsuitable for FAT
IHC = PCR results, so IHC is a good alternative to FAT
"IHC labelling of formalin fixed tissues offers an ideal method for rabies diagnosis"
FFPE is a disadvantage
HnRT- PCR all negative
causes RNA fragmentation (particularly at warm temperatures!!), due to formalin induced cross linking- sequences above 200-300bp long unlikely to succeed in FFPE tissues.
RT-PCR also failed ?? FFPE responsible?
usually 200 fold more sensitive than HnRT
however the positive control was positive
reasons for failure: age of tissue, mehtod of storage, wax embedding effect on RNA?
other studyformalin fixing is good, but processing is bad?
PCR is super reliable even when there is tissue degeneration. However HnRT does not work in FFPE.
hnRT-PCR was more sensitive than RT-PCR, and both techniques presented lower sensibility in decomposed samples.
Limitations of this investigation
Only tested canids for rabies. May be species variation. However useful in the context of transmission to humans, as most transmission occurs when dogs bite a human.
why does the article not consider rapid immunodiagnostic tests?
did not have every anatomical region for each case. cerebrum over represented. and brain stem under represented.
quality of samples is questionable.
: the common form of rabies that is characterized by anxiety, spasm of the muscles of the throat and diaphragm, hyperactive or violent behavior, hallucinations, excessive salivation, paralysis, coma, and death
HE examination is essential to define differential diagnoses of behaviour modifying conditions. THEY DID NOT PROVE THIS
should not be sole method for rabies diagnosis (OBVIOUSLY)
does not prevent IHC even when there is artefactual degeneration
could be a secondary diagnostic tool on field as preserve sample
not useful for HnRT but may be for Taq
brain stem represents the most viral immunoreactivity