Rift Valley fever (RVF) is a mosquito-borne zoonotic disease caused by a virus of the family
Bunyaviridae, genus Phlebovirus. It is responsible for extensive outbreaks of disease in
livestock in Africa with significant mortality and economic impact. Virus neutralization is
considered the gold standard for confirming Rift Valley fever virus (RVFV) infection but the
procedure is time consuming and expensive. Real-time reverse transcription-polymerase
chain reaction (rRT-PCR), histopathology, and immunohistochemistry (IHC) are the
diagnostic methods most often used in South Africa to confirm or exclude a diagnosis of
RVF in necropsied animals. Validated estimates of diagnostic accuracy of these tests, in
naturally infected livestock, however, have not been published. The objective of this study was to estimate the diagnostic sensitivity and specificity of rRT-PCR, histopathology, and
IHC using Bayesian latent class methods in the absence of a gold standard. A secondary
objective was to estimate stratum-specific values based on species, age, degree of
specimen autolysis, and the presence/absence of tissue pigments.
The Sensitivity (Se) and Specificity (Sp) of qRT-PCR were 97.4% (95% credibility interval
(CI): 95.2% - 98.8%) and 71.7% (95% CI: 65% - 77.9%) respectively. The extraordinary
analytical sensitivity of PCR makes this test very susceptible to false positive reactions, and
thus reduced specificity. This is more likely during large-scale epidemics due to crosscontamination
of specimens at necropsy facilities or testing laboratories.
The Se and Sp of histopathology were 94.6% (95% CI: 91% - 97.2%) and 92.3% (95% CI:
87.6% - 95.8%) respectively. Single cases of RVF could be confused with acute poisoning
with plants, bacterial septicaemias, and viral diseases such as infectious bovine
rhinotracheitis and Wesselsbron disease. Most of these conditions, however, can be
excluded using histological examination of the liver, special stains, bacterial culture, and
toxicological or serological investigations. The Se and Sp of IHC were 97.6% (95% CI: 93.9% - 99.8%) and 99.4% (95% CI: 96.9% -
100%) respectively. Immunohistochemistry is highly specific because characteristic positive
immunolabelling of the cytoplasm of hepatocytes can be correlated with the presence of
hepatocellular injury typical for RVFV infection. False negative results are sometimes
obtained with IHC because of reader error or loss of the antigenic epitopes due to advanced
autolysis. Scant positive immunolabelling might be missed or viral proteins might be absent
from sections of liver with advanced hepatocellular damage.
The stratified analysis suggested differences in test accuracy in foetuses and severely
autolysed specimens. The Sp of histopathology in foetuses (83.0%) was 9.3% lower than
the value obtained for the sample population (92.3%). Lesions in some foetuses are more
subtle and the typical eosinophilic intranuclear inclusions are often difficult to detect. In
severely autolysed specimens, the Se of IHC decreased by 16.1% and the Sp of rRT-PCR by 17.4%. There is no plausible biological explanation for this decrease in the Sp of rRTPCR
since the RNA of RVFV is resistant to degradation in autolysed tissues. Conversely,
the antibody used to detect RVFV using IHC detects epitopes raised against nucleoproteins
of the virus and it is possible that viral proteins become too widely dispersed and/or
degraded in autolysed tissues to detect by light microscopy. It is possible that the marked
decrease in Se of histopathology and IHC in severely autolysed specimens caused an
apparent decrease in Sp of rRT-PCR, due to the latent class method.
In conclusion, the high estimated Sp (99.4%) of IHC and the low Sp of rRT-PCR (71.3%)
suggests that the definitive diagnosis or exclusion of RVF should not rely on a single PCR
test and that IHC would be an effective confirmatory test for rRT-PCR positive field cases
necropsied during an epidemic. Immunohistochemistry results from severely autolysed
specimens, however, should be interpreted with caution and aborted foetuses in areas
endemic for RVF should be screened using a variety of tests. The diagnostic Se and Sp of
histopathology was much higher than expected confirming the value of routine post mortem
examinations and histopathology of liver specimens. The most feasible RVF testing option
in areas that do not have suitably equipped PCR laboratories, and where disease is often
not detected in livestock until after human cases have been diagnosed, would be routine
histopathology screening with IHC confirmation.
Key Words: Rift Valley fever; Rift Valley fever virus; Bayesian; latent-class model; real-time
reverse transcription-polymerase chain reaction; immunohistochemistry; histopathology;
diagnosis; sensitivity; specificity. / Dissertation (MSc)--University of Pretoria, 2014. / gm2014 / Paraclinical Sciences / unrestricted
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:up/oai:repository.up.ac.za:2263/40707 |
| Date | January 2014 |
| Creators | Odendaal, Lieza |
| Contributors | Clift, Sarah J., lieza.odendaal@up.ac.za, Fosgate, Geoffrey T. |
| Publisher | University of Pretoria |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Dissertation |
| Rights | © 2014 University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. |
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