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Rift Valley fever (RVF) belongs to the group of viral haemorrhagic fevers (VHFs), most of which are zoonotic diseases causing outbreaks in animals and humans all over Africa. In the absence of haemorrhagic or specific organ manifestations, these diseases are clinically difficult to diagnose. Rapid laboratory confirmation of cases is therefore essential for timely execution of supportive treatment, appropriate case management, infection control, and tracing of contacts. Rift Valley fever virus (RVFV), a mosquito-borne pathogen, is responsible for high mortality rates and abortion in domestic ruminants, resulting in significant socio-economic losses. Furthermore, the virus is potentially infectious by aerosol, can replicate in a wide range of mosquito species and poses a bioweapon threat. The recent spread of the virus outside of the African continent, demonstrates its ability to move northwards to RVF free regions, e.g. to Europe and Northern America. Such fears fuel the international demand for reliable and validated diagnostic tools for rapid diagnosis of RVF. The aim of this study was to develop a rapid and accurate molecular tool for the detection of RVFV. A real-time loop-mediated isothermal amplification assay (LAMP) targeting the L segment of RVFV, was developed and evaluated. The assay proved to be highly specific and able to detect RVFV strains representing the genetic spectrum of the virus. Furthermore, the assay did not amplify the RNA of other genetically and antigenically related phleboviruses. The sensitivity of the assay was compared to that of a previously published TaqMan RTD-PCR protocol and found to be equal. Similarly, the assay demonstrated very high diagnostic sensitivity and specificity in various clinical human and animal specimens, collected during natural outbreaks of the disease in Africa. The detection of specific viral genome targets in positive clinical specimens was achieved in less than 30 minutes. As a highly accurate, rapid and very simple nucleic acid detection format, the RT-LAMP assay has the potential to be used in less well equipped laboratories in Africa. The assay format can be adapted to a portable device that can be utilized during RVF outbreaks in remote areas, and can be a valuable tool for differential diagnosis of VHFs. / Dissertation (MSc)--University of Pretoria, 2010. / Microbiology and Plant Pathology / unrestricted

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