Real-time loop-mediated isothermal amplification assay for rapid detection of Rift Valley fever virus

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 Pathologyunrestricte

International External Quality Assessment of Molecular Detection of Rift Valley Fever Virus

Rift Valley fever (RVF) is a viral zoonosis that primarily affects animals resulting in considerable economic losses due to death and abortions among infected livestock. RVF also affects humans with clinical symptoms ranging from an influenza-like illness to a hemorrhagic fever. Over the past years, RVF virus (RVFV) has caused severe outbreaks in livestock and humans throughout Africa and regions of the world previously regarded as free of the virus. This situation prompts the need to evaluate the diagnostic capacity and performance of laboratories worldwide. Diagnostic methods for RVFV detection include virus isolation, antigen and antibody detection methods, and nucleic acid amplification techniques. Molecular methods such as reverse-transcriptase polymerase chain reaction and other newly developed techniques allow for a rapid and accurate detection of RVFV. This study aims to assess the efficiency and accurateness of RVFV molecular diagnostic methods used by expert laboratories worldwide. Thirty expert laboratories from 16 countries received a panel of 14 samples which included RVFV preparations representing several genetic lineages, a specificity control and negative controls. In this study we present the results of the first international external quality assessment (EQA) for the molecular diagnosis of RVF. Optimal results were reported by 64% of the analyses, 21% of the analyses achieved acceptable results and 15% of the results revealed that there is need for improvement. Evenly good performances were achieved by specific protocols which can therefore be recommended as an accurate molecular protocol for the diagnosis of RVF. Other protocols showed uneven performances revealing the need for improved optimization and standardization of these protocols

Human rabies associated with domestic cat exposures in South Africa, 1983–2018

Rabies is a fatal encephalitic disease caused by lyssaviruses belonging to the family Rhabdoviridae. At the time of this report, a total of 16 species of lyssaviruses, which included the prototype rabies virus (RABV), and 2 related but unclassified bat lyssaviruses, Taiwan and Kothalati, had been recognised by the International Committee on Taxonomy of Viruses (ICTV 2019). Globally RABV, also referred to as ‘classic rabies’, circulates in natural transmission cycles involving domestic dogs and various wildlife species. In the Americas, RABV is found in certain insectivorous and haematophagous bat species (Banyard et al. 2013). The public health burden of rabies is, however, very closely related to the occurrence of the disease in domestic dogs; thus, human cases of rabies are mostly reported from areas where dog rabies is uncontrolled (Hampson et al. 2015). An annual estimation of 59 000 human deaths occur worldwide with 95% of rabies cases occurring in Africa and Asia (Hampson et al. 2015). In South Africa, RABV circulates both in domestic animals and wildlife cycles, involving the canid and mongoose variants of the virus (Nel, Thomson & Von Teichman 1993). The urban cycle involves domestic dogs reported from various locations in the country, but particularly from the KwaZulu-Natal, Eastern Cape, Limpopo and Mpumalanga provinces (Cohen et al. 2007; Zulu, Sabeta & Nel 2009). Sylvatic cycles of the canid variant RABV in bat-eared foxes and black-backed jackal (Zulu et al. 2009) and the mongoose variant RABV in certain species of mongoose occur in South Africa (Van Zyl, Markotter & Nel 2010). Apart from the reservoir species, canid and mongoose RABV infections are reported in an array of domestic and wildlife species in the country, with these animals primarily serving as dead-end hosts (Sabeta et al. 2018). Laboratory-confirmed human rabies cases in South Africa are predominantly dogmediated, and seven cases of rabies linked to other domestic species and wildlife have been reported (Weyer et al. 2011).http://www.jsava.co.zaam2020Medical VirologyVeterinary Tropical Disease

South African Ebola diagnostic response in Sierra Leone : a modular high biosafety field laboratory

BACKGROUND : In August 2014, the National Institute for Communicable Diseases (NICD) in South Africa established a modular high-biosafety field Ebola diagnostic laboratory (SA FEDL) near Freetown, Sierra Leone in response to the rapidly increasing number of Ebola virus disease (EVD) cases. METHODS AND FINDINGS : The SA FEDL operated in the Western Area of Sierra Leone, which remained a ªhotspotº of the EVD epidemic for months. The FEDL was the only diagnostic capacity available to respond to the overwhelming demand for rapid EVD laboratory diagnosis for several weeks in the initial stages of the EVD crisis in the capital of Sierra Leone. Furthermore, the NICD set out to establish local capacity amongst Sierra Leonean nationals in all aspects of the FEDL functions from the outset. This led to the successful hand-over of the FEDL to the Sierra Leone Ministry of Health and Sanitation in March 2015. Between 25 August 2014 and 22 June 2016, the laboratory tested 11,250 specimens mostly from the Western Urban and Western Rural regions of Sierra Leone, of which 2,379 (21.14%) tested positive for Ebola virus RNA. CONCLUSIONS : he bio-safety standards and the portability of the SA FEDL, offered a cost-effective and practical alternative for the rapid deployment of a field-operated high biocontainment facility. The SA FEDL teams demonstrated that it is highly beneficial to train the national staff in the course of formidable disease outbreak and accomplished their full integration into all operational and diagnostic aspects of the laboratory. This initiative contributed to the international efforts in bringing the EVD outbreak under control in Sierra Leone, as well as capacitating local African scientists and technologists to respond to diagnostic needs that might be required in future outbreaks of highly contagious pathogens.S1 Video. ªHotº processing of Ebola clinical specimens, PPE and decontamination procedures in South African modular, field-operated biocontainment facility in Sierra Leone.Janusz T Paweska was supported by funding from National Research Foundation and the Global Disease Detection Programmehttp://www.plosntds.orgam2017Microbiology and Plant Patholog

Development and evaluation of a real-time reverse transcription-loop-mediated isothermal amplification assay for rapid detection of Rift Valley fever virus in clinical specimens

This paper reports on the development and validation of a real-time reverse transcription-loop-mediated isothermal amplification assay (RT-LAMP) targeting the genomic large RNA segment of Rift Valley fever virus (RVFV). The set of six designed RT-LAMP primers identified strains of RVFV isolated in geographically distinct areas over a period of 50 years; there was no cross-reactivity with other genetically related and unrelated arboviruses. When testing serial sera and plasma from sheep experimentally infected with wild-type RVFV, there was 100% agreement between results of the RT-LAMP, a TaqMan-based real-time PCR, and virus isolation. Similarly, the assay had very high levels of diagnostic sensitivity and specificity when testing various clinical specimens from humans and animals naturally infected with the virus during recent outbreaks of the disease in Africa. The detection of specific viral genome targets in positive clinical specimens was achieved in less than 30 min. As a highly accurate, rapid, and very simple nucleic acid detection format, the RT-LAMP has the potential to be used in less-well-equipped laboratories in Africa and as a portable device during RVF outbreaks in remote areas, and it can be a valuable tool for the differential diagnosis of viral hemorrhagic fevers

Results of the EQA for molecular detection of RVFV – Part 1.

<p>+: positive.</p><p>−: negative.</p><p>bold: quantified result.</p>*<p>: correct strain.</p><p>TOSV: Toscana virus.</p><p>RT: reverse transcription.</p><p>qRT: real-time RT-PCR.</p><p>(24): Drosten et al., 2002.</p><p>(25): Bird et al., 2008.</p><p>(31): Weidmann et al. 2008.</p><p>SC: SmartCycler from Cepheid.</p><p>LC: LightCycler from Roche Applied Science.</p><p>ABI: 7500 Real-Time PCR System from Applied Biosystems.</p><p>(ih): in house assay.</p

Risk factors associated with exposure to Crimean-Congo haemorrhagic fever virus in animal workers and cattle, and molecular detection in ticks, South Africa.

Crimean-Congo haemorrhagic fever (CCHF) is a severe tick-borne viral zoonosis endemic to parts of Africa, Europe, the Middle East and Central Asia. Human cases are reported annually in South Africa, with a 25% case fatality rate since the first case was recognized in 1981. We investigated CCHF virus (CCHFV) seroprevalence and risk factors associated with infection in cattle and humans, and the presence of CCHFV in Hyalomma spp. ticks in central South Africa in 2017-18. CCHFV IgG seroprevalence was 74.2% (95%CI: 64.2-82.1%) in 700 cattle and 3.9% (95%CI: 2.6-5.8%) in 541 farm and wildlife workers. No veterinary personnel (117) or abattoir workers (382) were seropositive. The prevalence of CCHFV RNA was significantly higher in Hyalomma truncatum (1.6%) than in H. rufipes (0.2%) (P = 0.002). Seroprevalence in cattle increased with age and was greater in animals on which ticks were found. Seroprevalence in cattle also showed significant geographic variation. Seroprevalence in humans increased with age and was greater in workers who handled livestock for injection and collection of samples. Our findings support previous evidence of widespread high CCHFV seroprevalence in cattle and show significant occupational exposure amongst farm and wildlife workers. Our seroprevalence estimate suggests that CCHFV infections are five times more frequent than the 215 confirmed CCHF cases diagnosed in South Africa in the last four decades (1981-2019). With many cases undiagnosed, the potential seriousness of CCHF in people, and the lack of an effective vaccine or treatment, there is a need to improve public health awareness, prevention and disease control

Results of the EQA for molecular detection of RVFV – Part 2.

<p>+: positive.</p><p>−: negative.</p><p>bold : quantified result.</p>*<p>: correct strain.</p><p>TOSV: Toscana virus.</p><p>PBV: Phlebovirus.</p><p>RT: reverse transcription.</p><p>qRT: real-time RT-PCR.</p><p>nRT: nested RT-PCR.</p><p>(co): commercial assay.</p><p>(ih): in house assay.</p><p>FN: false negative.</p><p>FP: false positive.</p><p>(22): Sall et al., 2002.</p><p>(23): Garcia et al. 2001.</p><p>(24): Drosten et al., 2002.</p><p>(25): Bird et al., 2007.</p><p>(26): Le Roux et al. 2009.</p><p>(27): Euler et al. 2012.</p><p>(31): Weidmann et al. 2008.</p><p>(32): Busquets et al. 2010.</p><p>(33): Mweango et al. 2012.</p><p>(34): Sanchez-Seco et al. 2003.</p><p>SC: SmartCycler from Cepheid.</p><p>LC: LightCycler from Roche Applied Science.</p><p>QR: qRT-PCR System from Qiagen Rotagen.</p><p>RT-LAMP: RT-loop-mediated isothermal amplification assay.</p><p>RPA: recombinase polymerase amplification assay.</p