Imadeldin E. Aradaib

Nosocomial Outbreak of Crimean-Congo Hemorrhagic Fever, Sudan

To confirm the presence of Crimean-Congo hemorrhagic fever in Sudan, we tested serum of 8 patients with hemorrhagic fever in a rural hospital in 2008. Reverse transcription-PCR identified Crimean-Congo hemorrhagic fever virus. Its identification as group III lineage indicated links to virus strains from South Africa, Mauritania, and Nigeria.

A multiplex PCR for simultaneous detection and differentiation of North American serotypes of bluetongue and epizootic hemorrhagic disease viruses

In the present study, a multiplex RT-PCR-based assay for simultaneous detection and differentiation of North American serotypes of bluetongue (BT) virus (BTV) and epizootic hemorrhagic disease (EHD) virus (EHDV) in cell culture and clinical samples was developed. Two pairs of primers (B1 and B4) and (E1 and E4) were designed to hybridize to non-structural protein 1 (NS1) genomes of (BTV-11) and (EHDV-1), respectively. The multiplex PCR-based assay utilized a single tube-PCR amplification in which EHDV and BTV primers were used simultaneously in a multiplex format. The BTV primers generated a 790 base pair (bp) specific PCR product from RNA samples of North American BTV serotypes 2, 10, 11, 13 and 17; whereas EHDV serotypes 1 and 2 or total nucleic acid extract from non-infected baby hamster kidney (BHK) cells failed to demonstrate the 790bp specific BTV PCR product. Likewise, the EHDV primers produced a 387bp specific PCR product from RNA samples of EHDV serotypes 1 and 2, but not from BTV serotypes 2, 10, 11, 13, 17 or from total nucleic acid extract of BHK cell controls. Two pairs of nested primers (B2 and B3) and (E2 and E3), internal to the annealing sites of primers (B1and B4) and primers (E1 and E4), produced a 520bp specific BTV and a 224bp specific EHDV PCR product from BTV and EHDV first amplification products, respectively. These nested amplifications increased the sensitivity of the PCR assay and confirmed the specificity of the first amplified EHDV or BTV PCR products. The described multiplex RT-PCR-based assay could be used to facilitate rapid detection and differentiation of North American BTV and EHDV serotypes and to provide a valuable tool to study the epidemiology of these orbivirus infections in susceptible animal populations.

Multiple Crimean-Congo Hemorrhagic Fever Virus Strains Are Associated with Disease Outbreaks in Sudan, 2008–2009

Background Crimean-Congo hemorrhagic fever (CCHF) activity has recently been detected in the Kordufan region of Sudan. Since 2008, several sporadic cases and nosocomial outbreaks associated with high case-fatality have been reported in villages and rural hospitals in the region. Principal Findings In the present study, we describe a cluster of cases occurring in June 2009 in Dunkop village, Abyei District, South Kordufan, Sudan. Seven CCHF cases were involved in the outbreak; however, clinical specimens could be collected from only two patients, both of whom were confirmed as acute CCHF cases using CCHF-specific reverse transcriptase polymerase chain reaction (RT-PCR). Phylogenetic analysis of the complete S, M, and L segment sequences places the Abyei strain of CCHF virus in Group III, a virus group containing strains from various countries across Africa, including Sudan, South Africa, Mauritania, and Nigeria. The Abyei strain detected in 2009 is genetically distinct from the recently described 2008 Sudanese CCHF virus strains (Al-fulah 3 and 4), and the Abyei strain S and L segments closely match those of CCHF virus strain ArD39554 from Mauritania. Conclusions The present investigation illustrates that multiple CCHF virus lineages are circulating in the Kordufan region of Sudan and are associated with recent outbreaks of the disease occurring during 2008–2009.

Detection of Epizootic Hemorrhagic Disease Virus Serotypes 1 and 2 in Cell Culture and Clinical Samples Using Polymerase Chain Reaction

The potential for the use of the polymerase chain reaction (PCR) in detecting epizootic hemorrhagic disease virus (EHDV) ribonucleic acid in cell cultures and clinical samples was studied. Using oligoribonucleotide primers selected from genome segment 6 of EHDV-2 (Alberta strain), the PCR-based assay resulted in a 387-base pair (bp) PCR product. EHDV RNA from US prototype serotypes 1 and 2 and a number of EHDV field isolates, propagated in cell cultures, were detected by this EHDV PCR-based assay. Amplification products were visualized on ethidium bromide-stained agarose gels or detected by chemiluminescent hybridization. The sensitivity of the PCR assay was 100 fg of virus RNA (equivalent to 6 × 103 virus particles) with ethidium bromide-stained agarose gels. Chemiluminescent hybridization increased the sensitivity of the PCR assay 1,000 times, and specific signals were detected from 0.1 fg of virus RNA (equivalent to 6 virus particles). Amplification product was not detected when the PCR-based assay was applied to RNA from the US bluetongue (BT) virus prototype serotypes 2, 10, 11, 13, and 17 or total nucleic acid extracts from uninfected baby hamster kidney-21 cells, Vero cells, and blood cells from deer that were EHDV seronegative and virus isolation negative. Application of this EHDV PCR-based assay to clinical samples resulted in detection of EHDV RNA from blood and spleen samples from a deer in California with clinical hemorrhagic disease. This EHDV PCR-based assay could provide a rapid, sensitive, and specific assay for detection of EHDV infection in susceptible ruminants.

Experimental epizootic hemorrhagic disease virus infection in calves: virologic and serologic studies

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Rift Valley Fever, Sudan, 2007 and 2010

Viral sequences analyzed indicate recent virus movement and support the need for surveillance.

A nosocomial transmission of crimean-congo hemorrhagic fever to an attending physician in North Kordufan, Sudan.

BackgroundCrimean-Congo hemorrhagic fever (CCHF), a tick-borne disease caused by Crimean-Congo hemorrhagic fever virus (CCHFV), is a member of the genus Nairovirus in the family Bunyaviridae. Recently, CCHFV has been reported as an important emerging infectious viral pathogen in Sudan. Sporadic cases and multiple CCHF outbreaks, associated with nosocomial chain of transmission, have been reported in the Kordufan region of Sudan.AimsTo confirm CCHF in an index patient and attending physician in North Kordufan region, Sudan, and to provide some information on virus genetic lineages.MethodsAntibody captured ELISA, reverse transcription PCR, partial S segment sequences of the virus and subsequent phylogenetic analysis were used to confirm the CCHFV infection and to determine the virus genetic lineages.ResultsCCHF was confirmed by monitoring specific IgM antibody and by detection of the viral genome using RT-PCR. Treatment with oral ribavirin, replacement with fluid therapy, blood transfusion and administration of platelets concentrate resulted in rapid improvement of the health condition of the female physician. Phylogenetic analysis of the partial S segment sequences of the 2 CCHFV indicates that both strains are identical and belong to Group III virus lineage, which includes viruses from Africa including, Sudan, Mauritania, South Africa and Nigeria.ConclusionFurther epidemiologic studies including, CCHFV complete genome analysis and implementation of improved surveillance are urgently needed to better predict and respond to CCHF outbreaks in the Kordufan region, Sudan.

Comparison of polymerase chain reaction and virus isolation for detection of epizootic hemorrhagic disease virus in clinical samples from naturally infected deer

We compared our recently reported reverse transcriptase polymerase chain reaction (PCR)-based assay for detection of epizootic hemorrhagic disease virus (EHDV) in clinical samples with different virus isolation (VI) procedures. Thirty-six blood samples and 1 spleen sample from deer were assessed by the EHDV PCR assay and VI in baby hamster kidney (BHK)-21 cells and embryonated chicken eggs (ECE). The EHDV PCR assay detected EHDV RNA from 6 blood samples obtained from deer during 1988–1989 outbreaks of epizootic hemorrhagic disease and from the spleen and blood samples of a deer with clinical hemorrhagic disease in 1992. The 6 blood samples from the 1988–1989 outbreaks and the spleen sample from the 1992 case were VI positive on BHK-21 cell culture. The blood from the same deer with the PCR- and VI-positive spleen was VI negative in BHK-21 cells and ECE. All EHDV isolates were identified as EHDV serotype 2 by a plaque inhibition test. The results of this study indicate that the sensitivity of the previously described EHDV PCR assay is comparable to or greater than that of the VI method in BHK-21 cell culture or ECE. The EHDV PCR assays could provide a superior diagnostic alternative to the current cumbersome and time-consuming VI procedures.

Development and evaluation of loop-mediated isothermal amplification assay for detection of Crimean Congo hemorrhagic fever virus in Sudan.

Crimean-Congo hemorrhagic fever (CCHF) virus (CCHFV) activity has been detected in Kordufan region of the Sudan in 2008 with high case-fatality rates in villages and rural hospitals in the region. Therefore, in the present study, a reverse transcription (RT) loop-mediated isothermal amplification (RT-LAMP) assay was developed and compared to nested RT-PCR for rapid detection of CCHFV targeting the small (S) RNA segment. A set of RT-LAMP primers, designed from a highly conserved region of the S segment of the viral genome, was employed to identify all the Sudanese CCHFV strains. The sensitivity studies indicated that the RT-LAMP detected 10fg of CCHFV RNA as determined by naked eye turbidity read out, which is more likely the way it would be read in a resource-poor setting. This level of sensitivity is good enough to detect most acute cases. Using agarose gel electrophoresis, the RT-LAMP assay detected as little as 0.1fg of viral RNA (equivalent to 50 viral particle). There was 100% agreement between results of the RT-LAMP and the nested PCR when testing 10-fold serial dilution of CCHFV RNA. The specificity studies indicated that there was no cross-reactivity with other related hemorrhagic fever viruses circulating in Sudan including, Rift Valley fever virus (RVFV), Dengue fever virus, and yellow fever virus. The RT-LAMP was performed under isothermal conditions at 63°C and no special apparatus was needed, which rendered the assay more economical and practical than real-time PCR in such developing countries, like Sudan. In addition, the RT-LAMP provides a valuable tool for rapid detection and differentiation of CCHFV during an outbreak of the disease in remote areas and in rural hospitals with resource-poor settings.

Rift Valley Fever among febrile patients at New Halfa hospital, eastern Sudan.

BackgroundSince the first isolation of the Rift Valley Fever virus (RVFV) in 1930s, there have been several epizootics outbreaks in the tropic mainly in Africa including Sudan. Recognition of cases and diagnosis of RVF are critical for management and control of the disease.AimsTo investigate the seroprevalence and risk factors for seropostive to RVFV IgG among febrile patients.MethodsAll febrile patients presented to New Halfa hospital in eastern Sudan during September through November 2007 were investigated to identify the cause of their fever including malaria and RFV.ResultsOut of 290 feverish patients presented to the hospital, malaria was diagnosis in 94 individuals. Fevers of unknown origin were diagnosed in 149 patients. Seropostive to RVFV IgG was detected by enzyme-linked immunosorbent assay in 122 (81.8%) of the sera from these 149 patients with fever of unknown origin. While socio-demographic characteristics (age, Job, education and residency) were not associated with seropostive to RVFV IgG, male (OR = 2.8, 95% CI = 1.0-7.6; P = 0.04) were at three times higher risk for seropostive to RVFV IgG.ConclusionThere was a high seropostive to RVFV IgG in this setting, more research is needed perhaps using other methods like PCR and IGM.