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

Journal of Virological Methods 190 (2013) 4–10

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Development and evaluation of loop-mediated isothermal amplification assay for detection of Crimean Congo hemorrhagic fever virus in Sudan Hana A.M. Osman a,b , Kamal H. Eltom a , Nasreen O. Musa a , Nasreldin M. Bilal b , Mustafa I. Elbashir c , Imadeldin E. Aradaib a,∗ a

Molecular Biology Laboratory (MBL), Department of Clinical Medicine, Faculty of Veterinary Medicine, University of Khartoum, P.O. Box 32, Khartoum North, Sudan Department of Medical Microbiology, Faculty of Medical Laboratory Sciences, University of Khartoum, P.O. Box 11081, Khartoum, Sudan c Department of Biochemistry, Faculty of Medicine, University of Khartoum, P.O. Box 32, Khartoum, Sudan b

a b s t r a c t Article history: Received 24 November 2012 Received in revised form 1 March 2013 Accepted 6 March 2013 Available online 28 March 2013 Keywords: Outbreaks Viral hemorrhagic fevers CCHFV RT-LAMP 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 10 fg 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 0.1 fg 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. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne zoonotic disease caused by Crimean-Congo hemorrhagic fever virus (CCHFV). CCHFV is a member of the genus Nairovirus in the family Bunyaviridae. The virus can be transmitted to humans by bites of Ixodid ticks, by contact with blood or tissue from infected livestock and from person to person by contact with blood or bodily fluids (Burney et al., 1980; Abu Salma, 1995; Khan et al., 1997; Rodriguez et al., 1997; Altaf et al., 1998; Ergonul, 2006; Avsic-Zupanc, 2007; Gurbuz et al., 2009; Aradaib et al., 2010). CCHFV has a genome composed of three segments (S, M and L genes), which codes for viral proteins (Schwarz et al., 1995; Rodriguez et al., 1997; Hewson et al., 2004; Lukashev, 2005; Deyde et al., 2006; Burt et al., 2009). Early diagnosis of CCHF would improve the ability to prevent subsequent spread of the infection to health-care workers in hospitals with

∗ Corresponding author. Tel.: +249 912380932. E-mail address: [email protected] (I.E. Aradaib). 0166-0934/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jviromet.2013.03.004

resource-poor settings and could protect relatives who provide nursing and medical assistance to hospitalized patients. In the past few years (2008–2010), CCHFV has been repeatedly reported as an important emerging infectious viral pathogen in the Kordufan region, Sudan. The first nosocomial outbreak of CCHF was reported in 2008 among health care workers in Alfulah rural hospital, West Kordufan (Aradaib et al., 2010). Subsequently, another outbreak was reported in 2009 in Donkup Village, Abyei District, South Kordufan (Aradaib et al., 2011). Very recently, a nosocomially acquired CCHFV infection was reported in an attending physician in Elobied hospital, North Kordufan, as a result of providing medical assistance to a an infected patient from an endemic area of Lagawa District, South Kordufan, Sudan (Elata et al., 2011). Currently, CCHFV is diagnosed by virus isolation, serology and by molecular-based techniques. Virus isolation requires high containment laboratory biosafety level (BSL-3), which is not available in most developing countries. Serology is useful in seroepidemiological surveys to identify recent or past infection by detecting CCHFV-specific IgM or IgG antibodies, respectively. However, CCHFV-specific IgM detection requires at least 3–5 days post

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Table 1 Design of LAMP primers for detection of Crimean Congo hemorrhagic fever virus based on the small RNA segment of Alfulah-3 strain isolated in Sudan (GenBank accession number GQ862371).

infection to be produced by the infected patient. Therefore, false negative results are likely to be obtained during early acute phase of the disease (Aradaib et al., 2011). Molecular-based techniques are useful for detection of the viral genome or viral proteins (Saijo et al., 2002, 2005; Garcia et al., 2006; Aradaib et al., 2010, 2011; Dowall et al., 2012). The combined use of RT-PCR for the detection of viral RNA and serological assays for the detection of specific IgM antibodies is considered to be the approach of choice for rapid and specific diagnosis of acute CCHF (Burt et al., 1998; Whitehouse, 2004; Aradaib et al., 2011; Elata et al., 2011). In previous studies, conventional RT-PCR assays were developed and evaluated for detection of CCHFV. However, most of the conventional RT-PCR assays utilize a second round of nested amplification or nucleic acid hybridization assays to increase the sensitivity and to confirm the identity of the primary amplified PCR product (Schwarz et al., 1996; Aradaib et al., 2011). It is well documented that nested PCR is time consuming, prone to error and is complicated by cross contamination due to multiple manipulations of the primary PCR products (Elata et al., 2011). Hybridization assays, despite their high specificity and relative sensitivity, are rather expensive, cumbersome and time consuming as they usually take over night (Aradaib et al., 1995; Burt et al., 1998). To address these problems, quantitative Taq-Man-based real-time PCR (qRT-PCR) were described for detection of CCHFV in biological specimens (Duha et al., 2006; Drosten et al., 2002). However, the developed real-time PCR assays are sophisticated techniques, which require expensive automated

thermal cycler and associated PCR kits. In addition, the application of real-time PCR requires a sophisticated level of training and infrastructure, which does not exist in many areas of Africa. Recently, the reverse transcription (RT) loop-mediated isothermal amplification (RT-LAMP) assay has been shown to be highly accurate for the detection of hemorrhagic fever viruses (HFVs) including Rift Valley fever virus (Peyrefitte et al., 2008; Le Roux et al., 2009), dengue virus serotypes (Parida et al., 2005), Chikungunya virus (Parida et al., 2007), West Nile virus (Parida et al., 2004), and Japanese encephalitis virus (Parida et al., 2006; Toriniwa and Komiya, 2006). However, no information is currently available on development and evaluation of the diagnostic potential of RT-LAMP assay for detection of CCHFV. In the present study, we evaluated the diagnostic potential of RT-LAMP assay for simple and rapid detection of the Sudanese strains of CCHFV using highly conserved primer sets derived from the small RNA (S) segment of Sudan Alfulah-3 strain (accession no. GQ862371). 2. Materials and methods 2.1. Collection of blood and sera from infected patients Fifteen CCHFV acute phase sera were collected from patients during disease outbreaks in different Districts of Alfulah, Abyei and Lagawa, Kordufan regions, Sudan. The samples were processed as described previously (Aradaib et al., 2010).

Table 2 Positions of the LAMP primers on the on the small RNA segment of Alfulah-3 strain isolated in Sudan (GenBank accession number GQ862371).

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H.A.M. Osman et al. / Journal of Virological Methods 190 (2013) 4–10 Table 3 Insertion of Eco R1 restriction sites between FIP and BIP LAMP primers based on the small RNA segment of Alfulah-3 strain isolated in Sudan (GenBank accession number GQ862371).

2.2. Extraction of viral nucleic acid from infected serum samples CCHFV RNAs were extracted from infected sera using QIAamp extraction kits (QIAamp, Hilden, Germany) as described by the manufacturer’s instructions. Details of the extraction procedure were described elsewhere by Aradaib et al. (2011). RNAs were quantified using a spectrophotometer at 260 nm wavelength. RNA extracts were then kept at −20 ◦ C until used for nested PCR or RT-LAMP assays. 2.3. Design of primers for RT-LAMP assay The primers used for CCHFV RT-LAMP amplification were designed from the nucleotide sequence of the S segment of Sudan-Alfulah-3 strain (Table 1). The nucleotide sequence was retrieved from GenBank accession number GQ862371 and aligned with the available sequences of cognate genes of other CCHFV strains to identify conserved regions by using CLUSTALW software version 1.83 (DNA Data Bank of Japan; http://clustalw.ddbj.nig.ac.jp/top-e.html). A potential target region was selected from the aligned sequences. A set of six primers comprising two outer (F3 and B3), two inner (FIP and FIB), and two loop primers (LF and LB) were selected. FIP contained F1c (complementary to F1), and the F2 sequence. BIP contained the B1c sequence (complementary to B1), and the B2 sequence as shown in (Table 2). LAMP primers were designed using software PrimerExplorer V4 (http://primerexplorer.jp/elamp4.0.0/index.html; Eiken Chemical Co., Japan), as described previously (Notomi et al., 2000).

mix was brought to 25 ␮l by adding nucleic acid-free water. Positive RNA controls (CCHFV Alfulah-3) and negative RNAs controls including Dengue virus (DEN-virus), Rift valley fever virus (RVFV), yellow fever virus and nucleic acid free water were included in each reaction assay. The control and test RNA samples were incubated at 63 ◦ C for 60 min for LAMP assay. For demonstration of the turbidity the RTLAMP assay was carried out in 50 ␮l volume. 2.6. Purification and digestion of LAMP products LAMP products generated by the modified primer mixtures containing restriction sites were purified by the QIAquick PCR Purification Kit (Qiagen, Germany) according to the manufacturer’s protocol. The products were then digested using EcoR1 enzyme (New England Biolabs, Japan) at 37 ◦ C for 2 h. 2.7. Visualization of LAMP products Visualization of LAMP products was made possible by the naked eye for detection of turbidity or change in color in the LAMP reaction mix using 1.0 ␮l of 50× SYBR green dye. The reaction was then visualized under UV light. LAMP products were also visualized by electrophoresis onto 2% ethidium bromide-stained agarose gel using gel documentation system (Uvitec, Cambridge, UK). In addition, the generated LAMP amplification products were also digested with ECoR1 (Roche, Mannheim, Germany) and analyzed with a 2% agarose gel electrophoresis. 2.8. Design of primers for nested PCR

2.4. Insertion of ECOR1 restriction sites in RT-LAMP assay Restriction enzyme recognition sites were inserted into each primer set. For each RT-LAMP assay the inner primers were modified by the insertion of an EcoR1 restriction site between the F1c and F2 segments of the FIP, and the B1c and B2 segment of the BIP primer (Table 3). 2.5. RT-LAMP reaction conditions The reaction condition for the RT-LAMP assay, used in this study, was performed in a final volume of 25 ␮l containing 12.5 ␮l 2× reaction buffer 40 mM Tris–HCl, 20 mM KCl, 16 mM MgSO4 , 20 mM (NH4 )2 SO4 , 0.2% Tween 20, 0.6 M betaine, 2.8 mM each dNTPs. The Bst DNA polymerase (New England Biolabs, Japan) was used at a concentration of 8 units per reaction. A volume of 1.3 ␮l primer mixture containing 40 pmol each of the FIP and BIP primers, 20 pmol each of the LF and LB primers, and 5 pmol each of the F3 and B3 primers was added to the RT-LAMP reaction mix. Fifty units of reverse transcriptase enzyme (Invitrogen, CA, USA) and 2 ␮l of the target RNA were added. The final volume of the RT-LAMP reaction

The primary and the nested primers used in this study were described basically in a previous study (Elata et al., 2011). Selection of the primers was based on a highly conserved fragment of the S RNA segment of Sudan, Alfulah-4 strain (GenBank accession number GQ862371). The primers were designed based on multiple sequence alignment of several published sequences of the gene using BioEidit software (Carlsbad, CA, USA). A forward primer CCHF1 5 -CTG CTC TGG TGG AGG CAA CAA-3 and a reverse primer CCHF2 5 -TGG GTT GAA GGC CAT GAT GTA T-3 were used to amplify a 425-bp primary PCR product. An internal pair of forward primer CCHFn15 -AGG TTT CCG TGT CAA TGC AAA-3 and a reverse primer CCHFn2 5 -TTG ACA AAC TCC CTG CAC CAG T-3 were used to amplify a 207-bp nested PCR product. 2.9. Nested RT-PCR conditions A single-tube RT-PCR assay was carried out for CCHFV RNAs amplification basically as described previously (Elata et al., 2011). Briefly, a standard 50 ␮l reaction mixture contained in final concentration of 1× enzyme mix reaction buffer, 5.0 ␮l of 10 mM dNTP

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mix, 5.0 ␮l of 25 mM Mg CL2, 5.0 U enzyme mix, 2.0 ␮l of 20 picomole of each primers (CCHF1 and CCHF2), 5.0 ␮l of target RNA, were used. The total volume was brought to 50.0 ␮l using RNase free water. Target genes were amplified in low-profile 0.2 ml tube (MJ Research, CA, USA). Rift Valley fever virus (RVFV), yellow fever virus (YFV) and Dengue virus RNA templates were used as negative controls. Thermal profiles were performed on a Techne PHC-2 thermal cycler (Techne, Princeton, NJ). The thermal cycling profiles were started with 30-min incubation at 50 ◦ C for reverse transcription of the CCHFV RNA templates into cDNA copies. The PCR tubes were then incubated at 95 ◦ C for 2 min to destroy the excess amount of RT enzyme and to activate the DNA polymerase. The synthesized cDNA copies in the PCR tubes were subjected to 40 cycles of denaturation at 95 ◦ C for 1 min, annealing at 56 ◦ C for 30 s and extension at 72 ◦ C for 45 s, and a final incubation at 72 ◦ C for 10 min. For the nested RT-PCR amplification, reaction mixtures similar to those of RT-PCR were used, except that 2 ␮l of the primary PCR products were used as a template DNAs for second round of PCR amplifications. The thermal cycling profiles were started with an initial denaturation at 95 ◦ C for 5-min, and the PCR tubes were subjected to 40 cycles of denaturation at 95 ◦ C for 1 min, annealing at 56 ◦ C for 30 s and extension at 72 ◦ C for 45 s, and a final incubation at 72 ◦ C for 10 min. Following amplification, 10 ␮l from each PCR amplification product were loaded onto 2% agarose gel and electrophoresed at 80 V for 1 h. The gels were stained with ethidium bromide, and a UV light source was used to visualize the primary and the nested PCR products. 2.10. Analytical sensitivities and specificity of the nested PCR and RT-LAMP

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Fig. 1. Detection of turbidity generated by Crimean Congo hemorrhagic fever virus. RT-LAMP reaction. The detection of turbidity was performed in 50 ␮l volume using serial dilutions of known concentration of CCHFV RNA obtained from acute phase sera of Alfula-3 infected patient. Tubes 1–4: 10-fold serial dilutions of 10 pg, 1 pg, 100 fg, 10 fg of CCHFV RNA, respectively.

3.2. Detection of turbidity and color change in the RT-LAMP products LAMP products were visualized by detection of turbidity and development of green color in the LAMP reaction mix using SYBR green intercalating dye (Fig. 1). 3.3. Analytical sensitivity of RT-LAMP The sensitivity of the RT-LAMP was determined by testing 10fold serial dilutions of RNA extracted from sera sampled during acute phase of the disease. The RT-LAMP products were visualized by ethidium bromide-stained agarose gel electrophoresis, which produced the typical ladder-like pattern with UV irradiation. The RT-LAMP has a detection limit, which span over 7 logs. High levels of analytical sensitivity were demonstrated by measuring decreasing numbers of RNA copies. The RT-LAMP assays had 100% sensitivity in detecting ≥0.1 fg of viral RNA (Fig. 2A).

To compare the analytical sensitivities of the nested PCR and RTLAMP for the detection of decreasing number of CCHFV RNA copies, 10-folds dilution series of the RNA standard, ranging from 107 to 101 per reactions, were tested in the nested assay. For evaluation of the specificity of the RT-LAMP assay, RNA extracted from closely related hemorrhagic fever viruses including Rift Valley fever virus (RVFV), Dengue virus, yellow fever virus (YFV), and nucleic acid-free water were used to determine the specificity of the nested RT-LAMP for specific detection CCHFV using specific primer sets.

Using simple water bath set at 63 ◦ C, and 1.0 pg of CCHFV RNA target, the LAMP product was detected from all Sudanese CCHFV strains, including Alfulah-3, Alfulah-4, Abyei and Lagawa strains using ethidium bromide-stained agarose gel electrophoresis (Fig. 2B).

2.11. Sequencing and sequence alignment

3.5. Specificity of RT-LAMP and conventional PCR assay

The specific 207 bp PCR products generated by the LAMP outer pair of primers (F3 and B3) or nested primers were purified using QIAquick purification Kit (Germany) according to the manufacture’s protocol and submitted for sequencing (Seqlab, Gottingen, Germany). Resulted sequences were edited using BioEdit software and the Basic Local Alignment Search Tool (BLAST) of NCBI (National Center for Biotechnology Information, Bethesda, MD) and used to confirm the identity of the generated sequences in the GenBank nucleotide database.

The specificity studies for both assays indicated that there were no amplification products when using the CCHFV-specific primer set with RNA extracted from closely related hemorrhagic fever viruses including Rift Valley fever virus (RVFV), Dengue virus, yellow fever virus and nucleic acid-free water (Fig. 2C).

3. Results 3.1. Optimization condition and visualization of RT-LAMP The optimization condition and visualization of RT-LAMP were determined using 10 pg of RNA extracted from the Sudan Alfulah3 strain, which was incubated at a range of 60–65 ◦ C. Optimum specific amplification for RT-LAMP assay was achieved at 63 ◦ C for 60 min.

3.4. Visualization of LAMP product from Sudanese CCHFV strains

3.6. Restriction endonuclease digestion with EcoR1 The specificity of the RT-LAMP amplification product was further confirmed by digestion of the LAMP product with Eco-R1 restriction enzyme, which resulted in the predicted amplified products. Multiple bands of 140, 93, 97, and 50 bp products were obtained, which correspond to the distance between F2 to B2c, F1c to B2c, F2 to B1 and F1c to B1 of the FIP and BIP primers, respectively (Fig. 3). 3.7. Conventional RT-PCR using LAMP outer primers (F3 and B3) The conventional RT-PCR, using LAMP outer pair of primers (F3 and B3), resulted in amplification of the specific 204-bp PCR

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Fig. 2. (A) Sensitivities of RT-LAMP for detection of Crimean Cong hemorrhagic fever virus using ethidium bromide-stained agarose gel electrophoresis. The RTLAMP assay was performed with serial dilutions of known concentration of CCHFV RNA obtained from acute phase sera of Alfula-3 infected patient. Lanes 1–7: 10-fold serial dilutions of 100 pg, 10 pg, 1 pg, 100 fg, 10 fg, 1.0 fg, 0.1 fg of CCHFV RNA, respectively. Lane 8: nucleic acid-free sample (negative control). (B) RT-LAMP detection of Sudanese strains of CCHFV onto 2% agarose gel using simple water bath. Lanes MW: Molecular marker; Lane 1: CCHFV Alfulah-3 RNA; Lane 2: CCHFV Alfulah-4 RNA; Lane 3: CCHFV Abyei strain; Lane 4: CCHFV Lagawa strain; Lane 5: CCHFV Lagawa strain; Lane 6: nucleic acid-free water. (C) Specificity of the LAMP primers for the detection of CCHFV RNA using Alfulah-3 CCHFV RNA as indicated and analyzed in a 2% agarose gel. Lanes MW: Molecular marker; Lane 1: Alfulah-3 CCHFV RNA (positive control); Lane 2: Rift Valley fever virus RNA; Lane 3: Dengue virus RNA; Lane 4: yellow fever RNA; Lane 5: nucleic acid-free water.

products. The specific PCR products were detected from 1 pg CCHFV RNA extracted from all Sudanese strains of CCHFV including, Alfulah, Abyei and Lagawa strains. No amplification products were obtained from other hemorrhagic fever viruses including, Rift Valley fever virus, Dengue virus and yellow fever virus (Fig. 4A).

Fig. 4. (A) Specificity of the RT-LAMP outer primers (F3 and B3) for amplification of the Sudanese CCHFV strains using conventional RT-PCR. Visualization of the 204-bp specific RNA PCR products on ethidium bromide-stained agarose gels. Lane MW: molecular weight marker; lanes 1: Alfulah-3 (positive control); Lane 2: Alfulah-4; Lane 3: Abyei-1; Lane 4: Lagawa strain; Lane 5: Rift Valley Fever virus; Lane 6: Dengue virus; Lane 7: yellow fever virus; Lane 8: nucleic acid-free water. (B) Amplification of the primary CCHFV-specific 425 bp product using Alfulah-3 CCHFV outer pair of primers. The RT-PCR assay was performed with serial dilutions of known concentration of CCHFV RNA obtained from acute phase sera of Alfula-3 infected patient. Lanes 1–7: 10-fold serial dilutions of 100 pg, 10 pg, 1 pg, 100 fg, 10 fg, 1.0 fg, 0.1 fg of CCHFV RNA, respectively. Lane 8: nucleic acid-free sample (negative control). (C) Nested RT-PCR showing the 204 bp product using the internal primers from Alfulah3 CCHFV primers. The nested RT-PCR assay was performed with serial dilutions of known concentrations of CCHFV RNA obtained from acute phase sera of Alfula-3 infected patient. Lanes 1–4: 10-fold serial dilutions of 100 fg, 10 fg, 1.0 fg, 0.1 fg of CCHFV RNA, respectively. Lane 5: nucleic acid-free sample (negative control).

cells (Fig. 4B). The internal (nested) pair of primers (CCHFn1 and CCHFn2) produced a 207-bp nested PCR product, internal to the annealing sites of primers (CCHF1 and CCHF2). The nested amplification increased the sensitivity and as little as 0.1 fg of RVFV RNA was detected by this nested RT-PCR assay. The nested PCR amplification products were visualized onto an ethidium bromide-stained agarose gels (Fig. 4C).

3.8. Sensitivity of the nested RT-PCR

4. Discussion

The outer pair of conventional primers (CCHF1 and CCHF2) produced a 425-bp primary PCR product from ≥100 fg RNA of CCHFV. The primary PCR amplification products were visualized onto an ethidium bromide-stained agarose gels. This level of sensitivity is 100 times less sensitive than that obtained by the RT-LAMP. The primers did not amplify RNA extracted from other hemorrhagic fever viruses such as Rift Valley fever; dengue virus; yellow fever virus RNA; or total nucleic acid extracts from non infected Vero

Viral hemorrhagic fevers, such as CCHF, often cause clusters of severe disease with high case fatalities. These can be devastating in remote areas, rural hospitals, and resource poor settings in the tropics when a nosocomial chain of transmission results in deaths of infected patients particularly, the highly qualified medical practitioners in these communities (Burney et al., 1980; Khan et al., 1997; Altaf et al., 1998; Ahmeti and Raka, 2006; Aradaib et al., 2010; Elata et al., 2011). It is, therefore, becoming increasingly obvious that the development of a molecular biological technique for early detection of CCHFV would be advantageous in a variety of circumstances including control of the disease and prevention of spread of infection to medical health workers and relatives who provide medical assistance or nursing to hospitalized patients. Recently, multiple outbreaks and sporadic cases of CCHF have been reported repeatedly in the Kordufan region, Sudan. (Aradaib et al., 2010, 2011; Elata et al., 2011). The genetic analysis of viruses associated with 2008 and 2010 disease outbreaks shows that several CCHFV strains are circulating and causing human outbreaks in Sudan, highlighting CCHFV as an emerging infectious viral pathogen. The Sudanese CCHFV strains are genetically unique members of Group III genetic lineage, which include viruses from across Africa including Sudan, Mauritania, South Africa and Nigeria (Aradaib et al., 2010, 2011; Elata et al., 2011). This indicates that these CCHFV strains are being grouped by geographical locations (Vesenjak-Hirjan et al., 1991;

Fig. 3. Restriction enzyme digestion of the LAMP product obtained from CCHFV strains. Visualization of the restriction patterns of the digested LAMP products using Eco R1 restriction enzyme. Lane MW: molecular weight marker; lanes 1: ECoR1 restricted Alfulah-3 LAMP product; Lane 2: Rift Valley Fever virus; Lane 3: Dengue virus; Lane 4: nucleic acid-free water; Lane 5: ECoR1 restricted Abyei strain LAMP product.

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Deyde et al., 2006). Rapid detection of emerging viral pathogens such as CCHFV is especially important in the Sudan given the large numbers of livestock in the country, and their importance to the economy and rural communities. Extensive research efforts have been made to improve the early diagnosis of CCHF in the Sudan. In previous studies, a number of RT-PCR assays for detection of CCHFV were described (Aradaib et al., 2010, 2011; Elata et al., 2011). The RT-LAMP assay should facilitate rapid detection and differentiation of an active CCHFV infection during disease outbreak in a resource-poor setting in the tropics. In this study, the potential of the RT-LAMP technique for rapid and accurate detection of CCHFV RNA in sera sampled during acute-phase of the disease was investigated, on a practical scale, for the first time. The RT-LAMP provides high levels of diagnostic sensitivity and specificity when testing a variety of acute-phase sera sampled during disease outbreaks in Kordufan region of the Sudan during 2008–2011. The time required for sample processing, extraction of viral RNA and application of RT-LAMP assay for detection of CCHFV RNA was estimated to be approximately 2 h, after arrival of the samples in the laboratory. In contrast, detection of CCHFV by conventional nested PCR requires 5 h from sample processing to final visualization of result. In addition, an important practical advantage of the LAMP technique is that it utilizes simple and relatively inexpensive equipment, which renders the assay promising for use in rural and remote areas with resourcepoor settings. Also, only basic molecular and technical skills are required for performance of the RT-LAMP assay procedure, and interpretation of the results may be as simple as a visual evaluation of turbidity or color change in the reaction mix. Furthermore, the development of LAMP assay requires the use of a set of multiple primers spanning a highly conserved genomic region of 204-bp. We demonstrated that the set of LAMP primers, targeting a highly conserved region of the S segment of Alfulah CCHFV, is well designed to detect all CCHFV strains used in this study. The sensitivity studies indicated that the RT-LAMP detected 10 fg of CCHFV RNA using 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 CCHF acute-phase cases. Using agarose gel electrophoresis, the RT-LAMP assay detected as little as 0.1 fg of viral RNA (equivalent to 50 viral particles). Results of the study illustrate that the sensitivities of the RT-LAMP and nested RT-PCR assays are in 100% agreement and both assays exhibit high levels of analytical sensitivity as measured by the detection of a known number of virus RNA copies. The specificity studies indicated that no cross reactivity was detected with 1.0 pg of RNA from related hemorrhagic fever viruses including Rift valley fever, Dengue fever and yellow fever; or non infected sera under the same stringency condition described in this study. This result confirms that the assay allows for rapid confirmation of clinical cases and early recognition of CCHF in an index patient before the appearance of the disease in an epidemic outbreak. Visualization of amplified products with the naked eye, fluorescence, or agarose gel electrophoresis would be appropriate for most laboratory settings in developing countries. In the present study the RT-LAMP assay was evaluated for detection of the Sudanese CCHFV variants and strains, which represent unique members of Group III genetic lineage. Since the RT-LAMP primers were designed based on multiple sequence alignment of several published sequences of the CCHFV S genes using BioEidit software (Carlsbad, CA, USA), and were selected from a highly conserved fragment of the S RNA segment, they would be expected to amplify RNAs from other CCHFV strains circulating globally. However, RNAs from other CCHFV strains are not available to be included in this RTLAMP assay. Therefore, additional research would be necessary to confirm this assumption. Like other molecular-based techniques, RT-LAMP could be used as supportive diagnostic assay for early

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detection of CCHFV RNA in clinical specimens. However, it should be noted that the gold standard conventional virus isolation will remain important for initial amplification of the virus in a susceptible cell line as large amount of viral RNA would be required for subsequent molecular characterization studies. In conclusion, RT-LAMP can be used for simple and early detection of CCHFV strains. There was 100% agreement between results of the RT-LAMP and nested RT-PCR when testing 10-fold serial dilution of CCHFV RNA. However, nested PCR is prone to error and is complicated by cross contamination due to multiple manipulations of the primary PCR products. The RT-LAMP provides very high levels of diagnostic sensitivity and specificity when testing a variety of acute-phase sera, sampled during CCHF outbreaks. The performance of the RT-LAMP under isothermal conditions without the need of special apparatus, and visualization of results by the naked eye, make the assay more economical and practical than nested RT-PCR in resource-poor settings in developing countries. Acknowledgements The authors would like to thank Dr. Stuart T. Nichol of the Molecular Biology Laboratory, Viral Special Pathogens Branch, Division of High Consequence Pathogens and Pathology, Centers for Disease Control and Prevention, USA, for his continuous support, encouragement and for the invaluable suggestion during the preparation of the draft manuscript. We would also like to thank Dr. Mohamed A. Bakheit for his assistance with the design of LAMP primers. The technical assistance of Afraa Elata, Reem Rabie is gratefully acknowledged. We would also like to thank Mr. Abdalla M. Fadlelmoula for his assistance in the laboratory. This study received partial financial support from the Scientific Research Directorate of the University of Khartoum, Sudan, and Centre for Disease Control and Prevention, Atlanta, USA. References Abu Salma, A.A., 1995. Meat hygiene in the Sudan: public health implications of edible offals. MVSc thesis. Faculty of Veterinary Medicine, University of Khartoum, Sudan. Ahmeti, S., Raka, L., 2006. Crimean-Congo haemorrhagic fever in Kosova: a fatal case report. Virol. J. 3, 85. Altaf, A., Luby, S., Ahmed, A.J., Zaidi, N., Khan, A.J., Mirza, S., McCormick, J., FisherHoch, S., 1998. Outbreak of Crimean-Congo haemorrhagic fever in Quetta, Pakistan: contact tracing and risk assessment. Trop. Med. Int. Health 3, 878–882. Aradaib, I.E., Wilson, W.C., Cheney, W., Pearson, J.E., Osburn, B.I., 1995. Application of the polymerase chain reaction for specific identification of epizootic hemorrhagic disease virus serotype 2. J. Vet. Diagn. Invest. 7, 388–392. Aradaib, I.E., Erickson, B.R., Mustafa, M.E., Khristova, M.L., Saeed, N.S., Elageb, R.M., Nichol, S.T., 2010. Nosocomial outbreak of Crimean-Congo hemorrhagic fever, Sudan. Emerg. Infect. Dis. 16, 837–839. Aradaib, I.E., Erickson, B.R., Karsany, M.E., Khristova, M.L., Elageb, R.M., Mohamed, M.E.H., Nichol, S.T., 2011. Multiple Crimean-Congo hemorrhagic fever virus strains are associated with disease outbreaks in Sudan, 2008–2009. PLoS Negl. Trop. Dis. 5, e1159, http://dx.doi.org/10.1371/journal.pntd.0001159. Avsic-Zupanc, T., 2007. Epidemiology of Crimean-Congo hemorrhagic fever in the Balkans. In: Ergonul, O.W.C.A. (Ed.), Crimean-Congo Hemorrhagic Fever: A Global Perspective. Springer, Dordrecht, pp. 75–88. Burney, M.I., Ghafoor, A., Saleen, M., Webb, P.A., Casals, J., 1980. Nosocomial outbreak of viral hemorrhagic fever caused by Crimean hemorrhagic fever-Congo virus in Pakistan in 1976. Am. J. Trop. Med. Hyg. 29, 941–947. Burt, F.J., Leman, P.A., Smith, J.F., Swanepoel, R., 1998. The use of a reverse transcription-polymerase chain reaction for the detection of viral nucleic acid in the diagnosis of Crimean-Congo haemorrhagic fever. J. Virol. Methods 70, 129–137. Burt, F.J., Paweska, J.T., Ashkettle, B., Swanepoel, R., 2009. Genetic relationship in southern African Crimean-Congo haemorrhagic fever virus isolates: evidence for occurrence of reassortment. Epidemiol. Infect. 137, 1302–1308. Deyde, V.M., Khristova, M.L., Rollin, P.E., Ksiazek, T.G., Nichol, S.T., 2006. CrimeanCongo hemorrhagic fever virus genomics and global diversity. J. Virol. 80, 8834–8842. Dowall, S.D., Richards, K.S., Graham, V.A., Chamberlain, J., Hewson, R., 2012. Development of an indirect ELISA method for the parallel measurement of IgG and IgM antibodies against Crimean-Congo haemorrhagic fever (CCHF) virus using recombinant nucleoprotein as antigen. J. Virol. Methods 2, 335–341.

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