526 research outputs found
Quantitative real-time PCR detection of Zika virus and evaluation with field-caught mosquitoes
No full textBACKGROUND Zika virus (ZIKV), a mosquito borne flavivirus is a pathogen affecting humans in Asia and Africa. ZIKV infection diagnosis relies on serology-which is challenging due to cross-reactions with other flaviviruses and/or absence or low titer of IgM and IgG antibodies at early phase of infection- virus isolation, which is labor intensive, time consuming and requires appropriate containment. Therefore, real-time RT-PCR (rRT-PCR) is an appealing option as a rapid, sensitive and specific method for detection of ZIKV in the early stage of infection. So far, only one rRT-PCR assay has been described in the context of the outbreak in Micronesia in 2007. In this study, we described a one step rRT-PCR for ZIKV which can detect a wider genetic diversity of ZIKV isolates from Asia and Africa. RESULTS The NS5 protein coding regions of African ZIKV isolates were sequenced and aligned with representative flaviviruses sequences from GenBank to design primers and probe from conserved regions. The analytical sensitivity of the assay was evaluated to be 32 genome-equivalents and 0.05 plaque forming unit (pfu). The assay was shown to detect 37 ZIKV isolates covering a wide geographic in Africa and Asia over 36 years but none of the 31 other flaviviruses tested showing high analytical specificity. The rRT-PCR could be performed in less than 3 hours. This method was used successfully to detect ZIKV strains from field-caught mosquitoes. CONCLUSION We have developed a rapid, sensitive and specific rRT-PCR for detection of ZIKV. This assay is a useful tool for detection of ZIKV infection in regions where a number of other clinically indistinguishable arboviruses like dengue or chikungunya co-circulate. Further studies are needed to validate this assay in clinical positive samples collected during acute ZIKV infection
One-step RT-PCR for detection of Zika virus
No full textBackground: Zika virus (ZIKV) is an emerging mosquito-borne flavivirus circulating in Asia and Africa. Human infection induces an influenza-like syndrome that is associated with retro-orbital pain, oedema, lymphadenopathy, or diarrhea. Diagnosis of Zika fever requires virus isolation and serology, which are time consuming or cross-reactive. Objective: To develop a one-step RT-PCR assay to detect ZIKV in human serum. Study design: An assay targeting the envelope protein coding region was designed and evaluated for its specificity, detection limit, repeatability, and capacity to detect ZIKV isolates collected over a 40-year period from various African countries and hosts. Results: The assay's detection limit and repeatability were respectively 7.7 pfu/reaction and 100% in serum and L-15 medium; none of 19 other flaviviruses tested were detected. Conclusions: The assay is rapid, sensitive, and specific to detect ZIKV in cell culture or serum, but needs to be validated for diagnosis using clinical samples
A recombinase polymerase amplification assay for rapid detection of rabies virus
No full textRabies is a generally fatal encephalitis caused by a negative-sense single-stranded RNA lyssavirus transmitted to humans mainly from dog bite. Despite the recommendation by WHO and OIE to use the direct immunofluorescence test as standard method, molecular diagnostic assays like reverse transcription quantitative polymerase chain reaction (RT-qPCR) are increasing as a confirmatory method. However, both technologies are inaccessible in resource-limited settings. Moreover, the available point-of-need molecular assay is of poor detection limit for African strains. Herein, we developed a reverse transcription recombinase polymerase amplification (RT-RPA) assay as potential point-of-need diagnostic tool for rapid detection of various strains of rabies virus including locally isolated African strains. The sensitivity and specificity of the method was evaluated using a molecular RNA standard and different Rabies-related viruses belonging to the Rhabdoviridea family, respectively. The RABV-RPA performances were evaluated on isolates representative of the existing diversity and viral dilutions spiked in non-neural clinical specimen. The results were compared with RT-qPCR as a gold standard. The RABV-RPA detected down to 4 RNA molecules per reaction in 95% of the cases in less than 10 min. The RABV-RPA assay is highly specific as various RABV isolates were identified, but no amplification was observed for other member of the Rhabdoviridea family. The sample background did not affect the performance of the RABV-RPA as down to 11 RNA molecules were identified, which is similar to the RT-qPCR results. Our developed assay is suitable for use in low-resource settings as a promising alternative tool for ante-mortem rabies diagnosis in humans for facilitating timely control decisions
Zahedan rhabdovirus, a novel virus detected in ticks from Iran
No full textBackground: Rhabdoviridaeinfect a wide range of vertebrates, invertebrates and plants. Their transmission can occur via various arthropod vectors. In recent years, a number of novel rhabdoviruses have been identified from various animal species, but so far only few tick-transmitted rhabdoviruses have been described. Methods: We isolated a novel rhabdovirus, provisionally named Zahedan rhabdovirus (ZARV), fromHyalomma anatolicum anatolicumticks collected in Iran. The full-length genome was determined using 454 next-generation sequencing and the phylogenetic relationship to other rhabdoviruses was analyzed. Inoculation experiments in mammalian Vero cells and mice were conducted and a specific PCR assay was developed. Results: The complete genome of ZARV has a size of 11,230 nucleotides (nt) with the typical genomic organization ofRhabdoviridae. Phylogenetic analysis confirms that ZARV is closely related to Moussa virus (MOUV) from West Africa and Long Island tick rhabdovirus (LITRV) from the U.S., all forming a new monophyletic clade, provisionally designatedZamolirhabdovirus, within theDimarhabdovirussupergroup. The glycoprotein (G) contains 12 conserved cysteins which are specific for animal rhabdoviruses infecting fish and mammals. In addition, ZARV is able to infect mammalian Vero cells and is lethal for mice when inoculated intracerebrally or subcutaneously. The developed PCR assay can be used to specifically detect ZARV. Conclusion: The novel tick-transmitted rhabdovirus ZARV is closely related to MOUV and LITRV. All three viruses seem to form a new monophyletic clade. ZARV might be pathogenic for mammals, since it can infect Vero cells, is lethal for mice and its glycoprotein contains 12 conserved cysteins only found in animal rhabdoviruses. The mammalian host of ZARV remains to be identified
Combination Random Isothermal Amplification and Nanopore Sequencing For Rapid Identification of the Causative Agent of an Outbreak
No full textBackground Outbreaks of fever of unknown origin start with nonspecific symptoms and case definition is only slowly developed and adapted, therefore, identifying the causative agent is crucial to ensure suitable treatment and control measures. As an alternative method for Polymerase Chain Reaction in molecular diagnostics diagnostic, metagenomics can be applied to identify the pathogen responsible for the outbreak through sequencing all nucleic acids present in a sample extract. Sequencing data obtained can identify new or variants of known agents. Objectives To develop a rapid and field applicable protocol to allow the identification of the causative agent of an outbreak. Study design We explored a sequencing protocol relying on multiple displacement isothermal amplification and nanopore sequencing in order to allow the identification of the causative agent in a sample. To develop the procedure, a mock sample consisting of supernatant from Zika virus tissue culture was used. Results The procedure took under seven hours including sample preparation and data analysis using an offline BLAST search. In total, 63,678 sequence files covering around 10,000 bases were extracted. BLAST search revealed the presence of Zika virus. Conclusion In conclusion, the protocol has potential for point of need sequencing to identify RNA viruses. The whole procedure was operated in a suitcase laboratory. However, the procedure is cooling chain dependent and the cost per sequencing run is still high
Chikungunya Outbreak in Kedougou, Southeastern Senegal in 2009–2010
No full textBackground In Senegal, Chikungunya virus (CHIKV), which is an emerging mosquito-borne alphavirus, circulates in a sylvatic and urban/domestic cycle and has caused sporadic human cases and epidemics since 1960s. However, the real impact of the CHIKV sylvatic cycle in humans and mechanisms underlying its emergence still remains unknown. Methodology One thousand four hundred nine suspect cases of CHIKV infection, recruited from 5 health facilities located in Kedougou region, south-eastern Senegal, between May 2009 to March 2010, together with 866 serum samples collected from schoolchildren from 4 elementary schools in May and November 2009 from Kedougou were screened for anti-CHIKV immunoglobulin (Ig)M antibodies and, when appropriate, for viral nucleic acid by real-time polymerase chain reaction (rPCR) and virus isolation. In addition, mosquitoes collected in the same area from May 2009 to January 2010 were tested for CHIKV by rPCR and by virus isolation, and 116 monkeys sera collected from March 2010 to May 2010 were tested for anti-CHIKV IgM and neutralizing antibodies. Results The main clinical manifestations of the CHIKV suspect cases were headache, myalgia, and arthralgia. Evidence for CHIKV infection was observed in 1.4% (20 of 1409) of patients among suspect cases. No significant difference was observed among age or sex groups. In addition, 25 (2.9%) students had evidence of CHIKV infection in November 2009. Chikungunya virus was detected in 42 pools of mosquitoes, mainly from Aedes furcifer, and 83% of monkeys sampled were seropositive. Conclusions Our findings further documented that CHIKV is maintained in a sylvatic transmission cycle among monkeys and Aedes mosquitoes in Kedougou, and humans become infected by exposure to the virus in the forest.Additional co-authors: Kathryn A. Hanley, Anta T Dia, Denis Malvy, Scott C. Weaver, Amadou Alpha Sal
Recombinase polymerase amplification assay for rapid detection of Monkeypox virus
No full textIn this study, a rapid method for the detection of Central and West Africa clades of Monkeypox virus (MPXV) using recombinase polymerase amplification (RPA) assay targeting the G2R gene was developed. MPXV, an Orthopoxvirus, is a zoonotic dsDNA virus, which is listed as a biothreat agent. RPA was operated at a single constant temperature of 42°C and produced results within 3 to 10 minutes. The MPXV-RPA-assay was highly sensitive with a limit of detection of 16 DNA molecules/μl. The clinical performance of the MPXV-RPA-assay was tested using 47 sera and whole blood samples from humans collected during the recent MPXV outbreak in Nigeria as well as 48 plasma samples from monkeys some of which were experimentally infected with MPXV. The specificity of the MPXV-RPA-assay was 100% (50/50), while the sensitivity was 95% (43/45). This new MPXV-RPA-assay is fast and can be easily utilised at low resource settings using a solar powered mobile suitcase laboratory
Mobile laboratory reveals the circulation of Dengue virus serotype I of Asian origin in Medina Gounass (Guediawaye), Senegal
No full textWith the growing success of controlling malaria in Sub-Saharan Africa, the incidence of fever due to malaria is in decline, whereas the proportion of patients with non-malaria febrile illness (NMFI) is increasing. Clinical diagnosis of NMFI is hampered by unspecific symptoms but early diagnosis is a key factor for better patient care and for disease control. The aim of this study was to determine the arboviral aetiologies of NMFI in a low resources settings using a mobile laboratory based on Recombinase Polymerase Amplification (RPA) assays. The panel of tests for this study was expanded to five arboviruses including (Dengue virus (DENV), Zika virus (ZIKV), Yellow fever virus (YFV), Chikungunya virus (CHIKV), and Rift valley fever virus (RVFV). One hundred and four children aged between one month and 115 months were enrolled and screened. Three of 104 blood samples of children < 10 years presenting at an outpatient clinic tested positive for DENV. The results were confirmed by real-time RT-PCR, partial sequencing, and non structural protein 1 (NS1) antigen capture ELISA (Biorad, France). Phylogenetic analysis of the derived DENV-1 sequences clustered them with sequences of DENV-1 isolates from Guangzhou in China in 2014. In conclusion, this mobile setup proved reliable for the rapid identification of the causative agent of NMFI, with results consistent with those obtained in the reference laboratory`s settings
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