In last five years, the Africa has faced two outbreaks of Zaire ebolavirus. These outbreaks have been the largest so far, and latest outbreak is still ongoing and affecting the Democratic Republic of the Congo. We tested in parallel three different Zaire ebolavirus (EBOV) realtime RT-PCRs targeting the nucleoprotein gene (EBOV NP-RT-qPCRs) described by Trombley et al. (2010), Huang et al. (2012) and Weidmann et al. (2004). These assays are used regularly in diagnostic laboratories. The limit of detection (LOD), intra-assay repeatability using different matrixes, sensitivity and specificity were determined. In addition, the primers and probes were aligned with the sequences available in ongoing and past outbreaks in order to check the mismatches. The specificity of all three EBOV NP-RT-qPCRs were excellent (100%), and LODs were under or 10 copies per PCR reaction. Intra-assay repeatability was good in all assays, however the Ct-values were bit higher using the EDTA-blood based matrix. All of the primers and probes in EBOV NP-RTqPCR assays have one or more mismatches in the probes and primers when the 2267 Zaire EBOV NP sequences, including strains Ituri from DRC outbreak (year 2018), was aligned. The EBOV strain of Bikoro (year 2018) circulating in DRC was 100% match in Trombley and Weidmann assay, but had one mismatch in Huang assay.
In last five years, the Africa has faced two outbreaks of Zaire ebolavirus. These outbreaks have been the largest so far, and latest outbreak is still ongoing and affecting the Democratic Republic of the Congo. We tested in parallel three different Zaire ebolavirus (EBOV) realtime RT-PCRs targeting the nucleoprotein gene (EBOV NP-RT-qPCRs) described by Trombley et al. (2010); Huang et al. (2012) and Weidmann et al. (2004). These assays are used regularly in diagnostic laboratories. The limit of detection (LOD), intra-assay repeatability using different matrixes, sensitivity and specificity were determined. In addition, the primers and probes were aligned with the sequences available in ongoing and past outbreaks in order to check the mismatches. The specificity of all three EBOV NP-RT-qPCRs were excellent (100 %), and LODs were under or 10 copies per PCR reaction. Intra-assay repeatability was good in all assays, however the Ct-values were bit higher using the EDTA-blood based matrix. All of the primers and probes in EBOV NP-RT-qPCR assays have one or more mismatches in the probes and primers when the 2267 Zaire EBOV NP sequences, including strains Ituri from DRC outbreak (year 2018), was aligned. The EBOV strain of Bikoro (year 2018) circulating in DRC was 100 % match in Trombley and Weidmann assay, but had one mismatch in Huang assay. ; Peer reviewed
Abstract: Despite the global zoonotic disease burden, the underlying exposures that drive zoonotic disease emergence are not understood. Here, we aimed to assess exposures to potential sources of zoonotic disease and investigate the demographics, attitudes, and behavior of individuals with sustained occupational animal contact in Vietnam. We recruited 581 animal workers (animal-raising farmers, slaughterers, animal health workers, and rat traders) and their families in southern and central Vietnam into a cohort. Cohort members were followed for 3 years and interviewed annually regarding (1) demography and attitudes regarding zoonotic disease, (2) medical history, (3) specific exposures to potential zoonotic infection sources, and (4) socioeconomic status. Interview information over the 3 years was combined and analyzed as cross-sectional data. Of the 297 cohort members interviewed, the majority (79.8%; 237/297) reported raising livestock; almost all (99.6%; 236/237) reported being routinely exposed to domestic animals, and more than a quarter (28.7%; 68/237) were exposed to exotic animals. Overall, 70% (208/297) reported slaughtering exotic animals; almost all (99.5%; 207/208) reported consuming such animals. The consumption of raw blood and meat was common (24.6%; 73/297 and 37%; 110/297, respectively). Over half (58.6%; 174/297) reported recent occupational animal-induced injuries that caused bleeding; the use of personal protective equipment (PPE) was limited. Our work demonstrates that individuals working with animals in Vietnam are exposed to a wide range of species, and there are limited procedures for reducing potential zoonotic disease exposures. We advocate better education, improved animal security, and enforced legislation of PPE for those with occupational animal exposure in Vietnam.
Despite the global zoonotic disease burden, the underlying exposures that drive zoonotic disease emergence are not understood. Here, we aimed to assess exposures to potential sources of zoonotic disease and investigate the demographics, attitudes, and behavior of individuals with sustained occupational animal contact in Vietnam. We recruited 581 animal workers (animal-raising farmers, slaughterers, animal health workers, and rat traders) and their families in southern and central Vietnam into a cohort. Cohort members were followed for 3 years and interviewed annually regarding (1) demography and attitudes regarding zoonotic disease, (2) medical history, (3) specific exposures to potential zoonotic infection sources, and (4) socioeconomic status. Interview information over the 3 years was combined and analyzed as cross-sectional data. Of the 297 cohort members interviewed, the majority (79.8%; 237/297) reported raising livestock; almost all (99.6%; 236/237) reported being routinely exposed to domestic animals, and more than a quarter (28.7%; 68/237) were exposed to exotic animals. Overall, 70% (208/297) reported slaughtering exotic animals; almost all (99.5%; 207/208) reported consuming such animals. The consumption of raw blood and meat was common (24.6%; 73/297 and 37%; 110/297, respectively). Over half (58.6%; 174/297) reported recent occupational animal-induced injuries that caused bleeding; the use of personal protective equipment (PPE) was limited. Our work demonstrates that individuals working with animals in Vietnam are exposed to a wide range of species, and there are limited procedures for reducing potential zoonotic disease exposures. We advocate better education, improved animal security, and enforced legislation of PPE for those with occupational animal exposure in Vietnam. ; Peer reviewed
Background: During the five decades since their discovery, filoviruses of four species have caused human hemorrhagic fever outbreaks: Marburg (MARV) marburgvirus, and Zaire (EBOV), Sudan (SUDV) and Bundybugyo (BDBV) ebolaviruses. The largest, devastating EBOV epidemic in West Africa in 2014-16, has been followed by outbreaks of MARV in Uganda, 2017, and EBOV in Democratic Republic of Congo, 2018, emphasizing the need to develop preparedness to diagnose all filoviruses. Objectives: The aim of this study was to optimize a new filovirus RT-qPCR to detect all filoviruses, define its limits of detection (LOD) and perform a field evaluation with outbreak samples. Study design: A pan-filovirus RT-qPCR targeting the L gene was developed and evaluated within the EbolaMoDRAD (Ebola virus: modern approaches for developing bedside rapid diagnostics) project. Specificity and sensitivity were determined and the effect of inactivation and PCR reagents (liquid and lyophilized format) were tested. Results: The LODs for the lyophilized pan-filovirus L-RT-qPCR assay were 9.4 copies per PCR reaction for EBOV, 9.9 for MARV, 1151 for SUDV, 65 for BDBV and 289 for Tai Forest virus. The test was set at the Pasteur Institute, Dakar, Senegal, and 83 Ebola patient samples, with viral load ranging from 5 to 5 million copies of EBOV per reaction, were screened. The results for the patient samples were in 100% concordance with the reference EBOVspecific assay. Discussion: Overall, the assay showed good sensitivity and specificity, covered all filoviruses known to be human pathogens, performed well both in lyophilized and liquid-phase formats and with EBOV outbreak clinical samples. ; Peer reviewed
Background During the five decades since their discovery, filoviruses of four species have caused human hemorrhagic fever outbreaks: Marburg (MARV) marburgvirus, and Zaire (EBOV), Sudan (SUDV) and Bundybugyo (BDBV) ebolaviruses. The largest, devastating EBOV epidemic in West Africa in 2014-16, has been followed by outbreaks of MARV in Uganda, 2017, and EBOV in Democratic Republic of Congo, 2018, emphasizing the need to develop preparedness to diagnose all filoviruses. Objectives The aim of this study was to optimize a new filovirus RT-qPCR to detect all filoviruses, define its limits of detection (LOD) and perform a field evaluation with outbreak samples. Study design A pan-filovirus RT-qPCR targeting the L gene was developed and evaluated within the EbolaMoDRAD (Ebola virus: modern approaches for developing bedside rapid diagnostics) project. Specificity and sensitivity were determined and the effect of inactivation and PCR reagents (liquid and lyophilized format) were tested. Results The LODs for the lyophilized pan-filovirus L-RT-qPCR assay were 9.4 copies per PCR reaction for EBOV, 9.9 for MARV, 1151 for SUDV, 65 for BDBV and 289 for Taï Forest virus. The test was set at the Pasteur Institute, Dakar, Senegal, and 83 Ebola patient samples, with viral load ranging from 5 to 5 million copies of EBOV per reaction, were screened. The results for the patient samples were in 100% concordance with the reference EBOV-specific assay. Discussion Overall, the assay showed good sensitivity and specificity, covered all filoviruses known to be human pathogens, performed well both in lyophilized and liquid-phase formats and with EBOV outbreak clinical samples.
In order to detect serum antibodies against clinically important Old and New World hantaviruses simultaneously, multiparametric indirect immunofluorescence assays (IFAs) based on biochip mosaics were developed. Each of the mosaic substrates consisted of cells infected with one of the virus types Hantaan (HTNV), Puumala (PUUV), Seoul (SEOV), Saaremaa (SAAV), Dobrava (DOBV), Sin Nombre (SNV) or Andes (ANDV). For assay evaluation, serum IgG and IgM antibodies were analyzed using 184 laboratory-confirmed hantavirus-positive sera collected at six diagnostic centers from patients actively or previously infected with the following hantavirus serotypes: PUUV (Finland, n = 97); SEOV (China, n = 5); DOBV (Romania, n = 7); SNV (Canada, n = 23); ANDV (Argentina and Chile, n = 52). The control panel comprised 89 sera from healthy blood donors. According to the reference tests, all 184 patient samples were seropositive for hantavirus-specific IgG (n = 177; 96%) and/or IgM (n = 131; 72%), while all control samples were tested negative. In the multiparametric IFA applied in this study, 183 (99%) of the patient sera were IgG and 131 (71%) IgM positive (accordance with the reference tests: IgG, 96%; IgM, 93%). Overall IFA sensitivity for combined IgG and IgM analysis amounted to 100% for all serotypes, except for SNV (96%). Of the 89 control sera, 2 (2%) showed IgG reactivity against the HTNV substrate, but not against any other hantavirus. Due to the high cross-reactivity of hantaviral nucleocapsid proteins, endpoint titrations were conducted, allowing serotype determination in .90% of PUUV- and ANDV-infected patients. Thus, multiparametric IFA enables highly sensitive and specific serological diagnosis of hantavirus infections and can be used to differentiate PUUV and ANDV infection from infections with Murinae-borne hantaviruses (e.g. DOBV and SEOV). ; Fil: Lederer, Sabine. EUROIMMUN Medizinische Labordiagnostika; Alemania ; Fil: Lattwein, Erik. EUROIMMUN Medizinische Labordiagnostika; Alemania ; Fil: Hanke, Merle. EUROIMMUN Medizinische Labordiagnostika; Alemania ; Fil: Sonnenberg, Karen. EUROIMMUN Medizinische Labordiagnostika; Alemania ; Fil: Stoecker, Winfried. EUROIMMUN Medizinische Labordiagnostika; Alemania ; Fil: Lundkvist, Åke. Karolinska Huddinge Hospital. Karolinska Institutet; Suecia ; Fil: Vaheri, Antti. University of Helsinki; Finlandia ; Fil: Vapalahti, Olli. University of Helsinki; Finlandia ; Fil: Chan, Paul K. S. Chinese University of Hong Kong; República de China ; Fil: Feldmann, Heinz. Canadian Science Centre for Human and Animal Health. Public Health Agency of Canada; Canadá ; Fil: Dick, Daryl. Canadian Science Centre for Human and Animal Health. Public Health Agency of Canada; Canadá ; Fil: Schmidt Chanasit, Jonas. Bernhard Nocht Institute for Tropical Medicine; Alemania ; Fil: Padula, Paula Julieta. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina ; Fil: Vial, Pablo A. Universidad del Desarrollo; Chile ; Fil: Panculescu Gatej, Raluca. CANTACUZINO National Institute for Research and Development in Microbiology and Immunology; Rumania ; Fil: Ceianu, Cornelia. CANTACUZINO National Institute for Research and Development in Microbiology and Immunology; Rumania ; Fil: Heyman, Paul. Queen Astrid Military Hospital; Bélgica ; Fil: Avšič Županc, Tatjana. Institute of Microbiology and Immunology; Eslovenia ; Fil: Niedrig, Matthias. Robert Koch Institute; Alemania
