Development of an immunochromatography strip test based on truncated nucleocapsid antigens of three representative hantaviruses (original) (raw)
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PLoS Neglected Tropical Diseases, 2013
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).
Rapid, whole blood diagnostic test for detecting anti-hantavirus antibody in rats
Journal of Virological Methods, 2013
Hantavirus is a causative agent of rodent-borne viral zoonoses, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome. Seoul virus (SEOV) is a causative agent of urban and laboratory rat-associated HFRS worldwide. Surveillance of rodents has been done mainly by serological detection of hantavirus-specific antibodies by enzyme linked immunosorbent assay (ELISA) and immunofluorescent antibody assay (IFA). An immunochromatographic (ICG) test was developed with the N-terminal 103 amino acids of nucleocapsid protein of Hantaan virus expressed by Escherichia coli as an antigen to detect IgG antibody specific to hantavirus in sera from Rattus sp. animals. Antibodydetecting sensitivity of the ICG test was the same as that of ELISA and about 100-times higher than that of IFA. Overall sensitivities and specificities of the ICG test in comparison to ELISA and IFA for sera from 192 urban rats and 123 laboratory rats were 99.3% and 100%, respectively. Diluted whole blood samples without separation could be used for the ICG test. The ICG test enabled detection of antibodies to SEOV, Hantaan, Dobrava/Belgrade, and Thailand viruses, which are causative agents of HFRS throughout Eurasia. The ICG test is a rapid, simple and safe method for diagnosis of SEOV infection in rats.
Competitive Homogeneous Immunoassay for Rapid Serodiagnosis of Hantavirus Disease
Journal of Clinical Microbiology, 2015
In this study we describe a competitive homogenous immunoassay making use of Förster 1 Resonance Energy Transfer (FRET) in rapid detection of pathogen-specific antibodies. The assay 2 principle is based on competition between a monoclonal antibody (MAb) and serum antibodies to a 3 given antigen. In the assay named as Competitive FRET Immuno Assay (CFRET-IA), FRET signal 4 is induced if MAb carrying a donor-label binds to an acceptor-labeled antigen. Specific antibodies 5 in serum compete for antigen binding, resulting in reduced FRET signal. 6 7
Journal of Virological Methods, 2003
Hantaviruses infecting humans in Eurasia include Hantaan, Seoul, Puumala and the closely related Dobrava and Saaremaa viruses. These viruses are causative agents of hemorrhagic fever with renal syndrome (HFRS), which is recognized as a severe health care problem in several countries. Diagnostics of hantavirus infections relies on serology, performed principally with enzyme immunoassay (EIA) or immunofluorescence assay (IFA). We developed four 5-min immunochromatographic IgM-antibody tests for diagnostics of acute Puumala, Dobrava and Hantaan virus infections and a similar combination test to detect all Eurasian pathogenic hantavirus infections. We evaluated the assays using 100 fingertip blood samples collected randomly from Finnish volunteers, 28 confirmed hantavirus IgM-negative sera, and 77 sera from patients with acute infections of various hantaviruses. The specificities and sensitivities of the Puumala-, Dobrava-and Hantaan virus-specific tests varied from 96 to 100%, whereas, the combination test showed 96% specificity and 80 to 93% sensitivity. Cross-reactions were observed commonly between the Dobrava and the Hantaan virus tests, but only rarely between the Puumala and the Hantaan virus, or the Puumala and the Dobrava virus, tests. Altogether, the rapid tests showed less cross-reactivity than the respective EIA tests. According to the results, the performance of these tests meets well the requirements for diagnostic use. Nevertheless, the specific one-antigen tests were markedly more sensitive than the combination test. However, if optimized, a combination test would be suitable for regions where several hantaviruses circulate. #
Laboratory diagnosis of human hantavirus infection: novel insights and future potential
Infections by Hantavirus (Bunyaviridae) can cause severe human diseases, such as hemorrhagic fever with renal syndrome in Eurasia and cardiopulmonary syndrome in the Americas. These diseases are emergent and became a serious public health problem worldwide. Thus, rapid, sensitive and reliable methods for diagnosis of hantavirus infection are necessary in order to manage patients and control this rodent-borne virosis. Serological methods, such as neutralization tests, immunoblots and enzyme immunoassays using hantavirus-recombinant proteins as antigens, are discussed in this article, as well new methods such as immunochromatographic test. Hantavirus genome detection, by different kinds of reverse transcription-PCR, including the real-time variant, is also discussed here.
Journal of Virological Methods, 2008
Indirect and capture enzyme-linked immunosorbent assays (ELISAs) for detection of Hantaan virus (HTNV)-specific immunoglobulins G (IgG) and M (IgM) in human serum samples were developed on the basis of recombinant yeast-expressed nucleocapsid (N) protein of HTNV. The sensitivities and specificities of the indirect and capture ELISAs were evaluated by comparing the reactivity of sera from patients with hemorrhagic fever with renal syndrome (HFRS) from China with that of a commercial IgG/IgM kit. The sensitivity of the indirect IgG and IgM ELISA tests was both 100% and the specificity of the indirect IgM and IgG ELISA test was 98% and 99%, respectively. The sensitivity and specificity of the capture IgM ELISA was 100% and 97%, respectively. The novel assays were found to detect HTNV-specific antibodies in acute phase sera from suspected HFRS patients in China. The results indicate that these novel ELISAs are suitable for the diagnosis of HTNV and for sero-epidemiological studies.
Quality control measures for the serological diagnosis of hantavirus infections
Journal of Clinical Virology, 2003
Background: With society's rapidly increasing mobility, patients infected with severe viral infections can become seriously ill at any place in Europe and elsewhere. Improving the diagnostics of these infections is the most important step in detecting the pathogens and dealing with them, and for this purpose, quality control measures are essential tools. Objectives: To assess the diagnostic reality for rare hantavirus infections in Europe by (1) running a pre-evaluation panel (four samples, sent out in 1999) to optimise sample preparation and shipping procedure and afterwards (2) starting an External Quality Assurance (EQA) program (20 samples, sent out in 2001). Study design: All samples sent out had to be tested for the presence of specific IgG and IgM antibodies against hantavirus. For the pre-evaluation panel, four samples were distributed (two samples IgG'//IgM(/, one sample IgG-borderline/IgM(/, one sample IgG(// IgM(/), for the EQA 20 samples (six samples IgG'//IgM'/, eight samples IgG'//IgM(/, one sample IgG(/borderline/ IgM(/, five samples IgG(//IgM(/). Thirteen laboratories took part in the pre-evaluation panel, 18 laboratories participated in the first EQA run. Results: For the pre-evaluation panel, the participants reported correct results for 64% of the IgG-positive samples (85% excluding borderline-positive sample), and 92% for the IgG-negative sample. IgM testing was correctly negative in all laboratories. For the EQA, the participants reported correct results for 76% of the IgG-positive samples, and 97% correct results for the IgG-negative samples. For the IgM-positive samples, 53% correct results were reported, and 98% correct results for the IgM-negative samples. Conclusions: The results presented here prove the importance of quality measures also for viruses only rarely suspected, like hantavirus, and they clearly demonstrate the need for improvement of the existing test systems.
Diagnostic Microbiology and Infectious Disease, 2007
The symptoms of hantavirus pulmonary syndrome may resemble those of other febrile illnesses. The development of an accurate diagnostic test should therefore improve clinical prognosis and be useful in epidemiologic studies. We evaluated the use of a recombinant antigen (rND 85 ) based on the S-segment sequences of a Brazilian hantavirus for detecting immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies against hantavirus in an indirect enzyme immunoassay (EIA). We assayed 613 serum samples (570 from humans and 43 from rodents). IgM EIA had a sensitivity of 94.1% and a specificity of 99.1%. IgG EIA had a sensitivity of 95.2% and a specificity of 98.4%. This evaluation confirms that rND 85 IgM and IgG EIA tests are potentially useful rapid, sensitive, and cost-effective tools for detecting antibodies against hantaviruses indigenous to Brazil and other South American countries, in patients with acute or convalescent hantavirus infection, and in rodent reservoirs. D
Journal of Clinical Virology, 2005
Background: The hantavirus cardiopulmonary syndrome (HCPS) was first recognized in 1993 after a cluster of acute respiratory distress syndrome deaths in the southwestern of the United States. The major causative agent of HCPS in North America is the Sin Nombre virus (SNV) carried by the deer mouse Peromyscus maniculatus. The first HCPS case imported to Europe was reported in 2002. Objectives: The objective of the study was to develop and evaluate ELISA and Western blot tests for the serological detection of human infections caused by SNV including those imported to Europe. Study design: A polyhistidine (His)-tagged recombinant nucleocapsid (rN) protein of SNV was expressed in Saccharomyces cerevisiae and purified by nickel chelation chromatography. On the basis of the purified SNV rN protein-capture and indirect IgM and IgG ELISAs and an IgG Western blot were developed. The evaluation of the tests was performed using a negative serum panel and a blinded serum panel from the US containing acute-phase sera from HCPS patients. Results: Based upon the results obtained using a panel of negative control sera the specificity for SNV-capture and indirect IgM and IgG ELISAs were found to be 100%. All 33 sera from SNV-infected HCPS patients included in the blinded panel were detected by the SNV-capture and indirect IgM ELISAs. Twenty-nine out of the 33 SNV-IgM positive sera reacted also in the SNV-IgG ELISA. An SNV-IgG Western blot confirmed the data of the SNV-IgG ELISA. Although the majority of anti-SNV positive sera cross-reacted with rN proteins of Puumala virus and Dobrava virus, the lacking reactivity of a few sera with these heterologous rN antigens in the corresponding IgM and IgG ELISAs demonstrates the value of virus-specific test formats for acute-phase sera. Conclusions: The novel SNV ELISA and Western blot tests represent a useful tool for the serological detection of SNV infections.
Journal of clinical microbiology, 1985
Antisera prepared against 16 strains of hantaviruses isolated from patients with hemorrhagic fever with renal syndrome (HFRS) or from rodents captured in HFRS-endemic and nonendemic regions were titrated against Hantaan virus strain 76-118, Puumala virus strain Sotkamo, and Prospect Hill virus strain Prospect Hill-I by using the indirect immunofluorescent antibody and plaque reduction neutralization tests. Isolates fell into one of four distinct groups or serotypes. Serotype 1 included Apodemus-derived strains, serotype 2 included Rattus-derived strains, serotype 3 included Clethrionomys-derived strains, and serotype 4 included Microtus-derived strains. Serotypic classification of hantavirus infections was possible for humans and rodents in widely varied geographical areas, but in a few instances, sera from patients with HFRS did not conform to any of the four serotypes, suggesting the existence of as yet unidentified serotypes. A definitive serological classification of hantaviruse...