RESUMO
BACKGROUND: Hamburg is a city state of approximately 1.9 Mio inhabitants in Northern Germany. Currently, the COVID-19 epidemic that had largely subsided during last summer is resurging in Hamburg and in other parts of the world, underlining the need for additional tools to monitor SARS-CoV-2 antibody responses. AIM: We aimed to develop and validate a simple, low-cost assay for detecting antibodies against the native coronavirus 2 spike protein (CoV-2 S) that does not require recombinant protein or virus. METHOD: We transiently co-transfected HEK cells or CHO cells with expression vectors encoding CoV-2 S and nuclear GFP. Spike protein-specific antibodies in human serum samples bound to transfected cells were detected with fluorochrome conjugated secondary antibodies by flow cytometry orimmunofluorescence microscopy. We applied this assay to monitor antibody development in COVID-19 patients, household contacts, and hospital personnel during the ongoing epidemic in the city state of Hamburg. RESULTS: All recovered COVID-19 patients showed high levels of CoV-2 S-specific antibodies. With one exception, all household members that did not develop symptoms also did not develop detectable antibodies. Similarly, lab personnel that worked during the epidemic and followed social distancing guidelines remained antibody-negative. CONCLUSION: We conclude that high-titer CoV-2 S-specific antibodies are found in most recovered COVID-19 patients and in symptomatic contacts, but only rarely in asymptomatic contacts. The assay may help health care providers to monitor disease progression and antibody responses in vaccination trials, to identify health care personnel that likely are resistant to re-infection, and recovered individuals with high antibody titers that may be suitable asplasma and/or antibody donors.
Assuntos
Anticorpos Antivirais/análise , COVID-19 , Glicoproteína da Espícula de Coronavírus , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , COVID-19/imunologia , Cricetinae , Cricetulus , Citometria de Fluxo , Células HEK293 , Humanos , Pessoa de Meia-Idade , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologiaRESUMO
The COVID-19 pandemic has caused significant morbidity and mortality. There is an urgent need for serological tests to detect antibodies against SARS-CoV-2, which could be used to assess past infection, evaluate responses to vaccines in development, and determine individuals who may be protected from future infection. Current serological tests developed for SARS-CoV-2 rely on traditional technologies such as enzyme-linked immunosorbent assays (ELISA) and lateral flow assays, which have not scaled to meet the demand of hundreds of millions of antibody tests so far. Herein, we present an alternative method of antibody testing that depends on one protein reagent being added to patient serum/plasma or whole blood with direct, visual readout. Two novel fusion proteins, RBD-2E8 and B6-CH1-RBD, were designed to bind red blood cells (RBCs) via a single-chain variable fragment (scFv), thereby displaying the receptor-binding domain (RBD) of SARS-CoV-2 spike protein on the surface of RBCs. Mixing mammalian-derived RBD-2E8 and B6-CH1-RBD with convalescent COVID-19 patient serum and RBCs led to visible hemagglutination, indicating the presence of antibodies against SARS-CoV-2 RBD. B6-CH1-RBD made in bacteria was not as effective in inducing agglutination, indicating better recognition of RBD epitopes from mammalian cells. Given that our hemagglutination test uses methods routinely used in hospital clinical labs across the world for blood typing, we anticipate the test can be rapidly deployed at minimal cost. We anticipate our hemagglutination assay may find extensive use in low-resource settings for detecting SARS-CoV-2 antibodies.
Assuntos
Anticorpos Antivirais/análise , Anticorpos Antivirais/imunologia , Teste Sorológico para COVID-19/métodos , COVID-19/sangue , COVID-19/imunologia , Testes de Hemaglutinação/métodos , Sistemas Automatizados de Assistência Junto ao Leito , SARS-CoV-2/imunologia , Antígenos Virais/imunologia , COVID-19/diagnóstico , COVID-19/virologia , Teste Sorológico para COVID-19/economia , Eritrócitos/imunologia , Testes de Hemaglutinação/economia , Humanos , Sistemas Automatizados de Assistência Junto ao Leito/economia , Proteínas Recombinantes de Fusão/imunologia , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Fatores de TempoRESUMO
Estimates of seroprevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies have been hampered by inadequate assay sensitivity and specificity. Using an enzyme-linked immunosorbent assay-based approach that combines data about immunoglobulin G responses to both the nucleocapsid and spike receptor binding domain antigens, we show that excellent sensitivity and specificity can be achieved. We used this assay to assess the frequency of virus-specific antibodies in a cohort of elective surgery patients in Australia and estimated seroprevalence in Australia to be 0.28% (95% Confidence Interval, 0-1.15%). These data confirm the low level of transmission of SARS-CoV-2 in Australia before July 2020 and validate the specificity of our assay.
Assuntos
Anticorpos Antivirais/análise , COVID-19/diagnóstico , Ensaio de Imunoadsorção Enzimática , Estudos Soroepidemiológicos , Antígenos Virais/imunologia , Austrália , COVID-19/imunologia , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , Humanos , Imunoglobulina G/análise , Fosfoproteínas/imunologia , Sensibilidade e Especificidade , Glicoproteína da Espícula de Coronavírus/imunologiaRESUMO
BACKGROUND: This study aimed to characterize sources of knowledge on the means of prevention of SARS-CoV-2 infections as well as to assess the methods of preventing SARS-CoV-2 infection among police employees in Poland and their potential impact on the risk of SARS-CoV-2 infection. METHODS: The study consisted of two phases: questionnaire and laboratory tests for SARS-CoV-2 infection. The questionnaire included 30 questions related to risk factors, knowledge about SARS-CoV-2, and methods of infection prevention. RESULTS: Data were obtained from 5082 police employees. The most common source of knowledge for a daily update on SARS-CoV-2 infection prevention was the Internet (42.6%), television (40.3%), and radio (39.7%). The most commonly used methods of SARS-CoV-2 infection included washing one's hands for at least 20 seconds (95.8%), wearing facemasks (82.9%), and physical distancing (74.9%). Results of IgG tests were lower in police units where the overall compliance with the preventive measures was higher (p < 0.01). Women were more likely to exercise SARS-CoV-2 infection prevention behaviors compared to men. Compliance with the recommended protective measures increased with age. CONCLUSIONS: Lower anti-SARS-CoV-2 IgG seropositivity rates were observed in police units with better overall compliance with the preventive measures, suggesting the key importance of group rather than individual behaviors.
Assuntos
COVID-19/prevenção & controle , Comportamentos Relacionados com a Saúde , Política de Saúde , Polícia , Adulto , Anticorpos Antivirais/análise , Controle de Doenças Transmissíveis , Feminino , Humanos , Imunoglobulina G/análise , Masculino , Pessoa de Meia-Idade , Pandemias , Polônia/epidemiologia , Inquéritos e Questionários , Adulto JovemRESUMO
INTRODUCTION: The COVID-19 pandemic caused by SARS-CoV-2 threatens global public health, and there is an urgent public health need to assess acquired immunity to SARS-CoV-2. Serological tests might provide results that can be complementary to or confirm suspected COVID-19 cases and reveal previous infection. The performance of serological assays (sensitivity and specificity) has to be evaluated before their use in the general population. The neutralisation capacity of the produced antibodies also has to be evaluated. METHODS AND ANALYSIS: We set up a prospective, multicentric clinical study to evaluate the performance of serological kits among a population of healthcare workers presenting mild symptoms suggestive of SARS-CoV-2 infection. Four hundred symptomatic healthcare workers will be included in the COVID-SER study. The values obtained from a control cohort included during the prepandemic time will be used as reference. A workflow was set up to study serological response to SARS-CoV-2 infection and to evaluate antibody neutralisation capacity in patients with a confirmed SARS-CoV-2 infection. The sensitivity and specificity of the tests will be assessed using molecular detection of the virus as a reference. The measurement of IgM and IgG antibodies will be performed once per week for 6 consecutive weeks and then at 6, 12, 18, 24 and 36 months after the diagnosis. The kinetics of IgM and IgG will determine the optimal period to perform serological testing. The proportion of false negative PCR tests in symptomatic subjects will be determined on the basis of subsequent seroconversions. ETHICS AND DISSEMINATION: Ethical approval has been obtained from the national review board for biomedical research in April 2020 (Comité de Protection des Personnes Sud Méditerranée I, Marseille, France) (ID RCB 2020-A00932-37). Results will be disseminated through presentations at scientific meetings and publications in peer-reviewed journals. TRIAL REGISTRATION NUMBER: NCT04341142.
Assuntos
Anticorpos Antivirais/análise , COVID-19/diagnóstico , Programas de Rastreamento/métodos , Pandemias , SARS-CoV-2/imunologia , COVID-19/epidemiologia , Feminino , Humanos , Masculino , Estudos Prospectivos , Testes SorológicosRESUMO
There is a clear requirement for an accurate SARS-CoV-2 antibody test, both as a complement to existing diagnostic capabilities and for determining community seroprevalence. We therefore evaluated the performance of a variety of antibody testing technologies and their potential use as diagnostic tools. Highly specific in-house ELISAs were developed for the detection of anti-spike (S), -receptor binding domain (RBD) and -nucleocapsid (N) antibodies and used for the cross-comparison of ten commercial serological assays-a chemiluminescence-based platform, two ELISAs and seven colloidal gold lateral flow immunoassays (LFIAs)-on an identical panel of 110 SARS-CoV-2-positive samples and 50 pre-pandemic negatives. There was a wide variation in the performance of the different platforms, with specificity ranging from 82% to 100%, and overall sensitivity from 60.9% to 87.3%. However, the head-to-head comparison of multiple sero-diagnostic assays on identical sample sets revealed that performance is highly dependent on the time of sampling, with sensitivities of over 95% seen in several tests when assessing samples from more than 20 days post onset of symptoms. Furthermore, these analyses identified clear outlying samples that were negative in all tests, but were later shown to be from individuals with mildest disease presentation. Rigorous comparison of antibody testing platforms will inform the deployment of point-of-care technologies in healthcare settings and their use in the monitoring of SARS-CoV-2 infections.
Assuntos
Anticorpos Antivirais/análise , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , Sistemas Automatizados de Assistência Junto ao Leito , Testes Sorológicos/métodos , Adulto , Idoso , Betacoronavirus , COVID-19 , Teste para COVID-19 , Técnicas de Laboratório Clínico , Serviços de Saúde Comunitária , Proteínas do Nucleocapsídeo de Coronavírus , Ensaio de Imunoadsorção Enzimática , Feminino , Hospitais , Humanos , Imunoensaio , Medições Luminescentes , Masculino , Pessoa de Meia-Idade , Proteínas do Nucleocapsídeo/imunologia , Pandemias , Fosfoproteínas , SARS-CoV-2 , Sensibilidade e Especificidade , Glicoproteína da Espícula de Coronavírus/imunologiaAssuntos
Betacoronavirus , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Programas de Rastreamento , Pneumonia Viral/diagnóstico , Sensibilidade e Especificidade , Anticorpos Antivirais/análise , Doenças Assintomáticas , Betacoronavirus/genética , Betacoronavirus/imunologia , Betacoronavirus/isolamento & purificação , COVID-19 , Teste para COVID-19 , Transmissão de Doença Infecciosa/prevenção & controle , Acessibilidade aos Serviços de Saúde , Humanos , Técnicas de Amplificação de Ácido Nucleico , Pandemias , Médicos de Atenção Primária , Valor Preditivo dos Testes , Prática de Saúde Pública , Quarentena , SARS-CoV-2 , Padrão de Cuidado , Fatores de TempoRESUMO
Infectious bursal disease (IBD) remains a potential worldwide threat to the poultry industry despite several vaccination approaches. Because maternally derived antibodies (MDA) constitute a critical problem for IBD vaccination, we examined the efficiency of the intracloacal vaccination approach in breaking through MDA. Experiment 1 determined the ability of the vaccinal strain to multiply in the bursa of Fabricius (BF) in chicks with a high level of MDA. Using real-time polymerase chain reaction, we quantified the strain in the bursae of vaccinated and non-vaccinated chicks. Experiment 2 was performed on three groups of chicks with high levels of MDA: group 1, non-vaccinated non-challenged; group 2, non-vaccinated challenged; and group 3, vaccinated challenged. Seroconversion to IBDV was measured using enzyme-linked immunosorbent assay. Groups 2 and 3 were challenged by vvIBDV at 25 days of age. Experiment 3 studied the effect of early IBD vaccinal strain multiplication on the immune response of vaccinated and non-vaccinated chicks to other vaccines. In experiment 1, the vaccinal strain showed progressive multiplication and reached the detectable titre in BF at 12â h post-vaccination despite high MDA titre. Experiment 2 showed that chicks in group 3 had significant seroconversion against IBDV. After challenge, group 3 showed significant improvements in several measured parameters compared with group 2. Moreover, results of experiment 3 proved that early multiplication of the vaccinal strain in the BF has no significant effect on the immune system or immune response to other vaccines. These results proved the promising success of this IBD vaccination approach.RESEARCH HIGHLIGHTS IBD vaccinal strain succeeded in multiplying in BF after intracloacal inoculation.Vaccinated chicks showed significant seroconversion of IBDV antibody titres.Vaccinated chicks showed a significant protection level against vvIBDV.Early IBD vaccination did not affect the immune response to other vaccines.
Assuntos
Infecções por Birnaviridae/veterinária , Vírus da Doença Infecciosa da Bursa/imunologia , Vacinação/veterinária , Animais , Anticorpos Antivirais/análise , Infecções por Birnaviridae/prevenção & controle , Bolsa de Fabricius/imunologia , Galinhas , Doenças das Aves Domésticas/prevenção & controle , Vacinação/métodos , Vacinas ViraisRESUMO
Coronavirus disease 2019 (COVID-19) is an acute infection of the respiratory tract that emerged in late 20191,2. Initial outbreaks in China involved 13.8% of cases with severe courses, and 6.1% of cases with critical courses3. This severe presentation may result from the virus using a virus receptor that is expressed predominantly in the lung2,4; the same receptor tropism is thought to have determined the pathogenicity-but also aided in the control-of severe acute respiratory syndrome (SARS) in 20035. However, there are reports of cases of COVID-19 in which the patient shows mild upper respiratory tract symptoms, which suggests the potential for pre- or oligosymptomatic transmission6-8. There is an urgent need for information on virus replication, immunity and infectivity in specific sites of the body. Here we report a detailed virological analysis of nine cases of COVID-19 that provides proof of active virus replication in tissues of the upper respiratory tract. Pharyngeal virus shedding was very high during the first week of symptoms, with a peak at 7.11 × 108 RNA copies per throat swab on day 4. Infectious virus was readily isolated from samples derived from the throat or lung, but not from stool samples-in spite of high concentrations of virus RNA. Blood and urine samples never yielded virus. Active replication in the throat was confirmed by the presence of viral replicative RNA intermediates in the throat samples. We consistently detected sequence-distinct virus populations in throat and lung samples from one patient, proving independent replication. The shedding of viral RNA from sputum outlasted the end of symptoms. Seroconversion occurred after 7 days in 50% of patients (and by day 14 in all patients), but was not followed by a rapid decline in viral load. COVID-19 can present as a mild illness of the upper respiratory tract. The confirmation of active virus replication in the upper respiratory tract has implications for the containment of COVID-19.
Assuntos
Betacoronavirus/imunologia , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Hospitalização , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Soroconversão , Replicação Viral , Anticorpos Antivirais/análise , Anticorpos Antivirais/imunologia , Sequência de Bases , Betacoronavirus/genética , Betacoronavirus/patogenicidade , Sangue/virologia , COVID-19 , Teste para COVID-19 , Técnicas de Laboratório Clínico , Proteínas do Envelope de Coronavírus , Infecções por Coronavirus/diagnóstico , Fezes/química , Fezes/virologia , Humanos , Imunoglobulina G/análise , Imunoglobulina G/imunologia , Imunoglobulina M/análise , Imunoglobulina M/imunologia , Pulmão/virologia , Pandemias , Faringe/virologia , Pneumonia Viral/diagnóstico , Polimorfismo de Nucleotídeo Único/genética , RNA Viral/análise , SARS-CoV-2 , Escarro/virologia , Urina/virologia , Proteínas do Envelope Viral/genética , Carga Viral/imunologia , Eliminação de Partículas ViraisRESUMO
We have previously reported the generation of the attenuated KNU-141112-S DEL5/ORF3 virus by continuous propagation of highly virulent G2b porcine epidemic diarrhea virus (PEDV) in Vero cells. The present study aimed to assess the safety of S DEL5/ORF3 and to evaluate its effectiveness as a live vaccine for prime-booster vaccinations. Reversion to virulence experiments revealed that the S DEL5/ORF3 strain retains its attenuated phenotype and genetic stability after five successive passages in susceptible piglets. Pregnant sows were primed orally with an S DEL5/ORF3 live vaccine and boosted intramuscularly twice with a commercial killed vaccine at 2-week intervals prior to parturition. This sow vaccination regimen completely protected nursing piglets against virulent G2b challenge, as evidenced by the increase in survival rate from 0% to 100% and the significant reduction in diarrhea intensity, including the amount and duration of PEDV fecal shedding. In addition, despite a 2-3 day period of weight loss in piglets from vaccinated sows after challenge, their daily weight gain was recovered at 7 days post-challenge and became similar to that of unchallenged pigs from unvaccinated sows over the course of the experiment. Furthermore, strong antibody responses to PEDV were verified in the sera and colostrum of immunized sows with the prime-boost treatment and their offspring. Altogether, our data demonstrated that the attenuated S DEL5/ORF3 strain guarantees the safety to host animals with no reversion to virulence and is suitable as an effective primary live vaccine providing durable maternal lactogenic immunity for passive piglet protection.
Assuntos
Infecções por Coronavirus/veterinária , Diarreia/veterinária , Doenças dos Suínos/prevenção & controle , Potência de Vacina , Vacinas Atenuadas/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/análise , Anticorpos Antivirais/sangue , Colostro/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Diarreia/prevenção & controle , Feminino , Genótipo , Imunização Secundária , Injeções Intramusculares , Vírus da Diarreia Epidêmica Suína/genética , Gravidez , Taxa de Sobrevida , Suínos , Doenças dos Suínos/imunologia , Vacinas Atenuadas/administração & dosagem , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de Produtos Inativados/imunologia , Vacinas Virais/administração & dosagem , Virulência , Eliminação de Partículas ViraisRESUMO
People who inject drugs (PWID) are a key high-risk group for Hepatitis C Virus (HCV) infection due to the sharing of needles and drug-preparation equipment. However, only approximately 50% of PWID are currently screened for HCV in Switzerland. At present, screening of PWID occurs in general practice via venepuncture. Compared to venepuncture, screening via rapid antibody saliva and dried blood spot (DBS) tests is well adapted to PWID, who typically have difficult venous access. The cost-effectiveness of an increased access screening programme of PWID (increased screening using rapid antibody saliva tests and DBS tests [semi-quantitative viraemia and viral genotype]) was analysed through a decision tree screening model combined with the outputs of a Markov treatment model. Sensitivity and scenario analyses examined the uncertainty of results. At a willingness to pay (WTP) threshold of CHF 100 000 (USD 105 000) per quality-adjusted life year (QALY), the increased access screening programme was cost-effective compared to current screening, with a base case incremental cost-effectiveness ratio of CHF 7 940 (USD 8337) per QALY. The net monetary benefit was CHF 959 802 668 (USD 1 007 792 801) for the PWID population and CHF 94 469 (USD 99 192) per person. The increased access screening programme had a 97.0% probability of being cost-effective compared to the current screening method at the WTP threshold of CHF 100 000 (USD 105 000). The results showed an increased access screening programme that uses tests which are better suited to the PWID population to be more cost-effective, due to the increased uptake that rapid antibody saliva and DBS tests generate.
Assuntos
Análise Custo-Benefício , Teste em Amostras de Sangue Seco/economia , Hepatite C/diagnóstico , Programas de Rastreamento/economia , Saliva/imunologia , Abuso de Substâncias por Via Intravenosa/epidemiologia , Adulto , Anticorpos Antivirais/análise , Usuários de Drogas/estatística & dados numéricos , Feminino , Custos de Cuidados de Saúde , Hepatite C/economia , Hepatite C/epidemiologia , Humanos , Masculino , Programas de Rastreamento/métodos , Abuso de Substâncias por Via Intravenosa/complicações , Suíça/epidemiologiaRESUMO
Chronic HCV infection, a highly endemic disease in Egypt, is usually asymptomatic for decades after infection. Prediction questionnaire tool was proofed to be a valuable, feasible and efficient instrument for the screening of several diseases. We previously developed an Egyptian HCV risk screening tool (EGCRISC). This study aims to validate/modify EGCRISC. A cross-sectional study testing 4579 individuals by EGCRISC as well as ELISA/PCR was performed. The sample was a stratified cluster sampling from urban and rural areas in Upper and Lower Egypt using a proportional allocation technique. The degree of agreement and positive and negative posttest probabilities were calculated. ROC curve was done and the cutoff points were customized for best performance. The total score was further classified into three levels according to the risk load. The mean age of the participants was 41.1±12.2 in whom HCV prevalence was 8.6%. EGCRISC, particularly after modifying the cutoff points, has a good discriminating ability. The degree of agreement was at least 68.1% and the positive posttest probability ranged from 5% to 37.2% whereas the negative posttest probability was in the range 1% to 17%. We conclude that EGCRISC is a valid tool that can potentially screen for HCV infection risk in Egypt and could diminish the demand for mass serologic screening in those apparently at minimal risk. Extensive use of electronic and self- or interviewer-administered risk-based screening strategy may simplify and promote overall screening and detection of HCV dissimilar communities.
Assuntos
Hepatite C Crônica/diagnóstico , Programas de Rastreamento/métodos , Adulto , Anticorpos Antivirais/análise , Área Sob a Curva , Estudos Transversais , Demografia , Egito/epidemiologia , Ensaio de Imunoadsorção Enzimática , Feminino , Hepacivirus/genética , Hepacivirus/imunologia , Hepacivirus/isolamento & purificação , Hepatite C Crônica/epidemiologia , Humanos , Masculino , Pessoa de Meia-Idade , Razão de Chances , Reação em Cadeia da Polimerase , Prevalência , RNA Viral/sangue , RNA Viral/metabolismo , Curva ROC , Medição de RiscoRESUMO
BACKGROUND: Rabies causes 55, 000 annual human deaths globally and about 10,000 people are exposed annually in Nigeria. Diagnosis of animal rabies in most African countries has been by direct microscopic examination. In Nigeria, the Seller's stain test (SST) was employed until 2009. Before then, both SST and dFAT were used concurrently until the dFAT became the only standard method. OBJECTIVE: This study was designed to assess the sensitivity and specificity of the SST in relation to the 'gold standard' dFAT in diagnosis of rabies in Nigeria. METHODS: A total of 88 animal specimens submitted to the Rabies National Reference Laboratory, Nigeria were routinely tested for rabies by SST and dFAT. RESULTS: Overall, 65.9% of the specimens were positive for rabies by SST, while 81.8% were positive by dFAT. The sensitivity of SST in relation to the gold standard dFAT was 81.0% (95% CIs; 69.7% - 88.6%), while the specificity was 100% (95% CIs; 76% - 100%). CONCLUSION: The relatively low sensitivity of the SST observed in this study calls for its replacement with the dFAT for accurate diagnosis of rabies and timely decisions on administration of PEP to prevent untimely deaths of exposed humans.
Assuntos
Anticorpos Antivirais/análise , Técnica Direta de Fluorescência para Anticorpo/métodos , Vírus da Raiva/imunologia , Vírus da Raiva/isolamento & purificação , Raiva/diagnóstico , Coloração e Rotulagem , Animais , Doenças do Cão , Cães , Humanos , Nigéria , Sensibilidade e EspecificidadeRESUMO
We recently showed that two immunochromatography point-of-care FIV antibody test kits (Witness FeLV/FIV and Anigen Rapid FIV/FeLV) were able to correctly assign FIV infection status, irrespective of FIV vaccination history, using whole blood as the diagnostic specimen. A third FIV antibody test kit, SNAP FIV/FeLV Combo (an enzyme-linked immunosorbent assay [ELISA]), was unable to differentiate antibodies produced in response to FIV vaccination from those incited by FIV infection. The aim of this study was to determine if saliva is a suitable diagnostic specimen using the same well characterized feline cohort. FIV infection status of these cats had been determined previously using a combination of serology, polymerase chain reaction (PCR) testing and virus isolation. This final assignment was then compared to results obtained using saliva as the diagnostic specimen utilizing the same three point-of-care FIV antibody test kits and commercially available PCR assay (FIV RealPCR). In a population of cats where one third (117/356; 33%) were FIV-vaccinated, both immunochromatography test kits accurately diagnosed FIV infection using saliva via a centrifugation method, irrespective of FIV vaccination history. For FIV diagnosis using saliva, the specificity of Anigen Rapid FIV/FeLV and Witness FeLV/FIV was 100%, while the sensitivity of these kits was 96% and 92% respectively. SNAP FIV/FeLV Combo respectively. SNAP FIV/FeLV Combo had a specificity of 98% and sensitivity of 44%, while FIV RealPCR testing had a specificity of 100% and sensitivity of 72% using saliva. A revised direct method of saliva testing was trialed on a subset of FIV-infected cats (n=14), resulting in 14, 7 and 0 FIV positive results using Anigen Rapid FIV/FeLV, Witness FeLV/FIV and SNAP FIV/FeLV Combo, respectively. These results demonstrate that saliva can be used to diagnose FIV infection, irrespective of FIV vaccination history, using either a centrifugation method (Anigen Rapid FIV/FeLV and Witness FeLV/FIV) or a direct method (Anigen Rapid FIV/FeLV). Collection of a saliva specimen therefore provides an acceptable alternative to venipuncture (i) in fractious cats where saliva may be easier to obtain than whole blood, (ii) in settings when a veterinarian or trained technician is unavailable to collect blood and (iii) in shelters where FIV testing is undertaken prior to adoption but additional blood testing is not required.
Assuntos
Anticorpos Antivirais/análise , Síndrome de Imunodeficiência Adquirida Felina/diagnóstico , Vírus da Imunodeficiência Felina/imunologia , Vírus da Imunodeficiência Felina/isolamento & purificação , Saliva/imunologia , Saliva/virologia , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/sangue , Gatos , Ensaio de Imunoadsorção Enzimática , Síndrome de Imunodeficiência Adquirida Felina/imunologia , Síndrome de Imunodeficiência Adquirida Felina/virologia , Feminino , Vírus da Imunodeficiência Felina/genética , Masculino , Sistemas Automatizados de Assistência Junto ao Leito/economia , Reação em Cadeia da Polimerase , Kit de Reagentes para Diagnóstico , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade , Vacinas Virais/administração & dosagemRESUMO
Our objective was to validate a commercially available ELISA to measure antibody titers against Epstein-Barr virus (EBV) in dried blood spots (DBS) to replace a previously validated assay for DBS that is no longer available. We evaluated the precision, reliability, and stability of the assay for the measurement of EBV antibodies in matched plasma, fingerprick DBS, and venous blood DBS samples from 208 individuals. Effects of hematocrit and DBS sample matrix on EBV antibody determination were also investigated, and the cutoff for seropositivity in DBS was determined. A conversion equation was derived to enable comparison of results generated using this method with the former DBS method. There was a high correlation between plasma and DBS EBV antibody titers (R(2) = 0.93) with very little bias (-0.07 based on Bland-Altman analysis). The assay showed good linearity and did not appear to be affected by the DBS matrix, and physiological hematocrit levels had no effect on assay performance. There was reasonable agreement between DBS EBV titer estimates obtained using this assay and the previously validated assay (R(2) = 0.72). The commercially available ELISA assay for EBV antibody titers that we validated for use with DBS will facilitate continued investigation of EBV antibody titers in DBS.
Assuntos
Anticorpos Antivirais/análise , Teste em Amostras de Sangue Seco , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/normas , Herpesvirus Humano 4/genética , Anticorpos Antivirais/sangue , Coleta de Amostras Sanguíneas/métodos , Coleta de Amostras Sanguíneas/normas , Humanos , Oregon , Reprodutibilidade dos TestesRESUMO
Quantitative point-of-care (POC) devices are the next generation for serological disease diagnosis. Whilst pathogen serology is typically performed by centralized laboratories using Enzyme-Linked ImmunoSorbent Assay (ELISA), faster on-site diagnosis would infer improved disease management and treatment decisions. Using the model pathogen Bovine Herpes Virus-1 (BHV-1) this study employs an extended-gate field-effect transistor (FET) for direct potentiometric serological diagnosis. BHV-1 is a major viral pathogen of Bovine Respiratory Disease (BRD), the leading cause of economic loss ($2 billion annually in the US only) to the cattle and dairy industry. To demonstrate the sensor capabilities as a diagnostic tool, BHV-1 viral protein gE was expressed and immobilized on the sensor surface to serve as a capture antigen for a BHV-1-specific antibody (anti-gE), produced in cattle in response to viral infection. The gE-coated immunosensor was shown to be highly sensitive and selective to anti-gE present in commercially available anti-BHV-1 antiserum and in real serum samples from cattle with results being in excellent agreement with Surface Plasmon Resonance (SPR) and ELISA. The FET sensor is significantly faster than ELISA (<10 min), a crucial factor for successful disease intervention. This sensor technology is versatile, amenable to multiplexing, easily integrated to POC devices, and has the potential to impact a wide range of human and animal diseases.
Assuntos
Técnicas Biossensoriais/instrumentação , Herpesvirus Bovino 1/isolamento & purificação , Rinotraqueíte Infecciosa Bovina/diagnóstico , Sistemas Automatizados de Assistência Junto ao Leito , Potenciometria/instrumentação , Animais , Anticorpos Antivirais/análise , Anticorpos Antivirais/sangue , Técnicas Biossensoriais/economia , Bovinos , Desenho de Equipamento , Infecções por Herpesviridae/sangue , Infecções por Herpesviridae/diagnóstico , Infecções por Herpesviridae/virologia , Humanos , Proteínas Imobilizadas/química , Imunoensaio/economia , Imunoensaio/instrumentação , Rinotraqueíte Infecciosa Bovina/sangue , Rinotraqueíte Infecciosa Bovina/virologia , Sistemas Automatizados de Assistência Junto ao Leito/economia , Potenciometria/economia , Testes Sorológicos/economia , Testes Sorológicos/instrumentação , Fatores de Tempo , Proteínas Virais/químicaRESUMO
Viruses that express reporter genes upon infection have been recently used to evaluate neutralizing antibody responses, where a lack of reporter expression indicates specific virus inhibition. The traditional model-based methods using standard outcome of percent neutralization could be applied to the data from the assays to estimate antibody titers. However, the data produced are sometimes irregular, which can yield meaningless outcomes of percent neutralization that do not fit the typical curves for immunoassays, making automated or semi-high throughput antibody titer estimation unreliable. We developed a type of new outcomes model, which is biologically meaningful and fits typical immunoassay curves well. Our simulation study indicates that the new response approach outperforms the traditional response approach regardless of the data variability. The proposed new response approach can be used in similar assays for other disease models.
Assuntos
Proteínas de Fluorescência Verde/química , Testes de Neutralização/métodos , Anticorpos Neutralizantes/análise , Anticorpos Antivirais/análise , Glicoproteínas de Hemaglutininação de Vírus da Influenza/análise , Modelos Estatísticos , Método de Monte CarloRESUMO
OBJECTIVE: Accurate laboratory testing is a critical component of dengue surveillance and control. The objective of this programme was to assess dengue diagnostic proficiency among national-level public health laboratories in the World Health Organization (WHO) Western Pacific Region. METHODS: Nineteen national-level public health laboratories performed routine dengue diagnostic assays on a proficiency testing panel consisting of two modules: one containing commercial serum samples spiked with cultured dengue viruses for the detection of nucleic acid and non-structural protein 1 (NS1) (Module A) and one containing human serum samples for the detection of anti-dengue virus antibodies (Module B). A review of logistics arrangements was also conducted. RESULTS: All 16 laboratories testing Module A performed reverse transcriptase polymerase chain reaction (RT-PCR) for both RNA and serotype detection. Of these, 15 had correct results for RNA detection and all 16 correctly serotyped the viruses. All nine laboratories performing NS1 antigen detection obtained the correct results. Sixteen of the 18 laboratories using IgM assays in Module B obtained the correct results as did the 13 laboratories that performed IgG assays. Detection of ongoing/recent dengue virus infection by both molecular (RT-PCR) and serological methods (IgM) was available in 15/19 participating laboratories. DISCUSSION: This first round of external quality assessment of dengue diagnostics was successfully conducted in national-level public health laboratories in the WHO Western Pacific Region, revealing good proficiency in both molecular and serological testing. Further comprehensive diagnostic testing for dengue virus and other priority pathogens in the Region will be assessed during future rounds.
Assuntos
Vírus da Dengue/isolamento & purificação , Dengue/diagnóstico , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Testes Sorológicos/normas , Anticorpos Antivirais/análise , Anticorpos Antivirais/sangue , Antígenos Virais/análise , Sudeste Asiático , Australásia , Dengue/virologia , Vírus da Dengue/genética , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoglobulina M/análise , Ilhas do Pacífico , Garantia da Qualidade dos Cuidados de Saúde , RNA Viral/análise , Organização Mundial da SaúdeRESUMO
Developing sophisticated statistical methods for go/no-go decisions is crucial for clinical trials, as planning phase III or phase IV trials is costly and time consuming. In this paper, we develop a novel Bayesian methodology for determining the probability of success of a treatment regimen on the basis of the current data of a given trial. We introduce a new criterion for calculating the probability of success that allows for inclusion of covariates as well as allowing for historical data based on the treatment regimen, and patient characteristics. A new class of prior distributions and covariate distributions is developed to achieve this goal. The methodology is quite general and can be used with univariate or multivariate continuous or discrete data, and it generalizes Chuang-Stein's work. This methodology will be invaluable for informing the scientist on the likelihood of success of the compound, while including the information of covariates for patient characteristics in the trial population for planning future pre-market or post-market trials.
Assuntos
Teorema de Bayes , Ensaios Clínicos Fase II como Assunto/estatística & dados numéricos , Ensaios Clínicos Fase III como Assunto/estatística & dados numéricos , Vacina contra Herpes Zoster/administração & dosagem , Herpes Zoster/prevenção & controle , Idoso , Análise de Variância , Anticorpos Antivirais/análise , Anticorpos Antivirais/imunologia , Ensaios Clínicos Fase II como Assunto/economia , Ensaios Clínicos Fase II como Assunto/métodos , Ensaios Clínicos Fase III como Assunto/economia , Ensaios Clínicos Fase III como Assunto/métodos , Simulação por Computador , Interpretação Estatística de Dados , Tomada de Decisões , Feminino , Herpes Zoster/imunologia , Vacina contra Herpes Zoster/imunologia , Herpesvirus Humano 3/imunologia , Humanos , Funções Verossimilhança , Modelos Lineares , Modelos Logísticos , Masculino , ProbabilidadeRESUMO
Stress and immune function may be important mediators of the strong association between social factors and health over the life course, but previous studies have lacked the data to fully explore these links in a population-based sample. This study utilizes data from Waves I-IV of the U.S. National Longitudinal Study of Adolescent Health (Add Health) to test the associations of race/ethnicity and socioeconomic status (SES) with levels of perceived stress and exposure to stressful life events (SLE) among 11,050 adult respondents aged 24-32 in 2008-2009. We further tested whether race/ethnicity and SES were associated with Epstein-Barr Virus (EBV) specific IgG antibodies, an indirect marker of cell-mediated immune function. Finally, we tested whether measures of stress were associated with EBV IgG and whether there was evidence that they explain any associations between race/ethnicity, SES and EBV IgG. We found strong associations between lower SES and higher levels of perceived stress (OR 2.07, 95% CI 1.73-2.48 for < high school vs. college or above) and a high level of stressful life events (OR 7.47, 95% CI 5.59-9.98 for < high school vs. college or above). Blacks had higher odds of a high level of stressful life events compared to whites (OR 2.00, 95% CI 1.63-2.47), but not higher perceived stress (OR 1.11, 95% CI 0.96-1.28). Blacks also had significantly higher EBV levels compared to whites (ß = 0.136, p < 0.01), but lower SES was not associated with higher EBV IgG. We found no evidence that stressful life events or perceived stress were associated with EBV IgG in this sample, and thus did not account for racial differences in EBV IgG. These results suggest consistent race/ethnic and SES differences in stressful life events, and confirm race/ethnic differences in markers of immune function that may have health implications across the life course.