A novel highly specific biotinylated MAC-ELISA for detection of anti-SARS-CoV-2 nucleocapsid antigen IgM antibodies during the acute phase of COVID-19.

The gold standard for diagnosing COVID-19 in the acute phase is RT-qPCR. However, this molecular technique can yield false-negative results when nasopharyngeal swab collection is not conducted during viremia. To mitigate this challenge, the enzyme-linked immunosorbent assay (ELISA) identifies anti-SARS-CoV-2 IgM antibodies in the initial weeks after symptom onset, facilitating early COVID-19 diagnosis. This study introduces a novel and highly specific IgM antibody capture ELISA (MAC-ELISA), which utilizes biotinylated recombinant SARS-CoV-2 nucleocapsid (N) antigen produced in plants. Our biotinylated approach streamlines the procedure by eliminating the requirement for an anti-N-conjugated antibody, circumventing the need for peroxidase-labeled antigens, and preventing cross-reactivity with IgM autoantibodies such as rheumatoid factor. Performance evaluation of the assay involved assessing sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy using 682 RT-qPCR-positive samples, categorized by weeks relative to symptoms onset. Negative controls included 205 pre-pandemic serum samples and 46 serum samples from patients diagnosed with other diseases. Based on a cut-off of 0.087 and ROC curve analysis, the highest sensitivity of 81.2% was observed in the 8-14 days post-symptom (dps) group (2nd week), followed by sensitivities of 73.8% and 68.37% for the 1-7 dps (1st week) and 15-21 dps groups (3rd week), respectively. Specificity was consistently 100% across all groups. This newly developed biotinylated N-MAC-ELISA offers a more streamlined and cost-effective alternative to molecular diagnostics. It enables simultaneous testing of multiple samples and effectively identifies individuals with false-negative results.

The prevalence of IGM antibodies to Zika virus in pregnant women in Northern Nigeria.

INTRODUCTION: Zika virus (ZIKV) infection during pregnancy can result in severe outcomes for both the pregnant woman and the developing fetus. The objective of this study was to investigate the prevalence of Zika virus infection among pregnant women who sought healthcare services at Ahmadu Bello University Teaching Hospital. MATERIALS AND METHODS: Serum samples were collected and analyzed using Enzyme Linked Immunoassay and RT-qPCR methods, while a structured questionnaire was used to gather relevant information about the participants. RESULTS: The results showed that 53 out of the 180 pregnant women tested positive for Anti-Zika IgM antibodies, which represents a 29.4% prevalence rate. Subsequent RT-qPCR analysis found that only 6 out of the 53 positive samples contained Zika virus RNA. Fever and headache were the most commonly reported symptoms related to the infection. CONCLUSION: These findings indicate a potential outbreak of Zika fever in Northern Nigeria emphasizing the importance for pregnant women to take precautions to avoid getting infected.

Dengue-chikungunya infection in the tertiary care hospital of northern India: Cross-sectional latent class cluster analysis in viral infection.

Cases of dengue and chikungunya fever are escalating all over India. Both viruses share a common vector, the "Aedes" mosquito. Due to similar clinical symptoms, both the dengue (DENV) and chikungunya (CHIKV) virus can circulate as co-infection. There is very limited data available on dengue-chikungunya co-infection in Uttarakhand, India. The purpose of this study was to determine the seroprevalence of dengue and chikungunya virus infections, as well as their co-infection, in patients presenting with clinical symptoms. Serum samples of clinically suspected patients from the tertiary care hospital of Uttarakhand were collected, and Latent Class Cluster Analysis was performed for clinical profiling. ELISA was performed for DENV and CHIKV. 279 cases were enrolled, out of which 222 (79.5%) came positive for dengue NS1 Ag, 143 (51.2%) for dengue IgM, 98 (35.1%) for IgG followed by 16 (5.7%) of CHIKV IgM, and 4 (1.4%) were NS1 Ag with CHIKV IgM. Among the clinical features, fever (n = 270, 96.8%) was the most common symptom in all suspected dengue and chikungunya cases. Other symptoms like chills (n = 254, 91.0%), arthralgia (n = 241, 86.4%), and headache (n = 240, 86.0%) were present in a significant number. Results showed fewer odds of getting both DENV and CHIKV infection simultaneously, but the risk is still not negligible. This study explores the clinical presentation of the suspected dengue-chikungunya case. The increasing incidence of dengue and chikungunya and their co-infection necessitate the authorities' active surveillance of endemic regions and effective patient care management.

Development of IgM-ELISA for diagnosis of recent infection of Japanese encephalitis virus in equines.

Japanese encephalitis (JE) is a re-emerging mosquito borne disease, for which equines are most susceptible amongst all animals. Detection of specific immunoglobulin 'M' (IgM) is considered as an ideal way to diagnose recent JE virus infection in equines due to low virus load and short-term viremia. The present study was undertaken to develop a sensitive and specific recombinant NS1 protein based indirect IgM-ELISA and IgM capture (MAC) ELISA to diagnose recent infection of JEV in equines. Indirect IgM ELISA was standardized with relative diagnostic sensitivity and specificity of 100% and 88.5%, respectively. The validation of indirect IgM-ELISA in different laboratories revealed excellent reproducibility with Cohen's kappa value ranging between 0.84 and 1. The standardization of MAC ELISA was attempted using checker board titration method and non-specific binding of polyclonal anti-equine IgM capture antibody with anti-porcine IgG conjugate and with hyperimmune serum raised in swine against the antigen was observed. Hence, the MAC ELISA was standardized with monoclonal capture antibody; however, its diagnostic performance could not meet the satisfactory limit. Due to better sensitivity and less turnaround time, indirect IgM-ELISA was employed to screen 821 equine serum samples revealing 33.73% positivity of IgM antibodies against JEV in equine population of India. The high JEV sero-positivity warrants the need for vaccination in Indian equine population along with the demand for research focused towards anti-viral therapy. The indirect IgM-ELISA developed in the present study could be useful to diagnose acute or recent infection of JEV in equines as well as in sero-epidemiological studies.

Serological Diagnosis of Dengue.

A reliable and specific diagnosis is imperative in viral diagnosis, both for clinical management and surveillance, and to ensure that early treatment and control measures are carried out. The number of days of illness is important to choose the most appropriate method to be used and for the correct interpretation of the results obtained. Specific IgM is elicited after that period, indicating an active infection and usually lasts up to 3 months. However, in DENV secondary infections, IgM levels may be significantly lower or undetectable. After 10-12 days, a lifetime specific IgG is produced. Routinely, the laboratory diagnosis of DENV infections can be performed by viral isolation and/or detection of viral nucleic acid, serological assays for the detection of specific antibodies (IgM/IgG), antigen (NS1) and the detection of viral antigens in tissues, which are suitable during certain phases of the disease. For serological diagnosis, serum, plasma, or cerebrospinal fluid (CSF) samples may be investigated. If the test is carried out a few days after collection, the specimens can be stored at 4 °C, since the immunoglobulins are stable in serum or plasma. If the storage period is extended, the material must be kept at -20 °C or -70 °C. In serology, several methods can be used to detect specific viral antigens and/or antibodies, produced by the host in response to DENV infection. Routinely, serological tests include the hemagglutination inhibition (HI) assay, the plaque reduction neutralizing test (PRNT), the gold standard assay for dengue immune response characterization, and ELISAs to detect IgM (MAC-ELISA) and IgG (IgG-ELISA).

Accuracy of the Zika IgM Antibody Capture Enzyme-Linked Immunosorbent Assay from the Centers for Disease Control and Prevention (CDC Zika MAC-ELISA) for Diagnosis of Zika Virus Infection.

Serological diagnosis of Zika virus (ZIKV) infection is challenging because of antigenic cross-reactivity with dengue virus (DENV). This study evaluated the accuracy of the Zika IgM antibody capture enzyme-linked immunosorbent assay (CDC Zika IgM MAC-ELISA) in differentiating between ZIKV and DENV infections. To determine sensitivity, we used acute- and convalescent-phase sera from 21 patients with RT-PCR-confirmed ZIKV infection. To determine specificity, we used acute- and convalescent-phase sera from 60 RT-PCR-confirmed dengue cases and sera from 23 blood donors. During the acute-phase of the illness, the assay presented a sensitivity of 12.5% (2/16) for samples collected 0-4 days post symptoms onset (DPSO), and of 75.0% (3/4) for samples collected 5-9 DPSO. During the convalescent-phase of the illness, the test sensitivity was 90.9% (10/11), 100% (2/2), and 0% (0/2) for samples obtained 12-102, 258-260, and 722-727 DPSO, respectively. Specificity for acute- and convalescent-phase samples from RT-PCR-confirmed dengue cases was 100% and 93.2%, respectively. Specificity for blood donor samples was 100%. The assay is an accurate method for Zika serological diagnosis and proved to be reliable for use during surveillance and outbreak investigations in settings where ZIKV and DENV cocirculate.

Assessment of Immunoglobulin M Enzyme-Linked Immunosorbent Assay Ratios to Identify West Nile Virus and St. Louis Encephalitis Virus Infections During Concurrent Outbreaks of West Nile Virus and St. Louis Encephalitis Virus Diseases, Arizona 2015.

West Nile virus (WNV) and St. Louis encephalitis virus (SLEV) are closely related mosquito-borne flaviviruses that cause clinical disease ranging from febrile illness to encephalitis. The standard for serological diagnosis is immunoglobulin M (IgM) testing followed by confirmatory plaque reduction neutralization test (PRNT) to differentiate the infecting virus. However, the PRNT is time-consuming and requires manipulation of live virus. During concurrent WNV and SLEV outbreaks in Arizona in 2015, we assessed use of a diagnostic algorithm to simplify testing. It incorporated WNV and SLEV ratios based on positive-to-negative (P/N) values derived from the IgM antibody-capture enzyme-linked immunosorbent assay. We compared each sample's ratio-based result with the confirmed WNV or SLEV sample result indicated by PRNT or PCR testing. We analyzed data from 70 patients with 77 serum and cerebrospinal fluid samples, including 53 patients with confirmed WNV infection and 17 patients with confirmed SLEV infection. Both WNV and SLEV ratios had specificity ≥95%, indicating a high likelihood that each ratio was correctly identifying the infecting virus. The SLEV ratio sensitivity of 30% was much lower than the WNV ratio sensitivity of 91%, likely because of higher cross-reactivity of SLEV antibodies and generation of lower P/N values. The standard for serological diagnosis of WNV and SLEV infections remains IgM testing followed by PRNT. However, these results suggest the ratios could potentially be used as part of a diagnostic algorithm in outbreaks to substantially reduce the need for PRNTs.

Zika Virus IgM 25 Months after Symptom Onset, Miami-Dade County, Florida, USA.

We assessed IgM detection in Zika patients from the 2016 outbreak in Miami-Dade County, Florida, USA. Of those with positive or equivocal IgM after 12-19 months, 87% (26/30) had IgM 6 months later. In a survival analysis, ≈76% had IgM at 25 months. Zika virus IgM persists for years, complicating serologic diagnosis.

Simultaneous detection of IgM antibodies against dengue and chikungunya: Coinfection or cross-reactivity?

BACKGROUND: Dengue and chikungunya sharing same mosquito vector are two most important arboviruses circulating in northern India including Delhi and are responsible for frequent outbreaks. Antigen and antibodies detection ELISA kits are the major tool to diagnose these viral illnesses, and are sometimes associated with cross-reactivity, giving a false picture of coinfection, although simultaneous harboring of both the viruses is not uncommon. Various studies have reported coinfection up to 25% from the same region. PROCEDURE: This study was conducted in the Department of Microbiology, Maulana Azad Medical College, New Delhi, during the month of September 2016 which included 200 blood samples from clinically suspected cases attending Medicine OPD of associated Lok Nayak Hospital, New Delhi. Diagnosis of dengue and chikungunya was made using NS-1 antigen and IgM MAC ELISA for dengue and IgM MAC ELISA for chikungunya as per manufacturer's instructions. RESULTS: Out of 200 suspected cases, 34 (17%) were positive for dengue serology, 77 (38.5%) were positive for chikungunya serology, and 29.9% of positive chikungunya cases were simultaneously affected with dengue. This higher percentage of coinfection might be because of cross-reactivity of the ELISA kits. DISCUSSION: India being a hyperendemic region for dengue and chikungunya, frequent outbreaks are quite common. Circulation of both the virus and huge susceptible population are the major causes for frequent outbreaks. Restricting our attention to diagnose one of them is not sufficient, and coinfection further complicates the illness. CONCLUSION: Simultaneous diagnosis of dengue and chikungunya is need of time to diagnose dual infection and prevent complications by starting supportive treatment well in time. Molecular technique if ever possible should be employed whenever the coinfection number is higher than expected to rule out cross-reactivity.

Comparison of Two Commercial ELISA Kits for the Detection of Anti-Dengue IgM for Routine Dengue Diagnosis in Laos.

The endemicity of Dengue virus (DENV) infection remains a major public health problem in Lao PDR. In this study, we compared two commercial anti-dengue IgM ELISA kits, Panbio® Dengue IgM Capture ELISA (Panbio Kit, Alere, Waltham, MA, USA) and DEN DetectTM MAC-ELISA (InBios kit, InBios International, Inc., Seattle, WA, USA), in the context of diagnosis of patients admitted to hospital with clinical dengue presentation. Two panels of paired blood samples were tested. Panel A was composed of 54 dengue confirmed patients (by DENV real-time RT-PCR) and 11 non-dengue dengue patients (other infections confirmed by corresponding PCR results). Panel B included 74 patients randomly selected from consecutive patients admitted to Mahosot Hospital in 2008 with suspicion of dengue fever according to WHO criteria. Results from panel A showed significantly better sensitivity for Panbio kit (64.8%; 95%CI: 50.6-77.3%) than for InBios kit (18.5%; 95%CI: 9.3-31.4%) when testing admission sera. Sensitivity was increased for both kits when combining results from admission and convalescent sera. Concordant results were obtained from panel B with fair agreement (κ = 0.29) between both kits when testing single admission samples, and moderate agreement (κ = 0.5) when combining results from admission and convalescent sera.

Zika Virus IgM Detection and Neutralizing Antibody Profiles 12-19 Months after Illness Onset.

Data on the duration of detectable Zika virus-specific IgM in infected persons are limited. Neutralizing antibody cross-reactivity occurs between Zika virus and related flaviviruses, but the degree to which this confounds diagnosis is uncertain. We tested serum specimens collected 12-19 months after illness onset from patients with confirmed Zika virus disease for Zika virus IgM and Zika virus and dengue virus neutralizing antibodies. Among 62 participants, 45 (73%) had detectable Zika virus IgM and 12 (19%) had an equivocal result. Although all patients tested had Zika virus neutralizing antibodies, 39 (63%) also had neutralizing antibodies against dengue virus; of those, 12 (19%) had <4-fold difference between Zika virus and dengue virus titers, and 5 (8%) had dengue virus titer >4-fold higher than Zika virus titer. Prolonged detection of IgM and neutralizing antibody cross-reactivity make it difficult to determine the timing of Zika virus infection and differentiate between related flaviviruses.

Evaluation of a Rapid Immunochromatographic Assay and Two Enzyme-Linked Immunosorbent Assays for Detection of IgM-Class Antibodies to Zika Virus.

There are currently five serologic assays available for detection of anti-Zika virus (ZIKV) IgM-class antibodies with U.S. Food and Drug Administration emergency use authorization. Among these are the Chembio DPP Zika IgM system (DPP Zika ICA; Chembio, Medford, NY), a rapid immunochromatographic assay (ICA), and the InBios ZIKV Detect 2.0 IgM antibody capture enzyme-linked immunosorbent assay (ZIKV 2.0 MAC-ELISA; InBios international, Inc., Seattle, WA), which has replaced the original InBios ZIKV Detect MAC-ELISA. We evaluated performance of these three serologic assays using 72 specimens characterized by plaque reduction neutralization testing (PRNT) for the presence or absence of neutralizing antibodies (NAbs) to ZIKV, dengue virus (DENV), and West Nile virus (WNV). The InBios ZIKV 2.0 MAC-ELISA was "presumptive Zika positive" in all 15 PRNT-confirmed ZIKV samples, while the Chembio DPP Zika ICA was nonreactive in three (20%) and the InBios ZIKV MAC-ELISA was negative in four (27%). The Chembio DPP Zika ICA and InBios ZIKV 2.0 MAC-ELISA showed >95% specificity in 22 ZIKV/DENV-seronegative specimens and in 13 samples positive for NAbs to non-ZIKV flaviviruses. Comparatively, the InBios ZIKV MAC-ELISA was "presumptive" or "possible Zika positive" in 8 of 12 WNV or DENV PRNT-positive samples and in 12 of 22 PRNT-seronegative sera. Our findings suggest that replacement of the InBios ZIKV MAC-ELISA with the InBios ZIKV 2.0 MAC-ELISA will lead to fewer samples requiring PRNT, minimizing unnecessary anxiety among patients ultimately determined to be seronegative for ZIKV and DENV by PRNT and alleviating some of the testing burden on laboratories performing PRNT.

Evaluation of 5 Commercially Available Zika Virus Immunoassays.

Because of the global spread of Zika virus, accurate and high-throughput diagnostic immunoassays are needed. We compared the sensitivity and specificity of 5 commercially available Zika virus serologic assays to the recommended protocol of Zika virus IgM-capture ELISA and plaque-reduction neutralization tests. Most commercial immunoassays showed low sensitivity, which can be increased.

Laboratory Diagnosis of Chikungunya Virus Infections and Commercial Sources for Diagnostic Assays.

Detection of chikungunya virus (CHIKV) or viral RNA is the primary laboratory test used to diagnose infection in serum collected <6 days after onset of illness. Two real-time reverse transcription-polymerase chain reaction (RT-PCR) kits are available commercially, but validity data are limited. There are 2 commercial sources of inactivated positive-control CHIKV RNA to be used with purchased primers. The Centers for Disease Control and Prevention provides viral RNA-positive controls and primer and probe nucleotide sequences for real-time RT-PCR testing. Detection of CHIKV-specific immunoglobulin M (IgM) antibody becomes a sensitive test for samples collected approximately >5 days of illness. Commercially available CHIKV IgM-detection assays include lateral flow rapid tests, IgM antibody capture enzyme-linked immunosorbent assays (MAC-ELISAs), and indirect immunofluorescence tests. Nine commercial CHIKV IgM detection assays were evaluated at 3 reference laboratories to provide guidance to public health diagnostic laboratories on their performance parameters. Sensitivity of the rapid tests and 3 MAC-ELISAs was <50%, and thus these assays are not recommended. Three of the MAC-ELISA kits and 1 indirect immunofluorescence kit had comparable performance to the reference assays. In summary, commercial assays with performance comparable to reference assays are available for molecular and serological diagnosis of CHIKV infections.

Production of a Sindbis/Eastern Equine Encephalitis chimeric virus inactivated cell culture antigen.

Eastern Equine Encephalitis virus (EEEV) is a medically important pathogen that can cause severe encephalitis in humans, with mortality rates ranging from 30 to 80%. Unfortunately there are no antivirals or licensed vaccines available for human use, and laboratory diagnosis is essential to differentiate EEEV infection from other pathogens with similar clinical manifestations. The Arboviral Diseases Branch (ADB) reference laboratory at the CDC Division of Vector-Borne Diseases (DVBD) produces reference antigens used in serological assays such as the EEEV immunoglobulin M antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA). However, EEEV is classified as a HHS select agent and requires biosafety level (BSL) three containment, limiting EEEV antigen production in non-select agent and BSL-2 laboratories. A recombinant Sindbis virus (SINV)/EEEV has been constructed for use under BSL-2 conditions and is not regulated as a select agent. Cell culture production of inactivated EEEV antigen from SINV/EEEV for use in the EEEV MAC-ELISA is reported here. Cell culture conditions and inactivation procedures were analyzed for SINV/EEEV using a recently developed antigen production algorithm, with the MAC-ELISA as the performance indicator.

Development of an algorithm for production of inactivated arbovirus antigens in cell culture.

Arboviruses are medically important pathogens that cause human disease ranging from a mild fever to encephalitis. Laboratory diagnosis is essential to differentiate arbovirus infections from other pathogens with similar clinical manifestations. The Arboviral Diseases Branch (ADB) reference laboratory at the CDC Division of Vector-Borne Diseases (DVBD) produces reference antigens used in serological assays such as the virus-specific immunoglobulin M antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA). Antigen production in cell culture has largely replaced the use of suckling mice; however, the methods are not directly transferable. The development of a cell culture antigen production algorithm for nine arboviruses from the three main arbovirus families, Flaviviridae, Togaviridae, and Bunyaviridae, is described here. Virus cell culture growth and harvest conditions were optimized, inactivation methods were evaluated, and concentration procedures were compared for each virus. Antigen performance was evaluated by the MAC-ELISA at each step of the procedure. The antigen production algorithm is a framework for standardization of methodology and quality control; however, a single antigen production protocol was not applicable to all arboviruses and needed to be optimized for each virus.

Comparative evaluation of the diagnostic potential of recombinant envelope proteins and native cell culture purified viral antigens of Chikungunya virus.

Despite the fact that Chikungunya resurgence is associated with epidemic of unprecedented magnitude, there are challenges in the field of its clinical diagnosis. However, serological tests in an ELISA format provide a rapid tool for the diagnosis of Chikungunya infection. Indeed, ELISAs based on recombinant proteins hold a great promise as these methods are cost effective and are free from the risk of handling biohazardous material. In this study, the performance of recombinant CHIKV antigens was compared in various ELISA formats for the diagnosis of Chikungunya. Two recombinant antigens derived from the envelope proteins of Chikungunya virus were prepared and evaluated by comparing their competence for detecting circulating antibodies in serum samples of patients infected with CHIKV using MAC-ELISA and indirect IgM-ELISA. The efficacy of the recombinant antigens was also compared with the native antigen. The indirect antibody capture IgM microplate ELISA revealed ≥90% concordance with the native antigen in detecting the CHIKV specific IgM antibodies whereas the recombinant antigen based MAC-ELISA showed 100% specificity. The recombinant antigens used in this study were effective and reliable targets for the diagnosis of CHIKV infection and also provide an alternative for native antigen use which is potentially biohazardous.

Concordancia de antígenos de dengue en el ELISA de captura de IgM (MAC ELISA) en el IICS-UNA / Concordance of dengue antigens M antibody-capture ELISA (MAC ELISA) in the IICS-UNA

El dengue es una enfermedad aguda grave considerada actualmente como infección reemergente, cuyo vector principal es el Aedes aegypti. En Paraguay en el 2007 fueron reportados 28.181 casos, 55 se clasificaron como fiebre hemorrágica del dengue de los cuales 7 fallecieron. El 90% de los casos fueron de Asunción y del Departamento Central,10% del resto del país. En los últimos años se han desarrollado diferentes sistemas inmunoenzimáticos para el diagnóstico del dengue, entre ellos el ELISA de captura de IgM (MAC ELISA). El objetivo de este estudio observacional analítico de corte transverso fu ecomparar la prueba del MAC ELISA desarrollada en el Instituto de Investigaciones en Ciencias de la Salud (IICS) utilizando antígenos suministrados por el Instituto Pedro Kouri(IPK) de Cuba y el Evandro Chagas de Brasil, con el kit comercial ELISA IgM por capturapara virus del dengue (Focus Diagnostics Inc. Cypress, CA, USA). Fueron seleccionados alazar 92 sueros de pacientes codificados que concurrieron al IICS con sospecha de dengue, respetándose la confidencialidad de los mismos. La concordancia obtenida fue del94.6% (Índice Kappa: 0.891) utilizando el antígeno del IPK y 96.7% (Índice Kappa:0.9350) con el antígeno del Evandro Chagas, mostrándose alta significancia estadística(p<0.00001) en ambos casos. La excelente concordancia obtenida con los dos antígenos indica que los mismos pueden ser utilizados indistintamente en la prueba del MAC ELISA desarrollada en el IICS, a fin de apoyar el diagnóstico del dengue a menor costo y quesería de producción local.

Dengue is an acute disease currently considered a re-emerging infection, whose main vector is Aedes aegypti. In 2007, 28,181 cases were reported in Paraguay, 55 were classified as dengue hemorrhagic fever and seven of them died. Ninety percent of the cases were from Asunción and the Central Department and the remaining 10% from the rest of the country. In recent years various immunoenzymatic systems have been developed immunoassay for the diagnosis of dengue, including the M antibody captureELISA (MAC ELISA). The aim of this cross-sectional observational study was to compare the MAC ELISA test developed at the Instituto de Investigaciones en Cienciad de la Salud(IICS) using antigens supplied by the Instituto Pedro Kouri (IPK) of Cuba and Evandro Chagas of Brazil with a commercial kit of M antibody capture ELISA for dengue virus (Focus Diagnostics Inc. Cypress, CA, USA). Ninety two coded serum samples wererandomly selected from patients who attended the IICS with suspected dengue, respecting their confidentiality. The concordance obtained was 94.6% (Kappa Index: 0.891) using the IPK antigen and 96.7% (Kappa index: 0.9350) with the antigen from Evandro Chagas showing high statistical significance (p<0.00001) in both cases. The excellent concordance obtained with the two antigens indicates that they can be used indistinctly in the MAC ELISA test developed in the IICS to support the diagnosis of dengue at a lower cost and would be locally produced.

Detecçäo de vírus Dengue sorotipos 1 e 2 pela técnica de immunoblotting utilizando proteínas recombinantes / Detection of dengue virus serotypes 1 and 2 by immunoblotting techniques using recombinant proteins

O vírus dengue säo patógenos que vêm afetando milhöes de pessoas durante os dois últimos séculos. No presente trabalho, comparou-se a técnica tradicional de MAC-ELISA e o teste de Immunoblotting, utilizando-se proteínas recombinantes, para pesquisa de anticorpos da classe M, contra os vírus Dengue sorotipos 1 e 2. Foram testadas 100 amostras de soro de pacientes, com suspeita clínica de dengue, por MAC-ELISA e Immunoblotting. Estes soros quando submetidos ao MAC-ELISA resultaram 56 positivos para dengue e 44 negativos. Estas mesmas amostras avaliadas por Immunoblotting erevelaram 56 soros negativos, 36 soros positivos para DEN-1, 6 para DEN-2 e 2 para DEN-1 e 2. Proteínas recombinantes (DEN-1 e DEN-2) utilizadas como antígeno em Immunoblotting possibilitam um diagnóstico diferencial dos sorotipos de dengue circulantes em área, enquanto o teste MAC-ELISA apesar de sensível e rápido näo tipifica o sorotipo de dengue, dado importante durante os períodos de epidemias. (AU)

Dengue viroses (DEN-1 to 4) are human pathogens affeeting millions of people In the last two eenturies. Results of a eomparative study between the traditional MAC-ELISA method and the Immunoblotting teehniques using reeombinant proteins of DEN-l and DEN-2 to deteet vírus speei- fie class M Immunoglobulins, are reported in this paper. One hundred sera samples were studied. Fifty six samples were positive to dengue by MAC-ELISA and 44 were positive by Immunoblotting teehni- que: 36 to DEN-1, 6 to DEN-2 and 2 to DEN-1 and 2 sorotypes. So, the serotype identifieation of the dengue vírus eireulantig in a determined área is possible by using these reeombinant proteins in Immunoblotting teehniques. These data are importante for epidemiologieal surveillanee mainly during the oeeurrenee of an epidemie. (AU)