Penelopie Koraka

Evaluation of Six Immunoassays for Detection of Dengue Virus-Specific Immunoglobulin M and G Antibodies

ABSTRACT The performance of six commercially available immunoassay systems for the detection of dengue virus-specific immunoglobulin M (IgM) and IgG antibodies in serum was evaluated. These included two IgM and IgG enzyme immunoassays (EIA) from MRL Laboratories and PanBio, a rapid immunochromatographic test (RIT) from PanBio, immunofluorescence assays (IFA) from Progen, a dot blot assay from Genelabs, and a dipstick EIA from Integrated Diagnostics (INDX). For this study a panel of 132 serum samples, including 90 serum samples from patients with suspected dengue virus infection and 42 serum samples from patients with other viral infections, was used. In addition, serial serum samples from two monkeys experimentally immunized and challenged with dengue virus type 2 were used. Results were considered conclusive when concordant results were obtained with four of the six antibody-specific assays. Based on this definition, the calculated overall agreement for the human serum samples for the respective IgM immunoassays was 97% (128 of 132), with 34% (45 of 132) positive serum samples, 63% (83 of 132) negative samples, and 3% of samples (4 of 132) showing discordant results. The calculated overall agreement for the IgG assays was 94% (124 of 132), with 49% (65 of 132) positive, 45% (59 of 132) negative, and 6% (8 of 132) discordant results, respectively. The sensitivities of the dengue virus-specific assays evaluated varied between 71 and 100% for IgM and between 52 and 100% for IgG, with specificities of 86 to 96% and 81 to 100%, respectively. The relative sensitivities of the respective IgM assays measured with the monkey serum samples were comparable with those obtained with 12 serial serum samples from humans. Overall performance, based on the sum of the agreement, sensitivity, specificity, and Kappa statistics of the IgM and IgG immunoassays, showed that the antibody detection systems from INDX and Genelabs and the MRL and PanBio EIA are useful and reliable assays for dengue virus serodiagnosis.

Reactivity of serum samples from patients with a flavivirus infection measured by immunofluorescence assay and ELISA

Flavivirus infections are a significant public health problem, since several members of the Flaviviridae family are highly pathogenic to humans. Accurate diagnosis and differentiation of the infecting virus is important, especially in areas where many flaviviruses are circulating. In this study we evaluated a newly developed commercially available immunofluorescence assay (IFA) (INDX, Baltimore, MD, USA) for the detection of IgM and IgG antibodies against dengue virus, yellow fever virus, Japanese encephalitis virus and West Nile virus. IFA was compared with standard diagnostic enzyme immunoassays (EIAs) specific for the detection of IgM and IgG antibodies against these viruses. Forty-seven serum samples from patients with a defined flavivirus infection were tested. As controls, serum samples from individuals with antibodies against tick-borne encephalitis virus and hepatitis C virus as well as healthy individuals were included. The results obtained from this study indicate that IFA showed a significantly better discrimination for flavivirus specific IgM antibodies than did the standard IgM specific EIAs (the overall cross-reactivity varied between 4 and 10% by IFA and 30-44% by EIA for the respective viruses). In contrast, the detection of flavivirus specific IgG antibodies showed high cross-reactions in both IFA and EIAs (overall cross-reactivity 16-71 and 62-84%, respectively). This study clearly stated the complexity of flavivirus diagnosis, showing that one cannot rely on one assay or search for one virus only. The flavivirus IFA is a useful tool for the identification of flavivirus infections during the acute stage of disease. In particular, IFA can be an important diagnostic tool for testing samples from travellers who have been accidentally exposed to these viruses.

Detection of immune-complex-dissociated nonstructural-1 antigen in patients with acute dengue virus infections.

ABSTRACT Accurate and timely diagnosis of dengue virus (DEN) infections is essential for the differential diagnosis of patients with febrile illness and hemorrhagic fever. In the present study, the diagnostic value of a newly developed immune-complex dissociated nonstructural-1 (NS-1) antigen dot blot immunoassay (DBI) was compared to a commercially available DEN antigen detection kit (denKEY Blue kit; Globio Co., Beverly, Mass.) and a reverse transcription-PCR (RT-PCR) kit. Serial serum or plasma samples (n = 181) obtained from 55 acute DEN-infected patients were used. In samples obtained from 32 of these 55 DEN-infected patients, viral RNA could be detected by RT-PCR. DEN antigen was detected in only 10 of these 55 patient samples by using the denKEY kit. When these samples were treated with acid to release the immune-complex-associated NS-1 antigen for detection by DBI, 43 of these 55 patients were found to be positive for DEN NS-1 antigen. In nondissociated samples, 22 of these patients were found to be positive by the DBI. In the presence of DEN-specific immunoglobulin M antibodies, both viral RNA and DEN (NS-1) antigen could be detected. The number of positive samples identified by RT-PCR and DBI from these patients with primary DEN infections varied between 28 and 78%. In secondary DEN infections, the number of samples that tested positive by the DBI after immune-complex dissociation (DIS-DBI) was 25% higher than the number of samples that tested positive by RT-PCR and was 35% higher than that determined by nondissociated antigen (NDIS-DBI) detection. We conclude that the denKEY kit has limited diagnostic value for acute DEN infections compared to the RT-PCR and the NDIS-DBI and DIS-DBI methods. We clearly demonstrate that in secondary DEN infections the dissociation of NS-1 immune complexes is essential for early diagnosis of DEN infections.

Kinetics of Dengue Virus-Specific Serum Immunoglobulin Classes and Subclasses Correlate with Clinical Outcome of Infection

ABSTRACT The kinetics of dengue virus (DEN)-specific serum immunoglobulin classes (immunoglobulin M [IgM] and IgA) and subclasses (IgG1 to IgG4) were studied in patients suffering from dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS). Serum samples from non-DEN febrile patients were included as controls. IgM, IgG1, and IgG3 serum antibodies were the predominant immunoglobulins throughout the course of illness in all three patient groups. In contrast, IgA antibodies were significantly higher in the acute phase in DSS patients compared to those in DF patients (P < 0.05). The levels of IgG1 differed significantly between patients with DF and those with DHF and DSS (P < 0.05). A significant difference was also found in IgG3 levels between DF patients and DHF patients (P< 0.05) but not between DF patients and DSS patients. Finally, levels of IgG4 antibodies differed significantly between DF patients and DSS patients (P < 0.05). Collectively, these data show that increased levels of DEN-specific IgA, IgG1, and IgG4 serum antibodies are risk markers for the development of DHF and DSS and that their measurement may provide valuable guidance for early therapeutic intervention.

Immunization with West Nile virus envelope domain III protects mice against lethal infection with homologous and heterologous virus.

Summary The Japanese encephalitis virus (JEV) serocomplex-group consists of mosquito-borne flaviviruses, which include West Nile virus (WNV) and JEV, and both may cause severe encephalitis in humans. WNV has spread rapidly across the United States since its introduction in 1999 and its geographical distribution within the western hemisphere is expected to further expand, whereas, JEV is the most common cause of viral encephalitis in Southeast Asia, China and India. Currently, there is no registered human vaccine or specific therapy to prevent or treat WNV infection. Here we describe the efficacy of recombinant domain III (DIII) of WNV glycoprotein E in a mouse model. It induces high neutralizing antibody titers, as well as, protection against lethal WNV infection in C57BL/6 mice. This vaccine preparation also afforded partial protection against lethal JEV infection.

DC-SIGN enhances infection of cells with glycosylated West Nile virus in vitro and virus replication in human dendritic cells induces production of IFN-α and TNF-α

The recent introduction of West Nile virus (WNV) into the Western hemisphere resulted in significant human outbreaks causing disease of variable severity. Previous studies classified WNV into two major lineages (L1 and L2) that differ in their virulence. Since most L1 strains are glycosylated, we investigated the role of dendritic cell-specific ICAM-3-grabbing non-integrin (DC-SIGN) in infection efficiency of glycosylated WNV strains. We showed that glycosylated strains, in contrast to non-glycosylated strains, infected DC-SIGN expressing cells more efficiently than DC-SIGN negative cells. Furthermore, WNV can productively infect cultured human dendritic cells (DCs) and infection of dendritic cells with the glycosylated WNV-NY99 L1 strain induced production of significantly more TNF-alpha and IFN-alpha in cultured DC, than infection with the non-glycosylated B956 L2 strain. Together, these results indicate that DC-SIGN enhances infection of cells by WNV glycosylated strains, which may at least in part explain the higher pathogenicity of glycosylated L1 strains versus most non-glycosylated L2 strains.

Dengue disease severity in Indonesian children: an evaluation of the World Health Organization classification system

BackgroundDengue disease severity is usually classified using criteria set up by the World Health Organization (WHO). We aimed to assess the diagnostic accuracy of the WHO classification system and modifications to this system, and evaluated their potential practical usefulness.MethodsPatients, admitted consecutively to the hospital with severe dengue, were classified using the WHO classification system and modifications to this system. Treating physicians were asked to classify patients immediately after discharge. We calculated the sensitivity of the various classification systems for the detection of shock and the agreement between the various classification systems and the treating physicians classification.ResultsOf 152 patients with confirmed dengue, sixty-six (43%) had evidence of circulatory failure. The WHO classification system had a sensitivity of 86% (95%CI 76–94) for the detection of patients with shock. All modifications to the WHO classification system had a higher sensitivity than the WHO classification system (sensitivity ranging from 88% to 99%). The WHO classification system was in only modest agreement with the intuitive classification by treating physicians whereas several modified classification systems were in good agreement.ConclusionThe use of the WHO classification system to classify dengue disease severity is to be questioned, because it is not accurate in correctly classifying dengue disease severity and it lacks sufficient agreement with clinical practice.

Differential gene expression changes in children with severe dengue virus infections.

Background The host response to dengue virus infection is characterized by the production of numerous cytokines, but the overall picture appears to be complex. It has been suggested that a balance may be involved between protective and pathologic immune responses. This study aimed to define differential immune responses in association with clinical outcomes by gene expression profiling of a selected panel of inflammatory genes in whole blood samples from children with severe dengue infections. Methodology/Principal Findings Whole blood mRNA from 56 Indonesian children with severe dengue virus infections was analyzed during early admission and at day −1, 0, 1, and 5–8 after defervescence. Levels were related to baseline levels collected at a 1-month follow-up visit. Processing of mRNA was performed in a single reaction by multiplex ligation-dependent probe amplification, measuring mRNA levels from genes encoding 36 inflammatory proteins and 14 Toll-like receptor (TLR)-associated molecules. The inflammatory gene profiles showed up-regulation during infection of eight genes, including IFNG and IL12A, which indicated an antiviral response. On the contrary, genes associated with the nuclear factor (NF)-κB pathway were down-regulated, including NFKB1, NFKB2, TNFR1, IL1B, IL8, and TNFA. Many of these NF-κB pathway–related genes, but not IFNG or IL12A, correlated with adverse clinical events such as development of pleural effusion and hemorrhagic manifestations. The TLR profile showed that TLRs were differentially activated during severe dengue infections: increased expression of TLR7 and TLR4R3 was found together with a decreased expression of TLR1, TLR2, TLR4R4, and TLR4 co-factor CD14. Conclusions/Significance These data show that different immunological pathways are differently expressed and associated with different clinical outcomes in children with severe dengue infections.

Increased PAI-1 plasma levels and risk of death from dengue: no association with the 4G/5G promoter polymorphism

BackgroundDengue virus infected patients have high plasminogen activator inhibitor type I (PAI-1) plasma concentrations. Whether the insertion/deletion (4G/5G) polymorphism in the promotor region of the PAI-1 gene is associated with increased PAI-1 plasma concentrations and with death from dengue is unknown. We, therefore, investigated the relationship between the 4G/5G polymorphism and PAI-1 plasma concentrations in dengue patients and risk of death from dengue.MethodsA total of 194 patients admitted to the Dr. Kariadi Hospital in Semarang, Indonesia, with clinical suspected severe dengue virus infection were enrolled. Blood samples were obtained on day of admission, days 1, 2 and 7 after admission and at a 1-month follow-up visit. Plasma concentrations of PAI-1 were measured using a sandwich ELISA kit. The PAI-1 4G/5G polymorphism was typed by allele-specific PCR analysis.ResultsConcentrations of PAI-1 on admission and peak values of PAI-1 during admission were higher than the values measured in healthy controls. Survival was significantly worse in patients with PAI-1 concentrations in the highest tertile (at admission: OR 4.7 [95% CI 0.9–23.8], peak value during admission: OR 6.3 [95%CI 1.3–30.8]). No association was found between the PAI-1 4G/5G polymorphism, and PAI-1 plasma concentrations, dengue disease severity and mortality from dengue.ConclusionThese data suggest that the 4G/5G polymorphism has no significant influence on PAI-1 concentrations in dengue virus infected patients and is not associated with the risk of death from dengue. Other factors contributing to the variability of PAI-1 plasma concentrations in patients with dengue need to be explored.

Serological Evidence of Human Hantavirus Infections in Indonesia

Here we describe the first evidence of hantavirus infection in humans in Indonesia. Hantaviruses belong to the family Bunyaviridae. Several serotypes have been described to cause disease in man. Hantaan (HNT), Seoul (SEO), Dobrava (DOB) and Puumala (PUU) hantaviruses cause severe to milder hemorrhagic fever with renal syndrome (HFRS) [1]. New World hantavirus serotypes (e.g. Sin Nombre [SNV],Andes) are associated with pulmonary syndrome (hantavirus pulmonary syndrome [HPS]) [1]. In Southeast Asia, evidence of hantavirus infections have been reported from Korea, the Philippines, Singapore and Thailand [2–5]. In Indonesia, a serological study indicated the presence of a Seoul-like virus in wild rats [6].So far no human cases of hantavirus infection have been reported from this country. Serum samples from 94 febrile patients initially suspected of a dengue virus infection were investigated for the presence of hantavirus-specific antibodies.All patients were residents of Yogyakarta or Semarang, Central Java, Indonesia and had presented as outpatients or had been admitted to hospital with symptoms of febrile illness between May 1995 and January 1996 during a dengue epidemic. Patients were divided into two age-groups; the first group included 69 patients in the age range 2–20 years (mean 13.9 years), the second group included 25 patients in the age range 21–47 years (mean 27.2 years). Dengue virus-specific serology in serum samples of these 94 patients was not indicative of recent infection with this virus.Differential diagnosis included measles, rubella, influenza and chikungunya viruses, as well as rickettsiosis and leptospirosis. Nevertheless, the etiology of their illness remained elusive. Serial serum samples of each patient were tested for the presence of hantavirus-specific IgM serum antibodies with a commercially available enzyme immunoassay (EIA) (Focus Technologies,Cypress,USA),using microplates coated with a mixture of SEO and SNV virus recombinant nucleocapsid proteins [7]. Positive EIA results were confirmed by immunofluorescence assay (using SEO, HNT and PUU serotypes) [8] and tested further for the presence of hantavirus-specific IgG serum antibodies by EIA [7], IFA [8] and immunoblotting using native hantavirus antigens, essentially as described earlier [9] (Table 1). In ten (11%) of the 94 patients, serology indicated a recent hantavirus infection.Five had hantavirus-specific IgM and IgG serum antibodies and five had hantavirus-specific IgM but no specific IgG serum antibodies. The ratio in the EIA for the positive samples ranged between 1.1 and 3.1 and between 1.6 and 4.6 for the IgM and IgG, respectively. In two patients only hantavirusspecific IgG serum antibodies were demonstrated, indicating a past infection. Seven sera were confirmed positive for hantavirus-specific IgG serum antibodies by immunoblotting.The serum samples of these patients reacted predominantly with the PUU serotype in the IFA (titers ranged between 64 and ≥ 256) and to a lesser extent with HNT and SEO serotypes (titers ranged between 16 and 128).The differences between IFA and ELISA pattern were probably caused by the fact that the antigen in the IFA is based on whole virus, whereas recombinant proteins were used in the ELISA.The scattered serological pattern in IFA and immunoblot might also indicate that an unknown hantavirus serotype is circulating in this region. Nine of the ten recently infected patients were under 20 years of age (mean 17.6 years), the youngest being 13 years old.This finding is in agreement with previous observations, showing the majority of hantavirus patients to be older than 15 years [10]. Previous epidemiological studies have demonstrated that the distribution of hantavirus infection in Europe is skewed towards the male population,whereas in Southeast Asia the distribution of hantavirus infections is almost equal among the sexes, which may be related to outdoor occupational activities [3,10]. In our study,eight of the patients were