Serological markers during dengue 3 primary and secondary infections.

BACKGROUND: The detection of the IgM antibody for the dengue virus in serum by ELISA has become one of the most important and useful methods for diagnosis of dengue using a single acute-phase serum sample. Currently, this system is an invaluable tool for the surveillance of dengue fever (DF) and dengue hemorrhagic fever (DHF). The usefulness of other serological markers such as IgA and IgE have been less studied. OBJECTIVE: To study the IgM, IgA and IgE specific antibody response in dengue 3 infected patients with different clinical picture and type of infection. STUDY DESIGN: One hundred and twenty-seven serum samples collected on days 5-7 at the onset of fever from clinically and serologically confirmed dengue cases were studied. Forty-two were classified as primary dengue fever cases, 48 as secondary dengue fever cases and 37 as secondary dengue hemorrhagic fever cases. All samples were tested by capture ELISA in order to detect dengue IgM, IgA and IgE antibodies. RESULTS AND CONCLUSIONS: In this study, significant differences were observed in the IgM, IgA and IgE response between the study groups. High IgA and IgE OD ratios in secondary dengue cases were found. The usefulness of serotype specific IgM antibody detection is also analyzed and discussed. A priority for future dengue research in terms of protection, recovery of infection and immunopathogenesis is to elucidate the role of these immunoglobulins. The cross reactivity response to IgM between dengue virus serotypes in primary and secondary cases should also be more studied.     

Comparison of strain-specific antibody responses during primary and secondary infections with respiratory syncytial virus

Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infection in infants. RSV repeatedly reinfects individuals: this may be due in part to the variability of the attachment (G) glycoprotein and changes in this protein have been shown to be under positive selection. Infants experiencing their primary infection show a genotype-specific antibody response with respect to the variable regions of the G protein. A prospective study of RSV infections in a birth cohort in rural Kenya identified infants experiencing repeat infections with RSV. The serum antibody responses of these infants were investigated with respect to their anti-RSV reactions in an enzyme-linked immunosorbent assay (ELISA) and the specificity of the response to a variable region of the G protein by ELISA and immunoblotting using bacterially expressed polypeptides representative of the currently circulating strains of RSV. The results presented here confirm that the primary antibody response to the variable regions of the G protein is generally genotype-specific, but show that the response may become cross-reactive (at least within group A viruses) during secondary infections even where the secondary infection is of the same genotype as the initial infection. Also, some infants who did not mount a detectable antibody response to whole RSV antigens during their primary infection nevertheless showed genotype-specific responses to the G protein. In conclusion, the strain-specific nature of the serum antibody response to the variable regions of the G protein of RSV observed in primary infections can become cross-reactive in subsequent reinfections.     

Cross-reactive IgM responses in patients with dengue or Japanese encephalitis.

BACKGROUND: Dengue and Japanese encephalitis viruses co-circulate in Thailand. IgM-capture enzyme-linked immunosorbent assay (ELISA) has been widely used for confirmation of dengue and Japanese encephalitis (JE). OBJECTIVES: To examine the cross-reactivity in IgM responses to dengue and JE viruses in serum and CSF samples from dengue and JE patients. STUDY DESIGN: Two hundred and fifty-eight serum samples from 177 confirmed dengue patients, and 99 serum samples and 37 cerebrospinal fluid (CSF) samples from confirmed JE patients were analyzed. RESULTS: Nine percent of serum samples from dengue patients were positive for anti-JE IgM. Thirteen percent of serum samples and 11% of CSF samples from JE patients were positive for anti-dengue IgM. Levels of cross-reactive IgM were lower than those of specific IgM in all the dengue and JE patients. CONCLUSIONS: Only specific IgM is detected in about 90% of dengue and JE patients, but cross-reactive IgM is also detected in the remainder. The presence of cross-reactive IgM responses should to be considered in the serodiagnosis of dengue and JE, especially in areas where dengue and JE viruses co-circulate.     

Dynamics of interleukin-21 production during the clinical course of primary and secondary dengue virus infections

Previous studies have revealed the clinical relevance of pro-inflammatory cytokine production during dengue virus (DENV) infections. In this study, we evaluated the production of interleukin-21 (IL-21), a key soluble mediator mainly produced by CD4+ T cells.     

Use of an immunoglobulin G avidity test to discriminate between primary and secondary dengue virus infections

An enzyme-linked immunosorbent assay-based immunoglobulin G (IgG) antibody avidity test was evaluated by using sera from 57 patients with acute dengue infection. Overall, 55 of 57 patients were correctly classified (27 of 27 with primary dengue and 28 of 30 with secondary dengue). We conclude that the IgG avidity test can be useful for differentiating between acute, primary, and secondary dengue infections.     

Immunity and immunopathology in dengue virus infections.

Dengue virus infections are a serious public health problem in tropical and subtropical areas of the world. Based on epidemiological data, it has been postulated that immune responses to dengue virus contribute to the pathogenesis of severe dengue illness, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Host immune responses are also important for controlling dengue virus infection. Therefore, dengue virus infections are an interesting model to explore the interactions between viruses and the immune system which result in immunopathology or recovery from infection. In this paper, we review immune responses to dengue viruses with an emphasis on the human T cell responses, and discuss possible roles of these immune responses in the control of dengue virus infection and in the pathogenesis of DHF/DSS.     

Correlation between increased platelet-associated IgG and thrombocytopenia in secondary dengue virus infections

Although the public health impact of dengue is increasing rapidly, the mechanism of thrombocytopenia in this disease remains unknown. To elucidate this mechanism, the relationship between platelet-associated IgG (PAIgG) and platelet count in 53 patients in the acute phase of secondary dengue virus infection was investigated in a prospective-hospital-based study. A significant inverse correlation between the two parameters was found in these patients, while no correlation was observed in healthy volunteers. The low baseline platelet counts during the acute phase in 12 patients with secondary dengue virus infection significantly increased during the convalescent phase, while the increased PAIgG levels during the acute phase in these patients significantly decreased during the convalescent phase. Anti-platelet IgG autoantibody was detected rarely in the plasma of 53 patients with secondary dengue infection. The involvement of anti-dengue virus IgG was also shown in platelets from all of 8 patients in the acute phase of secondary dengue virus infection. These findings suggest that PAIgG formation involving anti-dengue virus IgG plays a pivotal role in the induction of transient thrombocytopenia during the acute phase of secondary dengue virus infection.     

Utility of IgM/IgG ratio and IgG avidity for distinguishing primary and secondary dengue virus infections using sera collected more than 30 days after disease onset

Dengue virus (DV) IgM/IgG ratio and IgG avidity value (AV) can reliably distinguish between primary and secondary DV infections using sera collected within 30 days of disease onset, but little is known about their efficacies using sera collected >30 days after onset. To investigate this issue, we analyzed specimens submitted to our reference laboratory for DV antibody testing. We first classified patients as having primary (n = 55) or secondary (n = 58) infections based on seroconversion patterns in a comparison of two sera collected <30 days apart. We then evaluated IgM/IgG ratios and IgG AVs of the second specimens by using receiver operating characteristic curve analysis. The IgM/IgG ratio that best discriminated primary from secondary infection was 1.32; 95% of 55 primary infections exhibited ratios of >1.32, whereas 93% of 58 secondary infections exhibited ratios of ≤1.32. The discriminatory AV was 0.39; 95% of 41 primary infections exhibited AVs of ≤0.39, whereas 95% of 38 secondary infections exhibited AVs of >0.39. We then evaluated the IgM/IgG ratios and AV for primary-infection patients whose second serum samples were collected ≥30 days after the first serum samples; only 56% of 27 sera exhibited ratios of >1.32, whereas 81% of 21 sera exhibited AVs of ≤0.39. Assuming that the first specimens were collected within a week after symptoms appeared, these findings indicate that IgG AV is superior to the IgM/IgG ratio for distinguishing primary from secondary DV infections when using samples collected more than 5 weeks after disease onset.     

Laboratory diagnosis of primary and secondary dengue infection.

BACKGROUND: Dengue fever is routinely detected in many laboratories using commercial tests for the specific detection of dengue IgM antibodies. OBJECTIVES: We have studied the sensitivity of IgM antibody detection in paired serum samples of 43 patients with either with primary dengue (PD) or secondary dengue (SD). STUDY DESIGN: Two consecutive samples were drawn from 23 Vietnamese and 20 German patients. All patients were selected for a positive PCR and for the fact that consecutive serum samples were available. The diagnosis of PD was based on seroconversion to dengue antigen and in SD on the detection of virus RNA in the presence of anti-dengue IgG antibodies. RESULTS: In samples of patients with PD fever taken during days 1-3 of the disease no IgM antibody could be detected. During days 4-7 and after day 7, IgM antibody was detected in 55% and 94%, respectively. In patients with SD fever, even less positive IgM samples were found in samples taken during days 4-7 (47%) and after day 7 (78%). IgG titers were significantly higher in SD compared to PD patients, although high (>1280) titers were also found in some PD patients. CONCLUSION: In numerous acute dengue fever patients an early diagnosis will be obtained only by combining IgM antibody detection with detection of virus or virus RNA using RT-PCR.     

Association of increased platelet-associated immunoglobulins with thrombocytopenia and the severity of disease in secondary dengue virus infections

Severe thrombocytopenia and increased vascular permeability are two major characteristics of dengue haemorrhagic fever (DHF). To develop a better understanding of the roles of platelet-associated IgG (PAIgG) and IgM (PAIgM) in inducing thrombocytopenia and its severity of disease in patients with secondary dengue virus infection, the relationship between the PAIgG or PAIgM levels and disease severity as well as thrombocytopenia was examined in 78 patients with acute phase secondary infection in a prospective hospital-based study. The decrease in platelet count during the acute phase recovered significantly during the convalescent phase. In contrast, the increased levels of PAIgG or PAIgM that occurred during the acute phase of these patients decreased significantly during the convalescent phase. An inverse correlation between platelet count and PAIgG or PAIgM levels was found in these patients. Anti-dengue virus IgG and IgM activity was found in platelet eluates from 10 patients in an acute phase of secondary infection. Increased levels of PAIgG or PAIgM were significantly higher in DHF than those in dengue fever (DF). An increased level of PAIgM was associated independently with the development of DHF, representing a possible predictor of DHF with a high specificity. Our present data suggest that platelet-associated immunoglobulins involving antidengue virus activity play a pivotal role in the induction of thrombocytopenia and the severity of the disease in secondary dengue virus infections.     

Bispecific cells among IgM and IgG producers during the early phase of primary and secondary responses.

Simultaneous immunization of mice with sheep (SRBC) and horse (HRBC) erythrocytes regularly resulted in the appearance of hemolytic plaque-forming cells (PFC) specific for each type of erythrocyte and also of PFC lysing both types of erythrocytes. After primary stimulation the highest number of bispecific cells (42/10(6) cells) was found among PFC as revealed by the direct procedure (IgM producers). Among PFC enhanced with anti-mouse Fab serum (IgG producers), bispecific cells were less numerous (8/10(6) cells). In preparations enhanced by anti-mouse-gammaFc serum which reveals IgG producers without inhibiting IgM antibody, the number of bispecific PFC equalled the sum of bispecific cells revealed by direct and anti-Fab enhanced procedures. The number of direct bispecific PFC during primary and secondary response was approximately the same, whereas the number of IgG-producing, bispecific PFC increased considerably during the secondary response. Another difference was the time limitation of the appearance of bispecific cells: after primary immunization direct bispecific PFC were detected only on days 3, 4 and 5, but enhanced bispecific PFC were present from day 4 up to day 12. However, during the secondary reaction, bispecific PFC were detected by all three procedures only between days 3 and 6. Studies on the cross-reactivity between SRC and HRBC gave negative results at the humoral level, even when the mice were primed with a minimal amount of both erythrocytes and then two months later, boosted with one of them. Studies at the cellular level showed that after immunization with one antigen, only 0.4 to 0.7 direct or enhanced PFC/10(6) cells could simultaneously lyse both erythrocyte types. Thus, a hundred times more bispecific PFC were constantly found after double immunization of the animals. Moreover, sudden disappearance of all bispecific PFC on the 7th day after secondary stimulation makes it unlikely that all bispecific PFC are simply cross-reacting cells.     

Acute West Nile virus neuroinvasive infections: cross-reactivity with dengue virus and tick-borne encephalitis virus

Cross-reactions in serology are common among flaviviruses. During the outbreak of West Nile virus (WNV) infections in Greece in 2010, WNV IgM-positive serum and cerebrospinal fluid samples were tested for the presence of IgM and IgG antibodies against Dengue virus (DENV) and tick-borne encephalitis virus. Higher cross-reactivity was observed in IgM antibodies between WNV and DENV; however, the index of the WNV antibodies was in all cases higher than that of the DENV antibodies. There is a need for caution when evaluating serologic results of flaviviral infections, while efforts have to be focused on the development of diagnostic assays with increased specificity.     

Pathogenesis of equine herpesvirus-1 in specific pathogen-free foals: primary and secondary infections and reactivation

Summary Six specific pathogen-free foals shown to be free of equine herpesvirus-1 and 4 (EHV-1 and -4) and lacking in maternally-derived antibodies were used to investigate the pathogenesis of EHV-1 in horses. Following primary intranasal inoculation with EHV-1 all foals showed signs of a mild, self-limiting upper respiratory tract infection. A leucopenia was observed, comprising both a lymphopenia and neutropenia. Virus was isolated from nasal mucus and buffy coat cells over several days during the clinical episode and after the animals became clinically normal. Notwithstanding the mildness of the clinical disease, virus was not eliminated completely and intravenous administration of dexamethasone resulted in reactivation of latent EHV-1 in animals which had received only a single dose of the virus. In a second infection given to four foals, 61 days after the primary inoculation, no clinical signs were observed, haematological changes were minimal and viraemia was absent.     

Increased IgM and IgM immune complex-like material in the circulation of renal transplant recipients with primary cytomegalovirus infections.

Twelve of 60 consecutive adult recipients of cadaver kidney transplants had increased polyethyleneglycol (PEG) precipitable IgM immune complex-like material in their circulation in the first 4 months after transplantation. All 10 recipients with primary CMV and two of four with secondary CMV infections had significant elevations in PEG precipitable IgM that coincided with rises in their CMV antibody titres. Ultracentrifuge analysis demonstrated two peaks of PEG precipitable material with sedimentation rates of about 20S and 40S. Total IgM immunoglobulin levels also were increased in transplant recipients with CMV infections, but this was less specific and occurred in patients without CMV infections. The Clq binding assay, which is more sensitive for IgG than IgM containing complexes, was positive in only three of 10 patients with primary CMV and none of four with secondary CMV. Granular deposits of IgM, but not IgG, were detected in the glomeruli of six of seven transplants biopsied during CMV infection. The PEG-IgM assay was not influenced by rejection or prednisone therapy. Thus, transplant patients, who develop primary CMV infections, produce elevated levels of circulating IgM and IgM immune complex-like material. These findings may help to differentiate CMV infection from transplant rejection as well as to increase our understanding of the special pathogenic properties of CMV in transplant recipients.     

Comparison of capture immunoglobulin M (IgM) and IgG enzyme-linked immunosorbent assay (ELISA) and nonstructural protein NS1 serotype-specific IgG ELISA for differentiation of primary and secondary dengue virus infections

We have found that NS1 serotype-specific immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) can be used to differentiate primary and secondary dengue virus infections. This is due to the fact that the NS1-specific IgG antibody cannot be detected before day 9 of illness for primary infection, so the NS1-specific IgG antibodies measured in acute-phase sera must come from previous infection. Comparison of NS1 serotype-specific IgG ELISA with envelope- and membrane-specific capture IgM and IgG ELISA in the differentiation of primary and secondary dengue virus infections showed good correlation (95.90% agreement). Most important, we have found that the serotype of the dengue virus from the majority of patients with primary infection could be correctly identified when convalescent-phase or postinfection sera were analyzed by NS1 serotype-specific IgG ELISA. These findings suggested that NS1 serotype-specific IgG ELISA could be reliably applied for serodiagnosis and seroepidemiological study of dengue virus infection.     

Sensitivity and specificity of three ELISA-based assays for discriminating primary from secondary acute dengue virus infection.

BACKGROUND: Discrimination between primary and secondary dengue virus infection traditionally has been performed using the hemagglutination inhibition (HI) test. However, this test has practical limitations and disadvantages. OBJECTIVE: To evaluate the ability of three ELISA-based methods (IgG avidity test, IgM/IgG ratio and IgG titer) to discriminate primary from secondary dengue infection. STUDY DESIGN: Serum samples from convalescent-phase patients with confirmed acute, primary (n=46) or secondary (n=33) dengue virus infection were tested using three ELISA-based methods. A ROC curve was employed to establish the cut-off points and to evaluate the ability of the three methods to distinguish between acute, primary and secondary dengue virus infection. RESULTS: All three assays exhibited sensitivity and negative predictive values of 100% for defining secondary infection. The specificity and positive predictive values were respectively 97.8% and 93.7% for the IgG avidity test, 95.7% and 88.2% for the IgM/IgG ratio assays, and 97.8% and 93.7% for the IgG titer assay. CONCLUSION: All three ELISA-based assays proved reliable tools for discriminating between acute, primary and secondary dengue virus infection when using serum samples from convalescent-phase patients.     

The detection of specific IgM antibodies following infection with rubella virus.

A gel filtration technique using Sephadex G-200 has been used for the detection of specific IgM in sera from (a) 45 cases of clinical rubella in which diagnostic rises of rubella haemagglutination-inhibiting (HAI) antibody could be demonstrated; (b) 70 cases with clinical evidence of rubella in which a rising titre could not be demonstrated because the first serum sample already had high titre HAI antibodies; and (c) 100 patients in whom rubella was not suspected. The results indicate that the high specificity and sensitivity of the method described make it an appropriate technique for use in the routine diagnosis of acquired rubella.