Contribution to rapid diagnosis of infectious mononucleosis

By indirect immunofluorescence (IF) technique humoral antibodies to Epstein-Barr virus capsid antigen (EB-VCA) and to cytomegalovirus (CMV) were detected in 47% and 9% of persons with infectious mononucleosis (IM), respectively. In 23% of the patients examined, IgM antibodies to both viruses were detected, while in 8% of them high titres of IgG only were found in the absence of IgM class antibodies to EB-VCA or to CMV. The finding of IgM antibody to EB-VCA was in good correlation with the persisting symptoms of the disease. Discrepancy between the presence of specific IgM and the absence of heterophilic antibodies was observed in some children and in all persons with persistent or recurrent signs of IM. In the latter, specific IgM was found only during exacerbation of the disease, but during remissions IgG antibodies persisted in high levels. Antibodies to Epstein-Barr virus nuclear antigen (EBNA) were detected in all chronically ill persons and antibodies to the R-component of Epstein-Barr virus early antigen (EA) were present in the majority of them.     

Detection of specific immunoglobulin M antibody to different flaviviruses by use of enzyme-labeled antigens.

An enzyme immunoassay was developed for the detection of human immunoglobulin M (IgM) antibody to different flavivirus antigens. The IgM antibody of human sera was selectively bound to anti-IgM antibody-coated solid-phase plates. Flavivirus IgM antibodies were then detected by use of various enzyme-labeled antigens. The flavivirus antigens (dengue type 2 virus, West Nile virus, and tick-borne encephalitis virus) were produced in suckling mice. The antigens were labeled with horseradish peroxidase by adding the activated enzyme at alkaline pH to sucrose-acetone-treated antigens. Addition of unlabeled mouse brain suspension of uninfected animals to the diluted enzyme-labeled antigens effectively reduced nonspecific binding to the solid phase. In patients with acute flavivirus infections, viral IgM antibody could be demonstrated with high sensitivity. Furthermore, the enzyme-labeled antigen-IgM test showed greater specificity than the hemagglutination inhibition test.     

Evaluation of an Epstein-Barr Virus (EBV) Immunoglobulin M Enzyme-Linked Immunosorbent Assay Using a Synthetic Convergent Peptide Library, or Mixotope, for Diagnosis of Primary EBV Infection

An immunoglobulin M (IgM) enzyme-linked immunosorbent assay (ELISA) was developed by using a 24-amino-acid peptide of the 18-kDa Epstein-Barr virus (EBV) viral capsid antigen (VCAp18). IgM detection was increased by 23% by using this antigen. Detection of IgM antibodies to the EBV proteins in the new ELISA was 100% specific and 95% sensitive.     

Spontaneous Resolution of Hemophagocytic Syndrome Associated with Acute Parvovirus B19 Infection and Concomitant Epstein-Barr Virus Reactivation in an Otherwise Healthy Adult

Reported here is the case of a patient who spontaneously recovered from hemophagocytic syndrome associated with acute B19 infection and concomitant Epstein-Barr virus reactivation. The previously healthy 37-year-old-man was hospitalized after 10 days of high fever, arthralgia and arthritis and was determined to have hemophagocytic syndrome. Immunoglobulin (Ig) M antibodies to Epstein-Barr virus (EBV) capsid antigen, early antigen and parvovirus B19 (B19) were found. B19 DNA and low-level EBV DNA were detected in bone marrow, serum and peripheral blood mononuclear cells. The patient recovered spontaneously without any treatment. Two months later anti-B19 IgG antibodies were detected, while at 9-month follow-up, anti-B19 IgM antibodies were no longer detectable and B19 DNA had disappeared from serum. To the best of our knowledge, this is the first report of spontaneous resolution of hemophagocytic syndrome associated with acute B19 infection and concomitant EBV reactivation in an otherwise healthy adult. Electronic Publication     

A new principle for the detection of specific IgM antibodies applied in an ELISA for hepatitis A.

A new test principle for the detection of specific IgM-class antibodies was developed and applied in an Enzyme-Linked Immuno Sorbent Assay (ELISA) for the detection of hepatitis A IgM antibodies. A solid phase coated with anti-IgM was incubated successively with serum sample, specific antigen, and enzyme-labeled F (ab')2 fragments from IgG antibodies against the antigen and enzyme substrate. F(ab')2 fragments were used to avoid interference with rheumatoid factor. Specificity and sensitivity are very high. This test principle appears generally applicable in the diagnosis of infectious and parasitic diseases by testing only one serum sample.     

Comparison of two rapid diagnostic assays for detection of immunoglobulin M antibodies to dengue virus

Two easy-to-use commercial diagnostic assays, a dipstick enzyme-linked immunosorbent assay (ELISA) (Integrated Diagnostics, Baltimore, Md.) and an immunochromatographic card assay (PanBio, Brisbane, Australia) were evaluated for detection of immunoglobulin M (IgM) antibody to dengue virus with an in-house IgM antibody capture microplate ELISA as a reference assay. The dipstick ELISA was based on the indirect-ELISA format using dengue 2 virus as the only antigen and enzyme-labeled goat anti-human IgM antibody as the detector. The total assay time was 75 min. The immunochromatographic card assay was based on the antibody capture format and separately measured both anti-dengue virus IgM and IgG in the same test. Colloidal-gold-labeled anti-dengue virus monoclonal antibody bound with dengue virus 1 to 4 antigen cocktail was the detector, and anti-human IgM and IgG were the capture antibodies. The total assay time was <10 min. Sera from 164 individuals classified as either anti-dengue virus IgM positive (94) or anti-dengue virus IgM negative (70) in the reference microplate ELISA with a dengue virus 1 to 4 antigen cocktail were tested in the two commercial assays. The dipstick ELISA missed 7 of 94 positive samples, for a sensitivity of 92.6%, while the immunochromatographic card assay missed two positive samples, for a sensitivity of 97.9%. Of the 70 negative samples, four were false positive by the dipstick ELISA and two were false positive in the immunochromatographic card assay, resulting in specificities of 94.3 and 97.1%, respectively. Both commercial assays provide sensitive and specific detection of anti-dengue virus IgM antibody and could prove useful in settings where the microplate ELISA is impractical.     

Improved detection of virus-specific IgM antibodies. Elimination of non-specific IgM binding.

A non-specific reaction between human IgM and cytoplasmic structures of virus infected cells can often be observed if MgM antibodies to virus antigens are detected by indirect immunofluorescence or by immuno enzyme assays. Formaldehyde selectively inactivates the cytoplasmic receptors for human IgM without affecting the virus structural proteins. Alternatively, receptor-free antigens can be obtained by isolation of nuclei from virus infected cells. Due to reduced background, a more specific and more sensitive detection of IgM antibodies to Epstein-Barr virus, cytomegalovirus or central European encephalitis virus is possible.     

Epstein-Barr virus infections in childhood.

IgM and IgG antibodies against Epstein-Barr virus (EBV) capsid antigen and antibodies against EBV nuclear antigen and heterophil antibodies were investigated in 115 paired sera of children with acute infections and in 100 sera of healthy controls of the same age and sex. EBV-specific IgM antibodies could be recognized in 13.7% of the patients and in 7% of the controls. Antibodies against EBV nuclear antigen were not detected in the IgM-positive sera.     

Epstein-Barr virus infection and hepatic fibrin-ring granulomas

Hepatic fibrin-ring granulomas were the main histological finding in the liver of a 38-year-old man with Epstein-Barr virus primary infection. The patient presented with fever, hepatomegaly, icterus, abnormal liver tests, autoimmune hemolytic anemia, and mononucleosis syndrome. There was neither enanthema nor lymphadenopathy or splenomegaly. Serologic tests disclosed an Epstein-Barr primary infection profile: anti-viral capsid antigen IgM antibodies and anti-early antigen antibodies were present, whereas anti-Epstein-Barr nuclear antigen antibodies were absent. There was no evidence for Q fever, Hodgkin's disease, or allopurinol-induced hepatitis, which are recognized causes of hepatic fibrin-ring granulomas. It is suggested that Epstein-Barr virus infection might be an additional cause of these peculiar hepatic granulomas.     

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.     

Epstein-Barr virus-specific serum immunoglobulin A as an acute-phase antibody in infectious mononucleosis.

Immunoglobulin A (IgA) antibodies to Epstein-Barr virus viral capsid antigen were assayed serially in 19 patients with infectious mononucleosis and in 38 controls. Seventy-four percent of infectious mononucleosis patients demonstrated IgA antibody, whereas this was found in 13% of controls. This antibody appeared early in infectious mononucleosis and was virtually gone 10 weeks after onset. Comparison of IgA antibody kinetics was made with IgG and IgM antibodies to viral capsid antigen, heterophile antibody, and antibody to Epstein-Barr virus early antigen and nuclear antigen. Failure to demonstrate IgA antibody was associated with severe illness, prolonged illness, delay in IgG and anti-Epstein-Barr virus nuclear antigen antibody, and low or absent heterophile and anti-early antigen antibody. Assay of IgA antibody to viral capsid antigen is a potentially useful adjunct in the serodiagnosis of infectious mononucleosis or recent Epstein-Barr virus infection, as are the other antibodies tested, but in this study IgM viral capsid antigen antibody was the only acute-phase antibody present in all patients.     

Tissue polypeptide specific antigen for the detection of lymphoproliferative diseases induced by cyclosporin.

AIM--To evaluate the efficacy of tissue polypeptide specific (TPS) antigen for the early detection of cyclosporin A (CyA) induced post-transplant lymphoproliferative diseases. METHODS--Serum concentrations of TPS antigen were analysed using a monoclonal enzyme immunoassay and whole blood CyA concentrations were measured using high pressure liquid chromatography. Infection with Epstein-Barr virus and cytomegalovirus was detected by determining levels of IgM and IgG antibodies directed against viral capsid antigen (VCA). Immunohistochemistry and analysis of clonality were carried out on formalin fixed, paraffin wax embedded tissue. RESULTS--The mean serum concentration of TPS antigen in the eight transplant recipients investigated was 60 U/l during periods without complication (control), 101 U/l during infection, 166 U/l when the diagnosis of a lymphoma was confirmed, and 172 U/l when lymphoma and infection coincided. Increased TPS antigen concentrations were detected in six patients one month before detection of malignancy. After reduction of immunosuppression and the start of tumour regression, TPS antigen concentrations decreased. TPS antigen concentrations increased in the one patient who experienced a recurrence. CONCLUSIONS--Continuous monitoring of TPS antigen concentrations leads to the early discovery of CyA induced lymphoproliferative disease.     

Solid-phase antibody capture hemadsorption assay for detection of hepatitis A virus immunoglobulin M antibodies.

A solid-phase antibody capture hemadsorption (SPACH) assay was developed to detect hepatitis A virus (HAV)-specific immunoglobulin M (IgM) antibodies in sera from humans recently infected with hepatitis. The assay is performed with microtiter plates coated with anti-human IgM antibodies to capture IgM antibodies from the test sera. HAV-specific IgM antibody is detected by the addition of HAV hemagglutinating antigen and goose erythrocytes. Hemadsorption of erythrocytes to antigen-antibody complexes attached to the solid phase indicate the presence of IgM antibodies. The SPACH assay was compared to a commercial radioimmunoassay and was found to be equally or more sensitive and specific for the detection of HAV IgM antibodies. The SPACH assay is an alternative, rapid assay that doesn't require hazardous substrates or radioactivity for the detection of HAV-specific antibodies.     

A reverse passive haemagglutination test for detection of Japanese encephalitis virus antigens in cerebrospinal fluid

A rapid sensitive and specific reverse passive haemagglutination test (RPHA) was developed for the detection of Japanese encephalitis virus (JEV) antigens in human cerebrospinal fluid (CSF). Sheep red cells were sensitized with five monoclonal antibodies (109, 112, 203, 204 and 301) reactive with envelope glycoprotein of JEV. Viral antigens were detected in CSF from 35 of 58 (60%) clinical cases of JE when the five MAb coated cells were used in combination. An IgM capture ELISA detected JEV specific antibodies in CSF among 52 of these 58 cases (90%). While 29 specimens contained both antigen and IgM antibodies, 23 had only IgM antibodies and 6 had only antigen. RPHA proved valuable for detection of viral antigens in CSF samples obtained within 10 days after onset of clinical symptoms. Amongst the five MAbs used, the individual antigen detection rates were 44, 12, 43, 12 and 36%, for MAbs 109, 112, 203, 204 and 301, respectively.     

An enzyme-linked immunosorbent assay for IgG antibodies to human herpes virus 6

An indirect enzyme-linked immunosorbent assay (ELISA) with human herpes virus 6 (HHV6) membrane antigen was compared with indirect immunofluorescence assay (IFA) for measurement of HHV6 IgG antibodies. Five hundred serum samples from 403 Swedish patients with suspected symptomatic Epstein-Barr virus (EBV) infections were examined. The specificity of the ELISA compared with IFA was 98.7% and the sensitivity was 98.4%. In 90% of the patients, IgG antibodies to HHV6 were detected with both assays. The highest HHV6 IgG titers were found mainly in patients with EBV or CMV infections, but HHV6 mononucleosis was not diagnosed. The same HHV6 antigen was assessed for IgM ELISA but was found to be of limited value due to high IgM reactivity with the control antigen. The HHV6 IgM ELISA requires further investigation. The IgG ELISA described is a reliable alternative to IFA for measurement of HHV6 IgG antibodies and for large scale epidemiological studies.     

Kinetics of specific IgA, IgD, IgE, IgG, and IgM antibody responses in rubella

Rubella-specific IgD and IgE antibodies were determined with a solid-phase enzyme immunoassay using enzyme-labeled heavy-chain specific anti-immuno-globulins, and the antibody responses in rubella infection were compared to IgM, IgA, and IgG antibodies. IgD and IgE antibodies increased rapidly after the onset of infection, remained at a high level for at least 2 months, and declined slightly by 6 months. In comparison, the IgM antibodies decreased more rapidly, whereas the IgG antibodies persisted longer at a steady level. By 6 months the mean levels of the different antibodies had declined from their maximal mean levels as follows: IgM, 52%; IgA, 42%; IgE, 35%; IgD, 29%; and IgG, 8%. Thus IgD and IgE antibodies, in spite of their known short half lives, persisted longer than IgM and IgA antibodies, which limits their diagnostic value. The IgA antibody responses were found too variable to substitute for IgM antibody determination in diagnosis of a recent rubella virus infection from a single serum specimen. Comparison of maternal and cord blood sera indicated that, in addition to IgG antibodies, rubella-specific IgD antibodies were found to cross the placenta.     

The response to Epstein-Barr virus infection in Sj?gren's syndrome

To assess the response to Epstein-Barr virus (EBV) infection in patients with primary Sj?gren's syndrome (SS), the frequency of detection of EBV DNA was studied in salivary gland biopsies and the antibody and idiotypic response to the virus was compared with healthy controls and infectious mononucleosis (IM). Viral DNA, detected by in-situ hybridization, was found in biopsies from two out of 12 patients with SS and six out of 10 controls. IgG, IgA and IgM antibodies to the virus, measured by ELISA using synthetic peptides (early antigen and EBNA-1) and a cloned fusion protein (EBNA-1), were normal in sera from 20 patients with SS, whereas infectious mononucleosis patients showed an increase in IgM antibodies to EBNA-1 and IgG antibodies to early antigen. One similarity between infectious mononucleosis and Sj?gren's syndrome was a significant increase in the germline heavy chain idiotype G6 in both diseases, suggesting activation of similar B-cell subsets. It is possible that this is due to EBV, though the low frequency of EBV DNA in biopsies and the normal levels of EBV antibodies in SS does not lend any evidence that the virus itself is the causative agent.     

Measles virus-specific IgM antibodies in sera from patients with chronic active hepatitis

The occurrence of measles virus-specific IgM antibodies in sera from patients with chronic active hepatitis not caused by Hepatitis B virus was examined by a specific enzyme-linked immunosorbent assay (ELISA). Using whole serum, specific IgM antibodies were detected in 12 of 23 sera from patients with hepatitis B surface antigen (HBsAg)-negative chronic active hepatitis. In nine of these sera the finding of specific IgM antibodies was verified by separation of IgM by density gradient centrifugation and examination of the fractions by ELISA. Most of the sera from the patients with measles virus-specific IgM antibodies had an elevated level of specific IgG antibodies compared to the level of IgG found in control sera. The significance of these findings in view of a possible persistent measles virus antigen production in patients with chronic active hepatitis is discussed.