The use of semi-automated EBV IgG avidity determination for the diagnosis of infectious mononucleosis

The diagnosis of acute Epstein-Barr virus (EBV) infection is based frequently on the combination of positive viral capsid antigen (VCA) IgM antibodies and negative EB viral nuclear antigen 1 (EBNA-1) IgG antibodies. However, both VCA IgM and EBNA-1 IgG can provide false positive and false negative results. Therefore, situations in which the EBV serology remains unclear are not uncommon. Determination of EBV IgG avidity can clarify the EBV status in these patients. So far, mainly immunofluorescence assays have been used for this purpose. These tests are laborious, their evaluation is subjective, and automation is difficult. Therefore, two commercially available microtiter plate enzyme immunoassays (EIA) were compared for their usefulness for semi-automated EBV IgG avidity determination. One assay is based on a mixture of EBV antigens, the other assay uses a synthetic peptide of the VCA-complex. Patient sera of confirmed acute and past EBV infections were tested for avidity by both assays. The results with the antigen mixture assay proved to be highly sensitive (100%) and specific (100%). Avidity index calculations on the basis of one-point-quantification titers gave better results than calculations using OD values. Determination of EBV IgG avidity by the peptide assay was complicated by the fact that it was less sensitive than the antigen mixture assay for IgG detection in acute EBV infections. On the other hand, about 30% of the samples had to be retested with the peptide assay in a higher dilution because the IgG units in initial testing fell outside the range covered by the standard curve. Using OD values of the peptide EIA, the sensitivity was 99% but the specificity of detection of acute EBV infections was only 86%. Thus, while the peptide EBV avidity assay is unsuitable as a confirmatory assay, avidity testing with the antigen mixture assay is a useful tool to resolve equivocal EBV serologies. Avidity assays on the basis of EIA can be automated which should lead to wider use of this methodology. J. Med. Virol. 54:145–153, 1998. © 1998 Wiley-Liss,Inc.     

Comparison of IgG avidity assays in the confirmation of the diagnosis of cytomegalovirus primary infection

We have compared three ELISA techniques and one chemiluminescence immunoassay technique for determining cytomegalovirus (CMV) immunoglobulin G (IgG) avidity in serum samples from patients with recent and past CMV infections. Sensitivity varied from 84.6% to 100%; and specificity from 78.6% to 100%. IgG avidity assays appear to be adequate additional tools for characterizing CMV infections.     

Coupled particle light scattering: a new technique for serodiagnosis of Epstein-Barr virus infection

The Coupled Particle Light Scattering technique was evaluated for serological diagnosis of Epstein-Barr Virus (EBV) infection. Two hundred ninety-six patient sera selected from several clinical categories (acute infection, non-primary infection, interfering non-EBV infection, non-infected) were tested for IgM and IgG antibodies (anti-VCA, anti-EBNA and anti-EA). Determination of EBV IgG with Copalis multiplex was accurate when compared with Enzygnost Anti-EBV/IgG ELISA. Although the sensitivity of Copalis IgM for acute infections was 100% a positive IgM result did not always indicate an acute infection. Strong reactivity to IgG EA (ratio 3, 1) and IgG VCA (ratio 13, 3) correlated with persistent infection or reactivation. The CopalisI has many advantages over the existing methods, such as the possibility to measure three semi-quantitative IgG responses to three different EBV antigens simultaneously.     

Serum Epstein-Barr virus DNA load in primary Epstein-Barr virus infection

Specific viral laboratory diagnosis of primary Epstein-Barr Virus (EBV) infection is usually based on antibody-detection assays. During acute, lytic phase of infection, viral DNA can also be detected in serum. In the present study, the diagnostic utility of EBV DNA detection and quantitation in serum in primary EBV infection was investigated. The level of EBV DNA in the serum of 98 immunocompetent patients aged 1-47 years with symptomatic, antibody-confirmed EBV primary infection was assessed using a quantitative real-time PCR assay. The association between viral load and time after onset of disease, age and clinical and laboratory data was investigated. Quantitative PCR detected EBV DNA in 93 of 98 samples (94.9%), and the measured viral loads ranged from 3.8 x 10(1) to 6.6 x 10(4) copies/ml. EBV DNA detection exhibited a sensitivity of 94.9% and a specificity of 97.4% for primary EBV infection. EBV DNA was always detectable until day 12 after onset of symptoms, whereas no further positive PCR results were found after a period of 22 days after onset of disease. Detection of EBV DNA also showed a clearer association with the clinical manifestation of disease than the presence of EBV specific VCA IgG antibodies of low avidity. EBV DNA load was found to correlate inversely with the time after onset of disease (P < 0.001), and higher viral load levels were detected in younger (P = 0.009) and in hospitalized patients (P = 0.038). The results indicate that real-time PCR is a reliable tool for diagnosis of primary EBV infection early in the course of disease. In addition, EBV DNA detection may serve as a useful diagnostic supplement in serologically indeterminate EBV infections.     

Differentiation of primary cytomegalovirus infection from reactivation using the urea denaturation test for measuring antibody avidity

Cytomegalovirus (CMV) is probably the most common agent of prenatal infection of the newborn, and one of 20 congenitally infected newborns shows serious symptoms. It was therefore considered important to be able to differentiate primary CMV from reactivation in pregnant females. A urea denaturation test was used to distinguish primary from secondary rubella infection in which the urea is included in the wash step of the standard IgG ELISA. This resulted in the removal of low-avidity antibodies, which are the antibodies produced early in infection. A group of CMV IgM-negative and -positive sera were tested, and all but one showed moderate to high avidity, with an avidity index reading of more than 30%. Among a group of babies 3-12 months of age, who were CMV IgM positive, 55% (16 of 29) showed low-avidity CMV antibodies. A small group of renal transplant patients and patients with clinically and laboratory-confirmed CMV gave more or less predicted avidity index results. It appears that, with the method used at this laboratory, the urea denaturation test can be applied to CMV to determine primary infection or reactivation in the majority of cases.     

Measurement of antibody avidity for hepatitis C virus distinguishes primary antibody responses from passively acquired antibody

A new IgG antibody avidity test for hepatitis C virus (HCV) has been developed and was validated using sera from 12 renal dialysis patients infected with HCV. In primary HCV infection low avidity antibody (mean avidity index 24%) was detected within 50 days of seroconversion whereas in long-term infection (at least 300 days after seroconversion), the mean avidity index was high (88%); in five patients, the avidity index was shown to increase rapidly as time elapsed after primary infection, whereas immunosuppressive therapy was found to delay maturation of the immune reponse in two further patients. The assay was then employed to confirm that a spurious outbreak of primary HCV infection in eight bone marrow transplant patients was explicable by passive acquisition of high avidity anti-HCV after intravenous immunoglobulin therapy. It is concluded that this avidity test will have an important role in the investigation of HCV infection in patients. © 1994 Wiley-Liss, Inc.     

CD8 T cell immunodominance shifts during the early stages of acute LCMV infection independently from functional avidity maturation

Virus-specific T cell responses are often directed to a small subset of possible epitopes and their relative magnitude defines their hierarchy. We determined the size and functional avidity of 4 representative peptide-specific CD8⁺ T cell populations in C57BL/6 mice at different time points after lymphocytic choriomeningitis virus (LCMV) infection. We found that the frequency of different peptide-specific T cell populations in the spleen changed independently over the first 8 days after infection. These changes were not associated with a larger or more rapid increase in functional avidity and yet still resulted in a shift in the final immunodominance hierarchy. Thus, the immunodominance observed at the peak of an antiviral T cell response is not necessarily determined by the initial size or rate of functional avidity maturation of peptide-specific T cell populations.     

Immunoglobulin G antibody avidity assay for serodiagnosis of hepatitis C virus infection

It has been reported that the avidity of specific IgG antibody is lower in primary viral infection than in chronic viral infection. However, few studies have been reported on the IgG avidity in hepatitis C virus (HCV) infection. In the present study, 36 patients with antibody to HCV (anti-HCV) were examined for IgG avidity by an enzyme immunoassay with or without urea elution. The avidity index was significantly low in patients with primary HCV infection (7.7 +/- 6.8%, mean +/- SD), compared with patients with chronic HCV infection (77.0 +/- 21.8%) and individuals with past HCV infection (44.5 +/- 12.6%). Temporal changes of IgG avidity were examined in six patients with primary HCV infection. The avidity index was low in the acute phase of the infection and then increased with time. These results suggest that the avidity assay for IgG anti-HCV is a useful method for distinguishing primary HCV infection from chronic or past HCV infection.     

Antibody avidity: use for the diagnosis of HIV early infection

Determination of IgG avidity is useful to distinguish primary infection from reactivation or reinfection in viral, parasitic or bacterial infections. For diagnosis of HIV type 1 primary infection, the detection of IgM antibodies is often useless since they are also found in chronic infection. The usual serology (Elisa, western-blot, p24 antigen) may present no interest if done too late (more than 2 or 3 months after infection). Therefore, we have developed a test to determine the avidity of anti-HIV1 antibodies, using 1 M guanidine as denaturing agent. We have adapted the measurement of avidity to the Axsym automatic system for a routine use. Indeed, since requests for avidity determinations are sporadic, the use of microplates is not convenient. Using this assay, we found a low avidity (less than 50%) in immunocompetent and recent infected patients (less than 6 months), compared to old infected patients (more than 12 months) who had high avidity (80 to 100%). However, early treated patients (in the 6 months after contamination) had also low avidities but with a slower development of antibody maturation (8 to 27 months versus 2 to 8 months in non treated patients). To conclude, the determination of the anti-HIV1 avidity, according to the proper procedures explained here (notion of treatment and/or serious immunodepression), may help the physician to date the infection in each new infected patient who might benefit from an early treatment.     

Improving diagnosis of primary cytomegalovirus infection in pregnant women using immunoblots

Human cytomegalovirus (CMV) is the most common infectious cause of mental disability in newborns of developed countries. Transmission of CMV from mother to baby is more frequent in maternal primary infection, although CMV reactivation causes more congenital infections overall. Current diagnostic tests for distinguishing primary and reactivation CMV have problems with interpretation and immunoblots may assist with diagnosis. Sera from 60 pregnant women were analyzed using conventional serology in parallel with a commercial immunoblot assay (using Recomblot, Mikrogen Diagnostik). Comparison of detection of CMV IgG, IgM, IgG avidity in maternal primary infection showed the immunoblot relative to conventional serology had sensitivity and specificity of 100% for IgG identification. The detection of IgM on immunoblot showed sensitivity of 75%, specificity of 62.5%, positive predictive value (PPV) of 81.8% and negative predictive value (NPV) of 52.6%. The immunoblot IgG avidity assay had sensitivity of 94.1%, with a PPV of 100% when identifying low avidity serum samples, and sensitivity of 100% with a PPV of 97.1% for high avidity serum samples. Overall agreement between conventional serology (IgM, IgG avidity) and immunoblot (IgM, IgG avidity) for detection of primary CMV infection was 65%. Although the immunoblot is effective in detecting IgG and determining IgG avidity, it showed no significant benefits in performance or utility as a first line diagnostic technique for IgM or primary CMV infection in pregnant women. J. Med. Virol. 85:315–319, 2013. © 2012 Wiley Periodicals, Inc.     

Follow-up of avidity and titer of anti-myeloperoxidase antibodies in sera from patients with primary ANCA-associated vasculitis

Antineutrophil cytoplasmic antibodies (ANCA) are important serologic markers for ANCA-associated vasculitis (AAV). Our previous studies in propylthiouracil (PTU)-induced AAV demonstrated that withdrawal of PTU resulted in clinical remission and significant decrease of avidity of PTU-induced myeloperoxidase (MPO)-ANCA. This study investigated the changes in avidity and titer of MPO-ANCA in sequential sera from some patients with primary AAV with different disease activities. Sequential sera samples of seven patients with MPO-ANCA-positive vasculitis at their initial onset, remission and relapse were collected. The avidity of MPO-ANCA was assessed by antigen-inhibition enzyme-linked immunosorbent assay (ELISA). The titer of MPO-ANCA was determined by a two-fold dilution of sera in MPO specific ELISA. The titer of MPO-ANCA was not significantly different between initial onset and remission. The avidity constant (aK) of MPO-ANCA in active phase is not significantly different from that in remission (724.9 ± 828.4 l/mol vs. 353.4 ± 551.7 l/mol, p = 0.303). No significant correlation could be found between aK and the level of Birmingham Vasculitis Activity Score, times of relapse, the number of organ involvement, serum creatinine, or CRP. Avidity and titer of MPO-ANCA did not decreased significantly during remission in AAV, indicating the chronic repeated antigen stimulation was not removed, which might be the reason for recurrent relapses.     

Evidence of shared Epstein-Barr viral isolates between sexual partners, and low level EBV in genital secretions

Epstein-Barr virus is present in the saliva of most persistently infected individuals and is generally thought to be spread by close oral contact. However, there are now several reports of EBV in genital secretions, suggesting the possibility of sexual transmission between adults. The present study was undertaken to investigate the risk of sexual transmission of EBV. PCR analysis was used to examined the degree to which a group (n = 11) of patients with infectious mononucleosis (IM) shared the same viral isolates as their sexual partners, and compare this to the extent of isolate sharing among a different group (n = 18) of IM patients and their non-sexual contacts. There was significantly more sharing of EBV isolates among the IM/sexual-contact pairs than among the IM/non-sexual-contact pairs (P = 0.0012). Female cervical (n = 84), male urethral (n = 55), and semen (n = 30) samples from asymptomatic, unselected volunteers were analyzed for the presence of EBV DNA, revealing 7%, 5%, and 3% to be EBV positive, respectively. Fractionation of cervical and urethral samples into cellular and supernatant fluid components showed EBV to be mainly cell-associated. Quantitation of EBV in these samples gave levels of below 10 EBV genomes per microg of DNA. Overall the findings support the possibility that EBV could on occasions be transmitted sexually, however, the low levels detected in genital secretions compared to saliva suggest that this is not a major transmission route. The finding of small quantities of cell-associated virus suggests a latent infection; thus EBV is probably in the B lymphocyte rather than in the epithelial cell component of the secretions.     

The variability of the serological response to SARS‐corona virus‐2: Potential resolution of ambiguity through determination of avidity (functional affinity)

Data on the serological response toward severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) in 16 recent reports were analyzed and a high degree of variability was shown. Immunoglobulin M (IgM) responses were either found earlier than IgG, or together with IgG, later than IgG, or were missing. Therefore, clear distinctions between early, intermediate, and past infections are obviously not possible merely on the basis of IgM and IgG determinations. A review of publications on the serology of other virus groups shows that variable IgM responses can be found as well and therefore are not unique for SARS‐CoV‐2 infections. A model to explain this variability is proposed. The inclusion of avidity determination into regular diagnostic procedures has allowed to resolve such “atypical” serological constellations. The potential use of avidity determination for the diagnosis of COVID‐19, for risk assessment, epidemiological studies, analysis of cross reactions, as well as for the control of vaccination programs is suggested and discussed.     

Improved diagnosis of primary Toxoplasma gondii infection in early pregnancy by determination of antitoxoplasma immunoglobulin G avidity.

The ability to discriminate between primary Toxoplasma gondii infection acquired in early pregnancy and infection that occurred prior to pregnancy was assessed by an enzyme immunoassay (EIA) to determine the avidity of toxoplasma-specific immunoglobulin G (IgG). The results were compared to those of the Platelia Toxo-IgM EIA and the dye test. The mean IgG avidity of 73 serum samples collected within 20 weeks after the estimated time of infection was 5.9%. Among 26 serum samples showing latent infection (toxoplasma-specific IgG positive and IgM negative) and 56 IgM-positive serum samples with a low dye test titer (<300 IU/ml), the mean avidities were 51.3 and 57.5%, respectively. A total of 72.8% of 92 IgM-positive serum samples with a high dye test titer (>300 IU/ml), suggesting a recent toxoplasma infection, had an IgG avidity of >20%, indicating that the infection started more than 20 weeks earlier. By introducing high IgG avidity as a criterion in the first half of pregnancy to exclude the possibility that toxoplasma infection was acquired during gestation, many women will avoid unnecessary examinations, treatment, and anxiety.     

Alternatives to the tuberculin skin test: interferon-gamma assays in the diagnosis of mycobacterium tuberculosis infection

For nearly a century, there were no alternatives to the tuberculin skin test (TST) for diagnosing latent tuberculosis infection. Because of advances in immunology and genomics, for the first time, an alternative has emerged in the form of T cell based interferon-g (IFN-gamma) assays, a new generation of in vitro tests of cellular immunity. These assays measure cell mediated immune response by quantifying IFN-gamma released by T cells in response to stimulation by Mycobacterium tuberculosis antigens. Although early versions of IFN-gamma assays used purified protein derivative (PPD) as the stimulating antigen, newer versions use antigens that are significantly more specific to M. tuberculosis. These specific antigens include ESAT-6 and CFP-10. These proteins, encoded by genes located within the region of difference 1 (RD1) segment of the M. tuberculosis genome, are more specific to M. tuberculosis than PPD because they are not shared with any BCG substrains or several nontuberculous mycobacterial species. A review of current evidence on the performance of IFN-gamma assays and TST suggests that both the TST and IFN-gamma assays have advantages and limitations, and both tests appear to be useful at this time. The emergence of IFN-gamma assays is a much anticipated, welcome development that has, for the first time, increased the choice of tests available for diagnosing latent tuberculosis infection. Because both tests have their strengths and limitations, the decision to select one over the other will depend on the population, the goal of testing, and the resources available. To fully evaluate the utility of IFN-gamma assays in high burden countries such as India, long-term cohort studies are needed to determine the association between positive IFN-gamma results and the subsequent risk of active disease.     

Characterization of the expanded T cell population in infectious mononucleosis: apoptosis, expression of apoptosis-related genes, and Epstein-Barr virus (EBV) status

Infectious mononucleosis (IM), a manifestation of primary infection with EBV, is characterized by a massive expansion of the T cell population. In this study we examined this expanded T cell population regarding its EBV status, its proliferative and apoptotic activity, and its expression of apoptosis-related genes. Whereas previous studies were performed on ex vivo cultures or on peripheral blood, our investigations included in vivo analysis of IM tonsillectomy specimens (14 cases) by in situ hybridization for viral RNA (EBERs) combined with immunohistochemistry (IHC; CD3, CD45RO, CD20, CD79a, Ki-67, Bcl-2, Bax, Fas, FasL) and the TUNEL method. Of the EBER+ cells 50-70% showed expression of the B cell markers CD20/CD79a. The remainder of the EBER+ cells expressed neither B nor T cell antigens. No co-expression of EBERs and T cell antigens was detected in any of the specimens. In accordance with a high rate of apoptosis (up to 2.37%) within the expanded T cell population, Bcl-2 expression was drastically reduced and FasL expression remarkably increased. The levels of Bax and Fas expression showed no or moderate up-regulation. In conclusion, the massive expansion of IM T cells is not caused by EBV infection of these cells but merely represents an intense immune reaction. Through altered expression of Bcl-2/Bax and Fas/FasL, the activated T cells are subject to enhanced apoptosis while residing within the lymphoid tissue, which eventually allows the efficient silencing of this potentially damaging T cell response.