Concordance between Anti-CMV IgM ELISA and IgG Avidity testing of serum immunoglobulin in FUO patients

INTRODUCTION

Fever is a main ailment for which people need medical attention. Fortunately, fever is usually self-limiting, or the cause of fever can be determined. Fever of unknown origin (FUO) is described as a chronic, unexplained fever that persists despite a thorough examination. It is a common clinical problem. ¹

Fever of unknown origin (FUO) is considered as a fever of 101°F (38.3°C) or higher that remains unidentified after 3 days and for at least 2 days after microbiological culture incubation ².

Infectious, malignant/neoplastic,autioimmune  and miscellaneous other illnesses are the four etiologic categories of FUOs. About  a third of FUO cases are caused by infection. Infection with the human herpesvirus cytomegalovirus (CMV) is a frequent in healthy adults and children. Seroprevalence studies have found that in healthy people, the prevalence ranges from 50% to 90%, with the prevalence increasing with age.³

CMV infection is frequently asymptomatic or causes mild flu-like symptoms. A primary CMV infection could be the source of an unexplained fever.⁴

CMV is a common causative pathogen that causes serious morbidity and mortality in immunosuppressed individuals. Infection can cause a feotus to develop a symptomatic congenital illness if it occurs during pregnancy.
In immunocompetent people, however, initial CMV infection is generally asymptomatic or causes just a moderate illness. Recent illnesses in adults are frequently reported as mononucleosis.⁵

Anti-CMV IgM antibodies can last for months or even years following the original illness. In addition, they can also be detected after reactivation or reinfection with a new strain of CMV, making anti-CMV IgM's accuracy in predicting primary infection more difficult. ⁶

Furthermore, false-positive CMV IgM findings can arise during non-CMV diseases like primary Epstein-Barr virus infection. Furthermore, investigations have revealed that CMV IgM tests have a wide range of specificity and sensitivity, as well as a significant percentage of discordance.⁷

Antibody avidity, an effective indicator of how strongly an antibody attaches to its antigen of interest, has recently been discovered to rise in the weeks following initial infection. Low-avidity IgG antibodies to CMV last for up to 20 weeks following a primary CMV infection.  After that, high-avidity antibodies replace the low-avidity ones. ^{8 - 9}

The anti-CMV IgG avidity test is currently an important and robust test to determine primary CMV illness. During an acute or recent primary CMV infection, a low avidity index demonstrates the existence of low-avidity IgG antibodies in the serum. A high avidity index could be regarded as a reliable sign of previous infection. ²

In this study, we sought to evaluate the compatibility between the detection of serum anti-CMV IgM antibodies by ELISA and the results of the IgG avidity testing in patients with FUO, as well as to identify the possible frequency of CMV illness in FUO patients.

PATIENTS AND METHODS

This is a retrospective cross-sectional study that was performed on 170 cases that were presented with FUO and admitted to the inpatient section of a military fever hospital from May to November 2018. A detailed history has been taken, including personal data, the presenting complaint, and general and neurological symptoms. A thorough clinical examination, including general and neurological examination, was performed. Routine laboratory investigations include complete blood counts, liver function tests, renal function tests, and urine analysis. Routine imaging includes chest X-rays and abdominal ultrasonography. 56 patients were subjected to CMV IgG, IgM, and IgG avidity, and their age ranged between 16 and 46 years, with a mean age of 29.21 ± 7.55 years. 52 cases were males (92.9%) and 4 were females (7.1%)

The patients were enrolled according to the following criteria:

Inclusion criteria: Immune-competent Patients who have been hospitalised because they have fever > 38.3 for more than 7 days of unknown origin after performing routine investigations after hospitalization

Exclusion criteria: Patients aged ≤ 18 and ≥60, Patients with end-organ failure, Patients with absolute neutropenia and/or absolute leucopenia or any evidence of immune suppression, Patients with a history of immunosuppressive drugs, Patients with autoimmune disorders, Patients with malignancy, even during remission

Study design: Immune-competent Patients who were hospitalized with a fever greater than 38.3 for more than 7 days of unknown origin were identified after routine investigations.

Patients who were to be accepted in this study were selected after obtaining the ethical approval of the military fever hospital and El-Hussein University hospital. The effectiveness of the CMV avidity test using the fully automated Vidas analyzer (bioMérieux) as a function of the concentration of CMV-specific IgG in the patient's blood was investigated. ¹⁰ 56 patients were studied employing a modified ELISA test using an ELFA (enzyme-linked fluorescent assay) phase and immune concentration technology for the detection of CMV antibodies IgG, IgM, and IgG avidity.

The VIDAS is an automated instrument that performs an enzyme-linked fluorescent immunoassay (ELFA). The instrument controls all assay steps and assay temperature. Each CMV assay kit contains controls, standards, reagent strips, and SPR (Solid Phase Receptacle). After a sample dilution step, the sample is cycled in and out of the SPR for a specified length of time. Anti-CMV antibodies present in the specimen will bind to the purified CMV antigen coating the interior of the SPR. Unbound sample components are washed away. A monoclonal anti-human IgG conjugated with alkaline phosphatase is cycled in and out of the SPR and will attach to any human IgG bound to the SPR wall. A final wash step removes unbound conjugate. A fluorescent substrate, 4-methylumbelliferyl phosphate, is introduced into the SPR. Enzyme remaining on the wall of the SPR will catalyze the conversion of the substrate to the fluorescent product, 4-methylumbelliferone (450 nm). The intensity of the fluorescence is measured by the optical scanner in the instrument; it is proportional to the quantity of CMV IgG found in the sample. When the VIDAS CMV Assay is completed, the results are analyzed automatically by the computer. The quantity of anti-CMV IgG present in the sample is calculated in reference to a calibration curve stored in the instrument. A report is printed for each sample.

IgG antibodies with avidities of 50% were termed low avidity antibodies, those with avidities of 50.0 and 59.9% were regarded grey as zone antibodies, and those with avidities of 60% or more were termed high avidity antibodies ⁽²⁾.

Statistical analysis: The data were revised and analyzed using version 24 of the SPSS software package. The mean and standard deviation (SD) were used to express quantitative data. The frequency and proportion of qualitative data were calculated. Statistical analysis was done using the Mann–Whitney U test. The Fisher's extract was used to compare qualitative data and the Chi-square test to compare quantitative data.

A P-value of less than 0.05 was considered statistically significant.

Cohen's kappa is a measure of the agreement between two ratters who have recorded a categorical outcome for a number of individuals. Cohen's kappa factors out agreement due to chance and the two ratters either agree or disagree on the category that each subject is assigned to (the level of agreement is not weighted). Cohen's kappa is a metric often used to assess the agreement between two raters. It can also be used to assess the performance of a classification model .The Kappa statistic is calculated using the following formula:

A kappa value of 0.57 indicates moderate to good agreement between observers

RESULTS

This is a retrospective cross-sectional study that was performed on 170 cases that were presented with FUO and admitted to the inpatient section of a military fever hospital. 56 patients were tested to evaluate the IgG, IgM, and IgG avidity tests, and their age ranged between 16 and 46 years, with a mean age of 29.21 ± 7.55 years. 52 cases were males (92.9%) and 4 were females (7.1%). There is no important data in this table in terms of demographic outcomes (Table 1).

Table 1: Demographic analysis of results.

The results of complete blood counts, liver function tests, renal function tests, and urine analysis in relation to the CMV IgM positive and negative patients are presented in (Table 2). The blood transaminases and bilirubin levels of CMV IgM-positive patients are significantly elevated (Table 2).

Table 2: Comparison between CMV IgM positive and negative patients regarding laboratory investigations.

Out of 56 patients tested to evaluate the CMV IgM and IgG avidity tests, 10 patients (17.9%) were CMV-IgM positive, 6 patients (60%) were CMV low avidity, and the remaining 4 patients (40%) were CMV high avidity, which indicates that most patients with CMV IgM positive tests were significant low avidity tests (Table 3(. Hepatomegaly (20%) and hepatospienomegaly (20%) were highly significant imaging findings in patients with CMV low avidity (Table 3).

Table 3: Evaluation of CMV IgM positive patient according to specific laboratory investigation and imaging.

17.9% of patients were positive as regards the CMV IgM test, and 21.4% of patients were positive as regards the CMV avidity test. The degree of concordance between CMV IgM positive patients and CMV avidity according to kappa agreement was 44% (Table 4).

Table 4: Concordance of CMV IgM positive patient and CMV avidity according to kappa agreement.