Performance of a Cytomegalovirus IgG Enzyme Immunoassay Kit Modified To Measure Avidity

Abstract

bThe measurement of cytomegalovirus (CMV) IgG avidity accurately discriminates recent and past CMV infections. We sought to determine if the Wampole Laboratories CMV IgG enzyme immunoassay (EIA) could be modified to measure avidity. The evaluation panel consisted of 156 serum samples we used in 2002 to validate a laboratory-developed EIA, in which 78 serum samples exhibited low avidity, 7 exhibited intermediate avidity, and 71 exhibited high avidity. The qualitative agreement between the two avidity assays was 94% (147/156); all 9 sera with discordant results exhibited intermediate avidity in one of the assays. The avidity index (AI) values in the two assays showed excellent correlation (r ‫؍‬ 0.96, P < 0.0001). The definition of high avidity was verified for the Wampole assay by demonstrating high avidity in 91/93 (98%) recently collected CMV IgG-positive/IgM-negative serum samples. The performance of the Wampole avidity assay in a reference laboratory setting was assessed using 470 consecutive serum samples submitted for CMV IgG avidity testing. Surprisingly, 101 serum samples were negative when screened for CMV IgG using the Wampole kit per the package insert; 98 of these 101 serum samples were tested using a CMV IgG chemiluminescent immunoassay, and only 5 were positive. Of the 369 CMV IgG-positive samples, 6% exhibited low IgG avidity, 6% exhibited intermediate avidity, and 88% exhibited high avidity; CMV IgM detection rates were inversely related to AI levels. These findings show that (i) the Wampole CMV IgG EIA can be modified to measure CMV IgG avidity, (ii) many samples are apparently submitted for avidity testing without knowledge of their CMV IgG status, and (iii) most CMV IgG-positive sera submitted for avidity testing exhibit high avidity. T he accurate discrimination of primary cytomegalovirus (CMV)infection from reactivation or reinfection plays an important role in the clinical management of pregnant women (1-5). Primary CMV infection during pregnancy may lead to intrauterine infection of the fetus, which is associated with hearing loss, blindness, and mental retardation (6-8). In contrast, CMV reactivation or reinfection during pregnancy is rarely associated with intrauterine fetal infection (2, 7). The accurate identification of primary CMV infection is also important in managing solid organ transplant recipients, who exhibit an increased risk of organ rejection following primary CMV infection (9-11).Over the last decade, the assessment of CMV IgG avidity has become the preferred laboratory tool for identifying patients with primary CMV infection (4, 5). IgG avidity gradually matures over time, so that IgG detected by 5 to 6 months after a primary infection exhibits high avidity; thus, a low CMV IgG avidity result is a strong indicator of primary infection within the preceding 5 to 6 months (2, 3, 6, 12, 13). CMV IgG avidity is more accurate than CMV IgM detection for identifying primary infection, since CMV IgM persists for Ͼ6 months in some patients (2,7,14).In 2002, we validated a laboratory...

Role of Cytomegalovirus (CMV) IgG Avidity Testing in Diagnosing Primary CMV Infection during Pregnancy

“…This information, in conjunction with the CMV avidity maturation timeline discussed earlier, suggests that patients with IgM persistence will exhibit an IgM-positive but high-CMV-avidity result set if tested more than 5 to 6 months postinfection. This result pattern is common among patients being evaluated for possible primary CMV infection; core study groups found that about 50% of CMV IgM-positive at-risk patients had high CMV IgG avidity (27,28,33), a percentage confirmed by other investigators (13,15,22). Thus, some pregnant women with an IgM-positive/high-IgG-avidity result set may actually have nonrecent primary CMV infection in a setting of IgM persistence, rather than viral reactivation/reinfection.…”

“…Antigen-bound low-avidity IgG, but not high-avidity IgG, dissociates from the antigen in the presence of mild protein denaturants, such as urea, potassium thiocyanate, and guanidine chloride. The most common test format is an enzyme-linked immunosorbent assay (ELISA) utilizing urea as the dissociating agent (14,21,22). The patient's diluted serum is added to two microtiter wells coated with CMV antigen; after incubation, one well is washed with regular wash buffer, whereas the other well is washed with wash buffer containing urea.…”

“…The AI cutoff point for defining low avidity is typically 35% to 50%, whereas the AI cutoff point for defining high avidity is typically 50% to 65%. For example, in the assay used in our laboratory (22), low avidity is defined as an AI value of Յ50% and high avidity as an AI value of Ն60%; AI values of 51% to 59% are considered intermediate avidity.…”

Potential Impact of Different Cytomegalovirus (CMV) IgM Assays on an Algorithm Requiring IgM Reactivity as a Criterion for Measuring CMV IgG Avidity

“…The study utilized 369 consecutive CMV IgG-positive serum samples submitted to the Focus Diagnostics Reference Laboratory for CMV IgG avidity testing (16); 91% of these samples were supplied by women of childbearing age (15 to 49 years old), but no clinical data were available for any of the patients who supplied the serum samples. Following release of the avidity results, the samples were deidentified before the various IgM assays were performed.…”

“…The serum samples were tested for CMV IgG avidity using a commercially available CMV IgG EIA kit (Wampole Laboratories, Princeton, NJ) with modifications as described in the accompanying article (16). Briefly, a given sample was diluted per the package insert and added to two microtiter wells; after incubation, one well was washed with kit-supplied wash buffer and the other well was washed with dissociating buffer (wash buffer containing 6 M urea).…”