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Previous Article | Next Article 
Clinical and Diagnostic Laboratory Immunology, September 2005, p. 1123-1126, Vol. 12, No. 9
1071-412X/05/$08.00+0 doi:10.1128/CDLI.12.9.1123-1126.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Focus Diagnostics, Cypress, California,1 Blood Systems Research Institute, San Francisco, California,2 American Red Cross Blood Services, Gaithersburg, Maryland3
Received 21 April 2005/ Returned for modification 24 May 2005/ Accepted 15 June 2005
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In other viral infections, IgG avidity has proven to be a valuable tool for distinguishing recent from past infection (1, 2, 8-10, 12, 13, 15). Defined as the strength with which IgG attaches to antigen, IgG avidity matures with time following primary infection (2). When assessing cytomegalovirus (CMV) IgG avidity in pregnant women, for example, a high avidity result for a sample collected during the first trimester indicates that primary infection most likely occurred prior to conception, with little chance of CMV infection of the fetus (1, 10). Within the flavivirus family, dengue virus IgG avidity has proven useful for discriminating primary from secondary infections (6). We thus investigated the utility of WNV IgG for distinguishing recent from past infection. These studies utilized a panel of follow-up specimens collected from WNV-infected blood donors identified by WNV RNA detection (5). The viremic (RNA-positive) period begins within a few days of exposure and typically lasts about 20 days (3); WNV viremia is thus an excellent indicator of infection within the previous 4 weeks.
Specimens. WNV RNA-positive blood donors were identified by nucleic acid amplification test screening of donations in 2003 and 2004 (4, 5). Plasma from donations confirmed as WNV RNA positive (hereafter referred to as the index donations), as well as plasma or serum specimens collected during follow-up visits, was supplied by the Blood Systems Research Institute and American Red Cross Blood Services. Informed consent was obtained from all donors at the local blood donation site; protocols for nucleic acid amplification test screening and follow-up were approved by local institutional review boards and the Food and Drug Administration.
Methods. Specimens were tested for WNV IgG using a Food and Drug Administration-cleared enzyme-linked immunosorbent assay kit (Focus Diagnostics, Cypress, CA) (7, 11). IgG avidity was measured using this same enzyme-linked immunosorbent assay kit with a modified procedure (10). IgG-positive serum or plasma specimens were diluted per the package insert and added to duplicate microtiter wells. After an hour at room temperature, the well contents were discarded. Kit wash buffer was then added to one of each pair of duplicate wells, whereas dissociating buffer (kit wash buffer containing 6 M urea [ICN, Aurora, Ohio]) was added to the other well. After 5 min at room temperature, the well contents were discarded and the wash procedure was repeated (including the 5-minute incubation step). All wells were washed once more with kit wash buffer; the assay was then completed as described in the kit insert, and absorbance at 450 nm was measured. For a given specimen, the avidity ratio (AR) was calculated by dividing the absorbance value obtained for the well washed with urea buffer by the absorbance value obtained for the well washed with kit wash buffer.
Findings. Two samples with markedly different AR values (0.20 and 0.69) in the first avidity assay run were included in all 11 subsequent runs. The mean AR ± standard deviation over these 12 assay runs was 0.17 ± 0.02 for the first sample and 0.68 ± 0.04 for the second sample; the interassay coefficient of variation values were thus 12% and 6%, respectively. Intra-assay coefficient of variation values (eight replicates tested within a single assay run) were 6% and 4%, respectively.
Figure 1 shows the distribution of WNV IgG avidity values for 348 follow-up specimens from 170 viremic blood donors, plotted as a function of days postindex. Two major observations were apparent from visual examination of Fig. 1: (i) nearly all samples collected >90 days postindex exhibited AR values of 
0.50, and (ii) nearly all samples with AR values of <0.50 were collected 
90 days postindex. Based on these findings, an AR of 0.50 was defined as high avidity and an AR of <0.50 was defined as low avidity. The somewhat unexpected finding evident in Fig. 1 was that many samples collected 90 days postindex exhibited high avidity. The exact numbers of samples in the four quadrants evident in Fig. 1 were as follows: 95 high-avidity samples collected 90 days postindex, 61 high-avidity samples collected >90 days postindex, 3 low-avidity samples collected >90 days postindex, and 189 low-avidity samples collected 90 days postindex. Thus, of 64 samples collected >90 days postindex, 61 (95%) exhibited high WNV IgG avidity. Of 192 samples with low WNV IgG avidity, 189 (98%) were collected 90 days postindex. These 189 low-avidity specimens represented 67% of the 284 specimens collected 90 days postindex; i.e., 33% (95/284) of samples collected during this time frame exhibited high WNV IgG avidity.
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A cluster of 41 specimens with high WNV IgG avidity collected during the first 25 days of follow-up is evident in Fig. 1. The WNV IgG status of the index donations from the 20 donors contributing these 41 follow-up specimens was investigated. Ten index donations were WNV IgG negative, five were IgG positive with low avidity, and five were IgG positive with high avidity; thus, donors contributing early follow-up samples with high IgG avidity were heterogeneous with respect to the WNV IgG result of the index specimen.
We next asked if using dissociating buffer containing more urea would distinguish early-follow-up high-avidity samples from late-follow-up high-avidity samples. Seven samples with AR values of >0.70 collected <25 days postindex and seven samples with similar AR values collected >140 days postindex were retested in parallel using routine dissociating buffer (6 M urea) and 8 M urea as dissociating buffer. As expected, the distributions of AR values obtained using 6 M urea were similar in the early-follow-up and late-follow-up groups (Fig. 3); likewise, the distributions of AR values obtained using 8 M urea were similar in the two groups. When the group means obtained using a given urea concentration were compared, no significant differences were observed (data not shown). Thus, more stringent dissociating conditions did not distinguish early-follow-up high-avidity samples from late-follow-up high-avidity samples.
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Surprisingly, 33% of follow-up samples collected within 90 days of the index specimen exhibited high IgG avidity. Particularly noteworthy was a cluster of high-avidity specimens collected 25 days postindex. We hypothesized that these specimens were collected from the small percentage of viremic blood donors whose index specimens already contained WNV IgG; however, only half the donors contributing early-follow-up samples with high avidity were IgG positive at index. Next we hypothesized that early-follow-up and late-follow-up samples with similar high avidities when using 6 M urea would become distinguishable (i.e., early-follow-up sample avidities lower than late-follow-up sample avidities) using 8 M urea as dissociating buffer. However, the experimental results did not support this hypothesis; samples with similar AR distributions using 6 M urea also showed similar AR distributions using 8 M urea. Taken together, these findings indicate that WNV IgG avidity matures very rapidly in some donors following exposure to the virus. The explanation for this phenomenon remains unknown; one possibility is that these individuals were exposed to another flavivirus in the past and produce high-avidity WNV IgG as part of an anamnestic response to common flavivirus antigens. Further studies measuring neutralizing antibodies to a battery of flaviviruses in index and follow-up specimens are required to test this hypothesis; insufficient sample volumes precluded our conducting such studies using the existing specimen panel.
Measurement of WNV IgG avidity could be a useful tool for determining if WNV infection should be considered in the differential diagnosis for selected patients from areas of endemicity. For example, an elderly South Dakota resident presents in September with recent onset of fever and altered mental status; serologic tests detect both WNV IgM and WNV IgG, but the WNV IgM index of 1.93 (reference range of <0.90) is lower than typically observed in patients with recent WNV infection. A low WNV IgG avidity result would provide strong support for recent infection, indicating that WNV neuroinvasive disease should be included in the differential diagnosis. However, a high WNV IgG avidity value would not be useful for determining the time since WNV infection. The clinical utility of WNV IgG avidity data thus differs from the clinical utility of avidity results for other viral infections, where high avidity essentially excludes the possibility of recent infection. It will be important to ensure that clinicians and laboratorians understand this difference so that WNV IgG avidity results are appropriately interpreted.
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| Antimicrob. Agents Chemother. | Clin. Microbiol. Rev. | Infect. Immun. |
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| J. Clin. Microbiol. | J. Virol. | ALL ASM JOURNALS |
