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Clinical and Vaccine Immunology, April 2009, p. 587-588, Vol. 16, No. 4
1071-412X/09/$08.00+0     doi:10.1128/CVI.00462-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Frequency of Missed Cases of Probable Acute West Nile Virus (WNV) Infection when Testing for WNV RNA Alone or WNV Immunoglobulin M Alone{triangledown}

Harry E. Prince,* Jan Calma, Tiffany Pham, and Brent L. Seaton

Focus Diagnostics, Inc., Cypress, California 90630

Received 8 December 2008/ Returned for modification 11 January 2009/ Accepted 9 February 2009


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ABSTRACT
 
To estimate the frequency of missed cases of acute West Nile virus (WNV) infection if only WNV RNA or immunoglobulin M (IgM) testing is requested, we measured IgM in specimens negative for RNA and vice versa. Whereas 6 (5.5%) of 110 RNA-negative sera were IgM positive, only 3 (1.0%) of 299 IgM-negative sera were RNA positive (P < 0.05). Similarly, 11 (7.8%) of 141 RNA-negative cerebrospinal fluid specimens (CSF) were IgM positive, but 0 (0%) of 118 IgM-negative CSF were RNA positive (P < 0.05). WNV infections may be missed if only RNA or IgM testing is requested, with a higher frequency of missed cases if only RNA testing is requested.


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TEXT
 
Acute West Nile virus (WNV) infection remains a serious public health issue in the United States, with >1,300 cases reported to the Centers for Disease Control and Prevention (CDC) in 2008 (2). As recommended by the CDC (3), WNV immunoglobulin M (IgM) detection in serum or cerebrospinal fluid (CSF) is the major laboratory tool used to identify symptomatic individuals with acute WNV infection; the vast majority of acutely infected individuals are positive for WNV IgM at the time they first seek medical attention (4, 10). In addition, WNV RNA detection has emerged as another useful laboratory tool for identifying patients with acute WNV infection; although of limited utility due to the short viremic phase and low viral load (6, 7), the RNA assay may be the only test with a positive result for WNV-infected patients seeking medical attention very soon after symptom onset (6, 11). Individuals presenting with acute WNV infection may thus be positive for WNV IgM and RNA, WNV IgM only, or WNV RNA only. This finding raises concerns about the frequency of missed cases of acute WNV infection if only one of these tests is requested and the result is negative; in this situation, WNV infection may be incorrectly ruled out. We therefore sought to estimate the frequency of missed probable cases of WNV infection if only WNV IgM testing or only WNV RNA testing is requested.

The serum and CSF specimens utilized for this study were submitted to Focus Diagnostics, Inc., Cypress, California, by other laboratories for WNV RNA or WNV IgM testing during the 2008 North American WNV season; clinical information (e.g., time since symptom onset) was not provided for any of the samples. Specimens included 110 sera and 141 CSF samples submitted for RNA testing and found to be RNA negative, as well as 299 sera and 118 CSF samples submitted for IgM testing and found to be IgM negative. After the requested test was performed, specimens were deidentified and stored at or below –20°C for up to 2 weeks before further testing was performed.

WNV IgM was assayed using an enzyme-linked immunosorbent assay kit (5, 8) per the instructions of the manufacturer (Focus Diagnostics). This kit is FDA cleared for the testing of serum specimens only; in-house studies validated the kit for CSF testing (9). Index values of >1.1 were considered positive.

Nucleic acid extraction was performed using the MagNA Pure total nucleic acid isolation kit (Roche Applied Science, Indianapolis, IN) on the MagNA Pure liquid chromatograph (Roche Applied Science) automated extraction platform. A starting specimen volume of 200 µl was extracted and eluted into a final volume of 50 µl. All eluates were assayed using 10 µl of extracted DNA or RNA as a template. TaqMan real-time reverse transcription-PCR (6) was used to amplify and detect a 121-nucleotide sequence of the WNV genome that flanks the NS1 and NS2a genes.

Our findings are summarized in Table 1. Of 110 serum samples submitted for WNV RNA testing and found to be RNA negative, 6 (5.5%) were positive for WNV IgM. In contrast, of 299 serum samples submitted for WNV IgM testing and found to be IgM negative, only 3 (1.0%) were positive for WNV RNA. This difference in proportions was statistically significant, with a P value of 0.019 (significance was defined by a P value of <0.05). Similar findings were obtained for CSF samples; 11 (7.8%) of 141 CSF samples submitted for RNA testing and found to be RNA negative were positive for WNV IgM, whereas 0 (0.0%) of 118 CSF samples submitted for IgM testing and found to be IgM negative were positive for WNV RNA (P = 0.005).


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  		TABLE 1. Frequency of detection of WNV RNA or IgM in samples submitted for measurement of the other analyte

These results demonstrate that probable cases of acute WNV infection may be missed if either WNV RNA testing alone or WNV IgM testing alone is requested. Further, the likelihood of missing acute WNV cases is higher if only RNA testing is requested, particularly for CSF. These findings are consistent with our understanding of the timelines for WNV viremia and antibody production (1, 4, 7, 10). Levels of WNV RNA in serum typically peak before symptoms appear and then rapidly decline over several days as antibody production begins (1). By the time patients seek medical attention, RNA levels are often below detectable levels whereas IgM (and often IgG) is present at easily detectable levels (4, 7, 10). However, some patients, particularly those seeking medical attention within a week of symptom onset, may still be in the RNA-positive/antibody-negative window (11). Thus, to avoid missing cases of acute WNV infection, it may be appropriate to request both WNV RNA testing and WNV IgM testing, depending on the history the patient relates upon presentation and the specimen type(s) selected for testing.


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FOOTNOTES
 
* Corresponding author. Mailing address: Focus Diagnostics, Inc., 5785 Corporate Avenue, Cypress, CA 90630. Phone: (714) 822-2457. Fax: (714) 821-3364. E-mail: hprince{at}focusdx.com Back

{triangledown} Published ahead of print on 18 February 2009. Back


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Clinical and Vaccine Immunology, April 2009, p. 587-588, Vol. 16, No. 4
1071-412X/09/$08.00+0     doi:10.1128/CVI.00462-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.




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