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Development of a novel, rapid, and sensitive immunochromatographic strip assay specific for West Nile virus (WNV) IgM and testing of its diagnostic accuracy in patients suspected of WNV infection.

The West Nile virus (WNV), detected in the Western hemisphere in 1999, has since spread rapidly across North America into all 48 continental states of the US, 7 Canadian provinces, many Latin American countries, and throughout Mexico. More than 24 000 people in the US have tested positive for WNV infection; 9845 of these cases resulted in serious neuroinvasive disease and more than 959 were fatal (1). Guidelines for surveillance, prevention, and control of WNV are available (2).

WNV, a member of the Flaviviridae family, belongs to the Japanese encephalitis serocomplex that includes Japanese encephalitis and St. Louis encephalitis viruses (3). WNV is transmitted to humans by mosquitoes. The transmission cycle involves mosquitoes and birds. In rare instances, the virus may be transmitted from human to human through organ donation or blood transfusion or from pregnant mother to fetus (4-6). WNV infections in a pediatric population have also been reported (7). Many patients remain asymptomatic or have only mild symptoms. Others report having fatigue, rash, fever, headache, and muscle weakness and may require hospitalization (8). The more severe form of the disease is meningoen-cephalitis manifested by typical signs of central nervous system infection and, in a minority of cases, development of a flaccid paralysis (9).

Symptomatic patients will demonstrate an early antibody response of the IgM type during the 1st 4 days of illness, and nearly all patients will have detectable IgM antibodies by 7 to 8 days after the onset of illness (10). WNV-specific serum IgG is detectable by 3 weeks post-infection (11). The virus itself is usually no longer detectable by the time WNV-specific serum IgM appears, although both IgM and IgG may persist for more than a year (12). Laboratory-based ELISAs designed to detect WNV-specific IgM are now accepted as a sensitive early marker for WNV infection (13-15). However, current ELISA assays (e.g., Focus Technologies West Nile Virus IgM Capture ELISA, PanBio) are multistep procedures that require 5-6 h to perform, instrumentation to read the tests, and subsequent calculations to interpret the test results. We describe the development of a novel and a rapid WNV IgM strip test (RapidWN[TM]) that requires minimum sample preparation and produces results visibly in 15 min. We show that such results are substantially equivalent to currently used ELISA devices (e.g., CDC WNV IgM ELISA, Focus Technologies West Nile Virus IgM Capture ELISA).

Both the ELISA and the strip format assay use the same principle and the same WNV antigen (suckling mouse brain WNV protein E or recombinantly produced WNV protein E fraction) and flavivirus-specific monoclonal antibody 6B6C-1. The RapidWN uses solid-phase immuno-chromatographic strip technology to qualitatively detect the presence of WNV IgM antibodies in human serum or plasma. The test uses a fragment of recombinant WNV envelop protein (antigen) that was expressed in Escherichia coli, purified, and characterized. Antibody 6B6C-1, provided by the CDC as a hybridoma cell line, was cultured and purified. Goat-antihuman IgM (Jackson IRL) was biotinylated (biot-[alpha]h-IgM) and 6B6C-1 antibody was conjugated onto colloidal gold particles (detector). All 3 reagents were then dispensed onto the polyester pad and lyophilized. Streptavidin was recombinantly produced and immobilized onto a nitrocellulose (Sartorius) membrane strip at the test-band site. Rabbit antimouse IgG-Fc was immobilized at the control-band site.

When the specimen is dispensed into the sample well, it passes through the membrane, which contains antigen, detector, and biot-[alpha]h-IgM antibodies. The WNV IgM in the patient sample then forms a tertiary detector/antigen/IgM complex. The formed complex then migrates through the reaction strip and is captured at the test area. Excess, unreacted detector flows through the strip and is captured in the control area. The reactant concentrations are adjusted and optimized by analysis of calibrators, made from pooled WNV-positive sera, so that the test should produce a positive signal at WNV IgM Index value [greater than or equal to] 1.1 of an commercially available Focus Technologies West Nile Virus IgM Capture ELISA device (comparator device) and negative results below that number. Examples of positive, negative, and invalid test results are shown in Fig. 1.

Visible pinkish-purple horizontal bands appear in the test area if the concentration of the WNV IgM antibodies in the human serum sample is above the cutoff concentration in relation to the comparator device. A pinkishpurple band in the control area indicates that the test is working properly, and such a band must always appear, irrespective of the WNV IgM concentration, for the test to be considered valid.

RapidWN test precision studies were performed using a reproducibility panel made of different clinical serum specimens at 3 study sites during a 3-day period. Each day a new lot was used and a different operator was used. This 15-member reproducibility panel (blinded) consisted of 6 clinical specimens with a mean index value 25% above the cutoff, 6 clinical specimens with a mean index value 15% below the cutoff, and 3 clinical specimens with a mean index value 6 times the cutoff of the comparator device. All sites and operators produced the expected result for all panel members on every day of testing. Near-cutoff positive and negative samples also produced expected results when tested by 3 operators on the same lot for 10 consecutive days.

No significant interferences were found with common serum analytes such as human serum albumin, bilirubin, hemoglobin, or triglycerides at 2 to 3 times the normal expected values. No cross-reactivity with Japanese, Saint Louis, California, or Eastern equine encephalitis viruses was observed, but some cross-reactivity (10%) with Dengue virus was noted.

Reactivity with autoantibodies, such as antinuclear antibodies and rheumatoid factor, is common to all immunoassays (16), and the RapidWN test is not an exception. Care has been taken to block such activities at heterophile antibody titers commonly encountered in a population, but rare samples with higher titers may affect the test.

Clinical specificity of the RapidWN test was assessed on prospective specimens (n = 346) after institutional review board approval at 3 study sites from endemic regions of the US and Canada. Samples were from presumably healthy individuals (n = 67) or patients with non-WNV ailments (n = 279), including 61 febrile patients. The study group was 47% men and 53% women. There was 99% agreement between methods, with few incidences of heterophile antibody interferences (Table 1).

[FIGURE 1 OMITTED]

Studies of diagnostic accuracy were conducted following institutional review board approval at 3 sites on randomized blinded retrospective samples from patients with clinical symptomology consistent with WNV infection. Results presented in Table 1 showed that the RapidWN test produced 100% agreement of serological sensitivity for acute, CDC WNV IgM and IgG ELISA-positive and plaque neutralization reduction test (PRNT)-confirmed samples. There was >97% agreement with other positive samples (CDC WNV IgM and IgG ELISA positive and PRNT confirmed) and 96% agreement with the CDC WNV ELISA-negative specimens. Similar results were obtained when comparison was made with the comparator device. The RapidWN test produced 98% agreement with the WNV-negative and 95% agreement with WNV-positive specimens. The apparent decrease in serological sensitivity is partially due to the fact that ELISA-based devices may produce equivocal results for some samples (index values between 0.9 and 1.09 of the comparator device), whereas a strip test may show positive/negative results within the 10% limit of the cutoff values.

The RapidWN test can also be used on plasma samples. The device produced similar results on 63 cohort plasma and serum samples when tested concomitantly. This test can also be equally performed on cerebrospinal fluid samples (unpublished results).

The efficacy of ELISA-based or microsphere immunoassays for detecting WNV antibodies has been well documented and provided results confirmed by PRNT (1214,17,18). The RapidWN strip test described here also showed comparable results with the ELISA-based IgM tests and with the PRNT. However, the advantages of the strip test over currently marketed ELISA-based tests include rapid result generation (15 min), ease of use, room temperature stability, and visually readable results. In addition, the strip test can be used for a single sample or multiple samples. Hence, the cost:benefit ratio of a rapid strip test may be better than that of traditional ELISA devices because of decreased labor time, equipment costs, and transport times for samples and test results. Other commonly used immunochromatographic strip tests that generate results visibly have been clinically validated for patients suspected of myocardial ischemia (19). The data presented here show that the RapidWN strip test can be effectively used as a rapid visible test for patients suspected of WNV infections.

Grant/funding support: This study was supported in part by the Industry Canada Technology Partnerships Canada Project 720-488118.

Financial disclosures: None declared.

Acknowledgments: The excellent technical support of Benny Chen, Min Yuan Zhang, and Philip Lam is highly appreciated.

DOI : 10.1373/clinchem.2007.091140

References

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(2.) CDC. Epidemic/epizootic West Nile virus in the United States: revised guidelines for surveillance, prevention, and control, 3rd rev, 2003. http:// www.cdc.gov/ncidod/dvbid/westnile/lab-guidance.htm (accessed April 2007).

(3.) Brinton MA. The molecular biology of the West Nile virus. Annu Rev Microbiol 2002;56:371-402.

(4.) Iwamoto M, Jernigan DB, Guasch A, Trepka MJ, Blackmore CG, Hellinger WC, et al. Transmission of West Nile virus from an organ donor to four transplant recipients. N Engl J Med 2003;348:2196-203.

(5.) Pealer LN, Martin AA, Petersen LR, Lanciotti RS, Page PL, Stramer SL, et al. Transmission of West Nile virus through blood transfusion in the United States in 2002. N Engl J Med 2003;349:1236-45.

(6.) Chapa JB, Ahn JT, DiGiovanni LM, Ismail MA. West Nile virus encephalitis during pregnancy. AM Coll Obs Gyn 2003;102:229-31.

(7.) Civin R, Villacorte F, Robles DT, Dassey DE, Croker C, Borenstein L, et al. West Nile virus infection in the pediatric population. Pediatr Infect Dis J 2006;25:75-8.

(8.) Watson JT, Pertel PE, Jones RC, Siston AM, Paul WS, Austin CC, et al. Clinical characteristics and functional outcomes of West Nile Fever. Ann Intern Med 2004;141:360-5.

(9.) Sejvar JJ, Leis AA, Stokic DS, Van Gerpen JA, Martin AA, Webb R, et al. Acute flaccid paralysis and West Nile virus infection. Emerg Infect Dis 2003;9: 788-93.

(10.) Petersen LR, Roehrig JT, Hughes JM. West Nile virus encephalitis. N Engl J Med 2002;347:1225-6.

(11.) Petersen LR, Martin AA. West Nile virus: a primer for the clinician. Ann Intern Med 2002;137:173-9.

(12.) Roehrig JT, Nash D, Maldin B, Labowitz A, Martin DA, Lanciotti RS, et al. Persistence of virus-reactive serum immunoglobulin M antibody in confirmed West Nile virus encephalitis cases. Emerg Infect Dis 2003;9:376-9.

(13.) Tardei G, Ruta S, Chitu V, Rossi C, Tsai TF, Cernescu C. Evaluation of immunoglobin M (IgM) and IgG enzyme immunoassays in serologic diagnosis of West Nile virus infection. J Clin Microbiol 2000;38:2232-9.

(14.) Martin DA, Muth DJ, Brown T, Johnson AJ, Karabatsos CB, Roehrig JT. Standardization of immunoglobin M capture enzyme-linked immunosorbent assays for routine diagnosis of arboviral infections. J Clin Microbiol 2000; 38:1823-36.

(15.) Malan AK, Martins TB, Hill HR, Litwin CM. Evaluation of commercial West Nile virus immunoglobulin G (IgG) and IgM enzyme immunoassays show the value of continuous validation. J Clin Microbiol 2004;42:727-33.

(16.) Kricka U. Human anti-animal antibody interference in immunological assays. Clin Chem 1999;45:942-56.

(17.) Wong SJ, Demarest VL, Boyle RH, Wang T, Ledizet M, Kar K, et al. Detection of human anti-flavivirus antibodies with a West Nile virus recombinant antigen microsphere immunoassay. J Clin Microbiol 2004;42:65-72.

(18.) Johnson AJ, Noga AJ, Kosoy 0, Lancotti RS, Johnson AA, Biggerstaff BJ. Duplex microsphere-based immunoassay for detection of anti-West Nile virus immunoglobulin M antibodies. Clin Diagn Lab Immunol 2005;12:56674.

(19.) Hamm CW, Goldman BU, Heeschem C, Kreymann G, Berger J, Meinertz T. Emergency room triage of patients with acute chest pain by means of rapid testing for cardiac troponin T or troponin I. N Engl J Med 1997;337:164853.

A. Shaikh, [1,] *, Jun Ge, [2] Yi-Xue Zhao, [2] Paul Walker, [2] and Mike Drebot [3]

[1] Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada;

[2] Spectral Diagnostics, Inc., Toronto, Ontario, Canada;

[3] National Microbiology Laboratory, Winnipeg, Manitoba, Canada;

* address correspondence to this author at: 135-2 The West Mall, Toronto, Ontario, M9C 1C2 Canada; fax 1-416-626-7383, e-mail nshaikh@spectraldx.com)
Table 1. Serological sensitivity of RapidWN[TM] WNV Igm test with
other ELISA-based devices.

Specimen type (test devices) n % agreement (95% CI)

Non-flavivirus IgM serum specimens 346 99 (96.5%-99.9%
 (comparison with Focust Technologies
 West Nile Virus Igm Capture ELISA)
WNV WNV-suspected serum specimens
 Acute infection (plaque reduction 24 100 (88%-100%)
 neutralization test confirmed)
 CDC WNV Igm/Igm Elisa positives 64 97 (85.7%-99.7%)
 CDC WNV IgM/lgG ELISA negatives 80 96 (84.5%-99.7%)
 Focus WNV IgM ELISA negatives 151 98 (86.6%-99.5%)
 Focus WNV IgM ELISA positives 145 95 (83.8%-99.4%)
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Title Annotation:Abstracts of Oak Ridge Posters
Author:Shaikh, Nisar A.; Ge, Jun; Zhao, Yi-Xue; Walker, Paul; Drebot, Mike
Publication:Clinical Chemistry
Date:Nov 1, 2007
Words:2184
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