Utility of the focus technologies west nile virus immunoglobulin M capture enzyme-linked immunosorbent assay for testing cerebrospinal fluid

Focus Technologies has developed an immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay (ELISA) kit that utilizes recombinant West Nile virus (WNV) antigens to detect WNV IgM in serum. We evaluate here the utility of the kit for detecting WNV IgM in cerebrospinal fluid (CSF). The sensitivity was evaluated by using 52 CSF specimens from the 2002 WNV season that were positive in both the Public Health Service Laboratories WNV IgM ELISA and an in-house WNV IgM ELISA with native WNV antigen. The specificity was evaluated with two groups of specimens: (i). 73 CSF specimens submitted for in-house WNV IgM ELISA testing from February through April 2003 and yielding a negative WNV IgM result and (ii). 60 CSF specimens determined to be positive for another virus by PCR testing. Using these 185 CSF specimens at a screening dilution of 1:2, the kit was determined to be 100% sensitive and 100% specific. Endpoint titers were determined for 20 IgM-positive CSF specimens by testing serial twofold dilutions and ranged from 1:8 to 1:512. Index values (specimen absorbance value/calibrator absorbance value) for the screening dilution (1:2) showed no correlation with IgM titers, whereas index values for higher dilutions showed significant correlation with IgM titers. CSF screening dilutions of greater than 1:2 are not recommended, however, due to the risk of obtaining false-negative results. These findings show that the Focus Technologies WNV IgM capture ELISA, when utilized as recommended, offers accurate qualitative detection of WNV IgM in CSF specimens.     

Detection of West Nile virus (WNV)-specific immunoglobulin M in a reference laboratory setting during the 2002 WNV season in the United States

Between 1 June and 31 December 2002, 30,677 serum samples and 4,554 cerebrospinal fluid (CSF) samples were tested for West Nile virus (WNV)-specific immunoglobulin M (IgM) by an in-house enzyme-linked immunosorbent assay (ELISA); 1,481 serum samples (4.8%) and 345 CSF samples (7.6%) were positive for WNV IgM. Positive samples were forwarded to public health service laboratories (PHSLs) for further testing. PHSLs supplied results from their WNV IgM ELISAs for 654 samples; 633 (97%) were positive. PHSLs supplied WNV plaque reduction neutralization test results for 128 samples; 123 (96%) were positive. WNV IgM seroconversion and seroreversion trends were evaluated for 749 patients who each provided two serum samples that were tested during the study period. Of 574 patients whose first serum sample was IgM negative, 41 (7%) seroconverted (the second serum sample was IgM positive); of 175 patients whose first serum sample was IgM positive, 22 (13%) seroreverted (the second serum sample was IgM negative). The seroreversion rate was directly proportional to the time between serum sample collection; whereas only 1% of patients whose sera were collected <20 days apart showed seroreversion, 54% of patients whose sera were collected >60 days apart showed seroreversion. Conversion and reversion trends for CSF were evaluated for 68 patients. Of 54 patients whose first CSF specimen was IgM negative, 9 (17%) converted; none of 14 patients whose first CSF specimen was IgM positive reverted. Concomitant detection of WNV IgM in serum and CSF was assessed for 1,188 patients for whom paired serum and CSF specimens were available; for all 130 patients for whom IgM was detectable in CSF, IgM was also detectable in serum. These findings show that an in-house WNV IgM ELISA accurately identifies patients with WNV infection, document WNV IgM conversion and reversion trends, and demonstrate that WNV IgM detection in CSF is accompanied by WNV IgM detection in serum.     

Detection of West Nile Virus (WNV)-Specific Immunoglobulin M in a Reference Laboratory Setting during the 2002 WNV Season in the United States

Between 1 June and 31 December 2002, 30,677 serum samples and 4,554 cerebrospinal fluid (CSF) samples were tested for West Nile virus (WNV)-specific immunoglobulin M (IgM) by an in-house enzyme-linked immunosorbent assay (ELISA); 1,481 serum samples (4.8%) and 345 CSF samples (7.6%) were positive for WNV IgM. Positive samples were forwarded to public health service laboratories (PHSLs) for further testing. PHSLs supplied results from their WNV IgM ELISAs for 654 samples; 633 (97%) were positive. PHSLs supplied WNV plaque reduction neutralization test results for 128 samples; 123 (96%) were positive. WNV IgM seroconversion and seroreversion trends were evaluated for 749 patients who each provided two serum samples that were tested during the study period. Of 574 patients whose first serum sample was IgM negative, 41 (7%) seroconverted (the second serum sample was IgM positive); of 175 patients whose first serum sample was IgM positive, 22 (13%) seroreverted (the second serum sample was IgM negative). The seroreversion rate was directly proportional to the time between serum sample collection; whereas only 1% of patients whose sera were collected <20 days apart showed seroreversion, 54% of patients whose sera were collected >60 days apart showed seroreversion. Conversion and reversion trends for CSF were evaluated for 68 patients. Of 54 patients whose first CSF specimen was IgM negative, 9 (17%) converted; none of 14 patients whose first CSF specimen was IgM positive reverted. Concomitant detection of WNV IgM in serum and CSF was assessed for 1,188 patients for whom paired serum and CSF specimens were available; for all 130 patients for whom IgM was detectable in CSF, IgM was also detectable in serum. These findings show that an in-house WNV IgM ELISA accurately identifies patients with WNV infection, document WNV IgM conversion and reversion trends, and demonstrate that WNV IgM detection in CSF is accompanied by WNV IgM detection in serum.     

Intrathecal immune response and virus-specific immunoglobulin M antibodies in laboratory diagnosis of acute poliomyelitis.

The intrathecal immune response in 114 patients with clinically diagnosed acute poliomyelitis was studied by measuring poliovirus-specific immunoglobulin M (IgM) antibodies in cerebrospinal fluid (CSF) by a mu-capture immunoassay and by assessing the ratio between levels of poliovirus-neutralizing antibodies in serum and CSF. Fecal specimens were used for attempts to isolate the causative agents. Eighty-five percent of CSF specimens collected during the first 15 days of disease contained virus-specific IgM antibodies. Forty-five of 48 tested children (94%) also showed virus-specific IgM responses in their sera. Later on, the antibody levels decreased, and positive results after 30 days of onset of paralytic symptoms were rare. If the presence of poliovirus-specific IgM antibodies in the CSF was considered diagnostic, more cases were confirmed by this test than by virus isolation. A relative increase in poliovirus-neutralizing antibodies in the CSF was observed in about one-third of the cases; in all but three cases the increase was observed together with the presence of virus-specific IgM antibodies. A systemic virus-specific response can be seen and poliovirus can be isolated from a subclinically infected individual suffering from a concomitant poliomyelitis-like disease, while positive results by the two methods demonstrating an intrathecal immune response are likely to indicate a true causal relationship between infection and disease. Demonstration of poliovirus-specific IgM antibodies in the CSF thus appears to be a sensitive and specific method for laboratory confirmation of clinically diagnosed poliomyelitis.     

West Nile virus immunoglobulin A (WNV IgA) detection in cerebrospinal fluid in relation to WNV IgG and IgM reactivity.

BACKGROUND: Diagnostic criteria for neurologic involvement in WNV infection include WNV IgM detection in CSF; however, WNV IgM can persist in CSF >6 months. CSF IgA characterizes other flavivirus infections, but WNV IgA in CSF has not been evaluated. WNV IgM in CSF correlates with IgM in serum but the presence of WNV IgA in CSF compared to serum is unknown. OBJECTIVES: Evaluate WNV IgA detection in CSF as a marker of WNV neuroinvasive infection, initially with samples pre-selected based on WNV IgG and IgM reactivity and subsequently with all available CSF samples submitted for WNV antibody testing over an entire WNV season. STUDY DESIGN: Selected CSF samples and CSF/serum pairs previously tested for WNV IgG and IgM were assayed for WNV IgA. Subsequently, all available CSF samples tested for WNV antibodies during the 2005 season were tested for WNV IgA, including those where paired sera were available and tested for IgA, IgG and IgM. RESULTS: For most samples, including paired CSF and serum, the IgA result qualitatively agreed with the IgM result, regardless of the IgG result. CONCLUSION: IgA detection is equivalent to IgM detection as a marker of WNV infection in CSF.     

Persistence of West Nile virus immunoglobulin M antibodies, Greece

A major outbreak of West Nile virus (WNV) lineage 2 infections was observed in 2010 in Greece. In order to check the persistence of WNV IgM antibodies, a second serum sample taken 75-180 days after onset of the illness from 29 patients with WNV infection was tested. A third sample was obtained 181-270 days after onset of the illness from 8 of the 12 patients with IgM-positive second sample. Mixed effects linear regression analysis indicated that the approximate time at which IgM index became negative was 164 (95% confidence interval, 95% CI 99-236) days after the symptoms' onset. Persistence of IgM antibodies was observed in 12% of patients at 181-270 days of follow-up. A sharp decrease in the IgM levels was observed, mainly in patients who had high IgM index value in the acute phase. All patients were WNV IgG positive at the follow-up.     

Enzyme-linked immunosorbent assays using recombinant envelope protein expressed in COS-1 and Drosophila S2 cells for detection of West Nile virus immunoglobulin M in serum or cerebrospinal fluid

Humans infected with West Nile virus (WNV) develop immunoglobulin M (IgM) antibodies soon after infection. The microtiter-based assays for WNV IgM antibody detection used by most state public health and reference laboratories utilize WNV antigen isolated from infected Vero cells or recombinant envelope protein produced in COS-1 cells. Recombinant antigen produced in COS-1 cells was used to develop a WNV IgM capture enzyme immunoassay (EIA). A supplementary EIA using WNV envelope protein expressed in Drosophila melanogaster S2 cells was also developed. Both assays detected WNV IgM in the sera of experimentally infected rhesus monkeys within approximately 10 days postinfection. Human sera previously tested for WNV IgM at a state public health laboratory (SPHL) were evaluated using both EIAs. Among the sera from 20 individuals with laboratory-confirmed WNV infection (i.e., IgM-positive cerebrospinal fluid [CSF]) that were categorized as equivocal for WNV IgM at the SPHL, 19 were IgM positive and one was negative by the new EIAs. Of the 19 IgM-positive patients, 15 were diagnosed with meningitis or encephalitis; the IgM-negative patient was not diagnosed with neurological disease. There was 100% agreement between the EIAs for the detection of WNV IgM. CSF samples from 21 individuals tested equivocal for WNV IgM at the SPHL; all 21 were positive in both bead assays, and 16 of these patients were diagnosed with neurological disease. These findings demonstrate that the new EIAs accurately identify WNV infection in individuals with confirmed WNV encephalitis and that they exhibit enhanced sensitivity over that of the microtiter assay format.     

Enzyme-Linked Immunosorbent Assays Using Recombinant Envelope Protein Expressed in COS-1 and Drosophila S2 Cells for Detection of West Nile Virus Immunoglobulin M in Serum or Cerebrospinal Fluid

Humans infected with West Nile virus (WNV) develop immunoglobulin M (IgM) antibodies soon after infection. The microtiter-based assays for WNV IgM antibody detection used by most state public health and reference laboratories utilize WNV antigen isolated from infected Vero cells or recombinant envelope protein produced in COS-1 cells. Recombinant antigen produced in COS-1 cells was used to develop a WNV IgM capture enzyme immunoassay (EIA). A supplementary EIA using WNV envelope protein expressed in Drosophila melanogaster S2 cells was also developed. Both assays detected WNV IgM in the sera of experimentally infected rhesus monkeys within approximately 10 days postinfection. Human sera previously tested for WNV IgM at a state public health laboratory (SPHL) were evaluated using both EIAs. Among the sera from 20 individuals with laboratory-confirmed WNV infection (i.e., IgM-positive cerebrospinal fluid [CSF]) that were categorized as equivocal for WNV IgM at the SPHL, 19 were IgM positive and one was negative by the new EIAs. Of the 19 IgM-positive patients, 15 were diagnosed with meningitis or encephalitis; the IgM-negative patient was not diagnosed with neurological disease. There was 100% agreement between the EIAs for the detection of WNV IgM. CSF samples from 21 individuals tested equivocal for WNV IgM at the SPHL; all 21 were positive in both bead assays, and 16 of these patients were diagnosed with neurological disease. These findings demonstrate that the new EIAs accurately identify WNV infection in individuals with confirmed WNV encephalitis and that they exhibit enhanced sensitivity over that of the microtiter assay format.     

Use of immunoglobulin G avidity assays for differentiation of primary from previous infections with West Nile virus

Since its introduction in 1999, West Nile virus (WNV) infections have spread rapidly across the North American continent. Diagnosis of acute WNV infection by detection of WNV-specific immunoglobulin M (IgM) is complicated by the persistence of detectable IgM for more than 1 year in some patients. IgG antibody avidity testing was assessed as a supplemental assay in the diagnosis of current infections. Three groups of serum samples were assayed in parallel by two different IgG avidity test systems (indirect immunofluorescence test [IIFT] and prototype enzyme-linked immunosorbent assay [ELISA]; EUROIMMUN, Luebeck, Germany). Group I (40 sera taken between 2 and 9 days after the onset of influenza-like symptoms) and group II (40 sera taken between 10 and 43 days after onset) were acute and convalescent specimens from patients with a positive anti-WNV IgM test (ELISA; Focus Diagnostics, Cypress, CA). Group III consisted of 43 patient sera collected between 6 and 12 months after infection. IgG antibodies specific for WNV were detected in 38% (ELISA) and 50% (IIFT) of group I sera, in 90% (ELISA and IIFT) of group II sera, and in 100% (ELISA and IIFT) of group III sera. Low-avidity IgG antibodies were demonstrated in 86% (ELISA) and 95% (IIFT) of IgG-positive patient samples taken between 2 and 43 days after the onset of symptoms (groups I and II). High-avidity IgG antibodies were detected in 100% of group III sera obtained 6 months or more after the onset of symptoms (ELISA and IIFT). IgG avidity tests for WNV infections are rapid and simple to perform. The determination of IgG avidity provides additional diagnostic certainty in differentiating between recently acquired and previous infections with WNV.     

Antibody Capture Immunoassay Detection of Japanese Encephalitis Virus Immunoglobulin M and G Antibodies in Cerebrospinal Fluid

Immunoglobulin M (IgM) and IgG antibodies to Japanese encephalitis virus (JEV) were detected in acute-phase cerebrospinal fluid (CSF) specimens from patients with acute encephalitis by using a solid-phase radioimmunoassay of the antibody capture type. Of 12 patients with JEV infections subsequently proven by hemagglutination inhibition serology, 11 had JEV IgM antibodies, as measured by antibody capture radioimmunoassay, in the first CSF specimen (geometric mean titer, 1:2,500) compared with 0 of 8 patients with acute encephalitis proven not to be due to JEV. Specific IgM anti-JEV activity (units per microgram) was greater in CSF than in parallel serum specimens in all 11 positive cases by more than fourfold on the average (range, 1.4 to 13). Among seven patients with broadly reactive hemagglutination inhibition seroresponses typical of persons previously exposed to other flaviviruses, six had high levels of JEV IgG antibodies (as measured by antibody capture radioimmunoassay) in their acute-phase CSF (geometric mean titer, 1:26,000), whereas in five patients experiencing their first flavivirus infection, JEV IgG antibodies measured by antibody capture radio-immunoassay were either absent (one patient) or weakly reactive (four patients; geometric mean titer, 1:3,200). Specific IgG anti-JEV activity was greater in CSF than in parallel serum specimens in eight of the nine positive cases measured (range, 1.3- to 24-fold). The antibody capture solid-phase immunoassay approach is well suited for detecting specific antibody activity in CSF.     

Evaluation of a diagnostic algorithm using immunoglobulin M enzyme-linked immunosorbent assay to differentiate human West Nile Virus and St. Louis Encephalitis virus infections during the 2002 West Nile Virus epidemic in the United States

A diagnostic algorithm was developed to differentiate between human infections of West Nile virus (WNV) and St. Louis encephalitis virus (SLEV) using positive-to-negative (P/N) ratios derived from the immunoglobulin M capture enzyme-linked immunosorbent assay (MAC-ELISA). To validate this algorithm, we tested 1,418 serum and cerebrospinal fluid (CSF) samples from confirmed WNV and SLEV infections collected during the WNV epidemic of 2002 in the United States. WNV P/N-to-SLEV P/N ratios (W/S ratios) were calculated and used to identify the infecting virus. These results were compared to results from the plaque reduction neutralization test (PRNT), which is currently the standard assay used to discriminate between closely related flavivirus infections. If the W/S ratio was > or =1, the predictive value positive (PNP) for WNV was 97.8%, where 95% of flavivirus cases were due to WNV infection and only 3.7% of specimens would require PRNT to differentiate WNV from SLEV infection. Use of the W/S ratio as part of the testing algorithm to interpret MAC-ELISA results generates reportable probable cases quickly, alleviating the need for PRNT in most instances.     

Evaluation of a Diagnostic Algorithm Using Immunoglobulin M Enzyme-Linked Immunosorbent Assay To Differentiate Human West Nile Virus and St. Louis Encephalitis Virus Infections during the 2002 West Nile Virus Epidemic in the United States

A diagnostic algorithm was developed to differentiate between human infections of West Nile virus (WNV) and St. Louis encephalitis virus (SLEV) using positive-to-negative (P/N) ratios derived from the immunoglobulin M capture enzyme-linked immunosorbent assay (MAC-ELISA). To validate this algorithm, we tested 1,418 serum and cerebrospinal fluid (CSF) samples from confirmed WNV and SLEV infections collected during the WNV epidemic of 2002 in the United States. WNV P/N-to-SLEV P/N ratios (W/S ratios) were calculated and used to identify the infecting virus. These results were compared to results from the plaque reduction neutralization test (PRNT), which is currently the standard assay used to discriminate between closely related flavivirus infections. If the W/S ratio was ≥1, the predictive value positive (PNP) for WNV was 97.8%, where 95% of flavivirus cases were due to WNV infection and only 3.7% of specimens would require PRNT to differentiate WNV from SLEV infection. Use of the W/S ratio as part of the testing algorithm to interpret MAC-ELISA results generates reportable probable cases quickly, alleviating the need for PRNT in most instances.     

Clinical utility of commercial enzyme immunoassays during the inaugural season of West Nile virus activity, Alberta, Canada

West Nile virus (WNV) has spread rapidly across North America, creating a need for rapid and accurate laboratory diagnosis on a large scale. Immunoglobulin M (IgM) capture enzyme immunoassays (EIA) became commercially available in the summer of 2003, but limited data are available on their clinical performance. Consolidated human WNV diagnostic testing for the province of Alberta, Canada, at the public health laboratory permitted a large-scale evaluation of the assays, covering a wide clinical spectrum. Two thousand nine hundred sixty-nine sera were tested, from 2,553 Alberta residents, and 266 cases were identified. Sensitivities of the Focus assay and first-generation Panbio IgM capture EIA were 79 and 80%, respectively. During the first week of illness only 53 to 58% of cases were positive, but sensitivity was 96 to 97% after day 8. Sensitivity for neurological cases was 92% overall. Specificity was high for the Focus kit at 98.9%, but only 82.9% for the first Panbio kit. A positive Focus WNV IgG result with a twofold rise in IgG index was a reliable indicator of acute flavivirus infection (67/67 WNV). Agreement between the IgG test and hemagglutinin inhibition titers in paired sera was at least 82%. Commercial IgM and IgG EIA proved useful for WNV diagnosis, provided follow-up sera were collected after 8 days of illness.     

Detection of IgG and IgM to West Nile virus. Development of an immunofluorescence assay

West Nile virus (WNV) had its first recorded appearance in the western hemisphere in 1999 and has continued to spread across the United States, necessitating the development of serologic procedures to diagnose infection. We developed an immunofluorescence assay (IFA) protocol for the detection of WNV-specific IgG and IgM antibodies in serum and cerebrospinal fluid (CSF) specimens. We tested 82 serum and 16 CSF samples and compared the results with WNV IgG enzyme-linked immunosorbent assay (ELISA) and IgM antibody-capture (MAC) ELISA results. Agreement, clinical sensitivity, and clinical specificity for the IgG IFA were 92%, 100%, and 90%, respectively, and 98%, 96%, and 100% for the IgM IFA, respectively. Extensive arbovirus cross-reactions occurred in the IgG assays, but only minimal cross-reactions were observed in the IgM assays. The IFA protocol described herein is a cost-effective and sensitive alternative to ELISA and MAC-ELISA for the serologic diagnosis of WNV infection.     

Cerebrospinal fluid cytology in seasonal epidemic West Nile virus meningo-encephalitis

The incidence of West Nile Virus (WNV) infection has progressively increased in North America since the first epidemic in 1999. Formal scholarly documentation of cerebrospinal fluid (CSF) cytology changes in patients with WNV infection is limited. We report our experience with CSF cytospins from a population of consecutive patients with documented CSF WNV-specific IgM. Thirty-two patients (12 male, 20 female) with a median age of 52 yr (range, 19-88) diagnosed with WNV meningo-encephalitis were studied. Symptoms were present for a mean of 5 days (range, 1-14) prior to lumbar puncture. CSF proteins were elevated in 94% of patients (30/32) with a mean value of 79 mg/dl (range, 36-185). CSF glucose was normal to elevated in all cases. All cytomorphologically adequate samples demonstrated a pleocytosis with a mean of 156 cells/mm3 (range, 13-683). Nearly, all (26/28) patients showed increased CSF neutrophils--mean 43% (range, 1-83). Mean lymphocyte and monocyte fractions were 44% (range, 8-85) and 14% (range, 2-27), respectively. Three cases showed 1-4% plasma cells. Mean total leukocyte counts (TLC) (197 cells/mm3) and mean neutrophil fractions (50%) were greater in patients sampled within the first 3 days of symptoms than in those sampled beyond day 3 (mean TLC, 126 cells/mm3; mean neutrophil fraction, 37%). Relative lymphocyte proportions increased from a mean of 39 to 48% after 3 days of illness. WNV should be considered as a potential etiology of infectious CSF pleocytosis in the North American late summer and early fall seasons.     

Immunoglobulin G avidity in differentiation between early and late antibody responses to West Nile virus

In 1999 West Nile virus (WNV) surfaced in the United States in the city of New York and spread over successive summers to most of the continental United States, Canada, and Mexico. Because WNV immunoglobulin M (IgM) antibodies have been shown to persist for up to 1 year, residents in areas of endemicity can have persistent WNV IgM antibodies that are unrelated to a current illness with which they present. We present data on the use of IgG avidity testing for the resolution of conflicting data arising from the testing of serum or plasma for antibodies to WNV. Thirteen seroconversion panels, each consisting of a minimum of four samples, were used. All samples were tested for the presence of WNV IgM and IgG antibodies, and the avidity index for the WNV IgG-positive samples was calculated. Panels that exhibited a rise in the WNV IgM level followed by a sequential rise in the WNV IgG level were designated "primary." Panels that exhibited a marked rise in the WNV IgG level followed by a sequential weak WNV IgM response and that had serological evidence of a prior flavivirus infection were designated "secondary." All samples from the "primary" panels exhibited low avidity indices (less than 40%) for the first 20 to 30 days after the recovery of the index sample (the sample found to be virus positive). All of the "secondary" samples had elevated WNV IgG levels with avidity indices of > or =55%, regardless of the number of days since the recovery of the index sample. These data demonstrate that it is possible to differentiate between recent and past exposure to WNV or another flavivirus through the measurement of WNV IgG avidity indices.     

Persistence of antibodies to West Nile virus nonstructural protein 5

BACKGROUND: Because IgM antibody against West Nile virus (WNV) pre-membrane/envelope (preM/E) recombinant protein may persist for more than 1 year, an assay distinguishing recent from past WNV infection would be useful. Published findings for a single patient suggest that the presence of antibody against WNV nonstructural protein 5 (NS5) indicates recent infection. OBJECTIVES: To compare the persistence of WNV NS5 antibodies and preM/E IgM using plasma samples from blood donors who were viremic at the time of donation. STUDY DESIGN: Follow-up plasma samples from 35 viremic donors were tested for WNV NS5 antibodies using a microsphere immunoassay, and compared to WNV preM/E IgM antibodies determined on the same samples using an enzyme-linked immunosorbent assay (ELISA). RESULTS: At 90+/-14 days of follow-up, 20/26 donors (77%) were positive for NS5 antibodies; 6/25 (24%) were positive at 180+/-27 days, and 3/23 (13%) were positive at 365+/-55 days. The comparable values for preM/E IgM antibodies were 77%, 32% and 17%, respectively. CONCLUSION: Persistence of WNV NS5 antibody in plasma is similar to that of preM/E IgM antibody. WNV NS5 antibody cannot be used to distinguish recent from past WNV infection.     

Performance Characteristics of an In-House Assay System Used To Detect West Nile Virus (WNV)-Specific Immunoglobulin M during the 2001 WNV Season in the United States

During the 2001 U. S. West Nile virus (WNV) season, 163 specimens were reactive in an in-house WNV-specific immunoglobulin M (IgM) screening enzyme-linked immunosorbent assay (ELISA) and were referred to either the Centers for Disease Control and Prevention or the appropriate state public health laboratory (CDC/SPHL) for additional testing. CDC/SPHL supplied results for 124 specimens that could be further evaluated in-house: 70 specimens were nonreactive in the CDC/SPHL WNV-specific IgM screening assay, and 54 specimens were reactive. These specimens were used to evaluate a modified in-house WNV-specific IgM ELISA that incorporated background subtraction to identify nonspecific reactivity and thus improve assay specificity. Of the 70 CDC/SPHL nonreactive samples, 49 (70%) were nonreactive in the modified ELISA; of the 54 CDC/SPHL reactive samples, 51 (94%) were reactive in the modified ELISA. Confirmatory studies performed by CDC/SPHL indicated that 38 CDC/SPHL screen-reactive specimens represented true WNV infection; all 38 specimens were reactive in the modified in-house WNV-specific IgM ELISA. These findings demonstrate that an in-house ELISA system for WNV-specific IgM effectively identifies patients with WNV infection.     

Performance characteristics of an in-house assay system used to detect West Nile Virus (WNV)-specific immunoglobulin M during the 2001 WNV season in the United States

During the 2001 U. S. West Nile virus (WNV) season, 163 specimens were reactive in an in-house WNV-specific immunoglobulin M (IgM) screening enzyme-linked immunosorbent assay (ELISA) and were referred to either the Centers for Disease Control and Prevention or the appropriate state public health laboratory (CDC/SPHL) for additional testing. CDC/SPHL supplied results for 124 specimens that could be further evaluated in-house: 70 specimens were nonreactive in the CDC/SPHL WNV-specific IgM screening assay, and 54 specimens were reactive. These specimens were used to evaluate a modified in-house WNV-specific IgM ELISA that incorporated background subtraction to identify nonspecific reactivity and thus improve assay specificity. Of the 70 CDC/SPHL nonreactive samples, 49 (70%) were nonreactive in the modified ELISA; of the 54 CDC/SPHL reactive samples, 51 (94%) were reactive in the modified ELISA. Confirmatory studies performed by CDC/SPHL indicated that 38 CDC/SPHL screen-reactive specimens represented true WNV infection; all 38 specimens were reactive in the modified in-house WNV-specific IgM ELISA. These findings demonstrate that an in-house ELISA system for WNV-specific IgM effectively identifies patients with WNV infection.     

Development and persistence of West Nile virus-specific immunoglobulin M (IgM), IgA, and IgG in viremic blood donors

West Nile Virus (WNV) antibody development and persistence were investigated in blood donors who made WNV RNA-positive (viremic) donations in 2003. Plasma samples from the index donations and follow-up serum or plasma samples were tested for WNV immunoglobulin M (IgM), IgA, and IgG by using enzyme-linked immunosorbent assays. Antibody development was investigated with 154 samples collected from 84 donors 1 to 21 days after their RNA-positive, antibody-negative, index donation. WNV IgM and IgA were first detected on day 3, and all samples collected after day 9 were WNV IgM and IgA positive; WNV IgG was first detected on day 4, and all samples collected after day 16 were positive. Antibody persistence in this donor group (index donations antibody negative) was evaluated by using 128 samples collected from 89 donors on days 22 to 440 of follow-up; 88% of samples were WNV IgM positive, 86% were WNV IgA positive, and 100% were WNV IgG positive. In linear regression analysis, trendlines for WNV IgM and IgA reached the value discriminating positive from negative results at 218 days and 232 days of follow-up, respectively. Similar WNV IgM and IgA persistence trends characterized 27 donors whose index samples were positive for WNV IgM and IgA, as well as 14 donors whose index samples were positive for WNV IgG but negative for WNV IgM. These findings show that WNV IgG emerges after WNV IgM and IgA and that both WNV IgM and IgA typically persist for at least 6 months after infection. Thus, unlike some other flavivirus infections, WNV infection is not characterized by a relatively rapid disappearance of virus-specific IgA.