West Nile Virus Neurologic Disease in Humans, South Africa, September 2008–May 2009

We investigated West Nile virus (WNV) as a possible disease etiology in persons hospitalized in South Africa. Of 206 specimens tested, 36 had WNV neutralizing antibodies, significantly more than in similar earlier serosurveys. Seven probable acute WNV cases were identified, suggesting WNV may be overlooked as an etiology of severe disease in South Africa.


The Study
Serum and CSF samples were obtained from the National Health Laboratory Service, Thswane Academic Division, Thswane, South Africa, which serves public sector hospitals in northern South Africa. To select samples for testing, we reviewed laboratory submission requests for patients with clinical conditions consistent with WNV infection: fever, headache, rash, or neurologic signs (7,8). A total of 206 patient samples (15 CSF and 191 serum) were selected. During September 2008-May 2009, we screened samples for the presence of WNV by using real-time reverse transcription PCR (rRT-PCR) (9), virus neutralization assay, and IgM ELISA.
We detected WNV neutralizing antibodies in serum and CSF samples by using a modifi ed method (10). In brief, we mixed 50% tissue culture infective doses of Kunjin virus strain MRM61C (100 U/mL) in 2% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) with 2-fold dilutions of heat-inactivated patient serum (1:10-1:640) in equal volumes and incubated the mixture for 1 h at 37°C in 5% CO 2 . We then added 1 volume of Vero cells (1 × 10 5 cells/mL) in 2% fetal bovine serum, 100 U/mL of penicillin, and 100 μg/mL of streptomycin (Lonza, Basel, Switzerland) and incubated the mixture for 72 h at 37°C in 5% CO 2 . Samples were considered positive for WNV neutralizing antibodies if <25% of the cells/well displayed cytopathic effect. Comparative testing with Wesselsbron virus, a closely related fl avivirus, did not show cross-reactivity within the parameters of what we considered positive.
Of the 206 specimens, 190 were of suffi cient quantity to be subjected to RNA extraction (QIAamp Viral RNA Mini Kit; QIAGEN, Valencia, CA, USA) and subsequent WNV nested rRT-PCR (9). The presence of lineage 2 WNV RNA was identifi ed in 1 CSF specimen and confi rmed by sequencing (GenBank accession no. JX974605) (Figure).
To evaluate the sensitivity of these molecular and serologic tests for diagnosing WNV in humans, we used the same methods to test 9 archived sequential serum samples in parallel ( Table 2). The samples were from a patient with WNV encephalitis who became infected with neuroinvasive lineage 2 WNV strain in 2003 after a needlestick injury (11). Samples were collected 0-30 days after exposure. Initial symptoms developed on postexposure day 7 and persisted for 19 days; the patient completely recovered by day 26 (11).

Conclusions
We conducted a retrospective investigation of patients hospitalized with febrile illness or neurologic disease of unknown etiology in the Pretoria region of South Africa to determine whether some of the cases could be ascribed to WNV infection. Evidence of acute WNV infection was identifi ed in samples for 7 patients (Table 1). For 2 of the patients, WNV infection was identifi ed by the presence of IgM and neutralizing antibodies in serum samples; these patients had been hospitalized for febrile illness. For the other 5 patients, infection was identifi ed by a WNV-positive (by PCR) CSF sample (1 patient) and by the presence of neutralizing antibodies in CSF samples (4 patients); these patients had been hospitalized for neurologic signs and symptoms.
The 4 patients with neutralizing antibodies in CSF all had severe neurologic complications (Table 1). Samples from these patients were insuffi cient for performing IgM testing; thus, WNV infection cannot be defi nitively determined. However, the presence of WNV neutralizing antibody in CSF samples plus acute clinical signs and symptoms of WNV infection provide a high index of suspicion for WNV infection in these patients. Factors such as increased blood-brain barrier permeability and the persistence of WNV antibodies long after infection may also serve as explanations for the presence of neutralizing antibodies in their CSF.
Nested rRT-PCR results and phylogenetic analysis confi rmed the presence of lineage 2 WNV in the CSF sample from 1 patient (Figure); sequencing showed that the  virus is closely related to 2 neuroinvasive WNV lineage 2 strains identifi ed in South Africa (11,12) (Table 1). The low rate of PCR-positive cases was not entirely unexpected and may be explained by 2 factors: 1) PCR has limited success for detecting arboviruses because CSF contains low levels of virus and arbovirus-associated viremia is brief (13), and 2) false-negative test results may occur if samples are not properly stored to protect the integrity of potential viral RNA.
To evaluate the sensitivity of the 3 diagnostic methods used in our study, we conducted a time-trial experiment by using retrospective serum samples from a patient in whom WNV meningoencephalitis developed after a needlestick injury (11). Early samples (postexposure days 8 and 9) were positive for WNV by rRT-PCR only. Results for samples obtained >13 and >16 days after exposure were positive by neutralization assays and IgM ELISA, respectively ( Table  2). Experiments with horses have indicated that WNV neutralizing antibody assays show a positive result earlier than IgM ELISAs; the reasons for this are undetermined (14). Although our time-trial experiment refl ects fi ndings from only 1 patient and should ideally be performed on a cohort, the results, considered with those from the studies in horses (14), may imply that some cases of WNV infection in humans and animals may be missed if IgM ELISA is the only serologic test used.
Using virus neutralization assays, we identifi ed the presence of WNV antibodies in 36/204 serum samples from patients with febrile and neurologic illness in South Africa. This fi nding indicates that the patients were exposed to WNV. In addition, results were negative for the patients in our study who were tested for herpes simplex virus types 1 and 2, measles, mumps, and enteroviruses, and no other etiologic agent was found. Thus, infection with WNV should be included in the differential diagnosis of patients in this region with neurologic disease, especially considering the frequent detection of severe neurologic disease in horses in the region (15).
Our fi ndings confi rm that WNV is being overlooked as a cause of severe neurologic disease in South Africa, and they suggest a need for increased clinical awareness, enhanced prospective surveillance, and a more current serosurvey of WNV infection in humans. PCR may be a useful diagnostic method during early infection, but after seroconversion has taken place, serologic tests (e.g., IgM ELISA in conjunction with virus neutralization) are more likely to yield accurate results.