Figure 1. RT-PCR detection of West Nile virus RNA in cerebrospinal fluid. Lanes 1 and 3: negative controls; lanes 2 and 4: cerebrospinal fluid; lane M: 50-bp DNA ladder. Primer pairs used: lanes 1 and 2: CU9093/CL9279; lanes 3 and 4: D87F/D156R. ious genomic regions of WNV. PCR bands with the expected sizes were present in all four reactions (data not shown). The identity of the PCR bands was confirmed by sequence data obtained directly from the PCR amplicon. The diagnosis of WNV infection was based on the detection and sequence of the WNV genome in CSF. Although the serologic test (IgM capture enzyme-linked immunosorbent assay [ELISA]) on the CSF sample was negative for WNV, according to the interpretations set forth by CDC, the detection of viral genome sequence in CSF meets the definition of a confirmed case. RESEARCH TaqMan Assays To follow up this case, three whole-blood samples and one serum sample were examined by both standard RT-PCR (prim- ers in NS5 region) and TaqMan (primers in ENV region) assays; the results are summarized in Table 2. The highest RNA copy number, 2.5 x 106 copies/mL, was found in the blood sample that was collected 3 days after the patient’s neu- rologic symptoms appeared. WNV genome was also detected in a serum sample collected on day 19 after onset of symp- toms. Serologic tests (IgM capture ELISA and IgG ELISA) on serum for Eastern equine encephalitis virus, LACV, POWV, SLEV, and WNV were all negative. Other Clinical Data Laboratory studies of the CSF indicated the following val- ues: leukocyte (WBC) count 8 mm3 with 66% neutrophils, 4% lymphocytes, 4% atypical lymphocytes, and 26% monocytes; erythrocyte count 0; glucose level 76 mg/dL; total protein level 55 mg/dL, and lactate dehydrogenase level 35 IU/L. Fig- ure 2 presents fever curve, WBC curve, and viremia data. The serum immunoglobulins at the time of infection with WNV were IgG 492 mg/dL (normal level [nl]) 700–1,500 mg/dL), IgA 86 mg/dL (nl 65–450 mg/dL), and IgM 80 mg/dL (nl 45– 230 mg/dL). Virus Isolation The attempt to isolate WNV was carried out in a biosafety level 3 laboratory not routinely used for arbovirus work and located in a building separate from the facility where PCR test- ing was conducted. This procedure had the dual advantage of minimizing the possibility that any virus recovered originated from a source other than the human specimen and also ensur- ing that any isolate obtained would not lead to future spurious PCR results. A blood sample collected on September 24, 2001, and a CSF specimen collected on September 26, 2001, were chosen for recovery of the virus because of the presence of high-copy-number viral RNA. On day 6 postinfection, CPE was observed in the tubes inoculated with 0.01 mL and 0.05 mL of blood, but not in the tube inoculated with 0.1 mL of CSF. WNV was confirmed in the second-passage cell cultures by IFA by using WNV-specific monoclonal antibody H5-46. Specimen Blood Blood CSF Blood Serum 9/21/2001 9/24/2001 9/26/2001 10/02/2001 10/10/2001 2.2 x 103 2.5 x 106 1.1 x 106 5.4 x 104 3.7 x 103 CT 31.6 20.4 20.4 25.8 28.5 Rn 0.48 3.71 3.71 2.70 0.70 Positive Positive Positive Positive Positive n.d. n.d. Negative n.d. Negative n.d. Positive Negative n.d. n.d. Table 2. Detection of West Nile virus in human specimens by TaqMan and standard RT-PCR assaysa Collection date RNA (copy/mL) STD RT-PCR Serology Cell cultures aCT, threshold cycle number, the cycle number at which fluorescence increases above a fixed threshold value; Rn, normalized fluorescent signal, the fluorescent signal generated by the reporter dye; STD, standard; RT-PCR, reverse transcription–polymerase chain reaction; n.d.: not done, CSF, cerebrospinal fluid. Emerging Infectious Diseases • Vol. 8, No. 12, December 2002 1369