From January 1 through December 31, 2006, we tested 720 samples from 680 patients at the Bernhard-Nocht Institute for Tropical Medicine in Hamburg, Germany, for CHIKV infection. All had symptoms compatible with acute or recent CHIKV infection (sudden onset of fever, muscle and joint pain, headaches, rash) upon return to Europe (Germany, n = 515; Belgium, n = 99; Switzerland, n = 42; Denmark, n = 22; Poland, n = 2). For 189 patients, exact travel destinations were reported: Madagascar (n = 9), Mauritius (n = 92), the Seychelles (n = 23), Réunion Island (n = 18), Bali (n = 2), Indonesia (n = 6), Sri Lanka (n = 5), India (n = 28), Malaysia (n = 2), Kenya (n = 1), and Thailand (n = 3). Average ages of travelers to each country did not differ significantly (analysis of variance F-test, p>0.05). For 121 patients, exact dates of onset and sampling could be retrieved through voluntary questionnaires completed by telephone or fax after issuance of results. Age and sex distribution and travel histories for these patients are shown in Table 1. The day of onset of symptoms was defined as day 0. All samples with possible CHIKV in 2006 were tested for IgG and IgM by indirect immunofluorescence. During the first half of 2006, all samples were tested by real-time RT-PCR in addition; however, in the second half of the year RT-PCR testing was restricted to samples from patients with acute infection only (on the basis of experiences from the first 6 months as described below). Classification of patients as having laboratory-confirmed cases required either a positive RT-PCR or IgM result or an isolated IgG detection with at least a subsequent 4-fold increase in titer. The Statgraphics Plus 5.1 software package (Manugistics, Dresden, Germany) was used for all statistical analyses.

CHIKV strain S27 was grown on Vero cells at a multiplicity of infection of 0.5. Cells were spread on slides after 24 h, air dried, and fixed in ice-cold acetone. Serum samples were incubated for 1 h (IgG) or overnight (IgM) on fixed cells. Antibodies were detected by anti-human IgG or IgM labeled with fluorescein isothiocyanate (Sifin, Berlin, Germany). For IgM testing, IgG was absorbed before testing by Biosorb resin (Biomed, Munich, Germany). Specificity of antibodies was confirmed by plaque-reduction neutralization assay on a selection of samples (data not shown).

Two versions of real-time RT-PCR targeting the nsp1gene of CHIKV were used: 1 for CHIKV in general and 1 adapted specifically to the current epidemic strain. Reaction conditions and oligonucleotides are listed in Table 2. Quantified in vitro–transcribed RNA was generated by cloning the RT-PCR target region in plasmid pCR2.1 (Invitrogen, Karlsruhe, Germany). Inserts together with a 5′-T7 promotor sequence were amplified with M13 plasmid-specific primers and transcribed into RNA by use of Megascript T7 reagents (Ambion, Austin, TX, USA). After DNase digestion and affinity purification, transcripts were quantified by photometer and used as quantification standards.

Competitive internal controls were generated by overlap-extension PCR as described (25,26), by using ChikS/ChikAs primers and mutagenic primers 5′- ATCGTTCGTTGAGCGATTAGCAGCAGGAAGTACCACTGCGTTTGCC-3′and 5′- CTGCTAATCGCTCAACGAACGATGCACTACCAATATCCAGGATGGTTG-3′. Mutated constructs were cloned into pCR2.1 and transcribed into RNA. Both assays did not cross-react with RNA extracted from serum samples with high titers or undiluted cell culture supernatants of the following viruses: Barmah Forest, dengue, Epstein-Barr, hepatitis C, herpes simplex type 1, human immunodeficiency type 1, Japanese encephalitis, poliomyelitis type 1, Ross River, Sindbis, Venezuelan equine encephaltitis, West Nile, and yellow fever. According to series of parallel limiting-dilution experiments and probit analysis (25,26), the general CHIKV assay detected 3,844 RNA copies/mL of serum at >95% certainty (95% confidence interval [CI] 2,834–6,832 copies/mL); the assay adapted to the current Indian Ocean strain detected 2,285 copies/mL at 95% certainty (95% CI 1,694–5,326 copies/mL) (online Appendix, available from www.cdc.gov/EID/content/14/3/416-appG1.htm). On the whole panel of clinical samples described in this study, quantitative correlation between both assays was close (regression analysis slope = 1, SD 0.04, p<0.001; online Appendix, available from www.cdc.gov/EID/content/14/3/416-appG2.htm).

A fragment of the CHIKV E1 gene (positions 10265–11158, GenBank accession no. DQ443544) was amplified directly from patient plasma and sequenced. Sequencing was performed with a CEQ 8000 Genetic Analysis System (Beckman Coulter, Krefield, Germany) as recommended. Sequences were analyzed with Lasergene software package (DNASTAR, Madison, WI, USA).

Serum samples were tested for dengue antibodies by an IgM µ-capture assay (Pan-Bio, Sinnamon Park, Queensland, Australia) and by indirect IgG immunofluorescence assay as described (27). All samples that were reportedly taken during the first 12 days of illness were also tested for dengue virus by real-time RT-PCR (28).

CHIKV infection was laboratory-confirmed for 152 (22%) symptomatic patients and 188 (26%) samples. Median IgG titer for all 156 IgG-positive samples was 2,560 (range 40–40,960). Median IgM titer for 136 IgM-positive samples was 320 (range 20–10,240). Median viral load for 50 RNA positive samples was 1.7 × 10⁵ RNA copies/mL (range 1 × 10³–1.2 × 10¹⁰ RNA copies/mL). Table 3 summarizes cross-sectional testing results for individual patients at time of first visit.

In the first half of the year, the rate of confirmed infection for patients with suspected CHIKV was 24.4%; in the second half, 9.9%. Although the numbers of laboratory tests were comparable in the first and second half of the year (396 vs. 326), laboratory confirmation rates in travelers were significantly higher in the first half (χ² test, p<0.001).

We were able to retrieve 153 samples, exact information on travel destination, onset of symptoms, and date of sampling for 121 patients (Figure 1). CHIKV IgG was found for 79% of patients (125 [81.7%] of 153 samples). The earliest sample with a detectable IgG titer had been drawn on day 2 after onset; the latest, on day 252. An isolated IgG titer, i.e., no concomitant IgM, was detected in 15 samples, 14 of which had been collected late after onset (median 86.5 days, range 39–252 days). The remaining sample, collected on day 2, was positive by RT-PCR.

Demonstrable CHIKV IgM was found in 111 (72.5%) of 153 samples from 88 (72.7%) patients. The earliest IgM-positive result was obtained from a sample drawn on day 1; the latest was obtained on day 170, from 1 sample. The only IgM sample with no concomitant IgG titer was drawn on day 1 after onset of fever; it was concomitantly positive by RT-PCR.

For 11 patients whose first sample yielded antibodies, consecutive samples were available. All paired samples were drawn at least 14 days apart. The first samples were collected after a median of 13 days from onset (range 5–68 days), and all had IgM (median titer 320) and IgG (median titer 2,560). The second samples were collected after a median of 77 days from onset (range 20–207 days). Median IgG titer was 5,120; median IgM titer was 40. For 4 patients, IgM was not detectable in the second serum sample, the earliest of which was drawn 50 days after onset of symptoms.

Of the 121 patients with complete histories, 45 (37.2%) had positive real-time RT-PCR results. To compare the efficiency of diagnostic methods in acute disease, we selected all samples collected during the first 10 days of symptoms from patients in whom CHIKV was confirmed by virus isolation or subsequent seroconversion (n = 63). RT-PCR was 100% positive up to day 4, regardless of antibody results (Figure 2). IgG and IgM were 100% positive from day 5 onward. IgM was detected only 1 day earlier than IgG. Overlapping RT-PCR and antibody results occurred from day1 to day 7. No positive RT-PCR result was found after day 7.

To assess whether CHIKV RNA detection may be influenced by antiviral antibodies, we compared RNA concentrations in seronegative (n = 21) and seropositive (n = 6) samples, all collected within the first 4 days of illness (Figure 2). Median RNA concentration was 9.85 × 10⁷ copies/mL in seronegative samples, which was significantly higher than the 2.35 × 10⁵ copies/mL found as median concentrations in seropositive samples (p<0.017, analysis of variance F-test). To characterize the course of viremia in absence of antibodies, we plotted viral loads of all antibody-negative, PCR-positive samples against sampling days. The highest concentrations were found in samples drawn on day 0 with a mean of 1.2 × 10⁹ RNA copies/mL (95% CI 8.2 × 10⁸–1.6 × 10⁹ copies/mL) (Figure 2). Viral loads in later samples continuously decreased.

To reassess the diagnostic value of classic virus isolation techniques, we cultured 47 PCR-positive serum samples on Vero cells. All material had been frozen and thawed only 1 time before cell culture. Only 11 (23.4%) of 47 samples yielded a virus isolate. Virus was isolated only in samples containing >1 × 10⁷ RNA copies/mL (Figure 2). All culture-positive samples had been taken on or before the second day of fever, after a mean of 0.75 days from onset. Antibodies were not detectable in any of these samples, which suggests that infectivity in culture may be neutralized by antibodies.

Dengue virus is endemic to parts of the Indian Ocean region and resembles CHIKV infection. All serum samples received in the first half of 2006, including those from patients for whom travel and disease histories were incomplete, were tested for dengue virus. Only 3 of 368 patients had dengue IgG. Dengue IgM antibodies without IgG were found for 2 additional patients, which suggests acute infection. Both patients had traveled to Mauritius. One patient had dengue IgM and CHIKV RNA. This patient had a long period of fever (38°C–40°C for 11 days) and severe arthralgia. Another patient had dengue IgM and anti-CHIKV virus IgM and IgG. This patient had severe arthralgia for 1 month.

We sequenced 40 PCR-positive samples, of which 18 (45%) showed the original E1-226A genotype and 22 (55%) had the E1-226V mutation (2 from Réunion Island, 20 from Mauritius, all sampled in the first half of 2006). Samples from India (n = 2) and from Sri Lanka (n = 2), which were collected in the second half of 2006, displayed the original E1-226A genotype. CHIKV viral loads among genotypes were compared (Figure 3). Median concentrations (1.6 × 10⁷ copies/mL for E1-226A; 1.6 × 10⁵ copies/mL for E1-226V) were not significantly different at the 95% confidence level. To exclude an influence of CHIKV antibodies on this result, we analyzed samples without antibodies separately. Again, the difference was not significant (226A samples (n = 12), 5 × 10⁷ copies/mL; 226V samples (n = 12), 2.5 × 10⁶ RNA copies/mL).