On March 26, 2016, a 27-year-old woman visited the infectious diseases outpatient department because of persistent arthralgias and rash. She works as a stewardess on a cruise ship and she stayed in Costa Rica for 2 months where she developed symptoms; then she returned to Croatia on March 21. Six months before this illness, she was vaccinated against yellow fever. She remembered a mosquito bite on her left forearm because she noticed a little hematoma around the indurated papule which she had not experienced after earlier mosquito bites. Five days after the mosquito bite, on the first day of her disease, she felt symmetrical arthralgias in her ankles, knees, wrists, and elbows but also in small joints of her hands and feet. The third day, she developed high fever up to 38.5 C with chills. Her arthralgias deteriorated, so she could hardly walk. On the fifth day of her illness, an itchy rash appeared on her trunk, and the next day it extended to her face, extremities, as well as palms and soles. The erythematous papular rash subsided in the next 2 days. At the same time, she became afebrile, while arthralgias remained up to the tenth day of her illness after which she was feeling well, but exhausted. During the acute phase of her illness, she had nausea and she had a few loose stools too. Ten days after she had recovered, severe arthralgias in her ankles, elbows, right shoulder, and in the small joints of hands and feet reappeared. She also complained about morning stiffness lasting for 10-30 min.
On the initial visit (day 15 after the disease onset), the physical examination was normal except for rash on the trunk, extremities, palms, and soles (Figure 1). Routine laboratory tests were normal, except for slightly elevated liver transaminases, erythrocytes 4.35x1012/L (reference range [RR]: 3.86-5.08), leukocytes 8.4x109/L (RR: 83.4-9.7), lymphocytes 21.5% (RR: 20-46), C-reactive protein 1.3 mg/L (<5), platelets 219x109/L (RR: 158-424), BUN 5.4 mmol/L (RR: 2.8-8.3), creatinine 70 micromol/L (RR: 63-107), aspartate aminotransferase 32 U/L (RR: 8-30), alanine aminotransferase 51 U/L (RR: 10-36), gamma-glutamyl transferase 21 U/L (RR: 9-35), and lactate dehydrogenase 196 U/L (RR: 25-241).
According to the case definition proposed by the international chikungunya expert group (2015), which distinguishes 4 chikungunya case categories; acute clinical case, atypical case, severe acute case, and suspected and/or confirmed chronic case; our patient was classified as a confirmed chronic chikungunya case. The patient was treated with non-steroidal anti-inflammatory drugs and paracetamol. She was regularly followed up during the next few months in order to treat her arthralgias and also to observe the progress of her illness. Her symptoms ameliorated, but 3 months later she still reports arthralgias.
Due to similar geographical distribution and clinical symptoms, the serum sample (taken on day 20) was initially tested for CHIKV IgM/IgG antibodies, using indirect immunofluorescent assay (IFA; Euroimmun, Lubeck, Germany) as well as Zika virus (ZIKV) IgM/IgG and dengue virus (DENV) IgM/IgG antibodies using enzyme-linked immunosorbent assay (ELISA; Euroimmun). To exclude potential cross reactivity with other arboviruses, the sample was additionally tested for West Nile virus (WNV) and Usutu virus (USUV) antibodies using ELISA (Euroimmun), as well as yellow fever virus (YFV), Japanese encephalitis virus (JEV), and tick-borne encephalitis virus (TBEV) using IFA (Euroimmun). Serology results are presented in Table 1. CHIKV IgM and IgG antibodies were detected with titre 1:100 and 1:10,000, respectively (Figure 2). In addition, postvaccinal YFV IgG antibodies were found (titer 100). There was no cross reactivity with other tested arboviruses.
In addition, the sample was tested for the presence of CHIKV RNA using a qualitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) according to the protocol described by Smith et al. For nucleic acid isolation, an automated system QIAxtractor (QIAGEN, Hilden, Germany) was used. Real-time RT-PCR was performed using a single-tube RT-PCR test kit (Invitrogen SuperScript III Platinum  One-Step Qualitative Kit; Carlsbad, CA, USA). The amplification and detection were performed with 7500 Real Time PCR System (Applied Biosystems, Foster City, CA, USA). The test tube contained a 25-microL reaction mixture which included 5 microL of isolated RNA, 0.2 microM forward primer (CCGAAAGGAAACTTCAAAGCAACT), 0.2 microM reverse primer (CAGATGCCCGCCATTATTGAT), and 0.1 microM probe (FAM-GGGAGGTGGAGCATG-MGB). The reaction mixture was exposed to a 30 min 50 C reverse transcription step, 2 min of Taq activation at 95 C, and 50 cycles consisting of 95 C for 15 sec and 55 C for 32 sec. No CHIKV RNA was found in the tested sample. Sensitivity of the RT-PCR is reported to be high (0.3 plaque-forming units; PFU/mL), as is specificity (no cross reactivity with the most common alphaviruses that contained a minimum of 3 log10 PFU of heterologous viral RNA).