Introduction Chikungunya virus (CHIKV) is an arthropod-borne virus that belongs to the Alphavirus genus. Chikungunya disease is characterized by polyarthralgia, sometimes associated with rash. The articular symptoms, often debilitating, usually resolve within weeks, but have been reported to last for months, even though the natural history of this infection has not been precisely studied in prospective studies [1], [2], [3]. In 2005, CHIKV emerged in islands of Indian Ocean including La Réunion, a French overseas department, and approximately one third of the inhabitants (i.e. ∼300,000) was infected at the end of the outbreak in 2006 [4], [5]. Compared to earlier outbreaks, this episode occurred in a highly medicalized area. Moreover previously unreported severe forms of Chikungunya were observed, such as encephalopathy [6], [7], and mother-to-child CHIKV transmission was demonstrated, leading to severe neonatal CHIKV infection [5]. In the wake of this outbreak, CHIKV also re-emerged in India with over 1 million cases [8], [9]. In 2007, CHIKV emerged for the first time in Europe, causing an outbreak in Italy [10]. We have described the clinical and biological features of acute CHIKV infection in a prospective cohort of patients with positive blood CHIKV RT-PCR [11]. It included all patients referred to the Emergency Department in Saint-Pierre de la Réunion with febrile arthralgia between March and May 2006. As little is known about long-term outcome of CHIKV infection, we conducted a prospective longitudinal study to describe in details the specific clinical and biological features of chronic arthralgia, as well as clinical signs associated to this pathology. We evaluated the consequences of long-term arthralgia on patients' daily and social life, looked for risk factors associated with them and estimated their economic impact. Materials and Methods Ethics & STROBE statement Ethical clearance was obtained from the «Comité de Protection des Personnes Sud-Ouest et Outre-Mer III» of Saint-Pierre, La Réunion, Paris (CCP 2008/65, n° 2008-A00999-46). CHIK-IMMUNOPATH received approval from the ethical committee for studies with human subjects (CPP) of Bordeaux and the National Commission for Informatics and Liberty (CNIL). Written informed consent was obtained from patients included in the CHIK-IMMUNOPATH study. The study respects the STROBE statement (supporting information S1). Patients We studied a cohort of patients (n = 180) enrolled for febrile arthralgia to the Emergency Department of the Groupe Hospitalier Sud Réunion between March 2005 and May 2006 [11]. Patients were interviewed by telephone 4, 6, 14 and 36 months (M4, M6, M14 and M36) after the viremic phase, using the same questionnaire as that used at day 0 (D0) (supporting information S2). At M36 after the acute phase, all patients who agreed to participate to a complementary study (CHIK-IMMUNOPATH) and who were arthralgic were interviewed and underwent clinical examination. Among them, 22 patients with arthralgia (ART+) and 20 patients without persisting arthralgia (ART−) were randomly selected from the cohort. They signed a written consent and a blood sample was collected. Laboratory tests performed at M36 Blood cell count was performed and viremia was tested by qRT-PCR [12]. Both serum anti-CHIKV IgG and IgM specific antibodies were screened. An enzyme-linked immunosorbent assay (ELISA) was performed with CHIKV antigen [12]. The avidity of anti-CHIKV IgG was tested by ELISA in the presence or absence of urea 8 M [13]. Geometric Mean Antibody Titer (GMAT) was calculated as previously described [14]. Plasmatic protein electrophoresis was performed and C-reactive protein (CRP) concentration was measured. The presence of anti-nuclear, anti-dsDNA, anti-endomysium autoantibodies, anti-cyclic citrullinated peptide antibody (ACCP) and cryoglobulinemia were investigated. Samples were transported at 37°C to research cryoglobulins. Sera were sent to Myriad RBM (Austin,Texas) and analyzed by Luminex using the inflammation MAP. Assays are run according to CLIA guidelines and in all cases, >100 beads per analyte were measured with CV 2) (Figure 2C). Despite an increase of arthralgic sites at M36, there is an overall significant decrease of the number of painful joints during the study period (p 70 45 27 (60.0%) 0.62 (0.21–1.79) Hospitalization at D0 No 101 61 (60.4) 1 0.66 Yes 46 26 (56.5) 0.85 (0.42–1.73) Viral quantification at D0 (copies/mL) ≤100.000 70 39 (55.7) 1 0.41 >100.000 77 48 (62.3) 1.32 (0.68–2.54) CRP at D0 (unit) 10 126 79 (62.7) 3.12 (1.16–8.38) Missing 1 1 (100.0) No estimable Diabetes at D0 No 109 58 (53.2) 1 0.01 Yes 38 29 (76.3) 2.83 (1.23–6.54) Arthralgia at M4 No 26 5 (19.2) 0.10 (0.04–0.30) 35 Sex ratio (M/F) 1/1 Number of sites oligo or polyarthralgia Sites upper limbs : fingers. wrist lower limbs : knees. ankles Type symmetrical permanent or not migratory highly incapacitating morning stiffness duration average: ≈30 minutes General sign fever no fever Other clinical signs edema cutaneous lesion myalgia sleep and memory disorders Laboratory tests CRP normal ACCP normal antinuclear normal anti CHIKV IgGs positive Risk factor diabetes Our data reveal that more than 60% of CHIKV-infected patients suffer from arthralgia, 36 months after acute infection. This high percentage of patients with long-term symptoms was also reported by other studies of Italian cohorts and French cohorts of La Réunion Island or metropolitan France [17], [18], [19], [20], [21] but is dramatically higher than documented in India and Senegal [1], [22], [23], [24], [25]. While this discrepancy may result from particular features of the CHIKV strain responsible for the La Réunion outbreak, data from Italy following the 2007 outbreak resulted from a CHIKV strain more closely related to the viral strain present in India [10], with more than 60% of CHIKV patients in Italy having reported myalgia, asthenia or arthralgia 12–13 months after the acute disease [20]. Alternatively, reported differences may be a result of different genetic backgrounds of these populations. As joint pain is considered a subjective symptom, it might also reflect a difference in pain threshold of patients or reporting from physicians, thus reflecting differences in health care practices. Long-term CHIKV-associated arthralgia were mainly symmetrical, involving more than 2 different joints. Hand, wrist, ankle and knee were found to be the most affected, consistent with other studies [17], [18]. Moreover, 60–80% of patients had relapsing arthralgia, while 20–40% had unremitting arthralgia. While some patients reported “migrating” arthralgia, most disease symptoms mapped to joints that were most painful during acute Chikungunya disease. Thus, symptoms at the chronic phase may be indirectly associated to virus replication at the time of acute infection [26], [27], [28]. In addition to arthralgia, many patients suffered from myalgia and cutaneous lesions and several cognitive dysfunctions. Although study patients did not display neurological symptoms at the acute phase of disease, we cannot exclude that cognitive dysfunctions result from CHIKV spread in the CNS, as it has been reported that CHIKV disseminates to the CNS in humans and in animal models [12], [26], [27], [29]. Similar to other studies, chronic arthralgia are considered incapacitating for daily life tasks and impacted professional activities and quality of life [20], [21]. Beside this impact on patient, the economic burden of this long-term pathology is also very significant, independently of the cost of the acute disease [30]. The longitudinal design of our study enabled us to identify risk factors for development of long-term arthralgia. Individuals over the age of 35 years or with diabetes were more likely to suffer from chronic arthralgia. The age has been reported to be a risk factor with some cohorts [21], [31], [32], but not in others [18], [33]. None of our available parameters to measure the severity of acute disease were associated with long-term arthralgia. This may be explained by differences in the way to measure disease severity in other studies [18]. Importantly, we show that the presence and intensity of arthralgia at M4 after the onset of the acute disease is a good predictor of long-term arthralgia. Our study did not identify positive markers for autoimmune or rheumatoid arthritis. Additionally, we failed to identify systemic biomarkers associated with the arthralgic phenotype. Nevertheless, a slightly more elevated inflammatory status is found in a subset of arthralgic patients who have detectable serum level of IL1α, IL15 and slight elevation in Factor VII, C3 and CRP. This signature differs from that observed at the onset of the infection, when circulating virus is detectable and type I interferon, IP10, MCP1, ISG15 are highly elevated [34], [35], [36]. Others have identified IL6 and GM-CSF or IL12 as being specifically associated with long-term arthralgia [32], [33]. However, these studies were performed much earlier in the chronic phase (2–3 months and one year after disease onset). Our study shows that anti-CHIKV antibody titers were more elevated in ART+ patients than in ART- patients. This is in agreement with a recent study [37]. This higher level of antibodies could be associated with a more severe acute infection [37]. However, in our study, the level of antibody at M36 did not correlate with acute disease severity. Alternatively, this could reflect a persisting antigenic stimulation in ART+ patients (see below). Interestingly, it has been reported that viremia level at the acute phase correlates with a faster appearance of neutralizing antibodies and a better recovery 2–3 months after the acute phase [38]. Similarly to CHIKV, other so called “arthritogenic” alphaviruses, notably Ross River virus (RRV), are known to cause acute as well as chronic arthralgia [39]. Our data indicates that chronic symptoms are linked to the initial local joint inflammation and are not associated with markers of systemic inflammation or autoimmunity. A local inflammation of the joint could be maintained by the local persistence or delayed clearance of viral antigens. This is consistent with report of Hoarau et al. [32] who detected persistent CHIKV antigens within the synovial fluid of a patient suffering from chronic arthralgia. Moreover, experimental studies in CHIKV infected animal indicate that the joint is the most highly infected tissue, making it plausible that incomplete viral antigen clearance in this anatomical site may account for the long-term symptoms [26]. RRV has been shown to persist in vitro in mouse macrophages, and a model of RRV chronic arthritis suggests that viral persistence may account for chronic disease [40]. RRV and CHIKV have been shown to be weakly tropic for macrophages in vitro [41], [42] but the presence of antibodies dramatically increased RRV entry into macrophage [42]. Further studies will be required to assess the role of macrophages, as well the role of persistent infection and antibodies in chronic arthritis caused by CHIKV. In sum, this study furthers our understanding of the pathophysiology of CHIKV chronic arthralgia, a prerequisite for the development of efficient therapeutic strategies and for assessing the burden of disease inflicted upon populations affected by epidemic Chikungunya disease. Supporting Information Supporting Information S1 Strobe checklist for cohort study. (DOC) Click here for additional data file. Supporting Information S2 Questionnaire used for the study. (DOCX) Click here for additional data file. Supporting Information S3 Modelisation and estimation of spatiotemporal dynamics of arthralgia. (DOC) Click here for additional data file. Table S1 Likelihood ratio tests testing the complete model against different submodels. (DOC) Click here for additional data file. Table S2 Estimation of the annual economic impact of CHIKV long term arthralgia. (DOC) Click here for additional data file.
