Here we have reported a prospective study on Reunion Island of 54 adult outpatients, examined by general practitioners during the 2006 CHIK outbreak. These outpatients represent ‘standard’, ‘mild’, clinical presentations (the basis for inclusion was a recent presentation associating fever and arthralgia), which did not require hospitalisation or specific treatment for complications. Such patients represent the majority of cases: the proportion of hospitalised patients during the CHIKV 2005–2006 outbreak in Reunion Island was estimated to be 0.3% 
whilst the majority of medical investigations were dedicated to hospitalised patients. Thus our knowledge of severe presentations is currently more accurate than that of common presentations that do not require hospitalisation.
On Reunion Island, the epidemiological surveillance system was based, at first, on active and retrospective case detection around the reported cases, and then relied on a sentinel network of general practitioners 
. Accordingly, some clinical data regarding outpatients could be collected from cases notified through either the epidemiological surveillance system 
, or community based cross sectional survey 
. However, the unique aspect of the current study relies on the detailed clinical and virological follow-up of such patients. It included laboratory confirmation of CHIKV infection (based on RT-PCR and seroconversion), a clinical follow-up at the acute phase (associating daily auto-questionnaires and medical consultations), and late assessments (by telephone until day 300). The study took place at the end of the outbreak (i.e.
, the enrolment was performed by practitioners who had previously managed approximately one third of the population infected by the virus), and there is no evidence that another arbovirus (e.g.
dengue virus) has been circulating in Reunion Island during this period.
This study has obvious limitations. Firstly, a low patient count: 54 patients, with a confirmed CHIKV infection, was studied on day 1 (inclusion), but the follow-up was performed for only 27 of these patients, corresponding to the placebo arm of the clinical trial. Secondly we could not exclude a potential impact of a placebo effect during the follow-up. Thirdly, 22 patients with a negative diagnosis of CHIK (of whom 13 received placebo) were also included but, regarding outpatients and mild presentations, the aetiology of their disease could not be further investigated. Finally, the number of patients for which in depth analysis of intra-host viral genetic diversity was analysed was low (10 patients) and, despite interesting and statistically significant results, this specific aspect will deserve in the future analysis from a larger cohort.
Assessment at inclusion
The clinical presentation of CHIK at inclusion revealed a quite severe impact of the disease on quality of life, with more than half of the patients' scores <30/100. It conformed with the canonical presentation previously reported in Reunion Island and in the recent Indian reports, which included fever and symmetrical poly-arthralgia 
. However, this simplistic association (fever+polyarthralgia) seems to perform modestly for the specific diagnosis of CHIK: in a retrospective serologic survey of the CHIK outbreak in Mayotte Island 
, the PPV was as low as 74%. In our study, despite the great recent clinical experience of general practitioners, the PPV was similar (71%).
Looking into further details, it appears that arthralgia was most commonly observed in small joints (i.e.
, wrists, ankles, hands) and knees, as reported from both in- and outpatients 
. More precisely, this study highlighted the massive number of arthralgic joints (16, on average) and the specific importance of (i)
small joint pain such as MCP/PIP or wrist and (ii)
a minor or absent myalgia for the diagnosis of CHIK at the onset of the disease.
This strongly suggests that a convenient diagnostic score may profitably guide the diagnosis of CHIK at the acute stage of the disease. We proposed a very simple and purely clinical score () which reached 87% PPV in our population (i.e., outpatient 18 to 60 years old, examined before the second day of illness). For convenience, results were categorised as ‘probable’, ‘possible’ and ‘not probable’.
Our data also highlighted the high level of viral load (1.2×109
RNA copies/ml on average) at inclusion. It was slightly higher than in other reports 
, possibly due in the current study, to the very short delay between onset of disease and inclusion. Viral load at D1 significantly increased with age but no relationship with clinical presentation or co-morbidity could be identified. In contrast with previous studies dedicated to hospitalised patients 
, we did not identify a relationship between the level of CRP and transaminases, a reduction of the polymorphonuclear neutrophil level or other biological abnormalities, and the intensity and number of arthralgia or the quality of life at D1. However, as previously reported from inpatients 
, we observed that lymphopenia (<1G/L) was closely related to the level of viraemia. It constituted an important clue for the diagnosis of CHIK as illustrated by our clinico-biological score (), which again, classified the patients as ‘probable’, ‘possible’ or ‘not probable’, as a function of the probability to be infected by CHIKV, based on clinical presentation and lymphocyte count. The PPV of this score reached 94% in our population, for a threshold of 0.579.
Evolution of the disease
The most original input from the CuraChik protocol was the detailed information collected (patient self-assessment from D1 to D14, medical consultations (D1, D7, D25), biological analyses (D1, D6, D16)), which altogether provided an accurate description of the evolution of patients during the acute stage of the disease.
Deciphering these data indicated that the acute disease includes 2 distinct stages
- the first (D1–D4, ‘viral stage’) was associated with viraemia, i.e., clinical symptoms that reflect the viral ‘burst’ and the initiation of innate immunity, associated with high levels of pro-inflammatory cytokines , ,  such as interferon-α and IL-6 but also IL-1Ra, IL-12, IL-15, IP-10 and MCP-1. This is in coherence with results from Chow et al., who extended the ‘acute phase’ of the disease until D4 . In all cases in the current study, this response was associated with a rapid decrease of viremia from D1 to D3 (39.6% of patients remained viraemic at D3). In parallel, the clinical presentation improved promptly: the daily self-assessment, showed a rapid decrease of the number of arthralgic joints (from 16 to 9) () and all quality of life parameters dramatically improved ().
- From day 5 to day 14 (‘convalescent stage’), all patients had no detectable viraemia, but improvement was slower, considering both quality of life scores () or rheumatic parameters ( and supporting information figure S1 and movie S1). The initial symptoms (e.g., fever and shiver that fell from >80% at D1 to 10% at D7) receded but by the D7 medical visit >40% of patients had persistent arthralgia, asthenia, myalgia or headache. Dermatological signs were more often identified on D7 than D1 (), in coherence with studies reporting skin lesions mostly after disease onset , , . This observation may provide an explanation for the large range of frequency of dermatological signs in the literature (from 10% to 86%) , . Previous reports  showed that, by day 10, a number of inflammatory mediators (including interferon-α, IL-6, IL-1Ra, IL-12, IL-15, IP-10 and MCP-1) had significantly decreased, at least in patients with initial high viraemia.
However, despite clinical improvement, it is probable that immune mechanisms are still involved 
at this stage. The final outcome (complete clinical recovery or persistent pain and chronic joint inflammation) appears likely to depend upon a series of genetic, viral and immunologic factors that operate at the acute and convalescent stages. On Reunion Island 
and India 
but not Singapore 
, late complications were associated with severe acute disease. Here, in agreement with the clinical pattern observed on Reunion Island, patients with a high number of arthralgic joints at disease onset reported more frequently persistent arthralgia at D300.
The early and convalescent immune response may be, in addition to putative yet uncharacterised viral factors, modulated by innate (genetic) and acquired factors. The latter certainly include age, which appears in many studies to be a major determinant of the clinical presentation and outcome. Here, we found that an increase of age was an independent risk factor for symptomatic illness at the time of disease onset (number and intensivity of joint pains) and at D300 (number of cases with persistent arthralgia). At D300, the patients who did not report recovery and who reported persistent arthralgia were significantly older. These results are consistent with studies on hospitalised patients and Indian report which reported that elderly patients more frequently presented with atypical feature or a severe course 
with persistent arthralgia 
Genetic factors presumably trigger different immune responses which may account for the inter-individual and inter-ethnic variability of clinical presentation. Amongst them, gender is of specific interest. A single report mentioned a higher susceptibility of males, to CHIKV infection 
but globally, previously published data suggest that symptomatic CHIK is more frequent in women 
. On Reunion Island, women were over-represented based on reported cases 
whereas cross sectional studies, based on representative groups of the population, found similar seroprevalence values in females and males 
. In two other studies, at the late stage of the disease, female gender was associated with persistent arthralgia 
or light cerebral disorder or fatigue 
. In the current study, females were independently associated with a high number of painful joints at disease onset and at D300. They also reported non-recovery more frequently at D300. It is difficult at this stage to distinguish between gender-related specific clinical susceptibility and a different perception of the disease in males and females.
The interplay between the immune response and viral evolution most probably constitutes an important issue for disease outcome. A non-primate animal model 
showed that CHIKV could persist much longer than previously believed and such persistence may imply the existence of specific adapted variants, or at least depend on the kinetics of viral clearance by the immune system. To our knowledge, no study had previously described the intra-host genetic diversity of CHIKV in human samples. We observed that CHIKV was represented in serum by a variety of closely related genomes and that genetic diversity increased over time and was correlated with the decrease of viral load. This observation relied on the analysis of sequential serum samples from 2 patients, but was also supported by the analysis of other sera for which analysis of the intra-host genetic diversity of CHIKV was made available: the later the serum was sampled, the higher was the intra-host genetic diversity, based on percentage of mutant clones and average π aa and dN. These data are consistent with a mechanism in which acute infection produced an accumulation of mutations over time (resulting in an increased intra-host genetic diversity), associated with a lower number of virions and, possibly, an increased potential for persistence. Interestingly, we found that a higher amino-acid complexity at the acute stage was associated with increased reporting of arthralgia and intensity of sequelae at D300. This may indicate that the immunological processes associated with the initial viraemia decline or are partly circumvented, thus enhancing the opportunity for onset of virus persistence and long term clinical complications. In an experimental model, Coffey et al.
previously observed that a CHIKV variant with high fidelity polymerase produced truncated viraemia and lower organ titres and suggested that reduced genetic diversity impacts negatively on virus fitness in both invertebrate and vertebrate hosts 
. Moreover in the macaque model, long-term CHIKV infection was observed in joints, muscles, lymphoid organs, and liver which could explain the long-lasting CHIK symptoms observed in humans 
. Altogether, these data are consistent with the hypothesis that CHIKV displays increased intra-host diversity which may be associated with prolonged viraemia, higher organ viral load and an increased risk of chronic disease. If this is the case, such mechanisms appear to be quite different from those previously observed in the case of dengue fever, where lower intra-host diversity has been associated with more severe cases 
On the other hand, our results appear more closely related to previous reports on the relationship between intra-host genetic diversity, fitness and virulence in the examples of chronic infections by induced HIV or HCV 
. Some additional studies are needed to further characterise the intra-host genetic diversity of CHIKV at the acute phase of the disease. Sequential analysis provided by New Generation Sequencing tools may provide a more accurate picture of this diversity and allow a powerful analysis of the relationship between the structure and evolution of intra-host viral genetic diversity and the clinical evolution of CHIKV infected patients.