We carried out a prospective study of the clinical and biological features characterizing the acute viremic and post-viremic phases of human Chikungunnya infection in patients referred to the ED for febrile athralgia. The patients without Chikungunya entering the ED with fever and arthralgia during the study were used as a control group. The monthly numbers of newly admitted patients with CHIKV viremia in our study were consistent with the estimated incidence in La Réunion 
(March 2006 32,500, April 11,800, May 5,300). The percentages of viremic patients were stable along the study and represented 0.5% of the total estimated new cases in the Island in March, 0.45% in April and 0.45% in May. Consequently, the newly admitted viremic patients at the ED are representative of the epidemic. Yet, this study does not take into account asymptomatic infections, but they are thought to represent an extremely low proportion (5%) 
when compared to other arbovirosis such as dengue (80%) 
To our knowledge, this is the first prospective study on the clinical and biological aspects of acute Chikungunya infections in adults with differentiation between the viremic and non-viremic phases during an outbreak. Previously published clinical and biological data were obtained from retrospective analyses, usually in the absence of virological confirmation of acute infection. Several studies have used anti-CHIKV IgM as an acute phase criterion but they are still present in more than 50% of patients a year after the onset of the disease 
. Simon et al
. conducted a prospective observational clinical study during the early stages of Chikungunya infections (within 10 days of the disease onset) but did not differentiate between the viremic and non-viremic phases 
. In addition, they included in their study data from a retrospective questionnaire completed by patients examined 10 days after the onset of symptoms. Recent retrospective studies reviewing cases that occurred in the Indian Ocean were published. Taubitz et al
. described clinical and biological results for 20 travellers admitted in their department 2 to 73 days after the onset of symptoms 
. Borgherini et al
. published early biological and clinical features for 157 adult patients during the outbreak on Reunion Island 
. Borgherini's study displays the biases inherent to retrospective surveys. In particular, the patient's selection and data collection processes did not include frequent clinical signs such as myalgia. This study also differs from previous works since its patients constitute an unbiased representative sample of the population enrolling at the ED over a continuous three-month period, and describes the clinical and biological features of the viremic and post-viremic phases of the acute CHIKV infection, as assessed by RT-PCR and IgM assays in 216 consecutive patients.
Moreover, most of these retrospective studies were carried out in countries where co-infections with arboviruses such as Dengue and Yellow-fever viruses are likely to occur 
. We also searched for co-infections. No case of dengue fever was diagnosed in our study, and no cases of dengue fever or other arbovirosis was reported in 2006 in La Réunion, therefore reinforcing the specificity of our findings. Malaria and leptospirosis were not diagnosed in our patients infected with CHIKV. We found rare and benign hemorrhagic signs, in contrast to what previously reported in India and Southeast Asia —areas where Dengue is endemo-epidemic— where CHIKV was incriminated in haemorrhagic fever outbreaks 
. Hemorrhagic signs in other studies among adults were probably attributable to co-infection, especially with dengue or yellow fever viruses, even if Asian CHIKV genotypes differ from the one responsible for the Indian Ocean outbreak, which is of East African origin 
. During the outbreak in La Réunion, hemorrhagic signs were described in our prospective study of neonatal Chikungunya infection 
and thrombocytopenia and haemostasis troubles were also common in the neonatal context.
Since 1953, the classical clinical features of acute Chikungunya associate the triad fever, arthralgia and inconstant skin rash 
. We showed here that fever is concomitant of viremia, with the highest temperatures in the 2 days that follow the onset of the symptoms.
Arthralgia is key to the clinical diagnosis of acute CHIKV infection. However, forms with secondary arthralgia or without arthralgia have been described in Asian studies 
. In a random sample of the population of La Réunion during the post-epidemic phase, 9.7% of proven Chikungunya cases (i.e
. with a positive serology) reported no arthralgia, including the 5% asymptomatic forms 
. In our study, the presence of arthralgia was mandatory for inclusion, and asymptomatic forms were obviously not referred to the ED. CHIKV-associated arthralgia have not been characterized in detail so far 
. While in most cases, bilateral and symmetrical polyarthralgia were reported (although sometimes more intense on one side), we observed two cases of monoarticular arthralgia of the ankle. We recorded rare locations of arthralgia, such as of the temporo-mandibular joint, and forms that had never been reported in previous descriptions such as chondrocostal arthralgia, affecting up to 20% of the patients, and hip arthralgia (17%). CHIKV also appeared to revive pain along former fractures lines and ligament injuries, and clinical examination revealed entesopathies and talalgia, which had never been described previously.
The frequency of skin rash ranged from 20% to 85% 
. In our study, about half of the patients presented a skin rash, generally arising during the first three days of infection. Our study matches former studies in the location and aspect of the skin lesions but differs by the existence of vesicular and bullous forms as well as erysipela-like lesions of the lower limbs. By mean of RT-PCR, we could document the specificity of these skin manifestations by amplifying the viral genome within this vesicular fluid (our unpublished data).
Our study illustrates the variety of the other clinical manifestations that can be associated with CHIKV infection. Yet, these symptoms are non-specific as they can be present in a number of other viral infections. Some of these clinical symptoms may also correspond to decompensation of underlying conditions and to iatrogenic effects (e.g. paracetamol and hepatitis, non-steroidal anti-inflammatory and kidney failure).
Severe clinical forms with neurological 
and cardiac involvements 
have also been reported in the literature. In 1973, Obeyesekere 
described the first cases of acute pericarditis and myocardiopathy associated with CHIKV, based on serology. Thus, CHIKV may, as other viruses, trigger pericarditis and myocarditis. Lemant et al
, in a retrospective survey of CHIKV-infected patients admitted to intensive care, reported a case of fatal fulminant myocarditis for which analysis of post-mortem myocardial tissue samples pathological examination demonstrated the presence of cytoplasmic viral inclusions in myocytes. Cardiac complications and myocarditis have also been described in infected newborns on the Island of La Réunion 
Neurological symptoms (vertigo or confusion) observed in our study may be attributable to fever and dehydration. However, a neurotropism of CHIKV has several times been suspected 
. Mazaud 
reported a case of acute encephalitic syndrome, and Deller 
described the onset of vertigo and a vestibular syndrome. In India, Carey 
observed important neurological after-effects. In La Réunion, severe cases of meningo-encephalopathy among adults 
and newborns 
, and acute polyradiculoneuritis were also attributed to CHIKV 
. Consistent with these clinical observations, we have shown, in an animal model for Chikungunya, that CHIKV may disseminate to the central nervous system, and infect the meninges and the ependymal tissue 
observed biological similarities between dengue and chikungunya. Indeed, it was reported that these two arbovirosis are associated with brief leukoneutropenia (3 to 4 days) and thrombocytopenia 
. According to this author, leukoneutropenia and thrombocytopenia have great orientation value in favour of these two arboviroses. In other studies, no leukopenia was reported 
. In contrast to these retrospective studies in which the differential diagnosis between dengue and chikungunya could not be precisely assessed, we found in our series that 38% of patients were leukopenic and that in Group A1, lymphopenia dominated and was very closely associated with viremia; for 85% of the patients lymphocyte count was lower or equal to 1,000/mm3
and for 50% of the patients the level was lower than 500/mm3
. Thrombocytopenia was found in 42% of the patients, but to moderate levels, between 100,000 and 150,000/mm3
Cases of severe acute hepatitis occurring during the CHIKV infection have already been described in La Réunion. While it was suggested that this virus may target the liver –a finding confirmed in our animal model in the first hours of infection–, acute hepatitis seems to be mainly promoted by chronic ethylism, denutrition and paracetamol toxicity 
. The serum ASAT level was increased for 32% of the patients as opposed to only 7% for the ALAT. These results suggest that the increase in the serum level of ASAT was partially related to muscle injury, with 60% of the patients with increased ASAT also presented a rhabdomyolysis.
When using the two cardinal clinical signs in acute phase, fever and arthralgia, for the diagnosis, we found a specificity of 99,6% and a positive predictive value of 84.6%. Leucopenia and lymphopenia could be used to aid in the diagnosis of CHIKV infection in acute phase, but not for formal diagnosis. The positive predictive values for these parameters, found in our work, are inappropriate. Indeed, the high number of CHIKV-infected patients compared to controls tends to artificially raise the positive predictive values and decrease the negative predictive value. So, leucopenia/lymphopenia values are suggested as only one parameter that could be a part of a diagnosis algorithm. Other mosquito-transmitted viruses with overlapping geographic distributions, specifically Dengue Fever, can have similar hematologic abnormalities. However because of the risk of transmission of the Chikungunya fever in European countries free of Dengue Fever, these hematologic abnormalities are of interest and value.
Factors influencing disease severity are dominated by age and comorbidities. However, these two criteria are strongly linked. Among viremic patients, age under 65 was an excellent predictor of non-severity (severity negative predictive value 85.6%), with only functional impotence due to lower limbs pain and inability to walk leading to hospitalization in this group.
Little is known about the pathophysiology of CHIKV infection, in contrast to better characterized alphaviruses such as Sindbis and Ross River viruses. Sourisseau et al
have reported that CHIKV replicates in various human cultured cells, such as epithelial and endothelial cells, primary fibroblasts and, to a lesser extent, macrophages. CHIKV replication induces a significant cytopathic effect, and may also lead to apoptosis. CHIKV replication is significantly inhibited by type I IFNs, suggesting a prominent role of the innate immune system in the control of the infection and the rapid decline of viremia before the appearance of neutralizing antibodies during the acute phase of infection. It has also been reported that CHIKV may also infect muscle satellite cells in humans 
. In a mouse model of chikungunya, we have shown that fibroblasts of tissues in which seats symptoms in humans (muscles, joints, skin), are the target cells of CHIKV 
, these findings matching viral tropism in human as assessed by CHIKV immunolabeling of infected human tissues. The infection of muscles and joints, which contain nerve endings may account for the nociceptive pain observed in the course of human acute infection. The functional neurological manifestations complicating the course of acute CHIKV infection may reflect CHIKV dissemination to the central nervous system, consistent with the positive CHIKV RT-PCR in the cerebrospinal fluid of these patients and the observation that CHIKV infect the choroid plexuses, meninges and the ependyma in the mouse animal model 
The worldwide distribution of Aedes albopictus
and the adaptation of the virus to this vector 
may cause the emergence and spread of epidemic CHIKV in North and South America and in European countries, as illustrated recently in Ital 
. In addition to the basic original information it provides on this little known disease, this study should be of help to clinicians confronted to a CHIKV outbreak, for clinical and logistic management of acutely infected patients presenting at the emergency room.