Chikungunya virus (CHIKV) is responsible for acute febrile polyarthralgia and, in a proportion of cases, severe complications including chronic arthritis. CHIKV has spread recently in East Africa, South-West Indian Ocean, South-Asia and autochthonous cases have been reported in Europe. Although almost all patients are outpatients, medical investigations mainly focused on hospitalised patients. Here, we detail clinico-biological characteristics of Chikungunya (CHIK) outpatients in Reunion Island (2006). 76 outpatients with febrile arthralgia diagnosed within less than 48 hours were included by general practitioners during the CuraChik clinical trial. CHIK was confirmed in 54 patients and excluded in 22. A detailed clinical and biological follow-up was organised, that included analysis of viral intrahost diversity and telephone survey until day 300. The evolution of acute CHIK included 2 stages: the ‘viral stage’ (day 1–day 4) was associated with rapid decrease of viraemia and improvement of clinical presentation; the ‘convalescent stage’ (day 5–day 14) was associated with no detectable viraemia but a slower clinical improvement. Women and elderly had a significantly higher number of arthralgia at inclusion and at day 300. Based on the study clinico-biological dataset, scores for CHIK diagnosis in patients with recent febrile acute polyarthralgia were elaborated using arthralgia on hands and wrists, a minor or absent myalgia and the presence of lymphopenia (<1G/L) as major orientation criteria. Finally, we observed that CHIKV intra-host genetic diversity increased over time and that a higher viral amino-acid complexity at the acute stage was associated with increased number of arthralgia and intensity of sequelae at day 300. This study provided a detailed picture of clinico-biological CHIK evolution at the acute phase of the disease, allowed the elaboration of scores to assist CHIK diagnosis and investigated for the first time the impact of viral intra-host genetic diversity on the disease course.
Chikungunya virus (CHIKV) is an arbovirus (genus Alphavirus, family Togaviridae), transmitted by the bite of infected mosquitoes (Aedes aegypti = Stegomya aegypti and Aedes albopictus = Stegomya albopicta), that causes Chikungunya fever (CHIK), an acute febrile illness characterized by severe and often debilitating arthralgia [1]. CHIKV was first isolated in 1952 in East-Africa [2]. The name ‘Chikungunya’ refers to the stooped posture that develops in an individual as a result of arthritic symptoms and comes from the Bantu language of the Makonde people (Tanzania and Mozambique) [2]. Until recently, knowledge of CHIK clinical features was based on its first description in the 1970s [3], [4]. Since 2005, CHIK outbreaks of unprecedented magnitude have occurred in South-Asia and the Indian Ocean islands, including Reunion Island with an estimated 266,000 cases, accounting for roughly one third of the population [5]. In 2007, a viraemic traveller from India introduced CHIKV into northern Italy, resulting in an outbreak with 292 suspected cases [6]. Thus, CHIK constitutes a serious threat numerous tropical and temperate areas due to the fact that Aedes albopictus is now widespread, notably in Southern-Europe [6], [7], and displays seasonal synchronicity within potential endemic areas [8]. Although almost all CHIK patients are outpatients, most clinical and laboratory investigations of CHIK focused on hospitalised patients (i. e. mostly with severe presentations that represented a very small proportion of total infections) [9], [10], [11], [12]. Our attempts to describe the clinical and biological features of chikungunya acute disease took advantage of data collected during the CuraChik clinical trial, performed on Reunion Island during the 2006 Indian Ocean outbreak [13]. CuraChik provided a unique opportunity to collect detailed clinical and biological information from CHIKV infected patients with the most common forms of clinical presentation, recruited by general practitioners.
Amongst 76 patients included at D1, the diagnosis of CHIKV was confirmed in 54 patients (CHIKV+ve patients) and excluded in 22 patients (CHIKV−ve patients). Since the clinical and biological assessment at D1 was obtained prior to the beginning of the treatment, all CHIKV+ve patients could be used for analysis at the time of inclusion. By contrast, only the patients who received the placebo (27 CHIKV+ve and 13 patients CHIKV−ve, placebo group) were used to describe the evolution of the disease. Lymphopenia was frequent at inclusion (94% of cases with a value <1. 5 Giga per Litre (G/L); 79. 6% with a value <1 G/L). Thrombocytopenia (<150 G/L) was noted in 24% of cases and neutropenia (<2. 5 G/L, but always >1 G/L) in 33% of cases. Abnormal liver function (ALT >45 International Unit per Litre (IU/L) and AST >35 IU/L) was found in 14% and 28% respectively. C Reactive Protein (CRP) was >15 mg/L in 82% of cases, >50 mg/L in 33% and >100 mg/L in 12% (Table 1). There was no leukocytosis (>10 G/L). Five patients had anaemia (<12 g/dl), including one patient had sickle disease (8 g/dl). The average viral load at D1 was 1. 2×109 (3. 7×105–1. 4×1010, SD = 2. 3×109 RNA copies/ml). In multivariate analysis, a lower lymphocytosis was associated with a shorter time between onset of symptoms and inclusion (p = 0. 053, β = 0. 249, CI (−0. 03; 0. 502) ) and a higher viral load (p<0. 05, β = −0. 144, CI (−0. 284; −0. 004) ). A higher viral load was associated with an increase of age (p<0. 05, β = 0. 024, CI (0. 001; 0. 047) ) and a decrease of delay of inclusion (p<0. 05, β = −0. 608, CI (−1. 093; −0. 124) ).
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 [9], [10], [34], [35], [36], [37], [38]. 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 [28], 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 [9], [10], [29], [34], [39]. 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 (Figure 4) 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 [10], [40], 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 [9], [41], 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 [10], 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 (Figure 4), 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. 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 However, despite clinical improvement, it is probable that immune mechanisms are still involved [48] 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 [31], [43] and India [39] but not Singapore [44], 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 [10], [49], [50] with persistent arthralgia [31], [39], [43], [49], [50], [51]. 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 [52] but globally, previously published data suggest that symptomatic CHIK is more frequent in women [35], [38]. On Reunion Island, women were over-represented based on reported cases [24], [25] whereas cross sectional studies, based on representative groups of the population, found similar seroprevalence values in females and males [32], [53]. In two other studies, at the late stage of the disease, female gender was associated with persistent arthralgia [41] or light cerebral disorder or fatigue [27]. 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 [42] 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 [54]. 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 [42]. 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 [55] 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 [56], [57]. 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.
The mosquito-transmitted chikungunya virus is responsible for acute febrile polyarthralgia and, in a proportion of cases, complications including chronic arthritis. Since 2005, it has massively re-emerged in the Old World. Although the large majority of patients are outpatients, the most detailed studies have focused previously on hospitalised patients (i. e. , severe cases). Here, we report the detailed clinico-biological characteristics of ‘standard’ clinical presentations in patients followed-up by general practitioners in Reunion Island (2006) during the CuraChik clinical trial. At the onset of the disease, two stages were observed: (i) a ‘viral stage’ during the first 4 days, associated with an acute febrile polyarthralgic syndrome and a subsequent rapid clinical improvement; the main clinico-biological characteristics during that period were used to elaborate supportive chikungunya diagnostic scores, (ii) a ‘convalescent stage’ (days 5–14) with no detectable viraemia but a slower clinical improvement. Woman and elderly patients were found at risk for more symptomatic forms of the disease at both the acute and late stages (day 300) and we observed that the viral intra-host genetic diversity increased over time and that a higher viral amino-acid complexity at the acute stage was associated with more symptomatic illness at the late stage of the disease.
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Chikungunya Fever: A Clinical and Virological Investigation of Outpatients on Reunion Island, South-West Indian Ocean