The authors have declared that no competing interests exist.
Conceived and designed the experiments: SDT XDL AF. Performed the experiments: SDT VB. Analyzed the data: SDT XDL JG FS. Contributed reagents/materials/analysis tools: SDT XDL VB AF. Wrote the paper: SDT XDL.
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.
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 (
Chikungunya virus (CHIKV) is an arbovirus (genus
Although almost all CHIK patients are outpatients, most clinical and laboratory investigations of CHIK focused on hospitalised patients (
We aimed to provide a precise clinical and biological description of acute laboratory-confirmed CHIKV infection in outpatients and some information regarding follow-up until day 300.
We also compared CHIKV positive and negative patients recruited on the basis of clinical presentation with acute febrile arthralgia during an epidemic period.
We finally performed a comprehensive comparative analysis of intra-host viral genetic diversity.
The main details of the CuraChik trial have been reported elsewhere
This trial included adult patients (18–65 years old, men and women), who volunteered to take part in the study, residing in Reunion Island, having a typical presentation of acute CHIK (defined by acute febrile arthralgia) diagnosed within less than 48 hours. Exclusively general practitioners (GPs) enrolled the eligible patients.
Clinical data were collected from three sources:
Biological data were collected from the analysis of blood samples on D1, D3, D6 and D16. The extraction of nucleic acids and CHIKV specific RT-PCR
The case definition of CHIKV positive patients relied on the association of a CHIKV specific positive RT-PCR on D1 and seroconversion on D16. CHIK negative patients tested negative for CHIKV genome on D1 and showed no evidence of seroconversion on D16.
The study commenced on May 20, 2006, after obtaining authorisation from the French Health Products Safety Agency and Ethics Committee. All subjects provided informed written consent.
Ten patients with a variety of clinical and biological presentations and a positive CHIKV RT-PCR diagnostic test were selected within the placebo subgroup. The extraction of nucleic acids was performed from D1 and D3 sera using the EZ1 virus MiniKit (virus card 2.0) and an EZ1 biorobot (Qiagen, Germany) according to the manufacturer's protocol. For all patients (n = 10), a 692-nucleotide fragment within the E1 gene (positions 10138–10829) was amplified using the high fidelity Eppendorf One-Step RT-PCR kit and primers CV1F (
Sequences were analysed with the Sequencher software and aligned with ClustalX
To assess factors relating to clinical presentation and laboratory abnormality during CHIK on D1 and D300, we performed univariate analysis, for qualitative factors with Fisher's exact test and for continuous factors using the Mann Whitney nonparametric test. Correlations were assessed using the Spearman nonparametric test. Following the Mickey and Greenland approach
To compare the intra-host diversity and the available clinical and laboratory data, we performed correlation analyses using the Spearman nonparametric test.
Finally, we evaluated the possibility of generating a diagnostic score of CHIK on the first day of monitoring, by comparing the clinical and biological features of CHIKV positive and negative samples. Any factor for which a Pearson's chi2 or Fisher test was <0.2, was included in multivariate analysis (hierarchical log-linear model) to study the adjusted relationship between different variables and their interactions. Sensitivities (Se), specificities (Sp), predictive values - positive (PPV) and negative (NPV) - were estimated and yielded a Receiver Operating Characteristic (ROC) curve and the area under the curve (AUC).
All statistical analyses were performed with the IBM SPSS statistic 19 software.
For multivariate analysis, the alpha probability threshold of significance was 0.05.
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.
Amongst CHIKV+ve patients (54 patients), the mean age was 40 years old, the sex ratio (m/f) was 1.7 and the mean weight was 76.1 kg
CHIKV+ n (%) | CHIKV− n (%) | p-Value | Odd Ratio (95% CI) | |
|
||||
Mean (SD, min-max) | 40.1 (12.4, 18–66) | 41.4 (15.3, 20–66) | 0.92 | |
18–20 | 4 (7.4) | 1 (4.5) | ||
21–30 | 9 (16.7) | 9 (40.9) | ||
31–40 | 19 (35.2) | 2 (9.1) | ||
41–50 | 10 (18.5) | 3 (13.6) | ||
51–60 | 8 (14.8) | 3 (13.6) | ||
61–66 | 4 (7.4) | 4 (18.2) | ||
|
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Male | 34 (63) | 11 (50) | 0.3 | |
Female | 20 (37) | 11 (50) | ||
|
5 (9.3) | 5 (22.7) | 0.1 | |
|
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Metacarpophalangean (MCP) | 40 (74.1) | 9 (40.9) |
|
0.24 (0.09;0.69) |
Interphalangean (PIP) | 37 (68.5) | 9 (40.9) |
|
0.32 (0.11;0.89) |
Hands (MCP+PIP) | 43 (79.6) | 10 (45.5) |
|
0.21 (0.07;0.62) |
Wrist (W) | 39 (72.2) | 8 (36.4) |
|
0.22 (0.07;0.63) |
Ankles | 37 (68.5) | 11 (50) | 0.2 | |
Knees | 33 (61.1) | 17 (77.3) | 0.2 | |
Shoulders | 26 (48.1) | 11 (50) | 1. | |
Lombalgia | 25 (46.3) | 12 (54.5) | 0.6 | |
Feet | 23 (42.6) | 4 (18.2) |
|
0.30 (0.089;1.01) |
Cervicalgia | 21 (38.9) | 9 (40.9) | 1. | |
Elbows | 14 (25.9) | 10 (45.5) | 0.11 | |
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moderate or important | 25 (46.3) | 18 (81.8) |
|
5.22 (1.56;17.48) |
|
12 (22.2) | 2 (10) | 0.32 | |
|
43 (79.6) | 4 (20) |
|
0.06 (0.02;0.23) |
Mean (SD, min-max) | 0.8 (0.57, 0.3–4) | 1.5 (0,59, 0.6–2.7) |
|
|
|
14 (25.9) | 1 (5) |
|
0.15 (0.018;1.23) |
Mean (SD, min-max) | 185 (58.5, 104–348) | 217 (54,5, 148–362) |
|
|
|
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Mean (SD, min-max) | 52.4 (42.4, 7–195) | 51.8 (63.8, 0–264) | 0.26 | |
|
11 (22) | 0 (0) |
|
0.78 (0.67;0.9) |
The most common presenting clinical symptom was a febrile poly-arthralgia (16 joints on average, out of 34 on the proposed diagram, SD = 10, median = 17) with an intensity assessed as important (among 4 categories: absent, minimal, moderate, important) in 46.3%. Arthralgia was symmetrical (
Few other signs were noted: dermatological signs (28%), digestive disorders such as nausea/vomiting (44%), diarrhoea (22%) and dysgeusia (13%)
These clinical data were collected from three consultations with a general practitioner on day 1, day 7 (mean 6.4, SD = 1.4) and day 25 (mean 26.5, SD = 9.8) of the disease during the Reunion island outbreak 2005–2006. * Since clinical assessment during the first medical visit was obtained prior to the beginning of the treatment, all CHIKV+ve patients (N = 54) could be used for analysis. By contrast, only patients who received the placebo (N = 27) were included in the study of disease evolution (second and third medical visits).
Quality of life (assessed by visual analogical scales (VAS)) was severely impacted with the capacity to perform normal activities, health status and quality of sleep assessed on average at 33/100 (SD = 26), 28/100 (SD = 25) and 27/100 (SD = 13), respectively. More than half of the patients scored <30/100 in all aspects of quality of life evaluation
Three kind of quality of life (health status, capacity to perform normal activity and quality of sleep) were assessed by self reported visual analogic scale (VAS) from “very bad” (VAS = 0) to “very good” (VAS = 100) and are represented here by box plot diagrams. Box plot is a representative diagram of continuous variables. The bottom and the top of the box are the 25th and 75th percentile, the band near the middle is the median and the ends of the whiskers are the 1.5 inter-quartile of the lower and upper quartile. The data not included between the whiskers are plotted as an outlier with small circles (if between 1.5 to 3 inter-quartile of the lower or upper quartile) or with a star (if higher than 3 inter-quartile of the lower or upper quartile). The outliers are tagged with their patient numbers to follow them at different time period. * Clinical assessment at D1 was obtained from all CHIKV+ve patients (N = 54) during the Reunion island outbreak 2005–2006. Only patients receiving placebo (N = 27) were included in D2–D14 clinical assessment.
Demographic and biological data were compared as functions of clinical presentation at D1 using multivariate analysis. A higher number of arthralgic joints was independently associated with women (p<0.001, SIR = 1.361, CI (1.191;1.554)), with an increase of age (p<0.001, SIR = 1.017, CI (1.011;1.022)) and a decrease of leukocytosis (p<0.001, SIR = 0.881 CI(0.841;0.923)). Patients with important arthralgia were significantly older than others (p<0.05, OR = 1.073, CI (1.013;1.136)) and associated with a shorter time between onset of symptom and inclusion (p<0.05, OR = 0.288, CI (0.071;0.764)). Regarding quality of life no significant association, in multivariate analysis with any risk factor analysed, could be identified.
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%
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)).
There was no hospitalisation during the 14-day follow-up and two clinical stages of evolution were observed
From D1 to D4, a rapid improvement was observed (the percentage of patients with arthralgia decreased from 98% to 68%, the average number of arthralgic joints decreased from 16 to 9 (SD = 8,7), VAS quality of life scores increased more than twice and the percentage of VAS <50/100 decreased from 83% to 19%).
From D5 until D14, the evolution was slower (68% of patient still suffered from arthralgia on D14 with an average number of 6 painful joints per person (SD = 7,2), VAS quality of life was assessed at 82/100 on average, with less than 5% of VAS <50).
At the last medical visit (D25), 36% of patients reported residual asthenia; 65% reported arthralgia (corresponding to 71% with sporadic and 57% with permanent arthralgia respectively) while 44% and 32% of patients were assessed by GPs as recovered or improved in condition respectively.
The number of patients with positive viraemia decreased from 54 (100%) on D1 to 21 (39.6%) on D3 and the average viral load was 6.42 (4.43 to 9, SD = 1.19, log10 copies/ml). In all cases there was a decrease of viraemia from D1 to D3. At D6, all viral loads were negative. At D16, there was neither lymphopenia nor thrombocytopenia, while neutropenia and abnormal liver function persisted in 12% and 7% of patients respectively. CRP was >5 mg/L in 12% of cases without exceeding 50 mg/L.
During the D300 telephone interview, 5 out of 26 patients who could be contacted (19.2%) considered that they had not completely recovered; 6/26 (23%) declared residual arthralgia (7 joints on average, SD = 5.3); morning stiffness was assessed in 4/6 (66.7%) cases but arthralgia was always related to joint activity. Retrospectively, the duration of initial illness was estimated at more than 4 weeks by 12 out 21 patients who considered that they had completely recovered (57.1%). In univariate analysis, patients who did not report recovery at D300 were older (p<0.05, 52.80±12.61
Analysis of the CHIKV+ve and CHIKV−ve groups at baseline revealed no difference in the distribution of age, sex, weight or time of inclusion
more frequent pain in small upper joints (wrists, W) (p<0.01, OR = 4.55, CI(1.59;13.04)), metacarpophalangeal (MCP) (p<0.01, OR = 4.13, CI(1.45;11.74), proximal interphalangeal (PIP) (p<0.05, OR = 3.14 CI(1.13;8.77)),
more frequently minor or absent myalgia (MYOPAIN) (p<0.01, OR = 5.22, CI(1.56;17.48))
more frequently lymphopenia (<1 G/L) (p<0.0001, OR = 15.64, CI(4.35;56.25)).
Factors that showed a direct relationship in univariate and multivariate analyzes with CHIK diagnosis were W, MCP, MYOPAIN and lymphopenia. CHIKV+ve and CHIKV−ve patients were not statistically different for the impact on quality of life at the acute stage.
Finally, the probability (p) of having been infected by CHIKV has been estimated, using logistic regression, as follows:
p = 1/(1+exp(4.139)×exp(−3.666×lymphopenia)×exp(−1.940×MCP+)×exp(−2.341×W+)×exp(−2.700×MYOPAIN)). Each covariate was validated if equal to 1, otherwise 0. Tested on our cohort, the ROC curve had an AUC = 0.93 (p<0.001), with Se = 90%, Sp = 85%, PPV = 94% and NPV = 77% using a 0.579 probability threshold value.
Similarly, we could calculate a clinical score relying only on clinical symptoms (
Noticeably, in both scores, the variable Handpain (pain on MCP and/or PIP) could be used instead of the MCP variable with similar results.
Finally, these results were used to propose a clinical and clinico-biological score usable in ambulatory practice by GPs (see on
These scores were based on patients who reported fever and arthralgia for less than 48 hours. * MCP+: arthralgia on at least one metacarpophalangeal joint. * W+: arthralgia on at least one wrist. We propose a clinical score based exclusively on clinical data (W+, MCP+ and Myalgia absent or minor) and a clinico-biological score that further includes lymphopenia (<1G/L). The result of the score represents the predicted probability to have chikungunya (see calculation of scores in the main text).
To investigate the intra-host genetic diversity of CHIKV we sequenced, on average, 45 clones/serum (41–57) within the E1 gene
CHIKV samples #ID nb, D1 or D3, sex/age, time to inclusion, viral load, number of arthralgia at day 300 | N° of clonestested | % of mutant clones | % of variable nt sites | % of nt mutations | π nt | π aa | dN | dS | dN/dS |
|
47 | 27.66% | 2.02% | 0.05% | 0.0009 | 0.0022 | 0.00098 | 0.0005 | 1.93 |
|
40 | 32.5% | 1.45% | 0.05% | 0.0010 | 0.0024 | 0.00104 | 0.0006 | 1.74 |
|
40 | 24.4% | 1.73% | 0.04% | 0.0008 | 0.0019 | 0.00084 | 0.0009 | 0.96 |
|
38 | 42.5% | 3.18% | 0.09% | 0.0018 | 0.0041 | 0.00180 | 0.0018 | 1.00 |
|
43 | 37.21% | 2.89% | 0.07% | 0.0013 | 0.0026 | 0.00116 | 0.002 | 0.59 |
|
45 | 39.13% | 2.89% | 0.06% | 0.0013 | 0.0030 | 0.00133 | 0.0011 | 1.28 |
|
40 | 45.10% | 3.9% | 0.08% | 0.0016 | 0.0029 | 0.00129 | 0.0024 | 0.55 |
|
43 | 41.30% | 3.61% | 0.09% | 0.0018 | 0.0039 | 0.00174 | 0.0021 | 0.84 |
|
43 | 37.21% | 2.46% | 0.08% | 0.0015 | 0.0036 | 0.00159 | 0.0014 | 1.15 |
|
53 | 29.82% | 3.32% | 0.06% | 0.0012 | 0.0028 | 0.00125 | 0.0013 | 1.00 |
|
45 | 40.43% | 3.61% | 0.08% | 0.0017 | 0.0026 | 0.00115 | 0.0031 | 0.37 |
|
42 | 33.33% | 2.75% | 0.07% | 0.0013 | 0.0031 | 0.00137 | 0.0008 | 1.59 |
The percentage of variable nucleotide (nt) sites was calculated as the number of variable nt sites ×100 divided by the number of nt analysed (758 nt).
The percentage of nucleotide (nt) mutations was calculated as the number of nt mutations ×100 divided by the number of nt sequenced for each serum sample.
The average pairwise distance was calculated among the nucleotide (p nt) and amino acid (p aa) sequences from each serum.
The mean ratios of non-synonymous (dN) and synonymous (dS) substitutions per site were estimated using the pairwise method of Nei and Gojobori.
Sex: Male: male; F: female.
NA: not available.
Analysis of CHIKV intra-host genetic diversity was conducted according to demographic, clinical and biological data
An increased percentage of mutant clones and average π aa and dN was significantly correlated with an increased delay between onset of disease and inclusion (p = 0.045 for % of mutant clones, p<0.001 for π aa and dN).
A high viral load at D1 was significantly correlated with a low intra-host diversity (p<0.01 for % of mutant clones, % of nt mutations, % of variable nt sites and π nt, p<0.05 for dS).
A comparison of intra-host genetic diversity based on sequential serum at D1 and D3 from the same patients (n° 304 and n°4002) revealed an increase of genetic diversity over time
From the D300 telephone interview, intensity of sequelae and an increased number of reported arthralgia were significantly correlated with a higher amino-acid diversity at inclusion (π aa, dN; p<0.05).
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%
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
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.
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
Looking into further details, it appears that arthralgia was most commonly observed in small joints (
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 (
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
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,
From day 5 to day 14 (‘convalescent stage’), all patients had no detectable viraemia, but improvement was slower, considering both quality of life scores
However, despite clinical improvement, it is probable that immune mechanisms are still involved
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
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
The interplay between the immune response and viral evolution most probably constitutes an important issue for disease outcome. A non-primate animal model
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The authors would like to thank the general practitioners who have participated in the CuraChik study, F. Bureau (Stickstudio) for help in the design of the supplemental data, R. Dechesse for technical assistance and E. A. Gould for critical review of the article.