Increased CRP, anti-CCP antibody, IL-2R, COMP levels in prognosis of post-chikungunya chronic arthritis and protective role of their specific genotypes against arthritic manifestation

Highlights

• •Anti-CCP antibody, IL-2R, COMP high among post-chikungunya chronic arthritic patients.
• •AST, ALT, AST/ALT, bilirubin, ALP levels high among arthritic chikungunya patients.
• •Patients with COMP-rs144778694-GA were susceptible to arthralgic outcome.
• •Patients with certain genotypes of CRP, IL-2R, COMP were protected from arthralgia.
• •PCA analysis segregateed chronic arthritic group from acute and non-arthritic ones.

Abstract

Chikungunya infection leads to acute/chronic polyarthritis/polyarthralgia causing long-term morbidity among patients. Prognosis of post-chikungunya chronic arthritis (PCA) is of utmost necessity for proper disease management. Arthritic and hepatic biomarkers were evaluated among chikungunya patients without arthritis, with acute arthritis and with post-chikungunya chronic arthritis in the study. Serum levels of arthritic [CRP (C-reactive protein), anti cyclic-citrullinated-peptide (anti-CCP) antibody, soluble interleukin-2 receptor (sIL-2R), cartilage oligomeric matrix protein (COMP)] and hepatic [ALT (alanine aminotransferase), AST (aspartate aminotransferase), ALP (alkaline phosphatase), albumin and bilirubin] biomarkers of 167 chikungunya positive patients were determined by sandwich-ELISA/immunoturbidimetry/auto-analyser. 167 chikungunya-patients and 102 healthy controls were genotyped to understand role of CRP-rs3093059/rs3091244, IL-2R-rs743777 and COMP-rs144778694 polymorphisms towards chikungunya virus (CHIKV) infectivity and arthralgic manifestation. CRP, anti-CCP antibody, IL-2R and COMP levels significantly increased among PCA patients. Concentrations of AST, ALT, AST/ALT-ratio, bilirubin and ALP increased among arthritic chikungunya patients. Principal component analysis differentiated PCA groups from acute (AA) and non-arthritic groups. Patients with IL-2R-rs743777-GA, G-allele and COMP-rs144778694-GA genotypes were susceptible to chikungunya infection. Moreover, patients with CRP-rs3093059-CT, rs3091244-TT, IL-2R-rs743777-GA and COMP-rs144778694-AA genotypes were significantly protected from arthralgia, whereas, COMP-rs144778694-GA genotype was susceptible towards it. Patients with certain genotypes of CRP, IL-2R and COMP demonstrated significantly higher biomarker serum-levels among patients suffering from AA with/without PCA. Thus, both serum biomarker levels and polymorphic genotypes of infected patients play decisive role in development of PCA.

1. Introduction

Epidemics caused by chikungunya virus (CHIKV) have been frequently occurring in India 2005 onwards, after its disappearance for more than three decades (Naresh Kumar and Sai Gopal, 2010; Sengupta et al., 2020). This disease is characterized by abrupt onset of fever, frequently accompanied with joint pain, acute or chronic arthritis, causing stooped appearance of patients (Mohan et al., 2010). Acute arthritis might lead to development of devastating oligo- or polyarthritis involving 4 or more joints of both arms and legs (Alpay-Kanıtez et al., 2018). Approximately, 88-100% of infected patients might also experience post-chikungunya sub-acute arthritis (6 weeks) and 40–60% might demonstrate chronic arthritis even after 1.5 to 5 years of infection, indicating role of host genetics in determining chronic arthritis among infected patients (Javelle et al., 2015; Tritsch et al., 2020). There were reports of development of chronic inflammatory rheumatism and musculo-skeletal disease during post-chikungunya arthritis, which could be relapsing or unremitting (Tritsch et al., 2020). Clinical presentation of long term/post-chikungunya infection might mimic autoimmune rheumatological conditions viz. rheumatoid arthritis (Krutikov and Manson, 2016; Gauri et al., 2016). Hence, early identification of infected patients who might develop chronic arthralgia in long term could help clinicians in their pain management. Both DNA and serum level biomarkers might help in early differentiation between acute (AA) and post-chikungunya arthritic (PCA) patients (Flögel et al., 1998; Booth et al., 1998).

CHIKV primarily infects skin cells, thereby replicating in skin, liver, muscle and bone joints and simultaneously invading monocytes and blood vessels – resulting in bone erosion, degradation of extracellular matrix and release of pro-inflammatory cytokines (Srivastava et al., 2020). Increased level of C-reactive protein (CRP), an acute-phase hepatic protein has been reported among viraemic and arthritic infected patients (Gauri et al., 2016; Solanke and Karmarkar, 2014). Similarly, elevated ALT (alanine aminotransferase), AST (aspartate aminotransferase), ALP (alkaline phosphatase) levels has been reported among both viraemic and post-viraemic chikungunya patients – indicating involvement of hepatic anomalies in CHIKV infection (Danis-Lozano et al., 2017; Ng et al., 2009). Arthralgic manifestation during CHIKV infection resembles that of rheumatoid arthritis (RA), characterized by increased rheumatoid factor (RF) and anti cyclic-citrullinated-peptide (anti-CCP) antibody related articular damage (Imai et al., 2016). On the other hand, cartilage oligomeric matrix protein (COMP), an extracellular matrix protein, has been associated with cartilage turnover and joint destruction related to osteoarthritis and rheumatoid arthritis (Tseng et al., 2009). Soluble Interleukin-2 receptor (sIL-2R), a pro-inflammatory cytokine related to chikungunya infection, has also been implicated in development of synovitis and exacerbation of disease severity of rheumatoid arthritis (Wood et al., 1988). Moreover, RF, anti-CCP antibody and CRP has been recognized as serological and acute phase response markers for rheumatoid arthritis, according to 2010 American College of Rheumatology/European League Against Rheumatism (ACR/EULAR) classification criteria (Aletaha et al., 2010).

Besides protein levels, polymorphic genotypes of these genes might contribute to development of chikungunya induced arthritis among infected patients. Specific genotypes of cis-acting polymorphisms of CRP, viz. rs3091244, rs3093059, have been reported to contribute to variance in protein level among dengue, chikungunya, ankylosing spondylitic and osteomyelitic patients (Sengupta et al., 2022). Previously, mutations within COMP gene have been strongly associated with pseudoachondroplasia, multiple epiphyseal dysplasia and osteoarthritis (Deere et al., 1999; Mishra et al., 2019). Similarly, specific polymorphic genotypes of rs743777 (IL-2R), localized 6kb upstream of transcription start site (5′near gene region) and were associated with RA and peripheral arthritis of ankylosing spondylitic patients (Ruyssen-Witrand et al., 2014; Polo et al., 2019; Pál et al., 2010). But, contribution of these polymorphic variants towards development of chikungunya induced arthritis has not been studied. Moreover, prognostic value of these polymorphic genotypes and protein levels has not been evaluated in case of CHIKV induced arthritis.

Thus, this study explores prognostic values of these serum proteins and their genetic polymorphisms in determining acute and chronic arthritis among CHIKV infected patients.

2. Materials and methods

2.1. Ethics statement

Collection of blood from each of the febrile patients and healthy participants was performed in accordance with ethical standards of Clinical Research Ethical Committee of Calcutta School of Tropical Medicine, Kolkata, India (CREC-STM/53 dated 26.09.2013) and with that of 1964 Helsinki Declaration and its later amendments. Written consents were obtained from patients and healthy individuals.

2.2. Inclusion criteria

Patients with acute febrile illness and history of headache, body ache, myalgia, arthralgia, rash, with or without haemorrhagic manifestation of all age-groups and both sexes were reviewed by physician according to WHO criteria of chikungunya infection.

2.3. Exclusion criteria

Haematological malignancies, bleeding disorders, chronic liver disease, diabetes mellitus, and renal diseases.

2.4. Patients and healthy controls

Around 2ml of blood of all age groups and sexes were collected from each of 641 symptomatic patients during their first visit at Calcutta School of Tropical Medicine (CSTM), West Bengal, India from September 2014 to October 2016, within acute phase of infection. Febrile patients exhibiting any two of the following symptoms: headache, myalgia, arthralgia, nausea, vomiting, rash, fatigue was selected as per WHO criteria. Amongst them, 167 were CHIKV infected, as detected by anti-CHIKV IgM ELISA (NIV, Pune, India)/real-time qRT-PCR. To carry out age-matched case control study, blood from 102 healthy unrelated individuals of same ethnicity, who neither had any signs and history of other infections nor were detected with DENV, CHIKV, Japanese encephalitis, HIV and Hepatitis A/B/C infection - as tested by IgM and IgG ELISA/RT-PCR, were collected from same community. Symptomatic patients and healthy controls consisted of Bengali population of eastern India having similar ethnic genetic make up to that of Bengali Bangladeshis (BEB) group of SAS included within 1000 genome project of GWAS. Control population size was calculated using EpiInfo™ version 7.2 software of CDC, with 95% confidence interval and 24.64% CHIKV infection rate among eastern Indian patients (Sengupta et al., 2020).

All the following biochemical and genotypic experiments were performed on patient-blood collected during their first visit at CSTM. Detailed follow up study of arthralgic manifestations of CHIKV infected patients was performed both during their first visit and after 2 years from date of collection of blood and patients were grouped into acute and chronic chikungunya cases according to criteria of World Health Organization (WHO) (WHO, 2015). Patients were categorised into four groups: NA: Patients without any arthralgia; AA: patients with only acute arthralgia; AA+PCA: patients with both acute and post chikungunya arthralgia and PCA: patients developing only post-chikungunya arthralgia. Depending upon rheumatoid arthritic outcome, CHIKV-infected patients were also categorised according to 2010 American College of Rheumatology/European League Against Rheumatism (ACR/EULAR) classification criteria (Aletaha et al., 2010).

2.5. Extraction of viral RNA and determination of CHIKV

Viral RNA was extracted from 140μL of patients’ sera, using QIAamp Viral RNA Mini Kit according to manufacturer's protocol (Qiagen, Courtaboeuf, France). Genome presence of CHIKV was determined by real-time qRT-PCR using CHIKV Genesig kit (Primer Design Ltd., UK), according to manufacturers’ protocol. Limit of detection (LOD) of CHIKV Genesig kit was <100 copies of corresponding target genomes. Real-time PCR was performed on ABI Prism 7500 fast instrument. Each sample was loaded in triplicate.

2.6. Quantification of CRP

Sera collected from infected patients were subjected to CRP quantification. CRP level was determined by immunoturbidimetry method using AUTOSPAN turbi gold kit (SPAN diagnostics, India) according to manufacturer's protocols. Briefly, 3μl of patient serum/calibrator was mixed with 500μl ten times diluted latex reagent (containing latex particle coated with anti-human CRP antibodies) and absorbance was measured at 550nm wavelength after 10 s (A1) and 120 s (A2). CRP concentration (mg/l) in serum was measured using following formula:

Serum concentration of CRP (mg/l) = [(A2-A1) Serum sample/(A2-A1) Calibrator] x Concentration of calibrator

2.7. Quantification of serum COMP, Anti-CCP, IL-2R

Serum levels of COMP (LOD:0.085ng/ml, Invitrogen, MA, USA), Anti-CCP (LOD:0.582pg/ml, KINESISDx, CA, USA) and IL-2R (LOD:29pg/ml, Invitrogen, MA, USA) were determined by sandwich ELISA kits, according to manufacturers’ instructions. Absorbance was measured at 450nm wavelength. Standard-curve was drawn using GraphPad prism9 and levels of each protein were analysed.

2.8. Detection of RF

RF detection was done using RF-latex agglutination slide test kit (AUTOSPAN, UK), where patient serum was placed within circled area on special slide (provided with kit). One drop of Reagent-1 was added to it and mixed well. Agglutination was observed macroscopically under direct light source.

2.9. Quantification of biochemical parameters

Levels of AST, ALT, ALP, bilirubin and albumin were measured by using a standard automated clinical chemistry analyzer (ERBA Model no: EM360) according to manufacturer's instructions.

2.10. Identification of CRP, IL-2R and COMP genotypes susceptible to chikungunya related arthritis

To understand role of CRP, IL-2R and COMP polymorphisms (if any) towards CHIKV infectivity and arthralgic manifestation compared to healthy controls, genotyping of CRP (rs3093059 and rs3091244), IL-2R (rs743777) and COMP (rs144778694) polymorphisms was performed among 167 CHIKV infected and 102 healthy controls, based on their minor allele frequencies (MAF).

SNP (Single nucleotide polymorphism) genotyping was carried out by using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) based procedure. Briefly, genomic DNA was extracted from peripheral blood samples using QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany), according to manufacturer's instructions. Based on sequences available in GenBank database, four primer pairs were designed using Primer3 software, to amplify polymorphic region of CRP (rs3093059 and rs3091244), IL-2R (rs743777) and COMP (rs144778694) genes. PCR reaction was performed in 20µl volume, using 1xPCR buffer (Fermentas, USA), 1mM of each dNTP, 1unit of Taq DNA polymerase (Fermentas, USA), 1.5mM MgCl₂ and 20p.moles of previously mentioned primers (Table S1). Respective PCR products were digested with Tas I, BfaI, StuI, PspFI (Fermentas, USA) and Taq I (Himedia, India) accordingly. Different RFLP patterns were validated by sequencing of the respective PCR products using ABI Prism Big Dye Terminator v3.1 Cycle sequencing kit (Applied Biosystems, USA) in an ABI-Prism 3100 Avant Genetic Analyser (Applied Biosystems, USA).

2.11. Statistical analysis

Associations of protein levels and their SNP genotypes with CHIKV infectivity and arthralgic manifestations were analyzed and represented using GraphPad Prism 9. Allelic and genotypic frequencies were compared between different study groups using Pearson's Chi-square test, unpaired Welch's t-test and multi-variant analysis, using one way ANOVA. For genotypic associations; p-values, odds ratio (OR) and risk ratio were calculated. A p-value of <0.05 was considered statistically significant. Among healthy controls, Hardy–Weinberg equilibrium was analyzed for all the polymorphisms using Haploview program. Cut-off values, sensitivity and specificity of serum-markers associated with post-chikungunya chronic arthritis (PCA) were analyzed by ROC curve. A principal component analysis was conducted to identify the markers in different samples’ groups.

3. Results

In this study, male to female ratio of CHIKV infected patients:1.49:1 and mean age:33.06±14.7years, whereas, that of the control group was 1:1.01 and 36.2±11.8 years, respectively (Table 1). Major WHO-defined symptoms of febrile chikungunya patients were arthralgia, myalgia, persistent vomiting and joint swelling. According to arthralgic manifestation, as mentioned in materials and methods section, approximately, 14.10% (n = 24) of infected patients were NA (mean age:42.5±15.5years), 53.84% (n = 89) were AA (mean age:37.57±10.2years), 24.35% (n = 41) were AA+PCA (mean age:40.61±14.2years) and 7.69% (n = 13) were PCA (mean age:33.5±19.1years) groups. Younger and older patients were equally affected eliminating age biasness or any underlying medical condition. In addition, there was no significant difference in male-female distribution among the above mentioned groups. PCA patients complained about persistent joint pain even after 2 years of first visit blood collection. Whereas, AA+PCA patients reported arthralgia both during first visit and after 2 years of blood collection. 2010 ACR/EULAR scoring classification indicated majority of PCA and AA+PCA patients qualified for rheumatoid arthritis (score ≥6/10), with average score of 6 and 6.21, respectively. Principal component analysis of 2010 ACR/EULAR scoring parameters of CHIKV-infected patients indicated similar clustering of PCA and AA+PCA groups (Fig. S1).

Table 1.

Comparative demographics, symptomatic diversity and 2010 ACR/EULAR classification of CHIKV infected patients.

		Chikungunya infected patients (N = 167)	Healthy Controls (N = 102)
Mean age (in years)		33.06 ± 14.7 (range: 4-74 years)	36.2 ± 11.8(range: 18–63 years)
Sex	Male	59.88%	50.98%
	Female	40.11%	49.02%
	Male: Female ratio	1.49:1	1:1.01
Diagnostic Tools	Anti-CHIKV-IgM ELISA	44.31%
	Real time RT-PCR	82.03%
	Both anti-CHIKV-IgM ELISA and Real time RT-PCR	26.34%
Symptomatic prevalence	Fever	100%
	Myalgia	53.89%
	Arthralgia	67.06%
	Headache	22.15%
	Rash	19.76%
	Nausea	2.39%
	Persistent Vomiting	25.14%
	Abdominal Pain	8.98%
	Joint Swelling	21.55%
	Bleeding	0%
	Retro-Orbital Pain	0%
Patient's arthralgic manifestation	No arthralgia (NA) Mean age (in years)	14.10% 42.5±15.5years (range 7-62years)
	Acute arthralgia (AA) Mean age (in years)	53.84% 37.57±10.2years (range 12-72years)
	Acute arthralgia + Post-chikungunya arthritis (AA+PCA) Mean age (in years)	24.35% 40.61±14.2years (range 10-72years)
	Post-chikungunya arthritis (PCA) Mean age (in years)	7.69% 33.5±19.1years (range 11-46years)
% prevalence of patients with post-chikungunya rheumatoid arthritis according to 2010 ACR/EULAR scoring (score ≥6/10)*	NA	0%
	AA	2.43%
	PCA	83.33%
	AA+PCA	95.23%
Mean ACR/EULAR classification score	NA	3.09
	AA	4.66
	PCA	6
	AA+PCA	6.21

*2010 American College of Rheumatology/EuropeanLeague Against Rheumatism (ACR/EULAR) classification criteria

3.1. CRP, anti-CCP antibody, COMP and IL-2R levels among chikungunya patients with acute and chronic arthritis

Mean serum CRP concentrations significantly increased in AA (46.03mg/ml, p-value= 0.0291), PCA (60.71mg/ml, p-value=0.0212) and AA+PCA (77.05mg/ml, p-value<0.0001) groups when compared to NA which had a mean level of 19.60mg/ml (Fig. 1a). Moreover, CRP level of AA+PCA group was significantly higher than AA patients (p-value=0.0004). Anti-CCP antibody level was significantly high only among AA+PCA group when compared to both NA and AA patients (NA:133pg/ml, AA:190.1pg/ml, AA+PCA:419.7pg/ml, p-value=0.0003 and p-value<0.0001, respectively). On a similar trend, COMP level was significantly higher among AA+PCA and PCA groups when compared to both NA and AA groups (AA+PCA:40.35ng/ml, PCA:49.35ng/ml, AA:21.88ng/ml, NA:21.18ng/ml; p-value<0.0001). AA+PCA and PCA groups also showed higher IL-2R level compared to NA (AA+PCA:2421.22pg/ml and PCA:4882.45pg/ml vs NA:969.78pg/ml; p-value=0.0202 and p-value<0.0001, respectively). Also, PCA group showed highest mean IL-2R level compared to AA+PCA and AA groups (AA: 1128.96pg/ml; p-value<0.0001 and p-value=0.0005). RF was detected among 13.17% (22/167) CHIKV patients which was insignificant when compared to healthy individuals.

Fig. 1.

(a) Comparative analysis of serum concentration of arthritic biomarkers among AA+PCA, PCA, AA and NA groups of CHIKV-infected patients. (b) Comparative analysis of serum concentration of hepatic biomarkers among AA+PCA, PCA, AA and NA groups of CHIKV-infected patients.

Level of all four arthritic biomarkers was higher among chikungunya patients with chronic arthritis (PCA, AA+PCA) compared to those with only acute or no arthritis among all age groups (Fig. S2). Similar trend was followed among male and female chikungunya patients (Fig. S3).

3.2. AST, ALT, ALP, bilirubin and albumin levels among chikungunya patients with acute and chronic arthritis

Amongst hepatic markers, gradual increase of AST, ALT, albumin level and AST/ALT ratio was found in the groups according to following order: AA+PCA>AA>PCA>NA (Fig. 1b). AST levels were significantly elevated among AA+PCA (269.6IU/L) compared to NA (NA:41.50IU/L; p-value<0.0001), AA (AA: 102.3IU/L; p-value<0.0001) and PCA (PCA:82.36IU/L; p-value<0.0001) groups. Similar trend was followed for ALT among AA+PCA (AA+PCA:143.3IU/L) compared to all the other groups (NA:39.97IU/L, p-value<0.0001; AA:89.57IU/L, p-value=0.0016; PCA:71.49, p-value=0.0217). AST/ALT ratio was significantly high among AA+PCA (2.001) with respect to NA (1.102; p-value=0.0043) and AA (1.257; p-value=0.0008) groups. Also, AA+PCA had significantly higher bilirubin levels compared to AA (AA+PCA:1.173IU/L vs AA:0.6350IU/L; p-value=0.0004) and ALP compared to NA (AA+PCA=97.74IU/L, NA:34.12IU/L; p-value=0.0051). Single sample for each of AA and AA+PCA groups were exceptions with “maximum outlier” value for CRP, Anti-CCP antibody, COMP, IL-2R, AST, ALT, AST/ALT ratio, bilirubin and ALP. Among AA+PCA group, a single 72years old male patient with fever for 2 months exhibited these outlier values.

3.3. ROC curve analysis of biomarkers among chikungunya patients with and without chronic arthritis

A cut-off value of chikungunya induced chronic arthritis was analyzed by ROC curve to differentiate between patients with (PCA, AA+PCA) and without (AA, NA) chronic arthritis, using markers significantly altered amongst these two groups (Fig. 2). Cut off values of arthritic and hepatic biomarkers that significantly increased in case of chronic arthralgia were: CRP: >63.95mg/ml, Anti-CCP antibody: >186.0pg/ml, COMP: >28.88ng/ml, IL-2R: >1066pg/ml, AST: >133.6U/L, ALT: >90.75U/L, AST/ALT: >1.46U/L, Bilirubin: >0.83U/L, ALP: >70.43U/L (p-value: 0.0001-0.0051). High sensitivity and specificity values of COMP and IL-2R indicated their diagnostic potential for chronic arthralgia among infected patients. On the contrary, hepatic markers demonstrated lower sensitivity level compared to arthritic ones.

Fig. 2.

Receiver operating characteristic curve (ROC curve) analysis of arthritic and hepatic biomarkers with their sensitivity, specificity and cut-off value of chikungunya induced chronic arthritis.

3.4. Heatmap, correlation and principal component analysis of biomarkers among chikungunya patients with and without chronic arthritis

Heatmap analysis of arthritic and hepatic biomarkers revealed increase of CRP, anti-CCP antibody as well as COMP levels among four groups of infected patients compared to healthy controls (Fig. 3a). Serum albumin level increase was negligible. Principal component analysis of all analyzed biomarkers indicated gradual segregation of AA+PCA and PCA groups from AA and NA, which in turn overlapped with each other; NA was in proximity to healthy controls (Fig. 3b). Anti-CCP antibody, CRP, AST, ALT, AST/ALT ratio and bilirubin levels were linked with AA+PCA group, whereas, ALP and IL-2R levels were associated with PCA group. Highest proportion of variance of PC1 was 37.99%, PC2 was 51.72% and PC3 was 63.75%. Correlation studies of these biomarkers among these groups indicated a strong positive significant correlation between Anti-CCP antibody vs. CRP of AA+PCA (p-value=0.044), Anti-CCP antibody vs. CRP (p-value<0.001), COMP vs. CRP (p-value=0.023), COMP vs. Anti-CCP antibody (p-value=0.029) and COMP vs. IL-2R (p-value=0.004) of AA groups (Fig. 3a). For CRP and anti-CCP antibody, negative correlation was found between AA and NA, PCA and NA groups, respectively.

Fig. 3.

(a) Differential heatmap and correlation analysis of arthritic and hepatic biomarkers with AA+PCA, PCA, AA and NA groups of CHIKV-infected patients. (b) Principal component analysis representing linkage and variance among AA+PCA, PCA, AA, NA of CHIKV-infected patients and healthy groups, with arthritic and hepatic biomarkers.

3.5. Genotypic association of CRP, IL-2R, COMP polymorphisms with chikungunya susceptibility

Genotypic and allelic distribution of CRP, IL-2R and COMP polymorphisms was analyzed among 167 CHIKV infected patients and 102 healthy controls (Table 2a). Statistical analysis revealed individuals with CRP-rs3091244-TC genotype were less susceptible to CHIKV infection (p-value=0.0126). Additionally, according to additive model, rs3091244 was significantly associated with infection susceptibility (p-value=0.0286). Furthermore, patients with IL-2R-rs743777-GA genotype and G-allele were significantly susceptible to CHIKV infection (p-value=0.0198 and p-value=0.0079, respectively). Additive model suggested this IL-2R polymorphism to be significantly associated to CHIKV infection susceptibility (p-value=0.0179). Subsequently, COMP-rs144778694-GA genotype, A-allele and its additive model were positively associated with CHIKV infection when compared to healthy control population (p-values= 0.0032, 0.0020 and 0.0051, respectively).

Table 2.

(a) Genotypic and allelic distribution of CRP, COMP, IL-2R polymorphisms among CHIKV infected patients and healthy controls. (b) Genotypic and allelic distribution of CRP, COMP, IL-2R polymorphisms among CHIKV infected patients with or without arthralgia.

SNP Ref. No.	Genotype	Healthy Controls (%)	Chikungunya infected patients (%)	OR (95% C.I)	Relative risk (95% C.I)	p-value
	and allele distribution
		n = 102	n = 167
	CC	2	3	1.093 [0.1913 to 5.429]	1.056 [0.3082 to 2.098]	>0.9999
	CT	21	43	0.7476 [0.4144 to 1.374]	0.8304 [0.5526 to 1.196]	0.3776
	TT	79	121	ref
	C allele	25	49	0.8123 [0.4783 to 1.377]	0.8757 [0.6125 to 1.199	0.5191
CRP rs3093059	T allele	179	285	ref
	Additive	0.628
		n = 102	n = 167
	TT	3	11	2.327 [0.6932 to 7.942]	1.812[0.7990 to 5.176]	0.1915
	TC	39	40	1.965 [1.166 to 3.323]	1.489 [1.092 to 1.996]	0.0126*
CRP rs3091244	CC	60	116	Ref
	T allele	45	62	1.242 [0.8138 to 1.907]	1.140 [0.8736 to 1.452]	0.3243
	C allele	159	272	Ref
	Additive	0.0286*
		n = 102	n = 167
	GG	0	3	2.512 [0.4077 to 31.01]	1.929 [0.6057 to 10.69]	0.6523
IL-2R	GA	22	59	1.987 [1.139 to 3.422]	1.567 [1.079 to 2.353]	0.0198*
rs743777	AA	80	105	Ref
	G allele	22	65	1.999 [1.195 to 3.323]	1.596 [1.119 to 2.364]	0.0079*
	A allele	182	269	Ref
	Additive	0.0179*
		n = 102	n = 167
	AA	0	3	2.452 [0.3981 to 30.27]	1.900 [0.5966 to 10.54]	0.4103
COMP	GA	11	42	2.848 [1.409 to 5.897]	2.058 [1.241 to 3.630]	0.0032*
rs144778694	GG	91	122	Ref
	Aallele	11	46	2.822 [1.444 to 5.524]	2.088 [1.268 to 3.655]	0.0020*
	G allele	193	286	Ref
	Additive	0.0051*
(b): Genotypic and allelic distribution of CRP, COMP, IL-2R polymorphisms among CHIKV infected patients with or without arthralgia
SNP Ref. No.	Genotype	With Arthralgia (%)	Without Arthralgia (%)	OR (95% C.I)	Relative risk (95% C.I)	p-value
	and allele distribution
		n = 112	n = 55
	CC	3	0	2.036[0.3239 to 25.32]	1.207[0.5633 to 1.531]	0.6419
	CT	8	35	0.04396[0.01826 to 0.1106]	0.2218[0.1157 to 0.3914]	<0.0001*
	TT	101	20	Ref
	C allele	14	35	0.1429[0.07313 to 0.2746]	0.3878[0.2411 to 0.5797]	<0.0001*
CRP rs3093059	T allele	210	75	ref
		n = 112	n = 55
	TT	2	9	0.09293 [0.01981 to 0.3832]	0.2579 [0.07262 to 0.6821]	0.0004*
	TC	22	18	0.5025 [0.2510 to 1.029]	0.7761 [0.5532 to 1.012]	0.0626
CRP rs3091244	CC	88	28	Ref
	T allele	26	36	0.2699 [0.1519 to 0.4839]	0.5761 [0.4156 to 0.7555]	<0.0001*
	C allele	198	74	Ref
		n = 112	n = 55
	GG	1	2	0.2387 [0.01635 to 2.108]	0.4925[0.09066 to 1.187]	0.2097
IL-2R	GA	24	35	0.1558[0.07779 to 0.3111]	0.4992 [0.3534 to 0.6680]	<0.0001*
Rs743777	AA	87	18	Ref
	G allele	26	39	0.2391 [0.1356 to 0.4223]	0.5434[0.3908 to 0.7166]	0.0017*
	A allele	198	71	Ref
		n = 112	n = 55
	AA	0	3	0.1184 [0.009611 to 0.7509]	0.2947 [0.05328 to 0.9292]	0.0255*
COMP	GA	32	10	2.343 [1.024 to 5.225]	1.256 [1.003 to 1.516]	0.0471*
rs144778694	GG	80	42	Ref
	Aallele	32	16	0.9792 [0.5266 to 1.925]	0.9931 [0.7738 to 1.196]	0.9493
	G allele	192	94	Ref

*p<0.05 at 95% CI was considered as statistically significant.

“Ref”= reference genotype

3.6. Genotypic association of CRP, IL-2R, COMP polymorphisms with chikungunya induced arthritis

Role of these CRP, IL-2R and COMP polymorphisms was analyzed between patients with (n = 112) and without arthralgia (n = 55) (Table 2b). Analysis revealed CRP-rs3093059-CT, CRP-rs3091244-TT, IL-2R-rs743777-GA and COMP-rs144778694-AA genotypes were significantly associated with patients without any arthralgic manifestations (p-value<0.0001, p-value=0.0004, p-value<0.0001 and p-value=0.0255, respectively). In contrast, COMP rs144778694-GA genotype was significantly linked to patients with arthralgia (p-value=0.0471). Also, CRP-rs3093059-C, CRP-rs3091244-T and IL-2R-rs743777-G alleles were associated with patients without arthralgia (p-value<0.0001, p-value=0.0004 and p-value=0.0017, respectively). Interestingly, CRP-rs3093059-CT genotype was significantly more prevalent among patients who never developed PCA (p-value=0.0317) (Table S2). However, genotyping associations should be analysed upon larger patient cohorts for better clarity.

3.7. Association of CRP, IL-2R, COMP polymorphic genotypes with serum concentration among patients with and without chronic arthritis

Association between polymorphic genotypes and serum level of CRP, IL-2R and COMP was analyzed among AA, PCA and AA+PCA groups (Fig. 4). Serum level of CRP-rs3093059-TT genotype was significantly higher among patients of AA+PCA and AA groups when compared to CC genotype (AA+PCA-TT:77.25mg/l vs CC:55.33mg/l; p-value=0.0123 and TT:44.55mg/l vs CC:30.73mg/l; p-value=0.0037, respectively). CRP level was comparatively higher among patients with CT genotype (CT:91.43mg/l). Moreover, CRP level of rs3091244-CT and CC genotypes was significantly higher compared to patients with TT genotype among AA group (CT:53.27mg/l, CC:43.19mg/l vs TT:22.34mg/l; p-value=0.0089 and 0.0001, respectively). Similarly, patients with rs743777-GA and AA genotype demonstrated significantly higher IL-2R level compared to GG genotype among AA+PCA group (GA:3345pg/ml, AA:3050pg/ml, GG:1120pg/ml; p-value=0.0429, respectively). In case of COMP-rs144778694, patients with GA and GG genotypes showed remarkable increase in COMP level compared to AA genotype within AA+PCA group (GA:46.55ng/ml and GG:43.69mg/l vs AA:27.34ng/ml; p-value=0.0087 and 0.0143, respectively); but this trend was reversed among AA group (AA:34.07ng/ml vs GG:21.09ng/ml GA:22.45ng/ml; p-value=0.0033, p-value=0.0028).

Fig. 4.

Comparative genotypic distribution pattern of CRP, IL-2R, COMP polymorphisms and their serum levels among different arthritic groups of CHIKV-infected patients.

4. Discussion

CHIKV infection has been known to cause protracted illness among patients inducing acute and/or chronic polyarthritis/polyarthralgia, which might persist for at least one year, inviting chronic inflammatory rheumatism and musculo-skeletal disease during PCA (Imad et al., 2021). Thus, identification of certain biomarkers that might help in differentiating chikungunya patients with only acute arthritis from those who might develop chronic polyarthralgia would be useful in pain management of chronic arthritic patients. Both serum level and polymorphic genotypes of CRP, Anti-CCP antibody, IL-2R, COMP, RF and hepatic markers were analyzed in this regard.

CRP concentrations significantly increased among all patients with arthralgia (both acute and chronic) compared to those without. Also, its level was significantly high among AA+PCA patients compared to AA group. Increased level of CRP has been previously reported among chikungunya induced arthritic patients of Indian origin; but this is the first study to differentiate CRP level between acute and chronic arthritic patients with 81.13% sensitivity and 72% specificity (Solanke and Karmarkar, 2014). Increased CRP might act as inflammatory response to activate innate immune response against viral induced arthritis (Sproston and Ashworth, 2018). Anti-CCP antibody concentration was higher among AA+PCA and PCA groups with respect to NA and AA, but the increase was statistically significant for AA+PCA. Anti-CCP antibody was previously detected among La Reunion island-patients with post-chikungunya chronic arthritis, but its differential concentration among acute and chronic arthritic patients has not been previously demonstrated (Imai et al., 2016). Anti-CCP antibody was reported to cross-react with type II collagen, resulting in proteoglycan depletion and severe arthritis (Wu et al., 2020). Similar trend was followed for COMP with its noteworthy increase among AA+PCA and PCA. Current study indicated that COMP level could be used as differentiating diagnostic factor for chronic arthritis among CHIKV-infected patients with 86.79% sensitivity and 92% specificity. Previous reports also suggested COMP to be associated with osteoarthritis and rheumatoid arthritis (Deere et al., 1999; Mishra et al., 2019). COMP, found in articular cartilage, ligament, meniscus, synovial membrane, and tendon, have a role in endochondral ossification and its increased level has been linked to cartilage degradation (Tseng et al., 2009; Arellano et al., 2017). Increased IL-2R level among AA+PCA and PCA compared to NA and AA indicated its differential diagnostic role for chronic arthritic chikungunya patients with 88% sensitivity and 81.13% specificity. Elevated IL-2R concentration has been previously demonstrated among rheumatoid arthritic patients and was reported to be associated with synovitis (Wood et al., 1988; Symons et al., 1988). Thus, according to high sensitivity and specificity scores, current study is the first to demonstrate prognostic relevance of COMP, IL-2R and CRP levels in identifying chronic arthritic chikungunya patients (Fig. 2).

Hepatic anomalies have been previously reported among chikungunya patients of northern India, Singapore, Mexico and Sri Lanka; but their differential concentration between arthritic and non-arthritic chikungunya patients has not been studied earlier (Danis-Lozano et al., 2017; Ng et al., 2009; Singh et al., 2018; Premaratna et al., 2011). Increased concentrations of AST, ALT, AST/ALT ratio, bilirubin and ALP were reported among arthritic chikungunya compared to non-arthritic ones.

Principal component analysis segregated AA+PCA and PCA groups from overlapping AA and NA group of patients. There was a stark proximity of NA group with healthy controls. It further highlighted association of CRP, anti-CCP antibody, AST, ALT, AST/ALT ratio and bilirubin levels with AA+PCA group, whereas, IL-2R and ALP levels were associated with PCA group. Previous reports also associated high CRP, IL-2R levels with post chikungunya chronic arthritic patients (Patel et al., 2019; Teng et al., 2015).

Genotypic analysis implicated protection of individuals with CRP-rs3091244-TC genotype against CHIKV infection, whereas, those with IL-2R rs743777-GA, G-allele and COMP rs144778694-GA genotypes were susceptible to such infection. Cis-acting rs3093059 and tri-allelic rs3091244 has been previously associated with increased CRP levels in vitro, thus suggested to have functional role in transcription factor binding (Sengupta et al., 2022). Various genotypes of CRP-rs3091244 have been previously implicated for susceptibility towards CHIKV/DENV co/mono-infection, ankylosing spondylitis, haemorrhagic and ischaemic stroke among patients of eastern Indian, Turkish, Greek and Chinese origin, respectively. Previous report suggested patients with IL-2R rs743777-G allele to be susceptible to ankylosing spondylitis with peripheral arthritis (Polo et al., 2019). Moreover, infected patients with CRP-rs3093059-CT, CRP-rs3091244-TT, IL-2R rs743777-GA and COMP rs144778694-AA genotypes might have been safeguarded from arthralgic manifestations; whereas, those with COMP rs144778694-GA genotype were susceptible to arthralgia. CRP-rs3091244 variants have been previously reported to give protection against onset of pain among DENV-CHIKV co-infected Indian patients (Sengupta et al., 2022). A previous study on Dutch patients indicated IL-2R variants to be protective against severe forms of multiple sclerosis and RA (van Steenbergen et al., 2015).

In this study, patients with various genotypes of CRP-rs3093059 and rs3091244, IL-2R-rs743777 and COMP-rs144778694 manifested significantly higher protein levels among chikungunya patients suffering from acute arthralgia with/without chronic arthritis. Such elevated CRP levels were previously reported among ankolysing spondylitic and DENV-CHIKV co-infected patients with certain genotypes of CRP rs3091244 and rs3093059 (Sengupta et al., 2022). Indication for association with COMP levels was found with COMP genetic polymorphisms among osteo-arthritic patients of Dutch origin (Ramos et al., 2014).

5. Conclusion

In light of our findings, this study confirmed that both biomarker levels in serum and genetic factors played an important role in development of post-chikungunya chronic arthritis among infected patients. Upon diagnosis of CHIKV infected patients with/without acute arthralgia, these markers could be used to screen patients to identify their potential of developing chronic arthritis. This might be helpful for clinical management of PCA patients by a medical specialist. However, these factors should be validated among larger patient cohorts.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors.

Availability of data and material

All data generated and analysed during this study are included in this article.

Ethics approval

All procedures performed in this study involving collection of blood from all human participants were in accordance with ethical standards of Clinical Research Ethical Committee of Calcutta School of Tropical Medicine (CREC-STM/53 dated 26.09.2013). Written consents were received from patients and healthy control individuals prior to participation in the study.

Fig. S1: Principal component analysis representing linkage and variance among AA+PCA, PCA, AA, NA of CHIKV-infected patients according to 2010 ACR/EULAR classification.

Fig. S2: Comparative analysis of age groups and serum concentration of arthritic biomarkers among chronic arthritic, acute arthritic and non-arthritic groups of CHIKV-infected patients.

Fig. S3: Comparative analysis of gender and serum concentration of arthritic biomarkers among chronic arthritic, acute arthritic and non-arthritic groups of CHIKV-infected patients.

CRediT authorship contribution statement

Siddhartha Sengupta: Formal analysis, Investigation, Writing – original draft, Writing – review & editing. Nemai Bhattacharya: Resources. Anusri Tripathi: Conceptualization, Formal analysis, Investigation, Resources, Project administration, Writing – review & editing.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data Availability

• No data was used for the research described in the article.