2.1 Study Setting and Patient Enrollment

This study was conducted in the Cambodian provinces of Kampong Thom, Kandal, and Phnom Penh between 2021 and 2022 (Supporting Information S2: Figure S1). Patients presenting with acute CHIKV infection were enrolled from participating hospitals. The study received ethical approval from the Cambodian National Ethics Committee for Health Research (Approval Date: 26th October 2020, NECHR No. 262). All participants provided informed consent for the use of their blood samples and anonymized clinical data. For pediatric patients, parental consent was required.

Eligible participants included males and females aged 48 months and older who presented within 72 h of acute febrile illness (≥ 38.5°C) with sudden-onset joint pain and had CHIKV infection confirmed via RT-PCR. Written informed consent was required, and for patients under 18 years, consent was obtained from a parent or legal guardian, with inclusion contingent on the ability to collect an adequate blood sample and at least two follow-up sampling. Patients coinfected with dengue virus were excluded from the study, though prior dengue exposure did not disqualify participation.

2.2 Clinical and Laboratory Data Collection

At the initial hospital presentation, demographic details, clinical symptoms, and the timing of symptom onset relative to clinic presentation were recorded (Supporting Information S2: Table S1). Blood samples were collected at the first visit (Day 0) and at regular 2-day intervals for 10 days (Figure 1). Follow-up blood sampling as well as clinical symptoms after Day 0 were collected either during hospitalization or, for patients who were discharged, at their households. No further follow-up after Day 10 was performed. Each blood sample was tested for viral load, anti-CHIKV IgM and IgG antibodies, and C-reactive protein (CRP). Additionally, the total symptom burden was evaluated by counting the number of 14 prespecified symptoms reported by each patient at each visit (Table 1).

2.3 Laboratory Assays

All assays were conducted at the Virology Unit and Medical Biology Laboratory Unit of the Institut Pasteur du Cambodge (IPC) in Phnom Penh. Whole blood samples collected at local sites were centrifuged (1000–2000g for 10 min at 4°C) to separate serum, which was aliquoted into RNase- and DNase-free 1.5 mL polypropylene Eppendorf tubes. Serum samples were transported and stored at −80°C for subsequent testing.

Detection and quantification of CHIKV were performed on acute and subsequent serum samples. Viral RNA was extracted using the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) per manufacturer's instructions. The extracted RNA was tested for CHIKV using real-time RT-PCR (RT-qPCR) adapted from Pastorino et al. [30] targeting the E1 gene with SuperScript III Platinum One-Step Quantitative RT-PCR Kit (Invitrogen, Carlsbad, CA, USA). The reactions were conducted in 25 μL volume containing 5 μL of RNA, 12.5 μL of 2× Thermoscript Reaction Mix Buffer, 1 μL of 50 mM MgSO₄, 0.1 μM of the TaqMan probe, 0.2 μM of each primer, and 0.5 μL of the enzyme mix (Invitrogen). Real-time RT-PCR assays were carried out in the Bio-Rad CFX96 Real-Time Detection System (Bio-Rad, Hercules, CA, USA) using the following thermal cycling parameters: reverse transcription at 50°C for 15 min, inactivation at 95°C for 2 min followed by 45 cycles of fluorescence detection at 95°C for 15 s, and annealing and extension at 60°C for 1 min. Viral loads were expressed as RNA copy numbers based on a standard curve of known plasmid copy numbers. A cycle threshold (Ct) value > 40 indicated the absence of CHIKV.

CRP concentrations were measured using a commercially available EIA kit (Abcam ref. ab99995), with a lower limit of quantification of 5 mg/L. Anti-CHIKV IgM antibodies were quantified using an in-house MAC-ELISA method [31] with antigens derived from a Cambodian CHIKV strain isolated in 2012. Briefly 96-well microtiter plates were coated with goat anti-human IgM and 100 µL of serum samples and controls diluted 1∶100 in phosphate buffered saline + Tween 20 0.5% (PBS-T) + 5% nonfat dried milk (PBS-T-NDM) were added and incubated for 1 h at 37°C. After washing with PBS-T, CHIKV suckling mouse-brain derived antigen produced by sucrose acetone extraction was added to each well at a concentration of 16 hemagglutination units, and incubated at 4°C overnight. The plate was washed with PBS-T, monoclonal antibody to the antigens being detected was added, incubated at 37°C, washed and peroxidase conjugated anti-mouse Fab H + L was added. The final reaction was developed and read at 405 nm on an automated plate reader. A result was considered positive for CHIKV IgM antibodies when the optical density (OD) was higher than 0.1 (threshold determined by measuring the OD of CHIKV-negative human serum). IgG antibodies were measured using the CHIKJJ Detect IgG ELISA (InBios, Seattle, WA), with OD < 1.0 considered negative according to manufacturer's recommendation.

2.4 Statistical Analysis

Demographic and clinical characteristics of participants were summarized using absolute and relative frequencies for categorical variables and medians with ranges (minimum–maximum) for continuous variables. Comparisons between adults and children were performed using Pearson's χ² test or Fisher's exact test for categorical variables and the Mann–Whitney U test for continuous variables.

3.1 Patient Characteristics and Clinical Symptoms

A total of 46 acute CHIKV infection cases were identified, and among them 29 patients, all with Khmer ethnicity, who met all the inclusion and exclusion criteria with at least two follow-up sampling time-points were enrolled in the study. The cohort's demographic characteristics and clinical characteristics, stratified by age group, are shown in Table 1. The most frequently reported symptoms were similar in adults and in children, with both groups experiencing fever, headache, and arthralgia. However, gastrointestinal symptoms (nausea, vomiting, or diarrhea) were more frequent in children. In general, fever resolved within the first 7 days; however, some individuals experienced elevated temperatures up to 10–12 days after symptom onset. The evolution of the total symptom burden and individual symptom frequencies was summarized (Figure 2 and Supporting Information S2: Figure S2).

3.2 Viral Load Kinetics

A total of 17 subjects (59%) were sampled at all 6 time-points over a period of 11 days. Of the 29 subjects, 26 (90%) had complete data or no more than 1 missing sample. Using the TCL model, we constructed both population-average (Figure 3) and individual (Figure 4) viraemia-time profiles for all 29 subjects. The population-average profile revealed a peak viral load at ~1.87 day prior to symptoms onset, with significant variability observed at the individual level. Estimated population parameters (Supporting Information S2: Tables S2 and S3) were calculated using a nonlinear mixed effects model based on the TCL model. From these parameters, descriptive statistics of CHIKV viraemia across the 29 patients were derived and reported in Table 2.

Significant differences in the viral kinetics were identified between adults and children. Infants displayed a significantly lower target cell infection rate, but higher viral replication rate and maximum viral load leading to a shorter time to first detectable viraemia (Supporting Information S2: Table S4). The resulting higher peak viraemia in children (Supporting Information S2: Figure S3) did not translate into any differences in the frequency or number of symptoms. Viral kinetics were not associated with sex or number of symptoms (analyzed as a binary variable </≥ the median). Restricting this analysis to the number of acute musculoskeletal symptoms reported (arthralgia, arthritis, myalgia, and stiffness) also showed no statistically significant association with viral kinetics (Supporting Information S2: Table S4).

3.3 Kinetics of IgM, IgG, and CRP

IgM was first detectable in the majority of patients between 4 and 8 days post-symptom onset, with earlier detection in adults compared to children (p = 0.036; Figure 5A). In contrast, IgG was generally detectable after the first week, with no significant difference in time to detection between age groups (Figure 5B). Analysis by age category showed a more rapid appearance of IgM in adults with no difference in time to IgG appearance. In contrast, the amplitude of the IgM response was similar in the two age groups while the amplitude of the IgG response was greater in children (Table 3).

Elevated CRP levels (> 10 µg/L) were found in approximately half of patients at the first sample (17/29), typically during the first 3 days after symptom onset (Figure 5C). Most later samples showed normal serum concentrations, though a few patients had elevated levels in the second week. Only three individuals had markedly elevated CRP at multiple timepoints (Supporting Information S2: Figure S4).

It was noted that two individuals showed almost no detectable IgM or IgG during the sampling period (Supporting Information S2: Figure S4). The first was a 24-year-old female experiencing arthralgia and other symptoms throughout the 10-day follow-up, but with a maximum viral load of only 1.09 × 10³ copies/mL and no elevation of CRP. The second was a 70-year-old female experiencing few symptoms in this period, although viral load started high at 3.7 × 10⁸ declining to 7.3 × 10⁴ copies/mL 4 days later with elevated CRP between 8 and 14 mg/L.

3.4 Simulations of Antiviral Strategies

Simulation models indicated that an antiviral agent achieving a 99% reduction in target cell entry, such as an anti-CHIKV monoclonal antibody, when administered within the first 3 days of symptom onset, reduced the area under the viraemia curve (AUC) by 18%–23%, with no significant impact on the maximum viral load (data available upon request). However, prophylactic administration 3 days prior to symptom onset resulted in a 99% reduction in AUC, a 98% reduction in maximum viral load, and effectively prevented detectable viraemia.

4.1 Viraemia and Transmission Dynamics

Consistent with existing studies, our model suggests that viraemia can be detectable ~2–3 days before symptom onset. Presymptomatic viraemia is a critical factor in the transmission of CHIKV, similar to asymptomatic carriers, who might unknowingly contribute to outbreaks. Our findings align with previous reports by Riswari et al. [29], who observed prefebrile viraemia in clinical cases. This Indonesian study also highlighted variability in the timing of peak viral loads, with some patients showing detectable viraemia at Days 5–6 prior to symptom onset, potentially indicating a longer incubation period.

We observed that viral load typically peaks within 48–72 h post-symptom onset, with prolonged viraemia lasting up to 18 days in certain individuals. These results are consistent with prior population-level studies, which observed viraemia persisting beyond the first week of symptoms [25, 26]. Further research is needed to assess transmissibility, particularly during the extended period of detectable viremia.

4.2 Clinical Signs and Disease Progression

Our findings indicate that viraemia levels did not significantly correlate with the number or severity of symptoms in the acute infection phase, including musculoskeletal symptoms such as arthralgia and myalgia. This aligns with studies reporting no direct relationship between viral load and symptom severity, suggesting that symptom expression may be more strongly influenced by individual immune responses or other host factors [32, 33]. However, contrasting evidence highlights that higher viraemia levels and prolonged viraemia duration are associated with more severe manifestations, including neurological complications, some rare haemorrhagic features, and persistent joint pain. Animal models also suggest a potential link between CHIKV RNA detection in joint tissues and chronic arthralgia, indicating that localized viral persistence may contribute to disease severity and sequelae [34-37]. Furthermore, older age has been identified as a significant risk factor for severe disease, including atypical presentations and higher mortality during outbreaks [38-40]. These discrepancies underscore the need for further research into the interplay between host immune response, age, underlying conditions, viral load, and the persistence of CHIKV in tissues to better understand the determinants of symptom severity and disease progression.

4.3 Immune Response and Antibody Dynamics

In terms of immune response, anti-CHIKV IgM antibodies were first detectable between 4 and 8 days post-symptom onset, with adults showing earlier detection compared to children (p = 0.036). This finding is in line with previous studies [25, 41], which demonstrated a delay in IgM appearance in pediatric cases. The delayed IgM response in children, coupled with their higher viral replication rates and peak viral loads, could potentially extend the period of viraemia, making younger individuals more infectious for longer periods.

Interestingly, while there was no significant difference in the time to IgG detection between adults and children, children exhibited a stronger IgG response. This could reflect a more robust long-term immune response in pediatric cases, though the clinical significance of this remains unclear.

4.4 C-Reactive Protein (CRP) as an Inflammatory Marker

Our study found that CRP, a marker of systemic inflammation, was transiently elevated in about half of the patients during the acute phase of CHIKV infection, without strong associations with viral load or symptom severity. This aligns with previous studies suggesting CRP as an acute-phase inflammatory biomarker without clear predictive value for chronic complications [42, 43].

However, contrasting evidence indicates that elevated CRP during the acute phase may correlate with higher risk of chronic outcomes like postviral arthritis [44, 45]. These discrepancies highlight the need for further research to determine CRP's role in predicting long-term complications, accounting for variations in study populations and methodologies.

4.5 Monoclonal Antibodies as a Therapeutic Strategy

Our simulations of antiviral strategies demonstrated the significant potential of mAb treatments. Administering mAbs early—particularly within 3 days of symptom onset—was shown to reduce viraemia by up to 23%. These findings are consistent with recent advances in mAb therapy for CHIKV, where clinical and experimental studies have demonstrated their ability to neutralize the virus and reduce disease severity in animal models [17, 22, 23, 46, 47]. Moreover, other studies have confirmed the safety and efficacy of mAbs in reducing viraemia and alleviating joint inflammation [17, 48, 49].

Importantly, our simulation results indicated that by incorporating mAb prophylaxis into public health interventions, the risk of transmission during the presymptomatic phase could be significantly minimized. This approach may provide a powerful tool to curb outbreaks, limit community transmission, and protect public health. However, further clinical study is needed to understand its effectiveness in real world.