High COVID-19 virus levels and body inflammation linked to brain-related and muscle symptoms in long COVID

The coronavirus disease (COVID-19) pandemic was a public health concern because of its expansion worldwide, with 677 million cases and 6.9 million deaths reported worldwide to July 2024 [1]. Pakistan with its 220 million population, reported approximately 1.5 million cases and 31,000 COVID-19 related deaths [2]. While most individuals recover from the acute infection, a sizable portion of individuals report long-term varying symptoms lasting from weeks to several months following apparent recovery from the acute infection [3]. This condition is known as “Long COVID” as defined by World Health Organization (WHO) occurring typically after 3 months of acute COVID-19 infection [4].

Risk for Long COVID appears higher in females, advanced age, individuals with co-morbid conditions as well as those who experienced more symptoms during the acute infection (≥5 symptoms) [5]. Long COVID may involve multiple organ systems including neurological, respiratory, musculoskeletal, and reproductive systems with common manifestations such as fatigue, cognitive impairment, dyspnea, sleep disturbances, and musculoskeletal pain [6–9]. The underlying pathophysiology contributing to these symptoms is complex and includes micronutrient deficiency, autonomous dysfunction, chronic inflammation, immune dysregulation and occult viral persistence [10].

Emerging evidence suggests that inflammatory markers such as interleukin-6 (IL-6), C-reactive protein (CRP), and ferritin may remain elevated months after acute infection [11]. Micronutrient deficiencies have also been implicated in persistent symptomatology [12]. However, much of the available literature focuses on survivors of severe or critical illness, and data among individuals who experienced mild disease particularly in middle-aged populations remain limited.

The relationship between acute-phase viral load, reflected by reverse transcription polymerase chain reaction (RT-PCR) cycle threshold (Ct) values, and the subsequent development of Long COVID remains an area of ongoing investigation. Although higher viral loads have been associated with severe acute COVID-19 [13], evidence regarding their association with persistent symptoms following mild infection is scarce. Clarifying whether acute viral burden influences long-term symptom profiles may enhance understanding of Long COVID pathogenesis.

Understanding the biological determinants of persistent symptom clusters following mild COVID-19 is clinically important, as most infected individuals do not require hospitalization yet may experience prolonged morbidity. Identifying inflammatory and micronutrient alterations associated with specific symptom phenotypes may contribute to improved risk stratification and inform future mechanistic and interventional studies.

Therefore, this study aimed to estimate the frequency of Long COVID among middle-aged individuals recovering from mild infection and to determine whether inflammatory biomarkers, vitamin D and vitamin B12 levels, and acute-phase viral load were independently associated with dominant symptom clusters at ten months post-infection.

Spearman correlation analysis demonstrated a modest but statistically significant inverse association of acute-phase Ct values with IL-6 levels (rho= −0.337, p = 0.016), and ferritin levels (rho = −0.451, p = 0.001) indicating higher IL-6 and Ferritin levels among individuals with higher viral load (lower Ct values). No significant correlations were observed between Ct values and lymphocyte counts (p = 0.825).

In this age and gender matched case-control study of middle-aged individuals with mild or asymptomatic COVID-19 infection, we found that 59% had Long COVID at 10 months after the acute infection. Symptoms most commonly were related to neuropsychiatry, followed by musculoskeletal, respiratory, reproductive and gastrointestinal systems. It was observed that biochemical and hematological parameters, between controls and those with Long COVID, had several notable differences. Participants with Long COVID had significantly reduced hemoglobin, RBCs, with higher WBC count, lymphocytes, IL-6, and ferritin levels. Vitamin D3 and B12 were markedly lower in the Long COVID group. Our findings also showed that higher viral loads during acute infection were linked with neuropsychiatric and musculoskeletal symptoms of Long COVID.

At the 10-month follow-up, 59% of previously infected participants met criteria for Long COVID. The findings of our study align with and build on existing literature. It is broadly consistent with earlier reports indicating that roughly 40–70% of convalescent individuals exhibit persistent or newly emergent post-acute sequelae across varying follow-up intervals [20–22]. The findings of our study corroborate existing literature showing that neuropsychiatric, musculoskeletal and respiratory symptoms are the most frequently reported symptoms post-COVID, even in patients that had a mild acute infection [23]. In a comprehensive meta-analysis, which synthesized 63 studies and showed that predominant symptoms vary by follow-up interval: at 3– < 6 months, dyspnea, fatigue, sleep disturbance, and impaired concentration were common; at 6– < 9 months, effort intolerance, dyspnea, sleep disturbance, and fatigue predominated; and at 9–12 months, fatigue, dyspnea, sleep disturbance, and myalgia were most frequently reported [24].

We found hemoglobin was significantly lowered in Long COVID group along with RBC count and MCHC as compared to controls group. SARS-CoV-2 has been linked to alterations in erythrocyte morphology and rheology in severe disease, with changes persisting for up to six months after recovery [25]. Evidence in mild cases is limited. One study described lower hemoglobin, MCV, and MCHC at three months post-infection, whereas a study reported no group differences in hemoglobin, RBC count, or indices but did note morphological abnormalities relative to controls, acknowledging a small sample size [26].

Leukocyte findings in our study showed higher total WBC counts and a greater lymphocyte proportion in the Long COVID group. SARS-CoV-2 affects immune cell profiles during and after infection, and dysregulated differentials have been described. A study observed elevated neutrophils, lymphocytes, basophils, and eosinophils in recovered individuals, without differences in total WBC or monocytes versus healthy controls [26]. Conversely, lymphocytopenia during the acute phase and into follow-up has also been documented. Another study reported lymphocytopenia at six months post-acute COVID-19 illness [27]. The inflammatory markers, including IL-6, and ferritin were elevated in our study, aligning with previous studies [28,29] that reported persistent immune activation in Long COVID patients up to six months. This persistence of inflammatory markers is due to immunological dysregulation, following a viral infection. IL-6 is an important mediator of neuroinflammation and has been attributed to symptoms like brain fog, fatigue, cognitive dysfunction by disrupting the blood-brain barrier and altering neurotransmitter signaling [30]. Similarly, increased levels of ferritin indicate ongoing macrophage activation which might contribute to musculoskeletal symptoms via mitochondrial dysfunction and catabolic alterations [31]. These findings support our study and link to possible ongoing systemic inflammation even at 10 months post-acute COVID-19 infection.

While significant changes were observed in vitamin D and B12 levels in our study, micronutrient deficiency, especially that of vitamin D is significantly linked with fatigue, cognitive dysfunction and musculoskeletal symptoms [32]. An association was found between vitamin D levels and neuropsychiatric along with musculoskeletal symptoms in Long COVID. Vitamin D is vital for normal neuromuscular function and immunomodulation. Its deficiency inhibits T-regulatory cell activity, resulting in prolonged inflammation, contributing to fatigue, cognitive impairment and muscle weakness [33] which explains the associations found in our study.

While our study didn’t observe significant differences in lipid profile (total cholesterol, HDL, LDL, triglycerides), other studies have reported lipid profile alterations in Long COVID. A systematic review and meta-analysis reported significantly elevated levels of total cholesterol and triglycerides in long-COVID individuals in comparison to the controls, indicating increased atherogenicity [34]. These findings underscore the heterogeneity in lipid profiles amongst Long COVID individuals and emphasize the need for further research.

The association between SARS-CoV-2 viral load during acute infection and clinical phenotype in Long COVID remains incompletely characterized. While higher viral burden may carry prognostic value in individuals with compromised immune function, high titers are also observed in patients with mild or asymptomatic infection, limiting the potential use of viral load to serve a role as indicator of disease course [35]. We further explored if our findings have any correlation with SARS-CoV-2 viral load in acute phase. We found higher viral load in individuals with neuropsychiatric and musculoskeletal symptoms of Long COVID. Higher viral load of SARS-CoV-2 have been associated with immune activation, and a surge in cytokine levels (IL-6, TNF-α), leading to widespread inflammation and tissue injury. This elevated neuroinflammation may disrupt the integrity of blood-brain barrier, resulting in symptoms like brain fog, cognitive dysfunction and fatigue [36]. Similarly, myalgia and weakness can be attributed to inflammatory damage to the muscles and mitochondrial dysfunction [37] which might be attributed to higher viral load in initial COVID-19 illness.

In multivariable analyses, distinct biological patterns were observed across symptom clusters. Neuropsychiatric manifestations were independently associated with higher lymphocyte counts, IL-6, and ferritin levels, as well as vitamin D deficiency, suggesting a persistent inflammatory and immune activation profile. In contrast, musculoskeletal symptoms demonstrated a strong independent association with vitamin D deficiency, with a more modest contribution from ferritin and IL-6. These findings support the hypothesis that Long COVID may represent heterogeneous phenotypes characterized by differing inflammatory and micronutrient signatures rather than a uniform post-viral syndrome. Also, the observed inverse correlation of Ct values with IL-6 and ferritin supports the hypothesis that higher acute viral burden may contribute to sustained inflammatory activation [38].

The significant burden (59%) of long COVID beyond 10 months of acute mild infection calls for long-term follow-up. These observations emphasize the urgent need for comprehensive post-COVID systemic monitoring, the establishment of uniform clinical management protocols and standardized treatment guidelines. Persistent biochemical abnormalities also indicate that biological recovery may persist beyond symptom resolution, necessitating continued monitoring. Routine post-COVID biomarker assessment including inflammatory markers (IL-6, ferritin, CRP), lipid profile, hematological indices and micronutrient (vitamin D and B12) level assessments should be included in routine follow-up.

Moreover, raised inflammatory markers at ten months follow up and SARS-CoV-2 viral load during acute infection were also found to be associated with neuropsychiatry and musculoskeletal symptoms of Long COVID indicate the impact of viral load during acute infection on symptoms of Long COVID.

Future research using large scale, prospective, multi-center studies are essential to understand and identify physiological pathways involved in these individuals. These efforts are essential standardized clinical recommendations and optimizing long-term care plans for COVID-19 survivors.

The study was conducted in a single center restricting the general applicability of the results. Symptom assessment relied on patients themselves, raising concerns about reporting bias. We could not assess the symptoms of COVID-19 during acute infection due to possible recall bias. This could help in assessing if Long COVID symptoms correlate with previous symptoms or if they were more intense or prolonged. We did not have data regarding the biochemical markers of the participants during or before COVID-19 infection. Since pre-infection biochemical measurements were not available, we cannot exclude the possibility that some of the observed differences between Long COVID and control groups predated SARS-CoV-2 infection and were unrelated to COVID-19 itself.

Although our study identified statistically significant differences in several hematological and biochemical parameters, the clinical relevance of these findings must be interpreted with caution. Most of these parameters remained within established reference ranges hence should not be used as standalone markers of organ involvement. This suggests that isolated statistical significance may reflect normal physiological variation rather than pathophysiological effects of Long COVID. Emphasizing biologically plausible and consistently altered markers such as IL-6 and vitamin deficiencies provides a more meaningful framework for understanding persistent symptomatology and guiding future mechanistic and interventional studies.

The exclusion of individuals with pre-existing comorbidities such as diabetes, metabolic disorder, hypertension, may underestimate the true burden of Long COVID among a population where these conditions are common. Moreover, the laboratory parameters were measured from a single diagnostic facility, potentially introducing a selection bias.

We acknowledge and are grateful to all participants for their time and commitment. We also thank the faculty and staff of the Sindh infectious Disease Hospital and Research Center, and the Department of Physiology, Dow International Medical College, Karachi, Pakistan, for their sustained support throughout the study.

The data analyzed in manuscript is owned by Dow University of Health Sciences. The data can be obtained fromfor researchers who meet the criteria for access to confidential data. irb@duhs.edu.pk

The author(s) received no specific funding for this work.