Examining the Correlation between Serum Folate Levels and Disease Severity in Hospitalized Patients with COVID-19

¹ Cancer Research Center, Hamadan University of Medical Sciences, Hamadan, Iran

² Department of Infectious Disease, School of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran

³ Cardiovascular Research Center, Hamadan University of Medical Sciences, Hamadan, Iran

⁴ Department of Pathology, School of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran

⁵ Department of Paramedicine, Amol School of Paramedical Sciences, Mazandaran University of Medical Sciences, Sari, Iran

⁶ Department of Biostatistics, School of Public Health, Hamadan University of Medical Sciences, Hamadan, Iran

⁷ Department of Radiopharmacy, School of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran

*Corresponding author: Maryam Alvandi, Cardiovascular Research Center, Hamadan University of Medical Sciences, Hamadan, Iran. Email: maryamalvandi@yahoo.com

EXTENDED ABSTRACT

Background

Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, has produced a wide spectrum of clinical manifestations and substantial mortality worldwide [1]. Variation in disease severity is influenced by multiple host factors, including nutritional status and micronutrient availability. Vitamins and other micronutrients may contribute to antiviral defense through antioxidant, anti-inflammatory, and immune-modulating functions [2, 3]. Folate (vitamin B9) is essential for nucleic-acid and amino-acid synthesis and supports both innate and adaptive immunity; deficiency can impair cytotoxic T-cell, natural-killer-cell, and antibody responses [4, 5]. Experimental and clinical observations have suggested that folate status may influence susceptibility to SARS-CoV-2 infection through immune function, oxidative-stress control, regulation of the renin-angiotensin system, and interference with viral entry or replication [6-9]. However, published findings regarding the association between folate status and COVID-19 severity and prognosis have not been uniform [10]. The present study therefore examined serum folate levels in hospitalized patients with COVID-19, assessed their relationship with clinical severity, and evaluated whether folate concentration could have prognostic value for mortality.

Methods

This cross-sectional study was performed at Sina Hospital in Hamadan during Iranian calendar year 1401. A total of 117 hospitalized adults with COVID-19 were included. Eligible participants were at least 18 years old and had clinical manifestations compatible with COVID-19 together with chest CT findings and a positive PCR test. Patients with cancer, thalassemia, hepatic disorders, gastric ulcer, renal disease, major cardiac disease, known folate deficiency before COVID-19, pregnancy, or infection with hepatitis B or C virus, cytomegalovirus, or HIV were excluded. Demographic characteristics, comorbidities, clinical manifestations, vital signs, and laboratory data were extracted using a researcher-developed questionnaire. Hospitalization criteria included respiratory rate greater than 30 breaths/min, oxygen saturation below 93%, or pulmonary infiltration on chest imaging. Disease severity was classified according to the criteria used in the source article [11]: mild-to-moderate disease was characterized by oxygen saturation of 90%-93% with pulmonary infiltration below 50%; severe disease by oxygen saturation below 90%, pulmonary infiltration above 50%, and respiratory rate above 30/min; and critical disease by oxygen saturation below 88% with respiratory failure and multiorgan dysfunction. For folate assessment, blood samples were collected, processed by centrifugation, stored at -20 °C, and analyzed by ELISA using a Monobind kit and Stat Fax 3200 instrument. Data normality was evaluated with the Kolmogorov-Smirnov test. Depending on distribution and variable type, comparisons used ANOVA, chi-square, Mann-Whitney, or Kruskal-Wallis tests with appropriate post-hoc procedures. Binary logistic regression assessed the association of age, length of hospitalization, and folate with mortality. Receiver operating characteristic (ROC) analysis was used to evaluate folate as a discriminator of death versus survival and to determine a cut-off value. Analyses were conducted with SPSS version 26 and MedCalc, with statistical significance set at P<0.05. The study received ethics approval from Hamadan University of Medical Sciences (IR.UMSHA.REC.1401.582), and written informed consent was obtained.

Results

The 117 patients were classified as mild-to-moderate (n=47), severe (n=46), or critical (n=24). Mean age increased across these groups from 51.81±15.18 years to 63.11±13.38 years and 77.08±7.85 years, respectively. Mean body mass index also increased from 27.34±3.85 to 28.48±4.28 and 31.38±5.15 kg/m². The principal finding was a marked stepwise decline in serum folate with increasing disease severity: 25.28±2.37 ng/mL in the mild-to-moderate group, 16.14±1.42 ng/mL in the severe group, and 10.52±1.06 ng/mL in the critical group (P<0.001). The authors also reported significant associations between disease severity and several predisposing factors, including hypertension, dyslipidemia, smoking, and diabetes. Fever, chest pain, fatigue, and diarrhea differed significantly among severity groups, whereas several other symptoms did not. Oxygen saturation declined with greater severity (88.72±4.98%, 85.02±6.47%, and 82.71±8.11%; P<0.001), and mean hospital stay increased from 4.43±1.46 days to 6.93±4.71 and 8.63±4.99 days (P<0.001). No deaths occurred in the mild-to-moderate group, three deaths (6.5%) occurred in the severe group, and six deaths (25.0%) occurred in the critical group; mortality therefore increased significantly with disease severity (P=0.001).

Table 2. Clinical outcomes among patients with COVID-19 according to disease severity.

Binary logistic regression showed that longer hospitalization was significantly associated with mortality (odds ratio [OR]=1.24, P=0.007). Age was not independently associated with mortality in this model (OR=1.01, P=0.882). Mean folate concentration was 11.98±2.90 ng/mL among patients who died and 19.23±5.96 ng/mL among survivors. Each 1-unit increase in folate was associated with a 23% reduction in the odds of death (OR=0.77), but this adjusted association was not statistically significant (P=0.178). Despite this, ROC analysis showed good discriminatory performance for folate in distinguishing death from survival, with an area under the curve of 0.864 (95% CI: 0.788-0.921; P<0.001). A folate cut-off of 14.90 ng/mL yielded 88% sensitivity and 74% specificity for classifying mortality outcome. Thus, lower folate levels were strongly associated with more severe COVID-19, and folate concentration demonstrated useful unadjusted prognostic discrimination for mortality, although the adjusted logistic-regression coefficient for folate did not reach statistical significance.

Table 3. Binary logistic-regression analysis with mortality as the dependent variable.

Figure 1. Receiver operating characteristic (ROC) curve for serum folate in predicting mortality among patients with COVID-19.

Conclusion

Among hospitalized patients with COVID-19, lower serum folate levels were associated with progressively greater disease severity, longer hospitalization, lower oxygen saturation, and higher mortality across severity categories. Folate also showed good ROC discrimination for mortality at the reported 14.90 ng/mL cut-off, although its adjusted association with death was not statistically significant. The findings support assessment of folate status during hospitalization and consideration of supplementation when deficiency is identified, while recognizing the need for additional studies to clarify the clinical significance and causal role of folate in COVID-19 outcomes.

Keywords: COVID-19, Folic Acid, SARS-CoV-2, Vitamin B9

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