Abstract
Objective
To investigate the diagnostic value of peripheral blood lymphocyte testing in children with infectious mononucleosis (IM).
Methods
A total of 135 children with IM as the IM group and 100 healthy volunteers as the healthy group were included in this retrospective study. Peripheral blood lymphocyte subsets marked as CD3+, CD4+, CD8+, CD16 + CD56+, and CD19 + in the peripheral blood were quantified using flow cytometry. Statistical analysis was performed using the chi-square test, Kruskal-Wallis test, AUROC curve, and Kappa consistency test to assess the diagnostic value of these markers in IM.
Results
The AUROC curve for CD8 + cells and for CD4+/CD8 + ratios both achieved a value of 1 with the sensitivity and specificity of 100% (P<0.001). The Kappa coefficients were 1 for CD8+, CD4+/CD8 + ratios and the combined EBV analysis, indicating a 100% consistency with the clinical diagnosis. Significant differences were also observed in the CD3+, CD4+, CD16 + CD56+, and CD19 + lymphocyte subsets between the IM group and the healthy group (P<0.05).
Conclusion
The evaluation of CD8 + and CD4+/CD8 + ratios in peripheral blood lymphocytes represents a significant advancement in the diagnosis of IM. Peripheral blood lymphocyte testing offers a reliable, sensitive, and specific diagnostic tool to enhance the clinical management of children with IM.
Keywords: Peripheral blood lymphocyte testing, Infectious mononucleosis, CD8, CD4/CD8, AUROC
Introduction
Infectious mononucleosis (IM) is a contagious disease primarily associated with Epstein-Barr Virus (EBV) infection, commonly occurring in asymptomatic young children at age of 2–5 years old [1]. The primary clinical manifestations of IM encompass enlarged lymph nodes, fever, and pharyngitis [2]. It can lead to tonsillar enlargement, hepatomegaly, and splenomegaly, along with an elevated lymphocyte count in the peripheral blood of patients [2–5]. Although acute symptoms of IM are often self-limiting, serious complications such as airway obstruction and splenic rupture may occur if not treated promptly [6]. Early and accurate diagnosis of IM is in great need to reduce the incidence of severe cases.
The peripheral blood routine test has been widely used to diagnose IM in children [7]. The clinical positive diagnosis includes lymphocyte number in peripheral blood more than 50% of the total white blood cell (WBC), lymphocyte count >5 × 109 /L, or atypical lymphocytes over10% [5]. The serum EBV test is another common diagnostic tool including Epstein-Barr viral capsid antigen (VCA)-IgM, VCA-IgG, early antigen (EA)-IgG and Epstein-Barr virus nuclear antigen (EBNA)-IgG test and quantitative PCR-based detection of EBV-DNA load [8, 9]. Because of the immature immunity of children and the presence of maternal antibodies in infants, false negative and false positive results are prone to occur, leading to inaccurate results [6]. Therefore, more reliable tests will be required to confirm IM with the accurate clinical signs and symptoms.
Peripheral blood lymphocyte subsets fluctuate in response to EBV in the development of IM [10]. EBV primarily targets B cells leading to their necrosis or apoptosis. CD8 + T cells are activated to eliminate virus-infected cells [11–15], resulting in a decrease of the CD4+/CD8 + ratio [10, 16]. Both CD4 + and CD8 + T cells play a pivotal role in preventing the reactivation of the virus from latency although the mechanism remains unclear [17].
At the present, diagnosing IM requires a combination of clinical manifestations and laboratory diagnostic criteria [7]. A great percentage of children with IM initially present with nonspecific symptoms, which can lead to misdiagnosis or delayed diagnosis [7]. This study aimed to investigate the diagnostic value of lymphocyte subset classification by comparing the peripheral blood lymphocyte subsets between IM children and healthy ones, in order to develop an accurate method for early diagnosis of IM.
Materials and methods
General information
Ethical approval
for this study was provided by the Ethical Committee of the Suqian First Hospital between January 2019 and June 2023 and conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained before the patients’ parents or legal guardians participated in the study. The privacy rights of human subjects always be observed.
Inclusion and exclusion criteria
Inclusion criteria
Children in the IM group were diagnosed in accordance with the clinical and laboratory diagnostic criteria outlined in the Recommended Principles for the Diagnosis and Treatment of Diseases Associated with Major Non-Tumorigenic Epstein-Barr Virus (EBV) Infections in Children [18].
Exclusion criteria
(1) Children with autoimmune diseases or immunodeficiency disorders; (2) Children who have concurrent severe heart, liver, and kidney diseases; (3) Children diagnosed with malignant tumors; and (4) Children exhibiting mental illness or abnormal mental states.
Research methodology
Reagents and instruments
The fluorescent antibodies including CD3-FITC, CD16 + CD56-PE, CD45-PerCP-Cy5.5, CD4-PC7, CD19-APC, and CD8APC-Cy7 were acquired from Beijing Tongsheng Times Biotechnology Co. The samples were analyzed on a NAVIOS dual-laser, eight-color flow cytometer (Beckman, USA).
Specimen collection and testing procedure
A total of 2 mls of peripheral blood was collected from each child participating in the study. Using microsphere counting tubes, 10 ul of each of the following reagents were added: CD3, CD16 + CD56, CD45, CD4, CD19, and CD8. These reagents were diluted with 25 ul of solution. The reverse pipetting technique was employed to aspirate 50 ul of peripheral blood. The samples were then incubated in the dark for 25 min at room temperature. The erythrocyte lysing solution was added into the sample and incubated for 15 min. The samples were processed on a flow cytometer. Data was collected and analyzed using Navios software and the statistical software.
Cohen’s kappa coefficients were computed for each diagnostic index, taking into account the optimal threshold for diagnostic effectiveness and clinical diagnosis. Calculations were performed for EB-DNA (with a threshold of 400), MP-IgM, EB-IgG, EB-IgM, CMV-IgM, CMV-IgG, the combined EB (DNA + IgG + IgM), and clinical diagnosis. The consistency of the observed indicators, based on Cohen’s Kappa Coefficients, was categorized as follows: highest consistency ranging from 0.8 to 1.0, higher consistency ranging from 0.6 to 0.8, medium consistency ranging from 0.4 to 0.6, average consistency ranging from 0.2 to 0.4, and lower consistency with a Kappa value less than 0.2.
Statistical methods
Peripheral blood lymphocyte subpopulations were presented as the median and interquartile range (M, 25th (Q1) to 75th (Q3 percentile) and were compared via the Mann-whitney U-test. Significant variables between the IM group and the healthy group were conducted using Chi-square analysis. To determine the diagnostic value of lymphocyte subpopulation ratios, receiver operating characteristic (ROC) curves were generated using MedCalc 20.0 program (MedCalc Software, Belgium) and the area under the ROC curve (AUROC) with exact binominal 95% confidence intervals (CI) was compared by Z-test. Diagnostic accuracy was defined as excellent if an AUROC was greater than 0.8. Calibration was verified using calibration curves and the Hosmer Lemeshow goodness-of-fit test, and appropriate chi-squared values were calculated. Chi-squared values with p > 0.05 indicated a good fit. Subsequently, the threshold, sensitivity, specificity and accuracy were calculated. Cohen’s kappa coefficient was utilized for consistency testing. SPSS version 26.0 (IBM SPSS Statistics, USA) was used for statistical analysis, and a two-sided P value of < 0.05 was considered significantly different.
Results
Patient characteristics
A total of 86 boys and 49 girls with IM(median age: 3 years old) were included as study participants. Diagnostic criteria were referred to for inclusion. A total of 62 boys and 38 girls who underwent physical examination during the same period were allocated to the healthy control group(median age: 3 years old). Statistical analysis revealed no significant differences in age and gender distribution between the IM group and the healthy group (P < 0.05). These findings are summarized in Table 1.
Table 1.
Clinical characteristics of children with IM and healthy controls
| IM(n = 135) | Healthy(n = 100) | P-value | |
|---|---|---|---|
| Age M(years) | 2.35 ± 4.74 | 2.84 ± 0.37 | 0.359 |
| Sex (m/f) | 86/49 | 62/38 | 0.222 |
| WBC(×109/L) | 16.3 ± 6.84 | 7.19 ± 1.81 | 0.012 |
| Lymphocytes (×109/L) | 62.2 ± 9.92 | 51.50 ± 11.69 | 0.032 |
| heterogeneous lymphocyte(%) | 12.30 ± 6.74 | 0.24 ± 0.52 | 0.000 |
| CRP(mg/L) | 10.83 ± 12.19 | 3.41 ± 2.85 | 0.008 |
| PCT(ng/mL) | 0.28 ± 0.34 | 0.07 ± 0.04 | 0.029 |
| ALT(U/mL) | 105.98 ± 206.25 | 22.50 ± 11.75 | 0.000 |
| CD3 | 81.47 ± 10.45 | 68.89 ± 5.21 | 0.673 |
| CD4 | 14.55 ± 5.90 | 38.64 ± 5.96 | 0.027 |
| CD8 | 59.12 ± 12.93 | 20.97 ± 4.15 | 0.016 |
| CD16 + 56 | 9.03 ± 4.46 | 10.56 ± 4.56 | 0.397 |
| CD19 | 5.07 ± 3.07 | 17.75 ± 3.78 | 0.000 |
| CD4/CD8 | 0.29 ± 0.28 | 1.95 ± 0.67 | 0.000 |
| EBV-DNA(×105/L) | 5.61 ± 13.07 | 0 | 0.000 |
IM diagnosis by peripheral blood lymphocyte testing
The peripheral blood samples of 86 males and 49 females with IM were analyzed by flow cytometry to assess the immunological changes. The proportions of leukocytes, lymphocytes, and anisotropic lymphocytes were significantly elevated in the IM group compared with the healthy group (P < 0.001 for all subsets). Specifically, the number of CD3 + and CD8 + T cells were significantly increased in the IM group (P < 0.001 for both subsets). The levels of CD4 + and CD16 + CD56 + T cells and CD19 + B cells were substantially decreased in the IM group (P < 0.001, P < 0.05, and P < 0.001 for each subset). The CD4+/CD8 + ratio of the IM group was 0.29 which was significantly lower than 1.83 of the control group (P < 0.001). All the lymphocyte subset changes were detailed in Table 2.
Table 2.
Analysis of peripheral blood lymphocyte subset changes (%)
| IM M (Q1, Q3) | Healthy M (Q1, Q3) | Significance (P value) | |
|---|---|---|---|
| Leucocyte (109 /L) | 14.85 (10.91, 17.70) | 7.03 (5.80, 8.07) | 0.000 |
| Lymphocyte (%) | 66.00 (58.00, 71.75) | 53.00 (46.38, 59.45) | 0.000 |
| heterogeneous lymphocyte (%) | 11.00 (8.00, 15.00) | 0 (0.00, 0.00) | 0.000 |
| CD3 (%) | 84.20 (80.55, 87.40) | 69.50 (65.78, 73.25) | 0.000 |
| CD4 (%) | 17.00 (12.40, 22.95) | 37.50 (34.90, 43.25) | 0.000 |
| CD8 (%) | 58.50 (43.65, 67.40) | 21.30 (18.33, 23.60) | 0.000 |
| CD16 + 56 (%) | 8.00 (6.40, 10.80) | 9.55 (7.50,14.68) | 0.038 |
| CD19 (%) | 4.70 (3.00, 8.55) | 17.65 (15.30, 20.30) | 0.000 |
| CD4/CD8 | 0.29 (0.18, 0.49) | 1.83 (1.48, 2.18) | 0.000 |
Assessment of the diagnostic value of the ROC curves for IM
The ROC curves were used to validate the diagnostic values of the lymphocyte subset changes for IM (Fig. 1). The diagnostic values were evaluated in terms of sensitivity, specificity, and the area under the ROC curve (AUROC), as well as the highest Youden index with the optimal cut-off (Fig. 1). The AUROC and the Yoden index were both 1.000 (95%CI: 1～1) for the CD4+/CD8 + ratio of IM, indicating an excellent diagnostic performance. The sensitivity and specificity of using CD4+/CD8 + ratio with threshold of 1.115 were both 100% for IM diagnosis, as shown in Table 3; Fig. 1. These findings suggested that the CD4+/CD8 + ratio (the optimal threshold of 1.115) may be an accurate diagnostic indicator for IM. Further to validate the differences among other lymphocyte subsets, the data showed that the AUROC were 0.930, 0.969, 1.000, 0.621 and 0.964 for CD3+, CD4+, CD8+, CD16 + CD56 + T cells and CD19 + B cells, respectively with the corresponding optimal threshold of 75.4%, 27.4%, 29.9%, 13.1%, and 11.45%, respectively (Table 3). The sensitivity rates for these lymphocyte subsets were 87.8%, 100%, 100%, 28%, and 98% and the specificity rates were 96%, 85.7%, 100%, 93.9%, and 91.8%, respectively (Table 3).
Fig. 1.
The ROC curves for diagnosis of IM by each detection indicator
Table 3.
Detection efficacy of each index for diagnosing IM
| Diagnostic indicators (%) |
Area under the curve | 95% confidence interval | sensitivity | idiosyncrasy | optimal threshold | Jordon index |
|---|---|---|---|---|---|---|
| Leucocyte | 0.908 | 0.846 ~ 0.970 | 79.6% | 94% | 10.21 (%) | 0.736 |
| Lymphocyte | 0.805 | 0.719 ~ 0.891 | 77.6% | 72% | 57.5 (%) | 0.496 |
| heterogeneous lymphocyte | 0.997 | 0.991 ~ 1.000 | 95.9% | 100% | 3.5 (%) | 0.959 |
| CD3+ | 0.930 | 0.872 ~ 0.987 | 87.8% | 96% | 75.4 (%) | 0.838 |
| CD8+ | 1.000 | 1.000 ~ 1.000 | 100% | 100% | 29.9 (%) | 1.000 |
| CD4+ | 0.969 | 0.936 ~ 1.000 | 100% | 85.7% | 27.4 (%) | 0.857 |
| CD16 + CD56+ | 0.621 | 0.511 ~ 0.731 | 28% | 93.9% | 13.1 (%) | 0.219 |
| CD19+ | 0.964 | 0.927 ~ 1.000 | 98% | 91.8% | 11.45 (%) | 0.898 |
| CD4+/CD8+ | 1.000 | 1.000 ~ 1.000 | 100% | 100% | 1.115 (%) | 1.000 |
Assessment of the diagnosis consistency
The Kappa coefficient was analyzed to assess the consistency degree of using lymphocyte subset AUROC values for IM diagnosis. The Kappa coefficients of CD4+/CD8 + ratio was 1 (P < 0.001) as shown in Table 4, indicating a 100% of consistency with the clinical diagnosis (the combined EBV analysis). The Kappa coefficient for CD3+, CD4+, CD8+, and CD19 + were 0.838, 0.858, 1.000 and 0.899 respectively, all of which were greater than 0.8 suggesting a high degree of consistency with the clinical diagnosis.
Table 4.
Kappa coefficients for diagnosis of IM by indicators
| Kappa coefficients | Progressive standard error | approximation T | p | |
|---|---|---|---|---|
| leucocyte | 0.737 | 0.067 | 7.407 | 0.000 |
| lymphocyte | 0.495 | 0.087 | 4.936 | 0.000 |
| heterogeneous lymphocyte | 0.960 | 0.028 | 9.555 | 0.000 |
| CD3 | 0.838 | 0.055 | 8.368 | 0.000 |
| CD4 | 0.858 | 0.051 | 8.628 | 0.000 |
| CD8 | 1.000 | 0.000 | 9.950 | 0.000 |
| CD16 + 56+ | 0.217 | 0.073 | 2.886 | 0.004 |
| CD19 | 0.899 | 0.044 | 8.961 | 0.000 |
| CD4/CD8 | 1.000 | 0.000 | 9.950 | 0.000 |
| EB-DNA | 0.959 | 0.029 | 9.450 | 0.000 |
| MP-IgM | 0.124 | 0.053 | 2.479 | 0.013 |
| EB-IgG | 0.218 | 0.063 | 3.444 | 0.001 |
| EB-IgM | 0.855 | 0.052 | 8.510 | 0.000 |
| CMV-IgM | 0.066 | 0.037 | 1.815 | 0.070 |
| CMV-IgG | 0.113 | 0.048 | 2.394 | 0.017 |
| Combined EBVanalysis(DNA + IgG + IgM) | 1.000 | 0.000 | 9.849 | 0.000 |
Discussion
Pediatric infectious mononucleosis primarily caused by EBV infection represents a critical scenario that requires accurate diagnosis in clinical settings [19, 20]. IM is an immunopathologic disorder arising due to the response of host immunity to EBV [21]. Following the initial infection, the host’s intact immune system halts the virus dissemination and forestalls its reactivation, maintaining a state of homeostasis between the host and the virus [22]. The immunocompromised patients may experience aggressive immune response to EBV recurrence and significant diseases such as acute IM [23, 24]. Therefore, accurate diagnostic tools of IM are critical for pediatrians to help them make precise decisions on treatment.
In the present study, we compared the differences in lymphocyte subpopulations in the peripheral blood between IM children and healthy controls. Our results indicated that the percentage of CD8 + T cells was significantly elevated whereas CD4 + cells was notably decreased in the IM group. The CD4+/CD8 + ratio of IM group was significantly reduced, consistent with the previous studies [25–27]. We observed a decrease of CD19 + cells in the IM children compared with the healthy individuals, suggesting that the infected B cells may undergo necrosis or apoptosis following EBV infection. Previous animal studies demonstrated that NK cells play a regulatory role in the symptoms of EBV-induced lissencephalic infections [28]. A reduction of NK cells was associated with exacerbated symptoms of IM and promoting EBV-associated tumorigenesis [29]. Our results revealed a significant decrease of the CD16 + CD56 + NK cells in the IM children, suggesting that loss of NK cells during IM may play a role in regulating host immune response. Previous studies have found that during the acute phase of IM, CD3+, CD8 + lymphocytes were predominantly increased accompanied by a decrease of CD4+, CD16+, CD56+, and CD19 + cells [28, 30–32]. Our findings are in high consistency with the previous results, suggesting these lymphocyte subpopulations accurate diagnostic indicators.
Due to the complexity of clinical manifestations in IM, early and accurate diagnosis of pediatric IM is challenging. The existing diagnostic criteria for EBV-associated IM encompass fever and pharyngotonsillitis, along with any three of the following symptoms: cervical lymph node enlargement, hepatomegaly, splenomegaly, and eyelid edema. Laboratory diagnosis standard requires a peripheral blood heterogeneous lymphocyte ratio of ≥ 0.10 and/or an elevated lymphocyte count of ≥ 5 × 109/L [33]. Additionally, the produced IgM and IgG antibodies against the viral capsid antigen (VCA) in the host peripheral blood were measured for early diagnosis of atypical cases of IM [34, 35]. The predominant age group for IM onset in Chinese children is 4–6 years old and approximately half of children aged 2–5 years old exhibit a positive antibody response to EBV infection [36]. Therefore, the antibody test for diagnosing IM is largely limited among Chinese IM children [37]. Here, we evaluated the diagnostic value of the peripheral blood lymphocyte population for IM. Our findings revealed that the AUROC curve for CD8 + cells and for CD4+/CD8 + ratios both achieved a value of 1, suggesting a reliable and accurate diagnostic performance. The sensitivity and specificity reached 100% when the CD8 + and CD4+/CD8 + thresholds were set to be 29.9 and 1.115, respectively. The Kappa coefficients were 1 for CD8 + and CD4+/CD8 + ratios, which is as good as the combined EBV analysis, indicating a 100% consistency with the clinical diagnosis. These analyses have established the accuracy of using CD8 + and CD4+/CD8 + ratios as a diagnostic tool for IM, making peripheral blood lymphocyte testing more reliable compared with other methods [7].
This study had some limitations. First, this was a single-center study with a relatively small population. Secondly, the correlation between CD8 + and CD4+/CD8 + ratios and the severity of IM or other factors were not compared or analyzed.
In summary, CD8 + and CD4+/CD8 + ratios demonstrate excellent specificity and sensitivity for IM diagnosis and peripheral blood lymphocyte testing provides a novel laboratory basis for the early and accurate clinical diagnosis of IM. It provides clinicians a reliable and sensitive tool for diagnosing IM, especially in cases where the clinical symptoms are non typical or complex [38]. By incorporating peripheral blood lymphocyte testing into the diagnostic workflow, clinicians can improve their diagnostic accuracy and confidence, leading to earlier intervention and more effective patient management. Furthermore, the standardization of diagnostic criteria using these biomarkers has the potential to facilitate research and clinical trials, enabling the development of more targeted and effective therapies for IM.
Author contributions
Zhou JX and Zhang J were responsible for designing and running the experiments and writing the manuscript. Zhou D, Ma WT and Zhong Q were responsible for experiments and data analysis. Su J and Shen Q were responsible for the conception, design, and coordination of the study, acquisition and interpretation of the statistical data, and revision of the manuscript.
Funding
This work was supported by the Science and Technology Program Xuzhou Medical University (grant number XYFM202216), Suqian Sci&Tech Program (grant number K202309) and Research General Project of Jiangsu Provincial Health Commission (grant number H2023147).
Data availability
Data is provided within the manuscript. All the data used to support the findings of this study are available from the corresponding author upon request.
Declarations
Consent for publication
Not Applicable.
Human research ethics approval
The study was approved by the Medical Ethics Committee of the Suqian First Hospital and Xuzhou Medical University (2019-SL-0014) and conducted in accordance with the Declaration of Helsinki. All patients’ parents or legal guardians were informed with the study and the signed consent forms were obtained.
Consent to participate statement
All patients’ parents or legal guardians were informed with the study and the signed consent forms were obtained.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Jingxin Zhou and Jia Zhang co-first author, authors equally contributed to this study.
Contributor Information
Qin Shen, Email: shenqin2028@163.com.
Jing Su, Email: 928007387@qq.com.
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Associated Data
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Data Availability Statement
Data is provided within the manuscript. All the data used to support the findings of this study are available from the corresponding author upon request.