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Eight microliters of serially diluted plasmid was detected by qPCR and cdPCR. A total of 3.0 × 106, 3.0 × 105, 3.0 × 104, 3.0 × 103, and 3.0 × 102 copies/mL diluted plasmids were detected by qPCR and cdPCR. Thirty copies/milliliter of diluted plasmid was not detected by qPCR, but cdPCR showed that 30 copies/mL of the plasmid could be detected. The plasmid was doubly-diluted 1:2, 1:4, 1:8, 1:16, 1:32, and 1:64 using 3.0 × 102 copies/mL. The limit of detection (LOD) of EBV cdPCR was up to 110 copies/mL (2.1 copies/reaction) and the LOD of qPCR was 327 copies/mL for the plasmid dilutions. The results confirmed that the sensitivity of cdPCR was higher than that of qPCR. The consistency between cdPCR and the expected values of the diluted plasmid was r2 = 0.996, P = 0.84. The consistency between qPCR and the expected value of the diluted plasmid was r2 = 0.994, P = 0.89. Good consistency was observed between the diluted plasmid copies and those measured by cdPCR and qPCR.
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After EBV was 10-fold serially diluted from 10−1, 10−2, 10−3, 10 −4, 10 −5, 10−6, 10−7 to 10−8, 8 μL DNA of each dilution was used for detection by qPCR and cdPCR. A total of 10−1, 10−2, 10−3, 10−4, and 10−5 dilutions of the EBV B95-8 strain were detected by qPCR, and 10−1, 10−2, 10−3, 10−4, 10−5, and 10−6 dilutions were detected by cdPCR. The 10−6, 10−7, and 10−8 dilutions were not detected by qPCR. The EBV cdPCR did not detect the 10−7 or 10−8 dilutions. Together, the results showed that the sensitivity of EBV cdPCR was 10-fold higher than that of qPCR, and the LOD of EBV cdPCR was up to 121 copies/mL (2.3 copies/reaction).
To determine the specificity of cdPCR, EBV cdPCR was used to detect the other eight herpesviruses, including herpes simplex virus 1, herpes simplex virus 2, varicella zoster virus, human cytomegalovirus, human herpesvirus 6A, human herpesvirus 6B, and human herpesvirus 7. The results showed that EBV cdPCR did not react with seven herpesviruses except EBV. The results also showed that EBV cdPCR had no cross-reaction with the other herpesviruses.
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We then analyzed EBV infections in children after HSCT, based on the results of cdPCR. There were 41 males and 23 females, and 62 patients were allogeneic HSCT and two patients were autologous HSCT among the 64 children from May 2019 to September 2020. Among 64 children following HSCT, there were 22 cases of acute myelocytic leukemia (AML), 11 cases of acute lymphocytic leukemia (ALL), 7 cases of aplastic anemia (AA), 6 cases of myelodysplastic syndrome (MDS), 3 cases of lymphoma, 9 cases of Wiskott-Aldrich syndrome (WAS), and 6 cases of mucopolysaccharidosis (MPS) (Table 1). There were 4 cases of EBV infection among the 22 AML cases, with a viral load from 643 copies/mL to 1,530 copies/mL. There were 3 cases of EBV infection in the 11 ALL cases, and the viral load was from 397 to 3,209 copies/mL. There were 2 EBV-infected cases among the 6 MDS cases, with EBV viral loads of 617 and 797 copies/mL, respectively. Two cases of EBV infection were found in the nine WAS cases, and the viral loads were 140 and 1,551 copies/mL, respectively. There was only one EBV-infected among three lymphoma cases, with a viral load of 154 copies/mL. No EBV infections were found in both seven AA and six MPS cases. Complications and sequelae of 64 HSCT children included bacterial and fungal infections, rash, fever, agranulocytosis, GVHD, pneumonia, gastroenteritis, liver damage, secondary diabetes, hypertension, conjunctivitis, and sepsis.
Children characteristics No. of children (No. of EBV positive) Age (years) 0−6 39 (4) 7−12 17 (7) > 12 8 (1) Sex Male 41 (7) Female 23 (5) Disease AML 22 (4) ALL 11 (3) AA 7 (0) MDS 6 (2) Lymphoma 3 (1) WAS 3 (2) MPS 6 (0) HSCT Allogeneic 62 (12) Autologous 2 (0) Source of HSCT BM + PBSCT 58 (10) PBSCT 6 (2) Note. HSCT, hematopoietic stem cell transplantation; EBV, Epstein-Barr virus; AML, acute myelocytic leukemia; ALL, acute lymphocytic leukemia; AA, aplastic anemia; MDS, myelodysplastic syndrome; WAS, Wiskott-Aldrich syndrome; MPS, mucopolysaccharidosis; BM, bone narrow; PBHSCT, peripheral blood stem cell transplantation. Table 1. Characteristics of 64 children after hematopoietic stem cell transplantation
The incidence of EBV infection was 18.75% (12/64) in 64 children after HSCT, and the infection in males was 17.07% (7/41), and 21.74% (5/23) in females. The incidence of EBV infection was 10.26% (4/39) in the 0−6 years old age group, and the incidence of EBV infection was 41.18% (7/17) in the 7−12 years old age group. In the 0−6 years old age group, the incidence of males was 9.09% (2/22), and that of females was 11.76% (2/17). In the 7−12 years old age group, the incidence of males was 33.33% (5/15), and the incidence of females was 100% (2/2). For those over 12 years of age, the incidence of EBV was 12.50% (1/8), with only one female child infected (Table 2).
Age (years) No. EBV positive Positive rate
(%)Male Female Positive Negative Positive rate
(%)Positive Negative Positive rate
(%)0–6 39 4 10.26 2 20 9.09 2 15 11.76 7–12 17 7 41.18 5 10 33.33 2 0 100 > 12 8 1 12.50 0 4 0 1 3 25.00 Total 64 12 18.75 7 34 17.07 5 18 21.74 Table 2. Epstein-Barr virus infection in children after hematopoietic stem cell transplantation
The length of hospital stays were analyzed for 64 children after HSCT. The average hospital stay of children with EBV infection (184 ± 91 days) was longer than that of children without EBV infection (125 ± 79 days), P = 0.026 (Figure 1A).
Figure 1. The length of hospital stays of children after hematopoietic stem cell transplantation. (A) Comparison of hospital stays between infected and non-infected children after hematopoietic stem cell transplantation. (B) The comparison of hospital stays between low Epstein-Barr virus (EBV) load (< 784 copies/mL) group and high EBV viral load (≥ 784 copies/mL) group.
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For 12 positive children, the range of viral loads was from 140 copies/mL to 3,209 copies/ mL (median EBV viral load: 784 copies/mL). The viral loads of three children were less than 500 copies/mL, those of five children were between 500 copies/mL to 1,000 copies/mL, those of three children were between 1,000 copies/mL to 2,000 copies/ mL, and that of one patient was more than 3,000 copies/mL. Among 12 EBV positive children, except for patient 1, all other patients had complications and sequelae including GVHD (eight cases) and fever (two cases). The median viral load of EBV was 820 copies/mL for GVHD patients. Three children died after HSCT, with EBV viral loads of 820, 1,530 and 3,209 copies/mL. Co-infection with HCMV occurred in two of the three deaths (Table 3). Together, the results indicated that the high viral load of EBV may have caused more serious disease and even death for HSTC patients.
Cases Sex Age
(years)Disease Copies/mL The length of hospital
stay (days)Complications and sequelae Prognosis 1 F 5 AML 643 125 None 2 F 10 MDS 797 83 Secondary diabetes, GVHD 3 M 10 ALL 397 353 Secondary hypertension, GVHD 4 M 8 AML 820 196 Fungal pneumonia, acute upper respiratory tract infection, GVHD dead 5* M 7 ALL 3,209 320 Septic shock, pancreatitis, cholangitis, GVHD dead 6 M 5 Lymphoma 154 127 Liver damage 7* F 5 AML 1,530 302 Septicemia, type II respiratory failure, GVHD dead 8 M 10 WAS 140 106 Septicemia, liver damage, GVHD 9 F 15 MDS 617 123 Fever 10 M 7 ALL 771 151 Acute gastroenteritis, fever 11 M 2 WAS 1,551 166 Fungal pneumonia, enteritis, GVHD 12 F 9 AML 1,008 155 Thrombotic Microangiopathes, GVHD Note. F, female; M, male; GVHD, graft versus host disease. *Case 5 and case 7 involved co-infection with human cytomegalovirus. Table 3. Characteristics of 12 Epstein-Barr virus positive children after hematopoietic stem cell transplantation
The children following HSCT were divided into two groups according to the median EBV viral load of 784 copies/mL. The length of hospital stay of the low EBV viral load (< 784 copies/mL) group was 164 ± 94 days, and the length of hospital stay of the high EBV viral load (≥ 784 copies/mL) group was 203 ± 91 days. There was no significant difference between the two groups (P = 0.476) (Figure 1B).
The Viral Load of Epstein-Barr Virus in Blood of Children after Hematopoietic Stem Cell Transplantation
doi: 10.3967/bes2022.052
- Received Date: 2021-12-27
- Accepted Date: 2022-03-31
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Key words:
- Chip digital PCR /
- Epstein-Barr virus /
- Hematopoietic stem cell transplantation /
- Quantitative PCR
Abstract:
Citation: | WANG Wen Jun, FENG Shun Qiao, HE Feng, DU Hai Jun, FENG Miao, WANG Rui Fang, MEI Guo Yong, LIU Mi, LIU Rong, YAO Hai Lan, HAN Jun. The Viral Load of Epstein-Barr Virus in Blood of Children after Hematopoietic Stem Cell Transplantation[J]. Biomedical and Environmental Sciences, 2022, 35(9): 804-810. doi: 10.3967/bes2022.052 |