3.1 The sensitivity, specificity and repeatability of cdPCR
The linear dynamic range of the plasmid DNA containing the pUC57-UL83 gene was from 3.2⋅106 to 3.2⋅10 copies/ml. Eight µL of plasmid each dilution was detected by qPCR and cdPCR. The plasmid with dilution from 3.2⋅106 copies/ml to 3.2⋅102 copies/ml can be detected by qPCR and cdPCR simultaneously. The 32 copies/ml diluted plasmid could not be detected by qPCR and cdPCR. To accurately understand the sensitivity of qPCR or cdPCR, plasmids 3.2⋅102 copies/ml were diluted in a 2-fold series by1:2, 1:4, 1:8, 1:16, 1:32, 1:64. The limit of detection (LOD) of cdPCR was 103 copies/ml (2.0 copies/reaction) and the LOD of qPCR was 297 copies/ml. The results indicate that the sensitivity of cdPCR was higher than that of qPCR.
To understand the repeatability of cdPCR, a standard curve of HCMV DNA copy number was established using plasmids. Copies of serial diluted plasmids were detected by cdPCR and the coefficient of variations (CV, standard deviation /mean) of copy number detected by cdPCR was analyzed with standard curve (Fig. 1). The results showed the repeatability of cdPCR was good because the CV value was less than 15%. Good consistency was also observed between the expected value of diluted plasmid and those measured by cdPCR and qPCR (R = 0.979, P<0.05 for cdPCR and the expected value of diluted plasmid, R = 0.939, P<0.05, for qPCR and the expected value of diluted plasmid).
To determine the specificity of cdPCR for HCMV, 7 herpes viruses, including HSV-1, HSV-2, VZV, EBV, HCMV, HHV-6A, HHV-7, were tested by cdPCR, respectively. The results showed that cdPCR only detected HCMV AD169 strain but not the other 6 herpes viruses, suggesting that cdPCR for HCMV has no cross-reaction with other herpes viruses.
The black line shows standard curve of the plasmid DNA. Different scatter points are the HCMV DNA copies tested by cdPCR.
3.3 HCMV infection of children following HSCT
To verify the sensitivity of the cdPCR method and whether it can be used for HCMV detection in the blood of HSCT patients, 125 children's whole blood samples following HSCT were tested by qPCR and cdPCR. Thirty-four samples were positive through qPCR and cdPCR, ninety-one samples were negative through qPCR. However, 4 out of 91 qPCR negative samples were positive by cdPCR. The results showed that the method of cdPCR was more sensitive than qPCR for HCMV detection (Table 1).
Table 1
Comparisons between qPCR and cdPCR
qPCR
|
cdPCR
|
Total
|
P
|
Positive
|
Negative
|
Positive
|
34
|
0
|
34
|
<0.05
|
Negative
|
4
|
87
|
91
|
Total
|
38
|
87
|
125
|
Subsequently, one hundred twenty-five children's whole blood samples following HSCT were tested by cdPCR to investigate the viral load of HCMV infection in children following HSCT. The HCMV viral load was from 107 copies/ml to 6600 copies/ml by cdPCR. In 68 cases of D+ & R+, HCMV was detected in 28 cases (41.18%), and the viral load was 107–6600 copies/ml. HCMV was detected in 7 cases (22.58%) with a viral load of 437 to 5314 copies/ml in 31 D+ & R− cases. There were 18 cases of D− & R+, and three case (16.67%) of HCMV was detected, and the viral load was 463–883 copies/ml. There were 5 cases of D− & R−, and HCMV was not detected. The HCMV infection rate was 40.54% (15/37) among AML cases, and the HCMV viral load was from 107 copies/ml to 5314 copies/ml. The HCMV infection rate was 41.18% (7/17) in ALL cases, and the HCMV viral load was from 137 copies/ml to 1779 copies/ml. The HCMV infection rate was 22.73% (5/22) among AA cases, and the HCMV viral load was from 154 copies/ml to 6600 copies/ml. The HCMV infection rate was 40% (4/10) among MDS cases, and the HCMV viral load was from 999 copies/ml to 5957 copies/ml. The HCMV infection rate was 33.33% (1/3) among lymphoma cases, the HCMV viral load was 2494 copies/ml. There was no HCMV infection in NB cases. One case (16.67%) was infected with HCMV in WAS cases, and the HCMV viral load was 801 copies/ml. The HCMV infection rate was 20% (5/25) among MPS cases, and the HCMV viral load was from 351 copies/ml to 5100 copies/ml (Table 2). Due to the small number of cases, the HCMV infection rate of patients with different primary diseases is not statistically different, and the viral load of HCMV infection varies among different diseases group without significant variation.
Table 2 Characteristics of 125 children following HSCT
Children patient characteristics
|
No. of Children Patients
(No. of HCMV Positive)
|
Age (years)
|
0-6
|
70 (17)
|
|
7-12
|
43 (17)
|
|
≥12
|
12 (4)
|
Sex
|
Male
|
73 (22)
|
|
Female
|
52 (16)
|
HCMV IgG
|
D+ & R+
|
68 (28)
|
|
D+ & R-
|
31 (7)
|
|
D- & R+
|
18 (3)
|
|
D- & R-
|
5 (0)
|
Disease
|
AML
|
37 (15)
|
|
ALL
|
17 (7)
|
|
AA
|
22 (5)
|
|
MDS
|
10 (4)
|
|
Lymphoma
|
3 (1)
|
|
NB
|
5 (0)
|
|
WAS
|
6 (1)
|
|
MPS
|
25 (5)
|
HSCT
|
Allogeneic
|
122 (38)
|
|
Autologous
|
3 (0)
|
Source of HSCT
|
BM+PBSCT
|
98 (32)
|
|
PBSCT
|
18 (3)
|
|
UCB-HSCT
|
9 (3)
|
GVHD (grade)
|
0
|
83 (23)
|
|
1-2
|
30 (12)
|
|
3-4
|
12 (3)
|
Abbreviations: HSCT, hematopoietic stem cell transplantation. HCMV, Human Cytomegalovirus. D, donor patients. R, recipient patients. AML, Acute myelocytic leukemia. ALL, Acute lymphocytic leukemia. AA, Aplastic anemia. MDS, myelodysplastic syndrome. NB, Neuroblastoma. WAS, Wiskott-Aldrich syndrome. MPS, Mucopolysaccharidosis. BM, bone marrow. PBHSCT, peripheral blood stem cell transplantation. GVHD,graft vs. host disease.
The detection rate of HCMV was 30.40% (38/125) in 125 children following HSCT, and the range of HCMV viral load was from 107 copies/ml to 6600 copies/ml. The detection rate in the male group was 30.14% (22/73), in the female group was 30.77% (16/52). The detection rate of HCMV was 89.47% (34/38) in the HCMV positive children following HSCT aged 0-12. In the aged 0-6 group, the detection rate in males was 25.64% (10/39), in females was 22.58% (7/31). In the aged 7-12 group, the detection rate in males was 39.29% (11/28), in females was 40% (6/15). For over 12 years old children following HSCT, HCMV detection rate was 33.33% (4/12), the detection rate in males was16.67% (1/6), in females was 50% (3/6) (Table 3), suggesting that HCMV infection is mainly found in patients under 12 years of age following HSCT, and the gender does not affect HCMV infection rate.
The prognosis of 125 children following HSCT was analyzed retrospectively. GVHD was found in 13 children (the range of the HCMV viral load was from 437 copies/ml to 4457 copies/ml, and the median of viral load was 693 copies/ml) (34.21%) of 38 HCMV-positive children and 25 children (28.74%) of the 87 HCMV-negative children (P>0.05). HCMV-positive children were slightly more likely to develop GVHD than HCMV-negative children. Among the 38 positive children, five children died following HSCT. The co-infection with EBV occurred in 2 of the 5 deaths.
Table 3
HCMV infection rate by cdPCR in children following HSCT
Age
|
No.
|
HCMV Positive
|
Positive Rate (%)
|
Male
|
Female
|
Positive
|
Negative
|
Positive Rate (%)
|
Positive
|
Negative
|
Positive Rate (%)
|
0–6
|
70
|
17
|
24.28
|
10
|
29
|
25.64
|
7
|
24
|
22.58
|
7–12
|
43
|
17
|
39.53
|
11
|
17
|
39.29
|
6
|
9
|
40
|
≥ 12
|
12
|
4
|
33.33
|
1
|
5
|
16.67
|
3
|
3
|
50
|
Total
|
125
|
38
|
30.40
|
22
|
51
|
30.14
|
16
|
36
|
30.77
|