It has been confirmed that the HCMV viral load and growth in clinical samples can predict the risk of disease in patients. But the lack of well-established viral load thresholds limits HCMV RT-qPCR in clinical application. Due to the results of RT-qPCR cannot be directly compared, the results of HCMV viral load cannot be used after change hospital. No viral load value can be used to initiate preemptive therapy for patients infected HCMV without consensus standardization of HCMV RT-qPCR[10, 14]. There is widely variability of viral load in 33 laboratories using different HCMV RT-qPCR in a study. Though a World Health Organization (WHO) provide an international standard for calibration of HCMV RT-qPCR, viral load variability remains because of assay performance (limits of detection and quantification), sample type, method for nucleic acid extraction, gene target, and amplicon size, even the type of patients[19–21].
Digital PCR solves this problem well. Droplet digital PCR (ddPCR) and cdPCR are two types of commercial digital PCR platforms. Other studies have shown that the sensitive of digital PCR is significantly higher than that of RT-qPCR[22, 23]. Our results also gave a similar conclusion. ddPCR mainly forms water-in-oil droplets, and each droplet is an independent PCR reaction system. Furthermore, the sensitivity of ddPCR for HCMV is 100 copies/ml. And cdPCR limits detection of HCMV viral load is 15 copies/ml in clinical sample. cdPCR complete PCR reaction through 2D array of microchamber, and cdPCR can realize three-color multiplexing amplification[25, 26]. Due to simplified steps, cdPCR effectively reduce risk of contamination. Thus, we established a HCMV cdPCR method to evaluate HCMV infection in this study.
It is reported that RT-qPCR alone is inadequate for the accurate diagnosis of virus infection. In this study, the cdPCR effectively detected 7 HCMV positive samples with low copies nucleic acid, making the laboratory diagnosis rate of HCMV increased by 6.36% (7/110). Our results showed that the lowest copies of detection by cdPCR was 14.58 copies/ml. The results suggested that cdPCR was suitable for low loads nucleic acid detection.
In addition, we also found that HCMV was more easily detected in whole blood than in serum. In this study, both serum and whole blood samples were collected in 44 of 110 patients (31 of 59 HCT patients and 13 of 51 leukemia patients). We found there were 3(3/31 = 9.67%) cases of HCMV positive in whole blood not in the serum from 31 HCT patients. There was 1 (1/13 = 7.69%) case of HCMV positive in whole blood not in serum from 13 leukemia patients. The results made the laboratory diagnosis rate of HCMV increased by 3.63% (4/ 110).
We still found that cdPCR was more sensitive in the whole blood samples than in serum. And in our study, the lowest copies number is 15 copies/ml in whole blood samples and 27 copies/ml in serum samples, respectively. In a word, we found that the detection rate of HCMV in whole blood was slightly higher than in serum both in leukemia and HCT patients. The reason maybe that HCMV can replicate in many cells, including epithelial cells, endothelial cells and leukocytes of peripheral blood[28–30]. Only in the presence of viremia, high levels of HCMV viral load can be detected in the serum.
As we all known, HCMV is the most common virus infection after transplantation, it is considered to be the major risk factor for transplantation. Studies have confirmed that almost all HCMV viremia after bone marrow transplantation occurs in HCMV-positive recipients, and only in few patients can be transmitted from donor. In other words, it is necessary to pay attention to leukemia patients with HCMV and HCMV-infected bone marrow transplant patients. In our study, 34.54% of samples were positive for HCMV by cdPCR. More than 90% of HCMV infections occurred before the age of 12 in these patients with leukemia or HCT. Continuous monitoring and timely medication should be conducted to prevent postoperative viremia. Among HCT patients, the infection rate of HCMV in male 13.56% (8/59) is higher than that in female 10.17% (6/59). But in leukemia patients, the infection rate in female 17.65% (9/51) is nearly 3 times than that of males 5.88% (3/51). This phenomenon needs to be further confirmed by large samples.