To the Editor: Infection is one of the causes of death after chemotherapy in children with leukemia. About 20% to 30% acute lymphocytic leukemia (ALL) patients die from infection after chemotherapy. Patients with immune deficiency, under chemotherapy, or treated with immunosuppressor are more inclined to be infected with human cytomegalovirus (HCMV) which leads to severe systematic disorders. Cytomegalvirus retinitis (CMVR) is a major sight threatening disease which mainly occurs in patients with immune deficiency and results in blindness if treatment is delayed. The decrease of CD4+ T cells has been related to CMVR in immune deficiency syndrome like AIDS. However, little is known about the relation between the level of T cells with different markers and the onset of CMVR in ALL patients under chemotherapy. Therefore, this study was designed to investigate the diagnostic value of T cells with different markers for cytomegalovirus retinitis after chemotherapy in patients with ALL.
One hundred twenty-eight children with ALL who were under chemotherapy in the department of hematology from our hospital from January 2015 to September 2017 were included in this study. Inclusion criteria: (1) ALL was diagnosed by bone marrow examination; (2) ALL patients were under moderate risk and under uniform chemotherapy regimens; (3) Medical record was complete; (4) No HCMV infection before chemotherapy; (5) Patients were under ocular examination at regular intervals. All participants were divided into three groups based on whether they were infected with HCMV and whether they were present with CMVR. Group A included 99 patients who were HCMV negative. Group B included 18 patients who were HCMV positive but absent of CMVR. Group C included 11 patients who were HCMV positive and meanwhile present with CMVR.
Polymerase chain reaction (PCR) for CMV was used to determine blood CMV-DNA and urine CMV-DNA. When CMV-DNA<500U/mL, it was considered as CMV negative. When CMV-DNA>500U/mL, it was considered as CMV positive. After chemotherapy, blood sample was obtained from all patients and flow cytometry was used to determine the number of T cells with different markers, including CD3, CD4, CD8.
In 128 ALL patients, 11 patients showed CMVR after chemotherapy. Among them, three patients underwent vision loss (3/11), eight patients did not undergo eye discomfort and were diagnosed as CMVR when ophthalmologic examination was performed (8/11), seven patients underwent eye lesion in binoculus (7/11), and the other fourpatients underwent single eye lesion (4/11). Lesion in three eyes in two patients occurred in the center (3/18), seven eyes in five patients in the rim (7/18), and eight eyes in four patients both in the center and in the rim (8/18).
Lesion in seven eyes in four patients involved in yellow spot (7/18). Four eyes in two patients were involved in retinopathy and optic neuritis (4/18). In these two patients, hyperaemia and diffused hemorrhage could be seen in optic disk with obscure boundary, as shown in Figure 1A. Nine eyes in five patients showed extensive retinal vasculitis (9/18) and the vagina vasorum in retina was obvious [Figure 1B and 1C]. Two eyes in one patient showed preretinal hemorrhage (2/18) [Figure 1D].
Table 1 showed the counting results of T cells with different markers after chemotherapy in three groups. Except CD4+ T cells/CD8+ T cells ratio, the number of CD3+ T cells, CD4+ T cells, and CD8+ T cells were significantly different among three groups. Notably, the number of CD4+ T cells in group A was remarkably higher than that in group B, and remarkably higher in group B than in group C (A>B>C).
For all sample, when the number of CD4+ T cells was higher than 200/μL, 100–200/μL, and lower than 100/μL, the CMVR positive rate was 6/7, 26.31% (5/19), and 0 (0/102). In the 29 patients with HCMV infection, when the number of CD4+ T cells was higher than 200/μL, 100–200/μL, and lower than 100/μL, the HCMV infection rate was 0 (0/11), 45.45% (5/11), and 54.55% (6/11). The number of CD4+ T cells was negatively related to CMVR positive rate, and it showed a significant difference when χ 2 was performed (χ 2 = 70.187, P<0.01). That is to say, as the number of CD4+ T cells decreased, the positive rate of CMVR increased.
After chemotherapy, 11 out of 128 ALL patients showed CMVR positive, which count for 8.59% in all sample. The range of CD4+ T cells in CMVR positive patients was 32–192/μL, and the average was 113.72 ± 59.36/μL. Among these 11 patients, the number of CD4+ T cells in six patients was lower than 100/μL, and 100–200/μL in five patients. When the number of CD4+ T cells was higher than 200/μL, no patients were CMVR positive.
Based on the relationship between the number of CD4+ T cells and CMVR positive rate, we would like to further determine the diagnostic value of CD4+ T cells for CMVR after chemotherapy in ALL patients, ROC curve analysis was performed. As shown in Figure 2, the area values under the curve of the number of CD4+ T cells was 0.920 ± 0.029, which indicated that the number of CD4+ T cells possessed a high diagnostic value for CMVR after chemotherapy in ALL patients.
Cell-mediated immunity is the main force for anticancer, and T type lymphocytes play an important role in cell-mediated immunity. In our study, we measured the number of T cell subpopulation with different markers, including CD3, CD4, CD8, and investigated the relationship between CMVR positive rate and T cell-mediated immunity. We found that compared to the HCMV negative patients, HCMV positive patients showed a significantly lower level of CD3+, CD4, CD8+ T cells. And in patients with CMVR, those indexes were lower than patients without CMVR. Among that index, the level of CD4+ T cells was significantly lower in patients with CMVR than HCMV positive patients without CMVR, and the former was significantly lower than HCMV negative patients. It is well known that CD4+ T cell and CD8+ T cell are two important subpopulations of T type lymphocytes. CD4+ T cells can activate phagocytes and CD8+ T cells and thus enhance the ability to combat cancer.
In this study, comparing to the HCMV negative patients, the HCMV positive patients showed a lower level of CD3+ T cells, CD4+ T cells and CD8+ T cells and a lower ratio of CD4+ T cells to CD8+ T cells. This indicated that the decreased cell-mediated immunity might cause HCMV infection or HCMV from the status of inertia to activation. What is more, comparing to the HCMV negative patients and HCMV positive patients without CMVR, the patients with CMVR showed a significantly lower level of index discussed above. This indicated that the occurrence of CMVR might be caused by the further decreased immunity after HCMV infection.
The diagnosis of CMVR is mainly based on ophthalmologic examinationand medical history. When clinical feature shows no significant evidence of CMVR, laboratory examination is needed. On the one hand, CMV-IgM from the blood is the diagnostic index normally used for HCMV infection at present. However, for ALL patients who receive chemotherapy, their immunity has been impaired. When CMV-IgM can be detected in the blood, it is always found that CMVR is under a very severe condition. Therefore, its diagnostic value is evaluated. On the other hand, although sample from vitreous body and aqueous humor is much more accurate than that from blood, it becomes very unrealistic to obtain them because anesthesia or surgery is needed and the risk is comparatively high.
CMVR is an opportunistic infectious disease which can be normally found in ALL patients after chemotherapy. Lack of cytotoxic T lymphocytes and a low CD4+ lymphocyte count as considered to be risk factors for CMV disease. In this study, we found that the number of CD4+ T cells in CMVR was extremely low comparing to the other 2 groups. When the number of CD4+ T cells was lower than 200/μL, the occurrence of CMVR tended to be significantly higher. What is more, we have another strong evidence that the area values under the curve of the number of CD4+ T cells was 0.920 ± 0.029, and it indicated that the level of CD4+ T cells might be a potential biomarker for CMVR in ALL patients after chemotherapy.
The evidence discussed above suggest that when an ALL patient who is under chemotherapy appears eye discomfort and low level of CD4+ T cells, it is highly recommended that CMV-DNA from the blood and urine should be determined as the preliminary diagnosis for HCMV infection. And if the HCMV is positive, the ophthalmologic examination should be performed carefully to determine if CMVR is present and early treatment should be given in order to prevent vision loss.
Declaration of patient consent
The authors certify that they have obtained all appropriate patient consent forms. In the form, the patients have given their consent for their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
Conflicts of interest
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