Post-transplant monitoring of lymphocyte counts predict the occurrence of CMV DNAemia in early phase of kidney transplantation

Background The aim of this study was to assess whether monitoring of the number of lymphocytes in peripheral blood was helpful for evaluating the risk of cytomegalovirus (CMV) infection after kidney transplantation. Methods The total numbers of lymphocyte in peripheral blood were measured at baseline and posttransplant months 1, 3 and 6. Risk factors for DNAemia in KTRs were analyzed using univariate logistic regression analyses. Areas under receiver operating characteristic (ROC) curves were applied to assess the accuracy of lymphocyte counts for predicting CMV DNAemia. Results After follow-up 6 months, CMV replication was detected in 12 (31.6%) kidney transplant recipients (KTRs). The total lymphocyte counts were signicantly decreased in KTRs with CMV DNAemia in 1, 3 and 6 months. There was a negative correlation between CMV copies and the lymphocyte counts in 1, 3 and 6 months post-transplantation, and the decrease of lymphocyte counts in the 6 months post-transplantation was the risk factor of CMV DNAemia in the KTR. Patients with lymphocyte counts 1.085×10 9 cells/L had higher cumulative incidence of CMV DNAemia. The lymphocyte counts post kidney transplantation may be used as a simple and effective indicator for monitoring the CMV DNAemia status in KTR and for predicting the risk of CMV DNAemia.


Background
Cytomegalovirus (CMV) belongs to the b-herpesvirus family and can infect different species including rodents, non-human primates, and humans. Although CMV is widely distributed in the general population with a high prevalence, the primary CMV infection is usually asymptomatic and harmless [1]. In contrast, reactivation of latent CMV infection may cause CMV disease and be a major cause of morbidity and mortality in immunocompromised kidney transplant recipients (KTR). Cumulative data con rmed that CMV infection could result in the increase of the incidence of acute rejection and chronic histological changes of kidney such as interstitial brosis and tubular atrophy [2,3]. If without any form of preventive strategies, CMV infection primarily occurs in the rst 3 months following transplantation, and the incidence of symptomatic CMV infection reached to 20-60%. Moreover, the severity of CMV infection depends on the serostatus of the recipient (R) and donor (D) before transplantation, transplanted organ type, and immunosuppressive treatment after kidney transplantation [4].
The reactivation of CMV in KTR that commonly occurs shortly after transplantation is the consequence of a temporary disruption of an existing balance between immunological surveillance and viral replication by treated with immunosuppressive agents and by systemic infection and in ammation [5].
Clinical manifestations of CMV disease in KTR are non-speci c and commonly include fever with u-like syndrome, gastrointestinal disorders and hematologic and respiratory manifestations. Currently, use of speci c antiviral drugs such as valaciclovir and ganciclovir signi cantly improve the prognosis of CMV infection and graft survival. Both universal prophylaxis and preemptive therapy have alleviated the impact of CMV on graft function.
In recent years, some immune-monitoring strategies have been applied to evaluate the risk of CMV infection in KTR, the dynamics of certain cells in peripheral blood have been con rmed to be valuable for predicting opportunistic infection in recipients who received kidney and heart transplantation [6,7].
However, one more simple surrogate approach would be needed to monitor the state of immunosuppression, and further estimate the possibility of CMV infection in KRT. Therefore, we try to analyze the effect of CMV DNAemia within the rst 6 months on graft survival and function in KTR, and explore the latent value of routine monitoring of total lymphocyte counts in peripheral blood to predict the occurrence of CMV infection in the present study. CMV prevention and monitoring 38 patients were treated with ganciclovir (500mg, oral administration, tid) or valganciclovir (450mg, oral administration, qd) universal prophylaxis for 3 months. PCR for CMV DNAemia was performed at 2-week intervals for the rst 3 months, and 1-month intervals at 4, 5 and 6 months thereafter. CMV DNAemia was de ned by detection of a viral load of >500 copies/ml. All patients were followed up prospectively for 6 months or until death or graft failure.

Patients
Statistical analysis SPSS version 21.0 (SPSS Inc., Chicago, IL, USA) was applied to perform all statistical analyses.
Quantitative variables are expressed as mean and standard deviation and qualitative variables were described by frequency (percentage). Quantitative variables were compared between the two CMV infected and non-infected groups by using independent samples t-test and qualitative variables were analyzed by using Chi-square test. Risk factors for CMV DNAemia in KTR were analyzed using univariate logistic regression analyses. Receiver operating curve (ROC) analysis was performed to calculate the speci city and sensitivity of lymphocyte levels for detection of CMV infection. A P value <0.05 was considered signi cant.

Study population and incidence of CMV DNAemia
Data of 38 KTR receiving universal prophylaxis for anti-CMV treatment were included in this study. CMV replication was detected in 12 (31.6%) transplanted recipients within the rst 6 months, 6 patients were infected with CMV in the rst month, 6 patients were infected with CMV in the third month, and CMV DNAemia happened in 8 patients after 6 month kidney transplantation, respectively.
Demographic and baseline clinical characteristics of patients pre-transplantation are presented in Table  1. There was no comparable regard to the baseline characteristics including recipient age, gender, weight, underlying disease, induction therapy, last control immune suppression and incidence of delayed graft function (DGF) in patients with CMV DNAemia or not. Similarly, there was no signi cant difference in hemoglobin, white blood cells, neutrophil, lymphocyte, serum creatinine, blood urea nitrogen (BUN), albumin and uric acid between two groups ( Table 2).

Dynamics of lymphocyte during the 6 months after kidney transplantation
In order to determine the effect of CMV DNAemia on KTR, we further analyzed their biochemical parameters at 1, 3 and 6 months post-transplantation. As is shown in gure 1, we found that the lymphocyte counts were signi cantly higher in recipients free from CMV DNAemia in 1, 3 and 6 months when compared to those who developed CMV DNAemia. Meanwhile, the level of serum creatinine signi cantly increased gradually.
Lower lymphocyte counts were the risk factor of CMV DNAemia in the KTRs Table 3 showed that there was a negative correlation between CMV copies and the lymphocyte counts in 1, 3 and 6 months post-transplantation among the 38 KTR, although this was not statistically signi cant in the third month. In the univariate analysis, the decrease of lymphocyte counts in the 6 months posttransplantation was the risk factor of CMV DNAemia in the KTR ( Table 4).

Decrease of lymphocyte counts is a predictor of CMV DNAemia in the KTR
According to the analysis of ROC, the lymphocyte counts were con rmed to be the most accurate parameter at month 6 for predicting CMV DNAemia (AUC=0.78, 95% CI: 0.69-0.96, P = 0.005). On the basis of these statistical curves, we selected the cutoff value of 1.085×10 9 lymphocytes/L with the highest Youden's index. At the optimal cutoff value of lymphocyte counts, the sensitivity was 80.7% and speci city was 75.0% ( gure 2).

Discussion
In our present study, we demonstrated that monitoring of the number of lymphocytes in peripheral blood during the early period post kidney transplantation should be useful for predicting the occurrence of CMV DNAemia in KTRs. Especially, patients with a lymphocyte counts < 1.085×10 9 cells/L at month 6 experienced a higher incidence of CMV DNAemia compared with those above this cutoff, which can predict the occurrence of CMV DNAemia with sensitivity and speci city above 75%. In addition, we also found that there was a negative correlation between CMV copies and the lymphocyte counts in post kidney transplanted patients.
Monitoring of cell-mediated immunity (CMI) has been considered to be a promising strategy to reduce the incidence of opportunistic infection in KTRs. This monitoring could be based on quantitative measurement of the counts of total lymphocyte. A few studies have reported that low lymphocyte counts were associated with the adverse events in solid organ transplantation patients. Calarota SA et al. found that heart transplant recipients developing opportunistic infections had lower CD4 + and CD8 + T cells than those without infections, and the opportunistic infections only happened in the KTRs with low CD8 + T cells [6]. In liver transplantation recipients, the pretransplant total lymphocyte counts were signi cantly lower in patients with an infection compared with those without one, and there was a signi cant correlation between the risk of development of a posttransplant infection and pretransplant total lymphocyte counts<1.00×10 3 /μl [11]. Results from Fernández-Ruiz M et al. also showed that CD8 + T cell count <0.100×10 3 /μl at one month after transplantation can predict the subsequent occurrence of overall opportunistic infection and CMV disease in the non-ATG induction group, and CD4 + T cell count at month 1<0.050×10 3 /μl showed negative predictive potential for the subsequent occurrence of overall opportunistic infection and CMV disease in the ATG induction group respectively [7]. In addition, Ducloux D et al. found that lower CD4 + T cell level was associated with increased risk of noncutaneous neoplasia in KTRs [12], and they subsequently con rmed that CD4 + T cell lymphopenia persisting for >1 year resulted in a higher death rate in KTRs, which associated with a nearly ve-fold risk for death [13]. Various studies also reported the correlation of lymphocyte counts with pneumocystis pneumonia (PJP) infection. Lee SH et al. showed that the lymphocyte counts were relatively low in the PJP group, and the univariate analysis suggested the decrease of lymphocyte counts were associated with the development of PJP infection [14]. Thus, posttransplant kinetics of lymphocyte acted as a simple indicator for monitoring the immune condition in these solid organ transplanted patients.
Although the data from Nierenberg NE et al. suggested that pretransplant lymphopenia is a novel independent predictor of both CMV disease and non-CMV invasive infections after liver transplantation [15], Fernández-Ruiz M et al. also demonstrated that absolute lymphopenia evaluation pretransplantation was an independent risk factor for post-transplant infection in liver transplanted recipients [11]. An earlier study showed the combination of CMV infection and a previous episode of acute rejection was related with increased vasculopathic changes in 6-month protocol biopsy specimens [16]. Meanwhile, the other study con red that CMV DNAemia with viral load of more than or equal to 2000 copies/mL could result in increase of the risk of interstitial brosis and tubular atrophy in protocol biopsy within the rst 3 months after transplantation [17]. We did not found any differences in lymphocyte counts before kidney transplantation in our study, which indicated that there was no association between baseline lymphocyte counts and the risk of CMV DNAemia. Thus, we could not determine the predictive value of lymphocyte counts pre-transplantation.
There are some limitations in our study. We only evaluated the correlation of lymphocyte counts with the CMV DNAemia in early phase of kidney transplanted recipients, the long term affection lymphocyte counts of on CMV DNAemia did not be further elucidation. Moreover, we only detected the total lymphocyte counts, but we did not perform any functional studies of different lymphocytes subgroup. Finally, due to it was a retrospective observational study and single center cohort, the sample size was small, and making it unlikely to perform other subgroup analyses.

Conclusion
In conclusion, low lymphocyte counts early after renal transplantation was associated with CMV DNAemia, which may associate with signi cant loss of renal function. We suggest that post-transplant decreaseof lymphocyte counts in peripheral blood could be as a rapid, simple and effective biomarker, providing a convenient alternative way to notice the risk of CMV DNAemia.

Declarations
Ethics approval and consent to participate This study was approved by the ethics committee of Beijing Chao-Yang Hospital, Capital Medical University (2017--47) and was conducted in accordance with the Declaration of Helsinki.

Consent for publication
Not applicable.

Availability of data and materials
All data generated or analysed during this study are included in this published article [and its supplementary information les].

Competing interests
The authors declare that they have no competing interests.