Cytomegalovirus Viremia and Risk Factors in Renal Transplant Recipients in Iran: a Prospective Case-control Study

Background: In spite of effective anti-viral drugs and risk-balanced prophylaxis regimen, cytomegalovirus (CMV) remains a major reason of morbidity in kidney transplant patients. The aim of present study was to evaluate CMV viral load and laboratory ndings correlation with CMV viremia graft origin and investigation on late or early onset CMV infection in kidney transplant recipients with CMV viremia. Methods: This research designed as a prospective case-control study based on CMV PCR test and exclusion of other viral infection among renal transplant patients in Iran. Results: From 192 examined patients, 153 participants were qualied to enter the study: 43 in case (with CMV viremia) and 110 in control group (CMV negative test). Statistical analysis performed to identify the risk factors raising this viral viremia among kidney transplant patients. Conclusion: Receiving a renal graft from a deceased donor signicantly raise the chance of viremia in renal transplant patients. The median month of CMV viremia occurrence was month 4 after transplantation in both groups. Serum laboratory testing showed creatinine and platelets signicantly raised and reduced, retrospectively in the case compare to control group. Our results indicating the viremia has not affected the survival of the allograft or patient. CMV: Cytomegalovirus, BKV: BK polyomavirus, HHV6: Human herpesvirus-6, EBV: Epstein Bar Virus, PCR: Polymerase chain reaction, RT: Renal Transplant, EDTA: Ethylenediaminetetraacetic acid, SGOT: serum glutamic oxalacetic transaminase, SGPT: serum glutamic pyruvic transaminase, ALP: Alkaline phosphatase, WBC: White Blood Cell, FBS: Fasting Blood Sugar, DNA: Deoxyribonucleic Acid, RNA: Ribonucleic Acid, IQR: interquartile range.

transplantation in both groups. Serum laboratory testing showed creatinine and platelets signi cantly raised and reduced, retrospectively in the case compare to control group. Our results indicating the viremia has not affected the survival of the allograft or patient.

Background
In spite of effective anti-viral drugs and risk-balanced prophylaxis regimen, cytomegalovirus (CMV) remains a major reason of morbidity in kidney transplant patients (1). In the renal transplant (RT) patients, infection occurs primarily or as reactivation of latent virus depends on kidney donor or recipient CMV serostatus (2). CMV reactivation can result in CMV disease or direct effects of the disease related to the presence of high rates of viral replication and lytic virus production (3), and indirect effects caused by virus interaction with the most immune response acute rejection, graft dysfunction, opportunistic infections, diabetes mellitus and malignancies the indirect effects of CMV are assumed to be mediated by cytokine and chemokine production (4,5).
Re-emergence of CMV viremia could occur as a 'early onset' or 'late onset' infection (6). Ten to 50% of allograft recipients develop early onset CMV infection after or during receiving anti-viral prophylaxis. Approximately half of these patients will develop clinical manifestations of disease, and up to 30% of successfully treated cases of CMV disease will recur. CMV infection characteristically presents during the rst 6 months after organ transplantation, and recurrences occur within 3 months of completion of therapy for the initial episode. Late CMV disease is de ned as that which presents 16 months after organ transplantation, and it is most often related to the need to increase the level of immunosuppression because of late episodes of rejection (7,8).
Quantitation of CMV in RT patients allows for assessment of the degree of CMV replication, which is expressed as the absolute viral load value. Trends in viral loads over time (viral load kinetics) directly correlate with the likelihood of severe CMV disease (9). In a series of prospective studies, peak CMV load during active infection is identi ed as a major risk factor that correlates with the development of CMV disease (9).
There are reports in RT patients indicating the allograft origin might change the risk of CMV infection after transplantation. The studies show that outcome of living donor kidney transplantation has been better than that of deceased donor kidney transplantation (10) Deceased donors, particularly donors whose organs are accepted based on expanded criteria, might contribute to an increase in the infection risk through intensive immunosuppression or donor-derived nosocomial organisms (11). Recently, in a cohort study in the Europe, a deceased donor transplantation was identi ed to be associated with increased incidences of CMV viremia (12).
In Iran, a comprehensive study on CMV viral load and laboratory ndings correlation with CMV viremia, graft origin and investigation on late or early onset CMV infection in kidney transplant recipients with CMV viremia have not been performed. We therefore conducted a prospective case-control analysis to examine the above factors which could be affected by the virus viremia or have a correlation to increase the risk of developing the infection among renal transplant patients in Imam Khomeini hospital, one of the main center of kidney transplantation in Iran.

Study design and setting
The study design was prospective case-control, the recruited subjects comprised 153 RT patients referred to Imam Khomeini Hospital, Tehran, Iran between February 2019 and February 2021. To select the case group, the suspected or randomly found patients to CMV infection were sampled and the control group The study was evaluated and approved by the Ethical Committee of Tehran University of Medical Sciences. Before specimen collection, informed consent was obtained from all patients. The subjects' general data (age, gender, months after transplantation, donor source, and date of transplantation) and underlying diseases caused kidney loss of function recorded in a questionnaire. From each participant 3 ml aliquot of whole blood sample was withdrawn, and plasma was separated immediately from 1 ml using EDTA as anti-coagulant and were stored at -70˚C in RNase DNase free micro-tubes for viral nucleic acid extraction. Two ml of blood sent to the diagnostic laboratory for laboratory parameters including white blood cell count, platelet count, serum FBS, serum creatinine, serum uric acid, serum glutamic oxalacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), and Alkaline phosphatase (ALP) (Figure1).

Immunosuppressive Therapy
In IKH, most patients received routine triple immunosuppression with a calcineurin inhibitor (CyA/tacrolimus), mycophenolate sodium or mycophenolate mofetil and oral prednisolone. Those patients at risk received thymoglobulin induction. Target tacrolimus trough levels are typically between 5 and 10 μg/L for the rst 3 months, with the dose of prednisolone reduced to 5-10 mg daily by 3 months.
Biopsy-proven T cell mediated rejection episodes were managed with intravenous methylprednisolone, with T cell depleting antibody being prescribed for steroid-resistant rejection episodes. CMV prophylaxis with valganciclovir was administered for 100 days to patients.
DNA extraction and PCR for CMV, BKV, HHV6 and EBV Viral nucleic acids were extracted from 200ul of plasma samples using Roche viral nucleic acid extraction kit (Roche, Basel, Switzerland), according to the manufacturer's instruction (Supplementary1). BKV, HHV6 and EBV infection have examined by conventional PCR as described before (13,14). (Supplementary2)

Statistical analysis
Descriptive statistics including means and standard deviations of quantitative variables and frequencies (%) of qualitative variables were computed. Continuous and categorical variables were presented as median (IQR) and n (%), respectively. To compare differences between different groups we used the Wilcoxon rank-sum test, χ2 test, or Fisher's exact test where appropriate. To explore the risk factors associated with CMV viremia and allograft survival or rejection. Following analysis based on CMV PCR test, multivariate logistic regression models were used to control possible cofounders. A two-sided α of less than 0·05 was regarded statistically signi cant. Statistical analyses were done using R version 4.0.3 (2020-10-10).

Results
From among all the 192 surveyed patients, based on the qPCR test and CMV viral load, 43 patients had more than 200 copy of CMV genome in plasma. The study population included 43 patients and 110 controls. Univariate analysis revealed that CMV positive real time PCR test was signi cantly associated with having a lower platelets and higher blood creatinine (Supplementary 1). CMV infection has occurred signi cantly (P < 0.05) more in patients who received the kidney from a deceased donor. However, this univariate analysis did not show any signi cant differences between patients and controls regarding the months after transplantation between two groups (Table 1,2). The main reason of the patient's loss of function was blood pressure (72%) followed by diabetes (36 %) (Figure 2).

Demographic Pro le of Transplant Recipients
The descriptive variables categorized by CMV molecular test results are presented in table 1. Among the case-control participants, mean age was 51.3 years. The majority of patients were males (71%), and primary renal disease was chronic glomerulonephritis in 57% of CMV positive patients. About 27% of the kidney transplant recipients were from living related donors.

Risk Factors of CMV viremia
According to multivariate logistic regression models, age, WBC, platelets, FBS, creatinine, uric acid and donor source had P-values of less than 0.2 and entered to multivariable analysis. The result of models indicates that receiving organ from a deceased donor signi cantly augments the risk of CMV infection in the case group (Table 3).

Discussion
While CMV serostatus play an important role in CMV infection development in kidney Transplant recipients, it is yet not clear whether there are other risk factors could act as well. The present study aimed to investigate the risk factors which increase CMV infection occurrence in this group.
In the present study, we compare the results of serum laboratory tests in CMV positive patients to the negative patients to nd the tests which signi cantly differ between two groups. Besides, we investigate the incidence of early or late CMV infection, risk factors and consequences of CMV viremia among kidney transplant recipients compare to the control patients.
The presented results indicate that RT patients who received the allograft from deceased donor faced with higher risk of CMV viremia after transplantation mostly among the rst four months. Recent analysis among RT patients suggests grafting from a cadaveric would rise the risk of CMV viremia. CMV viremia in our study emerged mostly after 4 months after transplantation, likewise, in a cohort study in the Europe, among 19.2% of CMV + patients, viremia emerged within the rst three months after transplantation (12). given acute rejection episodes occurred within the rst month post transplantation, the association with CMV infection in this period may be attributed to excessive immunosuppression associated with acute rejection treatment. Infection recuurence probably promotes by the initial immunosuppression and cessation of anti-viral prophylaxis. Notably, it is suggested that during this time, patients be monitored and treated by antiviral prophylaxis in accordance with the higher risk of CMV infection. Even though, there was no signi cant different between the time of CMV detection among both CMV+/-groups, our results presenting that early CMV infection emerges in 4 months after transplantation that may consider an alarming time to examine the presence of CMV viremia in the RT patients.
The results show that late onset CMV infection has not occurred frequently since the late months among the investigated patients recorded on month 7 of receiving the kidney. In other studies, late-onset CMV disease still develops in approximately 18% of patients even in the presence of either prophylactic strategies (15, 16).
In the present study, CMV viremia did not signi cantly affect the allograft and patient's survival contrary to other studies (17). Given we investigated seropositive RT patients and in patients who are CMV seropositive, viral replication occurs in the context of pre-existing immunity, hence the observed replication rate is slower in such individuals. As a result of the widespread use of antiviral prophylaxis and preemptive therapy, the incidence and severity of CMV disease and its indirect effects are signi cantly reduced. The incidence of CMV in the renal transplant population is estimated to be between 8 and 32 percent (7).
Patients in the case group displayed worse serum creatinine values post transplantation, albeit without signi cant differences in graft and patient survivals (5). Serum Creatinine raised and platelets diminished. This result con rms the precedent facts which CMV infection should be considered in any renal transplant recipient who has a rise in creatinine even if symptom-free (18).
Our results did not investigate the local detection of CMV in the allograft, it is important to consider that the mere detection of CMV does not essentially exclude the presence of CMV in the blood. Indeed, lack of serum CMV positive test does not completely rule out CMV infection in these patients. Since transient periods of CMV viremia had been found in some cases due to the compartmentalized or localized CMV diseases (19, 20). In the present study, all subjects had received CMV antivirals including ganciclovir (1.25 mg/kg IV daily as induction for 1 month, which then was switched to oral valgancyclovir) or valcyte (450 mg, according to their plasma creatinine levels) for the rst 3 months post transplantation. In the cases of CMV DNAemia, some patients did not show any typical syndromes of CMV infection.
In our investigation, we selected CMV viremia detection by nding the virus DNA in the patients' blood. Given new diagnostic method based on the ampli cation of CMV RNA in blood samples has been commercialized, we recommend that future studies categories the case-control patients by these novel techniques (21, 22)

Conclusions
In conclusion, receiving a kidney from deceased donor signi cantly increased incidence of CMV viremia in studied RT patients.       The frequency of kidney loss of function in the investigated patients in both case-control patients.