Background: Cytomegalovirus (CMV) is the most common opportunistic viral infection in kidney transplant recipients. CMV classification is usually based on its glycoprotein B (gB) genotypes, which divides the virus into 4 strains (gB1â��4). Objective: To determine the incidence of CMV genotypes in Iran and their relation to various clinical factors. Methods: We studied 80 renal transplant recipients admitted to our transplant referral center between 2014 and 2015. All of the studied patients were monitored every 1â��2 weeks for CMV infection by immunofluorescence method. There were 34 CMV-infected patients whose sera were studied with sequencing technique to identify the 4 CMV genotypes. All patients were followed up to 6 months after transplantation. Results: gB1 was the most common genotype (35.3); it was followed by gB3 and gB4 (each with 17.6 ), gB2, and mixed gB1,3 and gB1,2 (each with 14.7). Age (p=0.037), time of infection after transplantation (p=0.011), and biopsy-proven rejection (p=0.012) were associated with CMV genotype. After adjusting for covariates, significant associations were found between genotype gB1 and family relationship (p=0.047) as well as HLA mismatch (p=0.014); genotype gB3 and family relationship (p=0.011); and genotype gB4 and age (p=0.019). Conclusion: The most common CMV gB genotype in CMV-infected kidney transplant recipients in Iran was gB1. We recommend considering related therapeutic applications in the management of such patients. Â© 2018 Iranian Society for Organ Transplantation. All rights reserved

Akbari, H.

Einollahi, B.

Jafari, M.

Nikoueinejad, H.

Piroozmand, A.

Soleimani, A.R.

kashan university of medical sciences

International Journal of Organ Transplantation Medicine
The Incidence of Cytomegalovirus Glycoprotein B 
Genotypes in Kidney Transplant Recipients in Iran
A. R. Soleimani1,  
M. Jafari2,  
A. Piroozmand3,  
H. Nikoueinejad4*,  
H. Akbari5,  
B. Einollahi4
1Internal Medicine Department, Kashan University of  Medical Sciences, 
Kashan, Iran 
2Student Research Committee, Kashan University of  Medical Sciences, 
Kashan, Iran 
3Autoimmune Diseases Research Center, Department of  Microbiology, 
Kashan University of  Medical Sciences, Kashan, Iran 
4Nephrology and Urology Research Center, Baqiyatallah University of  
Medical Sciences, Tehran, Iran  
5Trauma Research Center, Department of  Community Medicine, Kashan 
University of  Medical Sciences, Kashan, Iran
ABSTRACT 
Background: Cytomegalovirus (CMV) is the most common opportunistic viral infection in kidney trans-
plant recipients. CMV classification is usually based on its glycoprotein B (gB) genotypes, which divides 
the virus into 4 strains (gB1–4).
Objective: To determine the incidence of CMV genotypes in Iran and their relation to various clinical fac-tors.
Methods: We studied 80 renal transplant recipients admitted to our transplant referral center between 
2014 and 2015. All of the studied patients were monitored every 1–2 weeks for CMV infection by immu-
nofluorescence method. There were 34 CMV-infected patients whose sera were studied with sequencing 
technique to identify the 4 CMV genotypes. All patients were followed up to 6 months after transplanta-tion.
Results: gB1 was the most common genotype (35.3%); it was followed by gB3 and gB4 (each with 17.6 
%), gB2, and mixed gB1,3 and gB1,2 (each with 14.7%). Age (p=0.037), time of infection after trans-
plantation (p=0.011), and biopsy-proven rejection (p=0.012) were associated with CMV genotype. After 
adjusting for covariates, significant associations were found between genotype gB1 and family relation-
ship (p=0.047) as well as HLA mismatch (p=0.014); genotype gB3 and family relationship (p=0.011); and 
genotype gB4 and age (p=0.019).
Conclusion: The most common CMV gB genotype in CMV-infected kidney transplant recipients in Iran was 
gB1. We recommend considering related therapeutic applications in the management of such patients.
KEYWORDS: CMV infection; Glycoprotein B; Genotype; Renal transplantation
*Correspondence: Hassan Nikoueinejad, Nephrology 
and Urology Research Center, Baqiyatallah University of 
Medical Sciences, Baqiyatallah Hospital, Mollasadra Ave, 
Vanak Sq, PO Box: 19395-5487, Tehran, IR Iran
Tel: +98-913-161-5530
Fax: +98-21-8126-2073
E-mail: hnikuinejad@yahoo.com
INTRODUCTION
Cytomegalovirus (CMV) is a DNA virus with an estimated size of 200 nanome-ters and belongs to Herpes virus fam-
ily [1]. CMV infection continues to be a major 
clinical problem after solid organ transplanta-
tion with a significant morbidity and mortal-
ity. It causes symptomatic disease in 35% and 
death in 2% of renal transplant recipients [2]. 
Although gancyclovir and related drugs re-
duce almost 50%–70% of CMV disease inci-
dence as well as its mortality [3, 4], the toxicity 
associated with the use of currently available 
antiviral agents remains a significant problem 
[5]. Previous studies have shown that the im-
mune response against CMV may strongly be 
dependent on the virus strain [6] and that re-
infection with other strains may be completely 
different clinically from the relapse of the first 
Original Article
174 Int J Org Transplant Med 2018; Vol. 9 (4)    www.ijotm.com 
one [7]. Moreover, the presence of only one 
CMV strain compared to several ones in the 
first year after transplantation is associated 
with different clinical outcomes. For example, 
it has been demonstrated that mixed CMV-
strain infection in organ transplant recipients 
could be associated with a higher transplant 
rejection rate, delayed virus clearance from 
the blood and faster disease development [8].
The classification of CMV strains is usually 
done based on the virus glycoprotein B (gB) 
genotype [9], which could be done by either 
sequencing or restriction fragment length 
polymorphism (RFLP). Although not suit-
able for fast-screening large populations, such 
methods would be valuable in infected cases 
in whom different strains may have different 
pathogenicity needing different approaches 
and treatment [10].
In line with a previous study conducted in 
a local population of renal transplant recipi-
ents in Northwest of Iran [11], we designed 
this study in a general population of Iranian 
people to evaluate CMV gB distribution in 
CMV-infected renal transplant recipients. We 
also correlated different demographic as well 
as clinical characteristics of the patients to the 
studied genotypes.
MATERIALS AND METHODS
Out of 400 kidneys transplant recipients, 80 
were randomly enrolled into our cross-sec-
tional study conducted between 2014 and 2015 
in Baqiyatallah Hospital, a referral center for 
kidney transplant from all over Iran. The sam-
ple size was calculated based on an estimated 
incidence of CMV infection in kidney trans-
plant patients of 80%, and 5% type I error, and 
8% accuracy, using Cochran’s formula. All pa-
tients had a living donor and followed for a 
period of six months. The induction as well 
as maintenance immunosuppression protocol 
for all recipients included therapeutic adjusted 
doses of calcineurin inhibitors, mycophenolate 
mofetil, and steroids. All patients were fol-
lowed for six months on a monthly basis and 
were studied for the type of prescribed immu-
nosuppressive drugs (cyclosporine A or tacro-
limus), CMV infection defined according to 
the standard criteria, and biopsy-proven acute 
rejection. This study was approved by the lo-
cal Ethics Committee. Informed consent was 
obtained from all participants.
All of the studied patients were monitored ev-
ery 1–2 weeks for active CMV infection using 
antigenemia (AGM) assay. Using the Light 
Diagnostics CMV phosphoprotein (pp)65 An-
tigenemia Immunofluorescence Assay (IFA), 
we utilized an indirect immunofluorescence 
technique to identify the lower matrix protein 
pp65 of human CMV in cytospin preparations 
of peripheral blood leukocytes (Merck Mil-
lipore LIGHT DIAGNOSTICS CMV pp65 
Antigenemia IFA kit). 
Briefly, ethanol diamine tetra acetic acid 
(EDTA)-treated blood samples were fraction-
ated by erythrocyte lyses. Granulocytes were 
then centrifuged to prepare cytospin slides 
(2×105 granulocytes per slide). After air-dry-
ing and fixing the slides in formaldehyde, they 
were immunostained using sufficient CMV 
pp65 monoclonal antibody to detect the CMV 
lower matrix phosphoprotein (pp65), an early 
antigen in virus replication, which is abun-
dantly present in antigen-positive polymor-
phonuclear cells. Finally, the samples were ex-
amined with a fluorescence microscope under 
a magnification of 200–400×, for cells exhib-
iting the apple green fluorescence of fluores-
cent iso-thio cyanate (FITC).
CMV gB genotyping was done according to 
a method described elsewhere [12]. In brief, 
DNA extracted from 200 mL whole periph-
eral blood with the DNA blood and tissue ex-
traction kit (Qiagen, Chatsworth, CA, USA) 
was used as template. Then, polymerase chain 
reaction (PCR) was performed using primers 
gB1319 (5’-TGGAACTGGAACGTTTG-
GC-3’) and gB1604 (5’-GAAACGCGCG-
GCAATCGG-3’) [13], which amplify a re-
gion of unique long (UL)55 that encodes a 
variable part of gp55 yielding a 293–296 bp 
target, depending on the gB genotype. Each 
reaction was done in a final volume of 100 μL 
and contained 10 mM Tris-HCl (pH 8.3), 50 
A. R. Soleimani, M. Jafari, et al 
www.ijotm.com    Int J Org Transplant Med 2018; Vol. 9 (4) 175
mM KCl, 1.5 mM MgCl2, 0.01% gelatin, 200 
μM of each dNTP, 0.25 μM of each primer, 
2.5 units of Taq polymerase (Amplitaq; Perkin 
Elmer, Markham, Ont, Canada) and 10–30 μL 
of template DNA. Presence of the correct am-
plicon was verified by gel electrophoresis on 
2% agarose. 
Prior to be restricted and sequenced, the PCR 
products were also gel purified using the QIA-
quick gel extraction protocol (Qiagen Inc., Mis-
sissauga, Ont, Canada). The purified amplicon 
from each isolate was separately digested with 
HinfI and RsaI. Without running on gel, some 
of the isolates were sequenced to confirm that 
the restriction digests were producing reliable 
results. The sequences obtained in this study 
were aligned (Vector NTI 5.5) with those that 
were previously sequenced and typed by Chou 
and Dennison (Genbank accession numbers: 
M60928, M60930, M60931, and M60933). 
Statistical Analysis
The results were expressed as mean±SD. 
Normality test was performed for the exam-
ined variables. The groups were compared by 
χ2 test and ANOVA. In addition, we applied 
multiple logistic regression model with condi-
tional backward method to adjust the effect of 
independent variables on CMV genotypes. A 
p value <0.05 was considered statistically sig-
nificant. All analyses were made by the SPSS 
18 package.
RESULTS
CMV Infection Incidence
From all 80 renal transplant recipients exam-
ined, 34 (48%) showed CMV infection dur-
ing six months. The most infection rate with 
CMV happened during the first and second 
months (11% and 9%, respectively). 
Distribution of CMV gB Genotypes
Genotyping was examined in 34 viremic re-
cipients. The most common CMV gB geno-
type was gB1 (35.3%) followed by gB3 and 
gB4 (each with 17.6%), gB2 and mixed gB1,3 
and gB1,2 (each with 14.7%) (Table 1).
Evaluating the frequency of different vari-
ables according to the CMV gB genotypes, we 
found that rejection showed a significant rela-
tionship to gB strains (p=0.012) (Table 1); the 
most transplant rejection happened in gB mix 
(80%); the least was in those with gB1 (8.3%) . 
The relationship between CMV gB genotype 
and age (p=0.037) as well as the time of ge-
notyping after transplantation (p=0.011) was 
also statistically significant (Table 1).
After adjusting for covariates, the correlation 
between sex, age, family relation between the 
recipient and donor, and HLA mismatch, and 
different CMV gB genotypes, was studied. 
We found that some variables had a signifi-
cant relationship to some gB genotypes (Table 
Table 1: The frequency, mean and SD of studied variables stratified by gB CMV genotypes. Values are either 
mean±SD or n (%).
Variable
gB
1 2 3 4 mix p value
Sex
Female 5 (42) 3 (60) 3 (50) 3 (50) 3 (60)
0.968
Male 7 (58) 2 (40) 3 (50) 3 (50) 2 (40)
Mean±SD Age (yrs) 47.5±7.8 39.4±5.6 49.3±11.1 34.1±8.1 43.6±13.3 0.037
Mean±SD time of infection (months) 4.6±1.4 2.6±2.1 3.6±1.6 2.3±1.0 1.8±1.7 0.011
Rejection
No 11 (92) 2 (40) 5 (83) 2 (33) 1 (20)
0.012
Yes 1 (8) 3 (60) 1 (17) 4 (67) 4 (80)
Family relation between recipient and donor
No 8 (67) 4 (80) 2 (33) 5 (83) 3 (60)
0.432
Yes 4 (33) 1 (20) 4 (67) 1 (17) 2 (40)
CMV Glycoprotein B Genotypes in Kidney Tx Recipients
176 Int J Org Transplant Med 2018; Vol. 9 (4)    www.ijotm.com 
2). According to this regression model, fam-
ily relation (p=0.047) and HLA mismatch 
(p=0.014) were two independent predictors of 
gB1 infection; family relation was an indepen-
dent predictor of gB3 infection (p=0.011); age 
was an independent predictor of gB4 infection 
(p=0.019) (Table 2).
DISCUSSION
Considering that the immune response as well 
as clinical outcome of CMV infection after sol-
id organ transplantation may strongly be de-
pendent on the virus strain [6], we studied the 
distribution of CMV strains in Iranian kidney 
transplant recipients, based on virus glycopro-
tein B (gB) genotype. We also evaluated the 
association between different CMV genotype 
strains and a number of factors.
Similar to our findings, other studies on dif-
ferent populations such as those residing in 
northern Iran [11], Turkey [14], and Kuwait 
[15] showed that gB1 was the most prevalent 
CMV genotype (26.5%, 32.3%, and 59%, re-
spectively). This has also been shown in the 
study of Pang and colleagues on 121 plasma 
specimens from 47 solid organ transplant re-
cipients in whom the next most common gen-
otypes were also gB2, mixed gB, gB3 and gB4 
[16]. However, there were differences. For ex-
ample, in Kuwait, gB2 and gB3 (29% and 10%, 
respectively) were the most prevalent strains 
too [15] that was in contrast with our find-
gins most probably due to racial differences.
The association between different CMV geno-
types and different clinical characteristics of 
the transplant recipients has also been identi-
fied in other studies. Madi, et al, demonstrated 
that gB1 genotype is significantly associated 
with development of fever, leukopenia and se-
vere CMV disease compared with other gB 
genotypes [15]. In a study by Rosen and col-
leagues on 53 CMV-infected liver transplant 
recipients, it was shown that gB1 genotype is 
significantly associated with a higher number 
of acute rejection episodes but not with the re-
jection severity [17]. Correa revealed that gB4 
is the only genotype associated with clinical 
features of sepsis-like syndrome in newborns 
[18]. In contrast, Sarcinella, et al, in a study on 
58 liver transplant recipients with CMV infec-
tion showed that gB genotype does not corre-
late with peak CMV viral load, development of 
CMV disease, and acute rejection [12].
Age, time of genotyping after transplantation, 
and biopsy-proven rejection showed a signifi-
cant correlation with gB strains in our study. 
Although CMV infection was not an indepen-
dent risk factor for rejection, CMV gB geno-
type was associated with transplant rejection 
in CMV-infected patients. After adjusting for 
covariates, family relation and HLA mismatch 
were associated with gB1, family relation was 
associated with gB3, and age was associated 
with gB4.
Table 2: Association of variables with CMV geno-
types after adjusting for covariates
Genotype Variable p value OR
gB1
Sex (male) 0.532 0.675
Age 0.112 1.049
Family relation 
(yes) 0.047 4.126
HLA mismatch 0.014 2.240
gB2
Sex (male) 0.191 0.268
Age 0.300 0.940
Family relation 
(yes) 0.692 1.657
HLA mismatch 0.125 2.093
gB3
Sex (male) 0.245 0.314
Age 0.115 1.069
Family relation 
(yes) 0.011 17.087
HLA mismatch 0.193 1.957
gB4
Sex (male) 0.085 0.150
Age 0.019 0.838
Family relation 
(yes) 0.895 1.177
HLA mismatch 0.070 2.234
Mixed
Sex (male) 0.348 0.414
Age 0.972 0.998
Family relation 
(yes) 0.285 3.078
HLA mismatch 0.454 1.410
A. R. Soleimani, M. Jafari, et al 
www.ijotm.com    Int J Org Transplant Med 2018; Vol. 9 (4) 177
Further studies are needed to clarify the role 
of CMV gB genotypes in the clinical mani-
festations in transplant recipients and also to 
reveal the factors that influence the CMV gB 
genotype.
In conclusion, the most common CMV gB 
genotype in CMV-infected kidney transplants 
of Iran was gB1. Physicians should consider he 
related factors such as rejection in the man-
agement of kidney transplant recipients.
ACKNOWLEDGMENTS
This study was funded and supported by 
the Deputy of Research, Kashan University 
of Medical Sciences (kaums), grant number 
93212.
CONFLICTS OF INTEREST: None declared.
REFERENCES
1. Storch GA. Viral infection in immunocompromised 
patients. In: Diagnosis Virology. 1st ed. New York: 
Churchil-Livingstone, 2001: 203-32.
2. Yeung JS, Tong KL, Chan HWH. Clinical pattern, risk 
factors, and outcome of CMV infection in renal 
transplant recipients: Local experience. Transplant 
Proc 1998;30:3144-5.
3. Thomas DE. Hematopoietic cell transplantation. 
In: Blume KG, Forman SJ, Aprelbaum FR, editors. 
Cytomegalovirus infection. 3rd ed. J.A: Zaiz; 2004: 
700-26.
4. Boeckh M, Leisenring W, Riddell SR, et al. Late cy-
tomegalovirus disease and mortality in recipients 
of allogeneic hematopoietic stem cell transplants: 
importance of viral load and T-cell immunity. Blood 
2003;101:407-14.
5. Boeckh M, Boivin G. Quantitation of cytomegalo-
virus: methodologic aspects and clinical applica-
tions. Clin Microbiol Rev 1998;11:533-54.
6. Klein M, Schoppel K, Amvrossiadis N, Mach M. 
Strain-specific neutralization of human cytomega-
lovirus isolates by human sera. J Virol 1999;73:878-
86.
7. Grundy JE, Lui SF, Super M, et al. Symptomatic cy-
tomegalovirus infection in seropositive kidney re-
cipients: reinfection with donor virus rather than 
reactivation of recipient virus. Lancet 1988;2:132-
5.
8. Puchhammer-Stöckl E, Görzer I, Zoufaly A, et al. 
Emergence of multiple cytomegalovirus strains 
in blood and lung of lung transplant recipients. 
Transplantation 2006;81:187-94.
9. Van der Bij W, Schirm J, Torensma R, et al. Compar-
ison between viremia and antigenemia for detec-
tion of cytomegalovirus in blood. J Clin Microbiol 
1988;26:2531-5.
10. Grosjean J, Hantz S, Cotin S, et al. Direct genotyping 
of cytomegalovirus envelope glycoproteins from 
toddler’s saliva samples. J Clin Virol 2009;46:43-8.
11. Khalafkhany D, Makhdoomi K, Taghizadeh Afshari 
A, Motazakker M. Prevalence and genotype distri-
bution of cytomegalovirus UL55 sequence in renal 
transplant recipients in north west of Iran. J Med 
Virol 2016;88:1622-7.
12. Sarcinella L, Mazzulli T, Willey B, Humar A. Cyto-
megalovirus glycoprotein B genotype does not 
correlate with outcomes in liver transplant pa-
tients. J Clin Virol 2002;24:99-105.
13. Chou SW, Dennison KM. Analysis of interstrain 
variation in cytomegalovirus glycoprotein B se-
quences encoding neutralization-related epitopes. 
J Infect Dis 1991;163:1229-34.
14. Eren Dağlar D, Öngüt G, Çolak D, et al. Determina-
tion of cytomegalovirus glycoprotein B genotypes 
in different geographical regions and different pa-
tient groups in Turkey. Mikrobiyol Bul 2016;50:53-
62.
15. Madi N, Al-Nakib W, Pacsa A, Saeed T. Cytomega-
lovirus genotypes gB1 and gH1 are the most pre-
dominant genotypes among renal transplant re-
cipients in Kuwait. Transplant Proc 2011;43:1634-
7.
16. Pang X, Humar A, Preiksaitis JK. Concurrent ge-
notyping and quantitation of cytomegalovirus gB 
genotypes in solid-organ-transplant recipients 
by use of a real-time PCR assay. J Clin Microbiol 
2008;46:4004-10.
17. Rosen HR, Corless CL, Rabkin J, Chou S. Associa-
tion of cytomegalovirus genotype with graft rejec-
tion after liver transplantation. Transplantation 
1998;66:1627-31.
18. Correa C, Kourí V, Pérez L, et al. Diagnosis, gB gen-
otype distribution and viral load of symptomatic 
congenitally infected CMV patients in Cuba. J Peri-
natol 2016;36:837-42.
CMV Glycoprotein B Genotypes in Kidney Tx Recipients


The incidence of cytomegalovirus glycoprotein B genotypes in kidney transplant recipients in Iran

http://eprints.kaums.ac.ir/4060/1/ijotm-9-173.pdf

The incidence of cytomegalovirus glycoprotein B genotypes in kidney transplant recipients in Iran

Abstract

Similar works

Full text

Available Versions

kashan university of medical sciences