INTRODUCTION AND AIMS
Cytomegalovirus (CMV) or human herpesvirus is a double-stranded enveloped DNA virus of the Herpesviridae family. Primarily, the infections are asymptomatic and the virus resides in the body as latent infection is mostly restricted to white blood cell (WBC). Circulating viremia in the plasma is the most common presenting feature of primary infection or reactivation. CMV infections may be life threatening in premature newborns and severely immune-compromised patients.[ 1–3 ] Primary infection leads to lifetime seropositivity with CMV immunoglobulin G (IgG) while CMV IgM appears only for a brief period of time (few months) during primary infection, reactivation of latent infection, and during the re-infection with a different strain. CMV IgM positivity, although transient, has a higher possibility for transfusion transmissibility due to high concentration of CMV DNA in both whole blood and plasma.[ 4 ]
The oropharyngeal and genital routes are the most common ways of viral transmission in the newborn. The other modes include breastfeeding, organ transplantation, and blood transfusion. Primary infection (15%–50%) has a higher chance of transmission compared to secondary infection (0.15%–1%). Transfusion-transmitted CMV (TT-CMV) infections were first described by Kääriäinen et al . in 1966 and its molecular evidence was provided by Tolpin et al . in 1985.[ 5 , 6 ] Seroprevalence of CMV is variable depending on the demography and distribution of the population across the world. It is observed that developing countries have a higher CMV seroprevalence (80%–95%) compared to developed countries (40%–65%).[ 1 , 2 , 5 ] CMV transmission via blood transfusion can best be prevented by using blood from CMV seronegative donors. The high prevalence of CMV seropositivity among blood donors limits the availability of CMV-seronegative blood in hyperendemic countries like India. In India, a donor’s blood is not mandatorily screened for CMV. Studies have demonstrated that CMV is latent in cells of the monocyte/macrophage lineage and also these cells can support CMV replication.[ 3 , 7 ] The use of third-generation leuko-filters effectively removes approximately 3-log10 of the contaminating leukocytes in the blood, reducing the risk of TT-CMV.[ 8 ] To exclude the evidence of primary infection, CMV IgG reactive samples should be tested for CMV IgM and IgG avidity. A positive CMV IgM result in connection with low avidity is a strong indicator of primary CMV infection within the last 4 months.[ 9–11 ]
Aims
The present study primarily aimed to identify the overall seroprevalence of CMV in the healthy blood donors and the newborn who were transfused in the recent past with nonleukoreduced (non-LR) blood (control).
The impact of LR-blood transfusion in CMV prevention/control was observed by the pre- and posttransfusion seroprevalence of CMV or any new seroconversion in CMV-negative newborns.
Since prestorage LR facility was recently licensed at the transfusion center and the bedside LR was not routinely practiced in the nursery; it will estimate the importance of LR blood in TT-CMV.
METHODS
This prospective study was conducted on voluntary blood donors and newborns, from February 2021 to August 2022 by the department of transfusion medicine and neonatology at a government medical college in Kolkata, India. The study was accepted by the institution’s ethics committee (MC/KOL/IEC/NON-SPON/933/01/2021).
Study population and sample design
Cohort
A total of 852 healthy voluntary blood donors were chosen at random[ 2 , 12 ] to study the seroprevalence of CMV in the blood donor population. A total of 45 premature newborns (<37 weeks of gestation) who had received LR-blood transfusion for the first time were also randomly selected.[ 3 , 12 ]
Control
Forty (40) newborn who were transfused in the recent past (6 weeks post-transfusion) with non-LR blood[ 13 ] were chosen randomly as control from the neonatal outdoor clinic.[ 3 , 12 ]
Exclusion criteria
Blood donors who were deferred as per national guidelines[ 14 ] and newborns with recent history of transfusion or intrauterine exchange transfusion were excluded from the study. Newborns from the mother with bad obstetric history and past history of congenital malformed babies were also excluded.
Ethical approval and informed consent
The study was approved by the institutional ethics committee and informed consent was taken from all the participants and their parents (in case of newborns).
Sample collection and laboratory investigations
Blood donors from the age group of 18–65 years (selected according to the guidelines of the National Blood Transfusion Council, Ministry of Health and Family Welfare, Government of India)[ 14 ] and the newborn who require multiple (>l episode) blood transfusion in their course of treatment were chosen randomly. A volume of 3 ml blood was collected in plain vacutainers from blood donors aseptically, during blood donation procedures.
In case of newborns, 1 ml of blood sample was collected before blood transfusion as well as 6 weeks posttransfusion period.[ 13 ] Blood samples were centrifuged at 3000 rpm for 5 min to separate the serum/plasma. Serum/plasma was aliquoted and stored at −80°C.
The serum from the clotted blood sample of blood donors and newborns was tested for CMV IgG, IgM antibodies, and IgG avidity by Chemiluminescence Microparticle Immunoassay (CMIA, Abbott Architect Plus i2000sr, USA).[ 9–11 ]
Principle of immunoglobulin G, immunoglobulin M, and immunoglobulin G avidity assay
CMV IgG/IgM was two-step immunoassay for the qualitative detection and semi-quantitative determination of CMV antibodies in human serum and plasma on CMIA platform. In the first step; serum samples were combined with assay diluents, and CMV lysate (AD 169 strain)- coated paramagnetic microparticles. If anti-CMV antibody (IgG/IgM) was present in the sample, get bound to the microparticles. After thorough washing, murine acridinium-labeled anti-human IgG/IgM conjugate was added to create a reaction mixture. Following another wash cycle, pretrigger (1.32% hydrogen peroxide) and trigger solutions (0.35 N sodium hydroxide) were added to the reaction mixture. The resulting chemiluminescent reaction was measured as relative light units (RLU). If the chemiluminescent signal in the specimen was greater than or equal to the cutoff, the sample was considered positive for CMV antibodies (IgG/IgM).[ 9 , 10 ]
CMV IgM tests were done in all CMV IgG-negative blood donors. In the rest of the CMV IgG-positive blood donors, 27 were randomly selected along with 7 cohorts and 6 controls (newborns) to test CMV IgM antibodies. The reasons for such low sampling were due to the cost constraints and inadequate newborn blood sample.
CMV IgG avidity tests could be performed in 7 cohort and 10 control newborn subjects as well as those 27 CMV IgG-positive blood donors who were tested for CMV IgM.
Cytomegalovirus immunoglobulin G avidity test
CMV IgG avidity assay consists of 2 single tests that were both two-step immunoassays using CMIA technology. One aliquot of the sample (at least 100 ml) was pretreated with blocking agent (Pretreatment 2). A second aliquot of the sample (at least 100 ml) was pretreated with buffer (Pretreatment 1) instead of blocking agent. Each aliquot of the pretreated sample was combined with CMV virus lysate (strain AD169)-coated paramagnetic microparticles. After thorough washing, murine acridinium-labeled anti-human IgG conjugate was added. Following another wash cycle, pretrigger and trigger solutions were added to the reaction mixture. The resulting chemiluminescent reaction was measured as RLU.[ 11 ] The avidity of anti-CMV IgG in the sample was calculated using the RLU of both tests [Table 1 ]. The manufacturer’s instructions were strictly followed for CMV serology (IgG, IgM, and IgG avidity testing) as referenced.
Table 1: Algorithm of interpretation of test results of seroreactivity and avidity of cytomegalovirus
Prestorage leukoreduced and quality control of leukoreduced-packed red blood cell
Prestorage leukocyte removal was performed from freshly prepared packed red blood cell (pRBC) units collected from blood donors at the transfusion medicine department by the prestorage LR filter (BioR, Fresenius Kabi, Germany).
A quality control of the LR-pRBC was performed as per criteria.[ 15 ] i.e.
i) Residual leukocyte count: ≤5 ´ 106 / Unit
ii) At least 99.7% of WBC removed.
iii) Total volume of red cell removed was <15% of the orginal volume of pRBC unit prepared.
Analysis of data
Data collected were entered in MS Excel data sheet and analyzed by SPSS software (version 27.0, Chicago, IL, USA) and GraphPad Prism version 5. Subgroup comparison was made with Chi-square or Fisher’s exact test. For the evaluation of the association between CMV reactivity in newborns receiving LR blood during the pre- and posttransfusion periods, Wilcoxon sign rank test was applied. Spearman correlation test was applied to evaluate the correlation between age/sex of blood donors and CMV IgG semi-quantitative values (AU/ml) of plasma. P ≤0.05 was statistically significant.
RESULTS
Demographic distribution of the voluntary blood donors
Out of a total of 852 blood donors who were observed during the study period, 713 were male (83.7%) and the rest 139 (16.3%) were female. The age distribution of the study subjects is given in Table 2 .
Table 2: Distribution of blood donors according to their age (n =852)
Out of 46 blood donors, in the age group of 18–20 years, 33 were male and 13 were female. Among 311 blood donors, in the age group of 21–30 years, 253 were male and 58 were female. Within 250 blood donors, in the age group of 31–40 years, 209 were male and 41 were female. In the 173 blood donors, of the age group of 41–50 years, 153 were male and 20 were female. In the rest of 72 blood donors, of the age group of >50 years, 65 were male and 7 were female donors.
Seroprevalence of cytomegalovirus immunoglobulin G in the blood donor population and its significance
Among the 852 voluntary blood donors, 830 were CMV IgG positive and 22 were CMV IgG negative (overall prevalence rate of 97.4%). There was no significant difference in CMV seroprevalence rate observed in different age groups and blood groups in the donor population (P < 0.242 Chi-square test and P = 0.228 Fisher’s exact test, respectively). One of the blood donors was positive to both IgG and IgM.
On semi-quantitative estimation of CMV IgG across different age groups and sexes of the donors, there was no statistical significance (Spearman’s rho [r ] =0.061, two-tailed test P = 0.076) [Figures 1 , 2a and b ].
Figure 1: Scattered plot showing CMV IgG antibody Semi-quantitative concentration (AU/ml) versus donor’s age in years (n = 852). CMV: Cytomegalovirus, IgG: Immunoglobulin G.
Figure 2: (a) CMV IgG semiquantitative (AU/ml) among male blood donors (n = 713) (b) CMV IgG semiquantitative (AU/ml) ss among female blood donors (n = 139)
Seroprevalence of cytomegalovirus immunoglobulin M and avidity test results in the subgroup of blood donor population (n = 49)
In the present study, serological test for IgM CMV was done in all of the 22 IgG-seronegative (3.6%) blood donors and all of them were negative. In the remaining population of CMV IgG-seropositive donors, 27 were chosen at random. One of the blood donors showed seropositive results both in IgG and IgM. No blood donor was found to be CMV IgM positive only.
All of these 27 blood donors were tested for CMV IgG Avidity. The blood donor who was CMV IgG positive and IgM positive showed IgG low avidity suggestive of primary infection and high risk for transmission; whereas 4 blood donors were CMV IgG positive, IgM negative with IgG high avidity suggestive of past infection, low risk for transmission.[ 9–11 ] Rest of the 22 blood donors were CMV IgG positive, IgM negative with IgG low avidity; also suggestive of primary infection and high risk.[ 16–19 ]
The present study showed that almost 85.19% (23 out of 27) of CMV-seropositive individuals were of high risk for TT-CMV. There was no evidence of CMV transmission risk among CMV IgG-seronegative blood donors as none of them had IgM positivity.
The demography, blood group, and background characteristic distribution of newborn population in the cohort and control groups in the newborn population is detailed in Table 3 and annexure to Table 3 .
Table 3: Demography and background characteristics distribution of newborn population in cohort and control group
Annexure to Table 3: Background characteristic of the cohort and cases
Cytomegalovirus immunoglobulin M and immunoglobulin G avidity test results in cohort
CMV IgM and IgG avidity test was done among 7 premature newborns in the cohort group who were IgG positive. Among those, 5 were CMV IgG positive, IgM positive, and IgG low avidity suggestive of primary infection and possible for acquired in utero [ 13–15 ] [Table 4a ]. Two newborns were CMV IgG positive but IgM negative and IgG low avidity also suggestive of primary infection.[ 17–19 ]
Table 4a: Seroprevalence and avidity test in the preterm newborn cohort (7)
Cytomegalovirus immunoglobulin M and immunoglobulin G avidity test results in control
CMV IgG avidity test was done among 10 newborns in the control group [Table 4b ]. Among those, 4 were CMV IgG positive and showed CMV IgG low avidity. However, CMV IgM assay could not be done on them due to inadequate sample. The rest 6 babies were CMV IgG positive and IgM negative and showed IgG low avidity, suggestive of primary CMV infection.[ 17–19 ]
Table 4b: Seroprevalence and avidity test in control group
Results of CMV IgG antibody semi-quantitative value (AU/mL) in pre- and posttransfusion period (6 weeks) in 39 newborns in the cohort group (The posttransfusion test was done among 39 newborns in cohort group , as 5 were dead as the time of posttransfusion follow -up and CMV -negative newborns unchanged throughout the follow -up.) [Table 5 ].
Table 5: Pre and post transfusion CMV IgG antibody level (semiquantitative) in cohort*
Wilcoxon sign rank test showed that there was a statistically significant mean rank difference in CMV IgG antibody semi-quantitative determinant (semi-quantitative value decreases in posttransfusion than pretransfusion) of LR blood.
DISCUSSION
In the Indian scenario, there is always a challenge to provide universal LR blood to patient population due to its resource constraints. Several studies across the different parts of the country had observed that there is a high seroprevalence of CMV in our population in comparison to the Western studies.[ 1 , 2 , 20 , 21 ]
The present study had shown a very high seroprevalence rate (97.41%) in our blood donors, which is almost like the other observation across the country. The different contributing factors for high seroprevalence in our country may be related to low socioeconomic status and overcrowding.
CMV IgM test was performed among 49 donors. One donor out of 49 donors was found to be IgM positive in our study. CMV IgM seroprevalence in our study is 2.04%. Akinbami et al . in 2009 had observed 19.5% IgM reactivity among tested donors in Nigeria,[ 22 ] 2.6% was the IgM reactivity of donor population in a study done in Iran.[ 23 ] However, significantly lower results were obtained from studies done in other parts of India like Delhi (0%),[ 2 ] Kerala (0.44%),[ 1 ] Pune (0.071%),[ 24 ] and Thailand (0.006%).[ 25 ]
There were no statistically significant differences in CMV IgG reactivity among different age groups. However, reports from similar studies from Delhi[ 2 ] had the same results, but from the South Indian population, there was a high seroprevalence of CMV IgG in blood donors of 18–20 years of age.[ 1 ] This differs from Western studies which showed a significant increase in seropositivity with increasing age of blood donors.[ 21 ]
None of the blood donors who were seronegative to CMV IgG were positive to CMV IgM, but there was a high risk among the blood donors for TT-CMV as 85.19% of them were of low IgG avidity among the 27 blood donors on whom IgG avidity was done.
In case of the newborn population, we have observed that there was no significant difference in the CMV seroprevalence in both the cohort and control groups. The only newborn who was seronegative to the CMV in the cohort group remained same during the posttransfusion period. There was a reduction in CMV IgG (AU/ml) in the posttransfusion level in the cohort group, but its significance could not be ascertained. The avidity results in both the cohort (7) and control (10) groups of newborns was suggestive of recent infection or infection in utero .[ 11 , 19 ]
However, it is well recognized that leukoreduced blood not only limits CMV transmission, transmission of a number of other viruses can be prevented including Human T-lymphotropic virus (HTLV). Allergic and febrile reactions are also significantly reduced. The risks of number of other complications, specific to premature newborns such as bronchopulmonary dysplasia and retinopathy, are also reduced. Studies have shown that there is a reduction of neonatal intensive care unit stay by 11 days.[ 26 ]
Some of the limitations of the present study were the low number of CMV IgM and IgG avidity testing of the participants due to resource constraints/technicalities. CMV IgG titrations also could not be done in these subjects to quantify the IgG level.
CONCLUSION
The present study showed that there is a high prevalence of CMV infections in blood donors and the newborn. A low CMV IgG avidity value showed their high infectivity potential. The leukoreduced blood transfusion might be effective in prevention of other serious adverse outcomes except CMV infection.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
Acknowledgment
We acknowledge all the support from the postgraduate trainees, faculties, doctors, and laboratory technicians (transfusion-transmitted infection testing laboratory) in the Department of Immunohematology and Blood Transfusion, Medical College, Kolkata for their support.
Abbott Healthcare Private Limited and Mrs. Sonu Bhatnagar (Medical Director Abbott, Asia Pacific) had supported the CMV serology testing kits and reagents. Mr. Anupam Maity and Mr. Ranjit Bhowmick from Abbott Healthcare Private Limited also gave the technical and logistics support related to the CMV testing facility.
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