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1.  Comparison of Cytomegalovirus (CMV) Enzyme-Linked Immunosorbent Spot and CMV Quantiferon Gamma Interferon-Releasing Assays in Assessing Risk of CMV Infection in Kidney Transplant Recipients 
Journal of Clinical Microbiology  2013;51(8):2501-2507.
Assessing cytomegalovirus (CMV)-specific cell-mediated immunity (CMI) represents an appealing strategy for identifying transplant recipients at risk of infection. In this study, we compared two gamma interferon-releasing assays (IGRAs), Quantiferon-CMV and CMV enzyme-linked immunosorbent spot (ELISPOT), to determine the ability of each test to predict protective CMV-specific T-cell responses. Two hundred twenty-one Quantiferon-CMV and ELISPOT tests were conducted on 120 adult kidney transplant recipients (KTRs), including 100 CMV-seropositive transplant recipients (R+) and 20 CMV-seronegative transplant recipients of a CMV-positive donor (D+/R−). As a control cohort, 39 healthy adult subjects (including 33 CMV-seropositive and 6 CMV-seronegative subjects) were enrolled. CMV IgG serology was used as a reference for both tests. In the CMV-seropositive individuals, the ELISPOT and Quantiferon-CMV assays provided 46% concordance with the serology, 12% discordance, 18% disagreement between ELISPOT or Quantiferon-CMV and the serology, and 24% gray areas when one or both tests resulted in weak positives. None of the CMV-seronegative subjects showed detectable responses in the ELISPOT or the Quantiferon-CMV test. In transplant recipients, both the ELISPOT and Quantiferon-CMV assays positively correlated with each other and negatively correlated with CMV DNAemia in a significant way (P < 0.05). During the antiviral prophylaxis, all 20 D+/R− KTRs we examined displayed undetectable Quantiferon-CMV and ELISPOT results, and there was no evidence of CMV seroconversion. The receiving operator curve (ROC) statistical analysis revealed similar specificities and sensitivities in predicting detectable viremia (areas under the curve [AUC], 0.66 and 0.62 for Quantiferon-CMV and ELISPOT, respectively). ELISPOT and Quantiferon-CMV values of >150 spots/200,000 peripheral blood mononuclear cells (PBMCs) and >1 to 6 IU gamma interferon (IFN-γ) were associated with protection from CMV infection (odds ratios [OR], 5 and 8.75, respectively). In transplant recipients, the two tests displayed similar abilities for predicting CMV infection. Both the ELISPOT and Quantiferon-CMV assays require several ameliorations to avoid false-negative results.
doi:10.1128/JCM.00563-13
PMCID: PMC3719636  PMID: 23678073
2.  Acquisition of cytomegalovirus infection: an update. 
Clinical Microbiology Reviews  1989;2(2):204-216.
Human cytomegalovirus (CMV) is a ubiquitous deoxyribonucleic acid virus that commonly infects a majority of individuals at some time during their life. Although most of these CMV infections are asymptomatic, certain patient groups are at risk to develop serious illness. Understanding the epidemiology of this virus is a key element in the development of strategies for preventing CMV disease. However, a number of features of this virus complicate such understanding. Following infection, CMV can remain latent, with subsequent reactivation; the factors controlling latency and reactivation and those factors which determine whether a CMV infection will be symptomatic are unknown. CMV disease can be acquired by natural routes, including horizontal and vertical transmission. Due to the ubiquity of CMV, the delineation of CMV transmission by these natural routes is complicated by the myriad of possible sources. Moreover, concerns over the risk of CMV transmission to the seronegative pregnant female have been raised in relation to preventing CMV transmission. By using molecular biologic techniques, much knowledge has been gained regarding the transmission of CMV disease by natural routes; however, a number of questions remain unanswered. The transmission of CMV infection by natural routes is therefore reviewed and the issues are highlighted. Primary infection, reactivation, and reinfection are the types of active CMV infections that can occur in an immunocompromised patient. In addition to natural routes of infection, introduction of presumably latently infected organs and requirements for multiple blood transfusions increase potential exposure to CMV in the immunocompromised patient. Understanding the epidemiology of CMV infections in the immunocompromised patient is difficult and in some instances controversial due to the complexity and interdependency of a number of factors which lead to CMV infection. In an immunocompromised individual, a major risk factor in developing overt CMV-related disease is associated with the serological status of an organ donor, the recipient, and the blood product given to these patients. In addition, a large body of inferential data supports the transmission of CMV by blood products or organs from seropositive donors; however, the mechanisms by which transmission occurs remain unclear. The possible sources and mechanisms of transmission of CMV infections in the immunocompromised host are reviewed. Lastly, strategies for the ultimate prevention of CMV disease are discussed in light of the epidemiology of CMV infections. To date, these strategies have included use of CMV-seronegative blood products or organs, antiviral agents, and vaccines.(ABSTRACT TRUNCATED AT 400 WORDS)
PMCID: PMC358111  PMID: 2539899
3.  Cytomegalovirus pneumonitis and bone marrow transplantation: identification of a specific high risk group. 
Journal of Clinical Pathology  1993;46(5):415-419.
AIMS--To study the association between cytomegalovirus (CMV) excretion and interstitial pneumonitis in allogeneic bone marrow transplant (BMT) recipients, with reference to donor and recipient CMV antibody response. METHODS--The incidence of CMV excretion was prospectively studied in 62 allogeneic bone marrow transplantations performed on adults and children. All recipients received CMV seronegative blood products. Prophylaxis with high dose acyclovir and CMV immune globulin was given to high risk patients (donor or recipient, or both, CMV seropositive). RESULTS--CMV excretion was detected in eight of 26 (31%) high risk patients but in only one of 36 low risk patients (donor and recipient both CMV seronegative). Five of the eight (63%) excretors in the high risk category developed CMV, of whom four (80%) belonged to the seropositive recipient/seronegative donor group, and included the three CMV seropositive recipients whose CMV complement fixation antibody titres were 64 or greater before transplantation. CONCLUSIONS--These findings suggest that there is a subgroup of patients at especially high risk of developing CMV.
PMCID: PMC501249  PMID: 8391547
4.  Effects of oral valganciclovir prophylaxis for cytomegalovirus infection in heart transplant patients 
Background
Cytomegalovirus (CMV) infection is a serious complication following heart transplantation. This study (June 2003–January 2010) retrospectively assessed the effects of oral valganciclovir prophylaxis in adult heart transplant recipients during the first year after transplantation.
Methods
In patients with normal renal function, 900 mg of oral valganciclovir was administered twice daily for 14 days after heart transplant followed by 900 mg per day for following 6 months. In the event of renal insufficiency, valganciclovir was adjusted according to the manufacturer’s recommendations. Antigenemia testing for pp65 antigen and simultaneous polymerase chain reaction (PCR) were used to document exposure to CMV. From 2003 to 2010, 146 patients (74.0% men) of mean age 50.7 ± 10.3 years at the time of heart transplant were included.
Results
A total of 16 patients (11.0% of total, 75.0% male) had a positive pp65 and PCR result (ie, CMV infection) during the year following heart transplant; three of these patients had discontinued valganciclovir prophylaxis within the first 6 months following transplant because of leukopenia, including one patient developed CMV colitis. Two further patients developed CMV pneumonia during prophylactic valganciclovir therapy. Eight patients had positive pp65 and PCR tests in the 6–12 months after heart transplant following cessation of routine prophylaxis. One of these patients developed CMV pneumonia and another developed CMV colitis and CMV pneumonia. Thirty-seven of the 146 (25.3%) patients were CMV donor-seropositive/recipient-seronegative, and seven (18.9% of this subgroup) had a positive CMV test. In patients who were CMV donor-seropositive/recipient-seronegative, the risk of a positive CMV test (ie, CMV infection) was significantly elevated (P = 0.023).
Conclusion
CMV prophylaxis with oral valganciclovir for 6 months following heart transplant is clinically feasible. In line with previous studies, CMV donor-seropositive/recipient-seronegative patients have a significantly elevated risk of CMV infection. In patients who prematurely discontinue valganciclovir, close monitoring of CMV antigenemia appears warranted. No significantly elevated rate of CMV infection was observed after 6 months of valganciclovir prophylaxis.
doi:10.2147/DDDT.S36578
PMCID: PMC3472651  PMID: 23091373
cytomegalovirus infection; heart transplantation; valganciclovir prophylaxis
5.  Preformed Frequencies of Cytomegalovirus (CMV)–Specific Memory T and B Cells Identify Protected CMV-Sensitized Individuals Among Seronegative Kidney Transplant Recipients 
Monitoring preformed cytomegalovirus (CMV)–specific T- and B-cell memory immune responses, using novel functional immune assays, may allow accurate characterization of antiviral immune sensitization in kidney transplant recipients, regardless of CMV-specific serological profile, illustrating the risk for posttransplant CMV infection.
Background. Cytomegalovirus (CMV) infection remains a major complication after kidney transplantation. Baseline CMV risk is typically determined by the serological presence of preformed CMV-specific immunoglobulin (Ig) G antibodies, even though T-cell responses to major viral antigens are crucial when controlling viral replication. Some IgG-seronegative patients who receive an IgG-seropositive allograft do not develop CMV infection despite not receiving prophylaxis. We hypothesized that a more precise evaluation of pretransplant CMV-specific immune-sensitization using the B and T-cell enzyme-linked immunospot assays may identify CMV-sensitized individuals more accurately, regardless of serological evidence of CMV-specific IgG titers.
Methods. We compared the presence of preformed CMV-specific memory B and T cells in kidney transplant recipients between 43 CMV IgG–seronegative (sR−) and 86 CMV IgG–seropositive (sR+) patients. Clinical outcome was evaluated in both groups.
Results. All sR+ patients showed a wide range of CMV-specific memory T- and B-cell responses. High memory T- and B-cell frequencies were also clearly detected in 30% of sR− patients, and those with high CMV-specific T-cell frequencies had a significantly lower incidence of late CMV infection after prophylactic therapy. Receiver operating characteristic curve analysis for predicting CMV viremia and disease showed a high area under the receiver operating characteristic curve (>0.8), which translated into a high sensitivity and negative predictive value of the test.
Conclusions. Assessment of CMV-specific memory T- and B-cell responses before kidney transplantation among sR− recipients may help identify immunized individuals more precisely, being ultimately at lower risk for CMV infection.
doi:10.1093/cid/ciu589
PMCID: PMC4650765  PMID: 25048845
kidney transplantation; CMV infection; T- and B-cell ELISPOT assay; adaptive immunity
6.  Systematic Evaluation of Different Nucleic Acid Amplification Assays for Cytomegalovirus Detection: Feasibility of Blood Donor Screening 
Journal of Clinical Microbiology  2015;53(10):3219-3225.
Acute primary cytomegalovirus (CMV) infections, which commonly occur asymptomatically among blood donors, represent a significant risk for serious morbidity in immunocompromised patients (a major group of transfusion recipients). We implemented a routine CMV pool screening procedure for plasma for the identification of CMV DNA-positive donors, and we evaluated the sensitivities and performance of different CMV DNA amplification systems. Minipools (MPs) of samples from 18,405 individual donors (54,451 donations) were screened for CMV DNA using the RealStar CMV PCR assay (Altona Diagnostic Technologies), with a minimum detection limit of 11.14 IU/ml. DNA was extracted with a high-volume protocol (4.8 ml, Chemagic Viral 5K kit; PerkinElmer) for blood donor pool screening (MP-nucleic acid testing [NAT]) and with the Nuclisens easyMAG system (0.5 ml; bioMérieux) for individual donation (ID)-NAT. In total, six CMV DNA-positive donors (0.03%) were identified by routine CMV screening, with DNA concentrations ranging from 4.35 × 102 to 4.30 × 103 IU/ml. Five donors already showed seroconversion and detectable IgA, IgM, and/or IgG antibody titers (IgA+/IgM+/IgG− or IgA+/IgM+/IgG+), and one donor showed no CMV-specific antibodies. Comparison of three commercial assays, i.e., the RealStar CMV PCR kit, the Sentosa SA CMV quantitative PCR kit (Vela Diagnostics), and the CMV R-gene PCR kit (bioMérieux), for MP-NAT and ID-NAT showed comparably good analytical sensitivities, ranging from 10.23 to 11.14 IU/ml (MP-NAT) or from 37.66 to 57.94 IU/ml (ID-NAT). The clinical relevance of transfusion-associated CMV infections requires further investigation, and the evaluated methods present powerful basic tools providing sensitive possibilities for viral testing. The application of CMV MP-NAT facilitated the identification of one donor with a window-phase donation during acute primary CMV infection.
doi:10.1128/JCM.01091-15
PMCID: PMC4572558  PMID: 26202109
7.  Prevention of Primary Cytomegalovirus Infection in Pregnancy☆ 
EBioMedicine  2015;2(9):1205-1210.
Background
Cytomegalovirus (CMV) is the leading infectious agent causing congenital sensorineural hearing loss and psychomotor retardation. CMV vaccine is currently unavailable and treatment options in pregnancy are limited. Susceptible pregnant women caring for children are at high risk for primary infection. CMV educational and hygienic measures have the potential to prevent primary maternal infection.
Methods
A mixed interventional and observational controlled study was conducted to investigate the effectiveness of hygiene information among pregnant women at risk for primary CMV infection for personal/occupational reasons. In the intervention arm, CMV-seronegative women, identified at the time of maternal serum screening for fetal aneuploidy at 11–12 weeks of gestation, were given hygiene information and prospectively tested for CMV until delivery. The comparison arm consisted of women enrolled at delivery who were neither tested for nor informed about CMV during pregnancy, and who had a serum sample stored at the screening for fetal aneuploidy. By design, groups were homogeneous for age, parity, education, and exposure to at least one risk factor. The primary outcome was CMV seroconversion. Acceptance of hygiene recommendations was a secondary objective and was measured by a self-report.
Findings
Four out of 331 (1.2%) women seroconverted in the intervention group compared to 24/315 (7.6%) in the comparison group (delta = 6.4%; 95% CI 3.2–9.6; P < 0.001). There were 3 newborns with congenital infection in the intervention group and 8 in the comparison group (1 with cerebral ultrasound abnormalities at birth). Ninety-three percent of women felt hygiene recommendations were worth suggesting to all pregnant women at risk for infection.
Interpretation
This controlled study provides evidence that an intervention based on the identification and hygiene counseling of CMV-seronegative pregnant women significantly prevents maternal infection. While waiting for CMV vaccine to become available, the intervention described may represent a responsible and acceptable primary prevention strategy to reduce congenital CMV.
Highlights
•The rate of CMV seroconversion was 4/331 (1.2%) in the intervention group vs 24/315 (7.6%) in the comparison group•This study shows that hygiene information of CMV-seronegative pregnant women significantly prevents maternal infection.•93% participants in the study found hygiene recommendations worth suggesting to all pregnant women.
Cytomegalovirus is the leading infectious agent causing congenital sensorineural hearing loss and psychomotor retardation. Contacts with young children have been identified as the main source of virus transmission to mothers. While waiting for a CMV vaccine to become available, this study documents that an intervention based on the identification and hygiene counseling of pregnant women susceptible to be infected by CMV for the first time during pregnancy, significantly prevents maternal infection.
doi:10.1016/j.ebiom.2015.08.003
PMCID: PMC4588434  PMID: 26501119
Cytomegalovirus; Primary infection; Pregnancy; Prevention; Hygiene measures
8.  Cytomegalovirus inclusions in the gastroduodenal mucosa of patients after renal transplantation. 
Gut  1981;22(9):698-701.
Biopsies collected from gastroduodenal mucosa during endoscopic examination of 20 patients having undergone renal transplantation and subsequent immunosuppressive therapy showed cytomegalovirus (CMV) inclusion bodies in nine cases. CMV antibody titres were tested in all patients before and after the transplant procedure. Not all patients exhibited viraemia-related symptoms at the time of endoscopy. No correlation was found between the presence of CMV-type cells within the gastroduodenal mucosa, endoscopic and histological findings, the duration of the transplant, and the dosage of immunosuppressive drugs. The duodenum seems to be the elective site of CMV. The involvement of gastric mucosa seems to represents a worsening of the illness. Eight of nine patients with positive biopsies for CMV inclusion had negative pretransplant antibody titres to CMV. All nine patients were seropositive after transplantation and showed seroconversion. Five of 11 recipients with negative biopsies for CMV inclusion bodies, were seronegative before transplantation. Seroconversion occurred in five patients after the transplant; the other six had no rise in antibody titres. The lack of pre-transplant CMV antibody titre and its subsequent increase after transplantation identifies a greater risk of developing post-transplant CMV infection.
Images
PMCID: PMC1419872  PMID: 6271652
9.  Differences in immune responses between CMV-seronegative and -seropositive patients with myocardial ischemia and reperfusion 
CMV infection is responsible for acceleration of immune senescence and linked to systemic pathologies, including cardiovascular diseases. In this study, we investigated differences in the immune response between CMV-seropositive and seronegative patients undergoing primary percutaneous coronary intervention (PPCI) for acute myocardial infarction (MI). Peripheral blood samples were taken at six different time points: pre-, 15, 30, 90 min, 24 h after PPCI and at 3 months after MI. Absolute counts of lymphocyte subpopulations, immune response to specific and nonspecific stimulation, serum cytokines and levels of CMV-IgG, cardiolipin-IgG, and anti-endothelial cell antibodies were assessed. CMV-seropositive patients with MI showed a twofold higher IFN-γ production to PHA-stimulation, up to 2.5-fold higher levels of IP-10 in serum and up to 30% lower serum levels of IL-16 compared to CMV-seronegative individuals. CMV-seropositive patients could be divided into two subgroups with high (IL-10Hi) and low (IL-10Lo) IL-10 serum levels during the acute stage of MI. The IL-10Hi CMV-seropositive subgroup showed an increased exit of late-differentiated T lymphocytes, NK and NKT-like cells from the circulation, which may potentially enhance cytotoxic damage in the ischemic myocardium. Finally, we did not observe an acceleration of autoimmunity by MI in CMV-seropositive individuals. The immune response during acute MI showed characteristic differences between CMV seronegative and seropositive patients, with a stronger pro-inflammatory response in seropositive patients. The effects of IP-10, IL-16, and IL-10 on characteristics of acute immune responses and formation of different immune profiles in CMV-seropositive individuals require further investigation.
doi:10.1002/iid3.49
PMCID: PMC4444149  PMID: 26029366
CMV infection; cytokines; myocardial infarction; reperfusion injury; T cells
10.  Priorities for CMV vaccine development 
Vaccine  2013;32(1):4-10.
A multidisciplinary meeting addressed priorities related to development of vaccines against cytomegalovirus (CMV), the cause of congenital CMV (cCMV) disease and of serious disease in the immunocompromised. Participants discussed optimal uses of a CMV vaccine, aspects of clinical study design, and the value of additional research. A universal childhood CMV vaccine could potentially rapidly reduce cCMV disease, as infected children are sources of viral transmission to seronegative and seropositive mothers. A vaccine administered to adolescents or adult women could also reduce cCMV disease by making them immune prior to pregnancy. Clinical trials of CMV vaccines in women should evaluate protection against cCMV infection, an essential precursor of cCMV disease, which is a more practical and acceptable endpoint for assessing vaccine effects on maternal-fetal transmission. Clinical trials of vaccines to evaluate prevention of CMV disease in stem cell transplant recipients could use CMV viremia at a level triggering preemptive antiviral therapy as an endpoint, because widespread use of preemptive and prophylactic antivirals has rendered CMV-induced disease too rare to be a practical endpoint for clinical trials. In solid organ transplant patients, CMV-associated disease is sufficiently common for use as a primary endpoint. Additional research to advance CMV vaccine development should include identifying factors that predict fetal loss due to CMV, determining age-specific incidence and transmission rates, defining the mechanism and relative contributions of maternal reactivation and re-infection to cCMV disease, developing assays that can distinguish between reactivation and re-infection in seropositive vaccinees, further defining predictors of sequelae from cCMV infection, and identifying clinically relevant immune response parameters to CMV (including developing validated assays that could assess CMV antibody avidity) that could lead to the establishment of immune correlates of protection.
doi:10.1016/j.vaccine.2013.09.042
PMCID: PMC4623576  PMID: 24129123
cytomegalovirus; congenital CMV; viruses; clinical trial endpoints; meeting report
11.  Blood Transfusion and Breast Milk Transmission of Cytomegalovirus in Very Low Birth Weight Infants 
JAMA pediatrics  2014;168(11):1054-1062.
Importance
Postnatal cytomegalovirus (CMV) infection can cause serious morbidity and mortality in very low birth weight (VLBW) infants. The primary sources of postnatal CMV infection in this population are breast milk and blood transfusion. The current risks attributable to these vectors, and the efficacy of approaches to prevent CMV transmission, are poorly characterized.
Objectives
To estimate the risk of postnatal CMV transmission from 2 sources: 1) transfusion of CMV-seronegative and leukoreduced blood and 2) maternal breast milk.
Design
Prospective, multicenter birth-cohort study conducted from January 2010 to June 2013. CMV serologic testing of enrolled mothers was performed to determine their status. CMV nucleic acid testing (NAT) of transfused blood components and breast milk was performed to identify sources of CMV transmission. Enrolled VLBW infants underwent serum and urine CMV NAT testing at birth, to evaluate congenital infection, and surveillance CMV NAT testing at 5 additional intervals between birth and 90 days, discharge or death.
Setting
Three neonatal intensive care units (2 academically-affiliated and 1 private) in Atlanta, Georgia.
Participants
539 VLBW infants (birth weight ≤1500 grams) who had not received a blood transfusion were enrolled, with their mothers, within 5 days of birth.
Exposure
Blood transfusion and breast milk feeding
Main Outcomes and Measures
Cumulative incidence of postnatal CMV infection, detected by serum or urine NAT.
Results
CMV positive sero-prevalence among enrolled mothers was 76% (352/462). Among 539 enrolled VLBW infants, the cumulative incidence of postnatal CMV infection at 12 weeks was 6.9% (95% CI: 4.2%–9.2%); five infants with postnatal CMV infection developed symptomatic disease or died. Although 58% (310/539) of infants received 2061 transfusions, none of the CMV infections were linked to transfusion, resulting in a CMV infection incidence of 0.0% (95%CI: 0.0%–0.3%) per unit of CMV-seronegative and leukoreduced blood. Twenty-seven of 28 postnatal infections occurred among infants fed CMV-positive breast milk (12-week incidence: 15.3%; 95%CI: 9.3%–20.2%).
Conclusions and Relevance
Transfusion of CMV-seronegative and leukoreduced blood products effectively prevents transmission of CMV to VLBW infants. Among infants managed with this transfusion approach, maternal breast milk is the primary source of postnatal CMV infection.
Trial Registration
clinicaltrials.gov Identifier: NCT00907686
doi:10.1001/jamapediatrics.2014.1360
PMCID: PMC4392178  PMID: 25243446
12.  Seroprevalence of Cytomegalovirus among Voluntary Blood Donors 
Background
Primary cytomegalovirus (CMV) infection in immunocompetent host is self limiting infection, leading to latency of virus. However congenital CMV and CMV infections in immunocompromised patients are associated with high morbidity and mortality. Transfusion transmitted-cytomegalovirus (TT-CMV) infection in low birth weight neonate and immunocompromised transfusion recipients is being increasingly reported. Studies recommended transfusion of CMV free or CMV safe blood in prevention of TT-CMV. In this background, the study was undertaken to assess the CMV seroprevalence in blood donor.
Methods
A prospective study was conducted in which 431 voluntary blood donors were screened for CMV IgG and IgM by EIA (Enzyme Immuno Assay).
Result
A total of 379 (87.9 %) voluntary blood donors were seropositive for CMV IgG. There was no statistical difference of CMV seropositivity and age. Further, seven (1.6%) subjects were both CMV IgM and IgG seropositive.
Conclusion
High seroprevalence of CMV in our donor population is a threat to the blood safety. Strategies in reducing the risk of TT- CMV are discussed. Use of prestorage leucodepleted ‘CMV safe’ blood components along with judicious use of blood is recommended in prevention of TT-CMV in high risk recipients.
doi:10.1016/S0377-1237(09)80016-6
PMCID: PMC4921396  PMID: 27408259
Cytomegalovirus; Transfusion transmitted infections; Blood donors; Leucodepletion
13.  Prevention of Transfusion-Transmitted Cytomegalovirus in Low–Birth Weight Infants (≤1500 g) Using Cytomegalovirus-Seronegative and Leukoreduced Transfusions 
Transfusion medicine reviews  2011;25(2):125-132.
The transfusion-transmitted cytomegalovirus (TT-CMV) can cause serious morbidity and mortality in low–birth weight infants (LBWIs). Transfusion-transmitted cytomegalovirus can be minimized in LBWIs born to cytomegalovirus (CMV)–seronegative mothers with the use of CMV-seronegative blood components. Despite evidence that has independently shown that either leukoreduction or the use of CMV-seronegative components mitigates TT-CMV, the potential efficacy of combining these 2 strategies has not been substantiated in very LBWIs (<1500 g) born to either CMV-seronegative or CMV-seropositive mothers. Nonetheless, the serious risks of CMV infection posed by allogeneic transfusions and the broad implementation of universal leukoreduction have made this combination strategy the de facto clinical standard for transfusion of LBWIs. Although preferred, this combined approach has not been validated in clinical trials and, thus, warrants a large prospective study to determine whether this is the optimal transfusion tactic or if additional safety measures are necessary to prevent TT-CMV in LBWIs born to both CMV- seronegative and CMV-seropositive mothers. The aim of this prospective birth cohort study, therefore, is to estimate the incidence of TT-CMV in 1300 LBWIs (≤1500 g) who receive CMV-seronegative plus leukoreduced blood products to evaluate the effectiveness of this coupled strategy. Conducted in Atlanta, GA, this study has been registered at the US National Institutes of Health (ClinicalTrials.gov no. NCT00907686).
doi:10.1016/j.tmrv.2010.11.004
PMCID: PMC4793905  PMID: 21345642
14.  Limited Dissemination and Shedding of the UL128 Complex-Intact, UL/b′-Defective Rhesus Cytomegalovirus Strain 180.92 
Journal of Virology  2014;88(16):9310-9320.
ABSTRACT
The UL128 complex of human cytomegalovirus (CMV) is a major determinant of viral entry into epithelial and endothelial cells and a target for vaccine development. The UL/b′ region of rhesus CMV contains several open reading frames, including orthologs of the UL128 complex. We recently showed that the coding content of the rhesus CMV (RhCMV) UL/b′ region predicts acute endothelial tropism and long-term shedding in vivo in the rhesus macaque model of CMV infection. The laboratory-passaged RhCMV 180.92 strain has a truncated UL/b′ region but an intact UL128 complex. To investigate whether the presence of the UL128 complex alone was sufficient to confer endothelial and epithelial tropism in vivo, we investigated tissue dissemination and viral excretion following experimental RhCMV 180.92 inoculation of RhCMV-seronegative rhesus macaques. We show the presence of at least two virus variants in the RhCMV 180.92 infectious virus stock. A rare variant noted for a nontruncated wild-type-virus-like UL/b′ region, rapidly emerged during in vivo replication and showed high-level replication in blood and tissues and excretion in urine and saliva, features similar to those previously reported in naturally occurring wild-type RhCMV infection. In contrast, the predominant truncated version of RhCMV 180.92 showed significantly lower plasma DNAemia and limited tissue dissemination and viral shedding. These data demonstrate that the truncated RhCMV 180.92 variant is attenuated in vivo and suggest that additional UL/b′ genes, besides the UL128 complex, are required for optimal in vivo CMV replication and dissemination.
IMPORTANCE An effective vaccine against human CMV infection will need to target genes that are essential for virus propagation and transmission. The human CMV UL128 complex represents one such candidate antigen since it is essential for endothelial and epithelial cell tropism, and is a target for neutralizing antibodies in CMV-infected individuals. In this study, we used the rhesus macaque animal model of CMV infection to investigate the in vivo function of the UL128 complex. Using experimental infection of rhesus macaques with a rhesus CMV virus variant that contained an intact UL128 complex but was missing several other genes, we show that the presence of the UL128 complex alone is not sufficient for widespread tissue dissemination and virus excretion. These data highlight the importance of in vivo studies in evaluating human CMV gene function and suggest that additional UL/b′ genes are required for optimal CMV dissemination and transmission.
doi:10.1128/JVI.00162-14
PMCID: PMC4136270  PMID: 24899204
15.  A Prospective Monitoring Study of Cytomegalovirus Infection in Non-Immunosuppressed Critical Heart Surgery Patients 
PLoS ONE  2015;10(6):e0129447.
Background
Reactivation of cytomegalovirus (CMV) has been reported occasionally in immnunocompetent patients in the intensive care unit (ICU). The epidemiology and association of CMV infection with adverse outcome is not well defined in this population. Patients undergoing major heart surgery (MHS) are at a particularly high risk of infection. CMV infection has not been systematically monitored in MSH-ICU patients.
Methods
We assessed CMV plasma viremia weekly using a quantitative polymerase chain reaction assay in a prospective cohort of immunocompetent adults admitted to the MHS-ICU for at least 72 hours between October 2012 and May 2013. Risk factors for CMV infection and its potential association with continued hospitalization or death by day 30 (composited endpoint) were assessed using univariate and multivariate logistic regression analyses.
Results
CMV viremia at any level was recorded in 16.5% of patients at a median of 17 days (range, 3-54 days) after admission to the MHS-ICU. Diabetes (adjusted OR, 5.6; 95% CI, 1.8-17.4; p=0.003) and transfusion requirement (>10 units) (adjusted OR, 13.7; 95% CI, 3.9-47.8; p<0.001) were independent risk factors associated with CMV reactivation. Reactivation of CMV at any level was independently associated with the composite endpoint (adjusted OR, 12.1; 95% CI, 2.3-64; p=0.003).
Conclusion
Reactivation of CMV is relatively frequent in immunocompetent patients undergoing MHS and is associated with prolonged hospitalization or death.
doi:10.1371/journal.pone.0129447
PMCID: PMC4466502  PMID: 26070136
16.  Cytomegalovirus Infection Causes an Increase of Arterial Blood Pressure 
PLoS Pathogens  2009;5(5):e1000427.
Cytomegalovirus (CMV) infection is a common infection in adults (seropositive 60–99% globally), and is associated with cardiovascular diseases, in line with risk factors such as hypertension and atherosclerosis. Several viral infections are linked to hypertension, including human herpes virus 8 (HHV-8) and HIV-1. The mechanisms of how viral infection contributes to hypertension or increased blood pressure are not defined. In this report, the role of CMV infection as a cause of increased blood pressure and in forming aortic atherosclerotic plaques is examined. Using in vivo mouse model and in vitro molecular biology analyses, we find that CMV infection alone caused a significant increase in arterial blood pressure (ABp) (p<0.01∼0.05), measured by microtip catheter technique. This increase in blood pressure by mouse CMV (MCMV) was independent of atherosclerotic plaque formation in the aorta, defined by histological analyses. MCMV DNA was detected in blood vessel samples of viral infected mice but not in the control mice by nested PCR assay. MCMV significantly increased expression of pro-inflammatory cytokines IL-6, TNF-α, and MCP-1 in mouse serum by enzyme-linked immunosorbent assay (ELISA). Using quantitative real time reverse transcriptase PCR (Q-RT-PCR) and Western blot, we find that CMV stimulated expression of renin in mouse and human cells in an infectious dose-dependent manner. Co-staining and immunofluorescent microscopy analyses showed that MCMV infection stimulated renin expression at a single cell level. Further examination of angiotensin-II (Ang II) in mouse serum and arterial tissues with ELISA showed an increased expression of Ang II by MCMV infection. Consistent with the findings of the mouse trial, human CMV (HCMV) infection of blood vessel endothelial cells (EC) induced renin expression in a non-lytic infection manner. Viral replication kinetics and plaque formation assay showed that an active, CMV persistent infection in EC and expression of viral genes might underpin the molecular mechanism. These results show that CMV infection is a risk factor for increased arterial blood pressure, and is a co-factor in aortic atherosclerosis. Viral persistent infection of EC may underlie the mechanism. Control of CMV infection can be developed to restrict hypertension and atherosclerosis in the cardiovascular system.
Author Summary
Cytomegalovirus (CMV) infection is associated with cardiovascular diseases. The exact mechanisms, however, remain to be defined. Using both mouse model and cell culture analyses, we find that CMV infection alone causes an increase in blood pressure. Additionally, CMV infection augments the increased blood pressure induced by a high cholesterol diet. CMV infection alone, however, does not cause atherosclerosis in aortas. CMV infection along with a high cholesterol diet, however, causes the classic atherosclerotic plaque formation in the main artery connected to the heart. Further studies show that CMV infection induces renin and angiotensin II (Ang II) expression in blood and in vessel cells, in a persistent infection manner. An increased expression of renin and Ang II has been known to cause an increase in blood pressure or hypertension in humans. Expression of viral genes and viral persistent infection of blood vessel endothelial cells resulting in an increased expression of inflammatory cytokines, including renin and Ang II, may underpin the molecular mechanism by which CMV infection induces an increase in blood pressure.
doi:10.1371/journal.ppat.1000427
PMCID: PMC2673691  PMID: 19436702
17.  Seroprevalence of Cytomegalovirus Among Some Voluntary Blood Donors at the 37 Military Hospital, Accra, Ghana 
Ghana Medical Journal  2006;40(3):99-104.
Summary
Background
Cytomegalovirus (CMV) is one of the most significant pathogens infecting immunosuppressed individuals. CMV is transmissible through transfusion of blood components.
Aim
The goal of this study was to determine the seroprevalence of antibodies to CMV among blood donors seen at the 37 Military Hospital Blood Transfusion Unit, (MHBTU) Accra, Ghana.
Methods
The seroprevalence of antibodies specific for CMV was tested using CMV IgG/IgM particle agglutination test kit and ELISA.
Results
Of the 264 blood donors, 18 were negative and 246 were positive for CMV IgG antibodies, giving an overall CMV prevalence rate of 93.2%. None of the 264 blood donors was positive for CMV IgM antibodies. About 96% of the donors aged between 30 to 39 years were seropositive for CMV, as against 91.9% in those aged 20–29 years, 88.6% in 40 to 49 years, 75.0% (3 out of 4) in 50 to 59 years, and 100% (1 out 1) in 60–69 years. There was no statistically significant difference (P>0.05) in the CMV IgG status in different age groups. The blood donors comprised largely of male donors (236 out of 264), making sex comparisons statistically undesirable. However, all the female (n=28) donors were positive for CMV IgG.
Conclusion
Since about 93% of blood donors at the MHBTU are seropositive for CMV, it would be very useful to screen blood donors in Ghana for CMV to identify the very few CMV-seronegative blood donors, and maintain an inventory of them for use as donors.
PMCID: PMC1790848  PMID: 17299575
Cytomegalovirus; blood donors; seroprevalence
18.  Detection of human cytomegalovirus antigenaemia: a rapid diagnostic technique for predicting cytomegalovirus infection/pneumonitis in lung and heart transplant recipients. 
Thorax  1995;50(1):9-13.
BACKGROUND--New rapid diagnostic techniques offer the opportunity of early diagnosis of human cytomegalovirus (CMV) infection in immunocompromised patients at risk of developing CMV disease. The use of human CMV antigenaemia as a predictor of clinical CMV infection and disease in lung and heart transplant recipients was studied prospectively. METHODS--Twenty three heart and nine lung transplant recipients who survived 40 days were observed by standard CMV surveillance with serological testing, culture, and by sequential testing for CMV antigenaemia. CMV antigenaemia testing is a rapid and quantifiable technique in which a viral lower matrix protein is detected in cytospin preparations of peripheral blood polymorphonuclear leucocytes (PMNLs) by immunofluorescent staining. RESULTS--Eleven patients developed CMV infection and five developed CMV disease (four pneumonitis, one duodenitis). These clinical events occurred at a median of 65 days following transplantation. CMV antigenaemia occurred in 17 patients at a median of 35 days following transplantation. Detection of CMV antigenaemia had a sensitivity of 100%, a specificity of 93.7%, and a positive predictive value of 94.1% for CMV related illness. CMV antigenaemia was positive at a significant interval before the clinical event. High levels of CMV antigenaemia (> 50 CMV antigen positive cells/2 x 10(5) PMNLs) occurred in 11 patients and five of these developed disease. CMV antigenaemia of > 50 CMV antigen positive cells/2 x 10(5) PMNLs had a positive predictive value of 45.5% for disease but a negative predictive value of 100%. Patients with disease had higher levels of antigenaemia than those without disease. CONCLUSIONS--CMV antigenaemia is a rapid diagnostic technique which can identify patients likely to develop CMV disease, potentially allowing early treatment.
Images
PMCID: PMC473696  PMID: 7886659
19.  Management of cytomegalovirus antibody negative patients undergoing heart transplantation. 
Journal of Clinical Pathology  1990;43(5):373-376.
In a series of 61 consecutive patients undergoing heart, heart and lung, and lung transplantation, 24 patients were known to be cytomegalovirus (CMV) antibody negative on the day of transplantation. Enzyme linked immunosorbent assays (ELISA) for CMV IgG were performed on donor samples on the day of operation. In 16 of the 24 susceptible patients the test was negative and the only preventive measure taken was the use of blood and blood products from CMV-antibody negative blood donors. None of these patients acquired primary infection with CMV. In another six patients the donor serum was found to contain CMV specific IgG, and in these patients, including one heart and lung transplant recipient, prophylaxis with CMV specific hyperimmune globulin was given. All six patients developed CMV IgM antibodies and in five there was an associated but clinically mild illness. None of these patients required treatment. In the remaining two patients ELISA tests on the donor sera gave equivocal results and hyperimmune globulin was withheld. Both patients developed primary CMV infection of greater severity than those given hyperimmune globulin and one required treatment. Reference tests confirmed that the donor sera contained CMV antibodies. Primary CMV infection in susceptible patients after heart transplantation can be avoided by the use of screened blood and blood products where the organ donor is seronegative to CMV and it can be improved by the use of prophylactic hyperimmune globulin where the donor is CMV antibody positive.
PMCID: PMC502430  PMID: 2164531
20.  Detection of immunoglobulin M and G antibodies against cytomegalovirus early and late antigens by enzyme-linked immunosorbent assay. 
Journal of Clinical Microbiology  1984;20(4):763-771.
A sensitive and reproducible enzyme-linked immunosorbent assay (ELISA) is described for the detection of immunoglobulin M and antibodies with specifity for human cytomegalovirus (CMV) early (CMV-EA) and late (CMV-LA) antigens. The emphasis is on the production of high-quality CMV antigens, CMV-EA and CMV-LA separately, and conditions for their application in the ELISA. The induction of CMV-EA and -LA in infected cell extracts was studied in detail by using human sera with defined antibody specificity for CMV-EA and CMV-LA. This resulted in the development of a simple whole cell extraction procedure that provided a high yield of CMV antigens with reproducible antigen quality. The antigens were specific for the detection of anti-CMV antibodies. The influence of autoantibodies on the determination of CMV-specific antibodies was investigated. Parallel analysis of 322 human sera by indirect immunofluorescence and ELISA showed a high correlation between both assays (r = 0.9674 for CMV-EA and 0.9362 for CMV-LA). Antibody titers determined by ELISA were equal to (for CMV-EA) or slightly higher (for CMV-LA) that those determined by immunofluorescence but significantly higher (20- to 5,120-fold) than those determined by complement fixation. From 191 sera positive by ELISA (titer greater than or equal to 40) 4 (2.1%) were negative by immunofluorescence (titer less than 40), and from 61 ELISA-positive sera 12 (19.6%) were negative (titer less than 8) when tested by complement fixation. Consequently, ELISA for CMV may prove to be more reliable for the selection of CMV-seronegative blood donors than these other methods. The use of high-quality antigens allows more economic handling of large-scale serum determinations. Possibilities for further automation are discussed.
PMCID: PMC271427  PMID: 6208220
21.  Choice of induction regimens on the risk of cytomegalovirus infection in donor-positive and recipient-negative kidney transplant recipients 
Background
Late occurrence of cytomegalovirus (CMV) infection remains a concern in CMV-seronegative kidney and/or pancreas transplant recipients of CMV-seropositive organs (donor positive/recipient negative, D+/R −) despite the use of prophylaxis. We investigated the impact of various antibody induction regimens on CMV infection in this group of patients.
Methods
A total of 254 consecutive D+/R − kidney and/or pancreas transplant patients were studied. The induction agents rabbit anti-thymocyte globulin (rATG) or basiliximab were used according to the center practice. All patients received prophylaxis with valganciclovir (VGCV) for either 3 or 6 months. The occurrence of CMV infection was confirmed by positive DNA viremia. Multivariate Cox regression analyses were performed to determine risk factors for CMV infection.
Results
The cumulative incidence of CMV infection was 58, 112, and 59 cases per 1000 patient-years for patients who received no antibody induction, induction with rATG, or basiliximab induction, respectively (P=0.02). The use of rATG but not basiliximab was associated with an increased risk for CMV infection (adjusted hazard ratio [AHR] 2.13, 95% confidence interval [CI] 1.24–3.54, P=0.006). Acute rejection and its treatment with rATG were not associated with an increased risk for CMV infection when an additional course of VGCV was given following the treatment. Longer duration of prophylaxis was associated with a reduced risk for CMV infection (AHR 0.54, 95% CI 0.33–0.87, P=0.011).
Conclusions
Induction with rATG is associated with increased risk of CMV infection. Longer duration of prophylaxis is beneficial.
doi:10.1111/j.1399-3062.2010.00532.x
PMCID: PMC3831513  PMID: 20576019
cytomegalovirus; kidney transplant; pancreas transplant; donor/recipient mismatch; rabbit anti-thymocyte globulin; basiliximab; acute rejection
22.  Cytomegalovirus-specific T-cell responses and viral replication in kidney transplant recipients 
Background
Cytomegalovirus (CMV) seronegative recipients (R-) of kidney transplants (KT) from seropositive donors (D+) are at higher risk for CMV replication and ganciclovir(GCV)-resistance than CMV R(+). We hypothesized that low CMV-specific T-cell responses are associated with increased risk of CMV replication in R(+)-patients with D(+) or D(-) donors.
Methods
We prospectively evaluated 73 consecutive KT-patients [48 R(+), 25 D(+)R(-)] undergoing routine testing for CMV replication as part of a preemptive strategy. We compared CMV-specific interferon-γ (IFN-γ) responses of CD4+CD3+ lymphocytes in peripheral blood mononuclear cells (PBMC) using three different antigen preparation (CMV-lysate, pp72- and pp65-overlapping peptide pools) using intracellular cytokine staining and flow cytometry.
Results
Median CD4+ and CD8+T-cell responses to CMV-lysate, pp72- and pp65-overlapping peptide pools were lower in D(+)R(-) than in R(+)patients or in non-immunosuppressed donors. Comparing subpopulations we found that CMV-lysate favored CD4+- over CD8+-responses, whereas the reverse was observed for pp72, while pp65-CD4+- and -CD8+-responses were similar. Concurrent CMV replication in R(+)-patients was associated with significantly lower T-cell responses (pp65 median CD4+ 0.00% vs. 0.03%, p = 0.001; CD8+ 0.01% vs. 0.03%; p = 0.033). Receiver operated curve analysis associated CMV-pp65 CD4+ responses of > 0.03% in R(+)-patients with absence of concurrent (p = 0.003) and future CMV replication in the following 8 weeks (p = 0.036). GCV-resistant CMV replication occurred in 3 R(+)-patients (6.3%) with pp65- CD4+ frequencies < 0.03% (p = 0.041).
Conclusion
The data suggest that pp65-specific CD4+ T-cells might be useful to identify R(+)-patients at increased risk of CMV replication. Provided further corroborating evidence, CMV-pp65 CD4+ responses above 0.03% in PBMCs of KT patients under stable immunosuppression are associated with lower risk of concurrent and future CMV replication during the following 8 weeks.
doi:10.1186/1479-5876-6-29
PMCID: PMC2432058  PMID: 18541023
23.  Prevention of Transfusion-Transmitted Cytomegalovirus Infections: Which is the Optimal Strategy? 
Summary
Traditionally, leukoreduction and selection of blood products from seronegative donors have been used as alternative strategies to reduce the risk of transfusion-transmitted cytomegalovirus infections (TT-CMV) in atrisk patients. After the introduction of universal leukoreduction for red blood cell and platelet concentrates in Germany, a controversy evolved as to whether the additional selection of blood products from seronegative donors would reduce or even increase the risk of TT-CMV. This review summarizes the current knowledge about CMV infections in blood donors and the implications of this information on the effect of potential transfusion strategies. Even though there are conflicting data about the incidence of TT-CMV remaining after the introduction of leukodepletion, it has been clearly shown that both prevalence and concentration of CMV DNA in peripheral blood are highest in newly seropositive donors. Therefore, avoidance of blood products from these donors is the most important goal of any transfusion strategy. This goal can be reached by: i) selection of blood products from seronegative donors, ii) provision of CMV DNA-negative blood products, or iii) provision of blood from long-term seropositive donors. In cases of suspected TT-CMV, all implicated donors should be investigated carefully to gather further knowledge on which donors confer the lowest risk for TT-CMV.
doi:10.1159/000357102
PMCID: PMC3949610  PMID: 24659946
Human cytomegalovirus; CMV; Blood donor; Transfusion-associated infections; Transfusion
24.  Cytomegalovirus viral and antibody correlates in young children 
BMC Research Notes  2014;7:776.
Background
Young, healthy children shedding cytomegalovirus (CMV) in urine and saliva appear to be the leading source of CMV in primary infection of pregnant women.
Findings
We screened 48 children 6 months – 5 years old for CMV IgG and measured levels of CMV IgG, IgM and IgG avidity antibodies, frequency of CMV shedding, and viral loads in blood, urine, and saliva. Thirteen of the 48 children (27%) were CMV IgG positive, among whom 3 were also CMV IgM positive with evidence of recent primary infection. Nine of the 13 seropositive children (69%) were shedding 102-105 copies/ml of CMV DNA in one or more bodily fluid. Among seropositive children, low IgG antibody titer (1:20–1:80) was associated with the absence of shedding (p = 0.014), and enrollment in daycare was associated with the presence of CMV shedding (p = 0.037).
Conclusions
CMV antibody profiles correlated with CMV shedding. The presence of CMV IgM more often represents primary infection in children than in adults. Correlating antibodies with primary infection and viral shedding in healthy children adds to the understanding of CMV infection in children that can inform the prevention of CMV transmission to pregnant women.
doi:10.1186/1756-0500-7-776
PMCID: PMC4236479  PMID: 25367101
Cytomegalovirus; Viral load; Serology; Avidity
25.  Kinetics of specific immunoglobulins M, E, A, and G in congenital, primary, and secondary cytomegalovirus infection studied by antibody-capture enzyme-linked immunosorbent assay. 
Journal of Clinical Microbiology  1988;26(4):654-661.
Antibody-capture enzyme-linked immunosorbent assay (ELISA) using enzyme-labeled cytomegalovirus (CMV) nuclear antigen is a reliable and easily performed test suitable for routine use. As the serologic response to CMV infection may, however, vary considerably among patients, we have studied the kinetics of CMV-specific immunoglobulin M (IgM), IgE, IgA, and IgG antibodies in 352 sera from 61 patients by using antibody-capture ELISA and complement fixation (CF) tests. In a CMV mononucleosis group (n = 17), most patients had antibodies of all four immunoglobulin classes, but antibody levels decreased rapidly, with half the patients having a borderline-positive or a negative reaction for all classes, except IgG, 2 months after the appearance of symptoms. Twelve patients with a primary CMV infection after renal or bone marrow transplantation also developed all immunoglobulin-class antibodies. In only two patients did CMV IgM and IgE antibodies precede seroconversion of CF antibodies, and in one patient, these antibodies lagged months behind. Most patients had all classes of CMV antibodies, except IgA, for a year or more. Among 10 transplant patients with a secondary CMV infection, 50% had long-lasting IgM antibodies, and very few had IgE or IgA antibodies, but all had IgG antibodies to CMV. In 13 infected infants, the CMV-specific serologic response was also characterized by long-lasting IgM, IgE, and IgG antibodies. Two patients did not develop detectable IgM antibodies, and one of these did not show IgE antibodies either. The IgA response in infants as a whole was lacking; a few, however, were borderline positive. Of the nine acquired immunodeficiency syndrome patients with CMV infection studied during their last year of life, only one had antibodies in all four classes, the rest had only CF antibodies, and all except for one had IgG-class antibodies. All sera studied were also tested against a control antigen produced from noninfected cell nuclei. It was found that some patients developed antibodies to nuclear antigens in parallel with the rise in specific antibodies. The nonspecific antibodies occurred in all four classes, but most often they were of the IgM class. Addition of unlabeled control antigen to the conjugates was not always sufficient to abort this nonspecific reaction.
PMCID: PMC266401  PMID: 2835388

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