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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.
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 
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.
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.
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).
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.
PMCID: PMC3472651  PMID: 23091373
cytomegalovirus infection; heart transplantation; valganciclovir prophylaxis
5.  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.
PMCID: PMC1419872  PMID: 6271652
6.  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.
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.
PMCID: PMC4136270  PMID: 24899204
7.  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.
PMCID: PMC2673691  PMID: 19436702
8.  Seroprevalence of Cytomegalovirus Among Some Voluntary Blood Donors at the 37 Military Hospital, Accra, Ghana 
Ghana Medical Journal  2006;40(3):99-104.
Cytomegalovirus (CMV) is one of the most significant pathogens infecting immunosuppressed individuals. CMV is transmissible through transfusion of blood components.
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.
The seroprevalence of antibodies specific for CMV was tested using CMV IgG/IgM particle agglutination test kit and ELISA.
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.
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
9.  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.
PMCID: PMC473696  PMID: 7886659
10.  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
11.  Choice of induction regimens on the risk of cytomegalovirus infection in donor-positive and recipient-negative kidney transplant recipients 
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.
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.
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).
Induction with rATG is associated with increased risk of CMV infection. Longer duration of prophylaxis is beneficial.
PMCID: PMC3831513  PMID: 20576019
cytomegalovirus; kidney transplant; pancreas transplant; donor/recipient mismatch; rabbit anti-thymocyte globulin; basiliximab; acute rejection
12.  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
13.  Prevention of Transfusion-Transmitted Cytomegalovirus Infections: Which is the Optimal Strategy? 
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.
PMCID: PMC3949610  PMID: 24659946
Human cytomegalovirus; CMV; Blood donor; Transfusion-associated infections; Transfusion
14.  Cytomegalovirus-specific T-cell responses and viral replication in kidney transplant recipients 
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.
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.
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).
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.
PMCID: PMC2432058  PMID: 18541023
15.  Cytomegalovirus viral and antibody correlates in young children 
BMC Research Notes  2014;7(1):776.
Young, healthy children shedding cytomegalovirus (CMV) in urine and saliva appear to be the leading source of CMV in primary infection of pregnant women.
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).
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.
PMCID: PMC4236479  PMID: 25367101
Cytomegalovirus; Viral load; Serology; Avidity
16.  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
17.  Detection of cytomegalovirus in blood donors by PCR using the digene SHARP signal system assay: effects of sample preparation and detection methodology. 
Cytomegalovirus (CMV) is an important cause of transfusion-associated morbidity and mortality; however, only 0.4 to 12% of the blood products obtained from seropositive blood donors transmit infection. The effects of three commercially available whole-blood sample preparation kits on the detection of CMV PCR products by a semiquantitative adaptation of the Digene SHARP Signal System Assay (DSSSA) in samples from volunteer blood donors was assessed. Of 101 samples from seropositive blood donors, CMV was detected in 0 (0%) of the samples extracted with a QIAamp blood kit (QIAGEN), 1 (1%) of the samples extracted with an Amplicor whole-blood specimen preparation kit (Roche), and 8 (8%) of the samples extracted with an Isoquick nucleic acid extraction kit (modified by the addition of carrier tRNA) (Microprobe). CMV DNA was not detected in samples from seronegative blood donors (n = 13). Nested PCR of selected samples confirmed the detection of CMV in the sane eight samples extracted with the modified Isoquick nucleic acid extraction kit and detected an additional nine CMV-positive samples (n = 50). Samples from volunteer blood donors contain low copy numbers of CMV DNA. PCR amplification of such specimens can result in analytical sampling errors, giving results similar to the variations in titers recognized during determinations of the 50% tissue culture infective dose. The detection of CMV in blood samples from volunteer blood donors by PCR is a function of sample preparation, amplification conditions, and detection methodology. Accurate assessments of the clinical utility of CMV DNA detection by nucleic acid amplification for blood product screening and patients will require highly standardized and quantitative methodology.
PMCID: PMC228723  PMID: 8748266
18.  Relationship between cytomegalovirus (CMV) IgG serology, detectable CMV DNA in peripheral monocytes, and CMV pp65495–503-specific CD8+ T cells in older adults 
Age  2011;33(4):607-614.
In immunocompetent individuals, cytomegalovirus (CMV) is thought to persist in a latent state in monocytes and myeloid progenitor cells, establishing a lifelong infection. In CMV-seropositive older adults, aging has been associated with both expansion of CMV pp65495–503-specific CD8+ T cell clones and shrinkage of the T cell repertoire that characterize T cell immunosenescence. In fact it has been suggested that chronic CMV infection is a driving force in age-related T cell immunosenescence. In older adults, chronic CMV infection is conventionally diagnosed by positive IgG serology which does not distinguish between past and persistent infections. To better define the relationship between chronic CMV infection and expansion of CMV pp65495–503-specific CD8+ T cells, we directly assessed CMV viral DNA in monocyte-enriched peripheral blood mononuclear cells in 16 HLA-A2-positive elderly volunteers (mean age = 83 years). While all participants had positive CMV IgG serology by enzyme-linked immunosorbent assays, only nine (56%) had detectable CMV DNA by nested polymerase chain reaction. These nine individuals had significantly higher percentages of CMV pp65495–503 tetramer-positive CD8+ T cells (median = 1.3%) than those without detectable CMV DNA (median = 0.1%; p < 0.001). Absolute CMV IgG antibody titers did not differ between these two groups (median = 54.6 vs 44.2 EU/ml, respectively, p = 0.4). CMV IgM titers were negative for all 16 participants, suggesting that recent primary CMV infection was unlikely. These results demonstrate a strong association between the presence of CMV DNA in peripheral monocytes and the expansion of CD8+ T cells specific for the CMV immunodominant epitope pp65495–503. Although the sample size in this study is relatively small, these findings provide initial evidence suggesting the heterogeneity of CMV IgG-seropositive older adult population and CMV viral DNA detection in peripheral monocytes as an informative tool to better understand the relationship between chronic CMV infection and T cell immunosenescence.
PMCID: PMC3220402  PMID: 21274637
Monocytic CMV DNA; CMV pp65495–503-specific CD8+ T cells; CMV IgG serology; Older adults
19.  Seroprevalence and Risk Factors for Cytomegalovirus Infections in Adolescent Females 
Congenital cytomegalovirus (CMV) is a leading cause of disability, including sensorineural hearing loss, developmental delay, and mental retardation. Understanding risk factors for acquisition of CMV infection in adolescent females will help determine vaccine strategies.
Females (12–17 years) were recruited from primary care settings in Cincinnati, Galveston, Houston, and Nashville from June 2006 to July 2010 for a seroepidemiologic study, from which seronegative participants were recruited for a CMV vaccine trial. Participants (n = 1585) responded to questions regarding potential exposures. For those with young children in the home (n = 859), additional questions were asked about feeding and changing diapers, and for those > 14 years of age (n = 1162), questions regarding sexual activity were asked. Serum was evaluated for CMV antibody using a commercial immunoglobulin G assay.
Cytomegalovirus antibody was detected in 49% of participants. In the univariate analyses, CMV seroprevalence was significantly higher among African Americans, those with children < 3 years of age in the home, and those with a history of oral, anal, or vaginal intercourse. Among those with young children in the home, feeding children and changing diapers further increased the association with CMV infection. However, in the final multivariate analysis, only African Americans and household contact with young children were associated with CMV infection.
By age 12, evidence of CMV infection was common. Multiple factors regarding race and personal behaviors likely contribute to seroconversion earlier in life.
PMCID: PMC3656545  PMID: 23687583
Adolescents; Computer-Assisted Screening Interview; CASI; Cytomegalovirus; CMV; Epidemiology
20.  Frequent Occult Infection with Cytomegalovirus in Cardiac Transplant Recipients despite Antiviral Prophylaxis▿  
Journal of Clinical Microbiology  2007;45(6):1804-1810.
Despite antiviral prophylaxis, a high percentage (over 90%) of heart transplant patients experience active cytomegalovirus (CMV) infection, diagnosed by detection of viral DNA in peripheral blood polymorphonuclear leukocytes within the first few months posttransplantation. Viral DNA was detected in mononuclear cells prior to detection in granulocytes from CMV-seropositive recipients (R+) receiving a heart from a CMV-seropositive donor (D+). Based on assessment of systemic infection in leukocyte populations, both R+ subgroups (R+/D− and R+/D+) experienced a greater infection burden than the R−/D+ subgroup, which was aggressively treated because of a higher risk of acute CMV disease. Despite widespread systemic infection in all at-risk patient subgroups, CMV DNA was rarely (<3% of patients) detected in transplanted heart biopsy specimens. The R+ patients more frequently exceeded the 75th percentile of the CMV DNA copy number distribution in leukocytes (110 copies/105 polymorphonuclear leukocytes) than the R−/D+ subgroup. Therefore, active systemic CMV infection involving leukocytes is common in heart transplant recipients receiving prophylaxis to reduce acute disease. Infection of the transplanted organ is rare, suggesting that chronic vascular disease attributed to CMV may be driven by the consequences of systemic infection.
PMCID: PMC1933112  PMID: 17409205
21.  A prospective study comparing quantitative Cytomegalovirus (CMV) polymerase chain reaction in plasma and pp65 antigenemia assay in monitoring patients after allogeneic stem cell transplantation 
Low levels of Cytomegalovirus (CMV) viral load are frequently detected following allogeneic stem cell transplantation (SCT) and CMV disease may still develop in some allogeneic SCT patients who have negative pp65-antigenemia (pp65-Ag) or undetectable DNA. Pp65Ag is a sensitive method to diagnose CMV infection. Quantitative CMV-DNA PCR assay in plasma has been proposed to monitor CMV infection in SCT patients. We evaluated the clinical utility of pp65Ag and PCR assay in plasma of SCT recipients.
In a prospective longitudinal study, 38 consecutive patients at risk of CMV infection (donor and/or recipient CMV seropositive) were weekly monitored for CMV infection by both quantitative CMV-PCR in plasma (COBAS AMPLICOR CMV MONITOR) and pp65 Ag, during the first 100 days after SCT.
A total of 534 blood samples were simultaneously analysed for pp65Ag and PCR. Overall, 28/38 patients (74%) had active CMV infection within 100 days from SCT. In 16 patients, CMV was first detected by pp65 Ag alone; in 5 patients by both methods and in 6 by PCR assay alone; one patient had CMV biopsy-proven intestinal disease without pp65Ag and PCR assays positivity before CMV disease. Overall, three patients developed intestinal CMV disease (7.9%): one had negative both pp65Ag and PCR assays before CMV disease, one had disease and concomitant positivity of both methods, while in the remaining patient, only pp65Ag was positive before CMV disease.
Plasma PCR(COBAS AMPLICOR CMV MONITOR) and pp65Ag assays were effective in detecting CMV infection, however, discordance between both methods were frequently observed. Plasma PCR and pp65Ag assays may be complementary for diagnosis and management of CMV infection.
PMCID: PMC1664570  PMID: 17118205
22.  Comparison of immunoblotting with other serological methods and virus isolation for the early detection of primary cytomegalovirus infection in allograft recipients. 
Journal of Clinical Microbiology  1989;27(12):2672-2677.
Sequential specimens from nine allograft recipients were examined by using a variety of methods to detect primary cytomegalovirus (CMV) infection as rapidly as possible posttransplantation. Sera were examined for immunoglobulin G (IgG) and IgM antibodies by immunoblotting, enzyme immunoassay, and immunofluorescence and also by complement fixation, latex agglutination, and an immunofluorescence test for antibody to CMV early antigen. Urine and occasionally blood, tissue, and other specimens were centrifuged onto cell cultures to enhance CMV infectivity. Eight of the nine patients showed laboratory evidence of primary CMV infection, and CMV was isolated from seven of the eight: in no case was virus isolated before seroconversion had become evident. However, serological tests differed in their abilities to detect antibody response to CMV infection in different patients; while immunoblotting, latex agglutination, and enzyme immunoassay for IgG antibodies generally detected seroconversion before complement fixation, this was not invariably the case. At present, optimal laboratory detection of CMV infections in these patients can be achieved only by a combination of serological methods and virus isolation.
PMCID: PMC267106  PMID: 2556430
23.  Cytomegalovirus infection after liver transplantation: Current concepts and challenges 
Cytomegalovirus (CMV) is a common viral pathogen that influences the outcome of liver transplantation. In addition to the direct effects of CMV syndrome and tissue-invasive diseases, CMV is associated with an increased predisposition to acute and chronic allograft rejection, accelerated hepatitis C recurrence, and other opportunistic infections, as well as reduced overall patient and allograft survival. Risk factors for CMV disease are often interrelated, and include CMV D+/R- serostatus, acute rejection, female gender, age, use of high-dose mycophenolate mofetil and prednisone, and the overall state of immunity. In addition to the role of CMV-specific CD4+ and CD8+ T lymphocytes, there are data to suggest that functionality of the innate immune system contributes to CMV disease pathogenesis. In one study, liver transplant recipients with a specific polymorphism in innate immune molecules known as Toll-like receptors were more likely to develop higher levels of CMV replication and clinical disease. Because of the direct and indirect adverse effects of CMV disease, its prevention, whether through antiviral prophylaxis or preemptive therapy, is an essential component in improving the outcome of liver transplantation. In the majority of transplant centers, antiviral prophylaxis is the preferred strategy over preemptive therapy for the prevention of CMV disease in CMV-seronegative recipients of liver allografts from CMV-seropositive donors (D+/R-). However, the major drawback of antiviral prophylaxis is the occurrence of delayed-onset primary CMV disease. In several prospective and retrospective studies, the incidence of delayed-onset primary CMV disease ranged from 16% to 47% of CMV D+/R- liver transplant recipients. Current data suggests that delayed-onset CMV disease is associated with increased mortality after liver transplantation. Therefore, optimized strategies for prevention and novel drugs with unique modes of action are needed. Currently, a randomized controlled clinical trial is being performed comparing the efficacy and safety of maribavir, a novel benzimidazole riboside, and oral ganciclovir as prophylaxis against primary CMV disease in liver transplant recipients. The treatment of CMV disease consists mainly of intravenous (IV) ganciclovir, and if feasible, a reduction in the degree of immunosuppression. A recent controlled clinical trial demonstrated that valganciclovir is as effective and safe as IV ganciclovir for the treatment of CMV disease in solid organ (including liver) transplant recipients. In this article, the author reviews the current state and the future perspectives of prevention and treatment of CMV disease after liver transplantation.
PMCID: PMC2739936  PMID: 18756591
Cytomegalovirus; Outcome; Hepatitis; Transplantation; Valganciclovir; Maribavir; Prophylaxis; Treatment
24.  Comparison between viremia and antigenemia for detection of cytomegalovirus in blood. 
Journal of Clinical Microbiology  1988;26(12):2531-2535.
In a prospective study, 139 serial blood samples from 15 transplant recipients were assessed for the presence of cytomegalovirus (CMV) by virus isolation (CMV viremia) and by direct staining of CMV antigens (CMV Ag) in blood leukocytes (CMV antigenemia). CMV was isolated from 23 samples, whereas CMV Ag was detected in 44 specimens. All positive samples were from a total of nine patients who were diagnosed as having active CMV infections. In seven patients, active CMV infections were diagnosed by virus isolation from blood and urine and by a significant rise of CMV-specific antibodies. In these patients, 21 of the 23 blood samples which were positive for CMV by cell culture were also positive by direct CMV Ag detection. Moreover, CMV Ag were detected in 23 of the 116 culture-negative samples. Twenty of these samples were from the acute phase of infection in the same seven patients. The remaining three CMV Ag-positive specimens were from the other two patients, from whom CMV was not isolated but who had serological evidence of concomitant active CMV infections. These results suggest that direct detection of CMV Ag in peripheral blood leukocytes is as specific as and more sensitive than current isolation techniques. Furthermore, by its sensitivity and inherent rapidity the antigen detection test proved to be the earliest diagnostic marker of active CMV infection in eight of the nine patients. Finally, it was shown that monoclonal antibodies to CMV immediate early antigens are a prerequisite for demonstration of CMV antigenemia.
PMCID: PMC266939  PMID: 2852670
25.  Infection with cytomegalovirus is not associated with premature mortality 
Infectious Disease Reports  2011;3(2):e17.
Over 90% of the world's population acquires a cytomegalovirus (CMV) infection. This infection, although asymptomatic or self-limiting, is a major burden to the immune system. For this reason, and because CMV immunization is possible, determining whether CMV can cause reduced longevity, particularly among those with coronary artery disease, is important and previous reports have been conflicting. Thus our objective was to assess the association between CMV infection as defined serologically and antibody levels against CMV and long-term survival (18 years). We completed a prospective observational cohort study of 915 consecutive patients (mean age 58 years) undergoing coronary angiography. CMV immunoglobulin levels were measured at baseline using either a whole cell CMV antigen or a purified protein antigen (gB). After adjustment for potentially confounding variables (age, race, gender, body mass index, the presence or absence of coronary artery disease, the number of diseased vessels, diabetes, renal disease, hypertension, dialysis, congestive heart failure, and the maximum percent reduction in luminal diameter), Cox's proportional hazards models showed no association between CMV seropositivity or levels of antibodies against CMV by either assay and longevity for both patients with or without coronary artery disease (CAD) nor for those under or over 70 years of age at baseline. Our observations suggest that universal immunization against CMV may not improve longevity.
PMCID: PMC3892592  PMID: 24470914
cytomegalovirus; coronary artery disease; mortality.

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