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

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.     

The apparent paradox of maternal seropositivity as a risk factor for congenital cytomegalovirus infection: a population‐based prediction model

Because maternal seropositivity for CMV is associated with substantial protection against congenital CMV infection, prevention measures have focused mainly on seronegative pregnant women for decades. However, population‐wide insight in the contribution of nonprimary infection (reactivation and/or re‐infection with a different strain) on the most common sequela, hearing loss, is missing. A population‐based prediction model was developed to estimate the proportion of congenital CMV‐related hearing loss resulting from nonprimary maternal infection. Incorporated was a meta‐analysis of the risk of hearing loss, calculating pooled proportions of children with hearing loss after nonprimary and primary infection. Subsequently, the model was applied for worldwide present population seroprevalences (range 30–95%). It was estimated that, for all population seroprevalences, nonprimary maternal infections are responsible for the majority of congenital CMV infections. This proportion increased with seroprevalence, ranging from 57% (95%CI 24–85%) to 96% (95% CI 88–99%) for seroprevalences of 30% to 95%. Our meta‐analysis (six reports) showed that the risk of hearing loss after nonprimary infection was 11% (28/253 children, 95% CI 7–15%) versus 13% (50/385 children, 95% CI 10–16%) after primary infection. Incorporating this risk into our model, we estimated that nonprimary infections also accounted for the majority of CMV‐related hearing loss. This proportion ranged from 53% (95% CI 13–86%) to 95% (95% CI 62–99%) for seroprevalences of 30% to 95%. Our data underline the worldwide contribution of nonprimary infections in causing CMV‐related hearing loss. These results imply that prevention research such as vaccine and hygiene studies should not only be directed at seronegative but also seropositive pregnant women. Copyright © 2013 John Wiley & Sons, Ltd.     

Validation of an in-house assay for cytomegalovirus immunoglobulin G (CMV IgG) avidity and relationship of avidity to CMV IgM levels

Measurement of cytomegalovirus (CMV)-specific immunoglobulin G (IgG) avidity has proven to be a powerful tool for distinguishing primary from nonprimary CMV infection. An in-house enzyme-linked immunosorbent assay (ELISA) for measuring CMV IgG avidity was validated using 84 sera from pregnant women who had recently seroconverted following primary CMV infection and 74 sera from individuals with past CMV infection (IgG-positive and IgM-negative profile). Of the 84 sera from pregnant women, 73 sera were collected within 120 days of the last IgG-negative sample, and 72 of these 73 sera (99%) exhibited an avidity index (AI) of <50%. In contrast, 71 of 74 (96%) sera from individuals with past CMV infection exhibited CMV AI values of > 60%. Thus, low avidity in the in-house ELISA was defined as an AI of < or = 50%, whereas high avidity was defined as an AI of > or = 60%. In additional studies, the relationship between CMV IgG avidity and CMV IgM levels was examined using 64 CMV IgG-positive sera (time since seroconversion unknown) exhibiting equivocal or positive results in a CMV IgM capture ELISA (Diamedix). Of these 64 sera, 29 exhibited IgM index values of > or = 3.0, and 27 of these 29 (93%) exhibited low IgG avidity. A similar trend was observed when a subset of these 64 sera (n = 48) was tested in another CMV IgM capture ELISA (Trinity); of 18 sera with IgM index values of > or = 3.0, 17 (94%) exhibited low IgG avidity. These findings demonstrate the validity of an in-house ELISA for CMV IgG avidity and further show that strong reactivity of CMV IgG-positive sera in either of two CMV IgM capture assays is a reliable indicator of low CMV IgG avidity, and thus, recent CMV infection.     

Evaluation of the Abbott AxSYM Cytomegalovirus (CMV) Immunoglobulin M (IgM) Assay in Conjunction with Other CMV IgM Tests and a CMV IgG Avidity Assay

The measurement of the avidity of cytomegalovirus (CMV) immunoglobulin G (IgG) antibodies has been shown by several investigators to be useful in identifying and excluding primary CMV infections in pregnant women. In this work, we examined the diagnostic utility of reflex testing of CMV IgM-positive specimens from pregnant women by using a CMV IgG avidity assay. The utility of this approach was directly dependent on the sensitivity of the CMV IgM assay employed during the initial screen. The higher initial reactivity rate of the AxSYM CMV IgM assay was necessary in order to detect CMV IgM in specimens containing low-avidity CMV IgG antibodies, indicative of a primary CMV infection, which other CMV IgM assays (Behring, Vidas, Captia, and Eurogenetics) fail to detect in some cases. The use of the AxSYM CMV IgM assay, followed by an avidity test, should result in more accurate diagnosis of CMV infection in pregnant women.     

Evaluation of the Abbott AxSYM cytomegalovirus (CMV) immunoglobulin M (IgM) assay in conjunction with other CMV IgM tests and a CMV IgG avidity assay

The measurement of the avidity of cytomegalovirus (CMV) immunoglobulin G (IgG) antibodies has been shown by several investigators to be useful in identifying and excluding primary CMV infections in pregnant women. In this work, we examined the diagnostic utility of reflex testing of CMV IgM-positive specimens from pregnant women by using a CMV IgG avidity assay. The utility of this approach was directly dependent on the sensitivity of the CMV IgM assay employed during the initial screen. The higher initial reactivity rate of the AxSYM CMV IgM assay was necessary in order to detect CMV IgM in specimens containing low-avidity CMV IgG antibodies, indicative of a primary CMV infection, which other CMV IgM assays (Behring, Vidas, Captia, and Eurogenetics) fail to detect in some cases. The use of the AxSYM CMV IgM assay, followed by an avidity test, should result in more accurate diagnosis of CMV infection in pregnant women.     

Validation of an In-House Assay for Cytomegalovirus Immunoglobulin G (CMV IgG) Avidity and Relationship of Avidity to CMV IgM Levels

Measurement of cytomegalovirus (CMV)-specific immunoglobulin G (IgG) avidity has proven to be a powerful tool for distinguishing primary from nonprimary CMV infection. An in-house enzyme-linked immunosorbent assay (ELISA) for measuring CMV IgG avidity was validated using 84 sera from pregnant women who had recently seroconverted following primary CMV infection and 74 sera from individuals with past CMV infection (IgG-positive and IgM-negative profile). Of the 84 sera from pregnant women, 73 sera were collected within 120 days of the last IgG-negative sample, and 72 of these 73 sera (99%) exhibited an avidity index (AI) of <50%. In contrast, 71 of 74 (96%) sera from individuals with past CMV infection exhibited CMV AI values of >60%. Thus, low avidity in the in-house ELISA was defined as an AI of 50%, whereas high avidity was defined as an AI of 60%. In additional studies, the relationship between CMV IgG avidity and CMV IgM levels was examined using 64 CMV IgG-positive sera (time since seroconversion unknown) exhibiting equivocal or positive results in a CMV IgM capture ELISA (Diamedix). Of these 64 sera, 29 exhibited IgM index values of 3.0, and 27 of these 29 (93%) exhibited low IgG avidity. A similar trend was observed when a subset of these 64 sera (n = 48) was tested in another CMV IgM capture ELISA (Trinity); of 18 sera with IgM index values of 3.0, 17 (94%) exhibited low IgG avidity. These findings demonstrate the validity of an in-house ELISA for CMV IgG avidity and further show that strong reactivity of CMV IgG-positive sera in either of two CMV IgM capture assays is a reliable indicator of low CMV IgG avidity, and thus, recent CMV infection.     

In vitro cell-mediated immune responses of human immunodeficiency virus-infected and -uninfected individuals to whole cytomegalovirus antigens and their subunits

The aim of this study was to optimize the ability to detect cytomegalovirus (CMV)-specfic cell-mediated immunity (CMI) in human immunodeficiency virus (HIV)-infected individuals by comparing different assays (the lymphocyte proliferation assay [LPA] and assays for gamma interferon [IFN-gamma] and interleukin-2 [IL-2] production) and CMV antigenic preparations. Thresholds discriminating positive from negative CMI results were developed with specimens from 36 CMV-seropositive and 21 CMV-seronegative healthy individuals. The analysis showed that the CMI elicited by any of the four CMV whole lysates tested in this study tended to be more robust and sensitive than the responses to the subunit antigens gB and pp65. LPA and inducible IFN-gamma but not IL-2 were highly sensitive measures of CMV-specific CMI in HIV-infected and -uninfected individuals. The ability to detect CMV-specific LPA or IFN-gamma responses in HIV-infected individuals significantly increased with higher CD4 cell numbers. Nevertheless, the proportion of HIV-infected subjects with CD4 counts of >or=500 cells/mul who had a detectable CMV-specific CMI remained significantly lower than that of healthy adults. The ability to detect CMV-specific CMI in HIV-infected individuals decreased with higher levels of HIV replication, with discriminative thresholds of 10(3) to 10(4) HIV RNA copies/ml of plasma, for LPA or inducible IFN-gamma production elicited by different antigens. The LPA responses obtained with CMV whole lysate and phytohemagglutinin were significantly correlated in HIV-infected subjects but not uninfected controls, indicating a novel characteristic of the CMI defect caused by HIV. The intrasubject variabilities of the CMV-specific CMI were similar in HIV-infected and -uninfected individuals. These data show that LPA and the inducible IFN-gamma production elicited by CMV whole lysates may be used to assess modifications of the immune competency of HIV-infected individuals.     

Evaluation of the New Architect Cytomegalovirus Immunoglobulin M (IgM), IgG,and IgG Avidity Assays

A panel of new cytomegalovirus (CMV) assays for use on the Architect instrument has been developed, including a CMV avidity assay based on a new technology. The purpose of this study was to compare the performance characteristics of the fully automated CMV immunoglobulin M (IgM), IgG, and IgG avidity tests on the Architect instrument with those of other available assays. A total of 503 consecutive fresh patient serum specimens (routine serum specimens) and 96 serum specimens from 33 pregnant women with a recent CMV primary infection (seroconversion serum specimens) were tested for CMV IgM and IgG by the Architect (Abbott), Vidas (BioMérieux), and Enzygnost (Siemens) assays. The seroconversion sera and 100 preselected serum specimens IgM negative and IgG positive by the AxSYM assay were also tested by the IgG avidity tests on the Architect and Vidas instruments. The relative agreements for CMV IgM determination with routine sera between the Architect assay and the Vidas, Enzygnost, and AxSYM assays were 97%, 94%, and 93%, respectively, for the CMV IgM tests and 99%, 98%, and 98%, respectively, for the CMV IgG tests. The specificities of the CMV IgG avidity test were 98% for the Architect assay and 76% for the Vidas assay. No high CMV IgG avidity test results were found within the first 3 months after seroconversion by either of those assays. The correlation between the results of the newly developed CMV IgM and IgG tests on the Architect instrument with the Vidas and Enzygnost assays was excellent (≥94%). The CMV IgG avidity test reliably excluded patients with recent infections and showed an excellent specificity (98%).Human cytomegalovirus (CMV) is the most common cause of congenital infection. Primary infections occur in 0.15 to 2.0% of all pregnancies. The in utero transmission of CMV can take place during primary maternal infection or during nonprimary infection (reactivation and reinfection) of seropositive mothers, but the transmission rate to the fetus is much higher for nonimmune mothers (up to 40%) than for immune mothers (0 to 1%) (16). Fowler et al. showed that naturally acquired immunity results in a 69% reduction in the risk of congenital CMV infection in future pregnancies (2).Screening of pregnant women for CMV antibodies is a controversial issue and is not supported by international guidelines. Opponents of screening for CMV antibodies during pregnancy suggest that no clearly effective intervention is available. In addition, no tests can reliably predict which infected fetuses will have serious sequelae. The prognostic value of determination of the viral load in amniotic fluid is still a matter of debate and has the disadvantage of its invasive character (3, 5, 7, 10, 13, 15). On the other hand, many parents wish to have an antenatal diagnosis of intrauterine infections, and gynecologists generally offer screening for CMV antibodies. Prenatal screening certainly has advantages, such as the fact that the use of precautionary hygienic measures can be suggested to CMV-seronegative pregnant women. The knowledge of a primary CMV infection in a pregnant woman can lead to closer follow-up of the fetus by ultrasound and nuclear magnetic resonance.For the diagnosis of a primary CMV infection during pregnancy, it is of utmost importance that we have available reliable, noninvasive tests. CMV-specific immunoglobulin M (IgM) is produced during primary infection, but it is also detectable during reactivation and reinfection (9). A test that enables the discrimination of a primary infection from a nonprimary infection is important for counseling of the parents on the risk of congenital infection. CMV IgG avidity testing has been shown to be useful for distinguishing primary and nonprimary infections (1, 4, 6, 9). It measures the binding affinity of IgG antibodies. At the onset of infection, IgG antibodies of low avidity are produced. Over time, maturation of the antibody occurs, resulting in an increased binding affinity and, thus, a higher avidity. A diagnostic algorithm for CMV serology screening of pregnant women on the basis of CMV IgM, IgG, and IgG avidity testing was proposed by Munro et al. (12).Recently, a panel of new CMV assays for use on the Architect instrument has been developed, including a CMV IgG avidity assay. It is the first platform that enables the complete automation of avidity testing. While conventional avidity assays remove low-avidity antibodies by the use of a chaotropic agent, in tests with the Architect instrument, high-avidity antibodies are removed by neutralization with liquid antigen and low-avidity antibodies are detected directly. The purpose of the study described here was to evaluate the performance characteristics of the Architect CMV IgM, IgG, and IgG avidity assay and to compare the results with those of the CMV assays available from bioMérieux and Siemens.     

CMV antigenemia following bone marrow transplantation: risk factors and outcomes.

Cytomegalovirus (CMV) infection remains a major problem in blood and bone marrow transplant (BMT) recipients. Recent efforts have been directed at prevention, early diagnosis, and treatment of CMV disease following BMT. Assay for CMV early antigen pp65 on circulating leukocytes has been shown to be sensitive, and specific in detecting early CMV infection. We examined the frequency, risk factors, and outcomes of a positive CMV antigen assay in 118 consecutive BMT patients. Forty-three (36%) of the 118 patients developed CMV antigenemia a median of 26 days post-BMT (range, -6 to 209 days). The incidence of antigenemia in autologous, related donor, and unrelated donor BMT recipients was 15%, 50%, and 48%, respectively (P < .01) and was lower in CMV-seronegative patients (19% versus 51% in seropositive patients; P < .01). Patients with grade II to IV acute graft-versus-host disease (GVHD) had 2.2 times the risk of antigenemia of patients with no or only limited GVHD (P = .03). Age at transplantation, underlying disease, CMV prophylaxis regimen, and GVHD prophylaxis regimen did not affect the risk of CMV antigenemia. Ten of the 43 antigenemic patients, all CMV-seropositive allogeneic BMT (alloBMT) recipients, developed CMV organ disease a median of 101 days (range, 28-283 daya) post-BMT. These data suggest that CMV-seropositive alloBMT patients are at highest risk for CMV antigenemia and for organ disease as well. CMV disease may occur before antigenemia is detectable in leukopenic patients and may also develop late post-BMT, even in patients still receiving antiviral prophylaxis. In high-risk groups, intensive surveillance continuing for more than 6 months after BMT may be indicated.     

Low maternal CD4 count at inception of gestational cytomegalovirus (CMV) infection and impaired humoral response: effect on congenital CMV infection in the guinea pig.

In humans, the rate and clinical expression of disease in congenitally cytomegalovirus (CMV)-infected infants is modified by maternal immunity to CMV. We used the guinea pig model of congenital CMV infection to compare maternal CD4+ T-cell numbers in nonpregnant animals to those in pregnant dams just before and 7-14 days after inoculation with guinea pig CMV (gpCMV) very early, early, or late in gestation. We also examined ELISA antibody responses to gpCMV in the inoculated nonpregnant and pregnant animals. When compared to nonpregnant uninfected animals, CD4 counts were lower in very early and in late uninfected gestation. CD4 counts also dropped further in the postinoculation period. Compared to nonpregnant gpCMV-inoculated animals, initial antibody responses to gpCMV were also decreased in gpCMV-infected pregnant dams. The group of dams inoculated very early in pregnancy experienced delays in seroconversion to gpCMV, persisting low titers throughout gestation, in utero fetal resorptions, and CNS-infected pups. The group of dams inoculated late in gestation had the lowest geometric mean titers at delivery (almost 50% with no detectable antibody) and a high rate of vertical gpCMV transmission and postnatal pup death. Significantly lower rates of both congenital infection and postnatal pup deaths were observed in litters of late gestation-infected dams that had gpCMV antibody at delivery. Thus, decreased circulating maternal CD4+ T cells very early and late in gestation were further decreased after gpCMV inoculation and were associated with delayed and depressed maternal antibody responses, all of which were associated with poor outcome after primary maternal gpCMV infection, the expression of which varied by time in pregnancy when gpCMV was acquired.     

Congenital cytomegalovirus (CMV) epidemiology and awareness

This commentary highlights and discusses the implications of a number of recent studies that refine epidemiologic knowledge of CMV infection and assess awareness of congenital CMV among clinicians and the public. These studies highlight that: (1) congenital CMV results in a disease burden that is substantial and severe; (2) a high proportion of United States women of reproductive age are susceptible to CMV infection; (3) the majority of congenital CMV infections in the United States result from recurrent infections among pregnant women; (4) CMV seroprevalence and seroincidence are much higher among racial/ethnic minorities and persons of lower socioeconomic status (SES); (5) household transmission of CMV appears to be an important transmission route in the United States; (6) sexual transmission of CMV appears to be an important transmission route in some population sub-groups in the United States; (7) women have limited awareness and knowledge about congenital CMV; (8) most obstetrician/gynecologists do not counsel women about prevention of congenital CMV; (9) most women view CMV prevention messages positively.     

An enhancement by CMV seropositive serum or plasma of lymphocyte response in an in vitro CMV-specific lymphocyte transformation test

A lymphocyte transformation test for the in vitro assessment of cell-mediated immunity (CMI) to cytomegalovirus (CMV) was established. The test was specific for CMV and only lymphocytes from CMV seropositive donors responded. However, we observed that the response of lymphocytes from CMV seropositive donors was decreased or abolished in the presence of CMV seronegative plasma or serum. The reason for this apparent requirement for CMV seropositive serum, not reported by others performing similar tests, is not clear and suggests prudence in the interpretation of lymphocyte transformation assays as a measure of CMI.     

Cytomegalovirus (CMV)-specific lysis of CMV-infected target cells can be mediated by both NK-like and virus-specific cytotoxic T lymphocytes

The host response to cytomegalovirus (CMV) involves both humoral and cell-mediated immunity. Because virus-specific cytotoxicity appears to be associated with recovery from CMV infection, we have investigated spontaneous (NK) and cytotoxic T cell (Tc) activity against CMV-infected target cells in normal donors and infants with active CMV infection. Fresh mononuclear cells from seropositive donors expressed low-level, non-MHC-restricted cytotoxic activity that preferentially lysed CMV-infected but not uninfected autologous and allogeneic fibroblasts. No cytotoxic activity was observed when mononuclear cells from seronegative donors were studied. Mononuclear cells from infants with active CMV infection and mononuclear cells from seropositive adults that were pre-incubated with cell-free CMV antigen in bulk culture for 6 days had enhanced cytotoxic activity against CMV-infected target cells sharing one or more class I MHC determinants with the effector cell populations. Selective enrichment or depletion experiments were performed to characterize the effector cell populations involved in the augmented cytotoxic response following in vitro induction with CMV antigen. Purified E-rosette-forming cells expressed increased cytotoxic activity against both virus-infected fibroblasts and the NK target cell, K562. Depletion experiments with monoclonal antibodies and complement indicated that the CMV-specific cytotoxic response involves both an E-rosette-positive, T8+, M1- T-cell subset that most likely represents CMV-specific Tc, and an NK population that can be induced to differentiate by in vitro stimulation with CMV antigen following quantitative NK-cell depletion with monoclonal antibody, B73.1.     

Cell-Mediated Immunity to Cytomegalovirus in Pregnant Women

ABSTRACT: Employing the techniques of in vitro lymphocyte transformation (LTF) and complement fixation, cell-mediated immunity (CMI) and antibody to cytomegalovirus (CMV) were studied in pregnant and nonpregnant women. The LTF activity was determined by the whole blood microassay using four strains of CMV (AD-169 and its early antigen [EA], Davis, Veca, and Towne strains), and phytohemagglutinin (PHA). Lymphocyte transformation response to specific CMV antigens at 11–30 weeks of gestation and to nonspecific mitogen (PHA) in all pregnant and postpartum women were found to be significanty depressed compared with the nonpregnant women. The lower LTF responses to CMV antigen and PHA were found in specimens taken from pregnant women at 21–30 weeks of gestation. There were no significant differences in the mean complement-fixing (CF) antibody titers and the percentage of E-rosette-forming T lymphocytes between subjects in various stages of pregnancy. In addition, concanavalin A (Con A)-generated suppressor T cell activity was evaluated in pregnant and nonpregnant women. The suppressor effect of Con A-activated lymphocytes in pregnant women was somewhat higher than in nonpregnant women. These observations suggest that CMV-specific suppression of cellular immunity may play an important role in reactivation of CMV in pregnancy.     

Role of Cytomegalovirus (CMV) IgG Avidity Testing in Diagnosing Primary CMV Infection during Pregnancy

The risk of intrauterine transmission of cytomegalovirus (CMV) during pregnancy is much greater for women who contract primary CMV infection after conception than for women with evidence of infection (circulating CMV antibodies) before conception. Thus, laboratory tests that aid in the identification of recent primary CMV infection are important tools for managing the care of pregnant women suspected of having been exposed to CMV. CMV IgM detection is a sensitive marker of primary CMV infection, but its specificity is poor because CMV IgM is also produced during viral reactivation and persists following primary infection in some individuals. Studies conducted over the last 20 years convincingly demonstrate that measurement of CMV IgG avidity is both a sensitive and a specific method for identifying pregnant women with recent primary CMV infection and thus at increased risk for vertical CMV transmission. IgG avidity is defined as the strength with which IgG binds to antigenic epitopes expressed by a given protein; it matures gradually during the 6 months following primary infection. Low CMV IgG avidity is an accurate indicator of primary infection within the preceding 3 to 4 months, whereas high avidity excludes primary infection within the preceding 3 months. In this minireview, we summarize published data demonstrating the clinical utility of CMV IgG avidity results for estimating time since primary infection in pregnant women, describe commercially available CMV IgG avidity assays, and discuss some of the issues and controversies surrounding CMV IgG avidity testing during pregnancy.     

The value of CMV IgG avidity and immunoblot for timing the onset of primary CMV infection in pregnancy

BackgroundPrimary CMV infections in pregnancy are usually asymptomatic and only detected by serology. Estimating the onset of infection is a major diagnostic goal, since primary infections around conception and in early gestation hold a higher risk for congenital disease than those in later pregnancy.ObjectivesTo assess the ability of serological supplementary CMV assays to date the onset of primary infection.Study designFrom our routine diagnosis we identified 61 pregnant women (n = 188 serum samples) with precisely determined onset of CMV primary infection either by IgG seroconversion (n = 24) or by significant IgG antibody rise (n = 37). One hundred and forty-seven sera were investigated using the VIDAS^® CMV IgG avidity EIA (BioMèrieux) and 83 sera using the recomBlot CMV IgG with avidity (Mikrogen).ResultsBoth assays proofed to be reliable in terms of timing the onset of CMV primary infection. An avidity index (AI) in the VIDAS avidity EIA of <40% indicated primary infection within the last 20 weeks (positive predictive value 93.4%; 99/106), whereas an intermediate AI excluded primary infection within the last 12 weeks (negative predictive value 88.2%; 15/17). The recomBlot showed high reliability (PPV 96.9%; 31/33) for timing the onset of infection within the last 14 weeks. Avidity testing by blot however could not be interpreted in 11 of 47 sera (23.4%).ConclusionFor timing the onset of infection (before or in early pregnancy) CMV avidity testing is most helpful if carried out within the first trimester up to the beginning of second trimester.     

High TNF-alpha and IL-8 levels predict low blood dendritic cell counts in primary cytomegalovirus infection

BackgroundIn vitro studies suggest that human cytomegalovirus (CMV) modulates the functions of dendritic cells (DCs). However, there are limited data on DC homeostasis in CMV-infected patients.ObjectivesThe aim of this study was to characterize circulating DCs and plasma cytokine levels in immunocompetent patients with primary, symptomatic CMV infections.Study designThe study population consisted of 14 patients suffering of CMV mononucleosis and 14 healthy volunteers (11 CMV-seropositive and 3 CMV-seronegative subjects) included as controls. Peripheral blood mononuclear cells were isolated and used to characterize DCs and to quantify CMV in the blood. Plasma levels of pro-inflammatory and anti-inflammatory cytokines were also measured.ResultsWe observed that patients who were developing CMV mononucleosis presented lower myeloid and plasmacytoid DC counts in peripheral blood compared with healthy controls. We also noted elevated levels of inflammatory mediators, of which tumor necrosis factor-α (TNF-α)—which activates DCs and endothelial cells—was the highest. Notably, the decrease in blood DCs correlated with high TNF-α and IL-8 levels by a hyperbolic function.ConclusionsOur results suggest that increased levels of inflammatory factors facilitate alterations in DC homeostasis during primary CMV infection, which may contribute to viral-induced modulation of host immunity.     

Development and application of a PCR-based method including an internal control for diagnosis of congenital cytomegalovirus infection

Cytomegalovirus (CMV) is the most common cause of congenital infection in the developed world. We have designed and evaluated an assay that includes an internal control for amplification and detection of CMV DNA in amniotic fluid and neonatal urine samples. We present data on the use of this assay in the diagnosis of congenital CMV infection. A total of 145 amniotic and fetal fluid samples were examined by this assay; 83 were from healthy pregnant women and 62 were from women who were being investigated because of concerns over the pregnancy (diagnostic group). CMV DNA was detected in three amniotic fluid samples from the diagnostic group but was not detected in any samples taken from healthy pregnant women. Thirty-nine urine samples were obtained from 19 neonates with suspected congenital infection; CMV DNA was detected in urine from 6 of these patients. The assay provides useful information about CMV infection in the fetus and the neonate; when used in conjunction with other diagnostic tools it will enable mothers and obstetricians to make informed decisions about the management of pregnancies complicated by CMV infection.     

Frequency of human cytomegalovirus-specific T cells during pregnancy determined by intracellular cytokine staining

The characteristics of cytomegalovirus (CMV)-specific T-cell immunity was investigated in pregnant women with primary, latent, or reactivated CMV infection, and in a comparative group of non-pregnant women. Forty-six pregnant and 8 non-pregnant women were examined based on the presence of serum antibody activity against CMV and viral excretion in urine. The frequency of CMV-specific CD4(+) T cells in peripheral blood lymphocytes was determined by staining for intracellular cytokines, interferon (IFN)-gamma, and tumor necrosis factor (TNF)-alpha. There was no change in the frequencies of CMV-specific CD4(+) T cells in CMV-seropositive normal non-pregnant and pregnant women at any gestation. However, the frequency of CMV-specific CD4(+) T cells in pregnant women associated with CMV reactivation or reinfection was significantly higher than in CMV-seropositive normal pregnant and non-pregnant women. There were no CMV transmissions to the infants of all these women. These CMV-specific T cells responses in pregnant women may contribute some to block the intrauterine CMV infection in their infants.     

Detection of human cytomegalovirus-specific iga antibodies in colostrum by enzyme-linked lmmunosorbent assay (elisa)

Fifty women were examined after delivery for the prevalence of antibodies to human cytomegalovirus in colostrum and sera. Eighty percent of them had specific CMV IgG antibodies in the sera, as determined by the immunoperoxidase antibody to membrane antigen (IPAMA) technique. Of the CMV-seropositive women, 60% had specific CMV IgA antibodies in high titer in the colostrum as determined by enzyme-linked immunosorbent assay (ELISA). In only two of the seropositive women were specific CMV IgA antibodies detected in the sera as well. The significance of specific CMV IgA antibodies in colostrum as protection against perinatal infection and the mechanism of this production will be discussed.