Primary HCMV Infection Research Articles

Indeterminate/moderate IgG avidity during HCMV infection: comparison of methods

Background. The IgG avidity test is usually used for differentiating between primary and non-primary HCMV infection within 3 months. Weak avidity is highly suggestive of a primary infection, while high avidity tends to exclude it.An indeterminate or moderate avidity, however, does not allow a clear dating. Since there are several avidity tests with different performances, those tests that are able to minimize the results with indeterminate/moderate avidity are particularly useful. Objectives. The aim of our work was to evaluate the results obtained with two IgG avidity tests in IgG and IgM anti-HCMV positive patients. Study Design. 113 anti-HCMV IgG and IgM positive samples were tested with Enzime Linked Fluorescent Assay (ELFA) and Chemiluminescence Immuno Assay (CLIA) IgG avidity test. Results. 21 samples (18.6%), 50 (44.2%) and 42 (37.2%) with ELFA and 53 samples (46.9%), 10 (8.8%) and 50 (44.2%) with CLIA were found to have respectively low, indeterminate/moderate and high avidity. Of the 50 ELFA indeterminate avidity samples, 32 (64%), 10 (20%) and 8 (16%) were found to have respectively low, moderate and high CLIA.avidity. For 11 cases of the 32 ELFA indeterminate avidity and CLIA low avidity, there were previous data showing a seroconversion within three months. In a case of the 8 ELFA indeterminate avidity and CLIA high avidity, there were, instead, previous data of IgG and IgM positivity already four months earlier. Conclusions. It appears that the CLIA test for IgG avidity is more effective than ELFA for dating HCMV infection.

Aurora project: The IFIs in NICU in Southern Italy

congenital infections in industrialized countries, with incidence ranging between 0.3 and 2.4%. Complications of congenital HCMV infection may include deafness and mental retardation. Primary HCMV infection during pregnancy carries a high risk of vertical transmission. An uncontrolled, non-randomized study published in 2005 reported that the intravenous administration of anti-CMV immunoglobulin (Ig) reduced the maternal–fetal transmission of the virus from 40 to 16%. Design and population: This is an experimental non-randomized spontaneous monocentric study — Phase 2. Material and methods: Between 2003 and 2007: 26 pregnant women with primary CMV infection documented serologically and/ or virologically between the 6th and 22nd weeks of pregnancy were enrolled to receive anti-CMV IgG (Cytotect) monthly from the moment of diagnosis until birth. Results: Children born to 15 mothers (57.96%) were uninfected, while the newborns of 8 mothers (30.76%) presented CMV infection; 2 of these were symptomatic (1 with hearing impairment, 1 with encephalopathy), while the third, although asymptomatic, showed neuroradiological alterations. The remaining 3 pregnancies were interrupted by miscarriage in 1 case and voluntary abortion in 2 cases because of signs of CMV infection-induced alterations on ultrasonography. Overall, 42.3% of newborns/fetuses were infected. None of the expecting mothers presented side effects. Conclusions: Our study revealed no advantage in the use of antiCMV IgG. Nevertheless, controlled, and ideally, randomized trials are warranted to better clarify the usefulness of this therapeutic approach.

A model for HCMV infection in immunosuppressed patients

We propose a model for HCMV infection in healthy and immunosuppressed patients. First, we present the biological model and formulate a system of ordinary differential equations to describe the pathogenesis of primary HCMV infection in immunocompetent and immunosuppressed individuals. We then investigate how clinical data can be applied to this model. Approximate parameter values for the model are derived from data available in the literature and from mathematical and physiological considerations. Simulations with the approximated parameter values demonstrates that the model is capable of describing primary, latent, and secondary (reactivated) HCMV infection. Reactivation simulations with this model provide a window into the dynamics of HCMV infection in (D-R+) transplant situations, where latently-infected recipients (R+) receive transplant tissue from HCMV-naive donors (D-).

Human cytomegalovirus induces monocyte differentiation and migration as a strategy for dissemination and persistence.

Human cytomegalovirus (HCMV) pathogenesis is characterized by multiple organ system involvement due to viral spread to host organs after a cell-associated viremia. The cell type responsible for HCMV dissemination is unknown. Monocytes are the most likely candidate since they are the predominant cell type infected in the blood. However, monocytes are not productive for viral replication and are abortively infected. The results presented here provide a potential answer to this conundrum. We report that primary HCMV infection of monocytes induces transendothelial migration and monocyte-to-macrophage differentiation and that these HCMV-differentiated macrophages are productive for viral replication. Together, our data suggest a novel mechanism for HCMV pathogenesis; HCMV induces cellular changes in monocytes to promote viral replication and spread to host organs.

Identification of naive or antigen-experienced human CD8(+) T cells by expression of costimulation and chemokine receptors: analysis of the human cytomegalovirus-specific CD8(+) T cell response.

Human CMV (HCMV) infection provides an informative model of how long term human CD8(+) T cell memory is maintained in the presence of Ag. To clarify the phenotypic identity of Ag-experienced human CD8(+) T cells in vivo, we determined the expression of costimulation and chemokine receptors on Ag-specific CD8(+) T cells by quantifying individual virus-specific clones in different cell populations using TCR clonotypic probing. In healthy HCMV carriers, expanded CD8(+) clones specific for either HCMV tegument protein pp65 or immediate-early protein IE72 are found in both CD45RO(high) cells and the subpopulation of CD45RA(high) cells that lack the costimulatory molecule CD28. In contrast to previous suggested models of CD8(+) T cell memory, we found that in healthy virus carriers highly purified CD28(-)CD45RA(high)CCR7(-) cells are not terminally differentiated, because following stimulation in vitro with specific HCMV peptide these cells underwent sustained clonal proliferation, up-regulated CD45RO and CCR5, and showed strong peptide-specific cytotoxic activity. In an individual with acute primary HCMV infection, HCMV pp65-specific CD8(+) T cells are predominantly CD28(-)CD45RO(high)CCR7(-). During convalescence, an increasing proportion of pp65-specific CD8(+) T cells were CD28(-)CD45RA(high)CCR7(-). We conclude that naive human CD8(+) T cells are CD28(+)CD45RA(high), express CCR7 but not CCR6, and are predominantly CD27(+) and L-selectin CD62 ligand-positive. The phenotype CD27(+)CD45RA(high) should not be used to identify naive human CD8(+) T cells, because CD27(+)CD45RA(high) cells also contain a significant subpopulation of CD28(-)CD27(+) Ag-experienced expanded clones. Thus CD8(+) T cell memory to HCMV is maintained by cells of expanded HCMV-specific clones that show heterogeneity of activation state and costimulation molecular expression within both CD45RO(high) and CD28(-)CD45RA(high) T cell pools.

Discordant detection of human cytomegalovirus DNA from peripheral blood mononuclear cells, granulocytes and plasma: correlation to viremia and HCMV infection

There exist only few data about the HCMV infection of single positive leukocyte subtypes in immunosuppressed patients. Most reports describe HCMV coinfection of cells of the myelomonocytic line or even T- and B-cell populations. Correlation of positive PCR findings from two major leukocyte fractions and plasma to viremia and HCMV infection in general should contribute to select suitable sources of HCMV DNA for diagnostic purposes. The diagnostic value of qualitative leukoDNAemia of simultaneously isolated peripheral blood mononuclear cells (PBMC), granulocytes as well as plasmaDNAemia was evaluated by comparing the positive results of nested PCR from blood with virus isolation either from leukocytes or from any other sources, with serology and the clinical status of immunosuppressed patients. PBMC, granulocytes and plasma were prepared of a total of 220 blood samples of 75 immunosuppressed patients with clinically suspected primary or recurrent HCMV infection. In a collective of 35 patients consisting mainly of recipients of marrow or solid organ transplants positive results of leuko- or plasmaDNAemia were correlated with data from HCMV screening and the clinical status. For standardization, HCMV IE Exon 4 DNA was amplified from 100 ng cellular DNA of each leukocyte population. Cross contamination can be excluded. DNA from plasma was extracted by phenol/chloroform. Using this experimental design, HCMV DNA was not detectable in PBMC, granulocytes and plasma of 23 healthy HCMV seropositive blood donors. Leukocyte separation in a collective of 30 patients with positive leukoDNAemia revealed in only 12 cases (40%) double infection of PBMC and granulocytes. In the majority of cases (18 patients, 60%) however, HCMV DNA was detectable in only one leukocyte fraction, either in PBMC or granulocytes. LeukoDNAemia did not correlate to viremia. HCMV DNA amplified from plasma was shown to be cell free. Infectious virus from plasma was not isolated. The predictive value of qualitative nested PCR from blood to detect HCMV infection was high for plasma and decreased in the following sequence: plasma (0.92) > PBMC (0.83) > granulocytes (0.65). Qualitative nPCR from plasma and PBMC seems to be sufficient to detect (an ongoing) HCMV infection of immunosuppressed patients. However, the rate of single positive leukocyte fractions is approximately 60%. Therefore, viral leukoDNAemia in 40% of cases seems to be restricted to either PBMC or granulocytes. For diagnostic purposes the whole leukocyte population should be used for PCR analysis.

Specific IgE detected by ELISA and immunoblot after human cytomegalovirus infection (HCMV) in renal transplant (RT) recipients

Background: Specific HCMV IgE response has been reported by some authors, and was proposed as a valuable virologic marker of CMV infection. Objectives: we evaluated specific HCMV IgE in renal transplant patients with active (primary and secondary) HCMV infection with special interest to symptomatic infections. Study design: Specific IgE was tested retrospectively by ELISA and immunoblot (IB) on sera of 55 RT patients who were followed before and after transplantation with virologic markers of CMV infection. Results: Total serum IgE levels were similar in control group and in patients with primary and secondary HCMV infections. Anti-CMV specific IgE response by ELISA was more frequently found in patients with primary infection (76.9%) than in patients with secondary infection (47.1%). These specific IgE reacted on immunoblot with a 150 kDa protein in 84.6% of patients with primary infection and 94.1% with secondary infections; and reacted with rp52 (pUL44) in 76.9% of primary infection and 47.1% of secondary infection. Conclusions: Anti-CMV specific IgE tested by immunoblot and ELISA is a marker of CMV infection. It was clearly detected in cases of active infection (primary and secondary) and was present in cases with severe CMV clinical manifestations. In contrast, anti-CMV specific IgE, was consistently negative among healthy blood donors. This is the first report of CMV proteins detected by IgE immunoblot.

Histopathologic and Molecular Profile of Human Cytomegalovirus Infections in Patients with Heart Transplants

From November 1985 to December 1990, 2,552 endomyocardial biopsy specimens from 209 heart transplant patients were studied. Forty-four (21%) patients developed 45 episodes of major human cytomegalovirus infection (HCMV). Human cytomegalovirus infection was primary in 13 of 44 patients. Thirty-one patients developed episodes of recurrent major infection. One patient had both primary and recurrent infections. Conventional histopathologic and immunohistochemical study, in situ hybridization, and polymerase chain reaction were used to diagnose HCMV myocardial involvement on corresponding endomyocardial biopsy specimens performed during infection. Conventional morphologic study showed typical viral inclusion bodies in four biopsy specimens. Two cases had myocyte HCMV localization with necrotizing myocarditis, whereas two had endothelial cell involvement without any inflammatory reaction. In these four biopsy specimens, immunohistochemistry showed a higher number of infected cells than that recognized by conventional histopathologic study. In situ hybridization detected infected cells with no evidence of cytopathic effect. Polymerase chain reaction gave HCMV amplification products in two additional biopsy specimens otherwise interpreted as moderate and mild rejection, respectively. Therefore, 6 biopsies showed HCMV myocardial involvement (6 of 45; 13.3%): all were from patients with primary HCMV infection (6 of 13; 46%). None of 32 major recurrent infections showed any myocardial involvement. In conclusion, our study is the first to demonstrate that myocardial HCMV involvement preferentially occurs in primary infection and HCMV endothelial localization can be free from inflammatory reaction, whereas HCMV myocyte localization leads to necrotizing myocarditis. Polymerase chain reaction has a higher diagnostic sensitivity than in situ hybridization. However, polymerase chain reaction findings of HCMV DNA on otherwise negative endomyocardial biopsy specimens remains of questionable significance because polymerase chain reaction-positive biopsy samples do not necessarily indicate tissue infection. It is impossible to determine whether amplified sequences derive from circulating leukocytes or from tissue cells.

Influence of human cytomegalovirus on immune reconstitution after bone marrow transplantation.

HCMV infection diagnosed by the highly sensitive polymerase chain reaction (PCR) technology in blood, urine and skin biopsies of patients after bone marrow transplantation (BMT) correlated with the reconstitution of peripheral blood lymphocytes and dermal immunohistological alterations to evaluate the interaction of viral infection with the recovery of the immune system, as well as with the induction or aggravation of graft-versus-host disease (GVHD). In a prospective study 73% of 63 patients showed viremia at a median time of 25 days after BMT. Only 44% of these cases that also presented with a higher frequency of acute GVHD symptoms developed HCMB disease later on. In the skin, similar immunohistological alternations, as well as frequent primary local HCMV infection before the development of cutaneous signs of GVHD, was found, suggesting the direct involvement of anti-HCMV immune responses in the induction of GVHD-associated organ lesions.

Development and evaluation of a capture ELISA for IgM antibody to the human cytomegalovirus major DNA binding protein

A new capture ELISA (ELAb) for determination of the IgM antibody response to the human cytomegalovirus major DNA binding protein (p52) was developed by using a p52-specific monoclonal antibody. As a reference test, a capture ELISA using in parallel viral- and cell-control labeled antigens (ELA) was employed. General specificity, which was determined on 180 unselected IgM-negative sera from an adult population was 100%; stringent specificity, which was evaluated on 108 potentially interfering sera from patients with Epstein-Barr virus infectious mononucleosis, autoimmune diseases, rheumatoid factor or treated with radioimmunotherapy, was 96.3%; finally, clinical specificity, determined on 79 IgM-negative sera drawn prior to onset of primary HCMV infection, was 100%. Thus, the overall specificity was 98.9% ( 363 367 ) IgM negative tested sera). Sensitivity assayed on 277 IgM-positive sera was 100%. The study of the kinetics of the IgM antibody response in sequential blood samples from 9 immunocompetent and 9 heart transplanted patients showed that, while in the immunocompetent p52-specific IgM titer fell sharply 2–3 months after onset and was virtually undetectable 12 months after onset, in the immunocompromised the IgM response persisted for longer than a year. Recurrent HCMV infections were associated with a high titer IgM response in 6 (30%), and with a low IgM response in another 6 (30%) heart transplanted patients within a group of 20 patients sequentially examined. Finally, IgM antibodies were detected in all 4 infants with congenital infection and in 5 of 6 infants with neonatal infection. The results show that the HCMV p52-specific IgM antibody response parallels that obtained by ELA, thus representing a major component of it. ELAb is highly sensitive, specific and reproducible. It represents a major advance among capture ELISA techniques, allowing detection of IgM antibody reactive to a specific viral protein.