Human cytomegalovirus (HCMV) leukodnaemia correlates more closely with clinical symptoms than antigenemia and viremia in heart and heart-lung transplant recipients with primary HCMV infection

BACKGROUND: In the last few years, human cytomegalovirus (HCMV) viremia, pp65 antigenemia, and leuko- and plasma-DNAemia have been developed to quantitate virus in blood of immunocompromised patients with HCMV infection. However, thus far, no conclusive studies have been performed to define the correlation of each of the different assays with clinical symptoms in primary HCMV infections. METHODS: This correlation was investigated in a population of 20 heart and heart-lung transplant recipients with primary HCMV infection using standardized virological methods. RESULTS: Median peak HCMV viremia, antigenemia, and leukoDNAemia levels were 110 (0-2,000) p72-positive fibroblasts, 450 (27-2,000) pp65-positive leukocytes, and >10,000 (1,358-10,000) genome equivalents (GE) in the 14 symptomatic patients and 18 (1-130) p72-positive fibroblasts, 86.5 (5-350) pp65-positive leukocytes, and 248 (10-863) GE in the six asymptomatic patients, respectively. The difference was statistically significant for antigenemia (P=0.009) and leukoDNAemia (P<0.0001). However, on an individual basis, unlike viremia and antigenemia, all DNA peaks of the 6 asymptomatic patients were below the DNA range of the 14 symptomatic patients (<1,000 GE), while all the 14 symptomatic patients had DNA peaks higher than those of asymptomatic patients (>1,000 GE). Follow-up confirmed these results, showing that 1,000-2,000 GE was the threshold zone for emergence of clinical symptoms. Symptoms were never observed in patients with secondary DNA peaks, except for one patient suffering from an HCMV organ localization (HCMV gastritis). CONCLUSIONS: LeukoDNAemia is the viral parameter of choice for monitoring of primary HCMV infections and antiviral treatment in heart and heart-lung transplant recipients. In this patient population, antigenemia-guided preemptive therapy could be replaced by leukoDNAemia-based antiviral therapy.     

In vitro generation of human cytomegalovirus pp65 antigenemia, viremia, and leukoDNAemia

Immunocompromised patients with disseminated human cytomegalovirus (HCMV) infection have circulating PMN carrying HCMV pp65 (antigenemia), infectious virus (viremia), and viral DNA (leukoDNAemia). Because HCMV does not fully replicate in PMN, it is generally hypothesized that virions and viral materials are taken up by phagocytosis from fully permissive HCMV-infected endothelial cells. However, no experimental evidence has ever been provided for these PMN-endothelium interactions. PMN from 11 donors were cocultured with endothelial cells infected with an endothelium-adapted HCMV strain and with human fibroblasts infected with low-passaged clinical and laboratory-adapted HCMV strains. pp65-positive PMN were detected after coculture with both HCMV-infected endothelial and fibroblast cells, provided that wild and not laboratory-adapted strains were used. In addition, cocultured PMN carried infectious virus as demonstrated by virus isolation and presence of complete virus particles by electron microscopy. Moreover, high levels of viral DNA were consistently detected by quantitative PCR in cocultured PMN. Thus, we have generated in vitro the three most important viral parameters detected in patients with disseminated HCMV infection (antigenemia, viremia, and leukoDNAemia). The failure of laboratory-adapted HCMV strain to induce this phenomenon demonstrates that important modifications have occurred in attenuated viral strains affecting basic biological functions.     

Molecular analysis of human immunoglobulin heavy chain variable genes (IgVH) in normal and malignant B cells

The diagnostic value of monitoring human cytomegalovirus (HCMV) late pp67 mRNA expression by nucleic acid sequence-based amplification (NASBA) after renal-allograft transplantation was evaluated. RNAs were isolated from 489 whole-blood specimens of 42 patients for the specific amplification of the late pp67 (UL65) mRNA. NASBA results were compared to results from the pp65 antigenemia assay, virus isolation by cell culture, and serology. The sensitivity value for NASBA proved to be higher than that for the antigenemia assay (50 versus 35%) for the detection of HCMV infection, while the sensitivity values of cell culture and NASBA were comparable (54 and 50%, respectively). NASBA detected the onset of HCMV infection simultaneously with cell culture and the antigenemia assay. Both the antigenemia assay and NASBA are very specific (100%) and highly predictive (100%) for the onset of HCMV infection. Antiviral therapy with ganciclovir resulted in negative results for cell culture, the antigenemia assay, and NASBA. In conclusion, monitoring HCMV pp67 mRNA expression by NASBA is a highly specific method for the detection of HCMV infection in renal-allograft recipients and is more sensitive than the antigenemia assay. Furthermore, NASBA can be used to monitor the progression of HCMV infections and the effect of antiviral therapy on viral activity.     

A short region from the LEU2 gene of Saccharomyces cerevisiae functions as an ARS in the yeast Saccharomyces exiguus Yp74L-3

Human cytomegalovirus (HCMV) infection can result in neurological symptoms. In vitro replication of the HCMV was studied in primary cultures of microglial cells from the central nervous systems (CNS) of human embryos. The microglial cells were infected with various amounts of either the AD169 laboratory HCMV strain or a clinical HCMV isolate. A specific cytopathic effect occurred within 24 h and persisted for two months. Immunocytochemical tests for immediate early and late viral antigens done one and three days after the infection demonstrated that 60% to 80% of the microglial cells were infected and that 3% to 8% were the site of viral DNA replication. Kinetic studies showed accumulation of viral particles in the supernatant during the first two weeks after the infection. Prestimulation of the cells by PMA 24 h before the infection was associated with increased release of viral particles and with an increased percentage of cells expressing late viral antigens. The microglial cells of the human embryonic CNS are fully permissive targets for the HCMV. The in vitro HCMV model used in this study may prove useful for investigating the pathophysiology of HCMV encephalitis, in particular after mother-to-fetus transmission of the virus.     

Fine structure of extracellular matrix and basal laminae in two types of abnormal collagen production: L-proline analog-treated otocyst cultures and disproportionate micromelia (Dmm/Dmm) mutants

The diagnostic implications of different procedures of DNA extraction were examined for the detection of HCMV DNA from sera and plasma of immunosuppressed patients. The detection limit of HCMV plasmid DNA from cell free seronegative plasma and serum by limiting dilution nested PCR decreased in the following sequence: phenol/chloroform > NaI-single tube method > proteinase K digestion equal to amplification of native sera and plasma. Nested PCR from native sera and plasma performed well and surpassed the proteinase K method in sensitivity for detection of serum DNAemia. Semi-quantitative determination of HCMV DNA titer present in native sera of immunosuppressed patients did not seem to be correlated to HCMV disease. When compared to the persistence of leukoDNAemia, the viral DNA titer in native plasma could only be observed in the acute phase of HCMV infection, an important phenomenon for diagnostic purposes. Correlation of serum DNAemia to virus culture revealed low positive and high negative predictive values. Predictive values of nested PCR from native sera for HCMV infection were not lower than those following organic DNA extraction. Despite its low correlation to viremia and virus isolation from any site, nested PCR from organic DNA extracts of serum or plasma is the most sensitive diagnostic tool of an ongoing HCMV infection. Additionally, semi-quantitative end point dilution nested PCR from native serum or plasma promises to be a rapid and easy tool for the monitoring of antiviral therapy.     

Enhanced cytopathic effect of human cytomegalovirus on a retinal pigment epithelium cell line, K-1034, by serum-free medium

Although human cytomegalovirus (HCMV) predominantly infects epithelial cells in vivo, the majority of studies of HCMV gene expression and replication have been conducted using non-epithelial cell lines in part because of the absence of a good experimental system using epithelial cells. To address the nature of epithelial cell infection, we investigated the susceptibility of an epithelial cell line (K-1034) established from the retinal pigment epithelium to HCMV infection. This cell line exhibited high susceptibility to HCMV, as evidenced by detection of one of the immediate early antigens, IE2, in the nuclei of more than 80% of K-1034 cells at 24 h following inoculation at a multiplicity of infection of 3 plaque forming units per cell. However, the yield after one-step growth of HCMV in K-1034 cells was about twenty-fold less than that in human embryonic lung fibroblast cells. Cytopathic effect (CPE) on K-1034 cells was not prominent in medium supplemented with 10% fetal bovine serum and viral late antigens were detected in less than 5% of K-1034 cells. Interestingly, infected cells expressing late antigens and exhibiting CPE were markedly increased in serum-free medium, even though the yield of infectious HCMV and viral genome copy numbers were almost the same in the different serum concentrations, due to viral instability in the absence of serum. Thus, the progression of late antigens expression and the induction of CPE in infected epithelial cells is influenced by physiological conditions, and are negatively regulated by some serum factor.     

Viral serology and detection

Viral detection is an important part of clinical hepatology. For many years practical clinical tests have been serological but recently newer molecular techniques have become available for virus detection, although these have yet to become routine and some, such as PCR of viral nucleic acid in blood or tissue are not yet consistently reliable. Serology remains the mainstay at present for routine diagnosis. Hepatitis A testing in clinical practice is entirely serological, the IgM response representing acute infection and the IgG response immunity, although more sophisticated molecular techniques have been applied experimentally. A second agent of epidemic enteral hepatitis, the hepatitis E virus, has recently been cloned and sequenced and serological tests for this virus are available, although experience in their use is necessarily limited and a commercial IgM assay has yet to be produced. Serological tests for the hepatitis B virus are well developed. The IgM anticore response differentiates acute infection from chronic, the latter being characterized by the persistence of hepatitis B surface antigen for over six months. Chronic carriers are at risk of liver damage and this risk is best assessed by the amount of viral DNA circulating, which can be determined using a hybridization assay. More sensitive techniques such as the branched chain DNA assay or PCR can detect lower levels of viral DNA but their clinical relevance remains to be established. The hepatitis D virus is defective and relies on hepatitis B to replicate. Serology for antibody and antigen is well established although PCR for circulating viral genome may come to supplant hepatic viral antigen as a test for hepatitis D replication. For hepatitis C serology is feasible only for antibodies, not antigens; although early tests were prone both to false positives and false negatives, current versions are more reliable. PCR has been much used for detection of hepatitis C RNA in blood and tissues and a bDNA assay is now commercially available. Cytomegalovirus detection is confounded by the problem of distinguishing asymptomatic viral replication from disease. Serology is helpful, especially in primary infections, but viral culture is a widely used method. PCR (especially quantitative modifications) or the pp65 antigenaemia assay are experimental approaches which may prove specific enough for general use.     

Human cytomegalovirus-infected cells have unstable assembly of major histocompatibility complex class I complexes and are resistant to lysis by cytotoxic T lymphocytes

Viruses which cause persistence in the naturally infected host are predicted to have evolved immune evasion mechanisms. Human cytomegalovirus (HCMV) causes significant morbidity and mortality in immunocompromised patients yet persists without clinical manifestations in seropositive individuals who have normal immune function. We report that HCMV infection in vitro impairs major histocompatibility complex class I (MHC-I) assembly accompanied by resistance to killing by cytotoxic CD8+ T lymphocytes. Pulse-chase metabolic labelling experiments show that MHC-I complexes continue to be assembled by both uninfected and HCMV-infected cells. However, MHC-I molecules are unstable in HCMV-infected cells and are rapidly broken down. Endoglycosidase H treatment of immunoprecipitates indicates that the breakdown of MHC-I complexes in HCMV-infected cells occurs primarily in a pre-Golgi compartment. Interference with normal MHC-I assembly and expression, if relevant in vivo, may have implications for the restriction of the diversity of the CD8+ cytotoxic T lymphocyte repertoire directed against HCMV antigens and may be an important mechanism of viral persistence.     

Primary HIV infection

Up to 70% of individuals with primary HIV infection will develop symptoms of an acute illness. The most common symptoms reported are fever, generalized lymphadenopathy, arthralgia and myalgia, headache, pharyngitis, enanthema, skin rash, diarrhoea, and mucocutaneous ulcerations. More rarely, oesophageal candidiasis, meningoencephalitis, rhabdomyolysis and epiglottitis have been reported. The diagnosis of the acute HIV infection syndrome can be established by demonstrating antibodies to HIV or by demonstration of HIV antigen positivity. Detection of virus through culture or PCR may prove to be more sensitive, but are not yet used as routine methods. The course of the primary infection has prognostic importance for the subsequent course of HIV infection. This probably reflects the importance of both the viral phenotype and of the initial immune response to HIV. Primary HIV infection should be considered in any patient with possible exposure to HIV presenting with fever of unknown cause.     

Acute bronchitis

Acute bronchitis is a lower respiratory tract infection that causes reversible bronchial inflammation. In up to 95 percent of cases, the cause, is viral. While antibiotics are often prescribed for patients with acute bronchitis, little evidence shows that these agents provide significant symptomatic relief or shorten the course of the illness. In a few small studies, bronchodilators such as albuterol have been found to relieve some symptoms of acute bronchitis. Increased attention is being given to the role of Chlamydia species in acute bronchitis and adult-onset asthma. Studies in progress may help to clarify the importance of these organisms in acute bronchitis and to determine whether early treatment can prevent or ameliorate asthma.     

A rapid and sensitive radioimmunoassay for the detection of human cytomegalovirus binding and infection of human fibroblasts

A rapid and sensitive radioimmunoassay for the quantitation of HCMV binding and infection of human fibroblasts (HFF) was developed. The protocol involves the use of a monoclonal antibody (27-156) reactive with HCMV gB (alpha-gB), followed by an 125I-labeled second antibody to mouse IgG. Antibody to gB bound specifically to HFF inoculated with HCMV when compared to sham inoculated cells or cells inoculated with HSV (strain KOS). Antibody to gB also bound to HFF infected with HCMV 48 h prior to assay. The binding of antibody to HFF inoculated with HCMV was found to be dependent on antibody concentration and to demonstrate saturable kinetics. Moreover, antibody binding was directly dependent on the concentration of the virus inoculum, using either conventional viral preparations or gradient purified HCMV. The binding of antibody to HFF inoculated with HCMV at 4 degrees C was found to be dependent on antibody concentration and to demonstrate saturable kinetics. Displacement of HCMV binding to HFF with the proteoglycan heparin sulfate could be detected, thus allowing for competitive binding studies. This binding assay allows for the relative quantitation of HCMV binding to cells and will be useful for examining the early events of cell-viral interactions.     

Human cytomegalovirus persistently infects aortic endothelial cells

Endothelial cells (EC) have been implicated as constituting an important cell type in the pathogenesis of human cytomegalovirus (HCMV). Microvascular and macrovascular EC exhibit different biochemical and functional properties depending on the organ of origin. Phenotypic differences between microvascular and macrovascular EC may alter the ability of these cells to support HCMV replication. In this study, we compared the replication of HCMV in primary macrovascular aortic EC (AEC) with that in brain microvascular EC (BMVEC). An examination of IE72, pp65, and gB viral antigen expression in BMVEC and AEC by immunoflourescence revealed similar frequencies of infected cells. Intracellular production of virus was 3 log units greater in BMVEC than in AEC, while equal quantities of extracellular virus were produced in both cell types. HCMV infection of BMVEC resulted in rapid cellular lysis, while the virus was nonlytic and continuously released from HCMV-infected AEC for the life span of the culture. An examination of infected cells by electron microscopy revealed the formation of abundant nucleocapsids in both AEC and BMVEC. However, significant amounts of mature viral particles were only detected in the cytoplasm of BMVEC. These observations indicate that levels of HCMV replication in EC obtained from different organs are distinct and suggest that persistently infected AEC may serve as a reservoir of virus.     

Viral and immunologic aspects of Epstein-Barr virus infection in pediatric liver transplant recipients

Pediatric allograft recipients in particular are at increased risk for Epstein-Barr virus (EBV)-associated disorders. Early identification and diagnosis of EBV-associated disorders is critical, since disease progression can often be halted by reduction of immunosuppression. In this study we examined viral and immunologic parameters of EBV infection in the circulation of pediatric liver recipients to identify factors associated with disease. Peripheral blood DNA from pediatric liver recipients was analyzed by PCR for the EBV genes coding for the nuclear antigen 1 (EBNA-1) and the viral capsid antigen gp220. Sequences for these viral genes could be readily detected in the circulation of 36.5% of patients. Moreover, identification of the EBV genome was associated with symptomatic infection, suggesting that circulating EBV may be a useful marker of disease. Since EBV-infected B cells release the low-affinity IgE receptor (sCD23), we measured sCD23 in the circulation of pediatric liver recipients and found it to be elevated in patients with detectable virus or symptoms of infection. However, sCD23 was also elevated in cases where no EBV was detectable, suggesting that factors other than viral infection could stimulate release of sCD23. To further characterize the immune response to EBV infection, the peripheral levels of IL-4, IL-5, IL-10, and IFN-gamma were determined in pediatric liver recipients. Each of these cytokines was elevated in patients with symptoms or circulating virus compared with stable, age-matched liver recipients. IL-4, in particular, was significantly increased, indicating an important role for this cytokine in EBV infection. Together, these findings suggest that (1) monitoring circulating levels of EBV may be useful in patients at high risk and (2) cytokines that promote B cell growth and differentiation contribute to EBV-associated disorders.     

Recombinant mono- and polyantigens to detect cytomegalovirus-specific immunoglobulin M in human sera by enzyme immunoassay

Serological detection of human cytomegalovirus (HCMV)-specific antibody varies greatly because of antigen composition and the lack of antigen standardization. Antigenic materials composed of single well-characterized viral proteins or portions of them, produced via molecular biology, have proven to be promising tools in improving serodiagnosis. We constructed a recombinant protein containing two regions of ppUL32 (p150) and half of ppUL44 (p52) and compared the immunoglobulin M (IgM) reactivity of this triple-antigen fusion protein with that of a double-antigen fusion protein containing the two ppUL32 fragments and that of a monoantigen fusion protein containing half of ppUL44. We also constructed and tested two other monoantigen fusion proteins containing a large fraction of ppUL80a and a fraction of ppUL83. More than 700 serum samples from different groups of immunocompetent and immunosuppressed subjects were tested for the presence of HCMV IgM by recombinant enzyme immunoassay (rec-EIA) and by a commercially available EIA. Western blotting (immunoblotting) and (in the case of immunosuppressed individuals) antigenemia tests by immunofluorescence and PCR of polymorphonuclear leukocytes were also carried out. The results obtained demonstrate that (i) the triple-antigen fusion protein can replace the individual proteins; (ii) the triple-antigen fusion protein cannot be used alone to replace the virus or infected cells in the serological detection of anti-CMV IgM; (iii) the addition of the fusion proteins containing portions of ppUL83 and ppUL80a is essential for the formation of an antigenic mixture that can replace the virus for the search of HCMV-specific IgM; (iv) rec-EIA is very specific and is more sensitive than the commercially available EIA, and the results obtained are consistent with those obtained by Western blotting; and (v) rec-EIA can reliably be used to detect HCMV-specific IgM in different groups of patients with active HCMV infection.     

Use of recombinant human antibody fragments for detection of cytomegalovirus antigenemia

The determination and quantitation of peripheral blood leukocytes (PBLs) expressing human cytomegalovirus (HCMV) antigens is widely employed in clinical virology for rapid diagnosis of HCMV-related infections. We describe how CMV antigenemia may be accurately detected by means of human recombinant monoclonal Fab fragments rescued from a combinatorial phage display library prepared from an HCMV-infected donor. Fourteen recombinant Fabs were tested against HCMV-positive PBLs from a patient with ongoing HCMV infection. Three clones were found to react specifically with the nuclei of these cells. These three recombinant Fabs were subsequently tested, individually and pooled together, against 60 PBL samples taken from immunosuppressed patients. The reactivity observed was comparable to that obtained with mouse monoclonal antibodies commercially available for this purpose. The three recombinant Fabs were shown to react specifically with the 65-kDa viral tegument phosphoprotein encoded by UL83 (pUL83), which is the most abundant viral antigen in HCMV-infected PBLs.     

Summary of the II International Symposium on Cytomegalovirus

Human cytomegalovirus (HCMV) is a highly species-specific DNA virus belonging to the Betaherpesvirinae subfamily of the herpesviridae family. Like other herpesviruses, primary infection with HCMV is followed by persistence of the virus in a latent form. The sites of latency are still largely undefined, but they probably include bone marrow progenitor cells and peripheral blood monocytes. From these sites, the virus can reactivate, resulting in renewed shedding of the virus, or, in immunocompromized persons, development of disease. Humans are the only reservoir of HCMV and transmission occurs by person-to-person contact. Infection with HCMV is common. In most developed countries, HCMV seroprevalence steadily increases after infancy and 10-20% of children are infected before puberty. In adults, the prevalence of antibodies ranges from 40 to 100%. Although HCMV has a world-wide distribution, infection with HCMV is more common in the developing countries and in areas of low socioeconomic conditions, which is predominantly related to the closeness of contacts within these populations. Except for a mononucleosis-like illness in some persons, infection with HCMV rarely causes disease in immunocompetent individuals. However, HCMV can cause severe morbidity and mortality in congenitally infected newborns and immunocompromized patients, most notably transplant-recipients and HIV-infected persons. This article provides a review of the information presented at the Second International Symposium on Cytomegalovirus organized and convened by The Macrae Group (New York City, NY) in Acapulco, Mexico on 24-28 April 1998. During this symposium, the state-of-the-art knowledge on diagnosis, treatment and prophylaxis of HCMV infections were discussed, and, based on this information, attempts to highlight the future directions in basic and clinical research areas that need to be stimulated to facilitate advancement in prevention and treatment of CMV disease.     

Diagnosis of cytomegalovirus infections in patients with HIV infection. Comparison of the indirect immunofluorescence and viral isolation techniques

The technique for the detection of fluorescent early antigen was compared to the classical method of viral isolation in cells from the human lung. The study was performed with the use of 85 urine samples from 64 patients presenting with HIV infection. The technique for the detection of fluorescent early antigens showed a sensitivity of 91%, an specificity of 97%, and a coincidence of 94% with respect to viral isolation. The main advantage of the technique for the detection of fluorescent early antigens with respect to viral isolation is that the former provides a quicker diagnosis of cytomegalovirus infection within 48-72 hours, besides being easy to perform.