Antibody reactivity to human and vervet cytomegalovirus early antigen(s) in sera from patients with active cytomegalovirus infections and from asymptomatic donors.

Human fibroblasts were infected with vervet cytomegalovirus (VCMV) and cultured in medium containing 50 micrograms of cytosine arabinoside per ml for 72 h. Early antigens (EAg) were detected in the nuclei of infected cells by an indirect fluorescent antibody test with human sera having antibody to EAg of human cytomegalovirus (HCMV). Sera from patients with serological and/or virological evidence of active HCMV infection and from asymptomatic blood donors were examined for antibody activity with the HCMV and VCMV EAg's. The HCMV and VCMV EAg's were comparable in detecting levels of antibody activity and fluctuations in antibody titer of paired sera from patients. A total of 81% of patient sera reactive with HCMV EAg were also reactive with VCMV EAg. In contrast, only 14% of the asymptomatic donor sera reactive with HCMV EAg were also reactive with VCMV EAg. The VCMV EAg, therefore, appeared to differentiate latent from active infections in humans more effectively than did HCMV EAg.     

Detection of cytomegalovirus DNA in sera of liver transplant recipients.

We prospectively studied the utility of the amplification of cytomegalovirus (CMV) DNA in the sera of liver transplant recipients in order to predict symptomatic CMV infection, thus enabling preemptive therapy with antiviral agents. Serum samples obtained at biweekly intervals from 20 sequential liver transplant recipients for at least 8 weeks following transplantation were tested by the PCR amplification procedure. Results were correlated with blood and urine cultures, histopathological findings from infected organs, and clinical manifestations. Six patients (30%) developed symptomatic CMV infection; in five (83%) of these patients, CMV DNA was detected prior to symptomatic CMV infection, and in one (17%) of these patients, CMV DNA was detected at the time of symptomatic CMV infection. CMV DNA was detected a mean of 13 days (range, 0 to 23 days) prior to the onset of symptomatic CMV infection. In addition, CMV DNA was detected in the sera of four of five patients with asymptomatic viremia and two patients with asymptomatic viruria. Lastly, the PCR was negative for sera from seven patients with no evidence of CMV infection. We found that PCR was able to detect the presence of CMV DNA in the sera of liver transplant recipients at a sensitivity of 92% and a specificity of 100% for CMV infection, while the sensitivity and specificity for symptomatic infection were 100 and 57%, respectively.     

Immune complexes and antibodies to BCG in sera from patients with mycobacterial infections.

We have examined the prevalence of circulating immune complexes in sera from patients with mycobacterial infections. Sera from 68 percent of patients with active M. tuberculosis and 58 percent of patients with M. intracellulare infections had significantly elevated Clq binding activity (Clq-BA). In general there was a fall in Clq-BA with treatment. Only 15 percent of M. tuberculosis patient with a bacteriological cure, 22 percent of non-tubercular patients with chronic obstructive pulmonary disease, and 3 percent of normal individuals had an elevated Clq-BA. Antibodies to a BCG-derived antigen were demonstrated in most of the individuals studied in all groups but, significantly elevated levels were seen only in patients with mycobacterial infections. In certain patients there appeared to be an inverse relationship between Clq-BA and BCG binding, suggesting that perhaps BCG-related antigens participated in the immune complexes found. Other possible antigen-antibody complexes are discussed.     

Anti-spectrin in sera containing smooth muscle autoantibodies from patients with chronic active hepatitis.

Sera from patients with chronic active hepatitis containing anti-smooth muscle autoantibodies (SMA) react against rabbit muscle actin as well as human red cell spectrin. These anti-spectrin antibodies recognize the same antigen as rabbit anti-human spectrin antibodies and are not involved in the staining pattern given by SMA-containing sera tested with smooth muscle sections. These anti-spectrin antibodies probably recognize an antigenic structure common to both spectrin and another as yet undetermined molecule (which however is not likely to be myosin).     

Glomerulopathy in renal allografts from patients with and without active Cytomegalovirus infection

Cytomegalovirus (CMV) infection may affect renal graft survival, and a distinctive diffuse glomerulopathy has been described in renal grafts from patients with an active CMV infection. Fifty renal graft biopsies taken 30 to 180 days post-transplant from patients with primary, reactivated, inactive and no CMV infection were examined retrospectively for evidence of this glomerulopathy without knowledge of CMV status. The lesion was found in seven of 30 patients with reactivated CMV infection, one of 12 with primary infection, two of two with no evidence of active infection but evidence of previous infection, and in four of six with no evidence of past or present infection. The differences were not statistically significant. We concluded that the previously described distinctive diffuse glomerulopathy is not specifically associated with CMV infection, but may be a sign of graft rejection.     

Measles virus-specific IgM antibodies in sera from patients with chronic active hepatitis

The occurrence of measles virus-specific IgM antibodies in sera from patients with chronic active hepatitis not caused by Hepatitis B virus was examined by a specific enzyme-linked immunosorbent assay (ELISA). Using whole serum, specific IgM antibodies were detected in 12 of 23 sera from patients with hepatitis B surface antigen (HBsAg)-negative chronic active hepatitis. In nine of these sera the finding of specific IgM antibodies was verified by separation of IgM by density gradient centrifugation and examination of the fractions by ELISA. Most of the sera from the patients with measles virus-specific IgM antibodies had an elevated level of specific IgG antibodies compared to the level of IgG found in control sera. The significance of these findings in view of a possible persistent measles virus antigen production in patients with chronic active hepatitis is discussed.     

Immune response to herpes simplex virus infections: virus-specific antibodies in sera from patients with recurrent facial infections.

Radioimmunoprecipitation assays were used to identify antibodies against a number of herpes simplex virus type 1-specific antigens in serum samples from individuals with recurrent facial herpes virus infections and from seropositive individuals without recurrent infections. Individuals with recurrent infections contributed three sequential serum samples each: immediately after the appearance of lesions, 3 weeks later, and 3 months later. Antibodies against at least 18 viral polypeptides were present in all positive sera: these included antibodies against the major nucleocapsid polypeptide (approximate molecular weight, 150,000) and against two glycopolypeptides with molecular weights of 115,000 to 130,000. No significant differences were observed between the serum samples in regard to their virus-specific antibody composition. The high-molecular-weight glycopolypeptides were partially purified and used in quantitative titration experiments. All sera tested were equally reactive with this material. It was concluded that under the experimental conditions an individual's susceptibility to recurrent herpetic infections could not be correlated with quantitative or qualitative changes in the levels of virus-specific antibodies.     

Cytomegalovirus detection by biotinylated DNA probes

Clinical specimens from 317 patients suspected of cytomeglovirus infection were examined by immunofluorescence (IF) using monoclonal antibodies and by a biotinylated DNA probe kit after cell culture isolation. Of the 317 samples, 68 were positive by culture isolation. Of these 67 were IF positive when the cytopathic effect (CPE) was 1⁺ or less, whereas 56 gave positive results with DNA probes when the CPE was 2⁺. A further 83 specimens were examined directly by immunoperoxidase histopathology (IHP), IF and the DNA probe kit: 26 of these were positive by IHP examination, 25 by IF and only 6 by DNA probes. The sensitivity of the DNA probe kit was not satisfactory when the clinical tissue specimens were directly examined. However, the sensitivity improved considerably to 82% if the specimens were propagated first in cell culture. The IF method detected the virus before and after cell culture isolation equally well (96%–98.5%). Compared to the IF method, the DNA probe kit is costly and requires more labor and time.This paper was presented in part at the 88th annual meeting of the American Society for Microbiology, Miami Beach, FL, USA, 1988     

Cytomegalovirus infections in adult T-cell leukemia patients

Cytomegalovirus (CMV) infection frequently occurred in patients with malignant lymphoma of T-cell origin, especially with adult T-cell leukemia (ATL). This was evidenced by histopathological examination at autopsy, isolation of CMV, and detection of CMV antibodies that indicate recent or active infection. Cellular immune response was suppressed in most ATL patients when examined by skin hypersensitivity reaction to purified protein derivatives (PPD), streptococcal antigens (SuPs), and phytohaemoagglutinin (PHA). None of the CMV-positive patients reacted to them. Thus, the presence of tumor cells of T-cell origin and the absence of skin hypersensitivity reaction seem to be risk factors for CMV infection. Each CMV isolate exhibited unique DNA fingerprints, suggesting that cross-infection of CMV did not occur among the ATL patients on the same ward.     

Antibodies against membrane antigens of cytomegalovirus infected cells in sera of patients with a cytomegalovirus infection

The appearance of a new virus specific antigen was demonstrated by an indirect immunofluorescence technique on cell surfaces of CMV infected human fibroblasts, 48–72 hr after inoculation. The development of antibodies to these membrane antigens was followed in thirty-nine serial sera from twelve patients with a virologically and/or serologically confirmed CMV infection. In all patients except one, membrane antibodies could be detected. Sera from four patients collected before infection were negative, as were sera taken 1–13 days after onset of symptoms. From the end of the second week of illness CMV-membrane antibodies as well as CMV macroglobulin antibodies to intracellular antigens were detectable. The membrane antibodies persisted until 335 days after onset of illness. They were mainly of the IgM class. Most positive sera also reacted with non-infected fibroblasts but to a lesser degree. This reaction became negative after prior absorption of the sera with non-infected fibroblasts, whereas the reaction with CMV-infected cells remained positive.Controls consisted of forty-five sera from healthy persons and nine sera from patients suffering from other herpes virus infections with antibodies to herpes simplex, zoster/varicella and Epstein-Barr virus. All but two sera were negative in the membrane immunofluorescence test.     

Quantitative relationship between HBeAg and DNA polymerase activity in sera from patients with chronic active hepatitis during a three-month period

In eight patients with biopsy-confirmed chronic active hepatitis (CAH), hepatitis B e antigen (HBeAg) levels and DNA polymerase (DNA-P) activity were assayed three times a week for six weeks and once a week for another six weeks. HBeAg levels were rather constant, whereas DNA-P activity fluctuated. No correlation was observed between the quantities of HBeAg and DNA-P activity. An unexpected fluctuation in DNA-P activity was noted in all patients after an influenza vaccination.     

Immunoglobulin G subclasses and lymphocyte stimulatory responses to cytomegalovirus in transplant patients with primary cytomegalovirus infections.

The early activation of T- and B-cell responses to cytomegalovirus was studied in immunosuppressed patients. Primary lymphocyte stimulation to cytomegalovirus (CMV) antigen, a measure of T-helper activity, and anti-CMV IgG subclass responses were analyzed. Ten patients suffering from primary CMV infection following renal transplantation were studied. Of the ten, nine became positive for CMV induced lymphocyte proliferation 5-40 weeks after transplantation. Nine showed an almost simultaneous appearance of anti-CMV IgG1 and three at 3-32 weeks after transplantation, while one patient synthesized only low levels of anti-CMV IgG1. The lymphocyte proliferation assays have limited diagnostic value for primary CMV infection in renal transplant patients. The humoral and cellular immune responses seemed to be independent of each other.     

A recombinant topoisomerase I used for autoantibody detection in sera from patients with systemic sclerosis.

We reported three additional cases of a newly described syndrome called episodic angioedema with hypereosinophilia. In order to investigate its pathophysiological mechanisms, four parameters were concurrently investigated, including blood eosinophil density, serum chemoattractant activity, serum major basic protein (MBP) levels and the presence of anti-endothelial cell antibodies. Distribution of eosinophils through a metrizamide density gradient showed a preferential sedimentation of blood eosinophils in intermediate layers, clearly different from the hypodense cells (low-density layers) identified in a group of seven patients with idiopathic hypereosinophilic syndrome (HES). In two of the three patients with cyclic angioedema, a chemotactic activity towards eosinophils was detected in the serum (30 +/- 6 and 42 +/- 12 eosinophils per high-power field; P less than 0.05 compared with a control group). Serum MBP levels were at 1524, 619 and 1200 pg/ml. All three patients had circulating anti-endothelial cell antibodies, predominantly of the IgG isotype, in contrast to controls (P less than 0.01) or to patients with HES (P less than 0.01). Specificity of the antibody for endothelial cells was demonstrated in the three patients studied by the absence of binding to various blood cells, including monocytes, lymphocytes, eosinophils and platelets. In one case (patient 2), the levels of anti-endothelial cell antibodies, as well as the serum chemoattractant activity to eosinophils varied according to the successive acute phases of the disease. Although further investigations are needed to clarify the exact pathophysiology of this syndrome, and especially the possible participation of the anti-endothelial cell antibodies in the cutaneous lesions, these data suggest that angioedema observed in this syndrome could result from the combined effects of activated eosinophils and of immunologically induced endothelial lesions.     

Cytomegalovirus in urine: detection of viral DNA by sandwich hybridization.

A cytomegalovirus (CMV)-specific sandwich hybridization test was constructed by using two adjacent BamHI DNA fragments of CMV DNA as reagents. The fragments were cloned into two different vectors. One of the recombinants was attached to the filter, and the other was the labeled probe. When present in the sample, CMV DNA mediated labeling of the filter by hybridizing to both the filter-bound DNA and the probe. The sandwich hybridization test was applied for the detection of CMV DNA from urine. DNA was released from virus by 2% Sarkosyl, concentrated by 2-butanol extraction and isopropanol precipitation, denatured, and finally subjected to the sandwich hybridization test. As a result, 70 to 90% of the original viral DNA could be recovered and demonstrated by the quantitative hybridization reaction. Urine could be stored at room temperature in Sarkosyl for at least 2 days without affecting the detectability of CMV. The clinical applicability of the test was evaluated by studying urine samples from four infants excreting CMV. Sandwich hybridization demonstrated the presence of CMV DNA in all of the specimens. These contained originally 10(5) to 10(8) CMV DNA molecules per ml.     

Comparison of cytomegalovirus loads in plasma and leukocytes of patients with cytomegalovirus retinitis. The Cytomegalovirus Retinitis and Viral Resistance Study Group

Cytomegalovirus (CMV) DNA loads in paired leukocyte and plasma samples from 199 patient visits by 66 patients with CMV retinitis were determined. Leukocyte CMV load determinations had a greater range of values (mean, 24,587 copies/10(6) leukocytes; maximum, 539, 000) than did plasma CMV load determinations (mean, 10,302 copies/ml; maximum, 386,000), and leukocyte viral loads were detectable in a greater proportion of patients at the time of diagnosis of CMV retinitis prior to initiation of anti-CMV therapy (82%) than were plasma viral loads (64%) (P = 0.0078). Agreement with CMV blood cultures was slightly better for plasma (kappa = 0. 68) than for leukocytes (kappa = 0.53), due to a greater proportion of patients with detectable viral loads in leukocytes having negative blood cultures.     

Lack of correlation between virus detection and serologic tests for diagnosis of active cytomegalovirus infection in patients with AIDS.

The aim of this study was to evaluate the usefulness of serologic analysis for the diagnosis of active cytomegalovirus infection in patients with AIDS. Active cytomegalovirus infection was diagnosed by virus isolation from urine and saliva and detection of antigenemia. Serologic analysis was done by several conventional and innovative procedures. The results indicate no correlation between any of the most popular serologic procedures and virus detection by culture in urine or saliva or antigenemia.     

Cytomegalovirus infections in the human population.

Diagnostic and clinical experience gained in the period from 1966--1977 is summarized. Cytomegalovirus was isolated from 35 sucklings and 5 infants aged more than 1 year as well as from necropsy materials from 10 sucklings. The most frequently involved were the respiratory tract (77.8%), liver (53.5%) and haematopoetic apparatus (42.2%). Congenital defects were found in 22.2%. The incidence in the population varied from 20% in sucklings to 50--60% in persons aged over 40 years. The incidence of infection was higher in sucklings and infants in nurseries. Out of 37 adult patients with kidney transplantation, the virus was isolated from 10 (27%) and complement-fixing antibodies were found in 34 (91%) patients.