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.     

Standardization of the human cytomegalovirus antigenemia assay by means of in vitro-generated pp65-positive peripheral blood polymorphonuclear leukocytes

We generated in vitro human cytomegalovirus (HCMV) pp65-positive polymorphonuclear leukocytes (PMN) resembling those detected in vivo, following cocultivation of PMN from healthy donors and wild-type HCMV-infected endothelial cells or fibroblasts. After purification, PMN are suitable for preparation of cytospots which can be used for the antigenemia assay. Cytospin preparations containing a predetermined number of in vitro-generated pp65-positive PMN were used to test some of the major parameters involved in performing the antigenemia assay. The results showed or confirmed that (i) formalin fixation followed by permeabilization is the best fixation procedure developed to date, (ii) the test performance levels provided by different pools of pp65-specific monoclonal antibodies may be significantly different, and (iii) long-term storage (for an unlimited time) is best achieved by keeping fixed slides at -80 degreesC, whereas short-term storage (for up to 1 month) is best achieved by keeping unfixed slides at room temperature. This finding signifies that slides can be shipped all over the world at room temperature. In conclusion, the newly developed procedure for in vitro generation of pp65-positive PMN will provide the basis for standardization of the HCMV antigenemia assay and development of quality control programs.     

Novel DNA assay for cytomegalovirus detection: comparison with conventional culture and pp65 antigenemia assay

We compared conventional cytomegalovirus (CMV) isolation, rapid viral culture, a CMV pp65 antigenemia assay, and a novel CMV DNA hybrid capture system (HCS). A total of 309 blood samples from individuals in different risk groups were assessed by at least two of the methods mentioned above. Leukocytes were recovered either after centrifugation in Leucosep tubes containing 1.080 Ficoll for pp65 assay or after simple differential lysis steps for DNA detection. HCS was based on DNA hybridization with a CMV RNA probe and its capture by antibodies to DNA-RNA hybrids. The CMV pp65 lower matrix protein was detected by fluorescence with c10-c11 monoclonal antibody in formalin-fixed leukocytes. Concordant results were observed for 92.9, 78.3, and 82.7% of the patients when comparing (i) viral culture and the pp65 antigenemia assay, (ii) viral culture and HCS, and (iii) the pp65 antigenemia assay and HCS, respectively. Discordant results were observed between a positive HCS result and negative culture and/or pp65 results. These results were associated with relatively low DNA levels (< 20 pg/10(6) cells) and positive viruria. In conclusion, the pp65 antigenemia assay is a rapid and reliable method of detecting CMV and is preferable to culture, but the Murex HCS appears to be more sensitive for CMV detection.     

Comparison of PCR and pp65 antigenemia assay with quantitative shell vial culture for detection of cytomegalovirus in blood leukocytes from solid-organ transplant recipients

This study compared PCR and an assay for cytomegalovirus (CMV) pp65 antigenemia (CMV-vue; INCSTAR Corp.) with a quantitative shell vial culture (QSVC) technique for the detection of CMV in serial blood specimens from 46 solid-organ transplant recipients. In a comparison based on 535 specimens tested by PCR and QSVC, CMV was detected by PCR in 41 and by QSVC in 37 of 43 recipients at risk of CMV infection. The mean number of days after transplantation of initial detection of CMV was 29.9 for PCR and 34.0 for QSVC (P = 0.01). The antigenemia assay was performed on 395 specimens, including 304 of those also tested by PCR. In these specimens, CMV was detected by the antigenemia assay, QSVC, and PCR in 30, 32, and 35 (respectively) of 38 patients at risk, with no statistically significant difference in the time to detection. Each of the assays detected CMV in similar proportions of patients with and without clinically significant CMV infection. PCR stayed positive longer after transplantation than the other assays but frequently returned to negative when more than 6 months had elapsed after transplantation. The antigenemia assay and PCR stayed positive longer after institution of antiviral therapy than QSVC. PCR can provide highly sensitive detection of CMV viremia, but a PCR assay for CMV is not yet available in kit form. The pp65 antigenemia assay and shell vial culture are quantifiable and comparable in sensitivity. Either is recommended for rapid detection of CMV in blood specimens from solid-organ transplant recipients.     

Use of the cytomegalovirus antigenemia (CMV-Ag) assay for the detection of CMV in the blood of AIDS patients

Direct specimen testing was performed on 186 peripheral blood specimens to identify the presence of antigen to cytomegalovirus (viz., the cytomegalovirus antigenemia (CMV-Ag) assay). Confirmatory testing was performed using the shell vial indirect immunofluorescence assay (SVA-IFA), the indirect immunoperoxidase assay (TC-IPA), and conventional tube culture isolation (TC-CPE). The primary reagent for the CMV-Ag assay consisted of anti-CMV monoclonal antibody directed against the internal matrix structural phosphoprotein (1C3; Clonatec-Biosoft, France). The 72-kDa early nuclear antigen (Dupont) was utilized in the SVA-IFA and the TC-IPA. All test systems received an equal number of polymorphonuclear leukocytes in the inoculum. CMV was detected and isolated from 30% (55/186) of the specimens evaluated by either one or a combination of the tests. Detection and (or) isolation of CMV from blood by the CMV-Ag assay, SV-IFA, TC-IPA, and TC-CPE occurred at a rate of 17 (31/186), 12 (22/186), 16 (29/186), and 26% (49/186). Three of 55 positive specimens were identified only by the CMV-Ag assay; each patient in question, however, had at least one previous CMV isolate. No significant differences in sensitivity occurred between the CMV-Ag assay, the SVA-IFA, or the TC-IPA. However, TC-CPE including the blind passage of all negative tube cultures yielded a significantly larger number of positive blood specimens than either of the rapid detection methodologies. The CMV-Ag assay encompasses the benefits of a nonculture system, is simple to perform and easy to read, permits a same-day diagnosis, and requires less reagents than the routinely used SVA-IFA or TC-IPA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison between 2 monoclonal antibodies (clones 95/12 and 1C3 + AYm-1) for the detection of pp65 antigenemia associated with human cytomegalovirus

BACKGROUND: A prospective parallel and blind comparative study was carried out to evaluate the diagnostic efficacy of two available anti-pp65 monoclonal antibodies (clone 95/12 and the pool 1C3 + AYM-1) for the cytomegalovirus (CMV) antigenemia assay. MATERIAL AND METHODS: We carried out a comparative study of 107 blood samples from immunodepressed patients (renal transplant and AIDS patients) with suspected disseminated infection by CMV. The PMNLs were obtained using the method of sedimentation in saline dextran. Slides were stained by an indirect immunofluorescence assay with two commercially available monoclonal antibodies. RESULTS: Of the 107 blood samples studied 33 (30.8%) had a positive antigenemia test. The clone 95/12 detected 30 (90.9%) samples and the pool 31 (93.9%), no statistically significant difference was observed in the sensitivity of two reagents (p = 0.42). The values of the mean CMV-positive cell count obtained with the clone 95/12 was 60.6 vs 61.9 with the monoclonal pool (p = 0.026). CONCLUSIONS: No significant difference was detected between the two commercial monoclonal antibodies. However the pool detected a slightly superior CMV-positive cell count.     

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.     

Predictive value of cytomegalovirus (CMV) antigenemia and digene hybrid capture DNA assays for CMV disease in human immunodeficiency virus-infected patients

Oral ganciclovir prophylaxis decreases the incidence of cytomegalovirus (CMV) disease among persons infected with the human immunodeficiency virus (HIV), but universal prophylaxis is not cost-effective. We evaluated urine and peripheral blood mononuclear cell cultures, a qualitative and quantitative antigenemia assay, and a commercially available CMV DNA hybridization assay for their ability to predict CMV disease in 138 HIV-infected patients. During a median follow-up of 10 months, 23 patients (17%) developed CMV disease. The sensitivity, specificity, positive predictive value, negative predictive value, and mean lead times for the antigenemia assay (with use of a threshold of 8 positive cells per 10(5) peripheral blood mononuclear cells as a positive) were 74%, 91%, 63%, 95%, and 95 days, respectively. Corresponding figures for the DNA hybridization assay were 91%, 64%, 34%, 97%, and 152 days. These assays can identify patients at increased risk of CMV disease and should allow a strategy of preemptive therapy to be tested.     

Comparison of culture and the antigenemia assay for detection of cytomegalovirus in blood specimens submitted to a reference laboratory

We compared the antigenemia assay (AA) with tandem shell vial cultures (SVCs) and tube cultures (TCs) for detection of cytomegalovirus (CMV) in 343 blood specimens. For 249 specimens, the AA was performed in duplicate with two different commercially available monoclonal antibody reagents (Biotest Diagnostic Corporation and Argene Biosoft). Specimens considered true positives were positive in either culture system or both AAs. Only specimens which were negative in both cultures and positive in a single AA were tested retrospectively with a CMV PCR assay. CMV recovery rates were also calculated to determine if increased specimen age resulted in decreased positivity. CMV recovery rates for the AA and the combination of both cultures were 20.0 and 5.0% at 3 to 18 h, 20.2 and 14.0% at 18 to 35 h, 12.5 and 7.8% at 36 to 52 h, and 18.8 and 6.3% at 64 to 75 h, respectively. The sensitivities and specificities of the Biotest AA, the Argene AA, SVC, and TC were 84.4 and 100.0, 100.0 and 99.6, 44.4 and 100.0, and 46.0 and 100.0%, respectively. The AA was significantly more sensitive than either culture method alone and was also more sensitive than the two culture methods used in tandem (the tandem culture sensitivity was 63.5%); the Argene AA identified more positives than the Biotest AA.     

Characterization of the M(r) 65,000 lymphokine-activated killer proteins phosphorylated after tumor target binding: evidence that pp65a and pp65b are phosphorylated forms of L-plastin

Nursing, over the past 2 decades, has been concerned with the development of theories which will inform practice and help to establish nursing as an emerging discipline. The view that the individual is a biopsychosocial being has occupied a prominent position in the existing catalogue of nursing theories. The literature reveals that, although some existing theories do acknowledge that humans do have the advantage of a spiritual dimension, very little significant work appears to have been done in this area. This paper explores some of the difficulties in arriving at a definition of spirituality and goes on to examine the extent to which nursing theories incorporate the concept of spirituality. Additionally, nurse education does not appear to prepare practitioners adequately to meet the spiritual needs of patients. The paper concludes with a discussion of the potential for meeting the spiritual needs of patients in the context of the changes occurring in the British National Health Service.     

Comparative analysis of equine lymphocyte subsets in whole blood and gradient-purified samples

In the present study, two methods of lymphocyte preparation, whole blood lysis and Ficoll-Paque separation, prior to FACS analysis were compared. The comparison was done with single and dual-colour staining techniques. Monoclonal antibodies (mAb) against eCD4, eCD5, eCD8 and eMHC class II were used. There was no significant difference in the results obtained by these two methods.     

Problems found in the isolation of mycelial fungi (Fusarium solani) from blood culture (Bacter NR-860)

BACKGROUND: F. solani fungemia is unusual. Patients at risk are immunosuppressed, have underlying malignancy or severe debilitating diseases. METHODS: We report two cases of F. solani fungemia in two non neutropenic patients who had been treated with wide-spectrum antibiotics an/or systemic corticosteroids, parenteral nutrition and intravenous lines. Bactec NR-860 (Becton-Dickinson) system was used, and growth was detected in aerobic conditions (between 3-7 days of incubation). RESULTS: Removal of the catheters with or without i.v. amphotericin B were used successfully. CONCLUSION: The spectrum of Fusarium sp. fungemia is discussed. Current available antifungal therapy is also reviewed.     

Cytomegalovirus glycoprotein B genotyping in ocular fluids and blood of AIDS patients with cytomegalovirus retinitis

PURPOSE: To determine the frequency of cytomegalovirus glycoprotein B (gB) genotypes in clinical samples of ocular fluids of patients with acquired immune deficiency syndrome (AIDS) who have cytomegalovirus retinitis and to compare these with the cytomegalovirus gB genotype in paired peripheral blood leukocytes. METHODS: Glycoprotein B genotypes of cytomegalovirus genomic DNA were determined in 29 ocular and 9 paired blood samples of 27 patients, by polymerase chain reaction amplification followed by restriction fragment length polymorphism analysis. RESULTS: In the 29 ocular samples, 30 gB genotypes were determined: Glycoprotein B1 was found in 8 samples (27%), gB2 in 9 samples (30%), gB3 in 6 samples (20%), and gB4 in 3 samples (10%). In one sample, a mixed genotype was observed. In addition to these previously characterized gB genotypes, a new gB variant was observed in the ocular fluid of four patients. Partial sequence analysis revealed that this new gB genotype is closely related to gB3, and it was therefore named gB3'. In the blood samples, only gB1, gB2, and gB3 genotypes were observed. In the nine paired samples of ocular fluid and blood, four showed a difference in gB genotype between these compartments. CONCLUSIONS: The distribution of cytomegalovirus glycoprotein B genotypes gB1-gB4 in ocular fluids of patients with AIDS who have cytomegalovirus retinitis was determined in this study. The predominance of gB2, as described by others, was not confirmed. The glycoprotein B genotype in the eye can be different from the genotype found in the blood of the same patient. A new gB variant, gB3', was found in the ocular samples of 4 of 27 patients, but not in the blood samples tested.     

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.     

Comparative study of cytomegalovirus (CMV) antigenemia assay, polymerase chain reaction, serology, and shell vial assay in the early diagnosis and monitoring of CMV infection after renal transplantation

BACKGROUND: Early diagnosis of cytomegalovirus (CMV) infection, which is an important cause of morbidity and mortality in renal transplant recipients, remains of great importance. This prospective study was performed in kidney transplant recipients to determine the diagnostic value of the CMV antigenemia assay in comparison with polymerase chain reaction (PCR), serology, and shell vial assay. METHODS: Seventy-five consecutive renal transplant recipients were enrolled in this study and monitored by both antigenemia assay and serology. The initial 34 of the 75 patients were subjected to PCR and shell vial assay. RESULTS: Antigenemia, PCR, and shell vial assay became positive before the onset of CMV-related symptoms in 31/34 (89%), 13/16 (81%), and 2/16 (13%), respectively. None of the 34 patients who had symptomatic CMV disease showed a significant increase in IgG or IgM before the onset of symptoms. Antigenemia and PCR assays turned positive, 7 and 11 days (median), respectively, before the onset of clinical symptoms. Serology and shell vial assay became positive 21 and 25 days (median), respectively, after the onset of CMV-related clinical symptoms. To examine the clinical value of these assays, "good correlation" was defined based on the correlation between the clinical course and the results of the assays. Good correlation with the antigenemia assay was observed in 33 (96%) out of 34 renal transplant recipients who recovered from their CMV disease after ganciclovir therapy. Only one of 16 (7%) patients showed good correlation by shell vial assay, whereas PCR and serology did not show a good correlation. Consequently, antigenemia was considered the best way to monitor CMV infections after kidney transplantation. CONCLUSIONS: Only the CMV antigenemia assay can be successfully employed after renal transplantation for the early diagnosis and extensive monitoring of active CMV infection.     

A case of ganciclovir-resistant cytomegalovirus (CMV) retinitis in a patient with AIDS: longitudinal molecular analysis of the CMV viral load and viral mutations in blood compartments

OBJECTIVE: To study the temporal relationships between cytomegalovirus (CMV) viral load and specific UL97 mutations in polymorphonuclear leukocytes (PMNL) and plasma samples from a patient with AIDS who developed ganciclovir-resistant CMV retinitis. METHODS: Sequential PMNL and plasma samples were analysed for determination of the CMV viral load using non-molecular methods and a quantitative polymerase chain reaction (PCR) assay. Screening of the same samples for the most common mutations conferring ganciclovir resistance was performed using nested PCR and restriction enzyme analysis. RESULTS: At the time of progression of CMV retinitis (after 6 months of ganciclovir), a rapid increase in the CMV DNA load was found in both PMNL and plasma samples. This increase paralleled the emergence of a specific mutation (V594) in the same samples and recovery of ganciclovir-resistant blood isolates. In this patient, however, the only tests that substantially predicted the progression of CMV disease were the quantitative PCR assay using PMNL and to a lesser extent the pp65 antigenemia assay. CONCLUSIONS: Quantitative evaluation of the CMV viral load in PMNL using sensitive assays such as PCR appears to be a promising approach for monitoring antiviral therapy in subjects with AIDS. In addition, common mutations conferring ganciclovir resistance can be detected directly in PMNL and plasma samples.     

Moraxella (Branhamella) catarrhalis: its isolation in the respiratory secretions of adult patients

OBJECTIVE: We have designed a retrospective study in order to know the clinical significance of the isolation of Moraxella (Branhamella) catarrhalis (MC) in respiratory specimens of adult hospitalized patients. METHODS: We performed a Gram stain and culture on blood-agar, MacConkey media and quantitative culture in chocolate-agar to all respiratory samples. In patients with a clinical diagnosis of pneumonia BCYE-alpha was added. During 2 years (1992-1993) MC was isolated in respiratory specimens from 52 patients. We revised the clinical history of all these patients. RESULTS: MC was isolated in 60 respiratory specimens (sputum and/or tracheobronchial aspirates) from 52 patients. The Gram stain showed gram-negative cocci in 77% and gram-positive cocci in 17% of the cases. MC grew in pure culture in 28 specimens (46.6%). In 23% of cases MC was isolated with Streptococcus pneumoniae and in 21% with Haemophilus influenzae. Fifty-two stocks (86.6%) produced beta-lactamase. Twelve patients had a clinical diagnosis of pneumonia, 8 of them had an underlying chronic respiratory disease. Other 24 patients with an underlying chronic respiratory disease had a bronchial infection as a cause of exacerbation of their respiratory disease. Seven patients without an underlying chronic respiratory disease had a clinical episode of acute bronchitis. Finally, in 9 patients the isolation of MC was considered a colonization. CONCLUSIONS: In 17% cases MC was identified as a gram-positive cocci in the Gram stain, which may cause false diagnosis. The etiological importance of MC in episodes of acute exacerbation of patients with an underlying chronic respiratory disease is high.     

Technique for direct and quantitative evaluation of erythrocyte aggregability in blood samples

A group of young people ages 10 to 18, interviewed after basketball star Earvin "Magic" Johnson announced that he had tested positive for the human immunodeficiency virus (HIV), were asked for their reactions to the news. Their knowledge of and attitudes regarding AIDS were also compared to those of similar young people interviewed before the announcement. Reactions to the announcement were varied and were accompanied by only isolated changes in knowledge and attitudes, suggesting that news of this celebrity's HIV infection served primarily to reinforce or make temporarily more salient knowledge and attitudes that predated the announcement.     

Long-term predictive value of a single cytomegalovirus (CMV) DNA PCR assay for CMV disease in human immunodeficiency virus-infected patients

Universal prophylaxis with oral ganciclovir is not cost-effective for the prevention of cytomegalovirus (CMV) disease in human immunodeficiency virus infection. For a preemptive strategy to be considered, patients at highest risk for CMV disease need to be easily and accurately identified. In this study, the sensitivity, specificity, positive predictive value, and negative predictive value of a single CMV DNA PCR assay for the subsequent development of CMV disease were 0.75, 0.89, 0.75, and 0.89, respectively.