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.     

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A prospective virologic follow-up of solid organ transplant patients was designed to determine the usefulness of antigenemia and viremia as virologic markers for the diagnosis of cytomegalovirus (CMV) infections, and also for monitoring CMV disease and therapy control. A total of 629 blood samples from 127 patients (60 liver, 47 kidney, and 20 heart transplant recipients) were studied by tube and shell vial cultures, and by antigenemia assay. This later was carried out by an indirect immunofluorescent assay method for formalin-fixed cytospin slides containing 2 x 10(5) leukocytes, using a monoclonal antibody directed against the CMV pp65 antigen. CMV was detected by at least one of the three methods in 238 specimens (37.8%) from a total of 63 patients. The antigenemia assay was positive in 215 (90.3% of positive samples). A total of 94 samples were detected only by this marker, which occurred either in samples with low positive counts (70.2% with antigenemia counts < 10 positive cells/10(5) leukocytes) or in specimens from treated patients. There were 30 episodes of CMV disease in 23 patients. Antigenemia was positive in all these episodes, 27 of them with counts > 20 positive cells/10(5) leukocytes. With this cut-off, positive and negative predictive values for symptomatic CMV infection were 100% and 97.2%, respectively. The antigenemia assay is a rapid, sensitive, specific, and early marker of CMV infection in transplantees. Cultures became negative with antiviral therapy while remaining antigenemia detectable. There was an association between highest quantitative antigenemia test results and clinical symptoms in our patients. In its quantitative version, the assay is useful to detect symptomatic infection and appears to be a helpful tool in managing patients at risk and in guiding antiviral therapy.     

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The aim of the study was to compare the efficacy of pp65 antigenemia assay and the shell-vial culture (SVC; viremia) for the diagnosis of cytomegalovirus (CMV) infection in renal transplant recipients, comparing the results obtained in the first blood sample and the total number of blood samples analyzed in this group of patients. During the study period, 70 renal transplant recipients were studied: 44 (62.8%) with CMV infection. The method of sedimentation in a dextran solution for leukocyte extraction was used in the pp65 antigenemia assay. The MRC-5 shell-vial assay was used for CMV isolation from leukocytes (viremia). Eighty blood samples were examined from 70 renal transplant recipients: Of the 44 positive samples studied, in 77.5% of cases, both the antigenemia assay and the SVC were positive. In 16.2%, only the antigenemia assay was positive, and, in 6.2%, only the SVC was positive. In all blood samples studied, the antigenemia was present in 93.7% of cases, and the SVC was present in 83.7% (P = 0.04). If the results obtained in only the first blood sample taken for the diagnosis are studied, then we observe that the antigenemia assay was positive in 39 patients (88.6%), whereas the SVC was positive in 41 patients (93.1%), although the difference was not statistically significant (P = 0.39). It is concluded that the inoculation of all of the leukocytes extracted from blood samples in the SVC seems to produce a slight increase in the sensitivity of the cell culture and that the SVC becomes positive before the antigenemia for the detection of CMV in peripheral blood, especially in the first blood sample.     

Comparison of monoclonal antibodies for immunostaining in the cytomegalovirus shell vial assay on 4,388 specimens

The shell vial assay is a sensitive, rapid test for the detection of cytomegalovirus (CMV) in a variety of specimens. The sensitivity of this assay is dependent on a number of factors including the antibodies used for immunostaining. Monoclonal antibodies to the CMV major immediate-early antigen (p72) from Chemicon (MAB810) and Dupont (NEA-9221) were assessed side by side in duplicate vials on 4,388 specimens from a patient population consisting of > 90% organ transplant recipients. A total of 240 specimens (5.5%) were CMV positive in either one or both vials. Positivity rates were variable across different specimen types but highest (12.9%) in urine specimens. Of the positive specimens, 175 (72.9%) tested positive in both vials, 43 (17.9%) tested positive in the Chemicon-stained vial only, and 22 (9.2%) tested positive in the Dupont-stained vial only (P < 0.01, McNemar's chi-square test). This gave an overall positivity rate of 5.0% for Chemicon antibodies and 4.5% for Dupont. There was no difference in the fluorescent focus counts produced by the two antibody sets. It is concluded that use of the Chemicon antibodies provides increased sensitivity of detection of CMV in the shell vial assay above that afforded by the Dupont antibody.     

Quantitation of cytomegalovirus: methodologic aspects and clinical applications

Cytomegalovirus (CMV) is an important pathogen in transplant recipients and human immunodeficiency virus (HIV)-infected individuals. Major progress has been made in developing quantitative detection methods for CMV in recent years. Due to their high sensitivity, these assays can detect CMV early, and quantitation may be useful in predicting the patient's risk for disease and in monitoring the effect of antiviral therapy. This review discusses methodological aspects of currently used quantitative assays for CMV (i.e., viral culture techniques, antigen detection assays, DNA detection assays including PCR, branched-DNA assay, and the DNA hybrid capture assay) and addresses the correlation of systemic and site-specific CMV load and CMV disease in different populations of immunosuppressed patients as well as the response to antiviral treatment. To date, direct antigen detection and molecular techniques have largely replaced traditional culture-based techniques for CMV quantitation. In general, a high systemic CMV load is correlated with CMV disease. This correlation is strong in the HIV-infected population and in solid-organ transplant recipients but less clear in allogeneic marrow transplant recipients. Measuring the viral load at specific anatomic sites may be an alternative way to assess disease activity in situations where the systemic viral load correlates poorly with disease activity. A reduction of the systemic CMV load also correlates with a response to antiviral treatment, but more research is needed to evaluate the role of viral load as a surrogate marker for drug resistance. Due to the widespread use of quantitative CMV detection techniques to direct and monitor antiviral treatment, there is a great need for an assessment of the reproducibility of test results and better standardization of the assays.     

Diagnosis of cytomegalovirus infections by shell vial assay and conventional cell culture during antiviral prophylaxis

A total of 3,552 specimens for conventional cytomegalovirus (CMV) culture and shell vial assay for CMV immediate-early antigen were obtained during a prospective randomized trial for prophylaxis of CMV disease after liver transplantation. Prophylaxis with ganciclovir for 2 weeks and then high-dose acyclovir for 2.5 months was compared with high-dose acyclovir alone for 3 months. During the first 12 weeks after transplantation, when the patients were on prophylaxis, there were significantly more clinical samples positive by the shell vial assay and negative by standard culture in comparison with the number of samples obtained from weeks 13 to 24, after prophylaxis was discontinued, that were positive by the shell vial assay and negative by standard culture. In contrast, significantly fewer samples were positive by both the shell vial assay and standard culture during the first 12 weeks compared with the number obtained 13 to 24 weeks after transplantation that were positive by both methods. Samples positive by the shell vial assay only were obtained significantly more frequently from patients with asymptomatic than symptomatic CMV infections, while samples positive by both methods were obtained significantly more often from patients with symptomatic CMV infection. It was concluded that antiviral prophylaxis with high-dose acyclovir or ganciclovir and then high-dose acyclovir and asymptomatic CMV infection are associated with a decrease in the level of CMV isolation by standard cell culture in comparison with that by the shell vial assay.     

Use of laboratory assays to predict cytomegalovirus disease in renal transplant recipients

Eight laboratory assays, viz., the pp65 direct antigenemia test, a quantitative cytomegalovirus (CMV)-specific immunoglobulin G (IgG) assay (Biomerieux VIDAS), a CMV-specific IgM assay (Biomerieux VIDAS), the Hybrid Capture system (Murex), an in-house PCR with plasma (P-PCR) and leukocytes (L-PCR), and a commercial PCR (Roche AMPLICOR) with plasma (P-AMP) and leukocytes (L-AMP), were compared for their abilities to predict CMV disease before the onset of illness in a prospective study of 37 renal transplant recipients. By using an expanded criterion for active infection (two or more of the markers positive) and a clinical definition of disease, 22 (59%) patients were identified as having active CMV infection and 13 (35%) were identified as having CMV disease. Of the 13 CMV-seronegative recipients who received seropositive kidneys (R- group), 8 had active infection and disease. All assays were 100% specific and 100% predictive of CMV disease in the R- group. The leukocyte PCRs (L-PCR and L-AMP) were the most sensitive assays, had positive results an average of between 8 and 13 days before the onset of illness, and were the assays of choice. The performance of the assays was less satisfactory for the 24 patients who were CMV seropositive before transplantation (R+ group). A negative result was more useful for this group. Overall, P-AMP had the best results, and it could be the assay of choice for monitoring R+ patients. The non-PCR-based methods generally had high specificities but often gave late positive results and were not sensitive enough for use as prediction tools for either group of patients.     

The correlation between symptomatic CMV infection and CMV antigenemia in heart allograft recipients

Previous studies have demonstrated that CMV-specific antigens detected from peripheral blood leukocytes correlate with active CMV infection in transplant patients. However, the clinical diagnosis of CMV infection is difficult, and the significance of a positive blood finding is unclear, while CMV antigenemia and viremia may also occur in asymptomatic patients. To investigate the clinical significance of CMV antigenemia after heart transplantation, 68 heart allograft recipients were monitored weekly. Altogether 501 blood specimens were analyzed. CMV was demonstrated in blood leukocytes by a monoclonal antibody and immunoperoxidase staining, and the antigenemia level was expressed as CMV positive cells/50,000 leukocytes. CMV antigenemia occurred in 28/68 patients, and 12 of them developed a symptomatic infection. Of all blood specimens 88/501 were CMV positive, and 30 of them related to the clinical manifestation of CMV. When antigenemia level exceeded > 100/50,000, a significant correlation between antigenemia and CMV-related clinical manifestation was reached (P < 0.001). Of the 28 antigenemia positive patients 16 never developed any clinical signs of CMV infection. Their maximal antigenemia level was low (median 23, range 30-90) compared with those with clinical manifestation (median 500, range 30-1000) (P < 0.002). In conclusion, high antigenemia levels (> 100/50,000) correlate with clinical manifestations of CMV infection. Patients with lower levels (< 100/50,000) do not necessarily ever develop a symptomatic infection. Quantitative monitoring of CMV antigenemia may, thus, be helpful in the clinical diagnosis of CMV infection in heart transplant patients.     

Rapid cytomegalovirus pp65 antigenemia assay by direct erythrocyte lysis and immunofluorescence staining

A rapid cytomegalovirus (CMV) pp65 antigenemia assay with direct erythrocyte lysis (DL) with 0.8% NH4Cl, followed by indirect immunofluorescence staining (IF), was evaluated with 82 blood samples from renal transplant recipients, and the results were compared to those of the conventional antigenemia assay with dextran sedimentation and two-cycle alkaline phosphatase, anti-alkaline phosphatase staining (DS-APAAP). The DL-IF modification gave a higher leukocyte yield compared to DS-APAAP (75.4 versus 54.9%; P < 0.05), with similar leukocyte viability rates of >95%. The DL-IF methodology involved fewer technical steps, and the assay time was shortened from 5 h to less than 3 h. Nineteen of the 82 samples concordantly tested positive for pp65 antigenemia by both assays, and the readings showed a good correlation (r = 0.996; P < 0.01). No discordant results were observed. We conclude that the CMV pp65 antigenemia assay by this novel DL-IF modification is technically simpler, cheaper, and less time-consuming but yields results comparable to those of the conventional DS-APAAP assay. The shortened assay time and increased capacity to handle more samples confer distinct advantages in the rapid diagnosis and prompt treatment of CMV disease in immunosuppressed patients.     

Impact of high-dose oral acyclovir prophylaxis on cytomegalovirus (CMV) disease in CMV high-risk renal transplant recipients

Recent studies showed contradictory results concerning the efficacy of oral acyclovir in the prevention or amelioration of cytomegalovirus (CMV) disease after renal transplantation (TX). This study evaluated the incidence and severity of CMV disease within the first year after TX in high-risk renal transplant recipients (CMV-seropositive donor, seronegative recipient) treated prophylactically with oral acyclovir (800 to 3200 mg/day) over a period of 12 wk (ACY, N = 22), compared with high-risk patients randomly assigned as controls (CO, N = 10). Follow-up for CMV infection included serological determination of CMV-specific immunoglobulin G and immunoglobulin M antibodies, antigen detection in peripheral blood leukocytes (PP 65), shell vial culture (blood), and virus isolation/early antigen detection (urine). Severity of CMV disease was quantified by a scoring system for CMV-related symptoms. Nine patients (40.1%) in the acyclovir group and four patients (40%) in the control group developed CMV disease. Neither severity (ACY, 11.4 versus CO; 12.5 points score), nor duration of disease (ACY, 21 days; CO, 22 days), nor transplant function at the end of the observation period differed significantly. The onst of CMV disease was not delayed significantly in acyclovir-treated patients compared with controls (ACY, 47 +/- 34 days versus CO, 27 +/- 14 days after TX, not significant). Our results show no beneficial effect of oral acyclovir prophylaxis in CMV high-risk renal transplant recipients.     

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.     

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.     

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The optimal prophylactic strategy for cytomegalovirus (CMV) disease after allogeneic hematopoietic stem cell transplantation has not yet been established. The aim of this study was to analyze our single-center experience with a uniform protocol of CMV antigenemia-guided pre-emptive treatment with ganciclovir (GCV) after allografting. Fifty-two consecutive adult patients, 48 of them transplanted from HLA-identical matched related donors were included. T cell-depleted marrow or peripheral blood were used in 21 cases. After engraftment, weekly blood samples were tested for CMV pp65 antigenemia and viremia (conventional cultures) until day +100. GCV was started if CMV antigenemia and/or CMV viremia were detected. CMV infection (CMV-I) was found in 19 patients (37%). Seven patients suffered from CMV disease (CMV-D), three colitis and four pneumonias. There was one death directly related to CMV-D and three further cases died from refractory GVHD with CMV-D. Only one patient developed CMV pneumonia without any previous positive antigenemia and/or viremia. Multivariate analysis identified grades II-IV acute GVHD (P = 0.02) and peripheral blood stem cell transplantation (P = 0.03) to be risk factors for developing CMV-I. In conclusion, this monitoring protocol allowed early treatment of CMV-I without progression to CMV-D. Pre-emptive therapy had the additional advantage of avoiding GCV administration in most of our allograft recipients.     

Detection of cytomegalovirus (CMV) antigen for rapid diagnosis and monitoring of CMV diseases in AIDS

Ten to forty percent of the patients with acquired immunodeficiency syndrome (AIDS) develop sight- or life-threatening cytomegalovirus (CMV) infections. In some patients with AIDS, CMV is detected in the bronchoalveolar lavage fluid (BALF), urine, and other specimens, even when there are no symptoms of CMV disease. An indicator of active CMV infection is needed to facilitate the diagnosis of CMV disease in patients with AIDS or HIV infection and the evaluation of the efficacy of subsequent treatment. The present study was conducted during the period from 1993 to 1994. The subjects consisted of three patients with AIDS and a confirmed diagnosis of CMV disease (one case of retinitis, one case of gastrointestinal disease and one case of pneumonia), and five HIV-positive patients in whom CMV associated disease was ruled out. Those patients were monitored occasionally for the following parameters of active CMV infection and disease: expression of CMV antigen in the nucleus of polymorphonuclear leukocyte (CMV antigenemia), as it was determined with a monoclonal antibody against a lower matrix protein (p65); infectious CMV detected by shell vial method; CMV DNA detected by PCR; anti-CMV antibody titer; and histological findings. CMV p65 antigen was detected in the leukocytes of both the peripheral blood and BALF during the early phase of CMV disease in three out of three cases of the CMV disease group, and this antigen became negative in two out of two cases who responded to the therapy. All the five patients in the CMV-related-disease-negative group were negative for CMV antigenemia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of the AMPLICOR cytomegalovirus test with specimens from human immunodeficiency virus-infected subjects

The AMPLICOR cytomegalovirus (CMV) test, a new qualitative assay for the detection of CMV DNA in plasma, was compared to conventional methods and quantitative PCR (Q-PCR) assays by using leukocytes and plasma from 179 blood samples from subjects with AIDS. For the diagnosis of CMV disease, cell-based assays such as a Q-PCR with polymorphonuclear leukocytes (Q-PCR-PMNL) and a pp65 antigenemia assay had the highest sensitivities but suffered from a lack of specificity. The best agreement between the results of the Q-PCR-PMNL assay and those of the AMPLICOR test was found when a threshold diagnostic value of 690 copies per 10(5) cells was selected for the Q-PCR-PMNL assay. In that context, the AMPLICOR CMV test had a sensitivity of 96.4% and a specificity of 95.3% when results were compared to results of the cell-based PCR assay. This threshold was close to the one described as associated with the best sensitivity and specificity for the diagnosis of CMV disease in a recently published study (4). Blood samples that tested positive by the Q-PCR-PMNL assay but negative by the AMPLICOR CMV test were associated with viral loads (mean, 785 copies, median, 96 copies per 10(5) leukocytes) lower than the viral loads of blood samples that tested positive by both assays (mean, 21,452 copies; median, 9,784 copies per 10(5) leukocytes) (P = 0.003). The AMPLICOR CMV test gave positive results at least 48 days before the development of symptomatic CMV disease in a longitudinal analysis of a limited subset of patients (n = 6) from whom sequential specimens were available for testing. In conclusion, the AMPLICOR CMV test is a very convenient assay combining rapidity, simplicity, and the possibility of batch testing. A positive result by this test seems particularly important since this implies, in most instances, the presence or the imminence of CMV disease, although a negative test result does not rule out disease.     

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.     

Delayed acquisition of high-avidity anti-cytomegalovirus antibody is correlated with prolonged antigenemia in solid organ transplant recipients

Previous studies have demonstrated that maturation of cytomegalovirus (CMV)-specific antibodies in solid organ transplant recipients is delayed after primary CMV infection. To investigate the clinical significance of this finding, the avidity indices of anti-CMV antibody were determined in parallel with other serologic and virologic parameters in serial serum samples from 24 solid organ transplant recipients who had primary CMV infection that began during the first 3 months after transplantation. The data obtained show that a delay in antibody maturation is significantly correlated with a long persistence of positive antigenemia.     

Evaluation of Murex CMV DNA hybrid capture assay for detection and quantitation of cytomegalovirus infection in patients following allogeneic stem cell transplantation

Murex hybrid capture DNA assay (HCS) is a solution hybridization antibody capture assay for detection and quantitation of cytomegalovirus (CMV) DNA in leukocytes. To determine whether CMV HCS is sensitive enough to initiate and monitor antiviral therapy after allogeneic stem cell transplantation (SCT), 51 consecutive SCT recipients were prospectively screened for the appearance of CMV infection by HCS, PCR, and culture assays from blood samples. Preemptive antiviral therapy was initiated after the second positive PCR result in all patients, as previously reported, and HCS was not considered for clinical decision making. A total of 417 samples were analyzed. Of these, 21 samples were found to be positive by PCR and HCS, 88 samples were PCR positive but HCS negative, and 308 were negative by both assays. Concordance of results between PCR and HCS and between HCS and blood culture was observed in 78.9 and 95.9% of the samples assayed, respectively. PCR was found to be more sensitive than HCS, and HCS was more sensitive than the blood culture assay (P < 0.0001). Four patients with symptomatic CMV infection were PCR positive prior to the onset of CMV-related symptoms, whereas HCS detected CMV DNA in three patients prior to and one at onset of CMV disease. The numbers of genomes per milliliter of blood were higher in patients with symptomatic CMV infection than in those with asymptomatic CMV infection (P = 0.06). None of the HCS-negative patients developed CMV disease. Thus, all patients with CMV disease were correctly identified by HCS; however, the lower sensitivity limit of the HCS assay may still be insufficient to allow diagnosis of CMV infection early enough to prevent CMV disease in patients following allogeneic SCT.     

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)     

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.