Subtherapeutic ganciclovir (GCV) levels and GCV-resistant cytomegalovirus in lung transplant recipients.

BACKGROUND: Cytomegalovirus (CMV) infection results in significant morbidity and mortality in lung transplant recipients. Ganciclovir (GCV) has dramatically reduced complications caused by CMV infections. Unfortunately, GCV resistance is identified in 5-10% of CMV-infected patients. Mismatched CMV status and ongoing replication due to immunosuppression are risk factors for drug resistance. Whether subtherapeutic GCV levels contribute to resistance remains unknown. METHODS: A retrospective review was conducted in all 51 patients who underwent lung transplantation between March 2007 and June 2008 at Loyola University Medical Center. GCV resistance and outcome data of CMV-infected patients were analyzed to identify variables that may contribute to suboptimal response to CMV infection. RESULTS: During the 16-month period, CMV infection was identified in 21 of 51 lung transplant recipients. Ten of 21 patients (47.6%) had CMV infection with early response to GCV, and 11 patients (52.4%) had CMV infection with suboptimal response to GCV. GCV levels were obtained in the 11 CMV-infected patients with suboptimal response. In 6 patients, GCV levels were therapeutic; all 6 had delayed response to GCV. In 5 patients, GCV levels were subtherapeutic; each had persistent suboptimal response to GCV. Genotyping documented GCV-resistant (GCV-R) CMV in all 5 patients. Cystic fibrosis as the diagnosis requiring lung transplantation was associated with GCV-R CMV infection (P = 0.01). CONCLUSION: In our lung transplant recipient cohort, GCV levels were subtherapeutic in all patients with persistent suboptimal response to GCV, each of whom had GCV-R CMV infection. In contrast, GCV levels were therapeutic in CMV-infected patients with delayed GCV response.

Multilaboratory comparison of hepatitis C virus viral load assays.

We report a multilaboratory evaluation of hepatitis C virus (HCV) viral load assays to determine their linear range, reproducibility, subtype detection, and agreement. A panel of HCV RNA samples ranging in nominal concentration from 1.0 to 7.0 log10 IU/ml was constructed by diluting a clinical specimen (genotype 1b). Replicates of the panel were tested in multiple laboratories using the Abbott TaqMan analyte-specific reagent (Abbott reverse transcription-PCR [RT-PCR]), Roche TaqMan RUO (Roche RT-PCR), Roche Amplicor Monitor HCV 2.0 (Roche Monitor), and Bayer VERSANT HCV RNA 3.0 (Bayer bDNA) assays. Bayer bDNA-negative specimens were tested reflexively using the Bayer VERSANT HCV RNA qualitative assay (Bayer TMA). Abbott RT-PCR and Roche RT-PCR detected all 28 replicates with a concentration of 1.0 log10 IU/ml and were linear to 7.0 log10 IU/ml. Roche Monitor and Bayer bDNA detected 27 out of 28 and 13 out of 28 replicates, respectively, of 3.0 log10 IU/ml. Bayer TMA detected all seven replicates with 1.0 log10 IU/ml. Bayer bDNA was the most reproducible of the four assays. The mean viral load values for panel members in the linear ranges of the assays were within 0.5 log10 for the different tests. Eighty-nine clinical specimens of various genotypes (1 through 4) were tested in the Bayer bDNA, Abbott RT-PCR, and Roche RT-PCR assays. For Abbott RT-PCR, mean viral load values were 0.61 to 0.96 log10 greater than the values for Bayer bDNA assay for samples with genotype 1, 2, or 3 samples and 0.08 log10 greater for genotype 4 specimens. The Roche RT-PCR assay gave mean viral load values that were 0.28 to 0.82 log10 greater than those obtained with the Bayer bDNA assay for genotype 1, 2, and 3 samples. However, for genotype 4 samples the mean viral load value obtained with the Roche RT-PCR assay was, on average, 0.15 log10 lower than that of the Bayer bDNA. Based on these data, we conclude that the sensitivity and linear range of the Abbott and Roche RT-PCR assays enable them to be used for HCV diagnostics and therapeutic monitoring. However, the differences in the viral load values obtained with the different assays underscore the importance of using one assay when monitoring response to therapy.

Clinical characteristics of 13 solid organ transplant recipients with ganciclovir-resistant cytomegalovirus infection.

BACKGROUND: Ganciclovir-resistant (GCV-R) cytomegalovirus (CMV) is now being reported with increasing frequency in solid organ transplant recipients. OBJECTIVE: To describe the clinical characteristics and outcomes of all solid organ transplant patients with GCV-R CMV seen between 1990 and 2000 at a single center. METHODS: Patients with clinically suspected GCV resistance had viral isolates subjected to phenotypic analysis by plaque reduction assay, and also genotypic analysis. Medical records of the 13 patients with GCV-R CMV were reviewed for demographic, microbiologic, clinical, and pathologic data. RESULTS: Thirteen patients were identified, including 5 kidney, 1 heart, and 7 lung transplant recipients. All but one patient (92%) were CMV donor seropositive, recipient negative (D+/R-), and 11/13 (85%) had tissue-invasive CMV. CMV viremia was recurrent in 9/13 (69%); in 2 others, the first CMV episode was fatal. Overall, 9/13 (69%) of patients have died, all of CMV or its complications. Of the 10 who received foscarnet, only one survived. All patients had received GCV-based prophylactic regimens; 8/13 patients (62%) had received CMV hyperimmune globulin (CMVIG) as part of prophylaxis, 6/13 (46%) had received oral ganciclovir, and 5/13 (38%) had received intermittent (3 x/week) IV ganciclovir for prophylaxis. CONCLUSIONS: GCV-R CMV is associated with CMV D+/R- status, tissue-invasive disease, and high mortality even with foscarnet therapy. Exposure to less than fully therapeutic levels of GCV, in the form of oral or intermittent IV GCV, is common. The use of CMVIG in prophylaxis does not appear to prevent resistance. Further work remains to be done to elucidate the risk factors and optimal mode of prophylaxis and treatment for GCV-R CMV.

Sequencing of cytomegalovirus UL97 gene for genotypic antiviral resistance testing.

The widespread use of ganciclovir (GCV) to treat cytomegalovirus (CMV) infections in immunosuppressed patients has led to the development of drug resistance. Phenotypic assays for CMV drug resistance are presently too time-consuming to be therapeutically useful. To support the development of genotypic assays for GCV resistance, the complete sequences of the UL97 phosphotransferase genes in 28 phenotypically GCV-sensitive CMV clinical isolates were determined. The gene was found to be highly conserved, with nucleotide sequence identity among strains ranging from 98.6 to 100% and amino acid sequence identity of >99%. Primers for a genotypic assay were designed to amplify codons 400 to 707, because all known UL97 mutations conferring drug resistance occur at three sites within this region. This part of the UL97 gene was amplified from over 50 clinical isolates, and two sequencing reactions for the coding strand were successfully used to identify GCV resistance mutations. This genotypic assay can be performed in 48 h using genomic DNA extracted from cell monolayers at very low levels of virus infectivity, thus rapidly providing therapeutically useful results.

Comparison of quantitative and qualitative PCR assays for cytomegalovirus DNA in plasma.

We analyzed the performance characteristics of the qualitative AMPLICOR CMV Test (Roche Molecular Systems, Pleasanton, Calif.) and quantitative COBAS AMPLICOR CMV MONITOR Test (Roche Molecular Systems) assays and compared the performance of the AMPLICOR quantitative assay with an in-house-developed cytomegalovirus (CMV) DNA PCR assay. The quantitative AMPLICOR assay was found to be more sensitive than the qualitative AMPLICOR assay. The quantitative AMPLICOR assay has a lower limit of sensitivity of 400 CMV DNA copies/ml of plasma and is linear to 50,000 CMV DNA copies/ml of plasma. Compared to the in-house PCR assay, the AMPLICOR quantitative assay gave lower viral load values at all concentrations tested, but the difference between the two assays was not consistent across the entire dynamic range of the AMPLICOR quantitative assay. At the lower end of the assay, the viral load values obtained with the in-house PCR assay were three- to fivefold (0.5 to 0.7 log units) higher than those measured with the AMPLICOR assay. At higher input concentrations, the differences between the two assays approached 10-fold. This direct comparison of the in-house assay and the quantitative AMPLICOR assay provides the ability to compare previously published in-house data with an assay widely available for future research and clinical monitoring of patients with CMV infections.

Human cytomegalovirus UL144 open reading frame: sequence hypervariability in low-passage clinical isolates.

Human cytomegalovirus (HCMV) infects a number of organs and cell types in vivo, leading to the hypothesis that HCMV disease and tissue tropism may be related to specific sequence variants. A potential component of HCMV variant strains is the UL144 open reading frame (ORF), which encodes a homologue of the herpesvirus entry mediator, HveA, a member of the tumor necrosis factor receptor superfamily. Sequence analysis of the UL144 ORF in 45 low-passage clinical isolates demonstrated significant strain-specific variability. In individual isolates, nucleotide substitutions occur at up to 21% of the 531 positions, resulting in approximately the same percentage of substitutions in the predicted 176-amino-acid sequence. Phylogenetic analysis indicated that the nucleotide and amino acid sequences diverge into three major groups. For genotypic comparison, the known hypervariable region encompassing the proteolytic cleavage site of the glycoprotein B (gB) gene was also sequenced. All of the isolates could be typed according to the four known gB groups; however, the gB and UL144 sequence groups appeared to be phylogenetically unlinked. The predicted UL144 product homology with tumor necrosis factor receptor family members, along with the unexpectedly high level of sequence variability of the UL144 ORF, suggests that the predicted product may play a role in HCMV infectivity and subsequent host disease.

Novel mechanism of inhibition of cytomegalovirus by the experimental immunosuppressive agent leflunomide.

BACKGROUND: Despite progress in antiviral chemotherapy, cytomegalovirus (CMV) remains a major cause of morbidity and mortality among pharmacologically immunosuppressed organ transplant recipients, frequently engaging the clinician in a struggle to balance graft preservation with control of CMV disease. Leflunomide, an inhibitor of protein kinase activity and pyrimidine synthesis, is an experimental immunosuppressive agent effective against acute and chronic allograft rejection in animal models. Because a number of CMV proteins are known to be phosphorylated, we tested the hypothesis that this agent might exert inhibitory activity against CMV. METHODS AND RESULTS: Plaque assays demonstrated dramatic dose-dependent attenuation of production of multiple clinical CMV isolates in leflunomide-treated human fibroblasts and endothelial cells, common targets for CMV infection in vivo. As shown by Northern blot analysis and immunohistochemical staining, leflunomide neither interferes with transcription of immediate early or late viral genes, nor with expression of corresponding proteins. CMV-specific DNA dot blots and biochemical enzyme assays indicated that, in contrast to currently approved anti-CMV drugs, leflunomide exerts no inhibitory effect on the accumulation of viral DNA in infected cells, or on viral DNA polymerase activity. Rather, as visualized by transmission electron microscopy, this agent appears to act at a late stage in virion assembly by preventing tegument acquisition by viral nucleocapsids. Finally we have demonstrated equivalent inhibitory activity of leflunomide against multi-drug-resistant CMV isolates. CONCLUSIONS: These findings imply that leflunomide, an effective immunosuppressive agent, shows potential to concurrently attenuate a major complication of immunosuppression, CMV disease, by a novel mechanism of antiviral activity.

Cutting edge: a novel viral TNF receptor superfamily member in virulent strains of human cytomegalovirus.

The UL144 open reading frame found in clinical isolates of human CMV (HCMV) encodes a structural homologue of the herpesvirus entry mediator, a member of the TNFR superfamily. UL144 is a type I transmembrane glycoprotein that is expressed early after infection of fibroblasts; however, it is retained intracellularly. A YXXZ motif in the highly conserved cytoplasmic tail contributes to UL144 subcellular distribution. The finding that no known ligand of the TNF family binds UL144 suggests that its mechanism of action is distinct from other known viral immune evasion genes. Specific Abs to UL144 can be detected in the serum of a subset of HCMV seropositive individuals infected with HIV. This work establishes a novel molecular link between the TNF superfamily and herpesvirus that may contribute to the ability of HCMV to escape immune clearance.

Interstrain variation in the human cytomegalovirus DNA polymerase sequence and its effect on genotypic diagnosis of antiviral drug resistance. Adult AIDS Clinical Trials Group CMV Laboratories.

The polymerase (pol) coding sequence was determined for 40 independent clinical cytomegalovirus isolates sensitive to ganciclovir and foscarnet. Sequence alignments showed >98% interstrain homology and amino acid variation in only 4% of the 1, 237 codons. Almost all variation occurred outside of conserved functional domains where resistance mutations have been identified.

Inhibition of cytomegalovirus in vitro and in vivo by the experimental immunosuppressive agent leflunomide.

Despite progress in antiviral chemotherapy, cytomegalovirus (CMV) remains a major cause of morbidity and mortality among pharmacologically immunosuppressed transplant recipients, frequently engaging the clinician in a struggle to balance graft preservation with control of CMV disease. Leflunomide, an inhibitor of protein kinase activity and pyrimidine synthesis, is an experimental immunosuppressive agent effective against acute and chronic rejection in animal models. Herein we summarize our recent studies demonstrating that leflunomide inhibits the production of multiple clinical CMV isolates (including multi-drug-resistant virus) in both human fibroblasts and endothelial cells. In contrast to all other anti-CMV drugs currently in use, leflunomide does not inhibit viral DNA synthesis, but rather appears to interfere with virion assembly. Finally, preliminary studies in a rat model suggest that this agent reduces viral load in vivo. These findings imply that leflunomide, an effective immunosuppressive agent, shows potential to concurrently attenuate a major complication of immunosuppression, CMV disease, by a novel mechanism of antiviral activity.

Rapid ganciclovir susceptibility assay using flow cytometry for human cytomegalovirus clinical isolates.

Rapid, quantitative, and objective determination of the susceptibilities of human cytomegalovirus (HCMV) clinical isolates to ganciclovir has been assessed by an assay that uses a fluorochrome-labeled monoclonal antibody to an HCMV immediate-early antigen and flow cytometry. Analysis of the ganciclovir susceptibilities of 25 phenotypically characterized clinical isolates by flow cytometry demonstrated that the 50% inhibitory concentrations (IC50s) of ganciclovir for 19 of the isolates were between 1.14 and 6.66 microM, with a mean of 4.32 microM (+/-1.93) (sensitive; IC50 less than 7 microM), the IC50s for 2 isolates were 8.48 and 9.79 microM (partially resistant), and the IC50s for 4 isolates were greater than 96 microM (resistant). Comparative analysis of the drug susceptibilities of these clinical isolates by the plaque reduction assay gave IC50s of less than 6 microM, with a mean of 2.88 microM (+/-1.40) for the 19 drug-sensitive isolates, IC50s of 6 to 8 microM for the partially resistant isolates, and IC50s of greater than 12 microM for the four resistant clinical isolates. Comparison of the IC50s for the drug-susceptible and partially resistant clinical isolates obtained by the flow cytometry assay with the IC50s obtained by the plaque reduction assay showed an acceptable correlation (r2 = 0.473; P = 0.001), suggesting that the flow cytometry assay could substitute for the more labor-intensive, subjective, and time-consuming plaque reduction assay.

Flow cytometric determination of ganciclovir susceptibilities of human cytomegalovirus clinical isolates.

A flow cytometric assay has been developed for the measurement of susceptibilities to ganciclovir of laboratory strains and clinical isolates of human cytomegalovirus (HCMV). The assay uses fluorochrome-labeled monoclonal antibodies to HCMV immediate-early and late antigens to identify HCMV-infected cells and flow cytometry to detect and quantitate the number of antigen-positive cells. By this assay, the 50 and 90% inhibitory concentrations (IC50 and IC90, respectively) of ganciclovir for the AD169 strain of HCMV were 1.7 and 9.2 microM, respectively, and the IC50 for the ganciclovir-resistant D6/3/1 derivative of the AD169 strain was greater than 12 microM. The ganciclovir susceptibilities of 17 HCMV clinical isolates were also determined by flow cytometric analysis of the effect of ganciclovir on late-antigen synthesis in HCMV-infected cells. The average IC50 of ganciclovir for drug-sensitive HCMV clinical isolates was 3.79 microM (+/-2.60). The plaque-reduction assay for these clinical isolates yielded an average IC50 of 2.80 microM (+/-1.46). Comparison of the results of the flow cytometry assays with those obtained from the plaque-reduction assays demonstrated acceptable bias and precision. Flow cytometric and plaque-reduction analysis of cells infected with ganciclovir-resistant clinical isolates failed to show a reduction in the percentage of late-antigen-positive cells or PFU, even at 96 microM ganciclovir. The flow cytometric assay for determining ganciclovir susceptibility of HCMV is quantitative, and objective, and potentially automatable, and its results are reproducible among laboratories.

Molecular analysis of human cytomegalovirus strains from two lung transplant recipients with the same donor.

A total of four cytomegalovirus (CMV) isolates were obtained from two CMV seronegative patients, each of whom received a lung transplant from the same seropositive donor. CMV was isolated from Patient 1 from two bronchial alveolar lavage (BAL) specimens, one obtained during treatment with ganciclovir (GCV) and a second during later treatment with foscarnet. Both of these isolates are sensitive to GCV and foscarnet. CMV was isolated from Patient 2 from a blood and a BAL specimen obtained during treatment with GCV. Both of these isolates are resistant to GCV and show reduced GCV phosphorylation. Patient 1 is still alive 33 months posttransplant. Patient 2 died 6 1/2 months posttransplant. Although the four strains differ with respect to GCV susceptibility and phosphorylation, their DNA restriction fragment hybridization patterns and UL97 kinase gene sequences indicate that they are closely related. The restriction fragment hybridization patterns are identical among the strains, while these patterns differ markedly from those of unrelated strains. The DNA sequences of the UL97 genes of the strains from Patient 2 differ by only one nucleotide from those of Patient 1. The same comparison with unrelated strains shows a minimum of 12 nucleotide differences. The nucleotide change in the strains from Patient 2 produces an amino acid substitution of serine for leucine at residue 595, a substitution that was previously shown to transfer GCV resistance. Both patients, therefore, were apparently infected with the same donor strain, but during the course of GCV prophylaxis and treatment, a GCV-resistant mutant strain was selected in Patient 2.

Host cell-derived complement control proteins CD55 and CD59 are incorporated into the virions of two unrelated enveloped viruses. Human T cell leukemia/lymphoma virus type I (HTLV-I) and human cytomegalovirus (HCMV).

The current study was undertaken to determine whether the human T cell leukemia/lymphoma oncovirus type I (HTLV-I) and the herpesvirus human cytomegalovirus (HCM) incorporate host cell-derived C regulatory proteins. Our experiments showed that both CD59 and CD55 were associated with the external membrane of HTLV-I derived from MT2 cells, since virus could be captured by mAbs to these proteins, and antisera to CD55 and CD59 induced C-mediated lysis of HTLV-I virions. Additionally, both CD55 and CD59 were detected by immunoblot analysis of purified HTLV-I. Purified HCMV produced in human foreskin fibroblasts (HFF) also contained both CD55 and CD59, as detected by immunoblot analysis. However, treatment with anti-CD55, but not anti-CD59, reduced the HCMV infectious titer in the presence of C. Additional studies determined whether HTLV-I-associated CD55 and CD59 participated in the resistance of the virus to C-mediated lysis. Treatment of virus with phosphatidylinositol-specific phospholipase C (PI-PLC), which removes glycosylphosphatidylinositol-anchored CD55 and CD59, increased the sensitivity of HTLV-I to C-mediated destruction in the presence of anti-HTLV-I Abs. Reconstitution of PI-PLC-treated virus with purified CD55 and CD59 restored resistance to C. These experiments show that HTLV-I and HCMV acquire C control proteins from host cells. Together with our previous experiments showing that both CD55 and CD59 are present on HIV-1, these studies demonstrate a mechanism by which a variety of enveloped viruses may acquire resistance to C-mediated destruction.

Mutation in the UL97 open reading frame of human cytomegalovirus strains resistant to ganciclovir.

The same point mutation in the human cytomegalovirus UL97 open reading frame was found in three independently isolated ganciclovir-resistant mutants of strain AD169. Point mutations in the DNA polymerase genes of these strains have been previously identified (N.S. Lurain, K.D. Thompson, E.W. Holmes, and G.S. Read, J. Virol. 66:7146-7152, 1992). All three strains are, therefore, double mutants. To determine the contribution of the UL97 mutation to the high ganciclovir resistance of these mutants, the mutation from the ganciclovir-resistant strain D6/3/1 was transferred to the wild-type strain AD169 to produce the recombinant R6HS. The ganciclovir resistance of R6HS is 4-fold lower than that of D6/3/1 but 10-fold higher than that of AD169. R6HS, like AD169, is sensitive to the nucleotide analogs (S)-1-[(3-hydroxy-2-phosphonylmethoxy) propyl]adenine and (S)-1-[(3-hydroxy-2-phosphonylmethoxy)propyl]cytosine. Ganciclovir phosphorylation in R6HS-infected cells was at the same reduced level as that found in cells infected with the parental mutant D6/3/1. The same G-to-T transversion at nucleotide 1380 in the UL97 coding sequence is present in both R6HS and D6/3/1. This mutation results in the substitution of isoleucine for methionine at amino acid residue 460. In an alignment of the R6HS UL97 amino acid sequence with the amino acid sequences of a wide range of protein kinase family members, methionine 460 lies within a highly conserved region which may function in nucleotide binding and phosphate transfer.

Point mutations in the DNA polymerase gene of human cytomegalovirus that result in resistance to antiviral agents.

Three independently isolated mutants of human cytomegalovirus strain AD169 were found to be resistant to ganciclovir at a 50% effective dose of 200 microM. Phosphorylation of ganciclovir was reduced 10-fold in mutant-infected cells compared with AD169-infected cells. All three mutants were also determined to be resistant to the nucleotide analogs (S)-1-[(3-hydroxy-2- phosphonylmethoxy)propyl]adenine (HPMPA) and (S)-1-[(3-hydroxy-2-phosphonylmethoxy)propyl]cytosine (HPMPC) and hypersensitive to thymine-1-D-arabinofuranoside (AraT). Single base changes resulting in amino acid substitutions were demonstrated in the nucleotide sequence of the DNA polymerase gene of each mutant. The polymerase mutation contained in one of the mutants was transferred to the wild-type AD169 background. Ganciclovir phosphorylation in cells infected with the recombinant virus produced by this transfer was found to be equivalent to that of AD169-infected cells. The ganciclovir resistance of the recombinant was reduced fourfold compared with that of the parental mutant; however, the recombinant remained resistant to HPMPA and HPMPC and hypersensitive to AraT. The ganciclovir resistance of the mutants therefore appears to result from mutations in two genes: (i) a kinase which phosphorylates ganciclovir and (ii) the viral DNA polymerase.

A ganciclovir-resistant clinical isolate of human cytomegalovirus exhibiting cross-resistance to other DNA polymerase inhibitors.

Clinical isolates of human cytomegalovirus (HCMV) were screened for susceptibility to ganciclovir by plaque-reduction assay and in situ ELISA. A pretreatment isolate of HCMV obtained from the bronchial brushing of a heart transplant recipient contained both ganciclovir-susceptible and -resistant virus. Ganciclovir-susceptible (P8) and -resistant (D16) strains were further isolated by plaque purification. Both strains phosphorylated ganciclovir at levels similar to the ganciclovir-susceptible strain AD169. D16 was also resistant to phosphonoformic acid and to (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)adenine and cytosine. These data suggest that the resistance of D16 to these drugs results from a mutation in the viral DNA polymerase gene.

In situ ELISA for the evaluation of antiviral compounds effective against human cytomegalovirus.

An in situ ELISA was developed as an improved procedure over the plaque reduction assay for antiviral susceptibility testing of HCMV. Unlike the plaque reduction assay, the ELISA can be completed at 4-5 days post-infection. The effective dose (ED50) of ganciclovir (GCV), acyclovir (ACV), phosphonoacetic acid (PAA), or phosphonoformic acid (PFA), was determined using HCMV strain AD169. The resistance profiles of two laboratory-derived GCV-resistant mutants of HCMV strain AD169 and seven clinical isolates were determined using the ELISA. The ELISA results were confirmed by the plaque reduction assay. The ED50 for GCV with the AD169 control ranged from 3.1 to 6.2 microM with a mean inhibitory concentration of 5.4 +/- 1.4 microM. Six of the clinical isolates were susceptible to GCV (ED50 = 3.1-6.2 microM). The seventh isolate had an ED50 of 50 microM and was resistant to GCV. This ELISA assay is reproducible and relatively simple to perform. The ELISA endpoints are clearly determined and the assay works well with a variety of antiviral compounds.

Rapid detection of cytomegalovirus in clinical specimens by using biotinylated DNA probes and analysis of cross-reactivity with herpes simplex virus.

A method for rapid identification of human cytomegalovirus (HCMV) was developed with biotinylated DNA probes. BamHI restriction fragments from HCMV strain AD169 were selected and tested for their ability to detect virus in patient urine samples. All probes detected 30 pg of HCMV AD169 DNA. The BamHI B fragment detected 15 of 29 cell-culture-positive samples (sensitivity, 52%). There were four samples which were probe positive and cell culture negative (specificity, 87%). The D and H fragments used as combined probes detected 17 of 21 cell-culture-positive samples (sensitivity, 81%). There were five probe-positive and cell-culture-negative samples (specificity, 68.8%). The H fragment, when used alone, detected 11 of 14 culture-positive samples, and 5 samples were culture negative and probe positive. Sensitivity (78.6%) and specificity (76.2%) for the H fragment were similar to those for the combined probes, but the color intensity of the positive reactions detected by the H fragment alone was lower. There was unexpected cross-reactivity with herpes simplex type 1 and 2 controls when the combined D and H probes were used. Specific hybridization was demonstrated between subfragments of the HCMV BamHI D fragment and the herpes simplex virus type 1 EcoRI M fragment.