Biomedical subjects

V C Emery

Publications and source records attributed to V C Emery.

At least 55 records · Page 3Linked to original sources

PubMed1999

Quantity of human cytomegalovirus (CMV) DNAemia as a risk factor for CMV disease in renal allograft recipients: relationship with donor/recipient CMV serostatus, receipt of augmented methylprednisolone and antithymocyte globulin (ATG).

A prospective longitudinal study of 87 renal allograft recipients identified 31 patients with cytomegalovirus (CMV) viraemia. Previous studies have identified CMV viraemia, donor positivity, and CMV load in urine as independent risk factors for disease following renal transpl antation. We used quantitative-competitive polymerase chain reaction (QC-PCR) to quantify the CMV DNA load in blood from these patients, and report that it is a significant and independent risk factor for CMV disease. Patients with symptomatic CMV infection had significantly higher maximum CMV loads than those with no disease (P = .0003). We also found that peak loads were significantly higher in individuals experiencing primary CMV infection (P < .01), and CMV re-infection (P < .05) compared with recipients reactivating endogenous CMV. Univariate analysis revealed that CMV DNA load in blood, donor seropositivity, and receipt of antithymocyte globulin (ATG) were all significantly associated with disease (P = .005, .04, and .05, respectively). However, the association of donor/recipient serostatus, and receipt of ATG became nonsignificant in multivariate analyses whereas the significance of the quantity of CMV DNAemia was maintained, illustrating that CMV load plays a central role in the pathogenesis of CMV disease.

Adolescent↗

PubMed1999

Quantitative changes in cytomegalovirus DNAemia and genetic analysis of the UL97 and UL54 genes in AIDS patients receiving cidofovir following ganciclovir therapy.

Five AIDS patients with cytomegalovirus (CMV) retinitis who had received ganciclovir (GCV) therapy were followed with serial blood sampling to detectchanges both in CMV load and in the genetic composition of genes UL97 and UL54 whilst receiving cidofovir (CDV) therapy. CDV neither reduced CMV load in blood nor prevented its quantitative resurgence during therapy. These effects were not explained by the initial presence or development of CDV-associated drug resistance mutations in UL54. In two patients, UL97 genotypic resistance to GCV involving either a L595S mutation or a deletion of amino acids 590-603 were present at the initiation of CDV and, in both patients, repopulation of CMV strains with wild-type UL97 sequences occurred during CDV therapy. These data are consistent with GCV-resistant strains containing UL97 mutations being less fit than their wild-type counterparts and so being able to persist only with the selective pressure of GCV.

Acquired Immunodeficiency Syndrome↗

PubMed1999

Response of asymptomatic cytomegalovirus viraemia to oral ganciclovir 3 g/day or 6 g/day in HIV-infected patients.

Reactivation of cytomegalovirus (CMV) following immunosuppression may result in the development of CMV disease and is associated with an increased risk of death. CMV viraemia detected by the polymerase chain reaction (PCR) precedes CMV disease in HIV-infected patients and identifies individuals at high risk of disease. Pre-emptive ganciclovir (GCV) therapy in patients who have evidence of CMV viraemia is effective in preventing disease. An open study was conducted to assess the response of CMV viraemia to oral GCV at a dose of 3 or 6 g/day for 28 days. HIV RNA was measured to determine if CMV inhibition affected HIV viral load. Fourteen patients were studied, three of whom entered both phases of the study. None of the patients had evidence of CMV disease at the time of entry into the trial; two patients developed CMV retinitis after completion of the trial. Oral GCV at both 3 and 6 g/day caused a decrease in CMV viral load in individual patients. However, a rebound in CMV viral load occurred in patients receiving the 3-g/day dose. None of the patients receiving oral GCV 3 g/day became PCR negative after 21 days compared with six of eight patients receiving 6 g/day. Five of eight patients (63%) receiving GCV 6 g/day were concurrently taking protease inhibitors compared with two of nine (22%) receiving 3 g/day. Ten patients remained PCR negative throughout follow up. No change was found in HIV viral load during receipt of GCV at either dose. Thus, oral GCV is effective in reducing CMV viral load, but a dose of 3 g/day is insufficiently potent for pre-emptive therapy.

AIDS-Related Opportunistic Infections↗

PubMed1998

Cytomegalovirus polymerase chain reaction viraemia in patients receiving ganciclovir maintenance therapy for retinitis.

OBJECTIVES: To determine whether recurrence of polymerase chain reaction (PCR) viraemia during maintenance ganciclovir for cytomegalovirus (CMV) retinitis correlates with (i) CMV disease at a new anatomical site, (ii) progression of the presenting retinitis, or (iii) acquisition of genetic changes in gene UL97 associated with resistance to ganciclovir. DESIGN: A previously described cohort of 45 patients presenting with first episode retinitis was followed clinically using ophthalmoscopy and serial tests for PCR viraemia for a median of 7 months. CMV viral load and genetic markers of ganciclovir resistance were measured in PCR-positive samples. METHODS: PCR amplification of the glycoprotein B region of CMV and quantitative competitive PCR assays were employed. Genetic changes in UL97 were identified by sequencing/point mutation assay. RESULTS: PCR viraemia correlated significantly with new episodes of CMV disease (P=0.011) and a trend was seen for the association with progression of retinitis (P=0.07). Amongst the 14 patients PCR-positive during maintenance ganciclovir, 10 (71%) had genetic markers of resistance. None of these patients became PCR-negative in blood after reinduction ganciclovir therapy compared with three out of four without markers of resistance (P=0.022). CONCLUSIONS: CMV PCR viraemia correlated strongly with the development of new episodes of CMV disease. Most patients with progression of retinitis remained PCR-negative in blood, consistent with therapeutic failure due to poor intraocular penetration of ganciclovir. However, the minority who were PCR-positive in blood may have reinfected their eye, and frequently had markers of ganciclovir resistance. The implications of these findings for the management of patients with CMV disease are discussed.

Antiviral Agents↗

PubMed1998

Quantitative differences in the distribution of zidovudine resistance mutations in multiple post-mortem tissues from AIDS patients.

Replication of HIV introduces errors into the genome which are responsible for conferring a growth advantage over wildtype virus when drugs such as zidovudine (ZDV) exert a selective pressure. The molecular basis for HIV-1 resistance to ZDV has been mapped to codons 41, 67, 70, 215 and 219 of the reverse transcriptase gene both in vitro and in clinical samples of blood. This study has investigated the relationship between the quantitative prevalence of ZDV resistance in multiple organs of the same individual. Proviral HIV-1 load was measured by quantitative-competitive PCR in 90 samples from organs of 11 patients dying with AIDS. Nine of these patients had been prescribed zidovudine. The distribution of wildtype and mutant sequences at the positions 41, 67, 70, 215 and 219 of the reverse transcriptase was assessed using a point mutation assay. The results showed that the highest proviral loads were predominately found in lymph node, spleen and lung and there was a significant association between viral load and resistance to ZDV (P=0.008). Inter-organ distribution of wildtype and mutant sequences at codons 41, 67, 70, 215 and 219 was frequently not uniform and in some patients differed markedly between the lymphoreticular system and other organs. These results demonstrate that treatment of HIV-1 infection with zidovudine does not exert uniform selective pressures in multiple organs. These findings have implications for the interpretation of resistance data and design of treatment strategies for HIV, arguing in particular that alterations in therapeutic regimens should consider the likelihood of different resistance patterns being present in multiple sites within the same individual.

Acquired Immunodeficiency Syndrome↗

PubMed1998

Human complement factor I: its expression by insect cells and its biochemical and structural characterisation.

Factor I is a five-domain plasma serine protease which is essential for the regulation of the complement system. In order to express this, the factor I coding sequence was cloned into a recombinant baculovirus system, which was used to infect Trichoplusia ni cells. Using the native factor I leader sequence, recombinant factor I (rFI) was secreted into the culture medium. Purified rFI was recognised by polyclonal antisera and by the factor I-specific monoclonal antibody MRC-OX21. SDS PAGE showed that rFI was processed into two chains with molecular weights of 48,000 and 36,000. Amino acid sequence analysis showed that the N-terminal sequences of the rFI chains were the same as those of serum-derived factor I (sFI), confirming that processing was correct. Since both molecular weights were less than those observed for sFI, this is attributed to the replacement of complex-type oligosaccharides by high mannose ones in rFI. C3(NH,) cleavage assays showed that rFI had 55% the activity of sFI. Circular dichroism and Fourier transform infrared spectroscopy showed that the protein folding of rFI and sFI were very similar. Both had a secondary structure low in alpha-helix and high in beta-sheet, as expected from crystal structure and multiple sequence alignment analyses. It is inferred that the reduced activity of rFI is attributable to its changed glycosylation. The availability of rFI and structures for the domains in factor I makes possible new approaches to determine the molecular basis of its interactions with factor H and C3b.

Animals↗

PubMed1998

A multiplex PCR assay for the simultaneous detection of human herpesvirus 6 and human herpesvirus 7, with typing of HHV-6 by enzyme cleavage of PCR products.

A multiplex polymerase chain reaction (PCR) method was developed for the simultaneous detection of human herpesviruses 6 and 7 (HHV-6; HHV-7) in clinical samples, using primers which amplify a section of the HHV-6 U67 gene and the HHV-7 homologue of the HHV-6 U42 gene. Comparison of the multiplex assay with the respective single PCR assays, using cloned HHV-6 and HHV-7 sequences as targets for amplification, showed equivalent sensitivity and specificity for the assays. To demonstrate the use of multiplex PCR for the analysis of clinical samples, serum and saliva from infants were analysed using this technique. The results showed that a clear distinction can be made between the amplicons of HHV-6 and HHV-7, without loss of sensitivity or specificity. There was complete concordance between the respective single PCR assays, and the multiplex PCR. HHV-6 amplicons derived from the multiplex PCR analysis were typed by differential AvaII restriction endonuclease digestion, in which HHV-6 variant A amplicons are cleaved but those of variant B remain undigested. These results were compared to HHV-6 variant typing by an established method, the results of which showed complete concordance between assays. It is proposed that this multiplex assay, where HHV-6 positive samples may be typed directly from the reaction products, is an efficient and cost-effective approach to the analysis of large numbers of samples to determine the epidemiological importance of HHV-6 and HHV-7.

Child↗

PubMed1998

Longitudinal fluctuations in cytomegalovirus load in bone marrow transplant patients: relationship between peak virus load, donor/recipient serostatus, acute GVHD and CMV disease.

Quantitative competitive PCR was used to monitor the quantity of cytomegalovirus (HCMV) in 1647 blood samples from 110 BMT recipients. DNAemia was detected in 49/110 (45%) of the patients, of whom 15/49 experienced HCMV disease. Peak virus load during surveillance was elevated in symptomatic (median 4.5 log10 genomes/ml) vs asymptomatic patients (median 3.6 log10 genomes/ml, P=0.002) and was also significantly elevated in HCMV seropositive recipients of seronegative marrow, (R+D-, median 5.0 log10), compared to those in the R-D- and R+D+ groups (P < 0.01 and <0.005). Odds ratios for disease per 0.25 log10 increase in viral load, recipient seropositivity and aGVHD were 1.43 (P=0.004), 6.60 (P=0.05) and 3.17 (P=0.08), respectively. In multivariate logistic regression analysis only elevated viral load remained a significant risk factor for HCMV disease. The computed disease probability viral load curve showed a rapid increase in disease risk at viral loads between 3.8 and 5.5 log10 genomes/ml in blood, and odds ratios for disease were determined for different threshold viral loads. These data demonstrate the central role of viral load in the pathogenesis of HCMV in BMT recipients and provide an additional marker for targeting and monitoring therapy.

Acyclovir↗

PubMed1998

The effect of valaciclovir on cytomegalovirus viremia and viruria detected by polymerase chain reaction in patients with advanced human immunodeficiency virus disease. AIDS Clinical Trials Group Protocol 204/Glaxo Wellcome 123-014 International CMV Prophylaxis Study Group.

Samples of blood and urine were collected at baseline, week 4, and week 8 and then every 8 weeks from 310 patients entering a controlled trial of prophylaxis with valaciclovir versus acyclovir. Samples were tested under code by polymerase chain reaction (PCR) in one laboratory. The median number of samples collected from each patient was 5 for blood (range, 0-15) and 5 for urine (range, 0-15). Both baseline PCR viremia and PCR viruria were significantly associated with future cytomegalovirus (CMV) disease (P = .002 and P = .02, respectively). The greatest effect of valaciclovir on CMV disease was seen in patients who were PCR-positive in blood at baseline (P = .002), although a significant effect was also seen in those who were PCR-negative in urine (P = .02). Thus, PCR viremia provides prognostic information about CMV disease in AIDS patients, and valaciclovir showed activity as both a preemptive and prophylactic agent.

AIDS-Related Opportunistic Infections↗

PubMed1998

Transcription of the human cytomegalovirus natural killer decoy gene, UL18, in vitro and in vivo.

Multiplex RT-PCR analysis of human cytomegalovirus (HCMV) replication in human fibroblasts showed transcription of the natural killer (NK) cell decoy gene, UL18, from 72 h onwards. Transcription of glycoprotein B (gpUL55; a late gene) occurred from early time-points and peaked at 24 h post-infection. UL18 mRNA was also detected in the peripheral blood mononuclear cells of organ transplant recipients with HCMV viraemia, especially those with HCMV DNA virus loads greater than 10(5) genomes/ml whole blood. Thus, UL18 is produced via a low abundance transcript late during the infectious cycle at a time coincidental with the increased risk of NK cell lysis as a consequence of class I HLA down-regulation.

Base Sequence↗

PubMed1998

Cytomegalovirus drug resistance.

Clinical resistance of cytomegalovirus (CMV) against the currently licensed antiviral drugs is becoming an increasingly recognized problem. This review focuses on the molecular basis of resistance and describes mutations in the UL54 DNA polymerase leading to resistance against cidofovir, foscarnet and ganciclovir. The review highlights two important developments in our appreciation of resistance. Firstly, the use of more rapid molecular based assays to detect genotypic resistance and secondly, the relationship between resistance profiles in multiple organ systems of the same host. Finally, the changing face of CMV disease in the era of highly active antiviral chemotherapy is considered with respect to its impact on the frequency of CMV resistance in the clinic.

Antiviral Agents↗

PubMed1997

Prevalence of anti-vif antibodies in HIV-1 infected individuals assessed using recombinant baculovirus expressed vif protein.

A 630 base pair fragment of the HIV-1 genome encompassing the entire vif open reading frame has been produced by the polymerase chain reaction and cloned into the baculovirus transfer vector pAcYM1. Extracts from insect cells infected with a recombinant baculovirus expressing the HIV-1 vif gene product were used in a radioimmunoassay to analyse 238 sera from HIV infected individuals for the presence of anti-vif antibodies. The overall prevalence of anti-vif antibodies in this group of patients was 25.3%. Stratification of the group according to CD4 levels showed that anti-vif antibodies were more prevalent in patients with CD4 counts below the median of the group (155 x 10(6) cells/L; P = 0.005). A significant increase in anti-vif antibodies was observed in patients with CD4 levels less than 280 x 10(6) cells/L (P < 0.01) and in patients with symptomatic HIV infection (P = 0.0003). However, there was no significant difference in the prevalence of anti-vif antibodies in patients stratified according to p24 antigen status. The implications of these findings in the context of HIV replication are discussed.

Acquired Immunodeficiency Syndrome↗

PubMed1997

Development of a quantitative competitive polymerase chain reaction for human herpesvirus 8.

A quantitative competitive polymerase chain reaction (PCR) assay for human herpesvirus 8 (HHV8) was developed. The assay uses a competitive internal control sequence which differs from the wild type sequence by the presence of an EcoRI restriction endonuclease site. The quantitative method was validated by co-amplifying known amounts of control sequence DNA and wild type DNA, and shown to accurately quantify HHV8 within the range of 10(1)-10(6) genome copies (R = 0.998). The assay was used to quantify HHV8 in sequential blood samples of a renal transplant patient diagnosed with Kaposi's sarcoma (KS). A peak viral load of 28320 genomes/ml blood was present at diagnosis of KS, which reduced to 13680 genomes after 2 days and was undetectable 4 months later. Control of HHV8 replication in this patient parallelled the cessation of immunosuppressive therapy.

Calibration↗

PubMed1997

Development of a point mutation assay for the detection of human cytomegalovirus UL97 mutations associated with ganciclovir resistance.

A point mutation assay was developed to detect the quantitative prevalence of mutations at codons 460 (M to I; M to V), 520 (H to Q), 594 (A to V) and 595 (L to F; L to S) within the UL97 gene of human cytomegalovirus which segregate with ganciclovir resistance. Synthetic mixtures of wild-type and mutant plasmids containing the UL97 gene were amplified by nested polymerase chain reaction and the 700 base pair amplicon subsequently subjected to the point mutation assay. In plasmid reconstruction experiments, there was a high correlation between experimentally derived percentage mutant with the theoretical values. The assay was then used to assess the changes in the genetic composition of the UL97 gene in three patients on prolonged ganciclovir therapy. All three patients developed genotypic resistance against ganciclovir involving mutation at codon L595S, L595F and double mutation at codons L595F and M460I. In one patient, alteration of therapy to foscarnet did not affect the composition of UL97 and virus remained genotypically resistant to ganciclovir. In contrast, in two patients whose therapy was altered to cidofovir (HPMPC), repopulation with cytomegalovirus strains carrying the wild-type (ganciclovir-sensitive) codon at positions 595 and 460 occurred. The potential use of this assay for the rapid detection of cytomegalovirus resistance in patients on long-term ganciclovir therapy is discussed.

Acquired Immunodeficiency Syndrome↗

PubMed1997

Interrelationships among quantity of human cytomegalovirus (HCMV) DNA in blood, donor-recipient serostatus, and administration of methylprednisolone as risk factors for HCMV disease following liver transplantation.

Longitudinal analysis of 162 liver transplant recipients identified 51 patients who were viremic. Virus load was determined in 47 of these patients using quantitative-competitive polymerase chain reaction. Peak virus load was significantly higher in 20 symptomatic patients than 27 asymptomatic patients (P < .0001). Elevated virus load, donor seropositivity, and total methylprednisolone dosage were risk factors for human cytomegalovirus (HCMV) disease (odds ratio [OR], 2.22/0.25 log10 increase in virus load, P = .001; OR, 4.11, P = .05; OR, 1.30/1-g increment in methylprednisolone, P = .01). Methylprednisolone and virus load were independent risk factors in a multivariate analysis (OR, 2.70/1-g increase, P = .003; OR, 1.61/0.25 log10 increase, P = .03, respectively). Virus loads of 10(4.75)-10(5.25) genomes/mL of blood were associated with an increased disease probability; the latter was shifted to lower virus loads with increasing quantities of methylprednisolone. These data illustrate the central role of virus load in HCMV pathogenesis.

Adolescent↗

PubMed1997

Cytomegalovirus (CMV) viraemia detected by polymerase chain reaction identifies a group of HIV-positive patients at high risk of CMV disease.

BACKGROUND: Cytomegalovirus (CMV) disease is a major cause of morbidity in patients with HIV infection. Despite treatment, CMV retinitis causes substantial visual loss, especially in patients with CD4 cell counts below 50 x 10(6)/l. Although routine ophthalmological screening of these patients has been recommended, no controlled trials have evaluated how frequently it should be performed. The aim of this study was to assess whether CMV polymerase chain reaction (PCR) results could direct ophthalmological screening to patients at high risk of CMV retinitis. METHODS: In a prospective study of HIV-positive patients with CD4 cell counts below 50 x 10(6)/l, CMV viraemia was detected by qualitative PCR of whole blood. Patients who were CMV PCR-viraemic were allocated to monthly virological and ophthalmological follow-up; patients who were PCR-negative received 3-monthly virological and ophthalmological follow-up. CMV viral load was determined in all CMV-positive samples using a quantitative competitive PCR. RESULTS: Nineteen out of 97 patients developed CMV disease over the first 12 months of the study. Sixteen (59%) out of 27 patients who were CMV-positive developed disease compared with three (4%) out of 70 of patients who were PCR-negative (P = 0.0001). A positive CMV PCR result was significantly associated with the development of disease (P = 0.0001), with a relative hazard of 20.15 [95% confidence interval (CI), 5.80-69.98]. Median CMV viral load was significantly higher in those individuals who went on to develop CMV disease (P = 0.02). In PCR-positive patients, each 0.25 log10 increase in viral load increased the risk of disease (relative hazard, 1.37; 95% CI, 1.15-1.63; P = 0.0004). CONCLUSIONS: CMV PCR predicts the development of CMV disease and can be used to target ophthalmological resources to those patients at highest risk of retinitis. Asymptomatic patients who are PCR-positive represent a high-risk group in whom controlled trials of pre-emptive therapy could be conducted.

AIDS-Related Opportunistic Infections↗

PubMed1997

Long-term evolution of the 5'UTR and a region of NS4 containing a CTL epitope of hepatitis C virus in two haemophilic patients.

Haemophilic patients exposed to unsterilized clotting factor concentrates prior to 1985 have become infected with hepatitis C virus (HCV). We have studied the sequence evolution of the 5'UTR and a region of NS4 over 12 years in one human immunodeficiency virus (HIV) positive haemophilic patient and 14 years for one HIV negative haemophilic patient. One sample each year from the date of HCV infection to 1994 was analysed for genotype, virus load and nucleotide sequence of the two genetic loci. Both patients were infected with HCV genotype 1 throughout the study period. The virus load profiles were similar except that the profile for the HIV infected patient was displaced 4 years earlier relative to the other patient. Mean divergence of the quasispecies at both the 5'UTR and NS4 loci was higher in the HIV coinfected patient. Phylogenetic analysis indicated that evolution of the 5'UTR was host independent, whereas the NS4 region containing a CD8 restricted CTL epitope evolved in a host specific fashion.

Amino Acid Sequence↗

PubMed1997

Prospective study of human herpesvirus 6, human herpesvirus 7, and cytomegalovirus infections in human immunodeficiency virus-positive patients.

Blood samples from human immunodeficiency virus (HIV)-positive patients were monitored for cytomegalovirus (CMV), human herpesvirus 6 (HHV-6), and HHV-7 by PCR. We detected CMV in 17% of the patients, HHV-6 in 6%, and HHV-7 in 3%. The viral loads of CMV were significantly higher than those of HHV-6 (P = 0.007) or HHV-7 (P = 0.01). Detection of CMV and HHV-6 was associated with low and high CD4 counts, respectively.

CD4 Lymphocyte Count↗