Compartmentalized dynamics of cytomegalovirus replication in treated congenital infection.

BACKGROUND: Cytomegalovirus (CMV) is the most prevalent congenital infection in developed countries. A significant number of infected infants develop long-term neurodevelopmental and hearing impairment irrespective of whether disease is detectable at birth. Studies of viral load and replication dynamics have informed the treatment of CMV in adult populations but no similar data exist in neonates. OBJECTIVES: To study CMV virus kinetics in different body fluids of babies treated for congenital infection. STUDY DESIGN: CMV virus load was sequentially analyzed in blood, urine and saliva in 17 babies treated for symptomatic congenital CMV infection. RESULTS: Virus was detectable in the urine and saliva of all babies at baseline but in only 15/17 in blood. At the end of 6 weeks of antiviral treatment CMV remained detectable in 9/14 blood samples, 9/12 urine samples and 4/7 salivary swabs. Median half-life (T1/2) of virus decline in blood was 2.4 days (IQR 1.9-3.3) and basic reproductive number (Ro) was 2.3. Although T1/2 values were similar in urine and saliva to those observed in blood, virus dynamics differed both during and after treatment. CONCLUSIONS: T1/2 and Ro in blood in this group of neonates were similar to values derived from studies of immunocompromised adults. The persistent viremia observed in treated neonates cannot therefore be adequately explained by the virus dynamics early in treatment. The different dynamics exhibited in blood and urine suggests that studying changes in distinct body compartments may assist in further understanding long-term manifestations of disease.

Effects of donor/recipient human leukocyte antigen mismatch on human cytomegalovirus replication following liver transplantation.

BACKGROUND: Natural immunity against cytomegalovirus (CMV) can control virus replication after solid organ transplantation; however, it is not known which components of the adaptive immune system mediate this protection. We investigated whether this protection requires human leukocyte antigen (HLA) matching between donor and recipient by exploiting the fact that, unlike transplantation of other solid organs, liver transplantation does not require HLA matching, but some donor and recipient pairs may nevertheless be matched by chance. METHODS: To further investigate this immune control, we determined whether chance HLA matching between donor (D) and recipient (R) in liver transplants affected a range of viral replication parameters. RESULTS: In total, 274 liver transplant recipients were stratified according to matches at the HLA A, HLA B, and HLA DR loci. The incidence of CMV viremia, kinetics of replication, and peak viral load were similar between the HLA matched and mismatched patients in the D+/R+ and D-/R+ transplant groups. D+/R- transplants with 1 or 2 mismatches at the HLA DR locus had a higher incidence of CMV viremia >3000 genomes/mL blood compared to patients matched at this locus (78% vs. 17%; P = 0.01). Evidence was seen that matching at the HLA A locus had a small effect on peak viral loads in D+/R- patients, with median peak loads of 3540 and 14,706 genomes/mL in the 0 and combined (1 and 2) mismatch groups, respectively (P = 0.03). CONCLUSION: Overall, our data indicate that, in the setting of liver transplantation, prevention of CMV infection and control of CMV replication by adaptive immunity is minimally influenced by HLA matching of the donor and recipient. Our data raise questions about immune control of CMV in the liver and also about the cells in which the virus is amplified to give rise to CMV viremia.

Development of a novel single tube nested PCR for enhanced detection of cytomegalovirus DNA from dried blood spots.

Newborn screening for congenital cytomegalovirus (CCMV) using dried blood spots (DBS) has been proposed because many developed countries have DBS screening programmes in place for other diseases. The aim of this study was to develop a rapid, single tube nested polymerase chain reaction (PCR) method for enhanced detection of CMV from DBS compared to existing (single target) real time PCRs. The new method was compared with existing real time PCRs for sensitivity and specificity. Overall sensitivity of the single target PCR assays in both asymptomatic and symptomatic infants with laboratory confirmed congenital CMV was 69% (CMV PCR or culture positive before day 21 of life). In contrast, the single tube nested assay had an increased sensitivity of 81% with100% specificity. Overall the assay detected CMV from a DBS equivalent to an original blood sample which contained 500IU/ml. In conclusion this single tube nested methodology allows simultaneous amplification and detection of CMV DNA in 1.5h removing the associated contamination risk of a two step nested PCR. Owing to its increased sensitivity, it has the potential to be used as a screening assay and ultimately allow early identification and intervention for children with congenital CMV.

Human herpesvirus vaccines and future directions.

Over the last few years there has been an impressive increase in the virological and immunological tools available to detect both human herpesvirus (HHV) and immune control of replication post-solid organ transplantation. This has allowed a greater appreciation of pathogenesis, studies to be designed to evaluate potential vaccines, new approaches adopted for antiviral deployment and the success of interventions to be judged. This chapter aims to summarize the state-of-the-art in vaccine development and look forward to the role that vaccines, immune monitoring, viral kinetics and new antiherpesvirus agents may play in the future management of HHV infections after transplantation.

Cytomegalovirus replication kinetics in solid organ transplant recipients managed by preemptive therapy.

After allotransplantation, cytomegalovirus (CMV) may be transmitted from the donor organ, giving rise to primary infection in a CMV negative recipient or reinfection in one who is CMV positive. In addition, latent CMV may reactivate in a CMV positive recipient. In this study, serial blood samples from 689 kidney or liver transplant recipients were tested for CMV DNA by quantitative PCR. CMV was managed using preemptive antiviral therapy and no patient received antiviral prophylaxis. Dynamic and quantitative measures of viremia and treatment were assessed. Median peak viral load, duration of viremia and duration of treatment were highest during primary infection, followed by reinfection then reactivation. In patients who experienced a second episode of viremia, the viral replication rate was significantly slower than in the first episode. Our data provide a clear demonstration of the immune control of CMV in immunosuppressed patients and emphasize the effectiveness of the preemptive approach for prevention of CMV syndrome and end organ disease. Overall, our findings provide quantitative biomarkers which can be used in pharmacodynamic assessments of the ability of novel CMV vaccines or antiviral drugs to reduce or even interrupt such transmission.

Cytomegalovirus: recent progress in understanding pathogenesis and control.

Cytomegalovirus continues to be an important pathogen in a variety of patient groups especially the neonate and the transplant recipient, and has implicated in a range of pathologies including inflammatory disease and in contributing to early death in ageing populations. This review will focus on advances in understanding the virus-host interaction and options for the new therapeutic control measures.

The risk of early and late CMV DNAemia associated with Campath use in stem cell transplant recipients.

The risks associated with in vivo and ex vivo use of Campath-1H and -1G in a cohort of 206 stem cell transplant recipients for human CMV (HCMV) DNAemia have been quantified. DNAemia showed a biphasic incidence pattern with an inflexion at day 60. The first phase had a linear risk rate for HCMV DNAemia of 0.3% per day, whereas the second phase had a substantially lower risk rate of 0.058% per day. In multivariable analyses, risk factors for early DNAemia were HCMV serostatus, radiotherapy-based conditioning and CD34 stem cell dose, with the use of in vivo Campath-1H having the most significant risk (hazards ratio=3.68; 95% CI=2.02-6.72; P<0.001). Ex vivo use of Campath was not associated with an increased risk for HCMV DNAemia. Patients receiving either in vivo Campath-1H or -1G experienced HCMV DNAemia earlier (27 and 33 days, respectively) compared with patients receiving no Campath (time to DNAemia, 51 days; P=0.0006). Multivariable analysis of risk factors for HCMV DNAemia occurring beyond 100 days after transplant were older age, acute GVHD>grade II and a lower CD34 stem cell dose, whereas Campath-1H use was not associated with late HCMV DNAemia.

Polyfunctional cytomegalovirus-specific CD4+ and pp65 CD8+ T cells protect against high-level replication after liver transplantation.

To determine whether polyfunctional CD4+ T-cell responses coupled with CD8+ T-cell responses against human cytomegalovirus (HCMV) are key to the control of HCMV replication we prospectively analyzed 29 liver transplant recipients for CD4+ T-cell responses against soluble HCMV antigen, pp65 and IE1 proteins, CD8+ T-cell responses against pp65 and IE1 proteins and a range of T helper (Th) 1 and Th2 cytokines. Eleven patients (38%) developed HCMV DNAemia at a median of 21 days post-liver transplantation (range 17-31 days). There was a significantly lower frequency and absolute number of total HCMV CD4+ T cells producing IFNgamma, IFNgamma+IL2 and IL2 and pp65-CD8+ T cells producing IFNgamma in patients with DNAemia. The quantities of Th1 and Th2 cytokines present during the first 20 days posttransplant were not predictive of DNAemia. Cut-off levels during the first 20 days posttransplant of 0.1% of lysate stimulated CD4+ T cells producing IL2, and pp65-CD8+ T cells producing IFNgamma above 0.4% had positive and negative predictive values for DNAemia of 54% and 100% and 50% and 92%, respectively. Measuring polyfunctional CD4+ T cells against HCMV early posttransplant may allow targeted intervention to minimize the occurrence and acute and long-term consequences of HCMV replication.

Impact of HIV on host-virus interactions during early hepatitis C virus infection.

BACKGROUND: Human immunodeficiency virus (HIV) may influence the outcome and natural history of hepatitis C virus (HCV) infection through an impact on acute HCV-specific T cell responses. METHODS: Fifty-five HIV-positive males with acute HCV infection were identified; monoinfected individuals (n = 8) were used for peripheral blood mononuclear cell comparison. In 14 coinfected and 8 HCV-monoinfected patients, HCV-specific T cell responses against a range of HCV antigens were assessed using interferon (IFN)-gamma enzyme-linked immunospot (ELISpot) and proliferation assays. E1/E2 region genetic diversity and the selection pressure on the virus were measured in 8 coinfected patients by use of cloned sequences over time. RESULTS: HCV persisted in 52 (95%) coinfected individuals. HCV/HIV coinfection significantly reduced IFN-gamma ELISpot responses versus those in HCV-monoinfected individuals, especially against nonstructural proteins (1/10 vs. 5/8; P = .008). In coinfected patients, increased HCV genetic diversity was observed between the first and subsequent time points, with no evidence for positive selection in the E1/E2 region sequenced. CONCLUSION: HIV coinfection is associated with increased rates of HCV persistence and a lack of critical CD4 T cell responses, with no evidence of immune selection pressure during early HCV infection. Loss of key cellular immune responses against HCV during acute disease may contribute to the failure of early host control of HCV in HCV/HIV-coinfected patients.

Functional impairment of cytomegalovirus specific CD8 T cells predicts high-level replication after renal transplantation.

Human cytomegalovirus (HCMV) remains an important cause of morbidity after allotransplantation, causing a range of direct effects including hepatitis, pneumonitis, enteritis and retinitis. A dominant risk factor for HCMV disease is high level viral replication in blood but it remains unexplained why only a subset of patients develop such diseases. In this detailed study of 25 renal transplant recipients, we show that functional impairment of HCMV specific CD8 T cells in the production of interferon gamma was associated with a 14-fold increased risk of progression to high level replication. The CD8 T-cell impairment persisted during the period of high level replication and was more prominent in patients above 40 years of age (odds ratio = 1.37, p = 0.01) and was also evident in dialysis patients. Threshold levels of functional impairment were associated with an increased risk of future HCMV replication and there was a direct relationship between the functional capacity of HCMV ppUL83 CD8 T cells and HCMV load (R(2)= 0.83). These results help to explain why a subset of seropositive individuals develop HCMV replication and are at risk of end-organ disease and may facilitate the early identification of individuals who would benefit from targeted anti-HCMV therapy after renal transplantation.

Congenital cytomegalovirus: association between dried blood spot viral load and hearing loss.

AIM: To investigate the relation between cytomegalovirus (CMV) viral load on dried blood spots (DBS) from newborn biochemical screening ("Guthrie") cards, and sensorineural hearing loss (SNHL) in congenital CMV. DESIGN: Cross-sectional study with retrospective case-note review. SETTING: Seven paediatric audiology departments in the United Kingdom. PATIENTS: 84 children, median age 7 years: 43 with known congenital CMV, 41 with unexplained SNHL. INTERVENTIONS: Half a DBS was tested for CMV DNA viral load by quantitative real-time polymerase chain reaction (PCR). MAIN OUTCOME MEASURES: Pure tone average hearing thresholds (0.5-4 kHz). RESULTS: DBS CMV DNA viral load significantly correlated with hearing thresholds for the worse and better hearing ears (Spearman's rank correlations: r = 0.445, p = 0.008 and r = 0.481, p = 0.004 respectively). Multivariable logistic regression showed that the effect of DBS viral load on the risk of SNHL remained important, when age and central nervous system involvement had been taken into account (odds ratio (OR) 2.76, 95% confidence interval (CI) 1.14 to 6.63, p = 0.024). The mean log DBS viral load was significantly higher in children with SNHL than in those with normal hearing (2.69 versus 1.64, 95% CI -1.84 to -0.27, p = 0.01). 8/35 (23%) children with unexplained SNHL tested positive for CMV DNA on DBS. One false positive result was obtained. CONCLUSION: The risk of SNHL increased with DBS viral load. Further studies should investigate whether DBS CMV testing has a role in identifying asymptomatic congenitally infected neonates at risk of SNHL, and whether antiviral treatment can reduce this risk.

Kinetics of cytomegalovirus load decrease in solid-organ transplant recipients after preemptive therapy with valganciclovir.

The availability of valganciclovir (VGCV) has significantly simplified the treatment of human cytomegalovirus (HCMV) infection after solid-organ transplantation. We show that there was no difference in the kinetics of the decrease in HCMV load after preemptive therapy with VGCV in 22 solid-organ transplant recipients (T1/2=2.16 days), compared with that in 23 patients treated with intravenous ganciclovir (GCV) (T(1/2) = 1.73 days; P=.63). Preemptive therapy with VGCV provides control of HCMV replication that is comparable to that achieved with preemptive intravenous therapy with GCV.

Guidelines for laboratory monitoring of treatment of persistent virus infections.

A growing number of antiviral agents are available for treatment of persistent viral infections. This has increased the requirement for virology laboratories to undertake sophisticated assays for monitoring the efficacy of treatment and identifying drug failure at an early stage. The consensus guidelines within this article address the laboratory requirements for monitoring treatment of the herpes viruses, HIV-1, Hepatitis B and Hepatitis C.

Molecular correlates in AIDS patients following antiretroviral therapy: diversified T-cell receptor repertoires and in vivo control of cytomegalovirus replication.

OBJECTIVES: To evaluate whether successful, long-term immune reconstitution in vivo can be achieved in end-stage AIDS patients following antiretroviral therapy (ART). METHODS: A 1-year prospective study of changes of CD4+ and CD8+ T-cell surface phenotypes, T-cell receptor (TCR) repertoires and capacity to control in vivo replication of cytomegalovirus (CMV) was performed in five treatment-naive end-stage AIDS patients (median CD4+ T-cell counts of 19 cells/microL) following therapy. Proportions of CD45RA+, CD45RO+ and CD28+ cells within the CD4+ and CD8+ subsets, were determined by flow cytometry. Changes in TCR Vbeta repertoires within the CD4+ and CD8+ T-cell compartments were evaluated using CDR3 spectratyping. CMV replication was determined by a sensitive polymerase chain reaction (PCR) assay using whole blood. RESULTS: Following ART, proportionate increases in 'naive' (CD45RA+) and 'memory' (CD45RO+) T cells were observed within both CD4+ and CD8+ T-cell subsets, while increased numbers of CD28+ T cells were mainly observed within the CD4+ subset. Diversification of CD4+ and CD8+ TCR repertoires was established concomitantly with renewed in vivo control of CMV replication. CONCLUSIONS: An important degree of molecular and functional immune recovery is possible in end-stage AIDS patients introduced to therapy. Diversification of TCR repertoires and the in vivo restoration of immunocompetence to control opportunistic infections clearly show that an important degree of molecular immune reconstitution is established following the initiation of ART even in late-stage AIDS.

Viral infections and their impact on chronic renal allograft dysfunction.

Viral infections, particularly those involving HCMV, are an important complication of renal transplantation. Transplantation protocols and treatment regimens that increase HCMV infection and disease may promote the development of CRAD and impair long-term renal allograft survival. Investigators are beginning to illuminate the mechanisms by which HCMV infection may cause chronic rejection in general and transplant vascular sclerosis in particular. Migration and proliferation of SMCs within the intimal layer of blood vessels is an important component of transplant vascular sclerosis, and HCMV appears to facilitate both of these processes. Current management strategies for HCMV focus on prevention, either using a focal preemptive therapeutic approach or by administering antiviral therapies to all or at-risk patients.

Cytomegalovirus-specific cellular immune responses and viremia in recipients of allogeneic stem cell transplants.

The immune suppression inherent in allogeneic stem cell transplantation (SCT) offers a favorable environment for infection by opportunistic agents, such as human cytomegalovirus (CMV). Despite the application of potent antiviral prophylaxis, patients remain at risk for CMV infection until adequate immunity is restored. CMV-specific CD8(+) T cell counts were monitored, using HLA-A2 tetrameric complexes, to establish the level of immune response to the viral phosphoprotein UL83 in patients after allogeneic SCT. Correlating this with viral replication and clinical status shows that the level of tetramer-positive T cells provides an assessment of CMV immune reconstitution after stem cell transplantation. Most patients with seropositive donors did reconstitute long-term CMV immunity, unless prolonged immunosuppression to control graft-versus-host disease was induced. Together with polymerase chain reaction testing, this technique provides measurable parameters that can be a guide to therapeutic decision making and can form the basis of CMV immunotherapy.

Replicative fitness in vivo of HIV-1 variants with multiple drug resistance-associated mutations.

The relative fitness of HIV-1 viral variants containing a broad range of drug resistance-associated mutations has been little studied in vivo. Understanding the relative fitness associated with viruses containing mutations may aid future therapeutic management. The aim of this study was to investigate the relative fitness of mutant viruses by assessing a cohort of patients who had developed resistance to many drugs and subsequently stopped all therapy. Eleven patients were assessed for drug resistance associated mutations in the protease (PR) and reverse transcriptase (RT) genes before and, at multiple time points, after stopping therapy. Relative fitness was calculated as a function of the rate of disappearance of mutant viruses when therapy was stopped. The least fit viruses were associated with the RT mutation M184I/V (11.6% less fit) and the PR mutations D30N (12.4% less fit) and M46I/L (21% less fit). Mutations at these codons were associated with significant reductions in fitness levels compared to wild-type viruses. Mutations at codons 10, 20, 36, and 63 in the PR gene remained fairly constant when therapy was stopped and may not significantly reduce viral fitness. The rapid re-population of wild-type viruses may allow the recycling of antiretroviral drugs prescribed previously.

Quantity of cytomegalovirus DNA in different leukocyte populations during active infection in vivo and the presence of gB and UL18 transcripts.

The quantity of human cytomegalovirus (HCMV) DNA in the blood of immunocompromised individuals correlates with the development of HCMV disease. We wished to determine which leukocytes harboured DNA and whether this represented active viral replication. Magnetic bead separation techniques were used to obtain pure polymorphonuclear leukocyte (PMNL), monocyte, B and T cell fractions, and RT-PCR and quantitative-competitive PCR (QC-PCR) to detect HCMV glycoprotein B (gB; UL55) transcripts and quantify HCMV DNA levels, respectively, in each cell fraction. QC-PCR revealed that PMNLs contribute the greatest to the overall viral load in blood (median viral load: PMNLs, 10(5.37) genomes/ml of blood; monocytes, 10(4.40) genomes/ml; B cells, 10(3.70) genomes/ml; and T cells, 10(4.08) genomes/ml). However, monocytes have a viral burden of 0.65 genomes/monocyte which is greater than that within the other leukocyte populations (0.11 genomes/PMNL, 0.23 genomes/B cell, and 0.20 genomes/T cell). Glycoprotein B transcripts were detected in all four cell populations: 3/10 PMNL fractions, 6/13 monocyte fractions, 5/13 B cell fractions, and 4/13 T cell fractions. The data show that productive infection of these leukocyte subpopulations, including PMNLs, can occur in vivo. Furthermore, transcripts of gpUL18, the putative natural killer (NK) cell decoy, were detected in 2/6 monocyte fractions with active replication, and 1/4 T cell fractions but not in the other leukocyte fractions. The transient nature of UL18 gene expression, and the low abundance of the transcript relative to gB were confirmed.

Progress in understanding cytomegalovirus drug resistance.

The molecular basis for cytomegalovirus drug resistance against currently used antivirals comprises two genetic loci. In the case of ganciclovir, mutations in both the UL97 protein kinase and UL64 DNA polymerase can lead to resistance, whereas for cidofovir and foscarnet only mutations in UL54 give rise to resistance. Clinically, resistance strains of cytomegalovirus appear after prolonged periods of antiviral therapy especially when treatment has been interrupted or is at sub-optimal doses. Knowledge of the replication dynamics of cytomegalovirus in vivo can be used to predict the virologic course of patients who develop resistance virus. Using such models, a good agreement between experimentally determined viral load and resistance patterns is observed.