Marie-Christine Mazeron

Plasma cytomegalovirus DNA, pp65 antigenaemia and a low CD4 cell count remain risk factors for cytomegalovirus disease in patients receiving highly active antiretroviral therapy.

ObjectiveTo study the natural history and the current risk factors for cytomegalovirus (CMV) disease in the context of highly active antiretroviral therapy (HAART). SettingProspective multicentre cohort in 15 university hospitals in France. MethodsA group of 198 patients with CD4 cell count < 100 × 106 cells/l (or < 200 × 106 cells/l under HAART for at least 2 months), no previous CMV disease and CMV-positive serology were followed every 4 months clinically and for virological testing including HIV RNA and CMV blood markers (culture, pp65 antigenaemia, plasma CMV DNA and CMV late mRNA by the polymerase chain reaction). ResultsAt inclusion, median CD4 was 77 × 106 cells/l (0–308) and 85% of the patients received protease inhibitors. The percentage of patients receiving HAART reached 99% at 12 months. After a follow-up of 23.6 months, the incidence of CMV disease was 3.2/100 patient-years [95% confidence interval (CI) 1.3–5.0]. In univariate Cox models, all the CMV markers, a CD4 cell count remaining < 75 × 106 cells/l and an HIV viral load > 100 000 copies/ml were predictive for CMV disease. The hazard ratios for CMV disease were 11 for blood culture; 14 and 70 for pp65 antigenaemia of ⩾ 1 and ⩾ 100 nuclei/200 000 cells, respectively; 35 for plasma CMV DNA; 6 for CMV mRNA; 29 for CD4 < 75 × 106 cells/l; and 12 for HIV RNA > 100 000 copies/ml. In a stepwise multivariate analysis, only three covariates were independently associated with the occurrence of a disease: plasma CMV DNA, pp65 antigenaemia ⩾ 100 nuclei/200 000 cells and a CD4 count < 75 × 106 cells/l. ConclusionCMV blood markers and CD4 count < 75 × 106 cells/l remain risk factors for CMV disease in patients receiving HAART. Analysis of plasma CMV DNA by the polymerase chain reaction is a reproducible and standardized tool that could be used as a decision marker for initiating CMV pre-emptive therapy.

Failure Of Ganciclovir Treatment Associated With Selection Of A Ganciclovir-resistant Cytomegalovirus Strain In A Lung Transplant Recipient

We report the case of a lung transplant recipient with progressive cytomegalovirus (CMV) disease due to a resistant CMV strain emerging under ganciclovir (GCV) therapy. A discriminative polymerase chain reaction (PCR) assay, designed to detect the resistance-related V460 mutation within the viral enzyme UL97, revealed the presence of a mutated strain in a heterogeneous isolate 51 days after transplantation. The conventional antiviral susceptibility assay had failed to demonstrate resistance to GCV. Under prolonged GCV therapy, the mutated strain dominated the wild-type strain, as shown by the PCR assay. This domination led to laboratory resistance, associated with recurrent fever and progressively severe retinitis. As this discriminative PCR assay was shown to be effective in detecting mutated strains that constitute a minority in the virus load, it should allow better management of patients with CMV disease.

Natural Polymorphism of Cytomegalovirus DNA Polymerase Lies in Two Nonconserved Regions Located between Domains Delta-C and II and between Domains III and I

ABSTRACT We described the natural polymorphism of cytomegalovirus DNA polymerase in 42 unrelated isolates susceptible to ganciclovir, foscarnet, and cidofovir. All variations, including an eight-amino-acid deletion, were located between domains delta-C and II and between domains III and I, suggesting that these specific residues are not involved in enzymatic functions.

Maribavir Use in Practice for Cytomegalovirus Infection in French Transplantation Centers

Maribavir (MBV), a UL97 inhibitor, shows good oral bioavailability, low host cell toxicity, and theoretical benefits to inhibit cross-resistant viruses. We herein examined clinical and virological outcomes of 12 patients, including 3 bone marrow recipients and 9 organ recipients infected with resistant cytomegalovirus (CMV) and treated with MBV during 2011-2012. All received at least 800-mg daily doses. They had developed clinical (12/12) and/or virological (11/12) resistance to CMV infection. Based on a decrease of viral load in blood >1.5 log copies/mL half of them responded to MBV treatment. The individual changes varied from a rapid decrease in viral load (n = 4) to no response (n = 3) with some late response slowly decreasing viremia (n = 3). In 2 cases MBV was used as secondary prophylaxis. No clear parameter emerged as a clinical surrogate for nonresponse to MBV. These results contrast with the lack of efficacy in phase III trials of MBV prophylaxis among stem cell recipients, which were possibly due to low doses or inadequate timing of drug initiation in the study. Additional clinical and surrogate laboratory markers are needed to determine antiviral responses to guide MBV use. Dosage ranging studies might benefit future MBV use.

Favorable impact of natural killer cell reconstitution on chronic graft- versus -host disease and cytomegalovirus reactivation after allogeneic hematopoietic stem cell transplantation

Natural killer cells are the first lymphocyte subset to reconstitute, and play a major role in early immunity after allogeneic hematopoietic stem cell transplantation. Cells expressing the activating receptor NKG2C seem crucial in the resolution of cytomegalovirus episodes, even in the absence of T cells. We prospectively investigated natural killer-cell reconstitution in a cohort of 439 adult recipients who underwent non-T-cell-depleted allogeneic hematopoietic stem cell transplantation between 2005 and 2012. Freshly collected blood samples were analyzed 3, 6, 12 and 24 months after transplantation. Data were studied with respect to conditioning regimen, source of stem cells, underlying disease, occurrence of graft-versus-host disease, and profiles of cytomegalovirus reactivation. In multivariate analysis we found that the absolute numbers of CD56bright natural killer cells at month 3 were significantly higher after myeloablative conditioning than after reduced intensity conditioning. Acute graft-versus-host disease impaired reconstitution of total and CD56dim natural killer cells at month 3. In contrast, high natural killer cell count at month 3 was associated with a lower incidence of chronic graft-versus-host disease, independently of a previous episode of acute graft-versus-host disease and stem cell source. NKG2C+CD56dim and total natural killer cell counts at month 3 were lower in patients with reactivation of cytomegalovirus between month 0 and month 3, but expanded greatly afterwards. These cells were also less numerous in patients who experienced later cytomegalovirus reactivation between month 3 and month 6. Our results advocate a direct role of NKG2C-expressing natural killer cells in the early control of cytomegalovirus reactivation after allogeneic hematopoietic stem cell transplantation.

Clinical significance of cytomegalovirus viremia in bone marrow transplantation

Cytomegalovirus (CMV) viremia was systematically studied in 56 patients having undergone bone marrow transplantation for leukemia or aplastic anemia. Of the patients who survived at least three months, 57% had CMV viremia with a frequency peak between the 7th and the 9th weeks. We describe possible clinical signs associated with viremia, particularly late peripheral and/or central thrombocytopenia. The occurrence of viremia was studied according to the specific preexisting immune status of recipients and donors; granulocyte transfusions and graft-versus-host disease. The relationship between these parameters and viremia provides a basis for the analysis of prophylactic treatments of CMV infection.

Hepatitis E virus infection in HIV-infected patients with elevated serum transaminases levels.

Increases in aminotransferases levels are frequently encountered in HIV-positive patients and often remain unexplained. The role in this setting and natural history of hepatitis E in HIV-infected patients are unknown. The aim of the study was to assess HEV infection in HIV-infected patients attending a Parisian hospital, with a current or previous cryptogenic hepatitis.191 plasma samples collected from 108 HIV-infected patients with elevated aminotransferases levels were retrospectively tested for the presence of hepatitis E virus (HEV) infection markers: anti-HEV IgM antibodies, anti-HEV IgG antibodies, anti-HEV IgG avidity index and plasma HEV RNA.One acute infection, documented by positive tests for anti-HEV IgM antibody, low anti-HEV IgG avidity index and plasma HEV RNA (genotype 3e), and three past infections were diagnosed, without any observed case of persistent infection. The acute hepatitis was benign and resolved spontaneously within two weeks. This infection was probably contracted locally. Acute HEV hepatitis can occur in HIV-infected patients but rarely explains cryptogenic hepatitis, at least in an urban HIV population, regardless geographic origin and CD4 counts.

Rapid determination of antiviral drug susceptibility of human cytomegalovirus by real-time PCR.

A quantitative real-time PCR-based assay was developed for determination of cytomegalovirus (HCMV) susceptibility to antiviral drugs. After HCMV isolate-growth for 4 days, antiviral drug susceptibility was determined by measuring the reduction of intracellular HCMV DNA in the presence of increasing concentrations of either ganciclovir, or foscarnet or cidofovir. The 50% inhibitory concentration (IC(50)) was the drug concentration that reduced the number of HCMV genome copies by 50%. The IC(50) values were measured for seven HCMV reference strains sensitive or resistant to one or more antiviral drugs. The antiviral susceptibility of 21 HCMV isolates was then tested and the results were consistent with prior determination of their phenotype and/or genotype by plaque reduction assay and sequencing. The real-time PCR susceptibility assay reported here was found to be highly reproducible, simpler to perform than the plaque reduction assay, and amenable to use in the routine diagnostic virology laboratory.

Comparison of sequential cytomegalovirus isolates in a patient with lymphoma and failing antiviral therapy

BACKGROUND Long-term anti-cytomegalovirus (CMV) treatments in immunocompromised patients are hampered by resistance to antiviral drugs. Longitudinal changes in the resistance genotype may depend on changes in selective pressure and the complexity of CMV isolates. OBJECTIVE To evaluate longitudinal changes in the CMV resistance genotype and phenotype along with strain-specific variability in a patient with non-Hodgkins lymphoma in whom successive anti-CMV treatments failed. STUDY DESIGN The resistance phenotype and genotype of seven CMV isolates collected from one patient during a 2-year follow-up period were retrospectively analysed. In parallel, we used glycoprotein B (gB) genotyping, and a- and UL10-13-sequence analysis to study CMV interstrain variability. RESULTS The patient was infected by at least three CMV strains plus variants of the parental strains. Resistance to ganciclovir, cidofovir and foscarnet was successively detected during the follow-up period. UL97 protein kinase changes responsible for resistance to ganciclovir were initially detected at residues 591 and 592, and then at position 594. Decreased sensitivity to foscarnet coincided with the appearance of amino acid substitution N495K in DNA polymerase, whereas cross-resistance to ganciclovir and cidofovir was due to the L501I substitution. CONCLUSIONS The CMV isolates obtained from our patient were complex mixtures of strains. Changes in resistance genotypes depended on resistance selective pressure and were not linked to interstrain variation.

Eight flavonoids and their potential as inhibitors of human cytomegalovirus replication

The drugs currently available for treatment of severe human cytomegalovirus (HCMV) infections suffer from many drawbacks, particularly toxicity, and potential teratogenicity contraindicating their use in target populations such as pregnant women. The emergence of drug-resistant strains is still a problem for disease management, particularly in immunosuppressed populations where antivirals are used for extended periods of time. The flavonoid family of drugs contains promising candidates as they have low toxicity and inhibit different targets to currently available antivirals. We report here that, unlike their chalcon homologs, four flavonoids (baicalein, quercetin, quercetagetin and naringenin) inhibit various stages of HCMV replication, the most active anti-HCMV compound being baicalein and the less active and less selective being quercetagetin. These drugs could provide potential inhibitors of virus replication alone or in combination, without increased toxicity.