QuantiFERON-CMV assay by chemiluminescence immunoassay: Is it more suitable for real-live monitoring of transplant patients?

BACKGROUND: The QuantiFERONCMV (QF-CMV) assay is an interferon-gamma release assay (IGRA) used to monitor CMV-specific cell-mediated immunity (CMV-CMI) by ELISA in transplant patients. However, a chemiluminescent immunoassay (CLIA) has been developed to quantify IFNG in the QuantiFERON-Tuberculosis (TB) to detect latent TB infection. OBJECTIVES: The aim of this work is to compare the results of QF-CMV by ELISA with those obtained by CLIA in an automated Liaison XL analyzer using the QuantiFERON-TB Gold Plus reagents. STUDY DESIGN: The QF-CMV assay had been performed by ELISA in kidney and lung transplant patients between July 2019-April 2023 at the IMIBIC/Reina Sofía Hospital (Cordoba, Spain). The remaining QF-CMV supernatants had been preserved at -80 ºC from then. Now, the IFNG levels in the same samples were determined by CLIA. RESULTS: One hundred and three QF-CMV supernatants from kidney (n = 50) and lung (n = 53) transplant patients were selected. An agreement of 87.4 % (kappa coefficient 0.788) between CLIA and ELISA was observed. Thirteen (12.6 %) discrepant results were detected. Some Indeterminate results by ELISA converted to Non-reactive by CLIA (0.53-0.92 IU/mL for Mitogen-Nil values). Likewise, borderline Non-reactive results by ELISA were above the 0.2 IU/mL cut-off by CLIA and then were Reactive (0.21-0.31 for CMV-Nil values). CONCLUSION: CLIA shows substantial concordance with ELISA and acceptable discrepancies. The possible higher sensitivity of CLIA returns a higher number of Reactive results, which entails potential clinical consequences. Therefore, a new threshold to confer protection against CMV infection after transplantation needs to be defined.

Memory SARS-CoV-2 T-cell response in convalescent COVID-19 patients with undetectable specific IgG antibodies: a comparative study.

Background: During the COVID-19 pandemic, a variable percentage of patients with SARS-CoV-2 infection failed to elicit humoral response. This study investigates whether patients with undetectable SARS-CoV-2 IgG are able to generate SARS-CoV-2 memory T cells with proliferative capacity upon stimulation. Methods: This cross-sectional study was conducted with convalescent COVID-19 patients, diagnosed with a positive real-time PCR (RT-PCR) from nasal and pharyngeal swab specimens. COVID-19 patients were enrolled ≥3 months after the last PCR positive. Proliferative T-cell response after whole blood stimulation was assessed using the FASCIA assay. Results: A total of 119 participants (86 PCR-confirmed COVID-19 patients and 33 healthy controls) were randomly filtered from an initial cohort. Of these 86 patients, 59 had detectable (seropositive) and 27 had undetectable (seronegative) SARS-CoV-2 IgG. Seropositive patients were subclassified as asymptomatic/mild or severe according to the oxygen supplementation requirement. SARS-CoV-2 CD3+ and CD4+ T cells showed significantly lower proliferative response in seronegative than in seropositive patients. The ROC curve analysis indicated that ≥ 5 CD4+ blasts/µL of blood defined a "positive SARS-CoV-2 T cell response". According to this cut-off, 93.2% of seropositive patients had a positive T-cell response compared to 50% of seronegative patients and 20% of negative controls (chi-square; p < 0.001). Conclusions: This proliferative assay is useful not only to discriminate convalescent patients from negative controls, but also to distinguish seropositive patients from those with undetectable SARS-CoV-2 IgG antibodies. Memory T cells in seronegative patients are able to respond to SARSCoV-2 peptides, although at a lower magnitude than seropositive patients.

Polygenic Innate Immunity Score to Predict the Risk of Cytomegalovirus Infection in CMV D+/R- Transplant Recipients. A Prospective Multicenter Cohort Study.

Several genetic polymorphisms of the innate immune system have been described to increase the risk of cytomegalovirus (CMV) infection in transplant patients. The aim of this study was to assess the impact of a polygenic score to predict CMV infection and disease in high risk CMV transplant recipients (heart, liver, kidney or pancreas). On hundred and sixteen CMV-seronegative recipients of grafts from CMV-seropositive donors undergoing heart, liver, and kidney or pancreas transplantation from 7 centres were prospectively included for this purpose during a 2-year period. All recipients received 100-day prophylaxis with valganciclovir. CMV infection occurred in 61 patients (53%) at 163 median days from transplant, 33 asymptomatic replication (28%) and 28 CMV disease (24%). Eleven patients (9%) had recurrent CMV infection. Clinically and/or functionally relevant single nucleotide polymorphisms (SNPs) from TLR2, TLR3, TLR4, TLR7, TLR9, AIM2, MBL2, IL28, IFI16, MYD88, IRAK2 and IRAK4 were assessed by real time polymerase chain reaction (RT-PCR) or sequence-based typing (PCR-SBT). A polygenic score including the TLR4 (rs4986790/rs4986791), TLR9 (rs3775291), TLR3 (rs3775296), AIM2 (rs855873), TLR7 (rs179008), MBL (OO/OA/XAO), IFNL3/IL28B (rs12979860) and IFI16 (rs6940) SNPs was built based on the risk of CMV infection and disease. The CMV score predicted the risk of CMV disease with an AUC of the model of 0.68, with sensitivity and specificity of 64.3 and 71.6%, respectively. Even though further studies are needed to validate this score, its use would represent an effective model to develop more robust scores predicting the risk of CMV disease in donor/recipient mismatch (D+/R-) transplant recipients.

Humoral/Cellular Immune Discordance in Stem Cell Donors: Impact on Cytomegalovirus-Specific Immune Reconstitution after Related Hematopoietic Transplantation.

Cytomegalovirus (CMV) reactivation is an important cause of complications after hematopoietic stem cell transplantation (HSCT). Discrepancies between serologic and cellular CMV-specific immune response have been reported. This study evaluated the impact of lack of CMV-specific CD8+ T cell response in seropositive donors (ie, discordant donors) on the reconstitution of CMV-specific cell-mediated immunity (CMI) after related HSCT in seropositive recipients. CMV-CMI was assessed in donors and recipients using the QuantiFERON-CMV assay (QF). CMV-CMI was prospectively assessed for 1 year in 81 CMV-seropositive HSCT recipients with a haploidentical or matched related donor. A Cox proportional hazard regression analysis was performed. Of the 67 CMV-seropositive donors, 54 (80.6%) were D+QFpos. The remaining 13 CMV-seropositive donors (19.4%) had a QFneg result and thus were classified as discordant donors (D+QFneg). We found that patients with D+QFneg had a significantly higher risk of impaired CMV-CMI reconstitution compared with patients with D+QFpos (log-rank test, P = .001) or D- donors (log-rank test, P = .023). In addition, the D+QFneg group had a higher incidence of single-episode reactivation compared with D+QFpos or D- donors (69.2% versus 44.4% and 28.6%, respectively) but a lower incidence of CMV recurrence compared with the D- group (7.7% versus 57.1%; P = .003). After adjusting for other relevant variables, immune discordance in donors was independently associated with impaired CMV-CMI reconstitution compared with D+QFpos donors (adjusted hazard ratio [HR], 0.18; 95% confidence interval [CI], .06 to .52; P = .001) and D- donors (adjusted HR, .17; 95% CI, .05 to .59; P = .005). Discordant donors were associated with undetectable CMV-CMI during the 12-month follow-up period using the QF assay. The inability of these patients to become QFpos persisted even after CMV reactivation. This might be related to the low frequency of CMV recurrence in this group. CMV-CMI assessment, in conjunction with CMV serostatus, can be of utility to better classify stem cell donors as well as the risk of impaired CMV-CMI reconstitution after HSCT.

Immunoguided Discontinuation of Prophylaxis for Cytomegalovirus Disease in Kidney Transplant Recipients Treated With Antithymocyte Globulin: A Randomized Clinical Trial.

BACKGROUND: Antiviral prophylaxis is recommended in cytomegalovirus (CMV)-seropositive kidney transplant (KT) recipients receiving antithymocyte globulin (ATG) as induction. An alternative strategy of premature discontinuation of prophylaxis after CMV-specific cell-mediated immunity (CMV-CMI) recovery (immunoguided prevention) has not been studied. Our aim was to determine whether it is effective and safe to discontinue prophylaxis when CMV-CMI is detected and to continue with preemptive therapy. METHODS: In this open-label, noninferiority clinical trial, patients were randomized 1:1 to follow an immunoguided strategy, receiving prophylaxis until CMV-CMI recovery or to receive fixed-duration prophylaxis until day 90. After prophylaxis, preemptive therapy (valganciclovir 900 mg twice daily) was indicated in both arms until month 6. The primary and secondary outcomes were incidence of CMV disease and replication, respectively, within the first 12 months. Desirability of outcome ranking (DOOR) assessed 2 deleterious events (CMV disease/replication and neutropenia). RESULTS: A total of 150 CMV-seropositive KT recipients were randomly assigned. There was no difference in the incidence of CMV disease (0% vs 2.7%; P = .149) and replication (17.1% vs 13.5%; log-rank test, P = .422) between both arms. Incidence of neutropenia was lower in the immunoguided arm (9.2% vs 37.8%; odds ratio, 6.0; P < .001). A total of 66.1% of patients in the immunoguided arm showed a better DOOR, indicating a greater likelihood of a better outcome. CONCLUSIONS: Prophylaxis can be prematurely discontinued in CMV-seropositive KT patients receiving ATG when CMV-CMI is recovered since no significant increase in the incidence of CMV replication or disease is observed. CLINICAL TRIALS REGISTRATION: NCT03123627.

Human cytomegalovirus (HCMV)-encoded microRNAs: potential biomarkers and clinical applications.

HCMV-encoded microRNAs (hcmv-miRNAs) are non-coding and non-immunogenic molecules that target numerous cellular genes and allow the virus to modulate the host's signalling pathways, thus favouring viral survival and replication. Given their capacity to silence the human genes involved in various physiological processes, these hcmv-miRNAs have now emerged as a potential clinical biomarker in many human diseases. In this review, we summarize the evidence published on the diagnostic and prognostic value of hcmv-miRNAs in several human diseases and their clinical implications. Specifically, we discuss the role of hcmv-miRNAs in the development of cardiovascular diseases and cancer by silencing tumour suppressors. We also examine the current knowledge on the utility of some hcmv-miRNAs in predicting HCMV viraemia recurrence in transplant patients, as well as the interference of hcmv-miRNAs in the development of an appropriate immune response against other viral infections, which might have therapeutic implications.Abbreviations: HCMV, human cytomegalovirus; hcmv-miRNA, HCMV-encoded microRNAs.

Pretransplant CMV-Specific T-Cell Immunity But Not Dose of Antithymocyte Globulin Is Associated With Recovery of Specific Immunity After Kidney Transplantation.

BACKGROUND: This is a prospective, multicenter, observational study in cytomegalovirus (CMV)-seropositive kidney transplant recipients with pretransplant CMV-specific cell-mediated immunity (CMV-CMI) receiving antithymocyte globulin (ATG). We aimed to investigate posttransplant CMV-CMI over time and the impact of the dose-dependent ATG. METHODS: CMV-CMI was assessed at days +30, +45, +60, and +90 after transplantation with the QuantiFERON-CMV assay. A reactive result (interferon-γ [IFN-γ] ≥ 0.2 IU/mL) indicated a positive CMV-CMI. RESULTS: A total of 78 positive CMV-CMI patients were enrolled in the study, of which 59.5% had a positive CMV-CMI at day +30 and 82.7% at day +90. Multivariate logistic regression analysis showed that ATG dose was not associated with positive CMV-CMI at any point. However, pretransplant IFN-γ level (>12 IU/mL vs ≤12 IU/mL) was associated with positive CMV-CMI at day +30 (odds ratio, 12.9; 95% confidence interval, 3.1-53.3; P < .001). In addition, all the patients who did not recover CMV-CMI at day +90 had a pretransplant IFN-γ level ≤12 IU/mL. CONCLUSIONS: More than half of CMV-seropositive kidney transplant recipients receiving ATG recover (or maintain) CMV-CMI by the first month after transplantation. The pretransplant IFN-γ level, but not the ATG dose, shows a strong association with the kinetics of this recovery.

Lack of cytomegalovirus (CMV)-specific cell-mediated immune response using QuantiFERON-CMV assay in CMV-seropositive healthy volunteers: fact not artifact.

The QuantiFERON-CMV (QF) assay measures cell-mediated immunity against cytomegalovirus (CMV-CMI), which is particularly useful in individuals susceptible to CMV infection such as transplant patients. A positive QF result identifies patients that are better protected against CMV infection. However, the significance of a negative QF result in CMV-seropositive individuals needs to be clarified. CMV-CMI was analyzed in healthy subjects using the QF assay, and, in parallel, the Flow-cytometric Assay of Specific Cell-mediated Immune response in Activated whole blood (FASCIA). FASCIA assay measures T-cell proliferation using CMV lysate as stimulus whereas QF assay use a mix of peptides. A total of 93 healthy volunteers were enrolled, and 13/71 CMV-seropositive individuals (18.3%) showed humoral/cellular discordance using QF assay (CMV+ QF-). Interestingly, with FASCIA assay CD4+ and CD8+ T-cell proliferations were lower in CMV+ QF- than in CMV+ QF+ individuals. Furthermore, CMV+ QF- volunteers had a lower level of anti-CMV IgG than CMV+ QF+ subjects. Discordant CMV+ QF- volunteers can be defined as low responder individuals since they show lower CMV-specific humoral and cellular immune responses in comparison to CMV+ QF+ individuals. Immune discordance shows the high heterogeneity of immunity to CMV in healthy subjects.

Efficacy and safety of the combination of reduced duration prophylaxis followed by immuno-guided prophylaxis to prevent cytomegalovirus disease in lung transplant recipients (CYTOCOR STUDY): an open-label, randomised, non-inferiority clinical trial.

INTRODUCTION: Prolonged use of antivirals to prevent the development of cytomegalovirus (CMV) disease in lung transplant patients has been shown to have significant side effects, for which alternatives are being sought to reduce their use. The monitoring of cell immunity against CMV could be an alternative as it has shown to be useful in identifying transplant patients at low risk of infection, who could benefit from shorter prophylaxis. The aim of the CYTOCOR study is to demonstrate that the combination of a reduced prophylaxis strategy with subsequent CMV-specific immunological monitoring would allow CMV infection to be controlled in lung transplant patients as effectively as the usual strategy (prophylaxis followed by pre-emptive therapy), while reducing the side effects of antivirals due to the shorter duration of prophylaxis. METHODS AND ANALYSIS: Phase III randomised, open, multicentre, parallel, non-inferiority clinical trial to study the efficacy and safety of the combination of a prophylaxis strategy up to month +3 post-transplant followed by immuno-guided prophylaxis using the QuantiFERON-CMV technique up to month +12 post-transplant to prevent CMV disease in CMV-seropositive lung transplant recipients. This strategy will be compared with a combination of a usual prophylaxis strategy up to month +6 post-transplant followed by pre-emptive therapy up to month +12. To study the incidence of CMV disease, patients will be followed up to 18 months post-transplantation. A total of 150 patients are expected to be recruited for the study. ETHICS AND PUBLIC DISSEMINATION: The clinical trial has been approved by the Research Ethics Committees and authorised by the Spanish Agency of Medicines and Medical Devices (AEMPS).If the hypothesis of this clinical trial is verified, the dissemination of the results could change clinical practice by increasing knowledge about the safety and efficacy of discontinuing valganciclovir prophylaxis in lung transplant recipients. TRIAL REGISTRATION NUMBER: NCT03699254.

Analysis of spontaneous resolution of cytomegalovirus replication after transplantation in CMV-seropositive patients with pretransplant CD8+IFNG+ response.

This prospective study evaluates whether CMV-seropositive (R+) transplant patients with pretransplant CD8+IFNG+ T-cell response to cytomegalovirus (CMV) (CD8+IFNG+ response) can spontaneously clear the CMV viral load without requiring treatment. A total of 104 transplant patients (kidney/liver) with pretransplant CD8+IFNG+ response were evaluable. This response was determined using QuantiFERON-CMV assay. The incidence of CMV replication and disease was 45.2% (47/104) and 6.7% (7/104), respectively. Of the total patients, 77.9% (81/104) did not require antiviral treatment, either because they did not have CMV replication (n = 57) or because they had asymptomatic CMV replication that could be spontaneously cleared (n = 24). Both situations are likely related to the presence of CD8+IFNG+ response to CMV, which has a key role in controlling CMV infection. However, 22.1% of the patients (23/104) received antiviral treatment, although only 7 of them did so because they had symptomatic CMV replication. These patients developed symptoms in spite of having pretransplant CD8+IFNG+ response, thus suggesting that other immunological parameters might be involved, such as a dysfunctional CD4+ response or that they might have become QFNon-reactive due to the immunosuppression. In conclusion, around 80% of R+ patients with pretransplant CD8+IFNG+ response to CMV did not require antiviral treatment, although this percentage might be underestimated. Nevertheless, other strategies such as performing an additional CD8+IFNG+ response determination at posttransplant time might provide more reliable information regarding the patients who will be able to spontaneously clear the viremia.

Lack of evidence of association between IFNG and IL28B polymorphisms and QuantiFERON-CMV test results in seropositive transplant patients.

The aim of this study was to analyze the relationship between the IFNG +874 T/A and IL28B (rs12979860) C/T polymorphisms and the secretion of IFNG by CD8+ T cells after stimulation with cytomegalovirus (CMV) peptides, measured using QuantiFERON-CMV (QF-CMV) assay. A total of 184 CMV-seropositive solid organ transplant patients (108 kidney, 68 liver and 8 lung) were recruited. Of them, 151 patients were QF-CMV Reactive (IFNG ≥ 0.2 UI/mL) and 33 were Non-reactive. Genotype frequencies in the study population were TT (26.6%), AT (50.0%) and AA (23.4%) for IFNG +874 and CC (52.7%), CT (39.1%) and TT (8.2%) for IL28B (rs12979860). These frequencies did not significantly differ between QF-CMV Reactive and Non-reactive patients. Nor were any significant differences observed in the quantitative IFNG level among the genotypes in either the IFNG or the IL28 genes. When we analyzed whether these polymorphisms had any impact on the risk of CMV replication after transplantation, the adjusted analysis showed no association. In summary, our results showed that IFNG +874 T/A and IL28B (rs12979860) C/T polymorphisms are not associated with the IFNG response to CMV measured by the QuantiFERON-CMV assay, although these results should be confirmed with a higher number of patients.

Impact of age and cytomegalovirus on CD8+ T-cell compartment remodeling after solid organ transplantation: A one-year follow-up study.

Cytomegalovirus (CMV), a member of the ß-herpesvirus family, is a major complicating infection in transplant patients. CMV latency has a long-term impact on CD8+ T-cell differentiation. It is unclear, however, whether this effect can be detected in one-year period. To investigate this, we analyzed the remodeling of the CD8+ T-cell compartment during the first year after solid organ transplantation. A total of 55 kidney or lung transplant patients were recruited. CD8+ T-cell subsets were prospectively analyzed at pretransplant, at 3 or 6months and 12months after transplantation (mo post-Tx). A significant increase in the frequency of CD27-CD28-CD8+ T cells (from 32.8% to 42.3%; p=0.014) was observed from pretransplant to 12mo post-Tx. Further analysis, however, showed that the largest expansion was observed from 3/6 to 12mo post-Tx whereas small non-significant variations were observed from pretransplant to 3/6mo post-Tx. The adjusted analysis showed that age and CMV seropositivity were statistically associated with the baseline frequency of CD27-CD28-CD8+ T cells. Additionally, CMV replication was related to the posttransplant expansion of this subpopulation, since it was not observed in patients without CMV viremia (24% vs. 4.2%). The results indicate that the expanded frequency associated with late CMV replication is additive to the baseline frequency related to aging and CMV seropositivity. If the expanded frequency remains at this high level for a long period it might have clinical consequences related to the control of future reactivations of CMV or of other related viruses.

Prevention strategies differentially modulate the impact of cytomegalovirus replication on CD8(+) T-cell differentiation in high-risk solid organ transplant patients.

The present study aimed to determine whether antiviral prevention strategies against cytomegalovirus (CMV) infection used in high-risk D+R- solid organ transplanted patients can modulate the impact of CMV replication on CD8(+) T-cell differentiation. The different CD8(+) T-cell subpopulations were measured at a single point when at least one year had elapsed since transplantation. A total of 68 D+R- patients were included, of which 33 underwent pre-emptive therapy and 35 received prophylaxis. Multivariate analysis showed that CMV replication was associated with the expansion of CD28Ö¾ EMRA CD8(+) T cells in patients managed pre-emptively but not in patients under prophylaxis (21.4% vs. 3.6%). This finding is likely related to the higher frequency of CMV recurrence observed in patients under pre-emptive therapy compared to those under prophylaxis (75% vs. 14.3%; p < 0.001). In fact, multivariate analysis showed that having more than one replication episode was associated with a 17.2% increase (p = 0.001) in the percentage of CD28Ö¾ EMRA CD8(+) T cells compared to "no episode" and with a 10.9% increase with respect to "single episodes" (p = 0.025). Additionally, patients with IFNγ response to CMV (QuantiFERON-CMV Reactive) had a higher percentage of late-differentiated CD8(+) T cells than patients lacking this response. In summary, recurrent CMV replication in D+R- patients under pre-emptive therapy was associated with the expansion of CD28Ö¾ EMRA CD8(+) T cells, which might have a short-term beneficial effect related to the high functionality of this T-cell subpopulation. Nevertheless, we cannot rule out that this accumulation might have a long-term detrimental effect related to immunosenescence and inflammation.

Impact of the PROVAUR stewardship programme on linezolid resistance in a tertiary university hospital: a before-and-after interventional study.

BACKGROUND: There is little evidence of the impact of antimicrobial stewardship programmes on antimicrobial resistance. OBJECTIVES: To study the efficacy and safety of a package of educational and interventional measures to optimize linezolid use and its impact on bacterial resistance. METHODS: A quasi-experimental study was designed and carried out before and after implementation of a stewardship programme in hospitalized patients with Gram-positive infections treated with linezolid. RESULTS: The intervention reduced linezolid consumption by 76%. The risk of linezolid-resistant CoNS isolates (OR = 0.37; 95% CI = 0.27-0.49; P < 0.001) and Enterococcus faecalis (OR = 0.44; 95% CI = 0.21-0.90; P = 0.03) during the intervention period was lower than in the pre-intervention period. CONCLUSIONS: A programme to optimize linezolid use can contribute to reducing the resistance rate of CoNS and E. faecalis to this antibiotic.

Interferon-γ production by CMV-specific CD8+ T lymphocytes provides protection against cytomegalovirus reactivation in critically ill patients.

PURPOSE: To evaluate the usefulness of the secretion of interferon-γ (IFNγ) by cytomegalovirus (CMV)-specific CD8+ T cells to determine the risk of CMV reactivation in critically ill non-immunosuppressed patients. METHODS: Two-center prospective cohort study including critically ill non-immunosuppressed CMV-seropositive patients admitted between December 2012 and March 2013. The incidence of CMV reactivation by polymerase chain reaction (real-time PCR) in plasma was investigated. IFNγ secretion by CMV-specific CD8+ T lymphocytes was determined at the time of admission to the intensive care unit (ICU) by means of the QuantiFERON(®)-CMV (QF-CMV) test. Cox regression analyses were performed to investigate CMV reactivation risk factors. RESULTS: Fifty-three patients were included, of whom 13 (24.5%) presented CMV reactivation. Twenty-six patients (49.1%) were QF-CMV "reactive" (QF-CMV(R)). Of the 26 QF-CMV(R) patients, 11.5% (3/26) had CMV reactivation, whereas 37% (10/27) of QF-CMV "non reactive" patients (QF-CMV(NR)) presented reactivation (p = 0.03). By Cox regression, the presence of QF-CMV(R) at ICU admission (HR 0.09, 95% CI 0.02-0.44; p = 0.003) was associated with a decreased risk of CMV reactivation. The sensitivity, specificity, positive predictive value, and negative predictive value of QF-CMV were 77, 57, 37, and 88%, respectively. Eleven of the 53 patients (20.7%) died during the follow-up period. Mortality was more frequent in patients with CMV reactivation (6/13, 46.1 vs. 5/40, 12.5%; p = 0.015). CONCLUSIONS: In critically ill non-immunosuppressed patients, the presence of functional CMV-specific CD8+ T lymphocyte response at intensive care unit admission provides protection against CMV reactivation.

Persistence of pathological distribution of NK cells in HIV-infected patients with prolonged use of HAART and a sustained immune response.

OBJECTIVE: A prospective analysis of the distribution of NK subsets and natural cytotoxicity receptors (NKp30/NKp46) in HIV patients with long-term HAART use and sustained virological and immunological response. METHODS: The main inclusion criteria were: at least 3 years' receipt of HAART; current CD4+ count ≥ 500 cells/mm3; undetectable viral load for at least 24 months; no hepatotropic virus co-infection. Percentages of CD56dim, CD56bright NK cells and CD56neg CD16+ cells were obtained. Expression of the NCRs, NKp30 and NKp46 was analysed in CD56+ cells. Thirty-nine infected patients and sixteen healthy donors were included in the study. RESULTS: The percentages of total CD56+ and CD56dim NK cells were significantly lower in HIV-infected patients than in healthy donors (70.4 vs. 50.3 and 80.9 vs. 66.1 respectively). The percentage of total CD56+ NK cells expressing NCR receptors was lower in HIV patients than in healthy donors (NKp30: 25.20 vs. 58.63; NKp46: 24.8 vs. 50.59). This was also observed for CD56dim and CD56bright NK cells. Length of time with undetectable HIV viral load was identified as an independent factor associated with higher expression of NKp30 and NKp46. CONCLUSION: Despite the prolonged and effective use of HAART, HIV-infected patients do not fully reconstitute the distribution of NK cells. Length of time with an undetectable viral load was related to greater recovery of NKp30/NKp46 receptors.

Multifunctional cytomegalovirus (CMV)-specific CD8(+) T cells are not restricted by telomere-related senescence in young or old adults.

Antigen-specific multifunctional T cells that secrete interferon-γ, interleukin-2 and tumour necrosis factor-α simultaneously after activation are important for the control of many infections. It is unclear if these CD8(+) T cells are at an early or late stage of differentiation and whether telomere erosion restricts their replicative capacity. We developed a multi-parameter flow cytometric method for investigating the relationship between differentiation (CD45RA and CD27 surface phenotype), function (cytokine production) and replicative capacity (telomere length) in individual cytomegalovirus (CMV) antigen-specific CD8(+) T cells. This involves surface and intracellular cell staining coupled to fluorescence in situ hybridization to detect telomeres (flow-FISH). The end-stage/senescent CD8(+)  CD45RA(+)  CD27(-) T-cell subset increases significantly during ageing and this is exaggerated in CMV immune-responsive subjects. However, these end-stage cells do not have the shortest telomeres, implicating additional non-telomere-related mechanisms in inducing their senescence. The telomere lengths in total and CMV (NLV)-specific CD8(+) T cells in all four subsets defined by CD45RA and CD27 expression were significantly shorter in old compared with young individuals in both a Caucasian and an Asian cohort. Following stimulation by anti-CD3 or NLV peptide, similar proportions of triple-cytokine-producing cells are found in CD8(+) T cells at all stages of differentiation in both age groups. Furthermore, these multi-functional cells had intermediate telomere lengths compared with cells producing only one or two cytokines after activation. Therefore, global and CMV (NLV)-specific CD8(+) T cells that secrete interferon-γ, interleukin-2 and tumour necrosis factor-α are at an intermediate stage of differentiation and are not restricted by excessive telomere erosion.

Use of high-dose ganciclovir for the treatment of cytomegalovirus replication in solid organ transplant patients with ganciclovir resistance-inducing mutations.

BACKGROUND: Experience with high-dose ganciclovir for the management of resistant cytomegalovirus (CMV) replication in transplant patients is limited despite its adoption as an effective therapy by some consensus documents. METHODS: We studied six cases of CMV replication in solid organ transplant patients whose genotypic testing showed mutations associated with different levels of resistance to ganciclovir. All were treated with high-dose intravenous ganciclovir (7.5-10 mg/kg/12 hr) or oral valganciclovir (1350-1800 mg/12 hr) corrected according to creatinine clearance. The virologic response was considered positive if the CMV plasma viral load was undetectable. Safety was evaluated by clinical assessment, including the review of vital signs and laboratory tests. RESULTS: All patients had asymptomatic replication, except one who had digestive disease. Four patients received universal prophylaxis with valganciclovir. Two patients received preemptive therapy with valganciclovir for individual episodes of replication. Two of the six patients received steroid boluses before the episode of replication by resistant CMV. All patients responded to treatment, including those with mutations associated with a high level of ganciclovir resistance. Four patients had neutropenia (<1.5 × 10/L), but only one received treatment. CONCLUSIONS: High-dose ganciclovir/valganciclovir can be an option in the treatment of resistant CMV replication and could be considered an alternative treatment in nonsevere patients for whom the use of foscarnet should be avoided. The toxicity of this regimen does not appear to limit its use.

Clinical factors influencing phenotype of HCMV-specific CD8+ T cells and HCMV-induced interferon-gamma production after allogeneic stem cells transplantation.

Human cytomegalovirus (HCMV) infection causes significant morbidity and mortality after hematopoietic stem cell transplantation (HSCT). In this work, we characterized the phenotype and interferon-gamma (INF-γ) production of HCMV-specific T cells using QuantiFERON-HCMV assay in 26 patients 6 months after HSCT. We analysed whether these two parameters were associated with clinical variables. Our results showed that the patients receiving stem cells from donors ≥40 years old were 12 times more likely to have HCMV-specific CD8+ T cells with "differentiated phenotype" (CD45RA+CCR7+ ≤6.7% and CD28+ ≤30%) than patients grafted from donors <40 years old (OR = 12; P = 0.014). In addition, a detectable IFN-γ production in response to HCMV peptides (cutoff 0.2 IU/mL IFN-γ; "reactive" QuantiFERON-HCMV test) was statistically associated with HCMV replication after transplantation (OR = 11; P = 0.026), recipients ≥40 versus <40 years old (OR = 11; P = 0.026), and the use of peripheral blood versus bone marrow as stem cell source (OR = 17.5; P = 0.024). In conclusion, donor age is the only factor significantly associated with the presence of the "differentiated phenotype" in HCMV-specific CD8+ T cells, whereas HCMV replication after transplantation, recipient age, and stem cell source are the factors associated with the production of IFN-γ in response to HCMV epitopes.

Impact of cytomegalovirus on early immunosenescence of CD8+ T lymphocytes after solid organ transplantation.

The increasing number of elderly people eligible for solid organ transplants has made it necessary to reevaluate how the decline in immune function associated to ageing (immunosenescence) affects solid organ transplants. Some immunosenescence biomarkers, such as the expansion of CD28(-)CD8+ T lymphocytes, have been associated to cytomegalovirus infection and are related to a form of accelerated immune senescence in transplant recipients. However, the impact of cytomegalovirus replication on downregulation of CD28 on total CD8+ T cells is independent of patients' age, whereas downregulation on cytomegalovirus-specific CD8+ T cells depends on patients' age, inducing early immunosenescence of cytomegalovirus-specific CD8+ T cells in young but not elderly solid organ transplants recipients. Although immunosenescence in transplant recipients should be considered a two-edged sword as it is a risk factor for the development of tumors after transplantation, it has a beneficial effect in attenuating acute allograft rejection and correlates with better clinical outcomes.