Letermovir use may impact on the Cytomegalovirus DNA fragmentation profile in plasma from allogeneic hematopoietic stem cell transplant recipients.

Cytomegalovirus (CMV) DNA in plasma is mainly unprotected and highly fragmented. The size of the amplicon largely explains the variation in CMV DNA loads quantified across PCR platforms. In this proof-of-concept study, we assessed whether the CMV DNA fragmentation profile may vary across allogeneic hematopoietic stem cell transplant recipients (allo-SCT), within the same patient over time, or is affected by letermovir (LMV) use. A total of 52 plasma specimens from 14 nonconsecutive allo-SCT recipients were included. The RealTime CMV PCR (Abbott Molecular), was used to monitor CMV DNA load in plasma, and fragmentation was assessed with a laboratory-designed PCR generating overlapping amplicons (around 90-110 bp) within the CMV UL34, UL80.5, and UL54 genes. Intrapatient, inter-patient, and LMV-associated qualitative and quantitative variations in seven amplicons were observed. These variations were seemingly unrelated to the CMV DNA loads measured by the Abbott PCR assay. CMV DNA loads quantified by UL34_4, UL54.5, and UL80.5_1 PCR assays discriminate between LMV and non-LMV patients. Our observations may have relevant implications in the management of active CMV infection in allo-SCT recipients, either treated or not with LMV, although the data need further validation.

Immunovirology of cytomegalovirus infection in allogeneic stem cell transplant recipients undergoing prophylaxis with letermovir: A narrative review.

On November 7, 2017, the US Food and Drug Administration approved the use of letermovir (LMV) for prophylaxis of cytomegalovirus (CMV) infection in adult CMV-seropositive allogeneic stem cell transplant recipients. After 6 years of use, a large body of real-world experience has been accumulated that validates the Phase III clinical trial results, in which LMV was shown to significantly reduce the risk of clinically significant CMV infection-defined as CMV end-organ disease or CMV DNAemia requiring pre-emptive antiviral therapy (PET)-and increase survival up to Week 24 after treatment inception. Notwithstanding, several issues still need to be settled, thus further investigation is required. First, since viral DNA may accumulate as a result of LMV-driven abortive CMV infection, what is the optimal viral load threshold in the blood that would prompt LMV prophylaxis interruption and PET inception? Should this be adapted to the patient's risk? Second, what is the impact of LMV prophylaxis on the reconstitution of functional CMV-specific T-cell responses? Would it be a wise approach to individually tailor the duration of LMV treatment according to the number of peripheral blood CMV-specific T cells at the end of regular prophylaxis? Third, how frequently do LMV-resistant strains arise while patients are on LMV prophylaxis and how could this be minimized? Here, we discuss the literature addressing these topics.

T-lymphocyte subtyping: an early warning and a potential prognostic indicator of active cytomegalovirus infection in patients with sepsis.

Cytomegalovirus (CMV) infection is very common in patients suffering from sepsis and may cause poor prognosis. To explore the relationship between immune status of patients with sepsis and CMV infection, we assessed T lymphocyte subtyping and other commonly used clinical parameters in patients with sepsis upon admission to the intensive care unit (ICU) and evaluated their potential impact on diagnosis and outcomes of active CMV infection. In our study, 82 of 599 patients with sepsis were diagnosed with active CMV infection. The 28-day mortality was higher in active CMV-infected than nonactive CMV-infected patients (20.7% versus 9.9%); 51of 82 active CMV-infected patients with sepsis were assessed to have CMV-DNA-negative conversion, while 31 were persistently positive for CMV DNA. Higher CD8+ CD28+ T-cell counts at presentation were associated with CMV-DNA-negative conversion and lower 28-day mortality. The CMV-DNA-negative conversion and 28-day mortality of active CMV-infected patients with sepsis could be predicted using cutoff values of 151 (74.5% sensitivity and 87.1% specificity) and 64.5 (52.9% sensitivity and 92.3% specificity) CD8+ CD28+ T cells mL-1 at ICU admission, respectively. Higher CD8+ CD28+ T-cell count was significantly associated with active CMV infection, higher CMV-DNA-negative conversion and lower 28-day mortality, which may be a potential marker for early warning of active CMV infection and outcome prediction.

Features of Cytomegalovirus DNAemia Blips in Allogeneic Hematopoietic Stem Cell Transplant Recipients: Implications for Optimization of Preemptive Antiviral Therapy Strategies.

Cytomegalovirus (CMV) DNAemia occurs frequently in allogeneic hematopoietic stem cell transplant recipients (allo-HSCT). There is limited information about the incidence, features, and clinical impact of CMV DNAemia blips (episodes defined by an isolated positive PCR result) in this setting. In this retrospective study, 225 consecutive adult patients undergoing any modality of allo-HSCT at our center between May 2012 and July 2019 were included. Plasma CMV DNA load was monitored using a highly sensitive real-time PCR assay. In all, 187 of 225 patients had CMV DNAemia through day 365 after allo-HSCT (total number of episodes, n = 379). Eighty-three of the 187 patients had 1 or more blips (n = 104). Blips occurred as a first episode of CMV DNAemia as opposed to prolonged CMV DNAemia (≥2 consecutive positive PCR results) in 47 patients; in 20 of these patients, blips represented the only documented episode throughout the study period, and in 27 patients, blips preceded a prolonged CMV DNAemia episode. In the remaining 36 patients, blips developed as recurrences. Blips presenting as initial episodes occurred more frequently (P < .001) in patients receiving an allograft from a CMV-seropositive donor. The cumulative incidence of recurrent CMV DNAemia following initial blips, self-resolving prolonged CMV DNAemia episodes, or CMV DNAemia episodes treated preemptively with antivirals was not significantly different (P = .34). Receiver operating characteristic curve analysis indicated that a CMV DNA load cutoff of 48 IU/mL yielded the highest combined sensitivity (66%) and specificity (70.2%) for predicting a prolonged CMV DNAemia episode. The practical implications of our data in the optimization of preemptive antiviral therapy strategies are discussed.

Genetic variation in toll like receptors 2, 7, 9 and interleukin-6 is associated with cytomegalovirus infection in late pregnancy.

BACKGROUND: Maternal cytomegalovirus (CMV) infection and/or reactivation in pregnancy is associated with a myriad of adverse infant outcomes. However, the role of host genetic polymorphisms in modulating maternal CMV status is inconclusive. This study investigated the possible association of single nucleotide polymorphisms in toll-like receptor (TLR) and cytokine genes with maternal plasma CMV DNA status in black Zimbabweans. METHODS: In a cross-sectional study, 110 women in late gestation who included 36 CMV infected cases and 74 CMV uninfected, age and HIV status matched controls were enrolled. Twenty single nucleotide polymorphisms in 10 genes which code for proteins involved in immunity against CMV were genotyped using Iplex GOLD SNP genotyping protocol on the Agena MassARRAY® system. Statistical analyses were performed using Stata SE and the 'Genetics' and 'SNPassoc' packages of the statistical package R. RESULTS: The TLR7 rs179008A > T (p < 0.001) polymorphism was associated while the TLR9 rs352139T > C (p = 0.049) polymorphism was on the borderline for association with CMV positive (CMV+) status. In contrast, the interleukin (IL)-6 rs10499563T > C (p < 0.001) and TLR2 rs1816702C > T (p = 0.001) polymorphisms were associated with CMV negative (CMV-) status. Furthermore, allele frequencies of SNPs in TLR2, TLR4, TLR9, TLR7, IL-6, IL-10, IL-28B, IL-1A and interferon AR1 (IFNAR1) genes are being reported here for the first time in a Zimbabwean population. The allele frequencies in the Zimbabwean population are generally comparable to other African populations but different when compared to European and Asian populations. CONCLUSIONS: Toll-like receptor and interleukin genetic polymorphisms influence CMV status in late gestation among black Zimbabweans. This is attributable to possible modulation of immune responses to CMV reactivation in a population previously exposed to CMV infection.

Characterization of Natural Killer Cells in HIV Patients Beginning Therapy with a High Burden of Cytomegalovirus.

BACKGROUND: Active infections with cytomegalovirus (CMV) increase NK cell expression of the inhibitory receptor LIR-1 and the activating receptor NKG2C in transplant recipients. However, the effects of CMV on NK cells are different in HIV patients stable on antiretroviral therapy (ART) and have not been analyzed in young HIV patients beginning ART. METHODOLOGY: We followed a cohort of 78 Indonesian HIV patients beginning ART. CMV antibodies were measured in plasma before ART (baseline), and after 1, 3, 6, and 12 months. CMV DNA was sought in blood granulocytes at baseline by quantitative PCR assay and a deletion in the NKG2C gene was identified by PCR. NK cell profiles were monitored by flow cytometry in 19 patients stratified by the presence of CMV DNA. Healthy controls (n = 17) were assessed once. RESULTS: All 78 patients were CMV seropositive and 41 had detectable CMV DNA. CMV DNA+ patients had higher proportions of total NK cells and CD16+ NK cells at baseline, but similar expression of LIR-1 and NKp30 on NK cells on ART. However, levels of CMV antibody were inversely related to median LIR-1 expression on NK cells. A dramatic elevation in cells expressing NKG2C was restricted to CMV DNA+ patients heterozygous for the NKG2C deletion. Patients with High NKG2C expression had lower levels of CMV antibodies. CONCLUSION: A subpopulation of NK cells expressing NKG2C was induced by CMV replication in HIV patients heterozygous for a deletion in this gene. Individuals with an abundant NKG2C+ and LIR-1+ NK cells displayed lower levels of CMV reactive antibody.

Cytomegalovirus, Epstein-Barr virus and human herpesvirus 8 salivary shedding in HIV positive men who have sex with men with controlled and uncontrolled plasma HIV viremia: a 24-month longitudinal study.

BACKGROUND: This longitudinal study described Cytomegalovirus (CMV) DNA, Epstein-Barr (EBV) DNA and human herpesvirus 8 (HHV-8) DNA asymptomatic salivary shedding in HIV-positive men who have sex with men (MSM). We aimed to 1-analyze frequency and persistence of herpesvirus shedding, 2-correlate herpesvirus positivity and HIV viroimmunological parameters and 3-assess the association between HIV-RNA suppression and herpesvirus replication. METHODS: Herpesvirus DNA was tested with an in-house real-time PCR in 2 salivary samples obtained at T0 and T1 (24 months after T0). HIV-RNA was evaluated in the 24 months prior to T0 and in the 24 months prior to T1; MSM were classified as successfully suppressed patients (SSPs), viremic patients (VPs) and partially suppressed patients (PSPs). EBV DNA load was classified as low viral load (EBV-LVL, value ≤10,000 copies/ml) and as high viral load (EBV-HVL,> 10,000 copies/ml). Mann-Whitney U test tested the difference of the median between groups of patients. Chi-squared test and Fisher's exact test compared categorical variables according to the frequencies. Kruskal-Wallis test compared continuous data distributions between levels of categorical variables. RESULTS: Ninety-two patients (median CD4+ count 575 cells/mm3, median nadir 330 CD4+ cells/mm3) were included: 40 SSPs,33 VPs and 19 PSPs. The more frequently single virus detected was EBV, both at T0 and at T1 (in 67.5 and 70% of SSPs, in 84.8 and 81.8% of VPs and in 68.4 and 73.7% of SPSs) and the most frequently multiple positivity detected was EBV + HHV-8. At T1, the percentage of CMV positivity was higher in VPs than in SSPs (36.4% vs 5%, p < 0.001), the combined shedding of HHV-8, CMV and EBV was present only in VPs (15.1%, p = 0.01 respect to SSPs) and no VPs confirmed the absence of shedding found at T0 (vs 17.5% of SSPs, p = 0.01). EBV-HVL was more frequent in VPs than in SSPs: 78.6% at T0 (p = 0.03) and 88.9% at T1 (p = 0.01). CONCLUSIONS: The relationship between uncontrolled plasma HIV viremia and CMV, EBV, and HHV-8 shedding is multifaceted, as demonstrated by the focused association with EBV DNA load and not with its frequency and by the persistent combined detection of two oncogenic viruses as EBV and HHV-8 regardless of HIV virological control.

Determination of the Biological Form of Human Cytomegalovirus DNA in the Plasma of Solid-Organ Transplant Recipients.

Background: Whether cytomegalovirus (CMV) DNA exists in plasma as virion-associated or free DNA is uncertain. Methods: An assay combining DNase I digestion and CMV quantitative polymerase chain reaction (DNase-CMV-qPCR) was developed to differentiate free naked DNA from virion DNA. One hundred three frozen and 10 fresh CMV DNA-positive plasma samples from solid-organ transplant recipients (SOTRs) were tested. Three sets of paired qPCR (P-qPCR) assays with amplicons of variable length were used to study CMV DNA fragmentation in 20 SOTR plasma samples, viral stocks (Towne, Merlin, AD169) and the first World Health Organization (WHO) international standard (IS) for CMV DNA. Results: In all plasma samples, 98.8%-100% of CMV DNA was free DNA; this was the only form in 93 of 103 (90.3%) frozen and all 10 fresh samples tested using DNase-CMV-qPCR. Low levels of virion CMV DNA were found in 10 of 103 (9.7%) samples with higher total DNA load. Cytomegalovirus DNA results were highly reproducible for 3 CMV virus stocks and WHO IS (P > .80), tested by three sets of paired q-PCR. However, for the 20 SOTR plasma samples, the smaller amplicon assay result was 2.6-fold, 3.4-fold, and 6.5-fold higher than the longer amplicion result (P < .001). Conclusions: Cytomegalovirus DNA in SOTR plasma is almost exclusively free DNA, highly fragmented, and not virion associated.

Prediction of Congenital Cytomegalovirus Infection in High-Risk Pregnant Women.

BACKGROUND: This prospective study aimed to determine maternal clinical, laboratory, and ultrasound findings that effectively predict the occurrence of congenital cytomegalovirus (CMV) infection (CCI) in high-risk pregnant women. METHODS: Three hundred CMV immunoglobulin (Ig) M-positive pregnant women were enrolled. The maternal clinical and laboratory findings, including serum CMV IgM and IgG; IgG avidity index (AI); antigenemia assay (C7-HRP); polymerase chain reaction (PCR) for the detection of CMV-DNA in the maternal serum, urine, and uterine cervical secretion; and prenatal ultrasound findings, were evaluated. To determine predictive factors for the occurrence of CCI, logistic regression analyses were performed. RESULTS: In 22 of the 300 women, CCI was confirmed using PCR for CMV-DNA in newborn urine. Univariate analyses demonstrated that the presence of maternal flu-like symptoms, presence of ultrasound fetal abnormalities, serum titers of CMV IgM, positive results for C7-HRP, CMV IgG AI <40%, and positive PCR results in the uterine cervical secretion were statistically associated with the occurrence of CCI. Multivariable analysis revealed that the presence of ultrasound fetal abnormalities (odds ratio [OR], 31.9; 95% confidence interval [CI], 8.5-120.3; P < .001) and positive PCR results in the uterine cervical secretion (OR, 16.4; 95% CI, 5.0-54.1; P < .001) were independent predictive factors of CCI in CMV IgM-positive women. CONCLUSIONS: This is the first prospective cohort study to suggest that the presence of CMV-DNA in the maternal uterine cervical secretion and ultrasound fetal abnormalities are predictive of the occurrence of congenital CMV infection in high-risk pregnant women.

Analysis of Mutations in the Gene Encoding Cytomegalovirus DNA Polymerase in a Phase 2 Clinical Trial of Brincidofovir Prophylaxis.

Brincidofovir is an oral antiviral in development for prevention of cytomegalovirus disease. Cytomegalovirus genotyping results from a phase 2 trial comparing brincidofovir to placebo for prophylaxis against cytomegalovirus infection in hematopoietic cell transplant recipients provided initial data on the clinical resistance profile for brincidofovir. In this study, no known resistance-associated mutations were detected in brincidofovir-treated subjects; identified genotypic substitutions did not confer resistance to cytomegalovirus antivirals in vitro, suggesting that these changes represent polymorphisms unrelated to brincidofovir resistance. Lack of evidence for genotypic resistance during prophylaxis suggests that first-line use of brincidofovir for prevention of cytomegalovirus infection may preserve downstream options for patients.

Epstein-Barr and cytomegalovirus DNA salivary shedding correlate with long-term plasma HIV RNA detection in HIV-infected men who have sex with men.

The aim of the study was to evaluate cytomegalovirus (CMV) and Epstein-Barr virus (EBV) DNA salivary shedding in HIV-positive men who have sex with men (MSM) and to determine whether viro-immunological parameters and long-term (24 months) plasma HIV RNA (pHIV) detection may predict herpesviruses replication. A total of 193 HIV-positive MSM were consecutively recruited (mean CD4+ cell count 607 cells/mm(3) and mean nadir value 333 cells/mm(3) ); pHIV was analyzed for 24 months prior to saliva sampling: patients were categorized as successfully suppressed (SS) and not suppressed (NS). The EBV viral load was categorized as high viral load (HVL), intermediate (IVL), or low (LVL), CMV DNA as positive or negative. NS patients experienced both herpesviruses detectability more frequently respect to SS patients (P = 0.034); conversely, no salivary shedding was more frequent in SS patients (P = 0.014). HVL EBV was more frequent in NS patients than in SS subjects (P = 0.038 for isolated EBV detection and P = 0.001 when CMV shedding was associated). NS subjects with HVL EBV had a median pHIV of 43,820 copies/ml, significantly higher respect to IVL and LVL patients (P = 0.027 and P = 0.0005, respectively). CMV shedding was mostly associated to EBV shedding. NS patients showed a significantly higher frequency of saliva HVL EBV detection compared to SS patients; moreover, NS patients with HVL EBV had a higher pHIV respect to those with IVL and LVL shedding. Our results suggest that a successful pHIV suppression could reduce the burden of salivary EBV replication and likely the risk of herpesviruses-related cancers.

Comparison of two molecular assays for detection of cytomegalovirus DNA in whole blood and plasma samples from transplant recipients.

In immunosuppressed patients, pre-emptive therapy and a strict follow-up of CMV infection are the standard of care for the prevention of CMV disease. Several real-time PCR assays for CMV DNA quantification on whole blood (WB) and plasma (PL) are commercially available. This study compared and correlated CMV viral loads obtained by the Cobas AmpliPrep/Cobas TaqMan (CAP/CTM) platform on plasma specimens with those obtained on corresponding whole blood specimens by the real-time PCR assay (ELITe MGB-CMV) in 185 sequential samples from 41 immunosuppressed patients. Correlation between the two assays was good. Kinetics of CMV DNA within the same patient was similar, but PL viral load was constantly 1 log lower than WB. In patients under antiviral therapy, low level of CMV DNA persisted in WB, while it was absent in PL. The good correlation between CMV DNA detected on both PL and WB supports the reliability of the two matrices for viral monitoring and the therapeutic management of CMV infection. Nevertheless, due to significant quantification differences between PL and WB CMV DNA, the same biological specimen should be used for a sequential and reliable follow-up of patients at high risk of CMV infection.

Looking for biological factors to predict the risk of active cytomegalovirus infection in non-immunosuppressed critically ill patients.

The identification of non-immunosuppressed critically ill patients most at risk for developing cytomegalovirus (CMV) reactivation is potentially of great clinical relevance. The current study was aimed at determining (i) whether single nucleotide polymorphisms in the genes coding for chemokine receptor 5 (CCR5), interleukin-10 IL-10), and monocyte chemoattractant protein-1 (MCP-1) have an impact on the incidence rate of active CMV infection, (ii) whether serum levels of CMV-specific IgGs are associated with the risk of CMV reactivation, and (iii) whether detection of CMV DNA in saliva precedes that in the lower respiratory tract or the blood compartment. A total of 36 out of 78 patients (46%) developed an episode of active CMV infection. The incidence rate of active CMV infection was not significantly associated with any single nucleotide polymorphisms. A trend towards a lower incidence of active CMV infection (P = 0.06) was noted in patients harboring the IL10 C/C genotype. Patients carrying the CCR5 A/A genotype had high CMV DNA loads in tracheal aspirates. The serum levels of CMV IgGs did not differ significantly between patients with a subsequent episode of active CMV infection (median, 217 IU/mL) or without one (median, 494 IU/mL). Detection of CMV DNA in saliva did not usually precede that in plasma and/or tracheal aspirates. In summary, the analysis of single nucleotide polymorphisms in the IL10 and CCR5 genes might help to determine the risk of active CMV infection or the level of CMV replication within episodes, respectively, in non-immunosuppressed critically ill patients.

Diagnostic Utility of Cytomegalovirus (CMV) DNA Quantitation in Ulcerative Colitis.

Cytomegalovirus (CMV) colitis is a critical condition associated with severe complications in ulcerative colitis (UC). This study aimed to investigate the diagnostic value of the presence of CMV DNA in intestinal mucosa tissue and blood samples in patients with active UC. This study included 81 patients with exacerbated symptoms of UC. Patient data were obtained from the Hospital Information Management System. CMV DNA in colorectal tissue and plasma samples were analyzed using a real-time quantitative PCR assay. CMV markers were detected using immunohistochemistry and hematoxylin-eosin staining. Immunohistochemistry positivity was observed in tissue samples from eight (9.9%) patients. Only one (1.2%) patient showed CMV-specific intranuclear inclusion bodies. CMV DNA was detected in 63.0% of the tissues (median: 113 copies/mg) and in 58.5% of the plasma samples (median: 102 copies/mL). For tissues, sensitivity and the negative predictive value (NPV) for qPCR were excellent (100.0%), whereas specificity and the positive predictive value (PPV) were low (41.9% and 15.7%, respectively). For plasma, sensitivity and NPV were high (100.0%) for qPCR, whereas specificity and PPV were low (48.6% and 24.0%, respectively). CMV DNA ≥392 copies/mg in tissue samples (sensitivity 100.0% and specificity 83.6%) and ≥578 copies/mL (895 IU/mL) in plasma samples (sensitivity 66.7% and specificity 100.0%) provided an optimal diagnosis for this test. The qPCR method improved patient management through the early detection of CMV colitis in patients with UC. However, reliance on qPCR positivity alone can lead to overdiagnosis. Quantification of CMV DNA can improve diagnostic specificity, although standardization is warranted.

Update on Cytomegalovirus Infection Management in Allogeneic Hematopoietic Stem Cell Transplant Recipients. A Consensus Document of the Spanish Group for Hematopoietic Transplantation and Cell Therapy (GETH-TC).

Background: Cytomegalovirus (CMV) infection is a common complication following allogeneic hematopoietic stem cell transplantation (allo-HSCT) and in patients receiving novel hematological therapies. Its impact on morbidity and mortality necessitates effective management strategies. Despite recent advances in diagnostics and treatment, unresolved questions persist regarding monitoring and treatment, prompting the need for updated recommendations. Methods: A consensus was reached among a panel of experts selected for their expertise in CMV research and clinical practice. Key clinical areas and questions were identified based on previous surveys and literature reviews. Recommendations were formulated through consensus and graded using established guidelines. Results: Recommendations were provided for virological monitoring, including the timing and frequency of CMV DNAemia surveillance, especially during letermovir (LMV) prophylaxis. We evaluated the role of CMV DNA load quantification in diagnosing CMV disease, particularly pneumonia and gastrointestinal involvement, along with the utility of specific CMV immune monitoring in identifying at-risk patients. Strategies for tailoring LMV prophylaxis, managing breakthrough DNAemia, and implementing secondary prophylaxis in refractory cases were outlined. Additionally, criteria for initiating early antiviral treatment based on viral load dynamics were discussed. Conclusion: The consensus provides updated recommendations for managing CMV infection in hematological patients, focusing on unresolved issues in monitoring, prophylaxis, treatment, and resistance. These recommendations aim to guide clinical practice and improve outcomes in this high-risk population. Further research is warranted to validate these recommendations and address ongoing challenges in CMV management with emerging antiviral combinations, particularly in pediatric populations.

Virologic suppression measured by a cytomegalovirus (CMV) DNA test calibrated to the World Health Organization international standard is predictive of CMV disease resolution in transplant recipients.

BACKGROUND: Cytomegalovirus (CMV) load measurement is used to assess the efficacy of treatment of CMV disease, but lacks standardization. Using the World Health Organization (WHO) international standard for reporting, we correlated viral load with CMV disease resolution. METHODS: CMV load was quantified in plasma using a test calibrated to the WHO standard. Three predictive rules were predefined to determine association between CMV DNAemia and outcome: (1) pretreatment CMV DNA of <18,200 (4.3 log(10)) IU/mL; (2) viral load declines of 1.0, 1.5, 2.0, and 2.5 log(10) IU/mL from baseline to days 7, 14, and 21 of treatment, respectively; and (3) viral suppression <137 (2.1 log(10)) IU/mL at days 7, 14, and 21. Analysis was performed using Cox proportional hazard models. RESULTS: Of 267 patients, 251 had CMV disease resolution by day 49 of treatment. Patients with pretreatment CMV DNA of <18,200 (4.3 log(10)) IU/mL had faster time to disease resolution (adjusted hazard ratio [AHR], 1.56; P = .001). Patients with CMV load suppression (<137 IU/mL [<2.1 log(10)]) at days 7, 14, and 21 had faster times to clinical disease resolution (AHRs, 1.61, 1.73, and 1.64, and P = .005, <.001, and <.001, respectively). Relative CMV load reductions from baseline were not significantly associated with faster resolution of CMV disease. CONCLUSIONS: Patients with pretreatment CMV DNA of <18,200 (4.3 log(10)) IU/mL are 1.5 times more likely to have CMV disease resolution. CMV suppression (<137 [2.1 log(10)] IU/mL), as measured by a test calibrated to the WHO Standard, is predictive of clinical response to antiviral treatment. CLINICAL TRIALS REGISTRATION: NCT00431353.

Evaluation of cytomegalovirus (CMV)-specific T-cell immunity for the assessment of the risk of active CMV infection in non-immunosuppressed surgical and trauma intensive care unit patients.

The current study was designed to assess the predictive value of the evaluation of cytomegalovirus (CMV)-specific T-cell immunity early following admission to the intensive care unit for inferring the risk of active CMV infection in non-immunosuppressed surgical and trauma patients. A total of 31 CMV-seropositive patients were included. Patients were screened for the presence of CMV DNA in plasma and in tracheal aspirates by real-time PCR. Enumeration of CMV pp65 and IE-1-specific IFN-γ CD8(+) and CD4(+) T cells was performed by flow cytometry for intracellular cytokine staining. Virological and immunological monitoring was conducted once or twice a week. Active CMV infection occurred in 17 out of 31 patients. Undetectable levels of pp65 and IE-1-specific IFN-γ CD8(+) and CD4(+) T-cell subsets cells were observed in 10 patients who developed active CMV infection and in one who did not (at a median of 2 days following ICU admission). Peak CMV DNA loads in both tracheal aspirates and plasma were substantially higher (P = 0.018 and P = 0.091, respectively) in patients with undetectable IFN-γ T-cell responses than in patients with detectable responses. The expansion of both CMV-specific T-cell subsets following detection of active CMV infection was demonstrated in 9 out of 14 patients with active CMV infection. In conclusion, the evaluation of CMV pp65 and IE-1-specific IFN-γ-producing CD8(+) and CD4(+) T cells early following ICU admission may allow the identification of patients most at risk of either having or developing an episode of active CMV infection, particularly those associated with high-level virus replication.

Cytomegalovirus viremia in HIV-exposed and HIV-unexposed infants in Malawi.

In sub-Saharan Africa the great majority of infants acquire Cytomegalovirus (CMV) infection within the first year of life. Maternal long-term antiretroviral therapy (ART) has been suggested to reduce the rate of CMV acquisition in HIV-exposed infants. In the present study serum samples collected at 6 months of age from HIV-exposed and HIV-unexposed infants were analyzed for the presence of CMV DNA (with CMV positivity defined by levels of CMV DNA > 1000 UI/ml). Twenty out of 58 (34.5%) infants had CMV DNA > 1000 UI/ml. There was no difference in the prevalence of CMV viremia between HIV-exposed and -unexposed infants [33.3% (15/45) vs 38.5% (5/13), respectively, P = 0.488]. In the HIV-exposed group, mothers of CMV-negative infants had received a longer antiretroviral treatment before delivery in comparison to mothers of CMV-positive infants (28 vs 3 months, P = 0.187). No differences in weights and lengths at birth, and at 1, 6 and 12 months were observed between CMV-positive and CMV-negative infants. In this study, the prevalence of CMV viremia at six months of age was high in infants born to HIV-positive mothers receiving long-term ART, similar to that of HIV-unexposed infants. Considering the possible relevant impact of CMV on infant health, strategies for containment of the infection should be explored.

Changes in cytomegalovirus load in the breast milk of very/extremely premature infants and the effect of pasteurization and freeze-thawing on reducing viral load.

Objective: This study aimed to clarify the change in Cytomegalovirus (CMV) loads in breast milk (BM) of very/extremely premature infants (VPI/EPI) with birth weight < 1,500 g after birth, and to compare the effectiveness of pasteurization and freeze-thawing methods in reducing the CMV load of BM. Methods: Breast milk samples were collected and tested every 2 weeks by fluorescence quantitative polymerase chain reaction (FQ-PCR). We determined CMV load in BM before and after pasteurizing, and freeze-thawing. Results: Cytomegalovirus DNA can already be detected in colostrum. The viral load gradually increased in the first 4 weeks, peaked in the 4th to 6th weeks, and gradually decreased thereafter. The viral load gradually returned to the initial level approximately 10-12 weeks postpartum. During the peak period of the CMV load in BM, the viral load was higher in the EPI than the VPI (P < 0.05). The average CMV load (logarithmic [LG]) in the pasteurization group was significantly lower than that in the raw BM group. The average CMV load in the freeze-thawed BM group was significantly lower than that in the raw BM group. The mean CMV load in the pasteurized BM group was lower than that in the freeze-thawed BM group, but the difference was not statistically significant. The CMV-DNA clearance rate in pasteurized was higher than in freeze-thawed (P < 0.05). Conclusion: The CMV detoxification rate in BM is high and the peak load period is mainly between 4 and 6 weeks. The CMV load values detected are higher than the threshold values (7 × 103 copy number/mL) of CMV infection that are reported in the literature as a concern. Both the freeze-thaw and pasteurization techniques can effectively reduce the CMV load.

Universal Newborn Screening for Congenital Cytomegalovirus Infection - From Infant to Maternal Infection: A Prospective Multicenter Study.

Introduction: Most infants at risk for cytomegalovirus (CMV)-associated sensorineural hearing loss (SNHL) are unrecognized because of the absence of a universal neonatal CMV screening. The search of CMV-DNA by molecular methods in salivary swabs was demonstrated to be a reliable approach. This study describes the results obtained by carrying out a universal screening for congenital CMV (cCMV) infection including all live-born newborns in three Italian sites, as well as the therapeutic interventions and clinical outcome of the CMV-infected neonates. Moreover, CMV maternal infection's characteristics were evaluated. Methods: To confirm or exclude cCMV infection, a CMV-DNA-positive result on a first salivary swab was followed by repeated saliva and urine samples collected within 21 days of age. Breast milk samples were also collected. The search of CMV-DNA was performed with a single automated quantitative commercial real-time PCR assay, regardless of the type of samples used. Results: A total of 3,151 newborns were enrolled; 21 (0.66%) of them were congenitally infected (median saliva viral load at screening, 6.65 [range, 5.03-7.17] log10 IU/ml). Very low/low viral load in screening saliva samples (median value, 1.87 [range, 1.14-2.59] log10 IU/ml) was associated with false-positive results (n = 54; 1.7%). CMV-DNA was detected in almost half of the breast milk samples of mother-infant pairs with a false-positive result, suggesting that contamination from breast milk may not be the only explanation in the study population. cCMV infection confirmation with the search of CMV-DNA in a urine sample proved to be the gold standard strategy, since false-positive results were observed in 4/54 (7.5%) of the repeated saliva samples. Symptomatic cCMV infection was observed in 3/21 (14.3%) infants; notably, one (4.7%) developed moderate unilateral SNHL at 5 months after birth. Finally, two symptomatic cCMV infections were associated with primary maternal infection acquired in the first trimester of gestation; one newborn with severe cCMV symptoms was born to a mother with no CMV checkups in pregnancy. Conclusion: Without universal neonatal CMV screening, some infected infants who develop late neurological sequelae may not be recognized and, consequently, they are not able to benefit early from instrumental and therapeutic interventions to limit and/or treat CMV disease.