Human Cytomegalovirus Load Research Articles

Polyploid giant cancer cells, cytokines and cytomegalovirus in breast cancer progression

BackgroundBreast cancer is the most common cancer among women. Accumulated evidence over the past decades indicates a very high prevalence of human cytomegalovirus (HCMV) in breast cancer. High-risk HCMV strains possess a direct oncogenic effect displayed by cellular stress, polyploid giant cancer cells (PGCCs) generation, stemness, and epithelial-to-mesenchymal transition (EMT) leading to cancer of aggressive phenotype. Breast cancer development and progression have been regulated by several cytokines where the latter can promote cancer cell survival, help in tumor immune evasion, and initiate the EMT process, thereby resulting in invasion, angiogenesis, and breast cancer metastasis. In the present study, we screened cytokines expression in cytomegalovirus-transformed HMECs (CTH cells) cultures infected with HCMV high-risk strains namely, HCMV-DB and BL, as well as breast cancer biopsies, and analyzed the association between cytokines production, PGCCs count, and HCMV presence in vitro and in vivo.MethodsIn CTH cultures and breast cancer biopsies, HCMV load was quantified by real-time qPCR. PGCCs count in CTH cultures and breast cancer biopsies was identified based on cell morphology and hematoxylin and eosin staining, respectively. CTH supernatants were evaluated for the production of TGF-β, IL-6, IL1-β, and IL-10 by ELISA assays. The above-mentioned cytokines expression was assessed in breast cancer biopsies using reverse transcription-qPCR. The correlation analyses were performed using Pearson correlation test.ResultsThe revealed PGCCs/cytokine profile in our in vitro CTH model matched that of the breast cancer biopsies, in vivo. Pronounced cytokine expression and PGCCs count were detected in particularly CTH-DB cultures and basal-like breast cancer biopsies.ConclusionsThe analysis of cytokine profiles in PGCCs present mostly in basal-like breast cancer biopsies and derived from CTH cells chronically infected with the high-risk HCMV strains might have the potential to provide novel therapies such as cytokine-based immunotherapy which is a promising field in cancer treatments.

Detection of cytomegalovirus in bronchoalveolar lavage fluid from immunocompromised patients with pneumonitis by viral culture and DNA quantification

PurposeTo compare the detection of human cytomegalovirus (HCMV) in bronchoalveolar lavage (BAL) fluid by viral culture and quantitative polymerase chain reaction (qPCR), and to establish a viral load threshold that can identify cases of HCMV replication indicative of pneumonitis. There is currently no universal viral load cut-off to differentiate between patients with and without pneumonitis, and the interpretation of qPCR results is challenging. Methods176 consecutive BAL samples from immunosuppressed hosts with signs and/or symptoms of respiratory infection were prospectively studied by viral culture and qPCR. ResultsConcordant results were obtained in 81.25% of the BAL samples. The rest were discordant, as only 34% of the qPCR-positive BAL samples were positive by culture. The median HCMV load was significantly higher in culture-positive than in culture-negative BAL samples (5038 vs 178 IU/mL). Using a cut-off value of 1258 IU/mL of HCMV in BAL, pneumonia was diagnosed with a sensitivity of 76%, a specificity of 100%, a VPP of 100% and VPN of 98%, and HCMV was isolated in 100% of the BAL cultures. ConclusionWe found that a qPCR-negative was a quick and reliable way of ruling out HCMV pneumonitis, but a positive result did not always indicate clinically significant replication in the lung. However, an HCMV load in BAL fluid of ≥ 1258 IU/mL was always associated with disease, whereas < 200 IU/mL rarely so.

Prevalence of Human Cytomegalovirus Disease and its Related Factors in Renal and Bone Marrow Transplant Recipients in a Tertiary Hospital, Malaysia

Human cytomegalovirus (HCMV) infection may cause substantial morbidity and mortality after renal and bone marrow transplantation [1]. There are 3 major consequences of HCMV infection: HCMV disease with a wide range of clinical illnesses; superinfection with opportunistic pathogens; and injury to the transplanted organ [2]. Other than serological method to diagnose HCMV infection, viral load quantitation by real time polymerase chain reaction has been widely appreciated to diagnose and monitor the progress of viral infections. The aims of this study were to determine the incidence of HCMV infection in renal and bone marrow transplant recipients and to investigate its associations with HCMV disease, gender, and races.&#x0D; This retrospective cohort analysis involved 1520 blood samples from transplant recipients (renal, n = 164 and bone marrow, n = 182) from January 2020 to December 2021 collected from Virology Unit, Hospital Kuala Lumpur (NMRR ethical approval: NMRR-20-993-53201(IIR). The samples were analysed with quantitative polymerase chain reaction for HCMV DNA and the demographic, clinical and paraclinical aspects were evaluated. HCMV infection was present if the patient had positive HCMV viraemia and HCMV disease was diagnosed if HCMV infection was followed by clinical signs and symptoms. Statistical comparisons of patient demographics were performed with Chi-square tests for the categorical variables.&#x0D; The overall incidence of HCMV infection in the study group was 65% (225/346) where renal and bone marrow transplants account for 78.2% (176/225) and 21.7% (49/225) respectively. The incidence of HCMV infection in renal transplantation differed significantly by sex (p&lt;0.05) where it was higher in males (71.8%) than in females (28.2%) but there was not statistically significant by sex in bone marrow transplantation in which males and females account for 61.2% and 38.7% respectively. The incidence of HCMV differed significantly (p&lt;0.05) by races in both transplantation types as follows: 58% in Malay, 36% in Chinese, 5% in Indian and 1% in other indigenous races in renal transplantation while 59% in Malay, 29% in Chinese, 10% in Indian and 2% in other indigenous races in bone marrow transplantation. The incidence of HCMV disease differed significantly (p&lt;0.05) by type of transplantation where it is higher in renal transplantation (30.9%) than in bone marrow transplantation (20.2%). The most seen symptoms were fever, generalised lethargy, and headache. Viral load of HCMV has been shown to be a major determinant factor in the severity and the manifestation of the HCMV infection[3].&#x0D; It is significantly higher in patients who develop HCMV disease[3]. Various risk factors have been described for the progress of symptomatic HCMV infection in organ transplant recipient[3]. The incidence of HCMV infection was higher in renal transplant as compared to bone marrow transplant among Malaysian. This study has shown that HCMV viral load has a significant association with age, gender and HCMV disease. Various syndromes can be caused by HCMV ranging from a mild fever to severe end-organ diseases. Treatment with anti-HCMV therapy results in decline in HCMV load, usually to undetectable.

Chronic Lung Allograft Dysfunction is Associated with Acute Rejection and High Levels of Cytomegalovirus Load in Blood (but Not in Lung)

Purpose The potential association of human cytomegalovirus (HCMV) infection and acute rejection with the occurrence of chronic lung allograft dysfunction (CLAD) was analysed in lung transplant recipients not receiving anti-HCMV prophylaxis, but followed with a pre-emptive strategy. Methods We analysed 129 lung transplant recipients (Feb 2004 -feb 2018). HCMV infection in blood was monitored by HCMV DNA (101 patients) or pp65-antigenemia (28 patients, with retrospective confirmation of HCMV DNA), and in broncoalveolar lavage (BAL) by HCMV DNA. Protocol cut-off values for pre-emptive treatment initiation with Gancyclovir (GCV) or valganciclovir (VGCV) were; 100 pp65-positive /200,000 WBC or 300,000 HCMV DNA copies/ml blood, 100.000 HCMV DNA copies/ml BAL. Results HCMV infection was detected in 121 (94%) patients: 52 (40%) patients remained asymptomatic and resolved without treatment (peak HCMV DNA: 2,930, [ 100.000,10,000-100,000 or Conclusion By means of this retrospective analysis we can state that high levels of HCMV in blood, besides the occurrence of acute rejection, are associated with higher risk of CLAD occurrence. Although we cannot prove a causal relationship between HCMV load in blood and CLAD, on this basis cut-off for pre-emptive treatment must be lowered in order to decrease a potential risk for CLAD occurrence.

Virological and Immunological Monitoring of Human Cytomegalovirus (HCMV) Infection in Hematopoietic Stem Cell Transplantation Recipients (HSCTR)

Introduction HCMV infection is a significant viral complication in HSCTR, causing either a subclinical infection or a severe disease (systemic syndrome and localized tissue invasive disease (TID)). Pre-emptive therapy used for the management of HCMV infection and prevention of disease requires the monitoring of HCMV load in blood to start antiviral therapy after detection of a viral load threshold associated to the risk for HCMV severe infection and disease. In previous studies we observed that patients reconstituting both CD4+ and CD8+ HCMV-specific T cells are generally protected from HCMV disease. Thus, determination of specific T cells may discriminate patients lacking immunity and requiring stricter virological monitoring (negative T-cell response) from those early recovering T-cell immunity and thus not requiring antiviral interventions (positive T-cell response). Aims The aim of our work is to assess the possibility to manage HCMV infection by combined virological and immunological monitoring. The primary endpoint of this study is to compare the frequency of HCMV disease between two groups of HSCTR: in the first one, pre-emptive therapy was started according to combined virological/immunological methods; the second group consisted of an historical cohort of patients monitored with HCMV-DNA as the only guiding parameter for pre-emptive therapy. Methods We analyzed adult patients receiving allogeneic HSCT: the recipients, the donor or both had to be HCMV seropositive. Virological follow-up (HCMV-DNA determination in whole blood) was performed weekly for the first 3 months, then monthly during the first year. Immunological follow-up was performed at 1, 2, 3, 4, 6, 8, 10 and 12 months. Peripheral blood HCMV-specific T cells were determined by two assays: i) iCL (infected Cell Lysate)-ELISPOT, considering as "protective" immunity a blood count of specific T-cell ≥ 0.1/µL ii) iDC-CFC (infected Dendritic Cells-Cytokine Flow Cytometry), considering as "protective" immunity a blood count of CD4+ T-cell ≥ 1/µL and CD8+ T cells ≥ 3/µL. According to study, within the first 3 months following transplantation pre-emptive antiviral therapy was given to all patients presenting a viral load ≥30.000 copies/mL blood (severe infection). Beyond 3 months following transplantation, patients with negative T-cell response continued receiving a pre-emptive antiviral therapy as reported above. In patients with positive T-cell response confirmed by both assays ("protected" patients), antiviral therapy was deferred even if viral load would reach ≥30.000 copies/mL blood. Results We performed an analysis of 36 patients with a follow-up ≥4 months from HSCT (median follow-up 245 days, 120-397; see Table 1 for patients' characteristics). Among them, 32 HCMV-seropositive HSCTR developed HCMV infection whereas 4 HCMV-seronegative patient receiving HSCT from seropositive donors did not (nor developed specific immunity). Among the 32 HSCTR with HCMV reactivation, 13 (41%) resolved spontaneously HCMV reactivation, whereas 19 (59%) had a severe infection (11 [34%] controlled the infection after a single course of Valganciclovir therapy and the remaining 8 [25%] required multiple courses for relapsing episodes). Thirty days after HSCT, levels of total and HCMV-specific CD4+ and CD8+ T cells were significantly higher in the 13 patients spontaneously resolving the infection than in the 19 patients with severe infection. The two immunological assays were concordant in detecting time to protective immune reconstitution (median time: 177 vs 183 days for iDC and iCL-ELISPOT, respectively). HCMV-specific immune reconstitution was significantly delayed in patients with multiple infection episodes (median time: 250 days post-transplantation) vs patients with a single episode of severe infection (187 days) and patients with self-resolving infection (89 days). No patient developed HCMV severe infection after HCMV-specific immune reconstitution. Currently, no patient developed HCMV disease. Conclusion These results suggest that combined virological/immunological monitoring can be useful to identify patients not requiring strict virological controls and antiviral interventions. Disclosures No relevant conflicts of interest to declare.

Expression of human miR-200b-3p and -200c-3p in cytomegalovirus-infected tissues.

Human cytomegalovirus (HCMV) infection can cause inflammatory tissue-invasive end-organ diseases upon lytic replication. In humans, mature miR-200b-3p and -200c-3p suppress the synthesis of HCMV immediate early 2 (IE2) protein by binding to the 3′-UTR of the mRNA encoded by the unique long (UL) 122-123 region in human foreskin fibroblasts and pre-transplant peripheral blood mononuclear cells stimulated with HCMV. The present study aimed to quantitate the expression of Homo sapiens (hsa)-miR-200b-3p and 200c-3p in HCMV-infected tissues. We collected 240 HCMV-infected and 154 HCMV-non-infected, formalin-fixed, paraffin-embedded tissue samples of the gastrointestinal (GI) tract and bronchi/lungs. MiRNAs, HCMV, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were quantitated by quantitative reverse transcription-PCR (qRT-PCR) and quantitative PCR (qPCR) on the basis of standard curves generated using miRNA mimics, the HCMV strain from National Institute for Biological Standards and Control (NIBSC) 09/162, and GAPDH control. To avoid the effect of cell counts on the qRT-PCR and qPCR results, the data were normalized to GAPDH levels. HCMV-infected tissues had significantly lower levels of 200b-3p/GAPDH (3.03 ± 1.50 compared with 3.98 ± 1.08 log10 copies/μl, P<0.001) and 200c-3p/GAPDH (4.67 ± 1.84 compared with 6.35 ± 1.47 log10 copies/μl, P<0.001) than normal tissues. The values for 200b-3p/GAPDH (r = −0.51, P<0.001) and 200c-3p/GAPDH (r = −0.54, P<0.001) were significantly inversely correlated with HCMV load. Low tissue levels of 200b-3p and 200c-3p in humans are associated with cytopathic inflammation due to HCMV infection.

Latent Cytomegalovirus (CMV) Infection Does Not Detrimentally Alter T Cell Responses in the Healthy Old, But Increased Latent CMV Carriage Is Related to Expanded CMV-Specific T Cells.

Human cytomegalovirus (HCMV) primary infection and periodic reactivation of latent virus is generally well controlled by T-cell responses in healthy people. In older donors, overt HCMV disease is not generally seen despite the association of HCMV infection with increased risk of mortality. However, increases in HCMV DNA in urine of older people suggest that, although the immune response retains functionality, immunomodulation of the immune response due to lifelong viral carriage may alter its efficacy. Viral transcription is limited during latency to a handful of viral genes and there is both an IFNγ and cellular IL-10 CD4+ T-cell response to HCMV latency-associated proteins. Production of cIL-10 by HCMV-specific CD4+ T-cells is a candidate for aging-related immunomodulation. To address whether long-term carriage of HCMV changes the balance of cIL-10 and IFNγ-secreting T-cell populations, we recruited a large donor cohort aged 23–78 years and correlated T-cell responses to 11 HCMV proteins with age, HCMV IgG levels, latent HCMV load in CD14+ monocytes, and T-cell numbers in the blood. IFNγ responses by CD4+ and CD8+ T-cells to all HCMV proteins were detected, with no age-related increase in this cohort. IL-10-secreting CD4+ T cell responses were predominant to latency-associated proteins but did not increase with age. Quantification of HCMV genomes in CD14+ monocytes, a known site of latent HCMV carriage, did not reveal any increase in viral genome copies in older donors. Importantly, there was a significant positive correlation between the latent viral genome copy number and the breadth and magnitude of the IFNγ T-cell response to HCMV proteins. This study suggests in healthy aged donors that HCMV-specific changes in the T cell compartment were not affected by age and were effective, as viremia was a very rare event. Evidence from studies of unwell aged has shown HCMV to be an important comorbidity factor, surveillance of latent HCMV load and low-level viremia in blood and body fluids, alongside typical immunological measures and assessment of the antiviral capacity of the HCMV-specific immune cell function would be informative in determining if antiviral treatment of HCMV replication in the old maybe beneficial.

Human cytomegalovirus glycoprotein polymorphisms and increasing viral load in AIDS patients

BackgroundMultiple strains infection of human cytomegalovirus (HCMV) was found to be correlated with increased viral load in immunodeficient patients. However, the pathogenic mechanism underlying this correlation remains unclear. To evaluate genetic polymorphisms of HCMV glycoprotein and their potential role in its viral load, HCMV glycoprotein B, N, and O (gB, gN and gO) genotypes was studied in the population of HCMV infected acquired immune deficiency syndrome (AIDS) patients. The association between glycoprotein polymorphisms and HCMV viral load was analyzed.MethodsThe genetic polymorphisms of glycoprotein from sera of 60 HCMV infected AIDS patients was investigated by multiplex nested PCR and sequencing. HCMV viral load was evaluated by quantitative PCR.ResultsgB1, gO1a, and gN4a were the predominant glycoprotein genotypes in HCMV infected AIDS patients and composed 86.96%, 78.8%, and 49.2%, respectively. Only gN4a genotype infection significantly increased viral load (P = 0.048). 71% (43/60) of HCMV infected AIDS patients were found to carry multiple HCMV strains infection. A novel potential linkage of gO1a/gN4a was identified from multiple HCMV infected patients. It was the most frequent occurrence, accounted for 51.5% in 33 patients with gO and gN genotypes infection. Furthermore, the gO1a/gN4a linkage was correlated to an increased viral load (P = 0.020).ConclusionThe gN4a correlates to higher level HCMV load in AIDS patients. Interestingly, a novel gO1a/gN4a linkage is identified from the patients with multiple HCMV strains infection and is also associated with an increased viral load. Therefore, the pathogenic mechanism underlying glycoprotein polymorphisms and interaction of variants should be analyzed further.

Human cytomegalovirus detection in gastric cancer and its possible association with lymphatic metastasis

Increasing evidence suggests that human cytomegalovirus (HCMV) is associated with many human malignancies. However, its prevalence in gastric cancer (GC) and clinical association remain unknown. HCMV IgG and IgM antibodies in the sera of 80 GC patients and 80 healthy controls were detected using a microparticle enzyme immunoassay. The prevalence of HCMV UL47, UL55, UL56, and UL77 genes among 102 GC tumor tissues and adjacent normal specimens was measured by polymerase chain reaction (PCR) or nested PCR. Quantitative real-time PCR (Q-PCR) was used to determine viral load. Virus localization in neoplastic tissues was determined by immunohistochemistry. No significant difference of HCMV IgG and IgM seropositivity was found between GC patients and the healthy group. However, the overall HCMV DNA positivity rate was significantly higher in GC cancerous tissue compared with in paired normal tissue (P<0.01). HCMV infection was mainly localized in the tumorous epithelium. Q-PCR in HCMV-positive specimens indicated that the viral copy number was notably higher in GC tissues than in adjacent normal specimens (P<0.001). Clinical statistical analysis indicated that HCMV load in GC tumor tissue was positively associated with lymphatic metastasis (P=0.043), the area under the receiver operating characteristic (ROC) curve was 0.6638. Our data clearly provide the prevalence of HCMV in GC patients. We conclude that HCMV infection in malignant tissues might be associated with carcinogenesis or progression of GC and possibly relates to lymphatic metastasis.

Cytokine Production and Human Cytomegalovirus Load in Allogeneic Hematopoietic Stem Cell Transplantation Outcome

Objective: To investigate the impact of cytokine levels (IL-1β, IL-6, IL-10, IFN-γ and TNF-α) and Human cytomegalovirus (HCMV) load in the saliva and blood on the survival of allogeneic hematopoietic stem cell transplantation (allo-HSCT) recipients.

Study of human cytomegalovirus replication in body fluids, placental infection, and miscarriage during the first trimester of pregnancy.

Intrauterine infection caused by human cytomegalovirus (HCMV) can lead to embryo, fetal, and neonatal damage. The prevalence of HCMV replication in body fluids (blood, urine, and cervicovaginal secretion) was investigated, and its effects on HCMV vertical transmission and miscarriages in early pregnant women were evaluated. HCMV DNA in body fluids was detected in 1,064 early pregnant women (624 normal pregnancies and 440 miscarriages). There were 101 cases who were HCMV DNA positive in cervicovaginal secretion and the rates were 10.9% (48/440 cases) and 8.5% (53/624 cases) in miscarriages and normal pregnancies, respectively (P > 0.05). A total of 101 cases (63 and 38 cases with and without HCMV DNA in cervicovaginal secretion, respectively) were given HCMV DNA detection in placental villi/deciduas. There were five cases (7.9%; two normal pregnancies and three miscarriages) with HCMV DNA in placental villi/deciduas among the 63 cases with HCMV DNA in cervicovaginal secretion, whereas none of the other 38 cases were detected HCMV DNA positive in their placental villi/deciduas. The percentage of HCMV DNA in placental villi/deciduas was higher in miscarriage group (9.1% [3/33]) than that in the normal pregnancy group (6.7% [2/30]), but there was no statistical significance (P > 0.05). Two cases with a higher HCMV loads in cervicovaginal secretion and placental villi/deciduas had miscarriages. These findings suggest that HCMV replication in cervicovaginal secretion can involve in placental HCMV infection, and high HCMV DNA loads in cervicovaginal secretion and placental villi/deciduas are associated with miscarriage.

Quantitative analysis of human herpesvirus-6 and human cytomegalovirus in blood and saliva from patients with acute leukemia.

Human herpesvirus-6 (HHV-6) and human cytomegalovirus (HCMV) DNAs were quantified by real-time PCR assays in blood and saliva obtained from 50 patients with acute leukemia at the time of diagnosis (50 of each matrix), aplasia (65 of each matrix), remission (55 of each matrix), and relapse (20 of each matrix) to evaluate which biological matrix was more suitable to identify a viral reactivation, search for a possible link between HHV-6 and HCMV reactivations, and evaluate the relations between viral loads and count of different leukocyte types in blood. The median HHV-6 loads were 136; 219; 226, and 75 copies/million cells in blood at diagnosis, aplasia, remission and relapse, respectively. The HCMV loads were 193 and 317 copies/million cells in blood at diagnosis and remission. In the saliva samples, the HHV-6 loads were 22,165; 15,238; 30,214, and 17,454 copies/million cells at diagnosis, aplasia, remission, and relapse, respectively. The HCMV loads were 8,991; 1,461; 2,980, and 4,283 copies/million cells at diagnosis, aplasia, remission, and relapse, respectively. The HHV-6 load in the blood was correlated to the counts of polymorphonuclear leukocytes (R(2) = 0.5; P < 0.0001) and lymphocytes (R(2) = 0.4; P = 0.001) and was not correlated to the monocyte counts (R(2) = 0.07; P = 0.7). Saliva appears to be a more sensitive biological matrix than whole blood in the detection of HHV-6 or HCMV reactivations. The HHV-6 and HCMV reactivations were linked only in saliva.

The increased sensitivity of human cytomegalovirus (HCMV) PCR quantitation in whole blood affects reproductive rate (Ro) measurement

In order to determine the effect of the increase in sensitivity of HCMV detection in whole blood compared to plasma on reproductive rate (Ro) measurement, an optimized human cytomegalovirus (HCMV) quantitative PCR assay was developed. The results presented in this study are summarized by the following three methodological improvements: (i) at values below the limit of quantitation (LOQ) of 60copies/ml, determination of HCMV load was more sensitive with whole blood than plasma, (ii) for the determination of viral load, whole blood was more sensitive than plasma below 1000copies/ml but little difference was observed above 1000copies/ml and (iii) the measurement of “Reproductive Rate” can be affected by imprecise measurement of HCMV viral load in either plasma or whole blood compartments depending on whether samples were taken from patients on antiviral treatment or from patients where HCMV load was rising. Taken together this study provides methodological improvements suggesting that below HCMV viral load levels of 1000copies/ml (1640IU/ml) both plasma and whole blood should be tested.

High prevalence of HCMV and viral load in tumor tissues and peripheral blood of glioblastoma multiforme patients

Glioblastoma multiforme is the most prevalent and malignant tumor of the central nervous system. In the last few years, accumulating evidence has suggested an association between human cytomegalovirus (HCMV) infection and glioblastoma multiforme. In this study, tumor tissues and peripheral blood of patients with glioblastoma multiforme were examined for the presence of HCMV DNA. Twenty-two fresh surgical brain specimens and 20 peripheral blood samples were analyzed by real-time PCR (qPCR) and hemi-nested PCR (nPCR) for the presence of pp65 and (glycoprotein B) gB viral genomic regions, respectively. HCMV DNA was detected in the majority of the tumor samples analyzed (95% by qPCR and 91% by nPCR). About half of the patients with tumors positive for HCMV also had detectable viral DNA in their peripheral blood (47% by qPCR and 61% by nPCR). Genome copy numbers were determined and in the majority of the tumor samples cellular DNA outnumbers viral DNA (average of 1 infected cell in 33 cells). The gB genotypes were determined in HCMV-positive samples and gB2 was the most prevalent genotype in the tumor and blood samples. The results show a high prevalence of HCMV in glioblastoma multiforme samples reinforcing a possible association between HCMV infection and tumor development.

Human cytomegalovirus (HCMV) replication kinetics in stem cell transplant recipients following anti-HCMV therapy

Background Cytomegalovirus (HCMV) remains an important infection following stem cell transplantation (SCT) and is managed via pre-emptive therapy. In some patients HCMV loads continue to increase after therapy and they experience multiple episodes of replication. Objectives To identify the risk factors associated with failure to immediately control HCMV replication after antiviral therapy and for recurrence of replication. Study design Replication kinetics of human cytomegalovirus (HCMV) were studied a cohort of 153 T-cell depleted allogeneic SCT patients. Results In 57 patients (31%) who experienced HCMV DNAemia, the mean growth rate of HCMV was 0.35 day −1 equivalent to a doubling time of 2.2 days. In patients requiring anti-HCMV treatment with either ganciclovir or ganciclovir/foscarnet ( n = 49), HCMV load increased to a peak value of >2 days after initiation of therapy in 21 patients and only the growth rate prior to therapy was a risk factor (Odds ratio = 1.4 per 0.1 day −1 increase; p = 0.004). In patients where antiviral intervention occurred after peak virus load the decline rate of HCMV load was accelerated 4-fold if the patient was subsequently initiated on anti-HCMV therapy ( p = 0.02). A subset of patients (38%) experienced a recurrence of their DNAemia at a mean of 20 days after the cessation of their first replication episode and this was only associated with receiving stem cells from a seronegative donor (Odds ratio = 6.59; p < 0.001). Conclusions The kinetics of response to therapy is closely associated with HCMV replication kinetics prior to therapy while recurrence of replication is associated with HCMV serostatus of the donor.

High Cytomegalovirus Load and Prolonged Virus Excretion in Breast Milk Increase Risk for Viral Acquisition by Very Low Birth Weight Infants

Breast milk is the main source of postnatal human cytomegalovirus (HCMV) infection. The aim of this study was to assess the relationship between HCMV load in breast milk and viral transmission to very low birth weight (VLBW) infants. Breast-fed VLBW infants who were born to HCMV-seropositive mothers and who were managed in a neonatal intensive care unit were enrolled in the study. Blood from mothers and infants was tested for HCMV antibodies after birth. Breast milk was collected for viral culture and HCMV load measurement. Urine from the babies was obtained for HCMV-DNA detection. Symptoms of HCMV infection were recorded and evaluated by neonatologists. Of the 23 evaluated mothers during a 1-year period, 19 were HCMV seropositive; 17 of the women had detectable HCMV-DNA in their breast milk whey. Of the 23 infants born to the 19 seropositive mothers, 8 infants of 8 mothers had HCMV-DNA detected in the urine, indicating that they were infected, even though the breast milk was always frozen prior to feeding. Three infected infants had symptoms. At 4 weeks after delivery, the median viral load in breast milk from mothers of the 8 infected infants was significantly higher than that from mothers of the 15 noninfected infants (P = 0.04). HCMV was detectable in breast milk for a significantly longer period in mothers of infected infants (7.5 vs. 2.6 weeks P = 0.03). High HCMV load and prolonged virus excretion in breast milk are maternal risk factors for viral transmission to VLBW infants.

Use of the Roche LightCycler ® 480 system in a routine laboratory setting for molecular diagnosis of opportunistic viral infections: Evaluation on whole blood specimens and proficiency panels

The Roche LightCycler ® 480 (LC480) system was evaluated for quantitative molecular diagnosis of opportunistic viral infections caused by human cytomegalovirus (HCMV), Epstein–Barr virus (EBV), human herpesvirus 6 (HHV-6) and BK virus (BKV), in comparison with “in-house” real-time PCR assays. A total of 253 whole blood specimens obtained from transplant recipients were tested. Both the “in-house” and the LC480 methods were highly correlated (Spearman correlation coefficient Rho ≥ 0.85; p < 0.0001) with an excellent overall qualitative agreement (90.5%) and no significant quantitative difference between both techniques for the four viruses tested. The accuracy of the LC480 protocols were confirmed further by the results obtained with the 44 samples from the Quality Control for Molecular Diagnosis (QCMD) 2008 proficiency panel. The LC480 system constitutes a suitable and versatile real-time PCR platform in a routine laboratory setting for the diagnosis and monitoring of opportunistic viral infections in transplant recipients, by measuring HCMV, EBV, HHV-6, and BKV loads in whole blood samples.

Human cytomegalovirus-specific CD4+ and CD8+ T cell responses in primary infection of the immunocompetent and the immunocompromised host

The kinetics of primary human cytomegalovirus (HCMV) infection and specific T-cell responses were investigated in 16 immunocompetent pregnant women and 8 solid-organ transplant recipients (SOTR). T-cell responses to whole HCMV and to pp65 and IE-1 peptides were determined by flow cytometry evaluation of IFNgamma production. HCMV-specific CD4(+) and CD8(+) T-cells appeared earlier and simultaneously in immunocompetent subjects, whereas specific CD8(+) T-cells preceded CD4(+) T-cells in half of the SOTR examined. The magnitude of the HCMV-specific T-cell pool was comparable. HCMV load reached peak levels 100-1000 times higher in SOTR than in immunocompetent women, while the virus persisted for months in blood of both groups. T-cells directed to pp65 and IE-1 were only detected in a portion of subjects developing a full T-cell response to the whole virus. Thus, the development of cell-mediated immune response in primary HCMV infection may be missed when looking at pp65 and IE-1 peptide-stimulated T-cells only.

Management of Human Cytomegalovirus Infection in Transplant Recipients by the Pre-Emptive Therapy Approach

Human cytomegalovirus (HCMV) infections are still a major infectious complication in the post-transplant period of both solid organ transplant recipients (SOTRs) and hematopoietic stem cell transplant recipients (HSCTRs). For many years, the major diagnostic assay has been antigenemia, allowing semi-quantification of HCMV load in blood from transplanted patients with disseminated HCMV infection. More recently, the real-time PCR assay has replaced antigenemia for HCMV DNAemia quantification. Prevention of HCMV disease is based on either prophylaxis or pre-emptive therapy with antiviral drugs. The latter approach has been in use in our department for the last 15 years. A cut-off of 300,000 DNA copies/ml whole blood in SOTRs with either primary or reactivated infection, and a cut-off of 10,000 DNA copies/ml blood in HSCTRs proved to be safe and effective in prospective randomized, controlled trials. With this approach, HCMV disease is consistently prevented, except for a limited number of cases of organ localization in the absence of virus in blood. In these cases, HCMV infection/disease must be diagnosed by local biopsy samples.

High human cytomegalovirus loads and diverse linked variable genotypes in both HIV-1 infected and exposed, but uninfected, children in Africa

Human cytomegalovirus, HCMV, was analysed using real-time quantitative PCR in symptomatic or asymptomatic pediatric cohorts from HIV-1 infected, exposed (HIV-1+ mothers), or uninfected groups in Zambia, an HIV-1/AIDS endemic region of Africa. HCMV infections were identified in 94% samples from HIV-1+ respiratory pediatric mortalities, 50% with high DNA loads of 10 3–10 8 copies/10 6 cells. In comparison, HCMV viremia with high DNA loads, indicative of acute infections, were in 10% hospitalised febrile infants, with 50% HIV-1+. Whereas high sera loads were in 1% of asymptomatic infants, with 2% HIV-1+, and higher levels in both HIV-1 infected or exposed, but negative infants. All 8 linked-hypervariable glycoprotein gN–gO genotypes were shown, including identification of a new gN4d group with gO5 linkage (previously only Merlin reference strain), and samples with multiple infections. Overall, this shows global genotypes in Africa (unlike some herpesviruses) and acute pediatric HCMV infections in both HIV-1+ plus exposed, but uninfected infants, an emerging group.