Human cytomegalovirus multiple-strain infections and viral population diversity in haematopoietic stem cell transplant recipients analysed by high-throughput sequencing.

Human cytomegalovirus (HCMV) is an important opportunistic pathogen in allogeneic haematopoietic stem cell transplant (HSCT) recipients. High-throughput sequencing of target-enriched libraries was performed to characterise the diversity of HCMV strains present in this high-risk group. Forty-four HCMV-DNA-positive plasma specimens (median viral input load 321 IU per library) collected at defined time points from 23 HSCT recipients within 80 days of transplantation were sequenced. The genotype distribution for 12 hypervariable HCMV genes and the number of HCMV strains present (i.e. single- vs. multiple-strain infection) were determined for 29 samples from 16 recipients. Multiple-strain infection was observed in seven of these 16 recipients, and five of these seven recipients had the donor (D)/recipient (R) HCMV-serostatus combination D + R + . A very broad range of genotypes was detected, with an intrahost composition that was generally stable over time. Multiple-strain infection was not associated with particular virological or clinical features, such as altered levels or duration of antigenaemia, development of acute graft-versus-host disease or increased mortality. In conclusion, despite relatively low viral plasma loads, a high frequency of multiple-strain HCMV infection and a high strain complexity were demonstrated in systematically collected clinical samples from this cohort early after HSCT. However, robust evaluation of the pathogenic role of intrahost viral diversity and multiple-strain infection will require studies enrolling larger numbers of recipients.

Common Polymorphisms in the Glycoproteins of Human Cytomegalovirus and Associated Strain-Specific Immunity.

Human cytomegalovirus (HCMV), one of the most prevalent viruses across the globe, is a common cause of morbidity and mortality for immunocompromised individuals. Recent clinical observations have demonstrated that mixed strain infections are common and may lead to more severe disease progression. This clinical observation illustrates the complexity of the HCMV genome and emphasizes the importance of taking a population-level view of genotypic evolution. Here we review frequently sampled polymorphisms in the glycoproteins of HCMV, comparing the variable regions, and summarizing their corresponding geographic distributions observed to date. The related strain-specific immunity, including neutralization activity and antigen-specific cellular immunity, is also discussed. Given that these glycoproteins are common targets for vaccine design and anti-viral therapies, this observed genetic variation represents an important resource for future efforts to combat HCMV infections.

The Limitations of Cytomegalovirus DNA Detection in Cerebrospinal Fluid of Newborn Infants With Congenital CMV Infection: A Tertiary Care Neonatal Center Experience.

BACKGROUND: Congenital cytomegalovirus (cCMV) infection of the central nervous system (CNS) can cause ventriculomegaly, gliosis, calcifications and cortical defects. Detection of CMV DNA in cerebrospinal fluid by PCR (CSF-CMV-PCR) is a marker of CNS involvement. OBJECTIVE: To evaluate a diagnostic value of the positive CSF-CMV-PCR in cCMV. METHODS: Analysis of clinical, laboratory, neuroimaging and single-nucleotide polymorphisms (SNPs) data according to the results of CSF-CMV-PCR were performed in infants with cCMV. RESULTS: A total of 168 infants were included; 145 (86.3%) had negative and 23 (13.7%) had positive CSF-CMV-PCR results. Associations between the positive CSF-CMV-PCR results and prematurity (odds ratio [OR] = 3.24; 95% confidence interval [CI]: 1.30-8.07), microcephaly (OR = 5.67; 95% CI: 2.08-15.41), seizures (OR = 4.15; 95% CI: 1.10-15.67), sensorineural hearing loss (OR = 6.6; 95% CI: 2.49-17.46), splenomegaly (OR = 8.13; 95% CI: 3.12-21.16), hepatitis (OR = 10.51; 95% CI: 3.31-33.35), petechiae (OR = 10.21; 95% CI: 3.78-27.57) and heterozygous T/C genotype at TLR4rs4986791 (OR = 7.88; 95% CI: 1.55-40.12) were observed. When using a multivariate logistic regression analysis, only the presence of severe sensorineural hearing loss (OR = 7.18; 95% CI: 1.75-29.34, P = 0.006), cystic lesions on MRI (OR 5.29; 95% CI: 1.31-21.36, P = 0.02), and calcifications on MRI (OR = 7.19; 95% CI: 1.67-30.97, P = 0.008) remained as the significant independent predictors of the positive CSF-CMV-PCR results. CONCLUSIONS: The detection of CMV DNA in CSF is associated with a higher rate of CNS damage including abnormal MRI neuroimaging and severe hearing loss. Therefore, detection of CMV DNA in CSF may be considered as a marker of severe CNS injury in cCMV infection. However, the very low prevalence of the positive CSF-CMV-PCR results, even in infants with proven CNS involvement, may imply its limited role in clinical practice.

A Novel Strain-Specific Neutralizing Epitope on Glycoprotein H of Human Cytomegalovirus.

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that causes severe clinical disease in immunosuppressed patients and congenitally infected newborn infants. Viral envelope glycoproteins represent attractive targets for vaccination or passive immunotherapy. To extend the knowledge of mechanisms of virus neutralization, monoclonal antibodies (MAbs) were generated following immunization of mice with HCMV virions. Hybridoma supernatants were screened for in vitro neutralization activity, yielding three potent MAbs, 6E3, 3C11, and 2B10. MAbs 6E3 and 3C11 blocked infection of all viral strains that were tested, while MAb 2B10 neutralized only 50% of the HCMV strains analyzed. Characterization of the MAbs using indirect immunofluorescence analyses demonstrated their reactivity with recombinantly derived gH. While MAbs 6E3 and 3C11 reacted with gH when expressed alone, 2B10 detected gH only when it was coexpressed with gB and gL. Recognition of gH by 3C11 was dependent on the expression of the entire ectodomain of gH, whereas 6E3 required residues 1 to 629 of gH. The strain-specific determinant for neutralization by Mab 2B10 was identified as a single Met→Ile amino acid polymorphism within gH, located within the central part of the protein. The polymorphism is evenly distributed among described HCMV strains. The 2B10 epitope thus represents a novel strain-specific antibody target site on gH of HCMV. The dependence of the reactivity of 2B10 on the simultaneous presence of gB/gH/gL will be of value in the structural definition of this tripartite complex. The 2B10 epitope may also represent a valuable tool for diagnostics to monitor infections/reinfections with different HCMV strains during pregnancy or after transplantation. IMPORTANCE HCMV infections are life threatening to people with compromised or immature immune systems. Understanding the antiviral antibody repertoire induced during HCMV infection is a necessary prerequisite to define protective antibody responses. Here, we report three novel anti-gH MAbs that potently neutralized HCMV infectivity. One of these MAbs (2B10) targets a novel strain-specific conformational epitope on gH that only becomes accessible upon coexpression of the minimal fusion machinery gB/gH/gL. Strain specificity is dependent on a single amino acid polymorphism within gH. Our data highlight the importance of strain-specific neutralizing antibody responses against HCMV. The 2B10 epitope may also represent a valuable tool for diagnostics to monitor infections/reinfections with different HCMV strains during pregnancy or after transplantation. In addition, the dependence of the reactivity of 2B10 on the simultaneous presence of gB/gH/gL will be of value in the structural definition of this tripartite complex.

Extent of Cytomegalovirus Replication in the Human Host Depends on Variations of the HLA-E/UL40 Axis.

Human cytomegalovirus (HCMV) may cause severe infections in lung transplant recipients (LTRs). In response to HCMV infections, a subset of NKG2C+ NK cells expands, which limits HCMV replication and is characterized by high expression of the activating NKG2C/CD94 and absence of the inhibitory NKG2A/CD94 receptor. Both receptors bind to HLA-E, which is stabilized by HCMV-encoded UL40 peptides. HLA-E and UL40 occur as different genetic variants. In this study, we investigated the interplay between the human NK cell response and the infecting HCMV-UL40 strain, and we assessed the impact of HCMV-UL40 and of donor- and recipient-encoded HLA-E*0101/0103 variants on HCMV replication after lung transplantation. We included 137 LTRs displaying either no or low- or high-level (>1,000 copies/ml plasma) viremia. HCMV-UL40 and HLA-E*0101/0103 variants were determined. UL40 diversity was investigated by next-generation sequencing. UL40 peptide-dependent NK cell cytotoxicity was assessed by flow cytometry. Donor-encoded HLA-E*0101/0103 was significantly associated with development of high-level viremia after transplantation (P = 0.007). The HCMV-UL40 variant VMAPRTLIL occurred significantly more frequently in highly viremic LTRs, and the variant VMTPRTLIL occurred significantly more frequently in low-viremic LTRs (P = 0.004). This difference was associated with a better inhibition of NKG2A+ NKG2C- NK cells by VMAPRTLIL (P < 0.001). In LTRs with repeated high-level viremic episodes, HCMV strains with UL40 variants displaying low affinity to the patients' HLA-E variant emerged over time. The HLA-E-UL40 axis has a substantial impact on the level of HCMV replication in LTRs. The interplay between UL40 peptide variants, the recipient HLA-E status, and the activation of inhibitory NKG2A+ NKG2C- cells is of major importance for development of high-level viremia after lung transplantation.IMPORTANCE Infection with human cytomegalovirus (HCMV) is associated with substantial morbidity in immunosuppressed patients and after congenital infections. Therefore, development of a vaccine against HCMV is a main public health priority. Revealing the complex interaction between HCMV and host responses, is of utmost importance for understanding viral pathogenesis and for vaccine design. The present data contribute to the understanding of HCMV-specific host immune responses and reveal specifically the interaction between HLA-E and the virus-encoded UL40 peptide, which further leads to a potent NK cell response. We demonstrate that this interaction is a key factor for reduction of virus replication in immunosuppressed patients. We further show that distinct naturally occurring HCMV-UL40 variants reduce the activation of a specific subpopulation of host NK cells and thereby are associated with high-level viremia in the patients. These findings will allow the characterization of patients at risk for severe HCMV infection and contribute to strategies for HCMV vaccine development.

Distinct Properties of Human Cytomegalovirus Strains and the Appropriate Choice of Strains for Particular Studies.

Human cytomegalovirus is routinely isolated by inoculating fibroblast cultures with clinical specimens suspected of harboring HCMV and then monitoring the cultures for cytopathic effects characteristic of this virus. Initially, such clinical isolates are usually strictly cell-associated, but continued propagation in cell culture increases the capacity of an HCMV isolate to release cell-free infectious progeny. Once cell-free infection is possible, genetically homogenous virus strains can be purified by limiting dilution infections. HCMV strains can differ greatly with regard to the titers that can be achieved, the tropism for certain cell types, and the degree to which nonessential genes have been lost during propagation. As there is no ideal HCMV strain for all purposes, the choice of the most appropriate strain depends on the requirements of the particular experiment or project. In this chapter, we provide information that can serve as a basis for deciding which strain may be the most appropriate for a given experiment.

Using Diploid Human Fibroblasts as a Model System to Culture, Grow, and Study Human Cytomegalovirus Infection.

Primary human diploid fibroblasts are used routinely to study host/pathogen interactions of human cytomegalovirus (HCMV). Fibroblasts' ease of culture and tremendous permissiveness for infection allow the study of all facets of infection, an abbreviated list of which includes ligand-receptor interactions, activation of cell signaling responses, and dysregulation of the cell cycle and DNA repair processes. Another advantage to fibroblasts' permissiveness for HCMV is the capability to grow high titer stocks of virus in them. This chapter will discuss the production of viral stocks of HCMV in primary human fibroblasts, commencing with culturing and infection of cells and continuing through harvest, titration (determining the infectious capacity of a particular virus preparation), and storage of viral stocks for use in downstream experiments.

Donor Cytomegalovirus Transmission Patterns in Solid Organ Transplant Recipients With Primary Infection.

BACKGROUND: The epidemiology of single versus multiple cytomegalovirus (CMV) strain transmission from donor (D+) to seronegative solid organ transplant (SOT) recipients (R-) is uncertain, as is whether "relapsing" recipient infection represents changing strain predominance when multiple strains are transmitted. Here we characterized CMV strain transmission patterns in D+/R- SOT recipients. METHODS: We studied pairs or groups of D+/R- SOT recipients who received organs from a common donor (group A) and recipients who experienced ≥2 waves of CMV DNAemia (group B). CMV in plasma was characterized by genotype-specific real-time PCR for genes gB and gH. RESULTS: Single concordant genotypes were identified in 12 of 18 recipient pairs/group sharing a common donor (group A); at least 6 of 18 (33%) donors transmitted > 1 strain. A single CMV strain was detected in 14 of 15 recipients in group B; only 1 recipient had coinfection. A shift in CMV strain predominance occurred after the first posttransplant year in at least 4 recipients with coinfection. CONCLUSIONS: Using a common donor approach, we confirmed that multiple CMV strain transmission from donors to R- SOT recipients is not uncommon. D+/R- SOT recipients with CMV coinfection can undergo changes in strain predominance in late waves of CMV DNAemia.

Impact of porcine cytomegalovirus on long-term orthotopic cardiac xenotransplant survival.

Xenotransplantation using pig organs has achieved survival times up to 195 days in pig orthotopic heart transplantation into baboons. Here we demonstrate that in addition to an improved immunosuppressive regimen, non-ischaemic preservation with continuous perfusion and control of post-transplantation growth of the transplant, prevention of transmission of the porcine cytomegalovirus (PCMV) plays an important role in achieving long survival times. For the first time we demonstrate that PCMV transmission in orthotopic pig heart xenotransplantation was associated with a reduced survival time of the transplant and increased levels of IL-6 and TNFα were found in the transplanted baboon. Furthermore, high levels of tPA-PAI-1 complexes were found, suggesting a complete loss of the pro-fibrinolytic properties of the endothelial cells. These data show that PCMV has an important impact on transplant survival and call for elimination of PCMV from donor pigs.

Cytomegalovirus glycoprotein B and N genotypes in pediatric recipients of the hematopoietic stem cell transplant.

Clinical significance of the cytomegalovirus (CMV) genotypes in patients undergoing allogeneic hematopoietic stem cell transplant (HSCT) has been evaluated mostly in adults. The studies of diverse CMV glycoprotein B (gB) and N (gN) genotype variants in transplanted children and adolescents are lacking. We analyzed the investment of gB and gN genotype variants in the HSCTed children and their relation to clinical complications and disease outcome. The cohort included forty two pediatric recipients of the HSCT. Patients positive for CMV DNAemia (24/42, 57.1%) were genotyped. The gB4 and gN1 genotype variants predominated and were evidenced in 7/18 (38.9%) and 9/19 (47.4%) patients, respectively. The graft-versus-host disease (GvHD) predominated in children with viremia (p < 0.05). Frequencies of the gB and gN genotypes contrasted those reported in recent studies. The GvHD scaled strongly with CMV reactivation whereas viral loads were uncorrelated to medical complications and treatment outcomes.

[Comparison of Two Commercial Quantitative Cytomegalovirus (CMV) Polymerase Chain Reaction Tests Calibrated by World Health Organization International CMV Standard]. / Dünya Saglik Örgütü Uluslararasi Sitomegalovirüs (CMV) Standardi ile Kalibre Edilmis Iki Ticari Kantitatif CMV Polimeraz Zincir Reaksiyonu Testinin Karsilastirilmasi.

Cytomegalovirus (CMV) viral load quantitation is important in diagnosis, follow-up, and monitoring of antiviral therapy in transplanted patients. In this study, it was aimed to compare the results of the two commercial World Health Organization (WHO) International CMV standard calibrated polymerase chain reaction tests, CMV Cobas Ampliprep/Cobas Taqman (CMV-CAP/CTM) (Roche, Germany) and Artus CMV QIASymphony-Rotorgene (CMV-QS-RGQ) (Qiagen, Germany). Both tests were performed simultaneously on 244 plasma samples. The results were measured in copies/ml and converted to IU/ml by multiplying with 0.91 for CMV-CAP/CTM and 1.64 for CMV-QS-RGQ, as specified by the manufacturers. CMV DNA was detected in 174 (71.3%) and was not detected in 52 (21.3%) of the samples and eighteen (7.4%) samples had discordant results by both of the tests. In 16 out of 18 samples with discordance, the viral load was below the dynamic measuring ranges of both tests. In one sample, CMV DNA could not be detected by CMV-CAP/CTM but detected by CMV-QS-RGQ with 497 copies/ml, and 334 copies/ml CMV DNA was detected by CMV-CAP/CTM in another sample where it could not be detected by CMV-QS-RGQ. A high degree of agreement was found between the qualitative results of the both tests (kappa= 0.80, p< 0.001). For quantitative results in the dynamic measuring range of both assays (n= 129), the median viral load values measured by CMV-CAP/CTM and CMV-QS-RGQ were 1140 copies/ml (range: 151-254000) and 1826 copies/ml (range: 189-551521). When the results were converted to IU/ ml, the median viral load values measured by CMV-CAP/CTM and CMV-QS-RGQ were 1037 IU/ml (range: 137-231140) and 2993 IU/ml (range: 310-904133), respectively. There was a very strong correlation (r= 0.94, p< 0.001; r= 0.94, p< 0.001, respectively) between the log10 values of the quantitative results in the dynamic measuring ranges (n= 129) as copies/ml and IU/ml of both tests. CMV-QS-RGQ values corresponding to 150, 1000, 3000 copies/ml in CMV-CAP/CTM were as 94.5, 1571, 323.5 copies/ml and CMV-QS-RGQ values corresponding to 137, 910, 2730 IU/ml in CMV CAP/CTM were as 154, 2557.6, 6965.9 IU/ml, respectively. A variation of 0.45 log10 was determined between these values. In a total of 131 samples; 129 of them with the result of both tests in the dynamic measuring range and two of them which CMV DNA was not detected in one of the tests; it was found that 112 (85.5%) results for copy/ ml, 73 (56%) results for IU/ml were within the measurement difference of ± 0.5 log10 and 19 (14.5%) results for copy/ml and 58 (44%) results for IU/ml were greater than ± 0.5 log10. Bland-Altman analysis showed that CMV-CAP/CTM test made lower measurements than CMV-QS-RGQ and the average difference for copy/ml and IU/ml results were 0.22 log10 copies/ml and 0.47 log10 IU/ml. In conclusion; when the results were converted to IU/ml, the number of samples with an acceptable measurement difference between the two test results (≤ 0.5 log10) decreased and the number of samples with a measurement difference > 0.5 log10 increased and the difference was found as statistically significant (p< 0.001). Calibrating the Roche CMV CAP/CTM and Artus CMV-QS-RGQ tests with the WHO international CMV standard did not increase comparability between quantitative results in plasma samples, on the contrary, it was found that when the results were converted to IU/ml, a measurement difference indicating biologically significant viral replication was detected between the two test results.

The nucleic acid positive rate and genotype distribution of human cytomegalovirus in human milk banks in China.

To determine the status of human cytomegalovirus (HCMV) infection in human milk in China, a total of 510 human milk samples obtained from three provinces, including 211 donor human milk samples from human milk banks and 299 milk samples obtained from the mothers of premature infants, were tested to detect HCMV DNA. Overall, 46.4% of the donated milk samples and 59.2% of the samples obtained from mothers of premature infants were positive for HCMV DNA. The concentration of HCMV DNA was approximately 103 -104 copies/ml in the HCMV-DNA-positive human milk samples. Based on the nucleotide sequence of a 299- to 305-bp fragment of the glycoprotein B (gB) gene, three HCMV genotypes (gB1, gB2 and gB3) were identified in human milk samples. Mixed infection with genotypes gB1 and gB3 was also found in four milk samples from mothers. Genotype gB1 was the predominant genotype in the HCMV-DNA-positive human milk samples, and it could be subdivided into three lineages. There were also some characteristic nucleotides and amino acids in the three HCMV genotypes, which were helpful for distinguishing the genotypes. This is the first study to clarify the HCMV infection status and genetic characteristics of human milk obtained from banks in China, which will be helpful in preventing postnatal HCMV infections and ensuring the safety of human milk banks.

Human Cytomegalovirus Infection in Women With Preexisting Immunity: Sources of Infection and Mechanisms of Infection in the Presence of Antiviral Immunity.

Human cytomegalovirus (HCMV) infection remains an important cause of neurodevelopmental sequelae in infants infected in utero. Unique to the natural history of perinatal HCMV infections is the occurrence of congenital HCMV infections (cCMV) in women with existing immunity to HCMV, infections that have been designated as nonprimary maternal infection. In maternal populations with a high HCMV seroprevalence, cCMV that follows nonprimary maternal infections accounts for 75%-90% of all cases of cCMV infections as well as a large proportion of infected infants with neurodevelopmental sequelae. Although considerable effort has been directed toward understanding immune correlates that can modify maternal infections and intrauterine transmission, the source of virus leading to nonprimary maternal infections and intrauterine transmission is not well defined. Previous paradigms that included reactivation of latent virus as the source of infection in immune women have been challenged by studies demonstrating acquisition and transmission of antigenically distinct viruses, a finding suggesting that reinfection through exposure to an exogenous virus is responsible for some cases of nonprimary maternal infection. Additional understanding of the source(s) of virus that leads to nonprimary maternal infection will be of considerable value in the development and testing of interventions such as vaccines designed to limit the incidence of cCMV in populations with high HCMV seroprevalence.

Viral shedding, and distribution of cytomegalovirus glycoprotein H (UL75), glycoprotein B (UL55), and glycoprotein N (UL73) genotypes in congenital cytomegalovirus infection.

BACKGROUND: Children with congenital CMV infection (cCMV) shed virus in urine and saliva for prolonged periods of time. Outcome of cCMV varies from asymptomatic infection with no sequelae in most cases, to severe longterm morbidity. The factors associated with asymptomatic cCMV are not well defined. We evaluated the viral shedding in a cohort of infants with cCMV identified on newborn screening. In addition, we describe the distribution of viral genotypes in our cohort of asymptomatic infants and previous cohorts of cCMV children in the literature. METHODS: Study population consisted of 40 children with cCMV identified in screening of 19,868 infants, a prevalence of 2/1000. The viral shedding was evaluated at 3 and 18 months of age by real-time CMV-PCR of saliva and plasma, and CMV culture of urine. CMV positive saliva samples were analyzed for genotypes for CMV envelope glycoproteins gB (UL55), and gH (UL75) by genotype specific real-time PCR, and gN (UL73) by cloning and sequencing RESULTS: At 3 months age 40/40 saliva and urine samples, and 19/40 plasma samples were positive for CMV. At 18 months age all urine samples tested (33/33), 9/37 of saliva samples, and 2/34 plasma samples were positive for CMV. The genotype distribution did not differ from the published data CONCLUSIONS: The urinary virus shedding is more persistent than salivary shedding in children with cCMV. The genotype distribution was similar to previous literature and does not explain the low disease burden of cCMV in our population.

Epidemiological characteristics of human cytomegalovirus infection and glycoprotein H genotype in Chinese children.

BACKGROUND: Human cytomegalovirus (HCMV) is a common pathogen that causes many diseases in young children. HCMV glycoprotein H (gH) genotypes may be associated with the type and severity of some diseases. In this study, systematic surveillance was conducted on the prevalence of HCMV infections and HCMV gH genotypes in children. METHODS: Urine samples were collected from children admitted to the inpatient wards and outpatient departments between January 2015 and December 2016 in the Children's Hospital, Zhejiang University School of Medicine (Hangzhou, China), and these were tested by real-time PCR for HCMV DNA detection and HCMV gH genotyping. RESULTS: During the study period, a total of 32,542 urine samples were collected and analyzed using real-time PCR to confirm HCMV infection, and 5286 (16.2%) cases were positive for HCMV DNA. From our results, children aged less than one year were the most susceptible population to HCMV. Based on the data obtained from gH probes combined with real-time PCR assay, 964 HCMV-positive samples were genotyped for gH. Among them, 584 (60.6%) were positive for gH1 subtype, 307 (31.8%) for gH2 subtype, and 73 (7.6%) for both gH1 and gH2 subtypes. The gH2 rate of 42.9% indicated HCMV infection was the highest subtype in the group aged ≤28 days while gH1 rate of 77% was the highest in the group aged >3 years. The highest gH2 rate (36.4%) and lowest gH1 rate (50.0%) were detected in children with HCMV viremia, whereas the highest gH1 rates (65.0%) and lowest gH2 rates (28.8%) were found in children with HCMV hepatitis. CONCLUSION: The findings of our study show that children less than 1-year-old are the major population among HCMV-infected children. gH1 is the predominant genotype of HCMV in China, which occurs more frequently than gH2 genotype in the case of HCMV hepatitis. However, the opposite tendency is observed in HCMV viremia, where the gH2 genotype is predominant.

Prevalence of cytomegalovirus DNAemia and genotypic distribution among childbearing mothers and neonates in Taiwan.

BACKGROUND: Congenital cytomegalovirus (CMV) infection is the leading cause of neurologic disabilities and sensorineural hearing loss in children. However, in Taiwan, there is limited information on the genotypic diversity and prevalence of perinatal CMV infection in both mothers and neonates. The aim of this study was to screen samples from both mothers and umbilical cord blood for CMV at the time of delivery and to determine the CMV genotypic distribution. METHODS: Between June 2012 and July 2015, residual maternal and umbilical cord blood samples were collected from consenting participants admitted to the Chang Bing Show Chwan Memorial Hospital in central Taiwan. The blood samples were screened for CMV DNA using real-time PCR assay, and the genotypic classification of the CMV UL55, UL144, and US28 genes was determined by sequencing and phylogenetic analysis. RESULTS: A total of 1282 mother-neonate paired samples were enrolled in the study, 95.3% of whom were Taiwanese. CMV DNA was detectable in 6.2% of the maternal blood samples, with a significantly higher rate noted in non-Taiwanese mothers (11.7%,p=0.027). For the 1,282 umbilical cord blood samples, CMV DNA was detectable in 5.3% of the samples. The presence of CMV DNA in maternal blood was positively associated with the presence of CMV DNA in umbilical cord blood (p=0.01). In addition, the UL55, UL144, and US28 genotypic distribution was similar between mothers and neonates. CONCLUSION: The prevalence of CMV DNAemia in childbearing mothers and neonates is similar and their genotypic distribution implies potential CMV infection during pregnancy.

Cytomegalovirus Seroprevalence and Birth Prevalence of Congenital CMV Infection in Bosnia and Herzegovina: A Single-Center Experience.

BACKGROUND: Congenital cytomegalovirus infection (cCMV) is a leading cause of sensorineural hearing loss (SNHL) and neurodevelopmental disabilities in developed countries. Although high cCMV rates have been reported in populations with high seroprevalence, the cCMV prevalence in low/middle-income countries in Europe has not been defined. OBJECTIVE: To determine cytomegalovirus (CMV) seroprevalence and the cCMV prevalence in Bosnia and Herzegovina. METHODS: Between March 2010 and February 2019, 5222 sera samples from patients seen at the University Clinical Hospital Mostar were tested for CMV IgG. The cord blood samples collected from 2091 infants between July 2011 and January 2013 were analyzed for CMV IgG and CMV DNA. The cCMV prevalence was determined by testing saliva swabs from 1293 infants between November 2015 and October 2016. RESULTS: The overall CMV IgG prevalence was 81.4% (95% confidence interval: 0.8-0.82). Significantly higher prevalence was observed among females (84.9%) than in males (77.0%), and the rate increased from 50.8% in the 1 to 5 years group to 97.7% in the group > 65 years old. Most cord blood samples (2091/1925, 92.1%) were CMV IgG positive, and 2 (0.1%) were CMV DNA positive. Of the 1293 saliva swabs, 8 (0.62%; 95% confidence interval: 0.3-1.2) were CMV positive. All 8 infected infants had asymptomatic cCMV, and none had SNHL at 18 months of age. CONCLUSIONS: In a highly CMV seropositive population, the prevalence of cCMV was lower compared with that reported from other low/middle-income countries populations. None of the infected infants had symptomatic infection or SNHL at 18 months.

Age matters: older age as a risk factor for CMV reactivation in the CMV serostatus-positive kidney transplant recipient.

Evaluate risk factors for cytomegalovirus (CMV) reactivation during the first year after kidney transplantation in the CMV-seropositive older recipient. Retrospective single-center study. Between 2011 and 2015, 91 patients ≥ 65 years received a kidney transplant; these were matched with 91 controls, aged 40-60. Risk of CMV reactivation in the CMV-seropositive recipients was analyzed. Sixty-three older and 54 younger recipients were included; 50% had received CMV-directed prophylaxis. CMV reactivation was significantly more frequent in the older group (71.4% vs 44.4%, p = 0.003) and occurred earlier (p = 0.003). A multivariate model showed that only age was associated with CMV reactivation (OR 2.48, p = 0.03). After excluding patients that received thymoglobulin, older age group remained the only risk factor of CMV reactivation (OR 3.81, p = 0.014). Recurrent event analysis showed that the older cohort had an HR of 1.94 (p = 0.01) of CMV viremia; there was significant episode-cohort interaction (p < 0.01). While the older group had a higher risk of infection (HR = 2.43), after the initial episode the relative hazards were approximately equal (HR = 1.08, at period 2). This suggests that it is key to specifically avoid the first episode of reactivation. Universal prophylaxis or a hybrid prophylaxis model should be considered in the CMV-seropositive kidney transplant recipient aged ≥ 65 years.

Association of CMV genomic mutations with symptomatic infection and hearing loss in congenital CMV infection.

BACKGROUND: Congenital cytomegalovirus (cCMV) infection is the most common congenital infection and a leading cause of long-term neurological and sensory sequelae, the most common being sensorineural hearing loss (SNHL). Despite extensive research, clinical or laboratory markers to identify CMV infected children with increased risk for disease have not been identified. This study utilizes viral whole-genome next generation-sequencing (NGS) of specimens from congenitally infected infants to explore viral diversity and specific viral variants that may be associated with symptomatic infection and SNHL. METHODS: CMV DNA from urine specimens of 30 infants (17 asymptomatic, 13 symptomatic) was target enriched and next generation sequenced resulting in 93% coverage of the CMV genome allowing analysis of viral diversity. RESULTS: Variant frequency distribution was compared between children with symptomatic and asymptomatic cCMV and those with (n = 13) and without (n = 17) hearing loss. The CMV genes UL48A, UL88, US19 and US22 were found to have an increase in nucleotide diversity in symptomatic children; while UL57, UL20, UL104, US14, UL115, and UL35 had an increase in diversity in children with hearing loss. An analysis of single variant differences between symptomatic and asymptomatic children found UL55 to have the highest number, while the most variants associated with SNHL were in the RL11 gene family. In asymptomatic infants with SNHL, mutations were observed more frequently in UL33 and UL20. CONCLUSION: CMV genomes from infected newborns can be mapped to 93% of the genome at a depth allowing accurate and reproducible analysis of polymorphisms for variant and gene discovery that may be linked to symptomatic and hearing loss outcomes.