Evaluation of the indirect and IgM-capture anti-human cytomegalovirus IgM ELISA methods as confirmed by cytomegalovirus IgG avidity.

Primary cytomegalovirus (CMV) infection during pregnancy often results in congenital CMV infection with severe clinical complications. IgM antibodies are one of the indices of primary infection. The IgG avidity index (AI) is also known to remain low for 3 months after primary infection. Here, we evaluated and compared the performance of CMV IgM and IgG avidity assays. Because sensitivity and specificity reportedly differ between CMV IgM kits, CMV IgM detection was compared between the two commercially available ELISA kits that are most commonly used in Japan. Sera for CMV IgM were first screened using a traditional indirect ELISA kit. Selected samples were then tested for CMV IgM and CMV AI using a CMV IgM-capture ELISA kit and a CMV IgG avidity assay, respectively. The rate of concordance between the IgM kits was 89% (42/47), indicating the absence of any significant difference. Most of the CMV IgM-positive plasma samples showed high CMV IgG AI; however, 18 commercially available plasma samples with low CMV IgG AI were all CMV IgM-positive. One plausible explanation for this discrepancy is that the duration of low IgG AI is shorter than that of IgM positivity. Alternatively, CMV IgM tests may generate pseudo-positive readouts in cases of congenital infection. Nevertheless, our study confirms that CMV IgG AI can be a reliable indicator of CMV primary infection.

Congenital cytomegalovirus in Japan: More than 2 year follow up of infected newborns.

BACKGROUND: The aim of this study was to evaluate the outcome of congenital cytomegalovirus (CMV) infection identified on urine-filter screening assay at >2 years' follow up, and to observe the clinical outcomes after anti-CMV treatment. METHODS: Sixty of 72 congenital CMV patients were enrolled and clinically observed for >2 years. Forty-three were asymptomatic at birth; seven were symptomatic at birth but untreated with anti-CMV drugs; and 10 were symptomatic and treated with anti-CMV drugs. RESULTS: Of the 43 asymptomatic patients, three developed hearing loss or language disability for which association with congenital CMV has been repeatedly reported, and two had neurological sequelae of which the etiology was unclear, indicating that the rate of CMV-associated late-onset sequelae was 7-12%. All seven symptomatic infants without treatment developed sequelae, while three of the 10 treated patients were free from any sequelae. CONCLUSIONS: The rate of late-onset sequelae observed in Japan is similar to that reported in the USA and Europe. The treatment of symptomatic patients with antiviral agents results in favorable clinical outcomes. Thus, newborn urine-filter paper screening of congenital CMV infection is warranted.

A Thr72Ala polymorphism in the NKG2D gene is associated with early symptomatic congenital cytomegalovirus disease.

The potential risk factors for congenital cytomegalovirus (cCMV) infection or development of disease remain unclear. Here, we investigated the genetic polymorphisms in natural killer (NK) group 2, member D (NKG2D), an activating receptor expressed on NK cells, and in MHC class I-related chains A, the ligand of NKG2D, in 87 cCMV cases, and found that there was a significant association between cCMV disease and a single nucleotide polymorphism, Thr72Ala, in NKG2D.

A novel real-time PCR method for determination and quantification of each cytomegalovirus glycoprotein H subtype in clinical samples.

To investigate reinfection in patients with congenital cytomegalovirus (CMV) infection, we established a CMV subtype-specific real-time quantitative PCR method targeting the CMV gH epitope region that can be used for evaluating pathogenic CMV strains in cases of mixed CMV infection.

Dried umbilical cords in the retrospective diagnosis of congenital cytomegalovirus infection as a cause of developmental delays.

To clarify the impact of congenital cytomegalovirus (CMV) infection on developmental disabilities, 20 children with disabilities of unknown cause were analyzed. Five children were CMV positive and had no clinical manifestations at birth. Intracranial calcification was observed in 4 cases. Thus, congenital CMV infection is a significant cause of developmental disabilities.

Establishment of a cell-based assay for screening of compounds inhibiting very early events in the cytomegalovirus replication cycle and characterization of a compound identified using the assay.

To simplify the detection of infectious human cytomegalovirus (HCMV), we generated a cell line that produced luciferase in a dose-dependent manner upon HCMV infection. Using this cell line, we identified anti-HCMV compounds from a diverse library of 9,600 compounds. One of them, 1-(3,5-dichloro-4-pyridyl)piperidine-4-carboxamide (DPPC), was effective against HCMV (Towne strain) infection of human lung fibroblast cells at a 50% effective concentration of 2.5 microM. DPPC also inhibited the growth of clinical HCMV isolates and guinea pig and mouse cytomegaloviruses. Experiments using various time frames for treatment of the cells with DPPC demonstrated that DPPC was effective during the first 24 h after HCMV infection. DPPC treatment decreased not only viral DNA replication but also IE1 and IE2 expression at mRNA and protein levels in the HCMV-infected cells. However, DPPC did not inhibit the attachment of HCMV particles to the cell surface. DPPC is a unique compound that targets the very early phase of cytomegalovirus infection, probably by disrupting a pathway that is important after viral entry but before immediate-early gene expression.

Evaluation of screening tests for congenital cytomegalovirus infection.

Although the use of dried blood spots has been proposed for screening of newborns with congenital cytomegalovirus infection, viral loads in blood were significantly smaller than those in urine (P < 0.001), and DNA recovery from dried blood spots using the thermal shock procedure was inefficient. In contrast, our urine-based screening program identified asymptomatic cases with low viral loads in blood.

Newborn Congenital Cytomegalovirus Screening Based on Clinical Manifestations and Evaluation of DNA-based Assays for In Vitro Diagnostics.

OBJECTIVES: To establish a strategy for congenital cytomegalovirus (cCMV) screening and to establish confirmatory assays approved as in vitro diagnostics by the regulatory authorities, we evaluated the clinical risks and performance of diagnostic assays developed by commercial companies, since cCMV infection has significant clinical consequences. STUDY DESIGN: Newborns with clinical manifestations considered to be consequences of cCMV infection (n = 575) were screened for the presence of cytomegalovirus (CMV) DNA in urine specimens collected onto filter paper placed in their diapers using the polymerase chain reaction-based assay reported previously. Liquid urine specimens were obtained from all of 20 CMV-positive newborns and 107 of the CMV-negative newborns identified in the screening. We used these 127 specimens, as well as 12 from cCMV cases identified in a previous study and 41 from healthy newborns, to compare the performance of 2 commercial assays and 1 in-house assay. RESULTS: The risk-based screening allowed the identification of cCMV cases at least 10-fold more efficiently than our previous universal screening, although there appears to be a limit to the identification of asymptomatically infected newborns. Although CMV-specific IgM during pregnancy was found frequently in mothers of cCMV newborns, CMV-IgM alone is not an effective diagnostic marker. The urine-filter-based assay and the 3 diagnostic assays yielded identical results. CONCLUSIONS: Although risk-based and universal newborn screening strategies for cCMV infection each have their respective advantages and disadvantages, urine-filter-based assay followed by confirmatory in vitro diagnostics assays is able to identify cCMV cases efficiently.

Molecular comparison of bacterial isolates from blood with strains colonizing pharynx and intestine in immunocompromised patients with sepsis.

Most causative organisms of sepsis in immunocompromised patients are the same species as those that colonize their own nasopharynx or intestinal tract. To determine whether the strains recovered from blood originate mainly from patients' own flora, isolates from blood and throat and/or stool were investigated by genomic analyses. Surveillance cultures of throat and stool were taken prospectively from cancer patients being treated with intensive chemotherapy followed by haematopoietic stem-cell transplantation. In those cases of sepsis in which the isolate from blood was the same species as that from the throat and/or stool, the genomic profiles of the isolates were compared by PFGE. Ten cases of blood culture-positive sepsis were documented in six of 14 subjects during a 2 year period; isolates of Pseudomonas aeruginosa, Staphylococcus epidermidis, Enterococcus sp., viridans streptococci and Fusobacterium sp. were recovered from blood. In five of seven cases in which the blood isolate was the same species as that from the throat or stool, the genotypes of the isolates from both sites were identical. In the majority of immunocompromised patients, the causative organisms of bloodstream infections originated mainly from their own flora.

Construction of a scoring system for predicting the risk of severe gastrointestinal involvement in Henoch-Schönlein Purpura.

OBJECTIVE: To evaluate the parameters associated with significant gastrointestinal (GI) involvement in Henoch-Schönlein Purpura (HSP), and construct a scoring system for the identification of patients at high risk of gross blood in stools. STUDY DESIGN: Data for HSP patients hospitalized at each of seven institutes were retrospectively analyzed. Patients were divided into four groups according to the consequent severity of GI involvement. Identification of laboratory parameters at the time of admission were then used to differentiate the groups, and a scoring system to predict gross intestinal bleeding was constructed. Prognostic efficiency, correlation with the subsequent duration of abdominal pain, and association with manifestations excluding abdominal pain were also analyzed. RESULTS: An analysis of variance (ANOVA) test showed significant intergroup differences in white blood cell (WBC) count, neutrophil count, serum albumin, potassium, plasma D-dimer and coagulation factor XIII activity. A scoring system consisting of these parameters showed a good prognostic value for gross intestinal bleeding in a receiver operating characteristic (ROC) analysis, and a cut-off value of 4 points showed a sensitivity of 90.0% and specificity of 80.6%. The score was also correlated with the duration of abdominal pain after admission. A significantly higher score (s) was observed in patients presenting with nephritis, although the predictive value was poor. CONCLUSION: A scoring system consisting of generally available parameters was of use in predicting severe GI involvement in HSP patients. Although further study is needed, initial therapy in accordance with disease activity may be taken into consideration using this scoring system.

Polymorphisms in TLR-2 are associated with congenital cytomegalovirus (CMV) infection but not with congenital CMV disease.

BACKGROUND: Cytomegalovirus (CMV) is the most common cause of congenital virus infection. However, the risk factors for infection in utero and for progression to a severe clinical outcome remain uncertain. Recent studies have identified associations of specific single nucleotide polymorphisms (SNPs) in Toll-like receptor (TLR) genes with susceptibility to infections of some viruses and with their clinical outcome. METHODS: Genetic polymorphisms in the TLR-2, TLR-4, and TLR-9 genes were analyzed in 87 children with congenital CMV infections by the TaqMan allelic discrimination assay. The frequencies of genotypes in the general Japanese population were obtained from the National Center for Biotechnology Information (NCBI) databases. Associations between the analyzed SNPs and congenital CMV infection or disease were evaluated. RESULTS: The CC genotype at SNP rs3804100 in the TLR-2 gene was significantly associated with congenital CMV infection but not with congenital CMV disease. Furthermore, the AG genotype at SNP rs1898830 in the TLR-2 gene tended to be identified less frequently in children with congenital CMV infection. There were no statistically significant associations between SNPs in the TLR-4 and TLR-9 genes and congenital CMV infection or disease. CONCLUSION: TLR-2 polymorphisms may have some association with congenital CMV infection, although the mechanism underlying this effect remains to be clarified.

Cytomegalovirus (CMV) glycoprotein H-based serological analysis in Japanese healthy pregnant women, and in neonates with congenital CMV infection and their mothers.

BACKGROUND: Congenital cytomegalovirus (CMV) infection is caused by maternal primary infection as well as CMV reinfection or reactivation during pregnancy, although differences in the clinical impact between these modes of infection remain to be clarified. OBJECTIVES: To investigate the latest prevalence and risk of multiple CMV infection in healthy pregnant women, as well as the types of maternal CMV infection associated with congenital CMV infection. STUDY DESIGN: Seroprevalence against CMV and IgG subclasses were determined in 344 serum samples from healthy pregnant women in Japan. CMV genotype and serotype were also determined in 18 pairs of mothers and neonates with congenital CMV infection identified in our CMV screening program. RESULTS: Thirty-two percent of the pregnant women were seronegative, while 66% of CMV seropositive women had IgG3 antibodies against one epitope on glycoprotein H (gH) as the major subclass, and 52% had IgG1 antibodies against one epitope on glycoprotein B (gB). Only a single genotype determined by CMV gH neutralizing epitope was found in the urine from the 18 neonates with congenital CMV infection, even though one case possessed antibodies against multiple CMV strains. In that case, the antibodies against the strain not detected in the urine from the infant disappeared within one month after birth, whereas the antibodies against the infecting CMV strain continued to be detected at 12 months after birth. CONCLUSIONS: Two (11%) of 18 cases of congenital CMV infection occurred via maternal CMV reinfection. Maternal humoral immunity did not prevent congenital CMV infection with another gH subtype.

Sequential changes in pathophysiology of systemic inflammatory response in a disseminated neonatal herpes simplex virus (HSV) infection.

BACKGROUND: Disseminated neonatal herpes simplex virus (HSV) infection causes a typical systemic inflammatory response syndrome and has a high mortality rate. However, the validity of anti-inflammatory intervention against this condition remains unknown. OBJECTIVES: We sought to demonstrate the sequential changes in the pathophysiology of disseminated neonatal HSV infections. STUDY DESIGN: The HSV serum copy number as well as high-mobility group box 1 (HMGB1) and cytochrome c concentrations, which predict the severity and mortality rate of sepsis, were sequentially evaluated in a patient with disseminated neonatal HSV infection caused by HSV-2. RESULTS: As the patient presented with evidence of hyper-inflammation and severe illness, we empirically undertook anti-inflammatory intervention that included the administration of prednisolone, high-dose immunoglobulin, and blood exchange therapy in addition to high-dose acyclovir (ACV) therapy. The patient survived without significant neurological sequela. We found that (1) the serum concentrations of both HMGB1 and cytochrome c were extremely high, (2) temporal increases in these biomarkers were observed after admission, and (3) interestingly, the increase in HMGB1 level preceded that of cytochrome c. These results suggested that the pathophysiology of this condition changed sequentially in a dramatic manner, and the timing of our anti-inflammatory intervention was prior to the transition of pathological status from hyper-inflammation to massive apoptosis. CONCLUSIONS: Anti-inflammatory intervention may only be effective if it is undertaken during the early phase of disseminated neonatal HSV infections.

Screening for congenital cytomegalovirus infection using newborn urine samples collected on filter paper: feasibility and outcomes from a multicentre study.

Background As congenital cytomegalovirus (CMV) infection causes significant clinical consequences not only at birth but also later as neurological sequelae, it is critical to establish a strategy for screening congenitally infected newborns. Previous studies have identified an insufficient sensitivity in screening methods based on the use of dried blood spots (DBSs). Objectives To evaluate the feasibility of the authors' recently developed method for large-scale screening for congenital CMV infection and to identify risk factors for congenital infection. Methods More than 21 000 newborns were enrolled at 25 sites in six geographically separate areas of Japan. Urine was collected onto filter cards placed in the diapers, which were then analysed by quantitative PCR using the filter disc directly as a template. Clinical and physical findings of the newborns were extracted from their medical records. CMV strains from the cases and their siblings were genetically compared. Viral loads in DBSs obtained from some of the cases were compared with those in the urine filters. Results Congenital CMV infection was identified in 0.31% (95% CI 0.24% to 0.39%) of the newborns, and 30% of the cases (20/66) had typical clinical manifestations and/or showed abnormalities in brain images at birth. Although the positive predictive value of our screening was 94%, the lack of any comparison with a gold standard assay prevented calculation of the negative predictive value. Almost two-thirds of the cases had siblings, a significantly higher frequency than for uninfected newborns. Most of the cases (21/25) excreted CMV strains identical to those of their siblings. CMV DNA was undetectable in three out of 12 retrievable DBS specimens. Conclusions Implementation of an effective large-scale screening programme for congenital CMV infection is feasible. Siblings are the major risk factor for congenital CMV infection, which emphasises the need for education of mothers-to-be as well as vaccine development.

Single cytomegalovirus strain associated with fetal loss and then congenital infection of a subsequent child born to the same mother.

BACKGROUND: Intrauterine transmission of cytomegalovirus (CMV) can occur even in CMV-seropositive mothers. Previous studies demonstrated re-infection with a newly acquired CMV strain during pregnancy had a major role in such transmission. Although reactivation of latently infected CMV is another plausible cause, no direct evidence has been documented. OBJECTIVES: We sought to identify the route(s) and maternal risk factor of CMV infection that occurred in consecutive pregnancies and resulted in symptomatic congenital infections. STUDY DESIGN: A newborn identified with congenital CMV infection in our newborn screening program developed hearing loss and subsequent nystagmus. The mother had a history of an elective abortion due to a severe fetal CMV infection 32 months prior to delivery of this newborn. We analyzed maternal serological changes and compared CMV genomic sequences in specimens obtained from the aborted fetus and the present case. We also analyzed immunological functions of the mother. RESULTS: Our major findings were as follows: (1) the aborted fetus and the present case were infected with the same strain. (2) The congenital infection that resulted in the abortion was due to a primary infection. (3) CMV DNA was undetectable in the mother's blood from 3 months after the abortion. These results strongly suggested that maternal viral reactivation caused the congenital infection in the present case. However, we could not find impairment of immunological functions in the mother. CONCLUSIONS: Viral reactivation in an apparently immunocompetent mother can cause symptomatic congenital CMV infection.

Genetic variations in the gB, UL144 and UL149 genes of human cytomegalovirus strains collected from congenitally and postnatally infected Japanese children.

Human cytomegalovirus (CMV) is the leading cause of intrauterine viral infection. The association of genetic polymorphisms in some particular genes with the incidence and severity of congenital infection has been controversial. To address this issue, we analyzed the genotypes of the glycoprotein B (gB), UL144 and UL149 genes of CMV clinical strains obtained from 33 congenitally and 31 postnatally infected Japanese children. Our results demonstrated that (1) CMV strains with any combination of genotypes could be vertically transmitted from mother to fetus, potentially causing neurological abnormalities, (2) the gB3 genotype was more prevalent in the congenital cases than in postnatally infected children (P < 0.05), particularly in congenital cases with sensorineural hearing loss (P = 0.009), (3) there was no relationship between gB genotype and viral load in the urine and dried umbilical cord specimens in the congenital cases, and (4) the UL144 and UL149 genotype distributions had no bias for congenial infection. In future studies, it would be interesting to see whether the gB genotypes serve as a prognostic indicator of CMV-associated diseases.

Genetic linkage among human cytomegalovirus glycoprotein N (gN) and gO genes, with evidence for recombination from congenitally and post-natally infected Japanese infants.

Investigation of sequence polymorphisms in the glycoprotein N (gN; gp4273), gO (gp4274) and gH (gp4275) genes of human cytomegalovirus (HCMV) strains collected from 63 Japanese children revealed that their gO genotype distribution differed slightly from that of Caucasian populations and that there was a significant linkage between the gN and gO genotypes. Linkage of these genotypes in strains obtained from Caucasian populations has been reported, so our similar findings in Japanese infants are consistent with this, and suggest generality of this linkage. Sequence analysis suggests that recombination between two strains of different linkage groups occurred approximately 200 bp upstream of the 3'-end of the gO gene. Further studies are required to elucidate differences in biological characteristics among the linkage groups and the selective constraints that maintain the linkage.