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.