Detection of congenital cytomegalovirus infection using umbilical cord blood samples in a screening survey.

Easy screening and accurate diagnosis of congenital cytomegalovirus (CMV) infection are needed to predict and treat complications. We report the clinical course of two neonates with congenital CMV infection confirmed by real-time polymerase chain reaction (PCR) for CMV DNA in umbilical cord blood. A total of 1,010 neonates born at Yonaha Clinic from July 2005 to March 2007 were investigated. Umbilical cord blood was collected at birth, and DNA was extracted to screen for CMV DNA by real-time PCR. Head MRI and a developmental test were conducted for two cases (0.2%) in which CMV DNA was detected. Neither case showed clear abnormalities at birth, and head CT conducted at 1 month after birth revealed no abnormalities. Auditory brainstem responses were normal at both 1 and 12 months after birth in both cases. Head MRI at 12 months showed abnormalities in both cases. For both cases, development tests conducted at 12 months revealed mild developmental delays, particularly in posture and movement areas, which might have been caused by congenital CMV infection.

Polymorphisms of the factor VII gene associated with the low activities of vitamin K-dependent coagulation factors in one-month-old infants.

Despite administration of vitamin K (VK), some infants show lower activity of VK-dependent coagulation factors and they could develop intracranial hemorrhage. For preventing VK deficiency bleeding (VKDB) in infants, oral administration of VK and a screening test for VK deficiency are carried out in Japan. For the screening, the total activity of VK-dependent coagulation factors is measured using a commercial product, Normotest. This study was undertaken to clarify the importance of the following genetic and environmental factors on the coagulation status in one-month-old infants: two polymorphisms in the factor VII gene, -323P0/10 (a 10-bp insertion in the promoter region at position -323) and R353Q (the replacement of arginine [R] with glutamine [Q] at residue 353) and sex, age, gestational age, birth weight, and feeding regimen. Two hundred Japanese infants (34.6 +/- 4.0 days old) were screened for VK-dependent coagulation activity with Normotest and were genotyped for the two polymorphisms. Among the subjects screened, 18 infants (9%) carried the P10 allele and 26 (13%) carried the R353Q allele. Multiple regression analysis showed that the 10-bp inserted (P10) allele or the Q allele was associated with the lower coagulation activities. The coagulation activities for the R/Q genotype were significantly lower than those for the R/R genotype and those for the P0/P10 genotype were significantly lower than those for the P0/P0 genotype. Therefore, infants who carry the P10 allele or the Q allele show lower activity of VK-dependent coagulation factors. These infants may have a higher risk of VKDB manifestation.

Prevalence of hepatitis E virus infection in Japanese children.

BACKGROUND: Recently, sporadic cases of acute hepatitis and fulminant hepatitis caused by hepatitis E virus (HEV) have been reported in Japan. However, few reports have addressed the issue of HEV infection during childhood. METHODS: This study included 5 patients with fulminant hepatitis, 30 patients with acute hepatitis, and 309 patients without history of hepatic dysfunction or hepatitis in childhood as control. RNA was extracted from each serum sample, and HEV specific reverse-transcriptase polymerase chain reaction was performed. Anti-HEV immunoglobulin (Ig)M and IgG were measured by enzyme-linked immunoadsorbent assay. RESULTS: HEV RNA, anti-HEV IgM, and anti-HEV IgG were not detected in the sera of any of the five patients with fulminant hepatitis. In the 30 patients with acute hepatitis, only one (3.3%) was positive for anti-HEV IgG, and all were negative for anti-HEV IgM and HEV RNA. Of the 309 control patients, 8 (2.6%) were positive for anti-HEV IgG, and 2 (0.6%) were positive for anti-HEV IgM, respectively. CONCLUSION: The result of patients with fulminant hepatitis suggests that HEV is an unlikely cause of fulminant hepatitis in children. However, the detection rate of anti-HEV IgG shows that a history of HEV infection is not so rare among children in Japan.

Relationship between human cytomegalovirus glycoprotein B genotype and serum alanine aminotransferase elevation in infants.

The glycoprotein B (gB) region of the human cytomegalovirus (HCMV) is a major envelope glycoprotein that is a principal target of neutralizing antibodies and is known to stimulate the immune response of cytotoxic T lymphocytes. HCMV is currently classified into four genotypes on the basis of the nucleotide sequence of the gB region. The presence of HCMV in patients under 3 years of age was determined by subjecting urine samples taken from the patients to polymerase chain reaction (PCR) analysis. Analysis by direct sequencing of the gB region was carried out in 90 cases. These cases were grouped into the gB genotype 1 and gB genotype 3. Of 28 cases with a peak serum alanine aminotransferase (ALT) level(> or =100 IU/l), the duration of observed serum ALT elevation in the gB genotype 1 patients (166.7+/-126.7 days [mean+/-S.D.] [19 cases]) was significantly longer than that in the gB genotype 3 patients (39.7+/-31.7 days [9 cases]) (p<0.01). In the 54 cases with a serum ALT level(> or =50 IU/l), similar tendency was seen (p<0.05). These findings suggest that when serum ALT elevation is confirmed in young children infected with HCMV, analysis of the gB region is helpful for prediction of the duration of serum ALT elevation in the early stage of infection.

Prevalence of SEN virus among children in Japan.

Recently, a novel deoxyribonucleic acid (DNA) virus, designated SEN virus (SENV), was discovered and strong associations between the two SENV variants (SENV-D and SENV-H) and non-A to E hepatitis were reported. To clarify the character of SENV infection in children, we investigated the detection rates of serum SENV DNA by polymerase chain reaction (PCR) among children with non-A to C hepatitis, with histories of transfusions, with neither histories of transfusions nor liver diseases (control), and among pregnant women. SENV-D was detected in 60% of fulminant hepatitis, 5% of acute hepatitis, 11% of chronic hepatitis, 13% of controls, and 15% of pregnant women. SENV-H was detected in none of fulminant hepatitis, 5% of acute hepatitis, none of chronic hepatitis, 2% of controls, and 12% of pregnant women. No significant difference was found for SENV-D between acute or chronic hepatitis and controls, however SENV-D detection rate in fulminant hepatitis was significantly higher than that in controls (P < 0.05). No significant difference was found for SENV-H between any hepatitis and controls, however SENV-H detection rate in pregnant women was significantly higher than that in controls (P < 0.05). Neither SENV-D nor SENV-H was associated with acute or chronic hepatitis; however, SENV-D might be a risk factor of fulminant hepatitis.

Detection of PHKA2 gene mutation in four Japanese patients with hepatic phosphorylase kinase deficiency.

We analyzed the PHKA2 gene in four Japanese families with hepatic phosphorylase kinase (PhK) deficiency. Mutational analysis of PHKA2 cDNA was performed by reverse-transcribed polymerase chain reaction (RT-PCR) and direct sequencing, and each mutation was confirmed on the genomic DNA. In boys with low erythrocyte PhK activity (i.e., x-linked liver glycogenosis [XLG] type I), deletion of exon 2 (splice site mutation of 79-1 G > T) or nonsense mutation of Q1169X or R497X was identified. However, missense mutation of R295C was identified in one boy with normal erythrocyte PhK activity (i.e., XLG type II). This mutation was not found in 100 control alleles, and was considered responsible for presentation of the XLG type II phenotype. Excluding Q1169X, all mutations detected in this study represented novel mutations. All mothers were found to be heterozygous carriers of the mutations. Gene analysis was confirmed to represent a useful procedure for diagnosing XLG type II, for which liver biopsy had previously been required to detect hepatic PhK deficiency.

Bile salt export pump gene mutations in two Japanese patients with progressive familial intrahepatic cholestasis.

BACKGROUND: In recent years, progressive familial intrahepatic cholestasis has been classified into at least three types by genetic analysis: PFIC1, PFIC2, and MDR3. Liver transplantation is effective for treating patients with this intractable syndrome. Confirming the correct diagnosis is of paramount importance because prognosis after transplantation differs with the genetic type of the disease. METHODS: Synthesis of cDNA was accomplished using RNA extracted from liver tissue of two Japanese patients with progressive familial intrahepatic cholestasis. Polymerase chain reaction was performed using 13 primer sets designed for amplification of the bile salt export pump cDNA. Direct sequencing was undertaken, and identified sequences were compared with the sequence for bile salt export pump gene registered with GenBank. In addition, gene sequences for nonprogressive familial intrahepatic cholestasis patients were analyzed. RESULTS: Genetic analysis of patient 1 revealed that substitutions in bile salt export pump protein sequences, namely R575X and E636G, might be the cause of the disease. In patient 2, V330X and R487H might fulfill the same role. Results of gene analysis in parents and cholestatic controls supported these conclusions. CONCLUSIONS: Absence or presence of bile salt export protein gene mutations was confirmed as representing a useful prognostic marker for clinical course after liver transplantation.