Multiplex PCR for the Detection of 10 Viruses Causing Encephalitis/Encephalopathy and its Application to Clinical Samples Collected from Japanese Children with Suspected Viral.

BACKGROUND: Acute encephalitis is a serious neurological condition having a high mortality rate and affecting both children and adults. This study aimed to develop a multiplex PCR method for the simultaneous screening of clinical samples for the presence of the 10 viruses presently considered as the major viral causes of acute encephalitis/ encephalopathy in Asia. METHODS: Using previously published primers that have been widely used to screen for herpes virus-6, influenza A virus, human parechovirus, herpes simplex viruses 1 and 2, Japanese encephalitis virus, group A rotavirus, enterovirus, adenovirus, and dengue virus in clinical samples, a single-tube multiplex PCR assay was developed and was tested for its sensitivity and specificity. The method was then applied to screen 57 clinical samples, consisting of 13 fecal samples, 5 throat swabs, 3 post-nasal swabs, 18 serum samples, and 18 cerebrospinal fluid (CSF) samples, collected from 18 hospitalized Japanese children with suspected viral encephalitis/encephalopathy for the target viruses, and the results were compared with those of a monoplex PCR method. RESULTS: Positive viral controls of the 10 viruses were correctly typed using this multiplex PCR method. The multiplex PCR method showed high specificity with no unspecific amplification to non-target viruses. The results of applying this PCR method for screening clinical samples showed that 6 fecal samples, 2 serum samples, and 1 CSF sample collected from 7 patients were positive for a virus, specifically group A rotavirus (4 patients, 22.2%), enterovirus (2 patients, 11.1%), or adenovirus (1 patient, 5.6%). In comparison with monoplex PCR, for group A rotavirus, enterovirus, and adenovirus, the sensitivity of this multiplex PCR method decreased for serum, cerebrospinal fluid, and throat swab samples. CONCLUSIONS: This newly developed multiplex PCR method is a simple, rapid diagnostic tool and can be used to screen clinical samples for viruses causing acute encephalitis/encephalopathy in children in Asian countries.

Neuropsychological profiles of patients with 2q37.3 deletion associated with developmental dyspraxia.

Patients with 2q37 deletions manifest brachydactyly mental retardation syndrome (BDMR). Recent advances in human molecular research have revealed that alterations in the histone deacetylase 4 gene (HDAC4) are responsible for the clinical manifestations of BDMR. Here, we report two male patients with 2q37.3 deletions. One of the patients showed a typical BDMR phenotype, and HDAC4 was included in the deletion region. HDAC4 was preserved in the other patient, and he showed a normal intelligence level with the delayed learning of complex motor skills. Detailed neuropsychological examinations revealed similar neuropsychological profiles in these two patients (visuo-spatial dyspraxia) that suggested developmental dyspraxia. These observations suggested that some other candidate genes for neuronal development exist in the telomeric region of HDAC4.

Affinity for music in Wolf-Hirschhorn syndrome: two case reports.

BACKGROUND: Wolf-Hirschhorn syndrome is a congenital malformation syndrome resulting from deletion of the short arm of chromosome 4. Individuals with Wolf-Hirschhorn syndrome may have a "Greek warrior helmet" appearance, growth retardation, developmental delay, muscular hypotonia, epilepsy, and difficulty with language including verbal communication. An affinity for music has not previously been reported in these patients. PATIENTS: We describe two patients with Wolf-Hirschhorn syndrome who both have a strong affinity for music. One patient is a 20-year-old woman who likes to listen to music all day and can hum many tunes. The other patient is a 9-year-old girl who is calmed by music and received music therapy, with subsequent improvement in her communication skills (eye contact, joint attention, and vocalizations to request music). CONCLUSIONS: Individuals with Wolf-Hirschhorn syndrome may have a strong affinity for music and may benefit from music therapy. Additional studies are needed to investigate the interest in music in individuals with Wolf-Hirschhorn syndrome.

[Clinical efficacy and pharmacokinetics of lamotrigine for childhood-onset intractable epilepsy].

OBJECTIVE: We investigated the clinical efficacy and pharmacokinetics of lamotrigine (LTG) as an add-on therapy in childhood-onset intractable epilepsy. METHODS: We reviewed the charts of 28 outpatients who had received LTG as an add-on therapy. The data collected included epilepsy type, seizure frequency, concomitant anti-epileptic drugs, dosage of LTG and LTG serum levels. Furthermore, we reviewed the relationship between the LTG serum levels (microg/ml) and dosage of LTG (mg/kg/day), as well as the relationship between the LTG serum levels (microg/ml) and clinical efficacy in the following 2 groups:the valproate sodium (VPA) combination group and the non-VPA combination group. RESULTS: A reduction of 50% or more in seizure frequency was observed in 10 patients. In addition, there was a high correlation between the LTG serum levels and the dosage of LTG in each group. In the VPA combination group, the average of LTG serum levels in patients with adequate therapeutic response (50% reduction in seizure frequency) was higher than that in patients without adequate therapeutic response. In the non-VPA combination group, the average LTG serum level in adequate response patients was lower than that in patients without adequate therapeutic response. However, the epilepsy types of adequate response patients differed in the two groups. CONCLUSIONS: The LTG serum level is predictable based on the dosage of LTG. It was judged that the effective blood concentration of LTG differed when used with VPA, although factors other than the combined use of VPA should have been taken into consideration also.

A 3-year-old boy with Guillain-Barré syndrome and encephalitis associated with Mycoplasma pneumoniae infection.

Mycoplasma pneumoniae is a common cause of respiratory tract illness in children. Among the most common extrapulmonary manifestations are disorders of the central nervous system, including meningitis, meningoencephalitis, cerebellitis, polyneuropathy, acute disseminated encephalomyelitis, and Guillain-Barré syndrome. Guillain-Barré syndrome, also known as acute inflammatory demyelinating polyradiculoneuropathy, is an acute-onset, immune-mediated disorder of the peripheral nervous system. The central nervous system is usually intact in patients with Guillain-Barré syndrome. However, there have been some reports of an association of Guillain-Barré syndrome with central nervous system involvement in children. We report a 3-year-old boy with M. pneumoniae infection associated with Guillain-Barré syndrome and encephalitis. Both serum anti-GM1 ganglioside (IgG and IgM) and anti-galactocerebroside IgG antibodies were detected in our patient: the former in the earlier stage of the disease, and the latter in the later stage. We speculate that anti-GM1 ganglioside was associated more with encephalitis, and anti-galactocerebroside antibody was associated more with GBS in our case. Our patient is the youngest report of Guillain-Barré syndrome with central nervous system involvement, and the first report of a pediatric patient with associated M. pneumoniae infection. Such cases are rarely reported, but highlight the need for awareness of the association of the infection with Guillain-Barré syndrome with central nervous system involvement.

Acute encephalopathy with pandemic (H1N1) 2009 virus infection.

OBJECTIVES: In April 2009, a novel influenza A (H1N1) pdm virus was identified in Mexico and spread quickly around the world. However, the clinical features of acute encephalopathy associated with 2009 pandemic influenza have not yet been elucidated. METHODS: We treated 8 patients (3 boys and 5 girls) aged 4 to 11 years (average age, 8 y 3 months) with influenza virus-associated encephalopathy, who presented at our 2 hospitals between July 2009 and March 2010. We investigated the clinical characteristics, treatments, and outcomes in the patients. RESULTS: In all patients, brain computed tomography showed mild to severe diffuse cerebral edema, and electroencephalography revealed diffuse high-voltage slow waves. They were all treated with oseltamivir and methylprednisolone pulse therapy. Six patients recovered without any sequelae; however, the remaining 2 had residual neurological sequelae. These 2 patients presented with severe disturbance of consciousness, and their central nervous system symptoms appeared within 12 hours after the onset of fever. One patient had periventricular leukomalacia and symptomatic epilepsy by perinatal brain hypoxia, and the other patient had 1 complex febrile and 2 febrile seizures. CONCLUSIONS: This study showed that patients with influenza-associated encephalopathy caused by influenza A (H1N1) pdm infection were all older than those with seasonal influenza. Underlying neurological disease or history may be associated with poor prognosis.

Liver-specific mitochondrial respiratory chain complex I deficiency in fatal influenza encephalopathy.

We report on a 4-year-old boy who died from influenza encephalopathy. The clinical course and microscopic findings of the autopsied liver were compatible with Reye's syndrome. We examined the mitochondrial respiratory chain function by blue native polyacrylamide gel electrophoresis (BN-PAGE), western blotting, and respiratory chain enzyme activity assays. The activity of liver respiratory chain complex (CO) I was markedly decreased (7.2% of the respective control activity); whereas, the other respiratory chain complex activities were substantially normal (CO II, 57.9%; CO III, 122.3%; CO IV, 161.0%). The activities of CO I-IV in fibroblasts were normal (CO I, 82.0%; CO II, 83.1%; CO III, 72.9%; CO IV, 97.3%). The patient was diagnosed with liver-specific complex I deficiency. This inborn disorder may have contributed to the fatal outcome. We propose that relying only on fibroblast respiratory chain complex activities may lead to the misdiagnosis of liver-specific complex I deficiency.

Detection of group a rotavirus RNA and antigens in serum and cerebrospinal fluid from two children with clinically mild encephalopathy with a reversible splenial lesion.

We report on two children with mild encephalopathy with a reversible splenial lesion associated with group A rotavirus (GARV) infection. We examined stool, serum, and cerebrospinal fluid samples to determine the presence of the GARV VP7 gene and GARV antigen by reverse-transcription PCR and enzyme-linked immunosorbent assay, respectively. GARV antigen was detected in stool samples from both patients. The GARV G genotype was G9 in one child and G3 in the other. GARV antigens were also found in both serum samples. However, the GARV VP7 gene was detected in only one serum sample, which was collected on the first day of symptomatic illness. Neither GARV antigen nor the VP7 gene was detected in cerebrospinal fluid samples. Both patients had excellent outcomes. Our results suggest that the reversible splenial changes in our patients might have been caused by indirect effects to the central nervous system subsequent to viral infection.

Improvement of intractable childhood epilepsy following acute viral infection.

In this study, we report 11 patients with intractable childhood epilepsy that improved following acute viral infection. The patients were 8 boys and 3 girls. Six of the 11 children were diagnosed as West syndrome (5 of the symptomatic type and 1 of the cryptogenic type). The remaining 5 children were myoclonic seizures. The patients became seizure free within 6 days following acute viral infections without an exchange or addition of antiepileptic drugs (AEDs). The types of acute viral infections were Exanthema subitum (Roseola infantum) in 5 patients, Rotavirus gastroenteritis in 2 patients, Measles infection in 2 patients, Herpetic stomatitis in 1 patient and Common cold in the remaining patient. Salaam seizures and/or tonic spasms disappeared within 6 days after the onset of viral infections, and hypsarrhythmia evolved to localized spikes on electroencephalography (EEG) in the patients with West syndrome. Epileptic seizures disappeared rapidly and EEG gradually normalized or improved in patients with myoclonic seizures. Four patients became seizure free for 5 years to 20 years. In 6 patients, seizures relapsed within 14 days to 1 month after the disappearance of seizures. One child remained seizure free for 12 months after viral infection. Common factors in 4 children who were continuously seizure free include (1) normal or almost normal findings of brain CT/MRI, (2) normal development prior to the onset of epileptic seizures, and (3) a short time interval between the onset of seizures and the acute viral infection. We propose several hypotheses including an immunological effect for the improvement of intractable childhood epilepsy following acute viral infection. Further study may provide important information concerning the mechanism of seizure control and the applicable to treatment for intractable childhood epilepsy.

A case of D-lactic acid encephalopathy associated with use of probiotics.

A five year old girl was admitted to the hospital for evaluation of intermittent ataxia. She had undergone serial resections of the small intestine after birth, resulting in short bowel syndrome. Lactomin was prescribed for watery diarrhea at twice the regular dose 2 weeks before the onset of neurologic symptoms. D-lactic acidosis was diagnosed on the basis of a plasma D-lactate level of 5.537 mmol/l. Lactomin was discontinued, and she was treated with sodium bicarbonate and oral antibiotics. The probiotics the patient had taken were likely the cause of D-lactic acidosis and should therefore be avoided in patients with short bowel syndrome.

Detection of rotavirus RNA and antigens in serum and cerebrospinal fluid samples from diarrheic children with seizures.

Group A rotavirus (GARV) genes (the VP7 and NSP3 genes) in acute-phase cerebrospinal fluid (CSF), sera and stool samples from 6 children with convulsions accompanied by GARV gastroenteritis were investigated by reverse transcription-polymerase chain reaction (RT-PCR). When the VP7 gene was amplified from the samples, the G genotype (G type) of GARV was determined by RT-PCR. GARV genes were detected in the CSF samples of all 6 children, in 2 of the 3 blood samples, and in all of 4 stool samples. The G typing of GARV from 12 of a total of 13 samples indicated that G3 was the predominant G type in all samples. GARV antigens were detected by enzyme-linked immunosorbent assay in all of the 3 tested sera samples, while no GARV antigens were detected in any of the 5 tested CSF samples. We confirmed the presence of GARV genomes in the CSF samples from all of the children with rotavirus-associated seizures, including encephalopathy. However, the relationship between convulsions and the existence of GARV RNA in CSF remains unclear and further study is required.

Event-related potentials in response to 3-D auditory stimuli.

To evaluate auditory spatial cognitive function, age correlations for event-related potentials (ERPs) in response to auditory stimuli with a Doppler effect were studied in normal children. A sound with a Doppler effect is perceived as a moving audio image. A total of 99 normal subjects (age range, 4-21 years) were tested. In the task-relevant oddball paradigm, P300 and key-press reaction time were elicited using auditory stimuli (1000 Hz fixed and enlarged tones with a Doppler effect). From the age of 4 years, the P300 latency for the enlarged tone with a Doppler effect shortened more rapidly with age than did the P300 latency for tone-pips, and the latencies for the different conditions became similar towards the late teens. The P300 of auditory stimuli with a Doppler effect may be used to evaluate auditory spatial cognitive function in children.

Chronic fatigue syndrome after human parvovirus B19 infection without persistent viremia.

BACKGROUND: It is unclear how often chronic fatigue syndrome (CFS) appears after human parvovirus B19 (B19) infection and whether prolonged B19 viremia or some other factors cause CFS. OBJECTIVES: To determine how often CFS appears after B19 infection and whether prolonged B19 DNA presence, antibody production and persistently reduced complement levels occur in CFS patients after B19 infection. METHODS: Clinical findings were examined in 210 patients after B19 infection, and CH50, C3 and C4 levels were determined. B19 DNA and antibodies to B19 were also tested in 38 patients' sera including 3 with CFS. RESULTS: Serum B19 DNA disappeared after 4-5 months in all 18 patients tested. There are no differences in B19 DNA-positive period between patients with and without persistent symptoms. IgM antibody titers to B19 became reduced after 2 months in all 38 patients. Complement levels persistently decreased in a greater proportion of patients with persistent symptoms. CONCLUSIONS: The present study suggests that we should consider the possibility of CFS after B19 infection and that CFS may be derived from several aspects other than prolonged B19 DNA presence in sera.

Fish consumption and time to pregnancy in Japanese women.

The work described in this paper examined the possible relationship between fish consumption and human fecundity among Japanese women using hair mercury level and time to pregnancy (TTP) as indicators of fish consumption and fecundity. We hypothesized that hair mercury concentrations reflect the level of fish consumption and, consequently, can also be taken to indicate the level of intake of organochlorine compounds (OCs) such as dioxins and polychlorinated biphenyls (PCBs) for which fish are the primary source, and which can disrupt normal human reproductive processes. TTP was obtained by a self-administered questionnaire from women who had been delivered of a baby at either of two hospitals in Sendai, Japan, during the period of January 2002-March 2004. Total mercury concentration in their hair (0-3cm from the scalp) was determined by cold vapor atomic absorption spectrometry. Of the 298 women approached, 193 (65%) reported their TTP. The subjects were classified into two groups according to their TTP: group 1, 0-12 months TTP; and Group 2, >12 months TTP. A step-down procedure backward binominal logistic regression analysis was performed by using age, BMI, parity, frequency of intercourse, life-style parameters (smoking, drinking and dietary habits) and hair mercury level of the female subjects and their partners as independent variables. Two separate analyses were performed by including/excluding information on the partners of the subjects. The analyses did not extract hair mercury concentration as significant indicating that fish consumption did not prolong TTP, which was not consistent with the results of our previous study, i.e., in that study frequent fish eaters showed prolonged TTP. Possible reasons of the negative result are discussed and it is suggested that the hair mercury levels in the present study were not an appropriate indicator of fish consumption of the subjects or of their exposure to OCs. Further study on the relationship between fish consumption (and, ultimately, of OCs intake) and fecundity with more appropriate indicators of fish consumption and/or fish-mediated pollutants intake are warranted to characterize the health risk posed by fish consumption.

[Estimation of intake level of bisphenol A in Japanese pregnant women based on measurement of urinary excretion level of the metabolite].

OBJECTIVE: The daily bisphenol A (BPA) intake level of Japanese pregnant women was surveyed based on the measurement of the urinary excretion level of a BPA metabolite. METHODS: Spot urine samples were collected from 56 pregnant women who visited the gynecology division of a hospital for a routine health check between June and October 2003. The urinary concentrations of the BPA metabolite and creatinine were measured by GC/MS/MS and spectrophotometry, respectively. Daily BPA intake was assumed to be equal to daily excretion. RESULTS: The daily intake of BPA among Japanese pregnant women was estimated to be in the range of < 0.3 to 7.9 microg/day (median < 2.0 microg/day), being consistent with the levels in previous studies for non-pregnant Japanese women. This level was far below the current Acceptable Daily Intake (0.01 mg/kg/day) which was set by the European Commission. The maximum estimated intake per body weight (0.16 microg/kg/day) reached 1/10 of the Lowest Adverse Effect Level of BPA for pregnant mice for a reproductive effect on the offspring (2 microg/kg/day). CONCLUSION: It is desirable to lessen BPA intake from a precautionary viewpoint, particularly in pregnant women.

Daily urinary excretion of bisphenol A.

OBJECTIVES: Concerns over dietary exposure to bisphenol A (BPA), an endocrine disruptor, have been raised because BPA is contained in resins and plastics commonly used for the preservation of food and beverages. The purpose of the present study was to assess daily intake levels of BPA in a group of male subjects by measuring total urinary BPA (free BPA plus BPA released by treatment with ß-glucuronidase), as well as determining intra-individual variation in BPA excretion. METHODS: Twenty-four-hour urine was collected from 5 subjects for 5 consecutive days for the evaluation of between-day variation in urinary BPA excretion and from 36 male subjects for the estimation of the level of daily BPA intake. BPA in the urine samples was measured by GC/MS/MS following enzymatic hydrolysis of BPA glucuronate, solid phase extraction, and derivatization. RESULTS: A large between-day variation was found over 5 days for the daily excretion of urinary BPA in the 5 subjects. The daily excretion of urinary BPA was distributed log-normally in the 36 male subjects, with the median value being 1.2 µg/day (range: <0.21-14 µg/day), which was far below the Tolerable Daily Intake (0.01 mg/kg bw) recommended by a scientific committee in the European Commission in 2002. However, the maximum estimated intake per body weight (0.2 µg/kg/day) was only one order of magnitude lower than the reported lowest level for reproductive/behavioral effects in pregnant mice (2 µg/kg/day). CONCLUSIONS: Measuring urinary BPA in urine is a suitable approach for estimating short-term BPA intake levels in individuals and/or estimating the average exposure level of populations. Urine analyses will be increasingly important in the human health risk assessment of BPA.

[Preliminary study on measurement of human fecundity applicability of time to pregnancy with Japanese subjects].

BACKGROUND: Debate has been continuing whether endocrine disrupting chemicals (EDC) in the environment affect human reproduction. We are planning to use time to pregnancy (TTP) as a measure of human fecundity to relate EDC exposure in a future research. TTP is expressed as the number of non-contracepting menstrual cycles or months required for a couple to conceive. An extended TTP might be considered to reflect an injury to reproductive processes. Since this parameter has never been measured in Japan. the present preliminary study aimed at establishing a Japanese version of the TTP questionnaire and determining if TTP could be reliably measured in Japan. METHODS: We prepared a self-administered questionnaire that consisted of questions on TTP, sexual life and dietary habits. Women who visited a gynecology clinic in Tokyo for confirmation of pregnancy during the period October 2000 to March 2001 were the subjects asked to fill in the questionnaire. The results were statistically analyzed by chi-square test, t-test and binorminal logistic model after classifying the subjects into two groups: (a) Group I (TTP < or = 6 months; n = 53) (b) Group II (TTP > 6 months; n = 16) RESULTS: Of the 92 women approached, 69 (75.0%) could report TTP. The distribution of TTP, the mode being around 1-2 months with a positive skew toward longer value, was similar to those reported so far in Western countries. There were statistically significantly more smokers in Group II (smoker 62.5%) than in I (15.6%). Logistic regression analysis indicated prior gravidity and fish and milk consumption, in addition to cigarette smoking, to be related to TTP. CONCLUSIONS: The present preliminary study indicated that TTP surveys are feasible with Japanese based on the high response rate and plausible distribution of reported value. It also indicated that TTP was related to cigarette smoking, gravidity and dietary habits, again consistent with previous studies.