Takeshi Morisawa

A Novel Diagnostic Tool for Detecting Neonatal Infections Using Multiplex Polymerase Chain Reaction

Background: In newborns with infections, it is necessary to detect various pathogens rapidly and accurately, because the infections are often fatal when diagnosis is delayed. However, no diagnostic tools that rapidly detect pathogens causing neonatal infectious diseases are available. Objectives: To establish a rapid diagnostic tool using multiplex polymerase chain reaction (PCR) to detect 8 major pathogens that often cause neonatal infections, including Group B Streptococcus, Escherichia coli, Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus, Ureaplasma urealyticum, herpes simplex virus, Cytomegalovirus, and Candida albicans, and to validate this tool in the neonatal intensive care unit (NICU). Methods: One hundred and thirty clinical samples were obtained from newborns with any infectious signs or histories. DNA was extracted from these samples and multiplex PCR was performed with a mixture of 8 primer pairs, all designed to amplify pathogenic DNA and produce different sizes of amplicons. Seventy-seven samples with suspicion of bacterial infections were also examined by bacterial culture to evaluate the accuracy of the multiplex PCR results. Results: Six of the 8 pathogens could be rapidly detected by our multiplex PCR method, within 3.5–4.5 h. These positive results led us to immediately diagnose and select proper drugs against each pathogen. In comparison with culture results, our test characteristics were as follows: specificity: 93%, negative predictive value: 96%, and concordance rate: 90%. Conclusions: We have established and validated a rapid diagnostic tool for detecting pathogens using multiplex PCR, which may be useful for the confirmed diagnosis of neonatal infections in the NICU.

Novel double-deletion mutations of the OFD1 gene creating multiple novel transcripts

Oral-facial-digital syndrome type 1 (OFD1) is an X-linked dominant disease characterized by malformations of the face, oral cavity, and digits. Thus far, 18 small mutations in the OFD1 gene have been reported. Here, we describe, in one Japanese sporadic female OFD1 case, the presence of a novel pair of deletion mutations: a 4,094-bp deletion encompassing exon 7 to intron 9, and a 14-bp deletion in intron 9, both of which are present in her paternal X-chromosome. The first deletion, the largest known to affect OFD1, was revealed by identifying four novel transcripts that all lacked exons 7–9. The most likely cause of the double deletion is two unequal recombinations between homologous sequences. Identification of the 4,094-bp deletion was made possible only by analyzing OFD1 mRNA, underscoring the utility of mRNA analysis in the mutational analysis of OFD1.

Serum unbound bilirubin as a predictor for clinical kernicterus in extremely low birth weight infants at a late age in the neonatal intensive care unit

BACKGROUND This study aimed to evaluate peak serum total bilirubin (TB) and unbound bilirubin (UB) levels in preterm infants with clinical kernicterus (KI) who were diagnosed by clinical findings during infancy. DESIGN/SUBJECTS For this multicenter retrospective study, 18 Japanese extremely low birth weight (ELBW) infants with clinical KI were included. Clinical KI was diagnosed based on the presence of motor developmental impairment with/without athetosis, and abnormal magnetic resonance imaging or brainstem auditory evoked potential findings during infancy. High and low TB or UB levels were defined as serum TB levels ⩾ and <15 mg/dL or serum UB levels ⩾ and <0.8 μg/dL, respectively. The clinical characteristics of KI preterm infants were analyzed. The proportion of infants with high or low serum TB levels and with high or low serum UB levels was then investigated. Sensitivity and specificity were calculated. RESULTS In 18 KI infants, the median age when serum TB levels peaked was 28 days after birth. In eight KI infants with low serum TB levels, 88% of them had high serum UB levels. For comparison of the number of infants who had high or low serum TB and UB levels, the sensitivity was 90% and specificity was 13%. CONCLUSIONS Serum TB and UB levels peak at a later age than expected. Chronic serum UB monitoring may be helpful for identifying ELBW infants at risk for developing KI, even when they do not have high serum TB levels.

Novel treatment strategy for Japanese newborns with high serum unbound bilirubin

Serum unbound bilirubin (UB) is a measure of bilirubin not bound to albumin, and has been reported to be better than total bilirubin level at identifying infants at risk of developing bilirubin‐induced neurotoxicity, including auditory abnormalities. A detailed treatment strategy for newborns with high serum UB has not been established. The aim of this study was to assess auditory outcomes in newborns with serum UB ≥1.00 μg/dL who were treated according to a novel treatment protocol.

Thrombotic microangiopathy during the fetal period

Thrombotic microangiopathy (TMA), defined as renal injury, thrombocytopenia, and hemolysis, includes hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP). While TMA can develop because of various causes from the neonatal period to adulthood, no data are available on TMA during the fetal period. A female neonate diagnosed with fetal hydrops with neither polyhydramnios nor oligohydramnios was the second of dichorionic diamniotic twins delivered at 36 weeks of gestation. She had no family history of TMA. Birthweight was 2,398 g, with marked general edema (the first neonate was 2,515 g). Apgar score at 1 and 5 min after birth were 1 and 4, respectively. Laboratory evaluation indicated severe anemia (hemoglobin, 3.8 g/dL) and high lactate dehydrogenase (LDH; 3,664 IU/L). Platelet count and serum creatinine were 105 000/lL and 0.86 mg/dL, respectively. Peripheral blood smear immediately after birth contained schistocytes, and Heinz and Howell–Jolly bodies, suggestive of hemolytic anemia. Ultrasonography showed congestive heart failure (cardiomegaly, severe tricuspid and mitral insufficiency), but left ventricular contraction was not reduced. There were no anomalies of the kidney or reduced renal blood flow. At 2 days old, urine test indicated hematuria. At 4 days, she had decreased platelet count (79 000/lL), sustained hemolytic anemia, deteriorating renal function (serum creatinine, 2.6 mg/ dL), and concurrently developed pulmonary hemorrhage with abnormal coagulation function (activated partial thromboplastin time, 74.4 s; international normalized ratio of prothrombin time, 1.14; fibrinogen, 51 mg/dL). With regard to hemolytic anemia, blood type incompatibility was ruled out because the patient and her mother were both type A, and a test for irregular antibodies was negative. Autoimmune hemolytic anemia was unlikely on negative direct/indirect Coombs test. Finally, the shape of the red blood cells was not indicative of thalassemia or hereditary spherocytosis. Based on these results, the patient was diagnosed with TMA or pre-disseminated intravascular coagulation (DIC) and started on plasma infusion (PI) therapy without plasma exchange because of its invasive nature. Eculizumab therapy, which is effective only for TMA and not for DIC, and which carried a high risk of meningococcal infection, especially in the present neonate, who did not have a mature immune system, was avoided. While the lung injury, abnormal coagulation function and LDH improved after only one dose of PI, recovery of anemia and thrombocytopenia was temporary. The patient constantly required irradiated red cell concentrate transfusion for hemolytic anemia at approximately 20–30 days old. Additionally, after ligation for patent ductus arteriosus at 10 days old, serum creatinine was decreased at approximately 0.5 mg/dL, but did not improve to normal. Complement level was normal, and a disintegrin-like and metalloproteinase with thrombospondin type 1 motifs 13 (ADAMTS13) activation at 3 days old was 51%. Additionally, because of persistence of schistocytes on peripheral blood smear with normal coagulation function, hemolysis was still activated. Therefore, we finally concluded that the patient did not have congenital TTP, but instead had atypical HUS or secondary TMA. After commencement of PI twice per week again at 37 days old, the renal function and platelet count

Evaluation index for asymmetric ventricular size on brain magnetic resonance images in very low birth weight infants

OBJECTIVE Asymmetric ventriculomegaly is often evident on brain magnetic resonance imaging (MRI) in very low birth weight infants (VLBWI) and is interpreted as white matter injury. However, no evaluation index for asymmetric left-right and anterior-posterior ventricular sizes has been established. METHODS In this retrospective multicenter cohort study, brain T2-weighted MRI was performed at term-equivalent ages in 294 VLBWI born between 2009 and 2011. The value of a lateral ventricular index (LVI) to evaluate asymmetric ventricular size, as well as the relationship between the LVI value and walking at a corrected age of 18 months was investigated. At the level of the foramen of Monro in a horizontal slice, asymmetry between the left and right sides and between the anterior and posterior horns was identified by the corrected width and was detected by a low concordance rate and κ statistic value. An LVI representing the sum of the widths of the four horns of the lateral ventricle corrected for cerebral diameter was devised. RESULTS Asymmetric left-right and anterior-posterior ventricular sizes were confirmed. The LVI value was significantly higher in the non-walking VLBWI group (n = 39) than in the walking VLBWI group (n = 255; 18.2 vs. 15.8, p = 0.02). An LVI cut-off value of 21.5 was associated with non-walking. Multivariate analysis revealed that an LVI value >21.5 was an independent predictor of walking disability at the corrected age of 18 months (odds ratio 2.56, p = 0.008). CONCLUSIONS The LVI value calculated via MRI may predict walking disability at a corrected age of 18 months in VLBWI.