A Study of Gram Stained Specimens Obtained from Pediatric Inpatients of Kawasaki Medical School Hospital - Achievements from an elementary research experiment by a 2nd year medical student

　This study was designed as an elementary medical research experiment course for a 2nd year medical student. All Gram stained specimens deriving from upper respiratory tract specimens collected from pediatric inpatients admitted to the pediatric ward of our hospital during 2018 October 1st through 31st were examined; the result was assessed as to whether or not it was in concordance with past reports or not.　Gram-negative cocci (Moraxella catarrhalis -like), Gram-negative rods (Haemophilus influenzae -like), and Gram-positive diplococci (Streptococcus pneumoniae -like) were frequently encountered in this order; these three most encountered species were in concordance with previous reports, whereas their order was not. This difference in the order of frequency might be attributed to the legislation of vaccination against Haemophilus influenzae type b and Streptococcus pneumoniae

Home-based subcutaneous immunoglobulin after switch from intravenous immunoglobulin improved quality of life in pediatric patient with common variable immunodeficiency: A case report

　Common variable immunodeficiency (CVID) is one of the primary immunodeficiency. Regular immunoglobulin G (IgG) replacement therapy is often performed for patients with CVID.　We experienced a patient who was hospitalized in our hospital for repeated pneumonia and diagnosed CVID at the age of 10 years. He had often been absent from school due to infectious diseases. We were administered intravenous IgG (IVIG) two times and his serum level of IgG became over 1,000 mg/dL. Afterward, he was affected the hand-foot-and-mouth disease one week after discharge. At that time, his IgG level decreased to 751 mg/dL. To maintain stable IgG trough levels, we introduced subcutaneous IgG (SCIG). Since then, his IgG levels remained around 1,000 mg/dL, he has lived without suffering from infectious diseases.　There are some reports that IVIG and SCIG were compared and SCIG was able to obtain a stable IgG trough levels to prevent infection. In addition, because our patient is a mother and child family, it was difficult to visit the outpatient department frequently, so it was desirable to infuse at home.　We experienced a patient who had a stable trough levels with SCIG and improved quality of life, so we report this case with literature reviews

Reactive oxygen metabolites as a biomarker of congenital heart disease in children

　Brain natriuretic peptide (BNP), as a hematological biomarker, has been widely used in congenital heart disease (CHD). However, its sensitivity and specificity vary depending on age and pathological condition. In the present study, we assessed whether reactive oxygen metabolites (ROMs) and biological antioxidant potential (BAP), as oxidative stress indicators, could be new biomarkers in CHD.　Forty-two patients diagnosed with CHD were enrolled in this study. The levels of ROMs, BAP, BNP, cardiac muscle creatinine kinase, and heart-type fatty acid-binding protein were measured using the findings of echocardiography. The ROM and BNP levels were significantly higher than the standard reference levels. The estimated Qp/Qs correlated mildly with BNP and ROM levels. The medication caused a significant decrease in BNP and ROM levels. The optimal decision, Qp/Qs greater than 1.5, estimated from receiver operator characteristic (ROC) curves was 371 U.CARR (58% sensitivity, 90% specificity) for ROMs, and that for BNP was 28.4 pg/ml (97% sensitivity 45% specificity). Direct comparison of ROMs and BNP did not show significantly different area under the curve values.　ROM levels can be a new biomarker for oxidative stress evaluation in children with CHD at almost the same sensitivity as the previous biomarkers, and an effective indicator when combined with other biomarkers and indicators

The diagnostic accuracy of biomarkers for the prediction of bacteremia in patients with suspected infection: a prospective observational study

　Rapid recognition of bacteremia is important for critical care, especially in patients with suspected bloodstream infections. Procalcitonin and presepsin are widely used biomarkers in point-of care medical testing for identifying infectious diseases and sepsis; however, the diagnostic accuracy for the prediction of bacteremia is not well established. Therefore, this study aimed to evaluate the diagnostic accuracy of procalcitonin and presepsin for the prediction of bacteremia in patients with suspected bacteremia. We performed a prospective observational study at our hospital. A total of 210 patients (307 samples) who had been admitted from December 2014 through September 2016 with a suspected infection were included. Presepsin and procalcitonin were tested simultaneously with blood cultures and routine laboratory tests. One hundred and four blood samples were obtained at the emergency room (ER). Others were obtained during hospital admission. Blood cultures were positive in 34 samples; 25 samples were obtained in the ER. Presepsin and procalcitonin levels were significantly higher in patients with positive blood cultures than in those with negative blood cultures (1028.5 pg/mL vs. 485.0 pg/mL, P < 0.001 and 4.53 ng/mL vs. 0.33 ng/mL, P < 0.001, respectively). For predicting bacteremia, receiver operating characteristic curve analysis for presepsin showed an area under the curve (AUC) of 0.718 and negative predictive value (NPV) of 95%. The analysis for procalcitonin showed an AUC of 0.778 and NPV of 94.8%. C-reactive protein tests and the quick Sequential Organ Failure Assessment score in the ER failed to be useful tools for predicting bacteremia. Based on our results, procalcitonin and presepsin showed good diagnostic accuracy and NPV for predicting bacteremia among patients with suspected infection. Therefore, these biomarkers may be useful for ruling out bacteremia in patients with suspected infection

Diagnostic accuracy of 16S ribosomal RNA gene polymerase chain reaction in bacteremia: A prospective observational study

　The standard method for diagnosing bacteremia is blood culture. However, the sensitivity of blood culture is low when the number of bacteria in the blood is low or when antibiotics have already been administered. Furthermore, some bacteria are difficult to detect in blood cultures. 16S ribosomal RNA (rRNA) contains conserved sequences that are targeted for PCR amplification using universal primers. We investigated whether the threshold cycle (Ct) value of 16S rRNA real-time PCR in whole-blood samples can be used for early diagnosis of bacteremia. Ct values of the 16S rRNA real-time PCR in 307 collected specimens showed a bimodal distribution. Ct values of the blood culture-positive group were significantly lower than those of the blood culture-negative group (P < 0.001). The cutoff value of the receiver operating characteristic curve was 38.80, as determined using finite-mixture modeling and expectation-maximization algorithm. Analysis of the diagnostic accuracy at this cutoff value showed a sensitivity of 91.4%, specificity of 33.5%, positive predictive value of 15.0%, and negative predictive value of 96.8%. The Ct value of 16S rRNA real-time PCR shows high negative predictive value, it may be useful for excluding bacteremia when the cutoff value is set appropriately

Long-term survival with RAS-associated autoimmune leukoproliferative disorder with somatic KRAS mutation:A case report

　RAS -associated autoimmune leukoproliferative disorder (RALD) is a recently reported rare nonmalignant autoimmune disorder. The characteristic clinical findings of RALD include monocytosis, leukocytosis, lymphoproliferation, and autoimmune phenomena. RALD is defined by somatic mutations in KRAS or NRAS . It is a new disease that was reported by Niemela and Takagi in 2011. The prognosis and incidence are currently unknown and the treatment strategy has not yet been established.　Here we describe the long-term survival of a patient with who displayed a somatic KRAS G12D mutation. His clinical features and labolatory data were overlapped with juvenile myelomonocytic leukemia and chronic myelomonocytic leukemia. Mercaptopurine hydrate, hydroxycarbamide and azacitizine were administered to control white blood cell count and improve clinical symptoms. He had a long survival time without hematopoietic stem cell transplantation

Real-time RT-PCR detection method for SARS-CoV-2

　Emerging in December, 2019, SARS-CoV-2 is spreading worldwide, endangering the citizens of the globe. The authors sought to develop a feasible real-time RT-PCR method to detect RNA of the virus. The fluorescent probe the authors designed proved its potency and utility, by detecting in vitro transcribed RNA sequence at an estimated concentration approximating a single copy per reaction, used along with 3 sets of primers that flank the probe sequence. Though not tested against actual viral RNA, or clinical specimens, this method can serve as another choice of rapid detection of the plague-causing virus

A protocol to assay Anti-Yersinia pseudotuberculosis-derived mitogen human IgG by ELISA

　Our department has been continuing a research on the diagnosis of Yersinia pseudotuberculosis infections. This pathogen manifests in multifarious forms, including systemic vasculitis resembling Kawasaki Disease. Recently, a previously established ELISA assay for this infection was transferred from its originator at National Center for Child Health and Development. However, the protocol had to be modified for a different facility and equipment. Described herein is the protocol, successfully adapted for use at our laboratory. This protocol is contributing to the aforementioned research by improving analysis throughput of our laboratory

A feasible protocol for identifying macrolide resistant mutations in the 23S rRNA domain V sequence of Mycoplasma pneumoniae

　Continuing a nationwide surveillance of pediatric Mycoplasma pneumoniae infections since 2008, our department has been analyzing macrolide-resistance conferring mutations in the pathogen’s 23S rRNA gene sequence. There are three target positions, approximately 600 bases apart. We had been reading these positions by amplifying the flanking sequences of these target positions in two different PCR reactions, followed by sequencing of each PCR product, independently. Recent advances have boosted the expected read length of Sanger sequencing using dye-terminators from ~500 bases to ~1 Kb. Owing to a constant demand to process tens of specimens, the authors sought to refine the protocols with an aim to reduce handling time, complexity, and cost. Hereby presented is our refined procedure that enhanced our laboratory’s capacity enormousl

RT-LAMP detection method for SARS-CoV-2

　The COVID-19 (coronavirus disease 2019) pandemic is still endangering the globe since its emergence in December 2019. The authors sought to develop a feasible method to detect RNA of the causative virus, SARS-CoV-2 (severe acute respiratory syndrome corona virus 2), using RT-LAMP (reverse transcription loop-mediated isothermal amplification). The primers designed by the authors successfully detected in vitro transcribed RNA sequence (a portion from the surface glycoprotein coding sequence) at an estimated concentration of 16 copy per 20 μL reaction when used with fluorescence detection combined with melting curve analysis; 16 copy per 25 μL reaction when used with a real-time turbidimeter or end-point visual confirmation by the naked eye or fluorescence under UV illumination. Though not tested against actual viral RNA or clinical specimens, this method can serve as another option for rapid detection of the plague-causing virus. Taking into account the feasibility of LAMP, this method may prove its utility especially in resource-limited conditions