Clinical features, management, and prognosis of Bacillus cereus sepsis in premature neonates.

This study aimed to investigate the clinical characteristics, management and prognosis of Bacillus cereus sepsis in premature neonates. The clinical information of 8 premature neonates with B cereus sepsis who were treated in Shanghai Children Hospital from January 2015 to December 2019 was retrospectively collected from the medical records and analyzed. The neurodevelopment related conditions were collected at follow up visits at corrected age of 6 months and 12 months. Five patients developed meningitis, and cerebral magnetic resonance image showed abnormal in 5 patients. After treatment with meropenem and vancomycin, 1 patient died, and 7 patients survived and were smoothly discharged. At follow up visits, 1 patient was diagnosed with hydrocephalus and showed severely delayed neurodevelopment, 2 patients had mild delayed neurodevelopment, and the neurodevelopment was basically normal in remaining 4 patients. B cereus infection can cause severe complications of central nervous system, and affect neurodevelopmental outcome. Antibiotic treatment with meropenem and vancomycin is proven to be effective. Refreshing the central catheters is helpful for the prevention of B cereus sepsis and cerebral magnetic resonance image may be employed for the prognosis assessment.

Generation of an induced pluripotent stem cell line (SHCDNi007-A) from a patient with pyruvate carboxylase deficiency carrying compound heterozygous (c.182 T > C/ c.2581G > A) variants in PC.

Pyruvate carboxylase (PC) deficiency (PCD), due to biallelic PC variants, is a rare inherited metabolic disease, which is characterized by seizures, global developmental delay, as well as lactic acidosis, and elevated plasma pyruvate and alanine levels in affected individuals. In the present study, a new induced pluripotent stem cell line (SHCDNi007-A) was generated from the peripheral blood mononuclear cells of a 2-month-old male infant with biallelic PC mutations c.(182 T > C;2581G > A), i.e. p.(Ile61Thr;Val861Met). This cell line is expected to facilitate the in vitro modeling of the disease pathophysiology and the development of future therapeutics for PCD.

Antifungal Drugs and Drug-Induced Liver Injury: A Real-World Study Leveraging the FDA Adverse Event Reporting System Database.

Aims: We aimed to estimate the risk of drug-induced liver injury (DILI) from various antifungal treatments with azoles and echinocandins causing in real-world practice. Methods: We performed disproportionality and Bayesian analyses based on data from the first quarter in 2004 to the third quarter in 2021 in the Food and Drug Administration Adverse Event Reporting System to characterize the signal differences of antifungal drugs-related DILI. We also compared the onset time and mortality differences of different antifungal agents. Results: A total of 2943 antifungal drugs-related DILI were identified. Affected patients tended to be aged >45 years (51.38%), with more males than females (49.03% vs. 38.09%). Antifungal drug-induced liver injury is most commonly reported with voriconazole (32.45%), fluconazole (19.37%), and itraconazole (14.51%). Almost all antifungal drugs were shown to be associated with DILI under disproportionality and Bayesian analyses. The intraclass analysis of correlation between different antifungal agents and DILI showed the following ranking: caspofungin (ROR = 6.12; 95%CI: 5.36-6.98) > anidulafungin (5.15; 3.69-7.18) > itraconazole (5.06; 4.58-5.60) > voriconazole (4.58; 4.29-4.90) > micafungin (4.53; 3.89-5.27) > posaconazole (3.99; 3.47-4.59) > fluconazole (3.19; 2.93-3.47) > ketoconazole (2.28; 1.96-2.64). The onset time of DILI was significantly different among different antifungal drugs (p < 0.0001), and anidulafungin result in the highest mortality rate (50.00%), while ketoconazole has the lowest mortality rate (9.60%). Conclusion: Based on the Food and Drug Administration Adverse Event Reporting System database, antifungal drugs are significantly associated with DILI, and itraconazole and voriconazole had the greatest risk of liver injury. Due to indication bias, more clinical studies are needed to confirm the safety of echinocandins.

Initial use of voriconazole positively affects outcome of Candida parapsilosis bloodstream infection: a retrospective analysis.

BACKGROUND: Concerns have arisen regarding the optimal antifungal regimen for Candida parapsilosis (C. parapsilosis) bloodstream infection (BSI) in view of its reduced sensitivity to fluconazole. METHODS: The clinical characteristics of 58 C. parapsilosis BSI newborns who received treatment between June 2014 to December 2018 in the Shanghai Children's Hospital were retrospectively analyzed. Based on the initial antifungal drugs, these patients were divided into fluconazole group (n=30) and voriconazole group (n=21). After 7-10-day treatment, the antifungal drugs were replaced if blood culture still showed positive. The clinical characteristics and therapeutic effects were compared between two groups. RESULTS: There were no significant differences in the clinical characteristics between two groups (P>0.05). The median time to a negative culture in the voriconazole group was 7 [interquartile range (IQR), 6-10] days, which was significantly shorter than in the fluconazole group [9 (IQR, 7-18.5) days; P=0.034]. The overall median time to a negative culture was 8 days. After 8-day antifungal therapy, in the voriconazole group and fluconazole group, negative culture was observed in 16 and 12 patients, respectively; the positive culture was noted in 5 and 16 patients, respectively; the effective rate was 76.1% and 40%, respectively, showing marked difference (χ2=6.535, P=0.011). None died in the voriconazole group, but 4 died in the fluconazole group. The median time of treatment for fungal sepsis in the voriconazole group was 22 (IQR, 20-26) days, which was significantly shorter than in the fluconazole group [32 (IQR, 23.5-40) days; P=0.000]. CONCLUSIONS: The initial clinical manifestations of C. parapsilosis BSI vary among individuals, and voriconazole is superior to fluconazole in the treatment of C. parapsilosis BSI.

Clinical effect and safety of continuous renal replacement therapy in the treatment of neonatal sepsis-related acute kidney injury.

BACKGROUND: Sepsis is the leading cause of acute kidney injury (AKI) in the neonatal intensive care unit (NICU). The aim of the study is to explore the efficacy and security of continuous renal replacement therapy (CRRT) in the treatment of neonatal sepsis-related AKI. METHOD: Totally12 sepsis-related AKI neonates treated with CRRT were hospitalized in the NICU of Shanghai Children's Hospital between November 2012 and November 2019, and the clinical data of these 12 cases were retrospectively analyzed. Renal function, acid-base balance, electrolytes, blood pressure and hemodynamics indexes were recorded before CRRT initiation, 12/24/48 h after CRRT initiation and at the end of CRRT respectively. The efficacy of CRRT was evaluated and the clinical outcome was observed in these 12 sepsis-related AKI neonates. Repeated measurement analysis of variance was used for statistical analysis of the data. RESULT: (1) Continuous veno-venous hemodialysis filtration (CVVHDF) was used in 12 cases of sepsis-related AKI neonates. There were 6 cases with oliguria, 3 cases with fluid overload (FO), 3 cases with septic shock. The duration of CRRT was 49 ~ 110 h, average (76.2 ± 23.5) h. (2) The blood pressure (BP) of 12 sepsis -related AKI neonates could reach the normal level (40-60 mmHg) 12 h after CRRT initiation, and the normal BP level could be maintained during the CRRT treatment. After 12 h CRRT, the blood pH value increased to the normal range (7.35 ~ 7.45). After 12 h CRRT treatment, the oxygenation index of 12sepsis-related AKI neonates could reach 200 mmHg. After 24 h CRRT treatment, it could rise to more than 300 mmHg. Serum potassium, serum urea nitrogen and serum creatinine levels decreased significantly 12 h after CRRT initiation, and reached the normal range 24 h after CRRT initiation. The urine volume significantly increased 24 h after CRRT initiation. (3) Venous catheterization was performed successfully in all sepsis-related AKI neonates. We observed 2 cases of thrombocytopenia, 1 case of obstruction and 1 case of hypotension in the course of CRRT. There were no complications such as hypothermia, hemorrhage, thrombosis and infection.11 neonates were cured and discharged. One neonate was treated with CRRT and passed through the oliguria stage of AKI, but died after the parents gave up the treatment. CONCLUSIONS: It is safe and effective to treat neonatal sepsis-related AKI with CRRT, which should be an effective measure for the treatment of sepsis-related AKI neonates.

[Identification of a homozygous ASS1 mutation in a child with citrullinemia type â with high-melting curve method].

OBJECTIVE: To carry out rapid genetic diagnosis for a child affected with citrullinemia type Ⅰ. METHODS: Peripheral venous blood samples were obtained from the two-day-old child and his parents as well as 100 healthy controls. Serum ammonia and citrulline was determined by biochemical test and tandem mass spectrometry. Sixteen pairs of primers were designed for high-resolution melting (HRM) analysis of all exons and adjacent intronic sequences of the ASS1 gene in the proband, parents and healthy controls. Suspected mutations were confirmed by DNA sequencing, while the mRNA transcripts of the ASS1 gene were determined by reverse transcription (RT)-PCR. Functional impact of the mutation sites was predicted with PolyPhen-2 and SIFT Blink software. RESULTS: Blood ammonia and citrulline of the proband have respectively reached 286 µmol/L and 487.69 µmol/L, which far superseded the normal values. HRM analysis and DNA sequencing have identified in the child a homozygous c.380G>A (p.R127Q) mutation in exon 6 of the ASS1 gene, in addition with a homozygous IVS8+60G>A substitution in intron 8, while his parents were heterozygous carriers for both mutations. RT-PCR assay indicated that the IVS8+60G>A mutation did not result in abnormal splicing of the ASS1 gene transcripts. Bioinformatic analysis suggested that the site for p.R127Q was conserved among 45 species of vertebrates and may play a crucial role in citrulline metabolism. CONCLUSION: The severe urea cycle disorder in the proband was probably due to the compound homozygous R127Q and IVS8+60G>A mutations of the ASS1 gene.

Renal proteomic analysis of RGC-32 knockout mice reveals the potential mechanism of RGC-32 in regulating cell cycle.

This study aimed to investigate the exact function of RGC-32 in kidney diseases and explore the potential mechanism of RGC-32 in regulating cell cycle. RGC-32 knockout (RGC-32-/-) mice were generated from C57BL/6 embryonic stem cells. Differentially expressed proteins in the kidney were investigated with the isobaric tags for relative and absolute quantification (iTRAQ) technique. Gene ontology analyses (GO), Kyoto encyclopedia of genes and genomes (KEGG) pathway mapping analysis and functional network analysis were also performed. The expressions of Smc3, Smad 2-3, DNA-PK were further confirmed by qPCR. Results showed that 4690 proteins were quantified on the basis of 25165 unique peptides. Comparative proteomic analysis revealed 361 differentially expressed proteins in RGC-32-/- mice (knockout/wild ratio >+/- 1.2 and P<0.05). GO and KEGG pathway mapping analyses showed differentially expressed proteins were involved in spliceosome, fluid shear stress and atherosclerosis protein processing in endoplasmic reticulum, pathways in cancer, viral carcinogenesis, epithelial cell signaling in Helicobacter pylori infection, HTLV-I infection, PI3K-Akt signaling pathway, ubiquitin mediated proteolysis, Parkinson's disease, MAPK signaling pathway, carbon metabolism, Alzheimer's disease, NOD-like receptor signaling pathway, tight junction, Proteoglycans in cancer, phagosome, ribosome, mTOR signaling pathway, and AMPK signaling pathway. Differentially expressed proteins Smc3 (0.821), DNA-PK (0.761), Smad 2-3 (0.631) were involved in cell cycle regulation. mRNA expression of Smad2-3, DNA-PK, and Smc3 was consistent with that from iTRAQ. It is concluded that RGC-32 may affect the expression of many proteins (76 up-regulated and 285 down-regulated) in the kidney, and may regulate the expression of Smc3, DNA-PK and Smad 2-3 to affect the cell cycle.

P53 inhibitor pifithrin-α prevents the renal tubular epithelial cells against injury.

The injury and repair of renal tubular epithelial cells play an important role in the pathological process of acute kidney injury (AKI). This study aimed to clarify the role of cell cycle change in renal tubular epithelial cell injury and repair in vivo and in vitro. Sprague-Dawley rats received bilateral renal pedicle clamping for 45 min (ischemia) followed by reperfusion. Pifithrin-α, a p53 inhibitor, was administered at 24 h before renal ischemia and 3 and 14 days after reperfusion. Results showed the tubular epithelial cells in M phase increased significantly at 2 h to 72 h after ischemia/reperfusion (I/R), while pifithrin-α decreased them. Renal I/R caused renal tubular epithelial damage in rats, which was improved by pifithrin-α. The α-SMA mRNA expression was up-regulated significantly after I/R, while it was down-regulated by pifithrin-α.NRK-52E cells were cultured in vitro, cell damage was induced by addition of TNF-α, and then cells were treated with pifithrin-α. Cells treated with TNF-α alone in G2/M phase increased significantly, but they were reduced in the presence of pifithrin-α. In NRK-52E cells treated with pifithrin-α for 6 h, NGAL mRNA expression was significantly lower than in cells without pifithrin-α treatment. After NRK-52E cells were treated with pifithrin-α for 24 h, α-SMA and FN mRNA expression was significantly lower than in cells without the treatment. In summary, pifithrin-α can facilitate the progression of renal tubular epithelial cells through G2/M phase, protecting them against injury.

The expression of response gene to complement 32 on renal ischemia reperfusion injury in rat.

To investigate the expression of response gene to complement 32 (RGC32) in rat with acute kidney injury (AKI) and to explore the role of RGC32 in renal injury and repair induced by ischemia reperfusion. Rats were randomly divided into two groups, including sham operation group (n = 48) and acute ischemia reperfusion injury (IRI) group (n = 48). Rats were sacrificed following reperfusion 2 h, 6 h, 24 h, 48 h, 72 h, 1 week (w), 2 w, and 4 w. The distribution and expression of RGC32 in renal tissue were observed by means of immunohistochemistry. The mean density of the images detected by Image-Pro Plus 6 was designated as the representative RGC32 expression levels. Meanwhile, RGC32 mRNA expression was measured by qPCR. RGC32 mainly expressed in cytoplasm of proximal tubular epithelial cells. However, RGC32 did not express in renal interstitium and vessels. The expression levels of RGC32 measured by immunohistochemistry at different reperfusion time were 0.0168 ± 0.0029, 0.0156 ± 0.0021, 0.0065 ± 0.0013, 0.0075 ± 0.0013, 0.0096 ± 0.0014, 0.0132 ± 0.0016, 0.0169 ± 0.0014, 0.0179 ± 0.0022, respectively. Compared with the sham group, the level of RGC32 expression in IRI group was significant lower at 24 h, 48 h, 72 h after IRI (p < 0.05). The expression levels of RGC32 mRNA at different reperfusion time measured by qPCR were corroborated the immunohistochemistry finding. The in vitro experiments show the expression of α-SMA and extracellular matrix expression increased signification when the RGC32 was silenced. Our data showed that the RGC32 expression in AKI rat decreased significantly reduces with different reperfusion time and performs a time-dependent manner. RGC32 may play an important role in the pathogenesis of AKI following IRI and repair in rat.

Response gene to complement 32 regulates the G2/M phase checkpoint during renal tubular epithelial cell repair.

BACKGROUND: The aim of this study was to evaluate the influence of RGC-32 (response gene to complement 32) on cell cycle progression in renal tubular epithelial cell injury. METHODS: NRK-52E cells with overexpressed or silenced RGC-32 were constructed via transient transfection with RGC-32 expression plasmid and RGC-32 siRNA plasmid, and the cell cycle distribution was determined. The expression levels of fibrosis factors, including smooth muscle action (α-SMA), fibronectin (FN) and E-cadherin, were assessed in cells with silenced RGC-32. RESULTS: The cells were injured via TNF-α treatment, and the injury was detectable by the enhanced expression of neutrophil gelatinase-associated lipocalin (NGAL). RGC-32 expression also increased significantly. The number of cells at G2/M phase increased dramatically in RGC-32 silenced cells, indicating that RGC-32 silencing induced G2/M arrest. In addition, after treatment with TNF-α, the NRK-52E cells with silenced RGC-32 showed significantly increased expression of α-SMA and FN, but decreased expression of E-cadherin. CONCLUSIONS: The results of this study suggest that RGC-32 probably has an important impact on the repair process of renal tubular epithelial cells in vitro by regulating the G2/M phase checkpoint, cell fibrosis and cell adhesion. However, the exact mechanism needs to be further elucidated.

[Maple syrup urine disease caused by two novel BCKDHB gene mutations in a Chinese neonate].

OBJECTIVE: Maple syrup urine disease (MSUD) is an autosomal recessive metabolic disorder that is caused by mutations in the subunits of the branched chain α-ketoacid dehydrogenase (BCKD) complex. This report presents a Han ethnic Chinese newborn infant with the severe classic form of MSUD caused by two novel missense mutations in the BCKDHB gene. METHOD: The clinical and biochemical data of a Chinese neonate with classic form of MSUD were analyzed, and the DNA sequences of BCKDHA, BCKDHB, DBT and DLD genes were investigated for mutations. Then the DNA samples of the proband and the patient's parents were tested with Sanger sequencing. RESULT: The manifestations of this patient were poor feeding, low reaction, and compensatory metabolic acidosis. Tandem mass spectrometry (MS/MS) showed that leucine and valine were significantly higher than normal. Urine gas chromatography-mass spectrometry (GC/MS) showed significant abnormality. Brain CT scan showed white matter changes. We identified two previously unreported mutations in the BCKDHB gene, p.Leu194Phe (c.580 C>T) and p.Ser199Arg (c.597 T>G) in exon 5. Segregation analysis showed that the novel mutation p.Ser199Arg was maternally inherited and the novel mutation p.Leu194Phe was paternally inherited. Neither mutation was found in the 186 alleles of 93 normal Han ethnic Chinese individuals. In human BCKDHB protein crystal structure, the 194th and 199th amino acids changes are likely to affect the spatial structure of the protein. The 194th and 199th amino acid of human BCKDHB protein was conserved among species. PolyPhen protein function prediction indicated that the 194th and 199th amino acid changes were likely to affect protein function. CONCLUSION: Two novel missense mutations were identified in the BCKDHB gene in the Chinese patient with MSUD.

[Effects of response gene to complement 32 as a new biomarker in children with acute kidney injury].

OBJECTIVE: To investigate the new biomarkers of acute kidney injury, as well as to confirm the values of response gene to complement 32 (RGC-32) for early diagnosis of acute kidney injury by comparing the values of serum creatinine (Scr) and cystatin C (CysC) in children who had undergone cardiopulmonary bypass (CPB). METHOD: Sixty-seven patients who had accepted CPB were recruited from the cardiac surgery intensive care unit, Children's Hospital Affiliated to Shanghai Jiao Tong University from March to June 2013 and assigned to acute kidney injury group (group AKI) or non-acute kidney injury group (group non-AKI), on the basis of the definition by the pediatric RIFLE (pRIFLE) criteria. Also 30 healthy control children were recruited. Serum samples were taken regularly from each patient after CPB at 30 min, 2 h, 4 h, 24 h, 48 h and 72 h for RGC-32. Serum samples were tested by enzyme linked immunosorbent assay (ELISA) which was employed to determine the levels of serum RGC-32. Scr and CysC were analyzed by HITACHI 7180 automatic biochemical analyzer. All the data were analyzed by receiver operator characteristic curve (ROC) and area under curve (AUC). RESULT: The incidence of AKI was 34% (23/67), including 15 cases with risk stage AKI, 4 cases with injury stage AKI, 3 cases with failure stage AKI, 1 cases with loss stage AKI. Three out of four subjects with Failure stage AKI and the one case with Loss stage all accepted renal replacement therapy. CPB group had a higher level of serum RGC-32 than that of pre-operation after CPB 30 minute [(2.88 ± 0.68) µg/L vs. (1.39 ± 0.31) µg/L, P < 0.05]. At the same time, comparing with the non-AKI group, the levels of serum RGC-32 were higher than that of controls 30 min, 2 h, 4 h, 24 h and 48 h after CPB (t = 2.560, 2.180, 2.818, 2.226, 3.017; P < 0.05). The values for the AUC were determined for RGC-32 as 0.770, 0.707, 0.768, 0.728,0.723 and 0.770 after CPB 30 min, 2 h, 4 h, 24 h, 48 h and 72 h. The values for sensitivity of serum RGC-32 30 min, 2 h and 4 h after CPB was 0.914, 0.824, 0.824 and the values for specificity of serum RGC-32 was 0.619, 0.667, 0.810, respectively. But the values for sensitivity of CysC was 0.625, 0.813, 0.813, and specificity 0.571, 0.619, 0.571, respectively. The values for sensitivity of Scr was 0.625, 0.625, 0.813 and specificity was 0.571, 0.571, 0.524, respectively. CONCLUSION: The sensitivity of serum RGC-32 for detecting AKI was much higher than that of Scr and serum CysC in children who had accepted CPB, and that RGC-32 may be a new biomarker for early detection of AKI. However, the conclusion needs to be further elucidated.

[Rapid screening for MTHFR gene 677C>T polymorphism in Down syndrome using high resolution melting curve and pyrosequencing].

OBJECTIVE: To establish a rapid method for detecting MTHFR gene 677C>T polymorphisms with high-resolution melting curve method (HRM) and pyrosequencing. METHODS: Peripheral blood samples were collected from 155 Down syndrome patients and 182 normal controls from Children's Hospital of Shanghai. The accuracy of three methods including regular HRM, internal control HRM and artificial heterozygosity HRM was compared. Meanwhile, allele frequencies in 10, 30 and 50 mixed samples were measured with pyrosequencing, and the results were compared with that of HRM. RESULTS: Heterozygosity of 677C>T polymorphism could be distinguished by various HRM methods. However, homozygotes CC and TT were only identifiable by internal control HRM and artificial heterozygosity HRM. The accuracy of pyrosequencing for allele frequency has improved with increased sample number. When the number of mixed samples has exceeded 30, the difference between pyrosequencing results and actual values became less than 4%. TT genotype was more frequent in Down syndrome patients than controls (25.2% vs. 14.3%). No significant difference was found in T allele frequency between the two groups (44.9% vs. 40.1%). CONCLUSION: Respectively, internal control HRM and pyrosequencing may be ideal methods for determination of genotypic and allelic frequencies.