Objective:To establish and optimize a novel method, focus forming assay (FFA), for quantitation of influenza A virus (FluA) and compare its application performance with traditional plague forming assay (PFA).Methods:The foci chromogenic effects of three peroxidase substrates in immunostaining were compared. The PFA and FFA methods were used to explore FluA incubation times and plaque morphology on 12-well plates, and to determine optimal incubation times and virus adsorption volumes for different FluA subtypes on 96-well plates. The correlation between FFA and PFA was evaluated, and the optimized FFA was applied to the in vitro antiviral efficacy analysis of Favipiravir and neutralization test against different subtypes of FluA. Results:TRUEBLUE substrate was identified as the optimal substrate for foci visualization. Compared with the PFA, the FFA showed improved sensitivity and reduced detection time in FluA titration, and good correlation was shown between the two methods′ results. By replacing the 96-well plate with the 12-well plate for FFA titration of different subtypes of FluA, the detection time was shortened, and the amount of serum samples used could be further reduced by optimizing the virus adsorption volume. The half-maximal effective concentration of favipiravir against influenza viruses assessed by the FFA and PFA methods showed no significant difference, and was consistent with the results obtained from quantitative PCR. Additionally, the focus reduction neutralization test and hemagglutination inhibition assays demonstrated strong correlation in determining antibody titers against FluA in serum neutralization assays.Conclusions:The improved FFA method developed here provides a more efficient experimental tool for FluA titration, antiviral drug screening and broad-spectrum vaccine evaluation.

Objective:To explore the application value of three-dimensional high-resolution anorectal manometry(3D-HRAM)model in the diagnosis and localization of anal sphincter injury after surgery for complex anal fistula.Methods:A prospective study was con-ducted on 92 patients who had undergone surgery for complex anal fistula in the Department of Coloproctology of The People's Hospi-tal Affiliated to Fujian University of Traditional Chinese Medicine from January 2023 to January 2024.The study was conducted in two stages.In the first stage,92 patients underwent 3D-HRAM and endoanal ultrasonography(EAUS)to examine the sphincter,and the data were compared with intraoperative records to evaluate the diagnostic efficiency and localization accuracy of 3D-HRAM and EAUS.In the second stage,92 patients who had been examined were grouped according to the presence or absence of symptoms of anal dysfunction to further verify the diagnostic and localization values of 3D-HRAM and EAUS for postoperative compensated/decompen-sated sphincter injury.Results:Among 92 patients,41 and 57 patients were diagnosed with sphincter injury through 3D-HRAM and EAUS,with a sensitivity of 61.19%and 85.07%,a specificity of 88%and 76%,an area under the curve of 0.739 and 0.813 calcu-lated by plotting the receiver operating characteristic curve,and a localization accuracy of 44.77%and 71.64%,respectively,showing significant differences between 3D-HRAM and EAUS(P=0.002),indicating that 3D-HRAM had lower diagnostic efficiency and local-ization accuracy than EAUS for anal sphincter injury after surgery for complex anal fistula.After grouping by symptoms,the diagnostic efficiency and localization accuracy of 3D-HRAM for symptomatic sphincter injury(decompensated)were significantly better than those for asymptomatic sphincter injury,and were similar to those of EAUS(P=0.763).Conclusion:The application advantage of 3D-HRAM lies in the diagnosis and localization of decompensated anal sphincter injury after surgery for complex anal fistula,which provides effective technical support for the study of anal defecation control mechanisms and injury compensation mechanisms.

Objective:To establish and optimize a novel method, focus forming assay (FFA), for quantitation of influenza A virus (FluA) and compare its application performance with traditional plague forming assay (PFA).Methods:The foci chromogenic effects of three peroxidase substrates in immunostaining were compared. The PFA and FFA methods were used to explore FluA incubation times and plaque morphology on 12-well plates, and to determine optimal incubation times and virus adsorption volumes for different FluA subtypes on 96-well plates. The correlation between FFA and PFA was evaluated, and the optimized FFA was applied to the in vitro antiviral efficacy analysis of Favipiravir and neutralization test against different subtypes of FluA. Results:TRUEBLUE substrate was identified as the optimal substrate for foci visualization. Compared with the PFA, the FFA showed improved sensitivity and reduced detection time in FluA titration, and good correlation was shown between the two methods′ results. By replacing the 96-well plate with the 12-well plate for FFA titration of different subtypes of FluA, the detection time was shortened, and the amount of serum samples used could be further reduced by optimizing the virus adsorption volume. The half-maximal effective concentration of favipiravir against influenza viruses assessed by the FFA and PFA methods showed no significant difference, and was consistent with the results obtained from quantitative PCR. Additionally, the focus reduction neutralization test and hemagglutination inhibition assays demonstrated strong correlation in determining antibody titers against FluA in serum neutralization assays.Conclusions:The improved FFA method developed here provides a more efficient experimental tool for FluA titration, antiviral drug screening and broad-spectrum vaccine evaluation.

Objective:To construct a novel respiratory syncytial virus (RSV) vaccine based on a recombinant influenza virus vector and evaluate its immune protective effects in mice.Methods:A recombinant H1N1 influenza A virus (IAV) expressing the extracellular domain (Gecto) of RSV A2 G protein was constructed and rescued, named as PR8NAGecto/WSN. After in vitro verification of the Gecto expression and PR8NAGecto/WSN growth kinetics, a single dose of PR8NAGecto/WSN was used to immunize BALB/c mice through intranasal administration to evaluate the efficacy of PR8NAGecto/WSN by assessing humoral (IgG, neutralizing antibody), mucosal (IgA) and cellular immunity (IFN-γ ELISPOT). Four weeks after immunization, the mice were challenged with RSV A2 or RSV B9320 to evaluate the protective effects of PR8NAGecto/WSN by analyzing mouse body weight changes, lung tissue virus titers and pathological changes. Results:A single-dose intranasal immunization with PR8NAGecto/WSN induced robust humoral, mucosal and cellular immunity in mice. Moreover, the mice in the immunized group had lower lung virus loads and mild lung pathological damages following the challenge with RSV A or RSV B subtype as compared with the control group.Conclusions:A single-dose intranasal immunization with PR8NAGecto/WSN induces robust immunity and provide protection against RSV A and B challenges in mice. This study provides new ideas and reference for the development of novel mucosal vaccines against RSV.

Objective:To explore the potential genetic causes of unexplained neonatal encephalopathy.Methods:This retrospective study enrolled 113 infants diagnosed with unexplained neonatal encephalopathy and underwent genetic testing in the Children's Hospital of Hunan Province from January 2019 to May 2021. Perinatal data, clinical manifestations, electroencephalograph, brain MRI findings, genetic information, and prognosis of those patients were analyzed. T-test or Chi-square test were used for data analysis. Results:Of the 113 infants enrolled, 74 (65.5%) were males. The gestational age at birth was (38.6±1.5) weeks, and the birth weight was (2 957±561) g. The most common clinical manifestation was the disturbance of consciousness (83/113, 73.5%), followed by seizures (39/113, 34.5%). There were 38.2% (34/89) of the patients with abnormal brain MRI, and 80.4% (74/92) presented abnormal electroencephalography. Among the 113 infants, 60 (53.1%) had genetic abnormalities, including 48 with single nucleotide variations, eight with copy number variations, and four with chromosome abnormalities. Single nucleotide variations in the 48 patients were classified into syndromic ( n=18, 37.5%), metabolic ( n=16, 33.3%), epileptic ( n=11, 22.9%) and mitochondrial-related genes ( n=3, 6.3%), of which 14 were not included in any database. Among the 103 cases which were successfully followed up until December 31, 2021, 75 (72.8%) had a poor prognosis, including 52 (50.5%) death cases and 23 (22.3%) cases of development retardation. Birth weight and the incidence of seizures in the poor prognosis group were both lower than those in the non-poor prognosis group [(2 876±536) vs (3 254±554) g, t=3.15; 29.3% (22/75) vs 53.6% (15/28), χ2=5.20; both P<0.05], while the incidence of disturbance of consciousness was higher [80.0% (60/75) vs 53.6% (15/28), χ2=7.19, P<0.05]. The proportion of infants with genetic abnormalities in the poor prognosis group was higher than that in the non-poor prognosis group, but the difference was not statistically significant [53.3% (40/75) vs 46.4% (13/28), χ2=0.39, P=0.533]. Conclusions:Genetic abnormality is one of the leading causes of unexplained neonatal encephalopathy. Nucleotide variation is the most common genetic type. Syndromic, metabolic, and epileptic variants are frequently detected in these patients.

Objective:To summarize the situation of dead newborns and their parents after parents gave up treatment, and analyze the reasons and emotional needs of parents who gave up treatment, so as to provide reference for reducing neonatal mortality and negative emotions of parents.Methods:A retrospective study was conducted to collect the data of neonates and mothers who died after giving up treatment reported in Hunan Children′s Hospital from January 2019 to December 2021. The general information, perinatal risk factors, and the incidence of in-hospital diseases were analyzed. Then, semi-structured interviews were conducted with parents of newborns who died after giving up treatment from February to December 2021. Understand why parents give up treatment and their emotional needs.Results:A total of 172 newborns died after giving up were included in the analysis, including 103 males (59.88%) and 74 premature infants (43.02%); Umbilical cord, placenta and amniotic fluid abnormalities were 21 cases (12.21%), 39 cases (22.67%) and 25 cases (14.53%), respectively. Birth asphyxia was 31 cases (18.02%), including severe asphyxia in 18 cases (10.46%); There were 21 (12.21%), 35 (20.35%) and 30 (17.44%) cases of maternal infection in the third trimester, hypertension in pregnancy and diabetes in pregnancy, respectively. The top three causes of death were septicemia (18.02%), congenital malformation (16.86%) and severe pneumonia (10.47%). The main reason why parents give up treatment was that the child′s disease was critical and irreversible, and parents had strong emotional needs for hospice care in their hearts.Conclusions:There are many high risk factors of perinatal death of newborns after giving up treatment. Sepsis is the primary cause of death, and strengthening perinatal health care is fundamental. Parents have a strong demand for hospice care, so it is of practical significance to implement family-centered hospice care model for such special newborns.

Objective:To explore the relationship between the neural development of preterm infants and gut microbiota.Methods:66 premature infants who were hospitalized in the Neonatology Department of Hunan Children′s Hospital from September 2018 to September 2019 were included in the study. Their fecal samples and clinical data from the first admission were collected. According to the neurodevelopment, the patients were divided into normal neurodevelopment group and neurodysplasia group. The bacterial DNA of fecal samples was extracted by 16S rDNA high-throughput sequencing technology and bioinformatics analysis was conducted to compare the composition and diversity of gut microbiota between the two groups.Results:(1) The Shannon index of gut microbiota in normal neurodevelopmental group and neurodysplastic group was 0.89(0.41, 1.51) and 1.01(0.47, 1.31), respectively. There was no significant difference in diversity index between the two groups ( P>0.05). (2) Bifidobacterium, veronica and negativites in the gut microbiota of the normal neurodevelopmental group were significantly higher (all P<0.05), and streptococcus in the gut microbiota of the dysplastic group were significantly higher ( P<0.05). The gut microbiota of the two groups were mainly enterococcus and escherichia shigella. Conclusions:At the genus level, enterococcus and escherichia are the dominant flora of early gut microbiota in preterm infants. Gut microbiota is related to the neural development of preterm infants. The increased abundance of streptococcus, and the decreased abundance of bifidobacterium, veronicus, and negativites may be risk factors for neurodysplasia of preterm infants. The diversity of gut microbiota in early preterm infants may not be significantly related to neural development.

Objective:To investigate the clinical characteristics, treatment and prognosis of primary vitreoretinal lymphoma (PVRL) diagnosed and treated in our hospital during the past 10 years.Methods:A retrospective clinical study. From 2011 to 2021, 126 eyes of 67 patients with PVRL who were diagnosed and treated in Department of Ophthalmology, Eye-ENT Hospital, Fudan University were included in the study. Among them, there were 23 males (34.3%, 23/67) and 44 females (65.7%, 44/67); the average age was 57.1 years. There were 59 cases with both eyes (88.1%, 59/67) and 8 cases with one eye (11.9%, 8/67). At the initial eye diagnosis, 22 cases had a clear history of primary central nervous system lymphoma (PCNSL); 5 cases were found to have intracranial lesions by head imaging examination; 40 cases had no central nervous system involvement. Twenty cases were treated with glucocorticoids due to misdiagnosed uveitis. All patients received intravitreal injection of methotrexate (IVM) treatment. The treatment regimen was twice a week in the induction period for 2 weeks, once a week in the consolidation period for 1 month, and once a month in the maintenance period. Patients with PCNSL or both eyes received concurrent systemic chemotherapy (chemotherapy), and some in combination with radiation therapy to the brain (radiotherapy). The mean follow-up time was 39.3 months. The clinical manifestations, treatment and prognosis of the patients were retrospectively analyzed. The visual acuity before and after treatment was compared by t test. Results:Among the 22 cases with a clear history of PCNSL at the initial eye diagnosis, the average time from intracranial diagnosis to eye diagnosis was 22.9 months. Among the 40 cases without central nervous system involvement at first, 14 cases (20.9%, 14/67) developed central nervous system lesions during follow-up period. The mean time from ocular diagnosis to intracranial diagnosis was 9.9 months. Among the 126 eyes, 42 eyes (33.3%, 42/126) had anterior segment inflammation. vitreous inflammation type, retinal type, and vitreous retinal type were 58 (46.0%, 58/126), 7 (5.6%, 7/126), and 61 (48.4%, 61/126) eyes, and 9 of them (7.1%, 9/126) had optic nerve involvement at the same time. Patients received an average of 12 IVM treatments. IVM combined with systemic chemotherapy in 59 cases (88.1%, 59/67), of which 16 cases were combined with brain radiotherapy. All patients achieved complete remission after completing the treatment cycle (100.0%, 67/67). After treatment, 21 eyes (16.7%, 21/126) had ocular recurrence; 22 (32.8%, 22/67) had intracranial recurrence; 8 cases (11.9%, 8/67) died. The mean progression-free survival of patients was 23.7 months; the mean survival time was 43.6 months; the 5-year overall survival rate was 72.5%.Conclusions:The manifestations of PVRL are complex and diverse, and most of them are accompanied by involvement of the central nervous system. It can be divided into vitreitis type, retinal type and vitreoretinal type, and the optic nerve can be involved at the same time; IVM combined with systemic treatment can completely relieve the disease.

Objective:To compare the characteristics and differences of intestinal flora in premature infants with late-onset sepsis (LOS) and pneumonia by high-throughput sequencing technology, and to investigate the relationship between intestinal flora and LOS.Methods:Through the case-control method, premature infants with late-onset sepsis who were hospitalized in the neonatal department of Hunan Children′s Hospital from August 2018 to October 2019 were selected as the case group ( n=8). At the same time, premature infants diagnosed with pneumonia were selected as the control group ( n=8). The fecal samples of 16 premature infants were collected for the first time, and the DNA was extracted. The DNA of the target region was amplified by polymerase chain reaction(PCR). High-throughput sequencing was performed using NovaSeq 6000 platform to analyze the composition and diversity of intestinal flora between the two groups. Results:(1) Alpha diversity analysis showed that there was no significant difference in the richness and diversity of intestinal flora between the two groups(all P>0.05). (2) The intestinal flora in premature infants of LOS group and control group were dominated by Firmicutes and Proteobacteria, and facultative anaerobes such as Enterococcus and Escherichia-Shigella were the dominant flora at the genus level. Metastas statistical analysis showed that there was no statistically significant difference in flora composition between the two groups at the phylum level ( P>0.05). (3) Metastas statistical analysis was carried out at the level of class, order, family, genus, and species. The relative abundance of actinomycetes, digestive streptococcaceae and Clostridium in LOS group was higher than that in pneumonia group, and the difference was statistically significant (all P<0.05). (4) The relative abundance of Staphylococcus in the LOS group was significantly greater than that in the control group, but Metastas statistical analysis showed that there was no statistically significant difference in the relative abundance of staphylococcus between the two groups ( P>0.05). (5) Among the 8 cases of LOS, 3 premature infants had positive blood cultures, namely Streptococcus agalactiae, Streptococcus mitis, and Enterococcus faecalis. Enterococcus faecalis belongs to the genus Enterococcus, and Enterococcus belongs to the dominant genus in the LOS group. Conclusions:Different site infections have effects on intestinal microecology of premature infants. There were differences in intestinal flora composition between premature infants with LOS and premature infants with pneumonia.

Objective:To study the protective effect of polygalasaponin F (PGSF) on isoproterenol (ISO)-induced myocardial ischemia (MI) injury in neonatal rats and its possible mechanism.Methods:Fifty newborn Sprague Dawley (SD) rats were randomly divided into control group, model group, low, medium and high dose PGSF groups (5, 10, 50 mg/kg), with 10 rats in each group. The rats in the control group were treated with normal saline; Myocardial ischemia (MI) model was established in model group by subcutaneous injection of isoproterenol (ISO, 85 mg/kg, once a day); The MI model was established in rats of low, medium and high dose PGSF group after intraperitoneal injection of 5, 10 and 50 mg/kg PGSF for 7 days. The cardiac function of rats in each group was evaluated by echocardiography; pathological changes of myocardial tissue of rats in each group were observed by hematoxylin and eosin (HE) staining; The serum activities of troponin I (cTnI), creatine kinase isoenzyme (CK-MB), myoglobin (Mb) and lactate dehydrogenase (LDH) of rats in each group were detected by enzyme linked immunosorbent assay (ELISA); the content of malondialdehyde (MDA) and active oxygen species (ROS) in myocardial tissue were detected ; the expression of nuclear proliferation antigen (Ki67) and caspase-3 protein in myocardial tissue was detected by immunohistochemical staining; The expression of protein kinase B (AKT) and nuclear factor erythroid 2-related factor 2 (Nrf2) protein in myocardium was detected by Western blot.Results:In the model group, the myocardial structure was disordered, the cells were congested and swollen, and there were a lot of inflammatory cells infiltrating and large necrotic foci. The left ventricular wall thickness (LVWT), left ventricular ejection fraction (LVEF), fractional shortening (FS), heart rate (HR) and expression of Ki67 positive protein in the model group were lower than those in the control group (all P<0.05), while the left ventricular end systolic volume (LVESV), activities of cTnI, CK-MB, Mb, LDH in serum, content of ROS and MDA in myocardial tissue and caspase-3 positive protein in the model group were higher than those in the control group (all P<0.05). Compared with the model group, the degree of myocardial pathological changes in neonatal rats of the low, medium and high dose PGSF groups gradually decreased. Compared with model group, the LVWT, LVEF, FS, HR and expression of Ki67 positive protein increased in low, medium and high dose PGSF groups (all P<0.05), while the LVESV, activities of cTnI, CK-MB, Mb and LDH in serum, content of ROS and MDA in myocardial tissue and the expression of caspase-3 positive protein decreased (all P<0.05); Western blot results showed that the relative expression of phosphorylated(p)-AKT/AKT, p-Nrf2/Nrf2 protein in myocardium of model group was lower than that of control group (all P<0.05); The relative expression of p-AKT/AKT, p-Nrf2/Nrf2 protein in myocardium of low, medium and high dose PGSF groups were higher than that in the model group (all P<0.05). Conclusions:PGSF has protective effect on MI injury in neonatal rats, and its mechanism may be related to anti-apoptosis and anti-oxidative stress.