Objective:To investigate the effects of cryopreservation on the results of nucleic acid detection and culture for Bordetella pertussis in residual culture-positive nasopharyngeal swab specimens, aiming to provide the basis for specimens preservation, transport and centralized detection. Methods:In this cross-sectional study, the residual nasopharyngeal swab specimens which were culture-positive for Bordetella pertussis were collected in the Children′s Hospital, Zhejiang University School of Medicine from January to August in 2022. The specimens were placed at ?20 ℃ and?70 ℃ by random number table method, respectively. Re-detection by culture and PCR for Bordetella pertussis were conducted after these specimens were frozen for 114.3±31.9 days. The specimens were grouped according to the cryopreservation temperature and the semi-quantitative results by bacteria culture. The positive rates of the results were compared with χ 2 test between groups. Results:A total of 244 nasopharyngeal swabs specimens were included and 166 were culture-positive after cryopreservation, the positive rate decreased by 32%. Among them, the positive rate of re-culture of specimens containing low bacterial loads decreased by 56% after cryopreservation. However, there was no significant difference in the positive rate of culture between the specimens freezing at ?70 ℃ and ?20 ℃ (χ2=1.65, P=0.20). The positive rate of DNA detection decreased by 10.6% (88.9% vs 78.3%) after cryopreservation. The positive rate of the ?70 ℃ storage group was significantly higher than that of the ?20 ℃ group (χ2=5.11, P=0.02). The positive rate of the re-detection of DNA of nasopharyngeal swabs with low bacteria loads in ?70 ℃ storage group was significantly higher than that of the ?20 ℃ group (χ2=4.86, P=0.03). While for the samples with a bacterial load of "+" or more, there was no significant difference in the positive rate of DNA detection after cryopreservation between the ?20 ℃ and -70 ℃ (χ2=1.25, P=0.26) groups. The positive rate of nasopharyngeal swab culture after cryopreservation was 68.0% (166/244), which was significantly lower than the DNA detection positive rate of 78.3% (191/244, χ2=6.52, P=0.01). Conclusions:Cryopreservation nasopharyngeal swabs specimens could be used for Bordetella pertussis culture and nucleic acid detection. The bacterial load in the original sample affects the positive detection rate after cryopreservation. Cryopreservation has less influence on the positive rate of the result of nucleic acid detection when compared with culture. Preservation at ?70 ℃ is superior to ?20 ℃.

Objective:To analyze the biological characteristics and genomic features of a Streptococcus pyogenes strain isolated from the pus of a child with an atypical brain abscess. Methods:The Streptococcus pyogenes strain, named 21SPY7071 ( emm22 type), was isolated from the brain abscess specimen of a child undergoing surgery for brain abscess. The biological characteristics of this strain were analyzed using blood agar culture, Gram staining, growth curve measurement, and hemolytic analysis. After whole-genome sequencing, core genome multilocus sequence typing (cgMLST) was performed to analyze the cgST type of the strain, and the distribution of virulence genes and drug resistance genes were analyzed. qRT-PCR was used to detect the differences in the expression of virulence genes at mRNA level between the strain 21SPY7071 and two Streptococcus pyogene strains of ATCC 19615 ( emm80 type) and GAS029 ( emm22 type, clinical isolate). Results:The strain 21SPY7071 was a Gram-positive Streptococcus with atypical weak β-hemolysis. Whole-genome sequencing and cgMLST revealed that this isolate belonged to the cgST type of 65937, containing 187 virulence genes and 97 drug resistance genes, with high sequence similarity (95.16%~100.00%) to the main virulence factor-encoding genes in the Virulence Factor Database. Compared with the strains of ATCC 19615 and GAS029, the strain 21SPY7071 showed reduced expression of genes encoding streptolysin S ( sagABCDEFGHI), streptolysin O ( slo), and hyaluronate lyase ( endA/ sdaB). Besides, compared with the strain ATCC 19615, the strain 21SPY7071 also showed decreased expression of virulence genes including speG, ideS/ mac, hylA, smeZ, lmb, and scpA/ scpB. Conclusion:This Streptococcus pyogenes strain isolated from the pus of a child with atypical brain abscess exhibits weak hemolysis and lower expression of virulence genes, which may be related to the mild clinical symptoms observed in the child.

Objective:To analyze the biological characteristics and genomic features of a Streptococcus pyogenes strain isolated from the pus of a child with an atypical brain abscess. Methods:The Streptococcus pyogenes strain, named 21SPY7071 ( emm22 type), was isolated from the brain abscess specimen of a child undergoing surgery for brain abscess. The biological characteristics of this strain were analyzed using blood agar culture, Gram staining, growth curve measurement, and hemolytic analysis. After whole-genome sequencing, core genome multilocus sequence typing (cgMLST) was performed to analyze the cgST type of the strain, and the distribution of virulence genes and drug resistance genes were analyzed. qRT-PCR was used to detect the differences in the expression of virulence genes at mRNA level between the strain 21SPY7071 and two Streptococcus pyogene strains of ATCC 19615 ( emm80 type) and GAS029 ( emm22 type, clinical isolate). Results:The strain 21SPY7071 was a Gram-positive Streptococcus with atypical weak β-hemolysis. Whole-genome sequencing and cgMLST revealed that this isolate belonged to the cgST type of 65937, containing 187 virulence genes and 97 drug resistance genes, with high sequence similarity (95.16%~100.00%) to the main virulence factor-encoding genes in the Virulence Factor Database. Compared with the strains of ATCC 19615 and GAS029, the strain 21SPY7071 showed reduced expression of genes encoding streptolysin S ( sagABCDEFGHI), streptolysin O ( slo), and hyaluronate lyase ( endA/ sdaB). Besides, compared with the strain ATCC 19615, the strain 21SPY7071 also showed decreased expression of virulence genes including speG, ideS/ mac, hylA, smeZ, lmb, and scpA/ scpB. Conclusion:This Streptococcus pyogenes strain isolated from the pus of a child with atypical brain abscess exhibits weak hemolysis and lower expression of virulence genes, which may be related to the mild clinical symptoms observed in the child.

Objective:To investigate the effects of cryopreservation on the results of nucleic acid detection and culture for Bordetella pertussis in residual culture-positive nasopharyngeal swab specimens, aiming to provide the basis for specimens preservation, transport and centralized detection. Methods:In this cross-sectional study, the residual nasopharyngeal swab specimens which were culture-positive for Bordetella pertussis were collected in the Children′s Hospital, Zhejiang University School of Medicine from January to August in 2022. The specimens were placed at ?20 ℃ and?70 ℃ by random number table method, respectively. Re-detection by culture and PCR for Bordetella pertussis were conducted after these specimens were frozen for 114.3±31.9 days. The specimens were grouped according to the cryopreservation temperature and the semi-quantitative results by bacteria culture. The positive rates of the results were compared with χ 2 test between groups. Results:A total of 244 nasopharyngeal swabs specimens were included and 166 were culture-positive after cryopreservation, the positive rate decreased by 32%. Among them, the positive rate of re-culture of specimens containing low bacterial loads decreased by 56% after cryopreservation. However, there was no significant difference in the positive rate of culture between the specimens freezing at ?70 ℃ and ?20 ℃ (χ2=1.65, P=0.20). The positive rate of DNA detection decreased by 10.6% (88.9% vs 78.3%) after cryopreservation. The positive rate of the ?70 ℃ storage group was significantly higher than that of the ?20 ℃ group (χ2=5.11, P=0.02). The positive rate of the re-detection of DNA of nasopharyngeal swabs with low bacteria loads in ?70 ℃ storage group was significantly higher than that of the ?20 ℃ group (χ2=4.86, P=0.03). While for the samples with a bacterial load of "+" or more, there was no significant difference in the positive rate of DNA detection after cryopreservation between the ?20 ℃ and -70 ℃ (χ2=1.25, P=0.26) groups. The positive rate of nasopharyngeal swab culture after cryopreservation was 68.0% (166/244), which was significantly lower than the DNA detection positive rate of 78.3% (191/244, χ2=6.52, P=0.01). Conclusions:Cryopreservation nasopharyngeal swabs specimens could be used for Bordetella pertussis culture and nucleic acid detection. The bacterial load in the original sample affects the positive detection rate after cryopreservation. Cryopreservation has less influence on the positive rate of the result of nucleic acid detection when compared with culture. Preservation at ?70 ℃ is superior to ?20 ℃.

Objective:To study the clinical and microbiological characteristics of invasive Salmonella infection in children. Methods:Clinical data of children with invasive Salmonella infection diagnosed in the Children′s Hospital Zhejiang University School of Medicine and Women and Children′s Hospital of Ningbo University from January 2018 to June 2022 were retrospectively analyzed, and the serotypes and drug resistance of the pathogens were detected. The chi-square test was used to compare the resistance rates of different Salmonella serogroups to antibiotics. Results:A total of 105 invasive Salmonella infection cases were included, with ages of 20.0 (12.3, 43.5) months, and 67 cases(63.8%)were male. Fifty-seven cases occurred in summer (from June to August). Eighty-seven cases were confirmed by blood culture, 10 cases by bone marrow or bone marrow cavity pus culture, six cases by cerebrospinal fluid and other specimen culture, with an additional two cases where Salmonella was cultured from two types of specimens including blood, bone marrow or cerebrospinal fluid. Thirty-six patients had underlying diseases, of which 58.3%(21/36) were leukemia. The resistance rates of the strains to ampicillin, ceftriaxone, ceftazidime, piperacillin-tazobactam, imipenem, and trimethoprim-sulfamethoxazole were 48.5%(51/105), 12.3%(13/105), 9.5%(10/105), 1.9%(2/105), 0(0/105), and 29.5%(31/105), respectively. Ninety-nine strains (94.3%) belonged to invasive non-typhoidal Salmonella (iNTS), four strains were Salmonella typhi, and 13 strains were Salmonella typhimurium. The resistance rate of group D strains to ceftriaxone was lower than that of group B ( χ2=5.38, P=0.020) and group E ( χ2=5.74, P=0.017). The resistance rate of group D strains to trimethoprim-sulfamethoxazole was lower than that of group C ( χ2=5.67, P=0.017), group B ( χ2=13.02, P<0.001) and group E ( χ2=9.00, P=0.003), and the differences were all statistically significant. The symptoms of fever and diarrhea accounted for 94.3%(99/105) and 51.4%(54/105), respectively. The most common diagnosis was septicemia, accounting for 88.6%(93/105), pyogenic osteomyelitis with pyogenic arthritis accounted for 10.5%(11/105), and four patients (3.8%) developed purulent meningitis. All 105 patients received antibiotic treatment, 101 patients (96.2%) were cured or improved, and four cases (3.8%) died. Conclusions:iNTS infection is more common than typhoid fever in children. Different serotypes of strains have different resistance to antimicrobial drugs. Invasive strains such as bloodstream are more sensitive to the third-generation cephalosporin. The third-generation cephalosporin is still the first choice for the treatment of iNTS diseases.

Objective:To study the clinical and microbiological characteristics of invasive Salmonella infection in children. Methods:Clinical data of children with invasive Salmonella infection diagnosed in the Children′s Hospital Zhejiang University School of Medicine and Women and Children′s Hospital of Ningbo University from January 2018 to June 2022 were retrospectively analyzed, and the serotypes and drug resistance of the pathogens were detected. The chi-square test was used to compare the resistance rates of different Salmonella serogroups to antibiotics. Results:A total of 105 invasive Salmonella infection cases were included, with ages of 20.0 (12.3, 43.5) months, and 67 cases(63.8%)were male. Fifty-seven cases occurred in summer (from June to August). Eighty-seven cases were confirmed by blood culture, 10 cases by bone marrow or bone marrow cavity pus culture, six cases by cerebrospinal fluid and other specimen culture, with an additional two cases where Salmonella was cultured from two types of specimens including blood, bone marrow or cerebrospinal fluid. Thirty-six patients had underlying diseases, of which 58.3%(21/36) were leukemia. The resistance rates of the strains to ampicillin, ceftriaxone, ceftazidime, piperacillin-tazobactam, imipenem, and trimethoprim-sulfamethoxazole were 48.5%(51/105), 12.3%(13/105), 9.5%(10/105), 1.9%(2/105), 0(0/105), and 29.5%(31/105), respectively. Ninety-nine strains (94.3%) belonged to invasive non-typhoidal Salmonella (iNTS), four strains were Salmonella typhi, and 13 strains were Salmonella typhimurium. The resistance rate of group D strains to ceftriaxone was lower than that of group B ( χ2=5.38, P=0.020) and group E ( χ2=5.74, P=0.017). The resistance rate of group D strains to trimethoprim-sulfamethoxazole was lower than that of group C ( χ2=5.67, P=0.017), group B ( χ2=13.02, P<0.001) and group E ( χ2=9.00, P=0.003), and the differences were all statistically significant. The symptoms of fever and diarrhea accounted for 94.3%(99/105) and 51.4%(54/105), respectively. The most common diagnosis was septicemia, accounting for 88.6%(93/105), pyogenic osteomyelitis with pyogenic arthritis accounted for 10.5%(11/105), and four patients (3.8%) developed purulent meningitis. All 105 patients received antibiotic treatment, 101 patients (96.2%) were cured or improved, and four cases (3.8%) died. Conclusions:iNTS infection is more common than typhoid fever in children. Different serotypes of strains have different resistance to antimicrobial drugs. Invasive strains such as bloodstream are more sensitive to the third-generation cephalosporin. The third-generation cephalosporin is still the first choice for the treatment of iNTS diseases.

Objective:To study the drug resistance patterns of Bordetella pertussis in vitro, and to know the clinical characteristics of pediatric pertussis and evaluation the treatment outcomes, which may provide references for experiential diagnosis and treatment of this disease. Methods:Nasopharyngeal swabs of the hospitalized children with suspected pertussis in Children′s Hospital, Zhejiang University School of Medicine in 2017 were collected for culture. And the clinical data of the children were collected. The strains were identified by pertussis-specific antiserum agglutination and finally confirmed by mass spectrometry. The drug sensitivity test was performed using the E-test method. The efficacy of therapy with antibiotic was evaluated after two weeks of treatment. Statistical analysis was performed with Mann-Whitney U test and chi-square test. Results:Of 1 029 children, 211 (20.5%) nasopharyngeal swabs were positive for Bordetella pertussis culture, and the isolation rate of the specimens was highest (31.2%, 45/144) in July. Of the 211 pertussis patients, 105 (49.8%) were male and the age were 3.8 (2.2, 6.9) months, 114 (54.0%) were not vaccinated with pertussis diphtheria tetanus mixed vaccine and 192 (91.0%) were prescribed with previous antibiotics. There were 142 (67.3%) children from families with two or more than two children, and 136 (95.8%) of which were the youngest siblings. One hundred and fifty-nine (75.4%) patients had paroxysmal cough and 61 (28.9%) had whooping. The white blood cell counts were higher than 20×10 9/L in 94 (44.5%) patients, and the lymphocyte counts were higher than 10×10 9/L in 97 (46.0%) of patients. The drug susceptibility results showed that 138 (65.4%) strains were against erythromycin, azithromycin and clindamycin with minimum inhibitory concentration (MIC)>256.000 mg/L. The MIC 90 of the isolates to ampicillin, ceftriaxone, cefoperazone/sulbactam, meropenem and trimethoprim/sulfamethoxazole were 0.190 mg/L, 0.190 mg/L, 0.094 mg/L, 0.094 mg/L and 0.750 mg/L, respectively. All strains had a MIC of <0.016 mg/L for piperacillin/tazobactam. After treatment, symptoms were improved in 195(92.4%) patients when they were discharged from hospital. Seventy-six (57.1%) children whose symptoms did not improve after seven-day treatment with macrolides, were prescribed with other antibiotics or other antibiotic with macrolides in combination. Compared with the patients treated with macrolides, more patients treated with cefoperazone/sulbactam or piperacillin/tazobactam had negative nasopharyngeal culture results after two weeks of therapy (46/48(95.8%) vs 46/57(80.7%)), or on day seven (45/46(97.8%) vs 39/47(83.0%)) and on day 14 (45/45(100.0%) vs 41/47(87.2%)) since discharged. The differences were all statistically significant ( χ2=5.50, 5.86 and 6.15, respectively, P=0.019, 0.015 and 0.013, respectively). Conclusions:The majority of children with pertussis do not have whooping, and the resistant rate of Bordetella pertussis to macrolides is high. Further study is needed to evaluate the feasibility and reasonability of cefoperazone/sulbactam and piperacillin/tazobactam in treating pediatric pertussis caused by macrolides-resistant Bordetella pertussis.

Objective:To investigate the distribution of pathogen species isolated from cerebrospinal fluid culture (CSF) in children and analyze the antibiotic-resistance of the main isolates in vitro, which provides reference for interpreting the pathogens and choosing antibiotics in empiric therapy for pediatric patients. Methods:The results of cerebrospinal fluid culture were collected by checking laboratory information system of the Children′s Hospital of Zhejiang University and the clinical characteristics of these children were analyzed retrospectively by checking electronic medical record system.Results:A total of 1 312 isolates were detected, including 1 294 isolates of bacteria and 18 isolates of fungi. A total of 497 (37.9%) isolates were pathogenic microorganisms, of which 288 (57.9%) isolates were gram-positive, 200 (40.3%) isolates were gram-negative, and 9 (1.8%) isolates were fungi. The top 5 pathogens were Escherichia coli (102 isolates, 20.5%), Streptococcus pneumoniae (64 isolates, 12.9%), Streptococcus agalactiae (52 isolates, 10.5%), Enterococcus faecium (33 isolates, 6.6%) and Staphylococcus aureus (28 isolates, 5.6%). Most of the Streptococcus pneumoniae strains were isolated from children more than 1 year old (76.6%, 49/64), while the other top 4 bacteria were mainly isolated from infants less than 1 year old, with the rate of 95.1%(97/102) for Escherichia coli, 98.1%(51/52) for Streptococcus agalactiae, 81.8%(27/33) for Enterococcus faecium and 71.4% (20/28) for Staphylococcus aureus. A total of 815 (62.1%) isolates were considered to be contaminated pathogens according to the analysis on clinical manifestations and other laboratory findings in CSF, and coagulase-negative Staphylococcus (680 isolates), Micrococcus (50 isolates), Corynebacterium (28 isolates) and Enterococcus faecium (23 isolates), which accounted for 41.1% (23/56) of the total detected Enterococcus faecium, were the top 4 contaminated bacteria. During the study period, the isolation rate of the pathogenic microorganisms increased year by year (χ2=34.84, P<0.001), while the isolation rate of the contaminated pathogens, which detected mainly in summer and autumn, decreased year by year (χ2=13.26, P<0.001). Conclusions:The predominant bacteria causing pediatric purulent meningitis were Escherichia coli, Streptococcus pneumoniae, Streptococcus agalactiae, Enterococcus faecium and Staphylococcus aureus. Coagulase-negative Staphylococcus, Micrococcus, Corynebacterium and Enterococcus faecium were common contaminated bacteria in CSF culture, therefore clinicians should interpret the results of CSF culture cautiously according to the bacterial species and clinical manifestations.

The coronavirus disease 2019 (COVID-19) has caused a global pandemic. All people including children are generally susceptible to COVID-19, but the condition is relatively mild for children. The diagnosis of COVID-19 is largely based on the epidemiological evidence and clinical manifestations, and confirmed by positive detection of virus nucleic acid in respiratory samples. The main symptoms of COVID-19 in children are fever and cough; the total number of white blood cell count is usually normal or decreased; the chest imaging is characterized by interstitial pneumonia, which is similar to other respiratory virus infections and infections. Early identification, early isolation, early diagnosis and early treatment are important for clinical management. The treatment of mild or moderate type of child COVID-19 is mainly symptomatic. For severe and critical ill cases, the oxygen therapy, antiviral drugs, antibacterial drugs, glucocorticoids, mechanical ventilation or even extracorporeal membrane oxygenation (ECMO) may be adopted, and the treatment plan should be adjusted timely through multi-disciplinary cooperation.
Betacoronavirus ; isolation & purification ; Child ; Coronavirus Infections ; diagnosis ; pathology ; therapy ; Humans ; Pandemics ; Pneumonia, Viral ; diagnosis ; diagnostic imaging ; etiology ; pathology ; therapy

Betacoronavirus ; isolation & purification ; Child ; Clinical Laboratory Techniques ; Coronavirus Infections ; diagnosis ; drug therapy ; therapy ; Humans ; Pneumonia, Viral ; diagnosis ; therapy