The Clinical Analysis Of Neonatal Sepsis And Serotype Identification Of Group B Streptococcus By PCR And Sequencing

Part Ⅰ A Review of Sepsis Pathogenic Bacteria Distribution and Drug Resistance Analysis in Chinese Neonatal Care UnitsObjective:Even though perinatal care has been steadily improving, infection is still the main cause of neonatal morbidity and mortality. Due to the lack of neonatal immune defense, invasive operations such as indwelling gastric tube, endotracheal intubation, suctioning, etc, and application of broad-spectrum antibiotics, have made neonatal ward becoming a high incidence place of nosocomial infection. Thus it is of great importance to study the epidemic strain and their drug resistance of infection in neonatal ward including neonatal intensive care ward (NICU) for guiding neonatal clinicians to use antibiotic properly and reducing the incidence of nosocomial infection in newborns. Previous studies suggest that, with the change of the use of antibiotics, there would be corresponding changes in pathogens, and regional differences. Therefore, it is necessary for us to know pathogens distribution and drug resistance of bloodstream infection in neonatal intensive care units of our country.Methods:Papers published between 1990 and December 2013 on the core journals included by Chinese Biomedical Literature Database(CBM), China National Knowledge Infrastructure(CNKI) and VIP Chinese Periodical Database were systematically searched.Also We searched in PubMed (1990 to December 2013).Studies of bacteria distribution and drug resistance with a non-comparative, observational design were included. Relevant information was extracted.Document enrollment criteria:(1) study objects coming from the hospital environment in mainland China; (2) researches that should report neonatal bloodstream infection pathogens and drug resistance; (3) the type of research should be uncontrolled observational study to minimize the impact of the original case study enrollment criteria; and (4) blood culture positive cases is greater than or equal to 50.Excluded documents do not meet the above criteria, as well as literature, or the literature (1) repeated publication of periodic reports in a study, but there are other literature have reported complete results of the study; (2) basic laboratory research; (3) The article mentioning a bloodstream infection pathogens and drug resistance, but in fact is the study of other diseases; (4) research literature focusing on specific pathogens; (5) review, comment or lectures.Literature excerpts include:title, year of publication, type of research(retrospective/prospective), case collecting time and hospitals(province /city), the authors’ department, bloodstream infections and specific distribution of important pathogens drug resistance of pathogens.Use Z-test of SPSS13.0 and Excel 2010 table formulas and charts for data pooled analysis.Results:Overall,71 studies were included for this analysis with a total of 30346 strains (positive bacteria 23118, negative bacteria 6955, fungi,273).Strains are mainly from Beijing city, Zhejiang, Shanghai, Hubei, Guangdong, Hunan, Yunnan provinces. From 1983 to 2004, the first 3 gram-positive bacteria of CNICU bloodstream infections were coagulase-negative staphylococci (51.33±0.33)%, Staphylococcus aureus(13.01±0.22)%, Enterococcus(4.58±0.14)%; the first 3 gram-negative bacteria were Escherichia coli(7.67±0.18)%, Klebsiella(3.33±0.12)%, other Enterobacteriaceae (2.67±0.11)%; From2005 to 2012, the first 3 gram-positive bacteria of Chinese neonatal intensive care unit bloodstream infections were coagulase-negative staphylococci (61.35±0.57)%, Staphylococcus aureus(9.69±0.35)%%, Enterococcus(5.19±0.26)%; the first 3 gram-negative bacteria were Escherichia coli(6.33±0.28)%, Klebsiella(5.53±0.27)%, Acinetobacter(1.50±0.14)%. We applied Z-test to compare the two-stage bacterial composition ratio,with all P> 0.05 Two-stage bacterial constituent ratio difference was not statistically significant. It is considered as a whole that the distribution of neonatal septicemia no significant change.Bacteria resistance increased year by year, Gram-positive bacteria Staphylococcus aureus resistant to penicillin and ampicillin increased from 50% in the 1980s to more than 90% currently; to tetracycline, erythromycin resistance rate changes little by year,the former resistance now up to about 50%, while the latter amounted to 80%; oxacillin resistance increasing from 10% to around 65%, cefazolin, cefuroxime resistance was about 30%, gentamicin resistance decreased from 50% to about 30% and then rose to 60%, ciprofloxacin resistance has been low, currently only 30%, nearly all strains sensitive to vancomycin.Gram-negative bacteria Escherichia coli resistant rate to ampicillin, piperacillin rose from 60% to 80%; resistant rate to cefazolin, cefuroxime rose from less than 50% to more than 80%; resistant rate to cefazolin oxime, ceftriaxone had been no significant change, but it was between 50% -60%; resistant rate to cefoperazone, ceftazidime rose from 30% to nearly 50%; resistant rate to cefepime up to 45%; gentamicin, ciprofloxacin resistant rate is about 40%, the resistant rate to meropenem, imipenem was low, almost no resistance.In the reports there contain a small amount of bacteria resistance to vancomycin or Imipenem. For Vancomycin resistant bacteria, coagulase negative staphylococcus accounted for 72% while staphylococcus aureus accounted for 8%; For Gram-negative bacterium resistance to Imipenem, pseudomonas accounted for 35% while e. coli accounted for 17%.Conclusion:The bloodstream infection pathogen characteristic and drug resistance analysis showed that the bacterial drug resistance in China is serious. We need to strengthen the bacteria monitoring to guide clinical rational drug use and reduce the generation of drug-resistant bacteria.Part Ⅱ The Blood Culture Positive Rate of Newborn Who Suspected Suffer Bloodstream Infection in China:A Meta-analysisObjective:Sepsis is cause by pathogens invade the blood circulation, and in which growth and reproduction, produce toxins causing systemic infection, is a critical illness in the neonatal period, and one of the main causes of neonatal mortality. The incidence is approximately 0.1% to 1.0% of live births, accounting for 16.4% in very low birthweight infant (VLBW), the incidence of long-term hospitalization were up 30.0%, mortality was 10% -50%, survivors can may suffer complications, we should pay close attention to it. Currently,the gold standard for diagnosis neonatal sepsis is remains blood culture. However, blood culture positive rate of China’s suspected neonatal sepsis lack system, representative of data and correlation analysis. Based on this, we have our current published literature to conduct a meta-analysis of neonatal blood cultures positive rates and related factors.in suspicious bloodstream infection.Methods:Papers published between 1990 and March 2014 on the corn journals included by Chinese Biomedical Literature Database(CBM), China National Knowledge Infrastructure(CNKI) and VIP Chinese Periodical Database were systemetically searched.Also search the studies in PubMed (1990 to March 2014).Studies providing data of neonata blood culture positive rate with a non-comparative, observational design were included.Document inclusion criteria:(1) the study should come from the hospital environment in mainland China; (2) research should report the blood culture positive rate, or the data is available in the text report that can calculated the positive rate; (3) the type of research should be uncontrolled observational studies, in order to minimize the impact of the original case study inclusion criteria; and (4) blood culture positive cases is greater than or equal to 50.Exclude documentation does not meet the above criteria, as well as literature, or the literature (1) repeated publication of periodic reports in a study, but there are other literature have reported complete results of the study; (2) basic research laboratories; (3) The article mentions the blood culture positive rate, but in fact is the study of literature other diseases; (4) study only certain pathogens in blood culture positive rate of literature; (5) a review, comment or lectures.The following excerpt from the literature:title, year of publication, type of research (retrospective/prospective), time to collect cases, hospital patients collected their province (city), the authors’department, the number of cases of blood culture samples submission culture positive specimens, culture-positive rate, the volume of blood when blood culture collection, flasks species, specimens sent time, blood culture equipment models.Use R3.2.2 Meta software program package for the rate meta analysis,using logit method for rate conversion before meta-analysis, the first for heterogeneity test, if the result is P<0.05, explained that there are differences between the included studies, then conduct sensitivity test, exclud heterogeneity literature before using the fixed effects model for meta-analysis.Results:There were 16 literatures in the study, after heterogeneity and sensitivity test there remained seven documents, using a fixed effects model for meta-analysis and finally get Chinese newborns suspected bloodstream infections blood cultures positive rate of 12%(95%CI:11.2%-12.2%).Conclusion:Blood culture positive rate of newborn who suspected suffer bloodstream infection was at a low level in China, we need to regulate blood culture specimen collection, inspection and other processes in future.Part Ⅲ Clinical Analysis of Neonatal SepsisObjective:It is ideal to select appropriate antibiotics for neonatal sepsis treatment according to the results of blood culture and sensitivity test, but bacterial culture can not be achieved immediately. Thus experience-based choice of antibiotics empirically inevitably causes bacterial resistance. Studies have found that other than penicillin, quinolones and vancomycin, a significant variety of antibiotic resistance rates amount different hospitals exits.Therefore, the establishment of this unit of bacterial infectious diseases and antimicrobial resistance spectrum of clinical medication, anti-bacterial resistance plays an important role. The clinical features and etiological distribution and drug resistance of bloodstream infections in our hospital neonatal are discussed in this study, in order to guide clinical treatment.Methods:The clinical features and pathogenic bacteria distribution and drug resistance 106 neonatal sepsis which admitted in our hospital from January 2009 to December 2014 were retrospectively analyzed. Firstly analysis 6 years of general bacteria distribution was analyzed. Then, the clinical characteristics and distribution of pathogens were compared based on the gestational age and onset times (early onset and late onset). Finally,six years of neonatal sepsis were divided into two groups comparing two groups of pathogenic bacteria and drug resistance.Results:1.14281 cases of neonatal newborn were treated in our hospital from 1, January 2009 to December 2014,207 cases of neonatal sepsis include 106 cases of septicemia which were confirmed by blood culture positive, culture positive rate was 51.2%, patients diagnosed with neonatal sepsis accounted for a percentage of 0.74%;57 cases were male,49 cases were female; Birth weight (3025/900-4600) g, mean (2995±596) g; gestational age (39/27.14-42.14) w, mean (38.5±2.57) w; preterm (<37w) 22 cases, accounting for 20.75%, term (≥37w) 86 cases, accounting for 79.25%; 26 cases of cesarean section, accounting for 24.5%, 80 cases of vaginal delivery, accounting for 75.5%; 58 cases were born in our hospital,accounting for 54.7%,38 cases were born in other hospitals, accounting for 35.8%,10 cases were born at home, accounting for 9.4%; maternal age (28/ 19-42) years, mean (28.08±5.03) years; his mother had≥1 high-risk factors:28 cases had signs of premature, accounting for 26.4%,17 cases had fever before delivery of fever, accounting for 16%,17 cases of premature rupture of membranes, accounting for 16%; after treatment,59 cases were cured(55.7%),39 cases were improved (36.8%),8 cases died(7.5%). Gestational and weight of early-onset group and late-onset group were statistically significant differences. Birth weight of premature group and late onset group, the difference was not statistically significant (t = 4.452, P=0.215), compared gestational age between early-onset and late-onset group was statistically significant (t=3.941, P=0.015). The delivery way between premature group and late onset group was not statistically significant (χ2=0.005, P =0.944); compared the age of the mother between two group, the difference was no significant difference (t=0.351, P=0.782).2.The main pathogenic bacteria were Gram-positive bacteria, accounting for 83.9%, which Staphylococcus aureus accounted for 5.7%, coagulase-negative staphylococci accounted for 57.5%, Enterococcus faecalis accounting for 3.8%, Listeria accounting for 0.9%, Streptococcus agalactiae accounting for 16%; Gram-negative bacteria accounted for 16.1%, including 14.2% of Escherichia coli, 1.9% of Klebsiella pneumoniae. From January 2009 to December 2014, the neonatal sepsis pathogens of our hospital mainly were coagulase-negative staphylococci 57.5%, Streptococcus agalactiae 16%, Escherichia coli 14.2%.3.Based on gestational age, major pathogens of <37w were coagulase-negative staphylococci 41% (including Staphylococcus epidermidis 13.7%, Staphylococcus haemolyticus 18.2%, human subspecies aureus 9.1%), Escherichia coli 22.7%, Enterococcus faecalis 13.7%; major pathogens of>37w were coagulase-negative staphylococci 61.9%(including 25% Staphylococcus epidermidis, Staphylococcus haemolyticus 26.2%, human subspecies aureus 7.1%, Staphylococcus saprophyticus 1.2%, Wolfowitz aureus 1.2%, yield aureus 1.2%), Streptococcus agalactiae 17.8%, Escherichia coli 12%. The pathogens of different gestational age were coagulase-negative and E. coli.Enterococcus faecalis infecions in preterm infants compared with full-term infants increased significantly, the former was 13.6%,while the latter was 1.2%.In addition, GBS infections of full-term infants a little more than preterm infants, the former was 17.8%,the latter was 9.1%.4. Early-onset group predisposing factors mainly were respiratory infections 48%, preterm40%, premature rupture 28%, amniotic 20%, birth asphyxia 8%, mucocutaneous infection 8%; late-onset group predisposing factors mainly were respiratory infections 67.9%, skin infections37.1% (including impetigo skin 17.3%, omphalitis 19.8%), amniotic 23.5%, preterm 14.8%, premature rupture of membranes 12.3%, birth asphyxia 1.2%; the proportion of preterm factor in early-onset group was significantly higher than late-onset group, the difference was statistically significant (χ2=7.367, P=0.007); mucocutaneous infection of late-onset group (37.1%)higher than that of early-onset Group (8%), P value close to 0.05. Clinical manifestations of early-onset group had shortness of breath (40%), jaundice (24%), fever (20%), heart rate (20%), poor response (12%), refusal to milk (8%); late-onset group, the main clinical symptoms are fever (54.3%), heart rate (46.9%), shortness of breath (45.7%), jaundice (35.8%), convulsion (11.1%), refusal to milk 9.9%; two clinical manifestations are basically the same, but the late-onset group compared with early-onset group,had more fever(%2= 9.053, P=0.003),had more heart rate increased(χ2=5.739, P=0.017), the difference of the two groups was statistically significant..5. Distribution of early-onset group pathogens were Streptococcus agalactiae 40%, coagulase-negative staphylococci 16%(including Staphylococcus epidermidis 8%, S.haemolyticus 8%), Escherichia coli 12%, Staphylococcus aureus 12%, Enterococcus faecalis 8%, Klebsiella pneumoniae 8%; Distribution of late-onset group pathogens were coagulase-negative staphylococci 70.3%(including Staphylococcus epidermidis 27.2%, Staphylococcus haemolyticus 29.6%, people staphylococcus people subspecies 9.9%, Staphylococcus saprophyticus, Wolfowitz Staphylococcus aureus, Staphylococcus aureus produce each 1.2%), Escherichia coli 14.8%, Streptococcus agalactiae 8.6%, Staphylococcus aureus 3.7%; The former pathagens of early-onset group were Streptococcus agalactiae, coagulase-negative staphylococci, Escherichia coli; late onset sepsis pathogens were coagulase-negative staphylococci, Escherichia coli, Streptococcus agalactiae.6. Two-stage blood culture cultured 208 strains, from 2009 to 2011,there were 103 strains,the distribution of the strains were coagulase-negative staphylococci 78.6%(including Staphylococcus epidermidis38.8%, Staphylococcus haemolyticus 35%, Saprophytic、Staphylococcus aureus yield each 1.9%, Staphylococcus Klinefelter 1%), E. coli 8.7%, Staphylococcus aureus 5.8%, Streptococcus agalactiae3.9%, Enterococcus faecalis 1%, feces Enterococcus1%, Klebsiella pneumoniae 1%;from 2012 to 2014,there were 105 bacterial strains,the distribution of the strains were coagulase-negative staphylococci 41%(including Staphylococcus epidermidis 14.3% hemolysis Staphylococcus 8.6%,human grapes lactis subspecies 16.2%, Wolfowitz aureus 1.9%), Streptococcus agalactiae28.6%, Escherichia coli 19%, Enterococcus faecalis 5.7%, Staphylococcus aureus, Klebsiella pneumoniae, monocytogenes Scott Special bacteria each 1.9%;from of 2009 to 2011, the former pathagens were coagulase-negative staphylococci, Escherichia coli, Staphylococcus aureus; from 2012 to 2014, the former pathagens were coagulase negative staphylococci, Streptococcus agalactiae, Escherichia coli. Coagulase-negative staphylococci reduced, Streptococcus agalactiae, Escherichia coli increased significantly over the previous.7.ESBL-producing E. coli strains accounted for about 30%, recently three years, ampicillin resistance up to 60%, ampicillin/sulbactam resistance 20%, cephalosporin resistance about 30%,no resistance to imipenem, piperacillin/tazobactam. Compared Ciprofloxacin resistance of the two stage, the second Stage decreased significantly, the two-stage difference was statistically significant (P= 0.007); compared cotrimoxazole resistance of the two stage,the second stage increased significantly,the two-stage difference was statistically significant (P=0.011). Phase 13/9 ESBL-producing strains of the phase 1 was 3/9 (33.3%), Phase 2 was 6/20 (30%), the rates of two-phase were basically the same.MRSA of Staphylococcus aureus accounted for 50%, penicillin-resistant up to 100% recently three years. MRCNS of coagulase-negative staphylococci accounted for 86.4%, penicillin-resistant up to 90%, resistance to clindamycin, ring ciprofloxacin, erythromycin, cotrimoxazole were 44.2%,23.2%,62.8%,34.9%; gentamicin, levofloxacin, moxifloxacin resistance were 11.6%,2.3%,2.3%, no resistance to tigecycline, found a vancomycin-resistant strain. Comparison of the two-stage MRCNS proportion, the difference was not statistically significant (x2=0.547, P=0.460),Comparison of β-lactamase producing bacteria ratio of the two-stage, the difference was not statistically significant (χ2=1.904, P=0.168); Comparison of the two-stage drug resistance was no statistically significant; only the ciprofloxacin resistance of phase 2 decreased compared with phase 1, the difference was statistically significant (x2=10.355, P=0.001)Streptococcus agalactiae clindamycin resistance was significantly up 86.7%,no resistance to penicillin, ampicillin, vancomycin.Conclusions:The neonatal sepsis pathogens of our hospital in recent years were mainly coagulase-negative staphylococci, Streptococcus agalactiae and Escherichia coli; wherein Streptococcus agalactiae and Escherichia coli sepsis significantly increasing in recent years; Escherichia coli resisted to ampicillin up to 60%; coagulase-negative staphylococci and Staphylococcus aureus high proportion of MRCNS and MRSA, penicillin-resistant serious, amounting to about 90%, we should not use penicillin in these bacterial infections, should use enzyme-containing preparations or use vancomycin for serious infection therapy; Streptococcus agalactiae no resistance to penicillin, ampicillin, clindamycin resistance significantly, while infection we should choose penicillin or ampicillin for treatment, should not use clindamycin.Part IV The clinical analysis of neonatal streptococcus agalactiae sepsis and Serotype Identification of group B streptococcus by PCR and sequencingObjective:Our hospital neonatal bloodstream infection clinical study found that in recent years, our hospital neonatal S. agalactiae infection increased significantly, and higher than the domestic level reported in the literature. It is necessary to investigate the neonatal streptococcus agalactiae sepsis clinical characteristics and resistance in order to guide clinical treatment. No serotype indentification of Group B Streptococcus of human infection by PCR and Sequencing reported in our country,so we carried out this study.Method:The clinical features and resistance of 15 neonatal streptococcus agalactiae sepsis which admitted in our hospital from January 2012 to December 2014 were retrospectively analyzed, and Saved strains were cultured, purified. We extracted bacterial genome DNA, and identify the strains genotyping using PCR amplification and gene sequencing methods.Results:53 cases neonatal sepsis were diagnosed from 2012 to 2014, including 15 streptococcus agalactiae infections, accounting for 28.3%, mainly in 2012, while 25 cases diagnosed as neonatal sepsis, including 11 Streptococcus agalactiae infection, accounting for 44%;In 15 cases of Streptococcus agalactiae infection,9 cases of early-onset sepsis,6 cases of late-onset sepsis; clinical manifestations:6 cases got fever,5 cases got jaundice,4 cases got pneumonia (include 3 casesof early-onset infection),5 cases got intracranial infection (include 4 cases late-onset infection); All strains had no resistance to penicillin, ampicillin, tigecycline and vancomycin, linezolid,11.1% resistant to moxifloxacin,13.3% resistant to ciprofloxacin, levofloxacin; 86.7%resistant to Clindamycin,93.3%resistant to tetracycline. Serotype distribution:among 15 isolates from newborn, Ⅰ a 14.3%(n=2), Ⅰ b 28.6%(n=4), Ⅲ 57.1%(n=8).Among 10 isolates from Urology urinary tract infections, the serotype distribution were Ⅰ a 30%(n=3), Ⅰ b 30%(n= 3) Ⅲ 40% (n=4).Conclusion:The streptococcus agalactiae sepsis has a growing trend recently, especially in 2012. Early-onset infection is often associated with respiratory symptoms, late-onset infection is often associated with intracranial infection, we need to pay attention to improve the cerebrospinal fluid examination. Ⅰ a、Ⅰ b and Ⅲ were the main serotype. We can choose penicillin, ampicilin for treatment. Clindamycin and tetracycline are not suitable for clinical use because of high resistance. Currently newborn Streptococcus agalactiae infection is growing, neonatal clinical doctors from different hospitcals can use bioMerieux bacterial identification cards which have confirmed Streptococcus agalactiae for Streptococcus agalactiae genotyping detection, during detection,we should use nutritious broth such as brain heart infusion broth for bacterial culture, use 37℃ shaker overnight,in the logarithmic growth phase of bacteria for bacterial genomic DNA extraction. Primers used in this study for PCR and serotype sequencing meet almost all agalactiae serotypes. Only a handful of those which can not type, we should use another primer DSF2-DSR1 which based on 16S rRNA gene to diagnose strains.