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B族链球菌(group B streptococcus, GBS)是革兰阳性兼性厌氧菌,常存在于胃肠道和泌尿生殖道内,为条件致病菌[1]。围生期孕妇阴道或直肠内GBS的定植率为10%~35%[2-3]。约有50%的GBS携带孕妇会把该菌传播给新生儿,在没有产前抗菌药物预防的情况下,1%~2%的新生儿会出现早发型GBS感染,表现为重症肺炎、败血症及化脓性脑膜炎,如不及时治疗会造成新生儿死亡或神经系统后遗症等严重不良后果[4-5]。美国自20世纪90年代开始对围生期孕妇进行GBS筛查,对携带GBS的孕妇产前应用抗菌药物预防,极大降低了新生儿GBS感染率[6]。细菌培养法是GBS筛查的金标准,目前围生期孕妇阴道/直肠拭子筛查GBS的方法主要为血平板培养法、显色平板培养法。但培养法存在耗时长、灵敏度低、影响因素多等缺点。本研究拟通过比较荧光聚合酶链反应(PCR)法与显色平板培养法筛查围生期孕妇阴道/直肠拭子的阳性率,分析荧光PCR法筛查GBS的可行性。现作报道。
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GBS显色平板培养出146株GBS,阳性率为12.77%,荧光PCR法检出191例阳性标本,阳性率为16.71%,2种方法阳性率差异有统计学意义(P<0.01);荧光PCR法与显色平板培养法相比,敏感性为93.15%、特异性为94.48%、符合率为94.31%(见表 1)。显色平板培养法与荧光PCR法检测GBS阳性结果见图 1。
荧光PCR法 显色平板法 合计 χ2 P 敏感性/% 特异性/% 符合率/% 阳性 阴性 阳性 136 55 191 阴性 10 942 952 7.05 < 0.01 93.15 94.48 94.31 合计 146 997 1 143 表 1 荧光PCR法与显色平板培养法检测结果比较(n)
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显色平板培养法检出146株GBS,其中10株荧光PCR法检测为阴性,将这10份GBS菌株的增菌肉汤管取出重新鉴定,均为GBS。对这10株GBS、阴性对照、阳性对照进行CAMP试验,10株GBS和阴性对照菌株为阴性,阳性对照菌株为阳性。阴性对照选用粪肠球菌ATCC29212,阳性对照选用B族链球菌ATCC13183(见图 2)。
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进化树分析结果显示,10株CAMP阴性菌株(GBS1-GBS10)与GBS(AB596948)聚为一类(见图 3)。电泳结果显示,2株CAMP阳性GBS和阳性对照有特异性条带,10株CAMP阴性GBS和阴性对照未出现特异性条带。阳性对照测序结果提交到GenBank数据库中进行BLAST比对,与cfb基因符合率为100%(见图 4)。2株CAMP阳性菌株含有cfb基因,10株CAMP阴性GBS未扩增出cfb基因,证实10株GBS表现为CAMP试验阴性是由cfb基因缺失导致。
荧光PCR法在围生期孕妇B族链球菌筛查中的应用及CAMP试验阴性B族链球菌分子特征分析
Application of fluorescent PCR in screening of group B streptococcus in perinatal pregnant women and analysis of molecular characteristics of group B streptococcus with negative CAMP test
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摘要:
目的探讨荧光聚合酶链反应(PCR)在围生期孕妇B族链球菌(GBS)筛查中的应用价值及CAMP试验阴性GBS的鉴定分析。 方法收集产科门诊孕35~37周孕妇阴道/直肠拭子1 143例,将拭子放入GBS增菌肉汤管增菌,分别用GBS显色平板和荧光PCR法检测GBS。16SrDNA分子鉴定技术对CAMP试验阴性GBS进行种属鉴定,并采用PCR技术检测其cfb基因。 结果显色平板检出146份阳性样本,阳性率为12.77%,荧光PCR法检出191份阳性样本,阳性率为16.71%,2种方法的阳性率差异有统计学意义(P<0.01);荧光PCR法与显色平板培养法相比,敏感性为93.15%、特异性为94.48%、符合率为94.31%。146株GBS中有10株为CAMP试验阴性,检出率为6.85%,均为cfb基因缺失株。 结论荧光PCR法可有效提高围生期孕妇GBS筛查的阳性率;该地区CAMP试验阴性GBS检出率较高,CAMP试验不宜作为GBS的鉴别试验,因存在缺失cfb基因GBS,GBS分子检测试剂不应选择该段基因设计引物。 Abstract:ObjectiveTo explore the application value of fluorescent polymerase chain reaction (PCR) in screening of group B streptococcus (GBS) in perinatal pregnant women and the identification and analysis of GBS with negative CAMP test. MethodsA total of 1 143 vaginal/rectal swabs were collected from pregnant women aged 35-37 weeks in the obstetric clinic.The swabs were placed in a GBS enrichment broth tube to enrich the bacteria, and then GBS chromogenic plate and fluorescent PCR were used to detect GBS.16SrDNA molecular identification technology was used to identify the species of GBS with negative CAMP test, and PCR was used to detect its cfb gene. ResultsThe chromogenic plate detected 146 positive samples, with a positive rate of 12.77%, and the fluorescent PCR detected 191 positive samples, with a positive rate of 16.71%.The difference between the two methods was statistically significant (P<0.01).Compared with the chromogenic plate culture method, the fluorescent PCR method had a sensitivity of 93.15%, a specificity of 94.48%, and a coincidence rate of 94.31%.Among the 146 GBS strains, 10 strains were negative for CAMP test, with a detection rate of 6.85%, and all strains were cfb gene deletion. ConclusionsThe fluorescent PCR can effectively increase the positive rate of GBS screening for pregnant women during the perinatal period.The detection rate of GBS with negative CAMP test is relatively high in this area, and CAMP test should not be used as an identification test for GBS.Due to the deletion of cfb gene, GBS molecular detection reagents should not select this gene to design primers. -
Key words:
- group B streptococcus /
- fluorescent PCR /
- CAMP test /
- cfb gene
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表 1 荧光PCR法与显色平板培养法检测结果比较(n)
荧光PCR法 显色平板法 合计 χ2 P 敏感性/% 特异性/% 符合率/% 阳性 阴性 阳性 136 55 191 阴性 10 942 952 7.05 < 0.01 93.15 94.48 94.31 合计 146 997 1 143 -
[1] HEATH PT. Status of vaccine research and development of vaccines for GBS[J]. Vaccine, 2016, 34(26): 2876. doi: 10.1016/j.vaccine.2015.12.072 [2] RUSSELL NJ, SEALE AC, O'DRISCOLL M, et al. Maternal colonization with Group B Streptococcus and serotype distribution worldwide: systematic review and meta-analyses[J]. Clin Infect Dis, 2017, 65(suppl 2): S100. [3] GIZACHEW M, TIRUNEH M, MOGES F, et al. Streptococcus agalactiae maternal colonization, antibiotic resistance and serotype profiles in Africa: a meta-analysis[J]. Ann Clin Microbiol Antimicrob, 2019, 18(1): 14. doi: 10.1186/s12941-019-0313-1 [4] PUOPOLO KM, LYNFIELD R, CUMMINGS JJ. Management of infants at risk for Group B Streptococcal disease[J]. Pediatrics, 2019, 144(2): e20191881. doi: 10.1542/peds.2019-1881 [5] HUANG J, LIN XZ, ZHU Y, et al. Epidemiology of group B streptococcal infection in pregnant women and diseased infants in mainland China[J]. Pediatr Neonatol, 2019, 60(5): 487. doi: 10.1016/j.pedneo.2019.07.001 [6] NANDURI SA, PETIT S, SMELSER C, et al. Epidemiology of Invasive early-onset and late-onset Group B Streptococcal disease in the United States, 2006 to 2015: multistate laboratory and population-based surveillance[J]. JAMA Pediatr, 2019, 173(3): 224. doi: 10.1001/jamapediatrics.2018.4826 [7] CEZARINO BN, YAMAMOTO L, DEL NEGRO GM, et al. Diagnosis of neonatal Group B Streptococcus sepsis by nested-PCR of residual urine samples[J]. Braz J Microbiol, 2008, 39(1): 21. doi: 10.1590/S1517-83822008000100005 [8] SHABAYEK S, SPELLERBERG B. Group B Streptococcal colonization, molecular characteristics, and epidemiology[J]. Front Microbiol, 2018, 9: 437. doi: 10.3389/fmicb.2018.00437 [9] VERANI JR, MCGEE L, SCHRAG SJ. Prenvention of perinatal Group B Streptococcal disease-revised guidelines from CDC, 2010[J]. MMWR Recomm Rep, 2010, 59(RR 10): 1. [10] SCHRAG SJ, VERANI JR. Intrapartum antibiotic prophylaxis for the prevention of perinatal group B Streptococcal disease: experience in the United States and implications for a potential group B streptococcal vaccine[J]. Vaccine, 2013, 31(Suppl 4): D20. [11] KWATRA G, CUNNINGTON MC, MERRALL E, et al. Prevalence of maternal raniantion with group B Streptococcus: a systematic review and meta-analysis[J]. Lancet Infect Dis, 2016, 16(9): 1076. doi: 10.1016/S1473-3099(16)30055-X [12] DING Y, WANG Y, HSIA Y, et al. Systematic review and meta-analyses of incidence for group B streptococcus disease in infants and antimicrobial resistance, China[J]. Emerg Infect Dis, 2020, 26(11): 2651. doi: 10.3201/eid2611.181414 [13] LANG S, PALMER M. Characterization of Streptococcus agalactiae CAMP factor as a pore-forming toxin[J]. J Biol Chem, 2003, 278(40): 38167. doi: 10.1074/jbc.M303544200 [14] JIN T, BREFO-MENSAH E, FAN W, et al. Crystal structure of the Streptococcus agalactiae CAMP factor provides insights into its membrane-permeabilizing activity[J]. J Biol Chem, 2018, 293(30): 11867. doi: 10.1074/jbc.RA118.002336 [15] GOUDARZI G, GHAFARZADEH M, SHAKIB P, et al. Culture and real-time PCR based maternal screening and antibiotic susceptibility for group B Streptococcus: an ranian experience[J]. Glob J Health Sci, 2015, 7(6): 233. [16] TANAKA K, IWASHITA M, MATSUSHIMA M, et al. Intrapartum group B Streptococcus screening using real-time polymerase chain reaction in Japanese population[J]. J Matern Fetal Neonatal Med, 2016, 29(1): 130. doi: 10.3109/14767058.2014.989496 [17] HASSAN AA, AKINEDEN O, LAMMLER C, et al. Molecular characterization of phenotypically CAMP-negative Streptococcus agalactiae isolated from bovine mastitis[J]. J Vet Med B Infect Dis Vet Public Health, 2002, 49(5): 257. doi: 10.1046/j.1439-0450.2002.00553.x [18] RAJAGOPAL L. Understanding the regulation of Group B Streptococcal virulence factors[J]. Future Microbiol, 2009, 4(2): 201. [19] HENSLER ME, QUACH D, HSIEH CJ, et al. CAMP factor is not essential for systemic virulence of Group B Streptococcus[J]. Microb Pathog, 2008, 44(1): 84. doi: 10.1016/j.micpath.2007.08.005