新生儿败血症早期诊断指标的研究进展

1、新生儿败血症早期诊断指标的研究进展浙江大学医学院附属儿童医院 俞惠民 310003 杭州新生儿尤其是早产儿由于免疫功能不成熟，败血症发生率较高，目前仍是导致新生儿死亡或远期不良预后的重要原因。有资料表明，21%的VLBW至少有一次确诊败血症。由于新生儿败血症早期症状不典型、易于与其他疾病混淆，但病情进展迅速，故早期诊断和及时治疗是改善预后的关键。血培养是诊断的金标准，但阳性率低、需时较长，一些常用的血液学指标包括白细胞分类计数、I/T、血小板计数、微量ESR等敏感性和特异性均很差，不能满足临床诊断要求。理想的早期诊断指标应有明确的界值，其敏感性和阴性预报值接近100%，同时特异性和阳性预报值应

2、大于85%；能够鉴别病原菌的种类，如细菌或真菌、G阳性菌或阴性菌；能够检测疾病的进展或严重程度，以指导治疗并判断预后等。近年来，针对败血症实验室早期诊断指标的探索主要包括非特异性和特异性指标两大类。非特异性指标主要有急相反应蛋白、细胞因子和趋化因子、细胞表面抗原，特异性指标主要有细菌DNA检测。一、急相反应蛋白C反应蛋白（CRP）已广泛应用于临床，它由肝脏产生，细菌感染时水平显著升高可达1000倍以上，病毒感染一般不升高，特异性较高，检测方便快速（POCT）、价廉，但反应相对延迟，暴露于微生物产物6-8小时开始升高，敏感性相对较低，在严重的局部感染时可能不升高，而在其他情况也可见升高，如手术、

3、组织损伤和疫苗接种等。连续测定不升高有助于排除败血症。降钙素原（PCT）是降钙素的前体,主要在甲状腺滤泡旁细胞内合成,是由116个氨基酸组成的糖蛋白。PCT在酶的作用下逐步裂解成氨基末端PCT,32个氨基酸的CT和21个氨基酸的降钙蛋白。PCT是11号染色体上降钙素I基因(CALC-I)的表达产物。无感染时，甲状腺外CALC-1表达被抑制，限于甲状腺和肺的神经内分泌细胞有一定程度的表达。健康人血液中浓度非常低而稳定，< 0.05ng/ml。细菌感染时诱导全身各种组织(肝脏，其它如单核细胞、脾、肺或小肠的神经内分泌细胞等)多种类型细胞CALC-I表达和PCT连续性释放入血液循环反应快速，感

4、染开始后3小时即可测得，6-12小时后达到峰值。临床评价PCT敏感性较CRP为高，诊断新生儿败血症的敏感性和特异性分别可达81%和79%。PCT水平变化与病情有较好的相关性，可用于指导抗生素疗程。但新生儿出生后有一过性增高，<3天的新生儿正常界值应根据日龄进行矫正。血清淀粉样物质A（SAA）家族包含不同的表达产物，急性期SAA（A-SAA）和结构性SAA(C-SAA)是两种主要的表达产物。急性期SAA是一种急性时相蛋白，有104个氨基酸残基组成，在人体中由11号染色体上的SAA1和SAA2两个等位基因共同编码。生理情况下，人体血浆中A-SAA的含量极低，但当机体遭受感染、创伤时，急性期S

5、AA会在短时间内升高1000倍。在体内，SAA通过细胞因子IL-1、IL-6、TNF-介导的信号转导在肝脏生成。临床研究表明，SAA反应较CRP快，在早期诊断早产儿晚发型败血症中具有高的灵敏性（100）、特异性（93）以及阳性预测率(96), 具有较好的应用前景。二、细胞因子和趋化因子细胞因子是全身感染的“早期报警”指标，尤其是促炎细胞因子IL-6、IL-8等，早期即快速反应增高，然后刺激肝脏合成CRP等其他炎症介质，故其敏感性非常高，但由于半衰期非常短，不易掌握检测最佳时机，如与半衰期较长的IL-1ra同时检测，可在临床症状出现前诊断感染。其他相关促炎细胞因子或趋化因子尚有TNF-a、MCP

6、-1、RANTES等。抗炎细胞因子与促炎细胞因子的平衡或失衡可反映机体炎症的过程及其程度，IL-10/TNF-a比值升高可能提示重症败血症，预后不良。虽然细胞因子和趋化因子有诸多优势，但临床并没有得到普遍应用，主要原因是方法学上的障碍，如不能自动化检测、人工技术要求较高，不能随时按需检测，费用高等。三、细胞表面表记物(抗原)炎症细胞未遭激活时，其表面标记物表达水平极低，在暴露于病毒或细菌产物如内毒素、脂多糖等后数分钟即可表达显著增高，用流式细胞仪可定量或半定量检测到。表面标记物具有细胞特异性，反应快速，需用血量极少，4小时可出结果。研究较多的有：T淋巴细胞的CD45RA、CD45RO、CD13

7、、CD19、CD25、CD26、CD69、CD71、HLA-DR; 中性粒细胞的CD11b、CD11c、CD13、CD15、CD33、CD64、CD66b；NK细胞的CD69、HLA-DR其中以CD64 和CD11b最为重要。CD64诊断新生儿早发型和晚发型败血症的敏感性较高远远早于CRP，它还是腹腔内感染，包括NEC、肠穿孔和腹膜炎的良好指标。CD11b也是诊断新生儿早发型败血症的敏感指标，甚至在临床症状出现3天以前就显著升高，但在其他非感染疾病也有增高，如RDS。两者比较CD64要优于CD11b。细胞表面抗原的不足之处是流式细胞仪检测属于半自动方法，对技术员要求较高，尚不能在NICU常规开

8、展。四、分子生物学检测方法定量PCR可快速检测无菌体液中是否存在细菌DNA，尤其是其他传统微生物检测方法阴性时是较敏感的检测手段。分子技术如荧光原位杂交（FISH）可大大缩短阳性细菌鉴定时间，细菌只需18小时，真菌也只需42小时。定量PCR结合探针杂交技术尚可鉴别G阳性抑或G阴性菌，具有很高的特异性和阳性预报值，尤其是对G阴性菌，这对判断细菌毒力和感染严重程度、指导抗生素选择有较大的实用价值。细菌感染分子诊断的主要局限是：少见细菌的遗传序列没有包含在探针中会导致漏诊G阳性菌和真菌因有细胞壁不易降解和破坏，导致DNA的提取困难。我院用PCR方法检测细菌基因保守片段16SrRNA结合DNA基因芯片

9、技术诊断新生儿败血症敏感度可达100%，特异度也达97.8%。另外，采用微芯片技术检测耐细菌药基因数小时可出报告有利于早期选择敏感的抗生素，提高治愈率。基因组技术检测组织特异性mRNA可能为发现疾病特异的标记物奠定基础。质谱蛋白质组学技术提供了“自由假设”途径，可用于发现和识别宿主的反应蛋白作为各种病理情况的诊断标志，包括败血症，因此具有较好的应用前景，但仍需高质量的临床研究验证。总之，虽然近年来研究发现了一些早期实验室指标来诊断新生儿败血症，这些诊断方法对提高诊断敏感性、缩短诊断时间有很大帮助，但仍然不能取代传统的血培养。目前尚无理想的单一标志物能用于早期诊断，但根据病程将各种方法联合应用，

10、取长补短，可使新生儿败血症的诊断更加快速和敏感，有利于改善预后并减少不必要的抗生素使用。主要参考文献：1. Ng PC. Diagnostic markers of infection in neonates. Arch Dis Child Fetal NeonatalEd 2004;89(3):22935.2. Stoll BJ, Hansen N, Fanaroff AA, et al. Late-onset sepsis in very low birth weight neonates: the experience of the NICHD Neonatal Research Net

11、work. Pediatrics 2002;110(2):28591.3. Lam HS, Ng PC. Biochemical markers of neonatal sepsis. Pathology 2008;40(2): 1418.4. Chan KY, Lam HS, Cheung HM, et al. Rapid identification and differential of Gram-negative and Gram-positive bacterial bloodstream infections by quantitative polymerase chain rea

12、ction in preterm infants. Crit Care Med 2009;37(8): 24417.5. Ng PC, Li K, Leung TF, et al. Early prediction of sepsis-induced disseminated intravascular coagulation with interleukin-10, interleukin-6, and RANTES in preterm infants. Clin Chem 2006;52(6):11819.6. Rodwell RL, Leslie AL, Tudehope DI. Ea

13、rly diagnosis of neonatal sepsis using a hematologic scoring system. J Pediatr 1988;112(5):7617.7. Howard MR, Smith RA. Early diagnosis of septicaemia in preterm infants from examination of peripheral blood films. Clin Lab Haematol 1999;21(5):3658.8. Da Silva O, Ohlsson A, Kenyon C. Accuracy of leuk

14、ocyte indices and C-reactive protein for diagnosis of neonatal sepsis: a critical review. Pediatr Infect Dis J 1995;14(5):3626.9. Yang J, Yang M, Xu F, et al. Effects of oxygen-induced lung damage on megakaryocytopoiesis and platelet homeostasis in a rat model. Pediatr Res 2003;54(3): 34452.10. Ng P

15、C, Cheng SH, Chui KM, et al. Diagnosis of late onset neonatal sepsis with cytokines, adhesion molecule, and C-reactive protein in preterm very low birth weight infants. Arch Dis Child Fetal Neonatal Ed 1997;77(3):2217.11. Pourcyrous M, Korones SB, Yang W, et al. C-reactive protein in the diagnosis,

16、management and prognosis of neonatal necrotizing enterocolitis. Pediatrics 2005;116(5):10649.12. Philip AG, Mills PC. Use of C-reactive protein in minimizing antibiotic exposure: experience with infants initially admitted to a well-baby nursery. Pediatrics2000;106(1):E4.13. Polin RA. The ins and out

17、s of neonatal sepsis. J Pediatr 2003;143(1):34. 14. Ng PC, Lee CH, Fok TF, et al. Central nervous system candidiasis in preterm infants: limited value of biochemical markers for diagnosis. J Paediatr Child Health 2000;36(5):50910.15. Arnon S, Litmanovitz I, Regev R, et al. Serum amyloid A protein is

18、 a useful inflammatory marker during late-onset sepsis in preterm infants. Biol Neonate 2005; 87(2):10510.16. Arnon S, Litmanovitz I, Regev R, et al. The prognostic virtue of inflammatory markers during late-onset sepsis in preterm infants. J Perinat Med 2004;32(2): 17680.17. Dandona P, Nix D, Wilso

19、n MF, et al. Procalcitonin increase after endotoxin injection in normal subjects. J Clin Endocrinol Metab 1994;79(6):16058.18. Chiesa C, Panero A, Rossi N, et al. Reliability of procalcitonin concentrations for the diagnosis of sepsis in critically ill neonates. Clin Infect Dis 1998;26(3):66472.19.

20、Guibourdenche J, Bedu A, Petzold L, et al. Biochemical markers of neonatal sepsis: value of procalcitonin in the emergency setting. Ann Clin Biochem 2002;39(2):1305.20. Baek YW, Brokat S, Padbury JF, et al. Inter-alpha inhibitor proteins in infants and decreased levels in neonatal sepsis. J Pediatr

21、2003;143(1):115.21. Lim YP, Bendelja K, Opal SM, et al. Correlation between mortality and the levels of inter-alpha inhibitors in the plasma of patients with severe sepsis. J Infect Dis 2003;188(6):91926.22. Behrendt D, Dembinski J, Heep A, et al. Lipopolysaccharide binding protein in preterm infant

22、s. Arch Dis Child Fetal Neonatal Ed 2004;89(6):5514.23. Pavcnik-Arnol M, Hojker S, Derganc M. Lipopolysaccharide-binding protein in critically ill neonates and children with suspected infection: comparison with procalcitonin, interleukin-6, and C-reactive protein. Intensive Care Med 2004;30(7):14546

23、0.24. Ward PA. The dark side of C5a in sepsis. Nat Rev Immunol 2004;4(2):13342.25. Huber-Lang M, Sarma JV, Rittirsch D, et al. Changes in the novel orphan, C5a receptor (C5L2), during experimental sepsis and sepsis in humans. J Immunol 2005;174(2):110410.26. Franz AR, Bauer K, Schalk A, et al. Measu

24、rement of interleukin 8 in combination with C-reactive protein reduced unnecessary antibiotic therapy in newborn infants: a multicenter, randomized, controlled trial. Pediatrics 2004;114(1):18.27. Ng PC, Li K, Chui KM, et al. IP-10 is an early diagnostic marker for identification oflate-onset bacter

25、ial infection in preterm infants. Pediatr Res 2007;61(1):938.28. Ku¨ster H, Weiss M, Willeitner AE, et al. Interleukin-1 receptor antagonist and interleukin-6 for early diagnosis of neonatal sepsis 2 days before clinical manifestation. Lancet 1998;352(9136):12717.29. Orlikowsky TW, Neunhoeffer

26、F, Goelz R, et al. Evaluation of IL-8 concentrations inplasma and lysed EDTA-blood in healthy neonates and those with suspected early onset bacterial infection. Pediatr Res 2004;56(5):8049. Schultz C, Strunk T, Temming P, et al. Reduced IL-10 production and receptor expression in neonatal T lymphocy

27、tes. Acta Paediatr 2007;96(8):11225.31. van Dissel JT, van Langevelde P, Westendorp RG, et al. Anti-inflammatory cytokineprofile and mortality in febrile patients. Lancet 1998;351(9107):9503.32. Ng PC, Li K, Wong RP, et al. Proinflammatory and anti-inflammatory cytokine responses in preterm infants

28、with systemic infections. Arch Dis Child Fetal Neonatal Ed 2003;88(3):20913.33. Weinschenk NP, Farina A, Bianchi DW. Premature infants respond to early-onset and late-onset sepsis with leukocyte activation. J Pediatr 2000;137(3):34550.34. Ng PC, Li K, Wong RP, et al. Neutrophil CD64 expression: a se

29、nsitive diagnostic marker for late-onset nosocomial infection in very low birth weight infants. Pediatr Res 2002;51(3):296303.35. Ng PC, Li G, Chui KM, et al. Neutrophil CD64 is a sensitive diagnostic marker for early-onset neonatal infection. Pediatr Res 2004;56(5):796803.36. Bhandari V, Wang C, Ri

30、nder C, et al. Hematologic profile of sepsis in neonates: neutrophil CD64 as a diagnostic marker. Pediatrics 2008;121(1):12934.37. Weirich E, Rabin RL, Maldonado Y, et al. Neutrophil CD11b expression as a diagnostic marker for early-onset neonatal infection. J Pediatr 1998;132(3):44551.38. Nupponen

31、I, Andersson S, Jarvenpaa AL, et al. Neutrophil CD11b expression and circulating interleukin-8 as diagnostic markers for early-onset neonatal sepsis. Pediatrics 2001;108(1):E12.39. Turunen R, Andersson S, Nupponen I, et al. Increased CD11b-density on circulatingphagocytes as an early sign of late-on

32、set sepsis in extremely low-birthweight infants. Pediatr Res 2005;57(2):2705.40. Nupponen I, Pesonen E, Andersson S, et al. Neutrophil activation in preterm infants who have respiratory distress syndrome. Pediatrics 2002;110(1):3641.41. Hodge G, Hodge S, Haslam R, et al. Rapid simultaneous measureme

33、nt of multiple cytokines using 100 microl sample volumesassociation with neonatal sepsis. Clin Exp Immunol 2004;137(2):4027.42. Wu YD, Chen LH, Wu XJ, et al. Gram stain-specific-probe-base real-time PCR for diagnosis and discrimination of bacterial neonatal sepsis. J Clin Microbiol 2008; 46(8):26139

34、.43. Peters RP, Savelkoul PH, Simoons-Smit AM, et al. Faster identification of pathogensin positive blood cultures by fluorescence in situ hybridization in routine practice. J Clin Microbiol 2006;44(1):11923.44. Jordan J. Molecular Diagnosis of Neonatal Sepsis. Clin Perinatol 2010;37(2): 4119.45. Pe

35、rreten V, Vorlet-Fawer L, Slickers P, et al. Microarray-based detection of 90 antibiotic resistance genes of grampositive bacteria. J Clin Microbiol 2005;43(5): 2291302.46. Call DR, Bakko MK, Krug MJ, et al. Identifying antimicrobial resistance genes withDNA microarrays. Antimicrobial Agents Chemother