Berche P
Laboratoire de microbiologie, hôpital Necker-Enfants Malades, Paris.
Rev Prat. 1994 Apr 1;44(7):855-64.
The recognition and the identification of previously unrecognized infectious agents require a multidisciplinary approach to specify the nosologic entity of the disease and the epidemiological data, especially the modes of transmission and the risk factors, as well as to discover the microorganism in the laboratory. In the past 20 years, significant breakthroughs have been achieved in cellular cultures (growth factors), in immunology (monoclonal antibodies), and moreover in molecular biology, which have been widely used in the field of infectious diseases. Whereas the classical methods used to grow microorganisms remain of major interest in many cases, innovating strategies have been recently designed to identify previously unknown pathogens. The genomic amplification by polymerase chain reaction (PCR) of highly conserved bacterial genes (as those coding for ribosomal RNA), from tissue biopsies for example, allow to recognize unknown bacteria. The evolutionary distance between a newly recognized pathogen and known microorganisms can be calculated through sequencing of these genes, as described for Rochalimaea henselae or Tropheryma whipplelii. The constitution of cDNA banks from infected tissues is also a novel approach allowing to clone and sequence viral genes, such as those from hepatitis C or from hepatitis E. In the near future, noteworthy improvements will be achieved to rapidly detect microorganisms with highly sensitive and specific tests using monoclonal antibodies, molecular probes (including branched DNA) and with PCR (including Q beta replicase and ligase chain reaction), and to determine the genetic diversity of microbial pathogens by new methods as pulse field gel electrophoresis or arbitrarily primed PCR. This will result in a better knowledge of the pathophysiology of infectious diseases, in a better recognition of a typical, previously unrecognized clinical expression of pathogenicity, and also in a more precise assessment of the actual impact of a given pathogen in human populations by highly sophisticated diagnosis tests.
识别和鉴定以前未被认识的传染原需要采用多学科方法来确定疾病的病种实体和流行病学数据，尤其是传播方式和危险因素，同时还要在实验室中发现微生物。在过去20年里，细胞培养（生长因子）、免疫学（单克隆抗体）以及分子生物学领域都取得了重大突破，这些技术已在传染病领域广泛应用。尽管用于培养微生物的经典方法在许多情况下仍然至关重要，但最近已设计出创新策略来识别以前未知的病原体。例如，通过聚合酶链反应（PCR）对高度保守的细菌基因（如编码核糖体RNA的基因）进行基因组扩增，可从组织活检样本中识别未知细菌。正如对汉赛巴尔通体或惠普尔养障体所描述的那样，通过对这些基因进行测序，可以计算出新识别病原体与已知微生物之间的进化距离。从受感染组织构建cDNA文库也是一种新方法，可用于克隆和测序病毒基因，如丙型肝炎或戊型肝炎病毒的基因。在不久的将来，使用单克隆抗体、分子探针（包括分支DNA）和PCR（包括Qβ复制酶和连接酶链反应）等高度灵敏和特异的检测方法将取得显著改进，能够快速检测微生物，并通过脉冲场凝胶电泳或任意引物PCR等新方法确定微生物病原体的遗传多样性。这将有助于更好地了解传染病的病理生理学，更好地识别以前未被认识的典型致病性临床表型，还能通过高度精密的诊断测试更精确地评估特定病原体在人群中的实际影响。
