Developmental Studies on the Laboratory diagnosis at an early stage of Mycoplasam pneumoniae-infection (mycoplasmal pneumonia)

我们开发了一种间接免疫荧光试验（IF）和乳胶凝集试验（LAT），用于检测呼吸道渗出物中的肺炎支原体，作为肺炎支原体感染早期的快速诊断。此外，IF和LAT与dna探针测试（DP）进行了比较，这是唯一可用于快速检测生物体的商业测试。首先制备肺炎支原体特异性多克隆抗体。该抗体与多种支原体均无交叉反应，但与正常人血清及健康人呼吸道分泌物无交叉反应。当与马血清和酵母提取物吸收时，抗体与除Renitalium外的支原体的交叉反应性完全降低。培养基成分。- m。生殖器官与吸收抗体在LAT和DP中表现出交叉反应。然而，与生殖道支原体的交叉反应性，不太可能引起显著的诊断混淆，因为生殖道支原体滴度低于。即肺炎支原体，而生殖道支原体是生殖道的专门定植者。因此，使用吸收抗体的IF和LAT和DP具有足够的特异性，可用于呼吸道渗出液中肺炎支原体的检测。IF的检出限为2 × 10^5 cfu/ml， LAT的检出限为2 × 10^5 cfu/ml， cif DP的检出限为5 × 10^4 cfu/ml。三种方法中，DP的灵敏度最高。在血清学确诊的肺炎支原体感染患者的临床标本（咽喉涂片）中，85.7%的患者IF检测呈阳性。培养法检出肺炎支原体的临床标本中，IF阳性率为73.3%，LAT阳性率为63.3%，DP阳性率为26.6%。观察了肺炎分枝杆菌悬浮液的孵育效果。将悬浮液和混合物（肺炎分枝杆菌悬浮液和呼吸道渗出液）在37℃下孵育。随时间推移去除等分物，测定LAT和DP值。样品在LAT中三天内滴度相同。每个样品的滴度（样品-cpm对阴性对照-cpm的比率）在5 h内显著下降，认为LAT和IF中的靶SS分子在咽部积累。然而，DP（核糖体RNA）中的靶分子破坏要快得多，积累是不可预料的。临床标本中DP阳性率低的原因可能是靶分子分解快。因此，IF和LAT必须适用于临床实验室对患者呼吸道渗出液中的肺炎支原体的检测。特别推荐LAT，因为LAT的程序可以在半小时内完成，没有复杂的方式。临床标本中LAT的阳性率略低于IF。现在我们正在研究一种处理临床样品的方法，以提高LAT的检出限。少

We developed an indirect immunofluorescence test (IF) and a latex agglutination test (LAT) for detection of Mycoplasma pneumonias in respiratory exudated as rapid diagnosis at an early stage of M. pneumoniae-infection. Further, IF and LAT were compared with DNA-probe test (DP) which was the only commercially available test for the rapid detection of the organism.Firstly, we prepared polyclonal antibody specific to M. Pneumoniae. The antibody cross-react with many species of mycoplasmas, but not with normal human serum or with respiratory exudates from healthy person. Cross-reactivity of the antibody with species of mycoplasmas other than M. Renitalium was fully diminished when absorbed with horse serum and yeast extract. components of culture medium. -M. genitalium showed a cross-reaction in LAT with using the absorbed antibody and also in DP. However, the cross-reactivity with M. Renitalium, is unlikely to cause significant diagnostic confusion because the titer of M. genitalium was s … More ignificantly lower than., that of M. pneumonia, and M. genitalium is exclusively a genital tract colonizer. Therefore, IF and LAT with using the absorbed antibody, and DP are specific enough to be used for the detection of M. pneumonia in respiratory exudates.The detection limit of IF was about 2 x 10^5 cfu/ml, that-, of LAT was 2 x 10^5 cfu/ml and that cif DP was 5 x 10^4 cfu/ml in vitro. DP had the highest sensitivity among three methods.Among clinical specimens(throat smears)from patients with serologically confirmed M. pneumoniae-infection, 85.7% gave a positive test in IF. Among clinical specimens in which M. pneumoniae was detected by culture method, positive rate in IF was 73.3%, that in LAT was 63.3% and that in DP was 26.6%.A effect of the incubation of M. pneumonia suspension was examined. The suspension and the mixture (M. Pneumoniae suspension and respiratory exudates) were incubated at 37 ﾟC. An aliquot was removed with time course, and examinedin LAT and DP. The samples showed the same titer within three days in LAT. The titer (rate of sample-cpm to negativp- control-cpm) of each sample decreased remarkably within 5 h. It was considered that target SS'olecules in LAT and IF were accumulated in the pharyngeal portion. However, target molecule in DP (ribosomal RNA) was destructed much sooner, and the accumulation could not be expected. The reason f. or the low positive rate of DP in clinical'specimens may be due to the fast breakdown of target molecule.Consequently, IF and LAT must be applicable to the detection of M. pneumoniae in respiratory exudates from the patients in clinical laboratories. Especially, LAT was recommended because the procedure of LAT can be completed within a half hour without complicated manner. though the positive rate of LAT in clinical specimens was slightly lower than that of IF. Now we are examinating a process of treating clinical samples for rising the detection limit of LAT. Less