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

我们建立了间接免疫荧光试验(IF)和乳胶凝集试验(LAT)来检测呼吸道分泌物中肺炎支原体，作为肺炎支原体感染的早期快速诊断方法。此外，将IF和LAT与唯一商业化的快速检测微生物的DNA探针试验(DP)进行了比较。首先，制备了针对肺炎支原体的多克隆抗体。该抗体与多种支原体发生交叉反应，但不与正常人血清或健康人呼吸道分泌物交叉反应。当被马血清和酵母提取物吸收时，抗体与除雷氏支原体以外的其他支原体的交叉反应完全减弱。培养基组分。--M.生殖器在LAT中与吸附的抗体有交叉反应，在DP中也有交叉反应。然而，与雷氏分支杆菌的交叉反应不太可能引起重大的诊断混乱，因为生殖支原体的滴度是S…。比肺炎支原体低得多，生殖道支原体是唯一的生殖道定殖者。因此，IF、LAT和DP具有较高的特异性，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。在血清学确诊肺炎支原体感染患者的临床标本(咽拭子)中，间接免疫荧光试验阳性率为85.7%。在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%。将悬液和混合液(肺炎支原体悬液和呼吸道渗出物)在37ﾟC下孵育，按时间进程除去等量，并检测LAT和DP。在LAT中，样品在三天内显示相同的滴度。在5h内，各样本的滴度(样本-CPM与阴性-对照-CPM的比率)显著下降，认为LAT和IF中的靶SS‘分子聚集在咽部。然而，核糖体RNA(核糖体RNA)中的靶分子被破坏的速度要快得多，积聚也是意想不到的。临床标本中肺炎支原体阳性率低的原因可能与靶分子的快速分解有关，因此，IF和LAT必须适用于临床实验室患者呼吸道渗出物中肺炎支原体的检测。特别推荐使用LAT，因为LAT的过程可以在半小时内完成，不需要复杂的程序。虽然临床标本中LAT的阳性率略低于IF。现在我们正在研究一种处理临床样本的过程，以提高LAT的检测下限。较少

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