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 significantly 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.