ABSTRACT
Direct culture is the most common way to reliably detect mycoplasma, but it is not practical for the qualitative control of cell therapeutics because of the elaborate culture medium, the prolonged incubation time, and the large sample volumes. Here, we chose two alternative methods using commercial detection kits, the PCR mycoplasma detection kit with nested PCR and the selective biochemical method, MycoAlert(®), and validated them with the direct culture method as a reference. We tested eight mycoplasma species and five validation parameters: specificity, detection limit, robustness, repeatability, and ruggedness, based on the regulatory guidelines in the US Pharmacopoeia. All experiments were performed using fibroblasts spiked with mycoplasma. Specificity tests for both methods included all mycoplasma species, except Mycoplasma pneumonia and M. genitalium for the nested PCR and Ureaplasma urealyticum for the MycoAlert(®) assay. Regarding the detection limit, the nested PCR proved to be as sensitive as the direct culture method and more sensitive than the MycoAlert(®) assay. The predicted median for probit = 0.9 was 54 (44-76) CFU/ml for M. hyorhinis and 16 (13-23) CFU/ml for M. hominis by the nested PCR, but 431 (346-593) CFU/ml and 105 (87-142) CFU/ml, respectively, with MycoAlert(®). Changes in the concentration of reagents, reagent lot, or individual analysts did not influence the results of the examined methods. The results of this study support nested PCR as a valuable alternative for mycoplasma detection.
Subject(s)
Mycoplasma Infections/diagnosis , Mycoplasma/isolation & purification , Polymerase Chain Reaction/methods , Colony Count, Microbial , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Fibroblasts , Humans , Limit of Detection , Mycoplasma/enzymology , Mycoplasma/genetics , Mycoplasma Infections/microbiology , Polymerase Chain Reaction/standards , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
PURPOSE: The effects of air cleaners on the removal of airborne indoor allergens, especially house dust mites (HDM), are still controversial. The objective of this study is to evaluate the effect of an air cleaner with an electrostatic filter on the removal of airborne mite allergens. MATERIALS AND METHODS: A dried HDM culture medium that contained mite body particles and excretions was dispersed in a chamber equipped with an electrostatic air cleaner. The number of airborne particles was recorded continuously by a dust spectrometer for 60 minutes. Airborne particles in the chamber were collected on a sampling filter at a flow rate of 10 L/min and the Der f 1 concentration in the filter extracts was measured by two-site ELISA. RESULTS: The air cleaner efficiently removed airborne HDM particles. The air cleaner removed airborne HDM particles (size 2-12.5 µm) 11.4 ± 2.9 fold (cleaner operating for 15 minutes), 5.4 ± 0.7 fold (cleaner operating for 30 minutes), and 2.4 ± 0.2 fold (cleaner operating for 60 minutes) more than the removal of HDM particles by natural settle down. Removal kinetics differed according to the particle size of the airborne particles. The air cleaner decreased the concentration of Der f 1 in the extraction of airborne particles collected on the air sampling filter by 60.3%. CONCLUSION: The electrostatic air cleaner can remove airborne HDM allergens and may be useful as a supplementary environmental control tool for HDM sensitized respiratory allergic patients.
