New diagnostic method for pneumocystis using flow cytometry
Abstract number: p858
Pina Vaz C., Costa S., deOliveira G., Monteiro T., Carvalho A., Rodrigues
Pneumocystis jiroveci is an opportunistic fungal agent infecting immunocompromised hosts like AIDS and those receiving immunotherapy. Diagnosis of pneumonia due to Pneumocystis requires an accurate method. The laboratory diagnosis is based upon the detection of the agent using fluorescent monoclonal antibodies on respiratory samples using commercial kits. The organism does not grow in culture and nucleic acid amplification is still performed only in research setting. Flow cytometry has been used as a valuable tool on microbiology showing several advantages.
Materials and methods:
Two hundred and twenty respiratory samples (188 bronchial or bronchoalveolar samples and 32 traqueal aspirates secretions) were evaluated. The last were treated with N-acetil-cysteine prior to analysis. The samples were centrifuged and the sediment evaluated according two methods. For fluorescence microscopy the samples were smeared on a slide and stained with 25 ml of the monoclonal antibody of MerifluorR- Pneumocystis. To optimize the staining serial concentrations of the specific monoclonal (5, 10, 15, 20 and 25 ml), were used to stain positive samples. Thereafter, that all the samples were stained with 5 ml, centrifuged (10 min at 3000 rpm) and resuspended on deionized water for flow cytometry analysis (FC). FC acquisition protocol was optimized in order to define a scattergram and to determine the intensity of green fluorescence, FL1. A threshold of detection was evaluated after diluting a positive sample and performing both analysis, that is microscopy and FC analysis. Negative samples were contaminated with 25 ml of suspensions (0.5 McFarland) of rods, cocci and yeasts.
All the positives samples by microscopy were positive by flow cytometry. FC detected eight positive samples that were negative by microscopy. Such cases correspond to AIDS patients with CD4 count below 200/mm3, presenting fever and respiratory infection; they were treated as positive and seven improved. Samples contaminated with bacteria or fungi did not show increased fluorescence that is, no unspecific staining. FC using a specific fluorescent antibody, proved to be sensitive and useful to detect Pneumocystis.
|Session name:||XXIst ISTH Congress|
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