DEVELOPMENT OF A CONCENTRATION TECHNIQUE FOR PNEUMOCYSTIS CARINII

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Pneumocystis carinii (PC) is a significant cause of pneumonia in AIDS patients and other immunocompromised hosts. Particularly in the latter case, as well as in AIDS patients being treated for PCP, the number of organisms present may be very few. Finding these rare cysts is often time-consuming and difficult. It would thus be of benefit to both patient-care and laboratory efficiency to find a simple method of concentrating Pneumocystis, particularly in bronchoalveolar lavage specimens. Such a method could be extended to use on sputa to provide a less invasive mechanism of diagnosing PCP. Investigations into the use of cytospin prepared on lavage specimens resulted in the determination of optimal technical consideration for this method including: volume to spin, types filters to use, optimal speed, common technical problems. After resolving these questions, a parallel study using traditional centrifuge preparations compared to Cytospin prepared slides showed that examining one small circle of the Cytospin slide was equivalent to reading an entire slide prepared by the traditional centrifuge method. On the basis of these results, Cytospin slides are now used instead of traditionally prepared slides. Attempts were also made to develop methods utilizing sputum instead of lavage for diagnosis. Sputum quality is more variable than lavage and frequently contains more bacteria and yeast than lavages; they are also often more mocoid or thick. A method was develop using a combination of dithiothreitol digestion, rhozyme-tween digestion followed by cytospin preparation which concentrates the PC but also maintains sufficient morphology for reliable identification of the organism. This method has been introduced for routine use in order to obviate the need for bronchoscopy in patients who can be diagnosed from the sputum specimen instead.