Development and Evaluation of a Molecular Viability Assay for Pneumocystis carinii

Abstract

Despite recent declines in incidence, Pneumocystis carinii pneumonia (PCP) remains the most commonly occurring opportunistic illness among persons with AIDS in the United States. While P. carinii DNA has been detected in patient respiratory specimens and in air samples collected from various indoor environments housing PCP patients, the viability of these organisms is unknown. For this reason, we have developed and evaluated a molecular viability assay for P. carinii. This method is based upon the detection of P. carinii mRNA by a reverse transcription-PCR that employs specific primers from a member of the heat shock protein 70 family. Under optimal assay conditions, these primers were capable of detecting as few as 100 viable trophozoites as determined by ethidium bromide staining, while no signal was obtained from 106 trophozoites killed by heat, desiccation, or UV radiation. This assay was also capable of distinguishing P. carinii from other common fungi present in the air. Therefore, this molecular viability assay may be useful in conjunction with standard bioaerosol collection devices and procedures for the detection of viable P. carinii collected from various indoor environments. It may also be useful in confirming the presence of viable trophozoites in respiratory specimens collected by noninvasive techniques from putatively infected individuals.

SYTO-13, a Viability Marker as a New Tool to Monitor In Vitro Pharmacodynamic Parameters of Anti-Pneumocystis Drugs

“…Moreover, some authors used detection of Heat Shock Protein 70 transcripts by RT-PCR to monitor the viability of P . jirovecii in bronchoalveolar lavage (BAL) fluids from patients developing PcP [ 32 , 33 ] or in air samples collected from the environment of PcP patients [ 33 ]. However, the concentration-effect relationship ( i .…”

“…The main reasons for this situation are (i) the microscopic observation of Pneumocystis is time consuming and requires a great expertise [ 25 , 26 ] and (ii) the assessment of Pneumocystis viability with high confidence and reproducibility is lacking [ 27 ]. Many methods have been described such as the incorporation of classical vital stains or fluorescent indicator compounds [ 26 , 28 , 29 ], the ATP bioluminescent assay [ 27 , 30 ], the uptake of radiolabelled methionine, uracil or thymidine [ 31 ], and the RT-PCR [ 32 , 33 ]. However, none of these overcomes the host cell debris interferences and none has permitted to monitor the concentration-effect relationships (i.e.…”

Molecular detection of Candida krusei contamination in fruit juice using the citrate synthase gene cs1 and a potential role for this gene in the adaptive response to acetic acid

“…In light of this fact the detection of mRNA gene transcripts, through the exploitation of a reverse transcriptase PCR assay, has gained widespread acceptance as a means of assessing the viability of organisms from a range of bacterial and fungal origins (Klein and Juneja 1997; Vaitilingom et al. 1998; Maher et al. 2000; Okeke et al.…”

Pneumocystis carinii Cyst is Associated with Inflammation in the Host

“…Organisms were isolated from severely infected rats by the method of Chin et al [4]. Cysts were isolated from mixed populations of P carinii as described previously [10]. Infected animals were assessed for organism burden by lung impression smears stained with Giemsa for trophozoites and Gomori methenamine silver for cysts, and scored as previously described [1].…”