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 10(6) 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.

FIG. 1

Specific amplification of PcSA1 sequences from P. carinii mRNA. (A) Design of primers for the PcSA1 RT-PCR assay. The 5′ (coding orientation) primer crosses a 58-bp intron to prevent the amplification of contaminating genomic DNA derived from P. carinii. The exon sequences are shown in uppercase letters, and the intron sequence is shown in lowercase letters. (B) Results from the PcSA1 RT-PCR assay. Lane 1, RT-PCR carried out on total RNA extracted from 10⁶ P. carinii organisms derived from a spinner-flask culture; lane 2, control RT-PCR lacking reverse transcriptase carried out on total RNA extracted from 10⁶ P. carinii organisms derived from a spinner-flask culture; lane 3, PCR negative control. +RT, with reverse transcriptase; −RT, without reverse transcriptase.

FIG. 2

Sensitivity of the PcSA1 RT-PCR. RT-PCRs were carried out on total RNA preparations prepared from log-unit dilutions of viable P. carinii. Lane 6, 10⁶ P. carinii organisms; lane 5, 10⁵ P. carinii organisms; lane 4, 10⁴ P. carinii organisms; lane 3, 10³ P. carinii organisms; lane 2, 10² P. carinii organisms; lane 1, 10¹ P. carinii organisms; lane −RT, without reverse transcriptase (RNA from 10⁶ organisms); lane −, PCR negative control.

FIG. 3

Discrimination of viable from nonviable organisms by the PcSA1 RT-PCR. RT-PCR assays were carried out as described in Materials and Methods by employing RNAs extracted from treated cultures of P. carinii as templates. Lane 1, RNA derived from 10⁴ untreated P. carinii organisms; lane 2, RNA derived from 10³ untreated organisms; lane 3, RNA derived from 10² untreated organisms; lane 4, RNA derived from 10⁶ P. carinii organisms killed by desiccation; lane 5, RNA derived from 10⁶ organisms killed by heat; lane 6, RNA derived from 10⁶ organisms killed by UV light; lane 7, no RNA; lane 8, PCR negative control.

FIG. 4

Species specificity of the PcSA1 RT-PCR. RT-PCRs were carried out on fungal RNA preparations prepared as described in Materials and Methods. (A) EB-stained gel of PCR products; (B) Southern blot of the gel shown in panel A probed with the labeled PcSA1 PCR product. In each panel, lane 1 contains RNA extracted from P. carinii, lane 2 contains RNA extracted from S. cerevisiae, lane 3 contains RNA extracted from Penicillium chrysogenum, and lane 4 contains RNA extracted from A. niger.

FIG. 5

Detection of P. carinii cysts by the PcSA1 RT-PCR. Lane 1, poly(A)⁺ mRNA extracted from a cyst-enriched preparation; lane 2, RNA derived from a spinner-flask culture; lane 3, cyst-enriched preparation of RNA amplified in the absence of reverse transcriptase; lane 4, PCR negative control.