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Comparison of six different PCR methods for detection of Pneumocystis carinii
J J Lu1, C H Chen, M S Bartlett
1Department of Pathology, Tri-Service General Hospital and National Defense Medical Center, Taipei, Taiwan, Republic of China.
Abstract:
We have recently developed a nested PCR method which amplifies internal transcribed spacers (ITS) of the ribosomal RNA genes of Pneumocystis carinii. To determine whether this PCR method can be used to diagnose P. carinii infections, we examined 30 bronchoalveolar lavage (BAL) specimens that were shown microscopically to contain P. carinii organisms by the P. carinii ITS PCR (Pc-ITS-PCR) and five other PCR methods that have been described for detecting P. carinii in clinical specimens. The targets of these PCR methods are portions of 18S rRNA, mitochondrial (mt) rRNA, 5S rRNA, thymidylate synthase (TS), and dihydrofolate reductase (DHFR). We also examined five different fungi, including Saccharomyces cerevisiae, Candida albicans, Histoplasma capsulatum, Cryptococcus neoformans, and Aspergillus fumigatus to determine the specificity of these six PCR methods for P. carinii. All 30 BAL specimens were positive by both the Pc-ITS-PCR and the 18S rRNA gene PCR, whereas only 26 (87%), 18 (60%), 10 (33%), and 7 (23%) of 30 BAL specimens were positive by mt rRNA gene PCR, TS gene PCR, 5S rRNA gene PCR, and DHFR gene PCR, respectively. Although the 18S rRNA gene PCR had the same sensitivity as the Pc-ITS-PCR, it nonspecifically amplified S. cerevisiae and C. albicans. The TS gene PCR also produced false-positive PCR results with C. albicans and C. neoformans. None of the other PCR methods (Pc-ITS-PCR, mt rRNA gene, 5S rRNA gene, and DHFR gene PCR) amplified the control fungal DNA. Considering both sensitivity and specificity, we conclude that Pc-ITS-PCR is the most effective of the six PCR methods evaluated in this study for the detection of P. carinii in BAL specimens.
Insights
A new nested PCR method targeting Pneumocystis carinii internal transcribed spacers (ITS) offers superior sensitivity and specificity for diagnosing P. carinii infections. This Pc-ITS-PCR method is more effective than other PCR techniques for detecting the pathogen in bronchoalveolar lavage specimens.
Area of Science:
- Molecular Biology
- Mycology
- Infectious Diseases
Background:
- Accurate diagnosis of Pneumocystis carinii pneumonia (PCP) is crucial for effective treatment.
- Various PCR methods have been developed for P. carinii detection, but their diagnostic utility varies.
- The sensitivity and specificity of these PCR methods need rigorous evaluation.
Purpose of the Study:
- To evaluate a newly developed nested PCR method amplifying the internal transcribed spacers (ITS) of ribosomal RNA genes for P. carinii detection.
- To compare the diagnostic performance of this P. carinii ITS PCR (Pc-ITS-PCR) against five other established PCR methods.
- To assess the specificity of these PCR methods against common fungal contaminants.
Main Methods:
- A nested PCR assay targeting P. carinii internal transcribed spacers (Pc-ITS-PCR) was developed.
- Thirty bronchoalveolar lavage (BAL) specimens positive for P. carinii by microscopy were tested.
- Six PCR methods were evaluated: Pc-ITS-PCR, 18S rRNA, mitochondrial (mt) rRNA, 5S rRNA, thymidylate synthase (TS), and dihydrofolate reductase (DHFR) gene PCRs.
- Specificity was tested using five non-P. carinii fungal species.
Main Results:
- Pc-ITS-PCR and 18S rRNA gene PCR detected P. carinii in all 30 BAL specimens.
- Sensitivity varied for other methods: mt rRNA (87%), TS (60%), 5S rRNA (33%), and DHFR (23%).
- 18S rRNA gene PCR showed cross-reactivity with Saccharomyces cerevisiae and Candida albicans.
- TS gene PCR yielded false positives with C. albicans and Cryptococcus neoformans.
- Pc-ITS-PCR, mt rRNA, 5S rRNA, and DHFR gene PCRs demonstrated high specificity, with no amplification of control fungi.
Conclusions:
- The P. carinii ITS PCR (Pc-ITS-PCR) demonstrates high sensitivity and specificity for detecting P. carinii in BAL specimens.
- Pc-ITS-PCR is superior to the other evaluated PCR methods, including 18S rRNA gene PCR, for diagnosing P. carinii infections.
- The nested PCR method targeting ITS provides a reliable diagnostic tool for P. carinii detection.