Development and Characterization of a Molecular Viability Assay for<i>Pneumocystis carinii</i>f sp<i>hominis</i>

Maher, Nancy; Vermund, Sten H.; Welsh, David A.; Dillon, Hugh C.; Awooda, Abeer; Unnasch, Thomas R.

doi:10.1086/320738

Cited by 30 publications

(33 citation statements)

References 11 publications

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“…This observation established that P. jirovecii DNA can be exhaled by an infected patient (Sing et al, 1999a). P. jirovecii cDNA was also detected using RT-PCR in air samples from the rooms of patients who developed PCP, showing that the microorganism may still be viable and therefore potentially infectious in the patient's environment (Latouche et al, 2001;Maher et al, 2001). A report by Vargas and colleagues is consistent with transmission by the airborne route.…”

Section: Airborne Transmission Of P Jiroveciisupporting

confidence: 56%

NosocomialPneumocystis jiroveciiinfections

et al. 2008

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Summary :Airborne transmission of Pneumocystis sp. from host to host has been demonstrated in rodent models and several observations suggest that interindividual transmission occurs in humans. Moreover, it is accepted that the Pneumocystis organisms infecting each mammalian species are host specific and that the hypothesis of an animal reservoir for Pneumocystis jirovecii (P. jirovecii), the human-specific Pneumocystis species, can be excluded. An exosaprophytic form of the fungus cannot be strictly ruled out. However, these data point toward the potential for the specific host to serve as its own reservoir and for Pneumocystis infection in humans as an anthroponosis with humans as a reservoir for P. jirovecii. This review highlights the main data on host-to-host transmission of Pneumocystis in rodent models and in humans by the airborne route and provides a rationale for considering the occurrence of nosocomial infections and measures for their prevention KEY WORDS : Pneumocystis jirovecii, nosocomial infections, genotyping.

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Section: Airborne Transmission Of P Jiroveciisupporting

confidence: 56%

NosocomialPneumocystis jiroveciiinfections

et al. 2008

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“…Molecular viability assays targeting P. carinii f. sp. carinii mRNA have also been described recently (6,13). However, such assays are not suitable for quantifying the numbers of organisms, since the number of mRNA molecules per organism is unknown and since one cannot assume that the amount of mRNA per organism is the same under all conditions.…”

Section: Vol 40 2002mentioning

confidence: 99%

Development of a Rapid Real-Time PCR Assay for Quantitation ofPneumocystis cariniif. sp.carinii

Larsen

Kovacs

Stock³

et al. 2002

J Clin Microbiol

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A method for reliable quantification of Pneumocystis carinii in research models of P. carinii pneumonia (PCP) that is more convenient and reproducible than microscopic enumeration of organisms would greatly facilitate investigations of this organism. We developed a rapid quantitative touchdown (QTD) PCR assay for detecting P. carinii f. sp. carinii, the subspecies of P. carinii commonly used in research models of PCP. The assay was based on the single-copy dihydrofolate reductase gene and was able to detect <5 copies of a plasmid standard per tube. It was reproducibly quantitative (r ‫؍‬ 0.99) over 6 log values for standards containing >5 copies/tube. Application of the assay to a series of 10-fold dilutions of P. carinii organisms isolated from rat lung demonstrated that it was reproducibly quantitative over 5 log values (r ‫؍‬ 0.99). The assay was applied to a recently reported in vitro axenic cultivation system for P. carinii and confirmed our microscopy findings that no organism multiplication had occurred during culture. For all cultures analyzed, QTD PCR assays showed a decrease in P. carinii DNA that exceeded the expected decrease due to dilution of the inoculum upon transfer. In conclusion, a rapid, sensitive, and reproducible quantitative PCR assay for P. carinii f. sp. carinii has been developed and is applicable to in vivo as well as in vitro systems. The assay should prove useful for conducting studies in which quantification of organism burden or growth assessment is critical, such as in vitro antimicrobic susceptibility testing or in vivo immunopathological experiments.

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“…Routine qualitative RT-PCR assay has been used in assessing the viability of microorganisms by targeting the heat shock protein 70 mRNAs of Pneumocystis carinii (11), Giardia cysts, and Cryptosporidium oocysts (9) and the actin mRNA of C. albicans (14). In this study, we quantified C. albicans actin mRNA as a means of assessing the viability of C. albicans in a reconstituted-human-skin model of cutaneous candidiasis following the application of the antimycotic.…”

Section: Discussionmentioning

confidence: 99%

“…For this reason, reverse transcription (RT)-PCR of mRNA molecules has been used to assess the viability of microorganisms (9,11,14). However, RT-PCR is a qualitative assay, and this places a limitation on the inferences that can be drawn from the result; differences in the quantities of target mRNAs cannot easily be ascertained from the intensities of amplification bands.…”

Section: The Lightcycler System (Two-step Reverse Transcription-pcr-fmentioning

confidence: 99%

Quantification of Candida albicans Actin mRNA by the LightCycler System as a Means of Assessing Viability in a Model of Cutaneous Candidiasis

2001

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The LightCycler system (two-step reverse transcription-PCR-fluorescent hybridization [LC RT-PCR-FH]) was used to quantifyCandida species are frequently found as saprophytes that colonize the surfaces of certain mucous membranes in human. The superficial and disseminated infections caused by them are largely opportunistic, and Candida albicans accounts for most of the etiologic species isolated from cutaneous candidiasis. Microscopic demonstration of yeast elements in potassium hydroxide preparations of infected tissue and cultural isolation of yeast from skin scrapings are used to confirm clinical diagnosis of cutaneous candidiasis. Rapid and highly sensitive diagnostic assays, based on PCR of DNA, have also been used to identify candidal pathogens in tissue (3,4,5,7,8).The amplification of a targeted DNA sequence by PCR does not, however, indicate the viability of the source organism, important information for evaluating the efficacy of a particular therapy and thus for avoiding an unnecessary prolongation of therapy. For this reason, reverse transcription (RT)-PCR of mRNA molecules has been used to assess the viability of microorganisms (9,11,14). However, RT-PCR is a qualitative assay, and this places a limitation on the inferences that can be drawn from the result; differences in the quantities of target mRNAs cannot easily be ascertained from the intensities of amplification bands. A determination of quantitative differences is needed in an assessment of the potencies of different concentrations of an agent or the progressive efficacy of a particular treatment. Furthermore, with the template at low levels, bands are seldom visualized in gel, resulting in falsenegative interpretations (14).In this study, we estimated the viability of C. albicans in a reconstituted-skin model of cutaneous candidiasis by detection of C. albicans ACT mRNA using the LightCycler (LC) system (a two-step RT-PCR-fluorescent hybridization [FH] assay) (10). LC DNA-based identification of fungi has been shown to be highly sensitive and rapid (10), and we applied of this system to rapidly quantify C. albicans ACT mRNA. The ACT mRNA was specifically targeted because of the constitutive nature of the gene and the major role of actin in cell division (6,15).

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Development and Characterization of a Molecular Viability Assay forPneumocystis cariniif sphominis

Cited by 30 publications

References 11 publications

NosocomialPneumocystis jiroveciiinfections

NosocomialPneumocystis jiroveciiinfections

Development of a Rapid Real-Time PCR Assay for Quantitation ofPneumocystis cariniif. sp.carinii

Quantification of Candida albicans Actin mRNA by the LightCycler System as a Means of Assessing Viability in a Model of Cutaneous Candidiasis

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