- Email: [email protected]
PCR-based quantification of Pneumocystis carinii in in vitro systems
Microbes and Infection, 2, 2000, 737−743 © 2000 Éditions scientifiques et médicales Elsevier SAS. All rights reserved S1286457900003580/FLA
PCR-based quantification of Pneumocystis carinii in in vitro systems Ralph Hananoa,d, Joachim Fensterlea, Petra Nusserb, Kurt Reifenbergb, Stefan H. E. Kaufmanna,c* b
a Department of Immunology, University Clinics Ulm, Albert-Einstein-Allee 11, 89070 Ulm, Germany Laboratory Animal Research Unit, University Clinics Ulm, Albert-Einstein-Allee 11, 89070 Ulm, Germany c Max-Planck-Institute for Infection-Biology, Monbijoustr. 2, 10117 Berlin, Germany d Paul-Hartmann AG, Department of Science and Medicine, P.O.Box 1420, 89504 Heidenheim, Germany
(Received 13 January 2000; accepted 3 March 2000)
ABSTRACT – In many laboratories, PCR has become a routine method for the sensitive diagnosis of Pneumocystis carinii in patient samples. In contrast, quantification of fungal numbers in in vitro setups still largely relies on more conventional procedures such as histological stainings. These are time consuming and their applications are limited when dealing with small fungal numbers contaminated with tissue and cellular debris. This study presents a sensitive and rapid method for P. carinii quantification based on PCR analysis that can be easily integrated into standard detection procedures without requiring any major additional steps. P. carinii-specific PCR performed with total DNA extracted from both standard samples with known fungal numbers and experimental samples was quantified relative to PCR products of a standard concentration from a control plasmid added prior to DNA extraction. This measure controlled for variations in DNA extraction and PCR efficiency among the samples to be compared. The correlation between analyzed P. carinii-specific DNA and the actual fungal numbers employed was highly significant. © 2000 Éditions scientifiques et médicales Elsevier SAS plasmid / PCR / Pneumocystis carinii
1. Introduction Pneumocystis carinii pneumonia is a serious disease threatening immunocompromised patients, and it represents a major cause of premature mortality in AIDS patients [1, 2]. The ubiquitous nature of this fungus and the ease of its acquisition via the inhalatory route has promoted the establishment of reliable and sensitive detection assays. Frequently, respiratory specimens such as tissue samples or bronchoalveolar lavage (BAL) fluids contain abundant P. carinii organisms. Conventional detection procedures such as Gomori’s silver methenamine, Giemsa [3] and toluidine blue O staining [4] are usually sufficient for diagnosis. Less ambiguous and cumbersome determinations are achieved using antibodies directed against P. carinii [5], especially when directly labeled with fluorescence [6]. This approach allows detection of a relatively * Correspondence and reprints Address for correspondence: Max-Planck-Institute for Infection-Biology, Monbijoustr. 2, 10117 Berlin, Germany d
Current address
Microbes and Infection 2000, 737-743
small fungal load. With the development of P. cariniispecific PCR [7], a very sensitive diagnostic tool became available, and this was further fine-tuned with the application of nested PCR [8]. A major advantage of this method is the ability to detect minute fungal numbers in noninvasively collected samples, such as blood [9] and oral washes [10]. To determine total pulmonary fungal loads in small animal models, whole lungs are macerated in buffer, and tissue debris are removed by filtration and/or differential centrifugation. Subsequently, several dilutions are prepared, aliquots are transferred onto microscopic slides and stained appropriately. Usually P. carinii organisms are counted in a series of oil immersion fields, and average results are extrapolated to the whole lung. There are various modifications of this procedure, and the existing literature has proven these to be quite reliable as far as organ loads are concerned. When dealing with in vitro cultures or experimental setups, where it may be necessary to compare minute numbers of organisms, this method is less applicable [11]. Alternative methods used in previous reports include P. carinii-specific ELISA [12] or the 737
Original article
measurement of glucan synthase activity, which was correlated with P. carinii numbers [13]. These methods may be very accurate but require several methodological steps. In this report, we describe a reliable and reproducible procedure based on quantification of P. carinii-specific DNA that can determine P. carinii quantities in different in vitro setups. The addition of a standard concentration of commercially available plasmid DNA prior to total DNA extraction served as a control for DNA extraction and PCR efficiency. In addition to parasite detection, this method allows P. carinii quantification in parallel samples by a one-step procedure, without further need for cumbersome steps. Application of serial dilutions of a known P. carinii concentration served as a standard. The determination of actual fungal numbers is of particular value for comparing the effects of diverse in vitro treatments on parasite survival.
2. Materials and methods 2.1. Short-term cell culture
Ten diseased RAG-1–/– mutant mice (deficient in all mature T and B cells)(12th backcross to C57BL/6 mice) that had acquired disease naturally were lavaged with Dulbecco’s modified Eagle medium (DMEM; Gibco, Grand Island, NY). BAL fluids were combined and serial dilutions (undiluted; 1:4, 1:10, and 1:100) were prepared using DMEM. Dilutions served to obtain setups with various fungal numbers that differed by known factors (dilution factor) for comparative analysis. The precise fungal numbers were not relevant for our purpose and counting procedures were, therefore, unnecessary at this point. One ml of each dilution was transferred into 12-well plates (Nunc, Roskilde, Denmark) and cultured at 37 °C in a 7% CO2 humidified atmosphere for 3 h to allow macrophage adherence. This step was intended to simulate in vitro culture experiments. We assumed that parasite clearance would not commence at significant rates during this time, so that relative fungal numbers between the different dilutions would remain constant. Two replicates for each dilution were prepared. All animal experimentation protocols were cleared by the appropriate federal authorities. 2.2. DNA extraction
Supernatants from each well were transferred to separate centrifugation tubes and centrifuged at 700 g to pellet nonadherent cells that could potentially be associated with P. carinii. Supernatants were transferred to fresh tubes, which were centrifuged at 10 000 g for 20 min to pellet suspended P. carinii organisms. The following DNA extraction procedure was performed using a kit (QIAamp tissue kit 250; Quiagen, Hilden, Germany) according to the manufacturer’s instructions. Lysis buffer (100 µL) was added to each tube and pellets from both centrifugation steps were dissolved thoroughly. The tube contents of corresponding wells were then combined into the original well in the culture plate to join the complete fungal DNA from each sample. A further 100 µL lysis buffer was added to each tube to capture residues and then added to the corresponding wells, which now contained a total of 738
Hanano et al.
400 µL lysis buffer each. Using this approach, loss of P. carinii organisms/DNA was minimized. Into this mixture, 1 pg of the linearized plasmid pCI (Promega, Madison, WI) was added, and total DNA (composed of murine DNA, P. carinii DNA of an approximately 107-bp genome, with 13–20 chromosomes [14], and plasmid DNA [4008 bp]) was extracted to yield a final volume of 200 µL. A P. carinii standard was prepared from isolated parasites from pulmonary tissues of diseased RAG-1–/– mice. Parasites were counted using a standard protocol [15], and their concentration was adjusted to 3 × 106 nucleated organisms per mL. Serial dilutions were prepared (undiluted; 1:2; 1:4; 1:8; 1:10, 1:20), and subjected to total DNA extraction, including 1pg pCI. 2.3. Evaluation of optimal PCR cycle repetitions
To assess the optimal cycle number for analysis, DNA extractions of BAL fluids from five individual diseased –/– RAG-1 mice were used for P. carinii-specific PCR. Two PCR setups (P. carinii- and pCI-specific) of each BAL fluid sample, employing the same master-mix of PCR ingredients, were prepared simultaneously and PCR was performed with 10, 15, and 20 cycles. Following a previously established protocol [7], pAZ102-E (5’-GAT-GGCTGTTTCCAAGCCCA-3’) and pAZ102-H (5’-GTGTACGTTGCAAAGTACTC-3’), which amplify part of the gene encoding the large subunit of P. carinii mitochondrial rRNA, were employed as P. carinii-specific PCR primers. The PCR product is composed of 357 bp with a GC content of 41.6%. The pCI-specific PCR was performed using primers Amp-F (5’-TCCTGTTTTTGCTCACCC-3’) and Amp-R (5’-ACATGATCCCCCATGTTG-3’), amplifying part of the pCI plasmid encoding for ampicillin resistance (5’–2371–2785–3’). The sequences of these two primers were chosen using the computer program Prime (Genetic Computer Groups, Madison, WI). The following PCR protocol for pCI, using plasmid linearized through treatment with BamHI (MBI Fermentas Inc., Amherst, NY) was used: 10–20 cycles each comprised of 30 sec at 94 °C, 60 sec at 56 °C, and 90 sec at 72 °C. The PCR product is composed of 414 bp with a GC content of 47.6%. Amplified DNA amounts were determined in arbitrary units using a gel reader (Herolab, Wiesloch, Germany) with E.A.S.Y. (Enhanced Analysis System) software (Herolab), whereby band intensities and sizes were respected. To verify whether the DNA quantities measured in arbitrary units were directly proportional to the DNA amounts employed, three different dilutions of a commercially available DNA concentration standard (40 pg, 10 pg, and 5 pg, each in 20 µL distilled water) (Gibco) were resolved on 1% agarose gel. DNA quantities as measured by the gel reader were recorded. 2.4. Verification of the PCR product specificity
To assess whether the obtained PCR products were specific for P. carinii or pCI, they were blotted onto positively charged nylon membranes (Quiagen) and hybridized with specific 32P-labelled probes. The probes were derived from the plasmids pCI and pAZ102 (kindly provided by Dr A. Wakefield, Radcliffe Hospital, Oxford, UK), Microbes and Infection 2000, 737-743
Quantification of Pneumocystitis carinii
Original article
the latter containing rat derived P. carinii DNA (570 bp) cloned into the BamHI site of the vector pUC13. pAZ102 was treated with the restriction enzymes EcoRI and HindIII (MBI) to extract the inserted P. carinii DNA. The sequence of the P. carinii-specific PCR product lies within this fragment (5’–187–544–3’). The pCI was treated with XmnI and BmpI (MBI) to extract a DNA fragment (5’–2503–3034–3’) whose sequence largely overlaps with the pCI-specific PCR product sequence. Digestion products were separated on a 1% agarose gel and the respective fragments were extracted using a kit (QIAEX II gel extraction kit 150, Quiagen) according to the manufacturer´s instructions, and labeled with 32P, employing the Ready-to-go DNA labeling kit (-dCTP) (Pharmacia, Upsala, Sweden). Pure pCI and DNA from P. carinii-infected murine lung tissues served as positive controls for the respective PCR analyses, whereas DNA from immunocompetent murine blood cells or lung tissues served as negative controls. 2.5. Statistical analysis
Linear regression of obtained results is presented using Pearson’s correlation coefficient. Graphs and statistics were performed using the software Graph Pad Prism (Graph Pad Software Inc., San Diego, CA).
3. Results 3.1. P. carinii- and pCI-specific PCR
DNA extracted from lungs of diseased RAG-1–/– mice and healthy immunocompetent animals, supplemented with or without pCI, as well as pCI and DNA from diseased and healthy lungs alone, were subjected to PCR analyses using P. carinii- and pCI-specific primers in separate preparations. Samples containing DNA from infected lungs were always P. carinii-positive (single band of approximately 370 bp), and samples containing pCI always liberated a single band of approximately 400 bp. Samples containing neither nucleic acid were negative for the respective PCR (data not shown). Hybridizations with corresponding probes, which were derived from the respective plasmids, were performed to test for band specificity. PCR products hybridized positively with the corresponding probe only (figure 1). For P. carinii-specific PCR products, hybridization with a short probe, as used by Wakefield et al. [7], produced similar results (not shown). 3.2. Assessment of optimal PCR cycle repetitions for analysis and of optimal pCI concentrations for control purposes
DNA extracted from BAL fluids (in similar volumes to those in the experimental setups) of five individual diseased RAG-1–/– mice served as test samples for P. cariniispecific PCR performed with different cycle repetitions. Resolution on agarose gel demonstrated that at least 15 PCR cycles were necessary to visually detect PCR products using these samples (figure 2A). For analytical procedures of experimental samples 15, 17, and 20 PCR cycles were performed with each sample. PCR products obtained after 17 cycles appeared most appropriate, as mentioned in more detail below. To determine the amount of pCI to be Microbes and Infection 2000, 737-743
Figure 1. Hybridization of PCR products blotted onto nylon membranes. Samples containing pCI or P. carinii DNA were always positive for the corresponding probe. pCI samples from left to right: healthy BAL cells supplemented with pCI; medium plus pCI; diseased BAL cells with supplemented pCI. pAZ samples: BAL cells from 3 individual diseased RAG-1–/– mice. BAL cell numbers were not controlled for. Note that negative controls were always free of radioactivity. added to cultures prior to DNA extraction, PCR over 20 cycles was performed with a series of dilutions of the linearized plasmid (figure 2B). Judging from exhibited band intensities, application of 0.5 pg of pCI per sample appeared most appropriate. To account for possible DNA loss during extraction procedures, 1 pg of pCI was chosen for analysis. This was verified by running the extraction procedure with 1 pg of pCI, followed by PCR (figure 2C). 3.3. Measurement of standard DNA quantities
Purchased standard DNA was diluted to 40 pg, 10 pg, and 5 pg in 20 µL and resolved on agarose gel, and then DNA was determined by the gel reader in arbitrary units (figure 3). The correlation between the quantities of DNA employed and their corresponding amounts in arbitrary units was statistically significant (Pearson’s correlation coefficient = 0.999; P = 0.001). The DNA amounts measured in arbitrary units can, therefore, be regarded as directly proportional to the employed DNA quantities. 3.4. Assessment of P. carinii quantities in adherent cell cultures
Using extracted total DNA from adherent cell cultures, P. carinii- and pCI-specific PCR was performed using 15, 739
Original article
Hanano et al.
Figure 3. Measurement of DNA standards. A. Three concentrations of a commercially available DNA standard were transferred to an agarose gel (lane 1–3). B. Comparison of corresponding DNA quantities, as determined electronically by the gel reader in arbitrary units, whereby band sizes and intensities were respected, correlated well with applied DNA concentrations (Pearson’s correlation coefficient = 0.9999; P = 0.001).
Figure 2. Optimization of PCR cycle repetitions and concentrations of pCI to be added. A. DNA extracted from short-term cultures of BAL from five individual diseased RAG-1–/– mice was used for P. carinii-specific PCR using pAZ primers. B. Cycle numbers between 15 (vague signals) and 20 (clear signals) appear to be most appropriate for analysis. Ten cycle repetitions did not yield a visible signal. pCI-specific PCR over 20 cycles with different pCI amounts revealed addition of 0.5 pg – 1 pg pCI to be most appropriate for control purposes. C. This was verified when employing 1 pg of linearized pCI during DNA extraction and performing pCI-specific PCR for the same number of cycles as for P. carinii.
17, and 20 cycle repetitions. After 15 cycles (figure 4A), a P. carinii-specific band for the highest dilution could not be readily detected, although this was possible after 17 cycles (figure 4B). After 20 cycles, pCI-specific band intensities (and sizes) appeared to approach their plateau and could not be differentiated by the gel reader (not shown). Hence, PCR products obtained after 17 cycles, also using extracted total DNA from P. carinii standards (figure 5A), were used for analysis. From the pCI-specific bands it is evident that DNA extraction efficiencies differed between the samples. Respecting band intensities and sizes, specific DNA amounts within each band were calculated and expressed in arbitrary units using the gel reader (tables I and II). For comparison of relative P. carinii DNA amounts in the different culture wells, measured values of P. carinii740
specific DNA were divided by the corresponding values of pCI-specific DNA. Using these values with DNA from P. carinii standards, and plotting them against fungal numbers actually employed, a straight line was fitted (r2 = 0.995) (figure 5B). The slope (2 748 × 10–7) was computed and used in combination with the relative P. carinii DNA quantities to obtain corresponding P. carinii numbers as determined by PCR (table I). The numbers of P. carinii actually employed were determined by counting, and these correlated with the values calculated according to PCR analysis at a statistically significant level (Pearson´ s correlation coefficient = 0.997; P = 1.6 × 10–5) (table I). Similarly, when applying the calculations using PCR products from adherent cell culture samples, and comparing calculated fungal numbers with the relative P. carinii numbers within each dilution, correlations were also statistically significant (table II). Thus, the methodology employed is eligible for quantifying P. carinii numbers in experimental in vitro systems such as cell culture, in which it is additionally necessary to account for host cell-associated parasites.
4. Discussion With the outbreak of AIDS in the 1980s, opportunistic pathogens, particularly P. carinii, rapidly gained clinical significance [16, 17]. In recent years, sensitive assays for P. carinii-specific detection in patient samples have been developed, usually involving parasite-specific PCR procedures. However, efforts have mainly concentrated on qualiMicrobes and Infection 2000, 737-743
Quantification of Pneumocystitis carinii
Original article
Figure 4. PCR analysis of P. carinii amounts in adherent cell cultures. DNA was extracted from cultures of BAL from diseased RAG-1–/– mice supplemented with 1 pg pCI prior to extraction. Using DNA from each sample, P. carinii (using pAZ primers) and pCI-specific PCR was concomitantly performed with A. 15, B. 17, and 20 cycles (not shown). Numbers below bands indicate the dilution factor of BAL fluids derived from diseased mice. tative detection, as generally, quantitative assays are still based on counting stained organisms which are appropriate for most purposes, but tedious and time-consuming. Difficulties could be encountered in determining P. carinii in in vitro systems, especially in small-scale setups. Possible obstacles include small quantities of fungi which may not be readily distinguishable by histologic means [11] or failure to completely transfer fungal organisms from culture vessels, especially when associated with adherent cells such as macrophages, onto microscope slides. Unequivocal histological discrimination between mammalian cell constituents and associated P. carinii organisms may not always be possible, especially when only small fungal numbers are present. Undoubtedly, the ELISA system described by Durkin et al. [12] represents an accurate means for P. carinii quantification. However, it requires prior extraction of sera from convalesced animals that should preferably have been inoculated with the same stock of fungi to be used for both in vitro studies and antigen preparation. Furthermore, this ELISA takes several days before results are obtained. Similarly, specific measurements of glucan synthase activity, which represents a correlate of P. carinii numbers [13], are laborious. We report a reliable, reproducible, and rapid method based on P. carinii-specific PCR which allows direct quantification of P. carinii numbers in BAL samples (and presumably also in tissue specimens). This method is particularly applicable for experimental in vitro setups from which it may be difficult to isolate all fungal organisms for counting procedures. Due to the high sensitivity of PCR, small amounts of fungi can be quantified. In addition, the methods applied facilitate the capture of all P. carinii organisms from culture, including those associated with adherent cells, which are unlikely to be completely recovered from culture vessels. Since quantities of amplified PCR products are compared, adequate controls are necessary to account for (i) potential variations in the DNA extraction-efficiency, and (ii) PCR efficiency from each sample. A constant Microbes and Infection 2000, 737-743
Figure 5. PCR analysis of standard P. carinii concentrations. DNA was extracted from serial dilutions of known P. carinii numbers (lanes 1 - 6), supplemented with 1 pg pCI prior to extraction. A. P. carinii- and pCI-specific PCR was concomitantly conducted with 17 cycles. B. Relative P. carinii-specific DNA was calculated and plotted against the actual fungal numbers employed. A straight line was fitted (r2 = 0.995). P. carinii numbers employed for lanes 1–6: 0.15 × 106; 0.3 × 106; 0.375 × 106; 0.75 × 106; 1.5 × 106; 3 × 106, respectively. 741
Original article
Hanano et al.
Table I. Standard P. carinii numbers determined by counting and calculated from corresponding DNA quantities in PCR products after 17 amplification cycles.
a b
Counted P. carinii numbers (× 106)
pAZ102
3.0 1.5 0.75 0.375 0.3 0.15
33 370 39 777 15 897 4 543 4 233 1 300
pCI
Relative P. carinii DNA quantities pAZ102/pCI
Calculated P. carinii numbersb (× 106)
4 197 9 115 6 173 4 068 4 731 2 811
7.951 4.405 2.575 1.147 0.884 0.490
2.893 1.603 0.937 0.417 0.322 0.178
DNA quantitya
Arbitrary units; Counted and calculated P. carinii numbers correlate with high statistical significance (Pearson’s correlation coefficient = 0.997; P = 1.6 × 10–5).
Table II. DNA quantities and corresponding P. carinii numbers determined in PCR products after 17 amplification cycles. Relative P. carinii numbers (dilution factor) 1.00 (1) 0.25 (4) 0.10 (10) 0.01(100) a b
pAZ102
pCI
Relative P. carinii DNA quantities pAZ102/pCI
7 720 8 652 7 107 1 110
9 548 48 439 65 095 70 649
0.809 0.179 0.109 0.016
DNA quantitya
Calculated P. carinii numbersb 29.44×105 6.51×105 3.97×105 0.58×105
Arbitrary units; Relative and calculated P. carinii numbers correlate with high statistical significance (Pearson’s correlation coefficient = 0.998; P = 0.0016).
amount of a commercially available plasmid was added to each sample in the culture vessel prior to DNA extraction as an internal standard to control for variations in DNA extraction efficiency between sample mixtures. Murine host genes such as the gene encoding for β-actin should not be considered, even if equal cell numbers are used, because different culture treatments could affect cell viability. Possible PCR inhibition due to components present in respiratory specimens [18] is controlled for by simultaneous amplification of P. carinii DNA and control plasmid using the same PCR buffer master-mix, except for the primers, and the same number of PCR cycles. Considering the quantities of P. carinii-specific PCR products from each sample relative to the corresponding plasmid PCR products, a safe estimate of relative P. carinii numbers in each experimental sample is possible, provided that band intensities have not reached their upper plateau. Our data show a remarkable correlation between the computed fungal numbers and those actually employed in standard samples using this methodology. Similarly, computed P. carinii numbers in experimental samples containing unknown fungal loads correlated significantly with the relative fungal numbers (different dilutions) employed. Therefore, should it be necessary to not only diagnose P. carinii in various patient samples, but also determine the parasite loads, both tasks can be achieved using the same method. Additionally, abundant DNA is usually obtained to allow for further molecular-biological investigations employing the same samples. 742
Acknowledgments We thank Dr A. Wakefield for kindly providing helpful reagents and Dr P. Conradt for productive discussions. The superb assistance of the animal caretakers of the University of Ulm is acknowledged. Financial support was provided from Interdisciplinary Center of Clinical Research of the University of Ulm.
References [1] Masur H., Lane H.C., Kovacs J.A., Allegra C.J., Edman C.J., Pneumocystis pneumonia: from bench to clinic, Ann. Intern. Med. 111 (1989) 813–826. [2] Mills J., Pneumocystis carinii and Toxoplasma gondii infections in patients with AIDS, Rev. Infect. Dis. 8 (1986) 1001–1011. [3] Smith J.W., Bartlett M.S., Laboratory diagnosis of Pneumocystis carinii infection, Clin. Lab. Med. 2 (1982) 393–406. [4] Chalvardjian A.W., Grawe L.A., A new procedure for the identification of Pneumocystis carinii in tissue sections and smears, J. Clin. Pathol. 16 (1963) 383–384. [5] Kovacs J.A., Ng V.L., Masur H., Leoung G., Hadley W.K., Evans G., Lane H.C., Ognibene F.P., Shelhamer J., Parillo J.E., Gill V.J., Diagnosis of Pneumocystis carinii pneumonia: improved detection of an induced sputum with use of monoclonal antibodies, New Engl. J. Med. 318 (1988) 583–593. Microbes and Infection 2000, 737-743
Quantification of Pneumocystitis carinii
[6] Ng V.L., Virani N.A., Chaisson R.E., Yajko D.M., Sphar H.T., Cabrian K., Rollins N., Charach P., Krieger M., Hadley W.K., Hopewell P.C., Rapid detection of Pneumocystis carinii using a direct fluorescent mAb stain, J. Clin. Microbiol. 28 (1990) 2228–2233. [7] Wakefield A.E., Pixley F.J., Banerji S., Sinclair K., Miller R.F., Moxon E.R., Hopkin J.M., Detection of Pneumocystis carinii with DNA amplification, Lancet 336 (1990) 451–453. [8] Atzori C., Agostoni F., Gubertini G., Cargnela ., Diagnosis of PCP by ITSs nested PCR on noninvasive oropharyngeal samples, J. Eukaryot. Microbiol. 43 (1996) 41S.. [9] Lipschik G.Y., Gill V.J., Lundgren J.D., Andrawis V.A., Nelson N.A., Nielson J.O., Ognibene F.P., Kovacs J.A., Improved diagnosis of Pneumocystis carinii infection by PCR on induced sputum and blood, Lancet 340 (1992) 203–206. [10] Helweg-Larsen J., Jensen J.S., Benfield T., Svendsen U.G., Lundgren J.D., Lundgren B., Diagnostic use of PCR for detection of Pneumocystis carinii in oral wash samples, J. Clin. Microbiol. 36 (1998) 2068–2072. [11] Reddy L.V., Zammit C., Schuman P., Crane L.R., Detection of Pneumocystis carinii in a rat model of infection by polymerase chain reaction, Mol. Cell. Probes 6 (1992) 137–143.
Microbes and Infection 2000, 737-743
Original article
[12] Durkin M.M., Bartlett M.S., Queener S.F., Shaw M.M., Lee C.H., Smith J.W., An ELISA for enumeration of Pneumocystis carinii in vitro and in vivo, J. Clin. Microbiol. 30 (1992) 3258–3262. [13] Williams D.J., Radding J.A., Dell A., Khoo K.H., Rogers M.E.F., Richards F.F., Armstrong M.Y.K., Glucan synthesis in Pneumocystis carinii, J. Protozool. 38 (1991) 427–437. [14] Cushion M.T., Stringer J.R., Walzer P.D., Cellular and molecular biology of Pneumocystis carinii, Int. Rev. Cytol. 131 (1991) 59–107. [15] Hanano R., Kaufmann S.H.E., Pneumocystis carinii pneumonia in mutant mice deficient in both TCRα and TCRγ cells: cytokine and antibody responses, J. Infect. Dis. 179 (1999) 455–459. [16] Clarke J.R., Israel-Biet D., Interactions between opportunistic micro-organisms and HIV in the lung, Thorax 51 (1996) 875–877. [17] Mitchell D.M., Miller R.F., New developments in the pulmonary diseases affecting HIV-infected individuals, Thorax 50 (1995) 294–302. [18] Mathis A., Weber R., Kuster H., Speich R., Simplified sample processing combined with a sensitive one-tube nested PCR assay for detection of Pneumocystis carinii in respiratory specimens, J. Clin. Microbiol. 35 (1997) 1691–1695.
743
PCR-based quantification of Pneumocystis carinii in in vitro systems Ralph Hananoa,d, Joachim Fensterlea, Petra Nusserb, Kurt Reifenbergb, Stefan H. E. Kaufmanna,c* b
a Department of Immunology, University Clinics Ulm, Albert-Einstein-Allee 11, 89070 Ulm, Germany Laboratory Animal Research Unit, University Clinics Ulm, Albert-Einstein-Allee 11, 89070 Ulm, Germany c Max-Planck-Institute for Infection-Biology, Monbijoustr. 2, 10117 Berlin, Germany d Paul-Hartmann AG, Department of Science and Medicine, P.O.Box 1420, 89504 Heidenheim, Germany
(Received 13 January 2000; accepted 3 March 2000)
ABSTRACT – In many laboratories, PCR has become a routine method for the sensitive diagnosis of Pneumocystis carinii in patient samples. In contrast, quantification of fungal numbers in in vitro setups still largely relies on more conventional procedures such as histological stainings. These are time consuming and their applications are limited when dealing with small fungal numbers contaminated with tissue and cellular debris. This study presents a sensitive and rapid method for P. carinii quantification based on PCR analysis that can be easily integrated into standard detection procedures without requiring any major additional steps. P. carinii-specific PCR performed with total DNA extracted from both standard samples with known fungal numbers and experimental samples was quantified relative to PCR products of a standard concentration from a control plasmid added prior to DNA extraction. This measure controlled for variations in DNA extraction and PCR efficiency among the samples to be compared. The correlation between analyzed P. carinii-specific DNA and the actual fungal numbers employed was highly significant. © 2000 Éditions scientifiques et médicales Elsevier SAS plasmid / PCR / Pneumocystis carinii
1. Introduction Pneumocystis carinii pneumonia is a serious disease threatening immunocompromised patients, and it represents a major cause of premature mortality in AIDS patients [1, 2]. The ubiquitous nature of this fungus and the ease of its acquisition via the inhalatory route has promoted the establishment of reliable and sensitive detection assays. Frequently, respiratory specimens such as tissue samples or bronchoalveolar lavage (BAL) fluids contain abundant P. carinii organisms. Conventional detection procedures such as Gomori’s silver methenamine, Giemsa [3] and toluidine blue O staining [4] are usually sufficient for diagnosis. Less ambiguous and cumbersome determinations are achieved using antibodies directed against P. carinii [5], especially when directly labeled with fluorescence [6]. This approach allows detection of a relatively * Correspondence and reprints Address for correspondence: Max-Planck-Institute for Infection-Biology, Monbijoustr. 2, 10117 Berlin, Germany d
Current address
Microbes and Infection 2000, 737-743
small fungal load. With the development of P. cariniispecific PCR [7], a very sensitive diagnostic tool became available, and this was further fine-tuned with the application of nested PCR [8]. A major advantage of this method is the ability to detect minute fungal numbers in noninvasively collected samples, such as blood [9] and oral washes [10]. To determine total pulmonary fungal loads in small animal models, whole lungs are macerated in buffer, and tissue debris are removed by filtration and/or differential centrifugation. Subsequently, several dilutions are prepared, aliquots are transferred onto microscopic slides and stained appropriately. Usually P. carinii organisms are counted in a series of oil immersion fields, and average results are extrapolated to the whole lung. There are various modifications of this procedure, and the existing literature has proven these to be quite reliable as far as organ loads are concerned. When dealing with in vitro cultures or experimental setups, where it may be necessary to compare minute numbers of organisms, this method is less applicable [11]. Alternative methods used in previous reports include P. carinii-specific ELISA [12] or the 737
Original article
measurement of glucan synthase activity, which was correlated with P. carinii numbers [13]. These methods may be very accurate but require several methodological steps. In this report, we describe a reliable and reproducible procedure based on quantification of P. carinii-specific DNA that can determine P. carinii quantities in different in vitro setups. The addition of a standard concentration of commercially available plasmid DNA prior to total DNA extraction served as a control for DNA extraction and PCR efficiency. In addition to parasite detection, this method allows P. carinii quantification in parallel samples by a one-step procedure, without further need for cumbersome steps. Application of serial dilutions of a known P. carinii concentration served as a standard. The determination of actual fungal numbers is of particular value for comparing the effects of diverse in vitro treatments on parasite survival.
2. Materials and methods 2.1. Short-term cell culture
Ten diseased RAG-1–/– mutant mice (deficient in all mature T and B cells)(12th backcross to C57BL/6 mice) that had acquired disease naturally were lavaged with Dulbecco’s modified Eagle medium (DMEM; Gibco, Grand Island, NY). BAL fluids were combined and serial dilutions (undiluted; 1:4, 1:10, and 1:100) were prepared using DMEM. Dilutions served to obtain setups with various fungal numbers that differed by known factors (dilution factor) for comparative analysis. The precise fungal numbers were not relevant for our purpose and counting procedures were, therefore, unnecessary at this point. One ml of each dilution was transferred into 12-well plates (Nunc, Roskilde, Denmark) and cultured at 37 °C in a 7% CO2 humidified atmosphere for 3 h to allow macrophage adherence. This step was intended to simulate in vitro culture experiments. We assumed that parasite clearance would not commence at significant rates during this time, so that relative fungal numbers between the different dilutions would remain constant. Two replicates for each dilution were prepared. All animal experimentation protocols were cleared by the appropriate federal authorities. 2.2. DNA extraction
Supernatants from each well were transferred to separate centrifugation tubes and centrifuged at 700 g to pellet nonadherent cells that could potentially be associated with P. carinii. Supernatants were transferred to fresh tubes, which were centrifuged at 10 000 g for 20 min to pellet suspended P. carinii organisms. The following DNA extraction procedure was performed using a kit (QIAamp tissue kit 250; Quiagen, Hilden, Germany) according to the manufacturer’s instructions. Lysis buffer (100 µL) was added to each tube and pellets from both centrifugation steps were dissolved thoroughly. The tube contents of corresponding wells were then combined into the original well in the culture plate to join the complete fungal DNA from each sample. A further 100 µL lysis buffer was added to each tube to capture residues and then added to the corresponding wells, which now contained a total of 738
Hanano et al.
400 µL lysis buffer each. Using this approach, loss of P. carinii organisms/DNA was minimized. Into this mixture, 1 pg of the linearized plasmid pCI (Promega, Madison, WI) was added, and total DNA (composed of murine DNA, P. carinii DNA of an approximately 107-bp genome, with 13–20 chromosomes [14], and plasmid DNA [4008 bp]) was extracted to yield a final volume of 200 µL. A P. carinii standard was prepared from isolated parasites from pulmonary tissues of diseased RAG-1–/– mice. Parasites were counted using a standard protocol [15], and their concentration was adjusted to 3 × 106 nucleated organisms per mL. Serial dilutions were prepared (undiluted; 1:2; 1:4; 1:8; 1:10, 1:20), and subjected to total DNA extraction, including 1pg pCI. 2.3. Evaluation of optimal PCR cycle repetitions
To assess the optimal cycle number for analysis, DNA extractions of BAL fluids from five individual diseased –/– RAG-1 mice were used for P. carinii-specific PCR. Two PCR setups (P. carinii- and pCI-specific) of each BAL fluid sample, employing the same master-mix of PCR ingredients, were prepared simultaneously and PCR was performed with 10, 15, and 20 cycles. Following a previously established protocol [7], pAZ102-E (5’-GAT-GGCTGTTTCCAAGCCCA-3’) and pAZ102-H (5’-GTGTACGTTGCAAAGTACTC-3’), which amplify part of the gene encoding the large subunit of P. carinii mitochondrial rRNA, were employed as P. carinii-specific PCR primers. The PCR product is composed of 357 bp with a GC content of 41.6%. The pCI-specific PCR was performed using primers Amp-F (5’-TCCTGTTTTTGCTCACCC-3’) and Amp-R (5’-ACATGATCCCCCATGTTG-3’), amplifying part of the pCI plasmid encoding for ampicillin resistance (5’–2371–2785–3’). The sequences of these two primers were chosen using the computer program Prime (Genetic Computer Groups, Madison, WI). The following PCR protocol for pCI, using plasmid linearized through treatment with BamHI (MBI Fermentas Inc., Amherst, NY) was used: 10–20 cycles each comprised of 30 sec at 94 °C, 60 sec at 56 °C, and 90 sec at 72 °C. The PCR product is composed of 414 bp with a GC content of 47.6%. Amplified DNA amounts were determined in arbitrary units using a gel reader (Herolab, Wiesloch, Germany) with E.A.S.Y. (Enhanced Analysis System) software (Herolab), whereby band intensities and sizes were respected. To verify whether the DNA quantities measured in arbitrary units were directly proportional to the DNA amounts employed, three different dilutions of a commercially available DNA concentration standard (40 pg, 10 pg, and 5 pg, each in 20 µL distilled water) (Gibco) were resolved on 1% agarose gel. DNA quantities as measured by the gel reader were recorded. 2.4. Verification of the PCR product specificity
To assess whether the obtained PCR products were specific for P. carinii or pCI, they were blotted onto positively charged nylon membranes (Quiagen) and hybridized with specific 32P-labelled probes. The probes were derived from the plasmids pCI and pAZ102 (kindly provided by Dr A. Wakefield, Radcliffe Hospital, Oxford, UK), Microbes and Infection 2000, 737-743
Quantification of Pneumocystitis carinii
Original article
the latter containing rat derived P. carinii DNA (570 bp) cloned into the BamHI site of the vector pUC13. pAZ102 was treated with the restriction enzymes EcoRI and HindIII (MBI) to extract the inserted P. carinii DNA. The sequence of the P. carinii-specific PCR product lies within this fragment (5’–187–544–3’). The pCI was treated with XmnI and BmpI (MBI) to extract a DNA fragment (5’–2503–3034–3’) whose sequence largely overlaps with the pCI-specific PCR product sequence. Digestion products were separated on a 1% agarose gel and the respective fragments were extracted using a kit (QIAEX II gel extraction kit 150, Quiagen) according to the manufacturer´s instructions, and labeled with 32P, employing the Ready-to-go DNA labeling kit (-dCTP) (Pharmacia, Upsala, Sweden). Pure pCI and DNA from P. carinii-infected murine lung tissues served as positive controls for the respective PCR analyses, whereas DNA from immunocompetent murine blood cells or lung tissues served as negative controls. 2.5. Statistical analysis
Linear regression of obtained results is presented using Pearson’s correlation coefficient. Graphs and statistics were performed using the software Graph Pad Prism (Graph Pad Software Inc., San Diego, CA).
3. Results 3.1. P. carinii- and pCI-specific PCR
DNA extracted from lungs of diseased RAG-1–/– mice and healthy immunocompetent animals, supplemented with or without pCI, as well as pCI and DNA from diseased and healthy lungs alone, were subjected to PCR analyses using P. carinii- and pCI-specific primers in separate preparations. Samples containing DNA from infected lungs were always P. carinii-positive (single band of approximately 370 bp), and samples containing pCI always liberated a single band of approximately 400 bp. Samples containing neither nucleic acid were negative for the respective PCR (data not shown). Hybridizations with corresponding probes, which were derived from the respective plasmids, were performed to test for band specificity. PCR products hybridized positively with the corresponding probe only (figure 1). For P. carinii-specific PCR products, hybridization with a short probe, as used by Wakefield et al. [7], produced similar results (not shown). 3.2. Assessment of optimal PCR cycle repetitions for analysis and of optimal pCI concentrations for control purposes
DNA extracted from BAL fluids (in similar volumes to those in the experimental setups) of five individual diseased RAG-1–/– mice served as test samples for P. cariniispecific PCR performed with different cycle repetitions. Resolution on agarose gel demonstrated that at least 15 PCR cycles were necessary to visually detect PCR products using these samples (figure 2A). For analytical procedures of experimental samples 15, 17, and 20 PCR cycles were performed with each sample. PCR products obtained after 17 cycles appeared most appropriate, as mentioned in more detail below. To determine the amount of pCI to be Microbes and Infection 2000, 737-743
Figure 1. Hybridization of PCR products blotted onto nylon membranes. Samples containing pCI or P. carinii DNA were always positive for the corresponding probe. pCI samples from left to right: healthy BAL cells supplemented with pCI; medium plus pCI; diseased BAL cells with supplemented pCI. pAZ samples: BAL cells from 3 individual diseased RAG-1–/– mice. BAL cell numbers were not controlled for. Note that negative controls were always free of radioactivity. added to cultures prior to DNA extraction, PCR over 20 cycles was performed with a series of dilutions of the linearized plasmid (figure 2B). Judging from exhibited band intensities, application of 0.5 pg of pCI per sample appeared most appropriate. To account for possible DNA loss during extraction procedures, 1 pg of pCI was chosen for analysis. This was verified by running the extraction procedure with 1 pg of pCI, followed by PCR (figure 2C). 3.3. Measurement of standard DNA quantities
Purchased standard DNA was diluted to 40 pg, 10 pg, and 5 pg in 20 µL and resolved on agarose gel, and then DNA was determined by the gel reader in arbitrary units (figure 3). The correlation between the quantities of DNA employed and their corresponding amounts in arbitrary units was statistically significant (Pearson’s correlation coefficient = 0.999; P = 0.001). The DNA amounts measured in arbitrary units can, therefore, be regarded as directly proportional to the employed DNA quantities. 3.4. Assessment of P. carinii quantities in adherent cell cultures
Using extracted total DNA from adherent cell cultures, P. carinii- and pCI-specific PCR was performed using 15, 739
Original article
Hanano et al.
Figure 3. Measurement of DNA standards. A. Three concentrations of a commercially available DNA standard were transferred to an agarose gel (lane 1–3). B. Comparison of corresponding DNA quantities, as determined electronically by the gel reader in arbitrary units, whereby band sizes and intensities were respected, correlated well with applied DNA concentrations (Pearson’s correlation coefficient = 0.9999; P = 0.001).
Figure 2. Optimization of PCR cycle repetitions and concentrations of pCI to be added. A. DNA extracted from short-term cultures of BAL from five individual diseased RAG-1–/– mice was used for P. carinii-specific PCR using pAZ primers. B. Cycle numbers between 15 (vague signals) and 20 (clear signals) appear to be most appropriate for analysis. Ten cycle repetitions did not yield a visible signal. pCI-specific PCR over 20 cycles with different pCI amounts revealed addition of 0.5 pg – 1 pg pCI to be most appropriate for control purposes. C. This was verified when employing 1 pg of linearized pCI during DNA extraction and performing pCI-specific PCR for the same number of cycles as for P. carinii.
17, and 20 cycle repetitions. After 15 cycles (figure 4A), a P. carinii-specific band for the highest dilution could not be readily detected, although this was possible after 17 cycles (figure 4B). After 20 cycles, pCI-specific band intensities (and sizes) appeared to approach their plateau and could not be differentiated by the gel reader (not shown). Hence, PCR products obtained after 17 cycles, also using extracted total DNA from P. carinii standards (figure 5A), were used for analysis. From the pCI-specific bands it is evident that DNA extraction efficiencies differed between the samples. Respecting band intensities and sizes, specific DNA amounts within each band were calculated and expressed in arbitrary units using the gel reader (tables I and II). For comparison of relative P. carinii DNA amounts in the different culture wells, measured values of P. carinii740
specific DNA were divided by the corresponding values of pCI-specific DNA. Using these values with DNA from P. carinii standards, and plotting them against fungal numbers actually employed, a straight line was fitted (r2 = 0.995) (figure 5B). The slope (2 748 × 10–7) was computed and used in combination with the relative P. carinii DNA quantities to obtain corresponding P. carinii numbers as determined by PCR (table I). The numbers of P. carinii actually employed were determined by counting, and these correlated with the values calculated according to PCR analysis at a statistically significant level (Pearson´ s correlation coefficient = 0.997; P = 1.6 × 10–5) (table I). Similarly, when applying the calculations using PCR products from adherent cell culture samples, and comparing calculated fungal numbers with the relative P. carinii numbers within each dilution, correlations were also statistically significant (table II). Thus, the methodology employed is eligible for quantifying P. carinii numbers in experimental in vitro systems such as cell culture, in which it is additionally necessary to account for host cell-associated parasites.
4. Discussion With the outbreak of AIDS in the 1980s, opportunistic pathogens, particularly P. carinii, rapidly gained clinical significance [16, 17]. In recent years, sensitive assays for P. carinii-specific detection in patient samples have been developed, usually involving parasite-specific PCR procedures. However, efforts have mainly concentrated on qualiMicrobes and Infection 2000, 737-743
Quantification of Pneumocystitis carinii
Original article
Figure 4. PCR analysis of P. carinii amounts in adherent cell cultures. DNA was extracted from cultures of BAL from diseased RAG-1–/– mice supplemented with 1 pg pCI prior to extraction. Using DNA from each sample, P. carinii (using pAZ primers) and pCI-specific PCR was concomitantly performed with A. 15, B. 17, and 20 cycles (not shown). Numbers below bands indicate the dilution factor of BAL fluids derived from diseased mice. tative detection, as generally, quantitative assays are still based on counting stained organisms which are appropriate for most purposes, but tedious and time-consuming. Difficulties could be encountered in determining P. carinii in in vitro systems, especially in small-scale setups. Possible obstacles include small quantities of fungi which may not be readily distinguishable by histologic means [11] or failure to completely transfer fungal organisms from culture vessels, especially when associated with adherent cells such as macrophages, onto microscope slides. Unequivocal histological discrimination between mammalian cell constituents and associated P. carinii organisms may not always be possible, especially when only small fungal numbers are present. Undoubtedly, the ELISA system described by Durkin et al. [12] represents an accurate means for P. carinii quantification. However, it requires prior extraction of sera from convalesced animals that should preferably have been inoculated with the same stock of fungi to be used for both in vitro studies and antigen preparation. Furthermore, this ELISA takes several days before results are obtained. Similarly, specific measurements of glucan synthase activity, which represents a correlate of P. carinii numbers [13], are laborious. We report a reliable, reproducible, and rapid method based on P. carinii-specific PCR which allows direct quantification of P. carinii numbers in BAL samples (and presumably also in tissue specimens). This method is particularly applicable for experimental in vitro setups from which it may be difficult to isolate all fungal organisms for counting procedures. Due to the high sensitivity of PCR, small amounts of fungi can be quantified. In addition, the methods applied facilitate the capture of all P. carinii organisms from culture, including those associated with adherent cells, which are unlikely to be completely recovered from culture vessels. Since quantities of amplified PCR products are compared, adequate controls are necessary to account for (i) potential variations in the DNA extraction-efficiency, and (ii) PCR efficiency from each sample. A constant Microbes and Infection 2000, 737-743
Figure 5. PCR analysis of standard P. carinii concentrations. DNA was extracted from serial dilutions of known P. carinii numbers (lanes 1 - 6), supplemented with 1 pg pCI prior to extraction. A. P. carinii- and pCI-specific PCR was concomitantly conducted with 17 cycles. B. Relative P. carinii-specific DNA was calculated and plotted against the actual fungal numbers employed. A straight line was fitted (r2 = 0.995). P. carinii numbers employed for lanes 1–6: 0.15 × 106; 0.3 × 106; 0.375 × 106; 0.75 × 106; 1.5 × 106; 3 × 106, respectively. 741
Original article
Hanano et al.
Table I. Standard P. carinii numbers determined by counting and calculated from corresponding DNA quantities in PCR products after 17 amplification cycles.
a b
Counted P. carinii numbers (× 106)
pAZ102
3.0 1.5 0.75 0.375 0.3 0.15
33 370 39 777 15 897 4 543 4 233 1 300
pCI
Relative P. carinii DNA quantities pAZ102/pCI
Calculated P. carinii numbersb (× 106)
4 197 9 115 6 173 4 068 4 731 2 811
7.951 4.405 2.575 1.147 0.884 0.490
2.893 1.603 0.937 0.417 0.322 0.178
DNA quantitya
Arbitrary units; Counted and calculated P. carinii numbers correlate with high statistical significance (Pearson’s correlation coefficient = 0.997; P = 1.6 × 10–5).
Table II. DNA quantities and corresponding P. carinii numbers determined in PCR products after 17 amplification cycles. Relative P. carinii numbers (dilution factor) 1.00 (1) 0.25 (4) 0.10 (10) 0.01(100) a b
pAZ102
pCI
Relative P. carinii DNA quantities pAZ102/pCI
7 720 8 652 7 107 1 110
9 548 48 439 65 095 70 649
0.809 0.179 0.109 0.016
DNA quantitya
Calculated P. carinii numbersb 29.44×105 6.51×105 3.97×105 0.58×105
Arbitrary units; Relative and calculated P. carinii numbers correlate with high statistical significance (Pearson’s correlation coefficient = 0.998; P = 0.0016).
amount of a commercially available plasmid was added to each sample in the culture vessel prior to DNA extraction as an internal standard to control for variations in DNA extraction efficiency between sample mixtures. Murine host genes such as the gene encoding for β-actin should not be considered, even if equal cell numbers are used, because different culture treatments could affect cell viability. Possible PCR inhibition due to components present in respiratory specimens [18] is controlled for by simultaneous amplification of P. carinii DNA and control plasmid using the same PCR buffer master-mix, except for the primers, and the same number of PCR cycles. Considering the quantities of P. carinii-specific PCR products from each sample relative to the corresponding plasmid PCR products, a safe estimate of relative P. carinii numbers in each experimental sample is possible, provided that band intensities have not reached their upper plateau. Our data show a remarkable correlation between the computed fungal numbers and those actually employed in standard samples using this methodology. Similarly, computed P. carinii numbers in experimental samples containing unknown fungal loads correlated significantly with the relative fungal numbers (different dilutions) employed. Therefore, should it be necessary to not only diagnose P. carinii in various patient samples, but also determine the parasite loads, both tasks can be achieved using the same method. Additionally, abundant DNA is usually obtained to allow for further molecular-biological investigations employing the same samples. 742
Acknowledgments We thank Dr A. Wakefield for kindly providing helpful reagents and Dr P. Conradt for productive discussions. The superb assistance of the animal caretakers of the University of Ulm is acknowledged. Financial support was provided from Interdisciplinary Center of Clinical Research of the University of Ulm.
References [1] Masur H., Lane H.C., Kovacs J.A., Allegra C.J., Edman C.J., Pneumocystis pneumonia: from bench to clinic, Ann. Intern. Med. 111 (1989) 813–826. [2] Mills J., Pneumocystis carinii and Toxoplasma gondii infections in patients with AIDS, Rev. Infect. Dis. 8 (1986) 1001–1011. [3] Smith J.W., Bartlett M.S., Laboratory diagnosis of Pneumocystis carinii infection, Clin. Lab. Med. 2 (1982) 393–406. [4] Chalvardjian A.W., Grawe L.A., A new procedure for the identification of Pneumocystis carinii in tissue sections and smears, J. Clin. Pathol. 16 (1963) 383–384. [5] Kovacs J.A., Ng V.L., Masur H., Leoung G., Hadley W.K., Evans G., Lane H.C., Ognibene F.P., Shelhamer J., Parillo J.E., Gill V.J., Diagnosis of Pneumocystis carinii pneumonia: improved detection of an induced sputum with use of monoclonal antibodies, New Engl. J. Med. 318 (1988) 583–593. Microbes and Infection 2000, 737-743
Quantification of Pneumocystitis carinii
[6] Ng V.L., Virani N.A., Chaisson R.E., Yajko D.M., Sphar H.T., Cabrian K., Rollins N., Charach P., Krieger M., Hadley W.K., Hopewell P.C., Rapid detection of Pneumocystis carinii using a direct fluorescent mAb stain, J. Clin. Microbiol. 28 (1990) 2228–2233. [7] Wakefield A.E., Pixley F.J., Banerji S., Sinclair K., Miller R.F., Moxon E.R., Hopkin J.M., Detection of Pneumocystis carinii with DNA amplification, Lancet 336 (1990) 451–453. [8] Atzori C., Agostoni F., Gubertini G., Cargnela ., Diagnosis of PCP by ITSs nested PCR on noninvasive oropharyngeal samples, J. Eukaryot. Microbiol. 43 (1996) 41S.. [9] Lipschik G.Y., Gill V.J., Lundgren J.D., Andrawis V.A., Nelson N.A., Nielson J.O., Ognibene F.P., Kovacs J.A., Improved diagnosis of Pneumocystis carinii infection by PCR on induced sputum and blood, Lancet 340 (1992) 203–206. [10] Helweg-Larsen J., Jensen J.S., Benfield T., Svendsen U.G., Lundgren J.D., Lundgren B., Diagnostic use of PCR for detection of Pneumocystis carinii in oral wash samples, J. Clin. Microbiol. 36 (1998) 2068–2072. [11] Reddy L.V., Zammit C., Schuman P., Crane L.R., Detection of Pneumocystis carinii in a rat model of infection by polymerase chain reaction, Mol. Cell. Probes 6 (1992) 137–143.
Microbes and Infection 2000, 737-743
Original article
[12] Durkin M.M., Bartlett M.S., Queener S.F., Shaw M.M., Lee C.H., Smith J.W., An ELISA for enumeration of Pneumocystis carinii in vitro and in vivo, J. Clin. Microbiol. 30 (1992) 3258–3262. [13] Williams D.J., Radding J.A., Dell A., Khoo K.H., Rogers M.E.F., Richards F.F., Armstrong M.Y.K., Glucan synthesis in Pneumocystis carinii, J. Protozool. 38 (1991) 427–437. [14] Cushion M.T., Stringer J.R., Walzer P.D., Cellular and molecular biology of Pneumocystis carinii, Int. Rev. Cytol. 131 (1991) 59–107. [15] Hanano R., Kaufmann S.H.E., Pneumocystis carinii pneumonia in mutant mice deficient in both TCRα and TCRγ cells: cytokine and antibody responses, J. Infect. Dis. 179 (1999) 455–459. [16] Clarke J.R., Israel-Biet D., Interactions between opportunistic micro-organisms and HIV in the lung, Thorax 51 (1996) 875–877. [17] Mitchell D.M., Miller R.F., New developments in the pulmonary diseases affecting HIV-infected individuals, Thorax 50 (1995) 294–302. [18] Mathis A., Weber R., Kuster H., Speich R., Simplified sample processing combined with a sensitive one-tube nested PCR assay for detection of Pneumocystis carinii in respiratory specimens, J. Clin. Microbiol. 35 (1997) 1691–1695.
743