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Quantification of the detection of Pneumocystis carinii by DNA amplification
Molecular and Cellular Probes (1992) 6, 1 1 5-117
Quantification of the detection of Pneumocystis carinii by DNA amplification
Sarah E. Peters,'*, Ann E . Wakefield ; Suneale Banerji' and Julian M . Hopkin 2
'Molecular Infectious Diseases Group, Department of Paediatrics, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK, and Z OsIer Chest Unit, Churchill Hospital, Oxford, UK (Received 2 July 1991, Accepted 30 August 1991)
We have developed a highly specific and sensitive technique for the detection of Pneumocystis carinii DNA using DNA amplification by the polymerase chain reaction (PCR) . PCR products are detected by agarose gel electrophoresis and Southern hybridization to an oligonucleotide probe . Here we report the calibration of parasite numbers with amplification and hybridization signals and show that we can detect P. carinii to a lower limit of one to two organisms . The quantification of this diagnostic technique allows us to establish the number of organisms in a clinical sample which correspond to pneumocystis pneumonia or to sub-clinical pulmonary colonization .
KEYWORDS: Pneumocystis
carinii, DNA amplification, PCR, quantitative detection .
INTRODUCTION We have reported the cloning of mitochondrial rRNA genes from Pneumocystis carinii, an opportunistic, fungal pathogen of immunosuppressed individuals .', ' Using these sequences we have successfully developed a highly specific and sensitive technique for the identification of P. carinii by DNA amplification and subsequent internal oligonucleotide hybridization of the amplification products.',' Amplification of P. carinii DNA from human bronchoalveolar lavage and induced sputum samples in AIDS subjects suggests that a strong DNA amplification signal, visible after electrophoresis of the products on ethidium bromide stained agarose gels corresponds to the presence of clinical disease ; a weak signal, visible only after oligonucleotide hybridization and autoradiography, indicates sub-clinical colonization with fewer organisms, though may offer a guideline for the planning of prophylactic treatment .', ' Here we report the quantification of this diagnostic
technique by seeding uninfected human post-mortem lung with parasites isolated from infected rat lung, to establish the number of organisms corresponding to strong or weak amplification signals .
MATERIALS AND METHODS
Pneumocystis carinii infection,
documented by microscopy after Grocott's methenamine silver staining, was induced in Sprague-Dawley rats by steroid immunosuppression .' To isolate the parasites, one rat lung was homogenized through a nylon mesh with phosphate buffered saline (PBS) . The filtrate was overlayed onto a Ficoll gradient (5 . 7% Ficoll 400, 9 . 0% sodium diatrizoate, 0098% sodium azide) and centrifuged for 15 min at 1000 g . The flocculent interface containing the parasites was removed and the parasites were washed and resuspended in PBS . Slide
* Author to whom correspondence should be addressed . 0890-8508/92/020115 + 03 $03 .00/0
115
© 1992 Academic Press Limited
116
S. E . Peters et al.
preparations of parasites were stained with Giemsa and methenamine silver. Microscopy of 400 high power fields showed that the parasite fraction contained 2 . 3 x 10' cysts ml - ' and 1 . 4 x 109 trophozoites ml -1 . The average number of sporozoites per cyst was found to be 6 . 3 based on Giemsa staining . Hence, the P . carinii preparation contained 1 . 55 x 109 parasites ml -1 . A complete lobe of lung tissue was homogenized in a Waring blender and 100 µl aliquots of serial 10fold dilutions (10° to 10 -70) of the parasite preparation were added to 1 g samples of homogenized tissue . This lung had been previously shown, by DNA amplification, to carry no latent P . carinii.' Total DNA was extracted by proteinase K (Boehringer Mannheim) digestion (500 pg ml -1 ) at 50° C, in the presence of 0 . 25% SDS and 10 mm EDTA, followed by phenolchloroform extraction and ethanol precipitation . The oligonucleotide primers pAZ102-E and pAZ102-H were used to amplify P . carinii-specific DNA, as previously described .' Amplification reactions (in triplicate) were performed on 1/5 and 1/25 dilutions of template together with positive and negative controls . Products were electrophoresed on ethidium bromide stained agarose gels and underwent Southern transfer and hybridization with a 32 P end-labelled oligonucleotide, pAZ102-L1, which at high stringency (54 ° C) is specific for rat derived P . carinii .' Homogenization, parasite preparation, seeding, DNA extraction and preparation of amplification reactions were performed in a laminar flow cabinet to avoid contamination . All experimental stages were carried out in duplicate .
RESULTS
or the higher dilutions of parasite preparation (10 -6 to 10 _10) •
DISCUSSION AND CONCLUSIONS DNA amplification of ribosomal RNA gene sequences allows sensitive detection of P . carinii even in an excess of human DNA . We have demonstrated the calibration of the number of parasites with the quantity of amplified product and shown that (a) strong signals (visible after ethidium bromide stained agarose gel electrophoresis) denote the presence of 100 organisms or more in a clinical sample and (b) weak signals (visible after oligonucleotide hybridization) denote the presence of fewer than 100 organisms, to a lower limit of one to two parasites . The detection of such small numbers, indeed single organisms, is consistent with DNA amplification of other pathogens ."' Using our experimental conditions, strong signals are consistent with the diagnosis of pneumocystis pneumonia and weak signals indicate subclinical pulmonary colonization . The method is proving powerful in the diagnosis of clinical disease', ' and promises to be useful in monitoring treatment of and planning prophylaxis against pneumocystis pneumonia . This calibrated technique will also be useful for the examination of the epidemiology of P. carinii infection .
ACKNOWLEDGEMENTS This work was funded by the MRC (SEP), the Royal Society (AEW) and the Wellcome Trust (SB) . We are grateful to Dr K . Sinclair for helpful discussions, to Dr P . Millard for providing the post-mortem lung samples and to Mr N . White for the photographs .
Figure 1 shows the analysis, by agarose gel electrophoresis, of the products of DNA amplification obtained using template DNA isolated from seeded lung . Amplification products (355 base pairs) were visible after ethidium bromide staining at undiluted and 10 -2 dilutions of the parasite preparation but not at the 10 - ' dilution or in the unseeded control . Considering the sample volume from which DNA was extracted and the dilutions of template DNA used for amplification, band A corresponded to 775 parasites and band B to 155 parasites . Following oligonucleotide hybridization with pAZ102-L1, high stringency wash and autoradiography we were able to detect P. carinii to a level of one to two parasites (Fig . 1, Band D . Band C corresponded to seven to eight organisms) . No P . carinii DNA was detected in the unseeded lung as expected $
REFERENCES 1.
2.
Wakefield, A . E ., Hopkin, J . M., Burns, J ., Hipkiss, J . B ., Stewart, T . J . & Moxon, E . R . (1988) . Cloning of DNA from Pneumocystis carinii . Journal of Infectious Diseases 158, 859-62. Pixley, F . J ., Wakefield, A . E ., Banerji, S . & Hopkin, J . M . (1991) . Mitochondrial gene sequences show fungal homology for Pneumocystis carinii. Molecular Microbiology 5, 1347-51 .
3.
Wakefield, A . E ., Pixley, F . J ., Banerji, S . et al. (1990) . Amplification of mitochondrial ribosomal RNA sequences from Pneumocystis carinii DNA of rat and human origin . Molecular and Biochemical Parasitology 43, 69-76 .
117
Quantification of P . carinal detection by PCR
(a)
344(b)
(0) L
10-5
NT
10'
NT
10 -2
NT
NT RP L
L
10 0
NT NT RP L
344
(b)
f t C D
ft A B
Fig . 1 . Amplification reactions using P. carinii primers and template DNA isolated from post-mortem lung seeded with rat-derived parasites, (a) ethidium bromide stained agarose gel (1 . 5%) electrophoresis and (b) autoradiography of oligonucleotide hybridization (-80 ° C for 16 h) . Parasite preparation dilutions: undiluted (10 °), 10 -2, 10 -4, 10 -5, 10 -6 , 10', 10", 10 -9, 10" . For each parasite dilution, template DNA was diluted 1/5 (lefthand lanes) and 1/25 (righthand lanes) . NP: unseeded post-mortem lung DNA, RP: rat-derived P . carinii DNA positive control, NT : no template DNA negative controls, L : molecular weight markers (sizes shown in base pairs) . Arrows indicate position of specific amplification product .
4 . Wakefield, A . E ., Pixley, F . J ., Banerji, S. et al . (1990) . Detection of Pneumocystis carinii with DNA amplification . Lancet 336, 451-3 . 5 . Wakefield, A. E ., Guiver, L ., Miller, R. F . & Hopkin, J . M . (1991) . DNA amplification on induced sputum samples for diagnosis of Pneumocystis carinii pneumonia . Lancet 337, 1378-79. 6 . Savoia, D . & Carmello, P . (1989) . The microscopic identification of Pneumocystis carinii . Journal of Protozoology 36, 72S-74S . 7 . Sinclair, K ., Wakefield, A . E ., Banerji, S . & Hopkin, J . M . (1991) . Pneumocystis carinii organisms derived from rat and human hosts are genetically distinct . Molecular and Biochemical Parasitology 45, 183-4 . 8 . Peters, S . E ., Wakefield, A . E ., Sinclair, K ., Millard, P. R . &
Hopkin, J . M . A search for Pneumocystis carinii in Postmortem lungs by DNA amplification . Journal of Pathology (in press) . 9 . Wang, J .-T., Wang, T .-H ., Shen, J .-C., Shih, L.-N ., Lin, J .-T . & Chen, D .-S . (1991) . Detection of hepatitis B virus DNA by PCR in plasma volunteer blood donors negative for hepatitis B surface antigen . Journal of Infectious Diseases 163, 397-9 . 10. Cassol, S ., Salas, T., Arella, M., Neumann, P ., Schechter, M . T. & O'Shaughnessy, M . (1991). Use of dried blood spot specimens in the detection of HIV Type I by PCR . Journal of Clinical Microbiology 29, 667-71 . 11 . Valentine, J . L., Arthur, R. R ., Mobley, H . L . T . & Dick, J . D . (1991) . Detection of Helicobacter pylori by PCR . Journal of Clinical Microbiology 29, 689-95 .
Quantification of the detection of Pneumocystis carinii by DNA amplification
Sarah E. Peters,'*, Ann E . Wakefield ; Suneale Banerji' and Julian M . Hopkin 2
'Molecular Infectious Diseases Group, Department of Paediatrics, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK, and Z OsIer Chest Unit, Churchill Hospital, Oxford, UK (Received 2 July 1991, Accepted 30 August 1991)
We have developed a highly specific and sensitive technique for the detection of Pneumocystis carinii DNA using DNA amplification by the polymerase chain reaction (PCR) . PCR products are detected by agarose gel electrophoresis and Southern hybridization to an oligonucleotide probe . Here we report the calibration of parasite numbers with amplification and hybridization signals and show that we can detect P. carinii to a lower limit of one to two organisms . The quantification of this diagnostic technique allows us to establish the number of organisms in a clinical sample which correspond to pneumocystis pneumonia or to sub-clinical pulmonary colonization .
KEYWORDS: Pneumocystis
carinii, DNA amplification, PCR, quantitative detection .
INTRODUCTION We have reported the cloning of mitochondrial rRNA genes from Pneumocystis carinii, an opportunistic, fungal pathogen of immunosuppressed individuals .', ' Using these sequences we have successfully developed a highly specific and sensitive technique for the identification of P. carinii by DNA amplification and subsequent internal oligonucleotide hybridization of the amplification products.',' Amplification of P. carinii DNA from human bronchoalveolar lavage and induced sputum samples in AIDS subjects suggests that a strong DNA amplification signal, visible after electrophoresis of the products on ethidium bromide stained agarose gels corresponds to the presence of clinical disease ; a weak signal, visible only after oligonucleotide hybridization and autoradiography, indicates sub-clinical colonization with fewer organisms, though may offer a guideline for the planning of prophylactic treatment .', ' Here we report the quantification of this diagnostic
technique by seeding uninfected human post-mortem lung with parasites isolated from infected rat lung, to establish the number of organisms corresponding to strong or weak amplification signals .
MATERIALS AND METHODS
Pneumocystis carinii infection,
documented by microscopy after Grocott's methenamine silver staining, was induced in Sprague-Dawley rats by steroid immunosuppression .' To isolate the parasites, one rat lung was homogenized through a nylon mesh with phosphate buffered saline (PBS) . The filtrate was overlayed onto a Ficoll gradient (5 . 7% Ficoll 400, 9 . 0% sodium diatrizoate, 0098% sodium azide) and centrifuged for 15 min at 1000 g . The flocculent interface containing the parasites was removed and the parasites were washed and resuspended in PBS . Slide
* Author to whom correspondence should be addressed . 0890-8508/92/020115 + 03 $03 .00/0
115
© 1992 Academic Press Limited
116
S. E . Peters et al.
preparations of parasites were stained with Giemsa and methenamine silver. Microscopy of 400 high power fields showed that the parasite fraction contained 2 . 3 x 10' cysts ml - ' and 1 . 4 x 109 trophozoites ml -1 . The average number of sporozoites per cyst was found to be 6 . 3 based on Giemsa staining . Hence, the P . carinii preparation contained 1 . 55 x 109 parasites ml -1 . A complete lobe of lung tissue was homogenized in a Waring blender and 100 µl aliquots of serial 10fold dilutions (10° to 10 -70) of the parasite preparation were added to 1 g samples of homogenized tissue . This lung had been previously shown, by DNA amplification, to carry no latent P . carinii.' Total DNA was extracted by proteinase K (Boehringer Mannheim) digestion (500 pg ml -1 ) at 50° C, in the presence of 0 . 25% SDS and 10 mm EDTA, followed by phenolchloroform extraction and ethanol precipitation . The oligonucleotide primers pAZ102-E and pAZ102-H were used to amplify P . carinii-specific DNA, as previously described .' Amplification reactions (in triplicate) were performed on 1/5 and 1/25 dilutions of template together with positive and negative controls . Products were electrophoresed on ethidium bromide stained agarose gels and underwent Southern transfer and hybridization with a 32 P end-labelled oligonucleotide, pAZ102-L1, which at high stringency (54 ° C) is specific for rat derived P . carinii .' Homogenization, parasite preparation, seeding, DNA extraction and preparation of amplification reactions were performed in a laminar flow cabinet to avoid contamination . All experimental stages were carried out in duplicate .
RESULTS
or the higher dilutions of parasite preparation (10 -6 to 10 _10) •
DISCUSSION AND CONCLUSIONS DNA amplification of ribosomal RNA gene sequences allows sensitive detection of P . carinii even in an excess of human DNA . We have demonstrated the calibration of the number of parasites with the quantity of amplified product and shown that (a) strong signals (visible after ethidium bromide stained agarose gel electrophoresis) denote the presence of 100 organisms or more in a clinical sample and (b) weak signals (visible after oligonucleotide hybridization) denote the presence of fewer than 100 organisms, to a lower limit of one to two parasites . The detection of such small numbers, indeed single organisms, is consistent with DNA amplification of other pathogens ."' Using our experimental conditions, strong signals are consistent with the diagnosis of pneumocystis pneumonia and weak signals indicate subclinical pulmonary colonization . The method is proving powerful in the diagnosis of clinical disease', ' and promises to be useful in monitoring treatment of and planning prophylaxis against pneumocystis pneumonia . This calibrated technique will also be useful for the examination of the epidemiology of P. carinii infection .
ACKNOWLEDGEMENTS This work was funded by the MRC (SEP), the Royal Society (AEW) and the Wellcome Trust (SB) . We are grateful to Dr K . Sinclair for helpful discussions, to Dr P . Millard for providing the post-mortem lung samples and to Mr N . White for the photographs .
Figure 1 shows the analysis, by agarose gel electrophoresis, of the products of DNA amplification obtained using template DNA isolated from seeded lung . Amplification products (355 base pairs) were visible after ethidium bromide staining at undiluted and 10 -2 dilutions of the parasite preparation but not at the 10 - ' dilution or in the unseeded control . Considering the sample volume from which DNA was extracted and the dilutions of template DNA used for amplification, band A corresponded to 775 parasites and band B to 155 parasites . Following oligonucleotide hybridization with pAZ102-L1, high stringency wash and autoradiography we were able to detect P. carinii to a level of one to two parasites (Fig . 1, Band D . Band C corresponded to seven to eight organisms) . No P . carinii DNA was detected in the unseeded lung as expected $
REFERENCES 1.
2.
Wakefield, A . E ., Hopkin, J . M., Burns, J ., Hipkiss, J . B ., Stewart, T . J . & Moxon, E . R . (1988) . Cloning of DNA from Pneumocystis carinii . Journal of Infectious Diseases 158, 859-62. Pixley, F . J ., Wakefield, A . E ., Banerji, S . & Hopkin, J . M . (1991) . Mitochondrial gene sequences show fungal homology for Pneumocystis carinii. Molecular Microbiology 5, 1347-51 .
3.
Wakefield, A . E ., Pixley, F . J ., Banerji, S . et al. (1990) . Amplification of mitochondrial ribosomal RNA sequences from Pneumocystis carinii DNA of rat and human origin . Molecular and Biochemical Parasitology 43, 69-76 .
117
Quantification of P . carinal detection by PCR
(a)
344(b)
(0) L
10-5
NT
10'
NT
10 -2
NT
NT RP L
L
10 0
NT NT RP L
344
(b)
f t C D
ft A B
Fig . 1 . Amplification reactions using P. carinii primers and template DNA isolated from post-mortem lung seeded with rat-derived parasites, (a) ethidium bromide stained agarose gel (1 . 5%) electrophoresis and (b) autoradiography of oligonucleotide hybridization (-80 ° C for 16 h) . Parasite preparation dilutions: undiluted (10 °), 10 -2, 10 -4, 10 -5, 10 -6 , 10', 10", 10 -9, 10" . For each parasite dilution, template DNA was diluted 1/5 (lefthand lanes) and 1/25 (righthand lanes) . NP: unseeded post-mortem lung DNA, RP: rat-derived P . carinii DNA positive control, NT : no template DNA negative controls, L : molecular weight markers (sizes shown in base pairs) . Arrows indicate position of specific amplification product .
4 . Wakefield, A . E ., Pixley, F . J ., Banerji, S. et al . (1990) . Detection of Pneumocystis carinii with DNA amplification . Lancet 336, 451-3 . 5 . Wakefield, A. E ., Guiver, L ., Miller, R. F . & Hopkin, J . M . (1991) . DNA amplification on induced sputum samples for diagnosis of Pneumocystis carinii pneumonia . Lancet 337, 1378-79. 6 . Savoia, D . & Carmello, P . (1989) . The microscopic identification of Pneumocystis carinii . Journal of Protozoology 36, 72S-74S . 7 . Sinclair, K ., Wakefield, A . E ., Banerji, S . & Hopkin, J . M . (1991) . Pneumocystis carinii organisms derived from rat and human hosts are genetically distinct . Molecular and Biochemical Parasitology 45, 183-4 . 8 . Peters, S . E ., Wakefield, A . E ., Sinclair, K ., Millard, P. R . &
Hopkin, J . M . A search for Pneumocystis carinii in Postmortem lungs by DNA amplification . Journal of Pathology (in press) . 9 . Wang, J .-T., Wang, T .-H ., Shen, J .-C., Shih, L.-N ., Lin, J .-T . & Chen, D .-S . (1991) . Detection of hepatitis B virus DNA by PCR in plasma volunteer blood donors negative for hepatitis B surface antigen . Journal of Infectious Diseases 163, 397-9 . 10. Cassol, S ., Salas, T., Arella, M., Neumann, P ., Schechter, M . T. & O'Shaughnessy, M . (1991). Use of dried blood spot specimens in the detection of HIV Type I by PCR . Journal of Clinical Microbiology 29, 667-71 . 11 . Valentine, J . L., Arthur, R. R ., Mobley, H . L . T . & Dick, J . D . (1991) . Detection of Helicobacter pylori by PCR . Journal of Clinical Microbiology 29, 689-95 .