1378
DNA AMPLIFICATION AND SILVER STAINING FOR PNEUMOCYSTlS CARINI/IN PAIRED BRONCHOSCOPIC LAVAGE AND INDUCED SPUTUM SAMPLES
DNA amplification on induced sputum samples for diagnosis of Pneumocystis carinii pneumonia
*Other diagnoses in these cases and from 14 further respiratory episodes studied by lavage alone were Mycobacteriumavium-intracellulare (6), pulmonary tuberculosis (3), Staphylococcus aureus pneumonia (4), purulent bronchitis (3), lobar
pneumonia (2), aspiration pneumonia (1),cytomegalovirus pneumonitis (2), Kaposi’s scarcoma (3), pulmonary lymphoma (1),non-specific pneumonitis (open lung biopsy) (1),coryza (1),undiagnosed self-limiting illness (4).
endobronchial
subjected to electrophoresis in 1-5% agarose gel, and specific P carinii sequence (346 base pairs) was identified by visualisation with ultraviolet light after ethidium bromide staining or by oligohybridisation, after Southern transfer and autoradiography with the internal primer pAZ102-L2. Scoring of the DNA bands was done independently of the results of silver staining and of final clinical diagnosis, which was assessed by clinical features and response to treatment with co-trimoxazole or pentamidine.s
were
DNA
amplification and silver staining were used to identify Pneumocystis carinii in bronchoscopic lavage and induced sputum samples during 51 episodes of respiratory illness in 47 subjects with H IV infection. In 20 episodes, in which the clinical diagnosis was pneumocystis pneumonia (PCP), silver stain was positive in 14 lavage samples (70%) and 7 sputum samples (35%), whereas DNA amplification was positive in 19 lavage samples (95%) and 18 sputum samples (90%). DNA amplification was positive in only 1 of 31 patients without PCP (PCP developed in this patient within 10 weeks). DNA amplification on induced sputum offers a powerful technique for diagnosis of PCP. Pneumocystis carinii pneumonia is one of the most complications of the acquired immunodeficiency syndrome (AIDS). We have previously reported the development of oligonucleotide primers and probes, specific to the parasite, for its accurate identification by DNA amplification in bronchoscopic lavage samples.1,2 Though cough in pneumocystis pneumonia is almost invariably dry, sputum that may be useful for diagnostic purposes can be induced non-invasively by the inhalation of nebulised hypertonic saline.3,4 We here report the application of DNA amplification as a diagnostic technique for Pneumocystis carinii in induced sputum samples from HIV-positive patients presenting with acute respiratory illness. common
51 episodes of acute respiratory illness in 47 immunosuppressed HIV-infected individuals were studied. The patients presented with dyspnoea and cough and had at least one of the following: abnormal chest signs, arterial hypoxaemia, or abnormal chest radiograph. Bronchoscopic alveolar lavage was obtained from each patient and in 37 instances induced sputum was also obtained before bronchoscopy by inhalation of nebulised hypertonic saline for 15-20 min (’De Vilbiss 99’ ultrasonic nebuliser, De Vilbiss, Middlesex, UK) according to the method of Bigby et a] .3samples examined by routine microbiological and cytological methods, including methenamine silver staining for P carinii; part of each sample was reserved for DNA amplification. Our methods were essentially the same as previously described.2 Briefly, DNA was extracted from 1 ml lavage or sputum by proteinase K digestion (1 mg/ml final concentration of proteinase K, in the presence of 10 mmol/1 edetic acid [EDTA], pH 80 and 1 % (weight/volume) sodium dodecyl sulphate, at 50°C for 16 h) and phenol/chloroform extraction. DNA amplification was done with the oligonucleotide primers, pAZ102-E and pAZ102-H, with denaturation at 94°C for 90 s, annealing at 55°C for 90 s, and extension at 72°C for 2 min (40 cycles). The amplification products were
In 20 episodes, a final clinical diagnosis of pneumocystis pneumonia was based on diffuse radiographic changes and resolution of fever and dyspnoea within 7 days of starting treatment with pentamidine or high-dose co-trimoxazole. In the other 31 episodes, the final clinical diagnosis was not pneumocystis pneumonia; an alternative diagnosis was made, or the illness did not respond to pentamidine or to co-trimoxazole, or the episodes were self-limiting (see footnote table). The results from the paired lavage and sputum samples are sunnmarised in the table. All 14 patients who had a final clinical diagnosis of pneumocystis pneumonia and who also had a positive silver stain on lavage, had a strong signal of amplified pneumocystis DNA from both the lavage sample and sputum. Of 5 other patients with a final clinical diagnosis of pneumocystis pneumonia but negative silver stains, 4 were strongly positive by DNA amplification in alveolar lavage; 3 of these 4 were also positive in induced sputum. Silver stain was positive in only one-third of sputum samples from cases of pneumocystis pneumonia; in 2 patients, the sputum samples were, on microscopy, judged to be inadequate to identify P carinii since they showed only oral debris and squamous cells-the DNA amplification was positive in both samples. Among the 31 episodes with a final diagnosis of respiratory illness other than pneumocystis pneumonia, 7 patients had low levels of amplified pneumocystis DNA (detectable by oligoblot only) but only 1 patient had a strong DNA signal (seen on ethidium bromide stained gel as well as oligoblotting) in both lavage and induced sputum. This individual had a history of pneumocystis pneumonia and a further episode developed within 10 weeks of the study. In our first survey of DNA amplification in lavage samples,2 we emphasised that positive signals of amplified DNA could be categorised as strong (visible after ethidium bromide staining of the agarose gel) or weak (visible only on autoradiography after oligoblotting). Independent calibration experiments show that a strong signal indicates 100 organisms or more in a sample, whereas a weak signal implies many fewer organisms-as few as 1-2 organisms (unpublished). In our bronchoscopic lavage study,2 weak signals were found in 20% of samples from severely immunosuppressed patients without pneumocystis
’
1379
pneumonia but strong signals were found only in those with clinical disease. Our present study extends these observations and tests the efficacy of DNA amplification in sputum samples that may be of varying quality. In 20 cases
judged to have clinical pneumocystis pneumonia, a strong
Efficacy of quinine for falciparum malaria according to previous chloroquine exposure
DNA amplification signal was obtained in 19 (95%) of the
lavage samples and 18 (90%) of the paired sputum samples; by contrast, silver staining was positive in only 35% of sputum samples and 70% of lavage samples. Diagnostic by silver staining can be as high as 90% in lavage
rates
samples6 but may be lower in patients presenting earlier with their respiratory symptoms7 or in those receiving prophylactic nebulised pentamidine.8 The sensitivity of the DNA method is therefore excellent; it is unlikely that the single case, negative by both DNA amplification and silver staining on lavage, had pneumocystis pneumonia.2 The specificity of DNA amplification may be judged from the findings in lavage samples from the 31 patients in our present study and the 13 patients from our previous study in whom the final clinical diagnosis was of another respiratory illness and not of pneumocystis pneumonia and in whom silver stains were also negative. A strong amplification signal was obtained both in lavage and in sputum in only 1 of these 44 patients; this patient was receiving prophylaxis with dapsone for pneumocystis pneumonia, had had one previous episode, and returned with a further documented episode within 10 weeks of the current study. DNA amplification, with very simple calibration, offers a highly sensitive and specific technique for the diagnosis of pneumocystis pneumonia in both bronchoscopic alveolar lavage and non-invasively obtained induced sputum. This work was funded by the Wellcome Trust and Medical Research Council. A.E.W. is supported by The Royal Society. We thank Prof S.
Semple for allowing us to study his patients. REFERENCES 1. Wakefield AE, Pixley FJ, Banerji S, et al. Amplification of mitochondrial ribosomal RNA sequences from Pneumocystis carinii DNA of rat and human origin. Mol Biochem Parasitol 1990; 43: 69-76. 2. Wakefield AE, Pixley FJ, Banerji S, et al. Detection of Pneumocystis carinii by DNA amplification. Lancet 1990; 336: 451-53. 3. Bigby TD, Margolskee D, Curtis JL, et al. The usefulness of induced sputum in the diangosis of Pneumocystis carinii pneumonia in patients with the acquired immunodeficiency syndrome. Am Rev Resp Dis
1986; 133: 515-18. AE, Ganjei P, Torres A, et al. Sputum examination for the diagnosis of Pneumocystis carinii pneumonia in the acquired immunodeficiency syndrome. Am Rev Resp Dis 1986; 133: 226-29. 5. Miller RF, Millar AB, Weller IVD, Semple SJG. Empirical treatment
4. Pitchenik
without
bronchoscopy for Pneumocystis carinii pneumonia in the acquired immunodeficiency syndrome. Thorax 1989; 44: 559-64. 6. Broaddus C, Dake MD, Stulbarg MS, et al. Bronchoalveolar lavage and transbronchial biopsy for the diagnosis of pulmonary infections in the acquired immunodeficiency syndrome. Ann Intern Med 1985; 102: 747-52. 7. Griffiths MH, Kocjan G, Miller RF, Godfrey-Fausett P. Diagnosis of pulmonary disease in human immunodeficiency virus infection: role of transbronchial biopsy and bronchoalveolar lavage. Thorax 1989; 44: 554-58. 8. Jules-Elysee KM, Stover DE, Zaman MB, et al. Aerosolized pentamidine: effect on diagnosis and presentation of Pneumocystis carinii pneumonia. Ann Intern Med 1990; 112: 750-57.
ADDRESSES:
Department
Molecular
Infectious
Diseases
Group,
of Paediatrics, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, OX3 9DU (A. E. Wakefield, DPhil, L Guiver); Department of Medicine, University College and Middlesex School of Medicine, Middlesex Hospital, London (R. F. Miller, MRCP); and Osler Chest Unit, Churchill Hospital, Oxford (J. M. Hopkin, MD), UK. Correspondence to Dr A. E. Wakefield.
Chloroquine has been reported to antagonise the anti-parasitic action of quinine against Plasmodium falciparum in vitro. We looked for evidence of any such antagonism in vivo. In 123 Malawian children with cerebral malaria treated with parenteral quinine, the likelihood of survival and the rate of recovery were much the same in patients who had taken chloroquine and those who had not. In these circumstances we found no evidence of
chloroquine/quinine antagonism.
Falciparum malaria is a common cause of death in parts of the world where the infection is endemic, and is an important cause of illness in travellers. Chloroquine is still widely used as prophylaxis for travellers and as first-line treatment for people living in many endemic areas.2 As chloroquine resistance continues to spread, increasing numbers of people require alternative chemotherapy because prophylaxis or initial treatment with chloroquine has failed. Quinine is generally used in these situations, and is the drug of choice for severe or complicated malaria.3 In vitro, chloroquine antagonises the antiparasitic action of quinine and other antimalarial drugs on both chloroquine-sensitive and chloroquine-resistant isolates of P falciparum 4 We therefore looked for evidence of any such drug antagonism in vivo, by examining the efficacy of quinine in the treatment of severe malaria in Malawian children, some of whom had already received chloroquine. 123 children (54 boys) aged 7 months to 12 years (mean 34 years) with falciparum malaria (asexual P falciparum parasitaemia) and altered consciousness were investigated at the Queen Elizabeth Central Hospital, Blantyre, Malawi. Children were excluded if any cause of illness other than malaria was found. On admission, blood was taken into lithium/heparin tubes and immediately centrifuged, and plasma was stored in plain containers at - 20°C for subsequent measurement of chloroquine and quinine concentrations (high pressure liquid chromatography) 5.6All patients were treated with the same schedule of quinine dihydrochloride: the initial loading dose was 20 mg salt/kg body weight, and subsequent 8-hourly doses were 10 mg/kg. When patients were well enough for oral treatment, quinine was stopped and a therapeutic dose of oral pyrimethamine/ sulfadoxine (’Fansidar’) was given. Rectal temperature was recorded 4 hourly and peripheral parasitaemia was measured every 12 h. Fever clearance time was defined as the interval between start of treatment and fall of temperature to below 37°C without subsequent rise above 37°C for 24 h. Parasite clearance time was defined as the interval between start of treatment and the first of two consecutive negative blood films. Results were analysed by chi-squared test, paired t-test, or analysis of variance, as appropriate.
(67%) of the 123 patients, the initial plasma sample proved contain chloroquine, in concentrations ranging from 3 to 5079 ng/ml. Clinical features of these 82 patients were compared with those of the 41 patients having no chloroquine in the initial sample. Patients with and without In 82
to
DNA AMPLIFICATION AND SILVER STAINING FOR PNEUMOCYSTlS CARINI/IN PAIRED BRONCHOSCOPIC LAVAGE AND INDUCED SPUTUM SAMPLES
DNA amplification on induced sputum samples for diagnosis of Pneumocystis carinii pneumonia
*Other diagnoses in these cases and from 14 further respiratory episodes studied by lavage alone were Mycobacteriumavium-intracellulare (6), pulmonary tuberculosis (3), Staphylococcus aureus pneumonia (4), purulent bronchitis (3), lobar
pneumonia (2), aspiration pneumonia (1),cytomegalovirus pneumonitis (2), Kaposi’s scarcoma (3), pulmonary lymphoma (1),non-specific pneumonitis (open lung biopsy) (1),coryza (1),undiagnosed self-limiting illness (4).
endobronchial
subjected to electrophoresis in 1-5% agarose gel, and specific P carinii sequence (346 base pairs) was identified by visualisation with ultraviolet light after ethidium bromide staining or by oligohybridisation, after Southern transfer and autoradiography with the internal primer pAZ102-L2. Scoring of the DNA bands was done independently of the results of silver staining and of final clinical diagnosis, which was assessed by clinical features and response to treatment with co-trimoxazole or pentamidine.s
were
DNA
amplification and silver staining were used to identify Pneumocystis carinii in bronchoscopic lavage and induced sputum samples during 51 episodes of respiratory illness in 47 subjects with H IV infection. In 20 episodes, in which the clinical diagnosis was pneumocystis pneumonia (PCP), silver stain was positive in 14 lavage samples (70%) and 7 sputum samples (35%), whereas DNA amplification was positive in 19 lavage samples (95%) and 18 sputum samples (90%). DNA amplification was positive in only 1 of 31 patients without PCP (PCP developed in this patient within 10 weeks). DNA amplification on induced sputum offers a powerful technique for diagnosis of PCP. Pneumocystis carinii pneumonia is one of the most complications of the acquired immunodeficiency syndrome (AIDS). We have previously reported the development of oligonucleotide primers and probes, specific to the parasite, for its accurate identification by DNA amplification in bronchoscopic lavage samples.1,2 Though cough in pneumocystis pneumonia is almost invariably dry, sputum that may be useful for diagnostic purposes can be induced non-invasively by the inhalation of nebulised hypertonic saline.3,4 We here report the application of DNA amplification as a diagnostic technique for Pneumocystis carinii in induced sputum samples from HIV-positive patients presenting with acute respiratory illness. common
51 episodes of acute respiratory illness in 47 immunosuppressed HIV-infected individuals were studied. The patients presented with dyspnoea and cough and had at least one of the following: abnormal chest signs, arterial hypoxaemia, or abnormal chest radiograph. Bronchoscopic alveolar lavage was obtained from each patient and in 37 instances induced sputum was also obtained before bronchoscopy by inhalation of nebulised hypertonic saline for 15-20 min (’De Vilbiss 99’ ultrasonic nebuliser, De Vilbiss, Middlesex, UK) according to the method of Bigby et a] .3samples examined by routine microbiological and cytological methods, including methenamine silver staining for P carinii; part of each sample was reserved for DNA amplification. Our methods were essentially the same as previously described.2 Briefly, DNA was extracted from 1 ml lavage or sputum by proteinase K digestion (1 mg/ml final concentration of proteinase K, in the presence of 10 mmol/1 edetic acid [EDTA], pH 80 and 1 % (weight/volume) sodium dodecyl sulphate, at 50°C for 16 h) and phenol/chloroform extraction. DNA amplification was done with the oligonucleotide primers, pAZ102-E and pAZ102-H, with denaturation at 94°C for 90 s, annealing at 55°C for 90 s, and extension at 72°C for 2 min (40 cycles). The amplification products were
In 20 episodes, a final clinical diagnosis of pneumocystis pneumonia was based on diffuse radiographic changes and resolution of fever and dyspnoea within 7 days of starting treatment with pentamidine or high-dose co-trimoxazole. In the other 31 episodes, the final clinical diagnosis was not pneumocystis pneumonia; an alternative diagnosis was made, or the illness did not respond to pentamidine or to co-trimoxazole, or the episodes were self-limiting (see footnote table). The results from the paired lavage and sputum samples are sunnmarised in the table. All 14 patients who had a final clinical diagnosis of pneumocystis pneumonia and who also had a positive silver stain on lavage, had a strong signal of amplified pneumocystis DNA from both the lavage sample and sputum. Of 5 other patients with a final clinical diagnosis of pneumocystis pneumonia but negative silver stains, 4 were strongly positive by DNA amplification in alveolar lavage; 3 of these 4 were also positive in induced sputum. Silver stain was positive in only one-third of sputum samples from cases of pneumocystis pneumonia; in 2 patients, the sputum samples were, on microscopy, judged to be inadequate to identify P carinii since they showed only oral debris and squamous cells-the DNA amplification was positive in both samples. Among the 31 episodes with a final diagnosis of respiratory illness other than pneumocystis pneumonia, 7 patients had low levels of amplified pneumocystis DNA (detectable by oligoblot only) but only 1 patient had a strong DNA signal (seen on ethidium bromide stained gel as well as oligoblotting) in both lavage and induced sputum. This individual had a history of pneumocystis pneumonia and a further episode developed within 10 weeks of the study. In our first survey of DNA amplification in lavage samples,2 we emphasised that positive signals of amplified DNA could be categorised as strong (visible after ethidium bromide staining of the agarose gel) or weak (visible only on autoradiography after oligoblotting). Independent calibration experiments show that a strong signal indicates 100 organisms or more in a sample, whereas a weak signal implies many fewer organisms-as few as 1-2 organisms (unpublished). In our bronchoscopic lavage study,2 weak signals were found in 20% of samples from severely immunosuppressed patients without pneumocystis
’
1379
pneumonia but strong signals were found only in those with clinical disease. Our present study extends these observations and tests the efficacy of DNA amplification in sputum samples that may be of varying quality. In 20 cases
judged to have clinical pneumocystis pneumonia, a strong
Efficacy of quinine for falciparum malaria according to previous chloroquine exposure
DNA amplification signal was obtained in 19 (95%) of the
lavage samples and 18 (90%) of the paired sputum samples; by contrast, silver staining was positive in only 35% of sputum samples and 70% of lavage samples. Diagnostic by silver staining can be as high as 90% in lavage
rates
samples6 but may be lower in patients presenting earlier with their respiratory symptoms7 or in those receiving prophylactic nebulised pentamidine.8 The sensitivity of the DNA method is therefore excellent; it is unlikely that the single case, negative by both DNA amplification and silver staining on lavage, had pneumocystis pneumonia.2 The specificity of DNA amplification may be judged from the findings in lavage samples from the 31 patients in our present study and the 13 patients from our previous study in whom the final clinical diagnosis was of another respiratory illness and not of pneumocystis pneumonia and in whom silver stains were also negative. A strong amplification signal was obtained both in lavage and in sputum in only 1 of these 44 patients; this patient was receiving prophylaxis with dapsone for pneumocystis pneumonia, had had one previous episode, and returned with a further documented episode within 10 weeks of the current study. DNA amplification, with very simple calibration, offers a highly sensitive and specific technique for the diagnosis of pneumocystis pneumonia in both bronchoscopic alveolar lavage and non-invasively obtained induced sputum. This work was funded by the Wellcome Trust and Medical Research Council. A.E.W. is supported by The Royal Society. We thank Prof S.
Semple for allowing us to study his patients. REFERENCES 1. Wakefield AE, Pixley FJ, Banerji S, et al. Amplification of mitochondrial ribosomal RNA sequences from Pneumocystis carinii DNA of rat and human origin. Mol Biochem Parasitol 1990; 43: 69-76. 2. Wakefield AE, Pixley FJ, Banerji S, et al. Detection of Pneumocystis carinii by DNA amplification. Lancet 1990; 336: 451-53. 3. Bigby TD, Margolskee D, Curtis JL, et al. The usefulness of induced sputum in the diangosis of Pneumocystis carinii pneumonia in patients with the acquired immunodeficiency syndrome. Am Rev Resp Dis
1986; 133: 515-18. AE, Ganjei P, Torres A, et al. Sputum examination for the diagnosis of Pneumocystis carinii pneumonia in the acquired immunodeficiency syndrome. Am Rev Resp Dis 1986; 133: 226-29. 5. Miller RF, Millar AB, Weller IVD, Semple SJG. Empirical treatment
4. Pitchenik
without
bronchoscopy for Pneumocystis carinii pneumonia in the acquired immunodeficiency syndrome. Thorax 1989; 44: 559-64. 6. Broaddus C, Dake MD, Stulbarg MS, et al. Bronchoalveolar lavage and transbronchial biopsy for the diagnosis of pulmonary infections in the acquired immunodeficiency syndrome. Ann Intern Med 1985; 102: 747-52. 7. Griffiths MH, Kocjan G, Miller RF, Godfrey-Fausett P. Diagnosis of pulmonary disease in human immunodeficiency virus infection: role of transbronchial biopsy and bronchoalveolar lavage. Thorax 1989; 44: 554-58. 8. Jules-Elysee KM, Stover DE, Zaman MB, et al. Aerosolized pentamidine: effect on diagnosis and presentation of Pneumocystis carinii pneumonia. Ann Intern Med 1990; 112: 750-57.
ADDRESSES:
Department
Molecular
Infectious
Diseases
Group,
of Paediatrics, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, OX3 9DU (A. E. Wakefield, DPhil, L Guiver); Department of Medicine, University College and Middlesex School of Medicine, Middlesex Hospital, London (R. F. Miller, MRCP); and Osler Chest Unit, Churchill Hospital, Oxford (J. M. Hopkin, MD), UK. Correspondence to Dr A. E. Wakefield.
Chloroquine has been reported to antagonise the anti-parasitic action of quinine against Plasmodium falciparum in vitro. We looked for evidence of any such antagonism in vivo. In 123 Malawian children with cerebral malaria treated with parenteral quinine, the likelihood of survival and the rate of recovery were much the same in patients who had taken chloroquine and those who had not. In these circumstances we found no evidence of
chloroquine/quinine antagonism.
Falciparum malaria is a common cause of death in parts of the world where the infection is endemic, and is an important cause of illness in travellers. Chloroquine is still widely used as prophylaxis for travellers and as first-line treatment for people living in many endemic areas.2 As chloroquine resistance continues to spread, increasing numbers of people require alternative chemotherapy because prophylaxis or initial treatment with chloroquine has failed. Quinine is generally used in these situations, and is the drug of choice for severe or complicated malaria.3 In vitro, chloroquine antagonises the antiparasitic action of quinine and other antimalarial drugs on both chloroquine-sensitive and chloroquine-resistant isolates of P falciparum 4 We therefore looked for evidence of any such drug antagonism in vivo, by examining the efficacy of quinine in the treatment of severe malaria in Malawian children, some of whom had already received chloroquine. 123 children (54 boys) aged 7 months to 12 years (mean 34 years) with falciparum malaria (asexual P falciparum parasitaemia) and altered consciousness were investigated at the Queen Elizabeth Central Hospital, Blantyre, Malawi. Children were excluded if any cause of illness other than malaria was found. On admission, blood was taken into lithium/heparin tubes and immediately centrifuged, and plasma was stored in plain containers at - 20°C for subsequent measurement of chloroquine and quinine concentrations (high pressure liquid chromatography) 5.6All patients were treated with the same schedule of quinine dihydrochloride: the initial loading dose was 20 mg salt/kg body weight, and subsequent 8-hourly doses were 10 mg/kg. When patients were well enough for oral treatment, quinine was stopped and a therapeutic dose of oral pyrimethamine/ sulfadoxine (’Fansidar’) was given. Rectal temperature was recorded 4 hourly and peripheral parasitaemia was measured every 12 h. Fever clearance time was defined as the interval between start of treatment and fall of temperature to below 37°C without subsequent rise above 37°C for 24 h. Parasite clearance time was defined as the interval between start of treatment and the first of two consecutive negative blood films. Results were analysed by chi-squared test, paired t-test, or analysis of variance, as appropriate.
(67%) of the 123 patients, the initial plasma sample proved contain chloroquine, in concentrations ranging from 3 to 5079 ng/ml. Clinical features of these 82 patients were compared with those of the 41 patients having no chloroquine in the initial sample. Patients with and without In 82
to
