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Diagnosis of pneumocystis carinii pneumonia
222
ParasitologyToday,vol. 5, no. 7, I989
Diagnosis of Pneumocystis Pneumonia
carinii
J.M. Hopkin and A.E. Wakefield Pneumocystis carinii is the prime opportunistic pathogen ofour time, the leading cause of fatal pneumonia rn the increasing number ofimmunosuppressed subjects encountered on oncology and transplant programmes’ and in subj& with the acquired immunodeficiencysyndrome (AIOS)2,3. Seen in animal models of trypanosomiaSISin I9 IO (Ref. 4) P. carinii was formally recognized as a distinct parasite in Parisian rats in I 9 I4 (Ref. 5) and then as the causative agent of the remarkable European epidemic of interstitial plasma cell pneumonia that occurred in the I92OsI 940s affecting orphaned, malnourished infants6,7. As this epidemic declined with improving social conditions, the disease was recognized in its now familiar sporadic form in subjects who were chemotherapeutically immunosuppressed as a result of organ transplants or treatment for turnout-s. Later, recognition of Pneumocystis in young, previously well men in the USA led to the identification of the AIDS syndrome 2,3. The attack rate for Pneumocystis pneumonia is propot-tionate to the degree of immunosuppression, predominantly of T-lymphocyte function, varying from l-4% per year for patients on transplant programmes to 65% for AIDS patients in whom it is the leading cause of death’. Although the culture of P. carinii in cell-free medium has proved impossible, useful progress has been made in understanding its basic biology, but it remains uncertain whether the parasite is protozoan or fungal’. Work with the steroidimmunosuppressed rat model” has shown that Pneumocystis is a natural infector of normal mammalian lung, but produces disease only if immunosuppression supervenes. Serological data on
Fig. I. ‘Clockfoces’ of Pneumocystis cysts demonstrated by Giemsa staining and showing a ring offour to eight sporozoite nuclei.
immunoglobulin responses in man to a Pneumocystis fraction prepared from either human postmortem lungs or experimental mammal lungs, show that young healthy humans mount a progressive response. Starting from two years of age, 80% of most populations are seropositive by the age of ten, indicating the frequency of natural infection’ ’. Indeed it has been identified in very small numbers in the lungs of immunocompetent individuals dying from other diseases12. Light microscopy shows two phases of the organism, (I) the amoeboid trophozoite (2-5 pm) with its single punctate nucleus, and (2) the thick-walled cyst (4-6 pm) containing two to eight nucleated sporozoites. Gremsa staining demonstrates the punctate nuclei of the trophozoite and of the intracystic bodies deployed within the Giemsa negativestaining cyst wall (Fig. I). The wall itself, but not the nuclei within it, can be readily demonstrated by staining with toluidine blueI or methenamine silver’! (Fig. 2). Each method requires expertise when used in clinical diagnostic work, first to distinguish the parasite nuclei stained by Giemsa from host debris, and second to distinguish cyst walls stained by toluidine and silver from those of fungal spores or host red blood cells. The appearance of parenthesis-like thickening of the cyst wall and of empty cysts that have clearly discharged their intracystic bodies are typical (Fig. 2). Ultrastructural studies confirm the light microscopic evidence for two phases of the organism and also demonstrate the strictly extracellular nature of the parasite within the alveolar space of the lung15. The only apparent interaction with human tissue is adherence of the trophozoite to the type I (flat) pneumocyte of the alveolar wall. Under conditions of profound immunosuppression, the potential exists for parasite numbers to increase steadily so that the parasites progressively fill alveolar spaces and produce a diffuse pneumonia with the characteristic profound disturbance of oxygen transport in the lungs that leads to respiratory failure. Although Pneumocystis pneumonia presents a consistent cllnical picture
characterized by progressive breathlessness, dry cough, fever and variable degrees of diffuse radiographic change, these features are not specific in the immunosuppressed host. They may equally be caused by other infections, fungi, mycobacteria or cytomegalovirus, or by non-infective disease including pulmonary oedema or vasculitis, drug- or irradiation-induced pneumonitis”. The exclusively alveolar siting of the parasite has presented special problems for its definitive diagnosis. Sputum is rarely available and all the early studies’.6,7 show how rarely it provided an adequate specimen for direct visualization of the organism after staining with Giemsa or methenamine silver. This failure to obtain an adequate diagnostic sample from sputum, and then the development of effective chemotherapy for Pneumocystis I7 infection, made the need for precise diagnosis by alternative means pressing and this was originally undertaken by open lung biopsy. This of course proved to be a productive diagnostlc method but the associated trauma made it unacceptable as a first-choice procedure’*. Fine needle, percutaneous, transthoracic aspiration of the lung with subsequent staining of material by Giemsa or silver was able to provide the diagnosis in 70-80% of cases but in a quarter of those, deflation of the lung after puncture required tube drainage of the pleural space”. This technique has proved to be particularly traumatic for AIDS patients who suffer unusual and severe hypotensive reactions to the procedure due to underlying autonomic neuropathy*‘. The use of fibreoptic bronchoscopy with the passage of a slim (5 mm bore) flexible instrument under local anaesthesia has been a valuable development; it has provided a potential bedside test where samples could be taken from the patient in the form of saline lavage of the lung2’,22 or small transbronchial biopsies23. Staining of these samples, principally by silver or toluidine blue, has been remarkably effective in diagnosing Pneumocystis pneumonia, lavage and transbronchial biopsy each providing diagnostic sensitivity of approximately 90-95% (Ref. 24). A correlative postmortem study has shown no false positives for these techniques: where lavage samples have been positive, Pneumocystis pneumonitis is @ 1989,Elsev1erSc1ence Publtshers Ltd.(UK)OI694707/89,$03 50
223
Parasitology Today, vol. 5, no. 7, I989
Fig. 2. A number
ofcysts,
with their we//s, but
no contents, demonstrated by silver methenamine staining.
demonstrable at postmortem25. A small-scale bronchoscopic study of asymptomatic, HIV (human immunoindividuals, deficiency virus)-positive including some with CD4+-lymphocyte depletion showed no detectable Pneumocystis carinii in the lung after Giemsa and silver staining of alveolar lavage”. Alveolar lavage also offers the potential for the accurate diagnosis of other infections and also of non-infective processes27. A special phenomenon of concern in the AIDS population has been the persistence of the organism as demonstrated by lavage or biopsy after some weeks of treatment2”. It is as yet uncertain whether these visualized organisms are viable. If so, their demonstration may yet not be surprising since the chemotherapeutic agents for treating such heavy infections of Pneumocystis have static rather than tidal actions and the immunosuppressive statse of AIDS is unrelentingand progressive. Although bronchoscopy samplings have proved successful in diagnosis, a search has been made for less invasive diagnostic methods, and sorne of these seem likely to prove very effective. While serological investigations of antibody responses to Pneumocystis have been valuable in delineating the epidemiology of infection in normal individuals ‘, it has not been useful in diagnosing Pneumocystis carinii pneumonia in the immunosuppressed, nor has the use of polyclonal antibody raised to pneumocysts, in the search for circulating soluble antigen. The latter method has proved nons ecific, as well as variably sensitive’ B.30. The possibility of making available an expectorated or ‘coughed-up’ sample has been realized by the use of inhalations of nebulized hypertonic saline (3-5%)3 ’ which can induce sputum in
90% of subjects who have a nonproductive cough. Subsequent microscopy after Giemsa and silver staining provides the diagnosis in up to 50% or 60% of cases. The development of monoclonal antibodies to Pneumocystis by different groups32.33 and their application to induced sputum shows that diagnostic sensitivity may be improved to 60-90% and therefore be nearly equivalent to that of bronchoscopy. Induced sputum should also be valuable in screening for other microbes, The availability of monoclonal antibodies, and more recently cloned DNA from Pneumocystis34, now offers the chance for applying sensitive, potentially quantitative techniques in the identification of viable Pneumocystis organisms with the use of enzyme-linked immunosorbent assays and DNA hybridization analysis. These may be particularly valuable in the AIDS population for identifying and monitoring the progress of Pneumocystis infection thus providing guidelines for the use of chemoprophylaxis and formal treatment. References I Walzer, PD. et al. (1974) Ann Int. Med. 80, 83-93
2 Go’ctlieb, M. et al. ( I98 I ) New Eng1.j. Med. 305, 1425-1431
3 Masur, H. et al.
(I 98 I)
New Eng1.j. Med. 305,
1431-1438
4 Carlnii, A. ( I9 IO) Comm. SK Med. Sao Paolo
I6 Aug.,
Meuwrssen, J.H.E.T. et al. ( I977)j. I36,43-49 Sheldon, W.H. 287-297
I5 I6 I7 I8 I9 20 21 22 23 24 25 26 27 28 29 30 31 32
(1959)
Infect. Dis.
Am. 1, Dis. Chrld. 97,
Chalvardjan, A.M. and Grawe. L.A. (1963) /. Qn. Pothol. l6,383-384 Grocott, R.G. (1955) Am. /. Clin. Pathof. 25, 975-979 Barton, E.G. and Campbell, W.G. (I 969) Am.]. Pathol. 54,209-235 Hopkrn, J.M. (I 987) Advanced Medrcine pp I O+ I 17, Pitman Hughes. W.T. (1987) Parasitology Today 3. 332-335 Dominy, D.E. and Lucas, R.N. (I 965) Ann. Jhorac. Sure. I, 305-3 IO Chaudha< V.K. et al. ( 1977) Am. /. Dis. Chffd. I 3 I, 902-907 Craddock, C. et al. ( I987)Loncet 2, I6- I8 Hopkin, J.M. eta/. (I 983)Loncet 2.299-30 I Stover. D.E. et 01. ( 1984) Ann. Int. Med. IO I, l-7 Blumenfeld, W. et a/. ( 1984) Am./. C/in. Pathol. 81, l-5 Broaduss, C. et al. ( 1985) Ann. Int. Med. 102, 747-752 Gal, A.A. et al. (I 987) Arch. Pathol. Lob. Med. I I I,238241 Lundgren, J.D. et ol. (I 989) Thorax 44,68-69 Young, J.A. et ol. (I 984) /. C/in. Pathol. 37, 390-397 Shelhamer, J.H. et al. (I 984)Am. Rev. Resp. Dis. 130, 1161-l I65 Pifer. L.L. et of. (I 978) Pedidtncs 6 I, 35-4 I Hughes, W.T. (I 985) Chest 87,700 Pitchenik, A.E. et al. (1986)Am. Rev. Resp. Da. I33,22&229 Kovacs, ].A. et of. ( 1988) New Engl./. Med. 3 18,
589-593 33 Elvln, K.M. et al. ( l988)Br. Med. /. 297,38 I-384 34 Wakefield, A.E..et a/..( I988)].-/nfect. Dn. 153, 359-362
204
5 Delanoe, P. and Delanoe. M. 6 7 8 9 IO
(I 9 14) Bull. Sot. Pathol. Exot. 7,27 l-272 Vanek,].(l95l)Cos.Lek.Cesk90, 1121-l 124 Gadjusek. D.C. (I 957)Pedrotrrcs 19.543-565 Kovacs, ].A. et of. (I 984) Ann. Int. Med. 100. 66367 I Edman, J.C. etol. (I 988)Nature 334,5 19-522 Weller. R.W. (I 956) Z. Kinderherlkd. 78. 166-176 ’ ’
renewals
Churchill Hospital, Headington, Oxford OX3 7L_/,UK. Ann Wakefield is a Research Fellow at the Institute ofMolecular Medicine, John Radc/iffe Hospital, Oxford OX3 9DU, UK.
Today Subscriptions
Parasitology All new subscriptions,
julion Hopkin is o Consu/tont Physicion at the
and subscription
queries should be addressed
to our Barking office at the address below. Cl Personal Subscriptions (1989. the USA; f40.00 elsewhere. 0
12 monthly issues) f33.00
in the UK; $58.00 in
Library Subscriptions(I 989, I2 monthly issues plus hardcover bound compenin the UK; $294.00 in the USA; f 159.00 elsewhere. dium with index) f 159.00
? ?Student Subscriptions( 1989, I2 monthly issues) f27.00 USA; f 32.00 elsewhere.
in the UK; $46.00 in the
(Student Subscriptions must be accompanied by valid proof of student status.) All prices include postage (air delivery outside UK). All subscriptions can be paid by cheque, bank draft or UNESCO coupons. Personal and Library subscriptions can also be paid by credit card -Access/Mastercard, Eurocard, Visa or American Express. Subscription orders should be sent to the address below. Elsevier Science Publishers Ltd Crown House Linton Road Barking Essex IG I I SJU, UK Tel. (outside UK): +44 (I) 594 7272 Tel. (in UK): 0 I 594 7272
ParasitologyToday,vol. 5, no. 7, I989
Diagnosis of Pneumocystis Pneumonia
carinii
J.M. Hopkin and A.E. Wakefield Pneumocystis carinii is the prime opportunistic pathogen ofour time, the leading cause of fatal pneumonia rn the increasing number ofimmunosuppressed subjects encountered on oncology and transplant programmes’ and in subj& with the acquired immunodeficiencysyndrome (AIOS)2,3. Seen in animal models of trypanosomiaSISin I9 IO (Ref. 4) P. carinii was formally recognized as a distinct parasite in Parisian rats in I 9 I4 (Ref. 5) and then as the causative agent of the remarkable European epidemic of interstitial plasma cell pneumonia that occurred in the I92OsI 940s affecting orphaned, malnourished infants6,7. As this epidemic declined with improving social conditions, the disease was recognized in its now familiar sporadic form in subjects who were chemotherapeutically immunosuppressed as a result of organ transplants or treatment for turnout-s. Later, recognition of Pneumocystis in young, previously well men in the USA led to the identification of the AIDS syndrome 2,3. The attack rate for Pneumocystis pneumonia is propot-tionate to the degree of immunosuppression, predominantly of T-lymphocyte function, varying from l-4% per year for patients on transplant programmes to 65% for AIDS patients in whom it is the leading cause of death’. Although the culture of P. carinii in cell-free medium has proved impossible, useful progress has been made in understanding its basic biology, but it remains uncertain whether the parasite is protozoan or fungal’. Work with the steroidimmunosuppressed rat model” has shown that Pneumocystis is a natural infector of normal mammalian lung, but produces disease only if immunosuppression supervenes. Serological data on
Fig. I. ‘Clockfoces’ of Pneumocystis cysts demonstrated by Giemsa staining and showing a ring offour to eight sporozoite nuclei.
immunoglobulin responses in man to a Pneumocystis fraction prepared from either human postmortem lungs or experimental mammal lungs, show that young healthy humans mount a progressive response. Starting from two years of age, 80% of most populations are seropositive by the age of ten, indicating the frequency of natural infection’ ’. Indeed it has been identified in very small numbers in the lungs of immunocompetent individuals dying from other diseases12. Light microscopy shows two phases of the organism, (I) the amoeboid trophozoite (2-5 pm) with its single punctate nucleus, and (2) the thick-walled cyst (4-6 pm) containing two to eight nucleated sporozoites. Gremsa staining demonstrates the punctate nuclei of the trophozoite and of the intracystic bodies deployed within the Giemsa negativestaining cyst wall (Fig. I). The wall itself, but not the nuclei within it, can be readily demonstrated by staining with toluidine blueI or methenamine silver’! (Fig. 2). Each method requires expertise when used in clinical diagnostic work, first to distinguish the parasite nuclei stained by Giemsa from host debris, and second to distinguish cyst walls stained by toluidine and silver from those of fungal spores or host red blood cells. The appearance of parenthesis-like thickening of the cyst wall and of empty cysts that have clearly discharged their intracystic bodies are typical (Fig. 2). Ultrastructural studies confirm the light microscopic evidence for two phases of the organism and also demonstrate the strictly extracellular nature of the parasite within the alveolar space of the lung15. The only apparent interaction with human tissue is adherence of the trophozoite to the type I (flat) pneumocyte of the alveolar wall. Under conditions of profound immunosuppression, the potential exists for parasite numbers to increase steadily so that the parasites progressively fill alveolar spaces and produce a diffuse pneumonia with the characteristic profound disturbance of oxygen transport in the lungs that leads to respiratory failure. Although Pneumocystis pneumonia presents a consistent cllnical picture
characterized by progressive breathlessness, dry cough, fever and variable degrees of diffuse radiographic change, these features are not specific in the immunosuppressed host. They may equally be caused by other infections, fungi, mycobacteria or cytomegalovirus, or by non-infective disease including pulmonary oedema or vasculitis, drug- or irradiation-induced pneumonitis”. The exclusively alveolar siting of the parasite has presented special problems for its definitive diagnosis. Sputum is rarely available and all the early studies’.6,7 show how rarely it provided an adequate specimen for direct visualization of the organism after staining with Giemsa or methenamine silver. This failure to obtain an adequate diagnostic sample from sputum, and then the development of effective chemotherapy for Pneumocystis I7 infection, made the need for precise diagnosis by alternative means pressing and this was originally undertaken by open lung biopsy. This of course proved to be a productive diagnostlc method but the associated trauma made it unacceptable as a first-choice procedure’*. Fine needle, percutaneous, transthoracic aspiration of the lung with subsequent staining of material by Giemsa or silver was able to provide the diagnosis in 70-80% of cases but in a quarter of those, deflation of the lung after puncture required tube drainage of the pleural space”. This technique has proved to be particularly traumatic for AIDS patients who suffer unusual and severe hypotensive reactions to the procedure due to underlying autonomic neuropathy*‘. The use of fibreoptic bronchoscopy with the passage of a slim (5 mm bore) flexible instrument under local anaesthesia has been a valuable development; it has provided a potential bedside test where samples could be taken from the patient in the form of saline lavage of the lung2’,22 or small transbronchial biopsies23. Staining of these samples, principally by silver or toluidine blue, has been remarkably effective in diagnosing Pneumocystis pneumonia, lavage and transbronchial biopsy each providing diagnostic sensitivity of approximately 90-95% (Ref. 24). A correlative postmortem study has shown no false positives for these techniques: where lavage samples have been positive, Pneumocystis pneumonitis is @ 1989,Elsev1erSc1ence Publtshers Ltd.(UK)OI694707/89,$03 50
223
Parasitology Today, vol. 5, no. 7, I989
Fig. 2. A number
ofcysts,
with their we//s, but
no contents, demonstrated by silver methenamine staining.
demonstrable at postmortem25. A small-scale bronchoscopic study of asymptomatic, HIV (human immunoindividuals, deficiency virus)-positive including some with CD4+-lymphocyte depletion showed no detectable Pneumocystis carinii in the lung after Giemsa and silver staining of alveolar lavage”. Alveolar lavage also offers the potential for the accurate diagnosis of other infections and also of non-infective processes27. A special phenomenon of concern in the AIDS population has been the persistence of the organism as demonstrated by lavage or biopsy after some weeks of treatment2”. It is as yet uncertain whether these visualized organisms are viable. If so, their demonstration may yet not be surprising since the chemotherapeutic agents for treating such heavy infections of Pneumocystis have static rather than tidal actions and the immunosuppressive statse of AIDS is unrelentingand progressive. Although bronchoscopy samplings have proved successful in diagnosis, a search has been made for less invasive diagnostic methods, and sorne of these seem likely to prove very effective. While serological investigations of antibody responses to Pneumocystis have been valuable in delineating the epidemiology of infection in normal individuals ‘, it has not been useful in diagnosing Pneumocystis carinii pneumonia in the immunosuppressed, nor has the use of polyclonal antibody raised to pneumocysts, in the search for circulating soluble antigen. The latter method has proved nons ecific, as well as variably sensitive’ B.30. The possibility of making available an expectorated or ‘coughed-up’ sample has been realized by the use of inhalations of nebulized hypertonic saline (3-5%)3 ’ which can induce sputum in
90% of subjects who have a nonproductive cough. Subsequent microscopy after Giemsa and silver staining provides the diagnosis in up to 50% or 60% of cases. The development of monoclonal antibodies to Pneumocystis by different groups32.33 and their application to induced sputum shows that diagnostic sensitivity may be improved to 60-90% and therefore be nearly equivalent to that of bronchoscopy. Induced sputum should also be valuable in screening for other microbes, The availability of monoclonal antibodies, and more recently cloned DNA from Pneumocystis34, now offers the chance for applying sensitive, potentially quantitative techniques in the identification of viable Pneumocystis organisms with the use of enzyme-linked immunosorbent assays and DNA hybridization analysis. These may be particularly valuable in the AIDS population for identifying and monitoring the progress of Pneumocystis infection thus providing guidelines for the use of chemoprophylaxis and formal treatment. References I Walzer, PD. et al. (1974) Ann Int. Med. 80, 83-93
2 Go’ctlieb, M. et al. ( I98 I ) New Eng1.j. Med. 305, 1425-1431
3 Masur, H. et al.
(I 98 I)
New Eng1.j. Med. 305,
1431-1438
4 Carlnii, A. ( I9 IO) Comm. SK Med. Sao Paolo
I6 Aug.,
Meuwrssen, J.H.E.T. et al. ( I977)j. I36,43-49 Sheldon, W.H. 287-297
I5 I6 I7 I8 I9 20 21 22 23 24 25 26 27 28 29 30 31 32
(1959)
Infect. Dis.
Am. 1, Dis. Chrld. 97,
Chalvardjan, A.M. and Grawe. L.A. (1963) /. Qn. Pothol. l6,383-384 Grocott, R.G. (1955) Am. /. Clin. Pathof. 25, 975-979 Barton, E.G. and Campbell, W.G. (I 969) Am.]. Pathol. 54,209-235 Hopkrn, J.M. (I 987) Advanced Medrcine pp I O+ I 17, Pitman Hughes. W.T. (1987) Parasitology Today 3. 332-335 Dominy, D.E. and Lucas, R.N. (I 965) Ann. Jhorac. Sure. I, 305-3 IO Chaudha< V.K. et al. ( 1977) Am. /. Dis. Chffd. I 3 I, 902-907 Craddock, C. et al. ( I987)Loncet 2, I6- I8 Hopkin, J.M. eta/. (I 983)Loncet 2.299-30 I Stover. D.E. et 01. ( 1984) Ann. Int. Med. IO I, l-7 Blumenfeld, W. et a/. ( 1984) Am./. C/in. Pathol. 81, l-5 Broaduss, C. et al. ( 1985) Ann. Int. Med. 102, 747-752 Gal, A.A. et al. (I 987) Arch. Pathol. Lob. Med. I I I,238241 Lundgren, J.D. et ol. (I 989) Thorax 44,68-69 Young, J.A. et ol. (I 984) /. C/in. Pathol. 37, 390-397 Shelhamer, J.H. et al. (I 984)Am. Rev. Resp. Dis. 130, 1161-l I65 Pifer. L.L. et of. (I 978) Pedidtncs 6 I, 35-4 I Hughes, W.T. (I 985) Chest 87,700 Pitchenik, A.E. et al. (1986)Am. Rev. Resp. Da. I33,22&229 Kovacs, ].A. et of. ( 1988) New Engl./. Med. 3 18,
589-593 33 Elvln, K.M. et al. ( l988)Br. Med. /. 297,38 I-384 34 Wakefield, A.E..et a/..( I988)].-/nfect. Dn. 153, 359-362
204
5 Delanoe, P. and Delanoe. M. 6 7 8 9 IO
(I 9 14) Bull. Sot. Pathol. Exot. 7,27 l-272 Vanek,].(l95l)Cos.Lek.Cesk90, 1121-l 124 Gadjusek. D.C. (I 957)Pedrotrrcs 19.543-565 Kovacs, ].A. et of. (I 984) Ann. Int. Med. 100. 66367 I Edman, J.C. etol. (I 988)Nature 334,5 19-522 Weller. R.W. (I 956) Z. Kinderherlkd. 78. 166-176 ’ ’
renewals
Churchill Hospital, Headington, Oxford OX3 7L_/,UK. Ann Wakefield is a Research Fellow at the Institute ofMolecular Medicine, John Radc/iffe Hospital, Oxford OX3 9DU, UK.
Today Subscriptions
Parasitology All new subscriptions,
julion Hopkin is o Consu/tont Physicion at the
and subscription
queries should be addressed
to our Barking office at the address below. Cl Personal Subscriptions (1989. the USA; f40.00 elsewhere. 0
12 monthly issues) f33.00
in the UK; $58.00 in
Library Subscriptions(I 989, I2 monthly issues plus hardcover bound compenin the UK; $294.00 in the USA; f 159.00 elsewhere. dium with index) f 159.00
? ?Student Subscriptions( 1989, I2 monthly issues) f27.00 USA; f 32.00 elsewhere.
in the UK; $46.00 in the
(Student Subscriptions must be accompanied by valid proof of student status.) All prices include postage (air delivery outside UK). All subscriptions can be paid by cheque, bank draft or UNESCO coupons. Personal and Library subscriptions can also be paid by credit card -Access/Mastercard, Eurocard, Visa or American Express. Subscription orders should be sent to the address below. Elsevier Science Publishers Ltd Crown House Linton Road Barking Essex IG I I SJU, UK Tel. (outside UK): +44 (I) 594 7272 Tel. (in UK): 0 I 594 7272