- Email: [email protected]
ORGANISMS MIMICKING PNEUMOCYSTIS CARINII
1082 While
we
would agree with many of the indications for
cephalosporin therapy provided by Dr Selwyn, we would point of them is absolute. There are other agents with combinations, as required, of the properties that he listsbroad-spectrum, bactericidal, jMactamase-stable, non-toxicthat can be given to patients with penicillin allergy or infection with ampicillin-resistant gram-negative bacilli or coagulasenegative staphylococci, or indeed, other organisms. We often out
that
none
Department of Microbiology, St Thomas’ Hospital, London SE1
IAN PHILLIPS SUSANNAH EYKYN
SIR,-The response to my paper on the choice of p-lactam antibiotics (Sept. 18, p. 616) has been very stimulating. This applies both to the critical comments published in your columns and the expressions of appreciation I have received directly from many clinicians and microbiologists. Like Professor Garrod (Oct. 30, p. 960) I recorded my dissatisfaction at the inadequate information available about cephradine at the outset.’ But the main findings from my subsequent studies on this and other p-lactam antibiotics were presented considerably earlier than Professor Garrod has indicated--in July, 1975, at the 9th International Congress of Chemotherapy. The relatively poor activity of cephalosporins against H aemophilus influenza is mentioned by Professor Garrod, and I have also previously commented on this.2 However, the situation now appears in a fresh light with the increasing incidence of infections with strains that produce p-lactamase and are resistant to ampicillin and amoxycillin.3 Fortunately, the more enzyme-stable of the cephalosporins show no significant reduction in their activity against these ampicillin-resistant strains.
Although Dr Greenwood and Professor O’Grady (Nov. 6, p. 1026) may not fully share my enthusiasm for bacteriocidal
activity, their studies
on the artificial bladder system provide in-vitro model of the treatment failures and relapses observed with purely bacteriostatic drugs or with inadequate concentrations of bacteriocidal drugs. However, the comparatively slow bacterial lysis by cephalexin or cephradine which Dr Greenwood and Professor O’Grady observed in vitro has no counterpart in vivo, judging by the rapid cell destruction noted in serial specimens taken during my animal studies. Moreover, the relatively rapid excretion of these antibiotics does not prevent the achievement of excellent tissue levels, as shown by the extensive published data. At the same time, the very high urinary concentrations of these drugs maintain bacteriocidal levels for long periods. Thus after a single I g oral dose of cephradine the maximum and minimum concentrations over a twelve-hour period were 3260 and 90 pLg/ml, respectively4levels which are reflected in cure-rates of 88-93% obtained during therapeutic trials in urinary-tract infection.S an
The debate which my paper has provoked highlights coununcertainties about the correlation of in-vitro and invivo tests on antibiotics and poses questions about the therapeutic relevance of such tests. It is to be hoped that the impetus gained will lead to the solution of problems defined as long ago as 1871, when Joseph Lister first observed the antibacterial effects of Penicillium species in vitro as a prelude to his clinical use of culture extracts.6
tinuing
Department of Bacteriology, Westminster Medical School,
Selwyn, S. Br. med. J. 1973, ii, 239. Selwyn, S. ibid. 1974, i, 388. Medeiros, A. A., O’Brien, T. F. Lancet, 1975, i,
716. den Driessche, J. M. Ars Med., Brux. 1974, 29, 533. 5. Elkins, I., Montgomery, W. G., Cox, C. E. Proc. 8th int. Congr. Chemother.: suppl. Exerpta med. p. 35, Princeton, 1974. 6. Lister, J. Cited by W. Fraser-Moodie Proc. R. Soc. Med. 1971, 64, 87. 4 Hainaut, H.,
SIR,-Dr Maxted and his colleagues (Sept. 25, 692)
com-
the co-agglutination ’Phadebact’ kit (Pharmacy now available in the U.K. for streptococcal grouping. They report on cross-reaction problems with this technique and the slide agglutination method of Rosendall (when using antisera intended for use with the precipitation method), stating that trypsinised streptococcal suspensions are necessary for both methods. The problem of cross-reaction in the phadebact method has also been reported by Hahn and Nyberg2 and others. Hahn and Nyberg found no such problems from group-D streptococci. The phadebact method for streptococcal grouping has been in use at the Royal Free Hospital for 7 months. From the three hundred most recent streptococcal strains we have grouped here, eight strains cross-reacted, and of these, six were group D, these most commonly cross-reacting with the group G reagent. We have not so far found it necessary to trypsinise the streptococcal antigen. We use a four-hour culture of a streptococcal isolate in Todd Hewitt broth, and if no result is obtained at the time, we reincubate the culture overnight and repeat the test the next day. In a comparison with the Lancefield3 technique we are able to repeat the results using the phadebact method very successfully, and the technical staff who usually read the results, report no difficulty in detecting the coagglutination reaction. Pharmacia do not produce a reagent to detect group-D streptococci so some other method such as hydrolysis of bile aesculin agar should be used to exclude these organisms. We would also advise that the subcultures of the streptococci to be grouped are plated on to a blood-agar plate because in our experience some instances of so-called "cross-reaction" are due to the presence of more than one group of hsemolytic streptococcus, and we have recorded groups A and G and A with group C in combination on occasions. Other workers have demonstrated the presence of a second streptococcal group in lesions infected with group-A streptococci.4 It would be interesting to investigate the frequency of this in a wider study. ment on
Microbiology Department, Royal Free Hospital, London NW3 2QG
B. FARRELL I. AMIRAK
ORGANISMS MIMICKING PNEUMOCYSTIS CARINII
SIR,-Pneumocystis carinii often causes severe intra-alveopneumonia in immunosuppressed patients.’ This presumably protozoan organism can only be diagnosed by the characteristic cup-shaped cysts on staining with methenamine silver or by the demonstration of cysts, sporozoites, or trophozoites with polychrome methylene-blue, Wright’s, or Giemsa stain.: Gomori methenamine/silver-nitrate staining is the most sensitive technique for demonstrating Pneumocystis in human material in which the organisms are sparse. lar
Characteristic clinical manifestations and distinctive hison biopsy also aid diagnosis, but in their absence care must be taken to distinguish Pneumocystis from other organisms or cells which also stain positively with methenamine silver and may cause confusion-e.g., small fungi (Histoplasma, Candida, Cryptococcus), blood-cells, and parasites such as Nosema.Definitive diagnosis is most often in doubt
tology
SYDNEY SELWYN
London SW1
1. 2. 3.
AGGLUTINATION GROUPING OF STREPTOCOCCI
1. Rosendal K. Acta path. microbiol. scand. 1956, 39, 127. 2. Hahn, G., Nyberg, I. M., J. clin. Microbiol. 1976, 4,p. 99. 3. Lancefield R. C. J. exp. Med. 1933, 57, 571. 4. Anthony, B. F., Perlman, L. V., Wannamaker, L. W. Pediatrics, 1976, p.263, No. 2 (February 1976). 5. Goodell, B., Jacobs, J. B., Powell, R. D., DeVita, V. T. Ann. intern. Med.
1970, 72, 337.
van
6.
Kim, H., Hughes, W. T. Am. J. clin Path. 1973, 60, 462. 7. Margileth, A. M., Strano, A. J., Chandra, R., Neafie, R., Blum, McCully, R. M. Archs Path. 1973, 95, 145.
M.
1083 with material from sputum samples,8 percutaneous needle asof the lung,9 or brush catheter techniques’"—techniques often used in ante-mortem diagnosis of P. carin ii, so "methenamine masquerade" is of considerable clinical and
piration
therapeutic importance. Torulopsis glabrata, a yeast of the family Cryptococcaceae, itself occasionally an opportunistic pathogen in man," can mimic Pneumocystis in human material. A 59-year-old male with preleukaemia and peripheral pancytopenia was admitted with fever, cough productive of small amounts of blood-tinged sputum, and right anterior pleuritic chest pain He was not cyanotic or tachypnoeic. Bronchial breath sounds and a pleural rub were present over the right middle lobe. Chest X-ray revealed right-middle and upper-lobe infiltrates. Bronchoscopy revealed exudate coming from the right-middle and upper-lobe bronchi. Gram and acid-fastbacilli stains of sputum and bronchial washings were non-contributory. The patient was put on routine broad-spectrum antibiotic therapy. Cultures from bronchoscopy grew mixed bacterial flora. Methenamine silver stain of the bronchial brush material revealed cup-shaped 4-5 p’m diameter organisms typical of Pneumocystis (fig. 1). However, the patient was improving and physical findings were atypical so pentamidine was withheld. Pain, cough, and fever resolved over a 4-day period with continuing resolution of the pulmonary infiltrates at time of discharge 13 days later. Two separate fungal cultures of sputum obtained after bronchoscopy grew T. gla-
inhabitant of the tracheobronchial So in sputum it might be mistaken for P. carinii if one were not familiar with the similar appearance of the organisms stained with methenamine silver. It is important to be very critical of a diagnosis of Pneumocystis infection since the diagnosis demands the sometimes difficult therapy with pentamidine isethionate. Adequate tissuebiopsy material stained with methenamine silver and other stains rarely causes difficulty. In sputum, bronchial washings, or brush material differentiation of methenamine masqueraders from Pneumocystis can be accomplished by careful consideration of the differential diagnosis, by careful attention to size, by staining with multiple techniques, by careful search for budding, and, if necessary, by study of additional biopsy material. Clinical features atypical for Pneumocystis should always prompt consideration of a methenamine masquerade.
Torulopsis is not a and sputum.12 13
rare
tree
Medicine Branch and Laboratory of Pathology, National Cancer Institute, and Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases National Institutes of Health, Bethesda, Maryland 20014, U.S.A.
12. Dolan, C. T. Am. J.
clin. Path.
1971, 55,
ROBERT C. YOUNG JOHN E. BENNETT ELIZABETH W. CHU
580.
13. Mackenzie, D. W. R. Sabouraudia, 1961, 1, 8
brata.. The organism in the bronchial brushing material was probably T. glabrata and not Pneumocystis. Review of the brushing material revealed one instance of apparent budding thought to have the morphological appearance of Pneumocystis (fig. 2). Kidney tissue taken from a patient with necropsy-proven Torulopsis is shown in fig. 3. Coexistence of the two organisms cannot be excluded, but the importance of this case is that the two organisms are sufficiently similar on methenamine silver staining to be confused. Both are round or oval, diameters range from 1-5 p’m to 4-8 m, respectively, and both stain positively with methenamine silver. Torulopsis, unlike Pneumocystis, frequently demonstrates budding, and this should generally serve to distinguish Torulopsis as well as Candida and Cryptococcus from Pneumocystis, as would sufficient histological material. However, in this case budding was
not a
striking feature.
8. Fortuny, I. E., Tempero, K. F., Amsden, T. W.
Cancer 1970, 26, 911.
9. Johnson, H. D., Johnson, W. W. J. Am. med. Ass. 1970, 214, 1067. 10. Repsher, L. H., Schroter, G., Hammond, W. S. New Engl. J. Med. 1972,
Fig. 2-Photomicrograph of material in fig. 1. Arrow indicates apparent budding in organism stained with Gomori-
287, 340. 11. Marks, M. I., Langston, C., Eickoff, T. C. ibid. 1970, 283, 1131.
/methenamine-silver
Ftg. I-Photomicrograph of material obtained from bronchial brushing stained with Gomori/methenamine-silver. Arrow indicates 4-5 fLm diameter organisms consistent with P. ëaTJnli (x 1010).
(x 1010).
Fig. 3-Photomicrograph proven T. glabrata.
of human
kidney containing culturally
Gomori/methenamine-silver (x 1010).
we
would agree with many of the indications for
cephalosporin therapy provided by Dr Selwyn, we would point of them is absolute. There are other agents with combinations, as required, of the properties that he listsbroad-spectrum, bactericidal, jMactamase-stable, non-toxicthat can be given to patients with penicillin allergy or infection with ampicillin-resistant gram-negative bacilli or coagulasenegative staphylococci, or indeed, other organisms. We often out
that
none
Department of Microbiology, St Thomas’ Hospital, London SE1
IAN PHILLIPS SUSANNAH EYKYN
SIR,-The response to my paper on the choice of p-lactam antibiotics (Sept. 18, p. 616) has been very stimulating. This applies both to the critical comments published in your columns and the expressions of appreciation I have received directly from many clinicians and microbiologists. Like Professor Garrod (Oct. 30, p. 960) I recorded my dissatisfaction at the inadequate information available about cephradine at the outset.’ But the main findings from my subsequent studies on this and other p-lactam antibiotics were presented considerably earlier than Professor Garrod has indicated--in July, 1975, at the 9th International Congress of Chemotherapy. The relatively poor activity of cephalosporins against H aemophilus influenza is mentioned by Professor Garrod, and I have also previously commented on this.2 However, the situation now appears in a fresh light with the increasing incidence of infections with strains that produce p-lactamase and are resistant to ampicillin and amoxycillin.3 Fortunately, the more enzyme-stable of the cephalosporins show no significant reduction in their activity against these ampicillin-resistant strains.
Although Dr Greenwood and Professor O’Grady (Nov. 6, p. 1026) may not fully share my enthusiasm for bacteriocidal
activity, their studies
on the artificial bladder system provide in-vitro model of the treatment failures and relapses observed with purely bacteriostatic drugs or with inadequate concentrations of bacteriocidal drugs. However, the comparatively slow bacterial lysis by cephalexin or cephradine which Dr Greenwood and Professor O’Grady observed in vitro has no counterpart in vivo, judging by the rapid cell destruction noted in serial specimens taken during my animal studies. Moreover, the relatively rapid excretion of these antibiotics does not prevent the achievement of excellent tissue levels, as shown by the extensive published data. At the same time, the very high urinary concentrations of these drugs maintain bacteriocidal levels for long periods. Thus after a single I g oral dose of cephradine the maximum and minimum concentrations over a twelve-hour period were 3260 and 90 pLg/ml, respectively4levels which are reflected in cure-rates of 88-93% obtained during therapeutic trials in urinary-tract infection.S an
The debate which my paper has provoked highlights coununcertainties about the correlation of in-vitro and invivo tests on antibiotics and poses questions about the therapeutic relevance of such tests. It is to be hoped that the impetus gained will lead to the solution of problems defined as long ago as 1871, when Joseph Lister first observed the antibacterial effects of Penicillium species in vitro as a prelude to his clinical use of culture extracts.6
tinuing
Department of Bacteriology, Westminster Medical School,
Selwyn, S. Br. med. J. 1973, ii, 239. Selwyn, S. ibid. 1974, i, 388. Medeiros, A. A., O’Brien, T. F. Lancet, 1975, i,
716. den Driessche, J. M. Ars Med., Brux. 1974, 29, 533. 5. Elkins, I., Montgomery, W. G., Cox, C. E. Proc. 8th int. Congr. Chemother.: suppl. Exerpta med. p. 35, Princeton, 1974. 6. Lister, J. Cited by W. Fraser-Moodie Proc. R. Soc. Med. 1971, 64, 87. 4 Hainaut, H.,
SIR,-Dr Maxted and his colleagues (Sept. 25, 692)
com-
the co-agglutination ’Phadebact’ kit (Pharmacy now available in the U.K. for streptococcal grouping. They report on cross-reaction problems with this technique and the slide agglutination method of Rosendall (when using antisera intended for use with the precipitation method), stating that trypsinised streptococcal suspensions are necessary for both methods. The problem of cross-reaction in the phadebact method has also been reported by Hahn and Nyberg2 and others. Hahn and Nyberg found no such problems from group-D streptococci. The phadebact method for streptococcal grouping has been in use at the Royal Free Hospital for 7 months. From the three hundred most recent streptococcal strains we have grouped here, eight strains cross-reacted, and of these, six were group D, these most commonly cross-reacting with the group G reagent. We have not so far found it necessary to trypsinise the streptococcal antigen. We use a four-hour culture of a streptococcal isolate in Todd Hewitt broth, and if no result is obtained at the time, we reincubate the culture overnight and repeat the test the next day. In a comparison with the Lancefield3 technique we are able to repeat the results using the phadebact method very successfully, and the technical staff who usually read the results, report no difficulty in detecting the coagglutination reaction. Pharmacia do not produce a reagent to detect group-D streptococci so some other method such as hydrolysis of bile aesculin agar should be used to exclude these organisms. We would also advise that the subcultures of the streptococci to be grouped are plated on to a blood-agar plate because in our experience some instances of so-called "cross-reaction" are due to the presence of more than one group of hsemolytic streptococcus, and we have recorded groups A and G and A with group C in combination on occasions. Other workers have demonstrated the presence of a second streptococcal group in lesions infected with group-A streptococci.4 It would be interesting to investigate the frequency of this in a wider study. ment on
Microbiology Department, Royal Free Hospital, London NW3 2QG
B. FARRELL I. AMIRAK
ORGANISMS MIMICKING PNEUMOCYSTIS CARINII
SIR,-Pneumocystis carinii often causes severe intra-alveopneumonia in immunosuppressed patients.’ This presumably protozoan organism can only be diagnosed by the characteristic cup-shaped cysts on staining with methenamine silver or by the demonstration of cysts, sporozoites, or trophozoites with polychrome methylene-blue, Wright’s, or Giemsa stain.: Gomori methenamine/silver-nitrate staining is the most sensitive technique for demonstrating Pneumocystis in human material in which the organisms are sparse. lar
Characteristic clinical manifestations and distinctive hison biopsy also aid diagnosis, but in their absence care must be taken to distinguish Pneumocystis from other organisms or cells which also stain positively with methenamine silver and may cause confusion-e.g., small fungi (Histoplasma, Candida, Cryptococcus), blood-cells, and parasites such as Nosema.Definitive diagnosis is most often in doubt
tology
SYDNEY SELWYN
London SW1
1. 2. 3.
AGGLUTINATION GROUPING OF STREPTOCOCCI
1. Rosendal K. Acta path. microbiol. scand. 1956, 39, 127. 2. Hahn, G., Nyberg, I. M., J. clin. Microbiol. 1976, 4,p. 99. 3. Lancefield R. C. J. exp. Med. 1933, 57, 571. 4. Anthony, B. F., Perlman, L. V., Wannamaker, L. W. Pediatrics, 1976, p.263, No. 2 (February 1976). 5. Goodell, B., Jacobs, J. B., Powell, R. D., DeVita, V. T. Ann. intern. Med.
1970, 72, 337.
van
6.
Kim, H., Hughes, W. T. Am. J. clin Path. 1973, 60, 462. 7. Margileth, A. M., Strano, A. J., Chandra, R., Neafie, R., Blum, McCully, R. M. Archs Path. 1973, 95, 145.
M.
1083 with material from sputum samples,8 percutaneous needle asof the lung,9 or brush catheter techniques’"—techniques often used in ante-mortem diagnosis of P. carin ii, so "methenamine masquerade" is of considerable clinical and
piration
therapeutic importance. Torulopsis glabrata, a yeast of the family Cryptococcaceae, itself occasionally an opportunistic pathogen in man," can mimic Pneumocystis in human material. A 59-year-old male with preleukaemia and peripheral pancytopenia was admitted with fever, cough productive of small amounts of blood-tinged sputum, and right anterior pleuritic chest pain He was not cyanotic or tachypnoeic. Bronchial breath sounds and a pleural rub were present over the right middle lobe. Chest X-ray revealed right-middle and upper-lobe infiltrates. Bronchoscopy revealed exudate coming from the right-middle and upper-lobe bronchi. Gram and acid-fastbacilli stains of sputum and bronchial washings were non-contributory. The patient was put on routine broad-spectrum antibiotic therapy. Cultures from bronchoscopy grew mixed bacterial flora. Methenamine silver stain of the bronchial brush material revealed cup-shaped 4-5 p’m diameter organisms typical of Pneumocystis (fig. 1). However, the patient was improving and physical findings were atypical so pentamidine was withheld. Pain, cough, and fever resolved over a 4-day period with continuing resolution of the pulmonary infiltrates at time of discharge 13 days later. Two separate fungal cultures of sputum obtained after bronchoscopy grew T. gla-
inhabitant of the tracheobronchial So in sputum it might be mistaken for P. carinii if one were not familiar with the similar appearance of the organisms stained with methenamine silver. It is important to be very critical of a diagnosis of Pneumocystis infection since the diagnosis demands the sometimes difficult therapy with pentamidine isethionate. Adequate tissuebiopsy material stained with methenamine silver and other stains rarely causes difficulty. In sputum, bronchial washings, or brush material differentiation of methenamine masqueraders from Pneumocystis can be accomplished by careful consideration of the differential diagnosis, by careful attention to size, by staining with multiple techniques, by careful search for budding, and, if necessary, by study of additional biopsy material. Clinical features atypical for Pneumocystis should always prompt consideration of a methenamine masquerade.
Torulopsis is not a and sputum.12 13
rare
tree
Medicine Branch and Laboratory of Pathology, National Cancer Institute, and Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases National Institutes of Health, Bethesda, Maryland 20014, U.S.A.
12. Dolan, C. T. Am. J.
clin. Path.
1971, 55,
ROBERT C. YOUNG JOHN E. BENNETT ELIZABETH W. CHU
580.
13. Mackenzie, D. W. R. Sabouraudia, 1961, 1, 8
brata.. The organism in the bronchial brushing material was probably T. glabrata and not Pneumocystis. Review of the brushing material revealed one instance of apparent budding thought to have the morphological appearance of Pneumocystis (fig. 2). Kidney tissue taken from a patient with necropsy-proven Torulopsis is shown in fig. 3. Coexistence of the two organisms cannot be excluded, but the importance of this case is that the two organisms are sufficiently similar on methenamine silver staining to be confused. Both are round or oval, diameters range from 1-5 p’m to 4-8 m, respectively, and both stain positively with methenamine silver. Torulopsis, unlike Pneumocystis, frequently demonstrates budding, and this should generally serve to distinguish Torulopsis as well as Candida and Cryptococcus from Pneumocystis, as would sufficient histological material. However, in this case budding was
not a
striking feature.
8. Fortuny, I. E., Tempero, K. F., Amsden, T. W.
Cancer 1970, 26, 911.
9. Johnson, H. D., Johnson, W. W. J. Am. med. Ass. 1970, 214, 1067. 10. Repsher, L. H., Schroter, G., Hammond, W. S. New Engl. J. Med. 1972,
Fig. 2-Photomicrograph of material in fig. 1. Arrow indicates apparent budding in organism stained with Gomori-
287, 340. 11. Marks, M. I., Langston, C., Eickoff, T. C. ibid. 1970, 283, 1131.
/methenamine-silver
Ftg. I-Photomicrograph of material obtained from bronchial brushing stained with Gomori/methenamine-silver. Arrow indicates 4-5 fLm diameter organisms consistent with P. ëaTJnli (x 1010).
(x 1010).
Fig. 3-Photomicrograph proven T. glabrata.
of human
kidney containing culturally
Gomori/methenamine-silver (x 1010).