- Email: [email protected]
TRILOSTANE INTERFERENCE WITH STEROID ASSAYS
727 in the management of recurrent malignant effusions.1 Intrapleural injection of C. parvum after pleural aspiration induced a satisfactory pleurodesis in seven of the nine patients surviving long enough (more than four weeks) for adequate assessment of response, although two patients required a second intrapleural injection. This response was superior to that seen in the controls treated with intrapleural mustine. C. parvum was also effective in three out of four patients deemed "mustine failures" because of recurrent effusions uncontrolled by intrapleural mustine. Subsequent experience with a further five patients has confirmed this high success rate. Intrapleural C. parvum was well tolerated and we believe that, although its mode of action is uncertain, it offers a promising new approach to the difficult problem of recurrent malignant effusions. The mean survival times in our small series were 6 months (C. parvum) and 3 months (mustine). Further randomised studies of the various forms of treatment now available for malignant pleural effusion should include comparison of survival times, though this may be difficult to assess if several forms of treatment have been given.
drainage
Chest Unit,
City Hospital, Edinburgh EH10 5SB
J. W. MILLAR A. M. HUNTER N. W. HORNE
SERRATIA MARCESCENS CONTAMINATED DISINFECTANTS
StR,-In their investigation of antibiotic sensitive Serratia infections complicating cardiopulmonary operations in 1978 Dr Ehrenkranz and his colleagues (Dec. 13, p. 1289) tested the
marcescens
ability of S. marcescens to grow in four formulations of quaternary ammonium disinfectant, including one that we no longer market. The ’Hi-Tor’ referred to in the Lancet article carries Environmental Protection Agency registration number 303-94. In August, 1980, we introduced a revised formula (E.P.A. 303-91). Hi-Tor 303-94 contains a "quat" system ofn-alkyl(60% C14, 30% CI6’ 5% C18’ 5% CI2) dimethylbenzylammonium chloride 6.75% and n-alkyl (68% Ci2, 32% C14) dimethylethylbenzylammonium chloride 6-75%. Hi-Tor 303-91 contains didecyldimethylammonium chloride 7-5% and n-alkyl (Ct4 50%, C12 40%, C16 10%) dimethylbenzylammonium chloride 50%. The two formulations are completely different. The quat system in ’TOR’ (1 :64 dilution) (another Huntington product mentioned) is: n-alkyl (60% C14 30% C16, 5% Cl8’ 5% C12) dimethylbenzylammonium chloride 1 - 607o and n-alkyl (50% C12 30% C14 17% C16) 3% CIs) dimethylethylbenzylammonium chloride I6%, and the parts per million (p.p.m.) of quat in TOR required to kill the following organisms are: Pseudomonas aeruginosa 500, Staphylococcus aureus 400, and Salmonella choleraesuis 400. The quat system in Hi-Tor 303-94 (1 :256 dilution) is shown above and the p.p.m. required to kill are: Ps. aeruginosa 527, Staph. aureus 450, and S. choleraesuis 450. The quat systems for Hi-Tor 303-94 and TOR are much the same, the first quats being identical and the second identical in chemical nomenclature though differing in carbon chain distribution. In our experience of microbiology testing we have found that both TOR and Hi-Tor 303-94 disinfectants take approximately the same p.p.m.s consistently to kill certain test organisms, as indicated above. If I received a microbiology test report with the results as given in the Dec. 13 Lancet article I would ask for the tests to be done again. In use-dilution Hi-Tor 303-94 and TOR are too close to being identical, in regards to the quat system, to have the contrasting results described. The test procedure described by Ehrenkranz et al. consisted of taking 0 -05-0 -1 ml as an inoculum for 5-10 ml of disinfectant. We take that to mean that a certain number of organisms per ml was mixedwithup to 10 ml of disinfectant. The test procedure described not official or recognised, and if it is a modification of the 1 Millar JW, Hunter AM, Horne NW. Intrapleural immunotherapy with Corynebacterium parvum in recurrent malignant effusions. Thorax 1980; 35: 856-58
A.O.A.C. phenol coefficient test, this test is no longer recognised by the Environmental Protection Agency in that it does not simulate in-use conditions. E.P.A. requires the A.O.A.C. use-dilution confirmation test which does simulate in-use conditions. In other words, a disinfectant is designed to disinfect on hard non-porous surfaces. In the A.O.A.C. use-dilution confirmation test a hard non-porous surface is inoculated with the test organism (107- 109/ml) and then exposed to the disinfectant solution. Then we check for growth or no growth, and E.P.A. requires complete killing to pass the test. Once we have E.P.A. approval we can market that particular disinfectant with the disinfectant claims that we have established. Thus Ehrenkranz’s paper is misleading because the test procedure is not an official one while our test data are generated by official A.O.A.C. test methods required by the E.P.A. Even so, the test results are not consistent: TOR killed S. marcesens in all instances and Hi-Tor 303-94 killed S. marcescens only occasionally but killed the same organism along with three additional organisms in all instances in other hospitals. Furthermore Hi-Tor 303-94 and TOR are much the same in respect of "quat" chemical composition and p.p.m. of quat in the use-dilution. We would question not only the procedure but also the technique. Ehrenkranz et al. state that "The danger of using quaternary ammonium compounds as disinfectants rather than cleansers is reemphasised," and they refer to an article written by Center for Disease Control staff on Use of Quaternary Ammonium Compounds. The article consistently and specifically refers to benzylalkonium chlorides and concludes that this type of disinfectant should be used in non-critical areas of the hospital. We no longer use benzylalkonium chlorides in our disinfectant products; we use ammonium chlorides. Both types of chemicals are referred to as quats-therein lies the confusion. Benzylalkonium chlorides are not effective against some hospital pathogens, including Ps. aeruginosa. Ammonium chloride type quats are effective against hospital pathogens at 500 p.p.m. or less, including Ps. aeruginosa. The C.D.C. article on benzylalkonium chlorides seems out of date. The technology of quats for disinfection has improved with the advent of ammonium chlorides. -
Huntington Laboratories Inc., Huntington, Indiana 46750, U.S.A.
RICHARD D. SHEETS
TRILOSTANE INTERFERENCE WITH STEROID ASSAYS
SIR,-Like Professor Mattingly and Christine Tyler (March 7, p. 561) we have measured steroid hormone levels in serum and urine from patients being treated for Cushing’s syndrome with the new adrenal blocking agent trilostane and whilst we agree that the interference of trilostane with the fluorimetric assay for serum 11-hydroxycorticosteroids ("cortisol") may be eliminated by the inclusion of an alkali wash before the addition of fluorescence reagent we have evidence that interference with the fluorimetric assay for urinary free "cortisol" persists despite the alkali wash stage. Furthermore, patients being treated with trilostane show unexpected rises in serum levels of testosterone and androstenedione which may also result from interference of the drug with the assays. We have studied five patients (three male, two female), with Cushing’s syndrome taking trilostane in doses ranging from 240 to1 1080 mg per day. Serum "cortisol" results derived by fluorimetry, using the modification suggested by Mattingly, were lower than the results obtained by a radioimmunoassay (RIA) known not to crossreact with trilostane (’Immophase’; Corning Medical) and there was a reasonable correlation between methods: modified fluorimetry 0-71 RIA + 46(nmol/1); r = 0-75, n 28. Addition of trilostane to serum in concentrations up to 100 mol/1 did not alter the "cortisol" results obtained by either RIA or the modified fluorimetric technique. However, fluorimetric urinary free "cortisol" results were appreciably higher than the corresponding RIA results in the patients taking trilostane and there was a poor correlation between methods: =
=
1. Mattingly D. A simple fluorimetric method for the estimation 11-hydroxycorticoids in human plasma. J Clin Path 1962; 15: 374.
of free
728 SERUM ANDROGEN LEVELS IN PATIENTS TAKING TRILOSTANE
TABLE I-ORTHOPAEDIC PATIENTS WITH PSEUDOMONAS WOUND INFECTION IN THE TWO OUTBREAKS
=0-82 RIA + 1650 (nmol/24 h); r 0-19, n 24. Urine collected from the same patients before trilostane treatment showed a much closer agreement of free "cortisol" results between methods: fluorimetry =1-42 RIA - 7 (nmol/24 h); r 0 -98; n 8. We suggest that the routine alkali wash step (2 ml of 0.2mol/1 sodium hydroxide) in the fluorimetric urinary "cortisol" assay does not fully remove the high levels of trilostane and its metabolites found in the urine of patients treated with the drug, and in support of this we have shown that the addition of 1 mmol/1 trilostane to urine increases the measured "cortisol" result by 100 nmol/l. Serum testosterone rose in all patients tested and serum androstenedione rose in all but the patient taking the lowest dose of the drug (table). Rises in the serum levels of these steroids were not expected since both arise in the biosynthetic pathway after the 3(3-hydroxysteroid dehydrogenase enzyme which is blocked by trilostane.2 Since trilostane is structurally similar to these two androgens, interference in the RIA methods used must be a possibility. Preliminary results have confirmed that trilostane in concentrations of 10 tmol/1 increased the measured testosterone level by 5 -3nmol/1 although no such effect was observed on the androstenedione levels. Work is in progress to establish whether interference from trilostane or its metabolites is the sole cause of the increase in the measured levels of the two androgens or whether a true physiological increase also occurs, but for the present, all centres using trilostane should be alerted to the problems of interpreting steroid hormone levels in subjects taking the drug.
fluorimetry
=
=
=
=
*Strains
not distinguishable from Laboratory, P.H.L.S., Colindale).
one
another
(Cross-infection Reference
TABLE II-ENVIRONMENTAL SAMPLING
Part of this work will be presented in full by one of us (M. T.) in a thesis to be submitted shortly to the Institute of Medical Laboratory Sciences.
Departments of Steroid Biochemistry and
Medicine,
Royal Infirmary, Glasgow G4 OSF
G. H. BEASTALL W. A. RATCLIFFE M. THOMSON C. G. SEMPLE
OUTBREAK OF PLASTER-ASSOCIATED PSEUDOMONAS INFECTION
SIR,-At the end of1979, over a period of two months, eight male
patients on a 20-bed ward acquired Pseudomonas wound infection after orthopaedic surgery. Six serologically different strains of Ps. aeruginosa and one strain of Ps. fluorescens were isolated from their wound swabs (table i, first outbreak). Five patients had had replacesurgery and the infections were serious. Before that, Ps. aeruginosa wound infections in orthopaedic patients had been rare, there being one in June (in a male) and one in July (female). The theatre was closed for non-emergency surgery. The aseptic technique used in the theatre and on the ward before and after surgery was found to be satisfactory. All the infected patients had had a plaster cast applied in theatre, the wound having been covered with a sterile dressing and the rest of the limb having been covered with a thick layer of sterile plaster wool. Fresh tap water was drawn from the theatre scrub tap into two plastic buckets into which the plaster bandages (’Gypsona’) were immersed. Bacteriological sampling revealed that one tap in the washing-up room of the theatre suite and one tap on the ward yielded strains of Ps. aeruginosa of different serotypes (table II). The theatre was cleaned, the two taps were replaced, and the theatre was reopened. Although the water used for the preparation of plaster casts seemed the likely culprit, only male patients had ment
Swabs cultured on cetrimide
been infected and with Ps. aeruginosa strains of different serotypes. The plaster continued to be prepared as before, but it was with some apprehension that surgery was resumed. Over the following two weeks, five of the twenty-three patients who had orthopaedic surgery had wound infections soon after their operations (second outbreak). Ps. aeruginosa serotype 0:4, H:NT (outbreak strain) was isolated from wound swabs. All five patients had had plaster-of-paris casts applied in theatre. The outbreak strain was isolated from the original casts applied to two of these patients in theatre. The theatre was more extensively sampled bacteriologically, and the outbreak strain was isolated in pure heavy growth from the residual water in both plaster buckets but not from any other site (table 11). A laboratory study (to be published elsewhere) showed that plaster-of-paris takes a long time to dry thoroughly and that the outbreak strain could multiply in the drying plaster. In this study, on volunteers wearing wrist-to-elbow casts, skin contamination ensued when contaminated plaster was applied, despite a thick layer of sterile wool. Postoperative wound infections caused by Clostridium spp. have been reported as a result of using contaminated plaster-of-paris.1,2 The making of casts by immersing plaster bandages into freshly drawn tap water is common practice and it is surprising that plasterrelated infection caused by gram-negative rods has not been pans after a shelf procedure hip. New Engl J Med 1962; 267: 348-49. Murray EGD, Denton GD Plaster of paris as source of infection in tetanus and gas gangrene. Canad Med Assoc J 1949; 60: 1-4.
1. Kozinn HA. Clostridium welchri infections due to plaster of
of the
2.
Treatment of Cushing’s syndrome with trilostane, inhibitor of adrenal steroid biosynthesis. J Clin Endocrinol Metab 1978, 47: 1042.
Komamcky P, Spark RF, Melby JC an
agar and examined after 24 and 48 h.
- ]’0ther= strains of other serotypes not related to those from the patients. :f:Four 100 ml membrane-filtered water samples were also taken from orthopaedic theatre taps and one yielded Ps. aeruginosa, non-outbreak strain.
2.
drainage
Chest Unit,
City Hospital, Edinburgh EH10 5SB
J. W. MILLAR A. M. HUNTER N. W. HORNE
SERRATIA MARCESCENS CONTAMINATED DISINFECTANTS
StR,-In their investigation of antibiotic sensitive Serratia infections complicating cardiopulmonary operations in 1978 Dr Ehrenkranz and his colleagues (Dec. 13, p. 1289) tested the
marcescens
ability of S. marcescens to grow in four formulations of quaternary ammonium disinfectant, including one that we no longer market. The ’Hi-Tor’ referred to in the Lancet article carries Environmental Protection Agency registration number 303-94. In August, 1980, we introduced a revised formula (E.P.A. 303-91). Hi-Tor 303-94 contains a "quat" system ofn-alkyl(60% C14, 30% CI6’ 5% C18’ 5% CI2) dimethylbenzylammonium chloride 6.75% and n-alkyl (68% Ci2, 32% C14) dimethylethylbenzylammonium chloride 6-75%. Hi-Tor 303-91 contains didecyldimethylammonium chloride 7-5% and n-alkyl (Ct4 50%, C12 40%, C16 10%) dimethylbenzylammonium chloride 50%. The two formulations are completely different. The quat system in ’TOR’ (1 :64 dilution) (another Huntington product mentioned) is: n-alkyl (60% C14 30% C16, 5% Cl8’ 5% C12) dimethylbenzylammonium chloride 1 - 607o and n-alkyl (50% C12 30% C14 17% C16) 3% CIs) dimethylethylbenzylammonium chloride I6%, and the parts per million (p.p.m.) of quat in TOR required to kill the following organisms are: Pseudomonas aeruginosa 500, Staphylococcus aureus 400, and Salmonella choleraesuis 400. The quat system in Hi-Tor 303-94 (1 :256 dilution) is shown above and the p.p.m. required to kill are: Ps. aeruginosa 527, Staph. aureus 450, and S. choleraesuis 450. The quat systems for Hi-Tor 303-94 and TOR are much the same, the first quats being identical and the second identical in chemical nomenclature though differing in carbon chain distribution. In our experience of microbiology testing we have found that both TOR and Hi-Tor 303-94 disinfectants take approximately the same p.p.m.s consistently to kill certain test organisms, as indicated above. If I received a microbiology test report with the results as given in the Dec. 13 Lancet article I would ask for the tests to be done again. In use-dilution Hi-Tor 303-94 and TOR are too close to being identical, in regards to the quat system, to have the contrasting results described. The test procedure described by Ehrenkranz et al. consisted of taking 0 -05-0 -1 ml as an inoculum for 5-10 ml of disinfectant. We take that to mean that a certain number of organisms per ml was mixedwithup to 10 ml of disinfectant. The test procedure described not official or recognised, and if it is a modification of the 1 Millar JW, Hunter AM, Horne NW. Intrapleural immunotherapy with Corynebacterium parvum in recurrent malignant effusions. Thorax 1980; 35: 856-58
A.O.A.C. phenol coefficient test, this test is no longer recognised by the Environmental Protection Agency in that it does not simulate in-use conditions. E.P.A. requires the A.O.A.C. use-dilution confirmation test which does simulate in-use conditions. In other words, a disinfectant is designed to disinfect on hard non-porous surfaces. In the A.O.A.C. use-dilution confirmation test a hard non-porous surface is inoculated with the test organism (107- 109/ml) and then exposed to the disinfectant solution. Then we check for growth or no growth, and E.P.A. requires complete killing to pass the test. Once we have E.P.A. approval we can market that particular disinfectant with the disinfectant claims that we have established. Thus Ehrenkranz’s paper is misleading because the test procedure is not an official one while our test data are generated by official A.O.A.C. test methods required by the E.P.A. Even so, the test results are not consistent: TOR killed S. marcesens in all instances and Hi-Tor 303-94 killed S. marcescens only occasionally but killed the same organism along with three additional organisms in all instances in other hospitals. Furthermore Hi-Tor 303-94 and TOR are much the same in respect of "quat" chemical composition and p.p.m. of quat in the use-dilution. We would question not only the procedure but also the technique. Ehrenkranz et al. state that "The danger of using quaternary ammonium compounds as disinfectants rather than cleansers is reemphasised," and they refer to an article written by Center for Disease Control staff on Use of Quaternary Ammonium Compounds. The article consistently and specifically refers to benzylalkonium chlorides and concludes that this type of disinfectant should be used in non-critical areas of the hospital. We no longer use benzylalkonium chlorides in our disinfectant products; we use ammonium chlorides. Both types of chemicals are referred to as quats-therein lies the confusion. Benzylalkonium chlorides are not effective against some hospital pathogens, including Ps. aeruginosa. Ammonium chloride type quats are effective against hospital pathogens at 500 p.p.m. or less, including Ps. aeruginosa. The C.D.C. article on benzylalkonium chlorides seems out of date. The technology of quats for disinfection has improved with the advent of ammonium chlorides. -
Huntington Laboratories Inc., Huntington, Indiana 46750, U.S.A.
RICHARD D. SHEETS
TRILOSTANE INTERFERENCE WITH STEROID ASSAYS
SIR,-Like Professor Mattingly and Christine Tyler (March 7, p. 561) we have measured steroid hormone levels in serum and urine from patients being treated for Cushing’s syndrome with the new adrenal blocking agent trilostane and whilst we agree that the interference of trilostane with the fluorimetric assay for serum 11-hydroxycorticosteroids ("cortisol") may be eliminated by the inclusion of an alkali wash before the addition of fluorescence reagent we have evidence that interference with the fluorimetric assay for urinary free "cortisol" persists despite the alkali wash stage. Furthermore, patients being treated with trilostane show unexpected rises in serum levels of testosterone and androstenedione which may also result from interference of the drug with the assays. We have studied five patients (three male, two female), with Cushing’s syndrome taking trilostane in doses ranging from 240 to1 1080 mg per day. Serum "cortisol" results derived by fluorimetry, using the modification suggested by Mattingly, were lower than the results obtained by a radioimmunoassay (RIA) known not to crossreact with trilostane (’Immophase’; Corning Medical) and there was a reasonable correlation between methods: modified fluorimetry 0-71 RIA + 46(nmol/1); r = 0-75, n 28. Addition of trilostane to serum in concentrations up to 100 mol/1 did not alter the "cortisol" results obtained by either RIA or the modified fluorimetric technique. However, fluorimetric urinary free "cortisol" results were appreciably higher than the corresponding RIA results in the patients taking trilostane and there was a poor correlation between methods: =
=
1. Mattingly D. A simple fluorimetric method for the estimation 11-hydroxycorticoids in human plasma. J Clin Path 1962; 15: 374.
of free
728 SERUM ANDROGEN LEVELS IN PATIENTS TAKING TRILOSTANE
TABLE I-ORTHOPAEDIC PATIENTS WITH PSEUDOMONAS WOUND INFECTION IN THE TWO OUTBREAKS
=0-82 RIA + 1650 (nmol/24 h); r 0-19, n 24. Urine collected from the same patients before trilostane treatment showed a much closer agreement of free "cortisol" results between methods: fluorimetry =1-42 RIA - 7 (nmol/24 h); r 0 -98; n 8. We suggest that the routine alkali wash step (2 ml of 0.2mol/1 sodium hydroxide) in the fluorimetric urinary "cortisol" assay does not fully remove the high levels of trilostane and its metabolites found in the urine of patients treated with the drug, and in support of this we have shown that the addition of 1 mmol/1 trilostane to urine increases the measured "cortisol" result by 100 nmol/l. Serum testosterone rose in all patients tested and serum androstenedione rose in all but the patient taking the lowest dose of the drug (table). Rises in the serum levels of these steroids were not expected since both arise in the biosynthetic pathway after the 3(3-hydroxysteroid dehydrogenase enzyme which is blocked by trilostane.2 Since trilostane is structurally similar to these two androgens, interference in the RIA methods used must be a possibility. Preliminary results have confirmed that trilostane in concentrations of 10 tmol/1 increased the measured testosterone level by 5 -3nmol/1 although no such effect was observed on the androstenedione levels. Work is in progress to establish whether interference from trilostane or its metabolites is the sole cause of the increase in the measured levels of the two androgens or whether a true physiological increase also occurs, but for the present, all centres using trilostane should be alerted to the problems of interpreting steroid hormone levels in subjects taking the drug.
fluorimetry
=
=
=
=
*Strains
not distinguishable from Laboratory, P.H.L.S., Colindale).
one
another
(Cross-infection Reference
TABLE II-ENVIRONMENTAL SAMPLING
Part of this work will be presented in full by one of us (M. T.) in a thesis to be submitted shortly to the Institute of Medical Laboratory Sciences.
Departments of Steroid Biochemistry and
Medicine,
Royal Infirmary, Glasgow G4 OSF
G. H. BEASTALL W. A. RATCLIFFE M. THOMSON C. G. SEMPLE
OUTBREAK OF PLASTER-ASSOCIATED PSEUDOMONAS INFECTION
SIR,-At the end of1979, over a period of two months, eight male
patients on a 20-bed ward acquired Pseudomonas wound infection after orthopaedic surgery. Six serologically different strains of Ps. aeruginosa and one strain of Ps. fluorescens were isolated from their wound swabs (table i, first outbreak). Five patients had had replacesurgery and the infections were serious. Before that, Ps. aeruginosa wound infections in orthopaedic patients had been rare, there being one in June (in a male) and one in July (female). The theatre was closed for non-emergency surgery. The aseptic technique used in the theatre and on the ward before and after surgery was found to be satisfactory. All the infected patients had had a plaster cast applied in theatre, the wound having been covered with a sterile dressing and the rest of the limb having been covered with a thick layer of sterile plaster wool. Fresh tap water was drawn from the theatre scrub tap into two plastic buckets into which the plaster bandages (’Gypsona’) were immersed. Bacteriological sampling revealed that one tap in the washing-up room of the theatre suite and one tap on the ward yielded strains of Ps. aeruginosa of different serotypes (table II). The theatre was cleaned, the two taps were replaced, and the theatre was reopened. Although the water used for the preparation of plaster casts seemed the likely culprit, only male patients had ment
Swabs cultured on cetrimide
been infected and with Ps. aeruginosa strains of different serotypes. The plaster continued to be prepared as before, but it was with some apprehension that surgery was resumed. Over the following two weeks, five of the twenty-three patients who had orthopaedic surgery had wound infections soon after their operations (second outbreak). Ps. aeruginosa serotype 0:4, H:NT (outbreak strain) was isolated from wound swabs. All five patients had had plaster-of-paris casts applied in theatre. The outbreak strain was isolated from the original casts applied to two of these patients in theatre. The theatre was more extensively sampled bacteriologically, and the outbreak strain was isolated in pure heavy growth from the residual water in both plaster buckets but not from any other site (table 11). A laboratory study (to be published elsewhere) showed that plaster-of-paris takes a long time to dry thoroughly and that the outbreak strain could multiply in the drying plaster. In this study, on volunteers wearing wrist-to-elbow casts, skin contamination ensued when contaminated plaster was applied, despite a thick layer of sterile wool. Postoperative wound infections caused by Clostridium spp. have been reported as a result of using contaminated plaster-of-paris.1,2 The making of casts by immersing plaster bandages into freshly drawn tap water is common practice and it is surprising that plasterrelated infection caused by gram-negative rods has not been pans after a shelf procedure hip. New Engl J Med 1962; 267: 348-49. Murray EGD, Denton GD Plaster of paris as source of infection in tetanus and gas gangrene. Canad Med Assoc J 1949; 60: 1-4.
1. Kozinn HA. Clostridium welchri infections due to plaster of
of the
2.
Treatment of Cushing’s syndrome with trilostane, inhibitor of adrenal steroid biosynthesis. J Clin Endocrinol Metab 1978, 47: 1042.
Komamcky P, Spark RF, Melby JC an
agar and examined after 24 and 48 h.
- ]’0ther= strains of other serotypes not related to those from the patients. :f:Four 100 ml membrane-filtered water samples were also taken from orthopaedic theatre taps and one yielded Ps. aeruginosa, non-outbreak strain.
2.