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SELECTIVE CULTURE SCREENING FOR PENICILLINASE-PRODUCING NEISSERIA GONORRHŒA
605
regimens (6-5% at birth, 9-8% at 30 months2), very high. It is important to note that there has been no follow-up in the 179 apparently normal babies; not all malformations are detected at birth. The potential benefit of rifampicin, with its potent antituberculous action, must therefore be weighed against its possible teratogenic effects. Our advice to clinicians must depend on the Committee on Safety of Medicines regulations, which require warnings of teratogenic hazard when this has been revealed by animal studies, and we feel that such a warning on the data sheet is wise and should remain until we are certain that rifampicin does not damage the human fetus. It is only when large numbers antituberculous
was not
of well-documented cases have been obtained that we will be able to throw further light on this problem. We would be very NINE INFANTS WITH MALFORMATIONS BORN TO WOMEN RECEIVING
RIFAMPICIN DURING PREGNANCY
if those cases of patients receiving rifampicin pregnancy could be reported to the manufacturers.
grateful
during
Ciba
Laboratories, Horsham, West Sussex RH2 4AB
J. S. M. STEEN
Lepetit Pharmaceuticals Limited, Hounslow, Middlesex TW5 9QY
D. M. STAINTON-ELLIS
SELECTIVE CULTURE SCREENING FOR PENICILLINASE-PRODUCING NEISSERIA GONORRHŒA Neisseria SIR,-Penicillinase-producing gonorrhaeae (P.P.N.G.) strains, first reported by Phillips,! have since been isolated and confirmed in many parts of the world including the United States. More and more highly resistant P.P.N.G. strains were reported as surveillance increased.2 Several rapid laboratory tests have been described3 (iodometric, acidometric and chromogenic cephalosporin) which are useful in detecting &bgr;-lactamase production by cultures of N. gonorrhaeae and other bacterial species. In addition, although a modification of the disc diffusion test is recommended as a means of screening for high levels of penicillin resistance, p-lactamase may or may not be present, and the results must therefore be confirmed by one of the three more specific tests for p-lactamase. The disadvantage of these procedures is that pure subcultures must be obtained before the tests are performed, in order to avoid false-positive results. As presently designed, none of these tests can be performed di-
rectly
on
clinical
specimens (urethral exudate, vaginal
secre-
tions, or others). We wish
to
report
some
preliminary observations with a pri-
mary selective culture medium for
detecting P.P.N.G. Chocolate-agar biplates were prepared with one-half of each containing the inhibitors vancomycin (4 fLg/ml), colistimethate sodium (7-5 fLg/ml), a new antimycotic agent, anisomycin (20 g/ml), and trimethoprim (5 g/ml) (V.C.A.T.)4. The other half contained the same concentrations of the same inhibitors, except that penicillin G (2-5 units/ml) or ampicillin (2-5 g/ml) was substituted for the vancomycin, and 2% heat-inactivated horse serum was substituted for the 1% haemoglobin. In addition, 2-0 ml of a heavy suspension of Sarcina lutea5 (F.D.A. strain no. 1001) susceptible to <0-01 fLg/ml of penicillin was added to 100 ml of the molten (45 1 C) agar and mixed before the second half of the biplate was filled. The antimicrobics investigation section, bacteriology division, Center for Disease Control (C.D.C.), supplied us with 111 coded strains which were P.P.N.G. or non-p.p.N.G. These strains had been sent to the C.D.C. for identification, determination of &bgr;-lactamase production, and minimum-inhibitoryconcentration (M.l.c.) determinations. We grew each of the cultures overnight on chocolate-agar plates with 1% enrichment. The growth from each plate was harvested and separate inocula were prepared. The inocula were adjusted to approximately 108 c.F.u./ml, and concentrations of 106 c.F.u./ml and 104 c.F.u./ml were streaked in 3 mm loopsful on both halves of the biplate. The plates were incubated at 35-36°C in a candle extinction jar and were examined after 18, 42, and 66 hours. Of the 111cultures tested, 47were positive for penicillinase production when tested by the three established rapid methods. The 18-hours examination of the V.C.A.T. medium revealed growth of all 111 cultures with both inoculum concentrations,
rifampicin, E.M.B. ethambutol, LN.A.H. isoniazid. Maternal uncle has hydrocephalus. t It is most likely that R.M.P. was given before ovulation. Patient did not have tuberculosis. R.M.P.
=
=
=
*
t Abdominal X-rays. t This is probably a mechanically produced abnormality and unlikely to have been produced by R.M.P., particularly since it was given in 4th month of pregnancy.
1. Phillips, I. Lancet, 1976, ii, 656. 2. Morbid. Mortal. Wkly. Rep. 1977, 26, 29. 3. Thornsberry, C. Antimicrobial Susceptibility Tests for Hœmophilus influenzœ. Technical Improvement Service, no. 25, pp. 79-84. American Society for Clinical Pathology, Chicago, Illinois. 1976. 4. Martin, J. E., Jr., Lewis, J. S. Improved Anti-Mycotic Activity in Modified Thayer-Martin Medium. Public Health Laboratories, 35:53.1977. 5. Grove, D. C., Randall, W. A. in Assay Methods of Antibiotics (edited by H. Welch and F. Marti-Ibanez); p. 14. New York. 1955.
606 whereas 56 cultures had grown on the clear medium containing the substituted penicillin or ampicillin, indicating a resistant strain of N. gonorrhcece. Slight growth of the S. lutea after 18 hours was visible through a dissecting microscope (magnification x40) on these 56 cultures. The bright yellow colonies of S. lutea in and around the 56 cultures could usually be seen macroscopically after 42-66 hours of incubation. Thus, p-lactamase production was detected in 8-1% more P.P.N.G. isolates when coded pure cultures were grown on the described primary selective medium than when the isolates were evaluated with the combined established chemical tests for j3-lactamase. If the results of this limited study of the primary selective culture medium are supported by field trials now under way, this medium can be used to provide therapeutically useful information for the clinician within 18-24 hours and also to confirm the presence of plactamase-producing cultures for the microbiologist, the clinician, and the epidemiologist within 72 hours from.the time the clinical specimen is streaked on the selective culture medium. J. E. MARTIN, JR. Center for Disease Control, Atlanta, Georgia 30333, U.S.A. J. S. LEWIS
from rabid foxes and cows. Non-infected brains from these animals were treated by the same way as controls. The H.A. titre was proportional to the infectivity titre by intracerebral inoculation into mice. Trypsin treatment lowered the pH range of heemagglufination, as described by Ardoin et awl. with arboviruses. The maximum titre was obtained at pH 6.0 or 6.2 depending on the strain, and the range was very narrow. In foxes, the highest titres were obtained from salivary glands, as would be expected from their high infectivity titre (10’-10’). We are now attempting to develop a H.A.-inhibition test. The problem is to destroy non-specific inhibitors in serum. The best results, so far, have been obtained by trypsin treatment of the serum by the same technique as used with the antigen. We are also seeking the most sensitive antigen; Ardoin and Clarke described an increase in sensitivity using hydroxyapatite column chromatography’ for arbovirus haemagglutinins, and this will be tried. We have obtained H.A. from foxes, cattle, mice, and rabbits without concentration. Vaccines are now titrated by hxmagglutination. P. ARDOIN Service de la Rage, E. DE LALUN Centre National Institut Pasteur, A. GAMET 75004 Paris, France
HÆMAGGLUTININ FROM RABIES VIRUSES
SIR,-Rabies-virus hmmagglutinin has not hitherto been obtained from brain material of infected animals. Only virus strains adapted to tissue culture have shown a haemagglutinin when cultivated without serum: titres have been very low and have often required concentration.! For a hxmagglutinationinhibition test capable of replacing the cumbersome neutralisation test, a high-titre haemagglutinin is required. We used brains from animals infected artificially with fixed strains or naturally with street-virus strains. In large animals Ammon’s horn and bulb were homogenised; in mice or rabbits the entire brain was used. Tissuewas ground up in 1 mmol/1 disodium hydrogen phosphate in borate saline pH 9.0; the homogenate was then clarified by centrifugation at 3000 g for half an hour. The haemagglutination (H.A.) test was carried out by the method of Clarke and Casals2 for arboviruses in disposable plastic trays (96 SC, Limbro). The borate buffer used in the test had an adverse effect on the rabies hxmagglutinin so all antigens were diluted in virus-adjusting diluent at pHs ranging from 5-8to 7-0. Incubation was at 4°C. When this failed to reveal haemagglutination, trypsin treatment3 was used (Difco 1/250 crude at a final concentration of 256 .g/ml in borate saline). Before trypsin is used its potency should be assayed. We used an assay kit (’Dermatube’ TRY, Worthington) which measures the rate of splitting of the ether of N-benzoyl-N-Larginine. Based on this assay we used approximately 140 units/ml for antigen treatment. Antigen/trypsin mixtures were incubated at room temperature for 40 min; the trypsin action was then promptly terminated by addition of soybean trypsin inhibitor (Worthington) at a weight equal to the total weight of trypsin present. With larger amounts of trypsin a non-specific H.A., giving a pattern like rabies virus, was extracted from normal and infected brains. Its presence was indicated by hsemagglutination over a wide pH range inhibited by all immune or normal sera. The specific H.A. has a very narrow pH range and is inhibited only by specific antisera. All strains of rabies viruses used, whether fixed or street strains, showed haemagglutination. VP and CVS fixed strains were used in mice and rabbits. Street strains were obtained 1.
Kuwert, E. in Laboratory Techniques in Rabies; chap. 11. W.H.O., Geneva, 1973.
2.
Clarke, D., Casals, J. Am. J. trop. Med. Hyg. 1958, 7, 561.
CAT-SCRATCH DISEASE SKIN-TEST ANTIGEN PREPARATION
SIR,-The report by Bradstreet and Dighero’ regarding cobalt irradiation for "sterilising" cat-scratch disease skin-test antigen was of interest. While cobalt treatment seems to be an effective method for eliminating contaminating organisms, evidently some reduction in potency occurs. We would like to describe another method which may be simpler and which does not interfere with antigenicity. Aspirated fluids from skin-test-positive patients were collected and stored frozen (initially at -20°C, now we freeze at - 70°C) until a new antigen lot was needed-a period, at times, of several years. Generally, accumulations of pus from 15-20 patients were collected during that period. Pools of aspirated fluids, rather than fluids from single individuals, were preferred, because pools yield a broader spectrum of positives.2 When needed, accumulated aspirated fluids were prepared by pooling and homogenising in a mechanical blender all bacteriologically sterile fluids and diluting 1/5 in saline solution. The diluted antigen was autoclaved at 10 lb (4-5 kg) and 116 ° C for 10 min, sterility tested, and put into vials. To test for sterility we looked for bacteria (thioglycollate medium, 21 days at 37°C; aerobic and anaerobic blood-agar, 28 days at 37°C); mycoplasmas (P.P.L.o. agar-from P.P.L.O. medium, 21 days at 37°C); fungi (Sabouraud’s dextrose agar, 30 days at 37°C and room temperature); viruses (two passages on primary baboon kidney cells as well as on hela, human embryonic lung and SFRE-CL-1 [chimpanzee lung] cells); for animal pathology one litter of suckling mice was inoculated via three routes and observed for 14-21 days. The antigen may then be stored at 4 ° C, where it retains its potency for years. Activity of new antigen lots was determined by testing on 6-12 previously tested and known cat-scratch disease skin-test positive patients. The size of reaction was recorded and compared with those produced by other antigen preparations. In addition, another 6-12 new patients were tested with an old 3. Ardoin, P., Clarke, D., Hannoun, C. ibid. 1969, 18, 592. 4. Ardoin, P., Clarke, D. ibid. 1967, 16, 357. 5. Gordon Smith, C. E., Holt, D. Bull. Wld Hlth Org. 1961, 1. 2.
24, 749. Bradstreet, C. M. P., Dighero, M. W. Lancet, 1977, i, 913. Kalter, S. S. Ann. intern. Med. 1961, 55, 903.
regimens (6-5% at birth, 9-8% at 30 months2), very high. It is important to note that there has been no follow-up in the 179 apparently normal babies; not all malformations are detected at birth. The potential benefit of rifampicin, with its potent antituberculous action, must therefore be weighed against its possible teratogenic effects. Our advice to clinicians must depend on the Committee on Safety of Medicines regulations, which require warnings of teratogenic hazard when this has been revealed by animal studies, and we feel that such a warning on the data sheet is wise and should remain until we are certain that rifampicin does not damage the human fetus. It is only when large numbers antituberculous
was not
of well-documented cases have been obtained that we will be able to throw further light on this problem. We would be very NINE INFANTS WITH MALFORMATIONS BORN TO WOMEN RECEIVING
RIFAMPICIN DURING PREGNANCY
if those cases of patients receiving rifampicin pregnancy could be reported to the manufacturers.
grateful
during
Ciba
Laboratories, Horsham, West Sussex RH2 4AB
J. S. M. STEEN
Lepetit Pharmaceuticals Limited, Hounslow, Middlesex TW5 9QY
D. M. STAINTON-ELLIS
SELECTIVE CULTURE SCREENING FOR PENICILLINASE-PRODUCING NEISSERIA GONORRHŒA Neisseria SIR,-Penicillinase-producing gonorrhaeae (P.P.N.G.) strains, first reported by Phillips,! have since been isolated and confirmed in many parts of the world including the United States. More and more highly resistant P.P.N.G. strains were reported as surveillance increased.2 Several rapid laboratory tests have been described3 (iodometric, acidometric and chromogenic cephalosporin) which are useful in detecting &bgr;-lactamase production by cultures of N. gonorrhaeae and other bacterial species. In addition, although a modification of the disc diffusion test is recommended as a means of screening for high levels of penicillin resistance, p-lactamase may or may not be present, and the results must therefore be confirmed by one of the three more specific tests for p-lactamase. The disadvantage of these procedures is that pure subcultures must be obtained before the tests are performed, in order to avoid false-positive results. As presently designed, none of these tests can be performed di-
rectly
on
clinical
specimens (urethral exudate, vaginal
secre-
tions, or others). We wish
to
report
some
preliminary observations with a pri-
mary selective culture medium for
detecting P.P.N.G. Chocolate-agar biplates were prepared with one-half of each containing the inhibitors vancomycin (4 fLg/ml), colistimethate sodium (7-5 fLg/ml), a new antimycotic agent, anisomycin (20 g/ml), and trimethoprim (5 g/ml) (V.C.A.T.)4. The other half contained the same concentrations of the same inhibitors, except that penicillin G (2-5 units/ml) or ampicillin (2-5 g/ml) was substituted for the vancomycin, and 2% heat-inactivated horse serum was substituted for the 1% haemoglobin. In addition, 2-0 ml of a heavy suspension of Sarcina lutea5 (F.D.A. strain no. 1001) susceptible to <0-01 fLg/ml of penicillin was added to 100 ml of the molten (45 1 C) agar and mixed before the second half of the biplate was filled. The antimicrobics investigation section, bacteriology division, Center for Disease Control (C.D.C.), supplied us with 111 coded strains which were P.P.N.G. or non-p.p.N.G. These strains had been sent to the C.D.C. for identification, determination of &bgr;-lactamase production, and minimum-inhibitoryconcentration (M.l.c.) determinations. We grew each of the cultures overnight on chocolate-agar plates with 1% enrichment. The growth from each plate was harvested and separate inocula were prepared. The inocula were adjusted to approximately 108 c.F.u./ml, and concentrations of 106 c.F.u./ml and 104 c.F.u./ml were streaked in 3 mm loopsful on both halves of the biplate. The plates were incubated at 35-36°C in a candle extinction jar and were examined after 18, 42, and 66 hours. Of the 111cultures tested, 47were positive for penicillinase production when tested by the three established rapid methods. The 18-hours examination of the V.C.A.T. medium revealed growth of all 111 cultures with both inoculum concentrations,
rifampicin, E.M.B. ethambutol, LN.A.H. isoniazid. Maternal uncle has hydrocephalus. t It is most likely that R.M.P. was given before ovulation. Patient did not have tuberculosis. R.M.P.
=
=
=
*
t Abdominal X-rays. t This is probably a mechanically produced abnormality and unlikely to have been produced by R.M.P., particularly since it was given in 4th month of pregnancy.
1. Phillips, I. Lancet, 1976, ii, 656. 2. Morbid. Mortal. Wkly. Rep. 1977, 26, 29. 3. Thornsberry, C. Antimicrobial Susceptibility Tests for Hœmophilus influenzœ. Technical Improvement Service, no. 25, pp. 79-84. American Society for Clinical Pathology, Chicago, Illinois. 1976. 4. Martin, J. E., Jr., Lewis, J. S. Improved Anti-Mycotic Activity in Modified Thayer-Martin Medium. Public Health Laboratories, 35:53.1977. 5. Grove, D. C., Randall, W. A. in Assay Methods of Antibiotics (edited by H. Welch and F. Marti-Ibanez); p. 14. New York. 1955.
606 whereas 56 cultures had grown on the clear medium containing the substituted penicillin or ampicillin, indicating a resistant strain of N. gonorrhcece. Slight growth of the S. lutea after 18 hours was visible through a dissecting microscope (magnification x40) on these 56 cultures. The bright yellow colonies of S. lutea in and around the 56 cultures could usually be seen macroscopically after 42-66 hours of incubation. Thus, p-lactamase production was detected in 8-1% more P.P.N.G. isolates when coded pure cultures were grown on the described primary selective medium than when the isolates were evaluated with the combined established chemical tests for j3-lactamase. If the results of this limited study of the primary selective culture medium are supported by field trials now under way, this medium can be used to provide therapeutically useful information for the clinician within 18-24 hours and also to confirm the presence of plactamase-producing cultures for the microbiologist, the clinician, and the epidemiologist within 72 hours from.the time the clinical specimen is streaked on the selective culture medium. J. E. MARTIN, JR. Center for Disease Control, Atlanta, Georgia 30333, U.S.A. J. S. LEWIS
from rabid foxes and cows. Non-infected brains from these animals were treated by the same way as controls. The H.A. titre was proportional to the infectivity titre by intracerebral inoculation into mice. Trypsin treatment lowered the pH range of heemagglufination, as described by Ardoin et awl. with arboviruses. The maximum titre was obtained at pH 6.0 or 6.2 depending on the strain, and the range was very narrow. In foxes, the highest titres were obtained from salivary glands, as would be expected from their high infectivity titre (10’-10’). We are now attempting to develop a H.A.-inhibition test. The problem is to destroy non-specific inhibitors in serum. The best results, so far, have been obtained by trypsin treatment of the serum by the same technique as used with the antigen. We are also seeking the most sensitive antigen; Ardoin and Clarke described an increase in sensitivity using hydroxyapatite column chromatography’ for arbovirus haemagglutinins, and this will be tried. We have obtained H.A. from foxes, cattle, mice, and rabbits without concentration. Vaccines are now titrated by hxmagglutination. P. ARDOIN Service de la Rage, E. DE LALUN Centre National Institut Pasteur, A. GAMET 75004 Paris, France
HÆMAGGLUTININ FROM RABIES VIRUSES
SIR,-Rabies-virus hmmagglutinin has not hitherto been obtained from brain material of infected animals. Only virus strains adapted to tissue culture have shown a haemagglutinin when cultivated without serum: titres have been very low and have often required concentration.! For a hxmagglutinationinhibition test capable of replacing the cumbersome neutralisation test, a high-titre haemagglutinin is required. We used brains from animals infected artificially with fixed strains or naturally with street-virus strains. In large animals Ammon’s horn and bulb were homogenised; in mice or rabbits the entire brain was used. Tissuewas ground up in 1 mmol/1 disodium hydrogen phosphate in borate saline pH 9.0; the homogenate was then clarified by centrifugation at 3000 g for half an hour. The haemagglutination (H.A.) test was carried out by the method of Clarke and Casals2 for arboviruses in disposable plastic trays (96 SC, Limbro). The borate buffer used in the test had an adverse effect on the rabies hxmagglutinin so all antigens were diluted in virus-adjusting diluent at pHs ranging from 5-8to 7-0. Incubation was at 4°C. When this failed to reveal haemagglutination, trypsin treatment3 was used (Difco 1/250 crude at a final concentration of 256 .g/ml in borate saline). Before trypsin is used its potency should be assayed. We used an assay kit (’Dermatube’ TRY, Worthington) which measures the rate of splitting of the ether of N-benzoyl-N-Larginine. Based on this assay we used approximately 140 units/ml for antigen treatment. Antigen/trypsin mixtures were incubated at room temperature for 40 min; the trypsin action was then promptly terminated by addition of soybean trypsin inhibitor (Worthington) at a weight equal to the total weight of trypsin present. With larger amounts of trypsin a non-specific H.A., giving a pattern like rabies virus, was extracted from normal and infected brains. Its presence was indicated by hsemagglutination over a wide pH range inhibited by all immune or normal sera. The specific H.A. has a very narrow pH range and is inhibited only by specific antisera. All strains of rabies viruses used, whether fixed or street strains, showed haemagglutination. VP and CVS fixed strains were used in mice and rabbits. Street strains were obtained 1.
Kuwert, E. in Laboratory Techniques in Rabies; chap. 11. W.H.O., Geneva, 1973.
2.
Clarke, D., Casals, J. Am. J. trop. Med. Hyg. 1958, 7, 561.
CAT-SCRATCH DISEASE SKIN-TEST ANTIGEN PREPARATION
SIR,-The report by Bradstreet and Dighero’ regarding cobalt irradiation for "sterilising" cat-scratch disease skin-test antigen was of interest. While cobalt treatment seems to be an effective method for eliminating contaminating organisms, evidently some reduction in potency occurs. We would like to describe another method which may be simpler and which does not interfere with antigenicity. Aspirated fluids from skin-test-positive patients were collected and stored frozen (initially at -20°C, now we freeze at - 70°C) until a new antigen lot was needed-a period, at times, of several years. Generally, accumulations of pus from 15-20 patients were collected during that period. Pools of aspirated fluids, rather than fluids from single individuals, were preferred, because pools yield a broader spectrum of positives.2 When needed, accumulated aspirated fluids were prepared by pooling and homogenising in a mechanical blender all bacteriologically sterile fluids and diluting 1/5 in saline solution. The diluted antigen was autoclaved at 10 lb (4-5 kg) and 116 ° C for 10 min, sterility tested, and put into vials. To test for sterility we looked for bacteria (thioglycollate medium, 21 days at 37°C; aerobic and anaerobic blood-agar, 28 days at 37°C); mycoplasmas (P.P.L.o. agar-from P.P.L.O. medium, 21 days at 37°C); fungi (Sabouraud’s dextrose agar, 30 days at 37°C and room temperature); viruses (two passages on primary baboon kidney cells as well as on hela, human embryonic lung and SFRE-CL-1 [chimpanzee lung] cells); for animal pathology one litter of suckling mice was inoculated via three routes and observed for 14-21 days. The antigen may then be stored at 4 ° C, where it retains its potency for years. Activity of new antigen lots was determined by testing on 6-12 previously tested and known cat-scratch disease skin-test positive patients. The size of reaction was recorded and compared with those produced by other antigen preparations. In addition, another 6-12 new patients were tested with an old 3. Ardoin, P., Clarke, D., Hannoun, C. ibid. 1969, 18, 592. 4. Ardoin, P., Clarke, D. ibid. 1967, 16, 357. 5. Gordon Smith, C. E., Holt, D. Bull. Wld Hlth Org. 1961, 1. 2.
24, 749. Bradstreet, C. M. P., Dighero, M. W. Lancet, 1977, i, 913. Kalter, S. S. Ann. intern. Med. 1961, 55, 903.