995

associated with a 30-40% prevalence of P.P.N.G. among gonococcal infections in certain populations in the Philippines." In contrast, the absence of this plasmid in Eur-

opean-West African P.P.N.G. may explain why the P.P.N.G. epidemic in Liverpool disappeared in December 1976, after being found in 7.2% of gonococcal infections in the preceding 10 months; only three isolated cases have occurred in 1977 (A. Percival, personal communication). We believe, however, that a conjugative plasmid is likely to be present in some indigenous West African P.P.N.G. strains. This could be confirmed by prevalence

ferent parts of the world.6-12 Pneumococci were described as partially resistant, with the following minimum inhibitory concentrations (M.l.c.s): 0.1-2.0 mg/1 penicillin G, 2.5-10 mg/l methicillin/cloxacillin, 0 08-1-0 mg/1 ampicillin, 0.13-0-35 mg/l cephaloridine, and 0.62-2.0 mg/l cephalothin. 1-12 This report describes five cases (three fatal) of systemic infection in young African children admitted to the King Edward VIII Hospital, Durban, which were caused by strains of S. pneumonice serotype 19A resistant to penicillin/cephalosporin and chloramphenicol.

studies in the main endemic areas in West Africa. Requests for reprints should be addressed

to

Case-reports

P. L. P.

REFERENCES 1. Morbid. Mortal. wkly Rep. 1976, 25, 261. 2. Ashford, W. A., Golash, R. G., Hemming, V. G. Lancet, 1976, ii, 657. 3. Phillips, I. ibid. p. 656. 4. Percival, A., Corkill, J. E., Arya, O. P., Rowlands, J., Aleagant, C. D., Rees, E., Annels, E. H. ibid. p. 1379. 5. O’Callaghan, C. H., Morris, A., Kirby, S. M., Shingler, A. H. Antimicrob.

Ag. Chemother. 1972, 1, 283. 6. Jaffe, H. W., Biddle, J. W., Thornsberry, C., Johnson, R. E., Kaufman, R. E., Reynolds, G. H., Wiesner, P. J. New Engl. J. Med. 1976, 294, 5. 7. LaScolea, L. J., Jr., Young, R. E. Appl. Microbiol. 1974, 28, 70. 8. Meyers, J. A., Sanchez, D., Elwell, L. P., Falkow, S. J. Bact. 1976, 127, 1529. 9. Ödegaard, K., Solberg, O. Acta path. microbiol. scand. Sect. B., 1976, 84, 458. 10. Blog, F. B., Chang, A., DeKoning, G. A. J., Oranje, A. P., Stolz, A., Bosscher-Koetsier, G., DeJonge-Suy, M. P. E., Michel, M. F., O’Niel, E., DeWeerdt-Van Ameyden, S., Gaastra, L. Br. J. vener. Dis. 1977, 53, 98. 11. Kirven, L. A., Thornsberry, C. Antimicrob. Ag. Chemother. 1977, 11, 1004. 12. Eisenstein, B. I., Sox, T., Biswas, G., Blackman, E., Sparling, P. F. Science,

1977, 195, 998. 13. Roberts, M., Falkow, S. Nature, 1977, 266, 630. 14. Elwell, L. P., Roberts, M., Mayer, L. W., Falkow, S. Antimicrob.

Chemother. 1977, 11, 528. 15. Sparling, P. F., Holmes, K. K., Wiesner, 1977, 135, 865.

P.

J., Puziss, M. J. infect.

Case 1.-A 4-month-old infant developed meningitis 2 days after readmission for marasmus and gastroenteritis (initially treated with intravenous fluids, benzylpenicillin, kanamycin, and neomycin). Culture of blood and cerebrospinal fluid (c.s.F.) both yielded resistant pneumococci. Despite treatment

with penicillin G, kanamycin, chloramphenicol, and sulphadiazine the patient died 6 days after the onset of meningitis. Case 2.-A 5-month-old infant was admitted with suppurative otitis media and meningitis. Resistant pneumococci were isolated from the c.s.F. The patient died 36 h after admission, despite therapy with benzylpenicillin, sulphadiazine, chloramphenicol, and ampicillin. Case 3.-An infant of 22 months was recovering from kwashiorkor and hepatic amoebiasis when he developed measles complicated by pneumonia, septicaemia, and empyema. Treatment with penicillin G, kanamycin, cloxacillin, and chloramphenicol

avail, and resistant pneumococci were cultured from

was to no

on two occasions. Antimicrobial therapy was erythromycin, rifampicin, and isoniazid-the latter to cover the remote possibility of tuberculosis. On this regimen the patient improved and was discharged 61 days

the blood Ag. Dis.

changed

to

after admission. Case 4.-An infant of 3 months

being treated for marasmus gastroenteritis (with intravenous fluids, benzylpenicillin, neomycin, cloxacillin, and chloramphenicol) developed meningitis 37 days after admission. Resistant pneumococci were cultured from c.s.F., and the patient died a day later. Case 5.-A 2-year-old child developed measles, pneumonia, and

STREPTOCOCCUS PNEUMONIÆ RESISTANT TO PENICILLIN AND CHLORAMPHENICOL A. BHAMJEE A. F. HALLETT ROSEMARY C. COOPER

P. C. APPELBAUM J. N. SCRAGG ANNETTE J. BOWEN

septicaemia 2 weeks after admission with kwashiorkor and bronchopneumonia (the latter having been treated with penicillin and streptomycin). Therapy with cephazolin and tobramycin was ineffective and, after blood-culture had yielded resistant pneumococci, was changed to high doses of ampicillin. The child improved and was discharged 2 months after and

Departments of Microbiology and Pœdiatrics, Natal University Medical School, Durban, South Africa

admission.

Methods

Summary

Three

cases

septicæmia

of

were

and

of

meningitis caused by pneumococci two

the penicillins/cephalosporins and chloramNo phenicol. &bgr;-lactamase was demonstrated in any of the organisms. All three patients with meningitis died, but the patients with septicæmia recovered after being given appropriate antibiotic therapy.

resistant

to

Introduction

ANTiBIOTIc-sensitivity patterns of several bacterial groups have changed in the past few years. Enterobacteria and pseudomonads have become resistant to aminoglycosides,1,2 and &bgr;-Iactamase-producing strains of Haemophilus influenzae3 and Neisseria gonorrhaeae4 have appeared. The first report of penicillin resistance in Streptococcus pneumoniae came from Australia in 19675 and has since been followed by several others from dif-

Strains of S. pneumonice were isolated and identified by standard methods." Serotyping by the quellung reaction was done by Prof. H. J. Koornhof, of the South African Institute for Medical Research, Johannesburg, and Prof. Robert Austrian, of the department of research medicine, University of Pennsylvania Medical School, Philadelphia. Disc susceptibility was tested by the method of Stokes.14 Pneumococci were preliminarily screened for sensitivity to a 2-unit disc of penicillin G, after which the full range of antibiotics (see table) were used in disc-diffusion tests. M.I.C.S were determined in phenol-red serum broth containing suitable concentrations of antibiotics. Inocula were prepared by diluting an overnight culture in serum broth so as to yield 103, 104, or 107 colony-forming units (c.f.u.)/ml. One drop of culture was added to each tube. To measure minimum bactericidal concentrations (M.B.C.S), M.LC. cultures were plated on blood-agar. Penicillinase production was assayed by the chromogeniccephalosporin 11 and starch-iodinel6 methods. Nasopharyngeal swabs of ward contacts and outpatients

996 SENSITIVITY OF PNEUMOCOCCI TO ANTIMICROBIAL AGENTS ---7

I

I

._

l

l

I

I

i

R=resistant; PR=partially resistant; s=sensitive. screened for penicillin-resistant pneumococci by plating blood-agar containing 5 mg/1 tobramycinl7 and testing appropriate colonies with 2-unit discs of penicillin G.

were on

Results

Quellung reactions showed all organisms to be serotype 19A by the Danish classification, or type 57 by the American classification. The results of antimicrobial susceptibility tests (see table) were similar for all the organisms and were uninfluenced by inoculum strength. Pneumococci were resistant to benzylpenicillin, methicillin, carbenicillin, chlor-

amphenicol, streptomycin, neomycin, kanamycin, gentamicin, and tobramycin, partially resistant to ampicillin, cephalothin, and cephamandole, and sensitive to erythromycin, tetracycline, clindamycin, rifampicin, vancomycin, and co-trimoxazole. No p-tactamase production was demonstrated. Nasopharyngeal swabs from

188

ward

contacts

cleaning staff), 100 randomly selected women attending the antenatal clinic, and 100 patients being treated in the casualty department yielded no resistant pneumococci. (patients, doctors,

nurses,

Discussion The sensitivity pattern of strains described here differs from that of previously reported penicillin-resistant pneumococci. S-12 The pneumococci were resistant to carbenicillin and chloramphenicol as well as penicillin, and they showed higher M.l.c.s to streptomycin than is usual in these organisms.18 The organisms were less resistant to ampicillin than to other members of the penicillin group. This confirms reports by others.6-8.10.12 Lack of inoculum effect on penicillin and chloramphenicol M.l.c.s supports our failure to demonstrate p-lactamase in all our resistant S. pneumonice strains. Previous workers6.8.9 also failed to find this enzyme in pneumococci relatively resistant to penicillin. No difference in M.l.c.s was found between cephamandole and cephalothin. The advantages of cephamandole over cephalothin appear to be greater in gram-negative than grampositive resistant organisms. 19

patients who received appropriate antimicrobial therapy recovered. The drugs of choice for systemic nonmeningeal pneumococcal infections described here appear to be ampicillin (in high doses, to counteract partial resistance), erythromycin, tetracycline, clindamycin, co-trimoxazole, rifampicin, or vancomycin. Meningitis due to these resistant pneumococci poses a serious problem, and the drugs of choice here may be co-trimoxazole, rifampicin, and vancomycin. Three out of five patients from whom resistant pneuBoth

isolated developed their infections in the Of the other two, one developed meningitis 2 hospital. and the other was admitted witl1 after readmission, days two These patients were probably infected meningitis. outside the hospital. We plan to investigate the preva. lence of nasopharyngeal carriers of resistant pneumococci in the catchment of King Edward VIII Hospital Isolation from five patients of serologically identica’ pneumococci with similar resistance patterns points to a common exogenous source of infection. Failure to detect nasopharyngeal carriers of resistant pneumococci in a preliminary epidemiological survey does not rule out their presence inside or outside the hospital. More detailed studies are in progress to determine whether resistant pneumococci are present in a broader crosssection of the hospital population. All five patients had received penicillin and/or chloramphenicol for relatively long periods before admission or during their stay in hospital before resistant pneumococci were cultured. Similarly, Hansman and coworkers6.7reported extensive use of penicillin among New Guineans from whom pneumococci relatively resistant to penicillin were isolated. Pneumococcus type 19 is a common cause of suppurative otitis media in the United States.20 The presence of purulent otitis media in two infants may indicate the means of entry of the pneumococcus in these patients. Isolation from five patients of serologically identical pneumococci with similar resistance patterns to three unrelated antibiotic groups could reflect the presence of plasmids in these strains. Studies on the mechanism of mococci

were

antibiotic resistance in these strains are in progress. This report adds S. pneumonice to the growing list of

997

drug-resistant organisms which have appeared in recent If drug-resistant pneumococci become widespread the treatment of S. pneumoniae infections will need revears.

evaluation. We thank Prof. H.

J. Koomhof, Mr R. Robinson, and Dr R.

Robins-Browne, of the South African Institute for Medical Research, for invaluable help and discussion. We also thank Prof. Robert Austnan, of the department of research medicine, University of Pennsylvania School of Medicine, Philadelphia, for serotyping the pneumococci.

Requests for reprints should be addressed to P.C.A., Department of P.O. Box 17039, Congella 4013,

Microbiology, Faculty of Medicine, South Africa.

REFERENCES 1. Nonega, E. R.,

Leibowitz, R. E., Richmond, A. S., Rubinstein, E., Schaefler, S., Simberkoff, M. S., Rahal, J. J., Jr, J. infect. Dis. 1975, 131, suppl.

Introduction CONFIRMATION of the existence of the luteinising-hormone-releasing hormone (L.H.R.H.) by elucidation of its structure and its synthesis encouraged speculation about the eventual role of L.H.R.H. in clinical medicine.1.2 Much effort has been devoted to its diagnostic use in disorders of the hypothalamic-pituitary axis as well as to its therapeutic use in subfertility.3 Even more important, however, may be the new approaches L.H.R.H. offers to birth control.Prominent among these possibilities is the development of inhibitory analogues of L.H.R.H. active in man. This report demonstrates that a single injection a D-Phe-2,D-Trp-3,D-Phe-6- analogue of L.H.R.H. significantly suppresses the gonadotrophin response to

of

L.H.R.H.

for up to 24 hours.

p 45.

Materials and Methods

Meyer, R. D., Lewis, R. P., Halter, J., White, M. Lancet, 1976, i, 580. 3. Sykes, R. B., Matthew, M., O’Callaghan, C. H. J. med. Microbiol. 1975, 8, 2

437.

4. Phillips, I. Lancet,

1976, ii, 656. 5. Hansman, D., Bullen, M. M. ibid. 1967, ii, 264. 6 Hansman, D., Glasgow, H., Sturt, J., Devitt, L., Douglas, R. New Engl. J. Med. 1971, 284, 175. 7. Hansman, D., Glasgow, H. N., Sturt, J., Devitt, L., Douglas, R. M. Nature, 1971, 230, 407. 8. Hansman, D., Devitt, L., Riley, I. Br. med. J. 1973, iii, 405. 9. Naraqi, S., Kirkpatrick, G. P., Kabins, S. J. Pediat. 1974, 85, 671. 10. Dixon, J. M. S. Lancet, 1974, ii, 474. 11. Hansman, D., Devitt, L., Miles, H., Riley, I. Med. J. Aust. 1974, ii, 353. 12. Howes, V. J., Mitchell, R. G. Br. med. J. 1976, i, 996. 13. Austrian, R. in Manual of Clinical Microbiology (edited by E. H. Lennette, E. H. Spaulding, and J. P. Truant); p. 109. Washington, D.C., 1974. 14. Stokes, E. J. Clinical Bacteriology; p. 216. London, 1975. 15. O’Callaghan, C. H., Morris, A., Kirby, S. M., Shingler, A. H. Antimicrob. Ag. Chemother. 1972, 1, 283. 16 Perret, C. J Nature, 1954, 174, 1012. 17 Dilworth, J. A., Stewart, P., Gwaltney, J. M., Jr, Hendley, J. O., Sande, M. A. J. clin. Microbiol. 1975, 2, 453. 18. Garrod, L. P., Lambert, H. P., O’Grady, F. Antibiotic and Chemotherapy; p. 280. Edinburgh, 1973. 19. Eykyn, S., Jenkins, C., King, A., Phillips, I. Antimicrob. Ag. Chemother. 1973, 3, 657. 20. Sloyer, J. L., Jr, Howie, V. M., Ploussard, J. H., Amman, A. J., Austrian, R., Johnston, R. B., Jr, Infect. Immun. 1974, 9, 1028.

SUPPRESSION OF GONADOTROPHIN RELEASE IN MAN BY AN INHIBITORY ANALOGUE OF L.H.-RELEASING HORMONE DAVID GONZALEZ-BARCENA ABBA J. KASTIN DAVID H. COY KAROLY NIKOLICS ANDREW V. SCHALLY

Hospital General del Centro Medico Nacional, Mexico, D.F., Veterans Administration Hospital and Tulane University School of Medicine, New Orleans, Louisiana, U.S.A., and Semmelweis University Medical School, Budapest, Hungary

ability of an inhibitory analogue of L.H.-releasing hormone (L.H.R.H.) to suppress the release of luteinising hormone (L.H.) and folliclestimulating hormone (F.S.H.) after administration of a small dose of L.H.R.H. was tested in four men. A single intramuscular injection of 90 mg (D-Phe-2,D-Trp-3,DPhe-6)-L.H.R.H. diminished the gonadotrophin response to 25 µg of L.H.R.H. given 1, 4, 8, and 24 hours afterSummary

The

wards. Basal levels of L.H. and F.S.H. were not lowered. The results demonstrate that an inhibitory analogue of L.H.R.H. is active in man and suggest the possibility that inhibitors of L.H.R.H. might eventually form the basis of a new method of birth control.

(D-Phe-2,D-Trp-3,D-Phe-6)-L.H.R.H.4 was synthesised by the solid-phase method and purified. In this decapeptide, the histidine residue in position 2 of L.H.R.H. was replaced by D-phenylalanine, the tryptophan in position 3 by its stereoisomer, D-tryptophan, and the glycine in position 6 by D-phenylalanine. Four men without any known endocrine abnormalities volunteered for the study after it was fully explained to them. On the first day they received 25 µg of L.H.R.H. (Hoechst) intravenously. Heparinised blood was collected before and every 15 min after this injection for an hour. On the second day, 90 mg of (D-Phe-2,D-Trp-3,D-Phe-6)-L.H.R.H. was injected intramuscularly, and 25 µg L.H.R.H. was injected intravenously as a bolus 1, 4, 8, and 24 hours later. Blood was collected before and every 15 min for an hour after each injection of L.H.R.H. Plasma-levels of L.H. and F.S.H. were determined by radioimmunoassay with the kits obtained from CEA-IRE-SORIN. The results are expressed as ng/ml and can be converted to mi.u./ml of the second International Reference Preparation of human menopausal gonadotrophin by multiplying by 2 for L.H. and 3.5 for F.S.H. Release of gonadotrophin was determined from the absolute change (ng/ml) in plasma-levels from the basal value obtained at the time of each injection of L.H.R.H. to peak release. The total amount of gonadotrophins released in 1 hour by L.H.R.H. was measured with a Dietzgen compensating polar planimeter (DI803D), and the mean of three determinations of the area was used. Statistical evaluation was made by analysis of variance followed by Dunnett’s procedure.

Results The single intramuscular injection of 90 mg (D-Phe-2,DTrp-3,D-Phe-6)-L.H.R.H. suppressed the L.H.R.H.-induced release of L.H. 1, 4, 8, and 24 hours later. This inhibition was great enough to be highly significant (p