998
Unusual
non-toxigenic Corynebacterium diphtheriae in homosexual men
SiR,—Toxigenic Corynebacterium diphtheriae has almost disappeared from the general population in the UK and many hospitals no longer screen throat swabs routinely for this organism. Antitoxin reduces the survival of virulent strains but permits carriage of non-toxigenic ones, and the gene for virulence can be transferred.l Furthermore, where rates of immunisation are high, 50-70% of adults do not have the generally accepted protective level of antitoxin.2 Between December, 1988, and January, 1991, we cultured 9 isolates of a biochemically unusual non-toxigenic strain of C diphtheriae from 11000 routine throat swabs.3 7 patients were homosexual men. After the eighth patient a prospective study was mounted in two genitourinary medicine (GUM) clinics. Between Nov 1, 1990, and April 26, 1991, patients attending the clinics were entered in the study after giving informed consent. A history of sore throat or orogenital sex in the previous month, sexual orientation, and HIV seropositivity were recorded. A throat swab was inoculated onto Hoyle’s and blood agar containing nalidixic acid 15 mg/1. The Hoyle culture was incubated for 48 h at 37°C in air with identification by standard methods, toxigenicity of C diphtheriae being sought by the Elek test and guineapig inoculation. The other plate was incubated for 0-haemolytic streptococci. Any C diphtheriae carriers were re-examined, throat swabs were repeated, and urethral and rectal swabs were taken. The patients were given oral erythromycin stearate for 10 days and re-screened at 2 weeks. 12 isolates (6 from routine cultures and 6 from the prevalence study) and control strains NCTC 10356 and 10648 were subjected to polyacrylamide gel electrophoresis (SDS PAGE). Southern blot hybridisation patterns of BstEII digests of chromosomal DNA were compared and ribotyping was done with a biotin-labelled cDNA probe prepared from total rRNA of a C diphtheriae type strain. The prospective study added 7 more pharyngeal carriers of non-toxigenic C diphtheriae (NTCD). The 16 patients lived over a wide area of London. The 9 patients found by routine culture were aged 19-44 years. 7 were homosexual men. 3 of 5 men tested had antibodies to HIV. 3 had a tonsillar exudate and lymphadenopathy but only 1 had fever. Other symptoms were weakness and headache (2 patients), and malaise, night sweats, and arthralgia (1). The 6 treated patients responded to erythromycin or doxycyline, and the other 3 did not return for follow-up. In the prospective survey NTCD was isolated from 6 of 578 homosexual men (95% CI 0-4-2-2%) but from only 1 of 653 heterosexual men (p = 0-003). There were no isolates among 1043 women. The patients were aged 22-34 years and 2 of 4 patients tested were HIV seropositive. 4 had a sore throat, 5 pharyngitis, 1 lymphadenopathy, and 1 tonsillar exudate. Streptococcus pyogenes and Neisseria gonorrhoeae were each isolated in 2 patients. Urethral or rectal infection due to N gonorrhoeae was detected in 3. Pharyngeal inflammation was noted significantly more frequently in homosexual male carriers (table) but in other respects carriers were not significantly different from homosexual non-carriers:
Sign
CD+
(n=6)
CD-(n=572)
CD = C diphtheriae, *p=0 0 01. All carriers responded to treatment with erythromycin. Two of the carriers were sexual partners but all urethral and rectal swabs were negative for C diphtheriae. Carriage of Strep pyogenes in the throat was also more common among homosexual men (18/578 vs
8/1696, p < 0-005). The isolates formed small domed colonies with a smooth edge on tellurite agar, were non-haemolytic, and showed a brown halo on Tinsdale’s medium. The 8 isolates from homosexual men, when tested by ’API Coryne’ kit were identified as C diphtheriae vargravis (API profile 1010326) but acid was not produced from maltose in Hiss serum sugar; the other isolate was var belfanti. All isolates were non-toxigenic. SDS PAGE results (figure) are compared with
strains of similar biotype from a closed religious community and from other GUM clinics. 9 of the 12 produced an indistinguishable protein profile; 2 produced slightly different patterns; and the strain from 1 was distinct. Strains from other centres and the 2 control strains produced distinct patterns, with 1 exception. Ribotyping confirmed the results of SD S PAGE and showed that 11/12 isolates from the clinics had identical patterns.
SDS PAGE protein profiles of atypical C diphtheriae. Lanes 1-7=isolates from elsewhere (1, 6, 7 = homosexual patients; 2-5=outbreak of pharyngitis in close religious community) Lanes 8-10=patients in this survey (8, 9=routme culture, 10 = prospective) Lanes 11, 12=controls (11 = toxigenic, 12 = non-toxigenic) Lane 13 = low-molecular-weight marker (94-14-4kD).
Most isolates of NTCD were indistinguishable by SDS PAGE and ribotyping, which might suggest a common source, but no common factors other than sex and orientation were found. More strains should be investigated by molecular methods to establish the extent of variation in pattern. However, our findings do carry three messages--epidemiological, clinical, and microbiological. We can confirm that biotyping is of limited value in epidemiological investigations. Pharyngeal inflammation was more common in those carrying C diphtheriae, irrespective of other isolates (not shown). Non-toxigenic strains can produce sore throat and tonsillar membrane 4,5though we cannot be sure that in our series NTCD did cause pharyngitis. Variability in maltose fermentation is well recognised in C diphtheriae but the discrepancy in the maltose reaction between Hiss serum sugar and the API kit could result in an incorrect identification of this organism as C diphtheriae var gravis, which usually produces toxin. We would argue that routine culture of throat swabs for C diphtheriae is still justifiable. The absence of previous reports of carriage in homosexuals may reflect local laboratory practice. Department of Clinical Microbiology, University College and Middlesex Hospitals, London WC1 E 6AU, UK, Division of Hospital Infection, Central Public Health Laboratory, London NW9, and Department of Genitourinary Medicine,
University College and Middlesex Hospitals
A. P. R. WILSON A. EFSTRATIOU E. WEAVER E. ALLASON-JONES G. L. RIDGWAY J. BINGHAM D. MERCEY A. ROBINSON B. D. COOKSON G. COLMAN
1. Pappenheimer AM, Murphy JR. Studies on the molecular epidemiology of diphtheria. Lancet 1983; ii. 923-26 2. Rappouli R, Pergini M, Falsen E. Molecular epidemiology of the 1984-1986 outbreak of diphtheria in Sweden. N Engl J Med 1988; 318: 12-14. 3. Wilson APR, Ridgway GL, Gruneberg RN, Efstratiou A, Coleman G, CooksonB Routine screening for Corynebacterium diphtheriae. Lancet 1990; 336: 1199. 4. Barksdale L, Garmise L, Horibata K. Virulence, toxinogeny, and lysogeny in Corynebacterium diphtheriae Ann NY Acad Sci 1960; 88: 1093-108. 5. Jephcott AE, Gillespie EH, Davenport C, Emerson JW, Moroney PJ. Non-toxigenic Cornyebacterium diphtheriae in a boarding school. Lancet 1975; i: 1025-26.
Unusual
non-toxigenic Corynebacterium diphtheriae in homosexual men
SiR,—Toxigenic Corynebacterium diphtheriae has almost disappeared from the general population in the UK and many hospitals no longer screen throat swabs routinely for this organism. Antitoxin reduces the survival of virulent strains but permits carriage of non-toxigenic ones, and the gene for virulence can be transferred.l Furthermore, where rates of immunisation are high, 50-70% of adults do not have the generally accepted protective level of antitoxin.2 Between December, 1988, and January, 1991, we cultured 9 isolates of a biochemically unusual non-toxigenic strain of C diphtheriae from 11000 routine throat swabs.3 7 patients were homosexual men. After the eighth patient a prospective study was mounted in two genitourinary medicine (GUM) clinics. Between Nov 1, 1990, and April 26, 1991, patients attending the clinics were entered in the study after giving informed consent. A history of sore throat or orogenital sex in the previous month, sexual orientation, and HIV seropositivity were recorded. A throat swab was inoculated onto Hoyle’s and blood agar containing nalidixic acid 15 mg/1. The Hoyle culture was incubated for 48 h at 37°C in air with identification by standard methods, toxigenicity of C diphtheriae being sought by the Elek test and guineapig inoculation. The other plate was incubated for 0-haemolytic streptococci. Any C diphtheriae carriers were re-examined, throat swabs were repeated, and urethral and rectal swabs were taken. The patients were given oral erythromycin stearate for 10 days and re-screened at 2 weeks. 12 isolates (6 from routine cultures and 6 from the prevalence study) and control strains NCTC 10356 and 10648 were subjected to polyacrylamide gel electrophoresis (SDS PAGE). Southern blot hybridisation patterns of BstEII digests of chromosomal DNA were compared and ribotyping was done with a biotin-labelled cDNA probe prepared from total rRNA of a C diphtheriae type strain. The prospective study added 7 more pharyngeal carriers of non-toxigenic C diphtheriae (NTCD). The 16 patients lived over a wide area of London. The 9 patients found by routine culture were aged 19-44 years. 7 were homosexual men. 3 of 5 men tested had antibodies to HIV. 3 had a tonsillar exudate and lymphadenopathy but only 1 had fever. Other symptoms were weakness and headache (2 patients), and malaise, night sweats, and arthralgia (1). The 6 treated patients responded to erythromycin or doxycyline, and the other 3 did not return for follow-up. In the prospective survey NTCD was isolated from 6 of 578 homosexual men (95% CI 0-4-2-2%) but from only 1 of 653 heterosexual men (p = 0-003). There were no isolates among 1043 women. The patients were aged 22-34 years and 2 of 4 patients tested were HIV seropositive. 4 had a sore throat, 5 pharyngitis, 1 lymphadenopathy, and 1 tonsillar exudate. Streptococcus pyogenes and Neisseria gonorrhoeae were each isolated in 2 patients. Urethral or rectal infection due to N gonorrhoeae was detected in 3. Pharyngeal inflammation was noted significantly more frequently in homosexual male carriers (table) but in other respects carriers were not significantly different from homosexual non-carriers:
Sign
CD+
(n=6)
CD-(n=572)
CD = C diphtheriae, *p=0 0 01. All carriers responded to treatment with erythromycin. Two of the carriers were sexual partners but all urethral and rectal swabs were negative for C diphtheriae. Carriage of Strep pyogenes in the throat was also more common among homosexual men (18/578 vs
8/1696, p < 0-005). The isolates formed small domed colonies with a smooth edge on tellurite agar, were non-haemolytic, and showed a brown halo on Tinsdale’s medium. The 8 isolates from homosexual men, when tested by ’API Coryne’ kit were identified as C diphtheriae vargravis (API profile 1010326) but acid was not produced from maltose in Hiss serum sugar; the other isolate was var belfanti. All isolates were non-toxigenic. SDS PAGE results (figure) are compared with
strains of similar biotype from a closed religious community and from other GUM clinics. 9 of the 12 produced an indistinguishable protein profile; 2 produced slightly different patterns; and the strain from 1 was distinct. Strains from other centres and the 2 control strains produced distinct patterns, with 1 exception. Ribotyping confirmed the results of SD S PAGE and showed that 11/12 isolates from the clinics had identical patterns.
SDS PAGE protein profiles of atypical C diphtheriae. Lanes 1-7=isolates from elsewhere (1, 6, 7 = homosexual patients; 2-5=outbreak of pharyngitis in close religious community) Lanes 8-10=patients in this survey (8, 9=routme culture, 10 = prospective) Lanes 11, 12=controls (11 = toxigenic, 12 = non-toxigenic) Lane 13 = low-molecular-weight marker (94-14-4kD).
Most isolates of NTCD were indistinguishable by SDS PAGE and ribotyping, which might suggest a common source, but no common factors other than sex and orientation were found. More strains should be investigated by molecular methods to establish the extent of variation in pattern. However, our findings do carry three messages--epidemiological, clinical, and microbiological. We can confirm that biotyping is of limited value in epidemiological investigations. Pharyngeal inflammation was more common in those carrying C diphtheriae, irrespective of other isolates (not shown). Non-toxigenic strains can produce sore throat and tonsillar membrane 4,5though we cannot be sure that in our series NTCD did cause pharyngitis. Variability in maltose fermentation is well recognised in C diphtheriae but the discrepancy in the maltose reaction between Hiss serum sugar and the API kit could result in an incorrect identification of this organism as C diphtheriae var gravis, which usually produces toxin. We would argue that routine culture of throat swabs for C diphtheriae is still justifiable. The absence of previous reports of carriage in homosexuals may reflect local laboratory practice. Department of Clinical Microbiology, University College and Middlesex Hospitals, London WC1 E 6AU, UK, Division of Hospital Infection, Central Public Health Laboratory, London NW9, and Department of Genitourinary Medicine,
University College and Middlesex Hospitals
A. P. R. WILSON A. EFSTRATIOU E. WEAVER E. ALLASON-JONES G. L. RIDGWAY J. BINGHAM D. MERCEY A. ROBINSON B. D. COOKSON G. COLMAN
1. Pappenheimer AM, Murphy JR. Studies on the molecular epidemiology of diphtheria. Lancet 1983; ii. 923-26 2. Rappouli R, Pergini M, Falsen E. Molecular epidemiology of the 1984-1986 outbreak of diphtheria in Sweden. N Engl J Med 1988; 318: 12-14. 3. Wilson APR, Ridgway GL, Gruneberg RN, Efstratiou A, Coleman G, CooksonB Routine screening for Corynebacterium diphtheriae. Lancet 1990; 336: 1199. 4. Barksdale L, Garmise L, Horibata K. Virulence, toxinogeny, and lysogeny in Corynebacterium diphtheriae Ann NY Acad Sci 1960; 88: 1093-108. 5. Jephcott AE, Gillespie EH, Davenport C, Emerson JW, Moroney PJ. Non-toxigenic Cornyebacterium diphtheriae in a boarding school. Lancet 1975; i: 1025-26.
