Laboratory guidelines for the diagnosis of infections caused by Corynebacterium diphtheriae guidelines and C. ulcerans A Efstratiou, RC George Summary: These guidelines represent an application of the World Health Key words: Organization European Regions manual for the laboratory diagnosis of diphtheria Corynebacterium infections for laboratories in the United Kingdom (UK), but they could be applied to laboratories diphtheria overseas. The manual was rewritten in response to the re-emergence of diphtheria guidelines in eastern Europe and the emergence of other infections caused by Corynebacterium laboratory infection diphtheriae and C. ulcerans in the UK and overseas. The guidelines summarise microbiological our current recommendations and procedures for the microbiological diagnosis of techniques infections caused by toxigenic and non-toxigenic isolates of corynebacteria, with particular reference to C. diphtheriae and C. ulcerans. Commun Dis Public Health 1999: 2: 250-7. Introduction Background Guidelines for the laboratory diagnosis of diphtheria in Microbiology of infections caused by C. diphtheriae the World Health Organization (WHO) European and C. ulcerans Region1 were written in response to the re-emergence of Diphtheria is a rare disease caused by toxigenic strains diphtheria in eastern Europe1. This document applies of C. diphtheriae and, less often, C. ulcerans. C. diphtheriae the guidelines for use in laboratories in the United is a Gram positive, fermentative, pleomorphic rod, which Kingdom (UK). comprises four biotypes, var gravis, var mitis, var The guidelines present current recommendations for intermedius, and var belfanti. All biotypes, with the the microbiological diagnosis of infections caused by exception of the biotype belfanti, may produce the lethal potentially toxigenic isolates of corynebacteria, with diphtheria exotoxin. Diphtheria is usually classified particular reference to Corynebacterium diphtheriae and according to its site of manifestation; most cases are either C. ulcerans. They cover the following main areas: TABLE 1 Toxigenic C. diphtheriae isolates referred to laboratory safety issues SDRU: 1993 to 1998 the role of the diagnostic laboratory Number of Country Biotype the role of, and interaction with, the reference Year isolates of origin Source (var) laboratory procedures for presumptive identification of 1993 4 Africa skin mitis C. diphtheriae and C. ulcerans Pakistan skin gravis importance of toxigenicity testing Tunisia throat mitis no travel* throat mitis laboratory responsibility for reporting toxigenic C. diphtheriae, C. ulcerans, and C. pseudotuberculosis 1994 4 Bangladesh skin mitis susceptibility testing of coryneform bacteria Bangladesh throat mitis serological immunity testing India throat mitis specialised testing; molecular typing Pakistan throat gravis enhancing microbiological surveillance 1995 1 Thailand skin mitis A Efstratiou, RC George 1996 3 Nepal skin intermedius PHLS Respiratory and Systemic Infection Laboratory Thailand throat mitis no travel* throat mitis Address for correspondence: Dr Androulla Efstratiou 1997 3 eastern Europe throat gravis WHO Collaborating Centre for Diphtheria and Streptococcal Indonesia skin mitis Infections no travel throat gravis Respiratory and Systemic Infection Laboratory PHLS Central Public Health Laboratory 1998 3 Tanzania skin mitis 61 Colindale Avenue Tanzania throat mitis London NW9 5HT Tanzania nose mitis tel: 0208 200 4400, ext 4270 * contact with overseas person fax: 0208 205 6528 fatal case of diphtheria email: [email protected] laboratory acquired infection 250 VOL 2 NO 4 DECEMBER 1999 COMMUNICABLE DISEASE AND PUBLIC HEALTH TABLE 2 Isolates of toxigenic C. ulcerans referred to strains or whether laboratories are increasingly SDRU: 1993 to 1998 identifying corynebacteria in diseases other than Number of Country Age range respiratory diphtheria. It has been postulated that non- Year isolates of origin of patients Source toxigenic strains occur more frequently in people who have been immunised6. Non-toxigenic strains have been guidelines 1993 4 UK 9 to 54 years skin, throat (3) reported (albeit rarely) to cause systemic disease, both 2,7,8 1994 4* Italy 16 to 67 years nose, skin (2), throat in the UK and overseas , which makes the apparent 1995 4 Turkey 16 to 74 years throat increase in non-toxigenic biotypes of concern. The global incidence of infections caused by these organisms is 1996 5 UK 16 to 75 years skin, throat (4) unknown but it is worth recording. The isolation of a 1997 4 UK 5 to 51 years throat non-toxigenic isolate from a patient with pharyngitis 1998 1 UK 35 years throat without the presence of a membrane is not defined as a 1,5 * one case associated with ingestion of unpasteurised dairy products in Italy case of diphtheria . foreign travel in Turkey Laboratory based surveillance of non-toxigenic C. diphtheriae in England and Wales was enhanced in 1995 respiratory or cutaneous. Most cases in the UK are and 19965. Isolates came from patients in different travellers returning from areas where the disease is either 2 geographic locations and without any suggestion of endemic or epidemic . Toxin-producing strains of importation from overseas. The increased number of C. ulcerans as well as C. diphtheriae can cause respiratory 2,3 infections since 1990 could be due to improved diphtheria, but this is rare . ascertainment, but there has been no upward trend in Eighteen isolates of toxigenic C. diphtheriae were the numbers of toxigenic strains in the UK. This referred to the PHLS Streptococcus and Diphtheria suggests, therefore, that the increase in non-toxigenic Reference Unit (SDRU) from within the UK between 1993 strains is genuine2. and 1998 (table 1). One was from a 14 year old boy who had travelled from Pakistan to the UK and died of Characteristics of potentially toxigenic diphtheria4. Twenty-two isolates of toxigenic C. ulcerans corynebacteria were also referred to SDRU, some from patients noted The minimal characteristics for presumptive to have a diphtheritic membrane. Four patients identification include positive reactions for catalase, presented with infected skin lesions (table 2). nitrate reduction (except the biotype belfanti), cystinase From 1989 to 1992, SDRU received a total of 95 non- production, and glucose and maltose fermentation and toxigenic isolates of C. diphtheriae, most from cases of negative reactions for urease, sucrose, xylose, and severe and often recurrent throat infections2,5. Non- pyrazinamidase, which distinguish the biotypes and the toxigenic strains of C. diphtheriae have subsequently other potentially toxigenic species (table 3). The four emerged as potential pathogens in the UK. The numbers biotypes of C. diphtheriae differ biochemically: in of non-toxigenic C. diphtheriae isolates referred to SDRU particular, var intermedius is lipophilic, requiring lipids (particularly the gravis biotype) rose from 51 in 1993 to (for example, hydrolysis on tween 80 medium) for 178 in 1997. In 1998, 147 out of 163 non-toxigenic isolates optimal growth, as it grows as a tiny translucent colony were of the biotype var gravis, 15 var mitis, and one var on routine media. C. diphtheriae will grow on nutrient belfanti. Var gravis is the predominant biotype; isolates agar, but the presence of serum or blood will improve of var mitis are commonly isolated from cases of cutanous growth6. infection, as seen elsewhere in Europe, in particular, in areas of eastern Europe where the epidemic of diphtheria Significance of Corynebacterium sp isolates is declining (T Glushkevich, I Mazurova, personal Standard operating procedures for the identification of communication). This observation has also been made corynebacteria in diagnostic laboratories are being in other parts of Europe. It is not clear whether 9 established by the PHLS . It is advisable for laboratories immunisation selectively inhibits growth of toxigenic to establish protocols to ensure that appropriate action is taken, however, if corynebacteria are isolated in the TABLE 3 Biochemical identification of clinically significant corynebacteria following instances: from a respiratory specimen, notably throat and nose Species CYS PYZ Nitrate Urea Glucose Maltose Sucrose Glycogen swabs C. diphtheriae in a wound or skin swab/specimen from a person var gravis +- + - + + - + var mitis +- + - + + - - who has recently travelled abroad var intermedius +- + - + + - - as the predominant organism isolated from a var belfanti +- - - + + - - normally sterile site, or from a wound, abscess, or C. ulcerans +- - + + + - + C. pseudotuberculosis +- - + + + - - purulent sputum C. amycolatum -+ v v + v v - from more than two sets of blood cultures C. imitans -± - - + + ± - ≥ 4 C. pseudodiphtheriticum -v+ + - - - - at a level 10 colony forming units/mL in pure C. striatum -++ - + - v - culture from urine ±: weak reaction from multiple specimens v: variable reaction CYS: cystinase production on Tinsdale medium as significant growth of coryneforms with white PYZ: pyrazinamidase activity blood cells in the original Gram stain COMMUNICABLE DISEASE AND PUBLIC HEALTH VOL 2 NO 4 DECEMBER 1999 251 Laboratory safety issues microbiological report of C. diphtheriae (and rarely, Laboratory acquired diphtheria has been reported at C. ulcerans) in routine throat and other swabs taken least twice in the UK10,11. To minimise risks to staff, all from the respiratory tract may be the first indication that cultures of suspected C. diphtheriae and C. ulcerans should