# Non-toxigenic Corynebacterium diphtheriae in hallux ulceration

Gauthier Delvallez, Edgar Badell, Sokleaph Cheng, Soda Meng, Vivandet Tong, Judy Norman, Julie Toubiana, Koen Vandelannoote, Anne-Laure Bañuls, Mallorie Hide, et al.

► To cite this version:

Gauthier Delvallez, Edgar Badell, Sokleaph Cheng, Soda Meng, Vivandet Tong, et al. Non-toxigenic Corynebacterium diphtheriae in hallux ulceration. Journal of Infection in Developing Countries, 2022, 16 (06), pp.1118-1121. 10.3855/jidc.16153. pasteur-03929961

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# Non-toxigenic Corynebacterium diphtheriae in hallux ulceration

Gauthier Delvallez, Edgar Badell, Sokleaph Cheng, Soda Meng, Vivandet Tong, Judy Norman, Julie Toubiana, Koen Vandelannoote, Anne-Laure Bañuls, Mallorie Hide, et al.

► To cite this version:

Gauthier Delvallez, Edgar Badell, Sokleaph Cheng, Soda Meng, Vivandet Tong, et al. Non-toxigenic Corynebacterium diphtheriae in hallux ulceration. Journal of Infection in Developing Countries, 2022, 16 (06), pp.1118-1121. 10.3855/jidc.16153. pasteur-03929961

HAL Id: pasteur-03929961

https://pasteur.hal.science/pasteur-03929961v1

Submitted on 19 Jan 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
Case Report

Non-toxigenic Corynebacterium diphtheriae in hallux ulceration

Gauthier Delvallez<fnref n="1" />, Edgar Badell<fnref n="2" />,<fnref n="3" />, Sokleaph Cheng<fnref n="1" />,<fnref n="4" />, Soda Meng<fnref n="1" />, Vivandet Tong<fnref n="5" />, Judy Norman<fnref n="5" />, Julie Toubiana<fnref n="2" />,<fnref n="3" />,<fnref n="6" />, Koen Vandelannoote<fnref n="1" />, Anne-Laure Bañuls<fnref n="4" />,<fnref n="7" />,<fnref n="8" />, Mallorie Hide<fnref n="1" />,<fnref n="4" />,<fnref n="7" />,<fnref n="8" />, Sylvain Brisse<fnref n="2" />,<fnref n="3" />

Abstract

Introduction: Toxigenic *Corynebacterium diphtheriae* causes classical diphtheria. Skin infections by toxigenic or non-toxigenic *Corynebacterium diphtheriae* are prevalent in the tropics but are rarely reported.

Case presentation: We report the identification of a non-toxigenic *Corynebacterium diphtheriae* (biovar Gravis) isolate in a 52-year-old Cambodian male. The patient presented purulent and non-healing ulcerations on the right hallux. The wound has healed after 7 days of antibiotic therapy with a favourable outcome.

Conclusions: This case represents, to our knowledge, the first report of *Corynebacterium diphtheriae* in Cambodia in the last 10 years, and highlights the lack of diagnosis and notifications of diphtheria. It is important to raise awareness among clinicians and to set up diphtheria surveillance in Cambodia.

**Key words:** Cambodia; *Corynebacterium diphtheriae*; hallux ulceration.

J Infect Dev Ctries 2022; 16(6):1118-1121. doi:10.3855/jidc.16153

(Received 28 November 2021 – Accepted 22 January 2022)

Copyright © 2022 Delvallez et al. This is an open-access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Introduction

*Corynebacterium diphtheriae* (*C. diphtheriae*) was described in 1884 by Loeffler as the causative agent of diphtheria [1]. Toxigenic *C. diphtheriae* and non-toxigenic *C. diphtheriae* (NTCD), are also responsible for skin infections with a chronic, non-healing ulcerative clinical entity, prevalent in endemic areas of the tropics. Even though the diphtheria vaccine elicits the production of antibodies directed against the diphtheria toxin as well as various bacterial components, *C. diphtheriae* infections, caused by toxigenic and non-toxigenic strains, may re-emerge given the low vaccination coverage in some settings [2–5]. Here, we report the identification of a non-toxigenic *C. diphtheriae* isolate in a hallux ulceration of a Cambodian patient.

Case Report

A 52-year-old Cambodian male, with no travel history, living in Prey Veng province with a history of

type 2 diabetes and hypertension consulted his pharmacist for ulceration on the right hallux, which had been present for 5 days. The lesion was purulent, painful, and reddened but the patient did not present with a fever. The pharmacist empirically dispensed to the patient 250 mg ampicillin four times per day and 250 mg cloxacillin four times per day without establishing a diagnosis. After 3 days of treatment without apparent clinical response, the patient consulted his general practitioner at Mercy Medical Center Cambodia in Phnom Penh. Apart from the ulceration, the physical examination was unremarkable. A swab of the necrotic lesion was sent to the Medical Biology Laboratory of the Institut Pasteur du Cambodge (IPC) for bacteriological analysis. Polymicrobial growth was observed after 24 hours at 35 °C on both chocolate agar and selective sheep blood agar with nalidixic acid and colistin. Identification of different bacterial colonies initially performed by MALDI-Biotyper (Bruker Daltonics, Bremen,

<footnote n="1">¹ Medical Biology Laboratory, Institut Pasteur du Cambodge, Phnom Penh, Cambodia</footnote>

<footnote n="2">² Institut Pasteur, Université Paris Cité, Biodiversity and Epidemiology of Bacterial Pathogens, Paris, France</footnote>

<footnote n="3">³ National Reference Center for Corynebacteria of the Diphtheriae Complex, Institut Pasteur, Paris, France</footnote>

<footnote n="4">⁴ LMI Drug Resistance in South East Asia, Institut Pasteur du Cambodge, Phnom Penh, Cambodia</footnote>

<footnote n="5">⁵ Mercy Medical Center Cambodia, Phnom Penh, Cambodia</footnote>

<footnote n="6">⁶ Université Paris Cité, Service de Pédiatrie Générale et Maladies Infectieuses, Hôpital Necker-Enfants malades, Assistance Publique-Hôpitaux de Paris, AP-HP, France</footnote>

<footnote n="7">⁷ MIVEGEC, Univ. Montpellier, CNRS, IRD, Montpellier, France</footnote>

<footnote n="8">⁸ Centre de Recherche en Écologie et Évolution de la Santé (CREES), Montpellier, France</footnote>
Case Report

Non-toxigenic Corynebacterium diphtheriae in hallux ulceration

Gauthier Delvallez<fnref n="1" />, Edgar Badell<fnref n="2" />,<fnref n="3" />, Sokleaph Cheng<fnref n="1" />,<fnref n="4" />, Soda Meng<fnref n="1" />, Vivandet Tong<fnref n="5" />, Judy Norman<fnref n="5" />, Julie Toubiana<fnref n="2" />,<fnref n="3" />,<fnref n="6" />, Koen Vandelannoote<fnref n="1" />, Anne-Laure Bañuls<fnref n="4" />,<fnref n="7" />,<fnref n="8" />, Mallorie Hide<fnref n="1" />,<fnref n="4" />,<fnref n="7" />,<fnref n="8" />, Sylvain Brisse<fnref n="2" />,<fnref n="3" />

Abstract

Introduction: Toxigenic *Corynebacterium diphtheriae* causes classical diphtheria. Skin infections by toxigenic or non-toxigenic *Corynebacterium diphtheriae* are prevalent in the tropics but are rarely reported.

Case presentation: We report the identification of a non-toxigenic *Corynebacterium diphtheriae* (biovar Gravis) isolate in a 52-year-old Cambodian male. The patient presented purulent and non-healing ulcerations on the right hallux. The wound has healed after 7 days of antibiotic therapy with a favourable outcome.

Conclusions: This case represents, to our knowledge, the first report of *Corynebacterium diphtheriae* in Cambodia in the last 10 years, and highlights the lack of diagnosis and notifications of diphtheria. It is important to raise awareness among clinicians and to set up diphtheria surveillance in Cambodia.

**Key words:** Cambodia; *Corynebacterium diphtheriae*; hallux ulceration.

J Infect Dev Ctries 2022; 16(6):1118-1121. doi:10.3855/jidc.16153

(Received 28 November 2021 – Accepted 22 January 2022)

Copyright © 2022 Delvallez et al. This is an open-access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Introduction

*Corynebacterium diphtheriae* (*C. diphtheriae*) was described in 1884 by Loeffler as the causative agent of diphtheria [1]. Toxigenic *C. diphtheriae* and non-toxigenic *C. diphtheriae* (NTCD), are also responsible for skin infections with a chronic, non-healing ulcerative clinical entity, prevalent in endemic areas of the tropics. Even though the diphtheria vaccine elicits the production of antibodies directed against the diphtheria toxin as well as various bacterial components, *C. diphtheriae* infections, caused by toxigenic and non-toxigenic strains, may re-emerge given the low vaccination coverage in some settings [2–5]. Here, we report the identification of a non-toxigenic *C. diphtheriae* isolate in a hallux ulceration of a Cambodian patient.

Case Report

A 52-year-old Cambodian male, with no travel history, living in Prey Veng province with a history of

type 2 diabetes and hypertension consulted his pharmacist for ulceration on the right hallux, which had been present for 5 days. The lesion was purulent, painful, and reddened but the patient did not present with a fever. The pharmacist empirically dispensed to the patient 250 mg ampicillin four times per day and 250 mg cloxacillin four times per day without establishing a diagnosis. After 3 days of treatment without apparent clinical response, the patient consulted his general practitioner at Mercy Medical Center Cambodia in Phnom Penh. Apart from the ulceration, the physical examination was unremarkable. A swab of the necrotic lesion was sent to the Medical Biology Laboratory of the Institut Pasteur du Cambodge (IPC) for bacteriological analysis. Polymicrobial growth was observed after 24 hours at 35 °C on both chocolate agar and selective sheep blood agar with nalidixic acid and colistin. Identification of different bacterial colonies initially performed by MALDI-Biotyper (Bruker Daltonics, Bremen,

<footnote n="1">¹ Medical Biology Laboratory, Institut Pasteur du Cambodge, Phnom Penh, Cambodia</footnote>

<footnote n="2">² Institut Pasteur, Université Paris Cité, Biodiversity and Epidemiology of Bacterial Pathogens, Paris, France</footnote>

<footnote n="3">³ National Reference Center for Corynebacteria of the Diphtheriae Complex, Institut Pasteur, Paris, France</footnote>

<footnote n="4">⁴ LMI Drug Resistance in South East Asia, Institut Pasteur du Cambodge, Phnom Penh, Cambodia</footnote>

<footnote n="5">⁵ Mercy Medical Center Cambodia, Phnom Penh, Cambodia</footnote>

<footnote n="6">⁶ Université Paris Cité, Service de Pédiatrie Générale et Maladies Infectieuses, Hôpital Necker-Enfants malades, Assistance Publique-Hôpitaux de Paris, AP-HP, France</footnote>

<footnote n="7">⁷ MIVEGEC, Univ. Montpellier, CNRS, IRD, Montpellier, France</footnote>

<footnote n="8">⁸ Centre de Recherche en Écologie et Évolution de la Santé (CREES), Montpellier, France</footnote>
Germany) revealed the presence of *C. diphtheriae* along with methicillin-susceptible *Staphylococcus aureus* and high-level penicillinase-producing *Escherichia coli*.

The isolate was sent to the National Reference Center laboratory (France) for further characterization and confirmation of both identification and antibiotic susceptibility. The identification of *C. diphtheriae* was confirmed using both a real-time multiplex Polymerase chain reaction (PCR) assay [6] and a biochemical test (API Coryne, bioMérieux, France). The latter test also allowed determining that the biovar was of type Gravis. The multiplex PCR assay also tests for the diphtheria toxin gene (*tox*) presence and revealed that the isolate was non-toxigenic. These assays do not allow differentiating between *C. diphtheriae sensu stricto* and two novel species recently described, *C. belfantii* and *C. rouxii* [7,8]. Genomic sequencing was therefore performed. It showed that the isolate belonged to *C. diphtheriae sensu stricto*, and also confirmed that the isolate did not carry the *tox* gene. Further, the genomic sequence showed that the isolate (FRC1098) did not carry any resistance gene. Genotyping by the multilocus sequence typing (MLST) approach showed that the isolate was of MLST genotype ST779 [9].

Antibiotic susceptibility testing of the *C. diphtheriae* isolate was performed at IPC with the disk diffusion method according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) 2019 guidelines [10] and revealed resistance to penicillin (zone of inhibition diameter, 17 mm). The isolate was susceptible to erythromycin, clindamycin, vancomycin, ciprofloxacin, gentamicin, linezolid, tetracycline, co-trimoxazole, and rifampicin. Resistance to beta-lactam antibiotics was confirmed based on Etest (bioMérieux, France), which allowed determining the following minimal inhibitory concentrations (MIC): penicillin (0.25 mg/L), amoxicillin (0.25 mg/L), ceftriaxone (1 mg/L) and ceftazidime (8 mg/L).

Subsequent investigations regarding the patient's immunization status revealed he had never been vaccinated against diphtheria. The patient received 7 days of antibiotic therapy composed of co-trimoxazole twice a day and clindamycin 300 mg three times per day, a regimen covering all the bacterial species found in the culture. During a follow-up visit 8 weeks after ceasing antibiotic therapy, the wound had healed and the patient remained in good condition.

Discussion

We report on a cutaneous clinical case in a 52-year-
old patient with a *tox*-negative *C. diphtheriae* of biovar
Gravis and genotype ST779. Real-time PCR and

genomic sequencing were used to confirm the initial
identification by MALDI-TOF, as the latter does not
differentiate *C. belfantii* and *C. rouxii* due to the lack of
reference spectra in current commercial databases. The
strain is considered resistant to penicillin according to
breakpoints of EUCAST 2019 recommendations for
both the disk diffusion and Etest methods [10].
However, the interpretation of the diameters and MIC
values must be cautious, as the breakpoints for
corynebacteria were developed for species other than *C.
diphtheriae*. In addition, the genomic sequencing of the
isolate showed the absence of antibiotic resistance
genes and a recent study on the natural distribution of
MICs in *C. diphtheriae* revealed that the strain may be
considered susceptible [11]. Our investigations further
showed the MIC of ceftriaxone to be higher than that of
penicillin, which agrees with this same study. As in
most cases when isolating a non-toxigenic *C.
diphtheriae* strain from a skin wound, the bacterial
culture was polymicrobial, including in this case both
*Staphylococcus aureus* and *Escherichia coli*. The
polymicrobial character of the bacterial culture and the
absence of secretion of diphtheria toxin highlight the
difficulty of attributing the infection to the *C.
diphtheriae* isolate, as it could act as a co-pathogen or
colonizer of a pre-existing wound caused by another
pathogen. In addition, the first treatment dispensed by
the pharmacist based on antibiotics of the penicillin
family should have been effective on the *C. diphtheria*
isolate, which does not carry any resistance gene.
However, this antibiotic therapy was expected to be
ineffective on the two other isolated species. There is
currently no consensus on the definition of cutaneous
diphtheria, which has been defined as a chronic ulcer
growing *C. diphtheriae* from a wound specimen
regardless of toxigenic character [12], whereas others
consider that cutaneous diphtheria is caused specifically
by toxigenic strains [13].

Currently, the Cambodian national immunization
program provides diphtheria vaccination free of charge
at health centers to children at 6, 10, and 14 weeks of
age [14]. However, the 52-year-old patient of this report
did not receive any diphtheria vaccine during his
childhood, nor any boosters. The national expanded
program on immunization (EPI) was launched officially
in October 1986 in Cambodia [15].

Although diphtheria was endemic in Cambodia in
the 1980s, the latest report dates from 2010, with rare
cases reported to the World Health Organization
(WHO) between 2000 and 2010 [16]. However, several
diphtheria cases have been described during the last
decade in South-East Asia countries such as Thailand,
Germany) revealed the presence of *C. diphtheriae* along with methicillin-susceptible *Staphylococcus aureus* and high-level penicillinase-producing *Escherichia coli*.

The isolate was sent to the National Reference Center laboratory (France) for further characterization and confirmation of both identification and antibiotic susceptibility. The identification of *C. diphtheriae* was confirmed using both a real-time multiplex Polymerase chain reaction (PCR) assay [6] and a biochemical test (API Coryne, bioMérieux, France). The latter test also allowed determining that the biovar was of type Gravis. The multiplex PCR assay also tests for the diphtheria toxin gene (*tox*) presence and revealed that the isolate was non-toxigenic. These assays do not allow differentiating between *C. diphtheriae sensu stricto* and two novel species recently described, *C. belfantii* and *C. rouxii* [7,8]. Genomic sequencing was therefore performed. It showed that the isolate belonged to *C. diphtheriae sensu stricto*, and also confirmed that the isolate did not carry the *tox* gene. Further, the genomic sequence showed that the isolate (FRC1098) did not carry any resistance gene. Genotyping by the multilocus sequence typing (MLST) approach showed that the isolate was of MLST genotype ST779 [9].

Antibiotic susceptibility testing of the *C. diphtheriae* isolate was performed at IPC with the disk diffusion method according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) 2019 guidelines [10] and revealed resistance to penicillin (zone of inhibition diameter, 17 mm). The isolate was susceptible to erythromycin, clindamycin, vancomycin, ciprofloxacin, gentamicin, linezolid, tetracycline, co-trimoxazole, and rifampicin. Resistance to beta-lactam antibiotics was confirmed based on Etest (bioMérieux, France), which allowed determining the following minimal inhibitory concentrations (MIC): penicillin (0.25 mg/L), amoxicillin (0.25 mg/L), ceftriaxone (1 mg/L) and ceftazidime (8 mg/L).

Subsequent investigations regarding the patient's immunization status revealed he had never been vaccinated against diphtheria. The patient received 7 days of antibiotic therapy composed of co-trimoxazole twice a day and clindamycin 300 mg three times per day, a regimen covering all the bacterial species found in the culture. During a follow-up visit 8 weeks after ceasing antibiotic therapy, the wound had healed and the patient remained in good condition.

Discussion

We report on a cutaneous clinical case in a 52-year-
old patient with a *tox*-negative *C. diphtheriae* of biovar
Gravis and genotype ST779. Real-time PCR and

genomic sequencing were used to confirm the initial
identification by MALDI-TOF, as the latter does not
differentiate *C. belfantii* and *C. rouxii* due to the lack of
reference spectra in current commercial databases. The
strain is considered resistant to penicillin according to
breakpoints of EUCAST 2019 recommendations for
both the disk diffusion and Etest methods [10].
However, the interpretation of the diameters and MIC
values must be cautious, as the breakpoints for
corynebacteria were developed for species other than *C.
diphtheriae*. In addition, the genomic sequencing of the
isolate showed the absence of antibiotic resistance
genes and a recent study on the natural distribution of
MICs in *C. diphtheriae* revealed that the strain may be
considered susceptible [11]. Our investigations further
showed the MIC of ceftriaxone to be higher than that of
penicillin, which agrees with this same study. As in
most cases when isolating a non-toxigenic *C.
diphtheriae* strain from a skin wound, the bacterial
culture was polymicrobial, including in this case both
*Staphylococcus aureus* and *Escherichia coli*. The
polymicrobial character of the bacterial culture and the
absence of secretion of diphtheria toxin highlight the
difficulty of attributing the infection to the *C.
diphtheriae* isolate, as it could act as a co-pathogen or
colonizer of a pre-existing wound caused by another
pathogen. In addition, the first treatment dispensed by
the pharmacist based on antibiotics of the penicillin
family should have been effective on the *C. diphtheria*
isolate, which does not carry any resistance gene.
However, this antibiotic therapy was expected to be
ineffective on the two other isolated species. There is
currently no consensus on the definition of cutaneous
diphtheria, which has been defined as a chronic ulcer
growing *C. diphtheriae* from a wound specimen
regardless of toxigenic character [12], whereas others
consider that cutaneous diphtheria is caused specifically
by toxigenic strains [13].

Currently, the Cambodian national immunization
program provides diphtheria vaccination free of charge
at health centers to children at 6, 10, and 14 weeks of
age [14]. However, the 52-year-old patient of this report
did not receive any diphtheria vaccine during his
childhood, nor any boosters. The national expanded
program on immunization (EPI) was launched officially
in October 1986 in Cambodia [15].

Although diphtheria was endemic in Cambodia in
the 1980s, the latest report dates from 2010, with rare
cases reported to the World Health Organization
(WHO) between 2000 and 2010 [16]. However, several
diphtheria cases have been described during the last
decade in South-East Asia countries such as Thailand,
Laos, Myanmar, Vietnam, Malaysia, and Indonesia, including a similar case of foot infection with a non-toxigenic *C. diphtheriae* biovar Gravis in a 16-year-old girl, who had traveled to Thailand [17–23]. The present finding of *C. diphtheriae* in Cambodia highlights the fact that this pathogen should be considered for microbiological analyses in a country where the laboratory capacities are limited and where antibiotics are freely dispensed by pharmacies without bacteriological documentation and medical prescription. In the absence of laboratory capacity and appropriate identification and reporting of the cases, the burden of the disease is likely to be severely underestimated. It would be highly beneficial to improve laboratory capacity with the possibility to detect the *tox* gene, and ideally to test for the production of the diphtheria toxin locally. PCR alone cannot provide a definitive result about toxigenicity, as some *C. diphtheriae* isolates possess the *tox* gene, but do not express the toxin. Besides, PCR technology is not available in many Cambodian laboratories. Therefore, the implementation of the phenotypic Elek's test for defining toxin production would be advantageous, even though it is longer to obtain and less sensitive than PCR [24, 25]. In the present investigation, 6 weeks have elapsed between the initial strain identification in Cambodia and the molecular screening of the toxin in France, which is not useful for patient care in the event of a toxigenic strain. Rapid and reliable diagnosis is also critical to screen patients and their close contacts for *tox*-positive *C. diphtheriae* pharyngeal carriage. There is currently no national surveillance program for diphtheria in Cambodia, and this case report highlights the importance of setting up such a system, which would allow for prompt and effective patient care and could guide prevention and control actions. Finally, even though vaccine coverage for diphtheria in Cambodia is estimated at more than 90% as recommended by the WHO, only primary diphtheria immunization is performed and no booster dose is administered [14, 26]. In this context, a Cambodian seroprevalence study would be useful to evaluate the level of protection of the Cambodian population, given the high proportion of suboptimal protection even in the population of countries that provide several boosters [27].

## Acknowledgements

**Funding:** This work was supported by Institut Pasteur du Cambodge, Institut Pasteur (Paris, France), and Santé publique France (Saint Maurice, France). KV was supported by Institut Pasteur, Institut Pasteur du Cambodge, and The Peter Doherty Institute for Infection and Immunity.

## References

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4. Badell E, Alharazi A, Criscuolo A, The NCPHL diphtheria outbreak working group, Lefrancq N, Bouchez V, Guglielmini J, Hennart M, Carmi-Leroy A, Zidane N, Pascal-Perrigault M, Lebreton M, Martini H, Salje H, Toubiana J, Dureab F, Dhabaan G, Brisse S (2020) Epidemiological, clinical and genomic insights into the ongoing diphtheria outbreak in Yemen. medRxiv 2020.07.21.20159186.

5. Möller J, Kraner M, Sonnewald U, Sangal V, Tittlbach H, Winkler J, Winkler TH, Melnikov V, Lang R, Sing A, Mattos-Guaraldi AL, Burkovski A (2019) Proteomics of diphtheria toxoid vaccines reveals multiple proteins that are immunogenic and may contribute to protection of humans against *Corynebacterium diphtheriae*. Vaccine 37: 3061–3070.

6. Badell E, Guillot S, Tulliez M, Pascal M, Panunzi LG, Rose S, Litt D, Fry NK, Brisse S (2019) Improved quadruplex real-time PCR assay for the diagnosis of diphtheria. J Med Microbiol 68: 1455–1465.

7. Dazas M, Badell E, Carmi-Leroy A, Criscuolo A, Brisse S (2018) Taxonomic status of *Corynebacterium diphtheriae* biovar Belfanti and proposal of *Corynebacterium belfantii* sp. nov. Int J Syst Evol Microbiol 68: 3826–3831.

8. Badell E, Hennart M, Rodrigues C, Passet V, Dazas M, Panunzi L, Bouchez V, Carmi-Leroy A, Toubiana J, Brisse S (2020) *Corynebacterium rouxii* sp. nov., a novel member of the diphtheriae species complex. Res Microbiol 171: 122–127.

9. Institut Pasteur (2021) Full information on isolate FRC1098 (id: 1413). Available: https://big$sdb.pasteur.fr/cgi-bin/big$sdb/big$sdb.pl?page=info&db=pubmlst_diphtheria_isolates&id=1413. Accessed: 07 June 2022

10. Société Française de Microbiologie (2019) CASFM/EUCAST V2.0 Mai 2019. Available: https://www.sfm-microbiologie.org/2019/05/06/casfm-eucast-2019-v2/. Accessed: 03 March 2021

11. Marosevic DV, Berger A, Kahlmeter G, Payer SK, Hörmansdorfer S, Sing A (2020) Antimicrobial susceptibility of *Corynebacterium diphtheriae* and *Corynebacterium ulcerans* in Germany 2011-17. J Antimicrob Chemother 75: 2885–2893.

12. Lowe CF, Bernard KA, Romney MG (2011) Cutaneous diphtheria in the urban poor population of Vancouver, British
Laos, Myanmar, Vietnam, Malaysia, and Indonesia, including a similar case of foot infection with a non-toxigenic *C. diphtheriae* biovar Gravis in a 16-year-old girl, who had traveled to Thailand [17–23]. The present finding of *C. diphtheriae* in Cambodia highlights the fact that this pathogen should be considered for microbiological analyses in a country where the laboratory capacities are limited and where antibiotics are freely dispensed by pharmacies without bacteriological documentation and medical prescription. In the absence of laboratory capacity and appropriate identification and reporting of the cases, the burden of the disease is likely to be severely underestimated. It would be highly beneficial to improve laboratory capacity with the possibility to detect the *tox* gene, and ideally to test for the production of the diphtheria toxin locally. PCR alone cannot provide a definitive result about toxigenicity, as some *C. diphtheriae* isolates possess the *tox* gene, but do not express the toxin. Besides, PCR technology is not available in many Cambodian laboratories. Therefore, the implementation of the phenotypic Elek's test for defining toxin production would be advantageous, even though it is longer to obtain and less sensitive than PCR [24, 25]. In the present investigation, 6 weeks have elapsed between the initial strain identification in Cambodia and the molecular screening of the toxin in France, which is not useful for patient care in the event of a toxigenic strain. Rapid and reliable diagnosis is also critical to screen patients and their close contacts for *tox*-positive *C. diphtheriae* pharyngeal carriage. There is currently no national surveillance program for diphtheria in Cambodia, and this case report highlights the importance of setting up such a system, which would allow for prompt and effective patient care and could guide prevention and control actions. Finally, even though vaccine coverage for diphtheria in Cambodia is estimated at more than 90% as recommended by the WHO, only primary diphtheria immunization is performed and no booster dose is administered [14, 26]. In this context, a Cambodian seroprevalence study would be useful to evaluate the level of protection of the Cambodian population, given the high proportion of suboptimal protection even in the population of countries that provide several boosters [27].

## Acknowledgements

**Funding:** This work was supported by Institut Pasteur du Cambodge, Institut Pasteur (Paris, France), and Santé publique France (Saint Maurice, France). KV was supported by Institut Pasteur, Institut Pasteur du Cambodge, and The Peter Doherty Institute for Infection and Immunity.

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11. Marosevic DV, Berger A, Kahlmeter G, Payer SK, Hörmansdorfer S, Sing A (2020) Antimicrobial susceptibility of *Corynebacterium diphtheriae* and *Corynebacterium ulcerans* in Germany 2011-17. J Antimicrob Chemother 75: 2885–2893.

12. Lowe CF, Bernard KA, Romney MG (2011) Cutaneous diphtheria in the urban poor population of Vancouver, British
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13. Gordon CL, Fagan P, Hennessy J, Baird R (2011) Characterization of *Corynebacterium diphtheriae* isolates from infected skin lesions in the northern territory of Australia. J Clin Microbiol 49: 3960.

14. Hattasingh W, Pangsaa K, Thisyakorn U (2016) Report on: "The 1st workshop on national immunization programs and vaccine coverage in ASEAN countries, April 30, 2015, Pattaya, Thailand." Vaccine 34: 1233-1240.

15. National Maternal and Child Health Center, Ministry of Health (2016) National immunization program / Cambodia national immunization program strategic plan 2016-2020. Available: https://cdc-crdb.gov.kh/en/twg-jmi/sector_strategy/NIPSP_2016_2020.pdf. Accessed: 03 March 2021

16. World Health Organization (2021) Global health observatory data repository / Diphtheria / Reported cases by country. Available: https://apps.who.int/gho/data/node.main.WHS3_41?lang=en. Accessed: 28 October 2021

17. Depypere M, Verhaegen J, Derdelinckx I, Meersseman W (2013) A forgotten disease in a returning traveler from Thailand. Acta Clin Belg 68: 382-383.

18. Paveenkittiporn W, Sripakdee S, Koobkratok O, Sangkitporn S, Kerdsin A (2019) Molecular epidemiology and antimicrobial susceptibility of outbreak-associated *Corynebacterium diphtheriae* in Thailand, 2012. Infect Genet Evol J Mol Epidemiol Evol Genet Infect Dis 75: 104007.

19. Sein C, Tiwari T, Macneil A, Wannemuehler K, Soulaphy C, Souliphone P, Reyburn R, Ramirez Gonzalez A, Watkins M, Goodson JL (2016) Diphtheria outbreak in Lao people's democratic republic, 2012-2013. Vaccine 34: 4321-4326.

20. Weil LM, Williams MM, Shirin T, Lawrence M, Habib ZH, Aneke JS, Tondella ML, Zaki Q, Cassiday PK, Lonsway D, Farrque M, Hossen T, Feldstein LR, Cook N, Maldonado-Quiles G, Alam AN, Muraduzzaman AKM, Akram A, Conklin L, Doan S, Friedman M, Acosta AM, Hariri S, Fox LM, Tiwari TSP, Flora MS (2021) Investigation of a large diphtheria outbreak and co-circulation of *Corynebacterium pseudodiphtheriticum* among forcibly displaced Myanmar nationals, 2017-2019. J Infect Dis 224: 318-325.

21. Kitamura N, Le TTT, Le LT, Nguyen LD, Dao AT, Hoang TT, Yoshihara K, Iijima M, The TM, Do HM, Le HX, Do HT, Dang AD, Vien MQ, Yoshida LM (2020) Diphtheria outbreaks in schools in central highland districts, Vietnam, 2015-2018. Emerg Infect Dis 26: 596-600.

22. Mohd Khalid MKN, Ahmad N, Hii SYF, Abd Wahab MA, Hashim R, Liow YL (2019) Molecular characterization of *Corynebacterium diphtheriae* isolates in Malaysia between 1981 and 2016. J Med Microbiol 68: 105-110.

23. Arguni E, Karyanti MR, Satari HI, Hadinegoro SR (2021) Diphtheria outbreak in Jakarta and Tangerang, Indonesia: epidemiological and clinical predictor factors for death. PloS One 16: e0246301.

24. Elek SD (1949) The plate virulence test for diphtheria. J Clin Pathol 2: 250-258.

25. Engler KH, Glushkevich T, Mazurova IK, George RC, Efstratiou A (1997) A modified Elek test for detection of toxigenic corynebacteria in the diagnostic laboratory. J Clin Microbiol 35: 495-498.

26. World Health Organization (2013) Global vaccine action 2011-2020. Available: https://apps.who.int/iris/rest/bitstreams/110564/retrieve. Accessed: 03 March 2021

27. Berbers G, van Gageldonk P, Kassteele JV, Wiedermann U, Desombere I, Dalby T, Toubiana J, Tsiodras S, Ferencz IP, Mullan K, Griskevicius A, Kolupajeva T, Vestrheim DF, Palminha P, Popovici O, Wehlin L, Kastrin T, Maďarová L, Campbell H, Ködmön C, Bacci S, Barkoff A-M, He Q, Serosurveillance Study Team (2021) Circulation of pertussis and poor protection against diphtheria among middle-aged adults in 18 European countries. Nat Commun 12: 2871.

**Corresponding author**

Dr Gauthier Delvallez
Head of Medical Biology Laboratory
Institut Pasteur du Cambodge

5 Monivong Boulevard, P.O Box. 983, Phnom Penh, Cambodia
Tel: +85512802978

E-mail: gdelvallez@pasteur-kh.org

**Conflict of interests:** No conflict of interests is declared.
Columbia, Canada: a 10-year review. J Clin Microbiol 49: 2664-2666.

13. Gordon CL, Fagan P, Hennessy J, Baird R (2011) Characterization of *Corynebacterium diphtheriae* isolates from infected skin lesions in the northern territory of Australia. J Clin Microbiol 49: 3960.

14. Hattasingh W, Pangsaa K, Thisyakorn U (2016) Report on: “The 1st workshop on national immunization programs and vaccine coverage in ASEAN countries, April 30, 2015, Pattaya, Thailand.” Vaccine 34: 1233–1240.

15. National Maternal and Child Health Center, Ministry of Health (2016) National immunization program / Cambodia national immunization program strategic plan 2016-2020. Available: https://cdc-crdb.gov.kh/en/twg-jmi/sector_strategy/NIPSP_2016_2020.pdf. Accessed: 03 March 2021

16. World Health Organization (2021) Global health observatory data repository / Diphtheria / Reported cases by country. Available: https://apps.who.int/gho/data/node.main.WHS3_41?lang=en. Accessed: 28 October 2021

17. Depypere M, Verhaegen J, Derdelinckx I, Meersseman W (2013) A forgotten disease in a returning traveler from Thailand. Acta Clin Belg 68: 382–383.

18. Paveenkittiporn W, Sripakdee S, Koobkratok O, Sangkitporn S, Kerdsin A (2019) Molecular epidemiology and antimicrobial susceptibility of outbreak-associated *Corynebacterium diphtheriae* in Thailand, 2012. Infect Genet Evol J Mol Epidemiol Evol Genet Infect Dis 75: 104007.

19. Sein C, Tiwari T, Macneil A, Wannemuehler K, Soulaphy C, Souliphone P, Reyburn R, Ramirez Gonzalez A, Watkins M, Goodson JL (2016) Diphtheria outbreak in Lao people's democratic republic, 2012-2013. Vaccine 34: 4321–4326.

20. Weil LM, Williams MM, Shirin T, Lawrence M, Habib ZH, Aneke JS, Tondella ML, Zaki Q, Cassiday PK, Lonsway D, Farrque M, Hossen T, Feldstein LR, Cook N, Maldonado-Quiles G, Alam AN, Muraduzzaman AKM, Akram A, Conklin L, Doan S, Friedman M, Acosta AM, Hariri S, Fox LM, Tiwari TSP, Flora MS (2021) Investigation of a large diphtheria outbreak and co-circulation of *Corynebacterium pseudodiphtheriticum* among forcibly displaced Myanmar nationals, 2017-2019. J Infect Dis 224: 318-325.

21. Kitamura N, Le TTT, Le LT, Nguyen LD, Dao AT, Hoang TT, Yoshihara K, Iijima M, The TM, Do HM, Le HX, Do HT, Dang AD, Vien MQ, Yoshida LM (2020) Diphtheria outbreaks in schools in central highland districts, Vietnam, 2015-2018. Emerg Infect Dis 26: 596–600.

22. Mohd Khalid MKN, Ahmad N, Hii SYF, Abd Wahab MA, Hashim R, Liow YL (2019) Molecular characterization of *Corynebacterium diphtheriae* isolates in Malaysia between 1981 and 2016. J Med Microbiol 68: 105–110.

23. Arguni E, Karyanti MR, Satari HI, Hadinegoro SR (2021) Diphtheria outbreak in Jakarta and Tangerang, Indonesia: epidemiological and clinical predictor factors for death. PloS One 16: e0246301.

24. Elek SD (1949) The plate virulence test for diphtheria. J Clin Pathol 2: 250-258.

25. Engler KH, Glushkevich T, Mazurova IK, George RC, Efstratiou A (1997) A modified Elek test for detection of toxigenic corynebacteria in the diagnostic laboratory. J Clin Microbiol 35: 495-498.

26. World Health Organization (2013) Global vaccine action 2011-2020. Available: https://apps.who.int/iris/rest/bitstreams/110564/retrieve. Accessed: 03 March 2021

27. Berbers G, van Gageldonk P, Kassteele JV, Wiedermann U, Desombere I, Dalby T, Toubiana J, Tsiodras S, Ferencz IP, Mullan K, Griskevicius A, Kolupajeva T, Vestrheim DF, Palminha P, Popovici O, Wehlin L, Kastrin T, Maďarová L, Campbell H, Ködmön C, Bacci S, Barkoff A-M, He Q, Serosurveillance Study Team (2021) Circulation of pertussis and poor protection against diphtheria among middle-aged adults in 18 European countries. Nat Commun 12: 2871.

**Corresponding author**

Dr Gauthier Delvallez
Head of Medical Biology Laboratory
Institut Pasteur du Cambodge

5 Monivong Boulevard, P.O Box. 983, Phnom Penh, Cambodia
Tel: +85512802978

E-mail: gdelvallez@pasteur-kh.org

**Conflict of interests:** No conflict of interests is declared.