Non-toxigenic Corynebacterium diphtheriae cutaneous infections among two injecting drug-users with HIV/Tuberculosis coinfection from Bucharest, Romania

Dear Editor,

Diphtheria is a vaccine-preventable disease caused by Corynebacterium strains manifested primarily as localized upper respiratory or cutaneous infections and rarely as a systemic infection. Corynebacterium strains are categorized into toxigenic and non-toxigenic based on their ability to produce or not diphtheria toxin. Vulnerable populations for diphtheria are homeless people and alcohol and intravenous drug users (IDUs), clusters of diphtheria being previously described among IDUs [1].

In Romania, the vaccination against diphtheria was implemented in 1960, contributing to the elimination of toxigenic strains and a decline in the circulation of those non-toxigenic. According to the legal framework, all healthcare providers must report immediately any suspected case of diphtheria, and the laboratory any identified C. diphtheriae strain.

We report two patients admitted to the Hospital of Infectious and Tropical Diseases “Dr. Victor Babeș“ from Bucharest, Romania, in November 2023 who presented with skin infections linked to drug-injecting sites (Fig. 1 A and B). Both patients provided written informed consent for data publication. The patients shared similar demographic epidemiological and clinical features as shown in Table 1.

Fig. 1.

Patient 1 Extensive wounds at the right forearm after unsafe drug injections covered by a gelatinous secretion (A); Patient 2 Deep wound at the right inguinal region where he injected drugs and had previously an arterio-venous shunt covered by gelatinous secretion/membrane (B).

Table 1.

Characteristics of patients diagnosed with cutaneous diphtheriae.

	Patient 1	Patient 2
Date of admission	17th November 2023	22nd November 2023
Demographic characteristics
Gender	Male	Male
Age (years)	38	40
Intravenous drug use	For over 20 years (heroin, ethnobotanical drugs)	For over 20 years (heroin, ethnobotanical drugs)
Residence	Bucharest	Homeless in Bucharest
Travel history (last month)	No	No
Contact with migrants	No	No
Diphtheria vaccine history	Declared being vaccinated in childhood (no medical evidence)	UKN
Anti-diphtheria toxin antibodies (protective if > 1 Ul/mL)	0.438 UI/mL	0.066 UI/mL
Medical data
Chronic infections	HIV, hepatitis B ​+ ​D, hepatitis C	HIV, hepatitis C
Medical problems before admission		TB meningitis surgical intervention for an arteriovenous fistula on the right femoral vessels
Concomitant infections	●Pulmonary tuberculosis ●Pneumonia with methicillin-susceptible Staphylococcus ●Left pneumothorax	●TB meningitis ●Pulmonary tuberculosis ●Bronchopneumonia with respiratory failure ●Massive right pneumothorax ●Sepsis with Streptococcus pyogenes ●Clostridioides difficile enterocolitis
CD4 count (cells/ml)	181	336
HIV RNA	14.800 copies/mL	945.000 copies/mL
Germs isolated from ulcer
Corynebacterium diphtheriae	Non-toxigenic strain	Non-toxigenic strain
Streptococcus pyogenes	yes	yes
Staphylococcus aureus	yes

Of the samples collected from the skin lesions and the respiratory tract, the former yielded positive cultures for non-toxigenic C. diphtheriae in both patients with isolates expressing exposure/high dose susceptibility to Penicillin and high susceptibility to macrolides, rifampicin, and linezolid.

Both C. diphtheriae isolates were subjected to whole genome sequencing (WGS) using a NovaSeq 6000 instrument (Illumina San Diego, CA, USA) and the typical Illumina workflow with Nextera XT DNA Library Preparation kit and NovaSeq 6000 SP Reagent kit v1.5 (Illumina San Diego, CA, USA). Sequences were deposited in the European Nucleotide Archive with the accession numbers ERS17475334 (patient 1) and ERS17475335 (patient 2). WGS data were used for species identification (rMLST, https://pubmlst.org/species-id), prediction of virulence factors (VFDB, http://www.mgc.ac.cn/VFs/) and antimicrobial resistance phenotype (NCBI-AMRFinderPlus). Also, in silico multilocus sequencing typing (MLST) and core genome MLST (cgMLST) were peformed by using the SeqSphere ​+ ​tool (Ridom, Munster, Germany) with default options. Of note, cgMLST was performed using the scheme of 1553 loci as well as extended scheme with 601 more loci [2]. Both isolates were confirmed as C. diphtheriae representatives belonging to ST5 lineage, lacked the tox gene and did not harbor any of the antibiotic resistance markers found in AMRFinder Plus database. When compared, they showed 17 and 22 allele differences (ADs) in the cgMLST_1,553 and cgMLST₂₁₅₄, respectively. These results were higher than the default threshold of 14 AD used by the SeqSphere ​+ ​as warning for cluster detection.

Re-emerging infectious diseases (including diphtheria) are a concerning problem due to war, population growth, crowding, and poverty. Because of migration from endemic areas, and also decreased vaccination coverage in some countries, outbreaks of diphtheria might occur.

In this paper, we report two cases of cutaneous diphtheria sharing a common pattern. Both strains were non-toxigenic and determined localized infections. The wounds were linked to injection sites suggesting transmission by drug use. The local infection was polymicrobial as previously described [3]. Secondly, both patients had HIV infection and tuberculosis. The association of tuberculosis and diphteria has been historical described linked to poverty, malnourishment and overcrowding [4]. There are few reported cases of IDU’s with cutaneous diphtheria and HIV [5].

According to SeqSphere ​+ ​analysis of the cgMLST data, the two C. diphtheriae ST5 strains reported in our study were not part of a cluster. However, given that there are more cgMLST schemes available and no consensus threshold for cluster definition for this species, further WGS data analysis and epidemiological information are needed before providing a definitive answer regarding the relatedness of the cases.

In conclusion, we described the cases of two IDUs patients with cutaneous diphtheria and severe comorbidities and by providing the first WGS data for pathogen profiling raised concern about the emergence of a cryptic reservoir of non-toxigenic C. diphtheriae among a risk group in Bucharest. Thus, in order to prevent the reemergence of diphtheria, besides ensuring an adequate level of protection against the effects of exotoxin by vaccination and a proper case management with cross-collaboration we recommend the establishment of an enhanced laboratory-based approach to elucidate the local population structure and transmission of C. diphtheriae in our region.

CRediT authorship contribution statement

Simin Aysel Florescu: Conceptualization, Writing – original draft, Writing – review & editing. Irina C. Ianache: Data curation, Investigation, Writing – review & editing. Claudia Chirila: Data curation, Investigation, Writing – review & editing. Bianca Georgiana Enciu: Investigation, Methodology, Writing – review & editing. Mihaela Oprea: Investigation, Validation, Writing – review & editing. Sorin Dinu: Investigation, Validation, Writing – review & editing. Anca Sirbu: Investigation, Writing – review & editing. Maria Nica: Investigation, Methodology, Writing – original draft, Writing – review & editing. Corneliu Petru Popescu: Writing – original draft, Writing – review & editing. Cristiana Oprea: Data curation, Writing – review & editing. Luminița Ene: Conceptualization, Data curation, Writing – original draft, Writing – review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Corneliu Petru Popescu, corresponding author, on behalf of the authors of paper: “Non-toxigenic Corynebacterium diphtheriae cutaneous infections among two injecting drug-users with HIV/Tuberculosis coinfection from Bucharest, Romania”

Handling Editor: Patricia Schlagenhauf