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Background: Diphtheria, caused by Corynebacterium diphtheriae, reemerges in Europe since 2022. Genomic sequencing can inform on transmission routes and genotypes of concern, but currently, no standard approach exists to detect clinically important genomic features and to interpret emergence in the global C. diphtheriae population framework. Methods: We developed the bioinformatics pipeline DIPHTOSCAN (available at https://gitlab.pasteur.fr/BEBP/diphtoscan) to extract from genomes of Corynebacteria of the diphtheriae species complex, medically relevant features including tox gene presence and disruption. We analyzed 101 human C. diphtheriae isolates collected in 2022 in metropolitan and overseas France (France-2022). To define the population background of this emergence, we sequenced 379 additional isolates (mainly from France, 2018-2021) and collated 870 publicly-available genomes. Results: The France-2022 isolates comprised 45 tox-positive (44 toxigenic) isolates, mostly imported, belonging to 10 sublineages (

Antimicrobial therapy is important for case management of diphtheria, but knowledge on the emergence of multidrug-resistance in Corynebacterium diphtheriae is scarce. We report on the genomic features of two multidrug-resistant toxigenic isolates sampled from wounds in France 3 years apart. Both isolates were resistant to spiramycin, clindamycin, tetracycline, kanamycin and trimethoprim-sulfamethoxazole. Genes ermX, cmx, aph(3’)-Ib, aph(6)-Id, aph(3’)-Ic, aadA1, dfrA15, sul1, cmlA, cmlR and tet(33) were clustered in two genomic islands, one consisting of two transposons and one integron, the other being flanked by two IS6100 insertion sequences. One isolate additionally presented mutations in gyrA and rpoB and was resistant to ciprofloxacin and rifampicin. Both isolates belonged to sublineage 453 (SL453), together with 25 isolates from 11 other countries (https://bigsdb.pasteur.fr/diphtheria/). SL453 is a cosmopolitan toxigenic sublineage of C. diphtheriae, a subset of which acquired multidrug resistance. Even though penicillin, amoxicillin and erythromycin, recommended as the first line in the treatment of diphtheria, remain active, surveillance of diphtheria should consider the risk of dissemination of multidrug-resistant strains and their genetic elements.

ObjectivesCorynebacteria of thediphtheriaecomplex (Cdc) can cause diphtheria in humans and have been reported from companion animals. We aimed to describe animal infection cases caused byCdcisolates.Methods18 308 animals (dogs, cats, horses and small mammals) with rhinitis, dermatitis, non-healing wounds and otitis were sampled in metropolitan France (August 2019 to August 2021). Data on symptoms, age, breed, and the administrative region of origin were collected. Cultured bacteria were analyzed fortoxgene presence, for production of the diphtheria toxin, for antimicrobial susceptibility, and genotyped by multilocus sequence typing.ResultsC. ulceranswas identified in 51 cases, 24 of which were toxigenic. Rhinitis was the most frequent presentation (18/51). Eleven cases (6 cats, 4 dogs, 1 rat) were mono-infections. Large breed dogs, especially German Shepherds (9 of 28 dogs; p < 0.00001) were overrepresented.C. ulceransisolates were susceptible to all tested antibiotics.tox-positiveC. diphtheriaewas identified in 2 horses. Last, 11 infections cases (9 dogs, 2 cats; mostly chronic otitis, and 2 sores) hadtox-negativeC. rouxii, a recently defined species.C. rouxiiandC. diphtheriaeisolates were susceptible to most antibiotics tested, and almost all of these infections were polymicrobial.ConclusionsMonoinfections withC. ulceranspoint towards a primary pathogenic potential to animals.C. ulceransrepresents an important zoonotic risk, andC. rouxiimay represent a novel zoonotic agent. This case series provides novel clinical and microbiological data onCdcinfections, and underlines the need for management of animals and their human contacts.ImportanceWe report on the occurrence, clinical and microbiological characteristics of infections caused by members of the Corynebacteria of thediphtheriaecomplex (Cdc) in companion animals. This is the first study based on the systematic analysis of a very large animal cohort (18 308 samples), which provides data on the frequency of Cdc isolates in various types of clinical samples from animals. Awareness of this zoonotic bacterial group remains low among veterinarians and veterinary laboratories, among which it is often considered a commensal bacteria of animals. We suggest that in case of Cdc detection in animals, the veterinary laboratories should be encouraged to send the samples to a reference laboratory for analysis of the presence of thetoxgene. This work is relevant to the development of guidelines in case of Cdc infections in animals, and underlines their public health relevance given the zoonotic transmission risk.

International audience

ObjectivesAn increasing number of isolations ofCorynebacterium diphtheriaehas been observed in recent years in the archipelago of New Caledonia. We aimed to analyze the clinical and microbiological features of samples withC. diphtheriae.MethodsAllC. diphtheriaeisolates identified in New Caledonia from May 2015 to May 2019 were included. For each case, a retrospective consultation of the patient files was conducted. Antimicrobial susceptibility phenotypes,toxgene and diphtheria toxin expression, biovar and the genomic sequence were determined. Core genome multilocus sequence typing (cgMLST), 7-gene MLST and search of genes of interest were performed from genomic assemblies.Results58 isolates were included, with a median age of patients of 28 years (range: 9 days to 78 years). Cutaneous origin accounted for 51 of 58 (87.9%) isolates, andC. diphtheriaewas associated withStaphylococcus aureusand/orStreptococcus pyogenesin three quarters of cases. Half of cases came either from the main city Noumea (24%, 14/58) or from the sparsely populated island of Lifou (26%, 15/58). Six tox-positive isolates were identified, associated with recent travel to Vanuatu; 5 of these cases were linked and cgMLST confirmed recent transmission. Two cases of endocarditis in young female patients with a history of rheumatic fever involved tox-negative isolates. The 58 isolates were mostly susceptible to commonly used antibiotics. In particular, no isolate was resistant to the first-line molecules amoxicillin or erythromycin. Resistance to tetracycline was found in a genomic cluster of 17 (29%) isolates, 16 of which carried thetetOgene. There were 13 cgMLST sublineages, most of which were also observed in the neighboring country Australia.ConclusionsCutaneous infections may harbor non-toxigenicC. diphtheriaeisolates, which circulate largely silently in non-specific wounds. The possible introduction of tox-positive strains from a neighboring island illustrates that diphtheria surveillance should be maintained in New Caledonia, and that immunization in neighboring islands must be improved. Genomic sequencing uncovers how genotypes circulate locally and across neighboring countries.

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.

Corynebacterium diphtheriaeis highly transmissible and can cause large diphtheria outbreaks where vaccination coverage is insufficient. Sporadic cases or small clusters are observed in high-vaccination settings. The phylogeography and short timescale evolution ofC. diphtheriaeare not well understood, in part due to a lack of harmonized analytical approaches of genomic surveillance and strain tracking. We combined 1,305 genes with highly reproducible allele calls into a core genome multilocus sequence typing (cgMLST) scheme. We analyzed cgMLST gene diversity among 602 isolates from sporadic clinical cases, small clusters, or large outbreaks. We defined sublineages based on the phylogenetic structure withinC. diphtheriaeand strains based on the highest number of cgMLST mismatches within documented outbreaks. We performed time-scaled phylogenetic analyses of major sublineages. The cgMLST scheme showed high allele call rate inC. diphtheriaeand the closely related speciesC. belfantiiandC. rouxii. We demonstrate its utility to delineate epidemiological case clusters and outbreaks using a 25 mismatches threshold and reveal a number of cryptic transmission chains, most of which are geographically restricted to one or a few adjacent countries. Subcultures of the vaccine strain PW8 differed by up to 20 cgMLST mismatches. Phylogenetic analyses revealed a short-timescale evolutionary gain or loss of the diphtheria toxin and biovar-associated genes. We devised a genomic taxonomy of strains and deeper sublineages (defined using a 500-cgMLST-mismatch threshold), currently comprising 151 sublineages, only a few of which are geographically widespread based on current sampling. The cgMLST genotyping tool and nomenclature was made publicly accessible (https://bigsdb.pasteur.fr/diphtheria). Standardized genome-scale strain genotyping will help tracing transmission and geographic spread ofC. diphtheriae. The unified genomic taxonomy ofC. diphtheriaestrains provides a common language for studies of ecology, evolution, and virulence heterogeneity amongC. diphtheriaesublineages.

BackgroundCorynebacterium diphtheriaeis highly transmissible and can cause large diphtheria outbreaks where vaccination coverage is insufficient. Sporadic cases or small clusters are observed in high-vaccination settings. The phylogeography and short timescale evolution ofC. diphtheriaeare not well understood, in part due to a lack of harmonized analytical approaches of genomic surveillance and strain tracking.MethodsWe combined 1,305 genes with highly reproducible allele calls into a core genome multilocus sequence typing (cgMLST) scheme. We analyzed cgMLST genes diversity among 602 isolates from sporadic clinical cases, small clusters or large outbreaks. We defined sublineages based on the phylogenetic structure withinC. diphtheriaeand strains based on the highest number of cgMLST mismatches within documented outbreaks. We performed time-scaled phylogenetic analyses of major sublineages.ResultsThe cgMLST scheme showed high allele call rate inC. diphtheriaeand the closely related speciesC. belfantiiandC. rouxii. We demonstrate its utility to delineate epidemiological case clusters and outbreaks using a 25 mismatches threshold, and reveal a number of cryptic transmission chains, most of which are geographically restricted to one or a few adjacent countries. Subcultures of the vaccine strain PW8 differed by up to 20 cgMLST mismatches. Phylogenetic analyses revealed short timescale evolutionary gain or loss of the diphtheria toxin and biovar-associated genes. We devised a genomic taxonomy of strains and deeper sublineages (defined using a 500 cgMLST mismatches threshold), currently comprising 151 sublineages, only a few of which are geographically widespread based on current sampling. The cgMLST genotyping tool and nomenclature was made publicly accessible athttps://bigsdb.pasteur.fr/diphtheria.ConclusionsStandardized genome-scale strain genotyping will help tracing transmission and geographic spread ofC. diphtheriae. The unified genomic taxonomy ofC. diphtheriaestrains provides a common language for studies into the ecology, evolution and virulence heterogeneity amongC. diphtheriaesublineages.

Diphtheria is an infection that has been unreported for more than two decades in Mahajanga. A child, aged 4, presented with a pseudomembranous pharyngitis was associated with a dysphagia. He was from a rural municipality of Ambato Boeny at Mahajanga province and was admitted to the Pediatric Unit of the University Hospital Center. The child was not immunized against diphtheria. A throat swab was performed and cultured, from which Corynebacterium diphtheriae was identified. The strain, of biovar Mitis, was confirmed as diphtheria toxin (DT)-gene positive and produced DT (Elek test). Unfortunately, the child developed cardiac and neurological complications and died of respiratory and heart failure.

BackgroundAn outbreak of diphtheria, declared in Yemen in October 2017, is still ongoing. Methods. Probable cases were recorded through an electronic diseases early warning system. Microbiological culture, genomic sequencing, antimicrobial susceptibility and toxin production testing were performed.MethodsProbable cases were recorded through an electronic diseases early warning system. Microbiological culture, genomic sequencing, antimicrobial susceptibility and toxin production testing were performed.FindingsThe Yemen diphtheria outbreak developed in three epidemic waves, which affected nearly all governorates (provinces) of Yemen, with 5701 probable cases and 330 deaths (October 2017 - April 2020). The median age of patients was 12 years (range, 0.17-80). Virtually all outbreak isolates (40 of 43 tested ones) produced the diphtheria toxin. We observed low level of antimicrobial resistance to penicillin. We identified six separate Corynebacterium diphtheriae phylogenetic sublineages, three of which are genetically related to isolates from Saudi Arabia and Somalia. The predominant sublineage was resistant to trimethoprim and was associated with unique genomic features, more frequent neck swelling (p=0.002) and a younger age of patients (p=0.06). Its evolutionary rate was estimated at 1.67 × 10−6 substitutions per site year-1, placing its most recent common ancestor in 2015, and indicating silent circulation of C. diphtheriae in Yemen earlier than outbreak declaration.InterpretationWe disclose clinical, epidemiological and microbiological characteristics of one of the largest contemporary diphtheria outbreaks and demonstrate clinically relevant heterogeneity of C. diphtheriae isolates, underlining the need for laboratory capacity and real-time microbiological analyses to inform prevention, treatment and control of diphtheria.FundingThis work was supported by institutional funding from the National Centre of the Public Health Laboratories (Sanaa, Yemen) and Institut Pasteur (Paris, France) and by the French Government Investissement d ‘Avenir Program.

Corynebacterium diphtheriae, the agent of diphtheria, is a genetically diverse bacterial species. Although antimicrobial resistance has emerged against several drugs including first-line penicillin, the genomic determinants and population dynamics of resistance are largely unknown for this neglected human pathogen.Here we analyzed the associations of antimicrobial susceptibility phenotypes, diphtheria toxin production and genomic features inC. diphtheriae.We used 247 strains collected over several decades in multiple world regions, including the 163 clinical isolates collected prospectively from 2008 to 2017 in France mainland and overseas territories.Phylogenetic analysis revealed multiple deep-branching sublineages, grouped into a Mitis lineage strongly associated with diphtheria toxin production, and atox-negative Gravis lineage with fewtox+exceptions including the 1990s ex-Soviet Union outbreak strain. The distribution of susceptibility phenotypes allowed proposing ecological cutoffs for most of the 19 agents tested, thereby defining acquired antimicrobial resistance. Penicillin resistance was found in 17.2% of prospective isolates. Four isolates were multidrug resistant (>8 agents), including to penicillin and macrolides. Homologous recombination was frequent (r/m = 5) and horizontal gene transfer contributed to the emergence of antimicrobial resistance in multiple sublineages. Genome-wide association mapping uncovered genetic factors of resistance, including an accessory penicillin-binding protein (PBP2m) located in diverse genomic contexts. Genepbp2mis widespread in otherCorynebacteriumspecies and its expression inC. glutamicumdemonstrated its effect against several beta-lactams. A novel 73-kbC. diphtheriaemulti-resistance plasmid was discovered.This work uncovers the dynamics of antimicrobial resistance inC. diphtheriaein the context of phylogenetic structure, biovar and diphtheria toxin production, and provides a blueprint to analyze re-emerging diphtheria.

BackgroundCorynebacterium diphtheriae, the agent of diphtheria, is a genetically diverse bacterial species. Although antimicrobial resistance has emerged against several drugs including first-line penicillin, the genomic determinants and population dynamics of resistance are largely unknown for this neglected human pathogen.MethodsHere, we analyzed the associations of antimicrobial susceptibility phenotypes, diphtheria toxin production, and genomic features inC. diphtheriae. We used 247 strains collected over several decades in multiple world regions, including the 163 clinical isolates collected prospectively from 2008 to 2017 in France mainland and overseas territories.ResultsPhylogenetic analysis revealed multiple deep-branching sublineages, grouped into a Mitis lineage strongly associated with diphtheria toxin production and a largely toxin gene-negative Gravis lineage with few toxin-producing isolates including the 1990s ex-Soviet Union outbreak strain. The distribution of susceptibility phenotypes allowed proposing ecological cutoffs for most of the 19 agents tested, thereby defining acquired antimicrobial resistance. Penicillin resistance was found in 17.2% of prospective isolates. Seventeen (10.4%) prospective isolates were multidrug-resistant (≥ 3 antimicrobial categories), including four isolates resistant to penicillin and macrolides. Homologous recombination was frequent (r/m = 5), and horizontal gene transfer contributed to the emergence of antimicrobial resistance in multiple sublineages. Genome-wide association mapping uncovered genetic factors of resistance, including an accessory penicillin-binding protein (PBP2m) located in diverse genomic contexts. Genepbp2mis widespread in otherCorynebacteriumspecies, and its expression inC. glutamicumdemonstrated its effect against several beta-lactams. A novel 73-kbC. diphtheriaemultiresistance plasmid was discovered.ConclusionsThis work uncovers the dynamics of antimicrobial resistance inC. diphtheriaein the context of phylogenetic structure, biovar, and diphtheria toxin production and provides a blueprint to analyze re-emerging diphtheria.

A group of six clinical isolates previously identified asCorynebacterium diphtheriaebiovar Belfanti, isolated from human cutaneous or peritoneum infections and from one dog, were characterized by genomic sequencing, biochemical analysis and mass spectrometry (MALDI-TOF MS). The six isolates were negative for the diphtheria toxin gene. Phylogenetic analyses showed that the six isolates (including FRC0190T) are clearly demarcated fromC. diphtheriae, C. belfantii, C. ulceransandC. pseudotuberculosis. The average nucleotide identity of FRC0190TwithC. diphtheriaeNCTC 11397Twas 92.6%, and was 91.8% withC. belfantiiFRC0043T.C. diphtheriaesubsp.lausannensestrain CHUV2995Tappeared to be a later heterotypic synonym ofC. belfantii(ANI, 99.3%). Phenotyping data revealed an atypical negative or heterogeneous intermediate maltose fermentation reaction for the six isolates. MALDI-TOF MS differentiated the new group from the otherCorynebacteriumtaxa by the presence of specific spectral peaks.rpoBsequences showed identity to atypical, maltose-negativeC. diphtheriaebiovar Belfanti isolates previously described from two cats in the USA. We propose the nameCorynebacterium rouxiisp. nov. for the novel group, with FRC0190T(= CIP 111752T= DSM 110354T) as type strain.

International audience; Introduction. Diphtheria is caused by toxigenic strains of Corynebacterium diphtheriae, Corynebacterium ulcerans and Corynebacterium pseudotuberculosis. For diagnostic purposes, species identification and detection of toxigenic strains (diphtheria toxin (tox)-positive strains) is typically performed using end-point PCR. A faster quadruplex real-time PCR (qPCR) was recently developed (De Zoysa et al. J.Med.Microbiol. 2016;65(12):1521-1527).Aims. We aimed to improve the quadruplex method by adding a 16S rRNA gene target as an internal processing control, providing confirmation of the presence of bacterial DNA in the assays, thus avoiding the possibility of false-negative reporting.Methodology. Universal 16S rRNA gene primers and a probe were defined. The novel method was tested using 36 bacterial isolates and 17 clinical samples. Experimental robustness to temperature and reagent concentration variations was assessed.Results. The method allows detection of the tox gene and distinguishing C. diphtheriae (including the newly described species Corynebacterium belfantii) from C. ulcerans and C. pseudotuberculosis. Complete diagnostic specificity, sensitivity and experimental robustness were demonstrated. The lower limit of detection for C. diphtheriae, C. ulcerans and tox targets was 1.86 genome copies per 5 µl reaction volume. The method was successfully used on two distinct qPCR technologies (LightCycler 480, Roche Diagnostics and Rotor-Gene Q, Qiagen) and in two laboratories (Institut Pasteur, Paris, France and Public Health England - National Infection Service, London, UK).Conclusion. This work describes validation of the improved qPCR quadruplex method and supports its implementation for the biological diagnosis of diphtheria.

International audience; Introduction: Diphtheria due to Corynebacteriumdiphtheriae (C. diphtheriae) has become rare in developed countries. In France only 10 cases of toxigenic diphtheria have been reported since 1989, in all cases causing pharyngitis and all emanating from endemic countries with exception of one contact case. We report herein 13 cases with cutaneous diphtheria, in 5 of which diphtheria toxin was produced, and all imported into France between 2015 and 2018.Observations: Thirteen patients aged 4 to 77 years presented painful and rapidly progressive round ulcerations of the legs, that were superficial and in some cases purulent, with an erythematous-purple border covered with greyish membrane. Bacteriological sampling of ulcers revealed the presence of C. diphtheriae. Only 6 patients had been properly immunized over the preceding 5 years.Discussion: These cases underline the resurgence of cutaneous diphtheria and the circulation of toxigenic strains in France following importation from Indian Ocean countries. This may constitute an important reservoir for ongoing transmission of the disease. Re-emergence of this pathogen stems from the current migratory flow and decreased adult booster coverage.Conclusion: Cutaneous diphtheria should be considered in cases of rapidly developing painful skin ulcers with greyish membrane, especially among patients returning from endemic areas, regardless of their vaccination status. The clinician should order specific screening for C. diphtheriae from the bacteriologist, since with routine swabbing Corynebacteriaceae may be reported simply as normal skin flora. Vaccination protects against toxigenic manifestations but not against actual bacterial infection. Early recognition and treatment of cutaneous diphtheria and up-to-date vaccination are mandatory to avoid further transmission and spread of both cutaneous and pharyngeal diphtheria.

International audience; Cystic fibrosis (CF) patients are commonly colonized by bacterial pathogens, which can induce persistent lung inflammation and may contribute to clinical deterioration. Colonization of CF patients and cross-transmission by Corynebacterium diphtheriae have not been reported so far. The aim of this article was to investigate the possibility of a cross-transmission of C. diphtheriae biovar Belfanti between four patients of a CF center. C. diphtheriae biovar Belfanti (now formally called C. belfantii) isolates were collected from four patients in a single CF care center over a period of 6 years and analyzed by microbiological methods and whole-genome sequencing. Epidemiological links among patients were investigated. Ten isolates were collected from 4 patients. Whole-genome sequencing of one isolate from each patient showed that a single strain was shared among them. In addition, one patient was found to have the same strain in two consecutive samplings performed 9 months apart. The strain was nontoxigenic and was susceptible to most antimicrobial agents. Ciprofloxacin resistance was observed in one patient. The idea of transmission of the strain among patients was supported by the occurrence of same-day visits to the CF center. This study demonstrated colonization of CF patients by C. diphtheriae biovar Belfanti (C. belfantii), and the data suggest persistence and transmission of a unique strain during at least 6 years in a single CF patient care center.

Diphtheria is caused by toxigenic strains ofCorynebacterium diphtheriae, Corynebacterium ulceransandCorynebacterium pseudotuberculosis. For diagnostic purposes, species identification and detection of toxigenic strains (diphtheria toxin (tox)-positive strains) is typically performed using end-point PCR. A faster quadruplex real-time PCR (qPCR) was recently developed (De Zoysaet al. J Med Microbiol. 2016 65(12):1521-1527). Here, we present an improvement of the quadruplex method, in which a 16S rRNA gene target was added as an internal processing control, providing confirmation of the presence of bacterial DNA in the assays. This improved qPCR method was validated using 36 bacterial isolates and 16 clinical samples. The method allows detection of thetoxgene and distinguishingC. diphtheriae(including the newly described speciesC. belfantii) fromC. ulceransandC. pseudotuberculosis. Complete diagnostic specificity, sensitivity and experimental robustness of the method to temperature and reagent concentration variations were demonstrated. The lower limit of detection forC. diphtheriae, C. ulceransandtoxtargets was 1.86 genome copies per 5 μL reaction volume. Finally, the method was successfully used on two distinct qPCR technologies (LightCycler 480, Roche Diagnostics and Rotor-Gene Q, Qiagen) and in two laboratories (Institut Pasteur, Paris, France and Public Health England – National Infection Service, London, UK). This work describes validation of the improved qPCR quadruplex method and supports its implementation for the biological diagnosis of diphtheria.

Cystic fibrosis (CF) patients are commonly colonized by bacterial pathogens, which can induce persistent lung inflammation and may contribute to clinical deterioration. Colonization of CF patients and cross-transmission by Corynebacterium diphtheriae has not been reported so far. The aim of this article was to investigate the possibility of a cross transmission of C. diphtheriae biovar Belfanti between four patients of a CF center. C. diphtheriae biovar belfanti (now formally called C. belfantii) isolates were collected from four patients in a single CF care center over a 6 years period and analyzed by microbiological methods and whole genome sequencing. Epidemiological links among patients were investigated. Ten isolates were collected from 4 patients. Whole genome sequencing of one isolate from each patient showed that a single strain was shared among them. In addition, one patient had the same strain on two consecutive samplings nine months apart. The strain was non-toxigenic and was susceptible to most antimicrobial agents. Ciprofloxacin resistance was observed in one patient. Transmission of the strain among patients was supported by the occurrence of same-day visits to the CF center. This study demonstrates colonization of CF patients by C. diphtheriae biovar Belfanti (C. belfantii) and shows persistence and transmission of a unique strain during at least six years in a single CF patient care center.Previous meeting presentationsThe information in this work was not previously presented in any meeting.Accession numbersThe genomic sequence data generated in this work were submitted to the European Nucleotide Archive and are available from the International Nucleotide Sequence Database Collaboration (NCBI/ENA/DDBJ) databases under project accession number PRJEB28372 and run data accession numbers ERR2757916 to ERR2757921.

Cystic fibrosis (CF) patients are commonly colonized by bacterial pathogens, which can induce persistent lung inflammation and may contribute to clinical deterioration. Colonization of CF patients and cross-transmission by Corynebacterium diphtheriae have not been reported so far. The aim of this article was to investigate the possibility of a cross-transmission of C. diphtheriae biovar Belfanti between four patients of a CF center. C. diphtheriae biovar Belfanti (now formally called C. belfantii) isolates were collected from four patients in a single CF care center over a period of 6 years and analyzed by microbiological methods and whole-genome sequencing. Epidemiological links among patients were investigated. Ten isolates were collected from 4 patients. Whole-genome sequencing of one isolate from each patient showed that a single strain was shared among them. In addition, one patient was found to have the same strain in two consecutive samplings performed 9 months apart. The strain was nontoxigenic and was susceptible to most antimicrobial agents. Ciprofloxacin resistance was observed in one patient. The idea of transmission of the strain among patients was supported by the occurrence of same-day visits to the CF center. This study demonstrated colonization of CF patients by C. diphtheriae biovar Belfanti (C. belfantii), and the data suggest persistence and transmission of a unique strain during at least 6 years in a single CF patient care center.

International audience; BACKGROUND: Diphtheria is re-emerging in Europe. A total of 36 cases were reported in Europe in 2015 versus 53 cases between 2000 and 2009.PATIENTS: We report two cases of Corynebacterium diphtheriae infection in a French hospital in 2016: a cutaneous infection with negative toxin testing in a French traveller, and a respiratory diphtheria carriage with positive toxin testing in an Afghan refugee diagnosed with pulmonary tuberculosis. The vaccination history of the Afghan patient could not be retrieved.; Introduction : La diphtérie est une maladie réémergente en Europe. En 2015, 36 cas ont été rapportés contre 53 cas de 2000 à 2009.Patients : Nous rapportons deux cas d’infection à Corynebacterium diphtheriae survenus en 2016 dans un hôpital français : une infection cutanée avec recherche de toxine négative chez un voyageur et un portage respiratoire avec recherche de toxine positive chez un réfugié afghan atteint d’une tuberculose pulmonaire. L’histoire vaccinale était inconnue pour le patient afghan.

International audience; Clinical isolates belonging to Corynebacterium diphtheriae biovar Belfanti were characterized by genomic sequencing and biochemical and chemotaxonomic analyses. Phylogenetic analyses indicated that biovar Belfanti represents a branch that is clearly demarcated from C. diphtheriae strains of biovars Mitis and Gravis. The average nucleotide identity of isolates of biovar Belfanti with C. diphtheriae type strain NCTC 11397T (biovar Gravis) was 94.85 %. The inability to reduce nitrate differentiated biovar Belfanti from other strains of C. diphtheriae. On the basis of these results, we propose the name Corynebacterium belfantii sp. nov. for the group of strains previously considered as C. diphtheriaebiovar Belfanti. The type strain of C. belfantii is FRC0043T (=CIP 111412T=DSM 105776T). Strains of C. belfantii were isolated mostly from human respiratory samples.

Corynebacterium diphtheriae can cause various infections such as diphtheria, wound infections, septic arthritis, bacteraemia and endocarditis. Different virulence properties of the isolates might be related to different virulence factors expressed by the isolates. The objective of this study was to explore whether whole cell protein profiling might be useful in prediction of pathogenic properties of C. diphtheriae isolates.C. disphtheriae isolates collected from diphtheria, invasive and local infections and from asymptomatic carriers in Poland, France, New Caledonia and Canada in 1950-2014 were investigated using whole cell protein profile analysis.All the examined isolates were divided into two clades: A and B with similarity about 47%, but clade B was represented by only one isolate. The clade A was divided in two subclades A.I NS .II with similarity 53,2% and then into four groups: A.Ia, A.Ib, A.Ic and A.Id. The comparative analysis did not distinguish clearly toxigenic and nontoxigenic isolates as well as invasive and noninvasive isolates.Whole cell protein profile analysis of C. diphtheria exhibits good concordance with other genotyping methods but this method is not able to distinguish clearly invasive from non-invasive isolates.

Corynebacterium ulcerans is a bacterium able to infect humans by inducing a disease close to diphtheria. We describe the case of a 83-year-old patient hospitalized as a matter of urgency in intensive care for which C. ulcerans was isolated in pure culture in its bronchial samples. Even if the isolate was not secreting toxin in vitro, it possesses the tox gene which motivated the use of specific antitoxin serum. After two months of intensive care the patient went out of the service. It is about a remarkable case of clinicobiologic collaboration.

International audience; Epidemiology of diphtheria in the southwestern Indian Ocean is poorly documented. We analyzed 14 cases of infection with toxigenic Corynebacterium diphtheriae reported during 2007–2015 in Mayotte, a French department located in this region. Local control of diphtheria is needed to minimize the risk for importation of the bacterium into disease-free areas.

The species Corynebacterium renale, Corynebacterium pilosum, and Corynebacterium cystitidis were initially thought to be the same species C. renale, but with different immunological types. These bacteria are the causative agent of cystitis, urethritis and pyelonephritis and are found usually as constituents of the normal flora in the lower urogenital tract of cattle. Therefore, we present the draft genome sequences of two pathogenic Corynebacterium species: C. renale CIP 52.96 and C. pilosum CIP 103422. The genome sequences of these species have 2,322,762 bp with 2,218 protein encoding genes and 2,548,014 bp with 2,428 protein encoding genes, respectively. These genomes can help clarify the virulence mechanisms of these unknown bacteria and enable the development of more effective methods for control.

Cutaneous diphtheria is uncommon in Europe. In this study, we report a case of imported cutaneous infection due to a non-toxigenic but tox gene-bearing (NTTB) strain of Corynebacterium diphtheriae. The NTTB strains are recognized as emerging pathogens across Europe, and physicians and bacteriologists should be aware of the circulation of these strains.

UNLABELLED Selection of appropriate typing method depends on a number of factors, including the scale of the investigation, the rapidity required of the results and the financial and technical resources available. Several typing methods have been applied to Corynebacterium diphtheriae genotyping, but most are laborious and time-consuming or require expensive equipment. We report an evaluation of the utility of the PCR melting profile technique for simple and easy-to-perform genotyping of C. diphtheriae. We compared the method with ribotyping-the 'gold standard' for C. diphtheriae typing-and PFGE, MLST, AFLP, RAPD and spoligotyping. SIGNIFICANCE AND IMPACT OF THE STUDY Occurrence of Corynebacterium diphtheriae infections-in the form of diphtheria in endemic countries and in the form of invasive infections in countries with high antidiphtheria vaccination coverage-indicates the need for maintenance of ability to genotype this pathogen by laboratories. Application of an appropriate typing method is essential not only in outbreak investigations for understanding and predicting epidemics but also in monitoring of the evolution and spread of epidemic clones of C. diphtheriae. The PCR melting profile method presented in the study is a good alternative for C. diphtheriae typing.

Cutaneous diphtheria is uncommon in Europe. In this study, we report a case of imported cutaneous infection due to a non-toxigenic but tox gene-bearing (NTTB) strain of Corynebacterium diphtheriae. The NTTB strains are recognized as emerging pathogens across Europe, and physicians and bacteriologists should be aware of the circulation of these strains.

Corynebacterium diphtheriae is the etiological agent of diphtheria, a potential fatal disease caused by a corynephage toxin. The expression of this diphtheria toxin is controlled via an iron-dependent repressor with various functions (DtxR). Some mutations in the dtxR gene are associated with diminished activity or even with total loss of DtxR function. We conducted a molecular study to characterize the dtxR alleles harbored by 34 isolates of C. diphtheriae recovered from Romanian patients between 1961 and 2007. Three of the seven alleles identified in this study have not previously been described. Two new DtxR types were identified, one of which has an unusual polypeptide length. All the new DtxR types were found in toxigenic isolates, suggesting that they effectively regulate the expression of diphtheria toxin. Furthermore, one of the new DtxR identified was also found in a non-toxigenic isolate, making it a potential source of toxigenic isolates after lysogenic conversion.

Autochtonous toxigenic Corynebacterium diphtheriae have disappeared in mainland France, but non-toxigenic C. diphtheriae are still circulating. Using phenotypic and molecular tools, we retrospectively characterized 103 non-toxigenic C. diphtheriae collected in mainland France and highlight several changes. The proportion of C. diphtheriae belfanti increased between 1977 and 2011 and it is the most frequent biotype recovered in recent years. Resistance to ciprofloxacin has increased and most isolates with decreased sensitivity belong to the belfanti biotype. Using multilocus sequence typing, we demonstrate that French isolates are distributed in a large number of sequence types and identify three distinct lineages. C. diphtheriae mitis and gravis form lineage I while C. diphtheriae belfanti forms lineages II and III. Almost all isolates of lineage II are part of a unique clonal complex or are very close to it. Most French isolates have a dtxR sequence homologous to that of toxigenic isolates, suggesting that if lyzogenised by a corynephage, they can express diphtheria toxin.

Corynebacterium ulcerans is an emergent pathogen infecting wild and domesticated animals worldwide that may serve as reservoirs for zoonotic infections. In this study, we present the draft genome of C. ulcerans strain 03-8664. The draft genome has 2,428,683 bp, 2,262 coding sequences, and 12 rRNA genes.

Corynebacterium ulcerans is a pathogenic bacterium infecting wild and domesticated animals; some infection cases in humans have increased throughout the world. The current study describes the draft genome of strain 04-3911, isolated from humans. The draft genome has 2,492,680 bp, 2,143 coding sequences, 12 rRNA genes, and 50 tRNA genes.

Matrix-associated laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is a rapid and simple microbial identification method. Previous reports using the Biotyper system suggested that this technique requires a preliminary extraction step to identify Gram-positive rods (GPRs), a technical issue that may limit the routine use of this technique to identify pathogenic GPRs in the clinical setting. We tested the accuracy of the MALDI-TOF MS Andromas strategy to identify a set of 659 GPR isolates representing 16 bacterial genera and 72 species by the direct colony method. This bacterial collection included 40 C. diphtheriae , 13 C. pseudotuberculosis , 19 C. ulcerans , and 270 other Corynebacterium isolates, 32 L. monocytogenes and 24 other Listeria isolates, 46 Nocardia , 75 Actinomyces , 18 Actinobaculum , 11 Propionibacterium acnes , 18 Propionibacterium avidum , 30 Lactobacillus , 21 Bacillus , 2 Rhodococcus equi , 2 Erysipelothrix rhusiopathiae , and 38 other GPR isolates, all identified by reference techniques. Totals of 98.5% and 1.2% of non- Listeria GPR isolates were identified to the species or genus level, respectively. Except for L. grayi isolates that were identified to the species level, all other Listeria isolates were identified to the genus level because of highly similar spectra. These data demonstrate that rapid identification of pathogenic GPRs can be obtained without an extraction step by MALDI-TOF mass spectrometry.

International audience; The potent IFN-gamma inducing fusion antigen Ag8513-ESAT-6 (85136) is a lead subunit candidate to improve current vaccination against Mycobacterium tuberculosis (Mtb). The recombinant M. bovis BCG strain Myc3504 was constructed to secrete 85B6. It was based on commercial BCG strain Moreau Rio de Janeiro (BCG(MoWT)) which remains available for human oral administration. Myc 3504 induced higher levels of 85B6-specific IFN-gamma circulating T-cells as compared to BCG(MowT). A novel needle-free mucosal immunization regimen combining oral prime with Myc3504 or BCG(MoWT) With intranasal boost with LTK-63-adjuvanted 85136 was compared to subcutaneous prime-boost immunization. Strikingly whereas parenteral immunization induced sustained levels of 85B6-specific IFN-gamma secretion by circulating T-cells, rnucosal regimens induced barely detectable IFN-gamma. Despite this, mice and guinea pigs immunized with the mucosal regimens were as efficiently protected against aerosol Mtb challenge as parenterally immunized animals. After Mtb challenge, anti-ESAT-6 IFN-gamma responses sharply increased in non-vaccinated mice as a hallmark of infection. Parenterally immunized mice that controlled Mtb infection, displayed anti-ESAT-6 IFN-gamma responses as high as non-immunized infected mice, compromising the possible use of ESAT-6 as a diagnostic tool. Interestingly, in mucosally immunized mice that were equally protected, post-challenge ESAT-6-specific IFN-gamma T-cell response remained low. (c) 2008 Elsevier Ltd. All rights reserved.

International audience; Neutrophils are increasingly thought to modulate dendritic cell (DC) functions. We investigated the role of the neutrophil-DC partnership in the response to Mycobacterium bovis BCG-the vaccine used against tuberculosis. We compared neutrophil-DC crosstalk in humans and mice, searching for functional differences. In both species, neutrophils captured fluorescent BCG-enhanced green fluorescent protein (EGFP) and were more phagocytic than DC. Non-apoptotic BCG-infected neutrophils clustered with immature DC, establishing intimate contacts with dendrites, at which fluorescent intact bacilli were observed. Physical interactions between neutrophils and DC were required for DC activation. Human BCG-infected DC produced interleukin (IL)-10, an inhibitory cytokine, whereas DC exposed to BCG-infected neutrophils produced low to undetectable amounts of the cytokine. Mouse BCG-infected neutrophils induced sustained IL-2 production by DC. Human DC exposed to BCG-infected neutrophils stimulated recall T cell reactivity from vaccinated donors. Mouse DC infected with recombinant ovalburnin (OVA)-producing BCG (rBCG(ova)) elicited proliferation of TCR-OVA-transgenic CD4 and CD8 T cells. Moreover, exposing DC to rBCG(ova)-infected neutrophils enhanced OVA presentation. Thus, in mice and humans, neutrophils help DC to cross-present BCG antigens to T cells. Our results suggest that this "menage a trois" involving neutrophils, DC and T cells plays a major role in the immune response to BCG.

Here, we present the genome sequence of Corynebacterium ulcerans strain FRC11. The genome includes one circular chromosome of 2,442,826 bp (53.35% G+C content), and 2,210 genes were predicted, 2,146 of which are putative protein-coding genes, with 12 rRNAs and 51 tRNAs; 1 pseudogene was also identified.

Objective— Expression of human apolipoprotein (h-apo) A-IV in apoE-deficient (apoE 0 ) mice (h-apoA-IV/E 0 ) reduces susceptibility to atherosclerosis. Chronic infection mimicked by exposure to lipopolysaccharide (LPS) increases the size of atherosclerosis lesions in apoE 0 mice. Thus, we used h-apoA-IV/E 0 mice to determine whether h-apoA-IV plays a protective role after LPS administration. Methods and Results— We injected apoE 0 , h-apoA-IV/E 0 , and C57Bl/6 (wild-type) mice intraperitoneally with either LPS or phosphate-buffered saline (PBS) every week for 10 weeks. Atherosclerotic lesions were significantly smaller in h-apoA-IV/E 0 mice treated with LPS than in their apoE 0 counterparts. The titers of IgG2a and IgG2b autoantibodies to oxidized low-density lipoprotein (LDL) were higher in the LPS-group of h-apoA-IV/E 0 mice than in apoE 0 mice, suggesting that the Th1 response is stronger in the presence of h-apoA-IV. Lymphocytes from the blood, liver, spleen, and thymus of h-apoA-IV/E 0 mice treated with LPS produced less IL-4, INF-γ, and TNF-α proinflammatory cytokines than their apoE 0 counterparts. Furthermore, we demonstrated that recombinant h-apoA-IV blocks the LPS-induced stimulation of monocytes. Conclusions— The expression of h-apoA-IV in apoE 0 mice reduces the susceptibility to atherogenesis and decreases the secretion of proinflammatory cytokines after LPS administration.

International audience; The widely administered Mycobacterium bovis BCG is an attractive live vector for the development of AIDS vaccines. We explored immune responses induced in cynomolgus macaques tor BCG-SIV3, a mixture of three recombinant BCG strains expressing the SIVmac251 nef, gag and env genes. After a single intradermal (ID) inoculation, circulating blood cells from rBCG-SIV3-vaccinated monkeys exhibited CTL responses targeted against the three antigens and interferon-gamma (IFN-gamma) secretion was observed. A rectal or oral boosting dose of rBCG-SIV3 elicited anti-SIV IgAs in the rectum of vaccinated monkeys and increased IFNgamma secretion by circulating blood cells. Despite a good response against the vector, rBCG-SIV3 administration did not induce IgG antibody responses or lymphoproliferation against the SIV antigens in blood. This could be due to the lack of in vivo persistence of the recombinant BCG strains that were used. Rectal challenge with fully pathogenic SIVmac251-infected all animals. However, after viral challenge, anti-SIV cellular and antibody responses were higher in rBCG-SIV3 monkeys than in controls indicating that the vaccine induced anti-SIV CD4(+) T-cell memory. (C) 2003 Elsevier Ltd. All rights reserved.

Introduction Mayotte est un departement d’outre-mer geographiquement isole, expose au risque de maladies infectieuses et soumis a une forte migration sanitaire en provenance des iles voisines. La diphterie illustre l’importance d’un diagnostic microbiologique de proximite. En 2007, le dernier cas de diphterie ORL a Corynebacterium diphtheriae porteur du gene codant la toxine (tox +) est notifie chez un nourrisson de 2 mois, non vaccine, qui en est decede. L’annee suivante, le laboratoire de l’hopital met en place une procedure pour le diagnostic rapide des suspicions cliniques de diphterie. Materiels et methodes L’analyse porte sur les isolats de C. diphtheriae tox +identifies de 2008 a 2015 en associant microbiologie conventionnelle (milieu de culture specifique de Tinsdal) et biologie moleculaire (amplification du gene tox) avec confirmation secondaire du diagnostic par le CNR. Resultats Sur cette periode, 13 isolats de C. diphtheriae tox+ sont identifies au niveau de lesions cutanees chroniques evoluant depuis des semaines, voire des mois, souvent polymicrobiennes. Parmi ces cas : 10 hommes et 3 femmes, âge median de 11 ans (12 mois-39 ans), 8 cas importes et 7 sans antecedent vaccinal. Les patients ont recu une antibiotherapie adaptee, associee ou non a un geste chirurgical, sans serotherapie en absence de manifestations toxiniques. Le diagnostic par le laboratoire a permis une prise en charge adaptee et la mise en œuvre precoce des mesures de gestion preconisees par les autorites sanitaires pour les contacts (ecouvillonnage nasopharynge, antibioprophylaxie, vaccination ou rattrapage vaccinal). Parmi 168 contacts identifies, l’analyse a permis le diagnostic de 8 cas de portage pharynge asymptomatique dans l’entourage de 4 patients. Il est note que 25 isolats tox− ont egalement ete collectes de lesions cutanees chroniques. Conclusion Des cas de diphterie cutanee tox + surviennent episodiquement a Mayotte. L’absence d’une circulation plus importante de ces bacteries ne peut etre ecartee du fait de la frequence de plaies cutanees surinfectees et des difficultes d’acces aux soins d’une partie de la population. Face a l’insuffisance de couverture vaccinale chez l’adolescent et l’adulte, la poursuite de la surveillance microbiologique de la circulation de C. diphtheriae demeure une priorite.

To the Editor: Corynebacterium diphtheriae is the agent of pharyngeal and cutaneous diphtheria. We did a retrospective analysis of the antimicrobial drug susceptibilities of 46 C. diphtheriae isolates sent during 1993 through 2010 to the French National Reference Centre of Toxigenic Corynebacteria. The isolates came from metropolitan France and French overseas departments and territories. Only 1 isolate, C. diphtheriae biovar mitis, FRC24, expressed the following antimicrobial drug susceptibility profile: susceptible to penicillin, amoxicillin, ciprofloxacin, clindamycin, erythromycin, gentamicin, imipenem, kanamycin, rifampin, tetracycline, and vancomycin and resistant at an uncommonly high level to trimethoprim, sulfamethoxazole, and co-trimoxazole with Etest (bioMerieux, Marcy l’Etoile, France) MICs of >32, >1,024, and >32 mg/L, respectively. This FRC24 isolate was isolated in 2008 from a cutaneous wound on a vaccinated 11-month-old child in Mayotte, an overseas department located in the Indian Ocean. Cutaneous carriage of C. diphtheriae is frequent in tropical countries where cutaneous diphtheria is endemic; cutaneous carriage represents a common mode of transmission of the bacterium. FRC24 was identified by using the API Coryne strip (bioMerieux). FRC24 is a toxigenic isolate; toxigenicity was confirmed by both tox gene detection and Elek test (1). Multilocus sequence typing was performed, and the sequence type (ST) of the isolate is ST91. This ST contains only this isolate and is part of lineage II, as are all mitis and gravis biovars (2). To date, resistance to trimethoprim, sulfamethoxazole, or co-trimoxazole seems to be rare among the C. diphtheriae species, but few data are available (3). As trimethoprim resistance is often encoded by integron-driven dfr determinants, we looked for integrons. Integrons are bacterial genetic elements able to capture and express antimicrobial drug resistance gene cassettes (GCs) (4). GC movements are catalyzed by an integron-encoded integrase IntI. GCs, mainly promoterless, are usually expressed through a common Pc promoter (5). Only rare GCs contain their own promoter (cmlA, qac, ereA1). Three main classes of integrons have been described and are involved in the dissemination of antimicrobial drug resistance; class 1 is the most widely found in clinical isolates. Integrons have been mainly described among gram-negative bacteria; only a few studies have reported integrons in Corynebacterium spp (6,7). After bacterial genomic DNA extraction (DNeasy Blood & Tissue Kit; QIAGEN, Courtaboeuf, France), a multiplex Taqman-based quantitative PCR approach able to detect the 3 main classes of integrons was performed (8). We found that FRC24 harbored a class 1 integron. Analysis of the GC array showed that this integron harbored 2 GCs: dfrA16 of 588 bp conferring resistance to trimethoprim and qacH of 511 bp conferring resistance to quaternary ammonium compounds (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"FR822749","term_id":"323714042"}}FR822749). To our knowledge, this GC array has not been previously reported, even among reports of other gram-negative isolates. Moreover, a qac determinant has been found only once in a Corynebacterium species, C. pseudogenitalium (which harbors a qacH variant in the chromosome [GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"ABYQ02000013","term_id":"311303764"}}ABYQ02000013]), but not in an integron background. GC arrays were followed by the qacEΔ1 (which also confers resistance to quaternary ammonium compounds), sul1 (resistance to sulfamethoxazole), and orf5 determinants as found in most class 1 integrons (4). In class 1 integrons, 13 Pc variants have been described (5). In the FRC24 integron, the dfrA16 expression was mediated through a strong Pc variant (PcWTGN-10) (5) that enables the high-level resistance observed for trimethoprim. As previously demonstrated, the qacH GC possessed its own promoter (9). Trimethoprim is a commonly prescribed antimicrobial agent used in combination with sulfamethoxazole (co-trimoxazole) for the treatment of diarrheal diseases. This antimicrobial drug might have selected the emergence of such a strain expressing trimethoprim resistance. Furthermore, the FRC24 integron contains the antiseptic (quaternary ammonium compounds) resistance gene qacH. As cutaneous carriage of C. diphtheriae is frequent in tropical countries such as Mayotte, this bacterium could be exposed to quaternary ammonium compounds contained in disinfectants, hygienic hand washes, and cosmetic products. These products exert a selective pressure, which might play a role in selecting qac-containing strains, as has been suggested for Staphylococcus spp (10). For staphylococci, the MICs of quaternary ammonium compounds are >2 mg/L. With FRC24, we tested for the MIC of cetyltrimethylammmonium bromide and found a MIC of 4 mg/L, suggesting that qacH is expressed in FRC24. The sequencing of the genetic environment of this integron showed that it was framed by 2 copies of the insertion sequence IS6100 disrupting at the left-hand side the intI1 integrase gene (Figure A1). IS6100 has been described in a wide spectrum of host organisms, including Corynebacterium spp (6,7), thus enabling this integron to be efficiently transferred to various bacteria. Our findings show that C. diphtheriae is able to harbor integrons, which is of clinical relevance. Indeed, this genetic feature would give the isolates the capacity to easily acquire new GCs, such as ere GCs encoding resistance to erythromycin, which is one of the antimicrobial drugs recommended for diphtheria treatment.

We have recently designed a new Plasmodium falciparum mouse model and documented its potential for the study of immune effector mechanisms. In order to determine its value for drug studies, we evaluated its response to existing antimalarial drugs compared to that observed in humans. Immunocompromised BXN (bg/bg xid/xid nu/nu) mice were infected with either the sensitive NF54 strain or the multiresistant T24 strain and then treated with chloroquine, quinine, mefloquine, or dihydroartemisinin. A parallelism was observed between previously reported human responses and P. falciparum-parasitized human red blood cell (huRBC)‐ BXN mouse responses to classical antimalarial drugs, measured in terms of speed of decrease in parasitemia and of morphological alterations of the parasites. Mice infected with the sensitive strain were successfully cured after treatment with either chloroquine or mefloquine. In contrast, mice infected with the multiresistant strain failed to be cured by chloroquine or quinine but thereafter responded to dihydroartemisinin treatment. The speed of parasite clearance and the morphological alterations induced differed for each drug and matched previously reported observations, hence stressing the relevance of the model. These data thus suggest that P. falciparum-huRBC‐BXN mice can provide a valuable in vivo system and should be included in the short list of animals that can be used for the evaluation of P. falciparum responses to drugs. In the absence of a long-awaited effective vaccine, antimalarial drugs remain the main means by which to control morbidity and mortality due to Plasmodium falciparum malaria. Although several antimalarials are available, P. falciparum has gradually developed resistance to nearly all of them. Furthermore, the prevalence and degree of resistance are increasing and the pipeline of new antimalarial compounds is drying out (26). Thus, there is an urgent need to find either new combined therapies using available compounds or to develop new antimalarial drugs to replace failing ones. However, among the various reasons for the shortage of new drugs are the small number of animal species receptive to P. falciparum and their price. Immunodeficient mice have been widely used as xenogeneic transplantation models allowing in vivo investigations of human cells and organs. Recently, Badell et al. developed a model (1) in which P. falciparum-parasitized human red blood cells (P. falciparum-huRBC) can be grafted into immunodeficient (bg/bg xid/xid nu/nu) BXN laboratory mice (P. falciparumhuRBC‐BXN). In this mouse strain, which lacks T-cell, LAKC, and T-independent B-cell functions, a main factor in the survival of P. falciparum is the concomitant decrease in innate, nonadaptive immunity. Through chemical reduction of tissue macrophages and blood leukocytes, in vivo development of medium-grade P. falciparum parasitemia can be obtained in these mice in several weeks. This new model may eventually provide a valuable tool with which to investigate the biology of this malaria parasite under in vivo conditions and therefore facilitate research in the fields of chemotherapy and vaccine development. The goal of the present work was to investigate the potential value of this new animal model for antimalarial drug studies. This could be achieved only by using those drugs whose effects in humans are well established. Immunocompromised BXN mice were infected with the drug-sensitive NF54 strain or the multidrug-resistant T24 strain and treated with chloroquine, quinine, mefloquine, or dihydroartemisinin. We compared in vivo data obtained from the P. falciparum-huRBC‐BXN mouse model with the drug sensitivity of strains determined in vitro and with the response usually observed in natural infections of humans.

The development of a malaria preerythrocytic vaccine has been greatly influenced by the observation that sterile immunity could be experimentally induced in humans by immunization with Plasmodium falciparum radiation-attenuated sporozoites (9). The critical role of liver-stage trophozoites (reviewed in reference 16) led researchers to initiate a detailed study of the antigens expressed in P. falciparum preerythrocytic stages (22). To date, scientists have identified a series of new molecules expressed at sporozoite and/or liver stage (14). Liver-stage antigen 3 (LSA3), expressed on both the sporozoite surface and in the liver forms, was found to be of particular interest. LSA3 was identified by differential screening of immune responses from protected versus nonprotected volunteers (11). A number of dominant B- and T-cell epitopes to which a high prevalence of responses is detected in individuals exposed to malaria or in LSA3-immunized animals were identified (1, 2). The vaccine potential of this molecule has been recently demonstrated in the chimpanzee model, an animal susceptible and fully receptive to P. falciparum preerythrocytic stages and whose immune system is closest to that of humans. In this model, protection against successive challenges with P. falciparum sporozoites was obtained (11). However, the use of this primate for research purposes is severely hampered by cost and ethical constraints. Several homologues of P. falciparum antigens have been identified, through a variety of immunological and molecular cross-reactivity assays, in other Plasmodium species and particularly in those infecting rodents (5, 10, 12, 13, 27, 28). The identification of structurally and functionally conserved homologues of P. falciparum proteins in rodent malaria species might help to better understand the role of these molecules in the P. falciparum parasite life cycle. This is particularly true for preerythrocytic stages, where the relative ease of obtaining sporozoites and mouse primary hepatocytes would allow more extensive investigations to be carried out. In the process of identification of LSA3, a preerythrocytic subset of P. falciparum genomic fragments (22) was used to affinity purify antibodies (Abs) from sera of individuals with hyperimmunity to malaria. These Abs were consequently checked for immunofluorescence antibody test (IFAT) cross-reactivity with rodent malaria sporozoites, and in this assay Abs against all clones coding for various fragments of LSA3 proved to be strongly reactive with Plasmodium yoelii sporozoites but not with those from Plasmodium berghei. The present study was undertaken to investigate the extent of antigenic cross-reactivity between the LSA3 molecule and its putative homologue in P. yoelii and to determine whether specific human Abs are of biological significance in the mouse model. We show that such cross-reactivity exists for human anti-LSA3 Abs, which encompass four distinct LSA3 epitopes, including both nonrepeated and repeated domains, and extend to both sporozoite and liver stages of P. yoelii. A similar result was obtained using Abs from chimpanzees immunized with LSA3. Moreover, P. yoelii, but not P. berghei, sporozoites were able to induce dose-dependent proliferation of T cells collected from one of these animals. Finally, we have established that these homologous epitopes are targets for immune responses which have a biological consequence, since LSA3-specific human Abs inhibit the invasion of P. yoelii sporozoites into mouse hepatocytes both in vitro and in vivo.