Cutaneous ulcers revealing diphtheria: A re-emerging disease imported from Indian Ocean countries?

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.

Re-emergence of Corynebacterium diphtheriae.

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.

Microbiological and molecular characterization of Corynebacterium diphtheriae isolated in Algeria between 1992 and 2015.

The objectives of this study were to undertake the microbiological and molecular characterization of Corynebacterium diphtheriae isolates collected in Algeria during epidemic and post-epidemic periods between 1992 and 2015. Microbiological characterization includes the determination of biotype and toxigenicity status using phenotypic and genotypic methods. Antimicrobial susceptibility was determined by the E-test method. Molecular characterization was performed by multi-locus sequence typing. In total, there were 157 cases of C. diphtheriae isolates, 127 in patients with respiratory diphtheria and 30 with ozena. Isolates with a mitis biotype were predominant (122 out of 157; 77.7%) followed by belfanti (28 out of 157; 17.8%) and gravis biotype (seven out of 157; 4.5%). Toxigenic isolates were predominant in the period 1992-2006 (74 out of 134) whereas in the period 2007-2015, only non-toxigenic isolates circulated (23 out of 23). All 157 isolates were susceptible to erythromycin, gentamicin, vancomycin and cotrimoxazole. Reduced susceptibility to penicillin G, cefotaxime, tetracycline and chloramphenicol was detected in 90 (57.3%), 88 (56.1%), 112 (71.3%) and 90 (57.3%) isolates, respectively. Multi-locus sequence typing analysis indicates that sequence type 116 (ST-116) was the most frequent, with 65 out of 100 isolates analysed, in particular during the epidemic period 1992-1999 (57 out of 65 isolates). In the post-epidemic period, 2000-2015, 13 different sequence types were isolated. All belfanti isolates (ten out of 100 isolates) belonged to closely related sequence types grouped in a phylogenetically distinct eBurst group and were collected exclusively in ozena cases. In conclusion, the epidemic period was associated with ST-116 while the post-epidemic period was characterized by more diversity. Belfanti isolates are grouped in a phylogenetically distinct clonal complex.

Toxigenic Corynebacterium ulcerans in a fatal human case and her feline contacts, France, March 2014.

In March 2014, a person in their eighties who was diagnosed with extensive cellulitis due to toxigenic Corynebacterium ulcerans died from multiple organ failure. Environmental investigation also isolated C. ulcerans in biological samples from two stray cats in contact with the case. This finding provides further evidence that pets can carry toxigenic C. ulcerans and may be a source of the infection in humans.

The utility of the PCR melting profile technique for typing Corynebacterium diphtheriae isolates.

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.

Microbiological changes and diversity in autochthonous non-toxigenic Corynebacterium diphtheriae isolated in France.

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.

Evaluation of the Andromas matrix-assisted laser desorption ionization-time of flight mass spectrometry system for identification of aerobically growing Gram-positive bacilli.

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.

Characterization and comparison of invasive Corynebacterium diphtheriae isolates from France and Poland.

Corynebacterium diphtheriae, the agent of diphtheria, is rarely responsible for bacteremia. However, high numbers of bacteremia have been reported in countries with extensive immunization coverage. Here, we used molecular and phenotypic tools to characterize and compare 42 invasive isolates collected in France (including New Caledonia) and Poland over a 23-year period.

Diphtheria in the south of France, March 2011.

In March 2011, a 40 year-old French man was diagnosed with diphtheria caused by toxigenic Corynebacterium diphtheriae. Fifty-three close contacts were identified from whom throat samples were analysed. C. diphtheriae was found only in the asymptomatic partner of the index case. The two cases had travelled in Spain during the incubation period of the index case. Investigation around the second case identified 13 new close contacts.None of them was found to be infected.

Cell-mediated and not humoral immune responseis responsible for partial protection against toxoplasmosis in SCID mice reconstituted with human PBMC

El objetivo de este estudio fue profundizar en el conocimiento de la respuesta inmunologica especifica contra Toxoplasma en humanos. Usamos ratones SCID reconstituidos con PBMC de donantes sanos con y (..)(CNS) (AU)

The aim of this study was to further our understanding of the human Toxoplasma-specific immune response. We used severe combined immune deficiency (SCID) mice that had been reconstituted with peripheral blood mononuclear cells (PBMC) from healthy donors with and without serum Toxoplasma antigens(PBMC Toxo+ and PBMC Toxo-, respectively) and subsequently infected with parasite cysts. The specific lymphocyte proliferation rate (LPR) was higher for PBMC from Toxoplasma-immune donors and these PBMC secreted more Th1 cytokines than those obtained from Toxoplasma-non-immune donors. The engraftmentof human parasite-immune cells significantly increased the survival rate following infection compared to in unreconstituted animals,where as the engraftment of human non-parasite-immune cells did not alter the survival rate. Specific human anti-Toxoplasma antibodies were found in both groups of humanised animals, suggesting that the humoral immune response does not play a major role in this protection. Ten days after infection, cytometry revealed that there were more human CD45+ cells in the spleen and peritoneum of mice from the PBMC Toxo+ group than from the PBMC Toxo- group. Furthermore, plasma levels of nitric oxide(NO) peaked at an early stage of infection in resistant animals.Finally, no human CD4 mRNA or IFN-¦Ã mRNA was found in the brain during infection. All together, our results suggest that the human cell-mediated immune response provides partial protection against toxoplasmic encephalitis (TE) in this model. However,the effector mechanisms involved may be located outside of the central nervous system (CNS) (AU)

Cell-mediated and not humoral immune response is responsible for partial protection against toxoplasmosis in SCID mice reconstituted with human PBMC

El objetivo de este estudio fue profundizar en el conocimientode la respuesta inmunológica específica contra Toxoplasma enhumanos. Usamos ratones SCID reconstituidos con PBMC dedonantes sanos con y sin antígenos séricos de Toxoplasma (PBMCToxo+ and PBMC Toxo-, respectivamente) y subsecuentementeinfectados con cistos de parásito. La proliferación linfocitaria especifica(LPR) fue mayor en los PBMC de los donantes immunes aToxoplasma y éstos PBMC secretaron más citocinas Th1 que losobtenidos de donantes no immunes a Toxoplasma. La implantaciónde células inmunológicas de humanos infectados augmentósignificativamente la supervivencia después de la infección silo comparamos con la de los animales no reconstituidos. Sinembargo, la implantación de células inmunológicas de humanosno infectados no alteró la supervivencia. Se encontraron anticuerposespecíficos humanos anti- Toxoplasma en los dos gruposde animales humanizados, lo que sugiere que la respuesta inmunológicahumoral no juega un papel determinante en ésta protección.Diez días después de la infección, los estudios de citometríarevelaron que había más células humanas CD45+ en el bazoy peritoneo de los ratones del grupo PBMC Toxo+ que en los delgrupo Toxo-. Además, los niveles plasmáticos de óxido nítrico(NO) alcanzaron un máximo en un estadío más temprano de lainfección en animales resistentes. Finalmente, no se encontróRNAm de CD4 o IFN-gama humanos en el cerebro durante la infección.Nuestros resultados sugieren que la respuesta humana inmunológicacellular aporta una protección parcial contra la encephalitistoxoplásmica (TE) en este modelo. Sin embargo, los mecanismosefectores involucrados podrían estar localizados fuera delsistema nervioso central (CNS)

The aim of this study was to further our understanding of thehuman Toxoplasma-specific immune response. We used severecombined immune deficiency (SCID) mice that had been reconstitutedwith peripheral blood mononuclear cells (PBMC) fromhealthy donors with and without serum Toxoplasma antigens(PBMC Toxo+ and PBMC Toxo-, respectively) and subsequentlyinfected with parasite cysts. The specific lymphocyte proliferationrate (LPR) was higher for PBMC from Toxoplasma-immunedonors and these PBMC secreted more Th1 cytokines than thoseobtained from Toxoplasma-non-immune donors. The engraftmentof human parasite-immune cells significantly increased the survivalrate following infection compared to in unreconstituted animals,whereas the engraftment of human non-parasite-immunecells did not alter the survival rate. Specific human anti-Toxoplasmaantibodies were found in both groups of humanised animals, suggestingthat the humoral immune response does not play a majorrole in this protection. Ten days after infection, cytometry revealedthat there were more human CD45+ cells in the spleen andperitoneum of mice from the PBMC Toxo+ group than from thePBMC Toxo- group. Furthermore, plasma levels of nitric oxide(NO) peaked at an early stage of infection in resistant animals.Finally, no human CD4 mRNA or IFN-gamma mRNA was found in thebrain during infection. All together, our results suggest that thehuman cell-mediated immune response provides partial protectionagainst toxoplasmic encephalitis (TE) in this model. However,the effector mechanisms involved may be located outsideof the central nervous system (CNS)

Plasmidic versus insertional cloning of heterologous genes in Mycobacterium bovis BCG: impact on in vivo antigen persistence and immune responses.

Bivalent recombinant strains of Mycobacterium bovis BCG (rBCG) expressing the early regulatory nef and the structural gag(p26) genes from the simian immunodeficiency virus (SIV) SIVmac251 were engineered so that both genes were cotranscribed from a synthetic operon. The expression cassette was cloned into a multicopy-replicating vector, and the expression levels of both nef and gag in the bivalent rBCG(nef-gag) strain were found to be comparable to those of monovalent rBCG(nef) or rBCG(gag) strains. However, extrachromosomal cloning of the nef-gag operon into a replicative plasmid resulted in strains of low genetic stability that rapidly lost the plasmid in vivo. Thus, the nef-gag operon was inserted site specifically into the BCG chromosome by means of mycobacteriophage Ms6-derived vectors. The resulting integrative rBCG(nef-gag) strains showed very high genetic stability both in vitro and in vivo. The in vivo expression of the heterologous genes was much longer lived when the expression cassette was inserted into the BCG chromosome. In one of the strains obtained, integrative cloning did not reduce the expression levels of the genes even though a single copy was present. Accordingly, this strain induced cellular immune responses of the same magnitude as that of the replicative rBCG strain containing several copies of the genes.

CD4 T cell recovery is slower in patients experiencing viral load rebounds during HAART.

To determine whether viral load rebounds during HAART impact on CD4+ T cell recovery and immune reconstitution, we studied a prospective cohort of 355 antiretroviral naive patients enrolled to be randomized in a trial of three strategies of induction/maintenance HAART. The extent of immune reconstitution in blood through 72 weeks of antiretroviral treatment was evaluated. Lymphocyte subset markers (CD4, CD8, CD45RA, CD62L, CD16, CD19), activation markers (HLA-DR, CD38, CD25) were performed by cytometry analysis. Our results showed that plasma HIV-1 RNA was suppressed to below 500 copies per ml through week 72 in 240 patients (group 1) while the remaining 115 patients experienced at least one viral rebound (group 2). At baseline, CD4 cell count was higher and HIV-1 RNA was lower in group 1 than in group 2. Over 72 weeks, mean increase in CD4+ T cell count was 0.32 cell/mm3/day in group 1 and only 0.14 cell/mm3/day in group 2 (P < 0.0001). However, the patterns of changes in CD4+ and CD8+ T cell subsets during therapy were very similar across the two groups with only subtle and very limited differences. We conclude that permanent control of HIV replication could be necessary for faster immune reconstitution.

Human malaria in immunocompromised mice: new in vivo model for chemotherapy studies.

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.

Human antibodies against Plasmodium falciparum liver-stage antigen 3 cross-react with Plasmodium yoelii preerythrocytic-stage epitopes and inhibit sporozoite invasion in vitro and in vivo.

The Plasmodium falciparum liver-stage antigen 3 (LSA3), a recently identified preerythrocytic antigen, induces protection against malaria in chimpanzees. Using antibodies from individuals with hyperimmunity to malaria affinity purified on recombinant or synthetic polypeptides of LSA3, we identified four non-cross-reactive B-cell epitopes in Plasmodium yoelii preerythrocytic stages. On sporozoites the P. yoelii protein detected has a molecular mass similar to that of LSA3. T-cell epitopes cross-reacting with P. yoelii were also demonstrated using peripheral blood lymphocytes from LSA3-immunized chimpanzees. In contrast, no cross-reactive epitopes were found in Plasmodium berghei. LSA3-specific human antibodies exerted up to 100% inhibition of in vitro invasion of P. yoelii sporozoites into mouse hepatocytes. This strong in vitro activity was reproduced in vivo by passive transfer of LSA3 antibodies. These results indicate that the homologous epitopes may be biologically functional and suggest that P. yoelii could be used as a model to assess the antisporozoite activity of anti-LSA3 antibodies.

Human malaria in immunocompromised mice: an in vivo model to study defense mechanisms against Plasmodium falciparum.

We have recently described that sustained Plasmodium falciparum growth could be obtained in immunodeficient mice. We now report the potential of this new mouse model by assaying the effect of the passive transfer of antibodies (Abs) which in humans have had a well-established effect.Our results show that the total African adult hyperimmune immunoglobulin Gs (HI-IgGs) strongly reduce P. falciparum parasitemia similarly to that reported in humans, but only when mice are concomitantly reconstituted with human monocytes (HuMNs). In contrast, neither HI-IgGs nor HuMNs alone had any direct effect upon parasitemia. We assessed the in vivo effect of epitope-specific human Abs affinity-purified on peptides derived either from the ring erythrocyte surface antigen (RESA) or the merozoite surface protein 3 (MSP3). The inoculation of low concentrations of anti-synthetic peptide from MSP3, but not of anti-RESA Abs, consistently suppressed P. falciparum in the presence of HuMNs. Parasitemia decrease was stronger and faster than that observed using HI-IgGs and as fast as that induced by chloroquine. Our observations demonstrate that this mouse model is of great value to evaluate the protective effect of different Abs with distinct specificity in the same animal, a step hardly accessible and therefore never performed before in humans.

Plasmodium falciparum sporozoite invasion is inhibited by naturally acquired or experimentally induced polyclonal antibodies to the STARP antigen.

Antibody(Ab)-mediated inhibition of sporozoite invasion of hepatocytes is a mechanism that has been clearly demonstrated to act upon Plasmodium falciparum pre-erythrocytic stages in humans. Consequently we have analyzed the Ab response to a recently identified P. falciparum sporozoite surface protein, STARP, in malaria-exposed individuals and tested the inhibitory effect of these Ab upon hepatocyte invasion in vitro. STARP-specific IgG were detected in 90 and 61% of sera from regions where individuals were exposed to 100 and 1-5 infectious bites per year, respectively. These IgG were predominantly of the cytophilic IgG1 or IgG3 type. STARP and the major sporozoite surface protein, CS, elicited equivalent IgG levels in adults. When affinity purified from either African immune sera or the serum of an individual experimentally protected by irradiated sporozoite immunization, STARP-specific Ab prevented up to 90% of sporozoites from invading human hepatocytes. The dose-dependent and reproducible inhibition was more pronounced than that observed with human CS-specific Ab affinity purified under identical conditions. Substantial reduction of sporozoite invasion was also observed with Ab induced by artificial immunization with recombinant STARP protein and reactive with the native protein. Taken together with recent findings of human cytotoxic T lymphocytes specific for this antigen, these results promote the interest of studying the efficacy of STARP as a target for immune effector mechanisms operating upon pre-erythrocytic stages.

Human Plasmodium liver stages in SCID mice: a feasible model?

In a recent issue of Parasitology Today, Stanley and Virgin have stressed the potential of B- and T-cell deficient mice, among which severe combined immunodeficiency (SCID) mice are most frequently used, as models for the study of parasites. One of the most tantalizing prospects has been in the development of liver stages (LS) of human Plasmodium.