Deciphering Unexpected Vascular Locations of Scedosporium spp. and Lomentospora prolificans Fungal Infections, France.

Scedosporium spp. and Lomentospora prolificans are emerging non-Aspergillus filamentous fungi. The Scedosporiosis/lomentosporiosis Observational Study we previously conducted reported frequent fungal vascular involvement, including aortitis and peripheral arteritis. For this article, we reviewed 7 cases of Scedosporium spp. and L. prolificans arteritis from the Scedosporiosis/lomentosporiosis Observational Study and 13 cases from published literature. Underlying immunosuppression was reported in 70% (14/20) of case-patients, mainly those who had solid organ transplants (10/14). Osteoarticular localization of infection was observed in 50% (10/20) of cases; infections were frequently (7/10) contiguous with vascular infection sites. Scedosporium spp./Lomentospora prolificans infections were diagnosed in 9 of 20 patients ≈3 months after completing treatment for nonvascular scedosporiosis/lomentosporiosis. Aneurysms were found in 8/11 aortitis and 6/10 peripheral arteritis cases. Invasive fungal disease--related deaths were high (12/18 [67%]). The vascular tropism of Scedosporium spp. and L. prolificans indicates vascular imaging, such as computed tomography angiography, is needed to manage infections, especially for osteoarticular locations.

Intravenous Artesunate for the Treatment of Severe Imported Malaria: Implementation, Efficacy, and Safety in 1391 Patients.

BACKGROUND: Intravenous artesunate is the World Health Organization-recommended first-line treatment for severe malaria worldwide, but it is still not fully licensed in Europe. Observational studies documenting its safety and efficacy in imported malaria are thus essential. METHODS: We prospectively collected clinical and epidemiological features of 1391 artesunate-treated patients among 110 participant centers during the first 7 years (2011-2017) of a national program implemented by the French Drug Agency. RESULTS: Artesunate became the most frequent treatment for severe malaria in France, rising from 9.9% in 2011 to 71.4% in 2017. Mortality was estimated at 4.1%. Treatment failure was recorded in 27 patients, but mutations in the Kelch-13 gene were not observed. Main reported adverse events (AEs) were anemia (136 cases), cardiac events (24, including 20 episodes of conduction disorders and/or arrhythmia), and liver enzyme elevation (23). Mortality and AEs were similar in the general population and in people with human immunodeficiency virus, who were overweight, or were pregnant, but the only pregnant woman treated in the first trimester experimented a hemorrhagic miscarriage. The incidence of post-artesunate-delayed hemolysis (PADH) was 42.8% when specifically assessed in a 98-patient subgroup, but was not associated with fatal outcomes or sequelae. PADH was twice as frequent in patients of European compared with African origin. CONCLUSIONS: Artesunate was rapidly deployed and displayed a robust clinical benefit in patients with severe imported malaria, despite a high frequency of mild to moderate PADH. Further explorations in the context of importation should assess outcomes during the first trimester of pregnancy and collect rare but potentially severe cardiac AEs.

Genome editing in the yeast Nakaseomyces delphensis and description of its complete sexual cycle.

The environmental yeast Nakaseomyces delphensis is, phylogenetically, the closest known species to Candida glabrata, a major fungal pathogen of humans. C. glabrata is haploid and described as asexual, while N. delphensis is also haploid, but has been described as competent for mating and meiosis. Both genomes contain homologues of all the genes necessary for sexual reproduction and also the genes for Ho-dependent mating-type switching, like Saccharomyces cerevisiae. We first report the construction of genetically engineered strains of N. delphensis, including by CRISPR-Cas 9 gene editing. We also report the description of the sexual cycle of N. delphensis. We show that it undergoes Ho-dependent mating-type switching in culture and that deletion of the HO gene prevents such switching and allows maintenance of stable, separate, MATa and MATalpha haploid strains. Rare, genetically selected diploids can be obtained through mating of haploid strains, mutated or not for the HO gene. In contrast to HO/HO diploids, which behave as expected, Δho/Δho diploids exhibit unusual profiles in flow cytometry. Both types of diploids can produce recombined haploid cells, which grow like the original haploid-type strain. Our experiments thus allow the genetic manipulation of N. delphensis and the reconstruction, in the laboratory, of its entire life cycle.

Microsporidiosis after liver transplantation: A French nationwide retrospective study.

BACKGROUND: Microsporidiosis has been largely reported in patients with acquired immunodeficiency syndrome, but emerged as a cause of persistent diarrhea in solid organ transplant patients. METHODS: Through the French Microsporidiosis Network and the Groupe français de recherche en greffe de foie, we collected all microsporidiosis cases identified in liver transplant patients between 1995 and 2020 in France. RESULTS: We identified 24 liver transplant recipients with microsporidiosis. Sex ratio was balanced and median age was 58.8 (3.5-83.5) years (there were 4 children). Microsporidiosis occurred at a median time of 3.9 (0.1-18.9) years post-transplant. Median duration of diarrhea before diagnosis was 22 days (12-45). Therapeutic care included immunosuppressive therapy changes in 20 patients, as follows: stop cyclosporine or tacrolimus (n = 2), dose reduction of cyclosporine or tacrolimus (n = 12), stop MMF (n = 5), and dose reduction of corticosteroids (n = 1). In addition, 15 patients received specific therapy against microsporidiosis: fumagillin (n = 11) or albendazole (n = 4). Median duration of treatment was 14 days (8-45 days). Finally, 7 patients had immunosuppressive treatment tapering only. Microsporidiosis was complicated by renal failure in 15 patients, requiring dialysis in one case. Two patients had infection relapse. No patient presented proven rejection within the 3 months after microsporidiosis. None of the patients died within the 3 months after microsporidiosis. CONCLUSIONS: Microsporidiosis is a very rare infection after liver transplantation but can induce severe dehydration and renal failure. Therefore, it must be systematically sought in any case of persistent diarrhea after first line screening of frequent infectious causes.

Enterocytozoon bieneusi Microsporidiosis in Stem Cell Transplant Recipients Treated with Fumagillin

Enterocytozoon bieneusi microsporidiosis is an emerging disease in immunocompromised patients. We report 2 cases of this disease in allogeneic hematopoietic stem cell transplant patients successfully treated with fumagillin. Thrombocytopenia occurred but without major adverse events. Modifications of immunosuppression could be avoided when E. bieneusi is rapidly identified and fumagillin therapy is started promptly.

One small step for a yeast--microevolution within macrophages renders Candida glabrata hypervirulent due to a single point mutation.

Candida glabrata is one of the most common causes of candidemia, a life-threatening, systemic fungal infection, and is surpassed in frequency only by Candida albicans. Major factors contributing to the success of this opportunistic pathogen include its ability to readily acquire resistance to antifungals and to colonize and adapt to many different niches in the human body. Here we addressed the flexibility and adaptability of C. glabrata during interaction with macrophages with a serial passage approach. Continuous co-incubation of C. glabrata with a murine macrophage cell line for over six months resulted in a striking alteration in fungal morphology: The growth form changed from typical spherical yeasts to pseudohyphae-like structures - a phenotype which was stable over several generations without any selective pressure. Transmission electron microscopy and FACS analyses showed that the filamentous-like morphology was accompanied by changes in cell wall architecture. This altered growth form permitted faster escape from macrophages and increased damage of macrophages. In addition, the evolved strain (Evo) showed transiently increased virulence in a systemic mouse infection model, which correlated with increased organ-specific fungal burden and inflammatory response (TNFα and IL-6) in the brain. Similarly, the Evo mutant significantly increased TNFα production in the brain on day 2, which is mirrored in macrophages confronted with the Evo mutant, but not with the parental wild type. Whole genome sequencing of the Evo strain, genetic analyses, targeted gene disruption and a reverse microevolution experiment revealed a single nucleotide exchange in the chitin synthase-encoding CHS2 gene as the sole basis for this phenotypic alteration. A targeted CHS2 mutant with the same SNP showed similar phenotypes as the Evo strain under all experimental conditions tested. These results indicate that microevolutionary processes in host-simulative conditions can elicit adaptations of C. glabrata to distinct host niches and even lead to hypervirulent strains.

Postartesunate delayed hemolysis is a predictable event related to the lifesaving effect of artemisinins.

Patients with severe malaria treated with artesunate sometimes experience a delayed hemolytic episode. Artesunate (AS) induces pitting, a splenic process whereby dead parasites are expelled from their host erythrocytes. These once-infected erythrocytes then return to the circulation. We analyzed hematologic parameters in 123 travelers treated with AS for severe malaria. Among 60 nontransfused patients observed for more than 8 days, 13 (22%) had delayed hemolysis. The peak concentration of circulating once-infected erythrocytes was measured during the first week in 21 patients and was significantly higher in 9 patients with delayed hemolysis than in 12 with other patterns of anemia (0.30 vs 0.07; P = .0001). The threshold of 180 million once-infected erythrocytes per liter discriminated patients with delayed hemolysis with 89% sensitivity and 83% specificity. Once-infected erythrocyte morphology analyzed by using ImageStream in 4 patients showed an 8.9% reduction in their projected area, an alteration likely contributing to their shorter lifespan. Delayed clearance of infected erythrocytes spared by pitting during AS treatment is an original mechanism of hemolytic anemia. Our findings consolidate a disease framework for posttreatment anemia in malaria in which delayed hemolysis is a new entity. The early concentration of once-infected erythrocytes is a solid candidate marker to predict post-AS delayed hemolysis.

Inherited CARD9 deficiency in 2 unrelated patients with invasive Exophiala infection.

BACKGROUND: Exophiala species are mostly responsible for skin infections. Invasive Exophiala dermatitidis disease is a rare and frequently fatal infection, with 42 cases reported. About half of these cases had no known risk factors. Similarly, invasive Exophiala spinifera disease is extremely rare, with only 3 cases reported, all in patients with no known immunodeficiency. Autosomal recessive CARD9 deficiency has recently been reported in otherwise healthy patients with severe fungal diseases caused by Candida species, dermatophytes, or Phialophora verrucosa. METHODS: We investigated an 8-year-old girl from a nonconsanguineous Angolan kindred, who was born in France and developed disseminated E. dermatitidis disease and a 26 year-old woman from an Iranian consaguineous kindred, who was living in Iran and developed disseminated E. spinifera disease. Both patients were otherwise healthy. RESULTS: We sequenced CARD9 and found both patients to be homozygous for loss-of-function mutations (R18W and E323del). The first patient had segmental uniparental disomy of chromosome 9, carrying 2 copies of the maternal CARD9 mutated allele. CONCLUSIONS: These are the first 2 patients with inherited CARD9 deficiency and invasive Exophiala disease to be described. CARD9 deficiency should thus be considered in patients with unexplained invasive Exophiala species disease, even in the absence of other infections.

Delayed-onset hemolytic anemia in patients with travel-associated severe malaria treated with artesunate, France, 2011-2013.

Artesunate is the most effective treatment for severe malaria. However, delayed-onset hemolytic anemia has been observed in ≈20% of travelers who receive artesunate, ≈60% of whom require transfusion. This finding could discourage physicians from using artesunate. We prospectively evaluated a cohort of 123 patients in France who had severe imported malaria that was treated with artesunate; our evaluation focused on outcome, adverse events, and postartesunate delayed-onset hemolysis (PADH). Of the 123 patients, 6 (5%) died. Overall, 97 adverse events occurred. Among the 78 patients who received follow-up for >8 days after treatment initiation, 76 (97%) had anemia, and 21 (27%) of the 78 cases were recorded as PADH. The median drop in hemoglobin levels was 1.3 g/dL; 15% of patients with PADH had hemoglobin levels of <7 g/dL, and 1 required transfusion. Despite the high incidence of PADH, the resulting anemia remained mild in 85% of cases. This reassuring result confirms the safety and therapeutic benefit of artesunate.

Comparative genomics of emerging pathogens in the Candida glabrata clade.

BACKGROUND: Candida glabrata follows C. albicans as the second or third most prevalent cause of candidemia worldwide. These two pathogenic yeasts are distantly related, C. glabrata being part of the Nakaseomyces, a group more closely related to Saccharomyces cerevisiae. Although C. glabrata was thought to be the only pathogenic Nakaseomyces, two new pathogens have recently been described within this group: C. nivariensis and C. bracarensis. To gain insight into the genomic changes underlying the emergence of virulence, we sequenced the genomes of these two, and three other non-pathogenic Nakaseomyces, and compared them to other sequenced yeasts. RESULTS: Our results indicate that the two new pathogens are more closely related to the non-pathogenic N. delphensis than to C. glabrata. We uncover duplications and accelerated evolution that specifically affected genes in the lineage preceding the group containing N. delphensis and the three pathogens, which may provide clues to the higher propensity of this group to infect humans. Finally, the number of Epa-like adhesins is specifically enriched in the pathogens, particularly in C. glabrata. CONCLUSIONS: Remarkably, some features thought to be the result of adaptation of C. glabrata to a pathogenic lifestyle, are present throughout the Nakaseomyces, indicating these are rather ancient adaptations to other environments. Phylogeny suggests that human pathogenesis evolved several times, independently within the clade. The expansion of the EPA gene family in pathogens establishes an evolutionary link between adhesion and virulence phenotypes. Our analyses thus shed light onto the relationships between virulence and the recent genomic changes that occurred within the Nakaseomyces. SEQUENCE ACCESSION NUMBERS: Nakaseomyces delphensis: CAPT01000001 to CAPT01000179Candida bracarensis: CAPU01000001 to CAPU01000251Candida nivariensis: CAPV01000001 to CAPV01000123Candida castellii: CAPW01000001 to CAPW01000101Nakaseomyces bacillisporus: CAPX01000001 to CAPX01000186.

Longitudinal study assessing the return of chloroquine susceptibility of Plasmodium falciparum in isolates from travellers returning from West and Central Africa, 2000-2011.

BACKGROUND: Chloroquine (CQ) was the main malaria therapy worldwide from the 1940s until the 1990s. Following the emergence of CQ-resistant Plasmodium falciparum, most African countries discontinued the use of CQ, and now promote artemisinin-based combination therapy as the first-line treatment. This change was generally initiated during the last decade in West and Central Africa. The aim of this study is to describe the changes in CQ susceptibility in this African region, using travellers returning from this region as a sentinel system. METHODS: The study was conducted by the Malaria National Reference Centre, France. The database collated the pfcrtK76T molecular marker for CQ susceptibility and the in vitro response to CQ of parasites from travellers' isolates returning from Senegal, Mali, Ivory Coast or Cameroon. As a proxy of drug pressure, data regarding CQ intake in febrile children were collated for the study period. Logistic regression models were used to detect trends in the proportions of CQ resistant isolates. RESULTS: A total of 2874 parasite isolates were genotyped between 2000-2011. The prevalence of the pfcrt76T mutant genotype significantly decreased for Senegal (from 78% to 47%), Ivory Coast (from 63% to 37%), Cameroon (from 90% to 59%) and remained stable for Mali. The geometric mean of the 50% inhibitory concentration (IC50) of CQ in vitro susceptibility and the proportion of resistant isolates (defining resistance as an IC50 value > 100 nM) significantly decreased for Senegal (from 86 nM (59%) to 39 nM (25%)), Mali (from 84 nM (50%) to 51 nM (31%)), Ivory Coast (from 75 nM (59%) to 29 nM (16%)) and Cameroon (from 181 nM (75%) to 51 nM (37%)). Both analyses (molecular and in vitro susceptibility) were performed for the 2004-2011 period, after the four countries had officially discontinued CQ and showed an accelerated decline of the resistant isolates for the four countries. Meanwhile, CQ use among children significantly deceased in this region (fixed effects slope = -0.3, p < 10-3). CONCLUSIONS: An increase in CQ susceptibility following official withdrawal of the drug was observed in travellers returning from West and Central African countries. The same trends were observed for molecular and in vitro analysis between 2004-2011 and they correlated to the decrease of the drug pressure.

Invasive infections due to Candida norvegensis and Candida inconspicua: report of 12 cases and review of the literature.

Candida inconspicua and Candida norvegensis are two closely related species rarely involved in invasive infections. The purpose of this study was to depict the epidemiologic and clinical characteristics of candidemia due to these emerging fluconazole less susceptible species. A retrospective analysis of the epidemiology of C. inconspicua and C. norvegensis during the period 2006-2010 was initiated in six French University hospitals. From this, demographics, clinical, diagnostic and therapeutic data of C. inconspicua or C. norvegensis candidemia were recorded and compared to the observations reported in the literature. C. inconspicua was more frequently isolated compared to C. norvegensis (ratio 2.6) but from the same preferential body sites: mainly digestive (56.4% and 48.37%, respectively, for C. inconspicua and C. norvegensis) and respiratory (26% and 28.2%, respectively). Thirteen cases of candidemia were recorded and five additional cases were found in the literature. Hematogical malignancy was the main underlying disease (n = 12). Associated factors were the presence of a vascular catheter (n = 18), broad-spectrum antibiotics (n = 15), and neutropenia (n = 14). In 13 cases (72%), prior colonization was noted before the candidemia diagnosis. Combining the results for the two species, Minimal Inhibitory Concentrations (MIC50) of amphotericin B, fluconazole, voriconazole and caspofungin were 0.125, 48, 0.25, and 0.19 mg/l, respectively. These two species must be added to the growing list of emerging Candida species poorly susceptible to fluconazole.

Favorable outcome without corticosteroids during post-artesunate delayed hemolysis with positive direct antiglobulin test in severe imported Plasmodium falciparum malaria, France.

OBJECTIVES: Positive direct antiglobulin tests (DATs) have been reported in cases of post-artesunate delayed hemolysis (PADH), but the causal role of auto-immune hemolysis remains unclear. We aimed to analyze a cohort of patients with PADH and DAT during severe malaria. METHODS: We describe PADH and DAT results in a 7-year multi-center retrospective cohort of patients receiving artesunate for severe imported malaria. RESULTS: Of 337 patients treated with artesunate, 46 (13.6%) had at least one DAT result within 30 days of treatment initiation, and 25/46 (54.3%) had at least one positive DAT. Among 40 patients with available data, 17 (42.5%) experienced PADH. Patient characteristics were similar for patients with a positive or negative DAT, and DAT positivity was not associated with PADH occurrence (P = 0.36). Among patients, 5/13 (38.5%) with a positive DAT after day 7 experienced PADH, compared to 10/13 (76.9%) of those with a negative DAT after day 7 (P = 0.11). Overall, 41% of patients required blood transfusions, and outcome was favorable without corticosteroids, even in cases of PADH. CONCLUSIONS: DAT does not appear to be a marker of PADH, but rather an indirect marker of an immune-mediated mechanism. DAT positivity should not lead to the administration of systemic corticosteroids during PADH.

Chronic schistosomiasis imported in France: A retrospective multicentre analysis of 532 patients, calling for international recommendations.

BACKGROUND: Schistosomiasis is a major public health issue for migrants. This study aims to describe the clinical presentation and management of imported schistosomiasis in France. METHODS: We included all new cases of schistosomiasis in patients aged ≥18 years, defined by a positive specific Western blot and/or a positive parasitological analysis of urine, stool or biopsy, between January 1, 2016, and December 31, 2019, in 4 laboratories in Paris and Western France. RESULTS: Over the study period, 532 patients were included. Mean age was 37 years (18-91), and 461/532 (87 %) were men. Among 476/532 (89 %) patients born in an endemic area, 433 (91 %) were born in sub-Saharan Africa. Most of the patients (405/532, 76 %) had only a positive serology, and 127/532 (24 %) had ova on microscopic examination. Among 361/532 (68 %) who had at least one urine, stool or biopsy analysis, microscopic analysis was positive in 127 (35 %). Imaging showed lesions compatible with schistosomiasis in 88/164 (54 %) patients with clinical symptoms and 13/29 (45 %) patients without (p = 0.5). Patients who arrived in France less than one year before diagnosis were more likely to have clinical symptoms than those who arrived in France 1-5 years and >5 years prior to diagnosis (52 %, 41 % and 43 %, respectively, p = 0.03). Two-hundred and seventeen patients (40.8 %) were left untreated. CONCLUSION: Approximately 50 % of patients with imported chronic schistosomiasis have radiological abnormalities, whether they are symptomatic or not, and management is heterogeneous. Multidisciplinary international guidelines are requested to clarify the management of this neglected disease.

Coronavirus Disease 2019-Associated Mucormycosis in France: A Rare but Deadly Complication.

We studied COVID-19 associated mucormycosis based on 17 cases reported nationwide and assessed the differences with India. They differed by frequencies of diabetes mellitus (47% in France versus up to 95% in India), hematological malignancies (35% versus 1%), anatomical sites (12% versus >80% rhino-orbito-cerebral) and prognosis (88% mortality versus <50%).

Cryptococcal Meningitis in Kidney Transplant Recipients: A Two-Decade Cohort Study in France.

Cryptococcosis is the third most common cause of invasive fungal infection in solid organ transplant recipients and cryptococcal meningitis (CM) its main clinical presentation. CM outcomes, as well as its clinical features and radiological characteristics, have not yet been considered on a large scale in the context of kidney transplantation (KT). We performed a nationwide retrospective study of adult patients diagnosed with cryptococcosis after KT between 2002 and 2020 across 30 clinical centers in France. We sought to describe overall and graft survival based on whether KT patients with cryptococcosis developed CM or not. Clinical indicators of CNS involvement and brain radiological characteristics were assessed. Eighty-eight cases of cryptococcosis were diagnosed during the study period, with 61 (69.3%) cases of CM. Mortality was high (32.8%) at 12 months (M12) but not significantly different whether or not patients presented with CM. Baseline hyponatremia and at least one neurological symptom were independently associated with CM (p < 0.001). Positive serum cryptococcal antigen at diagnosis was also significantly associated with CM (p < 0.001). On magnetic resonance imaging (MRI), three patterns of brain injury were identified: parenchymal, meningeal, and vascular lesions. Although CM does not affect graft function directly, it entails a grim prognosis.

Candidal abscess of the parotid gland due to Candida glabrata: report of a case and literature review.

We report the case of an immunocompetent woman who developed a Candida glabrata abscess of the parotid gland and present a review of similar cases from the literature. Diagnosis was based on the isolation of C. glabrata in pure culture from the abscess pus. Examination of stained smears of the same material demonstrated small sized yeast cells, some being intra-macrophagic. Combination of a local drainage and oral fluconazole proved to be an efficient therapeutic strategy. Candidal abscesses are rare in immunocompetent patients and salivary gland localization has only been reported in five cases.

Uneven distribution of mating types among genotypes of Candida glabrata isolates from clinical samples.

In order to shed light on its basic biology, we initiated a population genetic analysis of Candida glabrata, an emerging pathogenic yeast with no sexual stage yet recognized. A worldwide collection of clinical strains was subjected to analysis using variable number of tandem repeats (VNTR) at nine loci. The clustering of strains obtained with this method was congruent with that obtained using sequence polymorphism of the NMT1 gene, a locus previously proposed for lineage assignment. Linkage disequilibrium supported the hypothesis of a mainly clonal reproduction. No heterozygous diploid genotype was found. Minimum-spanning tree analysis of VNTR data revealed clonal expansions and associated genotypic diversification. Mating type analysis revealed that 80% of the strains examined are MATa and 20% MATalpha and that the two alleles are not evenly distributed. The MATa genotype dominated within large clonal groups that contained only one or a few MATalpha types. In contrast, two groups were dominated by MATalpha strains. Our data are consistent with rare independent mating type switching events occurring preferentially from type a to alpha, although the alternative possibility of selection favoring type a isolates cannot be excluded.

Functional networks of co-expressed genes to explore iron homeostasis processes in the pathogenic yeast Candida glabrata.

Candida glabrata is a cause of life-threatening invasive infections especially in elderly and immunocompromised patients. Part of human digestive and urogenital microbiota, C. glabrata faces varying iron availability, low during infection or high in digestive and urogenital tracts. To maintain its homeostasis, C. glabrata must get enough iron for essential cellular processes and resist toxic iron excess. The response of this pathogen to both depletion and lethal excess of iron at 30°C have been described in the literature using different strains and iron sources. However, adaptation to iron variations at 37°C, the human body temperature and to gentle overload, is poorly known. In this study, we performed transcriptomic experiments at 30°C and 37°C with low and high but sub-lethal ferrous concentrations. We identified iron responsive genes and clarified the potential effect of temperature on iron homeostasis. Our exploration of the datasets was facilitated by the inference of functional networks of co-expressed genes, which can be accessed through a web interface. Relying on stringent selection and independently of existing knowledge, we characterized a list of 214 genes as key elements of C. glabrata iron homeostasis and interesting candidates for medical applications.