Nosocomial transmission of Aspergillus flavus in a neonatal intensive care unit: Long-term persistence in environment and interest of MALDI-ToF mass-spectrometry coupled with convolutional neural network for rapid clone recognition.

Aspergillosis of the newborn remains a rare but severe disease. We report four cases of primary cutaneous Aspergillus flavus infections in premature newborns linked to incubators contamination by putative clonal strains. Our objective was to evaluate the ability of matrix-assisted laser desorption/ionisation time of flight (MALDI-TOF) coupled to convolutional neural network (CNN) for clone recognition in a context where only a very small number of strains are available for machine learning. Clinical and environmental A. flavus isolates (n = 64) were studied, 15 were epidemiologically related to the four cases. All strains were typed using microsatellite length polymorphism. We found a common genotype for 9/15 related strains. The isolates of this common genotype were selected to obtain a training dataset (6 clonal isolates/25 non-clonal) and a test dataset (3 clonal isolates/31 non-clonal), and spectra were analysed with a simple CNN model. On the test dataset using CNN model, all 31 non-clonal isolates were correctly classified, 2/3 clonal isolates were unambiguously correctly classified, whereas the third strain was undetermined (i.e., the CNN model was unable to discriminate between GT8 and non-GT8). Clonal strains of A. flavus have persisted in the neonatal intensive care unit for several years. Indeed, two strains of A. flavus isolated from incubators in September 2007 are identical to the strain responsible for the second case that occurred 3 years later. MALDI-TOF is a promising tool for detecting clonal isolates of A. flavus using CNN even with a limited training set for limited cost and handling time.

Cutaneous aspergillosis is a rare but potentially fatal disease of the prematurely born infant. We described here several cases due to Aspergillus flavus and have linked them to environnemental strains using MLP genotyping and MALDI-TOF mass spectrometry coupled with artificial intelligence.

Surfaces and Air Contamination by Severe Acute Respiratory Syndrome Coronavirus 2 Using High-Flow Nasal Oxygenation or Assisted Mechanical Ventilation in Intensive Care Unit Rooms of Patients With Coronavirus Disease 2019.

BACKGROUND: Understanding patterns of environmental contamination by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for infection prevention policies. METHODS: We screened surfaces and air samples from single-bed intensive-care unit rooms of adult patients with coronavirus disease 2019 (COVID-19) for SARS-CoV-2 RNA and viable viruses. RESULTS: We evidenced viral RNA environmental contamination in 76% of 100 surfaces samples and in 30% of 40 air samples without any viable virus detection by cell culture assays. No significant differences of viral RNA levels on surfaces and in ambient air were observed between rooms of patients with assisted mechanical ventilation and those of patients with a high-flow nasal cannula system. Using an original experimental SARS-CoV-2 infection model of surfaces, we determined that infectious viruses may have been present on benches within 15 hours before the time of sampling in patient rooms. CONCLUSIONS: We observed that SARS-CoV-2 environmental contamination around patients with COVID-19 hospitalized in single-bed ICU rooms was extensive and that a high-flow nasal cannula system did not generate more viral aerosolization than a mechanical ventilation system in patients with COVID-19. Despite an absence of SARS-CoV-2 viable particles in study samples, our experimental model confirmed the need to apply strict environmental disinfection procedures and classic standard and droplet precautions in ICU wards.

Contribution of the anaerobic blood culture vial for the recovery of Candida glabrata: A retrospective multicentric study.

Although Candida spp are aerobic microorganisms, some Candida strains, mainly Candida glabrata, can be recovered from anaerobic blood culture vials. We assessed the contribution of the anaerobic vials for the diagnosis of candidemia, especially for C. glabrata. We conducted a multicenter retrospective study including eight university or regional hospitals. A single episode of monomicrobial candidemia per patient was included from September 1st, 2016, to August 31st, 2019. The characteristics of all aerobic and anaerobic blood culture vials sampled within 2 h before and after the first positive blood culture vials were recorded (type of vials, result, and for positive vials time-to-positivity and Candida species). Overall, 509 episodes of candidemia were included. The main species were C. albicans (55.6%) followed by C. glabrata (17.1%), C. parapsilosis (4.9%), and C. tropicalis (4.5%). An anaerobic vial was positive in 76 (14.9%) of all episodes of which 56 (73.8%) were due to C. glabrata. The number of C. glabrata infections only positive in anaerobic vials was 1 (2.6%), 1 (11.1%), and 15 (37.5%) with the BACT/ALERT 3D the BACT/ALERT VIRTUO and the BACTEC FX instrument, respectively (P < 0.01). The initial positivity of an anaerobic vial was highly predictive of the isolation of C. glabrata with the BACTEC FX (sensitivity of 96.8%). C. glabrata time-to-positivity was shorter in anaerobic vial than aerobic vial with all instruments. Anaerobic blood culture vials improve the recovery of Candida spp mainly C. glabrata. This study could be completed by further analyses including mycological and pediatric vials. LAY SUMMARY: Although Candida spp are aerobic microorganisms, C. glabrata is able to grow in anaerobic conditions. In blood culture, the time-to-positivity of C. glabrata is shorter in anaerobic than aerobic vials. Only the anaerobic vial was positive in up to 15 (37.5%) C. glabrata bloodstream infections.

The shift from pulmonary colonization to Pneumocystis pneumonia.

Pulmonary specimen pairs from five patients who presented with pulmonary colonization and later developed Pneumocystis Pneumonia (PcP) were retrospectively examined for P. jirovecii genotyping. A match of genotypes in pulmonary specimen pairs of three patients was observed, whereas a partial match and a mismatch were observed in the fourth and fifth patients, respectively. The genotyping results suggest that the colonization state can differ from PcP but can also represent the incubation period of PcP. Clinicians should not systematically rule out the treatment of putative colonized patients and should at least discuss the initiation of prophylaxis on a case-by-case basis.

The results suggest that clinicians should not systematically rule out the treatment of putative patients colonized by Pneumocystis jirovecii and should at least discuss prophylaxis initiation on a case-by-case basis.

Optimization of MALDI-ToF mass spectrometry for yeast identification: a multicenter study.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) is routinely used in mycology laboratories to rapidly identify pathogenic yeasts. Various methods have been proposed to perform routine MS-based identification of clinically relevant species. In this study, we focused on Bruker technology and assessed the identification performance of three protocols: two pretreatment methods (rapid formic acid extraction directly performed on targets and full extraction using formic acid/acetonitrile in tubes) and a direct deposit protocol that omits the extraction step. We also examined identification performance using three target types (ground-steel, polished-steel, and biotargets) and two databases (Bruker and online MSI [biological-mass-spectrometry-identification application]) in a multicenter manner. Ten European centers participated in the study, in which a total of 1511 yeast isolates were analyzed. The 10 centers prospectively performed the three protocols on approximately 150 yeast isolates each, and the corresponding spectra were then assessed against two reference spectra databases (MSI and Bruker), with appropriate thresholds. Three centers evaluated the impact of the targets. Scores were compared between the various combinations, and identification accuracy was assessed. The protocol omitting the extraction step was inappropriate for yeast identification, while the full extraction method yielded far better results. Rapid formic acid extraction yielded variable results depending on the target, database and threshold. Selecting the optimal extraction method in combination with the appropriate target, database and threshold may enable simple and accurate identification of clinically relevant yeast samples. Concerning the widely used polished-steel targets, the full extraction method still ensured better scores and better identification rates.

Detection methods and prevalence of transmission stages of Toxoplasma gondii, Giardia duodenalis and Cryptosporidium spp. in fresh vegetables: a review.

One of the ways of human parasitic infection is the accidental ingestion of vegetables contaminated with parasites, which represents a major human health hazard. This non-exhaustive review aims to evaluate studies carried out on five types of vegetables (lettuce, parsley, coriander, carrot and radish) since 2000, particularly the methods used for recovery, concentration, detection and identification of protozoan parasites such as Toxoplasma gondii, Giardia duodenalis and Cryptosporidium spp., and the results of each work. Various studies have determined the presence of pathogenic parasites in fresh vegetables with different rates; this variation in rate depends particularly on the detection method used which is related to each parasite and each vegetable type. The variation in parasitic prevalence in food could be due to different factors such as the geographical location, the size of analysed samples and the methods used for parasite detection.

Discovery of New Inhibitors of Toxoplasma gondii via the Pathogen Box.

Toxoplasma gondii is a cosmopolitan protozoan parasite which affects approximately 30% of the population worldwide. The drugs currently used against toxoplasmosis are few in number and show several limitations, such as drug intolerance, poor bioavailability, or drug resistance mechanism developed by the parasite. Thus, it is important to find new compounds able to inhibit parasite invasion or proliferation. In this study, the 400 compounds of the open-access Pathogen Box, provided by the Medicines for Malaria Venture (MMV) foundation, were screened for their anti-Toxoplasma gondii activity. A preliminary in vitro screening performed over 72 h by an enzyme-linked immunosorbent assay (ELISA) revealed 15 interesting compounds that were effective against T. gondii at 1 µM. Their cytotoxicity was estimated on Vero cells, and their 50% inhibitory concentrations (IC50) were further calculated. As a result, eight anti-Toxoplasma gondii compounds with an IC50 of less than 2 µM and a selectivity index (SI) value of greater than 4 were identified. The most active was MMV675968, showing an IC50 of 0.02 µM and a selectivity index value equal to 275. Two other compounds, MMV689480 and MMV687807, also showed a good activity against T. gondii, with IC50s of 0.10 µM (SI of 86.6) and 0.15 µM (SI of 11.3), respectively. Structure-activity relationships for the eight selected compounds also were discussed on the basis of fingerprinting similarity measurements using the Tanimoto method. The anti-Toxoplasma gondii compounds highlighted here represent potential candidates for the development of new drugs that could be used against toxoplasmosis.

Donor Derived Candida stellimalicola in a Clinical Specimen: Preservation Fluid Contamination During Pancreas Procurement.

We report here a case of possible donor-derived Candida stellimalicola infection after pancreas transplantation. Candida stellimalicola, an environmental non-filamentous yeast, was isolated from both the peritoneal fluid of the graft donor and the preservation fluid of the transplanted pancreas. Interestingly, this strain exhibited high minimum inhibitory concentrations to azoles. These results justified the use of echinocandins as therapy instead of fluconazole. This switch permitted a favorable outcome. To our knowledge, this is the first report of C. stellimalicola from clinical samples and therefore the first reported case of a possible human infection. This case report highlights the need for standardized microbiological procedures in solid organ transplant settings. Moreover, it underlines the importance of using molecular identification technique when routine techniques do not allow successful identification of the pathogen. It is of utmost importance to determine sensitivity profile, even in the absence of species-level identification, because resistance to fluconazole is not uncommon, especially in emergent species.

Features of cryptococcosis among 652 HIV-seronegative individuals in France: a cross-sectional observational study (2005-2020).

OBJECTIVES: We aimed to describe features and outcomes of cryptococcosis among HIV-seronegative individuals in a large surveillance network for cryptococcosis in France. METHODS: We included incident cases of cryptococcosis in HIV-seronegative individuals from 2005 to 2020. We compared patient characteristics, disease presentations, cryptococcal antigen results, and induction antifungal treatments according to underlying disease. We examined factors associated with 90-day mortality. Among patients with disseminated infections, we investigated whether receipt of flucytosine and polyene combination was associated with lower mortality. RESULTS: Among 652 individuals, 209 (32.1%) had malignancy, 130 (19.9%) were solid-organ transplant recipients, 204 (31.3%) had other immunocompromising conditions, and 109 (16.7%) had no reported underlying factor. The commonest presentations were disseminated infections (63.3%, 413/652) and isolated pulmonary infections (25.3%, 165/652). Solid-organ transplant patients were most likely to have disseminated infections and a positive serum cryptococcal antigen result. Patients with malignancy were older and less likely to receive a flucytosine-containing regimen for disseminated infections than others (58.7%, 78/133 vs. 73.2%, 194/265; p 0.029). The crude 90-day case-fatality ratio was 27.2% (95% CI, 23.5%-31.1%). Age ≥60 years (aOR: 2.75 [1.78-4.26]; p < 0.001), meningitis/fungaemia (aOR: 4.79 [1.80-12.7]; p 0.002), and malignancy (aOR: 2.4 [1.14-5.07]; p 0.02) were associated with higher 90-day mortality. Receipt of flucytosine and polyene combination was associated with lower 90-day mortality (aOR: 0.40 [0.23-0.71]; p 0.002) in multivariable analysis and inverse probability of treatment weighted analysis (aOR: 0.45 [0.25-0.80]; p 0.006). DISCUSSION: HIV-seronegative individuals with cryptococcosis comprise a wide range of underlying conditions with different presentations and outcomes, requiring a tailored approach to diagnosis and management.

Cryptococcus neoformans Infections Differ Among Human Immunodeficiency Virus (HIV)-Seropositive and HIV-Seronegative Individuals: Results From a Nationwide Surveillance Program in France.

Among 1107 cryptococcosis cases from the French surveillance network (2005-2020), the proportion of HIV-seronegative individuals has recently surpassed that of HIV-seropositive individuals. We observed marked differences in patient characteristics, disease presentations, cryptococcal antigen results, infecting species, and mortality according to HIV serostatus.

Malaria diagnosis in an emergency department before and after the COVID-19 pandemic: a retrospective study.

BACKGROUND: The COVID-19 pandemic impacted access to health facilities. METHODS: We assessed the number of blood smears sampled in the emergency department (ED) among all blood smears performed for malaria in Reims University Hospital before and after 2020. RESULTS: We showed a decrease in the number of blood smears performed after the onset of the COVID-19 pandemic, but only in 2020. The seasonal increase of cases during summer was preserved. All blood smears positive for malaria in 2020 were sampled in the ED. CONCLUSIONS: The ED played a key role in the diagnosis of malaria with the onset of the COVID-19 pandemic.

MALDI-TOF: A new tool for the identification of Schistosoma cercariae and detection of hybrids.

Schistosomiasis is a neglected water-born parasitic disease caused by Schistosoma affecting more than 200 million people. Introgressive hybridization is common among these parasites and raises issues concerning their zoonotic transmission. Morphological identification of Schistosoma cercariae is difficult and does not permit hybrids detection. Our objective was to assess the performance of MALDI-TOF (Matrix Assistated Laser Desorption-Ionization-Time Of Flight) mass spectrometry for the specific identification of cercariae in human and non-human Schistosoma and for the detection of hybridization between S. bovis and S. haematobium. Spectra were collected from laboratory reared molluscs infested with strains of S. haematobium, S. mansoni, S. bovis, S. rodhaini and S. bovis x S. haematobium natural (Corsican hybrid) and artificial hybrids. Cluster analysis showed a clear separation between S. haematobium, S. bovis, S. mansoni and S. rodhaini. Corsican hybrids are classified with those of the parental strain of S. haematobium whereas other hybrids formed a distinct cluster. In blind test analysis the developed MALDI-TOF spectral database permits identification of Schistosoma cercariae with high accuracy (94%) and good specificity (S. bovis: 99.59%, S. haematobium 99.56%, S. mansoni and S. rodhaini: 100%). Most misidentifications were between S. haematobium and the Corsican hybrids. The use of machine learning permits to improve the discrimination between these last two taxa, with accuracy, F1 score and Sensitivity/Specificity > 97%. In multivariate analysis the factors associated with obtaining a valid identification score (> 1.7) were absence of ethanol preservation (p < 0.001) and a number of 2-3 cercariae deposited per well (p < 0.001). Also, spectra acquired from S. mansoni cercariae are more likely to obtain a valid identification score than those acquired from S. haematobium (p<0.001). MALDI-TOF is a reliable technique for high-throughput identification of Schistosoma cercariae of medical and veterinary importance and could be useful for field survey in endemic areas.

On the systematics of Phlebotomus betisi and two new related species from Laos with proposal of the new subgenus Lewisius.

Phlebotomus betisi was described from Malaysia and classified after its description in the subgenus Larroussius. It was the only species to have a pharyngeal armature composed of dot-like teeth and an annealed spermatheca whose head is carried by a neck in females. Males were characterized by having a style bearing five spines and a simple paramere. The study of sandflies originating from a cave in Laos enabled us to discover and describe two sympatric species close to Ph. betisi Lewis & Wharton, 1963 and new for Science: Ph. breyi Vongphayloth & Depaquit n. sp., and Ph. sinxayarami Vongphayloth & Depaquit n. sp. They were characterized morphologically, morphometrically, geomorphometrically, molecularly, and proteomically (MALDI-TOF). All approaches converged to validate the individualization of these species whose morphological differential characters lay in the two genders by the observation of the interocular suture and by the length of the last two segments of the maxillary palps. In males, the length of the genital filaments discriminates these species. Females are distinguished by the length of the ducts of the spermathecae as well as by the narrow or enlarged shape of the neck bearing their head. Lastly, the particular position of the spines of the gonostyle coupled with molecular phylogeny led us to remove these three species from the subgenus Larroussius Nizulescu, 1931 and to classify them in a new subgenus: Lewisius Depaquit & Vongphayloth n. subg.

Title: Sur la systématique de Phlebotomus betisi et de deux nouvelles espèces apparentées du Laos avec proposition du nouveau sous-genre Lewisius. Abstract: Phlebotomus betisi a été décrit de Malaisie et fut classé après sa description dans le sous-genre Larroussius. C'était la seule espèce à posséder chez la femelle une armature pharyngienne composée de dents en forme de points et à avoir une spermathèque annelée dont la tête est portée par un cou. Les mâles se caractérisaient par un style porteur de cinq épines et par un paramère simple. L'étude de Phlébotomes originaires d'une grotte du Laos nous a permis de découvrir et de décrire deux espèces sympatriques proches de Ph. betisi Lewis & Wharton, 1963 et nouvelles pour la Science : Ph. breyi Vongphayloth & Depaquit n. sp., et Ph. sinxayarami Vongphayloth & Depaquit n. sp. Elles ont été caractérisées morphologiquement, morphométriquement, géomorphométriquement, moléculairement et protéomiquement (MALDI-TOF). Toutes ces approches convergent pour valider l'individualisation de chacune de ces espèces dont les caractères morphologiques différentiels reposent dans les deux sexes par l'observation de la suture interoculaire et par la longueur des deux derniers segments des palpes maxillaires. Chez les mâles, la longueur des filaments génitaux discrimine ces espèces. Les femelles sont distinguées par la longueur des conduits des spermathèques ainsi que par la forme étroite ou élargie du cou portant la tête de ces spermathèques. Enfin, la position particulière des épines sur le gonostyle couplée à une phylogénie moléculaire nous amène à extraire ces trois espèces du sous genre Larroussius Nitzulescu, 1931 pour les classer dans un nouveau sous-genre : Lewisius Depaquit & Vongphayloth n. subg.

Invasive fungal diseases in patients with autoimmune diseases: a case series from the French RESSIF network.

OBJECTIVES: We aimed to describe patients with autoimmune diseases (AID) developing invasive fungal disease (IFD) and identify factors associated with short-term mortality. METHODS: We analysed cases of IFD associated with AID from the surveillance network of invasive fungal diseases (Réseau de surveillance des infections fongiques invasives, RESSIF) registry of the French national reference centre for invasive mycoses. We studied association of AID-specific treatments with 30-day mortality. We analysed total lymphocyte and CD4-T cell counts in patients with Pneumocystis jirovecii pneumonia (PCP). RESULTS: From 2012 to 2018, 549 individuals with IFD and AID were included, mainly with PCP (n=227, 41.3%), fungemia (n=167, 30.4%) and invasive aspergillosis (n=84, 15.5%). Rheumatoid arthritis (RA) and anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitides (AAV) were the most frequent AID in PCP (n=55 and 25, respectively) and invasive aspergillosis (n=15 and 10, respectively), inflammatory bowel diseases (IBDs) were predominant in fungemia (n=36). At IFD diagnosis, 365 (66.5%) patients received glucocorticoids (GCs), 285 (51.9%) immunosuppressants, 42 (7.7%) tumor necrosis factor (TNF)-α blockers, 75 (13.7%) other biologics. Mortality at 30 days was 28.1% (143/508). Fungemia and high-dose GCs were independently associated with higher 30-day mortality. In PCP patients, lymphopenia <1500/mm3 was frequent (132/179, 73.7%) even if CD4+T cell count exceeded 200/mm3 in 56/78 patients (71.8%) (median 472.5/mm3, IQR 160-858). CONCLUSION: IFD associated with AID occurs primarily in RA, AAV and IBD, especially when treated with GCs and immunosuppressants. Mortality is high, especially for patients on high-dose GCs. Lymphopenia may help identify risk of PCP, but normal CD4+T cell count does not rule out the risk. Further studies are needed to assess the individual risk factors for IFD.

Gradient concentration strip-specific epidemiological cut-off values of antifungal drugs in various yeast species and five prevalent Aspergillus species complexes.

OBJECTIVES: To determine the epidemiological cut-off values (ECVs) of ten antifungal agents in a wide range of yeasts and Aspergillus spp. using gradient concentration strips. METHODS: The minimum inhibitory concentrations for amphotericin B, anidulafungin, caspofungin, micafungin, flucytosine, fluconazole, itraconazole, isavuconazole, posaconazole, and voriconazole, determined with gradient concentration strips at 35 French microbiology laboratories between 2002 and 2020, were retrospectively collected. Then, the ECVs were calculated using the iterative method and a cut-off value of 97.5%. RESULTS: Minimum inhibitory concentrations were available for 17 653 clinical isolates. In total, 48 ECVs (including 32 new ECVs) were determined: 29 ECVs for frequent yeast species (e.g. Candida albicans and itraconazole/flucytosine, and Candida glabrata species complex [SC] and flucytosine) and rare yeast species (e.g. Candida dubliniensis, Candida inconspicua, Saccharomyces cerevisiae, and Cryptococcus neoformans) and 19 ECVs for Aspergillusflavus SC, Aspergillusfumigatus SC, Aspergillusnidulans SC, Aspergillusniger SC, and Aspergillusterreus SC. CONCLUSIONS: These ECVs can be added to the already available gradient concentration strip-specific ECVs to facilitate minimum inhibitory concentration interpretation and streamline the identification of nonwild type isolates.

Broad respiratory virus detection in infants hospitalized for bronchiolitis by use of a multiplex RT-PCR DNA microarray system.

Newly available molecular tools allow a sensitive detection of a broad panel of viruses in respiratory tract specimens. In the present study, the application of a multiplex RT-PCR DNA microarray in diagnosis and epidemiological survey of viral infections in infants hospitalized for bronchiolitis was assessed. One hundred and thirty-eight nasopharyngeal aspirates collected from October 2007 to September 2008 were tested by direct immunofluorescence and viral culture, a combination of referenced RT-PCRs and the DNA microarray. One or more viruses were detected in 96, 126 and 126 of the specimens by direct immunofluorescence and viral culture, RT-PCRs and DNA microarray, respectively (70 vs. 91 vs. 91%, P < 10(-3)). The RT-PCRs and the DNA microarray yielded concordant results for 99% of specimens and identified mixed viral infections in 85 (62%). The most common associations were: human bocavirus and respiratory syncytial virus (32%), adenovirus and respiratory syncytial virus (30%), and parainfluenza virus type 3 and respiratory syncytial virus (23%). None of the bronchiolitis severity parameters including intensive care unit admission, O(2) supply, O(2) saturation percentage, O(2) length and length of stay at the hospital appeared to be significantly increased in multiple viral infections compared to single viral infections (P > 0.1). In conclusion, the use of this DNA microarray in clinical virology practice allows rapid and accurate identification of common and uncommon viral respiratory pathogens in infants hospitalized for bronchiolitis. It should improve the clinical management, the epidemiological survey, and the prevention of the nosocomial transmission of respiratory viruses in pediatric wards.

Identification of a clonal population of Aspergillus flavus by MALDI-TOF mass spectrometry using deep learning.

The spread of fungal clones is hard to detect in the daily routines in clinical laboratories, and there is a need for new tools that can facilitate clone detection within a set of strains. Currently, Matrix Assisted Laser Desorption-Ionization Time-of-Flight Mass Spectrometry is extensively used to identify microbial isolates at the species level. Since most of clinical laboratories are equipped with this technology, there is a question of whether this equipment can sort a particular clone from a population of various isolates of the same species. We performed an experiment in which 19 clonal isolates of Aspergillus flavus initially collected on contaminated surgical masks were included in a set of 55 A. flavus isolates of various origins. A simple convolutional neural network (CNN) was trained to detect the isolates belonging to the clone. In this experiment, the training and testing sets were totally independent, and different MALDI-TOF devices (Microflex) were used for the training and testing phases. The CNN was used to correctly sort a large portion of the isolates, with excellent (> 93%) accuracy for two of the three devices used and with less accuracy for the third device (69%), which was older and needed to have the laser replaced.

MALDI-TOF MS Limits for the Identification of Mediterranean Sandflies of the Subgenus Larroussius, with a Special Focus on the Phlebotomus perniciosus Complex.

Leishmania infantum is the agent of visceral leishmaniasis in the Mediterranean basin. It is transmitted by sandflies of the subgenus Larroussius. Although Phlebotomus perniciosus is the most important vector in this area, an atypical Ph. perniciosus easily confused with Ph. longicuspis has been observed in North Africa. MALDI-TOF MS, an important tool for vector identification, has recently been applied for the identification of sandflies. Spectral databases presented in the literature, however, include only a limited number of Larroussius species. Our objective was to create an in-house database to identify Mediterranean sandflies and to evaluate the ability of MALDI-TOF MS to discriminate close species or atypical forms within the Larroussius subgenus. Field-caught specimens (n = 94) were identified morphologically as typical Ph. perniciosus (PN; n = 55), atypical Ph. perniciosus (PNA; n = 9), Ph. longicuspis (n = 9), Ph. ariasi (n = 9), Ph. mascittii (n = 3), Ph. neglectus (n = 5), Ph. perfiliewi (n = 1), Ph. similis (n = 9) and Ph. papatasi (n = 2). Identifications were confirmed by sequencing of the mtDNA CytB region and sixteen specimens were included in the in-house database. Blind assessment on 73 specimens (representing 1073 good quality spectra) showed a good agreement (98.5%) between MALDI-TOF MS and molecular identification. Discrepancies concerned confusions between Ph. perfiliewi and Ph. perniciosus. Hierarchical clustering did not allow classification of PN and PNA. The use of machine learning, however, allowed discernment between PN and PNA and between the lcus and lcx haplotypes of Ph. longicuspis (accuracy: 0.8938 with partial-least-square regression and random forest models). MALDI-TOF MS is a promising tool for the rapid and accurate identification of field-caught sandflies. The use of machine learning could allow to discriminate similar species.

Species Identification and In Vitro Antifungal Susceptibility of Paecilomyces/Purpureocillium Species Isolated from Clinical Respiratory Samples: A Multicenter Study.

Paecilomyces spp. are emerging fungal pathogens, where Paecilomyceslilacinus and Paecilomyces variotii are the most reported species. Taxonomic and phylogenetic revisions in this genus have shown that P. variotii represents a species complex, whereas P. lilacinus is related to another genus called Purpureocillium. The aims of this study were to identify clinical isolates of Paecilomyces spp. at the species level, and to determine their antifungal susceptibility profiles. 70 clinical Paecilomyces spp. isolates were identified by MALDI-TOF Mass Spectrometry (MS) and by multilocus rDNA genes sequencing including ITS and the D1/D2 genes. Among the 70 Paecilomyces spp. isolates, 28 were identified as P. lilacinum, 26 as P. variotii stricto sensu, and 16 as P. maximus. For antifungal susceptibility testing, Minimal Inhibitory Concentrations (MICs) or Minimal Effective Concentrations (MECs) were determined for 8 antifungals. All P. lilacinum isolates had high MICs and MECs of amphotericin B and echinocandins, respectively, unlike P. variotii and P. maximus. For azole drugs, MICs were molecule- and species- dependent. The differences in in vitro susceptibility to antifungals underline the importance of accurate species identification. The MALDI-TOF MS can be a good alternative in routine laboratory to ensure fast identification of Paecilomyces spp. and P. lilacinum.

Case Report: Could Artemisinin-Based Combination Therapy Prevent Occupational Malaria following Blood Exposure?

Postexposure prophylaxis using artemisinin-based combination therapy (ACT) was prescribed to a malaria-naive nurse who experienced an injury with a hollow needle previously used on a patient admitted for severe imported Plasmodium falciparum malaria (blood parasitemia 10.8%). Artemether-lumefantrine, four tablets twice daily for 3 days, was started 12 hours after exposure, and no side effects were reported. During the six following months, she only developed one episode of fever that was associated with pyelonephritis. Biological follow-up, based on blood smears, molecular biology, and serology, did not evidence P. falciparum malaria. This case suggests that ACT can prevent occupational P. falciparum malaria following needle-stick injury. We found evidence of only one other unpublished similar case where a Turkish resident doctor did not develop malaria after postexposure prophylaxis using ACT. Such a prophylaxis could be prescribed not only in case of occupational exposure to Plasmodium spp. in nonvector-borne laboratory-acquired infections but also following blood exposure in healthcare setting.