Precise genome editing underlines the distinct contributions of mutations in ERG11, ERG3, MRR1, and TAC1 genes to antifungal resistance in Candida parapsilosis.

Candida parapsilosis has recently emerged as a major threat due to the worldwide emergence of fluconazole-resistant strains causing clonal outbreaks in hospitals and poses a therapeutic challenge due to the limited antifungal armamentarium. Here, we used precise genome editing using CRISPR-Cas9 to gain further insights into the contribution of mutations in ERG11, ERG3, MRR1, and TAC1 genes and the influence of allelic dosage to antifungal resistance in C. parapsilosis. Seven of the most common amino acid substitutions previously reported in fluconazole-resistant clinical isolates (including Y132F in ERG11) were engineered in two fluconazole-susceptible C. parapsilosis lineages (ATCC 22019 and STZ5). Each mutant was then challenged in vitro against a large array of antifungals, with a focus on azoles. Any possible change in virulence was also assessed in a Galleria mellonella model. We successfully generated a total of 19 different mutants, using CRISPR-Cas9. Except for R398I (ERG11), all remaining amino acid substitutions conferred reduced susceptibility to fluconazole. However, the impact on fluconazole in vitro susceptibility varied greatly according to the engineered mutation, the stronger impact being noted for G583R acting as a gain-of-function mutation in MRR1. Cross-resistance with newer azoles, non-medical azoles, but also non-azole antifungals such as flucytosine, was occasionally noted. Posaconazole and isavuconazole remained the most active in vitro. Except for G583R, no fitness cost was associated with the acquisition of fluconazole resistance. We highlight the distinct contributions of amino acid substitutions in ERG11, ERG3, MRR1, and TAC1 genes to antifungal resistance in C. parapsilosis.

Automatic patient-level recognition of four Plasmodium species on thin blood smear by a real-time detection transformer (RT-DETR) object detection algorithm: a proof-of-concept and evaluation.

Malaria remains a global health problem, with 247 million cases and 619,000 deaths in 2021. Diagnosis of Plasmodium species is important for administering the appropriate treatment. The gold-standard diagnosis for accurate species identification remains the thin blood smear. Nevertheless, this method is time-consuming and requires highly skilled and trained microscopists. To overcome these issues, new diagnostic tools based on deep learning are emerging. This study aimed to evaluate the performances of a real-time detection transformer (RT-DETR) object detection algorithm to discriminate Plasmodium species on thin blood smear images. The algorithm was trained and validated on a data set consisting in 24,720 images from 475 thin blood smears corresponding to 2,002,597 labels. Performances were calculated with a test data set of 4,508 images from 170 smears corresponding to 358,825 labels coming from six French university hospitals. At the patient level, the RT-DETR algorithm exhibited an overall accuracy of 79.4% (135/170) with a recall of 74% (40/54) and 81.9% (95/116) for negative and positive smears, respectively. Among Plasmodium-positive smears, the global accuracy was 82.7% (91/110) with a recall of 90% (38/42), 81.8% (18/22), and 76.1% (35/46) for P. falciparum, P. malariae, and P. ovale/vivax, respectively. The RT-DETR model achieved a World Health Organization (WHO) competence level 2 for species identification. Besides, the RT-DETR algorithm may be run in real-time on low-cost devices such as a smartphone and could be suitable for deployment in low-resource setting areas lacking microscopy experts.IMPORTANCEMalaria remains a global health problem, with 247 million cases and 619,000 deaths in 2021. Diagnosis of Plasmodium species is important for administering the appropriate treatment. The gold-standard diagnosis for accurate species identification remains the thin blood smear. Nevertheless, this method is time-consuming and requires highly skilled and trained microscopists. To overcome these issues, new diagnostic tools based on deep learning are emerging. This study aimed to evaluate the performances of a real-time detection transformer (RT-DETR) object detection algorithm to discriminate Plasmodium species on thin blood smear images. Performances were calculated with a test data set of 4,508 images from 170 smears coming from six French university hospitals. The RT-DETR model achieved a World Health Organization (WHO) competence level 2 for species identification. Besides, the RT-DETR algorithm may be run in real-time on low-cost devices and could be suitable for deployment in low-resource setting areas.

The Novodiag® Stool parasites assay, an innovative high-plex technique for fast detection of protozoa, helminths and microsporidia in stool samples: a retrospective and prospective study.

OBJECTIVES: We provide the first evaluation of the CE-IVD marked Novodiag® stool parasites assay (NVD), allowing rapid and high-plex detection of 26 distinct targets, encompassing protozoans, helminths and microsporidia in stool samples. METHODS: A total of 254 samples (n = 205 patients) were prospectively processed by the NVD and our routine procedure (RP). Performances of the NVD were compared with RP. Samples only positive by the NVD assay were investigated by external PCR assays. Sensitivity and specificity (Se/Sp) and time from sample receipt to results were determined for each method. The NVD was also evaluated against 77 additional samples positive for a wide range of parasites. RESULTS: Overall positivity rate was 16.9% for RP compared with 34% using the NVD assay, and 164 samples (66%) were negative by both methods. Only 30 positive samples (12%) showed full concordance between RP and NVD. Fifty-three discordant samples were sent for external investigations. Except for Giardia intestinalis and Trichuris spp., higher Se was observed for the NVD assay for Blastocystis spp. (100% vs. 63%), Dientamoeba fragilis (100% vs. 0%), Schistosoma spp. (100% vs. 17%), and Enterobius vermicularis (100% vs. 67%) but roughly similar to RP for the remaining parasites tested. False-positive results were identified for Blastocystis spp., G. intestinalis, and Trichuris spp. using the NVD assay. The NVD mostly provides a diagnosis on the day of sample receipt compared with a mean of three days with RP. CONCLUSIONS: Besides some limitations, the NVD is a new diagnostic strategy allowing rapid and high-plex detection of gastrointestinal parasites from unpreserved stools.

Title: Le test Novodiag® Stool parasites, une technique high-plex innovante pour la détection rapide des protozoaires, helminthes et microsporidies dans les échantillons de selles : une étude rétrospective et prospective. Abstract: Objectifs : Nous présentons la première évaluation du kit Novodiag® Stool parasite (NVD) marqué CE-IVD, permettant la détection rapide de 26 cibles distinctes dans les selles (protozoaires, helminthes et microsporidies). Méthodes : Un total de 254 échantillons (n = 205 patients) a été traité prospectivement par le NVD et notre procédure de routine (PR). Les performances du NVD ont été comparées à celles de la PR. Seuls les échantillons positifs au test NVD ont été étudiés par des PCR externes. La sensibilité et la spécificité (Se/Sp) ainsi que le temps écoulé entre la réception de l'échantillon et les résultats ont été déterminés pour chaque méthode. Le NVD a également été évalué par rapport à 77 échantillons supplémentaires positifs pour un large éventail de parasites. Résultats : Le taux de positivité global était de 16,9 % pour la PR contre 34 % avec le NVD, et 164 échantillons (66 %) étaient négatifs par les deux méthodes. Seuls 30 échantillons positifs (12 %) ont montré une concordance complète entre la PR et le NVD. Cinquante-trois échantillons discordants ont été envoyés pour des investigations externes. À l'exception de Giardia intestinalis et de Trichuris spp., des Se plus élevées ont été observées pour le test NVD pour Blastocystis spp. (100 % contre 63 %), Dientamoeba fragilis (100 % contre 0 %), Schistosoma spp. (100 % contre 17 %), Enterobius vermicularis (100 % contre 67 %) mais étaient à peu près similaires à la PR pour les autres parasites testés. Des faux positifs ont été identifiés pour Blastocystis spp., G. intestinalis et Trichuris spp. en utilisant le NVD. Le NVD fournit le plus souvent un diagnostic le jour de la réception du prélèvement contre une moyenne de trois jours avec la PR. Conclusions : Malgré quelques limites, le test NVD est une nouvelle stratégie de diagnostic permettant une détection rapide et high-plex des parasites gastro-intestinaux à partir de selles non conservées.

Medicopsis romeroi nodular subcutaneous infection in a kidney transplant recipient.

Phaeohyphomycosis is a set of fungal infections caused by various dematiaceous fungi such as coelomycetes. These infections can occur either in immunocompetent or immunocompromised patients like solid organ transplants. Here we describe a nodular lesion of the right hallux that occurred in a kidney transplant patient. Microscopic examination of the biopsy revealed fungal hyphae and culture was positive to a grey to black mould that lacked characteristic elements to be identified. Nucleic acid sequencing targeting the internal transcribed spacer of the ribosomal DNA identified this mould as Medicopsis romeroi. The patient benefited of an antifungal therapy with voriconazole associated with surgical excision of the lesion. No relapse of the lesion was observed during a six-month follow-up. In solid organ transplants, phaeohyphomycosis caused by Medicopsis romeroi are very rare with only 12 cases reported. The clinical history should be well assessed since the lesion can appear several years after a cutaneous trauma that happened in a tropical region. Therapy generally combines antifungals with surgical excision of the lesion in order to avoid any relapse or dissemination of the infection.