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Histopathology is the gold standard for fungal infection (FI) diagnosis, but it does not provide a genus and/or species identification. The objective of the present study was to develop targeted next-generation sequencing (NGS) on formalin-fixed tissue samples (FTs) to achieve a fungal integrated histomolecular diagnosis.

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Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM Objectives Histopathology is the gold standard for distinguishing between colonization and fungal infection, but it does not provide a precise diagnosis of genera/species. However, if the culture is negative or if no specimen is sent to the Mycology laboratory, only the specimen sent to the Pathology department is available. Formalin fixation and paraffin embedding (FFPE) cause DNA damage, making it difficult to perform molecular techniques. The objective was to develop and evaluate the contribution of massive parallel sequencing (MPS) to FFPE tissues. Methods Histopathological review of all cases was performed. Then, DNA extraction from FFPE tissues was optimized by: (1) macrodissecting the fungal-rich area on the paraffin block; (2) comparing the efficiency of two DNA extraction kits (QIAamp DNAmicro-kit, QIAGEN; Maxwell 16 LEVRNA FFPE Purification kit, Promega, optimized for RNA and DNA extraction), by comparison of Aspergillus fumigatus and Mucorale specific PCR results for 30 cases. For 124 other cases, the sensitivity of two primer pairs (ITS3/4 and MITS2A/2B) was tested for identification by Sanger sequencing and then MPS. Finally, a histomolecular comparison was performed. This work was funded by the Société de Pathologie Infectieuse de Langue Française (SPILF). Results To optimize extraction, DNA was extracted by both kits from samples of 16 mucormycoses and 14 A. fumigatus infections. PCR sensitivity was better with the QIAGEN extraction kit [100% (30/30)] compared to the Promega kit [86.7% (26/30)]. PCR amplification of fungal DNA from an additional 124 FFPE samples was performed. The primer pairs ITS3/4 and MITS2A/2B, allowed: (1) identification by Sanger sequencing-histopathological analysis in 38.7% (48/124) of the cases in total, and more specifically 33% (41/124) of cases with the ITS3/4 primers and 32.3% (40/124) of cases with the MITS2A/B primers; and (2) identification by integrated SMP-histopathological analysis in 75% (93/124) of all cases (primers ITS3/4 and MITS2A/2B), and more specifically 66.1% (82/124) for ITS3/4 and 62.1% (77/124) for MITS2A/B (both primer pairs did not detect/amplify the same fungal genera/species). The combination of all results from Sanger sequencing and MPS led to fungal identification in 75.8% (94/124) of cases. In total, the addition of NGS to Sanger sequencing increased the diagnostic proportion by 36.3% (45/124; P Conclusion The development of the fungal MPS on FFPE tissues is innovative and unprecedented for the achievement of an integrated histomolecular diagnosis in fungal pathology. It increases significantly the diagnostic proportion by 36.3%. This strategy can be used in hospitals and could improve patient management, especially when no sample is sent to the Mycology laboratory or when the culture is negative.

Occurrence of putative invasive pulmonary aspergillosis was screened in 153 consecutive adult intensive care unit (ICU) patients with respiratory samples addressed for mycological diagnosis during a 6-week period at the emergence of coronavirus disease 2019 (COVID-19) pandemic. Positive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) polymerase chain reaction (PCR) was observed for 106 patients (69.3%). Nineteen of them (17.9%) with positive Aspergillus results were considered as having putative invasive pulmonary aspergillosis. These observations underline the risk of pulmonary aspergillosis in COVID-19 patients, even in patients not previously known to be immunosuppressed, advocating active search for Aspergillus infection and prompt antifungal treatment. Standardized surveillance protocols and updated definitions for ICU putative invasive pulmonary aspergillosis are needed. Lay Abstract Adult ICU patients with respiratory samples addressed for mycological diagnosis were screened during the emergence of COVID-19 pandemic. Positive SARS-CoV-2 PCR was observed for 106 patients, nineteen of them (17.9%) having aspergillosis. This underlines the risk of aspergillosis in COVID-19 patients.

This study aims at evaluating fluconazole exposure in critically ill patients and identifying variables associated with the latter.This was a 2-year (2018-2019) retrospective multicenter cohort study. Adult patients 18 years-old with at least one fluconazole concentration measurement during their ICU stay were included.Twenty patients were included. Only 11 patients had a fluconazole trough concentration (Cmin) within the target range (≥15 mg/L). According to bivariable analysis, SOFA score, GGT, fluconazole clearance, Ke, and Vd, were independently associated with a decrease in fluconazole Cmin. The median loading dose required to achieve the Cmin target appeared to be greater in patients with higher SOFA or GGT level and in patients undergoing renal replacement therapy.This study supports recommendation for routine fluconazole therapeutic drug monitoring in ICU patients so as to avoid underexposure, especially if SOFA score is ≥ 7 and/or GGT is ≥ 100 U/L.

The emergence of azole resistant Aspergillus spp., especially Aspergillus fumigatus, has been described in several countries around the world with varying prevalence depending on the country. To our knowledge, azole resistance in Aspergillus spp. has not been reported in the West Indies yet. In this study, we investigated the antifungal susceptibility of clinical and environmental isolates of Aspergillus spp. from Martinique, and the potential resistance mechanisms associated with mutations in cyp51A gene. Overall, 208 Aspergillus isolates were recovered from clinical samples (n = 45) and environmental soil samples (n = 163). They were screened for resistance to azole drugs using selective culture media. The Minimum Inhibitory Concentrations (MIC) towards voriconazole, itraconazole, posaconazole and isavuconazole, as shown by the resistant isolates, were determined using the European Committee on Antimicrobial Susceptibility Testing (EUCAST) microdilution broth method. Eight isolates (A. fumigatus, n = 6 and A. terreus, n = 2) had high MIC for at least one azole drug. The sequencing of cyp51A gene revealed the mutations G54R and TR34/L98H in two A. fumigatus clinical isolates. Our study showed for the first time the presence of azole resistance in A. fumigatus and A. terreus isolates in the French West Indies.

The emergence of azole resistant Aspergillus spp., especially Aspergillus fumigatus, has been described in several countries around the world with varying prevalence depending on the country. To our knowledge, azole resistance in Aspergillus spp. has not been reported in the West Indies yet. In this study, we investigated the antifungal susceptibility of clinical and environmental isolates of Aspergillus spp. from Martinique, and the potential resistance mechanisms associated with mutations in cyp51A gene. Overall, 208 Aspergillus isolates were recovered from clinical samples (n = 45) and environmental soil samples (n = 163). They were screened for resistance to azole drugs using selective culture media. The Minimum Inhibitory Concentrations (MIC) towards voriconazole, itraconazole, posaconazole and isavuconazole, as shown by the resistant isolates, were determined using the European Committee on Antimicrobial Susceptibility Testing (EUCAST) microdilution broth method. Eight isolates (A. fumigatus, n = 6 and A. terreus, n = 2) had high MIC for at least one azole drug. The sequencing of cyp51A gene revealed the mutations G54R and TR34/L98H in two A. fumigatus clinical isolates. Our study showed for the first time the presence of azole resistance in A. fumigatus and A. terreus isolates in the French West Indies.

Resume Du 24 juin au 6 juillet 2005, neuf hommes se sont presentes successivement aux urgences de l’hopital de Fort-de-France (Martinique, Antilles francaise) avec une symptomatologie pulmonaire plus ou moins marquee associee dans deux cas a la presence de lesions cutanees. Ces neuf hommes avaient tous realise des travaux dans une maison abandonnee trois semaines auparavant. Le diagnostic d’histoplasmose a pu etre etabli grâce aux examens mycologiques realises sur les prelevements pulmonaires (l’examen direct et la culture), la biologie moleculaire et les serologies. L’interrogatoire et l’enquete environnementale sur la zone presumee de l’exposition aux spores d’ H . capsulatum var. capsulatum a permis de confirmer les lieux et les modalites de contamination.

We report a case of cerebral phaeohyphomycosis, a fungal brain infection due to a dark (dematiaceous) fungi in a 6-year-old French Guyanese boy. The child presented fever and drowsiness due to several paraventricular brain abscesses. Neurological surgeries were performed to reduce intracranial hypertension and to obtain abscess biopsies. Mycological cultures of intraoperative samples led to the diagnosis of cerebral phaeohyphomycosis due to Cladophialophora bantiana. The patient neurological status deteriorated and remained critical after several weeks of combination antifungal therapy with voriconazole 8 mg/kg/day, liposomal amphotericin B 10 mg/kg/day and flucytosine 200 mg/kg/day. A complete surgical resection was not possible because of multiple small abscesses. A multidisciplinary ethical staff decided on home medical care with palliative ventriculoperitoneal shunt, nasogastric feeding and analgesics. One year later, the patient's neurological condition had improved and cerebral lesions had regressed, while he had not received any antifungal treatment but only traditional medicines. Cerebral phaeohyphomycosis are rare diseases affecting immunocompromised but also apparently non-immunocompromised patients, as in this case. A complete surgical resection is not always possible and mortality rates are high in spite of treatments with a combination of antifungals. The diagnosis may be difficult because of these dematiaceous fungi's slowly growing and their potential pathogenicity for laboratory staff.

Neuroangiostrongyliasis is a parasitic disease caused by the accidental ingestion of the nematode Angiostrongylus cantonensis in its larval form. Human infection can lead to eosinophilic meningitis, sometimes complicated by life-threatening radiculomyelitis or encephalitis. Although some cases have been reported from other Caribbean Islands, no cases have been diagnosed in Martinique so far. Here, we report the first eight laboratory-confirmed cases of neuroangiostrongyliasis on the island of Martinique, French West Indies, between 1 January 2002 and 31 December 2017. One case was fatal and five resulted in neurological sequelae. The medical community should consider the risk of A. cantonensis infection in patients living in or returning from Martinique.Premiers cas d’angiostrongylose à Angiostrongylus cantonensis à la Martinique, de 2002 à 2017.L’angiostrongylose neuroméningée est une maladie parasitaire causée par l’ingestion accidentelle du nématode Angiostrongylus cantonensis sous sa forme larvaire. L’infection humaine peut conduire à une méningite à éosinophiles, pouvant évoluer en radiculomyélite ou encéphalite menaçant le pronostic vital. Bien que des cas aient été rapportés dans d’autres îles des Caraïbes, aucun cas n’avait été diagnostiqué à la Martinique jusqu’à présent. Dans cet article, nous caractérisons les huit premiers cas d’angiostrongylose neuroméningée biologiquement diagnostiqués à la Martinique, Antilles françaises, entre le 1

Scopulariopsis species are rarely but increasingly recognized as opportunistic pathogens in immunocompromised patients. We report on a patient suffering from cystic fibrosis who developed disseminated fungal infection due to a rare Scopulariopsis species, Microascus cirrosus , after heart and lung transplantation. Despite antifungal combination therapy with voriconazole and caspofungin, the patient died 4 weeks after transplantation. Diagnostic difficulties and optimal management of disseminated Scopulariopsis / Microascus infections are discussed.

Background The Caribbean is the second most affected region in the world by human immunodeficiency virus (HIV), and HIV prevalence is significantly higher among persons in jails and prisons than in the free population. The aim of our study was to assess the screening rates of HIV, hepatitis B and C, syphilis and human T cell leukaemia virus type 1 among newly-arrived persons in 2014, at Ducos facility in Martinique and the testing process performance. Methods This is an observational monocentric study conducted within the prison’s health unit. The study population consisted of all individuals incarcerated between 01/01/14 and 31/12/14. At the initial medical visit, HIV and STI testing were proposed to every newcomer. The rate of acceptance was calculated, as well as the screening process performance. Results In 2014 778 new persons were incarcerated, among those, 461 (59.3%) were tested. The main reasons for missing the testing opportunity were due to organization of the judiciary system (persons on electronic monitoring or day parole, transferred or quickly released before completion of the process) or to individual refusal. Finally, 75 persons did not get their results (all of them negative), 41 of them due to the medical staff work overload. Conclusions HIV and STI testing rates among newcomers at Ducos have notable room for improvement. The future availability of combined (HIV, HBV, HCV and syphilis) rapid tests may be very useful in case of short term incarceration, if their cost is not prohibitive. Reaching higher levels of testing will also require more resources.

Introduction L’angiostrongylose humaine est une zoonose transmise par un nematode du genre Angiostrongylus. Les 2 principales especes pathogenes identifiees chez l’Homme sont Angiostrongylus cantonensis ( A. cantonensis ) et Angiostrongylus costaricensis ( A. costaricensis ) responsables respectivement d‘angiostrongylose nerveuse (meningo-encephalite a eosinophiles) et d’angiostrongylose intestinale (pseudo-appendicite aigue avec hypereosinophilie). Cette pathologie est endemique sur le continent asiatique et en ocean pacifique mais reste peu decrite dans la Caraibe. La presence conjointe de ces 2 especes d’ Angiostrongylus dans un meme ecosysteme est exceptionnelle et n’a jamais ete rapportee, a notre connaissance, dans les petites Antilles. L’objectif de cette etude etait donc de decrire les donnees epidemiologiques sur l’angiostrongylose humaine en Martinique. Materiels et methodes Une etude observationnelle retrospective monocentrique a ete menee dans notre centre hospitalier entre le 01/01/2000 et le 31/12/2017. Tout patient ayant un diagnostic parasitologique (serologie ou PCR) ou anatomo-pathologique confirme d’angiostrongylose nerveuse ou intestinale etait inclus. Resultats Douze cas de d’angiostrongylose humaine ont ete identifies dans notre etude, incluant 8 cas d’angiostrongylose nerveuse a A. cantonensis et 4 cas d’angiostrongylose intestinale a A. costaricensis . L’âge median etait de 14 ans (IQR : 1–27) avec un sex-ratio homme/femme de 2. Une fievre (> 38 °C) etait observee dans 92 % des cas (11/12). Tous les patients presentaient une hypereosinophilie (> 0,5 G/L) avec une duree mediane de persistance de l’eosinophilie de 23,5 jours (IQR : 14,5–34,5). Tous les patients avaient une confirmation diagnostique serologique (sang ou LCR) ou anatomopathologique pour les cas d’ A. costaricensis . Le mode de transmission probable etait l’ingestion ou un contact etroit dans l’environnement avec Achatina fulica (hote intermediaire) dans 83 % des cas (10/12). Onze patients ont recu un traitement par albendazole (10/12) ou ivermectine (1/12). L’evolution etait favorable dans 11 cas sur 12. Un deces d’angiostrongylose neurologique a ete observe. Conclusion Nous decrivons la plus grande serie de cas d’angiostrongylose humaine dans la Caraibe. L’angiostrongylose, pathologie negligee, doit cependant etre evoquee devant toute hypereosinophilie avec atteinte neurologique ou abdominale en Martinique. Une meilleure connaissance de son hote intermediaire, Achatina fulica , devrait permettre d’ameliorer les strategies de prevention primaire et secondaire.

Diagnosis: lacaziosis, or lobomycosis [1]. The diagnosis was confirmed by direct examination of the skin biopsy, highlighting the presence of round or oval yeastlike organisms 6–12 μm in diameter, either isolated or in chains, with a birefringent membrane (Figure 1). The periodic acid-Schiff–stained and the Gomori-Grocott methenamine silver–stained sections of the sample revealed short chains of lemon-shaped fungal cells connected by thin, tubelike bridges (Figure 2). Morphology was consistent with the appearance of Lacazia loboi. There was a minor improvement of the lesion after pentamidine treatment, whereas the Leishmania research by microscopic examination, culture, and molecular biology was negative. However, these exams were done after treatment initiation, so an initial coinfection cannot be ruled out. Mycological culturesonSabouraud-gentamicin-cycloheximide withandwithout actidionewereperformedandwerenegativeafter 2 months. The patient is currently under treatment with terbinafine (250 mg twice daily), awaiting surgical excision of the lesion. Lacaziosis, or lobomycosis [1], is a chronic infectious disease that was first described in 1930 in Brazil by the dermatologist Jorge Lobo [2]. Since then, >500 human cases have been reported [3], mainly in Central and South America. The etiological agent, Lacazia loboi ( formerly known as Loboa loboi), an Onygenales dimorphic fungus [4], seems to be saprophytic in vegetation, soil, and water. It gains access to the skin following cutaneous traumatism, or animal bites [5]. After the initial lesion, there is a slow local dissemination period leading to pleomorphic lesions. The typical clinical aspect is generally multiple keloidal lesions, with a smooth, shiny aspect, but can also appear as papules, nodules, ulcers, sclerodermiform, or verrucous plaques [6]. These lesions are painless and hypoor hyperchromic. Clinical diagnosis is generally difficult because of pathology scarcity and frequently atypical cases. For example, infiltrated plaque-type lesions can be mistaken for cutaneous leishmaniasis; lesions with a keloidal aspect can be mistaken for lepromatous leprosy [7]; and lesions with a verrucous, vegetating, or nodular aspect can be mistaken for sporotrichosis, chromoblastomycosis, paracoccidioidomycosis, keloids, or neoplastic processes [6]. The diagnosis is based onmacroscopic examination of the lesions and on direct microscopic examination of biopsy or curettage. The diagnostic feature of Lacazia loboi is the chain of samesized buds, unlike Paracoccidioides, whose buds are smaller than the mother cell. Figure 1. Skin biopsy: round or oval yeast-like organisms from 6 to 12 μm in diameter, isolated or in chains with a bi-refringent membrane; direct microscopic examination (×1000 magnification). Figure 2. Skin biopsy section. Short chains of lemon-shaped fungal cells connected by thin, tubelike bridges (arrow). A, Periodic acidSchiff stain. B, Gomori-Grocott methenamine silver stain (×1000 magnification).

Objectif Preciser la repartition et la sensibilite des souches de levures isolees d’hemocultures de patients hospitalises dans le service de reanimation du CHU de la Martinique sur la periode 2001–2013. Methode Analyse retrospective des resultats de l’ensemble des candidemies (et levures apparentees) diagnostiquees au laboratoire de microbiologie entre le 1er janvier 2001 et le 31 decembre 2013. Identification a l’espece basee sur des caracteres morphologiques et biochimiques. Sensibilite aux antifongiques determinee par comparaison des CMI obtenues par bandelette E-test (bioMerieux) vis-a-vis des concentrations critiques actuellement definies par l’EUCAST et le CLSI, lorsque disponibles. Resultats Cent trente-deux souches de levures correspondant a 128 episodes septiques (moy. 9,8/an) furent isolees chez 121 patients. Candida albicans etait l’espece la plus prevalente (n = 40 ; 30,3 %), suivie de C. tropicalis (n = 30 ; 22,7 %), C. parapsilosis (n = 24 ; 18,2 %), C. glabrata (n = 17 ; 12,9 %), C. haemulonii (n = 13 ; 9,8 %) et S. cerevisiae (n = 5 ; 3,8 %). Candida krusei, C. dubliniensis et T. asahii furent chacune impliquees dans un episode septique (0,8 %). La proportion de souches fluconazole-R ou SDD etait de 6,1 %, 9,1 % et 13,6 % pour C. albicans, C. tropicalis et C. parapsilosis, respectivement, tandis que 76,9 % des souches de C. haemulonii presentaient une CMI elevee au fluconazole (> 2 μg/mL). Tous les isolats de C. albicans et C. tropicalis etaient sensibles au voriconazole contre 95 % des souches de C. parapsilosis, tandis que 91,7 % des souches de C. glabrata etaient considerees comme « sauvages » (CMI ≤ ECOFF). Le pourcentage de resistance a la caspofungine etait de 0 %, 3,6 % et 12,5 % pour C. tropicalis, C. albicans, C. parapsilosis, respectivement ; une proportion importante de C. glabrata (66,7 %) presentait une CMI superieure au breakpoint defini par le CLSI. Enfin, toutes les souches etaient sensibles a l’amphotericine B hormis la majorite des isolats de C. haemulonii (84,6 % avaient une CMI > 2 μg/mL). Discussion Nos resultats soulignent la frequence ainsi que les particularites epidemiologiques des candidemies survenues dans notre service de reanimation ces 13 dernieres annees. La part belle est faite aux levures autres que C. albicans (∼ 70 % contre 43 % dans l’etude AMARCAND), notamment C. tropicalis, C. glabrata, C. parapsilosis et la levure emergente C. haemulonii. Cette derniere, conformement a la litterature, presente des CMI souvent tres elevees pour l’amphotericine B et le fluconazole. La frequence de CMI elevees de C. glabrata a la caspofungine nous interpelle quant a sa capacite a predire la sensibilite aux echinocandines. Conclusion Cette etude met l’accent sur l’originalite des candidemies a la Martinique. Le role du laboratoire reste primordial dans leur prise en charge. Cela passe en priorite par une identification correcte des isolats afin d’orienter les cliniciens dans leur choix therapeutique dans l’attente des resultats de l’antifongigramme.

Introduction Les infections a Magnusiomyces capitatus (anciennement Geotrichum capitatum ) et a Geotrichum candidum sont rares. M. capitatus et G. candidum sont des levures cosmopolites. M. capitatus est considere comme pathogene emergent opportuniste chez les patients immunodeprimes. Nous decrivons le premier cas de co-infection pulmonaire a M. capitatus et a G. candidum chez une patiente presentant un adenocarcinome mammaire. Observation Une patiente de 65 ans etait hospitalisee pour un bilan de dyspnee d’apparition recente dans un contexte d’alteration de l’etat general. Cette patiente avait beneficie d’une mastectomie en 2012 pour un carcinome canalaire in situ droit. L’examen clinique revelait un syndrome cave superieur. Le scanner thoracique montrait des plages de condensation des lobes superieurs et moyens droits associees a une lymphangite carcinomateuse avec des polyadenopathies mediastinales et hilaires ainsi que des metastases hepatiques. Des epanchements pericardiques et pleuraux de grande abondance etaient egalement objectives. Les resultats anatomo-pathologiques de la biopsie ganglionnaire au niveau mediastinal confirmaient un adenocarcinome moyennement differencie en faveur d’une origine mammaire. Les bilans bacteriologiques et virologiques etaient negatifs. L’examen mycologique du lavage broncho-alveolaire a permis la visualisation de filaments myceliens arthrospores et de rares levures bourgeonnantes, a l’examen direct (coloration May-Grunwald Giemsa) et l’isolement en culture, sur Sabouraud chloramphenicol ± actidione, a 30 °C et 37 °C de M. capitatus et G. candidum . Un traitement antifongique n’a pas ete initie devant le refus de la patiente. Apres concertation pluridisciplinaire, la patiente a beneficie d’une prise en charge palliative. L’evolution etait rapidement defavorable conduisant au deces de la patiente 4 jours apres le diagnostic mycologique. Discussion Il existe moins de 100 cas rapportes d’infections disseminees a M. capitatus et a G. candidum . Ces champignons sont des levures ascomycetes pour lesquelles un portage pulmonaire est souvent retrouve. Leur pathogenicite dans les infections survient souvent sur un terrain d’immunodepression lie a une hemopathie pour les quelques cas decrits. Le diagnostic mycologique de ces deux levures est base sur la presence commune de filaments et d’arthrospores a l’examen direct. L’isolement en culture sur milieu de Sabouraud a 30 °C et 37 °C permet l’isolement de colonies blanches avec presence de filaments arthrospores de grand diametre, des arthrospores ± blastospores en fonction de l’espece. Le traitement antifongique repose generalement sur une association d’amphotericine B et de 5-fluorocytosine. Le role de la pathologie neoplasique sous-jacente et des pathogenes opportunistes dans l’evolution fatale de ce cas sont a discuter et renvoient au benefice d’une therapeutique antifongique systemique. Conclusion La co-infection a M. capitatus et G. candidum sur un terrain neoplasique est exceptionnelle et permet de nous sensibiliser sur l’emergence de mycoses profondes atypiques.

Background: Crohn's disease (CD) mainly affects young adults of reproductive age. Whereas a large amount of data is available concerning pregnancy in young CD women, no study has been conducted on their ovarian reserve status. This study aimed to investigate the potential effect of CD on ovarian reserve in young women in remission, as reflected by serum anti-Mullerian hormone (AMH). Methods: This retrospective case-control study was conducted in the University Hospital of Nantes, France. Serum levels of AMH were retrospectively measured in 50 women with CD in remission and in 163 control women with normal ovarian reserve, matched by age. Results: No statistical difference was found between mean serum AMH levels in CD and control women. Serum AMH levels remained comparable between CD and control women