Leprosy in French Guiana, 2015 to 2021: Dynamics of a Persistent Public Health Problem.

A resurgence of leprosy as a public health problem in French Guiana was reported over the period 2007 to 2014, particularly among Brazilians gold miners. Prolonged multidrug therapy and reversal reactions represent a therapeutic challenge. The objective of this study was to assess the evolution of leprosy in this European overseas territory. All patients with leprosy confirmed in histopathology between 1 January 2015 and 31 December 2021 were included. A total of 86 patients were included, including 64 new cases and 22 previously diagnosed cases. Sixty patients (70%) were male, 6 cases were paediatric. Brazilian gold miners represented 44.1% of reported occupations (15/34). Maroons represented the second community (13 patients, 15%). Multibacillary and paucibacillary forms were found in 53 (71%) and 22 (29%) patients, respectively. The annual prevalence never exceeded the threshold of 1/10,000. The mean incidence and prevalence were significantly lower than during the period 2007 to 2014 (p < 0.0001). Reversal reactions were found in 29 patients and almost always required a long course of steroids. Infliximab allowed a reduction in the length of treatment with steroids in 2/2 cases. In conclusion, the prevalence of leprosy has decreased significantly in French Guiana, but remains driven by the population of illegal gold miners. Anti-tumour necrosis factor (anti-TNF) drugs represent a promising option in the management of reversal reactions.

Using pentamidine to treat cutaneous leishmaniasis in children: a 10-year study in French Guiana.

There are little data on pentamidine as a treatment for paediatric cutaneous leishmaniasis (CL). The objective of this study was to describe the effectiveness and safety of pentamidine over a 10-year period. Every child seen in French Guiana between 2010 and 2020 with proven CL and treated with pentamidine was included. In total, 55 children met the inclusion criteria - 23 girls and 32 boys. There were 38 patients (38/55, 69%) with a > 50% improvement at 1 month after pentamidine treatment and a complete cure at 3 months; 16 children had a < 50% improvement at 1 month and were given a second dose. Of these 16, 8 showed a complete cure at 3 months, 5 were lost to follow-up and 3 showed therapeutic failure at 3 months. The overall cure rate was 84% (46/55) after one or two doses. In terms of the safety of pentamidine, no severe adverse events (grade ≥ 3) were reported.

Pulmonary Histoplasmosis in People Living with Human Immunodeficiency Virus in French Guiana: Clinical Epidemiology, Medical Imaging and Prognostic.

BACKGROUND: Histoplasmosis is mainly described as a disseminated disease in people living with HIV (PLHIV). Compared to historical descriptions in immunocompetent individuals, knowledge is lacking on the detailed clinical and radiological findings and outcomes of pulmonary histoplasmosis (PH). Overlooked or misdiagnosed with other AIDS-defining condition, prognostic of PLHIV may be at risk because of inappropriate care. METHODS: A retrospective multicentric study was conducted in PLHIV from French Guiana between January 1988 and October 2019. Proven PH were documented through mycological direct examination, culture, or histology. Patients with concomitant respiratory infections were excluded. RESULTS: Among 65 patients, sex ratio M:F was 2.4 with a median age of 39 years [IQR 25-75%: 34-44]. Median CD4 count was 24 cells/mm3 [11-71], with histoplasmosis as the AIDS-defining condition in 88% and concomitant AIDS-defining conditions in 29%. Clinical findings were fever (89%), cough (58%), dyspnea (35%), expectoration (14%), and hemoptysis (5%). Sixty-one X-rays and 24 CT-scans were performed. On X-rays, an interstitial lung disease was mainly found (77%). On CT-scans, a nodular pattern was predominant (83%): mostly miliary disease (63%), but also excavated nodules (35%). Consolidations were present in 46%, associated with miliary disease in 21%. Thoracic lymphadenopathies were found in 58%, mainly hilar and symmetric (33%). Despite antifungal treatment, case-fatality rate at one month was 22%. CONCLUSION: When faced with an interstitial lung disease on X-rays or a miliary pattern on CT-scans in advanced PLHIV, physicians in endemic areas, apart from tuberculosis or pneumocystosis, should include histoplasmosis as part of their differential diagnoses.

What about Current Diversity of Mycolactone-Producing Mycobacteria? Implication for the Diagnosis and Treatment of Buruli Ulcer.

The identification of an emerging pathogen in humans can remain difficult by conventional methods such as enrichment culture assays that remain highly selective, require appropriate medium and cannot avoid misidentifications, or serological tests that use surrogate antigens and are often hampered by the level of detectable antibodies. Although not originally designed for this purpose, the implementation of polymerase-chain-reaction (PCR) has resulted in an increasing number of diagnostic tests for many diseases. However, the design of specific molecular assays relies on the availability and reliability of published genetic sequences for the target pathogens as well as enough knowledge on the genetic diversity of species and/or variants giving rise to the same disease symptoms. Usually designed for clinical isolates, molecular tests are often not suitable for environmental samples in which the target DNA is mixed with a mixture of environmental DNA. A key challenge of such molecular assays is thus to ensure high specificity of the target genetic markers when focusing on clinical and environmental samples in order to follow the dynamics of disease transmission and emergence in humans. Here we focus on the Buruli ulcer (BU), a human necrotizing skin disease mainly affecting tropical and subtropical areas, commonly admitted to be caused by Mycobacterium ulcerans worldwide although other mycolactone-producing mycobacteria and even mycobacterium species were found associated with BU or BU-like cases. By revisiting the literature, we show that many studies have used non-specific molecular markers (IS2404, IS2606, KR-B) to identify M. ulcerans from clinical and environmental samples and propose that all mycolactone-producing mycobacteria should be definitively considered as variants from the same group rather than different species. Importantly, we provide evidence that the diversity of mycolactone-producing mycobacteria variants as well as mycobacterium species potentially involved in BU or BU-like skin ulcerations might have been underestimated. We also suggest that the specific variants/species involved in each BU or BU-like case should be carefully identified during the diagnosis phase, either via the key to genetic identification proposed here or by broader metabarcoding approaches, in order to guide the medical community in the choice for the most appropriate antibiotic therapy.

Kaposi Sarcoma with Mucocutaneous Involvement in French Guiana: An Epidemiological Study between 1969 and 2019.

Data on Kaposi sarcoma in French Guiana are scarce and out of date. This territory presents unique epidemiological features. The objectives of this retrospective study were to analyse the clinical features, outcome and incidence of the different forms of Kaposi sarcoma in patients diagnosed between 1969 and 2019. The study population comprised a total of 52 patients. Clinical forms included epidemic Kaposi sarcoma (n=30), endemic (n=18), iatrogenic (n=2), classic (n=1) and unclassified Kaposi sarcoma (n=1). The mean annual incidence rate of epidemic Kaposi reached a peak in the 1990s (0.93/100,000) then decreased in the 2000s (0.33/100,000), while the incidence of endemic Kaposi sarcoma reached a peak in the 1980s (0.82/100,000) before decreasing in the 2000s (0.12/100,000). Factors associated with the epidemic form were: sexual intercourse between men (p=0.0054) and Haitian origin (p=0.035). The presence of nodules and/or tumour, lesions limited to the lower limbs, and age >65 years were associated with the endemic form. While Creole populations seem to be as affected by endemic Kaposi sarcoma as their African counterparts, the dynamics of Kaposi sarcoma in French Guiana are now dominated by the epidemic form.

Validation of Swab Sampling and SYBR Green-Based Real-Time PCR for the Diagnosis of Cutaneous Leishmaniasis in French Guiana.

Recent studies have highlighted the interest in noninvasive sampling procedures coupled with real-time PCR methods for the detection of Leishmania species in South America. In French Guiana, the sampling method still relied on skin biopsies. Noninvasive protocols should be tested on a large annual cohort to improve routine laboratory diagnosis of cutaneous leishmaniasis. Therefore, we evaluated the performance of a new Leishmania detection and species identification protocol involving cotton swabs and SYBR green-based real-time PCR of the Hsp70 gene, coupled with Sanger sequencing. Between May 2017 and May 2018, 145 patients with ulcerated lesions compatible with cutaneous leishmaniasis were included in the study at the Cayenne Hospital and its remote health centers. Each patient underwent scrapings for a smear, skin biopsies for parasite culture and PCR-restriction fragment length polymorphism (RFLP) (RNA polymerase II), and sampling with a cotton swab for SYBR green-based PCR. The most accurate diagnostic test was the SYBR green-based PCR on swab samples, showing 98% sensitivity. The mean PCR cycle threshold (CT ) was 24.4 (minimum CT , 17; maximum CT , 36) and was <35 in 97.6% of samples. All samples positive by SYBR green-based real-time PCR were successfully identified at the species level by DNA sequencing. This new method should be considered for routine diagnosis of cutaneous leishmaniasis in South America and especially for remote areas, since noninvasive collection tools are easier to use and require fewer precautions for transportation.

Review of diagnostic methods and results for HIV-associated disseminated histoplasmosis: Pathologists are not sufficiently involved.

OBJECTIVES: Disseminated histoplasmosis is a major killer of HIV-infected persons in Latin America. Antigen detection, fungal culture and Polymerase Chain Reaction are often not available, but cytology and histology are present in most hospitals and may offer a diagnostic alternative. In this study, we review 34 years of clinical experience to describe the roles of cytology and histology in diagnosing disseminated histoplasmosis. METHODS: Retrospective multicentric study of 349 patients between 1 January 1981 and 1 October 2014 with confirmed disseminated histoplasmosis. RESULTS: Around 32/214 (14.9%) of samples were screened using cytopathology, as were 10/101 (9.9%) bronchoalveolar lavage samples and 5/61 (8.2%) of spinal fluid samples. The samples most commonly sent to pathology were liver biopsies, lower digestive tract and lymphnode biopsies; the greatest proportion of positive results were found in lower digestive tract (43/59 (72.9%) positives), lymph node (39/63 (66.1%)), and liver (38/75 (50.7%)) samples. Overall, 97.2% of bone marrow and 97% of bronchoalveolar lavage samples were directly examined by a mycologist. Positive direct examination was independently associated with death (aHR = 1.5 (95%CI = 1-2.2)). CONCLUSIONS: Opportunities for a rapid diagnosis were regularly missed, notably for bone marrow samples, which could have been examined using staining methods complementary to those of the mycologist.

Chromoblastomycosis in French Guiana: Epidemiology and Practices, 1955-2023.

Chromoblastomycosis (CBM) is a chronic neglected fungal disease, usually met in tropical areas. French Guiana is a South American territory with limited epidemiological data. This retrospective study concerned all patients with CBM proven by at least one paraclinical examination and diagnosed in French Guiana between 1950 and 2023. In total, 23 patients were included, mostly males (87%) of Creole origin, living in the coastal region (87%) and involved in outdoor occupations (74%). Lesions were mostly observed on the lower limbs (78.3%), with a median time to diagnosis of four years. Laboratory tests included positive direct microscopic examinations (78.3%) and mycological cultures (69.6%), identifying 14 cases of Fonsecaea pedrosoi and one case of Exophiala janselmei. Various treatments were employed, including antifungals, surgery and combinations of both. In conclusion, CBM in French Guiana involves a different population than other subcutaneous mycoses such as Lobomycosis or Paracoccidioidomycosis, mostly found in the forest hinterland. Surgery should be recommended for recent and limited lesions. Itraconazole and terbinafine should systematically be proposed, either in monotherapy or in combination with surgery or cryotherapy.

Migration in French Guiana: Implications in health and infectious diseases.

In French Guiana, more than a third of the population, and nearly half of the adults, are of foreign origin. This immigration is explained by the French standard of living, which is attractive to nationals of surrounding countries. Infectious diseases remain in the top 10 causes of premature death, often in the most precarious populations. In this context we aimed to synthesize the state of the knowledge regarding immigration and infectious diseases in French Guiana and the general implications that follow this diagnosis. For HIV, although the majority of patients are of foreign origin, estimates of the presumed date of infection based on CD4 erosion modelling and from molecular analyses suggest that the majority of transmissions in foreign-born individuals occur in French Guiana and that the Guiana shield has been a crossroad between Latin America and the Caribbean. Among key populations bridging these regions illegal gold miners are very mobile and have the greatest proportion B Caribbean HIV viruses. Gold miners have been a key vulnerable population for falciparum malaria and other tropical diseases such as leishmaniasis, leprosy, or leptospirosis. The complex history of migrations in French Guiana and on the Guiana Shield is also reflected in the fingerprinting of mycobacterium tuberculosis and the high incidence of tuberculosis in French Guiana, notably in immigrants, reflects the incidences in the countries of origin of patients. The high burden of infectious diseases in immigrants in French Guiana is first and foremost a reflection of the precarious living conditions within French Guiana and suggests that community-based proactive interventions are crucial to reduce transmission, morbidity, and mortality from infectious diseases.

Reply to Vilela, R.; Mendoza, L. Current Nomenclature of Paracoccidioides lobogeorgii's Disease Name. Comment on "Grotta et al. Fungal Density in Lobomycosis in French Guiana: A Proposal for a New Clinico-Histological and Therapeutic Classification. J. Fungi 2023, 9, 1005".

We have read with interest the comment sent by Raquel Vilela and Leonel Mendoza [...].

Fungal Density in Lobomycosis in French Guiana: A Proposal for a New Clinico-Histological and Therapeutic Classification.

BACKGROUND: Lobomycosis is a rare cutaneous tropical neglected disease caused by the fungal agent Lacazia loboi, recently renamed Paracoccidioides lobogeorgii. Our objectives were to present all cases of lobomycosis diagnosed in French Guiana, to offer a precise description of their histopathological features and to propose a new clinico-histological prognostic classification. METHODS: All cases of lobomycosis diagnosed in French Guiana between 1959 and 2022 were included. We looked for associations between the occurrence of relapses and the clinic-histological form. RESULTS: 31 patients diagnosed with lobomycosis were included. An epidemiological shift was observed in the 2000s as Brazilian patients became the most important ethnic group. Gold mining, forestry and fishing/sailing were reported as professional exposures. New histological features, such as inflammatory "rosettes" formations were described. We describe two clinic-histological patterns: a major form (high fungal density and/or multifocal lesions) and a minor form (low fungal density, unifocal lesions, association with fewer relapses). CONCLUSIONS: The changing epidemiology of lobomycosis in French Guiana is characterized by a shift towards Brazilian patients, mostly gold miners. Minor forms should be treated with surgery, major forms with a combination of surgery followed by nultiple drug therapy (MDT) or posaconazole.

Morphological and Molecular Characterizations of Cochliomyia hominivorax (Diptera: Calliphoridae) Larvae Responsible for Wound Myiasis in French Guiana.

Myiasis is an ectoparasitic infection caused by the larvae of true flies (Diptera). We came across a rather rare case of myiasis in an immunocompetent 34-year-old man from French Guiana with advanced wound myiasis masquerading as cavitary myiasis and a history of cholesteatoma surgery in the left ear. The Diptera larvae responsible for the disease were isolated and identified using morphological and molecular approaches as Cochliomyia hominivorax. We underline the importance of this parasitosis as the second case of myiasis caused by C. hominivorax and the first case of wound myiasis in this overseas department of France and its incidence in pre-urban areas of the capital, Cayenne, in South America.

Updated estimation of cutaneous leishmaniasis incubation period in French Guiana.

BACKGROUND: The cutaneous leishmaniasis (CL) incubation period (IP) is defined as the time between parasite inoculation by sandfly bite and the onset of the first CL lesion. IP distribution is difficult to assess for CL because the date of exposure to an infectious bite cannot be accurately determined in endemic areas. IP current estimates for CL range from 14 days to several months with a median around 30-60 days, as established by a few previous studies in both New and Old Worlds. METHODOLOGY: We estimated CL incubation period distribution using time-to-event models adapted to interval-censored data based on declared date of travels from symptomatic military personnel living in non-endemic areas that were exposed during their short stays in French Guiana (FG) between January 2001 and December 2021. PRINCIPAL FINDINGS: A total of 180 patients were included, of which 176 were men (97.8%), with a median age of 26 years. When recorded, the parasite species was always Leishmania guyanensis (31/180, 17.2%). The main periods of CL diagnosis spread from November to January (84/180, 46.7%) and over March-April (54/180, 30.0%). The median IP was estimated at 26.2 days (95% Credible Level, 23.8-28.7 days) using a Bayesian accelerated failure-time regression model. Estimated IP did not exceed 62.1 days (95% CI, 56-69.8 days) in 95% of cases (95th percentile). Age, gender, lesion number, lesion evolution and infection date did not significantly modify the IP. However, disseminated CL was significantly associated with a 2.8-fold shortening of IP. CONCLUSIONS: This work suggests that the CL IP distribution in French Guiana is shorter and more restricted than anticipated. As the incidence of CL in FG usually peaks in January and March, these findings suggest that patients are contaminated at the start of the rainy season.

Clinical and immunological spectra of human cutaneous leishmaniasis in North Africa and French Guiana.

Cutaneous leishmaniasis (CL) caused by infection with the parasite Leishmania exhibits a large spectrum of clinical manifestations ranging from single healing to severe chronic lesions with the manifestation of resistance or not to treatment. Depending on the specie and multiple environmental parameters, the evolution of lesions is determined by a complex interaction between parasite factors and the early immune responses triggered, including innate and adaptive mechanisms. Moreover, lesion resolution requires parasite control as well as modulation of the pathologic local inflammation responses and the initiation of wound healing responses. Here, we have summarized recent advances in understanding the in situ immune response to cutaneous leishmaniasis: i) in North Africa caused by Leishmania (L.) major, L. tropica, and L. infantum, which caused in most cases localized autoresolutives forms, and ii) in French Guiana resulting from L. guyanensis and L. braziliensis, two of the most prevalent strains that may induce potentially mucosal forms of the disease. This review will allow a better understanding of local immune parameters, including cellular and cytokines release in the lesion, that controls infection and/or protect against the pathogenesis in new world compared to old world CL.

[Overview of infectious and non-infectious diseases in French Guiana in 2022]. / Panorama des pathologies infectieuses et non infectieuses de Guyane en 2022.

Source of many myths, French Guiana represents an exceptional territory due to the richness of its biodiversity and the variety of its communities. The only European territory in Amazonia, surrounded by the Brazilian giant and the little-known Suriname, Ariane 6 rockets are launched from Kourou while 50% of the population lives below the poverty line. This paradoxical situation is a source of health problems specific to this territory, whether they be infectious diseases with unknown germs, intoxications or chronic pathologies.Some infectious diseases such as Q fever, toxoplasmosis, cryptococcosis or HIV infection are in common with temperate countries, but present specificities leading to sometimes different management and medical reasoning. In addition to these pathologies, many tropical diseases are present in an endemic and / or epidemic mode such as malaria, leishmaniasis, Chagas disease, histoplasmosis or dengue. Besides, Amazonian dermatology is extremely varied, ranging from rare but serious pathologies (Buruli ulcer, leprosy) to others which are frequent and benign such as agouti lice (mites of the family Trombiculidae) or papillonitis. Envenomations by wild fauna are not rare, and deserve an appropriate management of the incriminated taxon. Obstetrical, cardiovascular and metabolic cosmopolitan pathologies sometimes take on a particular dimension in French Guiana that must be taken into account in the management of patients. Finally, different types of intoxication are to be known by practitioners, especially due to heavy metals.European-level resources offer diagnostic and therapeutic possibilities that do not exist in the surrounding countries and regions, thus allowing the management of diseases that are not well known elsewhere.Thanks to these same European-level resources, research in Guyana occupies a key place within the Amazon region, despite a smaller population than in the surrounding countries. Thus, certain pathologies such as histoplasmosis of the immunocompromised patient, Amazonian toxoplasmosis or Q fever are hardly described in neighboring countries, probably due to under-diagnosis linked to more limited resources. French Guiana plays a leading role in the study of these diseases.The objective of this overview is to guide health care providers coming to or practicing in French Guiana in their daily practice, but also practitioners taking care of people returning from French Guiana.

Leprosy as immune reconstitution inflammatory syndrome in patients living with HIV: Description of French Guiana's cases over 20 years and systematic review of the literature.

BACKGROUND: HIV infection is highly prevalent in French Guiana, a territory where leprosy is also endemic. Since the introduction of Highly Active Antiretroviral Treatment (HAART) in the management of HIV, leprosy has been reported as part of the immune reconstitution inflammatory syndrome (IRIS). METHODOLOGY/PRINCIPAL FINDINGS: We aimed to present a general description of these forms of leprosy as IRIS, highlighting clinical and therapeutic specificities. A retrospective study was conducted in French Guiana, including patients living with HIV (PLHIV) with advanced infection (CD4 < 200/mm3) and developing leprosy or a leprosy reaction within six months of HAART initiation, from 2000 to 2020. Clinical, histological and biological data were collected for all these patients. Six patients were reported in French Guiana. A systematic review of the literature was conducted, and its results were added to an overall analysis. Overall, seventy-three PLHIV were included. They were mainly men (74%), aged 22-54 years (median 36 years), mainly from Brazil (46.5%) and India (32.8%). Most leprosy cases (56.2%) were borderline tuberculoid (BT). Leprosy reactions were frequent (74%), mainly type 1 reaction (T1R) (68.5%), sometimes intense with ulceration of skin lesions (22%). Neuritis was observed in 30.1% of patients. The outcome was always favorable under multidrug therapy (MDT), continuation of HAART and additional corticosteroid therapy in case of neuritis or ulceration. There was no relapse. CONCLUSION: Leprosy as IRIS in PLHIV mainly presents as a BT leprosy in a T1R state, sometimes with ulcerated skin lesions. Response to MDT is usually good. Systemic corticosteroids are necessary and efficient in case of neuritis.

Spectrum of skin diseases in Maroon villages of the Maroni area, French Guiana.

BACKGROUND: Due to their genetic characteristics and their high exposure to infectious diseases, Maroons are likely to suffer from a specific spectrum of skin diseases. However, skin disorders have never been explored in this population. We aimed to describe all skin diseases in Maroon villages of the Maroni region in French Guiana. METHODS: This retrospective study concerned all patients who consulted in the remote health centers of Apatou, Grand-Santi, Papaichton, and Maripasoula between October 5, 2017, and June 30, 2020. We included all patients registered with a skin disorder (International Classification of Diseases) in the medical database. We excluded patients whose diagnosis was invalidated after cross-checking by a dermatologist. RESULTS: A total of 4741 patients presented at least one skin disease, for 6058 different disorders. Nonsexually transmitted infections represented 71.6% of all diagnoses, followed by inflammatory diseases (9.8%) and bites/envenomations (4.6%). The three most frequent conditions were scabies, abscesses, and impetigo. Besides scabies, neglected tropical diseases (NTDs) were still prevalent as we reported 13 cases of leprosy and 63 cutaneous leishmaniasis. Atopic dermatitis (AD) represented only 2.5% of our diagnoses. CONCLUSIONS: With the exception of AD, which was less frequent among Maroons, these results are similar to those previously reported in Amerindians. Therefore, a common exposure to rainforest pathogens seems to induce a common spectrum of skin diseases dominated by infections. The high prevalence of NTDs requires specific public health actions.

Establishing the proportion of severe/moderately severe vs mild cases of progressive disseminated histoplasmosis in patients with HIV.

BACKGROUND: Progressive disseminated histoplasmosis remains a major but neglected cause of death among patients with advanced HIV. Recently, aiming to reduce avoidable deaths, the Pan American Health Organization issued the first diagnosis and treatment guidelines for HIV-associated histoplasmosis. But what proportion of progressive disseminated histoplasmosis in HIV-infected patients is severe is currently not known. Because this proportion influences treatment needs, we aimed to estimate this in a cohort of 416 patients in French Guiana. METHODS: We used the definition in the recent PAHO/WHO guidelines for severity. We used regression modelling to predict the impact of CD4 count on the proportion of severe cases. In a territory where treatment cost is not a limiting factor and where histoplasmosis is well known, we assumed that clinicians' initial treatment reflected their perception about the severity of the case and therefore, the needs for different treatments. RESULTS: Using these definitions, since the beginning, there were 274 (65.9%) severe/moderately severe cases and 142 (34.1%) mild cases. In practice 186 cases were treated with deoxycholate or liposomal amphotericin B (44.7%) and 230 (55.3%) cases treated with itraconazole as first line treatment. The Kappa concordance measure between the guideline definition and the actual treatment given was 0.22. There was a 9% risk difference for death within 30 days of antifungal treatment initiation between severe/moderately severe and mild cases. Over threequarters (77%) of early deaths were attributed to severe/moderately severe cases. CONCLUSIONS: This is the only rigorous estimate of the proportion of severe/moderately severe cases of progressive disseminated histoplasmosis in symptomatic HIV patients on the largest published cohort. These numbers may help defend budget needs for rapid diagnostic tests and liposomal amphotericin B.

Investigations upon the Improvement of Dermatophyte Identification Using an Online Mass Spectrometry Application.

Online MALDI-TOF mass spectrometry applications, such as MSI-2, have been shown to help identify dermatophytes, but recurrent errors are still observed between phylogenetically close species. The objective of this study was to assess different approaches to reduce the occurrence of such errors by adding new reference spectra to the MSI-2 application. Nine libraries were set up, comprising an increasing number of spectra obtained from reference strains that were submitted to various culture durations on two distinct culture media: Sabouraud gentamicin chloramphenicol medium and IDFP Conidia medium. The final library included spectra from 111 strains of 20 species obtained from cultures on both media collected every three days after the appearance of the colony. The performance of each library was then analyzed using a cross-validation approach. The spectra acquisitions were carried out using a Microflex Bruker spectrometer. Diversifying the references and adding spectra from various culture media and culture durations improved identification performance. The percentage of correct identification at the species level rose from 63.4 to 91.7% when combining all approaches. Nevertheless, residual confusion between close species, such as Trichophyton rubrum, Trichophyton violaceum and Trichophyton soudanense, remained. To distinguish between these species, mass spectrometry identification should take into account basic morphological and/or clinico-epidemiological features.