Proceedings of the Second International Meeting on Endemic Mycoses of the Americas (IMEMA), and First International Symposium on Implantation Mycoses (ISIM).

The Second International Meeting on Endemic Mycoses of the Americas (IMEMA) and the First International Symposium on Implantation Mycoses (ISIM) took place in Santiago del Estero, Argentina during September 25-27th, 2023. The conference provided a platform for researchers, clinicians, and experts to discuss the latest developments in the field of endemic and implantation mycoses. Topics included epidemiology, diagnostic advances, treatment strategies, and the impact of environmental factors in the spread of these fungal diseases. IMEMA and ISIM contributed to the regional discourse on the mycoses, emphasizing the importance of international cooperation in addressing these public health challenges.

IMEMA/ISIM, held in Santiago del Estero, Argentina, convened experts to discuss endemic and implantation mycoses, covering topics such as epidemiology, diagnostics, treatment, and advocacy. The event highlighted ongoing efforts in combating these diseases.

Patients with recurrent vulvovaginal candidiasis exhibit a decrease in both the fungicidal activity of neutrophils and the proliferation of peripheral blood mononuclear cells.

BACKGROUND: Recurrent vulvovaginal candidiasis (RVVC) is an important and underestimated fungal infection. OBJECTIVE: We aimed to determine the fungicidal and proliferative capacities of neutrophils and peripheral blood mononuclear cells (PBMCs), respectively and the clinical and microbiological characteristics of a cohort of Colombian patients diagnosed with RVVC. METHODS: A cross-sectional study was conducted. A total of 66 women were included (40 diagnosed with RVVC and 26 healthy women [HW]). Demographic and clinical data were recorded. Vaginal fluid samples were obtained for isolation, identification and antifungal susceptibility testing of Candida species using selective culture media and the Vitek 2.0® system. Blood samples were also obtained to evaluate cell subpopulations; furthermore, neutrophils and PBMCs were isolated to determine their fungicidal and proliferative capacities, respectively. RESULTS: The median age was 29 (IQR: 34-23) for RVVC and 24 (IQR: 30-23) for HW. Only two species of the genus Candida were identified: Candida albicans (92.5%) and Candida lusitaniae (7.5%). Resistance to fluconazole, voriconazole, flucytosine and amphotericin B was observed on six C. albicans isolates and one C. lusitaniae isolate. Only the family history of vulvovaginal candidiasis was associated with RVVC occurrence. The RVVC group exhibited a significantly higher number of neutrophils but with lower fungicidal activity in comparison to HW; likewise, PBMCs from RVVC patients presented a lower proliferation index when stimulated with C. albicans. CONCLUSION: Contrary to what has been reported worldwide, in Colombian patients with RVVC, C. albicans was the main isolated species without increased antifungal resistance. The diminished fungicidal and proliferative capacities of neutrophils and PBMCs, respectively, could suggest a possible alteration in the innate and adaptive immune responses.

Therapeutic Use of the Antimicrobial Peptide PNR20 to Resolve Disseminated Candidiasis in a Murine Model.

Invasive fungal infections (IFIs) caused by Candida species are an emerging threat globally, given that patients at-risk and antifungal resistance are increasing. Antimicrobial peptides (AMPs) have shown good therapeutic capacity against different multidrug-resistant (MDR) microorganisms. This study evaluated the activity of the synthetic peptide, PNR20, against Candida albicans ATCC 10231 and a MDR Colombian clinical isolate of Candida auris. Perturbation of yeast cell surface was evaluated using scanning electron microscopy. Cell viability of Vero cells was determined to assess peptide toxicity. Additionally, survival, fungal burden, and histopathology of BALB/c mice infected intravenously with each Candida species and treated with PNR20 were analyzed. Morphological alterations were identified in both species, demonstrating the antifungal effect of PNR20. In vitro, Vero cells' viability was not affected by PNR20. All mice infected with either C. albicans or C. auris and treated with PNR20 survived and had a significant reduction in the fungal burden in the kidney compared to the control group. The histopathological analysis in mice infected and treated with PNR20 showed more preserved tissues, without the presence of yeast, compared to the control groups. This work shows that the utilization of PNR20 is a promising therapeutic alternative against disseminated candidiasis.

In Vitro Antifungal Activity of Three Synthetic Peptides against Candida auris and Other Candida Species of Medical Importance.

Candidiasis is an opportunistic infection affecting immunosuppressed and hospitalized patients, with mortality rates approaching 40% in Colombia. The growing pharmacological resistance of Candida species and the emergence of multidrug-resistant Candida auris are major public health problems. Therefore, different antimicrobial peptides (AMPs) are being investigated as therapeutic alternatives to control candidiasis effectively and safely. This work aimed to evaluate the in vitro antifungal activity of three synthetic AMPs, PNR20, PNR20-1, and 35409, against ATCC reference strains of Candida albicans, Candida glabrata, Candida parapsilosis, Candida krusei, and Candida tropicalis, and clinical isolates of C. auris. Antifungal susceptibility testing, determined by broth microdilution, showed that the AMPs have antifungal activity against planktonic cells of all Candida species evaluated. In C. auris and C. albicans, the peptides had an effect on biofilm formation and cell viability, as determined by the XTT assay and flow cytometry, respectively. Also, morphological alterations in the membrane and at the intracellular level of these species were induced by the peptides, as observed by transmission electron microscopy. In vitro, the AMPs had no cytotoxicity against L929 murine fibroblasts. Our results showed that the evaluated AMPs are potential therapeutic alternatives against the most important Candida species in Colombia and the world.

Application of Real-Time PCR Assays for the Diagnosis of Histoplasmosis in Human FFPE Tissues Using Three Molecular Targets.

Histoplasmosis is a fungal infection caused by the thermally dimorphic fungus Histoplasma capsulatum. This infection causes significant morbidity and mortality in people living with HIV/AIDS, especially in countries with limited resources. Currently used diagnostic tests rely on culture and serology but with some limitations. No molecular assays are commercially available and the results from different reports have been variable. We aimed to evaluate quantitative real-time PCR (qPCR) targeting three protein-coding genes of Histoplasma capsulatum (100-kDa, H and M antigens) for detection of this fungus in formalin-fixed paraffin-embedded (FFPE) samples from patients with proven histoplasmosis. The sensitivity of 100-kDa, H and M qPCR assays were 93.9%, 91% and 57%, respectively. The specificity of 100-kDa qPCR was 93% when compared against samples from patients with other mycoses and other infections, and 100% when samples from patients with non-infectious diseases were used as controls. Our findings demonstrate that real-time PCR assays targeting 100-kDa and H antigen showed the most reliable results and can be successfully used for diagnosing this mycosis when testing FFPE samples.

Tackling Histoplasmosis Infection in People Living with HIV from Latin America: From Diagnostic Strategy to Public Health Solutions.

Histoplasmosis, caused by the thermally dimorphic fungus Histoplasma spp., is a disease with a broad clinical spectrum, presenting from asymptomatic/flu-like symptoms to progressive disseminated disease in people with immunosuppression. In recent years, the concept of histoplasmosis as a disease restricted to the American continent has changed, as now histoplasmosis is reported in many regions around the world. In Latin America, histoplasmosis represents a threat, especially in people with advanced HIV disease (AHD). Diagnosis of histoplasmosis in people living with HIV (PLHIV) is challenging due to the low index of suspicion of the disease, non-specificity of signs and symptoms, and limited access to specific laboratory testing, while the diagnostic delay is significantly associated with mortality. In the last decade, novel diagnostic tests have been developed for the rapid detection of histoplasmosis, such as commercial kits for antigen detection. Furthermore, advocacy groups were created that presented histoplasmosis as a public health problem, with emphasis on patients at risk of progressive disseminated disease. This review aims to discuss the impact of histoplasmosis associated with AHD in Latin America and the strategies employed to tackle histoplasmosis, from the implementation of laboratory testing to disease advocacy and public health interventions.

Histoplasma capsulatum modulates the immune response, affects proliferation and differentiation, and induces apoptosis of mesenchymal stromal cells.

Mesenchymal stromal cells (MSC) have been widely used not only for tissue regeneration but also for the treatment of various diseases; however, it has been shown that infection of MSCs by different pathogens can attenuate their intrinsic immunomodulatory properties, affecting the proliferation and differentiation of these cells. Currently, the mechanisms by which MSCs respond to pathogen invasion are poorly understood. Therefore, the objective of the present study was to determine if the infection of bone marrow-derived MSCs, with yeasts of the pathogenic fungus Histoplasma capsulatum affects the activation, differentiation and/or proliferation of the MSCs. The results indicate that MSCs have the ability to phagocytose H. capsulatum yeasts but do not exert a notable antifungal effect. On the contrary, the infection of the MSCs with this fungal pathogen not only modulates the expression of inflammatory mediators by a mechanism dependent on TLR2, TLR4 and Dectin-1 but also affects the viability and differentiation capacity of the MSCs. These findings suggest that infection of MSCs by H. capsulatum could not only affect haematopoiesis but also modulate the immune response in the infected host and, furthermore, these MSCs could provide a niche for the fungus, allowing it to persist and evade the immune response of the host.

Histoplasma capsulatum Activates Hematopoietic Stem Cells and Their Progenitors through a Mechanism Dependent on TLR2, TLR4, and Dectin-1.

Hematopoietic stem cells (HSCs), a multipotent and self-renewing population responsible for the generation and maintenance of blood cells, have been the subject of numerous investigations due to their therapeutic potential. It has been shown that these cells are able to interact with pathogens through the TLRs that they express on their surface, affecting the hematopoiesis process. However, the interaction between hematopoietic stem and progenitor cells (HSPC) with fungal pathogens such as Histoplasma capsulatum has not been studied. Therefore, the objective of the present study was to determine if the interaction of HSPCs with H. capsulatum yeasts affects the hematopoiesis, activation, or proliferation of these cells. The results indicate that HSPCs are able to adhere to and internalize H. capsulatum yeasts through a mechanism dependent on TLR2, TLR4, and Dectin-1; however, this process does not affect the survival of the fungus, and, on the contrary, such interaction induces a significant increase in the expression of IL-1ß, IL-6, IL-10, IL-17, TNF-α, and TGF-ß, as well as the immune mediators Arg-1 and iNOS. Moreover, H. capsulatum induces apoptosis and alters HSPC proliferation. These findings suggest that H. capsulatum directly modulates the immune response exerted by HPSC through PRRs, and this interaction could directly affect the process of hematopoiesis, a fact that could explain clinical manifestations such as anemia and pancytopenia in patients with severe histoplasmosis, especially in those with fungal spread to the bone marrow.

Current situation of endemic mycosis in the Americas and the Caribbean: Proceedings of the first international meeting on endemic mycoses of the Americas (IMEMA).

BACKGROUND: The Americas are home to biologically and clinically diverse endemic fungi, including Blastomyces, Coccidioides, Emergomyces, Histoplasma, Paracoccidioides and Sporothrix. In endemic areas with high risk of infection, these fungal pathogens represent an important public health problem. OBJECTIVES: This report aims to summarise the main findings of the regional analysis carried out on the status of the endemic mycoses of the Americas, done at the first International Meeting on Endemic Mycoses of the Americas (IMEMA). METHODS: A regional analysis for the Americas was done, the 27 territories were grouped into nine regions. A SWOT analysis was done. RESULTS: All territories reported availability of microscopy. Seventy percent of territories reported antibody testing, 67% of territories reported availability of Histoplasma antigen testing. None of the territories reported the use of (1-3)-ß-d-glucan. Fifty two percent of territories reported the availability of PCR testing in reference centres (mostly for histoplasmosis). Most of the territories reported access to medications such as trimethoprim-sulfamethoxazole, itraconazole, voriconazole and amphotericin B (AMB) deoxycholate. Many countries had limited access to liposomal formulation of AMB and newer azoles, such as posaconazole and isavuconazole. Surveillance of these fungal diseases was minimal. CONCLUSIONS: A consensus emerged among meeting participants, this group concluded that endemic mycoses are neglected diseases, and due to their severity and lack of resources, the improvement of diagnosis, treatment and surveillance is needed.

Evaluation of OIDx Histoplasma Urinary Antigen EIA.

A sandwich enzyme immunoassay (EIA) for the detection of Histoplasma antigens (Ag) in urine, developed by Optimum Imaging Diagnostics (OIDx) was evaluated. A verification using a standardized reference panel of urine samples found sensitivity of 92%, specificity of 32% and accuracy of 51%. In this study, the OIDx Histoplasma urinary Ag EIA displayed high sensitivity, however, in non-histoplasmosis cases this EIA displayed false-positive results in 68% of specimens tested.

Validation and Concordance Analysis of a New Lateral Flow Assay for Detection of Histoplasma Antigen in Urine.

Histoplasmosis is a major cause of mortality in people living with HIV (PLHIV). Rapid methods to diagnose Histoplasma capsulatum disease could dramatically decrease the time to initiate treatment, resulting in reduced mortality. The aim of this study was to validate a MiraVista® Diagnostics (MVD) Histoplasma urine antigen lateral flow assay (MVD LFA) for the detection of H. capsulatum antigen (Ag) in urine and compare this LFA against the MVista® Histoplasma Ag quantitative enzyme immunoassays (MVD EIA). We assessed the MVD LFA using a standardized reference panel of urine specimens from Colombia. We tested 100 urine specimens, 26 from PLHIV diagnosed with histoplasmosis, 42 from PLHIV with other infectious diseases, and 32 from non-HIV infected persons without histoplasmosis. Sensitivity and specificity of the MVD LFA was 96%, compared with 96% sensitivity and 77% specificity of the MVD EIA. Concordance analysis between MVD LFA and the MVD EIA displayed an 84% agreement, and a Kappa of 0.656. The MVD LFA evaluated in this study has several advantages, including a turnaround time for results of approximately 40 min, no need for complex laboratory infrastructure or highly trained laboratory personnel, use of urine specimens, and ease of performing.

Pulmonary Histoplasmosis.

Histoplasmosis is one of the most frequent causes of fungal respiratory infection in endemic regions, has a broad spectrum of clinical manifestations and can present in several forms. The extent of disease is determined by the number of conidia inhaled, the immune response of the host and the integrity of the respiratory tract. From an initial and most benign form, acute pulmonary histoplasmosis (an influenza-like illness that is typically asymptomatic or mild in persons without prior immune compromise), histoplasmosis can become a lifethreatening progressive disseminated infection (PDH) that affects mainly immunocompromised patients, with high morbidity and mortality. Chronic pulmonary histoplasmosis is an uncommon manifestation of Histoplasma infection, with features similar to pulmonary tuberculosis, and if it remains undiagnosed or untreated it also can cause significant morbidity. Some rare but serious complications may also occur that are produced by an excessive immune response, such as mediastinal fibrosis, histoplasmoma and broncholithiasis. Histoplasmosis is highly endemic in regions of North, Central and South America as well as being reported in parts of Asia and Africa. The risk of histoplasmosis is greatest in patients with HIV infection, especially those with CD4+ counts of <200 cells/µL. We review clinical manifestations, radiological findings and treatment options according to the clinical form (induction therapy and maintenance therapy), as well as different diagnosis tools and new laboratory tests that have been recently developed and validated and are becoming widely available. These should have an impact in reducing time for diagnosis and starting therapy and in reducing morbidity and mortality, especially in patients with HIV infection, where histoplasmosis is currently estimated to be responsible for 5-15% of AIDS-related deaths.

Pathogenicity Assessment of Colombian Strains of Candida auris in the Galleria mellonella Invertebrate Model.

Candida auris, first described in 2009, is an opportunistic pathogenic yeast that causes nosocomial outbreaks around the world, with high mortality rates associated with therapeutic failure. In this study, we evaluated the pathogenicity of 107 isolates from two cities in Colombia, associated with fungemia or colonization processes; to achieve this, we used the Galleria mellonella invertebrate model to compare pathogenicity. Our results showed that less than half of the total isolates of C. auris presented a high pathogenicity compared to the reference strain SC5314, and most of those highly pathogenic strains were from colonization processes. We observed that there was formation of large aggregates of cells that cannot be disrupted easily, without statistically significant differences between the pathogenicity of the aggregated and non-aggregated strains. In addition, protease activity was observed in 100% of the C. auris strains; phospholipase and hemolysin activity were observed in 67.3 and 68.2% of the studied strains, respectively. In conclusion, these results highlight the utility of determining survival using G. mellonella, which allowed us to provide new information on the pathogenicity, enzymatic activity, and the relationship of the aggregated and non-aggregated phenotypes of C. auris in this model.

Summary of Guidelines for Managing Histoplasmosis among People Living with HIV.

Histoplasmosis is a frequent fungal opportunistic infection in people living with HIV (PLHIV), associated every year to a total of 5% to 15% of AIDS-related deaths among this population. In 2020, the first global guidelines for diagnosing and managing disseminated histoplasmosis among PLHIV was published. This document recommends (1) detection of circulating Histoplasma antigens as the recommended laboratory assay to diagnose histoplasmosis among PLHIV; (2) the use of liposomal amphotericin for induction therapy in severe or moderately severe disease, followed by a maintenance therapy with itraconazole for 12 months; a shorter maintenance therapy could be considered if the patient is clinically stable and if immune status has improved; (3) antiretroviral therapy initiation as soon as possible among patients with histoplasmosis without involvement of central nervous system; and (4) that for the treatment of co-infection with histoplasmosis and tuberculosis (TB), treatment of TB should be initiated according to the World Health Organization treatment guidelines. Appropriate health education of providers, supportive supervision, and policy guidance for the care of PLHIV are required.

Cross-reactivity of a Histoplasma capsulatum antigen enzyme immunoassay in urine specimens from persons with emergomycosis in South Africa.

Histoplasma antigen detection in urine is a rapid diagnostic method for disseminated histoplasmosis, although cross-reactivity has been reported in specimens from patients with other thermally dimorphic fungal infections. We tested urine specimens, from persons with suspected invasive fungal infections, using a commercial monoclonal antibody Histoplasma enzyme immunoassay (EIA) at a South African national mycology reference laboratory from August 2014 through December 2018. Corresponding fungal culture and histopathology results were obtained from an electronic laboratory information system. In some cases, cultured fungal isolates were sent with the urine specimen for species-level identification by phenotypic and molecular methods. Cross-reactivity was confirmed using culture filtrates of several fungal pathogens. Of 212 referred cases, 41 (19%) were excluded since they had no recorded clinical history (n = 1), alternative diagnoses were confirmed (n = 2), or no fungal culture or histopathology results (n = 38). Eighty-seven of 212 (41%) had laboratory evidence of an invasive fungal disease, while 84 (40%) did not. Of the 87 cases, 37 (43%) were culture-confirmed mycoses: emergomycosis (n = 18), histoplasmosis (n = 8), sporotrichosis (n = 6), cryptococcosis (n = 2), talaromycosis (n = 1), and other fungi isolated (n = 2). The sensitivity and specificity of the EIA were calculated for two groups: culture-confirmed (n = 37) and histology-confirmed invasive fungal disease (n = 50). The sensitivity and specificity of the EIA for diagnosis of histoplasmosis compared to culture were 88% (7/8, 95%CI 47-100%) and 72% (21/29, 95%CI 53-87%), respectively, and for diagnosis of emergomycosis/histoplasmosis compared to histology was 83% (29/35, 95%CI 66-93%) and 93% (14/15, 95%CI 68-100%), respectively. Cross-reactions occurred in urine specimens of patients with Emergomyces africanus infection and in culture filtrates of E. africanus, T. marneffei and Blastomyces species. A commercial Histoplasma EIA had satisfactory accuracy for diagnosis of culture-confirmed histoplasmosis, but cross-reacted in urine specimens from patients with invasive disease caused by the closely-related pathogen, E. africanus and in culture filtrates of E. africanus and other related fungi. LAY SUMMARY: Emergomyces africanus and Histoplasma capsulatum are fungi that cause a multi-system disease among HIV-seropositive persons with a low CD4 cell count. Handling live cultures of these fungi to confirm a diagnosis requires specialized laboratory equipment and infrastructure which is infrequently accessible in low-resource settings. The features of the two diseases (i.e., disseminated histoplasmosis and emergomycosis) may be indistinguishable when infected tissue is prepared, stained, and examined under a microscope. Enzyme immunoassays (EIA) have been developed as rapid diagnostic tools for the detection of a cell wall component of H. capsulatum in urine specimens, although cross-reactions have been reported in specimens from patients with other fungal infections. We evaluated the accuracy of a commercial Histoplasma EIA to diagnose histoplasmosis and to assess cross-reactions in urine specimens from persons with emergomycosis and in cultures of E. africanus and related fungi. We report a sensitivity and specificity of 88% (95%CI 47-100%) and 72% (95%CI 53-87%) for diagnosis of histoplasmosis compared to culture and 83% (95%CI 66-93%) and 93% (95%CI 68-100%) for diagnosis of either histoplasmosis/emergomycosis compared to a diagnosis made by microscopic examination of infected tissue. The assay cross-reacted in urine specimens from patients with emergomycosis and in culture filtrates of related fungi. Although the EIA cross-reacted with other related fungi, this test can decrease the time to diagnosis and facilitate early treatment of emergomycosis and histoplasmosis in South Africa.

AIDS-Related Mycoses: Updated Progress and Future Priorities.

Serious fungal infections continue to devastate people living with HIV and remain a leading cause of infection-related deaths in this population, second only to tuberculosis. The third AIDS-related mycoses workshop updated progress in the field over the last 3 years and highlighted six key action points for the future.

Evaluation of a Histoplasma antigen lateral flow assay for the rapid diagnosis of progressive disseminated histoplasmosis in Colombian patients with AIDS.

BACKGROUND: Progressive disseminated histoplasmosis (PDH) is an important cause of mortality in persons living with HIV (PLHIV), especially in countries where patients have limited access to antiretroviral therapies and diagnostic testing. OBJECTIVE: A lateral flow assay (LFA) to detect Histoplasma capsulatum antigen in serum developed by MiraVista® was evaluated. METHODS: We tested 75 serum samples: 24 from PLHIV and culture-proven PDH and 51 from PLHIV with other fungal and bacterial infections as well as people without HIV. LFA devices were read manually (read by eye) and by an automated reader. RESULTS: When the LFA was read manually, sensitivity was 96% and specificity was 90%. When an automated reader was used, sensitivity was 92% and specificity was 94%. The Kappa index comparing manual and automated reader was 0.90. Cross-reactions were observed principally in samples from patients with proven diagnosis of paracoccidioidomycosis. CONCLUSIONS: The MiraVista® Diagnostics Histoplasma antigen LFA had high analytical performance and good agreement between manual and automated reader. This LFA allows Histoplasma antigen testing with minimal laboratory equipment and infrastructure requirements.

The Diagnosis of Fungal Neglected Tropical Diseases (Fungal NTDs) and the Role of Investigation and Laboratory Tests: An Expert Consensus Report.

The diagnosis of fungal Neglected Tropical Diseases (NTD) is primarily based on initial visual recognition of a suspected case followed by confirmatory laboratory testing, which is often limited to specialized facilities. Although molecular and serodiagnostic tools have advanced, a substantial gap remains between the desirable and the practical in endemic settings. To explore this issue further, we conducted a survey of subject matter experts on the optimal diagnostic methods sufficient to initiate treatment in well-equipped versus basic healthcare settings, as well as optimal sampling methods, for three fungal NTDs: mycetoma, chromoblastomycosis, and sporotrichosis. A survey of 23 centres found consensus on the key role of semi-invasive sampling methods such as biopsy diagnosis as compared with swabs or impression smears, and on the importance of histopathology, direct microscopy, and culture for mycetoma and chromoblastomycosis confirmation in well-equipped laboratories. In basic healthcare settings, direct microscopy combined with clinical signs were reported to be the most useful diagnostic indicators to prompt referral for treatment. The survey identified that the diagnosis of sporotrichosis is the most problematic with poor sensitivity across the most widely available laboratory tests except fungal culture, highlighting the need to improve mycological diagnostic capacity and to develop innovative diagnostic solutions. Fungal microscopy and culture are now recognized as WHO essential diagnostic tests and better training in their application will help improve the situation. For mycetoma and sporotrichosis, in particular, advances in identifying specific marker antigens or genomic sequences may pave the way for new laboratory-based or point-of-care tests, although this is a formidable task given the large number of different organisms that can cause fungal NTDs.