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20 pages, 6302 KiB

Open AccessArticle

Development of a Multiplex qPCR Assay for Fast Detection and Differentiation of Paracoccidioidomycosis Agents

by Breno Gonçalves Pinheiro, Ana Paula Pôssa, Giannina Ricci, Angela Satie Nishikaku, Ferry Hagen, Rosane Christine Hahn, Zoilo Pires de Camargo and Anderson Messias Rodrigues

J. Fungi 2023, 9(3), 358; https://doi.org/10.3390/jof9030358 - 15 Mar 2023

Viewed by 2078

Abstract

Classic paracoccidioidomycosis (PCM) is a potentially deadly neglected tropical systemic mycosis caused by members of the Paracoccidioides brasiliensis complex (P. brasiliensis s. str., P. americana, P. restrepiensis, and P. venezuelensis) and P. lutzii. The laboratorial diagnosis of [...] Read more.

Classic paracoccidioidomycosis (PCM) is a potentially deadly neglected tropical systemic mycosis caused by members of the Paracoccidioides brasiliensis complex (P. brasiliensis s. str., P. americana, P. restrepiensis, and P. venezuelensis) and P. lutzii. The laboratorial diagnosis of PCM relies on observing pathognomonic structures such as the “steering wheel” or “Mickey Mouse” shape in the direct mycological examination, fresh biopsied tissue in 10% KOH, histopathological analysis, and/or the isolation of the fungus in culture. However, these procedures are time-consuming and do not allow for the speciation of Paracoccidioides due to overlapping morphologies. Here, we propose a new one-tube multiplex probe-based qPCR assay to detect and recognize agents of the P. brasiliensis complex and P. lutzii. Primers (Paracoco-F and Paracoco-R) and TaqMan probes (PbraCx-Fam, Plu-Ned, and Paracoco-Vic) were developed to target the rDNA (ITS2/28S) in the Paracoccidioides genome. A panel of 77 Paracoccidioides isolates revealed a 100% specificity (AUC = 1.0, 95% CI 0.964–1.000, p < 0.0001) without cross-reacting with other medically relevant fungi or human and murine DNA. The lower limit of detection was 10 fg of gDNA and three copies of the partial rDNA amplicon. Speciation using qPCR was in perfect agreement with AFLP and TUB1-RFLP markers (kappa = 1.0). As a proof of concept, we assessed a panel of 16 formalin-fixed and paraffin-embedded specimens from histopathologically confirmed PCM patients to reveal a significant sensitivity of 81.25% and specificity of 100% (AUC = 0.906 ± 0.05, 95% CI = 0.756–0.979, p < 0.0001, Youden index J = 0.8125). Our assay achieved maximum sensitivity (100%) and specificity (100%) using fresh clinical samples (n = 9) such as sputum, bronchoalveolar lavage, and tissue fragments from PCM patients (AUC = 1.0, 95% CI 0.872–1.000, p < 0.0001, Youden index J = 1.0). Overall, our qPCR assay simplifies the molecular diagnosis of PCM and can be easily implemented in any routine laboratory, decreasing a critical bottleneck for the early treatment of PCM patients across a vast area of the Americas. Full article

(This article belongs to the Special Issue Young Investigator in Fungal Infections)

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9 pages, 1248 KiB

Open AccessCommunication

The Distinction between Dematiaceous Molds and Non-Dematiaceous Fungi in Clinical and Spiked Samples Treated with Hydrogen Peroxide Using Direct Fluorescence Microscopy

by Elchanan Juravel, Itzhack Polacheck, Batya Isaacson, Arie Dagan and Maya Korem

J. Fungi 2023, 9(2), 227; https://doi.org/10.3390/jof9020227 - 9 Feb 2023

Cited by 1 | Viewed by 1862

Abstract

Dematiaceous fungi are pigmented molds with a high content of melanin in their cell walls that can cause fatal infections in immunocompromised hosts. Direct microscopy is the main method for the rapid diagnosis of dematiaceous fungi in clinical specimens. However, it is often [...] Read more.

Dematiaceous fungi are pigmented molds with a high content of melanin in their cell walls that can cause fatal infections in immunocompromised hosts. Direct microscopy is the main method for the rapid diagnosis of dematiaceous fungi in clinical specimens. However, it is often difficult to distinguish their hyphae from non-dematiaceous hyphae and yeast pseudohyphae. Our aim was to develop a fluorescence staining method that targets melanin for the detection of dematiaceous molds in clinical specimens. Glass slide smears of clinical samples and sterile bronchoalveolar lavage spiked with dematiaceous and non-dematiaceous fungi were treated with hydrogen peroxide, and digital images were recorded using direct microscopy with different fluorescent filters. The images of fungi were compared for their fluorescence intensity using the NIS-Elements software. The fluorescent signal between dematiaceous and non-dematiaceous fungi demonstrated a markedly increased mean intensity for dematiaceous molds following hydrogen peroxide treatment (7510.3 ± 10,427.6 vs. 0.3 ± 3.1, respectively, p < 0.0001). No fluorescent signal was detected in the absence of hydrogen peroxide. “Staining” fungal clinical specimens with hydrogen peroxide, followed by fluorescence microscopy examination, can differentiate between dematiaceous and non-dematiaceous fungi. This finding can be used for the detection of dematiaceous molds in clinical specimens and enables the early and appropriate treatment of infections. Full article

(This article belongs to the Special Issue Young Investigator in Fungal Infections)

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17 pages, 3771 KiB

Open AccessArticle

O-Linked Glycans of Candida albicans Interact with Specific GPCRs in the Coronary Endothelium and Inhibit the Cardiac Response to Agonists

by Alejandro Ocaña-Ortega, Gabriela Pérez-Flores, David Torres-Tirado and Luis A. Pérez-García

J. Fungi 2023, 9(2), 141; https://doi.org/10.3390/jof9020141 - 20 Jan 2023

Cited by 1 | Viewed by 1629

Abstract

Candida albicans is an opportunistic fungal pathogen that may cause invasive infections in immunocompromised patients, disseminating through the bloodstream to other organs. In the heart, the initial step prior to invasion is the adhesion of the fungus to endothelial cells. Being the fungal [...] Read more.

Candida albicans is an opportunistic fungal pathogen that may cause invasive infections in immunocompromised patients, disseminating through the bloodstream to other organs. In the heart, the initial step prior to invasion is the adhesion of the fungus to endothelial cells. Being the fungal cell wall’s outermost structure and the first to come in contact with host cells, it greatly modulates the interplay that later will derive in the colonization of the host tissue. In this work, we studied the functional contribution of N-linked and O-linked mannans of the cell wall of C. albicans to the interaction with the coronary endothelium. An isolated rat heart model was used to assess cardiac parameters related to vascular and inotropic effects in response to phenylephrine (Phe), acetylcholine (aCh) and angiotensin II (Ang II) when treatments consisting of: (1) live and heat-killed (HK) C. albicans wild-type yeasts; (2) live C. albicans pmr1Δ yeasts (displaying shorter N-linked and O-linked mannans); (3) live C. albicans without N-linked and O-linked mannans; and (4) isolated N-linked and O-linked mannans were administered to the heart. Our results showed that C. albicans WT alters heart coronary perfusion pressure (vascular effect) and left ventricular pressure (inotropic effect) parameters in response to Phe and Ang II but not aCh, and these effects can be reversed by mannose. Similar results were observed when isolated cell walls, live C. albicans without N-linked mannans or isolated O-linked mannans were perfused into the heart. In contrast, C. albicans HK, C. albicans pmr1Δ, C. albicans without O-linked mannans or isolated N-linked mannans were not able to alter the CPP and LVP in response to the same agonists. Taken together, our data suggest that C. albicans interaction occurs with specific receptors on coronary endothelium and that O-linked mannan contributes to a greater extent to this interaction. Further studies are necessary to elucidate why specific receptors preferentially interact with this fungal cell wall structure. Full article

(This article belongs to the Special Issue Young Investigator in Fungal Infections)

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12 pages, 2309 KiB

Open AccessArticle

Aspergillus spp. Isolated from Lungs of Poultry (Gallus gallus) at the Mycology Laboratory, School of Veterinary Medicine, Universidad Nacional, Heredia, Costa Rica between 2008 and 2021 and Associated Factors

by Oscar Ulloa-Avellán, Alejandra Calderón-Hernández, Randall Rubí-Chacón and Bernardo Vargas-Leitón

J. Fungi 2023, 9(1), 58; https://doi.org/10.3390/jof9010058 - 30 Dec 2022

Cited by 1 | Viewed by 2212

Abstract

Aspergillosis is a disease caused by some species of the fungus Aspergillus, occurring in both mammals (including humans) and birds, the latter being the most susceptible group. Aspergillus must be considered a public health concern as it affects the poultry industry economically [...] Read more.

Aspergillosis is a disease caused by some species of the fungus Aspergillus, occurring in both mammals (including humans) and birds, the latter being the most susceptible group. Aspergillus must be considered a public health concern as it affects the poultry industry economically and is an occupational risk to its workers. A retrospective study of fungal isolates from the lungs of chickens (Gallus gallus), analyzed between 2008 and 2021 at the Mycology Laboratory, School of Veterinary Medicine, Universidad Nacional, Heredia, Costa Rica was performed to report the prevalence of Aspergillus spp. in poultry farms in Costa Rica and their associated factors. A total of 1113 cases were received, of which 31% (n = 392; 95% CI: 28.3–33.7) were positive for fungal isolation. Aspergillus was the most frequently detected genus, and the most frequent sections were Fumigati (n = 197/392, 50.3%), Flavi (n = 90/392, 22.9%), and Nigri (n = 50/392, 12.7%). Significant effects (p < 0.05) related to the year, geographical origin, purpose, and age were identified in relation to the Aspergillus infection. The identified factors are explained by climatic variations in the tropics and the particularities of the birds. Future research including molecular characterization and antifungal susceptibility tests in animals, humans, and the environment, are needed to better understand the risks of the diseases caused by those fungi in this country. Full article

(This article belongs to the Special Issue Young Investigator in Fungal Infections)

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12 pages, 3301 KiB

Open AccessArticle

In Vitro Antifungal Activity of LL-37 Analogue Peptides against Candida spp.

by Gladys Pinilla, Yenifer Tatiana Coronado, Gabriel Chaves, Liliana Muñoz, Jeannette Navarrete, Luz Mary Salazar, Carlos Pelleschi Taborda and Julián E. Muñoz

J. Fungi 2022, 8(11), 1173; https://doi.org/10.3390/jof8111173 - 7 Nov 2022

Cited by 8 | Viewed by 1684

Abstract

Fungal infections have increased in recent decades with considerable morbidity and mortality, mainly in immunosuppressed or admitted-to-the-ICU patients. The fungal resistance to conventional antifungal treatments has become a public health problem, especially with Candida that presents resistance to several antifungals. Therefore, generating new [...] Read more.

Fungal infections have increased in recent decades with considerable morbidity and mortality, mainly in immunosuppressed or admitted-to-the-ICU patients. The fungal resistance to conventional antifungal treatments has become a public health problem, especially with Candida that presents resistance to several antifungals. Therefore, generating new alternatives of antifungal therapy is fundamental. One of these possibilities is the use of antimicrobial peptides, such as LL-37, which acts on the disruption of the microorganism membrane and promotes immunomodulatory effects in the host. In this study, we evaluated the in vitro antifungal activity of the LL-37 analogue peptides (AC-1, LL37-1, AC-2, and D) against different Candida spp. and clinical isolates obtained from patients with vulvovaginal candidiasis. Our results suggest that the peptides with the best ranges of MICs were LL37-1 and AC-2 (0.07 µM) against the strains studied. This inhibitory effect was confirmed by analyzing the yeast growth curves that evidenced a significant decrease in the fungal growth after exposure to LL-37 peptides. By the XTT technique we observed a significant reduction in the biofilm formation process when compared to yeasts untreated with the analogue peptides. In conclusion, we suggest that LL-37 analogue peptides may play an important antimicrobial role against Candida spp. Full article

(This article belongs to the Special Issue Young Investigator in Fungal Infections)

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11 pages, 1027 KiB

Open AccessArticle

Combined Silymarin and Cotrimoxazole Therapy Attenuates Pulmonary Fibrosis in Experimental Paracoccidioidomycosis

by Victor Quinholes Resende, Karoline Hagata Reis-Goes, Angela Carolina Finato, Débora de Fátima Almeida-Donanzam, Amanda Ribeiro dos Santos, Jonatas Perico, Barbara Casella Amorim and James Venturini

J. Fungi 2022, 8(10), 1010; https://doi.org/10.3390/jof8101010 - 27 Sep 2022

Cited by 5 | Viewed by 1753

Abstract

Paracoccidioidomycosis (PCM), which mainly affects rural workers, is a systemic mycosis caused by the Paracoccidioides genus that induces pulmonary sequelae in most adult patients, causing serious disability and impairing their quality of life. Silymarin is herbal medicine with an effective antifibrotic activity. Considering [...] Read more.

Paracoccidioidomycosis (PCM), which mainly affects rural workers, is a systemic mycosis caused by the Paracoccidioides genus that induces pulmonary sequelae in most adult patients, causing serious disability and impairing their quality of life. Silymarin is herbal medicine with an effective antifibrotic activity. Considering that in PCM, antifibrotic treatment is still not available in pulmonary fibrosis, we aimed to evaluate combined silymarin and cotrimoxazole (CMX) therapy via the intratracheal route in BALB/c mice infected with P. brasiliensis yeast. After 12 weeks of treatment, the lungs were collected for the determination of fungal burden, production of OH-proline, deposition of collagen fibers, pulmonary concentrations of cytokines, and expression of fibronectin, α-SMA, MMP-2, MMP-9, and TIMP-2. Spleen cell cultures were also performed. Our results showed that infected mice treated with combined silymarin/CMX showed lower deposition of collagen fibers in the lungs and lower pulmonary concentrations of hydroxyproline than the placebo groups. Decreased levels of TGF-β1 and fibronectin and high levels of MMP-2 and IFN-γ were also observed in this group of mice. Collectively, our findings indicate that the combination of antifungal treatment with silymarin has a potent antifibrotic effect associated with an immunomodulatory effect that potentializes the antifungal immune response. Full article

(This article belongs to the Special Issue Young Investigator in Fungal Infections)

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12 pages, 1233 KiB

Open AccessArticle

COVID-19-Associated Pulmonary Fungal Infection among Pediatric Cancer Patients, a Single Center Experience

by Youssef Madney, Lobna Shalaby, Mahmoud Hammad, Mervat Elanany, Reem Hassan, Ayda Youssef, Ibrahim Abdo, Abeer Zaki and Reham Khedr

J. Fungi 2022, 8(8), 850; https://doi.org/10.3390/jof8080850 - 15 Aug 2022

Cited by 5 | Viewed by 2710

Abstract

Patients with COVID-19 are at risk of developing secondary complications such as invasive pulmonary aspergillosis and mucormycosis. This is a retrospective study including all cancer children diagnosed with COVID-19-associated pulmonary fungal infection (CAPFI) during the period 2020–2021. A total of 200 patients were [...] Read more.

Patients with COVID-19 are at risk of developing secondary complications such as invasive pulmonary aspergillosis and mucormycosis. This is a retrospective study including all cancer children diagnosed with COVID-19-associated pulmonary fungal infection (CAPFI) during the period 2020–2021. A total of 200 patients were diagnosed with COVID-19, out of which 21 (10%) patients were diagnosed with CAPFI, 19 patients (90%) with COVID-aspergillosis (CAPA), and 2 (10%) patients with COVID-mucormycosis (CAM). Patients with CAPFI were classified using the “2020 ECMM/ISHAM consensus criteria”; proven in 2 (10%) patients, probable in 12 (57%), and possible in 7 (33%) patients. Although the hematological malignancy patients were already on antifungal prophylaxis, breakthrough fungal infection was reported in 16/21 (75%), 14 (65%) patients had CAPA while on echinocandin prophylaxis, while 2 (10%) patients had CAM while on voriconazole prophylaxis. Overall mortality was reported in 8 patients (38%) while CAPFI-attributable mortality was reported in 4 patients (20%). In conclusion, clinicians caring for pediatric cancer patients with COVID-19 should consider invasive pulmonary fungal infection, even if they are on antifungal prophylaxis, especially with worsening of the clinical chest condition. A better understanding of risk factors for adverse outcomes may improve clinical management in these patients. Full article

(This article belongs to the Special Issue Young Investigator in Fungal Infections)

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11 pages, 1448 KiB

Open AccessArticle

Clinical and Eco-Epidemiological Aspects of a Novel Hyperendemic Area of Paracoccidioidomycosis in the Tocantins-Araguaia Basin (Northern Brazil), Caused by Paracoccidioides sp.

by Alessandra G. Krakhecke-Teixeira, Danielle H. Yamauchi, Alexsandra Rossi, Herdson R. de Sousa, Hans G. Garces, Joaquim L. Júnior, Antônio O. S. Júnior, Maria Sueli S. Felipe, Eduardo Bagagli, Heitor F. de Andrade, Jr. and Marcus de M. Teixeira

J. Fungi 2022, 8(5), 502; https://doi.org/10.3390/jof8050502 - 12 May 2022

Cited by 8 | Viewed by 1984

Abstract

Paracoccidioidomycosis (PCM) is the most prevalent systemic mycosis in Brazil. The disease is caused by dimorphic fungi nested within the Paracoccidioides genus. We described 106 PCM cases (47.1 cases/year) at the Tropical Diseases Public Hospital of Tocantins State. PCM was prevalent in males [...] Read more.

Paracoccidioidomycosis (PCM) is the most prevalent systemic mycosis in Brazil. The disease is caused by dimorphic fungi nested within the Paracoccidioides genus. We described 106 PCM cases (47.1 cases/year) at the Tropical Diseases Public Hospital of Tocantins State. PCM was prevalent in males and rural workers over 50 years; the chronic pulmonary form predominated in 67% of cases. The male-to-female ratio was 2.65:1, with more women affected than other endemic regions of Brazil. Urban or indoor activities were reported in women and are ascribed to disease urbanization. qPCR-based assays confirmed the identification of Paracoccidioides DNA in 37 biological specimens. Paracoccidioides sp. DNA was found in 53% of the environmental samples, suggesting autochthonous infections. Therefore, the Tocantins-Araguaia basin must be considered a novel hyperendemic area of PCM in Brazil, reinforcing the importance of including PCM as a notifiable disease, requiring specific diagnosis and health measures. Full article

(This article belongs to the Special Issue Young Investigator in Fungal Infections)

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5 pages, 241 KiB

Open AccessBrief Report

The Utility of (1→3)-β-D-Glucan Testing in the Diagnosis of Coccidioidomycosis in Hospitalized Immunocompromised Patients

by Mohanad M. Al-Obaidi, Parham Ayazi, Aishan Shi, Matthew Campanella, Elizabeth Connick and Tirdad T. Zangeneh

J. Fungi 2022, 8(8), 768; https://doi.org/10.3390/jof8080768 - 25 Jul 2022

Cited by 5 | Viewed by 1750

Abstract

Coccidioidomycosis is a fungal infection endemic to the Southwestern United States which is associated with high morbidity and mortality in immunocompromised hosts. Serology is the main diagnostic tool, although less sensitive among immunocompromised hosts. (1→3)-β-D-glucan (BDG) is a non-specific fungal diagnostic test that [...] Read more.

Coccidioidomycosis is a fungal infection endemic to the Southwestern United States which is associated with high morbidity and mortality in immunocompromised hosts. Serology is the main diagnostic tool, although less sensitive among immunocompromised hosts. (1→3)-β-D-glucan (BDG) is a non-specific fungal diagnostic test that may identify suspected coccidioidomycosis and other invasive fungal infections. We retrospectively investigated the utility of BDG between 2017 and 2021 in immunocompromised hosts with positive Coccidioides spp. cultures at our institutions. During the study period, there were 368 patients with positive cultures for Coccidioides spp.; among those, 28 patients were immunocompromised hosts, had both Coccidioides serology and BDG results available, and met other inclusion and exclusion criteria. Half of the patients had positive Coccidioides serology, and 57% had a positive BDG ≥ 80 pg/mL. Twenty-three (82%) had at least one positive test during their hospitalization. Among immunocompromised hosts with suspicion for coccidioidomycosis, the combination of Coccidioides serology and BDG can be useful in the initial work up and the timely administration of appropriate antifungal therapy. However, both tests failed to diagnose many cases, underscoring the need for better diagnostic techniques for identifying coccidioidomycosis in this population. Full article

(This article belongs to the Special Issue Young Investigator in Fungal Infections)

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