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Molecular Diagnostics of Fungal Infections
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Molecular Diagnostics of Fungal Infections

A special issue of Journal of Fungi (ISSN 2309-608X).

Deadline for manuscript submissions: closed (31 August 2019) | Viewed by 71292

Special Issue Editor

Prof. Dr. Cornelia Lass-Flörl

E-Mail Website
Guest Editor
Division of Hygiene and Medical Microbiology, Medical University of Innsbruck, Schöpfstraße 41, 6020 Innsbruck, Austria
Interests: mycology; bacteriology; hospital hygiene
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

Fungal infections affect immunocompromised hosts, patients hospitalized with severe underlying diseases, those requiring surgical procedures, and individuals who require support in intensive care units. Patients with profound neutropenia are at risk for invasive filamentous fungal infections, whereas patients in the intensive care unit with numerous indwelling intravascular catheters are at risk for yeast infections. A proper diagnosis with genus or at least species specification is of high importance to secure targeted treatment. Culture and microscopic examination are considered to provide definitive evidence of infection, but have a number of limitations. Antigen-based assays hold promise when used appropriately, but are restricted to specific patient populations and do not cover a wide range of fungi to be detected. Novel molecular techniques proved promising in trials and are currently under clinical evaluation. Given recent developments in this field the purpose of this Special Issue is to highlight new diagnostic approaches that are currently utilized or under development for invasive fungal infections.

Sincerely,

Prof. Cornelia Lass-Flörl
Guest Editor

Manuscript Submission Information

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Keywords

  • fungal diagnostics
  • fungal molecular diagnostics
  • invasive fungal infections

Published Papers (18 papers)

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Research

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9 pages, 241 KiB  
Article
Diagnosis of Pneumocystis jirovecii Pneumonia in Pediatric Patients in Serbia, Greece, and Romania. Current Status and Challenges for Collaboration
by Valentina Arsić Arsenijevic, Timoleon-Achilleas Vyzantiadis, Mihai Mares, Suzana Otasevic, Athanasios Tragiannidis and Dragana Janic
J. Fungi 2020, 6(2), 49; https://doi.org/10.3390/jof6020049 - 17 Apr 2020
Cited by 2 | Viewed by 2366
Abstract
Pneumocystis jirovecii can cause fatal Pneumocystis pneumonia (PcP). Many children have been exposed to the fungus and are colonized in early age, while some individuals at high risk for fungal infections may develop PcP, a disease that is difficult to diagnose. Insufficient laboratory [...] Read more.
Pneumocystis jirovecii can cause fatal Pneumocystis pneumonia (PcP). Many children have been exposed to the fungus and are colonized in early age, while some individuals at high risk for fungal infections may develop PcP, a disease that is difficult to diagnose. Insufficient laboratory availability, lack of knowledge, and local epidemiology gaps make the problem more serious. Traditionally, the diagnosis is based on microscopic visualization of Pneumocystis in respiratory specimens. The molecular diagnosis is important but not widely used. The aim of this study was to collect initial indicative data from Serbia, Greece, and Romania concerning pediatric patients with suspected PcP in order to: find the key underlying diseases, determine current clinical and laboratory practices, and try to propose an integrative future molecular perspective based on regional collaboration. Data were collected by the search of literature and the use of an online questionnaire, filled by relevant scientists specialized in the field. All three countries presented similar clinical practices in terms of PcP prophylaxis and clinical suspicion. In Serbia and Greece the hematology/oncology diseases are the main risks, while in Romania HIV infection is an additional risk. Molecular diagnosis is available only in Greece. PcP seems to be under-diagnosed and regional collaboration in the field of laboratory diagnosis with an emphasis on molecular approaches may help to cover the gaps and improve the practices. Full article
(This article belongs to the Special Issue Molecular Diagnostics of Fungal Infections)
8 pages, 507 KiB  
Article
Impact of ITS-Based Sequencing on Antifungal Treatment of Patients with Suspected Invasive Fungal Infections
by Sara Guenter, Gregor Gorkiewicz, Bettina Halwachs, Karl Kashofer, Andrea Thueringer, Phillip Wurm, Ines Zollner-Schwetz, Thomas Valentin, Juergen Prattes, Stefanie Wunsch, Elisabeth Ullrich, Christoph Zurl, Martin Hoenigl and Robert Krause
J. Fungi 2020, 6(2), 43; https://doi.org/10.3390/jof6020043 - 27 Mar 2020
Cited by 5 | Viewed by 3185
Abstract
Molecular techniques including the sequencing of fungal-specific DNA targets are increasingly used in the diagnosis of suspected invasive fungal infections. In contrast to established biomarkers like galactomannan or 1-3-β-d-glucan, the clinical impact of these methods remains unknown. We retrospectively investigated the [...] Read more.
Molecular techniques including the sequencing of fungal-specific DNA targets are increasingly used in the diagnosis of suspected invasive fungal infections. In contrast to established biomarkers like galactomannan or 1-3-β-d-glucan, the clinical impact of these methods remains unknown. We retrospectively investigated the impact of ITS1-sequencing on antifungal treatment strategies in 71 patients (81 samples) with suspected invasive fungal infections. ITS-sequencing either confirmed already ongoing antifungal therapy (19/71 patients, 27%), led to a change in antifungal therapy (11/71, 15%) or supported the decision to withhold antifungal treatment (34/71, 48%) (in seven of 71 patients, ITS-sequencing results were obtained postmortem). ITS-sequencing results led to a change in antifungal therapy in a relevant proportion of patients, while it confirmed therapeutic strategies in the majority. Therefore, ITS-sequencing was a useful adjunct to other fungal diagnostic measures in our cohort. Full article
(This article belongs to the Special Issue Molecular Diagnostics of Fungal Infections)
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9 pages, 495 KiB  
Article
Quantification of Pneumocystis jirovecii: Cross-Platform Comparison of One qPCR Assay with Leading Platforms and Six Master Mixes
by Sarah Dellière, Maud Gits-Muselli, P. Lewis White, Carlo Mengoli, Stéphane Bretagne and Alexandre Alanio
J. Fungi 2020, 6(1), 9; https://doi.org/10.3390/jof6010009 - 26 Dec 2019
Cited by 13 | Viewed by 3659
Abstract
Diagnosis of Pneumocystis jirovecii pneumonia relies on nucleic acid quantification in respiratory samples. Lack of standardization among molecular assays results in significant differences among assays/centers. To further promote standardization, we compared four thermocyclers and six master mixes for the detection of P. jirovecii [...] Read more.
Diagnosis of Pneumocystis jirovecii pneumonia relies on nucleic acid quantification in respiratory samples. Lack of standardization among molecular assays results in significant differences among assays/centers. To further promote standardization, we compared four thermocyclers and six master mixes for the detection of P. jirovecii. Whole nucleic acid (WNA) was extracted from broncho-alveolar lavages. Positive and negative sample extracts were pooled to get enough homogeneous materials. Three master mixes were tested to detect DNA by qPCR (D1, D2, and D3), and three to detect WNA by reverse transcriptase qPCR (W1, W2, and W3) manufactured by Roche, Eurogentec, Applied Biosystem, Invitrogen and Thermofischer Scientific. Experiments were performed on four thermocyclers (Roche LightCycler 480, Qiagen Rotor-Gene Q, Applied Biosystem ABI7500, and QuantStudio). Comparison of quantitative cycle (Cq) values between the methods targeting WNA versus DNA showed lower Cq values for WNA, independently of thermocycler and master mix. For high and low fungal loads, ∆Cq values between DNA and WNA amplification were 6.97 (±2.95) and 5.81 (±3.30), respectively (p < 0.0001). Regarding DNA detection, lower Cqs were obtained with D1 compared to D2 and D3, with median ∆Cq values of 2.6 (p = 0.015) and 2.9 (p = 0.039) respectively. Regarding WNA detection, no mix was superior to the others. PCR efficiency was not significantly different according to the qPCR platform (p = 0.14). This study confirmed the superiority of WNA over DNA detection. A calibration method (e.g., an international standard) for accurate comparative assessment of fungal load seems necessary. Full article
(This article belongs to the Special Issue Molecular Diagnostics of Fungal Infections)
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9 pages, 707 KiB  
Article
Serial Detection of Circulating Mucorales DNA in Invasive Mucormycosis: A Retrospective Multicenter Evaluation
by Toine Mercier, Marijke Reynders, Kurt Beuselinck, Ellen Guldentops, Johan Maertens and Katrien Lagrou
J. Fungi 2019, 5(4), 113; https://doi.org/10.3390/jof5040113 - 03 Dec 2019
Cited by 36 | Viewed by 4213
Abstract
Invasive mucormycosis is a fungal infection with high mortality. Early diagnosis and initiation of appropriate treatment is essential to improve survival. However, current diagnostic tools suffer from low sensitivity, leading to delayed or missed diagnoses. Recently, several PCR assays for the detection of [...] Read more.
Invasive mucormycosis is a fungal infection with high mortality. Early diagnosis and initiation of appropriate treatment is essential to improve survival. However, current diagnostic tools suffer from low sensitivity, leading to delayed or missed diagnoses. Recently, several PCR assays for the detection of Mucorales DNA have been developed. We retrospectively assessed the diagnostic and kinetic properties of a commercial Mucorales PCR assay (MucorGenius®, PathoNostics) on serial blood samples from patients with culture-positive invasive mucormycosis and found an overall sensitivity of 75%. Importantly, a positive test preceded a positive culture result by up to 81 days (median eight days, inter-quartile range 1.75–16.25). After initiation of appropriate therapy, the average levels of circulating DNA decreased after one week and stabilized after two weeks. In conclusion, detection of circulating Mucorales DNA appears to be a good, fast diagnostic test that often precedes the final diagnosis by several days to weeks. This test could be especially useful in cases in which sampling for culture or histopathology is not feasible. Full article
(This article belongs to the Special Issue Molecular Diagnostics of Fungal Infections)
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7 pages, 237 KiB  
Article
Detection of Fusarium Species in Clinical Specimens by Probe-Based Real-Time PCR
by Jan Springer, Grit Walther, Volker Rickerts, Axel Hamprecht, Birgit Willinger, Daniel Teschner, Hermann Einsele, Oliver Kurzai and Juergen Loeffler
J. Fungi 2019, 5(4), 105; https://doi.org/10.3390/jof5040105 - 12 Nov 2019
Cited by 6 | Viewed by 3191
Abstract
The mold Fusarium is a ubiquitous fungus causing plant, animal and human infections. In humans, Fusarium spp. are the major cause of eye infections in patients wearing contact lenses or after local trauma. Systemic infections by Fusarium spp. mainly occur in immunosuppressed patients [...] Read more.
The mold Fusarium is a ubiquitous fungus causing plant, animal and human infections. In humans, Fusarium spp. are the major cause of eye infections in patients wearing contact lenses or after local trauma. Systemic infections by Fusarium spp. mainly occur in immunosuppressed patients and can disseminate throughout the human body. Due to high levels of resistance to antifungals a fast identification of the causative agent is an urgent need. By using a probe-based real-time PCR assay specific for the genus Fusarium we analysed several different clinical specimens detecting Fusarium spp. commonly found in clinical samples in Germany. Also, a large collection of lung fluid samples of haematological patients was analysed (n = 243). In these, two samples (0.8%) were reproducibly positive, but only one could be confirmed by sequencing. For this case of probable invasive fungal disease (IFD) culture was positive for Fusarium species. Here we describe a rapid, probe-based real-time PCR assay to specifically detect DNA from a broad range of Fusarium species and its application to clinically relevant specimens. Full article
(This article belongs to the Special Issue Molecular Diagnostics of Fungal Infections)
10 pages, 885 KiB  
Article
First Isolation, Antifungal Susceptibility, and Molecular Characterization of Cryptococcus neoformans from the Environment in Croatia
by Donjeta Pllana-Hajdari, Massimo Cogliati, Ljiljana Čičmak, Sanja Pleško, Emilija Mlinarić-Missoni and Ivana Mareković
J. Fungi 2019, 5(4), 99; https://doi.org/10.3390/jof5040099 - 12 Oct 2019
Cited by 5 | Viewed by 2780
Abstract
The purpose of this study was to investigate the presence of Cryptococcus neoformans species complex isolates from environmental sources in Croatia and to determine their molecular types and antifungal susceptibility. Swab samples of tree hollows and bird excreta in the soil beneath trees [...] Read more.
The purpose of this study was to investigate the presence of Cryptococcus neoformans species complex isolates from environmental sources in Croatia and to determine their molecular types and antifungal susceptibility. Swab samples of tree hollows and bird excreta in the soil beneath trees were collected. Samples included 472 (92.73%) samples obtained from tree hollows and 37 (7.27%) samples from bird excreta. Four C. neoformans species complex isolates were recovered from tree hollow swabs along the Mediterranean coast, while there were no isolates recovered from bird excreta or from the continental area. Three isolates were identified as molecular types VNI and one as VNIV. All tested antifungals showed high in vitro activity against the four isolates. This is the first report proving the presence of C. neoformans species complex in the environment of Croatia. The results of the study suggest a major risk of exposure for inhabitants living along the Croatian coast and that both VNI and VNIV molecular types can be expected in clinical cases of cryptococcosis. Susceptibility to antifungals confirmed that no resistance should be expected in patients with cryptococcosis at the present time. Full article
(This article belongs to the Special Issue Molecular Diagnostics of Fungal Infections)
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20 pages, 3291 KiB  
Article
Evaluation of a Novel Mitochondrial Pan-Mucorales Marker for the Detection, Identification, Quantification, and Growth Stage Determination of Mucormycetes
by Rita Caramalho, Lisa Madl, Katharina Rosam, Günter Rambach, Cornelia Speth, Johannes Pallua, Thomas Larentis, Ricardo Araujo, Ana Alastruey-Izquierdo, Cornelia Lass-Flörl and Michaela Lackner
J. Fungi 2019, 5(4), 98; https://doi.org/10.3390/jof5040098 - 11 Oct 2019
Cited by 20 | Viewed by 3609
Abstract
Mucormycosis infections are infrequent yet aggressive and serious fungal infections. Early diagnosis of mucormycosis and its discrimination from other fungal infections is required for targeted treatment and more favorable patient outcomes. The majority of the molecular assays use 18 S rDNA. In the [...] Read more.
Mucormycosis infections are infrequent yet aggressive and serious fungal infections. Early diagnosis of mucormycosis and its discrimination from other fungal infections is required for targeted treatment and more favorable patient outcomes. The majority of the molecular assays use 18 S rDNA. In the current study, we aimed to explore the potential of the mitochondrial rnl (encoding for large-subunit-ribosomal-RNA) gene as a novel molecular marker suitable for research and diagnostics. Rnl was evaluated as a marker for: (1) the Mucorales family, (2) species identification (Rhizopus arrhizus, R. microsporus, Mucor circinelloides, and Lichtheimia species complexes), (3) growth stage, and (4) quantification. Sensitivity, specificity, discriminatory power, the limit of detection (LoD), and cross-reactivity were evaluated. Assays were tested using pure cultures, spiked clinical samples, murine organs, and human paraffin-embedded-tissue (FFPE) samples. Mitochondrial markers were found to be superior to nuclear markers for degraded samples. Rnl outperformed the UMD universal® (Molyzm) marker in FFPE (71.5% positive samples versus 50%). Spiked blood samples highlighted the potential of rnl as a pan-Mucorales screening test. Fungal burden was reproducibly quantified in murine organs using standard curves. Identification of pure cultures gave a perfect (100%) correlation with the detected internal transcribed spacer (ITS) sequence. In conclusion, mitochondrial genes, such as rnl, provide an alternative to the nuclear 18 S rDNA genes and deserve further evaluation. Full article
(This article belongs to the Special Issue Molecular Diagnostics of Fungal Infections)
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9 pages, 461 KiB  
Communication
Diagnostic Performance of a Novel Multiplex PCR Assay for Candidemia among ICU Patients
by Stefan Fuchs, Cornelia Lass-Flörl and Wilfried Posch
J. Fungi 2019, 5(3), 86; https://doi.org/10.3390/jof5030086 - 17 Sep 2019
Cited by 24 | Viewed by 4092
Abstract
Candidemia poses a major threat to ICU patients and is routinely diagnosed by blood culture, which is known for its low sensitivity and long turnaround times. We compared the performance of a novel, Candida-specific multiplex real-time PCR assay (Fungiplex® Candida IVD [...] Read more.
Candidemia poses a major threat to ICU patients and is routinely diagnosed by blood culture, which is known for its low sensitivity and long turnaround times. We compared the performance of a novel, Candida-specific multiplex real-time PCR assay (Fungiplex® Candida IVD Real-Time PCR Kit) with blood culture and another established diagnostic real-time PCR assay (LightCycler SeptiFast Test) with respect to Candida detection from whole blood samples. Clinical samples from 58 patients were analyzed by standard blood culture (BC) and simultaneously tested with the Fungiplex Candida PCR (FP) and the SeptiFast test (SF) for molecular detection of Candida spp. Compared to BC, the FP test showed high diagnostic power, with a sensitivity of 100% and a specificity of 94.1%. Overall diagnostic accuracy reached 94.6%. Using SF, we found a sensitivity of 60%, a specificity of 96.1%, and an overall diagnostic accuracy of 92.9%. The Fungiplex Candida PCR has shown good sensitivity and specificity on clinical samples of high-risk patients for direct detection of Candida species in whole blood samples. Together with conventional diagnostics (BC and antigen testing), this new multiplex PCR assay may contribute to a rapid and accurate diagnosis of candidiasis. Full article
(This article belongs to the Special Issue Molecular Diagnostics of Fungal Infections)
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12 pages, 1890 KiB  
Article
Minimal Inhibitory Concentration (MIC)-Phenomena in Candida albicans and Their Impact on the Diagnosis of Antifungal Resistance
by Ulrike Binder, Maria Aigner, Brigitte Risslegger, Caroline Hörtnagl, Cornelia Lass-Flörl and Michaela Lackner
J. Fungi 2019, 5(3), 83; https://doi.org/10.3390/jof5030083 - 04 Sep 2019
Cited by 12 | Viewed by 3876
Abstract
Antifungal susceptibility testing (AFST) of clinical isolates is a tool in routine diagnostics to facilitate decision making on optimal antifungal therapy. The minimal inhibitory concentration (MIC)-phenomena (trailing and paradoxical effects (PXE)) observed in AFST complicate the unambiguous and reproducible determination of MICs and [...] Read more.
Antifungal susceptibility testing (AFST) of clinical isolates is a tool in routine diagnostics to facilitate decision making on optimal antifungal therapy. The minimal inhibitory concentration (MIC)-phenomena (trailing and paradoxical effects (PXE)) observed in AFST complicate the unambiguous and reproducible determination of MICs and the impact of these phenomena on in vivo outcome are not fully understood. We aimed to link the MIC-phenomena with in vivo treatment response using the alternative infection model Galleria mellonella. We found that Candida albicans strains exhibiting PXE for caspofungin (CAS) had variable treatment outcomes in the Galleria model. In contrast, C. albicans strains showing trailing for voriconazole failed to respond in vivo. Caspofungin- and voriconazole-susceptible C. albicans strains responded to the respective antifungal therapy in vivo. In conclusion, MIC data and subsequent susceptibility interpretation of strains exhibiting PXE and/or trailing should be carried out with caution, as both effects are linked to drug adaptation and treatment response is uncertain to predict. Full article
(This article belongs to the Special Issue Molecular Diagnostics of Fungal Infections)
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5 pages, 964 KiB  
Communication
Hypoxia Decreases Diagnostic Biomarkers for Aspergillosis In Vitro
by Elisabeth Maurer, Maria Aigner, Cornelia Lass-Flörl and Ulrike Binder
J. Fungi 2019, 5(3), 61; https://doi.org/10.3390/jof5030061 - 11 Jul 2019
Cited by 1 | Viewed by 2796
Abstract
The aim of the study was to evaluate the influence of hypoxia on galactomannan and (1,3)-β-d-glucan release of clinically relevant Aspergilli in vitro. Hypoxia decreased biomass and consequently led to lower biomarker release. However, when normalized to biomass, hypoxia led to [...] Read more.
The aim of the study was to evaluate the influence of hypoxia on galactomannan and (1,3)-β-d-glucan release of clinically relevant Aspergilli in vitro. Hypoxia decreased biomass and consequently led to lower biomarker release. However, when normalized to biomass, hypoxia led to increased levels of biomarkers at early growth stages (24 h). Antifungals (amphotericin B and voriconazole) decreased the galactomannan amount of A. fumigatus, even more prominently in hypoxia. Full article
(This article belongs to the Special Issue Molecular Diagnostics of Fungal Infections)
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11 pages, 569 KiB  
Article
Inter-Specimen Imbalance of Mitochondrial Gene Copy Numbers Predicts Clustering of Pneumocystis jirovecii Isolates in Distinct Subgroups
by Cara Mia Dunaiski, Lena Janssen, Hannah Erzinger, Monika Pieper, Sarah Damaschek, Oliver Schildgen and Verena Schildgen
J. Fungi 2018, 4(3), 84; https://doi.org/10.3390/jof4030084 - 10 Jul 2018
Cited by 5 | Viewed by 3195
Abstract
The molecular detection of Pneumocystis jirovecii is an important therapy-relevant tool in microbiological diagnostics. However, the quantification of this pathogen in the past has revealed discordant results depending on the target gene. As the clinical variety of P. jirovecii infections ranges between life-threatening [...] Read more.
The molecular detection of Pneumocystis jirovecii is an important therapy-relevant tool in microbiological diagnostics. However, the quantification of this pathogen in the past has revealed discordant results depending on the target gene. As the clinical variety of P. jirovecii infections ranges between life-threatening infections and symptom-free colonization, the question arises if qPCRs are reliable tools for quantitative diagnostics of P. jirovecii. P. jirovecii positive BALs were quantitatively tested for the copy numbers of one mitochondrial (COX-1) and two nuclear single-copy genes (KEX1 and DHPS) compared to the mitochondrial large subunit (mtLSU) by qPCR. Independent of the overall mtLSU copy number P. jirovecii clustered into distinct groups based on the ratio patterns of the respective qPCRs. This study, which compared different mitochondrial to nuclear gene ratio patterns of independent patients, shows that the mtLSU gene represents a highly sensitive qPCR tool for the detection of P. jirovecii, but does not display a reliable target for absolute quantification. Full article
(This article belongs to the Special Issue Molecular Diagnostics of Fungal Infections)
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Review

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25 pages, 1499 KiB  
Review
An Overview on Conventional and Non-Conventional Therapeutic Approaches for the Treatment of Candidiasis and Underlying Resistance Mechanisms in Clinical Strains
by Sara B. Salazar, Rita S. Simões, Nuno A. Pedro, Maria Joana Pinheiro, Maria Fernanda N. N. Carvalho and Nuno P. Mira
J. Fungi 2020, 6(1), 23; https://doi.org/10.3390/jof6010023 - 10 Feb 2020
Cited by 27 | Viewed by 5324
Abstract
Fungal infections and, in particular, those caused by species of the Candida genus, are growing at an alarming rate and have high associated rates of mortality and morbidity. These infections, generally referred as candidiasis, range from common superficial rushes caused by an overgrowth [...] Read more.
Fungal infections and, in particular, those caused by species of the Candida genus, are growing at an alarming rate and have high associated rates of mortality and morbidity. These infections, generally referred as candidiasis, range from common superficial rushes caused by an overgrowth of the yeasts in mucosal surfaces to life-threatening disseminated mycoses. The success of currently used antifungal drugs to treat candidiasis is being endangered by the continuous emergence of resistant strains, specially among non-albicans Candida species. In this review article, the mechanisms of action of currently used antifungals, with emphasis on the mechanisms of resistance reported in clinical isolates, are reviewed. Novel approaches being taken to successfully inhibit growth of pathogenic Candida species, in particular those based on the exploration of natural or synthetic chemicals or on the activity of live probiotics, are also reviewed. It is expected that these novel approaches, either used alone or in combination with traditional antifungals, may contribute to foster the identification of novel anti-Candida therapies. Full article
(This article belongs to the Special Issue Molecular Diagnostics of Fungal Infections)
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14 pages, 299 KiB  
Review
Blood Aspergillus PCR: The Good, the Bad, and the Ugly
by Matthias Egger, Jeffrey D. Jenks, Martin Hoenigl and Juergen Prattes
J. Fungi 2020, 6(1), 18; https://doi.org/10.3390/jof6010018 - 27 Jan 2020
Cited by 28 | Viewed by 4875
Abstract
Invasive Aspergillosis (IA) is one of the most common invasive fungal diseases and is accompanied by high morbidity and mortality. In order to maximize patient outcomes and survival, early and rapid diagnosis has been shown to be pivotal. Hence, diagnostic tools aiding and [...] Read more.
Invasive Aspergillosis (IA) is one of the most common invasive fungal diseases and is accompanied by high morbidity and mortality. In order to maximize patient outcomes and survival, early and rapid diagnosis has been shown to be pivotal. Hence, diagnostic tools aiding and improving the diagnostic process are ambitiously searched for. In this context, polymerase chain reaction (PCR) may represent a potential candidate. Its additional value and benefits in diagnosis have been demonstrated and are scientifically established. Nevertheless, standardized and widespread usage is sparse because several factors influence diagnostic quality and need to be considered in order to optimize diagnostic performance and outcome. In the following review, the current role of PCR in the diagnosis of IA is explored, with special focus on the strengths and limitations of PCR in different settings. Full article
(This article belongs to the Special Issue Molecular Diagnostics of Fungal Infections)
15 pages, 709 KiB  
Review
Detecting Azole-Antifungal Resistance in Aspergillus fumigatus by Pyrosequencing
by Mireille H. van der Torre, Lilyann Novak-Frazer and Riina Rautemaa-Richardson
J. Fungi 2020, 6(1), 12; https://doi.org/10.3390/jof6010012 - 10 Jan 2020
Cited by 19 | Viewed by 6812
Abstract
Guidelines on the diagnosis and management of Aspergillus disease recommend a multi-test approach including CT scans, culture, fungal biomarker tests, microscopy and fungal PCR. The first-line treatment of confirmed invasive aspergillosis (IA) consists of drugs in the azole family; however, the emergence of [...] Read more.
Guidelines on the diagnosis and management of Aspergillus disease recommend a multi-test approach including CT scans, culture, fungal biomarker tests, microscopy and fungal PCR. The first-line treatment of confirmed invasive aspergillosis (IA) consists of drugs in the azole family; however, the emergence of azole-resistant isolates has negatively impacted the management of IA. Failure to detect azole-resistance dramatically increases the mortality rates of azole-treated patients. Despite drug susceptibility tests not being routinely performed currently, we suggest including resistance testing whilst diagnosing Aspergillus disease. Multiple tools, including DNA sequencing, are available to screen for drug-resistant Aspergillus in clinical samples. This is particularly beneficial as a large proportion of IA samples are culture negative, consequently impeding susceptibility testing through conventional methods. Pyrosequencing is a promising in-house DNA sequencing method that can rapidly screen for genetic hotspots associated with antifungal resistance. Pyrosequencing outperforms other susceptibility testing methods due to its fast turnaround time, accurate detection of polymorphisms within critical genes, including simultaneous detection of wild type and mutated sequences, and—most importantly—it is not limited to specific genes nor fungal species. Here we review current diagnostic methods and highlight the potential of pyrosequencing to aid in a diagnosis complete with a resistance profile to improve clinical outcomes. Full article
(This article belongs to the Special Issue Molecular Diagnostics of Fungal Infections)
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8 pages, 846 KiB  
Review
The Role of Molecular Tests in the Diagnosis of Disseminated Histoplasmosis
by Izadora Clezar da Silva Vasconcellos, Daiane Flores Dalla Lana and Alessandro C. Pasqualotto
J. Fungi 2020, 6(1), 1; https://doi.org/10.3390/jof6010001 - 18 Dec 2019
Cited by 19 | Viewed by 3702
Abstract
Histoplasmosis is an emerging fungal disease, with global distribution. The disseminated form of the disease is a more severe infection, generally associated with AIDS. Classic diagnostic methods for histoplasmosis consist of microscopy, culture, and histopathology. More recently, the importance of Histoplasma antigen detection [...] Read more.
Histoplasmosis is an emerging fungal disease, with global distribution. The disseminated form of the disease is a more severe infection, generally associated with AIDS. Classic diagnostic methods for histoplasmosis consist of microscopy, culture, and histopathology. More recently, the importance of Histoplasma antigen detection has dominated the literature on histoplasmosis diagnosis, but the relevance of molecular assays has not been as much studied. Here we describe the results of a systematic literature review focusing on studies that mainly compared immunological techniques (Histoplasma urine antigen detection) with molecular tests for the diagnosis of histoplasmosis. In addition to the review of comparative studies using such diagnostic techniques, the literature on polymerase chain reaction (PCR) tests in patients with disseminated histoplasmosis is also summarized. Two studies reported the comparison between immunological and molecular methods applied simultaneously for the diagnosis of disseminated histoplasmosis. PCR demonstrates a satisfactory performance assisting in the detection of Histoplasma spp. DNA in clinical samples. Full article
(This article belongs to the Special Issue Molecular Diagnostics of Fungal Infections)
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8 pages, 227 KiB  
Review
Molecular Diagnostics of Mucormycosis in Hematological Patients: A Literature Review
by Olga V. Shadrivova, Ekaterina V. Burygina and Nikolai N. Klimko
J. Fungi 2019, 5(4), 112; https://doi.org/10.3390/jof5040112 - 29 Nov 2019
Cited by 7 | Viewed by 2882
Abstract
Objectives: to analyze the results of molecular methods applying for the diagnosis of mucormycosis in hematologic patients based on a literature review. Data sources: A systematic search in databases PubMed, Google Scholar for August 2019. Review eligibility criteria: original articles published in English, [...] Read more.
Objectives: to analyze the results of molecular methods applying for the diagnosis of mucormycosis in hematologic patients based on a literature review. Data sources: A systematic search in databases PubMed, Google Scholar for August 2019. Review eligibility criteria: original articles published in English, studies of molecular methods for the diagnosis of mucormycosis in hematologic patients. Results. We analyzed the research data from 116 hematological patients with mucormycosis, including children (6%). Patients with localized forms of mucormycosis prevailed (72%), and lung involvement was diagnosed in 58% of these cases. For molecular verification of the causative agent of mucormycosis, blood serum was most often used, less commonly postoperative and autopsy material, biopsy specimens, formalin-fixed paraffin-embedded samples and bronchoalveolar lavage, pleural fluid and sputum. The sensitivity of molecular diagnostics of mucormycosis in a cohort of hematological patients was 88.2%. Conclusion. The use of molecular techniques along with standard mycological methods will improve the diagnostics of mucormycosis in hematologic patients. However, prospective studies of the effectiveness of molecular methods for the diagnosis of mucormycosis of various etiologies in hematological patients, including children, using bronchoalveolar lavage (BAL) and cerebrospinal fluid (CSF) are needed. Full article
(This article belongs to the Special Issue Molecular Diagnostics of Fungal Infections)
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23 pages, 320 KiB  
Review
Identification of Mycoses in Developing Countries
by Amir Arastehfar, Brian L. Wickes, Macit Ilkit, David H. Pincus, Farnaz Daneshnia, Weihua Pan, Wenjie Fang and Teun Boekhout
J. Fungi 2019, 5(4), 90; https://doi.org/10.3390/jof5040090 - 29 Sep 2019
Cited by 43 | Viewed by 5797
Abstract
Extensive advances in technology offer a vast variety of diagnostic methods that save time and costs, but identification of fungal species causing human infections remains challenging in developing countries. Since the echinocandins, antifungals widely used to treat invasive mycoses, are still unavailable in [...] Read more.
Extensive advances in technology offer a vast variety of diagnostic methods that save time and costs, but identification of fungal species causing human infections remains challenging in developing countries. Since the echinocandins, antifungals widely used to treat invasive mycoses, are still unavailable in developing countries where a considerable number of problematic fungal species are present, rapid and reliable identification is of paramount importance. Unaffordability, large footprints, lack of skilled personnel, and high costs associated with maintenance and infrastructure are the main factors precluding the establishment of high-precision technologies that can replace inexpensive yet time-consuming and inaccurate phenotypic methods. In addition, point-of-care lateral flow assay tests are available for the diagnosis of Aspergillus and Cryptococcus and are highly relevant for developing countries. An Aspergillus galactomannan lateral flow assay is also now available. Real-time PCR remains difficult to standardize and is not widespread in countries with limited resources. Isothermal and conventional PCR-based amplification assays may be alternative solutions. The combination of real-time PCR and serological assays can significantly increase diagnostic efficiency. However, this approach is too expensive for medical institutions in developing countries. Further advances in next-generation sequencing and other innovative technologies such as clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostic tools may lead to efficient, alternate methods that can be used in point-of-care assays, which may supplement or replace some of the current technologies and improve the diagnostics of fungal infections in developing countries. Full article
(This article belongs to the Special Issue Molecular Diagnostics of Fungal Infections)

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7 pages, 241 KiB  
Brief Report
Molecular Detection of Aspergillus: Application of a Real-Time PCR Multiplex Assay in Tissue Samples
by Raquel Sabino, Helena Simões and Cristina Veríssimo
J. Fungi 2020, 6(1), 11; https://doi.org/10.3390/jof6010011 - 10 Jan 2020
Cited by 5 | Viewed by 3116
Abstract
Diagnosis of invasive fungal infections is complex, and the lack of standardization of molecular methods is still a challenge. Several methods are available for the diagnosis of invasive aspergillosis, but their effectiveness will depend on the studied population, the patients’ comorbidities, and the [...] Read more.
Diagnosis of invasive fungal infections is complex, and the lack of standardization of molecular methods is still a challenge. Several methods are available for the diagnosis of invasive aspergillosis, but their effectiveness will depend on the studied population, the patients’ comorbidities, and the use of mold active prophylaxis, among others. The ability to determine the identity of the infecting Aspergillus species, and to detect mutations conferring specific resistance patterns directly from DNA extracted from the biological product, is an advantage of nucleic acid testing compared with antigen-based assays. In this study, we present laboratory cases where the diagnosis of aspergillosis was performed using a real-time multiplex PCR for the detection of Aspergillus DNA in tissue samples, showing its usefulness as one more tool in the diagnosis of aspergillosis in tissue samples. Aspergillus real-time multiplex PCR was also used to detect azole-resistance in some cases. In the majority of the PCR positive cases, cultures remained negative after 60 days. The PCR assay directed to Aspergillus gave positive signals for Aspergillus fumigatus sensu stricto. Results were confirmed by panfungal PCR, followed by sequencing, revealing 100% homology with Aspergillus fumigatus sensu stricto. Mutations conferring azole resistance were not detected. Full article
(This article belongs to the Special Issue Molecular Diagnostics of Fungal Infections)
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