Successful Medical Management of Recalcitrant Fusarium solani Keratitis: Molecular Identification and Susceptibility Patterns

Fungal keratitis is a rare but sight-threatening infection of the cornea that may be caused by several fungal pathogens. A delay in diagnosis and inadequate treatment may even lead to loss of the affected eye. Fungal keratitis is often misdiagnosed as bacterial keratitis because isolation and identification of the fungal pathogen is difficult and requires experience, and fungal growth in culture requires time. In this report, a 14-year-old boy with recalcitrant Fusarium solani keratitis, unresponsive to initial therapy, is presented. CLSI M38-A2 in vitro antifungal susceptibility tests demonstrated that only amphotericin B (0.5 μg/ml) had potent activity against F. solani; however, fluconazole (>64 μg/ml), itraconazole (>16 μg/ml), voriconazole (8 μg/ml), and posaconazole (>16 μg/ml) had high minimum inhibitory concentrations. In addition, caspofungin (>16 μg/ml) and anidulafungin (>16 μg/ml) exhibited high minimum effective concentrations. Repeated intrastromal voriconazole injections, topical voriconazole, and caspofungin combined with systemic antifungal agents improved of the corneal lesion with a significant increase in visual acuity. Intrastromal voriconazole injection may be used as an adjunctive treatment method for recalcitrant fungal keratitis with no prominent complications. The intrastromal route could be an effective route of administration of antifungal agents, especially for F. solani keratitis, as in this case. A combination of various antifungal agents administered by different routes prevented loss of the eye.     

Molecular Identification and Antifungal Susceptibility of Clinical Isolates of Sporothrix schenckii Complex in Medellin,Colombia

Background

Sporotrichosis is a subcutaneous mycosis that affects humans and other animals. Infection prevails in tropical and subtropical countries. Until a few years ago, it was considered that two varieties of Sporothrix schenckii caused this mycosis, but by applying molecular taxonomic markers, it has been demonstrated that there are several cryptic species within S. schenckii complex which varies in susceptibility, virulence, and geographic distribution.

Objective

This study aimed to identify the clinical isolates of Sporothrix spp. from patients with sporotrichosis in Medellin, Colombia, using two markers and to evaluate the in vitro susceptibility to itraconazole.

Methods

Thirty-four clinical isolates of Sporothrix spp. from Colombia, three from Mexico, and one from Guatemala were identified through sequencing of the noncoding region ITS-1?+?5.8SDNAr?+?ITS-2 and of the fragment containing exons 3 and 4 of the β-tubulin gene. Clinical isolate sequences were compared with GenBank reference sequences using the BLASTN tool, and then, phylogenetic analysis was performed. Besides, the in vitro susceptibility to itraconazole was evaluated by determining the minimum inhibitory concentrations according to the CLSI M38-A2 method.

Results

Clinical isolates were identified by morphology as Sporothrix spp. Using the molecular markers, ITS and β-tubulin, isolates were identified as S. schenckii sensu stricto (25) and Sporothrix globosa (13). Susceptibility to itraconazole was variable among clinical isolates.

Conclusion

This is the first scientific publication that identifies species that cause sporotrichosis in Colombia, along with the antifungal susceptibility to itraconazole.

     

黄瓜枯萎病抗性基因的连锁分子标记

黄瓜枯萎病是危害我国黄瓜的主要病害。本实验以黄瓜抗枯萎病亲本WIS2757和感枯萎病亲本津研2号及其F2代分离群体为试材,采用分离群体分组分析法(BSA)进行了与黄瓜抗枯萎病基因连锁的分子标记研究。AFLP分析表明:引物对P15M5扩增出的特异DNA片段P15M5-310与WIS2757黄瓜枯萎病抗性基因连锁,遗传距离为7cM。     

小麦赤霉病新抗源的发掘与抗性位点的检测分析

小麦赤霉病是由禾谷镰刀菌引起的世界性重要病害,发掘优异的抗性种质资源、培育抗病品种是持续防治赤霉病最经济且环境友好的措施。为发掘新的赤霉病抗源,本研究于2017—2021年在弥雾保湿大棚中,采用单花滴注法对642份小麦种质资源的赤霉病抗扩展性进行鉴定,同时利用已知抗赤霉病基因/位点Fhb1~Fhb7的分子标记对筛选出的抗性种质基因型进行分析。结果表明,不同年份间赤霉病病小穗率的相关性均达到极显著水平。筛选到3年及以上赤霉病抗性优于扬麦158的种质81份,主要来自长江中下游麦区,其中33份种质连续4年抗性优于扬麦158;筛选到3年及以上抗性与苏麦3号相当的种质9份,分别为望水白、Grandin、浩麦1号、剑子麦、魁小麦、农林26、软秆洋麦、苏麦2号和武农6号,其中剑子麦、软秆洋麦、苏麦2号和Grandin连续4年抗性与苏麦3号相当。对抗性种质携带的抗赤霉病基因/位点进行分析发现,浩麦1号、冀师7225-28、南农13Y110、石优17和武农6号不携带任何已知抗赤霉病基因/QTL,为小麦抗赤霉病研究和品种培育提供了新的种质资源和理论依据。     

Adipocytokine Profile,Cytokine Levels and Foxp3 Expression in Multiple Sclerosis: a Possible Link to Susceptibility and Clinical Course of Disease

Background

Adipocytokines may be involved in multiple sclerosis (MS) as well as other autoimmune and inflammatory-related diseases. This study aims to compare levels of resistin, visfatin and leptin in three subgroups of MS patients with healthy subjects and also to study their relationship with Foxp3 expression and levels of several pro-inflammatory mediators such as interleukine-1 β(IL-1 β),tumor necrosis factor-α (TNF-α) and human sensitive C-reactive protein (hs-CRP).

Methods

A total of 391 subjects including 200 healthy controls and 191 MS patients were recruited for this case-control study. Circulating adipocytokines and inflammatory mediators were measured using immunoassay methods. Foxp3 gene expression in peripheral blood mononuclear cells (PBMC) was determined by quantitative real-time PCR. Fat tissue mass was evaluated by using dual energy X-ray absorptiometery (DEXA).

Results

A significant difference was observed in levels of inflammatory mediators, adipocytokines, Foxp3 gene expression and adipose tissue mass between MS patients and healthy controls. All adipocytokines were positively correlated with levels of inflammatory mediators and negatively correlated with Foxp3 expression in MS patients. In controls, there were positive correlations between circulating leptin and resistin with TNF-α and IL-1β in subgroup analysis, the highest levels of TNF-α, IL-1β, hs-CRP, resistin and leptin were observed in primary progressive-MS (PP-MS) patients. Also, expression of Foxp3 and levels of visfatin in relapsing remitting-MS(RR-MS) patients were higher compared with the other subgroups.

Conclusions

Our findings suggest the potential role of adipocytokines in pathogenesis and severity of MS. Notably, the relationship of adipocytokines levels with inflammatory cytokines as well as clinical features of MS could be considerable in translational medicine and biomarker studies.     

Molecular Identification,Mycotoxin Production and Comparative Pathogenicity of Fusarium temperatum Isolated from Maize in China

A recently isolated Fusarium population from maize in Belgium was identified as a new species, Fusarium temperatum. From a survey of Fusarium species associated with maize ear rot in nineteen provinces in 2009 in China, ten strains isolated from Guizhou and Hubei provinces were identified as F. temperatum. Morphological and molecular phylogenetic analyses based on the DNA sequences of individual translation elongation factor 1‐alpha and β‐tubulin genes revealed that the recovered isolates produced macroconidia typical of four‐septate with a foot‐shaped basal cell and belonged to F. temperatum that is distinctly different from its most closely related species F. subglutinans and others within Gibberella fujikuroi complex species from maize. All the strains from this newly isolated species were able to infect maize and wheat in field, with higher pathogenicity on maize. Mycotoxin determination of maize grains infected by the strains under natural field condition by ultra‐high‐performance liquid chromatography–tandem mass spectrometry and gas chromatography–mass spectrometry analyses showed that among fifteen mycotoxins assayed, two mycotoxins fumonisin B₁ and B₂ ranging from 9.26 to 166.89 μg/g were detected, with massively more FB₂ mycotoxin (2.8‐ to 108.8‐fold) than FB₁. This mycotoxin production profile is different from that of the Belgian population in which only fumonisin B₁ was barely detected in one of eleven strains assayed. Comparative analyses of the F. temperatum and F. subglutinans strains showed that the highest fumonisin producers were present among the F. temperatum population, which were also the most pathogenic to maize. These results suggested a need for proper monitoring and controlling this species in the relevant maize‐growing regions.     

A Case of Relapsed Chromoblastomycosis Due to Fonsecaea monophora: Antifungal Susceptibility and Phylogenetic Analysis

Chromoblastomycosis is a chronic cutaneous and subcutaneous mycosis. The management of this infection continues to be challenging because there is no consensus on the therapeutic regimen. We report here a case of a 69-year-old male patient with cauliflower-like lesions on his left leg and foot. He had already been treated with itraconazole at a dose of 200 mg/day for 5 months, with mycological cure for all the affected areas. However, the lesions relapsed at both sites, and treatment with itraconazole was resumed at the dose previously used. Initially, direct mycological examination, cultural, and microculture slide observation were performed. Afterward, sequencing of the ITS1-5.8S rDNA-ITS2 region of the fungal DNA and evaluation of its susceptibility to antifungal agents alone and in combination were performed. In direct mycological examination, the presence of sclerotic cells was verified, and the fungus was identified as Fonsecaea based on cultural and microscopic examinations. Identification as Fonsecaea monophora was confirmed after sequencing of the ITS region and phylogenetic analysis. The isolate was susceptible to itraconazole and terbinafine. The combinations of amphotericin B and terbinafine and terbinafine and voriconazole were synergistic. The use of drugs for which the causative agent is susceptible to singly or in combination may be an alternative for the treatment of mycosis. Furthermore, the identification of the agent by molecular techniques is important for epidemiological purposes. To the best of our knowledge, this is the first case of relapsed chromoblastomycosis caused by F. monophora in Brazil.     

Relationship between Cell Wall Susceptibility to Cellulases and Pectinases of Fusarium oxysporum and Susceptibility of Date Palm Cultivars

Fusarium oxysporum f. sp. albedinis, the bayoud disease agent of date palm, grows on a mineral medium containing the cell walls of date palm roots as a sole carbohydrate source. The growth and development of pathogen under these conditions was related to the production of extracellular cell wall-degrading enzymes (CWDE): cellulases, polygalacturonases, polygalacturonate transeliminases, and pectinmethylesterases. The mycelial growth and the sporulation of pathogen were higher in the presence of cell walls of susceptible cultivars (BFG, JHL, BSK) than in the presence of those of resistant cultivars (IKL, SLY, BSTN). After 8 d of fungal culture, the activity of CWDE was equal whatever is the origin of the cell walls (resistant or susceptible cultivars). After 16 d of culture, the activity of these CWDE was higher when the parasite was cultivated on the cell walls of the susceptible cultivars than on those of the resistant cultivars. A positive correlation was observed between CWDE activities and the growth and the sporulation of F. oxysporum after 16 d of culture. These results clearly show a relation between the susceptibility/resistance of the cell walls of the roots of the date palm to the parasitic CWDE and the susceptibility/resistance of the cultivars.     

Developmental Regulation of Susceptibility and Tolerance to Fusarium Root Rot in Beans

The soil-borne fungus, Fusarium solani f. sp. phaseoli, attacks roots and hypocotyls of bean (Phaseolus vulgaris) plants causing a devastating disease called root and foot rot. In a study of the host-pathogen relationship it was found that young bean roots, with the radicle just emerging, were highly tolerant to the pathogen, whereas older bean seedlings, with a fully developed root system, were completely susceptible. Investigations by low-temperature scanning electron microscopy demonstrated that significantly fewer spores and hyphae were present on the root surface of young bean seedlings as compared to older ones. A similar pattern of attachment was found when bean roots were inoculated with spores of F. solani f. sp. pisi, a related pathogen causing disease on peas but not on beans. Light microscopic studies showed that F. solani f. sp. pisi did not penetrate the root but rapidly formed thick-walled resting spores on the root surface. F. solani f. sp. phaseoli on the other hand quickly penetrated the root and formed an extensive network of fungal hyphae. These results demonstrate that the ability of fungal propagules to adhere to and to penetrate host tissues are two distinct processes. Furthermore, the data indicate that young bean roots lack a surface component necessary for attachment of fungal spores which may help explain their tolerance to Fusarium root rot.     

Keratitis Due to the Wood Saprobic Ascomycete, Auerswaldia lignicola (Family Botryosphaeriaceae), in a Carpenter in India

Keratitis due to Auerswaldia lignicola in a 32-year-old Indian male carpenter is described. At presentation, the patient reported persistent pain and tearing (left eye) in spite of topical antimicrobial therapy for more than 3 weeks. Clinically, mycotic keratitis was suspected, and direct microscopy of corneal scrapings stained by lactophenol cotton blue and Gram stains revealed broad septate hyphae. Intensive topical antifungal therapy was then given for 15 days. The keratitis continued to progress, necessitating therapeutic penetrating keratoplasty. Following the keratoplasty, there was rapid reduction in inflammation and gradual quietening of the eye. Brown–black fungal colonies resembling Lasiodiplodia theobromae were isolated from corneal scrape and corneal button (post-surgery) material on Sabouraud glucose–neopeptone agar; however, sporulation did not occur, so the morphological identification could not be confirmed. Sequence analysis of the 18S rRNA region of extracted fungal genomic DNA yielded an identification of A. lignicola Ariyawansa, J.K. Liu & K.D. Hyde; the sequence data have been deposited in GenBank (A. lignicola strain DK/V4, accession number KC866317.1). Medical management of keratitis due to such rarely reported fungal species may be difficult, necessitating surgical procedures.     

Molecular Identification and Antifungal Susceptibility of Yeasts and Molds Isolated from Patients with Otomycosis

Mycopathologia - Fungal otitis externa, an infection of the external auditory canal caused by molds and yeasts, accounts for approximately 10–20% of ear canal infections accompanying high...     

Molecular Identification and Genetic Diversity of Fusarium species Associated with Onion Fields in Turkey

To identify Fusarium species associated with diseases of root and basal plate of onion, surveys were conducted in seven provinces of Turkey in 2007. Samplings were performed in 223 fields, and 332 isolates belonging to 7 Fusarium spp. were obtained. The isolates were identified as F. oxysporum, F. solani, F. acuminatum, F. equiseti, F. proliferatum, F. redolens, and F. culmorum based on morphological and cultural characteristics. Also, species‐specific primers were used to confirm the identity of Fusarium species. F. oxysporum was the most commonly isolated species, comprising 66.57% of the total Fusarium species. F. redolens was identified for the first time in onion‐growing areas of Turkey. Selected isolates of each species were evaluated for their aggressiveness on onion plant. F. oxysporum, F. solani, F. acuminatum, F. proliferatum, and F. redolens were highly pathogenic, causing severe damping‐off on onion plants cv. Texas Early Grano. Inter‐simple sequence repeats (ISSR) markers revealed a high degree of intra‐ and interspecific polymorphisms among Fusarium spp.     

Real-time PCR detection and quantification of Fusarium poae,F. graminearum,F. sporotrichioides and F. langsethiae in cereal grains in Finland and Russia

Abstract

TaqMan real-time quantitative PCR assays were developed for the accurate detection and quantification of DNA from Fusarium poae and F. graminearum species, which are able to produce trichothecenes. These and other PCR assays were used for the quantification of trichothecene-producing Fusarium fungi in cereal grains. A correlation was found between the levels of F. poae DNA and nivalenol and enniatins in barley and between the levels of F. graminearum DNA and deoxynivalenol in oats. The correlations between F. poae DNA and nivalenol and F. graminearum DNA and deoxynivalenol levels were higher than those between these mycotoxins and morphologically determined F. poae and F. graminearum/F. culmorum contamination levels. The use of F. poae specific primers and probe together with F. sporotrichioides/F. langsethiae specific primers and probe in a multiplex qPCR assay yielded results in accordance with those obtained using these primers and probes separately.     

Susceptibility of Barley Cultivars and Lines to Fusarium Infection and Mycotoxin Accumulation in Kernels

Heads of 12 barley genotypes (8 cultivars and 4 lines) were inoculated with conidial suspension of the following single isolates: F. culmorum no. 3, F. graminearum no. 122 and F. sporotrichioides no. ATCC 62 360. The number of kernels per head. 1000 Kernel weight and yield have been calculated for each genotype. Seed samples collected at harvest were analysed for each genotype. Seed samples collected at harvest were analysed for several trichothecene mycotoxins and zearalenone.The mycotoxin concentrations (mg/kg) in barley kernels inoculated with F. graminearum were as follows. deoxynivalenol (DON) 0.1 to 5.4 (av. 2.3). 3-acetyldeoxy-nivalenol (3-AcDON) 0.0–0.2 (av. 0.1), 15-acetyldeoxynivalenol (15-AcDON) 0.0–0.7 (av.0.2), nivalenol (NIV) 0.0–0.8 (av. 0.3). zearalenone (ZEA) 0.0–0.1 (av. 0.0); F. culmorum: DON 0.6 to 12.0 (av. 5.3), 3-AcDON 0.1 to 1.0 (av. 0.6). 15-AcDON nd. NIV 0.1–0.7 (av. 0.3). ZEA 0.1–0.5 (av. 0.2). F. sporotrichioides T-2 toxin 2.4–13.9 (av. 6.0), HT-2-toxin 0.1–0.8 (av.0.3) and neosolaniol 0.2–1.5 (av.0.7).     

Antifungals: mechanism of action and resistance, established and novel drugs

Serious fungal infections, caused mostly by opportunistic species, are increasingly common in immunocompromised and other vulnerable patients. The use of antifungal drugs, primarily azoles and polyenes, has increased in parallel. Yet, established agents do not satisfy the medical need completely: azoles are fungistatic and vulnerable to resistance, whereas polyenes cause serious host toxicity. Drugs in clinical development include echinocandins, pneumocandins, and improved azoles. Promising novel agents in preclinical development include several inhibitors of fungal protein, lipid and cell wall syntheses. Recent advances in fungal genomics, combinatorial chemistry, and high-throughput screening may accelerate the antifungal discovery process.     

Rapid Identification of Four Fusarium spp. Complex by High-Resolution Melting Curve Analysis and their Antifungal Susceptibility Profiles

Fusarium species are globally distributed filamentous ascomycete fungi that are frequently reported as plant pathogens and opportunistic human pathogens, leading to yield loss of crops, mycotoxin contamination of food and feed products as well as damage to human and livestock. Human infections of Fusarium spp. are difficult to treat due to broad antifungal resistance by members of this genus. Their role as disease-causing agents in crops and humans suggests a need for antifungal resistance profiles as well as a simple, rapid, and cost effective identification method. Fusarium strains were isolated from food and clinical samples. High-resolution melting curve (HRM) analysis was performed using specific primers targeting internal transcribed spacer (ITS) region, followed with evaluation of specificity and sensitivity. The antifungal susceptibility of four Fusarium species was studied using the Sensititre YeastOne method. HRM analysis revealed reproducible, unimodal melting profiles specific to each of the four Fusarium strains, while no amplification of the negative controls. The minimum detection limits were 100–120 copies based on a 2 µl volume of template. Clear susceptibility differences were observed against antifungal agents by different Fusarium isolates, with amphotericin B and voriconazole displayed strongest antifungal effects to all the tested strains. We developed a simple, rapid, and low-cost qPCR-HRM method for identification of four Fusarium spp. (F. oxysporum, F. lateritium, F. fujikuroi, and F. solani). The antifungal susceptibility profiles supplied antifungal information of foodborne and clinical Fusarium spp. and provided guidance for clinical treatment of human infections.

     

Antifungals,arthropods and antifungal resistance prevention: lessons from ecological interactions

Arthropods can produce a wide range of antifungal compounds, including specialist proteins, cuticular products, venoms and haemolymphs. In spite of this, many arthropod taxa, particularly eusocial insects, make use of additional antifungal compounds derived from their mutualistic association with microbes. Because multiple taxa have evolved such mutualisms, it must be assumed that, under certain ecological circumstances, natural selection has favoured them over those relying upon endogenous antifungal compound production. Further, such associations have been shown to persist versus specific pathogenic fungal antagonists for more than 50 million years, suggesting that compounds employed have retained efficacy in spite of the pathogens'' capacity to develop resistance. We provide a brief overview of antifungal compounds in the arthropods’ armoury, proposing a conceptual model to suggest why their use remains so successful. Fundamental concepts embedded within such a model may suggest strategies by which to reduce the rise of antifungal resistance within the clinical milieu.