Proven Invasive Pulmonary Aspergillosis in Stem Cell Transplant Recipient Due to <Emphasis Type="Italic">Aspergillus sublatus</Emphasis>, a Cryptic Species of <Emphasis Type="Italic">A. nidulans</Emphasis>

Invasive fungal disease represents one of the severe complications in haematopoietic stem cell transplant recipients. We describe a case of a patient treated for relapse of chronic lymphoblastic leukaemia 6 years after HSCT. The patient was treated for invasive pulmonary aspergillosis but died 3 months later from multiple organ failures consisting of haemorrhagic necrotizing fungal pneumonia, refractory chronic hepatic graft versus host disease and cytomegalovirus hepatitis. Autopsy samples revealed histopathological evidence of fungal hyphae and an unusual Aspergillus nidulans-like species was isolated in pure culture. More precise identification was achieved by using scanning electron microscopy of ascospores and sequencing of calmodulin gene, and the isolate was subsequently re-identified as A. sublatus (section Nidulantes) and showed good in vitro susceptibility against all classes of antifungals. Commonly used ITS rDNA region and β-tubulin gene fail to discriminate A. sublatus from related pathogenic species, especially A. quadrilineatus and A. nidulans. Although this is the first case of proven IPA attributed to A. sublatus, we demonstrated that at least some previously reported infections due to A. quadrilineatus were probably caused by this cryptic species.     

Subcutaneous Phaeohyphomycosis Caused by <Emphasis Type="Italic">Exophiala oligosperma</Emphasis> in an Immunocompetent Host: Case Report and Literature Review

We report a case of subcutaneous infection caused by Exophiala oligosperma. Erythematous ulcerated plaque with exudate was major clinical features. Histopathological examination showed yeast-like cells and fungal hyphae. Mycological and molecular identification revealed E. oligosperma as etiologic agent. Local debridement and oral itraconazole were effective. To the best of our knowledge, this is the first report of phaeohyphomycosis caused by E. oligosperma in mainland China. This report highlights the potential role of E. oligosperma as an emerging cause of infection in immunocompetent patients.     

Donor Derived <Emphasis Type="Italic">Candida stellimalicola</Emphasis> in a Clinical Specimen: Preservation Fluid Contamination During Pancreas Procurement

We report here a case of possible donor-derived Candida stellimalicola infection after pancreas transplantation. Candida stellimalicola, an environmental non-filamentous yeast, was isolated from both the peritoneal fluid of the graft donor and the preservation fluid of the transplanted pancreas. Interestingly, this strain exhibited high minimum inhibitory concentrations to azoles. These results justified the use of echinocandins as therapy instead of fluconazole. This switch permitted a favorable outcome. To our knowledge, this is the first report of C. stellimalicola from clinical samples and therefore the first reported case of a possible human infection. This case report highlights the need for standardized microbiological procedures in solid organ transplant settings. Moreover, it underlines the importance of using molecular identification technique when routine techniques do not allow successful identification of the pathogen. It is of utmost importance to determine sensitivity profile, even in the absence of species-level identification, because resistance to fluconazole is not uncommon, especially in emergent species.     

Coinfection with <Emphasis Type="Italic">Lichtheimia corymbifera</Emphasis> and <Emphasis Type="Italic">Aspergillus flavus</Emphasis> in an Immune-Competent Patient Mimicking as Pulmonary-Renal Syndrome

Lichtheimia corymbifera and Aspergillus flavus pulmonary coinfection has been rarely reported in immune-competent patients. We report case of a young male who presented with clinical features of pulmonary-renal syndrome and was later diagnosed to have bilateral polymicrobial fungal lung infection.     

Fatal <Emphasis Type="Italic">Talaromyces marneffei</Emphasis> Infection in a Patient with Autoimmune Hepatitis

Talaromyces marneffei, previously known as Penicillium marneffei, is the most important pathogenic thermally dimorphic fungus causing systemic mycosis in Southeast Asia. Traditionally, T. marneffei infection in human was mainly associated with acquired immunodeficiency syndrome caused by HIV infection. In recent years, there has been an increasing number of T. marneffei infections reported in non-HIV-infected patients with other immunocompromised conditions, including autoantibodies against interferon-gamma, systemic lupus erythematosis, solid organ transplantation, Job’s syndrome, hematological malignancies, and use of novel targeted therapies. In this article, we describe the first case of fatal T. marneffei infection in a patient with underlying autoimmune hepatitis, presented as fever without localizing features. The diagnosis of talaromycosis was confirmed with the identification of the fungi isolated from the blood culture specimen by conventional methods and using matrix-assisted laser desorption–ionization time-of-flight mass spectrometer. This case shows the importance of a high index of suspicion, particularly for such a highly fatal but potentially treatable fungal infection.     

Isolated Cutaneous Granuloma Caused by <Emphasis Type="Italic">Candida glabrata</Emphasis>: A Rare Case Report and Literature Review

The incidence of candidiasis due to non-albicans Candida species (especially Candida glabrata) has significantly increased in recent decades. Candida glabrata often invades immunocompromised hosts and causes systemic or mucosal infections, whereas cutaneous infections are rarely reported. We present a rare case of cutaneous infection caused by C. glabrata and review all similar cases available in the PubMed database. A patient was admitted to the hospital with a 2-month history of a plaque on the face. Histopathological examination displayed typical infectious granulomas in the deep dermis, and the pathogen was finally confirmed as C. glabrata using a series of microbial examinations (fungal culture, biochemical test, and PCR-directed sequencing). The patient was completely cured after 4 months of treatment with oral itraconazole combined with topical terbinafine. We reviewed similar reports of cutaneous infection caused by C. glabrata. All the data suggested that an accurate diagnosis of cutaneous candidiasis depends mainly on histological and fungal examinations, especially molecular biological assays. Antifungal agents based on microbial susceptibility tests are the first-line treatment choice for C. glabrata infection, but the prognosis might be more dependent on the basic condition of the host.     

Fungal Keratitis Secondary to <Emphasis Type="Italic">Trametes betulina</Emphasis>: A Case Report and Review of Literature

Purpose

We report the first case of human infection and keratitis secondary to Trametes betulina, a rare filamentous fungus.

Methods

Clinical examination including external and slit-lamp examination and corneal scrapings with microbiologic evaluation were performed on a patient with chronic allergic conjunctivitis, entropion and a long-standing corneal ulcer resistant to treatment.

Results

The culture from the corneal scraping revealed a basidiomycetous fungus which was submitted for identification. DNA extraction with sequencing and analysis of the ITS and D1/D2 regions were performed on the isolate and demonstrated 100% similarity to Lenzites betulina/Trametes betulina. Susceptibility testing demonstrated potent in vitro activity of voriconazole (MIC < 0.03 μg/ml). The patient was treated with voriconazole, and the corneal ulcer and infiltrate resolved. The infection resulted in corneal thinning and a dense central corneal scar. Penetrating keratoplasty was performed 5 months after diagnosis and treatment and revealed stromal scarring without fungal elements.

Conclusion

This is the first reported case of keratitis caused by Trametes betulina. This organism should be considered in the differential diagnosis for rare filamentous fungal keratitis and its treatment with voriconazole also noted.

     

Innate host defenses against <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis>

Cryptococcus neoformans, the predominant etiological agent of cryptococcosis, can cause life-threatening infections of the central nervous system in immunocompromised and immunocompetent individuals. Cryptococcal meningoencephalitis is the most common disseminated fungal infection in AIDS patients, and remains the third most common invasive fungal infection among organ transplant recipients. The administration of highly active antiretroviral therapy (HAART) has resulted in a decrease in the number of cases of AIDS-related cryptococcosis in developed countries, but in developing countries where HAART is not readily available, Cryptococcus is still a major concern. Therefore, there is an urgent need for the development of novel therapies and/or vaccines to combat cryptococcosis. Understanding the protective immune responses against Cryptococcus is critical for development of vaccines and immunotherapies to combat cryptococcosis. Consequently, this review focuses on our current knowledge of protective immune responses to C. neoformans, with an emphasis on innate immune responses.     

Fungal load in <Emphasis Type="Italic">Bradysia agrestis</Emphasis>, a phytopathogen-transmitting insect vector

Larvae of Bradysia agrestis, a phytopathogen-transmitting insect vector in East Asia, were sampled from geographically (ecologically) segregated regions to identify their intestinal fungal flora. A total of 24 fungal strains were isolated from the insect vectors and selected based on morphological differences. In addition, 38 fungal strains were isolated from the ulcerated parts of invaded host plants by the same method, revealing the impact of vector fungal flora on their host plants. For molecular identification of the fungi, internal transcribed spacer (ITS) regions were amplified and sequenced. Their sequences were compared with sequences of other fungal strains obtained from NCBI GenBank, and their phylogeny was determined. The dominant fungal genera in the insect vector were Penicillium (25%), Aspergillus (21%), and Cladosporium (13%). In plant scar lesions, most fungal isolates belonged to the genera Fusarium (31.6%), Phoma (7.8%), Didymella (7.8%), and Epicoccum (7.8%). Fungal genera in vectors or host plant lesions differed by study site. Furthermore, diversity indices by study site showed clear differences based on Margalef’s richness (2.06, 2.40, 3.04), and Menhinick’s (1.89, 2.12, 2.53), and Simpson’s indices (0.14, 0.07, 0.07). In addition, common fungal strains in insect vectors were found to be closely related to members of the genera Cladosporium, Penicillium, or Aspergillus. Among these strains, those showing the highest homology with Aspergillus terreus, which regarded as beneficial fungal genera could be considered ideal paratransgenesis candidates. Some other fungal strains from vectors or ulcerated plant parts from each study site after B. agrestis invasion may be harmful in terms of plant disease or agrifood safety. This study provides information on the fungal microbiota of B. agrestis, an emerging problem in East Asia, and proposes paratransgenesis candidates to control this insect vector. Furthermore, potential transferable pathogens or commensal fungi were revealed by comparing the fungal biota between the insect gut and the ulcerated parts of the invaded host plants.     

Spontaneous Remission of Subcutaneous Scedosporiosis Caused by <Emphasis Type="Italic">Scedosporium dehoogii</Emphasis> in a Psoriatic Patient

To date, only one case of post-traumatic endophthalmitis caused by Scedosporium dehoogii has been reported, but its contamination or colonization might not be precluded due to the absence of pathogenic isolation and/or pathological examination. We report the first case to our knowledge of S. dehoogii-induced subcutaneous scedosporiosis in a psoriatic patient. A 58-year-old man with 5-year history of psoriasis vulgaris and immunosuppressant therapy developed pyrexia and multiple subcutaneous abscesses on both knees. Direct microscopy of the yellowish pus showed masses of bright green short spores. Skin biopsy revealed some branched septate hyphae within the granuloma. Two aspirated pus specimens collected at a 1-week interval produced white cottony colonies on Sabouraud dextrose agar. Bacterial cultures of one blood and two purulent samples were negative, and fungal culture of blood sample was not performed. The isolate was identified as S. dehoogii using β-tubulin phylogeny and species-specific PCR with primer MSDE1/MSA2. Without addition of antifungal treatment, subcutaneous lesions disappeared spontaneously after immunosuppressant withdrawal and no relapse occurred during 64-month follow-up. The spontaneous recovery may result from immune reconstitution following immunosuppressant discontinuation.     

Immunohistochemical Cross-Reactivity Between <Emphasis Type="Italic">Paracoccidioides</Emphasis> sp. from Dolphins and <Emphasis Type="Italic">Histoplasma capsulatum</Emphasis>

Paracoccidioidomycosis ceti is a cutaneous disease of cetaceans caused by uncultivated Paracoccidioides brasiliensis or Paracoccidioides spp. Serological cross-reactions between paracoccidioidomycosis ceti and paracoccidioidomycosis, paracoccidioidomycosis and histoplasmosis, and paracoccidioidomycosis and coccidioidomycosis have been reported before. The present study aimed to detect immunohistochemical cross-reaction between antibodies to Paracoccidioides sp. and Histoplasma capsulatum, and vice versa. Thirty murine sera, obtained from experimental infections of 6 isolates of H. capsulatum, were reacted with paraffin-embedded yeast-form cells of Paracoccidioides sp. derived from a case of paracoccidioidomycosis ceti in Japan. The murine sera were also reacted with human isolates of H. capsulatum yeast cells, with P. brasiliensis yeast cells, and with fungal cells of Coccidioides posadasii. Three dolphins’ sera from cases of paracoccidioidomycosis ceti, two human sera from patients with paracoccidioidomycosis, and a serum from a healthy person with a history of coccidioidomycosis were used in order to determine that the tested fungal cells reacted properly. Sera derived from mice infected with an isolate of H. capsulatum reacted positively against yeast cells of Paracoccidioides sp., yeast cells of P. brasiliensis, and fungal cells of C. posadasii, while those derived from other strains were negative. The present study recorded for the first time the cross-reaction between the yeast cells of H. capsulatum and antibodies against Paracoccidioides spp., the yeast cells of Paracoccidioides sp. and antibodies against H. capsulatum, the yeast cells of Paracoccidioides sp. and antibodies against Coccidioides sp., and fungal cells of C. posadasii and antibodies against Paracoccidioides spp.     

ATMT transformation efficiencies with native promoters in <Emphasis Type="Italic">Botryosphaeria kuwatsukai</Emphasis> causing ring rot disease in pear

Botryosphaeria kuwatsukai is an important fungal pathogen affecting pear fruits. However, infection processes of this fungus are still unclear. This study seeks to develop the fungal transformation of B. kuwatsukai by Agrobacterium tumefaciens-mediated transformation (ATMT), assess the reliability of appropriate vectors and examine the infection processes in vitro using a GFP labeled strain of B. kuwatsukai. To establish a highly effective transformation system in B. kuwatsukai, binary vectors containing various lengths of H3 promoters and TEF promoters fused with GFP and hygromycin B resistance gene cassettes were constructed. These cassettes were integrated into the genomic DNA of B. kuwatsukai with high transformation frequency by the ATMT method. Transformants showed strong expression of GFP and hygromycin B resistance genes in cells. Furthermore, we investigated if native promoters are more suitable to govern marker genes than other general promoters used in other filamentous fungi. The results obtained herein demonstrate that the vectors constructed in this study can be utilized with high transformation rate. Microscopic examinations also reveal that fungal hyphae undergo morphological changes during the infection process resulting in biotrophic stage of infected host cells. Our results provide genetic insights to further explore the infection processes of B. kuwatsukai.     

Molecular profiling of fungal assemblages in the healthy and infected roots of <Emphasis Type="Italic">Decalepis arayalpathra</Emphasis> (J. Joseph &amp; V. Chandras) Venter,an endemic and endangered ethnomedicinal plant from Western Ghats,India

Decalepis arayalpathra, an endangered, endemic ethnomedicinal plant from southern Western Ghats, India, is targeted for its aromatic and medicinal properties. This study aimed at to identify fungal endophyte populations associated with healthy and diseased roots of this perennial shrub. Healthy and rotted root samples of D. arayalpathra were collected, fungal endophytes assemblages were identified both by culture-dependent and culture-independent approaches, further sequenced and the retrieved sequences were analysed with the reference sequences in GenBank to know their phylogenetic relationships. Analysis of the ITS rDNA region generated 24 different Ascomycota and three Basidiomycota taxa. Trichoderma sp. was most abundant in healthy and diseased root samples, while Penicillium and Aspergillus were confined to healthy roots. Furthermore, Fusarium solani, Fusarium oxysporum and Mucor velutinosus were found to be the most frequent fungi identified from the rotted root samples, thus substantiated to be the cause for D. arayalpathra decline in the wild. Interestingly, the strains assigned to Fusarium sp. were isolated from diseased roots showing typical clearly visible symptoms, such as a severe brown discolouration on the taproot. Molecular profiling of all the pure fungal isolates, viz., Trichoderma, Penicillium, Aspergillus, Fusarium and Mucor, revealed high sequence similarities (≥ 98 %) to corresponding reference sequences. Sequencing of Trichoderma pure cultures isolated from healthy and diseased roots revealed sequence similarities to Trichoderma harzianum, T. hamatum, T. koningiopsis, T. asperellum, T. pubescens and Hypocrea sp. This confirms the morphological examinations, as Hypocrea is the teleomorph stage of Trichoderma sp. This study signifies the first work pertaining to the taxonomy of the fungal endophytic community of D. arayalpathra, and the results reported in this work may help to ascertain the cause of root rot disease often perceived in D. arayalpathra. Also, it could be useful to identify the promising endophytic communities against the root rot diseases occurring in D. arayalpathra.     

Invasive Rhinosinusitis Caused by <Emphasis Type="Italic">Lasiodiplodia theobromae</Emphasis> in an Allogeneic Hematopoietic Cell Transplant Recipient Case Report and Review of Literature

Lasiodiplodia theobromae is a known plant pathogen in tropical and subtropical areas. Few cases have been reported in humans (usually keratitis and endophthalmitis) with only two cases of fungal sinusitis in immunocompromised and immunocompetent patients published to date. We report a case of invasive sinusitis secondary to L. theobromae in an allogeneic hematopoietic cell transplant recipient successfully treated with surgical debridement and triazole antifungals with a review of available literature.     

Genetic mapping of a barley leaf rust resistance gene <Emphasis Type="Italic">Rph26</Emphasis> introgressed from <Emphasis Type="Italic">Hordeum bulbosum</Emphasis>

Key message

The quantitative barley leaf rust resistance gene, Rph26, was fine mapped within a H. bulbosum introgression on barley chromosome 1HL. This provides the tools for pyramiding with other resistance genes.

Abstract

A novel quantitative resistance gene, Rph26, effective against barley leaf rust (Puccinia hordei) was introgressed from Hordeum bulbosum into the barley (Hordeum vulgare) cultivar ‘Emir’. The effect of Rph26 was to reduce the observed symptoms of leaf rust infection (uredinium number and infection type). In addition, this resistance also increased the fungal latency period and reduced the fungal biomass within infected leaves. The resulting introgression line 200A12, containing Rph26, was backcrossed to its barley parental cultivar ‘Emir’ to create an F₂ population focused on detecting interspecific recombination within the introgressed segment. A total of 1368 individuals from this F₂ population were genotyped with flanking markers at either end of the 1HL introgression, resulting in the identification of 19 genotypes, which had undergone interspecific recombination within the original introgression. F₃ seeds that were homozygous for the introgressions of reduced size were selected from each F₂ recombinant and were used for subsequent genotyping and phenotyping. Rph26 was genetically mapped to the proximal end of the introgressed segment located at the distal end of chromosome 1HL. Molecular markers closely linked to Rph26 were identified and will enable this disease resistance gene to be combined with other sources of quantitative resistance to maximize the effectiveness and durability of leaf rust resistance in barley breeding. Heterozygous genotypes containing a single copy of Rph26 had an intermediate phenotype when compared with the homozygous resistant and susceptible genotypes, indicating an incompletely dominant inheritance.

     

<Emphasis Type="Italic">Pyrola japonica</Emphasis>, a partially mycoheterotrophic Ericaceae,has mycorrhizal preference for russulacean fungi in central Japan

Mycorrhizal symbiosis often displays low specificity, except for mycoheterotrophic plants that obtain carbon from their mycorrhizal fungi and often have higher specificity to certain fungal taxa. Partially mycoheterotrophic (or mixotrophic, MX) plant species tend to have a larger diversity of fungal partners, e.g., in the genus Pyrola (Monotropoideae, Ericaceae). Preliminary evidence however showed that the Japanese Pyrola japonica has preference for russulacean fungi based on direct sequencing of the fungal internal transcribed spacer (ITS) region from a single site. The present study challenges this conclusion using (1) sampling of P. japonica in different Japanese regions and forest types and (2) fungal identification by ITS cloning. Plants were sampled from eight sites in three regions, in one of which the fungal community on tree ectomycorrhizal (ECM) tips surrounding P. japonica was also analyzed. In all, 1512 clone sequences were obtained successfully from 35 P. japonica plants and 137 sequences from ECM communities. These sequences were collectively divided into 74 molecular operational taxonomic units (MOTUs) (51 and 33 MOTUs, respectively). MOTUs from P. japonica involved 36 ECM taxa (96 % of all clones), and 17 of these were Russula spp. (76.2 % of all clones), which colonized 33 of the 35 sampled plants. The MOTU composition significantly differed between P. japonica and ECM tips, although shared species represented 26.3 % of the ECM tips community in abundance. This suggests that P. japonica has a preference for russulacean fungi.     

Fungi isolated from insects in strawberry crops act as potential biological control agents of <Emphasis Type="Italic">Duponchelia fovealis</Emphasis> (Lepidoptera: Crambidae)

Entomopathogenic fungi were collected from insects, belonging to orders Lepidoptera, Coleoptera, Hemiptera, Hymenoptera, and Diptera, with signs of infection. Single-spore colonies were identified based on morphological traits and ribosomal ITS sequences. The most commonly found fungi were Fusarium oxysporum Schlechtendal and Beauveria bassiana (Balsamo-Crivelli) Vuillemin. Isolation of Trichoderma atroviride Bisset, Aspergillus flavipes (Bainier and Sartory) Thom and Church, Aspergillus iizukae Sugiyama, Penicillium mallochii Rivera, Urb and Seifert, Penicillium adametzioides S. Abe ex G. Smith, and Mucor nidicola Madden, Stchigel, Guarro and Starks associated to insects is reported for the first time. Tests with B. bassiana isolates against Duponchelia fovealis Zeller (Lepidoptera: Crambidae), showed high larval mortality in vitro and in greenhouse, demonstrating its potential as biological control agent. Understanding the fungal microbiota from insects can provide promising isolates for use in integrated pest management programs. This is the first report characterizing the mycobiota in insects collected in strawberry crops, and evaluating the pathogenicity of B. bassiana against D. fovealis, a pest that causes severe losses to farmers.