Construction of a Human Immune Library from Gallbladder Cancer Patients for the Single-Chain Fragment Variable (scFv) Antibody Selection against Claudin 18.2 via Phage Display.

Gallbladder cancer (GBC) is a very aggressive malignant neoplasm of the biliary tract with a poor prognosis. There are no specific therapies for the treatment of GBC or early diagnosis tools; for this reason, the development of strategies and technologies that facilitate or allow an early diagnosis of GBC continues to be decisive. Phage display is a robust technique used for the production of monoclonal antibodies (mAbs) involving (1) the generation of gene libraries, (2) the screening and selection of isoforms related to an immobilized antigen, and (3) the in vitro maturation of the affinity of the antibody for the antigen. This research aimed to construct a human immune library from PBMCs of GBC patients and the isolation of scFv-phage clones with specificity against the larger extracellular loop belonging to claudin 18.2, which is an important biomarker overexpressed in GBC as well as gastric cancer. The immune-library-denominated GALLBLA1 was constructed from seven GBC patients and has a diversity of 6.12 × 1010pfu mL-1. After three rounds of panning, we were able to identify clones with specificity against claudin 18.2. GALLBLA1 can contribute to the selection, isolation, and recombinant production of new human mAbs candidates for the treatment of gastrointestinal cancers.

Molecular Study of Pneumocystis jirovecii in Respiratory Samples of HIV Patients in Chile.

Pneumocystis is an opportunistic fungus that causes potentially fatal pneumonia (PCP) in immunocompromised patients. The objective of this study was to determine the prevalence of P. jirovecii in HIV patients through phenotypic and molecular study, to investigate the genetic polymorphisms of P. jirovecii at the mitochondrial gene mtLSU and at the nuclear dihydropteroate synthase gene (DHPS), and by analysis of molecular docking to study the effect of DHPS mutations on the enzymatic affinity for sulfamethoxazole. A PCP prevalence of 28.3% was detected, with mtLSU rRNA genotypes 3 (33.3%) and 2 (26.6%) being the most common. A prevalence of 6.7% (1/15) mutations in the DHPS gene was detected, specifically at codon 55 of the amino acid sequence of dihydropteroate synthase. Molecular docking analysis showed that the combination of mutations at 55 and 98 codons is required to significantly reduce the affinity of the enzyme for sulfamethoxazole. We observed a low rate of mutations in the DHPS gene, and molecular docking analysis showed that at least two mutations in the DHPS gene are required to significantly reduce the affinity of dihydropteroate synthase for sulfamethoxazole.

Penicillium digitatum, First Clinical Report in Chile: Fungal Co-Infection in COVID-19 Patient.

Penicillium digitatum is one of the most important phytopathogens. It causes deterioration and rotting of citrus fruits, generating significant economic losses worldwide. As a human pathogen, it is extremely rare. We present a case of pulmonary co-infection in a patient diagnosed with pneumonia due to SARS-CoV-2. A 20-year-old female patient, primigravid, 36 weeks of gestation, without comorbidities, and diagnosed with severe pneumonia due to the SARS-CoV-2, showed rapid lung deterioration for which their pregnancy was interrupted by surgery. The patient was hospitalized in the Intensive Care Unit (ICU), connected to mechanical ventilation and receiving corticosteroids and antibiotics. The diagnosis of pulmonary fungal infection was made through bronchoalveolar lavage (BAL) culture, and the species identification was performed by sequencing of ß-tubulin. Phylogenetic analysis with related species was performed for the confirmation of species identification. Antifungal susceptibility tests were performed for itraconazole (4 µg/mL), voriconazole (2 µg/mL), and amphotericin B (2 µg/mL). The patient was successfully treated with itraconazole. This is the second worldwide report of pulmonary infection by P. digitatum and the first in Chile. Although it is a fungus that rarely infects humans, it could represent an emerging opportunistic fungal pathogen, with associated risk factors that should be considered in the differential diagnosis of Penicillium species isolated from infections in humans.

Novel species of Cladosporium from environmental sources in Spain.

Cladosporium is a monophyletic genus in Cladosporiaceae (Cladosporiales, Dothideomycetes) whose species are mainly found as saprobes and endophytes, but it also includes fungi pathogenic for plants, animals and human. Species identification is currently based on three genetic markers, viz., the internal transcribed spacer regions (ITS) of the rDNA, and partial fragments of actin (act) and the translation elongation factor 1-α (tef1) genes. Using this phylogenetic approach and from morphological differences, we have recognized six new species originating from soil, herbivore dung and plant material collected at different Spanish locations. They are proposed as Cladosporium caprifimosum, C. coprophilum, C. fuscoviride and C. lentulum belonging in the C. cladosporioides species complex, and C. pseudotenellum and C. submersum belonging in the C. herbarum species complex. This study revealed that herbivore dung represented a reservoir of novel lineages in the genus Cladosporium.

Three new Curvularia species from clinical and environmental sources.

Curvularia is a Pleosporalean monophyletic genus with a great diversity of species, including relevant phytopathogenic, animal and human pathogenic fungi. However, their microscopic identification is difficult due to overlapping morphological features amongst species. In recent years, multi-locus sequence analysis using the ITS region of the rDNA and fragments of the genes gapdh and tef1 revealed numerous cryptic species, especially in isolates that commonly produced 3-septate conidia. Therefore, based on sequence analysis of the above-mentioned DNA barcodes recommended for species delineation in Curvularia, we propose three novel species, C. paraverruculosa, C. suttoniae and C. vietnamensis, isolated from soil, human clinical specimens and plant material, respectively, collected in different countries. These new species are morphologically characterised and illustrated in the present study. Curvularia paraverruculosa differs from its counterparts, C. americana and C. verruculosa, mainly by its narrower conidia. Curvularia suttoniae and C. vietnamensis are closely related to C. petersonii, but the former two have larger conidia.

Heliocephala variabilis and Pseudopenidiella vietnamensis: Two New Hyphomycetous Species in the Microthyriaceae (Dothideomycetes) from Vietnam.

In a survey of microfungi from plant debris collected in Vietnam, two new hyphomycetous species were found, which belong to the genera Heliocephala and Pseudopenidiella and the family Microthyriaceae (Microthyriales, Dothideomycetes). Maximum Likelihood and Bayesian Inference sequence analyses of the internal transcribed spacers (ITS) and large subunit (LSU) of the ribosomal DNA barcodes allowed assessing the phylogenetic relationships of the new species with other species of the respective genera. Heliocephala variabilis sp. nov. was closely related to Heliocephala elegans, Heliocephala gracilis, and Heliocephala zimbabweensis, from which it was morphologically distinguished by its smaller conidiophores and non-rostrate conidia of up to four septa on the natural substratum. Pseudopenidiella vietnamensis sp. nov. was related to Pseudopenidiella piceae and Pseudopenidiella podocarpi and differed from the former principally by its lack of microcondiophores and from P. podocarpi by having larger macroconidiophores and smooth conidia. Key morphological features to distinguish the accepted species in Heliocephala and Pseudopenidiella are also provided. In addition, Pseudopenidiella pini was excluded from the genus on the basis of its morphological features.

Polyphasic identification of three new species in Alternaria section Infectoriae causing human cutaneous infection.

BACKGROUND: Cutaneous phaeohyphomycosis is an emerging disease in immunocompromised patients, being Alternaria one of the most common genera reported as a causative agent. Species identification is not carried out mainly due to the complexity of the genus. Analysis of the ITS barcode has become standard for fungal identification, but in Alternaria it is only able to discriminate among species-groups or sections. METHODS: We present three cases of cutaneous infection caused by Alternaria isolates morphologically identified as belonging to section Infectoriae. They have been morphologically characterised and phylogenetically delineated with five molecular markers (ITS, ATPase, gapdh, rpb2 and tef1). RESULTS: Mycotic infections have been diagnosed by repeated cultures and histopathological examination in two of the cases. The polyphasic approach has allowed to delineate three new species of Alternaria section Infectoriae, that is A anthropophila, A atrobrunnea and A guarroi. ATPase has been the only locus able to discriminate most of the species (29 out of 31) currently sequenced in this section, including A infectoria the commonest reported species causing alternariosis. Susceptibility test showed different antifungal patterns for the three species, although terbinafine was the most active in vitro drug against these fungi. CONCLUSIONS: The ATPase gene is recommended as an alternative barcode locus to identify Alternaria clinical isolates in section Infectoriae. Our results reinforce the relevance of identification of Alternaria isolates at the species level and the necessity to carry out antifungal susceptibility testing to determine the most adequate drug for treatment.

A human subcutaneous infection by Microascus ennothomasiorum sp. nov.

A 60-year-old woman presented with a nodular granulomatous skin lesion on her right thumb. It had developed after inoculation of a splinter of wood. Because it was resistant to various therapies, the nodule was finally excised. Complete healing followed this surgery and a melanised filamentous fungus with scopulariopsis-like morphology was recovered from the dermal tissue. Fitting with no known species, the fungus was subjected to extensive morphological, physiological and genetic investigations. It was characterised by resistance to cycloheximide, growth at 37°C, branched conidiophores with cylindrical annellides in brush-like groups producing dark conidia in basipetal chains, and cleistothecia with ellipsoidal to slightly reniform ascospores. Genetically it clustered in a well-supported clade together with Microascus (M.) brunneosporus, Microascus chinensis, Microascus intricatus, Microascus longicollis, Microascus micronesiensis and Microascus onychoides, but formed an independent branch distant from the other Microascus species. Based on its unique genetic characteristics and morphological findings, the isolate is proposed as a new species, Microascus ennothomasiorum. Morphologically it differs from its phylogenetically closest species by its branched conidiophores and ascomata with a peridium of textura intricata. Our observation once again emphasises that dermal granulomas can be caused by uncommon fungi; diagnostics should therefore include appropriate mycological investigations.

Neodendryphiella, a novel genus of the Dictyosporiaceae (Pleosporales).

In a survey of soil and herbivore dung microfungi in Mexico and Spain, several dendryphiella-like species were found. Phylogenetic analyses based on ITS and LSU sequences showed that these fungi belonged to the family Dictyosporiaceae (Pleosporales) and represent an undescribed monophyletic lineage distant from Dendryphiella. Therefore, the genus Neodendryphiella is proposed to accommodate three new species, N.mali, N.michoacanensis and N.tarraconensis. The novel genus shares morphological features with Dendryphiella such as differentiated conidiophores and polytretic integrated conidiogenous cells, that produce acropetal branched chains of conidia. Neodendryphiella differs in the absence of nodulose conidiophores bearing conidiogenous cells with pores surrounded by a thickened and darkened wall, typical features in the conidiogenous apparatus of Dendryphiella. In addition, the phylogenetic and morphological analysis of several reference strains of different Dendryphiella species, available for comparison, support the proposal of D.variabilissp. nov., which mainly differs from the other species of the genus by having conidia up to 7 septa and highlight that D.vinosa and D.infuscans are obscure species that require further taxonomic review.

Multiple species of Trichosporon produce biofilms highly resistant to triazoles and amphotericin B.

Invasive infections caused by Trichosporon spp. have increased considerably in recent years, especially in neutropenic and critically ill patients using catheters and antibiotics. The genus presents limited sensitivity to different antifungal agents, but triazoles are the first choice for treatment. Here, we investigated the biofilm production and antifungal susceptibility to triazoles and amphotericin B of 54 Trichosporon spp. isolates obtained from blood samples (19), urine (20) and superficial mycosis (15). All isolates and 7 reference strains were identified by sequence analysis and phylogenetic inferences of the IGS1 region of the rDNA. Biofilms were grown on 96-well plates and quantitation was performed using crystal violet staining, complemented with Scanning Electron Microscopy (SEM). Susceptibility tests for fluconazole, itraconazole, voriconazole and amphotericin B were processed using the microdilution broth method (CLSI) for planktonic cells and XTT reduction assay for biofilm-forming cells. Our results showed that T. asahii was the most frequent species identified (66.7%), followed by T. faecale (11.1%), T. asteroides (9.3%), T. inkin (7.4%), T. dermatis (3.7%) and one T. coremiiforme (1.8%). We identified 4 genotypes within T. asahii isolates (G1, G3, G4 and G5) and 2 genotypes within T. faecale (G1 and G3). All species exhibited high adhesion and biofilm formation capabilities, mainly T. inkin, T. asteroides and T. faecale. Microscopy images of high biofilm-producing isolates showed that T. asahii presented mainly hyphae and arthroconidia, whereas T. asteroides exhibited mainly short arthroconidia and few filaments. Voriconazole exhibited the best in vitro activity against all species tested. Biofilm-forming cells of isolates and reference strains were highly resistant to all antifungals tested. We concluded that levels of biofilm formation by Trichosporon spp. were similar or even greater than those described for the Candida genus. Biofilm-forming cells were at least 1,000 times more resistant to antifungals than planktonic cells, especially to voriconazole.