Ichthyophonosis in Peruvian rainbow trout Oncorhynchus mykiss: identification of endemic areas using molecular and histopathological tools.

Ichthyophonus infection was first detected in Peruvian Oncorhynchus mykiss in 1986, but the occurrence of ichthyophonosis disease in the region is unknown. This study investigated the presence and distribution of Ichthyophonus sp. in Peruvian rainbow trout using traditional and DNA sequencing tools. Between 2007 and 2008, 205 rainbow trout from 13 hatcheries in the Mantaro river basin were examined for the presence of Ichthyophonus, and at that time only 3 farms were positive. This early study confirmed the presence of Ichthyophonus sp. in the Tranca Grande lagoon for the first time, at a prevalence of 50%. In 2012, examination of 240 trout from 24 fish farms in 2 Peruvian Departments found 9 infected farms. More recently, in 2018, Ichthyophonus sp. was found in Lake Titicaca, infecting a trout in the Ichu area (in the Department of Puno). Our molecular analysis of the infected trout showed that ichthyophonosis disease in the Peruvian trout was caused by Ichthyophonus sp. Clade C. The finding of this pathogen in Lake Titicaca should be an alert for nearby farms and entities dealing with fish of economic importance in the rivers of Peru.

Human Pathogenic Entomophthorales.

The pathogenic entomophthoralean fungi cause infection in insects and mammalian hosts. Basidiobolus and Conidiobolus species can be found in soil and insect, reptile, and amphibian droppings in tropical and subtropical areas. The life cycles of these fungi occur in these environments where infecting sticky conidia are developed. The infection is acquired by insect bite or contact with contaminated environments through open skin. Conidiobolus coronatus typically causes chronic rhinofacial disease in immunocompetent hosts, whereas some Conidiobolus species can be found in immunocompromised patients. Basidiobolus ranarum infection is restricted to subcutaneous tissues but may be involved in intestinal and disseminated infections. Its early diagnosis remains challenging due to clinical similarities to other intestinal diseases. Infected tissues characteristically display eosinophilic granulomas with the Splendore-Höeppli phenomenon. However, in immunocompromised patients, the above-mentioned inflammatory reaction is absent. Laboratory diagnosis includes wet mount, culture serological assays, and molecular methodologies. The management of entomophthoralean fungi relies on traditional antifungal therapies, such as potassium iodide (KI), amphotericin B, itraconazole, and ketoconazole, and surgery. These species are intrinsically resistant to some antifungals, prompting physicians to experiment with combinations of therapies. Research is needed to investigate the immunology of entomophthoralean fungi in infected hosts. The absence of an animal model and lack of funding severely limit research on these fungi.

Development of esthetic prosthesis for a patient with severe stigmatizing facial lesions due to cancer: a pilot study.

PURPOSE: Severe physical facial deformities due to surgical interventions can have significant psychosocial consequences to patient's relationships with friends and family and thus, has a considerable impact on their quality of life. We have developed a 3D prosthesis for a 56-year-old woman diagnosed with epidermoid carcinoma at the right hemiface, to improve her quality of life. METHODS: The patient started radiotherapy with modulated intensity. To deal with the advance of the process, a maxilectomy of supra structure with modified radical cervical emptying on the right hemiface was performed. Reconstruction of areas surgically affected by the displacement of islands of skin and muscle (flaps) from healthy regions was initiated. Although the procedure occurred without intercurrences, the patient developed necrosis and loss of the myocutaneous flap. After the removal of the flap, the esthetic result of the treatment was evident causing exposure of subcutaneous and granulation tissues. RESULTS: A computational model was used to develop a 3D structure of the affected area and then used to construct the prosthesis. The prosthesis was applied over the affected area, and the patient was able see her face on the mirror for the first time in years. The patient was grateful and hopeful. CONCLUSION: We have found that the application of this new technology greatly improves the social interaction of patients with deformities due to surgical interventions.

Cutaneous Granulomas in Dolphins Caused by Novel Uncultivated Paracoccidioides brasiliensis.

Cutaneous granulomas in dolphins were believed to be caused by Lacazia loboi, which also causes a similar disease in humans. This hypothesis was recently challenged by reports that fungal DNA sequences from dolphins grouped this pathogen with Paracoccidioides brasiliensis. We conducted phylogenetic analysis of fungi from 6 bottlenose dolphins (Tursiops truncatus) with cutaneous granulomas and chains of yeast cells in infected tissues. Kex gene sequences of P. brasiliensis from dolphins showed 100% homology with sequences from cultivated P. brasiliensis, 73% with those of L. loboi, and 93% with those of P. lutzii. Parsimony analysis placed DNA sequences from dolphins within a cluster with human P. brasiliensis strains. This cluster was the sister taxon to P. lutzii and L. loboi. Our molecular data support previous findings and suggest that a novel uncultivated strain of P. brasiliensis restricted to cutaneous lesions in dolphins is probably the cause of lacaziosis/lobomycosis, herein referred to as paracoccidioidomycosis ceti.

Lagenidium giganteum pathogenicity in mammals.

Infections of mammals by species in the phylum Oomycota taxonomically and molecularly similar to known Lagenidium giganteum strains have increased. During 2013-2014, we conducted a phylogenetic study of 21 mammalian Lagenidium isolates; we found that 11 cannot be differentiated from L. giganteum strains that the US Environmental Protection Agency approved for biological control of mosquitoes; these strains were later unregistered and are no longer available. L. giganteum strains pathogenic to mammals formed a strongly supported clade with the biological control isolates, and both types experimentally infected mosquito larvae. However, the strains from mammals grew well at 25°C and 37°C, whereas the biological control strains developed normally at 25°C but poorly at higher temperatures. The emergence of heat-tolerant strains of L. giganteum pathogenic to lower animals and humans is of environmental and public health concern.

Treatment outcomes of surgery, antifungal therapy and immunotherapy in ocular and vascular human pythiosis: a retrospective study of 18 patients.

OBJECTIVES: Human pythiosis is a life-threatening disease for which no standard treatment protocols with proven efficacy exist. We present the results of our institutional pythiosis treatment protocol, composed of surgery, antifungal agents, iron chelator (only vascular cases) and immunotherapy. METHODS: We retrospectively analysed patients with proven vascular and ocular pythiosis in King Chulalongkorn Memorial Hospital from April 2003 to May 2013. Fisher's exact test and Wilcoxon's rank-sum test were used. The MICs of seven antifungal agents and combination drugs were investigated in eight clinical Pythium insidiosum strains. RESULTS: Eighteen patients were evaluated. Disease-free surgical margins were obtained in all surviving patients with vascular pythiosis (P = 0.08). Patients who underwent eye enucleation were significantly older than those who did not (P < 0.05). Patients with vascular or ocular pythiosis did not differ significantly in the median time from disease onset to first surgery or in the relationship between the type of P. insidiosum antigen and treatment outcomes. In vitro susceptibility profiles of all isolates demonstrated that no single agent or combination treatment was substantially more effective than the others. The highest MIC was detected for amphotericin B, followed in order by voriconazole, fluconazole, anidulafungin, caspofungin, itraconazole and terbinafine. No synergistic effects of the combination drug treatments were found. CONCLUSIONS: Surgery with adequate surgical margins is a crucial determinant of survival in patients with vascular pythiosis. Itraconazole and terbinafine do not have synergistic effects on Thai P. insidiosum strains. The role of immunotherapy remains inconclusive for both vascular and ocular pythiosis.

Etiological agents of fungal endophthalmitis: diagnosis and management.

Endophthalmitis caused by fungi is commonly diagnosed around the world in apparently healthy and immunocompromised individuals. An accurate clinical diagnosis for endophthalmitis confirmed by laboratory techniques is essential for early treatment with antifungal drugs, such as amphotericin B, imidazoles, and other antifungals. Here, we review endophthalmitis caused by fungi according to its classification into endogenous fungal endophthalmitis (EFE) and exogenous fungal endophthalmitis (EXFE). EFE is caused by endogenously acquired fungi, whereas the traumatic implantation of opportunistic fungal pathogens is the main feature of EXFE. We highlight the most important etiologies causing endophthalmitis and the steps required for a rapid diagnosis and management.

Itraconazole in human medicine and veterinary practice.

Diagnosis and management of fungal infections are challenging in both animals and humans, especially in immunologically weakened hosts. Due to its broad spectrum and safety profile when compared to other antifungals, itraconazole (ITZ) has been widely used in the treatment and prophylaxis of fungal infections, both in human and veterinary medicine. The dose and duration of management depend on factors such as the type of fungal pathogen, the site of infection, sensitivity to ITZ, chronic stages of the disease, the health status of the hosts, pharmacological interactions with other medications and the therapeutic protocol used. In veterinary practice, ITZ doses generally vary between 3 mg/kg and 50 mg/kg, once or twice a day. In humans, doses usually vary between 100 and 400 mg/day. As human and veterinary fungal infections are increasingly associated, and ITZ is one of the main medications used, this review addresses relevant aspects related to the use of this drug in both clinics, including case reports and different clinical aspects available in the literature.

Presumptive synchronized nuclear divisions without cytokinesis in the Rhinosporidium seeberi parasitic life cycle.

Despite numerous studies of the Rhinosporidum seeberi parasitic phase, the stages of its nuclear cycle leading to the formation of endoconidia have yet to be properly described. R. seeberi resists culture and can only be investigated on histological preparations. We have evaluated tissue sections collected from 35 host species with rhinosporidosis searching for the presence of mitotic figures during sporangia development. This study found that soon after endoconidia release, the prominent reddish vesicles typical of this stage vanished leading to the development of juvenile sporangia (JS) 12-70 µm in diameter. This stage possesses granular cytoplasm, a thick cell wall, and a central reddish nucleus with a conspicuous nucleolus. The first nuclear division takes place in the JS. It is a rarely encountered event characterized by the development of a distorted nucleus leading to the formation of two nuclei without cytokinesis. The finding of multiple nuclear divisions at prophase-, metaphase- and telophase-like stages without cytokinesis was detected in intermediate sporangia (IS). IS with multiple dividing nuclei seem to be at the same stage of nuclear partitioning, suggesting synchronized nuclear division. In these sporangia, the nuclei continue divisions without cytokinesis until the sporangia reach ≥300 µm in diameter. The last nuclear division prior to cytokinesis appears to take place in very large sporangia with thousands of nuclei. The build-up of cytoplasm around each nucleus and the formation of a thin cell wall lead to the formation of endoconidia. This study revealed the presence of several mechanisms of pathogenesis in R. seeberi that deserved further investigation.

Lagenidium sp. ocular infection mimicking ocular pythiosis.

This is a report of a Lagenidium sp. in a Thai patient who was diagnosed with severe keratitis that was unresponsive to antibacterial and antifungal drugs. Examination of a corneal biopsy specimen confirmed the presence of aseptate hyphae. The internal transcribed spacer DNA sequence of the strain isolated showed 97% identity with Lagenidium giganteum and other Lagenidium species.

Current Nomenclature of Paracoccidioides lobogeorgii's Disease Name. Comment on Grotta et al. Fungal Density in Lobomycosis in French Guiana: A Proposal for a New Clinico-Histological and Therapeutic Classification. J. Fungi 2023, 9, 1005.

We have read the article by Grotta and collaborators [...].

A taxonomic review of the genus Paracoccidioides, with focus on the uncultivable species.

Paracoccidioides species have always been surrounded by taxonomic uncertainties. The continuing nomenclatoral muddle was caused in part by the failure of Adolfo Lutz and Jorge Lôbo to name the etiologic agents of human paracoccidioidomycosis and Jorge Lôbo's diseases, respectively. Early in their history, it was postulated that the cultivable species causing systemic infections belonged in the genus Paracoccidioides, whereas the uncultivable species, causing skin disease, were not part of the genus. The taxonomy of these pathogens was further complicated when a similar skin disease with numerous yeast-like cells in infected dolphins was also reported. Due to its phenotypic similarities with that described by Jorge Lôbo in human and its uncultivable nature, it was assumed that the disease in dolphins was caused by the same fungus. Recent molecular and population genetic analysis, however, found the DNA extracted from the uncultivable yeast-like cells affecting dolphins shared common phylogenetic traits with cultivable Paracoccidioides species. The study revealed that the uncultivable pathogens comprised 2 different Paracoccidioides species, now known as P. ceti and P. loboi, correspondingly. To validate P. loboi binomial, a comprehensive historical critical review of Jorge Lôbo etiology was performed. This review showed the proposed binomial P. loboi was previously used, and, thus, a replacement name is introduced, Paracoccidioides lobogeorgii nom. nov. In addition, in this review, several cultivable human Paracoccidioides species are validated, and the generic type species, P. brasiliensis, is neotypified as the original material could not be traced.

Detection of Heavy Metals, Their Distribution in Tilapia spp., and Health Risks Assessment.

Concentrations of heavy metals (HMs) were assessed in Tilapia spp. from selected communities in Calapan City, Philippines. Eleven (11) inland farmed tilapia samples were collected and analyzed for HMs concentration using X-ray fluorescence (XRF). The 11 fish samples were cut into seven pieces, according to the fish body parts, constituting a total of 77 samples. These fish samples were then labeled as bone, fins, head, meat, skin, and viscera. Results showed that the mean concentration of Cd in all parts of tilapia exceeded the Food and Agriculture Organization/World Health Organization (FAO/WHO) limits. The highest concentration was recorded in the fins, which was sevenfold higher than the limit. The trend of the mean concentration of Cd in different parts of tilapia was fins > viscera > skin > tail > head > meat > bone. The target hazard quotient (THQ) recorded a value less than 1. This means that the population exposed to tilapia, within the area where fish samples originated, were not at risk to non-carcinogens. The concentrations of Cu, Pb, Mn, Hg, and Zn in different parts, particularly in skin, fins, and viscera, also exceeded the FAO/WHO limits. The calculated cancer risk (CR) in consuming the fish skin, meat, fins, bone, viscera, and head was higher than the USEPA limit. This indicated a possible carcinogenic risk when consumed regularly. Most of the correlations observed between HMs in various parts of the tilapia had positive (direct) relationships, which were attributed to the HM toxicity target organ characteristics. Results of the principal component analysis (PCA) showed that most of the dominating HMs recorded in tilapia were attributable to anthropogenic activities and natural weathering within the watershed of agricultural areas. The agriculture area comprises about 86.83% of the overall land area of Calapan City. The identified carcinogenic risks were associated with Cd. Therefore, regular monitoring of HMs in inland fishes, their habitat, and surface water quality shall be carried out. This information is useful in creating strategies in metals concentration monitoring, health risks reduction program, and relevant guidelines that would reduce the accumulation of HM in fish.

Molecular diagnosis of subcutaneous Pythium insidiosum infection by use of PCR screening and DNA sequencing.

Pythium insidiosum is an emerging human pathogen classified among brown algae and diatoms that can cause significant morbidity and mortality in otherwise healthy individuals. Here we describe a pediatric patient with pythiosis acquired in the southern United States, diagnosed by molecular screening and DNA sequencing of internal transcribed spacer region 1.

Vulvar pythiosis in two captive camels (Camelus dromedarius).

Two camels (Camelus dromedarius), 3- and 4-years-old, respectively, from an eastern Tennessee wildlife farm presented with persistent weight loss and large vulvar masses. An initial biopsy of the vulvar mass of one of the camels performed by a local veterinarian showed eosinophilic dermatitis. An allergic or parasitic dermatitis was suspected. The two camels were treated with one dose of sodium iodide (66 mg/kg, in 1.0 L of normosolR, IV) and ivermectin 1% (200 ug/kg PO). Upon presentation at the Veterinary Teaching Hospital, University of Tennessee, additional biopsies of the masses again revealed eosinophilic dermatitis. Microscopic examination of a Gomori methenamine silver (GMS)-stained section prepared from the biopsy of one of the camels revealed the presence of fungal-like hyphae of a mold which was suspected to be Pythium insidiosum. The vulvar masses were surgically debulked in both animals and sodium iodide and Pythium-immunotherapy prescribed. Pythium insidiosum was isolated in culture and hyphae elements were detected in histological sections confirming the diagnosis of pythiosis in both animals. Despite signs of progressive healing of the vulvar surgical areas, postoperative persistent weight lost in one of the camels suggested the possibility of gastro intestinal (GI) tract pythiosis. This camel died 5 months after the first onset of clinical signs and unfortunately a necropsy was not performed. The other camel responded well to the combination of surgery, iodides, and immunotherapy and has currently rejoined the other members of the herd.

Pythium insidiosum complex hides a cryptic novel species: Pythium periculosum.

Early phylogenetic analysis of Pythium insidiosum, the etiologic agent of pythiosis in mammals, showed the presence of a complex comprising three monophyletic clusters. Two included isolates recovered from cases of pythiosis in the Americas (Cluster I) and Asia (Cluster II), whereas the third cluster included four diverged isolates three from humans in Thailand and the USA, and one isolate from a USA spectacled bear (Cluster III). Thereafter, several phylogenetic analyses confirmed the presence of at least three monophyletic clusters, with most isolates placed in clusters I and II. Recent phylogenetic analyses using isolates from environmental sources and from human cases in India, Spain, Thailand, and dogs in the USA, however, showed the presence of two monophyletic groups each holding two sub-clusters. These studies revealed that P. insidiosum possesses different phylogenetic patterns to that described by early investigators. In this study, phylogenetic, population genetic and protein MALDI-TOF analyses of the P. insidiosum isolates in our culture collection, as well as those available in the database, showed members in the proposed cluster III and IV are phylogenetically different from that in clusters I and II. Our analyses of the complex showed a novel group holding two sub-clusters the USA (Cluster III) and the other from different world regions (Cluster IV). The data showed the original P. insidiosum cluster III is a cryptic novel species, now identified as P. periculosum. The finding of a novel species within P. insidiosum complex has direct implications in the epidemiology, diagnosis, and management of pythiosis in mammalian hosts.

Phylogenetic analysis of Pythium insidiosum Thai strains using cytochrome oxidase II (COX II) DNA coding sequences and internal transcribed spacer regions (ITS).

To investigate the phylogenetic relationship among Pythium insidiosum isolates in Thailand, we investigated the genomic DNA of 31 P. insidiosum strains isolated from humans and environmental sources from Thailand, and two from North and Central America. We used PCR to amplify the partial COX II DNA coding sequences and the ITS regions of these isolates. The nucleotide sequences of both amplicons were analyzed by the Bioedit program. Phylogenetic analysis using genetic distance method with Neighbor Joining (NJ) approach was performed using the MEGA4 software. Additional sequences of three other Pythium species, Phytophthora sojae and Lagenidium giganteum were employed as outgroups. The sizes of the COX II amplicons varied from 558-564 bp, whereas the ITS products varied from approximately 871-898 bp. Corrected sequence divergences with Kimura 2-parameter model calculated for the COX II and the ITS DNA sequences ranged between 0.0000-0.0608 and 0.0000-0.2832, respectively. Phylogenetic analysis using both the COX II and the ITS DNA sequences showed similar trees, where we found three sister groups (A(TH), B(TH), and C(TH)) among P. insidiosum strains. All Thai isolates from clinical cases and environmental sources were placed in two separated sister groups (B(TH) and C(TH)), whereas the Americas isolates were grouped into A(TH.) Although the phylogenetic tree based on both regions showed similar distribution, the COX II phylogenetic tree showed higher resolution than the one using the ITS sequences. Our study indicates that COX II gene is the better of the two alternatives to study the phylogenetic relationships among P. insidiosum strains.

Pythium insidiosum keratitis in Israel.

PURPOSE: To report with morphologic and phylogenetic speciation the first case from Israel of Pythium insidiosum keratitis associated with contact-lens wear. METHODS: Case report and literature review. RESULTS: A 21-year-old man with a history of contact-lens use and water exposure was hospitalized in Israel for a corneal ulcer. The ulcer progressed despite intensive antibiotics. He flew home to the United States for further care. Examination revealed a corneal ulcer with hypopyon. The infection progressed despite intensive medical therapy, and a therapeutic penetrating keratoplasty was performed. Histology and cornea cultures from the host cornea revealed sparsely septate, branching hyphae, consistent with P. insidiosum. DNA sequencing of the Pythium isolate supported the clinical history that the infection was acquired outside of the United States. Despite intensive medical therapy and a second corneal transplant, the ulcer progressed, ultimately requiring enucleation. CONCLUSION: This is the first reported case of culture-proven, contact lens-related Pythium keratitis originating from Israel. Pythium is a fungus-like, aquatic oomycete found in tropical climates. Human pythiosis is uncommon but associated with high morbidity. Case reports describe surgical cure of Pythium keratitis, but this case recurred despite two penetrating keratoplasties and maximal antifungal therapy. In cases of presumed fungal keratitis that do not respond to antifungals, the fungus should be sent for speciation because early surgical intervention is the only means to save the eye in ocular pythiosis.

The taxonomy of two uncultivated fungal mammalian pathogens is revealed through phylogeny and population genetic analyses.

Ever since the uncultivated South American fungal pathogen Lacazia loboi was first described 90 years ago, its etiology and evolutionary traits have been at the center of endless controversies. This pathogen infects the skin of humans and as long believed, dolphin skin. However, recent DNA analyses of infected dolphins placed its DNA sequences within Paracoccidioides species. This came as a surprise and suggested the human and dolphin pathogens may be different species. In this study, population genetic analyses of DNA from four infected dolphins grouped this pathogen in a monophyletic cluster sister to P. americana and to the other Paracoccidioides species. Based on the results we have emended the taxonomy of the dolphin pathogen as Paracoccidioides cetii and P. loboi the one infecting human. Our data warn that phylogenetic analysis of available taxa without the inclusion of unusual members may provide incomplete information for the accurate classification of anomalous species.