Maria da Luz Martins

Environmental distribution of Cryptococcus neoformans and C. gattii around the Mediterranean basin

In order to elucidate the distribution of Cryptococcus neoformans and C. gattii in the Mediterranean basin, an extensive environmental survey was carried out during 2012-2015. A total of 302 sites located in 12 countries were sampled, 6436 samples from 3765 trees were collected and 5% of trees were found to be colonized by cryptococcal yeasts. Cryptococcus neoformans was isolated from 177 trees and C. gattii from 13. Cryptococcus neoformans colonized 27% of Ceratonia, 10% of Olea, Platanus and Prunus trees and a lower percentage of other tree genera. The 13 C. gattii isolates were collected from five Eucalyptus, four Ceratonia, two Pinus and two Olea trees. Cryptococcus neoformans was distributed all around the Mediterranean basin, whereas C. gattii was isolated in Greece, Southern Italy and Spain, in agreement with previous findings from both clinical and environmental sources. Among C. neoformans isolates, VNI was the prevalent molecular type but VNII, VNIV and VNIII hybrid strains were also isolated. With the exception of a single VGIV isolate, all C. gattii isolates were VGI. The results confirmed the presence of both Cryptococcus species in the Mediterranean environment, and showed that both carob and olive trees represent an important niche for these yeasts.

Multiplex PCR identification of eight clinically relevant Candida species

Invasive fungal infections, specifically candidemia, constitute major public health problems with high mortality rates. Therefore, in the last few years, the development of novel diagnostic methods has been considered a critical issue. Herein we describe a multiplex PCR strategy allowing the identification of 8 clinically relevant yeasts of the Candida genus, namely C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, C. krusei, C. guilliermondii, C. lusitaniae and C. dubliniensis. This method is based on the amplification of two fragments from the ITS1 and ITS2 regions by the combination of 2 yeast-specific and 8 species-specific primers in a single PCR reaction. Results from the identification of 231 clinical isolates are presented pointing to the high specificity of this procedure. Furthermore, several Candida isolates were identified directly from clinical specimens which also attests to the methods direct laboratory application. The results from the multiplex reactions with other microorganisms that usually co-infect patients also confirmed its high specificity in the identification of Candida species. Moreover, this method is simple and presents a sensitivity of approximately 2 cells per ml within 5 hours. Furthermore, it allows discrimination of individual Candida species within polyfungal samples. This novel method may therefore provide a clinical diagnostic procedure with direct applicability.

Laboratorial diagnosis of paracoccidioidomycosis and new insights for the future of fungal diagnosis

Paracoccidioidomycosis (PCM) is the most prevalent mycosis in Latin-America. As for other mycosis, its importance of has been largely underestimated, partially due to the limited geographical distribution of the etiologic fungal agent (Paracoccidioides brasiliensis). However, the advent of AIDS and other immune suppressing conditions is creating an emergent need for improved diagnostic tests envisaging simpler, cheaper, faster and more sensitive and accurate detection of pathogenic fungi, especially those causing systemic and opportunistic diseases. Routine laboratorial diagnosis of PCM disease relies mainly on direct observation of the fungus. However, culture growing is slow and, too often, definite diagnosis can only be obtained at later growing stages. Immunodiagnosis is also widely employed, although usually cumbersome and complex. Enzyme-based immunoassays are more amenable to automation for high-throughput testing, but may lead to cross-reactivity with other fungi. Plus, molecular diagnosis relying on polymerase-chain reaction (PCR) and nucleic-acid hybridization, although still at early stages of application to routine diagnosis of P. brasiliensis, has triggered the development of techniques for its improved specific detection, thus contributing for epidemiological studies as well. In the future, microarrays and newer biosensing technologies, coupled to new bionanotechnological tools, will certainly improve diagnosis of PCM and other mycosis through very specific and sensitive pathogen biomolecular detection.

Direct and specific identification of Cryptococcus neoformans in biological samples using fluorescently labelled DNA probes

Fluorescence in situ hybridisation (FISH) is a suitable technique for the rapid, reliable and cultivation-independent identification of microbial pathogens. This study describes the development of fluorescently labelled rRNA-targeted oligonucleotides and a FISH assay to detect and identify Cryptococcus neoformans in culture and biological samples. All C. neoformans reference and clinical isolates gave positive signals with the specific oligonucleotide probes, whereas all non-target yeast species gave negative reactions with the same probes. The assay was also successfully applied to the detection of C. neoformans cells in cerebrospinal samples from patients with clinical diagnosis of cryptococcosis. The described FISH-based assay revealed to be practical, sensitive and specific for the detection and identification of C. neoformans yeasts.

Phaeohyphomycosis Caused by Alternaria Infectoria Presenting as Multiple Vegetating Lesions in a Renal Transplant Patient

The genus Alternaria is one of the most common black moulds and appears to be increasing as a causative agent of subcutaneous phaeohyphomycosis, particularly among immunosuppressed patients. A 53-year-old patient who had received a kidney transplant presented with multiple verrucous lesions on the distal extremities. Positive histopathology and cultures, in addition to rDNA ITS region sequencing, identified the fungal isolate as Alternaria infectoria. Oral itraconazole was administered for 10 months. A follow-up at 15 months demonstrated no signs of infection. Clinical manifestations of cutaneous alternariosis vary significantly and only a few cases have been described in the literature. Although optimal treatment options remain controversial, this case of phaeohyphomycosis was successfully treated with itraconazole monotherapy.

Genotypic diversity of environmental Cryptococcus neoformans isolates from Northern Portugal

The Cryptococcus neoformans/C. gattii species complex members are the main agents of systemic cryptococcosis. This disease is believed to be acquired from the environment via fungal cell inhalation. Often, isolates recovered from environmental and clinical sources have proven to be genotypically similar. We assessed the occurrence of C. neoformans and C. gattii in environmental substrates collected in a Portuguese region. Twenty‐eight isolates were identified as C. neoformans – five from decaying Eucalyptus leaves and 23 from domestic pigeon droppings. The isolates were genotyped using a URA5‐RFLP approach. The C. neoformans VNIV (53.6%, n = 15) and VNI (32.1%, n = 9) genotypes were abundantly present among environmental isolates. The hybrid VNIII (14.3%, n = 4) genotype was underrepresented and the VNII was not found. Cryptococcus gattii was also not found although some isolates yielded a positive canavanine–glycine–bromothymol blue test.

Atypical presentation of entomophthoromycosis caused by Conidiobolus coronatus

A male patient from Guinea-Bissau was admitted to Egas Moniz Hospital, Lisbon, complaining of fever and exhibiting a productive cough with mucopurulent discharge and weight loss. He had been using empirical medication with dexamethasone to treat his generalized facial swelling. At admission, he was cachectic and presented with soft facial edema, oropharyngeal thrush, and two fistulas of the palate. Acid-fast bacilli were detected in the sputum and were later identified as Mycobacterium tuberculosis. Cultures of the palatine exudate and biopsy resulted in the growth of Candida albicans. The patient was administered antituberculosis drugs and fluconazole, but his clinical situation deteriorated progressively. Extensive investigation of his clinical condition did not result in a conclusive diagnosis until he began to experience respiratory distress and subcutaneous nodules appeared on his face. Biopsies of the hypopharynx and nodules revealed the presence of Conidiobolus coronatus. After initiating combined antifungal and antibiotic therapy, the patients clinical condition improved significantly. We report an unusual presentation of entomophthoromycosis and describe the clinical difficulties that delayed this diagnosis.

Practicum experiences as sources of pre-service teachers’ self-efficacy

This study examines physical education pre-service teachers’ (PTs) self-efficacy and practicum experiences as self-efficacy sources through a mixed-method approach. For the quantitative phase, a self-efficacy questionnaire was applied to 141 PTs. Results showed a stronger self-efficacy in the relationship with students and discipline promotion. Lower self-efficacy was linked to instructional strategies. Concerning the qualitative phase, eight PTs were interviewed. PTs with higher self-efficacy reported professional experiences before practicum as mastery experiences. During the practicum they highlighted as mastery experiences: classes’ characteristics, planning and teaching practice; lesson observation as vicarious experiences; and post-lesson conversations as verbal persuasion. PTs with lower self-efficacy reported classes’ characteristics and teaching practice as failure experiences. Lesson observation was linked to negative vicarious experiences and post-lesson conversations were associated to negative emotions and the absence of verbal persuasion. This study’s results have implications inasmuch as they confirm the role of the practicum in teacher education programmes and the importance of training supervisors in the implementation and management of the training experience, thus contributing to PTs’ self-efficacy development.

Kerion caused by Microsporum audouinii in a child

Kerion celsi is rarely associated with Microsporum audouinii infection. We report the case of a 3-year-old girl with a kerion celsi caused by M. audouinii and successfully treated with oral terbinafine. Fungi identification was made by macro and microscopical colony morphology analyses and molecular (genotypic) studies.

Trichophyton erinacei skin infection after recreational exposure to an elephant in Southeast Asia.

Abstract Zoophilic fungal infections are a prevalent disease in tropical countries and clinicians must consider them in the differential diagnosis of pruritic skin lesions. We report a clinical case of Trichophyton erinacei skin infection after recreational exposure to an Asian Elephant. As far as we were able to search the literature, it is the first case described after contact with elephants