Silvana Sanna

Evaluation of the peptide nucleic acid fluorescence in situ hybridisation technology for yeast identification directly from positive blood cultures: An Italian experience

Summary Fungaemia is an increasing nosocomial pathology. The ‘gold standard’ for detection of fungaemia is blood culture, but it is time‐consuming and its sensitivity for early detection is low. On the other hand, yeasts present different antifungal sensitivity patterns to be quickly detected to allow an effective treatment. The aim of this study was to evaluate the diagnostic performances of PNA‐FISH to directly identify yeasts from blood cultures and to compare results with those obtained by culture. A total of 176 blood cultures positive for yeasts at direct Gram stain and 24 negative blood cultures as control collected from 15 Italian hospitals, included in a network coordinated by the Medical Mycology Committee, Italian Society of Clinical Microbiology (AMCLI), were examined both by culture and PNA‐FISH technology. Sensitivity of the PNA‐FISH technique evaluated for five Candida species was 99.3% and specificity, 100%. Distinguishing which yeast is implicated in fungaemia and whether the infection is caused by multiple species are important for the selection of antifungal therapy. The PNA‐FISH technique is a very useful approach because the test discriminates between groups of Candida species with different susceptibility pattern, particularly against azoles and echinocandins, with only a 90‐minute turn‐around time after the Gram‐stain reading.

Microarray technology for yeast identification directly from positive blood cultures. A multicenter Italian experience.

The authors evaluated the performance of the MycArray™ Yeast ID (Myconostica Ltd, UK) assay in the identification of a total of 88 yeast isolates recovered in culture as compared to that obtained through routine methods. The turn-around time for species identification directly from cultures by the MycArray was 6 hours, much quicker than classical methods and all yeasts were correctly identified. In two cases a double identification including Saccharomyces cerevisiae was noted, but it was not confirmed by culture. The results show that MycArray Yeast ID can be a potential tool for rapid detection and identification of Candida species.

Breakthrough cutaneous alternariosis in a patient with acute lymphoblastic leukemia: Clinical features and diagnostic issues

Cutaneous alternariosis, which is caused by opportunistic moulds of the genus Alternaria, is the most common form of phaeohyphomycosis. Alternaria infectoria is one of the six species which have been described as potential pathogens for humans. The clinical pattern of Alternaria infections is usually characterized by superficial cutaneous or subcutaneous involvement while organ infiltration is rare. The skin lesions can be extremely polymorphic, ranging from localized eczema-like lesions to chronic verrucous lesions and, occasionally, abscesses [1]. For this reason, the clinical diagnosis can be extremely challenging as in almost all cutaneous infections due to molds other than dermatophytes. Therefore, histological, cultural, and molecular investigations are often necessary. The incidence of Alternaria infections has been rising during the last years as a consequence of the increasing number of immunocompromised subjects. Several cases of cutaneous alternariosis have been reported in subjects who underwent solid organ transplant [2]. Conversely, this infection has been only occasionally described in patients with haematological malignancies. Almost all these cases were characterised by an exclusively cutaneous involvement [3 – 5]. Here we report on a case of alternariosis which occurred in a 24-year-old man presenting in September 2005 with acute T-lymphoblastic leukemia. After an early relapse during the consolidation phase and the failure of the second-line treatment, he was started on FLAG-Ida (fludarabine, cytosine arabinoside, G-CSF, and idarubicin) in March 2006. On day þ17 post-treatment, while he was pancytopenic, he developed a painful erythematoviolet nodular lesion on the dorsal surface of the right foot. After few days, the lesion became ulcerated (Figure 1). At that time and in the following period, the patient was apyretic and all the biochemistry – including inflammation indexes – was unremarkable. The histological examination of the skin lesion biopsy showed exclusively a perivascular inflammatory infiltrate without leukemic cells. For mycological examination, a sample from the biopsy specimen was cultivated onto Sabouraud dextrose agar and incubated at 308C and 378C. After 4 – 5 days, a slowgrowing fungus was isolated in both conditions. Microscopic examination of lactophenol cotton bluestained smears showed melanized septate hyphae, typical of dematiaceous fungi. To enhance sporulation, the fungus was subcultured on potato dextrose agar (PDA) and incubated at 258C for two weeks. The culture remained sterile and the isolated fungus could not be conclusively identified. Noticeably, he had been on antimycotic prophylaxis with itraconazole solution 200 mg/day for 3 months, and he had not been on steroids for more than 3 months. Notwithstanding the lack of a conclusive diagnosis, a treatment with intravenous voriconazole 400 mg/day was started, three weeks after the appearance of the lesion. Systemic dissemination of the fungal infection was excluded by serial systemic CT scans.

Madurella infection in an immunocompromised host

A 77‐year‐old farmer, born and living in Sardinia, affected by acute myelogenous leukemia and undergoing chemotherapy treatment with cytosine arabinoside, presented at the Institute of Dermatology of Sassari in March 1998 with multiple subcutaneous lesions on the legs ( Fig. 1 ) which had appeared 3 months previously. Scattered crusts were observed, and some nodules were necrotizing and ulcerated with a discharge of serosanguineous fluid. There was no history of trauma. A deep biopsy was performed and histologic examination showed suppurative inflammation in the middle and deep dermis with a mass of hyphae and numerous spores at periodic acid‐Schiff (PAS) reaction ( Fig. 2 ). Culture of a small skin sample at 37 °C on Saborauds dextrose agar resulted, 7 days later, in the development of flat, brownish, fluffy colonies. The reverse of the colony was black. Microscopically, the culture showed broad, segmented hyphae without conidia and with chlamydospores typical of Madurella. X‐Ray findings of the lower extremities showed no bony involvement.

Antifungal susceptibility testing of Aspergillus species complex in the Clinical Laboratory: how to do it, when to do it, and how to interpret it

The emergence of drug resistance in fungal pathogens has a profound impact on human health given limited number of antifungal drugs. Antifungal resistance in Aspergillus spp. infection can be encountered in the antifungal drug-exposed patient due to selection of intrinsically resistant species or isolates with acquired resistance belonging to species that are normally susceptible. Resistance to triazoles is not common in Aspergillus spp., however, triazole resistance in A. fumigatus appears to be increasing in several European countries in recent years and can be clinically relevant. The Clinical and Laboratory Standards Institute and European Committee on Antimicrobial Susceptibility Testing have developed breakpoints and epidemiological cutoff values that are now established for Aspergillus spp. Clinical microbiology laboratories will be employed commercial susceptibility assays, rather than reference broth microdilution methods and comparative studies are particularly important.

Breast Abscess by Nocardia

A 69-year-old woman reported the appearance of a painful lump in the lower outer quadrant of right breast for 10 days. The patient had history of relapsing uveitis poorly responsive to therapy, thus she underwent high-dose steroid therapy for about 6 months. Clinical examination showed Cushing-like aspect of the patient, peripheral edema of inferior limbs, subcutaneous lumps in the right lumbar region, and distal third of forearm, which appeared some weeks before. Breast lump was especially painfulness with inflamed skin.