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Clinical charts from 63 consecutive highly immunocompromised haematologic patients presenting with pulmonary nodular lesions on CT scan, classified as either probable or possible invasive fungal disease (IFD) according to the revised EORTC/MSG classification, were retrospectively studied. Histopathological analysis of lung tissues, available for 23 patients, demonstrated proven IFD in 17 cases (14 invasive aspergillosis and 3 invasive zygomycosis), diffuse alveolar damage in one and organising pneumonia (OP) in five cases. In the OP cases, three of which have been defined as probable IFD according to EORTC/MSG classification, extensive immunohistochemical, molecular and immunological analyses for fungi were negative. Our case descriptions extend the notion that OP may be encountered as a distinct histopathological entity in pulmonary nodular lesions in patients with leukaemia with probable/possible IFD.

The polysaccharide capsule is a major virulence factor of Streptococcus pneumoniae as it confers resistance to phagocytosis. The encapsulated serotype 4 TIGR4 strain was shown to be efficiently phagocytosed by the mouse microglial cell line BV2, whereas the type 3 HB565 strain resisted phagocytosis. Comparing survival after uptake of TIGR4 or its unencapsulated derivative FP23 in gentamicin protection and phagolysosome maturation assays, it was shown that TIGR4 was protected from intracellular killing. Pneumococcal capsular genes were up-regulated in intracellular TIGR4 bacteria recovered from microglial cells. Actual presence of bacteria inside BV2 cells was confirmed by transmission electron microscopy (TEM) for both TIGR4 and FP23 strains, but typical phagosomes/phagolysosomes were detected only in cells infected with the unencapsulated strain. In a mouse model of meningitis based on intracranic inoculation of pneumococci, TIGR4 caused lethal meningitis with an LD(50) of 2 × 10² CFU, whereas the LD(50) for the unencapsulated FP23 was greater than 10⁷ CFU. Phagocytosis of TIGR4 by microglia was also demonstrated by TEM and immunohistochemistry on brain samples from infected mice. The results indicate that encapsulation does not protect the TIGR4 strain from phagocytosis by microglia, while it affords resistance to intracellular killing.

Diagnosis of NonTuberculous Mycobacteria (NMT) infection frequently runs into difficulties regarding a precise definition of the strains. The use of molecular assays is the technique of choice for the identification of species, but commercial methods recognize only a limited number of species. Aim of this study was molecular identification of NonTuberculous Mycobacteria (NTM) in clinical specimens using commercial methods and automated sequencing. We analyzed 6192 clinical specimens for the isolation of Mycobacteria. One hundred and twelve strains of NTM were previously analyzed with GenoType Mycobacteria CM/AS kit and then with entire 16S rDNA and partial hsp65 sequencing. 100/112 NMT strains were identified with GenoType Mycobacteria CM/AS kit as: M. gordonae (n°25), M. xenopi (n°24), M. fortuitum (n°15), M. avium (n°12), M. intracellulare (n°7), M. chelonae (n°6), M. malmoense (n°4), M. peregrinum (n°3), M. mucogenicum (n°1), M. kansasii (n°1), M. abscessus (n°1), M. heckeshornense (n°1).Twelve unidentified strains were subjected to the entire 16S rDNA gene sequencing and nine were identified as M. arupense (n°7), M. avium complex (n°1), M. kumamotonense (n°1).Three unidentified strains were subjected to partial hsp65 gene sequencing and one was identified as M. arupense. Conclusions. Direct sequencing of entire 16SrDNA gene and of partial hsp65 gene appears to be a useful tool for the study of strains that are not identifiable by commercial methods.This new approach, applied to clinical diagnostic, allows also recognition of unusual strains or new species.

cation [1]. The patient persisted febrile, despite progressive withdrawal of corticosteroids, broad-spectrum antibiotic and antifungal therapy, including liposomal amphotericin-B, initially alone and subsequently in association with caspofungin. On day 134, after the introduction of posaconazole as salvage treatment, initially associated with caspofungin, and with a neutrophil count of 1.3 3 10 per liter, he became apyretic, showed improvement of pulmonary imaging on chest computed tomography (CT) and could resume the chemotherapeutic course. On day 164, after full hematologic recovery was obtained, with a neutrophil count of 4.2 3 10 per liter, the patient presented low-grade fever (37.78C) and left upper quadrant abdominal pain. Abdominal ultrasonography and contrast-enhanced CT disclosed a 6 cm solid, hypodense splenic lesion suggestive for abscess (Image 1A). The patient underwent laparotomic splenectomy, when receiving broad-spectrum antibiotic therapy, in addition to posaconazole. Cultural examinations on sple

Objective. Aim of this study was molecular identification in clinical specimens of NonTuberculous Mycobacteria (NTM) with commercial methods and automated sequencing. Materials and methods. Three thousand clinical specimens were analyzed for the isolation of Mycobacteria. Forty strains of NTM were previously analyzed with GenoType Mycobacteria CM/AS kit and then with partial 16S rDNA sequencing. Results. 38/40 NMT strains were identified with GenoType Mycobacteria CM/AS kit as: M. gordonae (15), M. intracellulare (8), M. xenopi (8), M. mucogenicum (2), M. kansasii (2), M. chelonae (1), M. avium (1), M. lentiflavum (1). Two unidentified strains were subjected to 16S rDNA sequencing and were identified as M. kumamotonense. Conclusions. Partial 16SrDNA sequencing is a valid assay to study NTM strains unidentified with commercial assays. This new approach, applied to clinical diagnostic, also permits the recognition of unusual strains or new species.