Microscopic, cultural and molecular evidence of disseminated invasive aspergillosis involving the lungs and the gastrointestinal tract

Abstract

A patient with acute promyelocytic leukaemia developed invasive aspergillosis post chemotherapy during a pancytopenic episode, clinically involving the lungs and the gastrointestinal tract. Dichotomously branched septate fungal hyphae were demonstrated microscopically in stools and sputa. Cultures of the samples yielded Aspergillus flavus, which were identical by RFLP and random amplification of polymorphic DNA analyses and antifungal MICs, proving disseminated disease. To the best of the author's knowledge, this is the first time that boluses of fungal hyphae have been demonstrated microscopically in the stools of a patient with gastrointestinal aspergillosis. IntroductionInvasive fungal infections (IFIs) are an important cause of morbidity and mortality in patients with haematological malignancies. Invasive aspergillosis (IA) is particularly common during neutropenic episodes following anticancer chemotherapy (Wright et al., 2003). The most common site of involvement is the respiratory tract. However, autopsy studies conducted in immunocompromised patients with disseminated IA have shown the gastrointestinal tract (GIT) to be a frequent site of sub-clinical involvement (Hori et al., 2002). We describe a patient with acute promyelocytic leukaemia (APML) who developed IA post chemotherapy during a pancytopenic episode. This was diagnosed as disseminated IA by conventional methods of both microscopy and culture. Dissemination was proven in addition by DNA fingerprinting analysis of the Aspergillus flavus isolates from both sputum and stool. The highlight of this case report is the finding of fungal hyphae in the direct microscopic examination of stools, which although unusual, may be a significant finding in an immunocompromised patient. Microbiologists, mycologists and pathologists involved in patient care may thus be able to offer improved support to clinicians by becoming proficient in recognizing and interpreting such forms in clinical samples. Case reportAn 18-year-old male who received consolidation chemotherapy consisting of daunomycin and cytosine arabinoside for acute promyelocytic leukaemia developed febrile neutropenia on the 11th day. Other associated symptoms were throat pain, cough and nasal discharge. Physical examination, however, was normal. His total leukocyte count (TLC) and absolute neutrophil count (ANC) were 300 ml 21 and zero, respectively. Chest X-ray was normal. Sputum culture grew Klebsiella pneumoniae sensitive to aminoglycosides and third-generation cephalosporins. The patient had no indwelling catheter. Despite receiving intravenous ceftazidime, amikacin and fluconazole, along with other supportive measures, fever persisted and his condition worsened with the development of haemoptysis and epistaxis. Hence an aggressive IFI was suspected, for which fluconazole was substituted with amphotericin B at a dose of about 1 mg kg 21 per day (50 mg per day). Two days after a transient improvement in symptoms, the patient's condition deteriorated. He developed diarrhoea and respiratory distress...

Fungal Biofilm Resistance

“…Aspergillary bronchitis has also been reported, which is characterized by bronchial casts containing mucus and mycelia [25]. Bronchopulmonary lavage (BAL) of patients with aspergillosis may also reveal the presence of numerous hyphae in the form of a complex multicellular mycetoma structure samples when examined histologically [26]. In addition to this, it has been reported to cause serious biomaterial-related infections of joint replacements, catheters, heart valves, cardiac pace makers, and breast augmentation implants [27–30].…”

Extracellular DNA Release Acts as an Antifungal Resistance Mechanism in Mature Aspergillus fumigatus Biofilms

“…ECM was extracted as previously described (35), with slight modifications. Briefly, standardized A. fumigatus conidia were inoculated into a 75-cm 2 tissue culture flask (Nunc, Rochester, NY) containing RPMI medium, yeast extract-peptone-dextrose (YPD), and yeast nitrogen base (YNB) (Sigma, United Kingdom) at 37°C for 8,12,24,48, and 72 h on a rocking platform. After incubation, the biofilm was removed from the flasks by use of sterile cell scrapers and then washed with PBS.…”

“…Different phases of biofilm (8,12, and 24 h), with or without azetazolamide (128 mg/liter; Sigma), a chitinase inhibitor (36), were formed at 37°C either in microtiter plates or in tissue culture flasks as described above. The quantity of eDNA within the ECM was measured using a microplate fluorescence assay (MFA) with a DNA-binding dye (SYBR green I) as previously described (37).…”

“…For example, aspergilli have been reported to cause serious biomaterial-related biofilm infections of catheters, joint replacements, cardiac pacemakers, heart valves, and breast augmentation implants (3)(4)(5)(6). Moreover, aspergillary bronchitis is characterized by bronchial casts containing mucus and mycelia (7), and bronchopulmonary lavage (BAL) fluid of some patients with aspergillosis reveals the presence of numerous hyphae in the form of a complex multicellular mycetoma structure (8). Collectively, these reports confirm that A. fumigatus does maintain the capacity to form biofilms, which are characterized by aggregates of multicellular hyphae enclosed within extracellular matrix (ECM) and which are clinically significant because they are resistant to host defenses and, particularly, antifungal drugs (2).…”

Fungal Biofilms in Human Disease

“…Filamentous fungi, mainly A. fumigatus, may cause a spectrum of respiratory disease including from a discrete lesion in a pre-existing cavity, aspergilloma, wheezing mediated by an immune response, allergic bronchopulmonary aspergillosis (ABPA) and invasive aspergillosis (IA) (Denning 1998 ). A bronchopulmonary lavage (BAL) of these individuals often reveals the presence of numerous intertwined hyphae in the form of a complex multicellular structure when examined histologically (Jayshree et al 2006 ), this is indicative of a biofi lm phenotype (Harding et al 2009 ;Mowat et al 2009 ). The recently described Aspergillus bronchitis may also be biofi lm associated and is characterized by bronchial casts containing mycelia forming compact masses (Young et al 1970 ).…”