Sharon C. -A. Chen

In vitro bacterial–fungal interaction studies in cystic fibrosis (CF) have mainly focused on interactions between bacteria and Candida. Here we investigated the effect of Pseudomonas aeruginosa on the growth of Scedosporium/Lomentospora spp. Standard suspensions of P. aeruginosa (16 non-mucoid and nine mucoid isolates) were dropped onto paper disks, placed on lawns of Lomentospora prolificans (formerly Scedosporium prolificans) strain WM 14.140 or Scedosporium aurantiacum strain WM 11.78 on solid agar. The median inhibitory activity (mIz) was calculated for each fungal–bacterial combination. As a group, mIz values for non-mucoid phenotype P. aeruginosa strains were significantly lower than those for mucoid strains (P < 0.001); 14/16 (87.5%) non-mucoid strains had mIz <1.0 against both fungi versus just 3/9 mucoid strains (33.4%) (P = 0.01). One non-mucoid (PA14) and one mucoid (CIDMLS-PA-28) P. aeruginosa strain effecting inhibition were selected for further studies. Inhibition of both L. prolificans and S. aurantiacum by these strains was confirmed using the XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide) reduction assay. Following incubation with XTT, inhibition of fungal growth was determined as the ratio of absorbance in liquid culture with Pseudomonas to that in control fungal cultures. An absorbance ratio of <1.0 consistent with bacterial inhibition of fungal growth was observed for all four P. aeruginosa–fungal combinations (P < 0.05). Fluorescence microscopy, subsequent to co-culture of either fungal isolate with P. aeruginosa strain PA14 or CIDMLS-PA-28 revealed poorly formed hyphae, compared with control fungal cultures. P. aeruginosa inhibits growth of L. prolificans and S. aurantiacum in vitro, with non-mucoid strains more commonly having an inhibitory effect. As P. aeruginosa undergoes phenotype transitions from non-mucoid to the mucoid form with progression of CF lung disease, this balance may influence the appearance of Scedosporium fungi in the airways.

Antifungal treatment of uncommon filamentous fungal infections is problematic. This study determined the in vitro susceptibility of miltefosine, as a single agent and in combination with posaconazole or voriconazole, against these pathogens. Susceptibility to miltefosine of 34 uncommon filamentous fungi was tested using CLSI broth microdilution M38-A2 methodology. Twenty isolates were studied for potential synergy using miltefosine/posaconazole and miltefosine/voriconazole combinations and the chequerboard microdilution assay. MICs of miltefosine were high (in general, >8 mg/L) for most isolates compared with amphotericin B, echinocandins and the azoles. Miltefosine had greatest activity against Scedosporium spp., Lichtheimia corymbifera and Rhizomucor sp. (MICs ≤4 mg/L). Miltefosine in combination either with posaconazole or voriconazole demonstrated synergy [fractional inhibitory concentration index (FICI) ≤0.5] in 12 instances (11 isolates): miltefosine/posaconazole combinations were synergistic against 3 of 4 Fusarium oxysporum strains (FICI range 0.37–0.5) and 5 of 10 mucormycete strains (FICI range 0.06–0.5). The combination of voriconazole with miltefosine showed synergy against one Scedosporium prolificans isolate and three mucormycetes—a single strain each of L. corymbifera, Rhizopus oryzae and Rhizomucor sp. No antagonism was observed. Miltefosine demonstrated synergy in 8/20 (40%) and 4/20 (20%) instances when combined with posaconazole and voriconazole, respectively. Synergy was most often observed against F. oxysporum and the mucormycetes. Study of miltefosine/azole combinations as a novel antifungal approach is indicated.

Background. We describe antifungal therapy and management of complications due to Cryptococcus gattii infection in 86 Australian patients followed for at least 12 months. Methods. Patient data from culture-confirmed cases (2000–2007) were recorded at diagnosis, 6 weeks, 6 months, and 12 months. Clinical, laboratory, and treatment variables associated with raised intracranial pressure (ICP) and immune reconstitution inflammatory syndrome (IRIS) were determined. Results. Seven of 10 patients with lung infection received amphotericin B (AMB) induction therapy (6 with 5-flucytosine [5-FC] for a median of 2 weeks); median duration of therapy including azole eradication therapy was 41 weeks, with a complete/partial clinical response in 78%. For neurologic disease, 88% of patients received AMB, 78% with 5-FC, for a median of 6 weeks. The median total course was 18 months. Nine patients receiving fluconazole induction therapy were reinduced with AMB plus 5-FC for clinical failure. Raised ICP (31 patients) was associated with initial abnormal neurology, and neurologic sequelae and/or death at 12 months (both P = .02); cerebrospinal fluid drains/shunts were placed in 58% of patients and in 64% of 22 patients with hydrocephalus. IRIS developed 2–12 months after starting antifungals in 8 patients, who presented with new/enlarging brain lesions. Risk factors included female sex, brain involvement at presentation, and higher median CD4 counts (all P < .05); corticosteroids reduced cryptococcoma-associated edema. Conclusions. Induction AMB plus 5-FC is indicated for C. gattii neurologic cryptococcosis (6 weeks) and when localized to lung (2 weeks). Shunting was often required to control raised ICP. IRIS presents with cerebral manifestations.

We developed a reverse line blot (RLB) hybridization-, and rolling circle amplification (RCA)-based assays for the identification of Trichoporon species and evaluated them with 48 isolates that had been previously recognized as belonging to eight species (Trichosporon asahii, T. cutaneum, T. dermatis, T. domesticum, T. inkin, T. japonicum, T. jirovecii, and T. laibachii). Results were compared to those obtained with DNA sequencing of three rRNA gene loci, i.e., the internal transcribed spacer (ITS) region, D1/D2 domain of the 28S rRNA gene and intergenic spacer 1 (IGS1) region. Using species-specific, or group-specific probes targeted at the ITS region and the D1/D2 domain, the RLB assay permitted accurate species identification of all 48 isolates with 100% specificity. Species-specific RLB probes correctly assigned 45/48 (94%) of the isolates (six species) with the exception of T. dermatis and T. japonicum isolates which were not targeted by the assay. Identification of T. dermatis relied on a positive hybridization result with the group-specific probe hybridizing with T. dermatis and T. jirovecii and the absence of a signal with the T. jirovecii-specific probe. T. japonicum strains were first assigned to the T. asahii-T. japonicum group by hybridization with the two species group-specific probe and then as T. japonicum by the absence of signal with a T. asahii-specific probe. Twelve species-specific RCA probes targeting the eight species studied detected templates of all 48 Trichosporon isolates and an artificial template of T. asteroides, all with good specificity. Both RLB and RCA are potential alternatives to DNA sequencing for the identification of Trichosporon species. The RLB approach is suited for the batched simultaneous analysis of large numbers of isolates, while RCA is more appropriate for the immediate study of single isolates. Comparative costs are US$7 and US$2 per assay for the RLB and RCA methods, respectively.

Background. Longer-term morbidity and outcomes of Cryptococcus gattii infection are not described. We analyzed clinical, microbiological, and outcome data in Australian patients followed for 12 months, to identify prognostic determinants. Methods. Culture-confirmed C. gattii cases from 2000 to 2007 were retrospectively evaluated. Clinical, microbiological, radiological, and outcome data were recorded at diagnosis and at 6 weeks, 6 months, and 12 months. Clinical and laboratory variables associated with mortality and with death and/or neurological sequelae were determined. Results. Annual C. gattii infection incidence was 0.61 per 10⁶ population. Sixty-two of 86 (72%) patients had no immunocompromise; 6 of 24 immunocompromised hosts had idiopathic CD4 lymphopenia, and 1 had human immunodeficiency virus/AIDS. Clinical and microbiological characteristics of infection were similar in immunocompromised and healthy hosts. Isolated lung, combined lung and central nervous system (CNS), and CNS only disease was reported in 12%, 51% and 34% of the cases, respectively. Complications in CNS disease included raised intracranial pressure (42%), hydrocephalus (30%), neurological deficits (27%; 6% developed during therapy) and immune reconstitutionlike syndrome (11%). Geometric mean serum cryptococcal antigen (CRAG) titers in CNS disease were 563.9 (vs 149.3 in isolated lung infection). Patient immunocompromise was associated with increased mortality risk. An initial cerebrospinal fluid CRAG titer of ≥256 predicted death and/or neurological sequelae (P = .05). Conclusions. Neurological C. gattii disease predominates in the Australian endemic setting. Lumbar puncture and cerebral imaging, especially if serum CRAG titers are ≥512, are essential. Long-term follow up is required to detect late neurological complications. Immune system evaluation is important because host immunocompromise is associated with reduced survival.