In vitro and in vivo evaluation of antifungal combinations against azole-resistant Aspergillus fumigatus isolates.

Azole resistance in Aspergillus fumigatus (Af) has become a widespread threat and a major concern for optimal management of patients with invasive aspergillosis (IA). Combination of echinocandins with azoles is an attractive alternative option for the treatment of IA due to azole-resistant Af strains. The aim of this study was to evaluate the in vitro and in vivo combination of caspofungin (CAS) with either voriconazole (VRZ) or posaconazole (PSZ). In vitro interactions were assessed by two methods, and an animal model of IA in Galleria mellonella was used for in vivo evaluation. Assessment of efficacy was based on larvae mortality. Groups of 10 larvae were infected by 3 clinical strains of Af (azole susceptible, AfS; PSZ resistant, AfR1; VRZ and PSZ resistant strain, AfR2). In vitro, combination of CAS and azoles was indifferent against AfS, and AfR2, and a synergy was found for AfR1. When compared to VRZ monotherapy, the combination of VRZ at 4 µg/larva with CAS at 4 µg/larva improved survival of AfR2-infected larvae (p=0.0066). Combination of PSZ at 4µg/larva with CAS at 4 µg/larva improved survival of AfR1-infected larvae compared to CAS (p=0.0002) and PSZ (0.0024) monotherapy. Antagonism was never observed. In conclusion, the combination of caspofungin with azoles is a promising alternative for the treatment of azole resistant strains of Af.

In Vivo Efficacy of Voriconazole in a Galleria mellonella Model of Invasive Infection Due to Azole-Susceptible or Resistant Aspergillus fumigatus Isolates.

Aspergillus fumigatus is an environmental filamentous fungus responsible for life-threatening infections in humans and animals. Azoles are the first-line treatment for aspergillosis, but in recent years, the emergence of azole resistance in A. fumigatus has changed treatment recommendations. The objective of this study was to evaluate the efficacy of voriconazole (VRZ) in a Galleria mellonella model of invasive infection due to azole-susceptible or azole-resistant A. fumigatus isolates. We also sought to describe the pharmacokinetics of VRZ in the G. mellonella model. G. mellonella larvae were infected with conidial suspensions of azole-susceptible and azole-resistant isolates of A. fumigatus. Mortality curves were used to calculate the lethal dose. Assessment of the efficacy of VRZ or amphotericin B (AMB) treatment was based on mortality in the lethal model and histopathologic lesions. The pharmacokinetics of VRZ were determined in larval hemolymph. Invasive fungal infection was obtained after conidial inoculation. A dose-dependent reduction in mortality was observed after antifungal treatment with AMB and VRZ. VRZ was more effective at treating larvae inoculated with azole-susceptible A. fumigatus isolates than larvae inoculated with azole-resistant isolates. The concentration of VRZ was maximal at the beginning of treatment and gradually decreased in the hemolymph to reach a Cmin (24 h) between 0.11 and 11.30 mg/L, depending on the dose. In conclusion, G. mellonella is a suitable model for testing the efficacy of antifungal agents against A. fumigatus.

Case Report: Allergic Bronchopulmonary Aspergillosis Revealing Asthma.

Allergic bronchopulmonary aspergillosis (ABPA) is an immunological pulmonary disorder caused by hypersensitivity to Aspergillus which colonizes the airways of patients with asthma and cystic fibrosis. Its diagnosis could be difficult in some cases due to atypical presentations especially when there is no medical history of asthma. Treatment of ABPA is frequently associated to side effects but cumulated drug toxicity due to different molecules is rarely reported. An accurate choice among the different available molecules and effective on ABPA is crucial. We report a case of ABPA in a woman without a known history of asthma. She presented an acute bronchitis with wheezing dyspnea leading to an acute respiratory failure. She was hospitalized in the intensive care unit. The bronchoscopy revealed a complete obstruction of the left primary bronchus by a sticky greenish material. The culture of this material isolated Aspergillus fumigatus and that of bronchial aspiration fluid isolated Pseudomonas aeruginosa. The diagnosis of ABPA was based on elevated eosinophil count, the presence of specific IgE and IgG against Aspergillus fumigatus and left segmental collapse on chest computed tomography. The patient received an inhaled treatment for her asthma and a high dose of oral corticosteroids for ABPA. Her symptoms improved but during the decrease of corticosteroids, the patient presented a relapse. She received itraconazole in addition to corticosteroids. Four months later, she presented a drug-induced hepatitis due to itraconazole which was immediately stopped. During the monitoring of her asthma which was partially controlled, the patient presented an aseptic osteonecrosis of both femoral heads that required surgery. Nine months after itraconazole discontinuation, she presented a second relapse of her ABPA. She received voriconazole for nine months associated with a low dose of systemic corticosteroid therapy with an improvement of her symptoms. After discontinuation of antifungal treatment, there was no relapse for one year follow-up.

Galleria mellonella as a screening tool to study virulence factors of Aspergillus fumigatus.

The invertebrate Galleria mellonella has increasingly and widely been used in the last few years to study complex host-microbe interactions. Aspergillus fumigatus is one of the most pathogenic fungi causing life-threatening diseases in humans and animals. Galleria mellonella larvae has been proven as a reliable model for the analysis of pathogenesis and virulence factors, enable to screen a large number of A. fumigatus strains. This review describes the different uses of G. mellonella to study A. fumigatus and provides a comparison of the different protocols to trace fungal pathogenicity. The review also includes a summary of the diverse mutants tested in G. mellonella, and their respective contribution to A. fumigatus virulence. Previous investigations indicated that G. mellonella should be considered as an interesting tool even though a mammalian model may be required to complete and verify initial data.

High Frequency of Enterocytozoon bieneusi Genotype WL12 Occurrence among Immunocompromised Patients with Intestinal Microsporidiosis.

Microsporidiosis is an emerging opportunistic infection causing severe digestive disorders in immunocompromised patients. The aim of this study was to investigate the prevalence of intestinal microsporidia carriage among immunocompromised patients hospitalized at a major hospital complex in the Tunis capital area, Tunisia (North Africa), and perform molecular epidemiology and population structure analyses of Enterocytozoon bieneusi, which is an emerging fungal pathogen. We screened 250 stool samples for the presence of intestinal microsporidia from 171 patients, including 81 organ transplant recipients, 73 Human Immunodeficiency Virus (HIV)-positive patients, and 17 patients with unspecified immunodeficiency. Using a nested PCR-based diagnostic approach for the detection of E. bieneusi and Encephalitozoon spp., we identified 18 microsporidia-positive patients out of 171 (10.5%), among which 17 were infected with E. bieneusi. Microsporidia-positive cases displayed chronic diarrhea (17 out of 18), which was associated more with HIV rather than with immunosuppression other than HIV (12 out of 73 versus 6 out of 98, respectively, p = 0.02) and correlated with extended hospital stays compared to microsporidia-negative cases (60 versus 19 days on average, respectively; p = 0.001). Strikingly, internal transcribed spacer (ITS)-based genotyping of E. bieneusi strains revealed high-frequency occurrence of ITS sequences that were identical (n = 10) or similar (with one single polymorphic site, n = 3) to rare genotype WL12. Minimum-spanning tree analyses segregated the 17 E. bieneusi infection cases into four distinct genotypic clusters and confirmed the high prevalence of genotype WL12 in our patient population. Phylogenetic analyses allowed the mapping of all 17 E. bieneusi strains to zoonotic group 1 (subgroups 1a and 1b/1c), indicating loose host specificity and raising public health concern. Our study suggests a probable common source of E. bieneusi genotype WL12 transmission and prompts the implementation of a wider epidemiological investigation.

Galleria mellonella for the Evaluation of Antifungal Efficacy against Medically Important Fungi, a Narrative Review.

The treatment of invasive fungal infections remains challenging and the emergence of new fungal pathogens as well as the development of resistance to the main antifungal drugs highlight the need for novel therapeutic strategies. Although in vitro antifungal susceptibility testing has come of age, the proper evaluation of therapeutic efficacy of current or new antifungals is dependent on the use of animal models. Mammalian models, particularly using rodents, are the cornerstone for evaluation of antifungal efficacy, but are limited by increased costs and ethical considerations. To circumvent these limitations, alternative invertebrate models, such as Galleria mellonella, have been developed. Larvae of G. mellonella have been widely used for testing virulence of fungi and more recently have proven useful for evaluation of antifungal efficacy. This model is suitable for infection by different fungal pathogens including yeasts (Candida, Cryptococcus, Trichosporon) and filamentous fungi (Aspergillus, Mucorales). Antifungal efficacy may be easily estimated by fungal burden or mortality rate in infected and treated larvae. The aim of the present review is to summarize the actual data about the use of G. mellonella for testing the in vivo efficacy of licensed antifungal drugs, new drugs, and combination therapies.