In vitro interaction between glabridin and voriconazole against Aspergillus fumigatus isolates

BackgroundVoriconazole (VRC) is widely recommended as the first-line therapy for invasive aspergillosis. However, surveillance studies have demonstrated that there is an increase in the frequency of azole resistance among Aspergillus fumigates isolates. In recent years, more studies on effective synergisms between natural agents and antifungal drugs have been published.AimsTo evaluate the synergistic antifungal effect of glabridin (Gla) and VRC against A. fumigatus isolates.MethodsPotential interactions between Gla and VRC were studied by using a microdilution checkerboard method based on the CLSI reference technique. To assess the interaction of drugs the fractional inhibitory concentration index (FICI) was calculated based on the Loewe Additivity model.ResultsThe minimum inhibitory concentrations (MIC) obtained with Gla alone were relatively high (MIC₅₀ 16 μg/ml). However, our results showed synergistic interaction between Gla and VRC against A. fumigatus strains, with FICI range values between 0.15 and 0.5.ConclusionsSynergistic activity of Gla and VRC against both VRC-sensitive and -resistant A. fumigatus isolates may lead to design new antifungal agents, especially for inhibiting those azole-resistant strains.     

Development of Azole Resistance in Aspergillus fumigatus during Azole Therapy Associated with Change in Virulence

Four sequential Aspergillus fumigatus isolates from a patient with chronic granulomatous disease (CGD) eventually failing azole-echinocandin combination therapy were investigated. The first two isolates (1 and 2) were susceptible to antifungal azoles, but increased itraconazole, voriconazole and posaconazole MICs were found for the last two isolates (3 and 4). Microsatellite typing showed that the 4 isolates were isogenic, suggesting that resistance had been acquired during azole treatment of the patient. An immunocompromised mouse model confirmed that the in vitro resistance corresponded with treatment failure. Mice challenged with the resistant isolate 4 failed to respond to posaconazole therapy, while those infected by susceptible isolate 2 responded. Posaconazole-anidulafungin combination therapy was effective in mice challenged with isolate 4. No mutations were found in the Cyp51A gene of the four isolates. However, expression experiments of the Cyp51A showed that the expression was increased in the resistant isolates, compared to the azole-susceptible isolates. The microscopic morphology of the four isolates was similar, but a clear alteration in radial growth and a significantly reduced growth rate of the resistant isolates on solid and in broth medium was observed compared to isolates 1 and 2 and to unrelated wild-type controls. In the mouse model the virulence of isolates 3 and 4 was reduced compared to the susceptible ones and to wild-type controls. For the first time, the acquisition of azole resistance despite azole-echinocandin combination therapy is described in a CGD patient and the resistance demonstrated to be directly associated with significant change of virulence.     

Possible Environmental Origin of Resistance of Aspergillus fumigatus to Medical Triazoles

We reported the emergence of resistance to medical triazoles of Aspergillus fumigatus isolates from patients with invasive aspergillosis. A dominant resistance mechanism was found, and we hypothesized that azole resistance might develop through azole exposure in the environment rather than in azole-treated patients. We investigated if A. fumigatus isolates resistant to medical triazoles are present in our environment by sampling the hospital indoor environment and soil from the outdoor environment. Antifungal susceptibility, resistance mechanisms, and genetic relatedness were compared with those of azole-resistant clinical isolates collected in a previous study. Itraconazole-resistant A. fumigatus (five isolates) was cultured from the indoor hospital environment as well as from soil obtained from flower beds in proximity to the hospital (six isolates) but never from natural soil. Additional samples of commercial compost, leaves, and seeds obtained from a garden center and a plant nursery were also positive (four isolates). Cross-resistance was observed for voriconazole, posaconazole, and the azole fungicides metconazole and tebuconazole. Molecular analysis showed the presence of the dominant resistance mechanism, which was identical to that found in clinical isolates, in 13 of 15 environmental isolates, and it showed that environmental and clinical isolates were genetically clustered apart from nonresistant isolates. Patients with azole-resistant aspergillosis might have been colonized with azole-resistant isolates from the environment.Invasive aspergillosis is a fungal disease caused by Aspergillus species that primarily affects immunocompromised patients, such as those treated for hematological malignancy. Patients may become infected by inhalation of ambient air that contains fungal spores. The Aspergillus conidia can penetrate into the alveoli and if not effectively removed, may germinate, proliferate, and cause invasive aspergillosis. Mortality and morbidity due to invasive aspergillosis remain a significant problem.Triazoles, such as itraconazole (ITZ), voriconazole, and posaconazole, are used increasingly in the management of patients with this disease. Although the risk of resistance due to the increased use of triazoles is considered low (11), we recently observed ITZ resistance rapidly emerging in clinical Aspergillus fumigatus isolates (19, 22, 24, 25). Azole resistance was observed in up to 6% of patients in our hospital and in up to 14.5% of isolates sent to our laboratory from other hospitals in The Netherlands, which were obtained from patients with aspergillus disease (19). Furthermore, azole resistance has been reported in other European countries (3, 13, 19). The ITZ-resistant isolates also showed significantly reduced susceptibility to the other mold-active medical triazoles voriconazole and posaconazole (19). A substitution of leucine for histidine at codon 98 (L98H), combined with a 34-bp tandem repeat (designated TR) in the promoter region of the cyp51A gene (TR/L98H), which is the target for antifungal azoles, was found in 94% of isolates (14, 19, 24).Azole resistance can develop through the exposure of the fungus to azole compounds, which may occur in azole-treated patients or through the use of azole compounds in the environment. The dominance of a single resistance mechanism is difficult to explain by resistance development in individual azole-treated patients, as one would expect multiple resistance mechanisms to develop. Also, spread by person-to-person transmission of any Aspergillus isolate is highly unlikely. As inhalation of airborne aspergillus spores is the common route of infection for aspergillus diseases, we hypothesized that the dominance of a single resistance mechanism in clinical ITZ-resistant isolates was more consistent with acquisition from a common environmental source (19). If azole-resistant A. fumigatus is present in our environment, patients could inhale resistant spores and subsequently develop azole-resistant disease. Indeed, azole-resistant aspergillosis was reported in azole-naïve patients, indicating that resistance does not exclusively develop during azole therapy (24).Favorable conditions for resistance development are exposure to azole compounds and the presence of reproducing fungus (1). A. fumigatus is abundantly present in our environment as saprophytic, reproducing fungi, most notably in soil and compost. Furthermore, azoles are commonly used for plant protection as well as material preservation. Therefore, it appears that resistance development in A. fumigatus is feasible in the environment, and isolates that develop resistance to fungicides might be cross-resistant to medical triazoles.We investigated if A. fumigatus isolates that are present in our environment are resistant to medical triazoles and if they are cross-resistant to azole fungicides. Furthermore, we characterized the isolates by microsatellite typing in order to determine if they were genetically related to clinical A. fumigatus isolates previously obtained from patients cared for in our University Medical Center.     

Emergence of azole resistance in Aspergillus fumigatus and spread of a single resistance mechanism

Background

Resistance to triazoles was recently reported in Aspergillus fumigatus isolates cultured from patients with invasive aspergillosis. The prevalence of azole resistance in A. fumigatus is unknown. We investigated the prevalence and spread of azole resistance using our culture collection that contained A. fumigatus isolates collected between 1994 and 2007.

Methods and Findings

We investigated the prevalence of itraconazole (ITZ) resistance in 1,912 clinical A. fumigatus isolates collected from 1,219 patients in our University Medical Centre over a 14-y period. The spread of resistance was investigated by analyzing 147 A. fumigatus isolates from 101 patients, from 28 other medical centres in The Netherlands and 317 isolates from six other countries. The isolates were characterized using phenotypic and molecular methods. The electronic patient files were used to determine the underlying conditions of the patients and the presence of invasive aspergillosis. ITZ-resistant isolates were found in 32 of 1,219 patients. All cases were observed after 1999 with an annual prevalence of 1.7% to 6%. The ITZ-resistant isolates also showed elevated minimum inhibitory concentrations of voriconazole, ravuconazole, and posaconazole. A substitution of leucine 98 for histidine in the cyp51A gene, together with two copies of a 34-bp sequence in tandem in the gene promoter (TR/L98H), was found to be the dominant resistance mechanism. Microsatellite analysis indicated that the ITZ-resistant isolates were genetically distinct but clustered. The ITZ-sensitive isolates were not more likely to be responsible for invasive aspergillosis than the ITZ-resistant isolates. ITZ resistance was found in isolates from 13 patients (12.8%) from nine other medical centres in The Netherlands, of which 69% harboured the TR/L98H substitution, and in six isolates originating from four other countries.

Conclusions

Azole resistance has emerged in A. fumigatus and might be more prevalent than currently acknowledged. The presence of a dominant resistance mechanism in clinical isolates suggests that isolates with this mechanism are spreading in our environment.     

Incidence of Cyp51 A Key Mutations in Aspergillus fumigatus—A Study on Primary Clinical Samples of Immunocompromised Patients in the Period of 1995–2013

As the incidence of azole resistance in Aspergillus fumigatus is rising and the diagnosis of invasive aspergillosis (IA) in immunocompromised patients is rarely based on positive culture yield, we screened our Aspergillus DNA sample collection for the occurrence of azole resistance mediating cyp51 A key mutations. Using two established, a modified and a novel polymerase chain reaction (PCR) assays followed by DNA sequence analysis to detect the most frequent mutations in the A. fumigatus cyp51 A gene conferring azole resistance (TR34 (tandem repeat), L98H and M220 alterations). We analyzed two itraconazole and voriconazole and two multi-azole resistant clinical isolates and screened 181 DNA aliquots derived from clinical samples (blood, bronchoalveolar lavage (BAL), biopsies, cerebrospinal fluid (CSF)) of 155 immunocompromised patients of our Aspergillus DNA sample collection, previously tested positive for Aspergillus DNA and collected between 1995 and 2013. Using a novel PCR assay for the detection of the cyp51 A 46 bp tandem repeat (TR46) directly from clinical samples, we found the alteration in a TR46/Y121F/T289A positive clinical isolate. Fifty stored DNA aliquots from clinical samples were TR46 negative. DNA sequence analysis revealed a single L98H mutation in 2010, two times the L98H alteration combined with TR34 in 2011 and 2012 and a so far unknown N90K mutation in 1998. In addition, four clinical isolates were tested positive for the TR34/L98H combination in the year 2012. We consider our assay of epidemiological relevance to detect A. fumigatus azole resistance in culture-negative clinical samples of immunocompromised patients; a prospective study is ongoing.     

Azole-resistant Aspergillus fumigatus in the environment: Identifying key reservoirs and hotspots of antifungal resistance

Aspergillus fumigatus is an opportunistic human pathogen that causes aspergillosis, a spectrum of environmentally acquired respiratory illnesses. It has a cosmopolitan distribution and exists in the environment as a saprotroph on decaying plant matter. Azoles, which target Cyp51A in the ergosterol synthesis pathway, are the primary class of drugs used to treat aspergillosis. Azoles are also used to combat plant pathogenic fungi. Recently, an increasing number of azole-naive patients have presented with pan-azole–resistant strains of A. fumigatus. The TR₃₄/L98H and TR₄₆/Y121F/T289A alleles in the cyp51A gene are the most common ones conferring pan-azole resistance. There is evidence that these mutations arose in agricultural settings; therefore, numerous studies have been conducted to identify azole resistance in environmental A. fumigatus and to determine where resistance is developing in the environment. Here, we summarize the global occurrence of azole-resistant A. fumigatus in the environment based on available literature. Additionally, we have created an interactive world map showing where resistant isolates have been detected and include information on the specific alleles identified, environmental settings, and azole fungicide use. Azole-resistant A. fumigatus has been found on every continent, except for Antarctica, with the highest number of reports from Europe. Developed environments, specifically hospitals and gardens, were the most common settings where azole-resistant A. fumigatus was detected, followed by soils sampled from agricultural settings. The TR₃₄/L98H resistance allele was the most common in all regions except South America where the TR₄₆/Y121F/T289A allele was the most common. A major consideration in interpreting this survey of the literature is sampling bias; regions and environments that have been extensively sampled are more likely to show greater azole resistance even though resistance could be more prevalent in areas that are under-sampled or not sampled at all. Increased surveillance to pinpoint reservoirs, as well as antifungal stewardship, is needed to preserve this class of antifungals for crop protection and human health.     

Correlation between broth microdilution and disk diffusion methods for antifungal susceptibility testing of caspofungin, voriconazole, amphotericin B, itraconazole and fluconazole against Candida glabrata

Candida glabrata is one of the most frequent organisms isolated from superficial and invasive fungal infections, after Candida albicans. This organism also exhibits intrinsically low susceptibility to azole antifungals and treatment often fails. The microdilution method is not very practical for use in routine susceptibility testing in the clinical laboratory, thus necessitating the use of other methods. In this study, we compared the in vitro activity of five antifungal agents in three different groups (echinocandin, polyene and azole) against 50 C. glabrata isolates by broth microdilution and disk diffusion methods recommended by Clinical Laboratory Standards Institute CLSI M27-A3 and CLSI M44-A, respectively. All the isolates were susceptible to amphotericin B (100%) and 98% of the isolates were susceptible to caspofungin by the broth microdilution method. Within the azole group drugs, voriconazole was the most active followed by fluconazole and itraconazole in vitro. The highest rate of resistance was obtained against itraconazole with a high number of isolates defined as susceptible-dose dependent or resistant. Although the disk diffusion method is easy to use in clinical laboratories, it shows very poor agreement with the reference method for fluconazole and itraconazole against C. glabrata (8% and 14%, respectively).     

Aspergilosis pulmonar en un paciente crítico no inmunodeprimido

BackgroundIn non-immunocompromised patients admitted to intensive care departments or units (ICU), it is difficult to establish a definitive diagnosis of pulmonary aspergillosis because the signs and symptoms of this infectious disease are non-specific, and serological techniques are not very specific as well. For this reason, a diagnosis of possible pulmonary aspergillosis is initially established, and the starting of the treatment is controversial.Case reportAn immunocompetent subject had a work-related accident after a fall, which resulted in multiple injuries (head, thorax, lower extremities). The patient required mechanical ventilation since admission. On the second week of ICU admission, he showed a clinical presentation of respiratory infection with fever, purulent secretions, bilateral pulmonary infiltrates and repeated isolation of Mucor and Aspergillus fumigatus in bronchial secretions and pharyngeal swabs. The patient was treated with amphotericin B lipid complex and voriconazole with an excellent clinical and radiological outcome.ConclusionsCombined treatment of antifungal agents, in this case amphotericin B lipid complex and voriconazole, is a therapeutic possibility to be considered in patients who failed to respond to initial antifungal monotherapy.     

Clonal Expansion and Emergence of Environmental Multiple-Triazole-Resistant Aspergillus fumigatus Strains Carrying the TR34/L98H Mutations in the cyp51A Gene in India

Azole resistance is an emerging problem in Aspergillus which impacts the management of aspergillosis. Here in we report the emergence and clonal spread of resistance to triazoles in environmental Aspergillus fumigatus isolates in India. A total of 44 (7%) A. fumigatus isolates from 24 environmental samples were found to be triazole resistant. The isolation rate of resistant A. fumigatus was highest (33%) from soil of tea gardens followed by soil from flower pots of the hospital garden (20%), soil beneath cotton trees (20%), rice paddy fields (12.3%), air samples of hospital wards (7.6%) and from soil admixed with bird droppings (3.8%). These strains showed cross-resistance to voriconazole, posaconazole, itraconazole and to six triazole fungicides used extensively in agriculture. Our analyses identified that all triazole-resistant strains from India shared the same TR₃₄/L98H mutation in the cyp51 gene. In contrast to the genetic uniformity of azole-resistant strains the azole-susceptible isolates from patients and environments in India were genetically very diverse. All nine loci were highly polymorphic in populations of azole-susceptible isolates from both clinical and environmental samples. Furthermore, all Indian environmental and clinical azole resistant isolates shared the same multilocus microsatellite genotype not found in any other analyzed samples, either from within India or from the Netherlands, France, Germany or China. Our population genetic analyses suggest that the Indian azole-resistant A. fumigatus genotype was likely an extremely adaptive recombinant progeny derived from a cross between an azole-resistant strain migrated from outside of India and a native azole-susceptible strain from within India, followed by mutation and then rapid dispersal through many parts of India. Our results are consistent with the hypothesis that exposure of A. fumigatus to azole fungicides in the environment causes cross-resistance to medical triazoles. The study emphasises the need of continued surveillance of resistance in environmental and clinical A. fumigatus strains.     

Lethal destructive sinusopathy due to amphotericin B-resistant Aspergillus flavus: A case report

BackgroundThe prevalence of pulmonary aspergillosis and the importance of its early diagnosis are recognized. However, non-pulmonary involvement, including the sinuses region, is not frequently reported, and an infection in this area can affect all paranasal sinuses (pansinusopathy), being a rare pathology that affects immunocompromised hosts. Recent studies have highlighted the occurrence of Aspergillus flavus resistant to antifungal therapy. Therefore, a nasal sinus infection by resistant Aspergillus strains in immunocompromised patients may be linked to a high risk of lethality.Case reportWe are reporting a resistant A. flavus infection in an allogeneic hematopoietic stem cell transplant recipient with episodes of febrile neutropenia, and prolonged use of various antibacterial drugs and antifungal prophylaxis. The patient underwent brain magnetic resonance, which showed the presence of pansinusopathy, and presented necrosis in the left nasal region. Direct microscopic examination of a sample taken from the nasal mucosa revealed the presence of septate hyphae and conidiophores resembling those of A. flavus, that species being the identification achieved with MALDI-TOF MS. Antifungigram was performed by microdilution in broth (EUCAST-E.DEF. 9.3.2) and E-test, and resistance to amphotericin B was shown in both tests. The patient died after septic shock and hemorrhage.ConclusionsInvasive fungal infections due to amphotericin-B resistant A. flavus may lead to the death of the patient due to an ineffective therapeutic management. Therefore, antifungal susceptibility testing are of utmost importance for administering the proper treatment.     

A Case Report of Penile Infection Caused by Fluconazole- and Terbinafine-Resistant <Emphasis Type="Italic">Candida albicans</Emphasis>

Candida albicans is the most common pathogen that causes balanoposthitis. It often causes recurrence of symptoms probably due to its antifungal resistance. A significant number of balanitis Candida albicans isolates are resistant to azole and terbinafine antifungal agents in vitro. However, balanoposthitis caused by fluconazole- and terbinafine-resistant Candida albicans has rarely been reported. Here, we describe a case of a recurrent penile infection caused by fluconazole- and terbinafine-resistant Candida albicans, as well as the treatments administered to this patient. The isolate from the patient was tested for drug susceptibility in vitro. It was sensitive to itraconazole, voriconazole, clotrimazole and amphotericin B, but not to terbinafine and fluconazole. Thus, oral itraconazole was administrated to this patient with resistant Candida albicans penile infection. The symptoms were improved, and mycological examination result was negative. Follow-up treatment of this patient for 3 months showed no recurrence.     

Renal abscess due to Aspergillus fumigatus as the only sign of disseminated aspergillosis in a patient with AIDS

BackgroundAspergillus fumigatus can cause a wide variety of clinical syndromes, especially in the three largest immunocompromised groups, such as HIV-infected patients. Primary renal aspergillosis is an extremely rare entity.AimsWe report an unusual case of renal abscess due to Aspergillus fumigatus in a patient with AIDS.MethodsWe review clinical and laboratory records, and provide follow up of the patient.ResultsA 38-year-old man, HIV seropositive, was admitted to our hospital with fever, lumbar pain and respiratory symptoms. Abdominal ultrasound and computerised tomography showed a single and large lesion consistent with an abscess located in the left kidney. Aspergillus fumigatus was isolated from clinical sample obtained by ultrasound-guided needle aspiration. Despite a correct treatment based on amphotericin B and drainage of the abscess, surgery was necessary and nephrectomy was carried out. Histopathological examination of the surgical specimen confirmed the diagnosis of renal aspergillosis. Systemic antifungal therapy based on intravenous and oral voriconazole and highly active antiretroviral therapy was started after surgery. The patient had a good response to the established treatment and he remains in a good clinical condition at one year of follow up.ConclusionsCombined medical and surgical treatment is the elective therapy for renal abscesses due to Aspergillus when percutaneous drainage and the administration of systemic antifungal drugs, such as amphotericin B and/or oral voriconazole or itraconazole, fail. This case emphasizes renal fungal infections should be included in the differential diagnosis of kidney abscesses in AIDS patients.     

Azole Resistance in <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> in Patients with Cystic Fibrosis: A Matter of Concern?

Aspergillus fumigatus is the most frequent filamentous fungus isolated from respiratory specimens from patients with cystic fibrosis (CF). Triazoles are the most widely used antifungals in the treatment of allergic bronchopulmonary aspergillosis (ABPA) and invasive aspergillosis (IA) in CF patients. Treatment success could be severely compromised by the occurrence of azole-resistant A. fumigatus (ARAf), which is increasingly reported worldwide from both clinical samples and the environment. In previous studies, ARAf has been detected in up to 8% of CF patients. Isolates from CF patients requiring antifungal treatment should therefore be routinely subjected to antifungal susceptibility testing. The optimal treatment of ABPA or IA in CF patients with azole-resistant isolates has not been established; treatment options include liposomal amphotericin B i.v. and/or echinocandins i.v.     

Contribution of galactofuranose to the virulence of the opportunistic pathogen Aspergillus fumigatus

The filamentous fungus Aspergillus fumigatus is responsible for a lethal disease called invasive aspergillosis that affects immunocompromised patients. This disease, like other human fungal diseases, is generally treated by compounds targeting the primary fungal cell membrane sterol. Recently, glucan synthesis inhibitors were added to the limited antifungal arsenal and encouraged the search for novel targets in cell wall biosynthesis. Although galactomannan is a major component of the A. fumigatus cell wall and extracellular matrix, the biosynthesis and role of galactomannan are currently unknown. By a targeted gene deletion approach, we demonstrate that UDP-galactopyranose mutase, a key enzyme of galactofuranose metabolism, controls the biosynthesis of galactomannan and galactofuranose containing glycoconjugates. The glfA deletion mutant generated in this study is devoid of galactofuranose and displays attenuated virulence in a low-dose mouse model of invasive aspergillosis that likely reflects the impaired growth of the mutant at mammalian body temperature. Furthermore, the absence of galactofuranose results in a thinner cell wall that correlates with an increased susceptibility to several antifungal agents. The UDP-galactopyranose mutase thus appears to be an appealing adjunct therapeutic target in combination with other drugs against A. fumigatus. Its absence from mammalian cells indeed offers a considerable advantage to achieve therapeutic selectivity.     

Composite Survival Index to Compare Virulence Changes in Azole-Resistant Aspergillus fumigatus Clinical Isolates

Understanding resistance to antifungal agents in Aspergillus fumigatus is of increasing importance for the treatment of invasive infections in immunocompromised patients. Although a number of molecular resistance mechanisms are described in detail, the potential accompanying virulence changes and impact on clinical outcome have had little attention. We developed a new measure of survival, the composite survival index (CSI) to use as a measure of the virulence properties of A. fumigatus. Using a novel mathematical model we found a strong correlation between the in vitro growth characteristics and virulence in vivo expressed as CSI. Our model elucidates how three critical parameters (the lag phase (τ), decay constant (λ), and growth rate (ν)) interact with each other resulting in a CSI that correlated with virulence. Hence, strains with a long lag phase and high decay constant were less virulent in a murine model of invasive aspergillosis, whereas high virulence for isolates with a high CSI was associated in vitro with rapid growth and short lag phases. Resistant isolates with cyp51A mutations, which account for the majority of azole resistant aspergillosis cases, did not show a lower virulence compared to azole-susceptible isolates. In contrast, the CSI index revealed that a non-cyp51A-mediated resistance mechanism was associated with a dramatic decrease in CSI. Because of its predictive value, the mathematical model developed may serve to explore strain characteristics in vitro to predict virulence in vivo and significantly reduce the number of experimental animals required in such studies. The proposed measure of survival, the CSI can be used more in a general form in survival studies to explore optimal treatment options.     

TR34/L98H Mutation in <Emphasis Type="Italic">CYP51A</Emphasis> Gene in <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> Clinical Isolates During Posaconazole Prophylaxis: First Case in Korea

Azole resistance in Aspergillus fumigatus is an emerging problem, especially in immunocompromised patients. It has been reported worldwide, including in Asia, but has not yet been reported in Korea. Here, we report a case of invasive pulmonary aspergillosis (IPA) caused by azole-resistant A. fumigatus that developed in a hematopoietic stem cell transplantation recipient during posaconazole prophylaxis for immunosuppressive therapy of graft-versus-host diseases. We identified TR34/L98H/S297T/F495L mutation in the CYP51A gene of A. fumigatus clinical isolate obtained from bronchial washing fluid. Minimal inhibitory concentrations for itraconazole, voriconazole, and posaconazole were >?16, 1, and 4 μg/mL, respectively. While IPA improved partially under voriconazole treatment, the patient died from carbapenemase-producing Klebsiella pneumoniae bacteremia. Further epidemiological surveillance studies are warranted.     

An Approach to Suspected Invasive Fungal Infection in Patients with Hematologic Malignancy and HCT Recipients with Persistent Neutropenic Fever Despite Mold-Active Prophylaxis

Purpose of Review

This review offers an approach to managing suspected invasive fungal infection (IFI) in a febrile neutropenic patient with hematologic malignancy or hematopoietic cell transplantation (HCT) while on mold-active prophylaxis. We take into consideration host characteristics, new diagnostic tools, and available therapeutics.

Recent Findings

Despite use of anti-Aspergillus prophylactic agents, invasive aspergillosis is the most commonly reported IFI breaking through common prophylactic agents including the newest azole, isavuconazole. While more fungal diagnostic modalities are available, how to best incorporate them in the work-up of IFI remains unclear, while sensitivity of any particular fungal biomarker or molecular test is low.

Summary

In a febrile neutropenic patient with hematologic malignancy or HCT and suspected IFI, consider particularly invasive aspergillosis, regardless of the mold-active prophylactic agent. Early diagnosis and intervention are especially important to a favorable outcome; treatment is directed based on the suspected IFI syndrome and suspected organism. Switching azoles, consideration of combination therapy, and reducing immunosuppression are proposed strategies for the management of breakthrough IFI, while surgical debridement remains crucial for Mucormycoses. More study is needed into the optimal antifungal approach in these clinical scenarios. Meanwhile, therapeutic drug monitoring and attention to drug-drug interactions are encouraged.

     

Aspergillus felis sp. nov., an Emerging Agent of Invasive Aspergillosis in Humans,Cats, and Dogs

We describe a novel heterothallic species in Aspergillus section Fumigati, namely A. felis (neosartorya-morph) isolated from three host species with invasive aspergillosis including a human patient with chronic invasive pulmonary aspergillosis, domestic cats with invasive fungal rhinosinusitis and a dog with disseminated invasive aspergillosis. Disease in all host species was often refractory to aggressive antifungal therapeutic regimens. Four other human isolates previously reported as A. viridinutans were identified as A. felis on comparative sequence analysis of the partial β-tubulin and/or calmodulin genes. A. felis is a heterothallic mold with a fully functioning reproductive cycle, as confirmed by mating-type analysis, induction of teleomorphs within 7 to 10 days in vitro and ascospore germination. Phenotypic analyses show that A. felis can be distinguished from the related species A. viridinutans by its ability to grow at 45°C and from A. fumigatus by its inability to grow at 50°C. Itraconazole and voriconazole cross-resistance was common in vitro.     

The Application of Laser Microdissection in Molecular Detection and Identification of Aspergillus fumigatus from Murine Model of Acute Invasive Pulmonary Aspergillosis

Invasive aspergillosis (IA) is a major concern in patients with severe immune deficiency. As antifungal susceptibility varies in different fungal pathogens, accurate and timely identification of species is becoming imperative for guidance of therapy and reducing high mortality rates in patients with IA. But, in fact, the diagnosis is challenging and new validated techniques are required for the detection and identification of clinically relevant isolates. The laser capture microdissection (LCM) system enables analysis of cytologically and/or phenotypically defined cell types from heterogeneous tissue and has been used in diagnosis and fungal species identification in pulmonary aspergillosis of white storks. To establish the experimental foundation for clinical application of the system, we microdissected and collected Blankophor-stained single hyphal strands from tissue cryosections of murine model of invasive pulmonary aspergillosis (IPA) with A. fumigatus by LCM, subsequently processed for DNA extraction, PCR sequencing, and species molecular identification. The sensitivity of LCM–PCR sequencing was 89 % (89/100), and the specificity was 100 %. Moreover, the positive predictive value and negative predictive value were 100 and 78.43 %, respectively. The result approved that the LCM-based methods had the potential for accurately diagnosis and rapidly identification fungal pathogens of IPA.