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Abstract Antimicrobial resistance (AMR) is a major public health threat, reducing treatment options for infected patients. AMR is promoted by a lack of access to rapid antibiotic susceptibility tests (ASTs). Accelerated ASTs can identify effective antibiotics for treatment in a timely and informed manner. We describe a rapid growth-independent phenotypic AST that uses a nanomotion technology platform to measure bacterial vibrations. Machine learning techniques are applied to analyze a large dataset encompassing 2762 individual nanomotion recordings from 1180 spiked positive blood culture samples covering 364 Escherichia coli and Klebsiella pneumoniae isolates exposed to cephalosporins and fluoroquinolones. The training performances of the different classification models achieve between 90.5 and 100% accuracy. Independent testing of the AST on 223 strains, including in clinical setting, correctly predict susceptibility and resistance with accuracies between 89.5% and 98.9%. The study shows the potential of this nanomotion platform for future bacterial phenotype delineation.

ABSTRACTCandida parapsilosis is known to cause severe and persistent outbreaks in clinical settings. Patients infected with multidrug-resistant C. parapsilosis (MDR Cp) isolates were identified in a large Turkish hospital from 2017–2020. We subsequently identified three additional patients infected with MDR Cp isolates in 2022 from the same hospital and two echinocandin-resistant (ECR) isolates from a single patient in another hospital. The increasing number of MDR and ECR isolates contradicts the general principle that the severe fitness cost associated with these phenotypes could prevent their dominance in clinical settings. Here, we employed a multidimensional approach to systematically assess the fitness costs of MDR and ECR C. parapsilosis isolates. Whole-genome sequencing revealed a novel MDR genotype infecting two patients in 2022. Despite severe in vitro defects, the levels and tolerances of the biofilms of our ECR and MDR isolates were generally comparable to those of susceptible wild-type isolates. Surprisingly, the MDR and ECR isolates showed major alterations in their cell wall components, and some of the MDR isolates consistently displayed increased tolerance to the fungicidal activities of primary human neutrophils and were more immunoevasive during exposure to primary human macrophages. Our systemic infection mouse model showed that MDR and ECR C. parapsilosis isolates had comparable fungal burden in most organs relative to susceptible isolates. Overall, we observed a notable increase in the genotypic diversity and frequency of MDR isolates and identified MDR and ECR isolates potentially capable of causing persistent outbreaks in the future.

Abstract The aromatic amino acid L-tryptophan (Trp) is essentially metabolized along the host and microbial pathways. While much is known about the role played by downstream metabolites of each pathways in intestinal homeostasis, their role in lung immune homeostasis is underappreciated. Here we have examined the role played by the Trp hydroxylase/5-hydroxytryptamine (5-HT) pathway in calibrating host and microbial Trp metabolism during Aspergillus fumigatus pneumonia. We found that 5-HT produced by mast cells essentially contributed to pathogen clearance and immune homeostasis in infection by promoting the host protective indoleamine-2,3-dioxygenase 1/kynurenine pathway and limiting the microbial activation of the indole/aryl hydrocarbon receptor pathway. This occurred via regulation of lung and intestinal microbiota and signaling pathways. 5-HT was deficient in the sputa of patients with Cystic fibrosis, while 5-HT supplementation restored the dysregulated Trp partitioning in murine disease. These findings suggest that 5-HT, by bridging host-microbiota Trp partitioning, may have clinical effects beyond its mood regulatory function in respiratory pathologies with an inflammatory component.

Candidemia is a major cause of morbidity and mortality in health care settings, and its epidemiology is changing. In the last two decades, the proportion of non-albicans Candida (NAC) yeasts in candidemia has increased. These yeasts more often display resistance to common antifungals. In many western countries, candidemia is mainly caused by susceptible C. albicans, while in resource-limited countries, including Iran, the candidemia species distribution is studied less often. Here, we investigated the species distribution, resistance levels, and characteristics of patients with candidemia in five hospitals in Mashhad (northeast Iran) for two years (2019–2021). Yeast isolates from blood were identified with MALDI-TOF MS and subjected to antifungal susceptibility testing (AFST) using the broth microdilution method, while molecular genotyping was applied to Candida parapsilosis isolates. In total, 160 yeast isolates were recovered from 160 patients, of which the majority were adults (60%). Candidemia was almost equally detected in men (48%) and women (52%). Almost half of patients (n = 67, 49%) were from intensive care units (ICUs). C. parapsilosis (n = 58, 36%) was the most common causative agent, surpassing C. albicans (n = 52, 33%). The all-cause mortality rate was 53%, with C. albicans candidemia displaying the lowest mortality with 39%, in contrast to a mortality rate of 59% for NAC candidemia. With microbroth AFST, nearly all tested isolates were found to be susceptible, except for one C. albicans isolate that was resistant to anidulafungin. By applying short tandem repeat (STR) genotyping to C. parapsilosis, multiple clusters were found. To summarize, candidemia in Mashhad, Iran, from 2019 to 2021, is characterized by common yeast species, in particular C. parapsilosis, for which STR typing indicates potential nosocomial transmission. The overall mortality is high, while resistance rates were found to be low, suggesting that the high mortality is linked to limited diagnostic options and insufficient medical care, including the restricted use of echinocandins as the first treatment option.

Summary: Complement is a fundamental innate immune response component. Its alterations are associated with severe systemic diseases. To illuminate the complement’s genetic underpinnings, we conduct genome-wide association studies of the functional activity of the classical (CP), lectin (LP), and alternative (AP) complement pathways in the Cooperative Health Research in South Tyrol study (n = 4,990). We identify seven loci, encompassing 13 independent, pathway-specific variants located in or near complement genes (CFHR4, C7, C2, MBL2) and non-complement genes (PDE3A, TNXB, ABO), explaining up to 74% of complement pathways’ genetic heritability and implicating long-range haplotypes associated with LP at MBL2. Two-sample Mendelian randomization analyses, supported by transcriptome- and proteome-wide colocalization, confirm known causal pathways, establish within-complement feedback loops, and implicate causality of ABO on LP and of CFHR2 and C7 on AP. LP causally influences collectin-11 and KAAG1 levels and the risk of mouth ulcers. These results build a comprehensive resource to investigate the role of complement in human health.

ABSTRACT Small colony variants are relatively common among some bacterial species and are associated with poor prognosis and recalcitrant infections. Similarly, Candida glabrata—a major intracellular fungal pathogen—produces small and slow-growing respiratory-deficient colonies, termed “petite.” Despite reports of clinical petite C. glabrata strains, our understanding of petite behavior in the host remains obscure. Moreover, controversies exist regarding in-host petite fitness and its clinical relevance. Herein, we employed whole-genome sequencing (WGS), dual-RNAseq, and extensive ex vivo and in vivo studies to fill this knowledge gap. WGS identified multiple petite-specific mutations in nuclear and mitochondrially encoded genes. Consistent with dual-RNAseq data, petite C. glabrata cells did not replicate inside host macrophages and were outcompeted by their non-petite parents in macrophages and in gut colonization and systemic infection mouse models. The intracellular petites showed hallmarks of drug tolerance and were relatively insensitive to the fungicidal activity of echinocandin drugs. Petite-infected macrophages exhibited a pro-inflammatory and type I IFN-skewed transcriptional program. Interrogation of international C. glabrata blood isolates (n = 1000) showed that petite prevalence varies by country, albeit at an overall low prevalence (0%–3.5%). Collectively, our study sheds new light on the genetic basis, drug susceptibility, clinical prevalence, and host-pathogen responses of a clinically overlooked phenotype in a major fungal pathogen. Importance Candida glabrata is a major fungal pathogen, which is able to lose mitochondria and form small and slow-growing colonies, called “petite.” This attenuated growth rate has created controversies and questioned the clinical importance of petiteness. Herein, we have employed multiple omics technologies and in vivo mouse models to critically assess the clinical importance of petite phenotype. Our WGS identifies multiple genes potentially underpinning petite phenotype. Interestingly, petite C. glabrata cells engulfed by macrophages are dormant and, therefore, are not killed by the frontline antifungal drugs. Interestingly, macrophages infected with petite cells mount distinct transcriptomic responses. Consistent with our ex vivo observations, mitochondrial-proficient parental strains outcompete petites during systemic and gut colonization. Retrospective examination of C. glabrata isolates identified petite prevalence a rare entity, which can significantly vary from country to country. Collectively, our study overcomes the existing controversies and provides novel insights regarding the clinical relevance of petite C. glabrata isolates.

IntroductionTo explore whether the reported lower pathogenicity in infected individuals of variant of concern (VoC) Omicron and its current subvariants compared to VoC Delta may be related to fundamental differences in the initial virus-tissue interaction, we assessed their ability to penetrate, replicate and cause damage in a human 3D respiratory model.MethodsFor this, we used TEER measurements, real-time PCR, LDH, cytokine and complex confocal imaging analyses.Results and discussionWe observed that Delta readily penetrated deep into the respiratory epithelium and this was associated with major tissue destruction, high LDH activity, high viral loads and pronounced innate immune activation as observed by intrinsic C3 activation and IL-6 release at infection sites. In contrast, Omicron subvariants BA.5, BQ.1.1 and BF7 remained superficially in the mucosal layer resulting merely in outward-directed destruction of cells, maintenance of epithelial integrity, minimal LDH activity and low basolateral release of virus at infection sites, as well as significantly smaller areas of complement activation and lower IL-6 secretion. Interestingly, also within Omicron subvariants differences were observed with newer Omicron subvariants BQ.1.1 and BF.7 illustrating significantly reduced viral loads, IL-6 release and LDH activity compared to BA.5. Our data indicate that earliest interaction events after SARS-CoV-2 transmission may have a role in shaping disease severity.

ABSTRACT Currently, SARS-CoV-2 Omicron BA.5 subvariants BF.7 and BQ.1.1 are rapidly emerging worldwide. To evaluate the SARS-CoV-2-neutralizing capacity of sera and saliva from triple vaccinated individuals, either boosted with an adapted bivalent COVID-19 vaccine or recovered from BA.4/BA.5 infection, we analyzed the sensitivity of replication-competent SARS-CoV-2 Omicron subvariants BA.4/5, BQ.1.1 and BF.7 to neutralization. Analysis of SARS-CoV-2-specific IgGs and IgAs showed increased serum IgG titers in the vaccinated group, while the serum and salivary IgA levels were comparable. Similar and efficient serum neutralization against the ancestral strain of SARS-CoV-2 and Omicron BA.4/BA.5 was detected in both cohorts, but critically reduced for BQ.1.1 and BF.7. In contrast, salivary neutralization against BA.4/BA.5 was increased in the convalescent compared to the vaccinated group, while salivary neutralizing capacity against BQ.1.1 and BF.7 was comparable in these groups. Further, personalized protective effects studied in a human 3D respiratory model revealed the importance of salivary protection against different Omicron subvariants. IMPORTANCE In BA.4/BA.5-convalescent versus vaccinated groups, salivary neutralization capacity increased against SARS-CoV-2 Omicron BA.4/BA.5. In contrast, it neutralized novel Omicron subvariants BQ.1.1 and BF.7 similarly. Salivary protection against various Omicron subvariants was even more evident when tested in a personalized approach using highly differentiated respiratory human 3D models.

Pancreatic ductal adenocarcinoma (PDAC) is still hampered by a dismal prognosis. A better understanding of the tumor microenvironment within the pancreas and of the factors affecting its composition is of utmost importance for developing new diagnostic and treatment tools. In this context, the complement system plays a prominent role. Not only has it been shown to shape a T cell-mediated immune response, but it also directly affects proliferation and apoptosis of the tumor cells, influencing angiogenesis, metastatic spread and therapeutic resistance. This makes complement proteins appealing not only as early biomarkers of PDAC development, but also as therapeutic targets. Fungal dysbiosis is currently the new kid on the block in tumorigenesis with cancer-associated mycobiomes extracted from several cancer types. For PDAC, colonization with the yeast Malassezia seems to promote cancer progression, already in precursor lesions. One responsible mechanism appears to be complement activation via the lectin pathway. In the present article, we review the role of the complement system in tumorigenesis, presenting observations that propose it as the missing link between fungal dysbiosis and PDAC development. We also present the results of a small pilot study supporting the crucial interplay between the complement system and Malassezia colonization in PDAC pathogenesis.

ABSTRACT Rising breakthrough infections with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron BA.4/5 led to the performance of various studies investigating systemic immunity and neutralizing antibodies in sera, but mucosal immunity remains understudied. In this cohort study, the humoral immune responses, including immunoglobulin levels and the presence of virus-neutralizing antibodies, of 92 vaccinated and/or BA.1/BA.2 convalescent individuals were investigated. Cohorts received two doses of ChAdOx1, BNT162b2, or mRNA-1273 and subsequent booster vaccination with either BNT162b2 or mRNA-1273, following BA.1/BA.2 infection. In addition, vaccinated and nonconvalescent or unvaccinated and BA.1 convalescent individuals were studied. Serum and saliva samples were used to determine SARS-CoV-2 spike-specific IgG and IgA titers and neutralizing activity against replication-competent SARS-CoV-2 wild-type virus and the Omicron BA.4/5 variant. Vaccinated/convalescent cohorts demonstrated strongest neutralization against BA.4/5, with 50% neutralization titer (NT50) values reaching 174.2; however, neutralization was reduced up to 11-fold, compared to wild-type virus. Both BA.1 convalescent and vaccinated nonconvalescent cohorts displayed the weakest neutralization against BA.4/5, with NT50 values being reduced to 4.6, accompanied by lower numbers of positive neutralizers. Additionally, salivary neutralization against wild-type virus was strongest in vaccinated and BA.2 convalescent subjects, but this elevated neutralization efficiency was lost when challenged with BA.4/5. Our data support the contention that current coronavirus disease 2019 (COVID-19) vaccines efficiently induce humoral immunity. However, antiviral effectiveness in serum and saliva is greatly reduced against novel variants of concern. These results suggest an adjustment of current vaccine strategies to an adapted or alternative vaccine delivery, such as mucosal booster vaccinations, which might establish enhanced or even sterilizing immunity against novel SARS-CoV-2 variants. IMPORTANCE Rising incidences of breakthrough infections caused by SARS-CoV-2 Omicron BA.4/5 have been observed. Although various studies were conducted investigating neutralizing antibodies in sera, mucosal immunity was barely evaluated. Here, we investigated mucosal immunity, since the presence of neutralizing antibodies at mucosal entry sites plays a fundamental role in disease limitation. We found strong induction of serum IgG/IgA, salivary IgA, and neutralization against SARS-CoV-2 wild-type virus in vaccinated/convalescent subjects but detected 10-fold reduced (albeit positive) serum neutralization against BA.4/5. Interestingly, vaccinated and BA.2 convalescent patients demonstrated the greatest serum neutralization against BA.4/5, but this advantageous neutralizing effect was not observed in the saliva. Our data support the contention that current COVID-19 vaccines are very efficient against severe/critical disease progression. Moreover, these results suggest an adjustment of the current vaccine strategy to adapted and alternative vaccine delivery, such as mucosal booster vaccinations, to establish robust sterilizing immunity against novel SARS-CoV-2 variants.

BackgroundThe role of respiratory viruses in chronic otitis media with effusion (COME) in children is not clearly defined. In our study we aimed to investigate the detection of respiratory viruses in middle ear effusions (MEE) as well as the association with local bacteria, respiratory viruses in the nasopharynx and cellular immune response of children with COME.MethodsThis 2017–2019 cross-sectional study included 69 children aged 2–6 undergoing myringotomy for COME. MEE and nasopharyngeal swabs were analyzed via PCR and CT-values for the genome and loads of typical respiratory viruses. Immune cell populations and exhaustion markers in MEE related to respiratory virus detection were studied via FACS. Clinical data including the BMI was correlated.ResultsRespiratory viruses were detected in MEE of 44 children (64%). Rhinovirus (43%), Parainfluenzavirus (26%) and Bocavirus (10%) were detected most frequently. Average Ct values were 33.6 and 33.5 in MEE and nasopharynx, respectively. Higher detection rates correlated with elevated BMI. Monocytes were elevated in MEE (9.5 ± 7.3%/blood leucocytes). Exhaustion markers were elevated on CD4+ and CD8+ T cells and monocytes in MEE.ConclusionRespiratory viruses are associated with pediatric COME. Elevated BMI was associated with increased rates of virus associated COME. Changes in cell proportions of innate immunity and expression of exhaustion markers may be related to chronic viral infection.

Wastewater-based epidemiology (WBE) is an effective approach for tracking information on spatial distribution and temporal trends of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the community level. Herein, the development, implementation, and operation of the wastewater monitoring program serving Tyrol – a federal province of Austria – are described. The development of this program was initiated by Tyrolean health authorities at the end of the first phase of the Coronavirus disease 2019 (COVID-19) pandemic (May 2020). In close co-operation with the water sector and academic institutions, efficient and effective workflows and processes for wastewater surveillance were established. The monitoring program went into operation in November 2020. By the end of July 2021, a total of 5,270 wastewater influent samples collected at 43 sites were analyzed. The monitoring program provided valuable insights into the development of the pandemic situation in Tyrol and fulfilled several tasks that are of importance in different phases of the pandemic. It represented an early-warning system, provided independent confirmation of temporal trends in COVID-19 prevalence, enabled the assessment of the effectiveness of measures, alerted about bursts of disease activity, and provided evidence for the absence of COVID-19. These findings underline the importance of establishing national wastewater monitoring programs as a complementary source of information for efficient and effective pandemic management. HIGHLIGHTS The development, implementation, and operation of the Tyrolean SARS-CoV-2 wastewater monitoring program are described.; Health authorities are receiving regular updates on the pandemic situation in 43 Tyrolean regions.; Turnaround times are

The rare, but emerging mold Aspergillus terreus is an important pathogen in some geographical areas, like Tyrol (Austria) and Houston (Texas). The reason for this high prevalence is unknown. The present serosurveillance study aimed to evaluate the trends in levels of A. terreus-specific IgG antibodies in various regions of Tyrol and to compare the results to the environmental spread of A. terreus in Tyrol. Therefore, 1058 serum samples from healthy blood donors were evaluated. Data revealed a significant difference between the Tyrolean Upland and Lowland. Moreover, female participants had higher A. terreus IgG antibody levels than male participants. The differences found in our study are consistent with the distributional differences in environmental and clinical samples described in previous studies, supporting that A. terreus IgG antibody levels reflect the environmental epidemiology of A. terreus in Tyrol.

There is an ongoing effort to optimize and revise antifungal susceptibility testing (AFST) methods due to the rising number of fungal infections and drug-resistant fungi. The rising antifungal resistance within Candida and Aspergillus species, which are common contributors to invasive fungal infections (IFIs), is a cause for concern, prompting an expanding integration of in vitro AFST to guide clinical decisions. To improve the relevance of in vitro AFST results to therapy outcomes, influential factors should be taken into account. The tested medium is one of several factors that could affect the results of AFST. The present study evaluated the effect of two complex media (Sabouraud dextrose and Columbia) versus the standard defined medium (RPMI 1640) on the AFST results of amphotericin B, posaconazole, and voriconazole against Candida spp. and Aspergillus spp. representatives, utilizing the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and the Etest methods. Overall, Candida species exhibited higher variability in minimum inhibitory concentration (MIC) across different media (more than three log2 dilutions) comparing to Aspergillus spp., while quality control isolates showed consistency regardless of tested media, antifungals, and methods. When comparing tested methods, MIC variation was mostly detected using EUCAST than it was using Etest.

ObjectivesThe identification of the SARS-CoV-2 Omicron variants BA.1 and BA.2 immediately raised concerns about the efficacy of currently used monoclonal antibody therapies. Here, we analyzed the activity of Sotrovimab and Regdanvimab, which are used in clinics for treatment of moderate to severe SARS-CoV-2 infections, and Cilgavimab/Tixagevimab, which are approved for prophylactic use, against BA.1 and BA.2 in a 3D model of primary human bronchial epithelial cells.MethodsPrimary human airway epithelia (HAE) cells in a 3D tissue model were infected with clinical isolates of SARS-CoV-2 Delta, BA.1 or BA.2. To mimic the therapeutic use of mAbs, we added Regdanvimab, Sotrovimab or Cilgavimab/Tixagevimab 6 h after infection. In order to mirror the prophylactic use of Cilgavimab/Tixagevimab, we added this compound 6 h prior to infection to the fully differentiated, pseudostratified epithelia cultured in air-liquid interphase (ALI).ResultsWe observed that Sotrovimab, but not Regdanvimab, is active against BA.1; however, both antibodies lose their efficacy against BA.2. In contrast, we found that BA.2 was sensitive to neutralization by the approved prophylactic administration and the therapeutic use, which is not yet permitted, of Cilgavimab/Tixagevimab.ConclusionImportantly, while the use of Tixagevimab/Cilgavimab is effective in controlling BA.2 but not BA.1 infection, monoclonal antibodies (mAbs) with efficacy against BA.1 are ineffective to reduce BA.2 virus replication in a human lung model. Our data may have implications on the variant specific clinical use of monoclonal antibodies.

The epidemiology of invasive fungal infections (IFIs) is currently changing, driven by aggressive immunosuppressive therapy, leading to an expanded spectrum of patients at risk of IFIs. Aspergillosis is a leading cause of IFIs, which usually affects immunocompromised patients. There are a limited number of antifungal medications available for treating IFIs, and their effectiveness is often hindered by rising resistance rates and practical limitations. Consequently, new antifungals, especially those with novel mechanisms of action, are increasingly required. This study assessed the activity of four novel antifungal agents with different mechanisms of activity, namely, manogepix, rezafungin, ibrexafungerp, and olorofim, against 100 isolates of Aspergillus section Terrei, containing amphotericin-B (AmB)-wildtype/non-wildtype and azole-susceptible/-resistant strains, according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) method. In general, all tested agents showed potent and consistent activity against the tested isolates, exhibiting geometric mean (GM) and minimum effective concentration (MEC)/minimum inhibitory concentration (MIC) ranges, respectively, as follows: manogepix (0.048 mg/L, 0.032–0.5 mg/L), rezafungin (0.020 mg/L, 0.016–0.5 mg/L), ibrexafungerp (0.071 mg/L, 0.032–2 mg/L), and olorofim (0.008 mg/L, 0.008–0.032 mg/L). In terms of MIC90/MEC90, olorofim had the lowest values (0.008 mg/L), followed by rezafungin (0.032 mg/L), manogepix (0.125 mg/L), and ibrexafungerp (0.25 mg/L). All the antifungals tested demonstrated promising in vitro activity against Aspergillus section Terrei, including A. terreus as well as azole-resistant and AmB-non-wildtype cryptic species.

BackgroundResidents of nursing homes are one of the most vulnerable groups during the severe acute syndrome coronavirus 2 (SARS-CoV-2) pandemic. The aim of this study was to characterize cellular and humoral immune responses in >70-year-old participants before vaccination, after first and second vaccination with BNT162b2, in contrast to second-dose-vaccinated participants younger than 60 years.MethodsPeripheral blood mononuclear cells of 45 elderly and 40 younger vaccinees were analyzed by IFNγ ELISpot, specific immunoglobulin G antibody titers against SARS-CoV-2 spike protein, and neutralization abilities against SARS-CoV-2 wild-type (WT) and Delta variant (B.1.617.2).ResultsOur results clearly demonstrate a significantly increased T cell response, IgG titers, and neutralization activities against SARS-CoV-2 WT and Delta between first and second vaccination with BNT162b2 in elderly vaccinees, thereby highlighting the importance of the second booster. Interestingly, similar cellular and humoral immune responses against SARS-CoV-2 WT and Delta were found after the second vaccine dose in the young and elderly groups.ConclusionsOur data demonstrate a full picture of cellular and humoral immune responses of BNT162b2-vaccinees in two age cohorts. In all vaccines, SARS-CoV-2 WT-specific antibodies with similar neutralizing activity were detected in all vaccinees. After the second vaccination, neutralization titers against SARS-CoV-2 Delta were impaired in both age groups compared with SARS-CoV-2 WT, thereby emphasizing the need for an additional booster to overcome rising variants of SARS-CoV-2.

With increasing frequency, clinical and laboratory-based mycologists are consulted on invasive fungal diseases caused by rare fungal species. This review aims to give an overview of the management of invasive aspergillosis (IA) caused by non-fumigatus Aspergillus spp.—namely A. flavus, A. terreus, A. niger and A. nidulans—including diagnostic and therapeutic differences and similarities to A. fumigatus. A. flavus is the second most common Aspergillus spp. isolated in patients with IA and the predominant species in subtropical regions. Treatment is complicated by its intrinsic resistance against amphotericin B (AmB) and high minimum inhibitory concentrations (MIC) for voriconazole. A. nidulans has been frequently isolated in patients with long-term immunosuppression, mostly in patients with primary immunodeficiencies such as chronic granulomatous disease. It has been reported to disseminate more often than other Aspergillus spp. Innate resistance against AmB has been suggested but not yet proven, while MICs seem to be elevated. A. niger is more frequently reported in less severe infections such as otomycosis. Triazoles exhibit varying MICs and are therefore not strictly recommended as first-line treatment for IA caused by A. niger, while patient outcome seems to be more favorable when compared to IA due to other Aspergillus species. A. terreus-related infections have been reported increasingly as the cause of acute and chronic aspergillosis. A recent prospective international multicenter surveillance study showed Spain, Austria, and Israel to be the countries with the highest density of A. terreus species complex isolates collected. This species complex seems to cause dissemination more often and is intrinsically resistant to AmB. Non-fumigatus aspergillosis is difficult to manage due to complex patient histories, varying infection sites and potential intrinsic resistances to antifungals. Future investigational efforts should aim at amplifying the knowledge on specific diagnostic measures and their on-site availability, as well as defining optimal treatment strategies and outcomes of non-fumigatus aspergillosis.

Providing timely antifungal treatment to patients suffering from life-threatening invasive fungal infections (IFIs) is essential. Due to the changing epidemiology and the emergence of antifungal resistance in Aspergillus, the most commonly responsible mold of IFIs, antifungal susceptibility testing (AFST) has become increasingly important to guide clinical decisions. This study assessed the essential agreement (EA) between broth microdilution methods (the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST)) and the Etest of amphotericin B (AmB), liposomal amphotericin B (L-AmB), and isavuconazole (ISA) against 112 Aspergillus section Terrei. An EA within ±2 dilutions of ≥90% between the two methods was considered acceptable. Excellent EA was found between EUCAST and CLSI of AmB and ISA (98.2% and 95.5%, respectively). The correlation of Etest results and EUCAST/CLSI was not acceptable (

The world is involved with a pandemic coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2. The clinical manifestations of reported COVID-19-associated pulmonary impairments range from asymptomatic infections to a pneumonia-induced acute respiratory distress syndrome that requires mechanical ventilation. Fungal superinfections complicating the clinical course remain underexplored. Angiotensin-converting enzyme 2, the receptor for COVID-19 that is mainly expressed in airway epithelia and lung parenchyma, is considered an important regulator of innate immunity. With regard to the viral-cell interaction, imbalanced immune regulation between protective and altered responses caused by the exacerbation of inflammatory responses should be considered a major contributor to secondary pulmonary aspergillosis. In addition, the complex inherited factors, age-related changes, and lifestyle may also affect immune responses. The complication and persistence of invasive aspergillosis have been well described in patients with severe influenza or COVID-19. However, there is a scarcity of information about the immunological mechanisms predisposing patients with COVID-19 to fungal co-infections. Therefore, this study was conducted to investigate the aforementioned domain.

Abstract Background Despite being associated with a high mortality and economic burden, data regarding candidemia are scant in Algeria. The aim of this study was to unveil the epidemiology of candidemia in Algeria, evaluate the antifungal susceptibility pattern of causative agents and understand the molecular mechanisms of antifungal resistance where applicable. Furthermore, by performing environmental screening and microsatellite typing we sought to identify the source of infection. Methods We performed a retrospective epidemiological-based surveillance study and collected available blood yeast isolates recovered from the seven hospitals in Algiers. To identify the source of infection, we performed environmental screening from the hands of healthcare workers (HCWs) and high touch areas. Species identification was performed by API Auxa-Color and MALDI-TOF MS and ITS sequencing was performed for species not reliably identified by MALDI-TOF MS. Antifungal susceptibility testing followed CLSI M27-A3/S4 and included all blood and environmental yeast isolates. ERG11 sequencing was performed for azole-resistant Candida isolates. Microsatellite typing was performed for blood and environmental Candida species, where applicable. Results Candida tropicalis (19/66) was the main cause of candidemia in these seven hospitals, followed by Candida parapsilosis (18/66), Candida albicans (18/66), and Candida glabrata (7/66). The overall mortality rate was 68.6% (35/51) and was 81.2% for C. tropicalis-infected patients (13/16). Fluconazole was the main antifungal drug used (12/51); 41% of the patients (21/51) did not receive any systemic treatment. Candida parapsilosis was isolated mainly from the hands of HCWs (7/28), and various yeasts were collected from high-touch areas (11/47), including Naganishia albida, C. parapsilosis and C. glabrata. Typing data revealed interhospital transmission on two occasions for C. parapsilosis and C. glabrata, and the same clone of C. parapsilosis infected two patients within the same hospital. Resistance was only noted for C. tropicalis against azoles (6/19) and fluconazole-resistant C. tropicalis isolates (≥8 μg/ml) (6/19) contained a novel P56S (5/6) amino acid substitution and a previously reported one (V234F; 1/6) in Erg11p. Conclusions Collectively, our data suggest an urgent need for antifungal stewardship and infection control strategies to improve the clinical outcome of Algerian patients with candidemia. The high prevalence of C. tropicalis joined by fluconazole-resistance may hamper the therapeutic efficacy of fluconazole, the frontline antifungal drug used in Algeria.

Objectives: Current knowledge on infections caused by Scedosporium spp. and Lomentospora prolificans in children is scarce. We therefore aim to provide an overview of risk groups, clinical manifestation and treatment strategies of these infections. Methods: Pediatric patients (age ≤18 years) with proven/probable Scedosporium spp. or L. prolificans infection were identified in PubMed and the FungiScope® registry. Data on diagnosis, treatment and outcome were collected. Results: Fifty-five children (median age 9 years [IQR: 5–14]) with invasive Scedosporium spp. (n = 33) or L. prolificans (n = 22) infection were identified between 1990 and 2019. Malignancy, trauma and near drowning were the most common risk factors. Infections were frequently disseminated. Most patients received systemic antifungal therapy, mainly voriconazole and amphotericin B, plus surgical treatment.Overall, day 42 mortality was 31%, higher for L. prolificans (50%) compared to Scedosporium spp. (18%). L. prolificans infection was associated with a shorter median survival time compared to Scedosporium spp. (6 days [IQR: 3–28] versus 61 days [IQR: 16–148]). Treatment for malignancy and severe disseminated infection were associated with particularly poor outcome (HR 8.33 [95% CI 1.35–51.40] and HR 6.12 [95% CI 1.52–24.66], respectively). Voriconazole use at any time and surgery for antifungal treatment were associated with improved clinical outcome (HR 0.33 [95% CI 0.11–0.99] and HR 0.09 [95% CI 0.02–0.40], respectively). Conclusions: Scedosporium spp. and L. prolificans infections in children are associated with high mortality despite comprehensive antifungal therapy. Voriconazole usage and surgical intervention are associated with successful outcome. Keywords: Scedosporium spp., Lomentospora prolificans, Invasive fungal infections, Pediatric patients, Children, Voriconazole, Surgery, FungiScope® registry

Background: Mucormycetes, a heterogeneous group of fungi, induce a life-threatening disease called mucormycosis. Immune deficiencies represent a major risk factor; hence, we wanted to illuminate the role of complement and platelets in the defense against mucormycetes. Methods: Rhizopus arrhizus (Ra), Rhizopus microsporus (Rm), Lichtheimia ramosa (Lr), Lichtheimia corymbifera (Lc), Rhizomucor pusillus (Rmp), and Mucor circinelloides (Mc) spores were opsonized with human and mouse serum, and C1q, C3c, and terminal complement complex (C5b-9) deposition was measured. Additionally, thrombocytopenic, C3-deficient, or C6-deficient mice were intravenously infected with selected isolates. Survival and immunological parameters were monitored, and fungal burden was determined and compared to that of immunocompetent and neutropenic mice. Results: In vitro experiments showed significant differences in complement deposition between mucormycetes. Mc isolates bound up to threefold more human C5b-9 than other mucormycetes. Lr, Lc, and Mc bound high levels of murine C3c, whereas human C3c deposition was reduced on Mc compared to Lr and Lc. Murine C3c deposition negatively correlated with virulence. Complement deficiencies and neutropenia, but not thrombocytopenia, were shown to be a risk factor for a lethal outcome. Conclusion: Complement deposition varies between mucormycetes. Additionally, we demonstrated that complement and neutrophilic granulocytes, but not platelets, play an important role in a murine model of disseminated mucormycosis.

(1) Background: Coronavirus disease 2019 (COVID-19)-associated pulmonary aspergillosis (CAPA) raises concerns to contribute to an increased mortality. The incidence of CAPA varies widely within hospitals and countries, partly because of difficulties in obtaining a reliable diagnosis. (2) Methods: Here, we assessed Aspergillus culture-positive and culture-negative respiratory tract specimens via direct fungal microscopy (gold standard) and compared the results with galactomannan enzyme immunoassay (GM-EIA) and Aspergillus PCR. (3) Results: 241 respiratory samples from patients suffering from SARS-CoV-2 pneumonia were evaluated. Results showed both diagnostic tools, Aspergillus PCR and GM-EIA, to be positive or negative displaying a sensitivity of 0.90, a specificity of 0.77, a negative predictive value (NPV) of 0.95, and a positive predictive value (PPV) of 0.58 in Aspergillus sp. culture and microscopic-positive specimens. Non-bronchoalveolar lavage (BAL) samples, obtained within a few days from the same patient, showed a high frequency of intermittent positive or negative GM-EIA or Aspergillus PCR results. Positivity of a single biomarker is insufficient for a proper diagnosis. A broad spectrum of Aspergillus species was detected. (4) Conclusions: Our study highlights the challenges of combined biomarker testing as part of diagnosing CAPA. From the results presented, we highly recommend the additional performance of direct microscopy in respiratory specimens to avoid overestimation of fungal infections by applying biomarkers.

Importantly, our study highlights an unusual target on DCs—the α chain of complement receptor 4 (CR4) (CD11c)—for therapeutic interventions in HIV-1 treatment. Targeting CD11c on DCs mediated a potent antiviral immune response via clustering of CR4 and CCR5 and subsequent opening of an antiviral recognition pathway in DCs via MAVS.

T cells play a fundamental role in the early control and clearance of many viral infections of the respiratory system. In SARS-CoV-2-infected individuals, lymphopenia with drastically reduced CD4+ and CD8+ T cells correlates with Coronavirus disease 2019 (COVID-19)-associated disease severity and mortality. In this study, we characterized cellular and humoral immune responses induced in patients with mild, severe and critical COVID-19. Peripheral blood mononuclear cells of 37 patients with mild, severe and critical COVID-19 and 10 healthy individuals were analyzed by IFNγ ELISpot and multi-color flow cytometry upon stimulation with peptide pools covering complete immunodominant SARS-CoV-2 matrix, nucleocapsid and spike proteins. In addition SARS-CoV-2 antibody levels, neutralization abilities and anaphylatoxin levels were evaluated by various commercially available ELISA platforms. Our data clearly demonstrates a significantly stronger induction of SARS-CoV-2 specific CD8+ T lymphocytes and higher IFNγ production in patients with mild compared to patients with severe or critical COVID-19. In all patients SARS-CoV-2-specific antibodies with similar neutralizing activity were detected, but highest titers of total IgGs were observed in critical patients. Finally, elevated anaphylatoxin C3a and C5a levels were identified in severe and critical COVID-19 patients probably caused by aberrant immune complex formation due to elevated antibody titers in these patients. Crucially, we provide a full picture of cellular and humoral immune responses of COVID-19 patients and prove that robust polyfunctional CD8+ T cell responses concomitant with low anaphylatoxin levels correlate with mild infections. In addition, our data indicates that high SARS-CoV-2 antibody titers are associated with severe disease progression.

Complement system and dendritic cells (DCs) form – beside neutrophils and macrophages – the first line of defense to combat fungal infections. Therefore, we here studied interactions of these first immune elements with Aspergillus fumigatus lacking ß-1,3-glucans (fks1tetOnrep under repressed conditions) to mechanistically explain the mode of action of echinocandins in more detail. Echinocandins are cell wall active agents blocking β-glucan synthase, making the A. fumigatus fks1tetOn mutant a good model to study immune-modulatory actions of these drugs. We now demonstrate herein, that complement was activated to significantly higher levels by the fks1-deficient strain compared to its respective wild type. This enhanced covalent linking of complement fragments to the A. fumigatus fks1tetOnrep mutant further resulted in enhanced DC binding and internalization of the fungus. Additionally, we found that fks1tetOnrep induced a Th1-/Th17-polarizing cytokine profile program in DCs. The effect was essentially dependent on massive galactomannan shedding, since blocking of DC-SIGN significantly reduced the fks1tetOnrep-mediated induction of an inflammatory cytokine profile. Our data demonstrate that lack of ß-1,3-glucan, also found under echinocandin therapy, results in improved recognition of Aspergillus fumigatus by complement and DCs and therefore not only directly affects the fungus by its fungistatic actions, but also is likely to exert indirect antifungal mechanisms by strengthening innate host immune mechanisms. Abbreviations: C: complement; CR:complement receptor; DC: dendritic cell; iDC: immature dendritic cell; DC-SIGN: Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin; ERK: extracellular signal–regulated kinases; JNK : c-Jun N-terminal kinases; MAPK: mitogen-activated protein kinase; NHS: normal human serum; PRR: pattern recognition receptor; Th :T helper; TLR :Toll-like receptor; WT: wild type.

Aspergillus spp and particularly the species Aspergillus fumigatus are the causative agents of invasive aspergillosis, a progressive necrotizing pneumonia that occurs in immunocompromised patients. The limited efficacy of currently available antifungals has led to interest in a better understanding of the molecular mechanisms underlying the pathogenesis of invasive aspergillosis in order to identify new therapeutic targets for this devastating disease. The Aspergillus exopolysaccharide galactosaminogalactan (GAG) plays an important role in the pathogenesis of experimental invasive aspergillosis. The present review article summarizes our current understanding of GAG composition and synthesis and the molecular mechanisms whereby GAG promotes virulence. Promising directions for future research and the prospect of GAG as both a therapy and therapeutic target are reviewed.

In contrast to mammalia, fungi are able to synthesize the branched-chain amino acid leucine de novo. Recently, the transcription factor LeuB has been shown to cross-regulate leucine biosynthesis, nitrogen metabolism and iron homeostasis in Aspergillus fumigatus, the most common human mold pathogen. Moreover, the leucine biosynthetic pathway intermediate α-isopropylmalate (α-IPM) has previously been shown to posttranslationally activate LeuB homologs in S. cerevisiae and A. nidulans. Here, we demonstrate that in A. fumigatus inactivation of both leucine biosynthetic enzymes α-IPM synthase (LeuC), which disrupts α-IPM synthesis, and α-IPM isomerase (LeuA), which causes cellular α-IPM accumulation, results in leucine auxotrophy. However, compared to lack of LeuA, lack of LeuC resulted in increased leucine dependence, a growth defect during iron starvation and decreased expression of LeuB-regulated genes including genes involved in iron acquisition. Lack of either LeuA or LeuC decreased virulence in an insect infection model, and inactivation of LeuC rendered A. fumigatus avirulent in a pulmonary aspergillosis mouse model. Taken together, we demonstrate that the lack of two leucine biosynthetic enzymes, LeuA and LeuC, results in significant phenotypic consequences indicating that the regulator LeuB is activated by α-IPM in A. fumigatus and that the leucine biosynthetic pathway is an attractive target for the development of antifungal drugs.

Aspergillus species account for the majority of invasive mold infections in immunocompromised patients. Most commonly, members of the Aspergillus section Fumigati are isolated from clinical material, followed by isolates belonging to section Terrei. The section Terrei contains 16 accepted species. Six species were found to be of clinical relevance and studied for differences in growth adaptability and virulence potential. Therefore, a set of 73 isolates (22 A. terreus s.s., 8 A. alabamensis, 27 A. citrinoterreus, 2 A. floccosus, 13 A. hortai, and 1 A. neoafricanus) was studied to determine differences in (a) germination kinetics, (b) temperature tolerance, (c) oxygen stress tolerance (1% O2), and (d) a combination of the latter two. Virulence potential of phialidic (PC) and accessory conidia (AC) was studied in G. mellonella larvae, using survival as read out. Further, the formation of AC was evaluated in larval tissue. All isolates were able to grow at elevated temperature and hypoxia, with highest growth and germination rates at 37°C. A. terreus s.s., A. citrinoterreus, and A. hortai exhibited highest growth rates. Virulence potential in larvae was inoculum and temperature dependent. All species except A. floccosus formed AC and germination kinetics of AC was variable. Significantly higher virulence potential of AC was found for one A. hortai isolate. AC could be detected in larval tissue 96 h post infection. Based on these findings, cryptic species of section Terrei are well adapted to the host environment and have similar potential to cause infections.

As the second leading etiological agent of candidemia in Turkey and the cause of severe fluconazole-non-susceptible (FNS) clonal outbreaks, Candida parapsilosis emerged as a major health threat at Ege University Hospital (EUH). Evaluation of microbiological and pertinent clinical profiles of candidemia patients due to C. parapsilosis in EUH in 2019–2020. Candida parapsilosis isolates were collected from blood samples and identified by sequencing internal transcribed spacer ribosomal DNA. Antifungal susceptibility testing was performed in accordance with CLSI M60 protocol and ERG11 and HS1/HS2-FKS1 were sequenced to explore the fluconazole and echinocandin resistance, respectively. Isolates were typed using a multilocus microsatellite typing assay. Relevant clinical data were obtained for patients recruited in the current study. FNS C. parapsilosis isolates were recovered from 53% of the patients admitted to EUH in 2019–2020. Y132F was the most frequent mutation in Erg11. All patients infected with C. parapsilosis isolates carrying Y132F, who received fluconazole showed therapeutic failure and significantly had a higher mortality than those infected with other FNS and susceptible isolates (50% vs. 16.1%). All isolates carrying Y132F grouped into one major cluster and mainly recovered from patients admitted to chest diseases and pediatric surgery wards. The unprecedented increase in the number of Y132F C. parapsilosis, which corresponded with increased rates of fluconazole therapeutic failure and mortality, is worrisome and highlights the urgency for strict infection control strategies, antifungal stewardship, and environmental screening in EUH.

Recurrent vulvovaginal candidiasis (RVVC) is one of the most prevalent fungal infections in humans, especially in developing countries; however, it is underestimated and regarded as an easy-to-treat condition. RVVC may be caused by dysbiosis of the microbiome and other host-, pathogen-, and antifungal drug-related factors. Although multiple studies on host-related factors affecting the outcome have been conducted, such studies on Candida-derived factors and their association with RVVC are lacking. Thus, fluconazole-tolerant (FLZT) isolates may cause fluconazole therapeutic failure (FTF), but this concept has not been assessed in the context of Candida-associated vaginitis. Iran is among the countries with the highest burden of RVVC; however, comprehensive studies detailing the clinical and microbiological features of this complication are scarce. Therefore, we conducted a 1-year prospective study with the aim to determine the RVVC burden among women referred to a gynecology hospital in Tehran, the association of the previous exposure to clotrimazole and fluconazole with the emergence of FLZT and fluconazole-resistant (FLZR) Candida isolates, and the relevance of these phenotypes to FTF. The results indicated that about 53% of the patients (43/81) experienced RVVC. Candida albicans and C. glabrata constituted approximately 90% of the yeast isolates (72 patients). Except for one FLZT C. tropicalis isolate, FLZR and FLZT phenotypes were detected exclusively in patients with RVVC; among them, 27.9% (12/43) harbored FLZR strains. C. albicans constituted 81.2% of FLZR (13/16) and 100% of the FLZT (13/13) isolates, respectively, and both phenotypes were likely responsible for FTF, which was also observed among patients with RVVC infected with fluconazole-susceptible isolates. Thus, FTF could be due to host-, drug-, and pathogen-related characteristics. Our study indicates that FLZT and FLZR isolates may arise following the exposure to over-the-counter (OTC) topical azole (clotrimazole) and that both phenotypes can cause FTF. Therefore, the widespread use of OTC azoles can influence fluconazole therapeutic success, highlighting the necessity of controlling the use of weak topical antifungals among Iranian women.

Upon entry of human immunodeficiency virus 1 (HIV-1) into the host, innate immune mechanisms are acting as a first line of defense, that considerably also modify adaptive immunity by the provision of specific signals. Innate and adaptive immune responses are intimately linked and dendritic cells (DCs) together with complement (C) play an important role in regulation of adaptive immunity. Initially, the role of complement was considered to primarily support – or COMPLEMENT - cytolytic actions of antibodies or antibody-complexed antigens (immune complexes, ICs) or directly kill the pathogens by complement-mediated lysis. Recently, the role of complement was revised and found to significantly augmenting and modulating adaptive immunity, in particular against viruses. Complement and DCs are therefore predestined to open novel avenues for antiviral research and potential therapeutic interventions. Recent studies on interactions of complement-opsonized HIV-1 with DCs demonstrated a high potential of such primed DCs to initiate efficient antiviral and cytotoxic anti-HIV-1 immunity and complement-coated viral particles shift DCs functions via CR3 and CR4 in an antithetic manner. This review will focus on our current knowledge of CR3 and CR4 actions on DCs during HIV-1 binding and the outcome of infection influenced by entry and signaling pathways.

Candidiasis is common in diabetic patients. Complement evasion is facilitated by binding complement factor H (FH). Since the expression of high-affinity glucose transporter 1 (Hgt1), a FH-binding molecule, is glucose-dependent, we aimed to study its relevance to the pathogenesis of Candida albicans. Euglycemic and diabetic mice were intravenously challenged with either Candida albicans lacking Hgt1 (hgt1-/-) or its parental strain (SN152). Survival and clinical status were monitored over 14 days. In vitro, Candida albicans strains were grown at different glucose concentrations, opsonized with human serum, and checked for C3b/iC3b and FH deposition. Phagocytosis was studied by fluorescein isothiocyanate-labeled opsonized yeast cells incubated with granulocytes. The murine model demonstrated a significantly higher virulence of SN152 in diabetic mice and an overall increased lethality of mice challenged with hgt1-/-. In vitro lower phagocytosis and C3b/iC3b deposition and higher FH deposition were demonstrated for SN152 incubated at higher glucose concentrations, while there was no difference on hgt1-/- at physiological glucose concentrations. Despite C3b/iC3b and FH deposition being glucose-dependent, this effect has a minor influence on phagocytosis. The absence of Hgt1 is diminishing this dependency on complement deposition, but it cannot be attributed to being beneficial in a murine model.

Invasive fungal infections caused by Aspergillus (A.) and Mucorales species still represent life-threatening diseases in immunocompromised individuals, and deeper knowledge about fungal interactions with elements of innate immunity, such as complement and platelets, appears essential for optimized therapy. Previous studies showed that galactosaminogalactan secreted by A. fumigatus and A. flavus is deposited on platelets, thereby inducing their activation. Since the altered platelet surface is a putative trigger for complement activation, we aimed to study the interplay of platelets with complement in the presence of fungal GAG. Culture supernatants (SN) of A. fumigatus and A. flavus both induced not only GAG deposition but also subsequent deposition of complement C3 fragments on the platelet surface. The SN of a Δuge3 mutant of A. fumigatus, which is unable to synthesize GAG, did not induce complement deposition on platelets, nor did the SN of other Aspergillus species and all tested Mucorales. Detailed analysis revealed that GAG deposition itself triggered the complement cascade rather than the GAG-induced phosphatidylserine exposure. The lectin pathway of complement could be shown to be crucially involved in this process. GAG-induced complement activation on the platelet surface was revealed to trigger processes that might contribute to the pathogenesis of invasive aspergillosis by A. fumigatus or A. flavus. Both pro-inflammatory anaphylatoxins C3a and C5a arose when platelets were incubated with SN of these fungal species; these processes might favor excessive inflammation after fungal infection. Furthermore, platelets were stimulated to shed microparticles, which are also known to harbor pro-inflammatory and pro-coagulant properties. Not only did early processes of the complement cascade proceed on platelets, but also the formation of the terminal complement C5b-9 complex was detected on platelets after incubation with fungal SN. Subsequently, reduced viability of the platelets could be shown, which might contribute to the lowered platelet numbers found in infected patients. In summary, fungal GAG initiates an interplay between complement and platelets that can be supposed to contribute to excessive inflammation, thrombocytopenia, and thrombosis, which are important hallmarks of fatal invasive mycoses.

Candida tropicalis is the fourth leading cause of candidemia in Turkey. Although C. tropicalis isolates from 1997 to 2017 were characterized as fully susceptible to antifungals, the increasing global prevalence of azole-non-susceptible (ANS) C. tropicalis and the association between high fluconazole tolerance (HFT) and fluconazole therapeutic failure (FTF) prompted us to re-evaluate azole susceptibility of C. tropicalis in Turkey. In this study, 161 C. tropicalis blood isolates from seven clinical centers were identified by ITS rDNA sequencing, genotyped by multilocus microsatellite typing, and tested for susceptibility to five azoles, two echinocandins, and amphotericin B (AMB); antifungal resistance mechanisms were assessed by sequencing of ERG11 and FKS1 genes. The results indicated that C. tropicalis isolates, which belonged to 125 genotypes grouped into 11 clusters, were fully susceptible to echinocandins and AMB; however, 18.6% of them had the ANS phenotype but only two carried the ANS-conferring mutation (Y132F). HFT was recorded in 52 isolates, 10 of which were also ANS. Large proportions of patients infected with ANS and HFT isolates (89 and 40.7%, respectively) showed FTF. Patients infected with azole-susceptible or ANS isolates did not differ in mortality, which, however, was significantly lower for those infected with HFT isolates (P = 0.007). There were significant differences in mortality (P = 0.02), ANS (P = 0.012), and HFT (P = 0.007) among genotype clusters. The alarming increase in the prevalence of C. tropicalis blood isolates with ANS and HFT in Turkey and the notable FTF rate should be a matter of public health concern.

Candida is a dominant fungal pathogen in immunocompromised hosts, leading to opportunistic infections. Complement with its multifaceted functions is involved in the immune defense against this yeast, and recently several novel aspects have emerged in this old battle. It is clear that Candida can adopt both roles as a colonizer or as a pathogen. In our article, we focus on the molecular mechanisms of the Candida-complement interplay, which occur in disseminated disease as well as locally on skin or on mucous membranes in mouth and vagina; the mechanisms can be supposed to be the same. Activation of the complement system by Candida is facilitated by directly triggering the three dominant pathways, but also indirectly via the coagulation and fibrinolysis systems. The complement-mediated anti-Candida effects induced thereby clearly extend chemotaxis, opsonization, and phagocytosis, and even the membrane attack complex formed on the fungal surface plays a modulatory role, although lysis of the yeast per se cannot be induced due to the thick fungal cell wall. In order to avoid the hostile action of complement, several evasion mechanisms have evolved during co-evolution, comprising the avoidance of recognition, and destruction. The latter comes in many flavors, in particular the cleavage of complement proteins by yeast enzymes and the exploitation of regulatory proteins by recruiting them on the cell wall, such as factor H. The rationale behind that is that the fluid phase regulators on the fungal cell surface down-regulate complement locally. Interestingly, however, evasion protein knockout strains do not necessarily lead to an attenuated disease, so it is likely more complex in vivo than initially thought. The interactions between complement and non-albicans species also deserve attention, especially Candida auris, a recently identified drug-resistant species of medical importance. This is in particular worth investigating, as deciphering of these interactions may lead to alternative anti-fungal therapies directly targeting the molecular mechanisms.

Objectives: Development and validation of a single-step and accurate reverse transcriptase loop-mediated isothermal amplification technique (RT-LAMP) for rapid identification of SARS-CoV-2 relative to commercial quantitative reverse transcriptase real-time PCR (qRT-PCR) assays to allow prompt initiation of proper medical care and containment of virus spread.Methods: Primers showing optimal in-silico features were subjected to analytical sensitivity and specificity to assess the limit of detection (LOD) and cross-reaction with closely- and distantly-related viral species, and clinically prominent bacterial and fungal species. In order to evaluate the clinical utility, our RT-LAMP was subjected to a large number of clinical samples, including 213 negative and 47 positive patients, relative to two commercial quantitative RT-PCR assays.Results: The analytical specificity and sensitivity of our assay was 100% and 500 copies/ml when serial dilution was performed in both water and sputum. Subjecting our RT-LAMP assay to clinical samples showed a high degree of specificity (99.5%), sensitivity (91.4%), positive predictive value (97.7%), and negative predictive value (98.1%) when used relative to qRT-PCR. Our RT-LAMP assay was two times faster than qRT-PCR and is storable at room temperature. A suspected case that later became positive tested positive using both our RT-LAMP and the two qRT-PCR assays, which shows the capability of our assay for screening purposes.Conclusions: We present a rapid RT-LAMP assay that could extend the capacity of laboratories to process two times more clinical samples relative to qRT-PCR and potentially could be used for high-throughput screening purposes when demand is increasing at critical situations.

Clonal expansion of fluconazole resistant (FLZ-R) Candida parapsilosis isolates is increasingly being identified in many countries, while there is no study exploring the antifungal susceptibility pattern, genetic diversity, and clinical information for Iranian C. parapsilosis blood isolates. Candida parapsilosis species complex blood isolates (n = 98) were recovered from nine hospitals located in three major cities, identified by MALDI-TOF MS, and their genetic relatedness was examined by AFLP fingerprinting. Antifungal susceptibility testing followed CLSI-M27-A3 and ERG11, MRR1 and hotspots 1/2 (HS1/2) of FKS1 were sequenced to assess the azole and echinocandin resistance mechanisms, respectively. Ninety-four C. parapsilosis and four Candida orthopsilosis isolates were identified from 90 patients. Only 43 patients received systemic antifungal drugs with fluconazole as the main antifungal used. The overall mortality rate was 46.6% (42/90) and death mostly occurred for those receiving systemic antifungals (25/43) relative to those not treated (17/47). Although, antifungal-resistance was rare, one isolate was multidrug-resistant (FLZ = 16 μg/ml and micafungin = 8 μg/ml) and the infected patient showed therapeutic failure to FLZ prophylaxis. Mutations causing azole and echinocandin resistance were not found in the genes studied. AFLP revealed five genotypes (G) and G1 was the main one (59/94; 62.7%). Clinical outcome was significantly associated with city (P = 0.02, α

Pathogenic mucormycetes induce diseases with considerable morbidity and mortality in immunocompromised patients. Virulence data comparing different Mucorales species and various underlying risk factors are limited. We therefore compared the pathogenesis of inhalative infection by Rhizopus (R.) arrhizus and Lichtheimia (L.) corymbifera in murine models for predominant risk factors for onset of infection. Mice with diabetes or treated with cyclophosphamide or cortisone acetate were challenged via the intranasal route with an isolate of R. arrhizus or L. corymbifera, respectively. Clinical, immunological and inflammation parameters as well as efficacy of posaconazole prophylaxis were monitored over 14 days. Whereas immunocompetent mice showed no clinical symptoms after mucormycete infection, mice treated with either cyclophosphamide (CP) or cortisone acetate (CA) were highly susceptible. Animals infected with the isolate of R. arrhizus showed prolonged survival and lower mortality, compared to those exposed to the L. corymbifera isolate. This lower virulence of R. arrhizus was risk factor-dependent, since diabetic mice died only after infection with Rhizopus, whereas all Lichtheimia-infected diabetic animals survived. Under posaconazole prophylaxis, both mucormycetes were able to establish breakthrough infections in CA- and CP-treated mice, but the course of infection was significantly delayed. Detailed analysis revealed that susceptibility of CA- and CP-treated mice could not be mimicked by exclusive lack or downmodulation of neutrophils, platelets or complement, but can be supposed to be the consequence of a broad immunosuppressive effect induced by the drugs. Both Lichtheimia corymbifera and Rhizopus arrhizus induce invasive mycoses in immunocompromised hosts after inhalative infection. Key parameters such as virulence and immunopathogenesis vary strongly according to fungal species and underlying risk group. Selected neutropenia is no sufficient risk factor for onset of inhalative mucormycosis.

Coronavirus disease 2019 (COVID-19)-associated pulmonary aspergillosis (CAPA) raises concerns as to whether it contributes to an increased mortality. The incidence of CAPA varies widely within hospitals and countries, partly because of difficulties in obtaining a reliable diagnosis. We implemented a routine screening of respiratory specimens in COVID-19 ICU patients for Aspergillus species using culture and galactomannan (GM) detection from serum and/or bronchoalveolar lavages (BAL). Out of 329 ICU patients treated during March 2020 and April 2021, 23 (7%) suffered from CAPA, 13 of probable, and 10 of possible. In the majority of cases, culture, microscopy, and GM testing were in accordance with CAPA definition. However, we saw that the current definitions underscore to pay attention for fungal microscopy and GM detection in BALs, categorizing definitive CAPA diagnosis based on culture positive samples only. The spectrum of Aspergillus species involved Aspergillus fumigatus, followed by Aspergillus flavus, Aspergillus niger, and Aspergillus nidulans. We noticed changes in fungal epidemiology, but antifungal resistance was not an issue in our cohort. The study highlights that the diagnosis and incidence of CAPA is influenced by the application of laboratory-based diagnostic tests. Culture positivity as a single microbiological marker for probable definitions may overestimate CAPA cases and thus may trigger unnecessary antifungal treatment.

Abstract Background Vancomycin resistant enterococci (VRE) are an emerging problem in health care settings. The purpose of the investigation was to assess the extent of the outbreak including environmental contamination and to limit further transmission. Methods We used retrospective patient and laboratory data including pulse field gel electrophoresis (PFGE) typing and virulence and resistance gene analysis. For comparison of medians the Mann-Whitney and for comparison of proportions the Fisher exact tests were used. Results PFGE typing of VRE strains of an outbreak of 15 VRE cases in a solid transplant unit revealed that nine of the cases belonged to one identical pattern (A), which was only found twice in the environment. Eleven further positive environmental samples showed a different, but identical PFGE pattern E. Only one patient was infected with this environmental strain. Two of nine (22.2%) PFGE A, but nine of eleven (81.2%) PFGE E samples were positive for gelatinase E (p = 0.01), which is described as enhancing biofilm production, suggesting a survival benefit for this strain on inanimate surfaces. Conclusion Routine disinfection was not able to stop the cluster, but after repeated enforcement of the infection prevention and control (IPC) bundle such as training, strict adherence to hand hygiene and surface disinfection no further cases were observed. We conclude that certain VRE strains predominate in the environment whereas others predominate in humans. Enforcement of the IPC bundle is essential for controlling VRE outbreaks and reducing further transmission.

Members of the order Mucorales may cause severe invasive fungal infections (mucormycosis) in immune-compromised and otherwise ill patients. Diagnosis of Mucorales infections and discrimination from other filamentous fungi are crucial for correct management. Here, we present an overview of current state-of-the-art mucormycosis diagnoses, with a focus on recent developments in the molecular field. Classical diagnostic methods comprise histology/microscopy as well as culture and are still the gold standard. Newer molecular methods are evolving quickly and display great potential in early diagnosis, although standardization is still missing. Among them, quantitative PCR assays with or without melt curve analysis are most widely used to detect fungal DNA in clinical samples. Depending on the respective assay, sequencing of the resulting PCR product can be necessary for genus or even species identification. Further, DNA-based methods include microarrays and PCR-ESI-MS. However, general laboratory standards are still in development, meaning that molecular methods are currently limited to add-on analytics to culture and microscopy.

In patients with cystic fibrosis, IL-9 signalling is increased. The authors describe an inflammatory loop in which IL-9 produced by Th9 cells drives mast cells to produce IL-2, resulting in ILC2 cell activation, and show inhibition of this loop with blocking antibodies to IL-9 in a mouse model of pulmonary infection.

Aspergillus are ubiquitous mold species that infect immunocompetent and immunocompromised patients. The symptoms are diverse and range from allergic reactions, bronchopulmonary infection, and bronchitis, to invasive aspergillosis. The aim of this study was to characterize 70 Aspergillus isolates recovered from clinical specimens of patients with various clinical conditions presented at Hamad general hospital in Doha, Qatar, by using molecular methods and to determine their in vitro antifungal susceptibility patterns using the Clinical and Laboratory Standards Institute (CLSI) M38-A2 reference method. Fourteen Aspergillus species were identified by sequencing β-tubulin and calmodulin genes, including 10 rare and cryptic species not commonly recovered from human clinical specimens. Aspergillus welwitschiae is reported in this study for the first time in patients with fungal rhinosinusitis (n = 6) and one patient with a lower respiratory infection. Moreover, Aspergillus pseudonomius is reported in a patient with fungal rhinosinusitis which is considered as the first report ever from clinical specimens. In addition, Aspergillus sublatus is reported for the first time in a patient with cystic fibrosis. In general, our Aspergillus strains exhibited low MIC values for most of the antifungal drugs tested. One strain of Aspergillus fumigatus showed high MECs for echinocandins and low MICs for the rest of the drugs tested. Another strain of A. fumigatus exhibited high MIC for itraconazole and categorized as non-wild type. These findings require further analysis of their molecular basis of resistance. In conclusion, reliable identification of Aspergillus species is achieved by using molecular sequencing, especially for the emerging rare and cryptic species. They are mostly indistinguishable by conventional methods and might exhibit variable antifungal susceptibility profiles. Moreover, investigation of the antifungal susceptibility patterns is necessary for improved antifungal therapy against aspergillosis.

Pathogenic bacteria and their microbial products activate dendritic cells (DCs) at mucosal surfaces during sexually transmitted infections (STIs) and therefore might also differently shape DC functions during co-infection with HIV-1. We recently illustrated that complement (C) coating of HIV-1 (HIV-C), as primarily found during the acute phase of infection before appearance of HIV-specific antibodies, by-passed SAMHD1-mediated restriction in DCs and therefore mediated an increased DC activation and antiviral capacity. To determine whether the superior antiviral effects of HIV-C-exposed DCs also apply during STIs, we developed a co-infection model in which DCs were infected with Chlamydia spp. simultaneously (HIV-C/Chlam-DCs or HIV/Chlam-DCs) or a sequential infection model, where DCs were exposed to Chlamydia for 3 or 24 h (Chlam-DCs) followed by HIV-1 infection. Co-infection of DCs with HIV-1 and Chlamydia significantly boosted the CTL-stimulatory capacity compared to HIV-1-loaded iDCs and this boost was independent on the opsonization pattern. This effect was lost in the sequential infection model, when opsonized HIV-1 was added delayed to Chlamydia-loaded DCs. The reduction in the CTL-stimulatory capacity of Chlam-DCs was not due to lower HIV-1 binding or infection compared to iDCs or HIV-C/Chlam-DCs, but due to altered fusion and internalization mechanisms within DCs. The CTL-stimulatory capacity of HIV-C in Chlam-DCs correlated with significantly reduced viral fusion compared to iDCs and HIV-C/Chlam-DCs and illustrated considerably increased numbers of HIV-C-containing vacuoles than iDCs. The data indicate that Chlamydia co-infection of DCs mediates a transient boost of their HIV-specific CTL-stimulatory and antiviral capacity, while in the sequential infection model this is reversed and associated with hazard to the host.

Aspergillus is the causative agent of human diseases ranging from asthma to invasive infection. Genetic and environmental factors are crucial in regulating the interaction between the host and Aspergillus. The role played by the enzyme indoleamine 2,3-dioxygenase 1 (IDO1), which catalyzes the first and rate-limiting step of tryptophan catabolism along the kynurenine pathway, is increasingly being recognized, but whether and how genetic variation of IDO1 influences the risk of aspergillosis in susceptible patients is incompletely understood. In addition, whether the closely related protein IDO2 plays a similar role remains unexplored. In the present study, we performed genetic association studies in two different cohorts of susceptible patients [cystic fibrosis (CF) patients and recipients of hematopoietic stem cell transplantation (HSCT)], and identified IDO1 polymorphisms that associate with the risk of infection in both cohorts. By using human bronchial epithelial cells and PBMC from CF and HSCT patients, respectively, we could show that the IDO1 polymorphisms appeared to down-modulate IDO1 expression and function in response to IFNγ or Aspergillus conidia, and to associate with an increased inflammatory response. In contrast, IDO2 polymorphisms, including variants known to profoundly affect protein expression and function, were differently associated with the risk of aspergillosis in the two cohorts of patients as no association was found in CF patients as opposed to recipients of HSCT. By resorting to a murine model of bone marrow transplantation, we could show that the absence of IDO2 more severely affected fungal burden and lung pathology upon infection with Aspergillus as compared to IDO1, and this effect appeared to be linked to a deficit in the antifungal effector phagocytic activity. Thus, our study confirms and extends the role of IDO1 in the response to Aspergillus, and shed light on the possible involvement of IDO2 in specific clinical settings.