Invasive pulmonary mucormycosis caused by Rhizopus microsporus is often fatal, yet analytical tools to monitor fungal burden and host responses in vivo remain limited. We developed a multimodal infection metallomics workflow combining ⁶⁸Ga-desferrioxamine B PET/CT, high-resolution MALDI mass spectrometry imaging (MALDI-MSI), and targeted LC-MS to map fungal siderophore production and host tissue remodeling in a neutropenic rat model and in human samples. ⁶⁸Ga-desferrioxamine B was rapidly taken up by R. microsporus in vitro and accumulated in infected lungs in vivo. MALDI-MSI visualized rhizoferrin (m/z 435.1259, [M−H]⁻) and homorhizoferrin (m/z 449.1420, [M−H]⁻) confined to hyphal foci and absent from control lungs, whereas heme b was depleted and spatially segregated. Neutrophil α-defensins (RatNP-2/3/4) increased 14–60-fold and formed halos around lesions, showing an inverse trend with siderophore abundance. Lipid MSI revealed remodeling of anionic surfactant lipids, with depletion of short-chain phosphatidylglycerols and phosphatidic acid–derived species and accumulation of long-chain polyunsaturated phosphatidylglycerols and phosphatidylinositols in infected regions. Targeted LC-MS of serial urine showed that rhizoferrin and homorhizoferrin emerged by day 2, peaked on day 4 (37.2 and 15.0 µg/mL), and declined with immune reconstitution; rhizoferrin was also detected in bronchoalveolar lavage from a patient with mucormycosis. Analytically, the workflow links radiotracer uptake, high-mass-accuracy spatial ion mapping of peptides with lipids, and matrix-matched urinary LC-MS quantitation within the same infection model. This integrated platform enables spatially resolved mechanistic readouts and supports non-invasive metallophore-based diagnostics of invasive mucormycosis.

Pseudomonas aeruginosa and Scedosporium/Lomentospora often coexist in the lungs of cystic fibrosis patients, where their interaction can affect disease outcomes. Our group has recently demonstrated that P. aeruginosa suppresses the growth of Scedosporium/Lomentospora species partly through mechanisms involving iron sequestration. In this study, we have investigated how molecules secreted by P. aeruginosa under high (36 µM) and low (3.6 µM) iron conditions affect the planktonic growth and biofilm formation by S. apiospermum, S. minutisporum, S. aurantiacum and L. prolificans. Although P. aeruginosa exhibited enhanced proliferation under high-iron conditions, spectrophotometric analyses revealed a marked increase in phenazine and pyoverdine production under low-iron conditions, with siderophore activity confirmed by Chrome Azurol S assays. Supporting these findings, supernatants from P. aeruginosa cells grown under iron limitation markedly inhibited fungal growth (≈30%) and biofilm formation (≈70%), whereas those from high-iron cultures were less effective. Notably, low-iron bacterial-free supernatants exhibited pronounced cytotoxic effects on mammalian cells, reducing metabolic activity by an average of 20% in A549 lung epithelial cells and 40% in THP-1 macrophages, and significantly compromising survival in the Tenebrio molitor infection model, resulting in 100% larval mortality within 7 days. Collectively, these results indicate that the antifungal activity of P. aeruginosa is closely coupled with increased host toxicity. Moreover, the results demonstrate that environmental iron availability plays a critical role in modulating both antifungal activity and toxicity, thereby shaping P. aeruginosa interactions with Scedosporium/Lomentospora species. Such iron-dependent dynamics may influence the progression and severity of respiratory co-infections, with important implications for patient management and therapeutic interventions.

ABSTRACT Clinically relevant infections commonly develop within polymicrobial environments where interkingdom interactions shape host responses and disease trajectories. Candida albicans and Klebsiella pneumoniae are critical pathogens that can co-exist in the respiratory tract, yet the consequences of their interaction in terms of fungal physiology, pathogenicity and impact on disease outcomes remain poorly understood. Here, we show that K. pneumoniae enhances C. albicans virulence traits suggesting that co-infections could exacerbate lung disease. Mechanistically, bacterial presence induces fungal hyphal morphogenesis via MAPK–CEK signaling, coupled to metabolic rewiring and alterations in cell wall remodeling and septation, resulting in highly elongated hyphae that escape faster from macrophages. At the host level, co-infection reprograms macrophages into a non-canonical state characterized by overlapping pro- and anti-inflammatory modules, integrating type I interferon and IL-10 signaling. This response contributes to tissue damage and facilitates fungal persistence. Our findings reveal that the bacterial–fungal interactions coordinately reprogram pathogen behavior and host immunity, promoting pathogenic synergy and potentially conferring a negative impact on disease outcomes. HIGHLIGHTS Candida–Klebsiella interactions modulate hyphal morphogenesis. Ectopic morphogenesis encompasses septation, cell wall remodeling and carbon metabolism. Candida–Klebsiella co-infections trigger tissue hyperinflammation and compensatory regulation. Candida-Klebsiella co-infection establishes a host environment facilitating microbial dissemination and tissue pathology. Candida–Klebsiella interactions enhance fungal virulence potentially impacting the severity of co-infections

Bone infections remain diagnostically challenging because current standard methods rely on microbiological and histopathological testing, which are time-consuming and may delay treatment decisions. This study evaluated handheld near-infrared spectroscopy for detecting spectral changes associated with staphylococcal inoculation in ex vivo human bone. An ex vivo bacterial inoculation model was established using human trabecular bone specimens inoculated with Staphylococcus aureus and Staphylococcus epidermidis. Spectra were acquired using a handheld NIR spectroscopy platform and analysed after standard normal variate preprocessing combined with either smoothing or first-derivative transformation. In total, 120 averaged spectra derived from 40 donors were included. Principal component analysis revealed partial clustering by inoculation status, particularly after derivative preprocessing, but substantial overlap remained. Linear discriminant analysis indicated stronger discrimination between inoculated and uninoculated bone than between the two staphylococcal species. However, classification performance depended on preprocessing and validation strategy; therefore, donor-wise dataset partitioning was used to reduce the risk of overly optimistic performance estimates caused by donor-level information leakage. These findings suggest that handheld NIR spectroscopy may provide rapid, non-destructive information on inoculation-associated spectral alterations in controlled ex vivo bone models. Further studies with sham-incubated controls, repeated donor-level cross-validation, independent external validation, additional pathogens, and clinically realistic confounders are required before clinical translation.

Background/Objectives: Fungal skin infections are commonly assessed using clinical examination and conventional histopathology, including hematoxylin-eosin (HE), periodic acid–Schiff (PAS), and Grocott methenamine silver (GMS) staining. However, these methods provide limited spatially resolved biochemical information. This proof-of-concept study investigated whether Fourier transform infrared (FTIR) chemical imaging can identify biochemical patterns associated with histologically defined fungal infection-associated tissue compartments in human skin. Methods: Archived formalin-fixed, paraffin-embedded skin samples with histological evidence of fungal infection were investigated. The study group comprised 19 patients, of whom 12 fulfilled the histological and technical eligibility criteria for quantitative FTIR analysis. These 12 independent biological cases yielded 46 histologically defined regions of interest (ROIs), comprising 16 fungal infection-associated ROIs, 14 keratosis/keratinised tissue ROIs, and 16 vital epidermis ROIs. ROI assignment was guided by corresponding HE-, PAS-, and GMS-stained sections. Results: Fungal infection-associated tissue compartments showed partially distinct spectral characteristics compared with vital epidermis and keratinised tissue. The most prominent exploratory differences occurred within the 900–1300 cm−1 fingerprint region. Case-level statistical analysis showed significant differences between fungal infection-associated tissue and vital epidermis at approximately 1185 and 1240 cm−1 after false discovery rate correction, whereas substantial overlap with keratinised tissue remained. Case-level PCA retained tissue-associated spectral structure after biological aggregation, although fungal infection-associated and keratinised tissue showed partial overlap. Unsupervised clustering further demonstrated spatially coherent spectral compartments corresponding to histologically identifiable tissue structures. Conclusions: FTIR chemical imaging may complement conventional histopathology by providing label-free, spatially resolved biochemical information on fungal infection-associated tissue compartments. Because fungal elements are embedded within surrounding keratinised and epithelial tissue, the observed spectral characteristics should be interpreted as exploratory infection-associated tissue signatures rather than fungal-specific diagnostic biomarkers. Larger independent studies with case-wise validation are required before diagnostic application can be considered.

Drug-resistant fungal disease must be addressed in the 2026 update to the Global Action Plan on Antimicrobial Resistance

Candida auris is an emerging fungal pathogen causing invasive infections in immunocompromised patients, with mortality rates reaching up to 60%. Pronounced drug resistance makes treatment failure common, and so does antifungal tolerance, a phenomenon enabling pathogen survival at supra-MIC concentrations without resistance mutations. Here, we uncover a mechanism of antifungal tolerance in C. auris engaging the mitochondrial cytochrome bc1 complex. Deletion of RIP1, a conserved catalytic complex subunit, does not significantly alter the susceptibility to caspofungin or voriconazole. However, rip1Δ mutants exhibit a marked reduction in antifungal tolerance across a wide range of supra-MIC concentrations. Transcriptomics reveals the dysregulation of multiple drug resistance and tolerance-related genes in the rip1Δ mutant, demonstrating a role for Rip1 in controlling antifungal susceptibility. Targeting cytochrome bc1 function with the fungal-specific inhibitor Inz-5 enhances antifungal action of both voriconazole and caspofungin. Additionally, ablation of RIP1 causes fitness defects, suggesting that cytochrome bc1 is a potential antifungal target against C. auris infections.

Azole antifungals are essential for controlling fungal diseases in medicine, veterinary care and agriculture. However, extensive cross-sector use has accelerated the emergence of resistant fungal pathogens, threatening human health, food security and ecosystem stability. This Review examines the dual role of azoles as life-saving therapeutics and drivers of antifungal resistance. We outline their development, mechanisms of action and applications across sectors, and highlight environmental and evolutionary pressures shaping resistance. Integrating perspectives from microbiology, agriculture and public health, we argue that coordinated One Health stewardship and sustainable antifungal strategies are urgently needed to preserve the efficacy of these critical compounds.

Bone infections, such as fracture-related and periprosthetic joint infections, present significant diagnostic and therapeutic challenges in orthopaedic surgery. Current diagnosic standards rely primarily on tissue cultures of intraoperatively obtained samples - a time-consuming approach with limited sensitivity and specificity and delayed clinical decision-making. This study investigates the use of hyperspectral imaging (HSI) in the visible and near-infrared (Vis-NIR), and short-wave infrared (SWIR) spectral ranges for the rapid detection of bone infections. Using ex vivo human bone samples, an in vitro biofilm model was established with Staphylococcus aureus and Staphylococcus epidermidis. Spectral data were analyzed using machine learning algorithms, including k-nearest neighbors (kNN), support vector machine (SVM), partial least squares discriminant analysis (PLS-DA), and soft independent modeling of class analogy (SIMCA). Vis-NIR-HSI models outperformed SWIR-based classification, achieving classification accuries of up to 99.58 % for distinguishing inoculated from uninoculated human bone samples, and enabling accurate bacterial species differentiation. These results highlight the diagnostic potential of Vis-NIR-HSI as real-time, label-free intraoperative tool for bone infection detection, bridging the gap between preoperative imaging and delayed microbiological results, and supporting immediate surgical decision making.

Background: Polymerase chain reaction (PCR) is highly sensitive and specific for the rapid diagnosis of invasive fungal disease (IFD) but is not yet widely implemented due to concerns regarding limited standardisation between assays, the lack of commercial options and the absence of clear guidance on interpreting results. Objectives and Methods: This review provides an update on technical and clinical aspects of PCR for the diagnosis of the most pertinent fungal pathogens, including Aspergillus, Candida, Pneumocystis jirovecii, Mucorales spp., and endemic mycoses. Summary: Recent meta-analyses have demonstrated that quantitative PCR (qPCR) offers high sensitivity for diagnosing IFD, surpassing conventional microscopy, culture and most serological tests. The reported specificity of qPCR is likely underestimated due to comparison with imperfect reference standards with variable sensitivity. Although the very low limit of detection of qPCR can generate false positive results due to procedural contamination or patient colonisation (particularly in pulmonary specimens), the rates are comparable to those observed for biomarker testing. When interpreting qPCR results, it is essential to consider the pre-test probability, determined by the patient population, host factors, clinical presentation and risk factors. For patients with low to moderate pre-test probability, the use of sensitive molecular tests, often in conjunction with serological testing or biomarkers, can effectively exclude IFD when all tests return negative results, reducing the need for empirical antifungal therapy. Conversely, for patients with high pre-test probability and clinical features of IFD, qPCR testing on invasive specimens from the site of infection (such as tissue or bronchoalveolar lavage fluid) can confidently rule in the disease. The development of next-generation sequencing methods to detect fungal infection has the potential to enhance the diagnosis of IFD, but standardisation and optimisation are essential, with improved accessibility underpinning clinical utility.

Background This systematic review and meta-analysis aimed to examine the performance of polymerase chain reaction (PCR) assays for diagnosing mucormycosis. Methods A standardised search was conducted from conception to December 3rd 2024 using PubMed, Embase, Global Health, and Cochrane library. Original studies that used PCR-based methods on any human specimen to diagnose mucormycosis were analysed for eligibility. Using a bivariate meta-analysis, the diagnostic performance of PCR was examined against the European Organisation for Research and Treatment of Cancer-Mycoses Study Group Education and Research Consortium 2020 (EORTC-MSGERC) definitions of proven and probable invasive mould disease, which was modified to include all patients at risk of mucormycosis. The study protocol was registered on the PROSPERO database (CRD42023478667). Findings Of 4855 articles, a total of 30 met inclusion criteria, including 5920 PCR reactions on 5147 non-duplicate specimens from 819 cases of proven/probable mucormycosis and 4266 patients who did not meet the EORTCMSGERC 2020 criteria. According to specimen type, sensitivity of PCR varied (p < 0.001) whereas specificity was similar (p = 0.662). Bronchoalveolar lavage fluid offered the highest sensitivity of 97.5% (95% CI 83.7-99.7%), specificity of 95.8% (95% CI 89.6-98.4%), positive likelihood ratio (LR+) of 23.5, and negative likelihood ratio (LR-) of 0.03. Tissue provided sensitivity of 86.4% (95% CI 78.9-91.5%), specificity of 90.6% (95% CI 78.1-96.3%), LR+ of 9.2, and LR- of 0.15. Blood provided reduced sensitivity of 81.6% (95% CI 70.1-89.4%), specificity of 95.5% (95% CI 87.4-98.5%), DOR of 95, LR+ of 18.3, and LR- of 0.19. Formalin-fixed paraffin- embedded specimens yielded the lowest sensitivity of 73.0% (95% CI 61.0-82.3%), highest specificity of 96.4% (CI 95% 87.5-99.0%), LR+ of 20.2, and LR- of 0.28. The covariates best explaining heterogeneity of the overall analysis were specimen type, study design (cohort versus case-control) and disease prevalence while patient population (COVID-19 versus other) and PCR (conventional versus quantitative) had less impact on heterogeneity. Interpretation This meta-analysis confirms the high performance of PCR for diagnosing mucormycosis and supports the instatement of PCR detection of free-DNA in blood, BALF and tissue into future updated definitions and diagnostic guidelines for mucormycosis. Copyright Crown Copyright (c) 2025 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

The fungal disease mucormycosis, while generally regarded as rare and not transmitted between individuals, has become increasingly prevalent in disaster areas, among the immunocompromised, and in diabetics especially in response to COVID-19. Treatment options are limited. These include debridement of necrotizing tissue followed by complicated multicomponent therapies with amphotericin B and selected azole drugs, usually having poor outcomes. Mucormycetes are intrinsically resistant to the widely used short-tailed azole drugs fluconazole and voriconazole, but susceptible to the long-tailed, though expensive, azole posaconazole. Knowledge of the crystal structure of Saccharomyces cerevisiae sterol 14α-demethylase (Erg11, Cyp51) led to the hypothesis that this pattern of intrinsic azole resistance and susceptibility is due to the Rhizopus arrhizus CYP51-F5 isoform residues F129 and A291, while the CYP51-F1 isoform residues Y127 and V291 confer susceptibility to both short- and long-tailed azole drugs. The heterologous overexpression of individual recombinant R. arrhizus CYP51 isoforms in a S. cerevisiae host, with or without the cognate NADPH-cytochrome P450 reductase (RaCPR), and selective genetic modification of CYP51-F5 have tested this hypothesis. Complementary gene deletion experiments in Rhizopus microsporus confirm that the amino acid residues that align with R. arrhizus CYP51-F5 F129 and A291 determine the resistance or susceptibility pattern of R. arrhizus to short-, medium-, and long-tailed azoles.

Bone infections caused by Staphylococcus aureus and Staphylococcus epidermidis are serious complications in orthopedic surgery. These infections commonly occur in joint replacements, fracture management, and bone grafting procedures. Rapid and accurate pathogen-specific diagnostic methods are urgently needed to support early clinical decisions. Current culture-based methods are slow and delay effective treatment. This study evaluated the diagnostic value of combining Raman microscopy with high-resolution micro-computed tomography (micro-CT). Human bone samples, either uninfected or inoculated with S. aureus or S. epidermidis, were analyzed. Raman spectroscopy detected distinct spectral changes in inoculated bones, including reduced intensity of phosphate (v1PO4 3-), Amide III, and CH2 deformation bands. A single principal component explained 96%-98% of the variance in these infection-related markers. Specifically, the v1PO4 3- and CH2 deformation bands effectively differentiated between S. aureus and S. epidermidis infections, capturing 99%-100% variance. Micro-CT analysis showed significant structural changes in inoculated bones. Trabecular volume, number, and spacing were particularly affected. Among these, VOX-BV/TV and Mean1 best differentiated between S. aureus and S. epidermidis infections (both p < 0.0001). Support vector machine (SVM) classification repeated stratified k-folg cross-validation accurately detected inoculation status. Combining Raman and micro-CT features yielded moderately improved classification performance in pathogen-specific discrimination. These findings demonstrate that combining molecular (Raman spectroscopy) and structural (micro-CT) methods allows rapid, non-destructive diagnosis of bone infections. This multimodal approach may improve diagnostic precision, supports timely clinical decisions, and ultimately improves patient outcomes in orthopedic and trauma surgery.

Mucormycosis is predominantly caused by members of the genera Lichtheima, Mucor, and Rhizopus. Here, we report the genome assemblies and comparative analyses of the clinically relevant species Mucor ardhlaengiktus (CBS 210.80), Mucor circinelloides (CBS 195.68), Mucor janssenii (CBS 205.68), and Mucor griseocyanus (CBS 116.08) to enable molecular analyses.

Fungi associated with humans include several Candida species that rely on phenotypic plasticity for persistence and pathogenicity. Key adaptive traits, such as adherence, stress resistance, and biofilm formation, enable survival in diverse host niches. However, the degree of intra- and interspecific phenotypic variation across human-associated Candida species has not been systematically characterized. We analyzed 1,366 clinical isolates representing 13 Candida species using high-throughput quantitative fitness profiling under controlled environmental stressors, antifungal exposure, and biofilm-inducing conditions. The resulting data set revealed both conserved and species-specific adaptive signatures. Isolates consistently segregated into three phenotypic archetypes: heat-resistant fast growers, osmo-sensitive strains, and slow growers. A robust inverse correlation was detected between basal growth rate and stress resistance, reflecting a fundamental physiological trade-off. In addition, distinct resistance profiles against antifungal agents and environmental stressors highlighted species-specific adaptive trajectories and ecological specialization. Despite genetic homogeneity, C. parapsilosis isolates displayed striking phenotypic heterogeneity. By contrast, the closely related C. albicans and C. dubliniensis exhibited divergent stress-response profiles. High-resolution fitness mapping of C. glabrata isolates revealed that temperature stress progressively disrupts multiple cellular functions, whereas osmotic stress exerts more discrete, pathway-specific effects. Our systematic phenotypic landscape analysis delineates conserved versus species-specific adaptive properties among human-associated Candida species, providing a comparative framework to interrogate evolutionary trends, ecological specialization, and pathogenic potential. IMPORTANCE:Human-associated fungi include multiple Candida species whose persistence relies on phenotypic plasticity enabling adherence, stress resistance, and biofilm formation. Yet, the extent of phenotypic variation within and across species remains poorly defined. We profiled 1,366 clinical isolates from 13 Candida species using high-throughput quantitative fitness assays under environmental stress, antifungal exposure, and biofilm-inducing conditions. The analysis uncovered both conserved and species-specific adaptive traits. Isolates segregated into three major phenotypic archetypes: heat-resistant fast growers, osmo-sensitive strains, and slow growers. A consistent inverse correlation emerged between basal growth rate and stress resistance, revealing a fundamental physiological trade-off. Species-specific resistance signatures further reflected ecological specialization and divergent adaptive trajectories. Our quantitative framework establishes, for the first time, a comparative phenotypic landscape across a multispecies collection of human-associated Candida, providing new insights into their ecological specialization and adaptive strategies.

The pronounced skin tropism and pan-antifungal resistance of Candida auris pose a serious global health threat. A key question in C. auris biology is how clinical isolates acquire amphotericin B resistance. Here we demonstrate that a carbonic sensing pathway (CSP) contributes to amphotericin B resistance by modulating mitochondrial energy functions in clinical C. auris isolates. Integrated transcriptomics and proteomics identify the carbonic anhydrase Nce103 and its transcription factors Rca1 and Efg1 as important regulatory components of the CSP. The conversion of CO2 into bicarbonate sustains energy metabolism required for colonization and fitness on human skin and in nutrient-limited microenvironments. We also show that bacterial skin colonizers engage urease to release CO2 that sustains C. auris fitness and skin colonization. These findings highlight therapeutic options to re-sensitize C. auris to antifungal treatments, as well as to prevent skin colonization by blocking the CSP.

OBJECTIVES:This study aims to characterize the possible contribution of eight pleiotropic drug resistance (PDR) transporters to the azole resistance phenotype of Mucor lusitanicus. METHODS:Gene expression analysis (RNA-sequencing and RT-qPCR) was performed on M. lusitanicus CBS277.49 cells exposed to three different types of azoles (4.0 mg/L). C-terminally GFP-tagged M. lusitanicus PDR transporters were overexpressed in the hypersensitive model host, Saccharomyces cerevisiae ADΔΔ. Their efflux pump functions were evaluated by determining the azole susceptibilities of the PDR transporter overexpressing cells and measuring their plasma membrane ATPase activities. RESULTS:M. lusitanicus PDR transporters separated into two phylogenetic clusters: A (pdr1, pdr6-8) and B (pdr2-5). RNA-sequencing and RT-qPCR revealed strong up-regulation of pdr1 and pdr6, but down-regulation of pdr7 and pdr8 in response to 80 min exposures of 4.0 mg/L voriconazole, isavuconazole or posaconazole. The expression of Pdr6 and Pdr7 in S. cerevisiae ADΔΔ increased its resistance to short- and mid-length tailed azoles. Pdr1 and Pdr8 expression, however, conferred pan-azole resistance including long-tailed azoles such as itraconazole and posaconazole. No efflux pump function and ATPase activity were detected for Pdr3 and Pdr5. The ATPase activities of Pdr1, Pdr6, Pdr7 and Pdr8 were comparable to Candida albicans Cdr1 expressed in ADΔΔ. CONCLUSIONS:All Mucor cluster A PDR transporters are multidrug efflux pumps, but Pdr1 and Pdr6 are possibly the major contributors to the innate azole resistance phenotype of M. lusitanicus.

ABSTRACT The aim of this study was to identify parameters influencing DNA extraction and PCR amplification efficiencies in an attempt to standardize Mucorales qPCR. The Fungal PCR Initiative Mucorales Laboratory Working Group distributed two panels of simulated samples to 26 laboratories: Panel A (six sera spiked with Mucorales DNA and one negative control serum) and Panel B (six Mucorales DNA extracts). Panel A underwent DNA extraction in each laboratory according to the local procedure and were sent to a central laboratory for testing using three different qPCR techniques: one in-house qPCR assay and two commercial assays (MucorGenius and Fungiplex). Panel B DNA extracts were PCR amplified in each laboratory using local procedures: nine in-house qPCR assays and two commercial kits (MucorGenius and MycoGENIE). All data were compiled and anonymously analyzed at the central laboratory. For Panel A, a total of six different automated platforms and five manual extraction methods were used. Positive rates were 64%, 70%, and 89%, for the MucorGenius, Fungiplex, and the in-house qPCR assay, respectively. Using a large volume of serum for DNA extraction provided the highest analytical sensitivity (82.5% for 1 mL compared with 62.7% for smaller volumes, P < 0.01). For Panel B, five in-house qPCR assays and two commercial kits had >78% positivity. Using larger PCR input volumes (≥7 µL) was associated with the highest sensitivity at 95.5% compared to 58.3% when lower input volumes were used ( P < 0.01). Using larger sample volumes for nucleic acid extraction and DNA template volumes for PCR amplification significantly improves the performance of Mucorales qPCR when testing serum. IMPORTANCE Mucormycosis is a life-threatening mold infection affecting immunosuppressed patients but also other patients with diabetes or trauma. Better survival is linked to shorter delays in diagnosis and treatment initiation. Detection of Mucorales-free DNA in serum or plasma using quantitative PCR allows a prompt diagnosis and earlier treatment. Several techniques and protocols of quantitative Mucorales PCR are used in Europe, and improving performance remains a common objective of laboratories participating in the fungal PCR Initiative Working Group. This study, which combined results from 26 laboratories in Europe, showed that the main parameters underpinning sensitivity are the preanalytical variables (volume of serum used for DNA extraction and DNA template volume), irrespective of the extraction platforms and qPCR assay/platform.