ABSTRACT Invasive fungal diseases are difficult to diagnose because of the limited sensitivity of culture. Panfungal PCR amplicon sequencing assays (targeting ribosomal RNA, such as 18S, 28S, ITS) are recommended for fungal identification in histopathology samples showing fungal elements. However, data describing its overall performance and consistency are lacking. This systematic literature review and meta-analysis assessed the performance of panfungal PCR on formalin-fixed paraffin-embedded (FFPE) and non-fixed (fresh or frozen) tissue samples. A systematic literature search was performed to include studies reporting the use of panfungal PCR for fungal identification in FFPE or non-fixed tissue samples. PCR sensitivity and specificity were assessed using the reference standard of histopathology showing fungal elements. Quality assessment was performed using the Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2) tool. Pooled estimates were obtained using random-effects meta-analysis. Twenty-eight studies were included. In FFPE samples (18 studies, 852 samples), sensitivity and specificity were 75.4% (95% confidence interval [CI], 59.2–86.6) and 93.5% (70.2–98.9), respectively. Sensitivity in non-fixed samples (13 studies, 207 samples) was 86.5% (74.7–93.3), while specificity could not be assessed (insufficient data). Comparative analyses showed a significantly higher sensitivity of panfungal PCR over culture (88.2%; 76–94.7 vs 52.2%; 39–65, P = 0.001). Sub-analyses could not demonstrate the superiority of one PCR target over another due to limited data. Panfungal PCR exhibited adequate sensitivity and good specificity in FFPE samples. Sensitivity was even higher in non-fixed samples and largely superior to culture. Nevertheless, large interstudy variability was observed, warranting interlaboratory studies to define the optimal PCR target and standardized protocols. IMPORTANCE Invasive fungal diseases are difficult to diagnose because of the low sensitivity of culture. Panfungal PCRs are widely used for fungal identification in tissue specimens but suffer from heterogeneous procedures and performance. This meta-analysis shows an acceptable sensitivity (75.4% and 86.5% in fixed and non-fixed samples, respectively) and good specificity (93.5%) of panfungal PCR, supporting its use, not only on histopathology-positive fixed samples but also in non-fixed samples concomitantly with other diagnostic tools (cultures and fungal-specific PCRs if available). These results provide a strong basis for further standardization of panfungal PCR techniques via interlaboratory assays to assess reproducibility and optimize analytical protocols. CLINICAL TRIALS This study is registered with PROSPERO as CRD42023461148 .

At the beginning of Friedrich Staib's academic career in the 1950s, medical mycology was a young discipline in Germany. He studied veterinary and human medicine, earning two doctorates. Initially fascinated by the changes in intestinal flora caused by the newly emerging broad-spectrum antibacterials, i.e., the selection of fungi. In 1953, he began setting up a mycological laboratory at the University of Würzburg and qualified as a professor in microbiology in 1962. In 1968, he became the head of the mycology department, which he established, at the Robert Koch Institute, Germany's public health Institute in Berlin. His work focused on improving the identification of fungi, both within hosts and the environment. His research on Cryptococcus neoformans (Staib agar indicator medium based on the virulence factor melanin for cultivation) gained him international recognition. He was the first to describe secreted proteases from Candida albicans as a virulence mechanism of the main human fungal pathogen. Later, nosocomial exposure of immunocompromised individuals to Aspergillus fumigatus became an issue for him. He cultivated intensive collaboration with clinicians and pathologists, which had a major influence on his research. Staib's broad interest in fungal diagnostics, virulence factors, and environmental niches of fungal pathogens was groundbreaking for medical mycology in Germany and beyond at a time when systemic mycoses were becoming increasingly important. With his systems thinking, he influenced doctoral students and was a role model for public health.

BACKGROUND: Here, we describe a case of an intracranial fungal granuloma in an adult, non-castrated female dromedary camel (Camelus dromedarius). CASE PRESENTATION: An 11-year-old female dromedary presented with progressive neurological signs over several weeks. Neurological examination suggested a forebrain lesion, and magnetic resonance imaging (MRI) revealed a mass in the frontal fossa. Owing to poor prognosis, the animal was euthanized. Histopathological examination demonstrated a granulomatous-necrotizing inflammatory process containing fungal structures. Broad-range polymerase chain reaction (PCR) and amplicon sequencing results were consistent with a member of the Aspergillus fumigatus species complex being the causative agent. CONCLUSIONS: Central nervous system involvement is rare in fungal infections. Intracranial fungal granulomas should be considered as a differential diagnosis in camels presenting with progressive neurological signs and space-occupying intracranial lesions.

This case report describes a disseminated Rasamsonia (R.) argillacea infection in a German shepherd dog involving internal and external lymph nodes, abdominal organs and one eye. R. argillacea is a member of the R. argillacea species complex, which is an emerging pathogen notable for azole resistance in the last decades. The dog was euthanised following 18 days of itraconazole therapy due to clinical deterioration.

Raccoon dogs (Nyctereutes procyonoides) originated from East Asia and are listed as invasive alien species of Union concern in the European Union. Apart from their potential negative impact on native fauna as predators and competitors, they are discussed as reservoirs and vectors for various infectious diseases. Between 2021 and 2022, comprehensive post-mortem examinations of 110 raccoon dogs were performed as part of a health and risk assessment study of selected invasive alien species in Schleswig-Holstein, Germany. Parasitic infections, often accompanied by eosinophilic and/or granulomatous inflammation, were one of the most common and significant findings. In total, 64% animals had an ectoparasitosis and up to 76% an endoparasitosis. Alaria alata and Trichinella spp were the most relevant endoparasite species found. Eggs of A. alata were detected in 30% of faecal samples, while the prevalence of Trichinella spp in muscle samples was 0.9%. Influenza A virus, canine distemper virus, rustrela virus, Borna disease virus 1 and severe acute respiratory syndrome corona virus 2 were not detected. Carnivore protoparvovirus 1 was detected with a prevalence of 3%, but was not associated with pathognomonic lesions. In general, most of the pathological findings were of minor significance regardless of whether pathogens were detected or not, and therefore did not appear to affect the overall health status of the examined animals. Our study shows that raccoon dogs may contribute to the spread and persistence of certain pathogens by acting as carriers of infectious diseases. This underpins the need to further investigate the risk they pose to endemic European wildlife, livestock and human health.

AbstractCryptococcosis causes a high burden of disease worldwide. This systematic review summarizes the literature on Cryptococcus neoformans and C. gattii infections to inform the World Health Organization’s first Fungal Priority Pathogen List. PubMed and Web of Science were used to identify studies reporting on annual incidence, mortality, morbidity, antifungal resistance, preventability, and distribution/emergence in the past 10 years. Mortality rates due to C. neoformans were 41%–61%. Complications included acute renal impairment, raised intracranial pressure needing shunts, and blindness. There was moderate evidence of reduced susceptibility (MIC range 16–32 mg/l) of C. neoformans to fluconazole, itraconazole, ketoconazole, voriconazole, and amphotericin B. Cryptococcus gattii infections comprised 11%–33% of all cases of invasive cryptococcosis globally. The mortality rates were 10%–23% for central nervous system (CNS) and pulmonary infections, and ∼43% for bloodstream infections. Complications described included neurological sequelae (17%–27% in C. gattii infections) and immune reconstitution inflammatory syndrome. MICs were generally low for amphotericin B (MICs: 0.25–0.5 mg/l), 5-flucytosine (MIC range: 0.5–2 mg/l), itraconazole, posaconazole, and voriconazole (MIC range: 0.06–0.5 mg/l). There is a need for increased surveillance of disease phenotype and outcome, long-term disability, and drug susceptibility to inform robust estimates of disease burden.

Cryptococcosis, an infection caused by Cryptococcus neoformans and Cryptococcus gattii, predominantly targets the central nervous system (CNS) in patients with AIDS but is not limited to this group. The disease can also occur in individuals with various immunosuppressive conditions, frequently involving the brain or lungs. Cryptococcal meningitis (CM) is the most common form of fungal meningoencephalitis, leading to intracerebral infections, cerebral infarction, or hydrocephalus. The clinical presentation of CM is nonspecific, and imaging features can vary significantly. This case report presents a patient with cerebral infarction, who was HIV-negative but had been on long-term cortisone therapy. Notably, the patient had a history of pulmonary cryptococcosis 15 years prior to cerebral involvement. When initially at our clinic, histology and culture results from brain biopsies were negative and the earlier pulmonary cryptococcosis history was unknown. Subsequently, cryptococcal antigen was detected in both serum and cerebrospinal fluid (CSF), and C. neoformans was cultivated from CSF. This case highlights the critical importance of maintaining a high index of suspicion for CM, particularly in patients with a history of previous cryptococcal infections, and it also demonstrates the possibility of false-negative brain biopsy results due to secondary vascular events associated with CM.

Cryptococcosis is the most prevalent fungal infection of the central nervous system worldwide. We performed a retrospective multicenter cohort study to gain insights into the epidemiology of cryptococcosis in Germany. We describe the use of diagnostic tests, clinical management and patient outcome. We included 64 patients with underlying HIV infection (55%) or other predispositions. Molecular typing by MLST documented 20 individual sequence types among 42 typed isolates. A fatal outcome was documented in 14% of patients in the first two months after diagnosis.

Hybrid AD strains of the human pathogenic Cryptococcus neoformans species complex have been reported from many parts of the world. However, their origin, diversity, and evolution are incompletely understood. In this study, we analyzed 102 AD hybrid strains representing 21 countries on five continents. For each strain, we obtained its mating type and its allelic sequences at each of the seven loci that have been used for genotyping haploid serotypes A and D strains of the species complex by the Cryptococcus research community. Our results showed that most AD hybrids exhibited loss of heterozygosity at one or more of the seven analyzed loci. Phylogenetic and population genetic analyses of the allelic sequences revealed multiple origins of the hybrids within each continent, dating back to one million years ago in Africa and up to the present in other continents. We found evidence for clonal reproduction and long-distance dispersal of these hybrids in nature. Comparisons with the global haploid serotypes A and D strains identified new alleles and new haploid multi-locus genotypes in AD hybrids, consistent with the presence of yet-to-be discovered genetic diversity in haploid populations of this species complex in nature. Together, our results indicate that AD hybrids can be effectively genotyped using the same multi-locus sequencing type approach as that established for serotypes A and D strains. Our comparisons of the AD hybrids among each other as well as with the global haploid serotypes A and D strains revealed novel genetic diversity as well as evidence for multiple origins and dynamic evolution of these hybrids in nature.

A 2-year-old Norwegian Forest cat was presented for evaluation of bilateral purulent nasal discharge and stertorous breathing. A computed tomography (CT) scan of the head revealed an intranasal mass of the left nasal cavity extending behind the tube openings and completely obstructing the nasopharynx. Rhinoscopy confirmed a pinkish, shiny mass. CT scan showed both compartments of the right middle ear filled with abnormal soft tissue attenuating material. There was no change in the bony outline of the middle ear. In the endoscopic examination, after endoscopically assisted tympanocentesis, this material in the accessible dorsolateral compartment proved to be classic polypous tissue in addition to highly viscous glue-like secretions. A secondary otitis media due to a drainage disorder was suspected.Using an endoscopic-interventional approach through the nostril, the nasopharyngeal mass was removed for histopathological examination, in order to restore the nasal airway, and to allow tube drainage. In contrast to cats with classical malignant nasal cavity masses, the cat showed several attachment points of the mass and multiple undulating elevations bilaterally in the nasopharyngeal mucosa.Cytological and histopathological examination identified the mass as a fungal granuloma in the context of a cryptococcus infection only rarely observed in Germany. Molecular genetic analysis confirmed an infection with Cryptococcus neoformans var. grubii.A single intranasal and nasopharyngeal endoscopic debridement resulted in a significant improvement of the clinical signs and a complete healing of the right middle ear (including the tympanic membrane) within 14 days, but not in a complete cure of the disease. The cat was therefore treated with oral itraconazole solution for several weeks.The case report shows that nasal cryptococcosis can also affect cats in Germany. Rhinoscopy reveals a nasopharyngeal mass with multiple attachment points, which is unusual for a neoplasia. In addition to the recommended removal of the mass, oral administration of systemic antimycotics is strongly advised.

We report a suspected case of acute pulmonary coccidioidomycosis contracted in Peru, where the disease is not known to occur, in a patient from Switzerland. Although not confirmed by direct diagnostic testing, the clinical manifestations and serologic testing results of this case are highly suggestive of coccidioidomycosis.

Cordyceps militaris-derived formulations are currently used for multiple purposes because of their medical properties, especially immune system modulation. This study analyzes the inhibitory effects of C. militaris aqueous extract on Candida albicans infections and the immune response in larvae of the greater wax moth Galleria mellonella (Lepidoptera: Pyralidae). Larvae exhibited melanization within 1 h of being infected with C. albicans inoculum at a concentration of 106 cells/larvae, and died within 24 h from a lethal dose. Aqueous extract of C. militaris proved to be nontoxic at concentrations of 0.25 and 0.125 mg/larvae, and had the greatest ability to prolong the survival of larvae infected with a sublethal dose of C. albicans at a concentration of 105 cells/larvae. Moreover, the number of hemocytes in the hemolymph of G. mellonella increased after infection with C. albicans and treatment with the aqueous extract of C. militaris at 1, 24, and 48 h by 1.21 × 107, 1.23 × 107, and 1.4 × 107 cells/100 µL, respectively. The highest number of hemocytes was recorded after treatment of infected G. mellonella with the extract for 48 h. Transcriptional upregulation of the immune system was observed in certain antimicrobial peptides (AMPs), showing that the relative expression of galiomicin, gallerimycin, and lysozyme genes were upregulated as early as 1 h after infection. Therefore, we conclude that C. militaris aqueous extract can modulate the immune system of G. mellonella and protect against infection from C. albicans.

Abstract Recognizing the growing global burden of fungal infections, the World Health Organization established a process to develop a priority list of fungal pathogens (FPPL). In this systematic review, we aimed to evaluate the epidemiology and impact of infections caused by Fusarium spp., Scedosporium spp., and Lomentospora prolificans to inform the first FPPL. PubMed and Web of Sciences databases were searched to identify studies published between January 1, 2011 and February 23, 2021, reporting on mortality, complications and sequelae, antifungal susceptibility, preventability, annual incidence, and trends. Overall, 20, 11, and 9 articles were included for Fusarium spp., Scedosporium spp., and L. prolificans, respectively. Mortality rates were high in those with invasive fusariosis, scedosporiosis, and lomentosporiosis (42.9%–66.7%, 42.4%–46.9%, and 50.0%–71.4%, respectively). Antifungal susceptibility data, based on small isolate numbers, showed high minimum inhibitory concentrations (MIC)/minimum effective concentrations for most currently available antifungal agents. The median/mode MIC for itraconazole and isavuconazole were ≥16 mg/l for all three pathogens. Based on limited data, these fungi are emerging. Invasive fusariosis increased from 0.08 cases/100 000 admissions to 0.22 cases/100 000 admissions over the time periods of 2000–2009 and 2010–2015, respectively, and in lung transplant recipients, Scedosporium spp. and L. prolificans were only detected from 2014 onwards. Global surveillance to better delineate antifungal susceptibility, risk factors, sequelae, and outcomes is required.

Zusammenfassung Eine 2-jährige Norwegische Waldkatze wurde aufgrund von bilateralem, eitrigem Nasenausfluss und Stertor vorgestellt. Eine Computertomografie (CT) des Kopfes zeigte eine intranasale Masse der linken Nasenhöhle, die sich bis hinter die Tubenöffnungen erstreckte und den Nasenrachen vollständig verlegte. Die Rhinoskopie bestätigte eine rosafarbene, speckige Masse. In der CT zeigten sich beide Kompartimente des rechten Mittelohres mit weichteilisodensem Material gefüllt. Die knöcherne Umrandung des Mittelohres war nicht verändert. Nach endoskopisch gestützter Eröffnung des Trommelfells (Tympanozentese) erwies sich die Füllung in dem einsehbaren, dorsolateralen Kompartiment neben weißlichem, hochviskosem Sekret als klassisches polypöses Gewebe. Es wurde der Verdacht einer sekundären Otitis media aufgrund einer Abflussstörung geäußert. Mit einem endoskopisch-interventionellem Zugang durch das Nasenloch wurde die nasopharyngeale Masse für die histopathologische Untersuchung sowie zur Wiederherstellung des nasalen Atemweges und zur Ermöglichung der Tubendrainage abgetragen. Im Gegensatz zu Katzen mit klassischen, malignen Neoplasien der Nasenhöhle zeigten sich bei dem Kater mehrere Ansatzpunkte der Zubildung und mehrere wellenförmige Erhebungen bilateral in der nasopharyngealen Schleimhaut. Die zytologische und histopathologische Untersuchung identifizierte die Masse als Pilzgranulom im Rahmen einer in Deutschland nur selten beobachteten Kryptokokken-Infektion. Die molekulargenetische Analyse bestätigte eine Infektion mit Cryptococcus neoformans var. grubii. Ein einmaliges intranasales und nasopharyngeales endoskopisches Débridement hatte innerhalb von 14 Tagen zu einer deutlichen Verbesserung der klinischen Befunde und einer vollständigen Ausheilung des rechten Mittelohres (inklusive des Trommelfells) geführt, jedoch nicht zu einer vollständigen Heilung der Erkrankung. Der Kater wurde deshalb nach Erhalt des Cryptococcus-positiven Befundes über mehrere Wochen mit oraler Itraconazol-Lösung therapiert. Der Fallbericht zeigt, dass auch in Deutschland die Kryptokokkose bei Katzen vorkommen kann. In der Endoskopie zeigt sich eine nasopharyngeale Masse mit multiplen Ansatzpunkten, was für eine Neoplasie untypisch ist. Für eine Therapie ist neben der empfohlenen Entfernung der Masse nach aktuellem Wissensstand die orale Gabe von systemischen Antimykotika dringend anzuraten.

Abstract Recognizing the growing global burden of fungal infections, the World Health Organization established a process to develop a priority list of fungal pathogens (FPPL). In this systematic review, we aimed to evaluate the epidemiology and impact of invasive infections caused by Aspergillus fumigatus to inform the first FPPL. The pre-specified criteria of mortality, inpatient care, complications and sequelae, antifungal susceptibility, risk factors, preventability, annual incidence, global distribution, and emergence were used to search for relevant articles between 1 January 2016 and 10 June 2021. Overall, 49 studies were eligible for inclusion. Azole antifungal susceptibility varied according to geographical regions. Voriconazole susceptibility rates of 22.2% were reported from the Netherlands, whereas in Brazil, Korea, India, China, and the UK, voriconazole susceptibility rates were 76%, 94.7%, 96.9%, 98.6%, and 99.7%, respectively. Cross-resistance was common with 85%, 92.8%, and 100% of voriconazole-resistant A. fumigatus isolates also resistant to itraconazole, posaconazole, and isavuconazole, respectively. The incidence of invasive aspergillosis (IA) in patients with acute leukemia was estimated at 5.84/100 patients. Six-week mortality rates in IA cases ranged from 31% to 36%. Azole resistance and hematological malignancy were poor prognostic factors. Twelve-week mortality rates were significantly higher in voriconazole-resistant than in voriconazole-susceptible IA cases (12/22 [54.5%] vs. 27/88 [30.7%]; P = .035), and hematology patients with IA had significantly higher mortality rates compared with solid-malignancy cases who had IA (65/217 [30%] vs. 14/78 [18%]; P = .04). Carefully designed surveillance studies linking laboratory and clinical data are required to better inform future FPPL.

Abstract In response to the growing global threat of fungal infections, in 2020 the World Health Organisation (WHO) established an Expert Group to identify priority fungi and develop the first WHO fungal priority pathogen list (FPPL). The aim of this systematic review was to evaluate the features and global impact of invasive infections caused by Pichia kudriavzevii (formerly known as Candida krusei). PubMed and Web of Science were used to identify studies published between 1 January 2011 and 18 February 2021 reporting on the criteria of mortality, morbidity (defined as hospitalisation and length of stay), drug resistance, preventability, yearly incidence, and distribution/emergence. Overall, 33 studies were evaluated. Mortality rates of up to 67% in adults were reported. Despite the intrinsic resistance of P. kudriavzevii to fluconazole with decreased susceptibility to amphotericin B, resistance (or non-wild-type rate) to other azoles and echinocandins was low, ranging between 0 and 5%. Risk factors for developing P. kudriavzevii infections included low birth weight, prior use of antibiotics/antifungals, and an underlying diagnosis of gastrointestinal disease or cancer. The incidence of infections caused by P. kudriavzevii is generally low (∼5% of all Candida-like blood isolates) and stable over the 10-year timeframe, although additional surveillance data are needed. Strategies targeting the identified risk factors for developing P. kudriavzevii infections should be developed and tested for effectiveness and feasibility of implementation. Studies presenting data on epidemiology and susceptibility of P. kudriavzevii were scarce, especially in low- and middle-income countries (LMICs). Thus, global surveillance systems are required to monitor the incidence, susceptibility, and morbidity of P. kudriavzevii invasive infections to inform diagnosis and treatment. Timely species-level identification and susceptibility testing should be conducted to reduce the high mortality and limit the spread of P. kudriavzevii in healthcare facilities.

AbstractHistoplasmosis, a significant mycosis primarily prevalent in Africa, North and South America, with emerging reports globally, poses notable health challenges, particularly in immunocompromised individuals such as people living with HIV/AIDS and organ transplant recipients. This systematic review, aimed at informing the World Health Organization’s Fungal Priority Pathogens List, critically examines literature from 2011 to 2021 using PubMed and Web of Science, focusing on the incidence, mortality, morbidity, antifungal resistance, preventability, and distribution of Histoplasma. We also found a high prevalence (22%–44%) in people living with HIV, with mortality rates ranging from 21% to 53%. Despite limited data, the prevalence of histoplasmosis seems stable, with lower estimates in Europe. Complications such as central nervous system disease, pulmonary issues, and lymphoedema due to granuloma or sclerosis are noted, though their burden remains uncertain. Antifungal susceptibility varies, particularly against fluconazole (MIC: ≥32 mg/l) and caspofungin (MICs: 4–32 mg/l), while resistance to amphotericin B (MIC: 0.125–0.16 mg/l), itraconazole (MICs: 0.004–0.125 mg/l), and voriconazole (MICs: 0.004–0.125 mg/l) remains low. This review identifies critical knowledge gaps, underlining the need for robust, globally representative surveillance systems to better understand and combat this fungal threat.

Background: In-house real-time PCR (qPCR) is increasingly used to diagnose the so-called endemic mycoses as commercial assays are not widely available.Objectives: To compare the performance of different molecular diagnostic assays for detecting Histoplasma capsulatum and Coccidioides spp. in five European reference laboratories.Methods: Two blinded external quality assessment (EQA) panels were sent to each laboratory that performed the analysis with their in-house assays. Both panels included a range of concentrations of H. capsulatum (n = 7) and Coccidioides spp. (n = 6), negative control and DNA from other fungi. Four laboratories used specific qPCRs, and one laboratory a broad-range fungal conventional PCR (cPCR) and a specific cPCR for H. capsulatum with subsequent sequencing.Results: qPCR assays were the most sensitive for the detection of H. capsulatum DNA. The lowest amount of H. capsulatum DNA detected was 1-4 fg, 0.1 pg and 10 pg for qPCRs, specific cPCR and broad-range cPCR, respectively. False positive results occurred with high concentrations of Blastomyces dermatitidis DNA in two laboratories and with Emergomyces spp. in one laboratory. For the Coccidioides panel, the lowest amount of DNA detected was 1-16 fg by qPCRs and 10 pg with the broad-range cPCR. One laboratory reported a false positive result by qPCR with high load of Uncinocarpus DNA.Conclusion: All five laboratories were able to correctly detect H. capsulatum and Coccidioides spp. DNA and qPCRs had a better performance than specific cPCR and broad-range cPCR. EQAs may help standardise in-house molecular tests for the so-called endemic mycoses improving patient management.