Molecular identification of Histoplasma capsulatum in patients with disseminated histoplasmosis and acquired immunodeficiency syndrome.

Histoplasmosis is caused by the fungus Histoplasma capsulatum and is often fatal for individuals with acquired immunodeficiency syndrome (AIDS). Delayed diagnosis is a major factor in worsening coinfection, as it can be mistaken for other diseases. Thus, rapid identification of Histoplasma in immunocompromised patients is essential. Molecular techniques, particularly polymerase chain reaction (PCR), were used in this study to identify H. capsulatum in patients coinfected with histoplasmosis and AIDS. Blood samples from 14 individuals with AIDS and disseminated histoplasmosis were collected and analyzed. The PCR method successfully amplified the fungal region in whole blood samples, while PCR-RFLP analysis confirmed a consistent profile in the samples. Genetic sequencing further confirmed the fungal species. Compared to clinical tests such as fungal culture and urinary antigen detection, molecular analysis proved faster, more sensitive, and cost-effective. These molecular markers can potentially be incorporated into routine diagnostics in the future. Further studies are needed to expand and enhance this diagnostic approach, particularly in patients with nonprogressive clinical forms of histoplasmosis.

Prevalence of fungal DNAemia mediated by putatively non-pathogenic fungi in immunocompromised patients with febrile neutropenia: a prospective cohort study.

Invasive fungal disease (IFD) presents a life-threatening condition in immunocompromised patients, thus often prompting empirical administration of antifungal treatment, without adequate mycological evidence. Over the past years, wide use of antifungal prophylaxis resulted in decreased occurrence of IFD but has contributed to changes in the spectrum of fungal pathogens, revealing the occurrence of previously rare fungal genera causing breakthrough infections. The expanding spectrum of clinically relevant fungal pathogens required the implementation of screening approaches permitting broad rather than targeted fungus detection to support timely onset of pre-emptive antifungal treatment. To address this diagnostically important aspect in a prospective setting, we analyzed 935 serial peripheral blood (PB) samples from 195 pediatric and adult patients at high risk for IFD, involving individuals displaying febrile neutropenia during treatment of hematological malignancies or following allogeneic hematopoietic stem cell transplantation. Two different panfungal-PCR-screening methods combined with ensuing fungal genus identification by Sanger sequencing were employed. In the great majority of PB-specimens displaying fungal DNAemia, the findings were transient and revealed fungi commonly regarded as non-pathogenic or rarely pathogenic even in the highly immunocompromised patient setting. Hence, to adequately exploit the diagnostic potential of panfungal-PCR approaches for detecting IFD, particularly if caused by hitherto rarely observed fungal pathogens, it is necessary to confirm the findings by repeated testing and to identify the fungal genus present by ensuing analysis. If applied appropriately, panfungal-PCR-screening can help prevent unnecessary empirical therapy, and conversely, contribute to timely employment of effective pre-emptive antifungal treatment strategies.

Multicenter comparative study of Enterocytozoon bieneusi DNA extraction methods from stool samples, and mechanical pretreatment protocols evaluation.

Nowadays, the use of qPCR for the diagnosis of intestinal microsporidiosis is increasing. There are several studies on the evaluation of qPCR performance but very few focus on the stool pretreatment step before DNA extraction, which is nevertheless a crucial step. This study focuses on the mechanical pretreatment of stools for Enterocytozoon bieneusi spores DNA extraction. Firstly, a multicenter comparative study was conducted evaluating seven extraction methods (manual or automated) including various mechanical pretreatment. Secondly, several durations and grinding speeds and types of beads were tested in order to optimize mechanical pretreatment. Extraction methods of the various centers had widely-varying performances especially for samples with low microsporidia loads. Nuclisens® easyMAG (BioMérieux) and Quick DNA Fecal/Soil Microbe Microprep kit (ZymoResearch) presented the best performances (highest frequencies of detection of low spore concentrations and lowest Ct values). Optimal performances of mechanical pretreatment were obtained by applying a speed of 30 Hz during 60 s with the TissueLyser II (Qiagen) using commercial beads of various materials and sizes (from ZymoResearch or MP Biomedicals). Overall, the optimal DNA extraction method for E. bieneusi spores contained in stool samples was obtained with a strong but short bead beating using small-sized beads from various materials.

Airborne DNA reveals predictable spatial and seasonal dynamics of fungi.

Fungi are among the most diverse and ecologically important kingdoms in life. However, the distributional ranges of fungi remain largely unknown as do the ecological mechanisms that shape their distributions1,2. To provide an integrated view of the spatial and seasonal dynamics of fungi, we implemented a globally distributed standardized aerial sampling of fungal spores3. The vast majority of operational taxonomic units were detected within only one climatic zone, and the spatiotemporal patterns of species richness and community composition were mostly explained by annual mean air temperature. Tropical regions hosted the highest fungal diversity except for lichenized, ericoid mycorrhizal and ectomycorrhizal fungi, which reached their peak diversity in temperate regions. The sensitivity in climatic responses was associated with phylogenetic relatedness, suggesting that large-scale distributions of some fungal groups are partially constrained by their ancestral niche. There was a strong phylogenetic signal in seasonal sensitivity, suggesting that some groups of fungi have retained their ancestral trait of sporulating for only a short period. Overall, our results show that the hyperdiverse kingdom of fungi follows globally highly predictable spatial and temporal dynamics, with seasonality in both species richness and community composition increasing with latitude. Our study reports patterns resembling those described for other major groups of organisms, thus making a major contribution to the long-standing debate on whether organisms with a microbial lifestyle follow the global biodiversity paradigms known for macroorganisms4,5.

Development of a Recombinase-Aided Amplification Assay for the Rapid Detection of Candida auris.

Candida auris (C. auris) was first discovered in Japan in 2009 and has since spread worldwide. It exhibits strong transmission ability, high multidrug resistance, blood infectivity, and mortality rates. Traditional diagnostic techniques for C. auris have shortcomings, leading to difficulty in its timely diagnosis and identification. Therefore, timely and accurate diagnostic assays for clinical samples are crucial. We developed a novel, rapid recombinase-aided amplification (RAA) assay targeting the 18S rRNA, ITS1, 5.8S rRNA, ITS2, and 28S rRNA genes for C. auris identification. This assay can rapidly amplify DNA at 39 °C in 20 min. The analytical sensitivity and specificity were evaluated. From 241 clinical samples collected from pediatric inpatients, none were detected as C. auris-positive. We then prepared simulated clinical samples by adding 10-fold serial dilutions of C. auris into the samples to test the RAA assay's efficacy and compared it with that of real-time PCR. The assay demonstrated an analytical sensitivity of 10 copies/µL and an analytical specificity of 100%. The lower detection limit of the RAA assay for simulated clinical samples was 101 CFU/mL, which was better than that of real-time PCR (102-103 CFU/mL), demonstrating that the RAA assay may have a better detection efficacy for clinical samples. In summary, the RAA assay has high sensitivity, specificity, and detection efficacy. This assay is a potential new method for detecting C. auris, with simple reaction condition requirements, thus helping to manage C. auris epidemics.

Global Spore Sampling Project: A global, standardized dataset of airborne fungal DNA.

Novel methods for sampling and characterizing biodiversity hold great promise for re-evaluating patterns of life across the planet. The sampling of airborne spores with a cyclone sampler, and the sequencing of their DNA, have been suggested as an efficient and well-calibrated tool for surveying fungal diversity across various environments. Here we present data originating from the Global Spore Sampling Project, comprising 2,768 samples collected during two years at 47 outdoor locations across the world. Each sample represents fungal DNA extracted from 24 m3 of air. We applied a conservative bioinformatics pipeline that filtered out sequences that did not show strong evidence of representing a fungal species. The pipeline yielded 27,954 species-level operational taxonomic units (OTUs). Each OTU is accompanied by a probabilistic taxonomic classification, validated through comparison with expert evaluations. To examine the potential of the data for ecological analyses, we partitioned the variation in species distributions into spatial and seasonal components, showing a strong effect of the annual mean temperature on community composition.

Carbon nanoparticle-based lateral flow assay for the detection of specific double-tagged DNA amplicons of Paracoccidioides spp.

To overcome the limitations of current methods for diagnosing paracoccidioidomycosis (PCM), it is critical to develop novel diagnostic strategies that can be implemented in low-resource settings and dramatically improve turnaround times. This study focused on the development of a portable molecular test to screen for Paracoccidioides spp. The proposed approach integrated double-tagging polymerase chain reaction (PCR) and a paper-based lateral flow assay (LFA) for readout, using carbon nanoparticles as a signal generation system. Primers tagged with biotin and digoxigenin were employed to conduct the double-tagging PCR, which can be conveniently carried out on portable thermocyclers. This method can generate billions of tagged DNA copies from a single target molecule, which can be rapidly detected by the LFA platform, providing results within minutes. Avidin-modified carbon nanoparticles served as a signal generation system, enabling detection in the immunochromatographic assay. The LFA demonstrated the capability to detect double-tagged amplicons as low as 0.21 ng or 0.10 ng, depending on whether the results were assessed visually or with a smartphone equipped with an image processor. These findings suggest that the proposed approach holds great promise as a point-of-care diagnostic tool for the early and accurate detection of PCM in low-resource settings. The diagnostic test is rapid and inexpensive, requires minimal handling and can be easily introduced into the general practitioner's armoury for ambulatory screening of infection. This innovative approach has the potential to make a substantial contribution to PCM diagnosis, ultimately reducing morbidity and mortality associated with this disease.

Research Note: Analysis of microbial diversity on the shell surface of eggs collected from geographically distinct farms in China.

Micro-organisms on the eggshell surface of affect the quality of the egg. Sometimes, these microbes even pose a serious threat to the health of the egg's consumer. Bacterial 16S rDNA and fungal internal transcribed spacer region were sequenced to analyze the microbial diversity on the shell surface of the eggs collected from 4 distinct regions of China: Guyuan (GY; 1.5 million hens), Langfang (LF; 0.1 million hens), Beihai (BH; 1.2 million hens), and Dongguan (DG; 0.2 million hens). The results showed a higher bacterial and fungal abundance on the eggs collected from the northern and southern China farms, respectively. The dominant bacterial phylum detected across all egg samples was Firmicutes. In addition, the shell surfaces of the DG and LF samples harbored abundant levels of Proteobacteria. The dominant fungal phyla detected across all egg samples were Ascomycota and Basidiomycota. The bacterial compositions on eggshell surfaces differed significantly across all geographic regions, and the fungal composition differed significantly between samples collected from the southern and northern farms (P < 0.05). The abundance and composition of microbial colonies on the eggshell surface varied based on their geographical location (climate and environment) and farming scale (management). Our findings provide an important reference for optimizing the cleaning and disinfection methods for fresh eggs collected from different sources.

THE CLINICAL VALUE OF ß-D-GLUCAN TESTING AND NEXT-GENERATION METAGENOMIC SEQUENCING FOR THE DIAGNOSIS OF FUNGAL ENDOPHTHALMITIS.

PURPOSE: To explore the clinical value of ß-D-glucan (BDG) testing and metagenomic next-generation sequencing (mNGS) for detecting the pathogens of fungal endophthalmitis (FE). METHODS: This study included 32 cases (32 eyes) with FE and 20 cases (20 eyes) with intraocular inflammation caused by other etiologies. All patients underwent extraction of aqueous humor or vitreous fluid samples for BDG testing and mNGS. The diagnostic performance and total clinical concordance rate of BDG testing and mNGS for FE were evaluated and calculated based on the results of the clinical diagnosis. RESULTS: Among the clinically diagnosed FE, the positivity rates of BDG testing and mNGS (90.63%) were both significantly higher ( P < 0.001) than that of microbial cultures (53.13%). There was 100% consistency in pathogen identification using mNGS and culture identification for culture-positive cases. The area under the curve was 0.927 for BDG testing and 0.853 for mNGS. When the two tests were combined, sensitivity (93.75%), specificity (100.00%), and total clinical concordance rate (96.15%) were all improved, compared with the single tests. CONCLUSION: The positive rates of BDG test and mNGS were markedly higher than those of cultures in FE identification. The combination of these two tests showed improved performance when compared with individual tests.

Novel B-DNA dermatophyte assay for demonstration of canonical DNA in dermatophytes: Histopathologic characterization by artificial intelligence.

We describe a novel assay and artificial intelligence-driven histopathologic approach identifying dermatophytes in human skin tissue sections (ie, B-DNA dermatophyte assay) and demonstrate, for the first time, the presence of dermatophytes in tissue using immunohistochemistry to detect canonical right-handed double-stranded (ds) B-DNA. Immunohistochemistry was performed using anti-ds-B-DNA monoclonal antibodies with formalin-fixed paraffin-embedded tissues to determine the presence of dermatophytes. The B-DNA assay resulted in a more accurate identification of dermatophytes, nuclear morphology, dimensions, and gene expression of dermatophytes (ie, optical density values) than periodic acid-Schiff (PAS), Grocott methenamine silver (GMS), or hematoxylin and eosin (H&E) stains. The novel assay guided by artificial intelligence allowed for efficient identification of different types of dermatophytes (eg, hyphae, microconidia, macroconidia, and arthroconidia). Using the B-DNA dermatophyte assay as a clinical tool for diagnosing dermatophytes is an alternative to PAS, GMS, and H&E as a fast and inexpensive way to accurately detect dermatophytosis and reduce the number of false negatives. Our assay resulted in superior identification, sensitivity, life cycle stages, and morphology compared to H&E, PAS, and GMS stains. This method detects a specific structural marker (ie, ds-B-DNA), which can assist with diagnosis of dermatophytes. It represents a significant advantage over methods currently in use.

Comparative analysis in diagnosis by real-time polymerase chain reaction versus direct microscopy, culture, and histology in fungal infections of the nails, scalp, and skin.

BACKGROUND: The diagnosis of superficial fungal infections is the subject of intensive research in many countries around the world. The diagnostic methods used are diverse, including both conventional and innovative techniques. METHODS: This study evaluates the sensitivity, specificity, and efficacy of the real-time polymerase chain reaction (PCR) methodology and compares them with those of the conventional methods - direct microscopic, cultural, and histological examinations of materials from hair, skin, and nails - in order to demonstrate the benefits and significance of real-time PCR for the diagnosis of dermatophytic infections. RESULTS: The values obtained for the sensitivity, specificity, and efficacy of direct microscopic, cultural, histological, and real-time PCR studies are as follows: 63.71, 88.89, and 72.96% (P < 0.001); 58.06, 100, and 73.47% (P < 0.001); 85.96, 100, and 90.70% (P < 0.001); 88.52, 100, and 92.63% (P < 0.001). CONCLUSION: The use of real-time PCR in the diagnosis of dermatophytic infections is a relatively new approach in mycology and is subject to testing and experience from its use. The results are promising, but the method has not yet established itself as a new gold standard in the diagnosis of superficial fungal infections caused by dermatophytes, though its application would be very useful in identifying isolates without conidiogenesis or absence of growth.

Microbiological culture combined with PCR for the diagnosis of onychomycosis: Descriptive analysis of 121 patients.

BACKGROUND: Onychomycosis is the most common nail pathology, involving various pathogens such as dermatophytes, moulds and yeasts. OBJECTIVE: The objective of this study was to observe the prevalence of onychomycosis, analyse the most appropriate diagnostic test, and assess the distribution of pathogens based on age, sex, quarter of the year, duration of symptoms and previous treatment. METHODS: Retrospectively, mycological culture and PCR data and results were collected from 121 patients. RESULTS: Of the 121 samples, 57% (69/121) tested positive when both microbiological study techniques were combined. The prevalence of onychomycosis was higher when PCR was performed (52.1%) compared to microbiological culture (33.1%). Among the 81 samples negative by microbiological culture, 31 were positive by PCR. Similarly, of the 58 samples negative by PCR, eight were positive by microbiological culture. Diagnostic accuracy data (with 95% confidence intervals) for PCR, using microbiological culture as the gold standard, were as follows: sensitivity of 0.8, specificity of 0.62, positive predictive value of 0.51 and negative predictive value of 0.86. The most frequently identified pathogen was Trichophyton rubrum, and the hallux nail plate was the most commonly affected location. However, no statistically significant associations were found between sex, age, quarter of the year and affected area with culture and PCR results. CONCLUSION: Combining microbiological culture and PCR can increase the detection rate of onychomycosis and help avoid false-negative results.

Improved RAPD Method for Candida parapsilosis Fingerprinting.

Recently, methods based on the analysis of arbitrarily amplified target sites of genome microorganisms have been extensively applied in microbiological studies, and especially in epidemiological studies. The range of their application is limited by problems with discrimination and reproducibility resulting from a lack of standardized and reliable methods of optimization. The aim of this study was to obtain optimal parameters of the Random Amplified Polymorphic DNA (RAPD) reaction by using an orthogonal array as per the Taguchi and Wu protocol, modified by Cobb and Clark for Candida parapsilosis isolates. High Simpson's index values and low Dice coefficients obtained in this study indicated a high level of interspecies DNA polymorphism between C. parapsilosis strains, and the optimized RAPD method proved useful in the microbiological and epidemiological study.

Mucormycosis-causing fungi in humans: a meta-analysis establishing the phylogenetic relationships using internal transcribed spacer (ITS) sequences.

Introduction. Mucormycosis is a severe angio-invasive fungal infection caused by mucormycetes, a group of fungi that are ubiquitous in the environment. The incidence of mucormycosis has been surging rapidly due to the global corona virus disease 2019 (COVID-19) pandemic.Gap Statement. The complete picture of the causative fungi associated with mucormycosis and their phylogenetic relationships are not well defined.Aim. This meta-analysis aimed to collate all confirmed fungal pathogens that cause mucormycosis, and assess their taxonomic relationships.Methodology. All types of articles in the PubMed database that report fungi as a cause of mucormycosis were reviewed. We summarized the fungal morphological characteristic up to the genus level. The internal transcribed spacer (ITS) nucleotide sequences of these fungi were retrieved from the National Center for Biotechnology Information (NCBI) and UNITE databases whenever available, and multiple sequence analysis was conducted using Clustal W. The phylogenetic tree was constructed using mega version 7.Results. Forty-seven fungal species were identified as pathogens causing mucormycosis in humans. Thirty-two fungal species were phylogenetically grouped into three clades, and it was evident that the ITS sequences have well-conserved regions in all clades, especially from the 400th to 500th base pairs.Conclusion. The findings of this work contribute to the descriptive data for fungi that cause mucormycosis, emphasizing the need for robust phylogenetic approaches when identifying clinical isolates from infected patients.

Time and cost-efficient identification of Trichophyton indotineae.

BACKGROUND: During the past 5 years, an outbreak of recalcitrant dermatophytosis due to a novel Trichophyton species generally resistant to terbinafine, T. indotineae, has spread out from South Asia to many countries around the World. These isolates cannot be reliably differentiated from other Trichophyton spp. on the basis of morphological traits and the current laboratory diagnostics relies on sequencing of ribosomal DNA ITS region. OBJECTIVES: In this study, we aimed to introduce two inexpensive and rapid PCR-based assays for differentiation between T. indotineae and other dermatophytes. METHODS: The first introduced assay is based on PCR-restriction fragment length polymorphism (PCR-RFLP) analysis, involving the amplification of TOP2 sequences and the digestion of PCR products by Cfr13I restriction enzyme. The second assay is proposed as conventional endpoint species-specific PCR amplification of the C120-287 intergenic locus. To validate the assays, a total of 191 Trichophyton spp. and 2 Microsporum canis strains with known ITS region sequences were used. From the T. mentagrophytes / T. interdigitale species complex (TMTISC), strains with 18 different ITS genotypes were tested. The sample of TMTISC isolates included 41 T. indotineae strains. RESULTS: TOP2 PCR-RFLP and T. indotineae-specific PCR were positive with testing on DNA of all 41 T. indotineae isolates and two strains of T. mentagrophytes belonging to ITS Types XIII and XVI, but negative with other species and other TMTISC ITS genotypes (n = 152). Therefore, the specificity of both new assays was 99%. CONCLUSION: The two developed diagnostic assays provide accurate and cost-effective means of identifying cultured T. indotineae isolates.

Pan-cancer analyses reveal cancer-type-specific fungal ecologies and bacteriome interactions.

Cancer-microbe associations have been explored for centuries, but cancer-associated fungi have rarely been examined. Here, we comprehensively characterize the cancer mycobiome within 17,401 patient tissue, blood, and plasma samples across 35 cancer types in four independent cohorts. We report fungal DNA and cells at low abundances across many major human cancers, with differences in community compositions that differ among cancer types, even when accounting for technical background. Fungal histological staining of tissue microarrays supported intratumoral presence and frequent spatial association with cancer cells and macrophages. Comparing intratumoral fungal communities with matched bacteriomes and immunomes revealed co-occurring bi-domain ecologies, often with permissive, rather than competitive, microenvironments and distinct immune responses. Clinically focused assessments suggested prognostic and diagnostic capacities of the tissue and plasma mycobiomes, even in stage I cancers, and synergistic predictive performance with bacteriomes.

Evaluation of different DNA extraction methods based on steel-bullet beating for molecular diagnosis of onychomycosis.

BACKGROUND: Considering increased trends toward molecular methods for detection/identification of fungi causing onychomycosis, the aim of this study is comparison three DNA extraction methods based on steel-bullet beating to extract DNA from nail. METHODS: Ex -vivo onychomycosis model was developed using bovine hoof with Candida albicans and Aspergillus flavus. For two models, total DNA was extracted using the three different methods. In method 1, the extraction and purification were performed by steel-bullet beating and phenol chloroform protocol, respectively. In method 2, a freezing step were applied before beating. The purification step in method 3 was carried out using a commercial kit, although DNA extraction was done similarly to method 1 in that approach. To evaluate the efficacy of each method, the extracted genomic DNA was amplified with Polymerase Chain Reaction (PCR) using Internal Transcribed Spacer (ITS) regions. Moreover, 50 nail samples were evaluated for onychomycosis using direct microscopy examination as well as PCR in order to evaluate the diagnostic efficiency of the optimal DNA extraction method. RESULTS: Regarding the desirable quality of the extracted DNA, cost effectiveness, and simplicity, method 1 could be used to extract DNA effectively. Additionally, the obtained data showed that PCR had a higher detection rate of fungal agents in the nail samples than direct microscopic examination. CONCLUSIONS: This study demonstrated that the mechanical disruption of the cell wall by steel-bullet beating is a useful and practical method to improve the quantity and quality of fungal DNA thorough the extraction process.

Toxigenicity of F. graminearum Residing on Host Plants Alternative to Wheat as Influenced by Environmental Conditions.

Fusarium graminearum is an important pathogen that causes Fusarium head blight (FHB) in several cereal crops worldwide. The potential of this pathogen to contaminate cereals with trichothecene mycotoxins presents a health risk for both humans and animals. This study aimed to evaluate the potential of different trichothecene genotypes of F. graminearum isolated from an alternative host plant to produce mycotoxins under different spring wheat grain incubation conditions. Fourteen F. graminearum strains were isolated from seven alternative host plants and identified as 3-acetyl-deoxynivalenol (3-ADON) and 15-acetyl-deoxynivalenol (15-ADON) genotypes. These strains were cultivated on spring wheat grains at 25 °C and 29 °C for 5 weeks. The mycotoxins produced were analysed with a high-performance liquid chromatograph (HPLC) coupled to a Thermo Scientific TSQ Quantiva MS/MS detector. The obtained results showed that the F. graminearum strains from alternative host plants could produce nivalenol (NIV), deoxynivalenol (DON), fusarenon-X (FUS-X), 3-ADON, deoxynivalenol-3-ß-d-glucoside (D3G), 15-ADON, and zearalenone (ZEA). F. graminearum strains produced DON and ZEA under both temperatures, with the mean concentrations varying from 363 to 112,379 µg kg-1 and from 1452 to 44,816 µg kg-1, respectively. Our results indicated the possible role of dicotyledonous plants, including weeds, as a reservoir of inoculum sources of F. graminearum-induced Fusarium head blight, associated with the risk of mycotoxin contamination in spring wheat.

Genotypes analysis of Candida albicans species complex from healthy individual saliva in Ahvaz, Iran.

Although Candida albicans is a part of the mycoflora of healthy individuals, it is causing mild to severe forms of candidiasis in patients with underlying diseases. The recent molecular investigations were classified three genotypes, A, B and C for C. albicans. The aim of the present study was to detect different genotypes of C. albicans complex species in a normal population in Iran. Saliva was randomly collected from a normal population, homogenized and cultured on CHROMagar Candida. Classical and molecular methods were used for the detection of isolates. Candida 25S rDNA gene was amplified using the primer pairs of CA-INT-L and CA-INT-R for ABC genotyping of C. albicans. Candida albicans complex was recovered from 103/194 (53·1%) normal papulation. Genotype A with a frequency of 41·7% was the most common isolate, followed by genotype C (34%), genotype B (20·4%) and genotype D (3·9%). Genotyping of C. albicans species complex from healthy individuals showed the presence of three A, B and C genotypes of C. albicans and one D genotype of C. dubliniensis.

Optimization of a Quantitative PCR Methodology for Detection of Aspergillus spp. and Rhizopus arrhizus.

INTRODUCTION: Multiplex quantitative polymerase chain reaction (qPCR) methods for the detection of Aspergillus spp. based only on SYBR Green and melting curve analysis of PCR products are difficult to develop because most targets are located within ITS regions. The aim of this study was to adapt our previously developed methodology based on a multiplex PCR assay coupled with GeneScan analysis to provide a qPCR method. METHODS: A SYBR Green-based real-time PCR assay was optimized to detect A. fumigatus, A. flavus, A. niger, A. terreus, and R. arrhizus in a multiplex assay and applied to cultured fungi and spiked plasma. RESULTS: Different melting temperatures allowed identification of all five pathogens and discrimination between them, even in samples with low amounts of fungal gDNA (from 1.3 to 33.0 pg/µL), which has been reported previously as problematic. No false-positive results were obtained for non-target species, including bacteria and human DNA. This method allowed detection of fungal pathogens in human plasma spiked with fungal DNA and in coinfections of A. niger/R. arrhizus. DISCUSSION: This work provides evidence for the use of a qPCR multiplex method based on SYBR Green and melting curve analysis of PCR products for the detection of A. fumigatus, A. flavus, A. niger, A. terreus, and R. arrhizus. The proposed method is simpler and less expensive than available kits based on fluorescent probes and can be used for aiding diagnosis of the most relevant invasive filamentous fungi, particularly in low-income health care institutions.