RESUMO
Milbemycin oximes are macrocyclic lactones that have a broad spectrum of activity against nematode infection in animals. They are known to block drug efflux, which increases the susceptibility of fungi to azoles. We investigated the effects of milbemycin on the azole susceptibility of fungi (Aspergillus fumigatus, Candida albicans, C. auris, Cryptococcus neoformans, and Trichophyton rubrum). To screen for changes in azole susceptibility, fungal growth was tested on a culture medium containing 1 µg/ml milbemycin. The results showed that milbemycin increased the azole susceptibility of azole-resistant strains of C. albicans, C. auris, C. neoformans, and T. rubrum. Thus, milbemycin might be useful against antifungal drug-resistant strains.
Milbemycin blocks drug efflux and increases the azole susceptibility of azole-resistant strains of Candida albicans, C. auris, Cryptococcus neoformans, and Trichophyton rubrum. This drug is expected to be a game changer against antifungal drug-resistant infections.
Assuntos
Cryptococcus neoformans , Macrolídeos , Micoses , Animais , Antifúngicos/farmacologia , Antifúngicos/uso terapêutico , Farmacorresistência Fúngica , Micoses/veterinária , Azóis/farmacologia , Testes de Sensibilidade Microbiana/veterináriaRESUMO
Candida auris is resistant to multiple antifungal agents. This study investigated its antifungal susceptibility and explored FKS1 mutations across the isolates from mice enterically colonized with wild-type C. auris and treated with echinocandin. Resistant C. auris with FKS1 mutations, including S639F, S639Y, D642Y, R1354H, or R1354Y, were isolated and found to be micafungin- and caspofungin-resistant in vivo; however, the MICs of isolates with mutation in R1354 remained below the micafungin breakpoint in vitro.
Assuntos
Candida auris , Equinocandinas , Animais , Camundongos , Antifúngicos/farmacologia , Antifúngicos/uso terapêutico , Farmacorresistência Fúngica/genética , Equinocandinas/farmacologia , Equinocandinas/genética , Trato Gastrointestinal , Micafungina/farmacologia , Testes de Sensibilidade Microbiana , Mutação/genéticaRESUMO
Candida auris is a health hazard because of its antifungal resistance and the potential to cause healthcare-associated outbreaks. To our knowledge, no previous cases of candidemia caused by C. auris have been reported in Japan. Herein, we report the first known case of clade I C. auris candidemia in a Japanese man with coronavirus disease 2019 (COVID-19) infection who was medically evacuated from the Philippines. A 71-year-old Japanese man traveled to Cebu Island in the Philippines 5 months before admission to our hospital. He contracted severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the Philippines and was admitted to the intensive care unit (ICU) in a local hospital. During his medical evacuation, we implemented precautions given his history of COVID-19 and pneumonia caused by multi-drug-resistant Acinetobacter baumannii complex. His blood culture revealed that C. auris infection was treated with antifungal agents but he did not survive. No evidence of nosocomial transmission was found among other patients in the ICU. This case study determines that accurate detection of C. auris, appropriate antifungal agent selection, precautions, and patient isolation are crucial to prevent nosocomial outbreaks, especially in patients with a history of multidrug-resistant organism (MDRO) colonization or international hospitalization. Medical professionals should recognize the risk of MDROs in international medical evacuation settings, considering the recent resumption of cross-border travel after the COVID-19 pandemic.
Assuntos
COVID-19 , Candidemia , Infecção Hospitalar , Masculino , Humanos , Idoso , Candidemia/microbiologia , Candida auris , Candida , COVID-19/epidemiologia , Pandemias , Japão , SARS-CoV-2 , Testes de Sensibilidade Microbiana , Filipinas , Antifúngicos/farmacologia , Antifúngicos/uso terapêutico , Infecção Hospitalar/microbiologiaRESUMO
Fungal keratitis is a corneal fungal infection that potentially leads to blindness and is mainly caused by filamentous fungi, such as Fusarium, with limited drug options available, such as natamycin and voriconazole. Therefore, this study aimed to evaluate the therapeutic effects of the imidazole antifungal drug-luliconazole-using a rabbit experimental model of fungal keratitis caused by Fusarium solani, which is the dominant causative agent of fungal keratitis. F. solani was inoculated into rabbit corneas. luliconazole 1% suspension or natamycin 5% eye drops were administered four times a day (N = 6 for each group) 3 days after inoculation. Signs were scored up to 14 days after inoculation to evaluate the efficacy of the drugs. Compared with the peak mean sign scores of the placebo control group, there was a significant decrease in the mean sign scores of both the treatment groups (P < 0.05). Sign score trends were similar between the two treatment groups. In conclusion, luliconazole demonstrated therapeutic efficacy comparable to that of natamycin in treating experimental fungal keratitis. This suggests that luliconazole can be a novel therapeutic agent for human fungal keratitis.
RESUMO
Fungal keratitis is a severe corneal infection, and the causative fungi include various rare fungal species. Fungal keratitis caused by Talaromyces species has yet to be reported, and there is no information about this fungus as a cause of keratitis. A 77-year-old man developed fungal keratitis while waiting for a donor cornea due to bullous keratopathy in his left eye. Fungal culture of a corneal scraping grew filamentous fungi, which were morphologically identified as Paecilomyces species. The corneal infection did not improve after topical administration of 1% voriconazole, and ribosomal DNA sequencing definitively verified the fungus to be Talaromyces coalescens. The lesion gradually improved after switching to topical 5% natamycin. Antifungal susceptibility tests determined the high minimum inhibitory concentrations of voriconazole to be > 8 µg/mL. This is the first report of Talaromyces fungal keratitis. Clinicians, especially those in ophthalmology, need to be aware of this rare fungus.
Assuntos
Úlcera da Córnea , Infecções Oculares Fúngicas , Ceratite , Talaromyces , Masculino , Humanos , Idoso , Voriconazol , Antifúngicos/farmacologia , Antifúngicos/uso terapêutico , Úlcera da Córnea/diagnóstico , Úlcera da Córnea/tratamento farmacológico , Infecções Oculares Fúngicas/diagnóstico , Infecções Oculares Fúngicas/tratamento farmacológico , Natamicina/uso terapêutico , Ceratite/diagnóstico , Ceratite/tratamento farmacológico , Ceratite/microbiologiaRESUMO
Skin colonization by the emerging pathogen Candida auris is common in outbreaks within medical settings. Culture-based screening of patients is an effective management strategy to control the pathogen, and the newly developed CHROMagar™ Candida Plus medium is claimed to enable the presumptive identification of C. auris. Here, we evaluated the use of this medium with 63 C. auris strains comprising its four well-established clades, as well as genetically related comparators, including species from the Metschnikowia clade. The colors and halos of both confluent growth and discrete colonies of all the tested strains were compared. It was found that on CHROMagar™ Candida Plus, C. auris formed characteristic white colonies with blue-green halos that were more evident after 72 hr of incubation at 35°C than after 48 hr. However, distinguishing between closely related species such as Candida haemulonii, Candida pseudohaemulonii, and Candida duobushaemulonii required the consideration of parameters other than color, including colony size and growth ability at 35°C. In conclusion, the novel chromogenic medium CHROMagar™ Candida Plus constitutes an easy screening tool for C. auris.
Assuntos
Candida , Candidíase , Ágar , Antifúngicos , Candida auris , Candidíase/diagnóstico , HumanosRESUMO
This study targets on-site/real-time taxonomic identification and metabolic profiling of seven different Candida auris clades/subclades by means of Raman spectroscopy and imaging. Representative Raman spectra from different Candida auris samples were systematically deconvoluted by means of a customized machine-learning algorithm linked to a Raman database in order to decode structural differences at the molecular scale. Raman analyses of metabolites revealed clear differences in cell walls and membrane structure among clades/subclades. Such differences are key in maintaining the integrity and physical strength of the cell walls in the dynamic response to external stress and drugs. It was found that Candida cells use the glucan structure of the extracellular matrix, the degree of α-chitin crystallinity, and the concentration of hydrogen bonds between its antiparallel chains to tailor cell walls' flexibility. Besides being an effective ploy in survivorship by providing stiff shields in the α-1,3-glucan polymorph, the α-1,3-glycosidic linkages are also water-insoluble, thus forming a rigid and hydrophobic scaffold surrounded by a matrix of pliable and hydrated ß-glucans. Raman analysis revealed a variety of strategies by different clades to balance stiffness, hydrophobicity, and impermeability in their cell walls. The selected strategies lead to differences in resistance toward specific environmental stresses of cationic/osmotic, oxidative, and nitrosative origins. A statistical validation based on principal component analysis was found only partially capable of distinguishing among Raman spectra of clades and subclades. Raman barcoding based on an algorithm converting spectrally deconvoluted Raman sub-bands into barcodes allowed for circumventing any speciation deficiency. Empowered by barcoding bioinformatics, Raman analyses, which are fast and require no sample preparation, allow on-site speciation and real-time selection of appropriate treatments.
Assuntos
Candidíase , beta-Glucanas , Antifúngicos/farmacologia , Candida auris , Quitina , Glucanos , ÁguaRESUMO
Oral candidiasis, a common opportunistic infection of the oral cavity, is mainly caused by the following four Candida species (in decreasing incidence rate): Candida albicans, Candida glabrata, Candida tropicalis, and Candida krusei. This study offers in-depth Raman spectroscopy analyses of these species and proposes procedures for an accurate and rapid identification of oral yeast species. We first obtained average spectra for different Candida species and systematically analyzed them in order to decode structural differences among species at the molecular scale. Then, we searched for a statistical validation through a chemometric method based on principal component analysis (PCA). This method was found only partially capable to mechanistically distinguish among Candida species. We thus proposed a new Raman barcoding approach based on an algorithm that converts spectrally deconvoluted Raman sub-bands into barcodes. Barcode-assisted Raman analyses could enable on-site identification in nearly real-time, thus implementing preventive oral control, enabling prompt selection of the most effective drug, and increasing the probability to interrupt disease transmission.
Assuntos
Candida , Candidíase Bucal , Candida/química , Candida/genética , Candida albicans , Candidíase Bucal/diagnóstico , Quimiometria , Análise Espectral Raman/métodosRESUMO
As part of a series of studies regarding the microbiota in manned space environments, we isolated the fungal strains from nasal and pharyngeal smears and saliva of 21 astronauts preflight, in-flight, and postflight. On the ground, 120 strains from 43 genera of environmental fungi were isolated from the astronauts. The dominant fungal genera were Cladosporium, Penicillium, and Aspergillus. Only 18 strains from four genera were isolated from the astronauts inside the International Space Station. These fungi are currently thought to be harmless, but regular screening and cleaning are necessary to prevent fungus-related health disorders.
Assuntos
Voo Espacial , Astronautas , Fungos , Humanos , Microbiota , SalivaRESUMO
The International Space Station (ISS) is a closed facility that orbits the earth carrying not only its crew but also microorganisms. We have participated in microbiota analysis projects for the Japanese Experiment Module KIBO (ISS; operations nomenclature: Microbe-I, II, III, and IV) and were in charge of fungal screening. The interior of KIBO was sampled using swabs and microbe detection sheets (MDSs) for fungal detection. The dominant genera obtained by culture were Aspergillus and Penicillium. DNA analyses of the fungal biota using a clone library showed that KIBO was dominated by Malassezia, a fungal inhabitant of human skin. Three fungal species, Aspergillus sydowii, Penicillium palitans, and Rhodotorula mucilaginosa, which grew under microgravity in KIBO were observed under a field emission-scanning electron microscope on the ground. No novel phenotypic characteristics were noted. The results of antifungal susceptibility testing of all isolates did not differ significantly from previous reports of corresponding fungi. In Microbe-I (August 2009), MDSs were culture negative, while in the next stages the CFU of MDSs were 10 for Microbe-II (February 2011), 24 for Microbe-III (October 2012), and 151 for Microbe-IV (February 2015). These results indicated that fungi inside KIBO are increasing and expanding over time, and therefore continuous surveillance is crucial.
Assuntos
Fungos , Astronave , Aspergillus , Fungos/genética , Humanos , Japão , Penicillium , RhodotorulaRESUMO
The prevalence of fungal otitis externa, or otomycosis, has been increasing in recent decades. Fungi may act as primary pathogens in this condition, or they may occur as secondary infections after prolonged ototopical treatment with antibiotics, which alters the flora of the external auditory canal (EAC) and enables overgrowth of its fungal inhabitants. We report here a case of otomycosis by Candida parapsilosis, Malassezia obtusa, and Malassezia furfur as a secondary infection following prolonged otic ofloxacin treatment. To the best of our knowledge, although isolation of C. parapsilosis and M. furfur from the EAC is not uncommon, the recovery of M. obtusa has not yet been reported. We also conducted a literature review of the searchable data on PubMed concerning the isolation of Malassezia species from the human EAC.
Assuntos
Malassezia , Otite Externa , Otomicose , Fungos , Humanos , Ofloxacino , Otite Externa/diagnóstico , Otite Externa/tratamento farmacológicoRESUMO
BACKGROUND: Candida auris is an emerging pathogen associated with outbreaks in clinical settings. Isolates of the pathogen have been geographically clustered into four clades with high intra-clade clonality. Pathogenicity varies among the clades, highlighting the importance of understanding these differences. OBJECTIVES: To examine the physiological and biochemical properties of each clade of C. auris to improve our understanding of the fungus. METHODS: Optimal growth temperatures of four strains from three clades, East Asia, South Asia and South Africa, were explored. Moreover, assimilation and antifungal susceptibility properties of 22 C. auris strains from the three clades were studied. RESULTS: The optimal growth temperatures of all strains were 35-37 °C. Assimilation testing demonstrated that the commercial API ID 32 C system can be used to reliably identify C. auris based on the biochemical properties of the yeast. Notably, C. auris can be uniquely differentiated from commonly clinical fungi by its ability to assimilate raffinose and inability to utilize D-xylose, suggesting a useful simple screening tool. The antifungal susceptibility results revealed that all strains are resistant against fluconazole (minimal inhibitory concentration (MIC) 4 to > 64 µg/mL) and miconazole (MIC 8 to > 16 µg/mL), with strains from the Japanese lineage showing relatively lower MIC values (1-4 µg/mL). Conversely, itraconazole, voriconazole, amphotericin B, micafungin and caspofungin were active against most of the tested strains. On the clade level, East Asian strains generally showed lower MICs against azoles comparing to the other clades, while they displayed MICs against flucytosine higher than those of strains from South Africa and South Asia clades. CONCLUSION: Our data suggest a simple identification approach of C. auris based on its physiological and biochemical properties and highlight aspects of C. auris population from various clades.
Assuntos
Antifúngicos , Candida , Anfotericina B , Antifúngicos/uso terapêutico , Ásia , Fluconazol , Testes de Sensibilidade MicrobianaRESUMO
Seborrheic dermatitis (SD) is a multifactorial disease in which Malassezia restricta has been proposed as the predominant pathogenic factor. However, experimental evidence supporting this hypothesis is limited. A guinea pig SD model using a clinical isolate of M. restricta was used to elucidate the pathogenicity of M. restricta. Also, the efficacy of 1% luliconazole (LLCZ) cream, a topical imidazole derivative, against M. restricta was compared with that of a 2% ketoconazole (KCZ) cream in the same guinea pig model. Dorsal skin hairs of guinea pig were clipped and treated with M. restricta by single or repeated inoculations without occlusion. Skin manifestations were examined macroscopically and histologically. A quantitative polymerase chain reaction (PCR) assay was also performed for mycological evaluation. An inflammatory response mimicking SD occurred after repeated as well as single inoculation but not in abraded skin. The inflammation score attained its maximum on day 11 and persisted until day 52. The yeast form of the fungal elements was distributed on the surface of stratum corneum and around the follicular orifices, and an epidermal and dermal histological reaction was observed. Application of 1% LLCZ or 2% KCZ cream significantly improved the skin manifestations and decreased the quantity of M. restricta rDNA in the skin lesions. The efficacy of topical antifungal drugs suggested that M. restricta is a pathogenic factor contributing to SD.
Assuntos
Antifúngicos/uso terapêutico , Dermatite Seborreica/tratamento farmacológico , Imidazóis/uso terapêutico , Malassezia/efeitos dos fármacos , Pele/efeitos dos fármacos , Administração Tópica , Animais , Antifúngicos/farmacologia , Dermatite Seborreica/microbiologia , Modelos Animais de Doenças , Epiderme/efeitos dos fármacos , Epiderme/microbiologia , Cobaias , Humanos , Imidazóis/farmacologia , Cetoconazol/farmacologia , Cetoconazol/uso terapêutico , Malassezia/isolamento & purificação , Masculino , Pele/microbiologia , Pele/patologia , Creme para a Pele/química , Creme para a Pele/uso terapêutico , Organismos Livres de Patógenos EspecíficosRESUMO
In vitro antifungal activity of luliconazole against nondermatophytic moulds causing superficial infections was compared with that of five classes of 12 topical and systemic drugs. The minimum inhibitory concentration (MIC) of the drugs against the genera of Neoscytalidium, Fusarium, Aspergillus, Scedosporium, and Alternaria was measured via modified microdilution method. In results, the nondermatophytic moulds were found to be less susceptible to drugs to which Neoscytalidium spp. and Fusarium spp. were typically drug resistant. However, luliconazole was effective against all the genera tested, including afore-mentioned two species, and had the lowest MICs among the drugs tested.
Assuntos
Antifúngicos/farmacologia , Fungos/efeitos dos fármacos , Imidazóis/farmacologia , Anfotericina B/farmacologia , Clotrimazol/farmacologia , Fluconazol/farmacologia , Fungos/classificação , Humanos , Itraconazol/farmacologia , Cetoconazol/farmacologia , Miconazol/farmacologia , Testes de Sensibilidade Microbiana , Morfolinas/farmacologia , Análise de Sequência de DNA , Terbinafina/farmacologia , Triazóis/farmacologia , Voriconazol/farmacologiaRESUMO
BACKGROUND: Dermatophytes are a group of keratinophilic fungi of medical importance. Despite a relatively long history of molecular taxonomic studies, there is still a need for information on genetic polymorphism in wider variety of genomic loci. OBJECTIVES: Our goal was to study partial DNA topoisomerase 2 gene (TOP2) polymorphism in dermatophytes. METHODS: We performed DNA sequencing of TOP2 in 26 dermatophyte species along with ribosomal internal transcribed spacer (ITS) sequencing. RESULTS: The number of polymorphic sites in TOP2 data set was similar to that one in ITS data set. Nannizzia species formed paraphyletic group in TOP2 tree. Trichophyton simii was paraphyletic in concatenated TOP2-ITS tree, one of its two clades contained solely Iranian isolates. CONCLUSIONS: Our results revealed several unresolved problems in the taxonomy of dermatophytes, including probable polyphyly of the genus Nannizzia and the species T simii.
Assuntos
Arthrodermataceae/enzimologia , Arthrodermataceae/genética , DNA Topoisomerases Tipo II/genética , DNA Fúngico/genética , Polimorfismo Genético , Arthrodermataceae/classificação , DNA Espaçador Ribossômico/genética , Irã (Geográfico) , Filogenia , Análise de Sequência de DNARESUMO
Nannizzia gypsea is a geophilic dermatophyte, previously known as Microsporum gypseum before renaming under the new taxonomy. This organism is distributed all over the world and is considered to be involved in keratin degradation in the soil. Generally, human infection involves direct contact with fertile soil. Tinea caused by geophilic dermatophytes is much rarer than that caused by anthropophilic dermatophytes. According to the latest survey in Japan, dermatophytosis due to N. gypsea accounted for only 0.4% of cases. Clinical presentations vary and may mimic other inflammatory dermatitis, leading to incorrect diagnosis and delayed treatment. According to that past report, distal parts of the upper and lower extremities were more commonly affected, followed by the trunk, face and scalp, and rarely the nail plate. A 38-year-old woman presented with an approximately 3-week history of an itchy, solitary erythematous lesion on the left medial angle of the eyelid. Direct microscopic examination of scales revealed fungal elements, and the causative agents was identified as N. gypsea by morphological and molecular biological diagnoses. The eruption improved with systemic itraconazole treatment at 100 mg/day for 8 weeks. No recurrence has been seen for a year. However, she had no history of contact with any infectious source. Herein, we report a case of tinea faciei due to N. gypsea with an uncommon site and route of infection.
Assuntos
Arthrodermataceae , Pálpebras , Tinha , Adulto , Pálpebras/microbiologia , Pálpebras/patologia , Feminino , Humanos , Japão , Microsporum , Tinha/microbiologiaRESUMO
BACKGROUND: Candida albicans is the most frequent pathogenic fungus in oral cavities. It adheres to dental tissues as part of dental plaques and contributes to caries formation. OBJECTIVES: To evaluate the effect of silver diamine fluoride (SDF) on reducing C. albicans adhesion on dentine surfaces. METHODS: Flat dentine surfaces were prepared from bovine dental disks, and samples were divided into three groups. The first and second groups were pretreated for 3 min with 299 mM or 2.99 M SDF, respectively, and the third group (control) did not undergo any SDF pretreatment. All samples were washed, inoculated with C. albicans suspension onto their dentine surface, incubated at 30 °C for 6 h, and washed again to remove any nonadherent cells. The abundance of adherent cells was investigated using colorimetric and real-time polymerase chain reaction approaches. Subsequently, the morphological changes in C. albicans by pretreatment with SDF were observed under a scanning electron microscope (SEM). RESULTS: SDF inhibited candidal growth at concentrations as low as 2.99 µM. Dentine disks pretreated with 299 mM or 2.99 M SDF displayed significantly fewer adhered cells as compared with the control group. Upon pretreatment with SDF, SEM images showed severe morphological changes in the cellular walls, in a dose-dependent manner, suggesting a fungicidal effect of SDF against the yeast. CONCLUSION: SDF should be considered for clinical applications aimed at inhibiting dental plaque caused by C. albicans, particularly in children and elderly individuals.
Assuntos
Candida albicans , Dentina/microbiologia , Compostos de Amônio Quaternário/farmacologia , Compostos de Prata/farmacologia , Animais , Candida albicans/efeitos dos fármacos , Bovinos , Fluoretos Tópicos/farmacologiaRESUMO
The mechanisms of terbinafine resistance in a set of clinical isolates of Trichophyton rubrum have been studied recently. Of these isolates, TIMM20092 also showed reduced sensitivity to azoles. The azole resistance of TIMM20092 could be inhibited by milbemycin oxime, prompting us to examine the potential of T. rubrum to develop resistance through multidrug efflux transporters. The introduction of a T. rubrum cDNA library into Saccharomyces cerevisiae allowed the isolation of one transporter of the major facilitator superfamily (MFS) conferring resistance to azoles (TruMFS1). To identify more azole efflux pumps among 39 ABC and 170 MFS transporters present within the T. rubrum genome, we performed a BLASTp analysis of Aspergillus fumigatus, Candida albicans, and Candida glabrata on transporters that were previously shown to confer azole resistance. The identified candidates were further tested by heterologous gene expression in S. cerevisiae Four ABC transporters (TruMDR1, TruMDR2, TruMDR3, and TruMDR5) and a second MFS transporter (TruMFS2) proved to be able to operate as azole efflux pumps. Milbemycin oxime inhibited only TruMDR3. Expression analysis showed that both TruMDR3 and TruMDR2 were significantly upregulated in TIMM20092. TruMDR3 transports voriconazole (VRC) and itraconazole (ITC), while TruMDR2 transports only ITC. Disruption of TruMDR3 in TIMM20092 abolished its resistance to VRC and reduced its resistance to ITC. Our study highlights TruMDR3, a newly identified transporter of the ABC family in T. rubrum, which can confer azole resistance if overexpressed. Finally, inhibition of TruMDR3 by milbemycin suggests that milbemycin analogs could be interesting compounds to treat dermatophyte infections in cases of azole resistance.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Antifúngicos/farmacologia , Azóis/farmacologia , Trichophyton/efeitos dos fármacos , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Antifúngicos/metabolismo , Azóis/metabolismo , Farmacorresistência Fúngica , Humanos , Macrolídeos/metabolismo , Macrolídeos/farmacologia , Testes de Sensibilidade Microbiana , Terbinafina/metabolismo , Terbinafina/farmacologia , Tinha/tratamento farmacológico , Tinha/microbiologia , Trichophyton/metabolismoRESUMO
Chronic pulmonary aspergillosis (CPA) is a common subtype of pulmonary aspergillosis and a life-threatening disease. However, its diagnosis remains difficult due to the lack of specific clinical features and radiologic findings, as well as the difficulty of isolating Aspergillus spp. We developed a novel species-specific detection method of medically important aspergilli using a loop-mediated isothermal amplification (LAMP) for CPA. Specific LAMP primer sets for Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus, and Aspergillus nidulans were designed. The use of the LAMP assay was validated using respiratory specimens (CPA cases, n = 21; nonaspergillosis cases, n = 23). A total of 15 cases were positive in the CPA group (A. fumigatus, n = 5; A. flavus, n = 1; A. niger, n = 1; A. terreus, n = 7; A. nidulans, n = 1), but only three in the non-CPA group (A. niger, n = 2; A. terreus n = 1). The sensitivity and specificity of the diagnosis of CPA by the LAMP system were 71.4% and 87.0%, respectively. In conclusion, we developed a species-specific detection approach for five medically important aspergilli using the LAMP method. The system showed high sensitivity and specificity for diagnosis of CPA.
Assuntos
Aspergillus/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico , Aspergilose Pulmonar/diagnóstico , Aspergilose Pulmonar/microbiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Aspergillus/classificação , Doença Crônica , Primers do DNA/genética , Feminino , Humanos , Pulmão/microbiologia , Masculino , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Especificidade da EspécieRESUMO
In Japan, an epidemiological survey of onychomycosis pathogens was performed using culture methods; however, the positive culture rate was 40% or less. As part of an epidemiological survey of dermatomycoses in Japan, we overcame this low positive rate by employing a real-time polymerase chain reaction (PCR) assay that allowed rapid and accurate detection and identification. In 2011, nail specimens were collected from patients at nine institutes in various prefectures in Japan and diagnosed as onychomycosis. For the detection and identification of the main pathogens causing onychomycosis, we performed real-time PCR using specific TaqMan® MGB probes and primer sets. Of the 496 onychomycosis samples, real-time PCR detected 382 cases (77.0%) caused by Trichophyton rubrum; 74 cases (15.0%) caused by Trichophyton interdigitale; and eight cases (1.6%) caused by Candida albicans. The real-time PCR positive rate was 96.2%. The most frequent pathogen was T. rubrum throughout life, with the number of patients affected peaking in the range of 60 to 69 years of age and no significant differences in the composition of causative pathogens by sex. We were able to detect and identify pathogens from almost all specimens and succeeded in analyzing the pathogens involved in onychomycosis cases in Japan. These data confirmed that our real-time PCR method was effective for detecting and identifying the main fungal pathogens from onychomycosis specimens.