African Histoplasmosis in HIV-Negative Patients, Kimpese, Democratic Republic of the Congo.

We describe a case series of histoplasmosis caused by Histoplasma capsulatum var. duboisii during July 2011-January 2014 in Kimpese, Democratic Republic of the Congo. Cases were confirmed by histopathology, immunohistochemistry, and reverse transcription PCR. All patients were HIV negative. Putative sources for the pathogen were cellar bats and guano fertilizer exploitation.

A matrix-assisted laser desorption/ionization time of flight mass spectrometry reference database for the identification of Histoplasma capsulatum.

The isolation of the pathogenic fungus Histoplasma capsulatum from cultures together with the visualization of typical intracellular yeast in tissues are the gold standard methods for diagnosis of histoplasmosis. However, cultures are time-consuming, require level 3 containment and experienced personnel, and usually call for an additional confirmation test. Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-ToF MS) has been established as a suitable tool for microbial identification in several clinical laboratories. A reference database has been constructed for the identification of H. capsulatum by MALDI-ToF MS by using six H. capsulatum strains previously identified by molecular methods. For validation, 63 fungal strains belonging to the Collection of the Spanish National Centre for Microbiology were tested against the new reference database combined with other commercial and in-house databases. In a blind assay, all H. capsulatum strains (n = 30) were correctly identified by the database and 86.6% had scores above 1.7. Considering both phases of the fungus for the same strain, the most reliable results were obtained with the mycelial phase, with only 13.3% of isolates having scores below 1.7. The new database was able to identify both morphological phases of the fungus. MALDI-ToF technology yields a prompt and simple identification from H. capsulatum yeast forms and early mycelial cultures. It allows for reducing response time and decreasing risk in fungus manipulation.

Usefulness of techniques based on real time PCR for the identification of onychomycosis-causing species.

Onychomycosis (OM) is a nail infection caused mainly by dermatophyte species but other species of yeast and moulds are frequently involved as well. Classical diagnosis has limitations thus empirical treatment is common. The usefulness of different real time PCR (RT-PCR) assays for identifying species causing OM was assessed in samples from seventy patients and fifteen controls. Conventional methods and four different RT-PCR assays were used: a panfungal, a pandermatophyte and two specific assays for detecting Candida spp. and Aspergillus spp. Fungal elements were visualised in 58% of the samples, and 54% of cultures were positive. Panfungal and pandermatophyte RT-PCR were positive in 28% and 60%, respectively, and the sensitivity relative to positive cultures was 47% and 90%. Candida spp. were detected in 76% of samples analysed and Aspergillus spp. in 60%. These species were also present in 80% of control cases. In conclusion, molecular techniques were useful but showed limitations. The panfungal assay showed a low sensitivity, the pandermatophyte assay was sensitive and specific but did not allow for differentiation among species of dermatophytes. Finally, the role of non-dermatophyte species detected by using specific RT-PCR techniques should be carefully analysed as these species were also present in healthy nails.

Cunninghamella bertholletiae infection in children: review and report of 2 cases with disseminated infection.

Mucormycosis is an emerging fungal infection affecting mainly immunosuppressed hosts. Cunninghamella bertholletiae causes the highest mortality among all mucormycetes. Infection by C. bertholletiae has rarely been reported in children. We present 2 children with acute leukemia and disseminated infection by C. bertholletiae, and review the relevant literature.

Analysis of strain relatedness using high resolution melting in a case of recurrent candiduria.

BACKGROUND: Several genotyping protocols have been described to study Candida albicans strains with different sensitivity values. In this study we have analyzed the genetic relatedness and the antifungal susceptibility of several Candida albicans strains isolated from a patient who from suffered recurrent candiduria for a period of five years. Strains were genotyped using Microsatellite Length Polymorphism (MLP) with three microsatellite markers (HIS 3, EF 3 and CDC 3), and a new method based on high resolution melting (HRM) was developed to analyze the microsatellite region. This method was compared with the conventional technique that uses capillary electrophoresis. RESULTS: MICs of the isolates showed the existence of fluconazole susceptible and resistant strains. An inter-colony test using single concentration (8 and 16 mg/l) of fluconazole revealed the coexistence of both fluconazole susceptible and resistant strains. Both genotyping analysis methods showed that all the patient's isolates had a clonal origin. HRM analysis method developed was able to accurately establish strain relatedness and presented a discriminatory power of 0.77. CONCLUSIONS: Although HRM analysis method presented a lower discriminatory power compared to methods based on capillary electrophoresis, it provided a more cost-effective and suitable alternative for genotyping C. albicans in a clinical laboratory.

Detection of invasive infection caused by Fusarium solani and non-Fusarium solani species using a duplex quantitative PCR-based assay in a murine model of fusariosis.

A duplex Real Time PCR (RT-PCR) assay for detecting DNA of members of the genus Fusarium has been developed and validated by using two mouse models of invasive infection. The duplex RT-PCR technique employed two specific molecular beacon probes targeting a highly conserved region of the fungal rDNA gene. This technique showed a detection limit of 10 fg DNA per µl of sample and a specificity of 100%. The sensitivity in a total of 48 samples from a murine model of Fusarium solani infection was 93.9% for lung tissues and 86.7% for serum samples. In comparison, the sensitivity in a total of 45 samples of a F. oxysporum murine model infection was 87% for lung tissues and 42.8% for serum samples. This molecular technique could be a reliable method for the quantification and the evaluation of the disease in animal models and for the clinical diagnosis of fusariosis.

[Current epidemiology and laboratory diagnosis of endemic mycoses in Spain]. / Epidemiología actual y diagnóstico de laboratorio de las micosis endémicas en España.

Histoplasmosis and paracoccidioidomycosis are emerging infections in Spain associated with immigration and travelling. In last three decades a total of 128 cases of histoplasmosis have been reported in Spain, 59 in travellers, 63 in immigrants, three associated to drug abuse, two in laboratory workers, and one in a solid organ transplant receptor. In 1969 the first Spanish case of paracoccidioidomycosis was published and a total of 21 cases have been reported so far. Those patients suffered from the chronic form of the disease with period of latency as long as 50 years. Other endemic mycoses such as blastomycosis, coccidioidomycosis, lobomycosis, pythiosis and sporotrichosis have not increased in frequency. Microbiological cultures of endemic fungi must be handled in facilities which comply with international biosafety regulations and must also be taken into account for cultures from patients with suspected endemic mycosis.

High-resolution melting analysis for identification of the Cryptococcus neoformans-Cryptococcus gattii complex.

We have developed a two-step method based on high-resolution melting (HRM) that reliably identifies species from the Cryptococcus species complex (Cryptococcus neoformans var. grubii, Cryptococcus neoformans var. neoformans, and Cryptococcus gattii). Our results indicate that HRM can provide a fast protocol to identify and distinguish among the main Cryptococcus species.

Analysis of performance of a PCR-based assay to detect DNA of Aspergillus fumigatus in whole blood and serum: a comparative study with clinical samples.

The performance of a real-time PCR-based assay was retrospectively analyzed (according to European Organization for Research and Treatment of Cancer/Mycosis Study Group criteria) in the samples of patients with invasive aspergillosis. A total of 711 serial samples (356 whole-blood and 355 serum samples) from 38 adult patients were analyzed. The Aspergillus fumigatus PCR assay results were positive for 89 of 356 (25%) whole-blood samples and 90 of 355 (25.35%) serum samples. Positive PCR results were seen in 29 of 31 (93.5%) patients for which serum was analyzed and in 31 of 33 (93.9%) cases with whole-blood specimens. Both blood and serum samples were available in 26 cases, and significant differences were not observed in this subgroup of cases. The average number of threshold cycles (C(T)) for positive blood samples was 37.6, and the average C(T) for serum was 37.4. The DNA concentration ranged between 2 and 50 fg per µl of sample, with average DNA concentrations of 10.2 and 11.7 fg in positive blood and serum samples, respectively (P > 0.01). The performance of this PCR-based quantitative assay was similar for both serum and blood samples. We recommend serum samples as the most convenient hematological sample to use for Aspergillus DNA quantification when serial determinations are done.

Global guideline for the diagnosis and management of the endemic mycoses: an initiative of the European Confederation of Medical Mycology in cooperation with the International Society for Human and Animal Mycology.

The global burden of the endemic mycoses (blastomycosis, coccidioidomycosis, emergomycosis, histoplasmosis, paracoccidioidomycosis, sporotrichosis, and talaromycosis) continues to rise yearly and these infectious diseases remain a leading cause of patient morbidity and mortality worldwide. Management of the associated pathogens requires a thorough understanding of the epidemiology, risk factors, diagnostic methods and performance characteristics in different patient populations, and treatment options unique to each infection. Guidance on the management of these infections has the potential to improve prognosis. The recommendations outlined in this Review are part of the "One World, One Guideline" initiative of the European Confederation of Medical Mycology. Experts from 23 countries contributed to the development of these guidelines. The aim of this Review is to provide an up-to-date consensus and practical guidance in clinical decision making, by engaging physicians and scientists involved in various aspects of clinical management.

Comparison of the Vitek 2 antifungal susceptibility system with the clinical and laboratory standards institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) Broth Microdilution Reference Methods and with the Sensititre YeastOne and Etest techniques for in vitro detection of antifungal resistance in yeast isolates.

The commercial technique Vitek 2 system for antifungal susceptibility testing of yeast species was evaluated. A collection of 154 clinical yeast isolates, including amphotericin B- and azole-resistant organisms, was tested. Results were compared with those obtained by the reference procedures of both the CLSI and the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Two other commercial techniques approved for clinical use, the Etest and the Sensititre YeastOne, were included in the comparative exercise as well. The average essential agreement (EA) between the Vitek 2 system and the reference procedures was >95%, comparable with the average EAs observed between the reference procedures and the Sensititre YeastOne and Etest. The EA values were >97% for Candida spp. and stood at 92% for Cryptococcus neoformans. Intraclass correlation coefficients (ICC) between the commercial techniques and the reference procedures were statistically significant (P<0.01). Percentages of very major errors were 2.6% between Vitek 2 and the EUCAST technique and 1.6% between Vitek 2 and the CLSI technique. The Vitek 2 MIC results were available after 14 to 18 h of incubation for all Candida spp. (average time to reading, 15.5 h). The Vitek 2 system was shown to be a reliable technique to determine antifungal susceptibility testing of yeast species and a more rapid and easier alternative for clinical laboratories than the procedures developed by either the CLSI or EUCAST.

Activity profile in vitro of micafungin against Spanish clinical isolates of common and emerging species of yeasts and molds.

A collection of 2,278 isolates belonging to 86 different fungal species was tested with micafungin and eight other drugs using the EUCAST procedures. Micafungin was active against species of Candida and Aspergillus (even azole-resistant species) as well as Penicillium spp., Scedosporium apiospermum, and Acremonium spp. It was inactive for species of Basidiomycota and Mucorales and for multiresistant species such as those of Fusarium.

Value of serial quantification of fungal DNA by a real-time PCR-based technique for early diagnosis of invasive Aspergillosis in patients with febrile neutropenia.

A study was designed to assess the reliability of the serial detection of Aspergillus sp. DNA to diagnose invasive aspergillosis (IA) in patients with febrile neutropenia. Two blood and two serum samples were taken weekly from 83 patients. A total of 2,244 samples were analyzed by real-time quantitative PCR. Twelve (14.4%) patients were diagnosed with IA. Taking two consecutive positive results as the diagnostic criterion, PCR detected 11 cases, with 4 false positives, giving sensitivity, specificity, positive, and negative predictive values of 91.6%, 94.4%, 73.3%, and 98.5%, respectively. On analyzing in conjunction with high-resolution chest tomography (HRCT) and galactomannan (GM) testing, the combination of serial PCR and GM detected 100% of aspergillosis cases, with a positive predictive value of 75.1%. This diagnostic strategy presented, according to CART analysis, a receiver-operator curve with an area under the curve of 0.97 (95% confidence interval, 0.895 to 1.032; P < 0.01), with a relative risk of IA 6.92 times higher than the control population and with predictive success of 95.2%. As regards early diagnosis, the serial detection of Aspergillus DNA took on average 21 days less than HRCT and 68 days less than GM. The serial detection of Aspergillus DNA using real-time quantitative PCR has great diagnostic applicability, which increases when combined with GM quantification.

Development and validation of a quantitative PCR assay for diagnosis of scedosporiosis.

Scedosporium apiospermum and Scedosporium prolificans are fungal pathogens that can cause severe human infections, including disseminated mycosis in immunocompromised patients. Two real-time PCR (RT-PCR) assays for the diagnosis of these species were developed and validated for the classification of clinical strains and for the detection of DNA in clinical samples by use of a murine model of invasive infection. A total of 14 clinical strains and 141 samples, including blood, serum, and lung samples from infected CD1 mice, were analyzed. Each RT-PCR methodology used a species-specific molecular beacon probe targeting a highly conserved region of the fungal ribosomal DNA gene. Results showed 100% specificity and a detection limit of 10 fg of DNA for both assays. The sensitivities for the S. prolificans-specific PCR assay were 100% for cultured clinical strains, 95.5% for lung tissues, 85% for serum, and 83.3% for blood. For S. apiospermum, the sensitivities were 100% for clinical strains and 97.2%, 81.8%, and 54.5% for lung tissues, serum, and blood, respectively. Both techniques can be useful for clinical diagnosis, and further studies are warranted.

Disseminated pulmonary adiaspiromycosis in a crested porcupine (Hystrix cristata Linnaeus, 1758).

Adiaspiromycosis is primarily a necrotizing granulomatous pneumonia caused by a dimorphic fungus of the genus Emmonsia. A young crested porcupine (Hystrix cristata) found dead showed multiple fractures, chronic pleuritis, and granulomatous pneumonia. Microscopically, cystic structures were consistent with adiaspiromycosis by Emmonsia crescens. The diagnosis was confirmed using molecular methods.

Histoplasmosis and paracoccidioidomycosis in a non-endemic area: a review of cases and diagnosis.

BACKGROUND: Histoplasmosis and paracoccidioidomycosis (PCM) have increased in Spain in recent years, due firstly to the migration from endemic regions and secondly to travelers returning from these regions. In non-endemic areas, diagnosis of both diseases is hampered by the lack of experience, long silent periods, and the resemblance to other diseases such as tuberculosis and sarcoidosis. METHODS: A total of 39 cases of imported histoplasmosis and 6 cases of PCM diagnosed in the Spanish Mycology Reference Laboratory since 2006 were analyzed. Microbiological diagnosis was performed using classical methods and also a specific real-time polymerase chain reaction (RT-PCR) assay for each microorganism. RESULTS: We had 9 cases of probable histoplasmosis in travelers and 30 cases in immigrants, 29 of whom were defined as proven. Paracoccidioidomycosis (PCM) cases were either immigrants or people who had lived for a long period of time in endemic regions, all of whom were classified as proven cases. Cultures showed a good sensitivity in detecting Histoplasma capsulatum in immigrants with proven histoplasmosis (73%); however, growth was very slow. The fungus was never recovered in traveler patients. Paracoccidioides brasiliensis was isolated in a culture only in one case of the proven PCM. Serological methods were not very reliable in immunocompromised patients with histoplasmosis (40%). A PCR-based technique for histoplasmosis detected 55.5% of the cases in travelers (probable cases) and 89% of the cases in immigrants (proven). The PCR method for PCM detected 100% of the cases. CONCLUSIONS: These kinds of mycoses are increasingly frequent in non-endemic areas, and newer and faster techniques should be used to reach an early diagnosis. The RT-PCR techniques developed appear to be sensitive, specific, and fast and could be helpful to detect those mycoses. However, it is also essential that physicians perform differential diagnosis in individuals coming from endemic areas.

In vitro activities of 35 double combinations of antifungal agents against Scedosporium apiospermum and Scedosporium prolificans.

Activities of 35 combinations of antifungal agents against Scedosporium spp. were analyzed by a checkerboard microdilution design and the summation of fractional concentration index. An average indifferent effect was detected apart from combinations of azole agents and echinocandins against Scedosporium apiospermum. Antagonism was absent for all antifungal combinations against both species.