Sara Gago

Global and Multi-National Prevalence of Fungal Diseases—Estimate Precision

Fungal diseases kill more than 1.5 million and affect over a billion people. However, they are still a neglected topic by public health authorities even though most deaths from fungal diseases are avoidable. Serious fungal infections occur as a consequence of other health problems including asthma, AIDS, cancer, organ transplantation and corticosteroid therapies. Early accurate diagnosis allows prompt antifungal therapy; however this is often delayed or unavailable leading to death, serious chronic illness or blindness. Recent global estimates have found 3,000,000 cases of chronic pulmonary aspergillosis, ~223,100 cases of cryptococcal meningitis complicating HIV/AIDS, ~700,000 cases of invasive candidiasis, ~500,000 cases of Pneumocystis jirovecii pneumonia, ~250,000 cases of invasive aspergillosis, ~100,000 cases of disseminated histoplasmosis, over 10,000,000 cases of fungal asthma and ~1,000,000 cases of fungal keratitis occur annually. Since 2013, the Leading International Fungal Education (LIFE) portal has facilitated the estimation of the burden of serious fungal infections country by country for over 5.7 billion people (>80% of the world’s population). These studies have shown differences in the global burden between countries, within regions of the same country and between at risk populations. Here we interrogate the accuracy of these fungal infection burden estimates in the 43 published papers within the LIFE initiative.

Candida parapsilosis, Candida orthopsilosis, and Candida metapsilosis virulence in the non-conventional host Galleria mellonella

The incidence of fungal infections due to C. parapsilosis and closely related cryptic species (-psilosis complex) has increased in the last few years, but differences in virulence among these species have not been widely studied. Fifteen clinical isolates of C. parapsilosis, C. orthopsilosis, and C. metapsilosis, including the type strains, were used to evaluate their virulence in Galleria mellonella larvae. Fluctuations in the hemocyte density and in the phagocytic activity were also tested. Differences in the median survival for these species were demonstrated at 37 °C (2.6 ± 1.02, 2.3 ± 0.92, and 4.53 ± 1.65 d for C. parapsilosis, C. orthopsilosis, and C. metapsilosis, respectively). Galleria mellonella hemocytes phagocytosed C. metapsilosis strains more effectively than did for C. orthopsilosis and C. parapsilosis (P < 0.05). The phagocytosis rate was lower for C. parapsilosis than for C. orthopsilosis (P < 0.05). The hemocyte density was increased in larvae infected with C. metapsilosis compared with those infected with C. parapsilosis or C. orthopsilosis (P < 0.05). Moreover, in vitro studies of virulence factors such as pseudohyphae production and hydrolytic enzyme secretion showed that the capability of C. metapsilosis strains to produce those virulence factors was reduced. Infections due to -psilosis complex species produced tissue damage in G. mellonella and pseudohyphae could be also observed during infection with C. parapsilosis.

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

ABSTRACT 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 (CT ) for positive blood samples was 37.6, and the average CT 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.

A Multiplex Real-Time PCR Assay for Identification of Pneumocystis jirovecii, Histoplasma capsulatum, and Cryptococcus neoformans/Cryptococcus gattii in Samples from AIDS Patients with Opportunistic Pneumonia

ABSTRACT A molecular diagnostic technique based on real-time PCR was developed for the simultaneous detection of three of the most frequent causative agents of fungal opportunistic pneumonia in AIDS patients: Pneumocystis jirovecii, Histoplasma capsulatum, and Cryptococcus neoformans/Cryptococcus gattii. This technique was tested in cultured strains and in clinical samples from HIV-positive patients. The methodology used involved species-specific molecular beacon probes targeted to the internal transcribed spacer regions of the rDNA. An internal control was also included in each assay. The multiplex real-time PCR assay was tested in 24 clinical strains and 43 clinical samples from AIDS patients with proven fungal infection. The technique developed showed high reproducibility (r 2 of >0.98) and specificity (100%). For H. capsulatum and Cryptococcus spp., the detection limits of the method were 20 and 2 fg of genomic DNA/20 μl reaction mixture, respectively, while for P. jirovecii the detection limit was 2.92 log10 copies/20 μl reaction mixture. The sensitivity in vitro was 100% for clinical strains and 90.7% for clinical samples. The assay was positive for 92.5% of the patients. For one of the patients with proven histoplasmosis, P. jirovecii was also detected in a bronchoalveolar lavage sample. No PCR inhibition was detected. This multiplex real-time PCR technique is fast, sensitive, and specific and may have clinical applications.

Recurrent Episodes of Candidemia Due to Candida glabrata with a Mutation in Hot Spot 1 of the FKS2 Gene Developed after Prolonged Therapy with Caspofungin

ABSTRACT We report two episodes of recurrent candidemia caused by echinocandin-resistant Candida glabrata in a 69-year-old patient who underwent repeated abdominal surgery. In the first episode of candidemia, an echinocandin-susceptible Candida glabrata strain was isolated, and the patient was treated with caspofungin. The isolates from the later episodes showed resistance to echinocandins. Analysis of the HS1 region of the FKS2 gene showed the amino acid substitution S663P. Microsatellite analysis demonstrated a strong genetic relationship between the isolates.

High-Resolution Melting Analysis for Identification of the Cryptococcus neoformans-Cryptococcus gattii Complex

ABSTRACT 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 strain relatedness using High Resolution Melting in a case of recurrent candiduria

BackgroundSeveral 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.ResultsMICs 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.ConclusionsAlthough 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.

Molecular identification, antifungal resistance and virulence of Cryptococcus neoformans and Cryptococcus deneoformans isolated in Seville, Spain.

Cryptococcal meningitis is one of the leading causes of death in HIV/AIDS patients. Our aim was to in order to characterise the epidemiology, antifungal susceptibility pattern and virulence of 28 Cyptococcus sp. strains recovered from 12 AIDS patients during two years in a Spanish single institution. Antifungal susceptibility testing was performed according to the CLSI protocols. Clinical strains were molecularly characterised by serotyping, mating type, PCR fingerprinting (M13 and GACA4 microsatellites) and analysis of two rDNA regions (IGS1 and ITS). Sequencing of the ERG11 gene was used to explore mechanisms of fluconazole resistance. Differences in virulence between species were studied in a Galleria mellonella infection model. Cryptococcus deneoformans and C. deneoformans x Cryptococcus neoformans hybrids were the most frequent variety (65%) followed by C. neoformans (35%). Strains were categorised according to 13 microsatellite genotypes and mixed infections could be detected in three patients. Twenty‐nine per cent of the strains were fluconazole resistant. In one of the patients, the fluconazole resistance phenotype was associated with a point mutation in the ERG11 gene responsible for the amino acid substitution G470R. C. neoformans strains were able to kill G. mellonella larvae more efficiently than C. deneoformans and hybrids between both species. Precisely molecular characterisation of C. neoformans species is important for an accurate patients management.

Ribosomic DNA intergenic spacer 1 region is useful when identifying Candida parapsilosis spp. complex based on high-resolution melting analysis

The epidemiology of Candida parapsilosis and the closely related species C. orthopsilosis and C. metapsilosis has changed in recent years, justify the need to identify this complex at the species level. In this study we investigate the intergenic spacer 1 (IGS1) of the ribosomal DNA (rDNA) to evaluate the utility of this gene region as a phylogenetic molecular marker and the suitability of a high-resolution melting (HRM) strategy based on this region for identification of members of the C. parapsilosis spp. complex. We sequenced the IGS1 and the internal transcribed spacer (ITS) regions of the rDNA from 33 C. parapsilosis sensu lato strains. Although both regions are useful in identifying species, comparative sequence analysis showed that the diversity in the IGS1 region was higher than in the ITS sequences. We also developed an HRM analysis that reliably identifies C. parapsilosis spp. complex based on the amplification of 70 bp in the IGS1 region. All isolates were correctly identified with a confidence interval >98%. Our results demonstrate that HRM analysis based on the IGS1 region is a powerful tool for distinguishing C. parapsilosis from cryptic species.

Development and validation of a quantitative real-time PCR assay for the early diagnosis of coccidioidomycosis ☆

A new real-time polymerase chain reaction (RT-PCR) assay based on a Coccidioides genus-specific molecular beacon probe was developed for the detection of coccidioidomycosis and validated with tissues from animal models and clinical samples. The assay showed high analytic reproducibility (r(2) > 0.99) and specificity for cultured strains (100%); the lower limit of detection was 1 fg of genomic DNA/μL of reaction. Fungal burdens in the organs of mice infected with Coccidioides posadasii strain Silveira were more accurately quantified by RT-PCR compared to colony-forming unit for all tissues. The RT-PCR assay was positive for 97.7% of spleen and 100% of liver or lung. Progression of infection in all organs was similar by both methods (P > 0.05). The sensitivity of the assay also was 100% for paraffin-embedded samples and samples from patients with positive cultures. Our RT-PCR assay is effective for the diagnosis and monitoring of Coccidioides infection, and its use also avoids the biohazard and time delay of identifying cultures in the clinical setting.