A multicentre external quality assessment: A first step to standardise PCR protocols for the diagnosis of histoplasmosis and coccidioidomycosis.

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

Low sensitivity of conventional fungal agars in fungemia by Rhodotorula mucilaginosa: description of two cases.

BACKGROUND: Although most bloodstream yeast infections are caused by Candida spp., infections by rare or less common species have increased in recent years. Diagnosis of infections caused by these species is difficult due to the lack of specific symptoms and adequate diagnostic tools. CASES PRESENTATION: We describe two cases of fungemia by Rhodotorula mucilaginosa within a few months of each other, in a secondary Spanish hospital. In both cases, diagnosis was challenging. Blood subcultures in conventional fungal media were persistently negatives and the use of non-conventional fungal media was essential for isolating the yeasts and achieving a correct diagnosis. 1-3 beta-D-glucan detection and a panfungal PCR assay were helpful techniques to confirm the diagnosis CONCLUSION: It is highly important to establish an early diagnosis for fungemia. The process is challenging because often non-specific symptoms are presents. When yeasts grow in blood cultures other genera than Candida spp. could be the cause of infection. Patient risk factors should be assessed to incorporate alternative culture media and the available rapid diagnostic test, in order to provide an early recognition of the pathogen.

New Panfungal Real-Time PCR Assay for Diagnosis of Invasive Fungal Infections.

The diagnosis of invasive fungal infections (IFIs) is usually based on the isolation of the fungus in culture and histopathological techniques. However, these methods have many limitations often delaying the definitive diagnosis. In recent years, molecular diagnostics methods have emerged as a suitable alternative for IFI diagnosis. When there is not a clear suspicion of the fungus involved in the IFI, panfungal real-time PCR assays have been used, allowing amplification of any fungal DNA. However, this approach requires subsequent amplicon sequencing to identify the fungal species involved, increasing response time. In this work, a new panfungal real-time PCR assay using the combination of an intercalating dye and sequence-specific probes was developed. After DNA amplification, a melting curve analysis was also performed. The technique was standardized by using 11 different fungal species and validated in 60 clinical samples from patients with proven and probable IFI. A melting curve database was constructed by collecting those melting curves obtained from fungal species included in the standardization assay. Results showed high reproducibility (coefficient of variation [CV] < 5%; r > 0.95) and specificity (100%). The overall sensitivity of the technique was 83.3%, with the group of fungi involved in the infection detected in 77.8% of the positive samples with IFIs covered by molecular beacon probes. Moreover, sequencing was avoided in 67.8% of these "probe-positive" results, enabling report of a positive result in 24 h. This technique is fast, sensitive, and specific and promises to be useful for improving early diagnosis of IFIs.

Galactomannan enzyme immunoassay and quantitative Real Time PCR as tools to evaluate the exposure and response in a rat model of aspergillosis after posaconazole prophylaxis.

INTRODUCTION: A steroid-immunosuppressed rat model of invasive pulmonary aspergillosis was use to examine the usefulness of galactomannan enzyme immunoassay (GM) and quantitative real time PCR (RT-PCR) in evaluating the association between response and exposure after a high dose of prophylactic posaconazole. METHODS: Two different strains of Aspergillus fumigatus with different in vitro posaconazole susceptibility were used. RESULTS: Serum concentrations demonstrated similar posaconazole exposure for all treated animals. However, response to posaconazole relied on the in vitro susceptibility of the infecting strain. After prophylaxis, galactomannan index and fungal burden only decreased in those animals infected with the most susceptible strain. CONCLUSION: This study demonstrated that both biomarkers may be useful tools for predicting efficacy of antifungal compounds in prophylaxis.

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.

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.

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.

Cutaneous Alternaria infectoria infection diagnosed by molecular techniques in a renal transplant patient.

Organ transplant recipients under immunosuppressive therapy have a highly increased risk of opportunistic fungal infections. Cutaneous infection caused by Alternaria species are relatively rare in humans and most cases reported in the literature are in immunocompromised individuals. We report here on a 33-year old male renal transplant patient with diabetes mellitus who presented with cutaneous alternariosis caused by Alternaria infectoria, two years after the transplant. The diagnosis was performed by real-time polymerase chain reaction assay and histopathologic examination. The extension of the lesion under itraconazole treatment required treatment consisting of a combination of surgical excision and liposomal amphotericin B.

A Phase I/IIa Prospective, Randomized, Open-Label Study on the Safety and Efficacy of Nebulized Liposomal Amphotericin for Invasive Pulmonary Aspergillosis.

Although nebulized liposomal amphotericin B (NLAB) is being used in invasive pulmonary aspergillosis (IPA) prophylaxis, no clinical trial has shown its efficacy as a therapeutic strategy. NAIFI is the inaugural randomized, controlled clinical trial designed to examine the safety and effectiveness of NLAB (dosage: 25 mg in 6 mL, three times per week for 6 weeks) against a placebo, in the auxiliary treatment of IPA. Throughout the three-year clinical trial, thirteen patients (six NLAB, seven placebo) were included, with 61% being onco-hematological with less than 100 neutrophils/µL. There were no significant differences noted in their pre- and post-nebulization results of forced vital capacity (FVC), forced expiratory volume in 1 s (FEV1), and oxygen saturation between the groups. Neither bronchospasm nor serum amphotericin B levels were reported in any patients given NLAB. 18F-Fluorodeoxyglucose positron emission tomography (FDG-PET-TC) was carried out at the baseline and after 6 weeks. A notable decrease in median SUV (standardized uptake value) was observed in NLAB patients after 6 weeks (-3.6 vs. -0.95, p: 0.039, one tail). Furthermore, a reduction in serum substance galactomannan and beta-D-Glucan was identified within NLAB recipients. NLAB is well tolerated and safe for patients with IPA. Encouraging indirect efficacy data have been derived from image monitoring or biomarkers. However, further studies involving more patients are necessary.

Molecular Diagnosis of Endemic Mycoses.

Diagnosis of endemic mycoses is still challenging. The moderated availability of reliable diagnostic methods, the lack of clinical suspicion out of endemic areas and the limitations of conventional techniques result in a late diagnosis that, in turn, delays the implementation of the correct antifungal therapy. In recent years, molecular methods have emerged as promising tools for the rapid diagnosis of endemic mycoses. However, the absence of a consensus among laboratories and the reduced availability of commercial tests compromises the diagnostic effectiveness of these methods. In this review, we summarize the advantages and limitations of molecular methods for the diagnosis of endemic mycoses.

Phenotypic characteristics of isolates of Aspergillus section Fumigati from different geographic origins and their relationships with genotypic characteristics.

BACKGROUND: Epidemiological studies worldwide have shown that A. fumigatus exhibits important phenotypic and genotypic diversity, and these findings have been of great importance in improving the diagnosis and treatment of diseases caused by this fungus. However, few studies have been carried out related to the epidemiology of this fungus in Latin America. This study's aim is to report on the epidemiology of the fungus by analyzing the phenotypic variability of Aspergillus section Fumigati isolates from different Latin American countries and the relationship between this variability, the geographical origin and genotypic characteristics. METHODS: We analyzed the phenotypic characteristics (macro- and micromorphology, conidial size, vesicles size, antifungal susceptibility and thermotolerance at 28, 37 and 48°C) of A. section Fumigati isolates from Mexico (MX), Argentina (AR), Peru (PE) and France (FR). The results were analyzed using analysis of variance (ANOVA) and Tukey's multiple comparison test to detect significant differences. Two dendrograms among isolates were obtained with UPGMA using the Euclidean distance index. One was drawn for phenotypic data, and the other for phenotypic and genotypic data. A PCoA was done for shown isolates in a space of reduced dimensionality. In order to determine the degree of association between the phenotypic and genotypic characteristics AFLP, we calculated the correlation between parwise Euclidean distance matrices of both data sets with the nonparametric Mantel test. RESULTS: No variability was found in the macromorphology of the studied isolates; however, the micromorphology and growth rate showed that the PE isolates grew at a faster rate and exhibited the widest vesicles in comparison to the isolates from MX, AR and FR. The dendrogram constructed with phenotypic data showed three distinct groups. The group I and II were formed with isolates from PE and FR, respectively, while group III was formed with isolates from MX and AR. The dendrogram with phenotypic and genotypic data showed the same cluster, except for an isolate from FR that formed a separate cluster. This cluster was confirmed using PCoA. The correlation between the phenotypic and genotypic data of the isolates revealed a statistically significant association between these characteristics. CONCLUSIONS: The PE isolates showed specific phenotypic characteristics that clearly differentiate them from the rest of the isolates, which matches the genotypic data. The correlation between the phenotypic and genotypic characteristics showed a statistically significant association. In conclusion, phenotypic and genotypic methods together increase the power of correlation between isolates.

Differential Diagnosis of Fungal Pneumonias vs. Tuberculosis in AIDS Patients by Using Two New Molecular Methods.

Opportunistic fungal pneumonias (OFP) are the main cause of death in AIDS patients worldwide. Diagnosis of these infections is often late as tuberculosis (TB) is frequently the first suspicion. In addition, diagnostic tools have limitations and are unavailable in disadvantaged regions. To perform the differential diagnosis of the main fungi causing OFP in AIDS patients (Histoplasma capsulatum, Cryptococcus neoformans/C. gattii and Pneumocystis jirovecii) vs. the Mycobacterium tuberculosis complex (MTBC), two new assays were developed: (i) a multiplex real-time PCR (MRT-PCR) and (ii) a simple and cost-effective method based on real-time PCR and the analysis of melting curves after amplification (MC-PCR). Both of the techniques were optimized and standardized "in vitro", showing a suitable reproducibility (CV ranged between 1.84 and 3.81% and 1.41 and 4.83%, respectively), a 100% specificity and detection limits between 20 and 2 fg of genomic DNA per 20 µL of reaction. A validation study was performed by retrospectively using 42 clinical samples from 37 patients with proven fungal infection or TB, and 33 controls. The overall sensitivity for the MRT-PCR assay and the MC-PCR assay was 88% and 90.4%, respectively. Both techniques were fast, sensitive and reproducible, allowing for the detection of these pathogens and the performance of a differential diagnosis.

Timely Diagnosis of Histoplasmosis in Non-endemic Countries: A Laboratory Challenge.

Human histoplasmosis is a fungal infection caused by the inhalation of microconidia of the thermally dimorphic fungi Histoplasma capsulatum. Autochthonous cases of histoplasmosis have been diagnosed in almost every country, but it is considered an endemic infection in specific areas of the world. Many of them are popular travel destinations or the source of migratory movements. Thus, the vast majority of the registered cases in non-endemic countries are imported. They correspond to people having been exposed to the fungus in endemic locations as immigrants, expatriates, transient workers or tourists, with reported cases also associated to organ donation. Misdiagnosis and delays in initiation of treatment are not uncommon in cases of imported histoplasmosis. They are associated to high fatality-rates specially in patients with compromised cellular immunity in which progressive disseminated forms develop. The diagnosis of this infection in non-endemic countries is hampered by the lack of clinical suspicion and a dearth of available diagnostic tools adequate to offer rapid and accurate results. Non-culture-based assays such as nucleic-acid amplification tests present as a suitable alternative in this situation, offering improved sensitivity and specificity, shortened turnaround time, and increased biosafety by avoiding culture manipulation. In non-endemic regions, molecular techniques are being used mainly in laboratories from countries that have registered an increase in the incidence of imported cases. However, the number of published techniques is limited and lack consensus. Efforts are currently under way to standardize nucleic acid amplification-based techniques for its implementation in areas registering a rising number of imported cases.

Multiplex PCR Based Strategy for Detection of Fungal Pathogen DNA in Patients with Suspected Invasive Fungal Infections.

A new and easy polymerase chain reaction (PCR) multiplex strategy, for the identification of the most common fungal species involved in invasive fungal infections (IFI) was developed in this work. Two panels with species-specific markers were designed, the Candida Panel for the identification of Candida species, and the Filamentous Fungi Panel for the identification of Aspergillus species and Rhizopusarrhizus. The method allowed the correct identification of all targeted pathogens using extracted DNA or by colony PCR, showed no cross-reactivity with nontargeted species and allowed identification of different species in mixed infections. Sensitivity reached 10 to 1 pg of DNA and was suitable for clinical samples from sterile sites, with a sensitivity of 89% and specificity of 100%. Overall, the study showed that the new method is suitable for the identification of the ten most important fungal species involved in IFI, not only from positive blood cultures but also from clinical samples from sterile sites. The method provides a unique characteristic, of seeing the peak in the specific region of the panel with the correct fluorescence dye, that aids the ruling out of unspecific amplifications. Furthermore, the panels can be further customized, selecting markers for different species and/or resistance genes.

Update on the epidemiology and diagnosis of invasive fungal infection.

In recent years, the most important advances in the treatment of transplant recipients, patients with haematological neoplasm and critically ill patients have been accompanied by an increase in the incidence of common fungal diseases and the emergence of some less common ones. Although new techniques (e.g. galactomannan detection) and new antifungals have appeared, these opportunistic infections remain difficult to diagnose and have a high mortality. New diagnostic techniques could improve this outlook, although they are far from becoming available in daily practice.

Copy Number Variation of Mitochondrial DNA Genes in Pneumocystis jirovecii According to the Fungal Load in BAL Specimens.

Pneumocystis jirovecii is an unculturable fungus and the causative agent of Pneumocystis pneumonia, a life-threatening opportunistic infection. Although molecular diagnosis is often based on the detection of mtLSU rRNA mitochondrial gene, the number of copies of mitochondrial genes had not been investigated. We developed and optimized six real-time PCR assays in order to determine the copy number of four mitochondrial genes (mtSSU rRNA, mtLSU rRNA, NAD1, and CYTB) in comparison to nuclear genome (DHPS and HSP70) and tested 84 bronchoalveolar fluids of patients at different stages of the infection. Unexpectedly, we found that copy number of mitochondrial genes varied from gene to gene with mtSSU rRNA gene being more represented (37 copies) than NAD1 (23 copies), mtLSU rRNA (15 copies) and CYTB (6 copies) genes compared to nuclear genome. Hierarchical clustering analysis (HCA) allowed us to define five major clusters, significantly associated with fungal load (p = 0.029), in which copy number of mitochondrial genes was significantly different among them. More importantly, copy number of mtLSU rRNA, NAD1, and CYTB but not mtSSU rRNA differed according to P. jirovecii physiological state with a decreased number of copies when the fungal load is low. This suggests the existence of a mixture of various subspecies of mtDNA that can harbor different amplification rates. Overall, we revealed here an unexpected variability of P. jirovecii mtDNA copy number that fluctuates according to P. jirovecii's physiological state, except for mtSSU that is the most stable and the most present mitochondrial gene.

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.

Comparison of PCR protocols for detecting Histoplasma capsulatum DNA through a multicenter study.

BACKGROUND: A multicenter study was conducted. A panel containing DNA from Histoplasma capsulatum, as well as negative and cross-reaction controls, was sent to five different laboratories, members of the MICOMOL network from CYTED Program. AIMS: The objective was to assess the accuracy of different PCR protocols to detect H. capsulatum DNA. METHODS: Seven different PCR protocols were tested. They were based on PCR techniques and used unicopy and multicopy targets. RESULTS: Most of these protocols (4/7) were able to detect the smallest amounts of fungal DNA (10(2)fg/µl). Overall sensitivity was 86% and specificity was 100%. The protocol based on a unicopy target (SCAR220) presented lower sensitivity (43%) but 100% specificity. The real-time protocols tested were highly reproducible, sensitive, and specific. Neither false positives nor cross-reactions were detected in any protocol. CONCLUSIONS: All laboratories were able to amplify H. capsulatum DNA, and real-time PCR seems to be a promising tool to efficiently detect this pathogen in clinical samples.

An unusual cutaneous tumor: African histoplasmosis following mudbaths: case report and review.

African histoplasmosis, caused by Histoplasma capsulatum var. duboisii, is endemic in Africa. The disease usually involves the skin, subcutaneous tissue, and bones. A case of African histoplasmosis presenting as a cutaneous tumor and non-healing wound in a 66-year-old immunocompetent male residing in Africa, the first ever reported following mudbaths and acupuncture, is hereby reported. Diagnosis was confirmed by means of polymerase chain reaction performed on tissue material. The patient was started on long-term itraconazole therapy and he responded well. African histoplasmosis should be included in the differential diagnosis of non-healing wounds or tumor-like lesions, especially in the context of mudbaths in an endemic area.

In vitro antifungal susceptibility pattern and ergosterol content in clinical yeast strains.

BACKGROUND: Absence or severe reduction in the amount of ergosterol in the fungal membrane and its replacement with other sterols have been described as potential antifungal resistance mechanisms in fungi. AIMS AND METHODS: The ergosterol content in a collection of 51 clinical yeast isolates, including susceptible and resistant strains to amphotericin B and azoles, was estimated by a simple chromatographic method (HPLC-UV). RESULTS: A high content of ergosterol was detected for several strains of Candida glabrata, Candida tropicalis or Pichia membranifaciens. In contrast, strains of Cryptococcus neoformans and Dipodascus capitatus had the lowest ergosterol concentrations. No significant correlation was observed between antifungal susceptibility patterns and ergosterol content. CONCLUSIONS: We conclude from this study that ergosterol content on yeasts may not be associated with specific resistant patterns.