Aspergillus PCR-Based Investigation of Fresh Tissue and Effusion Samples in Patients with Suspected Invasive Aspergillosis Enhances Diagnostic Capabilities

Although it is a severe complication in immunocompromised patients, diagnosing invasive fungal disease (IFD), especially invasive aspergillosis (IA), remains difficult. In certain clinical scenarios, examining tissue samples for identification of the infectious organism becomes important. As culture-based methods rarely yield results, the performance of an Aspergillus-specific nested PCR in fresh tissue or pleural effusion samples was evaluated. Fresh tissue (n = 59) and effusion (n = 47) specimens from 79 immunocompromised patients were subjected to an Aspergillus-specific PCR assay. Twenty-six patients had proven (n = 20) or probable (n = 6) IFD, according to the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) criteria, while the remaining patients were classified as having either possible IFD (n = 30) or no IFD (n = 23). IA was identified as the underlying IFD in 21/26 proven/probable cases. PCR positivity was observed for 18/21 proven/probable and 6 possible IA cases; cases classified as no IA did not show positive signals. Patients with proven IFD (n = 5) with cultures positive for non-Aspergillus molds also had negative Aspergillus PCR results. Aspergillus PCR performance analysis yielded sensitivity and specificity values of 86% (95% confidence interval [CI], 65% to 95%) and 100% (95% CI, 86% to 100%), respectively, thus leading to a diagnostic odds ratio of >200. In this analysis, good diagnostic performance of the PCR assay for detection of IA was observed for tissue samples, while effusion samples showed lower sensitivity rates. PCR testing represents a complementary tool; a positive PCR result strengthens the likelihood of IA, whereas IA seems unlikely in cases with negative results but findings could indicate non-Aspergillus IFD. Thus, PCR testing of these specimens enhances the diagnostic capabilities.     

Aspergillus PJI – A systematic analysis of all known cases and report of a new one

Fungi resemble less than one percent of all periprosthetic joint infections (PJI). While Candida PJI is well described, Aspergillus PJI has only been reported in a few cases without any systematic analysis present at this point. This review aims to systematically summarize and describe all cases of Aspergillus PJI. The systematic review used PubMed and Cochrane Library to identify case reports and studies eligible for inclusion. One additional case was reported by the authors. T-, Mann–Whitney U- and Fisher-exact tests were used for calculations. Overall, 11 cases of Aspergillus PJI were identified, and ten could be included for a detailed analysis (four hip, four knee, one elbow, one PIP-arthroplasty infection). A. fumigatus was identified in four, A. terreus in three, and A. niger in two cases. The average patient age at time of Aspergillus spp. diagnosis was 64.1 years (32–83) and the mean time from primary implantation to Aspergillus PJI 5.2 years (1–16). The calculated CCI was 2.7 (0–6). Surgery included one-, two-, three-stage-, and spacer-exchange, debridement and resection arthroplasty. Four patients were treated with a triazole for an average of three months, three with amphotericin (mean eight weeks), one with both amphotericin (six weeks) and triazole (seven months). In one patient, reinfection with Coagulase Negative Staphylococci following Aspergillus PJI treatment was noted after four years. A. terreus (p = .048) was associated with failed prosthesis reimplantation (n = 4). To give a resume, Aspergillus PJI is a rare, yet severe complication, with heterogeneous clinical presentation. Complete prosthesis removal is the treatment of choice.     

Validation of a New Aspergillus Real-Time PCR Assay for Direct Detection of Aspergillus and Azole Resistance of Aspergillus fumigatus on Bronchoalveolar Lavage Fluid

Azole resistance in Aspergillus fumigatus is increasingly reported. Here, we describe the validation of the AsperGenius, a new multiplex real-time PCR assay consisting of two multiplex real-time PCRs, one that identifies the clinically relevant Aspergillus species, and one that detects the TR34, L98H, T289A, and Y121F mutations in CYP51A and differentiates susceptible from resistant A. fumigatus strains. The diagnostic performance of the AsperGenius assay was tested on 37 bronchoalveolar lavage (BAL) fluid samples from hematology patients and 40 BAL fluid samples from intensive care unit (ICU) patients using a BAL fluid galactomannan level of ≥1.0 or positive culture as the gold standard for detecting the presence of Aspergillus. In the hematology and ICU groups combined, there were 22 BAL fluid samples from patients with invasive aspergillosis (IA) (2 proven, 9 probable, and 11 nonclassifiable). Nineteen of the 22 BAL fluid samples were positive, according to the gold standard. The optimal cycle threshold value for the presence of Aspergillus was <36. Sixteen of the 19 BAL fluid samples had a positive PCR (2 Aspergillus species and 14 A. fumigatus samples). This resulted in a sensitivity, specificity, and positive and negative predictive values of 88.9%, 89.3%, 72.7%, and 96.2%, respectively, for the hematology group and 80.0%, 93.3%, 80.0%, and 93.3%, respectively, in the ICU group. The CYP51A real-time PCR confirmed 12 wild-type and 2 resistant strains (1 TR34-L98H and 1 TR46-Y121F-T289A mutant). Voriconazole therapy failed for both patients. The AsperGenius multiplex real-time PCR assay allows for sensitive and fast detection of Aspergillus species directly from BAL fluid samples. More importantly, this assay detects and differentiates wild-type from resistant strains, even if BAL fluid cultures remain negative.     

Efficacy of DNA amplification in tissue biopsy samples to improve the detection of invasive fungal disease

The performance of a pan-fungal PCR-based technique was evaluated to assess the aetiology of invasive fungal diseases (IFDs). A total of 89 formalin-fixed paraffin-embedded biopsy samples from 84 patients with proven IFD were studied. Culture of tissue was performed in 68 (81%) patients. The sensitivities of the PCR-based technique and microbiological culture of tissues were 89% and 56%, respectively (p <0.01). According to PCR results, Aspergillus species accounted for 67%, Candida species for 13%, zygomycetes for 11%, and rare and emerging fungi for 9%. Aspergillus species were significantly associated with lung samples (79.6%, p <0.01), Mucorales were associated with skin/subcutaneous samples, and Candida species were associated with gastrointestinal samples. Regarding biopsy samples with Aspergillus species, Aspergillus fumigatus DNA was detected in 43 of 50 (86%), and Aspergillus flavus in six of 50 (12%). PCR was positive in 24 of 30 (80%) cases with negative culture. In nine of the 84 patients, the PCR technique failed to amplify the DNA. Six also had negative cultures, and in the remaining three cases culture was positive (Rhizopus microsporus, Rhizopus arrhizus, and Sakseneae vasiformis), suggesting that the PCR technique was not as effective in amplifying the DNA of some species of Zygomycetes. In five cases, there was no correlation between culture results and those obtained with DNA amplification, indicating the possibility of a mixed infection or the presence of colonizer/contaminant microorganisms. In summary, PCR-based techniques for DNA amplification should be implemented in histopathology and microbiology departments, as they appear to be complementary to conventional methods for IFD detection.     

Cryptic and Rare Aspergillus Species in Brazil: Prevalence in Clinical Samples and In Vitro Susceptibility to Triazoles

Aspergillus spp. are among the most common causes of opportunistic invasive fungal infections in tertiary care hospitals. Little is known about the prevalence and in vitro susceptibility of Aspergillus species in Latin America, because there are few medical centers able to perform accurate identification at the species level. The purpose of this study was to analyze the distribution of cryptic and rare Aspergillus species among clinical samples from 133 patients with suspected aspergillosis admitted in 12 medical centers in Brazil and to analyze the in vitro activity of different antifungal drugs. The identification of Aspergillus species was performed based on a polyphasic approach, as well as sequencing analysis of the internal transcribed spacer (ITS) region, calmodulin, and β-tubulin genes and phylogenetic analysis when necessary. The in vitro susceptibility tests with voriconazole, posaconazole, and itraconazole were performed according to the CLSI M38-A2 document (2008). We demonstrated a high prevalence of cryptic species causing human infection. Only three isolates, representing the species Aspergillus thermomutatus, A. ochraceus, and A. calidoustus, showed less in vitro susceptibility to at least one of the triazoles tested. Accurate identifications of Aspergillus at the species level and with in vitro susceptibility tests are important because some species may present unique resistance patterns against specific antifungal drugs.     

A comparison of the prevalence of sensitization to Aspergillus antigens among asthmatics in Cleveland and London

A survey of the frequency of sensitization to Aspergillus antigens was conducted in a group of asthmatics in Cleveland and compared with a group of asthmatics in London, using common antigens for testing purposes. The two groups were comparable except for earlier onset, longer duration of asthma, and a larger number of males in the London group. Twenty-eight per cent of the asthmatics from Cleveland and 23% from London had immediate skin reactivity to Aspergillus. Seven and one-half percent from the Cleveland group and 10.5% of the London group had Aspergillus precipitins in the serum. Aspergillus skin test reactivity was related to the severity of airways obstruction (p < 0.01) but was not influenced by other factors. We conclude that sensitization to Aspergillus antigens occur with equal frequency in both the United States and the United Kingdom.     

Comparison between PCR and Detection of Antigen in Sera for Diagnosis of Pulmonary Aspergillosis

We evaluated the usefulness of PCR and antigen detection for the diagnosis of pulmonary aspergillosis. Forty-four serum samples from patients with pulmonary aspergillosis (33 with pulmonary aspergilloma, 4 with allergic bronchopulmonary aspergillosis, 4 with invasive pulmonary aspergillosis, and 3 with aspergillus pyothorax) were used in this study. PCR detection of Aspergillus DNA in serum samples was successful in 39 patients. Galactomannan antigen was detected by sandwich enzyme-linked immunosorbent assay in 25 patients and by latex agglutination test in 13 patients. Detection of Aspergillus DNA in serum samples by nested PCR had the highest sensitivity of the three methods tested for the diagnosis of pulmonary aspergillosis.     

Evaluation of a Commercially Developed Semiautomated PCR–Surface-Enhanced Raman Scattering Assay for Diagnosis of Invasive Fungal Disease

Nonculture-based tests are gaining popularity in the diagnosis of invasive fungal disease (IFD), but PCR is excluded from disease-defining criteria because of limited standardization and a lack of commercial assays. Commercial PCR assays may have a standardized methodology while providing quality assurance. The detection of PCR products by a surface-enhanced Raman scattering (SERS) assay potentially provides superior analytical sensitivity and multiplexing capacity compared to that of real-time PCR. Using this approach, the RenDx Fungiplex assay was developed to detect Candida and Aspergillus. Analytical and clinical evaluations of the assay were undertaken using extraction methods according to European Aspergillus PCR Initiative (EAPCRI) recommendations. A total of 195 previously extracted samples (133 plasma, 49 serum, and 13 whole blood) from 112 patients (29 with proven/probable IFD) were tested. The 95% limit of detection of Candida and Aspergillus was 200 copies per reaction, with an overall reproducibility of 92.1% for detecting 20 input copies per PCR, and 89.8% for the nucleic acid extraction–PCR-SERS process for detecting fungal burdens of <20 genome equivalents per sample. A clinical evaluation showed that assay positivity significantly correlated with IFD (P < 0.0001). The sensitivity of the assay was 82.8% and was similar for both Candida (80.0%) and Aspergillus (85.7%). The specificity was 87.5% and was increased (97.5%) by using a multiple (≥2 samples) PCR-positive threshold. In summary, the RenDx Fungiplex assay is a PCR-SERS assay for diagnosing IFD and demonstrates promising clinical performance on a variety of samples. This was a retrospective clinical evaluation, and performance is likely to be enhanced through a prospective analysis of clinical validity and by determining clinical utility.     

Molecular characterization of Aspergilli isolated from outdoor air

Ubiquitous airborne conidia of the genus Aspergillus are responsible for a diverse group of human disorders from allergy to life treating invasive aspergillosis and mycotoxicoses. The aim of this study was to determine the population structure of Aspergillus isolated from outdoor air in Tehran by comparing the nucleotide sequences of ITS region and the PCR-RFLP molecular method. Internal transcribed spacer domains of 47 Aspergillus spp. were amplified and sequenced and PCR products were digested individually with restriction enzymes TaqI and EcoRI. For all species the PCR reaction produced a fragment of approximately 600 bp in length. All of the nucleotide sequences were highly similar with the corresponding reference sequences registered at the gene bank. The all isolates displayed same banding pattern on the basis EcoR1 cleavage. While Taq1 enzyme profiling provided 5 different banding pattern. The results show that the A. niger section has the highest frequency with 27 isolates (57.4%). Of these, 23 isolates (48.9%) belonged to the A. niger complex and 4 isolates (8.5%) to the A. aculeatus complex. The A. flavus complex was also placed in the next ranking with 9 isolates (19.1%). These results strongly support the need for using molecular markers as an auxiliary tool in differentiating Aspergillus species.     

Recombinant Allergens Combined with Biological Markers in the Diagnosis of Allergic Bronchopulmonary Aspergillosis in Cystic Fibrosis Patients

Allergic bronchopulmonary aspergillosis (ABPA) is a frequent complication in cystic fibrosis patients. The diagnosis remains difficult and requires a combination of clinical, radiological, biological, and mycological criteria. The aim of this study was to analyze the added value of two recombinant antigens, rAspf4 and rAspf6, associated with the detection of specific IgG; precipitins; total IgE; and Aspergillus fumigatus in sputum for the diagnosis of ABPA. In a retrospective study, we determined the specific IgE responses to these recombinants in 133 sera of 65 cystic fibrosis patients. We selected an average of five serum samples from each of the 17 patients with ABPA (13 proven and 4 probable ABPA) and from 3 patients with Aspergillus bronchitis and rhinosinusitis. One serum sample for the 45 patients without ABPA was tested. The sensitivity of specific IgE detection against rAspf4 calculated per patient (92.3%) was significantly higher (P < 0.05) than that of rAspf6 (53.8%). When rAspf4 IgE detection was associated with anti-Aspergillus IgG enzyme-linked immunosorbent assay (ELISA) and precipitin detection, the sensitivity rose to 100%. The specificities of rAspf4 and rAspf6 IgE detection were 93.7% and 91.6%, respectively. Other diagnostic criteria had slightly lower specificities (87.5% for anti-Aspergillus IgG ELISA, 89.6% for precipitins, 84.4% for total IgE, and 85.0% for positive A. fumigatus culture in sputum). In conclusion, this retrospective study showed the relevance of rAspf4 IgE detection, in combination with other biological markers (Aspergillus IgG ELISA, precipitins, and total IgE), for improving the biological diagnosis of ABPA.Allergic bronchopulmonary aspergillosis (ABPA) is a frequent complication in patients with cystic fibrosis that causes significant respiratory morbidity (1, 13, 18). The exact prevalence of ABPA is not clearly known; however, it ranges from 2 to 25% in patients with cystic fibrosis (22). ABPA may lead to acute worsening of the respiratory condition and ongoing decline in lung function. Without adequate treatment, ABPA ultimately progresses to a chronic state with lung fibrosis. ABPA is a hypersensitivity pulmonary disease associated with inflammatory destruction of airways in response to Aspergillus allergens (23). Thus, chronic airway colonization with Aspergillus induces strong inflammatory responses with high IgE levels (20). The factors leading to ABPA are not clearly understood, but it is believed that Aspergillus-specific, IgE-mediated type I hypersensitivity reactions and specific IgG-mediated type III hypersensitivity reactions play central roles in the pathogenesis of ABPA (18). Furthermore, host factors, including individual susceptibility, may contribute to the immunopathogenesis of ABPA. Early diagnosis and treatment of ABPA are important to prevent serious and potentially irreversible lung damage (9).ABPA is defined by five major diagnostic criteria, according to the Cystic Fibrosis Foundation Consensus Conference (19, 23): (i) acute or subacute clinical deterioration and decline in pulmonary function not attributable to another etiology, (ii) elevated serum IgE concentrations, (iii) immediate prick test reactivity to Aspergillus antigen or presence of specific IgE antibodies to Aspergillus fumigatus, (iv) precipitating antibodies to A. fumigatus or elevated serum IgG antibodies to A. fumigatus, and (v) history of pulmonary infiltrates (transient or fixed) and central bronchiectasis. Minor diagnostic criteria include repeated detection of Aspergillus species in sputum samples, a history of expectoration of brown plugs or flecks, and late skin reactivity to Aspergillus antigens. Despite these criteria, diagnosis of ABPA in cystic fibrosis patients remains particularly difficult (3, 16, 21, 22). Therefore, as stated in the most recent consensus document on the diagnosis and therapy of ABPA in cystic fibrosis patients (19), serological findings should strongly contribute to the confirmation or exclusion of clinically suspected ABPA.The demonstration that some recombinant antigens are specifically expressed only in hyphae explains why an IgE response to these antigens may occur in ABPA patients but not in A. fumigatus-sensitized patients. Thus, cloning, characterization, production, and clinical evaluation of A. fumigatus allergens have been major advances in the diagnosis of ABPA and help in the understanding of the pathophysiological mechanisms underlying the disease (9). The rAspf1, rAspf2, rAspf3, rAspf4, and rAspf6 recombinant allergens have been evaluated for their diagnostic performance in several serological studies concerning patients with both asthma and cystic fibrosis, with or without ABPA, and showed high specificity for the detection of A. fumigatus sensitization, as well as ABPA (14). Serological investigations involving rAspf4 and rAspf6 showed that allergen-specific IgE levels against these proteins increased almost exclusively in samples from patients with ABPA (9). However, de Oliveira et al. (10) showed that the determination of the IgE response against A. fumigatus recombinant antigens in asthmatic patients with immediate cutaneous reactivity to A. fumigatus was not helpful in diagnosing ABPA or in detecting sensitization to fungi.The aim of the present study was to evaluate the efficiencies of two recombinant allergens (rAspf4 and rAspf6) in IgE detection, combined with anti-Aspergillus IgG, precipitins, total serum IgE concentration, and fungal culture of sputum.     

Antifungal susceptibility of clinical isolates of 25 genetically confirmed Aspergillus species collected from Taiwan and Mainland China

BackgroundTo investigate the in vitro antifungal susceptibilities of 25 genetically confirmed Aspergillus species collected from Taiwan and Mainland China.MethodsA total of 390 non-duplicate and consecutive Aspergillus isolates representing of 25 species recovered from clinical sources at two teaching hospitals in Taiwan and Mainland china were preserved for this study. In vitro antifungal susceptibility testing (AFST) of those Aspergillus isolates against seven antifungal agents was performed using Sensititre YeastOne (SYO) system. The susceptibility profiles of isolates were analyzed according to the general interpretation code drawn from the SYO instruction. The CYP51A gene sequencing analysis was performed for triazole-resistant Aspergillus fumigatus isolates.ResultsAmong the 390 Aspergillus isolates, 76.7% (n = 299) exhibited complete susceptibility to all the tested antifungals, and 23.3% (91/390) of different Aspergillus species isolates showed resistance to one or two classes of antifungal agents with higher minimum inhibitory concentrations (MICs) of >2 mg/L. Resistance to amphotericin B was found in 91.2% (83/91) of those less susceptible isolates and most of them focused on species being resistant to Amphotericin B innately. The total rate of triazole resistance in this study was low (3.3%, 13/390), and only 3 (2.8%) A. fumigatus isolates were resistant to at least one of the triazoles with mutations of TR₃₄/L98H or TR₄₆/Y121F/T289A in CYP51A gene. The echinocandins were highly potent to the tested Aspergillus isolates.ConclusionThe existence of triazole resistance among A. fumigatus isolates in Taiwan and Mainland China indicates the need for continuous monitoring from now on.     

Species identification and antifungal susceptibility testing of Aspergillus strains isolated from patients with otomycosis in northern China

Background/PurposeThere are limited studies on species distribution and susceptibility profiles of Aspergillus strains isolated from patients with otomycosis in China.MethodsA total of 69 confirmed Aspergillus species isolates were obtained from ear swabs of patients diagnosed with otomycosis from 2017 to 2018 in northern China. Identification of these Aspergillus isolates at the species level was performed using conventional morphological methods and MALDI-TOF MS in combination with molecular sequencing, and in vitro susceptibility to nine antifungal agents was evaluated using the Sensititre YeastOne system.ResultsThe Aspergillus section Nigri had the greatest distribution of Aspergillus isolates. A. welwitschiae (n = 25) was the most predominant isolate in section Nigri, followed by A. tubingensis (n = 12) and A. niger (n = 11). Other Aspergillus species were also isolated, including A. terreus (n = 11), A. flavus/A. oryzae (n = 8), and A. fumigatus (n = 2). Amphotericin B, posaconazole, and echinocandins were highly in vitro active against all the isolates tested. 2.9% (2/69) of the isolates were resistant to azoles in our study, including one A. niger isolate with a high MIC value for itraconazole (ITR) (16 mg/L) and one A. tubingensis isolate cross-resistant to both voriconazole (VOR) (MIC >8 mg/L) and ITR (MIC >16 mg/L). One A. welwitschiae and one A. niger isolate both had increased MIC values of 4 mg/L against VOR.ConclusionsA. welwitschiae was the most prevalent Aspergillus species isolated from patients with otomycosis. Our findings also indicated that the azole-resistant Aspergillus section Nigri should be utilized to guide clinical medication for Otomycosis.     

Flore fongique du sable de deux plages à Casablanca (Maroc): Analyse et corollaires épidémiologiques

Due to the large number of people who spend time on beaches in summer, possible contamination of the sand by fungal spores could be a possible source of contamination of certain pathogenic fungi. The aim of this study was to identify the fungal flora present on two beaches in Casablanca, Morocco, by means of a mycological study carried out in the summer of 2004. Out of a total of 120 samples of sand, 70 species of fungus were identified in 56 infected samples. Different species were isolated including 19 strains of yeast (10 Candida albicans and nine other species), five Trichophyton rubrum, four Scytalidium dimidiatum, 25 Aspergillus spp., 13 Penicillium spp., two Cladosporium and two Scedosporium. These results confirm the presence of a wide range of fungal flora in the sand of beaches in Casablanca. In addition, keratinophile fungi were isolated mostly in humid sites which could favour the incidence of dermatomycoses among beachgoers.     

Performance of Galactomannan,Beta-d-Glucan,Aspergillus Lateral-Flow Device,Conventional Culture,and PCR Tests with Bronchoalveolar Lavage Fluid for Diagnosis of Invasive Pulmonary Aspergillosis

Galactomannan detection in bronchoalveolar lavage (BAL) fluid samples (GM test) is currently considered the gold standard test for diagnosing invasive pulmonary aspergillosis (IPA). The limitations, however, are the various turnaround times and availability of testing. We compared the performance of GM testing with that of conventional culture, an Aspergillus lateral-flow-device (LFD) test, a beta-d-glucan (BDG) test, and an Aspergillus PCR assay by using BAL fluid samples from immunocompromised patients. A total of 78 BAL fluid samples from 78 patients at risk for IPA (74 samples from Graz and 4 from Mannheim) collected between December 2012 and May 2013 at two university hospitals in Austria and Germany were included. Three patients had proven IPA, 14 probable IPA, and 17 possible IPA, and 44 patients had no IPA. The diagnostic accuracies of the different methods for probable/proven IPA were evaluated. The diagnostic odds ratios were the highest for the GM, PCR, and LFD tests. The sensitivities for the four methods (except culture) were between 70 and 88%. The combination of the GM (cutoff optical density index [ODI], >1.0) and LFD tests increased the sensitivity to 94%, while the combination of the GM test (>1.0) and PCR resulted in 100% sensitivity (specificity for probable/proven IPA, 95 to 98%). The performance of conventional culture was limited by low sensitivity, while that of the BDG test was limited by low specificity. We evaluated established and novel diagnostic methods for IPA and found that the Aspergillus PCR, LFD, and GM tests were the most useful methods for diagnosing the disease by using BAL fluid samples. In particular, the combination of the GM test and PCR or, if PCR is not available, the LFD test, allows for sensitive and specific diagnosis of IPA.     

Clinical Performance of Aspergillus PCR for Testing Serum and Plasma: a Study by the European Aspergillus PCR Initiative

Aspergillus PCR testing of serum provides technical simplicity but with potentially reduced sensitivity compared to whole-blood testing. With diseases for which screening to exclude disease represents an optimal strategy, sensitivity is paramount. The associated analytical study confirmed that DNA concentrations were greater in plasma than those in serum. The aim of the current investigation was to confirm analytical findings by comparing the performance of Aspergillus PCR testing of plasma and serum in the clinical setting. Standardized Aspergillus PCR was performed on plasma and serum samples concurrently obtained from hematology patients in a multicenter retrospective anonymous case-control study, with cases diagnosed according to European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) consensus definitions (19 proven/probable cases and 42 controls). Clinical performance and clinical utility (time to positivity) were calculated for both kinds of samples. The sensitivity and specificity for Aspergillus PCR when testing serum were 68.4% and 76.2%, respectively, and for plasma, they were 94.7% and 83.3%, respectively. Eighty-five percent of serum and plasma PCR results were concordant. On average, plasma PCR was positive 16.8 days before diagnosis and was the earliest indicator of infection in 13 cases, combined with other biomarkers in five cases. On average, serum PCR was positive 10.8 days before diagnosis and was the earliest indicator of infection in six cases, combined with other biomarkers in three cases. These results confirm the analytical finding that the sensitivity of Aspergillus PCR using plasma is superior to that using serum. PCR positivity occurs earlier when testing plasma and provides sufficient sensitivity for the screening of invasive aspergillosis while maintaining methodological simplicity.     

Respiratory specimens and the diagnostic accuracy of Aspergillus lateral flow assays (LFA-IMMY™): real-life data from a multicentre study

ObjectivesProper diagnosis of invasive aspergillosis is challenging because conventional methods lack sensitivity and are complicated by time-consuming incubation processes. To meet the requirement for early diagnosis the new Aspergillus-specific point-of-care test LFA-IMMY™ was evaluated with respect to the ability to accurately detect Aspergillus in bronchoalveolar fluids and sputa, and to clarify the potential of cross-reactivity with other fungal pathogens.MethodsRespiratory specimens (n = 398) from non-selected patients (n = 390) underwent either fungal microscopy, culture or both before Aspergillus lateral flow assay (LFA-IMMY) testing.ResultsFor Aspergillus culture- and microscopy-positive samples, sensitivity (48/52) and specificity (44/48) were 92% (95% CI 8.0%–9.7%) and 91% (95% CI 7.9%–9.7%), respectively; cross-reactivity was documented with non-Aspergillus pathogens.ConclusionLFA-IMMY is a reliable diagnostic tool for the detection of Aspergillus in respiratory samples.     

Aspergillus fumigatus conidia stimulate lung epithelial cells (TC-1 JHU-1) to produce IL-12, IFNγ, IL-13 and IL-17 cytokines: Modulatory effect of propolis extract

Aspergillus fumigatus conidia are the most prevalent indoors fungal allergens. The interaction between Aspergillus antigens and lung epithelial cells (LECs) result in innate immune functions. The association between Aspergillus conidia and allergic reactions, like allergic bronchopulmonary aspergillosis (ABPA) and asthma have been repeatedly reported. Since conventional therapies for allergy and asthma are limited, finding new promising treatments are inevitable. This study was designed to evaluate the effect of A. fumigatus conidia on IL-12, IFNγ, IL-13 and IL-17 release from mouse LECs and to investigate the effect of propolis on cytokines modulation. Cells were divided to two groups, one was exposed to 3 × 10⁴ conidia of Aspergillus fumigatus and another group was treated by propolis (25 μg/mL) as well as exposed to A. fumigatus conidia. Cytokines IL-13, IL-12, IFNγ and IL-17 were measured at times 0, 6 and 12 hours after exposure using ELISA assay. The results indicated that A. fumigatus could increase the release of the cytokines with IL-13 and IL-17 being the most affected ones whilst treatment with propolis decreased the effects of A. fumigatus on IL-13 and IL-17 production. The results showed that propolis has down regulatory effects on Th2 cytokine, IL-13, and IL-17 production, whereas it caused a significant induction of IL-12, as an important Th1 cytokines by LECs. With respect to the obtained results, propolis extract might be contributed to decrease Th2 responses in allergic asthma phenomenon. However more investigations must be done in future to fully understand its efficacy.     

Aspergillus citrinoterreus,a New Species of Section Terrei Isolated from Samples of Patients with Nonhematological Predisposing Conditions

The use of molecular identification techniques has revealed an increasing number of new species within Aspergillus section Terrei. We phenotyped a set of 26 clinical isolates that showed genetic differences from Aspergillus terreus sensu stricto by analyzing sequences from PCR-amplified β-tubulin and calmodulin genes and the internal transcribed spacer region. Since the isolates were phylogenetically and morphologically different from all of the members of Aspergillus section Terrei, they are described here as a new species, Aspergillus citrinoterreus, so named because it produces a diffusible yellowish pigment in agar. A. citrinoterreus isolates were significantly more susceptible to itraconazole, voriconazole, and posaconazole than A. terreus sensu stricto isolates were; in contrast, the amphotericin B MICs for both species were high. A. citrinoterreus was found in clinical samples from patients with proven or probable invasive aspergillosis and colonized patients, none of whom had hematological malignancies as predisposing conditions. However, they did have other underlying conditions such as chronic obstructive pulmonary disease, cirrhosis, and cancer or had received a solid organ transplants and presented not only with invasive pulmonary aspergillosis but also with mediastinitis. A. citrinoterreus isolates were detected for the first time in 2002. In all cases of invasive aspergillosis, A. citrinoterreus was found to be a copathogen, mostly with A. fumigatus.     

Online-Only Abstracts

Detection of Aspergillus IgG antibodies is important in the diagnosis of chronic pulmonary aspergillosis and allergic bronchopulmonary aspergillosis. Immunoprecipitation techniques to detect these antibodies appear to lack sensitivity and accurate quantitation compared with enzyme immunoassays (EIA). This study assessed the performance of two commercial EIAs compared with counterimmunoelectrophoresis (CIE). This was a prospective cohort study of 175 adult patients with chronic or allergic pulmonary aspergillosis. Aspergillus IgG antibodies were detected using CIE, Phadia ImmunoCap Aspergillus IgG and Bio-Rad Platelia Aspergillus IgG. Inter-assay reproducibility was determined for each method and 25 patients had two serum samples analysed within a 6-month interval. When compared with CIE, both ImmunoCap and Platelia Aspergillus IgG had good sensitivity (97 and 93%, respectively) for detection of Aspergillus IgG antibodies. The level of agreement between the two EIAs for positive results was good, but the concentration of antibodies was not correlated between the tests or with CIE titre. ImmunoCap IgG inter-assay coefficient of variation was 5%, whereas Platelia IgG was 33%. Median ImmunoCap IgG values for CPA and allergic aspergillosis were 95 and 32 mg/L, respectively, whereas Platelia IgG values were >80 and 6 AU/mL. The direction of CIE titre change over 6 months was mirrored by ImmunoCap IgG levels in 92% of patients, and by Platelia IgG in 72% of patients. Both ImmunoCap and Platelia Aspergillus IgG EIAs are sensitive measures of Aspergillus IgG antibodies compared with CIE. However, ImmunoCap appears to have better reproducibility and may be more suitable for monitoring patient disease.

Fast and specific dermatophyte detection by automated DNA extraction and real-time PCR

A. Bergman¹ D. Heimer² N. Kondori³ and H. Enroth¹1) Department of Clinical Microbiology, Unilabs AB, Skovde, 2) Department of Clinical Microbiology, Unilabs AB, Capio S:t Gorans Hospital, Stockholm and 3)Departmentof Clinical Bacteriology, Sahlgrenska University Hospital, Gothenburg, SwedenOriginal Submission: 12 September 2012; Revised Submission: 29 December 2012; Accepted: 2 January 2013Editor: E. BottieauArticle published online: 24 January 2013Clin Microbiol Infect 2013; 19: E205–E211

Abstract

The aim of this study was to develop and validate a rapid and sensitive real-time PCR method for detection of all known species of dermatophytes, including identification of Trichophyton rubrum and Trichophyton interdigitale. Fungal DNA was extracted directly from clinical samples by using a pre-lysis step, followed by automated DNA extraction on the MagNA Pure Compact. In total, 202 clinical samples were examined by both conventional culture and by the new PCR method. In 103 (51%) of the samples fungal nucleic acid was detected by PCR, while only 79 (39%) were found to be positive by culture. Out of 103 PCR-positive clinical samples, 94 (91%) were identified as T. rubrum and eight (8%) as T. interdigitale. This real-time PCR is far more sensitive and 2–4 weeks faster than conventional culture for detection of dermatophytes present in clinical samples.     

Evaluation of the Aspergillus Western Blot IgG Kit for Diagnosis of Chronic Aspergillosis

Immunoprecipitin detection (IPD) is the current reference confirmatory technique for anti-Aspergillus antibody detection; however, the lack of standardization is a critical drawback of this assay. In this study, we evaluated the performance of the Aspergillus Western blot (Asp-WB) IgG kit (LDBio Diagnostics, Lyon, France), a recently commercialized immunoblot assay for the diagnosis of various clinical presentations of chronic aspergillosis. Three hundred eight serum samples from 158 patients with aspergillosis sensu lato (s.l.) were analyzed. More specifically, 267 serum samples were derived from patients with Aspergillus disease, including 89 cases of chronic pulmonary aspergillosis, 10 of aspergilloma, and 32 of allergic bronchopulmonary aspergillosis, while 41 samples were from patients with Aspergillus colonization, including 15 cystic fibrosis (CF) and 12 non-CF patients. For blood donor controls, the Asp-WB specificity was 94%, while the kit displayed a sensitivity for the aspergillosis s.l. diagnosis of 88.6%, with a diagnostic odds ratio (DOR) of 119 (95% confidence interval [CI], 57 to 251). The DOR values were 185.22 (95% CI,78.79 to 435.45) and 43.74 (95% CI, 15.65 to 122.20) for the diagnosis of Aspergillus disease and Aspergillus colonization, respectively. Among the patients, the sensitivities of the Asp-WB in the diagnosis of Aspergillus colonization were 100% and 41.7% in CF and non-CF patients, respectively. The Asp-WB yielded fewer false-negative results than did IPD. In conclusion, the Asp-WB kit performed well for the diagnosis of various clinical presentations of aspergillosis in nonimmunocompromised patients, with an enhanced standardization and a higher sensitivity than with IPD, which is the current reference method.