Identification of Mytilus spp. and Pecten maximus in Irish Waters by Standard PCR of the 18S rDNA Gene and Multiplex PCR of the 16S rDNA Gene

Two molecular protocols for the identification of mussel and scallop have been developed using specific primers targeting the mitochondrial 16S ribosomal DNA gene and the nuclear 18S ribosomal DNA gene. Primers for the mitochondrial 16S ribosomal DNA gene in multiplex polymerase chain reaction (PCR) protocols yielded diagnostic DNA fragments for the mussels Mytilus edulis, Mytilus galloprovincialis, and the hybrid Mytilus edulis/galloprovincialis (335 bp), the king scallop Pecten maximus (382 bp) and the black scallop Mimachlamys varia (398 bp). DNA from the queen scallop Aequipecten opercularis showed no consistent PCR amplification of the 16S rDNA gene. Primers for the nuclear 18S rDNA gene in standard PCR protocols yielded similar-sized, diagnostic DNA fragments (approx. 190 bp) for the mussels Mytilus edulis, Mytilus galloprovincialis, and the hybrid Mytilus edulis/galloprovincialis, the king scallop Pecten maximus, the black scallop Mimachlamys varia, and the queen scallop Aequipecten opercularis. Both protocols have been tested with Mytilus spp., P. maximus, and 6 other bivalve species from a wide range of locations in Irish and European waters. Cross reaction of the specific primers with DNA template from any of the 6 other bivalve species was not observed. Rapid DNA extraction using FTA Card technology and the16S rDNA primers allowed for the detection of at least 10 mussel larvae in a subsample of natural plankton.     

Advances in molecular detection of Aspergillus: an update

Filamentous cosmopolitan fungi of the genus Aspergillus can be harmful in two ways, directly they can be opportunistic pathogens causing aspergillosis and indirectly due to aflatoxin production on food products which can lead to aflatoxicosis. Therefore, a number of methods have been proposed so far for detection of the fungi with lowest possible concentration at the earliest. Molecular methods such as PCR and/or in combination with certain techniques have been found to be useful for Aspergillus detection. We discuss here various technologies that have emerged in recent years and can possibly be used for the molecular detection of Aspergillus in an efficient way. These methods like RSIC, C-probe, and inversion probe with pyrosequencing or direct ss/dsDNA detection have been used for the identification of fungal or bacterial pathogens and thus formulate a ‘gold standard’ for Aspergillus detection.     

Toward systems metabolic engineering of Aspergillus and Pichia species for the production of chemicals and biofuels

Recently genome sequence data have become available for Aspergillus and Pichia species of industrial interest. This has stimulated the use of systems biology approaches for large-scale analysis of the molecular and metabolic responses of Aspergillus and Pichia under defined conditions, which has resulted in much new biological information. Case-specific contextualization of this information has been performed using comparative and functional genomic tools. Genomics data are also the basis for constructing genome-scale metabolic models, and these models have helped in the contextualization of knowledge on the fundamental biology of Aspergillus and Pichia species. Furthermore, with the availability of these models, the engineering of Aspergillus and Pichia is moving from traditional approaches, such as random mutagenesis, to a systems metabolic engineering approach. Here we review the recent trends in systems biology of Aspergillus and Pichia species, highlighting the relevance of these developments for systems metabolic engineering of these organisms for the production of hydrolytic enzymes, biofuels and chemicals from biomass.     

Prevention of invasive aspergillosis in high-risk patients: Universal versus preemptive, targeted treatment

Invasive aspergillosis (IA) is associated with increased morbidity and mortality, and there is a need for better preventative and therapeutic approaches. Successful treatment of documented IA remains difficult, often because of the inability to detect disease at an early stage. An important, recent advance in the management of aspergillosis is the availability of the newer broad-spectrum azoles, voriconazole and posaconazole, which have good activity against Aspergillus spp. In addition, newer diagnostic modalities including Aspergillus galactomannan, β-glucan, and polymerase chain reaction are more readily available. These diagnostic and treatment options have made new strategies possible for the management of IA. Prophylaxis and empirical therapy for high-risk patients have been popular for decades, and now a preemptive, targeted approach to IA management has become more attractive. This article reviews strategies for the prevention and management of IA and compares and contrasts universal prophylaxis to the preemptive, targeted approach for IA in high-risk patients.     

Nested PCR for the detection of Aspergillus species in maxillary sinus samples of patients with chronic sinusitis

Background

Fungal rhinosinusitis has become an increasingly recognized disease, being Aspergillus species responsible for most of the cases. Its diagnosis is quite difficult because of the non-specific symptoms and low sensitivity of the current diagnostic methods.

Galactomannan-Based and PCR-Based Assays in Bronchoalveolar Lavage to Diagnose Invasive Aspergillosis: Current Status and Future Prospects

Invasive pulmonary aspergillosis (IPA) is a life-threatening complication in patients receiving chemotherapy or undergoing allogeneic haematopoietic stem cell transplantation for acute leukemia. The existing tools to diagnose IPA lack specificity or sensitivity, or both; the search for improved diagnostic tools for IPA has focused on novel serologic and molecular methods. Aspergillus Galactomannan enzyme-linked immunosorbent assay (GM) analyses showed sensitivity rates in serum samples ranging in a wide span; testing GM in bronchoalveolar lavage (BAL) originated from the primary site of the infection seems to be more sensitive in patients with IPA. Other novel diagnostic markers to detect fungal DNA directly in clinical samples, rapidly, early, sensitively and specifically, are provided by polymerase chain reaction (PCR) based assays; higher sensitivity and specificity rates have been observed for BAL samples in IPA, even under antifungal treatment. The clinical place value of a diagnostic approach combining PCR and GM in BAL is unclear.     

Review of Epidemiology, Diagnosis, and Treatment of Invasive Mould Infections in Allogeneic Hematopoietic Stem Cell Transplant Recipients

Invasive mould infections are a major cause of morbidity and mortality in hematopoietic stem cell transplant recipients (HSCT). Allogeneic HSCT recipients are at substantially higher risk than autologous HSCT recipients. Although neutropenia following the conditioning regimen remains an important risk factor for opportunistic fungal infections, most cases of invasive mould infection in allogeneic HSCT recipients occur after neutrophil recovery in the setting of potent immunosuppressive therapy for graft-versus-host disease. Invasive aspergillosis is the most common mould infection. However, there has been an increased incidence of less common non-Aspergillus moulds that include zygomycetes, Fusarium sp., and Scedosporium sp. Reflecting a key need, important advances have been made in the antifungal armamentarium. Voriconazole has become a new standard of care as primary therapy for invasive aspergillosis based on superiority over amphotericin B. There is significant interest in combination therapy for invasive aspergillosis pairing voriconazole or an amphotericin B formulation with an echinocandin. There have also been advances in novel diagnostic methods that facilitate early detection of invasive fungal infections that include galactomannan and beta-glucan antigen detection and PCR using fungal specific primers. We review the epidemiology, diagnosis, and management of invasive mould infection in HSCT, with a focus on allogeneic recipients. We also discuss options for prevention and early treatment of invasive mould infections.     

Aspergillus Collagen-Like Genes (acl): Identification,Sequence Polymorphism,and Assessment for PCR-Based Pathogen Detection

The genus Aspergillus is a burden to public health due to its ubiquitous presence in the environment, its production of allergens, and wide demographic susceptibility among cystic fibrosis, asthmatic, and immunosuppressed patients. Current methods of detection of Aspergillus colonization and infection rely on lengthy morphological characterization or nonstandardized serological assays that are restricted to identifying a fungal etiology. Collagen-like genes have been shown to exhibit species-specific conservation across the noncollagenous regions as well as strain-specific polymorphism in the collagen-like regions. Here we assess the conserved region of the Aspergillus collagen-like (acl) genes and explore the application of PCR amplicon size-based discrimination among the five most common etiologic species of the Aspergillus genus, including Aspergillus fumigatus, A. flavus, A. nidulans, A. niger, and A. terreus. Genetic polymorphism and phylogenetic analysis of the aclF1 gene were additionally examined among the available strains. Furthermore, the applicability of the PCR-based assay to identification of these five species in cultures derived from sputum and bronchoalveolar fluid from 19 clinical samples was explored. Application of capillary electrophoresis on nanogels was additionally demonstrated to improve the discrimination between Aspergillus species. Overall, this study demonstrated that Aspergillus acl genes could be used as PCR targets to discriminate between clinically relevant Aspergillus species. Future studies aim to utilize the detection of Aspergillus acl genes in PCR and microfluidic applications to determine the sensitivity and specificity for the identification of Aspergillus colonization and invasive aspergillosis in immunocompromised subjects.     

De Novo synthesis of PCR templates for the development of SARS diagnostic assay

A novel coronavirus was identified as the cause for severe acute respiratory syndrome (SARS). The complete sequence of SARS genome has provided an opportunity for the development of molecular diagnostic assays. To restrain further outbreak of SARS, the World Health Organization has posted several pairs of polymerase chain reaction (PCR) primers for early diagnosis and urged more research to be done on PCR protocols. Here we report a strategy for the de novo synthesis of PCR templates complimentary to the SARS virus genome, which has the advantage of working on PCR templates without concern about viral infection and also has the advantage that it can be used by those who do not have access to the SARS virus. This highly efficient and safe strategy for obtaining SARS gene fragments is useful for the development of PCR assays, as well as for the preparation of reliable positive controls for PCR testing kits.     

Biosynthesis of D-mannitol from D-glucose by Aspergillus candidus

Mannitol has long been known as a product of glucose metabolism by some strains of Aspergillus. Apparently no concerted effort, has been made to develop a practical fermentation process to make mannitol. Work at the Northern Laboratory has shown that nearly all strains of white Aspergillus produce significant amounts of mannitol; many strains of black Aspergillus also have this characteristic. Aspergillus candidus NRRL 305 is an exceptionally good mannitol producer. Studies on a fermentation process were conducted in 20-1, stainless steel fermentors, without baffles. Czapek-Dox medium, modified by addition of corn meal, yeast extract, and enzymatically hydrolyzed casein was the most satisfactory medium tested. Suitable increments of glucose were fed daily to the fermentors. The duration of the fermentation was from 10 to 16 days. The effects of agitation, aeration, temperature, and pH of the medium were studied. Under optimal conditions yields of mannitol approached 50% of the glucose consumed.     

Einbau von Glycin und Serin in Sideramine vom Ferrichrom-Typ bei Pilzen der Gattung Aspergillus in vivo und in vitro

Zusammenfassung Markiertes Glycin und Serin wurde in Ferrichrom, Ferricrocin und Ferrichrysin von vier Stämmen der Gattung Aspergillus zu 1,6–11,8% in vivo eingebaut. Von Aspergillus fumigatus und A. quadricinctus wurden zellfreie Extrakte hergestellt, die Cyclohexapeptide synthetisieren können.

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Incorporation of glycine and serine in sideramines of the ferrichrome type with fungi of the genus Aspergillus in vivo and in vitro

A 1.6–11.8% incorporation of labelled glycine and serine into ferrichrome, ferricrocin and ferrichrysin has been achieved with four strains of Aspergillus in vivo. Cell-free extracts of Aspergillus fumigatus and A. quadricinctus capable of cyclohexapeptide biosynthesis have been prepared.

     

A Molecular Method for Detection of <Emphasis Type="Italic">Aspergillus carbonarius</Emphasis> in Coffee Beans

Ochratoxin A (OTA) is a carcinogenic and nephrotoxic mycotoxin that has been detected in a variety of food products, including green coffee beans. About 80% of Aspergillus carbonarius strains collected from coffee beans are able to produce OTA on this substrate. The rapid identification of this fungal species would be desirable. RAPD assays were applied to identify amplification products specific for A. carbonarius. One selected fragment, denoted OPX7₈₀₉, was cloned and sequenced. Based on the nucleotide sequence obtained, specific oligonucleotides (OPX7F₈₀₉ and OPX7R₈₀₉) were designed and used as primers for DNA amplification. One amplified band of 809 bp was obtained from A. carbonarius genomic DNA, whereas no amplified fragment from DNA of other Aspergillus species was detected. This PCR analysis was also successfully employed to detect A. carbonarius in coffee beans. This PCR assay could contribute to the early and rapid detection of the potential presence of OTA in coffee samples.     

A 76-year-old Man with a Right Lung Adenocarcinoma and Invasive Aspergillosis

A 76-year-old male with adenocarcinoma on the right lung underwent five cycles of chemotherapy with pemetrexed disodium, cisplatin, and dexamethasone. Imaging studies of control showed a node in a cavitary lesion on the left lung, and the main hypothesis was Aspergillus infection. PCR was utilized and contributed to establish the early diagnosis in this patient with invasive aspergillosis. Furthermore, the species Aspergillus fumigatus was characterized by its growing at 50 °C but not at 10 °C, typical culture features, and presence of subclavate vesicles. Diagnosis criteria for Aspergillus pulmonary infection include characteristic clinical and imaging findings, elevated C-reactive protein and erythrocyte sedimentation rate, positive specific serological test, and isolation of Aspergillus from bronchoalveolar cultures. Molecular methods, as PCR, have been useful to complement the conventional microbiological investigations in immunocompromised people with invasive fungal infections. The patient was successfully treated with a schedule of voriconazole 4 mg/kg intravenous infusion every 12 h for 21 days and then switched to oral administration of 200 mg twice a day. He has been comfortable, maintaining normal vital signs, and the results of the periodical microbiologic tests of control are negative. Pathogenesis of invasive aspergillosis in patients with lung cancer is not completely understood. Case studies may contribute to a better knowledge about Aspergillus infection in this setting.     

Aspergillus-specific antibodies – Targets and applications

Aspergillus is a fungal genus comprising several hundred species, many of which can damage the health of plants, animals and humans by direct infection and/or due to the production of toxic secondary metabolites known as mycotoxins. Aspergillus-specific antibodies have been generated against polypeptides, polysaccharides and secondary metabolites found in the cell wall or secretions, and these can be used to detect and monitor infections or to quantify mycotoxin contamination in food and feed. However, most Aspergillus-specific antibodies are generated against heterogeneous antigen preparations and the specific target remains unknown. Target identification is important because this can help to characterize fungal morphology, confirm host penetration by opportunistic pathogens, detect specific disease-related biomarkers, identify new candidate targets for antifungal drug design, and qualify antibodies for diagnostic and therapeutic applications. In this review, we discuss how antibodies are raised against heterogeneous Aspergillus antigen preparations and how they can be characterized, focusing on strategies to identify their specific antigens and epitopes. We also discuss the therapeutic, diagnostic and biotechnological applications of Aspergillus-specific antibodies.     

Homogenisation of cystic fibrosis sputum by sonication--an essential step for Aspergillus PCR

The importance of Aspergillus as a lung pathogen in cystic fibrosis (CF) is becoming increasingly recognised. However, fungal culture of CF sputum is unreliable and there is no consensus for identifying phenotypes beyond ABPA that may benefit from antifungal therapy. There are no published studies using real-time PCR to detect Aspergillus in CF sputum. The major barrier to sensitive detection of Aspergillus using PCR is sputum homogenisation. This study aimed to optimise sputum homogenisation utilising sonication to improve Aspergillus DNA extraction. Sonication amplitude and duration that enabled sputum homogenisation but ensured preservation of DNA integrity were first determined. 160 sputum samples were collected from CF patients. 49 of the sputum samples were split, one half was used for standard culture and the other half was homogenised with NALC-NaOH before undergoing DNA extraction. The subsequent 111 samples were homogenised with dithiothreitol plus sonication prior to culture and DNA extraction. Real-time PCR targeting a portion of the 18S rDNA of Aspergillus was performed on all DNA extractions. In the 49 samples with no sonication 8 (16%) were culture positive but only 4 of these were PCR positive. However, PCR was positive in 11 culture negative samples. PCR after sonication showed a significant improvement in sensitivity: 33 (30%) were culture and PCR positive, 48 (43%) were culture negative, but PCR positive (p < 0.0001) and 30 (27%) were culture and PCR negative. The combination of dithiothreitol and sonication to homogenise sputum increases PCR yield, with PCR being substantially more sensitive than culture.     

Aspergillus myositis in a patient with a myelodysplastic syndrome

We present the case of an elderly man who, while being treated with corticosteroids for a myelodysplastic syndrome, developed myositis of the calf due to Aspergillus fumigatus. Despite therapy with amphotericin B the myositis failed to resolve and he died. At autopsy, a localized necrotizing myositis of the right calf was found with no evidence of disseminated Aspergillus infection. Myositis in the setting of disseminated candidiasis or cryptococcosis has been previously reported. This case is unique in that it is the first reported case of localized fungal myositis and of myositis caused by Aspergillus.     

Bias due to methods of parasite detection when estimating prevalence of infection of Triatoma infestans by Trypanosoma cruzi

The study aimed to quantify the bias from parasite detection methods in the estimation of the prevalence of infection of Triatoma infestans by Trypanosoma cruzi, the agent of Chagas disease. Three common protocols that detect T. cruzi in a sample of 640 wild‐caught T. infestans were compared: (1) the microscopic observation of insect fecal droplets, (2) a PCR protocol targeting mini‐exon genes of T. cruzi (MeM‐PCR), and (3) a PCR protocol targeting a satellite repeated unit of the parasite. Agreement among protocols was computed using Krippendorff Kα. The sensitivity (Se) and specificity (Sp) of each protocol was estimated using latent class models. The PCR protocols were more sensitive (Se > 0.97) than microscopy (Se = 0.53) giving a prevalence of infection of 17–18%, twice as high as microscopy. Microscopy may not be as specific as PCR if Trypanosomatid‐like organisms make up a high proportion of the sample. For small T. infestans, microscopy is not efficient, giving a prevalence of 1.5% when PCR techniques gave 10.7%. The PCR techniques were in agreement (Kα = 0.94) but not with microscopy (Kα never significant with both PCR techniques). Among the PCR protocols, the MeM‐PCR was the most efficient (Se=1; Sp=1).     

In Vitro Interactions Between Antifungals and Methotrexate Against <Emphasis Type="Italic">Aspergillus</Emphasis> spp.

Methotrexate has been widely used in the treatment of osterosarcoma, intracranial lymphomas and leukemia. However, patients are also at high risk of opportunist pathogens such as Aspergillus spp. infection for their deeply depressed immunity. The optimal choice of antifungal agents during the infection of Aspergillus for these patients is necessary to be explored. In this study, we investigated the interactions between antifungals and methotrexate against Aspergillus in vitro. A total of 23 clinical isolates of Aspergillus spp. were studied. Microdilution checkerboard technique was performed to evaluate the interaction of methotrexate with voriconazole, itraconazole, terbinafine and amphotericin B. The highest rate of synergy was obtained for the combination of terbinafine and methotrexate, which exhibited synergy against 60.9% (14/23) of strains. No interaction was detected for the combinations of methotrexate plus itraconazole or amphotericin B against 95.7% (22/23) or 100% of strains, respectively. Although voriconazole exhibited indifferent against 87% (20/23) of strains when combined with methotrexate, antagonism effect was found against 13% (3/23) of strains. The positive interactions of terbinafine and methotrexate were also certified by disk diffusion assay. In addition, we observed the morphological changes for the interaction of methotrexate with terbinafine against Aspergillus. Further inhibition and distortion of growth were found after the combination of terbinafine and methotrexate compared with the drugs treated alone. Clinical studies are warranted to further elucidate optimal treatments for the immucompromised patients with Aspergillus infection.     

The air spora close to a compost facility in Northeast Oklahoma: Part I—spore trap sampling

A compost facility in northeast Oklahoma is located relatively close to a residential area and is the focus of complaints about smell and concerns about health effects. Several species of Aspergillus have been known to cause health problems, and at least one of these species is dominant in compost. The atmosphere surrounding the compost facility was monitored for 1 year using Burkard spore traps to determine if there was a significant difference in Penicillium/Aspergillus type spores concentration between a test and control site. Samplers were situated 710 m downwind for the test site and 6,085 m upwind at the control site. There was no significant difference in mean concentration of Penicillium/Aspergillus type spores between the two sites (t = 0.576 P > 0.05). The mean concentration of total spores was significantly higher at the upwind control site (t = −7.64, P < 0.01). Wind direction was examined to determine if the compost facility was a possible source for any spikes in concentration. No clear relationship was found between wind direction and mean Penicillium/Aspergillus concentration at the test site, but peak concentrations of Penicillium/Aspergillus seen at the test site were on days when it was downwind from the composting facility. However, these concentrations were no higher than those seen at the control site on other days. If the compost was releasing large amounts of Penicillium/Aspergillus type spores into the atmosphere they were generally diluted to background levels by the time they reached the test site.