Adherence of Aspergillus species to soft contact lenses and attempts to inhibit the adherence

The aim of this research was to assess the adherence of various Aspergillus species (A. niger, A. fumigatus, A. flavus) to contact lenses with different water content and to attempt to inhibit the adherence of Aspergillus spp. to the contact lenses by a chitin derivative (CSE). Adherence of Aspergillus spp. to lenses with higher water content was greater. Differences were found between the adherence levels of various Aspergillus species to contact lenses with different water content. CSE significantly inhibits the adherence in vitro of A. niger, A. fumigatus and A. flavus to soft contact lenses. These findings suggest a possibility for prevention of fungal ocular infections caused by Aspergillus spp. in wearers of contact lenses.     

In vitro susceptibilities of Aspergillus spp. causing otomycosis to amphotericin B, voriconazole and itraconazole

Otomycosis is worldwide in distribution and most commonly caused by Aspergillus species. Amphotericin B, itraconazole and voriconazole are used for the treatment of aspergillosis, but recently an increase in resistance to these agents has been reported. We aimed at investigating the in vitro activities of amphotericin B, voriconazole and itraconazole against Aspergillus isolates causing otomycosis. Mycological analysis of samples from the ear canals of patients was performed by culturing onto Sabouraud Dextrose Agar and by evaluating microscopically. Aspergillus species were identified with colony morphology and microscopic appearance, and tested for susceptibilities to amphotericin B, itraconazole and voriconazole by the CLSI reference broth microdilution method (M38-A document). A total of 120 isolates from 120 patients, comprising 57 Aspergillus niger, 42 Aspergillus fumigatus, nine Aspergillus flavus, six Aspergillus nidulans and six Aspergillus terreus strains were tested. No resistance was determined against amphotericin B and voriconazole, while six A. fumigatus and three A. niger isolates were resistant to itraconazole. In vitro data obtained in this study showed the resistance to itraconazole, while all of the isolates were susceptible to voriconazole and amphotericin B. Voriconazole seemed to be an alternative in the treatment of infections related to Aspergillus spp. but further studies are needed to learn more about the antifungal resistance of different species of Aspergillus to different agents.     

Aspergillus flavus: an emerging non-fumigatus Aspergillus species of significance

Invasive aspergillosis is rare in immunocompetent people but contributes to significant morbidity and mortality in immunosuppressed patients. The majority (approximately 80%) of invasive Aspergillus infections is caused by Aspergillus fumigatus . The second most frequent (approximately 15–20%) pathogenic species is Aspergillus flavus and to a lesser extent, Aspergillus niger and Aspergillus terreus . Aspergillus flavus has emerged as a predominant pathogen in patients with fungal sinusitis and fungal keratitis in several institutions worldwide. To date, there has not been any publication exclusively reviewing the topic of A. flavus in the literature. This article reviews the microbiology, toxigenicity and epidemiology of A. flavus as well as describes the clinical characteristics, diagnosis and management of infections caused by this organism.     

Experimental Aspergillosis in Mice

The untreated mice and the mice treated with cortisone, antibiotics and both cortisone and antibiotics, were exposed individually to the spores of A. fumigatus, A. flavus and A. niger. The cortisone but not the antibiotics seemed to enhance susceptibility of the mice to infection with Aspergillus species. The species of Aspergillus also affected the severity and progress of the lesion.     

Phenotypic and genotypic characterization of reference strains of the genus Aspergillus

Twenty-five culture collection strains from four Aspergillus species (A. fumigatus n = 8, A. flavus n = 8, A. niger n = 4, A. nidulans n = 5) were characterized by four methods: (i) determination of patterns in an assimilation assay; (ii) protein pattern of whole mycelial cell lysates in the sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE); (iii) reactivity of a pool serum obtained from cystic fibrosis patients with mycelial lysates in the immunoblot; and (iv) random amplification of polymorphic DNA (RAPD) with eight primers having arbitrary or repetitive sequences. In the assimilation assay the A. fumigatus strains showed identical patterns in contrast to the strains of the species A. flavus, A. niger, and A. nidulans, which each showed four patterns. In the SDS-PAGE no differences in the band patterns in the A. fumigatus strains were found, in contrast to the A. flavus (three patterns), A. nidulans (five patterns) and A. niger strains (two patterns). The immunoblot patterns were characteristic for each species with bands at 62 and 17/18 kDa in the A. fumigatus strains, at 51 and 18 kDa in the A. flavus strains, at 51 kDa in the A. niger strains, and at 51, 40 and 17/18 kDa in the A. nidulans strains allowing, however, no intraspecies typing. In the RAPD assay four out of eight primers gave interpretable patterns with 3-20 bands. None of the primers showed sufficient discriminatory power when used alone. However, when combining the results of two of the primers (5'-GTA TTG CCC T-3' and 5'-GAT AGA TAG ATA GAT A-3') all strains except two A. fumigatus strains could be clearly separated from each other. It is concluded that the the RAPD assay showed the most discriminatory power in all Aspergillus species investigated. In contrast to the phenotypically similar A. fumigatus strains, the strains of the species A. flavus, A. nidulans and A. niger differed in their phenotypic characteristics. The presented data of strains from international culture collections may serve as basis for interlaboratory standardization of typing methods.     

Rapid detection of common pathogenic Aspergillus species by a novel real-time PCR approach

Invasive aspergillosis is a major cause of morbidity and mortality in immunocompromised and critically ill patients. Standard culture based methods for the diagnosis of Aspergillus infections have limited sensitivity and specificity and are time consuming. The recent availability of novel molecular based diagnostic techniques offers the potential of rapid, highly sensitive and specific pathogen detection. In this study, we aimed to develop a diagnostic assay to detect simultaneously common pathogenic Aspergillus species including Aspergillus fumigatus , Aspergillus flavus , Aspergillus niger and Aspergillus terreus in whole blood specimen . A real-time PCR/probe set assay was designed to amplify the 18sRNA genomic region of Aspergillus. In addition to a previously probe set validated for the detection of A. fumigatus ('Asp.fum'), two other probe sets ('Asp.ter'; 'Asp.nig') were synthesised with 100% sequence homology to A. terreus or A. niger. Specificity testing demonstrated amplification of Aspergillus DNA, but not DNA from a panel of other common pathogenic fungi and bacteria and/or human DNA. Sensitivity testing in serial dilution assays revealed a lower detection limit of 1–5 CFU ml⁻¹ whole blood. However, no single probe set was most sensitive for all Aspergillus species tested and a combination of all three probe sets was necessary to achieve maximal overall assay sensitivity. Furthermore, inclusion of a subsequent probe melting temperature (Tm) analysis demonstrated species-specific Tm-patterns, useful to differentiate simultaneously between the different Aspergillus species detected. We describe a highly specific and sensitive real-time PCR approach to detect and differentiate the most common pathogenic Aspergillus species in a rapid 'same day' assay.     

Pulmonary Aspergillosis in Indian buffaloes

Five cases of pulmonary aspergillosis were recorded in buffaloes on histopathological basis. Aspergillus flavus from an aged buffalo and A. fumigatus from a 4 week old buffalo calf could be isolated. Only six out of 789 sera samples gave positive precipitin reaction with filtrate antigen of Aspergillus species. The sera samples from 3, 2 and 1 case reacted with antigen of A. flavus, A. fumigatus and A. niger respectively. Sixty four sera gave false positive lines due to C-reacting proteins. A. flavus isolated from one case was found to be highly pathogenic to the rabbits. The histopathological lesions in natural cases as well as experimentally inoculated rabbits consisted of mixed granulomatous foci in affected organs.

Zusammenfassung

Fünf Fälle einer pulmonalen Aspergillose wurden bei Büffeln aufgrund histopathologischer Untersuchungen diagnostiziert. Aspergillus flavus wurde bei einem alten Büffel und A. fumigatus bei einem 4 Wochen alten Büffelkalb aus den Lungen isoliert. Nur 6 von 789 Serumproben zeigten eine positive Präzipitationsreaktion auf filtriertes Aspergillus - Antigen. Sechs Serumproben reagierten positiv auf die Antigene wie folgt: A. flavus 3ma1, A. fumigatus 2mal und A. niger 1mal. 64 Seren zeigten falsch-positive Präzipitations- linien auf C-reaktive Proteine. Der in einem Fall isolierte A. flavus -Stamm erwies sich für Kaninchen als hoch pathogen. Die histopathologischen Läsionen sowohl bei natürlichen Erkrankungen als auch bei experimentell infizierten Kaninchen bestanden aus verschie- denen granulomatösen Herden in den befallenen Organen.     

Identification of medically important Aspergillus species by single strand conformational polymorphism (SSCP) of the PCR-amplified intergenic spacer region

The amplified 5.8S RNA coding DNA with the neighbouring internal transcribed spacers ITS I and ITS II (ITS I--5.8S rDNA--ITS II) of 27 culture collection strains of Aspergillus fumigatus, Aspergillus flavus, Aspergillus nidulans, Aspergillus niger, and Aspergillus terreus were investigated by single strand conformational polymorphism (SSCP) analysis. All strains showed a polymerase gel electrophoresis (PCR) product of 0.6 kb. Separation of DNA single strands of the PCR product in an acrylamide-bisacrylamide gel containing formamide SSCP resulted in individual patterns for each of the species. A minor variability within the species A. fumigatus and A. flavus did not affect the correct species identification. The results were confirmed when investigating 55 wild strains from patients and the environment. It is concluded that the analysis of the amplified ITS I--5.8S rDNA--ITS II region by SSCP allows the differentiation of the medically most relevant aspergilli. As the method does not require morphologically fully developed fungal colonies, it yields species diagnosis faster than the conventional macroscopic and microscopic identification.     

Antifungal activity of human polymorphonuclear and mononuclear phagocytes against non-fumigatus Aspergillus species

Human phagocytic defenses against non-fumigatus aspergilli were compared with those against Aspergillus fumigatus. Monocyte-derived macrophages exhibited lower phagocytic capacities against non-fumigatus aspergilli, particularly A. nidulans and A. niger, compared with A fumigatus (P < 0.05). Non-opsonized hyphae suppressed oxidative burst (as measured by superoxide anion production) of polymorphonuclear leukocytes (PMNs). Further, these cells responded with less vigorous oxidative burst to serum-opsonized hyphae of non-fumigatus Aspergillus species, particularly A. flavus and A. terreus, compared with A. fumigatus (P < or = 0.05). Similarly, PMNs induced less hyphal damage assessed by XTT colorimetric assay to non-fumigatus species, particularly A. flavus and A. nidulans, compared with A. fumigatus (P < 0.05). Thus, non-fumigatus aspergilli are generally more resistant to mononuclear and polymorphonuclear phagocytes than A. fumigatus, a finding which should be considered during management of invasive aspergillosis caused by these species.     

In vitro efficacy of 75 essential oils against Aspergillus niger

Aspergillus niger is an opportunistic human pathogen and a strong air pollutant. A study was conducted with 75 different essential oils for the inhibition of hyphal growth and spore formation in Aspergillus niger. Cinnamomum zeylanicum (bark), Cinnamomum zeylanicum (leaf), Cinnamomum cassia, Syzygium aromaticum and Cymbopogon citratus were the top five essential oils which demonstrated marked inhibitory effect against hyphal growth and spore formation of A. niger. The chemical composition of these five most active essential oils was investigated by gas chromatography-mass spectra (GC-MS). Most of the other essential oils were found challenging to combat A. niger, suggesting their use as strong aroma therapeutic agents.     

Molecular detection and identification of Aspergillus spp. from clinical samples using real-time PCR

The definite and rapid diagnosis of invasive aspergillosis is necessary because of the high mortality caused. The objective of this study was to evaluate a real-time PCR assay to detect Aspergillus spp. in clinical samples, based on the Light Cycler technology. Specificity was assessed by using DNA extracted from pathogenic and non-pathogenic bacteria/fungi from Spanish Collection including: two Aspergillus flavus , four Aspergillus fumigatus , two Aspergillus nidulans , two Aspergillus niger and two Aspergillus terreus isolates. The analytical sensitivity was evaluated with different inocula (10¹–10⁵ conidia ml⁻¹), and serially diluted DNA of A. fumigatus. To assess clinical applicability, samples from patients at risk were analysed. Species identification was determined by analysing the melting curves. Reactions using genomic DNA from other species of different genera than Aspergillus were negative (specificity: 100%). Analytical sensitivity was 60 fg using DNA and 5–20 conidia using conidial suspensions. The linear range was from 60 to 6 × 10⁷ fg. The Tm ranged from 67.34 to 70.7 °C for the different Aspergillus spp. studied. Nine hundred and forty-eight consecutive blood samples from 127 patients were processed. In total, 10 (1%) of 948 samples from blood samples were PCR-positive. The real-time PCR assay provides a high sensitivity and specificity for detection of fungal DNA and rapidly identifies most of clinically relevant Aspergillus species.     

Antimycotic Activity of Different Chemicals, Chaksine Iodide and Garlic/Antimykotische Aktivität von verschiedenen Chemikalien, Chaksinjodit und Knoblauch

Summary: Thirteen different fungi reported to produce keratomycosis in man, were tested for their susceptibility to in vitro antifungal action of alum, sulphacetamide, copper sulphate, silver nitrate, chaksine iodide and garlic. Interestingly, 2% alum, 30% sodium sulphacetamide, 0.2% copper sulphate and 0.1% silver nitrate inhibited the growth of all 13 fungi viz. Alternaria, Aspergillus flavus, Asp. fumigatus, Asp. niger, Candida albicans, Curvularia, Drechslera, Fusarium, Mucor, Penicillium, Rhizopus oryzae, Scopulariopsis and Syncephalastrum. Chaksine iodide, an alkaloid from an Indian medicinal herb, Cassia absus, at a concentration of 0.5% inhibited all fungi. Lower concentrations were either ineffective or effective against a few fungi only. Garlic (up to 1%) could inhibit none of the fungi. Repeated experiments yielded consistent findings.
Zusammenfassung: 13 verschiedene Pilze, die als Erreger einer Keratomykose des Menschen bekannt sind, wurden in vitro gegen folgende Substanzen getestet: Alaun, Sulfazetamid, Kupfersulfat, Silbernitrat, Chaksinjodit und Knoblauch.
Alle 13 Pilze wurden durch 2% Alaun, 30% Natriumsulfazetamid, 0,2% Kupfersulfat und 0,1% Silbernitrat gehemmt (Alternaria, Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Candida albicans, Curvularia, Drechslera, Fusarium, Mucor, Penicillium, Rhizopus oryzae, Scopulariopsis und Syncephalastrum). Chaksinjodit, ein Alkaloid aus einer indischen Heilpflanze, Cassia absus hemmte alle Pilze bei einer Konzentration von 0,5%. Niedrigere Konzentrationen waren entweder unwirksam oder nur gegen einige der Pilze wirksam. Knoblauch konnte in einer Konzentration bis zu 1% keine Pilze am Wachstum hindern. Die Wiederholung der Experimente führte zu entsprechenden Ergebnissen.     

Etiological Agents of Otomycosis in Nigeria Das Erregerspektrum von Otomykosen in Nigeria

A total of 159 suspected cases of otomycosis comprising 101 adults and 58 children, 61 males and 98 females were investigated. Of these, 36 cases were confirmed specifically of mycotic etiology on the basis of microscopic demonstration of fungal structures in epithelial debris/plugs and positive culture. Another 31 cases positive for fungi by culture but negative for direct microscopy were considered of doubtful fungal etiology. The predominant etiological agents in the confirmed cases were Aspergillus niger (13 cases), followed by A. flavus (6), Candida albicans (6) and C. parapsilosis (4). Other species represented were Pseudoallescheria boydii (2), C. guilliermondii (1), Aspergillus sp. (unidentified) (2), and Candida sp. (unidentified) (1). One case was of mixed infection due to A. niger and C. albicans. Fungal cultures from normal healthy ear canals of 46 persons were positive in 17 cases, the predominant fungus being Aspergillus (mainly A. niger).     

Expression pattern of aspartic proteinase antigens in aspergilli

Summary. Sporulating colonies of Aspergillus fumigatus, A. flavus , and A. niger were subjected to immunofluorescence using specific polyclonal antibodies against the aspergillopepsin PEP (EC 3.4.23.18), a secretory aspartic proteinase produced by A. fumigatus. The proteinase antigen was found mainly in developing conidiophores of aspergilli, in submerged mycelia and on the tips of growing aerial mycelia. Mature aerial hyphae and spores showed no immunofluorescence at all. Sporulating conidiophores revealed only weak activity in A. fumigatus and A. flavus . The distinct pattern of expression of the aspartic proteinase antigens suggests a role for such enzymes in the growth of hyphae and the development of conidiophores and thus for the sporulation process in aspergilli.
Zusammenfassung. Sporulierende Kolonien von Aspergillus fumigatus, A. flavus und A. niger wurden der Immunfluoreszenz mit spezifischen polyklonalen Antikörpern gegen das Aspergillopepsin PEP (EC 3.4.23.18), einer sekretorischen Aspartatprotease von A. fumigatus , unterworfen. Die Fluoreszenz fand sich hauptsächlich an jungen Konidiophoren. Reife Konidiophore zeigten abnehmende Fluoreszenz, während Konidien und reifes Luftmyzel von A. fumigatus und A. flavus keine Antigene exprimierten. Antigenaktivität fand sich auch am Nährmyzel und an den Spitzen wachsen-der Lufthyphen. Dieses charakteristische Antigenmuster deutet auf eine Rolle von Aspartat-proteasen beim Wachstum von Hyphen, der Entwicklung von Konidiophoren und somit auch der Sporulation von Aspergillen.     

Fungal Contamination of Market Goat Meat and its Public Health Importance

Summary: The fungual flora of 100 market goat meat samples comprising of 20 samples each of thigh muscles, chest muscles, liver, spleen and mesentric lymph nodes were studied with the object of assessing the fungual contamination of meat. The fungi isolated were Aspergillus niger, A. fumigatus, A. flavus, A. nidulans, Rhizopus sp., Penicillium sp., Absidia sp., Curvularia sp., Helminthosporium sp. and unidentified yeasts. No growth was observed in five samples. The mode of contamination and the public health importance of the isolated fungi were discussed.
Zusammenfassung: 100 Fleischproben (je 20 Proben Schenkelmuskulatur, Brust-muskulatur, Leber, Milz und Mesenteriallymphknoten) von geschlachteten Ziegen wurden kulturell auf das Vorhandensein von Pilzen untersucht. Aus 95 Proben gelang die Isolierung von Aspergillus niger, A. fumigatus, A. flavus, A. nidulans, Rhizopus sp., Penicillium sp., Absidia sp., Curvularia sp., Helminthosporium sp. und nicht näher bestimmte Hefen. Die Möglichkeiten der Kontamination sowie die Bedeutung der nachgewiesenen Schimmel- und Sproßpilze für die menschliche Gesundheit werden diskutiert.     

Fungal Contamination of Market Goat Meat and its Public Health Importance

Summary: : The fungual flora of 100 market goat meat samples comprising of 20 samples each of thigh muscles, chest muscles, liver, spleen and mesentric lymph nodes were studied with the object of assessing the fungual contamination of meat. The fungi isolated were Aspergillus niger, A. fumigatus, A. flavus, A. nidulans, Rhizopus sp., Penicillium sp., Absidia sp., Curvularia sp., Helminthosporium sp. and unidentified yeasts. No growth was observed in five samples. The mode of contamination and the public health importance of the isolated fungi were discussed.
Zusammenfassung: Fleischproben (je 20 Proben Schenkelmuskulatur, Brust-muskulatur, Leber, Milz und Mesenteriallymphknoten) von geschlachteten Ziegen wurden kulturell auf das Vorhandensein von Pilzen untersucht. Aus 95 Proben gelang die Isolierung von Aspergillus niger, A. fumigatus, A. flavus, A. nidulans, Rhizopus sp., Penicillium sp., Absidia sp., Curvularia sp., Helminthosporium sp. und nicht näher bestimmte Hefen. Die Möglichkeiten der Kontamination sowie die Bedeutung der nachgewiesenen Schimmel- und Sproßpilze für die menschliche Gesundheit werden diskutiert.     

In vitro studies on the activity of amphotericin B and lipid-based amphotericin B formulations against Aspergillus conidia and hyphae

The minimum inhibitory concentrations (MICs) of amphotericin B and lipid-based amphotericin B formulations against isolates of Aspergillus spp. were tested using a broth microdilution method. Twelve isolates of Aspergillus fumigatus, eight of Aspergillus flavus, six of Aspergillus niger and seven of Aspergillus terreus were examined. In addition, an assay for hyphae of Aspergillus spp. was performed since the invasive form is manifested by the appearance of hyphal structures. MICs of hyphae against lipid-based amphotericin B formulations were within three dilutions higher than those against conidia for almost all isolates of Aspergillus spp. (P < 0.01). In contrast, the differences in the in vitro efficacies of amphotericin B were the lowest for Aspergillus spp. This study demonstrates the importance of the type of inoculum used to test antifungal susceptibilities of Aspergillus spp. The significance of these results for in vivo outcome needs to be determined.     

Fungitoxic effect of some organic volatile substances against fungi causing otomycosis

Summary. The fungitoxic effect of seven volatile substances (ammonia, carbon disulphide, petroleum benzene, carbon dioxide, methanol, glacial acetic acid and hydrogen peroxide) against five fungi, i.e. Aspergillus niger, A. flavus, Absidia corymbifera, Penicillium nigricans and Candida albicans , was determined on the basis of their dry mycelial weight and sporulation or budding activity. These organisms were isolated from patients suffering from fungal infections of the ear. All the volatile substances tested were found to inhibit mycelial growth and sporulation or budding.
Zusammenfassung. Die fungitoxische Wirkung von sieben flüchtigen Stoffen (Ammoniak, Schwefelkohlenstoff, Benzin, Kohlendioxid, Methanol, Eisessig und Wasserstoffperoxid) auf fünf Pilzarten, nämlich Aspergillus niger, A. flavus, Absidia corymbifera, Penicillium nigricans und Candida albicans , wurde anhand des Trockengewichts und der Sporulations- bzw. Sprossungsfähigkeit untersucht. Alle Organismen waren Isolate von Otomykose-Patienten. Sämtliche getesteten flüchtigen Stoffe vermochten das Myzelwachstum wie auch Sporulation bzw. Sprossung zu hemmen.     

In vitro activity of terbinafine and itraconazole against Aspergillus species isolated from otomycosis

The minimum inhibitory concentrations (MIC, microg ml-1) of itraconazole and terbinafine against overall 34 Aspergillus isolates from the external ear canals with otomycosis have been determined with M38-P microdilution method suggested by National Committee for Clinical Laboratory Standards (NCCLS). MIC intervals in 48 h determined by taking MIC-2 value of itraconazole (the lowest drug concentration causing 50% inhibition of visible fungal growth) and MIC-0 value of terbinafine (the lowest drug concentration causing 100% inhibition of visible fungal growth) as a basis have been found as follows: 0.125-1 and 0.06-0.5 microg ml-1 for A. niger (22 strains), 0.06-0.25 and 0.06-0.125 microg ml-1 for A. flavus (10 strains), 0.125 and 0.125-0.5 microg ml-1 for A. terreus (two strains). It has been observed that both of the antifungal agents showed an in vitro activity against all Aspergillus species tested.