Use of Mycological, nested PCR, and Real-time PCR Methods on BAL Fluids for Detection of Aspergillus fumigatus and A. flavus in Solid Organ Transplant Recipients

Invasive aspergillosis continues to be a significant cause of morbidity and mortality in solid organ transplant (SOT) recipients. A reliable and early diagnostic method is needed to improve survival. In this study, four methods direct microscopy, culture, nested PCR on internal transcribed spacer region, and TaqMan real-time PCR targeted β-tubulin gene were examined for the detection of Aspergillus fumigatus and A. flavus in sixty-four bronchoalveolar lavage (BAL) fluids that were obtained from SOT recipients. Direct examination with 20 % KOH (potassium hydroxide) and culture on mycological media were also performed. Of the 64 samples, seven (10.9 %) were positive in direct examination (five with septate hyphae and two with aseptate hyphae), and 15 (23 %) had positive culture including five A. flavus, four A. niger, two Penicillium spp., two Rhizopus spp., one Fusarium spp. and one mixed A. flavus/A. niger. Twenty five (39 %) samples had positive nested PCR with A. flavus and 6 (9.4 %) with A. fumigatus-specific primers. Only eight (12.5 %) had positive real-time PCR for A. flavus and nine (14 %) for A. fumigatus. The incidence of aspergillosis in these patients included proven invasive pulmonary aspergillosis (IPA) in two (3 %), probable IPA in 14 (22 %), possible IPA in 38 (59 %), and not IPA in 10 (16 %). A. flavus was the most common cause of pulmonary aspergillosis (PA) in the study. The results suggest that because nested PCR is too sensitive it may increase the number of false-positive results and is not recommended for BAL samples for diagnosis of PA. Although further studies with significant number of proved positive/negative standard BAL samples are necessary for better evaluation, the novel multiplex real-time PCR developed in the study could be promising as a valid diagnostic method for IPA.     

Simple and Highly Discriminatory VNTR-Based Multiplex PCR for Tracing Sources of Aspergillus flavus Isolates

Aspergillus flavus is second only to A. fumigatus in causing invasive aspergillosis and it is the major agent responsible for fungal sinusitis, keratitis and endophthalmitis in many countries in the Middle East, Africa and Southeast Asia. Despite the growing challenge due to A. flavus, data on the molecular epidemiology of this fungus remain scarce. The objective of the present study was to develop a new typing method based on the detection of VNTR (Variable number tandem repeat) markers. Eight VNTR markers located on 6 different chromosomes (1, 2, 3, 5, 7 and 8) of A. flavus were selected, combined by pairs for multiplex amplifications and tested on 30 unrelated isolates and six reference strains. The Simpson index for individual markers ranged from 0.398 to 0.818. A combined loci index calculated with all the markers yielded an index of 0.998. The MLVA (Multiple Locus VNTR Analysis) technique proved to be specific and reproducible. In a second time, a total of 55 isolates from Chinese avian farms and from a Tunisian hospital have been evaluated. One major cluster of genotypes could be defined by using the graphing algorithm termed Minimum Spanning Tree. This cluster comprised most of the isolates collected in an avian farm in southern China. The MLVA technique should be considered as an excellent and cost-effective typing method that could be used in many laboratories without the need for sophisticated equipment.     

A simple PCR/RFLP analysis can differentiate between Candida albicans,Aspergillus niger,and Aspergillus fumigatus

The clinical management of immunocompromised patients depends on the rapid identification of infectious agents such as fungal pathogens. The procedure described here for accomplishing this uses a sensitive polymerase chain reaction method, previously reported, combined with restriction-enzyme digestion to distinguish between Candida and Aspergillus species and to classify Aspergillus strains.     

A simple PCR/RFLP analysis can differentiate between Candida albicans, Aspergillus niger, and Aspergillus fumigatus

The clinical management of immunocompromised patients depends on the rapid identification of infectious agents such as fungal pathogens. The procedure described here for accomplishing this uses a sensitive polymerase chain reaction method, previously reported, combined with restriction-enzyme digestion to distinguish between Candida and Aspergillus species and to classify Aspergillus strains.     

Relationships betweenAspergillus flavus,A. niger and some other fungi in the mycoflora of groundnut kernels

Summary The relationships betweenAspergillus flavus, A. niger, Penicillium funiculosum, P. rubrum, andFusarium solani have been studied in 234 samples and 5850 plates from fresh and stored kernels derived from 2 years' groundnut crops in Israel.Numerical relations between these fungi, as obtained by totalling the number of colonies developing in all the 25 platings made for each sample, did not always give reliable indications of potential antagonism between the species. But consideration of the number of colonies developing in individual plates showed pronounced antagonism betweenA. flavus andA. niger, and slightly less but still marked antagonism between each of these species andP. funiculosum, P. rubrum andF. solani.This research is supported by Grant Number FG-Is-161 of the United States Department of Agriculture to whom the author is indebted.     

Biosynthesis of extracellular and intracellular gold nanoparticles by Aspergillus fumigatus and A. flavus

Green chemistry is a boon for the development of safe, stable and ecofriendly nanostructures using biological tools. The present study was carried out to explore the potential of selected fungal strains for biosynthesis of intra- and extracellular gold nanostructures. Out of the seven cultures, two fungal strains (SBS-3 and SBS-7) were selected on the basis of development of dark pink colour in cell free supernatant and fungal beads, respectively indicative of extra- and intracellular gold nanoparticles production. Both biomass associated and cell free gold nanoparticles were characterized using X-ray diffractogram (XRD) analysis and transmission electron microscopy (TEM). XRD analysis confirmed crystalline, face-centered cubic lattice of metallic gold nanoparticles along with average crystallite size. A marginal difference in average crystallite size of extracellular (17.76 nm) and intracellular (26 and 22 nm) Au-nanostructures was observed using Scherrer equation. In TEM, a variety of shapes (triangles, spherical, hexagonal) were observed in both extra- and intracellular nanoparticles. 18S rRNA gene sequence analysis by multiple sequence alignment (BLAST) indicated 99 % homology of SBS-3 to Aspergillus fumigatus with 99 % alignment coverage and 98 % homology of SBS-7 to Aspergillus flavus with 98 % alignment coverage respectively. Native-PAGE and activity staining further confirmed enzyme linked synthesis of gold nanoparticles.     

Comparison of the Thermostability Properties of Three Acid Phosphatases from Molds: Aspergillus fumigatus Phytase, A. niger Phytase, and A. niger pH 2.5 Acid Phosphatase

Enzymes that are used as animal feed supplements should be able to withstand temperatures of 60 to 90°C, which may be reached during the feed pelleting process. The thermostability properties of three histidine acid phosphatases, Aspergillus fumigatus phytase, Aspergillus niger phytase, and A. niger optimum pH 2.5 acid phosphatase, were investigated by measuring circular dichroism, fluorescence, and enzymatic activity. The phytases of A. fumigatus and A. niger were both denatured at temperatures between 50 and 70°C. After heat denaturation at temperatures up to 90°C, A. fumigatus phytase refolded completely into a nativelike, fully active conformation, while in the case of A. niger phytase exposure to 55 to 90°C was associated with an irreversible conformational change and with losses in enzymatic activity of 70 to 80%. In contrast to these two phytases, A. niger pH 2.5 acid phosphatase displayed considerably higher thermostability; denaturation, conformational changes, and irreversible inactivation were observed only at temperatures of ≥80°C. In feed pelleting experiments performed at 75°C, the recoveries of the enzymatic activities of the three acid phosphatases were similar (63 to 73%). At 85°C, however, the recovery of enzymatic activity was considerably higher for A. fumigatus phytase (51%) than for A. niger phytase (31%) or pH 2.5 acid phosphatase (14%). These findings confirm that A. niger pH 2.5 acid phosphatase is irreversibly inactivated at temperatures above 80°C and that the capacity of A. fumigatus phytase to refold properly after heat denaturation may favorably affect its pelleting stability.     

Multiplex PCR assay for detection of Vibrio vulnificus biotype 2 and simultaneous discrimination of serovar E strains

In the present work we develop a multiplex PCR assay for the detection and identification of the fish pathogen Vibrio vulnificus biotype 2 with discriminating potential for zoonotic strains (serovar E). The PCR assay allowed the identification of two new biotype 2 serovar E human isolates from culture collections. Finally, the multiplex was successfully applied to both diagnosis and carrier detection in field samples.     

Preparation and properties of the endotoxins of Aspergillus fumigatus and Aspergillus flavus

Summary Endotoxins have been reported to occur inAspergillus fumigatus andA. flavus, but the data regarding them are incomplete. Optimal cultural conditions and methods of extraction are described here for the production of the toxins of both of these fungi. They are obtained from the well-washed mycelia; only minute amounts could be demonstrated in the concentrated culture fluids. Both are powerful nephrotoxins; a characteristic necrosis of the kidney cortex is evident grossly in mice, and histological study shows no difference between the effects on the kidney of the two toxins. However, the fumigatus extracts have strong hemolytic properties, especially for sheep erythrocytes, and marked dermonecrotic activity in rabbit skin. No hemolytic action has been demonstrated for the flavus extracts, which possess a powerful hemagglutinin for rabbit erythrocytes and only mild dermonecrotic properties. Immune sera have been prepared in rabbits for both toxins, and in each case these prevent all the observed effects of the toxin. Precipitin titers are closely correlated with neutralizing titers. Chemical differences are suggested by the different procedures applicable to concentration and purification of the toxins. The toxin fromA. fumigatus is precipitated by acetone and can be concentrated and purified to a considerable degree by acetone precipitation and dialysis. The flavus toxin has not been precipitated by any of the solvents tried but can be freed to some extent of inert substances by acid treatment and saturation with ammonium sulfate.Deceased.     

A Multiplex PCR Method for Rapid Identification of Brachionus Rotifers

Cryptic species are increasingly being recognized in many organisms. In Brachionus rotifers, many morphologically similar yet genetically distinct species/biotypes have been described. A number of Brachionus cryptic species have been recognized among hatchery strains. In this study, we present a simple, one-step genetic method to detect the presence of those Brachionus sp. rotifers that have been found in hatcheries. With the proposed technique, each of the B. plicatilis sensu stricto, B. ibericus, Brachionus sp. Nevada, Brachionus sp. Austria, Brachionus sp. Manjavacas, and Brachionus sp. Cayman species and/or biotypes can be identified with polymerase chain reaction (PCR) analysis. Based on 233 cytochrome c oxidase subunit I sequences, we reviewed all the available cryptic Brachionus sp. genetic polymorphisms, and we designed six nested primers. With these primers, a specific amplicon of distinct size is produced for every one of the involved species/biotypes. Two highly sensitive protocols were developed for using the primers. Many of the primers can be combined in the same PCR. The proposed method has been found to be an effective and practical tool to investigate the presence of the above six cryptic species/biotypes in both individual and communal (bulk) rotifer deoxyribonucleic acid extractions from hatcheries. With this technique, hatchery managers could easily determine their rotifer composition at the level of cryptic species and monitor their cultures more efficiently. Kalliopi Vasileiadou and Spiros Papakostas contributed equally to this work.     

Degradation of Ficus elastica rubber latex by Aspergillus terreus,Aspergillus flavus and Myceliophthora thermophila

Since isolates recovered on medium containing-Ficus elastica latex showed good growth on the respective natural rubber than those recovered on Euphorbia pulcherrima or Ficus nitida, 16 of these isolates were selected for further growth experiments on natural rubber to determine their protein content as well as rubber viscosity. Of these, the mesophilic strains Aspergillus terreus AUMC 4682, Aspergillus flavus AUMC 4795 and the thermophilic strain Myceliophthora thermophila AUMC 4653 showed low rubber viscosity and high mycelia protein content indicating high biodegradation ability of rubber. The strains were subjected for further analysis. They showed high ability to degrade poly (cis-1, 4-isoprene) rubber fig. The ability was also determined by measuring the increase in protein content of each fungus (mg g⁻¹ dry wt), reduction in molecular weight (g mol⁻¹) and inherent viscosity (dl g⁻¹). Moreover the degradation was characterized by determining aldehyde or keto group by Schiff reagent and observing the growth using scanning electron microscopy (SEM).     

多种食源性致病菌检测的多重PCR 方法的研究

目的:利用多重PCR技术,建立可以同时检测多种食源性致病菌的多重PCR方法。方法:分别选择沙门氏菌invA基因,志贺氏菌的ipaH基因,单核细胞增生李斯特氏菌的hlyA基因,大肠杆菌O157:H7的eaeA基因,副溶血弧菌的toxR基因,设计多重PCR引物,建立多重PCR检测体系,并对该体系进行特异性和灵敏度实验。结果:通过对19株菌株进行实验,所有的目标菌株均为阳性,而其余菌株为阴性。对多重PCR体系的灵敏度进行考察,沙门菌的灵敏度为5000 CFU/mL;志贺氏菌的灵敏度为5500CFU/mL;单核细胞增生李斯特氏菌的灵敏度为5200 CFU/mL;O157:H7的灵敏度为5000CFU/mL;副溶血弧菌的灵敏度为6300CFU/mL。结论:建立的多重PCR体系能实现多种致病菌同时检测。     

A Multiplex PCR for the Simultaneous Detection and Genotyping of the Echinococcus granulosus Complex

Echinococcus granulosus is characterized by high intra-specific variability (genotypes G1–G10) and according to the new molecular phylogeny of the genus Echinococcus, the E. granulosus complex has been divided into E. granulosus sensu stricto (G1–G3), E. equinus (G4), E. ortleppi (G5), and E. canadensis (G6–G10). The molecular characterization of E. granulosus isolates is fundamental to understand the spatio-temporal epidemiology of this complex in many endemic areas with the simultaneous occurrence of different Echinococcus species and genotypes. To simplify the genotyping of the E. granulosus complex we developed a single-tube multiplex PCR (mPCR) allowing three levels of discrimination: (i) Echinococcus genus, (ii) E. granulosus complex in common, and (iii) the specific genotype within the E. granulosus complex. The methodology was established with known DNA samples of the different strains/genotypes, confirmed on 42 already genotyped samples (Spain: 22 and Bulgaria: 20) and then successfully applied on 153 unknown samples (Tunisia: 114, Algeria: 26 and Argentina: 13). The sensitivity threshold of the mPCR was found to be 5 ng Echinoccoccus DNA in a mixture of up to 1 µg of foreign DNA and the specificity was 100% when template DNA from closely related members of the genus Taenia was used. Additionally to DNA samples, the mPCR can be carried out directly on boiled hydatid fluid or on alkaline-lysed frozen or fixed protoscoleces, thus avoiding classical DNA extractions. However, when using Echinococcus eggs obtained from fecal samples of infected dogs, the sensitivity of the mPCR was low (<40%). Thus, except for copro analysis, the mPCR described here has a high potential for a worldwide application in large-scale molecular epidemiological studies on the Echinococcus genus.     

Restriction enzyme analysis of mitochondrial DNA of the Aspergillus flavus group: A. flavus, A. parasiticus, and A. nomius.

Mitochondrial DNA restriction fragment length polymorphisms were identified that clearly distinguish Aspergillus flavus, A. parasiticus, and A. nomius. Mitochondrial DNAs of A. flavus and A. parasiticus were found to be circular, and their size was estimated size to be 32 kilobases. A restriction map was constructed for the mitochondrial genome of an A. parasiticus isolate by using four restriction endonucleases. Four genes tested were found to have the same order as in the mitochondrial genome of A. nidulans. The mitochondrial genome of A. nomius was estimated to be 33 kilobases.     

A PCR test for the identification and discrimination of Legionella longbeachae serogroups 1 and 2.

A PCR test has been developed for the specific identification of Legionella longbeachae. The test targeted sequence unique to both L. longbeachae serogroups 1 and 2 within the mip gene and permitted both species and serogroup identification. The test was trialed on a range of closely related species and 20 clinical isolates originating from Australia, the USA and Israel. Results were consistent with previous identification analyses. From 20 water samples known to contain Legionella spp. one sample yielded isolates which consistently tested positive by L. longbeachae serogroup 1 PCR. DNA sequencing of the PCR product, 5S rRNA gene sequence and hybridisation analysis with a specific oligonucleotide probe definitively identified one isolate as L. longbeachae serogroup 1. PCR testing was demonstrated as a superior method of identification to traditional seroagglutination reactions, which were ambiguous and could explain the previous failure to identify the presence of this microorganism in water.     

Multiplex PCR for joint amplification of carbapenemase genes of molecular classes A,B, and D

Here we present a method for joint amplification of genes of carbapenemases of molecular classes A, B, and D for hybridization analysis on DNA microarrays. Using new-generation DNA polymerase KAPA2G Fast (KAPA Biosystems, USA) together with optimization of the conditions for the multiplex PCR with 20 primer pairs allowed us to carry out joint amplification of full-length genes of seven different types of carbapenemases (KPC, VIM, IMP, SPM, SIM, GIM, and OXA) with simultaneous inclusion of biotin as a label. Yield of the labeled PCR product sufficient for further analysis by microarray hybridization was achieved 40 min after the start of the reaction. This reduced the total duration of DNA identification techniques, including sample preparation stage, to 4 h. The method for gene identification by DNA microarrays with the improved stage of amplification of specific carbapenemase genes was tested with clinical strains of gram-negative bacteria Pseudomonas aeruginosa, Acinetobacter baumannii, and Enterobacteriaceae spp. with different sensitivity towards carbapenems according to phenotyping tests. All clinical strains of A. baumannii resistant to carbapenems were found to have genes of OXA-type carbapenemases (subtypes OXA-51, OXA-23, OXA-40, and OXA-58), and clinical strains of P. aeruginosa resistant to carbapenems were found to possess the gene of VIM-type metallo-beta-lactamase (subtype VIM-2). When testing clinical strains sensitive to carbapenems, carbapenemase genes were not detected. Thus, the method of identifying carbapenemase genes on DNA microarrays is characterized by high accuracy and can be used in clinical microbiology laboratories for express diagnostics of resistance to carbapenems.     

Restriction enzyme analysis of mitochondrial DNA of the Aspergillus flavus group: A. flavus, A. parasiticus, and A. nomius

Mitochondrial DNA restriction fragment length polymorphisms were identified that clearly distinguish Aspergillus flavus, A. parasiticus, and A. nomius. Mitochondrial DNAs of A. flavus and A. parasiticus were found to be circular, and their size was estimated size to be 32 kilobases. A restriction map was constructed for the mitochondrial genome of an A. parasiticus isolate by using four restriction endonucleases. Four genes tested were found to have the same order as in the mitochondrial genome of A. nidulans. The mitochondrial genome of A. nomius was estimated to be 33 kilobases.     

产赭曲霉毒素A黑曲霉的PCR法检测

【目的】快速检测产赭曲霉毒素A(OTA)的黑曲霉。【方法】根据黑曲霉(Aspergillus niger)CBS513.88中An15g07920基因编码聚酮合酶的酰基转移酶(AT)域设计引物,建立针对产OTA黑曲霉的聚合酶链式反应(PCR)检测方法。【结果】对72株曲霉属菌株(黑曲霉、炭黑曲霉、赭曲霉、佩特曲霉、寄生曲霉和塔宾曲霉)进行检测,发现产OTA的黑曲霉能够扩增出特异性条带,而产OTA的其它菌株不能扩增出条带;检测出3株假阳性的产OTA黑曲霉,实时定量PCR分析此3株菌中An15g07920的同源基因表达情况,发现在产毒条件下可正常表达,排除了因基因无法表达导致假阳性的可能。本方法的检测灵敏度为25 pg的DNA含量,在污染所试农产品孢子浓度大于4.0×10~4–4.0×10~5个/g时可有效检测出产毒菌株。【结论】本方法虽会产生4%的假阳性结果,但是仍可作为产毒黑曲霉有效的快速检测方法,并在农产品污染产毒黑曲霉时进行有效预警。     

Multiplex real-time PCR (TaqMan) assay for the simultaneous detection and discrimination of potato powdery and common scab diseases and pathogens

Aims: To develop a multiplex real‐time PCR assay using TaqMan probes for the simultaneous detection and discrimination of potato powdery scab and common scab, two potato tuber diseases with similar symptoms, and the causal pathogens Spongospora subterranea and plant pathogenic Streptomyces spp. Methods and Results: Real‐time PCR primers and a probe for S. subterranea were designed based on the DNA sequence of the ribosomal RNA ITS2 region. Primers and a probe for pathogenic Streptomyces were designed based on the DNA sequence of the txtAB genes. The two sets of primer pairs and probes were used in a single real‐time PCR assay. The multiplex real‐time PCR assay was confirmed to be specific for S. subterranea and pathogenic Streptomyces. The assay detected DNA quantities of 100 fg for each of the two pathogens and linear responses and high correlation coefficients between the amount of DNA and C_t values for each pathogen were achieved. The presence of two sets of primer pairs and probes and of plant extracts did not alter the sensitivity and efficiency of multiplex PCR amplification. Using the PCR assay, we could discriminate between powdery scab and common scab tubers with similar symptoms. Common scab and powdery scab were detected in some tubers with no visible symptoms. Mixed infections of common scab and powdery scab on single tubers were also revealed. Conclusions: This multiplex real‐time PCR assay is a rapid, cost efficient, specific and sensitive tool for the simultaneous detection and discrimination of the two pathogens on infected potato tubers when visual symptoms are inconclusive or not present. Significance and Impact of the Study: Accurate and quick identification and discrimination of the cause of scab diseases on potatoes will provide critical information to potato growers and researchers for disease management. This is important because management strategies for common and powdery scab diseases are very different.