Aspergillus tubingensis and Aspergillus niger as the ... · were isolated from patients withsuspected fungal infections referred to diagnostic laboratories in Tehran, Iran, and additional

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MYCMED-592; No. of Pages 8

ORIGINAL ARTICLE/ARTICLE ORIGINAL

Aspergillus tubingensis and Aspergillus nigeras the dominant black Aspergillus, use ofsimple PCR-RFLP for preliminarydifferentiationAspergillus tubingensis et Aspergillus niger comme Aspergillus noirsdominants, l’utilisation d’une simple PCR-RFLP pour ladifferenciation preliminaire

H. Mirhendi a,*, F. Zarei b, M. Motamedi b, S. Nouripour-Sisakht c

aDepartment of Medical Parasitology and Mycology, School of Medicine, Isfahan University of MedicalSciences, Isfahan, IranbDepartment of Medical Parasitology and Mycology, School of Public Health, National Institute of HealthResearch, Tehran University of Medical Sciences, Tehran, IrancCellular and Molecular Research Center, Yasuj University of Medical Sciences, Yasuj, Iran

Received 3 July 2015; received in revised form 1st December 2015; accepted 7 December 2015

KEYWORDSBlack Aspergillus;Aspergillus niger;Aspergillus tubingensis;b-tubulin;PCR-RFLP;Iran

Summary This work aimed to identify the species distribution of common clinical andenvironmental isolates of black Aspergilli based on simple restriction fragment length polymor-phism (RFLP) analysis of the b-tubulin gene. A total of 149 clinical and environmental strains ofblack Aspergilli were collected and subjected to preliminary morphological examination. Totalgenomic DNAs were extracted, and PCR was performed to amplify part of the b-tubulin gene. Atfirst, 52 randomly selected samples were species-delineated by sequence analysis. In order todistinguish the most common species, PCR amplicons of 117 black Aspergillus strains wereidentified by simple PCR-RFLP analysis using the enzyme TasI. Among 52 sequenced isolates, 28were Aspergillus tubingensis, 21 Aspergillus niger, and the three remaining isolates includedAspergillus uvarum, Aspergillus awamori, and Aspergillus acidus. All 100 environmental and17 BAL samples subjected to TasI-RFLP analysis of the b-tubulin gene, fell into two groups,consisting of about 59% (n = 69) A. tubingensis and 41% (n = 48) A. niger. Therefore, the methodsuccessfully and rapidly distinguished A. tubingensis and A. niger as the most common species

Journal de Mycologie Médicale (2016) xxx, xxx—xxx

* Corresponding author.E-mail address: [email protected] (H. Mirhendi).

Available online at

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http://dx.doi.org/10.1016/j.mycmed.2015.12.0041156-5233/# 2016 Elsevier Masson SAS. All rights reserved.

Please cite this article in press as: Mirhendi H, et al. Aspergillus tubingensis and Aspergillus niger as the dominant black Aspergillus, use ofsimple PCR-RFLP for preliminary differentiation. Journal De Mycologie Médicale (2016), http://dx.doi.org/10.1016/j.myc-med.2015.12.004

http://dx.doi.org/10.1016/j.mycmed.2015.12.004



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among the clinical and environmental isolates. Although tardy, the Ehrlich test was also able todifferentiate A. tubingensis and A. niger according to the yellow color reaction specific toA. niger. A. tubingensis and A. niger are the most common black Aspergillus in both clinical andenvironmental isolates in Iran. PCR-RFLP using TasI digestion of b-tubulin DNA enables rapidscreening for these common species.# 2016 Elsevier Masson SAS. All rights reserved.

MOTS CLÉSAspergillus noir ;Aspergillus niger ;Aspergillus tubingensis ;b-tubuline ;PCR-RFLP ;Iran

Resume Ce travail avait pour but d’identifier la répartition des espèces des isolats cliniques etenvironnementaux communs d’Aspergillus noirs en se basant sur le simple polymorphisme delongueur des fragments de restriction (RFLP) du gène de la b-tubuline. Un total de 149 souchescliniques et environnementales d’Aspergillus noirs ont été prélevées et soumises à l’examenmorphologique préliminaire. L’ADN génomique total a été extrait et une PCR a été réalisée pouramplifier une partie du gène de la b-tubuline. Dans un premier temps, 52 échantillons choisis auhasard ont été identifiés en espèces par analyse de séquence. Afin de distinguer les espèces lesplus communes, des amplicons de 117 souches d’Aspergillus noirs ont été identifiés par simpleanalyse PCR-RFLP en utilisant l’enzyme TasI. Parmi 52 isolats séquencés, 28 étaient Aspergillustubingensis, 21 Aspergillus niger, et les trois isolats restants étaient : Aspergillus uvarum,Aspergillus awamori, et Aspergillus acidus. Tous les 100 échantillons de l’environnement et les17 LBA soumis à l’analyse TasI-RFLP du gène de la b-tubuline se sont retrouvés en deux groupes,composés pour 59 % (n = 69) de A. tubingensis et 41 % (n = 48) de A. niger. Ainsi la méthode rapidedistingue avec succès A. tubingensis et A. niger, espèces les plus communes chez les isolatscliniques et environnementaux. Bien que plus ancien, le test d’Ehrlich a également été enmesure de différencier A. tubingensis et A. niger selon la réaction de couleur jaune spécifique àA. niger. A. tubingensis et A. niger sont les Aspergillus noirs les plus communs dans les isolatscliniques et environnementaux en Iran. Une PCR-RFLP utilisant TasI avec digestion de l’ADN de lab-tubuline permet un dépistage rapide pour ces espèces communes.# 2016 Elsevier Masson SAS. Tous droits réservés.

2 H. Mirhendi et al.

Introduction

Aspergillus species are main members of environmentalsaprophytes and are typically included in fungal communitiesof both indoor and outdoor environments. They are normalcomponents of organic debris, but can be life-threateningopportunistic agents in debilitated or immunocompromisedpatients [3]. The genus Aspergillus includes several groups,including Aspergillus section Nigri with several species [16],some of which have been implicated in human disease [1].

The taxonomy of Aspergillus section Nigri (known as blackAspergilli) remains somewhat ill-defined. It comprises aclosely related group of organisms that are difficult to dis-tinguish based on morphological characteristics such ascolony morphology, conidial size, and ornamentation [29].Several approaches including morphological and physiologi-cal methods have been employed for studding this section[29]. Development of molecular DNA-based techniques suchas PCR-RFLP [7], RAPD-PCR [27], and nucleotide sequencing[23] for the identification of fungal strains has resulted inreclassification of black Aspergilli, and so these tools arenow being acknowledged as the gold standard [28]. About26 species have been recognized within this section, withsome of them (Aspergillus aculeatinus, Aspergillus aculea-tus, Aspergillus japonicus, Aspergillus uvarum, Aspergillusbrasiliensis, Aspergillus carbonarius, Aspergillus costari-caensis, Aspergillus ellipticus, Aspergillus foetidus, Asper-gillus heteromorphus, Aspergillus homomorphus,Aspergillus ibericus, Aspergillus lacticoffeatus, Aspergillus

Please cite this article in press as: Mirhendi H, et al. Aspergillus tubingesimple PCR-RFLP for preliminary differentiation. Journal De

med.2015.12.004

piperis, Aspergillus sclerotiicarbonarius, Aspergillus sclero-tioniger, Aspergillus tubingensis and Aspergillus vadensis)being only recently described [26].

Members of Aspergillus section Nigri are reported to bethe third most common Aspergillus species associated withinvasive disease and aspergilloma [4,8,22]. Aspergillus nigerhas also been reported as the most frequent etiological agentof otomycosis [15]; other species are rarely reported andmay be miss-identified as A. niger [34]. Since differentspecies may have dissimilar susceptibilities to antifungaldrugs, species identification informs the choice of antifungaltherapy [17,2,13]. In addition to their clinical significance,several black Aspergilli have agricultural importance, beingfood spoilage organisms [21]. Ochratoxin A, produced bysome Aspergillus species in the section Nigri, is a potentnephrotoxin and potential carcinogen, and concern has beenraised regarding the incorporation of this compound into thehuman and animal food chain [24].

We have already used the sequence analysis of b-tubulingenes for species delineation of black Aspergilli isolates [37].In the present study, the most common species of blackAspergilli, i.e. A. tubingensis and A. niger, isolated fromclinical and environmental samples, are differentiated bythe use of simple PCR-RFLP analysis.

Materials and methods

Strains. A total of 149 clinical and environmental isolates ofblack Aspergillus were used in this study. Forty-nine strains

nsis and Aspergillus niger as the dominant black Aspergillus, use ofMycologie Médicale (2016), http://dx.doi.org/10.1016/j.myc-



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PCR-RFLP to differentiate A. tubingensis and A. niger 3

were isolated from patients with suspected fungal infectionsreferred to diagnostic laboratories in Tehran, Iran, andadditional 100 strains were recovered from soil or air indifferent climatic areas of the country or from some foodproducts. Air sampling was performed in hospitals and publicplaces using the settled plate method on Dichloran Glycerol(DG-18) Agar. Strains were preliminarily identified as A. nigerbased on their macro-/microscopic colony appearance.

Total genomic DNA was isolated from each colony usingglass bead disruption [36]. Briefly, 5—10 mm of fresh colonieswas transferred to a 1.5-mL tube with 300 mg of glass beads(0.5 mm in diameter), 300 mg of lysis buffer (100 mM Tris-HCl, pH 8; 10 mM EDTA; 100 mM NaCl; 1% sodium dodecylsulfate (SDS); 2% triton X-100) and 300 mL of phenol-chloro-form (1:1). Samples were vortexed vigorously for 2 min,centrifuged for 5 min at 5000 rpm, and the supernatantwas transferred to a fresh tube in which DNA was extractedwith chloroform. An identical volume of isopropanol and a0.1-volume of 3M sodium acetate (pH 5.2) were added to thesupernatant, and after incubation at �20 8C for 30 min, themixture was centrifuged for 15 min at 12,000 rpm. The pre-cipitant was washed with cold 70% ethanol, dried in air,dissolved in 50 mL of water, and stored at �20 8C until use.

The b-tubulin gene was amplified using Bt2a and Bt2bprimers [12] as described previously [37]. A 5-mL aliquot of


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Figure 1 Microscopy of examples of black Aspergillus speciesA. tubingensis, A. acidus, and A. uvarum, respectively.Exemples microscopiques d’especes d’Aspergillus noirs etudies dans

A. acidus, et A. uvarum, respectivement.

the amplicons was electrophoresed using a 1.5% agarose gelin TBE buffer (90 mM Tris, 90 mM boric acid, 2 mM EDTA,pH 8.3) and visualized under UV irradiation after ethidiumbromide staining. Subsequently, PCR products from 52 sam-ples comprising 32 clinical and 20 environmental Aspergillusstrains were purified and sequenced followed by speciesidentifications by BLAST analysis (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

Restriction patterns of b-tubulin sequences of the blackAspergillus species were predicted for all known restrictionenzymes, using the BioEdit software version 7.2 (http://bioedit.software.informer.com/7.2). Predicted restrictionfragments were compared with each other in order to selectthose with the best discriminatory power. RFLP tests wereperformed for a total of 117 randomly selected environmen-tal and clinical isolates, including 27 random samples whichhad already been sequenced. Digestion was performed byincubating a 5-mL aliquot of each PCR product with 5 Uenzyme in a final reaction volume of 15 mL at 65 8C for2 h, and digested DNA was analyzed by electrophoresis usinga 2% agarose gel.

The Ehrlich test (detection of fungal alkaloids reacting withEhrlich reagent) was used by applying the filter paper method[11]. A 4-cm piece of Whatman filter paper wetted with Ehrlichreagent (2 g of 4-dimethylamino-benzaldehyde in 85 mL


examined in this study. Photos A to D represent A. niger,

ce travail. Les photos A a D representent A. niger, A. tubingensis,

http://blast.ncbi.nlm.nih.gov/Blast.cgi

http://blast.ncbi.nlm.nih.gov/Blast.cgi

http://bioedit.software.informer.com/7.2

http://bioedit.software.informer.com/7.2



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ethanol added to 15 mL 10 N HCl) was placed on the mycelialside of a 1 � 1 cm agar plug cut from colonies grown on CzapekYeast Extract agar (CYA) for one week [28]. A yellow ringappeared after about 10 min in isolates of A. niger, while nocolor change was observed for isolates of A. tubingensis.

Results

In this study, a total of 49 clinical and 100 environmentalstrains morphologically recognized as black Aspergillus weresubjected to molecular identification. In cultures, the strainspresented microscopic characteristics such as dark-brown toblack conidia, spherical vesicles, and hyaline or lightly pig-mented hyphae near the apex. These morphological featureswere generally shared among most strains (Fig. 1). Mostisolates, which were later identified as A. tubingensis,


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Figure 2 Examples of colonies of Aspergillus section Nigri isolatA. niger; E, F, G, and H represent A. tubingensis, and I representsExemples de colonies d’Aspergillus section Nigri isolees sur SDA apreet H representent A. tubingensis et I represente A. acidus.

A. niger, A. awamorii, and A. acidus by b-tubulin sequencing,produced biseriate phialides, while A. uvarum exhibiteduniseriate phialides. The ornamentation of the conidia wasalso characteristic for some species such as A. tubingensisand A. niger, which produced conidia with a spiny appea-rance, while A. awamori, A. acidus, and A. uvarum werecharacterized by smooth conidia. Fig. 2 shows the colonies ofsome isolated black Aspergillus on Sabouraud dextrose agar(SDA) after 5—7 days of incubation at 25 8C. The differentspecies exhibited slightly different growth characteristics.Strains identified as A. niger and A. tubingensis had sharedcolony characters in SDA and CYA that was not helpful todistinguish them from each other.

Using the universal fungal b-tubulin primer pair, a 500—550 base pair (bp) fragment was successfully amplified in alltested isolates, while no PCR-amplification was seen in nega-tive controls. Fig. 3 (A) shows agarose gel electrophoresis of


ed on SDA after 5 days of incubation. A, B, C, and D represent A. acidus.s 5 jours d’incubation. A, B, C et D representent A. niger ; E, F, G



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Figure 3 Agarose gel electrophoresis of b-tubulin PCR products. A. Before digestion with TasI: lanes 1—7 are example samples.Lanes 8 and 9 are negative controls, and lane M is a 100-bp molecular size marker. B. After digestion with TasI: lanes 1, 2, 4, and 5 areA. niger, lane 3 is A. tubingensis, and lane M is a 100-bp molecular size marker.Electrophorese en gel d’agarose des produits de la b-tubuline. A. Avant digestion avec TasI : les bandes 1 a 7 sont des exemplesd’echantillons. Bandes 8 et 9 sont des controles negatifs et la bande M est un marqueur de taille moleculaire de 100-pb. B. Apresdigestion par TasI : bandes 1, 2, 4 et 5 sont A. niger, bande 3 : A. tubingensis et bande M est un marqueur moleculaire de taille de100-pb.


PCR products from isolated black Aspergillus species. TheBLAST analysis of the sequences indicated that 28 (53.8%)and 21 (40.3%) isolates were A. tubingensis and A. niger,respectively. Accounting for about 6% of all sequenced sam-ples, three other sequences represented A. uvarum,A. awamori, and A. acidus (Table 1). The sequences weredeposited in GenBank and assigned as the accessionnumbers KT965680 to KT965724.


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Table 1 Summary of the results on species identification obtainResume des resultats de l’identification des especes des echantil

Source Identified by sequencin

Number oftested isolates

Id

Clinical samples (49)Nail (19) 19 A.

A.A.A.

BAL, sputum, palate and nose (24) 7 A.

Cerumen (4) 4 A.A.

Skin lesions (2) 2 A.Environmental samples (100)

Air (36) 10 A.A.

Spice (22) 6 A.A.

Grape (7) 2 A.A.

Soil (29) 2 A.A.

Dried fruit (4) 0 0

Grain (2) 0 0

Total number149 52 52

After analysis of nearly all commercially available res-triction enzymes, TasI was selected as one of the appropriateenzymes for differentiation between the two dominant spe-cies of black Aspergillus isolated in this study, A. niger andA. tubingensis. Fragment sizes of PCR products of all speciesidentified by sequencing in this study, before and afterdigestion with TasI, are shown in Table 2. A total of117 PCR products subjected to PCR-RFLP including all


ed for tested samples in this study.lons de cette etude.

g Identified by PCR-RFLP

entified species Number oftested isolates

Identified species

tubingensis (9) niger (8) uvarum (1) awamori (1)

0 —

tubingensis (7) 17 A. tubingensis (8)A. niger (9)

tubingensis (2) niger (2)

0 0

niger (2) 0 0


36 A. tubingensis (24)A. niger (12)





niger (1) acidus (1)




117 117



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Table 2 In silico TasI-RFLP analysis of b-tubulin for black Aspergillus species isolated in this study.In silico TasI-RFLP analyse de la b-tubuline pour les Aspergillus noirs isoles dans cette etude.

Species Example of GenBankaccession number

PCR product size in bpbefore digestion with TasI

PCR product size in bpafter digestion with TasI

A. niger JX4633191 555 78, 141, 336A. tubingensis KF434100 555 219, 336A. acidus KC4336731 558 221, 337A. uvarum JQ3179721 540 63, 118, 359A. awamori HQ2855991 557 78, 141, 338


100 environmental and 17 BAL samples fell into two groups,consisting of 59% (n = 69) A. tubingensis and 41% (n = 48)A. niger. A total of 27 isolates randomly selected from thosealready identified by b-tubulin sequencing were subjected toPCR-TasI-RFLP by which 26 of 27 samples had identicalresults. Only one isolate identified as A. acidus by sequenc-ing, was identified as A. tubingensis by TasI-RFLP analysis(Table 1). An example of agarose gel electrophoresis of PCR-RFLP products of representative isolates of Aspergillus isshown in Fig. 3 (B). As seen, the bands generated corres-ponded exactly to the predicted sizes (Table 2).

The Ehrlich test was performed on examples ofA. tubingensis and A. niger isolates since these were themost abundant species in our study. A clear difference inalkaloid production was observed between the species. Thetest yielded a yellow reaction (positive) for A. niger and nocolor (negative) for A. tubingensis (Fig. 4).

Discussion

Studies have suggested that a significant proportion of cli-nical isolates considered as A. niger are indeed other mem-bers of black Aspergilli, such as A. tubingensis,A. brasiliensis, and A. foetidus [2]. Some species havedistinct biochemical properties, such as those pertainingto nutritional growth conditions and hydrolase differences[20]. Production of secondary metabolites is often unique forspecies within Aspergillus section Nigri and could be used foridentification; however, it is not yet possible to differentiatethe species solely on metabolic properties. Meanwhile, thedevelopment of molecular diagnostic tools has facilitatedcorrect species determination of black Aspergilli [3].


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Figure 4 Ehrlich color reaction. A. Positive color reaction(yellow) in A. niger. B. Negative color reaction in A. tubingensis.Reaction coloree d’Ehrlich. A. Reaction positive (jaune) avecA. niger. B. Reaction negative avec A. tubingensis.

In the present study, the black Aspergilli isolated fromclinical and environment samples in Iran were identified usinga combination of different methods with a view to develop abetter understanding of the distribution of species profiles.Since b-tubulin is acknowledged as a valid marker for speciesdifferentiation of black Aspergilli, we carried out sequenceanalysis, targeting molecular identification of 52 isolates,among which A. tubingensis and A. niger were, by far, themost common species of black Aspergillus. The details of thissequence analysis have already been reported [37].

Therefore, we selected the enzyme for RFLP analysis,primarily with a view to discriminating between these twospecies. Our results confirm that these species display dif-ferent RFLP-based profiles (Fig. 3B). PCR products fromA. niger were cleaved into three fragments of 78, 141 and336 bp by TasI; meanwhile, there was only one restrictionsite for this enzyme in the sequence of A. tubingensis, forwhich only two fragments were produced (219 and 336 bp).Also, A. uvarum was cleaved into three fragments of 63, 118,359 bp. The enzyme digestion pattern was the same forA. tubingensis/A. acidus and A. niger/A. awamori. Giventhe fact that there was only one isolate of A. awamori andA. acidus among the 52 isolates sequenced, it appears that b-tubulin-PCR followed by TasI-RFLP generally successfullydifferentiates and identifies isolates as A. niger orA. tubingensis, being an easy and inexpensive tool for pre-liminary differentiation of black Aspergillus.

Recently, clinical isolates thought to belong to A. nigerwere re-classified by genetic tools as A. tubingensis,A. awamori, A. uvarum, and A. brasiliensis [13,19,25,31].According to our results, A. niger is no longer the dominantblack Aspergillus; instead, A. tubingensis comprises morethan half of the strains that have usually been assigned toA. niger. Likewise, Howard et al. examined 43 black Asper-gilli derived from various clinical sources by sequence ana-lysis of the internal transcribed spacer (ITS) region of theribosomal RNA gene, partial calmodulin, and b-tubulinsequences and found that 69.7% of the isolates belongedto A. niger and A. awamori, 18.6% to A. tubingensis, 7.0% toA. foetidus, and 4.7% to an undescribed species [14].

Otomycosis represents an infection, which is typicallycaused by black Aspergilli in tropical and semitropical cli-mates [10]. Vennewald et al. isolated fungal strains from themiddle ear of immunocompetent patients with chronic otitismedia, in which A. niger was observed in three out of fivecases [33]. Sequence data in this study indicated that inaddition to A. niger, A. tubingensis is also able to cause earinfections in Iran. Likewise, Szigeti et al. by using partialanalysis of the calmodulin gene sequence, suggested that inaddition to A. niger and A. tubingensis, A. awamori is also




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capable of causing otomycosis in Iran [32] and Hungary [31].In mycotic keratitis, although the primary causative agentwithin the genus is A. flavus, black Aspergilli appear to bethe most frequent pathogens in certain geographic regions[5]. From Aspergillus section Nigri, only A. niger has beenreported to date as a possible causative agent of fungalkeratitis [5]. The causative agents of two reported Indiancases of keratitis were identified as A. brasiliensis based onpartial sequence analysis of the b-tubulin gene [19], suggest-ing that this newly described species may be responsible for asignificant proportion of corneal infections caused by blackAspergilli.

Non-dermatophytic molds account for 1.5%—6% of ony-chomycosis [18] and are most frequently seen in elderly, inpatients with skin diseases, or in immunocompromisedpatients. Although representatives of the section Nigri areconsidered non-dermatophytic molds, their prevalence innail infections is low according to the total number ofculture-proven cases of onychomycosis [6]. However, theirproportion within non-dermatophytic onychomycosis can behigh [30]. Among Aspergillus species, A. niger predominatedin nail specimens according to a survey performed in NewDelhi [35]. A. niger was also found to be able to causesubungual onychomycosis in Italy [5]. According to English,non-dermatophytic mold can be considered as a pathogen ofonychomycosis only when hyphae or spores are seen onmicroscopic examination and the same strain is identifiedthrough repeated cultures [9]. In our study, the results ofsequencing of nail samples clearly indicated A. tubingensisand A. niger as predominating species. In the initial evalua-tion of a nail sample, hyphae were observed by the KOH test.Sequencing result of the grown colonies matched 100% withA. uvarum. The details of the case are reported elsewhere[38]. Also, A. acidus was isolated from a soil sample, aspecies that was identified as the cause of humaninfections [2].

Although micro-morphological structures can be helpful,in A. niger and its related taxa, it is difficult to distinguish thedescribed species. Nevertheless, A. carbonarius and theuniseriate species (A. uvarum, A. aculeatus, and A. japoni-cus) can be microscopically distinguished by vesicle andconidial size plus ornamentation [1]. Differentiation of clo-sely related species such as A. niger and A. awamori is achallenge because of the existence of very similar morpho-logical characters [5]. Fortunately, A. niger andA. tubingensis, can be easily but tardily differentiated bythe Ehrlich test, in which A. niger triggers a yellow reaction,while A. tubingensis does not.

Conclusion

A. tubingensis and A. niger are the most common blackAspergillus in both clinical and environmental isolates inIran. PCR-RFLP using TasI digestion of b-tubulin DNA enablesrapid screening of these two species. Although tardy, theEhrlich test was also able to differentiate A. tubingensis andA. niger.

Disclosure of interest

The authors declare that they have no competing interest.


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