A Review Molecular Typing Methods for Aspergillus flavusIsolates

Ines Hadrich • Fattouma Makni • Sourour Neji •

Fatma Cheikhrouhou • Hayet Sellami •

Ali Ayadi

Received: 25 February 2010 / Accepted: 16 February 2011 / Published online: 3 March 2011

� Springer Science+Business Media B.V. 2011

Abstract Aspergillus flavus is the second most

important Aspergillus species causing human infec-

tions. The importance of this fungus increases in

regions with a dry and hot climate. Small phylogenetic

studies in Aspergillus flavus indicate that the morpho-

logical species contains several genetically isolated

species. Different genotyping methods have been

developed and employed in order to better understand

the genetic and epidemiological relationships between

environmental and clinical isolates. Understanding

pathogen distribution and relatedness is essential for

determining the epidemiology of nosocomial infec-

tions and aiding in the design of rational pathogen

control methods. Typing techniques can also give us a

deeper understanding of the colonization pattern in

patients. Most of these studies focused on Aspergillus

fumigatus because it is medically the most isolated

species. To date, there has not been any publication

exclusively reviewing the molecular typing techniques

for Aspergillus flavus in the literature. This article

reviews all these different available methods for this

organism.

Keywords Molecular typing � RFLP �Microsatellites

Introduction

Human infections due to members of the genus

Aspergillus are recognized with increasing frequency

in immunocompromised patients. The majority

(approximately 80%) of invasive Aspergillus infec-

tions is caused by Aspergillus fumigatus. The second

most frequent pathogenic species is Aspergillus

flavus and to a lesser extent, Aspergillus niger and

Aspergillus terreus [1].

Aspergillus flavus is an ubiquitous mold. This

name is now used to describe a species as well as a

group of closely related species. Climate and geo-

graphical factors are important determinants of the

local prevalence of A. flavus infections. In countries

with semi-arid and arid dry weather conditions like

Saudi Arabia Sudan and Tunisia (Sfax), A. flavus is

the main etiological agent of invasive aspergillosis

[2–5]. It is also able to cause a number of other

diseases in immunocompetent patients such as

chronic indolent invasive sinonasal infection, kerati-

tis, otitis, and onychomycosis [6, 7]. Incrimination of

I. Hadrich (&) � F. Makni � S. Neji � F. Cheikhrouhou �H. Sellami � A. Ayadi (&)

Faculte de medecine, Laboratoire de biologie moleculaire

parasitaire et fongique, 3029 Sfax, Tunisie

e-mail: ineshadrich@yahoo.fr

A. Ayadi

Fungal and parasitic molecular biology laboratory,

School of Medicine Sfax, Sfax, Tunisia

e-mail: ali.ayadi@rns.tn

123

Mycopathologia (2011) 172:83–93

DOI 10.1007/s11046-011-9406-x

different environmental sources of Aspergillus infec-

tion has always been discussed [8–10].

The identification of A. flavus is not straightfor-

ward due to similarities with closely related species

(e.g., A. parasiticus and A. nomius). Aspergillus

subgenus Circumdati section Flavi, also referred to as

the Aspergillus flavus group, has attracted worldwide

attention for its industrial use and toxigenic potential.

Section Flavi is divided in two groups of species. One

includes the aflatoxigenic species A. flavus, A. para-

siticus, A. parvisclerotigenus, A. minisclerotigenes,

A. arachidicola, and A. nomius, which cause serious

problems worldwide in agricultural commodities, and

the other includes the non-aflatoxigenic species

A. oryzae, A. sojae, and A. tamarii, traditionally used

for the production of fermented foods [11]. Recently,

multiple molecular typing methods have been devel-

oped to trace the spread of particular subspecific

strains. Various DNA fingerprinting systems have

been described, for example, multilocus enzyme

electrophoresis [12], restriction fragment length

polymorphisms of total DNA [13–15], hybridization

of endonuclease digested DNA with DNA probes

[8, 16], interrepeat polymerase chain reaction [17],

and random amplification of polymorphic DNA

[18, 19]. Polymorphic microsatellite marker analyses

have been developed to delineate strains of Asper-

gillus species [20, 21]. In this review, we will discuss

the molecular tools for genotyping analyses of

A. flavus isolates to understand the epidemiology of

this opportunistic pathogenic fungus.

Random Amplification of Polymorphic DNA

(RAPD)

Using random primers of approximately 10 bases,

amplicons throughout the genome are targeted and

amplified. Amplified products are subsequently sep-

arated on an agarose gel and stained with ethidium

bromide. The genetic variation analysis based on

RAPD allows proper genetic diversity due to its

capacity to generate random markers from the entire

genome.

Diaz-Guerra TM et al. [22] have used RAPD with

three primers (R-108, R-151 and AP12 h) for typing

11 A. flavus isolates and they have found a satisfac-

tory discriminatory power. In one case, a genetic

similarity was noted between strains isolated from

patients and strains from the grilles of a dual-

reservoir cooler–heater used in the operating room

confirming the nosocomial origin of contamination

(Table 1).

Myoken Y et al. [23] have used RAPD with three

different PCR primers for typing 6 A. flavus isolates

obtained from leukemic patients with invasive

Aspergillus stomatitis. The molecular analysis

revealed that three isolates of A. flavus obtained from

three patients have the same type (Table 1).

A case of A. flavus nosocomial infection after

sternal surgery has been reported. RAPD with two

different primers (ERIC- 1, BG-2) to type A. flavus

strains from patients and from the hospital environ-

ment was used. All these strains showed the same

genotype, proving the clonal single-source of the

environmental contamination and the intraoperative

acquisition of A. flavus in the sternal surgical-site

infections outbreak (Table 1) [24].

The use of RAPD has allowed to differentiate

species of the A. flavus complex. This approves the

role of taxonomic studies. Batista PP et al. have used

six random primers giving a RAPD profile with very

different products for each A. flavus strain providing

evidence of its high genetic diversity. The primer

OPW-04 revealed low intraspecific variability and

high interspecific variability [21]. Among some strains

previously classified as A. flavus, one was reclassified

as A. oryzae, one as A. parasiticus, and two as

A. tamarii. But also one strain previously identified as

A. parasiticus was reclassified as A. flavus [21]. This

was similar to the report of Yuan et al. [25] who used

RAPD to differentiate two morphologically similar

species A parasiticus and A. sojae and who has

reclassified one strain as A. flavus.

The major problem in RAPD typing is a lack of

reproducibility of patterns (variation of number, size,

and intensity of bands) [15, 26] and the interlabora-

tory reproducibility.

Restriction Fragment Length Polymorphism

(RFLP)

The basic technique for detecting RFLPs involves

fragmenting a sample of DNA by a restriction

enzyme that can recognize and cut DNA wherever

a specific short sequence occurs in a process known

as a restriction digest. The resulting DNA fragments

84 Mycopathologia (2011) 172:83–93

123

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Mycopathologia (2011) 172:83–93 85

123

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86 Mycopathologia (2011) 172:83–93

123

are then separated by length by agarose gel electro-

phoresis and transferred to a membrane via the

Southern blot procedure. Hybridization of the mem-

brane to a labeled DNA probe determines the length

of the fragments that are complementary to the probe.

A RFLP occurs when the length of a detected

fragment varies between individuals. Each profile is

considered an allele and can be used in genetic

analysis.

RFLP has been used to distinguish between two

related species A. flavus and A. oryzae and to analyze

their phylogenetic relationships [27].

The characterization of mitochondrial DNA is a

useful adjunct to the standard morphological and

physiological characteristics to determine the taxo-

nomic status of isolates in Aspergillus flavus com-

plex. Moody et al. [28] identified mitochondrial

DNA RFLP and used them to propose the occurrence

of species within the A. flavus group: A. flavus,

A. parasiticus, and A. nomius (Table 1).

Mc Alphin et al. [29] constructed and character-

ized a DNA probe named pAF28 for distinguishing

strains of A. flavus (Table 1). This probe has been

used to type clinical and environmental strains

isolated from a neonatal intensive care unit [30].

James M et al. [30] demonstrated that DNA finger-

printing with the pAF28 repetitive probe is a highly

reproducible and discriminatory method for tracing

the transmission of human cases of A. flavus infection

expanding its utility beyond the agricultural purpose

for which it was developed (Table 1). These results

are consistent with the successful findings obtained

with the AfutI repetitive DNA sequence probe in

investigations of the nosocomial transmission of

A. fumigatus infection [31, 32].

Buffington J et al. [33] used RFLP analysis of Sma

I-digested DNA to discriminate A. flavus isolates

from outbreak- and non-outbreak-related situations

(Table 1). They combined the products from RAPD

and RFLP analysis of a tester strain of A. flavus to

produce a DNA probe for Southern blot analysis. So,

a high degree of discrimination among strain types

was achieved.

Although, complex RFLP patterns can be difficult

to interpret, RFLP remains a reproducible and high

discriminatory system.

Geiser et al. [34] have conducted PCR–RFLP

analysis of 11 nuclear genes and concluded that a

collection of A. flavus, A. parasiticus, and A. oryzae

strains could be divided into two clades (groups I and

II) with group I comprising isolates of A. oryzae

(Table 1).

The PCR–RFLP with Hae-III enzyme has been

used to analyze the genotypic differences existing

among A. flavus strains causing ocular infections.

This method showed the same profile of bands for all

isolates [35]. This approach is generally suitable for

discriminating among different species, but has

insufficient discriminatory power to distinguish

among unrelated isolates within a species.

Amplified Fragment Length Polymorphism

(AFLP)

This method combines the principle of the RFLP

analysis with highly specific PCR amplification.

Genomic DNA is usually cut with two restriction

enzymes, one with an average cutting frequency and

a second one with a higher cutting frequency, and

double-stranded adapters are ligated to the ends of the

DNA fragments to generate template DNA for

amplification. The sequence of the adapters and the

adjacent restriction site serve as primer binding sites

for subsequent amplification of the restriction frag-

ments. Selective nucleotides are included at the 30

ends of the PCR primers, which therefore can only

prime DNA synthesis from a subset of the restriction

sites. Only restriction fragments in which the nucle-

otides flanking the restriction site match the selective

nucleotides will be amplified. Variations between

different isolates originate by differences in the

number and the location of restriction enzyme

recognition sites in the genome.

AFLPs were used to explore genetic diversity

among twenty-four isolates of Aspergillus flavus

complex using twelve selective primer combinations.

This study demonstrated that AFLP can be an

excellent typing method to differentiate between

isolates of Aspergillus flavus complex [36]. It should

be generally useful in distinguishing between closely

related species or strains. It revealed a clear separa-

tion of A. flavus from A. parasiticus, and no

genotypic differences between aflatoxigenic and

non-aflatoxigenic producers could be detected

(Table 1) [36].

Mycopathologia (2011) 172:83–93 87

123

PCR–Single-Strand Conformation Polymorphism

(PCR–SSCP)

DNA material is denatured to single-stranded DNA

which is subjected to polyacrylamide gel electropho-

resis. The mobility of single-stranded DNA in the gel

is dependent on its secondary structure as determined

by the nucleotide sequence.

Kumeda Y and Asao T [37] optimized a PCR with

universal primers (ITS1 and ITS4) and a SSCP

conditions. PCR–SSCP analysis offers a reliable

method to distinguish between species of Aspergillus

flavus complex and is more simple and rapid than

some other methods based on DNA hybridization.

Non-radiolabeled PCR–SSCP analysis as well as

PCR–RFLP analysis is practical to perform without

any special apparatus or skill and should assist in

fungal morphological identification (Table 1) [37].

PCR Sequencing

PCR was a major breakthrough for molecular marker

research; any genomic region could be amplified and

analyzed in many individuals without the require-

ment for cloning and isolating large amounts of

ultrapure genomic DNA. PCR sequencing involves

the determination of the nucleotide sequence within a

DNA fragment amplified by the PCR, using primers

specific for a particular genomic site. Many genes

were studied for typing A. flavus isolates such as

cytochrome b gene, the beta-tubulin (BenA), cal-

modulin (CF), RNA polymerase II (RPB2), and aflR

genes.

DNA Sequencing of Cytochrome b Gene

The mitochondrial DNA cytochrome b sequence

analyses provided a reliable tool for classifying some

of the closely related species in Aspergillus flavus

complex [38].

Wang et al. described the use of partial sequences

of the mitochondrial cytochrome b gene (402 bp) to

differentiate 77 isolates in the Aspergillus flavus

complex into seven DNA types (D-1 to D-7): A. sojae

was defined as D-1, A. parasiticus as D-2, A. flavus

and A. oryzae were grouped together as D-4, A.

tamarii was defined as D-5, and A. nomius as D-7.

Furthermore, D-3 was found to be closely related to

A. parasiticus (D-2), also including one strain that

had been deposited as A. flavus var flavus. DNA

type D-6 included one strain that was identified

as A. flavus and was closely related to A. tamarii

(Table 1) [38].

PCR Sequencing of Internal Transcribed Spacer

(ITS) Region

The internal transcribed spacer (ITS) region is

located between the 18S and 28S rRNA genes and

offer distinct advantages over other molecular targets

including high sensitivity of detection due to the

existence of multiple copies per genome. The

sequence variation of ITS region has led to their

use in phylogenetic studies of different fungal

organisms [39]. Single-strand conformation polymor-

phism of ITS regions has also been used as a genetic

approach to differentiate species in the A. flavus

complex [37, 40].

The polymorphism of ITS regions confirmed that

the A. flavus complex seems to comprise distinct

clades [41]. The three main clades (P. alliaceus,

A. flavus, and A. tamarii) could also be distinguished

based on colony color and their ubiquinone system.

Based on ITS sequences, A. robustus, A. caelatus,

A. lanosus, A. albertensis, A. coremiiformis, A. flavo-

furcatis, A. toxicarius, A. terricola var indica,

and A. terricola were all located in Aspergillus

flavus complex. In addition, A. pseudotamarii and

A. bombycis were found to be closely related to

A. caelatus and A. nomius, respectively (Table 1) [41].

Rigo et al. [41] suggested that A. zonatus and A.

clavatoflavus should be excluded from Aspergillus

flavus complex (Table 1). This suggestion was pre-

viously made by Kozakiewicz and based on scanning

electron microscopic studies [42]. Recently, Frisvad

et al. found that A. toxicarius resembles A. parasit-

icus but differs in at least three nucleotide differences

in the ITS regions. Usually, the presence of three or

more sequence differences in ITS regions is an

indication of a different species [43]. A. zhaoqingen-

sis was considered the same as A. nomius in this study

[43].

Bagyalakshmi et al. used a PCR sequencing of ITS

regions to analyze the genetic similarity among seven

ocular isolates of A. flavus. The alignment of

contiguous fungal sequences demonstrated that both

single-nucleotide differences and short lengths of

88 Mycopathologia (2011) 172:83–93

123

sequence diversity due to insertion or deletion existed

in the ITS regions among the pathogenic A. flavus

strains (Table 1) [35].

Combined Gene Sequencing

Pildain MB et al. analyzed six species from Asper-

gillus flavus complex isolated from Argentinean

peanuts: A. caelatus, A. flavus, A. tamari, A. parasit-

icus, A. parvisclerotigenus, and A. parasiticus. For

the molecular analysis, two regions of the genome

were analyzed, namely parts of the calmodulin and

beta-tubulin genes [44]. Most of the sequenced

Argentinean isolates fell into one of two main clades,

represented by A. flavus and A. parasiticus. Six

Argentinean isolates from peanuts seeds formed

a well-defined clade related to A. flavus and

A. parvisclerotigenus on trees based on beta-tubulin

and calmodulin sequence data (Table 1). Further-

more, these Argentinean isolates belong to the same

vegetative compatibility groups (VCG) as described

by Pildain et al. [44]. This clade also includes four

isolates assigned to A. flavus group II by Geiser et al.

[34, 45] and three isolates producing small sclerotia

collected from soils from Australia. The calmodu-

lin and beta-tubulin sequence data indicate that

A. oryzae, A.thomii, A. kambarensis, A. asciculatus,

and A. subolivaceus are very closely related or

synonymous with A. flavus.

A total of 460 Aspergillus isolates were analyzed

by DNA sequencing of 4 loci: the beta-tubulin

(BenA), calmodulin (CF), ITS, and lsu rDNA and

RNA polymerase II (RPB2). Twelve lineages were

observed in Aspergillus flavus complex. The A. flavus

lineage included the ex-type cultures of A. oryzae,

A. flavus var columnaris, and A. thomii. One species

often held to be synonymous with A. flavus,

A. subolivaceus, was supported as a separate lineage.

The A. parasiticus lineage included the type strain of

A. terricola var. americana and an isolate of A. sojae.

The A. tamarii lineage included the type isolates of

A. flavofurcatis and A. terricola (Table 1) [46].

PCR Sequencing of the aflR Gene

Lee CZ et al. examined 34 strains of Aspergillus

flavus complex. The sequenced aflR genes from the

23 positive strains had greater than 96.6% similarity.

It was particularly conserved in the zinc-finger

DNA-binding domain. The aflR gene of A. sojae

has two obvious characteristics: an extra CTCATG

sequence fragment and a C to T transition that causes

premature termination of AFLR protein synthesis.

Differences between A. parasiticus/A. sojae and

A. flavus/A. oryzae aflR genes were also identified [47].

A detailed comparison of the aflR gene sequences

demonstrated that certain base variations can be used

to differentiate A. parasiticus/A. sojae from A. flavus/

A. oryzae. These differences comprise ten transitions,

three transversions, and one deletion (Table 1) [47].

Microsatellites

Microsatellites or short tandem repeats (STRs) are

short repetitive sequences that are abundantly present

in the genomes of most of the higher organisms and

to a lesser extent in several prokaryotic genomes [48].

Different isolates can be distinguished from each

other based on differences in repeat numbers.

Microsatellite markers are easily amplified by PCR

using primers based on their flanking sequences. If

one of the primers is fluorescently labeled they can be

sized very accurately using high-resolution electro-

phoresis platforms. The number of repeats in each

marker can be deduced from the sizes of the

fragments. All repeat numbers of the analyzed

markers form a genotype for each individual isolate.

These genotypes are easily compared to each other.

Tran-Dinh N and D Carter tested seven polymor-

phic microsatellite loci for 20 A. flavus and 15

A. parasiticus strains. Searches for microsatellite

motifs were performed on genomic sequences of

Aspergillus flavus complex. These seven markers

yielded two to eleven alleles for A. flavus and one to

nine alleles for A. parasiticus (Table 1) [49].

Guarro et al. used random amplified microsatel-

lites (RAMS) to type isolates of A. fumigatus and A.

flavus obtained from a supposed outbreak RAMS

combines microsatellite and RAPD analysis. A

discriminatory power of 0.9489 was obtained with

the combination of two different primers. A full

understanding of population of A. flavus and the

discriminatory power of these and other typing

systems awaits a full population genetics study

(Table 1) [20].

Batista et al. have used (GTG)5 and (GACA)4

primers that produced differential amplification

Mycopathologia (2011) 172:83–93 89

123

products varying both in size and band intensity.

(GACA)4 revealed higher genetic variability. The

number and size of (GTG)5 bands were in a charac-

teristic pattern in several strains of A. flavus. A high

interspecific variation was observed. Furthermore, four

strains initially classified as A. flavus displayed differ-

ential banding patterns that prompted to review their

taxonomic identification (Table 1) [21]. Microsatel-

lite-based approaches appear to be species specific and

it is able to recognize instantaneously mixed genotypes

by the presence of multiple peaks of different lengths of

the marker analyzed [50, 51].

Grubisha LC and Cotty PJ. investigated the distri-

bution of genetic variation in 243 samples of cotton

from three common vegetative compatibility groups

(VCGs) in Arizona and Texas. They used 24 micro-

satellite loci and the mating type locus to assess

population structure among A. flavus VCGs in sym-

patric populations. They found high levels of genetic

differentiation and no evidence of gene flow between

VCGs, including VCGs of opposite mating type. These

results suggest that these VCGs diverged before

domestication of agricultural hosts (Table 1) [52].

Hadrich et al. identified and selected suitable

microsatellite markers for A. flavus typing. 63

A. flavus isolates (48 from Sfax, Tunisia and 15 from

Marseille, France) were analyzed. The combination

of all 12 markers yielded 35 different haplotypes with

a 0.97 D value. A 5 markers combination (AFLA1,

AFLA3, AFLA7, AFM3, and AFM7) yielded 27

different alleles with a 0.952 D value. Isolates from

Tunisia and Marseille displayed distinct haplotypes,

indicating a highly significant geographical structur-

ing in A. flavus. The typing of clinical and environ-

mental A. flavus isolates in a hematology unit

provided insights into its hospital epidemiology.

From a heterogeneous genetic background, a cluster

indicative of a clonal propagation episode within the

unit could be identified. In two patients with invasive

aspergillosis, the same genotype was found in clinical

and environmental isolates, indicating hospital-

acquired colonization and infection (Table 1) [10].

Interpretation

The performance of typing techniques can be com-

pared to each other in respect of their practical

feasibility.

Specific advantages and disadvantages are evalu-

ated in terms of applicability, the ease of use, the

exchangeability, and the reproducibility within a

laboratory and between laboratories. The discrimina-

tory power of the typing methods will be also

evaluated. Table 1 gives an overview of the different

aspects for all typing techniques discussed before.

All techniques mentioned in this review can

roughly be divided into pattern-based approaches

and exact techniques. The patterns obtained with

band-based techniques can be extremely complex,

because they are composed of both strong and faint

bands, which make the interpretation of these meth-

ods difficult [15, 26]. In contrast, exact fingerprinting

techniques are much easier to interpret [10, 20].

RFLP analysis with Southern blotting may be

tedious and labor intensive [30]. RAPD analysis is

the most frequently applied method, although lack of

reproducibility is a well-known limitation of this

technique [15, 26]. Given the extensive polymor-

phism of microsatellites, they have proved to be

epidemiologically useful for typing A. flavus [10, 20].

Reproducibility refers to the ability of a technique to

yield the same result when a particular isolate is

repeatedly tested. For large scale and longitudinal

epidemiological studies, stable fingerprinting tech-

niques are required. The main source of ambiguity in

band-based approaches like RAPD, RFLP, SSDP, and

AFLP concerning reproducibility is the variable inten-

sity of bands, which is probably on account of small

variations in the various steps of the procedures that may

affect the final peak intensity [15, 26, 36, 37]. In contrast,

exact techniques, like microsatellites, are potentially

100% reproducible [10, 20]. Another important aspect

in evaluating typing techniques is the ability to exchange

data between different laboratories. Therefore, exact

techniques seem to be most suitable, because the data

generated with these techniques can be fully expressed

in a simple, digital format.

A clear advantage of microsatellite-based methods

compared to band-based approaches is its ability to

identify mixtures of isolates. Mixed genotypes in

microsatellite-based methods are recognized instan-

taneously by the presence of multiple peaks of

different lengths of the marker analyzed. In band-

based approaches, mixed genotypes are very difficult,

if not impossible, to recognize.

Another problem among the analysis of general

typing data is the subjective interpretation of a

90 Mycopathologia (2011) 172:83–93

123

genotype. The assignment of a genotype is user

dependent when using band-based approaches,

whereas the genotype resulting from exact techniques

is unambiguous.

Apart from the advantages and disadvantages of

practical feasibility and the interpretation of the

different techniques, another important factor, the

discriminatory power must be evaluated.

Buffington et al. combined the products from

RAPD analysis and RFLP analysis of a tester strain of

A. flavus to produce a DNA probe for Southern blot

analysis [33]. Although a high degree of discrimina-

tion among strain types was achieved, the probe and

target sequences remain undisclosed.

A discriminatory power of 0.9489 was obtained

with the combination of two different primers [20].

Grubisha LC and Cotty PJ used 24 microsatellite

loci. They found high levels of genetic differentiation

(ID = 0.86–1.0) [52]. The combination of all 12

markers yielded 35 different haplotypes with a 0.97 D

value [10].

Conclusion

The molecular typing of clinical and environmental

isolates of A. flavus will provide insights into

important epidemiological and public health issues

including tracing sources and routes of transmission,

identification of pathogenic or drug-resistant strains,

and the genetic relatedness of isolates. Assessing the

relatedness of strains isolated from patients and their

environment is instrumental in understanding the

epidemiology of this mold and documenting the

source of preventable health care-associated life-

threatening human infections. RAPD analysis may

not be appropriate as a tool for epidemiologic

tracking of isolates or for surveying the genetic

variation in natural populations because of many

artefactual variations [15]. Restriction endonuclease

analysis of total cellular DNA has not proved to be a

suitable method for the discrimination of strains of A.

flavus. RFLP analysis of A. flavus nuclear DNA

probed with recombinant DNA clones from

A. nidulans and Neurospora crassa supported the

results obtained with mitochondrial DNA but

revealed limited geographic correlations among

A. flavus strains. In recent years, it has been a

growing tendency to use exact typing methods for

discrimination between isolates. These methods are

advantageous over conventional methods in the

production of reproducible, portable, and exchange-

able typing data. The exact typing methods to date

are multilocus sequence typing (MLST) and micro-

satellite-based typing. A MLST scheme has been

developed for A. fumigatus typing (http://pubmlstorg/

afumigatus/) [53], but not for A. flavus. Most of the

techniques are developed to analyze A. fumigatus

isolates which is the most common species. The

sequencing of total genome of A. flavus may provide

more exact high-resolution fingerprinting techniques

in the future.

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