Molecular identification and antifungal susceptibility profile of Aspergillus flavus isolates recovered from clinical specimens in Kuwait
BMC Infectious Diseases
Molecular identification and antifungal susceptibility profile of Aspergillus flavus isolates recovered from clinical specimens in Kuwait
Faten Al-Wathiqi 0 1
Suhail Ahmad 0 1
Ziauddin Khan 0 1
0 Aspergillus species is being increasingly recognized [2-4]. Aspergillus flavus is the second most important species causing localized as well as systemic infections [2,5-7]. The species is of particular significance in North Africa, India and the Middle East, where it is predominantly associated with nasal/sinus infections [8-12]. In 2008, Clinical and Laboratory Standards Institute produced standard guidelines based on broth microdilution (BMD) method for determining antifungal susceptibilities of
1 Department of Microbiology, Faculty of Medicine, Kuwait University , P. O. Box 24923, Safat 13110 , Kuwait
Background: Within the genus Aspergillus, A. flavus is the second most important species of clinical significance. It is predominantly associated with infections involving sinuses, eye and skin, mostly in geographic regions with hot and arid climate, including the Middle East. Recent reports on emergence of resistance to triazoles among Aspergillus spp. is a cause of concern for treatment of patients with invasive aspergillosis. In this study we present data on genetic characterization and antifungal susceptibility profile of clinical and environmental isolates of A. flavus. Methods: Ninety-nine Aspergillus section Flavi isolates, originating from clinical (n=92) and environmental (n=7) sources, initially identified by morphological characteristics, were analyzed by partial sequencing of -tubulin and calmodulin gene fragments and their susceptibilities to six antifungal agents was determined by Etest on RPMI1640 and Muller-Hinton agar media. Etest minimum inhibitory concentrations (MICs) of amphotericin B and voriconazole were also compared with zone of inhibition diameters obtained by disc diffusion test on RPMI agar medium. Results: The identity of all clinical and environmental isolates was confirmed as A. flavus species by combined analysis of -tubulin and calmodulin genes. The mean MIC90 (g/ml) values on RPMI medium for amphotericin B, voriconazole, posaconazole, anidulafungin, micafungin and caspofungin were 3, 0.25, 0.25, 0.002, 0.002 and 0.032, respectively. No environmental isolate exhibited MIC value of >2 g/ml for amphotericin B. For clinical isolates, the zone of inhibition diameters for amphotericin B and voriconazole ranged from 7-16 mm and 24-34 mm, respectively. Linear regression analysis between Etest MIC values and disk diffusion diameters revealed a significant inverse correlation with amphotericin B (p <0.001) and voriconazole (p<0.003). Conclusions: The -tubulin and calmodulin gene sequences confirmed that all 92 clinical isolates identified phenotypically belonged to A. flavus taxon, thus suggesting that the other species within Aspergillus section Flavi are of little clinical significance. Triazoles and echinocandins showed very good in vitro activity against the A. flavus, however, 10% clinical isolates showed MICs of >2 g/ml for amphotericin B.
Aspergillus flavus; Molecular characterization; -tubulin and calmodulin genes; Antifungal susceptibility; Etest; Triazoles; Echinocandins; Amphotericin B
Among filamentous fungal pathogens, Aspergillus spp.
account for highest rates of morbidity and mortality
among severely immunocomnpromised patients .
Although A. fumigatus is the principal etiologic agent of
invasive aspergillosis, the etiologic role of non-fumigatus
spore-forming filamentous fungi . Recently,
epidemiologic cutoff values (ECVs) for triazoles and caspofungin
for wild-type strains of Aspergillus spp. have been
developed [14-16]. However, due to interspecies/inter-strain
differences in MICs, clinical breakpoints for each
Aspergillus spp. are not yet available. Occurrence of azole
resistance among wild-type strains of A. fumigatus due to
mutations in cyp51A gene and possibility of finding similar
resistance in other Aspergillus spp [17,18] have
necessitated the need to evaluate efficacy of simple agar-based
methods for antifungal susceptibility testing. Preliminary
studies have suggested that disk diffusion test and Etest
show comparable results with BMD method for
susceptibility testing with azoles [19-22]. Here, we present
molecular characterization and MIC results of six antifungal
agents tested against 99 A. flavus isolates by Etest
and compare amphotericin B and voriconazole MICs
with zone of inhibition diameters obtained by disk
Aspergillus flavus isolates
Ninety-two clinical and 7 environmental isolates of
A. flavus were included in the study (Table 1). The
clinical isolates were recovered from a variety of
specimens over an 18-year period (19932011). They were
deposited in the culture collection of the Mycology
Reference Laboratory and maintained by periodic sub-culture.
Since the isolates were obtained during routine
mycological investigations, there was no ethical requirement
to take approval from individual patients for their
subsequent use. The study (Project No. YM 03/10)
was approved by the Ethical Committee of Faculty of
Medicine, Health Sciences Center, Kuwait University. All
isolates were identified as A. flavus by typical colony and
microscopic characteristics as described by Klich . The
isolates were also characterized and speciated by
The genomic DNA from the isolates was prepared as
described previously and used as template for PCR
amplification . The ITS region (ITS-1, 5.8 S rRNA and ITS-2) of
rDNA was amplified with Aspergillus section Flavi-specific
AFLF and AFLR primers as described in detail previously
. The species-specific identification of all isolates was
studied by partial sequencing of -tubulin and calmodulin
gene fragments. The variable region of -tubulin gene was
amplified by using BTUBF (50-TGGTAACCAAATCGG
TGCTGCTT-30) and BTUBR (50-GCACCCTCAGTGT
AGTGACCCT-30) primers while the variable region of
calmodulin gene was amplified by using Cmd5 (50-GTCT
CCGAGTACAAGGAGGC-30) and Cmd6 (50-TCGCCG
Table 1 Sources of Aspergillus flavus isolates used for
in vitro antifungal susceptibility by Etest
*Include sputum, endotracheal secretion and bronchoalveolar lavage.
ATRGAGGTCATRACGTG-30) primers and the amplicons
were sequenced as described previously . GenBank
basic local alignment search tool (BLAST) searches (http://
blast.ncbi.nlm.nih.gov/blast/Blast.cgi?) were performed for
species identification. The DNA sequences for type strains
already available in GenBank were retrieved. The gene
sequences were analyzed individually or nucleotide
sequences of both, -tubulin and calmodulin gene fragments
were included in the combined analysis. Multiple sequence
alignments were performed with ClustalX version 2.0. The
phylogenetic trees were constructed using the
neighborjoining method with pair-wise deletion of gaps option.
Aspergillus parasiticus (CBS100926) was chosen as the
outlying taxon and the robustness of branches was assessed
by bootstrap analysis with 1,000 replicates.
Antifungal susceptibility testing
Preparation of inoculum
All isolates were freshly sub-cultured on potato dextrose
agar (PDA) slants to obtain good sporulation. The
culture tubes were flooded with 1 ml of 0.9% saline and
vortexed for 15 seconds to dislodge the conidia. The
growth suspensions were transferred to another sterile
tube containing 1.5-ml saline and 0.2% Tween 80. A
conidial suspension containing approximately 0.4 104 to
5 104 cells was used as inoculum . Reference
strains of A. flavus (CBS100927) and A. parasiticus
(CBS100926) were included with each batch of
susceptibility testing to ensure quality control.
Two media, namely, RPMI 1640 supplemented with 2%
glucose (pH adjusted to 7.0 with
morpholinepropanesulfonic acid buffer) and Mueller-Hinton (MH) II agar
(Becton, Dickinson and Company, Sparks, MD, USA)
supplemented with 2% glucose and methylene blue (0.5
g/ml) (MH-GMB) were used for Etest. Etest was
performed according to the manufacturers instructions
Briefly, each 150-mm petri plate containing 60 ml of
medium was inoculated by streaking the swab over the
entire surface of the medium. Etest antifungal
susceptibility strips for amphotericin B, voriconazole,
posaconazole, caspofungin, anidulafungin and micafungin were
obtained from bioMerieux Sa, Marcy-lEtoile, France.
Before applying Etest strips, the plates were allowed to
dry for about 15 min. MIC readings were taken after 24
h of incubation at 35C. The E-test MIC was defined as
the lowest drug concentration at which the border of the
elliptical inhibition zone intercepted the scale on the
antifungal strip, Microcolonies within the inhibition
zone were ignored. Considering that newer azoles are
the treatment of choice for Aspergillus infections,
the European Committee on Antifungal Susceptibility
Testing (EUCAST): (http://www.eucast.org/antifungal_
susceptibility_testing_afst/) has recently proposed the
following clinical break-points for A. fumigatus: for
itraconazole and voriconazole, 1 mg/L (susceptible) and
>2 mg/L (resistant) and for posaconazole, <0.12 mg/L
(susceptible) and >0.25 mg/L (resistant) . For A. flavus,
the clinical breakpoints for itraconazole are the same as
for A. fumigatus, whereas for other antifungal agents
there is insufficient evidence to recommend clinical
breakpoints. The epidemiologic cutoff values proposed
on the basis of MIC (mg/L) distribution of wild-type
strains of A. flavus are as follows: itraconazole, 1 (99.6%);
posaconazole, 0.25 (95%); voriconazole, 1 (98.1%);
caspofungin, 0.5 (99%) and amphotericin B, 4 (99%),
which are generally one step higher than A. fumigatus
Disk diffusion test
For the purpose of comparing zone of inhibition results,
the disk diffusion test was performed on RPMI 1640
medium supplemented with 2% glucose instead of
Mueller-Hinton agar medium. Disk diffusion disks for
voriconazole (1-g) and amphotericin B (100-g) were
obtained commercially (Bio-Rad Laboratories,
Marnesla-Coquette, France). After applying the discs, the plates
were incubated at 35C for 24 h. Zone diameters were
measured at the point where the growth significantly
decreased (80 to 100% inhibition) and were recorded to the
nearest millimeter. Recently, Espinel-Ingroff et al.
proposed for the first time quality control and reference zone
diameter limits for A. fumigatus (ATCC MYA-3626) for
amphotericin B (10-g disk), itraconazole (10-g disk),
posaconazole (10-g disk), and voriconazole (10-g disk)
. No quality control ranges were recommended for
A. flavus and A. terreus, because some results did not
meet M23-A3 document requirements . However, for
A. flavus, the acceptable zone diameter ranges for
voriconazole and posaconazole were proposed as 2536 mm
and 2737 mm, respectively.
The Spearman correlation test was performed to
determine the correlation between Etest MICs and zone of
inhibition diameters. A P value of <0.05 was considered
as significant. Independent samples T test was used to
compare mean MIC values of A. flavus strains isolated
in two different periods of 9-year each (19932001 and
All 99 A. flavus isolates included in this study were
identified as belonging to Aspergillus section Flavi by specific
amplification of a DNA fragment of expected size (~243 bp)
in PCR with Aspergillus section Flavi-specific primers
AFLF and AFLR . The partial -tubulin DNA
sequences from 94 isolates were completely identical and
showed no difference with sequence from reference
A. flavus (CBS100927) strain while the sequences of
remaining 5 isolates differed at only 1 nucleotide position.
The calmodulin gene sequences of the 99 isolates were
more variable (total 8 patterns) but still showed maximum
identity with sequence from reference A. flavus
(CBS100927) strain than with reference strains of other
species belonging to Aspergillus section Flavi. The -tubulin
and calmodulin gene sequences were combined and
the combined data set was compared with
corresponding combined data set from reference strains of
A. flavus CBS100927, A. parvisclerotigenus CBS121.62,
A. minisclerotigenes CBS117635, A. parasiticus CBS100926,
A. sojoe CBS100928, A. celatus CBS763.97, A. tamarii
CBS104.13 and A. nomius CBS260.88 strains. The combined
sequences from A. oryzae CBS100925 and other synonyms
of A. flavus (A. fasciculatus CBS110.55, A. thomii
CBS120.51, A. kambarensis CBS542.69 and A. subolivaceus
CBS501.65) were also used . The combined -tubulin
and calmodulin gene sequences of the 99 isolates also
showed maximum identity with sequence from
reference A. flavus (CBS100927) strain. Pair-wise sequence
comparisons showed that all 99 isolates exhibited 11
different patterns with three large clusters
corresponding to 3 patterns containing 78 isolates (Table 2). The
neighbor-joining phylogenetic tree based on combined
data set for -tubulin and calmodulin gene sequences
(using only one representative isolate from each of 11
patterns) is shown in Figure 1. All 11 representative A. flavus
isolates from Kuwait clustered together with A. flavus
strains on a separate branch. The unique DNA sequencing
data reported here have been submitted to EMBL under
accession nos. HF570030-HF570051.
The results of Etest antifungal susceptibility of clinical
isolates of A. flavus are presented in Table 3. In general, all
tested antifungal agents showed good in vitro activity
against A. flavus. The MIC range and MIC90 values on
RPMI 1640 medium were as follows: amphotericin B, 0.064
4 and 3 g/ml; voriconazole, 0.064 0.25 and 0.25 g/ml;
posaconazole, 0.016 0.38 and 0.25 g/ml; anidulafungin,
0.002 0.006 and 0.002 g/ml; micafungin, 0.002 0.008
and 0.002 g/ml and caspofungin, 0.002 0.125 and
0.032 g/ml, respectively. The MIC90 values obtained
on MH-GMB were similar to RPMI values for all the
tested antifungal agents except amphotericin B where
it was 4 g/ml.
The cumulative percentage of A. flavus isolates inhibited
at different MIC values are presented in Table 4. Of the 92
clinical isolates tested, 74.2% (n=69) and 59.1% (n=55)
isolates were inhibited by amphotericin B at MIC of 1.024
g/ml on RPMI and MH-GMB medium, respectively.
About 11% (n=10) of the isolates showed MICs of >2 g/ml
on RPMI medium. Among azoles, voriconazole inhibited
100% of the isolates at a concentration of 0.256 g/ml on
both test media. At this concentration, posaconazole
inhibited 100% of the isolates on RPMI and 93.5% of the
isolates on MH-GMB. For three echinocandins,
micafungin and anidulafungin demonstrated
comparable in vitro activity inhibiting 100% of the isolates at a
MIC of 0.008 g/ml on RPMI medium, whereas
caspofungin inhibited 63.4% of the isolates on RPMI
and 33.3% of the isolates on MH-GMB. A comparison
of the mean MIC values of A. flavus isolates recovered
in two 9-year periods (19932001 and 20022011) did
not show any significant difference for the antifungal
The mean and range of MIC values of seven
environmental A. flavus isolates on RPMI medium for the six
antifungal agents were as follows: amphotericin B, 1.25
g/ml 0.577 (range 0.75-2 g/ml); voriconazole, 0.138
g/ml 0.050 (range 0.094-0.25 g/ml); posaconazole,
0.079 g/ml 0.087 (range 0.008-0.25 g/ml);
anidulafungin, 0.002 g/ml 0.00 (range 0.002-0.002 g/ml);
micafungin, 0.002 0.00 (range 0.002-0.002 g/ml) and
caspofungin, 0.071 g/ml 0.082 (range 0.008-0.19 g/ml),
respectively. These mean MIC values were comparable
with those obtained on MH-GMB medium. None of the
environmental isolates exhibited MIC value of >2 g/ml
for amphotericin B.
Disk diffusion test
Using amphotericin B (100-g) and voriconazole (1-g)
disks, the mean values for zone of inhibition for clinical
isolates were 10.38 1.655 mm (range 716 mm) and
28.88 2.321 mm (2434 mm), respectively (Table 5).
For environmental isolates, the mean values for zone of
inhibition were 10.71 1.38 mm (range 812 mm) and
27.71 1.603 mm (2630 mm) for amphotericin B and
voriconazole, respectively. The differences in inhibition
zone diameters between clinical and environmental
isolates for voriconazole and amphotericin B were not
statistically significant. Linear regression analysis between
Etest MIC values and disk diffusion diameters obtained
with clinical isolates revealed a significant inverse
correlation, both with amphotericin B (R2 = 0.2536, p <0.001)
and voriconazole (R2 = 0.4043, p<0.003).
This study provides molecular characterization and
in vitro antifungal susceptibility data on clinical and
Table 2 Nucleotide sequence differences in combined -tubulin and calmodulin gene regions with the indicated
sequence from reference A. flavus strain CBS100927
Pattern No. of Representative *-tubulin region
isolates isolate no. 332 C 390 G
57 Ins 98 G 121 G 132 T 154 Ins 166 T 177 T 197 A 452 G 485 C 563 C
A A T T
*The nucleotide positions are relative to the 50-end of forward sequencing primers.
aFour environmental isolates belonged to this pattern.
bThree environmental isolates belonged to this pattern.
Figure 1 Neighbor-joining phylogenetic tree based on combined -tubulin and calmodulin gene sequence data for selected A. flavus
isolates, each representing the 11 unique patterns, from Kuwait together with reference strains of several species belonging to
Aspergillus section Flavi. Total number of isolates in various clusters are indicated within brackets. Numbers on the nodes branches are
bootstrap frequencies. Only values above 50% are indicated.
environmental isolates of A. flavus from Kuwait. All
clinical and environmental isolates were identified as
A. flavus strains based on the partial -tubulin and
calmodulin sequences. The phylogenetic tree derived from
combined -tubulin and calmodulin gene sequences also
showed that all 99 isolates analyzed in this study
clustered with reference strains of A. flavus or synonyms of
A. flavus  clearly distinct from A. parvisclerotigenus,
A. minisclerotigenes and A. parasiticus, the latter being
used as an out-group. These studies established
speciesspecific identity of all clinical and environmental isolates
used in this study.
The in vitro drug susceptibility testing data showed
that all the tested drugs exhibited good activity except
amphotericin B, where ~10% isolates showed MIC of
>2 g/ml on RPMI medium. Although no antifungal
susceptibility breakpoints are available for A. flavus,
there is a consensus that isolates demonstrating MIC values
of >1 g/ml may be considered resistant to amphotericin
B [26,30,31]. Badiee et al.  determined antifungal
Table 3 Comparative minimum inhibitory concentration (MIC) values of 92 clinical isolates of A. flavus on RPMI 1640
and Mueller-Hinton agar media read at 24 hours
SD- Standard deviation.
Table 4 Distribution of A. flavus isolates according to MIC values
Cumulative % of isolates inhibited at MIC of
Amphotericin B RPMI
susceptibility of 66 A. flavus isolates from Iranian patients
by Etest and CLSI methods. Thirty-six of the isolates were
resistant to amphotericin B by both the methods. In
another study from Tunesia, 31 of 37 isolates of A. flavus
obtained from 14 patients with hematological
malignancies were resistant (2 g/ml) to amphotericin B .
Notably, survival rate was much lower among patients
who yielded resistant strains (22% versus 67%) and were
treated with amphotericn B. Similar results were
obtained in a previous study . However, with the
introduction of voriconazole as a primary therapy for
invasive aspergillosis, the concerns about amphotericin
B resistance have been largely addressed in
All our A. flavus isolates were inhibited at a
concentration of 0.256 g/ml, which is lower or equal to the
recently proposed clinical breakpoints for resistance to
voriconazole (>2 g/ml) and posaconazole (>0.25g/ml)
for A. fumigatus complex isolates . Consistent with
our observations, none of the 98 A. flavus clinical
isolates tested in the Netherlands exhibited resistance to
itraconazole or voriconazole . So far, acquired
resistance to azoles in A. flavus is extremely rare .
However, recent reports of voriconazole resistance in an
A. flavus isolate cultured from lung specimen of a
patient in China and a case of voriconazole-refractory eye
infection in a patient from India have posed new
therapeutic challenges [35,36]. It is unclear why itraconazole/
voriconazole-resistance among A. flavus isolates is
so rare despite their wide-spread environmental
prevalence and exposure to same azole fungicides that are
apparently related to development of resistance in
A. fumigatus , more so, when voriconazole-resistant
strains of A. flavus can be readily obtained in the
laboratory . Although we tested only seven environmental
isolates of A. flavus, their mean MIC values were
marginally lower than clinical isolates for voriconazole and
posaconazole but higher for amphotericin B. In one
previous study, MIC values of environmental isolates
(n=59) for amphotericin B and itraconazole were found
to be significantly lower than the clinical isolates (n=29)
(p < 0.05) .
All three echinocandins showed good in vitro activity
against A. flavus by Etest. Consistent with several
Table 5 Comparative results of disk diffusion tests for amphotericin B and voriconazole
previous reports, anidulafungin and micafungin appeared
more potent than caspofungin [21,22,39,40]. Pfaller et al.
reported that, caspofungin, micafungin and anidulafungin
inhibited all A. flavus isolates (n=64) at a concentration
of 0.06 g/ml . Recently, Espinel-Ingroff et al.
determined epidemiological cut-off values (ECVs) for triazoles,
caspofungin and amphotericin B using wild-type isolates
of Aspergillus spp. employing CLSI microdilution
methodology [14-16]. Our Etest MICs obtained with A. flavus
isolates compared well with ECVs described in the above
studies, involving 100% of the isolates with voriconazole,
98.9% of the isolates with posaconazole, and 100% of the
isolates with caspofungin and amphotericin B (Table 6)
[14-16]. Although we used Etest, the data suggest that
there are no notable differences in the MICs of clinical and
wild-type isolates of A. flavus. Furthermore, there was no
significant difference in the mean MIC values of 14
A. flavus isolates recovered in the first 9-year period
(19932001) compared to 78 A. flavus isolates recovered
in the second 9-year period (20022011) for all the
antifungal drugs tested.
A limitation of our study is the non-availability of BMD
data for comparison with Etest MICs. Similar to some
other studies, we also ignored the presence of
microcolonies within the Etest inhibition zone [21,22]. A
good concordance has been reported between MICs
obtained by Etest and BMD test for posaconazole (84% to
98%) [41,42], itraconazole (100%) [43,44], and voriconazole
(85%) [44-46] against Aspergillus spp. at both, 24 and 48 h
readings in studies that have used these two methods.
Recently, Colozza et al. compared CLSI and Etest MICs for
determining Aspergillus spp. susceptibility to amphotericin
B and AmBisome . A high percentage of A. flavus
complex isolates were resistant (>1 g/ml) to AmBisome
(43.7%) than amphotericin B (16.7%) by BMD. This was in
contrast with the amphotericin B susceptibility results
obtained by BMD method for the same isolates, but not
with those obtained by the Etest. The authors inferred that
Etest may be a superior method than BMD for
determining amphotericin B-resistant Aspergillus strains, perhaps
Table 6 Comparison of epidemiologic cutoff values (ECV)
of A. flavus with Etest MICs obtained in the present study
because of the wider range of MIC distribution available
on the Etest strip.
There is paucity of data on correlation between
inhibition zone diameters and Etest MICs against Aspergillus
spp. A few studies available in the literature suggest that
agar diffusion methods have potential value for
evaluating susceptibility to antifungal agents [47,48]. In a
multicenter study, Espinel-Ingroff et al. evaluated agar
diffusion method for susceptibility testing of filamentous
fungi on plain Muller-Hinton agar medium . The
investigators suggested that using 5-g disk of
posaconazole and caspofungin, 1-g disk of voriconazole,
and 10-g disk for itraconazole, and an incubation time
of 24 h are optimal for determining susceptibility of
three Aspergillus spp. (A. fumigatus, A. flavus, and
A. niger). Recently, Espinel-Ingroff et al. , following
CLSI described guidelines , established zone
diameter limits for disk diffusion susceptibility testing for
A. fumigatus. No quality control ranges were
recommended for A. flavus and A. terreus as some of the
results did not satisfy the M23-A3 document requirements
. However, the zone diameter ranges of 27 to 37 mm
for posaconazole and 25 to 36 mm for voriconazole
obtained by the authors for A. flavus isolates were
considered acceptable. We obtained a comparable zone
diameter range of 24 to 34 mm with 1-g voriconazol
disk, whereas zone diameters for amphotericin B were
much narrower (7 to 16 mm) despite the fact that we
used 100-g disks. More recently, Martos et al. evaluated
disk diffusion method for determining susceptibility to
echinocandins . For all Aspergillus spp. (including 18
A. flavus isolates), caspofungin disk provided narrower zone
of inhibition (1429 mm) than micafungin (1440 mm)
and anidulafungin (2245 mm) at 24 h incubation,
which is in concordance with relatively higher Etest
MICs obtained with caspofungin than micafungin and
anidulafungin obtained in the present study.
All 92 clinical isolates collected over an 18-year period
in Kuwait and phenotypically identified as A. flavus,
were also characterized as A. flavus strains by partial
sequencing of -tubulin and calmodulin gene fragments.
The triazoles and echinocandins showed good activity
against clinical as well as environmental A. flavus
isolates, however, nearly 11% and 18% isolates showed MIC
of >2 g/ml against amphotericin B on RPMI agar
medium and Mueller-Hinton agar medium, respectively.
There was a significant inverse correlation between Etest
MICs and inhibition zone diameter values with
voriconazole and amphotericin B. To the best of our
knowledge, this is the first study reporting results of
molecular characterization of A. flavus isolates from the
The authors declare that they have no competing interests.
ZUK and SA conceived the study, supervised it and finalized the manuscript.
FAW did the work which formed part of her Master thesis and contributed
to writing of the manuscript. All authors have read and approved the final
version of the manuscript.
This work was supported by Research Administration grant YM 03/10 and
the College of Graduate Studies, Kuwait University. The authors are thankful
to Dr. Wassim Chehadeh for helping with statistical analyses.
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