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PCR Detection of Aspergillus flavus in Vaginal Specimens

Article · January 2016

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Baheeja Abees Hmood Al-Khalidi

University of Al-Qadisiyah
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 PCR Detection of Aspergillus flavus in Vaginal Specimens
Assistant prof. Baheeja Abees Hmood.College of Nursing ,Al-Qadissiyia university.

Abstract

Background: Cervical infections are commonly encountered problems occurring

in the women of the reproductive age group. Pap smear, a screening test for
carcinoma cervix is a simple, quick test that can also be used for diagnosing
cervical vaginal infections. The present study was undertaken to detection of
unusual agents in vulvovaginal infection and use (PCR) technique for rapid
detection of this agent in cervical specimens .in addition to the cultivability of the
target organism.

Methods:Fourth Vaginal samples were collected from vulvovaginal of infected

women during gynecological examination .To obtain specimens, a polyurethane
foam swab (Catch-All; Epicenter) was brushed against the lateral vaginal wall,
each swab were placed in 15-ml conical vials with 3 ml of saline and divided into
Two portion .first portion were investigated directly by lactophenoblue stain and
cultured on Sabouraud's agar (BD-Difco), ,Second portion was frozen until the
DNA extraction step begin for the performance of PCR assays for detection of
unusual fungi in vaginal swabs.

Results :Aspergillus flavus isolated from the vaginal mucosa was more frequent in
patients (35%) so Candida ssp isolated by rate (32.5%) and other agent (37.5%)(
these may be bacteria, parasites or viruses which not isolated in this study)
compared to uninfected women which don't appear any positive culture (p =
0.001).depending on direct and culture methods So the results of this study
depending on PCR test showed 8 specimens from infected women were had ITS2
region (380bp) which specific for A.flavus which served as the target organism for
this study

Keyword: Vaginitits, Aspergillus flavus, PCR

INTRODUCTION

Vaginitis with vaginal discharge is a common problem, causing 10 million

women each year to visit a physician's office for care (1).The most common causes
of vaginitis are bacterial and fungal. In as many as 75% of females with vaginitis,
vulvovaginal candidiasis and other fungi are the cause.(2) As many as 15%-20%
of females with vaginal yeast infections are asymptomatic.(3 ).Candida albicans is
able to adhere to vaginal epithelium more readily than other Candida species,
which is probably why it causes about 80% of yeast infections. It may be that
treatment failures point to the presence of a non-candida infection. Researchers
hypothesize that the widespread home use of nonprescription antifungal
medications has caused the emergence of others fungi ,such as Aspergillus genera
especially A.flavus and that the number of chronic and recurrent cases will
eventually increase as a result.(4).

Aspergillus flavus is a ubiquitous mold and the most common mold

contaminating foodstuffs. Many strains of A. flavus produce aflatoxins. In addition
it is an allergen and an opportunistic pathogen. A. flavus often is underestimated in
traditional culture analyses due to the expertise required and the cost associated
with speciation members of the genus Aspergillus.(5 ).

In clinical microbiology laboratories, traditional culture and biochemical

techniques remain the primary methodology for identifying most pathogens. With
the development of technology, automated microbiology system for the
identification of pathogens has been introduced in many laboratories. In recent
years, with the population aging, the number of patients with organ transplantation
and bone marrow transplantation increasing, and immunosuppressive agents,
anticancer drugs and invasive detection and treatment widely used, more and more
patients suffer from severe infection of fungi.( 6).

The definite and rapid diagnosis of invasive fungal infections is difficult due to the
lack of sensitive test methods. Therefore, efforts to improve diagnosis are ongoing
and need to be further intensified. Although proving the presence of infection by
histology and culture remains the cornerstone of diagnosis, non-culture-based
methods are being developed to allow early detection. Among the most promising
approaches are the detection of fungal antigens and PCR (7, 8, 9). The results of
some studies suggest that a combination of the Aspergillus galactomannan
enzyme-linked immunosorbent assay (GM-ELISA) in sterile human body fluids or
tissue samples to allow early diagnosis of fungal infections .The internal
transcribed spacer (ITS) region, ITS1-5.8-ITS2, located between the 18S and 28S
rRNA genes and the 5.8S rRNA gene, have proved sufficient for the discrimination
of most species of clinically important fungi. ITS regions, especially ITS2, have
emerged as the most common target of molecular-based identification (10,11).

PCR may provide improved diagnosis of invasive aspergillosis .Nevertheless, not

only the detection of fungi, but also their identification, which can be obtained only
by PCR or culture, is important for the optimal choice of antifungal and duration of
therapy. (12, 13, 14, 15).

The goal of this study was to detection of unusual agents in vulvovaginal infection
and use polymerase chain reaction (PCR) technique for rapid detection of this
agent in cervical specimens .in addition to the cultivability of the target organism
Material and methods

Fourth Vaginal samples were collected from vulvovaginal of infected women

during gynecological examination .To obtain specimens, a polyurethane foam
swab (Catch-All; Epicenter) was brushed against the lateral vaginal wall, each
swab were placed in 15-ml conical vials with 3 ml of saline and divided into Two
portion .first portion were investigated directly by lactophenoblue stain and
cultured on Sabouraud's agar (BD-Difco), incubated at 25ºC and examined
periodically with a microscope until substantial sporulation was observed (3–20 d).
and a direct examination was performed to diagnostic of fungi according to
morphological characteristic on Sabouraud's agar ,Second portion was frozen until
the DNA extraction step begin for the performance of PCR assays for detection of
fungi in vaginal swabs.Fourth vaginal swabs also collected from Healthy women
as(control group) in the same method above.( 16) .

DNA extraction

Vaginal swabs(second portion) for fungal PCR 1 to 2 ml of the specimen was

centrifuged for 5 min at 13,000 rpm. The pellet plus 200 μl supernatant was
incubated with(50 mM Tris [pH 7.6], 1 mM EDTA [pH 8.0], 0.2% 2-
mercaptoethanol) containing 20 U recombinant Lyticase, incubated at 37°C for 30
min, and then centrifuged at 13,000 rpm for 10 min. Finally, DNA was extracted
with a High Pure PCR template preparation kit(TaKaRa Bio Inc., Tokyo, Japan) by
following the instructions of the manufacturer. DNA was eluted with 100 μl
elution buffer.

ITS2 sequencing
 Aspergillus flavus served as the target organism for this study The ITS2
regions were amplified A.flavus DNA using conventional PCR with primers
ITS86-F (5′-GTGAATCATCGAATCTTTGAAC-3′)andITS4-R(5-
CCTCCGCTTATTGATATGC-3) (17), producing an amplicon of approximately
( 200-400 )bp. The PCR reaction mix, except for the primers, contained the same
ingredients described above. Thermal cycling parameters were 10 min at 94°C, 30
cycles of 30 s at 94°C, 40 s at 55°C, and 1 min at 72°C; a final extension step at
72°C was added for 5 min. The products from PCR amplification were examined
by gel electrophoresis .

Statistical analysis

Frequencies of fungi. infected women were compared to those of the control group
by using t tests for continuous variables and Chi-square tests .Statistical analysis
was done with the SPSS (version 11.0).
RESULTS

Aspergillus flavus isolated from the vaginal mucosa was more frequent in patients
(35%) so Candida ssp isolated by rate (32.5%) and other agent (37.5%)( these may
be bacteria, parasites or viruses which not isolated in this study) compared to
uninfected women which don't appear any positive culture (p = 0.001).depending
on direct and culture methods .table (1)

Table (1) frequency of fungal agents in cervical specimens

Groups Infected with Infected with Others agents(not

Aspergillus flavus Candida ssp isolated)

Women with vulvovaginal 14(35%) 13 (32.5%) 15(37.5%)

=40
Healthy women (Control ) None None None
=40

So the results of this study depending on PCR test showed 8 specimens from
infected women were had ITS2 region (380bp) which specific for A.flavus which
served as the target organism for this study .picture(1).
DISCUSSION

Cervical infections are commonly encountered problems occurring in the women

of the reproductive age group. Aspergillus spp is a fungus known to cause both
acquired and nosocomial infections in human beings. The most commonly affected
sites are the lungs, soft tissue, and skin(18,19) Infections of the genitourinary tract
are a common problem. Sullam et al.(18) reported a prevalence of 52.8% with a
spectrum consisting of Candida albicans (28.0%), Trichomonas vaginalis (8.7%),
Aspergillus species (7.4%), Streptococci (4.6%), and Chlamydia trachomatis
(4.2%).

However, apart from Candida albicans, fungal pathology is rarely seen in

cervico-vaginal smears. Most reported cases of female genital tract infections with
opportunistic fungi in cervical smears included Blastomyces dermatitidis,
Coccidioides immitis, Aspergillus flavus, Cryptococcus neoformans and Mucor,
with very few cases of Aspergillus spp(19).

We report here an unusual case in which Aspergillus flavus. was detected in the
cervical smear of female who presented with features of pelvic inflammatory
disease and was subsequently diagnosed as a case of cervical carcinoma this
finding agreement with case report record by (20) ,and (21) so (22) recorded
(0.15%) case of Aspergillus identified by the presence of condiophores and conidia
in pap smear.

Aspergillus flavus produce significant quantities of toxic compounds known as

Aflatoxins. he four major aflatoxins produced are B1, B2, G1, and G2. Aflatoxin
B1 is the most toxic and potent hepatocarcinogenic natural compound
characterized. A. flavus also produces other toxic compounds including
sterigmatocystin, cyclopiazonic acid, kojic acid, β-nitropropionic acid, aspertoxin,
aflatrem, gliotoxin, and aspergillic acid. (23).

In humans, A. flavus aflatoxin production can lead to acute hepatitis,

immunosuppression, mucosa carcinoma, and neutropenia. It is highly possible for
A. flavus to invade arteries of the lung or brain and cause infarction.( 24 ) Thus
found this fungi in unusual sites(such as vagina) may be lead to many
complication.

Molecular biological techniques have been used effectively for the enhanced
detection of microorganisms, overcoming the limitations associated with
traditional culture analysis and producing fast, sensitive results. The pathogenicity
of A. flavus and the ability of certain isolates to produce potent aflatoxins warrant
the development of a fast, reproducible and quantitative assay capable of detecting
this organism in clinical specimens, livestock feed and indoor environments.
However PCR-based methods currently available for the detection of A. flavus
employ conventional PCR and gel electrophoresis.( 25).

Of the hundreds of Aspergillus species A. flavus is one of several that has been
reported routinely as the causative agent of human infections.( 26 ) For the
identification of a unique A. flavus target region for the development of specific
PCR primers emphasis was placed on searching sequences of the 18S rRNA gene,
which contains both highly variable and highly conserved regions (27). Sequence
comparison results on the internal transcribed spacer regions of the indicate that
the GenBank database is lacking ITS sequences for some species, they
demonstrated that these same regions are more reliable than other regions for the
identification of medically important Aspergillus species. While(28) indicate that
both ITS1 and ITS2 were necessary to accurately identify Aspergillus species using
conventional PCR, more recently(29) observed interspecies variability in the ITS2
region and exploited this variation to design microarray probes for the
identification of pathogenic fungi.

Several investigators have published methods for the detection of A. flavus in

different materials using conventional PCR followed by agarose gel
electrophoresis (30, 31, 32), PCR and enzyme immunoassay (33) and multiplex
PCR assays with biprobes (34). Although faster and more sensitive than culture
analysis for detection, some of these methods require post-PCR manipulations and
are only semi quantitative.

Conclusion

The results of this research demonstrate there were an unusual case in which
Aspergillus flavus. was detected in the cervical smear of female who presented
with features of pelvic inflammatory disease and was subsequently diagnosed as a
case of cervical carcinoma so demonstrate the capabilities of PCR for the enhanced
detection and enumeration of fungi of significance to human health.
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