Documente Academic
Documente Profesional
Documente Cultură
, 3(1): 766-770
JANUARY- 2017
Address for Correspondence: Dr. Jaspreet Kaur, Senior Research Associate, Department of Biochemistry, Central
Research Laboratory, Shri Ram Murti Smarak Institute of Medical Sciences, Bareilly, India
Received: 22 August 2016/Revised: 19 September 2016/Accepted: 20 October 2016
ABSTRACT- Background & Objectives: Fungal infections of the eye are rampant in tropical regions and are
responsible for significant ocular morbidity and blindness. Prompt and specific identification of fungi is important in order
to define clinical treatment. However, in most cases conventional culture identification can be considered to be time
consuming, of low sensitivity and not without errors. The aim of the study is to evaluate a polymerase chain reaction
(PCR) based assay for fungal infections in the eye.
Methods: Polymerase chain reaction (PCR) for detection of fungal DNA was done with panfungal primers (ITS1 and
ITS4) for diagnosis of fungal aetiology. Ocular samples (conjunctival swabs, corneal scrapings, corneal biopsies, vitreous
aspirates and globe contents) from 30 patients with presumed fungal infection were evaluated using this assay, as well as
by standard microbiological techniques, and the results were compared.
Results: Thirty clinical specimens were evaluated by PCR and by other conventional techniques (culture and staining). Of
the 30 specimens analyzed, fungal keratitis was definitively diagnosed by culture in 18 (60%). 17 (57%) of these 18
specimens were PCR positive. One specimen (3% of the 30 total) was fungal culture positive but PCR negative. 12 (40%)
of 30 specimens were fungal culture negative, and 7 (23%) of these 12 were also PCR negative. Five (17%) were PCR
positive but fungal culture negative. Of the seven specimens negative by both PCR and fungal culture, all showed no
growth.
All 30 specimens were also examined by light microscopy with potassium hydroxide (KOH) and Grams staining. KOH
and/or Grams staining was found to be positive in 8 (27%) out of 30 cases. All positive smears revealed septate hyphae. 3
(38%) of these 8 specimens showed fungi on smear but were fungal culture negative, and two of these three specimens
were PCR positive which were clinically to have fungal keratitis.
Conclusions: PCR not only proved to be an effective rapid method for the diagnosis of fungal infections in eye but was
also more sensitive than staining and culture methods. Due to promising results of the DNA extraction protocol and PCR
assay in our laboratory, we were able to make early diagnosis. Hence, effective therapy was given, which had a major
impact on the improvement in the prognosis of subject with fungal infections.
Key-words- Fungal keratitis, Lyticase, Proteinase K, Polymerase chain reaction, PCR assay, DNA extraction
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INTRODUCTION
Fungal infections of the eye are on rise and are responsible
for ocular morbidity and blindness.
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DOI: 10.21276/ijlssr.2017.3.1.3
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(TE) buffer.
PCR Standardization
PCR was done with panfungal primers ITS1 (IDT,
Belgium) (5'TCC GTA GGT GAA CCT GCG G'3), and
ITS4 (IDT, Belgium) (5' TCC TCC GCT TAT TGA TAT
GC 3') to amplify ITSI, 5.8 S and ITSII region of the
rDNA. The 50 l PCR mixture contained 10 l of DNA
template, 5 l PCR buffer with 1.5 mM MgCl2, 200 M
each deoxynucleoside triphosphate (100 M Bangalore
Genei, Bangalore), 25 pmol of each primer, and 1.5 U of
Taq DNA polymerase (Himedia, Mumbai). Reaction
involved 1 cycle at 95C for 5 min, followed by 35 cycles
with a denaturation step at 95C for 30 sec, an annealing
step at 55C for 1 min, and an extension step at 72C for 1
min, followed by 1 cycle of 72C for 6 min (done in XP
thermal cycler, Bioer, Japan). Candida albicans (ATCC
204303), Aspergillus flavus (ATCC 90028), Fusarium
solani (clinical isolate), Staphylococcus aureus (ATCC
25923), Escherchia coli (ATCC 25922), Enterococcus
faecalis (ATCC 29212) and Pseudomonas aeruginosa
(ATCC 27853) were included as control in the present
study. Ten microliter of the PCR products, mixed with
loading buffer was then electrophoresed 1.5% agarose gel
with 0.5 g/ml ethidium bromide. A 100 base pair (bp)
DNA ladder (Himedia, Mumbai) was used for determining
the size of the amplicons. The gels were visualized using
ultraviolet illumination. Images were captured and stored
by using a UVP Gel Documentation System (UVP Ltd,
USA).
595 bp
Ocular samples
Thirty clinical specimens were evaluated by PCR and by
other conventional techniques (culture and staining). The
results are shown in Table 1. Of the 30 specimens analysed,
fungal keratitis was definitively diagnosed by culture in 18
(60%). 17 (57%) of these 18 specimens were PCR positive
(Fig. 2). One specimen (3% of the 30 total) was fungal
culture positive but PCR negative- an apparent false
negative PCR result. Twelve (40%) of 30 specimens were
fungal culture negative, and seven (23%) of these 12 were
also PCR negative.
RESULTS
Standard Fungal Isolate
PCR Specificity: The primers used in this study (ITSI
and ITS4) successfully amplified DNA from all the
standard fungal strains tested. A product of approximately
500-600 bp was obtained. No amplification products were
detected by using ITS1 and ITS4 primer pair with S.
aureus, E. coli, E. faecalis, and P. aeruginosa.
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PCR negative
for fungi
Culture negative
for fungi
5 (17%)
Clinical findings:
Fungal keratitis: 2
(7%)
Bacterial keratitis:
2 (7%)
Uncertain: 1 (3%)
7 (23%)
Culture results:
No growth: 7
(23%)
12 (40%)
DISCUSSION
In this report, we present our experience in the detection of
fungal pathogens in ocular samples to diagnose fungal
keratitis. This study demonstrates that fungi can be detected
in infected corneas using PCR techniques. The advantage
of PCR as shown is its rapidity. The PCR assay used in this
study required 6 hours to generate results, significantly
faster than the 2 days to 1 week required for any fungal
culture technique in confirming the culture growth. On the
other hand fungal smears also provide result quite quickly,
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CONCLUSION
We concluded in this study that ocular fungal infections are
increasing and gaining attention in clinical practice.
Therefore, newer research focused on improvement in
diagnostic techniques with cost efficiency would be
immensely helpful to the ophthalmologist in decreasing the
morbidity associated with ocular mycosis. Hence, this
method will be useful for fungal infections irrespective of
ocular samples in developing countries like India where the
diagnostic test should be cost and time effective. However,
there is a need to evaluate this protocol in larger number of
patients with fungal infection in the eye.
ACKNOWLEDGMENT
The authors thank SRMS IMS Trust, Bareilly for providing
the platform for this research work Authors also thank Dr
Arunaloke Chakrabarti, Professor, PGIMER, Chandigarh
for providing standard fungal cultures and Dr. Neelima
Mehrotra, Opthalmologist/Professor SRMS Hospital,
Bareilly for providing the ocular samples.
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