Iran J Parasitol: Vol. 11, No. 1, Jan -Mar 2016, pp.

19-23

Iran J Parasitol
Tehran University of Medical Open access Journal at Iranian Society of Parasitology
Sciences Publication http:// ijpa.tums.ac.ir http:// isp.tums.ac.ir
http:// tums.ac.ir

Original Article

Evaluation of a New Primer In Comparison With Microscopy for

the Detection of Giardia lamblia Infection in Stool Samples
Amir BAIRAMI 1, Sasan REZAEI 2, *Mostafa REZAEIAN 2, 3

1. Dept. of Medical Parasitology and Mycology, School of Medicine, Alborz University of Medical Sciences, Karaj, Iran
2. Dept. of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran
3. Center for Research of Endemic Parasites of Iran (CREPI), Tehran University of Medical Sciences, Tehran, Iran

Received 23 Jun 2015 Abstract

Accepted 18 Sep 2015 Background: Among the most important parasitic disease, causing diarrhea, Gi-
ardia lamblia is noteworthy. Nowadays detection methods for these parasites in-
clude parasitological methods such as microscopic examination. The sensitivity of
Keywords: these methods relies on the expertise and experience of examiners. In contrast,
Evaluation, molecular methods such as PCR are less dependent on the expertise of the exam-
PCR, iner. Here we developed a PCR for the detection of G. lamblia genome in stool
Detection, samples in comparison with microscopy, which is the gold standard.
Giardia lamblia, Methods: For the evaluation of primers, 22 positive samples and 47 negative
Stool samples were used. QIAamp DNA Stool Mini Kit (QIAGEN, Germany) was used
for DNA extraction from feces. Primers for PCR were designed using Primer-
BLAST which uses Primer 3 to designing specific primers (NCBI/ Primer-
*Correspondence BLAST).
Email: Results: Sensitivity of the PCR was done with 100% (95%CI: 84.56-100) for the
rezaiian@sina.tums.ac.ir detection of G. lamblia DNA isolated from patients stool samples which were posi-
tive for G. lamblia cysts and/or trophozoites using microscopy as gold standard. In
comparison with microscopy, PCR had showed the specificity of 97.87% (95%CI:
88.71-99.95).
Conclusion: We designed new primers for the Giardia, and PCR method for the
rapid and accurate identification of Giardia parasites established. With considera-
tion to the routine diagnosis techniques in medical parasitology and their limita-
tions such as time consuming, laborious, less sensitivity etc. This G. lamblia PCR is
a sensitive and specific application for the diagnosis of G. lamblia and provides us a
reliable method in the routine intestinal parasitic infection laboratory diagnosis.

19 Available at: http://ijpa.tums.ac.ir

 Bairami et al.: Evaluation of a New Primer In Comparison …

Introduction

A mong non-viral diarrhea, causing

agents Giardia lamblia Infection is
very prevalent (1). Microscopy is the
usual method has been used for diagnosis of
G. lamblia infections by using stool samples;
these techniques in routine laboratory plat-
forms for the detection of parasites. In this
regard, we developed a PCR for the detection
of G. lamblia genome in stool samples in com-
parison with microscopy, which is the gold
closely rely upon times of sampling, applica- standard.
tion of concentration methods and how much
expertise and experience has the technician Material and Methods
performing the test. Consequently, microscop-
ic test for the diagnosis of G. lamblia in stool More than 500 stool samples in the years
samples is behind hand and uneconomical (2), 2012 to 2013 were examined, among which 22
due to expensiveness of hand working in positive samples and 47 negative samples were
comparison to machine work in the developed used. The samples from different parts of Iran
countries. Over light microscopy, there are such as Tehran and Bandar Abbas were col-
some immunological methods for the diagno- lected. A standard diagnostic method in this
sis of G. lamblia cysts or trophozoites in bio- study (Golden Standard) was parasitological
logical samples. One is the detection of Giar- method, which is describing as follow. Micro-
dia antigens in the stool of patient (3). Appli- scopic examination was performed for all
cation of enzyme immunoassay for antigen samples. For loose, soft and formed speci-
detection does not eliminate the need to ana- mens formal-ether concentration was per-
lyze multiple stool specimens for accurate de- formed, then, wet mount prepared slides
tection of parasite (4). stained with temporary iodine stain and inves-
In general, conventional methods using mi- tigate for cyst and/or trophozoite of Giardia
croscopy, biochemistry and immunological using 400 × magnifications. For watery and
approaches have serious restrictions such as dysenteric samples, iodine stained wet mount
essential need to a high degree of expertise preparation had done also without a formal-
and experience in the diagnosis of Giardia (5). ether concentration for the detection of active
Therefore, molecular techniques with high trophozoites.
sensitivity, specify and workable there have We used QIAamp DNA Stool Mini Kit
been in demand. In recent for the detection of (QIAGEN, Germany) for DNA extraction
diarrhea causing agents such as viruses and from feces, 180 to 220 mg feces suspended in
bacteria many PCR based methods have been 1.4 ml of LSA buffer provided in kit and fol-
published but for the diagnosis of parasites it lowing the other commands recommended by
seems to be less addressed and, application of manufacturer, DNA was isolated with QI-
these methods for the diagnosis of parasitic Aamp DNA Stool Mini Kit spin columns.
diseases in routine laboratories is still very lim- Primers for PCR were chosen using Primer-
ited (2). DNA isolation from stool samples is BLAST. It uses Primer3 to design PCR pri-
very complicated and grinding. Nowadays, mers and then uses BLAST and global align-
there are some methods for nucleic acids iso- ment algorithm to design specific primers
lation from biological samples, which could (NCBI/ Primer-BLAST) (7), based on the G.
remove PCR inhibitors (6). lamblia Cathepsin L-like protease gene se-
Combination of simple and quick DNA iso- quence for G. lamblia (GenBank accession no.
lation methods with easy genome amplifica- XM_001706220.1) that G. lamblia DNA
tion and detection techniques lets us to use should be detected specifically.

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 Iran J Parasitol: Vol. 11, No. 1, Jan -Mar 2016, pp.19-23

The G. lamblia specific primers consisted of tested. Sequencing had done on the amplicon
forward primer GlF (5 - AATCTGTT- produced from Giardia samples achieved of
GACTTAAGGGAGTA-3 ; positions185–206), culture for analyzing, and verified to present
reverse primer GlR (5 -ATTGAGTCATT- image of the accurate sequence of the targeted
ATAGGGATTGT-3 ; positions 647–626), DNA.
product length: 463 base pairs (Fig. 1). Sensitivity of the PCR was done with 100%
The specificity of the PCR was checked out (95%CI: 84.56-100) for the detection of G.
by a range of DNA extracted of various mi- lamblia DNA isolated from patients stool sam-
croorganisms includeing E. histolytica, E. dispar, ples which were positive for G. lamblia cysts
Entamoeba coli, Blastocystishominis, Cryptosporidum and/or trophozoites using microscopy as gold
spp. and from Escherichia coli, Vibrio cholerae, standard. In comparison with microscopy,
and C. albicans. PCR showed 1 false positive of 47 samples
which were negative by microscopy in which
the specificity of the test calculated to be
97.87% (95%CI: 88.71-99.95). Thus, positive
predictive value (PPV) of the PCR was
achieved of 95.65% (95%CI: 78.05-99.89), and
the assay negative predictive value (NPV) was
100% (95%CI: 92.29-100). The accuracy of
the test was 98.5%.

Discussion
Prevalence of G. lamblia in developed coun-
tries is 2 to 7% and in developing countries is
about 20 to 30% (11, 12). Molecular methods
Fig. 1: 1% Agarosegel stained by ethidiumbromid
have high sensitivity against conventional
shows PCR product by Gl F&Gl R primers
M: size marker, Lane 1: G. Lamblia DNA, Lane 2: methods (13), among these approaches, PCR
Negative control have been used in studies for molecular epi-
demiology assessments and understanding
In DNase and RNase free PCR tubes Am- about zoonotic transmission possibility of Gi-
plification reactions were performed in a vol- ardia (14). Potentially in developed settings it
ume of 50 ml with 10x PCR buffer, 1mM could be used for diagnostic proposes.
MgCl2, 5 μl of the DNA template from the In our study, we used well-marked positive
stool samples, 1 unit/μl Taq DNA Polymerase and negative stool samples as controls. In
and 20 pmol/μl of each specific primer. PCR comparison with microscopy as the gold
was carried out by using a peqSTAR thermo- standard designed PCR results showed a sensi-
cycler (Peqlab, Germany) under the following tivity of 100% (95%CI: 84.56-100), the G.
conditions: 5 min at 94°C followed by 35 cy- lamblia PCR achieved specificity of 97.87%
cles of 60 s at 94 °C, 90 s at 55 °C, and 2 min (95%CI: 88.71-99.95) for G. lamblia. PCR
at 72 °C followed by 10 min at 72 °C. showed the positive predictive value (PPV),
and the negative predictive value (NPV) of
Results 95.65% (95%CI: 78.05-99.89) and 100%
(95%CI: 92.29-100) respectively.
DNA of 47 stool samples, which were nega- It is very challenging to elect the ‘gold stand-
tive for parasitic infections in microscopy, had ard’ in the studies like our study (2), but scien-

21 Available at: http://ijpa.tums.ac.ir

 Bairami et al.: Evaluation of a New Primer In Comparison …

tists stated that, microscopy is still the gold laborious, less sensitivity etc., this G. lamblia
standard for the detection of many protozoan PCR is a sensitive and specific application for
parasites (8). In this way, we used the micros- the diagnosis of G. lamblia and provide us a
copy as gold standard and compared our PCR reliable method in the routine intestinal para-
with it. Therefore, PCR showed high sensitivi- sitic infection laboratory diagnosis.
ty and specificity in comparison with micros- As well as possible, PCR proffer multiplex
copy for the diagnoses of G. lamblia infections. characterization of various objects in the same
These findings are alike with the findings of sample. Therefore, in subsequent steps au-
Verweij et al. (2) who used a real-time PCR for thors will try to develop a multiplex PCR with
the diagnoses of G. lamblia infections. One using this G. lamblia PCR in combination with
false positive result was observed by the PCR, PCR assays for other pathogens like E. histolyt-
occurrence of false positive results can be due ica and C. parvum.
to many various causes such as contamination
which could happen accidentally during the Conclusion
procedure or can occurs due to usage of pre-
viously contaminated reagents, even it could This PCR showed reliable sensitivity and
happen because of aerosol contamination. specificity for the detection of G. lamblia in
However, contamination is the Achilles‘ heel stool samples and has capability to perform in
in the open tube system tests like PCR in con- the routine diagnostic laboratories.
trast to those with closed tube systems such as
real-time PCR. Acknowledgements
There were no false negative results
achieved by the PCR, the main reason for this The authors declare that there is no conflict
as the case may be the existence of appropri- of interests.
ate DNA template in the reaction which we
had used. As it proved there were many PCR
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