International Journal of Current Microbiology and Applied Sciences 

ISSN: 2319-7706 Volume 4 Number 5 (2015) pp. 991-996 

http://www.ijcmas.com 

 

 
Original Research Article 
Evaluation of Different Techniques of Stool Examination for Intestinal 
Parasitic Infections in Sulaimani City - Iraq 
Hersh Ahmad Amin* and Shahnaz Abdulkader Ali 
Department of Microbiology, School of Medicine, Faculty of Medical Sciences, 
University of Sulaimani, Kurdistan region of Iraq 
*Corresponding author 


 
ABSTRACT 

 

 

This study was conducted: to determine the best diagnostic technique use for 
stool examination using three diagnostic methods, Zinc sulphate floatation, 
Formal-Ethyl Acetate sedimentation and direct wet mount techniques. In 
addition, to determine the prevalence of intestinal parasitic infections, 300 
samples were collected randomly from patients with different age groups 
from both sexes, who attended the central laboratory in Teaching Hospital in 
Sulaimani City/ Iraq from June to October 2014. The results showed that 
Zinc sulphate floatation technique had of the highest sensitivity rate 
148(49%), followed by Formal-Ethyl Acetate 130(433%) and Direct wet 
mount that showed lowest rate 68(22.6%). The prevalence of the intestinal 
parasitic infections were165 (55.6%) of which the most common parasite 
was Entamoeba histolytica(60.22%). The rate of infection for males was 
higher 118 (57.28%) than females 47 (50%).Children under 5 years of age 
had the highest prevalence of the parasitic infections 52(81.25%). 

 

 


Keywords 

 

Diagnostic 
techniques, 
Zinc sulphate 
floatation, 
Formal-Ethyl 
Acetate 
sedimentation, 
Direct wet 
mount 
technique, 
Sulaimani/Iraq 

 


 


 
Introduction 
Parasitic infections, caused by intestinal 
helminthes and protozoan parasites, are 
among the most prevalent infections in 
developing countries, while in developed 
countries, gastrointestinal infections are 
caused more commonly by protozoan 
parasites compared to helminthes. Intestinal 
parasites cause a significant morbidity and 
mortality in endemic countries. These 
infections are most prevalent in tropical and 
sub tropical regions of the developing world 


where adequate water and sanitation 
facilities are lacking (Savioli and Albonico, 
2004;Cappello,2004).The usual methods 
that are used for detection of intestinal 
parasites from stool specimens are the direct 
wet mount using saline and iodine, but the 
detection can be enhanced through using 
concentration procedures such as salt 
floatation, zinc sulphate centrifugal 
floatation, formal ether concentration 
(Ritchie, 1948) and modified Baerman 


sedimentation. These techniques increase 
the detection of protozoan cysts, helminthes 
eggs and larvae. However, the use of diethyl 
ether, an essential reagent of concentration 
technique, may be hazardous to laboratory 
workers, since it is explosive, of potential 
toxicity such as respiratory irritation and a 
cause of cardiovascular depression and 
narcosis (Boswell and Collins, 1996; Young 
et al., 1979).Therefore, a replacement for 
diethyl ether has been sought for this 
technique by introduced of ethyl acetate as a 
replacement. In view of the increasing 
polyparasitism in the developing countries, 
there is a need for sensitive diagnostic tools 
that are simple to apply and can detect 
different intestinal parasite species in the 
same stool sample. 
The objectives of this study wereto compare 
three different techniques in diagnosing 
parasitic infections, and to determine the 
prevalence of intestinal parasitic infections 
among the patients who attended the central 
laboratory in Teaching Hospital Sulaimani 
city. 
Stool Sample collection 


A total of 300 fresh stool samples were 
collected randomly from patient with 
different age groups, who attended the 
central laboratory in teaching hospital 
inSulaimanicity from June to October 2014. 
Specimens were collected in sterile 
containers and transported to the 
Department of Microbiology immediately. 
Both the formed and unformed stools were 
examined immediately. 
Material and methods 
Each stool specimen was examined by the 
following techniques 


Macroscopic examination: The stool 
specimens were examined by naked eye for 
presence of: The colour, consistency, blood, 
mucus, the nature of the faeces, and adult 
worms. 
Direct microscopic examination using 
saline and iodine preparations 
A small amount of the stool sample was 
emulsified in 1-2 drops of saline or iodine 
on a glass slide then a cover slip was placed 
on it and examined microscopically. 
Concentration microscopic examination 
A- Zinc Sulphate centrifugal flotation: 
Fresh faecal specimens were formalinised 
by adding few drops of 10% formalin to 1g 
of stool sample and then emulsified in 10 
parts of saline, strained through three folds 
of gauze. The filtrate was collected in a 
centrifuge tube and centrifuged at 1500rpm 
for 5 minutes. The supernatant was 
discarded and the sediment was re-
suspended in water. To the sediment,3-4 mL 
of 33% zinc sulphate solution was added, 
then mixed well and filled with ZnSO4 
solution to about half an inch of the rim. 
Several loopfuls of the supernatant fluid 
were removed with a bacteriological loop 
and put on slide, then covered with a cover 
slip and examined under 40X lens (Melvin 
and Brooke,1985). 
B-Formal-Ethyl Acetate concentration: 
Fresh faecal specimen was emulsified by 
mixing about 1g of stool with7 mL of 10% 
formol saline and was kept for 10 minutes 
for fixation, then strained through three 
folds of gauze. The filtrate was added to 3 
mL of ethyl-acetatand then centrifuged at 
1500rpm for 5 minutes and allowed to settle. 
The supernatant was removed and two drops 
of the sediments on a slide, covered with a 
cover slip then examined under 40X for 
parasite detection (Neimeister et al., 1987). 



Statistical analysis 
Chi-square test was used to evaluate the 
statistical significance, P-value <0.05 was 
considered as statistically significant. 
Results and Discussion 
A total of 300 stool samples were examined, 
out of which 165 (55.6%) samples were 
positive for intestinal parasites. Entamoeba 
histolytica was the most prevalent 
species110 (60.22%), other parasites 
observed were Blastocystis hominis (19%), 
Cryptosporidium spp (16.43%), Entamoeba 
coli (6%), Giardia lamblia (2%), 
Chilomastix mesnili (2.28%), Isospora spp 
(1.36%) and Enterobius vermicularis 
(0.45%) as shown in Table 1. 
From the results of three different diagnostic 
tests, demonstration of the parasitic 
infections with sensitivity of (49.3%) the 
Zinc sulfate flotation concentration 
technique was found to be most sensitive 
method in this study, then came Formal-
Ethyl acetate at rate of (43%) and direct wet 
mount (22.3%) as shown in Table 2. 
Interestingly males were more infected with 
intestinal parasites (57.28%) than females 
(50%).as shown in Table 3. Children under 
5 years of age had the highest prevalence of 
the parasitic infections as shown in Table 4. 
The rate of poly parasitism infections seen 
in 56(36%) samples were infection with 
Entamoeba histolytica and Blastocystis 
hominis cysts, Entamoeba histolytica and 
Giardia lamblia cysts that were detected by 
concentration methods. 


In the present study the prevalence of the 
intestinal parasitic infections was higher165 
(55.6%) and Entamoeba histolytica was the 
most prevalent species. This was agreement 
with the study conducted by (Hamad and 
Ramzy, 2012) who reported 30% in Arbil, 
61.1 % by (Farhan, 2012) in Al-Anbar and 
in Baghdad (23.44%) detected by (AL-
Kubaisyet al., 2014). Another study by 
(Jarallah, 2012) reported 28.87% in Basrah. 
Entamoeba histolytica was reported to be 
responsible for approximately 50 million 
cases of invasive amoebiasis and upwards of 
100,000 death/year (WHO, 1997). This may 
be due to that E. histolytica are highly 
resistant to chlorine disinfection and their 
small size (range 1–17 .m) enables them to 
penetrate water treatment systems and cause 
waterborne disease even following the 
consumption of treated drinking-water 
(Graczyk et al.,2005).Infection is also 
acquired primarily through the infection of 
infective cyst form present in faecally 
contaminated water and food (Petri and 
Singh, 1999). Second species recorded was 
Blastocystis hominis (19.60%).This was in 
disagreement with studies by (Ali, 2010) 
(28.81%), (Rezan, 2014) (22.15%) and 
(Raza, 2009) 6.3% in Sulaimani and7% in 
Baghdad by (Mahmood et al., 2014). 
The prevalence rate of parasitic infections 
was higher in males (57%) as compared to 
females (50%) with no significant 
difference. The reason for the male 
preponderance in our study may be related 
to the daily activity rather than the sex 
predominance. However, many studies from 
Iraq showed higher prevalence of these 
infections among males, in Sulaimani by 
(Rezan, 2014), (Mariwan, 2010) in kalar 
Sulaimani, (AL-Kubaisy et al., 2014) in 
Baghdad, while other studies showed 
increased infection among the females, 
(Hamad and Ramzy, 2012) in Erbil, (Raza, 
2009) in Sulaimani. Nevertheless, the sex 
predominance for the parasite infections has 
still not been confirmed. 


Statistically no significant difference in the 
percentage of intestinal parasites infections 
were according to the age of the patient 
(p>0.05). 


Table.1 Total Prevalence of parasitic infections 


parasites 

Number of (+ve) case 
(n=300) 

% 

Entamoeba histolytica 

110 

50.16 

Blastocystis hominis 

43 

19.6 

Cryptosporidium spp. 

36 

16.41 

Entamoeba coli 

15 

6.84 

Giardia lamblia 

6 

2.73 

Chilomastix mesnili 

5 

2.28 

Isospora spp. 

3 

1.36 

Enterobius vermicularis 

1 

0.45 

Total 

219 

 



 
Table.2 Sensitivity of different parasitic examination techniques 


Techniques 


No. positive for parasites 
(Total No. 300) 

% 

Zinc sulphate floatation 

148 

49.3 

Formal-Ethyl Acetate 

130 

43.3 

Direct wet mount 

68 

22.6 



 
Table.3 Sex prevalence of parasitic infections 


Sex 

Examined No. 

No. positive for parasites 

% 

Male 

206 

118 

57.28 

Female 

94 

47 

50 

Total 

300 

165 

55.6 



.2= 0.409, P-value >0.05 
Table.4 Age prevalence range of parasite infections 


Age 

Examined No. 

Positive 

% 

< 5 

64 

52 

81.25 

6-10 

14 

8 

57.14 

11-20 

58 

28 

48.27 

21-30 

102 

49 

48.03 

31-40 

55 

23 

41.8 

41-50 

7 

5 

71.8 

Total 

300 

165 

55.6 



.2 = 6.79, P-value >0.05 

 

The prevalence rate in all age groups is high, 
but children who were between 1-5 years of 
age had the highest prevalence rate of 
parasitic infections. The high prevalent rate 
could be attributed to poor personal hygiene 
and environmental exposure. Similar rates 
were recorded by (AL-Kubaisy et al., 2014), 
and (Farhan, 2012), which showed that the 


largest number of intestinal parasitic 
infections were found in children aged .5 
years and that infection declined 
progressively with increasing age groups. 
Conversely our results disagree with the 
result of (Raza, 2009) in Sulaimani, 
(Mohamed and Yahya, 1999) in kerkuk who 
recorded a higher prevalence rate of 
parasitic infection in children from 6-9 years 
old. 


The present study found that the Zinc 
sulphate floatation concentration technique 
was more sensitive with49.3% of the cases 
were detected, followed by Formal-Ethyl 
Acetate sedimentation (43.3%) and then 
direct wet mount technique showed the 
lowest detection rate (22.6%). Results of our 
study is in agreement with studies that found 
60% by (Mahmood et al., 2014) in Baghdad 
by the flotation, 58.7% by sedimentation and 
39.3% by direct wet mount. Other studies 
(Raza, 2009) in Sulaimai, (Allan et al., 
1981) in United states-Texas, found that the 
concentration by flotation methods were 
more sensitive in intestinal parasitic 
diagnosis. Flotation technique permits the 
separation of protozoan cysts and certain 
helminthes eggs from excess debris through 
the use of a liquid with a high specific 
gravity, where the parasitic elements are 
recovered from the surface film and the 
debris remains in the bottom of the tube. 
This technique yieldsa cleaner preparation 
and morphology of the parasites than the 
sedimentation procedure (Garcia, 2001), 
(Melvin and Brooks, 1985). A study from 
India by (Parameshwarappa et al., 2012) 
showed that the formol-ether concentration 
technique was more sensitive as compared 
to the other methods and found that 64.5% 
of the cases were detected by the formol-
ether method and 55% by Zinc sulphate 
floatation. This may be due to that 
Sedimentation procedure leads to the 
recovery of all protozoa, eggs and larvae 
present. However, the preparation contains 
more debris than the flotation procedure, 
though Ethyl acetate is used as an extractor 
of debris and fat from the faeces and leaves 
the parasites at the bottom of the suspension. 
Concentration by formalin-ethyl acetate 
sedimentation is recommended because it is 
the easiest to perform, allows recovery of 
the broadest range of organisms and is the 
least subject to technical error (Neimeister et 
al., 1987). In our study the direct wet mount 
showed the lowest sensitivity from the 
concentration methods, but the advantage of 
this method is to provide a quick diagnosis 
of a heavily infected specimen, to check 
organism motility and to diagnose parasites 
that may be lost in concentration techniques 
(Robyn, 1997). 
In conclusion, our results indicate that the 
Zinc sulphate floatation method is more 
sensitive technique from Formal-Ethyl 
Acetate sedimentation and direct wet mount 
but over all, these methods are important for 
diagnosis. The prevalence of parasitic 
infections is high and Entamoeba histolytica 
was the most prevalent species. 
Acknowledgments 
The authors would like to thank all staff in 
Parasitology Laboratory, Department of 
Microbiology, School of Medicine for their 
cooperation. Great thanks to the central 
laboratory in Sulaimani Teaching Hospital 
for their support and help in performing this 
study. 
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