THE EGYPTIAN JOURNAL OF MEDICAL SCIENCES VOL. 34-No. 2 – DECEMBER 2013 (ISSN: 1110-0540)

1. Egypt. J. Med. Sci. 34 (2) December 2013: 527-540.

DIAGNOSIS OF ACANTHAMOEBA KERATITIS IN CLINICALLY SUSPECTED

CASES AND ITS CORRELATION WITH SOME RISK FACTORS

By Mohamed Saad Younis

1, Azza Mohamed Salah-Eldin Elhamshary

1, Amina

Ibrahim Abd-Elmaboud1, Nagwa Mostafa El-Sayed

2, Shereen Magdy Kishik

1

Parasitology Departments, Faculty of Medicine- Benha University1

& Research Institute of Ophthalmology2, Egypt

ABSTRACT

This study aimed to detect Acan-

thamoeba infection in different speci-

mens obtained from patients with

keratitis and its correlation with vari-

ous host and risk factors. The study

was carried out on 110 patients who

were clinically suspected to have Acan-

thamoeba keratitis. The patients were

divided into 2 groups according to

contact lens use as 63 contact lens

wearers (CLW) and 47 non contact

lens wearers (NCLW). Obtained sam-

ples, including 110 corneal scrapings,

32 contact lenses, 32 contact lens stor-

age cases and solutions, were sub-

jected to cultivation on non-nutrient

agar overlaid with Escherichia coli,

direct smear and staining methods

using trichrome and Giemsa stains.

The results showed that Acan-

thamoeba infection was detected in 21

(19.1%) of clinically suspected cases;

17 (81%) of them were CLW and the

remaining 4 (19%) positive cases were

NCLW. These results revealed a sig-

nificant association between Acan-

thamoeba infection and wearing of

contact lenses (P <0.05). By examining

32 contact lenses, 32 contact lens stor-

age cases and 32 contact lens solutions,

there were 4(12.5%), 3 (9.4%) , 3

(9.4%) positive samples respectively.

The difference between sources of

sampling and detection of Acan-

thamoeba was statistically highly sig-

nificant (P =0.001). In addition, the

results revealed that the correlation

between host factors (age, sex and resi-

dence) and Acanthamoeba infection

among keratitic patients was statisti-

cally insignificant. The highest Acan-

thamoeba infection occurred in female

keratitic patients aging 20- 30 years

(47.6%) as most of them were CLW.

Regarding risk factors, there was a

significant correlation between Acan-

thamoeba infection and ocular trauma,

history of contact lens use, history of

swimming in swimming pools or ca-

nals (P = 0.02). In conclusion, Acan-

thamoeba keratitis is firmly associated

with the use of improperly sterilized

contact lenses, trauma or washing eyes

with contaminated water. Further

studies will be needed to realize the

actual association between them.

INTRODUCTION

Acanthamoeba keratitis (AK) is a

sight-threatening ocular infection caused

by free living Acanthamoeba species

which are abundant in the natural envi-

528 Younis et al.

ronment; soil, dust, air, natural and

treated water, seawater, domestic tap

water, hospitals and dialysis units, eye-

wash stations, and contact lens cases

(Johnston et al. 2009). Various species

have been implicated in human infec-

tions: Acanthamoeba castellanii (A. cas-

tellanii), A. astronyxis, A. culbertsoni, A.

polyphaga, A. hatchetti, A. rhysodes, A.

lugdunensis, A. palestinensis, A. griffini,

and A. quina (Khan, 2006).

Acanthamoeba has two stages in its

life cycle: a vegetative or trophozoite

stage that reproduces by binary fission

and feeds voraciously on bacteria and

detritus present in the environment, and a

non dividing cyst stage with a double

cyst wall, providing it with a high resis-

tance to unfavoured and adverse environ-

mental conditions, desiccation and disin-

fecting compounds, e.g. chlorine (Scheid

& Schwarzenberger, 2012).

Acanthamoeba keratitis was first

recognized in the mid 1970s. Then, a

dramatic increase in cases was associated

with the increasing use of soft contact

lens (in up to 93% of cases); this is

caused by improper lens handling and

poor hygiene (Zhang et al., 2004). Pa-

tients often present with pain, photopho-

bia, and irritation. By ophthalmic exami-

nation, there are clinical characteristics of

Acanthamoeba keratitis including a stro-

mal ring infiltrate, epithelial haze, and

radial keratoneuritis (Kumar & Lloyd,

2002). Delayed diagnosis has been asso-

ciated with poor visual outcome and

more-severe clinical progression

(Hammersmith, 2006).

The risk factors contributing to

Acanthamoeba keratitis includes swim-

ming especially while wearing contact

lenses, working with soil and rubbing

eyes, water-related activities (splashing

water) especially during or immediately

after contact lens wear, handling contact

lenses without proper hand washing and

using of home-made saline (or even chlo-

rine-based disinfectants) for contact lens

cleaning (Khan, 2006).

Definitive diagnosis requires cul-

ture of corneal scrapes, lenses and lens

cases solutions on non-nutrient agar

overlaid with Escherichia coli, histology,

or identification of Acanthamoeba de-

oxyribonucleic acid by polymerase chain

reaction (Dart et al., 2009).

This study aimed to detect Acan-

thamoeba infection in different speci-

mens obtained from patients with kerati-

tis and its correlation with some host and

risk factors.

MATERIALS AND METHODS

Subjects:

The present study was conducted at

Medical Parasitology Departments of

both Faculty of Medicine, Benha Univer-

sity & Research Institute of Ophthalmol-

ogy (RIO), Egypt during the period from

June 2011 to June 2012. The study was

carried out on 110 patients attending the

outpatient clinic of both Benha Univer-

sity hospitals & RIO who were clinically

suspected to have Acanthamoeba kerati-

tis e.g. suffering from different forms of

keratitis, corneal inflammation with se-

vere ocular pain and photophobia, kerato-

conjunctivitis, resistant corneal ulcers or

corneal abscesses and not responding to

antibacterial or antiviral treatment. The

patients were divided into 2 groups ac-

cording to contact lens use as contact

lens wearers (CLW), 63 cases (55 fe-

males and 8 males, and their age ranged

from 14 to 40 years) and non contact lens

Egypt. J. Med. Sci. 34 (2) 2013

Acanthamoeba 529

wearers (non CLW) 47cases (21 females

and 26 males, and their age ranged from

2 to 58 years). A questionnaire sheet was

prepared for each subject fulfilling the

following data: age, sex, residence and

risk factors including; ocular trauma,

history of contact lens use, history of

swimming in swimming pools or canals

and previous eye surgery.

Samples:

Samples including corneal scrap-

ings from all patients (110), 32contact

lenses, 32 contact lens storage cases and

solutions belonging to symptomatic

CLW were subjected to cultivation, di-

rect examination and staining methods

using trichrome and Giemsa stains.

Direct microscopic examination

(Boggild et al., 2009):

Corneal scrapings were suspended

into vials containing page's saline and

mixed well. With a sterile pipette, 2

drops of each specimen were placed onto

a glass slide, covered by cover slips and

examined by light microscope using x 40

magnification. Identification of Acan-

thamoeba was based on the characteristic

patterns of locomotion, morphologic fea-

tures of the trophozoite and cyst forms.

Trophozoites are characterized by the

presence of fine, tapering, and thorn-like

acanthopodia arising from the surface of

the body. They are uninucleate and the

nucleus has a centrally placed, large,

dense nucleolus (Figure 1a). Cysts are

double-walled. The outer cyst wall

(ectocyst) is wrinkled while the inner

cyst wall (endocyst) varied in shape

(stellate, polygonal, oval or spherical).

Cysts are uninucleate and the nucleus has

a centrally placed dense nucleolus

(Figure 1b) (Schuster, 2002).

For staining: 2 drops of each speci-

men were placed onto a glass slide and

allowed to air dry for 10 min. The slides

were then fixed in methanol and stained

by Giemsa (Ithoi et al., 2011) (Figure 1c)

and modified trichrome stains (Ryan et

al., 1993) (Figure 1d).

Cultivation of the corneal specimens

(Init et al., 2010):

The obtained corneal samples were

inoculated directly onto the plate surface

of non-nutrient agar (NNA) overlaid with

Escherichia coli (E.coli) and incubated at

30ºC for 7 days. The plates were exam-

ined daily by the inverted microscope for

Acanthamoeba growth. A drop of the

fluid overlying the agar surface was

placed on a glass slide, covered with a

cover slip and examined by light micro-

scope using x 40 magnification.

Cultivation of Contact lenses (Johns et

al., 1989):

A small piece of the contact lens

(soft or hard) was cut with a sterile scis-

sors and placed on a glass slide with a

drop of sterile distilled water and covered

with a cover slip, and then it was exam-

ined microscopically for the presence of

adherent Acanthamoeba trophozoites or

cysts. The remaining part of the contact

lens was cultivated on NNA-E.coli , in-

cubated at 30ºC for 7 days and examined

as mentioned in corneal specimens culti-

vation.

Cultivation of lens solutions (Johns et

al., 1989):

Contact lenses cases were shaken

and the solution inside each one was

transferred to a sterile test tube. A sterile

cotton swab was then rubbed against the

inner surface of the container and the

Egypt. J. Med. Sci. 34 (2) 2013

530 Younis et al.

cover. The swab was directly inoculated

onto NNA-E.coli. In addition, the solu-

tion obtained from the container was

centrifuged at 2000 rpm for 10 minutes.

The supernatant was aspirated and the

sediment was inoculated onto the same

NNA-E.coli plate. The same procedure

occurred on contact lens rinsing solution.

All incubated at 30ºC for 7 days and

examined as mentioned in corneal speci-

mens cultivation.

Statistical analysis: The positive find-

ings were expressed as a percentage, and

statistical analysis was conducted using

Z test, Fisher's exact test (FET) and Chi-

square (χ2) by SPSS V17. Probability

(P-value) < 0.05 was considered statisti-

cally significant and P=0.001 was con-

sidered statistically highly significant.

Ethical considerations: An informed

consent was taken from all patients after

explaining the aim of the study to them.

The study was approved by Research

Ethics Committee, Faculty of Medicine,

Benha University, Egypt.

RESULTS

Results are shown in tables (1-6)

and figure (1).

Table (1): Percentage of Acanthamoeba infection among contact lens wearers (CLW) and

non contact lens wearers (NCLW).

Acanthamoeba

Studied groups

Total

χ2

P- value NCLW CLW

№ % № % № %

Positive 4 19 17 81 21 19.1 5.947 < 0.05

Significant Negative 43 48.3 46 51.7 89 80.9

Total 47 42.7 63 57.3 110 100

Table (2): Results of detection of Acanthamoeba in different samples of contact lens

wearers (CLW).

CLW

Acanthamoeba Total Z -test P- value

+ve -ve

№ % № % № %

Corneal scrapings 17 27.0 46 73.0 63 100 4.12 0.001 HS

Contact lenses 4 12.5 28 87.5 32 100 6.41 0.001 HS

Contact lens storage

cases

3 9.4 29 90.6 32 100 7.88 0.001 HS

Lens solution 3 9.4 29 90.6 32 100 7.88 0.001 HS

HS: highly significant

Egypt. J. Med. Sci. 34 (2) 2013

Acanthamoeba 531

Table (3): Correlation between age distribution and the presence of Acanthamoeba infec-

tion among keratitic patients.

Age

Acanthamoeba Total FET P- value

+ve -ve

№ % № % № %

<20y 2 9.5 27 30.3 29 26.4 5.72 0.126

NS 20- 10 47.6 24 27.0 34 30.9

30- 6 28.6 20 22.5 26 23.6

≥40 3 14.3 18 20.2 21 19.1

Total 21 100 89 100 110 100

NS : Not significant

Table (4): Correlation between sex distribution and Acanthamoeba infection among

keratitic patients.

Sex

Acanthamoeba Total FET P-value

+ve -ve

№ % № % № %

Male 4 19.1 30 33.7 34 30.9 1.09 0.30

NS Female 17 80.9 59 66.3 76 69.1

Total 21 100 89 100 110 100

NS : Not significant

Table (5): Correlation between residence and Acanthamoeba infection among keratitic

patients.

Residence

Acanthamoeba Total χ2 P-value

+ve -ve

№ % № % № %

Urban 14 66.7% 45 50.6% 59 53.6 1.77 0.18

NS Rural 7 33.3% 44 49.4% 51 46.4

Total 21 100 89 100 110 100

NS : Not significant

Egypt. J. Med. Sci. 34 (2) 2013

532 Younis et al.

Table (6): Correlation between risk factors and the presence of Acanthamoeba infection

among keratitic patients.

Risk factors

Acanthamoeba Total χ2 P- value

+ve -ve

№ % № % № %

Contact lens 17 81.0 46 51.7 63 57.3 11.51 0.0214

Significant

Abrasion 1 4.76 0 0.0 1 0.9

Foreign body 1 4.76 14 15.7 15 13.6

Trauma 1 4.76 12 13.5 13 11.8

History of

swimming

1 4.76 17 19.1 18 16.4

Total 21 100 89 100 110 100

Figure (1): A) Trophozoites of Acanthamoeba spp. (wet preparation). B) Cysts of Acan-

thamoeba spp. (wet preparation). C) Cysts of Acanthamoeba spp. stained by trichrome

stain. D) Cysts of Acanthamoeba spp. stained by Giemsa stain (×40).

Egypt. J. Med. Sci. 34 (2) 2013

Acanthamoeba 533

DISCUSSION

The potential presence of AK is

most commonly recognized by the pres-

entation of free-living amoebae, by di-

rect observation of clinical specimens

under microscopy and culture from

amoebae inoculation on non-nutrient

agar (Lek-Uthai et al., 2009). However,

culture is the mainly used technique for

detecting Acanthamoeba in contact

lenses, corneal scrapes and lens solution

due to its low cost and simplicity

(Niyyati et al., 2009). It usually needs a

long incubation time (Lorenzo-Morales

et al., 2007) and may produce false nega-

tive results due to low parasitic load,

small sample volumes and the use of

antiseptic or antibiotics prior to sampling

(Petry et al., 2006). Additionally culture

of lenses may lead to false positive re-

sults as a result of lens case contamina-

tion with Acanthamoeba (Yera et al.,

2006).

Traditional direct smear analysis is

highly specific diagnostic method; it is

limited by high false-negativity rates for

the requirement for technical expertise

(Boggild et al., 2009). Although an ex-

perienced microscopist can occasionally

identify certain protozoa in a wet prepa-

ration both stained and unstained, most

identification should be considered ten-

tative until confirmed by the permanent

stained slide. Permanent stained smears

are reported to have many color ranges

that aided identification of organisms

within the samples (Pietrzak-Johnston et

al., 2000). So, in this study trichrome

and Giemsa stains were used to enhance

the visibility of Acanthamoeba. Giemsa

stain has been used successfully to detect

cysts of Acanthamoeba. Cysts stained by

Giemsa are clear, bright with polyhedric

or stellate shaped cysts, while tropho-

zoites, with the central nucleolus and

vacuoles, are more difficult to detect,

since they can resemble inflammatory

cells (Hammersmith, 2006). In addition,

Giemsa stain does not require time-

consuming techniques or special equip-

ment for evaluation (Grossniklaus et al.,

2003). In spite of modified trichrome

stain proved to be the best stain for iden-

tifying Acanthamoeba cysts (Mubareka

et al., 2006). It is costly, time consuming

and not suitable for survey study.

The results of the present study

showed that Acanthamoeba infection

was detected in 21 (19.1%) of clinically

suspected cases (110); 17 (81%) of them

were CLW and the remaining 4 (19%)

positive cases were NCLW. These re-

sults revealed a significant association

between Acanthamoeba keratitis and

wearing of contact lenses (P <0.05)

which appears to be an important risk

factor in infection. Our findings are in

agreement with Gupta and Aher (2009)

and Ibrahim et al., (2009) who reported

that wearing of contact lens was associ-

ated with 75% to 95% cases of AK.

Also, Wanachiwanawin et al., (2012)

diagnosed AK in 37.5% of NCLW and

in 62.5% of CLW. It is widely believed

that contact lenses serve as vectors for

transmitting infectious Acanthamoeba

trophozoites to the eye. Contact lenses

can stimulate the expression of glycopro-

teins on the corneal epithelium. This in

turn might exacerbate the infectious

process, as mannosylated proteins pro-

mote the binding of Acanthamoeba tro-

phozoites to the corneal epithelium via a

mannose-binding protein (mannose re-

ceptor) that is expressed on the Acan-

thamoeba cell membrane (Clarke, &

Niederkorn, 2006).

Egypt. J. Med. Sci. 34 (2) 2013

534 Younis et al.

Regarding to different samples

obtained from CLW, 32 contact lenses,

32 contact lens storage cases and 32 con-

tact lens solutions, there were 4(12.5%),

3 (9.4%), 3 (9.4%) positive samples for

Acanthamoeba respectively. The differ-

ence between sources of sampling and

detection of Acanthamoeba was statisti-

cally highly significant (P =0.001). The

presence of Acanthamoeba contamina-

tion of contact lens and lens storage

cases may be attributed to improper

ways of cleaning them; rinsing with tap

water (Jeong et al., 2007) most espe-

cially if the source were storage tanks

which promote growth of microorgan-

isms (Jeong and Yu, 2005), the use of

ineffective amoebicidal disinfectants,

i.e., lens care solutions and chlorine tab-

lets (Tzanetou et al., 2006), not rubbing

lenses while cleaning and reusing lens

care solutions (Joslin et al., 2007) or

even simply leaving the lens storage

cases wet (Seal et al., 1999) as well as

taking a shower or swimming while

wearing contact lenses. However, detec-

tion of Acanthamoebae from contact

lenses, solutions, or casings does not

strictly confirm the diagnosis; it is virtu-

ally diagnostic in the setting of a com-

patible clinical history (Marciano-Cabral

and Cabral, 2003).

In the current study, the correlation

between age distribution and Acan-

thamoeba infection among keratitic pa-

tients was statistically insignificant (P

=0.126). Out of 21 total positive cases,

there were 2 positive cases (9.5%) < 20

years, 10 positive cases (47.6%) between

20- 30 years, 6 positive cases (28.6%)

between 30- 40 years and 3 positive

cases (14.3%) >40 years. The highest

Acanthamoeba infection occurred in

keratitic patients aging 20- 30 years

(47.6%). This result agrees with Joslin et

al., (2006) who detected 40 AK cases,

their average age was 28 years. Also,

Bharathi et al., (2009) found that AK

was higher in the younger age group

(Mean, 20.3 years old). This may be

explained by Jansen et al. (2012) who

detected that wearers 18-25 years more

likely to replace soft contact lenses

(SCLs) only when there was a problem

with them and sleeping overnight while

wearing SCLs. Also, the authors re-

ported that this age group was signifi-

cantly lower rates of regular hand wash-

ing on lens insertion and removal and

significantly higher rates of ever sharing

contact lens solution. On the other hand,

Sharma et al., (2000) demonstrated that

the mean age of AK patients was 36.8

years. Also, Pacella et al., (2012) de-

tected AK at the age group ranged from

30 to 51 years old.

In this study, it was found that the

correlation between sex distribution and

Acanthamoeba infection among keratitic

patients was statistically insignificant (P

=0.30). The highest Acanthamoeba in-

fection observed in females as most of

them were CLW. Contact lens wearing is

gaining popularity worldwide, especially

with women (Rezaeian et al., 2007). This

agrees with Wanachiwanawin et al.,

(2012) who mentioned that 68.2% of AK

patients were females. On the other

hand, Joslin et al., (2006), Pacella et al.,

(2012) and Sharma et al., (2000) de-

tected that AK more frequent in males

than females. However, the exact reason

behind male prevalence in their studies

is not known.

In addition, the results of the pre-

sent study revealed no significant corre-

lation between Acanthamoeba infection

Egypt. J. Med. Sci. 34 (2) 2013

Acanthamoeba 535

among keratitic patients and residence of

these patients. Also, many investigators

though that Acanthamoeba keratitis is a

growing clinical problem in developed

as well as developing countries (Gupta

and Aher, 2009). In developed countries,

the single most important risk factor is

wearing of contact lens (Bharathi et al.,

2009). While, in developing countries

besides contact lens wearing, fall of dust

particles, trauma due to vegetable matter,

contact with contaminated water have

been found to be predominant risk fac-

tors of AK (Manikandan et al., 2004).

In this study, the most important

risk factor of Acanthamoeba infection in

keratitis patients (21) was contact lens

wearing (17), and risk factors among the

remaining 4 cases were abrasion, foreign

body, trauma, and history of swimming

in swimming canals. The firmly associa-

tion between AK and contact lens wear

may be attributed to several reasons.

Manipulation of the contact lens may

result in epithelial breaks that become

infected. Contact lenses cause chronic

hypoxic stress on the corneal epithelium

which leads to decreased corneal sensi-

tivity, decreased epithelial mitosis and

adhesion, premature desquamation of

epithelial cells, increased epithelial fra-

gility, epithelial microcystic oedema and

significant thinning of the epithelial cell

layer (Kolkailah et al., 1999). The main

finding that contact lens wear is the most

important risk factor for Acanthamoeba

keratitis was also observed by Rezaeian

et al., (2007) and Ibrahim et al., (2009).

This is because the parasite grows in

contact lens cases, contaminated cleaning

solution, and on the lens itself (Wynter-

Allison et al., 2005). In contrast, Sharma

et al., (2000) reported that contact lens

wear does not emerge as an important

risk factor for Acanthamoeba keratitis in

their population; this can probably be

attributed to the relatively few people

who are exposed to contact lens wear.

Regarding the other risk factors,

Rezaeian et al., (2007) found that most

amoebic keratitis in older individuals is a

result of trauma or eye surgery. In addi-

tion, Manikandan et al. (2004) reported

that corneal injury or fall of foreign body

from various sources, history of swim-

ming in swimming pools or canals

(Pacella et al., 2012) and the improper

uses of orthokeratology (Xuguang et al.,

2003) were the predisposing factors of

Acanthamoeba keratitis among the non

contact lens users.

In conclusion, Acanthamoeba

keratitis is firmly associated with the use

of improperly sterilized contact lenses,

trauma or washing eyes with contami-

nated water. Further studies will be

needed to realize the actual association

between them.

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الملخص العربي

وعالقته مع بعض عىامل الخطر بها إكلينيكيا في الحاالت المشتبه ألكنثاميبيتشخيص التهاب القرنية ا

دمحم سؼذ ٠س 1

ػزج دمحم صالح اذ٠ اشش- 1

أ١ح إتشا١ ػثذ اؼثد - 1

-

ج صطف اس١ذ2

ش١ش٠ جذ وشه & 1

جاؼح تا – و١ح اطة – لس اطف١١اخ1

ؼذ تحز أشاض اؼ١ & 2

اذف ز اذساسح اىشف ػ ػذ األوثا١ثا ف اؼ١اخ ار ذ احصي ػ١ا

اشظ از٠ ٠ؼا اراب امش١ح ػاللر غ اؼا اخاصح تاؼائ اع١ف ػا

.اخطشاخرفح

از٠ ٠ؼا أشىاي خرفح اشرث ف١ ش٠ط 110 لذ أجش٠د ز اذساسح ػ

ذ ذمس١ احاالخ إ جػر١ سئ١س١ر١ فما إلسرخذا اؼذساخ االصمح ػ اراب امش١ح

47شذذ اؼذساخ االصمح ػذد غ١ش . حاح63 ػذد شذذ اؼذساخ االصمح: اح ارا

32 ػة حفع اؼذساخ 32 اؼذساخ االصمح، 32 ػ١ح امش١ح، 110 ذ أخز .حاح

حي اؼذساخ

فحص اؼ١اخخعؼد ز اؼ١اخ زسع ػ أجاسغ١ش غز زسع ػ١ ا وال ،

. صثغا تصثغح ارش٠ىش اج١ساثاششج تؼذ

17 احاالخ اشرث تا إو١١ى١ا صاتح تاألوثا١ثا (٪19.1 )21لذ أظشخ ارائج

وشفد ز .شذذ اؼذساخ االصمحغ١ش (٪19 )4 شذذ اؼذساخ االصمح (81٪)

. (P <0.05)ارائج ػ جد إسذثاغ إحصائ ت١ ػذ األوثا١ثا اسذذاء اؼذساخ االصمح

حي اؼذساخ32ػثح حفع اؼذساخ 32 اؼذساخ االصمح 32ػ غش٠ك فحص

افشق ت١ صادس . ػ١اخ إ٠جات١ح ػ ارا (٪9.4 )3، (٪9.4 )3، (٪12.5 )4وا ان

وا اثثرد ارائج . P=0.001) )أخز اؼ١اخ اىشف ػ األوثا١ثا وا راخ دالح إحصائ١ح ػا١ح

Egypt. J. Med. Sci. 34 (2) 2013

540 Younis et al.

(اؼش اجس ىا اإللاح)أ ال ٠جذ إسذثاغ إحصائ ت١ اؼا اخاصح تاؼائ اع١ف

وا األوثش ػذ تاألوثا١ثا اإلاز ار . اؼذ تاألوثا١ثا ت١ شظ إراب امش١ح

ف١ا ٠رؼك تؼا . ػاا ؼظ شذذ اؼذساخ االصمح30 - 20ذرشاح أػاس ت١

اخطش وا ان إسذثاغ وث١ش ت١ ػذ األوثا١ثا اسذذاءاؼذساخ االصمح وذاخ اؼ١

.(P = 0.02)اسثاحح ف اثشن أ اماخ

ا ٠جذ اسذثاغ ث١ك ت١ اراب امش١ح تاالوثا١ثا اسرخذا ٠رث١ ارائج اساتمح

سف ذى ان حاجح إ ز٠ذ اذساساخ رحمك افؼ زا . اؼذساخ االصمح

.االسذثاغ

540-527 : 2013د٠سثش (2 )34اجح اصش٠ح ؼ اطث١ح .1

Egypt. J. Med. Sci. 34 (2) 2013