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EXPERIMENTAL STUDY ON THE USE OF

COBALT 60 IRRADIATION IN THE

INACTIVATION OF GIARDIA LAMBLIA CYSTS

Soad A Elrifaey1,Eman Y Shoeib1,Amira H Elnamaky2,Reta

M Kelada3Department of Parasitology,Faculty of Medicine,Cairo University1&Department of Parasitology and Animal Diseases,National ResearchCentre2 and Department of Parasitology, National HepatologyandTropical Medicine Research Institute3


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Presented ByEman Yassien ShoeibLecturer of Parasitology

Faculty of Medicine ,Cairo University


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Water pollution is a major problem worldwide.

It is one of the leading causes of deaths and diseases.

There has been a sharp increase in the number ofwaterborne outbreaks caused by ingestion of someintestinal protozoa.

Giardiasis has been one of the most frequently

occurring waterborne diseases in both developed anddeveloping countries.


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Waterborne outbreaks of giardiasis have beenreported all over the world; their studies have usuallyshown defective water treatment plans.

A need for low-maintenance, cost-effective treatment

for these systems has been recognized, andalternative technologies are being investigated.

Different types of radiation as ultraviolet radiation,

ozone and gamma rays are being considered aspotential alternatives to chlorine for disinfection insmall water systems.


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Aim of work:Experimental study to evaluate the effect of

cobalt 60 on the viability and infectivity of

Giardialamblia cysts for possible future use indisinfection purposes.


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Introduction:G.lamblia (syn. Giardia intestinalis, Giardia

duodenalis) is a flagellated unicellular eukaryoticmicroorganism that commonly causes diarrheal

disease throughout the world.G.lamblia is worldwide in distribution and is

especially common in areas with poor sanitaryconditions and insufficient water treatmentfacilities .It is the most common intestinal protozoan

causing diarrhea in children.


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Epidemiology:Giardiasis is the most common protozoal infection

of the intestine and occurs worldwide, from the

tropics to the arcticSeveral Giardia epidemics have been reported and

often frequentely manifested by diarrhea in man aswell as in animals

Giardiasis has an estimated prevelance of 280million cases annually & contributes to the 2.5million annual deaths from diarrheal disease


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Giardiasis:Effects ofGiardia infection range from the

asymptomatic to a severe malabsorption syndrome

Factors contributing to the variation in clinical

manifestations include: the virulence of the Giardia strain,

the number of cysts ingested,

the age of the host,

the state of the host immune system at the time ofinfection

Symptoms are nonspecific and resemble those of anumber of other gastrointestinal ailments.


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Life cycle


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After excystation, the trophozoites released in theupper part of the small intestine use their flagella andventral disc to move to the microvilli-covered surface ofthe duodenum and jejunum where they attach

themselves and play a role in the onset of thepathology.

The histopathological changes occurring range fromminimal to severe enough to cause enteropathy with

enterocyte damage, villus atrophy, and crypthyperplasia.


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Laboratory diagnosis of giardiasis:Microscopic examination of stool samples (gold

standard method).

The sensitivity of this method is low ,a series of even5or 6 consecutive stool specimens may not show anyparasite.

A minimum of 3 specimens on 3 consecutive days areusually examined to obtain an acceptable sensitivity.


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Concentration techniques can be performed&

Also Parasep, faecal parasite concentrator can be used.

Direct examination of small intestinal content may beuseful & can be done by:

Endoscopies with duodenal aspiration or,

Duodenal biopsy

The immunodiagnosis of giardiasis has received muchattention in the recent past.


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The introduction ofGiardia stool antigen tests has

improved giardiasis diagnostic capabilities and has shownto be more sensitive and more specific (>90%) than thetraditional microscopic examinations

Antigen tests is also rapid,simple to perform and can becombined with antigen testing for diagnosingCryptosporidium, making the antigen test more cost-effective.

The presence of antigen indicates active infection.It isproduced as G.lamblia multiplies in the intestine.Theantigen is stable and can be detected in fresh faeces.


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Prevention:Current efforts are largely directed for prevention and

control of giardiasis.

Free chlorine alone is unable to achieve desiredmicrobial inactivation levels without formingdisinfection byproducts.

Irradiation has been used as an alternative tochlorination for water treatment as it is more costeffective and need low maintenance.


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Cobalt 60: Type of Gamma irradiation, use high energy rays that

can penetrate deeply, making it possible to treat bulkfoods

These rays directlydamage the DNAof livingorganisms, making the organism unable to grow or

reproduce. They also interact with water molecules inthe organism generating free radicals that causeadditional damage to DNA.


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Treated food does not become radioactive, and the

sensory quality of foods is not significantly altered.

Irradiated food have proven to be safe in large scale trialsin animals and human volunteers;No ill effects were

observed, and, in particular, no teratogenic effects.

The actual dose required totreat food varries with thespecific pathogen and the specific circumstances of the

food.A higher radiation dose is required to inactivate thesame number of organisms in frozen food than it does tokill them in refrigerated food.


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Subjects and methods: 1- Sample collection:

Human stool samples positive for Giardia cysts wereobtained from NHTMRI.

2-Preservation and purification of protozoa:

Samples were centrifuged for 3 min at 5000rpm and thesediment was taken and mixed well with 50 mlphysiological saline ,then centrifuged again and the pelletcontaining the cysts were stored in distilled water at 4C.

3-Irradiation of the sample:

Half of the stored sample was irradiated by radioactivecobalt(Co-60) in a dose of 0.25KGy in NEMROCK and wasgiven to the experimental group while the other half wasntirradiated and was given to the control group.


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Experimental animals

100BALB/c mice(4-6 weeks old, male, 40-

50g)

Group A(CONTROL GROUP)Inoculated with non-irradiated G.lamblia

cysts

50 mice

Group B(EXPERIMENTAL

GROUP)Inoculated with

irradiated G.lambliacysts

50 mice

50 mice

Group C(RADIATION CONTROL

GROUP)Inoculated withirradiated water

10 mice

Group D(CONTROL NON

INFECTED GROUP)Didnt receive anything

10 mice

4-experimental animals:120 mice were divided into 4 groups and were orallyinoculated:


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Stool pellets from mice groups were examined from day 9post inoculation by 3 different methods:

Simple sedimentation Mini Parasep SF Rapid copro-antigen.


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Mini Parasep SF: Giardia strip test:


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5- Histopathologicalexamination:

All mice were sacrificed on the 28th

day and theirintestines were examined after staining with H&E.

6- statistical analysis of the results:

It was done using Chi square (2) test.All statistical calculations, data management and analysis

were performed using computer programs MicrosoftExcel version 7 and SPSS version 17.


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Results:

simple

parasep

antigens

dayday

dayday

day

simple

sedimentation

parasep

antigenspositive cases

among Group Aby differentdiagnosticmethods usedduring the study


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Technique

Time in

Days

% of positive cases

detected by the

simple

sedimentation

% of positive cases

detected by the

parasep device

% of positive cases

detected by the

copro- antigen

Group A Group B Group A Group B Group A Group B

Day 9 0% 0% 0% 0% 95.5% 0%

Day 10 0% 0% 37.7% 0% 95.5% 0%

Day 11 24.4% 0% 75.5% 0% 95.5% 0%

Day 12 73.3% 0% 82.2% 0% 95.5% 0%

Day 28 75.5% 0% 86.6% 0% 95.5% 0%


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section in the duodenum of a control normal mouse free of infection showingnormal intestine histology. (x100)

Histopathology:


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section in the duodenum of a mouse infected with irradiated G. lamblia

showing preserved architecture


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section in the duodenum of a mouse infected with non-irradiated G. lambliashowing trophozoites, oedema and shortening of villi. (x400)


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Intestinal giardiasis asdetected by H&E stain, Oil

immersion 1000.


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section in the intestine of a mouse inoculated with irradiated water showing

oedematous villi (upper arrow) and few non specific inflammatory cells (lower

arrow). (x 400)


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Findings in %

Group of mice infected

Group A Group B Group C

1. Villous changes

Shortening of villi

Epithelial erosions

2. Stromal changes

Edema

Vascular congestion

3.Submucosal layer

Vascular congestion

Cell infiltration

4-Presence of trophozoites

84.4%

73.3%

95.5%

95.5%

95.5%

95.5%

66.6%

Zero %

Zero%

60.4%

58.3%

60.4%

60.4%

Zero%

Zero%

Zero%

30%

zero%

zero%

40%

Zero%


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The present study is an experimental study conductedin Egypt that studies the ability of Cobalt-60 toinactivate G. lamblia cysts.

None of group B acquired infection with G. lamblia.

The irradiated cysts showed no morphologicaldifference from the non irradiated cysts.

These results go in agreement with Lenaghan andSundermann (2008) and Sundermann andEstridge (2010).


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Moreover, the results of the present study indicate

effectiveness of low dose Cobalt-60 in a dose lowerthan reported byLenaghan and Sundermann (2003)who used a dose of 5KGy to inactivate thetrophozoites.


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The laboratory diagnosis of irradiated G. lamblia cysts

was made using the simple sedimentation, MiniParasep SF and the rapid copro-antigen.

The most sensitive one was the copro- antigen ( 43

positive cases) followed by the Mini Parasep SF (39positive cases) and the least sensitive was the simplesedimentation method (34 positive cases).


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The immune-chromatographic assay beside being the most

sensitive method has the advantage of being rapid, easy,

doesn't require a medical expert and allows simultaneousdiagnosis of giardiasis in one step.

These results go in agreement with Minaketal. (2012) ,Selim etal. (2009) , Nagatyetal. (2007),Weitzeletal.

(2006) and Schunketal. (2001).

Parasep beside being more sensitive than the simplesedimentation is healthy, safe, easy to perform andfacilitates research work and field study where equipped

laboratory is not available.

This go in agreement with: Zeeshanetal. (2011), Lieretal.(2009), Levecke (2009) and NourElHodaetal. (2003)


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These results go in agreement withArevaloetal.(2010) , Kootetal. (2009) and Scottetal. (2000).

However, we disagree with Huh etal. (2009) andOberhuber etal. (1997)who reported an entirelynormal light microscopic appearance of the duodenalmucosa.

pathology

Group A66.6%

trophozoites

73.3% epithelialerosions

84.4% villouschanges

Group B

NO trophozoites

NO superficialerosions

NO villouschanges


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The present study highlights the effectiveness of lowdose radioactive cobalt -60 in prevention of G. lamblia

infection and indicates that Cobalt-60 can be used as acontrol measure to prevent infectivity and as a meansof water treatment.

We used three different diagnostic methods:

microscopic examination using the simplesedimentation technique, the Mini Parasep SF (fecalparasite concentrator) and the rapid Copro- Antigendetection kit (RIDAQUICK).

By comparing the results of the three methods it wasfound that the copro antigen gave the highestsensitivity followed by the Parasep and the leastsensitive was the microscopic examination using thesimple sedimentation technique.


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Therefore we can recommend the following:

Performing more studies to compare this newtechnology with other conventional methods used tocontrol G. lamblia and also to compare the costeffectiveness of this type of radiation and other typesof radiation as ultraviolet radiation.

The use of the Parasep on routine work as it is morecost benificial than repeated microscopical stoolexamination with the conventional methods.

The use of the Copro- antigen with cases clinicallysuspected of having giardiasis especially with repeatednegative stool examinations.


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