Evaluation of the Therapeutic Effect of Medium Chain Saturated Fatty Acid (Lauric Acid) against Giardia lamblia in Infected

Hamsters
Mona M. Aly2, Maisa A. Shalaby1, Samar S. Attia2, Shaimaa H. El Sayed1, Amira Helmy1, Olfat Hammam1, Soheir S. Mahmoud1, Soad I. Hassan1 Bilhars Research Institute, Imbaba, Giza, Egypt 2Medical Parasitology department, Kasr Al-Ainy Faculty of Medicine, Cairo University, Cairo, Egypt
1Theodor

Introduction.
Giardia duodenalis continues to be one of the most common intestinal parasitic protozoa reported in humans worldwide.

Recently, giardiasis has become a significant public health concern due to its association with multiple waterborne outbreaks. WHO reported that 3 billion people live in developing countries and the rate of giardiasis among them approaches 30%, contributing to 2.5 million deaths annually from diarrheal diseases.

Introduction
There are many problems associated with the currently used chemotherapeutic agents in treatment of giardiasis including treatment failure, unpleasant side effects, activity against normal intestinal flora, possible carcinogenicity and parasite resistance.

These adverse reactions had led to search for other suitable natural agents from different origins which are readily accepted, free of toxicity and may augment or even replace some of the standard chemotherapeutic agents presently employed in the treatment of giardiasis.

Introduction
Lauric acid is a naturally derived fatty acid belonging to the medium chain saturated fatty acids (MCSFAs) which is reported to has anti-giardial effect through accumulation within the parasite cytoplasm resulting in rupture of cell membrane and finally trophozoite death.

The best and richest natural sources of lauric acid are coconuts and coconut oil.

Aim of work

To evaluate the efficacy of lauric acid in treatment of giardiasis. To compare between metronidazole and lauric acid in treatment of giardiasis. To evaluate the combination of metronidazole and lauric acid as a line of treatment of giardiasis hopping to reduce the dose and long duration of treatment with metronidazole to avoid the resulting side effects.

MATERIAL & METHODS

Experimental Animals, Parasite & Drugs

Animals grouping Plan of work Assessment of Anti-giardial Activity

Experimental Animals, Parasite & Drugs

Experimental Animals: Laboratory bred Syrian hamsters with a weight range of 100-110 gm, provided by the Schistosome biological supply program (SBSP) in Theodor Bilharz Research Institute (TBRI). Parasite: Giardia lamblia cysts used for infection of hamsters were obtained from diarrheic patients attending the outpatient clinic of TBRI.
Flagy l Lauric acid

Drugs:

Metronidazole (Flagyl) was given orally in a suspension form in a dose of 7 mg/hamster/day for seven consecutive days

Animal Groups
Group A Group B (Normal ( Drug Control) control) Number of Hamster Infection Treatment Dose ( for 7 consecutive days
10 No No --15 No Yes Differs according to subgroup

Group C (Infected control)

10 Yes No ----

Group D
(Metronida zole treated)
10 Yes Yes

Group E (Lauric Acid treated)

10 Yes Yes

Group F
(Combined treated)

10 Yes Yes

7 mg per 20g per 3.5 mg Hamster/da Hamster/da (Metronidazo y y le + 10gLauric acid)per Hamster/day

Plan of Work
Oral Infection of hamsters with Giardia lamblia cysts in a dose of 10.00010 cysts/hamster. Three weeks later, Stool samples were collected and subjected to parasitological examination to detect Giardia lamblia cysts and to ensure that hamsters have been infected. Hamsters were given the corresponding medication for 7 consecutive days Two weeks later, stool samples were collected and subjected to parasitological examination to count the number of Giardia lamblia cysts per each gram of stool. Scarification of Hamsters duodenal contents were subjected to parasitological examination in order to count the number of Giardia lamblia trophozoites in five successive fields/animal Intestinal sections were dissected & subjected for histopathological & E. M. examination

Assessment of Anti-giardialActivity
Parasitological examination
I. Microscopic examination of stool samples
Direct wet smear

Merthiolate iodine formaldehyde concentration method (MIFC).

II. Examination of the duodenal contents in order to count the number of Giardia lamblia trophozoites in five successive fields/animal

Assessment of Anti-giardial Activity

Histopathological examination: Sections of small intestines of sacrified hamsters were done to detect the histopathological changes that occurred due to giardiasis and to assess the cure rates and the degree of healing of intestinal mucosa after drug administration.

Assessment of Anti-giardial Activity

Electron micrroscopic examination
Sections of small intestines of sacrified hamsters were subjected to TEM for higher and detailed magnification of the detected histopathological changes in the small intestine due to giardiasis.

leika ulta-microtome

Phillips 208S Transmission Electron Microscope

Results

Results

Parasitologica l results

Histopathological results

Electron microscopic results

stool examination

contents of small intestine examination

Parasitological Results

Parasitological results Results of stool examination

98.83%
93.77%

100.00%

Percentage of Reducton

95.00% 90.00% 85.00% 80.00% 75.00% 70.00%

82.03%

metronidazole (D)

lauric acid (E)

lauric acid + metronidazole (F)

Effect of treatment with metronidazole, lauric acid and combination of both drugs on the percentage of cysts reduction in hamsters infected with Giardia lamblia two weeks after administration of drugs.

Parasitological results
Results of examination of the contents of small intestine of hamsters
100.00% 95.00%

95.50%

96.95%

Percentage of Reduction

90.00% 85.00% 80.00% 75.00% 70.00% 65.00% 60.00%

78.76%

metronidazole (D)

lauricacid (E)

lauric acid + metronidazole (F)

Effect of treatment with metronidazole, lauric acid and combination of both drugs on the percentage of reduction in the number of Giardia trophozoites in the contents of the small intestines of infected hamsters two weeks after administration of drugs.

Histopathological Results

Histopathological results
GC

Ulceration of the lining intestinal epithelium (blue arrow) with Giardia trophozoites (red arrow) .

GD

GE

GF

Partial healing of the intestinal villi

Partial healing of villi with presence of few Giardia trophozoites in the intestinal

Complete healing with no Giardia trophozoites in the lumen

Electron microscopic results

Electron microscopic results

GC
Electron micrograph showing destructed intestinal cell projection (red arrow) with marked degeneration of intestinal submucosa (blue arrow) GD

GE

GF

partial repair of the intestinal cell projection &complete repair of the submucosa

complete repair of the partial repair of the intestinal intestinal cell projection and cell projection with submucosa with absence of degenerated trophozoite Giardia trophozoites (black arrow)

Conclusion
It was concluded that lauric acid (monolaurin) or coconut offers an alternative therapy to be used in place of, or concurrently with conventional antimicrobials used for treatment of giardiasis. The combination of lauric acid and metronidazole, the drug most currently used for the treatment of Giardia lamblia, revealed the highest percentage of reduction in cyst count in stool, vegetative forms in the small intestine, marked improvement of the histopathological and ultrastructural changes of the small intestine caused by Giardia lamblia infecton. This study may open fresh avenues for development of natural drug therapy in which food supplementation may augment or even replace some of the standard chemotherapeutic agents currently employed in the treatment of giardiasis and possibly other infectious intestinal diseases.

Acknowledgment
Theodor Bilhars Research Institute, Imbaba, Giza, Egypt. Soad I. Hassan. Maisa A. Shalaby. Soheir S. Mahmoud. Amira M. Helmy. Olfat A. Hammam. Medical Parasitology department, Kasr Al-Ainy Faculty of Medicine, Cairo University, Cairo, Egypt Mona M. Aly Samar S. Attia