Synergistic activity of azithromycin combined with metronidazole against toxoplasmosis in experimental mice

Objective: To evaluate the efficacy of synergistic activity of azithromycin combined with metronidazole against toxoplasmosis experimentally induced in murine model. Material & Methods: Albino mice model (balb/c mice) acutly infected with tachyzoites, were treated for ١٠days from day +١ postinfection with administration of azithromycin combined with metronidazol. Results: The results showed that prolonged survival of albino mice relative to that of untreated control, and the dead mice were found only in the control group. This experiment showed that a remarkable synergistic effect on toxoplasmosis was obsereved when azithromycin was adminstratered in combination with metronidazole at cuurative dosage i.e.(٢٥٠ and ٢٠٠ mg/kg/day), respectively. These results showed that azithromycin, which is remarkably active on toxoplasmosis, significantly potentiates the curative effect of metronidazol in the treatment. The treated mice in uncontrolled group were completely cured from toxoplasmosis as their brain were free of T. gondii cyst either microscopically or by bioassay. Conclusion: Combined drugs of azithromycin and metronidazole showed a cure rate of ١٠٠% in experimental murine toxoplasmosis.

ondigi is the one of the most common protozoan of mammals, birds and considered as an important human pathogens which is typically a brain infection and can also affect the eye and other organs.Only the cats play a major role in transmission of the disease because they harbor the reproductive forms of the parasite.Treatment of toxoplasmosis has not changed essentially during the past few decades, consist of various combination of T antibiotic drugs; the most common agents used are pyrimethamine, clindamycin, and the sulphonamides.These drugs are some times given in combination with corticosteroids to alleviate the inflammatory reaction especially in treatment of ocular toxoplasmosis which is responsible for the tragic loss of eye sight in this parasitic disease.٥ In addition, the combination of these drugs in the standard therapy regimen has been successful in most patients but may cause bone marrow suppression, hematological toxicity and or life threatening allergic reactions.٦ The relatively results in a lowering of the dosage or discontinuation of one or both drugs in the combination therapy predisposing to failure of treatment.٧ Thus the purpose of this study is to investigate alternative drugs with lower level of toxicity and increase potencies to treat toxoplasmosis.As far as we know the present was the first to evaluate the efficacy of the combination of azithromycin and metronidazole in the treatment of experimental toxoplasmosis.

Collection of samples
T.gondii was originally isolated from sixty suspected infected placentae taken from aborted and sixty normal delivery women attending Al-Batol, Al-Khansah and Al-Salam Teaching Hospitals in Mosul.All samples of placentae were examined microscopically by direct methods, and biopsy was done in normal (uninfected) mice to confirm the diagnosis.٨ The infected one processed according to the method described by previous workers.٩ From each placenta ٥٠ g were taken and cut into small pieces mixed with equal volume of normal saline and grinded with mortar and a pestle or homogenized by electric homogenizer.The suspension was filtered through cotton to avoid large particles .
One thousand units of penicillin and ١٠٠ mg of streptomycin were added to prevent contamination.١٠ ١.٠ ml of the filtered solution inoculated through intraperitoneal passage in laboratory bred, healthy Swiss albino mice (٨-١٠ weeks old) to maintain the infection of intraperitonial fluid with T.gondii.These infected mice were sacrificed within ٣-٤ days after inoculation.They were regularly inoculated intraperitonealy throughout the experiment by means of sterile tuberculine syringe, delivering ٠.١-٠.٢ml of fresh peritoneal exudate taken from a mouse inoculated intraperitonealy ٣ days before.١١  The exudates contained approximately ٥٠ organisms/١ml.Dilution of heavily infected exudate was done with sterile normal saline solution.١٢ The experimental white albino mice (Balb/c strain) ٨-١٠ weeks old of both sexes were used, and these mice were examined to be sure that they were not infected with toxoplasmosis by using serological tests Latex agglutination & ELISA.١٣ Induction of toxoplasmosis in mice: For evaluation to get infection orally or intraperitonally equal doses of tissue cysts (١ml.)contain ٥٠ cysts were given to ٢٠ mice.These mice were divided into two groups.The first group of mice were given orally, and in the second group, cysts were given by inoculation intraperitonally.The peritoneum exudates were examined for each group ٣-٦ days after inoculation for diagnosis of T. gondii.One month after inoculation the mice were sacrificed, their brains were examined to find the tissue cysts.The inoculation was also done in another ٢٠ mice by using equal doses (١ml) contain ٥٠ tachyzoites inoculated orally and intraperitonally.The mice were under observation for diagnosis of the parasite as mentioned before.Combined antimicrobial chemotherapy: ١.Azithromycin.This drug is an antibiotic belongs to the macrolide group, Its action is to bind to the ٥٠S subunit of bacterial ribosomes, blocking protein synthesis, breterostatic.١٤ It was also used against parasitic infection like cryptosporidiosis and angiomatosis, also has potent activity against chronic experimental toxoplasmosis.١٥ ٢.Metronidazole.(benzoyl metronidazol) It has fungicidal activity and also it has a direct action on parasitic infection like trichmoniasis, giardiasis cryptosporidiosis and amoebiasis .It is thought to enter the cells of microorganisms that contain nitroreductase.Unstable compounds are then formed that bind to DNA and inhibit its synthesis, causing cell death.١٦  The doses of combined drugs used in this experiment were as follows: Azithromycin (٢٥٠ mg/kg) + Metronidazole (٢٠٠mg).
Ten infected mice by intraperitoneal cysts were treated with metronidazole and azithromycin which was given before.The combined treatment was given ٢٤ hours after inoculation and continued for ١٤ days post-infection.The mice were under observation for ٣٠ days, and the experiment was terminated after ٤٥ days post-infection.Other١٠ mice was not given anytreatmentand served as control.At the end of the experiment all mice were sacrificed.Microscopical examination and bioassay were done for brain of mice, and the living mice were evaluated for efficacy of drugs used.Data were analyzed statistically using Ztest between two proportions to compare between methods and drugs.The results were considered significant at (P<٠.٠٥).Numbers, percentages were also calculated.

Synergistic activity of azithromycin and metronidazole
All ten mice were found dead in the control group.These mice had not been given any treatment from the ٧th day till the ١٤th day after inoculation with T. gondii.They showed signs and symptoms of illness (weakness, piloerction, ascitis of abdomen) which were not seen in treated group.After three days from the beginning of treatment the mice became active and their activity were normal in the period of treatment (١٤ days) then the treatment was stopped for all mice.The observation of these mice was continued for the period of ٣٠ days.The results revealed that the dead mice were found only in controlled group, but those mice which treated with azithromycin plus metronidazole were still living.This experiments showed that the synergistic activity of azithromycin and metronidazole had the ability to protect mice from acute toxoplasmosis, and there was a significant difference (P<٠.٠١) in the treated group in comparison with the control group (Table ١).

Discussion
Metronidazole is an antibiotic approved as a fungicidal ,and act against T. vaginalis, E.histolytica and works at both intestinal and extraintestinal amoebiasis, and this antiprotozoal drug enter into the microorganisms and bind to DNA and inhibited synthesis leading to cell death ١٧ , azithromycin, a similar drug, have been approved as antitoxoplasmosis,and nontoxic, and azithromycin cannot prevent recurrent disease ١٨ , so they might be useful in treating toxoplasmosis, and to bring attention to these treatment possibilities, so that others can investigate further and organize clinical trails to determine whether these drugs are valuable for treatment of toxoplasmosis and the possibility of preventing recurrence of ocular toxoplasmosis and for treating AIDS-related infections.
The results showed that all ١٠ mice in the control group died due to ascites.The parasites were detected in the peritoneal cavity and in the brain of dead mice.The result showed that all mice were alive in group which had been treated with azithromycin combined with metronidazole (Table ١).Others they showed that the combination of azithromycine with pyrimethamine resulted in a greater inhibition of Toxoplasma protein synthesis compared to azithromycin alone in vitro assay using mouse macrophages challenged with T. gondii tachyzoites ١٩ thus,our results showed that using azithromycin and metronidazole in experimental infection in mice with T. gondii is considered as fruitful work which gave a good result in the treatment.This study showed that the combined use of the drugs yielded better results in terms of cure rates.So these results indicates that evaluation of the efficacy of azithromycin and metronidazole, preferably by means of randomized studies is needed to determine the future role of these and other drugs in the treatment of toxoplasmosis and the possibility of preventing recurrence of ocular toxoplasmosis and for treating AIDSrelated infections.