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Efficacy of azithromycin in a murine toxoplasmosis model, employing a Toxoplasma gondii strain from Turkey
Acta Tropica 88 (2003) 45 /50 www.elsevier.com/locate/actatropica
Efficacy of azithromycin in a murine toxoplasmosis model, employing a Toxoplasma gondii strain from Turkey ¨ zgu¨r Kurt b, A. Taylan Tamay b, Kenan Deg˘erli a, Ali A. Kilimciog˘lu b, O ¨ zbilgin b,* Ahmet O a
Department of Microbiology and Clinical Microbiology, School of Medicine, Celal Bayar University, Manisa 45010, Turkey b Department of Parasitology, School of Medicine, Celal Bayar University, Manisa 45010, Turkey Received 9 August 2002; received in revised form 28 February 2003; accepted 15 May 2003
Abstract A murine toxoplasmosis model with Balb/C mice was used to investigate the therapeutic and prophylactic efficacy of azithromycin in a native strain of Toxoplasma gondii . Initially, seven groups */four studies and three controls */were established and 103 tachyzoites of this native strain of T. gondii were injected intraperitoneally to the mice in groups 1, 2, 3, 4 and 7. Azithromycin was given to groups 1 /4 at different times of infection orally between 100 and 300 mg/kg/ day for 10 days. Azithromycin was found to be effective at 200 mg/kg/day and above in the prophylaxis, at 250 mg/kg/ day and above in the treatment of toxoplasmosis. These results suggest that azithromycin is effective in the prophylaxis and early infection of a highly virulent strain of T. gondii , and it doubled the survival time in the late infection. Azithromycin could be an alternative treatment regimen for human toxoplasmosis, if supported by further clinical investigations. # 2003 Elsevier B.V. All rights reserved. Keywords: Azithromycin; Toxoplasma gondii ; Treatment and prophylaxis
1. Introduction Toxoplasma gondii is a coccidian parasite of felids with humans and other warm-blooded animals as alternative hosts. Epidemiological studies have shown that the infection has a worldwide distribution and is most common in moist tropical areas. Human infection generally occurs through
* Corresponding author. Tel.: /90-236-234-9070; fax: /90236-237-0213. ¨ zbilgin). E-mail address: [email protected] (A. O
the ingestion of raw or undercooked meat that contains cysts, through the ingestion of water or food contaminated with oocysts, or congenitally through transplacental transmission from a mother who acquired her infection during gestation. In immunocompetent individuals, toxoplasmosis is generally asymptomatic, however, it sometimes manifests with fever, malaise, headache, myalgia, asymptomatic lymph node enlargement, and chorioretinitis when it locates in the eye. In immunocompromised patients, T. gondii could cause life-threatening infections like encephalitis, pneumonia and chorioretinitis. Congenital toxo-
0001-706X/03/$ - see front matter # 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0001-706X(03)00194-3
46
K. Deg ˘ erli et al. / Acta Tropica 88 (2003) 45 /50
plasmosis develops only when nonimmune mothers are infected during pregnancy. It could lead to fatal complications for the fetus and manifest as chorioretinitis, calcification of the brain tissue, hydrocephalus and psychomotor or mental retardation. Today, the use of serological tests for the demonstration of specific antibody to T. gondii is the primary method of diagnosis. Immunocompetent patients with toxoplasmosis are not treated unless they acquired the infection by a laboratory accident or blood products. Since toxoplasmosis in immunodeficient patients is often lethal, treatment should be continued for 4/6 weeks after the resolution of all signs and symptoms (Montaya and Remington, 2000; Heyneman, 1991; Frenkel, 1991). Studies investigating the properties of azithromycin, a semi-synthetic macrolide antibiotic, report that azithromycin exerts its antimicrobial activity through the inhibition of protein synthesis by acting on the 70S unit of ribosomes. Its serum half-life is relatively short, being 11/14 h, indicating the initial rapid distribution of drug into tissues. High tissue concentrations are maintained for prolonged periods by once daily dosing, which were significantly related to good in vivo activity. Azithromycin is detectable in urine 7/14 days after a single dose (Lode, 1991; Lode et al., 1996; Girard et al., 1987). Azithromycin has activity against both tachyzoites and cyst forms of T. gondii . It is also effective against several other protozoa like Entamoeba histolytica and Giardia lamblia (Lode et al., 1996; Ravdin and Skilogiannis, 1989; Crouch et al., 1990). Today, the combination of pyrimethamine-sulphonamide is the standard treatment for toxoplasmosis. Both agents could cause toxic complications; pyrimethamine could depress the bone marrow, threatening the immunocompromised patients and its usage is not recommended during pregnancy due to its potential teratogenic effects. Thus, there is an increasing need for new drugs and treatment regimens against toxoplasmosis, which could safely be used both during pregnancy and in immunocompromised patients and that are active in toxoplasmosis located in the central nervous system (Araujo et al., 1988).
In this study. the therapeutic and prophylactic efficacy of azithromycin, as an antimicrobial agent with high intracellular concentration, was assessed against the native, highly virulent strain of T. gondii , was assessed.
2. Materials and methods 2.1. Mice Adult, male Balb/C mice, weighing 25 /30 g each, were used in the study. 2.2. Parasite strain The strain of T. gondii used in the study is considered to be the principle strain of toxoplasmosis in Turkey. It has been isolated from the mice infected with the intestinal extracts of stray cats in Ankara city. Our examinations revealed that it could survive for 10 days in saline at /4 8C, and it was still infective after 5 days in room temperature. In addition, its infectivity was maintained for 3 days in urine and 5 days in semen. This strain exhibited a 100% lethal dose (LD100) of a single viable parasite. When 103 tachyzoites were injected intraperitoneally to healthy Balb/C mice, they killed the mice by the end of the 4th day, proven by the detection of tachyzoites in the peritoneal ¨ zbilgin A. et al, unpublished data). fluid smears (O 2.3. Preparation of azithromycin Azithromycin was obtained in tablet form from the Pfizer Company (Zitromax† 500 mg), treaded to powder in a clean glass container, suspended in distilled water and given to mice in 0.3 ml of suspensions orally at the targeted drug concentration. 2.4. Method Initially, four study groups, groups 1, 2, 3 and 4 were formed; each group was consisting of nine dose subgroups and each subgroup contained 10 mice. Control groups, groups 5, 6 and 7, contained 10 mice each and group 6 received only azithro-
K. Deg ˘ erli et al. / Acta Tropica 88 (2003) 45 /50
47
mycin. 103 tachyzoites of the native strain of T. gondii were injected intraperitoneally (i.p) to infect the mice in groups 1, 2, 3, 4 and 7. Azithromycin was given to group 1 3 days before infection, group 2 together with infection, group 3 12 h after infection and group 4 24 h after infection via the oral route at all doses of 100, 125, 150, 175, 200, 225, 250, 275 and 300 mg/kg/day for 10 days. Group 1 and group 2 were assigned as ‘Prophylaxis Groups’, while groups 3 and 4 were assigned as ‘Therapy Groups’. Group 5 received neither infection nor azithromycin while group 6 was not infected but received 300 mg/kg/day of azithromycin for 10 days. Group 7 was infected but not treated with azithromycin. Physical conditions of all the mice in the study were examined regularly from the second day of the study at every other day. The peritoneal fluids of the mice with poor physical condition (abdominal swelling, decreased motility, poor appearance) were investigated for T. gondii tachyzoites. Detailed records, including the life durations and the groups of the mice that died during the study, were prepared meticulously. The remaining mice in all of the groups were sacrificed after being followed for 8 weeks. Then, T. gondii tachyzoites were investigated in Giemsa-stained preparations obtained from peritoneum, liver, spleen and brain smears. All the results of the study were analyzed with Fisher’s non-parametric x2-test and Fisher’s exact test.
gondii were detected only in their peritoneal fluid. The remaining mice in any of the groups 1/3 and non-infected controls, which were followed for 8 weeks, had no T. gondii tachyzoites in their Giemsa-stained preparations from peritoneum, liver, spleen and brain smears. There was no statistically significant difference between the ‘Prophylaxis Groups’, groups 1 and 2 (P ]/0.05). On the other hand, the differences between 175 mg/kg/day dose of azithromycin and 200 mg/kg/day and above doses of azithromycin were found statistically significant in groups 1 and 2 (P 5/0.05). It was also found that 200 mg/kg/day and above doses of azithromycin in groups 1 and 2 were statistically more effective in prophylaxis, compared to the same doses in the ‘Therapy Groups’, groups 3 and 4 (P 5/0.05). The result of the relative risk analysis between 200 mg/kg/day and above doses of azithromycin of the pair groups 1 and 2 and of the pair groups 3 and 4 was found as 7. The differences between 225 mg/ kg/day dose of azithromycin and 250 mg/kg/day and above doses of azithromycin were found statistically significant in group 3 (P 5/0.05). Two hundred and fifty mg/kg/day and above doses of azithromycin in group 3 were statistically more effective in therapy compared to the same doses in group 4 (P 5/0.05). Ten days of lifetime in group 3 and 8 days of lifetime in group 4 had statistically significant difference, compared to the 4 days of lifetime in group 7 (P 5/0.05).
3. Results
4. Discussion
Survival rates of the mice according to azithromycin doses are shown in Table 1. All the mice in groups 1 and 2 that received 100 /175 mg/kg/day of azithromycin as well as in group 3 that received 100/225 mg/kg/day of azithromycin died on the 7th or 8th day (Table 2). In group 3, the surviving mice died after the 10th day, while all the mice in group 4 died within 8 days, and the examination of their peritoneal fluids revealed T. gondii tachyzoites. However, liver, spleen and brain smear preparations of the dead mice revealed no tachyzoites. All the mice in group 7 died within 4 days and tachyzoites of T.
The course of toxoplasmosis in almost all immunocompetent individuals is relatively benign, but it is a serious and often life-threatening disease in immunodeficient patients (Montaya and Remington, 2000). Currently, the recommended therapy for toxoplasmosis is the synergistic combination of pyrimethamine and sulfadiazine, which may be toxic and insufficient, especially in immunocompromised patients. Pyrimethamine could be teratogenic and should not be used during the early months of pregnancy. (Rodriguez-Diaz et al., 1993). There is currently an increasing need for
K. Deg ˘ erli et al. / Acta Tropica 88 (2003) 45 /50
48
Table 1 The survival rates of the mice according to azithromycin doses Doses (mg/kg/day)
0 100 125 150 175 200 225 250 275 300
Mice groupsa Group 1
Group 2
Group 3
Group 4
0/10 0/10 0/10 0/10 10/10 10/10 10/10 10/10 10/10
0/10 0/10 0/10 0/10 8/10 10/10 10/10 10/10 10/10
0/10 0/10 0/10 0/10 0/10 0/10 4/10 5/10 5/10
0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10
Group 5
Group 6
10/10
Group 7 0/10
10/10
Group 1: azithromycin was given 3 days before infection; group 2, azithromycin was given together with infection; group 3, azithromycin was given 12 h after infection; group 4, azithromycin was given 24 h after infection; group 5, received neither infection nor azithromycin; group 6, received no infection but 300 mg/kg/day azithromycin for 10 days; group 7, received infection but not azithromycin. a Seven mice groups were formed in the study.
novel and more reliable treatment regimens for the treatment of toxoplasmosis. A new, prokaryotic ribosomal gene sequence has been discovered through DNA amplification of T. gondii with PCR. The ribosome coded with this new gene sequence is more sensitive to the macrolide-lincosamine group of antibiotics. Thus, it can be a functional target for azithromycin, clindamycin and other protein synthesis inhibitors (Beckers et al., 1995).
Azithromycin has activity against both tachyzoites and cyst forms of T. gondii . It can inhibit the growth of T. gondii tachyzoites in vitro, but the effect is only observed with prolonged incubation with the drug, reflecting the delayed mode of action of azithromycin on the parasite. In acute toxoplasmosis, elimination of the parasites from the brain tissue is not as pronounced as from blood and lungs of the mice. However, a better prophylaxis is maintained compared to the control
Table 2 Mean survival time (9/1 SD) in mice groups according to azithromycin doses Azithromycin doses (mg/kg/day)
0 100 125 150 175 200 225 250 275 300 a b
Mice Groupsa Group 1
Group 2
Group 3
Group 4
7.609/0.49b 7.609/0.49 7.709/0.46 7.809/0.40 56.00 56.00 56.00 56.00 56.00
7.709/0.46 7.709/0.46 7.809/0.40 7.909/0.30 10.509/0.50 56.00 56.00 56.00 56.00
7.809/0.40 7.809/0.40 7.809/0.40 7.809/0.40 7.909/0.30 8.00 11.309/0.75 12.209/0.75 12.809/0.75
7.109/0.83 7.209/0.75 7.409/0.66 7.609/0.49 7.609/0.49 7.709/0.46 7.709/0.46 7.809/0.40 7.909/0.30
Group 5
Group 6
56.00
n/10 for all cells. Days, mean9/1 SD.
Group 7 3.709/0.46
56.00
K. Deg ˘ erli et al. / Acta Tropica 88 (2003) 45 /50
group that receive no therapy. Thus, combination therapies of azithromycin could be used for therapy and/or prophylaxis of toxoplasmosis (Montaya and Remington, 2000; Derouin, 1995). Azithromycin has also a significant in vitro effect on the organisms that are not sensitive to beta-lactam antibiotics, resistant to benzyl penicillin and erythromycin, and that do not have effective treatment regimens, like T. gondii (Lode et al., 1996). Efficacy of azithromycin in murine models of toxoplasmosis, alone or in combination with other antimicrobial agents, has been investigated in several studies (Lode, 1991; Rodriguez-Diaz et al., 1993; Beckers et al., 1995; Chang, 1996). Results of these studies suggested that azithromycin, especially at 200 mg/kg/day, was effective in both prophylaxis and treatment of toxoplasmosis. Since azithromycin could reach high concentrations in the central nervous system, its usage in the primary and secondary prophylaxis of toxoplasmic encephalitis was also recommended (Montaya and Remington, 2000; Lode et al., 1996; Beckers et al., 1995). In addition, two groups of antibiotics, azalides and macrolides, have been drawing attention in T. gondii therapy for their lesser toxic effects, suggesting that azithromycin could be preferred in toxoplasmosis therapy of immunocompromised patients (Chang, 1996). In forty-two adult HIV infected patients diagnosed as toxoplasmic encephalitis (TE), the combination of azithromycin (900 /1200 mg a day) and pyrimethamine was reported as a reliable alternative therapy for treating TE of patients intolerant to sulphonamides and clindamycin (Jacobson et al., 2001). In this study with the rodent model, we found that there was no difference between the prophylaxis starting with the infection and 3 days before the infection; azithromycin doses of 200 mg/kg/day and above were effective in the prophylaxis of toxoplasmosis. Doses of 250 mg/kg/day and above doses of azithromycin were found to be the effective treatment doses in toxoplasmosis when treatment started early. However, it should be noted that the treatment given in the late periods (24 h after the infection) of infection doubled the lifetime of the mice, compared to the mice that had
49
not received any treatment. Azithromycin was found to be more effective in prophylaxis according to relative risk analysis. These data were obtained from our murine toxoplasmosis model. Since human infections become manifest later than the occurrence of natural infection, human studies should be planned to investigate the effects of the time gap between the natural infection and inception of treatment on the therapeutic prospects of azithromycin in toxoplasmosis. We conclude that azithromycin is a potent prophylactic agent even for the highly virulent strain of T. gondii in Turkey. It is also a drug of choice for the early periods of infection, while it doubles the lifetime at the late periods of the infection. Thus, with the support of further clinical trials, azithromycin could become an alternative, effective treatment protocol in toxoplasmosis.
References Araujo, F.G., Guptill, D.R., Remington, J.S., 1988. Azithromycin, a macrolide antibiotic with potent activity against Toxoplasma gondii . J. Antimicrob. Chemother. 32, 755 / 757. Beckers, C.J., Roos, D.S., Donald, R.G., Luft, B.J., Schwab, J.C., Cao, Y., Joiner, K.A., 1995. Inhibition of cytoplasmic and organellar protein synthesis in Toxoplasma gondii . Implications for the target of macrolide antibiotics. J. Clin. Invest. 95, 367 /376. Chang, H.R., 1996. The potential role of azithromycin in the treatment of prophylaxis of toxoplasmosis. Int. J. STD AIDS 7, 18 /22. Crouch, A.A., Seow, W.K., Whitman, L.M., Thong, Y.H., 1990. Sensitivity in vitro of Giardia intestinalis to dyadic combinations of azithromycin, doxycycline, mefloquine, tinidazole and furazolidone. Trans. R. Soc. Trop. Med. Hyg. 84, 246 /248. Derouin, F., 1995. New pathogens and mode of action of azithromycin: Toxoplasma gondii . Pathol. Biol. Paris 43, 561 /564. Frenkel, J.K., 1991. Toxoplasmosis. In: Gilles, H.M. (Ed.), Protozoal Diseases. Arnold Publishers, New York, pp. 530 / 547. Girard, A.E., Girard, P., English, A.R., 1987. Pharmacokinetic and in vivo studies with azithromycin (CP-62, 993), a new macrolide with an extended half-life and excellent tissue distribution. Antimicrob. Agents Chemother. 31, 1948 / 1954.
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Heyneman, D., 1991. Medical parasitology. In: Jawez, E., Melnick, J.L., Adelberg, E.A. (Eds.), Medical Microbiology. Appleton & Lange, New Jersey, pp. 332 /365. Jacobson, J.M., Hafner, R., Remington, J., Farthing, C., Holden-Wiltse, J., Bosler, E.M., Harris, C., Jayaweera, D.T., Roque, C., Luft, B.J., 2001. Dose-escalation, phase I/II study of azithromycin and pyrimethamine for the treatment of toxoplasmic encephalitis in AIDS. AIDS 15, 583 /589. Lode, H., 1991. The pharmacokinetics of azithromycin and their clinical significance. Eur. J. Clin. Microb. Infect. Dis. 10, 807 /812. Lode, H., Borner, K., Koeppe, P., Schaberg, T., 1996. Azithromycin */review of key chemical, pharmacokinetic
and microbiological features. J. Antimicrob. Chemother. 37 (Suppl. C), 1 /8. Montaya, J.G., Remington, J.S., 2000. Toxoplasma gondii . In: Mandell, G.L., Bennett, J.E., Dolin, R. (Eds.), Principles and Practice of Infectious Diseases. Churchill Livingstone, Philadelphia, pp. 2858 /2881. Ravdin, J.I., Skilogiannis, J., 1989. In vitro susceptibilities of Entamoeba histolytica to azithromycin (CP-63, 956), erythromycine and metronidazole. Antimicrob. Agents Chemother. 33, 960 /962. Rodriguez-Diaz, J.C., Martinez-Gueiro, M.M., Martinez-Fernandez, A.R., 1993. Comparative activity of several antibiotics against Toxoplasma gondii in a mouse model. Enferm. Infect. Microbiol. Clin. 11, 543 /546.
Efficacy of azithromycin in a murine toxoplasmosis model, employing a Toxoplasma gondii strain from Turkey ¨ zgu¨r Kurt b, A. Taylan Tamay b, Kenan Deg˘erli a, Ali A. Kilimciog˘lu b, O ¨ zbilgin b,* Ahmet O a
Department of Microbiology and Clinical Microbiology, School of Medicine, Celal Bayar University, Manisa 45010, Turkey b Department of Parasitology, School of Medicine, Celal Bayar University, Manisa 45010, Turkey Received 9 August 2002; received in revised form 28 February 2003; accepted 15 May 2003
Abstract A murine toxoplasmosis model with Balb/C mice was used to investigate the therapeutic and prophylactic efficacy of azithromycin in a native strain of Toxoplasma gondii . Initially, seven groups */four studies and three controls */were established and 103 tachyzoites of this native strain of T. gondii were injected intraperitoneally to the mice in groups 1, 2, 3, 4 and 7. Azithromycin was given to groups 1 /4 at different times of infection orally between 100 and 300 mg/kg/ day for 10 days. Azithromycin was found to be effective at 200 mg/kg/day and above in the prophylaxis, at 250 mg/kg/ day and above in the treatment of toxoplasmosis. These results suggest that azithromycin is effective in the prophylaxis and early infection of a highly virulent strain of T. gondii , and it doubled the survival time in the late infection. Azithromycin could be an alternative treatment regimen for human toxoplasmosis, if supported by further clinical investigations. # 2003 Elsevier B.V. All rights reserved. Keywords: Azithromycin; Toxoplasma gondii ; Treatment and prophylaxis
1. Introduction Toxoplasma gondii is a coccidian parasite of felids with humans and other warm-blooded animals as alternative hosts. Epidemiological studies have shown that the infection has a worldwide distribution and is most common in moist tropical areas. Human infection generally occurs through
* Corresponding author. Tel.: /90-236-234-9070; fax: /90236-237-0213. ¨ zbilgin). E-mail address: [email protected] (A. O
the ingestion of raw or undercooked meat that contains cysts, through the ingestion of water or food contaminated with oocysts, or congenitally through transplacental transmission from a mother who acquired her infection during gestation. In immunocompetent individuals, toxoplasmosis is generally asymptomatic, however, it sometimes manifests with fever, malaise, headache, myalgia, asymptomatic lymph node enlargement, and chorioretinitis when it locates in the eye. In immunocompromised patients, T. gondii could cause life-threatening infections like encephalitis, pneumonia and chorioretinitis. Congenital toxo-
0001-706X/03/$ - see front matter # 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0001-706X(03)00194-3
46
K. Deg ˘ erli et al. / Acta Tropica 88 (2003) 45 /50
plasmosis develops only when nonimmune mothers are infected during pregnancy. It could lead to fatal complications for the fetus and manifest as chorioretinitis, calcification of the brain tissue, hydrocephalus and psychomotor or mental retardation. Today, the use of serological tests for the demonstration of specific antibody to T. gondii is the primary method of diagnosis. Immunocompetent patients with toxoplasmosis are not treated unless they acquired the infection by a laboratory accident or blood products. Since toxoplasmosis in immunodeficient patients is often lethal, treatment should be continued for 4/6 weeks after the resolution of all signs and symptoms (Montaya and Remington, 2000; Heyneman, 1991; Frenkel, 1991). Studies investigating the properties of azithromycin, a semi-synthetic macrolide antibiotic, report that azithromycin exerts its antimicrobial activity through the inhibition of protein synthesis by acting on the 70S unit of ribosomes. Its serum half-life is relatively short, being 11/14 h, indicating the initial rapid distribution of drug into tissues. High tissue concentrations are maintained for prolonged periods by once daily dosing, which were significantly related to good in vivo activity. Azithromycin is detectable in urine 7/14 days after a single dose (Lode, 1991; Lode et al., 1996; Girard et al., 1987). Azithromycin has activity against both tachyzoites and cyst forms of T. gondii . It is also effective against several other protozoa like Entamoeba histolytica and Giardia lamblia (Lode et al., 1996; Ravdin and Skilogiannis, 1989; Crouch et al., 1990). Today, the combination of pyrimethamine-sulphonamide is the standard treatment for toxoplasmosis. Both agents could cause toxic complications; pyrimethamine could depress the bone marrow, threatening the immunocompromised patients and its usage is not recommended during pregnancy due to its potential teratogenic effects. Thus, there is an increasing need for new drugs and treatment regimens against toxoplasmosis, which could safely be used both during pregnancy and in immunocompromised patients and that are active in toxoplasmosis located in the central nervous system (Araujo et al., 1988).
In this study. the therapeutic and prophylactic efficacy of azithromycin, as an antimicrobial agent with high intracellular concentration, was assessed against the native, highly virulent strain of T. gondii , was assessed.
2. Materials and methods 2.1. Mice Adult, male Balb/C mice, weighing 25 /30 g each, were used in the study. 2.2. Parasite strain The strain of T. gondii used in the study is considered to be the principle strain of toxoplasmosis in Turkey. It has been isolated from the mice infected with the intestinal extracts of stray cats in Ankara city. Our examinations revealed that it could survive for 10 days in saline at /4 8C, and it was still infective after 5 days in room temperature. In addition, its infectivity was maintained for 3 days in urine and 5 days in semen. This strain exhibited a 100% lethal dose (LD100) of a single viable parasite. When 103 tachyzoites were injected intraperitoneally to healthy Balb/C mice, they killed the mice by the end of the 4th day, proven by the detection of tachyzoites in the peritoneal ¨ zbilgin A. et al, unpublished data). fluid smears (O 2.3. Preparation of azithromycin Azithromycin was obtained in tablet form from the Pfizer Company (Zitromax† 500 mg), treaded to powder in a clean glass container, suspended in distilled water and given to mice in 0.3 ml of suspensions orally at the targeted drug concentration. 2.4. Method Initially, four study groups, groups 1, 2, 3 and 4 were formed; each group was consisting of nine dose subgroups and each subgroup contained 10 mice. Control groups, groups 5, 6 and 7, contained 10 mice each and group 6 received only azithro-
K. Deg ˘ erli et al. / Acta Tropica 88 (2003) 45 /50
47
mycin. 103 tachyzoites of the native strain of T. gondii were injected intraperitoneally (i.p) to infect the mice in groups 1, 2, 3, 4 and 7. Azithromycin was given to group 1 3 days before infection, group 2 together with infection, group 3 12 h after infection and group 4 24 h after infection via the oral route at all doses of 100, 125, 150, 175, 200, 225, 250, 275 and 300 mg/kg/day for 10 days. Group 1 and group 2 were assigned as ‘Prophylaxis Groups’, while groups 3 and 4 were assigned as ‘Therapy Groups’. Group 5 received neither infection nor azithromycin while group 6 was not infected but received 300 mg/kg/day of azithromycin for 10 days. Group 7 was infected but not treated with azithromycin. Physical conditions of all the mice in the study were examined regularly from the second day of the study at every other day. The peritoneal fluids of the mice with poor physical condition (abdominal swelling, decreased motility, poor appearance) were investigated for T. gondii tachyzoites. Detailed records, including the life durations and the groups of the mice that died during the study, were prepared meticulously. The remaining mice in all of the groups were sacrificed after being followed for 8 weeks. Then, T. gondii tachyzoites were investigated in Giemsa-stained preparations obtained from peritoneum, liver, spleen and brain smears. All the results of the study were analyzed with Fisher’s non-parametric x2-test and Fisher’s exact test.
gondii were detected only in their peritoneal fluid. The remaining mice in any of the groups 1/3 and non-infected controls, which were followed for 8 weeks, had no T. gondii tachyzoites in their Giemsa-stained preparations from peritoneum, liver, spleen and brain smears. There was no statistically significant difference between the ‘Prophylaxis Groups’, groups 1 and 2 (P ]/0.05). On the other hand, the differences between 175 mg/kg/day dose of azithromycin and 200 mg/kg/day and above doses of azithromycin were found statistically significant in groups 1 and 2 (P 5/0.05). It was also found that 200 mg/kg/day and above doses of azithromycin in groups 1 and 2 were statistically more effective in prophylaxis, compared to the same doses in the ‘Therapy Groups’, groups 3 and 4 (P 5/0.05). The result of the relative risk analysis between 200 mg/kg/day and above doses of azithromycin of the pair groups 1 and 2 and of the pair groups 3 and 4 was found as 7. The differences between 225 mg/ kg/day dose of azithromycin and 250 mg/kg/day and above doses of azithromycin were found statistically significant in group 3 (P 5/0.05). Two hundred and fifty mg/kg/day and above doses of azithromycin in group 3 were statistically more effective in therapy compared to the same doses in group 4 (P 5/0.05). Ten days of lifetime in group 3 and 8 days of lifetime in group 4 had statistically significant difference, compared to the 4 days of lifetime in group 7 (P 5/0.05).
3. Results
4. Discussion
Survival rates of the mice according to azithromycin doses are shown in Table 1. All the mice in groups 1 and 2 that received 100 /175 mg/kg/day of azithromycin as well as in group 3 that received 100/225 mg/kg/day of azithromycin died on the 7th or 8th day (Table 2). In group 3, the surviving mice died after the 10th day, while all the mice in group 4 died within 8 days, and the examination of their peritoneal fluids revealed T. gondii tachyzoites. However, liver, spleen and brain smear preparations of the dead mice revealed no tachyzoites. All the mice in group 7 died within 4 days and tachyzoites of T.
The course of toxoplasmosis in almost all immunocompetent individuals is relatively benign, but it is a serious and often life-threatening disease in immunodeficient patients (Montaya and Remington, 2000). Currently, the recommended therapy for toxoplasmosis is the synergistic combination of pyrimethamine and sulfadiazine, which may be toxic and insufficient, especially in immunocompromised patients. Pyrimethamine could be teratogenic and should not be used during the early months of pregnancy. (Rodriguez-Diaz et al., 1993). There is currently an increasing need for
K. Deg ˘ erli et al. / Acta Tropica 88 (2003) 45 /50
48
Table 1 The survival rates of the mice according to azithromycin doses Doses (mg/kg/day)
0 100 125 150 175 200 225 250 275 300
Mice groupsa Group 1
Group 2
Group 3
Group 4
0/10 0/10 0/10 0/10 10/10 10/10 10/10 10/10 10/10
0/10 0/10 0/10 0/10 8/10 10/10 10/10 10/10 10/10
0/10 0/10 0/10 0/10 0/10 0/10 4/10 5/10 5/10
0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10
Group 5
Group 6
10/10
Group 7 0/10
10/10
Group 1: azithromycin was given 3 days before infection; group 2, azithromycin was given together with infection; group 3, azithromycin was given 12 h after infection; group 4, azithromycin was given 24 h after infection; group 5, received neither infection nor azithromycin; group 6, received no infection but 300 mg/kg/day azithromycin for 10 days; group 7, received infection but not azithromycin. a Seven mice groups were formed in the study.
novel and more reliable treatment regimens for the treatment of toxoplasmosis. A new, prokaryotic ribosomal gene sequence has been discovered through DNA amplification of T. gondii with PCR. The ribosome coded with this new gene sequence is more sensitive to the macrolide-lincosamine group of antibiotics. Thus, it can be a functional target for azithromycin, clindamycin and other protein synthesis inhibitors (Beckers et al., 1995).
Azithromycin has activity against both tachyzoites and cyst forms of T. gondii . It can inhibit the growth of T. gondii tachyzoites in vitro, but the effect is only observed with prolonged incubation with the drug, reflecting the delayed mode of action of azithromycin on the parasite. In acute toxoplasmosis, elimination of the parasites from the brain tissue is not as pronounced as from blood and lungs of the mice. However, a better prophylaxis is maintained compared to the control
Table 2 Mean survival time (9/1 SD) in mice groups according to azithromycin doses Azithromycin doses (mg/kg/day)
0 100 125 150 175 200 225 250 275 300 a b
Mice Groupsa Group 1
Group 2
Group 3
Group 4
7.609/0.49b 7.609/0.49 7.709/0.46 7.809/0.40 56.00 56.00 56.00 56.00 56.00
7.709/0.46 7.709/0.46 7.809/0.40 7.909/0.30 10.509/0.50 56.00 56.00 56.00 56.00
7.809/0.40 7.809/0.40 7.809/0.40 7.809/0.40 7.909/0.30 8.00 11.309/0.75 12.209/0.75 12.809/0.75
7.109/0.83 7.209/0.75 7.409/0.66 7.609/0.49 7.609/0.49 7.709/0.46 7.709/0.46 7.809/0.40 7.909/0.30
Group 5
Group 6
56.00
n/10 for all cells. Days, mean9/1 SD.
Group 7 3.709/0.46
56.00
K. Deg ˘ erli et al. / Acta Tropica 88 (2003) 45 /50
group that receive no therapy. Thus, combination therapies of azithromycin could be used for therapy and/or prophylaxis of toxoplasmosis (Montaya and Remington, 2000; Derouin, 1995). Azithromycin has also a significant in vitro effect on the organisms that are not sensitive to beta-lactam antibiotics, resistant to benzyl penicillin and erythromycin, and that do not have effective treatment regimens, like T. gondii (Lode et al., 1996). Efficacy of azithromycin in murine models of toxoplasmosis, alone or in combination with other antimicrobial agents, has been investigated in several studies (Lode, 1991; Rodriguez-Diaz et al., 1993; Beckers et al., 1995; Chang, 1996). Results of these studies suggested that azithromycin, especially at 200 mg/kg/day, was effective in both prophylaxis and treatment of toxoplasmosis. Since azithromycin could reach high concentrations in the central nervous system, its usage in the primary and secondary prophylaxis of toxoplasmic encephalitis was also recommended (Montaya and Remington, 2000; Lode et al., 1996; Beckers et al., 1995). In addition, two groups of antibiotics, azalides and macrolides, have been drawing attention in T. gondii therapy for their lesser toxic effects, suggesting that azithromycin could be preferred in toxoplasmosis therapy of immunocompromised patients (Chang, 1996). In forty-two adult HIV infected patients diagnosed as toxoplasmic encephalitis (TE), the combination of azithromycin (900 /1200 mg a day) and pyrimethamine was reported as a reliable alternative therapy for treating TE of patients intolerant to sulphonamides and clindamycin (Jacobson et al., 2001). In this study with the rodent model, we found that there was no difference between the prophylaxis starting with the infection and 3 days before the infection; azithromycin doses of 200 mg/kg/day and above were effective in the prophylaxis of toxoplasmosis. Doses of 250 mg/kg/day and above doses of azithromycin were found to be the effective treatment doses in toxoplasmosis when treatment started early. However, it should be noted that the treatment given in the late periods (24 h after the infection) of infection doubled the lifetime of the mice, compared to the mice that had
49
not received any treatment. Azithromycin was found to be more effective in prophylaxis according to relative risk analysis. These data were obtained from our murine toxoplasmosis model. Since human infections become manifest later than the occurrence of natural infection, human studies should be planned to investigate the effects of the time gap between the natural infection and inception of treatment on the therapeutic prospects of azithromycin in toxoplasmosis. We conclude that azithromycin is a potent prophylactic agent even for the highly virulent strain of T. gondii in Turkey. It is also a drug of choice for the early periods of infection, while it doubles the lifetime at the late periods of the infection. Thus, with the support of further clinical trials, azithromycin could become an alternative, effective treatment protocol in toxoplasmosis.
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Heyneman, D., 1991. Medical parasitology. In: Jawez, E., Melnick, J.L., Adelberg, E.A. (Eds.), Medical Microbiology. Appleton & Lange, New Jersey, pp. 332 /365. Jacobson, J.M., Hafner, R., Remington, J., Farthing, C., Holden-Wiltse, J., Bosler, E.M., Harris, C., Jayaweera, D.T., Roque, C., Luft, B.J., 2001. Dose-escalation, phase I/II study of azithromycin and pyrimethamine for the treatment of toxoplasmic encephalitis in AIDS. AIDS 15, 583 /589. Lode, H., 1991. The pharmacokinetics of azithromycin and their clinical significance. Eur. J. Clin. Microb. Infect. Dis. 10, 807 /812. Lode, H., Borner, K., Koeppe, P., Schaberg, T., 1996. Azithromycin */review of key chemical, pharmacokinetic
and microbiological features. J. Antimicrob. Chemother. 37 (Suppl. C), 1 /8. Montaya, J.G., Remington, J.S., 2000. Toxoplasma gondii . In: Mandell, G.L., Bennett, J.E., Dolin, R. (Eds.), Principles and Practice of Infectious Diseases. Churchill Livingstone, Philadelphia, pp. 2858 /2881. Ravdin, J.I., Skilogiannis, J., 1989. In vitro susceptibilities of Entamoeba histolytica to azithromycin (CP-63, 956), erythromycine and metronidazole. Antimicrob. Agents Chemother. 33, 960 /962. Rodriguez-Diaz, J.C., Martinez-Gueiro, M.M., Martinez-Fernandez, A.R., 1993. Comparative activity of several antibiotics against Toxoplasma gondii in a mouse model. Enferm. Infect. Microbiol. Clin. 11, 543 /546.