A Systematic Review of In vitro and In vivo Activities of Anti-Toxoplasma Drugs and Compounds (2006–2016)

The currently available anti-Toxoplasma agents have serious limitations. This systematic review was performed to evaluate drugs and new compounds used for the treatment of toxoplasmosis. Data was systematically collected from published papers on the efficacy of drugs/compounds used against Toxoplasma gondii (T. gondii) globally during 2006–2016. The searched databases were PubMed, Google Scholar, Science Direct, ISI Web of Science, EBSCO, and Scopus. One hundred and eighteen papers were eligible for inclusion in this systematic review, which were both in vitro and in vivo studies. Within this review, 80 clinically available drugs and a large number of new compounds with more than 39 mechanisms of action were evaluated. Interestingly, many of the drugs/compounds evaluated against T. gondii act on the apicoplast. Therefore, the apicoplast represents as a potential drug target for new chemotherapy. Based on the current findings, 49 drugs/compounds demonstrated in vitro half-maximal inhibitory concentration (IC50) values of below 1 μM, but most of them were not evaluated further for in vivo effectiveness. However, the derivatives of the ciprofloxacin, endochin-like quinolones and 1-[4-(4-nitrophenoxy) phenyl] propane-1-one (NPPP) were significantly active against T. gondii tachyzoites both in vitro and in vivo. Thus, these compounds are promising candidates for future studies. Also, compound 32 (T. gondii calcium-dependent protein kinase 1 inhibitor), endochin-like quinolones, miltefosine, rolipram abolish, and guanabenz can be repurposed into an effective anti-parasitic with a unique ability to reduce brain tissue cysts (88.7, 88, 78, 74, and 69%, respectively). Additionally, no promising drugs are available for congenital toxoplasmosis. In conclusion, as current chemotherapy against toxoplasmosis is still not satisfactory, development of well-tolerated and safe specific immunoprophylaxis in relaxing the need of dependence on chemotherapeutics is a highly valuable goal for global disease control. However, with the increasing number of high-risk individuals, and absence of a proper vaccine, continued efforts are necessary for the development of novel treatment options against T. gondii. Some of the novel compounds reviewed here may represent good starting points for the discovery of effective new drugs. In further, bioinformatic and in silico studies are needed in order to identify new potential toxoplasmicidal drugs.

The currently available anti-Toxoplasma agents have serious limitations. This systematic review was performed to evaluate drugs and new compounds used for the treatment of toxoplasmosis. Data was systematically collected from published papers on the efficacy of drugs/compounds used against Toxoplasma gondii (T. gondii) globally during 2006-2016. The searched databases were PubMed, Google Scholar, Science Direct, ISI Web of Science, EBSCO, and Scopus. One hundred and eighteen papers were eligible for inclusion in this systematic review, which were both in vitro and in vivo studies. Within this review, 80 clinically available drugs and a large number of new compounds with more than 39 mechanisms of action were evaluated. Interestingly, many of the drugs/compounds evaluated against T. gondii act on the apicoplast. Therefore, the apicoplast represents as a potential drug target for new chemotherapy. Based on the current findings, 49 drugs/compounds demonstrated in vitro half-maximal inhibitory concentration (IC 50 ) values of below 1 µM, but most of them were not evaluated further for in vivo effectiveness. However, the derivatives of the ciprofloxacin, endochin-like quinolones and 1-[4-(4-nitrophenoxy) phenyl] propane-1-one (NPPP) were significantly active against T. gondii tachyzoites both in vitro and in vivo. Thus, these compounds are promising candidates for future studies. Also, compound 32 (T. gondii calcium-dependent protein kinase 1 inhibitor), endochin-like quinolones, miltefosine, rolipram abolish, and guanabenz can be repurposed into an effective anti-parasitic with a unique ability to reduce brain tissue cysts (88.7, 88, 78, 74, and 69%, respectively). Additionally, no promising drugs are available for congenital toxoplasmosis. In conclusion, as current chemotherapy against toxoplasmosis is still not satisfactory, development of well-tolerated and safe specific immunoprophylaxis in relaxing the need of dependence on chemotherapeutics is a highly valuable goal for global disease control. However, with the increasing number of high-risk individuals, and absence of a proper vaccine, continued efforts are necessary for the development of novel treatment options against T. gondii. Some of the novel compounds reviewed here may represent good starting points for the discovery of effective new drugs. In further, bioinformatic and in silico studies are needed in order to identify new potential toxoplasmicidal drugs. Keywords: Toxoplasma gondii, toxoplasmosis, drugs, compounds, in vitro, in vivo
In the United States, the Centers for Disease Control and Prevention (CDC) reported that 22.5% of the population 12 years and older have been infected with Toxoplasma with 1.1 million new infections each year, making it the second most common cause of deaths due to foodborne diseases (an estimated 327 deaths) and the fourth leading cause of hospitalizations attributable to foodborne illness (an estimated 4428 hospitalizations). Also, an estimated 400-4000 infants are born with congenital toxoplasmosis in the United States each year (Jones et al., 2014).
T. gondii has three infectious stages of sporozoites (in oocysts), tachyzoites (rapidly multiplying form), and bradyzoites (tissue cyst form). Among them, tachyzoites are responsible for clinical manifestations and the acute phase of the disease. They are susceptible to the immune response of the host and to drug action. The resistant cyst form of the parasite is resistant to both the immune system and drugs (Hill and Dubey, 2002).
Acute toxoplasmosis in healthy individuals is usually subclinical and asymptomatic, but may lead to chronic infection. However, toxoplasmosis can lead to great morbidity and mortality rates in imunocompromised or congenitally infected individuals (Dubey and Jones, 2008;Ahmadpour et al., 2014). In AIDS patients, presence of the parasite causes necrotizing encephalitis and focal cerebral lesions in the central nervous system (CNS) from primary or recrudescent infection. In immunocompetent patients, latent toxoplasmosis occurs with the formation of cysts principally in the CNS (Martins-Duarte et al., 2006).
In the recent years, the development of well-tolerated and safe specific immunoprophylaxis against toxoplasmosis is a highly valuable goal for global disease control (Lim and Othman, 2014). Immunotherapeutics strategies for improving toxoplasmosis control could either be a vaccine which would induce strong protective immunity against toxoplasmosis, or passive immunization in cases of disease recrudescence. In the last few years, much progress has been made in vaccine research on DNA vaccination, protein vaccination, live attenuated vaccinations, and heterologous vaccination; while there were few candidates on passive immunization. New vaccine candidates have been tested, including in particular proteins from T. gondii ROP, MIC, and GRA organelles, multi-antigen vaccines, novel adjuvants but until now the researches could not access to a proper vaccine for prevention of toxoplasmosis in human (Zhang et al., 2013(Zhang et al., , 2015. The recommended drugs for treatment or prophylaxis of toxoplasmosis are pyrimethamine and sulfadiazine. Unfortunately, these drugs have side effects such as neutropenia, severe drop of platelet count, thrombocytopenia, leucopenia, elevation in serum creatinine and serum liver enzymes, hematological abnormalities, and hypersensitivity reactions (Bosch-Driessen et al., 2002;Silveira et al., 2002;Schmidt et al., 2006). In addition, other drugs, such as azithromycin, clarithromycin, spiramycin, atovaquone, dapsone, and cotrimoxazole (trimethoprim-sulfamethoxazole), have been used for clinical toxoplasmosis. However, these drugs are poorly tolerated and have no effect on the bradyzoite form (Araujo and Remington, 1992;Petersen and Schmidt, 2003;Serranti et al., 2011).
In a clinical trial, 24% of sera positive women treated with spiramycin and pyrimethamine plus sulfadoxine combination delivered Toxoplasma infected infants in France (Bessières et al., 2009). Spiramycin monotherapy can be effective during the early stage of pregnancy to prevent prenatal transmission (Julliac et al., 2010). More than 50% of patients treated with spiramycin retained T. gondii DNA in blood and remained infected (Habib, 2008).
In recent years, studies have focused on finding safe drugs with novel mechanisms of action against T. gondii. Accordingly, there is an urgent need to evaluate new drugs based on novel and innovative therapeutic strategies against T. gondii that are both efficacious and nontoxic for humans (Rodriguez and Szajnman, 2012;Vanagas et al., 2012;Angel et al., 2013). Therefore, the goal of the present systematic review was to retrieve published studies related to in vitro and in vivo evaluation of drugs and compounds for the treatment of toxoplasmosis (2006)(2007)(2008)(2009)(2010)(2011)(2012)(2013)(2014)(2015)(2016) in order to prepare comprehensive data for designing more accurate investigations in future.
Also, studies with at least one of the following criteria were excluded: (1) studies that were not relevant; (2) articles not available in English; (3) studies on treatments for ocular infection; (4) articles that were of review or descriptive study type; (5) articles which contained no eligible data; (6) case series reports; (7) the data were duplicated from other studies or we were unable to obtain them; (8) those that were on efficacy of anti-T. gondii medicines in humans; and (9) any drug with an IC 50 value > 10 µM.

Data Collection
All the experimental studies that were carried out to evaluate the efficacy of either drugs or compounds against T. gondii both in vitro and in vivo were included, and replicates were excluded. The inclusion criteria for selection of in vitro studies were important information about medication used for the experiments, type of cells used for culture, identification of the Toxoplasma strain, laboratory methods used for assessing drug activities, and main results comprising of the 50% inhibitory concentration (IC 50 ). We reported in vivo studies used animal models, Toxoplasma strain, route of infection, the treatment schedule (dosage, route of administration, duration of treatment), the criteria for assessing drug activity (mainly survival for acute toxoplasmosis, histology, and brain cyst burdens for chronic infection), and the main results.

Analysis of the Included Literature
A total of 118 papers (83 studies in vitro, 59 in vivo, 27 both in vitro and in vivo) published from 2006 to 2016, were included in the systematic review. Figure 1 briefly shows the search process in this systematic review article.

The Investigated Strains
T. gondii has three main clonal lineages in population structure; type I (including a highly virulent RH strain), Type II (including ME49 and PRU, avirulent strains), and Type III (including avirulent strains like NED), which is correlated with virulence expression in mice (Howe and Sibley, 1995).  Baramee et al., 2006;Ferreira et al., 2006Ferreira et al., , 2012Saleh et al., 2007;Meneceur et al., 2008;Bajohr et al., 2010;Doggett et al., 2012;Kul et al., 2013;de Lima et al., 2015 HDQ* b    In vitro and in vivo screening methods were used of type I T. gondii (mostly RH strain; 76 studies in vitro, and 36 in vivo). Because type I RH strain is highly virulent in mice, causing 100% mortality, but types II and III are relatively less virulent. Although in some studies, ME49 (7 studies in vitro, and 17 in vivo), Prugniaud, EGS, and VEG strains were used, which showed that the outcome of infections depends on the challenge dose and on the genotype of the host (Szabo and Finney, 2016). Details about the investigated strains in vitro and in vivo are shown in Tables 3, 4, respectively.

Laboratory Animals
T. gondii can infect most warm-blooded animals, and is studied in different animal models depending on the nature of the investigation (Szabo and Finney, 2016). The animal model used in studies was mostly mice (16 studies BALB/c and 19 studies Swiss-Webster). In murine models of acute toxoplasmosis, some medicines were protective even when administered at low dosages. But some drugs despite of their excellent in vitro activity were poorly protective in murine models with acute toxoplasmosis (Payne et al., 2013).

Diagnostic Tests and Evaluation Methods
The present review outlines the results of in vitro screening methods including morphological assay, incorporation of [3H] uracil assay, plaque assays, enzyme-linked immunosorbent assay (ELISA), colorimetric micro titer assay (b-galactosidase assay), flow cytometric quantification assay, and cell viability assay. Numerous versions of fluorescent proteins have been expressed in T. gondii (Kim et al., 2001). The reporter genes used in vitro and in vivo studies were the green fluorescent protein (GFP) and yellow fluorescent protein (YFP). Parasites expressing fluorescent proteins can also be analyzed and sorted by flow cytometry. This technology used for drugs screening in 10 studies.
Details about the diagnostic methods and drug dosage under in vivo conditions are shown in Table 4. Also, a comprehensive list of drugs/compounds evaluated against T. gondii with regard to IC 50 is illustrated in Table 5.

DISCUSSION
The aim of this systematic review was to investigate the in vitro and in vivo effects of anti-Toxoplasma drugs and synthetic compounds, from 2006 to 2016. The current anti-T. gondii chemotherapy is deficient; as it is not well-tolerated by immunocompromised patients and cannot completely eradicate tissue cysts produced by the parasite (Rodriguez and Szajnman, 2012). Therefore, developing new, safe, effective, and welltolerated drugs with novel mechanisms of action could be a global priority (Lai et al., 2012). An ideal drug for prophylaxis and/or treatment of toxoplasmosis would show effective penetration and concentration in the placenta, transplacental passage, parasiticidal properties vs. the different parasitic stages, penetration into cysts, and distribution in the main sites. No available drug fulfills these criteria (Derouin et al., 2000;Montoya and Liesenfeld, 2004).
Thus, the findings of the present systematic review article encourage and support more accurate investigations for future to select new anti-Toxoplasma drugs and strategies in designing new targets with specific activity against the parasite.
effectiveness of drugs, various changes have been made in construction of the clinically available medicines. Thus, the activity of new formulations of clinically available drugs against T. gondii should be evaluated to find alternative treatments for toxoplasmosis (da Cunha et al., 2010).
Interestingly, encapsulation of pyrimethamine improved the efficacy and tolerability of this drug against acute toxoplasmosis        in mice and can be considered as an alternative for reducing the dose and side effects of pyrimethamine (Pissinate et al., 2014). Recently, researchers reported that computational analysis of biochemical differences between human and T. gondii dihydrofolate reductase enabled the design of inhibitors with both improved potency and selectivity against T. gondii (Welsch et al., 2016). El-Zawawy et al. reported that incorporating triclosan into in the lipid bilayer of liposomes allowed its use in lower doses, which in turn, reduced its biochemical adverse effects (El-Zawawy et al., 2015b). In another study, sodium dodecyl sulfate (SDS)-coated atovaquone nanosuspensions (ANSs) considerably increased the therapeutic efficacy against experimentally reactivated and acquired toxoplasmosis by improving passage of gastrointestinal and blood-brain barriers. Accordingly, coating of ANSs with SDS may improve the treatment of toxoplasmic encephalitis and other cerebral diseases (Shubar et al., 2011). Also, various studies showed that a number of drugs were investigated for the mechanisms of action summarized in Table 2 and Figure 2. One study discussing the metabolic differences between the host and the parasite noted that dihydrofolate reductase, isoprenoid pathway, and T. gondii histone deacetylase are promising molecular targets (Rodriguez and Szajnman, 2012).
A large number of research papers suggested that the apicoplast represents a potential drug target for new chemotherapy, as it is essential to the parasite and it is absent in host cells. Functions of the apicoplast include fatty acid synthesis, protein synthesis, DNA replication, electron transport, and heme biosynthesis (Yung and Lang-Unnasch, 2004). Some of the drugs evaluated against T. gondii are shown to act in the apicoplast such as thiolactomycin, triclosan (TS), azithromycin, fusidic acid, ciprofloxacin, and quinoline derivatives (Costa et al., 2009;Martins-Duarte et al., 2009Payne et al., 2013;Kadri et al., 2014;El-Zawawy et al., 2015b).
In T. gondii, FAS-II enzymes are present in the apicoplast and are essential for its survival. The key enzyme in this process is the ENR enzyme, which is not found in mammals (Surolia and Surolia, 2001). This enzyme catalyzes the last reductive step of the type II FAS pathway. The TS, which inhibits type II FAS, significantly reduced mice mortality, parasite burden, as well as viability and infectivity of tachyzoites and cysts harvested from infected treated mice and their brains. Accordingly, TS is proved as an effective, promising, and safe preventive drug against acute and chronic murine toxoplasmosis. Liposomal formulation of TS enhanced its efficacy and allowed its use at a lower dose (Surolia and Surolia, 2001;El-Zawawy et al., 2015a,b). Among apicoplast pathways, DNA replication is an important potential chemotherapeutic target. Fluoroquinolones are the known DNA replication inhibitors that target prokaryotic type II topoisomerases (Collin et al., 2011). In two studies, researchers showed that derivatives of the antibiotic ciprofloxacin, a fluoroquinolone, are active against T. gondii tachyzoites both in vitro and in vivo (Neville et al., 2015). While all mice treated with ciprofloxacin died by day 10 post-infection, some mice treated with ciprofloxacin derivatives remained alive for at least 60 days, suggesting that ciprofloxacin derivatives cured T. gondii infection in treated mice (Dubar et al., 2011;Martins-Duarte et al., 2015).

Anti-Toxoplasma Activities of New Synthetic Compounds
There are numerous reports on efficacy of many new synthetic compounds with a focus on identifying drug candidates with innovative and acceptable profiles against T. gondii. The anticoccidial effect of 1-[4-(4-nitrophenoxy) phenyl] propane-1-one (NPPP), a synthetic compound, was studied both in vitro and in vivo. Treatment with NPPP showed anti-Toxoplasma activity in vitro with a lower EC 50 value than pyrimethamine. In ICR mice infected with T. gondii, oral administration of NPPP for 4 days showed statistically significant anti-Toxoplasma activity with lower number of tachyzoites than those of the negative control (Choi et al., 2015).
In a study by Kadri et al. anti-Toxoplasma properties of 58 newly synthesized quinoline compounds were evaluated. A significant improvement in anti-Toxoplasma effect among quinoline derivatives was detected in B11, B12, B23, and B24. Among these compounds, B23 was the most effective compound with the IC 50 value of <1 µM, displaying its anti-Toxoplasma effects and ability to cause the disappearance of the apicoplast (40-45% of the parasites lost their apicoplasts; Kadri et al., 2014).
In a study by Boyom et al. the strategy adopted was to repurpose the open access Malaria Box to identify chemical series active against T. gondii. The results showed that the most interesting compound was MMV007791, a piperazine acetamide, which has an IC 50 of 0.19 µM. This compound is novel for its anti-Toxoplasma activity, and of course, further studies on the rates and mechanisms of compound action will elucidate these considerations (Boyom et al., 2014).
Tetraoxanes, anti-cancer molecules, were tested in vivo against T. gondii. Subcutaneous, administration of a 10 mg/kg/day dose of derivative 21, for 8 days allowed the survival of 20% of infected mice, demonstrating the high potential of tetraoxanes for the treatment of T. gondii (Opsenica et al., 2015).
In another study by Moine et al. researchers evaluated in vitro anti-T. gondii activity of 51 compounds with a biphenylimidazoazine scaffold. Eight of these compounds displayed highly potent activity against T. gondii growth in vitro, with 50% effective concentration (EC 50 ) below 1 mM, without demonstrating cytotoxic effects on human fibroblastic cell at equivalent concentrations. However, these compounds have to be evaluated in animal models so as to confirm their in vivo activity (Moine et al., 2015a). Several pathways were characterized and shown to differ significantly from those of the mammalian host cells, thus, revealing an attractive area for therapeutic intervention. 1-Hydroxy-2-Alkyl-4 (1H) quinolone derivatives inhibit the fourth step of the essential de novo synthesis of pyrimidine, which uses ubiquinol reduction as an electron sink for dihydroorotate oxidation (Saleh et al., 2007). Also, newly synthesized bisphosphonates interfere with the mevanolate pathway, which leads to the synthesis of sterols and polyisoprenoid compounds that are important for parasite survival (Shubar et al., 2008).
Interestingly, Kamau et al. identified novel kinases that are integral to essential pathways, elucidating their mechanism of action and ultimately, identifying new drug targets (Kamau et al., 2012). In that study, 527 compounds were evaluated in vitro; also, they assessed the impact of the inhibitory compounds C1, C2, C3, and C5 in mouse models of toxoplasmosis. C2 was found quite effective in decreasing the parasite burden and increasing mice survival. These results should be considered with caution, since there are a number of factors are at play in whether a compound will be in vivo effective, such as solubility in vivo, access to different tissues, and host metabolic processes (Kamau et al., 2012). In a recent study, Dittmar et al. screened a collection of 1,120 compounds, 94 of which were blocked parasite replications with IC 50 of <5 µM. These data suggest that tamoxifen restricts Toxoplasma growth by inducing xenophagy or autophagic destruction of this parasite (Dittmar et al., 2016). According to a new study, in silico screening is useful, particularly in the identification of molecular targets in the laboratory. Fernandez et al. synthesized VAM2 compounds (7-nitroquinoxalin-2-ones), based on the design obtained from an in silico prediction with the software TOMOCOMD-CARDD. From the group of VAM2 compounds, Fernandez et al. chose VAM2-2 with an IC 50 of 3.3 µM against T. gondii. However, more studies are required to evaluate its effect on the cysts formed by of the parasite and in animal models of toxoplasmosis (Fernández et al., 2016).

Activity of Drugs, Compounds, and Combined Therapy against Cysts
An ideal drug against toxoplasmosis should not only be effective against the proliferative stage of the parasite but also exert dual activity against the tissue cyst stage and penetration into cysts (Benmerzouga et al., 2015). Currently, there is no approved therapy that eliminates the tissue cysts responsible for chronic infection (Innes, 2010). Derouin reported that among the drugs commonly used in humans, only atovaquone and azithromycin were found effective after long-term incubation. Besides, arpinocid-N-oxyde, an anticoccidial for veterinary use, was efficient at a high dosage (Derouin, 2005).
Recently, investigators have focused on guanabenz for in vivo studies, as guanabenz inhibitor of eIF2a dephosphorylation, is already an food and drug administration (FDA) approved drug and has excellent solubility with good penetration into the CNS. The results of that study show that guanabenz (5 mg/kg/day) not only protects mice against acute toxoplasmosis, but also reduces 69% of the number of brain cysts in chronically infected animals. This finding suggested that guanabenz can be repurposed into an effective antiparasitic with a unique ability to diminish tissue cysts in the brain (Benmerzouga et al., 2015).
Another study showed that miltefosine had no efficacy in controlling acute toxoplasmosis after 5 days of treatment; however, a 15-day treatment against the established chronic stage led to a 78% reduction of cysts in the brain. Additionally, the remaining cysts were noticeably smaller upon microscopic examination, suggesting that the drug effectively penetrates the blood-brain barrier, and that extension of treatment time may produce greater effects (Eissa et al., 2015).
In another study by Maubon et al. FR235222 and its derivatives were identified as new lead compounds for use against acute and chronic toxoplasmosis both in vitro and in vivo. In vivo experiments indicated that FR235222, as a histone deacetylase inhibitor, is able to access the bradyzoites within the cyst. The ability of FR235222 to permeate the membrane wall is a major advantage for crossing the blood-brain barrier and CNS tissue, where Toxoplasma cysts are located. This opens a promising way to develop drugs that are selective against Toxoplasma and those that have sterilizing activity, especially in patients with cysts, who are at risk for reactivating acute toxoplasmosis (patients with HIV infection, hematological malignancies, or transplantation). Still, effectiveness of FR235222 against chronically infected mice remains to be directly demonstrated in vivo (Maubon et al., 2010).
In a new study Vidadala et al. identified compounds 32 (T. gondii calcium-dependent protein kinase 1 inhibitor) a promising lead for the development of a new antitoxoplasmosis therapy. Compounds 32 is CNS-penetrant and highly effective in acute and latent mouse models of T. gondii infection, significantly reducing brain cysts by 88.7% (Vidadala et al., 2016).
Many studies reported anti-Toxoplasma effects of different drugs combination with novel compounds. The compound 2-hydroxy-3-(1 ′ -propen-3-phenyl)-1, 4-naphthoquinone (PHNQ6), (50 mg/kg/day) combined with sulfadiazine showed reduction or elimination of brain cysts in vivo (Ferreira et al., 2006). In another study that coadministered spiramycin and metronidazole, spiramycin, did not reach effective concentrations in the brain due to the presence of the efflux transporters multidrug-resistant protein 2, and P-glycoprotein. Metronidazole increased brain penetration of spiramycin, causing a significant reduction of T. gondii brain cysts, with potential clinical translatability for chronic toxoplasmosis treatment. According to the reports, combination therapy leads to faster recovery, less relapse, lower doses of drugs, and fewer side effects of the disease. Furthermore, such combinations are highly promising to develop a drug that is able to eliminate the cyst stage of the parasite, and thus, efficiently impairs relapse of the disease (Chew et al., 2012;Martins-Duarte et al., 2013).

Activity of Drugs against Congenital Toxoplasmosis
In pregnant women, current toxoplasmosis treatment is based on the administration of spiramycin or a drug combination such as sulphadiazine-pyrimethamine-folinic acid (SPFA) in cases of confirmed fetal infection. However, these drugs are not welltolerated and present many adverse effects due to their toxic effects to the host (Degerli et al., 2003).
Degerli et al. evaluated the effectiveness of azithromycin, artemisia annua infusion, spiramycin, and SPFA in Calomys callosus, such as model of congenital toxoplasmosis. The results demonstrated that the treatment of pregnant C. callosus with azithromycin was effective for inhibiting the vertical transmission of T. gondii ME49 strain, suggesting that it may be an alternative drug of choice for the treatment of congenital infection, since it is able to inhibit fetal infection and offers new perspectives for the treatment of congenital toxoplasmosis. Azithromycin is one of the new generation macrolides with numerous advantages. Mechanism of action of azithromycin is based on the inhibition of protein synthesis in both T. gondii tachyzoite and bradyzoite stages (Degerli et al., 2003), but it may present limited effectiveness against T. gondii, requiring high drug concentrations (Costa et al., 2009). In another study, Oz et al. reported that combined atovaquone and diclazuril therapy is a novel synergistic prophylactic and therapeutic approach to fetal maternal toxoplasmosis (Oz, 2014a). Atovaquone, an inhibitor of mitochondrial electron-transport processes, is an FDA-approved toxoplasmosis therapy but not for use in congenital toxoplasmosis treatment (Oz, 2014a). Another compound, diclazuril, and its related benzeneacetonitriles have long been used in the treatment and prevention of poultry and livestock coccidiosis. In addition, it is known to be a safe compound at therapeutic dose levels (Assis et al., 2010).

Adverse Effects of Drugs
However, anti-Toxoplasma effects of drugs/compounds were reported in many trials, but prednisolone increased the number of tachyzoites and bradyzoites in immunosuppressed infected mice (Puvanesuaran et al., 2012). In addition, betamethasone can escalate the invasion of tachyzoites, in cell culture. It could be suggested that patients under prolonged use of betamethasone and prednisolone should be protected against T. gondii infection. Also, if individuals receiving betamethasone are infected with T. gondii, interferon-gamma may be used to reduce the invasion of tachyzoites (Ghaffarifar et al., 2006).

CONCLUSIONS
As current chemotherapy against toxoplasmosis is still not satisfactory, the development of well-tolerated and safe specific immunoprophylaxis in relaxing the need of dependence on chemotherapeutics is a highly valuable goal for global disease control. Immunotherapeutics strategies for improving toxoplasmosis control could either be a vaccine which would induce strong protective immunity against toxoplasmosis, or passive immunization in cases of disease recrudescence. However, with the increasing number of high-risk individuals, such as immunocompromised patients, and absence of a proper vaccine, continued efforts are necessary for the development of novel treatment options against T. gondii. Some of the novel compounds reviewed here may represent good starting points for the discovery of effective new drugs. In further bioinformatic and in silico studies are needed in order to identify new potential toxoplasmicidal drugs.

AUTHOR CONTRIBUTIONS
AD and MS conceived the idea for this review. MM and SS searched the databases for potentially eligible articles based on their titles and abstracts. AD and MM participated in the study design and wrote the manuscript. SM and EA critically reviewed the manuscript. All authors read and approved the final manuscript for publication.