Introduction

Toxoplasma gondii, an obligate intracellular parasite belonging to the phylum Apicomplexa, is one of the most successful parasitic organisms in the world, which can infect many warm-blooded vertebrates including humans (Dubey and Jones 2008). It is estimated that up to one third of the human population is infected with T. gondii, which is acquired mainly by ingesting tissue cysts from undercooked meat or ingestion of food or water contaminated with oocysts shed from cats (as the definitive hosts). However, other routes of transmission include vertical transmission from mother to child, organ transplantation, blood transfusion, and inhalation of oocyst-contaminated dust. This ubiquitous parasitic protozoan is the etiologic agent of toxoplasmosis, which causes the greatest disease burden of foodborne pathogens in the developed countries. T. gondii, if not treated, is the second leading cause of death due to foodborne diseases in these countries (Havelaar et al. 2012; Scallan et al. 2011).

Tachyzoites, bradyzoites (tissue cyst form), and sporozoites are three infectious forms of the T. gondii parasite (Dubey et al. 1998). Tissue cysts, intracellular structures formed by bradyzoites, divide by endodyogeny (Ferguson et al. 1994). The size of a tissue cyst is variable, but on average, a mature cyst can range from less than 10 to 70 μm in diameter and consist of several to hundreds or even thousands of bradyzoites (Dubey 1977). The development of tissue cysts is more common in the brain, eyes, and muscles (e.g., skeletal and cardiac tissues); however, they can also develop in visceral organs (e.g., lungs, liver, and kidneys). Tissue cysts are especially prevalent in the central nervous system (CNS). They have been detected in neurons, astrocytes, and microglia (Dubey 1988). Stage conversion from tachyzoites to bradyzoites allows the life-long persistence of the T. gondii parasite in the host (Weiss and Kim 2000).

When chronically infected patients become immunocompromised with conditions such as AIDS or due to the medication process after organ transplantation, bradyzoites get released from tissue cysts, multiply, and spread to other organs, predominantly the brain and muscles, resulting in severe morbidity and mortality (Luft and Remington 1992). Unfortunately, cyst walls are resistant to both the immune system and drugs (Gormley et al. 1998). While several drugs are available that can control acute toxoplasmosis (Montazeri et al. 2017), there is no approved therapy that eliminates the tissue cysts responsible for chronic infections (Montazeri et al. 2016). Accordingly, the current systematic review was aimed at retrieving published studies related to in vitro and in vivo evaluation of antimicrobial agents for the treatment of chronic toxoplasmosis in humans or animals against tissue cysts in order to prepare comprehensive data for designing more accurate investigations in the future.

Methods

This review followed the Preferred Reporting Items for Systematic Reviews (PRISMA) guidelines (Moher et al. 2009). A protocol of this systematic review is available in PROSPERO International prospective register of systematic reviews (2013), CRD42017072655 (https://www.crd.york.ac.uk/prospero/).

Search strategy

English databases, including PubMed, Science Direct, Scopus, Google Scholar, and Web of Science, were systematically searched for articles related to in vitro or in vivo evaluation of anti-Toxoplasma activities of drugs and compounds published up to December 2017. The used keywords consisted of “Toxoplasma gondii,” “T. gondii,” “toxoplasmosis,” “drugs,” “compounds,” “tissue cysts,” “bradyzoites,” and “chronic infection.”

Study selection and data extraction

According to the inclusion criteria, papers written in English were selected and carefully reviewed for eligibility. Gray literature and abstracts of articles that were published in congresses were not explored. In addition, to avoid missing any articles, entire references of the papers were meticulously hand-searched. Among English articles that were found using the mentioned strategies, full-text papers that used laboratory methods both in vitro and in vivo for the detection of chronic infections were included. The included papers were precisely investigated, and the main information was extracted.

Results

Analysis of the included literature

A total of 55 papers (13 in vitro studies, 49 in vivo, and 7 both in vitro and in vivo) published in three decades from 1987 to 2017 were included in the systematic review. Figure 1 briefly shows this article’s search process.

Fig. 1
figure 1

The PRISMA flowchart describing the study design process

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In vitro and in vivo results

The efficacy of 50 drugs and several new compounds against T. gondii in vitro and in vivo was evaluated (Tables 1 and 2). Also, drugs or compounds with more than 20 pathways or mechanisms of action are shown in Table 3. Notably, several targets were identified against T. gondii including mitochondrial electron transport chain, calcium-dependent protein kinase 1, type II fatty acid synthesis, DNA synthesis, and protein synthesis. Our collected data indicated that many of the drugs or compounds evaluated against T. gondii cysts act on the mitochondria and apicoplast. Therefore, these organelles represent a potential drug target for new chemotherapy.

Table 1 Summary of in vitro studies that evaluated the anti-Toxoplasma activity of drugs/compounds against tissue cysts
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Table 2 Summary of in vivo studies that evaluated the anti-Toxoplasma activity of drugs/compounds against tissue cysts
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Table 3 Pathway/mechanism of action of drugs/compounds used against tissue cysts T. gondii
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Most of the in vitro and in vivo investigations on the activity of drugs against the T. gondii cyst were based on their infectivity by subinoculation to mice (5 studies) and counting the number of brain cysts (40 studies). Most of the surveys used the ME49 strain of T. gondii (4 in vitro studies and 28 in vivo) while in some studies, Prugniaud, EGS, and VEG strains were also used. The animal model used in the reviewed papers was mostly Swiss Webster mice (22 studies).

Discussion

The aim of this systematic review was to investigate the in vitro and in vivo effects of anti-Toxoplasma drugs and synthetic compounds against tissue cysts. The previous studies in 1992, 1994, and 1998 suggested that atovaquone had activity on cyst tissue in vivo (Araujo et al. 1992a; Ferguson et al. 1994; Gormley et al. 1998); however, clinical cases of resistance to this drug have been reported (Baatz et al. 2006; Megged et al. 2008). Unfortunately, Toxoplasma has a strong ability to spontaneously develop drug resistance by mutation of the atovaquone binding site on cytochrome b, and thus, this drug has never become a first-line treatment for chronic toxoplasmosis (Chirgwin et al. 2002).

In a new study, Vidadala et al. (2016) optimized compound 32 (T. gondii calcium-dependent protein kinase 1 inhibitor [CDPK1]), a promising lead for the development of a new antitoxoplasmosis therapy in the acute and latent stages of infection. Interestingly, this compound does not have human Ether-à-go-go-Related Gene (hERG) inhibitory activity. Moreover, compound 32 is a CNS-penetrant and can significantly reduce brain cysts by 88.7%.

In a recent study by Rutaganira et al. (2017), the effect of small molecule inhibitors of CDPK1 for the treatment of CNS toxoplasmosis was examined. In this study, compound 24 was effective in treating acute and chronic infection, reducing propagation to the CNS, and decreasing reactivation of chronic toxoplasmosis in immunocompromised mice. CDPK1, an essential enzyme in T. gondii, controls multiple processes that are critical for the intracellular replicative cycle of the parasite, including secretion of adhesins, motility, invasion, and egression. Based on these studies, CDPK1 inhibitors can be represented as the potential drugs for new chemotherapy methods.

In another study by Doggett et al. (2012), researchers showed the remarkable efficacy of endochin-like quinolone (ELQ-271 and ELQ-316) in decreasing T. gondii brain cysts by up to 88% at low doses, suggesting that they have the potential to eradicate latent infection at clinically applicable doses. ELQ-271 and ELQ-316 are inhibitors of the Qi site of the T. gondii cytochrome bc1 complex, and their mechanism of action differs from that of current clinically used anti-Toxoplasma therapies.

Recently, investigators have focused on miltefosine, an anticancer agent, which was demonstrated to result in significant reduction in brain cyst load in the chronic stage of toxoplasmosis. Also, the survived cysts were noticeably smaller upon microscopic examination, suggesting that this drug can effectively penetrate the blood–brain barrier and that the prolongation of treatment time may result in greater effects (Eissa et al. 2015). Additionally, future studies should focus on the mechanism of action of miltefosine against the T. gondii cyst form in chronic toxoplasmosis.

In two studies performed by Afifi et al., on the Toxoplasma brain cyst load, 74% reduction in cystic forms in the chronic phase of toxoplasmosis after treatment with rolipram, a phosphodiesterase-4 (PDE4) inhibitor, has been shown. Cyclic nucleotide phosphodiesterases are critical modulators of cellular levels of cAMP, which catalyzes cyclic nucleotide hydrolysis, since rolipram causing high cAMP levels can inhibit Toxoplasma’s conversion to the bradyzoite form. Additionally, rolipram could interfere with tachyzoite–bradyzoite interconversion due to suppression of cytokines TNF-α, IFN-γ, and IL-12. However, rolipram was also partially able to prevent progression to chronic toxoplasmosis. Clinical studies have reported adverse effects of this drug, mostly severe nausea and vomiting (Afifi and Al-Rabia 2015; Afifi et al. 2014; Eissa et al. 2015). It is suggested that investigators should focus on finding safe anti-Toxoplasma drugs in the future.

Guanabenz, a Food and Drug Administration (FDA)-approved drug, has excellent solubility and penetration into the CNS (Bougdour et al. 2009; Meacham et al. 1980). In a study by Benmerzougas et al. (2015) in chronically infected mice, guanabenz crossed the blood–brain barrier and reduced the number of brain cysts. Also, guanabenz inhibited the phosphorylation of T. gondii eukaryotic initiation factor 2α (eIF2 α), a novel antiparasitic drug target, and its ability to kill the Toxoplasma parasite did not involve the host’s eIF2α. T. gondii IF2α phosphorylation occurs in response to stresses, which induces conversion of tachyzoites to bradyzoites during the lytic cycle in tachyzoites (Konrad et al. 2013; Meacham et al. 1980).

The ability of compound 32, endochin-like quinolones, miltefosine, and guanabenz to penetrate the blood-brain barrier is the important criteria for therapeutic intervention as tissye cysts have a propensity to form in the brain (Benmerzouga et al. 2015; Doggett et al. 2012; Eissa et al. 2015; Vidadala et al. 2016).

The cyclopeptide FR235222 appears to be a bradyzoite to tachyzoite conversion inhibitor, and preventing the parasite differentiation process could be an effective way to prevent the parasite from spreading. FR235222 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 the CNS tissues where Toxoplasma cysts are located. It is shown that histone acetylation levels are controlled by histone acetylase (HAT) and histone deacetylase (HDAC) enzymes, and the specific inhibition of T. gondii histone deacetylase (TgHDAC3) by FR235222 disrupts the steady-state level of histone 4 (H4) acetylation across the genome, inducing derepression of stage-specific genes. Thus, acetylation of histones plays a substantial role in the control of gene expression during parasite interconversion (Bougdour et al. 2009; Maubon et al. 2010).

In a study by Ferreira et al. (2012), anti-Toxoplasma properties of new naphthoquinones (QUI-11, QUI-6, and QUI-5) were evaluated. In vitro incubation with QUI-11 resulted in the inhibition of infectivity of the bradyzoites; none of the surviving animals had detectable cysts in their brains. This suggests that this drug may be useful in treating chronic toxoplasmosis.

Recently, Murata et al. (2017) identified that tanshinone IIA and hydroxyzine represent novel lead compounds in preventing the reactivation of latent infection. These novel anti-Toxoplasma compounds can inhibit the growth of intermediately differentiated bradyzoites.

However, FR235222, QUI-11, tanshinone IIA, and hydroxyzine showed anti-Toxoplasma cyst effects in vitro (Ferreira et al. 2012; Maubon et al. 2010; Murata et al. 2017); their effectiveness in vivo against chronically infected mice remains to be directly demonstrated. Additionally, future studies should focus on the mechanism of action of QUI-11, tanshinone IIA, and hydroxyzine against the T. gondii cyst stage in chronic toxoplasmosis.

In a new study by El-Zawawy et al. (2015), it was shown that triclosan (TS) significantly reduced mice mortality, parasite load, as well as viability and infectivity of tachyzoites and the cysts that were harvested from infected mice and their brains in the treatment group. Accordingly, TS was proven as an effective, promising, and safe prophylactic drug against chronic murine toxoplasmosis. Liposomal formulation of TS enhanced its efficacy and allowed its use at a lower dose (El-Zawawy et al. 2015; Surolia and Surolia 2001). 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 cannot be found in mammals. This enzyme catalyzes the last reductive step of the type II FAS pathway. Significantly, TS inhibits type II FAS, suggesting that apicoplast represents a potential target for new chemotherapy drugs as it is essential for the parasite and it is absent in host cells (El-Zawawy et al. 2015; Surolia and Surolia 2001).

Interestingly, investigators in a study showed the effectiveness of toltrazuril treatment in lambs. The results of this study showed that muscle tissues of lambs receiving toltrazuril were free of tissue cysts (44.4%). The outcomes are promising as one of the paths of getting infected with this parasite is through consumption of undercooked or raw meat containing tissue cysts, and this could be used as a strategy to reduce the cyst exposure of humans (Kul et al. 2013). Given that Toxoplasma human infections depend on the prevalence of the parasite in animals and eating habits, production of T. gondii-free sheep, lambs, and goats for human consumption is important for public health.

Many studies described anti-Toxoplasma effects of different drugs in combination with novel compounds. The compound 2-hydroxy-3-(1′-propen-3-phenyl)-1, 4-naphthoquinone (PHNQ6), combined with sulfadiazine, showed reduction of the brain cysts in vivo (Ferreira et al. 2006).

In another study by Chew et al. (2012), administration of spiramycin and metronidazole, due to the presence of the efflux transporters multidrug-resistant protein 2 and P-glycoprotein spiramycin, did not result in an effective concentration in the brain. Importantly, metronidazole increased brain penetration of spiramycin causing a significant reduction of T. gondii brain cysts. According to the information, combination therapy leads to faster recovery, using lower doses of drugs, less relapse, and fewer side effects of the disease. Furthermore, such combinations are highly promising for the development of a drug that can eliminate the cyst form of the parasite and, thus, efficiently impair relapse of the disease in immunocompromised patients (Chew et al. 2012; Ferreira et al. 2006).

The particular resistance of cysts to drugs could be explained by two characteristics: the presence of the cyst walls and the low metabolism of bradyzoites compared to tachyzoites. Despite the importance of the tissue cyst in the life cycle of the parasite, only a few components of the T. gondii cyst wall and their functions have been identified. However, bradyzoite pseudokinase 1 (BPK1) is a component of the cyst wall. The expression of BPK1, specifically in the bradyzoite stage, suggests that it may have an important function in the bradyzoite biology and structure or the function of the tissue cyst in the life cycle of T. gondii (Buchholz et al. 2013).

Treatment of the T. gondii-infected cell cultures with atovaquone in combination with 3-bromopyruvate (3-BrPA), an inhibitor of cellular energy metabolism, led to fewer parasite-infected cells with no evidence of cystogenesis. However, the infection was not completely eliminated, and the apicoplast is possibly another energy source for T. gondii. This organelle is important in the parasite metabolism as it is the site of biosynthesis of fatty acid type II, isoprenoids, and some enzymes of carbohydrate metabolism. Based on these results, 3-BrPA can be used as a good tool for the study of cystogenesis in vitro and for gaining more knowledge regarding T. gondii parasite metabolism (de Lima et al. 2015).

Conclusions

In conclusion, as bradyzoites located inside the T. gondii cysts are resistant to all drugs, development of well-tolerated and safe specific immunoprophylaxis is a highly valuable goal for global disease control. Importantly, with the increasing number of high-risk individuals, and absence of a proper vaccine, persistent efforts are necessary for the development of novel treatments in patients with T. gondii cysts. Future studies should focus on the mechanisms of action of drugs or compounds that have sterilizing activity against the T. gondii cyst form in chronic toxoplasmosis in patients with cysts who are at risk for reactivating acute toxoplasmosis.