Review

Human toxocariasis: current advances in diagnostics, treatment, and interventions Gustavo Marc¸al Schmidt Garcia Moreira1, Paula de Lima Telmo1,2, Marcelo Mendonc¸a1, Aˆngela Nunes Moreira3, Alan John Alexander McBride1, Carlos James Scaini2, and Fabricio Rochedo Conceic¸a˜o1 1

Centro de Desenvolvimento Tecnolo´gico/Biotecnologia, Universidade Federal de Pelotas, CP 354, CEP 96010-900, Pelotas, RS, Brasil 2 Faculdade de Medicina/Laborato´rio de Parasitologia, Universidade Federal de Rio Grande, General Oso´rio, S/N, CEP 96200-190, Rio Grande, RS, Brasil 3 Faculdade de Nutric¸a˜o, Universidade Federal de Pelotas, CP 354, CEP 96010-900, Pelotas, RS, Brasil

Toxocariasis is a neglected zoonosis caused by the nematodes Toxocara canis and Toxocara cati. This disease is widespread in many countries, reaching high prevalence independently of the economic conditions. However, the true number of cases of toxocariasis is likely to be underestimated owing to the lack of adequate surveillance programs. Although some diagnostic tests are available, their sensitivity and specificity need to be improved. In addition, treatment options for toxocariasis are limited and are non-specific. Toxocariasis is listed as one of the five most important neglected diseases by the CDC. This review presents recent advances related to the control of toxocariasis, including new immunodiagnostics, therapies, and drug formulations, as well as novel interventions using DNA vaccines, immunomodulators, and probiotics. Toxocariasis spread and infection Human toxocariasis is a chronic parasitosis with a cosmopolitan distribution that is found mainly in developing countries with a tropical climate. However, the prevalence of this zoonotic disease and its impact on public health are underestimated [1], even in developed countries [2]. This is due to the lack of symptoms presented by the majority of infected individuals. Epidemiological studies performed in Latin America indicated high exposure of children to this disease, with a prevalence ranging from 28.8 to 62.3% [3–12]. Because contact with soil and animals is necessary for transmission, rural areas tend to exhibit higher prevalence (35–42%) than semi-rural (15– 20%) or urban (2–5%) areas. However, urban areas, particularly parks and town squares, have been shown to contain high numbers of Toxocara eggs [5,13]. This information has resulted in toxocariasis being considered one Corresponding author: Conceic¸a˜o, F.R. ([email protected]). Keywords: Toxocara canis; Toxocara cati; toxocariasis; immunodiagnostics; drug modification; probiotics. 1471-4922/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.pt.2014.07.003

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of the most important neglected diseases, mainly in the Americas, owing to the optimal climate for larval development and the high number of people living under conditions of poverty (see: http://www.cdc.gov/parasites/ toxocariasis/epi.html) [2]. Most cases of human toxocariasis have been associated with parasitism caused by Toxocara canis, an intestinal parasite of dogs. By contrast, the importance of T. cati, an intestinal parasite of cats, as an etiologic agent of this disease has been underestimated [7,8]. Thus, there is a need for more epidemiological studies regarding these two species and their importance. When dogs and cats consume embryonated eggs, many larvae are released inside the body and reach the small intestine [4,14], where they become adults and start to oviposit. Sandboxes, playgrounds, beaches, parks, and town squares contaminated with the feces of infected dogs and cats are the main source of human transmission. Humans are accidental hosts and are infected by ingesting Toxocara eggs, which hatch in the intestine and release larvae into the lumen. However, other sources of contamination have been reported, including the consumption of viscera and raw or undercooked meats from paratenic hosts, such as chickens [15] and cattle [16]. Furthermore, vertical transmission can also occur in humans, as seen in dogs and cats. The first record of congenital infection occurred in a premature neonate who developed retinopathy [17]. This highlights the need for prenatal diagnosis of pregnant women and in newborn children. Of note, vertical transmission in an experimental mouse model has provided new perspectives on the immunologic modulation in the mother and offspring, which will hopefully lead to more effective treatments [18–20]. Although the human intestine does not offer appropriate conditions for the development of adult parasites, the larvae can penetrate the small intestine and then reach the circulation where they spread by the systemic route. The larvae migrate throughout the body but cannot mature, and instead encyst as second-stage larvae [8]. Formation of cysts in the liver, lungs, heart, and/or lymph nodes is

Review Glossary ABZ/CH: chitosan-encapsulated albendazole. Chitosan is a polysaccharide composed by N-acetyl-D-glucosamine. Because it is hydrophobic in neutral environments, chitosan is ideal to carry the also insoluble ABZ drug. In acidic environments, chitosan becomes soluble and degrades, making it a promising option for protecting this drug in neutral and basic environments, and for performing efficient drug delivery to acidic locations such as the stomach. ABZ/PEG: pegylated albendazole. By combining the already used drug ABZ with PEG it is expected to increase the efficiency of the treatment because PEG increases drug stability by protecting it from macrophage degradation and thus extends the half-life of the drug. Furthermore, PEG increases ABZ bioavailability by augmenting its solubility. ABZ/PEG-LE: pegylated liposome-encapsulated albendazole. This formulation uses liposomes to trap the insoluble ABZ. Liposomes are biocompatible spherical lipid bilayers that can carry either soluble or insoluble molecules. ABZ is carried in the hydrophobic environment between the two layers. In addition, the liposomes are pegylated to protect the complex from macrophage degradation. Gradual release of ABZ from the complexes increases the effective half-life of the drug. ABZ: albendazole. A broad-spectrum anthelmintic drug used as the first option to treat different parasitic infections. C17/PEG-LE: pegylated liposome-encapsulated compound 17. This formulation uses a liposome to trap the insoluble C17. C17 is carried in the hydrophobic environment within the liposome bilayer. Liposome pegylation protects the complex from macrophage degradation, and gradual release of C17 increases the effective half-life of the drug. C17: a b-carboline alkaloid extracted from plants. In the described study, this compound was chemically synthesized and showed some effect against toxocariasis. Coproscopy: laboratorial analysis of feces. Because most human parasites infect through the intestine, this is the easiest way to verify infections. Detection can be carried out both by microscopy, with direct visual identification of the parasite, or by molecular procedures such as PCR to determine the presence of parasites in the samples. CT: covert or common toxocariasis. This is one of the four proposed forms of the disease and is characterized by mild symptoms such as abdominal pain, behavioral change, cough, headache, and sleeping problems. Similarly to another form of the disease, VLM, the parasite can reach almost any organ of the host, including muscle, liver, intestine, lungs, and heart. Dot-ELISA: enzyme-linked immunosorbent assay (ELISA) performed on nitrocellulose membranes. This technique is similar to conventional ELISA, but the tested antigens are adsorbed to a nitrocellulose membrane instead of onto an ELISA plate. In the case of Toxocara spp. Dot-ELISA, TES antigens obtained by the De Savigny method were adsorbed to the membrane and tested against human serum samples. Embryonation: a developmental process comprising the appearance of an embryo. In the case of Toxocara spp., embryonation can be observed when its eggs are incubated in appropriate conditions for parasite development and a small larva (the embryo) can be visualized under the microscope. When producing TES antigens, an embryonation rate of 70% or more is recommended for a high yield, meaning that at least 7 of 10 eggs must contain a small larva instead of a simple cellular mass when observed in a microscope. Enterococcus faecalis CECT 7121: a non-pathogenic strain of E. feacalis. It lacks most of the virulence genes, such as hemolysin and gelatinase, as well as the capsule. Furthermore, this strain has inhibitory activity against various enteropathogenic bacteria. In contrast to other strains, CECT 7121 contains only one plasmid that seems to play role on its activity against gut pathogens. These characteristics, together with the fact that it can colonize the intestinal epithelium, contribute to the safeness of its use as a probiotic. FBZ/PEG-LE: pegylated liposome-encapsulated fenbendazole. This formulation uses a liposome to trap the insoluble FBZ. FBZ is carried in the hydrophobic environment within the liposome bilayer. Liposome pegylation protects the complex from macrophage degradation, and gradual release of FBZ increases the effective half-life of the drug. FBZ: fenbendazole. A broad-spectrum anthelmintic drug used as an alternative treatment for different parasitic infections. Immunomodulator glucan: a polysaccharide composed of D-glucose monomers linked by b-glycoside bounds. Because they have atypical structures, they are recognized as foreign molecules and thus can activate the immune system. The goal of its use in treatment against toxocariasis is to improve the immune response against the parasite by increasing the activity of phagocytes. In the study described, this molecule was used together with ABZ or FBZ, conferring a second effect for the therapeutic formulation. Immunomodulatory compound: any molecule that can direct the immune response to a particular pathway. These molecules can be from the immune system itself, such as interleukins and cytokines, or from another source, such as those produced by microorganisms. In some cases, infection develops because the pathogen actively evades host immune responses. It is therefore necessary to manipulate the host immune system by using immunomodulatory compounds which activate the proper response against the pathogen.

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Larval recovery: the value (in %) of the number of larvae obtained after a simulated infection with a parasite. In experiments involving animal models, they are submitted to a simulated infection by ingestion of Toxocara spp. larvae. After a predetermined time, the animals receive the treatment for a specified period followed by necropsy. During necropsy, organs are taken, processed, and analyzed for the number of larvae recovered. The percentage is obtained by comparing the number of larvae between the treated and control (non-treated) groups. Mamilonated membrane removal: an essential step in native TES production. The mamilonated membrane is the outer and most important layer of Toxocara spp. eggs. Its structure is composed of protein–chitin, which serves as protection against the host and the environment. When a larva completes its formation inside the egg, the layer is naturally disrupted, thus allowing it to escape the egg. In native TES production, however, it is recommended to destabilize the mamilonated membrane, usually with sodium hypochlorite before hatching the eggs, such that release of larvae is more efficient. Microparticle: any particle or small object that measures 0.1–100 mm. In a biological field, these particles are primarily formed from biopolymers such as carbohydrates or lipids. These biopolymers are often used to create new drug formulations, which can increase the efficiency of an already used drug by, for example, decreasing toxicity or increasing the bioavailability and half-life. Native TES: native Toxocara spp. secretory-excretory antigens. These antigens are obtained by the De Savigny method, or its modifications, and consist of numerous glycoproteins with different molecular masses produced by the parasite and released into the body of the host. Moreover, they are highly immunogenic and are thus the best-studied components of Toxocara spp. NLM: neural larva migrans. One of the four proposed forms of the disease that is characterized by the parasite lodging in the brain. Because this organ is affected, location-related symptoms predominate, including fever, headache, and seizure. OLM: ocular larva migrans. One of the four proposed forms of the disease that is characterized by the parasite lodging in the eyes. Similarly to NLM, encapsulation of larva in this specific region triggers location-related symptoms including blindness, strabismus, retinal damage, and eye inflammation. Paratenic host: a host that can carry a parasite in the early stages of the life cycle but is not essential for its development. The paratenic host often accumulates high numbers of the parasite that are transferred to accidental hosts. In the case of Toxocara spp., paratenic hosts such as rabbits, chicken, or cattle can ingest embryonated eggs containing larvae, leading to accumulation of parasite burden in these species, that then serve as source of contamination for accidental hosts, such as human, for example by meat consumption. Pegylation: the addition of polyethylene glycol (PEG) to a drug or molecule. The covalent attachment of PEG can reduce the immunogenicity of a molecule and/or increase the half-life of a drug. Moreover, PEG is highly soluble in water and is thus widely used to prepare formulations with hydrophobic molecules. rTES-26, -30, and -120: recombinant Toxocara spp. secretory-excretory antigens with molecular masses of 26, 30, and 120 kDa, respectively. These glycoproteins are present in native TES extracts. TES-ELISA: enzyme-linked immunosorbent assay designed to detect Toxocara spp.. The method consists in the use of Toxocara spp. excretory-secretory (TES) antigens extracted by De Savigny method and adsorbed on an ELISA plate, allowing serum from patients to be tested for the presence of antibodies against the parasite. Vertical transmission: spread of a disease from mother to offspring. Also referred to as mother-to-child transmission, transmission can take place either during pregnancy or at the time of birth. VLM: visceral larva migrans. One of the four proposed forms of the disease in where, similarly to CT, the larva can encapsulate in many organs including the lungs, liver, and heart. VLM is characterized by more severe symptoms than CT and, moreover, is the only syndrome to have a higher prevalence in children aged 7 years (although adults also can be infected). The symptomatology related to VLM is highly variable because the parasite can lodge in different organs; major symptoms include abdominal pain, asthma, eosinophilia, fatigue, fever, headache, hepatomegaly, weight loss, diarrhea, and vomiting.

diagnosed as visceral larva migrans (VLM) (see Glossary). VLM is more common in children and the clinical symptoms include eosinophilia, hepatomegaly, asthma and its symptoms. If the Toxocara larvae migrate to the eye it is known as ocular larva migrans (OLM), but this is rare compared to VLM. OLM can include loss of vision, granulomas, and retinal damage. A more serious form of the disease is neural larva migrans (NLM), which presents with nonspecific symptoms such as fever, headache, and seizures [21]. Covert or common toxocariasis (CT) is similar to VLM, but its symptoms, including headache, abdominal pain, cough, sleeping problems, and behavioral 457

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change, are much less severe. These syndromes do differ in their symptomatic signals according to the affected tissue, and are independent of the infecting Toxocara spp. [4,22]. Following encapsulation of the larvae, reactivation of the encysted larvae is possible in immunocompromised individuals, and can lead to further migration and a new symptomatic phase. Within this context (asymptomatic, seropositive individual), ongoing clinical and serological monitoring of the patient is necessary for assessing the need for treatment because it can more precisely define the Toxocara spp. infection [23]. Diagnosis of toxocariasis Given the difficulty associated with the diagnosis and treatment of patients, some new approaches have been gaining attention in the control of human toxocariasis. Accordingly, this review discusses the current advances and perspectives related to the control of human toxocariasis. Coproscopy remains the preferred technique for the detection of gastrointestinal nematodes because it is rapid and affordable for most healthcare facilities. Molecular procedures associated with coproscopy recently allowed the detection of different nematode eggs in animal feces, including the discrimination of T. canis from T. cati [24– 26]. However, the development of adult parasites, which can oviposit in the gut lumen, does not occur in humans. Hence, the detection of eggs in human stool samples is redundant, even though the presence of other parasites

such as Ascaris lumbricoides and Trichuris trichiura is used to indicate fecal exposure and increases the likelihood of Toxocara larvae being present in host organs [27,28]. The diversity of clinical conditions associated with the different sites where Toxocara larvae can lodge makes it very difficult to diagnose toxocariasis. Thus, definitive diagnosis is performed by biopsy and visual detection of the parasite, and this is recognized as the gold standard [29]. Nonetheless, this procedure is generally not suggested because it is extremely invasive and depends on the larval load and the stage of the infection. Given this difficulty, different immunological methods have been developed. Immunodiagnosis with Toxocara spp. antigens The use of the Toxocara spp. excretory-secretory (TES) antigens in enzyme-linked immunosorbent assays (ELISA) [30] have long been used as a standard toxocariasis immunodiagnostic method [4]. The TES antigens are a group of glycoproteins secreted by the parasite larvae during its metabolism. They consist of diverse proteins that range from 30 to approximately 400 kDa and have a high content of carbohydrates [31,32]. Because they are highly immunogenic during infection, these proteins are the main target of studies involving diagnosis. However, their exact role in pathogenesis is not well known. Typically, positive results require confirmation by Western blotting [24]. DotELISA using native TES was recently described as an

Box 1. Traditional De Savigny method for TES antigen production The traditional method for obtaining TES antigens is based on the protocol described by De Savigny [25] and is depicted in Figure I. Although some modifications can be used, the basic process remains the same. It involves (1) the collection of female adult worms, usually after administering the anthelmintic piperazine that helps to expel live parasites from the body. (2) Female roundworms are then sorted in the laboratory and eggs are extracted from the uteri of pregnant roundworms. (3) Eggs are incubated in formalin solution to avoid contamination for approximately 1 month at 288C to allow embryonation to take place. This step requires approximately 70% efficiency (quantified by microscopy) (4) to provide sufficient native TES. After counting, mamilonated membrane removal with sodium hypochlorite (5) is necessary to allow the eggs

2

1

to hatch. After washing and centrifugation to remove sodium hypochlorite and mamilonated membrane, the hatching step (6) is performed in Roswell Park Memorial Institute (RPMI) medium at 378C, often in conjunction with vigorous agitation with glass beads to complete the disruption of the egg layers (6). Larvae are then separated in a Baermann apparatus (7), and the separated larvae further cultured (8) in RPMI medium for at least 7 days to generate TES antigens. The culture supernatant is then concentrated by ultrafiltration and dialyzed to remove residual medium that can interfere with diagnostic applications (9). The sample containing TES antigens is then filter-sterilized (10). Total proteins are then analyzed by SDS-PAGE and quantified before use. The entire process requires at least 60 days and specific training.

4

3

5

30–35 days

NaCIO 5% 5 min

CH2O 2–4% 28°C Collecon of adult parasites

6

Egg acquision

7

Embryonaon

Embryonaon count

8

9

Mamilonated membrane removal

10

Total ∼ 60 days

Filtraon

Protein quanficaon and use

At least 7 days

Hatching

Larvae separaon

Larvae culture

Concentraon and dialysis

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Figure I. Native TES antigen production by the method of De Savigny.

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Box 2. Generic process for recombinant protein production Escherichia coli is the preferred expression system because it is a well-known organism that can provide large amounts of recombinant protein in a relatively short period (Figure I). The standard process for protein expression involves the use of a plasmid containing the gene to be expressed. The plasmid, previously constructed by molecular biology techniques, is inserted into E. coli cells by heat-shock or electroporation (1). Transformed cells can be directly cultured in medium containing the proper selective agent, usually antibiotics, serving as a pre-inoculum to a larger culture. This larger culture then is used to perform protein expression (2), typically by adding an inducer of plasmid gene expression. After 3–16 h induction, cells are

disrupted (3) using chemical methods (e.g., lysozyme) and/or physical methods (sonication or French press) for harvesting of the recombinant protein. Soluble proteins may be present in the supernatant; insoluble proteins require solubilization with denaturing agents which may adversely effect protein folding. Recombinant proteins are typically expressed as fusions with a poly(His) tag, permitting rapid purification (4) by Ni2+ affinity chromatography; proteins are dialyzed before further use. Compared to De Savigny method for native TES production, the production of recombinant proteins in E. coli generally has a higher yield and is simpler and less timeconsuming, taking 5 days of work.

1

2

3

4

1 day

1 day

1 day

2 days

E. coli transformaon

Protein expression

Cell lysis and protein recovery

Protein purificaon

Total ∼5 days

Protein quanficaon and use

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Figure I. Obtaining recombinant TES using the E. coli system.

alternative but further investigation is needed [33]. The production of native TES antigen is laborious, time-consuming, requiring at least 2 months of intense work, and presents with a low yield [34]. Trained technicians [35] and the availability of adult female T. canis worms (Box 1) are necessary to perform the diagnosis. By comparison, the production of recombinant TES antigens (rTES) in an Escherichia coli system is much simpler, requiring no more than 5 working days, and represents a more controlled process (Box 2). Using this method, rTES production can be performed approximately six times in the same period as a single production process for native TES. Another drawback to using native TES is its cross-reactivity with antibodies generated against other helminthic infections [36,37], requiring prior incubation of sera with antigens derived from the related helminths to increase specificity [22]. The rTES have been produced and demonstrated [34,37,38] to provide better specificity, sensitivity, and applicability for the routine monitoring and diagnosing of toxocariasis. Western blotting based on the rTES-120 antigen produced in Pichia pastoris demonstrated cross-reactivity with sera from patients infected with parasites other than Toxocara spp. [39]. Although a secreted, glycosylated rTES antigen in P. pastoris seemed to be the logical choice, because native TES is glycosylated [40], the recombinant protein was produced in the cytoplasm, suggesting that the lack of glycosylation of the TES proteins does not affect test performance. However, because only a limited number of serum samples (n = 13) were evaluated, the potential of this recombinant antigen remains to be confirmed. Specificity improved when TES antigens with a lower molecular mass were used [36]. For example, rTES-30 produced in E. coli using cDNA from T. canis larvae showed promising results [41]. However, only 32 serum samples were evalu-

ated by Western blotting, and there was evidence of crossreactivity with Anisakis spp., another intestinal parasite. Another study with the same protein reported no crossreaction comparing rTES-30 with native TES in an ELISA format, using 153 serum samples from patients infected with 20 different helminths [37]. Of note, this study was carried out in Japan, where the Ascaris lumbricoides and hookworm (Ancylostoma spp. and Necator americanus), helminths that possess antigens similar to those from Toxocara spp., are not endemic. The sensitivity and specificity of TES- and rTES-ELISA was dependent on the combination of antigens used and the type of antibodies detected. Using commercially available TES-coated ELISA plates and the detection of IgG4 antibodies (a subclass of IgG), rather than total IgG, increased the specificity from 36 to 78.6% [42]. However, the sensitivity decreased from 97.1 to 45.7%. The use of rTES-30 and the detection of IgG4 also increased the specificity of the ELISA from 55.7 to 89.6% when compared to total IgG. Sensitivity was 92.3% and 100%, respectively [43]. In addition to rTES30, rTES-26 and -120, all produced in E. coli, were evaluated in an IgG4–ELISA [34]. The use of rTES-30 and rTES-120 resulted in 100% sensitivity (both proteins), with individual specificities of 93.9 and 92%, respectively. Although the rTES-26 ELISA had the highest specificity (96.2%), it had the lowest sensitivity (80.0%), suggesting that the combination of the rTES-30 and -120 antigens, together with IgG4 detection, was the best option for diagnosis of toxocariasis in an ELISA. However, because of the low sample sizes used in these studies, additional evidence is necessary to make conclusions concerning the specificities and sensitivities of the antigens. The use of a combination of semi-purified, native TES58 and TES-68 was evaluated [35] and, although only a small number of human samples (n = 20) were used, both 459

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Table 1. Detection of Toxocara spp. by ELISA Antigen a Native TES e rTES-30 Native TES Native TES rTES-30 Native TES rTES-26 rTES-30 rTES-120 TES-58 and -68 TCLA

Source b Produced in-house Escherichia coli, insoluble Cypress Diagnostics Cypress Diagnostics E. coli, soluble Cypress Diagnostics E. coli, soluble E. coli, soluble E. coli, soluble Produced in-house Produced in-house

Antibody detected IgG IgG IgG IgG4 IgG4 IgG IgG4 IgG4 IgG4 IgM or IgG IgG

Sensitivity c 100 (11/11) 100 (11/11) 97.1 (34/35) 45.7 (16/35) 92.3 (24/26) 100 (26/26) 80.0 (24/30) 93.3 (28/30) 93.3 (28/30) 100 (20/20) 92.2 (59/64)

Specificity d 57.0 (81/142) 97.9 (139/142) 36.0 (27/75) 78.6 (59/75) 89.6 (103/115) 55.7 (64/115) 96.2 (204/212) 93.9 (199/212) 92.0 (195/212) 100 (20/20) 86.6 (103/119)

Refs [36] [42] [43] [34]

[35] [44]

a

Native TES antigens include various glycoproteins obtained by the De Savigny method. Owing to concerns about its production, individual rTES antigens such as the 26, 30, and 120 kDa antigens have been produced and evaluated to improve the diagnostic value of the tests.

b

Knowing the source of the antigen is important in establishing the reproducibility of the results because each preparation of antigen can be different. The term ‘produced inhouse’ describes antigens (e.g., TES, TES-58, TES-68, or TCLA) obtained from parasites grown in the laboratory conducting the research. By contrast, the native TES from Cypress Diagnostics is a commercial ELISA plate with standard conditions for diagnosis, and this may increase reproducibility. The rTES antigens shown here were all produced in E. coli. This information, as well as the condition of the protein (soluble or insoluble), is important in maintaining the same parameters in further trials with these same antigens.

c

Sensitivity is expressed as a percentage (%). The value was reached by dividing the number of positive reactions (first number in parenthesis) by the number of true positive samples tested (second number in parenthesis).

d

Specificity is expressed as a percentage (%). The value was determined by dividing the number of negative reactions (first number in parenthesis) by the number of true negative samples tested (second number in parentheses).

e

Abbreviations: rTES, recombinant Toxocara spp. excretory-secretory antigen; TES, Toxocara spp. excretory-secretory antigen.

the sensitivity and specificity were 100% when detecting IgM or IgG (Table 1). A recent study described the use of crude antigens from T. canis larvae (TCLA) instead of TES. TCLA were tested using approximately twice the number of positive samples than other studies, and achieved 92.2 and 86.6% sensitivity and specificity, respectively [44]. This last study was more robust than those previously described, and was comparable to those using rTES in terms of the diagnostic values (Table 1). In terms of practicality, the production of TCLA eliminates the larval culture step, thereby decreasing the time required for antigen production compared to native TES (Box 1). However, once the larvae are dead, the acquisition of antigens is even more limited. As summarized in Table 1, the detection of IgG4 bound to native TES, rather than total IgG, increased specificity by 20–40%. Furthermore, the replacement of the native TES antigens with individual rTES further increased sensitivity by 30–50% and specificity by almost 20%. Substituting native TES with TCLA, with IgG detection, increased specificity in line with that reported for rTES (20%). The detection of IgG4 bound to TCLA could further improve specificity although this will require investigation with regard to the maintenance of sensitivity because these values are reduced when native TES is used. In addition, other antigens, such as TES-58 and 68, need further investigation with a larger panel of sera to be considered applicable. Considering that the use of rTES in an ELISA for the diagnosis of human toxocariasis appears to be the best option, further studies are required with a more representative panel of sera. Development of new therapies The treatment options for toxocariasis are limited and consist of the use of anthelmintics combined with 460

anti-inflammatories (http://www.cdc.gov/dpdx/toxocariasis/tx.html). There are two main goals while carrying out treatment: (i) to obtain a clinical resolution, and (ii) to reduce the level of larval migration to other organs, particularly the brain and eyes [23]. For treatment of VLM, anthelmintic drugs such as albendazole (ABZ, first option) and mebendazole (MBZ, second option) are indicated, despite their limited efficacy. Although anthelmintic treatment is still recommended in cases of OLM, antiinflammatory drugs are also necessary to reduce damage to the eyes [4,45]. Because the host immune response is hampered by the immune evasion mechanisms of the parasite, a novel intervention would be to overcome this evasion; however, the pathways of such suppression are not well elucidated [12,46,47]. The clinical studies evaluating the efficacy of ABZ and MBZ against Toxocara spp. were performed during the 1970s and 1980s, and the morerecent clinical studies did not evaluate any novel compounds for the treatment toxocariasis [48,49]. Owing to the lack of new drugs, efforts have focused on the formulation and delivery of anthelmintic drugs. To increase its effectiveness, a preparation of microparticles produced by a spraying technique using sodium lauryl sulfate containing chitosan-encapsulated ABZ (ABZ/CH) were evaluated in mice artificially infected with T. canis [45]. This formulation was able to eliminate completely Toxocara larval recovery from the brain and reduced larval migration to the liver and lungs by 72.22 and 75%, respectively [50] (summarized in Table 2). A variation using a polyethylene glycol (PEG)-conjugated (‘pegylated’) form of ABZ (ABZ/PEG) resulted in 100, 75.0, and 79.2% reductions in larval recovery from brain, liver, and lung, respectively [51]. In other studies, liposome-encapsulated ABZ stabilized with PEG (ABZ/PEG-LE) was able to lower the number of larvae in the liver and brain by 65.4 and 87.9%, respectively [52,53]. Moreover, the same strategy using fenbendazole

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Table 2. New drug formulation efficacies by organ and tissue Drug/vehicle a ABZ/CH d

ABZ/PEG

ABZ/PEG-LE ABZ/PEG-LE FBZ/PEG-LE ABZ/PEG-LE + glucan FBZ/PEG-LE + glucan C17/PEG-LE

Efficacy (%)b / Organ c 72.22 / liver 75.00 / lung 100.00 / brain 75.00 / liver 79.17 / lung 100.00 / brain 65.43 / liver 87.92 / brain 73.63 / skeletal muscle 92.22 / brain 91.55 / skeletal muscle 19.00 / brain 41.75 / skeletal muscle

Refs [50]

[51]

[52] [53]

[55]

a

Drug vehicles are structural components used widely to improve the effect of a particular therapeutic molecule by inhibiting its degradation, directing it to the correct site in the body, or by promoting long-lasting release and thus increasing the treatment time. In this table, different drug vehicles, used either alone or in combination, are shown.

b

The efficacy represents the percentage of the mean reduction in the larval recovery number in comparison to the controls of each study.

c

As described in the original studies, a treatment can also be effective in organs other than those shown in the table.

d

Abbreviations: ABZ, albendazole; CH, chitosan-encapsulated; PEG, polyethylene glycol (‘pegylated’); FBZ, fenbendazole; LE, liposome-encapsulated; C17, compound 17.

(FBZ/PEG-LE) showed interesting results for skeletal muscle, with 73.6% reduction [53]. Furthermore, including an immunomodulator (glucan) in the ABZ/PEG-LE or FBZ/ PEG-LE formulation improved the results for the brain and skeletal muscle: 92.2 and 91.6%, respectively. In addition to the established drugs, phytochemical compounds have been evaluated against toxocariasis. Studies using 17 different plant extracts from Picrasma quassioides and Ailanthus altissima (Simaroubaceae) [54] and the b-carboline alkaloids isolated from these extracts [55] found that one (compound 17, or C17) demonstrated the potential for treating toxocariasis. The C17/PEG-LE formulation resulted in larval reduction in the brain (19%) and skeletal muscle (41.75%) (Table 2). Comparison of ether and ethanol extracts from the leaves and fruits of Ficus obtusifolia Kunth (Moraceae) found that the ethanol extract was more effective against the adult parasite [56]. In another study, administration of a Nigella sativa (Ranunculaceae) extract (NSE) only, or NSE (100 mg/kg) plus ABZ (100 mg/kg), caused a reduction in inflammation and necrosis in the liver and lung [57]. Moreover, the systemic levels of the liver enzymes aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) were all reduced.

The ideal treatment for toxocariasis should eradicate all the larvae lodged in different organs – and not only decrease the intensity of infection, as occurs with ABZ, FBZ,and MBZ in animal models [51,53,55,58–60]. Furthermore, coadministration of immunomodulatory compounds (e.g., glycan) could be a good option to overcome the immunosuppression evoked after infection, but these formulations need to be explored further. New interventions against toxocariasis The real importance and the true prevalence of this neglected zoonosis can only be determined after the surveillance and reliable clinical diagnoses of a representative population [8]. Since the 1970s the World Health Organization (WHO) has provided recommendations for reducing the transmission of Toxocara spp. to humans. The main advice is to limit exposure to soil contaminated with Toxocara eggs, by reducing the presence of feces from infected dogs and cats, because they are the definitive hosts and the main epidemiological links in the chain of human toxocariasis [61]. Improved hygiene when preparing food can also help in avoiding toxocariasis [62]. This sanitary education is a slow but essential process for public awareness, and must involve both human healthcare as well as the control of stray dogs and cats [63]. Unfortunately, there is no vaccine available and the development of vaccines against toxocariasis remains a challenge. Probiotics represent a potential alternative for the prevention of toxocariasis, although to date most studies in this field are related to the prevention of bacterial diseases [64,65]. However, some studies reported a reduction in infection and protective effects against protozoan-related diseases, such as giardiasis and cryptosporidiosis [66,67], as well as helminthiases, such as trichinosis [68]. Moreover, a study that involved giardiasis found that a treatment consisting of both probiotics and anthelmintic drugs, such as ABZ, was better at reducing the establishment of the disease than the individual treatments alone [69]. A significant reduction in the number of larvae recovered in mice with acute toxocariasis was observed following treatment with the probiotic Enterococcus faecalis CECT 7121 for three consecutive days. The mice were challenged with 100 or 200 embryonated eggs of T. canis [70,71] (Table 3). The mechanism of action appears to be a consequence of a larvicide effect generated directly by E. faecalis CECT 7121, as demonstrated in vitro [71]. Although its safety and potential use in the control of toxocariasis have been confirmed, a probiotic comprising an E. faecalis strain may not be accepted by society because many pathogenic strains are known for this bacterium [72–74].

Table 3. New interventions and their main effects against toxocariasis Intervention DNA-based Probiotics a

pcDNA3-CpG pcDNA3-IL-12 Enterococcus faecalis CECT 7121 Saccharomyces boulardii

Effect Reduction of airway hyper-responsiveness Reduction of blood and lung eosinophilia 90% reduction of larvae number in liver and lungs 48 h after infection 36.84% reduction of larvae number in liver and lungs 72 h after infection 41.94% reduction of larvae number in the liver 2 days after infection 34.04% reduction of larvae number in the brain 60 days after infection

Refs [77] [69] [70] [75]

a

The dietary schedule varied depending on the study.

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Review By contrast, probiotics based on Saccharomyces boulardii, a GRAS (‘generally recognized as safe’) yeast, have already been used in human health for the prevention and treatment of gastrointestinal problems [75]. As reported recently, S. boulardii caused a reduction in the intensity of infection in mice with acute (36.7%) and chronic (35.9%) visceral toxocariasis [76]. Moreover, the use of this probiotic in an acute infection caused a 41.9% reduction in larval counts in the liver, and it reduced the number of larvae in the brain by 34.0% during chronic infection (Table 3). Instead of exerting a direct mechanism of action, such as larvicidal activity, it appears that the S. boulardii probiotic increased the integrity of the intestinal mucosal, thereby hindering the penetration of T. canis larvae [77]. Although only two species of probiotics have been evaluated to date, namely E. faecalis and S. boulardii, their use in the control of human toxocariasis appears promising. However, further studies with individual species or in combination or association with anthelmintic compounds are necessary. Another promising strategy for managing toxocariasis was based on the use of DNA-based vaccines together with immunomodulators. This strategy was evaluated in a mouse model of toxocariasis using mammalian expression vectors carrying unmethylated CpG motifs or interleukin 12 (IL-12) [78]. Vaccination significantly reduced airway hyper-responsiveness, whereas pcDNA3/IL-12 only blocked blood and lung eosinophilia after T. canis infection [76]. These data suggest that pcDNA3/CpG and pcDNA3/ IL-12 have distinct benefits in relation to eosinophilia and airway hyper-responsiveness, suggesting that the combination of the two agents has potential as an intervention against toxocariasis (Table 3). Concluding remarks and future perspectives The CDC considers toxocariasis to be one of the five most important neglected parasitic diseases – together with Chagas disease, neurocysticercosis, toxoplasmosis, and trichomoniasis. Although toxocariasis has a low incidence in humans, it is important to consider that the data related to this disease are probably underestimated and, not by chance, it is considered to be a neglected disease. Increasing contact with animals, mostly cats and dogs, allied to the lack of an efficient diagnosis, have contributed to the spread of Toxocara spp. Poor sanitation and the everincreasing number of individuals living in slum conditions throughout the world is another factor that will increase the potential for infection. Herein, innumerous efforts for the detection, treatment, and prevention of toxocariasis are presented and discussed. Advances in immunodiagnostic tools, focusing on recombinant antigens, seem to represent the best approach for the development of novel diagnosis assays for human toxocariasis. Furthermore, reports of new therapies and novel formulations of existing drugs for the efficient treatment and elimination of Toxocara larvae are promising. Lastly, recent studies using probiotics, immunomodulators, and DNA vaccination represent alternatives for the prevention of toxocariasis. Despite these recent studies on toxocariasis, more research is necessary to understand more fully the 462

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76 Avila, L.F. et al. (2013) Protective effect of the probiotic Saccharomyces boulardii in Toxocara canis infection is not due to direct action on the larvae. Rev. Inst. Med. Trop. Sao Paulo 55, 363–365 77 Malheiro, A. et al. (2008) pcDNA-IL-12 vaccination blocks eosinophilic inflammation but not airway hyperresponsiveness following murine Toxocara canis infection. Vaccine 26, 305–315 78 Woodhall, D.M. et al. (2014) Neglected parasitic infections in the United States: toxocariasis. Am. J. Trop. Med. Hyg. 90, 810–813