artigo toxocaras

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TREPAR-1305; No. of Pages 9
Human toxocariasis: current advances
in diagnostics, treatment, and
interventions
Gustavo Marçal Schmidt Garcia Moreira1, Paula de Lima Telmo1,2,
Marcelo Mendonça1, Ângela Nunes Moreira3, Alan John Alexander McBride1,
Carlos James Scaini2, and Fabricio Rochedo Conceição1
1 Centro de Desenvolvimento Tecnoló gico/Biotecnologia, Universidade Federal de Pelotas, CP 354, CEP 96010-900, Pelotas, RS,
Brasil
2 Faculdade de Medicina/Laborató rio de Parasitologia, Universidade Federal de Rio Grande, General Osó rio, S/N, CEP 96200-190,
Rio Grande, RS, Brasil
3 Faculdade de Nutrição, Universidade Federal de Pelotas, CP 354, CEP 96010-900, Pelotas, RS, Brasil
Review
Toxocariasis is a neglected zoonosis caused by the
nematodes Toxocara canis and Toxocara cati. This dis-
ease 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 ade-
quate surveillance programs. Although some diagnostic
tests are available, their sensitivity and specificity need
to be improved. In addition, treatment options for tox-
ocariasis are limited and are non-specific. Toxocariasis is
listed as one of the five most important neglected dis-
eases 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 cos-
mopolitan distribution that is found mainly in developing
countries with a tropical climate. However, the preva-
lence of this zoonotic disease and its impact on public
health are underestimated [1], even in developed coun-
tries [2]. This is due to the lack of symptoms presented by
the majority of infected individuals. Epidemiological
studies performed in Latin America indicated high expo-
sure 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, par-
ticularly parks and town squares, have been shown to
contain high numbers of Toxocara eggs [5,13]. This infor-
mation has resulted in toxocariasis being considered one
1471-4922/
� 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.pt.2014.07.003
Corresponding author: Conceição, F.R. (fabricio.rochedo@ufpel.edu.br).
Keywords: Toxocara canis; Toxocara cati; toxocariasis; immunodiagnostics; drug
modification; probiotics.
of the most important neglected diseases, mainly in the
Americas, owing to the optimal climate for larval devel-
opment 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 toxocar-
iasis have been associated with parasitism caused by
Toxocara canis, an intestinal parasite of dogs. By con-
trast, the importance of T. cati, an intestinal parasite of
cats, as an etiologic agent of this disease has been under-
estimated [7,8]. Thus, there is a need for more epidemio-
logical 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 contami-
nation 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 retinopa-
thy [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 provid-
ed 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 appropri-
ate 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
Trends in Parasitology xx (2014) 1–9 1
http://www.cdc.gov/parasites/toxocariasis/epi.html
http://www.cdc.gov/parasites/toxocariasis/epi.html
http://dx.doi.org/10.1016/j.pt.2014.07.003
mailto:fabricio.rochedo@ufpel.edu.br
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 macro-
phage 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 formula-
tion 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
ontoan 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 recom-
mended 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 formula-
tion 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 mono-
mers 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 immunomodula-
tory compounds which activate the proper response against the pathogen.
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.
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2
diagnosed as visceral larva migrans (VLM) (see Glossary).
VLM is more common in children and the clinical symp-
toms 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, granu-
lomas, 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 commontoxocariasis (CT) is simi-
lar to VLM, but its symptoms, including headache, abdom-
inal pain, cough, sleeping problems, and behavioral
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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 indi-
viduals, 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 toxocar-
iasis. 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
Box 1. Traditional De Savigny method for TES antigen productio
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 approxi-
mately 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
Collec�on of adult
parasites
Egg acquisi�on Embryona�on
Hatching Larvae
separa�on
Larvae
culture
CH2O 2–4%
28°C
At least 7 days
30–35 days
1 2 3 
8 76
Figure I. Native TES antigen productio
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 sug-
gested 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 de-
veloped.
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 immu-
nodiagnostic 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 immu-
nogenic 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]. Dot-
ELISA using native TES was recently described as an
n
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.
Concentra�on
and dialysis
Filtra�on
Protein
quan�fica�on
and use
NaCIO 5%
5 min
Embryona�on
count
Mamilonated
membrane removal
Total ∼ 60 days
TRENDS in Parasitology 
4 5
109
n by the method of De Savigny.
3
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 recombi-
nant 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 time-
consuming, taking �5 days of work.
Protein
quan�fica�on
and use
Protein
expression
Protein
purifica�on
Cell lysis and
protein recovery
E. coli
transforma�on
Total ∼5 days2 days1 day1 day1 day
TRENDS in Parasitology 
321 4
Figure I. Obtaining recombinant TES using the E. coli system.
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alternative but further investigation is needed [33]. The
production of native TES antigen is laborious, time-con-
suming, 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 draw-
back to using native TES is its cross-reactivity with anti-
bodies 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 pro-
duced 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. Speci-
ficity 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-
4
ated by Western blotting, and there was evidence of cross-
reactivity with Anisakis spp., another intestinal parasite.
Another study with the same protein reported no cross-
reaction 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 anti-
bodies (a subclass of IgG), rather than total IgG, increased
the specificity from 36 to 78.6% [42]. However, the sensitivi-
ty 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 rTES-
30, 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 combina-
tion 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 TES-
58 and TES-68 was evaluated [35] and, although only a
small number of human samples (n = 20) were used, both
Table 1. Detection of Toxocara spp. by ELISA
Antigena Sourceb Antibody detected Sensitivityc Specificityd Refs
Native TESe Produced in-house IgG 100 (11/11) 57.0 (81/142) [36]
rTES-30 Escherichia coli, insoluble IgG 100 (11/11) 97.9 (139/142)
Native TES Cypress Diagnostics IgG 97.1 (34/35) 36.0 (27/75) [42]
Native TES Cypress Diagnostics IgG4 45.7 (16/35) 78.6 (59/75)
rTES-30 E. coli, soluble IgG4 92.3 (24/26) 89.6 (103/115) [43]
Native TES Cypress Diagnostics IgG 100 (26/26) 55.7 (64/115)
rTES-26 E. coli, soluble IgG4 80.0 (24/30) 96.2 (204/212) [34]
rTES-30 E. coli, soluble IgG4 93.3 (28/30) 93.9 (199/212)
rTES-120 E. coli, soluble IgG4 93.3 (28/30) 92.0 (195/212)
TES-58 and -68 Produced in-house IgM or IgG 100 (20/20) 100 (20/20) [35]
TCLA Produced in-house IgG 92.2 (59/64) 86.6 (103/119) [44]
aNative 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.
bKnowing 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 in-
house’ 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.
cSensitivity 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).
dSpecificity 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).
eAbbreviations: rTES, recombinant Toxocara spp. excretory-secretory antigen; TES, Toxocara spp. excretory-secretory antigen.
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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 prac-
ticality, the production of TCLA eliminates the larval
culture step, thereby decreasing the time required for
antigen production compared to native TES (Box 1). How-
ever, 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 sensitiv-
ity 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
anti-inflammatories (http://www.cdc.gov/dpdx/toxocaria-
sis/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, par-
ticularly the brain and eyes [23]. For treatment of VLM,
anthelmintic drugs such as albendazole (ABZ, first option)
and mebendazole (MBZ, second option) are indicated, de-
spite their limited efficacy. Although anthelmintic treat-
ment is still recommended in cases of OLM, anti-
inflammatory drugs are also necessary to reduce damage
to the eyes [4,45]. Because the host immune response is
hamperedby 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 evalu-
ating the efficacy of ABZ and MBZ against Toxocara spp.
were performed during the 1970s and 1980s, and the more-
recent clinical studies did not evaluate any novel com-
pounds 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%, respec-
tively [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
5
http://www.cdc.gov/dpdx/toxocariasis/tx.html
http://www.cdc.gov/dpdx/toxocariasis/tx.html
Table 2. New drug formulation efficacies by organ and tissue
Drug/vehiclea Efficacy (%)b / Organc Refs
ABZ/CHd 72.22 / liver [50]
75.00 / lung
100.00 / brain
ABZ/PEG 75.00 / liver [51]
79.17 / lung
100.00 / brain
ABZ/PEG-LE 65.43 / liver [52]
ABZ/PEG-LE 87.92 / brain [53]
FBZ/PEG-LE 73.63 / skeletal muscle
ABZ/PEG-LE + glucan 92.22 / brain
FBZ/PEG-LE + glucan 91.55 / skeletal muscle
C17/PEG-LE 19.00 / brain [55]
41.75 / skeletal muscle
aDrug 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.
bThe efficacy represents the percentage of the mean reduction in the larval
recovery number in comparison to the controls of each study.
cAs described in the original studies, a treatment can also be effective in organs
other than those shown in the table.
dAbbreviations: ABZ, albendazole; CH, chitosan-encapsulated; PEG, polyethylene
glycol (‘pegylated’); FBZ, fenbendazole; LE, liposome-encapsulated; C17, com-
pound 17.
Review Trends in Parasitology xxx xxxx, Vol. xxx, No. x
TREPAR-1305; No. of Pages 9
(FBZ/PEG-LE) showed interesting results for skeletal mus-
cle, 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 aminotrans-
ferase (AST), alanine aminotransferase (ALT), and alka-
line phosphatase (ALP) were all reduced.
Table 3. New interventions and their main effects against toxoca
Intervention Effect 
DNA-based pcDNA3-CpG Reduction of a
pcDNA3-IL-12 Reduction of b
Probioticsa Enterococcus faecalis CECT 7121 90% reduction 
36.84% reducti
Saccharomyces boulardii 41.94% reducti
34.04% reducti
aThe dietary schedule varied depending on the study.
6
The ideal treatment for toxocariasis should eradicate all
the larvae lodged in different organs – and not only de-
crease the intensity of infection, as occurs with ABZ,
FBZ,and MBZ in animal models [51,53,55,58–60]. Further-
more, coadministration of immunomodulatory compounds
(e.g., glycan) could be a good option to overcome the im-
munosuppression evoked after infection, but these formu-
lations 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 sur-
veillance and reliable clinical diagnoses of a representative
population [8]. Since the 1970s the World Health Organi-
zation (WHO) has provided recommendations for reducing
the transmission of Toxocara spp. to humans. The main
advice is to limit exposure to soil contaminated with Tox-
ocara 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 toxocar-
iasis [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 pre-
vention 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]. More-
over, a study that involved giardiasis found that a treat-
ment 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 treat-
ment 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].
riasis
Refs
irway hyper-responsiveness [77]
lood and lung eosinophilia
of larvae number in liver and lungs 48 h after infection [69]
on of larvae number in liver and lungs 72 h after infection [70]
on of larvae number in the liver 2 days after infection [75]
on of larvae number in the brain 60 days after infection
Review Trends in Parasitology xxx xxxx, Vol. xxx, No. x
TREPAR-1305; No. of Pages 9
By contrast, probiotics based on Saccharomyces boular-
dii, 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, theuse of this probi-
otic 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 evaluat-
ed to date, namely E. faecalis and S. boulardii, their use in
the control of human toxocariasis appears promising. How-
ever, further studies with individual species or in combi-
nation 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 combi-
nation 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. Increas-
ing 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 ever-
increasing 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 dis-
cussed. 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 treat-
ment and elimination of Toxocara larvae are promising.
Lastly, recent studies using probiotics, immunomodula-
tors, 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
pathoetiology of the disease and develop effective treat-
ment and control strategies. However, full understanding
and characterization of the pathogen will only be possible
when surveillance programs are implemented to deter-
mine the disease burden, and this requires a reliable
and viable diagnostic test, which remains a limiting
factor. The information generated by this kind of epide-
miological study would not only remove toxocariasis from
the neglected disease listing but also would contribute
towards boosting basic and applied research on the
parasite.
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	Human toxocariasis: current advances in diagnostics, treatment, and interventions
	Toxocariasis spread and infection
	Diagnosis of toxocariasis
	Immunodiagnosis with Toxocara spp. antigens
	Development of new therapies
	New interventions against toxocariasis
	Concluding remarks and future perspectives
	References