Review

Relevance of and New

Developments in Serology for
Toxoplasmosis
Cline Dard,1,2,* Hlne Fricker-Hidalgo,1
Marie-Pierre Brenier-Pinchart,1,2 and Herv Pelloux1,2
Toxoplasmosis is a widespread parasitic disease caused by the intracellular
Trends
parasite Toxoplasma gondii with a wide spectrum of clinical outcomes. The
Serological tools play a key role in the
biological diagnosis of toxoplasmosis is often difcult and of paramount impor- diagnosis of toxoplasmosis and in epi-
tance because clinical features are not sufcient to discriminate between demiological studies worldwide.

toxoplasmosis and other illnesses. Serological tests are the most widely used Wide panels of serological techniques
biological tools for the diagnosis of toxoplasmosis worldwide. This review are available that need to be exploited
focuses on the crucial role of serology in providing answers to the most in the context of variable clinical and
immune features. Improvements are
important questions related to the epidemiology and diagnosis of toxoplasmo- still needed.
sis in human pathology. Notwithstanding their undeniable importance, serolog-
Recent developments in the serological
ical tools need to be continuously improved and the interpretation of the ensuing
diagnosis of toxoplasmosis mainly rely
results remains complex in many circumstances. on the discovery of new immunogenic
proteins and new methods of antigen
production. The development of
Toxoplasmosis: A Parasitic Disease of Great Importance Worldwide recombinant antigens and recombi-
T. gondii is an intracellular protozoan parasite discovered in 1908 by Nicolle and Manceaux that nant multiepitope chimeric peptides
greatly improves serological methods
causes toxoplasmosis [1]. Human infection generally occurs by the ingestion of cysts in under-
of diagnosis.
cooked meat or oocysts from the environment [2] (Figure 1). This parasite can evade the immune
system and persist throughout the life of its host by forming cysts, which are mostly found in the Serotyping is a promising way for typ-
brain, muscles, and retina [3]. Toxoplasmosis is a global health hazard. The worldwide sero- ing parasite lineages using less-inva-
sive approaches than genotyping.
prevalence is 3050% [4] but varies widely according to region [5,6] due to differences of climate,
diet, hygiene, and host susceptibility [2].

In contrast to the usually benign form found in immunocompetent people (IPs), toxoplasmosis
can cause a wide range of life-threatening clinical symptoms in cases of congenital infection and
in immunocompromised patients (ICs). Toxoplasmosis is particularly threatening for transplan-
tation and AIDS patients [4,7]. Early and accurate diagnosis using sensitive and specic
diagnostic tests is essential to prevent and treat severe toxoplasmosis [6]. The tests used to
diagnose toxoplasmosis depend on the immune status of the patient and the clinical setting [8].
Two biological approaches are currently used: (i) the direct detection of T. gondii using molecular
biology approaches or, less frequently, by mouse inoculation or microscopic examination; and (ii) 1
Laboratoire de Parasitologie-
indirect detection using serological assays [2]. Serology is the most convenient tool in most Mycologie, Centre Hospitalier et
cases, but some clinical situations, such as suspicion of disseminated disease or prenatal Universitaire de Grenoble Alpes,
Grenoble, France
diagnosis of congenital toxoplasmosis (CT), also require PCR analysis (Table 1) [9]. 2
Institut Albert Bonniot, INSERM
U1209 CNRS UMR 5309, Universit
Serological Tools Play a Key Role in the Diagnosis of Toxoplasmosis and Grenoble Alpes, Grenoble, France

Epidemiological Studies
The detection of specic antibodies remains the only way to address almost all of the important *Correspondence:
questions concerning T. gondii in human medicine (Table 1). What is the epidemiology of cdard@chu-grenoble.fr (C. Dard).

492 Trends in Parasitology, June 2016, Vol. 32, No. 6 http://dx.doi.org/10.1016/j.pt.2016.04.001

2016 Elsevier Ltd. All rights reserved.
 Cysts
Tachyzoites

Sporulated
oocysts Cysts

Sporulated
oocysts

Non-sporulated
oocysts
Cysts

Figure 1. Life Cycle of Toxoplasma gondii. The life cycle of T. gondii takes place between the denitive hosts, members
of the family Felidae (domestic and wild cats), and the intermediate hosts, mainly homeothermic animals such as birds and
mammals. The denitive hosts shed non-sporulated oocysts sexual forms in their feces. Oocysts become infective in the
environment after a 15-day sporulation phase. When infected, intermediate hosts can harbor cysts in their tissues. Felidae
are generally contaminated by ingesting cysts contained in their preys tissues but can also be contaminated by the
ingestion of mature oocysts from the environment. Intermediate hosts are generally contaminated by ingesting water or
plants contaminated with oocysts but can also be contaminated by ingesting cysts if they are carnivores. Humans can be
contaminated by ingesting cysts in meat or oocysts in water or vegetables, transplacentally from mother to fetus, and by
blood transfusion or organ transplantation. Oocysts and cysts generate tachyzoites shortly after ingestion by intermediate
hosts such as humans. Tachyzoites migrate through the blood and disseminate to the brain, muscles, and retina to
generate cysts in hosts.

T. gondii? Is a patient infected or not by the parasite? Is a pregnant woman susceptible to

primary infection? Is an infant congenitally infected? Is a HIV-infected patient at risk of reactiva-
tion of toxoplasmosis? Are transplanted organs a mismatch for T. gondii? Can a case of
retinochoroiditis a posterior uveitis be due to T. gondii? What is the risk of T. gondii
transmission via meat consumption? Is there a link between T. gondii chronic infection and
neurological disorders? These issues have been the subject of numerous studies as toxoplas-
mosis is a worldwide concern for the scientic and medical community. Despite major biological
innovations molecular biology, genotyping, and serotyping [1012] serological techniques
continue to be used and developed because of their key role in the diagnosis of toxoplasmosis
[13]. The purpose of this review is to highlight the enduring importance of and new developments
in serological tools for the diagnosis of toxoplasmosis.

Trends in Parasitology, June 2016, Vol. 32, No. 6 493

Table 1. Overview of Strategies for the Diagnosis of Toxoplasmosis in Humans
Clinical and Epidemiological Sample Technique Diagnostic Criteria Refs
Situation

Toxoplasmosis Determination of Serum Serologya,b IgG , IgM : no infection [2]

in IPs immune status IgG+, IgM or IgG+, IgM+,
high avidity: chronic
infection

Primary infection IgG /IgM+ or IgG+/IgM+:

acute infection

Toxoplasmosis OT Serum + AH Comparative Intraocular production of [6569]

in ICs WBb immunoglobulin

GWCd

AH qPCRb qPCR+
Vitreous uid

Monitoring Serum Serologya,b Seroconversion: primary [5462]

(primary infection
infection, Increase of IgG and/or
reactivation) IgM: toxoplasmosis
reactivation

Blood qPCRb qPCR+

Disseminated Blood qPCRb qPCR+

infection CSF Histologyb Toxoplasma gondii
BAL/AB biopsy on histology

CT Diagnosis Pregnant Serum Serologya,b IgG /IgM+ or IgG+/IgM+: [30]

and woman acute infection
Monitoring
Prenatal Amniotic uidc qPCRb qPCR+ [44,45]
diagnosis Mouse Seropositive mice
inoculationb
Diagnosis at Placenta and/or [46]
delivery cord blood

Post-natal Sera (cord blood Serologyb IgM+ and/or IgA+ in [24,30,31,

follow-up and/or newborn + newborn serum: 33,48]
mother) biological CT

Comparative Neosynthesized IgG

WBb and/or IgM by the
newborn

If no evidence for Serologyb Conrmation of the

CT: serological absence of CT by
follow-up total disappearance
of immunoglobulin

If evidence for Detection of serological

CT: monitor child rebound during follow-up
sera until the end of immunoglobulin levels
of the treatment

a
Methods for routine analysis.
b
Methods for reference laboratories.
c
Informed consent by the pregnant woman is required in some countries.
d
GWC = [anti-Toxoplasma IgG (IU/ml) in AH/anti-Toxoplasma IgG (IU/ml) in serum]  [serum total IgG (g/l)/AH total IgG (g/
l)].
AH, aqueous humor; BA, bronchoaspiration; BAL, bronchoalveolar lavage; CSF, cerebrospinal uid; CT, congenital
toxoplasmosis; OT, ocular toxoplasmosis.

494 Trends in Parasitology, June 2016, Vol. 32, No. 6

The Humoral Response to T. gondii Infection
T. gondii infection activates the host humoral and cellular immune response [14]. The humoral
response to T. gondii infection results in increased levels of specic circulating immunoglobulins:
IgA, IgM, IgE, and IgG (Figure 2). Each isotype appears with a specic kinetic prole after primary
infection [2]: (i) IgM and IgA appear during the rst week and their levels reach a peak after
approximately 1 month before decreasing to undetectable levels after several weeks to several
years [15]; (ii) IgE levels peak after approximately 3 months and then rapidly decrease [16]; and
(iii) IgG rst appears approximately 2 weeks after IgM, reaches a plateau after approximately 23
months, and then steadily decreases to a residual lifelong titer [2].

Qualitative, quantitative, and kinetic analyses of these antibodies (mostly IgM and IgG) are used
for the diagnosis, monitoring, and epidemiological studies of toxoplasmosis worldwide [2]. IgM
detection indicates recent exposure or an ongoing active infection. IgM may also be naturally
occurring or long lasting as it can persist for more than 18 months after the initial exposure to T.
gondii [17]. IgM may be undetectable during the reactivation of toxoplasmosis in ICs, CT in
newborns, and atypical seroconversion in IPs [8]. Some reference laboratories perform IgA
testing for the diagnosis of CT in newborns because of the higher sensitivity of IgA tests and
longer detection period of IgA than IgM [15]. However, there is little interest in testing adults for
IgA, especially ICs [18]. IgE titrations are rarely performed because they lack the sensitivity and
added value of the other isotypes. Nonetheless, the persistence of IgE may be a marker of active
toxoplasmosis and contribute to the diagnosis of lymphadenopathies [19]. The appearance of
IgG after IgM detection is required to conrm the infection. Serological results may vary
depending on the individual, his or her immune background, and the serological technique
used [20].

A Wide Panel of Serological Techniques Is Available for Diagnosis of

Toxoplasmosis
A wide range of serological techniques is available for the detection of specic T. gondii
immunoglobulins (Table 2). They vary depending on the methodology, antigens used, and
immunoglobulin isotype detected. There have been signicant improvements over the past few
years [13]. The principle methodologies are the SabinFeldman Dye Test (DT), enzyme-linked
immunosorbent assays (ELISAs), immunosorbent agglutination assay (ISAGA), indirect

Ab levels Figure 2. Anti-Toxoplasma-Specic

Immunoglobulin Kinetics after Pri-
Primary Acute infecon Chronic infecon
infecon mary Infection. A primary infection
induces a humoral response with the
appearance of different immunoglobulin
isotypes with various kinetics. IgM and
IgG
IgA appear during the rst week and their
levels reach a peak after approximately 1
month before decreasing to undetectable
levels after several weeks to several years
IgM [2,15]. IgE levels peak after approximately 3
months and then rapidly decrease [19]. IgG
IgE rst appears approximately 2 weeks after
IgA IgM, reaches a plateau after approximately
23 months, and then steadily decreases
0 1 2 3 4 5 6 7 8 9 10 11 12 24
to a residual lifelong titer. The appearance
Time (months)
of IgG after IgM detection allows the diag-
nosis of a primary infection with certainty.
This gure is presented for illustrative pur-
poses only. The kinetics of the immunoglo-
bulins is variable depending on the patient,
the antigens, and the techniques used for
the serological assays.

Trends in Parasitology, June 2016, Vol. 32, No. 6 495

496
Table 2. Serological Techniques for the Diagnosis of Toxoplasmosis: Principles, Advantages and Drawbacks (for Reviews See [8,11])
Trends in Parasitology, June 2016, Vol. 32, No. 6
Technique Principle
Techniques Using Entire Toxoplasma gondii
SabinFeldman DTb Addition of complement-free serum to a
Total specic suspension of live parasites and complement:
immunoglobulins parasite cytolysis if specic antibody is present
ISAGAb Microwell plates coated with anti-immunoglobulin
IgM, IgA, IgE antibodies incubated with serum and then
with formalin-xed tachyzoites: homogeneous
agglutination if reaction is positive and button-
shaped agglutination if reaction is negative
MAT Addition of serum to formalin-xed tachyzoites
in microwell plates: homogeneous agglutination
if reaction is positive and button-shaped
agglutination if reaction is negative
IFATb Addition of serum to formalin-xed
IgG, IgM tachyzoites on glass slides: revelation
with a uorescent anti-IgG or
IgM antibodies
Techniques Using Antigenic Extracts or Recombinant Antigens
ELISAa,b Addition of serum to T. gondii antigens and
IgG, IgM revelation by antibodies coupled to a detectable
label (i.e., enzyme or uorescent marker)
IHA Addition of serum to erythrocytes sensitized
with T. gondii soluble antigens: agglutination
if reaction is positive
WBb Addition of serum to resolved bands of T. gondii
IgG, IgM tachyzoite lysate or recombinant antigens on
nitrocellulose strips: detection of bands by
development of color if reaction is positive
ELIFAb Addition of serum to an electroimmunodiffusion of
IgG, IgM T. gondii soluble antigens and revelation of the
precipitin bands using an immunoenzymatic assay:
positive reaction if presence of one or several arcs
IgG aviditya,b Comparison of standard ELISA IgG test with
a modied IgG test using an agent that disrupts
IgGantigen binding: measure the afnity of IgG
for antigens through an index calculation
a
Methods for routine analysis.
b
Methods for reference laboratories.
Indications
Diagnosis of acute and chronic infection
IgM detection for infant monitoring
when CT is suspected
IgM-positive result conrmation
Serological assays in animals to
evaluate T. gondii transmission
via meat and for epidemiological studies
Diagnosis of acute and chronic infection
in humans
Epidemiological studies in the veterinary
eld
Diagnosis of acute or chronic infection
Screening of pregnant women
Mass screening in epidemiology surveys
Comparative serological proles:
Between mother and child sera in cases
of suspected CT
Between sera and AH in cases of
suspected retinochoroiditis suspicion
of early seroconversion diagnosis
Comparison of precipitating systems
between mother and child sera
in cases of CT suspicion
In cases of high IgG-avidity index, a
recent infection (less than 3
5 months) can be excluded
A weak index is not indicative
of the time of infection
Advantages
Early detection of immunoglobulin
in primary infection
Good sensitivity and specicity
Reference method for many studies
Simple, rapid
Good sensitivity and specicity
More sensitive than ELISA in neonates
Precocity of detection for acute
toxoplasmosis and CT
Good sensitivity and specicity in the
veterinary eld
Good sensitivity and specicity
Reference method for many studies
Automatable
Numerous commercial kits available
Possible to perform wide series
Simple, rapid
Good sensitivity and specicity
Early detection of IgG
Good sensitivity and specicity
Automatable commercial tests
Dating of infection without a
kinetics analysis
Drawbacks
Manual technique
Need to culture RH strain in laboratory
Need an external source of complement
Manual technique
Need technical skills for reliable results
Manual technique
Manual technique
Requires uorescence microscope and
technical skills for reliable results
Sensitivity and specicity not optimal
and dependent on the antigens used:
sometimes requires complementary
analysis
Manual technique
Lack of sensitivity and specicity
Manual technique
Manual technique
Not always discriminant: low avidity
is not indicative of the time of infection
hemagglutination assays (IHAs), indirect uorescent antibody tests (IFATs), the modied agglu-
tination test (MAT), western blotting (WB), and IgG avidity [11] (Table 2). The automation of
numerous techniques combined with commercially available kits has facilitated the performance
of serological assays, making them accessible in many countries worldwide. Local laboratories
generally perform serological screening using automated methods. Interpretation of serological
results is sometimes complex and may require complementary testing in reference laboratories
using manual and in-house techniques [8]. Such techniques are time consuming and require
additional personal and technical training, but often provide crucial results in critical clinical
situations. The highly variable antibody kinetics and titration rates measured using the various
techniques are explained by the wide diversity of the methodology. Early immunoglobulin
detection is achieved with techniques using whole parasites and surface antigens because
the immune response is rst directed against parasite surface antigens. The most common
methods that use whole parasites are DT [21], ISAGA [22], and IFAT (Table 2), whereas
recombinant surface antigens such as P30 (rSAG-1) are widely used in several commercial
IgG and IgM kits; for example, ARCHITECT (Abbott) and ELECSYS (Roche) [23,24]. Tests that
use mixtures of cytosolic or metabolic antigens based on numerous ELISA techniques detect
IgG a few days later [2]. Early IgM and IgG detection is important for early diagnosis and specic
follow-up. For example, ISAGA testing allows early detection of IgM in the sera of infants to
diagnose CT, whereas WB IgG II provides rapid detection of IgG to conrm seroconversion in
pregnant women [25]. However, the detection of long-lasting or natural IgM makes it difcult to
exclude a recent infection and may lead to serological misinterpretation [26]. Some tests have
been developed to help estimate the date of infection without analyzing sequential sera; this is
especially true for pregnant women. The most widely used test for this purpose is the IgG avidity
test [27], which measures the afnity of IgG antibodies for parasite antigens; that is, recombinant
P30 (rSAG1) for ELECSYS plus P35 (rGRA8) for ARCHITECT avidity testing [23,24]. A high-
avidity result excludes a recent infection (35 months) because mature antibodies have a
stronger afnity for antigens. However, a weak-avidity result is not proof of a recent infection.
Another technique called differential agglutination is used by a few reference centers [28].

Serological Tools Remain Important for the Diagnosis of Toxoplasmosis

The diagnostic strategy for each clinical situation is elaborated depending on the characteristics
of each technique [29]. This section presents the position of serological testing in critical clinical
situations. Serological tools are far from falling into obsolescence and cannot be replaced by
molecular biology-based techniques. However, they must be improved using new technological
methods, as no serological test is entirely satisfactory.

Diagnosis of Acute and Chronic Toxoplasmosis in IPs

Both acute and chronic T. gondii infections are diagnosed using serology in IPs. Primary infection
in IPs is asymptomatic in 80% of cases [4]. Some patients experience mononucleosis-like
syndrome with lymphadenopathy and fever, or pneumonia, depending on host susceptibility
and the virulence of the strain [2]. Serological assays aid in the differential diagnosis from other
infectious diseases such as cytomegalovirus and EpsteinBarr virus or hematological
malignancies. IgM detection is strongly indicative, but not a perfect marker, of an acute infection.
This must be conrmed with another serological test 2 weeks later to evaluate the antibody
kinetics [17,30]. An increase of the IgM titer favors the diagnosis of an acute infection whereas a
stable titer does not. When IgG is detected, a high IgG-avidity index excludes a recent primary
infection [24]. The detection of IgG without IgM suggests a chronic infection, whereas their
absence excludes T. gondii infection in IPs [9].

Evaluation of Primary Infection Risk in Pregnant Women

Primary infection during pregnancy can lead to CT through the vertical transmission of T. gondii,
resulting in serious fetal malformations or death [31]. Serological screening is indispensable for

Trends in Parasitology, June 2016, Vol. 32, No. 6 497

estimating the initial date of infection because T. gondii infection is generally asymptomatic in
pregnant women. Serological screening and monitoring makes it possible to: (i) exclude the risk
of CT in cases of infection acquired before gestation; (ii) provide prophylactic recommendations if
the woman is non-immunized; and (ii) detect seroconversion during pregnancy and conse-
quently propose antitoxoplasmosis treatment to prevent transmission of the parasite to the fetus
and antenatal monitoring and testing [32,33]. The screening and serological follow-up of
pregnant women varies according to country (e.g., USA [34], China [35,36], Colombia [37],
India [38], Kyrgyzstan [39], Thailand [40], Brazil [41,42]). Some countries have national recom-
mendations, including France, Italy, and Austria [9].

Serological interpretation of the results in pregnant women is subject to the same difculties
as those in non-pregnant IPs, and even more so. Some atypical antibody kinetics and
unusual serological proles must be taken into consideration, such as induction and increase
of IgG without IgM in previously seronegative women [20]. This can be explained by: (i)
residual IgG levels near the threshold indicating a past infection; (ii) the addition of exogenous
IgG through blood transfusion or immunoglobulin therapy [43]; (iii) the supply of antibody
subsets due to immune disorders [43]; or (iv) a true primary infection without IgM for
unknown reasons [20]. Serological misinterpretation in pregnant women can lead to emo-
tional distress and unnecessary intervention [17]. Therefore, sera for which the serological
interpretation is difcult are transmitted to reference laboratories to conrm and estimate the
date of maternal infection [17].

Diagnosis of CT in an Infant
Antenatal Diagnosis. Once maternal seroconversion is conrmed, fetal monitoring with ultra-
sound surveillance and biological antenatal diagnosis is generally performed by amniotic uid
PCR and mouse inoculation. The sensitivity of this approach is approximately 90% but varies
slightly depending on the center [44,45]. Serological tools are inappropriate for the diagnosis of
congenital infection because the fetal immune system is immature and intrauterine fetal blood
sampling is life threatening. A positive prenatal diagnosis directly affects the medical care of the
mother during pregnancy and the child at birth.

Postnatal Diagnosis. At birth, postnatal diagnosis is performed if CT is suspected. PCR and

mouse inoculation with placental tissue or cord blood are performed in some reference centers
for an early evaluation but are not sufcient to conrm or exclude the diagnosis of CT [46].
Serological analysis to detect IgG and IgM (and IgA in some centers) is routinely performed in
infants suspected to have CT. IgG is able to passively cross the placental barrier, contrary to IgM
and IgA, which are too large [47]. The detection of specic IgM and/or IgA in newborn or cord
blood sera is a marker of CT but has to be conrmed in another sampling because it may have
been caused by contamination with the mother's blood during delivery [48]. IgM and/or IgA are
not systematically detected in infants with CT. The use of adapted techniques, such as ISAGA,
improves the sensitivity, but it remains low (<60%) [2,49]. IgG detection in infants can be due to
transplacental transmission, which is not a marker of congenital infection, or the production of
antibodies by the neonate. Specic neonate isotype synthesis is investigated by comparative
qualitative analysis between sera of the child and of the mother by WB or enzyme-linked
immunoltration assay (ELIFA) [48] to discriminate between these two possibilities. In non-
infected infants, IgG antibodies transmitted by the mother normally disappear within 58 months
[8]. Longer persistence of IgG supports a delayed diagnosis of CT [2]. Imaging evaluation by
cranial transfontanellar ultrasound or computed tomography allows the detection of intracranial
calcication or hydrocephalus, which conrms symptomatic CT [50]. Fundus ocular examination
detects retinochoroiditis with typical retinal tissue damage [51]. These practices all vary depend-
ing on the center. The early diagnosis of toxoplasmosis allows earlier treatment to limit clinical
outcomes.

498 Trends in Parasitology, June 2016, Vol. 32, No. 6

Survey and Diagnosis of Toxoplasmosis in HIV Patients
The decrease of immune pressure in HIV-infected patients with chronic toxoplasmosis can lead
to cyst rupture and life-threatening disease [52]. The risk in HIV-infected patients is related to the
progression of immunodeciency and the number of CD4+ cells. The normal CD4+ cell level is
5001500/mm3. HIV-infected patients are at risk of cyst rupture when CD4+ cell levels fall below
100/mm3 [4,53]. The introduction of highly active antiretroviral therapy (HAART) in the late 1990s
has led to a large reduction of toxoplasmosis cases in this population [54]. The main clinical
feature in HIV patients is toxoplasmic encephalitis (TE), but retinochoroiditis, myocarditis, and
pulmonary and disseminated involvement can be observed [55]. The T. gondii serological status
of HIV-infected patients must be known. Regular serological screening of patients seronegative
for T. gondii with a low CD4+ count is recommended to detect any primary T. gondii infection. In
patients with neurological symptoms combined with imagery suggestive of TE, a diagnosis of TE
is unlikely if the serology is negative. If the serology is positive, clinical improvement under
antitoxoplasmic treatment may reinforce the TE diagnosis. Only PCR on tissue from a cerebral
biopsy or cerebrospinal uid (CSF), albeit with a lower sensitivity, is able to diagnose TE with
certainty [56].

Diagnosis of Toxoplasmosis in Solid Organ and Hematopoietic Stem Cell Transplant (HSCT)
Patients
Solid organ transplantation (SOT) and HSCT recipients present a risk of toxoplasmosis due to
immunosuppressive treatment to prevent organ rejection. The risk is mostly due to the trans-
mission of cysts contained in the graft for SOT and reactivation of a pre-transplantation latent
infection in seropositive HSCT patients [57]. Biological monitoring strategies differ depending on
the type of graft and the hospital. Patients are usually closely monitored during the 3 months
following transplantation, when immunosuppression is maximal [57]. Pre-graft evaluation of the
serological status of recipients and donors is required to interpret the post-graft serological
follow-up of the recipients [58,59].

For SOT, the risk is directly linked to T. gondii tropism for the grafted tissue (e.g., major for heart
transplant recipients). In mismatched cardiac transplantation (T. gondii-seropositive donor to
seronegative recipient), the transmission of T. gondii is approximately 75% [60] in heart recipients
without chemoprophylaxis, with a rare but serious clinical presentation [5860]. In cases of SOT
transplantation from a positive donor to a positive recipient (D+/R+) or from a negative donor to a
positive recipient (D /R+), an increase in IgG and the presence of IgM may indicate reactivation of
the toxoplasmosis. The immune challenges in HSCT patients are linked to intense immunosup-
pressive treatment during the conditioning period and the acquisition of a new immune system
from the donor during the rst 6 months after transplantation [2]. The low sensitivity of serology in
HSCT patients requires the use of PCR on peripheral blood [59]. Further investigation is necessary
when symptoms are suggestive of toxoplasmosis in HSCT patients. Positive PCR results from
blood, bronchoalveolar lavage (BAL), CSF, or other tissues can verify the involvement of T. gondii in
cases of disseminated, pulmonary, cerebral, or other localized toxoplasmosis [61,62]. A tabulated
form of serological follow-up of patients with SOT is given in Table 3.

Evidence of T. gondii Involvement in Ocular Toxoplasmosis (OT)

Cases of OT, mostly retinochoroiditis, have been reported in ICs, IPs, and CT patients. The
provenance of T. gondii is generally uncertain: cyst reactivation may occur in the eye or in another
site with dissemination to the eyes in the blood. Some virulent lineages can reactivate in ICs and
cause retinochoroiditis [63,64], particularly in South America [43]. The diagnosis of OT is
primarily based on the clinical examination when typical lesions are found in a patient seroposi-
tive for T. gondii and clinical improvement occurs on antitoxoplasmic treatment [65]. Laboratory
tests are helpful when atypical lesions are observed or antitoxoplasmic treatment is not effective
[66]. Biological diagnosis is based on the analysis of the aqueous humor (AH) using: (i)

Trends in Parasitology, June 2016, Vol. 32, No. 6 499

500

Table 3. Role of Serology in Monitoring SOT Recipients

Trends in Parasitology, June 2016, Vol. 32, No. 6

Pre-graft Monitoringa Risks Post-graft Monitoring Criteria for Positivity Limitations of Serology Refs
Toxoplasma No Consensual Recommendations, Only
gondii Serological Propositions from Individual Centers
Status Screening for
Donor and Recipient

Donor Recipient Recipients

Primary infection through food In case of clinical symptoms Seroconversion (IgM+ then IgG+) Negative serology in cases of very [57,58,6062]
or environment for all types of strong immunosuppression
grafts

+ Primary infection through donor
cysts and clinical toxoplasmosis
mainly for SOT grafts (mainly heart)
Prevention: chemoprophylaxisb
Serological follow-up in the rst Seroconversion (IgM+ then IgG+) Negative serology in cases of strong
6 months post-transplantation qPCR+ immunosuppression
qPCR in blood (CSF, BAL, (no humoral production)
biopsy according to the clinical Interpretation of seroconversion without
symptoms) any clinical signs

+ Clinical toxoplasmosis mainly for
HSCT (very rare for SOT)
+ +
Serological reactivation without
clinical signs is possible
mainly for SOT
High-risk periods (3 months post-graft,
strong immunosuppression) and
patients who do not receive prophylaxis
Increase of IgG level and/or
reappearance of IgMc
qPCR+d
Negative serology in cases of strong
immunosuppression, mainly for HSCT,
and when acquiring a new immune
system from a seronegative donor
Changes in serology: possibly difcult
interpretation due to immunodepression
and the clinical data

a
+, immunized patient (positive IgG serology); , non-immunized patient (negative serology).
b
Some drugs for the prevention of pneumocystosis are also effective in toxoplasmosis.
c
Changes in serology and/or positive PCR.
d
Positive PCR result can be an early sign of disease; positive PCR in blood without a change in kinetics of immunoglobulins is linked to T. gondii infection whereas positive PCR in blood and clinical symptoms with or
without a change in kinetics of immunoglobulins favors active toxoplasmosis.
 D0 D+10 D+30

Clinical
signs
PCR C +/- PCR
GWC WB
Immunocompromised

Foci > 2 opc disk Old scars

Mild or severe reacon in the
he
anterior chamber
Foci > 2 opc disk

Figure 3. Priority of Techniques to Use Depending on the Interval between the Onset of Symptoms of
Retinochoroiditis and Sampling. The combination of PCR, quantitative antibody titration using the GoldmannWitmer
coefcient (GWC), and western blotting (WB) offers the best diagnostic sensitivity regardless of the timing of the sampling
and the type of retinal lesion, with sensitivity approximately 90% and specicity approximately 95% [67,68]. The use of these
techniques might have to be prioritized if the quantity of aqueous humor is insufcient to perform all three. In this case, the
sensitivity of each method must be considered, depending on the time between the onset of clinical signs and sampling, the
immune status of the patient, and the size of the lesions. PCR performs best within the rst 10 days after the rst clinical
symptoms, particularly in immunocompromised (IC) patients and when the foci are large. The sensitivity of PCR is
approximately 50% and it is more informative than WB or GWC for IC patients [68]. Beyond 10 days (D + 10), the
combination of GWC and PCR appears to be the most appropriate. This combination has a sensitivity of approximately 80%
regardless of the timing of the rst appearance of clinical symptoms and sampling. GWC is particularly useful in cases where
there are old scars and a severe reaction in the anterior chamber and PCR when the foci are large. After 30 days (D + 30),
WB is the best tool, with a sensitivity of 72% [68]. The optic disk corresponds to the intraocular portion of the optic nerve and
serves as a point of reference for lesion size when foci are observed.

quantitative antibody detection by ELISA and establishment of the GoldmannWitmer coefcient

(GWC) (anti-Toxoplasma IgG in AH/total IgG in AH)/(anti-Toxoplasma IgG in serum/total IgG in
serum) to show intraocular specic Ig synthesis [67,68]; (ii) qualitative local antibody production
analysis by comparative WB between sera and AH [67,68]; and (iii) PCR analysis [68,69]. The
combination of these three techniques offers the best sensitivity. The sensitivity of each method
varies depending on the time between the onset of clinical signs and sampling, the immune
status of the patient, and the size of the lesions [68]. When the sample quantity is limited, these
elements must be considered to perform the best-adapted analysis (Figure 3).

Recent Developments in the Serological Diagnosis of Toxoplasmosis

Serological analyses are essential for the diagnosis of toxoplasmosis but have notable weak-
nesses that are only partially offset by other biological tools. The interpretation of the serological
results remains difcult, even for well-trained specialists. Improvements are necessary and
ongoing. The critical points are to: (i) distinguish between acute and chronic infection with only
one sample and date the infection; (ii) diagnose CT in infants and reactivation in ICs; (iii) type
lineages in a routine analysis; and (iv) distinguish the origin of infection oocysts or cysts. A
unique reference test that could determine the serological status for all patients and conrm or
exclude T. gondii in all clinical situations is still lacking.

New Methods of Antigen Production for Serological Analysis

The rst improvement needed is related to the antigens used for the IgG, IgM, and IgG avidity
assays in serum samples. Currently used techniques employ entire T. gondii antigenic extracts
and, more recently, single or combined recombinant antigens. The use of molecular biology
techniques to produce recombinant antigens has greatly improved the standardization, sensi-
tivity, specicity, and cost-effectiveness of serological tools [70]. Several recombinant antigens
seem promising (i.e., rGRA2 [71], rGRA5 [72], and rGRA7 [73]). The use of a stage-specic
antigen rCCp5A would help to identify an infection caused by oocysts with possibly higher
virulence [74].

Trends in Parasitology, June 2016, Vol. 32, No. 6 501

Work is ongoing to discover and study new T. gondii antigens that trigger the humoral response
[75]. Major developments such as bioinformatics tools [76], epitope mapping [77], and mono-
clonal antibodies now allow the identication of human immune-dominant B-cell epitopes for
various antigens (i.e., P30 (SAG-1) [78], GRA1 [79], and GRA4 [76]). This contributes to the
generation of promising recombinant multiepitope chimeric peptides (rMEPs) for use in immuno-
assays as vaccine candidates. rMEPs could be used to create a panel of selected epitopes that
represent the parasite antigenic variability related to the different T. gondii lineages, stages, and
pathophysiological mechanisms. Many combinations of epitopes are possible depending on the
intended diagnostic purpose. rMEPs could improve the sensitivity and specicity of serological
tools [8082] and allow the distinction between recent and past infection in pregnant women and
IPs [83] by, for example, using the chimeric antigen P35MAG1 [84]. Numerous combinations
remain to be explored as potential molecular markers to transcend the current limits of serology.
For example, rMEPs based on peptides that are expressed only in T. gondii-infected newborns
could help to diagnose CT.

The appropriate combination of epitopes with an avidity index directly proportional to the time of
infection would lead to the precise dating of infection because IgG avidity matures at different
rates according to the antigen. Further research to identify immunogenic epitopes that are stage
specic or expressed depending on the clinical situation is necessary before such serological
tools can be developed [75].

Serotyping Methods
There are at least 15 T. gondii haplogroups worldwide, which collectively dene six major clades
[85,86]. Recent studies have conrmed that lineages II (haplogroup 2, clade D) and III (hap-
logroup 3, clade C) have marked clonality and predominate in Europe and North America,
whereas lineage I (haplogroup 1, clade A) is rarely encountered [85]. By contrast, lineages in
South America are characterized by their greater genetic diversity, with none being clearly
dominant [86]. In Asia and Africa, T. gondii has a clonal population structure with a few dominant
lineages [2,85,86]. The genotype apparently inuences the disease severity in humans. In South
America, higher rates and severity of retinochoroiditis in CT and ICs are reported [87]. Several
hypothesis including modulation of the host immune response might be considered [87], but the
variability of genotypes with respect to virulence and disease outcome remains poorly under-
stood [86,88].

Recent advances in genotyping T. gondii that is, the use of restriction fragment length
polymorphism, microsatellites, or sequence-based markers and the study of isolates from all
continents have greatly improved the distinction of lineages [85,89]. However, genotyping has
some substantial limitations. Besides being time consuming and costly, genotyping requires
parasite isolation or a high quantity of DNA, necessitating invasive sampling with medical risks
and potentially biased sampling.

Serotyping potentially represents one of the most promising recent developments. It could be a
rapid, sensitive, and relatively noninvasive alternative to stringent genotyping [11]. Serotyping is
based on serological testing using specic immunogenic peptides from antigens with sequence
polymorphisms of the different clonal types, to detect strain-specic antibody proles. Such
peptides can be generated as described in the previous section. Serotyping is currently used
only for research and not in routine hospital practice and only allows the distinction of type II from
non-type II infections. Most serotyping tests are based on ELISAs using polymorphic peptides
derived from the antigens GRA5, GRA6, and GRA7 [12,90,91], sometimes in combination with
SAG2A and GRA3 [92]. Peptide microarray assays have also been tested with numerous
peptides [93]. They show better sensitivity than ELISA-based assays and conrm that peptides
derived from GRA5, GRA6, and GRA3 are the most reactive among those tested [93,94]. These

502 Trends in Parasitology, June 2016, Vol. 32, No. 6

studies conrmed that the type II lineage is predominant in Europe and only sporadically present Outstanding Questions
in South America [12,91,93]. Some studies have attempted to correlate the strain type with How can acute and chronic toxoplas-
clinical outcomes and showed, for example, that T. gondii serotype is associated with the mosis best be diagnosed?

development of OT [12,95]. Serotyping shows promise but the resolution and specicity need to How can we diagnose or exclude a
be improved. Sufcient antibody levels are required, thereby excluding patients who are strongly recent infection in one sample in cases
immunocompromised with a weak humoral response. Discrimination between type I and III of positive IgM  IgG? How can we
estimate the date of primary infection?
lineages remains challenging as they possess identical alleles for SAG2A and GRA3 and nearly
identical alleles for GRA6 and GRA7 [92]. Cross-reactions may occur between recombinant Which new method will allow rapid
lineages whereas rare and non-clonal lineages are not detected using polymorphic peptides diagnosis of reactivation of toxoplas-
[90,93]. A universal immunogenic marker such as SAG1 could be used to conrm the presence mosis in immunocompromised
patients?
of a non-clonal lineage if there is a strong SAG1 reaction and the absence of a reaction with the
other peptides [92]. How can we diagnose a congenital
infection?
Serotyping has the potential to become a routine analytical method for typing of lineages for: (i)
How can Toxoplasma gondii in retino-
epidemiological studies to screen the lineages present in a given population; and (ii) clinical
choroiditis cases best be diagnosed?
studies to predict the relationship between a given lineage and disease outcome. However,
serotyping relies largely on the existence of clonal lineages and constitutes a considerable How can we improve the sensitivity and
challenge in areas where the population structure of T. gondii is not clonal. specicity of toxoplasmosis serological
tests? Should serological tools based
on recombinant antigens, possibly
New Serological Techniques recombinant multiepitope peptides,
Saliva assays have shown promising results by detecting T. gondii-specic IgG in oral uid. The be considered?
use of recombinant SAG1 antigen in ELISA on pregnant women detected specic IgG in saliva
How can we distinguish the origin of
with 100% sensitivity and specicity [96]. Saliva testing using pre-coated antigen strips, with a infection (oocysts or cysts)?
negative predictive value of 99% [97], should be applicable for the follow-up of infants with
suspected CT. Although the performance of these tests needs to be improved, noninvasive Is testing of saliva, particularly for new-
borns suspected of having congenital
saliva sampling may facilitate the monitoring of infants with suspected CT and would facilitate the
toxoplasmosis, a viable option?
investigation of the epidemiology of toxoplasmosis [98]. Other promising techniques have been
developed, such as immunochromatographic tests [99] and piezoelectric immunoagglutination How can we reliably type lineages in a
assays [100], but are not routinely used. routine analysis in a less invasive way?
Should we consider serotyping
(through blood sampling) instead of
Concluding Remarks genotyping (requiring invasive
Serological techniques are tools of paramount importance for the diagnosis of toxoplasmosis sampling)?
and epidemiological studies. However, the diagnosis of toxoplasmosis remains challenging and
How can we predict the pathogenicity
serological results should be interpreted with caution and sometimes requires the opinion of an
of a strain in case of infection? How to
expert center. While many techniques are available none is perfect and a universal reference test detect atypical rare strains?
with high diagnostic performance in all clinical situations is still strongly sought after by the
parasitological community (see Outstanding Questions). New serological tests based on multi-
epitope chimeric peptides or antigens are the most promising for the diagnosis of toxoplasmo-
sis. Serotyping may become the method of choice for low-invasiveness typing of lineages in the
coming years, both as a prognostic test and for epidemiological studies. Further development of
serological tests will require further research to nd new strain- and clinical stage-specic
immunogenic peptides.

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