JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 2006, p. 1382–1389
0095-1137/06/$08.00ϩ0 doi:10.1128/JCM.44.4.1382–1389.2006Copyright 2006, American Society for Microbiology. All Rights Reserved.
Genotyping of Toxoplasma gondii by Multiplex PCR and Peptide-Based
Serological Testing of Samples from Infants in Poland Diagnosed
´n,3,4 Jack S. Remington,4,5 Michael Grigg,6 Elzbieta Golab,7
Department of Molecular Microbiology, Washington University School of Medicine, Saint Louis, Missouri 931101; Department ofFetal-Maternal Medicine and Gynecology, Research Institute Polish Mother’s Memorial Hospital, Lodz, Poland2; Department ofObstetrics and Gynecology, Stanford University School of Medicine, Stanford, California 943053; Department of Immunology andInfectious Diseases, Research Institute, Palo Alto Medical Foundation, Palo Alto, California 943014; Division ofInfectious Diseases and Geographical Medicine, Stanford University School of Medicine, Stanford, California 943055;Departments of Medicine and Microbiology & Immunology, University of British Columbia, Vancouver,British Columbia V5Z 3J5, Canada6; and Department of Medical Parasitology,National Institute of Hygiene, Warsaw, Poland7
Received 2 October 2005/Returned for modification 15 November 2005/Accepted 23 January 2006
Toxoplasma gondii has a clonal population genetic structure with three (I, II, and III) lineages that predominate in North America and Europe. Type II strains cause most cases of symptomatic human infections in France and the United States, although few other regions have been adequately sampled. Here we determined the parasite genotype in amniotic fluid and cerebrospinal fluid samples from congenital toxoplasmosis cases in Poland. Nineteen con- firmed congenital cases of toxoplasmosis were analyzed, including both severe and asymptomatic cases. The genotype of parasite strains causing congenital infection was determined by direct PCR amplification and restric- tion fragment length polymorphism analysis. Nested multiplex PCR analysis was used to type four independent polymorphic markers. The sensitivity of multiplex nested PCR was >25 parasites/ml in amniotic fluid and cerebral spinal fluid samples. Parasite DNA was successfully amplified in 9 of 19 samples (eight severely affected and one asymptomatic fetus). Only genotype II parasites were identified as the source of T. gondii infection based on restriction fragment length polymorphism analysis. Strains causing congenital infections were also typed indirectly based on detection of antibodies to strain-specific peptides. Serotyping indicated that 12 of 15 cases tested were caused by type II strains and these positives included both symptomatic and asymptomatic infections. Overall, the combined analysis indicated that 14 of the cases were caused by type II strains. Our results are consistent with the hypothesis that parasite burden is associated with severity of congenital toxoplasmosis and indicate that serological testing provides a promising method for genotypic analysis of toxoplasmosis.
Toxoplasmosis is a common parasitic disease caused by the
relatively avirulent in mice yet they readily establish chronic
protozoan parasite Toxoplasma gondii. Seroprevalence varies
infections characterized by tissue cysts that are highly infec-
between different geographic regions: in Poland, 2,200 (41.3%)
tious by the oral route (25, 27). Type I strains are more virulent
out of 4,916 pregnant women were found to have T. gondii-
in mice and have a greater capacity to cross tissue barriers in
specific anti-immunoglobulin G (IgG) antibodies (20, 20a).
Infection can be acquired by ingestion of viable tissue cysts in
Enhanced migration could potentially lead to greater capac-
undercooked meat or oocysts excreted by cats (23). T. gondii is
ity to cause congenital infection due to transplacental trans-
a major cause of morbidity and mortality in congenitally in-
mission, although such a relationship has not been directly
fected infants and immunodeficient and immunocompromised
demonstrated. A single study from Spain indicated that strains
possessing the type I allele at the SAG2 locus were found in 6
The population genetic structure of T. gondii is highly clonal,
of 13 cases of congenital infection (6). However, even strains
despite a sexual phase in the life cycle (14, 25, 26). Three
that are nonvirulent in the mouse model are capable of causing
predominant clonal types (I, II, and III) are recently derived
severe disease in humans, as shown by the prevalence of type
from recombination between two highly similar ancestral line-
II strains in congenital toxoplasmosis in France (1).
ages (8, 27). Recombinant genotypes are rarely found in na-ture, indicating infrequent sexual recombination between the
While the majority of genotyping studies have been based on
three lineages (1, 14). Type II strains have been identified as
polymorphic DNA markers, one of the primary limitations of
the cause of more than 70% of human cases of toxoplasmosis
this method is the inability to type strains causing chronic
in the United States and France (1, 5, 14). Type II strains are
infection. Strains of T. gondii are highly similar antigenically;however, the recent identification of serological epitopes thatare strain specific raises the possibility of genotyping even
* Corresponding author. Mailing address: Department of Molecular
chronic infections based on serological profile (17). Serological
Microbiology, Washington University School of Medicine, 660 S.
typing based on strain-specific peptides is capable of distin-
Euclid Ave., Saint Louis, MO 93110. Phone: (314) 362-8873. Fax: (314)362-3203. E-mail: email@example.com.
guishing type II strains from non-type II (typically I or III)
strains and offers the promise for determining the frequency of
homogenized and inoculated into another pair of mice that were then examined
strain types that cause both acute and chronic infections.
for Toxoplasma-specific antibodies as described above. Clinical samples. AF samples were obtained by amniopuncture under sono-
The presentation of congenital toxoplasmosis varies widely
graphic guidance in the Department of Ultrasound, RIPMMH, Lodz, Poland.
from subclinical to severe cases, which may cause fetal or
Additionally, in one case (number 18) fetal CSF was obtained when hydroceph-
neonatal death (23). The frequency and severity of fetal toxo-
alus was decompressed in utero by insertion of a ventriculoamniotic shunt (19).
plasmosis depends on the time when infection takes place
In two cases (nos. 1 and 10), CSF samples were collected postnatally for geno-
during pregnancy. Early in pregnancy, infections are less likely
typing. One neonate (no. 1) was confirmed infected by positive B1 PCR fromCSF taken a few days after birth (Table 1). In another case (no. 10), CSF was
to cross the placental barrier, yet those congenital infections
available from the infant after long-term antiparasitic treatment. For three ne-
that do result are more severe. While infection occurs more
onates (nos. 16, 17, and 18), AF was not available and instead, neonatal blood
readily late in pregnancy, the majority of such cases are mild or
was used for typing. Negative controls consisted of AF or CSF samples from
asymptomatic at birth (summarized in reference 23). High
pregnancies with negative anti-T. gondii serology after delivery. Samples werecollected and stored at Ϫ20°C at the Department of Fetal-Maternal Medicine
parasite concentrations in the amniotic fluid (AF) have been
and Gynecology, Research Institute Polish Mother’s Memorial Hospital, Lodz,
associated with severe outcome (24).
Poland and then sent frozen on dry ice to the Department of Microbiology,
In the present study we examined a set of well-characterized
Washington University School of Medicine, Saint Louis, MO.
cases of congenital toxoplasmosis from Poland. We deter-
Experimental samples. T. gondii tachyzoites were cultivated by 2-day passage
mined the genotypes of T. gondii found in AF and cerebral
in human foreskin fibroblast monolayers and purified from host cells as describedpreviously (14). Type strains consisted of RH (type I) (American Type Culture
spinal fluid (CSF) from cases of congenital toxoplasmosis using
Collection, Manassas, VA; ATCC 50174), PTG (type II) (ATCC 50841), and
a newly developed multiplex nested-PCR typing system (16).
CTG (type III) (ATCC 50842). T. gondii cells were harvested, counted, and
We also analyzed serological responses to strain-specific pep-
resuspended in phosphate-buffered saline (PBS). Two types of standards were
tides in order to serotype infections (17).
used for testing sensitivity. First, aliquots containing 1, 0.5, 2.5, 5, or 10 parasiteequivalents were made from a stock lysate of purified parasites prepared byincubation with 100 g/ml proteinase K for 1 h at 37°C and 2 h at 50°C, followedby heat inactivation at 90°C for 15 min. Second, aliquots (10 l) containing 5.0,
MATERIALS AND METHODS
10.0, 25.0, 50.0, or 100.0 parasites were added to 1 ml of negative AF and
Clinical cases of congenital toxoplasmosis. Nineteen cases of congenital toxo-
centrifuged at 3,000 ϫ g, and the pellet was used for analysis.
plasmosis diagnosed at the Research Institute Polish Mother’s Memorial Hospital
Multiplex PCR genotyping. We utilized a recently described multiplex nested
(RIPMMH, Lodz, Poland) between March 1999 and June 2003 were included in the
PCR for T. gondii genotyping based on four independent, unlinked markers:
study. The following classification of clinical forms of congenital toxoplasmosis was
5Ј-SAG2, 3Ј-SAG2, SAG3, GRA6, and BTUB (16). In the first round of PCR, all
used. Symptomatic toxoplasmosis was described when ventricular dilatation was
reverse and forward external primers were combined in one reaction tube. The
observed in repeated ultrasound scans and/or fetal and neonatal death was reported.
PCR mixture consisted of 5 l of 10ϫ PCR buffer (Sigma, St. Louis, MO)
Chorioretinitis was diagnosed during the neonatal period. Fetuses and/or neo-
containing 1.5 mM of MgCl2; 4 l of deoxynucleoside triphosphates (2.5 M
nates that did not present pathological symptoms were classified as asymptom-
each) (Roche Applied Sciences, Indianapolis, IN); 0.15 l of 50 M of each
atic. After birth, a team of specialists including neurologists and ophthalmolo-
forward and reverse primer (Integrated DNA Technologies, Coralvile, IA); 0.5
gists attended symptomatic and asymptomatic neonates. Congenitally infected
l of (5 U/l) Taq DNA polymerase (Sigma); and 31.0 l of distilled, DNase-
infants were treated with combined therapy with pyrimethamine and sulfadia-
and RNase-free water. PCR was conducted using a PTC-200 DNA engine system
zine. The studies were conducted with approval of the Ethical Committee of
Peltier thermal cycler (MJ Research Inc., Watertown, MA) programmed for
94°C for 30 seconds, 55°C for 60 seconds, and 72°C for 2 min during each of 35
Confirmation of congenital infection. Serological testing (anti-Toxoplasma
cycles. PCR products were digested with restriction enzymes (New England
IgG, IgM, IgA, and IgG avidity) was performed in the Department of Microbi-
BioLabs, Inc., Beverley, MA) and restriction fragment length polymorphisms
ology, RIPMMH, Lodz (20a). Prior to March 2000, screening of maternal sera
(RFLPs) were visualized using ethidium bromide staining of 2% agarose gels.
for Toxoplasma-specific IgG antibodies was performed with a latex agglutination
Sample processing. Clinical samples of AF and CSF were centrifuged for 10 min
´rieux) (positive cutoff: 4 IU/ml), or an indirect
at 3,000 ϫ g and pellets were extracted with a QIAamp DNA blood minikit
agglutination assay (Platelia Toxo-G; Diagnostics Sanofi Pasteur) (positive cut-
(QIAGEN Inc., Valencia, CA). Neonatal whole blood (200 l) was processed
off: Ͼ6 IU). Testing for Toxoplasma-specific IgM was conducted using an en-
directly using the QIAamp DNA blood minikit. In the final step, samples were
zyme-linked immunosorbent assay (ELISA) (Platelia Toxo M; Diagnostics
eluted with 25 l of buffer. Multiplex PCR was conducted using 12.5 l of the eluted
Sanofi Pateur). (cutoffs: 0 to 1 IU/ml, negative; 1 to 2 IU/ml, borderline; Ͼ2
samples as described above. Representative strains were used as positive controls.
IU/ml, positive). In cases where specific IgM was detected, an immunosorbent
Negative AF and CSF samples and distilled water served as negative controls.
agglutination assay was used for detecting specific IgM and IgA (bioMe
Serological typing. Strain-specific polymorphic peptides derived from the T.
(cutoffs: Ͻ6, negative; 6 to 7, borderline; Ͼ7, positive). In neonates, the Platelia
gondii dense granule (GRA) proteins GRA6 and GRA7 were coupled to keyhole
Toxo-M and Toxo-A tests were used with the same cutoff values as in the
limpet hemocyanin as described previously (17). Within the abbreviated names
mothers and the immunosorbent agglutination assay was used for IgM and IgA
of the peptides, 6 denotes peptides from GRA6 and 7 those from GRA7; I/III or
(cutoffs: Ͻ3, negative; 3 to 6, intermediate; Ͼ6, positive). IgG avidity was tested
II indicates the allele of the peptide, i.e., from which archetypal strain it was
using an ELISA (Labsystems) (cutoffs: low, Ͻ15%; borderline, 15 to 30%; high,
derived; and indicates a truncated version of the diagnostic peptide. Coupled
peptides were diluted to 2 g/ml in 0.1 M carbonate buffer, pH 8.5, and 50 l of
Beginning in March 2000, Toxoplasma-specific IgG was detected in sera from
each peptide solution was loaded into a polystyrene ELISA plate well overnight
mothers and their neonates by enzyme-linked immunofluorescent assay (VIDAS
at 4°C. Coated wells were blocked with 200 l of a 2% casein solution in PBS for
Toxo-IgG; bioMerieux) (positive cutoff: Ͼ8 IU/ml). Toxoplasma-specific IgM
was detected using VIDAS Toxo-IgM (bioMerieux) (positive cutoff: Ͼ0.65 IU/
Sera were tested by adding 50 l of diluted human serum (typically 1:100) to
ml) and Toxoplasma-specific IgA was detected using the Platelia Toxo-IgA
each well and incubating for 2 h at room temperature. ELISA plates were
method as described above. IgG avidity was tested using the enzyme-linked
washed four times with a PBS/0.1% Tween 20 solution before incubation with a
immunofluorescent assay test (VIDAS Toxo-avidity; bioMerieux) (cutoffs: low,
horseradish peroxidase-coupled monoclonal antibody against human IgG (BD
Ͻ0.2; borderline, 0.2 to 0.3; high, Ͼ0.3).
Pharmingen, San Diego, CA) for 1 h at room temperature. Plates were washed
PCR detection of parasite DNA was based on the B1 gene as described
in PBS and developed with 150 l of ABTS 2,2Ј-azinobis(3-ethylbenzthiazoline-
previously (7). PCR assays and bioassay by inoculation of mice were conducted
sulfonic acid) (ABTS) reagents (Kirkegaard and Perry Laboratories, Inc., Gaithers-
in the Department of Medical Parasitology National Institute of Hygiene, War-
burg, MD). Absorbance was read at 1, 2, and 4 h using a 405-nm filter. Two
saw. For bioassay, 1 ml of AF or CSF was injected intraperitoneally into labo-
control peptides served as negative controls to establish background reactivity for
ratory mice (CFW/Pzh strain). Six weeks after inoculation, blood samples were
normalization purposes. They consisted of a randomized sequence of the GRA6
examined with the Toxo-Screen DA test to detect seroconversion and establish
peptide and a mix of peptides derived from the human and Leishmania EF1␣
the titer of Toxoplasma-specific IgG antibodies. The brains of these animals were
proteins coupled to keyhole limpet hemocyanin.
TABLE 1. Clinical findings and genotype analysis in congenitally infected fetuses and infants with T. gondiiaa Abbreviations: GW, gestational week; NP, neonatal period; IUD, intrauterine death; MI, mouse inoculation; NS, neonatal serology; pos., positive result; neg.,
negative; ND, not done; NT, nontypeable; NA, not available; NB, neonatal blood. Mothers’ serology results are given for the first available test. CSF was used for PCRor mouse inoculation in cases 1 and 18 and AF was used in the remaining cases. Uncertain refers to low values with all peptides, such that a strain type could not bereliably determined. Atypical refers to the reaction with peptides from different strain types, reflecting either mixed infection or possibly unusual genotypes. The testsused and their cutoff values are given in the text.
ELISA data for each infection serum were normalized by dividing the
considered significant positive reactivity against the serotyping peptides, as
optical density (OD) value obtained at each of the eight serotyping peptides
defined previously (17). The four positive control sera utilized (two type II
by the mean of the OD readings for the two control peptides. Negative
and two type I/III) were from patients from whom parasites were recovered
reactivity thus yields a theoretical value of ϳ1.0: values of 1.4 or greater are
FIG. 1. Sensitivity of multiplex PCR analysis of T. gondii in AF (A) and CSF (B) samples. Multiplex PCR was performed for the four genetic
markers SAG2 (5Ј and 3Ј amplified separately), SAG3, GRA6, and BTUB followed by electrophoresis in agarose gels containing ethidium bromide. Lanes 1, 6, and 7 are negative controls, and lanes 2 to 5 are samples spiked with 0.5, 2.5, 5.0, and 10.0 T. gondii cells, respectively.
genital toxoplasmosis was confirmed by two of the followingcriteria: positive results by B1 PCR with AF or CSF; positive
Sensitivity of multiplex PCR in amniotic fluid. To establish
serology in mice after inoculation of AF or CSF samples into
the sensitivity of the multiplex PCR, 1-ml volumes of AF or CSF
mice; and presence of specific IgM and/or IgA in the neonate
samples from uninfected pregnancies were spiked with lysates of
(Table 1). Four neonates were classified as congenitally in-
T. gondii equivalent to 0.5 to 10 parasites per sample. Samples
fected based solely on demonstration of specific IgG, IgM,
were then directly processed for PCR. The sensitivity of detection
and/or IgA antibodies in repeated tests following birth (Table
ranged from 2.5 to 10 parasites per sample in both AF (Fig. 1A)
1). In the remaining case, only IgG was demonstrated, how-
and CSF (Fig. 1B). In the primary round of multiplex PCR (leftside of Fig. 1), no amplification products were seen, consistent
ever, it was included in the study based on inoculation of AF
with the low input of parasites. In the second round, specific
into naı¨ve mice and their subsequent positive serology (case
products were detected for each marker (right side, Fig. 1). Dif-
number 14). Ventricular dilatation (ventriculomegaly or hy-
ferences in the sensitivity of detection of individual markers likely
drocephalus) was detected prenatally in 11 cases (Table 1).
reflect efficiencies based on different PCR primers, since all of
Five out of 11 symptomatic cases died, two in utero and three
these targets are present as a single copy per genome.
in early postnatal periods. Chorioretinitis was diagnosed in
We also tested the sensitivity of multiplex PCR under con-
four neonates. Eight cases were asymptomatic.
ditions that more closely approximate sample processing. In
For 14 of the cases, AF samples were used for detection of
this instance, defined numbers of whole parasite cells were
parasite DNA by multiplex PCR (Table 1). However, for sev-
added to 1 ml of normal AF samples and centrifuged, and the
eral cases, only CSF (numbers 1 and 10), or only neonatal
pellet was extracted and analyzed. Under these conditions, the
blood (numbers 16, 17, and 18) was available. PCR amplifica-
sensitivity was approximately 25 parasites per sample (data not
tion of T. gondii genetic markers was only successful in 9 out of
shown). The decreased sensitivity under these conditions likely
19 samples; the remaining samples were negative in all tests
reflects inefficiency in recovery of parasites following centrifu-
(Tables 1 and 2). Eight of the nine cases that were positive
gation and/or extraction. However, since these conditions
were detected with AF samples and the remaining positive was
more closely simulate processing of clinical AF samples, they
likely reflect the real sensitivity of this method versus the more
Gene-specific amplification products were subjected to re-
efficient detection seen when small samples are spiked with
striction enzyme digestion to identify characteristic RFLPs and
the genotypes were determined based on the combination of
Genotypes of T. gondii by multiplex PCR-RFLP analysis.
alleles at different markers (16). For eight of the nine samples,
Nineteen cases of confirmed congenital toxoplasmosis were
the genotype was determined to be type II for all four markers
examined here, as summarized in Table 1. In 14 cases, con-
(Table 2). A representative example is shown for 3Ј-SAG2 in
TABLE 2. Genotypes of T. gondii in clinical samples from congenitally infected fetuses and newborns based on multiplex PCRaa Alleles are defined in reference 16. Genotype II is the result of allele 1 at 5Ј-SAG2 and allele 2 at 3Ј-SAG2. b Neg, negative. c Where sample type and volume are not specified for the case, the sample was 1 ml of AF.
Fig. 2. In one CSF sample (number 1), genotype II was deter-
peptides to define the genotype of parasites causing infection,
mined only for the 3Ј-SAG2 and GRA6 markers and the sam-
as described previously (17). A total of 28 mother and infant
ple was negative for the remaining markers (Table 2). Geno-
sera from 15 different cases of congenital toxoplasmosis (two
typing was not successful with the second CSF sample,
were not available as paired samples) were provided to the
suggesting a lower parasite concentration in CSF than in AF.
Toxoplasma Serology Laboratory (Palo Alto Medical Re-
In all three severe cases that were not successfully genotyped,
search Foundation) as a blinded set of samples without any
late referral complicated proper sampling and this may have
identifying information about strain genotype, clinical disease,
or dye test titer prior to analysis. Sera were tested against a set
Genotyping at the SAG1 locus and type X. The genotype at
of polymorphic peptides that have previously been shown to
SAG1 was also examined using gene-specific PCR to distin-
identify strain-specific antibodies present in patient serum
guish between parasite type II and newly described type X
(17). Reaction to these peptides can thus be used to genotype
(18). RFLP analysis does not discriminate between type X and
the strain responsible for infection.
type II strains at the markers (SAG2, SAG3, and GRA6) used
Positive control sera (Table 3) established that the assay was
here. However, type X can be distinguished from type II by the
reproducible and working within the detection and cutoff limits
presence of a type I RFLP pattern at SAG1 after digestion with
as previously established by Kong and colleagues (17). The
Sau961 (18). PCR-RFLP analysis of the nine clinical samples
results show that 20 of 28 sera (representing 12 of 15 cases)
studied here showed an RFLP pattern at SAG1 consistent with
produced a reactivity pattern consistent with infection by type
type II and distinct from type X (Table 2).
II strains (positive reaction with one or more type II peptides
Serological typing of clinical samples. Sera from both the
designated 6II, 6 d-II, 7 II, and 7 d-II) (Table 3). Two sera
mother and infant were tested for reaction to allele-specific
(maternal sera from cases 2 and 14 in Table 1) gave no clearindication of the type responsible for infection, as reactivityagainst all peptides was weak. However, in both cases, serafrom the infant showed reactivity to at least one type II pep-tide. These results for maternal and infant sera were consis-tently observed in repeated assays.
IgG antibodies detected in neonatal sera are almost cer-
tainly derived from transfer of maternal antibodies. Thus, thenegative results for the mothers of these infants is likely due todegradation of the sample during storage. Paired maternal andinfant sera from case number 13 reacted weakly with the 6d-I/III peptide but none of the other I/III diagnostic peptides. All type I/III infection sera previously characterized haveshown strong reactivity with peptide 6 I/III, so lack of reactivity
FIG. 2. RFLP analysis of PCR products amplified from AF and
with this peptide precludes assigning these two sera. Paired
CSF samples from cases of congenital toxoplasmosis. 3Ј-SAG2 ampli-
maternal and infant sera from case 15 and case 17 reacted
fication products digested with HhaI were resolved in 3% agarose gelsstained with ethidium bromide. The samples loaded in lanes I, II, and
strongly with both type II and type I/III peptides, suggesting a
III are representative of T. gondii strain types I, II, and III, respectively.
possible mixed infection. Mixed infections are highly unusual,
Samples from pregnancies with congenitally infected fetuses were
and thus their reactivity is listed as atypical. Based on the
loaded in lanes 1 to 9. Genotyping revealed all nine positive samples
serological testing, it was concluded that 12 of the 19 cases of
were type II strains of T. gondii. AF samples from uninfected pregnantwomen (neg) served as negative controls.
congenital toxoplasmosis were due to type II strains.
TABLE 3. Serological analysis of paired mother-infant samples with strain-specific peptidesaa Peptides utilized in testing are listed across the top of the table. The negative control value is the mean for two negative peptides (see text). Positive controls were
provided by patient sera from previously typed cases of toxoplasmosis (see text). Results are shown for sera from the cases listed in Table 1. Sera were reacted againstthe panel of eight strain-specific peptides to identify allele-specific antibodies present in infection serum. All data were normalized by dividing the A405 reading for eachserotyping peptide against the mean of the two control peptides. Values of Ͼ1.4 were considered significant (17). Uncertain refers to low values with all peptides, suchthat a strain type could not be reliably determined. Atypical refers to reaction with peptides from different strain types, reflecting either mixed infection or possiblyunusual genotypes. DISCUSSION T. gondii has a highly clonal population structure and most
human infections are caused by one of three main genotypes.
We used multiplex PCR to genotype strains of T. gondii
directly in AF and CSF samples from complicated congenital
Type II strains cause the majority (70 to 80%) of human cases
infections in Poland. We found exclusively type II strains of T.
of toxoplasmosis reported previously from North America and
gondii and eight of nine typeable cases represented severe
Europe (primarily France) (1, 5, 12–14). Type I is infrequent in
cases of toxoplasmosis. Serological typing was consistent with
nature, but has been shown to be more common both in con-
type II genotypes causing the majority of symptomatic and
genital toxoplasmosis (6) and in AIDS patients (14). Type III
asymptomatic infections. Our findings indicate that type II
strains are largely found in animals and only rarely cause hu-
strains can cause both benign and complicated cases and are
man infection for reasons that are unknown (14).
consistent with the hypothesis that the severity of infection is
In the present study, all of the cases typeable by nested PCR
primarily related to the burden of parasites.
were type II, and eight of nine positives were obtained from
severe cases. Five cases resulted in death in utero or early in
testing, which also identified five additional cases of type II
the neonatal period. A total of 14 of 19 cases studied in the
infections that were only typeable by serology. The failure to
present report were found to be due to type II strains, based on
detect type I/III samples among these samples is not due to
genotyping and/or serological typing. The samples studied here
assay bias, since both PCR-based and serological typing per-
come from a referral hospital that receives patients with com-
form well with standards that included samples from patients
plicated pregnancies from a wide area of Poland. Thus, our
with infections of known genotypes in the case of serology
studies provide a baseline for further analysis of congenital
cases of toxoplasmosis in Poland to determine if this pattern of
Severe symptoms of congenital toxoplasmosis occur
type II infections is widespread. Ajzenberg et al. also described
mostly as a result of infections that occur early in pregnancy.
type II in eight out of eight cases of fetal or newborn death and
Results of real-time PCR showed that severe outcome was
in a majority (73 of 86 samples) of all congenital cases studied
associated with high parasite load and the highest parasite
from France (1). In contrast to these findings, a study from
concentrations were found when seroconversion occurred
Spain based on single-locus PCR-RFLP analysis reported that
early in pregnancy (24). In our study, the timing of the
non-type II strains were found in 12 of 13 congenital infections
mother’s primary infection was uncertain due to the lack of
(6). This difference may be primarily related to geography,
serological monitoring. However, all of the mothers had
since any referral bias for severe cases would appear to be the
serological results suggesting primary infection during the
same in both studies. Collectively, these results indicate mul-
first or second trimester of pregnancy (IgM and/or IgA
tiple strain types can be associated with severe toxoplasmosis
and/or low IgG avidity). In Poland, serological screening in
pregnancy is not routinely performed, although anti-T. gon-
Clinical forms of congenital toxoplasmosis vary widely. Only
dii testing is recommended. Routine ultrasound screening
5 to 10% of all infected fetuses develop serious disease, and the
during pregnancy is well established in Poland.
majority of infections are asymptomatic (23). Chorioretinitis
In our study, detection of symptoms in a fetus was typically
can result from both congenital and acquired infection (11).
followed by prenatal diagnosis of congenital toxoplasmosis.
Human ocular toxoplasmosis is associated with different
The prevalence of T. gondii (41.3% seropositive) in pregnant
strains, including strains containing a mixture of type I and
women in Poland is high (20, 20a), and screening of cord blood
type III genotypes (9). Chorioretinitis was diagnosed in four
from neonates, which per se does not detect all congenitally
neonates presented in our study, all due to type II infections.
infected cases, indicates a frequency of infection of ϳ1 in 1,000
Recently, Miller et al. described T. gondii genotype X that was
to 2,000 live births (21, 22). Most of these cases are likely not
associated with brain lesions and mortality in sea otters in the
severe, and the high rate of fetal defects among the cases
western United States (18). Our samples from cases with se-
studied here is mainly due to the referral of complicated preg-
vere clinical signs of the disease (hydrocephalus and fetal/
nancies to RIPMMH, suggesting that the majority of asymp-
neonatal death) were found to match the type II, not the type
tomatic cases are not detected. Elective interruption of preg-
nancies in Poland is very rare, so the emphasis is placed on
Semiquantitative PCR with the B1 gene has been used pre-
possible treatment in utero and postnatally (19).
viously to estimate the parasite burden in AF during congenital
Strain type has been suggested to play a role in determining
infection (10, 24). The parasite load estimated using real-time
the outcome of T. gondii infection (3). In our study, T. gondii
PCR was less then 10 parasites/ml in 40 to 46% of AF samples
type II was identified in eight AF and one CSF sample from
and 11 to 100 parasites/ml in 30 to 40% of samples (4, 24)
Polish fetuses and neonates with severe forms of disease. Im-
Previous studies using real-time PCR in AF established the
portantly, for all samples for which PCR genotype data were
relationship between high parasite burden and severity of dis-
available, the serotyping assay accurately predicted infection
ease (24). PCR detection with B1 is more sensitive than the
by a type II strain (Tables 2 and 3). While most asymptomatic
methods used here; however, it is not sufficiently polymorphic
cases were generally not typeable by PCR, serological analysis
to allow simple RFLP typing of alleles. For these reasons, we
indicated a majority of these were also type II. The reaction
developed nested PCR-RFLP typing of several single-copy
between mothers and infants was highly correlated, likely re-
genes that are polymorphic (16). While these markers are well
flecting the fact that this assay detects IgG that is acquired by
suited for typing, they are less sensitive overall than detection
the infant by transfer across the placental barrier in utero.
Serum from several cases (13 and 15) reacted to peptides of
In our study, the sensitivity of parasite detection by multiplex
both type II and I/III, raising the possibility that these patients
nested PCR was Ն25 parasites/ml in AF and CSF. Thus, the
were multiply infected either with a mix of different strains or
negative status of many of our samples may have resulted from
by a novel strain(s) that possesses alleles at GRA6 and GRA7
small amounts of parasite DNA in these samples. Consistent
that induce antibodies that react to peptides of both alleles.
with this, most of those strains that were typeable by PCR (Ͼ25
Interpretation of these sera would require parasite genotype
parasites/ml) were from severe cases. These findings are con-
information, but unfortunately, insufficient parasite DNA was
sistent with an association between parasite burden and dis-
recovered from these patients to allow PCR-RFLP genotyping.
ease association established previously by more quantitative
Our findings demonstrate that type II strains can be associated
methods (10, 24). The failure to type some cases by nested
with either mild and severe disease and support the hypothesis
PCR-RFLP analyses also raises the possibility that the present
that high parasite concentration is primarily responsible for the
samples have an inherent bias. However, our conclusion that
severity of congenital disease. They also demonstrate a close
type II strains cause the majority of infections in this sample
concordance of parasite genotyping by PCR-based genetic mark-
group is supported by similar results obtained by serological
ers and serological testing using strain-specific epitopes. Ex-
panded use of serological typing should enable more comprehen-
14. Howe, D. K., and L. D. Sibley. 1995. Toxoplasma gondii comprises three
sive analysis of the contribution of parasite genotype to clinical
clonal lineages: correlation of parasite genotype with human disease. J. In- fect. Dis. 172:1561–1566.
15. Joynson, D. H., and T. J. Wreghitt. 2001. Toxoplasmosis: a comprehensive
clinical guide. Cambridge University Press, Cambridge, England. ACKNOWLEDGMENTS
16. Khan, A., C. Su, M. German, G. A. Storch, D. Clifford, and L. D. Sibley.
2005. Genotyping of Toxoplasma gondii strains from immunocompromised
D.N. was supported by a Fulbright Senior Fellowship Grant. This work
patients reveals high prevalence of type I strains. J. Clin. Microbiol. 43:5881–
was also partially supported by an NIH grant to L.D.S. (AI059176).
We thank Tadeusz H. Dzbenski for PCR testing and mouse inocu-
17. Kong, J. T., M. E. Grigg, L. Uyetake, S. F. Parmley, and J. C. Boothroyd.
lation studies conducted in the Department of Medical Parasitology,
2003. Serotyping of Toxoplasma gondii infections in humans using synthetic
National Institute of Hygiene, in Warsaw, Poland, Asis Khan for tech-
peptides. J. Infect. Dis. 187:1484–1495.
nical advice, and Julie Suetterlin and Dorota Stodolnik for technical
18. Miller, M. A., M. E. Grigg, C. Kreuder, E. R. James, A. C. Melli, P. R. Crosbie, D. A. Jessup, J. C. Boothroyd, D. Brownstein, and P. A. Conrad. 2004. An unusual genotype of Toxoplasma gondii is common in California REFERENCES
sea otters (Enhydra lutris nereis) and is a cause of mortality. Int. J. Parasitol.
1. Ajzenberg, D., N. Cogne´, L. Paris, M. H. Bessieres, P. Thulliez, D. Fillisetti, 34:275–284. H. Pelloux, P. Marty, and M. L. Darde ´. 2002. Genotype of 86 Toxoplasma
19. Nowakowska, D., M. Respondek-Liberska, E. Golab, B. Stray-Pedersen, K. gondii isolates associated with human congenital toxoplasmosis and correla-
Szaflik, T. H. Dzbenski, and J. Wilczynski. 2005. Too late prenatal diagnosis
tion with clinical findings. J. Infect. Dis. 186:684–689.
of fetal toxoplasmosis: a case report. Fetal Diagn. Ther. 29:190–193.
2. Barragan, A., and L. D. Sibley. 2002. Transepithelial migration of Toxo-
20. Nowakowska, D., M. Slaska, E. Kostrzewska, and J. Wilczynski. 2001. Anti- plasma gondii is linked to parasite motility and virulence. J. Exp. Med. Toxoplasma gondii antibody concentration in sera of pregnant women in the
´dz population. Wiad. Parazytol. 47(Suppl. 1):83–89.
3. Boothroyd, J. C., and M. E. Grigg. 2002. Population biology of Toxoplasma
20a.Nowakowska, D., B. Stray-Pedersen, E. Spiewak, W. Sobala, E. Malafiej, and gondii and its relevance to human infection: do different strains cause dif-
J. Wilczyski. Prevalence and estimated incidence of Toxoplasma infection
ferent disease? Curr. Opin. Microbiol. 5:438–442.
among pregnant women in Poland: a decreasing trend in the younger pop-
4. Costa, J. M., P. Ernault, E. Gautier, and S. Bretagne. 2001. Prenatal diagnosis
ulation. Clin. Microbiol. Infect., in press.
of congenital toxoplasmosis by duplex real-time PCR using fluorescence reso-
21. Paul, M., E. Petersen, Z. S. Pawlowski, and J. Szczapa. 2000. Neonatal
nance energy transfer hybridization probes. Prenat. Diagn. 21:85–88.
screening for congenital toxoplasmosis in the Poznan region of Poland by
5. Darde´, M. L., B. Bouteille, and M. Pestre-Alexandre. 1992. Isoenzyme anal-
analysis of Toxoplasma gondii-specific IgM antibodies eluted from filter pa-
ysis of 35 Toxoplasma gondii isolates and the biological and epidemiological
per blood spots. Pediatr. Infect. Dis. 19:30–36.
implications. J. Parasitol. 78:786–794.
22. Paul, M., E. Petersen, and J. Szczapa. 2001. Prevalence of congenital Toxo-
6. Fuentes, I., J. M. Rubio, C. Ramı´rez, and J. Alvar. 2001. Genotypic charac- plasma gondii infection among newborns from the Poznan region of Poland:
terization of Toxoplasma gondii strains associated with human toxoplasmosis
validation of a new combined enzyme immunoassay for Toxoplasma gondii-
in Spain: direct analysis from clinical samples. J. Clin. Microbiol. 39:1566–
specific immunoglobulin A and immunoglobulin M antibodies. J. Clin. Mi-
7. Golab, E. 1995. Detection of Toxoplasma gondii DNA in body fluids by
23. Remington, J. S., R. McLeod, P. Thulliez, and G. Desmonts. 2001. Toxo-
polymerase chain reaction. Wiad. Parazytol. 41:13–18.
plasmosis, p. 205–346. In J. S. Remington and J. O. Klein (ed.), Infectious
8. Grigg, M. E., S. Bonnefoy, A. B. Hehl, Y. Suzuki, and J. C. Boothroyd. 2001.
diseases of the fetus and newborn infant, 5th ed. W. B. Saunders Company,
Success and virulence in Toxoplasma as the result of sexual recombination
between two distinct ancestries. Science 294:161–165.
24. Romand, S., M. Chosson, J. Franck, M. Wallon, F. Kieffer, K. Kaiser, H. Grigg, M. E., J. Ganatra, J. C. Boothroyd, and T. P. Margolis. 2001. Unusual abundance of atypical strains associated with human ocular toxoplasmosis. Dumon, F. Peyron, P. Thulliez, and S. Picot. 2004. Usefulness of quantitative
polymerase chain reaction in amniotic fluid as early prognostic marker of
10. Hohlfeld, P., F. Daffos, J. Costa, P. Thulliez, F. Forestier, and M. Vidaud.
fetal infection with Toxoplasma gondii. Am. J. Obstet. Gynecol. 190:797–802.
1994. Prenatal diagnosis of congenital toxoplasmosis with a polymerase-
25. Sibley, L. D. 2003. Recent origins among ancient parasites. Vet. Parasitol.
chain-reaction test on amniotic fluid. N. Engl. J. Med. 331:695–699. 115:185–198.
11. Holland, G. N. 1999. Reconsidering the pathogenesis of ocular toxoplasmo-
26. Sibley, L. D., and J. C. Boothroyd. 1992. Virulent strains of Toxoplasma
sis. Am. J. Ophthalmol. 128:502–505. gondii comprise a single clonal lineage. Nature (London) 359:82–85.
12. Honore´, S., A. Couvelard, Y. J. Garin, C. Bedel, D. He´nin, M. L. Darde´, and
27. Su, C., D. Evans, R. H. Cole, J. C. Kissinger, J. W. Ajioka, and L. D. Sibley. F. Derouin. 2000. Genotyping of Toxoplasma gondii strains from immuno-
2003. Recent expansion of Toxoplasma through enhanced oral transmission.
compromised patients. Pathol. Biol. (Paris) 48:541–547.
13. Howe, D. K., S. Honore´, F. Derouin, and L. D. Sibley. 1997. Determination
28. Su, C., D. K. Howe, J. P. Dubey, J. W. Ajioka, and L. D. Sibley. 2002.
of genotypes of Toxoplasma gondii strains isolated from patients with toxo-
Identification of quantitative trait loci controlling acute virulence in Toxo-
plasmosis. J. Clin. Microbiol. 35:1411–1414. plasma gondii. Proc. Natl. Acad. Sci. USA 99:10753–10758.
Curriculum Vitae El Dr Ángel Luis Montejo (1959) es psiquiatra del Hospital Universitario de Salamanca y Profesor de Psiquiatría y Psicología Médica de la Facultad de Medicina de la Universidad de Salamanca. Es también Coordinador de Investigación de la sociedad Castellano Leonesa de Psiquiatría, creador y director del Grupo Español de Trabajo para el Estudio de la Sexualidad y Sa
E N E E S M I D D E L E N Actieve bewaking van nieuwe diabetesmiddelen Recent zijn er twee nieuwe geneesmiddelen terbehandeling van diabetes mellitus type 2 op de marktverschenen, sitagliptine en exenatide. Om een beeldte krijgen van het bijwerkingenprofiel van deze mid-delen, worden ze sinds februari 2008 gevolgd metCentrum voor Farmacie, basiseenheid Farmacotherapie en Farmaceutische