Abstract
purpose. Comparison of polymerase chain reaction (PCR) amplification of three Toxoplasma gondii genes in aqueous humor.
methods. Nested PCRs carried out using published methods were optimized for
maximum sensitivity and specificity. Five pairs of oligonucleotide
primers, directed against the B1, P30, and ribosomal genes, were used
and compared to determine which sequences were most effective in
detecting T. gondii DNA. Methods were developed with DNA
templates in water and were subsequently applied to both normal and
inflamed aqueous.
results. After one round of PCR amplification, P30 and ribosomal primers were
able to detect 1 pg genomic T. gondii DNA. However,
those directed against the B1 gene were able to detect 50 fg
(approximately single tachyzoite). This level of sensitivity was also
achieved using the P30 primers after a second round of PCR; however,
only primers based on the B1 gene maintained this level of sensitivity
in both normal and inflamed aqueous. B1-specific primers did not
amplify sequences from fungal, bacterial, or human lymphocyte DNA. The
sensitivity of T. gondii detection using B1
gene–specific primers was not compromised when large amounts of human
lymphocyte DNA were present, and application to an ocular sample or
retinal section from patients with toxoplasma chorioretinitis was
successful in confirming the presence of T. gondii DNA.
conclusions. The B1 PCR protocol appears to be the most sensitive protocol
in the detection of T. gondii DNA and has been
successful in identification of T. gondii DNA in ocular
fluids and retinal sections. This provides direct evidence of the
presence of T. gondii within the eye and may therefore
help in the management of toxoplasma
retinochoroiditis.
Diagnosis of Toxoplasma gondii retinochoroiditis can
be difficult in atypical cases, as might occur in AIDS patients or in
those cases where adequate funduscopy is precluded by overlying
vitreous opacity. Because the clinical picture is often that of a
panuveitis, associated with marked inflammatory activity in the
anterior chamber, and because anterior chamber taps are associated with
fewer complications than sampling from the vitreous cavity, an
investigation was undertaken to determine whether T.
gondii DNA could be detected in the aqueous humor. The
development of a highly sensitive polymerase chain reaction (PCR)
protocol to identify T. gondii DNA will help in the
early diagnosis of unusual retinochoroiditis and facilitate the
institution of appropriate treatment.
PCR amplification of sequences within the B1, P30, and ribosomal genes
of
T. gondii have been assessed in a variety of tissues,
in both immunocompetent and immunocompromised patients.
Blood,
1 2 bronchoalveolar lavage,
3 cerebrospinal fluid,
1 2 3 4 5 6 vitreous,
7 liver,
3 urine,
1 amniotic
fluid,
5 8 9 cardiac tissue,
10 cerebral
tissue,
3 11 12 and retina
13 14 15 have all been
investigated using these methods.
Several groups have reported the use of PCR analysis of aqueous humor
in the diagnosis of ocular toxoplasmosis using oligonucleotide primers
for DNA sequences within the B1 gene,
15 16 17 18 the P30
gene,
19 and the ribosomal gene
5 20 21 22 23 with
highly variable results.
In this study we have directly compared the sensitivity of previously
described primer sequences
4 24 25 in the amplification of
specific target sequences from these
T. gondii genes
both in water and aqueous humor and have developed a PCR-based protocol
that is capable of detecting a single
T. gondii tachyzoite.
Materials and Methods
Unless otherwise stated, all chemicals used were purchased from
Sigma Chemical (Poole, UK) and were of the highest grade available.
Preparation of T. gondii Tachyzoites, Inoculation of
Mice, and Isolation of DNA
Infection-free mice (MF1 strain) were inoculated intraperitoneally
with 0.5 ml of RH strain tachyzoites at a concentration of 500,000/μl
from a cell line that was originally isolated from a human
encephalopathy sample.
26 Tachyzoites isolated from
peritoneal fluid were counted using a modified Fuchs Rosenthal CSF
counting chamber. DNA was extracted after suspension in 2 ml lysis
buffer (50 mM Tris–HCl [pH 8.0], 0.1 M NaCl, 10 mM EDTA, 2% sodium
dodecyl sulfate, and 0.2 μg/ml proteinase K) and incubation at 50°C
for 3 hours. Samples were extracted with phenol:chloroform (1:1),
chloroform, precipitated with ethanol in the presence of 300 mM sodium
acetate (pH 5.2), and air-dried after washing with 70% ethanol. DNA
pellets were resuspended in TE buffer (10 mM Tris–HCl [pH 7.5], 1 mM
EDTA) and concentration assessed by both UV absorbance at 260 nm and by
comparison with
HindIII-digested lambda DNA standard. The
DNA was diluted to 10 ng/μl with water and stored at −20°C.
Collection of Aqueous Fluid
The extraocular environment was sterilized with 5%
povidone iodine solution before surgery. Approximately 100 to
200 μl of aqueous fluid was withdrawn using a 27-gauge (0.33-mm)
needle via a limbal paracentesis before routine cataract surgery in
patients with no evidence on funduscopy of T. gondii scars.
To test the effect, if any, of PCR amplification in the presence of
inflamed aqueous, 150 μl of aqueous was also obtained from a patient
with postoperative fibrinous uveitis.
Before sampling, informed consent was obtained from all patients. The
protocol for collection of aqueous samples was approved by the
institutional review board at Moorfields Eye Hospital. This research
followed the tenets of the Declaration of Helsinki at all times.
Retina
The sample of retinal tissue from an enucleation sample was
obtained from the Department of Pathology at the Institute of
Ophthalmology of Moorfields Eye Hospital (London, UK). Microscopy had
confirmed the presence of T. gondii cysts within the tissue
sections.
Nested PCR Protocols
One microliter of first-round product was used as a template for
subsequent nested amplification. The negative control sample from the
first-round amplification and a second-round negative control of
sterile water only was included in the nested amplification.
Amplification of the B1 Gene
Two pairs of oligonucleotide primers directed against the B1 gene
of
T. gondii 4 were used to perform a nested PCR
using purified
T. gondii DNA as a template
(Table 1) .
B1 Gene: First-Round Amplification.
PCRs contained 10 mM Tris–HCl, pH 8.3 (at 25°C), 50 mM KCl, 2 mM
MgCl2, 0.1 μM each primer, 0. 1 mM each dNTP,
1.25 U Taq DNA polymerase, and varying quantities of
purified T. gondii DNA. Reactions were cycled 40 times
with denaturation at 93°C for 10 seconds followed by annealing at
57°C for 10 seconds and finally an extension step at 72°C for 30
seconds. PCR negative control sample omitted template DNA, which was
substituted with sterile water.
B1 Gene: Nested Amplification.
Nested reactions contained 1 μl first-round product, 10 mM Tris–HCl,
pH 8.3 (at 25°C), 50 mM KCl, 3 mM MgCl2, 0.5μ
M each primer, 0.1 mM each dNTP, and 1 U Taq DNA
polymerase. Nested PCRs were cycled 40 times using a denaturation step
of 93°C for 10 seconds, followed by annealing at 62.5°C for 10
seconds and extension at 72°C for 15 seconds. Negative control
samples from first-round amplification and an additional second-round
negative control of sterile water were included in the nested
reactions.
P30 Gene
Nested primer sets identical to those used by Savva et
al.
24 and based on the published sequence of the
T.
gondii P30 gene
27 were used to amplify regions of the
P30 gene using a nested PCR approach
(Table 2) .
P30 Gene: First-Round Amplification.
Each 25-μl PCR mixture consisted of 10 mM Tris–HCl, pH 8.3 (at
25°C), 50 mM KCl, 2 mM MgCl2, 0.2 μM of each
primer, 0.1 mM of each dNTP, and 0.5 U Taq DNA polymerase
and varying concentrations of purified T. gondii DNA.
Reactions were cycled 35 times using a denaturation step of 95°C for
1 minute, annealing at 65°C for 1 minute, and extension at 74°C for
3 minutes. PCR negative control sample omitted template DNA, which was
substituted with sterile water.
P30 Gene: Nested Amplification.
One microliter of product was used as template for the nested
amplification. Each 25-μl reaction contained 10 mM Tris–HCl, pH 8.3
(at 25°C), 50 mM KCl, 1 mM MgCl2, 0.4 μM of
each primer, 0.1 mM of each dNTP, and 0.5 U Taq DNA
polymerase. Nested reactions consisted of 35 cycles using a
denaturation step at 95°C for 1 minute, followed by annealing at
66°C for 1 minute and extension at 74°C for 3 minutes. Negative
control samples from first-round amplifications and an additional
second-round negative control of sterile water were included in nested
PCRs.
Ribosomal Gene Amplification
Oligonucleotide primers used to amplify ribosomal DNA were based
on those used by Cazenave et al.
25 and on the published
sequence of the small subunit ribosomal RNA gene of
T.
gondii 28 (Table 3) . PCRs (25 μl) consisted of 10 mM Tris–HCl, pH 8.3 (at
25°C), 50 mM KCl, 2.5 mM MgCl
2, 0.2 μM of each primer,
25 μM of each dNTP, and 1.5 U
Taq DNA polymerase and
varying concentrations of purified
T. gondii DNA.
Reactions were heated at 95°C for 10 minutes, and cycled 35 times
using a denaturation step of 95°C for 10 seconds, followed by
annealing at 60°C for 30 seconds, and extension at 74°C for 1
minute. PCR negative control sample omitted template DNA, which was
substituted with sterile water.
PCR Amplification of Bacterial and Fungal Genomic DNA Using T. gondii B1 Gene Primers.
To show primer specificity, outer and nested B1 amplification reactions
were carried out on 25-μl reaction mixtures that contained 10 ng
genomic DNA from a variety of fungal and bacterial species. DNA from
the following species was used as template: Candida
albicans, Candida parapsilosis, Candida tropicalis, Aspergillus
fumigatus, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus viridans, Pseudomonas
aeruginosa, Serratia marcescens, Escherichia coli, Proteus mirabilis,
Klebsiella pneumoniae. Positive control consisted of 1 ng
genomic T. gondii DNA. The outer negative control
consisted of the 25-μl PCR without added DNA.
Amplification of Human Lymphocyte Genomic DNA Using T.
gondii B1 Gene–Specific Primers.
To ascertain that no human sequences were amplified with B1
gene–specific primers and that the presence of human lymphocyte DNA
did not inhibit detection of T. gondii B1 template, 1 ng of T. gondii genomic DNA was added to different amounts of
human DNA in sterile water and amplified using B1 gene primers.
Amplification of the B1 Gene in Inflamed Aqueous.
To establish the volume of inflamed aqueous responsible for inhibition
of B1 gene sequence, 1 ng of T. gondii genomic DNA was added
to various volumes of inflamed aqueous and subjected to PCR
amplification. To establish that the B1 PCR retained its sensitivity
even in the presence of inflamed aqueous, various quantities of T. gondii genomic DNA were added to 5-μl volumes of
aqueous that had been obtained from a patient with postoperative
fibrinous uveitis. Samples were subjected to direct PCR analysis
without prior DNA extraction.
Sample Preparation from Paraffin-Embedded Tissue
Retinal samples containing tissue cysts were deparaffinized
according to a technique described by Wright and
Manos.
29 Briefly, 15-μm sections of paraffin
embedded retina were cut, transferred to a 1.5-ml microcentrifuge tube,
and deparaffinized by two xylene extractions. One milliliter of xylene
was added to each tube, the closed tubes were then mixed at room
temperature for approximately 30 minutes. Tissue and residual paraffin
were then pelleted by centrifugation at 14,000 rpm for 5 minutes. After
a second xylene extraction the xylene was removed by pipette. The
samples were then washed twice with 100% ethanol to remove the organic
solvent. A 0.5-ml aliquot of 100% ethanol was added to each tube, the
contents of which were then mixed by inverting. Samples were then
centrifuged for 5 minutes at 14,000 rpm, and the ethanol removed by
pipette and the process repeated. The samples were resuspended in 100μ
l digestion buffer (50 mM KCl, 10 mM Tris–HCl [pH 8.3], 1 mM
EDTA, 2.5 mM MgCl
2, 0.1 mg/ml gelatin, 0.45%
octylphenol–ethylene oxide condensate, 0.45% polyoxyethylene sorbitan
monolaurate, 200 μg/ml proteinase K) and digested for 3 hours at
55°C. Proteinase K was inactivated by heating for 9 minutes at
95°C. After a 3-minute centrifugation at 14,000 rpm, 10 μl of
supernatant was used for PCR amplification. One in 2 and 1 in 10
dilutions of this material also underwent PCR amplification. In
addition 10 ng of genomic
T. gondii DNA in 10 μl
sterile water was also subjected to both the deparaffinizing process
and the digestion process so as to serve as positive controls for these
techniques. Negative extraction controls consisted of sterile water
that had undergone both the deparaffinizing and digestion processes.
Outer PCR positive controls consisted of 1 ng of genomic
T.
gondii DNA in 10 μl sterile water that underwent PCR without
prior extraction or digestion, and a final PCR negative control
consisted of sterile water that had not been subjected to either
extraction or digestion.
Additional positive and negative nested reaction controls consisted,
first, of 1 ng of genomic T. gondii DNA in 10 μl water
that had not been subjected to first-round PCR and, second, 10 μl
sterile water that had not been subjected to first-round PCR.
Visualization and Confirmation of PCR Amplification Products
Ten-microliter B1 and P30 amplification products were visualized
under UV illumination after electrophoresis on 1% to 2% TBE/agarose
gels and staining with ethidium bromide. Twenty microliters of
ribosomal gene amplification products was visualized on 4%
TBE/metaphor agarose gels (Flowgen Instruments, Lichfield, UK) or on
8% to 10% TBE/polyacrylamide gels (Bio-Rad Laboratories, Hemel
Hempstead, UK). A molecular weight marker was included in each run
(1.0-kb ladder, catalog No. 15615-016; GIBCO–BRL, Paisley, Scotland;
or 1.0-kb ladder, catalog No. G571A; Promega UK Ltd., Southampton, UK).
DNA Sequencing of PCR Products
Before sequencing of PCR products, amplified DNA from PCRs was
purified using the Geneclean II kit (BIO 101 Inc., Carlsbad,
California). PCR products were excised from agarose/TBE gels,
solubilized in sodium iodide and recovered into solution according to
the manufacturer’s instructions. PCR fragments were directly cycle
sequenced in both directions (using the outermost primers for each
gene) on an ABI prism automated DNA sequencer (model 377, version
2.1.1). DNA sequences were compared with target sequences and found to
be identical in all cases.
Results
Amplification of B1 Gene from T. gondii DNA
A single amplicon with a predicted size of 193 bp was amplified
using the first-round B1 gene primers. This reaction was capable of
detecting 50 fg of
T. gondii DNA after analysis on ethidium
bromide–stained TBE/agarose gels (
Fig. 1A ). It has been estimated that this is the amount of DNA contained within a
single
T. gondii organism.
29 When 1 μl of
first-round product was used as template in a nested amplification, a
96-bp target sequence was amplified but showed no increase in the level
of detection of
T. gondii DNA (50 fg). After the nested
reaction, however, there was a significant increase in the product
yield
(Fig. 1B) . The nested reaction did not yield product from either
the first- or the second-round negative controls. Both first-round and
nested PCR products were cycle-sequenced, which confirmed that the
amplified products were identical with the published sequence of the
T. gondii B1 gene.
4
B1 Gene: Amplification of T. gondii DNA in Aqueous
First-round amplification of
T. gondii DNA in 20%
aqueous (
T. gondii DNA spiked into 5-μl aqueous in a
total reaction volume of 25 μl) was able to detect 1 pg of
T.
gondii DNA
(Fig. 1C) . After second-round amplification using the
nested primers this protocol was effective in detecting 50 fg of
T. gondii DNA
(Fig. 1D) .
Amplification of T. gondii P30 Gene
Amplification of the P30 gene using outer primers resulted in a
single product of approximately 914 bp. Amplification of the P30 gene
using this protocol was able to detect 1 pg of purified
T.
gondii DNA after analysis on ethidium bromide–stained TBE/agarose
gels (
Fig. 2A ). When 1 μl of first-round PCR product was used as template in the
nested amplification, a single amplicon of approximately 520 bp was
observed. The sensitivity of the reaction was 50 fg of
T.
gondii DNA
(Fig. 2B) . Both the outer and nested products were
subject to cycle sequencing, and confirmed products were identical with
published sequences.
Amplification of the P30 Gene from T. gondii DNA in
Aqueous
First-round amplification of
T. gondii DNA in 20%
aqueous (
T. gondii DNA spiked into 5 μl aqueous in a total
reaction volume of 25 μl) was able to detect 1 ng of
T.
gondii DNA
(Fig. 2C) . However, second-round amplification using
the nested primers was only able to detect 1 pg
T. gondii DNA in the presence of 20% aqueous humor compared with 50 fg
T.
gondii DNA in the absence of aqueous humor
(Fig. 2D) .
Amplification of 18S rDNA Gene from T. gondii DNA
An 88-bp sequence from the 18S rDNA was used as the target
sequence for amplification.
25 Using oligonucleotide
primers based on those used by Cazenave et al. (1990),
25 amplification of the ribosomal gene from
T. gondii allowed
detection of 1 pg
T. gondii DNA (
Fig. 3A ). Sequence analysis confirmed the identity of the 88-bp amplicon.
Amplification of 18S rDNA Gene from T. gondii DNA in
Aqueous
When different concentrations of purified
T. gondii DNA
were supplemented with 5 μl aqueous, ribosomal gene amplification
detected only 1 pg of purified
T. gondii DNA
(Fig. 3B) .
B1 Gene–Specific Primer Sequences Do not Amplify Sequences from
Human, Bacterial, and Fungal Genomic DNAs
Primers directed against the
T. gondii B1 gene did not
produce an amplicon when the template genomic DNA was derived from
human lymphocytes (Lane 7,
Fig. 4B ), bacterial or fungal species.
Amplification of B1 Gene from 1 ng T. gondii DNA in
the Presence of Human Lymphocyte DNA
The presence of increasing amounts of human lymphocyte DNA
(10–150 ng) did not inhibit either the amplification of 193-bp B1 PCR
product from 1 ng purified
T. gondii genomic DNA in 20%
normal human aqueous or the nested amplification of 96-bp product
generated from 193-bp B1 PCR product
(Fig. 4) .
Amplification of the B1 Gene in Inflamed Aqueous
When 1 ng of genomic
T. gondii DNA was added to a
first-round PCR containing inflamed aqueous from a patient with
fibrinous uveitis, product inhibition was seen when the reaction mix
contained greater than 20% inflamed aqueous (
Fig. 5A ). When 1 μl of the outer reaction was added to a final nested reaction
mix of 25 μl, a 96-bp amplicon was produced even when the outer
reaction contained 60% inflamed aqueous. A decrease in the amount of
product however was seen in the nested reaction if the outer reaction
contained more than 40% by volume (10 μl) of inflamed aqueous
(Fig. 5B) . Because previous reports have indicated that 20% of aqueous fluid
per PCR can inhibit the reaction,
30 we have routinely used
5-μl samples of aqueous per 25 μl reaction for subsequent work.
After adding varying amounts of genomic
T. gondii DNA to 5μ
l of inflamed aqueous, a 193-bp amplicon was produced down to a
sensitivity of 1 pg
T. gondii DNA
(Fig. 5C) . When 1 μl of
this outer reaction was added to a nested reaction in a final volume of
25 μl, the reaction was capable of detecting 50 fg of
T.
gondii DNA, which is an identical sensitivity to the detection of
T. gondii DNA in water
(Fig. 5D) .
B1 Gene PCR Amplification of T. gondii DNA from a
Vitreous Sample in a Patient with Active Toxoplasmic Retinochoroiditis
Previous work in this laboratory has demonstrated equivalent
results for inhibition of PCR by aqueous and vitreous
fluids.
31 Therefore, the protocols described herein were
applied to a sample of vitreous taken from a patient with active
T. gondii retinochoroiditis. This sample was boiled at
95°C for 20 minutes and then divided into four 5-μl aliquots. To
the first three of these,
T. gondii genomic DNA was added.
No additional DNA was added to the fourth aliquot. The samples then
underwent PCR amplification using first-round primers directed against
T. gondii B1 gene in a 25-μl reaction mix. One microliter
of the outer reaction was then added to a 25-μl nested PCR.
First-round amplification of 193-bp B1 PCR product from vitreous
obtained from a patient with
T. gondii retinochoroiditis was
detected only in the positive control sample that had been spiked with
1 ng
T. gondii genomic DNA (
Fig. 6A ). However, nested amplification of 96-bp B1 PCR product generated from
vitreous obtained from a patient with
T. gondii retinochoroiditis was detected in all samples
(Fig. 6B) .
B1 Gene Amplification of T. gondii DNA from
Paraffin-Embedded Sections
Four deparaffinized retinal sections from a patient with
histopathologic evidence of
T. gondii cysts, typical of
Toxoplasma retinochoroiditis, were subjected to PCR using primers
directed against the B1 gene. After first-round amplification a single
193-bp product was visualized from reactions in three of the four
sections on 2% agarose gels (
Fig. 7A ). All negative controls remained negative. Positive and negative controls
that had been subjected to the deparaffinizing procedure all remained
positive and negative, respectively
(Fig. 7B) .
An identical product was visualized from all positive controls. After
nested amplification, 96-bp products were visualized from all retinal
sections
(Fig. 7C) . All positive and negative controls that
had been subjected to the deparaffinized procedure remained positive
and negative, respectively, after nested amplification (data not
shown).
Discussion
Ocular toxoplasmosis is the commonest cause of posterior uveitis
in the immunocompetent patient, accounting for some 30% to 50% of
cases worldwide.
32
T. gondii infection of the retina is characterized by
massive coagulative necrosis of the retina with inflammation of the
underlying choroid.
33 34 Toxoplasma retinochoroiditis is
usually a clinical diagnosis based on examination of the retina and
recognition of the characteristic lesions; however, this can be
difficult in atypical cases or where overlying vitreous opacity
precludes adequate funduscopy.
Current laboratory methods are limited by their lack of sensitivity and
specificity. Serology is of limited value in diagnosing
T.
gondii as the etiologic agent of disease in the eye. If the
patient is antibody negative,
T. gondii as the cause of a
retinal lesion is improbable,
35 36 although cases of
ocular toxoplasmosis with negative serum titers against
T.
gondii have been described.
37 38 Positive serology is
a sensitive test but is not specific, because serologic studies suggest
that asymptomatic toxoplasmosis occurs with great frequency in the
general population.
35 36 39 Analysis of local antibody
production to confirm a suspected clinical diagnosis of toxoplasma
retinochoroiditis is a valuable diagnostic tool in the immunocompetent
patient; however, false-positive
35 and
false-negative
36 results can be a problem.
A variety of different patients are at risk from this visually
debilitating disease. Immunocompromised hosts, as a result of
immunosuppressive therapy, malignancy, or AIDS, are at high risk of
disseminated toxoplasmosis. It has been estimated that 1% to 3% of
ocular infections in AIDS patients are due to
T.
gondii. 40 The disease, which may be bilateral, may be
the initial ocular infection in patients, sometimes preceding serologic
diagnosis of the HIV disease
41 42 and is often fulminant
and aggressive.
43 Prominent inflammatory reactions in both
the vitreous and anterior chamber are common
44 45 and in
proportion to the patient’ s CD4+ count.
46 Neither the
aqueous coefficient nor the serologic tests is helpful in making the
diagnosis of ocular toxoplasmosis in AIDS
patients.
41 45 46 The latter may remain negative
throughout the clinical course of the disease.
13
In the HIV patient population it is essential to establish the
diagnosis as rapidly as possible. Early and appropriate treatment of
ocular toxoplasmosis in AIDS patients is vital, because not only is the
ocular prognosis good
41 but also because ocular disease
often coexists with life-threatening cerebral
involvement
40 41 47 and may well precede neurologic
signs,
40 44 none of which are pathognomonic of
T.
gondii involvement in the central nervous system.
Molecular biological methods have been investigated to aid in the
clinical management of this condition. The use of PCR to amplify and
subsequently detect DNA within microorganisms in a range of tissues,
and particularly those that are difficult to culture
48 or
for which sample volumes are small, has proven extremely
valuable.
31 The ability to identify
T. gondii DNA in ocular samples will provide direct evidence of the presence of
the organism within the eye and therefore be helpful in determining the
diagnosis of
T. gondii infection and subsequent patient
management.
Primer Selection
The B1, P30, and ribosomal DNA genes are highly conserved in all
T. gondii strains tested to date,
4 8 49 and the
B1 and ribosomal genes are multiple copy genes within the
T.
gondii genome, making them ideal targets for PCR amplification.
The B1 gene is a 35-fold repetitive gene sequence with unknown
function. Within eukaryotes, ribosomal DNA is frequently repeated, and
within
T. gondii there are over 100 highly conserved copies
within the genome.
8 The P30 gene is expressed only by the
tachyzoite
50 and encodes for the most abundant surface
protein, comprising 5% of the total tachyzoite
protein.
27 51
Sensitivity
Nested amplification of the P30 and B1 genes allowed detection of
as little as 50 fg of T. gondii DNA in water. In the
aqueous, only amplification of the B1 gene yielded this level of
sensitivity. Nested amplification of the P30 gene in the aqueous
allowed detection only to a level of 1 pg T. gondii DNA.
The ribosomal gene was the least sensitive of these reactions, only
allowing detection of the predicted 88-bp target down to a DNA
concentration of 1 pg in water. This is perhaps surprising given that
it is the most highly repeated of the gene sequences studied, but may
be due at least in part to the fact that the ribosomal amplification
protocol used only a single reaction.
25 Nested reactions
increased the yield of product for both the other genes amplified.
Interlaboratory variability is a confounding factor when analyzing
results and trying to compare different methodologies used at different
centers. This study has used protocols published by others and
optimized all three PCRs in the one laboratory. We have concluded that,
at least in our hands, the B1 gene primers and PCR protocol are the
most sensitive of the three tested.
Selection of B1 Gene Primers
PCR amplification of DNA target sequences within the B1 gene had
several advantages. As with the P30 it allowed for greater sensitivity
than amplification of the ribosomal gene. However, it has been shown
that the P30 primers are less specific than those of the B1 gene,
because they have been demonstrated to amplify DNA targets of both the
Nocardia species
52 and
Mycobacterium
tuberculosis DNA.
53 We have shown that the
T.
gondii B1 primers do not amplify DNA from a variety of bacterial
and fungal species and that the reaction sensitivity remains unchanged
in the presence of increasing amounts of human DNA, and in the presence
of the increased levels of protein found in inflamed aqueous. It is,
however, possible that other parasites present in the tissue may be
amplified by these PCR techniques. To ensure the specificity of the PCR
primer, sequences were compared with all other sequenced DNA available
from current databases and were found to be noncomplementary to any
other known sequence. The possibility does exist, however, that a
related organism could be identified using these primers, an organism
whose gene sequence remains hitherto unknown. To minimize this
possibility the PCR is performed under stringent conditions. Reaction
specificity is facilitated by using the highest possible annealing
temperatures compatible with maximum reaction sensitivity. Finally,
sequencing of all amplified PCR products was performed and confirmed
the identity of the genes amplified.
Application to Clinical Samples
Although our initial investigations used spiked aqueous samples to
assess the efficacy of PCR detection of T. gondii, the first
clinical sample was vitreous obtained from a patient with suspected T. gondii retinochoroiditis. In this case a vitrectomy was
performed because the patient had developed a giant retinal tear, but
had an active lesion that was clinically suspicious of T.
gondii retinochoroiditis, and was already receiving prednisolone
EC 20 mg bid, and clindamycin 150 mg bid for ocular toxoplasmosis.
Previous work in this laboratory has demonstrated a lack of PCR
inhibition if the volume of vitreous does not exceed 20% of the final
reaction volume.
31 Because the B1 gene primers proved to
be both specific and the most sensitive of the primers tested, PCR
protocols using this primer set were applied to the detection of
T. gondii genes in ocular fluids and retinal sections. A
positive result was obtained with no “vitreous inhibition” of the
PCR, because all the vitreous samples spiked with genomic DNA from
T. gondii were positive with a sensitivity of 100 fg. The
applicability of these protocols to a large number of aqueous samples
has yet to be determined.
In conclusion, the B1 PCR protocol appears to be not only highly
specific in the amplification of T. gondii DNA but also, in
our hands, to be the most sensitive protocol in the detection of T. gondii and has been successful in the identification of T. gondii DNA in both vitreous and retinal sections. The
protocols described in this article have potential in aiding the future
management of patients with T. gondii infection.
Supported by grants from The Jules Thorn Trust Charitable Trust, London UK (CDJ); the Welcome Trust, London, UK (NO); and Fight for Sight, London, UK (PA).
Submitted for publication November 17, 1998; revised July 9, 1999; accepted August 23, 1999.
Commercial relationships policy: N.
Corresponding author: Susan Lightman, Department of Clinical Ophthalmology, Moorfields Eye Hospital, City Road, London, EC1V 2PD, UK.
[email protected]
| Oligonucleotide Primer | Sequence | Sequence Position |
| Outer primer (sense strand) | 5′-GGAACTGCATCCGTTCATGAG-3′ | 694–714 |
| Outer primer (nonsense strand) | 5′-TCTTTAAAGCGTTCGTGGTC-3′ | 887–868 |
| Inner primer (sense strand) | 5′-TGCATAGGTTGCAGTCACTG-3′ | 757–776 |
| Inner primer (nonsense strand) | 5′-GGCGACCAATCTGCGAATACACC-3′ | 853–831 |
Table 2. P30 Gene Primer Sequences
Table 2. P30 Gene Primer Sequences
| Oligonucleotide Primer | Sequence | Sequence Position |
| Outer primer (sense strand) | 5′-TTGCCGCGCCCACACTGATG-3′ | 405–424 |
| Outer primer (nonsense strand) | 5′-CGCGACACAAGCTGCGATAG-3′ | 1318–1299 |
| Inner primer (sense strand) | 5′-CGACAGCCGCGGTCATTCTC-3′ | 503–522 |
| Inner primer (nonsense strand) | 5′-GCAACCAGTCAGCGTCGTCC-3′ | 1024–1005 |
Table 3. Ribosomal Gene Primer Sequences
Table 3. Ribosomal Gene Primer Sequences
| Oligonucleotide Primer | Sequence | Sequence Position |
| Sense strand primer | 5′-CCTTGGCCGATAGGTCTAGG-3′ | 170–189 |
| Nonsense strand primer | 5′-TAGGCATTCGGGTTAAAGATTA-3′ | 253–231 |
The authors thank the following people for their advice: Ed Guy,
PhD, and David Joynson, FRCP, of Toxoplasma Reference Unit, Public
Health Laboratory, Singleton Hospital, Swansea, UK; and Bindya Patel,
MSc, Julie Johnson, MSc, and Rick Holliman, FRCP, of Toxoplasma
Reference Unit, Department of Medical Microbiology, St. George’s
Hospital, London, UK.
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