Specific, Sensitive, and Rapid Diagnosis of Active Toxoplasmosis by a Loop-Mediated Isothermal Amplification Method Using Blood Samples from Patients

Parasite culture.Tachyzoites of the T. gondii RH strain were obtained from the Institute for Medical Research (IMR), Kuala Lumpur, Malaysia. The tachyzoites were grown in MBDK cell monolayers in RPMI medium supplemented with 10% fetal calf serum at 37°C in a 5% CO₂ environment.

Clinical samples.A total of 105 individuals were studied. The first group consisted of 40 clinically suspected toxoplasmosis patients with acute toxoplasmosis (high Toxoplasma IgM antibody titers) serologically confirmed by Captia Toxoplasma gondii IgG and IgM commercial diagnostic kits (Trinity Biotech, Ireland) and Toxoplasma IgG avidity test (NovaLisa, Dietzenbach, Germany). The second group consisted of 40 healthy volunteers who did not show antibodies against Toxoplasma antigens on the basis of the results of the same tests. The third group consisted of 25 patients with other parasitic infections (confirmed by PCR or serology tests); among these patients, 15 were positive for malaria (Plasmodium spp.) and 10 were positive for filariasis (Brugia malayi).

Analytical sensitivity and specificity of LAMP.To determine the analytical sensitivity of the LAMP assay, T. gondii RH tachyzoites harvested from the cell culture were counted on a hemocytometer and serially diluted 10-fold to contain from 1 × 10⁶ tachyzoites to 1 tachyzoite in 200 μl of fresh human blood. The genomic DNA was extracted from the 200 μl of solution using a DNeasy blood and tissue kit (Qiagen, Germany), in accordance with the manufacturer's instructions. Genomic DNA was eluted with 200 μl of elution buffer. The reactions were done in triplicate. Genomic DNA of Cryptosporidium parvum (oocyst obtained from human feces), Neospora caninum (oocyst obtained from dog feces), and Toxocara canis (eggs obtained from dog feces) were also extracted using the same method to determine the specificity of the Toxoplasma LAMP assay.

LAMP assay.Three Toxoplasma-specific LAMP primer sets were designed, using the Primer Explorer program (http://primerexplorer.jp/elamp4.0.0/index.html ), to amplify the SAG1, SAG2, and B1 genes (Table 1). LAMP was optimized in 25 μl of a mixture containing 2 μl of the extracted DNA, 40 pmol (each) of primers FIP and BIP, 5 pmol (each) of primers B3 and F3, and 1 μl of Bst DNA polymerase (NEB) in 2.5 μl of buffer [20 mM Tris-HCl (pH 8.8), 10 mM KCl, 8 mM MgSO₄, 10 mM (NH4)₂SO₄, 0.1% Tween 20], 0.8 M betaine (Sigma-Aldrich), and 1.4 mM deoxynucleoside triphosphate (dNTP). The LAMP reaction was performed for 30 to 60 min at 65°C and was inactivated for 2 min at 80°C. The resulting amplicons were detected in real time by the fluorescence of SYTO-9 dye and visual observation after addition of SYBR green I. Twenty microliters of each of the LAMP products was analyzed by electrophoresis with 1.5% agarose gels, followed by ethidium bromide staining and photography.

DNA sequencing of LAMP products.To confirm the specificity of the LAMP assay for Toxoplasma detection, the PCR method was carried out with the outer primers B3 and F3. PCR amplification was performed in 25 μl of reaction mixtures containing 5 μl of PCR buffer (10×), 5 μl (200-μmol concentration) of each dNTP, 4 μl (200 nmol concentration) (each) of primers B3 and F3, and 0.25 μl (2.5 U) of AmpliTaq Gold polymerase (Applied Biosystems, Darmstadt, Germany). Initial denaturation was done at 95°C for 10 min, followed by 35 cycles of denaturation (45 s at 95°C), annealing (60 s at 60°C), and extension (90 s at 72°C). Five-microliter aliquots of the PCR products were subjected to electrophoresis on a 1.5% agarose gel and visualized under UV light. Purification of the PCR products was performed by a QIAquick PCR purification kit (Qiagen), using the microcentrifuge protocol, according to the manufacturer's instructions. PCR products were sent for sequencing (NHK Bioscience Solution, Malaysia).

Clinical sensitivity and specificity.The sensitivities and specificities of the LAMP assays in comparison to the results of the nested PCR were determined using 105 blood samples from the three groups of individuals. Genomic DNA was extracted from 100 μl of blood samples using a Qiagen blood and tissue purification kit (Qiagen), in accordance with the manufacturer's instructions. The purified DNA was dissolved in 30 μl of double-distilled water for subsequent LAMP and PCRs. Two microliters of the resulting supernatant was used as the template for nested PCR and the LAMP assay.

On the other hand, genomic DNA was also isolated by using an FTA card, which is a much cheaper and simpler method. For this approach, 100 μl of blood from the three groups of individuals was blotted and dried on an FTA card (Whatman, United Kingdom). The blotted blood was cut out with a 2-mm hole puncher (2.0-mm Harris micropunch; Whatman). A portion of the blotted blood was then washed three times with 100 μl of FTA purification reagent (Whatman) and eluted in 30 μl of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). Two microliters of the resulting supernatant was used as a template for the LAMP assay.

Sensitivity was calculated as (number of true positives)/(number of true positives + number of false negatives), and specificity was calculated as (number of true negatives)/(number of true negatives + number of false positives).

Nested PCR.Two pairs of oligonucleotide primers directed against the B1 gene of T. gondii (8) were used to perform a nested PCR. The first round of PCR amplification contained 10 mM Tris-HCl, pH 8.3 (at 25°C), 50 mM KCl, 1.5 mM MgCl₂, 0.1 μM each primer (Table 1), 0.2 mM each dNTP, 2.5 U Taq DNA polymerase, and 2 μl of extracted DNA. Reactions were cycled 35 times with denaturation at 94°C for 1 min, followed by annealing at 54°C for 30 s and finally an extension step at 72°C for 1 min. A PCR negative-control sample omitted template DNA, which was replaced by sterile water. Nested PCR mixtures contained 2 μl first-round product, 10 mM Tris-HCl, pH 8.3 (at 25°C), 50 mM KCl, 3 mM MgCl₂, 0.5 μM each primer (Table 1), 0.2 mM each dNTP, and 1 U Taq DNA polymerase. Nested PCR was cycled 35 times using a denaturation step of 94°C for 10 s, followed by annealing at 60°C for 10 s and extension at 72°C for 15 s. Negative-control samples from first-round amplification and an additional second-round negative control of sterile water were included in the nested PCRs. The PCR products were electrophoresed in a 1% TAE (Tris-acetate-EDTA) agarose gel and stained with ethidium bromide solution (1 μg/ml). The PCR amplification is expected to yield a product of 213 bp for the positive reaction.

Detection and confirmation of LAMP product.Three LAMP assays based on three genes, SAG1, SAG2, and B1, were developed (Table 1). All the positive LAMP reactions produced a typical ladder of multiple bands on the agarose gel (Fig. 1), indicating that stem-loop DNA with inverted repeats of the target sequence was produced. Moreover, positive reactions turned green on addition of SYBR green I to the PCR tube, while the negative ones remained orange (Fig. 2). PCR was used to amplify these LAMP products, which showed 100% identity with the target sequences after sequencing (data not shown).

• Open in new tab
• Download powerpoint

FIG. 1.

Electrophoresis of LAMP products amplified from T. gondii tachyzoite genomic DNA. Lanes, from left to right, respectively: no template (negative control of SAG1-LAMP, SAG2-LAMP, and B1-LAMP); SAG1-LAMP; SAG2-LAMP; B1-LAMP, and 100-bp DNA ladder.

• Open in new tab
• Download powerpoint

FIG. 2.

Detection of T. gondii in positive controls and clinical samples by LAMP. Turbidities of the LAMP reactions were inspected visually. Positive reactions turned green under UV light after the addition of SYBR green I. Tubes, from left to right, respectively: negative control, SAG1-LAMP, SAG2-LAMP, and B1-LAMP.

Optimization of LAMP assay.The best results were obtained when the reaction temperature was maintained at 65°C. The results of the LAMP assay were identical with either a water bath, a heater block, or a PCR machine, demonstrating its robustness.

Analytical sensitivities and specificities of LAMP assays versus results of nested PCR.The analytical sensitivities of all the LAMP assays were evaluated against a 10-fold serial dilution of T. gondii tachyzoite DNA. The detection limit of LAMP assays targeting SAG1, SAG2, and B1 is 0.1 tacyhzoite, whereas the detection limit of nested PCR targeted at the B1 gene is 1 tachyzoite (data not shown).

The LAMP assays were tested for their specificities using heterogeneous DNA samples as controls. No amplification was observed with DNA samples from C. parvum, N. caninum, and T. canis (data not shown).

Clinical sensitivities and specificities of LAMP assays versus results of nested PCR.The two DNA extraction methods are equally efficient in extracting DNA from T. gondii tachyzoites. Analysis of the samples by the LAMP assays and nested PCR revealed no difference in sensitivity between the two DNA extraction methods. The SAG2-LAMP produced positive results for 35 of the 40 blood samples from patients with active toxoplasmosis, while SAG1-LAMP and B1-LAMP produced positive results only for 32 of the cases. DNA fragments of the correct size were amplified by nested PCR from 25 of the 40 blood samples (Table 2). No amplification of DNA fragments by the LAMP assays and nested PCR was seen for all the negative controls, which included 40 blood samples from healthy volunteers and 25 blood samples from patients with other parasitic infections.