Development and Evaluation of a Sensitive and Specific Assay for Diagnosis of Human Toxocariasis by Use of Three Recombinant Antigens (TES-26, TES-30USM, and TES-120)

Abstract

Diagnosis of human toxocariasis currently relies on serologic tests that use Toxocara excretory-secretory (TES) antigen to detect immunoglobulin G (IgG) antibodies to the larvae. In general, however, these assays do not have adequate specificity for use in countries in which other soil-transmitted helminths are endemic. The use of recombinant antigens in these assays, however, is promising for improving the specificity of the diagnosis of toxocariasis. Toward this goal, we developed an IgG4 enzyme-linked immunosorbent assay (ELISA) involving three recombinant antigens: rTES-30USM (previously produced), rTES-26, and rTES-120. The latter two antigens were produced by reverse transcription-PCR cloning; subcloned into glutathione S-transferase (GST)-tagged and His-tagged prokaryotic expression vectors, respectively; and expressed in Escherichia coli. The recombinant proteins were subsequently purified by affinity chromatography using GST and His-Trap resins. The diagnostic potential of each purified recombinant antigen was tested with various immunoglobulin classes (IgG, IgM, and IgE) and IgG subclasses. The IgG4 ELISA was determined to have the highest specificity and was further evaluated using a panel of serum samples. The rTES-26 IgG4 ELISA showed 80.0% (24/30 samples positive) sensitivity, and both the rTES-30USM IgG4 ELISA and rTES-120 IgG4 ELISA had 93.0% (28/30) sensitivity. Combined use of rTES-120 and rTES-30 IgG4 ELISA for the diagnosis of toxocariasis provided 100% sensitivity. The specificities of rTES-26, rTES-30USM, and rTES-120 antigens were 96.2%, 93.9%, and 92.0%, respectively. These results indicate that the development of a diagnostic test using the three recombinant antigens will allow for more-accurate detection of toxocariasis.Human toxocariasis is a worldwide parasitic zoonosis, caused most commonly by the intestinal parasites the dog roundworm (Toxocara canis) and also the cat roundworm (Toxocara cati) (2). It commonly manifests as visceral larva migrans, ocular larva migrans, and covert toxocariasis. Toxocariasis is probably one of the most common zoonotic helminthiases in temperate climates and developed countries (20). In Malaysia, the seroprevalence rate is approximately 20% (6).Definitive diagnosis of toxocariasis is based on the detection of Toxocara larvae from biopsy tissues, but this test is timeconsuming and difficult to perform. Therefore, diagnosis is commonly based on clinical and serologic diagnosis. Currently, common routine serology tests are applied for detection, such as commercial immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) kits in which Toxocara excretorysecretory (TES) antigens obtained from culture of T. canis second-stage (L2) larvae are used (22). The use of the native TES antigen for serodiagnosis of human toxocariasis is a laborious and time-consuming technique, and the production capacity is limited by the culture volume (22). Further, the specificity is often low, especially in developing countries, where infections with helminths that cause c...

Producción y evaluación del antígeno recombinante Tes-30 de Toxocara canis para el inmunodiagnóstico de toxocariasis

“…Diversos autores han determinado, clonado y secuenciado los genes que codifican diferentes antígenos de las larvas de T. canis (29)(30)(31)(34)(35)(36) A pesar de la reacción encontrada en los sueros de pacientes con estrongiloidiasis y ascariasis, no se puede afirmar categóricamente que sea una reacción cruzada, debido a que estos sueros eran de pacientes provenientes de zonas endémicas para la toxocariasis, por lo que no se puede descartar de forma categórica una reacción contra este parásito.…”

“…Es así como los antígenos TES-26, TES-30 y TES-120 se han convertido en la alternativa recombinante para este antígeno. El TES-30 se ha evaluado exitosamente en varios países (29)(30)(31), incluido Brasil. Sin embargo, en Colombia aún no se ha evaluado ninguno de estos antígenos en su función de proteínas recombinantes alternativas a los antígenos de excreción-secreción.…”

Production of Toxocara cati TES-120 Recombinant Antigen and Comparison with its T. canis Homolog for Serodiagnosis of Toxocariasis

“…Conventional reverse transcriptase polymerase chain reaction (RT-PCR) was performed using Easy-A one tube RT-PCR System (Agilent Technologies, Santa Clara, CA), according to the manufacturer's instructions. The TES-120 forward and reverse primers were the same as that used previously 14 and were designed on the basis of the coding sequence of the T. canis TES-120 gene (GenBank: U39815), namely: TES-120 F, 5′-ATGCACGTCCTTACCGTCGCT-3′ and TES-120 R, 5′-ACAGAAGCCGCACGTCAGTGG-3′. The RT reaction mix was prepared in a 1.5-mL tube by adding 6.2 μL of RNase-free water, 0.8 μL of 10 × RT-PCR buffer, and 1 μL RT enzyme.…”

“…The recombinant protein was previously produced using T. canis TES-120 sequence 14 in pGEX-5X-1 expression vector (GE Healthcare).…”

“…This was incubated at 28-30°C for 30 days to allow for embryonation of the larvae. 12 Larvae were hatched and processed according to methods by Alcantara-Neves and others 13 and Mohamad and others 14 In brief, the formaldehyde was removed by washing five times with sterile PBS. An equal volume of 7-14% sodium hydrochloride (Sigma-Aldrich, St. Louis, MO) was added, and the solution was placed on a shaker at room temperature for 15 minutes until the eggs lost their external layer.…”