Detection of chronic toxoplasmosis in the brain of mice using loop-mediated isothermal amplification (LAMP) and conventional PCR.

BACKGROUND: Toxoplasma gondii is a common protozoan parasite that infects approximately one-third of the world's population. It is a disease with multiple manifestations. In immunocompetent individuals, symptoms are mild and flu-like, whereas, in immunocompromised patients, it often results in severe morbidity and mortality. Thus, studies for developing a simple, rapid diagnostic tool for early detection of Toxoplasma are emerging. Molecular diagnosis is highly accurate and helpful in congenitally infected and immunocompromised patients. The loop-mediated isothermal amplification (LAMP) technique was invented to improve nucleic acid amplification efficacy in terms of sensitivity and specificity. AIM OF THE STUDY: The study aimed to validate a LAMP protocol for detecting Toxoplasma DNA in the brain homogenates from mice experimentally infected with Toxoplasma's ME-49 (cyst-forming type II) strain in comparison to PCR. METHODS: In this study, the target DNA fragment was the Toxoplasma 529-bp, repeated 200-300 copies/genome. The sensitivity of both LAMP and conventional PCR techniques was estimated in brain homogenates in experimental mice at eight weeks post-infection and compared to the histopathology data. RESULTS: The LAMP reaction showed positive results in 18 of the 26 examined samples of brain homogenates. PCR showed the characteristic 529-bp band in 15 of the 26 examined samples. CONCLUSION: The LAMP showed a higher sensitivity over PCR in detecting Toxoplasma infection in brain homogenates of infected mice.

Validation of dried blood spots in monitoring toxoplasmosis.

Due to the growing importance of toxoplasmosis worldwide, simple methods of diagnosis are important for epidemiologic studies. Dried blood spot (DBS) is a useful tool that surpasses venipuncture. DBS-Toxoplasma testing via a finger-stick could also be used in setting where phlebotomies might not be feasible, such as worldwide prenatal and newborn screening for congenital toxoplasmosis. This study included 101 study subjects were occupationally at-risk to Toxoplasma gondii infection and 33 as controls. Serum was collected from both groups for the detection of anti-Toxoplasma IgG antibodies by ELISA as a reference gold standard test. For the occupational at-risk group, capillary finger stick derived blood was blotted onto five sets of Whatman protein saver cards. Discs were stored as four sets; three sets at 4°C and eluted 1, 2 and 3 months of storage and one set at -20°C for 3 months then eluted. Additionally, one set was eluted immediately. Anti-Toxoplasma IgG antibodies were evaluated by ELISA from DBS eluted samples and compared to matched sera. DBS elutes from discs that were freshly prepared for anti-Toxoplasma IgG showed 100% sensitivity, specificity and diagnostic accuracy. Serologic testing using DBS showed very good diagnostic accuracy under all mentioned conditions of storage. Higher stability was obtained when the blood discs stored at 4°C for 1 month and up to 3 months at -20°C, with 98.18% sensitivity, 100% specificity and 99% diagnostic accuracy. DBS-Toxoplasma testing is characterized by simplicity in performance, cost-effectiveness and the ease of handling, to store and to transport, with high diagnostic accuracy.

Loop mediated isothermal amplification (LAMP) of Toxoplasma DNA from dried blood spots.

INTRODUCTION: The cosmopolitan protozoan Toxoplasma gondii is a major parasite of warm-blooded animals including man. Early and accurate diagnosis is a must for proper treatment that prevents life threatening sequels. Loop-mediated isothermal amplification (LAMP) is a novel technique that can amplify DNA with high sensitivity and specificity under isothermal conditions. AIM OF THE STUDY: To validate a LAMP-specific protocol for detection of Toxoplasma DNA using dried blood spots (DBS) from mice experimentally infected with the cystogenic Toxoplasma ME-49 strain. METHODS: In this study, the target DNA fragment was the Toxoplasma 529-bp repeat element that exists in 200-300 copies per T. gondii genome. The sensitivity of both LAMP and conventional PCR techniques was estimated in DBS samples from experimental mice at 1-week and 8-weeks post-infection. RESULTS: Out of 20 blood samples gathered on Whatman filter paper from mice at 1-week post-infection, 18 and 16 were positive by LAMP and conventional PCR, respectively. Neither techniques detected parasite DNA in blood at 8th week of infection. CONCLUSION: Dried blood spots are easy source of material for molecular studies. LAMP assay proved higher sensitivity than the conventional PCR in detecting parasitemia in early infection with the cystogenic Toxoplasma strain.