In-house real-time PCR to detection Toxoplasma gondii in pregnant women candidates for amniocentesis and comparison with nested PCR and serologic methods in several clinical samples

Abstract Background: Toxoplasmosis is a common parasitic infection caused by Toxoplasma gondii (T. gondii), an obligate and intracellular parasite, that affects both humans and animals globally. It can cause severe damage to neonates during pregnancy and immunocompromised patients. Therefore, finding a new method with high sensitivity for the diagnosis of toxoplasmosis that involves low-risk sampling is crucial for patient management. This study aims to diagnose toxoplasmosis in pregnant women from plasma, amniotic fluid, buffy coat, and urine specimens using serological, nested PCR, and real-time PCR tests. Methods: We collected amniotic fluid, blood, and urine samples from 100 pregnant women referred to a gynecologist in Mazandaran province, northern Iran, who were candidates for amniocentesis. We performed anti- T. gondii IgM, IgG, and IgG avidity tests using enzyme-linked immunosorbent assay (chemiluminescence, ELISA), as well as nested PCR, real-time PCR, and multilocus PCR genotyping using RE and GRA6 target genes, respectively. Results: Based on the serologic test, 52% of subjects were seropositive (51 samples were positive for IgG, and 1 was positive for both IgG and IgM antibodies), and all cases showed high avidity. Among 100 amniotic fluid samples, 2 and 3 cases tested positive using nested and real-time PCR, respectively, while all samples from buffy coat and urine were negative. All positive DNA samples were genotyped as type I. Conclusions Our study results suggest that diagnosing congenital toxoplasmosis is achievable using a combination of serological and molecular tests. We also found that the real-time PCR method is more sensitive than the nested PCR.