Development of High Resolution Melting Analysis as a Diagnostic Tool for Molecular Detection of Toxoplasma Infection in Pregnant Women and HIV Positive Cases
Background: Toxoplasma gondii is an obligate intracellular protozoan with worldwide distribution. Diagnosis of toxoplasmosis is a very critical issue, especially in pregnant women and immunocompromised patients. The aim of this study was rapid detection of T. gondii DNA in peripheral blood samples (PBS) employing HRM technique and using RE gene.
Methods: Totally, 242 samples from pregnant women and human immunodeficiency virus (HIV) patients were collected from different hospitals and medical centers of Tehran during Oct 2017 to Dec 2018. High resolution melting analysis (HRM) using partial sequences of repetitive element (RE) gene was done and compared with ELISA test.
Results: Overall, 51 were positive for acute toxoplasmosis that among them, 12 and 20 reported as positive in pregnant women and HIV+ patients, respectively using HRM technique. Among 70 patients in chronic phase of disease, 10 and 3 samples were reported as positive for pregnant women and HIV+ patients respectively. From 121 negative control, 3 (4.62%) samples associated with HIV+ patients, showed positive real-time PCR and HRM analysis results.
Conclusion: For the first time, HRM technique via employing RE gene was used for detection of T. gondii infection in PBS. This method is suitable, helpful and in parallel with serological methods for early diagnosis of acute as well as active form of toxoplasmosis in pregnant women and HIV+ patients. The use of techniques based on melt curve and through employing next-generation dyes for diagnosis of T. gondii would be accessible for patients in developing countries.
2. Liu Q, Singla LD, Zhou H (2012). Vaccines against Toxoplasma gondii: status, challenges and future directions. Hum Vaccin Immunother, 8(9): 1305-1308.
3. Dubey JP (2016). Toxoplasmosis of animals and humans. CRC Press.
4. Tavalla M, Oormazdi H, Akhlaghi L, et al (2013). Genotyping of Toxoplasma gondii isolates from soil samples in Tehran, Iran. Iran J Parasitol, 8(2): 227-233.
5. Liu Q, Wang Z-D, Huang S-Y, et al (2015). Diagnosis of toxoplasmosis and typing of Toxoplasma gondii. Parasit Vectors, 8:292.
6. Moncada PA, Montoya JG (2012). Toxoplasmosis in the fetus and newborn: an update on prevalence, diagnosis and treatment. Expert Rev Anti Infect Ther, 10(7): 815-28.
7. Hosseini S, Amouei A, Sharif M, et al (2019). Human toxoplasmosis: a systematic review for genetic diversity of Toxoplasma gondii in clinical samples. Epidemiol Infect, 147: 1-9
8. Cuomo G, D’abrosca V, Rizzo V, et al (2013). Severe polymyositis due to Toxoplasma gondii in an adult immunocompetent patient: a case report and review of the literature. Infection, 41(4): 859-62.
9. Neves EdS, Kropf A, Bueno WF, et al (2011). Disseminated toxoplasmosis: an atypical presentation in an immunocompetent patient. Trop Doct, 41(1): 59-60.
10. Carme B, Bissuel F, Ajzenberg D, et al (2002). Severe acquired toxoplasmosis in immunocompetent adult patients in French Guiana. J Clin Microbiol, 40(11): 4037-44.
11. Candolfi E, De Blay F, Rey D, et al (1993). A parasitologically proven case of Toxoplasma pneumonia in an immunocompetent pregnant woman. J Infect, 26(1): 79-80.
12. Abhilash K, Roshine M, Vandana K, et al (2013). A probable case of acquired toxoplasmosis presenting as pyrexia of unknown origin in an immunocompetent individual. Int J Infect Dis, 17(11): e1067-8.
13. Mahmoudvand H, Ziaali N, Aghaei I, et al (2015). The possible association between Toxoplasma gondii infection and risk of anxiety and cognitive disorders in BALB/c mice. Pathog Glob Health, 109(8): 369-376.
14. Mahmoudvand H, Sheibani V, Shojaee S, et al (2016). Toxoplasma gondii infection potentiates cognitive impairments of Alzheimer's disease in the BALB/c mice. J Parasitol, 102(6):629-635.
15. Alipour A, Shojaee S, Mohebali M, et al (2011). Toxoplasma infection in schizophrenia patients: A comparative study with control group. Iran J Parasitol. 6 (2): 31-37.
16. Ramírez MdlLG, Orozco LVS, Ramírez CGT (2017). The Laboratory Diagnosis in Toxoplasma Infection. Toxoplasmosis, 6: 89-104.
17. Heydari SA, Keshavarz H, Shojaee S, et al (2013). Diagnosis of antigenic markers of acute toxoplasmosis by IgG avidity immunoblotting. Parasite, 20: 18.
18. Bastien P (2002). Molecular diagnosis of toxoplasmosis. Trans R Soc Trop Med Hyg, 1:S205-15.
19. Kotresha D, Noordin R (2010). Recombinant proteins in the diagnosis of toxoplasmosis. APMIS, 118(8): 529-42.
20. Kompalic-Cristo A, Frotta C, Suárez-Mutis M, et al (2007). Evaluation of a real-time PCR assay based on the repetitive B1 gene for the detection of Toxoplasma gondii in human peripheral blood. Parasitol Res, 101(3): 619-25.
21. Martino R, Bretagne S, Einsele H, et al (2005). Early detection of Toxoplasma infection by molecular monitoring of Toxoplasma gondii in peripheral blood samples after allogeneic stem cell transplantation. Clin Infect Dis, 40(1): 67-78.
22. Selseleh M, Modarressi MH, Mohebali M, et al (2012). Real-Time RT-PCR on SAG1 and BAG1 gene expression during stage conversion in immunosuppressed mice infected with Toxoplasma gondii Tehran Strain. Korean J Parasitol, 50(3): 199-205.
23. Kasper DC, Sadeghi K, Prusa A-R, et al (2009). Quantitative real-time polymerase chain reaction for the accurate detection of Toxoplasma gondii in amniotic fluid. Diagn Microbiol Infect Dis, 63(1): 10-5.
24. Santos GB, Espínola SM, Ferreira HB, et al (2013). Rapid detection of Echinococcus species by a high-resolution melting (HRM) approach. Parasit & Vectors, 6(1): 327.
25. Rostami A, Keshavarz H, Shojaee S, et al (2014). Frequency of Toxoplasma gondii in HIV positive patients from West of Iran by ELISA and PCR. Iran J Parasitol, 9(4): 474-481.
26. Hosseini-Safa A, Mohebali M, Hajjaran H, et al (2018). High resolution melting analysis as an accurate method for identifying Leishmania infantum in canine serum samples. J Vector Borne Dis, 55(4): 315-320.
27. Mousavi P, Mirhendi H, Mohebali M, et al (2018). Detection of Toxoplasma gondii in Acute and Chronic Phases of Infection in Immunocompromised Patients and Pregnant Women with Real-time PCR Assay Using TaqMan Fluorescent Probe. Iran J Parasitol, 13(3): 373-381.
28. Espy M, Uhl J, Sloan L, et al (2006). Real-time PCR in clinical microbiology: applications for routine laboratory testing. Clin Microbiol Rev, 19(1): 165-256.
29. Safa AH, Harandi MF, Tajaddini M, et al (2015). Rapid identification of Echinococcus granulosus and E. canadensis using high-resolution melting (HRM) analysis by focusing on single nucleotide polymorphism. Jpn J Infect Dis, 69(4): 300-5.
30. Gašparič MB, Tengs T, La Paz JL, et al (2010). Comparison of nine different real-time PCR chemistries for qualitative and quantitative applications in GMO detection. Anal Bioanal Chem, (6): 2023-9.
31. Mao F, Leung W-Y, Xin X (2007). Characterization of EvaGreen and the implication of its physicochemical properties for qPCR applications. BMC Biotechnol, 7(1): 76.
32. Monis PT, Giglio S, Saint CP (2005). Comparison of SYTO9 and SYBR Green I for real-time polymerase chain reaction and investigation of the effect of dye concentration on amplification and DNA melting curve analysis. Anal Biochem, 340(1): 24-34.
33. Costa J-M, Cabaret O, Moukoury S, et al (2011). Genotyping of the protozoan pathogen Toxoplasma gondii using high-resolution melting analysis of the repeated B1 gene. J Microbiol Methods, 86(3): 357-63.
34. Costa J-M, Alanio A, Moukoury S, et al (2013). Direct genotyping of Toxoplasma gondii from amniotic fluids based on B1 gene polymorphism using minisequencing analysis. BMC Infect Dis, 13(1): 552.
35. Aksoy U, Marangi M, Papini R, et al (2014). Detection of Toxoplasma gondii and Cyclospora cayetanensis in Mytilus galloprovincialis from Izmir Province coast (Turkey) by real time PCR/High-Resolution Melting analysis (HRM). Food Microbiol, 44: 128-35.
36. Marangi M, Giangaspero A, Lacasella V, et al (2015). Multiplex PCR for the detection and quantification of zoonotic taxa of Giardia, Cryptosporidium and Toxoplasma in wastewater and mussels. Mol Cell Probes, 29(2): 122-5.
37. Bispo PJ, Davoudi S, Sahm ML, et al (2018). Rapid Detection and Identification of Uveitis Pathogens by Qualitative Multiplex Real-Time PCR. Invest Ophthalmol Vis Sci, 59(1): 582-589.
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