Isothermal PCR for Feasible Molecular Diagnosis of Primary Toxoplasmosis in Women Recently Experienced Spontaneous Abortion
DOI:
https://doi.org/10.3889/oamjms.2018.227Keywords:
Isothermal, LAMP, Toxoplasma, qPCR, AbortionAbstract
AIM: The current study aimed to assess the practicability of a simple loop-mediated isothermal amplification (LAMP) about real-time quantitative PCR to diagnose primary toxoplasmosis among high-risk pregnant women.
METHODS: Cloned Toxoplasma samples were used to calculate the analytical sensitivity while specificity was assessed using pooled DNA samples extracted from other parasitic stages.
RESULTS: Both techniques showed 100% sensitivity and specificity and then applied to detect recent Toxoplasma infection in peripheral blood of 77 IgG negative women out of a total 139 women lately experienced spontaneous abortion. The 2 techniques obtained positive results in 8 samples confirming primary toxoplasmosis.
CONCLUSION: Generally, LAMP assay is a simple, cost-effective molecular technique can be completed in less than half an hour to diagnose primary Toxoplasma infection. The technique can be applied in a minimally equipped laboratory by ordinary workers to screen the vulnerable groups. Further analysis using larger samples with the quantitative approach is recommended to confirm the sensitivity of this emergent molecular technique.Downloads
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Lopes FM, Gonçalves DD, Mitsuka-Breganó R, Freire RL and Navarr IT. Toxoplasma gondii infection in pregnancy. Braz J Infect Dis. 2007; 11:496-506. https://doi.org/10.1590/S1413-86702007000500011 PMid:17962877
Robbins JR, Zeldovich VB, Poukchanski A, Boothroyd JC, Bakardjiev AI. Tissue Barriers of the Human Placenta to Infection with Toxoplasma gondii. Infection and Immunity. 2012; 80(1): 418–428. https://doi.org/10.1128/IAI.05899-11 PMid:22083708 PMCid:PMC3255695
Vado-SolÃs I, Suárez-SolÃs V, Jiménez-Delgadillo B, Zavala-Velázquez J, Segura- Correa J. Toxoplasma gondii Presence in Women with Spontanous Abortion in Yucatan, Mexico. 2013; 99(2):383-385.
Sultana M, Sazzad Hossain Md, Dewan F, Sultan J, Rashid M. Association of Toxoplasma gondii Infection with Spontaneous Abortion. Bangladesh. J Obstet Gynaecol. 2014; 29(2): 87-93.
Matin S, Shahbazi GH. Study on abortion associated with Toxoplasma gondii in women based on PCR detection of aborted placenta and maternal serology in Ardabil, Iran. J Med Microb Diagn. 2017; 6:2.
Saki J, Mohammadpour N, Khademvatan S. Seroprevalence of Toxoplasma gondii in women who have aborted in comparison with the women with normal delivery in Ahvaz, southwest of Iran. The Scientific World Journal. 2015; 2015: 764369. https://doi.org/10.1155/2015/764369 PMid:25699288 PMCid:PMC4325198
Robert-Gangneux, Belaz. Molecular diagnosis of toxoplasmosis. Curr Opin Infect Dis. 2016; 29:330–339. https://doi.org/10.1097/QCO.0000000000000275 PMid:27191201
Romand S, Chosson M, Franck J, Wallon M, Kieffer F, Kaiser K, Dumon H, Peyron F, Thulliez P, Picot S. Usefulness of quantitative polymerase chain reaction in amniotic fluid as early prognostic marker of fetal infection with Toxoplasma gondii. Am J Obstet Gynecol. 2004; 190:797–802. https://doi.org/10.1016/j.ajog.2003.09.039 PMid:15042017
Liu CY, Song HQ, Zhang RL, Chen MX, Xu MJ, Ai L, Chen XG, Zhan XM, Liang SH, Yuan ZG, Lin RQ, Zhu XQ. Specific detection of Angiostrongylus cantonensis in the snail Achatina fulica using a loop-mediated isothermal amplification (LAMP) assay. Mol Cell Probes. 2011; 25:164–167. https://doi.org/10.1016/j.mcp.2011.04.002 PMid:21515360
Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 2000; 28:e63. https://doi.org/10.1093/nar/28.12.e63 PMid:10871386 PMCid:PMC102748
Lau YL, Meganathan P, Sonaimuthu P, Thiruvengadam G, Nissapatorn V, Chen Y. Specific, sensitive, and rapid diagnosis of active toxoplasmosis by a loop-mediated isothermal amplification method using blood samples from patients. J Clin Microbiol. 2010; 48(10):3698-702. https://doi.org/10.1128/JCM.00462-10 PMid:20660217 PMCid:PMC2953084
Contini C, Seraceni S, Cultrera R, Incorvaia C, Sebastiani A, Picot S. Evaluation of a real-time PCR-based assay using the Lightcycler system for detection of Toxoplasma gondii bradyzoite genes in blood specimens from patients with toxoplasmic retinochoroiditis. Int J Parasitol. 2005; 35:275–283. https://doi.org/10.1016/j.ijpara.2004.11.016 PMid:15722079
Reis MM, Tessaro M, DAzevedo PA. Toxoplasma IgM and IgG avidity in single samples from areas with a high infection rate can determine the risk of mother to child transmission. Rev Inst med trop Sao Paulo. 2006; 48: 93 -98. https://doi.org/10.1590/S0036-46652006000200007 PMid:16699631
Ajzenberg D, Cogne N, Paris L, Bessières MH, Thulliez P, Filisetti D, Pelloux H, Marty P, Dardé ML. Genotype of 86 Toxoplasma gondii isolates associated with human congenital toxoplasmosis, and correlation with clinical findings. J Infect Dis. 2002; 186:684-689. https://doi.org/10.1086/342663 PMid:12195356
Marcolino PT, Silva DAO, Leser PG, Camargo ME, Mineo JR. Molecular Markers in Acute and Chronic Phases of Human Toxoplasmosis: Determination of Immunoglobulin G Avidity by Western Blotting. Clinical and Diagnostic Laboratory Immunology. 2000; 7(3):384– 389. https://doi.org/10.1128/CDLI.7.3.384-389.2000
Rahbari AH, Keshavarz H, Shojaee S, Mohebali M, Rezaeian M. IgG Avidity ELISA Test for Diagnosis of Acute Toxoplasmosis in Humans. Korean J Parasitol. 2012; 50(2):99–102. https://doi.org/10.3347/kjp.2012.50.2.99 PMid:22711919 PMCid:PMC3375462
Jenum PA and Stray-Pedersen B. Development of Specific Immunoglobulins G, M, and A Following Primary Toxoplasma gondii Infection in Pregnant Women. J Clin Microbiol. 1998; 36(10), 2907–2913. PMid:9738042 PMCid:PMC105086
Nagamine K, Hase T, Notomi T. Accelerated reaction by loop mediated isothermal amplification using loop primers. Mol Cell Probes. 2002; 16:223-229. https://doi.org/10.1006/mcpr.2002.0415 PMid:12144774
Alhassan A, Govind Y, Tam NT, Thekisoe OM, Yokoyama N, Inoue N, Igarashi I. Comparative evaluation of the sensitivity of LAMP, PCR and in vitro culture methods for the diagnosis of equine piroplasmosis. Parasitol Res. 2007; 100:1165-1168. https://doi.org/10.1007/s00436-006-0430-6 PMid:17216488
Thekisoe OM, Inoue N, Kuboki N, Tuntasuvan D, Bunnoy W, Borisutsuwan S, Igarashi I, Sugimoto C. Evaluation of loop-mediated isothermal amplification (LAMP), PCR and parasitological tests for detection of Trypanosoma evansi in experimentally infected pigs. Vet Parasitol. 2005; 30:327-330. https://doi.org/10.1016/j.vetpar.2005.04.019 PMid:15908123
Iseki H, Alhassan A, Ohta N, Thekisoe O]M, Yokoyama N, Inoue N, Nambota A, Yasuda J, Igarashi I. Development of a multiplex loop-mediated isothermal amplification (mLAMP) method for the simultaneous detection of bovine Babesia parasites. J Microbiol Methods. 2007; 71:281-287. https://doi.org/10.1016/j.mimet.2007.09.019 PMid:18029039
Bou G, Figueroa MS, Marti-Belda P, Navas E, Guerrero A. Value of PCR for detection of Toxoplasma gondii in aqueous humor and blood samples from immunocompetent patients with ocular toxoplasmosis. J Clin Microbiol. 1999; 37:3465-3468. PMid:10523535 PMCid:PMC85668
Teixeira LE, Kanunfre KA, Shimokawa PT, Targa LS, Rodrigues JC, Domingues W, Yamamoto L, Okay TS. The performance of four molecular methods for the laboratory diagnosis of congenital toxoplasmosis in amniotic fluid samples. Rev Soc Bras Med Trop. 2013; 46(5):584-8. https://doi.org/10.1590/0037-8682-0095-2013
Menotti J, Vilela G, Romand S, Garin YJ, Ades L, Gluckman E, Derouin F, Ribaud P. Comparison of PCR-enzyme-linked immunosorbent assay and real-time PCR assay for diagnosis of an unusual case of cerebral toxoplasmosis in a stem cell transplant recipient. J Clin Microbiol. 2003; 41(11):5313-6. https://doi.org/10.1128/JCM.41.11.5313-5316.2003 PMid:14605193 PMCid:PMC262488
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