Comparison of Identification of Toxoplasma gondii by Commercial Realtime PCR and Inhouse Realtime PCR Methods

Year 2019, , 79 - 84, 28.08.2019

Selma Usluca , Bekir Çelebi

https://doi.org/10.19127/mbsjohs.558436

Abstract

Objective: This study aimed to compare the diagnosis of toxoplasmosis with a
commercial kit and inhouse realtime PCR methods to determine molecular methods
with high diagnostic accuracy for use in addition to serologic tests for
routine diagnosis.

Methods: The study included a total of 116 samples of blood,
CSF or amniotic fluid with 19 identified positive and 97 negatives for T.
gondii sent to our laboratory. Due to the low number of positive samples,
DNA samples from an external quality control program that our laboratory
participates in were included in the study. First to all samples, realtime PCR
method were applied with commercial kit used primers for T. gondii
rep529 gene, and then inhouse realtime PCR were applied with TG-F and TG-R
primers and Taqman probe, targeting the insertion sequence region of T.
gondii B1 gene.

Results: The results for the total of 116 samples studied with both methods was that
17 were identified as positive with commercial realtime PCR and 19 were
determined as positive with inhouse realtime PCR. Accordingly, two cases with
the commercial realtime PCR method were determined as false negative. The limit
of detection for both methods used in our study was determined as 10-3 dilution
(0.028 copy/reaction). There was a high level of compatibility determined
between the inhouse and realtime PCR methods (kappa value: 0.934).

Conclusion: In conclusion, though there was perfect compatibility observed between the
results with the two methods, disadvantages of the commercial realtime PCR
method included isolates where the target gene was not found, deletion or
mutation of all or part of this gene or different numbers of repeats causing
false negative results and high cost. Considering this, our laboratory decided
to use the inhouse realtime PCR using primers for the B1 gene to research T.
gondii with molecular methods. A significant limitation of the study is the
low number of positive samples. For DNA samples belonging to the External
Quality Control Program, the commercial kit was 66.66% successful, while the
inhouse realtime PCR method was 100% successful.

Keywords

Inhouse realtime PCR, realtime PCR, T. gondii

References

• Belfort R, Isenberg J, Fernandes BF, DiCesare S, Belfort Jr R, Burnier Jr MN, Evaluating Different Methods of Toxoplasma Gondii Detection in Peripheral Blood, Invest Ophth Vis Sci, 2008;49(13):1-9.
• Calderaro A, Piccolo G, Gorrini C, Peruzzi S, Zerbini L, Bommezzadri S, Dettori G, Chezzi C, Comparison Between two Real-time PCR Assays and a Nested-PCR for the Detection of Toxoplasma gondii, Acta Biomed, 2006;77:75-80.
• Cardona N, Basto N, Parra B, Zea AF, Pardo CA, Bonelo A, G´omez-Marin JE, Detection of Toxoplasma DNA in the Peripheral Blood of HIV-Positive Patients with Neuro-opportunistic Infections by a Real-Time PCR Assay, J Neuroparasitology, 2011;2:1-6.
• Fallahi S, Mazar ZA, Ghasemian M, Haghighi A, Challenging Loop-Mediated Isothermal Amplification (LAMP) Technique for Molecular Detection of Toxoplasma gondii, Asian Pac J Trop Med, 2015;366-372.
• Hierl T, Reischl U, Lang P, Hebart H, Stark M, Kyme P, Autenrieth IB, Preliminary Evaluation of one Conventional Nested and two Real-time PCR Assays for the Detection of Toxoplasma gondii in Immunocompromised Patients, J Med Microbiol, 2004;53:629–632.
• Hill D, Dubey JP, Toxoplasma gondii: Tansmission, Diagnosis and Prevention, Clin Microbiol Infect, 2002;8:634–640.
• Ivović V, Vujanić M, Živković T, Klun I, Djurković-Djaković O, Molecular Detection and Genotyping of Toxoplasma gondii from Clinical Samples in: Toxoplasmosis – Recent Advances, Ed: Djurković-Djaković O, InTech Open Access Publisher, 2012;103-119.
• Kalantari N, Darabi ZA, Siadati S, Nikbakhsh N, Ghasemi M, Ghaffari T, Ghaffari S, Bayani M, Detection of Toxoplasma gondii DNA in Malignant Breast Tissues in Breast Cancer Patients, Int J Mol Cell Med, 2017;6(3):190-196.
• Lin MH, Chen TC, Kuo TT, Tseng CC, Tseng CP, Real-Time PCR for Quantitative Detection of Toxoplasma gondii, J Clin Microbiol, 2000;38(11): 4121–4125.
• Liu Q, Wang ZD, Huang SY, Zhu XQ, Diagnosis of Toxoplasmosis and Typing of Toxoplasma gondii, Parasites and Vectors, 2015;8:292-305.
• Mousavi M, Saravani R, Modrek MJ, Shahrakipour M, Sekandarpour S, Detection of Toxoplasma gondii in Diabetic Patients Using the Nested PCR Assay via RE and B1 Genes, Jundishapur J Microbiol, 2016;9(2):1-6.
• Rajendran C, Keerthana CM, Anilakumar KR, Satbige AS, Gopal S, Development of B1 Nested PCR for Assessing the Prevalence of Zoonotic Protozoan Disease Agent Toxoplasma Gondii among Food Animals from Karnataka State, Southern India, J Microbiol Lab Sci, 2018;1(1): 1-8.
• Robert-Gangneux F, Belaza S, Molecular Diagnosis of Toxoplasmosis in Immunocompromised Patients, Wolters Kluwer Health, 2016;29(4): 330-339.
• Robert-Gangneux F, Brenier-Pinchart MP, Yera H, Belaz S, Varlet-Marie E, Bastien P, Molecular Biology Study Group of the French National Reference Center for Toxoplasmosis, Evaluation of Toxoplasma ELITe MGB, Real-Time PCR Assay for Diagnosis of Toxoplasmosis, J Clin Microbiol, 2017;55(5):1369-1376.
• Rostami A, Karanis P, Fallahi S, Advances in Serological, Imaging Techniques and Molecular Diagnosis of Toxoplasma gondii Infection, Infection, 2018;1-13.
• Sails AD, Applications in Clinical Microbiology in: Real-time PCR: An Essential Guide, Eds: Edwards, Logan, Saunders, Horizon Scientific Press, Wymondham, UK, 2004:247-326.
• Sterkers Y, Varlet-Marie E, Cassaing S, Brenier-Pinchart MP, Brun S, Dalle F, Delhaes L, Filisetti D, Pelloux H, Yera H, Bastien P, Multicentric Comparative Analytical Performance Study for Molecular Detection of Low Amounts of Toxoplasma gondii from Simulated Specimens, J Clin Microbiol, 2010;48(9):3216–3222.
• Su C, Shwab EK, Zhou P, Zhu XQ, Dubey JP, Moving towards an Integrated Approach to Molecular Detection and Identification of Toxoplasma gondii, Parasitology, 2010;137, 1–11.
• Switaj K, Master A, Skrzypczak M, Zaborowski P, Recent Trends in Molecular Diagnostics for Toxoplasma gondii Infections, Clin Microbiol Infect, 2005;11: 170–176. https://assets.thermofisher.com/TFS-Assets/LSG/manuals/cms_042380.pdf
• 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):584-588.
• Wahab T, Edvinsson B, Palm D, Lindh J, Comparison of the AF146527 and B1 Repeated Elements, Two Real-Time PCR Targets Used for Detection of Toxoplasma gondii, J Clin Microbiol, 2010;48(2):591–592.
• Wastling JM, Nicoll S, Buxton D, Comparison of two Gene Amplification Methods for the Detection of Toxoplasma gondii in Experimentally Infected Sheep, Med Microbiol, 1993; 38:360-365.