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2020, Cilt 50, Sayı 1, Sayfa(lar) 056-062
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Identification of Leishmania spp., Plasmodium spp. and Toxoplasma gondii with Polymerase Chain Reaction: A Pilot Study for Limit Determination
Tuba Oyur1, Özgür Kurt2, İbrahim Çavuş1, Ahmet Özbilgin1
1Manisa Celal Bayar Tıp Üniversitesi Tıp Fakültesi, Tıbbi Parazitoloji Anabilim Dalı, Manisa
2Acıbadem Mehmet Ali Aydınlar Üniversitesi Tıp Fakültesi, Tıbbi Mikrobiyoloji Anabilim Dalı, İstanbul
Keywords: Leishmania spp., Plasmodium spp., Toxoplasma gondii, RT-PCR

Objective: Parasitic diseases that involve blood and tissues pose a significant threat to human health worldwide. Malaria, leishmaniasis and toxoplasmosis kill thousands of people worldwide annually, and impose a great burden on national economies due to treatment costs and loss of labour. The aim of this study are to determine the lowest number of parasites detectable by Real Time Polymerase Chain Reaction (RT-PCR) for Plasmodium spp., Leishmania spp. and T. gondii and to validate the RT-PCR test, and integrate RT-PCR in routine laboratories applications.

Methods: Leishmania tropica (both promastigotes and amastigotes), L. infantum (both promastigotes and amastigotes) and T. gondii were counted using a hemocytometer, while Giemsa-stained smears of P. falciparum and P. vivax were examined under the microscope to define their parasitic load. Then, dilutions as 1, 5, 10, 25, 50, 100, 500, 1.000, 2.500, 5.000 and 10.000 parasites/ul were prepared in PBS solutions. DNA isolation was performed from all parasite suspensions and the lowest parasite suspension which was positively detected by RT-PCR method was identified. This was followed by further dilutions between the previous negative and last positive dilutions, to further determine the lowest number of parasites that RT-PCR could eventually identify.

Results: Our assessments showed that the lowest number of parasites necessary for positive RT-PCR were 10 for P. falciparum and amastigotes and promastigote forms of L. tropica/L. infantum, while 12 for P. vivax and T. gondii.

Conclusion: These results show that RT-PCR is a reliable diagnostic option to be used successfully for the detection of Leishmania spp., Plasmodium spp. and T. gondii, even for infections with low parasitemia.


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