Diagnosis of leishmania sPP., plasmodium SPP. and toxoplasma gondii with polymerase chain reaction: Limit determination

AIM: Parasitic diseases that involve blood and tissues may pose a significant threat to human health. Among these, malaria, leishmaniasis and toxoplasmosis kill thousands of people worldwide annually, while bringing outstanding burden to developing economies for the treatment of patients and the loss of work power. Malaria is caused by the protozoa that belong to Plasmodium genus, and infects more than 300 million people worldwide, killing over 1 million, annually. Turkey's being a suitable habitat for Anopheles species, puts it under a continuous threat for malaria. Leishmaniasis is also a vector-borne disease that causes devastating public health effects in developing countries. According to the data of World Health Organization, there are 12 million people with leishmaniasis today in 98 countries, while 350 million are under the risk of the infection. Toxoplasmosis is a zoonotic disease caused by the intracellular protozoon, Toxoplasma gondii. It is transmitted to humans through various ways such as ingestion of the oocysts present in the stools of cats; ingestion of raw or undercooked meat; organ transplantation or laboratory infection. It may be a non-symptomatic infection in healthy individuals, but is associated with life-threatening brain infections in immunocompromised individuals. In addition, due to toxoplasmosis acquired in the first three months of pregnancy, fetus may die or born with neurological defects. Taking body samples for the diagnosis of such dangerous infections is sometimes difficult and the causative agent may not be detected with conventional diagnostic methods. Such cases are necessarily diagnosed with polymerase chain reaction (PCR) today. Advanced application of PCR, the Real Time PCR (RT-PCR), is preferred more common today compared to conventional PCR, due to its advantages such as faster application and lower contamination risk. Parasitic infections can also be diagnosed with RT-PCR owing to its specificity and sensitivity. However, since parasitic load may be extremely low in some cases, it is essential to know the limits of the diagnostic sensitivity of RT-PCR for each parasitic agent. The aim of the present study is to determine the lowest number of parasites at when the RT-PCR for each Plasmodium spp., Leishmania spp. and T. gondii turn from negative to positive results, and thus allocate the standards of these RT-PCR applications to routine health services after the conditions are settled. MATERIALS AND METHODS: Isolates of Leishmania tropica, Leishmania infantum, Plasmodium falciparum, Plasmodium vivax and Toxoplasma gondii kept in liquid nitrogen at -196°C in Celal Bayar University Faculty of Medicine Department of Parasitology were revived and suspensions of each were prepared with PBS having 107 parasites in a mililitre. Serial dilutions were then prepared from each isolate having 1, 5, 10, 25, 50, 100, 500, 1000, 2500, 5000 and 10.000 parasites in a mililitre. Real Time PCR (RT-PCR) tests were conducted with each dilution with an aim to determine the highest dilution with the positive outcome. The lowest number of positive RT-PCR test with each parasite were finally determined by making further dilution between the first positive and the following negative one. RESULTS: Our assessments showed that the lowest number of parasites necessary for positive RT-PCR were 10 for Plasmodium falciparum, Leishmania tropica (promastigotes), Leishmania infantum (promastigotes), Leishmania tropica (amastigotes), Leishmania infantum (amastigotes), while 12 for Plasmodium vivax and Toxoplasma gondii. CONCLUSIONS: These results show that, RT-PCR is a reliable diagnostic option for the detection of Leishmania spp., Plasmodium spp. and Toxoplasma gondii, even for infections with low parasitemia.