Use of polymerase chain reaction (nested) and loop mediated isothermal amplification (wet and lyophilized) for the detection of Toxoplasma gondii in clinical samples

Abstract

Toxoplasmosis is a disease in humans caused by a protozoan parasite known as Toxoplasma gondii. The widely used methods of diagnosis of T. gondii are mainly serological but have low sensitivity and are labor intensive. There is need for alternative diagnostic tests that are more sensitive and can be used when specific antibody titers are below detectable threshold levels. The amplification of repetitive 529 bp loci of T. gondii has been reported to be sensitive and specific and was amplified in this study. The objective of the study was to compare the performance of nested polymerase chain reaction (nPCR) with loop mediated isothermal amplification (LAMP) (wet and dry). A total of 87 human blood samples collected from the slaughter house workers between March 2013 and June 2013, and another 87 baboon blood samples collected between 2006 and 2011 were studied. The DNA extracted from the samples was analyzed using nested PCR and LAMP (wet and dry) based on the same target. In overall, 39.1% of the slaughter house workers and 33.3% of the baboons tested positive by nested PCR. T. gondii was detected in 33.3% and 42.5% of the slaughter house workers by wet LAMP and dry LAMP respectively. There was no significant difference in the performance between nested PCR and LAMP (wet and dry) and also between the wet LAMP and dry LAMP (p value >0.05). This study reports high prevalences of T. gondii infection in high risk group of workers and baboons in Thika District. This study recommends the need for public health awareness through education in the control of T. gondii in Thika District. Moreover, the baboon (P. anubis) can be used as an experimental model in the investigation of transmission, diagnosis and treatment of T. gondii infection. The study further demonstrates that lyophilized LAMP may be used in the field set up for the detection of T. gondii. However, further evaluation on reagent stability after storage at different temperature and duration should be investigated.