A Comparison of Parasitological Techniques (Microscopy) and Loop-mediated Isothermal Amplification (LAMP) of DNA in Diagnosis and Monitoring Treatment of Trypanosoma brucei rhodesiense Infection in Vervet Monkeys (Chlorocebus aethiops)

摘要

Microscopy and LAMP were compared in diagnosis and treatment follow-up of HAT with T. b. rhodesiense infected vervet monkeys ( Chlorocebus aethiops ) treated with diminazene aceturate (Berenil?) and Melarsoprol (Mel-B?). Saponin method and heat treatment were used to extract pure and crude DNA, amplified on thermocycler and water bath. SYBR-green and UV-illumination in agarose gel were used to assess results. Parasitaemia, CSF-parasitosis, PCV, WBC and CSF-protein concentration were determined. DNA was detected 7dpi in blood and serum and 21dpi in CSF. It cleared 56dpi in blood and serum of both monkeys and re-appeared 77dpi and 129dpi in blood and serum of monkey A and B respectively after Berenil? treatment. DNA cleared 40 and 90 days and 90 and 150 days in blood and serum and CSF of A and B respectively after Mel-B? treatment. Using blood and CSF, detection of microscopy was 28.21% and 21.18% while LAMP was 60.26%, 55.13% and 79.49% using blood, serum and CSF respectively. X2 was 16.734 (p=0.000) and 38.023 (p=0.000) between LAMP and microscopy in blood and CSF, pure DNA using thermocycler it was 60.27%, 55.13% and 78.12%, and 46.15%, 48.72% and 75.64% on water bath in blood, serum and CSF respectively. Crude DNA had %detection of 56.41%, 56.41% and 76.92% using thermocycler and 48.72%, 44.87% and 64.10% on water bath using blood, serum and CSF respectively. Crude DNA using thermocycler, LAMP and microscopy had Kappa (k) of 0.397 and 0.602 and X2 13.141 (p=0.000) and 35.247 (p=0000) using blood and CSF respectively. In late stages, k and X2 of 0.600 and 15.000 (p=0.000) were obtained using CSF. For treatment follow-up, k and X2 were 0.472 and 19.429 (p=0.000) and 0.527 and 21.346 (p=0.000) in blood and CSF respectively. Heat treatment and amplification using water bath may be used for sample preparation and amplification respectively.