EVALUATION OF THE SENSITIVITY AMONG CONVENTIONAL PCR, QUANTITATIVE PCR, FISH AND IHC FOR THE DETECTION OF LEISHMANIA SPP. USING LEISHMANIA SIAMENSIS CELL LINE

Abstract

Leishmania siamensis is a novel Leishmania species described recently in Thailand. Patients infected with L. siamensis can be presented with cutaneous leishmaniasis (CL), visceral leishmaniasis (VL) or combination of both CL and VL. Early diagnose is essential for therapeutic reasons and therefore decrease morbidity and mortality. Conventional diagnosis is usually based on microscopic examination by staining with haematoxylin and eosin or Giemsa. The technique requires expertise and shows low sensitivity. IHC and FISH are alternative techniques which used to detect Lesihmania parasites. These methods are known to use in routine laboratory for detection of other pathogens. Molecular techniques such as PCR and qPCR have been used to detect Leishmania DNA and have been shown high sensitivity for detection Leishmania spp. However, these techniques have higher cost than other conventional methods. The study is designed to compare the ability of IHC, FISH, PCR and qPCR for detection Leishmania parasites. The experiment sample was prepared from culture L. isamensis and was made to serial dilution of 107-101. IHC technique was used primary antibody anti-GP63, FISH technique was used probes designed label with fluorescent dye to hybridize the ITS1 gene region. PCR, and qPCR were used to detect the same extraction DNA from each dilution described previously. All result from 6 volunteers showed positive in low concentration (101) with both of PCR and qPCR and average of median are 4.00 can still observed in this concentration. While, IHC and FISH showed average of median are 5.71 and 5.87, respectively (P<0.05). However, they are lower sensitivity than molecular detections. The data obtained from this study can be used for detection promastigote of L. siamensis. These techniques can be applied for detection amastigotes form from clinical specimens. IHC and FISH is cheaper than molecular method that appropriates for a small laboratory. Moreover, qPCR developed from this study has been used to detect L.siamensis DNA in various clinical sample specimens. It showed positive in blood, buffy coat, saliva and urine. Techniques developed from this study therefore could be applied for epidemiological studies and follow up after treatment.