Advances in the Diagnosis of Human Opisthorchiasis: Development of Opisthorchis viverrini Antigen Detection in Urine.
BACKGROUND
Many strategies to control opisthorchiasis have been employed in Thailand, but not in the other neighbouring countries. Specific control methods include mass drug administration (MDA) and health education to reduce raw fish consumption. These control efforts have greatly shifted the epidemiology of Opisthorchis viverrini (OV) infection over the last decade from presenting as densely concentrated "heavy" infections in single villages to widespread "light" OV infections distributed over wide geographical areas. Currently, the "gold standard" detection method for OV infection is formalin ethyl-acetate concentration technique (FECT), which has limited diagnostic sensitivity and diagnostic specificity for light OV infections, with OV eggs often confused with eggs of minute intestinal flukes (MIFs) in feces. In this study, we developed and evaluated the diagnostic performance of a monoclonal antibody-based enzyme-linked immunosorbent assay for the measurement of OV excretory-secretory (ES) antigens in urine (urine OV-ES assay) for the diagnosis of opisthorchiasis compared to the gold standard detection FECT method.


METHODS
We tested several methods for pre-treating urine samples prior to testing the diagnostic performance of the urine OV-ES assay. Using trichloroacetic acid (TCA) pre-treated urine, we compared detection and quantification of OV infection using the urine OV-ES assay versus FECT in OV-endemic areas in Northeastern Thailand. Receiver operating characteristic (ROC) curves were used to determine the diagnostic sensitivity and specificity of the urine OV-ES assay using TCA pre-treated urine, and to establish diagnostic positivity thresholds. The Positive Predictive Value as well as the likelihood of obtaining a positive test result (LR+) or a negative test result (LR-) were calculated for the established diagnostic positivity threshold. Diagnostic risks (Odds Ratios) were estimated using logistic regression.


RESULTS
When urine samples were pre-treated with TCA prior to use in the urine OV-ES assay, the analytical sensitivity was significantly improved. Using TCA pre-treatment of urine, the urine OV-ES assay had a limit of detection (LoD) of 39 ng/ml compared to the LoD of 52 ng/mL reported for coprological antigen detection methods. Similarly, the urine OV-ES assay correlated significantly with intensity of OV infection as measured by FECT. The urine OV-ES assay was also able to detect 28 individuals as positive from the 63 (44.4%) individuals previously determined to be negative using FECT. The likelihood of a positive diagnosis of OV infection by urine OV-ES assay increased significantly with the intensity of OV infection as determined by FECT. With reference to FECT, the sensitivity and specificity of the urine OV-ES assay was 81% and 70%, respectively.


CONCLUSIONS
The detection of OV-infection by the urine OV-ES assay showed much greater diagnostic sensitivity and diagnostic specificity than the current "gold standard" FECT method for the detection and quantification of OV infection. Due to its ease-of-use, and noninvasive sample collection (urine), the urine OV-ES assay offers the potential to revolutionize the diagnosis of liver fluke infection and provide an effective tool for control and elimination of these tumorigenic parasites.
Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand; Liver Fluke and Cholangiocarcinoma Research Center, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand.
Chanika
C
Worasith
Department of Microbiology, Immunology and Tropical Medicine, and Research Center for Neglected Diseases of Poverty, School of Medicine & Health Sciences, George Washington University, Washington, D.C., United States of America.
Christine
C
Kamamia
Department of Microbiology, Immunology and Tropical Medicine, and Research Center for Neglected Diseases of Poverty, School of Medicine & Health Sciences, George Washington University, Washington, D.C., United States of America.
Anna
A
Yakovleva
Department of Public Health, Faculty of Science and Technology, Phetchabun Rajabhat University, Phetchabun, Thailand.
Kunyarat
K
Duenngai
Liver Fluke and Cholangiocarcinoma Research Center, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand; Biomedical Science Program, Graduate School, Khon Kaen University, Khon Kaen, Thailand.
Chompunoot
C
Wangboon
Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, Thailand.
Jiraporn
J
Sithithaworn
Department of Pre-Clinical Veterinary Sciences, Faculty of Veterinary Sciences, Mahasarakham University, Mahasarakham, Thailand.
Nattaya
N
Watwiengkam
Liver Fluke and Cholangiocarcinoma Research Center, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand; Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand.
Nisana
N
Namwat
Liver Fluke and Cholangiocarcinoma Research Center, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand; Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, Thailand.
Anchalee
A
Techasen
Liver Fluke and Cholangiocarcinoma Research Center, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand; Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand.
Watcharin
W
Loilome
Liver Fluke and Cholangiocarcinoma Research Center, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand; Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand.
Puangrat
P
Yongvanit
Centre for Biodiversity and Molecular Development of Therapeutics, Queensland Tropical Health Alliance, James Cook University, Cairns, Queensland, Australia.
Alex
A
Loukas
Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand; Liver Fluke and Cholangiocarcinoma Research Center, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand.
Paiboon
P
Sithithaworn
Department of Microbiology, Immunology and Tropical Medicine, and Research Center for Neglected Diseases of Poverty, School of Medicine & Health Sciences, George Washington University, Washington, D.C., United States of America.
Jeffrey M
JM
Bethony
D000328:Adult; D000818:Animals; D000947:Antigens, Helminth / Q000652:urine*; D003430:Cross-Sectional Studies; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D009889:Opisthorchiasis / Q000175:diagnosis*; D009891:Opisthorchis / Q000737:chemistry* / Q000276:immunology; D010274:Parasitology / Q000379:methods*; D011446:Prospective Studies; D012372:ROC Curve; D012680:Sensitivity and Specificity; D013048:Specimen Handling / Q000379:methods; D013785:Thailand; D016482:Urinalysis / Q000379:methods*; D055815:Young Adult
D000947:Antigens, Helminth