Comparison of a Urine Antigen Assay and Multiple Examinations with the Formalin-Ethyl Acetate Concentration Technique for Diagnosis of Opisthorchiasis

Abstract

Detection of worm antigen in urine is a sensitive diagnostic method for opisthorchiasis, particularly for light-intensity infections; however, the presence of eggs in feces is essential for validating results from the antigen assay. To address the issue of low sensitivity of fecal examination, we modified the protocol for the formalin-ethyl acetate concentration technique (FECT) and compared it against urine antigen measurements for detection of the parasite Opisthorchis viverrini. First, we optimized the FECT protocol by increasing the number of drops for examinations from the standard two drops to a maximum of eight. We were able to detect additional cases after examination of ≥ 3 drops, and the prevalence of O. viverrini saturated after examination of ≥ 5 drops. We then compared the optimized FECT protocol (examining five drops of suspension) against urine antigen detection for the diagnosis of opisthorchiasis in field-collected samples. The optimized FECT protocol detected O. viverrini eggs in 25 of 82 individuals (30.5%) who had positive urine antigen tests but were fecal egg negative by the standard FECT protocol. The optimized protocol also retrieved O. viverrini eggs in 2 of 80 antigen-negative cases (2.5%). In comparison with the composite reference standard (combined FECT and urine antigen detection), the diagnostic sensitivity of examining two and five drops of FECT and the urine assay was 58.2, 67, and 98.8%, respectively. Our results show that multiple examinations of fecal sediment increase the diagnostic sensitivity of FECT and thus provide further support for the reliability and utility of the antigen assay for diagnosis and screening of opisthorchiasis.

Figure 1.

Study design. Optimization (phase 1) was designed to adjust the number of drops for FECT examination in urine antigen-positive individuals. Validation (phase 2) applied the optimized FECT protocol to field-collected samples from an endemic area of opisthorchiasis to compare with results from the urine antigen test. FECT = formalin-ethyl acetate concentration technique.

Figure 2.

The correlation between O. viverrini antigen concentration (log-transformed values) determined by urine antigen assay and EPG (log-transformed values), calculated by using two-drop (A) and five-drop examinations (B) of the fecal sediments prepared by FECT from Na Mon District, Kalasin Province (N = 256). EPG = egg per gram feces; FECT = formalin-ethyl acetate concentration technique.