Prevalence of Ehrlichia-, Babesia-, and Hepatozoon-infected brown dog ticks in Khon Kaen Province, Northeast Thailand.

Background and Aim: The brown dog tick, Rhipicephalus sanguineus sensu lato, is the most common tick found on domestic dogs in Southeast Asia, including Thailand. Canine tick-borne pathogens are a public health concern worldwide. Tick-borne diseases are diagnosed by identifying pathogens based on the morphological or molecular analyses of dog blood samples. However, the collection of ticks, a non-invasive procedure, is easier than drawing blood. This study aimed to demonstrate the usefulness of collecting brown dog ticks for the diagnosis of tick-borne diseases and for estimating the prevalence of tick-borne pathogens among companion dogs in Khon Kaen, Northeast Thailand. Materials and Methods: Seventy brown dog ticks from 70 companion dogs in Khon Kaen Province, Thailand, were evaluated for molecular evidence of tick-borne pathogens, including Babesia spp., Ehrlichia canis, and Hepatozoon canis. Ticks were collected from dogs at a private animal hospital based on the presence of at least one of the three inclusion criteria: fever, anorexia, or lethargy. Molecular diagnosis was performed using conventional polymerase chain reaction for the detection of pathogens. Results: Of the 70 ticks collected from 70 sick dogs, 55 (78.57%) were positive for tick-borne pathogens. The most common infection was a single infection with H. canis (65.71%) followed by Babesia spp. (31.43%) and E. canis (30.00%). Coinfection was observed in 14 ticks (20.00%), and coinfection with Babesia spp. and E. canis was the most prevalent double infection (n = 6). The prevalence of coinfection was identical for H. canis mixed with Babesia spp. and H. canis mixed with E. canis (n = 4). Conclusion: The present study showed that tick-borne pathogens are highly prevalent among companion dogs in Khon Kaen Province. Therefore, we encourage an increase in tick control or the reduction and prevention of tick-borne diseases in this region. Furthermore, this study revealed that ticks are valuable samples for the molecular detection of tick-borne pathogens.

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. METHODOLOGY: 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. CONCLUSION: 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.

Improved performance and quantitative detection of copro-antigens by a monoclonal antibody based ELISA to diagnose human opisthorchiasis.

Copro-antigen detection has been advocated as a promising method for diagnosis of opisthorchiasis, particularly in people that harbored light infection or have had recent drug treatment. This study aimed to improve performance of a monoclonal antibody-based enzyme-linked immunosorbent assay (Mab-ELISA) for detection of Opisthorchis viverrini copro-antigen and assess the correlation between copro-antigen and intensity of infection. Four different treatment methods of 71 samples from the Lawa endemic area, Khon Kaen province were assessed for copro-antigen detection, namely (1) phosphate buffer saline (PBS), (2) heating (70°C 30min), (3) alkaline (pH 9.6 in carbonate buffer), and (4) trichloroacetic acid (TCA) treatment. Comparison of these protocols showed that the TCA method gave the best performance in discriminating O. viverrini positive and negative samples with high sensitivity (97.9%) and moderate specificity (54.2%) compared with other methods. Application of TCA-based Mab-ELISA method for antigen detection in fecal samples obtained from an endemic area of opisthorchiasis revealed that 86 of 141 samples (61.0%) were positive compared with 68 (48.2%) by PBS-based protocol, while the formalin ethyl-acetate concentration technique (FECT) yielded a positive proportion of 71.6%. Among 40 egg-negative samples confirmed by a gold standard parasitological method (FECT) from the same endemic area, 19 (47.5%) were positive by the TCA-based while only 6 (15%) were positive by PBS-based Mab-ELISA protocol. In addition, levels of antigen detection significantly correlated with intensity of infection (egg per gram feces). The results show that the improved Mab-ELISA method has high sensitivity and also quantifiable diagnosis of opisthorchiasis.