Study participants and sample collection
A cross-sectional survey was carried out from March to July 2018 in an endemic area for infection with O. viverrini and other helminth parasites in Na Mon district, Kalasin province, northeast Thailand (Fig. 1). Residents aged 15 years or older, both men and women, were recruited in this study. After written informed consent was obtained, the participants were registered for demographic information. Clean plastic containers were labeled with participants’ names and identifying numbers and were distributed to the villagers to collect the fecal samples. Approximately 10 g of fecal sample was obtained from each participant and the samples were kept in a chilled insulated box and transported from the study site to the laboratory at the Khon Kaen University. Each fecal sample was mixed and divided into 3 parts and processed for examinations by Mini Parasep SF stool concentrator kit method, FECT, and KK methods. The study population mostly comprised farmers working in paddy fields.
Methods and procedures for fecal examination
Stool kit method
The procedure for the Mini Parasep SF fecal parasite concentrator kit method (Diasys Europe, Berkshire, UK) was performed according to the manufacturer’s instructions. In short, a fecal sample (~0.5 g) was introduced into a tube containing 3.3 ml of 10% formalin solution, and one drop of Triton x-100 then was added. The sample was vortexed to emulsify the mixture. The tube was inverted and centrifuged at 500 g for 2 minutes and then the top layers of the supernatant were decanted. The pellet was resuspended in 1 ml of 10% formalin solution and two drops, mixed with Lugol’s iodine solution, were examined under a light microscope (100x – 400x magnification). Enumerations of egg per gram feces (EPG) were similar to the procedure in modified FECT (see below). Discrimination between eggs of O. viverrini and minute intestinal fluke (MIF) i.e., lecithodendrid eggs, was based on morphological characteristics as previously described [13, 20].
Formalin-ethyl acetate concentration technique (FECT)
Fecal examination using a modified FECT diagnostic method was conducted as reported previously [21, 22]. Briefly, fecal samples were homogenized, and two grams of fresh feces were weighed and diluted in 7 mL of 10% formalin solution and strained through two layers of gauze into the 15 mL centrifuge tube. The strained suspension was centrifuged at 2,500 rpm for 5 min, room temperature, and the supernatant was poured out. Thereafter, 7 mL of 0.85% saline were added to the tube and then vigorously mixed with 3 ml of ethyl acetate for 30-60 seconds to aid in the extraction of fat from the feces. The suspension was then centrifuged at 2,500 rpm for 5 min at room temperature and the pellet was resuspended and fixed with 1 mL of 10% formalin. The final fecal suspension was examined two times (two drops) per sample by the same microscopist using a compound microscope at 100x and 400x magnifications with the results combined and multiplied by the number of drops in the suspension and divided by the mass of stool in grams to calculate the number of eggs per gram of feces (EPG). Morphological differentiation between eggs of O. viverrini and minute intestinal fluke was done as previously described by Kaewkes .
After thorough homogenization of the samples, approximately one gram of feces was pressed through a mesh screen to remove large particles. Then a portion of the sieved sample was transferred to the standard template holding on a slide. After filling the hole, the template was removed, and the remaining sample was covered with a piece of cellophane pre-soaked (24 hours) in the 3% malachite green-glycerol solution. The slide was allowed to clear for 30 min before examination under a microscope. The number of eggs was counted and recorded for each helminth species separately and multiplied by 23 to calculate the EPG [23, 24].
This human subject protocol was approved by the Ethics Committee of Khon Kaen University, Khon Kaen Thailand (reference number HE601370). Permission for fieldwork was obtained from the Ministry of Public Health (MOPH) and the provincial, district, and sub-district health officers. Written informed consents were obtained from all participating subjects. At the end of the project, infected individuals were treated with appropriate anthelmintic drugs.
Data were recorded in case report forms, entered into an Excel worksheet (Microsoft), and analyzed using SPSS 26 (IBM, Chicago, IL, USA). Data from individuals with matched fecal samples by the three diagnostic tests were considered for analysis. Positive fecal examination results referred to the presence of parasite eggs or larva in the fecal specimen examined by any one of three methods: stool kit, FECT, or KK. In the analysis for diagnostic performance of each method, the gold standard (100 % specificity and sensitivity) was defined as a combined result from the other two methods. Diagnostic accuracy in terms of clinical sensitivity, clinical specificity, positive predictive values (PPV), negative predictive values (NPV), and 95% confidence intervals (95% CI) for total parasites and O. viverrini separately were determined using MedCalc (Med Calc Software, Ostend, Belgium). The agreement in the status of O. viverrini and other helminth infections between two different methods was evaluated using Cohen’s kappa coefficient. Cohen’s kappa values <0 were interpreted as indicating no agreement and 0-0.20 as slight, 0.21-0.40 as fair, 0.41-0.60 as moderate, 0.61-0.80 as substantial, and 0.81-1 as almost perfect agreement on the status of O. viverrini and other helminth infection of the study participants between different methods . McNemar’s chi-squared test was used to compare the prevalence of O. viverrini and other parasitic infections between diagnostic methods.
The intensity of O. viverrini infection determined as eggs per gram of feces (EPG) was categorized into three groups as 1-50, 51-100, and >100 . Kendall’s tau-b correlation test was used to determine the correlation of intensity (EPG) between two different methods. A statistically significant level was set at a P-value of <0.05.