Background: Intestinal parasitic infections can harm health by causing malnutrition, anemia, impaired growth and cognitive development, and alterations in microbiota composition and immune responses. Therefore, it is crucial to examine stool samples to diagnose parasitic infections. However, the traditional microscopic detection method is time-consuming, labor-intensive, and dependent on the expertise and training of microscopists. Hence, there is a need for a low-complexity, high-throughput, and cost-effective alternative to labor-intensive microscopic examinations. Methods: This study aimed to compare the performance of a fully automatic digital feces analyzer, Orienter Model FA280 (People’s Republic of China) with that of the formalin-ethyl acetate concentration technique (FECT). We assessed and compared the agreement between the FA280 and the FECT for parasite detection and species identification in stool samples. The first part of the study analyzed 200 fresh stool samples for parasite detection using the FECT and FA280. With the FA280, the automatic feces analyzer performed the testing, and the digital microscope images were uploaded and automatically evaluated using an artificial intelligence (AI) program. Additionally, a skilled medical technologist conducted a user audit of the FA280 findings. The second set of samples comprised 800 preserved stool samples (preserved in 10% formalin). These samples were examined for parasites using the FECT and FA280 with a user audit. Results: For the first set of stool samples, there was no statistically significant difference in the pairwise agreements between the FECT and the FA280 with a user audit (exact binomial test, P = 1). However, there were statistically significant differences between the pairwise agreements for the FECT and the FA280 with the AI report (McNemar’s test, P < 0.001). The agreement for the species identification of parasites between the FA280 with AI report and FECT showed fair agreement (overall agreement = 75.5%, kappa [κ] = 0.367, 95% CI 0.248–0.486). On the other hand, the user audit for the FA280 and FECT showed perfect agreement (overall agreement = 100%, κ = 1.00, 95% CI 1.00–1.00). For the second set of samples, the FECT detected significantly more positive samples for parasites than the FA280 with a user audit (McNemar’s test, P < 0.001). The disparity in results may be attributed to the FECT using significantly larger stool samples than those used by the FA280. The larger sample size used by the FECT potentially contributed to the higher parasite detection rate. Regarding species identification, there was strong agreement between the FECT and the FA280 with a user audit for helminths (κ = 0.857, 95% CI 0.82–0.894). Similarly, there was perfect agreement for the species identification of protozoa between the FECT and the FA280 with user audit (κ = 1.00, 95% CI 1.00–1.00). Conclusions: Although the FA280 has advantages in terms of simplicity, shorter performance time, and reduced contamination in the laboratory, there are some limitations to consider. These include a higher cost per sample testing and a lower sensitivity compared to the FECT. However, the FA280 enables rapid, convenient, and safe stool examination of parasitic infections. Graphical

Ethnic minority groups are often subjected to exclusion, social and healthcare marginalization, and poverty. There appears to be important linkages between ethnic minority groups, poor socioeconomic status, and a high prevalence of parasitic infection. Data regarding the prevalence and health effects of IPIs are necessary in the development and implementation of targeted prevention and control strategies to eradicate intestinal parasitic infection in the high-risk population. Thus, we investigated for the first time the intestinal parasitic infection status (IPIs), the socioeconomic status, and sanitary condition in the communities of Moken and Orang Laut, the ethnic minority peoples living on the coast of southwest Thailand. A total of 691 participants participated in the present study. The information concerning socioeconomic status and sanitary condition of the study population was obtained by personal interviews using a picture questionnaire. Stool samples were collected and examined for intestinal parasitic infection using direct wet smear and formalin-ethyl acetate concentration techniques. The results revealed that 62% of the study population were infected with one or more types of intestinal parasites. The highest prevalence of intestinal parasitic infections was found in the 11–20-year-old age range group. A statistically significant difference of IPIs among the three communities were observed (p < 0.0001). There was a statistical difference concerning 44 multiple infections of soil-transmitted helminths (STHs) (p < 0.001), whereas no statistically significant difference in multiple infections of protozoa was observed (p > 0.55). The results also displayed the significant difference in socioeconomic status and sanitary condition among the Moken living in Ranong and Phang Nga and the Orang Laut living in the Satun province (p < 0.001). Our study found no direct association between parasitic infection status and ethnic/geographic features; however, socioeconomic status is the key factor associated with prevalence of intestinal parasitic infection, with the observation that the higher prevalence of IPIs is due to a low socioeconomic status, consequently leading to poor hygiene and sanitation practices. The picture questionnaire played a major role in information gathering, especially from those of low or no education. Lastly, data pertaining to the species of the parasites and the mode of transmission assisted in the identification of group-specific vulnerabilities and shortcomings that can be utilized in education and corrected to reduce the prevalence of infection in the study areas.

The diagnosis of filariasis traditionally relies on the detection of circulating microfilariae (mf) using Giemsa-stained thick blood smears. This approach has several limitations. We developed a semi-automated microfluidic device to improve and simplify the detection of filarial nematodes. The efficiency and repeatability of the microfluidic device was evaluated. Human EDTA blood samples were ‘spiked’ with B. malayi mf at high, moderate, and low levels, and subsequently tested 10 times. The device was also used for a field survey of feline filariasis in 383 domesticated cats in an area of Narathiwat Province, Thailand, the endemic area of Brugia malayi infection. In the control blood arbitrarily spiked with mf, the high level, moderate level and low level mf-positive controls yielded coefficient variation (CV) values of 4.44, 4.16 and 4.66%, respectively, at the optimized flow rate of 6 µl/min. During the field survey of feline filariasis in Narathiwat Province, the device detected mf in the blood of 34 of 383 cats (8.9%) whereas mf were detected in 28 (7.3%) cats using the blood smear test. Genomic DNA was extracted from mf trapped in the device after which high-resolution melting (HRM) real-time PCR assay was carried out, which enabled the simultaneous diagnosis of filarial species. Among the 34 mf-positive samples, 12 were identified as B. malayi, 15 as Dirofilaria immitis and 7 as| D. repens. We developed a semi-automated microfluidic device to detect mf of filarial parasites that could be used to diagnose lymphatic filariasis in human populations. This novel device facilitates rapid, higher-throughput detection and identification of infection with filariae in blood samples.

We conducted a survey of canine microfilaraemia in 768 dogs in Chanthaburi, Samut Sakhon, and Narathiwat provinces of Thailand using a novel semi-automated, microfluidic device that is easy and rapid to perform. Microfilariae species were identified using High Resolution Melting real-time PCR (HRM real-time PCR). The prevalence of canine microfilaremia was 16.2% (45/278) in Chanthaburi and 5.5% (12/217) in Samut Sakhon. The prevalence of canine microfilaremia in Narathiwat was 22.7% (67/273). Brugia pahangi and Dirofilaria immitis were the predominant species of filariae found in the infected dogs from Chanthaburi and Narathiwat, respectively. The low prevalence of canine microfilaremia of Samut Sakhon may reflect the success of the Soi Dog foundation’s efforts and the establishment of veterinary control programs. An effective disease control and prevention strategies is needed in Chanthaburi and Narathiwat to reduce the risks of zoonotic transmission of the parasites. An appropriate drug treatment should be given to infected dogs and prophylactic drugs are suggested to be given to dogs age ≤1-year-old to prevent filarial infection. The novel microfluidic device could be implemented for surveillance of filariae infection in other animals.

Lymphatic filariasis is a neglected parasitic disease that is a leading cause of long-term disability. Information obtained from genome sequencing of filarial worm can help us identify systems in the worm that are likely to be useful for novel drug design. Brugia (B.) malayi is still the only lymphatic-dwelling filarial parasite with a nearly complete, fully annotated, and published genome. However, most previous studies were based on the FR3 strain of B. malayi, which originally was isolated from a human patient, and was adapted to the rodent model, then maintained in laboratories for more than 60 years. It is uncertain whether genetic variation exists, thus, sequencing of clinical isolates of lymphatic dwelling filarial parasites is a high priority. Here, we report for the first time the complete mitochondrial genome of B. malayi microfilariae from clinical isolate. Complete mitochondrial (mt) genome of the microfilariae isolated from a blood sample taken from a Thai subject living in Narathiwat Province, which is an endemic area of brugian filariasis, was assembled with sequencing reads obtained by Illumina sequencing. Gene annotation, phylogenetic analysis and single nucleotide polymorphism (SNP) were deployed. A complete 13,658-bp mt genome of B. malayi microfilaria was obtained, and it shows 68x coverage. Based on gene annotation, the mt genome consists of 12 protein-coding, two rRNA, and 23 tRNA genes. Phylogenetic analysis using all protein sequences of DNA sequences of mt genome or cytochrome c oxidase subunit I (COX1) revealed a close relationship among three lymphatic filariae (i.e., B. timori, zoonotic B. pahangi, and Wuchereria spp.). The SNPs in the COX1 gene can differentiate microfilariae of B. malayi in human from those found in canine. Furthermore, the number, order and transcription, and direction of B. malayi microfilariae mitochondrial genes were the same as those found in the FR3 strain of B. malayi. The comparison on mitochondrial genome of B. malayi could have important implications on the development of a new intervention or vaccine to treat or prevent this disease in endemic areas/regions around the world.