Conventional methods such as microscopy have been used to diagnose parasitic diseases and medical conditions related to arthropods for many years. Some techniques are considered gold standard methods. However, their limited sensitivity, specificity, and accuracy, and the need for costly reagents and high-skilled technicians are critical problems. New tools are therefore continually being developed to reduce pitfalls. Recently, three state-of-the-art techniques have emerged: DNA barcoding, geometric morphometrics, and artificial intelligence. Here, data related to the three approaches are reviewed. DNA barcoding involves an analysis of a barcode sequence. It was used to diagnose medical parasites and arthropods with 95.0% accuracy. However, this technique still requires costly reagents and equipment. Geometric morphometric analysis is the statistical analysis of the patterns of shape change of an anatomical structure. Its accuracy is approximately 94.0–100.0%, and unlike DNA barcoding, costly reagents and equipment are not required. Artificial intelligence technology involves the analysis of pictures using well-trained algorithms. It showed 98.8–99.0% precision. All three approaches use computer programs instead of human interpretation. They also have the potential to be high-throughput technologies since many samples can be analyzed at once. However, the limitation of using these techniques in real settings is species coverage.

Background: Wolbachia, obligate intracellular bacteria, infect the majority of arthropods, including many mosquito species of medical importance. Some Wolbachia strains interfere with the development of Plasmodium parasites in female Anopheles, a major vector of malaria. The use of Wolbachia as a means to block malaria transmission is an emerging vector control strategy in highly endemic areas. Hence, identification of native Wolbachia strains in areas where malaria transmission is low may uncover a particular Wolbachia strain capable of Plasmodium interference. This study aims to identify native Wolbachia strains in female Anopheles spp. that are predominant in a low-malaria transmission area in mainland Southeast Asia. Methods: Following a 2-year survey of malaria vectors in Umphang Valley of Tak Province, Thailand, DNA extracts of female An. minimus, An. peditaeniatus, and An. maculatus were subjected to amplification of the conserved region of the 16S rRNA-encoding gene. The DNA sequences of the amplicons were phylogenetically compared with those of known Wolbachia strains. Results: Among three Anopheles spp., amplification was detected in only the DNA samples from An. minimus. The DNA sequencing of amplicons revealed 100% similarity to Wolbachia pipientis, confirming the specificity of amplification. The Wolbachia-positive An. minimus samples were devoid of Plasmodium 18S rRNA amplification. The phylogenetic trees indicate a close relationship with Wolbachia strains in subgroup B. Conclusion: To the best of our knowledge, the data presented herein provide the first molecular evidence of a Wolbachia strain in An. minimus, hereinafter named wAnmi, in a low-malaria transmission area in the Umphang Valley of western Thailand. Further biological characterization is required to examine its potential for malaria transmission control in the field. Graphical

Many antiparasitic drugs have become obsolete and ineffective in treating parasitic diseases. This ineffectiveness arises from parasite drug resistance, high toxicity, and low drug efficacy. Thus, the discovery of novel agents is urgently needed to control parasitic diseases. Various strategies are employed in drug discovery, design, and development. This review highlights the paradigm of searching for bioactive molecules produced during inter- and intraspecific competition among organisms, particularly between microbes and parasites, as a strategy for de novo antiparasitic drug discovery. Competitive interactions occur when individuals of the same or different species coexist in overlapping niches and compete for space and resources. These interactions are well recognized. Therefore, bioactive molecules released during these interactions are promising targets for novel drug discovery. Compelling data indicate that microbes remain a potential source for the discovery of novel antiparasitic drugs because of their diversity. Many antimicrobial producers in nature have yet to be isolated and investigated. This body of evidence underscores the success of numerous therapeutic drugs, including penicillin, β-lactams, and tetracyclines, which have been successfully discovered and developed for treating infectious diseases. This review comprehensively covers these concepts, with a particular focus on inter- and intraspecific competition in the discovery of novel antiparasitic agents. This approach will pave the way for identifying alternative strategies to control and eradicate parasitic diseases that continue to threaten human health. Additionally, this review discusses current antiparasitic drugs and their mechanisms of action, limitations, and existing gaps. This discussion emphasizes the ongoing need to explore novel antiparasitic drugs.

Naegleria fowleri causes deadly primary amoebic meningoencephalitis (PAM) in humans. Humans obtain the infection by inhaling water or dust contaminated with amebae into the nostrils, wherefrom the pathogen migrates via the olfactory nerve to cause brain inflammation and necrosis. Current PAM treatment is ineffective and toxic. Seeking new effective and less toxic drugs for the environmental control of the amoeba population to reduce human exposure is logical for the management of N. fowleri infection. On the basis of the concept of competitive exclusion, where environmental microorganisms compete for resources by secreting factors detrimental to other organisms, we tested cell-free culture supernatants (CFSs) of three bacteria isolated from a fresh water canal, i.e., Pseudomonas aeruginosa, Pseudomonas otitidis, and Enterobacter cloacae, were tested against N. fowleri. The CFSs inhibited growth and caused morphological changes in N. fowleri. At low dose, N. fowleri trophozoites exposed to P. aeruginosa pyocyanin were seen to shrink and become rounded, while at high dose, the trophozoites were fragmented. While the precise molecular mechanisms of pyocyanin and products of P. otitidis and E. cloacae that also exert anti-N. fowleri activity await clarification. Our findings suggest that P. aeruginosa pyocyanin may have a role in the control of amphizoic N. fowleri in the environment.

During the COVID-19 pandemic, the parasitology laboratories dealing with fecal samples for the diagnosis of gastrointestinal parasitic infections are confronting the unsaved virus-containing samples. To allow for safe downstream processing of the fecal samples, a protocol for preparing a fecal smear is urgently needed. Formalin was tested with or without isotonic forms for virus inactivation using porcine epidemic diarrhea virus (PEDV) as a representative, as it belongs to the Coronaviridae family. The results revealed complete inactivation activity of 10% formalin and 10% isotonic formalin on coronavirus after 5 min of treatment at room temperature. Both also inhibited Naegleria fowleri growth after 5 min of treatment at 37 °C without disruption of the structure. In addition to these key findings, it was also found that isotonic formalin could stabilize both red and white blood cells when used as a solution to prepare fecal smears comparable to the standard method, highlighting its value for use instead of 0.9% normal saline solution for the quantification of blood cells without active virus. The 10% isotonic formalin is useful to safely prepare a fecal smear for the diagnosis of parasites and other infections of the gastrointestinal tract during the COVID-19 pandemic.

Sand flies are principal vectors of Leishmania spp. and Trypanosoma spp. Identifying precise vector species is crucial for effective control. We conducted a study on the species distribution of phlebotomine sand flies in cave-dwelling and non-cave-dwelling in four southern provinces of Thailand. In this study, we collected 621 sand flies (346 females and 275 males) and identified all specimens based on morphology and DNA barcoding, employing cytochrome c oxidase subunit 1 (cox1) and cytochrome b (cytb) genes. In female specimens, we also screened the small subunit 18S ribosomal RNA (18S rRNA) gene for Leishmania spp. and Trypanosoma spp. Morphologically, 467 (75.2%) sand flies were identified to species level, 47 (7.57%) to subgenus level, and 107 (17.23%) to genus level. These included Idiophlebotomus asperulus (43.48%), Sergentomyia khawi (26.73%), S. anodontis (2.25%), S. brevicaulis (2.25%), Grassomyia indica (0.48%), Phlebotomus (Euphlebotomus) spp. (4.83%), Phlebotomus (Lewisius) spp. (2.74%), Sergentomyia spp. (9.18%), and Phlebotomus spp. (8.05%). Among the 107 specimens identified to genus level, DNA barcoding further identified 49 (45.79%) as Sergentomyia barraudi (1.61%), S. bailyi (0.16%), Phlebotomus kiangsuensis (2.9%), and Ph. stantoni (1.61%). No Leishmania DNA was detected, but Trypanosoma DNA was found in females of S. khawi from Narathiwat Province. Expanding genetic reference databases of sand flies located in four provinces of southern Thailand will improve barcoding accuracy. Understanding sand fly species composition and distribution is imperative for vector control and disease prevention in Thailand.