In this research, we elucidated the species composition of Culicoides biting midges, infection prevalence, and genetic diversity of Leishmania parasites circulating in the affected community in Chiang Rai Province, being the most endemic area in Northern Thailand. A total of 146 parous and gravid females, belonging to at least twelve Culicoides species in five subgenera and one species group, were trapped from three collection sites with an overall Leishmania prevalence of 26.7% (39/146). Leishmania was detected, using ITS1-PCR, in C. mahasarakamense (15), C. guttifer (11), C. (Trithecoides) spp. (8), C. jacobsoni (2), C. oxystoma (2), and C. orientalis (1). The evidence of Leishmania infection in these last five species represents new records in Northern Thailand. Given a high infection rate in cavernicolous specimens, this indicates an increased risk of parasite exposure when visiting the cave. Using the nanopore amplicon sequencing, L. martiniquensis was ubiquitously identified in all positives, and more than half of these were also co-infected with L. orientalis. The genetic diversity analysis revealed 13 and 17 unique haplotypes for L. martiniquensis and L. orientalis, respectively. Higher haplotype diversity and relatively low nucleotide diversity were observed in both parasite populations, suggesting recent population divergence. Neutrality tests (Tajima’s D and Fu and Li’s D) showed to be significantly negative, indicating rapid population growth or a selective sweep. Moreover, dominant haplotypes of both Leishmania species were 100% identical to those in all leishmaniasis patients previously reported from Northern Thailand, strongly supporting the imperative role of Culicoides spp. in disease transmission. Essentially, this research provides the first entomological surveillance data representing the sympatric existence, transmission dynamics, and genetic complexity of two autochthonous Leishmania (Mundinia) parasites in several Culicoides species in the endemic area of Northern Thailand. This would contribute to a more complete understanding of the epidemiology of vector infection and facilitate the development of vector control programs to effectively reduce the transmission of this neglected tropical disease in endemic areas of Northern Thailand.

The World Health Organization has recently declared Thailand a leishmaniasis hotspot in Southeast Asia due to the continuous increase in new symptomatic and asymptomatic cases over the years. This emerging parasitic disease is known to be caused by two autochthonous species of Leishmania belonging to the newly described subgenus Mundinia, namely L. martiniquensis and L. orientalis. In Thailand, clinical cases due to L. martiniquensis typically present with visceral leishmaniasis, whereas L. orientalis mainly causes localized cutaneous leishmaniasis. Although Leishmania species confirmation is essential for clinical diagnosis and treatment planning, the availability of highly accurate and rapid diagnostic methods remains limited. In this study, we developed a duplex TaqMan quantitative PCR assay using newly designed species-specific primers and probes based on sequences from the nucleotide and genome databases of Leishmania spp. retrieved from GenBank. The duplex qPCR assay was optimized to specifically amplify the internal transcribed spacer 1 (ITS1) of L. martiniquensis and the heat shock protein 70 (type I) intergenic region (HSP70-I IR) of L. orientalis with high amplification efficiencies. The performance of the optimized duplex qPCR was evaluated by analyzing 46 DNA samples obtained from cultures, and clinical and insect specimens, consistent with the results of the previously validated 18S rRNA-qPCR and ITS1-PCR. The duplex qPCR could detect both species of Leishmania at a limit of detection of one copy per reaction and did not cross-amplify with other pathogen DNA samples. Standard curves of the singleplex and duplex assays showed good linearity with excellent amplification efficiency. Using conventional ITS1-PCR and plasmid sequencing as a reference standard assay, the duplex qPCR showed diagnostic sensitivity and specificity of 100% and positive and negative predictive values of 100% for both Leishmania species with a perfect level of agreement (kappa = 1.0). The novel duplex TaqMan-based qPCR has shown to be a rapid, cost-effective, and highly accurate diagnostic tool for the simultaneous detection and identification of two autochthonous Leishmania spp. in a variety of clinical and entomological samples. This will greatly facilitate early diagnosis, treatment monitoring, and surveillance, especially in leishmaniasis-endemic areas where sequencing-based diagnosis is not routinely available.

Bats act as hosts for various ectoparasites, including bat flies, bugs, fleas, ticks, and mites, which play crucial roles in the transmission of bat-borne pathogens. As obligate blood-feeding parasites, these ectoparasites can serve as direct vectors or indirectly influence pathogen dynamics in bat populations. In Thailand, molecular studies on bat ectoparasites are limited, with scarce data on their diversity, distribution, and role in zoonotic pathogen transmission. This study aims to provide the first molecular characterization of bat flies and other ectoparasites in Ratchaburi, Thailand, focusing on their blood meals and potential involvement in coronavirus transmission. Ectoparasites were identified using the cytochrome oxidase I (COI) gene, while host blood meals were confirmed using the cytochrome b (CytB) gene. A total of 37 bat flies and additional ectoparasites (ticks and flea) were analyzed. Coronavirus screening was conducted through targeted amplification of the RNA-dependent RNA polymerase (RdRp) gene, followed by sequencing and phylogenetic analysis of positive samples. Three bat fly genera were identified: Nycteribia sp. (20), Phthiridium sp. (15), and Raymondia sp. (2). Blood meal analysis indicated host associations with Rhinolophus coelophyllus for Nycteribia sp., R. pusillus and Chaerephon plicatus for Phthiridium sp., and R. pusillus for Raymondia sp. Additionally, two Ixodes sp. ticks and one Hystrichopsylla sp. flea were found, all associated with C. plicatus. Coronavirus screening identified positive samples, with sequencing revealing alphacoronaviruses and betacoronaviruses. This study highlights the importance of molecular tools in characterizing ectoparasites, their blood meal sources, and associated pathogens. It underscores the potential of ectoparasites as non-invasive tools for coronavirus detection in bats, addressing knowledge gaps and contributing to public health strategies for mitigating zoonotic spillover risks.

: Extralymphatic filariasis caused by filaria of zoonotic origins has been frequently reported in Thailand over recent years. Here, we report the first case of ocular filariasis in a 7.5-year-old Thai boy who initially presented with progressive conjunctival redness and blurred vision in his right eye. A small, slender, coiled worm was found and surgically removed from the right anterior chamber. Histopathological examination illustrated predominant eosinophilic inflammation surrounding the parasite, which showed smooth and thin cuticle, prominent lateral chords, flat and broad muscle cells, one intestine, and two reproductive tubes with unsegmented ova, typically characteristic of a female adult Brugia filarial nematode. The parasite was also molecularly identified as B. pahangi, based on mitochondrial cytochrome c oxidase subunit I sequence analysis. The patient was then empirically prescribed albendazole, systemic prednisolone, and topical methylprednisolone. Unfortunately, his vision did not recover after 2 months due to severe maculopathy, most likely resulting from parasitic infestation and subsequent vitreous inflammation. To the best of our knowledge, this is the first case of ocular infestation by B. pahangi with visual complications that occurred outside a filariasis-endemic area of Thailand. Furthermore, this report provides clinical data on preceding cases of B. pahangi filariasis formally reported in southeast Asian countries, including Thailand and Malaysia, which facilitate a better understanding of the epidemiology of this sporadic zoonotic infection for effective disease elimination.

Biting midges of the genus Culicoides have been identified as potential vectors for the transmission of species of the Leishmania subgenus Mundinia, the causative agents of autochthonous leishmaniasis in Thailand. Vector competence may be potentially affected by bacterial endosymbionts; however, there is no known correlation between these endosymbionts and Leishmania parasites in Culicoides biting midges. In this study, we aimed to explore the prevalence and association of bacterial endosymbionts and the detection of Leishmania DNA in Culicoides spp. Female midges were captured at five sampling sites in areas with autochthonous leishmaniasis in northern and southern Thailand. Culicoides species were identified using both morphological characteristics and cox1 sequencing. The presence of Wolbachia, Cardinium, and Leishmania DNA in individual midges was molecularly screened targeting the wsp and 16S rRNA genes, and the ITS1 region, respectively. All amplification products were sequenced and subjected to phylogenetic analysis. A total of 593 female midges were collected, comprising 21 species of Culicoides and one species of Culicoides (Trithecoides). The Wolbachia isolates from infected Culicoides spp. were phylogenetically classified into supergroups A, B, and F. Six Wolbachia putative strains belonged to clade Wol-b (Wol-b1 to Wol-b6), two to clade Wol-a (Wol-a1 and Wol-a2), and one to clade Wol-f (Wol-f1). Three Wolbachia strains were identified as wKerlac, wBeva_B, and wCauA. Moreover, both Cardinium groups A and C were identified. It is noteworthy that co-infections between bacterial endosymbionts and Leishmania spp. showed a significant association. To the best of our knowledge, this study provides the first evidence of Wolbachia and Cardinium in Culicoides spp. from leishmaniasis-affected areas in Thailand. Detecting bacterial endosymbionts co-occurring with Leishmania spp. in Culicoides biting midges may suggest a potential, but unconfirmed, antagonistic effect on Leishmania. This provides preliminary data that could inform the development of new vector control strategies for diseases transmitted by Culicoides spp. in Thailand.