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Abstract Background Leishmaniasis is an emerging vector-borne disease that occurs in Thailand. Although Leishmania (Mundinia) parasites, the causative agents of the disease have been identified, the vectors of the disease remain unidentified. In the present study, we collected sand flies from three caves located in endemic areas of leishmaniasis, including Lampang and Chiang Rai in northern Thailand, and Songkhla in southern Thailand. Methods Female sand flies were identified on the basis of morphological characteristics and confirmed by cytochrome c oxidase subunit I (COI) sequencing. Sand fly DNA samples were screened for Leishmania, Trypanosoma, and Bartonella DNA by polymerase chain reaction (PCR) on the basis of the ITS1 region of the ribosomal RNA (rRNA), SSU rRNA, and gltA genes, followed by phylogenetic relationships and haplotype diversity analysis. Results A total of 557 sand flies were identified, comprising four genera (Sergentomyia, Phlebotomus, Grassomyia, and Idiophlebotomus) and 11 species. Molecular detection of pathogens demonstrated that Leishmania DNA was not detected. However, Trypanosoma DNA was detected in 11 samples of Phlebotomus mascomai from Lampang (7 for T. noyesi), Se. anodontis from Chiang Rai (1 each for T. noyesi and Trypanosoma sp.), and Se. khawi from Songkhla (2 for Trypanosoma sp.). Bartonella DNA was detected in 16 samples of Se. anodontis and Se. barraudi s.l. from Chiang Rai, Se. anodontis from Lampang, and Se. khawi from Songkhla. The novel Bartonella sp. detected in Thai sand flies was phylogenetically related to Bartonella sp. from bats. Genetic diversity analysis showed high haplotype diversity in both Trypanosoma parasites and Bartonella bacteria. Conclusions The data from the present study indicate that phlebotomine sand flies could be potential vectors of zoonotic diseases caused by Trypanosoma sp. and Bartonella sp. To our knowledge, this is the first report of the natural infection of Bartonella associated with bats in Thailand, and the presence of T. noyesi and amphibian trypanosomes. However, further investigation is required to elucidate and enhance the understanding of potential vectors and transmission dynamics of pathogens in Thailand, particularly with regard to different seasonality, habitats, and host ranges. Graphical abstract

Abstract Background Sand flies, belonging to the Psychodidae family, represent small, hairy insects that serve as significant vectors in various important medical and veterinary diseases. Despite being recognized by the World Health Organization as an endemic area for leishmaniasis, Southeast Asia lacks comprehensive information on the species composition and biology of sand flies. To address this, the current study aimed to survey sand fly biodiversity. Methods Sand flies from six provinces in Southern Vietnam were collected using CDC light traps. Sand flies were subsequently identified morphologically and confirmed molecularly using mitochondrial cytochrome oxidase c subunit I (COI) and cytochrome b (cytb) sequences. BLASTN searches were conducted, and the species identity of sand flies was further confirmed through a Barcode of Life Database (BOLD) search utilizing COI sequences. Subsequently, nucleotide sequences were subjected to a panel of analyses including intraspecific variation, phylogenetic relationships and haplotype network. The average densities of collected sand flies (sand flies/trap/night) and species richness were also recorded. Results A total of 753 sand flies were collected. After excluding damaged specimens, six sand fly species, namely Phlebotomus stantoni, Sergentomyia khawi, Se. silvatica, Se. barraudi, Se. bailyi and Grassomyia indica, were identified. All conspecific sand fly sequences, including Ph. stantoni, Se. barraudi, Gr. indica, Se. bailyi, Se. khawi and Se. silvatica, clustered with their reference sequences, corroborating the results of morphology-based identification, BLASTN analysis and BOLD search. For intraspecific variation of sand flies obtained from the current study, COI diversity indices were consistently higher than those of cytb. Conclusions This study provides the first updates on morphological and molecular characterization of sand flies in Southern Vietnam. This acquired knowledge on sand fly species composition is essential for controlling sand fly-borne diseases in this potentially endemic region. Graphical Abstract

Abstract Background Clinical cases of leishmaniasis caused by Leishmania (Mundinia) parasites have been increasingly reported in Southeast Asia, particularly Thailand. Recent evidence has shown that Leishmania (Mundinia) parasites successfully developed into infective metacyclic promastigotes in Culicoides biting midges, strongly supporting their putative role in disease transmission. However, Culicoides diversity, host preference, and Leishmania prevalence in endemic areas remain largely unknown. Methods We investigated the seasonal dynamics, infection prevalence, and blood meal identification of Culicoides collected from the emerging focus of visceral leishmaniasis in Lampang Province, Northern Thailand, during 2021–2023. Midge samples were molecularly screened for Leishmania using SSU rRNA-qPCR and ITS1-PCR, followed by Sanger plasmid sequencing, and parasite haplotype diversity was analyzed. Host blood meal origins were comparatively identified using host-specific Cytb-PCRs and a nanopore-based metabarcoding approach. Results A total of 501 parous and gravid females and 46 blood-engorged ones belonging to at least 17 species of five subgenera (Remmia, Trithecoides, Avaritia, Hoffmania, and Meijerehelea) and two species groups (Shortti and Calvipalpis) were collected with temporal differences in abundance. Leishmania was detected by SSU rRNA-qPCR in 31 samples of at least 11 midge species, consisting of Culicoides oxystoma, C. guttifer, C. orientalis, C. mahasarakhamense, C (Trithecoides) spp., C. innoxius, C. shortti, C. arakawae, C. sumatrae, C. actoni, and C. fulvus, with the overall infection prevalence of 5.7%. The latter six species represent the new records as putative leishmaniasis vectors in Northern Thailand. The ITS1-PCR and plasmid sequencing revealed that Leishmania martiniquensis was predominantly identified in all qPCR-positive species, whereas L. orientalis was identified only in three C. oxystoma samples. The most dominant haplotype of L. martiniquensis in Thailand was genetically intermixed with those from other geographical regions, confirming its globalization. Neutrality test statistics were also significantly negative on regional and country-wide scales, suggesting rapid population expansion or selective sweeps. Nanopore-based blood meal analysis revealed that most Culicoides species are mammalophilic, with peridomestic and wild mammals (cow, pig, deer, and goat-like species) and humans as hosts, while C. guttifer and C. mahasarakhamense fed preferentially on chickens. Conclusions This study revealed seasonal dynamics and sympatric circulation of L. martiniquensis and L. orientalis in different species of Culicoides. Evidence of human blood feeding was also demonstrated, implicating Culicoides as putative vectors of human leishmaniasis in endemic areas. Further research is therefore urgently needed to develop vector control strategies and assess the infection status of their reservoir hosts to effectively minimize disease transmission. Graphical Abstract

Abstract Background Sand flies serve as crucial vectors in various medical and veterinary diseases. Sand fly-borne diseases pose a significant public health burden globally, as the causative agents can infect a diverse range of hosts, leading to severe consequences such as leishmaniasis and sand fly fever. Additionally, the widespread use of insecticides for agricultural purposes and mosquito control is not specifically targeted at sand flies, potentially leading to resistance development. We investigated sand fly species, their potential role as vectors of various parasitic agents, and insecticide resistance in the endemic regions of Natawi and Sadao districts in Songkhla, Thailand. Methods Sand flies were collected using CDC light traps. The collected sand flies were then identified to species level using molecular techniques. Subsequent analyses included the detection of pathogens and the identification of pyrethroid resistance mutations within the voltage-sensitive sodium channel (Vgsc) domain IIS6 gene, followed by sequence analysis. Results The study identified nine sand fly species belonging to the genera Phlebotomus and Sergentomyia. The DNA of Sergentomyia khawi was the only species found to test positive for one sample of Leishmania orientalis in Sadao district. This finding represents the first detection of L. orientalis in Thailand. Moreover, three samples of Leishmania martiniquensis and four samples of Trypanosoma sp. were found in the Natawi district. No I1011M, L1014F/S, V1016G, or F1020S mutations were detected in Vgsc gene. Conclusions The results of this study provide valuable information on sand fly species and the continuous circulation of Leishmania spp. and Trypanosoma spp. in Songkhla, southern Thailand. Moreover, the development of geo-spatial information on vectors, parasites, and insecticide resistance in sand flies has the potential to provide well-informed risk assessments and evidence-based guidance for targeted vector control in Thailand. These results can serve as a foundation for integrating the One Health approach, which is crucial for disease control, considering the diverse ecological interactions among human and/or animal reservoir hosts, parasites, and sand fly vectors. Graphic Abstract

Abstract Chikungunya virus (CHIKV) poses a significant global health threat, re-emerging as a mosquito-transmitted pathogen that caused high fever, rash, and severe arthralgia. In Thailand, a notable CHIKV outbreak in 2019–2020 affected approximately 20,000 cases across 60 provinces, underscoring the need for effective mosquito control protocols. Previous studies have highlighted the role of midgut bacteria in the interaction between mosquito vectors and pathogen infections, demonstrating their ability to protect the insect from invading pathogens. However, research on the midgut bacteria of Aedes (Ae.) aegypti, the primary vector for CHIKV in Thailand remains limited. This study aims to characterize the bacterial communities in laboratory strains of Ae. aegypti, both infected and non-infected with CHIKV. Female mosquitoes from a laboratory strain of Ae. aegypti were exposed to a CHIKV-infected blood meal through membrane feeding, while the control group received a non-infected blood meal. At 7 days post-infection (dpi), mosquito midguts were dissected for 16S rRNA gene sequencing to identify midgut bacteria, and CHIKV presence was confirmed by E1-nested RT-PCR using mosquito carcasses. The study aimed to compare the bacterial communities between CHIKV-infected and non-infected groups. The analysis included 12 midgut bacterial samples, divided into three groups: CHIKV-infected (exposed and infected), non-infected (exposed but not infected), and non-exposed (negative control). Alpha diversity indices and Bray–Curtis dissimilarity matrix revealed significant differences in bacterial profiles among the three groups. The infected group exhibited an increased abundance of bacteria genus Gluconobacter, while Asaia was prevalent in both non-infected and negative control groups. Chryseobacterium was prominent in the negative control group. These findings highlight potential alterations in the distribution and abundance of gut microbiomes in response to CHIKV infection status. This study provides valuable insights into the dynamic relationship between midgut bacteria and CHIKV, underscoring the potential for alterations in bacterial composition depending on infection status. Understanding the relationships between mosquitoes and their microbiota holds promise for developing new methods and tools to enhance existing strategies for disease prevention and control. This research advances our understanding of the circulating bacterial composition, opening possibilities for new approaches in combating mosquito-borne diseases.

Abstract Zika virus (ZIKV), a mosquito-borne flavivirus, has been continually emerging and re-emerging since 2010, with sporadic cases reported annually in Thailand, peaking at over 1000 confirmed positive cases in 2016. Leveraging high-throughput sequencing technologies, specifically whole genome sequencing (WGS), has facilitated rapid pathogen genome sequencing. In this study, we used multiplex amplicon sequencing on the Illumina Miseq instrument to describe ZIKV WGS. Six ZIKV WGS were derived from three samples of field-caught Culex quinquefasciatus mosquitoes (two males and one female) and three urine samples collected from patients in three different provinces of Thailand. Additionally, successful isolation of a ZIKV isolate occurred from a female Cx. quinquefasciatus. The WGS analysis revealed a correlation between the 2020 outbreak and the acquisition of five amino acid changes in the Asian lineage ZIKV strains from Thailand (2006), Cambodia (2010 and 2019), and the Philippines (2012). These changes, including C-T106A, prM-V1A, E-V473M, NS1-A188V, and NS5-M872V, were identified in all seven WGS, previously linked to significantly higher mortality rates. Furthermore, phylogenetic analysis indicated that the seven ZIKV sequences belonged to the Asian lineage. Notably, the genomic region of the E gene showed the highest nucleotide diversity (0.7–1.3%). This data holds significance in informing the development of molecular tools that enhance our understanding of virus patterns and evolution. Moreover, it may identify targets for improved methods to prevent and control future ZIKV outbreaks.

Culicoides biting midges were collected using UV light traps from nine livestock farms in Kanchanaburi Province, Thailand. Collections were made one night per month from June 2020 to May 2021 to determine the seasonal changes and spatial distribution of the Culicoides assemblage. The influence of four environmental factors (temperature, rainfall, humidity, and wind speed), types of animals in each shed (cattle, pigs, and avians), and neighboring animals (those in the other sheds and their proximity) were assessed. A subsample of 130,670 out of a total of 224,153 specimens collected was identified and counted. The collections were predominantly female (76.9%), though males were also commonly collected (23.1%). The dominant species included C. peregrinus (97,098 individuals; 43.0%), C. oxystoma (55,579; 24.6%), C. arakawae (45,996; 20.4%), and C. imicola (15,703; 7.0%), while other species accounted for the remaining 9777 individuals (4.4%). Trap captures were strongly influenced by seasons and animal sheds. Cattle exhibited the greatest effect on the community, likely due to their large biomass. Humidity and temperature had a positive effect on trap captures, whereas wind speed exhibited a negative effect. Cattle positively influenced all major species, except for C. arakawae, which showed a positive association with avians. A “neighboring effect” was also observed. Additionally, the lowest dispersal ability of C. arakawae was suggested.

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.

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.

BackgroundIn South-East Asia, Thailand is the country with the highest number of human autochthonous cases of leishmaniases mostly due to Leishmania martiniquensis. Their transmission remains unresolved to date even though sand flies are known vectors of leishmaniases. As such, we focused a study on the sand fly fauna of a cave in Thailand to explore the biodiversity of potential Leishmania vectors.Main resultsWe carried out an inventory in Pha Tong cave. We caught and identified 570 Phlebotomine sand flies (452 females and 118 males) and identified 14 species belonging to the genera Phlebotomus, Idiophlebotomus, Chinius, Sergentomyia and Grassomyia. Among these 14 species, two could not be related to known sand fly species. Herein, we propose the description of two new sand fly species, previously unknown to science. The first new species, Phlebotomus shadenae n. sp. is a sand fly of the subgenus Anaphlebotomus. It is morphologically close to Ph. stantoni, a species widely distributed throughout Southeast Asia. However, it differs by the length of the genital filaments in males or by the length of the ducts of the spermathecae in females as well as the high divergence of cytochrome b sequences. Additionally, we revised the systematics of the subgenus Anaphlebotomus and reinstated, by examination of its holotype, the validity of Ph. maynei, an Indian wrongly considered as a synonym of Ph. stantoni in the past. The second new species, Sergentomyia maiae n. sp., differs from a species in the same group, Se. barraudi, by an original cibarial double row of vertical teeth as well as by molecular data.ConclusionsWe propose the description of two new sand fly species for Science with morphological and molecular evidence. Ph. shadenae n. sp. was also found to be distributed in the south of Thailand and in Laos. Future studies need to determine whether these two species can play a role as vectors of Leishmania parasites, Trypanosomatids or Phlebovirus. Most of the species caught in the present study are strictly cavernicolous except Grassomyia sp. and a few Sergentomyia.

The prevalence of autochthonous leishmaniasis in Thailand is increasing but the natural vectors that are responsible for transmission remain unknown. Experimental in vivo infections in Culicoides spp. with Leishmania (Mundinia) martiniquensis and Leishmania (Mundinia) orientalis, the major causative pathogens in Thailand, have demonstrated that biting midges can act as competent vectors. Therefore, the isolation and detection of Leishmania and other trypanosomatids were performed in biting midges collected at a field site in an endemic area of leishmaniasis in Tha Ruea and a mixed farm of chickens, goats, and cattle in Khuan Phang, Nakhon Si Thammarat province, southern Thailand. Results showed that Culicoides peregrinus was the abundant species (>84%) found in both locations and only cow blood DNA was detected in engorged females. Microscopic examination revealed various forms of Leishmania promastigotes in the foregut of several C. peregrinus in the absence of bloodmeal remnants, indicating established infections. Molecular identification using ITS1 and 3’UTR HSP70 type I markers showed that the Leishmania parasites found in the midges were L. martiniquensis. The infection rate of L. martiniquensis in the collected flies was 2% in Tha Ruea and 6% in Khuan Phang, but no L. orientalis DNA or parasites were found. Additionally, organisms from two different clades of Crithidia, both possibly new species, were identified using SSU rRNA and gGAPDH genes. Choanomastigotes and promastigotes of both Crithidia spp. were observed in the hindgut of the dissected C. peregrinus. Interestingly, midges infected with both L. martiniquensis and Crithidia were found. Moreover, four strains of Crithidia from one of the clades were successfully isolated into culture. These parasites could grow at 37°C in the culture and infect BALB/c mice macrophages but no multiplication was observed, suggesting they are thermotolerant monoxenous trypanosomatids similar to Cr. thermophila. These findings provide the first evidence of natural infection of L. martiniquensis in C. peregrinus supporting it as a potential vector of L. martiniquensis.

Amphotericin B (AmpB) deoxycholate is the available first-line drug used to treat visceral leishmaniasis caused by Leishmania (Mundinia) martiniquensis, however, some cases of AmpB treatment failure have been reported in Thailand. Resistance to drugs is known to affect parasite fitness with a potential impact on parasite transmission but still little is known about the effect of resistance to drugs on L. martiniquensis. Here we aimed to gain insight into the fitness changes occurring after treatment failure or in vitro-induced resistance to AmpB. L. martiniquensis parasites isolated from a patient before (LSCM1) and after relapse (LSCM1-6) were compared for in vitro and in vivo fitness changes together with an in vitro induced AmpB-resistant parasite generated from LSCM1 parasites (AmpBRP2i). Results revealed increased metacyclogenesis of the AmpBPR2i and LSCM1-6 strains (AmpB-resistant strains) compared to the LSCM1 strain and increased fitness with respect to growth and infectivity. The LSCM1-6 and AmpBRP2i strains were present in mice for longer periods compared to the LSCM1 strain, but no clinical signs of the disease were observed. These results suggest that the AmpB-resistant parasites could be more efficiently transmitted to humans and maintained in asymptomatic hosts longer than the susceptible strain. The asymptomatic hosts therefore may represent “reservoirs” for the resistant parasites enhancing transmission. The results in this study advocate an urgent need to search and monitor for AmpB-resistant L. martiniquensis in patients with relapsing leishmaniasis and in asymptomatic patients, especially, in HIV/Leishmania coinfected patients.

Filariasis is classified as a vector-borne zoonotic disease caused by several filarial nematodes. The disease is widely distributed in tropical and subtropical regions. Understanding the relationship between mosquito vectors, filarial parasites, and vertebrate hosts is therefore essential for determining the probability of disease transmission and, correspondingly, developing effective strategies for prevention and control of diseases. In this study, we aimed to investigate the infection of zoonotic filarial nematodes in field-caught mosquitoes, observe the potential vectors of filaria parasites in Thailand using a molecular-based survey, conduct a study of host-parasite relationship, and propose possible coevolution of the parasites and their hosts. Mosquitoes were collected around cattle farms in Bangkok, Nakhon Si Thammarat, Ratchaburi, and Lampang provinces from May to December 2021 using a CDC Backpack aspirator for 20–30 minutes in each area (intra-, peri-, and wild environment). All mosquitoes were identified and morphologically dissected to demonstrate the live larvae of the filarial nematode. Furthermore, all samples were tested for filarial infections using PCR and sequencing. A total of 1,273 adult female mosquitoes consisted of five species: 37.78% Culex quinquefasciatus, 22.47% Armigeres subalbatus, 4.71% Cx. tritaeniorhynchus, 19.72% Anopheles peditaeniatus, and 15.32% An. dirus. Larvae of Brugia pahangi and Setaria labiatopapillosa were found in Ar. subalbatus and An. dirus mosquitoes, respectively. All mosquito samples were processed by PCR of ITS1 and COXI genes for filaria nematode species identification. Both genes showed that B. pahangi was found in four mosquitoes of Ar. subalbatus from Nakhon Si Thammarat, S. digitata was detected in three samples of An. peditaeniatus from Lampang, and S. labiatopapillosa was detected in one of An. dirus from Ratchaburi. However, filarial nematodes were not found in all Culex species. This study infers that this is the first data regarding the circulation of Setaria parasites in Anopheles spp. from Thailand. The phylogenetic trees of the hosts and parasites are congruent. Moreover, the data could be used to develop more effective prevention and control strategies for zoonotic filarial nematodes before they spread in Thailand.

Abstract Background The distribution of phlebotomine sand flies is changing rapidly due to climate change. This issue has implications for the epidemiology of sand fly-borne diseases, especially sand fly-associated viruses. Few studies concerning sand fly-associated viruses have been conducted in Thailand. Therefore, this study aimed to perform a molecular survey of groups of pathogenic RNA viruses belonging to the Orbivirus, Phlebovirus, and Flavivirus genera and family Rhabdoviridae in sand fly samples collected from southern Thailand. Methods Sand flies were collected at two locations in Trang and Songkhla provinces of southern Thailand, and individual sand fly samples were processed for species identification and virus detection. The Orbivirus, Phlebovirus, and Flavivirus genera and family Rhabdoviridae molecular determination was performed by RT-PCR, and positive samples were identified by cloning and sequencing, cell culture inoculation, and phylogenetic analysis. Results The results presented in this study were based on the analysis of a total of 331 female sand flies. This molecular study revealed evidence of Rhabdoviridae family virus presence in Phlebotomus papatasi (3/331, 0.9%). The findings demonstrated a new cluster of rhabdovirus that was closely related to Bactrocera dorsalis sigmavirus strain BDSV.abc5 and the lineages of insect-specific Rhabdoviridae. In addition, the Bayesian tree suggested that the common ancestor of this group was the dimarhabdovirus clade. It was assumed that the virus may have switched hosts during its evolution. However, the detection of Orbivirus, Phlebovirus, and Flavivirus genera using specific primers for RT-PCR was negative in the collected sand flies. Conclusions There is limited knowledge on the genetic diversity and ecology of Rhabdoviridae in Thailand. This is the first data regarding the circulation of Rhabdoviridae in Ph. papatasi from Thailand. We found a new cluster of rhabdoviruses that was close to the new B. dorsalis sigmavirus. It is possible that there is a great deal of diversity in this family yet to be discovered, and a more extensive survey for new rhabdoviruses may uncover viruses from a wide diversity of host taxa and broaden our understanding of the relationships among the Rhabdoviridae. Graphical abstract

Abstract Background Leishmaniasis is a human and animal disease caused by parasites of the genus Leishmania, which is now divided into four subgenera, Leishmania, Viannia, Sauroleishmania and Mundinia. Subgenus Mundinia, established in 2016, is geographically widely dispersed, its distribution covers all continents, except Antarctica. It consists of 5 species; L. enriettii and L. macropodum are parasites of wild mammals while L. martiniquensis, L. orientalis and an unnamed Leishmania sp. from Ghana are infectious to humans. There is very little information on natural reservoir hosts and vectors for any Mundinia species. Methods Experimental infections of guinea pigs with all five Mundinia species were performed. Animals were injected intradermally with 107 culture-derived promastigotes into both ear pinnae. The courses of infections were monitored weekly; xenodiagnoses were performed at weeks 4 and 8 post-infection using Lutzomyia migonei. The distribution of parasites in different tissues was determined post-mortem by conventional PCR. Results No significant differences in weight were observed between infected animals and the control group. Animals infected with L. enriettii developed temporary lesions at the site of inoculation and were infectious to Lu. migonei in xenodiagnoses. Animals infected with L. martiniquensis and L. orientalis developed temporary erythema and dry lesions at the site of inoculation, respectively, but were not infectious to sand flies. Guinea pigs infected by L. macropodum and Leishmania sp. from Ghana showed no signs of infection during experiments, were not infectious to sand flies and leishmanial DNA was not detected in their tissue samples at the end of experiments at week 12 post-inoculation. Conclusions According to our results, guinea pigs are not an appropriate model organism for studying Mundinia species other than L. enriettii. We suggest that for better understanding of L. (Mundinia) biology it is necessary to focus on other model organisms.

Emerging infectious disease has become the center of attention since the outbreak of COVID-19. For the coronavirus, bats are suspected to be the origin of the pandemic. Consequently, the spotlight has fallen on zoonotic diseases, and the focus now expands to organisms other than viruses. Microsporidia is a single-cell organism that can infect a wide range of hosts such as insects, mammals, and humans. Its pathogenicity differs among species, and host immunological status plays an important role in infectivity and disease severity. Disseminated disease from microsporidiosis can be fatal, especially among patients with a defective immune system. Recently, there were two Trachipleistophora hominis, a microsporidia species which can survive in insects, case reports in Thailand, one patient had disseminated microsporidiosis. This review gathered data of disseminated microsporidiosis and T. hominis infections in humans covering the biological and clinical aspects. There was a total of 22 cases of disseminated microsporidiosis reports worldwide. Ten microsporidia species were identified. Maximum likelihood tree results showed some possible correlations with zoonotic transmissions. For T. hominis, there are currently eight case reports in humans, seven of which had Human Immunodeficiency Virus (HIV) infection. It is observed that risks are higher for the immunocompromised to acquire such infections, however, future studies should look into the entire life cycle, to identify the route of transmission and establish preventive measures, especially among the high-risk groups.

Morphological characteristics of eggshells are important in sand fly ootaxonomy. In this study, eggshells from Phlebotomus stantoni Newstead, Sergentomyia khawi (Raynal), and Grassomyia indica (Theodor) sand flies collected in Chiang Mai province, Thailand were examined and characterized using light microscopy (LM) and scanning electron microscopy (SEM). Then, eggshell morphology of these three species was described for the first time. Each gravid female was forced to lay eggs by decapitation and the eggs were collected for SEM analysis. Egg laying females were identified by morphological examination and molecular typing using cytochrome b (Cytb) as a molecular marker. The chorionic sculpturing of Ph. stantoni eggs combines two patterns on the same egg: unconnected parallel ridges and reticular patterns. Sergentomyia khawi and Gr. indica have similar chorionic polygonal patterns, but their exochorionic morphology and aeropylar area are different. Results indicate that eggshell morphological characteristics such as chorionic pattern, exochorionic morphology, inter-ridge/boundary area, aeropylar area (including the number of aeropyles) and basal layer, can be useful to develop morphological identification keys of eggs. These can serve as an additional tool to distinguish species of sand flies. In addition, the chorionic sculpturing of the eggs of the three species of sand flies observed by LM is useful for species identification in gravid females with spermathecae obscured by eggs.

Leishmania (Mundinia) martiniquensis is responsible for visceral leishmaniasis in patients with no known underlying immunodeficiency, and visceral or disseminated cutaneous leishmaniasis in HIV-infected patients. The available anti-Leishmania drugs for treatment have limitations such as high toxicity and variable efficacy. To improve the therapeutic index of anti-Leishmania drugs, the search for a new drug or a new natural compound in combination therapy instead of using monotherapy to reduce drug side effect and have high efficacy is required. In this study, anti-Leishmania activity of 8-hydroxyquinoline (8HQN) and its synergistic effect with amphotericin B (AmB) against L. martiniquensis were evaluated in vitro for the first time. These results showed that 8HQN presented anti-Leishmania activity against L. martiniquensis with IC50 1.60 ± 0.28 and 1.56 ± 0.02 µg/mL for promastigotes and intracellular amastigotes, respectively. The selectivity index (SI) value of 8HQN was 79.84 for promastigotes and 82.40 for intracellular amastigotes, which highlight promising results for the use of 8HQN in the treatment of L. martiniquensis-infected host cells. Interestingly, four combinations of 8HQN and AmB provided synergistic effects for intracellular amastigotes and showed no toxic effects to host cells. These results provided information of using a combination therapy in treating this Leishmania species leads to further development of therapy and can be considered as an alternative treatment for leishmaniasis.

Leishmaniasis is a parasitic disease caused by Leishmania spp. with worldwide distribution. Autochthonous leishmaniasis has been reported to result from the infection by Leishmania martiniquensis in Thailand. This species was isolated in culture and subjected to high-throughput whole-genome sequencing. A total of 30.8 Mb in 36 chromosomes of the whole genome was assembled, annotated, and characterized. The L. martiniquensis under study was shown to segregate into the same clade and thus closely related to the previously identified L. martiniquensis (LU_Lmar_1.0), as determined by phylogenetic analysis of their genomic sequences along with those of representative kinetoplastid species. The total number of open reading frames genomewide predicts 8,209 protein-coding genes, of which 359 are putative virulence factors, including two previously known, e.g., cysteine proteinase C and superoxide dismutase B1. The results obtained from this study will be useful for further annotation and comparison with other Leishmania martiniquensis in the future.

Since 1996, autochthonous cases of emerging leishmaniasis caused by Leishmania (Mundinia) martiniquensis and Leishmania (Mundinia) orientalis have been more frequently reported, especially in the northern and southern parts of Thailand. However, the accurate identification of their natural vectors and reservoirs remains unconfirmed. Previous studies have suggested that these emerging parasites might be transmitted by other non-phlebotomine vectors. Herein, we speculated that Culicoides biting midges might act as the competent vectors responsible for autochthonous leishmaniasis in southern Thailand. In this research, 187 non-engorged, parous and gravid Culicoides females and 47 blood-engorged ones were trapped from the residences of two recently diagnosed visceral leishmaniasis patients in Sadao District and the unaffected site in Rattaphum District, Songkhla Province, southern Thailand. Species diversity and abundance of biting midges varied among the trapping sites. Using ITS1-PCR and BLASTn analysis, L. martiniquensis was predominantly detected in several Culicoides species, including C. peregrinus, C. oxystoma, C. mahasarakhamense, and C. huffi from the vicinity of patients’ houses; and in C. fordae and C. fulvus from the unaffected site. L. orientalis was also co-circulated in C. peregrinus and C. oxystoma caught near the second patient’s house. Additionally, Crithidia sp. were also detected using SSU rRNA-PCR across Culicoides spp. Host blood meal analysis of eight different Culicoides species from the unaffected site also revealed that all trapped Culicoides had fed on cows and goats, indicating the possible role of these mammalian species as reservoir hosts. Essentially, this study is the first entomological investigation, revealing the co-circulation of emerging trypanosomatids among several species of Culicoides biting midges and strongly supporting the potential role of this insect group as the main vectors responsible for the epidemiology of autochthonous leishmaniasis in southern Thailand.

Five hundred and fifty-nine female biting midges were collected, and seventeen species in six subgenera (Avaritia, Haemophoructus, Hoffmania, Meijerehelea, Remmia, and Trithecoides) and two groups (Clavipalpis and Shortti) were identified. The dominant Culicoides species was C. peregrinus (30.94%), followed by C. subgenus Trithecoides. From blood meal analysis of engorged biting midges, they were found to feed on cows, dogs, pigs, and avians. The majority of blood preferences of biting midges (68%; 49/72) displayed a mixed pattern of host blood DNA (cow and avian). The overall non-engorged biting midge field infectivity rate was 1.44 % (7/487). We detected Leucocytozoon sp. in three Culicoides specimens, one from each species: C. fulvus, C. oxystoma, and C. subgenus Trithecoides. Crithidia sp. was found in two C. peregrinus specimens, and Trypanosoma sp. and P. juxtanucleare were separately found in two C. guttifer. More consideration should be paid to the capacity of biting midges to transmit pathogens such as avian haemosporidian and trypanosomatid parasites. To demonstrate that these biting midges are natural vectors of trypanosomatid parasites, additional research must be conducted with a greater number of biting midges in other endemic regions.

Leishmania parasites, causative agents of leishmaniasis, are currently divided into four subgenera: Leishmania, Viannia, Sauroleishmania and Mundinia. The recently established subgenus Mundinia has a wide geographical distribution and contains five species, three of which have the potential to infect and cause disease in humans. While the other Leishmania subgenera are transmitted exclusively by phlebotomine sand flies (Diptera: Psychodidae), natural vectors of Mundinia remain uncertain. This study investigates the potential of sand flies and biting midges of the genus Culicoides (Diptera: Ceratopogonidae) to transmit Leishmania parasites of the subgenus Mundinia. Sand flies (Phlebotomus argentipes, P. duboscqi and Lutzomyia migonei) and Culicoides biting midges (Culicoides sonorensis) were exposed to five Mundinia species through a chicken skin membrane and dissected at specific time intervals post bloodmeal. Potentially infected insects were also allowed to feed on ear pinnae of anaesthetized BALB/c mice and the presence of Leishmania DNA was subsequently confirmed in the mice using polymerase chain reaction analyses. In C. sonorensis, all Mundinia species tested were able to establish infection at a high rate, successfully colonize the stomodeal valve and produce a higher proportion of metacyclic forms than in sand flies. Subsequently, three parasite species, L. martiniquensis, L. orientalis and L. sp. from Ghana, were transmitted to the host mouse ear by C. sonorensis bite. In contrast, transmission experiments entirely failed with P. argentipes, although colonisation of the stomodeal valve was observed for L. orientalis and L. martiniquensis and metacyclic forms of L. orientalis were recorded. This laboratory-based transmission of Mundinia species highlights that Culicoides are potential vectors of members of this ancestral subgenus of Leishmania and we suggest further studies in endemic areas to confirm their role in the lifecycles of neglected pathogens.

BackgroundPediculus humanus capitis or head louse is an obligate ectoparasite and its infestation remains a major public health issue worldwide. Molecular analysis divides head lice into six clades and intra-clade genetic differences have been identified. Several hypotheses have been formulated to elucidate the discrepancies of the variety of head lice among different regions of the world. It is currently concluded that head lice distribution might be associated with human migration history. This study aims to investigate genetic data of human head lice in Thailand. We believe that the analysis could help establish the correlation between local and global head lice populations.MethodWe investigated mitochondrial cytochrome b (cytb) gene of the collected 214 head lice to evaluate genetic diversity from 15 provinces among 6 regions of Thailand. The head lice genes were added to the global pool for the phylogenetic tree, Bayesian tree, Skyline plot, and median joining network construction. The biodiversity, neutrality tests, and population genetic differentiation among the 6 Thailand geographic regions were analyzed by DNAsp version 6.ResultsThe phylogenetic tree analysis of 214 collected head lice are of clade A and clade C accounting for roughly 65% and 35% respectively. The Bayesian tree revealed a correlation of clade diversification and ancient human dispersal timeline. In Thailand, clade A is widespread in the country. Clade C is confined to only the Central, Southern, and Northeastern regions. We identified 50 novel haplotypes. Statistical analysis showed congruent results between genetic differentiation and population migration especially with South Asia.ConclusionsPediculosis remains problematic among children in the rural areas in Thailand. Cytb gene analysis of human head lice illustrated clade distribution and intra-clade diversity of different areas. Our study reported novel haplotypes of head lice in Thailand. Moreover, the statistic calculation provided a better understanding of their relationship with human, as an obligate human parasite and might help provide a better insight into the history of human population migration. Determination of the correlation between phylogenetic data and pediculicide resistance gene as well as residing bacteria are of interest for future studies.

Following an outbreak of chikungunya virus (CHIKV) infections in Thailand in 2019, numerous cases of CHIKV infection have been diagnosed in Bangkok, the capital of the country. In our previous investigation of the vectors for disease transmission, we found natural infection of CHIKV in both male and female Aedes aegypti mosquitoes collected from the outbreak areas in Bangkok. Some reports mentioned the detection of CHIKV in Culex mosquitoes. In Thailand, the Culex quinquefasciatus Say mosquito is a common species found in urban and rural settings that coexists with Ae. aegypti. However, the role of Cx. quinquefasciatus mosquitoes in the spread of the Indian Ocean Lineage (IOL) of CHIKV in Thailand has never been investigated. In this study, Cx. quinquefasciatus were collected (16 males and 27 females) from an outbreak area in Bangkok. Eight of the 27 in field-caught female Cx. quinquefasciatus were positive for IOL CHIKV RNA, and 99-100% identity and full 100% coverage of sequences similar to CHIKV isolated from female Ae. aegypti in Bangkok, Thailand, whereas viral RNA was not detected in male samples using nested-RT-PCR. To determine whether CHIKV is able to replicate in Cx. quinquefasciatus, the laboratory strain of Cx. quinquefasciatus was allowed to feed on blood containing IOL CHIKV isolated from patient serum. The nested-RT-PCR, virus isolation, and immunofluorescence assay (IFA) were performed for CHIKV detection and replication. The results showed that CHIKV RNA was detected in Cx. quinquefasciatus until day 4 post infection. CHIKV did not produce any remarkable signs of infection, dissemination, or transmission in Cx. quinquefasciatus, and cytopathic effect (CPE) was not observed in C6/36 cells when infected with supernatant obtained from Cx. quinquefasciatus at days 7, 10, 14, and 21 post infection when compared to Ae. aegypti. The data from this study infer that CHIKV may be detected in Cx. quinquefasciatus but that the mosquito is not able to transmit CHIKV in Thailand.

Human head lice are blood-sucking insects causing an infestation in humans called pediculosis capitis. The infestation is more prevalent in the school-aged population. Scalp itching, a common presenting symptom, results in scratching and sleep disturbance. The condition can lead to social stigmatization which can lead to loss of self-esteem. Currently, the mainstay of treatment for pediculosis is chemical insecticides such as permethrin. The extended use of permethrin worldwide leads to growing pediculicide resistance. The aim of this study is to demonstrate the presence of the knockdown resistance (kdr) mutation in head lice populations from six different localities of Thailand. A total of 260 head lice samples in this study were collected from 15 provinces in the 6 regions of Thailand. Polymerase chain reaction (PCR) was used to amplify the α subunit of voltage-sensitive sodium channel (VSSC) gene, kdr mutation (C→T substitution). Restriction fragment length polymorphism (RFLP) patterns and sequencing were used to identify the kdr T917I mutation and demonstrated three genotypic forms including homozygous susceptible (SS), heterozygous genotype (RS), and homozygous resistant (RR). Of 260 samples from this study, 156 (60.00%) were SS, 58 (22.31%) were RS, and 46 (17.69%) were RR. The overall frequency of the kdr T917I mutation was 0.31. Genotypes frequencies determination using the exact test of Hardy-Weinberg equilibrium found that northern, central, northeastern, southern, and western region of Thailand differed from expectation. The five aforementioned localities had positive inbreeding coefficient value (Fis > 0) which indicated an excess of homozygotes. The nucleotide and amino acid sequences of RS and RR showed T917I and L920F point mutations. In conclusion, this is the first study detecting permethrin resistance among human head lice from Thailand. PCR-RFLP is an easy technique to demonstrate the kdr mutation in head louse. The data obtained from this study would increase awareness of increasing of the kdr mutation in head louse in Thailand.

Autochthonous leishmaniasis caused by Leishmania martiniquensis cases in Thailand have dramatically increased in the recent years. L. martiniquensis infection primarily occurs in immunocompromised patients, especially AIDS patients. In Thailand, amphotericin B is the only drug available for leishmaniasis treatment, and some patients relapse after amphotericin B therapy. Moreover, the efficacy of anti-leishmanial drugs against L. martiniquensis has not been evaluated to date. In this study, we determined the efficacy of various anti-leishmanial drugs against the promastigote and intracellular amastigote stages of L. martiniquensis using a colorimetric assay. Two strains (CU1 and CU1R1) were isolated from leishmaniasis HIV co-infected patient from Songkhla province, southern Thailand. The CU1 strain was isolated from the patient in 2011, and CU1R1 was isolated from the same patient in 2013, when he was diagnosed as relapse leishmaniasis. The third strain (LSCM1) used in this study has been isolated from immunocompetent patient from Lamphun province, northern Thailand. All strains were identified as L. martiniquensis by sequencing of ribosomal RNA ITS-1 and large subunit of RNA polymerase II gene. Bioassays have been conducted both with promastigote and intracellular amastigote stages of the parasite. All L. martiniquensis strains have been tested against amphotericin B, miltefosine and pentamidine to determine the efficacy of the drugs against the parasite by using a PrestoBlue. The efficacy of miltefosine and pentamidine exhibit no significant difference between each stage of L. martiniquensis among all strains. Surprisingly, the promastigote and intracellular amastigote of the CU1R1 isolate, which was isolated from a relapsed patient after amphotericin B treatment, exhibited a two-fold increased inhibitory concentration (IC50) against amphotericin B compared with other strains, and the difference was statistically significant (p

Abstract Background Leishmaniasis is an emerging disease in Thailand with an unknown incidence or prevalence. Although the number of properly characterized and clinically confirmed cases is about 20, it is suspected that this low number masks a potentially high prevalence, with clinical disease typically manifesting itself against an immunocompromised background, but with a substantial number of subclinical or cured cases of infection. To date leishmaniasis in Thailand has been mainly ascribed to two taxa within the recently erected subgenus Mundinia Shaw, Camargo & Teixeira, 2016, Leishmania (Mundinia) martiniquensis Desbois, Pratlong & Dedet, 2014 and a species that has not been formally described prior to this study. Results A case of simple cutaneous leishmaniasis was diagnosed in a patient from Nan Province, Thailand. Molecular analysis of parasites derived from a biopsy sample revealed this to be a new species of Leishmania Ross, 1908, which has been named as Leishmania (Mundinia) orientalis Bates & Jariyapan n. sp. A formal description is provided, and this new taxon supercedes some isolates from the invalid taxon “Leishmania siamensis”. A summary of all known cases of leishmaniasis with a corrected species identification is provided. Conclusions Three species of parasites are now known to cause leishmaniasis is Thailand, L. martiniquensis and L. orientalis n. sp. in the subgenus Mundinia, which contains the type-species Leishmania enriettii Muniz & Medina, 1948, and a single case of Leishmania infantum Nicolle, 1908. This study now enables epidemiological and other investigations into the biology of these unusual parasites to be conducted. It is recommended that the use of the taxonomically invalid name “L. siamensis” should be discontinued.

An entomological survey at rural and cavernicolous localities in four provinces in southern Thailand provided 155 blood-fed females of sand flies (Diptera: Psychodidae) that were identified based on morphological characters as Idiophlebotomus asperulus (n = 19), Phlebotomus stantoni (n = 4), P. argentipes (n = 3), Sergentomyia anodontis (n = 20), S. barraudi (n = 9), S. hamidi (n = 23), S. hodgsoni (n = 4), S. hodgsoni hodgsoni (n = 32), S. indica (n = 5), S. iyengari (n = 2), S. khawi (n = 17), S. silvatica (n = 11) and Sergentomyia sp. (n = 6). The dominant species in this study was S. hodgsoni hodgsoni, which was collected specifically in a Buddha cave. Screening for DNA of parasitic protozoans revealed eight specimens (5.16%) of four species (S. barraudi, S. indica, S. khawi and Id. asperulus) positive for Trypanosoma sp., while no Leishmania spp. DNA was detected. Blood meals of engorged females were identified by PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) assay on a fragment of cytochrome b (cyt b) gene with a success rate 36%, humans, dogs, and rats being determined as sources of blood. Bloodmeal analysis of two Trypanopsoma-positive females (S. barraudi and Sergentomyia sp.) identified blood from dogs and humans, respectively. Our findings indicate that S. barraudi, S. indica, S. khawi and Id. asperulus may be incriminated in circulation of detected Trypanosoma spp.

Over the years, cases of autochthonous leishmaniasis have been dramatically increasing in Thailand. Recently, several publications have claimed certain species of the phlebotomine sand flies and biting midges potentially serve as natural vectors of Leishmania and Trypanosoma species in this country. However, more information regarding the vector–parasite relationships, as well as their natural reservoirs in the country, still needs to be explored. Herein, we hypothesized that synanthropic reptiles in the leishmaniasis-affected area might be a natural reservoir for these parasites. In this present study, a total of nineteen flat-tailed house geckos were collected from the house of a leishmaniasis patient in Songkhla province, southern Thailand, and then dissected for their visceral organs for parasite detection. Small subunit ribosomal RNA (SSU rRNA) gene and internal transcribed spacer 1 (ITS-1)-specific amplifications were conducted to verify the presence of Trypanosoma and Leishmania parasites, respectively. Only Trypanosoma DNA was screened positive in eight gecko individuals by SSU rRNA-PCR in at least one visceral organ (4, 4, and 6 of the heart, liver, and spleen, respectively) and phylogenetically related to the anuran Trypanosoma spp. (An04/Frog1 clade) previously detected in three Asian sand fly species (Phlebotomus kazeruni, Sergentomyia indica, and Se. khawi). Hence, our data indicate the first detection of anuran Trypanosoma sp. in the flat-tailed house geckos from southern Thailand. Essentially, it can be inferred that there is no evidence for the flat-tailed house gecko (Hemidactylus platyurus) as a natural reservoir of human pathogenic trypanosomatids in the leishmaniasis-affected area of southern Thailand.

Culicine mosquitoes are medically important vectors. Therefore, mosquito control measures are a crucial strategy to interrupt disease transmission. Collection of data on mosquito feeding patterns is crucial for developing an effective vector control strategy. The objective of this study was to use molecular biology methods to identify the sources of DNA in mosquito blood meals. The DNA from blood meals in the mosquito stomachs was extracted and amplified with multiplex PCR, using specific primer sets based on the mitochondrial cytochrome b gene, to identify the DNA sources among human, pig, goat, dog, cow, and chicken. Among the 297 mosquito samples collected in the Songkhla province of Thailand, in Aedes spp. mosquitoes the percentages positive for human, dog, pig, chicken, cow, a mixture of 2 vertebrate DNAs, or of 3, and negative (no identified DNA) were 61.90, 2.38, 2.38, 0.60, 0.60, 4.18, 1.20 and 26.79% respectively. In Culex spp. blood meals the rank order was different: fractions positive for chicken, human, dog, cow, goat, pig, a mixture of 2 or 3 vertebrate DNAs, and negative were 40.83, 10.00, 5.00, 4.17, 1.67, 0.83, 8.32, 3.32 and 25.83% respectively. This study shows that feeding behaviors of the two species differ, with most Aedes spp. blood meals containing human blood, while Culex spp. had primarily consumed chicken blood. An improved understanding of the feeding behaviors of mosquitoes could contribute to new, more effective strategies for the control of mosquito populations.

The increasing global incidence of mosquito-borne infections is driving a need for effective control methods. Vector populations have expanded their geographical ranges, while increasing resistance to chemical insecticides and a lack of effective treatments or vaccines has meant that the development of vector control methods is essential in the fight against mosquito-transmitted diseases. This review will focus on Toxorhynchites, a non-hematophagous mosquito genus which is a natural predator of vector species and may be exploited as a biological control agent. Their effectiveness in this role has been strongly debated for many years and early trials have been marred by misinformation and incomplete descriptions. Here, we draw together current knowledge of the general biology of Toxorhynchites and discuss how this updated information will benefit their role in an integrated vector management program.

Termites of the genus Macrotermes (Termitidae: Macrotermitinae) are serious agricultural and structural pests, which also play vital roles in ecosystem functioning, and are crucial for the maintenance of tropical biodiversity. They are widely distributed, mainly in Southeast Asian countries; however, the parasitism of termites has been little researched. This research was conducted to identify and study the ecology of the parasitoids of termites at Kasetsart University, Kamphaeng Saen Campus, Nakhon Pathom, Thailand. Macrotermes gilvus (Hagen) soldier termites were collected from 25 mounds. In four of the 25 mounds, scuttle fly larvae were found inside the bodies of the soldier termites, and adult flies were found in all of the mounds. Some of the larvae successfully developed to pupae under laboratory conditions. The percentages of parasitized major soldier termites collected from the four mounds were 43.79%, 47.43%, 0.86%, and 3.49%, respectively, and the percentages of parasitized minor soldier termites were 0.64%, 0.00%, 0.21%, and 0.00%, respectively. Larvae, pupae, and adult flies were identified using both morphological and molecular identifications. Molecular identification used the partial nucleotide sequences of the mitochondrial cytochrome c oxidase I (COI) gene. The results of both identification methods identified the parasitic Diptera as the scuttle fly, Megaselia scalaris (Loew) (Diptera: Phoridae). The phylogenetic analysis of the 23 scuttle fly samples (11 larvae, 7 pupae, and 5 adults) classified them into two clades: (1) Those closely related to a previous report in India; (2) those related to M. scalaris found in Asia and Africa. This is the first discovery of M. scalaris in M. gilvus. Further investgation into termite parasitism by M. scalaris and its possible use in the biological control of termites is needed.

Abstract Background Leishmaniasis is a neglected tropical disease that is caused by an obligate intracellular protozoan of the genus Leishmania. Recently, an increasing number of autochthonous leishmaniasis cases caused by L. martiniquensis and the novel species L. siamensis have been described in Thailand, rendering an accurate diagnosis of this disease critical. However, only a few laboratories are capable of diagnosing leishmaniasis in Thailand. To expand leishmaniasis diagnostic capabilities, we developed a simple colorimetric loop-mediated isothermal amplification (LAMP) technique for the direct detection of Leishmania DNA. Methods LAMP was performed for 75 min using four primers targeting the conserved region of the18S ribosomal RNA gene, and the DNA indicator used was malachite green (MG). To simulate crude samples, cultured promastigotes of L. siamensis were mixed with blood or saliva. Also, clinical samples (blood, saliva, and tissue biopsies) were obtained from patients with cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL). All samples were boiled for 10 min and introduced directly into the LAMP reaction mixture without DNA purification. Results The use of MG resulted in an unambiguous differentiation of positive and negative controls. For L. siamensis, the detection limit was 103 parasites/mL or 2.5 parasites/tube. Saliva, tissue biopsies, and whole blood were indicative of active Leishmania infection, and their direct usages did not adversely affect the detection limit. In addition, this LAMP assay could detect DNA from multiple Leishmania species other than L. siamensis and L. martiniquensis, including L. aethiopica, L. braziliensis, L. donovani and L. tropica. Conclusions The simplicity and sensitivity of LAMP in detecting active Leishmania infection could enable the rapid diagnosis of leishmaniasis, thereby facilitating the survey and control of leishmaniasis in Thailand. However, our limited number of samples warranted a further validation with a larger cohort of patients before this assay could be deployed.

Abstract Background Head louse infestation, which is caused by Pediculus humanus capitis, occurs throughout the world. With the advent of molecular techniques, head lice have been classified into three clades. Recent reports have demonstrated that pathogenic organisms could be found in head lice. Head lice and their pathogenic bacteria in Thailand have never been investigated. In this study, we determined the genetic diversity of head lice collected from various areas of Thailand and demonstrated the presence of Acinetobacter spp. in head lice. Methods Total DNA was extracted from 275 head louse samples that were collected from several geographic regions of Thailand. PCR was used to amplify the head louse COI gene and for detection of Bartonella spp. and Acinetobacter spp. The amplified PCR amplicons were cloned and sequenced. The DNA sequences were analyzed via the neighbor-joining method using Kimura’s 2-parameter model. Results The phylogenetic tree based on the COI gene revealed that head lice in Thailand are clearly classified into two clades (A and C). Bartonella spp. was not detected in all the samples, whereas Acinetobacter spp. was detected in 10 samples (3.62%), which consisted of A. baumannii (1.45%), A. radioresistens (1.45%), and A. schindleri (0.72%). The relationship of Acinetobacter spp. and the head lice clades showed that Acinetobacter spp. was found in clade A and C. Conclusions Head lice in Thailand are classified into clade A and B based on the COI gene sequences. Pathogenic Acinetobacter spp. was detected in both clades. The data obtained from the study might assist in the development of effective strategies for head lice control in the future. Detection of pathogenic bacteria in head lice could raise awareness of head lice as a source of nosocomial bacterial infections.

Abstract Background Leishmania siamensis, a newly identified species, has been reported as a causative agent of leishmaniasis in Thailand. This organism has been identified and genetically characterized using PCR techniques based on several target genes. However, the sensitivities and specificities of these methods for the diagnosis of L. siamensis infection have never been evaluated. Methods To evaluate the sensitivities and specificities of PCR methods to detect L. siamensis infection, PCR for different genetic markers, i.e., the small subunit ribosomal RNA region (SSU-rRNA), the internal transcribed spacer 1 region (ITS1), cysteine protease B (cpb), cytochrome b (cyt b), heat shock protein 70 (hsp 70), the spliced leader mini-exon, and the triose-phosphate isomerase (tim) genes were compared. Results Both the ITS1-PCR and the SSU rRNA-PCR could detect promastigote of L. siamensis at concentrations as low as 0.05 parasites/μl or the DNA concentration at 2.3 pg/μl. Though the ITS1-PCR method only recognized 8 samples as positive, all of these could be assessed as true positive according to microscopic diagnosis and/or amplifying the results of the PCR and their sequencing, while other methods also produced false positive results. Compared with the ITS1-PCR method, the PCR amplified SSU-rRNA and cpb gene showed 100% sensitivity for the detection of L. siamensis in clinical specimens. The PCR amplified mini-exon and hsp 70 gene also gave a high sensitivity of 87.5%. In contrast, the PCR methods for cyt b and tim gene showed low sensitivity. The PCR methods for cyt b, mini-exon and tim gene showed 100% specificity compared with the ITS1-PCR. Conclusion As a result, the ITS1-PCR method is a suitable target for PCR-based detection of L. siamensis infection in clinical specimens due to its high sensitivity and specificity. The results of this study can be used for molecular diagnosis as well as in epidemiological studies of L. siamensis in affected areas.

Phlebotomine sand flies are tiny, hairy, blood-sucking nematoceran insects that feed on a wide range of hosts. They are known as a principal vector of parasites, responsible for human and animal leishmaniasis worldwide. In Thailand, human autochthonous leishmaniasis and trypanosomiasis have been reported. However, information on the vectors for Leishmania and Trypanosoma in the country is still limited. Therefore, this study aims to detect Leishmania and Trypanosoma DNA in field-caught sand flies from endemic areas (Songkhla and Phatthalung Provinces) and non-endemic area (Chumphon Province) of leishmaniasis. A total of 439 sand flies (220 females and 219 males) were collected. Head and genitalia dissection of female sandflies were done for morphology identification, and the remaining parts of those sand flies were then used for the detection of Leishmania and Trypanosoma parasites. The DNA was extracted from individual female sand flies. Polymerase chain reaction (PCR) anneal, specific to the ITS1 and SSU rRNA gene regions, was used to detect Leishmania and Trypanosoma DNA, respectively. The positive PCR products were cloned and sequenced. The results showed that the female sand fly species in this study consisted of Sergentomyia khawi (35.9%); Se. anodontis (23.6%); Phlebotomus betisi (18.6%); Ph. kiangsuensis (9.5%); Ph. asperulus (6.4%); Se. barraudi (2.3%); 0.9% of each Se. indica, Ph. stantoni, and Ph. major major; and 0.5% of each Se. sylvatica and Ph. mascomai. The PCR and sequence analysis were able to detect Leishmania and Trypanosoma DNA in sand fly samples, which were identified as L. martiniquensis, 1/220 (0.45%) in Se. khawi, 3/220 (1.36%) of T. noyesi in Se. anodontis, and Ph. asperulus. Fourteen (6.36%) of the unidentified trypanosome species in Se. khawi, Se. indica, Se. anodontis, Ph. asperulus, and Ph. betisi were found in all of the areas of this study. Interestingly, we found a 1/220 (0.45%) co-infection sample of L. martiniquensis and Trypanosoma in Se. khawi from Songkhla Province. These data indicate that several species of sand flies might be potential vectors of Leishmania and Trypanosoma parasites in southern Thailand. However, more extensive study for potential vectors using a larger number of sand flies should be conducted to prove whether these sand flies can be natural vectors of leishmaniasis and trypanosomiasis in both humans and animals. In addition, our study could be useful for the future study of infection prevention, including effective vector control for leishmaniasis and trypanosomiasis in Thailand.

Chikungunya virus (CHIKV) is a mosquito-borne virus belonging to the genus Alphavirus. The virus is transmitted to humans by the bite of infected female Aedes mosquitoes, primarily Aedes aegypti. CHIKV infection is spreading worldwide, and it periodically sparks new outbreaks. There are no specific drugs or effective vaccines against CHIKV. The interruption of pathogen transmission by mosquito control provides the only effective approach to the control of CHIKV infection. Many studies have shown that CHIKV can be transmitted among the Ae. aegypti through vertical transmission. The previous chikungunya fever outbreaks in Thailand during 2008−2009 were caused by CHIKV, the East/Central/South African (ECSA) genotype. Recently, there have been 3794 chikungunya cases in 27 provinces reported by the Bureau of Epidemiology of Health Ministry, Thailand during 1 January−16 June 2019; however, the cause of the re-emergence of CHIKV outbreaks is uncertain. Therefore, the aims of this study were to detect and analyze the genetic diversity of CHIKV infection in field-caught mosquitoes. Both female and male Ae. aegypti were collected from endemic areas of Thailand, and CHIKV detection was done by using E1-nested RT-PCR and sequencing analysis. A total of 1646 Ae. aegypti samples (900 females and 746 males) were tested. CHIKV was detected in 54 (3.28%) and 14 samples (0.85%) in female and male mosquitoes, respectively. Seventeen samples of female Ae. aegypti collected from the Ubon Ratchathani, Chiang Rai, Chiang Mai, Nakhon Sawan, and Songkhla provinces found mutation at E1: A226V. Interestingly, E1: K211E mutation was observed in 50 samples collected from Nong Khai, Bangkok, Prachuap Khiri Khan, and Krabi. In addition, the phylogenetic tree indicated that CHIKV in Ae. aegypti samples were from the Indian Ocean Clade and East/South African Clade. Both clades belong to the ECSA genotype. The information obtained from this study could be used for prediction, epidemiological study, prevention, and effective vector control of CHIKV. For instance, a novel CHIKV strain found in new areas has the potential to lead to a new outbreak. Health authorities could plan and apply control strategies more effectively given the tools provided by this research.

Canine filariasis is caused by several nematode species, such as Dirofilaria immitis, Dirofilaria repens, Brugia pahangi, Brugia malayi, and Acanthocheilonema reconditum. Zoonotic filariasis is one of the world’s neglected tropical diseases. Since 2000, the World Health Organization (WHO) has promoted a global filarial eradication program to eliminate filariasis by 2020. Apart from vector control strategies, the infection control of reservoir hosts is necessary for more effective filariasis control. In addition, many studies have reported that Wolbachia is necessary for the development, reproduction, and survival of the filarial nematode. Consequently, the use of antibiotics to kill Wolbachia in nematodes has now become an alternative strategy to control filariasis. Previously, a case of subconjunctival dirofilariasis caused by Dirofilaria spp. has been reported in a woman who resides in the center of Bangkok, Thailand. Therefore, our study aimed to principally demonstrate the presence of filarial nematodes and Wolbachia bacteria in blood collected from domestic dogs from the Bangkok Metropolitan Region, Thailand. A total of 57 blood samples from dogs with suspected dirofilariasis who had visited veterinary clinics in Bangkok were collected. The investigations for the presence of microfilaria were carried out by using both microscopic and molecular examinations. PCR was used as the molecular detection method for the filarial nematodes based on the COI and ITS1 regions. The demonstration of Wolbachia was performed using PCR to amplify the FtsZ gene. All positive samples by PCR were then cloned and sequenced. The results showed that the filarial nematodes were detected in 16 samples (28.07%) using microscopic examinations. The molecular detection of filarial species using COI-PCR revealed that 50 samples (87.72%) were positive; these consisted of 33 (57.89%), 13 (22.81%), and 4 (7.02%) samples for D. immitis, B. pahangi, and B. malayi, respectively. While the ITS1-PCR showed that 41 samples (71.93%) were positive—30 samples (52.63%) were identified as containing D. immitis and 11 samples (19.30%) were identified to have B. pahangi, whereas B. malayi was not detected. Forty-seven samples (82.45%) were positive for Wolbachia DNA and the phylogenetic tree of all positive Wolbachia was classified into the supergroup C clade. This study has established fundamental data on filariasis associated with Wolbachia infection in domestic dogs in the Bangkok Metropolitan Region. An extensive survey of dog blood samples would provide valuable epidemiologic data on potential zoonotic filariasis in Thailand. In addition, this information could be used for the future development of more effective prevention and control strategies for canine filariasis in Thailand.

Cattle lice are obligatory blood-sucking parasites, which is the cause of animal health problems worldwide. Recently, several studies have revealed that pathogenic bacteria could be found in cattle lice, and it can act as a potential vector for transmitting louse-borne diseases. However, the cattle lice and their pathogenic bacteria in Thailand have never been evaluated. In the present study, we aim to determine the presence of bacterial pathogens in cattle lice collected from three localities of Thailand. Total genomic DNA was extracted from 109 cattle louse samples and the Polymerase Chain Reaction (PCR) of 18S rRNA was developed to identify the cattle louse. Moreover, PCR was used for screening Bartonella spp., Acinetobacter spp., and Rickettsia spp. in cattle louse samples. The positive PCR products were cloned and sequenced. The phylogenetic tree based on the partial 18S rRNA sequences demonstrated that cattle lice species in this study are classified into two groups according to reference sequences; Haematopinus quadripertusus and Haematopinus spp. closely related to H. tuberculatus. The pathogen detection revealed that Bartonella spp. DNA of gltA and rpoB were detected in 25 of 109 samples (22.93%) both egg and adult stages, whereas Acinetobacter spp. and Rickettsia spp. were not detected in all cattle lice DNA samples. The gltA and rpoB sequences showed that the Bartonella spp. DNA was found in both H. quadripertusus and Haematopinus spp. closely related to H. tuberculatus. This study is the first report of the Bartonella spp. detected in cattle lice from Thailand. The finding obtained from this study could be used to determine whether the cattle lice can serve as a potential vector to transmit these pathogenic bacteria among cattle and may affect animal to human health.

Zika virus (ZIKV) infection is an emerging and re-emerging arbovirus disease that is transmitted to humans through the bite of infected mosquitoes. ZIKV infections were first described in Thailand in 1954 from the sera of indigenous residents and several travelers returning from Thailand in 2014. However, reported cases in Thailand have been increasing since 2015 and 2016, and epidemiological information about the vectors of ZIKV is unclear. We investigated the molecular epidemiology and genetic diversity of ZIKV from mosquitoes collected from different geographic regions experiencing ZIKV outbreaks in Thailand. Polymerase chain reaction was used to amplify the non-structural protein (NS5) gene of ZIKV, which was then sequenced. A total of 1026 mosquito samples (626 females, 367 males, and 33 larvae) were collected from active ZIKV patients’ houses. ZIKV was detected in 79 samples (7.7%), including Aedes aegypti (2.24% female, 1.27% male, and 0.19% larvae), Culex quinquefasciatus (1.85% female, 1.66% male, and 0.29% larvae), and Armigeres subalbatus (0.1% female and 0.1% male), whereas no ZIKV was detected in Aedes albopictus. Phylogenetic analysis of the 79 positive samples were classified into two clades: Those closely related to a previous report in Thailand, and those related to ZIKV found in the Americas. This is the first report of the detection of ZIKV in Ae. aegypti, Cx. quinquefasciatus, and Ar. subalbatus mosquitoes, and genetic variations of ZIKV in the mosquitoes collected from several geographic regions of Thailand were examined. Detection of ZIKV in male and larval mosquitoes suggests that vertical transmission of ZIKV occurred in these mosquito species. This study provides a more in-depth understanding of the patterns and epidemiologic data of ZIKV in Thailand; the data could be used for future development of more effective prevention and control strategies of ZIKV in Thailand.

Salivary gland proteins of the human malaria vector, Anopheles dirus B were determined and analyzed. The amount of salivary gland proteins in mosquitoes aged between 3 - 10 days was approximately 1.08 ± 0.04 µg/female and 0.1 ± 0.05 µg/male. The salivary glands of both sexes displayed the same morphological organization as that of other anopheline mosquitoes. In females, apyrase accumulated in the distal regions, whereas alpha-glucosidase was found in the proximal region of the lateral lobes. This differential distribution of the analyzed enzymes reflects specialization of different regions for sugar and blood feeding. SDS-PAGE analysis revealed that at least seven major proteins were found in the female salivary glands, of which each morphological region contained different major proteins. Similar electrophoretic protein profiles were detected comparing unfed and blood-fed mosquitoes, suggesting that there is no specific protein induced by blood. Two-dimensional polyacrylamide gel analysis showed the most abundant salivary gland protein, with a molecular mass of approximately 35 kilodaltons and an isoelectric point of approximately 4.0. These results provide basic information that would lead to further study on the role of salivary proteins of An. dirus B in disease transmission and hematophagy.Proteínas das glândulas salivares do Anopheles dirus B (Diptera: Culicidae), vetor da malária humana foram determinadas e analisadas. A quantidade de proteínas das glândulas salivares em mosquitos com três a 10 dias de idade foi de aproximadamente 1,08 ± 0,04 µg/ fêmea e de 0,1 ± 0,05 µg/macho. As glândulas salivares de ambos os sexos mostraram organização morfológica semelhante à de outros mosquitos anofelinos. Em fêmeas, apirase acumula-se nas regiões distais, enquanto alfa-glucosidase foi encontrada na região proximal dos lóbulos laterais. Esta distribuição diferencial das enzimas analisadas reflete a especialização de diferentes regiões para alimentação de açucares e sangue. Análise SDS-PAGE revelou que pelo menos sete proteínas foram encontradas nas glândulas salivares de fêmeas, das quais cada região morfológica continha diferentes proteínas principais. Perfis eletroforéticos de proteínas semelhantes foram detectados comparando-se mosquitos não alimentados e alimentados por sangue, sugerindo que não existe proteína específica induzida pelo mesmo. Análise por gel poliacrilamida bi-dimensional mostrou a mais abundante proteína de glândulas salivares com aproximadamente 35 kilodaltons de massa molecular e ponto isoelétrico de aproximadamente 4,0. Estes resultados dão informações básicas que levariam a estudos adicionais sobre o papel das proteínas salivares do An. dirus B na transmissão da doença e hematofagia.

BackgroundIn the past few decades, several researchers have proposed highly accurate prediction models that have typically relied on climate parameters. However, climate factors can be unreliable and can lower the effectiveness of prediction when they are applied in locations where climate factors do not differ significantly. The purpose of this study was to improve a dengue surveillance system in areas with similar climate by exploiting the infection rate in the Aedes aegypti mosquito and using the support vector machine (SVM) technique for forecasting the dengue morbidity rate.Methods and findingsAreas with high incidence of dengue outbreaks in central Thailand were studied. The proposed framework consisted of the following three major parts: 1) data integration, 2) model construction, and 3) model evaluation. We discovered that the Ae. aegypti female and larvae mosquito infection rates were significantly positively associated with the morbidity rate. Thus, the increasing infection rate of female mosquitoes and larvae led to a higher number of dengue cases, and the prediction performance increased when those predictors were integrated into a predictive model. In this research, we applied the SVM with the radial basis function (RBF) kernel to forecast the high morbidity rate and take precautions to prevent the development of pervasive dengue epidemics. The experimental results showed that the introduced parameters significantly increased the prediction accuracy to 88.37% when used on the test set data, and these parameters led to the highest performance compared to state-of-the-art forecasting models.ConclusionsThe infection rates of the Ae. aegypti female mosquitoes and larvae improved the morbidity rate forecasting efficiency better than the climate parameters used in classical frameworks. We demonstrated that the SVM-R-based model has high generalization performance and obtained the highest prediction performance compared to classical models as measured by the accuracy, sensitivity, specificity, and mean absolute error (MAE).

Since 1996, there have been several case reports of autochthonous visceral leishmaniasis in Thailand. Here we report a case in a 52-year-old Thai male from northern Thailand, who presented with subacute fever, huge splenomegaly and pancytopenia. Bone marrow aspiration revealed numerous amastigotes within macrophages. Isolation of Leishmania LSCM1 into culture and DNA sequence analysis (ribosomal RNA ITS-1 and large subunit of RNA polymerase II) revealed the parasites to be members of the Leishmania enriettii complex, and apparently identical to L. martiniquensis previously reported from the Caribbean island of Martinique. This is the first report of visceral leishmaniasis caused by L. martiniquensis from the region. Moreover, the majority of parasites previously identified as "L. siamensis" also appear to be L. martiniquensis.

Background: The ANT1 gene, encoding ADP/ATP translocase 1, was investigated in an adult patient with an autosomal recessive mitochondrial disorder characterised by congenital cataracts, hypertrophic cardiomyopathy, myopathy and lactic acidosis., Methods and Results: ANT1 sequencing showed that the patient was homozygous for a new nucleotide variation, c.111+1G→A, abolishing the invariant GT splice donor site of intron 1. The ANT1 transcript was undetectable in both muscle and skin fibroblasts. A markedly abnormal metabolic profile was found, and skeletal muscle showed a dramatic proliferation of abnormal mitochondria, increased mitochondrial mass, and multiple mitochondrial DNA deletions. No compensating increase in the transcript level of the ANT3 gene, which encodes the human ubiquitous isoform of the ADP/ATP translocase, was observed. The patient's heterozygous mother had normal clinical, biochemical and pathological features., Conclusions: Complete loss of expression of the ANT1 gene causes a clinical syndrome mainly characterised by cardiomyopathy and myopathy. This report expands the clinical spectrum of ANT1-related human diseases, and emphasises the crucial role of the mitochondrial ADP/ATP carriers in muscle function and pathophysiology of human myopathies.

The POLG genes were sequenced in two unrelated patients presenting with Alpers syndrome. The novel c.3626_3629dupGATA and the c.3643+2T>C alleles were associated in trans with p.A467T and p.[W748S;E1143G], respectively. POLG transcripts from skin fibroblasts showed complete exon 22 skipping for patient 2, but surprisingly partial exon 22 skipping from the c.3626_3629dupGATA for patient 1. The creation of a putative exonic splicing silencer could be responsible for the splicing anomaly observed in patient 1. Both c.3643+2T>C and c.3626_3629dupGATA create a premature termination codon and a low polymerase γ activity in skin fibroblasts is responsible for the severe phenotype in these patients., (Copyright © 2010 Mitochondria Research Society. Published by Elsevier B.V. All rights reserved.)

DGUOK [dG (deoxyguanosine) kinase] is one of the two mitochondrial deoxynucleoside salvage pathway enzymes involved in precursor synthesis for mtDNA (mitochondrial DNA) replication. DGUOK is responsible for the initial rate-limiting phosphorylation of the purine deoxynucleosides, using a nucleoside triphosphate as phosphate donor. Mutations in the DGUOK gene are associated with the hepato-specific and hepatocerebral forms of MDS (mtDNA depletion syndrome). We identified two missense mutations (N46S and L266R) in the DGUOK gene of a previously reported child, now 10 years old, who presented with an unusual revertant phenotype of liver MDS. The kinetic properties of normal and mutant DGUOK were studied in mitochondrial preparations from cultured skin fibroblasts, using an optimized methodology. The N46S/L266R DGUOK showed 14 and 10% residual activity as compared with controls with dG and deoxyadenosine as phosphate acceptors respectively. Similar apparent negative co-operativity in the binding of the phosphate acceptors to the wild-type enzyme was found for the mutant. In contrast, abnormal bimodal kinetics were shown with ATP as the phosphate donor, suggesting an impairment of the ATP binding mode at the phosphate donor site. No kinetic behaviours were found for two other patients with splicing defects or premature stop codon. The present study represents the first characterization of the enzymatic kinetic properties of normal and mutant DGUOK in organello and our optimized protocol allowed us to demonstrate a residual activity in skin fibroblast mitochondria from a patient with a revertant phenotype of MDS. The residual DGUOK activity may play a crucial role in the phenotype reversal.