Comparative analysis of midgut bacterial communities in Chikungunya virus-infected and non-infected Aedes aegypti Thai laboratory strain mosquitoes.

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.

Genetic diversity and phylogenetic analyses of Asian lineage Zika virus whole genome sequences derived from Culex quinquefasciatus mosquitoes and urine of patients during the 2020 epidemic in Thailand.

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.

Species diversity of phlebotomine sand flies and sympatric occurrence of Leishmania (Mundinia) martiniquensis, Leishmania (Leishmania) donovani complex, and Trypanosoma spp. in the visceral leishmaniasis focus of southern Thailand.

Autochthonous leishmaniasis in Thailand has recently been a public health concern due to an increasing number of new clinical cases. Most indigenous cases were diagnosed with Leishmania (Mundinia) martiniquensis, and Leishmania (Mundinia) orientalis. However, some doubts regarding vector misidentification have arisen and need to be elucidated. Accordingly, we aimed to assess the species composition of sand flies and determine the molecular prevalence of trypanosomatids in the transmission area of leishmaniasis in southern Thailand. In the present study, a total of 569 sand flies were caught from the vicinity of a visceral leishmaniasis patient's house in Na Thawi District, Songkhla Province. Of these, 229 parous and gravid females consisted of Sergentomyia khawi, Se. barraudi, Phlebotomus stantoni, Grassomyia indica, and Se. hivernus, accounting for 31.4%, 30.6%, 29.7%, 7.9%, and 0.4%, respectively. However, Se. gemmea, which has previously been proposed as the most abundant species and putative vector of visceral leishmaniasis, was not found in the present study. Based on ITS1-PCR and sequence analysis, two specimens of Gr. indica and Ph. stantoni showed positive amplification of L. martiniquensis and L. donovani complex, respectively, the first one being presumed indigenous and the second one being not. Anuran Trypanosoma was also molecularly detected using SSU rRNA-PCR and ubiquitously found in 16 specimens of four dominant sand fly species except for Se. hivernus. The obtained sequences could be phylogenetically categorized into the two major amphibian clades (An04/Frog1 and An01+An02/Frog2). The existence of the monophyletic subgroup and distinct lineage suggests them as novel Trypanosoma species. The TCS network analysis of these anuran Trypanosoma sequences also revealed high haplotype diversity (Hd = 0.925 ± 0.050), but low nucleotide diversity (π = 0.019 ± 0.009). Furthermore, the living anuran trypanosomes were microscopically demonstrated in a single specimen of Gr. indica, supporting the vectorial capacity. Importantly, our data confirmed the scarcity of Se. gemmea and also uncovered, for the first time, the co-circulation of L. martiniquensis, L. donovani complex, and suspected novel anuran Trypanosoma spp. in phlebotomine sand files, implicating their potential role as vectors of trypanosomatid parasites. Therefore, the novel data from this study would greatly facilitate the comprehension of the complexity of trypanosomatid transmission and the establishment of prevention and control measures for this neglected disease more effectively.

Description of potential vectors of zoonotic filarial nematodes, Brugia pahangi, Setaria digitata, and Setaria labiatopapillosa in Thai mosquitoes.

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.

Understanding dengue solution and larval indices surveillance system among village health volunteers in high- and low-risk dengue villages in southern Thailand.

Background: Successful dengue solutions require community collaboration between agencies engaged in human health, vector control and the environment. In Thailand, village health volunteers emphasize the need for a health working group to interact, collaborate, and coordinate actions. The objectives of this study were to acquire an understanding of dengue solutions, as well as the larval indices surveillance system of village health volunteers in high- and low-risk dengue villages. Methods: After 12 months of training in dengue prevention and setting larval indices surveillance systems, an analytical cross-sectional survey was conducted. A total of 117 villages were included in the 18 primary care facilities within one district in southern Thailand, and they were divided into 71 high-risk and 46 low-risk dengue villages. Sample size was determined using the G*power formula. The content validity index and reliability values of Cronbach's alpha coefficient for the questionnaires were 0.91 and 0.83, respectively. A random sampling approach was used to acquire data. The chi-square test, t-test, and odds ratio were used to assess the sample's level of understanding. Results: The study included 1302 village health volunteers, including 895 and 407 from high- and low-risk dengue communities, respectively. In total, 87.9% were female, 51.6% were 20-35 years old, 48.8% had worked as a village health volunteer for 11-20 years, 27.1% had an upper elementary education, and 59.1% had dengue in the previous 12 months. Understanding of the dengue solution and larval indices surveillance system varied across high- and low-risk dengue villages. Village health volunteers with a high level of understanding of the dengue solution and larval indies surveillance system were 1.064 and 1.504 times more likely to stay in high-risk dengue villages, respectively (odds ratio [OR] = 1.064, 95% confidence interval [CI]:0.798-1.419, p = 0.672 and OR = 1.504, 95% CI:1.044-2.167, p = 0.028). Conclusions: Village health volunteers require ongoing training to understand the prevention and control of dengue and larval indices surveillance systems, promote awareness, and monitor dengue in both high- and low-risk dengue villages.

Development and evaluation of a visible reverse transcription-loop-mediated isothermal amplification (RT-LAMP) for the detection of Asian lineage ZIKV in field-caught mosquitoes.

The Zika virus (ZIKV) infection is an emerging and re-emerging arbovirus infection that is transmitted to humans through the bite of infected mosquitoes. Early detection of ZIKV in mosquitoes is one of the prerequisite approaches for tracking the spread of the virus. Therefore, this study aims to develop and validate a visual reverse transcription-loop-mediated isothermal amplification (RT-LAMP) method called ZIKV-RT-LAMP, for detecting ZIKV in field collected mosquito samples from Thailand. A single-tube ZIKV-RT-LAMP assay was developed to detect Asian lineage ZIKV RNA. The detection limit and cross-reactivity of ZIKV were investigated. The hemi-nested RT-PCR (hn-RT-PCR) and the colorimetric LAMP kit (cLAMP kit) were performed as reference assays. The detection limit of the ZIKV-RT-LAMP assay was 10-6 ffu/ml or pfu/ml, making it highly specific and 100 times more sensitive than the hn-RT-PCR and cLAMP kits. The ZIKV-RT-LAMP assay detected the Asian lineage of ZIKV RNA without cross-reactivity with other arthropod-borne viruses. The sensitivity and specificity of the ZIKV-RT-LAMP assay were 92.31% and 100%, respectively. The ZIKV-RT-LAMP is a simple, rapid, and inexpensive method for detecting ZIKV in field-caught mosquitos. In the future, extensive surveys of field-caught mosquito populations should be conducted. Early detection of ZIKV in field-caught mosquitoes provides for prompt and effective implementation of mosquito control strategies in endemic areas.

Genome Assembly and Genome Annotation of Leishmania martiniquensis Isolated from a Leishmaniasis Patient in Thailand.

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.

First Report of Anuran Trypanosoma DNA in Flat-Tailed House Geckos (Reptilia: Gekkonidae) Collected from Southern Thailand: No Evidence as a Reservoir for Human Trypanosomatids.

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.

Culicoides Latreille (Diptera: Ceratopogonidae) as potential vectors for Leishmania martiniquensis and Trypanosoma sp. in northern Thailand.

Biting midges of genus Culicoides (Diptera: Ceratopogonidae) are the vectors of several pathogenic arboviruses and parasites of humans and animals. Several reports have suggested that biting midges might be a potential vector of Leishmania parasites. In this study, we screened for Leishmania and Trypanosoma DNA in biting midges collected from near the home of a leishmaniasis patient in Lamphun province, northern Thailand by using UV-CDC light traps. The identification of biting midge species was based on morphological characters and confirmed using the Cytochrome C oxidase subunit I (COI) gene. The detection of Leishmania and Trypanosoma DNA was performed by amplifying the internal transcribed spacer 1 (ITS1) and small subunit ribosomal RNA (SSU rRNA) genes, respectively. All the amplified PCR amplicons were cloned and sequenced. The collected 223 biting midges belonged to seven species (Culicoides mahasarakhamense, C. guttifer, C. innoxius, C. sumatrae, C. huffi, C. oxystoma, and C. palpifer). The dominant species found in this study was C. mahasarakhamense (47.53%). Leishmania martiniquensis DNA was detected in three samples of 106 specimens of C. mahasarakhamense tested indicating a field infection rate of 2.83%, which is comparable to reported rates in local phlebotomines. Moreover, we also detected Trypanosoma sp. DNA in one sample of C. huffi. To our knowledge, this is the first molecular detection of L. martiniquensis in C. mahasarakhamense as well as the first detection of avian Trypanosoma in C. huffi. Blood meal analysis of engorged specimens of C. mahasarakhamense, C. guttifer, and C. huffi revealed that all specimens had fed on avian, however, further studies of the host ranges of Culicoides are needed to gain a better insight of potential vectors of emerging leishmaniasis. Clarification of the vectors of these parasites is also important to provide tools to establish effective disease prevention and control programs in Thailand.

A new cluster of rhabdovirus detected in field-caught sand flies (Diptera: Psychodidae: Phlebotominae) collected from southern Thailand.

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.

Genetic characterization of chikungunya virus isolates from Aedes aegypti mosquitoes collected during a recent outbreak in Bangkok, Thailand.

Chikungunya virus (CHIKV) is a mosquito-borne emerging pathogen that is transmitted to humans through the bite of female Aedes mosquitoes. CHIKV infection has become a major public health concern worldwide, as it has a significant impact on the healthcare system. Since 2004, the virus has emerged in Africa and subsequently spread to countries located near the Indian Ocean, including India, and to Europe, the Americas, and Asia. In Thailand, a large CHIKV outbreak occurred during 2008-2009 and was caused by a virus originating from the east/central/south African (ECSA) CHIKV genotype. Since then, the ECSA genotype of CHIKV has continued to circulate and has caused sporadic cases in different areas in Thailand. Approximately 20,000 reported cases have been confirmed by the Bureau of Epidemiology, Ministry of Public Health, Thailand, from January 1, 2018 to July 31, 2020. However, the causes of this CHIKV re-emergence remain unclear. To obtain a better understanding of CHIKV circulation during the recent outbreak in Bangkok, Thailand, complete genome analysis of CHIKV isolates from field-caught mosquitoes collected in outbreak areas was performed. A total of 28 Ae. aegypti samples (21 females and 7 males) were collected, and individual mosquitoes were used for CHIKV detection and isolation. Eleven of 28 (39.29%) female and three of 28 (10.71%) male mosquitoes were positive for CHIKV by E1 nested RT-PCR. Four CHIKV isolates were successfully isolated from four female Ae. aegypti mosquitoes. Based on complete genome analysis, several amino acid substitutions were identified in the protein coding region. The E1:K211E and E2:V264A mutations in the background of the E1:226A mutation were observed in all four CHIKV isolates. An important observation was the presence of one amino acid substitution, leading to an E1:K245R change. This mutation was found in all four CHIKV isolates from mosquitoes in this study and in Thai patients described previously. Additionally, phylogenetic analysis indicated that the four CHIKV isolates belonged to the Indian Ocean clade of the ECSA genotype. The results obtained in this study provide detailed information on the molecular characteristics and evolution of currently circulating CHIKV strains in Thailand, which are useful for developing prevention and control strategies.

Molecular analysis of mitochrondrial cytb of Pediculus humanus capitis in Thailand revealed potential historical connection with South Asia.

BACKGROUND: Pediculus 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. METHOD: We 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. RESULTS: The 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. CONCLUSIONS: Pediculosis 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.

Case Report: First Report of Disseminated Trachipleistophora hominis Infection in an AIDS Patient from Thailand.

Microsporidial myositis caused by Trachipleistophora hominis is a life-threatening and emerging microsporidiosis among immunocompromised hosts. This article reports a case of disseminated microsporidiosis caused by T. hominis in southern Thailand. The patient had HIV and presented at the clinic with incapacitating muscle pain. She was diagnosed with disseminated microsporidiosis. Molecular identification revealed the sequence of 18S ribosomal RNA gene involving sequences sharing 99% nucleotide identity with T. hominis from an Australian patient. To our knowledge, this is the first study to report the detection of T. hominis microsporidia in an HIV patient in Thailand.

Molecular detection of Indian Ocean Lineage Chikungunya virus RNA in field collected Culex quinquefasciatus Say from Bangkok, Thailand but no evidence of virus replication.

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.

Detection of Changuinola virus (Reoviridae: Orbivirus) in field-caught sand flies in southern Thailand.

BACKGROUND: Phlebotomine sand flies are vectors for several pathogenic bacteria, parasites and viruses that have significant impacts on public health. Sand fly-associated viruses that cause diseases in humans and animals have recently received more attention. This study aimed to detect pathogenic viruses belonging to the Orbivirus genus, Phlebovirus genus, Flavivirus genus and family Rhabdoviridae in several field-caught sand fly species in southern Thailand. METHODS: Sand flies were collected in southern Thailand using CDC light traps. Each sample was processed individually for virus screening using RT-PCR and sequencing. RESULTS: Seven out of 60 sand fly samples (two samples of Idiophlebotomus spp., three of Phlebotomus papatasi and two of Sergentomyia khawi) were positive for the Orbivirus genus, which is closely related to Changuinola virus (CGLV). Phlebovirus genus, Flavivirus genus and family Rhabdoviridae were negative in all samples. CONCLUSIONS: CGLV causes Changuinola virus disease or Changuinola fever, a febrile illness in Central and South America. The virus has never been reported in Thailand. This study is the first report of the detection of CGLV in sand flies from Thailand. An extensive study of sand flies from other regions of the country and the associations between sand flies, viruses and vertebrate hosts in Thailand should be undertaken.

Presence of the knockdown resistance (kdr) mutations in the head lice (Pediculus humanus capitis) collected from primary school children of 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.

Detection of Leishmania infantum DNA by real-time PCR in saliva of dogs.

This study developed a real-time quantitative PCR (qPCR) assay to detect L. infantum kinetoplast DNA (kDNA) in canine saliva. The qPCR showed an efficiency of 93.8%, a coefficient of correlation of 0.996 and a detection limit of 0.5 fg/reaction (0.005 parasites), although it detected until 0.25 fg/reaction (0.0025 parasites). When samples from 12 dogs experimentally infected with L. infantum were collected, L. infantum kDNA was detected at 16-weeks post-infection (wpi) in 41.7% and 91.7% of saliva and bone marrow samples, respectively, and at 47-wpi in 75% of both samples. L. infantum kDNA can be detected by qPCR in canine saliva, with lower sensitivity in the early stages of infection and a lower parasite load estimation compared to bone marrow. However, saliva had similar sensitivities to bone marrow in the later stages of the infection and could be used to detect L. infantum kDNA being aware of its limitations.

Parasitism of Soldiers of the Termite, Macrotermes gilvus (Hagen), by the Scuttle Fly, Megaselia scalaris (Loew) (Diptera: Phoridae).

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.

Determination of anti-leishmanial drugs efficacy against Leishmania martiniquensis using a colorimetric assay.

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 < 0.05). Moreover, intracellular amastigotes isolated from CU1R1 exhibited slightly increased susceptibility to amphotericin B compared with the promastigote (p < 0.05). The result of this experiment is a scientific evident to support that in case of relapsed leishmaniasis caused by L. martiniquensis, increasing dosage of amphotericin B is essential. Moreover, this study also determined efficacy of other anti-leishmanial drugs for treatment the leishmaniasis in Thailand in case of these drugs are available in the country and the clinicians should have alternative drugs for treatment leishmaniasis in Thailand apart from amphotericin B.

Synthesis and antileishmanial activity of fluorinated rhodacyanine analogues: The 'fluorine-walk' analysis.

In a search for potent antileishmanial drug candidates, eighteen rhodacyanine analogues bearing fluorine or perfluoroalkyl substituents at various positions were synthesized. These compounds were tested for their inhibitory activities against Leishmania martiniquensis and L. orientalis. This 'fluorine-walk' analysis revealed that the introduction of fluorine atom at C-5, 6, 5', or 6' on the benzothiazole units led to significant enhancement of the activity, correlating with the less negative reduction potentials of the fluorinated analogues confirmed by the electrochemical study. On the other hand, CF3 and OCF3 groups were found to have detrimental effects, which agreed with the poor aqueous solubility predicted by the in silico ADMET analysis. In addition, some of the analogues including the difluorinated species showed exceptional potency against the promastigote and axenic amastigote stages (IC50 = 40-85 nM), with the activities surpassing both amphotericin B and miltefosine.