In vitro susceptibility to miltefosine of amphotericin B-resistant Leishmania (Mundinia) martiniquensis.

Leishmania (Mundinia) martiniquensis is a newly described species that causes human visceral, disseminated, and mucocutaneous leishmaniases. Amphotericin B deoxycholate (AmpB) is the first-line drug for the treatment of leishmaniasis in Thailand; however, several relapse cases of leishmaniasis caused by L. martiniquensis have been documented. In this study, in vitro susceptibility to AmpB and miltefosine (MIL) of wild-type (before treatment, LSCM1) and two AmpB-resistant L. martiniquensis strains (an in vitro-induced AmpB-resistant strain, AmpBRP2i, and a relapse strain, LSCM1-6) were determined. Results reveal that the IC50 value and resistance index against both drugs of promastigotes and intracellular amastigotes of the AmpBRP2i and LSCM1-6 strains were statistically significantly higher than those of the LSCM1 strain suggesting that cross-resistance with MIL occurred in both AmpB-resistant strains. The results of this study advocate further investigation into mechanisms that involve the complex nature of AmpB/MIL resistance in L. martiniquensis and development of effective methods for the identification of the AmpB-resistant parasites to help delivery of appropriate treatments for patients and for epidemiological surveys to survey the potential spread of drug-resistant strains.

Experimental infection of Leishmania (Mundinia) martiniquensis in BALB/c mice and Syrian golden hamsters.

Our objective was to investigate clinical progression, presence of parasites and DNAs, parasite loads, and histological alterations in BALB/c mice and Syrian golden hamsters after intraperitoneal inoculation with Leishmania (Mundinia) martiniquensis promastigotes with a goal to choosing an appropriate animal model for visceral leishmaniasis. Infections were monitored for 16 weeks. Infected BALB/c mice were asymptomatic during the infection course. Parasite DNAs were detected in the liver at week 8 of infection, followed by clearance in most animals at week 16; whereas in the spleen, parasite DNAs were detected until week 16. These results are correlated to those obtained measuring parasite loads in both organs. No parasite DNA and no alteration in the bone marrow were observed indicating that no dissemination occurred. These results suggest the control of visceralization of L. martiniquensis by BALB/c mice. In hamsters, weight loss, cachexia, and fatigue were observed after week 11. Leishmania martiniquensis parasites were observed in tissue smears of the liver, spleen, and bone marrow by week 16. Parasite loads correlated with those from the presence of parasites and DNAs in the examined tissues. Alterations in the liver with nuclear destruction and cytoplasmic degeneration of infected hepatocytes, presence of inflammatory infiltrates, necrosis of hepatocytes, and changes in splenic architecture and reduction and deformation of white pulp in the spleen were noted. These results indicate a chronic form of visceral leishmaniasis indicating that the hamster is a suitable animal model for the study of pathological features of chronic visceral leishmaniasis caused by L. martiniquensis.

Axenic amastigote cultivation and in vitro development of Leishmania orientalis.

Leishmania (Mundinia) orientalis is a recently described new species that causes leishmaniasis in Thailand. To facilitate characterization of this new species, an in vitro culture system to generate L. orientalis axenic amastigotes was developed. In vitro culture conditions of the axenic culture-derived amastigotes were optimized by manipulation of temperature and pH. Four criteria were used to evaluate the resulting L. orientalis axenic amastigotes, i.e., morphology, zymographic analysis of nucleases, cyclic transformation, and infectivity to the human monocytic cell line (THP-1) cells. Results revealed that the best culture condition for L. orientalis axenic amastigotes was Grace's insect medium supplemented with FCS 20%, 2% human urine, 1% BME vitamins, and 25 µg/ml gentamicin sulfate, pH 5.5 at 35 °C. For promastigotes, the condition was M199 medium, 10% FCS supplemented with 2% human urine, 1% BME vitamins, and 25 µg/ml gentamicin sulfate, pH 6.8 at 26 °C. Morphological characterization revealed six main stages of the parasites including amastigotes, procyclic promastigotes, nectomonad promastigotes, leptomonad promastigotes, metacyclic promastigotes, and paramastigotes. Also, changes in morphology during the cycle were accompanied by changes in zymographic profiles of nucleases. The developmental cycle of L. orientalis in vitro was complete in 12 days using both culture systems. The infectivity to THP-1 macrophages and intracellular growth of the axenic amastigotes was similar to that of THP-1 derived intracellular amastigotes. These results confirmed the successful axenic cultivation of L. orientalis amastigotes. The axenic amastigotes and promastigotes can be used for further study on infection in permissive vectors and animals.

Characterization of IgE-binding proteins in the salivary glands of Simulium nigrogilvum (Diptera: Simuliidae).

Simulium dermatitis is an IgE-mediated skin reaction in animals and humans caused by the bites of black flies. Although Simulium nigrogilvum has been incriminated as the main human-biting black fly species in Thailand, information on its salivary allergens is lacking. Salivary gland extract of S. nigrogilvum females was subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis, and the separated components were applied onto nitrocellulose membranes for immunoblotting, which was performed by probing the protein blots with sera from 17 individuals who were allergic to the bites of S. nigrogilvum. IgE-reactive protein bands were characterized further by liquid chromatography-mass spectrometry (LC-MS/MS) analysis. Nine protein bands (79, 42, 32, 25, 24, 22, 15, 13, and 11 kDa) were recognized in the serum of the subjects. Four of the nine protein bands (32, 24, 15, and 11 kDa) showed IgE reactivity in all (100%) of the tested sera, and they were identified as salivary secreted antigen 5-related protein, salivary serine protease, erythema protein, and hypothetical secreted protein, respectively. Three other proteins, salivary serine protease (25 kDa), salivary D7 secreted protein (22 kDa), and hypothetical protein (13 kDa), reacted with > 50% of the sera. The relevance of the identified protein bands as allergens needs to be confirmed by using pure recombinant proteins, either in the in vivo skin prick test or in vitro detection of the specific IgE in the serum samples of allergic subjects. This will be useful for the rational design of component-resolved diagnosis and allergen immunotherapy for the allergy mediated by the bites of black flies.

Early detection of novel Leishmania species DNA in the saliva of two HIV-infected patients.

BACKGROUND: Leishmaniasis caused by two new species of Leishmania; L. siamensis and L. martiniquensis have been recently described in Thailand. The disease has mainly been documented in AIDS patients from southern Thailand. In this study, polymerase chain reaction (PCR) was used to determine HIV-Leishmania co-infection in southern Thailand. METHODS: One ml of saliva and 3 ml of EDTA blood were collected from HIV-infected patients for PCR detection of Leishmania DNA, cloning and sequencing. The positive PCR samples were then cultured on Schneider's insect medium. RESULTS: Three out of 316 saliva samples collected from HIV-infected patients were found to be positive for Leishmania DNA (0.95%). Among the positive samples, one patient was observed with disseminated cutaneous lesions and also tested positive via saliva, whole blood and buffy coat in PCR. The second case presenting with nodular lesions also gave a positive saliva test via PCR two months prior to buffy coat. This diagnosis was confirmed by microscopic examination and a culture of biopsy samples from a nodule. The last case was an asymptomatic Leishmania infection which tested PCR positive only in saliva with a consecutive sample collection conducted for three months. CONCLUSIONS: The prevalence of Leishmania infection in HIV infected patients within this study is 0.95%. Leishmania DNA was detected in saliva by PCR prior to blood and buffy coat of two HIV infected patients. Early detection of Leishmania DNA in saliva would be beneficial for the follow up of asymptomatic Leishmania infected patients, the early treatment of leishmaniasis and for surveillance survey purpose. However, full evaluation of sensitivity and specificity of this technique with a large cohort of patients is required before deployment.

MIDGUT ULTRASTRUCTURE OF FOURTH INSTAR OCHLEROTATUS TOGOI (DIPTERA: CULICIDAE).

The ultrastructure of the midgut of fourth instar Ochlerotatus togoi was investigated by light, scanning and transmission electron microscopy. This study was performed to provide information to help devise future control efforts aimed at the larval stages of this vector of filariasis. The fourth instar midgut was approximately 2 mm in length and consisted of three morphologically distinct cell types: epithelial, regenerative, and endocrine cells. There was a monolayer of epithelial cells on the luminal surface of the midgut, with multiple folds of the plasma membrane where it adjoined the basement membrane. Regenerative cells were scattered throughout the basal portion of the epithelium, along with endocrine cells. No evidence of division or differentiation was seen in any of the cell types. Six layers of the peritrophic matrix were observed in the gut lumen which separated ingested food from the midgut epithelial cells. Cytoplasmic protrusions were seen in many areas of the luminal midgut surface and numerous autophagosomes were seen in the epithelial cells of both early and late fourth instar larvae, suggesting autophagy is involved in the degeneration process of the midgut in preparation for pupation. This study provides a basis for understanding normal Oc. togoi larval midgut development. Further studies are needed to determine the factors that control larval growth and the nutritional state. Such information could be used to reduce adult fecundity and develop biological control mechanisms.

Peritrophic matrix formation and Brugia malayi microfilaria invasion of the midgut of a susceptible vector, Ochlerotatus togoi (Diptera: Culicidae).

The mosquito midgut is the first site that vector-borne pathogens contact during their multiplication, differentiation, or migration from blood meal to other tissues before transmission. After blood feeding, the mosquitoes synthesize a chitinous structure called peritrophic matrix (PM) that envelops the blood meal and separates the food bolus from the midgut epithelium. In this study, a systematic investigation of the PM formation and the interaction of Brugia malayi within the midgut of a susceptible vector, Ochlerotatus togoi, were performed using scanning electron microscopy (SEM). SEM analysis of the midguts dissected at different time points post feeding on a B. malayi-infected blood meal (PIBM) revealed that the PM was formed from 45 min PIBM and gradually thickened and matured during 8-18 h PIBM. The PM degraded from 24 to 72 h PIBM, when digestion was completed. The invasion process of the microfilariae was observed between 3 and 4 h PIBM. In the beginning of the process, only sheathed microfilariae interacted with the internal face of the PM by its anterior part, and then the midgut epithelium before entering the hemocoel, after that they exsheathed. Microfilarial sheaths lying within the hemocoel were observed suggesting that they may serve as a decoy to induce the immune systems of the mosquitoes to respond to the antigens on the sheaths, thereby protecting the exsheathed microfilariae. These initial findings would lead to further study on the proteins, chemicals, and factors in the midgut that are involved in the susceptibility of O. togoi as a vector of filariasis.

Salivary gland proteome of the human malaria vector, Anopheles campestris-like (Diptera: Culicidae).

Anopheles campestris-like is proven to be a high-potential vector of Plasmodium vivax in Thailand. In this study, A. campestris-like salivary gland proteins were determined and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional gel electrophoresis, and nano-liquid chromatography-mass spectrometry. The total amount of salivary gland proteins in the mosquitoes aged 3-5 days was approximately 0.1 ± 0.05 µg/male and 1.38 ± 0.01 µg/female. SDS-PAGE analysis revealed at least 12 major proteins found in the female salivary glands and each morphological region of the female glands contained different major proteins. Two-dimensional gel electrophoresis showed approximately 20 major and several minor protein spots displaying relative molecular masses from 10 to 72 kDa with electric points ranging from 3.9 to 10. At least 15 glycoproteins were detected in the female glands. Similar electrophoretic protein profiles were detected comparing the male and proximal-lateral lobes of the female glands, suggesting that these lobes are responsible for sugar feeding. Blood-feeding proteins, i.e., putative 5'-nucleotidase/apyrase, anti-platelet protein, long-form D7 salivary protein, D7-related 1 protein, and gSG6, were detected in the distal-lateral lobes (DL) and/or medial lobes (ML) of the female glands. The major spots related to housekeeping proteins from other arthropod species including Culex quinquefasciatus serine/threonine-protein kinase rio3 expressed in both male and female glands, Ixodes scapularis putative sil1 expressed in DL and ML, and I. scapularis putative cyclophilin A expressed in DL. These results provide information for further study on the salivary gland proteins of A. campestris-like that are involved in hematophagy and disease transmission.

Enhancement of VGG16 model with multi-view and spatial dropout for classification of mosquito vectors.

Mosquitoes transmit pathogens that can cause numerous significant infectious diseases in humans and animals such as malaria, dengue fever, chikungunya fever, and encephalitis. Although the VGG16 model is not one of the most advanced CNN networks, it is reported that a fine-tuned VGG16 model achieves accuracy over 90% when applied to the classification of mosquitoes. The present study sets out to improve the accuracy and robustness of the VGG16 network by incorporating spatial dropout layers to regularize the network and by modifying its structure to incorporate multi-view inputs. Herein, four models are implemented: (A) early-combined, (B) middle-combined, (C) late-combined, and (D) ensemble model. Moreover, a structure for combining Models (A), (B), (C), and (D), known as the classifier, is developed. Two image datasets, including a reference dataset of mosquitoes in South Korea and a newly generated dataset of mosquitoes in Thailand, are used to evaluate our models. Regards the reference dataset, the average accuracy of ten runs improved from 83.26% to 99.77%, while the standard deviation decreased from 2.60% to 0.12%. When tested on the new dataset, the classifier's accuracy was also over 99% with a standard deviation of less than 2%. This indicates that the algorithm achieves high accuracy with low variation and is independent of a particular dataset. To evaluate the robustness of the classifier, it was applied to a small dataset consisting of mosquito images captured under various conditions. Its accuracy dropped to 86.14%, but after retraining with the small dataset, it regained its previous level of precision. This demonstrates that the classifier is resilient to variation in the dataset and can be retrained to adapt to the variation. The classifier and the new mosquito dataset could be utilized to develop an application for efficient and rapid entomological surveillance for the prevention and control of mosquito-borne diseases.

Natural infection with Leishmania (Mundinia) martiniquensis supports Culicoides peregrinus (Diptera: Ceratopogonidae) as a potential vector of leishmaniasis and characterization of a Crithidia sp. isolated from the midges.

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 resistance correlates with increased fitness in vitro and in vivo in Leishmania (Mundinia) 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.

Case Report: Simple Nodular Cutaneous Leishmaniasis Caused by Autochthonous Leishmania (Mundinia) orientalis in an 18-Month-Old Girl: The First Pediatric Case in Thailand and Literature Review.

We report an autochthonous case of simple, localized cutaneous leishmaniasis in a healthy 18-month-old girl from southern Thailand. The patient presented with a solitary chronic cutaneous nodular lesion on her left cheek for approximately 1 year. Histopathological dissection of the cheek skin biopsy demonstrated remarkably nodular and interstitial infiltrates of lymphocytes and histiocytes full of intracellular oval-shaped amastigotes, consistent with cutaneous leishmaniasis. The Leishmania promastigotes were also cultured successfully from the lesion biopsy and were designated with the WHO code MHOM/TH/2021/CULE5. Using internal transcribed spacer 1-specific polymerase chain reaction, the parasite DNA was demonstrated in both saliva and lesion biopsy. Based on the BLASTn and phylogenetic analysis, the parasite was identified as Leishmania orientalis, clustered in the Mundinia subgenus. The patient responded well to a 6-week course of oral itraconazole, without recurrence. To our knowledge, this is the fourth case of autochthonous leishmaniasis resulting from L. orientalis and the youngest patient of leishmaniasis ever reported in Thailand. More importantly, we also demonstrate the clinical course of the lesion according to the timeline before and after treatment, which can help physicians better understand and provide an accurate diagnosis with appropriate treatment of this emerging parasitic disease.

Cloning of a trypsin-like serine protease and expression patterns during Plasmodium falciparum invasion in the mosquito, Anopheles dirus (Peyton and Harrison).

Understanding specific gene regulation during responses to malaria infection is key to dissecting the mosquito defense mechanisms and host/parasite interactions. A full-length serine protease cDNA was isolated from the fat body of female Anopheles dirus, a major malaria vector in Thailand. The predicted amino acid sequence of SERF4 identifies it as a member of the serine protease family containing a single trypsin-like protease domain. Digestive trypsins function in the female mosquito midgut and are inducible in two phases in this tissue upon blood intake. However, the gene was highly upregulated in the midgut at day 3 postinfection by Plasmodium falciparum. In situ hybridization confirmed that SERF4 transcripts were located in the midgut epithelial cells rather than hemocytes or other tissues associated with the midgut. SERF4 was also strongly downregulated in the whole insects at day 16 after infection in comparison with the blood-fed control. Changes in the expression of the SERF4 gene in response to infection with this human malaria parasite suggest a role in parasite-specific innate immunity.

Proteomic analysis of salivary glands of female Anopheles barbirostris species A2 (Diptera: Culicidae) by two-dimensional gel electrophoresis and mass spectrometry.

Salivary gland proteins of adult female Anopheles barbirostris species A2, a potential vector of Plasmodium vivax in Thailand, were analyzed using a proteomic approach (two-dimensional gel electrophoresis followed by nanoLC-MS). Two-dimensional gel electrophoresis revealed approximately 75 well-resolved spots on the reference gel. Most of the protein spots displayed relative molecular masses from 14 to 85 kDa and isoelectric points ranging from 3.9 to 10. The proteome profiles of A. barbirostris species A2 female salivary glands were affected by aging. The typical electrophoretic pattern of the female salivary glands was reached in 48 h post emergence, suggesting the maturation of salivary glands and saliva contents for blood feeding. Proteins involved in blood feeding, i.e., putative 5' nucleotidase/apyrase, anti-platelet protein, long form D7 salivary protein, D7-related 1 protein, and gSG6 salivary protein, start to accumulate from emergence and gradually increase becoming predominant within 48 h. There are different salivary components expressed within each region of the female glands. The blood-feeding proteins were detected in the distal-lateral lobes and/or medial lobes. Proteins detected and/or identified by this approach could be tested in strategies developed to control pathogen and disease transmission. Moreover, the information of a 2D map of the female salivary gland could be used for comparison with other related species in the A. barbirostris complex to distinguish species members in the complex.

Stimulation of metacyclogenesis in Leishmania (Mundinia) orientalis for mass production of metacyclic promastigotes.

Leishmania (Mundinia) orientalis is a human pathogen causing leishmaniasis and studies on the properties of metacyclic promastigotes, the parasite's infective stage, are required for a better understanding of its transmission and infection. However, information on cultivation for mass production of L. orientalis metacyclic promastigotes and factors that stimulate their metacyclogenesis is limited. Therefore, the objective of this study was to develop a suitable methodology for generating promastigote cultures containing a high proportion and number of L. orientalis metacyclic promastigotes. Various media, i.e., Schneider's insect medium, Medium 199 and Grace's insect medium, supplemented with various quantities of dithiothreitol, Basal Medium Eagle vitamins, pooled human urine, and fetal bovine serum, were optimized for metacyclogenesis. The results revealed that the optimum culture medium and conditions of those tested were Schneider's insect medium supplemented with 100 µM dithiothreitol, 1% (v/v) Basal Medium Eagle vitamins, 2% (v/v) pooled human urine, and 10% (v/v) fetal bovine serum, pH 5.0 at 26°C. We also demonstrated that L. orientalis metacyclic promastigotes could be purified and enriched by negative selection using peanut lectin. Under these culture conditions, the highest yield of metacyclic promastigotes was obtained with a significantly higher percentage of parasite survival, resistance to complement-mediated lysis, and infection index in THP-1 macrophage cells compared to parasites cultured without media supplements at neutral pH. This is the first report providing a reliable method for mass production of L. orientalis metacyclic promastigotes for in vivo infections and other experimental studies of this emerging parasite in the future.

Significance of eggshell morphology as an additional tool to distinguish species of sand flies (Diptera: Psychodidae: Phlebotominae).

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.

Susceptibility of Anopheles campestris-like and Anopheles barbirostris species complexes to Plasmodium falciparum and Plasmodium vivax in Thailand.

Nine colonies of five sibling species members of Anopheles barbirostris complexes were experimentally infected with Plasmodium falciparum and Plasmodium vivax. They were then dissected eight and 14 days after feeding for oocyst and sporozoite rates, respectively, and compared with Anopheles cracens. The results revealed that Anopheles campestris-like Forms E (Chiang Mai) and F (Udon Thani) as well as An. barbirostris species A3 and A4 were non-potential vectors for P. falciparum because 0% oocyst rates were obtained, in comparison to the 86.67-100% oocyst rates recovered from An. cracens. Likewise, An. campestris-like Forms E (Sa Kaeo) and F (Ayuttaya), as well as An. barbirostris species A4, were non-potential vectors for P. vivax because 0% sporozoite rates were obtained, in comparison to the 85.71-92.31% sporozoite rates recovered from An. cracens. An. barbirostris species A1, A2 and A3 were low potential vectors for P. vivax because 9.09%, 6.67% and 11.76% sporozoite rates were obtained, respectively, in comparison to the 85.71-92.31% sporozoite rates recovered from An. cracens. An. campestris-like Forms B and E (Chiang Mai) were high-potential vectors for P. vivax because 66.67% and 64.29% sporozoite rates were obtained, respectively, in comparison to 90% sporozoite rates recovered from An. cracens.

Molecular identification of two newly identified human pathogens causing leishmaniasis using PCR-based methods on the 3' untranslated region of the heat shock protein 70 (type I) gene.

PCR-based methods to amplify the 3' untranslated region (3'-UTR) of the heat shock protein 70 (type I) gene (HSP70-I) have previously been used for typing of Leishmania but not with Leishmania (Mundinia) martiniquensis and L. (Mundinia) orientalis, newly identified human pathogens. Here, the 3'-UTRs of HSP70-I of L. martiniquensis, L. orientalis, and 10 other species were sequenced and analyzed. PCR-Restriction Fragment Length Polymorphism (RFLP) analysis targeting the 3'-UTR of HSP70-I was developed. Also, the detection limit of HSP70-I-3'-UTR PCR methods was compared with two other commonly used targets: the 18S small subunit ribosomal RNA (SSU-rRNA) gene and the internal transcribed spacer 1 region of the rRNA (ITS1-rRNA) gene. Results showed that HSP70-I-3'-UTR PCR methods could be used to identify and differentiate between L. martiniquensis (480-2 bp) and L. orientalis (674 bp) and distinguished them from parasites of the subgenus Viannia and of the subgenus Leishmania. PCR-RFLP patterns of the 3'-UTR of HSP70-I fragments digested with BsuRI restriction enzyme successfully differentiated L. martiniquensis, L. orientalis, L. braziliensis, L. guyanensis = L. panamensis, L. mexicana = L. aethiopica = L. tropica, L. amazonensis, L. major, and L. donovani = L. infantum. For the detection limit, the HSP70-I-3'-UTR PCR method could detect the DNA of L. martiniquensis and L. orientalis at the same concentration, 1 pg/µL, at a similar level to the SSU-rRNA PCR. The PCR that amplified ITS1-rRNA was more sensitive (0.01 pg/µL) than that of the HSP70-I-3'-UTR PCR. However, the sizes of both SSU-rRNA and ITS1-rRNA PCR amplicons could not differentiate between L. martiniquensis and L. orientalis. This is the first report of using HSP70-I-3'-UTR PCR based methods to identify the parasites causing leishmaniasis in Thailand. Also, the BsuRI-PCR-RFLP method can be used for differentiating some species within other subgenera.

Chromosome-Scale Assembly of the Complete Genome Sequence of Leishmania (Mundinia) martiniquensis, Isolate LSCM1, Strain LV760.

Leishmania (Mundinia) martiniquensis is a kinetoplastid parasite that was first isolated in 1995 on Martinique. We report the first complete genome for Leishmania martiniquensis from Asia, isolate LSCM1, strain LV760, which was sequenced using combined short-read and long-read technologies. This will facilitate greater understanding of the evolution of the geographically dispersed subgenus Mundinia.

Chromosome-Scale Assembly of the Complete Genome Sequence of Leishmania (Mundinia) orientalis, Isolate LSCM4, Strain LV768.

Leishmania (Mundinia) orientalis is a kinetoplastid parasite first isolated in 2014 in Thailand. We report the complete genome sequence of L. (M.) orientalis, sequenced using combined short-read and long-read technologies. This will facilitate greater understanding of this novel pathogen and its relationship to other members of the subgenus Mundinia.