Dual role of azo compounds in inhibiting Plasmodium falciparum adenosine deaminase and hemozoin biocrystallization.

Protein-ligand (GOLD) docking of the NCI compounds into the ligand-binding site of Plasmodium falciparum adenosine deaminase (PfADA) identified three most active azo compounds containing 4-[(4-hydroxy-2-oxo-1H-quinolin-3-yl) moiety. These compounds showed IC50 of 3.7-15.4 µM against PfADA, as well as inhibited the growth of P. falciparum strains 3D7 (chloroquine (CQ)-sensitive) and K1 (CQ-resistant) with IC50 of 1.8-3.1 and 1.7-3.6 µM, respectively. The identified compounds have structures similar to the backbone structure (4-N-(7-chloroquinolin-4-yl)) in CQ, and NSC45545 could mimic CQ by inhibiting the bioformation of hemozoin in parasitic food vacuole. The amount of in situ hemozoin in the ring-stage parasite was determined using a combination of synchrotron transmission Fourier transform infrared microspectroscopy and Principal Component Analysis. Stretching of the C-O bond of hemozoin propionate group measured at 1220-1210 cm-1 in untreated intraerythrocytic P. falciparum strains 3D7 and K1 was disappeared following treatment with 1.85 and 1.74 µM NSC45545, similar to those treated with 0.02 and 0.13 µM CQ, respectively. These findings indicate a novel dual function of 4-[(4-hydroxy-2-oxo-1H-quinolin-3-yl) azo compounds in inhibiting both PfADA and in situ hemozoin biocrystallization. These lead compounds hold promise for further development of new antimalarial therapeutics that could delay the onset of parasitic drug resistance.

Comparison of Recombinant Proteins of Kinesin 39, Heat Shock Protein 70, Heat Shock Protein 83, and Glycoprotein 63 for Antibody Detection of Leishmania martiniquensis Infection.

Leishmania martiniquensis, a zoonotic hemoflagellate, is a causative agent of cutaneous (CL) and visceral leishmaniasis (VL) among humans and animals. This organism, first reported in Martinique Island, now has become an emerging infectious agent in Thailand. Symptomatic cases of L. martiniquensis infection among humans have continuously increased. In the meantime, asymptomatic infection of this novel species has seriously created national public health awareness and concern to prevent and control disease transmission. The unsuccessful serological test using the commercial rK39 dipstick based on antigen from Leishmania donovani to detect the antibodies against VL among infected Thai patients has encouraged us to further explore a new sensitive and specific antigenic epitope. In this study, we determined the sequences and expressed recombinant proteins of kinesin 39 (k39), heat shock protein 70 (hsp70), heat shock protein 83 (hsp83), and glycoprotein 63 (gp63) of L. martiniquensis to evaluate the diagnostic efficiency to detect antibodies against L. martiniquensis in patient sera. The preliminary results from western blot analysis have suggested that K39 is the most sensitive recombinant protein to detect L. martiniquensis. Moreover, this recombinant protein reacts with antibodies against L. donovani and Leishmania infantum, making it a promising antigen for further development of a universal rapid diagnostic tool for VL.

Comparison of LAMP and PCR for molecular mass screening of sand flies for Leishmania martiniquensis infection.

BACKGROUND: Leishmaniasis caused by Leishmania martiniquensis infection has been reported in human and domestic animals of Martinique Island, Germany, Switzerland, USA, Myanmar and Thailand. The peculiar clinical features of disseminated cutaneous and visceral forms co-existence render the urgent need of specific diagnostic tool to identify the natural sand fly vectors for effective prevention and control strategies. Loop-mediated isothermal amplification (LAMP) of 18S rRNA gene as well as polymerase chain reaction (PCR) of minicircle kinetoplast DNA gene (PCR-mkDNA) have never been applied to detect L. martiniquensis and L. siamensis in sand fly vectors. OBJECTIVE: The present study was aimed to validate malachite green-LAMP (MG-LAMP) and PCR-mkDNA techniques to detect L. martiniquensis in sand fly vectors, compared with the conventional PCR of internal transcribed spacer 1 (PCR-ITS1). METHODS: We compared the validity of LAMP of 18S rRNA gene and PCR-mkDNA, to PCR-ITS1 in simulation model of L. martiniquensis infection in Sergentomyia gemmea sand flies. Attributable to the sensitivity and specificity, PCR-mkDNA was consecutively applied to detect L. martiniquensis in 380 female sand fly individuals captured in the newly identified affected region of Lamphun Province, Thailand. FINDINGS AND MAIN CONCLUSIONS: Results showed that PCR-mkDNA could detect at least one promastigote per sand fly, which was 10-time superior to LAMP and PCR-ITS1. In addition, PCR-mkDNA was more specific, able to differentiate L. martiniquensis from other viscerotropic Leishmania species, such as L. siamensis, L. (L.) donovani, and L. (L.) infantum. Consecutively, mass screening of L. martiniquensis in 380 female sand fly individuals by PCR-mkDNA was implemented in a new affected area of Thailand where a patient with leishmaniasis/HIV co-infection resides; however Leishmania DNA was undetected. In conclusion, PCR-mkDNA is a promising tool for molecular mass screening of L. martiniquensis infection in outbreak areas where several species of Leishmania and sand flies co-exist.

Development and evaluation of PCR methods based on cytochrome c oxidase subunit one (cox1) and NADH dehydrogenase subunit one gene (nad1) to detect Opisthorchis viverrini in human fecal samples.

Opisthorchis viverrini is highly prevalent throughout Southeast Asia. Chronic infection of this parasite leads to cholangiocarcinoma (CCA), a fatal bile duct cancer. The early and accurate detection of this parasite is very important; therefore, new PCR methods targeting cytochrome c oxidase subunit one and NADH dehydrogenase subunit one gene to detect O. viverrini in fecal specimens have been developed. Ninety O. viverrini-positive human fecal samples were used in this study. The PCR sensitivity of both genes was compared with internal transcribe spacer 2 (ITS2)-PCR. The sensitivity of cox1-PCR and nad1-PCR was 66.7 and 50 %, respectively. The sensitivity of cox1-PCR and nad1-PCR achieved 89.1 and 71.7 % in specimens containing O. viverrini eggs of >100 eggs per gram (EPG). Additionally, these primers can be used to provide the information on genetic diversity from mitochondrial genes of O. viverrini.

Distribution of pfmdr1 polymorphisms in Plasmodium falciparum isolated from Southern Thailand.

BACKGROUND: Drug resistance in Plasmodium falciparum is a major problem in malaria control especially along the Thai-Myanmar and Thai-Cambodia borders. To date, a few molecular markers have been identified for anti-malarial resistance in P. falciparum, including the P. falciparum chloroquine resistance transporter (pfcrt) and the P. falciparum multidrug resistance 1 (pfmdr1). However no information is available regarding the distribution pattern of these gene polymorphisms in the parasites from the Thai-Malaysia border. This study was conducted to compare the distribution pattern of the pfcrt and pfmdr1 polymorphisms in the parasites from the lower southern provinces, Thai-Malaysia border and the upper southern provinces, Thai-Myanmar border. In addition, in vitro sensitivities of anti-malarial drugs including chloroquine, mefloquine, quinine, and artesunate were determined. METHODS: In all, 492 P. falciparum-positive blood samples were collected from the lower southern provinces: Songkhla, Yala and Narathiwas. From the upper southern part of Thailand, Ranong and Chumphon, 66 samples were also collected. Polymorphisms of the pfcrt and the pfmdr1 gene were determined using PCR techniques. In vitro sensitivities of anti-malarial drugs were determined using radioisotopic method. RESULTS: All parasites from both areas contained the pfcrt 76 T allele. The pfmdr1 86Y allele was significantly more common in the parasites isolated from the lower southern areas. In contrast, the pfmdr1 184F allele was predominant among the parasites from the upper southern areas especially Ranong. In addition, the parasites from Ranong contained higher copy numbers than the parasites from other provinces. All adapted parasite isolates exhibited CQ-resistant phenotype. Neither QN nor MQ resistance was detected in these isolates. CONCLUSION: The parasites from Thai-Malaysia border exhibited different resistant patterns compared to other areas along the international border of Thailand. This information will be useful for anti-malarial drug policy in Thailand.

Incidence and risk factors of blastocystis infection in orphans at the Babies' Home, Nonthaburi Province, Thailand.

BACKGROUND: Blastocystis infection is one of the most common intestinal protozoan infections reported in Thai population of all age groups for which epidemiological information is important to understand patterns of transmission for developing methods ofprevention and control for each specific group. The authors aimed to study prevalence, incidence and riskfactors associated with Blastocystis infection in orphans and childcare workers. Additionally, subtypes ofBlastocystis were identified MATERIAL AND METHOD: A retrospective cohort study of Blastocystis sp. was conducted in orphans aged less than 5 years and their childcare workers at Babies' Home, Nonthaburi Province, Thailand. A base line survey was conducted in December 2009 and afollow-up survey was conducted in April 2010. A total of 336 and 331 stool samples were collected. Blastocystis infection was examined using short-term in vitro cultivation in Jones's medium supplemented with 10% fetal calf serum. To analyze subtypes ofBlastocystis sp., PCR-RFLP of the small subunit ribosomal RNA gene was performed. RESULTS: Theprevalence ofBlastocystis infection in December 2009 and April 2010 were 8.1% and 13.3%, respectively The inlcidence rate ofBlastocystis infection was 1.6/100 person-months. Subtype analysis ofBlastocystis sp. in December 2009 and in April 2010 showed that subtype 3 was the most predominant (76% and 76%), followed by subtype 1 (16% and 20%), and unidentified subtype (8% and 4%), respectively. Subtype 3 is of human origin, thus person-to-person transmission is considered a major route ofBlastocystis infection in this population. CONCLUSION: Person-to-person transmission of Blastocystis infection in orphans living in the same house had been proposed, thus the prevalence and incidence of Blastocystis infection could be used to reflect the hygienic condition in the orphanage. Infection prevention and control practice can be effectively implemented.

In vitro drug susceptibility using a parasite-rescue and transformation assay of Leishmania (Mundinia) martiniquensis and Leishmania (Mundinia) orientalis amastigotes against antileishmanial drugs.

Leishmaniasis is an emerging infectious disease in Thailand, with Leishmania martiniquensis and Leishmania orientalis identified as the primary causative agents among immunocompetent and immunocompromised individuals. Variations in drug susceptibility among different Leishmania species have been reported in different regions. Therefore, drug susceptibility assays are essential to assess the effectiveness of antileishmanial drugs used or potentially used in the affected areas. This study investigated the in vitro drug sensitivity of L. martiniquensis and L. orientalis, along with two reference species causing VL, namely L. donovani and L. infantum, against six antileishmanial drugs. Using a parasite-rescue and transformation assay, the results demonstrated that the IC50 values of amphotericin B (AmB), miltefosine (MIL), and sodium stibogluconate (Sb(III)) against all Leishmania species tested were within the sensitive range of each drug. On the contrary, the IC50 values of artemisinin (ART) and dihydroartemisinin (DHA), drugs primarily used for malaria treatment, were outside the sensitive range of the Leishmania species tested except L. infantum. This in vitro study highlights that AmB could effectively exhibit good sensitivity against the intracellular amastigotes of L. martiniquensis and L. orientalis. Also, MIL and Sb(III) could be considered alternative drugs for antileishmanial treatment in Thailand.

Multilocus characterization and phylogenetic analysis of Leishmania siamensis isolated from autochthonous visceral leishmaniasis cases, southern Thailand.

BACKGROUND: Visceral leishmaniasis (VL) caused by Leishmania siamensis is an emerging disease continuously reported in six southern provinces of Thailand. To date, the phylogenetic relationships among Leishmania isolates from Thai patients and other Leishmania species are still unclear and the taxonomic diversity needs to be established. In this study, the phylogenetic inference trees were constructed based on four genetic loci (i.e., SSU-rRNA, ITS1, hsp70, and cyt b), using DNA sequences obtained from autochthonous VL patients from southern Thailand and reference sequences of reported Leishmania isolates from other studies deposited in GenBank. RESULTS: Phylogenetic analyses of hsp70 and cyt b loci supported a clade comprised of L. siamensis isolates, which is independent to the other members in the genus Leishmania. In combination with genetic distance analysis, sequence polymorphisms were observed among L. siamensis isolates and two different lineages could be differentiated, lineages PG and TR. Phylogenetic analysis of the cyt b gene further showed that L. siamensis lineage TR is closely related to L. enrietti, a parasite of guinea pigs. CONCLUSION: The finding of this study sheds further light on the relationships of L. siamensis, both in intra- and inter-species aspects. This information would be useful for further in-depth studies on the biological properties of this important parasite.

Sergentomyia (Neophlebotomus) gemmea, a potential vector of Leishmania siamensis in southern Thailand.

BACKGROUND: Leishmaniasis, caused by Leishmania siamensis, is an emerging disease in Thailand. Although reported cases have been increasing, epidemiological information of the disease including host and vector aspects is not clearly known. This study was a preliminary survey of the potential vector of L. siamensis in an affected area of leishmaniasis, Trang Province, southern Thailand. METHODS: The collection of sandflies was performed around the area where a case of leishmaniasis was reported using CDC light traps. Species of sandfly were identified based on morphological characteristics according to Lewis's key. PCR amplification and sequencing of the heat shock protein 70 gene (hsp70) was used to identify L. siamensis DNA in sandflies. RESULTS: A total of 146 male and female sandflies were collected in the affected areas. Of 71 female sandflies, four species were identified, i.e., Sergentomyia (Neophlebotomus) gemmea, S. (Neophlebotomus) iyengari, S. (Parrotomyia) barraudi and Phlebotomus (Anaphlebotomus) stantoni. Among these species, S. (Neophlebotomus) gemmea was the most predominant species in all areas. DNA of L. siamensis was identified in S. (Neophlebotomus) gemmea. Nucleotide sequences of PCR products using DNA extracted from S. (Neophlebotomus) gemmea showed 99.8% identity to L. siamensis. CONCLUSION: S. (Neophlebotomus) gemmea might be a potential vector of L. siamensis in an affected area, Trang Province, southern Thailand. However further studies are needed to prove whether these sandflies can be natural vectors of leishmaniasis.

A survey of sandflies in the affected areas of leishmaniasis, southern Thailand.

Leishmania siamensis was firstly described as a causative agent of autochthonous visceral leishmaniasis in southern provinces of Thailand since 2008. The spread of leishmaniasis depends on the distribution of the vectors and reservoir hosts. Unfortunately, little is known about these vital factors. The objective of this study was to identify the distribution of sandfly species, their density, and their habitats in the affected areas of leishmaniasis, southern Thailand. A cross-sectional survey of sandflies was conducted in three provinces of southern Thailand where leishmaniasis cases were previously reported. The collection of sandflies was performed using CDC light traps for four consecutive months, from March to June 2009. A total of 2,698 sandflies were collected in the affected areas. Among 1,451 female sandflies, six species of genus Sergentomyia were identified, i.e., Sergentomyia gemmea, Sergentomyia iyengari, Sergentomyia barraudi, Sergentomyia indica, Sergentomyia silvatica, and Sergentomyia perturbans. S. gemmea (81.4 %) was the most predominant species in all areas. In addition, one species of the genus Phlebotomus, Phlebotomus argentipes, a known vector of leishmaniasis was also detected. The distribution of sandfly species in these leishmaniasis-affected areas was different from the previous studies in other areas of Thailand. Further studies are needed to proof whether these sandflies can be the natural vectors of leishmaniasis.

Identification of a unique conserved region from a kinetoplastid genome of Leishmania orientalis (formerly named Leishmania siamensis) strain PCM2 in Thailand.

Mitochondrial DNAs (mtDNAs) appear in almost all eukaryotic species and are useful molecular markers for phylogenetic studies and species identification. Kinetoplast DNAs (kDNAs) are structurally complex circular mtDNA networks in kinetoplastids, divided into maxicircles and minicircles. Despite several kDNAs of many Leishmania species being examined, the kDNAs of the new species, Leishmania orientalis (formerly named Leishmania siamensis) strain PCM2, have not been explored. This study aimed to investigate the maxicircle and minicircle DNAs of L. orientalis strain PCM2 using hybrid genome sequencing technologies and bioinformatic analyses. The kDNA sequences were isolated and assembled using the SPAdes hybrid assembler from the Illumina short-read and PacBio long-read data. Circular contigs of the maxicircle and minicircle DNAs were reconstructed and confirmed by BLASTn and rKOMICs programs. The kDNA genome was annotated by BLASTn before the genome comparison and phylogenetic analysis by progressiveMauve, MAFFT, and MEGA programs. The maxicircle of L. orientalis strain PCM2 (18,215 bp) showed 99.92% similarity and gene arrangement to Leishmania enriettii strain LEM3045 maxicircle with variation in the 12s rRNA gene and divergent region. Phylogenetics of the whole sequence, coding regions, divergent regions, and 12s rRNA gene also confirmed this relationship and subgenera separation. The identified 105 classes of minicircles (402-1177 bp) were clustered monophyletically and related to the Leishmania donovani minicircles. The kinetoplast maxicircle and minicircle DNAs of L. orientalis strain PCM2 contained a unique conserved region potentially useful for specific diagnosis of L. orientalis and further exploration of this parasite population genetics in Thailand and related regions.

Genetic variation and geographic distribution of Leishmania orientalis and Leishmania martiniquensis among Leishmania/HIV co-infection in Thailand.

Since 1999, the number of asymptomatic leishmaniasis cases has increased continuously in Thailand, particularly among patients with HIV who are prone to develop symptoms of cutaneous and visceral leishmaniasis further. The asymptomatic infection could play a key role in Leishmania transmission and distribution. Understanding population structure and phylogeographic patterns could be crucially needed to develop effective diagnoses and appropriate guidelines for therapy. In this study, genetic variation and geographic distribution of the Leishmania/HIV co-infected population were investigated in endemic northern and southern Thailand. Interestingly, Leishmania orientalis was common and predominant in these two regions with common regional haplotype distribution but not for the others. Recent population expansion was estimated, probably due to the movement and migration of asymptomatic individuals; therefore, the transmission and prevalence of Leishmania infection could be underestimated. These findings of imbalanced population structure and phylogeographic distribution patterns provide valuable, insightful population structure and geographic distribution of Leishmania/HIV co-infection to empower prevention and control of transmission and expansion of asymptomatic leishmaniasis.

Validation of quantitative loop-mediated isothermal amplification assay using a fluorescent distance-based paper device for detection of Escherichia coli in urine.

Uropathogenic Escherichia coli (UPEC) causes up to 90% of urinary tract infections (UTI) which is more prevalent among females than males. In urine, patients with symptomatic UTI usually have a high concentration of bacterial infection, ≥ 105 colony-forming units (CFU) per mL, in which the culture method is regularly the gold standard diagnosis. In this study, a simple and inexpensive distance-based paper device (dPAD) combined with the fluorescent closed tube LAMP assay was validated for simultaneously screening and semi-quantifying the infection level of E. coli in 440 urine samples of patients with UTI. The dPAD could measure the LAMP amplicons and semi-quantify the levels of E. coli infection in heavy (≥ 104 CFU/mL), light (≤ 103 CFU/mL) and no infection. The sensitivity and specificity had reliable performances, achieving as high as 100 and 92.7%, respectively. The one step LAMP assay could be performed within 3 h, which was 7.5 times faster than the culture method. To empower early UTI diagnosis and fast treatment, this inexpensive dPAD tool combined with the fluorescent closed tube LAMP assay is simple, reliably fast and practically portable for point-of-care settings, particularly in resource-limited areas, which can be set up in all levels of healthcare facilities.

Utilizing Quantitative Proteomics to Identify Species-Specific Protein Therapeutic Targets for the Treatment of Leishmaniasis.

Leishmaniasis is a tropical disease caused by Leishmania parasites, which are transmitted through the bites of infected sandflies. We focused on the emergence of leishmaniasis in Thailand caused by a species (Leishmania orientalis). Treatment by chemotherapy is not effective against L. orientalis. Hence, we intended to solve this issue using a proteomics approach to investigate protein profiles and in silico analysis for the identification of antigenic proteins from L. orientalis, Leishmania martiniquensis, and Leishmania donovani. Using principal component analysis (PCA), protein profile comparisons indicated that different species of Leishmania are different at the protein level. Proteomics analysis identified 6099 proteins. Among these proteins, 1065 proteins were used for further analysis. There were 16 proteins that were promising candidates for therapeutic aspects as they were abundantly expressed and common to all species. In silico analysis of protein's antigenicity revealed that eight proteins had the potential for the development of antigenic molecules. Protein profile information and these antigenic proteins may play key roles in the pathogeny of leishmaniasis and can be used as novel therapeutic targets against leishmaniasis in the future.

Comparative Draft Genomes of Leishmania orientalis Isolate PCM2 (Formerly Named Leishmania siamensis) and Leishmania martiniquensis Isolate PCM3 from the Southern Province of Thailand.

(1) Background: Autochthonous leishmaniasis, a sandfly-borne disease caused by the protozoan parasites Leishmania orientalis (formerly named Leishmania siamensis) and Leishmania martiniquensis, has been reported for immunocompromised and immunocompetent patients in the southern province of Thailand. Apart from the recent genomes of the northern isolates, limited information is known on the emergence and genetics of these parasites. (2) Methods: This study sequenced and compared the genomes of L. orientalis isolate PCM2 and L. martiniquensis isolate PCM3 with those of the northern isolates and other 14 Leishmania species using short-read whole-genome sequencing methods and comparative bioinformatic analyses. (3) Results: The genomes of the southern isolates of L. orientalis and L. martiniquensis were 30.01 Mbp and 32.39 Mbp, and the comparison with the genomes of the northern isolates revealed species-level similarity with a level of genome and proteome variation, suggesting the different strains. Comparative proteome analysis showed six protein groups with 53 unique proteins for the strain PCM2 and 97 for the strain PCM3. Certain proteins were related to virulence, drug resistance, and stress response. (4) Conclusion: Therefore, the findings could indicate the need for more genetic and population genomic investigation, and the close monitoring of L. orientalis and L. martiniquensis in Thailand and neighboring regions.

Identification of a conserved maxicircle and unique minicircles as part of the mitochondrial genome of Leishmania martiniquensis strain PCM3 in Thailand.

BACKGROUND: The mitochondrial DNA of trypanosomatids, including Leishmania, is known as kinetoplast DNAs (kDNAs). The kDNAs form networks of hundreds of DNA circles that are evidently interlocked and require complex RNA editing. Previous studies showed that kDNA played a role in drug resistance, adaptation, and survival of Leishmania. Leishmania martiniquensis is one of the most frequently observed species in Thailand, and its kDNAs have not been illustrated. METHODS: This study aimed to extract the kDNA sequences from Illumina short-read and PacBio long-read whole-genome sequence data of L. martiniquensis strain PCM3 priorly isolated from the southern province of Thailand. A circular maxicircle DNA was reconstructed by de novo assembly using the SPAdes program, while the minicircle sequences were retrieved and assembled by the rKOMIC tool. The kDNA contigs were confirmed by blasting to the NCBI database, followed by comparative genomic and phylogenetic analysis. RESULTS: We successfully constructed the complete circular sequence of the maxicircle (19,008 bp) and 214 classes of the minicircles from L. martiniquensis strain PCM3. The genome comparison and annotation showed that the maxicircle structure of L. martiniquensis strain PCM3 was similar to those of L. enriettii strain LEM3045 (84.29%), L. arabica strain LEM1108 (82.79%), and L. tarentolae (79.2%). Phylogenetic analysis also showed unique evolution of the minicircles of L. martiniquensis strain PCM3 from other examined Leishmania species. CONCLUSIONS: This was the first report of the complete maxicircle and 214 minicircles of L. martiniquensis strain PCM3 using integrated whole-genome sequencing data. The information will be helpful for further improvement of diagnosis methods and monitoring genetic diversity changes of this parasite.

Distance-based paper device using combined SYBR safe and gold nanoparticle probe LAMP assay to detect Leishmania among patients with HIV.

Asymptomatic visceral leishmaniasis cases increase continuously, particularly among patients with HIV who are at risk to develop further symptoms of leishmaniasis. A simple, sensitive and reliable diagnosis is crucially needed due to risk populations mostly residing in rural communities with limited resources of laboratory equipment. In this study, a highly sensitive and selective determination of Leishmania among asymptomatic patients with Leishmania/HIV co-infection was achieved to simultaneously interpret and semi-quantify using colorimetric precipitates (gold-nanoparticle probe; AuNP-probe) and fluorescence (SYBR safe dye and distance-based paper device; dPAD) in one-step loop-mediated isothermal amplification (LAMP) assay. The sensitivities and specificities of 3 detection methods were equivalent and had reliable performances achieving as high as 95.5%. Detection limits were 102 parasites/mL (0.0147 ng/µL) which were 10 times more sensitive than other related studies. To empower leishmaniasis surveillance as well as prevention and control, this dPAD combined with SYBR safe and gold nanoparticle probe LAMP assay is reliably fast, simple, inexpensive and practical for field diagnostics to point-of-care settings in resource-limited areas which can be set up in all levels of healthcare facilities, especially in low to middle income countries.

Design of a Chimeric Multi-Epitope Vaccine (CMEV) against Both Leishmania martiniquensis and Leishmania orientalis Parasites Using Immunoinformatic Approaches.

Leishmaniasis is a parasitic disease caused by protozoan flagellates of the genus Leishmania. Recently, Leishmania martiniquensis and Leishmania orientalis, emerging species of Leishmania, were isolated from patients in Thailand. Development of the vaccine is demanded; however, genetic differences between the two species make it difficult to design a vaccine that is effective for both species. In this study, we applied immuno-informatic approaches to design a chimeric multi-epitope vaccine (CMEV) against both L. martiniquensis and L. orientalis. We identified seven helper T lymphocyte (HTL) epitopes, sixteen cytotoxic T lymphocyte (CTL) epitopes, and eleven B-cell epitopes from sixteen conserved antigenic proteins found in both species. All these epitopes were joined together, and to further enhance immunogenicity, protein and peptides adjuvant were also added at the N-terminal of the molecule by using specific linkers. The candidate CMEV was subsequently analyzed from the perspectives of the antigenicity, allergenicity, and physiochemical properties. The interaction of the designed multi-epitope vaccine and immune receptor (TLR4) of the host were evaluated based on molecular dockings of the predicted 3D structures. Finally, in silico cloning was performed to construct the expression vaccine vector. Docking analysis showed that the vaccine/TLR4 complex took a stable form. Based on the predicted immunogenicity, physicochemical, and structural properties in silico, the vaccine candidate was expected to be appropriately expressed in bacterial expression systems and show the potential to induce a host immune response. This study proposes the experimental validation of the efficacy of the candidate vaccine construct against the two Leishmania.