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

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

Simple Summary: The number of leishmaniasis cases caused by autochthonous Leishmania (Mundinia) martiniquensis and Leishmania (Mundinia) orientalis has been continuously growing in Southeast Asia over the decades, particularly in Thailand. Recent evidence has suggested that Culicoides biting midges are the most likely natural vectors of autochthonous leishmaniasis in Thailand. Nevertheless, the epidemiology of vector infection and the genetic variation of Leishmania parasites in endemic areas have remained largely unknown. In this study, we report the high infection prevalence, sympatric circulation, and regional genetic diversity of two Leishmania (Mundinia) parasites in Culicoides spp. caught from the affected community and Tham Phra cave in Chiang Rai Province, Northern Thailand. These results provide us with a more complete understanding of the complexity of leishmaniasis transmission, which will be useful for establishing effective management and control measures for this neglected disease, especially in endemic areas of Northern Thailand. In this research, we elucidated the species composition of Culicoides biting midges, infection prevalence, and genetic diversity of Leishmania parasites circulating in the affected community in Chiang Rai Province, being the most endemic area in Northern Thailand. A total of 146 parous and gravid females, belonging to at least twelve Culicoides species in five subgenera and one species group, were trapped from three collection sites with an overall Leishmania prevalence of 26.7% (39/146). Leishmania was detected, using ITS1-PCR, in C. mahasarakamense (15), C. guttifer (11), C. (Trithecoides) spp. (8), C. jacobsoni (2), C. oxystoma (2), and C. orientalis (1). The evidence of Leishmania infection in these last five species represents new records in Northern Thailand. Given a high infection rate in cavernicolous specimens, this indicates an increased risk of parasite exposure when visiting the cave. Using the nanopore amplicon sequencing, L. martiniquensis was ubiquitously identified in all positives, and more than half of these were also co-infected with L. orientalis. The genetic diversity analysis revealed 13 and 17 unique haplotypes for L. martiniquensis and L. orientalis, respectively. Higher haplotype diversity and relatively low nucleotide diversity were observed in both parasite populations, suggesting recent population divergence. Neutrality tests (Tajima's D and Fu and Li's D) showed to be significantly negative, indicating rapid population growth or a selective sweep. Moreover, dominant haplotypes of both Leishmania species were 100% identical to those in all leishmaniasis patients previously reported from Northern Thailand, strongly supporting the imperative role of Culicoides spp. in disease transmission. Essentially, this research provides the first entomological surveillance data representing the sympatric existence, transmission dynamics, and genetic complexity of two autochthonous Leishmania (Mundinia) parasites in several Culicoides species in the endemic area of Northern Thailand. This would contribute to a more complete understanding of the epidemiology of vector infection and facilitate the development of vector control programs to effectively reduce the transmission of this neglected tropical disease in endemic areas of Northern Thailand. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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

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. [ABSTRACT FROM AUTHOR]

Abstract 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. Graphical abstract

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

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

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.

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. [ABSTRACT FROM AUTHOR]

Simple Summary: This study successfully sequenced the draft genomes of the southern isolates of Leishmania orientalis and Leishmania martiniquensis in Thailand. The comparison with the genomes of the northern isolates revealed species-level similarity with a level of genome and proteome variation, suggesting the different emerging strains. Comparing the proteins of these southern strains to those of the northern ones and 14 other Leishmania species showed six protein groups with numerous unique proteins: 53 for the southern strain PCM2 of L. orientalis and 97 for the strain PCM3 of L. martiniquensis. Some of these proteins were related to virulence, drug resistance, drug target, and stress response, which could be targeted for further experimental characterization. Therefore, the findings could initiate further genetic and population genomic investigation, and the close monitoring of L. orientalis and L. martiniquensis in Thailand and neighboring regions. (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. [ABSTRACT FROM AUTHOR]

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

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

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

Leishmania martiniquensis is a neglected cause of an emerging leishmaniasis in many countries, including France, Germany, Switzerland, the United States of America, Myanmar, and Thailand, with different clinical manifestations ranging from asymptomatic, cutaneous (CL), visceral (VL), and atypically disseminated CL and VL. The persistence of parasites and the recurrence of the disease after treatment are challenges in controlling the disease. To explore efficient prophylaxis and therapy, this study aimed to investigate infection outcome and organ-specific immune responses after inoculation with L. martiniquensis (MHOM/TH/2011/PG; 5 x 106 promastigotes) in BALB/c mice via intravenous and intraperitoneal routes. A quantitative PCR technique, targeting L. martiniquensis ITS1, was primarily established to estimate the parasite burden. We found that the infection in the liver resolved; however, persistent infection was observed in the spleen. Histopathology with Leishmania-specific immunostaining revealed efficient hepatic granuloma formation, while splenic disorganization with parasitized macrophages at different locations was demonstrated. The mRNA expression of Th1 cytokines (IFN-γ, TNF-α, IL-12p40) and iNOS in the liver and spleen was upregulated. In addition, high expression of IL-10 was observed in the spleen in the chronic phase, revealing a significant moderate correlation with the parasite persistence [r(12) = 0.72, P = 0.009]. Further clarification of the mechanisms of persistent infection and experimental infection in immunosuppressed murine models are warranted.

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

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

The article contains a literature review devoted to the history of the study of human leishmaniasis and one of the new pathogens — Leishmania martiniquensis. It is shown that leishmaniasis caused by this microorganism proceeds in the form of cutaneous, including diffuse in HIV-infected patients, and visceral leishmaniasis. The most typical clinical examples are given.

Leishmaniasis is a parasitic disease caused by flagellated protozoa of the genus Leishmania. It is transmitted by the bite of an infected sandfly. The 3 main clinical forms of the disease are cutaneous leishmaniasis (CL), visceral leishmaniasis (VL) and mucocutaneous leishmaniasis (MCL). Prior to 1996, all leishmaniasis cases were infected during the visit to the endemic areas. Thereafter, autochthonous leishmaniasis cases have been reported in Thailand. From 1996 to the present, at least 21 cases of autochthonous leishmaniasis have been confirmed in Thailand. Leishmania siamensis, a novel species of Leishmania, was suspected of being the causative pathogens in some of those cases, However, the data supporting the existence of this new species is limited. Until recently, in-depth investigation using molecular characterization and isoenzyme analysis revealed that this suspected novel species consists of 2 different, but closely related strains: L. siamensis and L. martiniquensis. L. martiniquensis, a rare species firstly discovered on Martinique Island, is the cause of leishmaniasis in the majority of cases. Meanwhile, L. siamensis, a true novel species firstly and only reported from Thailand, was confirmed as the cause of leishmaniasis in two autochthonous cases. Two clinical forms (CL and VL) have been observed in both L. martiniquensis and L. siamensis infection. The DNA of L. martiniquensis was found in black rats, suggesting their role as a natural reservoir. The presence of L. martiniquensis DNA in two sandfly species (Sergentomyia gemmea and Sergentomyia barraudi) that are commonly found in affected areas may also suggest their role as potential vectors. Here, we update the status of leishmaniasis in Thailand and its emergence as a potential public health concern.

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. [ABSTRACT FROM AUTHOR]

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

Leishmaniasis is a parasitic disease caused by flagellated protozoa of the genus Leishmania. It is transmitted by infection from the bite of an infected sandfly. The 3 main clinical forms of the disease are cutaneous leishmaniasis (CL), visceral leishmaniasis (VL) and mucocutaneous leishmaniasis (MCL). Prior to 1996, all leishmaniasis cases were infected during the visit to the endemic areas. Thereafter, autochthonous leishmaniasis cases have been reported in Thailand. During 1996 to the present, at least 21 cases of autochthonous leishmaniasis have been confirmed in Thailand. Leishmania siamensis, a novel species of Leishmania, was suspected of being the causative pathogens in some of those cases, although the data supporting the existence of this new species is limited. Until recently, in-depth investigation using molecular characterization and isoenzyme analysis revealed that a suspected novel species consists of 2 different, but closely related strains: L. siamensis and L. martiniquensis. L. martiniquensis, a rare species firstly discovered on Martinique Island, is the cause of leishmaniasis in the majority of cases. Meanwhile, L. siamensis, a true novel species first and only reported from Thailand, was confirmed as the cause of leishmaniasis in two autochthonous cases. Two clinical forms (CL and VL) have been observed in both L. martiniquensis and L. siamensis infection. DNA of L. martiniquensis was found in black rats, suggesting their role as a natural reservoir. The presence of L. martiniquensis DNA in two sandfly species (Sergentomyia gemmea and Sergentomyia barraudi) that are commonly found in affected areas may also suggest their role as potential vectors. Here, we update the status of leishmaniasis in Thailand and its emergence as a potential public health concern.

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

In this work, we describe the first Leishmania-infecting leishbunyavirus—the first virus other than Leishmania RNA virus (LRV) found in trypanosomatid parasites. Its host is Leishmania martiniquensis, a human pathogen causing infections with a wide range of manifestations from asymptomatic to severe visceral disease. This virus (LmarLBV1) possesses many characteristic features of leishbunyaviruses, such as tripartite organization of its RNA genome, with ORFs encoding RNA-dependent RNA polymerase, surface glycoprotein, and nucleoprotein on L, M, and S segments, respectively. Our phylogenetic analyses suggest that LmarLBV1 originated from leishbunyaviruses of monoxenous trypanosomatids and, probably, is a result of genomic re-assortment. The LmarLBV1 facilitates parasites’ infectivity in vitro in primary murine macrophages model. The discovery of a virus in L. martiniquensis poses the question of whether it influences pathogenicity of this parasite in vivo, similarly to the LRV in other Leishmania species.

Leishmania (Mundinia) martiniquensis is a causative agent of visceral leishmaniasis, but in HIV-infected patients both visceral and disseminated cutaneous leishmaniasis are presented. Recurrence of the disease after treatment has been reported in some cases indicating that improved chemotherapy is required. In this study, the susceptibility of L. martiniquensis to Amphotericin B deoxycholate (AmB), allicin, and andrographolide was evaluated and the synergistic effects of allicin or andrographolide combined with AmB against L. martiniquensis intracellular amastigotes in mouse peritoneal exudate macrophages (PEMs) were investigated in vitro for the first time. The results showed that L. martiniquensis was highly susceptible to AmB as expected, but allicin and andrographolide had selectivity index (SI) values greater than 10, indicating promise in both compounds for treatment of host cells infected with L. martiniquensis. Four AmB/allicin combinations presented combination index (CI) values less than 1 (0.58−0.68) for intracellular amastigotes indicating synergistic effects. The combination with the highest dose reduction index (DRI) allowed an approximately four-fold reduction of AmB use in that combination. No synergistic effects were observed in AmB/andrographolide combinations. The data provided in this study leads for further study to develop novel therapeutic agents and improve the treatment outcome for leishmaniasis caused by this Leishmania species.

New Leishmania isolates form a novel group of human parasites related to Leishmania enrietti , with cases in Ghana, Thailand, and Martinique; other relatives infect Australian and South American wildlife. These parasites apparently cause both cutaneous and visceral disease, and may have evolved a novel transmission mechanism exploiting blood-feeding midges. [ABSTRACT FROM AUTHOR]

• Five species of sand flies, i.e., Se. khawi, Se. barraudi, Ph. stantoni, Gr. indica , and Se. hivernus , were systematically identified in the VL focus of southern Thailand. • This is the first molecular evidence of L. martiniquensis and L. donovani complex in Gr. indica and Ph. stantoni , respectively. • Anuran trypanosomes were also ubiquitously detected and phylogenetically divided into two major clades (An04/Frog1 and An01+An02/Frog2). • The novel finding indicates the co-circulation of trypanosomatid parasites and the possible role of sand flies for autochthonous transmission in 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. [ABSTRACT FROM AUTHOR]

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

In this work, we describe the first Leishmania-infecting leishbunyavirus—the first virus other than Leishmania RNA virus (LRV) found in trypanosomatid parasites. Its host is Leishmania martiniquensis, a human pathogen causing infections with a wide range of manifestations from asymptomatic to severe visceral disease. This virus (LmarLBV1) possesses many characteristic features of leishbunyaviruses, such as tripartite organization of its RNA genome, with ORFs encoding RNA-dependent RNA polymerase, surface glycoprotein, and nucleoprotein on L, M, and S segments, respectively. Our phylogenetic analyses suggest that LmarLBV1 originated from leishbunyaviruses of monoxenous trypanosomatids and, probably, is a result of genomic re-assortment. The LmarLBV1 facilitates parasites' infectivity in vitro in primary murine macrophages model. The discovery of a virus in L. martiniquensis poses the question of whether it influences pathogenicity of this parasite in vivo, similarly to the LRV in other Leishmania species. [ABSTRACT FROM AUTHOR]

Leishmania (Mundinia) martiniquensis is a causative agent of visceral leishmaniasis, but in HIV-infected patients both visceral and disseminated cutaneous leishmaniasis are presented. Recurrence of the disease after treatment has been reported in some cases indicating that improved chemotherapy is required. In this study, the susceptibility of L. martiniquensis to Amphotericin B deoxycholate (AmB), allicin, and andrographolide was evaluated and the synergistic effects of allicin or andrographolide combined with AmB against L. martiniquensis intracellular amastigotes in mouse peritoneal exudate macrophages (PEMs) were investigated in vitro for the first time. The results showed that L. martiniquensis was highly susceptible to AmB as expected, but allicin and andrographolide had selectivity index (SI) values greater than 10, indicating promise in both compounds for treatment of host cells infected with L. martiniquensis. Four AmB/allicin combinations presented combination index (CI) values less than 1 (0.58–0.68) for intracellular amastigotes indicating synergistic effects. The combination with the highest dose reduction index (DRI) allowed an approximately four-fold reduction of AmB use in that combination. No synergistic effects were observed in AmB/andrographolide combinations. The data provided in this study leads for further study to develop novel therapeutic agents and improve the treatment outcome for leishmaniasis caused by this Leishmania species. [ABSTRACT FROM AUTHOR]

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.