Asymptomatic leishmaniasis cases have continuously increased, especially among patients with HIV who are at risk to develop further symptoms of cutaneous and visceral leishmaniasis. Thus, early diagnosis using a simple, sensitive and reliable diagnostic assay is important because populations at risk mostly reside in rural communities where laboratory equipment is limited. In this study, the highly sensitive and selective determination of Leishmania infection in asymptomatic HIV patients was achieved using dual indicators (SYBR safe and gold-nanoparticle probe; AuNP-probe) in one-step LAMP method based on basic instruments. The assay can be simply evaluated under the naked eye due to clear interpretation of fluorescent emission of LAMP-SYBR safe dye-complex and colorimetric precipitate of specific AuNP-probes. The sensitivities and specificities of fluorescent SYBR safe dye and AuNP-probe indicators were equal, which were as high as 94.1 and 97.1%, respectively. Additionally, detection limits were 10² parasites/mL (0.0147 ng/µL), ten times more sensitivity than other related studies. To empower leishmaniasis surveillance, this inexpensive one-step SYBR safe and AuNP-LAMP assay is reliably fast and simple for field diagnostics to point-of-care settings, which can be set up in all levels of health care facilities including resource limited areas, especially in low to middle income countries.

Background: Leishmaniasisis an emerging infectious disease reported in the north and south of Thailand of which patients with HIV/AIDS are a high risk group for acquiring the infection. A lack of information regarding prevalence, and the risk association ofLeishmaniainfection among asymptomatic immunocompetent hosts needs further investigation. Information on potential vectors and animal reservoirs in the affected areas is also important to control disease transmission.Methods: An outbreak investigation and a cross-sectional study were conducted following one index case of cutaneous leishmaniasis (CL) caused byL.martiniquensisin an immunocompetent male patient reported in August 2015, Chiang Rai Province, Thailand. From September to November 2015, a total of 392 participants at two study areas who were related to the index case, 130 students at a semi-boarding vocational school and 262 hill tribe villagers in the patient’s hometown, were recruited in this study. The nested internal transcribed spacer 1-PCR (ITS1-PCR) was performed to detectLeishmaniaDNA in buffy coat, and nucleotide sequencing was used to identify species. Antibody screening in plasma was performed using the Direct Agglutination Test (DAT), and associated risk factors were analyzed using a standardized questionnaire. Captured sandflies within the study areas were identified and detected forLeishmaniaDNA using nested ITS1-PCR. Moreover, the animal reservoirs in the study areas were also explored forLeishmaniainfection.Results: Of 392 participants, 28 (7.1%) were positive forLeishmaniainfection of which 1 (4.8%) wasL.martiniquensis, 12 (57.1%) wereL.orientalisand 8 (38.1%) wereLeishmaniaspp. Of 28, 15 (53.6%) were DAT positive. None showed any symptoms of CL or visceral leishmaniasis. Risk factors were associated with being female (adjusted odds ratio, AOR 2.52, 95%CI 1.01–6.26), increasing age (AOR 1.05, 95%CI 1.02–1.08), having an animal enclosure in a housing area (AOR 3.04, 95%CI 1.13–8.22), being exposed to termite mounds (AOR 3.74, 95%CI 1.11–12.58) and having domestic animals in a housing area (AOR 7.11, 95%CI 2.08–24.37). At the semi-boarding vocational school, sixSergentomyia gemmeasamples were PCR positive for DNA ofL.orientalisand oneS.gemmeawas PCR positive for DNA ofL.donovani/L.infantum. Additionally, onePhlebotomus stantoniwas PCR positive for DNA ofL.martiniquensis, and one black rat (Rattus rattus) was PCR positive for DNA ofL.martiniquensis.Conclusion: This information could be useful for monitoringLeishmaniainfection among immunocompetent hosts in affected areas and also setting up strategies for prevention and control. A follow-up study of asymptomatic individuals with seropositive results as well as those with positive PCR results is recommended.

In Thailand, leishmaniasis is an emerging vector-borne disease that has become a public health concern. In related epidemiological surveys to identify potential Leishmania vectors in the affected areas, DNA of Leishmania martiniquensis (Kinetoplastida: Trypanosomatidae) was detected in Sergentomyia (Neophebotomus) gemmea (Diptera: Psychodidae) and Sergentomyia (Parrotomyia) barraudi (Diptera: Psychodidae). Recently, a more elaborate study was conducted in the same areas that included sand fly species identification, screening sand flies for the presence of Leishmania DNA and blood meal analysis to identify potential reservoir hosts directed toward assessing the risk of human infection. Twenty-nine archived pools of sand flies collected in Hat Samran District, Trang Province were used in this study. Sand fly species were confirmed using PCR encompassing regions within the mitochondrial DNA. Leishmania DNA was detected using PCR of the heat shock protein 70 region (hsp70-PCR) and blood meal identification was performed using PCR of the cyt b gene of vertebrate mitochondrial DNA (cytb-nd1-PCR) and human-specific AluYb8 repeat (AluYb8-PCR). Four sand fly species were confirmed, i.e., Phlebotomus (Anaphlebotomus) stantoni (Diptera: Psychodidae), S. barraudi, Sergentomyia (Neophlebotomus) iyengari (Diptera: Psychodidae), and S. gemmea. Leishmania siamensis was detected in one female S. iyengari. Only human blood was detected in P. stantoni and S. gemmea, while both sun skink (Mabuya multifasciata) and human blood were detected in S. iyengari. In this study, we showed that S. iyengari could be a potential vector of L. siamensis infection among humans.

Human liver fluke infection caused by Opisthorchis viverrini increases the risk of cholangiocarcinoma (CCA) reported along the Mekong basin including Thailand, Lao People’s Democratic Republic (PDR), Cambodia, and Vietnam. The highest incidence of CCA has been reported in northeastern Thailand where liver fluke infection is prevalent. This study aimed to investigate the prevalence of O. viverrini infection in a northeastern-descendent community in rural Sa Kaeo Province, eastern Thailand, using stool examination and molecular technique. The Kato–Katz method was performed to determine eggs per gram (EPG) for infection intensity. Phosphate-buffered saline–ethyl acetate concentration was used to prepare specimens for polymerase chain reaction (PCR) and restriction fragment length polymorphism of the internal transcribed spacer 2 (ITS2) region of the ribosomal RNA. From 1,245 specimens, 105 (8.4%) samples were identified as Opisthorchis-like eggs from stool examination, and all positive specimens indicated light infection (< 1,000 EPG). From positive Opisthorchis-like egg samples, 55.2% (58/105) were identified as O. viverrini eggs from ITS2-PCR assay for which low infection intensity might result in a negative PCR result (44.8%). Using multiple logistic regression analysis, males were at 3.1 times higher risk of acquiring O. viverrini infection than females. From phylogenetic analysis, in eastern Thailand, nucleotide sequences of O. viverrini were grouped as a monoclade as those isolated from Greater Mekong, Vietnam, Myanmar, and west Siberia. The results revealed that the surveyed community is a low-grade endemic area of O. viverrini infection. Thus, data from this study can be used to improve health-promoting programs and activities to control the infection and its subsequent CCA.