Incidence and persistence of asymptomatic Leishmania infection among HIV-infected patients in Trang province, Southern Thailand: A cohort study.

Leishmaniasis poses a significant health burden, particularly among immunocompromised patients. In Thailand, Leishmania infection caused by Leishmania martiniquensis and Leishmania orientalis lacks information about the incidence and risk factors among HIV-infected populations. This longitudinal cohort study aimed to investigate the incidence and persistence of Leishmania infection among HIV-infected individuals in an affected area, Trang Province, Southern Thailand. The study also identified risk factors associated with the incidence of Leishmania infection. The study enrolled 373 participants in the HIV clinic, Trang Hospital, who initially tested negative for Leishmania infection during 2015-2016, and 133 individuals initially tested positive for Leishmania infection. Thus, follow-up visits of 506 participants occurred during 2018-2019. Direct Agglutination Test (DAT) and nested PCR (nPCR) identified incidents and persistent cases of Leishmania infection. Cox proportional-hazards regression analyses were performed to assess risk factors for the incidence of Leishmania infection. Among the initially negative group, 12 incident cases comprised one L. orientalis infection and 11 seropositive cases using DAT, resulting in a cumulative incidence of 3.2% and an incidence density of 10.38 per 1000 person-years. Increasing age was a significant predictor of the incidence of Leishmania infection. Five persistent cases comprised one Leishmania donovani complex and four seropositive cases using DAT in the initially positive group, with a cumulative persistence rate of 3.7% and a persistence density of 12.85 per 1000 person-years. All patients were asymptomatic. This study sheds light on the incidence and persistence of Leishmania infection among HIV-infected individuals in Trang Province, Southern Thailand, underscoring the importance of continued monitoring and tailored interventions to mitigate the impact of this co-infection.

Asymptomatic Leishmania Infection among Blood Donors in a Southern Province of Thailand.

Leishmaniasis poses significant public health challenges in endemic regions. Understanding the prevalence of asymptomatic Leishmania infection and identifying risk factors among blood donors is crucial. This study addressed a knowledge gap by evaluating the prevalence of asymptomatic Leishmania infection and pinpointing associated risk factors among blood donors in an endemic area in Thailand and aimed to enhance blood donation safety protocols and reduce the risk of transfusion-transmitted Leishmania infection. A cross-sectional study and a longitudinal follow-up were conducted among 500 blood donors in Trang Province, southern Thailand. A serological test was performed using the direct agglutination test (DAT), and DNA detection was performed using nested polymerase chain reaction (nPCR) to screen for Leishmania infection. Potential risk factors associated with the infection were also assessed. The study identified a 19.0% prevalence of asymptomatic Leishmania infection among blood donors, with nPCR proving more effective in detecting infections (13.0%) than DAT (6.4%). Notably, Leishmania martiniquensis was the predominant species identified, highlighting the local epidemiological profile of Leishmania infection. Furthermore, using multivariate analysis, living in stilt houses was independently associated with Leishmania infection (adjusted odds ratio = 1.85; 95% CI = 1.04-3.28; P = 0.035). A high prevalence of asymptomatic Leishmania infection among blood donors underscores the need for integrating comprehensive Leishmania screening protocols into blood donation processes, particularly in endemic regions. It advocates for using molecular diagnostics to enhance detection accuracy. Furthermore, living in stilt houses as a risk factor emphasizes the importance of environmental management in leishmaniasis control efforts.

Development of nucleic acid lateral flow immunoassay for duplex detection of Leishmania martiniquensis and Leishmania orientalis in asymptomatic patients with HIV.

Leishmaniasis, a neglected tropical disease caused by parasitic protozoa of the Leishmania genus, remains a global health concern with significant morbidity and mortality. In Thailand, the rising incidence of autochthonous leishmaniasis cases involving Leishmania (Mundinia) martiniquensis and novel Leishmania (Mundinia) orientalis underscores the critical need for accurate diagnosis and effective control strategies. This study presents a sensitive and specific nucleic acid lateral flow immunoassay (NALFIA) that integrates a duplex PCR assay with a lateral flow device (LFD) strip format. Targeting the internal transcribed spacer 1 (ITS1) region, known for its unique combination of conserved and variable sequences, this assay employs primers labeled with biotin, digoxigenin, and fluorescein isothiocyanate (FITC) markers, enabling precise species identification and differentiation of these two Leishmania species. Remarkably, the assay achieves a sensitivity that surpasses agarose gel electrophoresis, detecting as few as 10-2 parasite/µL for L. martiniquensis and 10-4 parasite/µL for L. orientalis. Notably, the assay exhibited reliable specificity, revealing no cross-amplification with other major viscerotropic Leishmania species or reference organisms. Evaluation using 62 clinical samples further confirms the effectiveness of the PCR-LFD assay, with a sensitivity of 100% for L. martiniquensis and 83.3% for L. orientalis, and an excellent agreement (κ value = 0.948) with nested PCR. This integrated assay represents a promising advancement in diagnostic tools, offering rapid and accurate results that can significantly contribute to effective disease management and control. Given the increasing relevance of these Leishmania species in current public health scenarios, this assay serves as a valuable tool for both diagnostic and research applications.

Leishmania Infection among HIV-Infected Patients in a Southern Province of Thailand: A Cross-Sectional Study.

Leishmaniasis, a neglected tropical disease, imposes a notable health burden, especially on immunocompromised individuals such as HIV patients. Recognizing its prevalence and risk factors in specific populations is vital for effective prevention. This study in Satun Province, southern Thailand, aimed to ascertain leishmaniasis prevalence and identify associated risks among HIV-infected patients. A cross-sectional study was conducted among 650 HIV-infected individuals at a tertiary care hospital. Data on demographic characteristics, clinical parameters, and potential risk factors were collected. Individual plasma, buffy coat, and saliva samples were collected. Leishmania infection was determined using the direct agglutination test and nested polymerase chain reaction (nPCR) of nPCR-buffy coat and nPCR-saliva. The association between risk factors and Leishmania infection was assessed with logistic regression analysis. The prevalence of Leishmania infection was 8.61% (56/650). Species was identified among 20 HIV-infected patients as follows: Leishmania orientalis (n = 14), Leishmania martiniquensis (n = 4), and Leishmania donovani complex (n = 2). The factors associated with Leishmania infection included age (adjusted odds ratio [OR] = 1.03), intravenous drug use (adjusted OR = 2.39), CD4 cell count <500 cells/mm3 (adjusted OR = 2.40), and a viral load ≥50 copies/mL (adjusted OR = 5.16). The prevalence of Leishmania infection among HIV-infected patients in Satun Province was considerable. These findings underscore the need for integrated care and targeted interventions to address this infection and improve public health outcomes. Further research and collaborative efforts are warranted to develop effective prevention and control strategies for Leishmania infection in the HIV-infected Thai population.

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.

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.

Efficiency of the Stool-PCR Test Targeting NADH Dehydrogenase (Nad) Subunits for Detection of Opisthorchis viverrini Eggs.

Opisthorchis viverrini infection is the major parasitic infection problem in Southeast Asian countries, and long-term infection will lead to cholangiocarcinoma (CCA), the bile duct cancer. The early diagnosis of O. viverrini infection may interrupt the progression of the opisthorchiasis and other related illnesses, especially CCA. The current diagnostic procedure is stool examination by microscope-based methods such as direct smear and concentration techniques but it is limited by low parasite egg numbers. The molecular diagnosis prompts the chance to evaluate the light infection with low number of parasite eggs but is currently inconvenient for routine use due to special equipment requirement and unstable sensitivities. Our present study aims to establish the efficiency of OvNad subunits, the mitochondrial gene, for introducing as a potential diagnostic target by conventional PCR, the cheapest and easiest molecular procedure. A total of 166 stool samples were investigated microscopically by the PBS-ethyl acetate concentration technique (PECT); 75 samples were O. viverrini positive with 28 samples that were positive with single parasite (hookworm, A. lumbricoides, S. stercoralis, Taenia spp., and T. trichiura), 11 samples were with mixed infection, and 52 samples were without parasite detection. The detection limits of OvNad subunits were evaluated in artificially spiked samples containing 0, 1, 5, 10, 20, 50, and 100 Ov-eggs. The result suggested that the best detection efficacy was of OvNad5 that had exact detection limits at only 5 eggs. In the PCR amplification of OvNad subunits, there exist 100% specificities with varied sensitivities from 64%, 88%, 80%, and 100% of OvNad1, OvNad2, OvNad4, and OvNad5, respectively. OvNad subunits were amplified specifically without cross reactivity with the other collected parasites. Our study established that OvNad subunits, especially OvNad5, are the potent candidates for PCR amplification of stool containing Ov-eggs with high confidential sensitivity, specificity, PPV, and NPV even in the light infection that would be a benefit for developing as a routine diagnosis of O. viverrini infection.

Zoonotic Transmission of Blastocystis Subtype 1 among People in Eastern Communities of Thailand: Organic Fertilizer from Pig Feces as a Potential Source.

Blastocystis sp., the most common intestinal protozoa, remains a public health problem among people in many countries, particularly in rural areas of developing countries. The infection usually reflects poor sanitation in communities by waterborne, zoonotic, and person-to-person transmission. Interestingly, at least 17 subtypes (STs) have been reported and are associated with a broad range of animal hosts, including humans. In this study, we reported potential evidence of zoonotic transmission of Blastocystis ST1 in rural communities of eastern Thailand where the overall prevalence of Blastocystis infection was 15.7%. Two major and three minor subtypes were found to be distributed unequally in this region. Of 5 STs, only ST1 was found to be associated with pig feces in an open farm system that produced organic fertilizer for agriculture uses in the community. This finding suggests that properly protective contact and standard production of organic fertilizer from pig feces by-products could be key factors for reducing the prevalence of Blastocystis infection and prevent Blastocystis reinfection among people in the community. IMPORTANCEBlastocystis sp. remains a public health problem among people, particularly in rural areas of many developing countries. The infection usually reflects poor sanitation in communities by waterborne, zoonotic, and person-to-person transmission. In this study, we reported potential evidence of zoonotic transmission of Blastocystis subtype 1 (ST1) in rural communities of eastern Thailand. Two major and three minor subtypes were found to be unequally distributed in this region. Interestingly, only ST1 was found to be associated with pig feces in an open farm system that produced organic fertilizer for agriculture uses in the community. The finding makes significant contributions to genetic and molecular investigations of microbial topics of practical value and suggest that properly protective contact and standard production of organic fertilizer from pig feces by-products could be key factors for reducing the prevalence of Blastocystis infection and prevent Blastocystis reinfection among people in the community.

Prevalence and associated risk factors of Leishmania infection among immunocompetent hosts, a community-based study in Chiang Rai, Thailand.

BACKGROUND: Leishmaniasis is 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 of Leishmania infection 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 by L. martiniquensis in 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 detect Leishmania DNA 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 for Leishmania DNA using nested ITS1-PCR. Moreover, the animal reservoirs in the study areas were also explored for Leishmania infection. RESULTS: Of 392 participants, 28 (7.1%) were positive for Leishmania infection of which 1 (4.8%) was L. martiniquensis, 12 (57.1%) were L. orientalis and 8 (38.1%) were Leishmania spp. 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, six Sergentomyia gemmea samples were PCR positive for DNA of L. orientalis and one S. gemmea was PCR positive for DNA of L. donovani/L. infantum. Additionally, one Phlebotomus stantoni was PCR positive for DNA of L. martiniquensis, and one black rat (Rattus rattus) was PCR positive for DNA of L. martiniquensis. CONCLUSION: This information could be useful for monitoring Leishmania infection 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.

Improvement of a PCR-based method for the detection of Opisthorchis viverrini eggs in human stool samples by targeting internal transcribed spacer-2 (ITS-2), cytochrome oxidase subunit 1 (cox1), and cytochrome b (cyb).

Opisthorchis viverrini infection causes various complications in patients, ranging from asymptomatic to severe chronic disease including cholangiocarcinoma (CCA). O. viverrini is endemic in Southeast Asia and acting as a risk for CCA. Early diagnosis of O. viverrini infection can reduce the number of CCA cases. The routine diagnosis for opisthorchiasis is direct wet smear, sometimes coupled with concentration techniques, which has limitations when investigating light infection or if done by laboratorians with lack of experiences. PCR-based methods have been established for the detection of O. viverrini egg DNA from stool samples, but have never fully succeeded for light infections when compared to wet smear concentration techniques. This study aims to improve the PCR-based method for detection of O. viverrini eggs in stool samples by targeting the genes ITS-2, cox1, and cyb. The results reveal higher sensitivity than conventional concentration techniques, with all newly designed primers. ITS-2 has an overall sensitivity of 76.9% with 66.7% in the samples with < 50 EPG, while cox1 has shown 96.2% overall sensitivity and 94.1% in the same EPG intervals. Interestingly, the new pointing target, cyb, has shown 100% sensitivity in all egg intervals in this study, particularly for light infections (EPG less than 100). No cross-reactivity was found in Taenia spp., Trichuris trichiura, Ascaris lumbricoides, Capillaria philippinensis, and hookworm. The procedure is convenient, with shorter steps compared to previous reports, and it appears appropriate for use in the diagnosis of light infection with O. viverrini. These three genes are good candidates for use in PCR-based detection of the parasite eggs. Further testing with a larger cluster of samples is however necessary.

Development of loop-mediated isothermal amplification (LAMP) assay using SYBR safe and gold-nanoparticle probe for detection of Leishmania in HIV patients.

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.

Heat-enhancing aggregation of gold nanoparticles combined with loop-mediated isothermal amplification (HAG-LAMP) for Plasmodium falciparum detection.

Malaria infection represents a major public health and economic issue that leads to morbidity and mortality globally. A highly effective and uncomplicated detection tool is required for malaria control in geographical hotspots of transmission. We developed a simple and more sensitive novel approach for the detection of the 18S rRNA gene of Plasmodium falciparum based on loop-mediated isothermal amplification (LAMP) and visualization using colorimetric, streptavidin-functionalized gold nanoparticles (SA-GNPs). Two loop primers of LAMP were biotinylated to produce biotin-containing products during amplification. After the addition of SA-GNPs, clusters of avidin-biotin complexes were established in the LAMP structure. While the positive reactions remained wine red, the negative reactions became colorless with partial aggregations induced by hydrochloric acid (HCl) under heat enhancement (60 °C). All steps of the assay were completed within 50 min, its detection limit was 1 parasite/µL, and it was highly specific for P. falciparum. This effortless detection system with high sensitivity and specificity could provide an alternative choice for malaria diagnostics in resource-limited regions.

Development of a fluorescent distance-based paper device using loop-mediated isothermal amplification to detect Escherichia coli in urine.

The highly sensitive and selective determination of Escherichia coli (E. coli) in urine was achieved using a SYBR™ safe loop-mediated isothermal amplification (LAMP) method with a distance-based paper device. New primers set specific to multi-copy the 16s rRNA gene of E. coli were designed and used in this study. The detection sensitivity of these primers was higher than in related work and they could be incorporated with a low-cost paper-based device to quantify E. coli in urine at a concentration lower than 101 CFU mL-1. Regarding standard artificial urine, a linear range of a 10-fold dilution of E. coli concentration (105-100 CFU mL-1) with an R-square value (R2) = 0.9823 was observed directly using a fluorescent migratory distance of the 4 µL reaction mixture in the detection zone under blue light without the need for postreaction staining process. Based on the device, E. coli infection could be significantly categorized into 3 groups; none, light, and heavy levels, which is beneficial for UTI diagnosis. Hence, this paper-based device is suitable for use with the SYBR™ Safe-LAMP assay to semi-quantify E. coli, especially in resource-limited settings due to advantages of low cost, simple fabrication and operation, and no requirement for sophisticated instruments, as well as its disposability and portability.

Simplified closed tube loop mediated isothermal amplification (LAMP) assay for visual diagnosis of Leishmania infection.

In Thailand, asymptomatic leishmaniasis cases have continuously increased, especially among patients with HIV at risk to develop further symptoms of cutaneous and visceral leishmaniasis. Thus, earlier diagnosis using a simple, sensitive and reliable diagnostic tool is needed because populations at risk mostly reside in rural communities where only basic laboratory equipment is available for health care services. In this present study, a closed tube loop mediated isothermal amplification (LAMP) was developed using a piece of parafilm placed between the dye and LAMP reaction mixture to form semi-layer that partially secured SYBR green I from spilling during amplification. No post-amplification preparation was required and accidental spill of the dye during LAMP amplification was prevented. The result could be visually interpreted under visible and UV lights after dye spinning down. The semi-layer modification of a closed tube LAMP showed successful amplification of Leishmania DNA with clear interpretation using both color and fluorescence dyes when observing by the naked eye. The sensitivity and specificity were as high as 94.4 and 96.9%, respectively whereas detection limits were 102 parasites/mL being ten fold more sensitive than other related studies. This user-friendly inexpensive approach is affordable and suitable for empowering leishmaniasis surveillance without the need of expensive devices in all levels of hospitals, including health services, as well as fieldwork, especially in low income countries.

Genetic Differentiation of Opisthorchis-Like Eggs in Northern Thailand Using Stool Specimens Under National Strategic Plan to Control Liver Fluke Infection and Cholangiocarcinoma.

Liver fluke infection caused by Opisthorchis viverrini is recognized as a potential risk factor for cholangiocarcinoma (CCA). The National Strategic Plan to Control Liver Fluke Infection and Cholangiocarcinoma has implemented microscopic-based stool examination screening. However, eggs of O. viverrini and minute intestinal flukes (MIFs) are nearly morphologically similar and could result in inaccurate O. viverrini diagnosis. Stool specimens were collected from eight districts of Chiang Mai Province in northern Thailand. Opisthorchis-like eggs were identified with the Kato-Katz technique and differentiated for O. viverrini and MIFs using molecular study by PCR and PCR-restriction fragment length polymorphism targeting the internal transcribed spacer 2 (ITS2) gene. Prevalence of Opisthorchis-like eggs was 5.9% from a total of 9,570 specimens. From PCR assays, all liver flukes were O. viverrini and all MIFs were Haplorchis taichui. The distribution of species was H. taichui (38.2%), O. viverrini (10.5%), coinfection of H. taichui and O. viverrini (37.2%), and 14.1% were negative from PCR. Totally, H. taichui was found in 75.4% of infections from Opisthorchis-like specimens. ITS2 nucleotide sequencing analysis showed a single variant of O. viverrini with no variation and two variants of H. taichui. This study first revealed the genetic background of Opisthorchis-like eggs in northern Thailand. Minute intestinal flukes are occasionally misdiagnosed as O. viverrini leading to misinterpretation and overestimation of the burden of O. viverrini infection. Molecular diagnosis such as PCR could effectively discriminate species of Opisthorchis-like eggs and help shape the robustness of epidemiological data to control liver fluke infection and raise awareness of other risk factors for CCA.

An Epidemiological Survey of Opisthorchis viverrini Infection in a Lightly Infected Community, Eastern Thailand.

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.

Phenotypic and genotypic characterization of Thai isolates of Plasmodium falciparum after an artemisinin resistance containment project.

BACKGROUND: In Thailand, artemisinin-based combination therapy (ACT) has been used to treat uncomplicated falciparum malaria since 1995. Unfortunately, artemisinin resistance has been reported from Thailand and other Southeast Asian countries since 2003. Malarone®, a combination of atovaquone-proguanil (ATQ-PG), has been used to cease artemisinin pressure in some areas along Thai-Cambodia border, as part of an artemisinin resistance containment project since 2009. This study aimed to determine genotypes and phenotypes of Plasmodium falciparum isolates collected from the Thai-Cambodia border after the artemisinin resistance containment project compared with those collected before. RESULTS: One hundred and nine of P. falciparum isolates collected from Thai-Cambodia border from Chanthaburi and Trat provinces during 1988-2016 were used in this study. Of these, 58 isolates were collected after the containment. These parasite isolates were characterized for in vitro antimalarial sensitivities including chloroquine (CQ), quinine (QN), mefloquine (MQ), piperaquine (PPQ), artesunate (AS), dihydroartemisinin (DHA), ATQ and PG and genetic markers for drug resistance including the Kelch13 (k13), Plasmodium falciparum chloroquine resistance transporter (pfcrt), P. falciparum multidrug resistance 1 (pfmdr1) and cytochrome b (cytb) genes. Mean CQ, QN, MQ, PPQ and AS IC50s of the parasite isolates collected from 2009 to 2016 exhibited significantly higher than those of parasites collected before 2009. Approximately 57% exhibited in vitro MQ resistance. Approximately 94% of the isolates collected from 2009 to 2016 contained the pfmdr1 184F allele. Mutations of the k13 gene were detected in approximately 90% of the parasites collected from 2009 to 2016 which were significantly higher than the parasite isolates collected before. No ATQ-resistant genotype and phenotype of P. falciparum were found among the isolates collected after the containment project. CONCLUSIONS: Although the containment project had been implemented in this area, the expansion of artemisinin-resistant parasites did not decline. In addition, reduced sensitivity of the partner drugs of ACT including MQ and PPQ were identified.

In Vitro Sensitivity of Pyronaridine in Thai Isolates of Plasmodium falciparum.

Pyronaridine, a Mannich base antimalarial agent with a high activity against chloroquine-resistant Plasmodium falciparum, has been combined with artesunate as a new artemisinin based combination therapy (ACT). Pyronaridine-artesunate combination could be one of the choices for the treatment of uncomplicated falciparum malaria in multidrug-resistant areas including Thailand. The aim of this study was to determine in vitro sensitivity and cross-resistance pattern of pyronaridine in Thai isolates of P. falciparum. In addition, the influence of resistant genes concerning in vitro pyronaridine sensitivity was determined. The mean pyronaridine 50% inhibitory concentration (IC50) of 118 parasite isolates was 5.6 ± 3.1 nM (range = 0.2-15.4 nM) with a significant positive correlation with artesunate IC50 (r = 0.246, P = 0.008) and amodiaquine IC50 (r = 0.220, P = 0.042) and a significant negative correlation with quinine IC50 (r = -0.185, P = 0.047). Parasites containing the pfmdr1 86Y allele exhibited significantly reduced pyronaridine sensitivity compared with those with the pfmdr1 N86 allele (7.6 ± 3.3 nM and 5.4 ± 3.0 nM, respectively, P = 0.032, independent t test); however, the difference may not be clinically relevant. Pyronaridine-artesunate could be the candidate ACT to treat multidrug-resistant falciparum malaria in Thailand with careful monitoring.

Molecular discrimination of Opisthorchis-like eggs from residents in a rural community of central Thailand.

BACKGROUND: Opisthorchis viverrini infection is a major public health problem in northern and northeastern Thailand. The chronic infection of O. viverrini is related to cholangiocarcinoma which causes high mortality in endemic areas. Therefore, the diagnosis, treatment, control and prevention of O. viverrini infection are necessary. The morphology of the egg is very similar to that of other species of human liver flukes (Opisthorchis felineus and Clonorchis sinensis) as well as that of small intestinal flukes in the family Heterophyidae. Thus, molecular characterization is crucially required to discriminate species of Opisthorchis-like eggs in fecal examination. METHODOLOGY/PRINCIPAL FINDINGS: We aimed to determine the prevalence of O. viverrini infection among villagers living in Sanamchaikate District, Chachoengsao Province, in central Thailand, where O. viverrini infection has previously been reported. A total of 2,609 fecal samples were examined for Opisthorchis-like eggs using microscopic examination. PCR-RFLP analysis of the ITS2 region was used to discriminate Opisthorchis-like eggs. The genetic structure of O. viverrini infection was demonstrated using nucleotide sequencing of cytochrome c oxidase subunit I (cox1) and NADH dehydrogenase subunit 1 (nad1). Testing of evolutionary neutrality of the cox1 and nad1 sequences of O. viverrini was performed using Tajima's D tests and Fu's Fs tests. Moreover, the haplotype networks and phylogenetic trees were constructed to study the relationships of O. viverrini isolated from different endemic areas. A high prevalence of O. viverrini infection is still observed in a rural community of Chachoengsao Province, central Thailand. The overall prevalence of Opisthorchis-like eggs using microscopic examination was 16.8%. PCR-RFLP profiles showed the predominant infection of O. viverrini (9.6%) including very low infections of other small intestinal flukes, Haplorchis taichui (0.08%) and Euparyphium albuferensis (0.08%). The genetic structure of O. viverrini populations in central Thailand was also described and revealed a non-significant difference in genetic diversity. In addition, the genetic background of the O. viverrini populations was closely related to the isolate from Lao PDR. CONCLUSIONS/SIGNIFICANCE: Our study highlighted the prevalence of O. viverrini infection in central Thailand indicating that control programs and health education regarding opisthorchiasis is still required in this endemic area. Additionally, the study demonstrated the genetic structure of O. viverrini, in central Thailand which could provide information on the molecular epidemiology of this parasite.