Molecular identification and genetic diversity of zoonotic hookworm infections in domestic dogs from northeastern, Thailand.

Hookworm infections remain a significant public health concern in tropical and subtropical regions, including Thailand. This study investigated the species and genetic diversity of hookworm infections in domestic dogs from northeastern Thailand. The molecular analysis focused on amplifying and sequencing specific regions of ribosomal RNA genes (ITS1-5.8S-ITS2 region) and the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene in hookworm larvae recovered from 21 domestic dog stool samples. Among 21 larvae (one larva per infected dog) analyzed, 14 had sequences identical to Ancylostoma caninum, and 7 showed sequences almost identical to Ancylostoma ceylanicum. Phylogenetic analysis of cox1 sequences placed A. caninum and A. ceylanicum in separate clades. The median-joining network of A. caninum cox1 sequences from Thailand showed high haplotype diversity and belonged to the same cluster as sequences from Australia while forming separate clusters from those of A. caninum samples from the USA. The available published A. ceylanicum cox1 sequences (n = 33), in combination with seven sequences in the present study, represented 15 haplotypes distributed among three clusters. Interestingly, A. ceylanicum sequences from dogs and humans shared the same haplotypes. These findings are crucial for recognizing the potential for zoonotic transmission, highlighting the necessity for targeted control measures, and increasing awareness among pet owners and healthcare professionals to mitigate the risk of hookworm transmission to humans.

Preliminary findings and molecular characterization of thin-walled Sarcocystis species in hearts of cattle and buffaloes in Thailand, Lao PDR, and Cambodia.

Cattle and buffaloes, popular protein sources worldwide, are intermediate hosts for several Sarcocystis species. These coccidian protozoans cause sarcocystosis resulting in subclinical and chronic infections in striated muscles by forming macrocysts or microcysts. In Thailand, Lao People's Democratic Republic, and Cambodia, Sarcocystis species have been reported, but molecular identification has been lacking. This study investigates the prevalence of infection, histo-morphology, and molecular identification of Sarcocystis species in hearts of cattle and buffalo sold in local markets. A phylogenetic tree inferred from a portion of the 18S ribosomal (r) RNA gene was used to identify the genus and species of Sarcocystis. The mitochondrial cytochrome c oxidase subunit 1 (cox-1 gene) was sequenced to confirm the species of host tissue. In Thailand, Sarcocystis was detected in 66.7% (14/21) of samples. In Lao People's Democratic Republic, 90% (9/10) of samples were infected and in Cambodia 100% (8/8). For the first time from these countries, we report Sarcocystis cruzi, Sarcocystis heydorni, and Sarcocystis levinei found in taurine cattle (Bos taurus) and water buffalo (Bubalus bubalis). Zoonotic protozoan transmission needs to be controlled by inspection activities by local health inspectors, and appropriate action is required at all points in the food chain by competent authorities to protect consumer health and prevent sarcocystosis in cattle and water buffaloes.

Comparative assessment of immunochromatographic test kits using somatic antigens from adult Opisthorchis viverrini and IgG and IgG4 conjugates for serodiagnosis of human opisthorchiasis.

Chronic infections of humans with Opisthorchis viverrini and Clonorchis sinensis spanning decades may lead to life-threatening pathology prior to cholangiocarcinoma (CCA), which usually has a poor prognosis. Serological tools can support the parasitological examination in clinical diagnosis and support screening for risk of CCA. We developed novel immunochromatographic test kits using a soluble, somatic tissue extract of adult O. viverrini worms as an antigen and colloidal gold-labeled conjugates of IgG and IgG4 antibodies, and evaluated the diagnostic values of both the OvSO-IgG and OvSO-IgG4 kits. For diagnosis of human opisthorchiasis individually, the diagnostic sensitivity, specificity, and positive and negative predictive values with 95% confidence intervals in the OvSO-IgG kit were 86.6% (78.9-92.3), 89.5% (84.2-93.5), 82.9% (74.8-89.2), and 91.9% (87.0-95.4), respectively, while the 75% (65.9-82.7), 98.4% (95.5-99.7), 96.6% (90.3-99.3), and 87% (81.7-91.2), respectively, for the OvSO-IgG4 kit at the prevalence of infection of 37.1%. Twenty-three (76.7%) and 14 (46.7%) of 30 clonorchiasis sera showed positive reactivity with the OvSO-IgG and OvSO-IgG4 kits, respectively. There was 84.1% (κ-value = 0.649) concordance between the two kits, which was statistically significant (p < 0.001). Both ICT kits can be employed as quick and easy point-of-care diagnostic tools, and hence, the OvSO-IgG and OvSO-IgG4 kits can support expanded capacity for clinical diagnosis of human opisthorchiasis and clonorchiasis. These kits may find utility in large-scale surveys in endemic areas where there are limited sophisticated medical facilities or capacity.

Development of immunochromatographic device as a point-of-care tool for serodiagnosis of human strongyloidiasis cases.

Human strongyloidiasis is an important gastrointestinal disease with an estimated 30 to 100 million people infected. Prevalence is generally underestimated since many infections are asymptomatic, and traditional diagnostic tests based on parasitological examination of stool samples are not adequately sensitive. Serological tests are useful and supportive but are still only available in a reference research setting. We made an immunochromatographic test (ICT) kit for rapid serodiagnosis of human strongyloidiasis. The antigen used in the ICT kit was extracted from larvae of Strongyloides stercoralis. Diagnostic efficacy of the kit was evaluated using human serum samples from strongyloidiasis patients, healthy persons, and those with other parasitoses. When using a cutoff level of 0.5 or above, the diagnostic sensitivity, specificity, and positive and negative predictive values at the prevalence of infection of 34.4%, were 93.3%, 83.7%, 76.7%, and 95.6%, respectively. This ICT kit is easy to use at the point-of-care and a result can be obtained in 15 min. Sophisticated instruments and highly trained staff are not required. It can be used in several diagnostic and public-health settings, e.g., prevalence surveys in endemic areas, confirmation and monitoring of cure post-treatment, diagnosis and screening of infected but asymptomatic individuals, and populations "at risk" for hyperinfection syndrome or disseminated strongyloidiasis if they are given immunosuppressive treatment for other conditions.

Development of an immunochromatographic device to detect antibodies for rapid diagnosis of human angiostrongyliasis.

Cerebral angiostrongyliasis is a central nervous system disease caused by Angiostrongylus cantonensis and can produce eosinophilic meningitis or meningoencephalitis in humans. Sero-immunological techniques, such as the enzyme-linked immunosorbent assay (ELISA) and immunoblotting, are most commonly used for the diagnosis of human angiostrongyliasis. However, diagnosis in remote areas remains problematic because sophisticated equipment and specialized skills are required. To overcome, we have developed the immunochromatographic test (ICT) kit for rapid serological diagnosis of angiostrongyliasis through the detection of anti-A. cantonensis-specific antibodies in human serum. A recombinant A. cantonensis galectin-2 (rAcGal2) from young adult female worms was used as an antigen for the ICT kit development. Diagnostic values were evaluated and compared using the ELISA. The sensitivity, specificity and positive and negative predictive values of the ICT kit were 87.0, 96.5, 94.6 and 91.4%, respectively, and those of the ELISA were 91.0, 97.2, 95.8 and 94.0%, respectively. The concordance of the ICT kit was 93.9%. We, thus, determined that the ICT kit is sensitive and specific and provides reliable diagnostic results. It is rapid and simple to perform and can be utilized for both point-of-care diagnosis in the bedside laboratory and epidemiological surveys in endemic regions where access to diagnostic equipment is limited.

Effectiveness of Fasciola gigantica excretory-secretory and recombinant cathepsin L antigens for rapid diagnosis of human fascioliasis using immunochromatographic devices.

Fascioliasis, a food- and water-borne trematodiasis, has been identified as a public health threat by the World Health Organization, with millions of people estimated to be infected or at risk of infection worldwide. We developed an immunochromatographic test (ICT) as a point-of-care (POC) tool for the rapid serodiagnosis of human fascioliasis caused by Fasciola gigantica and evaluated their diagnostic ability. Two tests were developed using antigens from adult F. gigantica excretory-secretory (ES) product and recombinant F. gigantica cathepsin L (rFgCL). Sera from 12 patients with parasitologically proven fascioliasis caused by F. gigantica, 18 with clinically suspected fascioliasis, 65 with other parasitic infections, and 30 healthy controls were used. Using a cutoff of > 0.5 for antibody detection, the sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of the ES-based ICT method were 100%, 98.9% 96.8%, 100%, and 99.2%, respectively, and those of the rFgCL-based ICT method were 86.7%, 93.7%, 81.3%, 95.7%, and 92.0%, respectively. The concordance between the two methods was 91.2%. Tests using F. gigantica ES and rFgCL antigens can be employed quickly and easily as POC diagnostic tools. They can be used to support the clinical diagnosis of human fascioliasis gigantica and in large-scale surveys in endemic areas throughout tropical regions without necessitating additional facilities or ancillary supplies.

Evaluating the in vitro efficacy of gatifloxacin, levofloxacin and gentamicin against Acanthamoeba cysts.

PURPOSE: To evaluate the in vitro efficacy of three commercial ophthalmic solutions (gatifloxacin, levofloxacin and gentamicin) against cysts of Acanthamoeba species. DESIGN: Experimental study METHODS: Acanthamoeba cysts belonging to genotypes T3, T4 and T5 were incubated with three ophthalmic solutions for different periods of time; 1, 24, 48 and 72 h at 37 °C. After incubation, treated cysts were stained with trypan blue and counted to express the percent of growth inhibition. Additionally, the viability of treated cysts was assessed by culturing them in PYG medium at 30 °C for 72 h as well as on non-nutrient agar plates at 30 °C for 1 month. RESULTS: Acanthamoeba cysts of all genotypes were susceptible to gentamicin and gatifloxacin after exposure for 1 h and 24 h, respectively, and for levofloxacin, cysts of all genotypes were resistant to levofloxacin even after 72 h of incubation. Gentamicin and gatifloxacin showed statistically highly significant difference (P < 0.001), and levofloxacin showed statistically significant difference (P < 0.05) in comparison to non-treated control. CONCLUSIONS: Gentamicin and gatifloxacin were highly effective against Acanthamoeba cysts. Although our results should be confirmed in animal models, this result will guide the choice of the appropriate ophthalmic drugs for early treatment of eye infection caused by Acanthamoeba spp.

Molecular identification and genetic diversity of Gnathostoma spinigerum larvae in freshwater fishes in southern Lao PDR, Cambodia, and Myanmar.

Gnathostomiasis, an emerging food-borne parasitic zoonosis in Asia, is mainly caused by Gnathostoma spinigerum (Nematoda: Gnathostomatidae). Consumption of raw meat or freshwater fishes in endemic areas is the major risk factor. Throughout Southeast Asia, including Thailand, Lao PDR, Cambodia, and Myanmar, freshwater fish are often consumed raw or undercooked. The risk of this practice for gnathostomiasis infection in Lao PDR, Cambodia, and Myanmar has never been evaluated. Here, we identified larvae of Gnathostoma species contaminating freshwater fishes sold at local markets in these three countries. Public health authorities should advise people living in, or travelling to, these areas to avoid eating raw or undercooked freshwater fishes. Identification of larvae was done using molecular methods: DNA was sequenced from Gnathostoma advanced third-stage larvae recovered from snakehead fishes (Channa striata) and freshwater swamp eels (Monopterus albus). Phylogenetic analysis of a portion of the mitochondrial cytochrome c oxidase subunit I gene showed that the G. spinigerum sequences recovered from southern Lao PDR, Cambodia, and Myanmar samples had high similarity to those of G. spinigerum from China. Sequences of the nuclear ribosomal DNA internal transcribed spacer 2 region closely resembled sequences of G. spinigerum from Thailand, Indonesia, the USA, and central Lao PDR. This is the first molecular evidence of G. spinigerum from freshwater fishes in southern Lao PDR, Cambodia, and Myanmar.

Current high prevalences of Strongyloides stercoralis and Opisthorchis viverrini infections in rural communities in northeast Thailand and associated risk factors.

BACKGROUND: Two important helminths, Strongyloides stercoralis (an intestinal roundworm) and Opisthorchis viverrini (a liver fluke), are endemic in northeast Thailand. There have been national campaigns in place aimed at the control and eradication of soil-transmitted helminthiasis and opisthorchiasis in Thailand for several decades. However, these helminths still exist and raise concerns regarding public health. This study aimed to evaluate the current prevalence of S. stercoralis and O. viverrini infections in rural communities in northeast Thailand. The data from this study will be useful to improve strategies for future helminth prevention and control. METHODS: A cross-sectional study was conducted from December 2016 to June 2017 in Mueang Khon Kaen district in Khon Kaen, Thailand. The participants were selected using a simple random sampling method. Demographic data were collected using a questionnaire. Stool samples were collected and processed using agar plate culture to determine the presence of S. stercoralis infection and an in-house formalin-ethyl acetate concentration technique to determine the presence of O. viverrini and other intestinal parasite infections (IPIs). RESULTS: In total, 602 persons were enrolled. However, only 526 were analyzed for S. stercoralis and 387 for O. viverrini risk factors. The overall prevalence of S. stercoralis infection was 23.0% (95% confidence interval [95%CI]: 19.4 to 26.6). The prevalence of O. viverrini infection and IPIs other than S. stercoralis was 20.4% (95%CI: 16.5 to 24.8). The prevalence of O. viverrini infection was 19.4% (95%CI: 15.6 to 23.7). Male sex was significantly associated with S. stercoralis infection [Adjusted Odds Ratio (aOR) 4.0; 95%CI: 2.5 to 6.2; P-value < 0.001]. Males were significantly more likely to be infected with O. viverrini and other IPIs (aOR 4.1; 95%CI: 2.3 to 7.2, P-value < 0.001). CONCLUSIONS: This study demonstrated that the updated prevalence of intestinal parasite infections is still high in rural communities in northeast Thailand, especially that of strongyloidiasis and opisthorchiasis.

Impact of the health education and preventive equipment package (HEPEP) on prevention of Strongyloides stercoralis infection among rural communities in Northeast Thailand: a cluster randomized controlled trial.

BACKGROUND: Strongyloidiasis is prevalent in northeast Thailand. This study aimed to evaluate the impact of the Health Education and Preventive Equipment Package (HEPEP), a package we developed to improve awareness and aid in the prevention of Strongyloides stercoralis infection among rural communities in northeast Thailand. METHODS: This was an intervention trial conducted in 12 villages (six interventions and six controls) in rural areas of northeast Thailand from March 2016 to September 2017. Single stool sample was collected from each participant and examined using agar plate culture (APC) technique. Each participant was interviewed using a pre-tested questionnaire, treated with single dose of ivermectin (200 µg/Kg), and allocated to either the intervention or control group. Members of the intervention group were given "Practices to Prevent Strongyloidiasis" poster and vinyl boards containing information aimed at raising awareness of S. stercoralis and strongyloidiasis. In addition, they were given a poster lecture regarding the lifecycle of S. stercoralis before being treated with ivermectin. Aside from that, they were also given a protective equipment package. Monthly refresher courses were provided by village health volunteers (VHVs) regarding the health information they had received and proper equipment usage. The control group, on the other hand, was only provided with a five-minute lecture regarding strongyloidiasis. Assessment of new infection was conducted 3 months later in 327 and 318 participants in the intervention group and control group, respectively. RESULTS: The HEPEP had 41% greater efficacy in preventing S. stercoralis infection in the intervention group than the measures taken in the control group (adjusted Odds Ratio (aOR) = 0.59; 95%CI: 0.41 to 0.85, P-value = 0.005). The intervention group also scored significantly higher on all aspects of a test of S. stercoralis knowledge compared with the control group (mean difference (mean dif.) = 2.89, P-value = < 0.05). CONCLUSIONS: The HEPEP was the first model that has been found to be effective in controlling of S. stercoralis in rural communities in the northeast Thailand. The results should encourage policy makers and public health personnel to improve control programs, as well as health promotion, with regard to parasites. TRIAL REGISTRATION: Thai Clinical Trials Registry (TCTR), Medical Research Foundation of Thailand, Medical Research Network of the Consortium of Thai Medical Schools: MedResNet (Thailand) (identification number: TCTR20180404002 ) Registered 4 April 2018 (retrospectively registered).

Immuno-proteomic analysis of Trichinella spiralis, T. pseudospiralis, and T. papuae extracts recognized by human T. spiralis-infected sera.

The present study explored potentially immunogenic proteins of the encapsulated (Trichinella spiralis) and non-encapsulated (T. pseudospiralis, T. papuae) species within the genus Trichinella. The somatic muscle larval extracts of each species were subjected to immunoblotting analysis using human T. spiralis-infected serum samples. Fifteen reactive bands of all three species were selected for further protein identification by liquid chromatography-tandem mass spectrometry, and their possible functions were ascertained using the gene ontology. Our findings showed immunogenic protein patterns with molecular mass in the range of 33-67 kDa. Proteomic and bioinformatic analysis revealed a wide variety of functions of 17 identified proteins, which are associated with catalytic, binding, and structural activities. Most proteins were involved in cellular and metabolic processes that contribute in the invasion of host tissues and the larval molting processes. The parasite proteins were identified as actin-5C, serine protease, deoxyribonuclease-2, and intermediate filament protein ifa-1. This information may lead to alternative tools for selection of potential diagnostic protein markers or aid in the design of vaccine candidates for prevention and control of Trichinella infection.

Molecular identification of Ascaris lumbricoides and Ascaris suum recovered from humans and pigs in Thailand, Lao PDR, and Myanmar.

Ascaris lumbricoides is the largest roundworm known from the human intestine while Ascaris suum is an internal parasite of pigs. Ascariasis, caused by Ascaris lumbricoides, has a worldwide distribution. Here, we have provided the first molecular identification of Ascaris eggs and adults recovered from humans and pigs in Thailand, Lao PDR, and Myanmar. We amplified and sequenced nuclear ribosomal DNA (ITS1 and ITS2 regions) and mitochondrial DNA (cox1 gene). Sequence chromatograms of PCR-amplified ITS1 region revealed a probable hybrid genotype from two human ascariasis cases from Chiang Mai Province, northern Thailand. All complete ITS2 sequences were identical and did not differ between the species. Phylogenetic trees and haplotype analysis of cox1 sequences showed three clusters with 99 haplotypes. Forty-seven samples from the present study represented 14 haplotypes, including 7 new haplotypes. To our knowledge, this is the first molecular confirmation of Ascaris species in Thailand, Lao PDR, and Myanmar. Zoonotic cross-transmission of Ascaris roundworm between pigs and humans probably occurs in these countries.

An eleven-year retrospective hospital-based study of epidemiological data regarding human strongyloidiasis in northeast Thailand.

BACKGROUND: Human strongyloidiasis is a chronic and persistent gastrointestinal disease caused by infection with soil-transmitted helminths of the genus Strongyloides. The aim of this research was to obtain diagnostic prevalence regarding strongyloidiasis in northeast Thailand through a hospital-based study. METHODS: Patients' demographic data and the results of stool examinations conducted using the formalin ethyl acetate concentration technique were collected from the parasitology laboratory records at Srinagarind Hospital in Khon Kaen, Thailand. The relevant information from years 2004 to 2014 was collected and descriptively analyzed. RESULTS: Of a total of 22,338 patients, 3889 (17.4%) had stool samples that tested positive for Strongyloides larvae. The highest prevalence was 22.8% (95% CI = 19.6-26.2%) in the year 2004. This percentage progressively decreased, reaching 11.2% (95% CI = 10.2-12.4%) in 2013 and remaining stable at 12.9% (95% CI = 11.8-14.1%) in 2014. Males (2741 cases) had double the positivity rate of females (1148 cases). The prevalence of infection was highest (25.9%; 95% CI = 24.5-27.3%) among patients that were 51-60 years of age. CONCLUSIONS: Areas endemic for strongyloidiasis should be emphasized under the national helminth control program and health education campaigns. Nationwide assessments should also be performed regarding Strongyloides infection, including risk factors, treatment, and prevention. The diagnostic laboratory data presented here identify the geographical focus of disease to be the northeastern region of the country. Further targeted surveillance using more sensitive methods will almost certainly reveal a higher individual disease burden than found in this report.

Identification of antigenic proteins in Strongyloides stercoralis by proteomic analysis.

Strongyloides stercoralis is an intestinal helminth that infects people worldwide. Hyperinfection or disseminated human strongyloidiasis can involve vital organs, leading to lethal outcomes. We analyzed immunoproteomics of antigenic spots, derived from S. stercoralis third-stage larvae and reacted with human strongyloidiasis sera, by two-dimensional gel electrophoresis and immunoblotting. Of 26 excised antigenic spots analyzed by liquid chromatography-electrospray ionization-tandem mass spectrometry, 20 proteins were identified. Most proteins were associated with enzymes involved in the metabolic process, energy generation, and oxidation-reduction. The proteins relate to promotion of worm development, cell division, cell signaling and transportation, and regulation of muscular contraction. Identification of antigenic proteins shows promise in helping to discover potential diagnostic protein markers or vaccine candidates for S. stercoralis infection.

Development and evaluation of a rapid diagnostic immunochromatographic device to detect antibodies in sera from intestinal capillariasis cases.

Intestinal capillariasis, a fish-borne nematodiasis, is an important emerging zoonotic disease. Patients present clinical symptoms of borborygmus chronic diarrhea, intermittent abdominal pain, weight loss, and several degrees of painless lower-leg edema. Death may occur in cases of misdiagnosis and improper treatment. Diagnosis is difficult because of the atypical clinical symptoms and diagnostic confusion with diarrhea caused by gastrointestinal cancer, opportunistic infections in human immunodeficiency virus patients, and hyperthyroidism. In addition, parasite eggs are not always found in stool examination. Serology can provide a supportive diagnostic tool. We have produced a rapid and simple immunochromatographic test (ICT) kit for diagnosis of intestinal capillariasis by detection of diagnostic antibodies in human sera. Serum samples from healthy volunteers and patients with proven intestinal capillariasis and other parasitic diseases were evaluated with the Trichinella spiralis larval extract antigen absorbed ICT strips. The diagnostic sensitivity, specificity, and positive and negative predictive values were 100, 96.6, 90.2, and 100%, respectively. The ICT kit is simple and rapid to use and can supplement stool examination in clinical diagnosis of intestinal capillariasis. The test can be completed in 15 min without a need for any sophisticated instruments or reagents.

First molecular identification and genetic diversity of Strongyloides stercoralis and Strongyloides fuelleborni in human communities having contact with long-tailed macaques in Thailand.

The parasitic nematodes, Strongyloides stercoralis and Strongyloides fuelleborni, can infect humans and non-human primates. We amplified and sequenced a portion of the 18S ribosomal RNA gene (rRNA) and of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene of Strongyloides from humans in the study area in Thailand, where people have frequent contact with long-tailed macaques. Fresh stool samples were obtained from 213 people and were examined using the agar plate culture method. The overall prevalence of Strongyloides infection was 8.92% (19/213). From a total of 19 worms (one per infected person), 18 adult males had 18S rRNA sequences identical with that of S. stercoralis and one adult female had a sequence almost identical with that of S. fuelleborni. A median-joining network of cox1 sequences revealed nine new haplotypes from S. stercoralis, and an overall haplotype diversity (Hd) of 0.9309. The single haplotype of S. fuelleborni was also new and contributed to an overall haplotype diversity for that species of 0.9842. This is the first molecular identification of S. stercoralis and S. fuelleborni in a human community having contact with long-tailed macaques in Thailand. It is also the first report of S. fuelleborni infecting a human in Thailand.

Detection and quantification of Wuchereria bancrofti and Brugia malayi DNA in blood samples and mosquitoes using duplex droplet digital polymerase chain reaction.

Lymphatic filariasis, a mosquito-borne disease, is still a major public health problem in tropical and sub-tropical countries. Effective diagnostic tools are required for identification of infected individuals, for epidemiological assessment, and for monitoring of control programs. A duplex droplet digital polymerase chain reaction (ddPCR) was conducted to differentiate and quantify Wuchereria bancrofti DNA by targeting the long DNA repeat (LDR) element and Brugia malayi DNA by targeting the HhaI element in blood samples and mosquito vectors. The analytical sensitivity and specificity were evaluated. Our results indicated that the duplex ddPCR assay could differentiate and quantify W. bancrofti and B. malayi DNA from blood samples and mosquitoes. DNA from a single larva in 50 µl of a blood sample, or in one mosquito vector, could be detected. The analytical sensitivity and specificity for W. bancrofti are both 100 %. Corresponding values for B. malayi are 100 and 98.3 %, respectively. Therefore, duplex ddPCR is a potential tool for simultaneous diagnosis and monitoring of bancroftian and brugian filariasis in endemic areas.

Restoration of hookworm egg development after prolonged storage in stool suspension.

Hookworm infection is still prevalent in southern Thailand despite control measures. Hookworm eggs submerged for an extended period under water from rainfall or in latrines may not survive, but they may recover their ability to develop into infective larvae when exposed to atmospheric air. This study examined the survival of the hookworm eggs in stool suspension and the restoration of development capability after prolonged storage. In stool mass, eggs developed normally and yielded infective filariform larvae (FL) in 7 days. On the contrary, in 1:10 stool suspension, hookworm eggs were found to remain at the 4-8 cell stage; degenerated eggs were observed after 15 days of storage, and the number of degenerated eggs reached 80 % on day 30. Aeration of the suspension, or transferring to a Petri dish or agar plate, restored the capacity of eggs stored for up to 15 days to develop into FL; thereafter, the capacity declined sharply. Retardation of egg development under water or in stool suspension may be due to a lack of atmospheric air. Use of "night soil" from latrines as fertilizer may be one factor in maintaining hookworm transmission, as worm eggs can undergo normal development upon exposure to atmospheric air.

First molecular identification and report of genetic diversity of Strongyloides stercoralis, a current major soil-transmitted helminth in humans from Lao People's Democratic Republic.

Strongyloidiasis is a major soil-transmitted helminth (STH) disease that affects people worldwide. We present updated data on prevalence in the Lao People's Democratic Republic (Lao PDR) in 2015, arising from a community cross-sectional helminthiasis survey. Fecal samples were collected from 327 individuals across three provinces in Lao PDR (Luang Prabang in the north, Khammouane in the center, and Champasack in the south). Agar plate culture and Kato-Katz methods were used to examine duplicate stool samples from each participant to detect Strongyloides stercoralis and co-infecting helminths. Overall prevalences of S. strercoralis human hookworm, Taenia spp., Trichuris trichiura, Ascaris lumbricoides, and Enterobius vermicularis were 41.0, 28.1, 4.9, 4.0, 1.5, and 0.9 %, respectively. The prevalence of miscellaneous trematodiases (including opisthorchiasis) was 37.9 % and of Schistosoma mekongi infection was 0.3 %. Strongyloidiasis is a current major STH disease in Lao PDR. We also report the molecular-phylogenetic identification of S. stercoralis adult males collected from 40 representative human strongyliodiasis fecal samples. DNA was extracted, amplified, and sequenced from a portion of the mitochondrial cox1 gene and the nuclear 18S ribosomal DNA. Phylogenetic analyses indicated that all specimens sequenced belonged to S. stercoralis (Bavay, 1876) Stiles and Hassall, 1902. The cox1 sequences exhibited great diversity (24 haplotypes) in Lao PDR. This is the first molecular identification and report of genetic diversity of S. stercoralis in humans from Lao PDR. An effective parasite control program is needed to reduce the serious health impacts.

Strongyloides stercoralis diagnostic polypeptides for human strongyloidiasis and their proteomic analysis.

Human strongyloidiasis is a deleterious gastrointestinal disease mainly caused by Strongyloides stercoralis infection. Strongyloides stercoralis is a soil-transmitted helminthiasis that is distributed around the globe. Although definitive diagnosis is carried out through the detection of parasite objects in human stool samples, the development of reliable immunological assays is an important alternative approach for supportive diagnosis. We characterized the two sensitive and specific bands of S. stercoralis filariform larvae that reacted with human strongyloidiasis sera based on immunoblot analysis. Serum samples obtained from strongyloidiasis patients showed a sensitivity of 90 and 80 % at the approximate molecular mass of 26 and 29-kDa polypeptide bands, respectively. The reactive specificity of the 26-kDa band was 76.5 % while for the 29-kDa band was 92.2 %. Proteomic analysis identified the 26-kDa band protein was 14-3-3 protein zeta, while the 29-kDa band protein was ADP/ATP translocase 4. The results provided a basic framework for further studies regarding the potential of the S. stercoralis recombinant antigen to become a leading to diagnostic tool.