Development of nucleic acid lateral flow immunoassay for molecular detection of Entamoeba moshkovskii and Entamoeba dispar in stool samples.

Entamoeba moshkovskii, recently known as a possible pathogenic amoeba, and the non-pathogenic Entamoeba dispar are morphologically indistinguishable by microscopy. Although PCR was used for differential diagnosis, gel electrophoresis is labor-intensive, time-consuming, and exposed to hazardous elements. In this study, nucleic acid lateral flow immunoassay (NALFIA) was developed to detect E. moshkovskii and E. dispar by post-PCR amplicon analysis. E. moshkovskii primers were labeled with digoxigenin and biotin whereas primers of E. dispar were lebeled with FITC and digoxigenin. The gold nanoparticles were labeled with antibodies corresponding to particular labeling. Based on the established assay, NALFIA could detect as low as 975 fg of E. moshkovskii target DNA (982 parasites or 196 parasites/microliter), and 487.5 fg of E. dispar target DNA (444 parasites or 89 parasites/microliter) without cross-reactivity to other tested intestinal organisms. After testing 91 stool samples, NALFIA was able to detect seven E. moshkovskii (87.5% sensitivity and 100% specificity) and eight E. dispar samples (66.7% sensitivity and 100% specificity) compared to real-time PCR. Interestingly, it detected three mixed infections as real-time PCR. Therefore, it can be a rapid, safe, and effective method for the detection of the emerging pathogens E. moshkovskii and E. dispar in stool samples.

Inhibitory effects of anthracyclines on partially purified 5'-3' DNA helicase of Plasmodium falciparum.

BACKGROUND: Plasmodium falciparum has been becoming resistant to the currently used anti-malarial drugs. Searching for new drug targets is urgently needed for anti-malarial development. DNA helicases separating double-stranded DNA into single-stranded DNA intermediates are essential in nearly all DNA metabolic transactions, thus they may act as a candidate for new drug targets against malarial parasites. METHODS: In this study, a P. falciparum 5' to 3' DNA helicase (PfDH-B) was partially purified from the crude extract of chloroquine- and pyrimethamine-resistant P. falciparum strain K1, by ammonium sulfate precipitation and three chromatographic procedures. DNA helicase activity of partially purified PfDH-B was examined by measuring its ability to unwind 32P-labelled partial duplex DNA. The directionality of PfDH-B was determined, and substrate preference was tested by using various substrates. Inhibitory effects of DNA intercalators such as anthracycline antibiotics on PfDH-B unwinding activity and parasite growth were investigated. RESULTS: The native PfDH-B was partially purified with a specific activity of 4150 units/mg. The PfDH-B could unwind M13-17-mer, M13-31-mer with hanging tail at 3' or 5' end and a linear substrate with 3' end hanging tail but not blunt-ended duplex DNA, and did not need a fork-like substrate. Anthracyclines including aclarubicin, daunorubicin, doxorubicin, and nogalamycin inhibited the unwinding activity of PfDH-B with an IC50 value of 4.0, 7.5, 3.6, and 3.1 µM, respectively. Nogalamycin was the most effective inhibitor on PfDH-B unwinding activity and parasite growth (IC50 = 0.1 ± 0.002 µM). CONCLUSION: Partial purification and characterization of 5'-3' DNA helicase of P. falciparum was successfully performed. The partially purified PfDH-B does not need a fork-like substrate structure found in P. falciparum 3' to 5' DNA helicase (PfDH-A). Interestingly, nogalamycin was the most potent anthracycline inhibitor for PfDH-B helicase activity and parasite growth in culture. Further studies are needed to search for more potent but less cytotoxic inhibitors targeting P. falciparum DNA helicase in the future.

Seasonal prevalence, risk factors, and One Health intervention for prevention of intestinal parasitic infection in underprivileged communities on the Thai-Myanmar border.

BACKGROUND: Tha Song Yang District, located on the Thai-Myanmar border, contributes to the second highest cases of amoebic dysentery due to intestinal parasitic infections (IPI). However, there were limited disease prevalence data, specific surveillance systems, and interventions available. OBJECTIVE: This study aimed to explore the epidemiological features of the IPIs and apply the One Health (OH) approach to solve IPI-related problems. METHODS: Prevalence of asymptomatic infections in human and animals, yearly symptomatic cases, and associated risk factors were investigated. The OH intervention included improving the knowledge, attitude, and practice (KAP) of the community, microscopic diagnosis training, and stakeholder engagement for IPI prevention designs. RESULTS: The prevalence of asymptomatic cases was much higher than that of the symptomatic cases. Infective stages of the intestinal parasites were discovered in animal stool and water samples, indicating possible transmission routes. One year after the intervention, there were significant declines in asymptomatic IPIs and symptomatic cases of amoebic dysentery. Significant improvements in KAP and awareness regarding water and manure-waste management of the community were observed. CONCLUSION: We reported the successful application of the OH intervention in reducing the IPI prevalence and mitigating disease-related risks. The intervention might be applied to address other infectious diseases in the future.

Efficacy of Ageratum conyzoides extracts against Giardia duodenalis trophozoites: an experimental study.

BACKGROUND: Giardia duodenalis causes giardiasis in humans, particularly in developing countries. Despite the availability of treatments, resistance to some of the commercial anti-Giardia drugs has been reported in addition to their harmful side effects. Therefore, novel treatments for giardiasis are required. In this study, we aimed to assess the in vitro activity of crude extracts of Ageratum conyzoides against G. duodenalis trophozoites. METHODS: Plants were classified into three groups based on their flower colors: white (W), purple (P), and white-purple (W-P). Plants were separately cut into leaf (L) and flower (F) parts. Changes in internal organelle morphology of trophozoites following exposure to crude extracts were assessed using transmission electron microscopy (TEM). In subsequent experiments, efficacy of the most active essential oils from crude extracts [half maximal inhibitory concentrations (IC50) ≤ 100 µg/mL] against G. duodenalis trophozoites was tested. In vitro anti-Giardia assays using essential oils were performed in the same way as those performed using crude extracts. RESULTS: LW-P and FP extracts showed high activity (IC50 ≤ 100 µg/mL) against G. duodenalis trophozoites, with IC50 ± SD values of 45.67 ± 0.51 and 96.00 ± 0.46 µg/mL, respectively. In subsequent experiments, IC50 ± SD values of LW-P and FP essential oils were 35.00 ± 0.50 and 89.33 ± 0.41 µg/mL, respectively. TEM revealed the degeneration of flagella and ventral discs of G. duodenalis trophozoites following exposure to crude extracts. CONCLUSION: Crude LW-P and FP extracts of A. conyzoides showed the highest activity against G. duodenalis. Exposure to crude extract induced changes in the flagella and ventral discs of G. duodenalis trophozoites, which play important roles in attachment to the surface of mucosal cells. Our results suggest that the tested extracts warrant further research in terms of their efficacy and safety as giardiasis treatment.

Inhibitory effects of Thai plants beta-glycosides on Trichomonas vaginalis.

Trichomoniasis is now an important health problem in developing countries. Although metronidazole has so far been widely used to treat this disease, the prevalence of metronidazole-resistant protozoa and unpleasant adverse effects have been found. In this study, natural products purified from Thai plants were, therefore, investigated for their effectiveness against Trichomonas vaginalis. The minimal inhibitory concentrations for all beta-glycosides against Trichomonas vaginalis at 24 h were in a range of 6.25-12.5 microM. In addition, torvoside A and H were found to be more potent than their corresponding aglycones, deglucosylated torvoside A and H, while other beta-glycosides were generally as active as their corresponding aglycones. The cytotoxicity of these compounds was also determined. Except for dalcochinin, none of the tested compounds showed cytotoxicity against Vero and cancer cell lines (KB and MCF-7), having IC(50) values greater than 50 microg/ml. In conclusion, beta-glycosides and several aglycones showed selective inhibition against Trichomonas vaginalis without harmful effect to mammalian cells.