Comparison of Three Methods to Extract Plasmodium falciparum DNA from Whole Blood and Dried Blood Spots.

This study aimed to compare the effectiveness of three DNA extraction methods: the GF-1 Blood DNA Extraction Kit (GF-1 BD Kit), which employs a spin column along with lysing and washing buffers; the tris-ethylenediaminetetraacetic acid and proteinase K (TE-pK) method, which utilizes a combination of TE buffer and proteinase K for cell lysis; and DNAzol® Direct (DN 131), a single reagent combined with heating for the extraction process. Plasmodium falciparum DNA was extracted from both whole blood and dried blook spots (DBSs), with consideration of DNA concentration, purity, cost, time requirement, and limit of parasite detection (LOD) for each method. The target gene in this study was 18S rRNA, resulting in a 395-bp product using specific primers. In the comparative analysis, the DN 131 method yielded significantly higher DNA quantities from whole blood and DBSs than the GF-1 BD Kit and TE-pK methods. In addition, the DNA purity obtained from whole blood and DBSs using the GF-1 BD Kit significantly exceeded that obtained using the TE-pK and DN 131 methods. For LOD, the whole blood extracted using the DN 131, GF-1 BD Kit, and TE-pK methods revealed 0.012, 0.012, and 1.6 parasites/µL, respectively. In the case of DBSs, the LODs for the DN 131, GF-1 BD Kit, and TE-pK methods were 1.6, 8, and 200 parasites/µL, respectively. The results revealed that the TE-pK method was the most cost-effective, whereas the DN 131 method showed the simplest protocol. These findings offer alternative approaches for extracting Plasmodium DNA that are particularly well-suited for large-scale studies conducted in resource-limited settings.

Comparison of 3 DNA extraction methods for extracting DNA from an adult Culex quinquefasciatus (Diptera: Culicidae).

The aim of this study was to compare 3 DNA extraction methods: the PureLink Genomic DNA kit, DNAzol Direct reagent, and a microwave-based method, for extracting DNA from an adult Culex quinquefasciatus by focusing on the quantity and purity of DNA, cost, and time required. Ten mosquitoes were individually used for DNA extraction by each method. Based on the results obtained, DNA was extracted from each method using specific primers, resulting in a polymerase chain reaction (PCR) product with a length of 274 bp. The DNA quantity extracted using the DNAzol Direct (179.08 ±â€…3.77 ng/µl) differs significantly from that of the commercial kit (115.98 ±â€…4.57 ng/µl) and a microwave-based method (119.26 ±â€…3.06 ng/µl). The absorbance ratio of DNA extracted using the PureLink Genomic DNA kit, the DNAzol Direct, and the microwave-based methods was 1.92 ±â€…0.02, 1.79 ±â€…0.01, and 1.87 ±â€…0.01, respectively. Among the 3 methods evaluated, the microwave-based method is simpler, less expensive, and more time efficient. This is the first evaluation of the microwave-based method for extracting DNA from an adult mosquito. This study provides a useful guide for alternative DNA extraction methods for PCR-based assays, especially in low-resource settings.

Zoonotic Helminthiases in Rodents (Bandicota indica, Bandicota savilei, and Leopoldamys edwardsi) from Vientiane Capital, Lao PDR.

Zoonotic helminths of three rodent species, Bandicota indiaca, Bandicota savilei, and Leopoldamys edwardsi, were investigated in Vientiane capital, Lao PDR. A total of 310 rodents were infected with 11 species of helminth parasites. There were 168 (54.2%) of 310 rodents infected with zoonotic helminths. From our results, there are six recorded zoonotic helminth species, and the highest prevalence was exhibited by Raillietina sp. (30.7%), followed by Hymenolepis diminuta (17.7%), Hymenolepis nana (2.6%), Echinostoma ilocanum (1.9%), Echinostoma malayanum (1.3%), and Angiostrongylus cantonensis (1%). This is the first study of zoonotic helminths in L. edwardsi and the first report of H. diminuta, H. nana, E. ilocanum, and E. malayanum in Bandicota indica and B. savilei, and the first demonstration of A. cantonenensis in B. indica in Lao PDR. From our results, these three rodents are potentially important reservoir hosts of zoonotic helminths. Thus, effective control programs should be considered for implementation to prevent the transmission of these zoonoses in this area.

Investigation of the in vitro cysticidal activity of miltefosine against Acanthamoeba spp.

The present study evaluated the in vitro efficacy of miltefosine against cysts of Acanthamoeba spp. belonging to genotypes T3, T4 and T5. Each genotype was incubated with miltefosine at the concentration of 2.42, 4.84, 9.68, 19.36, 38.72 and 77.44 mM for different periods; 1, 3, 5, 7 d at 37 °C. The viability was assessed by staining with 0.4% trypan blue and culturing on NNA medium at 30 °C for 1 month. The results showed 100% eradication of cyst stage of all concentrations, but exhibited a different degree of activity against different genotypes. The MCC of 38.72 mM could kill genotype T4 and T5 after 1 d of incubation, whereas the killing of T3 needed MCC of 77.44 mM at the same incubation time. Miltefosine showed statistically highly significant difference (P < 0.001) in comparison to non-treated control. Although our finding needs to confirm in animal models, this information may be the guideline for optimizing therapy or considered to combine with the other drugs for effective treatment.

Current status of Opisthorchis viverrini, minute intestinal fluke and Taenia spp. infections among inhabitants in the Kenethao district of northern Lao PDR.

BACKGROUND: This study reports the prevalence of Ov/minute intestinal fluke (MIF) and Taenia infections among inhabitants of the Kenethao district, northern Lao PDR. METHODS: Fecal samples from 580 inhabitants were examined using the formalin-ethyl acetate concentration technique. RESULTS: The prevalence of Ov/MIF, Taenia spp. and coinfection was 45.3, 11.9 and 6.1%, respectively. There was no significant difference between males and females for Ov/MIF (p=0.813) and Taenia infection (p=0.759). The prevalence of Ov/MIF was significantly associated with age (p=0.005), but not for Taenia infection (p=0.836). Consumption of raw fish (p=0.001) and raw meat (p=0.046) was significantly associated with parasitic infections. CONCLUSIONS: The results suggest that Ov/MIF and Taenia spp. are highly endemic in this area and there is a need for projects to eliminate these parasites.

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.