Human rotavirus VP4 and VP7 gene sequences were amplified by reverse transcription-PCR from 53% (322 of 607) of fecal specimens collected from children with severe diarrhea who visited hospitals in six urban areas of South Korea in 2000 and 2001. G2 was the most frequently found G type (constituted 50.6%), followed by G1 (30.1%) and G4 (13.0%). Although the P types of high incidence were P[4] (53.1%) and P[8] (21.4%), a significant incidence of P[6] (20.2%) was also noticeable. The commonest G-and P-type combination found in this study was G2P[4], rather than G1P[8], the most prevalent type known worldwide.Rotaviruses are the major etiological agent of gastroenteritis and cause vomiting, diarrhea, and fever in infants and young children worldwide (8). Rotaviruses are divided into seven groups (groups A to G) on the basis of their antigenic properties. Group A rotaviruses are further divided into the G and the P subtypes according to the antigenic property of the VP7 protein (glycoprotein) and the VP4 protein (protease-susceptible protein), respectively. To date, 10 G types have been identified in humans, but most cases of human infection are associated with four G types (types G1 to G4), of which G1 is the most prevalent worldwide (5, 10). It is well established that the G serotypes coincide with the G genotypes, while P serotypes are classified by a system different from that used to classify P genotypes. Types P[4] and P[8] are most frequently found in humans (according to convention, P genotypes are indicated with brackets). Recent studies, however, indicated the emergence of novel P types in different parts of world (3,11) Rotavirus infection is the most common cause of acute diarrhea in infants and young children in South Korea (14). To monitor the diversity of rotavirus strains circulating in the country, we carried out an analysis of 607 fecal specimens collected from infants and young children with acute diarrhea who visited 18 urban hospitals and four clinical laboratories scattered around the six provinces of the country from January 2000 to April 2001. The fecal suspension (10% in phosphatebuffered saline) was centrifuged at 10,000 ϫ g for 10 min, and the double-stranded viral RNA was extracted by treatment of the supernatant with phenol-chloroform-1% sodium dodecyl sulfate, as described previously (6). Reverse transcription (RT)-PCR was performed with the consensus primers Beg9 and End9 to amplify the VP7 gene sequence in full (1,062 bp) and primers Con2 and Con3 to amplify the VP4 gene sequence (877 bp). A second-round, multiplex PCR was performed with primers specific for G types (G1, G2, G3, G4, G8, and G9) and P types (P[4], P[6], P[8], P[9], and P[10]), as described previously (4, 6). The G and P types were determined from the migration rates of the amplicons in a 1.2% agarose gel. Among the 607 stool samples 322 (53.0%) were positive for rotavirus RNA by RT-PCR. The presence of rotaviral antigens was determined by a latex agglutination assay (Biomerieux, Marcy l'Etoile, France) and an enzyme-li...
Niclosamide, which is an anti-tapeworm drug, was developed in 1958. However, recent studies have demonstrated the antiviral effects of niclosamide against the SARS-CoV-2 virus, which causes COVID-19. In this study, we developed and validated a quantitative analysis method for the determination of niclosamide in rat and dog plasma using liquid chromatography–tandem mass spectrometry (LC–MS/MS), and used this method for pharmacokinetic studies. Biological samples were prepared using the protein precipitation method with acetonitrile. Ibuprofen was used as an internal standard. The mobile phase used to quantify niclosamide in rat or dog plasma consisted of 10 mM ammonium formate in distilled water-acetonitrile (30:70, v/v) or 5 mM ammonium acetate-methanol (30:70, v/v). An XDB-phenyl column (5 µm, 2.1 × 50 mm) and a Kinetex® C18 column (5 µm, 2.1 × 500 mm) were used as reverse-phase liquid chromatography columns for rat and dog plasma analyses, respectively. Niclosamide and ibuprofen were detected under multiple reaction monitoring conditions using the electrospray ionization interface running in the negative ionization mode. Niclosamide presented linearity in the concentration ranges of 1-3000 ng/mL (r =0.9967) and 1-1000 ng/mL (r =0.9941) in rat and dog plasma, respectively. The intra- and inter-day precision values were <7.40% and <6.35%, respectively, for rat plasma, and <3.95% and <4.01%, respectively, for dog plasma. The intra- and inter-day accuracy values were <4.59% and <6.63%, respectively, for rat plasma, and <12.1% and <10.9%, respectively, for dog plasma. In addition, the recoveries of niclosamide ranged between 87.8-99.6% and 102-104% for rat and dog plasma, respectively. Niclosamide was stable during storage under various conditions (three freeze-thaw cycles, 6 h at room temperature, long-term, and processed samples). A reliable LC–MS/MS method for niclosamide detection was successfully used to perform pharmacokinetic studies in rats and dogs. Niclosamide presented dose-independent pharmacokinetics in the dose range of 0.3-3 mg/kg after intravenous administration, and drug exposure in rats and dogs after oral administration was very low. Additionally, niclosamide presented high plasma protein binding (>99.8%) and low metabolic stability. These results can be helpful for further developing and understanding the pharmacokinetic characteristics of niclosamide to expand its clinical use.
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