Development of an in vitro microtest to assess drug susceptibility of Babesia bovis and Babesia bigemina.

Continuous cultivation of the bovine hemoparasites Babesia bovis and Babesia bigemina was developed as an in vitro microtest to assess parasite susceptibility to babesicidal compounds. Reproducibility of parasite multiplication rates was independent of culture size, making it possible to use a microscale of 100 microliters for each test sample. Inhibitory concentrations (IC50s) of a commonly used babesicide, quinuronium sulfate, evaluated by this in vitro method were found to be 5 x 10(-8) g/ml for B. bovis and 2 x 10(-9) g/ml for B. bigemina.     

Alternative method for determination of pyrimethamine in plasma by high-performance liquid chromatography

A rapid, selective, sensitive and reproducible reversed-phase high-performance liquid chromatography (HPLC) procedure for the quantitative determination of pyrimethamine (PYR) in plasma is described. The procedure involved the two-step extraction of PYR and the internal standard, quinine (QN) with acetonitrile and dichloromethane at basic pH. Chromatographic separation consisted of the mobile phase (methanol-water containing 0.005 M octanesulfonic acid, 50:50, v/v) running through the column (Techopak-10 C₁₈) at a flow-rate of 1.6 ml/min. Detection was at UV wavelength of 240 nm. The mean recoveries of PYR and QN at a concentration range of 50 and 500 ng/ml were 98.9 and 89%, and 94.7 and 96% for PYR and QN. The within-day coefficients of variation were 2.1–5.1% for PYR and 5.9% for QN. The day-to-day coefficients of variation were 2.1–4.1% for PYR and 5% for QN. The minimum detectable concentrations for PYR and QN in plasma were 3 and 10 ng/ml. The method was found to be suitable for use in clinical pharmacokinetic study.     

Comparison of Recombinant Proteins of Kinesin 39, Heat Shock Protein 70, Heat Shock Protein 83, and Glycoprotein 63 for Antibody Detection of Leishmania martiniquensis Infection

Leishmania martiniquensis, a zoonotic hemoflagellate, is a causative agent of cutaneous (CL) and visceral leishmaniasis (VL) among humans and animals. This organism, first reported in Martinique Island, now has become an emerging infectious agent in Thailand. Symptomatic cases of L. martiniquensis infection among humans have continuously increased. In the meantime, asymptomatic infection of this novel species has seriously created national public health awareness and concern to prevent and control disease transmission. The unsuccessful serological test using the commercial rK39 dipstick based on antigen from Leishmania donovani to detect the antibodies against VL among infected Thai patients has encouraged us to further explore a new sensitive and specific antigenic epitope. In this study, we determined the sequences and expressed recombinant proteins of kinesin 39 (k39), heat shock protein 70 (hsp70), heat shock protein 83 (hsp83), and glycoprotein 63 (gp63) of L. martiniquensis to evaluate the diagnostic efficiency to detect antibodies against L. martiniquensis in patient sera. The preliminary results from western blot analysis have suggested that K39 is the most sensitive recombinant protein to detect L. martiniquensis. Moreover, this recombinant protein reacts with antibodies against L. donovani and Leishmania infantum, making it a promising antigen for further development of a universal rapid diagnostic tool for VL.     

PBS incubation, a simple technique to release miracidia of Opisthorchis-like eggs for DNA extraction

This study describes a simple technique for releasing miracidia from Opisthorchis-like eggs before DNA extraction by keeping PBS washed specimens at 4 °C for two days. Using PCR amplification, the sensitivity of the PBS incubation technique was as good as that obtained from freeze-thaw technique. Moreover, this low-cost technique is less laborious.     

Continuous cultivation and improved drug responsiveness of Plasmodium falciparum in p-aminobenzoic acid-deficient medium

Long term cultivation of three culture lines of Plasmodium falciparum was achieved in Waymouth and RPMI media, both supplemented with 10% heat-inactivated human serum. Observations on parasite multiplication over a 6-mo period showed no difference in the rate of parasite population increase. Growth of asexual erythrocytic stages in Waymouth medium was as good as that in RPMI when measured by the rate of glucose utilization and extent of 3H-isoleucine and 3H-methionine incorporation. Responsiveness of the parasites to pyrimethamine and sulfadoxine was demonstrated to increase when tested in Waymouth medium because it contained no p-aminobenzoic acid to compete with the drugs.     

Observation on Complete Schizogony of Plasmodium vivax in Vitro1

Attempts to grow Plasmodium vivax in vitro were made on 43 isolates in three different culture media. Complete schizogony occurred in the new medium SCMI 612 in which 34 out of 43 isolates produced merozoites. The RPMI 1640 and Waymouth media suitable for the cultivation of P. falciparum were also used with markedly less success. Results of the experiments indicate differences in nutritional requirements between the two species of Plasmodium.