Incidence and risk factors of Giardia duodenalis infection in an orphanage, Thailand.

A cohort study was performed to evaluate the incidence and risk factors of Giardia duodenalis infection in an orphanage in suburban area outside Bangkok, central Thailand. Stool specimens were examined for the presence of G. duodenalis in January 2007, May 2007 and January 2008. Of 892 stool specimens from 481 individuals, simple wet preparation, PBS ethyl-acetate sedimentation and PCR amplification of the SSU-rRNA gene were performed to detect G. duodenalis. Using PCR of the glutamate dehydrogenase gene and sequence analysis, G. duodenalis assemblages were identified. Associated risk factors were analysed using Fisher's exact test which revealed significant infection of G. duodenalis in boys and specific rooms where orphans aged 25-48 months old lived. Genotypic characterization of G. duodenalis revealed that assemblage A subtype AII was the most predominant found in orphans living in the specific rooms, thus the transmission was likely to occur via person-to-person. Other modes of transmission were less likely to occur. This study showed that the incidence rate of Giardia infection gradually decreased significantly after the implementation of health education and appropriate treatment of infected orphans.

Epidemiology of giardiasis and genotypic characterization of Giardia duodenalis in preschool children of a rural community, central Thailand.

A cross-sectional study was conducted to determine the prevalence and risk factors of Giardia duodenalis infection in 189 preschool children at Sanamchaiket District, Chachoengsao Province, central Thailand in February 2007. Stool specimens were examined for the presence of Giardia using simple wet preparation and PBS-ethyl acetate concentration technique. The prevalence of G. duodenalis in preschool children was 5.8%. Using PCR-RFLP analysis of the glutamate dehydrogenase gene (gdh), genotypes of G. duodenalis revealed assemblage AII (3, 30.0%), BIII (1, 10.0%), and BIV (6, 60.0%). Using multivariate analysis, children who kept cat(s) at home were at 5.1 times (95% CI; 1.3- 20.3) greater risk of acquiring giardiasis. This study possibly represents the information supporting the potential zoonotic transmission of G. duodenalis between cats and preschool children. Unfortunately, in this study, we did not determine G. duodenalis infection in cats, so further studies in cats should be performed to confirm this postulation.

Detection of lymphatic Wuchereria bancrofti in carriers and long-term storage blood samples using semi-nested PCR.

The semi-nested PCR was conducted for detection of Wuchereria bancrofti in patients' blood. The primers were designed based on the repetitive DNA sequences of the parasite. The results demonstrated that the semi-nested PCR could detect as little as 0.001 fg of parasite DNA. In addition, the primers showed no PCR amplification from human and other hemoparasites such as Brugia malayi, Plasmodium falciparum and P. vivax DNAs. This technique was used for detection of 18 W. bancrofti infected blood samples with a long-term storage, the data revealed that all samples were positive. The results obtained from this study clearly indicated that the semi-nested PCR is specific, sensitive, and suitable for detection of the disease carriers.

Therapeutic efficacy of artesunate in Plasmodium vivax malaria in Thailand.

Our previous study showed that in vitro susceptibility of Plasmodium vivax to chloroquine has significantly decreased in Thailand within the past two decades. Thus, the evaluation of alternative antimalarials for treatment of vivax malaria is needed. The aim of this study was to examine parasitological and clinical efficacy of an artemisinin derivative (artesunate) for the treatment of vivax malaria in patients who were admitted to the Bangkok Hospital for Tropical Diseases. We randomly allocated patients aged 12-56 years to receive 3.3mg/kg (adult dose 200 mg) on the first day, and for the next four days each patient was given 1.65 mg/kg orally (adult dose 100 mg), total dose = 600 mg. After the five-day course of artesunate, primaquine was given: a single oral dose of 15mg for 14 days. A total number of 42 patients received treatment. All participants were followed up for 28 days. In all the cases, both parasitemia and fever were resolved rapidly; the mean fever clearance time and parasite clearance time, 14.6 and 36.7 hours, respectively, showed that therapeutic response to artesunate was better than that of chloroquine. The 14-day cure rate was 100%, but reappearance of parasitemia was seen in two patients on days 21 and 25 following treatment, respectively. These two cases of failure rate should be considered as true relapse rather than recrudescence, since the relapse interval in Southeast Asian vivax malaria according to recent findings seems to be 3 weeks after start of treatment, if primaquine is not given or an inadequate amount is given. In conclusion, artesunate might be useful in treatment of vivax malaria, causing a good blood schizontocidal effect. However, to prevent emerging resistance it should never be used alone.

Monitoring of Plasmodium vivax sensitivity to chloroquine in vitro in Thailand.

The sensitivity of Plasmodium vivax to chloroquine in vitro was investigated in patients admitted to the Bangkok Hospital for Tropical Diseases, Thailand, between September 2001 and May 2002. Of 42 isolates, 34 were successfully tested for parasite sensitivity to chloroquine in vitro; the results showed a significant decrease in sensitivity compared with data published in 1989 and 1995: the IC50 and IC90 were 187.2 and 1217.9 ng/mL blood, respectively, an approximate 4-fold decrease in sensitivity in comparison with other data from the past 2 decades. A number of in vitro tests were performed simultaneously using both WHO microplates and our own laboratory-prepared pre-dosed microplates under the same conditions and there was no significant difference between the results.

Production and characterization of a monoclonal antibody against recombinant fatty acid binding protein of Fasciola gigantica.

In Fasciola parasites fatty acid binding proteins (FABPs) are the carrier proteins that help in the uptake of fatty acids from the hosts' fluids. Attempts have been made to utilize both native and recombinant FABP (rFABP) for immunodiagnosis and vaccine development for fasciolosis. In this study, we have produced a number of monoclonal antibodies (MoAbs) against rFABP of Fasciola gigantica. These MoAbs were initially screened against rFABP by ELISA and then tested for their specificities by immunoblotting. Five stable clones were selected and characterized further: four of them were of the isotype IgG(1) while one clone was IgG(2a). All the MoAbs reacted with rFABP which has a molecular weight (MW) of 20 kD and with at least two isoforms of native proteins at MW 14.5 kD that were present in the tegumental antigen (TA) and crude worm extracts, and the excretion-secretion materials. Immunoperoxidase staining of frozen sections of adult parasites by using these MoAbs as primary antibodies indicated that FABP were present in high concentration in the parenchymal cells and reproductive tissues, in low concentration in the tegument and caecal epithelium. All MoAbs cross-reacted with a 14.5 kD antigen present in the whole body (WB) extract of Schistosoma mansoni, while no cross-reactivities were detected with antigens from Eurytrema pancreaticum and Paramphistomum spp.

Plasmodium vivax malaria in Southeast Iran in 1999-2001: establishing the response to chloroquine in vitro and in vivo.

Chloroquine-resistant Plasmodium vivax is emerging in Oceania, Asia and Latin America. The drug sensitivity of P. vivax to chloroquine both in vivo and in vitro in the southern part of Iran was assessed; chloroquine-resistant Plasmodium falciparum has already been documented in this area. The in vitro sensitivity of 39 P. vivax isolates was assessed: the mean IC50 and IC90 were 189 ng/ml and 698 ng/ml blood respectively; for in vivo testing, all 39 vivax malaria patients were treated with a standard regimen of chloroquine and followed-up at 28 days: the mean parasite clearance time was 67.2 +/- 22.5 hours. The in vitro development of young parasites to mature schizonts in standard test medium was compared with that obtained in McCoy's 5A medium: no significant difference was observed. Synchronization of the blood-stage parasites was performed according to Lambros' method: the method was not suitable because it was detrimental to the parasites. A number of in vitro tests were performed using both our own laboratory-predosed microplates and WHO microplates: there was no significant difference between the results.

Pharmacokinetic and pharmacodynamic interactions of mefloquine and quinine.

This study was carried out to investigate the pharmacokinetic and pharmacodynamic interactions between two antimalarial drugs, mefloquine and quinine. A randomized, comparative, three-way crossover study was performed in seven healthy male Thais after the administration of three drug regimens on three occasions i.e., a single oral dose of quinine sulfate (600 mg), mefloquine (750 mg) alone, or the combination of mefloquine (750 mg) and quinine (600 mg given 24 h after mefloquine). QTc interval was significantly prolonged in subjects following the combination regimen (at 2.5, 3, 4, 6, 8, 12, 18, 24 h after the quinine dose) but no abnormal clinical signs or symptoms were found. There were no significant changes in vital signs or routine laboratory values in any of the subjects. The pharmacokinetics of mefloquine and quinine were influenced by the presence of the other drug. Greater blood schizonticidal activities were collected from the sera of subjects on the combination regimen than from the sera of subjects the quinine or mefloquine regimens. The minimum inhibitory concentrations (MICs) of the equivalent concentrations (Eqs) of quinine or mefloquine, which completely inhibited the growth of the K1 strain of Plasmodium falciparum in vitro (MICs of quinine Eq and mefloquine Eq) were significantly lower in the sera of subjects on the combination regimens, than in the sera of subjects on mefloquine or quinine alone [MICs of quinine Eq: 41.2 (21.25-73.5) vs. 135 (118-150) ng/ml; MICs of mefloquine Eq: 18.2 (17-19.2) vs. 25.2 (24.4-26.8) ng/ml].

Ex vivo blood schizontocidal activities of artemisinin derivatives against Plasmodium falciparum.

Serum samples collected at intervals from eight healthy volunteers after the administration of the six regimens of artemisinin derivatives were investigated for their ex vivo blood schizontocidal activities against K1 strain Plasmodium falciparum. The regimens included single doses of (a) 300 mg oral artemether; (b) 300 mg intramuscular artemether; (c) 100 mg suppository artemether; (d) 300 mg oral artesunate (Guillin formulation); (e) 300 mg oral artesunate (Arenco formulation); (f) 300 mg oral dihydroartemisinin. Sera collected after various regimens of artemisinin derivatives showed distinct degree of ex vivo blood schizontocidal activities. Activity of sera after suppository dosing was remarkably low and variable comparing to the other two formulations (oral, intramuscular). Median values for Amax (the maximum activity normalized with dose) of sera from oral dosing were 2.4- and 118-fold, while AUA (the area under activity-time curve, normalized with dose) were 0.82- and 2,370-fold of that after the intramuscular and suppository dosing, respectively. Sera from artesunate-Arenco dosing exhibited significantly higher Amax and AUA (medians: Amax 12.4 vs 5.13 nmol/l/mg dose; AUA: 21.9 vs 8.8 nmol x h/ml/mg dose), compared to that from artesunate-Guillin dosing. Among the oral formulations of artemisinin derivatives investigated (artemether, artesunate, dihydroartemisinin), sera collected following a single dose of oral dihydroartemisinin exhibited lowest bioactivity (Amax 2.35 nmol/l/mg dose; AUA: 44 nmol x h/ml/mg dose).

Pharmacokinetic interactions of artemether and pyrimethamine in healthy male Thais.

The pharmacokinetics of a single oral dose of artemether (300 mg) and pyrimethamine (100 mg) given as each individual drug alone or as a drug combination (artemether 300 mg plus pyrimethamine 100 mg), were investigated in 8 healthy male Thai volunteers. Both artemether and pyrimethamine were rapidly absorbed after oral administration. Elimination of pyrimethamine was however, a relatively slow process compared with artemether, and thus resulted in a long terminal phase elimination half-life (50-106 hours). Pharmacokinetics of artemether and dihydroartemisinin following a single oral dose of artemether alone or in combination with pyrimethamine were similar. In contrast, coadministration of artemether resulted in significantly increased Cmax (medians of 818 vs 1,180 ng/ml) and contracted the apparent volume of distribution (medians of 3 vs 2.56 l/kg) of pyrimethamine.

Incidence of antimalarial pretreatment and drug sensitivity in vitro in multidrug-resistant Plasmodium falciparum infection in Thailand.

Blood samples for determination of baseline antimalarial levels and sensitivity testing in vitro were collected from 411 patients with uncomplicated multidrug-resistant Plasmodium falciparum malaria (365 males, 46 females) before starting antimalarial treatment (62 in hospital and 349 as out-patients). Three hundred and eighty-two were successfully tested, and 110 (28.8%) and 20 (5.2%) patients, respectively, had detectable baseline blood mefloquine and quinine levels. Thirty-nine (10.2%), 44 (11.5%), 23 (6.0%) and 4 (1.1%) cases, respectively, had mefloquine concentrations in whole blood of < 100, 100-500, > 500-1000 and > 1000 ng/mL; the corresponding values for baseline plasma quinine levels were 0 (0%), 9 (2.4%), 3 (0.8%) and 9 (2.4%). None had detectable baseline artemether or artesunate. Sensitivity tests in vitro of pretreatment P. falciparum isolates showed the median IC50, IC90 and IC99 values (ranges in parentheses) for mefloquine, quinine and artemisinin to be 0.121 (0.046-0.715), 0.333 (0.085-3.0) and 0.64 (0.16-1.28) microM, 0.256 (0.064-1.315), 1.10 (0.154-20.49) and 2.56 (0.64-5.12) microM, and 0.02 (0.003-0.382), 0.112 (0.015-4.3) and 0.3 (0.03-3.0) microM, respectively. There was no difference in the sensitivity of P. falciparum isolates to these antimalarial compounds, regardless of the areas where patients had contracted the infection. Previous treatment with mefloquine or quinine was not statistically associated with a high incidence of resistance to these compounds.

Plasma containing artemether-pyrimethamine has ex vivo blood schizonticidal activity against Plasmodium falciparum.

Plasma samples collected at intervals from healthy volunteers, after administration of 3 drug regimens [artemether (ART) 300 mg, pyrimethamine (PYR) 100 mg, and ART 300 mg plus PYR 100 mg] were examined for blood schizonticidal activity against K1 strain and T(9/94) clone of Plasmodium falciparum ex vivo. A synergistic effect against T(9/94), a pyrimethamine sensitive clone, was observed in plasma collected after ART+PYR administration, when the test was carried out in low p-aminobenzoic acid, low folic acid medium. The maximum activity (Amax), expressed as equivalent dihydroartemisinin concentration, for plasma samples collected after the combined ART+PYR regimen [6,935 (1,330-13,400) nmol/l] was significantly higher than those for the single ART or PYR regimens [935 (397-2,000) and 9.9 (5.6-15.6) nmol/l, respectively]. In addition, the area under the activity curve (AUA) for the combined regimen [12,8397 (39,274-19,7901) nmol.h/l] was significantly higher than those for the single ART or PYR regimens [(3618 (1406-5597) or 334 (82.3-733.3) nmol.h/l, respectively]. Microscopic observation revealed that ART in the combined regimen exerted its inhibitory effect against all erythrocytic stages and that this occurred before effects of PYR activity. Prolongation of inhibitory effects for the combined ART+PYR regimen was shown to be due to PYR activity by comparison to the activity from the single ART regimen. Results clearly demonstrated no PYR activity against K1, a pyrimethamine resistant strain, in plasma samples collected after the single PYR regimen and the ART+PYR regimen. Microscopic examination confirmed that growth inhibition of K1 was caused by ART activity only.

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.

In vitro blood schizonticidal activity of sera containing mefloquine-quinine against Plasmodium falciparum.

Serum samples collected at intervals from healthy volunteers, after the administration of 3 drug regimens (quinine (QN) 600 mg, mefloquine (MQ) 750 mg, and MQ 750 mg plus QN 600 mg) were investigated for their blood schizonticidal activities against K1 strain Plasmodium falciparum in vitro. Superiority of activity was shown in the sera collected after the combination regimen. In the diluted sera of the QN regimen, a complete inhibitory effect was observed for only 24 hours, whereas the effect was sustained for 72 hours in the sera collected after MQ in either regimen (MQ alone or MQ/QN). The pattern of minimum inhibitory concentrations (MICs) of QNEq of the sera from QN alone was constant throughout a 24-hour period, with significantly higher concentrations than that from the combination regimen (118-150 vs 21.25-73.5 micrograms/l). In sera collected after the combination regimen, however, the MIC gradually decreased from 0.5 until 2.5 and 4 h, and thereafter gradually returned to the same levels again during a period of 6-24 hours. The MICs of MQEq when given as MQ alone or in combination appeared constant, with a significantly higher value in the former regimen (24.4-26.8 vs 17-19.2 micrograms/l).

Artemether-pyrimethamine in the treatment of pyrimethamine-resistant falciparum malaria.

In vitro susceptibility and clinical response of multidrug resistant Plasmodium falciparum to the combination artemether-pyrimethamine were evaluated in patients with acute uncomplicated falciparum malaria. Sixty patients were randomized to receive 3 oral regimens of the combination artemether-pyrimethamine as follows: Regimen-I: artemether (300 mg) plus pyrimethamine (100 mg) on the first day, then placebo on the two consecutive days; Regimen-II: artemether (300 mg) plus pyrimethamine (100 mg) on the first day, then artemether (150 mg) plus pyrimethamine (50 mg) on the second day, and placebo on the third day; Regimen-III: artemether (300 mg) plus pyrimethamine (100 mg) on the first day, then artemether (150 mg) plus pyrimethamine (50 mg) on the second and third days. All patients had a rapid initial response to treatments with 95% of parasitemia being cleared within the first 24 hours. PCT24hours and PCT48hours were similar among the three drug regimens (11 vs 4, 6 vs 12, and 9 vs 11 patients for a 1-day, 2-day, and 3-day combination regimen, respectively). Fever was cleared within 48 hours in all patients in either group. Transient mild nausea, vomiting and loss of appetite were found in a few patients during the first 2 days of treatment. Seven patients did not complete the 28 day follow-up period (5 vs 2 in a 1-day vs 2-day regimen), the reason for withdrawal was not associated with drug-related adverse effects. Only 53 patients were therefore qualified for the efficacy assessment. There was 15, 13 and 5 patients in a 1-day, 2-day and 3-day combination regimens, respectively, who had reappearance of the parasitemia between days 11 and 21. The cure rates of the 3 treatment groups were statistically significantly different (0, 27.8, and 75% for a 1-day, 2-day and 3-day combination regimen, respectively). Two patients developed P. vivax malaria on days 20 and 24. All of the isolates were highly resistant to pyrimethamine, with MIC of 10(-5) M. There is potential advantage of this combination therapy in reducing the dosage and treatment period of artemisinin derivative, which is therefore likely to improve complaince in clinical practice. The use of a 3-day combination regimen (300 mg artemether plus 100 mg pyrimethamine on the first day, then 150 mg artemether plus 50 mg pyrimethamine on the second and third days) seems to be a good alternative regimen to sulfadoxine/ pyrimethamine in areas where P. falciparum is sensitive to pyrimethamine eg in Africa.

Clinical response and susceptibility in vitro of Plasmodium vivax to the standard regimen of chloroquine in Thailand.

The clinical effectiveness of the standard regimen of chloroquine (CQ) (a total dose of 1500 mg, given over 48 h at 0, 6, 24 and 48 h) for the treatment of Plasmodium vivax malaria in Thailand was investigated in 57 patients in an endemic area of Thailand (Chantaburi Province, eastern Thailand). For radical treatment, an additional course of a tissue schizontocidal agent, primaquine, was given following the complete course of CQ. With this regimen, satisfactory whole blood concentration-time profiles of CQ and its major metabolite desethylchloroquine (DECQ) were achieved. Mean whole blood levels of CQ and DECQ always much exceeded the reported therapeutic level of CQ (90 ng/mL) during the first 7 d of treatment. All patients responded well to the treatment; in most cases, complete and rapid clearance of parasitaemia was observed within the first 48 h. No reappearance of the parasitaemia was detected in peripheral blood films of any patient within 14 d of the evaluation period. In 6 patients, however, reappearance of P. vivax parasitaemia was observed after 30 d; 2 of them had not completed the course of primaquine. There was no difference in whole blood concentrations of CQ and DECQ, admission parasitaemia, susceptibility of the isolates to chloroquine in vitro, and parasite clearance time between patients with or without reappearance of parasitaemia. A prominent trend of deteriorating sensitivity of the parasite to the drug was, however, suggested.

The pharmacokinetics of chloroquine in healthy Thai subjects and patients with Plasmodium vivax malaria.

The pharmacokinetics of chloroquine (CQ) and desethylchloroquine (DECQ) were studied in seven male Thai patients with P. vivax malaria and seven healthy male Thais, after the standard oral dosage regimen of CQ (a total dose of 1500 mg given over 3 days). All patients showed a rapid initial response to the treatment with median (range) values of fever and parasite clearance times of 13.7 (2-47) and 58 (33-38) h, respectively. In the patients, the median range Cmax value was significantly higher (1547 (996-2446) vs 838 (656-1587) ng ml-1), and AUC(0,28d) was greater (281 (250-515) vs 122 (103-182) micrograms ml-1 h). In addition, the median (AUC(0,28d) of DECQ was significantly greater (170 (72-265) vs 77 (49-140) micrograms ml-1 h). The AUC(0,28d) ratio of DECQ to CQ in patients was significantly higher than that in healthy subjects (0.67 (0.43-0.90) vs 0.51 (0.29-0.61)).

A specific DNA probe which identifies Babesia bovis in whole blood.

A genomic library of Babesia bovis DNA from the Mexican strain M was constructed in plasmid pUN121 and cloned in Escherichia coli. Several recombinants which hybridized strongly to radioactively labeled B. bovis genomic DNA in an in situ screening were selected and further analyzed for those which specifically hybridized to B. bovis DNA. It was found that pMU-B1 had the highest sensitivity, detecting 25 pg of purified B. bovis DNA, and 300 parasites in 10 microliters of whole infected blood, or 0.00025% parasitemia. pMU-B1 contained a 6.0 kb B. bovis DNA insert which did not cross-hybridize to Babesia bigemina, Trypanosoma evansi, Plasmodium falciparum, Anaplasma marginale, Boophilus microplus and cow DNA. In the Southern blot analysis of genomic DNA, pMU-B1 could differentiate between two B. bovis geographic isolates, Mexican strain M and Thai isolate TS4. Thus, the pMU-B1 probe will be useful in the diagnosis of Babesia infection in cattle and ticks, and in the differentiation of B. bovis strains.

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.