Nematocidal effect of Piper retrofractum Vahl on morphology and ultrastructure of Strongyloides stercoralis third-stage infective larvae.

In a previous research work aimed at discovering natural helminthicides as alternatives to conventional synthetic drugs, Piper retrofractum fruit hexane extract (PHE) has been shown to possess promising nematocidal activity against the third-stage infective larvae of Strongyloides stercoralis. Thus, this study was designed to evaluate the chemical composition and the impact of PHE on symptom and structural alterations of S. stercoralis. Chemical analysis of PHE by gas chromatography-mass spectrometry demonstrated 26 different compounds, constituting 100% of the total composition. The main components were 4-acetylphenyl (4-benzoylphenoxy) acetate (14.86%) and octyl methoxycinnamate (12.72%). Nematocidal bioassays revealed promising potential of PHE against S. stercoralis larvae, with an LC50 value of 0.059 mg/ml, while the reference drug ivermectin exerted higher efficacy, with an LC50 value of 0.020 µg/ml. Behavioural observations under light microscopy revealed that PHE-treated S. stercoralis larvae moved slowly, became paralysed and eventually died during 24 h of incubation. The dead larvae appeared under light microscope as straight worms with unknown vacuoles of different sizes inside their internal bodies. Morphological alterations of the PHE-treated S. stercoralis larvae, such as straight bodies with swollen cuticle, faded transverse annulations and faded longitudinal striations, as well as shallow and smooth lateral longitudinal grooves, were seen clearly under scanning electron microscopy. Ultrastructural changes in the treated larvae, such as protruded lateral longitudinal grooves, loose muscle with vacuolation, dissociation between the hypodermis and cuticle and marked intracellular disorganization with vacuolation, were detected under transmission electron microscopy. The results of this study provide evidence that PHE is toxic against S. stercoralis and also a potential new alternative for anti-Strongyloides chemotherapy.

Relative developmental and reproductive fitness associated with F1534C homozygous knockdown resistant gene in Aedes aegypti from Thailand.

The effect of permethrin resistance, conferred by a homozygous mutation (F1534C) in the voltage-gated sodium channel protein, upon the reproductive fitness of Aedes aegypti (PMD-R strain) from Thailand was evaluated by comparing with a pyrethroid-susceptible sub colony (PMD strain). The parameters evaluated included larval development time, pupation success, adult emergence, egg production and hatchability, mating ability, female wing length and adult longevity. Larval development times were similar with very low mortality of larvae, pupae and emerging adults among either strain. However, PMD produced significantly fewer females than PMD-R. The mean numbers of eggs laid by PMD (54.2±15.9) and PMD-R (54.6±14.5) strains were not significantly different but the hatchability of PMD eggs (53.7%) was lower than PMD-R eggs (71.2%). The mean wing length of PMD females (2.85±0.15 mm) was longer than PMD-R females (2.74±0.09 mm). The insemination rates for both strains were 100%. The longevity of both strains was mostly not significantly different, over 90% of both sexes surviving at day 30. Our results suggest that the presence of the homozygous F1534C mutation does not lead to fitness reductions. This is in accordance with the high frequency of this allele found among wild populations of Ae. aegypti in many countries. These results also suggest that the removal of pyrethroid insecticide selection pressure may not lead to a regression of 1534C alleles in pyrethroid resistant Ae. aegypti.

Repellent activity of selected essential oils against Aedes aegypti.

Essential oils extracted from ten plant species were screened for repellency against Aedes aegypti mosquitoes. Three oils; Zanthoxylum piperitum, Anethum graveolens and Kaempferia galanga, exerted protection against A. aegypti, with median complete-protection times of 1, 0.5 and 0.25 h, respectively. The protection times were increased significantly by incorporating 10% vanillin. The highest potential was established from Z. piperitum oil +10% vanillin (2.5 h, range=1-2.5 h). Mixtures from pairs of the effective oils possessed slight repellency that ranged from 0-0.5 h. None of the oil combinations repelled A. aegypti for longer than their constituent oil alone. With vanillin added, however, each oil mixture provided improved protection, which was approximately equal to oil on its own. GC/MS analysis revealed that the main component of Z. piperitum fruit oil was limonene (37.99%), with minor amounts of sabinene (13.30%) and beta-myrcene (7.17%). Repellent testing of stored samples of Z. piperitum fruit oil against A. aegypti demonstrated that repellent activity of those kept at -20 degrees C or 4 degrees C was present for a period of at least 3 months. Therefore, the essential oil of Z. piperitum fruit may prove useful in the development of mosquito repellents as an effective personal protection measure against mosquito bites.

Aromatic plant-derived essential oil: an alternative larvicide for mosquito control.

Five aromatic plants, Carum carvi (caraway), Apium graveolens (celery), Foeniculum vulgare (fennel), Zanthoxylum limonella (mullilam) and Curcuma zedoaria (zedoary) were selected for investigating larvicidal potential against mosquito vectors. Two laboratory-reared mosquito species, Anopheles dirus, the major malaria vector in Thailand, and Aedes aegypti, the main vector of dengue and dengue hemorrhagic fever in urban areas, were used. All of the volatile oils exerted significant larvicidal activity against the two mosquito species after 24-h exposure. Essential oil from mullilam was the most effective against the larvae of A. aegypti, while A. dirus larvae showed the highest susceptibility to zedoary oil.

Effectiveness of Zanthoxylum piperitum-derived essential oil as an alternative repellent under laboratory and field applications.

Recently, there were considerable efforts made to promote the use of environmentally friendly and biodegradable natural insecticides and repellents, particularly from botanical sources. In this study, Zanthoxylum piperitum-derived essential oil isolated by steam distillation was investigated and compared to the standard synthetic repellent, N,N-diethyl-3-methylbenzamide (DEET), for repellency against mosquitoes under laboratory and field conditions. The oil of Z. piperitum alone and also with 5% vanillin added repelled laboratory-reared female Aedes aegypti with the median protection times of 1.5 and 2.5 h, respectively. These repellency times were proven to be significantly lower than those of DEET-based products: 25% DEET and 25% DEET + 5% vanillin demonstrating 3.5 and 5.5 h, respectively. However, when applied under field conditions, Z. piperitum oil + 5% vanillin was found to provide better protection against a wide range of natural mosquito populations than 25% DEET + 5% vanillin. While the product of DEET was effective in reducing bites with 99.7% protection, the protective effect of Z. piperitum oil product appeared complete (100%). It had a protective effect against Aedes gardnerii, Anopheles barbirostris, Armigeres subalbatus, Culex tritaeniorhynchus, Culex gelidus, Culex vishnui group, and Mansonia uniformis. The better repellency against a wide range of field mosquitoes derived from Z. piperitum oil products suggested an advantage of efficacy by providing a broad spectrum of activity. Therefore, Z. piperitum could have potential for use in the development of combined repellents as a natural active ingredient, synergist, or additive to conventional synthetic chemicals, particularly in situations when DEET is ineffective and impractical.

Pharmacokinetics of the four combination regimens of dihydroartemisinin/mefloquine in acute uncomplicated falciparum malaria.

The pharmacokinetics of oral dihydroartemisinin and mefloquine were investigated in 40 patients (aged 16-30 y, weighing 45-60 kg) with acute uncomplicated falciparum malaria following the four combination regimens of dihydroartemisinin/ mefloquine [regimen-I: 300 mg dihydroartemisinin (h-0) plus 750 mg mefloquine (h-0); regimen-II: 300 mg dihydroartemisinin (h-0) plus 750 mg mefloquine (h-24); regimen-III: 300 mg dihydroartemisinin (h-0) plus 750 and 500 mg mefloquine (h-24 and 30); regimen-IV: 300 mg dihydroartemisinin (h-0) plus 750 and 500 mg mefloquine (h-0, 24)]. The four combination regimens were well tolerated. Patients in all treatment groups had a rapid initial response. However, 9 patients (4, 4, and 1 cases in regimens-I, II, and IV) had reappearance of parasitemia during the follow-up period. Significant changes in the pharmacokinetic parameters of both mefloquine and dihydroartemisinin were observed in patients with malaria compared with healthy subjects reported in a paralleled study. For mefloquine, Cmax (mg per dose), AUC0-day1 (mg per dose), and AUC0-day7 (mg per dose) were significantly higher in patients. Furthermore, tmax, was prolonged while V/F contracted and tl/2,z, MRT shortened in patients with malaria. For dihydroartemisinin, Cmax AUC, tmax and Vz/F were changed in the same direction as mefloquine, whereas t1/2z and MRT were prolonged. CL/F was also significantly reduced in patients with malaria. Absorption/disposition kinetics of oral dihydroartemisinin were similar among the various regimens. On the other hand, AUC0-day1 (mg per dose) of mefloquine after regimen-III was significantly higher than the other three regimens. Combination regimens with two divided doses of mefloquine (regimens-III and IV) resulted in a significantly delayed tmax (especially regimens-IV) compared with those with single dose regimens (regimens-I and II).

Mosquito repellency of the seeds of celery (Apium graveolens L.).

When the mosquito repellencies of four fractions of Apium graveolens seeds (one hexane, two dichloromethane and one methanolic) were investigated in the laboratory, all four were found to offer human volunteers some protection against female, adult Aedes aegypti. The hexane fraction, however, was found to exhibit the highest repellency in the laboratory, with median effective doses (ED50) and ED95 of 0.41 and 2.93 mg/cm2 skin, respectively. Only this fraction, which was also found to provide protection against mosquito bites for 3.5 h when applied, in the laboratory, at a concentration of 250 mg/ml, was then investigated for its repellency in the field and its stability. In storage, it was found to retain its repellency for at least 2 months, although significant reductions in its repellency were observed (in terms of shortened complete-protection times) after 3 months, whatever the temperature of storage (-20 degrees C, 4 degrees C, or room temperature). When applied to the skin of volunteers under field conditions, the hexane fraction showed strong repellent activity against a wide range of mosquito species belonging to various genera (Ae. gardnerii, Ae. lineatopennis, Armigeres subalbatus, Culex tritaeniorhynchus, Cx. vishnui group, Cx. quinquefasciatus and Mansonia uniformis). It appeared not to cause dermal irritation or any other adverse effect, either during 6 months of use or in the following 3 months of follow-up. Mosquito repellents based on extracts of Ap. graveolens seeds could be developed commercially, as an effective personal-protection measure against mosquito bites and the diseases caused by mosquito-borne pathogens.

Polygamy: the possibly significant behavior of Aedes aegypti and Aedes albopictus in relation to the efficient transmission of dengue virus.

The polygamous behavior of male Aedes aegypti (L.) and Ae. albopictus (Skuse) was investigated by co-habiting a newly-emerged male and females in a 30 cm3 cage (1 male: 20 females) for up to 5 consecutive days. As determined by insemination rates, the results indicated that one Ae. aegypti and Ae. albopictus male could successfully mate with 1.10 (0-4), 4.10 (1-8), 5.40 (4-8), 5.10 (2-8), 5.15 (3-9) and 0.20 (0-3), 1.70 (0-3), 2.35 (1-4), 2.30 (0-4), 2.35 (1-4) Ae. aegypti and Ae. albopictus females, respectively on day 1,2,3,4 and 5 consecutively. The possibly significant role of their polygamy in relation to dengue virus transmission is discussed.

Comparative morphometry, morphology of egg and adult surface topography under light and scanning electron microscopies, and metaphase karyotype among three size-races of Fasciola gigantica in Thailand.

Comparative morphometry of eggs and adults under light microscope, and morphology of adults under scanning electron microscope (SEM) were undertaken in the three size-races (< 25 mm, 25-35 mm, > 35 mm) of Fasciola gigantica (Thailand strain). Morphometric examination revealed intraspecific variation with respects to the dimensions of eggs and adults, whereas surface topography of the three size-race adults under SEM was morphologically similar. The observations on mitotic metaphase chromosomes of spermatogonial cells from testes of the three size-races revealed 2n=20 (diploid type), and no karyotypic difference was observed among them. The meiotic metaphase chromosome was 10 bivalents in primary spermatocyte in diplotene to diakinesis, and many mature spermatozoa were seen in the testicular preparations.

Screening of ten plant species for metaphase chromosome preparation in adult mosquitoes (Diptera: Culicidae) using an inoculation technique.

The screening of 10 plant species (Aloe barbadensis Mill., Asparagus officinalis L., As. plumosus Bak., As. racemosus Willd., As. sprengeri Regel, Codyline fruticosa Goppert, Dracaena loureiri Gagnep., Gloriosa superba L., Hemerocallis flava L., and Sansevieria cylindrica Bojer) for colchicine-like substance(s) using a mosquito cytogenetic assay revealed that a 1% solution of dried Gl. superba rhizome extracted in 0.85% sodium chloride solution could be used instead of a 1% colchicine in Hanks' balanced salt solution. The metaphase rates and average number of metaphase chromosomes per positive mosquito of Aedes aegypti (L.) after intrathoracic inoculation with 1% Gl. superba-extracted solution were 100% and 29.80 in females, and 90% and 25.78 in males, whereas the inoculation with 1% colchicine solution yielded 100 and 90% metaphase rates, and 20.90 and 12.22 average number of metaphase chromosomes per positive mosquito in females and males, respectively. The application of Gl. superba-extracted solution for metaphase chromosome preparation in other mosquito genera and species [e.g., Culex quinquefasciatus Say, Toxorhynchites splendens (Wiedemann), and Anopheles vagus (Döenitz)] also has yielded the satisfactory results.

Pharmacokinetic and pharmacodynamic interactions of mefloquine and dihydroartemisinin.

Pharmacokinetic and pharmacodynamic interactions between dihydroartemisinin and mefloquine were investigated in 10 healthy Thai males. The study was of a three-way crossover design. Subjects were randomized to receive three drug regimens on three separate occasions as follows: regimen I: a single oral dose of 300 mg dihydroartemisinin; regimen II: a single oral dose of 750 mg mefloquine; regimen III: a single oral dose of 300 mg dihydroartemisinin, given concurrently with a single oral dose of 750 mg mefloquine. All regimens were well tolerated. Oral dihydroartemisinin was rapidly absorbed and disappeared from systemic circulation within 8-10 h. Mefloquine absorption and disposition were relatively slow processes. Pharmacokinetics of dihydroartemisinin and mefloquine when given concurrently were similar, except for the absorption rate of mefloquine which was faster in the presence of dihydroartemisinin. Pharmacodynamically, the combination of dihydroartemisinin and mefloquine resulted in a synergistic effect on ex vivo blood schizontocidal activity. Maximum activity (Amax) and area under effect-time curve (AUEC) of dihydroartemisinin and mefloquine were increased approximately two- and 20-fold (Amax), and four- and twofold, respectively, compared with each individual drug alone. AUEC of mefloquine during the first 24 h (AUEC 0-24 h) was increased approximately 50-fold in the presence of dihydroartemisinin.

Investigations of incidence of pretreatment, drug sensitivity in vitro, and plasma levels of pyrimethamine in patients with multidrug resistant falciparum malaria following the three combination regimens of artemether/pyrimethamine.

The study was carried out to investigate the status of in vitro susceptibility of Plasmodium falciparum to pyrimethamine (PYR) in multidrug resistant area of the Thai-Myanmar border, the incidence of unregulated use of the combination of PYR with sulfadoxine (Fansidar) in this area and the relevance of pharmacodynamic and pharmacokinetic factors in determining the treatment outcome from the three combination regimens of ART/PYR (1-, 2- and 3-day regimens), in patients with acute uncomplicated falciparum malaria. The majority of patients had baseline PYR concentrations in the range of 1-100 (50.6%) or 100-500 (34.8%) ng/ml, while concentrations of more than 500 ng/ml were found in only 1.1%. All of the isolates exhibited high grade resistance to PYR with the minimum inhibition concentration (MIC) of as high as 10(-5) M. No association was observed between treatment outcome and the presence of baseline plasma PYR concentrations. In addition, lack of association between plasma concentrations during the acute phase (day-1 and -2) and treatment outcome was found.

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.