Comparative insecticidal power of three pyrethroids on netting.

Adult mosquitoes, Anopheles gambiae Giles and Culex quinquefasciatus Say (Diptera: Culicidae), were exposed for 3 min to replicate samples of polyester netting cut from replicate bednets treated with pyrethroid insecticide formulations at the recommended concentration (alphacypermethrin SC at 40mg ai/m2; cyfluthrin EW at 50 mg ai/m2; deltamethrin WT at 25 mg ai/m2), or treated with only a quarter of those dosages. After 4 months domestic use of the bednets in Malawi, chemical assays showed that pyrethroid deposits on the netting were somewhat less than the target concentrations. Comparing the pyrethroid bioassay results with Anopheles at both treatment concentrations, deltamethrin gave significantly higher mortality (99.7-100%) than the other compounds (alphacypermethrin 94-96%, cyfluthrin 80-89%). Culex bioassay mortality was lower (alphacypermethrin 56-74%; cyfluthrin 63-65%; deltamethrin 50-81 %) and results with the three pyrethroid insecticides at their recommended doses did not differ significantly.

Authentication of artemether, artesunate and dihydroartemisinin antimalarial tablets using a simple colorimetric method.

The recent and widespread appearance of counterfeit antimalarial tablets in South-east Asia prompted the search for simple field assays to identify genuine drugs. In a recently described colorimetric assay for artesunate, Fast red TR salt reacted with an alkali-decomposition product of artesunate to produce a distinct yellow colour. However, that assay is specific for artesunate and it cannot be used to test for artemether. Because of potential concerns over artemether tablet counterfeiting, the colorimetric assay was modified to detect artemether, dihydroartemisinin and artesunate tablets. Other common antimalarials (artemisinin, chloroquine diphosphate, mefloquine HCl, sulphadoxine and pyrimethamine), as well as aspirin and acetaminophen, were negative in the assay, indicating its specificity for artemether, dihydroartemisinin and artesunate. The colorimetric method can be used to obtain a rapid visual assessment of tablet authenticity. The method can also be used to quantify the drug content of tablets, when used in conjunction with a spectrophotometer.

A colorimetric field method to assess the authenticity of drugs sold as the antimalarial artesunate.

Artesunate is the most widely used of the artemisinin derivatives. These drugs are being used increasingly throughout the tropical world, and are an essential component of the treatment of multi-drug resistant malaria. The recent and widespread appearance of counterfeit artesunate tablets in several countries in Southeast Asia poses a serious threat to health in this region. We have developed a simple, inexpensive colorimetric test to determine artesunate authenticity in tablets. The test is based on a reaction between an alkali decomposition product of artesunate and a diazonium salt, fast red TR (FRTR). The appearance of a yellow color indicates the presence of artesunate. The specificity of the test is dependent on the pH of the reaction. Among other antimalarials tested, (i.e. artemisinin, artemether, chloroquine, quinine, primaquine, sulfadoxine, and pyrimethamine) only artesunate produced a positive color reaction at pH 4. The assay requires only 1% of the total weight of a standard tablet containing 50 mg of artesunate and can be completed within 10 min. The method was tested on six genuine artesunate tablets and six counterfeit artesunate tablets obtained in Southeast Asia. The average amount of artesunate in the genuine tablets was determined to be 50.8 +/- 2.9 mg while the counterfeit tablets were found to contain no artesunate.

Improved validated assay for the determination of mefloquine and its carboxy metabolite in plasma, serum and whole blood using solid-phase extraction and high-performance liquid chromatography.

An improved high-performance liquid chromatography method using a low silanol activity octadecylsilica column and a solid-phase extraction technique is validated for the simultaneous analysis of mefloquine and its carboxy metabolite in whole blood, plasma and serum. An octadecylsilica column with high silanol activity is compared to a column of low activity in terms of pH dependent variability of chromatographic retention times for mefloquine and its carboxy metabolite. The low silanol activity column showed a relatively large mobile phase pH range where retention times for both components are consistent. The solid-phase extraction procedure consists of a simple protein precipitation step followed by sample concentration and extraction using a C18 membrane disk. The inter- and intra-assay variability for a therapeutic concentration of mefloquine (1000 ng/ml) is less than 2% in whole blood, plasma and serum while carboxymefloquine (1000 ng/ml) is 2.3% or less. At concentrations as low as 100 ng/ml the inter-assay variability is 6.2% or less for both analytes. This method shows a robust analytical procedure for the simultaneous analysis of mefloquine and its carboxy metabolite where precise measurements are useful in pharmacokinetic studies and in estimating drug compliance.

Prevalence of malaria parasitemia and accuracy of microscopic diagnosis in Haiti, October 1995.

In October 1995 the Ministry of Public Health and Population in Haiti surveyed 42 health facilities for the prevalence and distribution of malaria infection. They examined 1,803 peripheral blood smears from patients with suspected malaria; the overall slide positivity rate was 4.0% (range, 0.0% to 14.3%). The rate was lowest among 1- to 4-year-old children (1.6%) and highest among persons aged 15 and older (5.5%). Clinical and microscopic diagnoses of malaria were unreliable; the overall sensitivity of microscopic diagnosis was 83.6%, specificity was 88.6%, and the predictive value of a positive slide was 22.2%. Microscopic diagnoses need to be improved, and adequate surveillance must be reestablished to identify areas where transmission is most intense. The generally low level of malaria is encouraging and suggests that intensified control efforts targeted to the areas of highest prevalence could further diminish the effect of malaria in Haiti.

Field detection of sulfonamides in urine: the development of a new and sensitive test.

A new, field-adapted, colorimetric method for detecting sulfonamide drugs in urine is described. The method uses the color reagent, p-dimethylaminocinnamaldehyde, and has a detection limit of about 1 microgram/ml. Analysis of 35 samples collected in the field, comparing results obtained with the colorimetric field test with those obtained using high-performance liquid chromatography, indicated a calculated sensitivity value of 94% and a specificity value of 94% for the test to detect the presence of sulfonamides. The field test can be modified to allow quantitation of sulfonamides in urine in field situations, using a hand-held, portable photometer for measuring the absorbance of test solutions. For this test, calculated coefficients of variation for day to day reproducibility were < or = 5% at sulfonamide concentrations > or = 3 micrograms/ml. This new test for detecting the presence of sulfonamides in urine is more sensitive and reliable than the presently used Bratton-Marshall test.

Long-term use of permethrin-impregnated nets does not increase Anopheles gambiae permethrin tolerance.

Previous use of permethrin-impregnated bednets (mosquito nets) and curtains in four Kenyan villages for one year, 1990-91, raised the permethrin LT50 of Anopheles gambiae to 2.4-fold above its baseline value, designated permethrin tolerance (PT), as measured by exposure to 0.25% permethrin-impregnated papers in W.H.O. test-kits. During 1992-93, with ongoing use of permethrin-impregnated nets and curtains, PT regressed slightly compared with the contemporary susceptibility level of An.gambiae from non-intervention villages, to 1.8-fold in 1992 and only 1.6-fold in 1993. Thus the selection pressure of impregnated nets for PT in An.gambiae appears to be minimal in our study villages, although the impact of permethrin was demonstrated by a significantly lower parous-rate of An.gambiae females in the intervention (63-66%) than in non-intervention (79%) villages, and by reduced malaria transmission (reported elsewhere). In a selected stock of An.gambiae from the study area, PT did not affect the susceptibility to deltamethrin, fenitrothion, propoxur or DDT. Bioassays described herein provide easy procedures for field-monitoring of mosquito susceptibility/tolerance/resistance to insecticides used for net impregnation in operational programmes.

Validity of mother's history regarding antimalarial drug use in Malawian children under five years old.

History obtained from parents and carers is an important, and often the only, source of information for health workers treating children for malaria, but its validity has not been well evaluated. At 2 hospitals in Malawi, we obtained malaria treatment histories from mothers of 973 ill children reported to have had fever as part of the illness. Urine samples were collected from 755 of the 973 children (78%). Of the 755, 457 (61%) were reported to have received some kind of treatment. Among those who reportedly received treatment, 79 (17%) were said to have received chloroquine and 23 (5%) a sulphonamide-containing medicine; however, when urine specimens were tested for antimalarial drugs, chloroquine was found in 182 specimens (40%) and a sulphonamide in 148 (32%). Among urine specimens collected from 291 children who were reported to have received no treatment (no report was recorded for 7 children), chloroquine was detected in 56 (19%) and a sulphonamide in 44 (15%). Although not statistically significant, mothers often reported a child as not having received an antimalarial drug if the child was younger than 12 months or had been sick for more than 3 d. The mothers' information regarding home treatment of fever in children was highly inaccurate. Malaria treatment histories, whether collected at health facilities or in surveys of knowledge, attitudes, and practices, must be interpreted with caution.

Determination of sulfadoxine concentrations in whole blood using C18 solid-phase extraction, sodium dodecyl sulfate and dimethylaminocinnamaldehyde.

A simple method is described for the extraction and subsequent analysis of sulfadoxine in human whole blood using a solid-phase extraction technique and colorimetric reaction. This procedure utilizes the micellar properties of sodium dodecyl sulfate to: (1) extract sulfadoxine from a C18 solid-phase sample-preparation column; (2) enhance the colorimetric reaction produced by the addition of p-dimethylaminocinnamaldehyde (DMAC); and (3) provide stability to the coloured product generated by the reaction of sulfadoxine with DMAC. The intense, violet-red colour reaction can be conveniently used for qualitative and semiquantitative visual interpretations of sulfadoxine levels. Under the assay conditions, drug concentrations in the blood of subjects receiving sulfadoxine were determined from absorbance measurements. These results correlated well with the sulfadoxine levels determined from high-performance liquid chromatographic analysis. Important advantages of the procedure include the ability to evaluate small samples of whole blood (100 microliters), the minimal use of organic solvents, no sophisticated instrumentation, and formation of a stable, coloured reaction product. The method proved to be a suitable field assay for determining whole-blood levels of sulfonamides in the concentration range from 5 to 100 micrograms ml-1.

Packed-column supercritical fluid chromatography of artemisinin (qinghaosu) with electron-capture detection.

In this preliminary report, a supercritical fluid chromatographic method is described for the determination of artemisinin in whole blood. The chromatography is carried out on a 20 cm x 1 mm I.D. Deltabond cyano supercritical fluid chromatographic column with detection of the artemisinin via an electron-capture detector. The sample work-up uses a liquid-liquid extraction with hexane, giving a recovery of 82%. The current limit of detection using 1 ml of blood is 20 ng/ml. We speculate that the endoperoxide moiety accounts for the response to the electron-capture detector and thus provides a new approach by which this class of compounds may be analyzed.

Chemiluminescent detection of artemisinin. Novel endoperoxide analysis using luminol without hydrogen peroxide.

A novel method for artemisinin quantitation employing high-performance liquid chromatography (HPLC) with chemiluminescence (CL) detection in the absence of hydrogen peroxide (H2O2), is reported. After elution from the HPLC column, artemisinin is combined with an alkaline solution of hematin and luminol. The resulting CL signal is detected by use of a spectrofluorometer with the excitation lamp disabled, and is proportional to artemisinin concentration. The CL method was optimized and applied to the analysis of artemisinin in spiked human serum. CL in the absence of H2O2 or other known oxidizing species is remarkable since such oxidizers are usually required to produce CL from luminol under alkaline conditions. Artemisinin, a naturally occurring sesquiterpene, is one of several natural products that contain an endoperoxide functional group. Since H2O2 is not needed in the analysis, the endoperoxide moiety on artemisinin is implicated as a contributing source of superoxide radicals required for the light-producing reaction with luminol.

Effects of permethrin-impregnated bed nets on malaria vectors of northern Guatemala.

The authors evaluated the effects on malaria vectors of bed nets impregnated with permethrin over the course of a 16-month controlled study in four communities of Northern Guatemala. Anopheles albimanus and An. vestitipennis were the known malaria vectors in the area. Households were allocated to one of three experimental groups: those receiving bed nets impregnated with 500 mg/m2 of permethrin, those receiving untreated bed nets, and those where no intervention measures were taken. The impact of the treated and untreated bed nets on mosquito abundance, behavior, and mortality was determined by indoor/outdoor night-bite mosquito collections, morning pyrethrum spray collections, inspection of bed net surfaces for dead mosquitoes, and capture-release-recapture studies. The duration of the treated nets' residual insecticide effect was assessed by modified WHO cone field bioassays, and their pyrethrin content was estimated by gas-liquid chromatography analysis. The most important observation was that fewer mosquitoes were found to be resting in the households with treated bed nets. The treated nets probably functioned by both repelling and killing vector mosquitoes. Capture-release-recapture studies showed exit rates from houses with treated nets were higher (94%) than those from control houses (72%), a finding that suggests repellency. However, no significant differences were noted between the indoor night-bite mosquito collections at houses with and without treated nets. The horizontal surfaces of treated bed nets were nearly 20 times more likely to contain dead anopheline mosquitoes than were the comparable surfaces of untreated nets. the bioassays indicated that unwashed permethrin-impregnated bed nets retained their insecticidal activity for 6 months after treatment.

Hardness anisotropy of acetaminophen crystals.

The anisotropy of acetaminophen hardness was demonstrated using both Vickers and Knoop indentation hardness measurements. Based on a model of Knoop hardness anisotropy proposed by Brookes et al. (1), it was concluded that plastic flow in acetaminophen crystals occurs primarily as a result of slip in the (010)<001> system. This conclusion was corroborated with the results of the Vickers indentation tests. The apparent brittleness of acetaminophen was rationalized because only one slip system appeared to be operative. Under these conditions generalized plastic flow cannot occur, since this requires the operation of at least five independent slip systems (2). The high stress concentrations that result from flow lead to fracture. Therefore acetaminophen is more precisely classified as being semiductile. When a material deforms plastically as a result of slip in only one slip system, considerable crystal realignment can occur during compaction. This in turn can facilitate capping during decompression and ejection, since the cleavage plane, (010), would become aligned with the direction of highest tensile stress.

Reduced susceptibility of Anopheles gambiae to permethrin associated with the use of permethrin-impregnated bednets and curtains in Kenya.

Susceptibility of the malaria vector Anopheles gambiae to permethrin decreased following the installation of mosquito nets impregnated with 0.5 g permethrin per square metre in four villages near Kisumu, Kenya. During the first year that permethrin-impregnated bednets and curtains were in place, the exposure time to 50% mortality (LT50) increased 2.5-fold from 13 to 33 min, while the LT50 for An.gambiae was unchanged in two other villages where no intervention measures were used. Two years after permethrin-impregnated mosquito nets were distributed the LT50s for An.gambiae were 28, 28 and 16 min, respectively, in the villages with bednets, curtains and with no such intervention. Using a colony of An.gambiae derived from females collected in the villages using permethrin-impregnated mosquito nets, we lengthened the LT50 from 28 to 41 min in two generations by exposing all females to permethrin-treated papers for 60 min and rearing offspring of the survivors. Permethrin-impregnated bednets and curtains are intended to reduce vectorial capacity. Reduced susceptibility to permethrin could counter this beneficial effect.

Effectiveness of permethrin-impregnated bed nets and curtains for malaria control in a holoendemic area of western Kenya.

The effectiveness of village-wide use of permethrin-impregnated bed nets or eave, window, and door curtains as control measures for Plasmodium falciparum malaria was evaluated during two successive high-transmission seasons in western Kenya. Pairs of villages were assigned to one of three study groups: bed net, curtain, or control. Clinical, parasitologic, and entomologic measures were made from March to July 1990 and again 12 months later. When compared with the controls in 1990 and 1991, we observed a marked reduction in the incidence of P. falciparum infections in children less than six years old in the bed net villages (reduced by 40% and 48%) and a smaller but still significant reduction in the curtain villages (10% and 33%). Significant reductions were also seen in the incidence of P. falciparum parasitemias greater than 2,500/mm3 in the bed net group (reduced by 44% and 49%) and curtain group (16% and 32%). Additionally, we observed significant reductions in the incidence of documented fevers in association with P. falciparum parasitemia in bed net (reduced by 63%) and curtain villages (53%) when compared with controls. Entomologic inoculation rates in both bed net and control villages decreased by more than 50% below control values during both high transmission seasons. The results of this study, together with a 1988 study in the same area during the low transmission season, show that bed nets offer greater year-round of protection against P. falciparum infection than curtains. However, during the high transmission season, this technique reduces the frequency of P. falciparum infection rather than preventing it entirely.

Vivax malaria resistant to treatment and prophylaxis with chloroquine.

Chloroquine has been the treatment of choice for vivax malaria for more than 40 years. Lately, several case-reports have suggested the emergence of resistance to chloroquine in Plasmodium vivax in Papua New Guinea and Indonesia. We undertook prospective treatment and prophylaxis trials of chloroquine in children and adults with vivax malaria living in Irian Jaya (Indonesia New Guinea). 46 villagers with P vivax parasitaemia were treated with chloroquine by mouth (25 mg base/kg body weight divided over 3 days) and followed up for 14 days. Parasitaemia cleared initially but recurred within 14 days in 10 (22%) subjects. All recurrences were in children younger than 11 years, 7 of whom were younger than 4 years; the failure rate among children under 4 was 70%. 7 of the patients with recurrences were given a second course of chloroquine. In all, the infections initially cleared but recurrent parasitaemia developed in 5 (71%) within 14 days. Whole-blood chloroquine concentrations were consistently above those previously shown to cure P vivax blood infections (90 micrograms/L whole blood). Subjects whose initial infections cleared and who had no parasitaemia on day 14 received weekly prophylaxis with chloroquine. Despite the presence of expected blood chloroquine concentrations, P vivax parasitaemia developed in 9 of 17 subjects receiving prophylaxis during 8 weeks of follow-up (median time to parasitaemia 5.3 weeks). Chloroquine can no longer be relied upon for effective treatment or chemoprophylaxis of P vivax blood infections acquired in this part of New Guinea.

Determination of mefloquine in blood, filter paper-absorbed blood and urine by 9-fluorenylmethyl chloroformate derivatization followed by liquid chromatography with fluorescence detection.

We describe a method for determination of mefloquine (MQ) in 100-microliters samples of urine, whole blood, and capillary blood collected on filter paper; quantification is by liquid chromatography with fluorescence detection at 475 nm of the 9-fluorenylmethyleneoxycarbonyl derivative. Whole blood and urine samples were prepared by extraction of MQ and internal standard from aqueous base with methyl tert.-butyl ether (MTBE), separation and evaporation of the MTBE layer, and derivatization using a solution of 9-fluorenylmethyl chloroformate in acetonitrile. Filter paper spots were immersed for 16 h in 0.1 M hydrochloric acid, followed by extraction with MTBE from aqueous sodium carbonate. The separated and evaporated organic layer was treated with the derivatizing solution. An aliquot was injected onto a high-performance liquid chromatography system using a C18 reversed-phase column and acetonitrile-water (72:28) mobile phase for filter paper spot extracts as for whole blood and urine extracts. The method has a limit of determination in blood, blood spots, and urine of 50 ng/ml with 100 microliters sample size (coefficient of variation = 16%). Linearity and precision (within-day and between-day) for the method are good. The MQ derivative was isolated and characterized spectroscopically. Values for MQ concentrations in filter paper blood spots compared favorably with values found in corresponding whole blood samples analyzed by a published method.

Gas chromatographic method for determination of p,p'-DDT in DDT technical and formulated products: collaborative study.

A gas chromatographic (GC) method for determination of p,p'-DDT in technical and formulated products was developed and it performed well in an initial small collaborative study among 4 laboratories. Samples are dissolved in chloroform, and p,p'-DDT is separated on an OV-210 column and determined by GC analysis with flame ionization detection. 2,2'-Dinitrobiphenyl is used as an internal standard. The method was subjected to an international collaborative study with 10 participating laboratories. Collaborators received matched pairs of technical DDT products and of water-dispersible powder, emulsifiable concentrate, and dustable powder formulations. Relative standard deviations for reproducibility (RSDR) for the paired samples were 1.16, 1.48, 2.08, and 1.80%, respectively. The method has been approved interim official first action by AOAC as a CIPAC-AOAC method.

Determination of mefloquine in blood by supercritical fluid chromatography with electron-capture detection.

Supercritical fluid chromatography (SFC) with electron-capture detection is described for the sensitive quantification of mefloquine in 0.1-ml blood samples. The method is internally standardized and incorporates partitioning into methyl tert.-butyl ether (MTBE) from aqueous base, back-extraction into dilute aqueous acid and final partitioning into MTBE from aqueous base. SFC conditions include a silica-gel-packed, glass-lined steel column and a mobile phase of 0.15% n-butylamine and 1% methanol in supercritical n-pentane. The method has a detection limit of 7.5 ng/ml in 0.1-ml blood samples and exhibits good linearity and precision. The method compares favorably with a published high-performance liquid chromatographic procedure in the analysis of blood from volunteers who received mefloquine hydrochloride (15 mg as base per kg body weight).

Adaptations of the Saker-Solomons test: simple, reliable colorimetric field assays for chloroquine and its metabolites in urine.

Two field-adapted colorimetric methods for measuring the antimalarial drug chloroquine in urine are described. Both are modifications of the method of Saker and Solomons for screening urine for phencyclidine and other drugs of abuse, using the colour reagent tetrabromophenolphthalein ethyl ester. One method is semiquantitative, detecting the presence of chloroquine (Cq) and its metabolites in urine with a 1 microgram/ml detection limit; it is more sensitive and reliable than the commonly used Dill-Glazko method and is as easy to apply in the field. The second method uses a hand-held, battery-operated filter photometer to quantify Cq and its metabolites with a 2 microgram/ml detection limit and a linear range up to 8 micrograms/ml. The first method was validated in the field using a published quantitative colorimetric method and samples from a malaria study in Nigeria. The second method was validated in the laboratory against high-performance liquid chromatographic results on paired samples from the Nigerian study. Both methods may be used in remote locations where malaria is endemic and no electricity is available.