Dwight L. Mount

Evaluation of long-lasting insecticidal nets after 2 years of household use.

Development of long‐lasting insecticidal nets (LLINs) may eliminate the need for insecticide retreatment of ITNs. While two LLINs (Olyset®, Sumitomo Chemical Co., Japan; and PermaNet® 1.0, Vestergaard‐Frandsen, Denmark) have received recommendations from the World Health Organization Pesticide Evaluation Scheme, field‐testing under normal use has been limited. We used a survival analysis approach to compare time to net failure of conventional polyester bednets treated only with deltamethrin to two LLINs and two candidate LLINs (Olyset®; PermaNet®; Insector, Athanor, France; and Dawa®, Siamdutch Mosquito Netting Co., Thailand). Additionally, we evaluated nets treated with a process designed to increase the wash‐durability of permethrin‐treated nets through the addition of cyclodextrin (a starch) in the treatment process. Houses in western Kenya were randomly assigned to one of the six net types and nets were distributed to cover all sleeping spaces. Households were visited monthly to assess reported side effects in inhabitants and washing frequency. Nets were evaluated for insecticidal activity by periodic WHO cone bioassays with mortality assessed at 24 h. Nets with bioassay mortality <70% were assayed monthly until failure, defined as the first of two consecutive bioassay mortality rates <50%. Time to failure was analyzed using an extended Cox Proportional Hazards model controlling for the cumulative number of washes. We distributed 314 nets to 177 households in June–July 2002; 22 nets (7.0%) were lost to follow‐up and 196 (62.4%) failed during the first 2 years of the evaluation. Controlling for cumulative number of washes, PermaNet® 1.0 [Hazard Ratio (HR) 0.14, 95% Confidence Interval (CI) 0.06–0.31] had a significantly lower risk of failure than conventional nets while Insector had a significantly higher risk of failure (HR 2.57, 95% CI 1.06–4.15). The risks of failure of the remaining nets (Olyset®: HR 1.29, 95% CI 0.79–2.10; Dawa®: HR 0.58, 95% CI 0.32–1.18; cyclodextrin: HR 0.65, 95% CI 0.40–1.1) were not significantly different from that of a conventional net. PermaNet® 1.0 performed significantly better than conventional nets and should be recommended to malaria control programs.

Laboratory wash resistance of long-lasting insecticidal nets

Long‐lasting insecticidal nets (LLINs) may eliminate the need for retreatment of mosquito nets used for the control of malaria and other vector‐borne diseases. The efficacy of LLINs after repeated washing under laboratory conditions has been used to predict long‐lasting efficacy under field conditions. We evaluated under laboratory conditions the wash resistance of two LLINs (PermaNet® 1.0, Vestergaard‐Frandsen, Denmark; Olyset®, Sumitomo Chemical Co., Japan), two candidate LLINs (Dawa®, Siamdutch Mosquito Netting Co., Thailand; Insector, Athanor, France) and a net treated with a process designed to increase its wash resistance and compared them with conventionally treated nets (deltamethrin, 25 mg/m2). Nets of all six types were washed using a standard protocol and tested weekly using WHO cone bioassays with Anopheles gambiae (Kisumu strain). The PermaNet® 1.0 was the most wash resistant with >50% mosquito mortality in WHO cone bioassays after as many as 20 washes. The Dawa® net also retained some activity after repeated washing but exhibited wide variation in insecticide retention and biological activity. The remaining nets lost >90% of their biological activity after six washes as measured by 24‐h mortality of A. gambiae in WHO cone tests. After 20 washes, all nets lost >50% of their initial insecticide concentrations except for the Olyset® net. After 20 washes, nets were heated for 4 h at 60 °C to determine whether biological activity could be restored by heat‐assisted regeneration. Only the Olyset® net was regenerated by heating, with average mosquito mortality and knockdown in WHO cone tests rising to >90% after heating for 4 h at 60 °C. However, regeneration of the biological activity of Olyset® nets that had been washed three times did not occur at 30 °C or 35 °C after 12 weeks. The wash resistance of these LLINs corresponded well to their retention of biological activity observed in a field trial, suggesting that wash resistance may be a good predictor of the longevity of insecticidal activity of LLINs under field conditions.

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.

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.

High-performance liquid chromatographic assay for the simultaneous determination of sulfadoxine and pyrimethamine from whole blood dried onto filter paper.

A method using solid-phase extraction and high-performance liquid chromatography is evaluated for the simultaneous determination of sulfadoxine and pyrimethamine from 0.1 ml of whole blood dried onto filter paper. Extraction recoveries are about 60% for both drugs. The coefficient of variation for intra-assay precision, inter-assay precision and accuracy is less than 10% for sulfadoxine (10-100 microg/ml) and pyrimethamine (1-10 microg/ml).

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

Abstract. 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 Angambiae appears to be minimal in our study villages, although the impact of permethrin was demonstrated by a significantly lower parous‐rate of Angambiae females in the intervention (63–66%) than in non‐intervention (79%) villages, and by reduced malaria transmission (reported elsewhere).

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.

Comparative insecticidal power of three pyrethroids on netting

Abstract 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 40 mg 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.

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.

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.