Practices Regarding the Use of Antimalarial Medications among Inhabitants of the Buea Health District, Southwestern Cameroon: Implications for Malaria Treatment Policy

Background: Malaria treatment policy recommends continuous monitoring and reporting of therapeutic efficacy of antimalarial medications for early detection of resistant strains. Patient adherence to policies regarding the use of antimalarial medications is critical to success of global malaria elimination. This study assessed the practices regarding the use of antimalarial medications in the Buea Health District, Southwest Cameroon. Methods: A descriptive cross-sectional survey of a random sample of 495 people living in the district with episodes of malaria in the last one year prior to the study was conducted between February and August, 2015. Questionnaire was designed to obtain information from participants on the general knowledge of malaria and practices regarding to the use of antimalarial medications. Results: Knowledge on malaria symptoms, transmission and prevention was reasonable among 80.6% (399) of the respondents (p < 0.07). Only 31.3% (155) of the respondent could attribute cause of malaria to protozoan of genus Plasmodium species. Majority of the respondents 56.9% (283) frequently treat malaria with ACT, 32.4% (161) with monotherapy, < 15% with other non-ACTs. Presumptive diagnosis was commonly practiced by 67.3% (333) of the respondents. The prevalence of self-medication in the study population was 18.4%. Only 57.2% (283) of respondents took prescribed antimalarials. Majority of self-medicated respondents (63%) obtained antimalarials from drugstores and friends. About 50.9% (252) of the respondents took medications regularly and 57.6% (258) completed the treatment regimen. Respondents whose treatments were based on laboratory diagnosis adhered better than those on self-medication or recommended at the pharmacy (p < 0.02). Conclusion: The findings revealed a high knowledge of malaria with poor practices regarding the use of antimalarials. Efforts are needed to educate inhabitants of the district on practices regarding the use of antimalarials to prevent early emergence of drug resistance. Keywords: Antimalarials, Drug resistance, Presumptive diagnosis, Self-medication, Adherenc

Juglone-induced GSR-1 expression is not regulated by DAF-16.

<p>Worms carrying the <i>Pgsr-1::GFP</i> reporter construct were grown on HT115 bacteria carrying empty pL4440 vector (A, B) or subjected to <i>daf-16(RNAi)</i> (C, D). The respective effects on GFP-expression were monitored under standard culture conditions (A, C) or after induction by juglone (B, D) (quantified by ImageJ, Scale bars = 100 µm).</p

The Glutathione Reductase GSR-1 Determines Stress Tolerance and Longevity in <i>Caenorhabditis elegans</i>

<div><p>Glutathione (GSH) and GSH-dependent enzymes play a key role in cellular detoxification processes that enable organism to cope with various internal and environmental stressors. However, it is often not clear, which components of the complex GSH-metabolism are required for tolerance towards a certain stressor. To address this question, a small scale RNAi-screen was carried out in <i>Caenorhabditis elegans</i> where GSH-related genes were systematically knocked down and worms were subsequently analysed for their survival rate under sub-lethal concentrations of arsenite and the redox cycler juglone. While the knockdown of γ-glutamylcysteine synthetase led to a diminished survival rate under arsenite stress conditions, GSR-1 (glutathione reductase) was shown to be essential for survival under juglone stress conditions. g<i>sr-1</i> is the sole GSR encoding gene found in <i>C. elegans</i>. Knockdown of GSR-1 hardly affected total glutathione levels nor reduced glutathione/glutathione disulphide (GSH/GSSG) ratio under normal laboratory conditions. Nevertheless, when GSSG recycling was impaired by <i>gsr-1(RNAi)</i>, GSH synthesis was induced, but not vice versa. Moreover, the impact of GSSG recycling was potentiated under oxidative stress conditions, explaining the enormous effect <i>gsr-1(RNAi)</i> knockdown had on juglone tolerance. Accordingly, overexpression of GSR-1 was capable of increasing stress tolerance. Furthermore, expression levels of SKN-1-regulated GSR-1 also affected life span of <i>C. elegans</i>, emphasising the crucial role the GSH redox state plays in both processes.</p></div

Northern blot analyses.

<p><i>C. elegans</i> N2 wild-type worms were cultured on NGM agar under standard conditions (lane 1) or exposed to 150 µM (lane 2) and 300 µM juglone for 4 h, before total RNA was isolated and applied to Northern blot analyses using a radiolabeled <i>gsr-1</i> probe. Since there is only a 40 bp difference in transcript size (C46F11.2a, C46F11.2b1-3), only one band can be observed.</p

Analyses of GSR-1 and GCS-1 dependent stress tolerance.

<p>Wild-type eggs/L1 were placed on RNAi plates (A) <i>gsr-1(RNAi)</i> or (B) <i>gcs-1(RNAi)</i> and cultured until they have reached the L4/young adult stage, before being transferred to corresponding RNAi plates containing the indicated stressors. As control, plates with empty control vector pL4440 were used. Following 16 h incubation at 20°C, the survival rate was determined. Data represent means of at least three independent triplicate determinations (n≥120 animals, p values from Fisher test).</p

Regulation of <i>gsr-1</i> promoter activity by SKN-1.

<p>Worms carrying the <i>Pgsr-1::GFP</i> reporter construct were grown on HT115 bacteria carrying empty pL4440 vector (A, C) or subjected to <i>skn-1(RNAi)</i> (B, D). The respective effects on GFP-expression were monitored under standard culture conditions (A, B) or after induction by juglone (C, D) (quantified by ImageJ, Scale bars = 100 µm).</p

Determination of γ-glutamylcysteine, GSH and GSSG levels in lysate of synchronised 3-days-old worms.

<p>(<b>A</b>) Worms were cultured on RNAi-plates containing (i) HT115 bacteria carrying empty pL4440 vector or (ii) HT115 bacteria carrying empty pL4440 vector plus 150 µM juglone or (iii) <i>gsr-1(RNAi)</i> feeding bacteria or (iv) <i>gsr-1(RNAi)</i> feeding bacteria plus 150 µM juglone. (<b>B</b>) Analysis of the respective GSH/GSSG ratios.</p

GSR-1 expression pattern under standard and stress conditions.

<p>(<b>A</b>) The GSR-1 expression pattern of all stages was examined under standard culture conditions using <i>the Pgsr-1::GFP</i> worms. L4/young adults were transferred to (<b>B</b>) NGM plates containing 0.15 mM juglone. (<b>C</b>) Effect of dietary deprivation on <i>gsr-1</i> promoter activity. <i>Pgsr-1::GFP</i> worms were grown under standard culture conditions until they reach the L4/young adult stage. Animals were then transferred to NGM agar plates without <i>E. coli</i> food for 16 h, before being analysed by fluorescence microscopy (quantified by ImageJ). Note the different scale bars for L1–L3 (50 µm), for L4 and adult (100 µm).</p

Analyses of the interrelation between GSH synthesis and GSH redox cycling.

<p>Applying RNAi, GCS-1 expression was suppressed in the <i>C. elegans Pgsr-1::GFP</i> reporter strain (left) or GSR-1 expression in the <i>Pgcs-1::GFP</i> (middle) or <i>Pgst-4::GFP</i> reporter strain (right). RNAi-treated worms were analysed by fluorescence microscopy and GFP-expression was compared with control worms grown on HT115 bacteria carrying empty pL4440 vector. Fluorescence intensities for <i>Pgsr-1::GFP</i> reporter strain were scored as low (A), medium (B) and high (C) according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060731#pone.0060731-Tullet1" target="_blank">[16]</a>. Fluorescence intensity scoring for <i>Pgcs-1::GFP</i> and <i>Pgst-4::GFP</i> reporter strains is given in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060731#pone.0060731.s004" target="_blank">Figure S4</a></b>. Significant alterations in GFP expression was detected only in <i>Pgcs-1::GFP</i> (p<0.001) and <i>Pgst-4::GFP</i> worms (p<0.001) exposed to GSR-1(RNAi). Results are the means of at least three independent assays (n = 100 worms; p values from Fisher test).</p