Research ArticleVECTOR CONTROL

Efficacy of the Olyset Duo net against insecticide-resistant mosquito vectors of malaria

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Science Translational Medicine  14 Sep 2016:
Vol. 8, Issue 356, pp. 356ra121
DOI: 10.1126/scitranslmed.aad3270

Figures

  • Fig. 1. Knockdown of susceptible mosquitoes of the A. gambiae Kisumu strain in cone bioassays.

    One hundred fifty unfed 3- to 5-day-old female mosquitoes of the susceptible A. gambiae Kisumu strain were tested in cone bioassays with each bed net and for each wash point for 3 min in replicates of five mosquitoes per cone. Knockdown is shown for pyriproxyfen-treated net, Olyset Net, and Olyset Duo net as percentage. A total of four replicate net samples were tested for each wash point (50 mosquitoes per replicate). Knockdown was recorded after 1 hour. Each bar represents percentage of knockdown at each wash point (0, 5, 10, 15, and 20 washes) for the different bed net types tested. The red dotted line indicates WHO cutoff for knockdown (95%) after 20 washes in cone bioassays. Error bars represent 95% confidence intervals (CI). Knockdown with Olyset Duo was >95% after 20 washes, demonstrating that Olyset Duo meets WHO criteria for efficacy in cone bioassays.

  • Fig. 2. Mortality of susceptible mosquitoes of the A. gambiae Kisumu strain in cone bioassays.

    One hundred fifty unfed 3- to 5-day-old susceptible A. gambiae Kisumu female mosquitoes were tested for 3 min per cone for each type of bed net (pyriproxyfen-treated net, Olyset Net, and Olyset Duo) at each wash point in replicates of five mosquitoes. A total of four replicate net samples were tested at each wash point (~40 mosquitoes per replicate). The number of dead mosquitoes was recorded after a 24-hour holding period. Each bar represents percent mortality at each wash point (0, 5, 10, 15, and 20 washes) for the different bed net types tested. For each net type, bars bearing the same letter label are not significantly different at the 5% level (P > 0.05) (generalized linear mixed models). Error bars represent 95% CIs. The red dotted line indicates WHO cutoff for mortality (80%) in cone bioassays after 20 washes. Mortality with Olyset Duo was >80% after 20 washes, showing that the net meets WHO criteria for efficacy in cone bioassays.

  • Fig. 3. Mortality of the pyrethroid-resistant A. gambiae VKPer strain in tunnel tests.

    About 200 adult female pyrethroid-resistant mosquitoes of the A. gambiae VKPer strain (aged 5 to 8 days) were tested in four replicate tunnel tests for each type of netting (control untreated, pyriproxyfen-treated net, Olyset Net, and Olyset Duo) at three washing intervals (0, 10, and 20 washes). Tunnels were kept overnight in a dark room at 25° to 27°C and 75 to 85% relative humidity. Mosquitoes were collected from the tunnel in the morning, and mortality was recorded after a 24-hour holding period. Each bar represents percentage of mortality at each wash point for the different bed net types. For each net type, bars bearing the same letter label are not significantly different at the 5% level (P > 0.05) (generalized linear mixed models). The red dotted line indicates WHO cutoff for mortality (80%) after 20 washes in tunnel tests. Error bars represent 95% CIs. Mortality with Olyset Duo was >80% after 20 washes, showing that the net meets WHO criteria for efficacy in tunnel tests.

  • Fig. 4. Inhibition of blood feeding by the pyrethroid-resistant A. gambiae VKPer strain in tunnel tests.

    About 200 adult female pyrethroid-resistant mosquitoes of the A. gambiae VKPer strain (aged 5 to 8 days) were tested in four replicate tunnel tests for inhibition of blood feeding by each type of netting (pyriproxyfen-treated, Olyset Net, and Olyset Duo) at three washing intervals (0, 10, and 20 washes). Tunnels were kept overnight in a dark room at 25° to 27°C and 75 to 85% relative humidity. Mosquitoes were collected from the tunnel in the morning, and blood-feeding rates were recorded. Each bar represents percentage of blood-feeding inhibition (relative to the control untreated net) at each wash point for the different bed net types. For each net type, bars bearing the same letter label are not significantly different at the 5% level (P > 0.05) (generalized linear mixed models). The red dotted line indicates WHO cutoff for blood-feeding inhibition (80%) after 20 washes in tunnel tests. Error bars represent 95% CIs. Blood-feeding inhibition with Olyset Duo was >80% after 20 washes, showing that the net meets WHO criteria for efficacy in tunnel tests.

  • Fig. 5. Blood feeding by wild free-flying pyrethroid-resistant A. gambiae mosquitoes in experimental field huts.

    Consenting adult human volunteers slept in experimental huts in Cove, Benin, from 8:00 p.m. to 5:00 a.m. each night of the study to attract mosquitoes into the huts. Three replicate nets in each hut were tested, and these nets were rotated in the hut every 2 days for each week of the trial. Mosquitoes were collected in the morning and were scored for blood-feeding status. Each bar represents percentage of blood-fed mosquitoes (relative to the control untreated net) at each wash point (0 and 20 washes) for the different bed net types (pyriproxyfen-treated net, Olyset Net, and Olyset Duo). Bars bearing the same letter label are not significantly different at the 5% level (P > 0.05) (generalized linear mixed models). Error bars represent 95% CIs. Blood-feeding rates with Olyset Duo either unwashed or after 20 washes were much lower than with Olyset Net (P < 0.05).

  • Fig. 6. Mortality of wild free-flying pyrethroid-resistant A. gambiae mosquitoes in experimental field huts.

    Consenting adult human volunteers slept in experimental huts in Cove, Benin, from 8:00 p.m. to 5:00 a.m. each night of the study to attract mosquitoes into the huts. Three replicate nets in each hut were tested, and these nets were rotated in the hut every 2 days for each week of the trial. Mosquitoes were collected in the morning, and mortality was recorded after a 24-hour holding period. Each bar represents percentage of mortality at each wash point (0 and 20 washes) for the different bed net types [control (untreated net), pyriproxyfen-treated net, Olyset Net, and Olyset Duo]. Bars bearing the same letter label are not significantly different at the 5% level (P > 0.05) (generalized linear mixed models). Error bars represent 95% CIs. Mortality rates with Olyset Duo either unwashed or after 20 washes were higher than with Olyset Net (P < 0.05).

  • Fig. 7. Mortality of susceptible A. gambiae Kisumu mosquitoes exposed to net pieces treated with permethrin and/or pyriproxyfen.

    Fifty 3- to 5-day-old mosquitoes of the A. gambiae Kisumu strain were exposed (in batches of 10) for 3 min to net samples hand-treated with pyriproxyfen, permethrin, or a mixture of the two insecticides in WHO cylinders. Mortality was recorded after a 24-hour holding period. The doses of active ingredients applied (y axis) are expressed in mg/m2. Each bar represents the percentage of mortality for each dose or combination of doses applied. Bars bearing the same letter label are not significantly different at the 5% level (P > 0.05) (generalized linear mixed models). Error bars represent 95% CIs. Mortality rates with nets hand-treated with a mixture of pyriproxyfen and permethrin did not increase compared to nets treated with either active ingredient alone (P > 0.05).

Tables

  • Table 1. Oviposition rate for surviving pyrethroid-resistant A. gambiae VKPer mosquitoes in tunnel tests.
    Net typeNo. of washesNo. of females observed
    (blood-fed alive at 24 hours)
    No. of females
    laying eggs
    % Blood-fed females
    laying eggs (95% CI)*
    % Oviposition
    inhibition (95% CI)
    Control17615286 (81–91)a
    Pyriproxyfen net0198116 (3–9)b94 (90–97)
    1017510560 (51–69)c,d30 (20–41)
    201884826 (20–32)e70 (63–77)
    Olyset Net10282486 (73–98)a0 (0–15)
    20161275 (54–96)a,c13 (0–37)
    Olyset Duo1021943 (22–64)d50 (26–74)
    2012975 (51–100)a,c13 (0–41)

    *Values bearing the same superscript are not significantly different at the 5% level (P > 0.05). Generalized linear mixed models.

    • Table 2. Offspring of surviving pyrethroid-resistant A. gambiae VKPer mosquitoes in tunnel tests.
      Net typeNo. of washesTotal eggs laidEggs per
      female observed
      (95% CI)*
      % Reduction
      in fecundity
      Total larvae
      hatched
      Hatch
      rate (%)
      Larvae per
      female observed
      (95% CI)*
      % Reduction
      in offspring
      (95% CI)
      Control14,01280 (75–85)a98567056 (49–61)a
      Pyriproxyfen net05303 (1–4)b97 (95–99)131251 (0–2)b99 (96–100)
      1012,40071 (65–75)c11 (3–19)41253324 (21–27)c58 (53–63)
      208,20644 (40–48)d45 (40–50)37654620 (18–23)c64 (60–68)
      Olyset Net102,828101 (94–109)e0 (0–18)17406262 (51–71)a0 (0–9)
      2092458 (48–69)f28 (16–40)7297946 (34–56)a19 (0–39)
      Olyset Duo1062530 (25–35)g63 (56–69)4116620 (14–25)c65 (55–75)
      2042535 (19–49)dg56 (36–76)3244727 (18–33)c52 (36–68)

      *Values down a column bearing the same superscript are not significantly different at the 5% level (P > 0.05). Kruskal-Wallis test.

      • Table 3. Results for wild free-flying pyrethroid-resistant A. gambiae mosquitoes in experimental huts in Cove, Benin.
        Net typeControlPyriproxyfen netOlyset NetOlyset Duo
        No. of washes020020020
        Total females caught2874300728973804378238403293
        Average catch per night68726991909178
        % Deterrence000000
        Total exiting1325136712862493217724581943
        % Exiting (95% CI)*46 (42–48)a45 (42–47)a44 (41–47)a65 (62–68)b58 (56–60)c64 (62–68)b59 (57–61)c
        % Inside net (95% CI)21152651136
        Total blood-fed mosquitoes10047201031444875281497
        Inhibition of blood feeding (%)*31 (28–34)a0 (0–8)b66 (61–71)c34 (31–37)a79 (75–83)d63 (58–68)c
        Personal protection (%)*28a0b58c13d72e50c
        No. of dead after 24 hours1598686601228101215361314
        Killing effect (%)*26a18b39c31d51e43c

        *Values along a row bearing the same superscript are not significantly different at the 5% level (P > 0.05). Generalized linear mixed models.

        • Table 4. Oviposition rate for surviving wild blood-fed pyrethroid-resistant A. gambiae mosquitoes in experimental hut studies.
          Net typeNo. of washesNo. of females observed
          (blood-fed alive at 24 hours)
          No. of females
          laying eggs
          % Blood-fed females laying eggs (95% CI)*% Oviposition
          inhibition (95% CI)
          Control40121955 (50–60)a
          Pyriproxyfen net02943412 (8–16)b78 (71–85)
          2044420145 (40–50)c18 (9–27)
          Olyset Net022912555 (49–61)a0 (0–11)
          2036121058 (53–63)a0 (0–4)
          Olyset Duo01203731 (23–39)d44 (30–58)
          2022710647 (41–53)c15 (4–25)

          *Values down a column bearing the same superscript are not significantly different at the 5% level (P > 0.05). Generalized linear mixed models.

          • Table 5. Offspring of surviving wild blood-fed pyrethroid-resistant A. gambiae mosquitoes in experimental hut studies.
            Net typeNo. of
            washes
            Total
            eggs laid
            Eggs per
            female
            observed*
            % Reduction in
            fecundity
            Hatch
            rate (%)
            Larvae per
            female
            observed*
            % Reduction in
            offspring
            Reproductive
            rate R0
            % Reduction in
            R0 (95% CI)
            Control25,06763 (61–65)a3321 (18–24)a7.1
            Pyriproxyfen
            net
            02,83310 (6–13)b84 (78–90)222 (0–4)b90 (81–100)0.593 (86–100)
            2020,27346 (39–51)c27 (19–38)3013 (10–15)c38 (29–52)4.636 (33–39)
            Olyset Net013,99461 (57–65)a3 (0–10)2616 (12–21)c24 (0–42)1.875 (71–79)
            2023,91866 (58–74)a0 (0–8)2617 (15–19)c19 (10–29)3.748 (44–53)
            Olyset Duo02,96525 (19–30)d60 (50–70)236 (4–8)b71 (62–80)0.494 (88–100)
            2012,44055 (51–58)e13 (7–19)2212 (8–15)c43 (29–62)1.678 (74–82)

            *Values down a column bearing the same superscript are not significantly different at the 5% level (P > 0.05). Kruskal-Wallis test.

            • Table 6. Tunnel test results for susceptible A. gambiae Kisumu mosquitoes exposed to net pieces hand-treated with permethrin and/or pyriproxyfen.

              Net treatment (mg/m2)No. of exposed% Penetration*% Blood-fed*% Blood-feeding inhibition% Mortality*
              Control98100a97a3a
              95% CI95–10094–1000–6
              Pyriproxyfen (250)10199a98a08b
              95% CI97–10095–1003–13
              Permethrin (150)10540b30b6946c
              95% CI31–4921–3936–55
              Permethrin (150) + pyriproxyfen (250)10443b41b5835c
              95% CI31–5533–5226–44

              *Values down each column sharing the same superscript are not significantly different at the 5% level (P > 0.05). Generalized linear mixed models.

              • Table 7. Analysis of total active ingredients in Olyset Duo and pyriproxyfen-treated nets.
                No. of
                wash cycles
                Permethrin
                (g/kg)
                % Retention
                of
                permethrin
                Pyriproxyfen
                (g/kg)
                % Retention
                of
                pyriproxyfen
                Olyset Duo
                Unwashed19 ± 0.210 ± 0.2
                515 ± 0.3797 ± 0.370
                1014 ± 0.4746 ± 0.460
                1513 ± 1.8686 ± 1.860
                2013 ± 0.2685 ± 0.250
                Pyriproxyfen
                net
                Unwashed9 ± 0.2
                55 ± 0.155
                104 ± 0.144
                154 ± 0.144
                203 ± 0.133

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