Entomological status of Anopheles sergentii and the first molecular investigation of its insecticide-resistant genes, kdr and ace-1 in Morocco.

BACKGROUND & OBJECTIVES: Prior to their elimination in 1974 and 2004, respectively, Plasmodium falciparum and P. vivax were the main native malaria parasites involved in disease transmission in Morocco. Imported cases of human malaria are still reported from the country. Anopheles labranchiae in northern Morocco and An. sergentii in the southern regions are the main malaria vectors. The bionomics and insecticide susceptibility of An. sergentii are poorly understood and need to be further studied to enhance the epidemiological surveillance of this important malaria vector. METHODS: The adults and larvae of Anopheles sergentii were collected during the mosquito breeding season in 2015 and 2016 and environmental characteristics of their breeding sites were recorded. Blood meals were analyzed using PCR-RFLP. Alongside, the WHO routine susceptibility tests with DDT (4%) and malathion (5%) were conducted and An. sergentii specimens were screened for knockdown resistance (kdr) and acetyl cholinesterase encoding (ace-1) gene mutations. RESULTS: Anopheles sergentii was observed during the summer and autumn seasons, feeding mainly on sheep, cows and also on humans. The WHO bioassays revealed complete susceptibility to DDT and malathion. Analysis of the sequences of the voltage-gated sodium channel gene revealed the absence of the kdr "Leu-Phe" mutation and PCR-RFLP revealed the absence of the G119S mutation in the ace-1. INTERPRETATION & CONCLUSION: With the increasing number of imported cases of human malaria in Morocco, the indiscriminate feeding behavior of this species may pose an infectious medical threat. Fortunately, the absence of insecticide resistance can ensure, for now, the efficiency of insecticides, as a part of the vector control program in controlling An. sergentii in Morocco.

Insect repellents and associated personal protection for a reduction in human disease.

Personal protection measures against biting arthropods include topical insect repellents, area repellents, insecticide-treated bednets and treated clothing. The literature on the effectiveness of personal protection products against arthropods is mainly limited to studies of prevention of bites, rather than prevention of disease. Tungiasis was successfully controlled by application of topical repellents and scrub typhus was reduced through the use of treated clothing. Successful reduction of leishmaniasis was achieved through the use of topical repellents, treated bednets and treated clothing in individual studies. Malaria has been reduced by the use of insecticide-treated bednets (ITN), certain campaigns involving topical repellents, and the combination of treated bednets and topical repellents. Although area repellents such as mosquito coils are used extensively, their ability to protect humans from vector-transmitted pathogens has not been proven. Taken together, the literature indicates that personal protection measures must be used correctly to be effective. A study that showed successful control of malaria by combining treated bednets and topical repellents suggests that combinations of personal protection measures are likely to be more effective than single methods. Implementation of successful programmes based on personal protection will require a level of cooperation commonly associated with other basic societal functions, such as education and food safety.

A new module for quantitative evaluation of repellent efficacy using human subjects.

A new module for quantitative evaluation of arthropod repellents in human subjects was designed, constructed, and protocols for use of the module were developed. Doses of 3 arthropod repellents, 1-[3-cyclohexen-1-ylcarbonyl] piperidine (AI3-35765), 1-[3-cyclohexen-1-ylcarbonyl]-2-methylpiperidine (AI3-37220), and N,N-diethyl-3-methylbenzamide (deet), were evaluated using the mosquito, Anopheles stephensi Liston. Biting responses to varied doses of the repellents on human skin were quantified, and the effectiveness of the 3 repellents was compared. The new module consists of 6 test cells and permits the simultaneous comparison of up to 5 repellent doses or chemical types and a control using a complete randomized block design with minimal treatment interaction and with > or = 6 replicates per human subject.

Stereochemical effects in an insect repellent.

Racemic 1-[3-cyclohexen-1-ylcarbonyl]-2-methylpiperidine repels blood-feeding arthropods such as mosquitoes, chiggers, and ticks. The compound contains two asymmetric carbon atoms and the racemate consists of four stereoisomers. Quantitative mosquito bioassays using Aedes aegypti (L.) showed that (1S,2'S) and (1R,2'S) configurations were 2.8-3.1 and 1.6-1.8 times more effective, respectively, than the other two stereoisomers in reducing mosquito bites. (1S,2'S) was 2.5 more repellent than the racemate. Biological data show that an interaction of the (2'S)-2-methylpiperidine configuration with a repellent receptor system in A. aegypti is apparently important to repellent activity. Nuclear magnetic resonance spectra and molecular mechanics calculations for the stereoisomers provided insight into the conformation of the (2'S)-group. Results indicate that enhanced repellent effects can be realized through formulation of the most active stereoisomers of the compound.

Comparative activity of deet and AI3-37220 repellents against the ticks Ixodes scapularis and Amblyomma americanum (Acari: Ixodidae) in laboratory bioassays.

The repellents N,N-diethyl-3-methylbenzamide (deet) and racemic 2-methylpiperidinyl-3-cyclohexene-1-carboxamide (AI3-37220) were evaluated using two different laboratory bioassays to determine their relative effectiveness against host-seeking nymphs of the blacklegged tick, Ixodes scapularis Say, and the lone star tick, Amblyomma americanum (L.). In a petri dish bioassay, ticks were released within a ring of repellent on a horizontal filter paper disk. In the second bioassay, ticks were allowed to climb a vertical strip of filter paper whose central portion was treated with a repellent. Deet and AI3-37220 were more effective against I. scapularis than A. americanum nymphs. In the petri dish bioassay, none of the concentrations of deet or AI3-37220 tested confined A. americanum within the treated ring. However, in the vertical bioassay, both species exhibited avoidance of the repellents, and I. scapularis was repelled by much lower concentrations than A. americanum. I. scapularis were repelled by lower concentrations in the vertical bioassay than in the petri dish bioassay. Deet was slightly more effective against I. scapularis than AI3-37220 in both bioassays, but AI3-37220 was significantly more effective than deet against A. americanum in the vertical bioassay.

Field evaluation of deet and a piperidine repellent against Aedes communis (Diptera: Culicidae) and Simulium venustum (Diptera: Simuliidae) in the Adirondack Mountains of New York.

Repellent efficacy of N,N-diethyl-3-methyl-benzamide (deet), the piperidine, 1-[3-cyclohexen-1-ylcarbonyl]-2-methylpiperidine (AI3-37220), and a 1:1 ratio of deet + AI3-37220 were evaluated topically (0.25 mg/cm2 applied in ethanol solution) on human volunteers against the mosquito Aedes communis (DeGeer) and the black fly Simulium venustum Say. The average repellency of all three formulations was > 95% at 4 h. For both mosquitoes and black flies, deet alone provided < 90% protection at 6 h, whereas AI3-37220 provided > 95% protection. Although repellent treatments were not significantly different overall, the contrasts between AI3-3720 versus deet were significant at 6 and 8 h. The 95% confidence interval on percent repellency at 6 h ranged from 90.1 to 98.9% for AI3-37220 versus 64.3 to 82.2% for deet, and at 8 h ranged 76.1 to 88.5% for AI3-37220 versus 47.8 to 64.0% for deet. Similarly, the confidence interval for protection against black flies at 6 h by (AI3-37220 ranged from 86.3 to 99.5% and did not overlap with the confidence interval provided by deet alone (51.2 to 78.8%). There was no evidence of synergistic repellency from a combination of the two compounds; i.e., protection from combined compounds was no better than either repellent used alone.

Laboratory and field trials of four repellents with Culex pipiens (Diptera: Culicidae).

During Operation "Desert Shield," 16 volunteers field-tested four insect repellents (deet, the lactone CIC-4, and the piperidine compounds AI3-37220 and AI3-35765) against biting mosquitoes at King Fahd Airport, Eastern Province, Saudi Arabia. CIC-4 and AI3-37220 (25% wt/vol) provided effective (> 90%) protection against bites for 4 h. Deet and AI3-35765 protected for only 2 h. The compounds subsequently were evaluated for repellency against laboratory-reared Culex pipiens L. CIC-4 was more effective than deet, AI3-37220, or AI3-35765 at the ED50 but not at the ED95 level in initial sensitivity tests using human volunteers. At the ED95 level, deet provided significantly better protection than either piperidine compound. In laboratory duration tests, AI3-37220 provided 8 h of effective (> 90%) protection against Cx. pipiens bites, deet and AI3-35765 7 h of protection, and CIC-4 2 h of protection.

Laboratory evaluation of AI3-37220, AI3-35765, CIC-4, and deet repellents against three species of mosquitoes.

Four repellents, N,N-diethyl-3-methyl-benzamide (deet), 2-hydroxy-methyl-cyclohexyl acetic acid lactone (CIC-4), and 2 piperidines (1-[3-cyclohexen-1-ylcarbonyl] piperidine [AI3-35765] and 1-[3-cyclohexen-1-ylcarbonyl]-2-methylpiperidine [AI3-37220]) were evaluated alone and in combination against Aedes aegypti, Anopheles stephensi, and Culex quinquefasciatus using a modified in vitro test system. This method was a valuable tool for comparing effective concentrations of the new compounds. Because of the controlled conditions of the test, it was possible to use the results of assays that had been conducted over a 5-year period and to perform the many replications necessary to evaluate combinations of compounds. The new candidate repellents were generally as effective as deet. Although speculative at this time, there was some evidence of synergistic interaction. Repellent combinations of CIC-4/AI3-37220/AI3-35767, deet/AI3-35765, and deet/AI3-37220/AI3-35765 against An. stephensi and CIC-4/AI3-35765, deet/AI3-37220/AI3-35765, AI3-37220/AI3-35765, and CIC-4/AI3-37220 against Ae. aegypti were more effective than the component compounds alone.

Military preventive medicine support: the Balkan experience.

Preventive medicine (PVNTMED) support to deployed forces is as varied as the circumstances for force deployment. The Bosnia-Herzegovina deployment of the 1st Armored Division as part of the Dayton Peace Accords Implementation Forces proved to be no exception to this premise. PVNTMED units, both in the field and at the U.S. Army-Europe support base, were challenged to provide mission support under significant mobility restrictions and in arenas of public health practice not previously thought to be of tactical significance, specifically environmental pollution. New to this operation was the deployment of a Theater Army Medical Laboratory with a mission to assist deployed PVNTMED units with the capability to rapidly diagnose infectious disease agents and provide an expanded array of environmental monitoring support. Vector-borne diseases were also a threat to health, and an innovative base camp sanitation assessment and reporting system was created to alert leaders to the risk of disease transmission to soldiers.

Soldier acceptability of a camouflage face paint combined with DEET insect repellent.

Under operational conditions, current doctrine requires separate application of the standard military insect repellent and camouflage face paint. Working with an industrial partner, Amon Re, Inc., we developed a combined camouflage face paint and insect repellent that offers excellent protection from arthropod disease vectors. We undertook a study to determine whether the new product was acceptable to soldiers under field conditions. A new formulation of camouflage face paint containing the insect repellent N,N-diethyl-3-methyl-benzamide (DEET) was tested for user acceptability during a joint-service, multinational, military field training exercise (Operation Cobra Gold 1999) in Thailand. Soldiers testing the camouflage face paint were members of one of three companies (A, B, and C) of 1st Battalion, 21st Infantry Regiment, 25th Infantry Division. Soldiers in A company (N = 98) received standard military camouflage face paint and the U.S. military's Extended Duration Topical Insect and Arthropod Repellent containing 33% DEET, soldiers in B company (N = 75) received a new formulation of camouflage face paint without DEET, and soldiers in C company (N = 88) received the new formulation of camouflage face paint containing 30% DEET. Every soldier who volunteered to participate completed the study and submitted responses to the questionnaire. The different treatments were evaluated while soldiers spent 4 days conducting simulated combat exercises in a hot, tropical environment in central Thailand. Soldiers were provided the test materials, given a briefing on the study, and completed an initial questionnaire on May 19, 1999. Soldiers completed a final questionnaire after they returned from the training exercise on May 24, 1999. Results of the study indicated that soldiers found the new formulation of 30% DEET camouflage face paint easier to apply (88% of respondents) and remove (77%) than the current standard issue camouflage face paint. Soldiers liked the new 30% DEET camouflage face paint formulation better (61%) and were more likely to recommend it (67%) than the old camouflage face paint formulation. Although the addition of DEET to the camouflage face paint occasionally caused minor irritation after application for a short period (less than 15 minutes), the new camouflage face paint formulation with 30% DEET was evaluated most often (72%) as either good or excellent. The new 30% DEET camouflage face paint formulation offers significant operational advantages: protection from disease-transmitting arthropods, elimination of the need to carry separate insect repellent and camouflage face paint, and reduction in the time required to apply repellent and camouflage face paint separately.

Twelve-hour duration testing of cream formulations of three repellents against Amblyomma americanum.

The repellent efficacies of the U.S. military repellent 33% N,N-diethyl-3-methylbenzamide (deet), 10% and 20% (1S, 2'S) 2-methylpiperidinyl-3-cyclohexene-1-carboxamide (SS220) and 10% and 20% 1-methyl-propyl-2-(hydroxyethyl)-1-piperidinecarboxylate (Bayrepel) cream formulations on human volunteers against the lone star tick Amblyomma americanum (L.) were evaluated in a simulated forest floor environment over a 12-h testing period. At 2-h intervals, volunteers, with repellent applied in a 5-cm-wide band around each ankle, stood for 5 min in plastic tubs containing leaf litter and 100 host-seeking A. americanum nymphs. Ticks were allowed to remain on a volunteer's feet and ankles for an additional 5 min after the volunteer exited the tub. All repellent formulations provided high levels of protection for the entire 12 h. No ticks crossed 5-cm-wide bands of 20% SS220 and Bayrepel during any challenge, and thus 100% protection was afforded throughout the test. These formulations showed a long-lasting efficacy hitherto unknown in tick repellents intended for use on human skin.

Repellency of deet and SS220 applied to skin involves olfactory sensing by two species of ticks.

Responses of host-seeking nymphs of the blacklegged tick, Ixodes scapularis Say and lone star tick, Amblyomma americanum (Linnaeus) (Acari: Ixodidae) to the repellents N,N-diethyl-3-methylbenzamide (deet) and (1S, 2'S)-2-methylpiperidinyl-3-cyclohexene-1-carboxamide (SS220) were studied using fingertip laboratory bioassays. Ethanol solutions of both compounds applied to the skin strongly repelled both species of ticks at 0.8 and 1.6 micromole of compound/cm2 skin. The ticks were also repelled when two layers of organdie cloth covered the portion of a finger treated with either deet or SS220. Gas chromatographic analyses of the outer layer of cloth that had covered skin treated with 1.6 micromole compound/cm2 skin revealed only 0.1 nmole SS220/cm2 cloth and 2.8 nmole deet/cm2 cloth. However, in bioassays in which a single layer of cloth was treated with a dose of deet or SS220 equivalent to the amount found in the outer layer of cloth, ticks were not repelled. Results unequivocally demonstrated that these ticks responded to the repellents in the vapour phase when repellent treated skin was covered with cloth to obviate tactile contact with them, and made it clear that the ticks detect the repellents by olfactory sensing. Heretofore, the mode of action of deet and SS220 was unclear.

The effect of sunscreen on the efficacy of insect repellent: a clinical trial.

Sunscreen loses efficacy when used with the insect repellent N,N -diethyl-3-methylbenzamide (diethyltoluamide; deet). A previous study demonstrated a 33.3% decrease in sun protection factor when insect repellent and sunscreen were applied sequentially to the skin. The purpose of this study was to determine whether the efficacy of insect repellent is affected by two different formulations of sunscreen. The combination of sunscreen and insect repellent was tested in 8 groups: a control group (no sunscreen or insect repellent), sunscreen alone (two groups [gel and cream]), insect repellent alone, insect repellent applied before sunscreen (two groups), and insect repellent applied after sunscreen (two groups). The results of this study showed that insect repellent has the same efficacy even when sunscreen is applied with it. This indicates that in the formulations tested, sunscreen does not reduce the efficacy of insect repellent.