Evaluation of efficacy and duration of the stick camouflage face paint with 30% deet against mosquitoes in Belize.
Subject: Wetlands (Health aspects)
Soldiers (Surveys)
Soldiers (Health aspects)
Zinc industry (Health aspects)
Authors: Lawrence, Kendra L.
Benante, John Paul
Close, Nicole C.
Achee, Nicole L.
Pub Date: 07/01/2009
Publication: Name: U.S. Army Medical Department Journal Publisher: U.S. Army Medical Department Center & School Audience: Professional Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2009 U.S. Army Medical Department Center & School ISSN: 1524-0436
Issue: Date: July-Sept, 2009
Accession Number: 242963593
Full Text: Introduction

Historically, disease and nonbattle injuries, particularly vector-borne diseases, have resulted in more casualties than have combat operations. (1) The impact of vector-borne disease on military operations is well-documented from the Revolutionary and American Civil Wars. (2) The incidence of these diseases in recent operations (3-8) demonstrates that the threat has not diminished. Currently, the US military has a presence in over 130 countries (9) and is involved in a wide-range of operations, including stability and support operations, small scale contingency operations, and major theater wars. Military personnel remain exposed to a wide range of vectors and vector-borne diseases. The very nature of military operations (rapid deployments, troop movements, fluidity of the modern battlefield) often makes the conduct of area-wide vector control operations impossible. In many tactical situations (eg, assault, bivouac, and entrenchment), vector control measures cannot be instituted, or are insufficient to prevent disease transmission. Thus, personal protective measures (PPMs) have been, and remain the first and best line of defense for military personnel against all vector-borne diseases.

There are many types of PPMs available to military personnel, including permethrin for uniform and bed net treatment, and the Extended Duration Topical Insect and Arthropod Repellent (EDTIAR) with 33% N,N-diethyl-3-methylbenzamide (deet) in a polymer formulation. (10) The Department of Defense Insect Repellent System, consisting of a permethrin-treated uniform, EDTIAR on exposed skin, and a properly worn uniform, is a system that, when employed properly, is very effective at protecting Soldiers from vector-borne disease threats. (11) The EDTIAR is the standard military repellent which is designed to be used in all situations for protection against vector threats.

Since concealment is important in many tactical situations, Soldiers use camouflage face paint. Although EDTIAR can be used in conjunction with the standard camouflage face paint, it requires 2 separate applications, an initial application of EDTIAR followed by the camouflage face paint. (12) Recognizing the special needs of tactical situations, the Walter Reed Army Institute of Research (WRAIR) Repellent Program, in collaboration with Amon Re, Inc (Thomasville, Georgia), developed combined camouflage face paint with 30% deet. (13) The addition of insect repellent to the camouflage face paint replaces 2 items with one, saves space, reduces the weight of the individual survival kit, and saves application time.

An Operational Requirements Document (ORD) dated May 28, 1999 (internal use document) was issued to guide development of camouflage face paint combined with 30% deet in both compact and stick form. The compact form, with 5 colors of face paint (black, green, loam, sand, and white) was developed and tested for efficacy and Soldier acceptability, (14) and endorsed by the Armed Forces Pest Management Board. It was recommended for assignment of a national stock number (6840-01493-7334) in 2001. Development of the stick formulations to meet the requirements of the ORD began in 2006.


The purpose of the study was to evaluate the repellent efficacy and duration of the stick camouflage face paint formulated with 30% deet (SCFPwD). The study was essential for further advanced development and processing of this product into the military supply system.

Materials and Methods

Study Site

Belize (formerly British Honduras) is a Central American country with a geographic area of 22,966 sq km and a population of approximately 290,000. (15) The climate and general ecology of Belize is favorable for year round transmission of malaria. Extensive marshes, swamps, and rivers provide continuous larval habitats for malaria vector species, even during the dry seasons. Malaria incidence is significantly higher in the southern and western provinces of Toledo, Cayo, and Stann Creek than in the northern provinces of Corozal and Orange Walk. (16) The attack rate of malaria is less than 1% per month, concentrated primarily in the south. (17) The study was conducted in the town of San Roman in the northern Belize province of Orange Walk (Figure 1) from February 20 to February 22, 2007. The study site in San Roman (18 [degrees] 17.8' N, 88[degrees] 30.6' W) was located about 100 m from the Rio Nueve (New River) in an open field (approximately 300 [m.sup.2]) bordered on one side by a semiwooded area. Weather conditions during the 3 nights of collection were seasonal, with an average temperature of 24[degrees]C, 87% relative humidity, and wind speeds about 2 kph.

Test Material

The SCFPwD is a camouflage face paint in a hardened, tube-shaped form, comprised of the following ingredients: Puresyn ME300, Puresyn 1000 (Polydecene), 7V,7V-diethyl-3-methylbenzamide, Multiwax W835 (microcrystalline wax), white beeswax, light candelilla wax, propylparaben, Cab-O-Sil M5 (silica), Thixcin R (trihydroxystearin), Tenox 6 (Zea mays (corn) oil, glyceryl oleate, propylene glycol, BHA, BHT, propyl gallate, citric acid), titanium dioxide, black iron oxides, red iron oxides, yellow iron oxides, chromium oxide greens, ultramarines, and zinc oxide. The manufacturer of this product, Iguana, Inc (Thomasville, GA), received a formulation amendment dated July 11, 2007, from the Environmental Protection Agency (EPA). A formulation amendment is intended for products that are identical or substantially similar to a currently registered product. This product has the same EPA registration number (66306-11) and label as the camouflage face paint with 30% deet in the compact, originally approved on March 27, 2001.


Volunteers in this study were recruited, screened, and enrolled under a human-use protocol reviewed and approved by the WRAIR Institutional Review Board. (18) Volunteers were recruited from the villages of Orange Walk, August Pine Ridge, and San Roman, with the assistance of bilingual field liaisons. Interested parties were briefed on the nature of study participation. Participating volunteers signed informed consent forms prior to any study related procedures in accordance with research guidelines for studies involving humans. (19) Volunteer recruitment, screening, and enrollment occurred February 17 through February 20, 2007.

Study Design and Procedure

All 5 colors of the camouflage face paint with 30% deet were evaluated on human volunteers at 6 time points: 2, 4, 6, 8, 10, and 12 hours postapplication. Peak biting activity occurred between 6 pm and 9 pm; therefore, only 2 mosquito challenges could occur each night, one at 6 pm and one at 8 pm. Three study nights were required in order for each volunteer to be challenged at all 6 time points. Therefore, a staggered application design was employed. Application times were at 8 am, 12 noon, and 4 pm each day for 3 days. At the start of the study, volunteers were randomly assigned to a treatment group: black, green, loam, sand, or white camouflage face paint (n = 5 per treatment). Each volunteer rotated through all of the treatment times during the course of the study to measure all postapplication time points during peak biting activity.

A 600 [cm.sup.2] treatment area from above the ankle to below the knee was determined for each volunteer. Five equally spaced circumference measurements were taken along each lower leg, averaged and divided into 600 to get the length of the exposed area which was defined by an indelible marker on both legs. Each day of the study, the marked area from ankle to knee on one leg was treated with the assigned SCFPwD color and the same marked area on the opposite leg was left untreated to serve as a control. The EPA Product Performance Test Guidelines (19) recommend using between 1.0 g and 1.5 g of lotion or cream product over 600 [cm.sup.2] of skin surface area for testing repellents. Using these guidelines, 1.5 g of the SCFPwD was applied evenly on the treatment leg, which was similarly repeated on subsequent nights of testing. Treatment application alternated between the left and right legs of each volunteer every night of the trial to minimize the number of bites on any one leg that served as the control.

From February 20 through February 22, 2007, volunteers performed landing collections at the study site in San Roman starting at 6 pm and ending at 8:30 pm each night. During the mosquito challenges, volunteers were covered (long sleeve shirts, long pants, jackets of Bug-Out(R) insect barrier material (Bug-Out Outdoorwear, Inc, Cedarville, IA), and footwear) except for the exposed experimental areas (treatment and control) on each leg. All mosquitoes landing in the marked areas of the exposed lower legs were counted and mouth-aspirated for a 20-minute test period or until 20 mosquitoes were counted and collected on the control leg. If 20 mosquitoes were collected from the control leg before the 20 minutes elapsed, the time was recorded and the challenge completed for the volunteer at that challenge point. If mosquito landing rates were low (<20 mosquitoes in 20 minutes), the testing period was extended for an additional 10 minutes. Collected mosquitoes were placed into screen-topped cartons marked with collection data (date, time of collection, collector number, etc). All insects were killed, labeled, and stored for identification. One of the coauthors, Dr Achee, identified the anophelines, and a taxonomist of the WRAIR Biosystematics Unit performed the remaining identifications.


Data were analyzed by calculating percent protection (PP) of a particular subject, at a particular time point, using a particular camouflage paint stick color. Percent protection was calculated for each volunteer at each time point as: PP=100x ((LC-LT)/(LRC)) where LC represents the number landing on the bare skin control and LT the number landing on the leg treated with SCFPwD. The PP values were not normally distributed; therefore, the arcsine transformation was applied to the calculated PP values to stabilize the variance. The transformed data were then analyzed using a mixed effects model, with both fixed and random effects. Fixed effects were stick color and duration of repellent. A variable representing the association of data within subjects was included to account for the variability of repeated measurements on subjects (random effects). The model was run first with the interaction term: stick color x duration. If the interaction term was not significant, the model was run again without the interaction term. The data analysis was generated using SAS System for XP Pro Platform, Version 9.1.3 (SAS Institute Inc, Cary, NC).

Results and Discussion

Twenty-four male and 2 female volunteers participated in this study. Of those enrolled, 46% were hispanic, 31% mestizo, and 23% other. The mean age of the volunteers was 29.7 years (range, 18-57 years). No adverse reactions or events were reported during the study and there were no deviations from the approved human-use protocol.


During the 3 nights of collection (February 20 through February 22, 2007), over 3,600 mosquitoes from 20 species were collected from human volunteers, with Anopheles albimanus Wiedemann constituting the majority (75%) of those collected (Table 1). The number of mosquitoes landing per volunteer was consistent over each night and between challenge points (Table 2). The biting pressure, expressed as landing rate (number landing as a function of time), was also consistent in the control over each night and between challenge points (Table 2). The overall landing rates on control legs, averaged over all 3 nights, were 1.10 [+ or -] 0.04 (mean [+ or -] SE) and 1.29 [+ or -] 0.08 (mean [+ or -]SE) at 6 pm and 8 pm, respectively. Thus, the biting pressure under which the study was conducted exceeds that recommended by the EPA.20

Percent protection (PP) of the SCFPwD was at least 85%, with 4 of the 5 colors demonstrating an overall PP of at least 90% (Figure 2). Overall PP was the highest for green (0.94 [+ or -] 0.03, mean [+ or -] SE) and the lowest for sand (0.86 [+ or -] 0.04, mean [+ or -] SE). Percent protection was near 100% at 2 hours and 4 hours (Table 3), gradually declining over time (Figure 3) to 0.85 [+ or -] 0.05 (mean [+| or -] SE) at 12 hours (Table 3). Percent protection was lowest (0.80 [+ or -] 0.06, mean [+ or -] SE) at the 10 hour time point (Table 3) due to a decrease for 3 of the colors, as shown in Figure 2. A decrease in PP over time is expected as the product's effectiveness declines; however, a more marked decrease in only 3 of the colors (green, sand, black) was unexpected. Furthermore, PP increased for those 3 colors at the 12 hour time point (Figure 2) and overall PP increased to 85% (Table 3). This decrease and subsequent increase in PP could be explained by the fact that all 10-hour challenges took place at 6 PM, the very beginning of the activity period. Landing rates on the controls were slightly lower at 6 pm than at 8 PM on all 3 nights (Table 2). Therefore, as the repellent wears off or loses effectiveness over the course of the day, small differences in landing rates, such as seen at 10 versus 12-hour challenges, will be more obvious or amplified giving the appearance of a possible interaction between the 2 fixed effects, color and duration (time). However, in the mixed effects model with the interaction term, duration was the only significant factor ([F.sub.5,120] = 5.44, P < 0.001) while neither color ([F.sub.4,120] = 1.26, P = 0.29) nor duration x color ([F.sub.20, 120] = 0.53, P = 0.95) were significant (Table 4). When the model was run without the interaction term, duration was again highly significant (F5,140 = 4.54, P< 0.0001) and color was not significant ([F.sub.4,140] = 1.35, P = 0.25) (Table 4).


The new SCFPwD meets the threshold specifications of the ORD, to provide a minimum of 8 hours of protection. It provided maximum protection for 4 hours and substantial protection for 8 hours when tested against the malaria vector Anopheles albimanus under field conditions in Belize. Data collected during this field study, along with a laboratory study with mosquitoes and sand flies and Soldier acceptability surveys, will be compiled and submitted to the Armed Forces Pest Management Board for recommendation for a national stock number. Upon approval, the product, shown in Figure 5, will be available to all military personnel as part of the personal protective measures used to protect against arthropod bites.


We thank Ireneo Briceno and Russell King of the Ministry of Health, Belize, for their field assistance and role in data collection, and Jim Pecor of the Walter Reed Biosystematics Unit, WRAIR, for performing mosquito identifications.

Funding for this study was provided by the US Army Medical Materiel Development Activity.

The SCFPwD used in this study was supplied by Iguana, Inc at no cost to WRAIR.



(1.) National Research Council. Health Effects of Permethrin-Impregnated Army Battle-Dress Uniforms. Washington, DC: National Academy Press; 1994.

(2.) Macedo PA, Peterson RKD, Davis RS. Risk assessments for exposure of deployed military personnel to insecticides and personal protective measures used for disease-vector management. J Toxicol Environ Health Part A. 2007;70:1758-1771.

(3.) Cutaneous leishmaniasis in U.S. military personnel-southwest/central Asia, 2002-2003. MMWR Morb Mortal Wkly Rep. 2003;52:1009-1012.

(4.) Sharp TW, Wallace MR, Hayes CG, et al. Dengue fever in U.S. troops during Operation Restore Hope, Somalia, 1992-1993. Am J Trop Med Hyg. 1995;53:89-94.

(5.) Wallace MR, Sharp TW, Smoak B, et al. Malaria among United States troops in Somalia. Am J Med. 1996;100:49-55.

(6.) Dengue fever among U.S. military personnel-Haiti, September--November, 1994. MMWR Morb Mortal Wkly Rep. 1994:43(46):845-848.

(7.) Sanders JW, Putnam SD, Frankart C, et al. Impact of illness and noncombat injury during Operations Iraqi Freedom and Enduring Freedom (Afghanistan). Am J Trop Med Hyg. 2005;73:713-719.

(8.) Coleman RE, Burkett DA, Putnam JL, et al. Impact of phlebotomine sand flies on U.S. military operations at Tallil Air Base, Iraq: 1. background, military situation, and development of a "leishmaniasis control program". J Med Ent. 2006;43:647-662.

(9.) GlobalSecurity.org website. Where are the legions? global deployments of US forces. Available at: http://www.globalsecurity.org/ military/ops/global-deployments.htm. Accessed August 15, 2009.

(10.) Gupta RK, Rutledge LC. Laboratory evaluation of controlled-release repellent formulation on human volunteers under three climactic regimens. J Am Mosq Control Assoc. 1990;5:52-55.

(11.) Soto J, Medina F, Dember N, Berman J. Efficacy of permethrin-treated uniforms in the prevention of malaria and leishmaniasis in Colombian soldiers. Clin Infect Dis. 1995;21:599-602.

(12.) Personal Protective Measures Against Insects and Other Arthropods of Military Significance. Technical Guide No. 36. Washington, DC: Armed Forces Pest Management Board; April 18, 2002.

(13.) Debboun M, Strickman DA, Klun JA. Repellents and the military: our first line of defense. J Am Mosq Control Assoc. 2005; 21:4-6.

(14.) Debboun M, Coleman RE, Sithiprasasna R, et al. Soldier acceptability of a camouflage face paint combined with deet insect repellent. Mil Med. 2001;166:777-782.

(15.) The World Factbook. Central Intelligence Agency website. Available at: https://www.cia.gov/library/ publications/the-world-factbook/geos/BH.html. Accessed August 15, 2009.

(16.) Hakre S, Masouka P, Vanzie E, Roberts DR. Spatial correlations of mapped malaria rates with environmental factors in Belize, Central America. International Journal of Health Geographics. 2004;3:6-18.

(17.) National Center for Medical Intelligence. Defense Intelligence Assessment, 6 March 2009: Food and Water-borne Diseases, Malaria. Available at: http:// www.intelink.gov/ncmi/product/idra.php? id=65768#malaria. Accessed August 14, 2009.

(18.) WRAIR Protocol #1324: Field Efficacy Study of Stick Camouflage Paint with 30% deet (SCFPwD) using Human Volunteers. Silver Spring, MD: Walter Reed Army Institute of Research; 2006. Internal use document.

(19.) Guidelines for Investigators: Requirements for US Army Medical Research and Materiel Command Headquarters Review and Approval of Research Involving Human Volunteers, Human Anatomical Substances, and/or Human Data. Fort Detrick, MD: Human Research Protection Office, US Army Medical Research and Materiel Command; October 1, 2007. Available at: https://mrmc.amedd.army.mil/ docs/rcq/guidelinesForInvestigators.doc. Accessed July 16, 2009.

(20.) Environmental Protection Agency. Product Performance Test Guidelines: OPPTS 810.3700 Insect Repellents for Human Skin and Outdoor Premises. December 1999. Available at: http:// www.epa.gov/opptsfrs/publications/OPPTS_ Harmonized/810_Product_Performance_Test_Guide lines/Drafts/810-3700.pdf. Accessed July 20, 2009.

MAJ Kendra L. Lawrence, MS, USA

John Paul Benante

Nicole C. Close, PhD

Nicole L. Achee, PhD

MAJ Lawrence is Chief, Vector Control Department, Division of Entomology, Walter Reed Army Institute of Research, Silver Spring, Maryland.

Mr Benante is a Biologist in the Vector Control Department, Division of Entomology, Walter Reed Army Institute of Research, Silver Spring, Maryland.

Dr Close is Chief of Biostatistics, Division of Regulated Activities and Compliance, US Army Medical Research and Materiel Command, Ft. Detrick, Maryland.

Dr Achee is a Medical Entomologist at the Henry M. Jackson Foundation for the Advancement of Military Medicine, Rockville, Maryland.
Table 1. Distribution of species across total
mosquitoes collected from human volunteers
at San Roman, Orange Walk, Belize (20-22
February 2007).

Species                        Collected     %

Anopheles albimanus               2,742     75.02
Mansonia titillans                  243      6.65
Anopheles vestitipennis             137      3.75
Anopheles punctimacula              124      3.39
Aedes scapularis                     87      2.38
Anopheles gabaldoni                          2.22
Culex erraticus                      54      1.48
Anopheles darlingi                   49      1.34
Psorophora confinnis                 33      0.90
Coquillettidia venezuelensis         32      0.88
Anopheles crucians                   23      0.63
Culex sp                             15      0.41
Culex coronator                      11      0.30
Aedes serratus                        5      0.14
Psorophora varipes                    5      0.14
Psorophora sp                         5      0.14
Aedes sp                              4      0.11
Culex corniger/lactator               2      0.05
Culex quinquefasciatus                2      0.05
Aedes aegypti                                0.03
Total                             3,655    100.00

Table 2. Number of mosquitoes landing and landing rate (no.
landing/minute) on treatment and control legs at each collection
point over three nights of testing in San Roman, Orange Walk,
Belize (2007).

                                               Feb 20

6 PM   No. landing (mean [+ or -] SE)
         Treatment                        1.88 [+ or -] 0.76
         Control                         23.04 [+ or -] 1.19
       Landing rate (mean [+ or -] SE)
         Treatment                        0.09 [+ or -] 0.04
         Control                          1.08 [+ or -] 0.06
       Total landing                             623

8 PM   No. landing (mean [+ or -] SE)
         Treatment                        0.80 [+ or -] 0.26
         Control                         20.88 [+ or -] 0.44
       Landing rate (mean [+ or -] SE)
         Treatment                        0.08 [+ or -] 0.03
         Control                          1.62 [+ or -] 0.15
       Total landing                             542

                                               Feb 21

6 PM   No. landing (mean [+ or -] SE)
         Treatment                        2.32 [+ or -] 0.67
         Control                         22.36 [+ or -] 1.39
       Landing rate (mean [+ or -] SE)
         Treatment                        0.17 [+ or -] 0.05
         Control                          1.50 [+ or -] 0.06
       Total landing                             617

8 PM   No. landing (mean [+ or -] SE)
         Treatment                        3.96 [+ or -] 1.67
         Control                         21.72 [+ or -] 0.84
       Landing rate (mean [+ or -] SE)
         Treatment                        0.22 [+ or -] 0.07
         Control                          1.76 [+ or -] 0.13
       Total landing                             642

                                               Feb 22

6 PM   No. landing (mean [+ or -] SE)
         Treatment                        3.32 [+ or -] 1.46
         Control                         25.56 [+ or -] 1.44
       Landing rate (mean [+ or -] SE)
         Treatment                        0.11 [+ or -] 0.05
         Control                          0.97 [+ or -] 0.05
       Total landing                             722

8 PM   No. landing (mean [+ or -] SE)
         Treatment                        2.00 [+ or -] 0.91
         Control                         21.76 [+ or -] 0.85
       Landing rate (mean [+ or -] SE)
         Treatment                        0.14 [+ or -] 0.06
         Control                          1.42 [+ or -] 0.16
       Total landing                             594

                                             All Nights

6 PM   No. landing (mean [+ or -] SE)
         Treatment                        2.51 [+ or -] 0.59
         Control                         23.65 [+ or -] 0.78
       Landing rate (mean [+ or -] SE)
         Treatment                        0.12 [+ or -] 0.03
         Control                          1.18 [+ or -] 0.04
       Total landing                            1962

8 PM   No. landing (mean [+ or -] SE)
         Treatment                        2.25 [+ or -] 0.65
         Control                         21.45 [+ or -] 0.42
       Landing rate (mean [+ or -] SE)
         Treatment                        0.14 [+ or -] 0.03
         Control                          1.60 [+ or -] 0.08
       Total landing                            1,778

Table 3. Number of mosquitoes landing on treatment
and control legs and percent protection
averaged over each postapplication collection

Time *   N ([dagger])   Variable              Mean [+ or -] SE

2             25        Control              23.28 [+ or -] 1.51
                        Treatment             0.44 [+ or -] 0.18
                        Percent Protection    0.98 [+ or -] 0.01
4             25        Control              20.40 [+ or -] 0.28
                        Treatment             0.44 [+ or -] 0.15
                        Percent Protection    0.98 [+ or -] 0.01
6             25        Control              23.08 [+ or -] 1.33
                        Treatment             2.20 [+ or -] 0.77
                        Percent Protection    0.92 [+ or -] 0.02
8             25        Control              21.48 [+ or -] 0.64
                        Treatment             2.12 [+ or -] 0.92
                        Percent Protection    0.91 [+ or -] 0.04
10            25        Control              24.60 [+ or -] 1.25
                        Treatment             4.88 [+ or -] 1.48
                        Percent Protection    0.80 [+ or -] 0.06
12            25        Control              22.48 [+ or -] 1.03
                        Treatment             4.20 [+ or -] 1.66
                        Percent Protection    0.85 [+ or -] 0.05

* Number of hours after application of face paint

([dagger]) Number of volunteers who participated

Table 4. The results of the mixed-effects
model ANOVA run with and without the
interaction term.

Effect                   df     F-value      P

Model with
    Duration            5,120    5.44     0.0002
    Color               4,120    1.26     0.2905
    Duration x Color   20,120    0.53     0.9491
Model without
    Duration            5,140    5.84    <0.0001
    Color               4,140    1.35     0.2551
Gale Copyright: Copyright 2009 Gale, Cengage Learning. All rights reserved.