Introduction

Globally, there is an increasing trend in the occurrence of vector-borne diseases, particularly those transmitted by Aedes mosquitoes (WHO 2014). Originally restricted to tropical and subtropical areas, these mosquitoes have now spread to most continents, and the threat is increasing as there is potential for further expansion. These day-biting mosquitoes spread a wide range of diseases including dengue, Zika, chikungunya and yellow fever (Kittayapong et al. 2017). Rapid urbanisation, international travel, migration, improper waste management practices and climate change etc. are attributed to the emergence of these diseases. An estimated 390 million dengue cases are reported yearly, and about 4 billion people around the world are at risk for dengue infections (Shim 2017). Chikungunya resurgence has been reported in 2003 onwards, and Pacific islands and India have witnessed disease outbreaks in the recent past (Cecilia 2014). Zika is spreading rapidly to new geographical locations, and the virus activity is reported from across the continents (Tilak et al. 2016).

Overall, there is substantial loss of man days and financial loss due to morbidity and mortality associated with vector-borne infections across the world (WHO 2014). Vector control and bite protection are often the most effective methods for preventing the incidence and spread of vector-borne diseases. The use of personal protective measures, which prevent or reduce man–vector contact, assumes great significance in this context. These include the use of topical repellents, long-lasting insecticidal nets (LLIN) and insecticidal fabrics (IF). LLIN prevent contact between mosquitoes and users and are toxic to mosquitoes on contact (Wanzira et al. 2016). These are widely being used in malaria endemic regions as per the World Health Organization (WHO) recommendations (WHO 2013b). LLIN are designed to last for 20 washes or more, thereby avoiding the need for reapplication of the insecticide onto the nets (Bhatt et al. 2012; Pennetier et al. 2013). However, LLIN cannot provide protection from day-biting mosquitoes transmitting dengue, chikungunya and Zika since bed nets are mostly used during the night hours. The rapid geographical expansion and the consequent threat posed by Aedes mosquitoes necessitate the development and use of products and technologies that can be used effectively during the daytime (Kittayapong et al. 2017).

The use of IF is an important but often neglected method of personal protection. Unlike LLIN, IF can provide protection during day and night and in both indoor and outdoor environments. With the recent outbreaks and geographical spread of dengue and chikungunya and with the emerging threat of Zika virus infections, there is a renewed interest in insecticide-treated fabrics in the civilian market as well. Recently, trials of permethrin-impregnated school uniforms were carried out in Thailand to assess the impact on Aedes mosquitoes and dengue infections in school children. Since children are vulnerable to dengue infections and they are in school uniforms during the peak biting hours of Aedes, the use of impregnated uniforms could be pursued as a dengue control strategy if found useful (Kittayapong et al. 2017). Athletes from South Korea were reportedly using ‘Zika-proof’ uniforms during the Rio Olympics 2016 (Sheikh 2016). Fabrics factory-treated with permethrin for civilian use and permethrin aerosols for treating fabrics to make them insecticidal are now commercially available under different brand names (Faulde et al. 2016). IF are useful for protection from night-biting mosquitoes and also in situations were sleeping under a bed net is not possible as in the case of troops deployed in field operations or civilians engaged in outdoor recreational activities. Studies have proven the efficacy of IF against different disease vectors and for reducing vector-borne infections such as malaria and leishmaniasis (Faulde et al. 2009; Rowland et al. 1999; Soto et al.1995). IF are effective against many of public health pests such as mosquitoes, ticks, mites, tsetse flies, sand flies and body lice (Banks et al. 2014).

Other synthetic pyrethroid insecticides like deltamethrin, bifenthrin and cyfluthrin were used in the past for treatment of fabrics. However, permethrin still remains to be the most preferred and widely used active ingredient in IF. It is used in the form of skin creams for the treatment of human head lice and scabies. However, skin application makes it ineffective as an insecticide/repellent as it does not bond to human skin (Kitchen et al. 2009). Characteristics such as excito-repellency, ‘hot-feet’, knock-down (KD) effect, lethality and residual efficacy of permethrin have made it a preferred active ingredient in IF (Faulde et al. 2016). Permethrin with a cis:trans ratio of 25:75 is commonly used for fabric impregnation (Faulde et al. 2003).

Currently, there are no WHO guidelines for testing the bioefficacy of insecticidal fabrics (Most et al. 2017; Faulde et al. 2016; Banks et al. 2014). Also, no international guidelines exist for washing and determination of residues from IF. The German Armed Forces is using TL 8305-0331 licencing procedure for permethrin-treated uniforms (Faulde et al. 2016; Most et al. 2017), whereas the Dutch standard for permethrin-treated clothing, NEN 8333:2018 nl, was released recently (NEN 2018). The goal of the present study was to describe and compare the test methods currently used and to highlight the need for the development of standard guidelines specific to IF. IF have been made using many insecticides and are effective against a large number of biting arthropods. However, the present review focuses on permethrin-treated fabrics and their bioefficacy against mosquitoes, which are the major disease vectors.

Insecticidal fabrics for military personnel

Although IF are now available for civilian use, these were originally developed for use by the military personnel. Troops stationed in the tropical regions are at the risk of contracting vector-borne infections, and often more service days are lost due to vector-borne infections than to combat (Macedo et al. 2007; Pages et al. 2010). The exposure of troops to disease vectors is more as they train and operate mostly outdoors. Unlike native populations, the lack of acquired immunity makes the forces more vulnerable to diseases during operations in endemic areas (Macedo et al. 2007). Disease outbreaks in harsh environments might have a debilitating effect on troops thereby adversely affecting field operations in sensitive areas (Dhiman et al. 2011). Civilians travelling to endemic areas for work or leisure may also be exposed to the risk of contracting infections. The travellers may carry these infections back to their native places leading to further transmission among the local population. IF are more useful in the hot and humid regions wherein the regular use of repellents on skin and sleeping under a bed net may not be convenient at all times.

Repellent-treated uniforms have been used by the forces since the Second World War though the earlier used repellents such as dimethyl phthalate and DEET (N, N-diethyl-m-toluamide) were later replaced by permethrin—a synthetic pyrethroid insecticide (Pennetier et al. 2010). Currently, the armed forces of many countries across the world are using permethrin-treated military uniforms to protect their personnel from vector-borne infections (Frances et al. 2014; Kitchen et al. 2009; Most et al. 2017; Pennetier et al. 2010). Permethrin treatment of clothing is recommended by WHO as a method of protection from disease vectors (Rozendaal 1997). The United States Environmental Protection Agency (USEPA) has approved permethrin for clothing sprays and for commercial factory treatment of clothing, and many consumer-oriented, permethrin factory-treated clothing products were registered with the EPA since 2003 (USEPA 2017). The US Centers for Disease Control and Prevention (CDC) instructs travellers to use permethrin-treated clothing (Mutebi et al. 2017). Clothing application of repellents is preferred to topically applied repellents so as to reduce the occurrence of allergic reactions (Rozendaal 1997).

Initially, insecticidal treatment of military uniforms was done by spraying or dipping and consequently, the residual efficacy was low (Most et al. 2017). Evaluation of permethrin as a clothing repellent was initiated by the US Department of Defense (DoD) in association with the Department of Agriculture (USDA) (Kitchen et al. 2009). Studies were carried out on the efficacy of permethrin sprays on clothing against the lone star tick (Schreck et al. 1982). Permethrin is being used by the US forces for treating military uniforms since 1991 (McCain and Leach 2007). As ticks are the most common disease vectors in the USA, IF are primarily being used to combat tick bites and tick-borne infections such as Lyme disease and Rocky Mountain spotted fever (Vaughn and Meshnick 2011). Clothing factory-treated with permethrin was approved for defence use in 1990 (Kitchen et al. 2009). The US Marine Corps is currently using uniforms factory-treated with permethrin. The US defence personnel are trained on permethrin treatment methods and are instructed to apply a DEET formulation on exposed skin. These procedures are followed when the forces are deployed in high-risk areas (Kitchen et al. 2009). Permethrin-treated uniforms have been used by the Australian Defence Forces (ADF) since 1990s (Frances et al. 2003). Currently, permethrin-treated long-lasting military uniforms have been developed for many armed forces (Most et al. 2017). The standardised testing and licencing algorithm TL 8305-0331 was adopted by the German Armed Forces (Bundeswehr) so as to ensure compliance of the permethrin-treated battle-dress uniforms (PTBDU) with the minimum quality requirements regarding safety and bioefficacy (Most et al. 2017). Permethrin-treated long-lasting battlefield uniforms (BFU) were developed for the French Army (Pennetier et al. 2010).

Methods of permethrin treatment

Currently, the commonly used methods for treatment of fabrics with permethrin include spraying, dipping, polymer coating and microencapsulation (Faulde et al. 2016). The various methods of treatment and the bioefficacy of the treated fabrics against different mosquito species are summarised in Table 1. In the spraying and dipping methods, the fabrics are individually treated with permethrin. The US forces use pressurised aerosols containing 0.5% permethrin to treat the uniforms. The uniforms are then dried under shade before wearing (AFPMB 2009). The uniforms are also treated using a 2 gal sprayer wherein the spray solution is made using a 40% EC formulation of permethrin. In this method, a dose of 0.52% by weight of permethrin is ensured. The dipping method used by the US military is known as individual dynamic absorption (IDA) wherein the personnel are provided with a kit containing a permethrin 40% EC, gloves and treatment bags. A permethrin emulsion is prepared, and the fabric to be treated is rolled, tied and kept immersed in the permethrin solution for 3 h after sealing the treatment bag. Once dry, the treated fabric is ready to be worn (AFPMB 2009).

Table 1 Bioefficacy of permethrin-treated fabrics against different mosquito species
Full size table

The polymer-coating method involves the incorporation of permethrin into a polymer matrix and polymerisation onto the fabrics (Faulde et al. 2003; Faulde et al. 2016). This method is commonly used for the factory treatment of fabrics with permethrin. The factory treatment ensures long-lasting insecticidal efficacy, and the user is not required to apply or reapply the insecticide unlike in the case of spray and dip methods. Thus, the hazards due to unintentional exposure of the personnel during the treatment process are avoided. Specific polymerisation of permethrin onto the fibre surface helps in achieving long-lasting bioefficacy lasting up to 100 washings or more (Faulde et al. 2016; Most et al. 2017). The polymerisation process is identified to be critical to the bioefficacy and resistance to washing (Faulde et al. 2003). Trials were conducted in Thailand to test the efficacy of factory-dipped clothing and school uniforms against dengue vectors. These were prepared by factory treatment with permethrin 0.52% w/w and were superior in bioefficacy as compared to microencapsulated clothing (Banks et al. 2015). Permethrin-treated (1250 mg/m2) BFU of the French Army were found to be effective in protecting their personnel (Pennetier et al. 2010). Fabrics polymer-coated with permethrin at 542 mg/m2 which was tested wherein the effect of skin coverage (full or partial) by IF on protection from mosquitoes was studied (Osborne et al. 2016). Factory-treated school uniforms (0.054 mg permethrin/m2) were evaluated in Thailand for their impact on mosquitoes and dengue incidence (Kittayapong et al. 2017). The microencapsulation method of permethrin treatment aims to achieve a continuous and enduring discharge by the penetration of permethrin into the fibres (Banks et al. 2015). However, in the trials conducted in Thailand, the fabrics treated by this method provided lower mosquito KD and mortality compared to dipping or factory treatment. The lower efficacy reported here might be due to the low availability of permethrin on the fabric surface (Banks et al. 2015).

Guidelines on insecticidal fabrics

The currently available guidelines on IF include TL 8305-0331 used by the German Armed Forces (Faulde et al. 2016; Most et al. 2017) and the Dutch NEN 8333: 2018 nl (NEN 2018). WHO guidelines are available for the testing of LLIN (WHO 2013b) but not for IF. The method of use, method of treatment, type of fibres and the frequency of washing of IF differ from that of the insecticidal nets. Thus, there is a need to develop internationally accepted guidelines specific to IF. The development of guidelines for the bioefficacy testing, chromatographic determination and washing of IF would help in quality control thereby ensuring adequate bioefficacy against the target pests and safety to the user.

The LLIN test guidelines by WHO specify the bioefficacy criteria for nets washed 20 times as mosquito mortality not less than 80% or mosquito KD not less than 95% in cone bioassay. Alternatively, tunnel tests can be employed wherein the bioefficacy criteria are mortality not less than 80% or blood-feeding inhibition not less than 90% (WHO 2013b). As per TL 8305-0331 for PTBDU implemented in 2002, cone tests carried out wherein 10 mosquitoes are constantly exposed to the test fabric, and the time required to achieve 99% KD (KD99) is recorded (Faulde et al. 2016; Most et al. 2017). The tests are conducted in 10 replicates, and the treated fabrics should provide a mean KD99 time of 71.5 min or less after 100 washings (Faulde et al. 2016; Most et al. 2017). These tests are carried out using Aedes aegypti mosquitoes since it is the most appropriate disease vector for testing the bioefficacy of IF. Additionally, a minimum permethrin content of 200 mg/m2 is required to be maintained after 100 washings (Faulde et al. 2016; Most et al. 2017). The Dutch standard NEN 8333: 2018 nl developed by The Netherlands Standardisation Institute is applicable to clothing factory-treated with permethrin for protection from ticks (NEN 2018).

Bioefficacy test methods

In the absence of standard guidelines, different methods including WHO tube bioassay, WHO cone bioassay and arm in cage bioassays have been used for the testing of IF. The test methods used along with a brief description and the mosquito species used are given in Table 2.

Table 2 Methods for testing the efficacy of insecticidal fabrics against mosquitoes
Full size table

Tube bioassays

The plastic tubes (cylinders) from the WHO insecticide susceptibility kit are used for conducting tube bioassays (WHO 2016). The tubes lined with the test fabrics were used to study the effect of washing and exposure time on the efficacy of impregnated military uniforms to Culex pipiens mosquitoes (Fryauff et al. 1996). The percent KD 15-min post-exposure was 98 (unwashed) and 9 (3 washings), whereas the percent mortality 24-h post-exposure was 74 (unwashed) and 17 (3 washings) (Fryauff et al. 1996). In another study employing the tube bioassay method, Ae. aegypti mosquitoes were continuously exposed to the test fabrics within the tube. After 100 launderings, the 100% KD time of mosquitoes was only 38.3 min indicating that the factory-treated fabrics were still effective against mosquitoes. The results of the tube bioassay were supported by the chromatographic analysis wherein 280 mg/m2 permethrin was detected after 100 washings (Faulde et al. 2003).

Tube bioassays with Anopheles farauti and Ae. aegypti mosquitoes were performed to evaluate the clothes impregnated with bifenthrin and permethrin. For 3-min exposure, the mortality was 94.2–100% initially but reduced to < 28% after 2 washes, whereas the KD reduced from 100% to < 25% after 3 washes (Frances et al. 2003). We have reported tube bioassay with Aedes albopictus mosquitoes to study the persistence of permethrin in the treated fabrics after repeated washings (Gopalakrishnan et al. 2014a). We observed that for unwashed fabrics, the percent KD 15-min post-exposure was 77.6 (5-min exposure) and 100 (10-min exposure), whereas there was no KD after 4 washings. The percent mortality dropped from 100 initially (5 and 10 min) to 88 (5 min) and 95.4 (10 min). The median knock-down times (KT50) were 5.9 min (5 min) and 4.61 min (10 min) on exposure to unwashed fabric whereas 71.8 min (5 min) and 59.7 min (10 min) after the fifth washing (Gopalakrishnan et al. 2014a). In another study, we have used tube bioassays to evaluate the durability of permethrin-impregnated fabrics against Ae. albopictus in the hot and humid environment of northeastern India (Gopalakrishnan et al. 2014b). We found that the percent mosquito KD 15 min after exposure was 100 at the beginning but was reduced to 26 after 2 years of weathering, whereas the percent mortality was 100 initially and 97.8 after 2 years. The treated fabric in storage provided 73% KD and 100% mortality after 2 years. The KT50 was 4.22 min at the beginning whereas 28.5 min after 2 years of weathering, whereas the fabric under storage for 2 years provided KT50 of 6.47 min (Gopalakrishnan et al. 2014b). Tube bioassay was carried out against An. farauti and Ae. aegypti mosquitoes for comparative evaluation of factory-treated ADF uniforms (Frances et al. 2014). Significantly higher KD of Ae. aegypti was obtained on 3-min exposure to the factory A and dipping treatments. For An. farauti, KD was significantly higher with factory A-treated fabric. The lower bioefficacy of factory B-treated fabric may be due to a stronger binding of permethrin in the fabric, which makes the insecticide unavailable to biting and foraging mosquitoes (Frances et al. 2014).

Cone bioassay

Cone bioassay uses WHO standard plastic cones for evaluating the residual activity of insecticides on different substrates including insecticidal nets (WHO 2006). We reported the evaluation of wash resistance of permethrin-treated fabrics using cone bioassays. The mosquito KD was 99 ± 1% initially, which was reduced to 22.9% after the fifth washing, whereas the mortality dropped from 100 to 70.5% (Gopalakrishnan et al. 2014a). In another study using fabrics dipped in permethrin, we observed that the KD of Ae. aegypti mosquitoes was reduced from 98.3% initially to zero after 55 cycles of washing, whereas the mortality was 100% even after 55 cycles of washing (Sukumaran et al. 2014). We tested the durability of permethrin-impregnated fabrics against Ae. albopictus in the hot and humid environment of northeastern India using cone bioassay. The KD and mortality were both 100% at the beginning but were reduced to 37.3% and 67.6% after 2 years. However, the treated fabric sample not exposed to weathering retained bioefficacy even after 2 years of storage (Gopalakrishnan et al. 2014b). WHO cone bioassays with 3-min exposure were employed for studies on the bioefficacy of permethrin-treated fabrics in Thailand. The KD percentage for factory-dipped clothing, microencapsulated clothing and factory-dipped school uniforms was 96.5, 50.8 and 98.2, whereas the percent mortality was 97.1, 73.3 and 98.6, respectively (Banks et al. 2015).

Cone bioassay was employed to test the efficacy of different commercially available permethrin-treated clothes against Ae. aegypti, Anopheles stephensi and Culex pipiens wherein the mosquitoes were continuously exposed to the test fabrics. For BDU, the mean KD99 times were found to be 38.3, 44 and 98 min against the three species (Faulde et al. 2016). The bioefficacy of permethrin-treated battle-dress uniforms (PTBDU) after field use was tested using cone bioassays. The results showed that the mean KD99 time against Ae. aegypti was 47.7 min for PTBDU blouses and 60.2 min for trousers. This was supported by the chromatographic determination wherein the mean remaining permethrin was slightly higher (743.9 mg/m2) in blouses than that in trousers (720.2 mg/m2). The KD times of the blouses washed 218 times and the trousers washed > 54 times exceeded the KD99 cut-off value of 71.5 min as per TL 8305-0331 licencing protocol (Most et al. 2017). Cone bioassay was used to test the bioactivity of permethrin-treated school uniforms in Thailand against Ae. aegypti mosquitoes. It was found that KD and mortality were close to 100% initially but declined quickly after 4 washes indicating the loss of bioefficacy (Kittayapong et al. 2017).

Biting and contact bioassays

Mosquito feeding through IF or contact with IF and the resultant KD or mortality is recorded in biting and contact bioassays. However, there was no uniformity in the test equipments and the methods used by various authors in conducting these assays. In one such study, the effect of weathering on permethrin-treated fabrics was investigated using biting bioassay wherein the test fabrics subjected to weathering were more effective against An. stephensi than Ae. aegypti (Gupta et al. 1989). Permethrin-treated military uniforms were evaluated in northeastern Thailand using contact and biting bioassays. In the contact bioassay, the KD of Anopheles dirus mosquitoes was 99.4% after 1 day and zero after 180 days of treatment, whereas in the biting bioassay, not more than 10% of An. dirus mosquitoes were observed to feed through the treated fabrics (Eamsila et al. 1994). The bioefficacy of permethrin-impregnated clothes against Ae. aegypti was tested using biting bioassay wherein less than 6.1% of mosquitoes were found to feed through the treated clothes (Frances et al. 2003). In another study, the treated military uniforms were evaluated against An. farauti and Ae. aegypti mosquitoes. The blood-feeding percentage of Ae. aegypti was up to 31.8%, whereas no feeding was observed in An. farauti up to 50 launderings. This showed that Ae. aegypti is more efficient in obtaining a blood meal through impregnated clothes than An. farauti (Frances et al. 2014).

Arm-in-cage bioassay

Arm-in-cage bioassays are described in the WHO guidelines on testing of topical repellents (WHO 2009). This method was used by some authors to study the biting behaviour of mosquitoes on arms covered with IF. However, studies on repellent-treated bed nets using arm-in-cage bioassay have shown that permethrin in an IF is not acting as a spatial repellent but as a non-volatile contact insecticide with excito-repellency (Faulde et al. 2010). The biting behaviour of mosquitoes in an arm-in-cage bioassay is different towards a topical repellent and an IF (Faulde et al. 2010); and hence, these tests might not provide valid conclusions on the bioefficacy of IF.

The efficacy of permethrin-treated fabrics was tested against Ae. albopictus mosquitoes using arm-in-cage bioassay in which the treated fabric provided complete protection from bites even after 5 washings. The KD was 60% in unwashed fabric and 26% after 5 washings (Schreck and McGovern 1989). Arm-in-cage tests were carried out by Faulde et al. (2010) to compare the landing and biting behaviour of Ae. aegypti mosquitoes on bed net fabrics polymer coated with topical repellents (DEET and IR3535) and on a LLIN containing 500 mg/m2 permethrin. Repellent-coated bed net fabrics provided complete protection from mosquito bites, whereas the LLIN failed to provide any bite protection. This showed that the host-seeking instinct of the test mosquitoes could offset the hot-feet and excito-repellency effects of permethrin in a treated fabric (Faulde et al. 2010). We used arm-in-cage bioassay to evaluate the bioefficacy of fabrics dipped in permethrin against Ae. aegypti (Sukumaran et al. 2014). The percentage of mosquitoes landing in the unwashed-treated and control fabrics was 32 and 86 whereas 51.2 and 75.2 after 55 washings. This indicated that the landings were significantly less on the treated fabrics. We observed that almost all mosquitoes landing on the treated fabrics died until the 25th washing, and there was a trend in reduction in the percent mortality after successive cycles of washing. Hence, mortality/landing ratio was estimated as an indicator of bioefficacy, and the ratio decreased from 0.96 in the unwashed fabric to 0.47 in the fabric subject to 55 washings (Sukumaran et al. 2014).

Arm-in-cage bioassay with 90-s exposure was used for testing the bioefficacy of permethrin-treated clothes in Thailand (Banks et al. 2015). The percent bite protection against Ae. aegypti was 79.9, 65.5 and 91.5 for factory-dipped clothing (FDC), microencapsulated clothing (MC) and HDC (home-dipped clothing), whereas the percent landing protection was 40.9 and 49.9 for FDC and HDC. FDC and HDC provided the highest protection from mosquito biting and landing (Banks et al. 2015). Arm-in-cage bioassays with factory-treated clothing were carried out to study the difference in protective efficacy of fully covered (FCT) and partly covered-treated clothes (PCT). FCT provided better protection from Ae. aegypti landing compared to PCT, whereas PCT reduced biting significantly compared to the control (Osborne et al. 2016).

Free flight rooms

Free flight rooms or testing areas are used for testing the efficacy of spatial repellents as per WHO guidelines (WHO 2013a). Since permethrin in an IF is not acting as a spatial repellent (Faulde et al. 2010), tests conducted in free flight rooms might not be helpful in establishing the bioefficacy of IF. Osborne et al. (2016) studied the difference in protective efficacy of fully covered (FCT) and partly covered factory-treated clothes (PCT) in free flight rooms. Coverage was not found to be a significant factor affecting mosquito-landing inhibition though the reduction in blood feeding by FCT (91.4%) was more in comparison to PCT (49.3%) (Osborne et al. 2016).

Experimental huts

The use of experimental huts is recommended by WHO for small-scale field trials (Phase II) of LLIN (WHO 2013b). The efficacy of permethrin-treated French BFU was tested using experimental huts in which the volunteers wearing the uniforms were stationed singly. Ae. aegypti mosquitoes were released into the huts at dusk and collected back 2 h later. The blood-feeding inhibition (BFI) by the unwashed-treated fabric was 56.25%, which was reduced to 37.5 after 20 washings (Pennetier et al. 2010).

Human-landing catches

Human-landing catches is the most direct method of sampling mosquitoes biting humans wherein the mosquitoes landing and attempting to feed on the collectors are captured (Kenea et al. 2017). A combination of permethrin-treated uniforms along with two topical repellent formulations was evaluated in Thailand using human-landing catches. The protection provided by the treated clothing alone was about 37% for Culex sitiens while about 43% for Aedes vigilax (Harbach et al. 1990). In another field study involving human-landing catches, the impregnated uniforms were observed to provide 89% protection from the bites of Cx. pipiens mosquitoes (Khoobdel et al. 2005).

Permethrin quantification

The WHO recommended dose of permethrin is 1250 mg/m2 (Rozendaal 1997). Permethrin is currently registered with USEPA for fabric impregnation at a rate not to exceed 1250 mg/m2, whereas GFIFRA recommends polymer coating of the uniforms with permethrin (cis: trans ratio 25:75) at the rate of 1300 ± 300 mg/m2 (Faulde et al. 2016). The toxicological studies performed indicated that, under normal use, permethrin at the recommended dose would not have any adverse effect on human health while providing adequate bioefficacy against biting arthropods (Faulde et al. 2016; Appel et al. 2008). However, recent studies have shown that some of the fabric samples contained initial (unwashed) doses up to 4300 mg/m2 far exceeding the recommended maximum (Faulde et al. 2016). In order to ensure the safety of users, the permethrin dose in the treated fabric should be within the recommended limit as too high concentrations may lead to adverse health effects. On the other hand, too low concentrations may not provide protection from arthropod bites. Moreover, sublethal concentrations may cause undesirable behavioural effects or the development of insecticide resistance on the target pests. The minimum effective dose of permethrin on fabrics may differ markedly among different species of arthropods (Most et al. 2017). The uniforms should retain at least 200 mg/m2 permethrin after 100 washings as per TL 8305-0331 licencing procedure of the German Armed Forces (Faulde et al. 2016). Hence, quality control during the fabric production process is required to monitor permethrin concentrations and release rates in factory-treated IF (Faulde et al. 2016; Appel et al. 2008). The content of permethrin gets reduced over a period of time due to usage and washing. Quantification is required to ascertain that the permethrin content is in the specified range before and after washing. Thus, along with the bioefficacy testing, permethrin quantification forms another important aspect of quality control of IF. Permethrin from the IF is extracted by an organic solvent, and chromatographic methods are used for its quantification.

In one of the methods, permethrin was extracted from the treated fabrics using Soxhlet apparatus wherein the fabric specimens were uniformly cut into 2.5 cm2 subsamples and the extraction was done with acetone. The estimation was done using a GC having a glass column and flame ionisation detector (Gupta et al. 1989). As per another method described, the fabric samples were extracted with toluene in an ultrasonic bath, and gas chromatography–mass spectrometry (GCMS) with selected ion-monitoring mode was used for the analysis (Faulde et al. 2003). The same method was used in another study for the quantification of permethrin from PTBDU and other commercially available factory-impregnated fabrics from the civilian market (Most et al. 2017; Faulde et al. 2016). Permethrin quantification from IF using HPLC was described wherein the extraction was carried out with acetone in an ultrasonic bath. In this method, photodiode array detector was used, and the mobile phase was acetonitrile (Frances et al. 2003). In another study, HPTLC method was employed to quantify permethrin from military uniforms. The samples were extracted with acetone, spotted on a silica gel and then scanned by TLC scanner in absorption/reflection measurement mode at 207 nm (Khoobdel et al. 2005). We have reported a method of permethrin quantification using HPLC in which a C-18 analytical column and acetonitrile–water mobile phase were used, and the fabric samples were extracted in acetonitrile (Gopalakrishnan et al. 2014a). A GCMS method of quantification was described by Frances et al. (2014) wherein permethrin was extracted in dichloromethane and bifenthrin was used as an internal standard. Two recent studies have used HPLC method for permethrin quantification from IF in which a C-18 column was used, and the mobile phase was water-acetonitrile (Banks et al. 2015; Osborne et al. 2016).

Washing methods

Obviously, there will be clear differences in the wash resistance of fabrics washed using different methods (hand washing or machine washing) and washing agents (soaps and detergents). Since an internationally accepted standard procedure for washing of IF is not available, different washing methods were employed in the trials for evaluating the wash resistance. The absence of a common protocol makes the comparison of the trial outcomes in terms of residual permethrin content and bioefficacy difficult and irrelevant. As per the WHO criteria of evaluation, the bioefficacy of LLIN is required to last for 20 washes or more (WHO 2013b). However, long-lasting insecticidal fabrics (LLIF) should ideally be able to withstand 100 or more washings as the frequency of washings of IF by the user is more compared to that of LLIN. Apart from the frequency of washings, the type and concentration of the soap/detergent used and the method of washing will affect the wash resistance of the fabric.

In the standard laboratory-washing procedure recommended by WHO for LLIN, the samples are washed in beakers containing a soap solution (2 g/l) in a water bath. One washing followed by two rinsing is done for 10 min each at 30 °C and 155 rpm. Similarly, during field trials, the washing and rinsing are recommended to be carried out in non-plastic bowls with agitation at 20 rpm (WHO 2013b). The washing method developed by the Collaborative International Pesticides Analytical Council (CIPAC) involves the preparation of a washing agent containing polyoxyethylene glycol monostearate, water and sodium oleate. The washing agent is added to a glass bottle into which the samples are introduced. For washing and rinsing, the bottle is kept in a water bath at 30 °C for 10 min each. The wash resistance index (w) can be estimated as w = 100 × n√(tn/t0) where, n is the wash number, tn and t0 stands for the content of the active ingredient (g/kg) before and after n washes (WHO 2013b).

A hand-washing method using 4 g/l of a household detergent was used for the evaluation of permethrin-treated Thai military uniforms. Bioassays against An. dirus mosquitoes indicated complete loss of bioactivity after 3 washes, although up to 60% of the initial permethrin content was detected from the test samples (Eamsila et al. 1994). Another study employed machine washing of permethrin-treated fabrics at 60 °C as per EN 26330:1993/ISO 6330:1984 (Faulde et al. 2003). Similarly, permethrin-treated French BFU were washed as per EN ISO 6330 in a washing machine (Pennetier et al. 2010). Permethrin-impregnated Australian DPCU were washed as per AS2001.5.4-2005 standard, which involved wash and rinse cycles of 10 min duration at 30 °C followed by drying at 50 °C (Frances et al. 2014). WHO standard washing and machine washing were compared for their effects on the bioefficacy of IF (Banks et al. 2015). The machine washing procedure used here involved 30 min cycles at 30 °C and 800 rpm. For fabric pieces washed using the WHO procedure, the mosquito KD and mortality dropped from 96.9% and 86.9% (unwashed) to 7.9% and 11.4% (30 washes). In the case of machine washing, the KD and mortality reduced from 96.0% and 89.3% initially to 57.9% and 40.9% after 30 washes. The wash number corresponding to 50% reduction in mortality (LW50) was 14.4 with the WHO method whereas 25.6 with the machine washing suggesting that the former method was comparatively more rigorous. However, the difference spotted here could be due to the different washing agents used in the two methods (Banks et al. 2015). EN ISO 6330:2000 standard machine washing was also used to compare the commercially available factory-treated fabrics (Faulde et al. 2016). Another study described the use of machine washing for evaluating the efficacy of clothing treated with permethrin using polymer-coating method (Osborne et al. 2016). Hand washing followed by shade drying was carried out for evaluating Thai school uniforms factory-treated with permethrin. The bioefficacy rapidly declined after 4 washes contrary to the manufacturer’s claim that the treated fabrics can withstand up to 70 washes. The mosquito KD and mortality were well below 20% after 20 washes. The unexpected loss of bioactivity reported here might be due to the rigorous washing method adopted simulating the actual field conditions in the tropical regions (Kittayapong et al. 2017).

Prospects and challenges ahead

With the rapid expansion in the geographical range of arthropod vectors especially Aedes mosquitoes, there is an ever increasing threat of vector-borne diseases worldwide. More than half of the world is currently under the threat of Aedes-transmitted infections including dengue, Zika and chikungunya (Kittayapong et al. 2017). The incidence and local transmission of these diseases are being increasingly reported from new areas aided by the introduction of vector mosquitoes through air travel and maritime freight. Illegal cross border migration, inadequate housing and poor vector control activities are also augmenting the spread of Aedes-transmitted infections across continents (Gardner et al. 2017; Tilak et al. 2016). The primary vector Ae. aegypti is mainly distributed in the tropics, whereas Ae. albopictus has a much wider global distribution and has spread to areas from which it has not been previously reported (Gratz 2004). Ae. albopictus is known to be a secondary vector of dengue and chikungunya and is suspected to be involved in Zika virus transmission (Gardner et al. 2017). The spread of these diseases is widely regarded as a serious public health threat and requires urgent interventions in the form of vector control and personal protective measures. IF are the intervention of choice for protection from the day-biting mosquitoes and the pathogens transmitted by them (Banks et al. 2014).

Mosquitoes are unable to land on the permethrin-treated fabric for considerable time due to ‘hot-feet’ effect, and most of the mosquitoes which land are killed due to contact toxicity (Osborne et al. 2016; Sukumaran et al. 2014). Quick KD and lethality on contact with impregnated clothing could have a major effect on disease transmission. For example, dengue vectors are capable of disease transmission only after 4.7–6.5 days, which corresponds to the incubation period of the virus (Osborne et al. 2016). The KD and mortality of the vectors before they become capable of disease transmission could greatly reduce the potential transmission in an area. The uptake levels of permethrin from the impregnated fabrics to skin are well below the limits specified by WHO and USEPA (Appel et al. 2008; Rossbach et al. 2010). Consequently, this residual bioefficacy might protect the user for some more time even after removal of the impregnated clothing (Osborne et al. 2016).

The technology to ensure long-lasting efficacy through factory treatment of fabrics with permethrin has been evolved and is being used by the armed forces of the USA, Germany and France (Kitchen et al. 2009; Pennetier et al. 2010; Faulde et al. 2016). Factory-treated LLIF are now available for military and civilian use for protection from arthropod bites during work or recreation. IF are different from insecticidal nets in many aspects including the type of material used and the technology for impregnation (Table 3). In comparison to the nets, IF need to withstand higher number of washes in order to make the technology cost-effective and popular. LLIF are prepared by factory treatment of fabrics with permethrin using a polymer-coating method to ensure long-lasting efficacy. In the polymer-coating method, the fibre surfaces are coated with a polymer layer containing permethrin (Faulde and Uedelhoven 2006), and there is reduced environmental impact of washing-treated clothing due to less run-off of permethrin in the wash liquid (Banks et al. 2014). Another technique of factory impregnation is microencapsulation wherein permethrin is enclosed in a polymer shell. A thin layer of polymer is used here, whereby a constant release rate, low dermal absorption and high stability are ensured. This technique does not change the comfort properties of the clothing, which have an impact on user compliance in hot and humid environments (Banks et al. 2014). Further improvements in the long-lasting technology including microencapsulation techniques could make the LLIF technology simple, scalable and cost-effective thereby making it an attractive and effective method for combating vector-borne infections.

Table 3 Comparison between long-lasting insecticidal nets (LLIN) and long-lasting insecticidal fabrics (LLIF) based on some important features
Full size table

In order to ensure safety to the user and efficacy against target pests, standardised testing and licencing procedures for IF should be formulated. This would also prevent the manufacturers from making misleading claims of product efficacy. As per TL 8305-0331 licencing protocol adopted for the evaluation of German PTBDU, cone bioassay is the test method wherein Ae. aegypti mosquitoes are continuously exposed to the fabric pieces (Faulde et al. 2016). This method is markedly different from the WHO method for testing LLIN wherein the mosquito mortality or KD percentage in cone bioassays is set as the bioefficacy criteria (WHO 2013b). In the German method, the mosquitoes are continuously exposed until 99% KD, and the time required for 99% KD is measured. The time of exposure is only 3 min in the WHO method wherein KD% 1-h post-exposure and mortality (%) 24-h post-exposure are recorded (WHO 2013b; Faulde et al. 2016). Further studies on the suitability of these two methods in evaluating the bioefficacy of IF are required, and standard international guidelines for testing and licencing need to be formulated. The licencing procedure should differentiate between insecticidal nets and IF. The only function of an insecticidal net is to protect from mosquitoes; and hence, it is to be registered as an insecticidal product. However, the primary function of an IF is clothing for military or civilian use, and protection from arthropod bites is only an additional characteristic imparted to the fabric. Hence, ‘treated articles’ such as IF might be exempted from registering as insecticidal products but should bear labels indicating the insecticidal action and the active ingredient used. However, the insecticidal formulation used for treating the fabrics is required to be registered as an insecticidal product (Wünsche 2017).

The claims of bioefficacy include the number of washes, which can be endured by the treated fabrics. Hence, it is evident that the washing method, washing frequency, washing agent, drying conditions and ironing may affect the outcome of the wash resistance studies. The LLIF worn by the military personnel under the deployment conditions usually do not sustain repeated machine launderings. Hence, it is expected that the LLIF are effective throughout their lifetime. It is assumed that hand washing results in higher retention of permethrin compared to machine washing, probably due to less mechanical disruption of the polymer layer during hand washing (Faulde et al. 2003). Currently, there are no standard methods for washing of IF, which makes the comparison of the results of various trials difficult. The LLIF prepared through polymer-coating method are reported to last for 70–100 or more washes (Faulde et al. 2016; Vaughn and Meshnick 2011). However, a field trial in Thailand showed that the bioactivity lasted only up to 4 washes, whereas the manufacturer’s claim was 70 washes. The comparatively rigorous tropical washing conditions along with open air drying and ironing are obviously much different from the test conditions adopted by the manufacturers. Such differences between laboratory test conditions and the conditions of actual field use might lead to lower bioefficacy in user trials than what is claimed (Kittayapong et al. 2017). The WHO standard washing procedure for LLIN (WHO 2013b), which is also used for washing clothing (Banks et al. 2014), could be employed for meaningful comparison of wash resistance of LLIF in the absence of specific guidelines for IF.

Permethrin content in the IF is of utmost importance in determining the bioefficacy, safety and durability. Two commercial products were reported to have extremely high doses of permethrin (up to 4300 mg/m2), whereas two others could not provide bioefficacy up to 100 washes (Faulde et al. 2016). The availability of standardised methods of residue analysis is important for quality control and consumer safety. The CIPAC methods for quantification of the active ingredient are available for LLIN. However, there is no internationally accepted protocol available for the residue analysis of IF. A method developed by the Federal Armed Forces Research Institute is being used for permethrin quantification of PTBDU of the German Armed forces (Faulde et al. 2016; Faulde et al. 2003).

In India, there is high incidence of malaria and Japanese encephalitis in many regions. The outbreaks of dengue and chikungunya are reported frequently from the urban areas including the national capital region. The use of LLIF by the troops and civilians could provide protection from disease vectors thereby reducing the disease transmission. LLIF is currently not available in India, and a simple, cost-effective, safe and durable technology needs to be developed so as to complement the existing disease control measures. Since the active ingredient permethrin is an insecticide, the regulatory aspects concerning the manufacture, sale and use of LLIF need to be taken care of, and specific guidelines for the evaluation, licencing and quality control of LLIF need to be formulated.

Test procedure for insecticidal fabrics

In the absence of widely accepted guidelines on IF, we have formulated a test procedure, which could be used for the evaluation of IF especially LLIF until standard guidelines are available. This procedure is derived from the TL 8305-0331 testing algorithm (Faulde et al. 2016; Most et al. 2017) and the WHO guidelines on testing of LLIN (WHO 2013b) with suitable modifications based on the outcomes of our previous studies on IF. Adult female, non blood-fed, susceptible Ae. aegypti mosquitoes (2–5 day old) in batches of five are used for the testing, and the tests are replicated ten times for each sample. The test mosquitoes are continuously exposed to the IF sample under a standard WHO cone, and the time required for complete (100%) KD is recorded. For the LLIF to be effective, the sample should provide a mean complete knock-down time (CKDT) of ≤ 71.5 min initially (unwashed) as well as after one hundred WHO standard washings (WHO 2013b). The CKDT could be easily determined unlike the 99% KD time, which requires a series of observations on the KD time and probit analysis.

In the present method, the KD time of only the fifth (the last one to be knocked-down) mosquito is to be recorded, and the test outcomes are obtained within a maximum duration of 80 min. There is no need to wait for 24 h to obtain the mortality data unlike in the WHO cone bioassay method for LLIN. Thus, the present method is a lot easier and more practically useful for testing and quality control of IF. Furthermore, we have standardised a method for permethrin quantification from IF, which involves the extraction of IF samples in toluene and the addition of tributyl phosphate as the internal standard. The samples are then analysed in a GCMS in select ion-monitoring mode. In order to be effective, the LLIF sample should contain an initial (unwashed) permethrin content of 1300 ± 300 mg/m2 and ≥ 200 mg/m2 after one hundred WHO standard washings (Faulde et al. 2016; Most et al. 2017; WHO 2013b). The test procedure described here could help in meaningful comparison of the performances of IF manufactured using different permethrin recipes or treatment processes.

Conclusions

The safety and efficacy of IF have been established by various studies worldwide, and these fabrics are being used worldwide for protection from disease vectors. Long-lasting technology for fabric impregnation has resulted in the development of IF with high wash resistance. LLIF are emerging as an important tool for fighting diseases transmitted by day-biting mosquitoes. A review of literature indicates that there is no uniformity in the methods adopted for bioefficacy testing, washing and residue analysis, which often makes comparison of the results irrelevant. There is a need to formulate standard guidelines for testing of IF in the context of emergence and rapid spread of dengue and Zika virus infections.