Assessing the insecticide susceptibility status of field population of Phlebotomus papatasi (Diptera: Psychodidae) in a hyperendemic area of zoonotic cutaneous leishmaniasis in Esfahan Province, Central Iran

Acta Tropica 176 (2017) 316–322

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Assessing the insecticide susceptibility status of field population of Phlebotomus papatasi (Diptera: Psychodidae) in a hyperendemic area of zoonotic cutaneous leishmaniasis in Esfahan Province, Central Iran

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Leila Shirani-Bidabadia, Alireza Zahraei-Ramazania, Mohammad Reza Yaghoobi–Ershadia, Yavar Rassia, Amir Ahmad Akhavana, Mohammad Ali Oshaghia, Ahmad Ali Enayatic, ⁎ Zahra Saeidia, Reza Jafarid, Hassan Vatandoosta,b, a

Department of Medical Entomology and Vector control, School of Public Health,Tehran University of Medical Sciences, Tehran, Iran Institute for Environmental Research, Tehran University of Medical Sciences, Tehran, Iran Department of Medical Entomology and Vector control, School of Public Health, Mazandaran University of Medical Sciences, Mazandaran, Iran d Esfahan Health Research Station, National Institute of Health Research, Tehran University of Medical Sciences, Tehran, Iran b c

A R T I C L E I N F O

A B S T R A C T

Keywords: Susceptibility test Phlebotomus papatasi Zoonotic Cutaneous Leishmaniasis Iran

Leishmaniasis is a Neglected Tropical Disease (NTD) and emerging parasitic infection that affect mainly poor regions around the world. This study aimed to determine the baseline susceptibility of Phlebotomus papatasi to commonly used insecticides in a hyper endemic area using WHO standard procedure in central Iran. A total of 4–5 replicates containing 120–200 sand flies were used for each insecticide. Baseline susceptibility to DDT and pyrethroids was assessed on 5326 specimens collected from the study area. The LT50 and LT90 values were measured according to the World Health Organisation test using probit analysis and regression lines. The test results against males P. papatasi revealed that LT50 values to DDT 4%,Permethrin 0.75%, Deltamethrin 0.05%, Cyfluthrin 0.15% and Lambdacyhalothrin 0.05% were 564.07, 38.08, 1.95, 0.60 and 9.78 s and the figures for females were 584.44, 110.10, 11.64, 1.53 and 16.91 s, respectively. Our results indicated that P. papatasi as the main cutaneous leishmaniasis vector was susceptible to Cyfluthrin 0.15%, Lambdacyhalothrin 0.05%, Permethrin 0.75% and Deltamethrin 0.05% and tolerant to DDT 4%. This study was carried out in one out of many Leishmaniasis foci in Iran. We recommend that future studies incorporate other regions and use the same procedure for monitoring and evaluating sand fly resistance. Also, WHO can provide a specific guideline and create a test kit for sand fly resistance monitoring and for applying susceptibility test because the tubes prepared for mosquitoes are not actually fit for sand flies.

1. Introduction World Health Organization (WHO) has classified Leishmaniasis among Neglected Tropical Diseases (NTDs), and an emerging parasitic disease that affect mainly poor regions around the world. The disease is endemic in 88 countries (72 are developing countries) with approximately 350 million individuals at risk of contracting the disease and an annual incidence of 1.5–2 million new cases (WHO, 2016). Cutaneous Leishmaniasis (CL) is seen in two forms: the rural or wet (zoonotic) and the urban or dry (anthroponotic) forms. Ninety percent of CL cases occur in 7 countries; Afghanistan, Algeria, Brazil, Iran, Peru, Saudi Arabia and Syria (Desjeux 2004; Assimina et al., 2008) and 90% of Visceral Leishmaniasis (VL) cases occur in rural and suburban areas in 7 countries; Bangladesh, India, Nepal, Sudan and Brazil (Gramiccia and

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Gradoni 2005; Chappuis et al., 2007). About 80% of Leishmaniasis cases reported in Iran are Zoonotic Cutaneous Leishmanias (ZCL) and is endemic in many rural areas of 17 out of 31 provinces in the country, posing a great health problem (Yaghoobi-Ershadi and Javadian, 1993; Yaghoobi-Ershadi et al., 2001b, 2003; Akhavan et al., 2007). The trend of CL in Iran indicates that the number of cases has decreased from 20718 in 1994–19693 in 2015 (Norouzinezhad et al., 2016; Fig. 1). Phlebotomine sand flies are known to transmit various diseases including Leishmaniasis, Bartonellosis and Sand fly fever that sometimes affect humans and animals (Zahraei-Ramazani and Leshan Wannigama, 2016). Approximately 1000 species of sand flies have been identified, and around 70 of them act as Leishmaniasis vectors (Bates, 2008). Vector control helps to reduce or interrupt transmission of disease by controlling sand flies, especially in domestic areas. Control methods

Corresponding author at: Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, P.O Box 6446-14155, Tehran, Iran. E-mail addresses: [email protected], [email protected] (H. Vatandoost).

http://dx.doi.org/10.1016/j.actatropica.2017.08.035 Received 19 November 2016; Received in revised form 28 August 2017; Accepted 31 August 2017 0001-706X/ © 2017 Published by Elsevier B.V.

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Fig. 1. Number of leishmaniasis cases in Iran (CL trend between 1994 and 2015; Norouzinezhad et al., 2016).

(southeast) showed that the field population species was still susceptible to these insecticides in different studied areas (Yaghoobi-Ershadi et al., 2003, 2006; Akhavan et al., 2007; Aghaei-Afshar et al., 2013; Saeidi et al., 2011). The activity of Phlebotomus papatasi (P. papatasi) in Badrood, an endemic area in central Iran, starts usually from late April and extends to mid-Octobor with two peaks, one in around mid June and the second in around mid-September (Yaghoobi-Ershadi and Akhavan, 1999). In the study area there are diffenet methods for disease control including spraying residential buildings with Propoxur (Baygon 0.2%), spraying with pyrethroides, baiting with anticoagulant rodenticides, changing the vegetative cover of the region, improving the environmental condition to reduce the breeding places nad hiding sites of sandflies such as, filing mesh on all doors and windows of buildings in residential parts (Nilforoushzadeh et al., 2012). Rodent control operation using Zinc phosphide once a month in the two villages of AbbasAbad and Matin-Abad (Yaghoobi-Ershadi et al., 2005). The objective of this study was to determine the baseline susceptibility of P. papatasi to the commonly used insecticides in an endemic area of ZCL in central part of Iran. The results generated will be used for the evaluation of mechanisms of resistance of this vector in Esfahan province as hyperendemic area in Iran.

include insecticide spray, use of insecticide–treated nets, environmental management and personal protection (WHO, 2015). Insecticidal control of adult sand flies and insecticide-based approaches such as impregnated bed nets are still considered the most effective methods for personal protection and rapidly reducing populations of sand flies involved in Leishmaniasis transmission (WHO, 2015). In some foci of the disease (including Esfahan), DDT (75%) at a dosage of 2 mg/m2 as residual spraying was used from 1952 until 1959 for malaria control, but from1959 onwards, owing to malaria consolidation (malaria was controlled), the use of DDT was stopped (Yaghoobi-Erashadi and Javadian, 1995). Another survey was carried out using WHO standard method on wild caught P. papatasi from human dwellings in Mashhad (northeast), Esfahan (central Iran) and Khuzestan (southwest) between 1985–1988. The results showed that the LC50 value for DDT had increased to 2.3% and 3% in Mashhad and Esfahan respectively, disclosing the existence of selection pressure in the lack of public health insecticide use (Seyedi-Rashti et al., 1992). Yaghoobi- Ershadi (1993) provided evidence of tolerance of P. papatasi to 4.0% DDT following a standard WHO technique in Esfahan. LT50 and LT90 values were calculated to be 18.12 and 63.31 min, respectively. The mortality rate after one hour exposure to DDT 4.0% and the 24 h recovery period was 88.8%. (Yaghoobi-Ershadi and Javadian, 1993). In 2001, the susceptibility test using WHO technique which was performed in the rural district of Borkhar (Esfahan) showed that the susceptibility of P. papatasi to DDT 4.0% has returned because the mortality rate after one hour exposure was calculated to be 100%. This was achieved due to the low use of Chlorinated insecticides in agriculture, during the last 9 years in the area (Yaghoobi-Ershadi, Tehran University of Medical Sciences, unpublished data). Zahraei-Ramazani and Javadian (1993) provided evidence of susceptibility of P. papatasi to DDT 4.0% following a standard WHO technique in Esfahan (Zahraei-Ramazani, 1993). Between 1993–2001 the same study was carried out in the provinces of Fars (southwest), Kerman (southeast) and Badrood area in Esfahan and LT50 ranged between 5.5–29.7 min and LT90 between 18.5–58 min, respectively. Phlebotomus papatasi was found to be susceptible to DDT in all of these areas (Aghasi, 1996; Yaghoobi-Ershadi and Akhavan, 1999; Rassi et al., 2000). Susceptibility test which were performed again between 2002 and 2013 on P. papatasi against DDT 4%, deltamethrin 0.05%, 0.025% and propoxur 0.1% by WHO standard method in Sabzevar (northeast), Esfahan (Central), Bam, Baft and Dehbakri

2. Materials and methods 2.1. Study area This survey was conducted in summer of 2015. Sand flies were collected from the rural district of Badrood, Natanz county, Esfahan province, central Iran (Fig. 2). This area is located in the foothills of the Karkas Mountains with an altitude 3895 m in the mountaintop. The area has a semi-desert climate. 2.2. Sand fly collection All sand flies were collected from Matin- Abad tourist camp in Badrood district, Natanz County, Esfahan province, situated in altitude of 978 m (51° 59′ E, 33° 45′ N). Sand flies were collected by hand aspirator and flash light from 8.00 pm until 4.00 am at differernt time intervals. The adults were caught from outdoor during dawn and dusk 317

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Fig. 2. Study area in Badrood rural district, Natanz County, Esfahan Province, Iran, 2015 (http://www.wyattfamilyreunion.com/map-of-iran-provinces/).

2.4. Insecticide papers and their concentration

from parked car in the tourist camps, near their breeding places. Balck parked car attract the adult sandflies and they could be collected directly by aspirator. Additionally, the inside of a car was used for trapping sand flies by aspiration tool. Aftre collection, all adults were transferred to cages with a hanging piece of wet cloth for supplying suitable humidity. Adults were fed with 20% sucrose solution soaked on cotton pads. Subsequently the cages were transported covered with wet cloth to increase humidity to the laboratory at Esfahan Health Research Station, National Institute of Health Research, Tehran University of Medical Sciences. They were kept in appropriate condition (27 ± 2° C and 60 ± 10% relative humidity). Temperature was maintained by electric heaters and photoperiod of 14:10 h L:D was maintained in the insectary.

The WHO test-kit tubes and impregnated papers were procured from collaborating center of WHO in Malaysia. This papers with different diagnostic dosages were DDT 4% (the expiry date: September 2014–2019 and batch number: DD214), Deltamethrin 0.05% (the expiry date: October 2014–2015 and batch number: DE381), Permethrin 0.75% (the expiry date: September 2014–2015 and batch number: PE289), Lambdacyhalothrin 0.05% (the expiry date: September 2014–2015 and batch number: LA183) and Cyfluthrin 0.15% (the expiry date: September 2014–2015 and batch number: CY089), as well as the control paper. 2.5. Statistical analysis

2.3. Susceptibility test The mortality rates of sand flies to insecticides at different time intervals were subjected to Finney’s test for calculation of regression lines. We calculated LT50, LT90 and 95% confidence intervals with probit analysis. For plotting of regression lines, Excell 2013 was used.

After 1 h of resting and feeding by sucrose solution 20%, sand flies were tested according to the standard test procedures of WHO (WHO, 2013). A total of 4–5 replicates containing 120–200 sand flies were used for each insecticide. The sand flies (15–20) were transferred into the exposure tubes at different time intervals (7, 14, 28, 56, 113, 225, 450, 900, 1800, 3600 s) and mortality rate was then scored after 24 h recovery period. There was one group of control for each insecticide in each test. All mortality rates were corrected according to the results of control with Abbott,s correction formula, in case the control mortality was between 5 and 20% that was expressed as corrected percent mortality. After each test, all dead and alive sand flies were mounted separately by pouri’s medium for species identification according to the available keys (Theodor and Mesghali, 1964). Males and females were counted separately. The same procedure used for the male tests was also used for the female. After selection with DDT, we are looking the mechanism of resistance to DDT and cross resistance to pyrethroids. For evaluation of genetics of resistance, there is need for cross and backcross among male and female. So that males are used for susceptibility tests. Both sex of sand flies were confined together for oviposition and rearing of developmental stages for obtaining its next generation. Inherited DDT resistant character of progenies of tolerant parent was estimated and compared by mortality rate and DDT resistant ratio through the insecticide susceptibility test conducted at its subsequent generations.

3. Results In this study 5326 sand fly specimens were mounted and identified. Susceptibility test against specimens collected in Matin-Abad Badrood, were carried out in summer of 2015. Table 1 shows the probit regression line parameters for females P. papatasi to different insecticides at the discriminative dose. The results of the data for females were 584.44, 110.10, 11.64, 1.53 and 16.91 s, respectively. Table 2 presents results of the experiment against male P. papatasi and reveals LT50 values to DDT 4%, Permethrin 0.75%, Deltamethrin 0.05%, Cyfluthrin 0.15% and Lambdacyhalothrin 0.05% of 564.07, 38.08, 1.95, 0.60 and 9.78 s, respectively. Table 3 presents the results of mortality in 60 min to insecticides against female P. papatasi. Figs. 3, 4 show the probit regression lines. Males were more susceptible than females to all insecticides tested at LT50 level (Fig. 5). The results also show a significant difference between the male and female sand flies for LT50 calculated to DDT (P value = 0.011, p < 0.05). However, there was no significant difference between the males and females to Permethrin 0.75% (Pvalue = 0.288), Deltamethrin 0.05% (P value = 0.166), Cyfluthrin 0.15% (P value = 0.262) and Lambdacyhalothrin 0.05% (P value = 0.394)(Fig. 5). Three thousands sixty seven (3067) females P. 318

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Table 1 Parameters of probit regression lines of different insecticides against female Phlebotomus papatasi, Esfahan, Iran, 2015. Insecticide

Aa

B ± SEb

LT50c, 95%Cl

LT90d, 95%Cl

Χ2(df)

Heterogeneity P-value

Y = a + bx

DDT 4%

−5.4268

1.9614 ± 0.523

P < 0.05

Y = −5.4268 + 1.9614 X

−1.0028

0.4911 ± 0.110

4.228(4)

P > 0.05

Y = −1.0028 + 0.4911 X

Deltamethrin 0.05%

−0.9956

0.9339 ± 0.164

17.143 (5)

P < 0.05

Y = −0.9956 + 0.9339 X

Cyfluthrin 0.15%

−0.1050

0.5662 ± 0.097

1.115 (4)

P > 0.05

Y = −0.1050 + 0.5662 X

Lambdacyhalothrin 0.05%

−0.9049

0.7367 ± 0.250

990.08 2631.11 30412256.70 6754.81 44791.73 5094966.88 98.64 274.31 2515.60 106.84 281.05 1630.87 195.81 928.80 –

17.699 (3)

Permethrin 0.75%

164.08 584.44 3819.86 53.54 110.10 220.25 3.32 11.64 26.79 0.41 1.53 3.34 0.0029 16.91 51.04

23.031 (5)

P < 0.05

Y = −0.9049 + 0.7367 X

a b c d

A = Intercept. B ± SE = Slope and its Standard Error. LT50,95%C.L. = Leathal time cause 50% mortality and its 95% Confidence Interval. LT90,95%C.L. = Leathal time cause 90% mortality and its 95% Confidence Interval.

papatasi were exposed to 5 insecticides, in which 2160 (70.4%) females exposed died and 907 (29.6%) survived. Chi-square test did not show any significant relation between the response to insecticide and abdominal condition of the sand flies (Pvalue = 0.852,df = 3) (Fig. 6).

Table 3 The mortality rate of female of Phlebotomus papatasi after 60 min contact with some insecticides, Esfahan, Iran, 2015.

4. Discussion Insecticide resistance in vectors is a major public health problem especially in the tropical regions. There have been several reports of decrease in sand flies population as a collateral benefit of malaria control programmes, although, these sand flies have developed resistance to insecticide, especially to DDT and less to other insecticides such as malathion and pyrethroids (Dinesh et al., 2010). Therefore, it is of great value in view of control of sand flies and leishmaniasis to establish a baseline data and to assess the extent of insecticide susceptibility and resistance in sand fly vectors, in order to design an effective control programme. Several studies have investigated the susceptibility of sand flies to insecticides around the world. However, the methods used in those studies were not identical i.e. insecticide concentrations and time of exposure varied. Most experiments were performed on reared sand fly colonies using dose-mortality bioassays, or time-mortality bioassays

Insecticide

No. tested

No. Dead

MR ± EB*

Resistance status**

Deltamethrin 0.05% Control Lambdacyhalothrin 0.05% Control Cyfluthrin 0.15% Control Permethrin 0.75% Control DDT4% Control

50 279 49

49 18 48

97.86 ± 1.66 – 97.78 ± 1.66

S** – S**

267 67 250 84 235 108 271

20 67 18 83 18 104 16

– 100 – 98.7 ± 0.5 – 96.05 ± 0.59 –

– S** – S** – RC –

*Mortality Rate ± Error Bar. R**: Resistance, RC: Resistant Candidate (exeistance Resistance), T: Tolerance, S **: Susceptible.

(Alexander et al., 2009). However, there are few studies that have focused on sand flies collected from the field and adopted the discriminating concentration (Singh et al., 2001).

Table 2 Parameters of probit regression lines of different insecticides against male Phlebotomus papatasi, Esfahan, Iran, 2015. Insecticide

Aa

B ± SEb

LT50c, 95%C.L.

LT90d, 95%C.L

X2 (df)

Heterogeneity P value

Y = a + bx

DDT 4%

−2.7676

1.0059 ± 0.480

P < 0.05

Y = −2.7676 + 1.0059 X

−1.2786

0.8089 ± 0.304

10.994 (4)

P < 0.05

Y = −1.2786 + 0.8089 X

Deltamethrin 0.05%

−0.1590

0.5448 ± 0.113

5.001 (5)

P > 0.05

Y = −0.1590 + 0.5448 X

Cyfluthrin 0.15%

0.1707

0.7915 ± 0.199

0.600 (4)

P > 0.05

Y = 0.1707 + 0.7915 X

Lambdacyhalothrin 0.05%

−0.6631

0.6693 ± 0.176

– 10602.31 – – 1462.91 – 151.83 440.87 3914.76 11.54 25.32 122.78 246.19 804.27 25375.72

13.132 (3)

Permethrin 0.75%

– 564.07 – – 38.08 – 0.25 1.95 5.41 0.04 0.60 1.72 1.88 9.78 19.82

3.388 (4)

P > 0.05

Y = −0.6631 + 0.6693 X

a b c d

A = Intercept. B ± SE = Slope and its Standard Error. LT50,95%C.L. = Leathal time cause 50% mortality and its 95% Confidence Interval. LT90,95%C.L. = Leathal time cause 90% mortality and its 95% Confidence Interval.

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Fig. 3. Probit regression lines of different insecticides against female Phlebotomus papatasi, Esfahan, Iran, 2015.

Lambdacyhalothrin, Permethrin and Deltamethrin were 34.56, 5.3 and 50.2 folds, respectively (Fig. 5). The high LT50 level of this vector to DDT was attributed to the longterm use of insecticides for malaria vector control in the region that was transmitted genetically to their progeny. According to the report of the branch of the Ministry of Jahad in Esfahan province, several herbicides, fungicides and inesticides have been used for agriculture and veterinary pest control in the region, including Methalaxile, Carbaryl, Permethrin, Cypermethrin, Deltamethrin, Metasystox-R and Mancozeb. The susceptibility test of a wild strain of P. papatasi to DDT and pyrethroids in Badrood shows that males were more susceptible than females to all the insecticides tested at LT50 level. In comparing our results and the study by Saeidi et al. (2012), LT50 values of male to DDT

The WHO susceptibility test recommended for mosquitoes were followed. The discriminative doses of DDT and pyrethroids were used in our study. In accordance with WHO (WHO, 1998), the bioassay results for malaria vectors were summarized in three resistance classes according to mortality rate: susceptible (< %98); possibly resistant, so called ‘tolerant’ (90–97%) and resistant (< 90%). Results of the present study using ststistical analysis revealed that at the LT50 level females need more time than males to be killed at the same concentration (p < 0.05). For both males and females, the susceptibility levels to DDT were greater than to pyrethroids. For example, at LT50 level, the required time to gain the same mortality for females against DDT in comparison with Cyfluthrin was 381 times higher. The figures for

Fig. 4. Probit regression lines of different insecticides against male Phlebotomus papatasi, Esfahan, Iran, 2015.

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Fig. 5. LT50 values of different insecticides against male and female Phlebotomus papatasi, Esfahan, Iran, 2015.

Fig. 6. Abdominal condition of females Phlebotomus papatasi tested with the insecticides, Matin-Abad, Badrood, Esfahan, Iran, 2015.

Fars province, southern Iran and the results indicating highly susceptibility level of this species to DDT in this area (Seyedi-Rashti et al., 1992). Yaghoobi-Ershadi et al. (2006) also proposed that P. papatasi susceptible to Deltamethrin in Borkhar district, Esfahan province (Yaghoobi-Ershadi et al., 2006), and susceptible to DDT in Badrood district (Yaghoobi-Ershadi and Akhavan, 1999). In Sabzevar district susceptible to DDT (4%), Permethrin (0.25%) and Propoxure (0.1%) (Yaghoobi-Ershadi et al., 2003) and in Orzuie district in Kerman province susceptible to DDT (4%) (Akhavan et al., 2007). Tolerance of P. papatasi to DDT has been reported from some parts of Iran (YaghoobiErshadi and Javadian, 1993; Seyedi-Rashti and Nadim, 1974). Faraj et al. (2012) reported that P. sergenti and P. papatasi were susceptible to all insecticides tested in Morocco (Faraj et al., 2012). Karakus et al. (2016) showed that sand fly populations from Mugla province in Turky were resistance against deltamethrin and permethrin where both insecticides have been applied for long time while no resistance was found in the insecticide free area, Aydin Province (Karakus

4% was greater than LT50 in this study, males were more susceptible to pyrethroids and the sand flies were more tolerant compared to our results. LT50 values in males and females in our study to Permethrin, Lambdacyhalothrin, Cyfluthrin were also smaller compared to their results, but LT50 values in males and females to Deltamethrin in our study were greater than theirs (Saeidi et al., 2012). This difference was due to the presence of Deltamethrin in agriculture fields in recent years in Matin-Abad. Several factors were also involved in the insecticide resistant. There are several reports of susceptibility of leishmaniasis vectors to different insecticides in Iran. Seyedi-Rashti et al. (1992) showed that P. papatasi from Esfahan was more tolerant to DDT than other parts and probably a manifestation of DDT resistance (Seyedi-Rashti et al., 1992). Yaghoobi-Ershadi et al. (1995) also showed that the tolerance in P. papatasi population to DDT(4%) at 1 h exposure time with mortality rate of 88.8% (Yaghoobi-Ershadi et al., 1995). The susceptibility status of P. papatasi shown at zoonotic cutaneous leishmaniasis (ZCL) foci of 321

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et al., 2016). Sand flies were susceptible to DDT in 1980 in India, and resistance was first detected in 1993, a year after the third cycle of DDT use to prevent visceral leishmaniasis was started. Insecticide susceptibility to DDT (4%) and Deltamethrin (0.05%) WHO-impregnated papers was determined with wild-caught sand flies. Coleman et al. (2015) also proposed that sand flies were highly resistant to DDT but susceptible to deltamethrin in eight visceral leishmaniasis (VL) endemic districts in Bihar State, India (Coleman et al., 2015). Denlinger et al. (2015) determine lethal concentrations (LCs) and lethal exposure times (LTs) to assess levels of susceptibility of laboratory Lutzomyia longipalpis and P. papatasi to 10 insecticides using a modified version of the WHO exposure kit assay and Centers for Disease Control and Prevention (CDC) bottle bioassay. The LCs and LTs from the study is now used as a baseline reference point for future studies using the CDC bottle bioassays to assess insecticide susceptibility of sand fly populations in the field (Denlinger et al., 2015). Three species of sand flies, P. papatasi, P. argentipes and Sergentomyia shorti were resistant to DDT in India, although there are reports of increased tolerance to this compound in some other countries (WHO, 1986; Maroli and Khoury, 2004). 5. Conclusion In this study, the susceptibility of P. papatasi to different insecticide was compared. The fact that the adult P. papatasi population of field tested is tolerant (existence resistance) to DDT 4% and was still highly susceptible to Permethrin 0.75%, Deltamethrin 0.05%, Cyfluthrin 0.15% and Lambdacyhalothrin 0.05% raises the possibility of using these pyrethroids in sand fly control programmes in foci of leishmaniasis. This study was carried out in one out of many areas of leishmaniasis foci in Iran, future studies should incorporate other regions.The results indicate the ability of developing insecticide resistance in P. papatasi. The authors recommend that the same procedure be used in different parts of Iran.WHO can provide a specific guideline and create device for sand fly resistance monitoring and for applying susceptibility test because of the tubes prepared for mosquitoes are not actually fit for sand flies. This guideline will help countries in monitoring and evaluation of insecticides resistance for implementation of control measures in infected regions. Acknowledgements Authors wish to thank all staff of National Institute of Health Research, Tehran University of Medical Sciences, special thanks to Niloufar Shareghi, Mohamad Hossein Arandian, Mohsen Mahmoodi. We would also like to thank Fatemeh Nikpour, for providing the WHO kit-test. This research was financially supported by School of Public Health, Tehran University of Medical Sciences (TUMS). Under code number 95-01-27-30104. The authors declare that there is no conflict of interest. References Aghaei-Afshar, A., Vatandoost, H., Sharifi, I., Rassi, Y., Abai, M.R., Oshaghi, M.A., 2013. First determination of impact and outcome indicators following indoor Residual spraying (IRS) with deltamethrin in a new focus of anthroponotic cutaneous leishmaniasis (ACL) in Iran. Asian Pac. J. Trop. Dis. 3 (1), 5–9. Aghasi, M., 1996. Present Status of Anthroponotic Cutaneous Leishmaniasis in Kerman, Southeast Iran (MSPH Thesis). School of Public Health, Tehran University of Medical Sciences, No. 1968, Tehran, Iran (in Persian). Akhavan, A.A., Yaghoobi-Ershadi, M.R., Hasibi, F., Jafari, R., Abdoli, H., Arandian, M.H., et al., 2007. Emergence of cutaneous leishmaniasis due to Leishmania major in a new focus of southern Iran. Iran. J. Arthropod-Borne Dis. 1 (1), 1–8. Alexander, B., Barros, V.C., DeSouza, S.F., Barros, S.S., Teodoro, L.P., Soares, Z.R., et al., 2009. Susceptibility to chemical insecticides of two Brazilian populations of the visceral leishmaniasis vector Lutzomyia longipalpis (Diptera:Psychodidae). Trop. Med. Int. Health 14 (10), 1272–1277. Assimina, Z., Charilaos, K., Fotoula, B., 2008. Leishmaniasis: an overlooked public health concern. Health Sci. J. 2, 196–205. Bates, P.A., 2008. Leishmania sand fly interaction: progress and challenges. Curr. Opin. Microbiol. 11, 340–344. Chappuis, F., Sundar, S., Hailu, A., Ghalib, H., Rijal, S., Peeling, R.W., et al., 2007.

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