Entomological investigations of dengue vectors in epidemic-prone districts of Pakistan during

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1 Entomological investigations of dengue vectors in epidemic-prone districts of Pakistan during Muhammad Mukhtar, a# Zarfishan Tahir, b Taj Muhammad Baloch, c Faisal Mansoor d & Jaleel Kamran e a Department of Zoonotic and Vector-Borne Diseases, Public Health Laboratories Division, National Institute of Health, Islamabad, Pakistan. b Bectoriologist Laboratory, Institute of Public Health, 6-Abdul Rehman Choughtai Road, Lahore, Pakistan. c Directorate of Malaria Control, Wahadat Colony, Hyderabad, Pakistan. d Pakistan Medical Research Council, Islamabad, Pakistan. e Epidemic Investigation Cell, National Institute of Health, Islamabad, Pakistan. Abstract Intensive entomological investigations were carried out in seven dengue epidemic-prone districts of Pakistan, classifying them into three geographical regions, viz. southern, central and northern Pakistan. A total of 5132 water habitats from 2136 households in and around dengue-positive houses were sampled. Additionally, 264 samples each at least 30 metres away from dengue-positive houses were also collected from outdoor habitats. Only indoor samples data were used for the estimation of entomological indices. House Index, Container Index and Breteau Index were estimated at 39.42%, 27.96% and respectively. Underground water tanks showed the highest (42.38%) positivity, followed by earthen pots (36.97%), drums (33.38%) and the least (4.58%) from discarded containers. From outdoor sites, only 5.05% (n=14) samples were found positive. Aedes aegypti and Aedes albopictus species exhibited a distinct association with different geographical regions. In the south of the country only Ae. aegypti was recorded in all (n=452) positive habitats while in the central part, both Ae. aegypti and Ae. albopictus were reported from 88.2% (n=253) and 11.8% (n=34) of the total 287 positive habitats respectively. In the north/submountainous region, 88.45% (n=628) of 710 positive samples were found infested with Ae. albopictus. Both species showed a significant population-rising trend from September to November, similar to the dengue case-load trend. Keywords: Entomological investigations; Aedes aegypti; Aedes albopictus; Dengue; Pakistan; # mukasbilumm@gmail.com Dengue Bulletin Volume 35,

2 Introduction Dengue fever (DF) and dengue haemorrhagic fever (DHF) are considered important reemerging arboviral diseases in more than 100 tropical and subtropical countries of the world. [1] The disease epidemiology is complex in nature and requires understanding of a variety of factors that include weather and environmental changes, [2,3,4] vector species composition and behaviour, [5,6,7] population dynamics and degree of immunity in local population. [8,9,10] In Pakistan, dengue is emerging as one of the major public health problems, particularly since 2005, threatening the lives of millions of people due to prevailing peculiar socioeconomic conditions and epidemiological situation. Historically, dengue has been endemic in the southern parts of the country. In Pakistan, dengue was recognized for the first time in 1994 in Karachi and one patient out of 145 cases died. [11] In October 1995, 57 out of 76 persons were found positive for antibodies against the dengue virus in Hub, southern Balochistan. In October 2003, dengue outbreaks were detected for the first time in submountainous areas of Haripur district, Khyber Pakhtoonkhawa province and Khushab district, Punjab province, claiming six lives among 717 cases. In October 2005, dengue hit Karachi again after 10 years and 21 deaths out of the total 103 confirmed cases were recorded. [12,13] Since then, the disease has become widely prevalent and has been accepted as one of the major public health problems in Pakistan. Until 2010, cases and 156 deaths have been reported (Epidemic Investigation Cell, National Institute of Health, 2010 Unpublished data). Aedes aegypti and Aedes albopictus have been considered as the major vectors of dengue in South-East Asia, including Pakistan. [14,15,16] Both species have been closely associated with human dwellings due to their breeding preference for clean-water domestic habitats. [17,18] Entomological surveillance, particularly based on larval surveys, provides vital information for better dengue disease management. However, in Pakistan, at present there is no systematic entomological surveillance system, particularly after the 1980s to update the knowledge of vector species and their bionomics. In view of the deteriorating situation with regard to dengue/dhf in the country and poor knowledge of vector(s), systematic and intensive entomological surveys were conducted in seven high-risk districts during dengue outbreaks to identify the potential breeding sites of dengue vector(s) and to determine the levels of vector infestations at each affected area by using the commonly used larval indices (House, Container and Breteau). Finally, in order to estimate the transmission potential for dengue outbreaks in the country, the new knowledge generated through these investigations will provide a technical basis to design evidencebased, community-friendly and sustainable preventive and control measures against dengue in Pakistan. 100 Dengue Bulletin Volume 35, 2011

3 Materials and methods Study type and selection of study areas The study was descriptive in nature and all seven dengue epidemic-prone districts in the country were selected for entomological surveys and were classified into three geographical areas, viz. Southern (Karachi), Central plain (Lahore), and Northern/submountainous areas (Rawalpindi, Islamabad, Attock, Chakwal and Haripur). Detail of descriptions of selected districts are given in Figure 1 and Table 1. Figure 1: Dengue epidemic-prone districts in Pakistan Dengue Bulletin Volume 35,

4 Table 1: Description of all dengue epidemic-prone districts of Pakistan District Geographic region Lat (N) Geography Meteorology Area (sq km) Population (2010) Density (Pop/sq km) Long (E) Elev (m) Temp C (Max) (Temp) Min R/H (%) Ppt (mm) Total Urban Rural Total (m) Urban (%) Rural (%) Total Urban Rural Karachi Southern Lahore Central ,604 Attock Northern (Submountain) Rawalpindi Northern (Submountain) Islamabad Northern (Submountain) Haripur Northern (Submountain) Chakwal Northern (Submountain) Dengue Bulletin Volume 35, 2011

5 Population estimation The population in 2010 and other characteristics of the selected districts have been calculated on the basis of the estimation provided by the Population Census Organization and the Federal Bureau of Statistics, Government of Pakistan. [19,20] Sampling strategies In each selected district, maps of dengue cases were prepared and most-affected Union Councils (basic administrative unit/area in the country) were identified for entomological investigations. The basic sampling unit was the household and at least 10 households around each selected dengue case were searched for water-holding containers as possible breeding sites. All indoor habitats were classified into underground cemented water tanks, overhead water tanks, earthen pots, discarded containers and drums. Additionally, some potential outdoor breeding sites which include open ponds (including street pools, drains, irrigated fields, etc.) and used tyres, at least 30 metres away from patients homes, were also included in the surveys. These habitats were examined during an outbreak for the presence of Aedes larvae using larval net or dipper. Collected specimens were preserved in 70% formalin for species identification in laboratory using Leopoldo (2004) key. [21] To estimate the entomological indices (House Index (HI), Container Index (CI), and Breteau Index (BI)), only the data of indoor entomological surveys was used. Outdoor surveys data were used only as a reference to compare the breeding preferences of Aedes species between outdoor and indoor habitats. Results On an average, the highest number of water-holding containers in each household were found in Karachi (n=3.0), followed by Rawalpindi (n=2.6) and Attock (n=2.5). Out of the total 2136 households surveyed, 23.36% (n=499) and 19.10% (n=408) were in Karachi and Lahore respectively. The least number of households were surveyed in Chakwal (7.82%) and Attock (8.80%). Of the total households surveyed, 39.42% (n=842) were found positive for Ae. aegypti and Ae. albopictus; the highest HI was found in Karachi (46.5%), followed by Haripur (42.0%), Lahore (41.7%) and Islamabad (41.4%). Of the total 5132 indoor samples collected, 29.17% (n=1497) and 15.90% (n=816) were collected in Karachi and Lahore respectively, of which 27.96% (n=1435) were found positive with Aedes species. The highest CI was observed in Lahore (34.6%), followed by Karachi (30.2%) and Islamabad (29.9%). The highest BI was recorded in Karachi (90.6), followed by Lahore (69.1) and Islamabad (65.8). Overall, the HI, CI and BI of the seven districts were estimated at 39.42%, 27.96% and 67.2 respectively. Details of the households sampled, and the HI, CI and BI are given in Table 2. Dengue Bulletin Volume 35,

6 Table 2: District-wise details of number of households, average number of containers, positivity rate, HI, CI, and BI District Province Total HH House Index (HI) Positive Index (%) Av. no of containers Container Index (CI) Containers inspected Positive Index (%) Breteau Index (BI) Karachi Sindh Lahore Punjab Attock Punjab Rawalpindi Punjab Islamabad Capital Territory Haripur K. Pakhtoonkhawa Chakwal Punjab Total Among the indoor water-holding containers, underground cemented water tanks showed the highest positivity rate (42.38%), followed by earthen pots (36.97%) and drums (33.38%). Only 4.58% (n=16) samples from discarded containers were found positive. However, no sample from overhead water tanks was found positive. Among the outdoor breeding sites, only 5.30% (n=14) of the total 264 samples were found positive, of which all were from used tyres and no sample from open ponds was found positive with Aedes species. Details of the district-wise samples collected and the positivity rate are given in Table 3. In the southern part of the country (Karachi), all (n=452) indoor positive samples were positive only with Ae. aegypti and no Ae. albopictus positive sample was recorded. However, in central plains (Lahore), 89.7% (n=253) and 10.3% (n=34) of the positive samples were positive with Ae. aegypti and Ae. albopictus respectively. In the northern/submountainous areas (Haripur, Rawalpindi, Islamabad and Attock), 93.4% (n=158), 89.1% (n=169), 87.1% (n=170) and 86.1% (n=68) respectively were found positive with Ae. albopictus and the rest were positive with Ae. aegypti. From the outdoor collections, all (n=14) samples were positive with only Ae. albopictus. District-wise details of the association of Ae. aegypti and Ae. albopictus with individual habitat under different geographical areas are given in Table 4. Among the 1435 indoor positive water containers, 84.88% (n=1218) were found uncovered or poorly covered at the time of sample collection. However, only 15.12% (n=217) habitats which were covered properly were found positive. Details of the correlation between the sample positivity rate and covering of water containers are given in Table Dengue Bulletin Volume 35, 2011

7 Table 3: District-wise number of samples collected and positivity rate of water containers with Aedes species in Pakistan Indoor breeding sites Outdoor breeding sites Underground water tank Overhead water tank Drum Earthen pot Discarded container Sub total Used tyre Open pond Grand total Total +ve % Total +ve % Total +ve % Total +ve % Total +ve % Total +ve % Total +ve % Karachi Lahore Attock Rawalpindi Islamabad Haripur Chakwal Total Dengue Bulletin Volume 35,

8 Table 4: District-wise number of positive containers with Aedes aegypti and A. albopictus in Pakistan Indoor breeding sites Outdoor breeding sites Underground water tank Overhead water tank Drum Earthen pots Discarded containers Total Used tyres Open ponds Grand total A. ag A. alb T A. ag A. alb T A. ag A. alb T A. ag A. alb T A. ag A. alb T A. ag A. alb T A. ag A. alb T A. ag A. alb T A. ag A. alb T Karachi Lahore Attock Rawalpindi Islamabad Haripur Chakwal Total Dengue Bulletin Volume 35, 2011

9 Table 5: Association between covering of containers and positivity with Aedes species in Pakistan Indoor breeding sites Outdoor breeding sites Underground Water Tanks Overhead Water Tanks Drums Earthen Pots Discarded Containers Total Used Tyres Open Ponds Grand Totals Coveredness Coveredness Coveredness Coveredness Coveredness Coveredness Coveredness Coveredness Coveredness Un Poorly Properly Un Poorly Properly Un Poorly Properly Un Poorly Properly Un Poorly Properly Un Poorly Properly Un Poorly Properly Un Poorly Properly Un Poorly Properly Karachi Lahore Attock Rawalpindi Islamabad Haripur Chakwal Totals Percentages Dengue Bulletin Volume 35,

10 Out of the total 1449 indoor and outdoor positive samples, 35.5% (n=524) were found positive during the month of November, followed by 34.3% (n=434) and 27.3% (n=277) in October and September respectively. Very low vector densities were recorded during the cold (December February) and hot months (May July). Among the districts, there was no notable difference in the population-building trend of both vector species (data not shown). Month-wise, the case-load in the entire country during the study period also exhibited the same rising trend, and 96.00% (n=13 925) of the total dengue cases and 96.79% (n=151) deaths due to dengue were reported during September November (Table 6). Discussion The occurrence of mosquito immatures in different habitats reflects both oviposition preference of females as well as the ability of the immatures to survive in a particular habitat. Changes in the physio-chemical and biotic characteristics of the habitat may create conditions either favourable or unfavourable for their breeding success, depending upon the Table 6: Month-wise details of case-load, deaths and number of containers examined and positivity rate Dengue cases ( ) Entomological investigations Suspected Confirmed %age Death Containers examined Positive %age January February March April May June July August September October November December Total Dengue Bulletin Volume 35, 2011

11 range of tolerance of different species. [22,23] The present investigations explain the breeding preference of Ae. aegypti and Ae. albopictus to different water-holding containers in different geographical regions. In South-East Asia, both these species are important vectors of dengue and dengue haemorrhagic fever and, traditionally, both species, particularly Ae. aegypti, are believed to be associated with man-made artificial habitats in shaded places in human dwellings. [14,17,18] Our results confirm these associations as 100% positive habitats were man-made and domestic in nature. In Thailand, a distinct endophilic behaviour of Ae. aegypti and exophilic behaviour of Ae. albopictus were recorded. [16,24] Present investigations also support the indoor preference of Ae. aegypti. Similarly, Chen et al (2006) [25] and Sulaiman et al (1991) [26] reported a dominant endophilic behaviour of Ae. albopictus in addition to outdoor breeding preferences in Malaysia. Our finding also supported these findings. Furthermore, we, in agreement with other researchers, [17,24,27,28,29] confirm that the most attractive indoor breeding sites of Ae. aegypti and Ae. albopictus and their sequence was underground cemented water tanks, earthen pots and drums (Figure 2). Normally, a underground cemented water tank (Figure 2a) is 8 x10 in size having 8 12 feet depth and is in common use in these districts, and we observed that these tanks were never emptied completely and cleaned ever since their construction. Most people make a small hole in their metallic lid to insert a pipe for filling and taking out water, which makes an easy access of Aedes mosquitoes to the water for breeding. The use of narrow-neck earthen pots (Figure 2b) is also very common for water storing and these pots are mainly used in rural communities for drinking purposes and are kept in shaded places with extreme care taken to cover them. However, we observed that refrigerators were in common use even among poor communities which greatly reduced the use of these earthen pots for drinking water storage, but more for general domestic use. In northern parts of Pakistan, particularly in Haripur and Chakwal districts, we observed a common use of a very particular type of wide-neck earthen pot (Figure 2c) which is feet tall and kg in weight, having a storage capacity of litres of water. However, these pots are Figure 2: Different water-holding containers and their physical conditions at the time of sampling Figure 2a: Underground water tanks Figure 2b: Narrow-neck earthen pots Figure 2c: Wide-neck earthen pots Figure 2d: Drums Figure 2e: Overhead water tanks Dengue Bulletin Volume 35,

12 usually covered with a very poorly maintained lid made of wood. To keep the water cool, these pots are kept dug in the ground up to half of their height. All these characteristics and household habits make it almost impossible to wash and clean them properly, which results in profound breeding of Aedes mosquitoes. Despite the large number of samples that were collected, we did not find any positivity in the overhead water tanks (Figure 2e), which are mainly made of plastic and are placed in open sunlight on the roofs of houses. The use of these tanks has become very common in the country due to rapid urbanization, particularly since the late 1980s. During the sampling we also observed that these tanks were properly covered and there was a high temperature inside due to their location in open sunlight, which most probably prevents the Aedes mosquito from breeding while other water containers in the same household placed in shaded places were found positive. These findings further confirm the breeding preferences of both the Aedes species for shaded indoor habitats. In contrast with some previous findings, [30,31,32] interestingly, our investigations indicated a distinct negative association of Ae. aegypti particularly, and Ae. albopictus generally, with used tyres and discarded containers. Out of the 349 samples from discarded containers, only 2 and 14 containers were found positive with Ae. aegypti and Ae. albopictus respectively. However, we could not find any association of Ae. aegypti with used tyres. In disagreement with Cruz et al (2008), [30] and in agreement with Kittayapong et al (2006), [33] our investigations revealed that proper covering of water-holding containers make a direct impact on the reduction in the densities of both species. However, the use of larvicides in these habitats should also be a top most priority for better management of dengue vectors. During our interviews with the sampled households, we noticed that most of them did not know about the breeding of mosquitoes inside these water-holding containers and they had no idea about their control through washing, cleaning or proper covering of these containers and through the use of chemicals. We also noticed that due to the uncertainty of water supply, communities did not remove even small quantities of the water remaining in these containers before refilling them when there was water again; so, this remaining water contained eggs, larvae and pupae. Since Aedes mosquitoes require only a small quantity of water for breeding, once a water container becomes infected with Aedes mosquitoes, it remains infected till complete emptying and proper washing. Similarly, Aedes eggs can also stick to the coarse walls of these containers even when there is no or little water, and these eggs hatch later after refilling and, most probably, due to this reason, some covered containers were also found positive at the time of sampling. These findings further indicate the need of use of larvicides for proper control of dengue vectors breeding in these manmade habitats. 110 Dengue Bulletin Volume 35, 2011

13 Some researchers have reported the influence of some life-limiting factors of latitude, altitude, temperature, rainfall, humidity, etc., on the geographical distribution and densities of Aedes species. [4,34,35,36] Consistent with these findings we also recorded a discernible demarcation in the occurrence of both species in different geographical areas of Pakistan. In Karachi (southern area), which is located at 24 metres above sea level, only Ae. aegypti was the prevalent species whereas in the northern/submountainous areas ( metres above sea level with upper limit of 2500 metres), Ae. albopictus showed a significant dominance. However, in the central part of the country, Ae. aegypti showed a dominance while Ae. albopictus also showed reasonably high densities. In India, Ae. aegypti has been recorded in 1968 at an altitude of 2500 metres above sea level at Mcleodganj, western Himalayas. [37] In Pakistan, the submountainous areas are mostly covered with thick vegetation, while in Lahore, there is also a very large area covered by the Changa Manga National Forest Park. Chen et al (2006) [25] and Aslam Khan and Sulman (1969) [38] also reported a significant association of Ae. albopictus with thickly vegetated areas. Similar to the malaria vectors in Pakistan, overall, both the Aedes species exhibited a well-defined rising trend in their population in the post-monsoon season (September to November) in all selected areas. Li et al (1985) [39] revealed a positive correlation between a dengue outbreak and rainfall due to increased number of breeding habitats of Aedes vectors in Malaysia. However, we noticed that none of the indoor positive breeding habitats has any direct link with rainwater. In our study areas, particularly in Karachi, the water supply is very erratic, irregular and it is available for a very short period of minutes in a day without any fixed timings (sometimes on alternate days). Therefore, water has to be stored in drums, buckets, underground tanks, earthen pots, etc. All positive samples were collected from these water containers placed inside the houses. Similarly, the average rainfall in Karachi is also very low and the water pools in the streets become organically polluted and support the breeding of only Culex mosquitoes. Though in the submountainous region, the annual rainfall is high, even then all samples collected from the open-field habitats, including rainwater pools, were found negative, while all positive habitats were man-made inside the houses which have no link with rainwater. These findings indicate that there is no correlation between rain and number of Aedes breeding sites in Pakistan. However, the month-wise data of dengue cases showed a strikingly rising trend after the monsoon months (September November) parallel to the vector density data. During the study period, 95.53% of the dengue case-load was reported during these months, and there was a rapid decline in the cases during and after December, which indicated a positive correlation between the vector densities and the disease incidence. Our investigations reported high levels of HI, CI and BI. The high BI revealed a significant direct relationship between positive containers and houses and confirms a high transmission potential for dengue outbreaks in the study districts. [6,31] A very high dengue case-load in the selected districts (>80% of total case-load of the county) further confirms our findings. Dengue Bulletin Volume 35,

14 Conclusions and recommendations The present five-year entomological investigations of the dengue outbreaks during showed a distinctly high level of vector(s) infestation in the man-made shaded habitats in human dwellings in the districts covered by our study, particularly during September November, in parallel with the disease trends. Since there is a rising incidence of dengue/dhf in Pakistan, particularly since 2005, there is an urgent need: (i) to establish a separate Dengue Control Cell within the Ministry of Health as part of overall health system strengthening; (ii) for a mass health information and promotion campaign for the sensitization of local communities for better acceptance of intervention(s), particularly the use of personal protective measures and also to change their behaviour for employing improved water-storage practices like proper covering of water-holding containers, use of larvicides, symptoms recognition for prompt treatmentseeking, etc.; (iii) for establishing a functional intersectoral mechanism of coordination between all stakeholders for implementation of an integrated vector management approach; (iv) for sensitization of local authorities for regular water supply and proper solid waste management; and (v) for regular capacity building programmes. Operational research on insecticide resistance in dengue vector(s), characteristics of virus, vector(s) densities and bionomics between high- and low-affected areas, rural and urban areas, frequency of hostvector contact and disease epidemiology is also strongly recommended, which ultimately would lead to the development of an evidence-based, community-friendly and sustainable disease management strategy in the country. Acknowledgement We gratefully acknowledge the support received from the district health offices and district governments of the selected districts. The efforts for data management made by Mr Muazzam Abbas and Dr Mumtaz Ali Khan of the Epidemic Investigation Cell, National Institute of Health, are highly appreciated. We especially would like to thank Mr Muhammad Shafiq, Mr Imam Bukhsh Keerio, Mr Mukhtiar Ahmed Channa and Mr Mir Ali Talpur for their great dedication to facilitate our mission. The authors would also like to express their appreciation to the people of the Union Councils of these districts for their kind cooperation throughout the study. References World Health Organization. [1] Dengue and dengue haemorrhagic fever. Fact sheet no.117 March Geneva: WHO, accessed 12 January Dengue Bulletin Volume 35, 2011

15 [2] Foo LC, Lim TW, Lee HL, Fang R. Rainfall, abundance of Aedes aegypti and dengue infection in Selangor, Malaysia. Southeast Asian J Trop Med Public Health. 1985; 16: [3] [4] [5] [6] [7] [8] Hales S, de Wet N, Maindonald J, Woodward A. Potential effect of population and climate changes on global distribution of dengue fever: an empirical model. Lancet. 2002; 360 (9336): Hii YL, Rocklöv J, Ng N, Tang CS, Pang FY, Sauerborn R. Climate variability and increase in incidence and magnitude of dengue incidence in Singapore. Global Health Action Nov 11; 2. doi: / gha.v2i Bartley LM, Donnelly CA, Garnett GP. The seasonal pattern of dengue in endemic areas: mathematical models of mechanisms. Trans Roy Soc. Trop Med Hyg. 2002; 96(4): Reisen WK, Milby MM. Population dynamics of some Pakistan mosquitoes: changes in adult relative abundance over time and space. Ann Trop Med Parasitol. 1986; 80: Yang HM, Macoris MLG, Galvani KC, Andrighetti MTM, Wanderley DMV. Assessing the effects of temperature on the population of Aedes aegypti, the vector of dengue. Epidemiol Infect. 2009; 137: Eisenhut M, Schwarz TF, Hegenscheid B. Seroprevalence of dengue, chikungunya and Sindbis virus infections in German aid workers. Infection. 1999; 27(2): [9] Halstead SB. Global epidemiology of dengue hemorrhagic fever. Southeast Asian J Trop Med Public Health. 1990; 21: [10] Humaira Z, Dhodhy M, Hayyat A, Akhtar N, Rizwan F, Chaudhary B, Zareef S. Seroprevalence of dengue viral infection in healthy population residing in rural areas of district Rawalpindi. International J Pathology. 2010; 8(1): [11] Qureshi JA, Notta NJ, Salahuddin N, Zaman V, Khan JA. An epidemic of dengue fever in Karachi: associated clinical manifestations. J Pak Med Assoc. 1997; 47: [12] Ali N, Nadeem A, Anwar M, Tariq W, Chotani RA. Dengue fever in malaria endemic areas. J Coll Phy Surg Pak. 2006; 16: [13] Riaz MM, Mumtaz K, Khan MS, Patel J, Tariq M, Hilal H, Sadiqui AS, Shahzad F. Outbreak of dengue fever in Karachi 2006: A clinical perspective. J Pak Med Assoc. 2009; 59(6): [14] Gubler DJ. Aedes aegypti and Aedes aegypti borne disease control in the 1990s: top down or bottom up. Am J Trop Med Hyg. 1989; [15] Kittayapong P, Strickman D. Distribution of container-inhabiting Aedes larvae (Diptera: Culicidae) at a dengue focus in Thailand. J Med Entomol. 1993; 30: [16] Thavara U, Tawatsin A, Phan-Urai P, Ngamsuk W, Chansang C, Liu M, Li Z. Dengue vector mosquitoes at a tourist attraction, Ko Samui, in Southeast Asian J Trop Med Public Health. 1996; 27(1): [17] Sharma SK. Entomological investigations of DF/DHF outbreak in rural areas of Hissar District, Haryana, India. Dengue Bulletin. 1998; 22: [18] Strickman D, Kittayapong P. Dengue and its vectors in Thailand: introduction to the study and seasonal distribution of Aedes larvae. Am J Trop Med Hyg. 2002; 67(3): Government of Pakistan [19]. Federal Bureau of Statistics. Planning & Development Division II- Ministry of Economic Affairs and Statistics pp 102. Dengue Bulletin Volume 35,

16 [20] Government of Pakistan. Population Census Organization php - accessed 13 January [21] Leopoldo MR. Pictorial key for the identification of mosquitoes (Diptera: Culicidea) associated with dengue virus transmission. Zootaxa ; Pp 60. [22] Mukhtar M, Jeroen E, Wim van der Hoek, Felix PA, Konradsen F. Importance of waste stabilization ponds and wastewater irrigation in the generation of vector mosquitoes in Pakistan. J Med Entomol. 2006; 43(5): [23] Reisen WK, Siddiqui TF, Aslamkhan M, Malik GM. Larval interspecific associations and physicochemical relationships of the ground-water breeding mosquitoes of Lahore. Pak J. Sc. Research. 1981; 3: [24] Thavara U, Tawatsin A, Chansang C, Kongngamsuk W, Paosriwong S, Boon-Long J, Rongsriyam Y, Komalamisra N. Larval occurrence, oviposition behavior and biting activity of potential mosquito vectors of dengue on Samui Island, Thailand. J Vector Ecol. 2001; 26(2): [25] Chen CD, Seleena B, Nazni WA, Lee HL, Masri SM, Chiang YF, Azirum MS. Dengue vector surveillance in endemic areas in Kuala Lumpur City Center and Selangor State, Malaysia. Dengue Bulletin. 2006; 8: [26] Sulaiman S, Pawanchee ZA, Jeffert J, Ghauth I, Busparani V. Studies on the distribution and abundance of Aedes aegypti and Aedes albopictus (Diptera: Culicidae) in an endemic area of dengue/dengue haemorrhagic fever in Kuala Lumpur. Mosquito-Borne. Diseases Bulletin. 1991; 8 (2): [27] Abdalmagid MA, Alhusein SH. Entomological investigation of Aedes aegypti in Kassala and Elgadarief States, Sudan. Sudanese J. Public Health. 2008; 3 (2): [28] Isaacs N. Measuring Inter Epidemic Risk in Dengue Endemic Rural Area Using Aedes larval indices. Indian J. Com Medicine. 2006; 31(3): [29] Seng CM, Jute N. Breeding of Aedes aegypti (L.) and Aedes albopictus (Skuse) in urban housing of Sibu town, Sarawak. Southeast Asian J Trop Med Public Health. 1994; 25(3): [30] Cruz EI, Salazar FV, Porras E, Mercado R, Orais V, and Juancho, B. Entomological survey of dengue vectors as basis for developing vector control measures in Barangay Poblacion, Muntinlupa City, Philippines, Dengue Bulletin. 2008; 32: [31] Higa Y, Yen TN, Kawada H, Son TH, Hoa TN, Takagi M. Geographic Distribution of Aedes aegypti and Aedes albopictus Collected from Used Tires in Vietnam. J Am Mosq Cont Assoc. 2010; 26(1):1-9. [32] Tsuda, Y, Suwonkerd W, Chawprom S, Prajakwong S, Takagi M. Different spatial distribution of Aedes aegypti and Aedes albopictus along an urban-rural gradient and the relating environmental factors examined in three villages in northern Thailand. J Am Mosq Control Assoc. 2006; 22: [33] Kittayapong P, Uruyakorn C, Chitti C, Amaret B. Community participation and appropriate technologies for dengue vector control at transmission foci in Thailand. J Am Mosq Cont Assoc. 2006; 22(3): Ishak H, Miyagi I, Toma T, Kamimura K. Breeding habitats of [34] Aedes aegypti (L) and Aedes albopictus (Skuse) in villages of Barru, South Sulawesi, Indonesia. Southeast Asian J Trop Med Public Health. 1997; 28(4): Dengue Bulletin Volume 35, 2011

17 [35] Schultz GW. Seasonal abundance of dengue vectors in Manila, Republic of the Philippines. Southeast Asian J T Med Public Health. 1993; 24(2): [36] Suleman M, Khan K, Khan S. Ecology of mosquitoes in Peshawar valley and adjoining areas: species composition and relative abundance. Pak J Zool. 1993; 25(4): [37] Kalra NL, Wattal BL, Raghvan NGS. Distribution pattern of Aedes (Stegomyia) aegypti in India Some ecological considerations. Bull Ind Soc Mal Com Dis. 1968; 5(3) [38] Khan MA, Sulman C. The bionomics of the mosquitoes of the Changa Manga National Forest, West Pakistan. Pak. J. Zoology. 1969; 1(2): Li CF, Lim TW, Han LL, Fang R. Rainfall, abundance of [39] Aedes aegypti and dengue infection in Selangor, Malaysia. Southeast Asian J Trop Med Public Health. 1985; 16(4): Dengue Bulletin Volume 35,