The reduction of the infestation rates of the mosquito Aedes aegypti (Linnaeus, 1762) (Diptera: Culicidae), is a priority task of vector control programs in Cuba. The province of Santiago de Cuba has been severely hit in the last decades from the local transmission of dengue and more recently of zika virus. In this scenario, intensive control was carried out with mainly pyrethroid insecticides, but deltamethrin had never been used. Hence, the objective of this study was to determine the susceptibility status to deltamethrin as an alternative insecticide and the impact of two strategies (insecticides impregnated curtains and insecticides residual treatments) added to Ae. aegyptimosquito control program in Santiago de Cuba municipality was the objective of this study .

First, we determined the state of resistance /susceptibility to the insecticide deltamethrin in a strain of Ae. aegypti from Santiago de Cuba. Subsequently, the effectiveness of insecticides impregnated curtains with deltamethrin (ITC) and the insecticides residual treatment (IRT) with K-Othrine 250 WG was evaluated through pupal indexes. The container with the highest pupal production and most involved in the infestation by Ae aegypti were identified. .

The evaluated populations of Ae. aegypti from Santiago de Cuba were considered susceptible with 100% mortality at 24 hours. When assessing the effectiveness of ITC and IRT at resting sites with K-Othrine 250 WG, although there was no significant difference in any type of treatment  the use of IRT was the most effective with a reduction of pupal infestation by 87% (p = 0.89) while CP only reduced 71% (p = 0.09) and ITC 14.3% (p = 0.48) respectively. The containers with the highest pupal production were the low tanks, which together with the useful and not useful artificial containers are the most involved in the infestation by Ae. aegypti. The most effective option was the IRT in the study period.

The IRT added to the routine activities of the program is an option taking into account the entomological indicators, while the ITC should not stand as a control method to be used in Santiago de Cuba.

               Cuba has suffered several dengue epidemics, since 1981 when, the first major dengue hemorrhagic epidemic reported in the Americas was caused by dengue 2 (Guzmán et al, 1989), and later in 1997, when an outbreak was detected in Santiago de Cuba City related to the same serotype (Kouri et al., 1998). Afterwards there have been outbreaks and epidemics of greater or lesser magnitude (Peláez et al., 2004). Subsequent to 2004, transmissions have been reported from imported cases that have been controlled and eliminated; in 2006 an epidemic was reported that was also eliminated ().Active surveillance and established control actions have made it possible to detect the early presence of the virus and generate timely actions to deal with epidemic outbreaks (Guzmán et al., 2006). Gutiérrez et al 2017 in a study carried out in Cuba in a locality of Havana City with severalAe. aegypti mosquito populations. Showed the existence of vertical transmission of dengue viruses in these.

Control of the vector that transmits the dengue virus is the only method available for the disease prevention (TDR 2006). No specific medication is yet available for the treatment of the disease. Although potentially effective vaccines against the four dengue serotypes are being developed and tested, it will take some time before they are ready for public health use. Even at that time, they will only complement and not replace vector control measures (Ooi et al., 2006).

               Usually the Ae.aegypti control programs use several methods. These can be the coupling of chemical and biological larvicides in water tanks in-house with space fumigation with non-residual insecticides and residual insecticides application in mosquito resting sites, complemented with physical control by destruction of the deposits that constitute potential breeding sites.

They are also impregnated surfaces such as walls of all kinds, wood, formic, marble, glass, plastics among others, which not only act as a barrier to prevent the mosquitoes bite, but also, as a repellent, inhibitor or annihilator of vectors and materials impregnated with insecticides, particularly impregnated curtains with deltamethrin (WHO, 2005).

               In 1981, the current Ae. aegypti National Eradication Program in Cuba, which develops intensive control activities, is based on regular monitoring and destruction of breeding sites, use of larvicides in water tanks, non-residual insecticides and residuals to reduce populations of adult mosquitoes, the application of health legislation and educational campaigns.

Since then, the program has used different insecticides formulations with different chemical groups, and its use has increased yearly (Montada et al., 2005, Montada et al., 2012). The groups of pyrethroid insecticides have been used since 1986. However, neither residual deltamethrin nor permanently insecticide impregnated curtains with (deltamethrin) have been used.

               Santiago de Cuba City in 1997 was affected by a major dengue outbreak (Valdés et al., 1999). ). After 1996, there is sustained infestation by Ae. aegypti that in recent years has been one of the most important in the country. Although it is low compared to other endemic countries (between 0 and 2.4 of Index House, in some specific blocks it can be much higher, Toledo et al, 2011. Among the factors associated with this, is the possession of an average of 5 water deposits per household. Despite these efforts, in Santiago de Cuba City, the persistent infestation by Ae. aegypti represent one of the main health problems in the province. As a result of this situation, it was considered appropriate to test whether the use of insecticide-impregnated curtains and the residual application with deltamethrin, treatments added to routine control activities, resulted in an increase in effectiveness in terms of impact on the entomological index. The objective of this study is to determine the impact of two control strategies added to the Ae.aegypti Mosquito control program in Santiago de Cuba municipality, Cuba.

Materials And Methods

               The study had an experimental type design (community trial) by a completely randomized conglomerate. It consisted in the comparison of the effectiveness of the routine activities of Ae aegypti control program. (Control arm) with the insecticides impregnated curtains (ITC) and the insecticides residual treatment in Aedes aegyptiresting sites (IRT) added to these routine activities (intervention arms), in Santiago de Cuba City, between March 2011 and March 2012.

               This study was conducted in 8 urban areas of the Santiago de Cuba municipality.For this, all blocks that had an accumulated Breteau Index above 2, collected in routine entomological information records between January to December 2010 were included.

               Then the assignments to control, the intervention of ITC or IRT group were randomly. An initial pupal survey by vector control workers was conducted in March 2011, which was considered as a pre-intervention survey (T0). The pupal intervention surveys were scheduled with the same persons, in October 2011 at 6 months (T1) and in March 2012 at 12 months (T2) of the study started. Surveys were conducted in 20% of the universe selected at random, which corresponded to 23 blocks / study arm. As a defining requirement before selecting the areas for field study, a study of susceptibility level of Aedes aegypti population from Santiago de Cuba which was collected in immature stages (larvae and pupae) from a natural population from the different areas of Santiago de Cuba itself in March 2010 to achieve a representative strain was made before with deltamethrin impregnated papers supplies by WHO.The susceptibility bioassays in adults were carried out by the recommended procedure of the World Health Organization (WHO / VBC.81.805) WHO 1981

Effectiveness Analysis:

                For this analysis, the reduction of the pupal index per person in intervention areas and control was used as a measure of effectiveness. The reduction of pupal x person index in each of the arms was measured and compared before the implementation (March 2011), at 6 months (October 2011) and after one year (March 2012). The different indexes used in the study were as follow: Pupae x person = quantity pupae / amount of person housing

Pupal production (%) = No Pupae deposits / no. Total deposits with pupae

Pupae / deposit = Pupae collected same deposit / no. total deposits with pupae. The statistical program Epidat 3.0 and X² was applied to determine if there was a significant difference between the treatments. The containers with the highest pupal production were determined at the beginning of the study, at 6 months and at 12 months. In addition, the percentage difference of the pupal index between the stages (% pupal reduction) was calculated using the coefficient of percentage variation  (CVr ) . Ae. aegypti positivity by deposit at all stages, the number of pupae and the proportion of pupae per container was also computed.

Routine Ae aegypti Control Program (CPAe.). Control ARM.

               The vector control program in Santiago de Cuba is directed centrally by the MINSAP (Health Ministry), but at the Provincial and Municipal levels decisions are made according to the specific situation of each health area. Its activities are based on the periodic review of each dwelling (at this time every 24 days) for Aedes aegyptilarvae identification, destruction and larvicides addition (Abate) in water tanks. When a vector focus or a case of dengue is identified, it is fumigated inside the houses of the blocks in question and blocks adjacent to it.

ROUTINE Ae.aegypti CONTROL PROGRAM + INSECTICIDES IMPREGNATED CURTAINS (CPAe + ITC).

               In this arm the routine activities of the program were maintained and distributed up to a maximum of three curtains per dwelling. The ITC that were used contain in their polyester fibers the residual insecticide pyrethroid deltamethrin , which is added during its manufacture, and which maintains its presence two years after being used, so its re-impregnation is not required ( PermaNet®, company Vestergaard-Frandsen). It has UV protection. The ITCs were placed on internal doors, behind the beds, in closets without doors or on curtains already placed in the houses, where they had minimal contact with the direct rays of the sun. Although the maximum number of curtains distributed per dwelling was 3, they were also distributed in schools and labor centers.

ROUTINE Ae. aegypti CONTROL PROGRAM + INSECTICIDE RESIDUAL TREATMENT (CPAe + IRT). RESIDUAL ARM

               In the residual treatment arm, the routine activities of vector control program were maintained. The K-Othrine 250 WG (deltamethrin), supplied by Bayer Environmental Sciences co, was applied by spraying every 4 months with portable spray equipment (three times / year) (WHO 2007).The insecticide was applied in the intra-domestic areas (rest sites of Aedes adult, for example under the beds and inside the closets etc and peri domestic (on the surface of the water tanks and the walls near them where it usually rests the mosquitoes).

I.- Susceptibility and / or resistance of Aedes aegypti mosquito

            When evaluating the susceptibility with deltamethrin impregnated papers  0.05% in adults with Santiago de Cuba strain and Rockefeller insecticide-susceptible strain of the Caribbean, colonized during the 1930s and was provided by the CDC laboratory in San Juan, Puerto Rico, both showed a very similar behavior in terms of susceptibility with 100 % mortality.

II.- Effectiveness based on the indicator Pupas / person

The initial study period with ITC and IRT in the health areas began in March 2011. In this period, the pre-intervention pupae survey (T0) was conducted in all arms. These measurements showed that, in IRT, the pupae index per person was 0.015, while in the control program arm it was 0.007, in the selected one for curtains it was 0.007.

               However, at 6 months, corresponding analysis period from March 2011 to October 2011, in the 2 arms (ITC and IRT) there was reduction of pupae indicator per person. The residual  treatment arm showed a marked reduction in the values ​​of the pupae per person (PP) index with respect to the Control Program with a Coefficient of percentage variation  (CVr) of 80%, with p = 0.04 (Table 1 ).

Table 1. Relative variation coefficient CVr (%) for each arm in the study period

               In the case of malaria, Figueredo et al., 1998 conducted other studies to measure the residual potential of deltamethrin aqueous solution of (EW 25 mg ai / m²) in raffia curtains. The results showed that the residual power of deltamethrin in the curtains was high. The percentage of mortality was greater than 85% after 360 days and was reduced to approximately 50% at 420 days post treatment. In this regard, in the state of Khartoum, Sudan, they showed that curtains impregnated with 0.5; 1.0 or 1.5 g / m² of permethrin produced a 100% mortality within 24 hours of exposure (Elnaiem et al., 1999.)

Pupas / Persona Index:                      

               With residual treatment application in the post hematophagicsites the indicator of pupae per person decreased significantly. Although relatively low vector densities are reported, it is inferred that they are not sufficient to avoid epidemic outbreaks, since it has climatic conditions favorable to the vector and is available in areas of low popular consciousness of the masses to eliminate mosquito breeding sites in the community.

               The results of the present study differ from (Focks et al., 2000) when they determined that, with temperatures of 28 degrees Celsius, threshold levels were estimated that could range between approximately 0.5 and 1.5 pupae per person, for the dengue-endemic or dengue-susceptible areas in the Caribbean, Central America and Southeast Asia, these scientists found that they can range between 0.3 and> 60. With the exceptions of Cuba and Singapore, where efforts have been made in the reduction of foci and pupal indicators have been worked on, threshold levels of pupal index per person have not been estimated that indicate risk of dengue transmission. Practical advice is provided on the use of these methods for operational control projects (Focks et al., 2000).

               In Malindi, Kenya, Midega et al., 2006, they obtained higher average figures of pupae / person (7.61) and pupae / housing (18.12). Which were registered in the rural area. In Thailand, (Barbazan et al., 2008) obtained 2.3 and 0.8 Ae. aegypti pupae per person in rural and urban areas, respectively. Lezcano et al., 2008, in Corrientes City , Argentina, obtained 0.84 and 0.74 pupas per person, respectively.

Pupal productivity and deposits involved in the infestation by Ae. aegypti.

               In the present study the low tank is the most important container in Aedes aegypti pupae production and contributes to the highest positivity of this dengue vector , both in the rainy season and in the dry season, This results demonstrate the role that this type of deposit plays in the maintenance and pupae productivity of this species. In second place in importance were the artificial containers not useful and useful both in rainy and dry period. These artificial ones, which are usually found outside the dwellings that are not destroyed by the personnel of the program, show poor identification, destruction and elimination uring their inspection visits to the houses. In Colombia, Romero-Vivas, 2002 found that water storage tanks and drums constituted 79% of positive containers by Ae. aegypti larvae , which contributed 93% and 92% of the total production of fourth-stage larvae and pupal populations, respectively.

               These results coincide with a wide range of studies carried out by various researchers in Cuba and beyond. One of these studies was carried out by Bisett et al., 2006, in Playa municipality in Havana City , where the low tank is the most productive reservoir. These produced 74.1% of the total pupae, with another 19% found in several small containers. In another study carried out by the same author, in other areas of the mentioned municipality, they found that the largest number of pupae detected was in the low tanks (88.6%, 100% and 56.6% in each area); of which 90.9% were uncovered or only partially covered, while in the remaining areas small artificial deposits were the most frequent with 85.7%.

               In La Lisa municipality, Havana ity , Bisett et al., 2008 showed that the low tanks and the useful small containers turned out to be the most positive and produced the largest pupal productions in the study period. They also found a positive relationship between temperature, rainfall, number of habitats and high pupal production in large water storage containers, low tanks, elevated tanks and cisterns.

               In addition Marquetti et al., 2010 in la Lisa municipality Havana City and Cienfuegos municipality in the south central region of the country, found that, the low tank was the most positive container. In La Lisa the second corresponded to the cans, while in Cienfuegos it was the diverse group. In this last municipality , Carrazana et al., 2010 its study showed that the deposits with the highest Ae. aegypti positivity were the low tanks, the various ones and the larvitraps and the highest positivity was reported during August-November period.

               In Cuba it has been shown that infestation by Ae. aegypti presents a seasonal pattern conditioned by climatic fluctuations, and its trends show changes from one period to another; the highest rates of infestation correspond to the period of maximum energy potential and warmer and wetter conditions within the rainy and temperature ranges. (Orozco et al., 2010).

               In the case of the marked decrease in the pupal productivity in the residual arm and to a lesser extent in the curtain arm, it is that in addition to the actions of the program, other control strategies were intervened. Therefore, these results support pupal surveillance as an indicator to determine the most productive recipients in an area and thus focus control measures. However, under these conditions, the routine personnel of the program should be worked on to improve the sample taking, fundamentally of the immature stage of pupa.

March 2011 to October 2011

               There was also a slight reduction (CVr) of 28.5% in the index (PP) in the curtain arm with respect to the Control Program 6 months after the study began. This reduction was not significant (p = 0.6), with PP values ​​that varied from 0.007 before to 0.005 after the intervention (Graphic 1).

                    In the pupal survey of October 2011, in the program control arm remained unchanged, its values ​​remained at 0.007 pupae per person.

In the period from October 2011 to March 2012, in the residual arm it showed a smaller reduction than the previous period, only 33% of relative variation coefficient. These variations were not significant with p = 0.92. The values ​​ranged from 0.003 to 0.002 (PP).

                    The curtain arm showed an increased risk of pupae infestation by 20% (CVr -20%) and p = 0.31. Pupae values ​​per person in this arm increased from 0.005 in October 2011 to 0.006 in March 2012 (Graphic 1).

However, the Control Program arm had a 71% reduction. The PP index values ​​ranged from 0.007 to 0.002 in this period (Graphic 1). In general in this period, the treatments showed a p = 0.34 not significant.

                    When analyzing the study period that includes dates of March 2011 to March 2012, the IRT arm was the most effective because it had a pupae reduction infestation of 87% CVr, with p = 0.89. Followed by Control Program arm of 71% CVr with p = 0.09 and the curtain arm of 14.3% CVr, with p = 0.48 respectively.

III.- Pupal productivity and deposits more involved in infestation by Ae. aegypti.

               The carrying out of the pre-intervention pupae survey corresponding to March 2011 coincided with the dry season. During the inspection, a total of 13 positive pupae deposits were recorded, which were grouped into 6 categories, low tank, elevated tank, useful artificial, not useful, others and in some cases cistern. .

               The low tank with the highest positivity (11) stands out for its type of container and, as a consequence, the highest pupae production (84.6%) in the residual arm with 10 pupae and 100% pupae production, while the arm program (CPAe) had 14 positivity and 60.9 pupae percentage . In second place of productivity by type of deposit we have the artificial one useful in the control arm with 30.4%. Also the artificial useful constitutes the highest proportion of pupae per deposit with a value of 7.0 in the initial stage (T0) in the CPAe

               In October 2011, another pupae survey was conducted, which corresponded to the measurement of pupae indicators at 6 months of the initial survey that coincided with the rainy season. These surveys showed an increase in positive recipients with Ae. aegypti due to inadequate inspection of them, due to the high infestation rates of the dengue vector in the areas. During the dwellings inspection, a total of 125 positive Ae. aegypti deposits were detected, grouped into 6 categories (low tanks, elevated tanks, artificial useful, not useful and cisterns). In the analysis by branch control program had the highest positive pupae deposits 73 (58.4%) of the total and most pupae (115) for 67.6% of the total. By type of deposit, the low tank is again highlighted as the tank with the highest positivity (59) and the largest number of pupae (81) for 70.4% in the control program arm, followed by the residual with 11 positivity low tanks with an amount of 6 pupae and for 60.0% pupae. For the curtain arm with 17 positive low tanks, 21 pupae and 46.7% pupae production in descending order. The breeding site that reaches the second place in pupae productivity is the elevated tank in the residual with 40%. No less important, were the non-useful artificial ones that reached the third place in the productive containers of pupae and contributed at highest proportion of pupae per deposit 4,5 at 6 months (T1) in the control program. This increase in positive deposits was possibly due to those who were outside the houses, poorly covered and with an inadequate inspection of them.

               Six months after the trial start (T1) there was a greater positivity in the deposits and more pupae. In the residual  and curtain arms there was a marked reduction of the pupae productivity and in its more generators containers due to the influence of these added actions (IRT and (ITC) which did not turn out well for the control program arm, where only vector surveillance and control activities were carried out.

In March 2012, when the corresponding annual pupal survey was conducted, a marked increase in pupal productivity was detected per type of deposits in curtain and residual treatments arms. The curtain arm showed the highest percentage in the pupae stage with 48.8%, followed by the residual arm with 32.6% and finally the control program with 18.6% of productivity. This last arm reduced its figures from 67.6% in October (6 months study) to 18.6% of pupae in March 2012 (12 months) and in terms of the number of pupae per type of deposit, the tank category shows the highest values, 8.0 in artificial not useful, followed by the ITC arm with 5.0 in this type of deposit.

               In terms of productivity by type of deposit, the low tank is the main producer of pupae with 92.9% in the residual arm followed by a curtain with 52.4% and finally a control program with 50%. Second, by type of productive deposit, we have tanks that are added to the list of breeding sites that produce more pupae in this period.

I. Susceptibility and / or Resistance of Ae. aegypti

            When evaluating the deltamethrin 0.05% insecticide by susceptibility bioassays in adults with Santiago de Cuba and Rockefeller strains, both showed a very similar behavior in susceptibility.Susceptibility to deltamethrin in Ae. Aegypti adult populations is widely documented. The above is demonstrated in resistance studies conducted by Bisset et al., 2003 with 2 Ae. aegypti strains from Panama where total susceptibility to this pyrethroid was detected. Studies conducted in Cuba by Bisset 2014, when evaluating the temephos-resistant strain, SAN-F6 showed resistant to deltamethrin but was susceptible in the adult stage. In contrast, adults of  deltamethrin-resistant strain (SAN-F12) showed resistance to all the pyrethroids evaluated (deltamethrin, cypermethrin, and lambda-cyhalothrin). In addition Rodriguez et al 2017 in a study conducted to determine the diagnostic doses of 5 insecticides using the CDC method of bottles impregnated with insecticides showed similar results, based on WHO criteria. The temephos-resistant strain (SAN-F6) was susceptible (90–97% mortality) to deltamethrin and lambdacyhalothrin in both assays. The deltamethrin-resistant strain (SAN-F12) showed mortalities between 90% and 97%, indicating that resistance might be developing. These results coincide with those made by (Maestre and cols. 2010), in Colombia, where evaluated Ae. aegypti adult populations versus impregnated bottles, which showed susceptibility to malathion and deltamethrin, but not to the other chemical insecticides evaluated. Santacoloma et al., 2010, compared impregnated bottles and with insecticides impregnated papers methodology, obtaining susceptibility to deltamethrin without significant differences between the results obtained by boths techniques.

                Other studies carried out with 2 Ae. aegypti strains from Santiago de Cuba with different resting habitat, by Bisett et al., 2005,found high resistance to deltamethrin in Santiago de Cuba Ceiling strain compared to Santiago de Cuba wall strain. The results revealed that genetic polymorphism existed among the populations under study, which could have an implication in the ecology and epidemiology of the vector.

               Our results suggest that the probable cause of susceptibility found to deltamethrin is due to the fact thatAe.aegypti mosquito strain  was formed from larvae and pupae collected in all health areas in Santiago de Cuba municipality, which was established as a defining requirement for the beginning in our objectives in the field study, so that undoubtedly the population collected can be considered heterogeneous, a condition that can influence in the susceptibility results to any insecticide.

Effectiveness of treatments with (ITC) and (IRT) and Control Program for Surveillance and Vector Control.

 Residual treatment with deltamethrin

               The present study shows a positive impact in the reduction of Ae. aegypti pupal index , by applying residual treatment with deltamethrin ( K-Otrina 250 WG) in resting sites s added to the control program. Other study in Cuba carried out by Montada et al., 2012, who obtained a reduction to zero Ae. aegypti focality in the dwellings treated during the following three months after treatment with this same chemical formulation. According to the results of the present study, it is inferred that, the residual treatment in Ae. aegypti resting sites, meets the requirements according to the ecology of the vector, as it is applied on attractive surfaces where this mosquito species rests.

               However, the results obtained by Ansari and Razdan, 2003 differ from the study conducted. They measured the bioefficacy of deltamethrin in indoor residual spraying against Aedes genus vectors,in this case, the residual spraying with deltamethrin was not effective because Ae. aegypti and Ae. albopictus population in this area was not reduced. In addition, Rosilawati et al., 2005 in an investigation found a residual effect of six weeks, when evaluating the efficacy of spraying outside houses with deltamethrin against Ae. aegypti in an urban area of ​​Kuala Lumpur.

 Curtains Impregnated with Insecticides:

               The PermaNet curtains demonstrated in this study that they do not reduce the pupal indicators of Ae. aegypti. There were no significant differences between CPAe + ITC areas and CPAe alone  According to the results obtained it is inferred that, in Cuba, with Ae. aegypti mosquito densities relatively low there is no impact on the decline of this vector populations in dwellings. (Toledo et al., 2011) obtained similar results using this control variant in Guantánamo. Although in Venezuela, (Vanlerberghe et al., 2011) and in Thailand (Vanlerberghe et al.,2013),they found significant reductions in Ae. aegypti in the treated areas, with respect to the untreated ones of the same municipality, the curtain coverage treated in homes must be at least 50% to reduce 50 % infestation. by Ae aegypti .

1. The Santiago de Cuba strain collected during the study period was susceptible to deltamethrin through susceptibility and / or resistance tests, so it is inferred that its use in the control program is effective.

2. Low tanks are the containers of larger pupal productions. These attached to the useful and not useful artificial containers  are the most involved in the infestation by Ae. aegypti, which is why its monitoring should be a priority for the control program.

3. Curtains impregnated with insecticide are not a cost-effective option in an environment with a low Ae aegyptiinfestation in a control program with intensive activities, such as Santiago de Cuba.

4. The residual treatment in the rest sites with deltamethrin, added to the routine activities of the program is a cost-effective option taking into account the entomological indicators.