AN INVESTIGATION INTO IMPACT OF UNSPRAYED SURFACES ON MOSQUITO BEHAVIOUR AND MALARIA TRANSMISSION IN AN AREA UNDERGOING INDOOR RESIDUAL SPRAYING IN NORTHERN GHANA BY OTUBEA ANSAH MANTE 10395542 THIS THESIS IS SUBMITTED TO THE UNIVERSITY OF GHANA, LEGON IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF THE MPHIL ENTOMOLOGY DEGREE INSECT SCIENCE PROGRAMME* UNIVERSITY OF GHANA LEGON *JOINT INTERFACULTY INTERNATIONAL PROGRAMME FOR THE TRAINING OF ENTOMOLOGISTS IN WEST AFRICA. COLLABORATING FACILITIES OF SCIENCE AND COLLEGE OF AGRICULTURE AND CONSUMER SCIENCE JULY, 2014 University of Ghana http://ugspace.ug.edu.gh ii DECLARATION This thesis is the result of research work undertaken by Otubea Ansah Mante towards the reward of MPhil Entomology in the African Regional Postgraduate Programme in Insect Science (ARPPIS), University of Ghana, under the supervision of Dr. Maxwell Appawu and Dr. Samuel Dadzie. ________________________________ Otubea Ansah Mante (10395542) Student ______________________ _____________________ Dr. Maxwell Appawu Dr. Samuel Dadzie (Supervisor) (Supervisor) __________________________ Dr. Rosina Kyerematen (Coordinator) University of Ghana http://ugspace.ug.edu.gh iii DEDICATION This work is dedicated to my Almighty God, my family and my friends. University of Ghana http://ugspace.ug.edu.gh iv ACKNOWLEDGEMENTS I am grateful to God for His relentless support through these times. I am grateful to my supervisors, Dr Maxwell Appawu and Dr Samuel Dadzie of the Parasitology department of the Noguchi Memorial Institute for Medical Research (NMIMR) for always being ready to guide me and supervising my work. I thank the leadership and staff of the Abt-IRS project in Tamale and the Noguchi Memorial Institute for Medical Research (NMIMR) in Legon, Accra for making their facilities available to me throughout the period of this study. I am endlessly grateful especially to Mr. Sylvester Coleman of the Abt-IRS project for his input and technical advice. Also Ms. Sellase Pi-Bansa, Mr. Kwadwo Kyereme Frempong and the service personnel of the NMIMR for their support and contributions to this study. I thank the ProMPT project/USAID and Malaria Care project/USAID for funding parts of my MPhil education. Finally, thank you to my family and friends for the encouragement and support especially during the difficult times of this study. University of Ghana http://ugspace.ug.edu.gh v TABLE OF CONTENTS DECLARATION ....................................................................................................................... ii DEDICATION ...........................................................................................................................iii ACKNOWLEDGEMENTS ...................................................................................................... iv LIST OF TABLES ....................................................................................................................vii LIST OF FIGURES ................................................................................................................. viii LIST OF APPENDICES ........................................................................................................... ix LIST OF ABBREVIATIONS .................................................................................................... x ABSTRACT..............................................................................................................................xii CHAPTER ONE ...................................................................................................................... 1 GENERAL INTRODUCTION ............................................................................................... 1 1.0 Introduction ...................................................................................................................... 1 1.1 Rationale........................................................................................................................... 4 1.2 Objectives ......................................................................................................................... 6 1.2.1 Specific Objectives........................................................................................................ 6 CHAPTER TWO ..................................................................................................................... 7 LITERATURE REVIEW ....................................................................................................... 7 2.0 Introduction ...................................................................................................................... 7 2.1 History of Malaria ............................................................................................................ 7 2.2 Epidemiology of Malaria ................................................................................................. 7 2.3 The Parasite .................................................................................................................... 10 2.4 The Vector ...................................................................................................................... 14 2.6 Life Cycle of the Vector ................................................................................................. 24 2.7 Socio-Economic Impact of Malaria ............................................................................... 26 2.8 Malaria Control .............................................................................................................. 30 CHAPTER THREE ............................................................................................................... 38 MATERIALS AND METHOD ............................................................................................ 38 3.1 Study Area ...................................................................................................................... 38 3.2 Mosquito Collections ..................................................................................................... 41 3.2.1 Pyrethrum Spray Collections ................................................................................... 43 University of Ghana http://ugspace.ug.edu.gh vi 3.2.2 Exit Trap Catches .................................................................................................... 44 3.3 Processing of Mosquitoes............................................................................................... 47 3.3.1 Morphological Identification of mosquitoes ........................................................... 47 3.3.2 Molecular Identification of Mosquitoes .................................................................. 48 CHAPTER FOUR .................................................................................................................. 56 RESULTS ............................................................................................................................... 56 4.0 Vector Species Composition & Distribution .................................................................. 56 4.2 Indoor Resting Densities of Anopheles .......................................................................... 63 4.3 Movement of Anopheles Mosquitoes ............................................................................. 65 4.4 Mortality of Anopheles Mosquitoes in Exit Traps ......................................................... 67 4.5 Entomological Parameters.............................................................................................. 68 CHAPTER FIVE.................................................................................................................... 70 DISCUSSION ......................................................................................................................... 70 5.0 Discussion .......................................................................................................................... 70 CHAPTER SIX....................................................................................................................... 75 CONCLUSION ...................................................................................................................... 75 6.0 Conclusion.......................................................................................................................... 75 6.1 Limitations of the Study ..................................................................................................... 75 6.2 Recommendations .............................................................................................................. 76 REFERENCES ....................................................................................................................... 77 APPENDICES ........................................................................................................................ 96 Appendix I................................................................................................................................ 96 Appendix II .............................................................................................................................. 97 University of Ghana http://ugspace.ug.edu.gh vii LIST OF TABLES Table 1: Ribosomal DNA (rDNA) intergenic spacer species-diagnostic primers, their melting temperatures (Tm) and species represented (Scott et al., 1993). ....................... 49 Table 2: Alignment from the 3‟ to 5‟ end of the 561- 600 fragment of the IGS regions in M and S of the Anopheles gambiae s.s. complex. The arrow indicates the restriction site of the Hha I enzyme in An. gambiae s form............................................................. 50 Table 3: Species composition of Anopheles mosquitoes collected by the Pyrethrum Spray and Exit Trap methods from Tarikpaa and Kulaa in the Savelugu-Nanton and Tamale districts of Northern Ghana................................................................................ 58 Table 4: Species composition and abundance of Anopheles species collected by PSC and ETC in rooms with and without hanging clothes in an IRS (Tarikpaa) and a Non- IRS (Kulaa) areas in the Savelugu-Nanton and Tamale districts of Northern Ghana. ... 59 Table 5: Species composition and abundance of Anopheles species collected from ETC in rooms with and without hanging clothes in an IRS (Tarikpaa) and a Non-IRS (Kulaa) areas in the Savelugu-Nanton and Tamale districts of Northern Ghana......................... 60 Table 6: Species composition and abundance of indoor resting Anopheles species collected by PSC in rooms with and without hanging clothes in an IRS (Tarikpaa) and a Non-IRS (Kulaa) areas in the Savelugu-Nanton and Tamale districts of Northern Ghana ......................................................................................................................................... 61 Table 7: Indoor resting density and gonotrophic stages of An. gambiae s.s. and An. funestus collected from IRS (Tarikpaa) and Non-IRS (Kulaa) areas in Nothern Gana. . 64 Table 8: The abundance and gonotrophic stages of An. gambiae s .l. collected by the Exit trap from IRS (Tarikpaa) and Non-IRS Areas (Kulaa) in the Northern Ghana. ..... 66 Table 9: Mortality rates for exiting of An. gambiae s .l. from rooms in IRS (Tarikpaa) and non-IRS areas (Kulaa) in Northern Ghana ............................................................... 67 Table 10: Entomological parameters for An. gambiae and An. funestus collected from the IRS (Tarikpaa) and Non-IRS (Kulaa) areas .............................................................. 69 University of Ghana http://ugspace.ug.edu.gh viii LIST OF FIGURES Figure 1: World distribution of malaria ........................................................................... 9 Figure 2: Life Cycle of Malaria Parasite........................................................................ 12 Figure 3: World distribution of Anopheles mosquitoes Source: Kiszewksi et al., 2004. ......................................................................................................................................... 15 Figure 4: Differences in banding pattern between the wings of Anopheles gambiae complex (top) and Anopheles funestus complex (bottom).............................................. 21 Figure 5: Diagrams of An. gambiae showing the A) Palps and B) legs of An. gambiae ......................................................................................................................................... 22 Figure 6: Life Cycle of Anopheles Mosquito ................................................................. 25 Figure 7: Map of malaria burden and poverty levels ..................................................... 28 Figure 8: Map showing areas of mosquito collection .................................................... 40 Figure 9: Rainfall pattern for study districts .................................................................. 41 Figure 10: Mosquitoes being collected from white sheet after spraying rooms ............ 44 Figure 11: An exit trap fixed in a window ..................................................................... 45 Figure 12. Mosquitoes being picked from exit traps with aspirator into paper cups ..... 45 Figure 13: Illustration of the study design ..................................................................... 46 Figure 14: Illustration of 96 we ll micro titer plate ..................................................... 52 Figure 15: Microtiter plate showing positive colour change and negative and positive controls............................................................................................................................ 54 Figure 16: Molecular identification of An. gambiae s.s. from rooms with and without hanging clothes in Tarikpaa (IRS) and Kulaa (non IRS) areas ....................................... 62 University of Ghana http://ugspace.ug.edu.gh ix LIST OF APPENDICES Appendix I: WHO recommended insecticide for Indoor Residual Spraying against malaria vectors…………………………………………….....…………….…. 100 Appendix II: Preparations of solutions for ELISA tests...........………………. 101 University of Ghana http://ugspace.ug.edu.gh x LIST OF ABBREVIATIONS AFRO WHO African Regional Office AGA AngloGold Ashanti DDT Dichlorodiphenyltrichloroethane DNA Deoxyribonucleic Acid EDTA Disodium ethylene diamine tetra acetate. 2HsO EIR Entomological Inoculation Rate ETC Exit Trap Collections IPT Intermittent Preventive Treatment IRD Indoor Resting Densities IRS Indoor Residual Spraying ITN Insecticide Treated Nets LLINS Long Lasting Insecticide Treated Nets MAB Monoclonal Antibody MBR Man Biting Rate MICS Multiple Indicator Cluster Survey MPA Mosquito/Plasmodium Antigen NMCP National Malaria Control Program PBS Phosphate Buffered Saline PCR Polymerase Chain Reaction PMI President‟s Malaria Initiative PSC Pyrethrum Spray Collections RAPD Random Amplified Polymorphic DNA RDT Rapid Diagnostic Test RFLP Restriction Fragment Length Polymorphism University of Ghana http://ugspace.ug.edu.gh xi SDDH2O Sterile Double Distilled Water SEARO South East Asia Regional Office SNP Single Nucleotide Polymorphism SSA Sub-Sahara Africa WHO World Health Organization WHOPES WHO Pesticide Evaluation Scheme University of Ghana http://ugspace.ug.edu.gh xii ABSTRACT Indoor Residual Spraying (IRS) is one of the malaria vector control tools used in the control of malaria and has helped to reduce malaria transmission in many areas in Sub- Saharan Africa. This involves spraying the walls of rooms with insecticides so as to kill any mosquito that rest on the insecticide treated walls after feeding. However, recent discussions have been centered on the role unsprayed surfaces such as hanging cloths play in sprayed rooms in malaria transmission. In this study, the effect of hanged clothes on mosquito resting and exiting behaviour as well as on malaria transmission was investigated using mosquitoes captured by pyrethrum spray (PSC) and window exit trap (ETC) collections. The study was undertaken in Tarikpaa, in the Savelugu-Nanton district in Northern Ghana, an area undergoing Indoor Residual Spraying. Kulaa in the Tamale district of Northern Ghana was used as the control area with no IRS. Mosquitoes were sampled from August to December 2013. Anopheles gambiae s.l., Anopheles funestus, Anopheles nili and Anopheles rufipes were the Anopheles species collected during the period of the study making up 97.1%, 2.0%, 0.3% and 0.6% of the species respectively. Both An. coluzzi (M form) and S molecular forms occurred in all rooms in both areas. An. gambiae s.l. collected in rooms with hanging clothes in the IRS community (Tarikpaa) was lower (14.68%) compared to rooms with no hanging clothing (85.32%). The same was observed for the non-IRS community (Kulaa). A total of 18 and 100 Anopheles mosquitoes were collected from rooms with hanging clothes and rooms without hanging clothes in Tarikpaa respectively. The mean Indoor Resting Density (IRD) of Anopheles collected from rooms in the IRS area without hanging clothes was higher than that of rooms with hanging clothes (2.7 mosquitoes/room vs 0.36 mosquitoes/room). However, this difference was not significant (F (1, 12) = 2.078, P = 0.175). The same trend was observed for the unsprayed community, Kulaa. A Fed University of Ghana http://ugspace.ug.edu.gh xiii to Gravid ratio of 0.9:1 and 1:1 was estimated for mosquitoes collected exiting rooms with hanging clothes and without hanging clothes respectively. The sporozoite rate for both rooms with hanging clothes and rooms without hanging clothes in Tarikpaa was zero. The only positive mosquito samples were from the rooms without hanging clothes in the non-sprayed area (1.83%). An Entomological Inoculation Rate (EIR) of 0.0043 infective bites/man/night was estimated for An. gambiae collected from Kulaa. The fed: gravid ratio also indicated that more blood fed mosquitoes left the rooms immediately after feeding in the presence of insecticide on the walls. The number of Anopheles species collected by ETC from rooms with hanging clothes in the IRS area was significantly higher than in rooms without hanging clothes. The practice of hanging clothes in the area undergoing indoor residual spraying did not affect malaria transmission. Anopheles mosquitoes tend to exit rooms in the morning after feeding. This has implications for the effectiveness of the IRS in the area because many of the mosquitoes will not rest on the insecticide treated walls to be killed. University of Ghana http://ugspace.ug.edu.gh 1 CHAPTER ONE GENERAL INTRODUCTION 1.0 Introduction Control of malaria represents one of the world's greatest public health challenges, especially in sub-Saharan Africa where most of the disease occurs (Mabaso, 2004). Globally, an estimated 3.3 billion people were at risk of malaria in 2011, with populations living in Sub-Saharan Africa having the highest risk of acquiring malaria: approximately 80% of cases and 90% of deaths are estimated to occur in the WHO African Region, with children under five years of age and pregnant women most severely affected (World Malaria Report, 2012). Worldwide malaria led to 216 million clinical episodes, and 655,000 deaths in 2011 (World Malaria Report, 2011). In Ghana, the National Malaria Control reports over 4,154,261 cases and 3,259 deaths (NMCP Annual Report, 2011). These estimates render malaria the pre-eminent tropical parasitic disease and one of the top three killers among communicable disease (Breman, 2001, Breman et al., 2004). Malaria is an acute disease caused by the protozoa, Plasmodium species and transmitted through the bite of the female Anopheles mosquito. Mainly, five species of Plasmodium are incriminated to cause malaria in humans: P. vivax, P. malaraie, P. ovale, and P. falciparum. P. knowlesi (the relatively new parasite), which until recently was diagnosed as the more benign P. malaraie (Cox-Singh et al, 2007). P. falciparum has been identified as the most virulent strain causing about 800,000 deaths of malaria cases every year (John, 2010). In Africa, it accounts for 90-98% of all malaria cases. Several species of Anopheles are responsible for transmitting malaria and this varies according University of Ghana http://ugspace.ug.edu.gh 2 to location. Much variation can be observed between villages and also in different micro-ecological zones a few kilometers apart (Appawu et al., 2004). In Central Africa and even in Ghana, it is not rare to capture two to four different vectors (e.g. An. gambiae, An. funestus) during the same night (Fontenille and Lochouarn, 1999; Appawu et al., 2001). The disease is characterized mainly by fever, severe headache, joint aches, and also by respiratory distress, neurological disease and haemolysis. Malaria is an entirely preventable and treatable disease, provided the currently recommended interventions are properly implemented. Though these interventions have been proven to work in reducing malaria morbidity and mortality, they do not achieve significant progress as stand-alone projects. Most effective programs have adopted the approach of integrating a number of interventions. These include preventive interventions such as vector control through the use of Insecticide-Treated Nets (ITNs), Indoor Residual Spraying (IRS) and, in some specific settings, larval control, and chemoprevention for the most vulnerable populations, particularly pregnant women and infants. There are also treatment-based approaches such as confirmation of malaria diagnosis through microscopy or Rapid Diagnostic Tests (RDTs) for every suspected case, and timely treatment with appropriate antimalarial medicines. Indoor Residual Spraying (IRS) is one of the primary vector control interventions for reducing and interrupting malaria transmission and remains a powerful vector control tool (World Malaria Report, 2012). IRS is the application of long-acting chemical insecticides on the walls of all houses and domestic animal shelters in a given area, in order to kill the adult vector mosquitoes that land and rest on these surfaces. In 2011, 80 countries, including 38 in the African Region, recommended IRS for malaria control. In that year, 153 million people were protected by IRS worldwide, or 5% of the global University of Ghana http://ugspace.ug.edu.gh 3 population at risk. In the African Region, the proportion of the at-risk population that was protected rose from less than 5% in 2005 to 11% in 2010 and remained at that level in 2011, with 77 million people benefiting from the intervention (World Malaria Report, 2012). Different countries use different insecticides based on what works and what does not work with regards to the resistance status of the insecticide in that country. Depending on the kind of insecticide, the application is sprayed once annually, such as with Deltamethrim or Fendona or twice yearly such as with Ficam a carbamate insecticide (Reddy et al., 2011). The primary effects of IRS towards curtailing malaria transmission are to reduce the life span of vector mosquitoes so that they can no longer transmit malaria parasites from one person to another thus reducing transmission and also to reduce the density of the vector mosquitoes. Some insecticides used also repel mosquitoes and by so doing reduce the number of mosquitoes entering the sprayed room, and thus human-vector contact. Scientific evidence of IRS efficacy in reducing or interrupting malaria transmission in different epidemiological settings has been available since the 1940s and 1950s (Russel, 1955). Numerous studies have shown that IRS has substantially reduced infant and child mortality. In Africa, malaria eradication pilot projects were initiated from the 1950s to the 1970s in Benin, Burkina Faso, Burundi, Cameroon, Kenya, Liberia, Mada- gascar, Nigeria, Rwanda, Senegal, Uganda and the United Republic of Tanzania. These projects demonstrated that malaria was highly responsive to control by IRS with significant reduction of Anopheline vector mosquitoes and malaria, although in most cases, transmission could not be interrupted (Beales, 1989). IRS is an ideal tool when majority of vector population feeds and rests inside houses, where the vectors are University of Ghana http://ugspace.ug.edu.gh 4 susceptible to insecticide in use, where night sleeping of people is predominantly indoors, where prevailing structures are suitable for spraying and also in settlements furthest from health facilities. In Ghana, IRS began with the AngloGold Ashanti Malaria Control Programme in Obuasi in 2005. It has been shown to reduce malaria cases from about 12,000 in 2005 to about 4,000 in 2008. The Presidents‟ Malaria Initiative (PMI) of the United States of America in 2008 undertook IRS in five districts of the Northern Region; Tolon- Kumbungu, Savelugu-Nanton, Karaga, Gushiegu and West Mamprusi. Ghana has two main season which affects the pattern of the malaria disease in the country; the rainy and dry seasons. In the northern Ghana, there is a prolonged rainy season. In the south, two rainy seasons occur, from April to July and from September to November. 1.1 Rationale IRS as a tool in malaria control has been used since the 1931‟s in Kwazula-Natal a district in South Africa. A dramatic reduction in the number of malarial cases was observed after the first in the KwaZulu-Natal province indoor spraying with pyrethrum in 1932 (le Sueur et al., 1993). In Ghana, IRS in Obuasi records a decrease in malaria cases at the Edwin cade hospital in Obuasi from about 5800 in 2005 to 1000 in 2008 (AGA report, 2010). The premise of IRS is that the malaria causing mosquitoes, in Ghana, An. gambiae and An. funestus bite indoors and rest indoors. After biting, mosquitoes tend to rest on walls which in an IRS area has been sprayed with an insecticide. While resting on the walls, the insect picks a lethal dose of the insecticide and eventually dies. The resting surfaces for indoor resting mosquitoes range from sprayable structures such as walls, roofs, University of Ghana http://ugspace.ug.edu.gh 5 windows to unsprayable structures such as household objects (Nagpal et al., 2012) including hanged clothes. It has been observed that most people tend to hang clothes and also possess other materials in their rooms which are not sprayed. These hanging clothes are usually clothes that have been worn a number of times by residents and thus contain body odour which serve as attractants for mosquitoes. Chemicals from human body odour, sweat, and breath have been identified and proven to attract a variety of mosquito species (Cork and Park 1996, Healy and Copland 2000, Bernier et al., 2002, Healy et al., 2002) These hanging clothes and other unsprayed surfaces may serve as alternative resting places for mosquitoes even in the presence of the insecticide on the walls, thus avoiding the insecticides on the wall. This study aims to investigate the impact of unsprayed surfaces on mosquito behaviour and malaria transmission in an area undergoing indoor residual spraying. This investigation was done in an area undergoing Indoor Residual Spraying and an area where Indoor Residual Spraying has not been undertaken. University of Ghana http://ugspace.ug.edu.gh 6 1.2 Objectives To evaluate the impact of unsprayed surfaces on mosquito behaviour in an area with IRS activities 1.2.1 Specific Objectives  To determine the species composition and distribution of Anopheles mosquitoes collected resting from rooms with sprayed and unsprayed surfaces  To determine the room density of Anopheles mosquitoes in rooms with and without hanging clothes  To investigate movement of mosquitoes from rooms with sprayed and unsprayed surfaces  To determine the man biting rates of Anopheles mosquitoes in rooms with and without hanging clothes.  Determine the sporozoite rates of mosquitoes from the two study areas. University of Ghana http://ugspace.ug.edu.gh 7 CHAPTER TWO LITERATURE REVIEW 2.0 Introduction 2.1 History of Malaria Humans began to take notice of the malaria disease millions of years ago. It has been postulated to have been transferred from gorillas to humans (Lui et al., 2010). Mention is made of malaria as far back as 2700 BCE when the Chinese were drawing relationships between fever and enlarged spleens. However, in 400BC, Hippocrates known later as the father of medicine was the first to describe malaria fever and draw a close to precise relationship between malaria and stagnant water. He is reported to have said "Those who drink [stagnant water] have always large, stiff spleens and hard, thin, hot stomachs, while their shoulders, collarbones, and faces are emaciated; the fact is that their flesh dissolves to feed the spleen” (Lehrer, 1979). Malaria became an important and dominant disease when the number of deaths attributed to malaria became more or equal to the number of people killed in a war due to death from weapons. In those days, a lot was unknown about the disease. However, enough was known about its effects and in some cases were even used as biological weapons. 2.2 Epidemiology of Malaria Malaria epidemiology is concerned with the study of the patterns and factors that affect the distribution of malaria and also the use of these patterns to produce an effective control of the disease. Malaria is an acute disease of humans characterised by chills, University of Ghana http://ugspace.ug.edu.gh 8 fever, headache, anaemia etc. caused by the Plasmodium parasite through the bite of an infective female Anopheles mosquito. It is prevalent between latitude 450 N and 400 S around the world and is responsible for more deaths per year than any other arthropod- transmitted disease. (Foote and Cook, 1959) Globally, malaria is known as a dangerous disease that causes malaise, other health complications and also death amongst people of all ages, the highest number of deaths recorded in children. However, malaria has been eradicated in some parts of the world. The decline or ``natural recession'' of indigenous malaria in industrialized countries during the middle of the 19th century (Hackett, 1995) has been attributed to several factors, such as drainage of swampy areas, better animal husbandry, improved housing, greater availability of quinine, and general socioeconomic improvement. Indeed, the achievement of malaria eradication on the continent of Europe is viewed as a major success in the chequered history of global malaria eradication (Bruce-Chwatt; 1980, 1985). As seen in Figure 1, In Africa, malaria can be found in mainly in West and East Africa, the incidence being greater than 200 cases. In South and North Africa, malaria incidence is between 0 – 4 cases. Across 107 malaria-endemic countries, estimated incidence in 2004 totalled 402 million (range 350-500 million) cases, of which around 57% occurred in World Health Organization Africa Regional Office (AFRO) and 30% in World Health Organization South East Asia Regional Office (SEARO). This includes an estimated 311 million (range 270-400 million) cases of falciparum malaria, of which around 72% occurred in AFRO and 19% in SEARO. These numbers are around 6-fold higher than cases globally recorded in national health information systems, or around 17-fold higher for non-African countries. University of Ghana http://ugspace.ug.edu.gh 9 Figure 1: World distribution of malaria Source: http://www.cdc.gov/malaria/about/distribution.html 02/06/2014 In Ghana, malaria is endemic and stable and constitutes one of the leading causes of morbidity and mortality, especially among pregnant women and children under the age of five. The Ministry of Health (MOH) estimates that 3 to 3.5 million cases of suspected malaria are reported each year in public health facilities, representing 30-40% of outpatient attendance. Of this figure, over 900,000 are children under the age of five. Malaria also accounts for about 61% of hospital admissions of children under five years and 8% of admissions of pregnant women. It is estimated that malaria accounts for 22% of under-five mortality and 9% of maternal deaths (The President‟s Malaria Initiative, 2007). Ghana‟s efforts to control malaria date back to the pre-independence era, when various strategies were employed at different times. In 1999, the country adopted the Roll Back University of Ghana http://ugspace.ug.edu.gh 10 Malaria initiative and has since been implementing a combination of curative and preventive interventions. Ghana subscribed to the Abuja Accord of the year 2000, by African Heads of States, which sought to achieve 60% coverage of malaria interventions by the year 2005, focusing particularly on pregnant women and children under five in need of access to suitable and affordable combinations of personal and community protective and curative measures such as insecticide-treated mosquito nets (ITNs) and prompt, effective treatment for malaria. The Abuja Accord sought to ensure that at least 60% of all pregnant women at risk of malaria, especially those in their first pregnancies, have access to appropriate chemoprophylaxis or Intermittent Preventive Treatment (IPT) (DHS, 2008). 2.3 The Parasite Malaria is caused by the parasite, Plasmodium which belongs to the family Plasmodiidae, Order Haemospiridia, Class Aconoidasida, Phylum Apicomplexa, Subphylum Alveolata and Kingdom Chromoalveolata. There are several species of Plasmodium affecting a range of organisms including birds, lizards, monkeys and man. In man, five species of Plasmodium have been identified: P. falciparum, P. vivax, P. malariae, P. ovale, and in recent times, P. knowlesi. P. falciparum is known as the most virulent species; causes most of the severe cases of malaria. In Africa, P. falciparum is the most prevalent and the most virulent, but in most malaria endemic regions multiple sympatric species are found and co-infection within individual human hosts or the mosquito vector population is common (Gnémé et al., 2013). The effect of mixed species infections on clinical outcome has been described as both beneficial (Smith et al., 2001) and adverse (May et al., 2000). P. falciparum is found mostly in Sub-Saharan Africa. Malaria episodes caused by P. falciparum in Ghana accounts for 90-95% of University of Ghana http://ugspace.ug.edu.gh 11 total malaria cases (MICS, 2013). The distribution of P. vivax is concentrated in the Horn of Africa, covering Djibouti, Eritrea, Ethiopia, Somalia, and Sudan. It is estimated malaria cases in these areas annually can be attributed to P. vivax infections (Mendis et al., 2001). In eastern and southern Africa, P. vivax represents around 10% of malaria cases but 1% of cases in western and central Africa. In Ghana and sub-Saharan Africa in general, P. vivax malaria infections have not yet been detected. This can be attributed to the presence of the Duffy negative trait in people of West Africa. The Duffy negative trait is an inherited red cell phenotype that lacks the receptor for invasion of the human red cell by the merozoites of P. vivax (Sanger et al., 1955). P. malariae is known to cause a more benign form of malaria, also known as the quartan malaria parasite because paroxysms occur every three days. P. malariae is rare but still exists in Central and Western Africa. It is usually seen as mixed infections with P. falciparum. In Ghana, infections with P. malariae account for less than 10% of the malaria cases annually (MICS, 2013). P. ovale is found primarily in West Africa, though in recent years, P. ovale infections have been recorded in the Western Pacific and Asian mainland. In Ghana, the MICS survey reports of less than 2% of malaria infections annually are due to P. ovale. Initially, most P. knowlesi malaria infections were misdiagnosed as P. malariae; the early blood stages of P. knowlesi morphologically resemble P. falciparum; the mature blood stages and gametocytes are similar to those of P. malariae (Singh et al., 2004). P. knowlesi has been reported from South East Asian countries such as Malaysia, Thailand, Vietnam, Myanmar and Philippines. University of Ghana http://ugspace.ug.edu.gh 12 Figure 2: Life Cycle of Malaria Parasite Source: Aboagye-Antwi, F., 2010. Most parasites infect one or two hosts to complete its life cycle. The malaria parasite possesses an indirect or complex life cycle. The parasite infects both humans (the host) and the mosquito (the vector). The parasite changes morphologically throughout the various stages of its life cycle. When a mosquito infected with the Plasmodium parasites bite a human being, the parasites are introduced into man as sporozoites and invades the liver cells; hepatocytes. In the liver, the sporozoites undergo asexual reproduction, dividing into numerous cells, a process known as exo-erthrocytic shizogony. At this stage, the parasite is asymptomatic and will remain in the liver for about 7 to 10 days. In certain species, P. vivax and P. ovale, the sporozoites develop into the hypnozoites which can stay in the liver from weeks to years. The parasite now University of Ghana http://ugspace.ug.edu.gh 13 in the form of merozoites are released into the blood stream where they invade red blood cells. The invasion of red blood cells by the merozoites is by means of specific receptors on the erythrocyte membranes and ligands on the surface membrane (Fujioka and Aikawa, 2002), a process known as erythrocytic schizogony. Merozoites develop into trophozoites then to shizonts, which rupture releasing merozoites that infect more erythrocytes. Clinical symptoms are manifested at this stage (NIH, 2007). Concomitantly, small portions of the parasites differentiate from newly invaded merozoites into sexual forms, which are macrogametocyte (female) and microgametocyte (male) (Fujioka and Aikawa, 2002). During a blood meal, the mosquito ingests macrogametocyte and microgametocyte. In the gut of the mosquito, the microgamates exflagellate, forming eight haploid motile microgametes. One microgamete fuse with a macrogamete to form the zygote. Within 18–24 hours, the non-motile zygotes transform into motile ookinetes. The ookinetes cross two barriers: the peritrophic matrix and midgut epithelium. After traversing the midgut epithelium, the ookinete reaches the extracellular space between the midgut epithelium and the overlaying basal lamina, and transforms into an oocyst. Ten to 24 days after infection, depending on the Plasmodium species and ambient temperature, thousands of sporozoites are released into the hemocoel and the motile sporozoites invade the salivary gland epithelium (Fujioka and Aikawa, 2002). This process inside the mosquito is known as the sporogonic cycle (CDC, 2006). When an infected mosquito bites a susceptible vertebrate host, the Plasmodium life cycle begins again. University of Ghana http://ugspace.ug.edu.gh 14 2.4 The Vector Female mosquitoes belonging to the genus Anopheles transmit malaria parasites. Other mosquitoes, Aedes and Culex are responsible for transmitting other diseases. There are about 490 species of which only about 60-70 have been incriminated as vectors of malaria under natural conditions; and about 30-40 species are of major importance. (Fig 3) The Anopheles belong to the order Diptera, An. gambiae s.s. a member of the homonymous species complex comprising six recognized sibling species, is the most anthropophilic malaria vector worldwide (Coluzzi, 1992). Together with its sibling species An. arabiensis, they are responsible for more than three-quarters of all P. falciparum inoculations (Coluzzi, 1984, 1994). In Ghana, the major vectors are the An. gambiae species complex and the An. funestus complex. These species generally bite late in the night, are indoor resting, and are commonly found in the rural and peri-urban areas where socio-economic activities lead to the creation of breeding sites (GHS, 2008). The An. gambiae complex is made up of the An. gambiae s.s. An. merus, An. melas, An. quadriannulatus A., An. quadriannulatus B., An. arabiensis, and An. bwambae (Coetzee et al., 2000). In addition to the An. gambiae complex, large parts of Africa are also home to other dominant vector species, including An. funestus, An. nili and An. moucheti (Sinka et al., 2010) University of Ghana http://ugspace.ug.edu.gh 15 Figure 3: World distribution of Anopheles mosquitoes Source: Kiszewksi et al., 2004. University of Ghana http://ugspace.ug.edu.gh 16 2.4.1 Anopheles gambiae complex The complex consists of seven closely related, morphologically indistinguishable sibling species originally defined by polygene chromosome analysis (Fanello et al., 2002) but varying in their ecological, behavioural and vector competence (Djogbénou et al., 2010). These however, can occur in sympatry with each other (Lindsay, 1998; Coluzzi et al., 2002). 2.4.1.1 Anopheles gambiae sensu stricto (s.s.) An. gambiae s.s. is the most efficient vector of malaria. An. gambiae Giles sensu stricto (s.s.) indicate the existence, in West Africa, of five chromosomal forms designated Bamako, Bissau, Forest, Mopti and Savanna (Coluzzi et al., 1985). An. gambiae s.s. is usually found in sympatry with An. arabiensis, An. gambiae predominated in saturated environments, whilst An. arabiensis in sites subject to desiccation (Lindsay, 1998). Larval habitats are generally small, temporary, sunlit, clear and shallow fresh water pools (Giming, 2001). 2.4.1.2 Anopheles arabiensis An. arabiensis is described as zoophilic, exophagic and exophilic (Sinka et al., 2010), however the feeding and biting patterns of An. arabiensis varies depending on geographical location (Gilles and de Million, 1968). Larval habitats are similar to those of An. gambiae s.s. although An. arabiensis is able to utilize a greater variety of locations than An. gambiae s.s., including slow flowing, partially shaded streams (Giming, 2001). University of Ghana http://ugspace.ug.edu.gh 17 2.4.1.3 Anopheles melas/merus An. melas is largely anthropophilic, even in the presence of animals, this species readily feed on man (de Meillon, 1947). This species is a brackish water-breeding member of the An. gambiae complex that is distributed along the coast of West Africa and is a major malaria vector within its range (Deitz et al., 2012), Although it withstands very high salinity, it usually found breeding in saline waters which is the same or lower than seawater (Akogbeto, 1995). An. melas is not an effective carrier of the malaria parasite, in endemic malaria zones, observed sporozoite indices are at least ten times lower than that of An. gambiae s.s. (Bryan, 1983). 2.4.1.4 Anopheles bwambae This species is known only form the vicinity of Burunga hot springs in Bwamba Country, Toro District, Uganda, where it breeds in brackish water from geothermal springs and other halophilic mosquitoes such as Aedes albocephalus, Ae. natronius and Culex tenagius (White, 1985). An. bwambae can also be found in the Semiliki forest of Uganda, where the adult is found resting on the buttress bases of large trees, on fallen logs and sticks and loose dry leaves (White, 1985). An. bwambae is described largely as anthropophilic with endophilic tendencies, although in the absence of man, it tends to be zoophilic (White, 1985). 2.4.1.5 Anopheles quadriannulatus A/B An. quadriannulatus species A and B, found in southern Africa and Ethiopia, respectively, are considered medically unimportant: human malaria parasites have never been detected in wild caught An. quadriannulatus females (Coetzee et al., 2000). An. University of Ghana http://ugspace.ug.edu.gh 18 quadriannulatus A and B display characteristics that are believed to have existed in ancestral forms of the complex, i.e. standard chromosomal arrangements, disjointed distribution and adaptation to temperate climates (Coluzzi et al., 2002). An. quadriannulatus A and B are considered strictly zoophilic although recent laboratory and field studies report equal feeding preference for human and cattle (Pates et al., 2001; 2005; 2006). 2.4.2 Anopheles funestus An. funestus, in some cases, have a greater impact on malaria transmission even than An. gambiae (Gilles and de Million, 1968; Coetzee and Fontenille, 2004). An. funestus is anthropophilic; Charlwood et al. surmised that An. funestus may have been the first Anopheline species to specialize on biting humans; that their preferred larval sites (permanent water bodies in savannah-like environments) are likely to have been areas where humans first settled. An. funestus is a member of the funestus Subgroup (Garrous et al., 2005), which includes: An. aruni, An. confusus, An. funestus, An. parensis and An. vaneedeni. The members of this subgroup exhibit important variation in their biology and behaviour, especially in regard to malaria vectorial capacity and are only morphologically distinguishable during certain stages in their development (Gillies and de Meillon, 1968; Gillies and Coetzee, 1987; Coetzee and Fontenille, 2004). Only An. funestus is regarded as an important vector of malaria in this subgroup Coetzee and Fontenille, 2004), however Wilkes et al., 1996 recently showed by salivary gland dissection that An. rivulorum (from the An. funestus group) from Tanzania was infected with P. falciparum. University of Ghana http://ugspace.ug.edu.gh 19 2.5 Identification of members of Anopheles species complex Correct species identification is the starting point for studying the epidemiological role of vectors (Bortel et al; 2001). Species recognition are scientifically valid and meaningful if they refer to taxonomic units having biological significance, that is to species defined according to the concepts developed by evolutionary biologists and generally applicable to sexually reproducing organisms (Dobzhansky, 1970; Mayr, 1963; White, 1978). Morphologically distinct organism are thus easily identified with differences in morphology – larval and pupa anatomy, wing spots, legs and abdomen. Reproductive isolation may be acquired with or without morphological divergence (unpublished, 2005), i.e. species undetectable by morphological differences. Identification is mostly achieved using morphological criteria, but this fails when sibling species and species with overlapping morphological characters are involved (Van Bortel, W., 2001). A number of methods and markers have been established to distinguish between morphologically indistinct species including cytogenetic; crossing experiments, the study of chromosomal complement with particular reference to differential banding patterns of the polygene chromosomes from ovarian nurse cells of semi gravid of adult females (Coluzzi and Sabatini, 1967), isozyme analysis, classical genetic markers, mitochondrial DNA, ribosomal DNA (rDNA), microsatellite DNA and Random Amplified Polymorphic DNA (RAPD). 2.5.1 Morphological Identification University of Ghana http://ugspace.ug.edu.gh 20 Preliminary identification of species in the field is still largely based on the use of morphological features. Gillies and de Meillon, (1968) and Gillies and Coetzee, (1987) provide detailed information on the morphological identification of Anopheline mosquitoes. Unfortunately, the proliferation of species groups and complexes within many of the Anopheline taxa is rendering the use of morphological characteristics invalid for the identification of important malaria vectors (Beebe and Cooper, 2000). 2.5.1.2 Morphological Identification of Anopheles gambiae s. l and the Anopheles funestus group Gillies and De meillon, (1968) and Gillies and Coetzee, (1987) provide detailed information on the morphological identification of Anopheline mosquitoes. Anopheles gambiae s.l. and Anopheles funestus are identified morphological by their palps, wings, legs and abdomen. The general coloration of Anopheles gambiae s.l. is yellowish brown to grayish brown and could be darker depending on the area of collection. Females usually have three pale bands on palps. The apical band is broader enveloping the whole of the 5th and apex of the 4th segment. (Evans, 1938; Roberts and Janovy, Jr., 2000) The femora, tibia and 1st tarsal segment of the hind legs are speckled to variable degrees, while the 5 th tarsus is all black. The wings have pale scales that are creamy white and tinged with yellow. The 3rd dark area (preapical dark spot) on vein 1 with a pale interruption, sometimes fused with preceding pale area (Gilles and Coetzee, 1987) (Fig 4) The abdomen is mainly light brown in colour with hairs, but the eighth tergite usually has scales, which may extend to the seventh. University of Ghana http://ugspace.ug.edu.gh 21 Female An. funestus have three pale bands on palps with rather variable width that are generally narrow. The abdomen is usually dark brown devoid of scales. Legs are generally dark; tibia with small, apical, white spots. Wing markings of An. funestus species show the characteristic four pale spots on the costa (Hervey et al., 1998). Figure 4: Differences in banding pattern between the wings of Anopheles gambiae complex (top) and Anopheles funestus complex (bottom). Source: Professor Rolph Garms, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany. University of Ghana http://ugspace.ug.edu.gh 22 A) Figure 5: Diagrams of An. gambiae showing the A) Palps and B) legs of An. gambiae 2.5.2 Cuticular analysis for identification Interests in the cuticular lipids of insects has grown steadily in recent years encouraged partly by the discovery that these substances play a part in species recognition (Howard et al., 1982; Bonavita-Cougourdan et al., 1987). Carlson and Service, (1980) reports of using cuticular compounds to establish a significant difference between An. gambiae and An. arabiensis. 2.5.3 Molecular Identification This involves the use and analysis of DNA for identification. This has been found to be the most direct analysis of genetic material possible and provides an easy to use method (Post and Boakye, 1992). The use of DNA overcomes previous challenges to the identification of species. Some techniques involve specific parts or stage of the insect (karyotyping of polytene chromosomes, cytogenetic). Isozyme analysis also involves A) B) University of Ghana http://ugspace.ug.edu.gh 23 the samples being used fresh or kept frozen until analysis. Classical genetic markers involve targeting a specific genetic fragment using techniques such as single Nucleotide Polymorphism (SNPs), Single-strand conformation polymorphism (SSCP) and restriction fragment length polymorphism (RFLP). Other analysis involves the use of mitochondrial DNA, ribosomal DNA (using the intergenic spacer and internal transcribed spacers) as is used in Polymerase Chain Reaction (PCR)-based diagnostic tools that differentiate between cryptic taxa, microsatellite DNA and random amplified polymorphic DNA (RAPD) that have been used to distinguish between An. gambiae and An. arabiensis. 2.5.4 PCR Technology Polymerase Chain Reaction (PCR) is an in vitro technique that involves the exponential amplification of a specific DNA fragment of interest by repeating cycles of polymerase- mediated oligonucleotide primer extension (Mullis and Faloona, 1987; Saiki et al., 1988; Ochman et al., 1988; Innis et al., 1990) The development of polymerase chain reaction (PCR) has facilitated the diagnosis of number of viral infections. It possesses several obvious advantages, such high specificity, sensitivity, rapidity, etc. (Morita, 1994). PCR is based on the ability of DNA polymerase to synthesize exponential amounts of strands of target DNA complementary to the target strand using repetitive series of cycles. The reaction components, DNA template, DNA polymerase, oligonucleotide primers, deoxynucleotides, Taq polymerase and the reaction buffer are all included in a reaction mixture and the amplification reaction is carried out by simply cycling the temperature within the reaction tube (Saiki et al., 1988) University of Ghana http://ugspace.ug.edu.gh 24 One cycle of PCR is composed of three steps, that is, denaturing the template, cooling to allow annealing of primers to their complementary sequence and extension of the annealed oligonucleotide by the DNA polymerase when warmed. Denaturing the template is carried out by heating the reaction mixture to a temperature, 92-950C. The temperature is reduced (to between 50-550C) to allow the primers which are oriented at their 3‟ ends pointing towards each other to anneal to template (White et al., 1989). The temperature is raised to between 720C to allow the DNA polymerase to add nucleotides unto the annealed primers. This ends one cycle, temperature is increased again to 92-950C to repeat cycles. The accurateness and yield is influenced by numerous components of PCR, including the buffer conditions, the PCR cycling regime (i.e., temperature and duration of each step), and DNA polymerases. (Cha and Thilly, 1993). 2.6 Life Cycle of the Vector The mosquito‟s life cycle is in four stages; egg, larvae, pupae and adult. The mosquito thus undergoes a complete metamorphosis. The female mosquito needs a blood meal to develop its eggs. Prior to this, the female mates once in its lifetime. During which a store of sperms needed for further fertilisation is stored in the spermatheaca of the female‟s abdomen. When the eggs are developed, they are laid on the surface of water bodies. The Anopheles will oviposit in relatively clean water; hoof-prints and rain pools to streams, swamps, canals, rivers, ponds, lakes, rice fields, and sometimes even dirty water. (WHO Entomology Manual, 2013). Mosquitoes of different species and genera have different oviposition sites. A mosquito lives for 3 to 4 weeks. During these weeks, about 7 batches of eggs are laid. Each batch contains about 100 to 150 eggs. The eggs University of Ghana http://ugspace.ug.edu.gh 25 of the Anopheles which are boat-shaped and about 1mm long are laid singly with floats on each side of the egg. Eggs also possess a network of outer layer of chorion that holds a film of gas and contributes to the buoyancy of the egg, provides it with a part of its coloration and serves as a plastron when the egg is submerged (Hinton, 1967). In the tropics, viable eggs will hatch in 2-3 days into larvae but in cooler temperate regions, it may take 4 to7 days or longer to hatch (Service, 1980) and much longer in colder regions. Figure 6: Life Cycle of Anopheles Mosquito Source: J. Brunhes, 1998 The larvae lie just below the surface of the water and respire by lying parallel to the water surface to obtain supply of oxygen and also feed by filtering food particles from Adult Pupa Egg Larvae University of Ghana http://ugspace.ug.edu.gh 26 the water. When disturbed, the larvae move below into the water but immediately move back to the surface. There are four larval stages or instars. After 1 to 2 days, the first instar sheds its skin and becomes the second instar, followed by the third and fourth instars at further intervals of about two days each (WHO Entomology Manual, 2013). The larva remains in the fourth instar stage for 3 to 4 more days before changing to become a pupa. The total time spent in the larval stage is generally 8 to10 days (WHO Entomology Manual, 2013) at mean water temperatures of 25 to 280C. The pupa is comma shaped and bears respiratory trumpets. This stage is an inactive stage where prominent changes take place (pupa loses its larval features and begins to turn into an adult). Pupal development takes 2 to 3 days in the tropics. At the end of pupa life, the skin on the dorsal surface splits and the adult mosquito emerges. The adult waits awhile for its wings and body to dry and harden before it flies off. 2.7 Socio-Economic Impact of Malaria Malaria affects directly and indirectly the millennium development goals. Malaria affects six out of the eight of the goals; achieve universal primary education, promote gender equality and empower women, reduce child mortality, improve maternal health, combat HIV/AIDS, malaria and other disease and to ensure environmental sustainability. Currently malaria is confined to Africa, South America and Asia, prevalent in 110 countries and territories. Ankomah Asante and Asenso-Okyere reports in 2003 that in Sub-Saharan Africa (SSA), malaria is ranked second after HIV/AIDS accounting for 10.6% of the disease burden. Many other serious diseases predominantly found in poor countries clearly are a consequence of poverty, caused by inadequate sewage treatment, unsafe drinking water, poor hygiene, or substandard housing. University of Ghana http://ugspace.ug.edu.gh 27 Malaria, though, does not follow this pattern – its severity, and the difficulty in eradicating it, are determined mainly by climate and ecology (Gallup and Sachs, 1998). Sachs and Malaney (2002) have also observed that where malaria prospers most, human society have prospered least. Globally, malaria endemic regions, the regional and sub- regional zones, mark similar boundaries as the world‟s poorest countries as shown in Figure 7. University of Ghana http://ugspace.ug.edu.gh 28 Figure 7: Map of malaria burden and poverty levels Source: Sachs, 1999 University of Ghana http://ugspace.ug.edu.gh 29 Malarial countries are not only poor, but economic growth in malarial countries over the past quarter century has been dismal. Malaria, reduces economic growth rate by 1.3% in malaria and takes about 40% of public health expenditure in endemic countries (Gallup and Sachs, 2001). Growth of income per capita from 1965 to 1990 for countries with severe malaria has been 0.4% per year, while average growth for other countries has been 2.3%, over five times higher (Gallup and Sachs, 1998). The cost of malaria cannot be quantified only in terms of cost spent on the illness. The costs involved are both direct (covering the cost of payment for illness) and indirect (covering the effects of illness on family and the society). It is hard to evaluate the cost of lost opportunities of household members who help out a person with malaria. Most attempts to directly measure the lost work due to malaria (which ignore these problems) find small or no impacts (Chima and Mills, 1998). Direct and indirect costs of illness for malaria were less than 10% of the household income, but still significant when combined with the costs of other illnesses (Russell, 2004). The direct and indirect costs of malaria in Africa exceed US$2 billion a year (GHS, 2008). Iron deficiency anaemia per se has been shown to affect the cognitive skills of children as well as their cognitive abilities in later life (Pollit et al., 1989, and Lozoff et al., 1991). Malaria is a known cause of anaemia especially in children, though malaria alone cannot be credited with this; P. falciparum infection is believed to be a major contributory factor to the etiology of severe anaemia seen in malaria endemic areas (Koram, 2000). Incidence of severe anaemia and age-specific rates of anaemia strongly correlate with the intensity of P. falciparum transmission (Clark and Tomlison, 1949). The socioeconomic impact of malaria tends to be difficult to quantify, however, it cannot be overlooked. Malaria affects everyone irrespective of social or economic University of Ghana http://ugspace.ug.edu.gh 30 standing, thus well to do individuals and investors are affected by it. As long as malaria protection is imperfect and cumbersome, well-to-do foreign investors and tourists may stay away from malarial countries (Gallup and Sachs, 2008). In most African societies, the success of a man is measured by his ability to produce healthy children. In most communities in Northern Ghana for example, men are considered successful by the number of wives and most importantly, the number of children these wives have produced. Every year at least 30 million women in malarious areas of Africa become pregnant; most of these women live in areas of relatively stable malaria transmission (WHO, 2013). Malaria in pregnancy, however, in endemic areas of Ghana causes a myriad of problems including low birth weight, anaemia in mother and child, maternal deaths and poorer infant survival. As reported by Asante and Asenso-Okyere 2003, malaria contributes to the loss of productive time not only to the economically active patients but also the caretakers of sick children. The value of productive time lost to the households amounted to ¢75,681.21 (US$ 8.92) per case of malaria. The indirect cost represented 56.48% of the total cost of illness to the household. About 9 workdays were lost by economically active patients while more than 5 workdays were lost by their caretakers. School children also lost about four school days on the average due to the malaria illness (Asante and Asenso-Okyere, 2003). 2.8 Malaria Control Malaria control involves a group of curative and preventive measures employed to bring down the burden of the disease until they are no longer of a public health concern. Malaria control can be defined as reducing malaria morbidity and mortality to a locally University of Ghana http://ugspace.ug.edu.gh 31 acceptable level through deliberate efforts using the preventive and curative tools available today. These measures are usually interrelated and multipronged. Interventions for malaria control differ from country to country depending on the epidemiology and topography of the country and largely on logistics available. The control of malaria dates back to the period with Ronald Ross' demonstration in 1897 that mosquitoes transmit malarial parasites, efforts to control malaria were naturally focused on attacking the mosquito vector by various measures, mainly directed against the aquatic stages, for example, coating marshes with paraffin (to kill Anopheles mosquito larvae) and draining stagnant water. Although the results were spectacular in some areas, there was a growing realisation that effective control of malaria depended on other factors, including the availability of better drugs than quinine and a greater understanding of the epidemiology of the disease under various environmental conditions. With the discovery of Dichlorodiphenyltrichloroethane (DDT), an all-out effort was made to eradicate malaria by attacking adult mosquitoes. Eradication was achieved in a few countries such as Spain, Portugal, Yugoslavia, Hungary, Bulgaria, Romania, Poland, Italy and Netherlands. However, in most countries, eradication was not accomplished in, mainly because of inadequate health infrastructures, social and political factors. In more recent years, control efforts have been marred by drug-resistant parasites, marking the era of more effective drugs and drug combinations and the monitoring of resistance in these new drugs. Insecticide- impregnated bed nets have become the preferred method of vector control. Although the search for better tools continue today, the experience from the past emphasise the need to strengthen health systems to ensure that they are capable of delivering effective interventions and of assessing their effectiveness in controlling malaria. University of Ghana http://ugspace.ug.edu.gh 32 Currently, tools for malaria control available (known broadly as vector control), include the use of Long lasting Insecticide-Treated Nets, Indoor Residual Spraying, and source reduction by larviciding. Intermittent preventive treatment of malaria in infants, young children and those attending school, Intermittent preventive treatment for pregnant women and prompt access to effective treatment. These strategies are deployed with effective education and communication. 2.8.1 Long lasting Insecticide-Treated Nets (LLINs) Treating mosquito nets and other fabrics with insecticides makes them an effective method of reducing malaria attacks and deaths (Alonso et al., 1993; Lengeler, 1998). The premise for the effectiveness of LLIN use is that the vectors that cause malaria bite and rest indoors and also have peak biting times between dawn to dusk. Thus the use of ITNs at night serves as a barrier between the human host and the mosquito whilst the insecticide kills and repels the mosquito. LLINs available now are made from polyester, polyethylene and more currently polypropylene. The long lasting insecticide in mosquito nets are mainly pyrethroids; deltamethrin, permethrin and alpha-cypermethin. A Cochrane review concluded that ITNs reduce overall mortality by about 20% in Africa (range 14%-29%) and that, for every 1,000 children aged 1-59 months protected by ITNs, about six lives are saved each year. The review also concluded that ITNs reduce clinical episodes of uncomplicated malaria caused by P. falciparum and P. vivax infections by 50%, as well as reducing parasitaemia. For the effect of ITNs on malaria in pregnancy, Ter Kuile et al., (1999) reported that women in their first to third pregnancies living in insecticide treated bed net villages University of Ghana http://ugspace.ug.edu.gh 33 were significantly less likely to develop malaria parasitaemia compared to those living in control villages, and to become anaemic. The incidence of low birth weight and low birth weight combined with stillbirths, abortions and intra-uterine growth retardation were significantly reduced by 28% and 25% respectively. A study in The Gambia (D‟Alessandro et al., 1995) also indicates insecticide treated bed nets reduce morbidity and mortality in pregnant women. The study, carried out in an area of high malaria endemicity with seasonal transmission and parasite inoculation rate of 1-10 demonstrated that during the rainy season there were significantly fewer primigravidae with parasitaemia in villages with insecticide treated bed nets than in control villages even though there was no difference in the prevalence of severe anaemia or in the mean haemoglobin level. Thus insecticide treated bed net had an effect on malaria prevalence but not on severe anaemia which was demonstrated to be low during the dry season in women using insecticide bed nets than in control villages but no difference between the two villages during the rainy season. On the other hand, during the rainy season there were fewer preterm deliveries among women who used insecticide treated nets than women who used no nets. The mean birth weight of children born in villages with treated bed nets was 130g higher than that of children born in control villages and at the individual level. A study carried out in Ghana by Binka et al., (1996) indicated that the distribution of impregnated bednets was associated with an overall reduction in all- cause mortality of 17% in children aged 6 – 59 months and reduced malaria-specific mortality by 22%. Binka et al., also stated a 17% protective efficacy, although this was lower in other comparative studies carried out in other countries such as The Gambia (D‟Alessandro et al., 1995) and Kenya (Nevill et al., 1996) which reported 25% and 33% respectively. Recent studies have shown that such nets are a cheap and effective method of reducing University of Ghana http://ugspace.ug.edu.gh 34 man-vector contact and child morbidity (Curtis et al. 1992; Bermejo & Veeken, 1992) Though ITNs have been found to be effective, coverage must be high, it is suggested that children sleeping without nets may even receive fewer infective bites in a village where bed net coverage is high (Clarke et al., 2001) and nets used every night to ensure this effectiveness. Thus net distribution campaigns and projects should be followed by extensive, effective communicative messages. 2.8.2 Larviciding In recent years, larviciding has received more attention and focus as a response to growing concern with drug and insecticide resistance. ITNs and IRS are currently the most effective vector control options, however, these tools will not be optimally effective in areas where mosquitoes exhibit outdoor resting and biting behaviours, or where the widespread use of ITNs and IRS has controlled endophilic mosquitoes, but left a smaller, more intractable population of exophilic and exophagic mosquitoes (Bayoh et al., 2010, Lindblade et al., 2006). The sustainability of IRS and ITNs is further threatened by the appearance of pyrethroid resistance in some mosquito populations (Read, 2009). Larviciding forms a part of interventions on the breeding sites of malaria vectors by larval source management. Larval source reduction involves the regular application of chemical or biological agents to kill mosquito larvae in their aquatic habitats (WHO, 2012). Larviciding is usually used as a part of integrated vector control approaches and not as a stand-alone intervention. Although, if used alone has the potential to affect both indoor and outdoor resting mosquitoes as compared to IRS or ITNs (WHO, 2011), but larvicides, unlike IRS and ITNs, act on a single, non-transmitting stage in the mosquito University of Ghana http://ugspace.ug.edu.gh 35 lifecycle and can only impact disease by reducing vector abundance (Kouznetsov, 1977). Larvicides used range from contact poisons, stomach poisons to hormones, insect growth regulators and other biological agents. Currently, the recommended agent/insecticide for larviciding include Bacillus thuringiensis isrealensis (Bti), Chlorpyrifos, Diflubenzuron, Novaluron, Pyriproxyfen, Fenthion, Pirimiphos-methyl, Temephos, and Spinosad (WHOPES, 2013). These insecticides are available in different formulations. The appropriate agent/insecticide, right formulation, timing and quantity are based largely on the biology of the targeted species and the ecology of the water body. Larvicides can be applied from either the ground by truck, boat, and hand held devices or by aerial application. Larviciding is recommended to be used as a compliment to the other core interventions such as the use of ITNs or IRS and should never be used as a substitute for ITNs or IRS in areas with significant malaria risk. Also its use is normally restricted to areas where breeding sites are few, fixed and findable especially in urban communities. 2.8.3 Indoor Residual Spraying (IRS) Indoor Residual Spraying (IRS) is one of the primary vector control interventions for reducing and interrupting malaria transmission (World Malaria Report, 2012). Historically, IRS was largely responsible for the tremendous accomplishments of malaria programmes in Europe, Asia and the Americas that resulted in hundreds of millions of lives being saved between the 1940s and the 1980s. In recent years, pilot projects have demonstrated that malaria was highly responsive to control by IRS with insecticides (mainly DDT). Significant reductions in anopheline vectors and malaria were recorded but transmission could not be interrupted (Kouznetsov, 1977). University of Ghana http://ugspace.ug.edu.gh 36 IRS is a standardised, well-controlled method of regulating vectors of malaria. Indoor Residual Spraying (IRS) is the application of a long-lasting, residual insecticide to potential malaria vector resting surfaces such as internal walls, eaves, and ceilings of all houses or structures (including domestic animal shelters) where such malaria vectors might come into contact with the insecticide (WHO, 2013). IRS reduces mosquito life span, reduces mosquito density, indirectly reduces the number of mosquitoes biting and thus reduces malaria transmission. In 2011, 80 countries, including 38 in the African Region, recommended IRS for malaria control. In the same year, 153 million people were protected by IRS worldwide, or 5% of the global population at risk. In the African Region, the proportion of the at- risk population that was protected rose from less than 5% in 2005 to 11% in 2010 and remained at that level in 2011, with 77 million people benefiting from the intervention (World Malaria Report, 2012). IRS works on the premise that the Anopheles mosquitoes that come indoors to feed at night, after feeding on humans, is more likely to rest on a sprayed surface and pick up a lethal dose of insecticide, and die eventually. This prevents further bites by the infective mosquito and thus breaks transmission. However, effective IRS requires good coverage of all sprayable surfaces, epidemiological and entomological monitoring, and bioassays for insecticide susceptibility of the local vector populations. It also includes the selection and use of appropriate insecticides. Selection of insecticide should be made after information on the local transmission, ecology and epidemiology, e.g. length of transmission season, levels of transmission, nature of sprayable surfaces, susceptibility or otherwise of mosquitoes to insecticide obtained and analysed. The insecticide should be safe, efficacious, cost-effective, available and with acceptable levels of residual University of Ghana http://ugspace.ug.edu.gh 37 effect. Insecticides approved by the World Health Organization for use in IRS can be found in Appendix I. The spraying technique involves the use of pressure pumps fitted with nozzles to deliver the insecticide, at the recommended dosage, with the spray lance kept at 45 cm away from the wall surface, spraying should be done from roof to the floor using a downward motion with a spray discharge rate of 740 to 860 ml per minute. University of Ghana http://ugspace.ug.edu.gh 38 CHAPTER THREE MATERIALS AND METHOD 3.1 Study Area The study was carried out in Tarikpaa and Kulaa in the Savelugu-Nanton and Tamale districts of Northern region respectively (Figure 8). The Northern Region is the largest region in Ghana in terms of land mass, occupying an area of 70, 384 square kilometers with a population of 2,479,461. It is bordered on the north by the Upper East and Upper West Regions, on the south by the Volta and Brong Ahafo Region, the Republic of Togo to the east and Cote d‟Ivoire to the west. The topography of the Region is mainly flat except for areas such as Gambaga escarpment where the land rises to about a height of 400 to 450 meters above sea level. The vegetation cover is savannah with grass and sparsely distributed drought resistant trees such as the baobab and the Shea tree. The region has a tropical climate. Temperatures range from 14°C (59°F) at night to 40°C (104°F) during the day. The rainy season lasts for about 5 months from June to October. The Northern Region is also characterized by a cold dry season from November to February and a hot dry season from March to May. Tarikpaa is a rural community, located: (9°35ʹ11.30˝N, 0°54ʹ11.39˝W), which has been a beneficiary community of the PMI IRS program since 2008. The community was sprayed for four years with alphacypermethrin (a pyrethroid insecticide) from 2008 to 2011. In 2012 however the community was sprayed with an organophosphate insecticide (pirimiphos methyl CS formulation) due to the declining susceptibility of the predominant vector species in the Savelugu-Nanton district to pyrethroid insecticides. University of Ghana http://ugspace.ug.edu.gh 39 Kulaa, at (9°26ʹ56.32˝N, 0°43ʹ41.37˝W), on the other-hand has no history of indoor residual spraying as a malaria control intervention, and so served as control site for comparison. Though it is located within the Tamale municipality, Kulaa is a rural community with fairly similar characteristics as Tarikpaa. Figure 9 shows the average rainfall pattern of Savelugu-Nanton and Tamale districts for the year 2013. It also indicates peak rainy period between July and November. The average annual rainfall is 71mm and 76mm for Savelugu-Nanton and Tamale districts respectively. The study sites experience stable, endemic malaria transmission (MICS, 2013). A peak in transmission often follows the long rains. The communities have huts made of mud and roofs of thatch or roofing sheets. The huts selected for this study had roofs of thatch and no eaves. The compounds are made of several huts, joined into a wall that fully encloses the compound, usually representing one family. The huts are mostly round and sometimes square. Huts are smoothed over with a water-proof mixture. The people in the study site are mostly farmers. University of Ghana http://ugspace.ug.edu.gh 40 Figure 8: Map showing areas of mosquito collection University of Ghana http://ugspace.ug.edu.gh 41 Figure 9: Rainfall pattern for study districts 3.2 Mosquito Collections Mosquito collections were done using two methods for mosquito sampling: Pyrethrum Spray Collections (PSC) and Exit Trap Collections (ETC). PSC was used to collect indoor resting mosquitoes whilst ETC was used to collect mosquitoes that exit the rooms during the night. PSC can be used to determine directly the indoor resting density (the number of mosquitoes resting indoors during the day), the human-biting density (indirectly) and the seasonal changes in indoor resting densities. Exit Trap Collections can be used to determine the species that rest outdoors, the relative numbers of mosquitoes resting outdoors, seasonal changes in outdoor resting habits and any alteration in the relative numbers of mosquitoes resting outdoors following the application of insecticides in houses and other buildings. Collections were undertaken between August and December 2013. 0 20 40 60 80 100 120 140 160 180 R ai n fa ll/ m m Graph showing rainfall in Savelugu-Nanton and Tamale districts for 2013 Savelugu Nanton Tamale University of Ghana http://ugspace.ug.edu.gh 42 In each community, two (2) compounds were selected randomly. The distance between compounds were about 150-200m. In each compound, two rooms were used, one of the rooms were provided with a wardrobe into which all hanging clothes that would have otherwise been hanged on lines in the room were kept enclosed in the wardrobe throughout the period of the collections. The other room was left with clothes hanging as is normally done in the hut. Thus in each community, two compounds were chosen and two rooms in each compound were used. (Figure 13). The same design was used in the other community (Figure 13). In every month, two sessions of collections were carried out. In each session, four collections were made using both the PSC and ETs. This was done simultaneously for the two communities University of Ghana http://ugspace.ug.edu.gh 43 3.2.1 Pyrethrum Spray Collections Pyrethrum Spray Collections (PSC) involves using a pyrethrum space spray to knock down mosquitoes resting inside a house and collecting them on white sheets spread on the floor and other surfaces in the house. Using PSC, it is possible to collect all mosquitoes from a well-enclosed room. The collection allows for quantitative studies including measurement such as indoor resting densities and human biting densities (WHO, 2013). PSCs were done within the hours of 6.00 and 8.00 in the morning. After the inhabitants have left the room, the floor of the room was covered with white sheets. The doors were closed and the room sprayed with Raid® Insecticide containing pyrethroids (prallethrin and sumithrin). The rooms were vacated after spraying. After 10 minutes, the white sheets were carefully lifted and contents were all placed on one sheet. This sheet was then held at its four corners and brought outside. The mosquitoes from the sheets were picked with forceps and placed in petri dishes and sent to the laboratory. (Figure 10) These petri dishes were lined with damp cotton wool and filter paper to prevent the mosquitoes from drying up. The number of people who slept in the room the previous night was also recorded. Pyrethrum Spray Catches were carried out in all of the settings described (Figure 13). Collections made were stored in paper cups, labelled appropriately according to the date, time, method and place of collection and taken to the laboratory. In the laboratory, the number of catches were noted and species identified. University of Ghana http://ugspace.ug.edu.gh 44 Figure 10: Mosquitoes being collected from white sheet after spraying rooms 3.2.2 Exit Trap Catches Some mosquitoes tend to be endophagic but exophilic. Exit or window traps are commonly used to capture mosquitoes leaving houses (WHO, 2013). Thus mosquitoes obtained from traps are used to determine the species that bite indoors but rest outdoors, the effect of control measures such as indoor residual spraying on movement and feeding habits of the mosquitoes and effect of insecticides as shown by the numbers of dead mosquitoes collected and by mortality rate of those caught alive (WHO, 2013). The exit traps cages were fixed on the window in each of the rooms; with hanging clothes or without hanging clothes. Cages were fixed on the windows (Figure 11) in the evening between the hours of 1800 and 1900 prior to the PSC the next morning. Mosquitoes trapped in cages were removed with an aspirator in the morning and placed into paper cups covered with mesh and labelled appropriately (Figure 12). The paper cups were subsequently placed carefully in boxes and transported to the laboratory for processing. The cups were observed within a 24 hour period and mortalities were recorded. University of Ghana http://ugspace.ug.edu.gh 45 Figure 11: An exit trap fixed in a window Figure 12. Mosquitoes being picked from exit traps with aspirator into paper cups University of Ghana http://ugspace.ug.edu.gh 46 Figure 13: Illustration of the study design Compound 2 Compound 2 IRS Community (Tarikpa) Compound 1 Room 1 (T01) (Room without hanging) Room 4 (T04) (Room with hanging) Room 3 (T03) (Room without hanging) Room 2 (T02) (Room with hanging) PSC ET PSC ET PSC ET PSC ET Compound 1 Room 1 (K01) (Room without hanging) Room 4 (K04) (Room with hanging) Room 2 (K02) (Room with hanging) Room 3 (K03) (Room without hanging) PSC ET PSC ET PSC ET PSC ET Non-IRS Community (Kulaa) University of Ghana http://ugspace.ug.edu.gh 47 3.3 Processing of Mosquitoes Mosquitoes obtained from Pyrethrum Spray Catches and those captured with Exit Traps were sorted out morphologically into various genera (Gillies and De Meillon, 1968). The physiological states of the abdomen were classified as unfed, blood fed, half gravid and gravid. Unfed females in which the abdomen was completely empty (without blood), narrow and collapsed. Freshly-fed females in which at least the first six abdominal segments, viewed ventrally, were filled with red blood. Half gravid females in which the first three to five abdominal segments were filled with dark-coloured blood at various stages of digestion. Gravid females in which the blood was completely digested or remained narrow blackish line in the first abdominal segment and the abdomen was distended with eggs. The mosquitoes after morphological identification were placed in 1.5µl microcentrifuge tubes and appropriately labelled with species of mosquitoes, place, date, and method of collection. These tubes were kept in zip lock bags containing silica gel for further analysis. 3.3.1 Morphological Identification of mosquitoes With the aid of a light microscope and identification keys, (Gillies and Coetzee, 1987; Gillies and de Mellion, 1968) the mosquitoes were morphologically identified. Culex species was identified by its brownish, dull coloured appearance, short palps and the absence of patches or spots on legs and wings. Aedes species possess distinctly black and white rings, on legs and abdomen. An. gambiae had pale patches, 3 bands on palps and wings with 3rd main dark area of vein 1 with pale interruption, sometimes fused with University of Ghana http://ugspace.ug.edu.gh 48 preceding pale spots. An. funestus was identified as mosquitoes with no pale interruption on 3rd main dark area of vein 1 on wings, palps with less than 4 bands and pale at apex and legs without pale patches. An. rufipes were identified as mosquitoes with hind tarsal segments 4 and 5 entirely white; 3rd main dark area on 1st vein of wings with a pale interruption, or with a short extension of the subcostal pale spot into the dark area on first vein. An. nilli had palps with pale apex and no other pale band on palp. 3.3.2 Molecular Identification of Mosquitoes 3.3.2.1 Polymerase Chain Reaction 3.3.2.1.1 DNA Extraction Using Cochran‟s formula (Bartlett et al., 2001) for sample size determination, mosquitoes were selected to be processed for DNA extraction. The legs of the selected desiccated mosquitoes were removed into 1.5µl microcentrifuge tubes. 15µl of double distilled water (ddH2O) was placed in a tube per leg. The legs were ground with a plastic pestle and homogenate placed in a water bath at 900C for 15min. After 15min, homogenate was removed, centrifuged for 5min at 16000g and used immediately or stored at -400C. 3.3.2.1.2 Amplification of Ribosomal DNA The amplification followed the protocol used by Scott et al., (1993) and oligonucleotide primers universal (UN), gambiae (GA), merus/melas (ME) and arabiensis (AR) Table 1 shows the oligonucleotide sequences in the 5‟-3‟ direction and their melting temperatures. The PCR reaction mix of 20μl contained 1X PCR buffer, 2µl (Invitrogen, USA), 1.5 mM MgCl2 (Promega, USA), 0.25 mM each of the 4 deoxyribonucleotide triphosphates (dATP, University of Ghana http://ugspace.ug.edu.gh 49 dTTP, dGTP and dCTP), 0.15μM of oligonucleotide primers, 0.5µ of Taq polymerase enzyme (Promega, USA) and 3μl of the extracted DNA supernatant. Sterile double distilled water (sddH2O) was added to make up the volume to 20μl. Table 1: Ribosomal DNA (rDNA) intergenic spacer species-diagnostic primers, their melting temperatures (Tm) and species represented (Scott et al., 1993). The amplification reaction was carried out with a PTC 100 thermo cycler (MJ Research Inc, USA). The PCR was carried out with a program of initial denaturation at 94°C for 3min, followed by 33 cycles of, 94ºC for 30 sec, annealing at 50°C for 30 sec, and extension at 72°C for 30 sec. Thermo cycler was also set to store at 4°C until removed. 7µl of each PCR product was then mixed with 1µl Orange-G (5x) gel loading dye and electrophoresed through a 2% agarose Tris-borate-EDTA gel containing 0.5µg/ml of Ethidium bromide in a 1x TAE buffer. The amplified fragments were visualized with short wave ultraviolet light. The results were photographed with a UV illuminator Sony Toyobo Primer Sequence (5’-3’) Melting Temp (TmºC) Size (bp) Species UN GTG TGC CCC TTC CTC GAT 58.3 468 - GA CTG GTT TGG TCG GCA CGT TT 59.3 390 An. gambiae s.s. ME TGA CCA ACC CAC TCC CTT GA 57.2 464 An. melas/merus AR AAG TGT CCT TCT CCA TCC TA 47.4 315 An. arabiensis University of Ghana http://ugspace.ug.edu.gh 50 FASAII. The size of each sample was estimated by comparison with a standard 100bp molecular weight ladder (Promega, USA) and results recorded. 3.3.2.2 Identification of An. gambiae M and S Forms The PCR amplification of a 1.3 kb rDNA fragment is followed by digestion with restriction enzymes Hha I and a specific pattern is obtained for each form (Favia et al., 1997). This was done using the protocol of Fanello et al., 2002 and on the principle that the restriction site for the enzyme Hha1 lies within the An. gambiae-specific fragment (Scott et al., 1993). After the amplification, a master mix of 1.5µl of 10x enzyme buffer, 0.15µl of 100x Bovine Serum Albumin (BSA), 0.1µl of Hha1 (10µ/µl) and sddH2O to make up a volume of 10µl. 5µl of the master mix was added directly to the PCR product, centrifuges and incubated at a temperature of 37oC for 5 hours. Table 2: Alignment from the 3‟ to 5‟ end of the 561- 600 fragment of the IGS regions in M and S of the Anopheles gambiae s.s. complex. The arrow indicates the restriction site of the Hha I enzyme in An. gambiae s form. Molecular Forms Sequence An. gambiae S form GTGCCCCTTCCTCGATGGCGCAACGAACCATCTTGGTCTG An. gambiae M form GTGCCCCTTCCTCGATGGCATAACGAACCATCTTGGTCTG University of Ghana http://ugspace.ug.edu.gh 51 After digestion, fragments were run through a 2% Ethidium bromide agarose gel and photographed under ultraviolet light illumination. The An. gambiae S-form digestion profile was characterized by two fragments, 257and110bp long, which are due to the presence of the Hha I restriction site. The An. gambiae M form does not have this restriction site and thus is characterized by a single 367bp fragment (Fanello et al., 2002). 3.4 Sporozoite Detection by Enzyme Linked Immunosorbent Assay (ELISA) The head and thorax of each sample were placed into a 1.5μl microcentrifuge tube and appropriately labeled. The tube was filled with 50μl of Grinding Solution. The sample is ground with a plastic pestle to obtain a homogenous solution. Grinding solution was used to wash pestle to obtain a 200μl of the Mosquito/Plasmodium Antigen (MPA) suspension. This was stored at -400C until ready to use. A plate map bearing Identification number of samples were recorded on a plate map as indicated in Figure 14. Using the protocol of Wirtz et al., 1987, the 96 well polyvinyl chloride, u-shaped micro titration plate was coated with a capture monoclonal antibody (MAb) at a coat rate of 0.1μg/50μl/well. Plate left overnight at 40C. The following solutions were prepared for the ELISA: Phosphate Buffered Saline (PBS), Grinding solution, blocking buffer and washing buffer. The protocols for these are attached as Appendix II. Plate was flipped empty and banged on paper towels. The remaining sites for protein binding on the micro titer plate was saturated with blocking buffer 200μl per well and incubated for an hour at room temperature. After an hour, plates were flipped empty, banged and washed twice with the washing buffer (PBS/Tw-20). Mosquito extracts and controls were loaded and incubated at University of Ghana http://ugspace.ug.edu.gh 52 50μl/well at room temperature for two hours. Plates were flipped and washed with washing buffer three times. The second labeled antibody, MAb-peroxidase conjugate (0.05μg/50μl/well) in blocking buffer was incubated for an hour in darkness. Plates were flipped empty, washed and banged three times. 100μl/well of substrate was incubated at room temperature for 50min. Plates were read between 30 to 60 min. Plates with change in color were recorded on plate map as positive whilst the others were recorded as negative. 1 2 3 4 5 6 7 8 9 10 11 12 Figure 14: Illustration of 96 well micro titer plate sam-sample +ve – positive control -ve – negative control BLK - block A sam sam sam sam sam sam sam sam sam sam sam sam B sam sam sam sam sam sam sam sam sam sam sam sam C sam sam sam sam sam sam sam sam sam sam sam sam D sam sam sam sam sam sam sam sam sam sam sam sam E sam sam sam sam sam sam sam sam sam sam sam sam F sam sam sam sam sam sam sam sam sam sam +ve +ve G sam sam sam sam sam sam sam sam sam sam BLK BLK H sam sam sam sam sam sam sam sam sam sam -ve -ve University of Ghana http://ugspace.ug.edu.gh 53 University of Ghana http://ugspace.ug.edu.gh 54 Figure 15: Microtiter plate showing positive colour change and negative and positive controls University of Ghana http://ugspace.ug.edu.gh 55 3.5 Data Analysis Completed forms were organized into date, site and capture method. These forms were entered into database fields using Ms Excel datasheets for use in statistical analysis. Data was transported into IBM SPSS (Version 12) Analysis of Variance will be used to test for significant differences between densities of mosquitoes from PSC obtained in rooms without hanging clothes and rooms with hanging clothes. Statistically significant differences were based on comparisons that yielded P values <0.05 and 95% confidence interval. The Man Biting Rate (MBR) was obtained from an index of the indoor resting blood fed females per inhabitant. The Entomological Inoculation Rate (EIR) is a measure of the intensity of malaria transmission, it is an indirect measure of the number of infective bites of the mosquito per man per night. This was determined by the multiplication of the sporozoite rate and the MBR. The assumption made here is that the vector is predominantly endophagic and endophilic in behaviour (Garrett-Jones and Shidrawi, 1969). The sporozoite rate was calculated as the number of mosquito samples positives for P. falciparum obtained from ELISA tests divided by the total number of mosquito samples examined. University of Ghana http://ugspace.ug.edu.gh 56 CHAPTER FOUR RESULTS 4.0 Vector Species Composition and Distribution Anopheles gambiae s.l., An. funestus, An. nili and An. rufipes were the Anopheles mosquito species collected during the period of the study using both the Pyrethrum Spray Collection (PSC) and Exit trap Collections (ETC) methods. An. gambiae s.l. was the predominant vector species collected from all the sites making up about (642/661) 97.1% of the total number of Anopheles species (Table 3). An. funestus, An. nili and An. rufipes made up (13/661) 2.0%, (2/661) 0.3% and (4/661) 0.6% respectively of the total Anopheles species. The majority of the mosquitoes collected in all the study areas were obtained from Exit Trap Collections (569/661) 86.1% compared to PSC (92/661) 13.9% Table 3. The largest number of all Anopheles species collected were from the non-IRS area, 82% (543/661) compared to IRS area, 23.7% (118/661) Table 4. An. gambiae s.l. was collected in high numbers in all the months of the study period whilst An. funestus was collected towards the end of the rainy season, October and November. An. gambiae s.l. collected in rooms with hanging clothes in the IRS community (Tarikpaa) was lower, 14.68% compared to rooms with no hanging clothing, 85.32% (Table 4). The same was observed for the non-IRS community (Kulaa) where An. gambiae from rooms with hanging clothes was 18.3% compared to 81.61% for rooms with no hanging clothes. The number of Anopheles species collected by ETC from rooms without hanging clothes in the IRS area was significantly higher (P<0.05) 15, (79.2%) than in rooms with hanging clothes, 57, (20.8%) Table 5. The same trend was observed in the non-IRS area, although much higher numbers were collected than non-IRS area (Table 5). The number of indoor University of Ghana http://ugspace.ug.edu.gh 57 resting Anopheles collected by PSC also indicated that less mosquitoes were resting in rooms with hanging clothes, 5 (11.1%) compared to rooms without hanging clothes, 40 (88.9%) in the IRS area (Table 6). However, in the non-IRS area more mosquitoes were collected in rooms with hanging clothes than from rooms without hanging clothes although the difference was not significant. The mean number of An. gambiae obtained from Tarikpaa for rooms with no hanging clothes was not statistically different from rooms with hanging clothes. (F (1, 12) =3.981; p= 0.072). In Kulaa, also, difference between the mean numbers obtained from rooms with no hanging clothes was not statistically different from those from rooms with hanging clothes. University of Ghana http://ugspace.ug.edu.gh 58 Table 3: Species composition of Anopheles mosquitoes collected by the Pyrethrum Spray and Exit Trap methods from Tarikpaa and Kulaa in the Savelugu-Nanton and Tamale districts of Northern Ghana. Anopheles species Pyrethrum Spray Collections Exit Trap Collections Total No. of Anopheles collected by PSC & ET Tarikpaa (IRS) Kulaa (Non IRS) Total No. (%) Tarikpaa (IRS) Kulaa (Non IRS) Total No. (%) An. gambiae s.l. 43 42 85.0 (92.4%) 66 491 557.0 (97.9%) 642.0 (97.1%) An. funestus 2 1 3.0 (3.3%) 6 4 10.0 (1.8%) 13.0 (2.0%) An. nili 1 1 2.0 (2.2%) - - 2.0 (0.3%) An. rufipes - 2 2.0 (2.2%) - 2 2.0 (0.4%) 4.0 (0.6%) Total 46 46 92 (13.9%) 72 497 569.0 (86.1%) 661.0 University of Ghana http://ugspace.ug.edu.gh 59 Table 4: Species composition and abundance of Anopheles species collected by PSC and ETC in rooms with and without hanging clothes in an IRS (Tarikpaa) and a Non-IRS (Kulaa) areas in the Savelugu-Nanton and Tamale districts of Northern Ghana. Species of Anopheles IRS Area (Tarikpaa) Non-IRS Area (Kulaa) Total No. of Mosquitoes collected Rms WH1 No. (%) Rms NoH2 No. (%) Total Rms WH1 No. (%) Rms NoH2 No. (%) Total An. gambiae 16 (14.68) 93 (85.32) 109 98 (18.38) 435 (81.61) 533 642 An. funestus 2 (33.3) 6 (75.0) 8 2 (40) 3 (60) 5 13 An. nili 0 1 (100) 1 0 1 (100) 1 2 An. rufipes 0 0 0 4 (100) 0 4 4 Total 18 100 118 104 439 543 661 Rms WH1= Rooms with hanging, Rms NoH2 = Rooms with no hanging, No. = Number University of Ghana http://ugspace.ug.edu.gh 60 Table 5: Species composition and abundance of Anopheles species collected from ETC in rooms with and without hanging clothes in an IRS (Tarikpaa) and a Non-IRS (Kulaa) areas in the Savelugu-Nanton and Tamale districts of Northern Ghana. Species of Anopheles IRS Area (Tarikpaa) Non-IRS Area (Kulaa) Total No. of Mosquitoes collected Rms WH1 No. (%) Rms NoH2 No. (%) Total Rms WH1 No. (%) Rms NoH2 No. (%) Total An. gambiae 11 (16.67) 55 (83.33) 66 76 (15.48) 415 (84.52) 491 557 An. funestus 4 (66.67) 2 (33.33) 6 3 (75.00) 1 (25.00) 4 10 An. nili 0 0 0 0 0 0 0 An. rufipes 0 0 0 2 (100) 0 2 2 Total 15 (20.8%) 57 (79.2%) 72 104 (20.9%) 439 (88.3%) 497 661 Rms WH1= Rooms with hanging, Rms NoH2 = Rooms with no hanging, No. = Number University of Ghana http://ugspace.ug.edu.gh 61 Table 6: Species composition and abundance of indoor resting Anopheles species collected by PSC in rooms with and without hanging clothes in an IRS (Tarikpaa) and a Non-IRS (Kulaa) areas in the Savelugu-Nanton and Tamale districts of Northern Ghana. Species of Anopheles IRS Area (Tarikpaa) Non-IRS Area (Kulaa) Total No. of Mosquitoes collected Rms WH1 No. (%) Rms NoH2 No. (%) Total Rms WH1 No. (%) Rms NoH2 No. (%) Total An. gambiae 5 (11.63) 38 (88.37) 43 22 (52.38) 20 (47.62) 42 85 An. funestus 0 2 (100) 2 1 (100) 0 1 3 An. nili 0 0 0 0 0 0 0 An. rufipes 0 0 0 2 (100) 0 2 2 Total 5 (11.1%) 40 (88.9%) 45 25 (55.5%) 20 (44.4%) 45 90 Rms WH1= Rooms with hanging, Rms NoH2 = Rooms with no hanging, No. = Number University of Ghana http://ugspace.ug.edu.gh 62 4.1 Identification of Sibling Species and Molecular Forms of An. gambiae A total number of 242 of Anopheles gambiae s.l., (83, Tarikpaa; 159, Kulaa) were analyzed. In all the study areas, An. gambiae s.s. was the only sibling species identified. A total of 65 samples from IRS area were successfully amplified by PCR and out of this, 20% (13/65) of An. gambiae s.s were from rooms with hanging clothes whilst 80% (52/65) An. gambiae s.s were from the rooms with no hanging clothes. The same trend was observed in the non-IRS area. Out of a total of 164 An. gambiae s.s analyzed, An. coluzzi (M form) was the predominant species found in both the IRS and non-IRS areas. The same trend was observed in rooms with hanging clothes and rooms without hanging clothes in all the study areas (Figure 16) Figure 16: Molecular identification of An. gambiae s.s. from rooms with and without hanging clothes in Tarikpaa (IRS) and Kulaa (non IRS) areas 0 10 20 30 40 50 60 Rooms with hanging Rooms without hanging Rooms with hanging Rooms without hanging IRS Area (Tarikpaa) Non-IRS Area (Kulaa) N u m b e r o f A n . g a m b ia e s. s M S University of Ghana http://ugspace.ug.edu.gh 63 4.2 Indoor Resting Densities of Anopheles Table 7 provides a summary of Indoor Resting Densities (IRD) of An. gambiae s.s. and An. funestus with their gonotrophic stages. The mean IRD for Tarikpaa (1.54 mosquitoes/room) and Kulaa/unsprayed community (1.50 mosquitoes/room) were not statistically different (F (1, 12) =0.001; p= 0.973). The mean IRD of Anopheles collected from rooms in the IRS area without hanging clothes was higher than that of rooms with hanging clothes (2.7 mosquitoes/room vs. 0.36 mosquitoes/room). However, this difference was not significant (F (1, 12) = 2.078, P = 0.175). There was also no significant differences in IRDs between rooms in the unsprayed community, Kulaa with hanging clothes and rooms without hanging clothes (1.42 mosquitoes/room vs. 1.57mosquitoes/room) (F (1,12) = 0.011, P = 0.917) A blood fed to gravid ratio of 4.5:1(Table 7) was estimated for Anopheles collected from rooms with no hanging clothes in the IRS area. In the rooms with hanging in the same area, a blood fed to gravid ratio of 5:1 was also estimated for Anopheles mosquitoes. A similar observation was made in Kulaa where mosquitoes from rooms with no hanging clothes had the blood fed to gravid ratio of 16:1(Table 7), indicating that they are more likely to leave the room after feeding. The rooms with hanging clothes in the non-IRS area however had a blood fed to gravid ratio of 1.2:1. University of Ghana http://ugspace.ug.edu.gh 64 Table 7: Indoor resting density and gonotrophic stages of An. gambiae s.s. and An. funestus collected from IRS (Tarikpaa) and Non-IRS (Kulaa) areas in Nothern Ghana. IRDs1 = Indoor resting densities, RWH 2= Rooms with hanging, RNoH 3 = Rooms with no hanging, UF=Unfed, BF=Blood Fed HG=Half Gravid, G=Gravid Study Site IRDs1 (sd) Abdominal Condition BF: G Ratio UF BF HG+G Total IRS Area (Tarikpaa) RWH 2 0.36 (± 0.63) 0 5 1 6 5:1 RNoH 3 2.71 (± 4.28) 5 27 6 38 4.5:1 Non IRS Area (Kulaa) RWH 2 1.57 (± 3.11) 0 12 10 22 1.2:1 RNoH 3 1.43 (± 1.67) 3 16 1 20 16:1 University of Ghana http://ugspace.ug.edu.gh 65 4.3 Movement of Anopheles Mosquitoes The results from the Exit trap collections (ETC) are presented in Table 8. The abdominal composition of mosquitoes collected were analyzed to assess their post-feeding behavior on the basis of the relative proportions of blood fed and gravid individuals. Table 8 shows that for the IRS Area (Tarikpaa), mosquitoes collected after exiting the rooms during the night from rooms with hanging and no hanging showed almost the same F to G ratio of. (0.9:1 and 1:1). This suggests an equal tendency for mosquitoes to rest indoors or move outside after feeding for both rooms. In the non-IRS area, Kulaa, rooms with no hanging showed F: G ratio of 0.09:1. This indicates that the mosquitoes stayed in rooms and develop their eggs post feeding than before attempting to go out for oviposition. A similar behaviour was observed in the mosquitoes exiting from rooms with hanging with F: G ratio of 0.24:1. This indicates that the mosquitoes complete their gonotrophic cycles before exiting the rooms. This shows that most of the mosquitoes will rest indoors before exiting the rooms. University of Ghana http://ugspace.ug.edu.gh 66 Table 8: The abundance and gonotrophic stages of An. gambiae s .l. collected by the Exit trap from IRS (Tarikpaa) and Non-IRS Areas (Kulaa) in the Northern Ghana. Study Site Abdominal Condition BF: G Ratio UF BF HG+G Total IRS Area (Tarikpaa) RWH 1 1 5 5 11 1:1 RNoH 2 11 21 23 55 0.91:1 Non IRS Area (Kulaa) RWH 1 39 9 28 76 0.24:1 RNoH 2 273 12 130 415 0.09:1 RWH 1= Rooms with hanging, RNoH 2 = Rooms with no hanging, UF=Unfed, BF=Blood Fed HG=Half Gravid, G=Gravid University of Ghana http://ugspace.ug.edu.gh 67 4.4 Mortality of Anopheles Mosquitoes in Exit Traps The mortalities of An. gambiae s.l. that were caught exiting rooms is shown in Table 9. A total of 55 exiting mosquitoes were collected from rooms with no hanging clothes and 11 from the rooms with hanging clothes in the IRS area (Tarikpaa). The immediate mortality of Anopheles collected from rooms with hanging clothes (3.63%) was significantly lower than in rooms without hanging clothes (23.6%) Table 9. The reverse was the case in the non-IRS area. The delayed mortality (mortality after 24hrs) of Anopheles collected in rooms with hanging clothes was higher (22.2%) than in rooms without hanging clothes, 9.5% in the IRS area. The actual mortalities of the species were similar in both environments (36.3% and 30.9%) respectively. Table 9: Mortality rates for exiting of An. gambiae s .l. from rooms in IRS (Tarikpaa) and non-IRS areas (Kulaa) in Northern Ghana Study Site Total No. Exiting Mosquitoes Immediate Mortality (%) Delayed Mortalities (%) Actual Mortalities (%) IRS Area (Tarikpaa) RWH 1 11 3.63 22.22 36.36 RNoH 2 55 23.63 9.52 30.90 Non IRS Area (Kulaa) RWH 1 76 28.57 10.71 34.21 RNoH 2 415 0.96 9.58 10.36 RWH 1= Rooms with hanging, RNoH 2 = Rooms with no hanging, No. = Number University of Ghana http://ugspace.ug.edu.gh 68 4.5 Entomological Parameters The Man Biting Rates (MBR), Sporozoite Rates (SR) and Entomological Inoculation Rates (EIR) of both An. gambiae s.l. and An. funestus is shown in Table 10. It was observed that when the rooms in Tarikpaa were compared, a person is likely to be bitten 13.57 times in a 100 days in rooms without hanging clothes whilst in rooms with hanging clothes, a person is more likely to be bitten 1.6 times in 100 days. The non-IRS area, Kulaa recorded the reverse of the results found in the IRS area, Tarikpaa. A person is more likely to be bitten 39.3 times in the room with hanging clothes whilst in the rooms without hanging clothes, a person is more likely to be bitten 23 times. The mosquitoes obtained from Kulaa were more than that obtained from Tarikpaa thus as the results indicate, there is a greater likelihood of being bitten in Kulaa than in Tarikpaa. The sporozoite rate was calculated as the number of positives obtained from ELISA tests divided by the total number examined. The rooms with hanging clothes and without hanging clothes in the spray area, Tarikpaa both had a sporozoite rate of zero. There were no mosquito samples from these two rooms that tested positive for P. falciparum. Thus the rate of infectivity in rooms with hanging clothes and rooms without hanging clothes in the IRS area, Tarikpaa are the same. The non-spray area, however, had a sporozoite rate of 1.83 and 0 from rooms without hanging clothes and rooms with hanging clothes respectively. The EIR is an indirect measure of how many bites of the mosquito will be infective with the Plasmodium parasite per man per night. It was observed that the EIR estimated for rooms with hanging clothes and without hanging clothes in the spray area was zero (Table 10). However, in the non-IRS area, EIR of 0.0043 infective bites per man-night was University of Ghana http://ugspace.ug.edu.gh 69 estimated for Anopheles in rooms without hanging clothes translating to an annual EIR of 1.57 (Table 10). Table 10: Entomological parameters for An. gambiae and An. funestus collected from the IRS (Tarikpaa) and Non-IRS (Kulaa) areas Entomological Parameters MBR* (bite/man/night) SR (%) EIR (Infect. bites/man/night) IRS Area (Tarikpaa) 1RWH 0.0163 0 0 2RNoH 0.1357 0 0 IRS Area (Kulaa) 1RWH 0.393 0 0 2RNoH 0.2380 1.83 0.0043 MBR= Man Biting Rate, SR= Sporozoite Rate, EIR=Entomological Inoculation Rate, *Biting rate was calculated from PSCs, 1RWH= Rooms with hanging, 2RNoH = Rooms with no hanging, No. = Number University of Ghana http://ugspace.ug.edu.gh 70 CHAPTER FIVE DISCUSSION 5.0 Discussion Over the past decade, there has been a massive scale-up of antimalarial interventions including insecticide-treated nets, Artemisinin-Combination Treatments (ACTs), rapid diagnostic tests kits and in selected areas, indoor residual spraying (Mueller et al., 2011). The impact of these scaling up campaigns is gradually being observed. The WHO World Malaria Report, 2013 reports that between 2000 and 2012, the scale-up of interventions helped to reduce malaria incidence rates by 25% globally, and by 31% in the WHO African Region. Effective malaria control involves the integration of a number of control tools against the parasite and the vector. Vector control is a cornerstone of malaria control and it remains the most generally effective measure to prevent malaria transmission (WHO, 2013). Indoor Residual Spraying remains one of the most useful vector control measures. Data from a meta-regression analysis of 13 studies suggest indoor residual spraying reduced malaria prevalence by 62% in the developing world (Kim et al., 2012). It is suspected that when an intervention is introduced in an area to control mosquito vectors of malaria, the behaviour of the mosquito may be altered after a period of time in aversion to the control method. Shifts in biting cycle from the early hours of the morning to the evening having been shown following DDT indoor spraying in the Solomon Islands for An. gambiae s.l. (Taylor, 1975) and for An. minimus in forested terrain in northern Thailand (Ismail et al., 1978). In addition, Nagpal 2012 reports of An. stephensi changing its behaviour from University of Ghana http://ugspace.ug.edu.gh 71 resting on sprayed walls to resting on unsprayed surfaces such as utensils, hanged clothes, and stored clothes some years post spraying. This study was carried out to investigate the impact of unsprayed surfaces on the resting and exiting behaviour of Anopheles mosquito in rooms which have had indoor residual spraying (IRS). An. gambiae s.l. was found as the dominant vector collected from all study sites and in all collection methods employed: pyrethrum spray and exit trap catches. Other species collected were An. funestus, An. nili and An. rufipes which were however in much smaller numbers. This is consistent with findings from a similar study carried out in Northern Ghana, where An. gambiae was the most predominant species found in that area (Appawu et al., 2004). Majority of the Anopheles mosquitoes obtained were collected between September and October, two out of four of the months in which rainfall was at its highest for that particular year. In this study, An. gambiae s.s. was the only sibling species of the An. gambiae complex recorded. Studies carried in similar settings, (Appawu et al., 2004, Taylor et al., 1993) recorded An. gambiae s.s. and An. arabiensis as the only species recorded with An. gambiae s.s. being the dominant species (Appawu et al., 2004). In another study, An. arabiensis was found as the dominant sibling species (Taylor et al., 1993). An. coluzzi (M form) and S forms of An. gambiae s.s. were both found in the study area, contrary to studies by Yawson et al., 2004 which indicates that the M form was the only species found in northern Ghana. The proportions of An. coluzzi were high in both rooms with or without hanging clothes in both IRS and non-IRS area. This implies that the occurrence of University of Ghana http://ugspace.ug.edu.gh 72 molecular form of An. gambiae s.s. were not dependent on the presence or absence of hanging clothes in the rooms. The number of indoor resting Anopheles mosquitoes captured indicated that less mosquitoes were resting in rooms with hanging clothes compared to rooms without hanging clothes in the IRS area. It is expected that in the rooms with hanging clothes, mosquitoes will use the clothes as alternative resting places since the walls have been sprayed and thus more mosquitoes should be found there. The fed: gravid ratio also indicated that more blood fed mosquitoes left the rooms immediately after feeding in the presence of insecticide on the walls. This is because the insecticide on the walls may have exerted some excito-repellency effect on the mosquitoes in the rooms and so many leave the rooms after feeding. Also as expected, the IRD of Anopheles mosquitoes were significantly higher in the non-IRS than IRS area. The number of Anopheles species collected by ETC from rooms without hanging clothes in the IRS area was significantly higher than in rooms with hanging clothes. It is however, expected that the number of Anopheles species collected from rooms without hanging clothes will be higher since the resting surfaces for the mosquito will be reduced with the absence of hanging clothes compared to rooms without hanging clothes. This will corroborate studies by Nagpal et al., 2012 which showed that household objects like cupboards, furniture, hanging clothes, goods stacked on loft, stored clothes, cobwebs and floors can be dominant resting sites in area sprayed with insecticide, although, in this study, the focus was on the hanging clothes because in the study area, the practice of hanging clothes in the rooms was very common. And also studies by Mutinga et al., 1995 which suggested that the storage of clothes in bedroom uncovered could serve as favoured resting sites from where mosquitoes would University of Ghana http://ugspace.ug.edu.gh 73 freely feed on the inhabitants of the house. Results from this study is however, indicates that, mosquitoes after feeding left the room in the presence of the insecticide on the wall, in spite of the presence of hanging clothes. The low delayed mortality of An. gambiae and An. funestus in exit traps from rooms with hanging clothes showed that many of the mosquitoes do not rest on the insecticide treated walls before exiting the rooms. However, this findings need to be further investigated because in the non-IRS area, the immediate and delayed mortality of Anopheles was higher than in the IRS area. This means that the mortalities observed may not be due the effect of the insecticide but environmental conditions that prevailed outside the rooms where the traps were fixed. In this study, P. falciparum was tested because it constitutes over 98% of malaria parasite infections in the study area (Binka et al., 2004). Adungo et al., (1991) and Fontenille et al., (2001) have also justified the use of CS-ELISA for measuring sporozoite rates in mosquitoes after comparing with salivary gland dissections. The P. falciparum sporozoite infection was only found for the non-sprayed area for rooms without hangings. In the spray area, infectivity rate was found to be zero for both rooms. The intensity of malaria transmission as normally expressed as the entomological inoculation rate (EIR) was recorded for only Anopheles collected from the non-IRS area. This is not surprising because, this area has not been sprayed for the past six years and therefore malaria transmission is likely to be higher than the non-IRS area. This has been University of Ghana http://ugspace.ug.edu.gh 74 corroborated by a study in the same area where malaria transmission in the non-IRS area was significantly higher than the IRS area (PMI report, 2012). University of Ghana http://ugspace.ug.edu.gh 75 CHAPTER SIX CONCLUSION 6.0 Conclusion  The study identified An. gambiae s.s. as the most prevalent vector in the all the study areas with An. coluzzi being the most predominant molecular form  A study showed that An. stephensi shifted their behavior by avoiding sprayable surfaces and rested on unsprayable surfaces during all its movement rhythms. It was concluded that the changed behaviour of resting of An. stephensi on unsprayable objects in sprayed rooms largely accounted for failure of malaria control in India (Nagpal et al, 2012). However, data from this study indicated that the practice of hanging clothes in the area undergoing indoor residual spraying did not affect malaria transmission, although a more longitudinal study is needed to confirm the findings from this study. The study also showed that in both the IRS and non-IRS area, Anopheles mosquitoes tend to exit rooms in the morning after feeding. This has implications for the effectiveness of the IRS in the area because many of the mosquitoes will not rest on the insecticide treated walls to be killed. 6.1 Limitations of the Study The limitations of the study were that:  The sample size of mosquitoes were relatively low and these may have affected the findings of the study University of Ghana http://ugspace.ug.edu.gh 76  The study was done only in the rainy season and therefore the seasonal effect on interaction between hanging clothes, IRS and malaria transmission was not available 6.2 Recommendations  A more longitudinal study is recommended to increase sampling times and size of mosquito samples that were collected and analyzed  It is recommended that future studies will also focus on other unsprayable surfaces such as tables, chairs, cobwebs, stored clothes, utensils etc. since all these can serve as resting surfaces for Anopheles inside rooms  Since IRS is known to target endophilic mosquito species, the seemingly exophilic tendencies of Anopheles shown in this study needs to be investigated further to ensure the effectiveness of IRS in the area. 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University of Ghana http://ugspace.ug.edu.gh 96 APPENDICES Appendix I University of Ghana http://ugspace.ug.edu.gh 97 Appendix II PREPARATION OF SOLUTIONS FOR ELISA TESTS Protocol used for detecting circumsporozoite antigen in Anopheles mosquitoes PHOSPHATE BUFFERED SALINE a) Calibration of Flask 1. Pour 1, 2 or 3 litres of tap water into the flask. 2. Gently slide a stirring rod to be used along the side into the flask. 3. Mark the final volume of water in the flask. b) Preparation of buffer 1. Discard the water in (a) above and rinse the flask with distilled water 2. Pour about 750ml of distilled water into calibrated flask 3. Rinse stirring rod with distilled water and slide into flask 4. Place flask on electronic magnetic stirrer and stir 5. Empty one bottle of Dulbecco‟s Phosphate Buffered Saline (DPBS) powder (for one litre) into flask while content is stirring 6. Rinse DPBS bottle thoroughly and add to contents of flask 7. Adjust pH to manufacturer‟s specifications if necessary 8. Add more distilled water to the content of the flask to the eve of the calibrated mark. Store at 40C; Shelf life – 2 weeks University of Ghana http://ugspace.ug.edu.gh 98 WASHING BUFFER Washing Buffer = PBS plus 0.05% Tween 20 1. Follow the procedure for preparation of PBS above to the stage of pH adjustment 2. Add 0.5ml Tween 20 (to 1 litre PBS) while stirring 3. Add more distilled water to the level of the calibrated mark BLOCKING BUFFER Reagent Amount in 0.50 litre 1.00 litre Casein 2.50g 5.00g 0.1N NaOH 50.00ml 100.00ml PBS ph 7.4 450.00ml 900.00ml 1. Suspend casein in 0.1N NaOH and bring to boil 2. After casein is dissolved, slowly add the PBS, allow to cool and adjust the pH 7.4 with HCl SUBSTRATE SOLUTION 1. Mix KP & L ABTS (Solution A) and hydrogen peroxide (Solution B) 1:1 Immediately before use (incubate at 100µl/well) University of Ghana http://ugspace.ug.edu.gh 99 Preparation of solution for coating one micro-titre plate (summary) 1. Stock capture MAb=0.5µg/µl 2. Coat rate is 0.1µg/50µl/well 3. One micro-titre plate contains 96*4 (100 wells). Provision against spillage 4. 10 (100x0.1)µg MAb is needed to coat one plate 5. From 1 above 10µg MAb is contained in 10 x 1/0.5 = 20µl solution 6. From 2,5000 (50 x 100) µl of MAb solution is needed to coat one micro-titre plate 7. Add 4,980 µl PBS to 20µl (10µMAb) solution 8. Coat immediately, 40C overnight or store frozen until use. Coat at 50µl (0.1µg MAb)/well. PLASMODIUM CIRCUMSPOROZOITE ELISA TEST (PROTOCOL) 1. Coat MAb [50µl/well (96-well plate) 30min, Room Temperature (RT) or 40C overnight at a coat rate of 0.1µg/50µl/well 2. Flip plate empty and bang on tissue paper 3. Fill wells with blocking buffer (BB); 200µl/well, 1hr, RT 4. Flip plate empty, bang, wash and incubate with the test mosquito extracts and controls (50µl/well; RT, 2hrs) 5. Wash plate twice with washing buffer (PBS/Tw-20), flip empty and bang 6. Incubate MAb-peroxidase conjugate (0.05µg/50µl/well) in BB; 1hr; in darkness 7. Flip plate empty, wash and bang 3 times 8. Incubate substrate [100µl/well: (30-60mm)] RT. 30-60min: Start reading plate after 30min and finish by 60th minute University of Ghana http://ugspace.ug.edu.gh