Opportunistic feeding behaviour and partial exophily make An. arabiensis much more difficult to control by indoor residual spraying than any other vector in the Afro-tropical region. The persistent malaria outbreaks in Zimbabwe despite decades of indoor house spraying prompted this investigation into the role of An. arabiensis in malaria transmission and assessment of the possible impact of this control measure. The study was conducted in the malaria endemic districts of Binga and Gokwe. An. gambiae complex mosquitoes were collected from artificial outdoor resting sites, and from human dwellings by i) daytime hut searches, ii) pyrethrum spray catches and iii) exit window traps. Mosquito components were processed to enable: i) the distinction of An. arabiensis from An. quadriannulatus and An. merus on the basis of the pale band at the junction of the hind leg 3/4 tarsomeres; ii) species identification and scoring of inversion polymorphism on the basis of the X chromosome and autosomes respectively; iii) the determination of blood meal sources using the Ouchterlony precipitin test; and iv) identification of An. gambiae s.l. using polymerase chain reaction (PCR) and enzyme electrophoresis techniques. Entomological assessment of residual spraying included determining: the vector resting densities indoors and outdoors, bioassay and insecticides susceptibility tests. Data were also collected on hut profiles, knowledge-attitudes-practices surveys, and household malaria prevalence . surveys. An. arabiensis and An. quadriannulatus were found in sympatry in Binga and Gokwe, and in addition, An. merus was found in Gokwe. Most species identifications were made using PCR; which was found to have 7.5% and 41.6% levels of error for An. arabiensis and An. quadriannulatus respectively, using the cytogenetic technique as benchmark. The pale band technique yielded > 80% correct identification for An. arabiensis but the extent of overlap in the pale band lengths between An. arabiensis and An. quadriannulatus renders the method unsuitable for distinguishing these two species. Inversions 2Rb and 3Ra were found floating in An. arabiensis, with 60% frequency in the former. The Wright's F statistic value of -0.0416 indicated an excess of heterozygotes, and a state of panmixis in the vector population. No significant differences were observed between 2Rb karyotypes in host choice. Human blood indices among indoor (0.82), exit trap (0.98) and outdoor resting (0.30) specimens suggested exophilic behaviour. This was corroborated by the high fed:gravid ratios of 6.8: 1 and 11.6: 1 in sprayed and non-sprayed dwellings respectively. This was worsened by a high feeder-survivor index (FSI) of 93 % among exit trap specimens. The susceptibility to deltamethrin coupled with residual efficacy nine weeks post-spray indicated the suitability of the insecticide. Rural dwellings were suitably built for spraying but had no mosquito proofing. Personal protective measures are hardly known; sleeping outdoors occurs in Siabuwa. While An. arabiensis bites humans indoors the partial exophily it exhibits is a threat to indoor residual insecticide spraying. An integrated malaria control approach is recommended. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1996.

Malaria remains an important cause of morbidity and mortality among children in sub-Saharan Africa despite recent reductions in malaria incidence in some parts of Africa. Current control tools face threats such as the emergence of drug resistant parasites and insecticide resistant mosquitoes. A malaria vaccine is needed to complement and/or replace existing tools in order to achieve better malaria control and eventually eliminate the disease. RTS,S/AS01E is the most clinically advanced pre-erythrocytic malaria vaccine candidate and is currently being tested in a phase III trial. The short-term efficacy of RTS,S/AS01E is known but the duration of protection is unknown. Furthermore, although RTS,S is protective, it is unclear which immunological assays predict efficacy: hence there are no known correlates of vaccine-induced protection against clinical malaria. In a randomized controlled trial, I assessed the efficacy of RTS,S/AS01E in children (5-17 months old) residing in Kilifi, Kenya, over 4 years of follow-up and determined the correlates of protection against clinical malaria. In order to examine the effect of variations in malaria exposure on vaccine efficacy, I developed an individual marker of malaria exposure calculated as distance-weighted prevalence of malaria infection within 1 km radius of every child. Over 4 years of follow-up, RTS,S/AS01E had an efficacy of 29.9% (95%CI: 10.3% to 45.3%, p=0.005) and 16.8% (95%CI: -8.6% to 36.3% p=0.18) against first and all malaria episodes, respectively (by intention to treat analysis). Vaccine efficacy waned over time and with increasing malaria exposure. RTS,S/AS01E efficacy was 43.6% (95% CI, 15.5 to 62.3) in the first year but was -0.4% (95% CI, -32.1 to 45.3) in the fourth year. Vaccine efficacy was 45.1% (95%CI 11.3% to 66.0%) among children with lower than average malaria exposure index, but 15.9% (95%CI -11.0 to 36.4%) among children with higher than average malaria exposure index. Despite waning in efficacy, RTS,S/AS01E averted 65 cases of malaria per 100 vaccinated children, with more cases averted among the children in the higher malaria-exposure cohort (78 cases per 100 vaccinated children) than those the low exposure cohort (62 cases per 100 vaccinated children). RTS,S/AS01E induced high titres of anti-CS protein antibodies and CD4+ T cell but not CD8+ T cell responses. Anti-CS antibody titres and the frequency of TNF-α producing CD4+ T cell responses were independently associated with protection from clinical malaria, and the combination of both anti-CS titers and TNF-α producing CD4+ T cell response satisfied the Prentice criteria for surrogate markers of protection. There was no association between avidity of RTS,S-induced anti-CS protein antibodies and protection from clinical malaria. Conclusions: RTS,S/AS01E efficacy against all episodes is 16.8% over the 4 years of follow-up. The vaccine efficacy wanes over time and with increasing malaria exposure. RTS,S/AS01E-induced TNF-α producing CD4 T cell and anti-CS protein antibody responses were independently associated with protection from clinical malaria. Anti-CS avidity did not predict protection from clinical malaria. Long-term follow-ups of malaria vaccine trials are essential in the evaluation of the longevity of vaccine efficacy.

Background The years 1985 to 1988 and 1997 to 2001, were periods of high morbidity and mortality due to malaria in KwaZulu-Natal, South Africa. One reason for the increased burden of disease was the emergence of drug resistant Plasmodium falciparum. The parasite was resistant initially to chloroquine and then to sulphadoxine-pyramethamine, the medication of choice for the treatment and prevention of malaria in different periods of time. The changing epidemiology of malaria in Mrica was exacerbated by policy makers not making timely and rational change to the failing malaria drug regimens to newer and effective ones. Purpose ofthe study This study was conducted to determine the impact of delayed introduction of sulphadoxine-pyramethamine (Fansidar®) and artemether-lumefantrine (Coartem®) as a first-line drugs for malaria in KwaZulu-Natal from 1985 to 1988 and 1997 to 2001 respectivel y, Study Design Observational, Analytic, Ecological Method The incidence of malaria in KwaZulu-Natal was compared during different phases of the period when chloroquine was the first line treatment. The baseline phase (1982 to 1984) was taken when chloroquine correctly should have been used and this was compared with the delayed phase (1985 to 1988), when it should have been replaced by of sulphadoxinepyramethamine. During the second period sulphadoxine-pyramethamine was the first line treatment of malaria, the baseline phase (1993 to 1996) when it correctly should have been used was compared to the delayed phase (1997 to 2001) of introduction of the alternate treatment of malaria with artemether-Iumefantrine. Ethical approval for this study was obtained from the Biomedical Research Ethics Committee, of the University of KwaZulu-Natal. Statistical Methods The relative association of malaria infection during the chloroquine baseline and change phases and the sulphadoxine-pyrametharnine baseline and change phases were compared with statistical significance at 0.05. Results The risk of malaria infection was 4.5 times (Incidence Risk Ratio = 4.5; 95% Confidence Interval: 4.1 to 5.0; P < 0.0001) higher in chloroquine change phase relative to the baseline phase. During the sulphadoxine-pyrametharnine period, the malaria risk was 3.5 times greater (Incidence Risk Ratio = 3.50; 95% Confidence Interval: 3.40- 3.60; p < 0.0001) in the change phase. In the chloroquine period, the malaria mortality risk was 9.1 times higher (95% Confidence Interval: 2.1 to 38.5; p=0.0003) and the case fatality rate was increased 1.3 times more (95% Confidence Interval: 1.0 to 1.7; p< 0.001) in the change period. The risk of death during the sulphadoxine-pyramethamine change phase was 4.8 times (95% Confidence Interval: 3.3 to 7.0; p<O.OOl) and case fatality rate of2 times (95% Confidence Interval: 1.5 to 2.7; p <0.001) relative to the baseline phase. Conclusions The dramatic change in the malaria epidemiology in Africa in recent times was exacerbated by delay in replacing first line failing antimalarial drugs. The establishment of sentinel sites for assessing drug resistance or failure and the application of World Health Organisation standards in drug resistance studies will go a long way to achieving the Roll Back Malaria target by 2010. / Thesis (MMed.)-University of KwaZulu-Natal, Durban, 2007.

This study investigated the effects of temperature and relative humidity (both controlled and natural) on the lifecyle and morphology of adults of members of the Anopheles gambiae complex in northern KwaZulu-Natal, South Africa. Laboratory investigations into the effects of simulated temperature and relative humidity regimes concentrated on seasonal differences in longevity, egg hatchability, reproductive potential and adult survivorship of An. arabiensis. Differences were found in the life table parameters when these mosquitoes were reared under conditions of seasonal temperature and relative humidity. During the cool season the lifespan and adult survivorship of mosquitoes were greater than those reared during the warm season. In summer, the egg hatchability and reproductive potential were greatest whereas in winter An. arabiensis underwent gonotrophic dissociation although these females were found to take blood meals readily. The influence of seasonal temperature and relative humidity on the body size of An. arabiensis was investigated, both in the laboratory and under field conditions. In both environments, these factors were found to significantly influence body size. In winter, there was a 13% increase in wing size compared to summer bred mosquitoes. A comparison of body size of An. arabiensis, An. merus and An. gambiae reared under laboratory conditions of seasonal temperature and relative humidity showed that the wing size of An. arabiensis was greater than that of An. merus and An. gambiae. The effect of temperature and relative humidity on morphological criteria used in species separation was also investigated. Seasonal differences in wing spot size were compared for An. arabiensis, An. merus and An. gambiae. From this investigation it was concluded that the pale and dark spots on the wing of Anopheles mosquitoes could not be used in species identification due to the large degree of inter-species overlap in the wing spot measurements. The measurement of the pale band at the junction of the 3rd and 4th tarsomere on the hind leg was also investigated for its use in species separation and were found to be useful within the An. gambiae complex. The implications of this study on the transmission and control of malaria are discussed with reference to the late season transmission during March to May that is characteristic of the region. / Thesis (Ph.D)-University of Natal, Pietermaritzburg, 1996.

The irrigation farming methods on the Makhathini Flats are thought to be responsible for recent increase in malaria cases in the Mamfene area of northern KwaZulu-Natal despite ongoing malaria control activities. Their coincidence with the period of intensive fanning is an interesting one. This study was therefore carried out to determine the relationship between larval habitats and adult mosquito population in malaria transmission using Geographic Information System (GIS). Four types of breeding sites were utilised by malaria vectors in Mamfene, viz; types 1, II, III and IV. Habitat type I was tap pools, type II was irrigation spillage in agricultural land, type III was spillage outside but adjacent to the agricultural land and type IV was depression pools located both in the Balamhlanga swamp and inland. The cumulative larval density in all habitats peaked in March 1995 (20/man-hr), with irrigation spillage (type III) recording the highest density index (33/man-hr) as compared to tap pools (type I) which recorded 32.8/man-hr while other waterbodies recorded I3 .4/man-hr (type II) and O.5/man-hr (type IV) respectively. Subsamples of both larvae and adults of An. gambiae group were identified by the Polymerase Chain Reaction (PCR) technique. Of the larvae identified, 94.1 % and 5.9% (n=289) were An. arabiensis and An. quadriannulatus respectively while in the adult component, the composition was 98.7% and 1.3% (n=303) for An. arabiensis and An. quadriannulatus respectively. This confirmed An. arabiensis as the dominant malaria vector in Mamfene area while the exophilic behaviour of An. quadriannulatus was portrayed. Of the An. gambiae group dissected for parity, 51 .5%) were found parous. This is an indication that the population was old and was able to maintain transmission locally despite ongoing vector control measures. The Global Positioning System (GPS) was used to position larval and adult mosquito sampling sites. The spatial distribution of adult mosquitoes from the breeding sites were plotted using GIS soft ware (Map Info ) and the distance between the breeding sites and study houses were measured using a utility distance tool. With the aid of GIS, the adult mosquito density in houses could be used as an indicator to locate the breeding sites in the vicinity. The importance of these findings in terms of application in cost-effective malaria control cannot be over-emphasized. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1995.

Mosquitoes spread diseases that shorten and worsen the lives of many people, chiefly children in poor countries, around the world. Since Ronald Ross' discovery at the end of the nineteenth century that mosquitoes transmit malaria, field entomologists have collected a great deal of information about mosquito ecology. Despite this tremendous effort, there still remain significant gaps in our knowledge of mosquito ecology, in part, reflecting the significant variation in mosquito ecology across species and geographies. The main aim of this thesis is an attempt to synthesise the substantial information that field entomologists have collected on mosquito lifespan. In Chapters 2 and 3, I conduct meta-analyses of the two predominant approaches used to estimate mosquito lifespan: mark-release-recapture experiments and female mosquito dissection-based studies, respectively. These analyses produce estimates of mosquito lifespan by species and genus, and more broadly, allow for an appraisal of these two experimental approaches. In Chapter 4, I describe a recently developed approach, known as near-infrared spectroscopy, which enables estimation of the age of individual mosquitoes, and then perform an in silico analysis to explore the use of this technology for estimating the average lifespan of wild populations of mosquitoes. The emergence of mosquito resistance to the main insecticides used in vector control, along with the concerning recent discovery that the malaria parasites in Asia are becoming resistant to arteminisin - an important drug used to treat malaria - highlight the need for novel approaches to control disease transmission. Some recently-proposed approaches involve genetic modification of the mosquito vectors, for example, to render them incapable of acting as hosts for disease or to reduce their fecundity. In Chapter 5, I model the impact of a release of mosquitoes carrying a genetic construct known as a homing endonuclease, which has been constructed to bias the sex of mosquito offspring towards males, in computational environments that capture some facets of the real life landscapes where mosquito borne disease is rife. About a century ago, the famous Italian Malariologist Giovanni Grassi declared that malaria was a "giant with clay feet"; reflecting the optimism, in some academic circles at the time, that eradication of this terrible disease would soon occur. Unfortunately, a century of often unsuccessful attempts to control and eradicate malaria, and other mosquito borne diseases, would follow Grassi's statement, meaning that this fight is likely to continue throughout the twenty-first century. We now know much more about mosquitoes and mosquito borne disease than we did a generation ago, but there is still crucial information that we do not. In this thesis, I argue that in order to make significant inroads to disease eradication, further research on mosquito ecology is crucial. Only when we better understand our unwitting mosquito foe, can we design and implement effective disease control measures that are so desperately needed in those most desperate parts of the world.

Os diversos aspectos da cadeia de transmissão da malária autóctone são importantes para o estabelecimento de estratégias de intervenção. Entre abril de 2001 e março de 2004, 65 pacientes e 1.777 habitantes foram avaliados em nove municípios da região montanhosa do Espírito Santo. Foram realizados: gota espessa, esfregaço fino, PCR Multiplex, reação de imunofluorescência indireta (IFI) para detecção de anticorpos contra antígenos de estágios eritrocitários de Plasmodium e ELISA para detecção de anticorpos contra peptídeos sintetizados a partir da porção repetitiva da proteína circunsporozoíta (CSP) das variantes de P. vivax e do P. malariae. Foram capturados anofelíneos no peridomicílio, com pesquisa, por PCR Multiplex, de DNA de Plasmodium. O mesmo foi pesquisado também em alguns símios locais. Os pacientes tinham 35,11 + 16 anos, em média. A maioria era do gênero masculino (51 ou 78,5%), 42 (64,6%) residiam em área rural, 23 (35,4%) eram agricultores e oito (12,3%) estudantes. Não houve viagens relevantes. Sessenta e dois (95,4%) nunca haviam tido malária. Vinte e quatro (36,9%) declararam ter entrado na mata. Predominaram a febre, a cefaléia e os calafrios. A febre era episódica em 63 (96,9%), a cada 48 horas em 48 (73,8%) e a cada 24 horas em 15 pacientes (23,1%). O baço foi impalpável em 26 (42,6%). Foi evidenciado o P. vivax em 47 de 48 pacientes e o P. malariae naquele restante, por características morfológicas e pela PCR Multiplex. Esta foi positiva para P. vivax em 45 dos 48, para P. malariae em um e negativa em dois. A IFI foi positiva, para P. malariae, em seis de sete testados, para IgM, e em todos os sete para IgG. Para o P. vivax, entre 50, 47 (94%) foram positivos para IgM e 48 (96%) para IgG. Entre 50 pacientes, pelo ELISA, 25 (50%) tinham anticorpos contra variantes do P. vivax ou contra o P. malariae. As freqüências individuais foram: 22 (44%) para a VK 210, 11 (22%) para a VK 247, 10 (20%) para o P. vivax-like e 10 (20%) para o P. malariae. Entre 253 amostras dos habitantes testadas na IFI para o P. malariae, o resultado foi positivo em 15,8% (40/253) para IgM e em 44,6% (113/253) para IgG. Para o P. vivax , em 1.701, foram 6,2% (105/1701) para IgM e 37,7% (641/1.701) para IgG. Foram detectados anticorpos contra a CSP em 615 de 1.702 amostras (36,1%). Foram 433 (25,4%) para a VK210, 258 (15,1%) para P. malariae, 108 (6,3%) para a VK 247 e 182 (10,7%) para P. vivax -like. A PCR Multiplex, em 1.527 amostras, detectou P. vivax em 23, P. malariae em 15, P. falciparum em nove e P. falciparum e P. malariae em um. Entre 785 espécimes de anofelíneos, com 10 espécies, foi encontrado DNA de P. vivax em um conjunto de exemplares de A. evansae. O P. malariae/brasilianum foi identificado pela PCR Multiplex em dois de cinco símios da região, em um também pelo esfregaço fino. Existem dois possíveis cenários para a transmissão. No primeiro, ela seria inter-humana, com vetores Nyssorhynchus secundários. Em um segundo, viria do reservatório símio, por indivíduos adentrando o ambiente florestal. / The several aspects of the transmission cycle of the indigenous malaria are important to base on the intervention strategies. From April 2001 to March 2004, 65 patients and 1,777 inhabitants were evaluated in nine Municipalities of the highlands of Espírito Santo State. Laboratory methods included: thick and thin smears, Multiplex PCR, imunnofluorescent assay to detect antibodies against crude blood-stages antigens of the Plasmodium genus (IFA) and ELISA to detect antibodies against synthetic peptides corresponding to the repetitive region of the Circumsporozoite protein of P. vivax variants and P. malariae. Anopheline mosquitoes were captured nearby the houses, being screened by Multiplex PCR in the search for Plasmodium DNA. The same test was also applied to some local wild monkeys. Patients had 35.11 + 16 years old in average. Most of them were males (51 or 78.5%), 42 (64,6%) lived in the rural environment, 23 (35.4%) were farmers and eight (12.3%) were students. There was no relevant history of travel. Sixty-two (95.4%) of them had never experienced malaria before. Twenty- four (36.9%) of them informed excursions inside the forest. The predominant symptoms were fever, headache and chills. Fever was periodic in 63 patients (96.9%), recurring each 48 hours in 48 of them (73.8%) and each 24 hours in 15 (23.1%). Spleen was not palpable in 26 patients (42.6%). Morphologic aspects and PCR results disclosed P. vivax as the agent involved in 47 of the 48 cases so screened. Multiplex PCR was positive for P. vivax in 45 of 48 tested, for P. malariae in another one and negative for the two remaining. IFA tested positive for IgM against P. malariae in six of seven evaluated samples, and for IgG against the same parasite in all of the seven. For P. vivax , the figures were 47 of 50 (94%) for IgM antibodies and 48 of 50 (96%) for IgG antibodies. From fifty patients whose samples were screened by ELISA, 25 (50%) were positive for P. vivax variants or P. malariae. The results considering each one of the tested peptides were: 22 (44%) for VK 210, 11 (22%) for VK 247, 10 (20%) for P. vivax -like e 10 (20%) for P. malariae. Among 253 population samples screened in search for P. malariae antibodies at IFA, 40 (15.8%) were positive for IgM antibodies and 113 (44,6%) for IgG antibodies. The search for P. vivax antibodies by the same technique in1,701 samples, resulted in 105 (6.2%) positive for IgM antibodies and in 641 positive for IgG antibodies. Anti-CSP antibodies were detected in 615 of 1,702 tested samples (36.1%). Among these 615, the positive results for each one of the tested peptides were: 433 (25,4%) for VK210, 258 (15,1%) for P. malariae, 108 (6,3%) for VK 247 e 182 (10,7%) for P. vivax-like. Multiplex PCR detected P. vivax DNA in 23 out of 1,527 tested samples, as it did for P. malariae in 15 of them, for P. falciparum in nine of them and both for P. malariae and P. falciparum in one of them. Among 785 mosquito specimens, representing 10 Anopheline species, P. vivax DNA was found in a set of some A. evansae specimens. P. malariae/brasilianum was identified by Multiplex PCR in two of five wild monkeys screened, in one of them also by thin smear. There are two possible scenarios to explain this transmission cycle. The first one bears malaria as a disease transmitted exclusively among human beings by secondary Nyssorhynchus vectors present nearby the houses. In a second scenario, the malaria is acquired after the simian reservoir when the human beings make excursions inside the forest.

Um estudo foi desenvolvido numa coorte localizada em um assentamento agrícola no Acre, Brasil, com o objetivo de identificar fatores de risco de malária. Ao longo de 70 meses, acompanharam-se 531 indivíduos, que contribuíram com 2.281,53 pessoas-ano. Registraram-se 443 casos de malária por Plasmodium vivax, 129 por P. falciparum e 27 de malária mista (P. vivax e P. falciparum). A análise espacial de varredura identificou clusters de malária no final da área de estudo, próximo ao limite da floresta. Nessa análise, 69,1% de todos os casos de malária ocorreram em apenas 27 domicílios (22,3% do total), os quais estavam dentro de um raio de 2,2 Km ao centro do cluster. Quatro estudos transversais foram realizados ao longo do seguimento e infecções assintomáticas foram detectadas pela PCR aninhada. Modelos de fragilidade identificaram o local de moradia como a principal covariável relacionada ao risco de aquisição de malária tanto nos modelos não ajustado quanto nos ajustado. Este tipo de análise pode otimizar as ações de combate à malária e reduzir custos operacionais. / A study was developed in a cohort located in an agricultural settlement in Acre, Brazil, aiming to identify risk factors of malaria. Over 70 months followed up 531 individuals who contributed 2281.53 people-years. It was recorded 443 cases of malaria by Plasmodium vivax, 129 by P. falciparum and 27 mixed malaria (P. vivax and P. falciparum). The spatial analysis canning identified clusters of malaria at the end of the study area, near the forest border. In this analysis, 69.1% of all malaria cases occurred in only 27 households (22.3% of total), which were within a radius of 2.2 km from the clusters center. Four cross-sectional studies were conducted along the follow-up and asymptomatic infections were detected by nested PCR. Frailty models identified the housing location as the main covariate related risk of acquiring malaria in both unadjusted and adjusted models. This type of analysis may optimize actions against malaria, and reduce operational costs.

Os diversos aspectos da cadeia de transmissão da malária autóctone são importantes para o estabelecimento de estratégias de intervenção. Entre abril de 2001 e março de 2004, 65 pacientes e 1.777 habitantes foram avaliados em nove municípios da região montanhosa do Espírito Santo. Foram realizados: gota espessa, esfregaço fino, PCR Multiplex, reação de imunofluorescência indireta (IFI) para detecção de anticorpos contra antígenos de estágios eritrocitários de Plasmodium e ELISA para detecção de anticorpos contra peptídeos sintetizados a partir da porção repetitiva da proteína circunsporozoíta (CSP) das variantes de P. vivax e do P. malariae. Foram capturados anofelíneos no peridomicílio, com pesquisa, por PCR Multiplex, de DNA de Plasmodium. O mesmo foi pesquisado também em alguns símios locais. Os pacientes tinham 35,11 + 16 anos, em média. A maioria era do gênero masculino (51 ou 78,5%), 42 (64,6%) residiam em área rural, 23 (35,4%) eram agricultores e oito (12,3%) estudantes. Não houve viagens relevantes. Sessenta e dois (95,4%) nunca haviam tido malária. Vinte e quatro (36,9%) declararam ter entrado na mata. Predominaram a febre, a cefaléia e os calafrios. A febre era episódica em 63 (96,9%), a cada 48 horas em 48 (73,8%) e a cada 24 horas em 15 pacientes (23,1%). O baço foi impalpável em 26 (42,6%). Foi evidenciado o P. vivax em 47 de 48 pacientes e o P. malariae naquele restante, por características morfológicas e pela PCR Multiplex. Esta foi positiva para P. vivax em 45 dos 48, para P. malariae em um e negativa em dois. A IFI foi positiva, para P. malariae, em seis de sete testados, para IgM, e em todos os sete para IgG. Para o P. vivax, entre 50, 47 (94%) foram positivos para IgM e 48 (96%) para IgG. Entre 50 pacientes, pelo ELISA, 25 (50%) tinham anticorpos contra variantes do P. vivax ou contra o P. malariae. As freqüências individuais foram: 22 (44%) para a VK 210, 11 (22%) para a VK 247, 10 (20%) para o P. vivax-like e 10 (20%) para o P. malariae. Entre 253 amostras dos habitantes testadas na IFI para o P. malariae, o resultado foi positivo em 15,8% (40/253) para IgM e em 44,6% (113/253) para IgG. Para o P. vivax , em 1.701, foram 6,2% (105/1701) para IgM e 37,7% (641/1.701) para IgG. Foram detectados anticorpos contra a CSP em 615 de 1.702 amostras (36,1%). Foram 433 (25,4%) para a VK210, 258 (15,1%) para P. malariae, 108 (6,3%) para a VK 247 e 182 (10,7%) para P. vivax -like. A PCR Multiplex, em 1.527 amostras, detectou P. vivax em 23, P. malariae em 15, P. falciparum em nove e P. falciparum e P. malariae em um. Entre 785 espécimes de anofelíneos, com 10 espécies, foi encontrado DNA de P. vivax em um conjunto de exemplares de A. evansae. O P. malariae/brasilianum foi identificado pela PCR Multiplex em dois de cinco símios da região, em um também pelo esfregaço fino. Existem dois possíveis cenários para a transmissão. No primeiro, ela seria inter-humana, com vetores Nyssorhynchus secundários. Em um segundo, viria do reservatório símio, por indivíduos adentrando o ambiente florestal. / The several aspects of the transmission cycle of the indigenous malaria are important to base on the intervention strategies. From April 2001 to March 2004, 65 patients and 1,777 inhabitants were evaluated in nine Municipalities of the highlands of Espírito Santo State. Laboratory methods included: thick and thin smears, Multiplex PCR, imunnofluorescent assay to detect antibodies against crude blood-stages antigens of the Plasmodium genus (IFA) and ELISA to detect antibodies against synthetic peptides corresponding to the repetitive region of the Circumsporozoite protein of P. vivax variants and P. malariae. Anopheline mosquitoes were captured nearby the houses, being screened by Multiplex PCR in the search for Plasmodium DNA. The same test was also applied to some local wild monkeys. Patients had 35.11 + 16 years old in average. Most of them were males (51 or 78.5%), 42 (64,6%) lived in the rural environment, 23 (35.4%) were farmers and eight (12.3%) were students. There was no relevant history of travel. Sixty-two (95.4%) of them had never experienced malaria before. Twenty- four (36.9%) of them informed excursions inside the forest. The predominant symptoms were fever, headache and chills. Fever was periodic in 63 patients (96.9%), recurring each 48 hours in 48 of them (73.8%) and each 24 hours in 15 (23.1%). Spleen was not palpable in 26 patients (42.6%). Morphologic aspects and PCR results disclosed P. vivax as the agent involved in 47 of the 48 cases so screened. Multiplex PCR was positive for P. vivax in 45 of 48 tested, for P. malariae in another one and negative for the two remaining. IFA tested positive for IgM against P. malariae in six of seven evaluated samples, and for IgG against the same parasite in all of the seven. For P. vivax , the figures were 47 of 50 (94%) for IgM antibodies and 48 of 50 (96%) for IgG antibodies. From fifty patients whose samples were screened by ELISA, 25 (50%) were positive for P. vivax variants or P. malariae. The results considering each one of the tested peptides were: 22 (44%) for VK 210, 11 (22%) for VK 247, 10 (20%) for P. vivax -like e 10 (20%) for P. malariae. Among 253 population samples screened in search for P. malariae antibodies at IFA, 40 (15.8%) were positive for IgM antibodies and 113 (44,6%) for IgG antibodies. The search for P. vivax antibodies by the same technique in1,701 samples, resulted in 105 (6.2%) positive for IgM antibodies and in 641 positive for IgG antibodies. Anti-CSP antibodies were detected in 615 of 1,702 tested samples (36.1%). Among these 615, the positive results for each one of the tested peptides were: 433 (25,4%) for VK210, 258 (15,1%) for P. malariae, 108 (6,3%) for VK 247 e 182 (10,7%) for P. vivax-like. Multiplex PCR detected P. vivax DNA in 23 out of 1,527 tested samples, as it did for P. malariae in 15 of them, for P. falciparum in nine of them and both for P. malariae and P. falciparum in one of them. Among 785 mosquito specimens, representing 10 Anopheline species, P. vivax DNA was found in a set of some A. evansae specimens. P. malariae/brasilianum was identified by Multiplex PCR in two of five wild monkeys screened, in one of them also by thin smear. There are two possible scenarios to explain this transmission cycle. The first one bears malaria as a disease transmitted exclusively among human beings by secondary Nyssorhynchus vectors present nearby the houses. In a second scenario, the malaria is acquired after the simian reservoir when the human beings make excursions inside the forest.

Um estudo foi desenvolvido numa coorte localizada em um assentamento agrícola no Acre, Brasil, com o objetivo de identificar fatores de risco de malária. Ao longo de 70 meses, acompanharam-se 531 indivíduos, que contribuíram com 2.281,53 pessoas-ano. Registraram-se 443 casos de malária por Plasmodium vivax, 129 por P. falciparum e 27 de malária mista (P. vivax e P. falciparum). A análise espacial de varredura identificou clusters de malária no final da área de estudo, próximo ao limite da floresta. Nessa análise, 69,1% de todos os casos de malária ocorreram em apenas 27 domicílios (22,3% do total), os quais estavam dentro de um raio de 2,2 Km ao centro do cluster. Quatro estudos transversais foram realizados ao longo do seguimento e infecções assintomáticas foram detectadas pela PCR aninhada. Modelos de fragilidade identificaram o local de moradia como a principal covariável relacionada ao risco de aquisição de malária tanto nos modelos não ajustado quanto nos ajustado. Este tipo de análise pode otimizar as ações de combate à malária e reduzir custos operacionais. / A study was developed in a cohort located in an agricultural settlement in Acre, Brazil, aiming to identify risk factors of malaria. Over 70 months followed up 531 individuals who contributed 2281.53 people-years. It was recorded 443 cases of malaria by Plasmodium vivax, 129 by P. falciparum and 27 mixed malaria (P. vivax and P. falciparum). The spatial analysis canning identified clusters of malaria at the end of the study area, near the forest border. In this analysis, 69.1% of all malaria cases occurred in only 27 households (22.3% of total), which were within a radius of 2.2 km from the clusters center. Four cross-sectional studies were conducted along the follow-up and asymptomatic infections were detected by nested PCR. Frailty models identified the housing location as the main covariate related risk of acquiring malaria in both unadjusted and adjusted models. This type of analysis may optimize actions against malaria, and reduce operational costs.