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This research report has been submitted to the Faculty of Health Sciences, University of the Witwatersrand , in partial fulfillment of the requirements for the award of the Masters of Science in Medicine (Biology and Control of African Disease Vectors)degree. May 2015 / Background: Ongoing residual malaria transmission is increasingly mediated by outdoor-biting mosquito populations, especially in communities where insecticidal interventions like indoor residual insecticides (IRS) and long-lasting insecticide treated nets (LLINs), are used. Often, the vectors are also physiologically resistant to the insecticides, making this a major against malaria elimination.

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A identificação de focos de transmissão pode ser de grande utilidade no controle da malária. Por esse motivo, hospitais em regiões endêmicas buscam saber os locais que foram visitados anteriormente por pacientes. No entanto, tais informações, obtidas através de questionários fornecidos aos pacientes, são geralmente vagas e muitas vezes imprecisas. Isto torna o processo manual, lento e de pouca valia em estudos epidemiológicos de larga escala. Baseando-se no fato de que uma parcela significativa da população possui celulares com GPS, o objetivo deste projeto é melhorar a acurácia, organização e dinâmica do processo de coleta de dados de geolocalização de pacientes infectados. Um sistema (https://sipos.fcf.usp.br) foi desenvolvido para que pacientes que chegam aos hospitais possam, sob consentimento voluntário, fornecer os dados de GPS dos seus celulares. Os dados dos usuários, que são tratados de forma anônima, são automaticamente processados e armazenados de forma segura. Através do sistema SiPoS Explorer, epidemiologistas e especialistas em saúde pública podem explorar e analisar os dados de geolocalização, permitindo, desta forma, que regiões vulneráveis sejam priorizadas durante campanhas de controle. / The identification of regions with high rates of infection can be of great use in the control of malaria. For this reason, hospitals in endemic regions seek to know the places previously visited by patients. However, such information, obtained through questionnaires provided to patients, is usually vague, inaccurate and not integrated into databases. This makes the process manual, slow and of little value in large-scale epidemiological studies. Based on the fact that a significant portion of the population has smartphones equipped with GPS, this project aims to improve the accuracy and organization of the process of collecting geolocation data from infected patients. The Sickness Positioning System (https://sipos.fcf.usp.br) was developed so that patients who arrive at hospitals can, with voluntary consent, provide the GPS data collected by their smartphones. User data, which is handled anonymously, is automatically processed and securely stored. Through the SiPoS Explorer system (https://sipos.fcf.usp.br/explorer), epidemiologists and public health experts can explore and analyze geolocation data, thereby allowing vulnerable regions to be prioritized during control campaigns.

A identificação de focos de transmissão pode ser de grande utilidade no controle da malária. Por esse motivo, hospitais em regiões endêmicas buscam saber os locais que foram visitados anteriormente por pacientes. No entanto, tais informações, obtidas através de questionários fornecidos aos pacientes, são geralmente vagas e muitas vezes imprecisas. Isto torna o processo manual, lento e de pouca valia em estudos epidemiológicos de larga escala. Baseando-se no fato de que uma parcela significativa da população possui celulares com GPS, o objetivo deste projeto é melhorar a acurácia, organização e dinâmica do processo de coleta de dados de geolocalização de pacientes infectados. Um sistema (https://sipos.fcf.usp.br) foi desenvolvido para que pacientes que chegam aos hospitais possam, sob consentimento voluntário, fornecer os dados de GPS dos seus celulares. Os dados dos usuários, que são tratados de forma anônima, são automaticamente processados e armazenados de forma segura. Através do sistema SiPoS Explorer, epidemiologistas e especialistas em saúde pública podem explorar e analisar os dados de geolocalização, permitindo, desta forma, que regiões vulneráveis sejam priorizadas durante campanhas de controle. / The identification of regions with high rates of infection can be of great use in the control of malaria. For this reason, hospitals in endemic regions seek to know the places previously visited by patients. However, such information, obtained through questionnaires provided to patients, is usually vague, inaccurate and not integrated into databases. This makes the process manual, slow and of little value in large-scale epidemiological studies. Based on the fact that a significant portion of the population has smartphones equipped with GPS, this project aims to improve the accuracy and organization of the process of collecting geolocation data from infected patients. The Sickness Positioning System (https://sipos.fcf.usp.br) was developed so that patients who arrive at hospitals can, with voluntary consent, provide the GPS data collected by their smartphones. User data, which is handled anonymously, is automatically processed and securely stored. Through the SiPoS Explorer system (https://sipos.fcf.usp.br/explorer), epidemiologists and public health experts can explore and analyze geolocation data, thereby allowing vulnerable regions to be prioritized during control campaigns.

Section 1: Construction and screening of a genomic library for the mosquito Aedes aegypti (Diptera, culicidae). A genomic library has been constructed for this important vector of arboviral disease. Total genomic DNA and various classes of RNA from Ae. aegypti were used to screen this library. The results obtained indicate that this species has a short period interspersion pattern of repeated sequences. Transcription of these repeats could not be detected using total cytoplasmic RNA, hnRNA or mRNA as hybridisation probes. Section 2: Sequence organisation of ribosomal DNA in Aedes aegypti. The Aedes aegypti genomic library was used to isolate clones containing the intact ribosomal DNA (rDNA) repeat of this species. This has been restriction mapped and the transcribed regions have been identified. The rDNA repeat is 9.0 Kb in length and is present as approximately 500 head-to-tail tandemly repeated copies. A low level of intraspecies polymorphism of Ae. aegypti rDNA is evident. Two restriction polymorphisms have been identified within the rDNA repeat. Section 3: Analysis of ribosomal DNA variation within Ae. aegypti'and between closely related species. Four variant rDNA clones have been isolated. One of these' may contain the end of a tandem array of ribosomal genes. Another variant contains a duplication of rDNA within the internal transcribed spacer region of the ribosomal repeat. Sequence analysis of this clone has identified regions at the 3' end of the 18S rRNA gene of Ae. aegypti which show very strong homology with the corresponding regions in other species. Some repeated sequences have been identified downstream of the 18S rRNA gene in this clone. Preliminary analysis of the two other rDNA variants indicates that one contains a duplication or insertion of DNA in the 28S rRNA coding region and one contains non-transcribed spacer homologous sequences which are not associated with rRNA coding regions. Section 4: DNA probes for species identification of mosquitoes in the Anopheles gambiae complex. DNA sequences have been isolated which distinguish four of the morphologically identical members of the An. gambiae species complex. Two sequence classes were obtained. Class 1 homologues are highly reiterated in the genomes of An. arabiensis and An. merus, present in low copy number in An. melas and were not detected in An. gambiae s. s. Class 1 sequences are male specific in An. arabiensis. Class 2 homologues are highly reiterated in the genomes of An. merus and An. melas and present in low to middle copy number in An. gambiae s. s, and An. arabiensis. Sex specificity of Class 2 homologues does not occur in the species tested (An. gambiae s. s. and An. arabiensis). Hybridisation of these species specific DNA sequences to mosquitoes squashed directly onto nitrocellulose provides a simplified method of species identification.

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