The lymphatic system has fundamental physiological roles in maintaining fluid homeostasis, immune cell trafficking and lipid transport from the small intestine to the venous circulation. Lymphatic vessels are the main functional organ responsible for the diverse transport roles the system plays. Unlike the blood vasculature, the lymphatic system does not have a central pump, such as the heart, and relies on a variety of factors to move lymph through. It was long thought that only external factors, such as skeletal muscle contraction and lymph formation, played a role in the functional transport capacity of these vessels. With the advancement of imaging capabilities (both hardware and software), it has become clear in the past two decades or so that the main factor in driving lymph transport is the ability of these vessels to intrinsically contract whereby each vessel is comprised of a chain of ‘mini pumps’ in series. The functional capacity of these vessels is thus now understood to be primarily determined by this pumping activity that has been shown to be regulated by various mechanical and biochemical cues. Lymphatic vessel dysfunction has been implicated in a variety of diseases including many lipid related pathologies and a neglected tropical disease known as lymphatic filariasis. While it has been possible to study the vessel function in the context of fluid drainage and immune cell trafficking, the capability to understand the role of lymphatic vessels in lipid transport has not been available due to the lack of experimental animal models and acquisition systems. As part of this thesis, we sought to develop an experimental animal model along with hardware and software tools to investigate the interplay between lymphatics and their lipid content. We report the first functional measurements of how vessels respond to elevated lipid loads. We further utilized our engineering expertise to develop an experimental platform allowing us to further understand the parasite known as B. malayi that migrates to and resides in lymphatic vessels.

Lymphatic filariasis is an infection caused in the Americas by the helminth species Wuchereria bancrofti. This parasite exclusively infects humans, the only source of infection for the mosquito vector Culex quinquefasciatus. In Brazil a national programme to eliminate Bancroftian filariasis was implemented and currently distribution of the infection is almost restricted to Recife and its Greater Metropolitan area, in Pernambuco State. In Maceió-Alagoas, and Belém-Pará, lymphatic filariasis transmission are about to be eliminated. As part of the assessment of the effectiveness of control strategies for filariasis in these two cities, molecular xenomonitoring was conducted in order to verify interruption of Wuchereria transmission. Xenomonitoring is polymerase chain reaction (PCR)-based method for the detection of W. bancrofti in mosquitoes. This technique detects parasite DNA by PCR in blood engorged female mosquitoes collected in an endemic area. Xenomonitoring is a useful and sensitive marker to estimate natural infection with filarial larvae in the vector mosquitoes. It is also a tool for assessing indirectly filariasis prevalence in the human population because it reveals blood microfilariae recently ingested by the insect. Field collected mosquitoes, mainly through the xenomonitoring, from Maceió and Belém, two areas under the filariasis control programme in Brazil, were processed by PCR assay for the detection of infection. A simple and rapid DNA extraction method was standardized. Identification of the parasite was based on amplification of the SspI repeat DNA sequence to W. bancrofti, using species specific NV1 and NV2 primers. A total of 2,583 mosquitoes from the three city sectors endemic for lymphatic filariasis in Maceió, and 59,583 from the eight administrative districts of Belém, were submitted to PCR analysis. No W. bancrofti-infected mosquitoes were found, suggesting an interruption of filariasis transmission in the studied areas. The objective of the World Health Organization (WHO) is to eliminate filariasis as a public health problem in the world by the year 2020. Brazil is among the countries that joined the WHO global efforts. According to the present data, Belém and Maceió will achieve that goal antecipating WHO target date for the global elimination. Nevertheless, to maintain this trend, surveillance measures must be conducted in order to detect and promptly treat patients, to prevent the risk of resurgence of foci apparently under control. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A filariose linfática é uma infecção causada nas Américas por helmintos da espécie Wuchereria bancrofti. O parasito infecta exclusivamente seres humanos, que são as únicas fontes da infecção para os mosquitos vetores da espécie Culex quinquefasciatus. No Brasil um programa nacional para eliminação da bancroftose foi implementado, e atualmente a distribuição da parasitose está praticamente restrita à Recife e sua região metropolitana, em Pernambuco. Em Maceió-Alagoas, e Belém-Pará, a transmissão está em vias de ser considerada eliminada. Como parte da avaliação da eficácia das estratégias de controle adotadas nestas duas cidades, utilizou-se o xenomonitoramento molecular a fim de verificar a interrupção da transmissão da Wuchereria. O xenomonitoramento é um método baseado na reação em cadeia da polymerase (PCR), para detecção de W. bancrofti em mosquitos. Esta técnica consiste na captura e exame pela PCR de fêmeas de mosquitos recém ingurgitados provenientes de uma área endêmica. O xenomonitoramento é um marcador sensível e útil para estimar os níveis de infecção natural em mosquitos. É igualmente uma ferramenta que permite avaliar indiretamente a prevalência da infecção humana, porque revela a presença de microfilárias recém ingeridas pelos insetos. Mosquitos capturados, preferencialmente através de xenomonitoramento, em Maceió e em Belém, duas áreas sob controle no Brasil, foram processados pela PCR para detecção da infecção. Um método simples e rápido para extração do DNA foi padronizado. A identificação do parasito foi baseada na amplificação de um fragmento gênico repetitivo de W. bancrofti SspI, utilizando os iniciadores espécie-específicos NV1 e NV2. Foram submetidos a análise pela PCR 2.583 mosquitos provenientes dos três bairros endêmicos para filariose linfática na cidade de Maceió, e 59.126 mosquitos, provenientes de todos os oito distritos administrativos da cidade de Belém. Não foi encontrado DNA de W. bancrofti em nenhum dos mosquitos investigados. A infecção natural negativa dos insetos sugere a interrupção da transmissão de W. bancrofti nas áreas trabalhadas. É objetivo da Organização Mundial de Saúde (OMS) a eliminação da filariose linfática como problema de saúde pública no mundo até 2020. O Brasil está entre os países que se juntaram aos esforços da OMS. Baseado nos dados obtidos, Belém e Maceió atingirão essa meta antes da data programada. No entanto, para manter esta tendência medidas de vigilância devem ser conduzidas a fim de detectar e tratar precocemente pacientes, para evitar o risco de ressurgimento de focos, aparentemente já controlados.

Lymphatic filariasis is a highly impact disease because it can impair workers and make difficult their social life. Early diagnosis is very important for efficacy of the treatment. The aim of this study was to establish, in a defined population, the prevalence of Wuchereria bancrofti, the causative agent of lymphatic filariasis, using rapid immunochromatographic card test and thick blood smears; compare the immunochromatographic test with the thick blood smear parasitological method, and evaluate different parasitological techniques. Inhabitants of lymphatic filariasis endemic area were examined in Maceió, Alagoas State, Northeasth Brazil (at the edge of a channel - Reginaldo Canal - that crosses the city sectors Feitosa, Jacintinho and Pitanguinha). The examination of 3,000 children between 5 and 10 years old by immunochromatographic test revealed 10 (0,33%) antigen-positive, and of 411 individuals older than 10 years of age only one (0,24%) was antigen-positive. By thick blood smear method, in the same endemic area, among 5,261 children examined between 5 and 10 years old 4 (0,076%) microfilaremic were detected, and 25 (0.13%) from 19,875 individuals older than 10 years of age were found to be microfilaraemic. The parasite antigen assay using rapid immunochromatographic for diagnosis made possible to carry out tests during daytime, in opposition of the traditional thick blood smears method. Comparing the results from immunochromatographic test and thick smears, a increased ability for parasite detection by the immunological method was observed when children between 5-10 years old were examined, being odds relative 4.4 (I.C. 95% = 1.3 - 16.6). However, with individuals older than 10 years of age, no significant difference on parasites detection ability between these techniques was observed. All antigen-positive individuals by immunochromatographic test were examined by membrane blood filtration and none of them were microfilaraemic. Positive individuals by immunochromatographic test and thick blood smears were also positive when submitted to the enzyme-linked immunosorbent assay. The enzyme-linked immunosorbent assay was better than immunochromatographic test in detected W. bancrofti antigen. From the results of immunochromatographic test with children between 5 and 10 years old (age group preconized by WHO to verify active transmission of lymphatic filariasis) it was possible to show a very low prevalence of parasitism in the delimited area, meaning that in a short time Maceió can be free from lymphatic filariasis transmission / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A filariose linfática é uma doença de grande impacto, pois pode incapacitar seus portadores para o trabalho e dificultar seu convívio social, sendo de grande importância o diagnóstico ainda na fase inicial para que o tratamento seja eficaz. O objetivo do presente trabalho foi identificar em população definida, a prevalência de Wuchereria bancrofti, causadora de filariose linfática, utilizando testes de imunocromatografia rápida em cartão e gota espessa de sangue; comparar a imunocromatografia rápida frente ao método parasitológico da gota espessa de sangue, e avaliar diferentes técnicas parasitológicas. Foram examinados moradores da região endêmica de filariose linfática, definida anteriormente em Maceió, AL (parte dos bairros centrais e contíguos Feitosa, Jacintinho e Pitanguinha, ao longo do canal do Reginaldo). Através da imunocromatografia rápida foram avaliadas 3.000 crianças com idade entre 5-10 anos, sendo 10 (0,33%) antígeno-positivo; e entre 411 indivíduos > 10 anos examinados, apenas um (0,24%) foi antígeno-positivo. Através da gota espessa de sangue e na mesma área endêmica, foram examinadas 5.261 crianças com idade entre 5-10 anos, sendo 4 (0,076%) microfilarêmicas, e examinados 19.875 indivíduos > 10 anos, sendo 25 (0,13%) microfilarêmicos. A utilização de imunocromatografia rápida como forma de diagnóstico, através da pesquisa de antígenos do parasito, possibilitou a realização dos exames em horários matutino e vespertino, fato que se opõe ao método tradicional através de gota espessa. Fazendo-se uma comparação entre os resultados obtidos pela imunocromatografia rápida com os da gota espessa pode-se observar uma maior capacidade de detecção dos parasitados pelo método imunológico quando avaliadas crianças de 5-10 anos de idade sendo a odds relativa igual a 4,4 (I.C. 95% = 1,3 16,6); já em indivíduos com > 10 anos não houve diferença significativa na capacidade de detecção de parasitados entre essas técnicas. Todos os indivíduos antígeno-positivo pela imunocromatografia rápida foram submetidos a exames pela filtração de sangue em membrana e nenhum foi microfilarêmico. Os indivíduos positivos através da imunocromatografia e da gota espessa de sangue, foram submetidos ao teste imunoenzimático, sendo todos positivos por essa última técnica. A técnica imunoenzimática mostrou igual capacidade na detecção de antígenos circulantes de W. bancrofti quando comparada com a imunocromatografia rápida. Através dos exames realizados, utilizando a imunocromatografia rápida na faixa etária de 5 a 10 anos (idade preconizada pela OMS para verificação de transmissão ativa da filariose linfática), foi detectada uma prevalência muito baixa da parasitose na área demarcada, mostrando que em pouco tempo a cidade de Maceió-AL poderá estar livre da transmissão da filariose linfática

Filariasis and onchocerciasis are debilitating diseases affecting 120 million people globally. The massive socio-economic impact of these diseases energized the international community to declare a goal of eliminating filariasis 2020. This resulted in a dramatic increase in the efforts to eliminate filariasis and onchocerciasis, employing a strategy of mass drug administration (MDA). However, these programs rely upon the small arsenal of drugs. This leaves these programs vulnerable to failure in the face of developing resistance and local intolerance to the current drug regimens. Thus, new drugs against these infections are critically needed. A homologue of the ecdysone receptor (EcR), a master regulator of development in insects, has been identified in B. malayi. The potential of the EcR as a drug target has been underscored by work in the agricultural industry, where insecticides targeting the ecdysone developmental pathway are effective and non-toxic to non-target species. As the EcR is absent in humans, it represents an attractive potential chemotherapeutic target. The first study investigates the hypothesis that the ecdysone receptor controls the embryogenesis and molting in the filarial parasite. In-vitro embryogram and in-vivo phenotypic studies were conducted to delineate the effect of 20-hydroxyecdysone on the Brugia malayi parasites. The results suggest that the hormone accelerates embryogenesis and causes precocious molts, resulting in the death of the parasite. Further, transcriptomic and proteomic analysis of the ecdysone treated worms provided evidence that the up-regulated genes participate in embryogenesis. Based upon the validation of the ecdysone receptor as a potential drug target, subsequent studies focused on the development of a drug discovery model to screen for agonists and antagonists of the B. malayi ecdysone receptor. A stable cell line was created to aid the high throughput screening to rapidly identity agonist and antagonist compounds. A total of 7 agonists and 2 antagonists were identified. A homology model of the BmEcR ligand-binding domain was created as an alternate method for virtual screening of small molecules as well as to study the ligand-receptor interactions. The hits identified with the assay were docked in the active site of the BmEcR homology model providing an excellent correspondence of data between the molecular assay and the virtual screening method.

Lymphatic filariasis (LF) is an incapacitating disease caused by three filarial nematodes belonging to the family Onchocercidae, namely Brugia timori, Wuchereria bancrofti and Brugia timori. An estimated 90% of lymphatic filariasis cases globally are caused by Wuchereria bancrofti. To evaluate the success of the Global Program to eliminate Lymphatic Filariasis it is essential to monitor the frequency of larval infection in the mosquito vector. Molecular methods to detect Wuchereria bancrofti DNA in mosquitoes have been in existence since 1996. However these methods have not been widely adopted due to the high cost associated with them and the inability of these assays to distinguish between immature and infectious stages in the mosquito vector. The overall aim of this project was to modify, as previously described in literature, the Laney real time PCR assay to permit it to be used in an end point Reverse Transcriptase (RT)-PCR ELISA format. The endpoint PCR-ELISA uses inexpensive conventional thermocyclers and inexpensive reagents and probes. To accomplish this overall goal the specific objectives were to produce a positive control RNAs for Wb-cut-1.2 L3 specific RT-PCR and Wb-TPH RT-PCR that detects any stage of the parasite, and to adapt the detection of both transcripts to a PCR-ELISA format. Positive RNA controls were prepared and purified using template cDNA made available through FR3, subsequent development and optimization of the RT PCR ELISA was achieved through the adaptation of the Onchocerca volvulus O150 PCR ELISA protocol. We found a 16-fold difference in the limit of detection between the ELISA assay and conventional end point RT-PCR when we did a 2-fold dilution series of PCR products for both Wb-Cut-1.2 and Wb-TPH. This indicates that our assay was 16 times more sensitive than the use of regular agarose gel electrophoresis to analyze PCR products. The limit of detection with ELISA and gel analysis were comparable when a 10-fold dilution series of the positive control RNA template was done. The RT-PCR ELISA takes a day to complete and up to three 96 well plates a day can be processed compared to the limited number of samples that can be analyzed by gel electrophoresis a day. It is anticipated that our assay will be used in the molecular xenomonitoring of Wuchereria bancrofti providing earlier time-point assessments of LF infection in endemic areas. In areas that were once endemic, our diagnostic tool will play a pivotal role in monitoring LF resurgence.

Estudo preliminar da redução da microfilaremia in vivo de Mansonella ozzardi (Manson, 1897) com uso de ivermectina, utilizando a técnica de filtração em membrana de policarbonato, Lábrea, Amazonas, Brasil. Estudou-se a eficácia do uso de dose única de ivermectina 0,15 mg/kg de peso no tratamento de 74 pacientes com infecção por Mansonella ozzardi. Inicialmente foi realizado a coleta de sangue para o diagnóstico parasitológico utilizando a filtração de sangue em membrana de policarbonato e para análises bioquímicas e hematológicas. Foi realizado a quantificação de microfilárias antes e depois do tratamento seguindo-os até 1 ano. Foi observado uma redução estatisticamente significativa em relação à densidade de microfilárias (&#967;2 de Friedman = 159,00; valor-p < 0, 0001) após 1 ano do uso da medicação, e que não houve alterações laboratoriais e efeitos adversos que comprometessem o uso da ivermectina. Concluiu-se que o fármaco nesta dose é eficaz e seguro, e mantém o \"clearance\" de microfilaremia pelo menos 1 ano após o uso. / Preliminary study of the reduction of microfilaremia in vivo of Mansonella ozzardi (Manson, 1897) with use of ivermectin, using the blood filtration polycarbonate membrane technique, in Labrea, Brazilian Amazon, Brazil. The study focused in the efficacy and tolerability of a single-dose use of ivermectin 0.15 mg / kg body weight in the treatment of 74 patients infected by the filarial worm M. ozzardi. Before and after the parasitological diagnosis by a blood filtration polycarbonate membrane and treatment, anamnesis, clinical examination and blood collection for quantification of microfilariae, biochemical and hematological analysis were done, comparing the outcomes of patients the in first day and after 72 hours and at least following up the patients for 1 year. The study concluded that there was a statistically significant reduction in microfilariaemia density (&#967;2 Friedman = 159, 00; p-value <0, 0001) after 1 year of use of the medication, and that there were no laboratory abnormalities and clinical symptoms that compromised the use of ivermectin. In conclusion, the use of ivermectin is an effective microfilaricide and maintains a suppressive effect for at least 1 year and the adverse reactions are not an obstacle for the treatment.

archives@tulane.edu / 1 / Kimberly Michelle Koporc

A dirofilariose canina é uma parasitose de distribuição mundial cujo agente etiológico é um verme nemátoda filarióide. Em sua fase adulta, os vermes produzem microfilárias por meio de reprodução sexuada. Sua transmissão ocorre por meio de mosquitos que atuam como hospedeiros intermediários. No Brasil, até o momento, a dirofilariose canina foi encontrada em 15 estados de todas as regiões. A maioria dos estudos está concentrada nas regiões sudeste e sul. A prevalência nacional é de 2%, embora alguns trabalhos tenham encontrado prevalências locais mais elevadas. Neste trabalho apresentamos o primeiro registro de dirofilariose canina para o estado de Rondônia com mapas de distribuição dos cães positivos e análise de mosquitos. Amostras de sangue de 727 cães foram coletadas aleatoriamente na cidade de Porto Velho. As amostras forma analisadas em busca de microfilárias e antígenos circulantes usando duas técnicas: microscopia ótica de gota espessa corada com Giemsa e imunocromatografia de fluxo lateral (ICT). As amostras positivas foram também testadas por PCR, as negativas forma testadas na mesma técnica em pools. Mosquitos foram coletados no domicilio e peridomicílio de todos os casos de cães positivos, estes mosquitos foram testado por PCR em busca de DNA de Dirofilaria immitis. Um mapa de distribuição dos casos de cães positivos foi elaborado. Noventa e três amostras de sangue foram positivas no ICT, representando 12,8% da amostra total, nenhuma amostra foi positiva na gota espessa. O PCR das amostras de sangue resultou em 10% para as positivas no ICT e 0% nas negativas no mesmo teste. Entre os 93 cães positivos, 89 (95,7%) nasceram em Porto Velho. Nenhuma diferença estatística foi observada entre cães que moram em quintais ou intradomicílio. O mapa de distribuição indica um hotspot na região norte da cidade. O PCR dos mosquitos resultou em apenas um pool positivo. A transmissão de dirofilariose canina ocorre na cidade de Porto velho e a frequência que ocorre nos cães é considerada moderada. A técnica de imunocromatografia e PCR são mais eficazes na detecção de dirofilariose comparadas a gota espessa. A confirmação de transmissão de dirofilariose canina em Porto Velho, coloca esta doença no ranking de diagnóstico diferencial de nódulos pulmonares em Porto Velho. / Heartworm is an infectious disease with worldwide distribution. Mosquitoes acts as intermediary host and vectors. In Brazil, until now, heatworm was found in 15 states with an national average of 2%. This present work aims to report for the first time canine heartworm in the state of Rondônia and confirms the transmission of the disease in the state. Blood samples were randomly collected from 727 dogs in the municipality of Porto Velho. The samples were analyzed for the presence of microfilariae and circulating antigens using two different techniques: thick blood microscopy stained with Giemsa and immunochormatography for the detection of filarial antigens. Aiming to test the efficacy of the immunoassay test, all the positive cases were examined by PCR and pools of negative samples were also examined. Mosquitoes were collected at the domiciles presenting positive cases and analyzed by PCR. A distribution map was made with positive cases. Ninety three blood samples out of 727 (12.8%) were positive by the immunoassay technique and none by the thick smear method. All the positive cases by the immunoassay technique were examined by PCR and pools of all negatives samples were also examined resulting in 10% and 0% positivity frequency, respectively. Among the 93 positive dogs, 89 (95.7%) were born in Porto Velho. No differences in the frequency of infection were observed between dogs raised indoor or in the yards. The distribution map indicates a hotspot in the north area of the city. Mosquitoes were analyzed by PCR, resulting in only one positive pool. This result shows that the transmission of canine heartworm is occurring in the municipality of Porto Velho and has moderate prevalence among dogs. The immunoassay technique and the PCR method are more efficient in detecting D. immitis infections in dogs when compared to the blood smear technique. The confirmation of heartworm transmission in Porto Velho also includes this disease among the ranks of differential diagnosis of pulmonary nodules in humans in Rondônia.

Every year, through mass drug administration (MDA), hundreds of millions of the world’s poorest people receive a single annual dose of one or more drugs to eliminate certain parasitic worm or bacterial infections. Some of these infections, mostly prevalent in tropical areas, have traditionally been neglected from the public health and research point of view. These conditions, collectively known as the neglected tropical diseases (NTDs), still cause, at the cusp of the second decade of the 21st century, a significant amount of morbidity and mortality. The existing control measures for NTD have an enormous potential, although there are still some challenges that require further investigation. For some diseases, alternative strategies may be needed, including longer duration of MDA programmes or modified drug regimens. For other diseases, such as yaws, the work must start almost from scratch, since little has been achieved in terms of control of this disease in the past 50 years. Although eight NTDs affect the region, two diseases pose a major public health problem in the South Pacific Islands, namely yaws and lymphatic filariasis and are the basis for his thesis. These two infections were selected for a number of reasons. First, they affect the South Pacific region disproportionately. Secondly, little research has been conducted in the past years. And third, but more importantly, several epidemiological, technological and historical factors make these two diseases amenable to elimination. Safe and effective tools and interventions to achieve these targets are available and concerted efforts to scale them up are likely to lead to success. Yaws is one of the most neglected of the NTDs. Yaws was one of the first diseases to be targeted for eradication on a global scale, efforts which almost led to the disease disappearance as a result of a massive treatment program started in the 1950s. After the successful eradication campaigns the primary health care systems were supposed to give the last push towards eradication of yaws. However a combination of various factors including poor political commitment and limited funding resulted in a progressive abandonment of efforts and the resurgence of the disease. Every new case of yaws was the disappointing confirmation that the public health world had missed a great opportunity. Today yaws has resurged in many tropical areas and presents new challenges including its unknown epidemiological situation, the attenuated clinical forms of the disease, a poor awareness and knowledge among health care workers, the lack of knowledge about the effectiveness of classic treatment with penicillin and, an obvious need for research into simplified administration schemes or new antibiotic treatments, particularly oral ones. There is an enormous knowledge gap regarding current reliable epidemiological information about the disease. Certainly we know little about the burden in the three Melanesian countries where the disease is highly endemic, Papua New Guinea, Solomon Islands and, Vanuatu. In Solomon Islands and Vanuatu there are indications that Yaws is widespread and prevalent, but we know that the diagnosis is unreliable. This takes us to the next point, what does a diagnosis of yaws mean? Overall the natural history of the disease in this era, where it is often subject to inadequate antibiotic pressure, is very unclear. Some authors have suggested that yaws appears to be attenuated in both Solomon Islands and Vanuatu. They state that bone involvement in yaws is now rare and implies that yaws is a mild disease not requiring efforts for elimination. However, the first paper of this thesis describes the epidemiology of yaws in Lihir Island (Papua New Guinea, PNG) and shows a high rate of classical primary ulcers (almost 60%) and significant bone and periosteal involvement (more than 15%), suggesting that “attenuation” is not an important issue. When we look at the diagnostic criteria for yaws, signs and symptoms alone are still used often in many areas to diagnose the disease. This reliance on clinical findings was the result of the difficulty of performing serological tests in remote areas. Today, available rapid serological tests are simple, rapid, inexpensive and useful for guiding confirmation of cases, making them adequate tools for the diagnosis and monitoring of the disease. The clinical diagnosis of yaws is complicated because its clinical manifestations may be unspecific. Thus, it is possible that a significant proportion of yaws cases may in fact have been falsely diagnosed. We show, in the first article, that in our experience only 60% of the cases with a clinical suspicion of yaws were finally confirmed by serologic tests. Therefore, a proper diagnosis of yaws requires the interpretation of clinical findings with reference to laboratory results and the epidemiologic history of the patient. Serological testing in yaws is not only important for diagnostic accuracy, but also is very helpful in defining the disease’s evolution and eventual cure after treatment. Rapid plasma regain (RPR) titres should decline within 6-12 months, becoming negative in less than 2 years. The second article of this thesis combines a clinical and serological approach to assess the response after treatment with benzathine benzylpenicillin, and it identifies an overall 20% treatment failure. This could be related to resistance to the antimicrobial drug used or to re-infection caused. The distinction between re-infection and true resistance to antibiotic treatment is difficult to make but these failures are worrisome. This article also proposes a multivariate model performed to identify independent determinants of failure that affected the outcome after treatment. The risk for reinfection caused by repeated contact with infected children seems to be a pivotal predictor of failure. Low baseline titters (<1:32) of RPR are also an important and independent predictor of failure, possibly as a result of the greater difficulty in resolving chronic infections which are usually accompanied by low titters. With yaws re-emerging, the development of new strategies against this infection aimed at simplifying its treatment and potentially re-focussing strategies towards its eradication seems essential. Injectable penicillin is still effective but management with an oral drug that can be easily administered on a large scale should be the preferred method for treatment. To date, there had been no studies that directly compared the efficacy of penicillin with any of the potentially alternative agents shown to work in the treatment of the non-venereal treponematoses. The fourth paper in this thesis has shown that a single-dose of oral azithromycin is non-inferior to benzathine benzylpenicillin for the treatment of yaws in children in PNG. In an open-label randomised trial, at 6-month follow-up, 96% of patients treated with azithromycin were cured, as were 93% in the benzathine benzylpenicillin group. The prospects of eliminating and eventually eradicating yaws may now be enhanced by the use of a single-dose of oral azithromycin in mass drug administration campaigns. Community based mass administration of azithromycin has been widely used in many locations for the control of trachoma, which, like yaws, is a disease of poor rural communities in developing countries, and has been used in a more limited way to control granuloma inguinale and outbreaks of venereal syphilis. Elimination of yaws and lymphatic filariasis in the South-Pacific Islands is now considered biologically feasible and programmatically attainable. The Global Programme to Eliminate Lymphatic Filariasis (GPELF) has expanded quickly to reach the target of elimination by 2020. On the other hand the strategy to eliminate yaws is again at the centre of discussions and given that infected humans are the only source of disease, its eradication could be achieved within a very relatively short time. The fifth article of the thesis comprehensively reviews antimicrobial treatments and elimination strategies against yaws. In order to control yaws and push it towards elimination, we propose to move away from penicillin to azithromycin and use mass treatment campaigns of the entire population in endemic communities irrespective of the prevalence. Also, to make sure all cases are tracked down and treated, strict follow-up measures and selective mass treatment will be required until zero case prevalence is reached. Importantly, we suggest testing the principle of interrupting transmission in pilot implementation studies, including prevalence surveys to assess the impact of the intervention and macrolide resistance monitoring which in our opinion will be essential evaluation tools to guide us towards a sustainable elimination. Lymphatic filariasis (LF), caused by the mosquito-borne nematode Wuchereria Bancrofti, is a major public-health problem in the Melanesian countries. Annual MDA over five years is currently the WHO’s recommended strategy to eliminate lymphatic filariasis. This approach aims to suppress microfilaraemia in infected individuals and bring the infection below a threshold that leads to interruption of transmission. However theoretical work and clinical field experience has highlighted how the ecological diversity between different endemic regions can result in elimination thresholds that vary between local communities. This means that the duration required might be different for different areas. Other variables have also been previously identified as potentially having an influence on the outcome of the program, including baseline prevalence of infection, vector density or the treatment coverage. The last article of this thesis provides data about the impact of a five-year filariasis control program in Papua New Guinea. The findings reported support this strategy for areas with low-to-moderate rates of transmission in regions where anopheline mosquitoes transmit this infectious disease. Additional measures or longer periods of treatment may be necessary in areas with a high rate of transmission. The experience acquired on Lihir Island in MDA programs during the campaigns for the elimination of filariasis, will be very valuable when implementing a pilot strategy for yaws control. Also, in the near future it might be important to link yaws mass treatment with other mass programmes to increase efficiency. The plan for elimination of lymphatic filariasis in PNG was approved as a pilot project in 2005 but the program still needs to be extended to the total of 20 provinces in the country where filariasis is endemic. In this context, an integrated approach to NTD control could represent an important global public health solution in PNG and other South Pacific Islands. Little has been achieved in the past decade in NTDs. We are now in a good position to translate into policies the results of our research projects. A new elimination policy for yaws around the azithromycin pillar has been sketched a WHO consultation meeting held in Morges, Switzerland last March. In the intentions of the organization, a last global mass campaign to tackle yaws should permit to reach zero cases in 2017, and the subsequent certification of worldwide interruption of transmission by 2020. / Cada año, a través de la administración masiva de medicamentos (MDA), cientos de millones de personas, las más pobres del mundo, reciben una dosis única de uno o más medicamentos para eliminar ciertas infecciones, parasitarias o bacterianas. Algunas de estas infecciones, frecuentes sobre todo en las zonas tropicales, han sido tradicionalmente desatendidas desde el punto de vista de salud pública e investigación. Estas enfermedades, conocidas comúnmente como las enfermedades tropicales desatendidas (ETD), aún causan, en el inicio de la segunda década del siglo 21, una cantidad significativa de morbilidad y mortalidad. Las medidas de control actuales para ETDs tienen un enorme potencial, pero todavía existen algunas cuestiones que requieren investigación. Para algunas de estas infecciones, son necesarias estrategias alternativas, incluyendo una mayor duración de los programas de MDA o regímenes modificados de medicamentos. Para otras enfermedades, como la enfermedad de pián, el trabajo debe comenzar casi desde cero, ya que poco se ha logrado, en términos de control de esta enfermedad, en los últimos 50 años. Aunque ocho ETDs afectan a la región, dos enfermedades constituyen un problema importante de salud pública en las Islas del Pacífico Sur, a saber: el pián y la filariasis linfática y son la base de esta tesis. Estas dos infecciones fueron elegidas por muchas razones. En primer lugar, afectan a la región del Pacífico Sur de forma desproporcionada. En segundo lugar, pocas investigaciones se han llevado a cabo en los últimos años. Y en tercer lugar, pero lo más importante, varios factores epidemiológicos, tecnológicos e históricos hacen que estas dos enfermedades sean susceptibles de eliminación. Existen armas terapéuticas seguras y eficaces para lograr este objetivo, y esfuerzos coordinados para ejecutar los programas de control pueden conducir al éxito. El pián es una de las más olvidadas de las ETDs. Ésta fue una de las primeras enfermedades en ser objetivo de erradicación a escala global. Los esfuerzos de un programa de tratamiento masivo, que se inició en la década de 1950, casi llevaron a la desaparición de la enfermedad. Después de las exitosas campañas de erradicación, los sistemas de salud de atención primaria debían dar el último empujón hacia la erradicación del pián. Sin embargo, una combinación de varios factores, incluyendo un pobre compromiso político y una financiación limitada, dieron como resultado el abandono progresivo de los esfuerzos y el resurgimiento de la enfermedad. Cada nuevo caso de pián era la decepcionante confirmación de que el mundo de la salud pública había perdido una gran oportunidad. Hoy la enfermedad de pián ha resurgido en muchas áreas tropicales con nuevos desafíos: una situación epidemiológica desconocida, formas clínicas atípicas o atenuadas, poco conocimiento de la enfermedad entre el personal sanitario, la falta de datos acerca de la eficacia del tratamiento clásico con penicilina inyectable y la necesidad de desarrollar esquemas terapéuticos simplificados o investigar en nuevos tratamientos antibióticos, en especial de administración oral. Actualmente hay una enorme brecha de conocimiento entorno a la información epidemiológica fiable sobre la enfermedad. Ciertamente, sabemos poco acerca de la incidencia en los tres países melanesios, donde la enfermedad es altamente endémica, Papúa Nueva Guinea (PNG), Islas Salomón y Vanuatu. En las Islas Salomón y Vanuatu, las cifras de incidencia son muy altas lo que demuestra que el pián es una enfermedad frecuente y ampliamente extendida, pero sabemos que el diagnóstico no es muy fiable. Esto nos lleva al siguiente punto: ¿Cuáles son los criterios diagnósticos del pián? En general, la historia natural de la enfermedad en la época actual, donde la bacteria es objeto de presión antibiótica inadecuada, no es muy clara. Algunos autores han escrito que el pián parece presentar manifestaciones “atenuadas” en las Islas Salomón y Vanuatu. Afirman que la afectación ósea en el pián es poco frecuente, lo que implica que el pián es una enfermedad leve que no requeriría esfuerzos para su eliminación. Sin embargo, el primer trabajo de esta tesis describe la epidemiología del pián en la Isla de Lihir (Papúa Nueva Guinea) y muestra una alta tasa de úlceras primarias clásicas (casi el 60% de casos) y una afectación significativa del hueso y periostio (más del 15%) que sugiere que la "atenuación" no es un tema importante. Cuando nos fijamos en los criterios diagnósticos, únicamente signos y síntomas todavía se utilizan en muchas áreas para el diagnóstico de la enfermedad. Esta confianza en los hallazgos clínicos fue el resultado de la dificultad de realizar pruebas serológicas en las zonas remotas. Hoy en día, las pruebas serológicas rápidas son simples, rápidas, económicas y útiles para orientar la confirmación de los casos. El diagnóstico clínico del pián es complicado debido a que sus manifestaciones pueden ser inespecíficas. Así, es posible, que una proporción significativa de los casos de pián puedan haber sido falsamente diagnosticados. En el primer artículo, presentamos que, en nuestra experiencia, sólo el 60% de los casos con sospecha clínica de pián fueron finalmente confirmados por pruebas serológicas. Por lo tanto, un diagnóstico adecuado del pián requiere la interpretación de los hallazgos clínicos con referencia a los resultados de laboratorio y la historia epidemiológica de los pacientes. Las pruebas serológicas en el pián no sólo son importantes para el diagnóstico de la enfermedad, también son muy útiles en la definición de curación después del tratamiento. En la prueba de la Reagina plasmática rápida (RPR) los títulos deben descender a los 6-12 meses, llegando a ser negativa en menos de 2 años. El segundo artículo de esta tesis combina un enfoque clínico / serológico para evaluar la respuesta a bencilpenicilina benzatina, e identifica una tasa de fracaso terapéutico del 20% a los 12 meses del tratamiento. Esto podría estar relacionado con resistencia al fármaco antimicrobiano, o bien indicar una re-infección por re-exposición. La distinción entre la re-infección y la resistencia verdadera al tratamiento es difícil, pero estos fracasos terapéuticos son preocupantes. En este artículo se describe un modelo multivariante realizado para identificar los factores determinantes del fracaso terapéutico. El riesgo de re-infección causado por el contacto repetido con otros niños infectados parece ser un predictor fundamental de fracaso. También es un factor de riesgo, los títulos basales bajos (< 1:32) de RPR. Este último factor podría estar relacionado con la mayor dificultad para resolver infecciones crónicas (en estadio secundario), habitualmente acompañadas de títulos bajos. Con la enfermedad de pián re-emergiendo, el desarrollo de nuevas estrategias contra la infección para hacer más fácil los esfuerzos de erradicación es esencial. La penicilina inyectable sigue siendo eficaz, pero el tratamiento con un fármaco por vía oral que pueda ser fácilmente administrado a gran escala es el método preferido para el tratamiento, prevención y finalmente eliminación en todas las regiones endémicas del mundo. Hasta la fecha, no ha habido estudios que comparen directamente la eficacia de la penicilina con cualquiera de los agentes alternativos en el tratamiento de las treponematosis no venéreas. El cuarto artículo de esta tesis ha demostrado que una dosis única de azitromicina por vía oral no es inferior a la bencilpenicilina benzatina intramuscular, para el tratamiento del pián en niños en Papúa Nueva Guinea. En un ensayo abierto, aleatorio, el 96% de los pacientes tratados con azitromicina estaban curados a los 6 meses de seguimiento, al igual que el 93% en el grupo de bencilpenicilina benzatina. Las perspectivas de finalmente erradicar el pián son ahora mayores, mediante el uso de una dosis única de azitromicina oral en campañas masivas de tratamiento. El tratamiento masivo con azitromicina ha sido ampliamente utilizado para el control del tracoma, que, al igual que el pián es una enfermedad de comunidades rurales pobres de países en desarrollo. También se ha utilizado de una manera más limitada para controlar el granuloma inguinal y brotes de sífilis venérea. En general, el uso de azitromicina ha demostrado ser seguro, y de hecho ha habido beneficios inesperados de salud en algunos programas. La eliminación del pián y la filariasis linfática en las Islas del Pacífico Sur se considera ahora biológicamente factible y operacionalmente alcanzable. El Programa Global para Eliminar la Filariasis Linfática (GPELF) se ha expandido rápidamente para alcanzar la meta de eliminación en el año 2020. Por otro lado la estrategia para eliminar el pián es nuevamente centro de atención. Además, dado que los seres humanos infectados son la única fuente de la enfermedad, su eliminación podría lograrse en un plazo relativamente corto. El quinto artículo de la tesis revisa de forma integral el tratamiento con antimicrobianos y las estrategias de eliminación contra el pián. Con el fin de controlar el pián hasta la erradicación, se propone pasar de la penicilina a la azitromicina, y el uso de campañas de tratamiento masivo de toda la población en todas las comunidades endémicas. Además, para asegurar que todos los casos son encontrados y tratados, serán necesarias medidas estrictas de seguimiento y tratamiento masivo selectivo hasta llegar al objetivo de cero casos clínicos. Es importante destacar que el principio de interrupción de la transmisión se debe probar en estudios piloto, incluyendo estudios de prevalencia, para monitorizar el impacto de la intervención, y también la valoración de resistencia a macrolidos, que en nuestra opinión, serán herramientas fundamentales que nos guíen en el camino hacia una eliminación sostenible La filariasis linfática (FL), causada por el nematodo Wuchereria bancrofti, es otro de los grandes problemas de salud pública en los países de la Melanesia. Un curso de MDA anual, durante cinco años, es la estrategia que la OMS recomienda para eliminar la FL. Este enfoque tiene como objetivo suprimir la microfilaremia en los individuos infectados y disminuir los niveles de infección por debajo de un umbral que conduzca a la interrupción de la transmisión. Sin embargo, trabajo teórico y experiencia práctica clínica han puesto de relieve cómo la diversidad ecológica, entre diferentes regiones endémicas, puede resultar en que los umbrales de eliminación varíen en diferentes comunidades. Esto significa que la duración requerida podría ser diferente para diferentes áreas. Algunas variables que han sido previamente identificadas como potenciales determinantes en el resultado de un Programa para la eliminación de FL (PELF) son la prevalencia basal de infección por filariasis, la densidad de vectores (mosquitos) o la cobertura del tratamiento en la población. El último artículo de esta tesis, proporciona datos sobre el impacto de un PELF de cinco años en PNG. Los resultados obtenidos apoyan la estrategia descrita para las zonas con baja a moderada tasas de transmisión en regiones donde mosquitos anofelinos transmiten la infección (pe. Melanesia, África). Medidas adicionales o períodos más largos de tratamiento pueden ser necesarios en áreas con una alta tasa de transmisión. La experiencia adquirida en la Isla de Lihir en los programas de tratamiento masivo durante las campañas para la eliminación de la filariasis, será muy valiosa en la aplicación de una estrategia piloto para el control del pián. Además, en un futuro próximo podría ser importante vincular los programas para el control del pián con otros programas de tratamiento masivo (por ejemplo, filariasis) para aumentar la eficiencia y reducir los costos. El plan para la eliminación de la filariasis linfática en PNG fue aprobado como proyecto piloto en 2005 en la provincia de Milne Bay. El programa todavía tiene que ser extendido a un total de 20 provincias en el país, donde la filariasis es endémica. En este contexto, un enfoque integrado para el control de enfermedades tropicales olvidadas podría representar una importante solución global de salud pública en PNG. Poco se ha logrado en la última década en enfermedades tropicales desatendidas. Ahora estamos en una buena posición para traducir los frutos de nuestra investigación en políticas de salud. Durante una consulta celebrada en la sede de la OMS en Ginebra el pasado mes de marzo, ya se ha esbozado una nueva política de eliminación para el pián que toma como pilar el tratamiento con azitromicina. La intención de la OMS es que una última campaña global debe permitir llegar a cero casos de pián en 2017, y la posterior certificación de la interrupción de la transmisión en todo el mundo en el año 2020.

Previous studies have indicated that the promoters of the human filarial parasite Brugia malayi are unusual in that they do not exhibit the CAAT or TATAA sequences usually found in the core domains of promoters of most eukaryotic organisms. Analysis of the promoters of the ribosomal proteins showed that the region flanking the splice leader (SL) addition site plays an important role in transcription and may function as the core promoter domain in B. malayi. To test the hypothesis that the SL addition domain is the most important essential region of the ribosomal protein promoters, the SL addition site of the BmRPL13 gene was replaced with the SL addition domains from other ribosomal protein genes from B. malayi. The promoter activity of the replacement constructs were tested using a transient transfection dual luciferase assay. Promoter activity with RPL13 replacement constructs was correlated with that seen in the wild type promoters, suggesting that roughly 80% of the variations seen in promoter activity among ribosomal protein promoters is due to variation in the SL core promoter domain.