Foram testadas, com o teste não treponêmico VDRL e com o teste treponêmico imunoenzimático de competição, 23.531 amostras de soros, coletados em todas as regiões do Brasil, com o objetivo de verificar o comportamento do teste imunoenzimático treponêmico na triagem de amostras. A prevalência obtida foi de 0,63% com o VDRL e de 0,84% para o teste imunoenzimático. A análise dos dados foi feita comparando-se os resultados dos dois testes com os resultados do teste treponêmico de imunofluorescência indireta (FTA-ABS), considerado como teste de referência. No total, 1120 amostras foram submetidas ao teste FTA-ABS, incluindo todas as que foram reagentes em qualquer um dos testes de triagem e 872 amostras negativas. Amostras com resultados discordantes entre os testes foram submetidas a um teste imunoenzimático do tipo Western blot. Nas amostras por nós estudadas, o teste imunoenzimático apresentou sensibilidade de 89,95% e especificidade de 99,78%, muito superior aos 55,11% de sensibilidade e 97,43% de especificidade que encontramos para o VDRL. Os resultados dos testes detectaram positividade em amostras diferentes portanto, recomendamos utilizar a associação dos dois testes, como método de triagem, quando se trata de populações de baixa prevalência. Resultados preliminares do Western blot sugerem a participação doas proteínas de 43 kD, 17 kD e 15,5 kD na reação de ELISA treponêmico competitivo. / The VDRL, a non treponemal test, and a treponemal competitive ELISA were used to test 23,531 serum samples, collected from conscript men throughout Brazil, with the objective of assessing the performance of the competitive ELISA on the screening of serum samples. The VDRL showed a prevalence of 0.63% contrasting with a 0.84% prevalence showed by the competitive ELISA. The results obtained with the two tests were then compared to those obtained by fluorescent treponemal antibody absorption (FTA-ABS) test which is considered the gold standard method for detection of antibodies for syphilis. A total number of 1,120 samples, which included all that were reagent in at least one of the screening test plus 872 that were negative in both tests, were submitted to the FTA_ABS test. In addition, some of the samples that presented discrepant results between the two tests studied were also submitted to the Western blot test. The results of the screening tests showed an 89.95% sensitivity and a 99.78% specificity for the competitive ELISA, which are much higher than the 55.11% sensitivity and 97.43% specificity presented by the VDRL. Also, the tests detected positivity in different samples. In conclusion, we recommended the use in tandem of both tests as screening for syphilis antibodies in low prevalence populations. In addition, the results of the Western blot seemed to suggest the positivity of the ELISA becoming non reactive after treatment of the patient and that the 43 kD, 17 kD and 15 kD proteins are the main proteins involved in the ELISA competitive reaction.

A sífilis adquirida e a congênita continuam aumentando e preocupando as autoridades sanitárias do mundo, ao verem que as metas estabelecidas para o seu controle estão longe de serem atingidas. Apesar dos esforços feitos na descoberta de novas ferramentas para o diagnóstico e monitoramento da sífilis, vemos um despreparo muito grande, principalmente entre os laboratoristas, em processar corretamente os reagentes disponíveis e melhor selecionar aqueles que apresentem qualidade para serem usados na rotina do laboratório. O que constatamos neste estudo é um espelho da realidade do diagnóstico da sífilis e mostra a dificuldade que os clínicos enfrentam ao receberem um laudo do laboratório. Trabalhando em colaboração com diferentes serviços e grupos de pesquisa, selecionamos aqueles que trabalhavam com: gestantes atendidas no pré-natal, doadores de sangue, pacientes infectados pelo HIV e pacientes de laboratório clínico e fizemos um estudo crítico da sorologia utilizada em cada serviço. Verificamos discrepância dos resultados obtidos nos testes não-treponêmicos VDRL e RPR, principalmente entre os soros de baixa reatividade, e nos treponêmicos FTA-abs, TPHA e ELISA. Decidimos aplicar o método de Western blotting e analisar o seu comportamento em todas as amostras de soros ensaiadas. Para obtenção de resultados reprodutíveis, fizemos a industrialização do método e formatamos um reagente na forma de um kit diagnóstico, WB Tp-IgG, que pudesse ser facilmente utilizado e interpretado. Os resultados obtidos mostraram que o WB Tp-IgG pode ser utilizado como método confirmatório da sífilis, substituindo o FTA-abs, tradicionalmente recomendado para essa finalidade e que pudesse fazer parte de uma proposta de algoritmo para o diagnóstico sorológico da sífilis. / Acquired and congenital syphilis have been increased and worried the worldwide health authorities, mainly because the WHO targets for syphilis control are far from to be held. Much effort had been made for development of new tools to be used in syphilis diagnosis and following up, however we noticed a lack of ability of laboratory workers in the correctly choosing and using the reagents in laboratory routine. In this study what we observed were the reality of syphilis diagnosis and the difficulties that physicians have in how to procedure when they received the results from the laboratory. Working in collaboration with different settings and research groups we choose some of them that attend pregnant women, blood donors, HIV patients and patients from clinical laboratory. With these group individuals we made a critical study of the serology methods used in each one. We verified high level of discordant results between nontreponemal tests VDRL and RPR, mainly in serum samples with low reactivity and between treponemal tests FTA-abs, TPHA and ELISA, in all services. To obtain reproducibility of the results we made the industrialization of the method and set up a reagent as a kit diagnosis, easily to performer. Appling the western blotting method we evaluated the performance of the test in all sera samples assayed. The results showed that the WB Tp-IgG can be useful as confirmatory test for syphilis, replacing FTA-abs, traditionally recommended for this and that could be included in algorithm propose for serological diagnosis of syphilis.

Os principais marcadores sorológicos para o diagnóstico da toxoplasmose aguda ou recente são os anticorpos IgM específicos e anticorpos IgG de baixa avidez. Entretanto em alguns pacientes, anticorpos IgM e baixa avidez de anticorpos IgG podem persistir, ultrapassando o período da fase recente aguda contribuindo para erros de interpretação diagnóstica. No presente estudo, a eficiência diagnóstica do ensaio imunoenzimático foi avaliada, com o uso de frações antigênicas ou peptídeos sintéticos originados do antígeno ESA de T.gondii, denominados de SAG-1, GRA-1 e GRA-7. Foram estudadas frações isoladas e combinadas em múltiplos peptídeos antigênicos (MAP), visando estabelecer um perfil confiável para definição sorológica de toxoplasmose recente aguda em amostra única de soro. A melhor eficiência diagnóstica do ensaio foi encontrada com o uso da combinação de peptídeos SAG- 1,GRA-1 e GRA-7, denominada MAP1. A detecção de anticorpos IgG e IgM anti- MAP1 apresentou a melhor definição entre a fase recente aguda da fase recente não aguda na toxoplasmose. Nossos resultados mostraram que IgM anti-MAP1 poderá se constituir um marcador sorológico importante no aumento da eficiência diagnóstica da toxoplasmose recente aguda / The main serological marker for the diagnosis of recent toxoplasmosis is the specific IgM antibody, along with IgG antibodies of low avidity. However, in some patients these antibodies may persist long after the acute/recent phase, contributing to misdiagnosis in suspected cases of toxoplasmosis. In the present study, the diagnostic efficiency of ELISA was evaluated, with the use of peptides derived from T. gondii ESA antigens, named SAG-1, GRA-1 and GRA-7. In the assay referred to, we studied each of these peptides individually, as well as in four different combinations, as Multiple Antigen Peptides (MAP), aiming to establish a reliable profile for the acute/recent toxoplasmosis with only one patient serum sample. The diagnostic performance of the assay using MAP1, with the combination of SAG-1, GRA-1 and GRA-7 peptides, demonstrated better discrimination of the acute/recent phase from non acute/recent phase of toxoplasmosis. Our results show that IgM antibodies to MAP1 may be useful as a serological marker, enhancing the diagnostic efficiency of the assay for acute/recent phase of toxoplasmosis

Na prática hemoterápica em todo mundo, a triagem e o diagnóstico da infecção pelo HCV continua sendo um desafio. Desta forma, considerando as variáveis genéticas do vírus da Hepatite C e as variáveis populacionais de cada região, o conhecimento mais profundo da realidade da epidemiologia molecular do vírus da Hepatite C na população de doadores de sangue em regiões específicas se torna cada vez mais imprescindível. Desta forma, este estudo visou realizar a caracterização sorológica e molecular da infecção pelo Vírus da Hepatite C (HCV) em doadores de sangue do Estado do Amazonas a fim de subsidiar conhecimentos para interpretação dos diferentes perfis laboratoriais e clínicos, além de contribuir para o conhecimento das rotas de transmissão e da epidemiologia da infecção do Amazonas. Foram estudados 154 doadores de sangue do Estado do Amazonas com sorologia repetidamente reativa para anti-HCV. Foram realizados testes sorológicos por ELISA e Imunoblot, testes de detecção de RNA viral plasmático por Nested PCR, determinação da carga viral e genotipagem do vírus por sequenciamento. O estudo foi realizado no período de setembro de 2005 a abril de 2007 quando o índice de descarte por anti-HCV reativo foi de 0,37%. Foi observado que 50% dos casos de Imunoblot indeterminado apresentaram positividade no Nested PCR indicando a presença de RNA plasmático. A carga viral baixa foi um fator limitante para a determinação do genótipo viral pelo método de sequenciamento de algumas amostras. A freqüência de casos presumidos de clearance viral plasmático na amostra estudada foi de 18,8%. O genótipo mais prevalente do HCV nos doadores do Estado do Amazonas foi o genótipo 1 (87,5%) seguido do genótipo 3 (12,5%). Considerando as nuances da história natural da Hepatite C e os diversos momentos clínicos que os doadores possam se encontrar devem ser adotadas mudanças de condutas do acompanhamento dos doadores sororeativos para anti-HCV no banco de sangue do Amazonas e no fluxograma de diagnóstico da infecção. / The screening and diagnostic of HCV infection continue to be a challenge on the hemotherapic practice because the unique characteristics of each population and the molecular variability of the virus. The aim of this study was to characterize the serologic and molecular profile of 154 anti-HCV+ Amazon blood donors from the Amazon Hematology and Hemoterapy Foundation. Screening for anti HCV antibody was performed using ELISA and recombinant Imunoblot assay. Seropositive samples were assessed further with Nested PCR, viral load and genotyping techniques. In the study period, 2005-2007 the anti-HCV discard rate was 0,37%. We observed that 50% of the indeterminate Immunoblot cases showed HCV RNA presence in plasma by Nested PCR. The most prevalent genotype between subjects of this study was genotype 1 (87,5%), followed by genotype 3 (12,5%). The presumed plasma clearance frequency was 18,8%. The low viral load was a determinant critical factor to the genotype determination in some samples. Considering the different stages of the HCV natural infection different conducts may be adopted with inclusion of molecular testes as the first option for confirmation to the follow up of the positive anti-HCV blood donors in the Amazon blood bank and in the algorithm of the infection diagnostic, saving resources and providing a better counseling for the reactive donors.

Das Cherry leaf roll virus (CLRV) ist ein weltweit verbreitetes Pflanzenvirus, das eine Vielzahl an Laubgehölzen und Stauden infiziert. In der vorliegenden Arbeit wurden phylogenetische und serologische Analysen der Populationsstruktur des CLRV durchgeführt und ihre Korrelation mit epidemiologischen Faktoren, wie der geographischen Verbreitung und der Wirtspflanzenart, untersucht. Der Nachweis von CLRV erfolgte mittels Immunocapture - Reverse Transkription -Polymerase Kettenreaktion (IC-RT-PCR). Dabei konnte CLRV in 20 verschiedenen Gehölzarten an 33 Standorten in Deutschland nachgewiesen werden. Außerdem wurden Isolate aus 6 anderen Ländern, die von Kollegen zur Verfügung gestellt worden waren, in die Untersuchungen einbezogen. Der Vergleich der Symptomausprägungen auf verschiedenen Testpflanzen zeigte keine auffälligen biologischen Unterschiede bei den gewonnenen CLRV-Isolaten. Untersuchungen zur RNA-Populationsstruktur basierend auf einem 380 bp langen Teilbereichs der 3’-nicht-translatierten Region (3’UTR) der genomischen RNA1 und RNA2 zeigte eine homogene Basenzusammensetzung innerhalb der Virusisolate. Hingegen traten zwischen den 73 untersuchten CLRV-Isolaten Sequenzunterschiede bis zu 15,5 % auf. Die phylogenetische Analyse der 3’UTR deckte eine Gruppierung der Virusisolate nach den natürlichen Wirtspflanzenarten auf, die durch statistischen Analysen mittels GST-Koeffizient bzw. Mantel-Test verifiziert werden konnten. Der Vergleich der phylogenetischen mit der serologischen Gruppierung, die unter der Verwendung monoklonaler Antikörper analysiert wurde, zeigte für 24 CLRV-Isolate eine hohe Korrelation in Bezug auf die Gruppenzuordnung. Auch beim phylogenetischen Vergleich der Hüllprotein-Sequenzen für 9 CLRV-Isolate ergaben sich die gleichen Gruppen. Innerhalb der 380 bp langen 3’UTR wurde mittels Computer-Modellierung der Sekundärstruktur unter Verwendung von 67 CLRV-Sequenzen zwei konservierte Stemloops identifiziert, die die Ergebnisse anderer Autoren bestätigen und die funktionelle Bedeutung der 3’UTR belegt. / Cherry leaf roll virus (CLRV) is worldwide distributed and is infecting a variety of deciduous trees and shrubs. In this study phylogenetic and serological analyses of the population diversity of CLRV and the correlation with the epidemiological factors geographical distribution and host plant species, have been investigated. During a survey in Germany plants were tested by IC-RT-PCR and virus isolates recovered from a range of woody plants from different geographical regions. CLRV was detected in 20 different plant species from 33 locations in Germany. Also isolates from 6 other countries received from colleagues were included in the study. Comparison of symptom expression on different indicator plants did not show obvious biological differences between the recovered CLRV isolates. Investigations of the RNA population structure based on a 380 bp long fragment of the 3''-non-translated region (3''UTR) of genomic RNA1 and RNA2 revealed a homogeneous base composition for single isolates. However, between 73 CLRV isolates 3’UTR sequences showed up to 15.5 % divergence. Phylogenetic analysis of the 3''UTR uncovered a grouping of the virus isolates according to the natural host plant species, which was verified by statistic analyses using GST coefficient and Mantel test. The comparison of the phylogenetic grouping with the serological grouping of 24 selected CLRV isolates, which was analyzed using a set of seven monoclonal antibodies, showed a high correlation regarding the group arrangement. The phylogenetic comparison of the coat protein sequences for 9 CLRV isolates also revealed the same group arrangement. Secondary structure analysis by computer modelling of the consensus sequence of the 380 bp long 3''UTR using 67 CLRV sequences identified two conserved stem loop regions supporting the results of other authors and indicating the functional significance of the 3''UTR.

<p>This thesis analyses several factors affecting the control of triatomines in Guatemala. There are three synantropic triatomines in Guatemala, i.e., <i>Rhodnius prolixus</i>, <i>Triatoma dimidiata</i> and <i>T. nitida</i>. Their distibution is mainly at an altitude between 800 and 1500 m a.s.l. <i>R. prolixus</i> and <i>T. nitida</i> have localized but scaterred distibution while <i>T. dimidiata</i> is present in 21 of the 22 departments in the country. Several investigations have shown that <i>R. prolixus</i> could be relatively easily eradicated while <i>T. dimidiata</i> may be more difficult to control, since it is present in domestic, peridomestic and sylvatic environments showing high diversity and a variety of epidemiological characteristics. Based on the incidence of <i>Trypanosma cruzi</i> infection in humans in the distributional areas of the triatomines, <i>R. prolixus</i> appear to be a more competent vector than <i>T. dimidiata</i>. This is despite the fact that these vectors have similar infection rates. Inside houses, <i>R. prolixus</i> and <i>T. dimidiata</i> and in artificial environments, <i>T. ryckmani</i> and <i>T. dimidiata</i>, preferred the northern side of the walls. Therefore, selective application of insecticides should focus on walls and furniture located in the northern part of the house. House improvements reduced the infestation of triatomines, and could be used as a complement to insecticidal spraying. Although <i>T. dimidiata</i> is not an efficient vector its wide distribution, versatility in occupying different habitats and capacity to disperse render this species difficult to control in Central America. Thus, only few months after insecticidal spraying <i>T. dimidiata</i> had reinfested the domestic environments. Morphometic methodology and genetic markers have been developed to differentiate within-species populations of <i>T. dimidiata</i> and <i>T. nitida</i>. Studies on the migration patterns of sylvatic <i>T. dimidiata</i> and <i>T. ryckmani</i> have been performed in order to clarify the colonization patterns. The adults migrate, in particular, in the dry part of the year. This finding may be of help in attempts to control <i>T. dimidiata</i>.</p>

Groundnut or peanut (Arachis hypogaea L.) is an important legume cultivated in several developing countries in the tropics and subtropics. It plays a significant role as a food crop in regions with alarming population growth rates. The disease “peanut clump”, which is caused by viruses in the genus Pecluvirus, has been reported from India and from several countries of West Africa. In India, the causal agent is the Indian peanut clump virus (IPCV), which is transmitted by a soil-borne root parasite, Polymyxa graminis. The virus is also transmitted by infected seed and so far no economical method of control has been found. Therefore efforts have been concentrated on understanding the epidemiology of peanut clump disease with the aim of devising cultural methods of control. The work addressed in this thesis describes how investigation in various aspects of clump disease epidemiology, including identification of alternative hosts of the virus and the vector, and of factors that contribute to survival and spread of inoculum, has led to formulation of simple cultural practices that could reduce disease incidence.

Groundnut or peanut (Arachis hypogaea L.) is an important legume cultivated in several developing countries in the tropics and subtropics. It plays a significant role as a food crop in regions with alarming population growth rates. The disease “peanut clump”, which is caused by viruses in the genus Pecluvirus, has been reported from India and from several countries of West Africa. In India, the causal agent is the Indian peanut clump virus (IPCV), which is transmitted by a soil-borne root parasite, Polymyxa graminis. The virus is also transmitted by infected seed and so far no economical method of control has been found. Therefore efforts have been concentrated on understanding the epidemiology of peanut clump disease with the aim of devising cultural methods of control. The work addressed in this thesis describes how investigation in various aspects of clump disease epidemiology, including identification of alternative hosts of the virus and the vector, and of factors that contribute to survival and spread of inoculum, has led to formulation of simple cultural practices that could reduce disease incidence.

This thesis analyses several factors affecting the control of triatomines in Guatemala. There are three synantropic triatomines in Guatemala, i.e., Rhodnius prolixus, Triatoma dimidiata and T. nitida. Their distibution is mainly at an altitude between 800 and 1500 m a.s.l. R. prolixus and T. nitida have localized but scaterred distibution while T. dimidiata is present in 21 of the 22 departments in the country. Several investigations have shown that R. prolixus could be relatively easily eradicated while T. dimidiata may be more difficult to control, since it is present in domestic, peridomestic and sylvatic environments showing high diversity and a variety of epidemiological characteristics. Based on the incidence of Trypanosma cruzi infection in humans in the distributional areas of the triatomines, R. prolixus appear to be a more competent vector than T. dimidiata. This is despite the fact that these vectors have similar infection rates. Inside houses, R. prolixus and T. dimidiata and in artificial environments, T. ryckmani and T. dimidiata, preferred the northern side of the walls. Therefore, selective application of insecticides should focus on walls and furniture located in the northern part of the house. House improvements reduced the infestation of triatomines, and could be used as a complement to insecticidal spraying. Although T. dimidiata is not an efficient vector its wide distribution, versatility in occupying different habitats and capacity to disperse render this species difficult to control in Central America. Thus, only few months after insecticidal spraying T. dimidiata had reinfested the domestic environments. Morphometic methodology and genetic markers have been developed to differentiate within-species populations of T. dimidiata and T. nitida. Studies on the migration patterns of sylvatic T. dimidiata and T. ryckmani have been performed in order to clarify the colonization patterns. The adults migrate, in particular, in the dry part of the year. This finding may be of help in attempts to control T. dimidiata.

Borrelios är den vanligaste fästingburna infektionen på norra halvklotet, och orsakas av spiroketer tillhörande Borrelia burgdorferi sensu lato-komplexet. Dessa bakterier kan spridas till flera organ och ge upphov till olika symptom i bland annat hud, nervsystem, leder och hjärta. Omkring 15 % utvecklar neurologiska symptom, så kallad neuroborrelios. Den bästa indikatorn på aktiv neuroborrelios är framförallt karakteristiska neurologiska symptom samt tecken på en inflammatorisk förändring i cerebrospinalvätskan (CSV) i kombination med lokalt producerade antikroppar mot Borrelia burgdorferi s.l. i CSV. Nuvarande metod för diagnostik av neuroborrelios är en immunokemisk metod, en ELISA (enzyme-linked immunosorbent assay) som bygger på en jämförelse av Borrelia-antikroppsnivåer i CSV och i serum genom beräkning av antikroppsindex (AI). Beräkning av AI kompenserar för en eventuell ospecifik överföring av antikroppar från serum, till följd av en skada på blod-hjärnbarriären. Det finns dock tecken på att den nuvarande analysmetoden har för låg sensitivitet med falskt negativa resultat, framförallt tidigt i infektionsförloppet. För diagnostik av andra former av borrelios än neuroborrelios används en typ av ELISA baserad på C6-peptid. C6-peptid ELISA visar god känslighet för detektion av B. burgdorferi s.l.-specifika antikroppar i serum. C6-antigenet utgör en starkt immunogen och konserverad region av bakteriens VlsE-ytprotein. Syftet med den här studien var att undersöka om detektion av antikroppar mot C6-peptid i CSV kan komplettera den nuvarande använda metoden och därmed förbättra den totala sensitiviteten för diagnostik av neuroborrelios. I studien analyserades 169 patientprover från unga personer, samt 18 oklara patientfall som tidigare bedömts negativa med den nuvarande metoden. Antikroppar mot C6-peptid detekterades hos åtta unga patienter samt två oklara patientfall. Av dessa hade åtminstone tre unga patienter sannolikt neuroborrelios. Resultat från den här studien tyder på att C6-peptid-ELISA på CSV-prover kan fungera som ett komplement till befintlig metod för diagnostik av neuroborrelios. En kombination av båda metoderna kan sannolikt ge en betydligt högre sensitivitet. Vid tolkning av resultat från C6-peptid-baserade analysmetoder på CSV ska hänsyn tas till eventuell ospecifik överföring av B. burgdorferi s.l.-specifika antikroppar genom blod-hjärnbarriären. / Lyme Borreliosis, caused by spirochetes of the Borrelia burgdorferi sensu lato-complex, is the most common tick-borne infection in the temperate regions of the northern hemisphere. The bacteria can infect many different organs, this can give rise to a variety of symptoms in skin, the nervous system, joints and heart. Approximately 15 % of the infected individuals show neurological symptoms referred to as neuroborreliosis. An active neuroborreliosis is indicated by inflammatory changes in the cerebrospinal fluid (CSF) and local synthesis of anti-Borrelia antibodies in CSF. The current method to diagnose neuroborreliosis is an enzyme-linked immunosorbent assay (ELISA) which compares levels of anti-Borrelia antibodies in CSF and serum by calculating an antibody index (AI). Calculations of AI compensate for unspecific leakage of antibodies from serum to CSF following an injury of the blood-brain barrier. The drawback of the current method is a low sensitivity with a high rate of false negative results in samples collected early during an infection. Another type of ELISA, based on the use of a C6 peptide, has earlier shown good sensitivity for detection of B. burgdorferi s.l.-specific antibodies in serum. The C6 antigen corresponds to a highly immunogenic and conserved region of the bacterial surface protein VlsE. The aim of this study was to investigate whether a detection of antibodies against the C6 peptide in CSF could improve the total sensitivity for the diagnostics of neuroborreliosis. In the current study, 169 samples with negative AI from young patients and 18 samples from special cases were analyzed. Antibodies against the C6 peptide were found in 8 young patients and in 2 samples from special cases. Out of these, 3 young patients were stated positive for neuroborreliosis. Results of this study show that the C6 peptide ELISA on CSF samples could act as a complement to the current serological method for diagnosing neuroborreliosis. A combination of both methods could possibly increase the overall sensitivity. However, the blod-brain barrier injury issue is a problem in the analysis and interpretation of the results of the C6 peptide-based method on CSF should take into consideration a possible dysfunction of the blood-brain barrier. In conclusion, a combination of both the current method and the C6 peptide ELISA could give a markedly improved sensitivity in diagnostics of neuroborreliosis.