Historically, diagnosis has relied on clinical signs of disease, microscopy and serological testing. However, these approaches have a number of drawbacks for example, differential diagnosis, low sensitivity (microscopy) and the inability to differentiate past from current infections (serology). In the past decade the use of molecular techniques, such as the polymerase chain reactions (PCR) have gained favour. Many research groups have used these techniques to study the molecular epidemiology of diseases in sub-Saharan Africa. Such methodologies rely on the detection of genetic materials and as such are reliant on the specificity of their components and the quality of the starting materials. It is the aim of this thesis is to demonstrate improvements that can be made to sample collection that will help to enhance the reliability of these tests and highlight the importance of the diagnostic parameters. The model that I will use to demonstrate these improvements are African trypanosomes, these are the causative agents of sleeping sickness in humans and nagana in animals, and are wide spread across much of sub-Saharan Africa. My work will be presented as three main sections: Firstly, a comparison of the suitability of various different approaches to cattle blood sample collection – including the genetic materials prepared directly in the field and the use of Whatman FTA®cards – in terms of the provision of appropriate materials for molecular screening will be presented. It was found that uneven distribution of genetic materials occurs across the surface of the FTA®cards due to the matrix chemistry. Therefore suggestions for improvements for the preparation of materials to be stored on these cards and their downstream application are made. Secondly, a comparison between the specificity of the pan-Trypanosoma ITS-PCR reaction and the species-specific reactions is made. The ITS-PCR has gained favour in recent years as it is reported to be capable of identifying a wide range of trypanosomes, as this is a single nested PCR reaction the reduction in time and cost has been very appealing to researchers in this field. My work suggests that this test is not reliable in terms of the accurate detection of trypanosomes species, and in fact on a direct comparison of 969 samples, 37 parasitic events where identified by this approach compared to 197 when species-specific tests were applied. Thirdly, based on my findings from the previous two chapters I present two case studies, the first of which looks to evaluate the impact on the prevalence of trypanosome species in cattle after drug treatment during the Ugandan, Stamp Out Sleeping sickness (www.sleepingsickness.com) campaign. The results of this case study highlight the importance of understanding the relationship that occurs between trypanosome species in mixed infections, my second case study therefore looks to quantifying the infection load of Trypanosoma brucei and T. congolense within the midgut of their insect vector (Glossina morsitans morsitans) using qPCR.

Background Patient safety is an undisputable part of healthcare. The use of clinical practice guidelines, usually based on evidence-based practice/best practice, promotes patient safety and high quality care, reduces unnecessary patient suffering, and healthcare costs. Analysing results from venous blood specimen collection is one of the most commonly used services within healthcare, and a substantial number of decisions on diagnosis, treatment, and treatment evaluation are based on the results. Hence, the accuracy of these tests are vitally important. Earlier research has demonstrated that healthcare staff report suboptimal adherence to venous blood specimen collection guidelines together with the need for improved practices. Blood sample collection is carried out by several professionals, among them registered nurses and, as a consequence, nursing students too. University nursing students learn and practice venous blood specimen collection in one of their first semesters. After initial skill training at clinical skill laboratories, they continue to perform the task during clinical placements in various clinical settings. Few or no studies have been performed on nursing students, hence it seemed important to assess guideline adherence to venous blood specimen collection among university students as well as to further explore adherence to guidelines among healthcare staff. Therefore, the overall aim for this thesis was to explore adherence to, and factors influencing venous blood specimen collection guidelines practice among university nursing students and healthcare staff. Methods The thesis includes four studies. Study I-III had a quantitative, cross-sectional design, study IV had a qualitative approach. Study I included 164 healthcare staff from 25 primary healthcare centres. Study II included 101 nursing students in their 5th and 6th semesters, and study III included 305 nursing students in their 2nd, 4th, and 6th semesters. To assess adherence to venous blood specimen collection guidelines, data were collected using the Venous Blood Specimen Questionnaire, completed with background variables (I, II, III) and additional scales (III). Descriptive statistics, multilevel and multiple logistic regression analyses were used to analyse the data. In study IV, data were collected through five focus group interviews among 6th semester nursing students (n=26). Data were analysed using qualitative content analysis. Results Workplace affiliation was found to explain variances in reported adherence between different primary healthcare centres. Associations between reported venous blood specimen collection practices and individual as well as workplace factors were revealed. Nursing students were found to increasingly deviate from guideline adherence during their education. Also among students, several associations between guideline adherence and other iv factors were revealed. Reported research use at clinical practice was associated with higher levels of adherence, as were higher capability beliefs regarding both evidence-based practice and academic ability. Analyses from focus group interviews summarised students’ reflections on deviations from VBSC guidelines in the overall theme ‘Striving to blend in and simultaneously follow guidelines’. Conclusion Both healthcare staff at primary healthcare centres and nursing students demonstrate decreasing levels of guideline adherence with time. Factors influencing adherence are both individual as well as contextual. This indicate that both students and staff are subjected to socialisation processes that influences levels of adherence. In order to enhance venous blood specimen collection practices and thereby patient safety, actions must be taken - both in healthcare clinical contexts and by educators. The use of models in practical skill training, and in the ambition to bridge the theory-practice gap may be the path to success. It is reasonable to assume that collaboration between, on the one hand, education representatives and on the other, supervising RNs in clinical settings, will be fruitful. Finally, by empowering students their self-efficacy may be strengthened, and hence their ability to maintain guideline adherence.

A punção venosa periférica é a inserção de dispositivos por veia periférica para acesso à corrente sanguínea, como na coleta de sangue para exames; embora seja um procedimento comum no tratamento intra-hospitalar, é complexo e exige competência profissional. A realização desse procedimento de forma inadequada pode colocar em risco a saúde do paciente, podendo provocar diversas complicações ao seu tratamento. A pesquisa teve como objetivo desenvolver e validar a primeira versão de um simulador de realidade virtual (RV) imersiva para o procedimento de punção venosa periférica para coleta de sangue a vácuo no paciente adulto. Revisão integrativa da literatura permitiu verificar as estratégias de ensino de punção venosa periférica na enfermagem. Procedeu-se, em seguida, um estudo com delineamento metodológico para desenvolver e validar a primeira versão do simulador. O uso de recursos tecnológicos como estratégias de ensino em enfermagem está em expansão, porém a simulação com RV, principalmente no ensino de punção venosa periférica, ainda é um vasto campo a ser explorado. Foi desenvolvida a primeira versão de um simulador de RV para a coleta de sangue à vácuo no adulto - o VIDA-Enfermagem v1.0, o qual foi avaliado por profissionais e graduandos de enfermagem. Foram considerados válidos 79,6% dos itens avaliados pelos profissionais e 66,7% dos itens avaliados pelos graduandos. As sugestões propostas pelos sujeitos da pesquisa para melhorias do sistema, em sua maioria, são passíveis de aceitação no decorrer do incremento das versões seguintes. Mesmo havendo necessidade de revisão de diversos itens, na avaliação dos participantes, o simulador foi considerado uma ferramenta promissora e inovadora para o ensino do procedimento de punção venosa periférica para coleta de sangue a vácuo em paciente adulto, para graduandos de enfermagem que estão se iniciando no estudo da temática e da técnica, como estratégia a ser combinada com os recursos já utilizados atualmente no ensino do procedimento. É relevante que se invista em estratégias inovadoras e motivadoras para aplicação no ensino de enfermagem, principalmente com o uso da realidade virtual imersiva / Peripheral venipuncture is the insertion of a peripheral venous device to access bloodstream, such as in blood collection for tests; although it is a common procedure in in-hospital treatment, it is complex and requires professional competence. Failure to perform this procedure may put the patient\'s health at risk, and may cause several complications to his/her treatment. This study aimed to develop and validate the first version of an immersive virtual reality (VR) simulator for the peripheral venipuncture procedure for vacuum blood collection in adult patient. Teaching strategies of peripheral venipuncture in nursing were verified through an integrative literature review. Then, a study with a methodological design was performed to develop and validate the first version of the simulator. The use of technological resources such as teaching strategies in nursing is expanding; however, the simulation with VR, mainly in the teaching of peripheral venipuncture, is still a vast field to be explored. The first version of a VR simulator for vacuum blood collection in adult patient was developed - VIDA-Enfermagem v1.0, which was evaluated by professionals and undergraduate nursing students. 79.6% of the items evaluated by the professionals and 66.7% of the items evaluated by the undergraduate students were considered valid. The suggestions proposed by the research subjects for system improvements, for the most part, may be included in the following versions. Although it was necessary to review several items, in the evaluation of the participants, the simulator was considered a promising and innovative tool for the teaching of the peripheral venipuncture procedure for vacuum blood collection in adult patients, for nursing undergraduate students who are beginning the study of the subject and technique, as a strategy to be combined with the resources already used in the teaching of the procedure. It is relevant to invest in innovative and motivating strategies for application in nursing teaching, especially with the use of immersive virtual reality

O diagnóstico rotineiro da dengue é realizado com amostra de sangue dos casos suspeitos. A coleta tradicional de sangue (por punção venosa) é um procedimento que dificulta a realização de exames e pesquisas por ser um procedimento invasivo que nem sempre é prático para crianças e bebês, requer pessoal especializado e necessita de um local para armazenamento da amostra sob refrigeração ou congelamento. O propósito deste estudo foi coletar amostras por punção digital com uma nova tecnologia (FTA Card) e compará-la com amostras coletadas por punção venosa, avaliando-as através de uma técnica molecular de PCR em tempo real. Sendo o PCR em Tempo Real a técnica molecular atualmente disponível de maior rapidez, sensibilidade e especificidade, padronizamos uma metodologia de passo único de PCR em Tempo Real com SYBR green baseando-se na região 3 não codificante do vírus e utilizando primers degenerados, capazes de detectar os quatro sorotipos de uma só vez. A avaliação das técnicas de coleta e amplificação foram feitas com amostras suspeitas de dengue, obtidas em Goiânia durante surto ocorrido no ano de 2008. O limite de detecção da reação padronizada no presente estudo foi de aproximadamente 100 cópias/ml e uma especificidade de 100%. Para tipagem das amostras positivas a técnica empregada foi o PCR multipex. Dentre as 89 amostras coletadas 60 (67%) foram positivas para àquelas coletadas por punção venosa e 14 (16%) para àquelas coletadas por punção digital. Dentre as 89 amostras para o PCR em Tempo Real, apenas 29 (32%), foram tipadas pelo método de PCR multiplex, sendo 3 casos do vírus da dengue 1 (10%), 16 casos do vírus da dengue 2 (55%), e 10 casos do vírus da dengue 3 (35%). Descritores: Dengue/diagnóstico, coleta de amostras sanguíneas, reação em cadeia da polimerase, corantes fluorescentes / The collection by venipuncture is a procedure that is difficult to carry out in diagnosis and research because it is an invasive procedure that is not always practical for children and babies , requires specialized staff, needs a place to store the sample under refrigeration or forzen, This study aimed to collect samples by fingerstick puncture with a new technology named FTA card and compare it with samples collected by venipuncture, using a realtime PCR to evaluate if FTA card collection would have a similar performance to standard blood sampling. Towards that, we obtained viral load values in order to estimate the differences not only qualitatively (e.g. pos or neg) but also in numbers.. We used an one-step SYBR Green I Real-Time PCR based on the region 3 \'noncoding virus using degenerate primers which was able to detected all four serotypes of dengue virus. Among the 89 samples collected 60 (67%) were positive for those collected by venipuncture and 14 (16%) to those collected by fingerstick, Only 29 (32%) were possible to be typed by PCR multiplex. Three cases were dengue virus 1 (10%), 16 cases were dengue virus 2 (55%) and 10 cases were dengue virus 3 (35%). The limit of detection obtained was approximately 100 copies / ml and aspecificity of 100% was observed. Keywords: Dengue / diagnosis, collection of blood samples, polymerase chain reaction, fluorescent dyes

Background Venous blood tests constitute an important part in the diagnosis and treatment of patients. However, test results are often viewed as objective values rather than the end result of a complex process. This has clinical importance since most errors arise before the sample reaches the laboratory. Such preanalytical errors affect patient safety and are often due to human mistakes in the collection and handling of the sample. The preanalytical performance of venous blood testing in primary health care, where the majority of the patients contact with care occurs, has not previously been reported. Aims To investigate venous blood sampling practices and the prevalence of haemolysed blood samples in primary health care. Methods A questionnaire investigated the collection and handling of venous blood samples in primary health care centres in two county councils and in two hospital clinical laboratories. Haemolysis index was used to evaluate the prevalence of haemolysed blood samples sent from primary health care centres, nursing homes and a hospital emergency department. Results and discussion The results indicate that recommended preanalytical procedures were not always followed in the surveyed primary health care centres. For example, only 54% reported to always use name and Swedish identification number, and 5% to use photo-ID, the two recommended means for patient identification. Only 12% reported to always label the test tubes prior to blood collection. This increases the possibility of sample mix-up. As few as 6% reported to always allow the patient to rest at least 15 minutes before blood collection, desirable for a correct test result. Only 31% reported to have filed an incident report regarding venous blood sampling, indicating underreporting of incidents in the preanalytical phase. Major differences in the prevalence of haemolysed blood samples were found. For example, samples collected in the primary health care centre with the highest prevalence of haemolysed samples were six times (95% CI 4.0 to 9.2) more often haemolysed compared to the centre with the lowest prevalence. The significant variation in haemolysed samples is likely to reflect varying preanalytical conditions. Conclusions This thesis indicates that the preanalytical procedure in primary health care is associated with an increased risk of errors with consequences for patient safety and care. Monitoring of haemolysis index could be a valuable tool for estimating preanalytical sample quality. Further studies and interventions aimed at the preanalytical phase in primary health care are clearly needed.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2009. / Härtill 6 uppsatser.

Background: About 60%–80% of decisions regarding diagnosis and treatment are based on laboratory test results. Low adherence to venous blood specimen collection (VBSC) guidelines may lead to erroneous or delayed test results, causing patient harm and high healthcare costs. Educational intervention programs (EIPs) to update, improve and sustain VBSC practices are seldom evaluated. After testing a self-reported venous blood sampling questionnaire, the overall aim of this thesis was to evaluate the impact of a large-scale EIP on healthcare personnel’s VBSC practices. Methods: The study settings were primary healthcare centres (PHCs) in northern Sweden. Participants were VBSC personnel. Data consisted of a VBSC questionnaire of self-reported practices, records of low-level haemolysis index in serum samples (specimen quality indicator), and interviews reflecting VBSC practices. First, experts on questionnaires and VBSC were consulted, and test-retest statistics were used when testing the VBSC questionnaire for validity and reliability. Thereafter, we evaluated the impact of a short, large-scale EIP with a before-after approach comparing self-reported VBSC questionnaire of two county councils. The personnel of the county councils (n = 61 PHCs) were divided into an intervention group (n = 84) and a corresponding control group (n = 79). In order to test changes in blood specimen quality we monitored haemolysis in serum samples (2008, n = 6652 samples and 2010, n = 6121 samples) from 11 PHCs. Finally, 30 VBSC personnel from 10 PHCs reported their experiences. The interview questions were open-ended with reflective elements and the interviews were analysed by qualitative content analysis. Results: The VBSC questionnaire was found to be valid and could be used to identify risk of errors (near misses) and evaluate the impact of an EIP emphasising VBSC guideline adherence. The intervention group demonstrated several significant improvements in self-reported practices after the EIP, such as information search, patient rest, test request management, patient identification, release of venous stasis, and test tube labelling. The control group showed no significant improvements. In total, PHCs showed minor differences in blood specimen quality. Interviews summarized VBSC personnel experiences in the overall theme: education opened up opportunities for reflection about safety.   Conclusion: This thesis is, to our knowledge, the first to evaluate the impacts of a large-scale EIP on VBSC practices. The VBSC questionnaire and monitoring for low-level haemolysis reflected VBSC practices. The frequently occurring near-miss markers made it possible to compare and benchmark VBSC practices down to the healthcare unit and hospital ward. The short, general EIP opened up opportunities for reflection about safety and improved VBSC practices in PHCs with larger deviations from guidelines. EIPs that provide time for reflection and discussion could improve VBSC further. Directed EIPs focused on specific VBSC flaws might be more effective for some near misses in VBSC practices, while some near misses must be changed at a different level in the system. Clinical relevance: Our results indicate that monitoring and counteracting the near misses in VBSC practices is a well-functioning preventive action. We propose that the VBSC monitoring instruments (VBSC questionnaire & haemolysis index) we used and the EIP strategy proposed should be tested in additional countries with different healthcare settings. It is suggested that a national program intended to identify near misses and prevent VBSC errors be developed in the healthcare system. General e-learning programs may be cheaper than, and as effective as, the EIP program and may be performed everywhere and any time. Systematic planning, useful for reflection and with focus on the specific elements in a skill, together with VBSC guidelines, could probably increase improvements. Our studies have led to deeper and extended knowledge of the impact of an EIP on VBSC practices. Our results can be used when considering future VBSC practice interventions. Using a model for practical skills in nursing to describe VBSC in a more holistic and less technical way might highlight VBSC as a practical nursing skill. / Bakgrund: Av kliniska beslut angående diagnostik och behandling baseras 60%–80% på laboratorieresultat. Därför är det helt nödvändigt att laboratorieresultat är tillförlitliga. Låg följsamhet till provtagnings anvisningar kan leda till felaktiga och fördröjda analysresultat, förorsaka skada och lidande för patienter och utgöra en stor kostnad för hälso- och sjukvården. Felaktiga provsvar beror till stor del på felaktig provtagning och provhantering och går oftast att undvika. Interventioner som avser att uppdatera och säkra korrekt venprovtagning kan leda till förbättringar men genomförda interventioner har sällan utvärderats. Efter att en enkät för självrapporterad venprovtagning testats för validitet och reliabilitet genomfördes ett omfattande interventionsprogram som utvärderades med hjälp av den testade enkäten och andra utvärderingsmått. Det övergripande syftet var att utvärdera i vilken utsträckning interventionsprogrammet påverkade provtagande personals praktiska utförande av venprovtagning. Metoder: Studierna i denna avhandling omfattade provtagande personal vid hälsocentraler i norra Sverige. För datainsamling användes en enkät som mäter självrapporterad venprovtagning, förekomst av låggradig hemolys (indikator på blodprovets kvalitet) och intervjuer. Initialt testades enkätens förmåga att mäta vad som avsetts (validitet) och testades enkätens förmåga att vid upprepade mätningar vara tillräckligt stabil (reliabilitet) för att användas i interventionsstudier. Därefter utvärderades ett kort men storskaligt interventionsprogram i preanalys inkluderande venprovtagning med före och efter mätningar. Vi jämförde provtagande personal från två landsting vid 61 hälsocentraler. Landstingens personal delades upp i en interventionsgrupp (n=84) och en motsvarande kontrollgrupp (n = 79). För att mäta kvaliteten av blodproverna extraherades uppgifter om hemolys i serumprover (2008, n = 6652 blodprov) och (2010, n = 6121 blodprov) från elva hälsocentraler i ett landsting. Slutligen, intervjuades 30 provtagande personal från 10 hälsocentraler efter att de deltagit i interventionsprogrammet. Intervjuerna var öppna och genererade korta berättelser och analyserades med innehållsanalys. Resultat: Venprovtagningsenkäten befanns vara valid och kan användas för att utvärdera personalens följsamhet till provtagningsanvisningar i venprovtagning och identifiera riskhändelser. Interventionsgruppen visade flera signifikanta förbättringar i självrapporterat utförande av venprovtagning såsom förbättrad informationssökning, vila inför provtagning, remissförfarande, kontroll av patientidentitet, användning av stas och etikettering av provrör. Kontrollgrupen visade inga signifikanta förbättringar. Blodprovskvaliteten visade små skillnader. Provtagande personals erfarenheter från intervjuerna sammanfattades i ett övergripande tema; utbildningsinsatsen öppnade upp möjligheter för reflektion om säkerhet.   Slutsats: Avhandlingen är så vitt vi vet den första att utvärdera effekten av ett storskaligt interventionsprogram med hjälp av självrapporterat utförande av venprovtagning och blodprovers kvalitet (låggradig hemolys). Med dessa metoder ökar andelen riskhändelser så att jämförelser kunde göras även på enhetsnivå och avdelningsnivå. Utbildningsprogrammet öppnade upp för reflektioner om säkerhet och förbättrade utförande av venprovtagning vid enheter med större brister. Utbildningsprogram som öppnar upp för reflektion och diskussion kan leda till ökad patientsäkerhet i hälso- och sjukvården. Trots utfallet av resultaten, är riktade utbildningsinsatser för sjukvårdsenheter som uppvisar specifika brister troligtvis mer effektiva än breda utbildningsinsatser. Klinisk betydelse: Interventionsprogram avseende preanalys och venös provtagning förbättrade personalens praktiska utförande. Monitorering av och åtgärder för att minska riskhändelser är väl fungerande preventiva åtgärder. Instrumenten (självrapporterande enkät och hemolys) bör också testas i andra kontexter inom hälso- och sjukvården. Ett externt nationellt program för att identifiera och förebygga riskhändelser bör utvecklas i hälso- och sjukvården. Interventioner i form av e-lärande kan då vara ett alternativ som är billigt och effektivt. Dessutom kan systematisk planering och genomförande med fokus på reflektion av specifika delar i en färdighet vara effektivt för att uppnå förbättringar. Våra studier har bidragit till en djupare och utökad kunskap om effekten av ett interventionsprogram på utförande av venprovtagning. Resultaten kan användas vid framtida planering av utbildningsinsatser. Modeller för praktiskt färdighetsutövande inom omvårdnad kan beskriva venprovtagning ur ett helhetsperspektiv och synliggöra venprovtagning som en viktig praktisk färdighet inom omvårdnad. / Preanalys

O diagnóstico rotineiro da dengue é realizado com amostra de sangue dos casos suspeitos. A coleta tradicional de sangue (por punção venosa) é um procedimento que dificulta a realização de exames e pesquisas por ser um procedimento invasivo que nem sempre é prático para crianças e bebês, requer pessoal especializado e necessita de um local para armazenamento da amostra sob refrigeração ou congelamento. O propósito deste estudo foi coletar amostras por punção digital com uma nova tecnologia (FTA Card) e compará-la com amostras coletadas por punção venosa, avaliando-as através de uma técnica molecular de PCR em tempo real. Sendo o PCR em Tempo Real a técnica molecular atualmente disponível de maior rapidez, sensibilidade e especificidade, padronizamos uma metodologia de passo único de PCR em Tempo Real com SYBR green baseando-se na região 3 não codificante do vírus e utilizando primers degenerados, capazes de detectar os quatro sorotipos de uma só vez. A avaliação das técnicas de coleta e amplificação foram feitas com amostras suspeitas de dengue, obtidas em Goiânia durante surto ocorrido no ano de 2008. O limite de detecção da reação padronizada no presente estudo foi de aproximadamente 100 cópias/ml e uma especificidade de 100%. Para tipagem das amostras positivas a técnica empregada foi o PCR multipex. Dentre as 89 amostras coletadas 60 (67%) foram positivas para àquelas coletadas por punção venosa e 14 (16%) para àquelas coletadas por punção digital. Dentre as 89 amostras para o PCR em Tempo Real, apenas 29 (32%), foram tipadas pelo método de PCR multiplex, sendo 3 casos do vírus da dengue 1 (10%), 16 casos do vírus da dengue 2 (55%), e 10 casos do vírus da dengue 3 (35%). Descritores: Dengue/diagnóstico, coleta de amostras sanguíneas, reação em cadeia da polimerase, corantes fluorescentes / The collection by venipuncture is a procedure that is difficult to carry out in diagnosis and research because it is an invasive procedure that is not always practical for children and babies , requires specialized staff, needs a place to store the sample under refrigeration or forzen, This study aimed to collect samples by fingerstick puncture with a new technology named FTA card and compare it with samples collected by venipuncture, using a realtime PCR to evaluate if FTA card collection would have a similar performance to standard blood sampling. Towards that, we obtained viral load values in order to estimate the differences not only qualitatively (e.g. pos or neg) but also in numbers.. We used an one-step SYBR Green I Real-Time PCR based on the region 3 \'noncoding virus using degenerate primers which was able to detected all four serotypes of dengue virus. Among the 89 samples collected 60 (67%) were positive for those collected by venipuncture and 14 (16%) to those collected by fingerstick, Only 29 (32%) were possible to be typed by PCR multiplex. Three cases were dengue virus 1 (10%), 16 cases were dengue virus 2 (55%) and 10 cases were dengue virus 3 (35%). The limit of detection obtained was approximately 100 copies / ml and aspecificity of 100% was observed. Keywords: Dengue / diagnosis, collection of blood samples, polymerase chain reaction, fluorescent dyes