Thesis (MMed)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Background: Sputum is a non-invasive, economic investigation whereby bronchogenic carcinoma can be identified. Manual cytological screening is labour intensive, time-consuming and requires a continuous high level of alertness. Automation has recently been successfully introduced in gynaecological cytology. Since sputum samples are similar to cervical smears, the question arises as to whether they are also suitable for automated screening. Objective: This study presented with various objectives: 1) To test automated sputum screening using the BD FocalPoint™ Slide Profiler (FP) and compare with manual sputum screening. 2) To determine the sensitivity and specificity of sputum in identification of bronchogenic carcinoma. 3) To ascertain if any clinical, radiological or bronchoscopy findings would be predictors for bronchogenic carcinoma. 4) To determine the significance of adequacy. Method: Sputum samples were collected prospectively from patients attending the Division of Pulmonology at Tygerberg hospital for a transbronchial fine needle aspiration biopsy (TBNA) or a transthoracic fine needle aspiration biopsy (TTNA) for the period from 2010 to 2012. A pre-bronchoscopy sputum was collected and submitted for processing. Stained slides were put through the FP for automated screening. After slides were qualified, sputum slides were put back in the routine screening pool. Correlation was done using the TBNA/TTNA result as the standard to evaluate the sputum results. Results: 108 sputum samples were included in this study. Of the 84.3% malignant (n=91) and 15.7% benign (n=17) cases confirmed with a diagnostic procedure, sputum cytology had a sensitivity of 38.5% (35/91 malignant cases), and a specificity of 100% (17/17 benign cases). Automated screening had a better sensitivity of 94.3% (33/35 positive sputum cases), while manual screening showed a sensitivity of 74.3% (26/35 positive sputum cases) when compared to the final sputum result. Individual parameters with a significant association with positive sputum included the presence of an endobronchial tumour, partial airway obstruction / stenosis, round mass, spiculated mass (negative association), loss of weight (negative association) and squamous cell carcinoma as the histological subtype. Adequacy was not as significant as hypothesised since 85.3% of true positive sputum, but also 65.5% of false negative sputum, had large numbers of alveolar macrophages present. Conclusion: Sputum cytology remains an important part of the screening programme for bronchogenic carcinoma in the public health sector of South Africa. Results confirm that sputum cytology is very specific, and automated screening improves sensitivity. Automated screening proved to be more time efficient, resulting in 83.1% reduction (p<0.0001) in the screening time spent per case by a cytotechnologist. Results confirm that the quantity of alveolar macrophages is not directly proprtional to pathology representation. Positive sputum results did however improve with sputum adequacy, but had no significant association. Recommendations from this study include adopting automated sputum screening. / AFRIKAANSE OPSOMMING: Agtergrond: Die verkryging van ‘n sputummonster is ‘n nie-indringende, ekonomiese ondersoek waardeur bronguskarsinoom identifiseer kan word. Nie-geoutomatiseerde sitologiese ondersoek is arbeidsintensief, tydrowend en vereis ‘n deurlopende hoë vlak van konsentrasie en fokus. Outomatisering is onlangs suksesvol geïmplementeer in ginekologiese sitologie-ondersoeke. Aangesien sputummonsters soortgelyk aan servikale monsters is, het die vraag ontstaan of sputummonsters ook geskik sou wees vir geoutomatiseerde sifting. Doelwit: Hierdie studie het verskeie doelwitte gehad: 1) Om geoutomatiseerde sifting van sputummonsters te toets deur gebruik te maak van BD Focal Point ™ Slide Profiler (FP), en te vergelyk met nie-geoutomatiseerde sputum sifting. 2) Om die sensitiwiteit en spesifisiteit van sputum in die identifikasie van bronguskarsinoom te bepaal. 3) Om vas te stel of enige kliniese, radiologiese of brongoskopiese bevindings bronguskarsinoom sou kon voorspel. 4) Om die belang van ‘n verteenwoordigende monster te bepaal. Metode: ‘n Prospektiewe studie van die pasiënte wat die Divisie van Pulmonologie by Tygerberg Hospitaal vir transbrongiale nodale aspirasie (TBNA) of ‘n transtorakale aspirasie (TTNA) vanaf Julie 2010 tot Mei 2012 bygewoon het, is gedoen. ‘n Prebrongoskopiese sputum is geneem en gestuur vir prosessering. Die gekleurde skuifies is deur die FP gestuur vir geoutomatiseerde ondersoek. Indien die sputumskuifies gekwalifiseer het vir geoutomatiseerde sifting, is hulle in die groep vir ondersoek ingesluit. ‘n Korrelasiestudie, om die sputumresultate te evalueer, is uitgevoer deur die TBNA/TTNA bevindings as standaard te gebruik. Resultate: Vir hierdie studie is 108 sputummonsters ingesluit. Vanuit die 84.3% maligne (n=91) en 15.7% benigne (n=17) gevalle, bevestig deur ‘n diagnostiese prosedure, het sputumsitologie ‘n sensitiwiteit van 38.5% (35/91 maligne gevalle) en ‘n spesifisiteit van 100.0% (17/17 benigne gevalle), getoon. Geoutomatiseerde sifting het ‘n beter sensitiwiteit met 94.3% (33/35 maligne gevalle), terwyl nie-geoutomatiseerde (ondersoek) ‘n sensitiwiteit van 74.3% (26/35 maligne gevalle) wanneer met die finale resultaat vergelyk, gevind. Individuele parameters met ‘n betekenisvolle assosiasie het die teenwoordigheid van ‘n endobrongiale tumor, gedeeltelike lugwegobstruksie / stenose, ronde massa, ‘n spekuleerde massa (negatiewe assosiasie), gewigsverlies (negatiewe assosiasie) en plaveiselkarsinoom as die histologiese subtipe, ingesluit. Geskiktheid van die monster was nie so betekenisvol as wat in die hipotese gestel is nie: aangesien 85.3% van ware positief gediagnoseerde sputummonsters, maar ook 65.5% van die vals negatiewe sputummonsters, groot hoeveelhede alveolêre makrofae ingesluit het. Gevolgtrekking: Sputumsitologie bly steeds ‘n belangrike deel van die siftingsprogram vir bronguskarsinoom in die openbare gesondheidssektor in Suid-Afrika. Resultate van hierdie studie bevestig dat sputumsitologie baie spesifiek is en dat geoutomatiseerde sifting die sensitiwiteit verbeter. Ge-outomatiseerde sifting het bewys dat dit meer tydsbesparend is, met ‘n 83.1% vermindering (p<0.0001) in die siftingstyd wat deur een sitotegnoloog per geval bestee word. Resultate het bevestig dat die hoeveelheid alveolêre makrofae nie direk proporsioneel verwant is tot die patologie nie. Hoe meer verteenwoordigend die sputummonster was, hoe groter was die kanse om ‘n akkurate positiewe diagnose te maak. Die assosiasie van die geskiktheid van die sputummonster en die positiewe resultate het egter nie ‘n statisties betekenisvolle resultaat getoon nie. Aanbevelings vir hierdie studie sluit in die aanwending van geoutomatiseerde sputumondersoeke.

Thesis (M Med.(Family Medicine))--University of Limpopo, 2010. / OBJECTIVE: In order to obtain optimal results using sputum smear microscopy for acid-fast bacilli (AFB) detection in the diagnosis of pulmonary tuberculosis (PTB), good quality sputum with an adequate volume of at least 5.0ml is required. An inadequate amount of sputum sample may result cases of active PTB being missed. This study was aimed at showing that a single sputum sample of at least 5.0ml would lower the chances of missing active PTB cases, and increase sputum smear positivity by microscopy thus enabling prompt treatment of PTB, and restricting its transmission. METHODS: An analytical cross sectional study was carried out at Dr George Mukhari Hospital in Pretoria, South Africa. Two sputum samples, one of 5.0ml and the other with 2.0ml were collected from each adult patient suspected of having active PTB. Sputum collection was supervised and patients were given instructions on how to enhance sputum expectoration. Sputum samples were processed using the N-acetyl-L-cysteine (NaLc-NaOH) method and stained with Auramine O. Sputum analysis was done with the aid of fluorescence microscopy. Following microscopy, both the 2.0ml and 5.0ml specimens were sent for culture using the Middlebrook broth medium, and culture results were available after 6-8 weeks. Using the culture results as gold standard, the yield through microscopy of the 2.0ml specimen versus the 5.0ml specimen for each patient were compared and analysed. RESULTS: A Total of 330 sputum samples were analysed of which 77 were found to be culture positive. A sensitivity of 76.6% and specificity of 99.6% was obtained for AFB test in the 5.0ml specimens; while in the 2.0ml samples the sensitivity was 75.3%, with a specificity of 99.2%. The difference in the smear positivity rate of 76.6% obtained using the 5.0ml sputum specimen compared to the 75.3% obtained using the 2.0ml specimen from patients suspected of having TB in this study was statistically insignificant – ascribable to the small sample size. CONCLUSION: In this study, the volume of sputum collected did not determine a better AFB test yield in the diagnosis of pulmonary tuberculosis in patients suspected of having TB.

Thesis (MPH.) -- University of Limpopo, 2017. / The present study presents data about the prevalence and determinants of sputum smear non-conversion in smear positive tuberculosis patients. Despite the intervention by the Sekhukhune District Department of Health through continual training and workshops of professional nurses in respect of the NTCP, there are still more challenges observed in terms of TB management. Aim: To investigate the prevalence and determinants of sputum smear non-conversion in smear positive PTB patients after intensive phase of treatment. Method: Quantitative, descriptive retrospective study of TB records was conducted. Data collection was done by extracting data from ETR.net and exporting it to excel. Data cleaning was done before analysis. Data analysis was done using the computer Statistical Package Software for Social research (SPSS) volume 23.1. Findings: 834 TB patients’ records were extracted from the ETR.net database. 34% of records were available at 2 – months; 57% of the patients were males; also, 81% of the patients were diagnosed/treated at PHC facilities; 52% of the patients were HIV positive; 69% percent of the patients who were smear positive grading p+++ failed to convert after two months. In the univariate logistic regression patients with age 20 – 29 were observed to be 4.9 times likely (O.R. = 4.97) to be sputum positive (P = 0.142).Sputum grade 3(p+++) at the time of diagnosis was found to be significantly associated (P = 0.031) with sputum non – conversion after intensive phase of treatment. Conclusion: Two month sputum smear non-conversion is associated with pre-treatment sputum smear grading.

Cystic fibrosis (CF) lung disease is characterised by early airways infection and inflammation, chronic suppuration, frequent infective exacerbations and an increased influx of acute, and chronic inflammatory cells. The inflammatory process involves activation of many cell types including neutrophils, macrophages and epithelial cells, and leads ultimately to the development of progressive respiratory failure and death. Accurate assessment of the inflammatory process is a crucial part of disease monitoring and should allow appropriate evaluation of therapeutic interventions so as to maximize control of the respiratory sequelae of the disorder. Lung function markers such as FEV1 are insensitive and indirect. Direct but invasive methods such as fibreoptic bronchoscopy and biopsy are limited in application, repeatability and safety. Non-invasive methods of assessment are, therefore, attractive. Exhaled Breath Gases, Exhaled Breath Condensate and Induced Sputum provide potential for such measures. These techniques are safe, simple, repeatable and could assess all airways and can be used in children as young as 6 years. We hypothesised that biomarkers of inflammation in Cystic Fibrosis Lung Disease are measurable in samples collected noninvasively, and can be developed into clinically useful assays. These assays would have the ability to reflect the level of inflammation in the CF lungs as well as holding the potential to act as surrogate markers of CFTR function. Methods Non-invasive markers of inflammation in Cystic Fibrosis lung disease Methods. Exhaled breath gases, exhaled breath condensate, bronchoalveolar lavage fluid and induced sputum were investigated using a number of analysis techniques to identify the markers which best discriminated CF from non CF subjects. Analysis techniques used were electrochemical cells, chemiluminescene, ELISA, EIA, ion selective probes and mass spectrometry. Results Markers found to discriminate CF from non CF subjects were EBC pH and ammonium, and 38 proteomic markers were found in induced sputum. 21 proteomic markers were found in bronchoalveolar lavage fluid. One biomarker has been identified with confidence, Calgranulin A. Discussion A large component of the work of this thesis was focussed on exhaled breath condensate. Two markers, pH and Ammonium were different between the CF and control groups. The measurement of EBC pH and ammonium as markers of inflammation should be used in future gene therapy trials as they are cheap, quick and simple to perform Using clean techniques free from contamination, no proteins are repeatedly detectable in EBC using highly sensitive SELDI techniques. This technique reflects the highest sensitivity of any available proteomics instrument and therefore until new technologies become available, it would be incorrect to assay any proteins in EBC. The induced sputum proteomics study identified 38 independent markers of CF lung inflammation Therefore, sampling by collection of induced sputum should be used in gene therapy trials. The endpoints should be assessed by a combination of SELDI as an endpoint and by ELISA where this is available. The marker Calgranulin is likely to report on neutrophil recruitment to the lung. It is anticipated that this will be a sensitive marker of inflammation in the lung and it also has the potential to report on successful of gene transfer as it is raised in heterozygote carriers as well as homozygotes with CF. Therefore, the non-invasive technique induced sputum coupled to proteomic analysis would have the ability to reflect the level of inflammation in CF subjects and may also report on CFTR function.

Tuberculosis remains a communicable disease of major public health importance in South Africa. The purpose of this study is to search for trends in management of tuberculosis at Hillbrow Health Centre from 2000 to 2002. Furthermore, to assess completeness of routine records, compare performance of tuberculosis control in this clinic with others in region 8 during 2002 and identify residential areas with high volumes of tuberculosis patients. Data was obtained from tuberculosis documents at the facility. Information provided by the district office was compared with research findings and used to evaluate performance of Hillbrow Health Centre against the other four clinics in the region. The main findings from the study conducted at Hillbrow Health Centre were the large number of patients diagnosed with tuberculosis annually and cure rates below 40% during the study period. In 2002, the clinic reported the highest proportion of TB patients and the lowest cure rate compared to the other four clinics reporting tuberculosis in region 8. “Hot spots” for the disease were identified in Hillbrow and Joubert Park suburbs and this is where Esselen, Hillbrow and Urban Health Clinics are situated. There is room for improvement of tuberculosis control. More research needs to be done to determine factors contributing to the high incidence of TB in Hillbrow and Joubert Park Suburbs.

Sputum induction by inhalation of hypertonic saline has been used for more than15 years. It has become one of the most intriguing methods to study airway inflammation. It is the only direct, non-invasive method for measuring airway inflammation indices.Sputum induction has been used in the diagnosis of many respiratory illnesses including asthma, chronic pulmonary obstructive disease, tuberculosis, chronic cough, lung cancer and Pneumocystis Carinii on patients who are unable to produce sputum spontaneously. There are currently many different methods used worldwide to induce sputum, but there is a lack of one generally accepted gold standard method. The proposed protocols for sputum induction proved to be safe, simple and produced satisfactory amount of expectorate. However, it did not contain enough cells from the lower respiratory tract and was contaminated by squamous cells when compared to another method based on the work of F. E. Hargreave. Investigation demonstrated that the use of impulse oscillometry, which requires no effort from patients, needs further research with larger study samples before it could be used instead of spirometry to evaluate airway obstruction. Initial methylene blue stain of the fresh expectorate smear was shown to be useful tool for identifying grossly contaminated sputum samples by squamous epithelial cells.Our first study group included 20 volunteers in good health. Sputum was induced by inhalation of 3% saline mist created by ultrasonic nebulizer at maximum output (4ml/min). Sputum induction intervals lasted 4-5 minutes with cumulative duration of induction about 4-15 minutes which was tolerated well. Lung function was evaluated for obstruction at baseline and every 5 minutes with spirometry and impulse oscillometry.The whole expectorated sample was processed and slides were stained with HEMA 3stain. With this method we were able to collect a mean of 6.1 ml expectorate. The mean total cell count was 804 000 with high proportion of squamous cells. The second study group included 5 volunteers in good health. This method utilized 3%, 4% and 5% saline mist for inhalation, 7 minutes each. Ultrasonic nebulizer was set at low output of 0.9 ml/min. This procedure was also tolerated well without major adverse effects. Lung function was evaluated at baseline and every 7 minutes for obstruction. Only dense portions of expectorate were selected and processed. Slides were stained with Wright stain. This method produced much more total cells with a mean of 3 385 000 per gram of sputum which came from the lower airways and were not contaminated by squamous cells. The second method was far superior producing adequate sputum sample with cells from the lower airways and minimal squamous cell contamination and will be used in our Breath Lab.

Thesis (Ph. D.)--University of Texas at Austin, 2008. / Vita. Includes bibliographical references.

Made available in DSpace on 2015-04-20T12:31:40Z (GMT). No. of bitstreams: 1 Tese Final Luciana Botinelly.pdf: 2352589 bytes, checksum: 2dfaff58148566cdae4e7f8988cc79f7 (MD5) Previous issue date: 2010-10-27 / Fundação de Amparo à Pesquisa do Estado do Amazonas / In almost all Tuberculosis control programs in the world, sputum smear analyze is the first choice diagnosis tool of the disease. The World Health Organization estimates that 150 million people, between the years 2002 and 2020, would have Tuberculosis, and this would be the minimum number of sputum smears to be performed during this period. The most widely stain used in smear staining is Ziehl-Neelsen s, described for over 125 years. This stain is based on the property that mycobacterias have, including Mycobacterium tuberculosis main causing tuberculosis, to withhold a dye solution composed of fuchsin and phenol (carbolfuchsin) in its cell wall, even after washing with acid-alcohol solution. However, carbolfuchsin is recognized as a carcinogenic substance in rodents, when introduced into diet. There is reference to danger of great harm to human health by prolonged exposure, with irreversible consequences. It is flammable, but heating is recommended for implementation of main coloring technique, which releases combustion gases and vapors hazardous if inhaled. No other less toxic substance was proposed yet. Thus, experimental study of evaluation of twelve alternate dyes on coloring smears was carried out, aimed at implementing in clinical samples suspected of Tuberculosis. In-test dyes solutions were prepared, at different concentrations and without phenol addition. For testing, positive acid-fast bacilli sputum smears and clinical isolate of M. tuberculosis smears were stained without heating, for 5 minutes, bleached with alcohol-acid solution for 1 minute, and counterstained with methylene blue, for 1 minute. The microscopic readings were held in transmitted light and in fluorescence (three filters), at immersion objective (100 x). The most promising dye was the alkaloid from vegetable species (AEV), which allowed fluorescence reading, at green and blue filters, without excessive aggregation to background cells and/or to mucous material present in sputum samples. Sensitivity and specificity tests, and comparison with current bacteriological method confirmed the potential use of AEV. Optimization tests for traditional microscopy reading were made with no success, a fact that indicates other testing. Three purified alkaloids and four alkaloids fractions from amazonian plant species were tested and considered promising for use in mycobacterial stain. They could be added to AEV, providing the ideal dye for Tuberculosis diagnosis, allowing both traditional and fluorescence microscopy. / Em quase todos os programas mundiais de controle da Tuberculose, a avaliação baciloscópica é adotada como primeira opção de exame diagnóstico da doença. A Organização Mundial de Saúde estima que 150 milhões de pessoas, entre os anos de 2002 e 2020, adoecerão por Tuberculose, e este seria o número mínimo de baciloscopias a serem realizadas neste período. A coloração mais utilizada na baciloscopia é a de Ziehl-Neelsen, descrita há mais de 125 anos. Tem como princípio a propriedade que as micobactérias possuem, incluindo o Mycobacterium tuberculosis principal causador da Tuberculose, de reter uma solução corante composta por fucsina básica e fenol (carbolfucsina) em sua parede celular, mesmo após lavagem com solução álcool-ácida. Entretanto, a carbolfucsina é considerada indutora de tumores em roedores, quando introduzida em sua dieta, e há referência a perigo de grande dano para saúde humana em caso de exposição prolongada, com possíveis efeitos irreversíveis. É produto combustível, que em caso de aquecimento, como o recomendado para a execução da principal técnica de coloração, forma gases de combustão e vapores perigosos para inalação. Nenhuma opção de substituto menos tóxico que a carbolfucsina foi até o momento proposto. Desta forma, foi realizado estudo experimental de avaliação de doze corantes alternativos na execução de coloração baciloscópica para micobactérias, visando aplicação em amostras clínicas submetidas ao diagnóstico laboratorial da Tuberculose. Para tanto, foram preparadas soluções dos corantes em teste - em diferentes concentrações e sem adição de fenol. Para os testes, baciloscopias positivas para BAAR, obtidas a partir de amostras de escarro e de isolado clínico de M. tuberculosis, foram coradas sem aquecimento, por 5 minutos, descoradas com solução álcool-ácido por 1 minuto, e contra-coradas com azul de metileno, por 1 minuto. As leituras microscópicas foram realizadas em campo claro e em fluorescência (três filtros), objetiva de imersão (aumento de 100x). O corante mais promissor foi o alcalóide de espécie vegetal (AEV), o qual permitiu visualização bacilar, em microscopia de fluorescência, filtros azul e verde, sem agregação excessiva às células de fundo e/ou à secreção mucóide presente em amostras de escarro. Testes de sensibilidade de detecção bacilar, de especificidade, e comparativo com método bacteriológico tradicional confirmaram o potencial de utilização do AEV. Testes de otimização visando sua utilização para leitura em microscopia tradicional foram realizados, sem obter sucesso, fato que indica a realização de novos testes. Três alcalóides purificados e quatro frações alcaloídicas de possíveis fontes amazônicas do AEV foram testados e considerados como promissores para uso na coloração de micobactérias. Estes podem ao serem agregados ao AEV, propiciar o corante ideal para o diagnóstico da Tuberculose, permitindo visualização tanto à microscopia tradicional, quanto à de fluorescência.

Background: The clinical and pathological presentation of COPD is heterogeneous. ‘Chronic bronchitis’ is a phenotype of COPD, which is a clinical diagnosis of a productive cough of ≥ 3 months for ≥ 2 consecutive years. Chronic bronchitis is associated with worse lung function, frequent exacerbations, recurrent hospitalisations and premature death in patients with COPD. Chronic bronchitis sufferers can be further subphenotyped into those who produce sputum during exacerbation or during winter months only and those who are ‘persistent sputum producers,’ who experience mucous hypersecretion throughout the year. An improved understanding of persistent sputum producers is the object of this thesis. Aims: 1) To compare the clinical characteristics and airway inflammatory biomarker profile of COPD persistent sputum producers to that of COPD sputum non-producers 2) To investigate the short term repeatability of sputum parameters in COPD persistent sputum producers 3) To study the expression and relationship of mucins, hypoxia inducible factor (HIF-1α) and carbonic anhydrase IX (CAIX) in COPD persistent sputum producers. Methods: 1) Lung physiology, health status, sputum inflammatory biomarkers and sputum culture results were compared between COPD persistent sputum producers and sputum non-producers 2) Repeatability of spontaneous and induced sputum parameters at 8 weeks was assessed in COPD persistent sputum producers 3) Immunohistochemistry was performed on bronchial biopsies of COPD persistent sputum producers and control groups (COPD sputum non-producers, smokers with normal lung function and lifelong healthy non-smokers with normal lung function) to study the expression of MUC5AC, MUC5B, HIF-1α and CAIX 4) The association between HIF-1α and MUC5B expression was investigated in vitro. Results and Conclusions: The findings suggest that 1) COPD persistent sputum producers have clinically more severe disease, increased airway inflammation, increased impact on health status, increased rate of bacterial colonization and higher number of exacerbations compared to COPD sputum non-producers 2) Induced sputum is repeatable over short term in COPD persistent sputum producers 3) Expression of MUC5B, HIF-1α and CAIX is increased in COPD persistent sputum producers compared to COPD sputum non-producers, smokers with normal lung function and healthy non-smokers 4) HIF-1α can potentially cause increased MUC5B expression. This work reveals potential targets for the development of novel therapies to limit mucous hypersecretion in COPD.

No description available.