Thesis (MSc (Med)(Microbiology)) -- University of Limpopo, 2012. / Mycobacterium tuberculosis is the most common cause of death world-wide and its incidence has been steadily increasing, which is more evident when comparing the global tuberculosis (T8) incidence of 9.24 million in 2006 to 9.27 million cases in 2007. African countries are the second most affected by the epidemic and South Africa is among the 22 highest burden countries most affected by T8 with a very high number of cases relative to the total population. The early diagnosis of tuberculosis and screening of contacts is the cornerstone for controlling spread of active T8 infection. T8 diagnosis becomes even more challenging in patients with immunosuppression (for example in human immunodeficiency virus (HIV) infected), in the case of latent infection and extra pulmonary T8 such as pleural T8. The definitive diagnosis of pleural T8 depends on the demonstration of M. tuberculosis in sputum, pleural fluid and pleural biopsy. Although acid fast bacilli (AF8) microscopy is a rapid, inexpensive and relatively simple method, it has low sensitivity. The culture method is more sensitive than AF8 microscopy, detecting 25-37% of all pleural tuberculosis cases however it takes 4 to 8 weeks for a visible growth on a solid medium. Therefore it is important to find a rapid and reliable test for the diagnosis of pleural T8 particularly in developing countries such as South Africa where there is a high T8 incidence and HIV infection rate.

Two molecular forms of adenosine deaminase have been isolated from bovine livers. These two forms were found to be interconvertible. Adenosine deaminase extracted from human tissues exhibited similar properties as well. Previous studies have shown the presence of a conversion factor which formed an aggregate with the small form (the C-form) of the enzymes, and the resulting enzyme complex was identified to be the large form (the A-form) of adenosine deaminase. Studies have been made with rat tissues. The C-form of the adenosine deaminase is widely distributed in the various tissues, while the large form of the enzyme is absent in those tissues examined.The outline of this study is summarized below: (1) The isolation of the conversion factor from human liver.(2) The isolation of the adenosine deaminase from the various tissues.(3) The coversion of human adenosine deaminase C-form to the A-form.(4) Quantization of the conversion of the C-form to the A-form.(5) Testing the effect of conversion on nonhuman enzymes.(6) The optimum temperature and time to yield greatest amount of conversion.The results from this study would verify any differences that might exist between the small forms of the enzyme present in different species.

Thesis (Ph. D. in Pharmacology)--Vanderbilt University, Dec. 2005. / Title from title screen. Includes bibliographical references.

A Thesis submitted to the Faculty of Medicine, University of the Witwatersrand, Johannesburg, for the Degree of Master of Science JOHANNESBURG 1986 / The adenosine deaminase enzyme from a Xhosa tribesman has been characterized. Red blood cell activity levels were 6-9% of normal whereas his white cell ADA levels were about 30% of normal. The enzyme's stability at 57°C was shown to be greatly reduced suggesting a mutation resulting in an enzyme with reduced stability in vivo. It was concluded that the discrepancy in ADA activity levels between red and white blood cells was due to the red cells being anucleate. The proband's residual ADA was found to have a Michaelis Constant (K ) for adenosine m of 47.9 ♦ IS.BuM, a value which is not significantly different from that of normal ADA (51.7 ± 11.4ufl). Red cell deoxy-ATP levels were measured and found to be elevated two-to-three times over normal levels. Red cells from ADA-deficient patients with severe combined immunodeficiency (SCID) have been reported with deoxy-ATP levels elevated about 1 000 times. It was concluded that the slight elevation of deoxy-ATP levels in the proband were too low to have any noticeable effect on functions of his immune system. Starch gel electrophoresis of red cell ADA from members of the proband's family in conjunction with red cell ADA activity levels suggested that both parents carried a gei e for 'partial' ADA deficiency, both of which had been inherited by the proband as well as one of his sibs. Isoelectric focusing studies suggested that the two, parental AUA partial deficiency genes were different from one another. It was also found that another rare allele of ADA, possibly ADA ',was segregating within the same family although this event appaars to be unconnected with the ADA partial deficiency.

Recently, we discovered patients with inherited adenosine deaminase (ADA) deficiency are predisposed to pulmonary alveolar proteinosis (PAP). PAP is characterized by the accumulation of surfactant in the alveoli. To overcome ethical issues and limited patient samples, animal models are often utilized. Here, I investigated the lung abnormalities in ADA deficient (ADA -/-) mice, which suffer from severe hypoxia, till their death at 3 weeks. I hypothesized that, similar to ADA-deficient patients, ADA -/- mice demonstrate evidence of PAP. Indeed, electron microscopy showed thickening of type I cells, accumulation of apoptotic foamy alveolar macrophages, cholesterol and lipoproteinaceous material that is periodic-acid Schiff (PAS) positive and diagnostic of PAP. Moreover, the pulmonary abnormalities were corrected with supplementation of ADA. In conclusion, we demonstrated evidence of PAP in ADA -/- mice for the first time and their suitability to study pathogenesis of PAP in ADA deficiency.

Recently, we discovered patients with inherited adenosine deaminase (ADA) deficiency are predisposed to pulmonary alveolar proteinosis (PAP). PAP is characterized by the accumulation of surfactant in the alveoli. To overcome ethical issues and limited patient samples, animal models are often utilized. Here, I investigated the lung abnormalities in ADA deficient (ADA -/-) mice, which suffer from severe hypoxia, till their death at 3 weeks. I hypothesized that, similar to ADA-deficient patients, ADA -/- mice demonstrate evidence of PAP. Indeed, electron microscopy showed thickening of type I cells, accumulation of apoptotic foamy alveolar macrophages, cholesterol and lipoproteinaceous material that is periodic-acid Schiff (PAS) positive and diagnostic of PAP. Moreover, the pulmonary abnormalities were corrected with supplementation of ADA. In conclusion, we demonstrated evidence of PAP in ADA -/- mice for the first time and their suitability to study pathogenesis of PAP in ADA deficiency.

The hydrolysis of adenosine, 2-deoxyadenosine and 6-chloropurine riboside by adenosine deaminase preparations from human spleen has been investigated, and Km and Vm values have been determined. The effect of the substrate on the reaction velocity was followed over a 250-fold range Results showed no deviation from Michaelis-Menten kinetics. The effect of pH on Vm, Vm/Km, and the apparent activation energy was examined. Inactivation of the enzyme by p-chloromercuribenzoate suggests the involvement of one or more SH groups. Competitive inhibition by inosine and the fact that ammonia is not, an inhibitor support a reaction mechanism involving a ternary complex. The apparent activation energy of the a parameter was smaller or less sensitive to temperature than the ß parameter.Ball State UniversityMuncie, IN 47306

Previous studies of serum adenosine deaminase have neglected consideration of the two molecular forms of this enzyme that exist in human tissues. The purpose of this study was to survey the distribution of these forms in normal and pathological human sera. Both molecular forms were present in normal serum, though the small form predominated. This form also predominates in lymphocytes, erythrocytes, and in tissues with high specific activity of this enzyme. The ratio of the two forms is different for plasma and serum and can change with sample storage. The activity of the small form varied over a wider range than the activity of the large form in normal serum. Many pathological samples showed an altered distribution of the two forms. This study demonstrates the potential usefulness of serum forms of adenosine deaminase for distinguishing some pathological conditions.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / IntroduÃÃo: A leishmaniose visceral (LV) à uma doenÃa causada por protozoÃrios do gÃnero Leishmania e transmitida por insetos flebotomÃneos. O Brasil à uma das mais importantes Ãreas endÃmicas para a doenÃa, sendo necessÃrio o acompanhamento da ocorrÃncia dos casos atravÃs de uma vigilÃncia epidemiolÃgica ativa. A adenosina desaminase (ADA) à uma enzima que catalisa a desaminaÃÃo da adenosina produzindo inosina e amÃnia. Em humanos, està presente como duas isoenzimas e trÃs isoformas (ADA1, ADA1-CD26 e ADA2), sendo fundamental para o funcionamento do sistema imunolÃgico, uma vez que a deficiÃncia genÃtica da ADA1 provoca uma imunodeficiÃncia severa e combinada (SCID), bem como o vÃrus HIV à capaz de promover uma diminuiÃÃo na contagem dos linfÃcitos T via interaÃÃo com a ADA1-CD26. Objetivos: Descrever o perfil epidemiolÃgico da LV no Brasil (de 2001 a 2011) e no Cearà (de 2007 a 2011), bem como a atividade de ADA em pacientes acometidos pela LV. Metodologia: Realizou-se um estudo observacional descritivo da LV no Brasil e no estado do Cearà utilizando os dados secundÃrios disponibilizados pelo SINAN/MS, sendo categorizadas as Ãreas de transmissÃo; faixa etÃria, sexo e escolaridade dos casos; incidÃncia, prevalÃncia e evoluÃÃo da doenÃa; co-infecÃÃo HIV-LV e casos em gestantes. A ADA e suas isoenzimas foram determinadas em plasma de pacientes acometidos por LV utilizando o mÃtodo de Giusti, EHNA e por eletroforese. Os valores de cutoff para a ADA foram determinados utilizando a curva ROC. Resultados: A LV à endÃmica no Brasil, presente em 26 dos 27 estados, com uma mÃdia anual de ~3.600 casos, incidÃncia de ~1,79 casos/100.000hab e prevalÃncia de ~1,96 casos/100.000hab. No CearÃ, està presente em ~88% dos municÃpios, com mÃdia anual de ~600 casos, incidÃncia de 6,1casos/100.000hab e prevalÃncia de 7,1casos/100.000hab. A ADA està significativamente aumentada na LV, com uma atividade mÃdia de 106,8Â4,5U/L (vs controle 21,1Â0,6U/L). Este aumento ocorre com ambas isoenzimas, contudo a ADA2 à a principal isoenzima presente em pacientes acometidos por LV, com um valor de cutoff de 47,6 U/L para ADA total e 29,6U/L para a ADA2, utilizando adenosina. ConclusÃo: A LV à uma doenÃa endÃmica presente em todos os estados brasileiros, a exceÃÃo do Acre. Os estados do CearÃ, MaranhÃo e Minas Gerais apresentaram a maior quantidade de casos e o estado de Tocantins a maior incidÃncia e prevalÃncia. O Cearà à uma Ãrea endÃmica para a LV e a cidade de Fortaleza à o municÃpio que registrou a maior quantidade de casos no paÃs. A ADA pode ser utilizada como um marcador da resposta inflamatÃria na LV, e a determinaÃÃo da isoenzima ADA2 pode ser utilizada para avaliar a ativaÃÃo ou participaÃÃo de monÃcitos e macrÃfagos no processo infeccioso. / Introduction: Visceral leishmaniasis (VL) is a disease caused by protozoa of the genus Leishmania and transmitted by sandflies. Brazil is one of the most important endemic areas for the disease, being necessary monitoring of the occurrence of the disease through active epidemiological surveillance. Adenosine deaminase (ADA) is an enzyme that catalyzes the deamination of adenosine to produce inosine and ammonia. In humans, it is present as two isoenzymes and three isoforms (ADA1, ADA2 and ADA1-CD26) and is pivotal to immune system function, since ADA1âs genetic deficiency causes a severe combined immunodeficiency (SCID), as well as HIV is able to promote a decrease in the count of T lymphocytes via interaction with ADA1-CD26. Objectives: To describe the epidemiology of VL in Brazil (2001-2011) and Cearà (2007-2011); and the ADA activity in patients suffering from VL. Methods: We conducted an observational descriptive study of VL in Brazil and in the state of Cearà using secondary data provided by SINAN/MS, being categorized the transmission areas, age, sex and education of cases, incidence, prevalence and evolution of disease, co-infection HIV-VL and cases among pregnant women. The ADA and its isoenzymes were determined in plasma of patients suffering from LV using the method of Giusti, EHNA and electrophoresis. The cutoff values for the ADA were determined using the ROC curve. Results: VL is endemic in Brazil and is present in 26 of 27 states, with an annual average of ~ 3,600 cases, incidence of ~1.79 cases/100.000 inhabitants and prevalence of ~1.96 cases/100.000 inhabitants. In CearÃ, it is present in ~88 % of the municipalities, with an annual average of ~ 600 cases, incidence of 6.1 cases/100.000 inhabitants and prevalence of 7.1 cases/100.000 inhabitants. The ADA activity is significantly increased in LV, with an average activity of 106.8  4.5 U / L (vs. control 21.1  0.6 U / L). This increase occurs with both isoenzymes, but the ADA2 is the major isoenzyme present in patients suffering from LV, with a cutoff value of 47.6 U / L for total ADA activity and 29.6 U/L for ADA2 activity, using adenosine. Conclusion: LV is an endemic disease present in all Brazilian states, with the exception of Acre. The states of CearÃ, MaranhÃo and Minas Gerais had the highest number of cases and the state of Tocantins a higher incidence and prevalence. Cearà is an endemic area for VL and the city of Fortaleza is the municipality that the highest number of cases in the country. ADA can be used as a marker of the inflammatory response in VL, and the determination of isoenzyme ADA2 can be used to evaluate the activation or participation of monocytes and macrophages in the infectious process.

El texto completo de este trabajo no está disponible en el Repositorio Académico UPC por restricciones de la casa editorial donde ha sido publicado. / Objectives: Diagnosis of tuberculous meningitis (TM) is a challenge in countries with a high burden of the disease and constrained resources and clinical prediction rules (CPRs) could be of assistance. We aimed at developing a CPR for diagnosis of TM in a Latin American setting with high tuberculosis incidence and a concentrated HIV epidemic. Methods: We enrolled adult patients with clinical suspicion of TM attending two hospitals in Lima, Peru. We obtained information on potential anamnestic, clinical and laboratory predictive findings that are easy to collect and promptly available. We independently diagnosed TM according to a composite reference standard that included a series of microbiological tests. We performed bivariate analysis and constructed a logistic regression model to select the predictive findings associated with TM. With the selected predictors included in the model, we developed a score-based CPR. We assessed its internal validity and diagnostic performance. Results: Of 155 analysed patients, 59 (38%) had TM. The CPR we derived includes three predictors: cough for 14 days or more, 10–500 cells in CSF and adenosine deaminase ≥ 6 U/l in CSF. It classifies patients into high-, moderate- or low-score groups and has an overall area under the ROC curve of 0.87. 59% of patients were assigned to either the high- or the low-score group, permitting prompt decision-making. In patients in the high-score group, it attains a positive likelihood ratio for TM of 10.6 and in patients with low scores, a negative likelihood ratio of 0.10. Bootstrap analysis indicated high internal validity. Conclusion: This CPR could support decision-making in patients with clinical suspicion of TM. External validation and further assessment of its clinical impact are necessary before application in other settings. / Revisión por pares