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  2. 2. 2 BIOCHEMISTRY  What is? Biochemistry, is the study of chemical processes in living organisms, governs all living organisms and living processes. By controlling information flow through biochemical signalling and the flow of chemical energy through metabolism, biochemical processes give rise to the incredible complexity of life. Much of biochemistry deals with the structures and functions of cellular components such as proteins, carbohydrates, lipids, nucleic acids and other biomolecules although increasingly processes rather than individual molecules are the main focus . Today the main focus of pure biochemistry is in understanding how biological molecules give rise to the processes that occur within living cells which in turn relates greatly to the study and understanding of whole organisms.
  3. 3. 3  Basic BiochemicalTechniques As an experimental science of biochemistry requires numerous instrumental techniques that enable the development andexpansion,some of them are useddailyin any laboratory and others are very exclusive. o Subcellular fractionation, including multiple techniques o Centrifugation o Chromatography o Electrophoresis o Radioisotope Techniques o PCR o Flow cytometry o Immunoprecipitation o ELISA o Electron Microscope o X-ray Crystallography o Nuclear Magnetic Resonance o Mass Spectrometry
  4. 4. 4  THERAPEUTICAPPLICATIONS Radioisotopic The most known is the use of radioiodine (Iodine-131) in the treatment of hyperthyroidism and differentiated thyroid cancer. It is based on the emission Iodine-131 beta, high energy, short range, which, when incorporated into the thyroid cell, produces its destruction without much systemic damage. Immunoprecipitation Is a technique that uses specific antibodies to a protein to remove these proteins from the solution. The complexesof the antibody-proteinprecipitate outof solutionwith the additionof an insolubleformof the antibody bindingproteins.Examples include protein A, protein G, Zysorbin, Immunoprecipitin, or adding a second antibody to the solution.
  5. 5. 5 MEDICAL DIAGNOSIS In medicine,diagnosisorclinical propaedeuticisthe procedure bywhich a disease is identified or any condition of health-disease. The medical diagnosisisbasedsymptoms,signsandfindingsof additional teststodetermine what disease a person suffers. Diagnostic Tools o Symptoms Physical experiences are reported by the patient. Not be observed. Example:  Pain  Dyspnea  Fatigue
  6. 6. 6 o Signs The sings are findings in the patient and are detected by the doctor. Can be seen Example:  Tachypnea  Fever  Edema o Physical Examination It consists of various maneuvers performed by the doctor in the patient.  Inspection: is the method of physical examination is done by sight.
  7. 7. 7  Palpation: isthe processof examiningthe body using the sense of touch. Provides information on shape, size, texture, surface moisture, tenderness and mobility.  Percussion: Percussion is a method that is tapped certain body parts during a physical examination with fingers, hands or small instruments to assess the size, consistency, borders and presence or absence of fluid in the body's organs.
  8. 8. 8  Auscultation: is to listen, either directly or through instruments like the stethoscope, normal or pathological sounds produced by the human body. o Supplementary Examinations Theyare a set of studiesthatprovide valuable informationto medical analysis, and eithertoconfirm or give more certainty tothe diagnosisof a disease. Biopsy:procedure inwhichtissue samplesunderamicroscope toobserve.
  9. 9. 9  Radiograph:noninvasive procedure,whichshowssoftandsolidstructuresof the body. Ultrasound:usessoundwavestocreate imagesof internal oraganos.
  10. 10. 10 SCIENTIFIC ARTICLE Diagnosis and Treatment of Tuberculous Pleural Effusion in 2006 Tuberculous (TB) pleural effusion occurs in approximately 5% of patients with Mycobacterium tuberculosis infection. The HIV pandemic has been associated with a doubling of the incidence of extrapulmonaryTB,whichhasresultedinincreased recognition of TB pleural effusions even in developed nations. The diagnosis can be established in a majority of patients from the clinical features,pleural fluidexamination,includingcytology, biochemistry, and bacteriology, and pleural biopsy. The definitive diagnosis of TB pleural effusions depends on the demonstration of acid-fast bacilli in the sputum, pleural fluid, or pleural biopsy specimens. The treatment of TB pleural effusions in patients with HIV/AIDS is essentially similar to that in HIV-negative patients.
  11. 11. 11 Tuberculosis (TB) Is a leading cause worldwide of preventable morbidity and mortality from an infectious agent. o Epidemiology A total of nine millionnew casesandapproximately two milliondeathsfromTBwere reported in 2004. Although the African region has the highest estimated incidence (356 per 100,000 population per year), the majority of patients with TB live in the most populous countries of the Asian subcontinent, which accounts for nearly half of the newcasesthat arise yearly. TB pleural effusion is the second most common form of EPTB, only less frequent than lymph node TB. The incidence of TB pleural effusionsinHIV/AIDShasbeen variablyreportedfrom15 to 90%,8,9 withan effusionbeingmore common in patients with higher CD4_ counts. o Pathogenesis TB pleural effusionscanmanifestasprimaryorreactivateddisease.Rupture of a subpleural caseousfocusin the lung into the pleural space is consideredthe initial eventinthe pathogenesis of primaryTB pleural effusions.1The accumulationof fluidin pleural cavity results predominantly from increased capillary permeability and secondarily from impairment of lymphatic clearance of proteinsandfluidfromthe pleural space because of occlusion of pleural stomata. In contrast, reactivationdiseaseis frequently associated with parenchymal lesions. The delayed hypersensitivity reaction responsible for the pathogenesis of TB pleural effusionsismediatedbyT-helper type 1 (Th1) cells that activate macrophages to switch on mechanisms responsible for the killing of mycobacteria. Clinical Features In contrast to pulmonary TB, most TB pleural effusions manifest as an acute illness, with approximately one third of patients being symptomatic for 1 week and two thirds for 1
  12. 12. 12 month. The most common presenting symptoms are pleuritic chest pain (75%) and nonproductive cough (70%). Therefore,pleural tuberculosis should be considered in any adult or elderly patient with a unilateral pleural effusion.TBpleural effusions,typicallyunilateral andsmall tomoderate in size, usually occupy less than two thirds of a hemithorax. o Diagnosis The definitive diagnosis of TB pleural effusions depends on the demonstration of M tuberculosis in sputum, pleural fluid, or pleural biopsy specimens. Supportive evidence includes demonstration of classical TB granulomas in the pleura and elevated adenosine deaminase (ADA) and IFN-_ levels in pleural fluid. o Sputum Examination Traditional dogma has stated that patients with pleural TBwithoutconcomitant pulmonary disease are sputumnegative and,therefore,noncontagious. However, one study reported a remarkably high yield of mycobacterial culture (52%) in a single specimen of induced sputum with sputum smear positivity in 12% of cases. o Tuberculin Skin Test A positive tuberculinskintestresult is supportive evidence in the diagnosis of TB pleural effusions in areas of low prevalence (or no vaccination); however, a negative tuberculin skin test result may occur inapproximatelyone thirdof patients. A negative test result could result from the following: (1) Anergy secondary to immunosuppression or Malnourishment. (2) Recent infection. (3) Circulating mononuclear cells suppressing the specifically sensitized circulating T- lymphocytes in the peripheral blood. (4) Sequestration of purified protein derivative-reactive T-lymphocyte in pleural space. However,resultsof atuberculinskintestrepeated6to 8 weekslater will almost always be positive.
  13. 13. 13 o Thoracocentesis Pleural Fluid Examination A TB pleural effusion is typically clear and straw colored; however, it can be turbid or serosanguinous but is virtually never grossly bloody. The effusion is virtually always an exudate.14 Pleural fluid pH is usually between 7.30 and 7.40 (rarely ever _ 7.40), although it can be _ 7.30 in approximately 20% of cases.32 Pleural fluid glucose concentration is _ 60 mg/dL in 80 to 85% of cases.22 Pleural fluid glucose is _ 30 mg/dL inapproximately15%of cases. Although in the initial stage of illness(uptofirst 2 weeks), the differential cell count may reveal predominantly neutrophils,18 serial thoracenteses show a shift toward lymphocyte predominance.9 The older literature suggestedthat _ 5% mesothelial cellsinpleural fluidwere rarely compatible with TB pleural effusions. Pleural Fluid Smear and Culture Direct examination of pleural fluid by Zeihl- Neelsen staining requires bacillary densities of 10,000/mL and, therefore,detectsacid-fast bacilli (AFB) in 10% of cases. However, in patients with HIV coinfection, the yield of pleural fluid microscopy is 20%. Culture requires a minimum of 10 to 100 viable bacilli and, therefore, is more sensitive with a yield ranging from 12 to 70%, with the majority of series showing diagnostic yields of 30%. Sensitivityof mycobacterial culture canbe improvedbybedside inoculation of pleural fluid and by using liquid culture media or BACTEC system. ADA (adenosine deaminase) ADA is an enzyme inthe purine salvage pathwaythatcatalyzesthe conversionof adenosine and deoxyadenosine to inosine and deoxyinosine with the release of ammonia. Although ubiquitousindistribution,itplaysanimportantrole indifferentiation of lymphoid cells and is most abundant in lymphocytes (predominantly active T-lymphocytes) whose concentration is inversely related to the degree of differentiation.
  14. 14. 14 Pirasand colleaguesforthe firsttime in1978 reportedhighlevelsof ADA inpatientswithTB pleural effusions.Subsequently,several studieshave exploredthe usefulness of estimation of ADA activityinthe diagnosisof TBpleural effusions.Itwasdeterminedthatapleural fluid ADA level 70 IU/L is highly suggestive of TB, while a level 40 IU/L virtually excludes the diagnosis. A metaanalysis of 40 studies published from 1966 to 1999 concluded that the test performance of ADA (sensitivity range 47 to 100%, and specificity range 0% to 100%) in diagnosing TB pleural effusions is reasonably good (adequate to avoid pleural biopsy in young patients from areas with high prevalence of TB). IFN-GAMMA: Produced by T-lymphocytes, is capable of activating macrophages, increasing their bactericidal capacityagainst Mtuberculosis andis involvedingranulomaformation. Several studieshave foundelevatedconcentrations of INF in TB pleural effusions, which is related to increased production at the disease site by effector T cells. Other diseases that can cause elevated INF levels in pleural fluid include hematologic malignanciesandempyema.Immunosuppressedpatients(HIV orafterrenal transplant) had INFlevelssimilartoimmunocompetentindividualsretainingitsefficacy as a diagnostic test.
  15. 15. 15 PCR:
  16. 16. 16 Polymerase chainreaction(PCR)isbasedonamplificationof mycobacterial DNA fragments. As TB pleural effusionis paucibacillarydisease,the sensitivitycould be improved by PCR, as it can detect as few as 10 mycobacteria. Advantages of PCR include rapid diagnosis, improved specificity and sensitivity, and no requirement of intact immunity. Numerousstudies(Table)evaluatingthe efficacyof PCRfordiagnosisof pleural tuberculosis have shown a sensitivity ranging from 20 to 90% and specificity from 78 to 100%. o Other diagnostictests Inmunodiagnosis: The utilization of immunodiagnostics is hindered by its low sensitivity. The table lists the detailsregarding various studies using immunologic markers in the diagnosis of tb pleural effusions. Further studies are required to address the clinical utility of these markers.
  17. 17. 17 Cytokines: Cytokine-producing cells and various cytokines have been demonstrated in pleural fluid frompatientswithTB pleural effusions. The emerging role of IFN has been discussed (vide supra). Significantly higher levels of IL-6 have been demonstrated in TB effusions compared to parapneumonic and malignant pleural effusions. o Treatment:  ANTITUBERCULOSIS DRUGS: The natural history of an untreated tuberculous pleural effusion is characterized by spontaneous resolution in 4 to 16 weeks with subsequent development of active pulmonary TB or EPTB in 43 to 65% of cases over the next several years. These data emphasize the importance of proper diagnosis and treatment of tuberculous pleural effusions.Accordingtothe directlyobservedtreatmentshortcourseguidelines, severely ill patientswithextensive or bilateral pleural effusions and sputum positivity are given treatment under category I (treated during intensive phase with four drugs: isoniazid, rifampin,pyrazinamide,andethambutol for2 monthsfollowedbycontinuationphase of 4 monthswithisoniazidandrifampin).Those withasolitaryTBpleural effusionshouldbe treatedwithisoniazid,rifampin,andpyrazinamidefor2 monthsfollowedby 4 months of twodrugs, isoniazidandrifampin.Especially in loculated TB pleural effusions, there can be delayedresorption of pleural fluid even after completion of 6 months of treatment.
  18. 18. 18  CORTICOSTEROIDS: A hypersensitivity reaction to M tuberculosis results in granulomatous pleuritis. Corticosteroids through their antiinflammatory action may hasten fluid resorption and preventpleural adhesionsduringhealing.Threerandomizedtrialshave investigated the possible role of adjunctive oral corticosteroidsin TB pleural effusion. A dose of 0.75 to 1 mg/kg/d was used for a period ranging from 4 to 12 weeks. Early resolution of clinical symptoms and signs including fever, chest pain, and dyspnea was observed. Although there wasa trendtowardlessresidual pleural fluidatthe endof treatment,there wasno difference inthe developmentof residual pleural thickening or adhesions on follow-up. Residual lungfunctionwassimilarbetween steroid and control groups at completion of treatment. All three studies had insufficient power to examine death as an outcome; nevertheless, no deaths occurred in the study groups.
  19. 19. 19 UNRESOLVED ISSUES AND CONCLUSIONS There have been significant advances in the understanding of pathophysiology of TB pleural effusions. The more frequent use of imaging modalities, such as CT, and bronchoscopic techniques, such as BAL, has lead to detection of underlying pulmonary TB in a larger proportionof patients.However,the implicationof these findings in the treatment of patients withTB pleural effusionisunclear.Likewise,the estimationof ADA and IFN in pleural fluid has gained wide acceptance in the diagnosis of TB pleural effusion. However, the use of these investigations may be limited because of availability and quality control, especially in developing nations. Porcel and Vives found effective prediction models to differentiate tuberculous from malignant pleural effusions based on readily available clinical (age, temperature, history of malignancy) and pleural fluid (RBC, protein, ADA, pleural to serum lactate dehydrogenase ratio) data, although further validation is needed. The proper use of corticosteroids in the treatment of TB pleural effusions remains unresolved. It is hoped that further research to clarify these issues will result in better diagnostic and therapeutic approaches to TB pleural effusions.