Diagnostic Value of Tuberculin Test, Adenosine Deaminase and ...

Egyptian Journal of Medical Microbiology, April 2008 Vol. 17, No. 2Diagnostic Value of Tuberculin Test, Adenosine Deaminase andPolymerase Chain Reaction in Tuberculous and non tuberculousPleural EffusionMona Abdel monem Esmail*, Hala Abdel Hameed**, Salama Rabea***,YehiaShaker **** Atef Elakad***** Microbiology and Immunology**Chest, *** Bio Chemistry and ****Internal Medicine Department,Faculty of Medicine, Minia University.ABSTRACTBackground: Tuberculosis (TB) is a major cause of pleural effusion, which in TB usually haslymphocytic and exudative characteristics. The final diagnosis of TB pleurisy was based on combining clinicaljudgment with radiological findings, and microbiologic tests, In cases of suspected pleural tuberculosis, a rapidand accurate laboratory diagnosis through ADA activity and PCR detection is of prime importance, sincetraditional techniques of detecting acid- fast bacilli have limitations.Aim of the work: To examine the diagnostic efficiency of Ziehl-Neelsen (Z.N) staining, tuberculin test, ADAactivity, and PCR in patients with pleural effusions and related the results to clinical signs and symptoms in apopulation with a high prevalence of TB.Patients: The study included 92 patients with pleural effusion of different etiologies taken from ChestDepartment, Minia University Hospital and Minia Chest Hospital between July and 2007 and March 2008.The diagnosis of pleural (p) TB was confirmed in any patient presenting with positive findings ofMycobacterium tuberculosis, either on the sputum or pleural fluid and also, in the absence of negativelaboratory results, those patients with clinical improvement after empirical treatment.Methods: A single specimen of pleural fluid (50to100ml) was submitted for appearance, and protein content,Ziehl-Neelsen staining, pleural ADA activity determination and PCR .Also 10 ml blood for estimation ofhemoglobin, ESR and serum ADA activity. The sputum was examined for acid fast bacilli by Z.N. staining.Results: Out of 92 cases studied there were 40 cases (43%) of highly suspicious of tuberculous pleural effusion.Examination of pleural effusion by Z&N stain in all cases was negative. On the other hand, +ve PCR wasfound only in 16 cases (40 %) of TB pleural effusion and 3 cases of empyema. All 40 cases of tubercular pleuraleffusion showed greatly significant elevated ADA levels in pleural fluid, with a mean value of 57.4±7.7 U/L incomparison with other nonTB pleural effusion ADA levels including 25 cases of Para pneumonic effusionswith a mean value of 27.5 ± 9 U/L., 7 cases of empyema with a mean value of 24.8 ± 3 U/L, and, of 18.5 ± 6.5U/L was the mean value of pleural fluid ADA among cases of liver cell failure. A significant +ve. correlationwas observed between sputum examination by ZN and tuberculin test, and PCR in TB pleural effusion group(p

Egyptian Journal of Medical Microbiology, April 2008 Vol. 17, No. 2permeability and secondarily from impairment oflymphatic clearance of proteins and fluid from thepleural space because of occlusion of pleuralstomata (11) .In contrast, reactivation disease isfrequently associated with parenchymal lesions.(10) (1).TB pleural effusion was considered a disease ofthe young, with a mean age of 28 years, comparedto 54 years for parenchymal tuberculosis (12)However, in recent years, several authors reportedthat mean patient age has gradually risen (13) andthat tuberculous pleurisy is becoming apredominantly reactivated form of tuberculosis, atleast in industrialized nations.Diagnosis of pulmonary tuberculosis isconfirmed mainly by sputum examination of AFB.However, the diagnosis of extra pulmonarytuberculosis requires investigation of pleural fluidbiochemistry, cytology and pleural biopsy.Positivity of AFB and Histopathological (HP)study of pleura is very low and culture is very timeconsuming. ELISA, PCR & Interferon are veryexpensive tests. Adenosine deaminase has beenproposed to be a useful surrogate marker fortuberculosis in pleural, pericardial and peritonealfluids.(14)ADA is an enzyme in the purine salvage pathwaythat catalyzes the conversion of adenosine and deoxyadenosineto inosine and deoxyinosine withthe release of ammonia. Although ubiquitous indistribution, it plays an important role indifferentiation of lymphoid cells and is mostabundant in lymphocytes (predominantly active T-lymphocytes) whose concentration is inverselyrelated to the degree of differentiation.(15).Studies have confirmed high sensitivity andspecificity of adenosine deaminase for earlydiagnosis of extra pulmonary tuberculosis (14).The aim of the work:It was to examine the diagnostic efficiency ofconventional, (Z.N staining and tuberculin test)and alternative diagnostic methods (ADA activityand PCR) in patients with tuberculous pleuraleffusions individually and in combination andrelated the results to clinical signs and symptomsin a population with a high prevalence ofTB.Subsequently, we determined the sensitivity,specificity, positive predictive values (PPVs) andnegative predictive values (NPVs) of each methodin patients with pleural effusions.PATIENTS AND METHODSPatientsThe study included 92 patients with pleuraleffusion seen in Chest Department, MiniaUniversity Hospital and Minia Chest Hospitalbetween July and 2007 and March 2008.Clinicalsymptoms and signs, demographic data andradiological results were recorded. The response tointradermal purified proteins derivative (PPD) wasevaluated, and an induration of >10 mm wasconsidered to be positive result.The following four groups were evaluated:1-Group 1 pleural TB (40 cases)2-Group 2 Para pneumonic effusions (25 cases)3-Group 3 empyema (7cases)4- Group 4 pleural effusions due to liver cellfailure (20 cases)Case Definition of TuberculosisCases of pTB were defined as those patients withclinical manifestations suggestive of tuberculosis,i.e. the presence of productive cough, low-gradefever, night sweats, weight loss, and chest pain,especially if these symptoms lasted 4 weeks andpresenting with a positive Ziehl-Neelsen result, insputum and or the pleural fluid .Patients withclinical and radiological findings that lackedmicrobiological confirmation but respondedpositively to empirical anti TB therapy were alsoconsidered to be tuberculosis cases. We defined apositive response to therapy as the improvementof clinical and radiological findings after 2 monthsof the therapy.Laboratory methodsA single specimen of pleural fluid (50to100ml)was submitted for appearance, examinationprotein content, Ziehl-Neelsen staining, pleuralADA activity determination and PCR Also 10 mlblood for estimation of hemoglobin, ESR andserum ADA activity. Pleural fluid samples wereconcentrated by centrifugation for 20 min at10.000g at4ْ C. The pellet was resuspended in 1.5 mlDNAse-free buffer (PH, 8.0).The sputum wasexamined for acid fast bacilli by Z.N. staining.Tuberculin skin test was also done.Adenosine deaminase assay in serum andPleural effusion:Assay procedure:The ADA assay kit was obtained fromDIAZYME. Serum and pleural effusion sampleswere analyzed and monitored kinetically for 10minutes at 550 nm (16)Principle of assay:The ADA assay is based on the enzymaticdeamination of adenosine to inosine which isconverted to hypoxanthine that converted to uricacid by xanthine oxidase and H 2 O 2 generated usedto monitor assay kinetically at 550 nm.DNA Extraction and PCR:50µl pleural fluid was added to a sterile1.5 mlmicrofuge tube containing 250µl Cell LysisSolution then it was incubated at65 ْc for 15 minutes to complete lysis. 1.5 µlRNase A Solution was added to the cell lysate.The sample was mixed by inverting the tube 25times and was incubated at 37 ْc for 15-60 minutes.288

Egyptian Journal of Medical Microbiology, April 2008 Vol. 17, No. 2It was cooled to room temperature.100 µl ProteinPrecipitation Solution was added to the lysate, wasvortexed, was placed into an ice bath and wascentrifuged at 13.000-16.000 xg for 3 minutes.The precipitated proteins formed a tight pellet.The supernatant containing the DNA (leavingbehind the precipitated protein pellet) was pouredinto a clean 1.5 ml microfuge tube containing300µl 100% Isopropanol.0.5 µl Glycogen Solution was added per 300 µlIsopropanol.The sample was mixed by inverting gently 50times and was incubated at room temperature forat least 5 minutes. The sample was centrifuged at13.000-16.000 xg for 5 minutes. The supernatantwas poured off and tube was drained briefly onclean absorbent paper. 300 µl 70%Ethanol wasadded and the tube was inverted several times towash the DNA pellet. The tube was centrifugedfor 1 minute. The tube was inverted and drainedon clean absorbent paper and was allowed to airdry10-15 minutes. 10 µl of DNA HydrationSolution was added. DNA was rehydrated byincubating sample for one hour at 65ْC. Tube wasvortexed and DNA was stored at 4ْC (17) (18)(Gentra, USA)Thermal cycling parametersA standard three step endpoint PCR cyclingprotocol consists of multiple cycles ofdenaturation(95ْ C),annealing(40-60ْC) andextension(72ْ C ).An initial denaturation step (95ْ Cfor 5 min) is recommended to ensure completedenaturation of the template DNA. In some cases,it may be possible to amplify a target sequenceusing two –step PCR, the denaturation step(95ْC)is followed by a combined annealing/elongationstep(50-65ْ C). For most standard, three-stepPCRs, 35 cycles produce5 9a 10 -10 fold amplification of the target sequence.PCR product yield can be improved by increasingthe number of cycles to 40 (19)(20) (AmershamBiosciences, England).Thereafter Gel was prepared with 2% agarose gel.The samples were placed in the gel chambers, andthen power was supplied on 105 volt for1 hours.Finally, the bands were detected by UV lamp (Fig:3).Statistical analysis:Data were coded, entered and analyzed by SPSSpackage version 13.0.under windows. Mean,standard deviation, frequency distribution andcross tabulation were estimated for study groups.Tests of significance used during comparisonamong study groups were one-way ANOVA forquantitative data and Chi-square test forcategorical data. Pearson correlation coefficientwas to correlate between; correlation coefficientwas to correlate between quantitative data whileSpearman correlation coefficient was to correlatebetween categorical data. P value of

Egyptian Journal of Medical Microbiology, April 2008 Vol. 17, No. 2Table 2: Comparison between groups regarding laboratory data:laboratory data:TB pleuraleffusion(n= ٤٠)Parapneumonic(n= ٢٥)Empyema(n= 7)Liver cellfailure(n= 2٠)Total(n=92)TestPTuberculintestZNsputumRange 15-18 6-14 6-11 8-10 6-18Mean ± SD 16.2±1.2 9.6±3.4 9.14±1.57 9.2±0.76 12.4±3.9+ve 5( 12.5%) 0 0 0 5(5.4%)-ve 35(87.5%) 25(100%) 7(100%) 20(100%) 87(94.6%)F=83.20.0001F=38.1 0.07PCRPositive 16(40%) 0(0%) 3(42.8%) 0(0%) 19(20.6%)Negative 24(60%) 25(100%) 4(57.2%) 20(100%) 73(79.4%)X 2 =22.950.0001ADA effusionRange 48-65 19-40 19-28 14.7-31 14.7-65Mean ± SD 57.4±7.7 27.5±9 24.8±3 18.5±6.5 38.3±18.6F=152.380.0001ADA serumRange 24-53 12-22 14-20 22-26 12-53Mean ± SD 37.4±9.4 17.7±3.4 18.9±2.3 24.2±1.9 27.8±10.9F=54.570.0001HbRange 9-12.5 8.5-10.5 12.5-12.5 9-11 8.5-12.5Mean ± SD 10.6±1.2 9.6±0.8 12.5±0 ±0.7١٠٫٢ 10.4±1.2F=9.160.0001ESR1stRange 60-106 60-100 47-63 60-95 47-106Mean ± SD 89.6±16.3 77.6±17.7 56.4±5.7 75±16.9 80.64±18.6F=10.230.00012ndRange 105-126 68-90 62-72 50-90 50-126Mean ± SD 110.2±8.24 79.8±9.24 69±4.3 68.4±18.6 89.7±21.6F=80.60.0001Table 3: Statistical analysis Tuberculin test (TT) readings in p TB effusionP(TT) Sensitivity Specificity Positive Pred.Cutoff point%%value %Negative Pred.value %>6>8>9100.00100.00100.0017.3125.0061.5448.250.666.7100.0100.0100.0>10>11>14 *>15>16>17>18100.00100.00100.0060.0040.0020.000.0080.7782.69100.00100.00100.00100.00100.0080.081.6100.0100.0100.0100.00100.0100.0100.076.568.461.956.5291

Egyptian Journal of Medical Microbiology, April 2008 Vol. 17, No. 2230 pbof isolatedband23 4 5 6 7 81100 pbla d d e rFig .3. Lane1 is 100 pb ladder. Lane2 is the-ve control. Some positive samples employ lanes 3-8.1.00.75.50Sensitivity.250.00ADA serumADA pleural fluidTuberculin test0.00.25.50.751.001 - SpecificityFig 1: Roc curve in tuberculus pl. effusion group at the best cut off1.00.75.50Sensitivity.250.00ADA serumADA pleural fluidTuberculin test0.00.25.50.751.001 - SpecificityFig 2: Roc curve in non tuberculous group (n=52) at the best cut off293

Egyptian Journal of Medical Microbiology, April 2008 Vol. 17, No. 2pleural fluid with a mean value of 57.4±7.7 U/Litand were significantly raised compared to othercases of pleural effusions Piras et al for the firsttime in 1978 reported high levels of ADA inpatients with TB pleural effusions (34).Subsequently, several Studies (35) (37) haveexplored the usefulness of estimation of ADAactivity in the diagnosis of TB pleural effusions. inthe study of (35) the mean value of a pleural fluidADA ( 77.20 ± 30.63 U/Lit,) was significantlyhigher as compared to other types of pleuraleffusions.However, elevated ADA in lymphocyte-richpleural effusions has been reported in otherdiseases, such as rheumatoid arthritisbronchoalveolar carcinoma, mesothelioma,Chlamydia pneumonia,, brucellosis,Mediterranean fever (2). (coccidioidomycosis andin most patients with empyema(11) .None of ourtuberculosis patients had coexisting diseases suchas rheumatoid arthritis or chronic lymphaticleukemia, etc since the patients were randomlyincluded in the control group, there was no bias.to The ADA cutoff value indicative oftuberculosis is subject debate , since the literaturepresents a great variation of these values,It is difficult to define a universal cutoff value forADA activity. This test has to be validated foreach region, and eventually for every servicewhere the test is to be used. (28) .The presentstudy showed that at > 40 U Lit was the best cutoff point, the sensitivity and specificity ,apositive predictive value ,and negative predictivevalue were 100% for pTB group and very low fornon tuberculous effusion.The high sensitivity and specificity of ADA inthe differential diagnosis of pleural TB have beensubstantiated in studies conducted in other areas ofhigh TB prevalence, such as Mexico, Spain, andBrazil (24) (35)show that the best cut off point is60 U negative predictive value 80%, which is alsoconfirmed by plotting ROC curves. O’cana (39)reported a specificity of 97 % and sensitivity of100% at cut off point of 45 U/Lit. Nevertheless,the sensitivity and robustness of ADA activity inthediagnosis of pleural TB, together with itssimplicity speed, and low cost, urge thewidespread implementation and routine utilizationof the method in populations with a highprevalence of TB and its evaluation in areas of lowprevalence (33).This study showed a significant elevation theserum ADA of TB pleural effusion with the meanvalue (37.4± 9.4) as compared to other types ofpleural effusion; this is similar to (35). Al-Shmmary, 1997 reported that negative Ziehl-Neelsen staining for acid fast bacilli and PurifiedProtein Derivative skin test results were obtainedin 32.3% and 11.1% of the examined patientsrespectively. All these patients showedsignificantly elevated serum or pleural fluid ADAlevels. It is hereby suggested that serum andpleural fluid ADA levels can in conjunction withother tests, serve as a marker in patients ofpulmonary tuberculosis (40).In contrast with the indirect nature of ADAactivity, PCR, which is based on the amplificationof a specific genomic sequence of M tuberculosis,is theoretically highly specific (33)In our study,+ ve PCR was found only in 16 cases(40 %) of TB pleural effusion and 3 cases ofempyema. Kuan et al2007 (28) reported that, PCRwas positive in 43.4%of patients with TB pleurisy.Among probable TB cases, the 31% that could beconfirmed as due to M tuberculosis by PCR (42).Many studies have reported experiences of PCRamplification with pleural fluid and found themonly positive in 17.1% of TB pleurisy, andpositive culture in 24.6% of patients (41)Babu et al.,2001(43)reported a possibility of afalse positive PCR finding due to the presence ofold healed tuberculosis infection in a patienthaving non tubercular effusion due to otherdiseases (43) . Contamination either withextraneous DNA or with amplification products ofprevious analysis is the principal source of falsepositiveresults (44)In the study of Lang et al., 1998,the frequency of afalse negative PCR results 24 cases(60%)) is mostlikely attributable to the inefficient recovery ofgenomic DNA from the characteristically lownumber of bacilli in patients with pleural TB. Theextraction of DNA from frozen samples, asoccurred in this study, can be less efficient thanextraction from fresh samples (45)The results of Babu et al.,2001 suggest that thePCR is more sensitive than other existingmethods, but still not nearly good enough toidentify all cases. The results are not surprisingconsidering the fact that pleural effusion inpatients with tuberculosis mainly occurs as a resultof a delayed hypersensitivity reaction totuberculous proteins, and the presence of bacilli inpleural fluid is not always necessary (43)All of the patients with positive PCR results hadresponded very well to ATT. It was shown in aprevious study (46) that patients withinflammatory uveitis who were treated with ATTbased on a PCR test result showed resolution ofinflammation with no recurrence for 2 years offollow-up (46)Advantages of PCR include rapid diagnosis,improved specificity and sensitivity, and norequirement of intact immunity Thedisadvantages of PCR include high cost, risk ofcontamination, and the technology involved in theprocedure does not permit routine diagnostic useat present .However, the limitation of relying onPCR alone is the low sensitivity (43.4%) yield due295

Egyptian Journal of Medical Microbiology, April 2008 Vol. 17, No. 2to the low number of TB bacilli in the pleuralfluid, which was demonstrated by the low culture(28.3%) yield from pleural fluid (1).In the present study a significant +ve. correlationbetween sputum examination by ZN andtuberculin test , and PCR in TB pleural effusiongroup and the mean value of pleural fluid ADA(62.7±2.3) was significantly higher in +ve PCRcases in comparison with that of -ve PCR cases ofTB pleural effusion group ,while in empyema thedifference is insignificant he combination ofPCR and ADA activity determination allowedthe selective increase of sensitivity andspecificity for probable and confirmed casescompared to individual methods. Positive andnegative predictive values for these individualor combined methods were maintained over awide range of prevalence of pleural TB in thepatient (36).In conclusionAlthough PCR, tuberculin test and ADA activityhave been evaluated individually in the diagnosisof pleural TB, the results ofusing all three methods in the same patients havenot been availablepreviously, and their combined use has not beenexplored.Our findings show that together, measure cost andexpertise are also considerations in adoptingtechnologies for use at different levels ofhealth service. ADA and tuberculin testmeasurement are simple and comparatively lowcostprocedures whereas PCR is a moredemanding and expensive method yet has theadvantage of being a rapid and direct means ofdetecting M tuberculosis in pleural fluid. 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Egyptian Journal of Medical Microbiology, April 2008 Vol. 17, No. 2القيمة التشخيصية لاختبارمنى عبد المنعم اسماعيل*‏ ، هالة عبد الحميدالتيبروآلين الدرنى ، أدينوسين دي أميناز والتفاعل البللوميريزي المتسلسلفي السائل البللوري الدرني والغير درني** ، سلامة ربيع***‏ ، يحيى شاآر****‏ ، عاطف العقاد ****‏*قسم الميكربيولوجي والمناعة ، ‏**قسم الصدر ، ‏***قسم الكيمياء الحيوية ، **** قسم الباطنة العامة-‏ آليةالطب-‏ جامعة المنيا.‏مرض الدرن هو السبب الرئيسي لتكوين السائل البللوري وفي مرض الدرن يكون السائل البللوري لها خاصيةليمفاوية وارتشاحية،التشخيص النهائي لالتهاب البللوري الدرني يعتمد على التشخيص الاآلينكي مع الأشعةوالاختبارات الميكروبيولوجية.‏ في حالات الالتهاب البللوري الدرني(‏ المشتبه فيهم)‏ ويكون التشخيص المعمليالسليم والسريع من خلال أدينوسين دي أميناز،‏ الاختبار البلوميريزي المتسلسل ولذلك الطرق التقليدية لتعيينالميكروبات العصوية الدرنية أصبحت أقل أهمية.‏الهدف من البحث:‏ هو اختبار القدرة التشخيصية لصبغة الزيل نيلسن ، اختبار حساسية الجلد للدرن ، أدينوسين ديأميناز والتفاعل البللوميريزي المتسلسل في مرض السائل البللوري ومرجعية النتائج الى الأعراض والعلاماتالاآلينيكية في الناس الذين يتمتعون بزيادة نسبة انتشار الدرن بينهم.‏المرضى:‏ تشمل هذه الدراسة ٩٢ مريض بالانسكاب البللوري بأسبابه المختلفة وهؤلاء المرضى من مستشفى المنياالجامعي ، مستشفى الصدر من يوليو ٢٠٠٧ الى مارس . ٢٠٠٨ وقد تأآدنا من تشخيص الانسكاب البللوريالدرني في المرضى عن طريق تواجد الميكروب الدرني في عينة البصاق وعينة السائل البللوري وفي المرضىالسلبيين في الاختبارات المعملية بعد تحسنهم بالعلاج.‏الطرق والوسائل:‏ ٥٠ الى ١٠٠ مللي من السائل البللورى تخضع الى عدة اختبارات ا مظهرالسائل البللورى ،نسبه البروتين ، صبغة الزيل نيلسن،‏ أدينوسين دي أميناز والاختبار البللوميريزي المتسلسل و أيضا ١٠ مللي دملتعيين نسبة الهيموجلوبين ، سرعة الترسيب وايضا نسبة الأدينوسين دي أميناز في المصل يختبر البصاق لتعيينميكروب الدرن عن طريق صبغة الزيل نيلسن.‏النتائج:‏ من خلال الدراسة على ٩٢ مريض قد تواجد ٤٠ مريض بنسبة ٤٣% من مرضى السائل البللوري الدرني‏(المشتبه فيهم).‏نتائج السائل البللوري لصبغة الزيل نيلسن سلبية جميعا ‏.على الوجه الآخر نتائج اختبار التفاعل البللوميريزيالمتسلسل الموجبة آانت في ١٦ حالة في مجموعة السائل البللوري الدرني و ٣ حالات ايجابية فيمجموعةالانسكاب البللورى نتيجة الالتهاب الصديدي.‏.(%٤٠ )آل المرضى(‏ ٤٠) في مجموعة السائل البللوري الدرني أظهرت زيادة في أدينوسين دي أميناز في السائلالبللوري ومتوسطه ٧٫٧±٥٧٫٤ وحدة/لتر وبالمقارنة بمجموعة السائل البللوري الغير درني ومتوسط الأدينوسيندي أمينازهو ٩±٢٧٫٥ وحدة/لترفي حالات السائل البللوري المصاحب للالتهاب الرئوي.‏ في حالات السائل البللوري الصديدي متوسط الأدينوسين)٣±٢٤٫٨ وحدة/لتر.‏في حالات الفشل الكبدي متوسط الأدينوسين ٦٫٥±١٨٫٥ وحدة/لتر.‏ وقد لوحظ وجود توافق ايجابي بين البصاقاختبر بصبغة زيل نيلسن)‏ واختبار الدرن واختبار التفاعل البللوميريزي المتسلسل في مجموعة السائل البللوريالدرني.‏نستخلص من هذا البحث:‏أن تحليل الأدينوسين دي أميناز وتعيين الحمض النووي الديؤآسي ريبوزي الدرني عن طريق اختبار التفاعلالبللوميريزي المتسلسل في العينات هو أفضل وسيلة تشخيصية لكي نحصل على تشخيص سريع ودقيق في حالاتالالتهاب البللوري الدرني.‏الكلمات الهامة:‏ الدرن – أدينوسين دي أميناز – التفاعل البللوميريزي المتسلسل.‏298