Contents - Medicinski Fakultet u Sarajevu - University of Sarajevo

FOLIA MEDICA FACULTATIS MEDICINAE UNIVERSITATIS SARAEVIENSISJournal of Medical Faculty University of Sarajevo, Bosnia&Herzegovina2011 Vol. 46, No. 1ContentsORIGINAL PAPERSSerotyping Streptococcus Pyogenes of Preschool and Primary School ChildrenAida Koso, Šukrija Zvizdić . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3Frequency and Sex Distribution of Omphalocele in Surgically Treated Infantsin Sarajevo Region of Bosnia And HerzegovinaSelma Aličelebić, Amela Vilić . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9Acute Toxicity Study of Combination of Tridecactide and Met-EnkephalinMaida Rakanovic-Todic, Nedzad Mulabegovic, Fahir Becic, Mirjana Mijanovic,Svjetlana Loga-Zec, Jasna Kusturica, Lejla Burnazovic-Ristic, Aida Kulo, Elvedina Kapic . . . . . . . . . . . 13Brucellosis – Emerging Zoonosis in Bosnia and HerzegovinaSajma Dautović-Krkić, Edina Bešlagić, Meliha Hadžović-Čengić, Snježana Mehanić,Adnan Karavelić, Sead Ahmetagić, Ibrahimpašić Nevzeta, Eldira Hadžić, Ivo Curić,Nazif Derviškadić, Janja Bojanić. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22Diagnostic Significance of Platelet Count in Patients With Lung CancerSelma Arslanagić, Hasan Žutić, Nedžad Mulabegović,Rusmir Arslanagić, Reuf Karabeg, Naima Arslanagić. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28Frequency and Antibiotic Susceptibility of Methicillin-Resistant Staphylococcus Aureus(MRSA) in Canton Sarajevo; Bosnia and HerzegovinaEdina Bešlagić , Sabaheta Bektaš, Mufida Aljičević, Snježana Balta, Sadeta Hamzić . . . . . . . . . . . . . . 35Detection of Cytokines IFN γ, TNF α and IL 10 in Acute and Chronic Human ToxoplasmosisSabaheta Bektaš, Snježana Balta , Mufida Aljičević, Edina Bešlagić . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41REVIEW PAPERSUse Of Homeostatic Model Assessment In Managing Type 2 DiabetesDamira Kadić, Davorka Dautbegović-Stevanović . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48Paragangliomas of the NeckJasminka Alagić-Smailbegović, Kamenko Šutalo, Edina Hadžić . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54Recommendations for Treatment of Active and Latent Tuberculosis Before and DuringThe Tumor Necrosis Factor Antagonists Therapy in Bosnia and HerzegovinaHasan Žutić, Nenad Prodanović, Šekib Sokolović, Mladen Duronjić,Suada Mulić Bačić, Nađa Borovac, Nedžad Mulabegović . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60Preporuke za tretman aktivne i latentne tuberkuloze prije i tokom terapije antagonistimafaktora tumorske nekroze u Bosni i HercegoviniHasan Žutić, Nenad Prodanović, Šekib Sokolović, Mladen Duronjić,Suada Mulić Bačić, Nađa Borovac, Nedžad Mulabegović . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73Instructions to authors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

FOLIA MEDICA FACULTATIS MEDICINAE UNIVERSITATIS SARAEVIENSISJournal of Medical Faculty University of Sarajevo, Bosnia&HerzegovinaEditor-in-ChiefNedžad MulabegovićExecute EditorMaida Rakanović TodićEditorial BoardJasminko HuskićDamir AganovićAmela BegićSlavica IbruljSemra ČavaljugaNermin SarajlićAlmira Hadžović DžuvoLejla Burnazović RistićRadivoj JadrićLectorised byDubravko VaničekTechnical Editor and PrintSaVart SarajevoDTPNarcis PozderacAdress of the Editorial board:71000 Sarajevo, Čekaluša 90Bosnia & HerzegovinaPhone: 00387 33 226 472Fax: 00387 33 203 670Published byFaculty of Medicine,University of Sarajevowww.mf.unsa.ba/foliaISSN 0352-9630EBSCO Publishing (EP) USAhttp://www.epnet.comPrinted on acid-free paper

Folia Medica 2011; 46 No 1:1-8Figure 1. Representation of certain M typesAmong patients who had the streptococciisolated M typing in relation to sex andage there are no statistically significant differencesin M - types of streptococci.Also was done the testing blood samplesfor the presence of anti-streptolysin Oantibody (ASO) titer in 19 or 27.1% of respondents.The results which derived fromASO tests indicate that it is positive in 9 or47.4% of respondents, and that its value of200 were found in 3 or 33.3% of total positivetests, and the titer of 400 at 6 or 66.7%of total ASO positive tests. Most patientswith positive findings ASO 200 have theM-6 type, and most patients with ASO valueof 400 is M-3 type. ASO value 400 is foundwith M-1 and M-6 type.DiscussionThis study of severe forms of streptococcaldisease came to the conclusion that it occurin cycles that are caused by certain prevalenttypes of streptococci. European studies haveshown that the distribution of ß-streptococcusgroup A haemolyticus is representeddifferently in different countries. In severalEuropean countries, the types of M-1, M-3and M-28 predominates, but there is a tendencyof increase of new invasive types (M-77, M-81, M-82, M-89) (17-19). It is knowthat in certain regions of the United States,during the twentieth century, was found alarge number of patients with acute rheumaticfever, of which had as a previous illness,infection with Streptococcus pyogenes,type M-1, M-3 and M 18 (20). Reducementin the incidence of rheumatic fever in theUnited States explains the changing representationof virulent strains of streptococcus-reducednumber rheumatic types(significantly reduce the number of isolatedtype M-3, M-5 and M-6 streptococci, from49.7% to 10.6%) (21). Tests in the Republicof Croatia indicate that the occurrence ofsevere forms of diseases caused by streptococciand the occurrence of complicationsare associated with the isolation and identificationof highly virulent strains of mucousM-serotypes of Streptococcus pyogenes(22). Testing (during the six year period) inpatients treated for streptococcal disease atthe Clinic for Infectious Diseases (Dr “FranMihaljević”) in Zagreb, Republic of Croatia,has shown that patients with severe streptococcaltoxic and invasive infections in 45.5%of cases have isolated invasive M-1 and M-3type, with documented types of M-6, M-28also M-76 M-4, M-12 and M-60, but in asmaller percentage (23).In the Microbiological laboratory of theDepartment of Public Health of SarajevoCanton, was isolated by standard methodsand typed Streptococcus pyogenes, which6

Aida Koso et al.: Serotyping Streptococcus Pyogenes of Preschool and Primary School Childrenis governed by the microbiology laboratoryof the National Reference Laboratoryin Prague, Czech Republic. Of the 72 typedstrains of streptococcus, in 70 or 97.2% ofthe samples type was confirmed by isolationof Streptococcus pyogenes, which indicatesthe high quality and validity of routine microbiologicaldiagnostics in bacteria that isperformed in the microbiological laboratoryof the Department of Public Health ofthe Canton Sarajevo. Serotyping of streptococciin our country does not exist, and thesamples were serotyped in Prague, invasivetypes of M 1 and M 3 were isolated in 35.7%of samples. Considering the total number ofM type sorts, 75.7% of samples were M-1,M-3, M-6, M-12. Other types which areisolated was M-28, M-4, M-5, M-27G, M-50.62, M-75, M-77, M-87 and M-89.Epidemiological data on streptococcalinfections in developing countries are ofcrucial importance, although it turned outthat they are very scarce. Consequently isdeveloped the Strep-EURO project, whichaims to improve our knowledge of streptococcusgroup A. In order to prevent streptococcaldisease it is necessary continuouslycollect information and knowledge aboutthe cause of the diseases that are its consequence.It is necessary to continually monitorthe presence of particular antigenic types(10.24).ConclusionStudies in the world have pointed out theimportance of isolation, identification andserotyping of streptococcus, the importanceof identifying the occurrence of mucoustypes greater incidence of acute rheumaticfever and the importance of introducing anactive control over the movement of certainM-types of streptococci, as a risk factorin the possible development of rheumaticheart disease, or acute glomerulonephritis.In the future in our country it is necessary tocreate a program similar to the Strep-EUROprogram for identification and serotypingStreptococcus pyogenes in order to establisha permanent expert network of referenceand other relevant centers. Role of referencecenters would be to develop and applymodern methods in the field of molecularbiology, genetics, microbiology and otherbranches of medicine, with the aim of theresearch of streptococci and streptococcalinfections. The results should be evaluatedand coordinated with other European centersin order to take appropriate measures toprevent streptococcal infection.References:1. Kalenić S, Mlinarić–Missoni E. Medicinska bakteriologijai mikologija. Merkur A.B.D. Zagreb,2005; 142-160.2. Bisno AL, Brito MO, Collins CM. Molecular basisof group A streptococcal virulence. Lancet InfectDis 2003; 3(4): 191-2003. Kriz P, Mottova J. Analysis of active survellenceand passive notification of streptococcal diseasesin The Czech Republic. Adv Exp Med Biol 1997;418: 217-19.4. Veasy LG, Tany LY, Daly JA, Korgenski K, et all.Temporal association of the appearance of mucoidstrains of Streptococcus pyogenes with a continuinghigh incidence of rheumatic fever in Utah. PubMed Pediatrics 2004; 113(3 ): 168-72.5. Martin JM, Green M, Barbadora KA, Wald ER.Group A Streptococci among school-aged children:clinical characteristics and the carrier state.Pub Med Pediatrics 2004; 114(5): 1212-9.6. Stephenson KN. Acute and chronic pharyngitisacross lifespan. Pub Med Lippincots Prim CarePract 2000; 4 (5) : 471-89.7. Bisno AL, Brito MO, Collins CM. Molecular basisof group A streptococcal virulence. Lancet InfectDis 2003; 3(4): 191-200.8. Facklam R, Beall B, Efstratiou A, Fischetti V, et all.Emm typing and validation of provisional M typesfor group A streptococci. Emerg Infect Dis 1999;5(2): 247-53.9. Stollerman GH. Rheumatic fever as we enter the21 century. Boston University. Archived Reports,20007

Folia Medica 2011; 46 No 1:1-810. Lamagni TL, Efstratiou A, Vuopio-Varkila J, JasirA, Schalen C.Strep-EURO. The epidemiology ofsevere Streptococcus pyogenes associated diseasein Europe. Indexed in Medline as Euro Euroveill2005; 10(9): 179-84.11. Kapetanović O, Ohranović M. Zbirka sanitarnihpropisa. Bosanska riječ Sarajevo, 2005 : 40-59.12. Službeni list SRBiH 36/87 Pravilnik o načinu prijavljivanjazaraznih bolesti: 6113. Knjige evidencije zaraznih oboljenja u KantonuSarajevo, 2006.14. Milovanović J. Etiološka identifikacija akutnihtonsilofaringitisa kod dojenčadi i djece s posebnimosvrtom na terapiju streptokokih infekcija.Magistarski rad. Univerzitet u Sarajevu, Medicinskifakultet, Sarajevo, 1987.15. Dinarević S, Mulaosmanović V, Subašić D,Selimović A, i saradnici. Correlation of Streptococcusgroup A in throat culture and antistreptolysintitre in post-war Bosnian pediatric population.Paediatric clinic Sarajevo, Microbiology InstituteCCS Sarajevo, Bosnia and Herzegovina Acta PaediatricaEspanola vol 60; (8)(381-2) ; 2002.16. Bernard Beall. Streptococcus pyogenes emm sequencedatabase Centers for Disease Control andPrevention, Atalanta, 2008. http:/www.cdc.gov/ncidod/biotech/strep/protocol_emm-type.htm17. Colman G, Tanna A, Gaworzewska ET. Changesin the distribution of serotypes of Streptococcuspyogenes. Int J Med Microbiol 1992; 277-8(Supp22):14-6.18. Kaplan EL, Wotton JT, Johnson DR. Dynamic epidemiologyof group A streptococcal serotypes associatedwith pharyngitis. Lancet 2001; 358(9290):1334-7.19. Siljander T, Toropainen M, Muotiala A, Hoe NP,et all. Emm- typing of invasive T28 group A streptococci,1995-2004. Finland. 15th European Congressof Clinical Microbiology and Infectious DiseasesClin Microbiol Infect 2005; 11(Suppl 2): 567.20. Benenson A. Priručnik za spriječavanje i suzbijanjezaraznih bolesti. Beograd, 1995; 459-66.21. Shulman ST, Stollerman GH, Beal B, Dale JB, etall. Temporal changes in streptococcal M proteintypes and the near-disappearance of acute rheumaticfever in the United States. Clin Infect Dis2006; 15: 42 (4): 441-7.22. Begovac J. Kliničko značenje serotipizacije streptokokau Zagrebu. Medicinski fakultet, Zagreb,1995. SVIBOR- Projekt broj: 3-01-29.23. Bejuk D. Učestalost tipova beta-hemolitičkihstreptokoka grupe A u bolesnika liječenih u kliniciza infektivne bolesti Dr Fran Mihaljević od 1990.-1996. godine. Doktorska disertacija, Medicinskifakultet Zagreb, 2002.24. Smith A, Lamagni TL, Oliver I, Efstratiou A, etall. Invasive groupA streptococcal disease: shouldclose contacts routinely receive antibiotic prophylaxis?Lancet Infect Dis 2005; 5(8): 494-5008

Original PapersFrequency and sex distribution of omphalocele in surgicallytreated infants in Sarajevo region of Bosnia and HerzegovinaSelma Aličelebić 1 , Amela Vilić 21, 2Institute of Histology and Embryology,University of Sarajevo, School of Medicine,Čekaluša 90, 71000 Sarajevo,Bosnia and HerzegovinaCorresponding authorSelma AličelebićInstitute of Histology and EmbryologyUniversity of SarajevoSchool of MedicineČekaluša 90, 71000 SarajevoBosnia and HerzegovinaE-mail: alicelebicselma@gmail.comOmphalocele is a prolapse (herniation) of abdominal organs,through an anterior abdominal wall defect at the base of theumbilical cord. Herniated abdominal organs (liver, small andlarge intestine, stomach, spleen, gallbladder) are covered withthe amnion and sometimes also with the peritoneum. Theaim of this research is to determine the frequency and sexdistribution of surgical cases of omphalocele treated at theClinic for Children’s Surgery; Clinical Center, University ofSarajevo. It is a retrospective study that included 9 cases ofsurgically treated omphalocele, in the period of time from2000 until 2009. These data are obtained from the medicalprotocols and patient histories at the Clinic for the Children’sSurgery. Analysis of data found that in the period of timefrom 2000 - 2009 at the Clinic for Children’s Surgery, 9 patientswith omphalocele were treated surgically. From these9 patients, 6 of them were males (70%), and 3 females (30%).5 (55.55%) of these patients had associated anomalies, whichof those the most frequent were chromosomal abnormalitiespresent at 3 patients (33.33%). Based on these data wecan conclude that omphalocele is a rare congenital anomalywhose frequency doesn’t vary a lot through the years.Key words: omphalocele, frequencyIntroductionDuring the 4 th to 5 th week of development,the flat embryonic disk folds in four directionsand/or planes: cephalic, caudal, andright and left lateral. Each fold converges atthe site of the umbilicus, thus obliteratingthe extra embryonic coela. The lateral foldsform the lateral portions of the abdominalwall and the cephalic and caudal folds makeup the epigastrium and hypogastrium (1,2). Rapid growth of the intestines and liveralso occurs at this time. During the 6 thweek of development (or eight weeks fromthe last menstrual period), the abdominalcavity temporarily becomes too small to9

Folia Medica 2011; 46 No 1:9-12accommodate all of its contents, resultingin protrusion of the intestines into the residualextra embryonic coela at the base ofthe umbilical cord. This temporary herniationis called physiologic midgut herniation.Reduction of this hernia occurs by the 12 thpostmenstrual week; beyond the 12 th week amidgut herniation is no longer physiological(3). A simple midline omphalocele developsif the extra-embryonic gut fails to return tothe abdominal cavity and remains coveredby the two-layer amniotic-peritoneal layerinto which the umbilicus inserts (1,2,4). Incontrast to fetal bowel, the liver does nothave a physiological migration outside ofthe abdominal cavity during development.Therefore, the liver is never present in physiologicmidgut herniation. However, if thelateral folds fail to close, a large abdominalwall defect is created through which theabdominal cavity contents, including theliver, can be herniated (1,2). The result is aliver-containing omphalocele. The omphalocelemay be small, with only a portionof the intestine protruding outside the abdominalcavity, or large, with most of theabdominal organs (including intestine, liver,and spleen) present outside the abdominalcavity. A translucent membrane covers theprotruding organs. A “small” type omphalocele(involving protrusion of a small portionof the intestine only) occurs in one out ofevery 5.000 live births. A “large” type omphalocele(involving protrusion of the intestines,liver, and other organs) occurs inone out of every 10.000 live births. Isolatedomphalocele occurs in approximately 1 in5000 live births (5). Many babies born withan omphalocele also have other abnormalities.Associated malformation ranges from30 to 88% (6). Babies with omphalocele haveabnormalities of other organs or body parts,most commonly the central nervous system,digestive system, heart, urinary system, andlimbs. More boys than girls are affected withomphalocele. The aim of this study was toobtain the frequency and sex distribution ofthe omphalocele among cases hospitalizedat the Clinic for Children’s Surgery; ClinicalCenter, University of Sarajevo, Bosnia andHerzegovina, during the period from January2000 to December 2009.Patients and methodsRetrospective study was carried out on thebasis of the clinical records in the Clinic forChildren’s Surgery; Clinical Center, Universityof Sarajevo, Bosnia and Herzegovina.From 1 st January 2000 to 31 st December2009, a total of 7529 patients were hospitalizedand out of that number nine cases(0.119%) were diagnosed as some type ofomphalocele malformation. Standard methodsof descriptive statistics were performedfor the data analysis.ResultsA total of 9 cases were treated in the Clinicfor Children’s Surgery; Clinical Center, Universityof Sarajevo in the period from 2000to 2009. The number of omphalocele casesdoes not vary much in the observed period(Table 1).Table 1. Frequency of omphalocele in theobserved periodYear N°2000 02001 12002 12003 22004 02005 12006 02007 02008 22009 2∑ = 910

Selma Aličelebić et al.: Frequency and sex distribution of omphalocele in surgically treated infants...At the Clinic for Children’s Surgery;Clinical Center, University of Sarajevo surgicallywere treated cases of omphalocelefrom the whole Federation of Bosnia andHerzegovina and their geographical distributionis shown in Table 2.Table 2. Frequency of omphalocele in theobserved geographical regionCanton N°Sarajevo 3Una-Sana 2Zenica-Doboj 2Central Bosnia 1Herzegovina-Neretva 1∑ = 9The structure of patients with omphaloceletreated according to the gender is shownin Table 3. Out of that num ber 6 (66.66%)were male patients, while 3 (33.33%) werefemale; sex ratio 2:1.Table 3. Total number and gender of treatedomphaloceleGENDER N° %MALE 6 66.66 %FEMALE 3 33.33%TOTAL 9 100%Among surgically treated patients wereeight cases of “small” type and one case of“large” type omphalocele.Isolated omphalocele occurs in four cases,multiple malformations were found infive cases, three cases were with one and twocases were with two associated malformations(Figure 1). Chromosome aberrationswere present in three patients, one patienthad gastrointestinal and one had musculoskeletalmalformation.DiscussionIn the period from 1 st January 2000 to 31 stDecember 2009 a total of 7529 patientswere hospitalized. Out of that number 9were omphalocele cases (0.119%) and outof that num ber 6 (66.66%) were male patients,while 3 (33.33%) were female; sexratio 2:1. Small omphalocele was presentin 8 (88.88%) of 9 surgically treated cases,while the large omphalocele was present inonly one patient (11.11%). Anomalies associatedwith omphalocele were present in55.55% patients and the most common werechromosome aberrations (33.33%). Ourdata was consistent with the other studiesthat have shown similar results. It was reportedthat the prevalence of omphalocelewas 1:3400 in the period 1994-2002 and1:2709 from 2000 to 2002 in the UnitedStates, sex ratio was 1.7: 1 and 76 % patientsFigure 1 – Frequency of omphalocele with associated anomalies11

Folia Medica 2011; 46 No 1:9-12had associated anomalies (7). Prevalence ofomphalocele was 4.08:10,000 newborns inEngland, Wales and Scotland (8). Between1993-2002 there were 100 cases of omphalocele(82.6%) in Singapore, with an incidenceof 2.63: 10,000 live births and 40%of omphalocele cases were accompanied byassociated anomalies (9). It was reportedthat the omphalocele incidence in Norwaywas 5.4:10,000 and 5.1:10,000 in Russia.The data covered the period 1995-2004 inArkhangelskaja Oblast (AO) in Russia and1999-2003 in Norway which were obtainedfrom the malformation register in AO andthe Medical Birth Registry of Norway (10).However, based on our data we can concludethat omphalocele is a rare congenitalanomaly whose frequency doesn’t vary a lotthrough the years. In conclusion, our studyis not consistent with world-wide trends inshowing the increasing incidence of anteriorabdominal wall defects. However, our studysuggests that omphalocele is not on the risingtrend and that more studies should bedone to elucidate this phenomenon.References:1. Duhamel, B., Embryology of exomphalos and alliedmalformations. Arch Dis Child 1963; 38:142.2. Hutchin, P. Somatic anomalies of the umbilicusand anterior abdominal wall. Surg Gynecol Obstet1965; 120:1075.3. Cyr, DR, Mack, LA, Schoenecker, SA, Patten, RM.Bowel migration in the normal fetus: US detection.Radiology 1986; 161:119.4. Margulis, L. Omphalocele (amnicele). Am J ObstetGynecol 1945; 49:695.5. Townsend. Abdomen: In Sabiston Textbook ofSurgery (16th ed.) Philadelphia. WB SaundersCo. 2001. p. 1478.6. Gilbert WM, Nicolaides KH. Fetal omphalocele:associated malformations and chromosomal defects.Obstet Gynecol. 1987 Oct;70(4):633-5.7. Goldkrand JW, Causey TN, Hull EE. The changingface of gastroschisis and omphalocele in southeastGeorgia. J Matern Fetal Neonatal Med. 2004May;15(5):331-5.8. Tan KH, Kilby MD, Whittle MJ, et al. Congenitalanterior abdominal wall defects in England andWales 1987-93: retrospective analysis of OPCSdata. BMJ 1996; 313: 903-6.9. Tan KBL, Tan KH, Chew SK, Yeo GSH Gastroschisisand omphalocele in Singapore: a tenyearseries from 1993 to 2002. Singapore Med J2008;49(1) :31.10. Petrova JG, Vaktskjold A. The incidence and maternalage distribution of abdominal wall defectsin Norway and Arkhangelskaja Oblast in Russia.Int J Circumpolar Health. 2009 Feb;68(1):75-83.12

Original PapersAcute Toxicity Study of Combination of Tridecactide andMet-EnkephalinMaida Rakanovic-Todic 1 , Nedzad Mulabegovic 1 , Fahir Becic 1 , Mirjana Mijanovic 1 ,Svjetlana Loga-Zec 1 , Jasna Kusturica 1 , Lejla Burnazovic-Ristic 1 , Aida Kulo 1 , ElvedinaKapic 11Institute of Pharmacology, ClinicalPharmacology and Toxicology,Medical Faculty of SarajevoCorresponding author:Maida Rakanovic-TodicInstitute of Pharmacology,Clinical Pharmacology and Toxicology,Medical Faculty,University of SarajevoČekaluša 90, 71 000 SarajevoPhone/fax: +387 33 227 018E-mail: maida@dic.unsa.baIntroductionIntroduction: Acute toxicity study was done as the entry stepof prospective drug toxicological evaluation. The fixed combinationof two peptide components: met-enkephalin and tridecactideis newly registered drug in Bosnia and Herzegovina(Enkorten ® , Farmacija d.o.o. Tuzla). Materials and methods:Acute toxicity testing was performed on Wistar albino rats,as the single dose testing in accordance with the Limit Testmethodology. Both peptides cannot be absorbed after theoral application and the combination was individually appliedto male and female rats by intravenous (IV), subcutaneous(SC) and intraperitoneal (IP) route. For each route ofapplication three dose ranges were calculated, as multiplicationsof expected human therapeutic dose (met-enkephalin5-10 mg and tridecactide 1-2 mg). The administered multiplicationswere: IV 50, 100 and 200 times; SC 100, 250 and500 times; and IP 100, 250, 500 times plus 1000 times in additionalgroup of males. Results and conclusions: Neither lethalitynor significant macroscopic and microscopic changesat necropsy following planned sacrificing were observed.The main clinical signs observed at administered doses wereslight changes in motor activity and intensive phonation inIV treated groups. No statistically significant differences inpostmortem organ weights were noted. The tested combinationinduced no lethality and demonstrated low level of toxicityin rationally high doses.Key words: tridecactide, met-enkephalin, 1-13-corticotropine,alpha-melanocyte-stimulating hormone, acute toxicityThe newly registered drug in Bosnia andHerzegovina, Enkorten ® (Farmacija d.o.o.Tuzla), consists of two neuropetides: metenkephalinand tridecactide (a 1-13-corticotropine).Tridecactide was previously nameda-melanocyte-stimulating hormone-like(a-MSH-like). Tridecactide is deamidatedand deacetylated a-MSH. In the model ofethanol induced gastric lesions in rats, metenkephalinand a-MSH exhibited statisticallysignificant additive cyto-protective effect(1,2), achieving the best effect in 5:1 doses13

Folia Medica 2011; 46 No 1:13-21proportion. The practical consequence ofthe latter observation was estimation of theexpected human dose (for entering the clinicaltrials) on 5-10 mg of met-enkephalin and1-2 mg of tridecactide, administered (intramuscularlyor subcutaneously) five times aweek and gradually reduced to once a week(2). Both peptides cannot be absorbed followingoral application.Alpha-MSH exerts antipyretic, antiinflammatoryand antimicrobial effects(3,4,5,6,7). Alpha-MSH is being produced inthe human pituitary and by extra-pituitarycells including gastrointestinal cells, monocytesand keratynocytes. Research suggeststhat a-MSH anti-inflammatory effect couldbe mediated by melanocortin receptorsand regulatory circuits in macrophages,and through descending anti-inflammatorypathways originating from the neurons(8,9). Combined use with met-enkephalinencourages the presence of both amino-acidsequences in the same precursor molecule,proopiomelanocortin. Processing yields a-MSH and b-endorphin, while met-enkephalinis mainly derived from preproenkephalin(10). Met-enkephalin is one of the simplestopioid peptides, with prominent role in investigationof enkephalins engagement inimmunity. Opioid receptors are detectedon the human leucocytes, lymphocytes andthrombocytes. Also, evidence suggests lymphocytesproduction of enkephalins andendorphins (11). Growth factors role wassuggested for opioid peptides (12,13), implicatingcontrol of cell differentiation andproliferation.Investigational pharmaceuticals toxicitytesting aims on evaluation of the toxicologicalpotential and risks of the human exposure.Acute toxicity provides the importantsafety parameters for the prospective humanoverdose scenario and its expectedclinical presentation (14,15). Traditionally,international authorities and governmentalagencies had adopted the mean lethal doseas the sole measurement of acute toxicity.Currently, the significance is given to theinformation on toxicity signs, mechanism oftoxicological action and dose-response relationshipfor non-lethal parameters, whilelethality should not be intended endpoint inacute toxicity studies (14,15). Use of the rationallyhigh doses in acute toxicity testingis considered as acceptable, and complieswith the Limit Test concept. The use of dosesup to the maximum tolerated dose, a doseachieving large exposure multiplies or maximumfeasible dose, are considered appropriatefor the general toxicity testing. The dosesof 1000 mg/kg/day are considered appropriatefor acute toxicity testing, excluding thesituations when the mean exposure marginof 10-fold is not covered. Also, assessmentfollowing the intravenous (IV) route of applicationmaximizes the exposure to the testsubstance.Our study aims on detecting toxicitypotential of the met-enkephalin and tridecactidecombination following three differentways of application, identifying targetorgans for toxicity, reversibility of toxic effectsand symptoms of acute intoxication. Apreclinical safety evaluation, with an acutetoxicity testing as a first step, would enablethe prospective drug entering the clinicalevaluation.Materials and methodsExperimental AnimalsAdult, healthy, Wistar albino rats, of bothsexes, were randomized in three treatmentand one control group for IV application(3M + 3F), subcutaneous (SC) application(5M + 5F), and intraperitoneal (IP) dosing(treatment groups 5M + 5F and control 3M+ 3F). The additional group of males (4M)was IP dosed with highest dose. Averagebody mass at randomization was 401.75 g,14

Maida Rakanovic-Todic et al.: Acute Toxicity Study of Combination of Tridecactide and Met-Enkephalinfor males, and 264.42 g, for females. Animalsspent minimally 21 day in quarantine beforerandomization. Standardized care, nutritionand water supply were provided during thestudy. This research was conducted followingthe Guide for the Care and Use of LaboratoryAnimals, US National Institutes ofHealth.CompoundsThe test samples of a) met-enkephalin andb) tridecactide were manufactured by CommonwealthBiotechnologies, USA, and deliveredas lyophilized powder in closed flacons.Compounds were dissolved in physiologicalsaline (0.9% NaCl) upon the application,and applied as combination in fixedproportion of individual substances.Experimental design and ConductAcute toxicity testing by IV, SC and IP applicationwas performed as the single dosetesting in accordance with the Limit Testmethodology. The animals were randomlyassigned to cages and the individual animalwas fur marked with picric acid. The animalswere dosed with the test combination individuallyand dose was adjusted towards theanimal’s body mass. Dose ranges were calculatedas the multiplication of expected humantherapeutic dose (a+b as met-enkephalin+ tridecactide). Control animals receivedthe solvent only (physiological saline).Following multiplications were providedfor IV dosing:– 50 times: 3.55 mg/kg + 0.7 mg/kg(group code IV1, sex subgroups IVF1,IVM1)– 100 times: 7.1 mg/kg + 1.4 mg/kg(group code IV2, sex subgroups IVF2,IVM2)– 200 times: 14.2 mg/kg + 2.8 mg/kg(group code IV3, sex subgroups IVF3,IVM3)– Controls (group code IVC, sex subgroupsIVFC, IVMC)Following multiplications were providedfor SC dosing:– 100 times: 7.1 mg/kg + 1.4 mg/kg(group code SC1, sex subgroupsSCF1, SCM1)– 250 times: 17.7 mg/kg + 3.5 mg/kg (group code SC2, sex subgroupsSCF2, SCM2)– 500 times: 35.5 mg/kg + 7.1 mg/kg (group code SC3, sex subgroupsSCF3, SCM3)– Controls (group code SCC, sex subgroupsSCFC, SCMC)Following multiplications were providedfor IP dosing:– 100 times: 7.1 mg/kg + 1.4 mg/kg(group code IP1, sex subgroups IPF1,IPM1)– 250 times: 17.7 mg/kg + 3.5 mg/kg(group code IP2, sex subgroups IPF2,IPM2)– 500 times: 35.5 mg/kg + 7.1 mg/kg(group code IP3, sex subgroups IPF3,IPM3)– 1000 times: 71.0 mg/kg a + 14.3 mg/kg (additional group code IP4)– Controls (group code IPC, sex subgroupsIPFC, IPMC).Doses were applied in constant volumeof 0.001 ml/g IV, 0.0005 ml/g SC, and 0.005ml/g IP (excluding additional group).Observation period was set to 14-days.Clinical observations were set on 90, 150and 210 minutes and 4 hours after dosingduring the first day and thereafter on thedaily basis. Animal weight, food and waterconsumption were recorded before dosingand thereafter on the weekly basis. Intensityof phonation was assessed subjectively duringmanipulation and handling of animals,after the IV and SC application. Plannedsacrificing and necropsy were set at the endof the study. Following the macroscopic examination,the organ weights were recorded(as percentage of animal’s body weight). The15

Folia Medica 2011; 46 No 1:13-21samples of liver, kidneys, lungs, heart, brainand organs with noted macroscopic changeswere examined histopathologicaly.Statistical analysis was performed withmodification for small samples by Kruskal-Wallis test and Dunnett test with multiplecomparisons of distributions.ResultsNo lethality was observed during or after IV,SC and IP application. Animal body massincreased during the study in all treatmentand control groups, compared to the bodymass before the application (Figure 1). Thestagnation and decrease in body mass wasnoted only for females following SC application(Table 1). Statistically significant differencein body mass was noted for IVM1(p=0.037812), compared to control group.Statistical analysis of water and food consumptiondata (Table 2) showed no statisticallysignificant difference, except for IVM1(p = 0.037812; p = 0.032902).The most prominent clinical signs werenoted on the day 1 of the observation periodfollowing all three application routes. Slightlyreduced motor activity was observedin all animals treated with the highest IVdose, males treated with the medium dose,and males and one female treated with thelowest dose. On the contrary, females treatedwith the medium and the lowest doseseemed more active. There was a slight ptosis(eyelids down ¼) in two males treatedwith the highest dose, two males and onefemale treated with medium dose, and allmales and one female treated with the lowestdose. Horizontally positioned tail wasnoted in all animals treated with the highestdose, two males and one female treated withmedium dose, one male and two femalestreated with the lowest dose. Slight cyanosisand general vasoconstriction was noted inone male treated with the highest and onemale treated with medium dose.Following the SC application (day 1)slightly slower motor activity of all treatedand control animals was observed. Also,slight muscular hypotonia was noted on day3 in three males treated with highest doseand one male treated with the lowest dose.Following the IP application (day 1), irregularbreathing, decrease in motor activity,somnolence, ataxia, catalepsy and muscularhypotonia were observed in all malesfrom group IP4. A proportion of the effectsnoted in IP4 group could be attributed toTable 1. Median of growth of animal body mass during the study periodGroup Median Group Median Group MedianIVMC 6.96 SCMC 3.47 IPMC 4.25IVM1 1.19 SCM1 3.08 IPM1 2.86IVM2 3.47 SCM2 3.11 IPM2 0IVM3 5.13 SCM3 3.70 IPM3 2.67IPM4 2.40IVFC 2.89 SCFC 0.44 IPFC 3.21IVF1 4.81 SCF1 -0.65 IPF1 2.00IVF2 4.17 SCF2 0 IPF2 5.16IVF3 4.00 SCF3 -1.24 IPF3 1.6116

Maida Rakanovic-Todic et al.: Acute Toxicity Study of Combination of Tridecactide and Met-EnkephalinFigure 1. Body mass ratio before application and upon the sacrificationTable 2. Median weekly water and food consumption of the animals during the study periodGroup X maxX minMedian Group X maxX minMedian Group X maxX minMedianWater consumptionIVMC 16.78 14.15 15.10 SCMC 12.25 7.09 8.43 IPMC 12.68 11.03 12.21IVM1 11.66 9.56 10.62 SCM1 13.97 10.07 11.87 IPM1 13.84 9.21 11.72IVM2 13.89 11.23 13.42 SCM2 15.18 8.98 12.80 IPM2 14.63 12.85 14.58IVM3 12.82 10.70 12.46 SCM3 15.50 9.66 11.43 IPM3 13.40 11.73 13.08IPM4 11.37 9.60 10.11IVFC 16.57 15.09 16.09 SCFC 12.92 9.35 10.98 IPFC 20.05 16.61 18.35IVF1 16.85 15.34 15.73 SCF1 19.81 8.92 16.39 IPF1 21.80 14.70 18.62IVF2 16.63 13.53 14.34 SCF2 14.06 12.19 13.57 IPF2 15.58 14.48 14.71IVF3 13.22 11.90 12.27 SCF3 15.00 9.02 11.45 IPF3 15.38 13.82 14.68Food consumptionIVMC 7.28 6.10 6.13 SCMC 6.46 4.43 6.02 IPMC 7.08 5.73 6.62IVM1 5.13 4.71 4.74 SCM1 6.93 5.96 6.92 IPM1 8.29 7.16 7.50IVM2 5.82 5.25 5.79 SCM2 6.24 5.63 5.81 IPM2 6.80 6.18 6.34IVM3 6.49 5.76 5.88 SCM3 6.39 5.34 6.38 IPM3 7.94 6.57 6.70IPM4 6.25 5.90 6.03IVFC 7.05 5.99 6.79 SCFC 7.06 5.78 6.54 IPFC 7.38 5.70 6.89IVF1 6.25 5.91 6.03 SCF1 6.43 5.89 6.22 IPF1 6.02 5.58 5.88IVF2 6.90 6.13 6.59 SCF2 8.32 6.78 6.86 IPF2 6.02 5.52 5.76IVF3 6.98 5.32 6.26 SCF3 7.34 5.48 7.28 IPF3 6.22 5.24 5.5917

Folia Medica 2011; 46 No 1:13-21IP application of the large amount of fluid,since the constant volume was not respectedfor this group. Somnolence and muscularhypotonia was noted in male from groupIPM3, and only somnolence in male fromgroup IPM1. Afterwards, frequency andcharacter of breathing were observed onceweekly and remained in physiological limits.The intensive phonation during the manipulationwith animals was registered forIV dosed groups between 9 and 14 day ofobservation period (Figure 2), and wasn’tnoted in control animals. Statistically significantdifference was detected for increasedphonation in all treatment groups of males,IVF3 and IVF2, compared to controls (p£0.000001). Following the SC applicationeach phonation during the manipulationwith animals was noted (Figure 3). Thereis no prominent difference in variation inphonation in males. Statistically significantdifference in phonation was noted for groupSCF3 (P=0.000001), compared to the control.Necropsy and histopathology did not revealsignificant changes in organs and tissuesmacroscopic and microscopic structure.Following the SC application, macroscopicchanges were detected in one control maleand one female in group SCF2 (hyperemiaand dilatation of tubas uterine). The macroscopicchanges were supposed not to berelated with application of the test combination.Also, no abnormal changes were notedin postmortem organ mass with respect tobody mass, in experimental groups in comparisonwith controls (Table 3).DiscussionThe purpose of this study was to gain theinsight in the acute toxicity profile of thetested combination. Applied combinationinduced no lethality and demonstrated lowlevel of toxicity in rationally high doses.The use of doses achieving large exposuremultiplies is considered appropriate for thegeneral toxicity testing, indicating that testingof higher doses just to induce lethality innot necessary according to the current regulatorydemands. Also, available data showsthat met-enkephalin (in doses up to 10 mg/kg) and a-MSH (in dose range 1-3.3 g/kg)were applied without lethality in animal experiments(1,2). However, no evaluation hasbeen made so far regarding acute toxicity oftridecactide individually nor in combinationwith met-enkephalin.Increase in animals body mass confirmedlow toxic potential. Similar foodconsumption of animals in control and experimentalgroups, as the true indicator ofanimal growth rate, indicates the feed intakeand utilization was not affected. Also, detectingneither organ or tissue damage nororgan mass ratio differences in control andexperimental groups, indicate that applicationof the tested combination to the rats didnot result in permanent adverse toxicologicaleffect on examined organs. Reversiblesigns of toxicity disappear with substanceelimination and usually are not followed bypermanent tissue damage.Met-enkephalins high doses produceeffects on OP3 (m) receptors with sedation,mood changes, miosis, respiratory depressionand gastrointestinal disturbances(10,11). Registration of ptosis, motor activitychanges, horizontally positioned tail orincreased phonation could suggest opioidpathway engagement. Increased intensity ofphonation was clearly noted for all IV dosedtreatment groups, while not noted in controlanimals. Following SC application, all phonationswere registered and effect was not sowell defined. Nevertheless, effects involvinganimal’s emotional reaction are very unstablefield for interpretation, while sensitivityof animal tests is generally high and specificityis low.The toxicological studies aim to revealthe type of toxicological action, what18

Original PapersBrucellosis – Emerging Zoonosis in Bosnia and HerzegovinaSajma Dautović-Krkić¹, Edina Bešlagić², Meliha Hadžović-Čengić¹, SnježanaMehanić¹, Adnan Karavelić¹, Sead Ahmetagić³, Ibrahimpašić Nevzeta 4 , EldiraHadžić 5 , Ivo Curić 6 , Nazif Derviškadić 6 , Janja Bojanić 7¹ Clinic for Infectious Diseases,Clinical Center of Sarajevo University, Sarajevo² Institut for Microbiology Medical FacultyUniversity of Sarajevo³ Clinic for Infectious Diseases,University Clinical Center Tuzla4Department for Infectious Diseases,Cantonal Hospital Bihać5Department for Infectious Diseases,Cantonal Hospital Zenica6Department for Infectious Diseases,Clinical Hospital Mostar6Department for Infectious Diseases,Hospital Južni logor, Mostar7Institut for Epidemiology, Banja LukaCorresponding author:Clinic for infectious diseases,Clinical Center of Sarajevo University, SarajevoBolnička 25, 71000 SarajevoBosnia and HerzegovinaE-mail: sajmadautovic@gmail.comBrucellosis is a zoonotic disease present across the world.Disease in humans has an acute, sub acute or chronic course,whereas in animals, usually, occurs as asymptomatic chronicinfection. In Bosnia and Herzegovina (B&H), brucellosis isimported by livestock donated to refugees and displaced personsafter aggression on Bosnia and Herzegovina and the warfrom 1992–1995 and since then has spread throughout thecountry in both Entities.AIM of this study was to make comparable analysis of researchresults on brucellosis data in the period 2000/2005,with the results reported for the period 2006/2009 and toencourage professionals of veterinary and human medicineto take active action and cooperation with government structuresin order to found an optimal solution to control anderadicate this anthropozoonosis.Results of research showed that during the 2006/2009 (44months) was hospitalized six times more cases than in theprevious period 2000/2005 (72 months), the increasing numberof sick children, as well as recurrence, regardless of thetherapy. Mode of expansion and maintenance of the diseasein some regions in the last ten years indicates its endemicity.The authors CONCLUDE that brucellosis in B&H is emergentzoonotic disease that has become endemic, based onnature of continuing epidemic outbreaks in some areas, andthat the work of state institutions was not nearly enough tocombat the disease and place it under control. Likewise, therewas no and there is no planned and programmed and supportedcooperation with experts from human and veterinarymedicine.Key words: brucellosis, epidemic, endemia, emergent zoonosis22

Dautović-Krkić S. et al.: Brucellosis - Emerging Zoonosis in Bosnia and HerzegovinaIntroductionBrucellosis is the most widespread zoonosis,that affects more than a half million peopleper year, all over the world. Causative agentis Brucella species, domestic and wild animalsare reservoirs, and it affects mostly humans(1).In humans, Brucella causes acute, subacute or chronic disease. In a first 3-6 weeks,brucellosis is manifested with general flulikesymptoms, and thereafter is localized incertain organs, most commonly the jointsand intervertebral discus, then the liver,lungs, testicles, less on the heart valves, andother organs (2, 3, 4, 5, 6, 7, 8, 9). Often, thedisease manifests in humans, only whenbecame chronic and has consequences oncertain organs. In older patients the diseasecan be mistaken for rheumatoid or reactivearthritis of other etiology, thus creating diagnosticdifficulties, delayed early therapyand leaves the possibility of disability (5).The disease is widespread in livestockregions of the world and in most countriesof the Mediterranean region is endemic. InBosnia and Herzegovina disease appearedafter the war 1992-1995 and since then hasbeen steadily spreading among animals andhumans, with no signs of reduction in thenumber of infected and affected. Brucellosiswas imported from neighboring countries,with donated livestock to refugees and displacedpersons, since, due to political reasons,there was not adequately controled ofimport of livestock on the borders.Objectives– To make a comparative analysis ofhospitalized patients with brucellosisin Bosnia and Herzegovina in twoconsecutive periods: 2000–2005 and2006–2009.– To make conclusions and guidelinesfor suppression and surveillance ofbrucellosis– To encourage better cooperationamong clinicians, veterinarians andauthorities in order to engage allavailable potentials in suppressionand surveillance of brucellosisFigure 1. Distribution of human brucellosis in Bosnia and Herzegovina 1999–2009.23

Folia Medica 2011; 46 No 1:22-27Material and methodsWe performed a retrospective study of brucellosisfiles from three clinics and four departmentsfor infectious diseases from bothBosnian entities (Federation of Bosnia andHerzegovina and Republic of Srpska). Diagnosiswas confirmed either by ELISA andRose-Bengal test or by isolation of Brucellaspecies in blood. We used following statisticaltests: Student T-test and Chi-square test.ResultsBrucellosis was imported in Bosnia andHerzegovina after the war 1992–1995 withimported sheep and cattle that were donatedto refugees and returnees. First cases wereconfirmed in Veterinarian institute “VasoButozan” in Banja Luka (Republic of Srpska)in 1999. Tested sera had been taken from returneesliving in Trebinje region. There werethree Brucella-positive sera, but only onewas officially reported. In period 2000–2005human brucellosis was spreading acrossFederation of Bosnia and Herzegovina andslowly becoming a continuous epidemic.Spreading of human brucellosis was closelyconnected to spreading among the animalson a certain territory.Figure 2. Brucellosis in Republic Bosnia andHerzegovina 1946–1999.DiscussionThere were only four isolated cases of brucellosisreported in period 1946–1992 inBosnia and Herzegovina. All four of themwere reported in military camps (Manjača,Konjic, Han Pijesak, and Bihać). Infectedsheep were euthanized, with all precautionsand epidemiological measures undertaken.Number of infected people was limited, referringto those who were in contact with infectedanimals on daily basis; cattle-breedersTable 1. Distribution of human brucellosis by the place of hospitalization, 1999–2005.FB&H* RS** B&H***Year Sarajevo Tuzla Mostar Zenica Bihać1999. 0 0 0 0 0 1 12000. 8 0 8 0 0 0 162001. 3 0 1 0 0 3 72002. 4 0 3 7 0 0 142003. 28 0 5 14 0 1 482004. 47 0 10 31 1 0 892005. 65 0 6 64 0 0 135Total 155 0 33 116 1 5 310*Federation of Bosnia and Herzegovina, **Republic of Srpska, ***Bosnia and Herzegovina24

Dautović-Krkić S. et al.: Brucellosis - Emerging Zoonosis in Bosnia and HerzegovinaFigure 3. Brucellosis in Bosnia andHerzegovina 1999–2009and soldiers (10). Brucellosis emerged againin 2000 after the war in Bosnia and Herzegovina(1992–1995). It emerges simultaneouslyin three cantons of Federation ofBosnia and Herzegovina: Sarajevo, CentralBosnia, Herzegovina-Neretva. In CantonZenica-Doboj first case of brucellosis wasreported in 2002, and in Canton Una-Sanain 2004 (11, 12). There were 305 casesof human brucellosis reported in Federationof Bosnia and Herzegovina at the endof 2005. Mean age of patients was 40 years(0.8-85). Men were four times more affectedthen women. The most vulnerable populationwere cattle-breeders, then pupils andhousewives. Osteoarticular form of brucellosiswas dominant clinical form. There wasone death case due to Brucella endocarditis.In the same period (1999-2005) therewere only five reported cases of human brucellosisin Republic of Srpska. All reportedcases were among returnees to their pre-warhomes. Infection was transmitted from infectedcattle that were donated through differentaid-programs. There was no significantspread of the diseases among the cattle,since there was not much contact betweenreturnee’s and local people’s cattle.During the following four years (2006–2009) human brucellosis was rapidly spreadingamong people in all areas of Bosnia andHerzegovina. There were six times morereported cases of human brucellosis (1797)than it was in former period (1999–2005)in Federation of Bosnia and Herzegovina.During this period the disease spread allacross Bosnia and Herzegovina. There were323 cases reported in Republic of Srpska,with 216 of them reported in 2008. Meanage of the patients was 36 years (1.5–82years). Men were 2–4 times more affectedthen women. Concerning fact was increasednumber of infected children (one tenth ofreported cases), as well as increased numberof relapsing diseases regardless of therapy.There was a possibility that one part of therelapses is a reinfection, since people hasn’tbeen educated of self protection.Table 2. Distribution of human brucellosis by the place of hospitalization, 2006–2009.FB&H* RS** B&H***Year Sarajevo Tuzla Mostar Zenica Bihać2006. 64 0 4 84 6 4 1622007. 119 0 22 124 207 24 4962008. 133 113 49 319 139 216 9692009. 87 44 12 136 105 79 493Total 403 157 87 663 457 323 2120*Federation of Bosnia and Herzegovina, **Republic of Srpska, ***Bosnia and Herzegovina25

Folia Medica 2011; 46 No 1:22-27Analysis of disease spreading shows thatbrucellosis has become and endemic diseasefor the last ten years (12,13,14,15). From thefirst reported case it has had characteristicsof continuing epidemic in Canton Sarajevo,Canton Herzegovina-Neretva and CantonCentral Bosnia. In Canton Tuzla, and CantonUna-Sana disease emerged in form ofepidemic in 2007 and 2008 respectively, withmore than a hundred of infected individuals.All this leads to conclusion that thereis extremely high proportion of infectedcattle that were managed by poorly educatedcattle-breeders, which anticipate diseasespreading among people and animals. Noserious preventive precautions have beenundertaken in the past time in order to stopmixing of infected and uninfected cattle.Due to after-war division of Bosnia andHerzegovina, and lack of political will forthe past ten years, the disease has not beeneradicated or controlled, even though Bosniaand Herzegovina has State VeterinaryInstitute. Only palliative measures havebeen undertaken. Cattle have been testedfor brucellosis in both Bosnian Entities, butonly 10-30% of total population.There was not a single cattle cemetery inBosnia and Herzegovina before 2008, andeuthanized cattle ended at garbage dumps,in rivers or improperly buried. Two mobilecrematories started to operate at the end of2007. Bosnia and Herzegovina still properbutcheries. Veterinary Institute started cattlevaccination program at the end of 2008 inboth Bosnian Entities. Vaccination is obligatoryfor all cattle (young, old, pregnant, etc).Major concern is that there is no continuousand systemic cooperation between animaland human medicine specialists. Cooperationis based on personal contacts, and it’snot supported in any way.Conclusions– Human and animal brucellosis inBosnia and Herzegovina was reportedonly as a sporadic and isolateddisease, with no spreading before thewar 1992-1995.– After the war, brucellosis was confirmedfor the first time in 1999.– After emerging, brucellosis keptspreading, gathering characteristicsof continuous epidemic.– From 2006 the disease emerged incertain areas in form of high scaleepidemic.– Brucellosis in Bosnia and Herzegovinahas been emerging zoonosis since1999/2000.– For the past ten years, brucellosis hasbecome an endemic disease in Bosniaand Herzegovina.– Government of Bosnia and Herzegovinahasn’t started sufficient andproper actions over the past ten yearsto eradicate or control the disease becauseof lack of political will.– There wasn’t any systematic cooperationbetween human and animalmedicine specialists.– Government and related institutionsmust take full responsibility for endangeringof human and animalhealth on national level.– A cohort of experts and scientistsmust be formed in order to start seriousand systematic work, and createnational programs for eradicatingbrucellosis, as well as other infectiousdiseases.26

Dautović-Krkić S. et al.: Brucellosis - Emerging Zoonosis in Bosnia and HerzegovinaReferences1. Godfroid J, Cloeckaert A, Liautard JP, Kohler S,Fretin D, et al. From the discovery of the Maltafever’s agent to the discovery of a marine mammalreservoir, brucellosis has continuously beena re-emerging zoonosis. Vet Res. 2005 May-Jun;36(3):313-26.2. Husseini AS, Ramlawi AM. Brucellosis in theWest Bank, Palestine. Saudi Med J. 2004 Nov;25(11):1640-3.3. Mantur BG, Akki AS, Mangalgi SS, Patil SV, GobburRH, Peerapur BV. Childhood brucellosis-amicrobiological, epidemiological and clinicalstudy. J Trop Pediatr. 2004 Jun; 50(3):153-7.4. Kalaycioglu S, Imren Y, Erer D, Zor H, ArmanD. Brucella endocarditis with repeated mitralvalve replacement. J Card Surg. 2005 Marapr;20(2):189-92.5. Ozden M, Demirdag K, Kalkn a, Ozdemir H, YuceP. A case of brucella spondylodiscitis with extended,multiple-level involvement. South Med J. 2005Feb; 98(2):229-31.6. Seidel G. Pardo Ca, Newman-Toker D. OliviA, Eberhart CG. Neurobrucellosis presentingas leucoencephalopathy: the role of cytotoxicT lymphocytes. Arch Pathol Lab Med. 2003Sep;127(9):e374--7.7. Cesur S, Ciftci A, Sozen TH, Tekeli E. A case ofepididymo-orchitis and paravertebral abscess dueto brucellosis. J Infect. 2003 May;46(4):251-3.8. Hesseling AC, Marais BJ, Cotton MF. A child withneurobrucellosis. Ann Trop Paediatr. 2003 Jun;23(2):145-8.9. Almuneef M, Memish ZA, Al Shaalan M, Al BanyanE, Al-ALOLA s, Balkhy HH. Brucella melitensisbacteremia in children: review of 62 cases. JChemother. 2003.Fe;15(1):76-80.10. Zavod za Javno Zdravstvo Kantona Sarajevo, Zavodiza Javno Zdravstvo Entiteta države Bosne iHercegovine.11. Dautović-Krkić S, Lukovac E, Mostarac N,Hadžović M, Gazibera B, Muratović P. Zoonosisin infectious practica. Prvi simpozijum o zoonozamasa međunar. učešć. April 2005, 88.12. Sajma Krkić-Dautović. Snježana Mehanić. MerdinaFerhatović. Semra Čavaljuga. Brucellosis epidemiologicaland clinical aspects (Is brucellosis amajor public health problem in Bosnia and Herzegovina?).Bosnian J of basic Medic Scienc 2oo6;6(2) 11-15..13. Bosilkovski M., Dimzova M., Grozdanovski K,Natural history of brucellosis in an endemic regionin different time periods. Acta clin Croat.2009 Mar;48 (1):41-6.14. Bosilkovski M, Krteva L, Dimzova M, Kondova I.Brucellosis in 418 patients from the Balkan Peninsula:exposure related differences in clinicalmanifestation, laboratory test results, and therapyoutcome. Int J Infect Dis. 2007 Jul; 11 (4):342-7.Epub 2007 Jan 22.15. Muhammad N, Hossain MA, Musa AK, MahmudMC, Paul SK, Rahman MA, Haque N, Islam NT,Parvin US, Khan SI, Nasreen SA, Mahmud NU.Seroprevalence of human brucellosis among thepopulation at risk in rural area. Mymensingh MedJ. 2010 Jan; 19(1):1-4.27

Original PapersDiagnostic Significance of Platelet Count in Patients WithLung CancerSelma Arslanagić 1 , Hasan Žutić 2 , Nedžad Mulabegović 3 , Rusmir Arslanagić 4 ,Reuf Karabeg 1 , Naima Arslanagić 51Clinic of Plastic surgery Clinical Centerof Sarajevo University2Clinic of Pulmonal disease Clinical Centerof Sarajevo University3Institute of Pharmacology,Clinical Pharmacology and toxicology,Medical Faculty University of Sarajevo4Clinic of Ear, Throat, Nose diseases,Clinical Center of Sarajevo University5Clinic of Dermatology, Clinical Centerof Sarajevo UniversityCorrespondence to:Selma Arslanagić,Clinic of Plastic surgery,Clinical Center of Sarajevo UniversityBolnička 25, 71000 SarajevoBosnia and HerzegovinaTel: 033 297 366E-mail: arslas@hotmail.comA number of studies have shown that reactive thrombocytosiscan be associated with malignant disease. Thrombocytosiscan be inexpensive and easy indicator of solid cancer. It isconfirmed to be a paraneoplastic symptom. There is increasingevidence that interaction between tumor cells, endothelialcells and platelets may contribute to the development ofmetastases resulting in poor prognosis.Aim of our study was to analyze thrombocytosis in lung cancerpatients before any kind of therapy.Our research showed that there is significant difference inprevalence of thrombocytosis between lung cancer patientsand control patients group. Elevated platelets count was morecommon in patient with advanced stages lung cancercancer,bat not significantly. There were no significant differences inthe occurrence of thrombocytosis between the various histologicallung cancer types.Key wordsLung cancer, thrombocytosisIntroductionIn last decade thrombocytosis is consideredas an inexpensive and simple indicator ofmalignancy. Despite numerous studies andopinions, many authors don’t agree aboutquestion whether thrombocytosis is theultimate result of the actions of growth factorsthat create cell cancer. Opinions on thefrequency of thrombocytosis in lung cancerare divided. Clinicians who have studiedthe significance of thrombocytosis in lung28

Arslanagić S et al.: Diagnostic Significance of Platelet Count in Patients With Lung Cancercancercancer patients, consider that thethrombocytosis is clinically useful analysisto assess the diagnosis of malignancy in patientswith radiographically suspicious lungcancer.OBJECTIVES OF RESEARCH is to examinethe prevalence of thrombocytosis inpatients with lung cancercancer. To analyzerelationship of platelets count in relation tothe histopathological type and TNM stagecancerlung cancer. To investigate a differencein platelets count between non smalllung cell cancer (NALCO) and small celllung cancer (SCLC).Material and methodsThe study involved two groups: group ofpatients with a confirmed diagnosis of lungcancer and control group.The group of patients with lung cancerIn the retrospective hospital based- cohortstudy was analysed thrombocytes 239 patientswith lung cancer, before any kind oftherapy, hospitalized at the Clinic for PulmonaryDiseases, in the period since the1 st of January 2005 to 31 st December 2007.Classification of cancer was made accordingto the recommendations of the WorldHealth Organization (1).The study did not include patients withreactive thrombocytosis due to other diseasesas:– Inflammatory disease of lung;– Autoimmune disease;– Any other malignancy.In this study, we didn’t include any patientswho were under chemotherapy and/or are subjected to surgical intervention.The control groupDermatovenerology, which practically canbe considered healthy population. Theseparticipants were tested on possible allergieson medicaments. According to the protocolfor testing, all symptoms of allergic reactionsdisappeared before at least a monthbefore investigation and such person duringthe test did not take any therapy. The controlgroup according to sex and age correspondedto patients with lung cancer.Statistical analysis was performed usingChi- square 2x2 and Student-t test, utilizingStatistical Package for the Social Science,SPSS, (Version 13). The difference betweentwo groups was evaluated using ANOVAtest at 95% confidence interval. Data wereanalyzed descriptively and expressed as rawfrequencies and percentages, and 95% confidenceintervals (95%CI) were calculatedwith CIA. The significance level was set atp

Folia Medica 2011; 46 No 1:28-34Table 1. Gender distribution of patients by histopathologic type of cancerHISTOLOGICALTYPE OFCANCERTotal patientsMALESEXFEMALENumber Percent Number Percent Number PercentAdenocarcinoma 105 43.8 85 35.6 20 8.4Squamous cellcarcinoma89 37.2 62 25.9 27 11.3Large cell carcinoma 3 1.5 2 0.8 1 0.4Micro cell carcinoma 42 17.5 34 14.2 8 3.4Total 239 100.0 183 76.5 56 23.5Table 2. Distribution of patients with NSCLC according to the TNM classificationHISTOPATHOLOGICALTYPE OF CANCERTotalpatientsTNM CLASSIFICATIONIIA IIB IIIA IIIB IVNum. Perc. Num. Perc. Num. Perc. Num. Perc. Num. Perc. Num. Perc.Adenocarcinoma 105 100.0 6 5.7 14 13.3 34 32.4 14 13.3 37 35.3Squamous cell carcinoma 89 100.0 3 3.3 6 6.7 40 44.9 21 23.7 19 21.4Large cell carcinoma 3 100.0 0 0.0 0 0.0 1 33.3 1 33.3 1 33.3Total 197 100.0 9 4.6 20 10.1 75 38.0 36 18.4 57 28.9The distribution of SCLC patients accordingto TNM classification is shown onTable 3. In the extended stage of lung cancerwere more affected (55.0%) compared to thelimited stage (45.0%).Table 3. Distribution of micro cell carcinomapatients according to tnm classificationHistopathological typeof cancer according toTNM classificationNumber ofpatientsPercentageLIMITED 20 45.0EXTENDED 22 55.0TOTAL 42 100.0Statistical analysis, shown on Figure 1,ANOVA test, demonstrated that there is statisticallysignificant differences mean valuesof platelets count between lung cancer patientsand control group at the level of significanceof p

Arslanagić S et al.: Diagnostic Significance of Platelet Count in Patients With Lung CancerFigure 14. The mean values platelets count for eachgroups of lung cancer group are significantlydifferent from the mean values of plateletscount of the control group at the significancelevel of p0.05. The meanplatelets values of different SCLC stages isshown on Table 5.Table 6 presents the statistical analysis ofmean values in platelets count of earlier stagesNSCLC (IIA and IIB) to advanced stages(IIIA, IIIB and stage IV). There were noTable 4. Analysis of mean values of platelet* specific histopathological lung cancer and control groupsPatients Xmin XmaxaverageX95* % CIstandarddeviation(+/-)medianPatients suffering fromadenocarcinoma134 687 385 366-404 117.0 396controlgroup120 373 222 197-246 50.9 218Patients with squamouscell carcinoma182 811 375 356-393 106.0 391controlgroup120 373 222 199-244 50.9 218Patients suffering fromlarge cell carcinoma238 393 318 257-378 77.6 323controlgroup120 373 222 199-244 51.1 218Patients with small celllung cancer134 793 324 294-354 143.0 282controlgroup120 373 222 196-247 51.1 218LEGEND: platelet* = platelet count x 10 9 /LCI = confidence intervallikelysignificancep

Folia Medica 2011; 46 No 1:28-34Table 6. Analysis of the mean platelet* count of patients with NSCLS according to the TNM classification (IIAand IIB in relation IIIA, IIIB and IV)TNMCLASSIFICATIONPatients with NSCLC(IIA, IIB)Patients with NSCLC(IIIA, IIIB i IV)XminXmaxaverageX95 % CIstandarddeviation(+/-)median207 601 374 331-415 108 350134 811 408 389-426 120 408likelysignificancep>0.05r.n.sLEGEND: platelet* = platelet count x 10 9 /LTable 7. Analysis of mean values of platelet* counts in patients with NSCLS and SCLSPatients Xmin XmaxPatients withNSCLCPatients withSCLCaverageX95 % CIstandarddeviation(+/-)median134 811 377 360-393 112 390134 793 324 228-360 143 282likelysignificancep>0.05r.n.s*LEGEND: platelet* = platelet count x 10 9 /Lstatistically significant differences in meanvalues of platelets count between two testedgroups at the significance level p>0.05.Statistical analysis of mean values ofplatelets count NSCLC compared to SCLCis shown on Table 7. There was no significantdifference at the significance level p>0.05.DiscussionOne of the most common blood abnormalitiesof patients with solid tumors is thrombocytosis(2-4,5-8,13). Several studies haveconfirmed thrombocytosis as paraneoplasticsymptom (2-6,8-12).In a certain percentage, reactive thrombocytosiswas observed, in the diagnosis oflung cancer, prior chemotherapy and/or surgery(2-4).Pathogenesis of reactive thrombocytosisassociated with malignancy has not yet beencompletely solved. The precise mechanismof reactive thrombocytosis may be due tomediators secreted by tumor cells, whichdirectly affect production bone marrowmegakaryocytes. Mediators of tumor cells,such as IL-6, IL-1, IL-11 and macrophagecolony-stimulating factor (14) are probablyresponsible for the thrombocytosis asparaneoplastic symptom (13,15). In addition,some tumors, such as ovarian cancer(15,16), hepatoblastoma and hepatocellularcancer produce thrombopoietin, which hasthe effect on thrombocytosis (7,17).Recent studies indicate interaction betweentumor cells, endothelial cells andplatelets which contribute to the developmentof metastases and poor prognosis onsurvival (13,18,19,20). However, the prognosticsignificance of thrombocytosis onsurvival time has not yet been elucidated.Platelets help tumor spread by increasingthe adhesion of tumor cells in the endotheliumof blood vessels and accumulating inthe tumor cells. In this way, at the same time,platelets prevent the detection and removalof tumor cells by the immune system (21).The largest number of patients with lungcancer in our study was from 51 to 60 yearsof age (No=92, or 38.5%). Number of men32

Arslanagić S et al.: Diagnostic Significance of Platelet Count in Patients With Lung Cancer(No. = 183, or 76.6%) was three times greaterthan the number of women (No = 56, or23.4%). Ratio of men to women was 3.2:1.Pedersen LM and Milman N (3), like GarciaPrim JM et al. (21), had similar gender ratio.About 43.8% ofNSCLC was adenocancer,37.2% squamous cell cancer and 1.5%large cell lung cancer. Similar results havebeen reported in study Pedersen LM. andMilman N. (3.5), Garcia Prim JM et al. (22)and Moyer P (23).In our study, from the 42 patients sufferingfrom SCLC, 20 patients (45.0%) were inlimited stage according to TNM classification,and 22 patients (55.0%) in the expandedstage. We found that SCLC men fourtimes more than women. Pedersen LM andMilman N (3,5), Garcia Prim JM et al. (22)and Moyer P (23) have found similar results.The mean platelets value of lung cancerpatients in our study was 395x10 9 /l, standarddeviation of 122.0. The lowest plateletsvalues was 134x10 9 /l. The largest number ofplatelets was 811x10 9 /l.Different authors found different resultsof thrombocytosis in lung cancer patientsas results a different time when authors determinednumber of platelets. Aoe K et al.(2) determined the number of platelets atthe first hospital admission. The prevalenceof thrombocytosis at that time was lower,about 16%. Survival time of lung cancer patientswith thrombocytosis was 7.5 monthsand was significantly lower to 10.1 monthsto lung cancer patients without thrombocytosis.Statistical analysis of mean platelets valuesof specific histopathological types ofcancer (adenocancer, squamous cell cancer,large cell cancer and small cell lung cancer)to the control group, for each type of cancer,were statistically significant difference.On the basis of our research , we can concludethat thrombocytosis was more presentin advanced TNM stages lung cancer , butnot significantly, compared to less advancedstages of lung cancer. Results supporting ourfindings have been reported in study Aoe Ket al (2), Pedersen LM and Milman (3,5),Engan T, Hannisdal E (11), Gislason T et al.(24).Unlike these results, Inoue K et al. (25)found a positive association of thrombocytosisas tumor size as like TNM stage of renalcell cancer. The same authors not foundpositive correlation between thrombocytosisand histological type of cancer. They alsofound that the number of platelets, after renalcell cancer nephrectomy, normalized inall patients with thrombocytosis.We must emphasize that there is verylittle data in the literature examined thepresence of thrombocytosis in certain histopathologicaltypes of lung cancer.There were no significant difference betweenmean values of platelets count NSCLCand SCLC in our research. Pedersen LM andMilman N (5) found significantly higherthrombocytosis in patients with SCLC inrelation to thrombocytosis in patients withNSCLC. The same authors attach great importanceon present thrombocytosis in patientswith lung cancer. They concluded thatthe tumors markers cannot provide moreinformation about the malignant nature ofsome changes in relation to the increasednumber of platelets.ConclusionThere are significant differences in meanvalues of platelets count among lung cancerpatients in relation to the control group.We did not found a statistically significantdifference between mean values of plateletscount in NSCLC in relation to SCLC.We haven’t found a significant differencein the variability of the mean values of plateletscount between different TNM stages oflung cancer.33

Folia Medica 2011; 46 No 1:28-34References1. Kreyberg L, Liebow AA, Uehlinger EA. InternationalHistologic Classification of Tumours: No.1.Histological Typing og Lung Tumours. Geneva:World Health Organization, 2 nd ed. 1981.2. Aoe K, Hiraki A, Ueoka H, Kiura K, Tabota M,Tanaka M, Tanimoto M.: Thrpmbocytosis as aUseful Prognostic Indicator in Patients with LungCarcinoma. Resp 2004;71:170-1733. Pedersen LM, Milman N.: Prognostic significanceof thrombocytosis in patients with primary lungcarcinoma. Eur Respir J., 1996,9:1826-1830.4. Hamilton W, peters TJ, Round A and SharpD.What are the clinical features of lung carcinomabefore the diagnosis is made? A population basedcase-control study. Thorax 2005;60:1059-1065.5. Pedersen LM and Milman N. Diagnostic significanceof platelet count and other blood analyses inpatients with lung carcinoma. Oncology reports2003,10: 213-216.6. Ikeda M, Furukawa H, Imamura H, Shimizu J,Ishida H, Masutani S. et al. Poor Prognosis AssociatedWith Thrombocytosis in Patients WithGastric carcinoma Ann Surg Oncol 2002;9:287-2917. Hwang SJ, Luo JC, Li CP et al. Thrombocytosis:a paraneoplastic syndrome in patients with hepatocellularcarcinoma. World J Gastroenterol 2004;10:247-2477.8. Sambas NP, Townesend R, El-Galley R, Keane TE,Graham SD and Petros JA. Poor prognosis associatedwith thrombocytosis in patients with renalcell carcinoma. BJU Intern .2000;86:203-207.9. Inoue K, Kohashikawa K, Suzuki S, Shimada Mand Yoshida H.Prognostic significance of thrombocytosisin renal cell carcinoma patinets. JUrol.2004; 11364-36710. Bensalah K, Leray E, Fergelot P:Prognostic valueof thrombocytosis in renal cell carcinoma. J Urol2006;175:859-86311. Engan T, Hannisdal E: Blodd analyses as prognosticfactors in primary lung carcinoma. Acta Oncol.1990;29(2):151-154.12. Taucher S, Salat A, Gnant M, Kwasny W, MlineritschB, Menzel RC et al.: Impact of pretreatmentthrombocytosis on survival in primary breast carcinomaThromb Haemost 2003;98: 1098-110613. Hillen HFP. Thrombosis in carcinoma patients.Ann Oncol 2000;11(3):273-27614. Watanabe M, Ono K, Ozeki Y et al. Production ofgranulocytemacrophage colony stimulating factorin a patient with metastatic chest wall large cellcarcinoma. J J of Clin.Oncol. 2007; 28(9): 559-562.15. Gastl G, Plante M, Finstad CL, Wong GY, FedericiMG,Bander NH et al. High IL-6 levels in asciticfluid corerelate with reactive thrombocytosis inpatients with epithelial ovarian carcinoma. Br JHaemet. 1993;83:433-44116. John Li A and Karlan BY. Androgene Mediation ofThrombocytosis in Epithelial Ovarian CarcinomaBiology. Clin Canc Res 2005, 11:8015-801817. Chen cc, Chang JY, Liu KJ, Chan C, Ho CH, LeeSC and Chen LT. Hepatocellular carcinoma associatedwith acquired von Willebrand disease andextreme thrombocytosis. Ann Oncol 2005;16(6)988-98918. Roth GH. Plateles and blood vessels: the adhesionevent. Immunol.Today. 1992;13:224-23019. Hsu-Lin SC, Berman CL, Furie BC et al. A plateletmembrane protein expresseed during plateletactivation and secretion. J.Biol.Chem. 1984;259:9121-912720. Klerk CP, Smorenburg SM, Otten HM, LensingAW, Prins MH, Piovella P et al. The effect of lowmolecular weight heparin on survival in patientswith advanced malignancy. J Clin Oncol 2005;23:2130-2135.21. Palumbo JS, Talmage KE,Massari JV, La JeunesseCM, Flick MJ, Kombrinck KW et al. Plateles andfibrin (ogen) increase metastatic potential by impedingnatural killer cell-mediated elimination oftumor cells. Blood 2005;105(1):178-18022. Garcia Prim JM, Gonzales Barcala FJ , MoldezRodrigez M et al. Impact of hemoglobin levelon lung carcinomas survival. Med Clin(Barc)2008;131(6):612-61323. Moyer P. Low hemoglobin level in non-small-celllung carcinoma a sign of poor prognosis: presentedat MASCC-ISOO, www. docguide.com/news/content.nsf/news.found 9.6.2009.24. Gislason T, Nou E. Sedimentation rate, leukocytes,platelet count and hemoglobin in bronchial carcinoma:an epidemiological study. Eur J Respir Dis1985;66(2):141-14625. Inoue K, Kohashikawa K, Suzuki S et al. Prognosticsignificance of thrombocytosis in renal cellcarcinoma patients. Intern. J of Urology 2004; 11:364-36734

Original PapersFrequency and Antibiotic Susceptibility of Methicillin-Resistant Staphylococcus Aureus (MRSA) in Canton Sarajevo;Bosnia and HerzegovinaEdina Bešlagić 1 , Sabaheta Bektaš 2 , Mufida Aljičević 1 , Snježana Balta 2 , Sadeta Hamzić 11Department of Microbiology,Faculty of Medicine, University of Sarajevo2Department of Microbiology,Institute for Public Health of Canton SarajevoCorresponding Author:Sabaheta BektašDepartment of Microbiology,Institute for Public Health of Canton SarajevoObjective: To assess frequency and antibiotic susceptibility patternof Methicillin Resistant Staphylococcus Aureus (MRSA)in Canton Sarajevo. Materials and methods: This study used19983 various specimens collected from health care workers,employees who are subject of sanitary inspection and outpatients.In this research 1129 Staphylococcus aureus strains wasisolated. Study was performed at Department of Microbiologyof Institute for Public Heath of Canton Sarajevo during 3months period, September-November 2010. The strains wereidentified by conventional microbiological tests. Sensitivitytesting to 8 antimicrobial drugs was performed according toCLSI guidelines, while MRSA strains were confirmed by E testreference. Results: Out of 1129 Staphylococcus aureus isolates,83 (7.3%) were penicillin sensitive and therefore excluded fromfuture analysis of the frequency of MSSA (Methicillin SensitiveStaphylococcus Aureus) and MRSA isolates. Out of 1442 testedhealth care workers, 6 (0.4%) had nasal colonization and only 1health care worker had MRSA. Out of 4220 routinely screenedemployees, 74 (1.8%) had nasal Staphylococcus aureus colonizationand 2 employees had MRSA. Out of 14321 varioussamples from outpatients, 966 (6.75 %) persons had identifiedStaphylococcus aureus out of which 178 persons had MRSAisolates. The greatest proportion of MSSA isolates (82.7%) wasresistant to penicillin only. Remaining isolates (17.3%) exhibitedadditionally variable resistance to antibiotics, including gentamicin,chloramphenicol, ciprofloxacin and erythromycin.MRSA isolates were tested to 8 antibiotics with 7.4% resistantto penicillin, cefoxitin and erythromycin and 5.9% resistant topenicillin and cefoxitin. Conclusion: MRSA in general populationof Canton Sarajevo has non-multidrug resistance profile,providing increased options for empirical and directed therapyof infections caused by these strains. Continued monitoring ofepidemiology and emerging drug resistance data is critical forthe effective management of these infections.Keywords: MSSA (Methicillin Sensitive Staphylococcus Aureus),MRSA (Methicillin Resistant Staphylococcus Aureus),antimicrobial resistance35

Folia Medica 2011; 46 No 1:35-40IntroductionStaphylococcus aureus is one of the mostcommonly isolated pathogenic bacteria responsiblefor many human infections, bothin hospital and in outpatient conditions.Under the influence of antibiotic pressureStaphylococcus aureus becomes resistantfirst to the penicillin, acquisition and disseminationof β lactamase plasmid, andthen to methicillin acquisition of mecAgene, which encodes altered PBP2a protein,which resulted in the appearance of strainsof MRSA (Methicillin-Resistant StaphylococcusAureus) (1). Consequently, MRSAstrains have spread in health institutionsaround the world so that it recorded a veryhigh percentage of nosocomial infectionscaused by this type of bacteria (2).At the end of the nineties of the lastcentury, there is a separate entity the CA-MRSA, acquired in outpatient conditionsand which in addition to the similarity withHA-MRSA (hospital acquired MRSA) hassignificant differences in the epidemiologicaland clinical sense. Center for DiseaseControl and Prevention, in Atlanta - USA, isdefined outpatient MRSA (CA-MRSA) andhospital MRSA (HA-MRSA) (3). Commonto all reports of infections caused by MRSAof community is this: the disconnect withrisk factors that are present in patients infectedwith MRSA strains originating fromhospital (4). Patients who were infected orcolonized with MRSA, after hospitalizationcan spread those strains in community andconversely CA-MRSA can be got in hospitalconditions (5).Although in our country there are somestudies on the incidence of MRSA infectionsthat are mainly related to HA-MRSA, itwould be important to determine the prevalenceof MRSA in the general populationthat is particularly applicable to the carrierand infections caused by CA-MRSA, as wellas the occurrence of hospital strains in thegeneral population after a stay in hospital.Material and methodsIn this study, 1129 Staphylococcus aureusstrains were isolated out of total 19983 variousspecimens collected from health careworkers, employees who are subject of sanitaryinspection and outpatients. Study wasperformed at Department of Microbiologyof Institute for Public Heath of Canton Sarajevoduring 3 months period, September-November 2010.All samples were cultured and identifiedusing internal protocols, approved for routineapplication of the general bacteriologicalprocedures in the microbiological laboratory.The samples were incubated at 37°Cfor 24 hours on blood agar and after that,identification was performed with tube coagulaseand mannitol tests. The tubes wereincubated at 37°C for 24 hours and observedfor clot formation and positive mannitol test(red color). Sensitivity testing to 8 antimicrobialdrugs: penicillin (PEN), cefoxitin(FOX), erythromycin (ERY), trimethoprimsulfamethoxazole (TSX), ciprofloxacin(CIP), gentamicin (GEN), chloramphenicol(CHL) and fucidic acid (FA) was performedaccording to Clinical Laboratory StandardsInstitute (CLSI) guidelines and interpretationstandards. For the identification ofMRSA strains was used in routine phenotypicmethods of disk diffusion with cefoxitina representative beta-lactam antibiotics,which showed a certain advantage in a seriesof studies in relation to oxacillin. MRSAstrains were confirmed by E test reference.ResultsTotally 19983 specimens including 1442,4220 and 14321 are corresponding to nasalswabs in health care workers and employeesand various clinical samples in outpatients.Out of total number of specimens, 1129 isolatesof Staphylococcus aureus were dividedto 6 (0.4%) in care workers and 74 (1.8%)in employees as nasal colonization and 96636

Edina Bešlagić et al.: Frequency and Antibiotic Susceptibility of Methicillin-Resistant Staphylococcus...(6.75 %) in outpatients from various clinicalspecimens. Out of total 1129 Staphylococcusaureus isolates, 83 (7.3%) were penicillinsensitive and therefore excluded from futureanalysis of the frequency of MSSA andMRSA isolates. (Figure 1).Table 1. presents the frequency of MSSAand MRSA strains related to isolates ofStaphylococcus aureus by groups of respondentswho were divided into three groups ashealth workers and workers who are subjectto sanitary inspection (by the Law on Protectionof Population from Infectious DiseasesFederation B&H Official Gazette no.29/05) and patients with identified Staphylococcusaureus whether carriers or to havea staphylococcal infection.Nasal colonization was identified in 6health workers where 1 (16.6%) person wasthe carrier of MRSA and in 74 workers underthe sanitary control of where the two(2.7%) individuals were carriers of MRSA.Out of total 966 patients in 178 (18.4%)cases MRSA strains were identified, in which105 isolates belonged to staphylococcal carriers(nasal swabs and certain areas of theskin) and the remaining 73 strains of MRSAwere identified as the causative agents of infection.Table 1. The frequency of MSSA and MRSA strainsof the respondentsRespondents MSSA % MRSA % TotalHealth CareWorkers5 (83.3) 1 (16.6) 6Employees 72 (97.3) 2 (2.7) 74Patients 788 (81.6) 178 (18.4) 966Total 865 (82.7) 181 (17.3) 1046In this study, MRSA was isolated withthe highest frequency of nasal swabs andswabs of certain areas of the skin (perineum,axillae, gluteal region of infants) and as thecarriage as a cause of suppurative skin infectionsand ear infections. (Table 2)Table 2. Sources of MSSA and MRSA strainsSource MSSA % MRSA % Totalnasal swab 653 (62.4) 68 (6.5) 721 (68.9)sputum 5 (0.5) 4 (0.4) 9 (0.9)swabs and skinchanges70 (6.7) 75 (7.2) 145 (13.9)wound swab 22 (2.1) 3 (0.3) 25 (2.4)swabthe outer ear68 (6.5) 23 (2.2) 91 (8.7)conjunctivalswab40 (3.8) 4 (0.4) 44 (4.2)urethral swab 1 (0.1) 0 (0.0) 1 (0.1)vaginal swab 1(0.1) 1 (0.1) 2 (0.2)cervical smear 2 (0.2) 0 (0.0) 2 (0,2)navel swab 3 (0,3) 3 (0,3) 6 (0,6)total 865 (82.7) 181 (17.3) 1046 (100.0)Figure 1. The frequency of penicillin sensitiveStaphylococcus aureus in relation to the total numberof isolated Staphylococcus aureusAll isolates of Staphylococcus aureuswere tested by disc diffusion technique to 8antibiotics: penicillin, cefoxitin, erythromycin,sulfamethoxazole trimethoprim, ciprofloxacin,gentamicin, chloramphenicol andfucidic acid. MSSA strains with the highest37

Folia Medica 2011; 46 No 1:35-40Figure 2. Antimicrobial resistance of the isolated strains of MSSA and MRSAfrequency showed resistance to penicillin(82.7%) and a smaller percentage to gentamicin(4.2%). Cefoxitin resistant strainswere confirmed by E test as MRSA strains.They show cross resistance to penicillinand cefoxitin in 17.3% of cases and associatedwith resistance to macrolides (7.8%),particularly in isolates of Staphylococcusaureus from swabs of the nose, throat, skin,changes in the skin, ears, eyes and navel innewborns delivered in the local maternityhospitals (HA-MRSA) and some strainsshowed a much smaller percentage of theassociated resistance to trimethoprim sulfamethoxazole(0.4%), ciprofloxacin (0.4%)and gentamicin (3.5%) in those older agewho have a history of recent hospitalizationor some other risk factor that connects themwith HA-MRSA.Antibiotic type predominant amongMSSA cells is characterized by resistanceto penicillin in 78.2% of cases, and the restof the MSSA strains were distributed in 7more strains in addition to penicillin resistanceand associated with resistance togentamicin, chloramphenicol, ciprofloxacinand erythromycin in various combinations.MRSA cells were divided into 7 antibiotictypes with predominantly antibiotic typesresistant to penicillin and cefoxitin (5.9%)and penicillin, cefoxitin and erythromycin(7.4%).DiscussionResults of our investigation indicate thatMRSA might be en emerging pathogen inCanton Sarajevo. To our knowledge, this isone of the first reports of routine surveillancefor this organism in Bosnia and Herzegovina.Prior studies of MRSA have been reportedonly from hospital condition (6,7).It is assumed that the majority of invasivestaphylococcal infections originated fromnasal carriers. Data from the literature showthat the rate of nasal carriers of 1.5- 9.0%among adults in the community anywherein the world (8,9). Among health workersin hospital conditions that rank ranges from6.0-17.8%, although there are reference dataand higher rates of nasal carriers (10,11).38

Edina Bešlagić et al.: Frequency and Antibiotic Susceptibility of Methicillin-Resistant Staphylococcus...Table 3. Resistant phenotypes isolated MSSA and MRSAMSSA n=865PEN FOX ERY SXT CIP GEN CHL FA Total %R S S S S S S S 818 (78.2)R S S S S R S S 18 (1.7)R S S S S S R S 4 (0.4)R S S S S R R S 9 (0.9)R S S S R R S S 3 (0.3)R S S S R R R S 1 (0.1)R S R S R R S S 7 (0.7)R S R S S R R S 5 (0.5)MRSA n=181PEN FOX ERY SXT CIP GEN CHL FA Total %R R S S S S S S 62 (5.9)R R R S S S S S 77 (7.4)R R S S S R S S 35 (3.3)R R S R S S S S 3 (0.3)R R R S R S S S 2(0.2)R R R S R R S S 1(0.1)In this study, the rate of MRSA was 16.6%, 2.7 % and 18.4 % and corresponds tohealth care workers, employees and symptomaticpatients which are correlated in resultsobtained by other researches.MRSA in the Sarajevo Canton have nota multidrug resistance profile, providing increasedoptions for empirical and directedtherapy of infections caused by these strains.Global increase of Staphylococcus aureusresistance to beta-lactam and other group ofantimicrobial agents shows significantly differencesin hospital centers and communityacquired of infections among regions andcoutries (12).Certain limitations are associated withthis investigation. First, because not allhealthcare workers and employees werescreened during the tested period of 3months and because we reviewed laboratoryrecords from outpatient facilities thattested this organism only among symptomaticpatients, data might be underreported.Second, because we didn’t review medicalrecords of outpatients on their prior hospitalization,data might not be applicableto all community. Third, because nitrocefintest for antimicrobial resistance to penicillinhas not been performed, susceptibility topenicillin might be reported lower than it isactually.In conclusion, MRSA in general populationof Canton Sarajevo does not have amultidrug resistance profile, providing increasedoptions for empirical and directedtherapy of infections caused by these strains.Continued monitoring of epidemiology andemerging drug resistance data is critical forthe effective management of these infections.References1. Barber M. Methicillin-resistant Staphylococci. JClin Pathol., 1963:1:308-11.2. Boerlin P, Reid-Smith R. J. Antimicrobial resistance:its emergence and transmission. AnimHealth Res Rev. 2008;9:115-26.39

Folia Medica 2011; 46 No 1:35-403. Community-associated Methicillin-ResistantStaphylococcus aureus (CA-MRSA) http://www.cdc.gov/ncidod/dhqp/ar_mrsa ca.html.2005.4. Naomi TS, LeDell KH, Como-Sabetti K. Comparisonof community- and health care-associatedmethicillin-resistant Staphylococcus aureus infection.Jama 2003;290(22):2976-84.5. Petrović Jeremić LJ. Učestalost meticilin-rezistentnihStaphylococcus aureus (MRSA) sojevakod zdravih kliconoša u Pomoravskom okrugu.Acta Medica Medianae 2010;49(1):33-36.6. Šiširak M, Zvizdić A, Hukić M. Methicillin-resistantStaphylococcus aureus (MRSA) as a cause ofnosocomial wound infections. Bosnia Journal ofBasic Medical Sciences 2010;10(1):34-37.7. Nurkić M. Causes of infection surgical wound.The fourth Symposium of Hospital Infection Controlof Bosnia and Herzegovina, Tuzla, Bosniaand Herzegovina, Juny 14 th -16 th , 2006. Proceedings:9-19.8. Bischoff WE, Wallis ML, Tucker KB, ReboussinBA, Shertz RJ. Staphylococcus aureus nasal carriagein a student community: prevalence clonalrelationships and risk factors. Infect Control HospEpidemiol 2004; 25(6):485-91.9. Wertheim HF, Melles DC, Vos MC, van LeeuwenW, van Belkum A, Verbrugh HA et al. The roleof nasal carriage in Staphylococcus aureus infections.Lancet Infect Dis 2005; 5(12):751-62.10. Eveillard M, Martin Y, Hidri N, Boussougant Y,Joly-Guillou ML. Carriage of methicillin-resistantStaphylococcus aureus among hospital employees:prevalence, duration, and transmission tohouseholds. Infect Control Hosp Epidemiol 2004;25:114-20.11. Cesur S, Cokca F. Nasal carriage of methicillinresistantStaphylococcus aureus among hospitalstaff and outpatients. Infect Control Hosp Epidemiol2004; 25:169-71.12. Hacek DM, Suriano T, Noskin GA, Kruszynski J,Reisberg B, Peterson LR. Medical and economicbenefit of a comprehensive infection control programthat includes routine determination of microbialclonality. Am J Clin Pathol. 1999; 111:647-654.40

Original PapersDetection of Cytokines IFN γ, TNF α and IL 10 in Acute andChronic Human ToxoplasmosisSabaheta Bektaš 1 , Snježana Balta 1 , Mufida Aljičević 2 , Edina Bešlagić 21Department of Microbiology,Institute for Public Health of Canton Sarajevo2Department of Microbiology,Faculty of Medicine, University of SarajevoCorresponding Author:Edina BešlagićDepartment of Microbiology,Faculty of Medicine, University of SarajevoObjective: Cytokines are low-level molecular proteins secretedby wide spectrum of cells and included in the mediationand the regulation of non-specific and specific acquired immuneresponse. T. gondii infection is strong trigger of naturaland acquired immunity regulated by vast range of protectiveand regulatory cytokines. The aim of this research is to analyzeand verify the level of production of cytokines (IFN-γ,TNF-α and IL 10). Materials and methods: This research wasclinical-experimental study which involved experimental andcontrol group. First group of subjects was represented by 40patients seropositive to T. gondii, out of which 26 patients hadacute toxoplasma infection and 14 ones had chronic toxoplasmainfection. Second group consisted of 30 subjects seronegativeto T. gondii. Their serums were tested for the presenceand concentration of specific cytokines (IFN-γ, TNF-αand IL10). The levels of mentioned cytokines were verifiedby the Elisa test (enzyme-linked immunosorbent assay) usingcommercial kits: IFN-γ-EASIA (Biosource Europe S.A,Nivelles, Belgium), Human TNF-α/TNFSF1A (R&D SystemsInc, Minneapolis, USA) and Human IL-10 (R&D SystemsInc, Minneapolis, USA). Results: Acute phase of infectionis expressed by significant increase of IFN-γ level related toothers cytokines (p

Folia Medica 2011; 46 No 1:41-47IntroductionCytokines are low level-molecular proteinssecreted by wide spectrum of cells and includedin the mediation and the regulationof non-specific and specific acquired immuneresponse. Their function is connectionto specific receptors in the membrane oftarget cells which induce signal pathways viaother mediators, often tyrosine kinase, andleads to change in the expression of genesencode the functions and the behaviour ofcells (1). On the basis dominant biologicalactivity in the toxoplasma infection, theycan be divided into 2 main types: protectiveand regulatory cytokines (2). T. gondii infectionis strong trigger of natural immunitywith non-specific activation of macrophagesand along with other hematopoietic andnon-hematopoietic cells (3). They produceIL 12 that then activates NK and T cells tosecrete IFN-γ which early burst is crucial inshaping of resistance to this infection. IFN-γsynergizes with TNF-α in mediating to killT. gondii tachyzoites by macrophage microbicidalfunction (4). Combination of theseboth cytokines leads to production of reactivenitrogen intermediates which are capableof reducing parasite proliferation.Non-specific immune response leadsto differentiation of macrophages and Blymphocytes into APC. T lymphocytes arestimulated by APC presenting specific antigento TCR (T cell receptor) (5). This resultsin the Th1 differentiation of CD4+ TLwith production protective cytokines IFN-γand IL 2, and also Th2 differentiation withproduction regulatory cytokines IL 4, IL 5,IL 6 and IL 10. These cytokines synergeticpromote the regulation of the protectiveimmunity (6,7,8). CD8+ TLs, activated byIL 2 from Th1 type cells, show cytotoxicactivity against parasite and infected cells.Finally, the control of this infection is resultedby synergetic action between CD4+and CD 8+ TL (9). Acute phase of infectionis characterized by high levels of IFN-γ andproinflammatory cytokine, but also of synthesisof IL 10 to avoid an excessively strongcell mediated immunity and then to allowthe parasite to survive and establish chronicinfection (10). Chronic phase of infection ischaracterized by decrease of level of IFN-γand proinflammatory cytokines, while thelevel of IL 10 rises (11). Most of the datain the field of immunity of Toxoplasma infectionhave been obtained from studies inanimals, in particular, in mice. Although,the mice model represents a vast spectrumof immune response related to controlledvariation of the genetic background, in essence,these data acquired in animals cannotbe directly applied to humans. The aim ofthis research is to measure and analyze thelevel of cytokines (IFN-γ, TNF-α and IL 10).Material and methodsThis research was clinical-experimentalstudy and involved experimental and controlgroup. First group of subjects was representedby 40 patients seropositive to T.gondii, out of which 26 patients had acutetoxoplasma infection and 14 had chronictoxoplasma infection. Second group consistedof 30 subjects seronegative to T. gondii.Their serums were tested for the presenceand concentration of specific cytokines(IFN-γ, TNF-α and IL10). This survey wasconducted at Institute for Microbiology andpathology of the University Clinical CenterTuzla during 2007.The level of mentioned cytokines wasverified by the Elisa test (enzyme-linked immunosorbentassay) using commercial kits:IFN-γ-EASIA (Biosource Europe S.A, Nivelles,Belgium), Human TNF-α/TNFSF1A(R&D Systems Inc, Minneapolis, USA) andHuman IL-10 (R&D Systems Inc, Minneapolis,USA). The plates with the samples, standardsand controls were examined at 450nmin the «Metertech 960» reader during 3042

Sabaheta Bektaš et al.: Detection of Cytokines IFN γ, TNF α and IL 10 in Acute and Chronic ...minutes after test was over. Results are measuredby interpolation at standard curve.ResultsTable 1. Demographic characteristic of patientsand control subjects.numbersexagemale(%)female(%)patientscontrolsubjects40 3014 (35.0) 12 (40.0)26 (65.0) 18 (60.0)interval 1-66 21-38median 25 22Prospective research involved 40 patientswho are infected with T. gondii confirmedby serological testing to specific antibody.Out of total number, 24 patients had clinicalstatement of acute toxoplasmosis confirmedpositive serological results (IgM positive,IgG positive) and prior that other diseaseswere excluded (mononucleosis, granulomatosis,lymphomas etc). All these patients hadperiphery lymphadenopathy (cervical, nuchaland inguinal) and 9 of them had morecomplex clinical states with significant hepatosplenomegaly,mediastinal and abdominallymphadenopathy followed by a rise in bodytemperature, cough and prostration. Rest of16 patients had chronic form of toxoplasmosisconfirmed by serological results (IgMnegative, level of IgG antibodies over 200IU/ml). Other diseases were excluded. Inthis group there were 14 male and 26 femalepersons at age range 1-66 years.Control subjects involved 30 persons, 12men and 18 women, aged 21-38 years, clinicallyasymptomatic and did not had antitoxospecific antibody.Serums of these examinees were testedfor the presence and concentration of specificcytokines (IFN-γ, TNF-α and IL10).In patients acutely infected with T. gondiithe mean level (0.68±0.17) was eightfoldas high as the mean level in healthy personswhat is statistically significant (p

Folia Medica 2011; 46 No 1:41-47Table 3. Comparison of level IFN-γ, TNF-α and IL 10 between patients chronic infected with T. gondii andcontrol subjects.cytokines examinees N° mean±SE median rangt studenttestdfsignificancelevelIFN-γinfected 16 0.47± 0. 09 0.45 0.03-1.20control 30 0.08 ± 0.01 0.07 0.03-0.2047.56 52 p0.05IL 10infected 16 8.04 ± 0.67 8.10 3.9-13.00control 30 4.06 ± 0.42 4.15 0.00-7.606.36 52 p

Sabaheta Bektaš et al.: Detection of Cytokines IFN γ, TNF α and IL 10 in Acute and Chronic ...Figure 2. Comparison of concentration TNF- α between acute and chronic phase of infectionStudent t= 1.18 DF=38 p>0.10Level of TNF-α, strong proinflammatory cytokine, in essence, did not change significantly. Although therewere some extreme values of this cytokine, the mean level this cytokine between acute and chronic phasedoesn´t show statistically significant difference (p>0.10).Figure 3. Comparison of concentration IL 10 between acute and chronic phase infection.Student t=1.47 DF=38 p>0.10Concentration of IL 10 in acute and chronic phase of toxoplasma infection doesn´t show statisticallysignificance (p>0.10) although the comparison of mean level of this cytokine between infected and healthywas twice enhancement in the chronic infection (influence of extreme values).45

Folia Medica 2011; 46 No 1:41-47The level IFN-γ in chronic toxoplasmosis(0.47± 0.09) was 5.8 fold as high as in thecontrol group (0.08 ± 0.01) what is statisticallysignificant (p0.05). Comparison of themean level of IL 10 between infected (8.04 ±0.67) and control group (4.06±0.42) revealsstatistically significant differences (p

Sabaheta Bektaš et al.: Detection of Cytokines IFN γ, TNF α and IL 10 in Acute and Chronic ...exhibits powerful polarization towards Th1cell immune response with overproductionof IFN-γ in acute and in chronic infection.References1. Lieberman LA, Hunter CA. The role of cytokinesand their signaling pathways in the regulation ofimmunity to Toxoplasma gondii. 2002;21:373-403.2. Filisettti D, Candolfi E. Immune response to Toxoplasmagondii. Ann Ist Super Sanita. 2004;40(1):71-80.3. Sher A, Sousa CR. Ignition of the Type I responseto intracellular infection by dendritic cell derivedinterleukin 12. Eur Cytokine Netw. 1998;9:65-8.4. Abbas AK, Lichtman AH. Basic Immunology, II ed.2006-2007.5. Reichmann G, Walker W, Villegas EN, Craig L, CaiG, Alexeder J, Hunter CA. The CD40/CD40 ligandinteraction is required for resistance to toxoplasmicencephalitis. Infect Immun. 2000;68(3):1312-8.6. Janeway CA, Travers P, Walport M, Shlomichik M.Immunobiology, V th ed. Garland Publishing, NewYork, 2005.7. McHeyzer-Wiliams M, McHeyzer-Wiliams L, PanusJ, Pogue-Caley R, Bikah G, Driver D, EisenbraunM. Helper T-cell-regulated B-cell imunity. Microbesand Infection. 2003;5:205.12.8. Hanna N, Hanna I, Hleb M, Wagner E, DoughertyJ, Balkundi D, Padbury J, Sharma S. Gestational agedependentexpression of IL10 and its receptor in humanplacental tissue and isolated cytotrophoblasts.JImmunol.2000;164:5721-8.9. Reichmann G, Walker W, Villegas EN, Craig L, CaiG, Alexeder J, Hunter CA. The CD40/CD40 ligandinteraction is required for resistance to toxoplasmicencephalitis. Infect Immun. 2000;68(3):1312-8.10. Mordue DG, Monroy F, regina ML, DinarelloCA, Sibley D. Acute toxoplasmosis leads to lethaloverproduction of Th1 cytokines. J Immunol.2001;167:4574-84.11. Soete M, Dubremetz JF. Toxoplasma gondii: kineticsof stage specific protein expression during tachyzoite-bradyzoiteconversion in vitro.Curr. Top. Microbiol.Immunol. 1996;219:76–80.12. Johnson J, Suzuki Y, Mack D, Mui E, Estes R, DavidC, Skamene E, Forman J, McLeod R. Genetic analysisof influences on survival following Toxoplasmagondii infection. Int J Parasitol 2002;32(2):179-85.13. Lieberman LA, Hunter CA. The role of cytokinesand their signaling pathways in the regulation ofimmunity to Toxoplasma gondii. 2002;21:373-403.14. Hunter C, Subauste C, Van Cleave V, RemingtonJ. Production of gamma interferon by naturalkiller cells from Toxoplasma gondii-infected SCIDmice: regulation by interleukin-10, interleukin-12,and tumor necrosis factor alpha. Infect Immunity1994;62(7):2818-24.15. Gazzinelli RT, Hieny S, Wynn T, Wolf S, Sher A.Interleukin 12 is required for the T-lymphocyteindependentinduction of interferon gamma byan intracellular parasite and induces resistancein T-cell-deficient hosts. Proc Natl Acad Sci USA1993;90:6115-9.16. Cai G, Kastelein R, Hunter CA. Interleukin-18(IL-18) enhances innate IL-12 mediated resistanceto Toxoplasma gondii. Infect Immun2000;68(12):6932-8.17. Sarciron ME, Gherardi A. Cytokines involved inToxoplasmic encephalitis. Scandinavian J Immunol.2008;52(6):534-43.18. Cai G, Radzanowski T, Villegas EN, Kastelein R,Hunter CA. Identification of STAT4-dependent andindependent mechanisms of resistance to Toxoplasmagondii. J Immunol 2000;165(5):2619-27.19. Gazzinelli RT, Wysocka TM, Hayashi EY, DenkersEY, Hieny S, Caspar P, Trinchieri G, Sher A. Parasite-inducedIL-12 stimulates aerly IFN-γ synthesisand resistance during acute infection with T. gondii.J Immunol. 1994;153;2533-43.20. Gazzinelli RT, Amichay D, Scharton-Kersten T,Grunvald E, Farber JM, Sher A. Role of macrophagederivedcytokines in the induction and regulation ofcell mediated immunity to Toxoplasma gondii. CurrTop Microbiol Immunol. 1996;219:127–40.21. FiorentinoDF, Zlotnick A, Mosmann T, Howard M,O’Garra A. IL-10 inhibits cytokine production byactivated macrophages. J. nol.1991;147:3815–3822.22. Hanna N, Hanna I, Hleb M, Wagner E, DoughertyJ, balkundi D, Padbury J, Sharma S. Gestational agedependentexpression of IL10 and its receptor in humanplacental tissue and isolated cytotrophoblasts. JImmunol. 2000;164:5721-8.23. Khan IA, Matsura T, Kasper LH. IL 10 mediates immunosuppressionfollowing acute infection with T.gondii in mice. Parasite Immunol. 1995;17:185-95.24. Pfaff AW, Abou-Bacar A, Letscher-Bru V, Villard O,Senegas A, Mousli M, Candolfi E. Cellular and molecularphysiopathology of congenital toxoplasmosis:The dual role of IFNγ. ogy. 2007;134(13):1895-902.25. Yamamoto JH, Vallohi AL, Silveira C, Filho JK, NussenblattRB, Cunha-Neto E, Gazzinelli RT, Belfort RJr, Rizzo LV. Discrimination between patients withacquired toxoplasmosis and congenital toxoplasmosison the basis of the immune response to parasiteantigens. J Infect Dis. 2000;181(6):2018-247

Review PapersUse of Homeostatic Model Assessment in Managing Type 2DiabetesDamira Kadić¹, Davorka Dautbegović-Stevanović²¹Department of Laboratory Diagnostics²Department of Internal DiseasesCantonal Hospital ZenicaCorresponding AuthorDamira KadićCantonal Hospital ZenicaCrkvice 6772000 Zenica, B&HPhone: +387 32 405 133E-mail: damira.kadic@gmail.comThe prevalence of type 2 diabetes has reached epidemic proportionsworldwide, and the incidence of the life-threateningcomplications of diabetes is increasing. It is of great importanceto make a good assessment of newly diagnosed diabeticsand to select an appropriate treatment. Without theprecise knowledge of the insulin sensitivity and pancreaticβ-cell function, choosing and adjusting glucose-loweringagents may lead to suboptimal results in some circumstances.Several methods are used for evaluation of the function ofβ-cells and the degree of insulin resistance. Although, clamptechnique is considered as a reference technique, several noninvasive,fasting or „homeostatic“ models have been used.Homeostatic model assessment is a mathematical modelwhich allows values for insulin sensitivity, insulin resistanceand β-cell function to be obtained if fasting plasma glucoseand insulin concentrations are known. The HOMA approachhas been widely used because it is simple, cheap, low laborintensive,low time-consuming and acceptable to patients andclinicians.Key words: type 2 diabetes, pancreatic β-cell function, insulinsensitivity, insulin resistance, HOMA.IntroductionDiabetes mellitus has been defined by theWHO (World Health Organization) as astate of chronic hyperglycemia. It is a syndromecomprised of heterogeneous clinicalfeatures and hyperglycemia which leads toits diagnosis and may be the result of variousdiseases and disorders. Absolute or relativeinsulin deficiency or an inadequate insulineffect on the cells is the underlying causes ofhyperglycemia. The clinical classification ofthe diabetes mellitus syndrome (Table 1) followsthe WHO recommendations (1).More than 90 % of diabetics fall intothe type 2 category. The prevalence of type2 diabetes has reached epidemic proportionsworldwide, and the incidence of thelife-threatening complications of diabetessuch as cardiovascular disease, retinopathy,nephropathy and lower-limb amputation,through continued exposure of tissues tohigh glucose levels is increasing (2,3).According to the WHO, the number ofpeople with type 2 diabetes worldwide wasapproximately 170 million and 280 million in48

Damira Kadić et al.: Use of Homeostatic Model Assessment in Managing Type 2 Diabetes2000 and 2010, respectively, which is expectedto increase to 430 million by 2030 (2,4).These data suggest that diabetes is a hugeeconomic, social and medical problem. Althoughwe recently pay more and more attentionto primary prevention, in practice,we mainly deal with secondary prevention.It is therefore of great importance to make agood assessment of newly diagnosed diabeticsand to select an appropriate treatment, inorder to prevent or delay late complicationsof diabetes.Type 2 diabetes mellitusType 2 diabetes mellitus is a multifactorialdisease. Polygenic hereditary characteristicsand environmental factors are important forthe appearance of clinical manifestations.From a laboratory diagnostic point of view,the disease pathogenesis can be followed asa transition from normal to impaired glucosetolerance and subsequently to the continuouspresence of hyperglycemia.Pathobiochemically, type 2 diabetes mellitusis characterized by disorders involvingboth the secretion as well as the cellular effectsof insulin. This pathobiochemical processis enhanced by progressive hyperglycemia.In particular, the individual underlyingdisorders which can already be found yearsprior to the clinical manifestation of type 2diabetes mellitus:- Decreased cellular effect of insulin atthe target organ sites also referred toas insulin resistance. This term describesthe disturbed insulin effecton cells such as adipocytes, skeletalmuscle cells and hepatocytes. In simplifiedterms, glucose uptake by theseinsulin-dependent cells is reduced. Ascompensation, a rise in insulin secretionwith hyperinsulinemia ensues.As part of the further course, whencompensatory β-cell production ofinsulin is exhausted, hyperglycemiawill set in.- Impaired insulin secretion by thepancreatic β-cells in response to glucoseresulting in absolute or relativeinsulin deficiency. The pulsatility ofspontaneous insulin secretion andthe early phase of the glucose-mediatedinsulin secretion are already disturbedduring the early normoglycemicstage of type 2 diabetes mellitus.The glucose-mediated insulin secretionof the late phase, in contrast, isincreased thereby causing hyperinsulinemia(1).Diagnosis of diabetes is set exclusivelyby confirming of hyperglycemia, so thateach finding of hyperglycemia in a randomsample taken during the day is necessaryto confirm with additional determinationon the next day. It is important to note thatwe use recommendation of the WHO andADA (American Diabetes Association) forthe glucose concentrations in the diagnosisof diabetes. Other diagnostic and laboratorytests yield to the classification of disease andthe assessment of already caused complicationsat the time of the diagnosis (5).Evaluation of the β-cell function andthe degree of insulin resistanceDiabetic complications can be prevented bystrict glycaemic control. Glycaemic controldepends mainly on the degree of residualpancreatic β-cell function, insulin sensitivityand other factors such as compliance totreatment and glycaemic loads. In order tomanage diabetic patients effectively, suchdata should be readily available to physicians.However, choosing and adjusting glucose-loweringagents in the routine care ofdiabetic patients are largely empirical. Withoutthe precise knowledge of the underlyinginsulin sensitivity and pancreatic β-cell49

Folia Medica 2011; 46 No 1:48-53function, it may lead to suboptimal resultsin some circumstances (6).Several methods are used for evaluationof the function of β-cells and the degree ofinsulin resistance such as clamp technique,frequently sampled intravenous glucosetolerance test (FSIVGTT) and continuousinfusion of glucose with model assessment(CIGMA) (8).Clamp technique is considered as a referencetechnique for the determination ofinsulin sensitivity in humans, because itdirectly measures the metabolic effect ofinsulin in precisely set conditions. It can beimplemented in euglycaemic, isoglycaemicand hyperglycaemic conditions. However,it is also the most complex technique as itrequires the simultaneous administrationof the glucose and insulin, multiple bloodsampling and experienced operator (a physicianor a technician) over a course of 3 and6 hours, respectively (8).In the quest for noninvasive measurementtechnique for insulin sensitivity, severalfasting or “homeostatic” models havebeen proposed such as fasting glucose/insulinratio (FGIR), quantitative insulin sensitivitycheck index (QUICKI) and homeostaticmodel assessment (HOMA). Thesemethods have been the most frequentlyused techniques in clinical investigations.The fact that these tests require only a singlevenipuncture in fasting state and do not callfor concomitant intravenous access makesthem particularly attractive to patients andclinicians alike (9).Types of HOMA modelHOMA 1: The original HOMA modelHomeostatic model assessment was firstdescribed in 1985 (Matthews et al.). It is astructural mathematical model which allowsvalues for insulin sensitivity (which isreciprocal of insulin resistance) and β-cellfunction, expressed as a percentage of normal,to be obtained if simultaneous fastingplasma glucose and fasting plasma insulinor C-peptide concentrations are known (6).These parameters can be used by means ofthe following formulas (10):HOMA-IR= (FPI x FPG)/22.5HOMA-%B= HOMA-IR – (20 x FPI)/(FPG-3.5)(HOMA-%S= 100/HOMA-IR)IR= insulin resistanceFPI= fasting plasma insulin level; FPG=fasting plasma glucose level% B= % of functional β-cells in the pancreas;% S= % of insulin sensitivityThe relationship between glucose and insulinin the basal state reflects the balancebetween hepatic output and insulin secretion,which is maintained by feedback loopbetween the liver and β-cell. The predictionused in the model arises from experimentaldata in humans and animals (11).The approximating equation, in earlymodel, used a fasting plasma sample, andwas derived by use of the insulin-glucoseproduct, divided by a constant (assumingnormal weight, normal subjects

Damira Kadić et al.: Use of Homeostatic Model Assessment in Managing Type 2 Diabetesof proinsulin secretion into the model andthus allows use of total or specific insulin assays(11).Use of HOMA modelsHOMA can be used either in normal subjectsor in those with varying degrees of glucosetolerance. It has been validated in diabeticpatients treated with diet alone, or withoral hypoglycemic agents (6).HOMA measurement is important forestimation of the severity of insulin resistance,to detect patients with an absolute lossof β-cell function and as an aid in choosingthe best therapeutic agent for an introductorytreatment in patients with type 2 diabetes(5). It is also an appropriate method for assessinglongitudinal change in insulin resistanceand β-cell function with time duringtreatment of diabetes (6).Some studies demonstrate that productof HOMA-%S and HOMA-%B, the so-calleddisposition index, was significantly relatedto glycaemic control as assessed by HbA1c.This implies that assessing the dispositionindex is likely to be useful in clinical decisionmaking for taking care of patients withdiabetes (6).If the low disposition index is due mainlyto low HOMA-%S, the underlying problemis predominantly insulin resistance andthe addition or the increase in the doses ofinsulin sensitizer can be of benefit. Manystudies reported that thiazolidinediones,using alone or in combination with otherhypoglycemic agents, increase HOMA-%Sby 9-37% and may increase HOMA-%B upto 28%, which suggests their beneficial effecton the pancreatic β-cells. On the otherhand, if HOMA-%B is markedly reduceddespite maximum doses of insulin secretagogue,secondary drug failure is likely andinsulin therapy should be initiated (6). Tavernareported (7) that subjects with HOMA-%B

Folia Medica 2011; 46 No 1:48-53The first HOMA RIA calculator was originallydesigned for insulin radioimmunoassay.Main disadvantage of this assay refers tothe risk of environmental pollution due toimproper handling with radioactive materials.Later computer models have been recalibratedto modern insulin assays.The (Architect) Insulin assay, that we use,is one-step immunoassay to determine thepresence of human insulin in human serumor plasma, using CMIA (chemiluminescentmicro particle assay) technology. Sample,anti-insulin coated paramagnetic microparticles, and anti-insulin acridinium-labeledconjugate are combined. Insulin presentin the sample binds to the anti-insulincoated micro particles and anti-insulin acridinium-labeledconjugate. After washing,pre-trigger and trigger solutions are thenadded to the reaction mixture. The resultingchemiluminescent reaction is measuredas relative lights units (RLUs) by the opticalsystem which convert them to assay-specificanalyte concentration units.calculator are considered non-steady stateand HOMA is considered to be inappropriate.Input limits for insulin are 20-400pmol/l (2.9-57.6 µU/ml) (12).ConclusionHomeostatic model assessment is a methodfor the evaluation of the function of pancreaticβ-cells and the degree of insulin resistance.The HOMA approach has been widelyused because it is simple, cheap, low laborintensive,low time-consuming and acceptableto patients and clinicians (9). HOMAhas been compared with other tests for thedetermination of the degree of insulin resistance.Although euglycaemic and hyperglycemicclamps are referred to as “the goldenstandards” studies have demonstrated excellentcorrelation between HOMA and euglycaemicclamp (13).Limits of homa modelingAs HOMA is based on steady-state physiology,the model includes limits for glucose.Values outside these limits, 3-25 mmol/l forTable1. Classification of diabetes mellitus and impaired glucose tolerance according to the WHOrecommendations– Type 1 diabetes mellitus (formerly also known as insulin-dependent diabetes mellitus=IDDM)– Type 2 diabetes mellitus (formerly also known as non insulin-dependent diabetes mellitus=NIDDM)a) Noneobese type 2a diabetes mellitusb) Obese type 2b diabetes mellitusc) MODY=maturity-onset diabetes of the young– Diabetes mellitus secondary to certain other diseases and distrubances (formerly also referred to assecondary diabetes mellitus), e.g. pancreatic diseases, endocrine disorders, iatrogenically induced orcaused by toxic substances– Gestational diabetes mellitus– Impaired glucose tolerance52

Damira Kadić et al.: Use of Homeostatic Model Assessment in Managing Type 2 DiabetesReferences1. Lothar T, M.D. Clinical laboratory diagnostics.First edition. Frankfurt/Main, Germany: TH-Books Verlagsgesellschaft GmbH,1998.2. Shaw JE, Sicree RA, Zimmet PZ. Global estimatesof the prevalence of diabetes for 2010 and 2030.Diabetes Res Clin Pract 2010; 87(1): 4-14.3. Campbell RK. Type 2 diabetes:where we aretoday:an overview of disease burden,currenttreatments,and treatment strategies.J Am PharmAssoc 2009; 49(1):3-9.4. Wild S, Roglic G, Green A, Sicree R, King H.Global prevalence of diabetes: estimates for theyear 2000 and projections for 2030. Diabetes care2004; 27 (5): 1047-53.5. Topić E, Primorac D, Janković S. Medicinskobiokemijskadijagnostika u kliničkoj praksi. Zagreb:Medicinska naklada, 2004.6. Dansuntornwong B, Chanprasertyothin S, JongjaroenprasertW, Ngarmukos C, Bunnag P, PuavilaiG, Ongphiphadhanakul B. The relation betweenparameters from homeostasis model assessmentand glycemic control in type 2 diabetes.J Med Assoc Thai 2007; 90 (11): 2284-90.7. Taverna MJ, Pacher N, Bruzzo F, Slama G, SelamJL. Beta-cell function evaluated by HOMA as apredictor of secondary sulphonylurea failure intype 2 diabetes. Diabet med 2001; 18: 584-8.8. Muačević-Katanec D. Dijagnoza metaboličkeinzulinske rezistencije. Medicus 2004; 13 (2): 91-4.9. Keskin M, Kurtoglu S, Kendirci M, Atabek M.E,Yazici C. Homeostasis model assessment is morereliable than the fasting glucose/insulin ratio andquantitative insulin sensitivity check index for assessinginsulin resistance among obese childrenand adolescents. Pediatrics 2005; 115; e500-3.10. Matthews DR, Hosker JP, Rudenski AS et al. Homeostasismodel assessment: measurement of insulinresistance and beta-cell function from fastingplasma glucose and insulin concetrations inman. Diabetologia 1985; 28:412-419.11. Wallace T.M, Levy J.C, Matthews D.R. Use andabuse of HOMA modeling. Diabetes Care 2004;27:1487-95.12. Manley S.E, Luzio S.D, Stratton I.M, Wallace T.M,Clark P.M.S. Preanalytical, analytical, and computationalfactors affect homeostasis model assessmentestimates. Diabetes care 2008; 31(19): 1877-83.13. Muačević-Katanec D, Profozić V, Metelko Ž. Theimportance of determining the presence and degreeof insulin resistance in the general populationand in some clinical states. Diabetol croat 2005;34-1.53

Review PapersParagangliomas of the NeckJasminka Alagić-Smailbegović 1 , Kamenko Šutalo 1 , Edina Hadžić 11 Department of Otolaryngology,University of Sarajevo, Clinical CenterBolnička 25, 71000 Sarajevo,Bosnia and HerzegovinaCorresponding authorJasminka Alagić-SmailbegovićE-mail: orlkonges@gmail.comParagangliomas are the most common type of benign vascularneoplasm of the neck. Multicentric paragangliomas occurin approximately 10% overall. The etiology of paragangliomasseems to be multifactorial. The characteristic feature ofcarotid body tumors is slow growth rate, which is reflectedclinically by the delay between the first symptoms and the diagnosis,averaging between 4 and 7 years. MRI and CT angiographyare the most useful imaging studies. Surgery remainsthe mainstay of treatment for carotid body tumors.Key words: paragangliomas, multicentric, diagnosis, treatmentIntroductionParagangliomas represent the most commontype of benign vascular neoplasm ofthe neck. These tumors arise from extra adrenalparaganglionic cells derived from neuralcrest. The controversy over the propernomenclature of paragangliomas is based onlocation, so there are „carotid paragangliomas“,„jugulotympanic paragangliomas“ and„vagal paragangliomas“, although the termscarotid body tumor, glomus tympanicumand glomus jugulare still persist. Glomus tumorsof the head and neck are benign but locallyinvasive tumors that arise from glomuscells. These are chemoreceptor cells thatdetect levels of carbon dioxide and oxygenin the blood and send corresponding signalsto the part of the brain that regulates breathing.In the head and neck, glomus tissue isfound in the middle ear and/ or brain(1).The normal carotid body measures 3 to5 mm in diameter but is often larger in personsliving at higher altitudes. The averageweight of the normal adult gland is 12 mg,with a wide range previously reported as 1to 47 mg (2).The anatomist von Haller first describedthe carotid body in 1743; its function, however,was unknown at the time. Histological54

Jasminka Alagić-Smailbegović et al.: Paragangliomas of the Neckstudies of the carotid body revealed glandularacini, and so the carotid body was renamedthe carotid gland. Von Luschkafirstdescribed a tumor of the carotid body in1862. In 1880, Reigner performed the firstresection of a carotid body tumor, but thepatient did not survive. Six years later, Maydlresected a carotid body tumor; the patientsurvived but had postoperative hemiplegiaand aphasia (1).Tumors originating in glomus cells of themiddle ear are called glomus tympanicumtumors. Though rare, these are the mostFigure 1. Middle ear paraganglioma CT scancommon tumors of the middle ear. Carotidbody glomus tumors arise at the place inthe neck where the carotid artery branches.Glomus tumors may also arise in deep neckspace along the course of the vagus nerve andthese are called glomus vagale tumors. Theyusually present as a slow growing painlessneck mass located at the anterior border ofthe sternocleidomastoid muscle just lateralto the tip of the hyoid bone. They may bulgeinto the pharynx or extend upward into theneck (parapharyngeal area). Patients usuallyhave noted the mass for several years, onaverage of 2-8 years. The patient may presentwith hoarseness, vocal cord paralysis, ordysphagia(1).Multicentric paragangliomas occur inapproximately 10% overall. About 20% ofparagangliomas are hereditary. If there is afamily history of paragangliomas, the tendencyfor multicentricity is much higher -about 40-50%. The causes of solitary glomustumors are not fully understood though theyare believed to result from an over-responseto change in body homeostasis. There issome evidence for a link between glomus tumorsand oxygen deprivation. Cases involvingmultiple tumors are the result of a seriesof inherited genetic mutations. Risk factorsinclude: female sex (for glomus vagale ratio55

Folia Medica 2011; 46 No 1:54-59of affected females to males is 3:1 and forglomus jugulare 5:1), long term exposureto high altitudes, family history of multipleand/or carotid body glomus tumors. Carotidparagangliomas can occur at any age butthe mean patient age is 45-50 years (1).EtiologyFigure 2. Right-sided glomus caroticum tumor in thesame patientThe etiology of paragangliomas appearsto be multifactorial. Most paragangliomasare solitary head and neck paragangliomasare recognized to occur in two forms: sporadicand familial forms (3). It is estimatedthat the familial incidence of head and neckparagangliomas is approximately 5%. If sucha history is present, there is a 78–87% possibilityof multiple tumors (3). In additionto these associations, a syndrome of familialparagangliomas characterized by multipleparagangliomas, especially in the head andneck region, has been described and occursin approximately 10% of cases, most commonlyas bilateral carotid body tumors.Malignant paragangliomas have been reportedin 6% of carotid body paragangliomasby Batsakis (1).Clinical presentation and diagnosisFigure 3. Left-sided glomus caroticum tumorin the same patientThe characteristic feature of carotid bodytumors is slow growth rate, which is reflectedclinically by the delay between the firstsymptoms and the diagnosis, averaging between4 and 7 years.A carotid body tumor usually presentsas a lateral cervical mass, which is mobilelaterally but less mobile in the craniocaudaldirection because of its adherence to the carotidarteries. This physical finding has beencalled a positive Fontaine sign (1).Patients with jugular paragangliomasmay present with pulsatile tinnitus, hearingloss, or with cranial neuropathies of cranialnerves IX, X, XI, or XII. Otoscopic examinationmay demonstrate a vascular mass in themiddle ear.56

Jasminka Alagić-Smailbegović et al.: Paragangliomas of the NeckImaging studiesThere are various diagnostic imaging modalitiesavailable in the evaluation of carotidbody tumors. Noninvasive duplex ultrasonographydemonstrates a hypervascularmass and the tumor’s relationship to the carotidartery. Ultrasonography may also delineateany intrinsic carotid artery disease.CT scanning with intravenous contrastdemonstrates a hypervascular mass at thecarotid bifurcation, splaying the internaland external carotid arteries. CT angiographymay be performed to demonstrate therelationship of the carotid vessels to the enhancingneck mass.MRI may be the most useful imagingstudy for evaluating carotid body tumorsbecause it offers triplanar views and superiorsoft tissue contrast without the needfor ionizing radiation, compared with CTscanning (4). Carotid angiography has beenreplaced by MR angiography, but carotidangiography is useful when preoperativeembolization is necessary. In the middle ear,most glomus tumors are readily noted byphysical examination, appearing as reddishbluemass behind the eardrum.ManagementFigure 4. Glomus jugulare – intraoperative viewTreatment options for paragangliomas include1) observation, or “watchful waiting”,where the patient has CT and/or MRI scansperiodically to monitor the growth of thetumor, 2) radiation therapy, or 3) surgicalremoval (6). Surgery remains the mainstayof treatment for carotid body tumors. However,controversy persists in certain cases,particularly as it relates to multicentric tumorsor for patients with advanced diseaseor significant comorbidity.Treatment options:– Surgical treatment– Method of choice in the younger patientswith a good ability for a compensationof possible neurologic disorders.– Preoperative embolization of theblood vessels that supply tumorproves as very useful at jugular at vagalparagangliomas; surgery is performedwithin seven days.– Risks include somewhat higher incidenceof facial nerve paralysis as wellas some other cranial nerves.– Irradiation– Indirect effect that causes vasculitisleading to blood vessels obliteration.– Inhibits tumor growth.– Gama-knife or LINAC.– Rare complications.– Shortfalls are the absence of direct effecton tumor cells and questionablelong-term results.– Wait and scan– Justified at slow growing tumors andin the elderly patients.– Asymptomatic patient.– Shortfalls include irreversible neuralparalysis, possibility of malignant alteration(7).57

Folia Medica 2011; 46 No 1:54-59Table 1. Glasscock-Jackson classification of glomus tumorsGlomus TympanicumType I — Small mass limited to the promontoryType II — Tumor completely filling the middle earType III — Tumor filling the middle ear andextending into the mastoidType IV — Tumor filling the middle ear, extendinginto the mastoid or through the tympanicmembrane to fill the external auditory canal; mayextend anterior to the internal carotid arteryGlomus JugulareType I — Small tumor involving the jugular bulb,middle ear, and mastoidType II — Tumor extending under the internalauditory canal; may have intracranial extensionType III — Tumor extending into the petrous apex;may have intracranial extensionType IV — Tumor extending beyond the petrousapex into the clivus or infratemporal fossa; mayhave intracranial extensionDiscussionIn a report from Memorial by Lack andothers 39 of 43 patients with carotid bodytumors were treated surgically; one patientreceived definitive radiation therapy, andthree others were observed but not treated.In this cohort of patients, 24 of 39 patientswere free of disease after surgery, at an averagefollow-up interval of 12 years (6 monthsto 38 years) (8). Local recurrence occurredin 4 of 39 patients (10%). Regional or distantmetastases occurred in four of 39 patients(10%). All 4 of these patients were dead ofdisease within 6 years. If the tumor is completelyremoved, the recurrence rate is about10%. McCaffrey found that carotid paragangliomasthat measure greater than 5 cmcarry an operative complication rate of 67%compared with those less than 5 cm whichcarry a risk of 14% (6).Preoperative considerations should includeage, the medical condition of thepatient, and the size of the tumor and themulticentricity of the tumor. Tumor size is avery important indicator of operative morbidity.If the patient elects for surgical therapy,he or she should be properly informed ofall risks. These historically have included anoverall operative mortality of approximately2-8%, a stroke rate of up to 20% and a cranialnerve damage (mainly CN X and XII)rate of 40-50%.In the patient with bilateral paraganglioma,every effort must be made to save at leastone vagus nerve and its laryngeal branches.Should a patient present with bilateral carotidbody tumors, the case may be madeto resects the smaller carotid body tumorfirst, with the intent of preserving the vagusand hypoglossal nerves on the less challengingside, before embarking on resecting thelarger tumor of the other side. At a plannedsecond stage, resection of the larger carotidbody tumor can be undertaken.Therapeutic embolization is the othertreatment option (9), however, Myers andJohnson do not recommend preoperativeembolization of carotid body tumors becausethese lesions can be dissected free ofcarotid artery without major blood loss (10).The incidence of permanent cranialnerve impairment as a complication of surgeryhas been reported in the literature tooccur in approximately 20% of cases. In thisreport by Lack and colleagues, cranial nerve58

Jasminka Alagić-Smailbegović et al.: Paragangliomas of the Necksacrifice of the vagus or hypoglossal nervewas necessary in 15% (6 of 39) of patients.An additional patient developed Horner’ssyndrome postoperatively. Although notquantified, the superior laryngeal nerve,supplying sensory innervation to the larynxand motor innervation to the cricothyroidmuscle, may be the nerve most frequentlyinjured during carotid body resection. Paralysisof one superior laryngeal nerve canresult in some degree of aspiration, althoughisolated palsy of one superior laryngeal nervetypically requires no additional rehabilitation.Furthermore, denervation of one cricothyroidmuscle can result in pitch changesin singers, but changes in voice may not beperceptible. Injury to the cervical sympatheticchain will result in Horner’s syndrome,with ipsilateral ptosis, miosis, and anhydrosis.Netterville and others have describedfirst bite syndrome resulting from injury tothe cervical sympathetic chain, causing lossof sympathetic input to the parotid gland.Another recently described and rarely recognizedproblem after bilateral carotid bodyexcision is baroreflex failure syndrome (8).Carotid body tumors were originallythought not to be radiosensitive because theeffects of radiation therapy were cytostaticbut not cytotoxic. Radiation therapy for paragangliomaswill arrest growth but not shrinktumor size. Furthermore, potential tumor regrowthafter radiation therapy remains a possibility.Risks associated with radiation therapyfor paragangliomas include carotid atherosclerosis,radiation-induced malignancy,and associated morbidity, including mucositis.RT has been reported to provide excellentcontrol for unresectable lesions and to be anadjunct to operation for incompletely excisedtumors or metastases. Furthermore, there isno advantage of subtotal resection along withradiotherapy compared with radiotherapyalone. Therefore, the decision to use radiotherapyor surgery is based primarily on therisk of treatment complications (11).ConclusionThe keys to successful surgery are carefulpreoperative planning, proximal and distalcontrol of the vasculature with vessel loops,careful identification and preservation ofneural structures, such as the vagus nerveand hypoglossal nerve, and dissection in theperiadventitial plane with meticulous attentionto hemostasis.Radiotherapy without surgery should beconsidered in the patients with unresectablelesions or relapse.References1. Cummings C.W.: Benign neoplasms of the neck.Otolaryngology—Head & Neck Surgery. Mosby:2005.2. Heath D., Edwards C., Harris P.: Post-mortem sizeand structure of the human carotid body. Thorax1970; 25:129-140.3. Sobol S.M., Bailey J.C.: Familial multiple cervicalparagangliomas:report of a hundred and reviewof the literature. Otolaryngol Head Neck Surg1990; 102: 382–390.4. Kaman L., Singh R., Aggarwal R., Kumar R.,BeheraA., Katariya R.N.: Diagnostic and therapeutic approachesto carotid body tumors:report of threecases and review of the literature. Aust N Z J Surg1999; 69: 852–855.5. Netterville J.L.: Carotid body tumors: a reviewof 30 patients with 46 tumors. Laryngoscope1995; 105:115-126.and others6. http://www.american-hearing.org/disorders/tumors/glomus_tumor.html7. Katić V., Prgomet D. Et all.:Otorinolaringologijai kirurgija glave i vrata.Naklada Ljevak. Zagreb,2009:91-928. Lack E.E.: Paragangliomas of the head and neckregion: a clinical study of 69 patients. Cancer1977; 39:397-409.9. Dimakakos P.B., Kotsis T.E.: Carotid bodparaganglioma:review and surgical management.10. Eur J Plast Surg 2001; 24: 58–65.11. Myers E.N., Johnson J.T.: Neoplasms; in CummingsCW, Fredricson JM, Harker LA,12. Krause CJ, Richardson MA, Schuller DE(eds):Otolaryngology – Head and Neck Surgery, ed 2. StLouis, Mosby-Year Book, 1993, 1590–1604.13. Isik A., Imamoglu M, Erem C, Sari A: Paragangliomasof the Head and Neck. Med Princ Pract2007;16:209–21459

Review PapersRecommendations for Treatment of Active and LatentTuberculosis Before and During The Tumor Necrosis FactorAntagonists Therapy in Bosnia and HerzegovinaHasan Žutić 1 , Nenad Prodanović 2 , Šekib Sokolović 3 , Mladen Duronjić 4 ,Suada Mulić Bačić 5 , Nađa Borovac 6 , Nedžad Mulabegović 71Clinic of Lung Diseases and Tuberculosis,Clinical Center, University of Sarajevo,Bardakčije 90, 71000 Sarajevo2Department of Rheumatology and ClinicalImmunology, Clinical Center of Banja Luka,Ul. 12 beba bb, 78000 Banja Luka3Clinic of Heart and Rheumatic diseases,Clinical Center, University of Sarajevo,Bolnička 25, 71000 Sarajevo4Lung Clinic, Clinical Center of Banja Luka,Zdrave Korde 1, 78000 Banja Luka5Department of Rheumatology, Clinic for Internalmedicine, University Clinical Center,Trnovac bb, 75000 Tuzla6Clinic of Gastroenterohepatology,Clinical Center, University of Sarajevo,Bolnička 25, 71000 Sarajevo7Faculty of Medicine, University of Sarajevo,Čekaluša 90, 71000 SarajevoPatients with autoimmune diseases such as rheumatoid arthritis,ankylosing spondylitis, psoriatic arthritis, juvenileidiopathic arthritis, Crohn disease, ulcerative colitis and psoriasis,during the administration of anti-TNF (tumor necrosisfactor alpha) medication are at an increased risk of developingsevere infections, including tuberculosis. This is a consequenceof their immunocompromised status caused by theprimary disease and previous treatment with immunosuppressivedrugs. To reduce the risk of tuberculosis, all patientsshould undergo tests to diagnose this disease prior to the administrationof anti-TNF therapy. The experiences from thecountries that have introduced the recommendations for thediagnosis of tuberculosis over the previous several years haveshown a significant reduction in the incidence of this diseasein patients treated with anti-TNFα drugs. In the detection ofMycobacterium tuberculosis, IGRA (Interferon-Gamma ReleaseAssay) test is used. Alternatively, PPD (purified proteinderivative) skin test can be used. Induration larger than 5 mmin diameter is considered a positive PPD test. In case that thefirst PPD result is negative, it is advisable to repeat PPD testonce again after 1 to 3 weeks. Diagnosed active tuberculosisis a contraindication for anti-TNFα medications use. Such patientsshould be referred to treatment, and six months aftercompletion of such treatment, the possibilities to introducethe aforementioned therapy may be considered. Patients diagnosedwith latent tuberculosis infection can not immediatelystart treatment with anti-TNFα, but must be referred to theuse of chemoprophylaxis against tuberculosis. Isoniazid chemoprophylaxisis indicated, 5 mg / kg of body weight daily for6 months. Anti-TNF therapy can be started no earlier than60 days after starting chemoprophylaxis. The first control ontuberculosis during administering the anti-TNFα medicationsis recommended six months after starting the treatment andsubsequently, on an annual basis.Keywords: Tuberculosis, treatment, tumor necrosis factor(TNF), IGRA test, PPD test60

Hasan Žutić et al.: Recommendations For Treatment of Active and Latent Tuberculosis Before...Introduction – tumor necrosis factor(TNF)Tumor Necrosis Factor (TNF) was isolatedin mid-80’s of the 20 th century and wasoriginally described as a protein with theability to cause death of tumor cells in vitro,i.e. hemorrhagic necrosis of transplantabletumors in mice (Old, 1985; Bazzoni andBeutler, 1996). The serum protein called ‘cachectin’that was isolated in rabbits chronicallyinfected with Trypanosoma Brucei waslater proved to have the same structure asTNF (Beutler, 1989). The term ’TNF’ refersto two very close cytokines: TNFα i TNFβ(also called lymphotoxin-α). Both interactwith the same receptors and trigger a multitudeof reactions and are therefore, implicitlyidentified as mediators in various pathologicalconditions in humans (Papadakisand Targan, 2000, Tracey and Cerami, 1994,Sacco et al. 1998).The TNF biological effects are manifestedthrough anti-tumor activity, antiviralactivity and mediation of shock and cachexia.It is a pluripotent pro-inflammatorycytokine primarily produced by activatedmonocytes / macrophages in response todifferent stimuli, including lipopolysaccharide,viral infections, Gram negative andGram positive pathogens (Papadakis andTagran, 2000; Giacomini et al. 2001). TNFαalso may be manifested through activatedT and B lymphocytes, killer cells, as well assome tumor cells (Papadakis and Tagran,2000). There are two forms of TNF: solubleand transmembrane. Response to TNFα ismediated through two receptors (TNFR1and TNFR2) on the surface of many types ofcells to which TNF binds as a ligand (Traceyet al. 2008). Stimulation of TNFR1 (55 kDa,p55) causes apoptosis and proliferation,while TNFR2 (75 kDa, p75) causes only theproliferation and appears to be less potentreceptor (Sprang, 2003, Jacobs et al. 2000).Tests in animals have shown that TNFR1 isimportant in granuloma formation duringM. tuberculosis infection (Bean et al. 1999)and susceptibility to intracellular pathogens(Pfeffer et al. 1993). Because of its high toxicitydemonstrated in animals and in humans,TNF did not meet early expectations that itcould be used in the treatment of malignantdiseases (Bazzoni and Beutler, 1996). However,over time the increasing evidence confirmedthat excessive or disproportionateTNF creation has been responsible for thepathogenesis of many chronic inflammatoryautoimmune diseases such as rheumatoidarthritis (RA), ankylosing spondylitis (AS),psoriatic arthritis (PsA), Crohn’s disease(CD ) ulcerative colitis, uveitis and psoriasis(Valesini et al. 2007). Tumor necrosis factoris the most important pro-inflammatory cytokinein the biology of rheumatoid arthritis(RA) and inflammatory bowel disease. Thephysiological role of TNFα is the regulationof immune responses, while in inflammatorydiseases it is responsible for flaring upand maintaining inflammation: it causesapoptosis, cell proliferation and differentiationand sustains inflammation.The biotechnological development hasenabled the creation of TNFα inhibitors thathave changed the outcome of the treatmentof autoimmune inflammatory diseases. Sofar, few therapeutic agents have been developedfrom this group; presently (July 2011)in Bosnia and Herzegovina, two TNFα antagonistsare used and those are the onesthat have been successfully applied in thetreatment of RA and other systemic autoimmunediseases throughout the world for almosta decade now. Adalimumab and infliximabare monoclonal antibodies that inhibitthe soluble and transmembrane TNFα bybinding to TNFα and blocking its biologicalfunctions. Infliximab is a chimeric mouse &human monoclonal antibody, while adalimumabis fully a human one. The difference isin the application as well: Infliximab is administratedintravenously and adalimumab61

Folia Medica 2011; 46 No 1:60-72subcutaneously. They are equally effectivein RA treatment (Kristensen et al. 2007;Alonso-Ruiz et al. 2008). Binding antibodies(infliximab, adalimumab) to transmembraneTNF triggers the complement cascadeand cell apoptosis. The complement cascadeinduces the release of intracellular microorganisms(Scallon et al. 1995), and the apoptosisof cells destroys most of the intracellularbacteria (Lugering et al. 2006). Thisleads to reduced levels of C-reactive protein(CRP), erythrocyte sedimentation rate andlevels of cytokines in serum. TNFβ or lymphotoxinis responsible for local granulomaformation and its neutralization can causedelayed granulomatous response and exposureto infection (Harasho et al. 2001).These medications, the so-called blockers,inhibitors, or TNFα antagonists, have asignificant role in the modern treatment ofautoimmune diseases because they lead tosignificant clinical response and halt diseaseprogression, which makes them superiorto other existing types of therapy (Lin et al.2008). With regard to rheumatic diseases,TNF inhibitor therapy ensures maintainingthe functionality and mobility of joints, thusenabling the patients to freely carry out theirdaily activities without assistance.TB infectionOne-third of Earth’s population is infectedwith M. tuberculosis. Each year, new 8 millionpatients are diagnosed with tuberculosisand about 3 million die (WHO, 2010). Evennowadays tuberculosis belongs to the groupof 10 leading diseases in the world. After encounteringan infected patient, tuberculosisinfection occurs in 30% of cases. Out of thetotal number of infected persons, 5-10% willdevelop manifest disease; half of them willdevelop it within two years; and the otherhalf sometime later in life. Whether the latentinfection will develop and become themanifest disease or not depends on the sizeof the infectious dose of tubercle bacilli andthe defense mechanisms of infected persons.Bosnia and Herzegovina belongs to thegroup of countries with medium-high incidenceof tuberculosis. The number of newTB cases in the Federation of Bosnia andHerzegovina was 1080 in 2009 (FBiH PublicHealth Institute, 2009), i.e. the incidenceis about 46.5 per 100,000 population, andin Republika Srpska, there were 479 newcases, i.e. about 35 per 100,000 population(RS Institute for the Protection of Health,2008). In 1993, the World Health Organization(WHO) created a new tuberculosiscontrol strategy, called DOTS (DirectlyObserved Therapy Short Course) strategy(World Health Organization, 1993). Thisstrategy is extremely important in the preventionof MDR-TB (Multi-drug resistanttuberculosis), as well as new forms that areeven more resistant - XDR-TB (Extensively/Extreme drug-resistant tuberculosis), beingresistant not only to the two main anti-tuberculosisdrugs (isoniazid and rifampicin),but also to at least one of parenteral drugsand to some of the fluoroquinolone drugs(Futin, 2007). It is the risk-bearing behavior,which has led to a rapid spread of AcquiredImmunodeficiency Syndrome (AIDS), diseasesof modern civilization (diabetes, cancer,kidney failure, autoimmune disease)and finally a number of chronic diseases thatrequire immunosuppressive therapy (rheumaticdisease, inflammatory bowel disease,interstitial lung disease, psoriasis, etc.) thatput tuberculosis in the focus of the world’sinterest as a significant global public healththreat that requires reaction by mobilizingall resources.Anti-TNF therapy and tuberculosisIn patients with autoimmune diseases thereis an increased risk of infection due to theirimmunocompromised condition that occursas a consequence of autoimmune62

Hasan Žutić et al.: Recommendations For Treatment of Active and Latent Tuberculosis Before...diseases and the use of immunosuppressivedrugs such as corticosteroids and anti-rheumaticdrugs that modify the course of thedisease (disease modifying antirheumaticdrugs – DMARDs), which include methotrexate,azathioprine, leflunomide, cyclophosphamideand cyclosporine (Mandic etal. 2009; Patkar et al. 2008; Winthrop, 2006).Any immunosuppressive drug, and thus theTNF inhibitors, can lead to reactivation oflatent tuberculosis infection or progressionof a recently acquired mycobacterial infectionto active tuberculosis. The developmentof tuberculosis, being one of the unintendedconsequences that may occur during thetreatment is indicated in the instructions onthe application of these drugs. TNF plays animportant role in the protection against infectionwith M. tuberculosis. In vitro and invivo studies have shown that TNFα providesprotective mechanisms against infectionwith M. tuberculosis in macrophages (Flynnet al. 1995). Furthermore, the studies haveshown that the absence of TNFα has a detrimentalimpact on the ability to form andmaintain granuloma, i.e. to stop the replicationof tubercle bacilli. In tuberculosis infection,TNFα increases the phagocytic abilityof macrophage to neutralize mycobacteriaand participates in the formation of granulomas,which sequester the affected tissueand constrain the spread of tubercle bacilli.Granuloma formation, which consists ofmacrophages and lymphocytes, is essentiallyimportant for the control of tuberculosis infection.In a series of complex mechanisms,TNFα inhibitors may lead to impaired homeostasisof tuberculous granuloma and activationof latent tuberculosis (Ehlers, 2005,Jacobs et al. 2007; Hernandez et al. 2008).Active tuberculosis is an absolute contraindicationto the use of anti- TNFα drugs.In latent tuberculosis infection the patientmust first start chemoprophylaxis, followedby a treatment with anti-TNFα medications.Failure to perform screening for tuberculosisprior to the application of TNF inhibitorscan have serious consequences both to thehealth of the patient and to the health of thegeneral population (Hernandez et al. 2008).In patients on anti- TNFα therapy, the incidenceof tuberculosis is higher by severaltimes, and it is considered to be 449 per100,000 patients (Popovic-Grle and Babic-Naglic, 2008). There is a higher proportionof extra-pulmonary forms of tuberculosisthat occur during the treatment with anti-TNFα (over 50% of cases) and those that aredisseminated (in about 25% of cases). Themost critical period for reactivation of latentTB is the first three months of applicationof monoclonal antibodies. The bioavailabilityof TNFα is the main protective factor inthe treatment with anti-TNF drugs becausethe infection control requires at least smallquantities of TNF. The correct evaluation ofpatients/candidates for anti-TNF therapy iscrucial for safe treatment. In the pre-treatmentpreparation of patients, it is importantto define the potential for reactivationof latent TB, and potential sources of newinfection, to implement preventive anti-tuberculosistherapy in vulnerable patients, todetermine the optimal timing for the introductionof anti-TNF therapy and possibly tomodify the dose of TNF inhibitors (Alonso-Ruiz et al. 2008).Tuberculosis diagnosis and anti-TNFtherapyTo avoid the application of anti-TNFα drugsin patients with latent tuberculosis infection,it is necessary to take the appropriatemeasures for its detection. Both in countrieswith high and in countries with low incidenceof tuberculosis, there are guidelinesfor the diagnosis of tuberculosis before theapplication of anti-TNFα therapy in theform of recommendations, guides or guidelinesthat have been developed by medicalorganizations or groups of experts, whose63

Hasan Žutić et al.: Recommendations For Treatment of Active and Latent Tuberculosis Before...the tuberculin reactivity may weakenover time. The repeated test would revealthat there are more people withlatent tuberculosis (reduced numberof false negative tests).– BCG vaccination at birth is not relevantfor the interpretation of PPDtest results (Wang et al. 2002); it isknown that PPD response to BCGvaccine weakens over time, so thatafter only 15 years of vaccine administration,it becomes insignificant. InBosnia and Herzegovina, it is regulatedby law that the BCG vaccine is administeredonly at birth. Therefore, inthis case, BCG vaccine administeredat birth is likely to be irrelevant forthe interpretation of PPD test resultsin patients older than 18 years of age.Diagnosis of active tuberculosis inadult patients prior to treatment withTNFα inhibitorsThe algorithm for the diagnosis of active tuberculosis(Scheme 1) shows the handlingprocedures for each patient in the diagnosticphase, before the application of anti-TNFαdrug. IGRA or PPD test are not necessaryfor the diagnosis of active tuberculosis. Inthe case of confirmed diagnosis of this disease,the application of anti-TNFα therapyis contraindicated, and the patient shouldbe referred to the treatment of tuberculosisin a reference health-care center. Only sixmonths after the end of that treatment, theintroduction of anti-TNFα therapy may beconsidered.Application of Interferon-Gamma ReleaseAssay (IGRA) And PPD Tests in TheDiagnosis Of Active/Latent Tuberculosis InfectionIGRA is a laboratory blood test that isused in the diagnosis of latent and active tuberculosis.It measures the patient’s immunereactivity to M. tuberculosis. Leukocytesof majority of the patients infected withM. tuberculosis release interferon-gamma(IFN-γ) when combined with M. tuberculosisantigens. In order to carry out the test,a blood sample is mixed with the antigens,i.e. control antigen. There are several differentIGRA tests (QuantiFERON® - TB Gold,QuantiFERON® - TB Gold In-Tube; T-SPOT® .TB, among others) and the antigens, testingmethods and criteria for interpretationof the test vary to some extent.The advantagesof IGRA test in relation to the PPD testare as follows:– It requires only one visit of the patient;– BCG vaccination cannot cause falsepositive results of IGRA test;– The test result is available within 24hours;– It does not cause the so-called “boosterphenomenon”, i.e. it does not increasethe possibility that any subsequenttests become positive if theywere initially negative;– There is less subjectivity in readingcompared with the PPD test;The IGRA test has also certain disadvantages:– The blood sample must be processedwithin a period of 8 to 16 hours aftertaking;– Some factors may reduce the accuracyof testing (such as errors whentaking a blood sample, errors made intransport, or in carrying out and interpretationof the test);– Limited data in terms of predictiverole of IGRA for any possible developmentof tuberculosis infection inthe future;– Limited data for specific populations(children younger than 5 years ofage, patients recently exposed to M.tuberculosis, immunocompromisedpatients, serial testing);– Relatively high cost of the test;65

Folia Medica 2011; 46 No 1:60-72Scheme 1: Algorithm for the diagnosis of active tuberculosisGenerally, the IGRA test can be recommendedfor patients who are expected tohave poor turnout on the retest and thosewho were vaccinated with BCG vaccine. Onthe other hand, PPD is recommended forchildren younger than 5 years of age due tolimited data on the reliability of the IGRAtest at this age. For the rest of the population,either one or the other test (IGRA or PPD)can be used without special preferences.In any case, the routine use of both testsin the same patient is not recommended.However, the results of both tests can be usefulin certain specific situations such as– The initial test is negative and thereis a high risk of infection, diseaseprogression or poor outcome (HIVinfected persons, children under 5years of age exposed to a person withinfectious TB);– The initial test is negative but there isclinical suspicion of infection (symptoms,radiological test results, etc.)66

Hasan Žutić et al.: Recommendations For Treatment of Active and Latent Tuberculosis Before...Scheme 2: Algorithm for the diagnosis of latent tuberculosis– The initial test is positive but furtherevidence is required to confirm theinfection, i.e. to encourage patient’sacceptance and adherence to thetherapy (e.g. patients who believe thattheir PPD test is positive due to BCGvaccination);– The initial test is positive in a personwith low risk of tuberculosis infectionor disease progression;– If the initial IGRA test is ambiguousor borderline, but the reasons for performingthe testing persist.67

Folia Medica 2011; 46 No 1:60-72The algorithm for the diagnosis of latenttuberculosis in patients prior to administrationof anti-TNFα therapy is given below.As an alternative to IGRA test, PPD testcan be used in the detection of M. tuberculosis.Induration larger than 5 mm after theinitial PPD test is considered a positive test.If the result of the first PPD test is negativei.e. if the induration is smaller than 5 mm,it is necessary to repeat this test after one tothree weeks. If this test is also negative, theresult is considered to be conclusively negative,i.e. the latent tuberculosis is excludedand the therapy with TNF inhibitors can beimmediately prescribed to the patient. If theresult of the repeated PPD test is positive,this means that there is a latent tuberculosisinfection. In that case, chemoprophylaxiscontrolled by a medical doctor in the referencehealth-care center for treating tuberculosisshould be prescribed to the patient.Six-month isoniazid chemoprophylaxisis the most frequently applied therapy.Already after 60 days from the commencementof the chemoprophylaxis, anti-TNFαtherapy can be introduced with caution.In addition to the positive personal history,the positive family history of tuberculosisis an important factor that must be takeninto account when assessing the patients.Risk factors, other than immunosuppressivetherapy, for activation of latent tuberculosisinfection are:– Birth or residence in endemic tuberculosisregion– Recent contact with persons sufferingfrom tuberculosis– An employee in a microbiologicallaboratory– High-risk persons enviroment– Diabetes– Silicosis– Organ transplantation– Chronic renal insufficiency– Gastrectomy or jejunal-ileal bypass– Malignant disease of the head orneck, leukemia, lymphoma– Immunosuppression due to otherreasons (primary or secondary immunodeficiency)The above can be summarized as follows:If at least one of the following is positive fora patient:– Personal / family history or– IGRA test results positive or– PPD test shows induration of ≥ 5 mmin diameter or– Lungs x-ray test results show signs oflatent tuberculosis infection,Table 1: Recommendations for latent tuberculosis treatmentIGRA / PPD testLungs x-raytestRecommendations for latent infection treatmentnegative / < 5 mm Normal Not recommended*positive / ≥ 5 mm Pathological Consultations with an expertnegative / < 5 mm PathologicalConsultations with an expert / If the microbiological test is negative,isoniazid chemoprophylaxis for 6 to 9 months is recommended. Anti-TNFtherapy can be administrated, with caution, starting 60 days after thecommencement of chemoprophylaxispositive / ≥ 5 mm NormalIf the microbiological test is negative, isoniazid chemoprophylaxis for 6 to9 months is recommended. Anti-TNF therapy can be administrated, withcaution, starting 60 days after the commencement of chemoprophylaxis*Consultations with an expert are, however, recommended in patients with negative test, who have risk factors for tuberculosisinfection68

Hasan Žutić et al.: Recommendations For Treatment of Active and Latent Tuberculosis Before...Schema 3: Algorithm for the tuberculosis control (prevention) during anti-TNF therapythe latent tuberculosis treatment should beadministered according to the recommendationsgiven in Table 1.Control (Prevention) Of TuberculosisDuring Treatment With TNFαInhibitorsAlgorithm of tuberculosis control (prevention)(Scheme 3) shows the recommendedprocedures during anti-TNFα therapy. Thefirst control on tuberculosis should be donesix months after the commencement of theanti-TNFα drug administration and thenafter a year as of the commencement of thetreatment. The checkup includes both radiographyand IGRA or PPD test, because inaddition to the lung tuberculosis, tuberculosisof other locales occurs as well in thesepatients in a much higher proportion thanin the general population (around 50%, asopposed to 15%). Treatment of the patientswith active tuberculosis and the chemoprophylaxisof the patients with latent tuberculosisinfection should be carried out by apneumophtisiologist, or a pulmonologist, inan institution specialized for the treatmentof this disease, and in accordance with theadopted professional and methodologicalguide. The diagnosis is also done in specializedhealth-care institutions.ConclusionAnti-TNFα medications have an importantrole as a modern biological therapy for certainautoimmune diseases. In patients withautoimmune diseases, there is an increasedrisk of developing serious infections, includingtuberculosis. It is therefore necessary toperform appropriate diagnostic proceduresaiming to detect active or latent tuberculosisbefore the administration anti-TNFα medications.In patients with active form of thisdisease the use of Anti-TNFα medications iscontraindicated, and they must be referredto treatment in an appropriate health-carecenter. Patients diagnosed with latent tuberculosisinfection should be prescribed chemoprophylaxis.Based on the experiences ofcountries worldwide where similar guidesor guidelines are in place, it is expected thatacting in accordance with the recommendationsfor the diagnosis of TB would significantlyreduce the risk of developing activetuberculosis in the patients in Bosnia andHerzegovina who are receiving anti-TNFαtherapy.69

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Preporuke za tretman aktivne i latentne tuberkuloze prije itokom terapije antagonistima faktora tumorske nekroze uBosni i HercegoviniHasan Žutić 1 , Nenad Prodanović 2 , Šekib Sokolović 3 , Mladen Duronjić 4 ,Suada Mulić Bačić 5 , Nađa Borovac 6 , Nedžad Mulabegović 71Klinika za plućne bolesti,Klinički centar univerziteta u Sarajevu,Bardakčije 90, 71000 Sarajevo2Odjeljenje za reumatologijui kliničku imunologiju, Klinički centar Banja Luka,Ul. 12 beba bb, 78000 Banja Luka3Klinika za bolesti srca i reumatizam,Klinički centar univerziteta u Sarajevu,Bolnička 25, 71000 Sarajevo4Klinika za plućne bolesti,Klinički centar Banja Luka,Zdrave Korde 1, 78000 Banja Luka5Odjeljenje za reumatologiju,Klinika za interne bolesti,Univerzitetski klinički centar,Trnovac bb, 75000 Tuzla6Klinika za gastroenterohepatologiju,Klinički centar univerziteta u Sarajevu,Bolnička 25, 71000 Sarajevo7Medicinski fakultet Univerziteta u Sarajevu,Čekaluša 90, 71000 SarajevoOsobe s autoimunim bolestima, kao što su reumatoidni artritis,ankilozirajući spondilitis, psorijatički artritis, juvenilniidiopatski artritis, Kronova (Crohn) bolest, ulcerozni kolitisi psorijaza, tokom primjene anti-TNFα (engl. tumor necrosisfactor alpha) lijekova su pod povećanim rizikom od pojaveteških infekcija, uključujući i tuberkulozu. To je posljedicanjihovog imunokompromitovanog stanja usljed primarnebolesti i prethodnog liječenja imunosupresivnim lijekovima.Da bi se smanjio rizik od pojave tuberkuloze, sve bolesnikebi prije primjene anti-TNFα terapije trebalo podvrgnuti pretragamaza dijagnozu ovog oboljenja. Iskustva iz zemalja ukojima su prethodnih nekoliko godina uvedene preporukeza dijagnostiku tuberkuloze pokazala su značajno smanjenjepojave ove bolesti kod osoba liječenih anti-TNFα lijekovima.U detekciji Mycobacterium tuberculosis koristi se IGRA (Interferon-GammaRelease Assay) test. Kao alternativa, možese koristiti PPD (engl. purified protein derivate) kožni test.Pozitivnim PPD testom smatra se induracija prečnika većegod 5 mm. U slučaju negativnog prvog PPD nalaza, uputno jenakon 1 do 3 sedmice ponoviti PPD test još jednom. Dijagnostikovanaaktivna tuberkuloza je kontraindikacija za primjenuanti-TNFα lijekova i takve bolesnike treba uputiti naliječenje, a šest mjeseci po završenom liječenju mogu se razmotritimogućnosti uvođenja pomenute terapije. Bolesnici sadijagnostikovanom latentnom tuberkuloznom infekcijom nemogu odmah započeti liječenje sa anti-TNFα, već moraju bitiupućeni na primjenu hemoprofilakse tuberkuloze. Indiciranaje hemoprofilaksa izonijazidom, 5 mg/ kg tjelesne težine svakodnevnokroz 6 mjeseci. Anti-TNF terapija može se započetinajranije 60 dana po otpočinjanju hemoprofilakse. Prvakontrola na tuberkulozu tokom primjene anti-TNFα lijekovapreporučuje se šest mjeseci od početka liječenja, a zatim nasvakih godinu dana.Ključne riječi: tuberkuloza, tretman, faktor nekroze tumora(TNF), IGRA test, PPD test73

Hasan Žutić et al.: Preporuke za tretman aktivne i latentne tuberkuloze prije i tokom terapije...Kaskada komplementa inducira oslobađanjeintracelularnih mikrooorganizama (Scalloni sar. 1995), a apoptozom ćelija uništava se iveći dio intracelularnih bakterija (Lugering isar. 2006). To dovodi do smanjenja nivoa C–reaktivnog proteina (CRP), brzine sedimentacijeeritrocita i nivoa citokina u serumu.TNFβ ili limfotoksin je odgovoran za lokalnostvaranje granuloma i njegova neutralizacijamože izazvati odgođen granulomskiodgovor organizma te izloženost infekciji(Harashima i sar. 2001). Ovi lijekovi – tzv.blokatori, inhibitori ili antagonisti TNFα,imaju značajno mjesto u savremenom liječenjuautoimunih bolesti jer dovode do značajnogkliničkog odgovora i zaustavljaju napredovanjeoboljenja što ih čini superiornimu odnosu na drugu postojeću terapiju (Lin isar. 2008). Kada su u pitanju reumatske bolesti,terapija TNF inhibitorima obezbjeđujeodržavanje funkcionalnosti i pokretljivostizglobova, omogućavajući pacijentima danesmetano obavljaju svakodnevne aktivnostibez tuđe pomoći.TBC infekcija1/3 stanovništva Zemlje je zaražena bacilomtuberkuloze. Svake godine oboli novih8 miliona bolesnika od tuberkuloze, a oko 3milijuna umire (WHO, 2010). I u današnjevrijeme tuberkuloza pripada krugu od 10vodećih oboljenja u svijetu. Nakon susretas infektivnim bolesnikom infekcija tuberkulozomnastaje u 30% slučajeva. Od ukupnogbroja inficiranih u 5–10 % će se razviti manifestnabolest, kod polovine njih unutar dvijegodine, a kod druge polovine nekad kasnijetokom života. Da li će se latentna infekcijapokrenuti i postati izražena bolest zavisi odveličine infektivne doze bacila tuberkulozete o odbrambenim mehanizmima inficiraneosobe. Bosna i Hercegovina pripada kruguzemalja sa srednje visokom incidencom tuberkuloze.Broj novih slučajeva tuberkulozeu Federaciji BiH iznosio je 1080 u 2009.godini (ZZJZ FBiH, 2009) tj incidenca jeoko 46,5 na 100.000 stanovnika, a u RepubliciSrpskoj zabilježeno je 479 novooboljelihodnosno oko 35 na 100.000 stanovnika (Insitutza zaštitu zdravlja RS, 2008). Svjetskazdravstvena organizacija (SZO) je od 1993.godine kreirala novu strategiju kontrole tuberkuloze,nazvanu DOTS (Directly observedtherapy short course) strategija (WorldHealth Organization, 1993). Ova strategijaje iznimno važna u sprječavanju multirezistentnihbacila tuberkuloze (MDR–TB Multidrug-resistant tuberculosis), kao i novih jošotpornijih oblika (eng. XDR-TB Extensively(ili Extreme) drug-resistant tuberculosis), rezistentnihne samo na oba glavna antituberkulotika(izonijazid i rifampicin), nego i nanajmanje jedan od parenteralnih lijekova tena neki od fluorokinolonskih lijekova (Futin,2007). Upravo rizična ponašanja, kojasu dovela do brzog širenja sindroma stečeneimunodeficijencije (AIDS), bolesti modernecivilizacije (dijabetes, maligne bolesti, zatajivanjebubrega, autoimune bolesti) i na krajuniz hroničnih bolesti koje zahtjevaju imunosupresivnuterapiju (reumatske bolesti,upalne bolesti crijeva, intersticijske bolestipluća, psorijaza, itd.) stavili su tuberkulozuu žarište svjetskog interesa kao značajnuglobalnu javnozdravstvenu prijetnju kojojtreba odgovoriti mobilizacijom svih resursa.ANTI–TNF terapija i tuberkulozaKod bolesnika sa autoimunim bolestima povećanje rizik od infekcije zbog imunokompromitovanogstanja koje nastaje kao posljedicaautoimune bolesti i primjene imunosupresivnihlijekova kao što su kortikosteroidii antireumatski lijekovi koji modificirajutok bolesti (engl. disease modifying antirheumaticdrugs – DMARDs), u koje se ubrajajumetotreksat, azatioprin, leflunomid,ciklofosfamid i ciklosporin (Mandić i sar.2009; Patkar i sar. 2008; Winthrop, 2006).Svaki imunosupresivni lijek, pa time i TNF75

Folia Medica 2011; 46 No 1:73-84inhibitori, mogu dovesti do reaktivacije latentnetuberkulozne infekcije ili progresijunedavno stečene infekcije mikobakterijemu aktivnu tuberkulozu. Razvoj tuberkuloze,kao jedna od neželjenih posljedica kojase može javiti tokom liječenja navedena je uuputstvima o primjeni pomenutih lijekova.TNF igra važnu ulogu u zaštiti od infekcijesa M. tuberculosis. In vitro i in vivo istraživanjapokazala su da TNFα osigurava protektivnemehanizme protiv infekcije sa M.tuberculosis u makrofagima (Flynn i sar.1995). Nadalje, studije su pokazale da odsustvoTNFα ima štetan uticaj na sposobnostformiranja i održavanja granuloma odnosnozaustavljanja replikacije tuberkuloznogbacila. U tuberkuloznoj infekciji TNFα povećavafagocitnu sposobnost makrofaga daneutralizira mikobakterije te učestvuje uformiranju granuloma, koji sekvestrirajuzahvaćeno tkivo i ograničavaju širenje bacilatuberkuloze. Stvaranje granuloma, kojise sastoji od makrofaga i limfocita, suštinskije značajno za kontrolu tuberkulozne infekcije.Inhibitori TNFα mogu nizom složenihmehanizama dovesti do narušanja homeostazetuberkuloznog granuloma i aktivacijelatentne tuberkuloze (Ehlers, 2005; Jacobs isar. 2007; Hernandez i sar. 2008). Aktivnatuberkuloza je apsolutna kontraindikacija zaprimjenu anti–TNFα lijekova. Kod latentnetuberkulozne infekcije bolesnik mora prvozapočeti hemoprofilaksu, a zatim liječenjeanti–TNFα lijekovima. Izostanak screeningana tuberkulozu prije početka primjeneTNF inhibitora može imati teške posljedicekako na zdravlje samog bolesnika, tako i pozdravlje opšte populacije (Hernandez i sar.2008). Kod bolesnika na anti–TNFa terapijiincidenca tuberkuloze povećana je nekolikoputa, te se smatra da iznosi 449 na 100.000bolesnika (Popović-Grle i Babić-Naglić,2008). Oblici tuberkuloze koji se javljaju tokomterapije anti–TNFa su u većem postotkuekstrapulmonalni (više od 50% slučajeva)i diseminirani (u oko 25%). Najkritičnijiperiod za reaktivaciju latentne TB su prva 3mjeseca primjene monoklonalnog antitijela.Bioraspoloživost TNFα je osnovni zaštitnifaktor tokom liječenja anti–TNF lijekovimajer su za kontrolu infekcije neophodnemakar i male količine TNF-a. Ispravna evaluacijabolesnika – kandidata za anti–TNFterapiju presudna je za sigurno liječenje. Upreterapijskoj pripremi bolesnika važno jedefinisati mogućnost reaktivacije latentneTBC i potencijalne izvore nove zaraze, provestipreventivnu antituberkulotsku terapijukod ugroženih bolesnika, odrediti optimalnovrijeme za uvođenje anti–TNF terapijei možda modificirati dozu TNF inhibitora(Alonso-Ruiz i sar. 2008).Dijagnostika tuberkulozei anti–TNF terapijaDa ne bi došlo do primjene anti–TNFα lijekovakod bolesnika sa latentnom tuberkuloznominfekcijom, neophodno je primijenitiodgovarajuće mjere radi njenog otkrivanja.Kako u zemljama visoke, tako i u zemljamaniske incidencije tuberkuloze, postojeuputstva za dijagnostiku tuberkuloze prijepočetka primjene anti–TNFα terapije u vidupreporuka, vodiča ili smjernica koje su pripremilemedicinske organizacije ili grupeeksperata, a čiji je cilj smanjenje rizika odrazvoja aktivne tuberkuloze (Mandić, et al.2009; CDC, 2006; Furst, et al. 2002; Doherty,et al. 2008; Elkayam, et al. 2007; Fonseca, etal. 2006; Koike, et al. 2007; Salmon-Ceron,2002; Popović-Grle and Babić-Naglić, 2008;Maritte, et al. 2002; Gomez-Reino, et al.2003). U velikom broju zemalja u kojima seprimjenjuju ove preporuke uočeno je značajnosmanjenje pojave tuberkuloze kod bolesnikana anti–TNFα terapiji. Prema analizirezultata iz registra BIOBADASER bolesnikaliječenih ovom terapijom u Španiji, stopapojave aktivne tuberkuloze kod bolesnikasa reumatoidnim artritisom smanjena je za83% (Carmona, et al. 2005).76

Hasan Žutić et al.: Preporuke za tretman aktivne i latentne tuberkuloze prije i tokom terapije...Izrada i primjena preporuka namećuse kao potreba i u zemljama u kojima preporukekao takve ne postoje. S obzirom načinjenicu da u Bosni i Hercegovini ne postojipisani vodič za dijagnostiku tuberkulozekod kandidata za anti–TNFα terapiju,a imajući u vidu objektivan rizik od pojaveovog oboljenja kod tih bolesnika, grupastručnjaka iz centara koji liječe reumatskebolesti biološkom terapijom (Klinički centarUniverziteta u Sarajevu, Klinički centarBanjaluka i Univerzitetski klinički centarTuzla) postigla je konsenzus za navedenelokalne preporuke. Naš konsenzus oslanjase na preporuke Centara za kontrolu i prevencijubolesti (Centers for Disease Controland Prevention – CDC) Guide for PrimaryHealth Care Providers: Targeting TuberculinTesting and Treatment of Latent TuberculosisInfection iz 2005. godine (CDC, 2005) ikonsenzus dokumenta sa Wolfheze konferencijeodržane 2008. godine pod nazivomTuberculosis contact investigation in lowprevalence countries (Wolfheze Conference,2008) pri čemu smo uvažavali lokalne okolnostii specifičnosti.Opšte preporuke za dijagnostikutuberkuloze prije liječenja TNFinhibitorima– Svi bolesnici koji su kandidati zaanti–TNFα terapiju moraju proći dijagnostikutuberkuloze (Perez i sar.2005) – u svim postojećim dostupnimvodičima (preporukama), bez obzirana to da li se anti–TNFα lijek koristiu okviru svakodnevne prakse ili u kliničkimstudijama, stav je da bolesnicikandidati za ovu vrstu terapije morajuproći dijagnostiku za tuberkulozu.– Definicija pozitivnog PPD testa: induracijaveća od 5 mm 72 sata poslijeprimjene PPD-a (Perez i sar. 2005;CDC, 2000; ATS, 2000) – obzirom načinjenicu da su bolesnici sa reumatoidnimartritisom već imunokompromitovanizbog prirode bolesti iranije primijenjene terapije koja djelujeimunosupresivno, te činjenicuda sama anti–TNF terapija povećavarizik od aktivacije latentne tuberkuloze,ovi bolesnici se svrstavaju u kategorijuvisokog rizika prema preporuciCDC za tumačenje rezultata PPD testa.Stoga je prihvaćeno da se induracija,nastala nakon intradermalneaplikacije 0.1 ml tuberkulina, očitananakon 72 sata, veća od 5 mm, smatrapozitivnim odgovorom na PPD testu.Još jednom napominjeno da se alternativnomože koristiti i IGRA test.– Bolesnicima koji su inicijalno postiglinegativan nalaz PPD testa trebaponoviti ovaj test (ATS, 2000; Gomez-Reinoi sar. 2007) – na osnovupreporuka CDC, ukoliko je rezultatprvog testa negativan, nakon jednedo tri sedmice trebalo bi ponoviti test.Cilj je otkriti lažno negativan rezultat,jer reaktivnost na tuberkulin vremenommože da oslabi. Ponovljeni testotkrio bi veći broj osoba sa latentnomtuberkulozom (smanjen broj lažnonegativnih testova).– BCG vakcinacija na rođenju nijerelevantna za interpretaciju rezultataPPD testa (Wang i sar. 2002) –poznato je da PPD odgovor na BCGvakcinu vremenom slabi, tako da većposlije 15 godina od primjene vakcinepostaje neznatan. U Bosni i Hercegovinije zakonom regulisano da seBCG vakcina obavezno daje samo narođenju. Stoga je u ovom slučaju zainterpretaciju rezultata PPD testa kodbolesnika starijih od 18 godina BCGvakcina dobijena na rođenju vjerovatnoirelevantna.77

Folia Medica 2011; 46 No 1:73-84Dijagnosticiranje aktivne tuberkulozekod odraslih bolesnika prije početkaliječenja inhibitorima TNFαAlgoritam za postavljanje dijagnoze aktivnetuberkuloze (shema 1) prikazuje postupkeobrade svakog bolesnika u okviru faze dijagnostike,prije početka primjene anti–TNFαlijeka. IGRA ili PPD test nisu neophodni zapostavljanje dijagnoze aktivne tuberkuloze.U slučaju potvrđene dijagnoze ovog oboljenja,kontraindicirana je primjena anti–TNFαterapije, a bolesnika treba uputiti na liječenjetuberkuloze u referentni zdravstveni centar.Tek šest mjeseci po završetku tog liječenjamože se razmotriti mogućnost uvođenjaanti–TNFα terapije.Primjena IGRA(eng. Interferon–Gamma ReleaseAssay) i PPD testa u dijagnozi aktivne/ latentne tuberkulozNe INFEKCIJEIGRA je laboratorijski test krvi koji se koristiu dijagnozi i latentne i aktivne tuberkuloze.Mjeri imunu reaktivnost pacijenta naM.tuberkuloze. Leukociti većine osoba inficiranihsa M.tuberkuloze otpuštaju interferongama (IFN-γ) kada se spoji sa antigenimaM.tuberkuloze. Da bi se test sproveo,uzorak krvi se miješa sa antigenima odnosnokontrolom. Postoji nekoliko različitih IGRAtestova (QuantiFERON® - TB Gold, Quanti-FERON® - TB Gold In-Tube; T-SPOT ® .TB,između ostalih) – antigeni, metode testiranjai kriteriji interpretacije testa se izmeđunjih donekle razlikuju.Prednosti IGRA testa u odnosu na PPDtest su :– Potrebna je samo jedna posjeta pacijenta– BCG vakcinacija ne može uzrokovatilažno pozitivne rezultate IGRA testa– Rezultat je dostupan u roku od 24 sata– Ne uzrokuje tzv ‘booster fenomen’ tjne povećava mogućnost da eventualnanaredna testiranja postanu pozitivnaako su prvobitno bila negativna– Manja je subjektivnost u očitavanjunego kod PPD testaIGRA test ima i određene nedostatke:– Uzorak krvi mora se obraditi najkasnijeu periodu od 8 do 16 sati nakonuzimanja– Pojedini faktori mogu umanjiti tačnosttestiranja (pogreške pri uzimanjuuzorka krvi, transporta, provođenjai interpretacije testa)– Limitirani su podaci u smislu prediktivneuloge IGRA za eventualni razvojinfekcije tuberkulozom u budućnosti– Limitirani podaci za pojedine populacije(djeca mlađa od 5 godina, pacijentinedavno izloženi m.tuberkuloze,imunokompromitirani pacijenti, serijskatestiranja)– Relativno visoka cijena testaGeneralno, IGRA se može preporučitikod pacijenata kod kojih se očekuje slab odzivna ponovno testiranje, te kod onih kojisu vakcinisani sa BCG vakcinom. S drugestrane, PPD se preporučuje kod djece mlađeod 5 godina zbog limitiranih podatakao pouzdanosti IGRA testa u ovom uzrastu.Za ostale populacije ili jedan ili drugi test(IGRA ili PPD) mogu biti korišteni bez posebnihpreferencija.– U svakom slučaju, rutinska primjenaoba testa kod istog pacijenta se nepreporučuje. Međutim, rezultati obatesta mogu biti od koristi u pojedinimspecifičnim situacijama kao što sunpr: Inicijalni test je negativan a postojivisoki rizik za infekciju, progresijuoboljenja ili loš ishod (HIV inficiraneosobe, djeca mlađa od 5 godinaizložena kontaktu sa tuberkuloznimbolesnikom)– Inicijalni test je negativan ali postojiklinička sumnja na infekciju (simptomi,radiološki nalaz, itd)78

Hasan Žutić et al.: Preporuke za tretman aktivne i latentne tuberkuloze prije i tokom terapije...Shema 1: Algoritam za postavljanje dijagnoze aktivne tuberkuloze– Inicijalni nalaz je pozitivan, ali jepotreban dodatni dokaz za potvrduinfekcije odnosno prihvatanje postojanjainfekcije kao i pridržavanje terapijeod strane pacijenta (npr osobekoje smatraju da je njihov PPD testpozitivan usljed BCG vakcinacije)– Inicijalni test je pozitivan kod osobesa malim rizikom od infekcije tuberkulozomili progresije oboljenja– Ako je incijalni IGRA test neodređen,graničan ali postoje argumenti da setestiranje obaviAlgoritam za dijagnostiku latentne tuberkulozekod bolesnika prije primjeneanti–TNFα terapije naveden je ispod.Kao alternativa IGRA testu, u detekcijiM.tuberkuloze može se koristiti PPD test.Induracija veća od 5 mm poslije inicijalnogPPD testa smatra se pozitivnim nalazom.Ako je rezultat prvog PPD testa negativan,tj. induracija manja od 5 mm, neophodnoje ponoviti ovaj test poslije jedne do tri sedmice.Ako je i ovaj nalaz negativan, rezultatse smatra zaista negativnim, odnosno isključenoje postojanje latentne tuberkuloze79

Folia Medica 2011; 46 No 1:73-84Shema 2: Algoritam za dijagnostiku latentne tuberkulozei bolesniku se može odmah propisati terapijaTNF inhibitorom. Ukoliko je rezultatponovljenog PPD testa pozitivan,znači da postoji latentna tuberkuloznainfekcija. Tada bolesniku treba uključitihemoprofilaksu pod kontrolom ljekara ureferentnoj zdravstvenoj ustanovi za liječenjetuberkuloze. Najčeće se primjenjujehemoprofilaksa izoniazidom u trajanju od6 mjeseci. Već poslije 60 dana od početkahemoprofilakse bolesniku se može uključitiantiTNFα terapija.80

Hasan Žutić et al.: Preporuke za tretman aktivne i latentne tuberkuloze prije i tokom terapije...Osim pozitivne lične anamneze, pozitivnaporodična anamneza na tuberkulozuje važan faktor koji se mora uzeti u obzirprilikom procjene bolesnika. Faktori rizika,osim imunosupresivne terapije, za aktivacijulatentne tuberkulozne infekcije su :– Rođenje ili boravak u endemičnimtuberkuloznim regijama– Nedavni kontakt sa oboljelim od tuberkuloze– Zaposlenik mikrobiološkog laboratorija– Boravak unutar okruženja u kome suosobe sa visokim rizikom– Diabetes– Silikoza– Transplantacija organa– Hronična bubrežna insuficijencija– Gastrektomija ili jejuno-ilaealni bajpas– Maligno oboljenje glave ili vrata, leukemija,limfom– Imunosupresija usljed nekih drugihrazloga (primarna ili sekundarnaimunodeficijencija)Ukratko se može sažeti – ako pacijentima pozitivno bar jedno od navedenih:– ličnu / porodičnu anamnezu ili– rezultati IGRA testa su pozitivni ili– PPD test pokazuje otvrdnuće ≥ 5 mmu promjeru ili– Rtg nalaz pluća pokazuje znake latentnetuberkulozne infekcijetretman latentne tuberkulozne infekcijetreba započeti po preporukama navedenimu tabeli 1.Kontrola (PREVENCIJA) tuberkulozetokom liječenja inhibitorima TNFαAlgoritam kontrole (prevencije) tuberkuloze(Shema 3) prikazuje preporučene proceduretokom anti–TNFα terapije. Prvukontrolu na tuberkulozu treba obaviti šestmjeseci od početka primjene anti–TNFαlijeka, a zatim nakon godinu dana od početkaliječenja. Kontrolni pregled podrazumijevai radiografiju i IGRA ili PPD test, jerse pored plućne, kod ovih bolesnika javlja ituberkuloza drugih lokalizacija u mnogo većemprocentu nego u opštoj populaciji (oko50% u odnosu na 15%). Liječenje bolesnikas aktivnom tuberkulozom i hemoprofilaksubolesnika s latentnom tuberkuloznom infekcijomtreba da sprovede pneumoftiziolog,odnosno pulmolog, u okviru ustanovespecijalizovane za liječenje ovog oboljenja,a prema usvojenom stručno-metodološkomuputstvu. Dijagnostika se takođe vrši u specijalizovanimzdravstvenim ustanovama.Tabela 1: Preporuke za tretman latentne tuberkulozeIGRA / PPD test Rtg pluća Preporuka za tretman latentne infekcijenegativan / < 5 mm normalan Nije preporučena*pozitivan / ≥ 5 mm patološki Konsultacije sa ekspertomnegativan / < 5 mmpozitivan / ≥ 5 mmpatološkinormalanKonsultacije sa ekspertom / Ukoliko je mikrobiološki nalaz negativan,preporučena profilaksa izonijazidom u trajanju 6 do 9 mjeseci. AntiTNFterapija može, uz oprez, započeti 60 dana nakon početka hemoprofilakseUkoliko je mikrobiološki nalaz negativan, preporučena je profilaksaizonijazidom u trajanju 6 do 9 mjeseci. AntiTNF terapija može, uz oprez,započeti 60 dana nakon početka hemoprofilakse* konsultacije sa ekspertom su ipak preporučene kod pacijenata sa negativnim testom koji imaju faktore rizika za infekciju tuberkulozom81

Folia Medica 2011; 46 No 1:73-84Shema 3: Algoritam za kontrolu (prevenciju) tuberkuloze tokom anti-TNFα terapijeZaključakAnti–TNFα lijekovi imaju značajno mjestokao savremena biološka terapija određenihautoimunih bolesti. Kod bolesnika saautoimunim oboljenjima povećan je rizikod razvoja teških infekcija, uključujući ituberkulozu. Stoga je neophodno prije početkaprimjene anti–TNFα lijeka obavitiodgovarajuće dijagnostičke postupke kakobi se otkrila aktivna ili latentna tuberkuloza.Kod bolesnika s aktivnim oblikom ovogoboljenja kontraindikovana je primjenaanti–TNFα lijekova i oni se moraju uputitina liječenje u odgovarajući zdravstvenicentar. Bolesnicima sa dijagnostikovanomlatentnom tuberkuloznom infekcijom morase propisati primjena hemoprofilakse. Naosnovu iskustava zemalja u svijetu u kojimapostoje slični vodiči ili smjernice, očekuje seda će postupanje u skladu sa preporukamaza dijagnostiku tuberkuloze značajno smanjitirizik od pojave aktivne tuberkuloze kodbolesnika u Bosni i Hercegovini koji primajuanti–TNFα terapiju.Reference1. Alonso-Ruiz A, Pijoan JI, Ansuategui E, CaladozoM, Urkaregi A, Quintana A. (2008). Tumor necrosisfactor alpha drugs in rheumatoid arthritis:systematic review and metaanalysis of efficacy andsafety. BMC Musculosceletal Disord 9:52.2. American Thoracic Society (2000). Targeted TuberculinTesting and Treatment of Latent TuberculosisInfection. Am J Respir Crit Care Med 161:S221–47.3. Bazzoni F, Beutler B (1996). Seminars in Medicineof the Beth Israel Hospital, Boston: the tumor necrosisfactor ligand and receptor families. N Engl JMed 334:1717-1725.4. Bean AG, Roach DR, Briscoe H, et al (1999).Structural deficiencies in granuloma formation inTNF genetargeted mice underlie the heightenedsusceptibility to aerosol Mycobacterium tuberculosisinfection, which is not compensated for bylymphotoxin. J Immunol 162: 3504–11.5. Beutler B, Cerami A (1989). The biology of cachectin/TNF--aprimary mediator of the host response.Annu Rev Immunol 7:625-655.6. Carmona L, Gomez-Reino JJ, Rodriguez-ValverdeV, et al (2005). BIOBADASER group. Effectivenessof recommendations to prevent reactivation of latenttuberculosis infection in patients treated withtumor necrosis factor antagonists.Arthritis andRheumatism 52(6):1766-7282

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Instructions to AuthorsThe Journal “Folia Medica” is publishing original scientific articles, professional articles, review articles,case reports, books reviews, information articles, professional informations and other articles fromall medical fields.Authors are responsible for all data and statements in their articles. If the article is written by multipleauthors and co-authors, it is necessary to provide the exact address (with the phone number and e-mail)of the corresponding author for the cooperation with the editorial board in editing of the text beforepublishing.If the research on humans is presented in the article, must be stated whether they are conducted inaccordance with the principles of medical deontology and Declaration from Helsinki/Tokyo.If the article is presenting the research on animals, must be stated whether they are conducted in accordancewith the ethical principles.When using measurement units it is necessary to obey the rules listed in the SI system.Articles must be sent to the Editorial on following address:COVERING LETTER“Folia Medica”,Medical Faculty of Sarajevo University,Čekaluša 90,71 000 Sarajevo,Bosnia and HerzegovinaWith the papers, the authors must send the covering letter to the Editorial of the journal “FoliaMedica”. This covering letter must contain signed statement by all authors:1. That the article is not published or in process of publishing in some other journal.2. That all authors agree about the article content3. That during the research guidelines from the Helsinki Declarations is followed.4. That the respondents give their written consent to participate in the research;5. That the authors have written permission for publishing the pictures which can reveal the patientsidentity.6. That the authors have written permission of the publisher, owning the copyrights, to publishilustrations, schematics or tables.7. That the accepted paper will become the property of the “Folia Medica” journal.SIZE AND SHAPE OF THE TEXTThe papers should not exceed ten pages prepared on a computer, including illustrations, charts, tablesand references. Electronic form of the document is obligatory (Word for Windows).85

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Folia Medica 2011; 46 No 1:85-88Quotation examples– example of the article in a journal:McGuigan C, Davies M, Pathirana R et al. Synthesis and anti –HIV activity of some novel diarylphosphate derivatives of AZT. Antiviral Res 1994; 24: 69-77– example of the article in a journal, anonymous author:Anon. An enlarging neck mass in a 71-year-old woman. Am J Med 1989; 86: 459-464.– example of the article in a journal, organization as author:Americal College of Physicians. Clinical ecology. Ann Int Med 1989; 111: 168-178.– example of article in journal supplement:Miller GJ. Antithrombotic therapy in the primary prevention of acute myocardial infarction. Am JCardiol 1989; 64: Suppl 4: 29B-32B.– example when quoting a book:Sperof L, Glass R, Kase N. Clinical gynecologic endocrinology and infertility. Williams & Wilkins.London 1989: 25-28– example for a single chapter within a book:Bjersing L. Ovarium histochemistry. In: Zuckerman L, Weir BJ (eds.). The ovary. Vol 2. New York:Academic Press, 1977: 45-47– example for a summary from proceedings:Schneider W. Platelet metabolism and membrane function. In: Ulutin ON, Vinazzer H eds. Proceedingof 4 th international meeting of Danubian league against thrombosis and haemorrhagic diseases.Istanbul: Goezlem Printing and Publishing Co, 1985: 11-15.These instructions are written in accordance with the Vancouver quotation rules.www.mf.unsa.ba88