Imunodijagnostičke i molekularne metode u proceni efikasnosti terapije obolelih od tularemije

Abstract

Introduction: Tularaemia is a severe bacterial zoonosis caused by the highly infectious agent Francisella tularensis. Microbiological diagnosis of tularemia mainly relies on serology. Within the last decade great efforts have been made to develop more sensitive and specific immunological and molecular techniques for the laboratory diagnosis of tularemia. Occurance of tularemia epidemic in the Southeast of Serbia in 1998/1999. initiated epidemiological as well as microbiological and clinical research in this area. That was the motive to start applying and improving immunodiagnostic tests, and a real challenge for the clinicians to fight the infectious agents, bacteria F. tularensis. Objective: 1. Determination of the correlation between the test results done by immunodignostic, histological, immunohistochemic and molecular methods; assesment of significance of the noted tests in diagnosis and the efficiency assesment in therapy of tularemia patients. 2. Monitoring and evaluation of the serological antibody finding of tularemia patients within the period from 1 to 11 years since the beginning of the disease. 3. Assesment of efficiency of the applied antibiotic therapy on patients suffering from tularemia. Methods: Testing was performed on a group of 113 patients suffering from tularemia in a period starting in 1999.until the end of 2011. Control group included 111 examinees, out of which 31 patient with differential diagnosis of lymphadenopathy of different origin, 20 seropositive brucella patients, 10 seronegative brucella patients, 30 seropositive yersinia patients and 20 voluntary blood donors. During the diagnostic processing of patients, following materials were used: serum and bioptat of lymph node. Total of 270 serum samples were examined, out of which 189 serums suspicious of tularemia, 111 serums from the control groupe and 26 lymph node bioptats. There were 8 diagnostic techniques used on the material mentioned, out of which 6 serological, imunohistochemical method of indirect immunoperoxidase (IIP) and molecular method (PCR). Serological methods included immunoensyme assays: Serion ELISA IgG i IgM, Serazym ELISA and ELISA in house; imunohromatografic test (ICT) and Western-blot. On patohistological material ,(IIP) was done with the use of primary monoclonal antibodies on F. tularensis, and the molecular method done, was PCR. Results: Serums of the patients monitored in the interval of 1 to 11 years starting with the occurrence of the disease, indicated positive finding of IgG, IgM class antibodies, as well as total antibodies on F. tularensis, in most of the tests. Analysis of the results obtained in the used tests, confirmed that most of the tests indicate high sensitivity, specificity and mutually similar results. IgG Serion test showed the highest sensitivity (97,4%) and specificity(93,1%). IHH method of the indirect imunoperoxidase (IIP) proved the highest specificity (100%), but low sensitivity (65%). Complications were noticed in 43,4% patients mostly with suppurative lymphadenitis and recidivism of the disease. Two thirds of the complications occurred after late onset of appropriate treatment, 3 weeks after the beginning of illness. The highest percentage of healing and the least complications were noted with the use of gentamycin (83,3%), then ciprofloxacin (75%) and finally, in the successive therapy with gentamycin and doxycycline (70,0%). The biggest failure in children patients was noted in the use of amikacin with cephtriaxon which is related to occurance of suppurrative lymphadenitis in all treated children.. Conclusion: The use of immunodiagnostic tests confirmed the old F.tularensis infection and established retrograde diagnosis on previously undiagnosed or serologically unconfirmed tularemia. Serological finding must be interpreted only within clinical diagnosis of tularemia. Finding of IgM and IgG class antibodies or total antibodies on F.tularensis in serums of the patients without clinical manifestations of the disease, even many years after the beginning of the infection, does not indicate acute illness, but the cured illness, which once existed. Test which proved highest sensitivity and specificity is IgG Virion ELISA, therefore it is suggested as the most reliable method for diagnosis and monitoring of tularemia patients. Immunohistochemical method IIP can be used in confirmative diagnosis of tularemia due to its highest specificity. PCR method is not a method of choice izbora for diagnosis of tularemia on byopsy samples of the tissue mouldedin parafin. The success of therapy was proved in healing of all patients monitored within the period from 1 to 11 years (96.5% of the patients) except for persistent lymphadentopathy in 4 patients (3.5%). There was no influence of the therapy on serological analysis of the patients.