Aleksandra Ignjatović

Seroreactivity to Dirofilaria antigens in people from different areas of Serbia

BackgroundThe Northern part of Serbia is hyperendemic-endemic for canine dirofilarioses. Considering this fact, many human dirofilarial infections could be expected, however only about 30 cases in Serbia have been described until today. Aims of this survey were to assess the people reactivity to the antigens of D. repens and D. immitis and to identify risk factors for the contact exposure.MethodsInvestigation included sera taken from 297 people (179 women and 118 men) living in different areas of Serbia (Pančevo, Novi Sad, Zaječar, Leskovac, Vranje, Niš, Pirot). Sera were analysed by means of two indirect enzyme-linked immunosorbent (ELISA) home-designed that use as antigens adult somatic/metabolic polyproteins of D. repens (DR) and D. immitis (DI), respectively. The results were elaborated using the statistical method of descriptive and quantitative analysis.ResultsSignificant differences by area in the reactivity of human sera to dirofilarial antigens were not observed (p = 0.056). A high seroreactivity was demonstrated in people from the towns of northern Serbia (Pančevo = 27,1%; Novi Sad = 16,3%), as well as in people from Zaječar (eastern Serbia = 15,8%) and Vranje (southern Serbia = 15,1%). No differences were evidenced between people reactivity to polyproteins of the two dirofilarial species, nor differences related to the gender of examinees. Factor risks evidenced were: i) place of residence; ii) spending work time outdoors during the mosquito season; iii) spending time outdoors and nearby rivers, lakes, swamps or canals; unespectedly, iv) cat owning.ConclusionThe findings emerging from this investigation indicate that clinicians and public health authorities should pay greater attention to this zoonosis. Continuing education and training of physicians will greatly contribute to the knowledge of the actual impact of filarial worms on animal and public health, and allow for the planning of suitable measures to prevent the infections.

Canine Dirofilaria Infections in Two Uninvestigated Areas of Serbia: Epidemiological and Genetic Aspects

In 2009 canine filarial infections were investigated in two northern areas of Serbia (Pančevo and Veliko Gradište), applying morphometry, biochemical staining, and immunological kit to detect Dirofilaria immitis antigens, and two home-made ELISAs to detect antibodies to D. repens and D. immitis somatic/metabolic polyproteins. Moreover, molecular tools were applied to analyze the phylogenetic relationships of the isolates. The microfilariae detected in 21/122 dogs (17.2%) were identified as D. repens (n=21) and D. immitis (n=2). D. immitis antigens were found in another 13 animals with occult infection. All of the 15 heartworm-positive dogs also had antibodies to this parasite, which were detected in another 13 subjects, indicating an overall D. immitis seroprevalence rate of 22.9%. Serology for D. repens revealed evidence of antibodies in 42.6% of the dogs, but was negative for 4 microfilaremic dogs. As for the two different areas, the prevalence of microfilariae and/or D. immitis antigens, mainly due to D. repens microfilaremic animals, was not significantly higher in Veliko Gradište (33.3%) than in Pančevo (22%). However, serology showed a different epidemiological picture. Heartworm infection occurred more often in both areas, and antibodies to dirofilarial nematodes were detected in 72.9% of dogs living in Pančevo, a rate higher than in those living in Veliko Gradište (57.1%). No risk factors for infection were found, confirming the uselessness of prophylactic drugs against D. repens, and suggesting the presence in these areas of sunrise- or sunset-biting mosquitoes as important vectors. The results indicate the need for both appropriate entomological studies and further research on the intra-species variability shown by D. repens.

The prevalence of Candida onychomycosis in Southeastern Serbia from 2011 to 2015.

Despite the increasing of onychomycosis caused by Candida spp., in referent literature, there is still data insufficiency about this nail infection. The objectives of this retrospective study were to determine epidemiological characteristics of Candida onychomycosis, the antifungal susceptibility of isolated species in vitro, and to compare the results of antifungal susceptibility testing with conducted treatment in period from 2011 to the end of March 2015. Out of 761 patients who were underwent clinical and mycological examinations, 137 had Candida species isolated from nails. The dominant species was Candida albicans (C. albicans) (36.59%) followed by C. parapsilosis (23.78%), C. krusei (9.76%), and C. guilliermondii (6.71%). Antifungal susceptibility in vitro testing showed good susceptibility to antimycotics, except C. krusei, which was resistance to fluconazole (FCZ) and isolates of C. tropicalis and C. glabrata which were dose dependent to itraconazole (ITZ) and fluconazole. Evaluation of medical histories determined that combined therapy, which included pulsed systemic regimen of ITZ with topical application of clotrimazole, had better clinical outcomes regarding the proscribed only topical application of clotrimazole. Multidisciplinary approach of dermatologists and mycologists is required in solving the problem of onychomycosis, which is the dominant nail disease.

Dimethylarginine – biomarkers in progression of kidney disease / Dimetilarginini – biomarkeri u progresiji bubrežnih oboljenja

Summary Decreased nitric oxide (NO) production and/or impaired NO bioavailability may occur in patients with the chronic kidney disease (CKD), and could contribute to elevation of blood pressure, cardiovascular disease (CVD) and progression of renal injury in these patients. Free guanidinomethylated arginine residues occur endogenously as a result of proteolysis of post-translational methylated tissue proteins. The asymmetric dimethyl arginine (ADMA) is a competitive inhibitor of the nitric oxide synthase (NOS) enzymes. The kidney has a predominant role in ADMA elimination by combining two mechanisms; urinary excretion and metabolization of ADMA The degradation of ADMA is accomplished intracellularly by the enzyme dimethylarginine dimethylaminohydrolase (DDAH). ADMA is not only a uremic toxin, but also a strong marker of the endothelial dysfunction and atherosclerosis and a stronger independent predictor of all-cause mortality and cardiovascular outcome in patients with the chronic renal failure. There are at least four mechanisms that may explain the accumulation of ADMA in CKD: increased methylation of proteins, increased protein turnover, decreased metabolism by DDAH and impaired renal excretion. A strong positive correlation between symmetric dimethyl arginine (SDMA) and creatinine suggests that SDMA might be of value as a marker of the renal function. Reduced NO elaboration secondary to accumulation of ADMA and elevated inflammation may be important pathogenic factors for endothelial dysfunction in patients with the renal disease. Elevation of ADMA may be a missing link between CVD and CKD. Kratak sadržaj Smanjenje koncentracije NO i/ili nedovoljna raspolo`ivost ovog molekula kod pacijenata sa bubre`nim bolestima mo`e biti razlog pove}anja krvnog pritiska, kardiovaskularnih bolesti (KVS) i progresije bubre`nog o{te}enja. Metilarginini nastaju u procesu proteolize posttranslaciono metilisanih argininskih rezidua u proteinima. Asimetri~ni dimetilarginin (ADMA) je kompetitivni inhibitor azot oksid sintaze (NOS). Najva`nija uloga bubrega u eliminaciji ADMA podrazumeva procese urinarne ekskrecije i razgradnju pod uticajem dimetilarginin dimetilaminohidrolaze (DDAH). Na aktivnost DDAH uti~u oksidativni stres i inflamacija. ADMA nije samo uremijski toksin ve} i zna~ajan marker endotelne disfunkcije i ateroskleroze, kao i nezavisni prediktor mortaliteta i kardiovaskularnih bolesti kod pacijenata sa HBI. Osnovni uzroci koji dovode do akumulacije ADMA su pove}ana metilacija proteina, njihov pove}an metabolizam, smanjena aktivnost DDAH i smanjena urinarna ekskrecija. Klirens simetri~nog dimetilarginina (SDMA) u plazmi zavisi samo od renalne funkcije (pozitivna korelacija sa kreatininom) i njena akumulacija predstavlja nespecifi~ni indikator uremijskih toksina. Redukovana koli~ina NO pra}ena akumulacijom ADMA, udru`ena sa inflamacijom mo`e biti va`an patogeni faktor endotelne disfunkcije kod bubre`nih pacijenata. Porast koncentracije ADMA mo`e biti veza izme|u KVS i HBI.

Seroprevalence and Risk Factors for Toxocara canis Infection in Serbia During 2015.

We report data on the Toxocara seroprevalence evidenced in 2015 from samples of 40 children and 298 adults of the population living in different areas of Serbia, and on possible association of certain variables with infection. Detection of specific antibodies was performed using an enzyme-linked immunosorbent assay; all ambiguous results and part of the positive and negative sera were further analyzed by confirmatory Western blot test. An overall 23.5% seroprevalence was noticed, which was confirmed in 13.0% of the examined population with no significant difference regarding the age (children = 10.0%; adults = 13.4%) or by country area (East = 18.2%; North = 15.5%, Southeastern = 9.5%; p = 0.005). In contrast, the group of adult women proved more reactive than men (p = 0.001), and subjects both who spend spare time in square/parks (p = 0.041) and with positive onychophagy (p = 0.001) habit turned out more exposed to the infection. Possible reasons of these differences were analyzed, and the medical, veterinary, and economic impact of this soil-transmitted zoonosis were discussed.

Oxidative and Nitrosative Stress in Stable Renal Transplant Recipients with Respect to the Immunosuppression Protocol – Differences or Similarities? / Oksidativni I Nitrozativni Stres U Odnosu Na Imunosupresivni Protokol Kod Pacijenata Sa Stabilnom Funkcijom Presađenog Bubrega – Razlike I Sličnosti

Summary Background: The aim of the study was to evaluate parameters of oxidative and nitrosative stress as well as antioxidative parameters in a group of renal transplant recipients with stable graft function and no clinical signs of cardiovascular disease. We also aimed to determine the correlations among these parameters and to evaluate potential differences in all the biomarkers with regard to the immunosuppression protocol. Methods: We enrolled 57 renal transplant recipients and 31 controls who were age and sex matched with the renal transplant recipients. All of the patients included in this study had post-renal transplant surgery at least 12 months earlier and were on standard immunosuppressive therapy. In this study, we determined thiobarbituric acid-reactive substances in plasma and red blood cells and advanced oxidation protein products, nitrosative stress parameters (asymmetric and symmetric dimethylarginine - ADMA and SDMA), and antioxidative parameters (total SH groups and catalase activity). Results: The results of our study demonstrated that the levels of oxidative and nitrosative stress were significantly increased compared to the healthy population (p<0.01 except for plasma catalase activity p<0.05). Correlation analysis showed significant positive correlations between: ADMA and SDMA (p<0.01); ADMA and nitrates (p<0.05); SDMA and nitrates (p<0.05); between OS parameters in the experimental group; AOPP and SH groups (p<0.05) and TBARS in plasma and SH groups (p<0.01), SDMA and AOPP (p< 0.05); SDMA and TBARS in plasma (p<0.05); SDMA and SH groups (p<0.01); nitrates and SH groups (p<0.05). Conclusion: There was no significant difference in oxidative and nitrosative stress parameters with respect to the immunosuppressive protocol. Kratok sadrzaj Uvod: Transplantacija bubrega sama po sebi popravlja bubrežnu funkciju, ali ne dovodi do potpunog oporavka. Cilj ovog rada bio je da se odrede parametri oksidativnog i nitrozativnog stresa kao i parametri antioksidativne zastite u populaciji pacijenata sa presacfenim bubregom sa stabilnom funkcijom grafta i bez kliničkih znakova kardiovasku- larne bolesti. Takođe, naš cilj je bio da se utvrdi povezanost među ispitivanim parametrima i procene potencijalne razlike između svih ispitivanih biomarkera u odnosu na imunosupresivni protokol. Metode: U istraživanje jeuključeno 57 pacijenata sa presadenim bubregom i 31 kontrolni subjekt, koji su po go- dinama i polu odgovarali pacijentima sa presađenim bubregom. Svi pacijenti uključeni u istrazivanje imali su transplantaciju bubrega najmanje 12 meseci pre početka istraživanja i bili su na standardnoj imunosupresivnoj terapiji. U ovom radu određivali smo reaktivne supstance tiobar- bituratne kiseline (TBARS) u plazmi i eritrocitima, uznapre- dovale produkte oksidacije proteina (AOPP), parametre nitrozativnog stresa (asimetrični i simetrični dimetilarginin- ADAAA i SDMA) i antioksidativne zastite (ukupne SH grupe i aktivnost katalaze). Rezultati: Rezultati naše studije su pokazali znatno više vrednosti parametara oksidativnog i nitrozativnog stresa kod pacijenata sa presađenim bubregom u odnosu na zdrave dobrovoljce (p<0,01 osim za aktivnost katalaze u plazmi, kada je p<0,05). Korelaciona analiza pokazala je znacajnu pozitivnu korelaciju između ADMA i SDMA (p<0,01); ADMA i nitrata (p<0,05); SDMA i nitrata (p<0,05); između parametara oksidativnog stresa u eksperimentalnoj grupi; AOPP i SH grupe (p<0,05), kao i TBARS u plazmi i SH grupe (p<0,01), SDMA i AOPP (p<0,05); SDMA i TBARS u plazmi (p<0,05); SDMA i SH grupe (p<0,01); nitrata i SH grupe (p<0,05). Zakljudak: Naši rezultati nisu ukazali na statistički značajnu razliku u ispitivanim parametrima među pacijentima na ciklosporinu A i takrolimusu

Superficial mycoses in the Nis region, Southeast-Serbia

The aim of the study is to investigate the most frequent cause of superficial mycoses in patients from the territory of city Niš Southeast Serbia in the period from 1998 to 2010. A total of 3223 samples from 2887 patients with suspected dermatomycoses were examined. Superficial mycoses were diagnosed using standard microbiology techniques (conventional microscopy and cultivation). Dermatophytes were determined on the basis of their macroscopic and microscopic morphological and morphometric characteristics. Morphometric characteristics were obtained by Laboratory Universal Computer Image Analysis system (Lucia M, 1996). Species of genus Candida were identified using the test of production of germ tube in sera, by growth on comertial chromatogen medium (Chromotogenic Candida, Liofichem/Bacteriology products, Italy) and by using Auxacolor TMBioRad, France. The results were elaborated with the statistical method of descriptive and quantitative analysis (SPSS 14.0 for Windows 2003). The prevalence of superficial mycoses was 25,1%. Dermatophytes were identified in 67.6% of all positive cultures. Microsporum canis was the most prevalent (50.3%) dermatophyte isolated, followed by Trichophyton metagrophytes var. mentagrophytes (35.4%). Yeast genus Candida has become a more frequent cause of superficial fungal infection since 2001. and C. albicans was the dominant yeast (61.1%).

ADMA and C-reactive protein as mortality predictors in dialysis patients

There is a higher mortality between patients with end-stage renal disease than patients in the general population. These circumstances have led to a search for risk factors as predictors of mortality in dialysis patients. Amongst those, inhibitors of the nitric-oxide (NO) synthesis deserve special attention, since patients with end-stage renal disease are also characterized by accelerated atherosclerosis. Asymmetric-dimethylarginine (ADMA) and symmetric-dimethylarginine (SDMA), as well as C-reactive protein (CRP), have also been recognized as predictors of mortality in patients on dialysis. The aim of our study was to compare the prediction power of ADMA, SDMA and CRP for all-cause mortality in patients with end stage renal disease during the fourteen month follow-up. In total 162 patients on hemodialysis were included. ADMA and SDMA were measured by the high-performance liquid chromatography (HPLC); CRP was measured using immunonephelometric assays. During the 14-month period 28 patients (34.1%) died from all-cause mortality. Using univariate analysis, hazard ratios (HR) of the potential independent predictors of mortality in hemodialysis patients were ADMA (HR 1.39 (1.01–1.91) p=0.043) and CRP (HR 1.024 (1.009–1.1.040) p=0.001). Further, multivariate analysis (MVA), however, showed that ADMA is the only predictor of all-cause mortality (HR 1.76 (1.002–3.11) P=0.049), while SDMA failed to predict death in this population. Therefore, our data shows that ADMA is an independent and better marker of all-cause mortality compared with CRP.

Progress of statistical analysis in biomedical research through the historical review of the development of the Framingham score

BackgroundThe interest in developing risk models in medicine not only is appealing, but also associated with many obstacles in different aspects of predictive model development. Initially, the association of biomarkers or the association of more markers with the specific outcome was proven by statistical significance, but novel and demanding questions required the development of new and more complex statistical techniques.MethodsProgress of statistical analysis in biomedical research can be observed the best through the history of the Framingham study and development of the Framingham score.ResultsEvaluation of predictive models comes from a combination of the facts which are results of several metrics. Using logistic regression and Cox proportional hazards regression analysis, the calibration test, and the ROC curve analysis should be mandatory and eliminatory, and the central place should be taken by some new statistical techniques. In order to obtain complete information related to the new marker in the model, recently, there is a recommendation to use the reclassification tables by calculating the net reclassification index and the integrated discrimination improvement. Decision curve analysis is a novel method for evaluating the clinical usefulness of a predictive model. It may be noted that customizing and fine-tuning of the Framingham risk score initiated the development of statistical analysis.ConclusionClinically applicable predictive model should be a trade-off between all abovementioned statistical metrics, a trade-off between calibration and discrimination, accuracy and decision-making, costs and benefits, and quality and quantity of patient’s life.

Immune Response in Infections Caused by Helminthes

Summary The first line of defence in parasitic infection is the innate immune system. On the other hand, adaptive immune system posseses numerous mechanisams of hummoral and cellular immunity. Cellular immunity in a helminth infection is characterised by Th2 immune response. Considering the fact that the aim of a parasite is not to kill its host, the majority of parasites are highly addapted to the life inside the host, and succesefully avoid or limit its deffences. A special signifficance of the parasite as a potential pathogen is its possibility to escape immunity. Numerous helminths are releasing different substances that are acting as lymphocyte suppressors and macrophage inactivators and they are capable of destroying antibodies. They have a possibility of camouflage, sequestration and surface shell peeling with the aim to avoid immune response. Latest research in the field of immunology has revealed the significance of CD40 co-stimumlating protein of antigen presenting cells in the immune response to parasitic infection. Immune response in the course of parasitic infestion is important in pathogenesis of helminthioses. Sažetak Pored urođene otpornosti, u borbi protiv parazita, organizam domaćina raspolaže i specifičnim humoralnim i celularnim mehanizmima zaštite. Celularna imunska reaktivnost u toku helmintoza uspostavlja se preko Th2 imunskog odgovora. Imajući u vidu činjenicu da cilj parazita nije da ubije svog domaćina, većina parazita je visoko adaptirana na život unutar domaćina i uspešno izbegava ili ograničava njegove odbrambene sposobnosti. Helminti, paraziti čoveka, produkuju određene supstance koje deluju kao supresori limfocita i inaktivatori makrofaga i sposobni su da izvrše destrukciju produkovanih antitela. Takođe, helminti mogu maskiranjem, sekvencioniranjem i gubitkom eksponiranih antigena kutikule da izbegnu mehanizme odbrane nosioca. Najnovija istraživanja na polju imunologije otkrivaju značaj CD40 ko-stimulirajućeg proteina antigen prezentujućih ćelija u okviru imunološkog odgovora u toku parazitske infekcije. Pored zaštitne uloge, imunološki odgovor u toku parazitske infekcije značajno utiče i na patogenezu helmintoza.