TABLE OF CONTENT

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1. NEUTROPHIL RESPIRATORY BURST ACTIVITY IN NEONATAL INFECTION

G. Zhelezova, I. Altankova, P. Zacharieva, S. Simeonova, D. Dyankova, R. Georgieva, E. Christova

ABSTRACT
To determine the neutrophil respiratory burst activity (NRBA) in neonates with infection, and correlate the obtained results with the IL-6 levels. Thirty-nine neonates (23 preterm and 16 term) were studied. They were: neonates with infection and positive blood culture (n=20), neonates with infection and negative blood culture (n=10) and neonates without infection, as controls (n=9). Group of 21 healthy adults were studied, too. The NRBA was measured in whole blood flow cytometric assay. The test was performed without stimulation and with stimulation with fMLP. IL-6 levels were determined by ELISA. Unstimulated neutrophils of the control neonates without infection demonstrated greater NRBA, compared with the healthy adults (P <0.05). After fIVILP stimulation control neonates had similar results to adults. Nonstimulated burst activity in the neonates with infection (with positive blood culture, as well as with negative blood culture) was significantly higher than those in control neonates (P<0.05). After fMLP stimulation, the neonates with infection had also elevated NRBA, but the difference was statistically significant only in neonates with negative blood culture (P<0.05). The IL-6 were elevated in neonates with infection (with positive and with negative blood culture (P<0.01), but there is no correlation with the NRBA. These data demonstrate that in neonates with infection (with positive and with negative blood culture) NRBA was elevated, compared with the noninfected neonates, with an exception of the group with positive blood culture after fMLP stimulation. There is no correlation between the NRBA and elevated IL-6 levels.


2. PREVENTION OF SURGICAL SITE INFECTION INSTITUTIONALLY - PROJECT PHASE I

E. Keuleyan, G. Kirov, D. Vezeva, I. Lozev, V. Stephanov, G. Markov, R. Benchev

ABSTRACT
Surgical site infections (SSI) constitute an important group of nosocomial infections. In aim of reducing their rate at our institute, several important steps have been undertaken. After consulting the contemporary literature, we re-enforced the Infection control Committee and created an Antibiotic policy Committee within the Drug Therapeutic Commission. The Surgery department was renovated, we prepared Institutional programs for Infection control and Rational antibiotic policy. We introduced an audit of antibiotic prescriptions, both for Antibiotic prophylaxis in surgery, and therapeutic antibiotic usage. The rate of SSI was stable < 3-5 % during the last 3 years.


3. INVESTIGATION ON THE IMMUNE STATUS OF THE POPULATION AGAINST WHOOPING COUGH DURING THE PERIOD 2001 -2004

R. Alexiev, K. Hadjiisky, S. Malchanova, V. Demireva, PI. Nenkov

ABSTRACT
The specific immunoprophylaxis of humans with pertussis, as a component of combine bacterial vaccine leads to production of specific antibodies that is indicator of the whooping cough prevention. For evolution of immunization procedures and the vaccine itself, antibody levels against pertussis are useful to show the immune status of the population. An enzyme-linked immunosorbent assay was used for measuring immunoglobolin G pertussis antibodies in human sera. The assay was done in plastic plates coated with inactivated bacterial cells. For investigation were tested 3 711 human sera in different age groups. The comparison of different groups of age showed that the best protection against whooping cough have in people younger than 25 years old. In these groups of age the sera without antibodies against pertussis are 7% and 11% of patients have titer of antibody more than 1:321, which titer is used as a criteria of disease. Aging of people leads to reduction of the percent of protected persons and to increasing the percent of non-protected people. Despite this, the percent of protected population in-groups of age between 16 to 55 years old is better than in some European countries and in the USA. The patients over 56 years old have low levels of antibodies against pertussis because the vaccination process in Bulgaria is began since 1958 and in about 55% lacked protection against whooping cough. The annual differences of the rate of protection levels during the investigation period were not observed. Present results indicate a good protection against pertussis in Bulgaria. This fact is a result of specific immunoprophylaxis by pertussis vaccine, as a component of combined bacterial vaccine, produced by BB - NCIPD, Ltd.


4. DIAGNOSTICS OF THE FIRST SUSPECT HUMAN CASES OF AVIAN INFLUENZA A/H5N1/ VIRUS IN BULGARIA

T. Hadzhiolova, S. Pavlova, R. Kotseva

ABSTRACT
The first diagnostic investigations have been made, demonstrating avian flu А/Н51М1/ virus etiological role. For the period January-March 2006 twenty six suspect patients, having been in close contact with ill or dead birds, and with a subsequent respiratory illness, were tested. Specific A/H5N1/ assays were applied /GeNet Bio rapid antigen detection test and Sacace RT-PCR kit/. Viral isolation was performed using IVIDCK cell lines and chicken embryos. Avian flu А/Н51М1/ virus as a causative agent of respiratory disease was neither isolated, nor detected in any one of the tested patients. Subtype A/H1N1/ human influenza virus, identifiable by HIT and RT-PCR, was isolated in three of the patients tested.


5. EXTENDED SPECTRUM BETA-LACTAMASE (ESBL) PRODUCERS AMONG ENTEROBACTERIACAE FROM PATIENTS IN BULGARIAN HOSPITALS

R. Markovska, E. Keuleyan, M. Sredkova, D. Ivanova, B. Markova, K. Rachkova, E. Dragijeva, I .Haydouchka, T. Kostayanev, I. Mitov

ABSTRACT
To preliminary characterize the main types of ESBLs among Bulgarian Enterobacteriaceae strains and to determine their rate of resistance. Methods: 458 Enterobacteriaceae (K.pneumoniae -243, K. oxytoca - 9, E.coli - 156, C. freundii - 12, Enterobacter spp - 18, Proteus spp - 4, Serratia spp -15, Salmonella enterica Serotype Corvallis - 1) strains identified as ESBL producers were collected from 8 medical centers in Sofia, Pleven, Stara Zagora and Plovdiv during 1996-2003. They were confirmed by phenotypic tests (DDS, NCCLS method). MICs were determined by an agar dilution technique (NCCLS, 2002). Conjugative plasmid transfer was performed, followed by an isoelectric focusing according to Mathew/Bauernfeind. The hydrolytic activity of the bands was proved by Bioassay (Bauernfeind).PCR experiments were used to confirm the type of ESBL Results: The rate of resistance was: amoxicillin/clavulanate - 86%, ceftazidime - 85% , cefotaxime-95%, ceftriaxone - 94%, aztreonam - 74%, cefoxitin - 14%, ceftibuten - 19%, cefepime - 37%, imipenem - 0%, tobramycin - 95%, gentamicin - 84%, amikacin - 41%, ciprofloxacin -56%, tetracycline -86%, co-trimoxazole - 53% chloramphenicol - 58%. MIC of ceftazidime (CAZ) ranged 1 - >512 mg/L and of cefotaxime (CTX) -2-512 mg/L The strains were divided into two main groups: the first one- MICCAZ > MICcre - 279 strains and the second: MICCAZ < MIC -179. In all strains sulbactam in combination with CAZ and CTX showed an inhibitory effect. CAZ and/or CTX resistance was transferable in 185 from 216 mating experiments. IEF analysis of 247 strains showed the presence of 3 clusters. The pi data were from transconjugants and wild type strains. The SHV type was predominant among ESBLs Enterobacteriaceae producers in Bulgarian hospitals. TEM type was proved in one hospital. CTX-M types ESBLs have increased from 2001 after their first detection and become emerging problem in Bulgaria. All strains were highly polyresistant.


6. ANTIHELMINTIC ACTIVITY OF NEW PIPERAZINE - CONTAINING BENZIMIDAZOLES

K. Anichina, D. Vutchev, A. Mavrova

ABSTRACT
Three new synthetic analogues of the benzimidazole and piperazine hexahydrate have been developed. Experiments on white mice, infected with Syphacia obvelata show antihelminthic activity over 50% intens-efficacy /1Е/. This result is promising and further investigation is necessary.


7. ANTIPROTOZOAL EFFECT OF SOME NOVEL BIS-BENZIMIDAZOLES - AN EXPERIMENTAL STUDY

D. Vutchev, K. Anichina, A. Mavrova

ABSTRACT
Movel bis-benzimidazole compounds with confirmed antiparasitic activity in helminthoses are synthesized. Experimental investigations on the antiprotozoal effect “in vitro” on Paramaecium caudatum are carried out Chemotherapeutical tests “in vivo” on with mice infected with Lamblia muris are performed with the most effective bis-bensimidasoles. Some of the tested compounds have a well expressed antiprotozoal activity.


8. CORRELATION BETWEEN FIVE METHODS FOR ANTIFUNGAL SUSCEPTIBILITY TESTING OF FLUCONAZOLE, VORICONAZOLE, ITRACONAZOLE, KETOCONAZOLE, 5-FLUOROCITOZINE AND AMPHOTERICINE B AGAINST CANDIDA SPP

Z. Ivanova, A. Kouzmanov, L Boyanova

ABSTRACT
Antifungal susceptibility testing may be an important aid in the treatment of patients with life-threatening yeast infections. The activity of Fluconazole, Itraconazole, Ketoconazole, Voriconazole, 5-Fluorocitosine, Amphotericine B against 71 clinical isolates of Candida spp. were tested by the E-test, disk diffusion, microdilulution method NCCLS M27-A2, ATB Fungus and Merlin. We determined comparatively high per cent of resistant to asole antifungals strains and good activity of Amphotericine B. The IMCCLS method was found to be too complex and labor-intensive for routine testing. The agar-based E-test and disk diffusion methods are reliable alternative to the NCCLS M27-A2 reference microdilution method, but experience in determining MICs and careful attention to procedural details are critically important. Continued surveillance and refinement of broth - and agar-based test methods will help to identify susceptibility trends and improve the laboratory capability for antifungal susceptibility testing.


9. COMPARATIVE STUDY OF LABORATORY METHODS FOR IDENTIFICATION OF MEDICALLY IMPORTANT YEASTS

L Boyanova, A. Kouzmanov, Z. Ivanova

ABSTRACT
Advances in antifungal therapy necessitate the need for accurate identification of fungi, especially yeasts to their species level for more effective management. The identification of yeasts is best accomplished using a combination of colonial, microscopic and biochemical methods. The colonial morphological features are variable and often are not helpful. The microscopic morphological features of yeasts are helpful, but don’t give 100% correct identification. Biochemical methods include fermentation and assimilation patterns of various carbohydrates, the new commercialized yeast identification systems are simpler, rapid and are particularly easy to interpret.


10. PRELIMINARY CHARACTERIZATION OF THE TYPES OF BETA-LACTAMASES PRODUCED BY CEFTAZIDIME-RESISTANT PSEUDOMONAS AERUGINOSA STRAINS

T. Strateva, R. Markovska, I. Mitov

ABSTRACT
Objective:
To preliminary characterize the main types of beta-lactamases among ceftazidime-resistant strains of P. aeruginosa and to determine their rate of resistance.

Materials and methods: A total of 30 ceftazidime (CAZ)-resistant P. aeruginosa strains were collected from four large hospitals in Sofia during 2001-2005. Antimicrobial resistance towards 17 antimicrobial agents among the selected strains has been surveyed according to NCCLS-2003. The antibacterial activity of some beta-lactams, aminoglycosides and ciprofloxacin has been tested by determination of their minimum inhibitory concentrations (MICs) using Etest (AB Biodisk). The prevailing resistance mechanisms were studied as previously described by Jarlier et al. and Lee et al. Isoelectric points (pis) of beta-lactamases produced by these strains were determined by isoelectric focusing (IEF) according to Mathew/Bauernfeind. The hydrolytic activity of the bands was proved by Bioassay (Bauernfeind).

Results: The rates of resistance were: to carbenicillin- 100 %, azlocillin- 96.7 %, piperacillin-93.3 %, piperacillin/tazobactam - 56.7 %, ceftazidime - 100 %, cefoperazone - 100 %, cefepime - 100 %, cefpirome - 100 %, aztreonam - 96.7 %, imipenem - 56.7 %, meropenem - 56.7 %, amikacin - 93.3 %, gentamicin - 93.3 %, tobramycin - 96.7 %, netilmicin - 76.7 %, ciprofloxacin - 90 % and polymyxin B - 0 %. MICs of CAZ ranged from 32 to >256 mg/l. 14 strains of all 30 CAZ-resistant P. aeruginosa (46.7 %) were presumptive producers of extended-spectrum beta-lactamases (ESBLs) according to the double disk synergy test of Jarlier et al. 12 strains (40 %) were resistant to all beta-lactams, including to carbapenems, but they did not show a positive Hodge test for metallo-beta-lactamases. The strains demonstrated four different beta-lactamases with pis: 5.7; 6.1; 7.4 and 8.2. 25 strains of all studied CAZ-resistant P. aeruginosa (83.3 %) expressed CAZ hydrolytic bands with pi value of 7.4 (positive Bioassay) likely corresponded to the pi of VEB-1 ESBL The other presumptive beta-lactamases were: CARB-enzymes (pi 5.7), OXA-enzymes (pi 6.1) and AmpC cephalosporinase (pi 8.2). In 5 from investigated 30 strains (16.7 %) resistance to CAZ was associated with the overproduction of chromosomal AmpC beta-lactamase, or with non-enzymatic mechanisms such as drug efflux or outer membrane impermeability.

Conclusion: In the present study resistance to extended-spectrum cephalosporins, including to CAZ, was related to the production of VEB-1 type ESBL from Ambler class A. VEB-1 is widespread mostly in Asia and is rarely in European countries. Nevertheless it appears to have a significant presence among CAZ-resistant P. aeruginosa isolates in Bulgarian hospitals and causes serious impediments in antimicrobial treatment.


11. CHARACTERIZATION OF EXOCELLULAR NEURAMINIDASE FROM ERYSIPELOTHRIX RHUSIOPATHIAE

I. Abrashev

ABSTRACT
A method for isolation and purification of neuraminidase from Erysipelothrix rhusiopathiae is described. Some of the characteristics of the enzyme as molecular mass, elecrophoretic mobility, isoelectric point, chemical nature etc. are presented.


12. CHARACTERIZATION OF AN EPIDEMIC METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS STRAIN SPREAD IN BULGARIAN HOSPITALS

D. Nashev, K. Toshkova, S. Salasia, A. Hassan, C. Lammler, M. Zschock

ABSTRACT
The present study was designed to investigate the clonal relationship of methicillin-resistant Staphylococcus aureus (MRSA) strains isolated from different hospitals in Bulgaria. The isolates were analyzed by pulsed-field gel electrophoresis (PFGE) after Smal digestion and by PCR amplification of the genes encoding coagulase (coa), the gene segments encoding X region and the IgG binding region of protein A (spa) and the genes encoding staphylococcal enterotoxin A (sea), B (seb), C (sec), D (sed), E (see), G (seg), H (seh), I (sei) and J (sej) and the toxic shock syndrome toxin 1 (tst). The isolates could be classified to PFGE profile type A with 2 subtypes, type B with 5 subtypes and to type C. All three types were considered to represent different MRSA strains. This classification could generally be confirmed by the indistinguishable PCR profiles of the genes spa and coa and by identical toxin gene patterns. The prevalent MRSA type A strain caused outbreaks in three hospitals in 1995, 1998 and 1999. The same strain could also be isolated from sporadic cases of infection and colonization in two other hospitals and from outpatient clinic. This PFGE type A MRSA strain was isolated from more than two patients and from more than two hospitals and could be defined as epidemic MRSA strain. The isolates belonging to PFGE type B caused an outbreak in one hospital and occurred sporadically in a second hospital. The third PFGE type C was found in sporadic cases in one hospital and in outpatient clinic. According to the data of the present study, a single epidemic MRSA strain seems to be responsible for infections of patients in at least five major Bulgarian hospitals.


13. EXPRESSION LIBRARY IMMUNISATION REVEALING IMMUNO-DOMINANT DETERMINANTS OF FRANCISELLA TULARENSIS USING PLASMID DNA IMMUNIZATION TO SCREEN SUB-LIBRARIES FOR INDUCTION OF ANTI-FRANCISELLA TULARENSIS RESPONSES IN MICE

P. Padeshki, T. Kantardjiev, M. Mincheff, I. Tone

ABSTRACT
The Expression-Library Immunization (ELI) technique was used to take advantage of the relatively small genomes of the pathogen F.tularensis and the already established protocols for plasmid DNA immunization in rodents. Two separate representative genomic libraries have been created and tested for that pathogen. The first - encodding secreted products to be processed via the class II MHC presentation pathway and stimulated CD4, CD8 T cell and humoral immunity. Alternatively, products from the second one targeted to the proteasome and processed via the class I MHC presentation pathway for primarily CD8 T cell stimulation. Immunization studies were conducted in a DBA mouse model and the expression libraries were screened for protection against F.tularensis. Mice were immunized with expression libraries containing the entire genome of the pathogen. Partial (prolongation of survival) protection from pathogen-induced disease in immunized animals identified sub-libraries that contain plasmids encoding for reactive antigenic epitopes. In this way, expression library immunization provides an unbiased, systemic approach for isolating vaccine candidates.


 

Editor-in-Chief
Prof. B. Petrunov, MD, DSc

Production Manager
Theodor Koshev

Editorial Board
Prof. H. Taskov, MD, DSc
Prof. P. Nenkov, MD, DSc
Assoc. Prof. R. Kurdova, MD, PhD
Assoc. Prof. R. Kotzeva, MD, PhD
Assoc. Prof. N. Gatcheva, MD, PhD
Assoc. Prof. T. Kantardjiev, MD, PhD

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