TABLE OF CONTENT

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1. IN VITRO ANTIMICROBIAL SUSCEPTIBILITY OF FRANCISELLA TULARENSIS ISOLATED IN BULGARIA

T. Velinov , T. Kantardjiev, M. Nicolova and A. Kuzmanov

ABSTRACT
Francisella tularensis is the causative agent of tularemia, a zoonosis of the northern hemisphere. MIC50 and MIC90 values of gentamicin,  streptomycin, amikacin, penicillin, ceftriaxone, piperacillin/tazobactam, doxycycline, erythromycin, levofloxacin, ciprofloxacin and chloramphenicol  for 21 isolates of Francisella tularensis subsp. holarctica were determined using the E-test. All strains were resistant to all tested β-lactams and  rythromycin. Our in vitro susceptibility results can be applied for the detection and comparison of resistance development and provide in vitro data for  the guidance of therapy. The establishment of a standardized antibiotic susceptibility method for Francisella spp. would be useful for resistance  determination in these bacteria and possible evaluation of bioterrorism risks. These in vitro results show that fluoroquinolones are  promising antimicrobial agents for the treatment of human tularemia.


2. FIRST ISOLATION AND MICROBIOLOGICAL CHARACTERIzATION OF RARE AND UNIQUE SEROTYPES OF SALMONELLA ENTERICA IN BULGARIA, 2011

G. Asseva, P. Petrov, K. Ivanova, S. Ljubenova, K. Parmakova, M. Nikolova, B. Panova, G. Mateva, T. Kantardjiev

ABSTRACT
This is the first report of rare and unique Salmonella enterica serotypes isolated in Bulgaria in 2011 from human cases of salmonellosis. None of these four serotypes: Grampian, Szentes, Leeuwarden and II 48: z10 : [1,5] has previously occurred in the WHO data summaries and there are few publications in literature available. We defined S. Szentes as “reemerging” since it was first isolated in the 1950-s in Bulgaria and the remaining as “unique”. Susceptible profiles to a set of 12 antimicrobial agents have been determined. Strains are available at the NRL of Enteric Pathogens, Sofia, Bulgaria for further molecular and epidemiological investigations.


3. TOXINOTYPING OF STRAINS OF C. DIFFICILE

V. Tolchkov, M. Marina, T. Kantardjiev, R. Argirova

ABSTRACT
Clostridium difficile is both a human pathogen and a member of the normal human intestinal flora with clinical significance, causative agent of Clostridium difficile-associated diarrhea (1). Determination of the strains and related information about their virulence is very important for the correct diagnosis and therapy of the patients, the prophylaxis and the control of the spreading of the infection in the society (2). Precise phenotyping of Clostridium is complicated because of relative low sensitivity of classical microbiological methods and low vitality of the anaerobe Clostridium difficile in oxidative atmosphere (3). The methods developed in the last decade for PCR-techniques directly from clinical samples are not useful for sequencing and need a special equipment and transport conditions to be taken, kept and preserved until their genotyping (4). Development of quick and easy to be performed methods is important for the epidemiology and understanding the spread of infections within the society. Most important factors of virulence are citotoxins A, B and C (5). We have genotyped 17 strains from clinical isolates from Bulgaria and control strains from Netherlands in the present study and developed fast protocol for extraction of clostridial DNA from plate culture without anaerobic laminar box and additional equipment. Immunochromatography used in our laboratory is capable just to determine whether the sample contains toxigenic strain or not. It is unable to give any information about the gene expression. PCR-typing showed prevalence of gene for citotoxin C in Bulgarian hospital population.


4. IS THERE A PRESENCE OF SYNERGISM BY COMBINING OF HYBRID MATERIALS WITH INCLUDED SILVER NANOPARTICLES WITH ANTIMICROBIAL SUBSTANCES?

D. Pencheva, R. Bryaskova, T. Kantardjiev

ABSTRACT
It was investigated the presence of synergism by combining of different antibiotics and two synthesized hybrid materials with included silver nanoparticles. For this purpose two different methods were employed: Disk Diffusion Method (DDM) and Chess method. Initially, control and further clinical microbial strains were tested by DDM. The results showed the presence of synergism between disks impregnated with PVA/TEOS/AgNps and some of the tested 28 kinds of antimicrobial disks. The existence of synergism between a liquid hybrid material based on PVA/AgNps and antimicrobials was tested by the chess method as a reference method using two clinical strains with established resistance to piperacillin and cefepime.


5. DETECTION OF ANTIBODIES TO GERM TUBE OF CANDIDA ALBICANS BY MICROSCOPIC INDIRECT IMMUNOFLUORESCENCE METHOD FOR DIAGNOSIS OF SYSTEMIC CANDIDOSIS

T. Kantardjiev

ABSTRACT
We introduced a method for detection of antibodies of different subclases by microscopic immunofluorescence and worked to improve the diagnostic capabilities of tests to prove invasive infections caused by Candida albicans. Applying of the indirect immunofluorescence method for detection of antibodies to the germ tube of Candida albicans provides significant advantages in distinguishing colonization from systemic infection.


6. HANTAVIRUS INFECTIONS IN SOUTH-WESTERN REGIONS OF BULGARIA IN THE LAST 5 YEARS

M. Stoycheva, M. Pishmisheva, I. Christova, N. Vatev, C. Venchev

ABSTRACT
The aim of the study is to present epidemiological and clinical features of patients with Hemorrhagic fever with renal syndrome, in order to help early diagnosis and appropriate treatment regimen. The study involved 22 diseased of Hemorrhagic fever with renal syndrome (HFRS). They were hospitalised in Infectious diseases clinics in Plovdiv and Pazardzik –south-western Bulgaria regions, endemic for Hanta viruses, between years 2005-2009. The methods of clinical investigation, laboratory tests and epidemiological analyses were used. The serologic diagnosis was made by indirect immunofluorescent antibody test in National Center of Infectious and Parasitic Diseases, Sofia. Fever, chills, kakesthesia to prostration, headache were universally present. Intense back pain was associated in 82% of the patients. Vision disturbances as dyplopia, acute myopia had 9/22 (41%). Haemorrhagic diathesis occured in 77 %, acute renal failure in 40 %. Typical early laboratory findings are thrombocytopenia (100%), abnormal urinalysis, with markant proteinuria, hyper aminotransferasaemia. 11 patients (50 %) had to be dialysed. All patients but one recovered. Mortality rate was 4,54%. All parameters in survivers return to their normal value and a normal renal function without sequels. Clinical course of HFRS in south-western Bulgaria regions is moderate to severe. Acute renal failure is leading clinical syndrome. When dealing with patients with fever, prostration, back pain, blurred vision and thrombocytopenia, renal disturbancy and high aminotransferase level, Hantavirus infection should be kept in mind. History should be south for any possible contact with rodents.


7. CARRIERS OF HEPATITIS B AND HEPATITIS C AMONG MEDICAL PERSONNEL AND PROTECTION BY VACCINE

A. Kevorkyan, P. Teoharov, N. Petrova,Y. Stoilova, A. Plachkova, N. Angelova. Angelov

ABSTRACT
Medical personnel are at increased risk of nosocomial infection with hepatothropal viruses, mainly hepatitis B.

Objective: To assess the carriage of hepatitis B and hepatitis C and vaccination coverage with recombinant hepatitis B vaccine among medical staff to improve prevention.

Materials and methods: 100 serum samples from non-immune healthcare workers were studied for HBsAg and anti-HCV, and data on the immunization status of 1885 persons working in the University Hospital St. George, Plovdiv analyzed. Conventional serological methods were used, the complex epidemiological method and statistical method - an alternative analysis.

Results: Carriers of HБsAg were found in 4% of the subjects with low immunization coverage – 59 % in 2009 and 62 % in 2010. There are no proven anti-HCV positive samples.

Conclusion: The protection of medical personnel from occupational exposure to biological agents must be among the priorities in the overall set of measures to control hepatitis B in health care facilities.


8. PROLIFERATION PATTERNS OF CD8 T CELL SUBSETS IN HIV+ PATIENTS ARE ASSOCIATED WITH THE LEVEL OF REGULATORY CD25HIGH CD4 T CELLS

M. Nikolova, M. Muhtarova, D. Beshkov, I. Elenkov, h. Taskov

ABSTRACT
Chronic HIV infection profoundly perturbs CD8 T cell responses, although the mechanisms for this are not completely elucidated. We hypothesised that persistent antigen burden affects the proliferation potential and differentiation state of the general CD8 T cell pool. Using CFSE staining and multicolour flow cytometry, we compared the proportions and proliferation responses of naïve (CD45RA+CD27+), memory (CD45RA-CD27+,M), effector–memory (CD45RA-CD27-, EM), and terminally-effector (CD45RA+CD27-,TE) CD8 T subsets in HIV+ patients and HIV- controls. Two patterns of CD8 T cell proliferation and differentiation were observed in HIV+ patients: subgroup A (N=13) with expanding M (81% vs. 16% on D0), and steady EM (12 vs. 9% on D0) CD8 subsets, comparable to HIV- controls, and subgroup B (N=11): with predominant differentiation and accumulation of EM CD8 T cells (48% vs. 22%). No significant differences between subgroups A and B regarding CD4 AC or HIV-specific response were established. However, group A presented with a significantly lower VL (4.6 vs. 2.0 log HIV RNA copies/ml), and a higher level of circulating Treg cells (8.4% vs. 4.9%, p<0.05), combined with a significantly higher proportion of naïve CD8 T cells and a lower proportion of TE. Thus, HIV-induced Treg might determine the differentiation state and functional fitness of the CD8 T cell pool in the settings of long-lasting chronic infection and should be considered during immune monitoring.


9. CIRCULATING CD8+ T CELL SUBSETS AND CD39+ T REGULATORY CELLS IN PATIENTS WITH HBEAG NEGATIVE CHRONIC HEPATITIS B

M. Nikolova, M. Petrova, M. Muhtarova, D. Nikolovska, Z. Krastev, H. Taskov

ABSTRACT
CD4+CD25high FoxP3+ regulatory T cells (Treg) are a heterogeneous population that maintains immune tolerance but may suppress antigen-specific T-cell responses in chronic infection. Here we investigated the role of CD39+ T regulatory subset in 28 HBeAg(–) hepatitis B patients. Serum HBV DNA was quantified by real-time PCR. T-cell subsets were determined by flowcytometry. HBeAg(–) hepatitis B patients presented increased effector-memory (CCR7-CD45RA-), (p<0.001) and decreased naïve (CCR7+CD45RA+), (p<0.001) and cytotoxic (CD27-CD28-CD160+) CD8 T (p<0.01), as compared to controls. A significantly higher effector-memory / central-memory ratio was observed in patients with high viral load as compared to those with low viral load (p<0.01). The increased percentage of patient’s CD39+ T regulatory cells correlated directly with the percentage of effector-memory CD8 (R= 0.8, p<0.01), and the level of liver enzymes (R=0.7, p<0.05). Furthermore, an increased density of CD39 molecules on T reg correlated with HBV viral load (R=0.8, p<0.01). The increased level of CD39+ Treg might prevent the extreme differentiation of antigen-specific T-cell clones and should be further studied and monitored in hepatitis B patients.


 

Editor-in-Chief
Prof. T. Kantardjiev, MD, DSc

Editorial Board
Acad. B. Petrunov, MD, DSc
Prof. H. Taskov, MD, DSc
Prof. M. Kojuharova, MD, PhD
Prof. R. Kurdova, MD, PhD
Assoc. Prof. I. Christova, MD, DSc
Assoc. Prof. I. Rainova, MD, PhD
Assoc. Prof. P. Teoharov, MD, PhD

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