TABLE OF CONTENT

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1. SEROTYPE DISTRIBUTION AND ANTIBIOTIC SUSCEPTIBILITY OF S. PNEUMONIAE CAUSING INVASIVE INFECTIONS AND ACUTE OTITIS MEDIA IN BULGARIA BEFORE INTRODUCTION OF PNEUMOCOCCAL CONJUGATE VACCINE

D. Nashev, T. Velinov, A. Kuzmanov, L. Setchanova, N. Petrov, L. Bizeva, I. Pavlina, Z. Ivanova, L. Boyanova, D. Merdzhanov, T. Kantardjiev

ABSTRACT
We report data on serotype distribution and antibiotic susceptibility of 36 invasive S. pneumoniae isolates and 50 isolates from acute otitis media (AOM). The invasive isolates were collected in 14 hospitals located in 10 Bulgarian cities between June 2009 and December 2010. Sixteen isolates were obtained from infections in children ≤5 years and 20 – from children >5 years and adults. The AOM isolates were collected between October 2009 and November 2010 from children aged ≤8 years. The universal infant immunization in Bulgaria with PCV10 was initiated in April 2010, so we considered the time period to the end of 2010 still representative for the pre-vaccine serotype distribution in Bulgaria.

The most frequently encountered serotype among invasive isolates was 19F (13,9%), followed by 1 (11,1%), 4 (8,3%), 6B, 23F, 9V and 3 (5,6% each). Serotypes 6A, 19A, 18C and 7F were represented by one isolate each. Among the 16 IPD isolates from children <5 years the most frequent serotypes were 19F (n=4) and 1 and 4 (n=3 each), followed by 6B, 23F, 9V and 18C (n=1 each). The most commonly encountered serotype among AOM isolates was 19F (24%), followed by 6B (14%), 14 (8%), 9V and 3 (6% each) and 23F and 18C (4% each).

According to CLSI breakpoints, among meningitis isolates penicillin-resistance rate was 36,8%. All of the non-meningitis isolates were penicillin susceptible with the exception of one isolate which was intermediate. Among invasive isolates erythromycin, clindamycin and TMP/SMX resistance rates were 17%, 17% and 33%, respectively. The majority of AOM isolates (52%) were penicillin non-susceptible, of which 12% were penicillin-resistant and 40% penicillin-intermediate. AOM isolates resistant to erythromycin, clindamycin and TMP/SMX were 44%, 32% and 56%, respectively. Among both invasive and AOM strains, resistance to levofloxacin and rifampin was not found. Most of the penicillin non-susceptible and polyresistant S. pneumoniae isolates belonged to serotypes 19F, 6B and 14.


2. STUDY OF BACTERIAL ETIOLOGY OF ACUTE OTITIS MEDIA IN BULGARIA BEFORE INTRODUCTION OF PNEUMOCOCCAL CONJUGATE VACCINE

D. Nashev, T. Velinov, A. Kuzmanov, L. Setchanova, N. Petrov, L. Bizeva, I. Pavlina, Z. Ivanova, L. Boyanova, T. Kantardjiev

ABSTRACT
We report microbiological data on 119 middle ear specimens collected from children aged ≤8 years diagnosed with AOM between October 2009 and November 2010. Among culture positive samples (105/119; 88%) the most frequently isolated pathogen was S. pneumoniae (47,6%), followed by H. influenzae (27,6%), M. catarrhalis (8,6%) and S. pyogenes (6,7%). All H. influenzae isolates, except one, were non-typable (97%). The most commonly encountered serotype among pneumococcal isolates was 19F (24%), followed by 6B (14%), 14 (8%), 9V and 3 (6% each) and 23F and 18C (4% each). The prevalence of penicillin non-susceptible S. pneumoniae was relatively high (52%) and associated mainly with serotypes 19F, 6B and 14.


3. ANTIMICROBIAL ACTIVITY OF TIGECYCLINE AGAINST MULTIDRUG-RESISTANT STRAINS COLLECTED IN BULGARIA

T. Velinov, B. Todorova, M. Petrov, V. Dimitrova T. Kantardjiev

ABSTRACT
Background:
Tigecycline is a new injectable glycylcycline antibacterial product with potent activity and broad spectrum against Gram-positive and Gram-negative bacteria currently used for the treatment of complicated skin and skin structure-infections and intra-abdominal infections. We evaluated the in vitro activity of this compound against clinical strains of Gram-positive and Gram-negative species collected from Bulgarian medical centers in the National Reference Laboratory for “Control and Monitoring of Antimicrobial Resistance” at the NCIPD.

Methods: A total of 187 non-duplicate clinical strains from different medical centers were tested for susceptibility by reference broth microdilution methods according to Clinical and Laboratory Standards Institute guidelines and interpretative criteria.

Results: All S. aureus strains were inhibited at a tigecycline MIC of ≤0.5 µg/ml. Among the tested Enterococcus strains the highest tigecycline MIC value was 0.25 µg/ml. Similarly ESBL producing E. coli and K. pneumoniae exhibited uniformly susceptibility to this compound with MIC values reaching 2 µg/ml only for some strains K. pneumoniae. Among the multiple drug resistant A. baumannii strains collected, our assessment indicated MIC 90 value of 2 µg/ml.

Conclusion: Tigecycline showed highly potent in vitro activity against all clinical strains tested and appears to be an excellent therapeutic option for serious infections caused by difficult-to-treat microorganisms. The present study did not detect any tigecycline-resistant strains with the exception of a single A. baumannii isolate with MIC value of 8 µg/ml.


4. PCR-RIBOTYPING AND PCR METHODS FOR DETECTION OF TOXIN CODING GENES IN CLOSTRIDIUM DIFFICILE STRAINS

E. Dobreva, I. Ivanov, R. Vatcheva-Dobrevska, K. Ivanova, M. Marina, P. Petrov, T. Kantardjiev, E. Kuijper

ABSTRACT
C. difficile infections (CDI) are associated with patients who have contact with health care settings and with antibiotic exposures. This anaerobic bacterium causes asymptomatic colonization to severe diarrhea; pseudomembranous colitis, toxic megacolon, intestinal perforation and death.
C. difficile is recognized as a gut colonizer and a cause of diarrhea in several animal species. The enteropathogen produces enterotoxin A and cytotoxin B. The majority of strains with changes in coding genes tcdA and tcdB produce a binary toxin CDT.

PCR-ribotyping method and PCR methods for detection of toxin coding genes were presented for characterization of C. difficile strains. Ninety stool samples from patients (65/90) and animals (25/90) were investigated for C. difficile. 20% (18/90) of all samples were positive for C. difficile. 23% (15/65) from clinical samples and 12% (3/25) from horses were positive for C. difficile by culture test. 21, 5% (14/65) of clinical isolates produce toxins A and B by EIA. 86,7% (13/15) from clinical samples were PCR positive for tcdA gene. Deletion in tcdA gene (714bp) was detected in 40% (6/15) of the clinical strains. 93,3% (14/15) C. difficile clinical strains were positive for tcdB gene. Three toxigenic variants C. difficile have been distinguished among clinical strains by PCR: 46,67% (7/15) toxin A+B+; 46,67% (6/15) A–B+ and 6,67% (2/15) A-B-. The binary-toxin genes cdtA and cdtB was PCR detected in one of the A+B+ strains. The genes tcdA, tcdB and cdtA/cdtB were not detected in C. difficile isolates from horses by PCR.

The most prevalent ribotypes among C. difficile clinical strains were: 017- 40% (6/15); 002- 13%; 014/020-13% and 012, 046, 078 were represented by 7% each. Patterns were compared to reference ECDC C. difficile collection. Thirteen percent of C. difficile clinical strains were corresponded to unknown PCR-ribotypes. PCR-ribotyping patterns of the C. difficile isolates from horses were different from patterns of the clinical strains.
The significant number of cases C. difficile diagnosed with outbreak ribotypes may represent a significant problem in the future.


5. LYME BORRELIOSIS IN PATIENTS OVER 60-YEARS-OLD

M. Gospodinova, I. Christova

ABSTRACT
Lyme borreliosis is the most common tick-borne infection in Europe. All age groups are susceptible. Problems of persons over 60 years (60 +) are the focus of WHO and relative institutions. Various aspects of infectious diseases in patients over 60s are not investigated. Our goal was to investigate the course of Lyme borreliosis (LB) in elderly patients. 74 patients with Lyme borreliosis aged 61 to 85 years and 89 controls aged 20 to 57 years for the period 2000 – 2007 were examined. Routine clinical and epidemiological methods, hematological and biochemical tests, serological methods for detection of anti-borrelial antibodies in serum and CSF of patients were used. Variation, alternative, parametric, correlation and graphical analysis were used for statistical analysis. Within this period in Varna region, 497 people were infected with LB, of whom 344 were hospitalized. Of these, 149 were aged over 60 years (32.55 percent). 77.02% of the adults have accompanying illnesses. Clinical course, laboratory findings, seropositivity and therapeutic approaches in patients with LB at 60 + are described. Conclusions: Lyme borreliosis proceeds as normal in patients over 60 years; in patients at 60 + late LB was not observed; the most common form of early LB in elderly patients was EM; in 44.59% of the patients wound, crusta, necrosis or bula were found in the central part of EM; regional lymphonodulitis was established in 48.64% of the patients over 60 years with LB; initial seropositivity was present in 18.90% of the patients at 60 +. Early and adequate treatment of the old patients conducted in the early stage of LB is beneficial and a guarantee of a good prognosis.


6. CHLAMYDIA TRACHOMATIS IN SYMPTOMATIC PATIENTS FROM SOFIA, BULGARIA: A FOUR YEAR SURVEY, 2006-2009

S. Panaiotov, N. Brankova, V. Levterova, K. Tankova

ABSTRACT
In total 512 samples were examined for Chlamydia trachomatis. All samples were analyzed by nucleic acid amplification (PCR) test. The specimen of choice for the men was first-catch urine and cervical swab sample for the women, as recommended. We identified prevalence of C. trachomatis of 3.71% in the examined samples. The prevalence of Chlamydia infection for the town of Sofia could not be deduced from these figures. We identified higher prevalence of C. trachomatis among men, thus men possibly provide a reservoir for continued transmission to women. Screening more men with nucleic acid amplification test assays may provide the possibility of reducing the overall burden of Chlamydia in both men and women.


7. EVALUATION OF A NEW MOLECULAR TEST FOR THE IDENTIFICATION OF DRUG RESISTANCE IN MYCOBACTERIUM TUBERCULOSIS CLINICAL ISOLATES

I. Bergvala, S. Sengstakea, E. Bachyiskab, N. Brankovab, K. Tankovab, V. Levterovab, A. Ivanovab, Y. Atanasovab, S. Yordanovab, E. Abadiac, C. Solac, M. Akhaliad, R. Aspindzelashvilid, D. van Soolingene, R.M. Anthonya, T. Kantardjievb, S. Panaiotovb

ABSTRACT
Mycobacterium tuberculosis is a clonal organism, once a single nucleotide polymorphism (SNP) is introduced into the genome it will be passed on to the next generation. Drug resistance in M. tuberculosis strains is to a great extent caused by the presence of SNPs in genes conferring resistance to tuberculostatics. SNPs can be used as discriminative markers for drug resistance as well as to generate robust phylogenetic trees. SNP based species identification is also possible. A new molecular method was recently developed for the analysis of drug resistance and strain typing of M. tuberculosis strains, based on the detection of informative genetic markers such as deletions or single nucleotide polymorphisms (SNPs).

The method evaluated in this study, multiplex ligation-dependent probe amplification (MLPA) [Schouten, 2002] contains three essential steps, a hybridization step followed by a ligation step and an amplification step. The final read-out is performed on the MAGPIX device using the Luminex xTAG bead assay. The multiplexing capacity of the assay allows the simultaneous analysis of up to 47 different molecular markers within one sample and therefore provides complex information from a single assay. 13 markers provided information about resistance to first- and second line drugs based on the detection of specific mutations in the bacterial genome. We have analysed DNA isolated from bacterial culture by MLPA and compared the results to the current molecular method for the detection of drug resistance in TB strains, the MTBDRplus and MTBDRsl assays (HAIN Lifesciences).


8. MOLECULAR-GENETIC METHODS IN THE DIAGNOSTICS OF DENTAL INFECTIOUS CAUSED BY OBLIGATELY ANAEROBIC BACTERIA

V. Tolchkov, M. Marina, T. Kantardjiev

ABSTRACT
The diagnostics of human dental pathogens is essential for the correct treatment of gingivitis, periodontitis and other diseases of the oral cavity. The most often causative agents are anaerobic bacteria such as Fusobacterium nucleatum, Porphyromonas gingivalis and others. Their cultivation is time and work consuming and expensive as well. In the last 20 years a variety of molecular methods for fast diagnosis and typing appear that allow exact determination on species, strain and biovar level. On the other hand development of the quantitative PCR gives a possibility to estimate the ratio of bacterial cells from the normal flora and pathogenic strains – very important for the correct diagnosis.


9. PERIODONTAL ANAEROBIC INFECTIONS AND CARDIOVASCULAR DISEASES

V. Tolchkov, M. Marina, T. Kantardjiev

ABSTRACT
For a long time the anaerobic bacteria and other periodontal pathogens were considered as causative agents of the infections of the teeth and oral cavity. In the past years series of studies of different investigators showed relation between the oral microflora and dental and cardiovascular diseases. Therefore in the present days diagnostics of the oral bacteria (mostly anaerobes) is important both for dentists and cardiologists to make a correct decision for the prophylaxis and treatment of dental and cardiovascular diseases. In this review we discuss some important aspects about the health problems for humans related with the oral anaerobes.


10. TESTING OF THE CONTROL STRAIN ESCHERICHIA COLI O 104 USING HYBRID MATERIALS BASED ON SILVER NANOPARTICLES

D. Pencheva, R. Bryaskova, T. Kantardjiev

ABSTRACT
The Minimal Bactericidal Concentration and the zone of inhibition of two types of hybrid materials containing silver nanoparticles have been determined. These results could increase the possibilities of their usage for diagnostic, prevention and treatment of Enterohemorragic Escherichia coli infections.


 

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

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

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