TABLE OF CONTENT

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1. INVESTIGATING THE RELATIONSHIPS BETWEEN ANTIBIOTIC CONSUMPTION AND RESISTANCE (MINIREVIEW)

B. Todorova

ABSTRACT
All methods for investigating and evaluating the impact of antimicrobial use on resistance pursue the goal of predicting future behaviour of resistance rates, identifying risk factors in the face of available treatment options and developing strategies for combating the emergence and spread of difficult-to-treat pathogens. There is a perception of hospital environment as the main reservoir of antibiotic resistance since patient population is highly exposed to antibiotic pressure. The strongest evidence for the actual relationship between antibiotic consumption and resistance could be drawn from data collected on each individual patient's exposure to antibiotics and subsequent emergence of resistance. Due to different constraints, most hospital researchers have only aggregated microbiology and pharmacy data available and this kind of analysis provides only correlations but not direct causal evidence. However ecological studies allow the formation of an overall view of the total effect of antimicrobials and encompass consequences of both individual antibiotic therapy courses and transmissibility of resistance.


2. CARBAPENEMASES – TYPES AND DETECTION

K. Ivanova

ABSTRACT
The spread of carbapenemase-producing bacteria is a threat to healthcare and patient safety. In Bulgaria carbapenem resistance is relatively new phenomenon, which has to be studied, especially when it occurs with outbreaks in hospital settings and in the society. The need of such investigation is related to identification of new and circulating carbapenemases and in-depth examination of the phenotype and genetic characteristics of the carbapenemase-producing bacteria detected in our country. Although carbapenemases belonging to Ambler’s classes A (KPC-2), B (VIM, NDM-1), and D (OXA-23, OXA-24, OXA-58) have been reported, still there is no full and comprehensive data on the epidemiology of the carbapenemase positive strains in our country. Another issue of major importance is tracking the emergence and the spread of carbapenem-resistant Enterobacteriaceae, which have been increasingly reported worldwide. For a period of 5 months (May-September 2014) at the National Reference Laboratory “Control and Monitoring of Antibiotic Resistance” in the Department of Microbiology at NCIPD were identified several carbapenemase-producing Enterobaceriaceae: five KPC-producing K.pneumoniae isolates, one NDM-producing K.pneumoniae, and VIM-producing S.marcescens.


3. RAPIDLY GROWING MYCOBACTERIA IN SUSPECTED FOR TUBERCULOSIS PATIENTS IN BULGARIA

Y. Atanasova, E. Bachiyska, S. Yordanova, Y. Todorova, A. Baykova, T. Kantardjiev

ABSTRACT
The aim of this study was to describe Nontuberculous Mycobacteria (NTM), in particular Rapidly Growing Mycobacteria (RGM), isolated from clinical specimens of suspected for tuberculosis patients in Bulgaria. Notification of NTM is not mandatory in Bulgaria, according to Regulation 21 of the MoH (in contrast to registration of TB infections) (2). The survey was conducted at the National Reference Laboratory of Tuberculosis, National Centre of Infectious and Parasitic Diseases (NRL-TB, NCIPD) and covers the five year period – from 2009 to 2013. NTM were identified by conventional phenotypic methods and line probe assays. In total 245 NTM strains were identified to species level. More than one third of them were RGM - 32.65% (n = 80) including the clinically significant species: M. abscessus, M. chelonae and M. fortuitum - 73.75% (n = 59).


4. DEVELOPMENT AND PRELIMINARY EVALUATION OF A NOVEL METHOD FOR DNA EXTRACTION FROM SERUM

K. Dimitrov, D. Dimitrov, I. N. Ivanov

ABSTRACT
Molecular methods became indispensable tool for diagnosis of various infectious diseases. In cases of bacteremia and sepsis, DNA detection assays provide the speed and sensitivity required for initiation of an early appropriate antimicrobial treatment. While blood is still the preferred diagnostic specimen, a number of studies brought evidence for the value of serum as an alternative sample for PCR-based diagnosis of sepsis. Thus in this study our efforts were focused on developing a rapid and efficient single tube (ST) method for DNA isolation from simulated serum samples. Evaluation of the method was accomplished by two separate Real-time PCR assays on simulated serum samples. In a side by side comparison, the optimized DNA extraction procedure performed equally or with better efficiency than a commercial DNA extraction kit.


5. WEST NILE VIRUS INFECTION IN A BULGARIAN MAN: A CASE REPORT

M. Baymakova, E. Panayotova, I. Christova, S. Dakova, R. Mihaylova-Garnizova, K. Ramshev, K. Plochev

ABSTRACT
West Nile virus belongs to the family Flaviviridae. Birds are the natural reservoir, whereas humans and horses are dead-end hosts. The transmission is maintained by mosquito biting. The enzootic/epizootic cycle is linked to the activity of mosquito vectors of the genus Culex. The clinical presentation varies from subclinical to acute febrile illness and/or neurological manifestation. We present a case report of a young man with acute febrile syndrome and CNS disorder. Testing of acute and convalescent serum samples showed high level of IgM antibodies against West Nile virus.


6. DETECTION AND PHYLOGENETIC ANALYSIS OF ERYTHROVIRUS B19 (EVB19) IN PATIENTS WITH HAEMATOLOGIC DISORDERS (PRELIMINARY RESULTS)

St. Ivanova, A. Toshev, Z. Mihneva

ABSTRACT
Erythrovirus B19 (EVB19) is a pathogen associated with a wide range of disease manifestations, syndromes and pathological conditions with acute or chronic course, some of which depend largely on the condition of the immune system and the haematological status of the patient.
The aim of this study is to prove the involvement of EVB19 as the etiological and/or persistent agent in patients with haematological disorders using different laboratory diagnostic tools.

Materials and Methods: The present study included 33 serum samples from patients with haematological diseases. Group 1 included the following diagnoses: aplastic anemia, erythroblastopenia, thrombocytopenia and pancytopenia (n=10); group 2 included transfusion-dependent anaemic patients (thalassemia major and others haemolytic anemia) and haemorrhagic diathesis (n=21), and group 3 included patients with unidentified fever (n=2). Etiological presence of EVB19 was demonstrated by serological, specific B19-IgM/IgG antibodies (indirect ELISA assay), and molecular (extraction and amplification of viral DNA, sequencing and phylogenetic analysis) methods.

Results: The presence of acute infection (B19-IgM marker) was demonstrated in 4/33 (12.12%) of the tested patients. Specific B19-IgG were found in 18/33 (54.55%) as evidence of past/ persistent infection or obtained from donor blood. Positive PCR signal of B19 -DNA was reported in 14/33 (42.42%) patients. The combination of B19-IgG positive diagnostic marker and B19 DNA positive result was detected in 12/33 (36.36%) patients. 14 PCR products were subjected to sequencing and determination of EVB19 genotype. Phylogenetic analysis showed dominance of genotype 1, subgenotype 1a.

Conclusion: EVB19 is proved as a pathological factor in a number of haematological diseases for which frequent blood transfusions are indicated. This pilot study focuses on testing of donor blood for this blood-transmissible agent (demonstrated by serological and molecular markers), which is especially necessary due to asymptomatic infection with erytrovirus B19 in about 30-40% of cases.


7. HEPATOTROPIC CO-INFECTIONS IN HIV-POSITIVE PATIENTS: IMPACT ON CELLULAR IMMUNITY PARAMETERS

M. Alexandrova, M. Muhtarova, O. Angelova, E. Golkocheva-Markova, I. Elenkov, P. Teoharov, I. Alexiev, M. Nikolova

ABSTRACT
Background: Due to shared transmission routes, the prevalence of co-infections with HIV and hepatotropic viruses increases worldwide, leading to complications, unpredictable response to combined antiretroviral therapy (cART) and increased mortality.
We evaluated the impact of hepatotropic infections in HIV+ patients subjected to cART, using routine immune monitoring data. Our aim was to identify phenotypic indicators of impaired cellular immunity and therapy response in co-infection.

Materials and methods: Three patients groups were studied: A. Monoinfected (HIV+, n=15), B. HCV-coinfected (HIV+HCV+, n=15), C. HCV and HBV co-infected (HIV+HCV+HBV+, n=13). Peripheral blood samples were obtained at baseline, and after 12 months of cART. Absolute counts (AC) and percentage (%) of lymphocyte (Ly) subsets were determined by flow cytometry using Multitest6-ColorTBNK reagent and TRUCountTubes (BDBiosciences, FACSCantoII).

Results: Basic immunologic parameters and HIV viral load (VL) did not differ significantly between groups at baseline. Co-infected patients were distinguished with poor immunologic response to cART, and specific NK subset dynamics. While progression of HIV monoinfection (A) was associated with decreased levels of cytotoxic CD56lo subset, the latter was stable and correlated with viral supression in co-infected patients (B, C). Hepatotropic co-infection affected significantly the regulatory CD56hi subset: it increased in HIV+HCV+ group (B) and lastingly decreased in double co-infection (C), regardless of cART.

Conclusions: Hepatotropic co-infection significantly affects cART mediated immune recovery. This effect is independent from influence on viral load, and is mediated at least in part through disturbed differentiaton and balance of CD56hi/CD56loNK subsets. Extending the routine immune monitoring to peripheral blood NK subsets is instrumental in HIV/hepatotropic co-infection.


8. SUITABILITY OF WEEKLY CASE DATA ON TOTAL ACUTE HEPATITIS FOR THE DETECTION OF VARIATIONS IN HEPATITIS A INCIDENCE

S. O. Stoitsova, A. Minkova, M. Kojouharova, A. Kurchatova

ABSTRACT
The development of a model for early detection of unexpected increases in hepatitis A incidence can facilitate timely risk communication and other public health measures, aimed at reducing further infection transmission. In Bulgaria, the absence of weekly reporting on hepatitis A cases complicates the design of this model. Due to the technical time it takes to identify the causative agent of an acute hepatitis case, weekly reporting is aimed at providing only the total number of acute hepatitis cases, without specifying the virus type. Acute hepatitis cases are then broken down by causative agent in the monthly and annual reports. This study evaluates whether acute hepatitis case numbers in weekly reports can be used to estimate hepatitis A case numbers. The reliability of weekly and monthly reports is assessed through careful consideration of error rates. The relationship between weekly acute hepatitis and monthly hepatitis A case numbers at national and regional levels is assessed through correlation. The analysis demonstrates that weekly operational data on acute hepatitis cases is suitable for further modelling of variations in hepatitis A virus incidence.


9. STRIVING FOR BETTER COMMUNICATION WITH UNDERSERVED COMMUNITIES IN BULGARIA - A STEP TOWARDS IMPROVING IMMUNISATION COVERAGE

L. Marinova, K. Parmakova, M. Kojouharova

ABSTRACT
With regard to the immunisation uptake, Roma communities in Bulgaria are still underserved. Evidence of the inadequate immunisation coverage in this population is the large measles outbreak occurred in the country in 2009-2011 which affected predominantly Roma people who were about 89% from all measles cases. The international project “Let’s talk about protection” was developed with the aim to identify a proper way for communication with parents, especially from so called “hard-to-reach” groups, to motivate them to vaccinate their children and thus to increase the immunisation uptake. It started in 2012 and finished in 2014. Experts from the National Centre of Infectious and Parasitic Disease in collaboration with specialists from other institutions (European Centre for Disease Prevention and Control, World Health Communication Associates and Bulgarian Association “National Network of Health Mediators”) adapted a Practical Guide, created and issued a Flipbook on vaccines and immunisations with focus on the communication between health care providers and parents. As a result of the project, it is expected that there will be increased awareness among parents with regard to benefits from timely vaccination of their children.


 

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

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

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