TABLE OF CONTENT

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1. DEVELOPMENT OF A MODIFIED MULTI-LOCUS VARIABLE NUMBER OF TANDEM REPEATS ANALYSIS FOR TYPING OF B. PERTUSSIS APPLICABLE TO CLINICAL SAMPLES

N. Brankova, S. Panaiotov, I. Ivanov, V. Levterova, I. Simeonovski, R. Durmaz, A. Karagoz, T.Kantardjiev

ABSTRACT
Pertussis is one of the major vaccine - preventable diseases with global distribution. In recent years the world has seen an increase in both the total number of cases and in those resulting in death of newborns. The purpose of this paper is to present the development of a new method for MLVA typing of B. pertussis applicable both to pure cultures and clinical samples. A new VNTR locus VNTR11 is described and evaluated for typing of B. pertussis. VNTR11 is characterized by seven alleles and seems to be to be highly polymorphic and hence useful for typing. In addition we have modified the method with the usage of unlabeled primers as to be applicable to agarose electrophoresis. From the 51 samples tested, 32 MLVA genotypes were registered. The results obtained by using this method show that the B. pertussis strains circulating in Bulgaria during the last decade are genetically similar but somewhat divergent from the strain used for the production of the whole cell vaccine in Bulgaria.


2. RECURRENT RESPIRATORY PAPILLOMATOSIS: THE IMMUNOLOGICAL POINT OF VIEW

E. Vetzkova, M. Nikolova

ABSTRACT
Recurrent respiratory papillomatosis (RRP) is a clinical manifestation of chronic HPV infection characterized by a significant morbidity and an unpredictable clinical course. Inefficient antiviral response in RRP is determined by impaired Th1/Th2/Th17 cytokine balance, expansion of Treg cells with inhibitory function, and viral mechanisms to avoid immune control. Bulgaria preparation “Calgevax” (BCG) modulates cellular immune mechanisms and has an antitumor activity. Preliminary results from combined surgical/ “Calgevax” treatment of RRP patients suggest that BCG restores the balance of effector and regulatory T cell subsets through changes in cytokine background.


3. LABORATORY DIAGNOSIS OF INVASIVE FUNGAL DISEASES

L. Boyanova, A. Kouzmanov

ABSTRACT
The aim of this review is to present the common laboratory diagnostic methods for diagnosis of invasive fungal diseases. The majority of those diseases are caused by Candida and Aspergillus species but many studies show an increasing range of infection agents such as Cryptococcus, Trichosporon.
The most common serological tests for antigen and antibody-detection in body fluids (serum, plasma, BAL) are: Indirect immunofluorescense (IIF), detection of 1, 3 – β D-glucan, ELISA – Platelia (enzyme linked immunosorbent assay), latex-agglutination and PCR – molecular techniques. Our study has the aim to approve the methods for rapid diagnosis of invasive fungal diseases.


4. CORRELATION OF THE HEPATITIS B SURFACE ANTIGEN (HBSAG) LEVELS WITH HBV DNA CONCENTRATION IN PATIENTS WITH HBV INFECTION

E. Golkocheva-Markova , I. Aleksiev, L. Karamacheva, P. Teoharov

ABSTRACT
It has been shown that hepatitis B surface antigen (HBsAg) titers correlate with serum HBV DNA levels but they vary in different phases of hepatitis B viral infection. Patients were selected according the presence of HBV DNA in the present study. Hepatitis B surface antigen was detected by ELISA and HBV DNA – by real time PCR. One of the follow up patients was HBV inactive chronic carrier with strong tendency of HBsAg seroclearance. The strong correlation of HBsAg serum titers with HBV DNA was observed for HBV mono-infection during process of HBsAg seroclearance (with low HVB DNA concentration) and in cases of acute hepatitis (with high HBV concentration). However when the levels of HBsAg in patients with medium HBV DNA concentration were evaluated a correlation was not observed. HBsAg levels were higher for HIV/HBV co-infected patients but there was not correlation with HBV DNA concentration. Data show that serum HBsAg levels correlate with HBV replication in a case of HBV mono-infection and can be used for predicting low and high serum HBV DNA levels.


5. NEURAMINIDASE INHIBITORS SUSCEPTIBILITY TESTING OF INFLUENZA VIRUSES CIRCULATING IN BULGARIA DURING THE FIRST THREE POSTPANDEMIC FLU SEASONS

N. Korsun, A. Teodossieva, M. Jordanova

ABSTRACT
The aim
of this study was to determine the neuraminidase inhibitors susceptibility of influenza viruses circulating during the 2010/2011, 2011/2012 and 2012/2013 flu seasons in Bulgaria.
Materials and methods: Screening of 152 influenza A(H1N1)pdm09 viruses was carried out using Real Time RT-PCR discrimination assay for detection of the H275Y oseltamivir resistance point mutation. Three influenza A(H1N1)pdm09 and 11 A(H3N2) viruses were evaluated by phenotypic method using the fluorоgenic substrate MUNANA.
Results: No influenza A(H1N1)pdm09 viruses carrying a H275Y substitution were found. None of Bulgarian A(H1N1)pdm09 and A(H3N2) viruses evaluated for neuraminidase inhibitors susceptibility showed genetic or phenotypic (IC50) evidence for resistance or reduced susceptibility.
Conclusion: The Real Time RT-PCR assay described in this report can be used to screen large numbers of clinical A(H1N1)pdm09 virus positive samples for resistance to oseltamivir. The article demonstrates the importance of continued influenza antiviral susceptibility monitoring in clinical specimens.


6. ANTIFUNGAL SUSCEPTIBILITY TESTING OF MEDICALLY IMPORTANT YEASTS

Z. Ivanova, A. Kouzmanov

ABSTRACT
The aim of this review is to present the new approaches in antifungal susceptibility testing of medically important yeasts. The most commonly used standardized reference methods for yeasts are CLSI M27-A3 broth dilution method and similar to it EUCAST method and CLSI M44-A2 disk diffusion method. The alternative agar based approaches for yeasts are NeoSensitabs tablets and E-test. Etest is easy to use in the routine setting and have been recently incorporated into clinical laboratory practice and allows determination of minimal inhibitory concentration of the most commonly used antifungal agents.
Molecular analyses of antifungal drug resistance are focused mainly on the different mechanisms of azole resistance of Candida spp.


7. OPPORTUNISTIC AND COINFECTIONS IN HIV POSITIVE PATIENTS AT THE CLINIC OF INFECTIOUS DISEASES, “ST.GEORGE” UNIVERSITY HOSPITAL, PLOVDIV

V. Georgieva, A. Dineva, V. Nikolov, N.Vatev, M.Stoycheva

ABSTRACT
Objective:
to be determined the type and frequency of the opportunistic and coinfections in HIV positive patients as well as their connection with the degree of the immune deficiency.
Methods: the study included 170 patients with verified HIV infection- 146 men and 24 women. They were seperated in 3 groups: stage A- 16; stage B- 104; stage C- 50. The used methods are clinical observation, laboratory and microbiological analysis. In all patients HIV was confirmed by present diagnostic methods ( ELISA, Western-Blot, PCR, flow cytometry).
Results: The opportunistic infections in HIV positive patients were: candidiasis - 32 ( 4- stage A; 8- stage B; 20- stage C); pneumocystis pneumonia- 14 patients (stage C); tuberculosis – 8 ( 1 – stage B; 7- stage C); HHV8 – 2 patients- both of them in stage C; 1 patient with salmonella sepsis; 1 patient with toxoplasma meningitis.
Coinfections with hepatotropic viruses: 110 patients (64.7%) infected with hepatitis C ; 23 patients infected with hepatitis B; 15 patients were positive for both hepatitis B and C. Intravenous drug users in these groups were 68%; 65.21% and 80% respectively.
Conclusions: The most common opportunistic infections are candidiasis (18.8%) and pneumocystis pneumonia (8.2%). The most common coinfection is hepatitis C. Coinfections with hepatotropic viruses correlate with the mechanism of transmission. The intravenous drug users are at a very high risk.


8. HEPATITIS A IN PLOVDIV REGION, 2007-2012

N. Vatev, А. Petrov, М. Тroiancheva, М. Stoychevа, M. Pishmishev

ABSTRACT
Introduction:
Viral hepatitis А is an infectious disease showing one of the highest levels of incidence rate in Bulgaria. In the mid nineties of the previous century several highly effective vaccines for protection against the disease were licensed. Many countries have introduced these vaccines as a routine practice and thus managed to achieve significant decrease in the incidence. This vaccine is not yet included in the immunization calendar of Bulgaria and this is the reason why the morbidity of hepatitis A in Bulgaria has the highest rate compared to other European Union member countries. The aim of the study is to conduct a clinical and epidemiological analysis of hepatitis А cases in Plovdiv region for the period 2007-2012.
Materials and methods: A total of 2 427 patients with hepatitis А were studied in the research. In all cases the diagnosis was confirmed with a positive test for anti-HAV IgM. The complex method of epidemiological research, routine clinical, biochemical and laboratory methods have been used.
Results: For the period 2007-2012, 2 427 confirmed cases of hepatitis A occurred. The incidence rate during the years ranged from 2.98/100 000 to 173.58/100 000. The average yearly incidence for the entire period was 57.77/100 000, the lowest rate was in 2009, and the highest – in 2011. The highest percentage of cases and the highest incidence were observed for the age group 5-9 years (297.24/100 000). During the research period 17 epidemic outbreaks were registered, respectively: 2007 – 5; 2010 – 4; 2011 – 4 and 2012 - 4. Clinically the course of the disease remained predominantly mild or moderate. Relative increase of cases in a moderate course was established as compared to these in the mild forms.
Conclusions: 1.Viral hepatitis А maintains its epidemic endemic occurrence in Bulgaria. 2. The incidence of hepatitis A in Plovdiv region (as well as in Bulgaria) remains still at a high level. 3. We find appropriate the introduction of a routine immunization against the disease.


 

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
Prof. R. Kurdova, MD, PhD
Assoc. Prof. I. Rainova, MD, PhD
Assoc. Prof. P. Teoharov, MD, PhD

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