TABLE OF CONTENT

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1. RAPID TESTS IN THE DIAGNOSTICS OF SOME FASTIDIOUS MICROORGANISMS

K. Ivanova, M. Marina

ABSTRACT
Rapid tests for hydrolysis of DNA, nitrate reduction, phosphatase and urease were developed. We applied the tests for more fastidious and biochemically inactive bacteria like Campylobacter /242 strains/, Helicobacter /55 strains and 42 biopsies/, and for Bilophila wadsworthia /10 strains/ - an anaerobic microorganism recently isolated. Reading the results was for 30 minutes. We compared the rapid tests with conventional ones and they proved to be as exact as them. Rapid tests also exceed conventional by the rapidity of reading the results and easiness to perform.


2. DETECTION OF EAF PLASMID IN ENTEROPATHOGENIC ESCHERICHIA COLI (ЕРЕС) ISOLATES BY PCR

P. Petrov, T. Kantardjiev, E. Dobreva, G. Asseva

ABSTRACT
The aim of this study was to demonstrate the EAF plasmid in Escherichia coli diarrheagenic isolates, which is responsible for the localized adherence (LA) and the full virulence of ЕРЕС. A total of 53 clinical isolates of ЕРЕС were investigated by PCR using specific primers and amplicons visualized by capillary electrophoresis. Five out of 53 (9.4%) of ЕРЕС strains were pEAF positive. Conclusion: PCR technique is reliable method for detection of pEAF in ЕРЕС isolates. More isolates from different ЕРЕС serotypes should be tested to find out the true prevalence of the plasmid among them. The first investigation for pEAF among ЕРЕС in Bulgaria is presented.


3. INCIDENCE AND PREVALENCE OF HCV INFECTION IN THE THRACIA REGION IN BULGARIA FOR 15 YEARS PERIOD

O. Boykinova, S. Novakov, Y. Stoilova

ABSTRACT
From the time of the virus isolation HCV infection progressively expands and conquers new risk groups. The aim of the study was to examine the prevalence of HCV infection for 15 years period among 597 people from following risk contingents: 161 patents on chronic haemodialysis, 120 haemophiliacs, 86 patients with burn injuries and 230 intravenous drug abusers (IDUs), as well as the incidence and risk factors in 178 patients with acute hepatitis C. Clinic and laboratory methods, anti HCV antibodies, PCR were made as well as genotyping of fifty patients. HCV positive were 45.34% from the patients on haemodialysis, 96.67% of the haemophiliacs, 33.38% from the patients with burn injuries and 70% of IDUs. The rate of infection among the studied groups was very high, especially in haemophiliacs and IDUs. From 2001 the incidence of HCV infection shows rising tendency. The risk of posttrasfusion hepatitis C has significantly decreased while there is a relative increase of cases with unknown way of transmission.


4. EMERGENCE OF QNR GENES AMONG EXTENDED-SPECTRUM BETA-LACTAMASE-PRODUCING ENTEROBACTERIAL ISOLATES IN A CANCER HOSPITAL IN BULGARIA

S. Sabtcheva, T. Kantardjiev, M. Ivanova, M. Kaku

ABSTRACT
Objectives:
To evaluate the presence of qnr genes among enterobacterial isolates carrying extended-spectrum beta-lactamases (ESBLs).

Methods: Screening for the qnrA, qnrB and qnrS genes was carried out by PCR amplification with specific primers in 163 nonduplicate, clinically relevant ESBL-producing enterobacterial isolates collected between 2000 and 2005. ESBLs from all qnr-positive isolates were identified by PCR amplification and DNA sequencing. Pulsed-field gel electrophoresis was used to investigate the clonality of qnr-carrying isolates.

Results: Seven Citrobacter freundii isolates, each from a different individual, carried qnrB. Among them, 4 had qnrB17,2 had qnrB10 and 1 had qnrB12. None of the isolates carried qnrA and qnrS. Among the qnrB17-carrying isolates, 3 possessed blaTEM-3 alone and 1 had both blaCTX-M-15 and blaOXA-1. The isolates with qnrB1O possessed blaTEM-3. The qnrB12-carrying isolate had blaCTX-M-15, blaOXA-1 and blaTEM-1.

Conclusions: The prevalence of qnr among enterobacterial clinical isolates carrying ESBLs between 2000 and 2005 was low (4.3%). Only qnrB was found and only in C. freundii isolates dating since 2002. qnrA and qnrS were not detected.


5. PESTICIDES AND IMMUNITY

J. Radenkova-Saeva

ABSTRACT
Most pesticides used today are acutely toxic to humans. Pesticides cause poisonings and deaths every year. Acute pesticide poisonings frequently involve organophosphate pesticides, or sometimes their close relatives, the n-methyl carbamates. Symptoms of organophosphate or carbamate poisoning include blurred vision, salivation, diarrhea, nausea, vomiting, wheezing, and sometimes seizures, coma, and death.

Many pesticides damage the immune system. The most immediately noticeable immune reaction to pesticide exposure is an increase in allergic reactivity, often including multiple chemical hypersensitivity. People whose immunity is suppressed by pesticides may also be unable to fight off viral infections or may experience a reactivation of one or more of the herpes viruses. Immunological studies reveal that pesticide-exposure can cause a decrease in the number of B and Tcells.

Lots of clinical, epidemiological and experimental studies show reductions or disruptions in cell-mediated, humoral and non-specific immunity.


6. CASE REPORTS OF HELICOBACTER PYLORI INFECTION ASSOCIATED WITH MULTI-DRUG RESISTANT STRAINS IN CHILDREN

L. Boyanova, G. Gergova, C. Jelev, E. Lazarova, E. Panteleeva, P. Yaneva, I. Mitov

ABSTRACT
Successful eradication of H. pylori is curative. However, antibacterial resistance is the major cause for treatment failure. The present study focuses on two cases of H. pylori infection in children (0.6% of 357 children evaluated since 1996), associated with strains exhibiting uncommon multi-drug resistance to amoxicillin, macrolides and metronidazole.


7. PREVALENCE OF THE FLUOROQUINOLONE - MODIFYING ACETYLTRANSFERASE GENE AAC(6’)-IB-CR AMONG EXTENDED-SPECTRUM BETA-LACTAMASE-PRODUCING ENTEROBACTERIAL ISOLATES IN A CANCER HOSPITAL IN BULGARIA

S. Sabtcheva, T. Kantardjiev, M. Ivanova, M. Kaku

ABSTRACT
Objectives:
To determine the prevalence of the fluoroquinolonemodifying aminoglycoside acetyltransferase-encoding gene aac(6’)-lb-cr among enterobacterial isolates carrying extended-spectrum beta-lactamases (ESBLs).

Methods: One hundred and sixty three non-duplicate, clinically relevant ESBL-producing enterobacterial isolates collected between 2000 and 2005 were screened for aac(6’)-lb by PCR. aac(6’)-lb-cr was identified by sequencing. PCR amplification and DNA sequencing identified beta-lactamase genes in all aac(6’)-lb-cr-positive isolates. Pulsed-field gel electrophoresis (PFGE) was used to investigate the clonality of aac(6’)-lb-cr-carrying isolates.

Results: Ninety-nine of 163 ESBL-producing isolates (60.7%) were positive for aac(6’)-lb including Escherichia coli (65/94), Klebsiella pneumoniae (21 /40), Citrobacter freundii (7/10), Enterobacter cloacae (1/7), Klebsiella oxytoca (1/2) and single isolates of Enterobacter aerogenes, Morganella morganii, Providencia rettgeri and Escherichia hermannii. The aac(6’)-lb-cr variant was identified in 52 (31.9% of all) isolates including 47 E. coli, 2 C. freundii, and 1 each of K. pneumoniae, E. aerogenes and M. morganii. Among the aac(6’)-lb-cr-positive isolates, 32 had blaCTX-M-5, blaOXA-1 and blaTEM-1, 16 had blaCTX-M-5blaOXA-1, and 2 had blaCTX-M-15, blaOXA-1, blaSHV-12 and blaTEM-1. The remaining 2 isolates had blaTEM-1 and ESBL of none of the studied types. PFGE of 47 E. coli digests displayed 30 different patterns, suggesting the limited importance of clonal spread of aac(6’)-lb-cr gene. Conclusions: The aac(6’)-lb-cr variant was present in various species in the family Enterobacteriaceae and highly prevalent in E. coli isolates (50%). The majority of aac(6’)-lb-cr-carrying isolates co-produced CTX-M-15 and OXA-1 beta-lactamases. The spread of multiresistant isolates expressing the aminoglycoside and fluoroquinolonemodifying enzyme AAC(6’)-lb-cr together with ESBLs is a worrisome development requiring continuous monitoring.


8. ACANTHAMOEBA CULBERTSONI PROMOTES GROWTH AND SURVIVAL OF HELICOBACTER PYLORI

E. Tzvetkova-Georgieva, P. Petrov, K. Marinov, K. Ivanova, K. Katzarov

ABSTRACT
On the basis of free-living Acanthamoeba culbertsoni strain investigation was made of the invasion, growth, and survival of reference strain H. pylori CCUG 17874 and 3 clinical isolates of H. pylori. The interaction of amoebae and bacteria has been studied by cultivating them in proteose yeast glucose (PYG) medium and proteose peptone horse serum (PPHS} medium in microaerophilic conditions and aerobic atmosphere. The survival of H. pylori in amoebae has been demonstrated by routine methods of its cultivation. The growth of H. pylori and its survival in media containing amoebae was found to be considerably longer for a period of 14 days (period of observation) when compared with the bacterial cultures which did not contain amoeba cells. The latter did not survive even an overnight incubation. By immunofluorescent staining of samples from mixed cultures using H. pylori antisera it was shown that on the fourth day amoeba cells contained vacuoles filled up with fluorescein stained bacteria and the bacterial number rose up to 8-12 day. In conclusion: The presence of H. pylori in amoeba cells could be interpreted as a mechanism of survival in the environment.


9. CLONING AND EXPRESSION OF RECOMBINANT BORRELIA BURGDORFERI PROTEINS

I. Christova, M. Lesseva, G. Miloshev

ABSTRACT
Development of ELISA tests with recombinant antigens is a new stage in improving serological diagnosis of Lyme brreliosis, especially in terms of its specificity. Major B. burgdorferi proteins that induce immune response OspA and OspC (outer surface proteins) were cloned. OspA and ospC genes were amplified from genomic DNA of B. burgdorferi sensu stricto strain B31 using specific, especially designated for the purpose, primers. Each gene was integrated in the plasmid expression vector pGEX-2T. DIMA constructs were used to transform E. coli strain DH5 6. Specificity of the obtained proteins was confirmed by specific anti-OspA and anti-OspC monoclonal antibodies in immunoblot.


10. PCR AS A METHOD FOR PRESUMPTIVE DIAGNOSTICS OF INVASIVE CANDIDOSIS

P. Angelov, T. Kantardjiev, R. Vacheva, E. Zamfirova, M. Lesseva, E. Shopova, S. Bobcheva, B. Markova

ABSTRACT
Candida spp. are ubiquitous yeasts, which is known as a most frequent causative agents of systemic fungal infections in humans. Systemic candidosis is life-threatening infection which occurs in severely immunocompromised individuals, and most of the cases have an lethal outcome. The rapid detection and identification of Candida spp. in clinical laboratories are extremely important for the management of patients with systemic candidosis. The routinely used culture and immunological species identification are time-consuming and lack the required sensitivity and specificity. Molecular methods for diagnostics of systemic candidiasis are mostly PCR-based and use serum or blood specimens. However, there are many issues due to insufficient sensitivity of the method, and the occurrence of false-positive results. In this study we developed conventional PCR - based diagnostic method, using universal primers for detection of Candida spp. in serum specimens, which significantly improves the sensitivity of such methods, without any false-positive results.


11. ANTI-INFLUENZA VIRUS EFFECTS OF A PLANT POLYPHENOLIC EXTRACT AND E-AMINOCAPROIC ACID APPLIED ALONE AND IN COMBINATION

E. Nikolova, A. Teodosieva, I. Roeva, J. Serkedjieva

ABSTRACT
Influenza Is a major public health problem because of its wide spread, high morbidity rate and considerable social and economic implications. Despite the achievements of antiviral chemotherapy, the need of new potent antiviral agents continues to exist alongside with the necessity of novel methods and approaches. The combined application of natural and synthetic viral inhibitors may be used successfully to potentate the antiviral efficacy of the plant preparations and may enable dose reduction of their toxic components. The aim of the study is to investigate the combined in vitro and in vivo anti-influenza virus effects of a plant polyphenol-rich extract, obtained from Geranium sanguineum L. (PC) and the protease inhibitor e-aminocaproic acid (ACA). The in vitro antiviral activity was determined by the difference in the infectious titres of control and treated viruses and the combined effect was defined on the base of infectious viral yields. The combined protective activity in vivo was determined on the base of the reduction of mortality rates. The combined use of PC with ACA resulted in additive to synergistic inhibition of the A/Aichi/2/68 (H3IM2) (А/ Aichi) virus replication in MDCK cells; however some of the combinations were antagonistic. The joint application of PC with ACA in the murine experimental influenza A/Aichi-a virus infection produced a synergistic protective effect with no enhancement of host toxicity. Survival rate and mean survival time were markedly increased; the index of protection reached 91.2%. The presented results support the combined application of natural and synthetic viral inhibitors with different modes of action.


12. ANALYZING AND EVALUATION OF VARIOUS ANTIGENS FROM TRICHINELLA SPIRALIS LARVAE USING WESTERN BLOT

I. Rainova, M. Ivanova, R. Kurdova

ABSTRACT

The accurate diagnosis of trichinellosis depends largely on the antigens used in serological tests. Nowadays there are two kinds of antigens, isolated from T. spiralis larvae which are used in diagnostic methods. One kind is crude extract from T.spiralis larvae and the other - excretory/secretory products from T.spiralis larvae, cultivated in virto.

The aim of the present study was to analyze these two kinds of antigens, obtained from T. spiralis larvae by SDS-PAGE and Western blot. Electrophoresis was performed in 4% stacking and 12% separating gel under reducing conditions. Western blot was performed in Towbin’s buffer on nitrocellulose paper.

The results obtained showed a very complex antigen profile. Crude extract (CE) yielded in Western blot 7-11 bands with molecular weight (MW) between 28-198 kDa, while excretory/secretory antigen (E/S) revealed bands with MW between 35-208 kDa.

The presence of antigen fractions with molecular weight 45-55 kDa corresponds with the opinion of most authors who consider that these fractions are specific for Trichinella spiralis.

These results implicate that the antigens examined in our study are suitable for evaluation of antibody response in trichinellosis. This paper was supported by National fond „Scientific Research“ from the Ministry of Education and Science.


13. SHIGELLOSIS: PECULIARITIES AND MANAGEMENT

P. Padeshki, T. Kantardjiev

ABSTRACT
Infectious diseases kill about 11 million children each year and 99 per cent of these deaths occur in the developing countries. Notably, of the 1 1 million deaths, 4 million die within the first year of their life (1) Acute diarrhoeal diseases rank second amongst all deaths due to infectious diseases accounting for 3.1 million deaths in under 5 children; 80 per cent of these deaths occur in children below 2 yr of age. In recent years etiology of salmonelloses was profoundly described. (2) It was introduced in context of WHO-Global Salm Surveillance system. (3) Shigellosis is an important cause of diarrhoeal deaths. It has been reported that no less than 140 million cases of shigellosis occur worldwide with 600,000 deaths annually; 60 per cent of such deaths are seen in children under 5 (4). In this review, attempts have been made to highlight the molecular epidemiology, epidemic and pandemic potential, current case management strategies including drug resistance problem and preventive aspects of shigellosis.


 

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

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