TABLE OF CONTENT

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1. VIROLOGICAL SURVEILLANCE OF ACUTE FLACCID PARALYSES IN BULGARIA IN THE PERIOD 2001-2006

N. Korsun, Z. Mladenova, Sn. Gyurova

ABSTRACT
After the certification of Europe as polio-free region the risk of wild poliovirus importation from polio-endemic countries as well as the risk of appearance of vaccine-derived poliovirus continues to exist. In connection with this the virological surveillance of cases of acute flaccid paralyses in children aged under 15 years is very important. The results of virological investigations of patients with suspected poliovirus etiology of diseases conducted in the National Reference Enterovirus Laboratory during 2001-2006 are presented. In this article the performance of main indicators of quality of AFP surveillance in Bulgaria is also shown.


2. ACTIVE BK VIRUS INFECTION IN A PATIENT WITH MULTIPLE-TYPES HPV ASSOCIATED GENITAL LESIONS

S. Slavov, I. Nenkov, P. Draganov, G. Nenkova, D. Dimova and Z. Kalvathcev

ABSTRACT
Human polyoma-(BKV) and papilloma-(HPV) viruses are oncogenic agents, associated with different urological disorders (nephropathies, ureteritis and cystitis) and/or benign or malignant lesions. Here we report on a 45-year-old man, who had suffered from condylomata acuminata and idiopathic uroinfection and present the results on elucidation the etiological agents of the diseases. PCR of the material taken from the lesion, revealed multiple HPV types. Patient’s urine was also investigated by cyto-logical staining and pathognomonic “Decoy cells", characteristic for reactivated BK virus infection were found. We discuss eventual pathogenetic relations between both infections.


3. DIAGNOSTICS OF THE FIRST SUSPECT HUMAN CASES OF AVIAN INFLUENZA A/H5N1/VIRUS IN BULGARIA

T. Hadzhiolova, S. Pavlova, R. Kotseva

ABSTRACT
The first diagnostic investigations have been made, demonstrating avian flu A/H5N1/ virus etiological role. For the period Janu-ary-March 2006 twenty six suspect patients, having been in close contact with ill or dead birds, and with a subsequent respiratory illness, were tested. Specific A/H5N1/ assays were applied /GeNet Bio rapid antigen detection test and Sacace RT-PCR kit/. Viral isolation was performed using MDCK cell lines and chicken embryos. Avian flu A/H5N1/ virus as a causative agent of respiratory disease was neither isolated, nor detected in any one of the tested patients. Subtype A/H1N1/ human influenza virus, identifiable by HIT and RT-PCR, was isolated in three of the patients tested.


4 PROFILE OF NEWLY EMERGING MONOPHASIC SALMONELLA ENTERICA SUBSP. ENTERICA SEROVAR 9,12:L,V:- STRAINS IN BULGARIA CAUSING ENTERIC INFECTIONS

P. Petrov, T. Kantardjiev, G. Asseva

ABSTRACT
Strains of newly emerging in Bulgaria Salmonella enterica subsp. enterica serovar 9,12:1,v:- causing enteric infections were examined. The biochemical profile of the organisms is consistent with that of Salmonella enterica subsp. enterica (subspecies I) serovars. All strains showed susceptibility to ampicillin, amoxicillin-clavulanic acid, cephalothin, cefuroxime, cefotaxime, ceftazidime, chloramphenicol,ciprofloxacin, gentamicin, tetracycline, and trimethoprim-sulfamethoxazole. All but one were susceptible to nalidixic acid. On pulsed- field gel electrophoresis (PFGE), the strains showed 15 genomic fragments with one PFGE profile. Causing a cluster of salmonellosis it certainly represents an evolved pathogen of clinical significance. To establish its taxonomy additional genotypic methods are needed.


5 CHARACTERIZATION OF SOME VIRULENCE DETERMINANTS OF AEROMONAS SPP. ISOLATED FROM MEAT, DRINKING WATER AND PATIENTS IN BULGARIA

P. Orozova, P. Petrov, R. Nenova-Poliakova, I. Abrashev

ABSTRACT
In the present study 46 Aeromonas spp. strains isolated from meat, drinking water and patients in Bulgaria were tested for pathogenicity by studying its hemolysis, enterotoxin production, Congo red absorbtion, neuraminidase activity and cytotoxicity. Hemolysis, tested on sheep erythrocytes, was frequently seen with water isolates and human isolates as with food isolates too. Neuraminidase activity was detected in all Aeromonas hydrophila isolates and it was the highest to those Aeromonas hydrophila strains isolated from water and meat. Cytotoxicity, evaluated on Vero cells was frequently observed with all meat isolates (100%), with 13 water isolates (65%) and with 10 human isolates (50%). About 60% of the water isolates were positive for enterotoxin activity. These results indicate that the occurrence of Aeromonas spp. in the environment represents a potential risk for humans. These results suggest that Aeromonas species are potential enteric pathogens in our geographical region.


6. ANTIMICROBIAL RESISTANCE OF P. AERUGINOSA ISOLATES FROM DIFFERENT WARDS IN VARNA UNIVERSITY HOSPITAL OVER A 3-YEAR PERIOD

K. Bojkova, T. Stoeva

ABSTRACT
During the period 2002- 2004, 272 nonduplicate P.aeruginosa strains, isolated from different clinical specimens, were studied. The identification was done by conventional methods, semiautomated and automated terms. The strains were tested to Piperacillin (PIP), Piperacillin/tazobactam (TZP), Ceftazidime (CAZ), Cefepime (FEP), Amikacin (AM), Ciprofloxacin (CIP), Imipenem (IMP) and Meropenem (MEM). The most active antimicrobials for the studied period were: TZP>IMP>PIP>FEP>AM>CIP>CAZ. During 2004, the resistance of P.aeruginosa showed an important increase against CAZ, but significant decreases in resistance were noted for AM and CIP during the same period. IMP resistance has stayed in constant levels. Among 247 isolates, MEM and IMP showed similar activity. For the period 2002-2004, multi-resistant (resistance to at least 4 of the tested drugs) were 11.4 % of the strains. The agent with the lowest level of resistance among multi-drug resistant isolates was IMP (34.5%).


7. A CASE OF LYME CARDITIS WITH ATRIOVENTRICULAR BLOCK AND REVIEW OF THE LITERATURE

I. Christova, T. Gladnishka, E. Tasseva

ABSTRACT
A case of a 39-year old patient with Lyme carditis manifested as atrioventricular block grade III was presented. Carditis occurred in the absence of erythema migrans and the patient did not recall a tick bite, only a skin inflammation about a month ago and arthralgia now. He was hospitalized because of dizziness, presyncope and bradycardia. ELISA showed significant levels of IgIVI antibodies to B. burgdorferi. After treatment with Famotidine, Methylprednisolon and Rocephin, the AV block evolved through grade II to grade I. Serology was more positive after the second 15 day-course with Rocephin. At that time the patient felt only significant weakness but had no cardie abnormalities. Specific antibodies decreased 2 months later and after a 25-day course with doxy-cycline p.o. The patient was clinically healthy and had no subjective complaints. Lyme carditis cases are usually underestimated not only in our country. The principal manifestations of Lyme carditis are conduction abnormalities, pericarditis, myocarditis, tachyarrythmias, and congestive heart failure. They should be considered as possible Lyme borreliosis even in patients without anamnesis of tick bite and erytema migrans rash and Lyme serology should be ordered. Reliable diagnosis and adequate early treatment are guarantee for avoiding complications and progression of the disease.


8. DETECTION OF PARODONTOPATHOGENS IN A DENTAL PLAQUE USING FISH METHOD

M. Belcheva, R. Gmuer, Tz. Uzunov

ABSTRACT
The aim of the author was to study the FISH method in order to apply this modern method in Bulgaria. FISH method was used for a detection of parodontopathogens in the dental plaque for first time in our country. The samples from 8 patients with periodontal disease were tested with double stranded DIMA probes for some of the most frequent parodontopathogens: Actinobacillus actinomycetemcomitans, Prevotella spp., Porphyromonas gingivalis, Fusobacterium nucleatum, Tannerella forsythia, Veillonella spp., Selenomonas spp., Campylobacter spp.


9. SPECIFYING CLINICAL FORMS OF TOXOCAROSIS AFTER TESTING SEROLOGICALLY SUSPECTED PATIENTS

I. Rainova, R. Kurdova

ABSTRACT
Toxocarosis is helminthic disease with various clinical pictures. Few years ago the main clinical forms were visceral and ocular. Recently some authors separated neurological, skin form and toxocarosis of mussels and disseminated in immunocomprommised patients. The aim of the study was to determine clinical forms of toxocarosis in Bulgaria after testing sera samples from suspected patients in ELISA and Western blot. The results showed that in suspected patients from our country is possible to obtain all described clinical forms of toxocarosis. Patients with visceral form were the largest number while tested patients from groups with other clinical features were not sufficient for radical conclusions. When the results were confirmed by Western blot the number of positive patients from all tested groups was significant lower than tested in ELISA. These differences could be explained with cross reactions due to other helminthic diseases.


10. INVESTIGATION ON THE POTENCY TEST OF BACTERIAL COMBINED VACCINES PRODUCED IN BB-NCIPD LTD

J. Cenkova, M. Klisarova, S. Todorov

ABSTRACT
Whole animal assays for potency of bacterial combined vaccines provides very important information for their activity. Nevertheless their expense, the large number of animals used, the time spend and their variability, the whole animal assays for potency and process of establishing of accuracy, precision, specificity, linearity and reliability are performed for the final product release and stability testing. The information is of a great importance, because of the ability to estimate even the procedure of potency testing is correct and the results are between the limits of precision performance. It is possible the data are acceptable, but at the level that gives a warning for eventual future problems or the precision of the test is not satisfactory. On the basis of revue of the potency test results for a long period the levels of acceptance of the results for each component of the combined vaccines, produced in Bui Bio - NCIPD Ltd., are established. In the study all produced lots are included - Tetatox since 1996, Tetadif since 1998, Diftet since 1997 and Diftetkok since 1997. This information gives permission for making a decision about improving the process.


11. ADSORPTION POWER OF ALUMINIUM HYDROXIDE USED AS ADJUVANT IN BACTERIAL VACCINES

K. Veleva, J. Cenkova, V. Radenkova

ABSTRACT
Aluminium hydroxide is commonly used as an adjuvant in the production of bacterial vaccines. It serves as a carrier of the immunogen thus bringing it in the most effective form to the organism. The capacity of the aluminium hydroxide to accumulate the antigen is an important characteristic, which must be estimated in order to evaluate its adjuvant properties. The adsorption power of the aluminium hydroxide is a useful measure of its capacity. Therefore the adsorption of bovine serum albumin (BSA) on aluminium hydroxide produced in BB - NCIPD was studied. The relationship between concentration of BSA and percentage of adsorption on aluminium hydroxide was established. The adsorption capacity of the aluminium hydroxide produced at BB-NCIPD was characterized.


12. PROTECTION AGAINST WHOOPING COUGH IN CHILDREN BETWEEN 0-6 YEARS OLD

R. Alexiev, K. Hadjiisky, S. Malchanova, V. Demireva and PI. Nenkov

ABSTRACT
The specific immunoprophylaxis of humans with pertussis, as a component of combine bacterial vaccine leads to production of specific antibodies that is indicator of the whooping cough prevention. For evolution of immunization procedures and the vaccine itself, antibody levels against pertussis are useful to show the immune status of the population. An enzyme-linked immunosorbent assay was used for measuring immunoglobolin G pertussis antibodies in human sera. The ELISA involves the blinding of bacterial cells to polystyrene tubes. Results of the direct ELISA test are highly reproducible. It is believed that a pertussis antibody level of 1:81 provides protection against disease. The titters of antibody from 1:161 to 1:320 showed full protection of people against whooping cough and titters over 1:321 are used as a criteria of disease or used as a criteria of passed disease. The results of estimation of immune status of population in Bulgaria were based on the same criteria.

The assay was done in plastic plates coated with inactivate^ bacterial cells. For investigation 279 human sera were tested in age group between 0-6. The tested sera showed that 54% of persons have full protection against whooping cough and in 42% of children the obtained titters showed basic immunity. In 4% of patients the titter of antibody is more than 1:321, which titter is used as a criteria of disease of human sera. All of sera have a level enough for protection against whooping cough and non-protected patients were not found.

Present results indicate a good protection against pertussis in Bulgaria in children till 6 years old. This fact is a result of specific immunoprophylaxis of infants up to 2 years of age by pertussis vaccine, as a component of DIFTETKOK (DTP) combined bacterial vaccine, produced by BB - NCIPD, Ltd., Sofia Bulgaria.


13. STABILITY STUDIES OF BACTERIAL VACCINES PRODUCED IN “BUL BIO - NCIPD” LTD. TETATOX (TETANUS VACCINE ADSORBED) - ACCELERATED TEST

J. Cenkova, S. Todorov, V. Radenkova, K. Veleva, M. Klisarova

ABSTRACT
To provide evidence on how the quality of the bacterial vaccines varies under the influence of environmental factors such as temperature and humidity, and to conform established shelf-life for the products in different dosage forms and confirm the recommended storage conditions accelerated tests were performed. The accelerated testing covers the attributes that are likely to influence the quality, safety and/or efficacy of the bacterial vaccines. The analytical procedures for the parameters are validated. The storage conditions and the lengths of studies chosen are sufficient to cover storage, shipment and subsequent use. The stability studies are performed with vaccines filled in different dosage forms (vials - 10 and 20 doses) with rubber stoppers and aluminum caps. The testing of the parameters is performed by approved methods at the Laboratory for Control of Human Medicines.


 

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
Assoc. Prof. T. Kantardjiev, MD, PhD

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