TABLE OF CONTENT

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1. DIAGNOSIS OF TUBERCULOSIS - 5 YEARS EXPERIENCE AFTER THE ESTABISHMENT OF THE TB NATIONAL REFERENCE LABORATORY, NATIONAL CENTER OF INFECTIOUS AND PARASITIC DISEASES

E. Bachiyska, T. Kantardjiev, A. Ivanova, Y. Atanasova, D. Merdzhanov

ABSTRACT
Today, 5 years after the establishment of the National Reference laboratory of Tuberculosis as a part of the Microbiology Department of the National Center of Infectious and Parasitic Diseases, it is a modern laboratory with as well trained staff as other TB NRL in EU. TB NRL, NCIPD provided for first time in Bulgaria new, modern and rapid microbiological methods for diagnosis of tuberculosis. TB NRL, NCIPD covered the whole TB laboratory network in the country through an External Quality Assessment scheme and training of the personnel of all TB laboratories in Bulgaria.


2. LIMITED EVIDENCES FOR SPREAD OF MYCOBACTERIUM TUBERCULOSIS “BEIJING" GENOTYPE IN BULGARIA

St. Panaiotov, E. Bachyiska, V. Levterova, N. Brankova, A. Ivanova, D. Merdjanov, Y. Atanasova, T. Kantardjiev

ABSTRACT
In 1995, IS6110 DNA fingerprinting identified in Mycobacterium tuberculosis isolates from China, a genetically closely related group of bacteria - the Beijing genotype family. Strains of this genotype family dominate in Southeast Asia and are globally spread. This genotype is associated with higher pathogenicity and multidrug resistance. In this study we show that Beijing genotype of Mycobacterium tuberculosis is not diffused in Bulgaria as evidenced by spoligotyping of M. tuberculosis sensitive and multidrug resistant strains collected across the country during the last 6 years. Only two strains with the characteristic Beijing spoligotype were identified. Our results suggest, that in Bulgaria, although the active exchange contacts with countries where Beijing genotype is predominant we do not observe wide spread of Beijing genotype, as observed in other West European and Central Asian countries. We suppose that genetic and environmental factors should be investigated to prove unpredisposition of the local population to this globally diffused Mycobacterium tuberculosis genotype.


3. CLONING AND EXPRESSION OF OSP (OUTER SURFACE PROTEIN) C FROM BORRELIA BURGDORFERI SENSU STRICTO

I. Trifonova, I. Christova, T. Gladnishka, E. Tasseva, V. Ivanova

ABSTRACT
One of major outer surface proteins of Borrelia burgdorferi was cloned and expressed. OspC C is a major immunodominant antigen and induces a strong immune response during the early stages of Lyme disease. Specific antibodies against OspC are detectable on 20-30 day after infection. Our main purpose was cloning and expression of this protein as a first step in developing more specific and sensitive ELISA and immunoblot tests for diagnosis of the disease. The ospC gene was amplified in PCR reaction with specific primers. The product was inserted in pET TOPO® vector. This construct was used for transformation of E.coli BL 21 strain. Expression of the protein was stimulated by IPTG. Production of the specific recombinant protein was detected by immunoblot with anti-OspC monoclonal antibody.


4. HUMAN SERUM REACTIVITY AGAINST BORRELIA OSPC, OSPA, FLAB AND VLSE PROTEIN ANTIGENS IN EARLY AND DISSEMINATED LYME BORRELIOSIS

I. Trifonova, T. Gladnishka, E. Tasseva, V. Ivanova, I. Christova

ABSTRACT
Reliable laboratory diagnostic of Lyme disease is still a non solved problem. We tested 116 serum samples from patients with Lyme disease by ELISA tests based on one or more Borrelia antigens in different combinations. Four Borrelia proteins – OspC, FlaB, VlsE and OspA, were cloned, expressed and applied as antigens. The aim of this study was to compare humoral immune response against these proteins in the corse of infection. In the group of patients with erythema migrans, reaction to at least one antigen was found in 48/68 (71%). Of them, IgM class antibodies were found in 28/48 (58%) of the patients and IgG antibodies were detected in 40/48 (83%). Among patients with disseminated Lyme disease, a total of 75% (36/48) were found positive, IgM antibodies only were detected in 72% (26/36) of the patients and IgG antibodies in 83% (30/36) of the patients. The most commonly detectable antigen by IgG antibodies and quite often by IgM antibodies in patients with early and disseminated Lyme borreliosis was VlsE. OspC and FlaB antigens were recognized in similar amounts of serum samples from early Lyme borreliosis. FlaB was more often found in disseminated than in early stage of the disease. OspA was detectable at low rate in all patients despite the stage of the disease. We concluded that no single antigen could ensure appropriate diagnosis. Hence, a combination of antigens to obtain optimal balance in specificity and sensitivity is required.


5. CLINICAL MANAGEMENT OF TRAVEL-ASSOCIATED BRUCELLOSIS CASES IN BULGARIA

I.N. Ivanov , R. Nenova, S. Panaiotov, M. Tiholova, K. Marinov, T. Kantardjiev

ABSTRACT
Bulgaria is free from brucellosis and no resident human cases have been documented for the last 50 years. In 2005 a relatively large number of cases with brucellosis compatible symptoms were reported and investigated. This work describes the epidemiological, clinical and diagnostic findings in these cases. The study also illustrates a simple technique for treatment of serum prior to PCR without the need for DNA purification. A total of 63 individuals were clinically and laboratory studied for brucellosis by applying a number of methods including: epidemiological investigations, hematological and microbiological methods- blood cultures, Rose Bengal, SAT, Coombs IgG, ELISA IgM and IgG. A novel direct PCR assay with serum samples was adapted and evaluated as a rapid confirmative test. This approach excludes any DNA purification steps resulting in significantly shorter turnaround time. In addition, PCR was successfully applied in the post-mortem diagnosis in one patient. Combining multiple diagnostic methods a total of 21 out of the 63 people were confirmed to be infected with Brucella spp. and classified as imported cases. The epidemiological data corroborated that 20 out of the 21 confirmed brucellosis patients had been working in sheep-breeding farms in endemic region in Greece and one in Cyprus. The delayed sampling and/or prior antibiotic treatment resulted in zero positive cultures. The ELISA IgG provided the best diagnostic results with respect to sensitivity (100%), followed by PCR (95%) and Coombs IgG (90%). All of the applied serological methods resulted in compromised specificity. Significant titers were observed with samples from non-confirmed but occupationally exposed individuals. In contrast, PCR yielded 100% specificity. Post-mortem diagnosis was achieved in one patient by means of PCR amplification of Brucella spp. DNA from different body locations. Mainly due to the delayed diagnosis and inadequate treatment 15 patients developed chronic forms and presented with a number of focal complications. Brucellosis still remains a diagnostic challenge especially in non-endemic countries where most of the reported cases are imported and difficult to trace. The presented direct serum PCR assay is rapid, simple and could represent an important alternative to serology especially when studying patients who are occupationally exposed to Brucella and also in culture negative cases.


6. LEMIERRE'S SYNDROME AFTER AN OROPHARYNGEAL INFECTION: TWO CASES WITH ISOLATION OF ANAEROBES AND REVIEW OF THE LITERATURE

M. Marina, K. Ivanova

ABSTRACT
We present two cases (from a period of about an year – June 2008-July 2009) with isolation of anaerobes in patients with an oropharyngeal infection that developed Lemierre’s syndrome. Lemierre’s syndrome is a severe illness caused by Gram-negative anaerobes such as Fusobacterium sp. and mainly F. necrophorum and F.nucleatum. The infection originates from the throat or other oropharyngeal infections and spreads via a septic thrombophlebitis of the internal jugular vein. The ensuing bacteraemia is complicated by infections of the lung /abscess or empyema/, central nervous system /meningitis/, joints and bones, liver, kidneys, etc. Although rare in our days which differs from the past, there is evidence of increasing its frequency in the last years, possibly associated with reduced use of antibiotic therapy for sore throats. The typical clinical picture is characteristic but many clinicians are unaware of the condition and diagnosis is often delayed with potentially fatal consequences. Early diagnosis and the adequate and prolonged antibiotic therapy are extremely important for reducing the mortality of this forgotten from the preantibiotic era disease.


7. FIRST CASES OF SEVERE HOSPITAL-ACQUIRED CLOSTRIDIUM DIFFICILE INFECTIONS IN SOFIA, BULGARIA

K. Ivanova, P. Petrov, G. Asseva, E. Dobreva, I. Ivanov, R. Vatcheva-Dobrevska, M. Marina, V. Tolchkov, T. Kantardjiev, D. W. Notermans and E. J. Kuijper

ABSTRACT
Background:
Clostridium difficile has become a critically important pathogen worldwide, particularly in hospitalized patients. In 2008, the European Center for Disease Prevention and Control (ECDC) initiated a pan European surveillance study to collect epidemiological and microbiological data of C. difficile associated diseases (CDAD). Since Bulgaria has joint this study a number of CDAD cases were discovered. The objective of the present work is to report the first cases of CDAD occurring in Sofia, Bulgaria.

Material and Methods: For the period November 2008 - February 2009, thirty six fecal samples from patients with severe enterocolitis and previous antibiotic treatment have been investigated. The stool samples derived from 3 hospitals in Sofia. Strains were typed and further characterized for the presence of toxins A (TcdA), B (TcdB)_and binary toxins (CdtA and CdtB).

Results: No outbreaks were reported and the incidence of C. difficile infection (CDI) in the hospitals was 3.12 per 10,000 patient admissions (0.7 per 10,000 patient-days). Four patients with severe CDI were identified of which two patients died due to complications of the infection. Two of the isolates belonged to PCR ribotypes 017 (TcdA-; TcdB+; CdtA/B-), one was 046 (TcdA+; TcdB+) and one was 078 (TcdA+; TcdB+; CdtA/B+). All of these ribotypes have been reported to cause oubreaks worldwide.

Conclusion: These are the first well documented cases of CDI in Sofia, Bulgaria. Despite the fact that none of the Bulgarian isolates belonged to the hypervirulent C. difficile NAP1/BI/027, C. difficile ribotypes 078, 046 and 017 were found to be associated with a severe CDI.


8. COMPARISON OF DIFFERENT METHODS FOR DETECTION OF HELICOBACTER PYLORI INFECTION

K. lvanova, M. Marina, B. Vladimirov, J. Churchev, I. Tersiev

ABSTRACT
Helicobacter pylori colonizes the human stomach and is associated with active chronic gastritis, peptic ulcers, gastric carcinoma and MALT-lymphomas. The different methods, used to detect H. pylori, are either invasive (histology, culture, PCR) or non-invasive (serology, urea breath test).

The aim of this study was to compare a non-invasive H. pylori antibody detection test with invasive ones - histological and bacteriological (examination of gastric biopsy samples with direct microscopy, rapid urease test and culture).

33 patients (17 male and 16 female) submitted to endoscopy because of gastrointestinal disorders were studied. Serums for serological examination was obtained from all patients.

30 (91%) of all patients were infected with H. pylori. H. pylori positive rates by microbiological methods were 88% (with diagnostic sensitivity /DS/ -

97%), by histological examination - 70% (DS - 77%) and by serology - 76% (DS - 83%). Diagnostic accuracy of different bacteriological methods was: for direct rapid urease test - 93%, direct microscopy examination - 63% and culture - 51%.

The combination between histological and microbiological methods was with highest efficiency for detection of Helicobacter pylori infection - 98%.


9. TESTING THE BACTERICIDAL ACTIVITY OF PVA/TEOS/AG-NP HYBRID THIN FILMS ONTO CLINICAL STRAINS WITH PROVEN RESISTANCE TOWARD ONE OR MORE ANTIMICROBIAL AGENTS

D. Pencheva, R. Bryaskova, T. Kantardjiev

ABSTRACT
The synthesized novel PVA/TEOS/Ag-Nps hybrid materials were tested for bactericidal activities onto 86 clinical isolates from patients with urinary tract infections, surface skin infections and wound infections. Disk diffusion method (DDM) was used in order to determine the resistance of the strains towards antimicrobial agents and their sensitivity to the disks impregnated with PVA/TEOS/Ag-Nps. The zones of inhibitions were determined using the main criteria of CLSI and for their interpretation and the EUCAST expert rules in antimicrobial susceptibility testing are taken into account. Strains of the three examined groups of bacteria – Staphylococcus aureus (gram-positive bacteria), Escherichia coli (gram-negative bacteria), Pseudomonas aeruginosa (non-fermentative gram-negative bacteria) showed resistance toward different groups of antimicrobial agents and some of them were also multiresistant. There was no strain to show resistance towards the disks impregnated with PVA/TEOS/Ag-Nps.


10. NEURAMINIDASE FROM ENVIRONMENTAL AND CLINICAL ISOLATES OF AEROMONAS SPP. - BIOCHEMICAL STUDIES ON THE ENZYME PRODUCTION

S. Engibarov, R. Eneva, R. Abrashev, I. Abrashev

ABSTRACT
A total of 40 Aeromonas spp. strains were screened for neuraminidase production and 72.5% of them were positive. No correlation was observed between the source of the strains and their ability to produce neuraminidase. Enzyme production depending on the growth phases, media and cultivation conditions was studied using the strain with the highest enzyme activity - Aeromonas sp.40/02. Neuraminidase production was found to be predominantly intracellular. It was most intensive at static aerobic cultivation – 14.7 U/ml for the exogenous and 40 U/ml for the endogenous form of the enzyme. No activity was detected at anaerobic conditions. Significant variations of the cultural liquid pH were observed during the cultivation at all aeration conditions. The possible role of neuraminidase in Aeromonas pathogenesis, nutrition and adaptation is discussed.


11. DIAGNOSTIC CAPACITY OF CFA AND ELISA METHODS FOR DETECTION OF ANTIBODIES AGAINST CRIMEAN-CONGO HAEMORRHAGIC FEVER VIRUS IN PATIENTS SERUM

N. Kalvatchev, I. Christova, M. Pishmisheva, M. Marinova, S. Jeliazkova, L. Andonova

ABSTRACT
Complement Fixation Assay (CFA) and Enzyme-linked Immunosorbent Assay (ELISA) were used to test 16 serum samples from patients divided in three basic groups: 1st group included nine patients suspected to be infected with Crimean-Congo haemorrhagic fever virus (CCHFV) according to their clinical history and status; 2nd group - six patients immunized with a vaccine against CCHFV; 3rd group - one patient who has recovered from CCHF. The results obtained by the two methods for detection of specific antibodies against CCHFV were compared. Specific IgG antibodies against CCHFV were found in 4 patients. IgM specific antibodies were found in only one patient. The coincidence in diagnostic capacity of CFA and ELISA was achieved in small percentage of the examined samples.


12. FEATURES OF CRIMEAN-CONGO HAEMORRHAGIC FEVER IN PATIENTS WITH FEBRILE SYNDROME IN BULGARIA

E. Taseva, I. Christova, T. Gladnishka, I. Trifonova, V. Ivanova

ABSTRACT
Crimean-Congo haemorrhagic fever (CCHF) is an acute tick-borne viral disease, affecting only humans and newborn mice, with haemorrhagic manifestations and considerable mortality in humans. A series of investigations have been conducted regarding circulation of CCHF virus (CCHFV) in Bulgaria. General epidemiological and ecological investigations have been completed in different geographic regions of Bulgaria. The aim of this study was to reveal to what extent the infection with Crimean Congo hemorrhagic fever virus (CCHFV) could be the cause for an acute febrile syndrome in Bulgaria. A total of 302 paired serum samples (acute and reconvalescent) from 149 patients from Sofia, Bourgas, Sliven and Plovdiv area were tested by ELISA for antibodies against CCHFV. Serological data were obtained for 9 (6,04%) patients from districts of Bourgas, Sliven and Sofia. Most of the CCHF cases were reported in summer with a peak in July - 3/9 (33,33%). People aged 10-19 years were more affected. An analysis of the frequency of the clinical signs was made. Only one patient had bleeding (11,11%). Four patients (44,44%) had a rash. Seven of the studied patients (77,78%) recovered completely. Continued surveillance is required to monitor suspected CCHF cases, not only within known foci, but also outside endemic areas as reported in this study.


13. COMPARISON OF A COMPLEMENT FIXATION ASSAY, ELISA AND IMMUNOBLOT FOR SEROLOGIC DIAGNOSIS OF HANTAVIRUS INFECTIONS IN BULGARIA

I. Christova, V. Ivanova, I. Trifonova, T. Gladnishka, E. Taseva

ABSTRACT
We introduced species-specific ELISAs and confirmatory immunoblot (lineblot) to determine hantavirus types that cause infections in Bulgaria and to compare their abilities for serologic diagnosis with those of routinely used complement fixation assay (CFA). A total of 44 serum samples from patients suspected to have hantavirus infection were tested in paralel by ELISAs and CFA and 26 of them were tested by immunoblot. Species-specific ELISAs revealed Dobrava reactivity. We found coincidence of the results between ELISA and CFA in 77,27%. Positive predictive value of ELISA and CFA was 90% and 69,6% when negative predictive value was 100% and 33,3% resp. Immunoblot confirmed all but two ELISA-positive and 9 CFA-positive results. ELISA-positive result revealed uniform antibody reactivity against Dobrava viruses but no reactivity to Puumala hantavirus. We concluded that with a very high reliability, ELISA could be used as confirmatory assay to reduce false-positive and false-negative results by CFA.


 

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

Editorial Board
Acad. B. Petrunov, MD, DSc
Prof. T. Kantardjiev, MD, DSc
Prof. P. Nenkov, 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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