TABLE OF CONTENT

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1. ETIOLOGICAL DIAGNOSIS OF MENINGOCOCCAL MENINGITIS AND SEROGROUP TYPING OF NEISSERIA MENINGITIDIS BY REAL-TIME PCR IN BULGARIA

V. Levterova, I. Simeonovski, A. Kurchatova, S. Panaiotov, I. Philipova, T. Kantardjiev

ABSTRACT
Neisseria meningitidis is a leading cause of bacterial meningitis globally. The present study focused on diagnosing and serotyping of N. meningitidis in Bulgaria. We applied adapted protocols for DNA isolation with Chelex 100 resin from cerebospinal fluid (CSF) and diagnostic real-time PCR technique. One hundred and thirty CSF samples, collected from all over Bulgaria were tested for detection or confirmation the etiology of bacterial meningitis by ctrA and sodC marker genes. N. meningitidis accounted for 14.8% of the meningitis cases in Bulgaria. N. meningitidis was confirmed in 42% of patients over 20 years of age. We targeted sacB, synD, synE, xcbB, and synF marker genes for A, B, C, W135, X, and Y serogroup typing by real-time PCR. Serogroups B and C were predominant, accounting for 74% and 19% of the isolates, respectively. Our results confirm that in Bulgaria serogroups B and C are major causes of invasive meningococcal disease, in agreement with data from other European countries. The real-time PCR technique is useful for confirmation of isolates and detection of meningococci in clinical samples, regardless of the encapsulation status.


2. DISTRIBUTION OF CHLAMYDIA TRACHOMATIS SEROTYPES BY DNA SEQUENCING OF OMPA GENE IN SOFIA, BULGARIA

I. Philipova, I. Ivanov, A. Kouzmanov, Z. Ivanova, V. Levterova, I. Simeonovski, V. Kantardjiev

ABSTRACT
In order to detect and characterise Chlamydia trachomatis serotypes in patients from Sofia, Bulgaria, 1124 endocervicaland urethral swabs, and first-void urine samples were collected for cryptic plasmid PCR analysis. C. trachomatis serotypes were identifiedby performing DNA sequencing of the ompA gene. The phylogenetic tree was designed by alignment of the obtained sequences with chlamydial reference genotypes D-K sequences available in the GenBank database. The prevalence of C. trachomatis was 5.87% (66/1124). DNA sequencing results revealed the following distribution of serotypes: E (32%), G (20%), F (12%), D (11%), K (8%), Ia (7%), J, Ja, and H (4% each).
This is the first study providing information about the distribution of C. trachomatis genotypes among patients from Sofia, Bulgaria.


3. CHLAMYDIA TRACHOMATIS GENOTYPING BY HIGH-RESOLUTION MELTING ANALYSIS

I. Philipova, I. Ivanov, A. Kouzmanov, Z. Ivanova, V. Kantardjiev

ABSTRACT
High-resolution melting analysis (HRMA) is an inexpensive, single-step, closed-tube method using real-time PCR technology to investigate DNA sequence variation. Recent studies suggest that HRMA could be applied as a cost-effective alternative to ompADNA sequencing for C. trachomatis genotyping. Here we provide the first data on the successful implementation of HRMA for C. trachomatis genotyping in Bulgaria directly from clinical specimen.


4. DNA SEQUENCING OF ITS REGION FOR RAPID AND ACCURATE IDENTIFICATION OF MEDICALLY IMPORTANT FUNGI

A. Kouzmanov, I. Philipova, Z. Ivanova, I. Ivanov, V. Kantardjiev

ABSTRACT
Infections caused by fungi have increased during the last decades mainly due to the increasing population of immunocompromised patients. Therefore, the number of medically important yeast species, not covered by conventional identification kits, and human infections associated with moulds that are difficult to identify with classical methods, also increased. These issues created the necessity for rapid and robust molecular identification techniques. We have recently implemented successfully ITS sequencing at the National Reference Mycology Laboratory in Sofia, Bulgaria as a rapid and accurate method to facilitate conventional identification of medically important fungi.


5. HUMAN LEPTOSPIROSIS IN BULGARIA, 2010-2014

I. Christova, E. Tasseva

ABSTRACT
The study revealed current trends in incidence, epidemiology, clinical symptoms, circulating Leptospira serogroups, mode of transmission, and source of infection in laboratory confirmed human leptospirosis cases in Bulgaria between 2010 and 2014. Serogroup Icterohaemorrhagiae had the leading role in aetiological structure in 2010-2013 and serogroup Sejroe was the most common in 2014. Following heavy rainfall and floods in 2014, 5-time increase in leptospirosis cases was observed in comparison with 2013. Water was found as the main source of infection. Leptospirosis in Bulgaria, located in temperate climate region of southeastern Europe, is endemic with low incidence but significant case fatality rate. General understanding of specific relations between aetiology, epidemiology and clinical manifestations of leptospirosis would help in prevention and management of the disease.


6. CHOLESTASIS SYNDROME IN HEPATITIS E – TWO-YEAR PERIOD ANALYSIS (2014-2015)

M. Pishmisheva, P. Teoharov, E. Naseva, N. Vatev,M. Stoycheva, M. Karamisheva, D. Velkova, I. Ivanov

ABSTRACT
Introduction: Hepatitis E is a viral disease with the clinical and morphological characteristics of acute hepatitis. In endemic areas the disease is a major health problem. The increase in the number of patients with indigenous hepatitis in developed countries also becomes an issue. Hepatitis E clinically manifests most often with ictericform, and subjective complaints do not differ from those of hepatitis caused by other hepatotropic viruses. Caroli et al. report for the first time cholestatic form of acute viral hepatitis in 1942. Cholestasis syndrome is observed in all types of viral hepatitis. According to Caroli, 80% of cases of intrahepatic cholestasis are associated with viral hepatitis B and C, and only 20% - with other etiological factors. HBV, HCV, CMV, EBV can cause serious cholestasis in immunocompromised patients, as well as transplant patients. They can develop severe infection known as fibrosing cholestatic hepatitis, which can progress to liver failure if not treated. Unlike the above mentioned viruses, enterally transmitted hepatotropic viruses HAV and HEV can cause significant cholestasis in immunocompetent individuals.The aim of this paper is to analyse the clinical manifestations in patients with hepatitis E and cholestasis.

Material and methods: Ninety patients diagnosed with acute hepatitis E were treated at the Infectious Diseases ward of the Regional Hospital - Pazardzhik during the period 1 January 2007 – 30 June 2016, with the majority (83) being hospitalised between 1 January 2014 – 30 June 2016. Patients were subjected to clinical monitoring and observing the objective condition,combined with paraclinical and instrumental methods of examination. The diagnosis was confirmed at the NRL for hepatitis viruses, National Inspectorate - Sofia, with detection of anti-HEV IgM and anti-HEV IgG antibodies with ELISA methodology.

Results: Out of 90 patients with hepatitis E, 25 (27.77%) were with signs of cholestasis. Patients with cholestasis were aged between 40-76 years (average 58.04). Gender distribution was 20 (80%) male patients and 5 (20%) female patients. The average age of men was 58.75 years, ranging from 40 to 76, and for women - 55.2, with a range from 47 to 60 years. Of all patients with cholestasis, 23 (92%) had comorbidities. Subjective complaints of patients with hepatitis E (with or without cholestasis) were not different from those of patients with hepatitis caused by other hepatotropic viruses. We observed the following symptoms: astheno-adynamia - in 17 (68%), anorexia - in 13 (52%); pruritus - in 17 (68%), dyspeptic symptoms - in 7 (28%) patients. The most common symptoms were astheno-adynamia and itching. Patients with cholestatic form of hepatitis E had longer hospital stay compared to patients with other forms, with an average length of stay of 20.8 days. In addition to the long hospitalisation period, recovery period was longer.

Conclusion: As with other types of viral hepatitis, cholestasis is commonly observed in hepatitis E. Comorbidities (e.g. diabetesmellitus, alcohol, etc.) are likely to contribute to the development of cholestasis. Hepatitis E with cholestasis syndrome occurred with significant deviations in the biochemical profile that persisted longer than usual and the recovery period is prolonged. Development of cholestasis is associated with prolonged hospitalisation and treatment with greater financial cost.


7. COMPARATIVE STUDY OF METHODS FOR DETECTION OF CLOSTRIDIUM DIFFICILE DIRECTLY FROM STOOL

M. Pavlova, E. Dobreva, K. Ivanova, G. Asseva, I. N. Ivanov, P. Petrov, E. Savov, I. Todorova, A. Trifonova, T. Kantardjiev

ABSTRACT
Objective: In this study, we compared various methods for determination of Clostridium difficile antigen and toxins directly from stool.

Material and methods: Тhirty-four faecal specimens from patients with diarrhoeal syndrome were examined. The following methods were applied directly on the samples yielding results within a few minutes to a few hours: immunochromatographic assay QuikChek Complete for determination of gluDantigen and toxins A/B, EvaGreen Real-Time PCR for gluD and tcdB, C.difficile Verigene test (Nanosphere, Northbrook, IL, USA) for detecting tcdA, tcdB, and the binary toxin. Culture methods were performed to confirm the results for detection of C.difficile from stools.

Results: Twenty-three out of 34 (23/34, 68%) faecal specimens were positive for C. difficile by culture methods. The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of the different methods were as follows: QuikChek Complete - 90.9%, 75%, 95.2%, 60%, and 88.9%; EvaGreen Real-Time PCR – 83.3%, 100%, 100%, 100 %, and 88.9%; Verigene C.difficile test – 90.9 %, 100%, 100%, 100%, and 100%.

Conclusion: According to the results obtainedin this study, the most effective method for detection of C. difficile antigen and toxins directly from stool samples was the Verigene C.difficile test.


8. CASE OF ICTEROHAEMORRHAGIC LEPTOSPIROSIS WITH PREMORBID LIVER DISORDER

G. Gancheva, T. Doichinova, I. Pakov

ABSTRACT
Liver involvement is a common feature of leptospirosis. It varies from mild to severe hepatic dysfunction. Our aim was to analyse a case of icterohaemorrhagic leptospirosis and to assess the impact of premorbid liver disorder on severity of leptospirosis.

Case report: sixty-seven-year-old, frequently fishing veterinary worker was admitted to the Clinic of Infectious Diseases at University Hospital – Pleven with one-week history of fever, myalgia, weakness, vomits, and oliguria.

Past history: viral hepatitis A (1974); “hepatic inflammation” (1983). On admission, the patient was in severe condition, afebrile, adequate, with severe adynamia, intensive jaundice, conjunctival suffusions and haemorrhages. Decreased breathing, basal crackles, tachyarrhythmia, hypotension, bloated abdomen, and hepatosplenomegaly were found. Laboratory results: RBC 3.8; Hg 119; WBC 23.7; granulocytes 0.88; platelets 45; total/direct serum bilirubin 153/145 µmol/L; ASAT 42; ALAT 27 IU/L; total protein 61.3; albumins 27.2; fibrinogen 8.0; prothrombin index 82%; С-reactive protein 278; blood-urea-nitrogen 16; creatinine 308. Immuno-phenotypisation of lymphocytes subsets: granulocytosis with lymphopenia (markedly decreased total T-lymphocytes, T-helpers and T-helpers/cytotoxic T-cells ratio). Abdominal ultrasonography: mild hepatic steatosis; partitioned gallbladder. Microagglutination test: positive for L. icterohaemorrhagiae (1:1600) (National Reference Laboratory – NCIPD, Sofia). Prompt intensive treatment was performed, but along with clinical improvement the intensity of jaundice extremely increased. Optical hallucinations and disturbed spatial perceptions appeared. Delirium resolved after increasing the doses of hepatoprotectors. Patient was discharged (on the 14th day) in improved condition with moderate jaundice and hepatomegaly. During the two months follow-up control, favourable recovery was registered.

Conclusions: Liver involvement in leptospirosis is an important factor for severity. Premorbid liver disorder poses an additional risk for prognosis.


9. BACTERIOPHAGE THERAPY

A. Trifonova, I. Todorova, E. Savov

ABSTRACT
The increasing problem of antimicrobial resistance is an issue of major concern to physicians and clinical practice, and treatment failure of infections becomes more likely. The prevention of antibiotic-resistant infections requires a multifaceted approach. One of the alternatives in this respect is to use bacteriophage therapy - an “old-new” method, known before the appearance of the first antibiotics. In this review, we present data on the current state and approaches in the use of bacteriophages as a tool for the effective treatment of infections in an era of resistance.


10. CELL SURFACE COMPONENTS DETERMINING AGGREGATION IN LACTOBACILLI (MINIREVIEW)

T. Yungareva

ABSTRACT
The species from the Lactobacillus genus are the most common microorganisms exploited as probiotics and food preservatives. Processes such as aggregation, co-aggregation, adhesion, and biofilm formation are important mechanisms that allow colonisation of intestinal and urogenital tract, the oral cavity and different food surfaces. Aggregation is one of the desirable characteristics of probiotic bacteria as it could increase bacterial capacity to compete and it could play an important role in human health. Due to co-aggregation with pathogenic bacteria such as Escherichia coli, Salmonella Typhimurium , Campylobacter coli, and Campylobacter jejuni, lactobacilli prevent pathogenic colonisation and assist in the removal of pathogens from the human mucosal surfaces. In some lactobacilli aggregation is the first step of adhesion or biofilm formation. Their ability to form aggregates is a species- and strain-specific feature. The process is a result of a complex interaction between cell surface components, such as proteins, glycoproteins, lipotechoic and techoic acids, secreted factors and enzymes. In some Lactobacillus strains the components involved in autoaggregation and adherence or in autoaggregation and co-aggregation are identical, while in other strains each process is determined by a different set of components. The present work presents a brief overview of cell components in lactobacilli considered to be involved in aggregation and co-aggregation.


 

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

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

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