TABLE OF CONTENT

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1. GENETIC SURVEY OF INVASIVE S. PNEUMONIAE SEROTYPES IN BULGARIA FOR A 5-YEAR PERIOD

I. Simeonovski, V. Levterova, M. Malcheva, T. Kantardjiev

ABSTRACT
Streptococcus pneumoniae colonises the mucosal lining of the upper respiratory tract and is an important cause of invasive infections affecting young children, adults over 65 years of age, the immunocompromised and individuals with chronic diseases. Recent studies have shown variations in virulence based on the high rate of pneumococcal recombination. PCR-based molecular methods are highly sensitive, specific and are becoming the preferred tool for quick and accurate diagnosis of bacterial meningitis which is required to be defined within 2-3 hours. During the 5-year survey period (2013-2017), 202 materials received as cerebrospinal fluid samples and pneumococcal strains isolated from patients diagnosed with meningitis, were examined by Real-time PCR in the reference laboratory at NCIPD. Serotyping of S. pneumoniae-positive materials was performed with conventional multiplex PCR and Real-time PCR with primers for 41 serotypes/serogroups. There is a high incidence of S. pneumoniae serotypes not covered by the pneumococcal conjugate vaccine (PCV10) currently used in Bulgaria. It was found that all cases of meningitis caused by S. pneumoniae vaccine serotypes occurred in patients that were not vaccinated.


2. DISTRIBUTION OF MYCOBACTERIUM AVIUM COMPLEX IN BULGARIA

Y. Atanasova, S. Yordanova, Y. Todorova, A. Baikova, E. Bachiyska

ABSTRACT
The members of Mycobacterium avium complex (MAC) are opportunistic microorganisms, common in the environment (soil and water) and cause infections in birds, mammals and humans. The objective of the present study was to determine the prevalence of MAC representatives among suspected for tuberculosis patients in Bulgaria over an eight-year period – from 2010 to 2017. The exact epidemiology of pulmonary non-tuberculous mycobacterial (NTM) disease was difficult to determine because unlike cases of infection with M. tuberculosis complex, notification of NTM and MAC in particular, is not mandatory in terms of Ordinance 21 of the Ministry of Health (1) and there is no any accurate information about their prevalence in Bulgaria. The survey was conducted in the National Reference Laboratory of Tuberculosis at the National Centre of Infectious and Parasitic Diseases. MAC was identified using phenotypic methods, biochemical features and molecular genetic assay (LPA). A total of 596 NTM strains were identified during the study period and 16.8% (n=100) of them were MAC. In EU countries, MAC representatives are prevalent among NTM isolates, but in Bulgaria they are significantly less in number.


3. UNEXPECTEDLY HIGH NUMBER OF WEST NILE NEUROINVASIVE DISEASES IN BULGARIA IN 2018

I. Christova, E. Panayotova, I. Trifonova, E. Taseva, T. Gladnishka

ABSTRACT
West Nile virus (WNV) is an emerging arbovirus in Europe and America which disseminated widely in recent years. This study analysed epidemiological data and laboratory findings of probable and confirmed human WNV cases in Bulgaria in 2018. A total of 15 patients with WNV infection were detected in 2018, with 2 deaths among them. All patients were diagnosed in August or September. Four patients were from Sofia district, 4 from Burgas, 3 from Plovdiv and 1 from each of the following districts: Shumen, Yambol and Pazardzhik. Laboratory diagnosis for 7 of the patients was based solely on detection of specific antibodies in the serum samples. Eight patients met the criteria for confirmed WNV case. In 6 of them WNV genome was detected by RT-PCR in blood or urine sample and in 3 patients specific IgM antibodies were found in CSF samples which confirmed WNV neuroinvasive infection. The high number of diagnosed human WNV cases in 2018 in Bulgaria is a good sign of increasing recognition of the disease. More efforts are needed in proper transportation of clinical samples undergoing genetic detection of the viral genome. WNV neuroinvasive infection should be suspected and included in the differential diagnosis of viral encephalitis or meningo-encephalitis especially in late summer and early autumn.


4. DETECTION OF MEASLES AND RUBELLA ANTIBODIES IN DRIED BLOOD SPOTS

S. Krumova, E. Golkocheva-Markova, A. Pavlova, S. Angelova, I. Georgieva, P. Genova-Kalou

ABSTRACT
This study aimed to determine the frequency of detection of measles and rubella antibodies in dried blood spots in Bulgaria.
Material and Methods: Two types of clinical material, serum samples and dried blood spots (DBS), were tested from a total of 101 patients. Serological methods (indirect ELISA) were used for detection of specific viral markers (IgM and IgG antibodies) indicating acute or past measles and rubella infection.
Results: In the present study, the patients were with median age of 39 years and divided into 11 age groups. The majority of patients were under 30 years of age and from the capital of Sofia. In 3 patients acute measles infection was confirmed by positive ELISA-IgM results for the serum samples and DBS. No acute rubella infection was detected. Measles and rubella IgG seroprevalence was determined as 83/101 (82%, 95% CI: 74.51÷89.49) and 79/101 (78%, 95% CI: 69.92÷86.08) in serum samples, and 79/101 (78%, 95% CI: 69.92÷86.08) and 73/101 (72%, 95% CI: 63.25÷80.75) in DBS, respectively. In combination with immunoenzymatic testing for measles and rubella IgM/IgG markers, coincidence of results for both types of clinical material was found in >90% of cases. No significant differences were found in the results in terms of gender and age.
Conclusion: In recent years a variety of new and innovative applications of DBS are introduced in medicine, neonatology, virology, microbiology, etc. The optimisation of the DBS technique as an alternative approach to venepuncture in virology is very important for conducting seroepidemiological studies and to a certain extent for the surveillance of epidemic outbreaks.


5. MIXED INFECTION - HANTAVIRUS HAEMORRHAGIC FEVER WITH RENAL SYNDROME AND HERPES SIMPLEX VIRUS ENCEPHALITIS: A CASE REPORT

P. Argirova, I. Boev, M. Stoycheva

ABSTRACT
Introduction: This case report describes a patient treated at the Clinic of Infectious Diseases of University Hospital “St. George” Plovdiv, with two infections occurring at the same time – haemorrhagic fever with renal syndrome (HFRS) and herpes simplex virus (HSV) encephalitis.
Results: A 53-year-old male presented with fever, flu-like syndrome and renal impairment with mild nitrogen elevation. The patient was treated at the Clinic of Nephrology for one week. Several hours after discharge he had three generalised tonic-clonic seizures and was admitted to the intensive care unit with status epilepticus and acute respiratory failure requiring intubation and mechanical ventilation for 4 days. Lumbar puncture results showed: white blood cells – 1.10 6 /l, glucose – 5.3 mmol/l, total protein – 1.6 g/l, increased immunoglobulin levels. The patient was transferred to the Clinic of Infectious Diseases with suspicion of viral encephalitis. HSV type 1 was detected in the cerebrospinal fluid by PCR. Due to suspected hantavirus infection, serum was examined and IgM and IgG antibodies against the HFRS virus were detected by ELISA. Specific treatment with acyclovir was started and there was a rapid improvement in the clinical condition. Convulsions did not recur. Nitrogen levels normalised and there was a short period of polyuria. Thrombocytopenia quickly resolved without occurrence of haemorrhagic syndrome. In the course of the disease, the patient developed mild diarrhoeal syndrome and left thrombophlebitis. He was discharged clinically well with negative PCR results for HSV.
In conclusion, we can assume that as a result of immunosuppression due to HFRS, HSV type 1 reactivated and led to herpes encephalitis.


6. EXITUS LETALIS OF A GIRL WITH HIV/AIDS INFECTION AND HIV-ASSOCIATED CARDIOMYOPATHY (CLINICAL CASE REPORT)

P. Vasilev, T. Velyanova, L. Chochkova, I. Baltadzhiev, A. Dineva, I. Alexiev, P. Gardjeva, M. Ivanovska, M. Murdjeva, M. Stoycheva

ABSTRACT
AThis report describes the clinical case of an 8-year-old girl with very late HIV diagnosis, AIDS-defining diseases and fatal outcome.
Material and methods: Patient examination was performed with clinical and epidemiological analysis, haematological, biochemical, microbiological tests and computed tomography (CT) scan. Virological and immunological monitoring of HIV was conducted by ELISA, western-blot, Real-time PCR and flow cytometry.
Results: An 8-year-old girl with anaemia was hospitalised at the Clinic of Paediatrics, University Hospital “St. George”- Plovdiv. The child was with delayed neurophysiological development, wasting syndrome, severe oropharyngeal candidiasis, hepatomegaly and diarrhoeal syndrome. On 28 March 2018 she was diagnosed with HIV and hospitalised at the Clinic of Infectious Diseases in poor general condition, with fever and progressive cardiac failure. Blood tests showed anaemia, neutrophilia, elevated aminotransferase activity, hypoalbuminemia, viral load of more than 10,000,000 cop/mL and  CD4 + T-lymphocyte count of 1/mm 3 . CT scan revealed congestive changes in the lungs, hepato-splenomegaly and para-aortic lymphadenopathy. Antiretroviral therapy was initiated with zidovudine, lamivudine and lopinavir/ritonavir. On 14 May 2018 the child was discharged with improved general condition and laboratory results. On 3 July 2018 she was hospitalised again with fever, sore throat and inability to swallow. On 4 July 2018 the grandmother took the girl and left the Clinic without leave. On 5 July 2018 she was admitted in a terminal condition. Exitus letalis was registered within hours.
Conclusions: The factors that led to this rapid, progressive course of HIV infection were the late diagnosis and the delayed antiretroviral treatment. The  direct cause of the fatal outcome was HIV-associated cardiomyopathy. Regular screening for HIV in families of HIV-positive patients is required together with well-timed diagnosis and therapy.


7. CHANGES OF THE GALL BLADDER WALL IN CHILDREN WITH SALMONELLA GASTROENTERITIS

V. Velev, M. Pavlova, M. Popov, M. Karageorgiev, E. Aleksandrova

ABSTRACT
This study examined 102 children admitted to the hospital with acute gastroenteritis. Salmonella spp. was identified as the etiological agent in 38 of them. Abdominal ultrasonography monitoring changes of the gall bladder was performed in all patients. Thickening of the gall bladder wall was found in 6 children, 2 of whom developed acute acalculous cholecystitis. All children with thickening of the gall bladder wall in the course of acute Salmonella gastroenteritis were with prolonged hospital stay.


8. SYSTEMIC MYCOSES - SEROLOGICAL AND MOLECULAR DIAGNOSTIC METHODS IN LABORATORY PRACTICE

L. Boyanova

ABSTRACT
The aim of this review is to present the main laboratory methods for diagnosis of the majority of invasive fungal diseases – candidosis, aspergillosis and cryptococcosis. Some studies show an increasing range of infectious agents such as Trichosporon and the endemic fungal pathogens Histoplasma, Blastomyces and Coccidioides. The most common methods for detection of antigens and antibodies in body fluids (serum, plasma, BAL) are indirect immunofluorescense, ELISA Platelia (enzyme-linked immunosorbent assay), latex agglutination, immunodiffusion and PCR-based molecular techniques.


9. DETECTION OF ORAL MICROBIAL FLORA USING POLYMERASE CHAIN REACTION

V. Tolchkov, L. Stefanov, G. Hristova, T. Bolyarova

ABSTRACT
Periodontitis is an inflammatory disease of the oral cavity caused by anaerobic bacteria. In some cases it is related with other common chronic diseases, including cardiological and rheumatological diseases and therefore its diagnosis and therapy is very important. Some of the most common periodontal pathogens are Porphyromonas gingivalis, Prevotella intermedia and Treponema denticola. Classical microbiological methods for their cultivation and detection are cultural methods, which are time consuming, need expensive equipment and trained staff with specific skills. In this study we developed PCR-based technique using dead bacteria useful for diagnosis of these three species. In 83 patients we identified in 8 all of these 3 species, in 11 P. gingivalis and P. intermedia, in 7 P. intermedia and T. denticola, in 6 P. gingivalis and T. denticola, in 7 P. gingivalis, in 10 P. intermedia, in 4 T. denticola. DNA isolation for PCR was optimized without transport media for anaerobes in first time in this study and satisfactory results were observed. We developed a protocol for identification of these three pathogens which is much cheaper that the commercial available kits.


IN MEMORIAM

Assoc. Prof. Irina Haydushka
Prof. Dimitar Strahilov
Prof. Moritz Albert Yomtov


 Editor-in-Chief
Prof. Todor Kantardjiev, MD, PhD, DSc– NCIPD, Sofia, Bulgaria

Editorial Board:
Prof. Iva Christova, MD, DSc – NCIPD, Sofia, Bulgaria
Richard Anthony, PhD – RIVM, The Netherlands
Dale Barnard, PhD – Utah State University, USA
Roger Hewson, PhD – WHO Collaborating Centre for Virus Research & Reference, Porton Down, UK
Judith M. Hübschen – Luxembourg Institute of Health, Luxembourg
Emad Mohareb, PhD– U.S. Naval Medical Research Unit No.3, Egypt
Bruno Gottstein – Institute of Parasitology, University of Berne, Switzerland
Anton Andonov, PhD – Public Health agency, Canada
Anna Papa-Konidari, PhD- Aristotle University, Thessaloniki, Greece
Anna Erdei, DSc - Eötvös L. University, Budapest, Hungary
Lanfranco Fattorini – Instituto Superiore di Sanita, Italy
Hannes Stocklinger, PhD – Medical University, Vienna, Austria
Acad. Bogdan Petrunov, MD, DSc – NCIPD, Sofia, Bulgaria
Assoc. Prof. Iskra Rainova, MD, PhD – NCIPD, Sofia, Bulgaria
Assoc. Prof. Lubomira Nikolaeva-Glomb, MD, PhD – NCIPD, Sofia, Bulgaria

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