TABLE OF CONTENT

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1. ROUTINE LABORATORY DETECTION OF KLEBSIELLA PNEUMONIAE CARBAPENEMASE-PRODUCING ENTEROBACTERIACEAE

S. Sabtcheva, B. Todorova, I. N. Ivanov, E. Dobreva, K. Ivanova, V. Dobrinov, T. Kantardjiev

ABSTRACT
The prompt and accurate detection of carbapenemase-producing Enterobacteriaceae is essential for patient care and infection control procedures. That is why, the introduction of standardised method for routine carbapenemase detection in clinical diagnostic laboratories seems to be mandatory. According to EUCAST methodology carbapenemase inhibition tests with boronic acid derivatives provide a reliable phenotypic confirmation for class A enzymes. We evaluated the performance of a combined disc test, the KPC&MBL&OXA-48 disc kit, for detection of Klebsiella pneumoniae carbapenemase (KPC) production in clinical K. pneumoniae strains recovered at Bulgarian hospitals. Our results indicated that the KPC&MBL&OXA-48 combined disc test is rapid, easy to perform, simple to interpret, and cost-effective method for routine laboratory detection of KPC production with 100% sensitivity and specificity.


2. MDR-TB WITH ADDITIONAL FLUOROQUINOLONE RESISTANCE IN BULGARIAN

S. Yordanova, E. Bachiyska, Y. Atanasova, Y. Todorova, A. Baikova, S. Panaiotov, T. Kantardjiev

ABSTRACT
The aim of this retrospective study was to examine the multidrug-resistant tuberculosis with additional fluoroquinolone resistance in Bulgaria, its distribution in the country and further evolution. The study covered eight-year period of time from 2007 to 2014.
Clinical isolates from 32 patients were confirmed at the NRL TB, NCIPD, as MDR-TB with additional resistance to ofloxacin. Conventional drug susceptibility testing was performed by BАСТЕС MGIT 960 System to first- and second-line drugs using testing concentrations of 0.1 μg/ml for isoniazid, 1.0 μg/ml for rifampicin, and 2.0 μg/ml for ofloxacin. Mutations in the relevant genes were detected by GenoType® MTBDRplus/sl. Genotyping was performed by 24 loci MIRU-VNTR and spacer olygonucleotide typing (spoligotyping).
The most common mutation in the rpoB gene was S531L (96.15%). Isoniazid resistance was attributed to the C15T mutation in the regulatory region of the inhA gene in 80.76% of cases and the A90V mutation in the gyrA gene was found in 70% of cases.
The clustering rate for MIRU-VNTR was 0.73, and for spoligotyping – 0.78.
The highest prevalence of fluoroquinolone-resistant MDR-TB cases was in Dobrich District.
Further evolution of the resistance to XDR-TB was identified in four cases.
Timely detection of fluoroquinolone resistance in MDR-TB is essential for the treatment regimen and its effectiveness.


3. EVALUATION OF THE CARBA NP TEST FOR DETECTION OF CARBAPENEM-PRODUCING ENTEROBACTERIACEAE: PRELIMINARY RESULTS

K. Ivanova, I. N. Ivanov, S. Sabtcheva, B. Todorova, E. Dobreva, V. Dobrinov, T. Kantardjiev

ABSTRACT
Rapid and accurate detection of carbapenemase-producing Enterobacteriaceae (CPE) is of critical important for patient care and for preventing their further dissemination. Although molecular detection of carbapenemase genes is considered the gold standard, no commercial PCR assay has the ability to detect all CPE since the existing phenotypes mutate and new ones emerge. Recently the biochemical Carba NP test has been developed for rapid identification of carbapenemase production in Enterobacteriaceae. The objective of this study was to evaluate the performance of an updated version of the Carba NP procedure for detection of carbapenemase production in Enterobacteriaceae isolated in Bulgaria. Among the 51 isolates tested, 36 produced carbapenemase according to PCR and sequencing. The Carba NP test detected all carbapenemase producers of KPC-type, NDM-type and VIM-type except the sixVIM-1-producing Proteus mirabillis (17% of all carbapenemase-producing Enterobacteriaceae in this study). The carbapenemase negative isolates were correctly identified as negative. The Carba NP test had an overall 83% sensitivity and 100% specificity. The false negative test results with VIM-1-positive P. mirabilis need further investigations.


4. IMPLEMENTATION OF THE MULTILOCUS SEQUENCE TYPING METHOD FOR TYPING OF CARBAPENEMASE-PRODUCING KLEBSIELLA PNEUMONIAE IN BULGARIA

V. Dobrinov, I.N. Ivanov, S. Sabtcheva, B. Todorova, E. Dobreva, K. Ivanova, T. Kantardjiev

ABSTRACT
With the global increase of infections caused by multidrug resistant organisms, there is a growing need for high resolution and reliable typing method able to track those strains. Multilocus Sequence Typing (MLST) is an internationally recognised high-resolution typing method based on DNA sequencing of house-keeping genes providing data that are readily exportable between laboratories. In the recent years, multidrug high-risk MLST clones are considered one of the main vehicles for spreading antimicrobial resistance. These clones have acquired numerous adaptive determinants accompanied by plasmids encoding multidrug resistance and their tracking is vital for preventing further dissemination. In this pilot study, we report the implementation of the MLST technique for Bulgarian Klebsiella pneumoniae strains producing various types of carbapenemases. The results revealed that NDM-1 producers belonged to sequence type ST11, VIM-1 producer was classified as ST147, KPC-2 producers linked to both ST15 and ST258, while OXA-48 producer was determined as ST530. The detection of exclusively high-risk international clones such as ST11, ST15, ST147 and ST258 is a worrisome finding that needs further investigations.


5. METHOD FOR DETECTION OF GROWTH PROPERTIES OF NUTRIENT MEDIA AFTER BEING USED IN SEDIMENTATION TEST IN CLEAN ROOMS

D. Pencheva, M. Iliev, E. Velichkova, P. Genova-Kalou, T. Kantardjiev

ABSTRACT
Requirements for the absence of microbial contamination in the air of premises from a certain class of purity, as well as in laminar flows, situated in different types of laboratories were confirmed by the sedimentation test. The specific conditions in these facilities, however, questioned whether the nutrient media used in the test, keep their growing qualities and are able to register the presence of microbes in the air. A comparative method was successfully developed to assess the growth properties of manufactured batch culture media as a percentage following their use in the sedimentation method. The preserved growth properties show the reliability of the media as an indicator of microbial contamination.


6. LACK OF TYPE-SPECIFIC ANTIBODIES TO HERPES SIMPLEX VIRUS IN A PATIENT WITH GENITAL HERPES: A CASE REPORT

E. Shikova, S. Kyosseva, S. Raleva, A. Kumanova, M. Muhtarova, M. Nikolova

ABSTRACT
We present a woman with clinically manifested HSV2 infection, as evidenced by PCR, while repeatedly seronegative for HSV2IgG and HSV2IgM by gG-based ELISA. Immunodeficiency affecting antiviral immunity was excluded. Our results underline the advantage of combined type-specific serology and viral DNA detection for accurate diagnosis and management of HSV infection.


7. TRYPANOSOMA INFECTION IN MEDITERRANEAN MOUSE (MUS MACEDONICUS PETROV & RUŽIĆ, 1983) IN BULGARIA

H. Dimitrov, Ts. Chassovnikarova, V. Mitkovska

ABSTRACT
Trypanosoma musculi is a non-pathogenic stercorarian trypanosome which is infective only to mice. The present study reveals a Trypanosoma infection in Mediterranean mouse (Mus macedonicus Petrov & Ružić, 1983) from the rice fields in Plovdiv region, Bulgaria. The average established prevalence of the parasite in Mus macedonicus was 17.1% with higher infection rate in male (21.1%) compared to female (12.5%) species. All trypomastigotes exhibited morphological features typical of the subgenus Herpetosoma (Stercoraria section) to which T. lewisi–like parasites belong. These features included: size of approximately 25 ± 5 μm, free flagellum, characteristic “C shape”, visible undulating membrane, oval-shaped subterminal kinetoplast and a nucleus at the anterior end. The characteristic morphology and the presence of infection only in individuals of Mus macedonicus allow us to make an evidence-based assumption that the observed parasite is Trypanosoma musculi. Future characterisation should include molecular methods to confirm the registered species.


8. GENETIC STABILITY OF BCG VACCINE PRODUCTION IN BULGARIA

Tz. Stefanova

ABSTRACT
A century ago, Albert Calmette and Camille Guérin began a daunting task, which is unmatched even today, that led to the most widely used vaccine in human history – BCG vaccine. There is little doubt that BCG will continue to play a role in TB control. It remains part of upcoming clinical trials as an integral component of new vaccinesor a primer to be boosted by new components. In the last years the importance of BCG characterisation and control has been strongly emphasised especially in the light of new knowledge based on the development of sophisticated genome analysis techniques.

The paper describes the results from the molecular typing performed on Bulgarian BCG strain Sofia SL222 and commercial BCG Lots focusing on their specific genetic characteristics in regard to monitoring the consistency of production and checking for genetic variations arising during the maintenance of the sub-strain for more than 30 years.

The genetic stability of Bulgarian BCG vaccine was confirmed by using two different and powerful tools of the post-genomic era – VNTR and DNA microarray hybridisation.


9. GENOTOXICITY BIOMONITORING OF ANTHROPOGENIC POLLUTION IN RICE FIELDS USING THE MICRONUCLEUS TEST IN STRIPED FIELD MOUSE (APODEMUS AGRARIUS PALLAS, 1771)

H. Dimitrov, V. Mitkovska, P. Koleva, Ts. Chassovnikarova

ABSTRACT
Zoomonitoring of small mammal populations, exposed to potential mutagens, can provide an early detection system for the initiation of cell dysregulation. The striped field mouse (Apodemus agrarius Pallas, 1771) is an appropriate zoomonitor species suitable for genotoxicological research, especially due to its wide distribution, r-type reproductive strategy, relatively small home range, high trophic chain position and metabolic rate. The present study was carried out in differently polluted areas. The striped field mice were collected in the rice fields located near Plovdiv (Southern Bulgaria) and in background region of Strandzha Nature Park (Southeastern Bulgaria). The rice fields are exposed to different anthropogenic pollutants like heavy metals and polycyclic aromatic hydrocarbons, due to the nearby located highway and the use of various fertilizers in agricultural practices. The results showed that anthropogenic pollution in rice fields induces DNA and chromosomal lesions in striped field mouse’s cells, which was well demonstrated by the micronucleus assay. The mean frequency of micronuclei in the individuals from the rice fields was significantly higher compared to the mean frequency of the individuals from the background region of Strandzha Nature Park. This proves the existence of geno- and cytotoxic effect in the region of the paddies. The micronucleus assay showed no gender differences. The statistically significant differences in mean frequencies of micronuclei in striped field mice both from the impact and from the background area demonstrated the good genomic sensitivity of the species against anthropogenic pollution. The obtained results confirm the importance of Apodemus agrarius as a zoomonitor species for biomonitoring studies in the species` characteristic habitats – wetlands.


 

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

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

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