TABLE OF CONTENT

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1. PHENOTYPIC DETECTION OF 16S RRNA METHYLTRANSFERASE-PRODUCING ENTEROBACTERIACEAE BY ROUTINE ANTIBIOGRAM

S. Sabtcheva

ABSTRACT
16S rRNA methyltransferase (16S-RMTase)-mediated aminoglycoside resistance, formerly confined to aminoglycoside producers as a mechanism of self-defence, has recently been identified in Gram-negative pathogens. The genes encoding these determinants are usually associated with extended-spectrum β-lactamases (ESBLs), but recently they have been found in association with carbapenemases resulting in difficult-to-treat multidrug-resistant bacteria. In this study, we analysed resistance phenotypes of 120 well-characterised, consecutive 16S-RMTase-producing Enterobacteriaceae, collected at a cancer hospital during an 11-years survey period (2004-2015). Our results suggest that concomitant use of gentamicin, amikacin, and apramycin provide a reliable screening for 16S-RMTase production in all Enterobacteriaceae. Moreover, the addition of meropenem as an indicator of carbapenemase production and the double-disc synergy test with cefepime for ESBL detection provided a good tool for further surveillance of 16S-RMTase-producing Enterobacteriaceae at the hospital level. In conclusion, concomitant high level resistance to gentamicin and amikacin, but susceptibility to apramycin could be a sensitive screening tool for rapid detection, improved surveillance, and appropriate treatment of 16S-RMTase-producing multidrug-resistant enterobacterial pathogens.


2. COMPARISON OF TWO COMBINATION DISC TESTS FOR PHENOTYPIC DETECTION OF CARBAPENEMASE- PRODUCING ENTEROBACTERIACEAE

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

ABSTRACT
Background: The prompt and accurate detection of carbapenemase-producing Enterobacteriaceae is essential for patient care and infection control procedures. Therefore, the introduction of a standardised method for routine carbapenemase detection in clinical diagnostic laboratories seems to be mandatory. The aim of the present study was to compare the performance of two commercially available combination disc tests for confirmation of carbapenemase production in Enterobacteriaceae, isolated in Bulgaria.

Material and methods: Two commercial combination disc tests (CDTs): the MAST Carbapenemase Detection Set (MAST-CDS) and the KPC&MBL&OXA-48 disc kit were evaluated in comparison with molecular detection of carbapenemase genes. Test strains comprised 58 well-characterised Enterobacteriaceae from the collection of the National Reference Laboratory for Control and Monitoring of Antibiotic Resistance. Of these, 40 Enterobacteriaceae were confirmed to harbour various carbapenemase genes as follows: 17 blaKPC-positive Klebsiella pneumoniae, 3 blaOXA-48-positive K. pneumoniae, 11 blaNDM-positive strains (4 Escherichia coli and 7 K. pneumoniae), and 9 blaVIM-positive strains (6 Proteus mirabilis, 2 Serratia marcescens, and 1 K. pneumoniae). Furthermore, 18 carbapenem-non-susceptible and carbapenemase-non-producing Enterobacteriaceae were also included as negative controls. The tests were performed following the manufacturer’s instructions.

Results: Each of the two CDTs had 100% sensitivity for identification of class A (KPC) and class D (OXA-48) carbapenemases. In non-Proteus species, the sensitivity for class B carbapenemase detection was 100% when MAST-CDS was applied, while KPC&MBL&OXA-48 disc kit failed to detect the class B carbapenemase in VIM-positive S.marcescens strains. Both CDTs in this study failed to detect VIM production in P. mirabilis strains. The overall specificity was 100%.

Conclusions: Our results indicate that the MAST-CDS combined with temocillin disc and the KPC&MBL&OXA-48 disc kit provide reliable phenotypic confirmation for class A, B, and OXA-48 carbapenemases in non-Proteus Enterobacteriaceae species isolated in Bulgaria. For phenotypic detection of VIM-producing P. mirabilis strains another method should be considered.


3. PHENOTYPIC DETECTION OF AAC(6’)-IB-CR-PRODUCING ENTEROBACTERIACEAE BY ROUTINE ANTIBIOGRAM

S. Sabtcheva

ABSTRACT
Plasmid-mediated quinolone resistance (PMQR) in Enterobacteriaceae has increased in the last years, seriously compromising the management of life-threatening infections. PMQR genes (qnr, aac(6 ')-Ib-cr, qepA and oqxAB) confer low-level quinolone resistance and are frequently co-transmitted with extended-spectrum β-lactamase genes. AAC(6)-Ib-cr-mediated aminoglycoside and fluoroquinolone resistance was first detected in 2006, but is now recognised to be widely disseminated. In the present study, we attempted to develop a disc-based method to screen for AAC(6)-Ib-cr producers, based on the peculiar substrate profile of the AAC(6’)-Ib-cr enzyme. We analysed resistance phenotypes of 52 well-characterised aac(6 ')-Ib-cr-positive Enterobacteriaceae, collected at a cancer hospital. Our results show that resistance to tobramycin along with reduced susceptibility to amikacin and ciprofloxacin, but susceptibility to gentamicin, nalidixic acid and levofloxacin provide a reliable screening for AAC(6’)-Ib-cr producion in Enterobacteriaceae. The proposed antibiotic disc configuration reflects the substrate profile of AAC(6’)-Ib-cr enzyme and could be useful for early detection of AAC(6)-Ib-cr-positive/quinolone mutation-negative isolates. We conclude that the developed disc-based method is a simple cost-effective screening tool for rapid detection and appropriate treatment of AAC(6’)-Ib-cr-producing multidrug-resistant enterobacterial pathogens.


4. COMPARISON OF EUROPEAN COMMITTEE ON ANTIMICROBIAL SUSCEPTIBILITY TESTING AND CLINICAL LABORATORY STANDARDS INSTITUTE CRITERIA FOR THE INTERPRETATION OF EXTENDED-SPECTRUM Β-LACTAMASE-PRODUCING ENTEROBACTERIACEAE ISOLATED AT A CANCER HOSPITAL

S. Sabtcheva, B. Todorova, T. Kantardjiev

ABSTRACT
Background: In 2010 European Committee on Antimicrobial Susceptibility Testing (EUCAST) and Clinical and Laboratory Standards Institute (CLSI) eliminated the need for routine extended-spectrum β-lactamase (ESBL) detection and reporting β-lactam susceptibility testing results accordingly. The aim of this study was to determine how many ESBL-producing Enterobacteriaceae test susceptible using EUCAST and CLSI clinical breakpoints.

Material and methods: One hundred consecutive Enterobacteriaceae with ESBL phenotype were collected at the Specialised hospital for active treatment in oncology between August 2015 and June 2016. ESBL production was systematically determined on the basis of observation of a synergy zone between amoxicillin/clavulanic acid and cefepime and/or ceftazidime and/or cefotaxime as described in the EUCAST guidelines for detection of resistance mechanisms. The collection comprised 40 Escherichia coli, 25 Klebsiella pneumoniae, 12 Enterobacter cloacae, 12 Serratia marcescens, 7 Klebsiella oxytoca, 2 Citrobacter freundii, and single isolates of Enterobacter aerogenes and Morganella morganii. Disc diffusion antimicrobial susceptibility testing was performed and interpreted according to EUCAST 2015 and CLSI 2015 guidelines.

Results: All 100 ESBL-producing isolates were non-susceptible to ceftriaxone and cefotaxime if EUCAST and CLSI zone diameter clinical breakpoints were applied, whereas 6% of all (5/40 E. coli and 1/25 K. pneumoniae) were susceptible to cefepime according to both breakpoints. However, significant differences in the susceptibility rates of ceftazidime and aztreonam were demonstrated applying both guidelines. According to EUCAST 2015 breakpoints 4% of all ESBL-producers (3/40 E. coli and 1/25 K. pneumoniae) were susceptible to ceftazidime and aztreonam, whereas, according to CLSI 2015 clinical breakpoints 22% of all (18/40 E. coli, 2/12 S. marcescens, 1/25 K. pneumoniae, 1/1 M. morganii) were susceptible to ceftazidime and 19% (17/40 E. coli, 1/25 K. pneumoniae, 1/1 M. morganii) were susceptible to aztreonam.

Conclusions: All ESBL-producing Enterobacteriaceae isolated at the cancer hospital would be reported to be non-susceptible to cefotaxime and ceftriaxone applying the EUCAST and CLSI 2015 clinical breakpoints, but a substantial number of ESBL-producing E. coli strains would be reported to be susceptible to ceftazidime and aztreonam, according to CLSI 2015 clinical breakpoints. Further harmonisation is needed to cease the controversial reporting of ceftazidime and aztreonam in therapy recommendations when using the EUCAST and CLSI guidelines.


5. SUCCESSFUL TREATMENT OF PERITONITIS CAUSED BY GLYCOPEPTIDE-RESISTANT ENTEROCOCCUS FAECIUM, AND EXTENDED - SPECTRUM Β-LACTAMASE-PRODUCING ESCHERICHIA COLI, ENTEROBACTER CLOACAE, KLEBSIELLA PNEUMONIAE, AND KLEBSIELLA OXYTOCA. CASE REPORT

B. Todorova, S. Sabtcheva, K. Neykov, E. Raicheva, B. Tzingilev, V. Tabakov, T. Kantardjiev

ABSTRACT
Background: Considering the high burden of morbidity and mortality, postoperative peritonitis is a feared complication of intra-abdominal surgery. Typically this is a polymicrobial infection and the most common isolates include aerobic Gram-negative bacilli, anaerobes, and enterococci. Here, we describe a case of postoperative complication involving multidrug-resistant pathogens in a cancer patient.

Case presentation: 79-year-old male patient diagnosed with cancer of the bladder and prostate was admitted to the urology ward at a 242-bed oncology hospital in Sofia, Bulgaria, for radical cystoprostatectomy. Due to small bowel adhesion to the pelvis and ileus causing peritonitis, two re-operations ensued. Empirical therapy was initiated with piperacillin/tazobactam, teicoplanin, and metronidazole. The first positive abdominal drainage fluid cultures yielded ESBL-producing Klebsiella pneumoniae and K. oxytoca followed by isolation of glycopeptide-resistant Enterococcus faecium, and ESBL-producing Escherichia coli, and Enterobacter cloacae. Piperacillin/tazobactam was replaced with administration of meropenem and linezolid, together with metronidazole until clinical improvement and negative cultures.

Conclusion: We report a clinical case of successful treatment of peritonitis caused by glycopeptide-resistant E. faecium, and ESBL-producing E. coli, E. cloacae, K. pneumoniae, and K. oxytoca. Revealing the evolution of underlying resistance mechanisms and changes in microbial etiology during the course of therapy will improve the management of patients undergoing surgical interventions and suffering from comorbidities.


6. MULTIPLE VIRAL PATHOGENS CAUSE “UNDIFFERENTIATED” CNS INFECTIONST

I. Christova, I. Trifonova, R. Vatcheva, V. Ivanova, T. Gladnishka, N. Korsun, A. Stoyanova, L. Nikolaeva-Glomb

ABSTRACT
In an attempt to elucidate etiology, we tested 110 serum samples from patients with “undifferentiated” viral CNS infections for antibodies against herpes simplex virus (HSV), varicella zoster virus (VZV), Epstein-Barr virus (EBV), Coxsackie B viruses (CVB), influenza viruses, parainfluenza viruses, tick-borne encephalitis virus (TBE), and West Nile fever virus (WNV). A total of 21 (19.1%) of the samples were reactive showing presence of IgM antibodies against at least one of the tested viruses. The most common reactivity was found against either EBV, Coxsackie B viruses or influenza viruses – 11 (10%) of the samples reacted with one of the three viruses, followed by HSV-2, TBEV, VZV, and parainfluenza viruses – 6 (5.5%) of the samples were reactive to one of the latter four viruses. Cases of HSV-1 and WNV infections were not detected. The vast majority of the annually recorded viral meningitis and encephalitis cases in Bulgaria are diagnosed only on clinical manifestations and the diagnosis is usually not supported by detection of virus species as the etiological agent. In this study, we succeeded to identify a possible viral etiological agent in one fifth of the cases, showing that investigation for multiple virus species should be pursued in an attempt to confirm the viral etiology of the CNS infection.


7. MEDITERRANEAN SPOTTED FEVER (MSF) WITH UNUSUAL PORTAL OF ENTRY – CASE REPORT

T. Doichinova, G. Gancheva, I. Pakov

ABSTRACT
Background: Mediterranean spotted fever (or Boutonneuse fever) is re-emerging tick-borne infectious disease, caused by Rickettsia conorii. The trend of disease distribution in Bulgaria is increasing in the last two decades. Pleven region is not endemic and only sporadic cases have been registered. The aim was to report a case of R. conorii infection with unusual portal of entry.

Case presentation: 58-years-old woman crushed a tick manually followed by conjunctival exposure of the left eye to the tick`s blood. Six days later there was a sudden onset of fever and shivering. Intensive conjunctival hyperaemia, swollen eyelids, and worsened vision ensued. On the 5th day after hospital admission generalised papulous rash developed involving the palms and the soles. The fever persisted, the woman was in a state of nervous excitement with headache and hepatomegaly; there were no neurological signs. Laboratory investigations revealed normocytosis with granulocytosis (WBC 6.9 x 109/L, granulocytes 0.76, respectively), C-reactive protein 16.1 mg/dL, fibrinogen level 5.14 g/L, and normal liver biochemical tests. Later on, the degree of leucocytosis and granulocytosis increased and the C-reactive protein rose up to 90.2 mg/l. Haemoculture was negative and serological investigation was positive for R. conorii. Complex etiologic and supportive treatment was administered, including ciprofloxacin, lincomycin, and metronidazole intravenously, eye-drops, and symptomatic drugs. The patient improved and discharged after twelve days of hospital treatment.

Conclusions: The case is interesting because of the unusual portal of entry. An increased awareness of MSF could eliminate the risk of delayed and incorrect diagnosis and also improve the prognosis.


8. CASE REPORT WITH PULMONARY AND NEURAL TUBERCULOSIS

G. Gancheva, T. Doichinova, I. Pakov

ABSTRACT
Background: Neurotuberculosis is the most hazardous type of systemic tuberculosis (TB) because of high mortality and possible serious neurological complications. Early diagnosis and prompt treatment are crucial for favourable outcome.

Case report: A 36-year-old female patient was admitted to suburban Infectious Diseases Ward with a one-week history of fever, headache, and vomiting. Primary diagnosis was “viral encephalitis”. Four days later, she became unconscious and was transported to the Clinic of Infectious Diseases at the University Hospital – Pleven. On the physical examination, there was syndrome of meningeal irritation, depressed tendon reflexes, and a positive Babinski’s sign bilaterally. Investigation of cerebrospinal fluid (CSF) revealed increased protein level (3.15 g/L), leucocytes count 80/μL (30% neutro¬phils and 70% mononuclears), decreased glucose level (0.61 mmol/L). Mycobacterium tuberculosis was confirmed by culture of CSF. CT-scan revealed brain edema and subarachnoid cyst suboccipitally. The first X-ray of the lungs was considered as “negative”, the second as „pleuropneumonia”. Tuberculostatic (streptomycin, isoniazid, rifampicin, pyrazinamide) and supportive treatment was performed but the patient’s condition worsened, oculomotor and abducens nerves were involved, respiratory disorders appeared requiring mechanic ventilation. The patient died on the 20th day after admission. On autopsy, infiltrative-pneumonic TB, fibrinous-purulent pneumonia, and bronchiolitis were found. Subsequently tuberculous meningoencephalitis was found, visualised morphologically by lymphocytic basal meningitis and parenchymal vessel vasculitis with microthrombosis. Cortical and basal multifocal ischemia and pulmonary disorders were the direct cause of the lethal outcome.

Conclusion: The globally increased incidence of TB and co-existence of extra-neural tuberculosis and neurotuberculosis require diagnostic improvement and specific therapy even in suspected cases.


 

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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