LUNG COLLAPSE DURING MINITHORACOTOMY REDUCES PENETRATION

OF CEFUROXIME TO THE TISSUE – INTERSTITIAL MICRODIALYSIS STUDY

IN ANIMAL MODEL

Martin Děrgel1,

1 Department of Cardiac Surgery, Charles University, Faculty of Medicine and University

Hospital Hradec Králové; 2 Department of Pharmacology, Charles University, Faculty of

Medicine in Hradec Králové; 3 Department of Anesthesiology, Resuscitation and Intensive

Medicine, Charles University, Faculty of Medicine and University Hospital Hradec Králové; 4 Institute of Clinical Biochemistry and Diagnoses, Charles University, Faculty of Medicine

and University Hospital Hradec Králové; 5 Animal Research Facility, Faculty of Military

Health Sciences, University of Defense, Hradec Králové

Co-authors: M. Voborník1, M. Pojar1, M. Karalko1, J. Gofus1, J. Chládek, 2*, Z. Turek3, J.

Maláková4, Š. Studená2, V. Radochová5, J.Manďák1

*Correspondingauthor: J.Chládek, Department of Pharmacology, Charles University,

Faculty of Medicine in Hradec Králové

Tutor: Prof. Jiří Manďák, M.D., Ph.D.

Background

One-lung ventilation facilitates surgical exposure during minimally invasive cardiac surgery.

However, deeper knowledge on antibiotic distribution within a collapsed lung is mandatory

for an effective antibiotic prophylaxis of pneumonia.

Methods

The pharmacokinetics/pharmacodynamics (PK/PD) of cefuroxime were compared between

the plasma and interstitial fluid (ISF)of collapsed and ventilated lungs in ten anesthetized

pigs, which were ventilated through a double-lumen endotracheal cannula. Cefuroxime (20

mg/kg)was administered in single 30-min i.v. infusion. Samples of blood and lung

microdialysatewere collecteduntil 6 hourspostdose.Ultrafiltration, in-vivoretrodialysis and

high-performance liquid chromatography-tandem mass spectrometry were used to determine

plasma and ISF concentrations of free drug. The concentrations were subjected to non-

compartmental analysis and compartmental modelling.

Results

The concentration of freecefuroxime in ISF was lower in the non-ventilated lung than the

ventilated one, evidenced by a lung penetration factor of 47% vs. 63% (P<0.05),the ratio

between maximum concentrations (65%, p<0.05) and the ratio between the areas under the

concentration-time curve (78%, p=0.12).The time needed to reach a minimum inhibitory

concentration (MIC) was 30-40% longer for a collapsed lung than for a ventilated one. In

addition, a delay of 10-40 minutes was observed for lung ISF when compared to plasma. The

mean residence time of cefuroxime was longer in the former than inthe latter. Regarding

sensitive strains (MIC≤4 mg/L), the percentage of the inter-dose interval with a concentration

above MIC (%fT>MIC) was comparable in all fluids.

Conclusion

The concentration of cefuroxime in the ISF of a collapsed lung is lower thanin a ventilated

one; further, its equilibration withplasma isdelayed. Prolonging the time window between

cefuroximedosage and surgery is recommended, as well as intensifying the dose prophylaxis

against pathogens with higher MICs. PK/PD models enablean optimal prophylactic regimen

combining initial and continuous infusions.

MINISTERNOTOMY FOR AORTIC VALVE REPLACEMENT: IS IT BETTER IN

TERMS OF PULMONARY FUNCTIONS AND QUALITY OF LIFE?

Gofus Ján1

1 Department of Cardiac Surgery, Charles University, University hospital and Faculty of

Medicine in Hradec Kralove, Hradec Kralove, Czech Republic; 2 Department of Pulmology,

Charles University, University Hospital and Faculty of Medicine in Hradec Kralove, Hradec

Kralove, Czech Republic

Co-Authors: M. Voborník1, A. Myjavec1, V. Koblížek2, J. Vojáček1, M. Pojar1

Tutor: Marek Pojar, M.D., Ph.D1

Introduction

Ministernotomy (i.e. upper hemisternotomy) is commonly used as a miniinvasive approach for

aortic valve replacement worldwide. It was shown to provide shorter artificial ventilation time,

shorter ICU stay and lower blood loss comparing with standard median sternotomy approach.

Apart from that, preservation of lower half of thoracic cage could lead to better pulmonary

functions and quality of life postoperatively. There is no recent prospective randomised study

proving this hypothesis.

Aims

To show that ministernotomy leads to better outcomes in pulmonary function and quality of

life testing in short-term postoperatively.

Methods

Between 05/2017 and 05/2019, 30 patients undergoing aortic valve replacement at our

department were prospectively randomised to ministernotomy and full sternotomy group in 1:1

ratio. Standard perioperative data were recorded together with pulmonary function testing

(PFT), 1 minute Sit-to-Stand test (1m-STST) and SF-36 Health Survey. PFT and 1m-STST

were performed one day before surgery, one week after and three months after the surgery. SF-

36 Health survey was filled out preoperatively and three months postoperatively.

Results

There was no difference in standard preoperative characteristics between the two groups.

Ministernotomy group had better preoperative values of obstructive pulmonary parameters

(p<0.001 for FEV1, p<0.001 for FEV1/FVC, p<0.01 for MEF50%). 2 patients (13.3%) from

miniinvasive group required conversion to standard approach. We found no difference in

operative and early postoperative outcomes, except significantly higher blood loss in

sternotomy group (p<0.01). Ministernotomy was shown to cause significantly greater

obstructive defect of pulmonary functions (p=0.02 for FEV1, p=0.03 for FEV1/FVC, p=0.03

for MEF50%). On the other hand, ministernotomy lead to greater improvement in quality of

life postoperatively in terms of physical functioning and general health. Outcomes of 1m-STST

were equal in both groups. No other significant difference was found.

Discussion

In accordance with other studies, our study showed that ministernotomy is non-inferior to

standard full sternotomy approach in terms of standard postoperative outcomes. It lead to lower

blood loss in early postoperative period. Shortening of arteficial ventilation time or ICU stay

was not proved in our conditions.

Stoliński et al. (2016) examined change of pulmonary functions after minithoracotomy

approach for aortic valve replacement and showed superiority of minimally invasive approach.

Results of our study (concentrating on ministernotomy) favor standard full sternotomy

approach. However, the outcomes are disputable because of different values of obstructive

parameters between the two groups preoperatively.

To our knowledge, 1m-STST was never used in cardiac surgery before. It shows tendence to

drop in first week postoperatively and to improve beyond preoperative values after three

months. However, the test was not suitable for comparison of the two surgical approaches.

Quality of life seems to be better in ministernotomy group postoperatively in accordance with

other papers.

Conclusion

Ministernotomy approach seems to be at least as safe as full sternotomy regarding standard

postoperative outcomes. Quality of life could be better than in full sternotomy group. It can lead

to more notable drop in PFT outcomes but clinical implication of this finding remains

questionable. 1m-STST is probably not suitable for comparison of surgical approaches in

cardiac surgery.

References

1. Kirmani BH, Jones SG, Malaisrie SC, Chung DA, Williams RJ. Limited versus full

sternotomy for aortic valve replacement. Cochrane Database Syst Rev

2017;4:CD011793.

2. Phan K, Xie A, Tsai Y-C, Black D, Di Eusanio M, Yan TD. Ministernotomy or

minithoracotomy for minimally invasive aortic valve replacement: a Bayesian network

meta-analysis. Ann Cardiothorac Surg. 2014;4(1):3-14.

3. Stoliński J, Plicner D, Gawęda B, et al. Function of the Respiratory System in Elderly

Patients After Aortic Valve Replacement. J Cardiothorac Vasc Anesth.

2016;30(5):1244-53.

MUTATION IN THE PBP3 GENE AND THEIR EFFECTS ON RESISTANCE TO

BETA-LACTAM ANTIBIOTICS IN HAEMOPHILUS INFLUENZAE STRAINS

Vladislav Jakubů 1,2

1Centre for Epidemiology and Microbiology, National Institute of Public Health, Prague 2Faculty of Medicine in Hradec Králové, Charles University, Czech Republic

Co-authors: L. Mališová1, M. Musílek1, H. Žemličková1,2

Tutor: Assoc. Prof. Helena Žemličková, M.D., Ph.D.

Introduction, objectives

β-lactam antibiotics are the first choice drug in the treatment of Haemophilus influenzae

infections. Since 2010, the National Reference Laboratory for Antibiotics (NRL for ATB) has

been registering an upward trend in the incidence of H. influenzae strains with a non-enzymatic

type of resistance to these antibiotics. Non-enzymatic resistance is due to a decrease in β-

lactams binding to the cell wall due to a mutation of the penicillin-binding protein 3 (PBP3)

encoded by the ftsI gene. This type of resistance is difficult to detect by phenotypic methods.

The aim of this study was to determine the occurrence of mutations, their effect on resistance

to β-lactam antibiotics and identify the epidemiological relationship between strains.

Methods

During 2014-2018, 229 H. influenzae strains were sent to NRL for ATB from 39 clinical

laboratories to confirm non-enzymatic resistance to β-lactams. Detection of ftsI gene mutations

was performed by sequencing a fragment of the ftsI gene (621bp). The obtained nucleotide

sequence was converted into an amino acid sequence and compared to the amino acid sequence

of a reference non-mutated strain (sw Bionumerics). Multilocus sequence typing (MLST) was

used for epidemiological typing followed by the Single locus variant analysis of clonal

complexes (sw Goeburst). The sensitivity of detection of non-enzymatic resistance by

individual β-lactam antibiotics was determined by the disc diffusion method (EUCAST

methodology).

Results

Comparative analysis revealed 23 different amino acid (AA) substitutions in PBP3: V329I,

D350N, S357N, A368T, M377I, S385T, A388V, L389F, P393L, A437S, I449V, I491V,

G490E, R501L, A502S, A502T5, A502T5, A502T5 I519L, N526K, A530, T532S. 37

combinations of these AA substitutions were found (Figure 1). The most common combination

was “D350N, M377I, A502V, N526K”, which was carried by 35% of strains from the tested

group. When substitutions occur in combinations, a “basic” substitution N526K or R517H is

always present. In no combination are N526K and R517H substitutions together. Strains

resistant to the 3rd generation cephalosporins always contained at least the S385T and L389F

substitutions. The rest of the strains analyzed showed a wide variety of mutations. The strains

were epidemiologically diverse, 81 MLST types found (Figure 2).

Figure 1.

Figure 2. Clonal complex analysis

Discussion

The contribution of individual mutations to resistance levels is difficult to determine, only two

(S385T and L389F) have a clear causal relationship to resistance (resistance to the 3rd

generation cephalosporins). Clonal experiments to confirm the contribution of individual AA

substitutions to resistance have not been performed and may possibly be subject to further

study. There is no single clone spread in the Czech Republic, as it results from the analysis of

clonal complexes (Figure 2).

Conclusions

Isolates with non-enzymatic resistance due to mutation of the ftsI gene can be reliably detected

in routine practice only by simultaneous examination of penicillin, ampicillin, amoxicillin /

clavulanate and cefuroxime by the disk diffusion method. A third of strains carried combination

“D350N, M377I, A502V, N526K”, but these strains are not epidemiologically homogenous,

belong to 14 ST.

Blue ellipses – clonal complexes (CC)

with more than 10 strains: CC1218 – 23

strains, CC103 – 18 strains, CC396 – 11

strains.

Red ellipse – the most extensive,

CC1034 – 64 strains.

"Supported by MH CZ - DRO (" National Institute of Public Health - NIPH, 75010330 ")

References

1. Pracovní skupina pro monitorování rezistence (PSMR). Respirační studie ATB

rezistence. Dostupné http://www.szu.cz/respiracni-studie-atb-rezistence

2. Ubukata K, Shibasaki Y, Yamamoto K, el al. Association of amino acid substitutions in

penicillin-binding protein 3 with β-lactam resistance in β-lactamase-negative

ampicillin-resistant Haemophilus influenzae. Antimicrob Agents Chemother 2001; 45;

6: 1693–1699.

3. Witherden EA, Kunde D, Tristram G. PCR screening for the N526K substitution in

isolates of Haemophilus influenzae and Haemophilus haemolyticus. J Antimicrob

Chemother 2013; 68: 2255-2258.

4. Identification of PBP3 mutations in invasive isolates. Dostupné na

https://pubmlst.org/hinfluenzae/.

5. The European Committee on Antimicrobial Susceptibility Testing. Breakpoint tables

for interpretation of MICs and zone diameters, version 7.1, 2017 Dostupné na

http://www.eucast.org/clinical_breakpoints/Clinical breakpoints - bacteria (v 9.0).

NANO-FORMULATED CURCUMIN IN THE TREATMENT OF THE SPINAL

CORD INJURY

Petr Krůpa

Department of Neurosurgery, Faculty of Medicine in Hradec Králové, Charles University in

Prague, Institute of Experimental Medicine v.v.i., The Czech Academy of Sciences

Co-authors: B. Svobodova, J. Dubisova, S. Kubinova, P. Jendelova, L. Machova Urdzikova

Tutor: Prof. Svatupluk Řehák, M.D., Ph.D.

Co-tutor: Assoc. Prof., Dr. Pavla Jendelová, Ph.D.

Introduction

Severe traumatic spinal cord injury (SCI) even in the 21st century remains a challenging medical

condition. Current therapies in the best medical praxis are limited to spinal cord decompression,

high dose corticosteroids, and rehabilitation, all of which result in modest and often very limited

recovery. A permanent or long-lasting neurological deficit is the result of the cascade of

pathophysiological processes, which involve primary and secondary injury. Secondary injury

promotes a series of cellular and molecular events, developing an inflammatory response and

cytokine production, followed by the formation of oedema and free radicals and finally resulting

in ischemia, apoptosis and necrosis of neural and glial cells

Curcumin (1,7-bis[4-hydroxy-3-methoxyphenyl]-1,6-heptadiene-3,5-dione) is a polyphenolic

component derived from the Indian spice - curcuma longa. Curcumin has a wide range of

pharmacological activities, including amongst others anti-inflammatory, antioxidant,

antiinfectious and specially neuroprotective properties (1). Curcumin reduces the production of

reactive oxygen species (ROS), lipid peroxidation and subside the inflammatory responses by

inhibiting the NF-κB pathway. More importantly, the above-mentioned result in a functional

improvement after SCI (2). However, clinical use of the curcumin remains challenging because

of his poor bioavailability. Recently, complexation of the curcumin with various molecules

have been successfully attempted with improved systemic distribution. In our study we used

high purity Curcumin contained within LipodisqTM - biodegradable lipid-based discoidal nano-

particle (Nanocurcumin).

Aims

The main purpose of this study was to investigate the neuroprotective potential of the novel

synthetized drug nanocurcumin on the functional recovery, tissue repair and levels of

inflammatory interleukins after SCI. We chose repetitive, daily, systemic subcutaneous delivery

in combination with local delivery to the site of the lesion, weekly for the first four weeks after

SCI. The effect was assessed by series of behavioural, histological, immunohistochemical and

qPCR analysis.

Methods

Experiment was performed on 10-weeks-old male Wistar rats with the body weight varying

270-300 g. SCI was introduced with the Balloon-induced ischemic-compression model of SCI.

After the surgery, rats were randomly divided into two groups - Nanocurcumin treated group

(n=21) and Nanocarrier (control) treated group (n=11). Nanocurcumin was applied both

directly in situ and systemic (subcutaneously). A direct application of Nanocurcumin around

the dura mater was performed immediately after SCI and then by minimal invasive approach

weekly 7., 14. and 28. day after SCI followed by daily application into the subcutaneous tissue

of the rats back.

Through the experiment the behavioural assessment was regularly performed (BBB, beam walk

test, motoRater). After 9 weeks of behavioural testing, all the animals were perfused with 4 %

paraformaldehyde in PBS and their spinal cords were removed, embedded in paraffin and cut

into cross sections. Histological and immunohistochemical analysis of the tissue sparing, glial

scar and axonal sprouting was performed. To assess the inflammatory dynamic response, qPCR

of selected genes and cytokines was performed 1st and 14th day and 9th week after SCI.

Results

1. Behavioural testing: In the basic locomotor tests (BBB, beam walk) we observed a

trend towards the improved locomotor skills of injured rats in the nanocurcumin treated

group. Significantly improved motor biomechanics of the paw and better self-carrying

ability was observed in the advance biomechanical testing using the motoRater.

2. Histological assessment: In the nanocurcumin treated group was achieved significantly

greater sparing of the white matter tissue cranially to the centre of the lesion.

Nanocurcumin also reduced the total GFAP+ area representing the glial scar as well as

the number of the protoplasmatic astrocytes in the centre of the lesion. Axonal sprouting

of GAP43+ fibers was insignificantly increased in the nanocurcumin treated groups.

3. qPCR: Significant changes in the dynamics of the inflammatory cytokines were found.

1 day after the treatment there was a significantly lower expression of IL-11 whereas 14

days after the treatment, there was significant up-regulation in the expression of

RANTES (CCL5), IL-11 and IL-12 and.

Conclusions/Summary

In conclusion, our study proved the feasibility of the new delivery system of curcumin with a

beneficial role on recovery after SCI. We found significant changes in the locomotor patterns

of the hind-limbs. Histology and immunohistochemistry proved significant beneficial role in

protection of the white matter and reduction of the glial scar. We also observed

immunomodulatory properties with ameliorating of the local immune response in the early

stage of SCI.

References

1. Ormond DR, Peng H, Zeman R, Das K, Murali R, Jhanwar-Uniyal M. Recovery from

spinal cord injury using naturally occurring antiinflammatory compound curcumin:

laboratory investigation. Journal of neurosurgery Spine. 2012;16(5):497-503.

2. Machova Urdzikova L, Karova K, Ruzicka J, Kloudova A, Shannon C, Dubisova J, et

al. The Anti-Inflammatory Compound Curcumin Enhances Locomotor and Sensory

Recovery after Spinal Cord Injury in Rats by Immunomodulation. International journal

of molecular sciences. 2015;17(1).

A PORCINE MODEL OF SKIN WOUND INFECTED WITH

A POLYBACTERIAL BIOFILM

Jan Kucerab,d,e

aBiomedical Center, Faculty of Medicine in Pilsen, Charles University; bContipro , a.s., Dolni

Dobrouc; cFaculty of Medicine, Slovak Medical University, Bratislava, SK; dInstitute

of Histology and Embryology, Faculty of Medicine in Hradec Kralove, Charles University; eCzech Centre for Phenogenomics, Institute of Molecular Genetics/BIOCEV, Vestec;

fDepartment of Dermatology, Third Faculty of Medicine, Charles University, Prague; gInstitute

of Molecular and Translational Medicine, Palacky University, Olomouc; hUniversity

of Pardubice, Faculty of Chemical Technology, Department of Analytical Chemistry, Pardubice; iInstitute of Animal Science, Dept. of Pig Breeding, Kostelec nad Orlici

Co-authors: P. Kleina,b*, M. Sojkab,c, J. Matonohovab , V. Pavlikb,f, J. Nemecb, G. Kubickovab,

R. Slavkovskyb,g, K. Szuszkiewicz b,h, P. Daneki, M. Rozkoti, V. Velebnyb.

Tutor: Prof. Jaroslav Mokry, M.D., Ph.D.

Introduction

It is estimated that 1–2% of people in developed countries experience a chronic wound that will

not heal within four weeks of standard wound care during their life. Although the impaired healing

of such wounds is multifactorial, presence of bacterial biofilms is a proved and important factor.

The eradication of mature biofilm from the wound bed is problematic as bacteria in this form are

highly tolerant to same doses of antibiotics that are effective against planktonic bacteria. Therefore,

there is a growing need to develop and test specially designed treatments for chronically infected

wounds. There is a lack of appropriate in vivo models for studying the healing of chronic infected

wounds, which would closer resemble human-type of wound healing. The skin of pigs is similar

with human skin anatomically, physiologically and biochemically and like human skin, it heals

predominantly by re-epithelization which makes the pig a very valuable in vivo wound healing

model.

Methods

The model was established in 10 castrated male minipigs (Minnesota – type breed) of 13 months

of age. Five wounds reaching the subcutaneous fat (25 × 25 × 5 mm) were excised on each flank.

Four animals served as uninfected saline-treated controls and wounds of six individuals were

infected with an in vitro pre-cultured modified Lubbock type chronic wound multi-species biofilm

consisting of Staphylococcus aureus, Enterococcus faecalis, Bacillus subtilis and Pseudomonas

aeruginosa isolated from chronically infected wounds of clinical patients, and assessed for biofilm

production ability and presence of several virulence factors such as leukocidins and haemolysins

production, and gelatinase and hyaluronidase activity. All wounds were re-bandaged and

documented twice a week. On days 3, 7, 10, 14, and 24 post induction/infection one of five wounds

on each flank was sampled for quantitative microbiology cultivation, histology examination and

gene expression analyses.

Results

The presence of a biofilm significantly affected wound closure defined as the percentage of healed

area compared to the initial wound area measured on day 0. The largest differences (control /

inoculated) were observed on days 7, 10, and 14 (45 %/ 21 %, 66 %/ 37 %, and 90 % / 57 %).

Bacterial counts of the four species used for wound infection behaved differently. E. faecalis slowly

decreased during the course of the experiment. P. aeruginosa was the most abundant bacterial

species in the first 10 days p.i., while a consecutive decrease in the species load was observed from

day 3 until day 24 p.i. In contrast, S. aureus counts appeared to be relatively stable in the first 14

days p.i. and B. subtilis was not detected in the wounds after two weeks. Histological evaluation of

infected wounds revealed necrotic changes, significant increase and extension of both acute and

chronic inflammatory reaction and notably delayed onset (day 10) of decreased quality granulation

tissue formation. Histochemical staining and immunohistochemical detection of individual

bacterial strains confirmed presence of bacteria in a form of biofilm with corresponding production

of specific extracellular polymeric substances. Despite the delay of proliferative phase onset in

infected animals, the wounds were almost or completely epithelised by day 24 in both groups. Gene

expression analysis detected significant elevation of inflammatory mediators interleukin 8, C-X-C

motif chemokine 13 and arginase 1 on days 7, 10 and 14, accompanied by increase of oxidative

stress modulating genes expression such as superoxide dismutase 2 and angiopoetin-like 4.

Enhanced expression of matrix metalloproteinases 1 and 3 and decreased production of collagen 1

and laminin 2 were also found in infected wounds. Factors evaluated suggest prolonged

inflammation, enhanced oxidative stress and perturbed granulation tissue formation.

Discussion

The pig has been selected as a more clinically relevant wound healing model animal due to similar

wound healing mechanisms. Limitations of this model are 1) an availability of only young and

healthy animals without systemic illness typical for patients with chronic wounds, and 2) wound

localisation on animal non-exudating flanks versus usually exudating defects of lower limbs in

human patients. In order to increase the likelihood of successful experimental wound infection, the

bacteria were selected on bases of certain virulence factors presence. The presence of biofilm

caused severe infection and overall impairment of wound healing. The biofilm persisted at least for

two weeks and the rate of wound closure was significantly affected between days 7 and 17. Both

histology evaluation and gene expression profiles corresponded to increased inflammatory and

proteolytic condition and perturbed granulation, resembling the state in clinical chronic wounds.

Conclusion

We were able to successful set up a porcine excisional skin wound model infected with multi-

species biofilm. This model is suitable for studying of host-pathogen interactions occurring during

wound healing events. Within the one-week window this model could be useful also for monitoring

of impact of potential novel antimicrobial/anti-biofilm agents intended for wound biofilm

elimination and wound healing effects.

CYTOLOGICAL-ENERGY ANALYSIS OF PLEURAL EFFUSIONS FROM PATIENTS

WITH CHEST EMPYEMA AND OTHER IMPAIRMENT WITH PREDOMINANCE OF

NEUTROPHILS IN PLEURAL EFFUSION

Inka Matuchová1,2

1Department of Clinical Immunology and Allergology, Faculty of Medicine in Hradec Králové, Charles

University; 2Biomedical Centre, Masaryk Hospital Ústí nad Labem; 3Department of Thoracic Surgery,

Masaryk Hospital Ústí nad Labem

Co-authors: J. Kubalík1,3, E. Hanuljaková2. I. Staněk3, P. Kelbich1,2

Tutor: prof. RNDr. Jan Krejsek, CSc.1

This work was supported by Charles University, Faculty of Medicine in Hradec Králové, Czech

Republic, project ‘PROGRES Q40/10’ and by the Internal Grant of the Krajská zdravotní, a.s. in Ústí

nad Labem, Czech Republic ‘IGA-KZ-2017–1-5’.

Introduction

Pleural infections by extracellular bacteria can be very serious. It affects approximately 100,000

patients in the Czech Republic every year. Some causes develop to the empyema with high mortality

[1,2]. Its essence is a purulent inflammation with oxidative burst on neutrophils. This type of

inflammatory response consumes a lot of oxygen and leads to the fast development of anaerobic

metabolism in the pleural cavity. We assess this process using frequency of neutrophils and coefficient

of energy balance (KEB) values in pleural effusions. The KEB values present a theoretical average

number of adenosine triphosphate (ATP) produced from one molecule of glucose under set conditions

in the pleural cavity. Concentrations of lactate dehydrogenase (LD) and aspartate aminotransferase

(AST) catalytic activities in pleural effusions are useful parameters for evaluation of tissue damage in

the same locality [3, 4, 5].

Aim

Our aim was to compare intensity of local immune response and seriousness of tissue damage in

pleural cavity of patients with chest empyema and patients with transudative pleural effusions, with

pneumonia and after chest surgery. We used a cytological-energy analysis and assessment of LD and

AST catalytic activities in pleural effusions.

Methods

We investigated in total 634 samples of the pleural effusions taken from 283 patients with chest

empyema, 58 patients with heart failure and systemic sepsis (transudative effusions), 89 patients after

chest surgery without inflammation, 108 patients after chest surgery with inflammation and 96 patients

with bacterial pneumonia. Evaluated parameters were relative frequencies of neutrophils, KEB values

and concentrations of LD and AST catalytic activities in pleural effusions. We assessed cut-off values

in pleural effusions for LD = 500.0 IU/L and for AST = 40.0 IU/L. Statistical analysis was performed

using the Mann-Whitney U test (p < 0.01 was considered as significant) via STATISTICA 13.3

software (StatSoft Inc., USA).

Results

Significant differences between patients with chest empyema and patients of other subgroups are

presented in the table.

Table: Cytological-energy analysis and LD and AST catalytic activities in pleural effusions

Groups of patients

N.gr. (%)# KEB LD (IU/L) AST (IU/L)

Median

(1th - 3thquartile)

Median

(1th - 3thquartile)

Median

(1th - 3thquartile)

Median

(1th - 3thquartile)

Transudative effusions

n = 58

58.0*

(49.5 – 73.5)

32.7*

(30.5 – 34.2)

150.6*

(110.1 – 290.7)

15.6*

(9.6 – 27.6)

Chest surgery without

inflammation; n = 89

77.0*

(60.0 – 87.0)

26.7*

(23.7 – 29.6)

685.2*

(410.4 – 1,212.0)

113.4*

(54.0 – 193.8)

Chest surgery with

inflammation; n = 108

87.0

(80.0- 92.0)

4.8*

(-38.2 – 14.1)

1,776.0*

(1,096.8 – 2,889.0)

222.9

(110.9 – 569.9)

Pneumonia

n= 96

84.5*

(75.0 – 91.3)

-169.6*

(-1,771.1 to -5.1)

1,430.4*

(869.2 – 3,212.1)

69.9*

(46.0 – 142.6)

Chest empyema

n = 283

88.0

(78.0 – 94.0)

-2,334.4

(-5,701.8 to -50.8)

4,170.9

(1,495.8 – 13,980.0)

236.4

(76.8 – 570.0)

# N.gr – relative frequencies of neutrophils (%); *significant difference p < 0,01

Discussion

Predominance of neutrophils in pleural effusions is typical for all presented subgroups of patients. The

type and intensity of local immunity response in the pleural cavity correlate with activation of these

immunocompetent cells. Reliable parameter for their specification is the KEB [4, 5]. Its lowest values

in patients with chest empyema, bacterial pneumonia and partly after chest surgery are typical for

purulent inflammation. The most intensive purulent inflammation in patients with chest empyema

correlates with the highest LD and AST catalytic activities in the pleural effusions. Their lower values

were found in the cases of lower intensity of purulent inflammation in patients with bacterial

pneumonia. The high KEB values in patients with transudative effusions and in the 2nd part of patients

after chest surgery excluded the purulent type of inflammation in the pleural cavity. Concentrations of

LD and AST catalytic activities under cut-off values in patients with transudative effusions show

absence of structural tissue damage in their pleural cavity. The lowest intensity or absence of purulent

inflammation and high LD and AST catalytic activities in pleural effusions from patients after chest

surgery shows surgical intervention as very important cause of tissue damage in the pleural cavity.

Conclusion

The KEB values in pleural effusions are useful parameter for determination of the type and intensity of

local immunity response in the pleural cavity. Concentrations of LD and AST catalytic activities in

pleural effusion are suitable parameters for evaluation of structural tissue damage in the same locality.

We found the most intensity of purulent inflammation simultaneously with the most serious tissue

damage in the pleural cavity of patients with chest empyema. We observed the milder intensity of

purulent inflammation and milder tissue injury in the pleural cavity of patients with bacterial

pneumonia. Absence of local inflammation and absence of structural tissue damage in the pleural

cavity were found in patients with transudative pleural effusions. Main reason of tissue injury in the

pleural cavity of patients after chest surgery is a surgical intervention.

References

1. Kašák, V. (2004): Diagnostika a léčba komunitní pneumonie v ambulantní praxi. Interní

Med., 6, 1, 31-33. 2. Garvia V., Paul M. (2019): Empyema. Treasure Island (FL): StatPearls Publishing, available at:

https://www.ncbi.nlm.nih.gov/books/NBK459237.

3. Krejsek J. (2016): Imunologie člověka. Hradec Králové, Garamon s.r.o, 495 stran.

4. Kelbich P, Hejčl A, Selke Krulichová I., et al. (2014): Coefficient of energy balance, a new parameter

for basic investigation of the cerebrospinal fluid. Clin Chem Lab Med, 52, 7, 1009-17.

5. Kelbich P., Malý V., Matuchová I. et al. (2019): Cytological-energy analysis of pleural

effusions. Ann Clin Biochem, 16, doi: 10.1177/0004563219845415.

CRYOPRESERVATION OF DENTAL PULP STEM CELLS

Nela Pilbauerová

Department of Dentistry, Faculty of Medicine and University Hospital in Hradec Králove

Tutor: Jakub Suchánek, M.D., Ph.D.

Introduction

Dental pulp stem cells (DPSCs) have opened a promising future in regenerative medicine,

because they are easily accessible from a dental pulp. As other stem cells, they have

remarkable features known as self-renewal, high proliferation activity and multilineage

differentiation. One of the principal goals in cell based therapies is to deliver individual

treatment to repair or regenerate tissue that has been lost due to an accident or disease. To

achieve this, isolated stem cells have to be amplified and stored in sub-zero temperatures and

thus preserved for future clinical applications.

Cryopreservation is a process of sustaining the viability of cells and tissues by freezing and

storing them at sub-zero temperatures, when biochemical reactions do not occur [1].

However, DPSCs could be a subject to an irreversible damage during the freezing or thawing

process, known as a freezing injury [2]. The exact mechanism is poorly understood, but in

general, irreversible changes to the DPSCs are explained by extracellular and intracellular

formation of ice crystals. To avoid that, there is a cryoprotective agent (CPA) incorporated

into the freezing medium to protect DPSCs during both the freezing and thawing process.

The most frequent freezing medium contains dimethyl sulfoxide (DMSO) as the CPA.

However, there are some documented concerns about its cytotoxicity and associated side

effects [3].

Aim

The purpose of this study was to cryopreserve DPSCs for 6 and 12 months using

uncontrolled-rate freezing technique and observe its effect on biological properties of the

stored stem cells.

Materials and methods

For this study we successfully isolated seven dental pulp stem cell lineages using an

enzymatic digestion method. The isolated populations of DPSCs were cultivated in a

modified cultivation medium (Alpha-MEM), for mesenchymal adult progenitor cells,

containing 2 % fetal bovine serum (FBS) and supplemented with growth factors and Insulin-

Transferrin-Selenium supplement (ITS). The cell viability in the 2nd and 8th passage, cell

count and cell diameter in every passage were examined using a Vi-Cell analyzer and Z2-

Counter. The phenotype analysis in the 3rd and 7th passages was performed using a flow

cytometer Cell Lab Quanta. For differentiation in chondroblasts and osteoblasts we used

commercially available differentiation media.

To assess the effect of uncontrolled-rate cryopreservation, we cryopreserved 1.5 x 106 cells

from the 1st passage for 6 and 12 months . We stored them in -82 °C using uncontrolled-rate

freezing technique and 10 % DMSO as the CPA. After their thawing in 37 °C thermal bath,

we repeated the same cultivation protocol as for the controlled unfrozen group.

Results

Unfrozen DPSCs kept the vitality over 90 % for the entire cultivation. We observed spindle-

like shaped cells. We cultivated them over 46.3 ± 3.2 cumulative population doublings (PD)

in average. The average population time (PT) was 48.9 hours ± 17.0 hours. They showed

high positivity for mesenchymal stem cell markers CD29, CD44, CD90, and negative

expression or low positivity for hematopoietic markers CD34, CD45. We were able to induce

chondrogenesis and osteogenesis.

In compassion to controlled group, DPSCs cryopreserved for 6 months had lower viability

after thawing, but they reached over 90 % viability at the end of the cultivation. The

phenotype analysis showed the phenotype for mesenchymal stem cells and they kept the

ability to differentiate into chondroblast-like and osteoblast-like cells. We cultivated them

over 42.8 ± 4.3 PD in average. The PT was 50.1 hours ± 11.3 hours.

On the other hand, we observed different trend in viability of DPCSs cryopreserved for 12

months. After thawing, we observed high viability over 90 %. However, it dropped to 88 % at

the end of the cultivation. Also, we did not observe any changes in the morphology or

phenotype or ability to differentiate in mature cells. The PD was 41.2 ± 4.1 and PT was 62.2

hours ± 18.0 hours.

Conclusion

The uncontrolled-rate freezing technique is not technically demanding and it is effective for

DPCSs cryopreservation. Cryopreserved cells kept the same morphology, phenotype of

mesenchymal stem cells and the ability to differentiate into mature cells as the controlled

group. They also remained proliferatively active after thawing process. However, we

observed a negative effect on viability with increasing period of time, which DPCSs were

cryopreserved for.

References

[1] Mullen F, Critser JK. The science of cryobiology. Cancer Treat Res 2007; 138:83–109

[2] Zhurova M, Woods EJ, Acker JP. Intracellular ice formation in confluent monolayers of

human dental stem cells and membrane damage. Cryobiology 2001; 61:133–41

[3] Zambelli A, Poggi D, Da Prada G et al. Clinical toxicity of cryopreserved circulating

progenitor cells infusion. Anticancer Res 1998; 18(6B): 4705–8

A NEW APPROACH FOR NEXT-GENERATION SEQUENCING OF BCR-ABL1

KINASE DOMAIN IN PATIENTS WITH CHRONIC MYELOID LEUKEMIA

Michaela Řehounková

Institute of Clinical Biochemistry and Diagnostics, Charles University, Faculty of Medicine in

Hradec Králové and University Hospital Hradec Králové

Co-authors: K. Hrochová, L. Petrová, F. Vrbacký, P. Bělohlávková, P. Žák

Tutor: prof. MUDr. Vladimír Palička, CSc., dr. h. c.

Introduction

Every year, more than 60 patients in Czech Republic [1] are newly diagnosed with chronic

myeloid leukemia (CML). Typical for all CML cases is the occurrence of t(9:22) translocation

known as the Philadelphia chromosome, that can also occur in some cases of acute lymphatic

leukemia. This translocation is responsible for forming a fusion BCR-ABL1 gene. ABL1 gene

normally codes ABL1 protein with a character of tyrosine kinase, that is tightly regulated.

Pathological fusion BCR-ABL1 protein loses its autoinhibitive function and become

unregulated, which is the main driver of a malignant transformation. Nowadays this excessive

kinase activity can be successfully inhibited by tyrosine kinase inbihitors (TKIs) such as

imatinib as first-line therapy, or dasatinib, nilotinib, bosutinib and ponatinib as a TKIs of second

and third generation. But even after 15 years of safe and effective therapy, some patients don’t

reach the expected response. Insensivity to the therapy can be caused by mutations in ABL1

kinase domain, due to the conformational changes in mutated protein. Mutational analysis of

BCR-ABL1 kinase domain can be an important step in the decision of choosing the right TKI

when current response to the therapy is not optimal. [2-4]

Aims

Aim of this study is to develop a new method for detection of mutations in BCR-ABL1 kinase

domain that will be more sensitive and accurate than currently used method based on a Sanger’s

sequencing of ABL kinase domain.

Methods

Because of the required higher sensitivity and accuracy, we have chosen sequencing of the

BCR-ABL1 kinase domain with the Illumina platform for next generation sequencing (NGS)

and we prepared the entire library to be compatible with this system. As a primers for capturing

the fusion transcript we chose one primer in BCR gene and second in ABL1 gene [5], to prevent

amplification of normal ABL1 transcripts. Then we chose over 800bp-long area in ABL1 part

of gene, where all important mutations occur, and we designed 3 pairs of primers to cover this

area, in order to make a 3 overlapping amplicons with a maximal length of 450bp. These

amplicons were sequenced by an Illumina sequencer, but we also performed sequencing by

classical Sanger’s method, to compare the results.

We already analyzed 44 samples from 23 patients. Each sample was sequenced by NGS at least

2 times in a separate analyzing runs, to ensure sufficient accuracy of the results. Samples for

validation of the method were chosen as a screening from a clinical samples tested for a quantity

of BCR-ABL1 transcripts in years 2017-2019 in FNHK. Required relative quantification level

of BCR-ABL1 transcript was from 0,1% to 10% of the International scale (IS). Patients were

chosen because of long-term non-optimal response to the therapy, suddenly relapsed patients

with suspected mutations in ABL1 domain or patients with unstable levels of BCR-ABL1

transcript.

Results

Reads obtained from Illumina sequencer were analyzed through the NextGene software and

compared with reference sequence. Threshold level for confirming the presence of the mutation

was determined as 3%. From a screening cohort of samples were 23 samples analysed with a

mutation or insertion. From that was 12 samples from 5 patients harboring a clinically

significant mutation. Two samples were selected from a given group of patients, from which

we developed a control to ensure the correctness of analysis between individual runes. The first

sample included 2 mutations with high and low mutation levels, and the second sample was

without mutation. These controls were used in each run.

Conclusion

From the obtained data we showed a higher sensitivity of the method compared to classical

Sanger sequencing, which is a great advantage especially in the early stages of disease

development or relapse. Although the methodology is more demanding financially and

manually, it provides more reliable and accurate data. But the question remains, that need to be

further investigated, at what level can the mutation be considered significant for clinical

outcome. We would like to continue in screening in patients for mutations from retrospective

samples, but we also want to perform mutational analysis of BCR-ABL1 kinase domain that is

requested from hematologists in clinical samples from patients in actual need.

This study was supported by MH CZ - DRO (UHHK, 00179906).

Resources

1. Folber F., Koriťáková E. Portál české leukemické skupiny [online]. 2016 [cit. 2018-10-

01]. Available from: http://www.leukemia-cell.org.

2. Soverini, S., et al., In chronic myeloid leukemia patients on second-line tyrosine kinase

inhibitor therapy, deep sequencing of BCR-ABL1 at the time of warning may allow

sensitive detection of emerging drug-resistant mutants. BMC Cancer, 2016. 16: p. 572.

3. Soverini, S., et al., Best Practices in Chronic Myeloid Leukemia Monitoring and

Management. Oncologist, 2016. 21(5): p. 626-33.

4. Branford, S., Molecular monitoring in chronic myeloid leukemia-how low can you go?

Hematology Am Soc Hematol Educ Program, 2016. 2016(1): p. 156-163.

5. Strhakova, L., et al., Use of direct sequencing for detection of mutations in the BCR-

ABL kinase domain in Slovak patients with chronic myeloid leukemia. Neoplasma, 2011.

58(6): p. 548-53.

IMPROVEMENT IN THE QUALITY OF LIFE IN PATIENTS WITH STABLE

MACULOPATHY BY IMPLANTING INTRAOCULAR MACULAR LENS AND

MODULATING VISUAL PLASTICITY BY TRANSCRANIAL ELECTRICAL

STIMULATION

Marketa Stredova

Ophthalmology Clinic Faculty University Hospital Hradec Kralove; Constitution Pat.

Physiology and Biophysics, Medical Faculty in Hradec Králové, Charles University

Co-authors: J. Kremlacek, J. Nekolova, J. Langrova, J. Szanyi, R. Sikl, J. Lukavsky,

D. Moravkova, K. Drtilkova, M. Kuba, Z. Kubova, F. Vit, N. Jiraskova

Tutor: Prof. Naďa Jirásková, M.D., Ph.D.

Introduction

Maculopathy is a dysfunction of macula and is characterized by a progressive loss of central

vision that impairs visual functions and thus unables patients to do normal daily activities. In

developed countries, we most often encounter the age-related macular degeneration (ARMD).

Patients suffering with maculopathy are dependent on the use of optical aids (internal or

external) to be able to read. The external optical aids are used to enlarge the image so that it is

recognizable by the perimacular area of the retina. Unfortunately, they have many limitations.

Therefore, recent emphasis has been placed on the development of intraocular optical aids. In

our study we use the Scharioth intraocular macular lens (SML). SML is a one-piece, add-on

arteficial intraocular lens developed by professor Scharioth. The lens is bifocal, its periphery is

optically neutral or can be individually optically adapted. In the centre of the lens there is an

area of 1.5 mm diameter with addition of plus 10 diopters. The lens is used to improve near

vision, at the same time, the distant vision remains unaffected. Also implantation does not affect

intraocular pressure values or does not narrow the field of view.

Aims

An essential goal of this study is to improve the quality of life of patients with stable

maculopathy. Secondary aims are to verify the effect of transcranial electrical stimulation on

visual rehabilitation and depicting changes in brain plasticity due to both SML implantation and

transcranial electrical stimulation.

Methods

A total of 20 patients will be implanted within 3 years. The follow-up period of one patient is 6

months. To this date, 14 patients has been implanted already, a whole follow-up period is

finished at 10 of them. To join the study patients had to meet strict inclusion and exclusion

criterias. Examinations included assessment of visual acuity, central retinal thickness, contrast

sensitivity, intraocular pressure, visual plasticity in the form of evoked potentials (EP) and

magnetic resonance (MR) and patients‘ subjective satisfaction. The examinations are

performed: preoperatively and then at the day 3, month 1, 2 and 6 after surgery. Only MR

examintation and questionnaires are performed only twice - at the time of indication and at the

last visit. The third day day after implantation a visual rehabilitation and transcranial electrical

stimulation starts and continues for up to another 21 days. Transcranial electrical stimulation is

a low current density electrical current applied to the electrodes placed on the head surface for

several tens of minutes. It serves to modulate brain activity and promote so-called associative

plasticity, it causes subliminal excitability changes in nearby cortical areas, but does not induce

direct reactions or behavioral changes. Side effects, that include pruritus, pricking, redness of

the skin and mild headache, can occur in about 10 % of people, however these symptoms occur

equally frequently in patients with mock stimulation. In our study, transcranial electrical

stimulation is performed in double-blinded design. Visual rehabilitation helps the patient to

learn how to look with a newly implanted lens. The quality of life is assessed using two

questionnaires (MaCDQoL - Macular Disease Dependent Quality of Life and NEI-VFQ -

National Eye Institute Visual Function Questionnaire).

Results

The results of 10 patients are following. In eight patients we found improvement in reading

ability without additional aids or glasses (p = 0.006). The mean distance vision was not

significantly changed, which was also confirmed by the comparison of pre- and post-operative

electrophysiological examinations. Modulation of cortical plasticity by tDCS did not show a

statistically significant effect on reading ability. The questionnaire survey showed that in the

vast majority of patients (9) the positive effects of intervention outweighed the demands of

treatment and stimulation burden.

Conclusion: Intraocular lens implantation and visual rehabilitation have a positive impact on

the capabilities of AMD patients. The results so far have not shown the effect of tDCS

intervention on the measured parameters of visual and cognitive performance. Whether the

effect of current stimulation is at least moderate force will be known after completion of the

second part of the project.

Literature

[1] http://www.medicontur.com/files/For_professionals/A45SML/SML-brochure-doctors-A4-

18-printable.pdf

[2] Grzybowski, A., Wasinska-Borowiec, V., Alio, J. L., a kol.: Intraocular lenses in age-related

macular degeneration. Graefes Arch Clin Exp Ophthalmol (2017) 255: 1687-1696.

[3] Nekolová, J., Rozsíval, P., Šín, M., Jirásková, N.: Scharioth Macula Lens: A new intraocular

implant for low-vision patients with stabilized maculopathy – first experience. Biomed Pap

[4] Med Fac Univ Palacky Olomouc Czech Repub. 2017 Jun; 161 (2): 206-209.

Supported by grant AZV NV18-06-00484.

UNCOMMON BACTERIAL SPECIES ISOLATED IN VARIOUS CLINICAL

SPECIMENS IDENTIFIED IN THE NATIONAL REFERENCE LABORATORY

(NRL) FOR ANTIBIOTICS AND IN THE CZECH NATIONAL COLLECTION OF

TYPE CULTURE (CNCTC) IN 2016-2018

Retana Šafránková1,3

1National Institute of Public Health, Prague, 2Regional Hospital Příbram, 3Faculty of Medicine in Hradec Králové, Charles University

Co-authors: L. Mališová1, M. Musílek1, P. Ježek2, H. Žemličková1,3, V. Jakubů1,3

Tutor: Assoc. Prof. Helena Žemličková, M.D., Ph.D.

The Czech National Culture of Type Cultures (CNCTC) was repeatedly asked for identification

of bacterial strains in the last three years. They were not successfully identified in community

(urban or rural) hospitals by standard phenotypic (i.e. microbiological and biochemical) or by

current/modern methods. Strains were isolated in clinical laboratories and they were sent to the

NRL for Antibiotics / the CNCTC as secondary isolates. They were obtained from blood (n=6),

urethral swab (n=1), pus (n=1), ear swab (n=5) and foreskin swab (n=1).

Strains were initially put under extended biochemical tests (API-Biomerieux) and identified by

matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF

MS; instrument Bruker Microflex). Samples for API were prepared according to manufacturer

instructions applying to particular API sets. Samples for MALDI-TOF MS were prepared by

both direct and extract methods according to the Bruker´s instructions. Complete identification

of strains (if previously mentioned methods were unsuccessful, went wrong or failed) was

achieved with using 16S rDNA-Sequential Analysis. The sequences were evaluated in the

program BioNumerics 7.6.2 and compared with a publicly accessible database (Nucleotide

BLAST).

Strains were identified as: Lactobacillus sakei, Sphingomonas paucimobilis, Corynebacterium

tuberculostearicum, Moraxella atlantae, Gemella morbillorum, Lactobacillus rhamnosus

(blood), Pasteurella dagmatis (urethral swab), Dialister pneumosintes (pus), Cupriavidus

gilardii, Bacillus siralis, Kerstersia gyiorum, Oligella urethralis, Rhizobium radiobacter (ear

swab), Corynebacterium glucuronolyticum (foreskin swab). Isolates belonged to either rare

taxa or taxa uncommonly associated with given diagnosis. Etiological importance of studied

isolates was not evaluated in relation to their accounting for anticipated or determined

diagnoses.

All strains were deposited in the CNCTC.

The MALDI-TOF MS method is accurate, sensitive, rapid and reproducible method for the

identification and characterization of bacteria, but is not always sufficient, especially for

unusual agents. 16S rDNA-Sequential Analysis is a standard for the classification and

identification of bacterial strain; it can be used to create phylogenetic trees showing the possible

relationship of studied microorganisms.

References

1. Zagorec M. et al.: Lactobacillus sakei: A starter for sausage fermentation, a protective

culture for meat products. Microorganisms, 2017, 5(3), 56.

2. Toh H.S. et al.: Risk factors associated with Sphingomonas paucimobilis infection. J

Microbiol Immunol Infect, 2011, 44(4):289-95.

3. Hinic V. et al.: Corynebacterium tuberculostearicum: a potentially misidentified and

multiresistant Corynebacterium species isolated from clinical specimens. J Clin

Microbiol, 2012, 50(8):2561-7.

4. De Baere T. et al.: Bacteremia due to Moraxella atlantae in a cancer patient. J Clin

Microbiol, 2002, 40(7):2693-5.

5. Zakir R.M. et al.: Mitral bioprosthetic valve endocarditis caused by an unusual

microorganism, Gemella morbillorum, in an intravenous drug user. J Clin Microbiol,

2004, 42(10): 4893-6.

6. Gouriet F. et al.: Lactobacillus rhamnosus bacteremia: an emerging clinical entity. Eur J

Clin Microbiol Infect Dis, 2012, 31(9):2469-80.

7. Xiong J. et al.: Bacteremia due to Pasteurella dagmatis acquired from a dog bite, with a

review of systemic infections and challenges in laboratory identification. Can J Infect Dis

Med Microbiol, 2015, 26(5): 273–6.

8. Rousée J.M. et al.: Dialister pneumosintes associated with human brain abscesses. J Clin

Microbiol, 2002, 40(10): 3871–3.

9. Tena D. et al.: Muscular abscess caused by Cupriavidus gilardii in a renal transplant

recipient. Diagn Microbiol Infect Dis, 2014, 79(1):108-10.

10. Pettersson B. et al.: Bacillus siralis sp. nov., a novel species from silage with a higher

order structural attribute in the 16S rRNA genes. Int J Syst Evol Microbiol, 2000, 50 Pt

6:2181-7

11. Pence M.A. et al.: Two cases of Kerstersia gyiorum isolated from sites of chronic

infection. J Clin Microbiol, 2013, 51(6): 2001–4.

12. Ježek P. et al.: Isolation of Oligella urethralis, a rare species, from a labial cyst of a

gynecological patient. Zprávy CEM (SZÚ, Praha), 2012, 21(1): 19–21.

13. Lai C.C. et al.: Clinical and microbiological characteristics of Rhizobium radiobacter

infections. Clin Infect Dis, 2004, 38(1): 149-53

14. Gherardi G. et al.: Corynebacterium glucuronolyticum causing genitourinary tract

infection: Case report and review of the literature. IDCases, 2015, 2(2): 56–8.

15. Bøvre K. et al.: Moraxella atlantae sp. nov. and its distinction from Moraxella

phenylpyruvica. Int J Syst Bacteriol, 1976, 26, 511-21.

Acknowledgement: Project was supported by SVV 260398.

SINGLE CONTROLLED VISUAL EVOKED POTENTIALS (VEP) DISPLAY

Petr Voda

Co-authors: A. Bezrouk, J. Kremlacek

Tutor: Assoc. Prof. Jan Kremlacek, Ph.D.

Introduction

Reversal pattern visual stimulation in conjunction with evaluating visually evoked potentials

(VEP) is a common examination method used for the diagnosis of disability of visual

sensorial pathways from the peripheral receptors up to the primary visual cortex and is

commonly used to diagnose different disabilities of this pathway, [1]. Optical stimulus for this

examination is to swap the contrasting elements of the displayed structure, usually reversing

black and white blocks of chess (reversal pattern VEP).

Stimulator for these examinations may be the classic mechanical-optical system or the screen

(computer monitor), what brings some problems [2], [3], [4], [5]. Experiments with LED

matrix have been made [6]. Ideal properties of VEP monitors are defined in ISCEV standard

to obtain systems were comparable [7], [8].

The main imperfection common to all referred imaging systems is a long time of reversing of

all the structure in the entire area of the image, the gradual picture arising and reversing of the

entire structure taking certain time [5].

Aims

The aim of the study is to design a device, that displays the image not gradually (by

scanning), but at the same time, having no inertia. To measure properties of such device,

compare to currently used devices (screens) and compare potentials evoked by this device to

potentials evoked using the screen.

Methods

There has been created Single Controlled LED device (SCLED), that contains a very

contrasting array of 144 white light LED elements the size of a 5 mm cubes with flat matte

face placed close side to side with no space between. These elements are custom made to

achieve full opacity between the individual elements. That's a big improvement over the

problems caused by shady bulkheads and dispersion plate [6]. These LEDs are individually

connected to the system of drivers driving each LED individually. The display is controlled

by a microcontroller, what allows to achieve a reversal of the whole structure at once with a

single synchronization pulse coming to all drivers synchronously and allows to create any

(black and white) image consisting of the basic 5mm square elements.

Three types of measurements have been made. 1) Validating electrical and optical properties

of designed SCLED system, 2) measurements on some LCD, TFT and CRT screens to

compare designed SCLED display, 3) testing of the SCLED device on persons in

experimental conditions of real physiological laboratory and comparison of the device with

CRT monitor used now for analysis.

Results

1) The measured rise time of designed SCLED is 20 us, what is 100 times faster than

commonly used LCD display. Easing all LEDs in the entire area of the display is synchronous

and the time of lightning of all LEDs is the same.

Additionally, the brightness, its settling and its

stability during the time was measured and

compared.

2) Measurements of conventional LCD TFT and

CRT monitors with two matching calibrated probes

show that changes brightness of individual parts of

the image and its stability in time at various types

of display is much worse compared to designed

LED display.

3) The results of measurements of persons were

designed into graphs marked with the letters of

persons. Red line in graph represents using CRT

screen, blue line is designed SCLED stimulator.

First column is obtained using 5mm square

checkboard, second column represents 10mm

square checkboard.

Discussion

Measurements implies that the whole required structure of SCLED device lights up during

20us what is 100 times faster than commonly used LCD display. The stabilization of

brightness (however in 10% fluctuation only) takes 5ms and brightness of the display is stable

all the time. That is also much better than common LCD display, what is 4 times slower. The

voltage of the probe when LED is off, is not measurable, thus device has a very high contrast.

Measurements by two probes taken on CRT monitor show very quick easing of brightness,

but also just a very short time of illumination. One can see very clearly the low contrast of the

monitor and the time difference between easing the element at the top and bottom of the

image (up to 10 ms). Flickering can be seen clearly on CRT tube recording. With

conventional LCD (TFT) is the full brightness rising time 20 ms, descending time 2ms. Rising

time of the structure using mechanical-optical VEP system is 2 ms [5].

Measured electrical and optical characteristics of the designed and completed apparatus are

significantly better than the existing types of stimulators. The most important parameter (time

of easing and the speed of reversal of the whole structure) is several orders of magnitude

better. Also the stability of the brightness and speed of the exchange of individual images is

significantly higher than for the other types. Differences in the measured responses for few

persons are not significant, but it can be stated, that the realized SCLED stimulator gives a

slightly better results. Due to the small size of the stimulator the only a small part of the

Visual field is stimulated, so latency of displaying distant parts of the image not apply much.

The results of our measurements correspond with the findings in [4], [2].

Conclusion

There was created a new prototype of Single controlled LED (SCLED) pattern shift VEP

Stimulator. Its electrical and optical characteristics are significantly better than the existing

types of stimulators. Pilot testing on the small group of tesing people shows that this SCLED

stimulator is suitable for examination of the VEPs and gives at least similar, maybe slightly

better, results than conventional CRT monitors.

Therefore, it has a sense to think about the construction of larger display assembled from

several here designed SCLED stimulators used as serialized modules controlled by one

common microcontroller. It can be assumed, that such a very synchronous stimulator, featured

with zero latency of individual parts of the image can provide better results than conventional

monitors used to stimulation, whether the CRT or now used LCD or OLED displays.

Acknowledgement

This work was supported by the programme PROGRES Q40-09

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[2] Zhang, G., Li, A., Miao, C., He, X., Zhang, M., & Zhang, Y. (2018). A consumer-grade

LCD monitor for precise visual stimulation. Behavior Research Methods.

doi:10.3758/s13428-018-1018-7

[3] Nagy, B. V., Gemesi, S., Heller, D., Magyar, A., Farkas, Á, Ábraham, G., & Varsanyi, B.

(2011). Comparison of pattern VEP results acquired using CRT and TFT stimulators in

the clinical practice. Documenta Ophthalmologica,122(3), 157-162. doi:10.1007/s10633-

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[4] Cooper, E.A., Jisng, H., Vildavski, V., Farrell, J.E., &Nortica, A.M. (2013) Assessment

of OLED displays for vision research. Journal of Vision,13(12):16, 1-12.

doi:10.1167/13.12.16

[5] Kubova, Z., Kuba, M., Spekreijse, H., & Blakemore, C. (1995). Contrast dependence of

motion-onset and pattern-reversal evoked potentials. Vision Research, 35(2), 197-205.

doi:10.1016/0042-6989(94)00138-c

[6] Link, B., Rühl, S., Peters, A., Jünemann, A., & Horn, F. K. (2006). Pattern Reversal ERG

and VEP – Comparison of Stimulation by LED, Monitor and a Maxwellian-view System.

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