MEDICAL AND BIOLOGICAL SCIENCES

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1 UNIWERSYTET MIKOŁAJA KOPERNIKA w TORUNIU COLLEGIUM MEDICUM im. LUDWIKA RYDYGIERA W BYDGOSZCZY MEDICAL AND BIOLOGICAL SCIENCES (dawniej ANNALES ACADEMIAE MEDICAE BYDGOSTIENSIS) TOM XXIII/4 październik grudzień ROCZNIK 2009

2 REDAKTOR NACZELNY Editor-in-Chief Grażyna Odrowąż-Sypniewska ZASTĘ PCA REDAKTORA NACZELNEGO Co-editor Jacek Manitius SEKRETARZ REDAKCJI Secretary Beata Augustyńska REDAKTORZY DZIAŁ ÓW Associate Editors Mieczysława Czerwionka-Szaflarska, Stanisław Betlejewski, Roman Junik, Józef Kałużny, Jacek Kubica, Wiesław Szymański KOMITET REDAKCYJNY Editorial Board Aleksander Araszkiewicz, Beata Augustyńska, Michał Caputa, Stanisław Dąbrowiecki, Gerard Drewa, Eugenia Gospodarek, Bronisław Grzegorzewski, Waldemar Halota, Olga Haus, Marek Jackowski, Henryk Kaźmierczak, Alicja Kędzia, Michał Komoszyński, Wiesław Kozak, Konrad Misiura, Ryszard Oliński, Danuta Rość, Karol Śliwka, Eugenia Tęgowska, Bogdana Wilczyńska, Zbigniew Wolski, Zdzisława Wrzosek, Mariusz Wysocki KOMITET DORADCZY Advisory Board Gerd Buntkowsky (Berlin, Germany), Giovanni Gambaro (Padova, Italy), Edward Johns (Cork, Ireland), Massimo Morandi (Chicago, USA), Vladimir Palička (Praha, Czech Republic) Adres redakcji Address of Editorial Office Redakcja Medical and Biological Sciences ul. Powstańców Wielkopolskich 44/22, Bydgoszcz Polska Poland tel. (052) Informacje w sprawie prenumeraty: tel. (052) medical@cm.umk.pl, annales@cm.umk.pl ISSN X UNIWERSYTET MIKOŁAJA KOPERNIKA W TORUNIU COLLEGIUM MEDICUM im. LUDWIKA RYDYGIERA BYDGOSZCZ 2009

3 Medical and Biological Sciences, 2009, 23/4 REVIEWS CONTENT p. Katarzyna Skonieczna, Marcin Woź niak, Urszula Rogalla, Patrycja Daca, Marta Mielnik, Katarzyna Linkowska, Marta Gorzkiewicz, Jarosł aw Bednarek, Edyta Rychlicka, Anna Czarnecka, Tomasz Grzybowski Genome sequencers towards personalized genomics and medicine Maciej Ś n i e g o c k i Image analysis medical and technical problem ORIGINAL ARTICLES Anna Bednarek, Beata Ję d r u s z a k Health threats in children in the selected rural environment 17 M a r t a C e b u l a k, A n n a K s y k i e w i c z - D o r o t a Structure of utilization of nursing time in psychiatric ward preliminary studies Jerzy Eksterowicz, Marek Napierał a Morphological build of physical education students at the Kazimierz Wielki University in Bydgoszcz Mirosł awa Felsmann, Elż bieta Kę dzierska Patients suffering from acute coronary syndromes and the accompanying anxiety Mariusz Klimczyk Sport profiles of 13-year-old pole vault jumpers Monika Wił kość, Beata Augustyń ska, Aleksander Araszkiewicz, Kinga Sobieralska-Michalak, Anna Dudzic-Koc, Piotr Bijakowski Cognitive functions in alcohol dependent patients. A description of a new cognitive battery QMT (Quick Mind Testing) CASE REPORT Mał gorzata Ł ukowicz, Jan Pawlikowski, Paweł Zalewski, Magdalena Weber-Zimmermann, Katarzyna Ciechanowska, Agnieszka Pawlak Body weight supoort during treadmill therapy in patients after SCI case study

4 Medical and Biological Sciences, 2009, 23/4 PRAGE POGLĄDOWE SPIS TREŚCI str. Katarzyna Skonieczna, Marcin Woź niak, Urszula Rogalla, Patrycja Daca, Marta Mielnik, Katarzyna Linkowska, Marta Gorzkiewicz, Jarosł aw Bednarek, Edyta Rychlicka, Anna Czarnecka, Tomasz Grzybowski Sekwenatory genomowe narzędzia spersonalizowanej genomiki i medycyny Maciej Ś n i e g o c k i Analiza obrazu wspólny problem lekarza i inżyniera PRACE ORYGINALNE Anna Bednarek, Beata Ję druszak Zagrożenia zdrowotne u dzieci w wybranym środowisku wiejskim M a r t a C e b u l a k, A n n a K s y k i e w i c z - D o r o t a Struktura wykorzystania czasu pracy pielęgniarek na oddziałach psychiatrycznych badania wstępne Jerzy Eksterowicz, Marek Napierał a Budowa morfologiczna studentów Uniwersytetu Kazimierza Wielkiego w Bydgoszczy z kierunku wychowania fizycznego Mirosł awa Felsmann, Elż bieta Kę dzierska Pacjenci z ostrymi stanami wieńcowymi a towarzyszący im lęk Mariusz Klimczyk Profile sportowe 13-letnich skoczków o tyczce Monika Wił kość, Beata Augustyń ska, Aleksander Araszkiewicz, Kinga Sobieralska-Michalak, Anna Dudzic-Koc, Piotr Bijakowski Funkcje poznawcze u pacjentów uzależnionych od alkoholu. Opis nowej baterii testów poznawczych QMT (Quick Mind Testing) OPIS PRZYPADKU Mał gorzata Ł ukowicz, Jan Pawlikowski, Paweł Zalewski, Magdalena Weber-Zimmermann, Katarzyna Ciechanowska, Agnieszka Pawlak System dynamicznego odciążenia w terapii chodu na bieżni u pacjenta po urazie rdzenia kręgowego prezentacja przypadku Regulamin ogłaszania prac w Medical and Biological Sciences

5 Medical and Biological Sciences, 2009, 23/4, 5-10 REVIEW / PRACA POGLĄDOWA Katarzyna Skonieczna, Marcin Woźniak, Urszula Rogalla, Patrycja Daca, Marta Mielnik, Katarzyna Linkowska, Marta Gorzkiewicz, Jarosław Bednarek, Edyta Rychlicka, Anna Czarnecka, Tomasz Grzybowski GENOME SEQUENCERS TOWARDS PERSONALIZED GENOMICS AND MEDICINE SEKWENATORY GENOMOWE NARZĘDZIA SPERSONALIZOWANEJ GENOMIKI I MEDYCYNY Department of Molecular and Forensic Genetics, Chair of Forensic Medicine, Nicolaus Copernicus University Collegium Medicum in Bydgoszcz Head: PhD Tomasz Grzybowski Summary In recent years, the development in the DNA sequencing technology has resulted in the invention of new platforms for DNA sequence analysis. The achievements in sequencing technology offer promising opportunities for a fast and cheap, full genome determination which spur research development in biodiversity studies, agriculture or personalized medicine. This review provides information on both whole genome sequencers and sequencing technology applied in each instrument to determine the nucleotide sequence of complete genomes. Streszczenie W ostatnich latach rozwój technologii sekwencjonowania DNA zaowocował powstaniem nowych urządzeń umożliwiających poznanie sekwencji DNA. Osiągnięcia dokonane w dziedzinie sekwencjonowania niosą obiecujące możliwości szybkiego i taniego poznania sekwencji pełnych genomów, a tym samym rozwoju badań nad zróżnicowaniem organizmów, rozwojem rolnictwa czy też tzw. medycyny spersonalizowanej. Niniejsza praca dostarcza informacji o sekwenatorach genomowych oraz wykorzystywanych w nich metodach sekwencjonowania stosowanych do określania sekwencji nukleotydowej pełnych genomów. Key words: Automated Capillary Array Analyzer, FLX Sequencer, Illumina/Solexa Analyzer, SOLiD Analyzer, Heliscope Sequencer, SMRT Sequencer, Nanopore DNA Sequencer Słowa kluczowe: sekwenator kapilarny, sekwenator FLX, sekwenator Illumina/Solexa, sekwenator SOLiD, sekwenator Heliscope, sekwenator SMRT, sekwenator Nanopore INTRODUCTION Information about the whole genomic DNA sequence of individuals could provide us with a better understanding of its influence on a phenotype of organisms. This would have a great impact on and application to many areas of our life, such as energy production (selection and/or modification of organisms like bacteria or fungi to produce energy), agriculture (selection and/or modification of plants, which would boost crop yields) and especially to our health as information about the genome sequence and its variation could provide information on disease risk, diagnosis and proper treatment. In 1977 two methods of sequencing, proposed by Sanger and Maxam and Gilbert respectively, were

6 6 Katarzyna Skonieczna et al. announced and brought the opportunity to determine the DNA sequence [1, 2]. Since then, development in sequencing technology, which allows to improve the sequencing speed, has led to the introduction of new instruments for DNA sequence determination to the market. AUTOMATED CAPILLARY ARRAY ANALYZER The chain termination sequencing method developed by Frederick Sanger is based on the DNA chain termination by dideoxyribonucleotide triphosphates (ddntp s). The original Sanger method relied on oligonucleotide chain elongation with deoxyribonucleotide triphosphates (dntp s) by DNA polymerase, which was terminated by incorporation of 32 P labeled ddntp. Four separate reactions were performed for one DNA template, each containing unlabeled dntp s and one of four ddntp terminators: ddatp, ddctp, ddgtp, ddttp. As the DNA polymerase was not able to distinguish between dntp s and ddntp s, and lack of 3 OH group of ddntp stopped the elongation of DNA, a mixture of fragments of different size was obtained in a single experiment. The determination of the DNA sequence was then elucidated from denaturing acrylamide gel electrophoresis of the four sequencing reactions running simultaneously on the same gel [1]. The modification of the Sanger method based on the usage of four ddntp s labeled with fluorescent dyes of different colors in a single reaction is called dye terminator sequencing. The separation of the DNA fragments of varying lengths and DNA sequence determination could be further provided by slab gel or capillary electrophoresis; however, capillary electrophoresis is much less time-consuming [3]. The first automated 1-capillary array analyzer for capillary electrophoresis was developed by Applied Biosystems and commercialized in Three years later, in 1998, Applied Biosystems introduced the highthroughput automated 96-capillary sequencer to the market [4]. Since then, the dye terminator sequencing technology with the application of an automated capillary array sequencer has been applied for whole genome sequencing of many species from bacteria [5] to a human [6, 7]. The highest throughput of currently available capillary sequencers can be achieved on ABI 3730xl instrument, capable of sequencing 96 DNA fragments (each with reads longer than 900 bp) in a single run time of 3 hours [4]. Despite the above, the method is still too time- and cost-consuming for large genome sequencing projects, given the growing need for sequence data from many fields of molecular biology. To put the matter of costs and time in a perspective one should realize that sequencing of the entire human genome with one high-throughput 96- capillary analyzer would take about 60 years and cost over 30 million US dollars [8]. Thus, invention of new sequencing technologies was necessary to speed up the generation of new large sets of sequence data from either genomes or transcriptomes of different organisms. Below we present a few of those new sequencing techniques, some of them currently available and some announced to arrive to the market in the near future. FLX GENOME SEQUENCER The method used to elucidate the DNA sequence with the FLX instrument relies on an emulsion polymerase chain reaction (empcr), which allows the clonal amplification of a 400 bp long DNA fragment and a subsequent pyrosequencing reaction (sequencing by synthesis method) performed in a PicoTiterPlate (PTP) on an FLX instrument [9]. Emulsion PCR is a reaction of DNA amplification in a water-in-oil emulsion. During empcr a single DNA fragment is linked to the specific adaptor and bound to a single streptavidin-coated bead. Thereafter DNA carrying beads are suspended in a water-in-oil emulsion, so that each bead with a single DNA fragment resides in an individual emulsion droplet, and DNA fragments are amplified in the polymerase chain reaction. As a result of empcr each bead is coated with millions of copies of a single DNA fragment. Subsequently beads carrying amplified DNA are placed into fiber-optic wells on PTP where one bead resides in one well (which is determined by the size of a well), and a DNA sequence is determined through a pyrosequencing reaction on the FLX sequencer [10]. During pyrosequencing the universal sequencing primer anneals to the DNA fragment and afterward is elongated in repeated cycles of sequential dntp incubations and washing with apyrase enzyme (which degrades nucleotides) between each dntp flow. dntp incorporation to the growing DNA strand (whenever it happens) causes the release of inorganic pyrophosphate, which is measured chemiluminescently by a charge-coupled device (CCD) camera and allows the DNA sequence to be determined [11]. The FLX analyzer was developed by 454 Life Science and

7 Genome Sequencers - towards personal genomics and medicine 7 Roche Diagnostics and commercialized in The highest throughput of the FLX instrument could be achieved with Titanium reagents, which allow to generate 1 million reads of up to 400 bp long in a single sequencing run that takes less than 10 hours [9]. The disadvantage of the FLX genome sequencer is low quality of reads of nucleotide homopolymers longer than eight bases [10]. During the last two years the FLX analyzer has made it possible to sequence and resequence whole genomes of bacteria, viruses, plants, and humans [12, 13]. High throughput and ability to perform reads from a single molecule, whose length covers the average length of DNA extracted from fossils, allowed the application of FLX instrument for sequence determination of degraded ancient DNA (adna). The analysis of adna, which could be extracted from hair shafts or bones would have a great impact on evolutionary studies. To date, the FLX analyzer has enabled adna sequence of Neanderthal [14], mammoth [15] or Tasmanian Tiger [16] to be determined. The FLX instrument was also applied in the investigation of the methylation status of CpG sites, transcriptome analysis, metagenomics studies or small RNA molecules characterization [9]. ILLUMINA/SOLEXA ANALYZER Sequencing DNA with the Illumina/Solexa Analyzer is based on sequencing by synthesis method that employs reversible terminators with removable fluorescent dyes. To determine the sequence of DNA, the molecule is fragmented and ligated to adaptors. Subsequently DNA fragments are attached to the optically transparent surface, known as flow cell, via adaptors that bind to the adaptor complementary sequence that resides on a flow cell. During bridge PCR, DNA fragments that are bound by adaptors to the flow cell to create a bridge are amplified to obtain approximately 1000 clonal copies of an individual DNA molecule that forms a local cluster on a flow cell. Up to 100 million clusters of different DNA fragments could be obtained on a single flow cell. Sequencing of DNA molecules in the clusters is carried out on the Illumina/Solexa Analyzer with a universal sequencing primer. Throughout the DNA sequencing one of four reversible terminators labeled with different removable fluorescent dyes is incorporated to the growing DNA chain and the laser detection enables the determination of the DNA sequence. Next, the fluorescent dye is removed and the 3 OH group of the incorporated nucleotide is regenerated allowing DNA elongation with the next reversible terminator [17, 18]. The Illumina/Solexa Genome Sequencer was designed by Illumina, Inc. and introduced to the market in The analyzer is able to generate at least 1 Gb of sequence composed of 35 bp long reads in a single run that takes approximately 3 days [17]. Application of the Illumina/Solexa Sequencer to whole genome resequencing enabled the determination of the DNA profiles of many species, for example, human [18], Caernohabditis elegans [19] or Bacillus subtilis [20]. The Illumina/Solexa sequencer was also applied for transcriptome analysis, metagenomics or characterizing the DNA methylation patterns [17]. SOLiD ANALYZER The SOLiD (Sequencing by Oligonucleotide Ligation and Detection) system, which was designed and commercialized in 2007 by Applied Biosystems, takes advantage of sequencing by ligation technology to determine the sequence of DNA [4]. Preparation of DNA fragments for sequencing with the SOLiD analyzer begins with sequencing library construction through the empcr process. Thereafter the 3 OH end of DNA fragments is chemically modified so that the molecules bind covalently to the glass surface. The sequencing step of the DNA template on the SOLiD analyzer begins with sequencing primer hybridization and subsequent repeated cycles of ligation of fluorescently labeled di-base probes and color detection [4, 21]. To date, the application of the SOLiD Analyzer to whole genome sequencing has been published only for Escherichia coli [22]; however, Applied Biosystems announced the re-sequencing of the whole human genome with this instrument early this year. The SOLiD instrument was also applied to whole transcriptome analysis of undifferentiated mouse embryonic stem cells, mouse blastomere, colon cancer cell line HT-29 or Bacillus anthracis. The highest throughput of the SOLiD sequencing platform could be achieved with the SOLiD 3 System, which enables the generation of up to 20 Gb data of more than 50 bp long reads in a single sequencing run that takes approximately 7 days [4]. HELISCOPE SINGLE MOLECULE SEQUENCER Sequencing DNA templates with the Heliscope Sequencer relies on True Single Molecule Sequencing (tsms) technology. The tsms technology begins with DNA library preparation through DNA shearing and

8 8 Katarzyna Skonieczna et al. addition of poli(a) tail to generated DNA fragments. Next, DNA fragments hybridized to the poli(t) oligonucleotides are attached to the flow cell and simultaneously sequenced in parallel reactions. The sequencing cycle consists of DNA extension with one out of four fluorescently labeled nucleotides, which is followed by nucleotide detection with the Heliscope sequencer. Subsequent chemical cleavage of fluorophores allows the next cycle of DNA elongation to begin with another fluorescently labeled nucleotide and so enables the determination of the DNA sequence [23]. The Heliscope sequencer was developed by Helicos Biosciences Corporation and is capable of sequencing up to 28 Gb in a single sequencing run that takes about 8 days. The Heliscope sequencer generates short reads with a maximal length of 55 bases [24]. So far the Heliscope sequencer has been applied for genome sequencing of the M13 virus [23]. SMRT SEQUENCER DNA sequencing with the SMRT (Single Molecule Real Time) sequencer relies on single molecule real time sequencing by synthesis method provided on the sequencing chip containing thousands of zero-mode waveguides (ZMWs). The sequencing reaction of a DNA fragment is performed by a single DNA polymerase molecule, which is attached to the bottom of each ZMW, so that each DNA polymerase resides at the detection zone of ZMW. During the sequencing reaction the DNA fragment is elongated by DNA polymerase with dntp s that are fluorescently labeled at the terminal phosphate moiety (each nucleotide is labeled with a fluorophore of different color). DNA sequence is determined on the basis of fluorescence nucleotide detection with CCD array, which is performed before nucleotide incorporation, while the labeled dntp forms a cognate association with the DNA template. The fluorescence pulse is stopped after phosphodiester bond formation, which causes the release of a fluorophore that diffuses out of ZMW. Subsequent labeled nucleotide incorporation and detection allows to determine the DNA sequence [25, 26, 27]. The SMRT sequencer was designed and is still being developed by the Pacific Bioscience company. Although the SMRT instrument is not available on the market, the company claims that the SMRT analyzer (capable of obtaining 100 Gb per hour with reads longer than 1000 in a single run) will be commercialized in 2010 [25]. NANOPORE DNA SEQUENCER In contrast to all DNA sequencers mentioned above, sequencing a DNA molecule with the Nanopore DNA sequencer is free of nucleotide labeling and detection. DNA sequencing with the Nanopore instrument is based on converting the electrical signal of nucleotides passing through a nanopore into the DNA sequence. The nanopore is an α-hemolysin pore with a covalently attached cyclodextrin molecule the binding site for nucleotides. During the sequencing process the ionic current that passes through the nanopore is blocked by the nucleotide (previously cleaved by exonuclease from a DNA strand) that interacts with cyclodextrin. The time period of current block is characteristic for each base and enables the DNA sequence to be determined [28, 29, 30]. The Nanopore sequencer is still under development by Oxford Nanopore Technologies [28]. CONCLUSIONS A wide range of genome sequencers is currently available (3730xl sequencer, FLX sequencer, Illumina/Solexa sequencer, SOLiD sequencer, Heliscope sequencer) or will be soon available (SMRT sequencer) on the market (Tab. I). Genome sequencers reviewed in this paper allow to generate high quality sequencing data from many thousands or even millions of single sequence reads varying from 35 to more than 1000 bp. All of the genome analyzers are capable of obtaining short reads, whereas long reads are obtainable only with the 3730xl capillary analyzer, FLX genome sequencer or SMRT sequencer. Although short reads are sufficient for whole genome re-sequencing, they might be inadequate for de novo sequencing, due to problems related to sequence assembly in segments of genomic DNA containing sequence repeats. The current commercially available highthroughput genome sequencers such as the FLX, Illumina or SOLiD platforms allow the whole human genome to be re-sequenced for a fraction of costs required to perform the same task with Sanger technology. The goal is to determine the human genome sequence for no more than 1000 US dollars [30], which would enable individual genomes to be analyzed on a routine basis. To promote the development of human genome sequencing technology the X PRIZE Foundation established the Archont X

9 Genome Sequencers - towards personal genomics and medicine 9 Table I. Properties of sequencers Tabela I. Charakterystyka sekwenatorów GENOME SEQUENCER TYPE (RODZAJ SEKWENATORA GENOMOWEGO) 3730xl Capillary Analyzer (sekwenator kapilarny 3730xl) FLX Sequencer (sekwenator FLX) Illumina/Solexa Analyzer (sekwenator Illumina/Solexa) SOLiD 3 System (system SOLiD 3) Heliscope Sequencer (sekwenator Heliscope) SMRT Sequencer (sekwenator SMRT) Nanopore Sequencer (sekwenator Nanopore) SEQUENCING TECHNOLOGY (TECHNOLOGIA SEKWENCJONOWANIA) dye terminator sequencing (sekwencjonowanie z wykorzystaniem znakowanych terminatorów ) pyrosequencing (pirosekwencjonowanie) Sequencing-by-synthesis with reversible terminators (sekwencjonowanie poprzez syntezę z wykorzystaniem odwracalnych terminatorów ) sequencing-by-ligation (sekwencjonowanie poprzez ligację) single molecule sequencingby-synthesis (sekwencjonowanie pojedynczych cząsteczek poprzez syntezę) single molecule real-time sequencing-by-synthesis (sekwencjonowanie pojedynczych cząsteczek poprzez syntezę w czasie rzeczywistym) Nanopore sequencing (nanosekwencjonowanie*) MAXIMAL READ LENGTH (MAKSYMALNA DŁUGOŚĆ ODCZYTU) BASES/RUN (ILOŚĆ ZASAD\ EKSPERYMENT) RUN TIME (CZAS TRWANIA EKSPERY- MENTU) > 900 bp 96 Kb 3 hours (3 godziny) 400 bp 400 Mb 10 hours (10 godzin) 35 bp 1 Gb 3 days (3 dni) 50 bp 20 Gb 7 days (7 dni) 55 bp 28 Gb 8 days (8 dni) > 1000 bp 100 Gb 1 hour (1 godzina) No information (brak informacji) No information (brak informacji) No information (brak informacji) COMMERCIALLY AVAILABLE (DOSTĘPNOŚĆ NA RYNKU) YES (TAK) YES (TAK) YES (TAK) YES (TAK) YES (TAK) NO (NIE) NO (NIE) * - nanosekwencjonowanie to proponowana przez autorów niniejszego artykułu nazwa techniki polegającej na ustalaniu sekwencji DNA na podstawie sygnałów generowanych przez nukleotydy przechodzące przez otwór o średnicy kilku nanometrów PRIZE for Genomics in Ocotber The 10 million US dollar award will be given to the first team that will be able to sequence 100 human genomes in 10 days for no more than 10,000 dollars per genome [31]. None of the currently available sequencers presented above could reach the 1000 dollar genome or the Archont X PRIZE. REFERENCES 1. Sanger F., Nicklen S., Coulson A.R.: DNA sequencing with chain-terminating inhibiotrs. Proc. Natl. Acad. Sci. USA (1997) 74: Maxam A.M., Gilbert W.: A new method for sequencing DNA. Proc. Natl. Acad. Sci. USA (1997) 74: Huang X.C., Quesada M.A., Mathies R.A.: DNA Sequencing Using Capillary Array Electrophoresis. Anal. Chem. (1992) 64: Oh J.D., Kling-Backhed H., Giannakis M. et al.: The complete genome sequence of a chronic atrophic gastrisis Helicobacter pylori strain: Evolution during disease progression. Proc. Natl. Acad. Sci. USA (2006) 103: Lander E.S., Linton L.M., Birren B. et al.: Initial sequencing and analysis of the human genome. Nature (2001) 409: Venter J.C., Adams M.D., Myers E.W.: The sequence of the human Genome. Science (2001) 291: Bennett S.T., Barnes C., Cox A. et al.: Toward the 1,000 dollars human genome. Pharmacogenomics (2005) 6: Margulies M., Egholm M., Altman W.E. et al.: Genome sequencing in microfabricated high-density picoliter reactors. Nature (2005) 437: Ronaghi M., Karamohamed S., Pettersson B. et al.: Real- Time DNA Sequencing Using Detection of Pyrophosphate Release. Anal. Biochem. (1996) 242: Ronaghi M., Shokralla S., Gharizadeh B.: Pyrosequencing for discovery and analysis of DNA sequence variations. Pharmacogenomics (2007) 8: Rounsley S., Marri P.R., Yu Y., et al.: De Novo Next Generation Sequencing of Plant Genomes. Rice (2009) 2: Green R.E., Malaspinas A.S., Krause J. et al.: A Complete Neandertal Mitochondrial Genome Sequence Determined by High-Throughput Sequencing. Cell (2008) 134:

10 10 Katarzyna Skonieczna et al. 15. Gilbert M.T., Drautz D.I., Lesk A.M., et al.: Intraspecific phylogenetic analysis of Siberian woolly mammoths using complete mitochondrial genomes. Proc. Natl. Acad. Sci. USA (2008) 105: Miller W., Drautz D.I., Janecka J.E. et al.: The mitochondrial genome sequence of the Tasmanian tiger (Thylacinus cynocephalus). Genome Res. (2009) 19: Bentley D., Balasubramanian S., Swerdlow H.P., et al.: Accurate whole human genome sequencing using reversible terminator chemistry. Nature (2008) 456: Hillier L.W., Marth G.T., Quinlan A.R., et al.: Wholegenome sequencing and variant discovery in C. elegans. Nat. Methods (2008) 5: Srivatsan A., Han Y., Peng J., Tehranchi A.K., et al.: High-Precision, Whole-Genome Sequencing of Laboratory Strains Facilitates Genetic Studies. PLoS Genet. (2008) 4:e Ondov B.D., Varadarajan A., Passalacqua K.D., et al.: Efficient mapping of Applied Biosystems SOLiD sequence data to a reference genome for functional genomic application. Bioinformatics (2008) 24: Durfee T., Nelson R., Baldwin S., et al.: The Complete Genome Sequence of Eschrichia coli DH10B: Insights into the Biology of a Laboratory Workhorse. J. Bacteriol. (2008) 190: Harris T.D., Buzby P.R., Babcock H., et al.: Single- Molecule DNA Sequencing of a Viral Genome. Science (2008) 320: Levene M.J., Korlach J., Turner S.W., et al.: Zero-Mode Waveguides for Single-Molecule Analysis at High Concentrations. Science (2003) 299: Eid J., Fehr A., Gray J., et al.: Real-Time DNA Sequencing from Single Polymerase Molecules. Science (2009) 323: Astier Y., Braha O., Bayley H.: Toward Single Molecule DNA Sequencing: Direct Identification of Ribonucleoside and Deoxyribonucleoside 5 - Monophosphates by Using an Engineered Protein Nanopore Equipped with a Molecular Adapter. J. Am. Chem. Soc. (2006) 128: Rusk N.: Cheap third-generation sequencing. Nat. Methods (2009) 6: Address for correspondence: PhD Marcin Woźniak ul. Skłodowskiej-Curie Bydgoszcz tel. (052) marcinw@cm.umk.pl Received: Accepted for publication:

11 Medical and Biological Sciences, 2009, 23/4, REVIEW / PRACA POGLĄDOWA Maciej Śniegocki IMAGE ANALYSIS MEDICAL AND TECHNICAL PROBLEM ANALIZA OBRAZU WSPÓLNY PROBLEM LEKARZA I INŻYNIERA Department of Neurotraumatology, Nicolaus Copernicus University Collegium Medicum in Bydgoszcz Summary Not only is medicine full of humanism, but also it counts on many technological modernizations in this field of science. Modern technologies are frequently used in therapy. However, the most important is to present illnesses and monitor therapy. Technical development gives access to many pieces of information, which can be called a media chaos. Technological development has changed the way in which radiologists analyze images. They used to analyze only one available picture, whereas nowadays they are able to compare as many pictures as were taken. If research on digital analysis is stopped, therapy and diagnostics will not achieve the highest level of advance. The mainstream of researches on digital way of analyzing images is presented in this essay. The most important problem which is taken into consideration in this article is cooperation between doctors and engineers. Streszczenie Współczesna medycyna, mimo przepajającego ją humanizmu, opiera się na zdobyczach techniki. Nowoczesne technologie znajdują codzienne zastosowanie w terapii. Najważniejszą wydaje się obrazowanie procesów chorobowych i monitoring terapii. Postęp technologiczny umożliwia dostęp do wielkiej ilości informacji, które stanowią szum informacyjny. Radiolog, który dotychczas w zaciszu gabinetu analizował pojedynczą kliszę fotograficzną, jest obecnie zasypany setkami obrazów w jednym badaniu, ponadto, istnieje możliwość wykonania setek takich badań w ciągu doby. Zatrzymanie prac nad cyfrową analizą obrazu nie jest możliwe, ponieważ spowoduje automatycznie ograniczenie diagnostyki i terapii chorych z wszystkimi tego następstwami. Praca przedstawia główne kierunki badań i opracowań inżynierów i lekarzy w zakresie cyfrowej analizy obrazu. Szczególnie podkreślona jest rola bliskiej współpracy pomiędzy nimi oraz konieczność wypracowania wspólnego języka zrozumiałego dla obu stron uczestniczących w tym procesie. Key words: image analysis, CT, MRI, USG, co-operation Słowa kluczowe: analiza obrazu, tomografia komputerowa, rezonans magnetyczny, ultrasonografia, współpraca Although modern medicine is very humanistic, technical achievements have a great influence on its development. Biotechnical, metallurgical, optical, scientific, chemical and other inventions are often used in diagnostics and therapy. Imaging diseases and monitoring therapy seem to be the most important in medicine. Radiologists frequently use new techniques and technologies which help them to gather a great amount of information which is called information noise. Doctor, who used to analyse simple photo on his own, now is able to carry out analysis of many pictures at the same time. It is possible to make hundreds of such medical examinations during 24 hours. If research on digital picture analysis stops, diagnostics and therapy will be bound to cease automatically. Digital radiology has a very serious problem with collecting data because of having no more storage space for modern pictures and photos. A widely used method of changing classical picture into a digital one is the use of a digital camera or a laser scanner (1). Famous program Adobe Photoshop 6.0 makes editing radiological photos easier and more popular. What is more, it also makes them more legible. Editing radiological photos and data is possible with a simple computer with 256 Mb RAM at least. Modern digital cameras have resolution close to 2-3 mega pixels. For

12 12 Maciej Śniegocki radiology it is important to use cameras with lens which allow to focus on things which are close to the camera. The transfer of photos from the camera to a computer is done by USB. It is not done automatically, but manually. The effect depends on person s knowledge and skills. Digital pictures are available in a few types which are called formats: bits maps (.bmp), a standard type of pictures in the Windows system, GIF (.gif) Graphical Interchange Format, with 256 colors only, JPEG (.jpg) Joint Photographic Experts Group Format mostly used in the Internet, TIFF (.tif) Tagged Image File Format elastic bits maps, commonly used in printing and publishing. A Photoshop user has many possibilities to edit picture in an appropriate way to achieve its highest quality. This program allows to rotate picture in any way that is needed, select and delete inappropriate parts of the picture it is possible to change the shape, size, color, contrast and brightness of the photo. It is also possible to add some comments, footnotes and elements such as arrows or overlapping pictures. Digital camera and graphical programs let people convert traditional radiological photos into digital data with a small cost, which leads to achieving an acceptable standard of the pictures. It is important that any change in an original radiogram must be accepted by the owner of the photo. Converting and manipulating the data may create an opportunity and simultaneously a danger to change pictures or introduce fake data. 25 years ago one of the most important American medical organizations ACR (American College of Radiology) signed an agreement with NEMA (National Electrical Manufacturers Assosiation). The aim of this agreement was to establish a standard of medical imaging. No one expected how important it would be for the development of digital radiology in the XXIst century (2). Looking back, there are some doubts if DICOM solved all problems connected with digital visualization of medical data. The medical imaging standard, which was created in 1985, guarantees now compatibility between all active departments of radiology, as well as in a whole hospital. The main advantage and the main aim of imaging standard is providing criteria according to which such process must be conducted. Nowadays, when technological changes are so often, the biggest problem is to rate if it is not a disadvantage for digital radiology. There is a race between producers of medical software (PACS/RIS/HIS), whose aim is to offer users as many functions as possible and use the biggest number of possibilities which are available by using medical hardware that is already in hospital (3.4). However, to make it commonly available on the market, it has to satisfy the requirements given by e.g. DICOM s standards. Assessment of the problem from a doctor s and an economist s point of view is definately positive independent body takes care of compatibility between devices from different producers, which prevents monopolizing. We have to face a lot of challenges the importance of telemedicine and work at distance grows, the amount of medical digital data increases in geometric rate and technology of recording data on mobile media constantly develops. How is DICOM s standard in such a situation? DICOM is updated every 2 months. However, it may seem not to be up-to-date as the parts 10 or 12 of the Declaration of compliance with DICOM, which were written more than 10 years ago, are barely changed, which may be perceived as obvious archaism even by a medic (5,6). It is not a mystery that with progressive specialization of medical units their requirements are becoming less and less common. To avoid DICOM as an obstacle in their development, it should be incorporated into the Integration Profiles, which were developed by IHE (Intergrating the Healthcare Enterprise). The organization, which does not create any new standards, puts them appropriately in practice. An example of such a conception is the product Agfa a specific system of digital radiography dedicated to radiology. This system co-operates with X-ray cameras, which produce radiation with small or big energy and also may prepare digital simulation for small or big dose of radiation. The new system of computed radiotherapy is a complete solution, which allows to make radiological pictures by digital radiography methods. In contrast to traditional technology, which was based on photographic film, digital photos may be edited, which allows to improve contrast and visibility of details. The new system of radiotherapy can support a few irradiation stands. It is offered with the CR 25.0 system. The system is a supplement and can even replace electronic stands which are prepared for EPID pictures, because quality of the photos is better than the EPID system. Fields of exposure and resolution of pictures are also bigger. It is possible to co-operate with a common type of picture cassettes, which allows to manipulate photos easily. Thanks to not using traditional plate and chemical processing in a darkroom any more, the new system of radiotherapy is really economical. Pictures taken by DICOM can be put into an information network, which allows to transmit and share them. The main point is the fact, that this system was created in connection with specific requirements of

13 Image analysis - medical and technical problem 13 radiotherapist, which are different than medical requirements. Software system is easy to use and transmission of photos can be done by DICOM. Nearly 60-70% of examinations are based on radiology, which similarly to other sciences wants to use new digital techniques. Radiology changes the way in which pictures are saved from analog to digital. It also benefits from electronic transmission and data saving. Lack of highly qualified radiological staff and the need to increase the effectiveness of work cause using digital techniques. Another factor is the PACS system (Picture Archiving and Communication System). Digital techniques are to improve work in a small hospital departments. If use of digital radiology is encouraged, possible challenges should also be presented. In analog radiology it is hardly possible that all processors break down at the same time, whereas in digital radiology, for example in the DR technique, such a security no longer exists. Systems should be connected with different installations which allow to save and store data at least for 24 hours. In the case when the PACS system fails, functionality gradually decreases as it is in the CT/MR system. At the same time radiologists carry on their work with CT or MR photos on consoles. Of course, discussion about computed systems which let analyze pictures is a part of academic research until their importance and practical purpose is realised. All medical techniques are to help take therapeutic decisions and decrease possibility of making a mistake. This goal can be achieved by imaging structures a doctor is interested in. Fig. 2. Haemorrhagic zone presented in brain tumour Fig. 3. The reason of bleeding presented in brain tumour Fig. 1. Brain tumour Figures 1, 2, 3 present brain tumour. Picture analysis is needed. In the Figure 1 the change in brain is not clear enough to assess its topography properly. Use of an appropriate technique allows to locate it exactly. In the Figure 3 the reason of bleending in brain tumour is presented. It is important for operation planning.

14 14 Maciej Śniegocki Fig. 4. LS spine injury Fig. 6. LS spine injury. 3D picture Figures 4, 5, 6 present spine just after injury and show the possibility to assess effects of treatment. It is achieved by an assessment of bone s union and 3D visualization of spine s deformation. Final diagnosis and treatment not always can be based only on classical radiography. Figure 7 presents compression fracture of thoracic vertebra. It is not possible to say if it is better to operate or not. Figure 8 presents an analysis of radiogram by digital methods, which shows spinal cord compression and makes it easier to decide if it is necessary to operate or not. Fig. 5. LS spine injury. Assessment of bone union after operation Fig. 7. Thoracic vertebra fracture

15 Image analysis - medical and technical problem 15 Fig. 8. Spinal cord compression. The same as in the Figure 7 Classical radiograms are not able to solve every problem. Is the bone cement in Figure 9 in the right place or not? The answer cannot be based only on this picture. A method of image analysis should also be used to decide if vertebroplasty was successful. Fig. 10. Patient after vertebroplasty procedure. Example of use of picture analysis methods Picture analysis methods are very useful to assess the effect of an operation. They are used to assess if implants which stabilize spine are put correctly. Fig. 11. Incorrect setting of TPF screw operation is needed Fig. 9. Classical radiogram after vertebroplasty procedure Fig. 12. TPF screw is put improperly. Operation is needed

16 16 Maciej Śniegocki The doctor who helps immediately after accident often has difficulties to assess radiograms quickly, because he/she does not have time to do some more radiological projections. Figure 13 presents craniovertebral junction damage not very clearly. Only the use of digital methods lets choose a proper kind of treatment. The main aim of this essay is to encourage medical higher education institutions to co-operate with technical ones. Based on my experience I can tell that a co-operation between a doctor, an engineer and a scientist allows to use equipment efficiently. Doctor expects to find appropriate data to decide about treatment. The main problems with medical techniques which must be solved by scientists are: making sick cells more evident, making healthy tissue which is located improperly more evident, imaging changes in a body after accident, imaging location of implants, including these which cannot be seen in classical radiology. I hope the co-operation between doctors and scientists is open to such new methods, because their aim is worthy it is people s health and life. LITERATURE Fig. 13. Damage of cranio-vertebral junction after accident 1. 1.Chalazonitis AN, Koumarianos D, Tzovara J, Chronopulos P. Journal of Digital Imaging, 2003, 16, PACS users wise to maintain DICOM object integrity, Erik L., Ridley, Exploring PACS Secrets -- How to fix DICOM, Michael J., Cannavo, The Dicom Standard, Dicom Analyser,, Digital Imaging and Communications in Medicine; NEMA PS ed.; Global Engineering Documents, Englewood CO, DICOM Basics, Oosterwijk, Herman and Paul T. Gihring; 3nd ed.; OTech, Inc., Aubrey, TX; Fig. 14. Fracture of the first cervical vertebrae All the problems reviewed are difficult for both the doctor who faces them during diagnosis and treatment, and the engineer who is interested in medical techniques. The article shows how important an analysis of pictures is. Nowadays, every medical examination is connected with many pictures, from which any may be the most important to save patient s life. Only computed method, which allows to analyze many pictures, gives a solution to this problem and lets assess health status and choose appropriate data. If modern digital methods are not used, much of the potential of diagnostic equipment will be wasted, which can result in difficulties with curing and restoring patient s efficiency. Address for correspondence: Katedra i Klinika Neurochirurgii i Neurotraumatologii UMK w Toruniu Collegium Medicum im. L. Rydygiera ul. M. Curie Skłodowskiej Bydgoszcz tel.: (52) fax: (52) Received: Accepted for publication:

17 Medical and Biological Sciences, 2009, 23/4, ORIGINAL ARTICLE / PRACA ORYGINALNA Anna Bednarek 1, Beata Jędruszak 2 HEALTH THREATS IN CHILDREN IN THE SELECTED RURAL ENVIRONMENT ZAGROŻENIA ZDROWOTNE U DZIECI W WYBRANYM ŚRODOWISKU WIEJSKIM 1 Head of the Chair and Department of Paediatric Nursing, Faculty of Nursing, Medical University in Lublin 2 Nurse working in the educational environment in the Łęczna county Summary Introduction. The quality of health in the population of the developmental age is shaped from the babyhood under the influence of diverse factors, i.e. intracorporeal and environmental ones as well as the health culture of the family and school. Patterns of health behavior are developed. Biologically negative patterns bring about threats or loss of health of an organism. Health education and health promotion among children and young people are included in essential tasks of nurse in an educational environment. T h e o b j e c t i v e. Identification of health threats in the selected school community in the rural area. M a t e r i a l s a n d m e t h o d s. There were analyzed 823 cards of preventive examination of pupils of the primary school in the Łęczna town near Lublin, in the years 2007 and 2008, with regard to health threats. 85.0% of pupils attending the school come from the rural environment. The primary school in Łęczna belongs to the All-Poland Network of Schools Promoting Health. R e s u l t s. Essential health threats observed in 38.9 % of cases result from different dysfunctions of the osseoarticular system. An often observed illness of the sight organ in the analyzed school community is first of all shortsightedness (35.7%). At the primary school in classes of the Ist-IIIrd grade allergoses are a quite common health threat (6.0%), including bronchial asthma, which constituted 3.4% of the allergy cases. In the IVth-VIth grade classes additional complications are: eating disorders (3.8%), excess weight (4.9 %), short height (8.8 %), chronic illness, i.e. epilepsy - 1.4%, diabetes - 2.3% and mental handicap 1.0%. C o n c l u s i o n s. 1. On the basis of an analysis of medical documentation of pupils of the primary school the number and character of health threats were stated. Among the health threats illnesses of the locomotor system, eyesight defects as well as allergoses prevailed. 2. The knowledge about the health situation of pupils allows to define the educational priorities and develop effective programs of prevention of the identified threats. Streszczenie Wstę p. Jakość zdrowia w populacji wieku rozwojowego kształtuje się od wczesnego dzieciństwa pod wpływem różnorodnych czynników, tj. wewnątrzustrojowych, środowiskowych oraz kultury zdrowotnej rodziny i szkoły. Bionegatywne wzorce zachowań zdrowotnych wywołują zagrożenia bądź utratę zdrowia organizmu. Edukacja zdrowotna oraz promocja zdrowia wśród dzieci i młodzieży należy do istotnych zadań pielęgniarki w środowisku nauczania i wychowania. C e l p r a c y. Identyfikacja zagrożeń zdrowotnych wybranej społeczności szkolnej dzieci wiejskich. Materiał i m e t o d y. Przeanalizowano 823 karty profilaktycznego badania zdrowia uczniów szkoły podstawowej w miejscowości Łęczna k/lublina, w roku 2007 i 2008, w zakresie występowania zagrożeń zdrowotnych. Do szkoły uczęszcza 85,0% uczniów ze środowiska wiejskiego. Szkoła Podstawowa w Łęcznej należy do Ogólnopolskiej Sieci Szkół Promujących Zdrowie. Wyniki. Istotne zagrożenia zdrowotne, zaobserwowane w 38,9% przypadków, wynikają z różnych dysfunkcji układu kostno-stawowego. Często obserwowane choroby narządu wzroku w analizowanej społeczności szkolnej to przede wszystkim krótkowzroczność (35,7%).

18 18 Anna Bednarek, Beata Jędruszak W szkole podstawowej w klasach I-III dość powszechnym zagrożeniem zdrowotnym są choroby alergiczne (6,0%), w tym astma oskrzelowa, która stanowi 3,4% przypadków. W klasach IV-VI dodatkowymi problemami są zaburzenia odżywiania (3,8%), nadwaga (4,9%), niskorosłość (8,8%), choroby przewlekłe, tj. epilepsja 1,4%, cukrzyca 2,3% oraz upośledzenie umysłowe 1,0%. W n i o s k i. 1. Na podstawie analizy dokumentacji medycznej uczniów szkoły podstawowej zaobserwowano ilości i jakość zagrożeń zdrowotnych, wśród których przeważały problemy związane z chorobami narządu ruchu, wadami wzroku oraz chorobami alergicznymi. 2. Wiedza na temat sytuacji zdrowotnej uczniów umożliwia określenie priorytetów edukacyjnych oraz wypracowanie skutecznych programów w zakresie profilaktyki rozpoznanych zagrożeń. Key words: health threats, children, rural environment Słowa kluczowe: zagrożenia zdrowotne, dzieci, środowisko wiejskie INTRODUCTION Conditionings of the health in the population of children and young people are strictly influenced by diverse environmental modifiers. Among them the progressing degradation of the natural and social environment, of the status and socio-economic situation of the family as well as working and learning conditions do not support maintaining optimal attitudes towards the care of one s own health [1]. Eliminating harmful factors from the environment for the sake of correct functioning and shaping the health culture belongs to significant tasks of the social policy implemented by different public sectors and in the indirect way monitored by educational and promotional actions of health service employees [2]. A lifestyle, i.e. a system of behaviours and health attitudes, is the most important factor conditioning the health of an individual. Biologically negative health behaviours trigger threats or loss of one's health. Effects of the deficiency of pro-health attitudes and occurrence of behaviours risky for the health usually appear after many years. Children and young people do not usually notice the direct connection between their behaviour and health and, in general, are not very susceptible to educational influences [3]. Review of the medical literature concerning the health status of the Polish society, including children and young people, isn't satisfactory. Studies reveal that over half of the Polish society displays wrong health behaviours, and about 90% of Poles do not notice the connection between worsening of their health and lack of care for it [4]. The evaluation of pro-health behaviours and resulting from them health threats is an important component of preventive and emergency actions carried out in the population at the developmental age, which is supposed to encourage children and young people to make deliberate, prudent and healthy choices [5, 6]. The aim of the study was to identify health threats in the selected school community of countryside children. MATERIAL AND METHOD There were analyzed 823 cards of preventive examination of pupils of the primary school in the town Łęczna near Lublin, in 2007 and 2008, with regard to health threats. The health card of a pupil consists of four parts. First of them includes information obtained from parents about the health and social functioning of the child in the family and surroundings. Second part includes data obtained from the class tutor concerning identified school problems of the pupil. The third and fourth part concern the information obtained from the school nurse, being the result of conducted screening tests, and the information obtained from a primary health care doctor, being a general evaluation of health and results of physical examination. The statistical software SPSS 14.0PL with the use of numbers, percentage calculations, chi square test of compliance and chi square test of independence, were used. RESULTS 823 pupils attend the school in the Łęczna town, icluding Ist-IIIrd grade classes pupils (207 boys and 204 girls) and the IVth VIth grade classes pupils (212 boys and 209 girls). The school is located in the centre of the town. The school operates in a twoshift system of work and the school nurse s room and the dentist's surgery are in the school building. The school is attended by 85.0% of pupils from the rural environment. The primary school in Łęczna belongs to the All-Poland Network of Schools Promoting Health. Health threats observed in pupils of the Ist-IIIrd grade classes of the primary school (Figure 1) result from different dysfunctions of the osseo-articular

19 system and they predominate in boys (33.4%). Among the dysfunctions pravail flat and lopsided feet % of pupils of the Ist-IIIrd grade classes of primary education and abnormal spinal curvatures - 3.9% of pupils, also of the upper grade classes of the primary school. About 2.0% of pupils have flat foot, and 1.5% asymmetry of shoulder blades. Slouch, coming off shoulder blades and round back are found in 1.0% of pupils. Hyperkyphosis do not appear among the examined population of school children. 35% 30% 25% 20% 15% 10% 5% Health threats at children in the chosen country environment 19 p < 0,001 0% p < In the IVth-VIth grade classes (Figure 2) the structure of health threats looks similar, and additional problems are: eating disorders (3.8 %), excess weight (4.9 %), short height (8.8 %), chronic illnesses, mainly epilepsy % and diabetes % and low-grade mental deficiency 1.0 % 60% 50% 40% 30% 20% 10% chłopcy p < 0,001 dziewczęta Wady narządu ruchu - Defects in the motor organ Wady narządu wzroku - Defects in the sight organ Choroby alergiczne - Allergoses Niskorosłość - Short height Niedobór m. ciała - Deficiency of the body weight Nadwaga - Exscess weight Otyłość - Obesity Wady serca - Heart defects Wady mowy - Flows in the speech Wady słuchu - Hearing defects Epilepsja - Epilepsy Cukrzyca - Diabetes Upośledzenie umysłowe w stopniu lekkim - Mental Handicap in the slight grade 0% chłopcy boys dziewczęta girls Wady narządu ruchu - Deffects in the motor organ Wady narządu wzrok u - Defect in the sight organ Choroby alergiczne - Allergos es Nisk orosłość - Short he ight Nadwaga - Excess weight Otyłość - Obes ity Wady serca - Heart defects Wady mowy - Speech defects Wady słuchu - Hearing defects Epile ps ja - Epilepsy Fig. 1. Structure of health threats amongst pupils of the lower grade (I-III) classes of primary school Ryc. 1. Struktura zagrożeń zdrowotnych wśród uczniów klas młodszych (I-III) szkoły podstawowej Another problem is short height in 12.1% of pupils and eyesight defects in 11.2% of boys and 9.8% of girls. Often observed illnesses of the sight organ in the analyzed school community are mainly shortsightedness, and in 1.4% long-sightedness. Squint occures in 1.7% of children, but disorders connected with seeing colours and astigmatism in 1.0% of the pupils. At the primary school in classes of Ist-IIIrd grade allergoses are a common health threat 6.0%), including the bronchial asthma, which constitutes 3.4% of cases in the analyzed group. Over 4.4 % of pupils of the primary education are obese, and 1.5 % are overweight. The next health threat is connected with speech disorders (4.2%), while in 1.2% of pupils hearing defects appear. To sum up, health threats in pupils of the lower grade classes of the primary school are connected first of all with different dysfunctions of the locomotor system, with sight and speech defects, metabolic disorders and physical development, as well as allergoses of the respiratory tract. Fig. 2. Health threats in pupils of upper grade (IV-V) classes Ryc. 2. Zagrożenia zdrowotne uczniów klas starszych (IV-V) In the group of priority health threats in pupils of the primary school (Figure 3) there prevail: shortsightedness %, short height - 5.6%, obesity - 4.9%, scoliosis %, bronchial asthma - 2.8% and diabetes - 2.3%. Both amongst younger pupils, as well as the upper grade primary school pupils (Figure 4) health threats appeared more often in boys than in girls. Skolioza Scoliosis 4,6%* Krótko- Wzroczność Shortsightedness 18,4%* Astma oskrzelowa Bronchial asthma 2,8%* Problemy zdrowotne Heath problems *p<0,05 Niski wzrost Short height 5,6%* Otyłość Obesity 4,9%* Cukrzyca Diabetes 2,3%* Fig. 3. Priority health threats of pupils of the primary school Ryc. 3. Priorytetowe zagrożenia zdrowotne uczniów szkoły podstawowej

20 20 Anna Bednarek, Beata Jędruszak Fig. 4. Distribution of health threats depending on the sex of the study subjects Ryc. 4. Podział zagrożeń zdrowotnych ze względu na płeć badanych DISCUSSION I-III IV-VI p < 0,001 chłopcy boys dziewczynki girls School age is a period of rapid development of children, when they develop or deepen health irregularities and when health behaviours and abilities of the children children are shaped [7]. Over years substantial and organizational assumptions as to the health care for pupils at school underwent many changes. At first health of a pupil was associated exclusively with providing appropriate sanitary-hygienic conditions at school. The pupil himself/herself and his/her health problems were treated as secondary issue. In recent years a single pupil has become the priority interest of the school Health Service, along with his health status and development and the quality of his/her family environment. Therefore, monitoring parameters of the biological, psychological and social development, conducted by a nurse in the educational environment, constitutes the essential element of the identification of children s disorders and undertaking preventive actions, and of health promotion in the population of school children [8]. Since 1999 a new system of health care for pupils at school has caused that the nurse in the educational environment has been working independently in the partner cooperation with a primary health care doctor. Simultaneously the duty of promoting health amongst pupils is also assigned to the teaching staff of the school and children s parents [9.10]. The present organization of the school system gives the possibility of providing children and young people with comprehensive preventive health care, particularly screening tests, preventive medical check-ups, health education and psychological and pedagogical care. Participation of educational environment nurses in the preventive care of pupils is becoming more and more significant. This tendency reveals the need to accept by nurses specific professional tasks related to health promotion and health education [1.2]. Data obtained during the analysis of medical documentation of the study pupils allows to define their health status as unsatisfactory. Numerous health threats result from different disorders and dysfunction mainly of the locomotor system and eyesight. Growing adverse ecological influences decide about the growing prevalence of allergoses. Also of the growing number of metabolic disorders manifesting itself in excess weight and obesity is alarming. Health threats observed at the primary school in Łęczna are consistent with the health problems characteristic for the entire population of school children. Studies by Wojnarowska [11] and Felińczak et al. [12] also show that about 39.7% of children have different irregularities concerning health, above all dysfunctions of the locomotor system, eyesight defects, metabolic disorders and allergoses. Own studies clearly show that a high percentage of children (82.5%) are categorized to many sorts of unhealthy groups due to their various health problems. Similar data is presented in analyses of other authors. Examinations conducted amongst children from educational institutions in Bielsko Biała show that health problems already occur in nursery schools in 23.3% of children, and at primary schools and lower secondary schools the most common irregularities concern the locomotor system %, eyesight defects %, somatic development - 7.8% and allergoses of the respiratory system - 5.8% [13]. Unhealthy lifestyle of modern societies is bringing about more and more negative effects on health, of which consequences are particularly harmful to the youth. An analysis of the health situation at the primary school in Łęczna gives guidelines concerning the scope of duties of the nurse working in an educational environment, which should include both the identification of health problems in individual pupils and help in solving them, as well as the health education and setting oneself an example to follow [14, 15].