<p><strong>Introduction. </strong>Antibiotic resistance is a major threat to public health globally. The aim was to examine the impact of the COVID-19 pandemic on the distribution and antimicrobial resistance of pathogenic microorganisms isolated from samples&nbsp;<br />obtained during standard hospital care in one hospital center. &nbsp;<br /><strong>Methods.</strong> Data were obtained retrospectively from a database of the hospital microbiology laboratory. Study sample consisted of 3012 samples tested before the pandemic in 2019 and 3130 samples from the pandemic period in 2021.&nbsp;<br /><strong>Results.</strong> There was no statistically significant difference in the occurrence of agents that were resistant to all antibiotics between the observed years, but there was the difference in the occurrence of those agents between departments, with the highest frequency in the intensive care unit and the COVID-19 department (p&lt;0.001). Isolation of Acinetobacter bacteria increased 2.7 times, and Clostridioides difficile 6.4 times during 2021 compared to 2019. Statistically significant differences were registered in resistance to: imipenem, piperacillin-tazobactam, ceftayidime, cefepime, glycopeptides, aminoglycosides, levofloxacin and ciprofloxacin in 2021 compared to 2019.&nbsp;<br /><strong>Conclusion. </strong>Our results suggest possible influence of COVID-19 on antimicrobial resistance and input a need for a new larger study addressing this issue.</p>

<p><strong>Introduction.</strong> Vitamin D is a liposoluble vitamin that has many important roles in the human body. Daily requirements for vitamin D are met through intake of food and exposure to sunlight. The high frequency of vitamin D deficiency is a public health problem that can be corrected using food supplements (FS), which is why its consumption is increasing. The quality of FS, including the content of active components, depends on the good manufacturing practice which is not strictly regulated for the production of FS, as well as the formulation, packaging and storage. Because of that, the quality of FS can be variable. The aim of our pilot study was to examine the conformity of the actual determined content of vitamin D and the declared content in 49 FS, in the form of tablets and capsules, present on the market in Republic of Srpska. <strong>Method.</strong> Determination of vitamin D content was performed by high-performance liquid chromatography (HPLC). <strong>Results.</strong> The range of the determined content of vitamin D in relation to the declared content, expressed as a percentage, was from 64,4% to 188,8%, whereby the deviation is not statistically significant (mean: 100.50%; Cl 95% -0.54 to 0.17; p=0.313 tablets; mean: 98.02%; Cl 95% -3.00 to 0.42; p=0.127 capsules). When measurement uncertainty is taken into account, only one sample (2.0%) was outside the legally allowed range (88.8% more than labeled). <strong>Conclusion.</strong> Therefore, it has been shown that most of the examined products are of satisfactory quality in terms of vitamin D content, but it is necessary to continuously monitor the food supplements that are on the market.</p>

Water is one of the most frequently used raw materials in pharmaceutical industry. Water for pharmaceutical purposes includes the two primary water types: purified water and water for injection. Drinking water used for obtaining purified water is not official in pharmacopoeia. Depending on quality prescribed for a certain product preparation, various water types and procedures have been used to prepare pharmaceutical industry water. Possible ways to obtain water for pharmaceutical purposes are: reverse osmosis, demineralization, electrodeionization, ultrafiltration, distillation. Reasons for the widespread use of water lie in the facts that it is capable of dissolving a great number of therapeutic substances, compatible with a large number of substances, appropriate to be used from a physiological aspect given that it is an integral part of the cell and the major component of body fluids and whenever the drug is administered in the form of an aqueous solution, reabsorption is rapid and complete, it also has suitable physical-chemical properties. In this study, analysis results of 15 samples of water were obtained using pharmacopoeial methods for pharmaceutical purposes. Results showed that 86.6% of water samples were accurate and 13.3% did not have appropriate calcium content, magnesium content and total hardness values.

Abstract Introduction: Antibiotics are indispensable to maintaining human health; however, their overuse has resulted in resistant organisms, increasing morbidity, mortality and costs. Increasing antimicrobial resistance (AMR) is a major public health threat, resulting in multiple campaigns across countries to improve appropriate antimicrobial use. This includes addressing the overuse of antimicrobials for self-limiting infections, such as upper respiratory tract infections (URTIs), particularly in lower- and middle-income countries (LMICs) where there is the greatest inappropriate use and where antibiotic utilization has increased the most in recent years. Consequently, there is a need to document current practices and successful initiatives in LMICs to improve future antimicrobial use. Methodology: Documentation of current epidemiology and management of URTIs, particularly in LMICs, as well as campaigns to improve future antimicrobial use and their influence where known. Results: Much concern remains regarding the prescribing and dispensing of antibiotics for URTIs among LMICs. This includes considerable self-purchasing, up to 100% of pharmacies in some LMICs. However, multiple activities are now ongoing to improve future use. These incorporate educational initiatives among all key stakeholder groups, as well as legislation and other activities to reduce self-purchasing as part of National Action Plans (NAPs). Further activities are still needed however. These include increased physician and pharmacist education, starting in medical and pharmacy schools; greater monitoring of prescribing and dispensing practices, including the development of pertinent quality indicators; and targeted patient information and health education campaigns. It is recognized that such activities are more challenging in LMICs given more limited resources and a lack of healthcare professionals. Conclusion: Initiatives will grow across LMICs to reduce inappropriate prescribing and dispensing of antimicrobials for URTIs as part of NAPs and other activities, and these will be monitored.

Introduction. Arsenic exists in various forms in nature and living organisms. Toxic elements, including arsenic, which are present in some plants, can severely damage haemopoietic, immune, nervous and reproductive systems. For this reason, a content of heavy metals is one of the criteria for the assessment of the safe use of plant material in the production of traditional medicines and herbal infusions. This instigates the need for constant and organized safety control of plants that are used as raw materials in pharmaceutical industry. The aim of this study is to determine the arsenic content in selected teas which are available on the market of the Republic of Srpska. Methods. The 10 g samples of 13 herbal and 3 fruit teas were mineralized by dry ashing and arsenic contents were determined by the atomic absorption spectrophotometer Agilent Technologies Series 200 with an air-acetylene burner and D2 background correction. Results. Mean arsenic concentrations in the herbal tea samples ranged from 0.009 to 0.145 mg/kg. The lowest arsenic concentration in a single sample of 0.007 mg/kg was found in Chamomile tea and Uva ursi collected as a wild plant at elevation above 1200 m. The highest arsenic concentration was found in the sample of Sambucus nigra tea (0.145 mg/kg). In fruit teas, the arsenic concentration ranged from 0.014 mg/kg (Cranberry) to 0.027 mg/kg (Fruit mix). Conclusion. Arsenic content in all analyzed tea samples is below the value stipulated by the national legislation.

Background: Monitoring and measuring of the medicine utilisation enables to assess the quality of use of medicines, providing the evidence-based data for the improvement of the prescribing practice and a more rational use of medicines. The aim of this study was to analyse utilisation patterns of medicines and to compare the results with other countries. Methods: A retrospective, observational study to analyse outpatient medicines utilisation in the Republic of Srpska between 2009 and 2017. Data of medicines utilisation were retrieved from the national database in the Public Health Institute of the Republic of Srpska and calculated and analysed by using the Anatomical Therapeutic Chemical/Defined Daily Dose (ATC/DDD) methodology. The results were expressed as Defined Daily Doses (DDDs) per 1,000 inhabitants per day. Results: Total medicines utilisation increased, from 448 DDDs in 2009 to 1,036 DDDs in 2017. Cardiovascular medicines (group C) were the most used medicines, and their share in the total utilisation increased from 36.6% in 2009 to 44.4% in 2017. Among them, the most frequently used were angiotensin-converting enzyme inhibitors, plain and in combinations with diuretics, namely enalapril. The share of medicines used in diabetes in the total utilisation increased from 3.9% in 2009 to 5.1% in 2017. Metformin and glimepiride accounted for about 83% of the blood glucose lowering medicines group (A10B). Among the antithrombotic medicines, the most frequently used were platelet aggregation inhibitors (B01AC), mainly acetylsalicylic acid whose use tripled since 2009. Diclofenac was the most frequently used non-steroidal anti-inflammatory and antirheumatic drug (M01). Conclusion: The trend of increased medicines utilisation was observed in this study. This finding is comparable with other countries. Variations between countries in the preferred medicines within a class as well as the extent of medicines use were observed. These differences were probably consistent, but not solely attributable, to differences in local guidelines and reimbursement policies.

Introduction: There are increasing concerns world-wide with growing rates of antibiotic resistance necessitating urgent action. There have been a number of initiatives in the Republic of Srpska in recent years to address this and improve rational antibiotic prescribing and dispensing despite limited resources to fund multiple initiatives. Objective: Analyse antibiotic utilization patterns in the Republic of Srpska following these multiple initiatives as a basis for developing future programmes in the Republic if needed. Methods: Observational retrospective study of total outpatient antibiotic utilization from 2010 to 2015, based on data obtained from the Public Health Institute, alongside documentation of ongoing initiatives to influence utilization. The quality of antibiotic utilization principally assessed according to ESAC, ECDC, and WHO quality indicators and DU 90% (the drug utilization 90%) profile as well as vs. neighboring countries. Results: Following multiple initiatives, antibiotic utilization remained relatively stable in the Republic at 15.6 to 18.4 DIDs, with a decreasing trend in recent years, with rates comparable or lower than neighboring countries. Amoxicillin and the penicillins accounted for 29–40 and 50% of total utilization, respectively. Overall, limited utilization of co-amoxiclav (7–11%), cephalosporins, macrolides, and quinolones, as well as low use of third and fourth generation cephalosporins vs. first and second cephalosporins. However, increasing utilization of co-amoxiclav and azithromycin, as well as higher rates of quinolone utilization compared to some countries, was seen. Conclusions: Multiple interventions in the Republic of Srpska in recent years have resulted in one of the lowest utilization of antibiotics when compared with similar countries, acting as an exemplar to others. However, there are some concerns with current utilization of co-amoxiclav and azithromycin which are being addressed. This will be the subject of future research activities.

Objective: Irrational use of antimicrobials is a major driver of antimicrobial resistance, exacerbated by dispensing antibiotics without a prescription. Our previous study suggested this was a problem in the Republic of Srpska despite legislation. Since then, a number of activities have been initiated. Consequently, the study aimed to ascertain whether these multiple initiatives had reduced this. Methods: Patients visiting all community pharmacies in the Republic from October 2014 to July 2015 presenting with symptoms typical of an acute, viral and mostly uncomplicated upper respiratory tract infection, with results compared to the previous study. If an antibiotic was suggested, the maximum allowance was Euro 3/ pack. Findings: Self-medication with antibiotics significantly decreased from 58% to 18.5% of pharmacies. In both studies, most patients were offered over-the-counter medication. The most common reason for not dispensing an antibiotic was ‘antibiotics can be dispensed with a prescription only’. The penicillins were the most dispensed antibiotic. Fewer patients than the previous study were given instructions about antibiotic use and no discussion on their sideeffects. Conclusion: Whilst encouraging that self-medication decreased significantly, 18.5% was disappointing given recent initiatives Fewer instructions about antibiotics if an antibiotic was dispensed was also disappointing. This suggests the need for even stronger enforcement of the laws as well as further training of pharmacy personnel to ensure future appropriate use of medicines.

Objective: Irrational use of antimicrobials is a major driver of antimicrobial resistance, exacerbated by dispensing antibiotics without a prescription. Our previous study suggested this was a problem in the Republic of Srpska despite legislation. Since then, a number of activities have been initiated. Consequently, the study aimed to ascertain whether these multiple initiatives had reduced this. Methods: Patients visiting all community pharmacies in the Republic from October 2014 to July 2015 presenting with symptoms typical of an acute, viral and mostly uncomplicated upper respiratory tract infection, with results compared to the previous study. If an antibiotic was suggested, the maximum allowance was Euro 3/ pack. Findings: Self-medication with antibiotics significantly decreased from 58% to 18.5% of pharmacies. In both studies, most patients were offered over-the-counter medication. The most common reason for not dispensing an antibiotic was ‘antibiotics can be dispensed with a prescription only’. The penicillins were the most dispensed antibiotic. Fewer patients than the previous study were given instructions about antibiotic use and no discussion on their side-effects. Conclusion: Whilst encouraging that self-medication decreased significantly, 18.5% was disappointing given recent initiatives Fewer instructions about antibiotics if an antibiotic was dispensed was also disappointing. This suggests the need for even stronger enforcement of the laws as well as further training of pharmacy personnel to ensure future appropriate use of medicines.

Objective: Irrational use of antimicrobials is a major driver of antimicrobial resistance, exacerbated by dispensing antibiotics without a prescription. Our previous study suggested this was a problem in the Republic of Srpska despite legislation. Since then, a number of activities have been initiated. Consequently, the study aimed to ascertain whether these multiple initiatives had reduced this. Methods: Patients visiting all community pharmacies in the Republic from October 2014 to July 2015 presenting with symptoms typical of an acute, viral, and mostly uncomplicated upper respiratory tract infection, with results compared to the previous study. If an antibiotic was suggested, the maximum allowance was €3/pack. Findings: Self-medication with antibiotics significantly decreased from 58% to 18.5% of pharmacies. In both studies, most patients were offered over-the-counter medication. The most common reason for not dispensing an antibiotic was “antibiotics can be dispensed with a prescription only.” The penicillins were the most dispensed antibiotic. Fewer patients than the previous study were given instructions about antibiotic use and no discussion on their side effects. Conclusion: While encouraging that self-medication decreased significantly, 18.5% were disappointing given recent initiatives. Fewer instructions about antibiotics if an antibiotic was dispensed were also disappointing. This suggests the need for even stronger enforcement of the laws as well as further training of pharmacy personnel to ensure the future appropriate use of medicines.

Introduction: Controlling the presence of contaminants or harmful substances in milk and dairy products provides early detection of risks since their presence, even in legally permitted concentration, increases the risk of damaging human health, especially children's health, such as allergic manifestations, and they can have potentially toxic, carcinogenic and genotoxic effects. Aim of the Study: The aim is to determine the frequency of the presence of contaminants in samples of milk and dairy products in the Republic of Srpska from 2010 to 2012 (metals, radionuclides, aflatoxins and residues of antibiotics and pesticides), and especially to point out their public health significance because of possible health risks. The aim of the paper is to emphasize the need for monitoring all the other contaminants in milk and dairy products specified in regulations. Material and Methods: The frequency of the presence of contaminants in the samples of milk and dairy products (n= 407) was determined on the basis of legally prescribed methodology of sampling, chemical analyses and preparation of expert opinion on food safety in accordance with current food regulations. Descriptive statistical indicators were used (a number of the samples, minimum and maximum concentrations). Chi square test (χ2) of contingency was used for testing the significance of differences in presence of contaminants and residues analyzed and recommended by regulations in samples of milk and dairy products. Results: The results of public health research of contaminants in samples of milk and dairy products indicate that no health defective food samples were determined at tested parameters - metals, radionuclides, aflatoxins, and residues of antibiotics and pesticides. A statistically significant number of samples examined on heavy metal content (83.29% or 339 samples) was determined comparing to the number of samples examined on the other contaminants and residues recommended by regulations in milk and dairy products (χ2=1000.776, p<0.001) Conclusion: The data obtained can serve as a basis for further analysis in the context of milk and dairy product sample monitoring. Although samples of milk and dairy products are safe, a long-term exposure to residues of harmful substances results in cumulative effect and damage health, meaning that each early detection of food risk found through continuous controls has a public health significance for preservation and promotion of population health in the Republic of Srpska.

Introduction. Antimicrobial resistance is a very serious public health threat worldwide. The main cause of antimicrobial resistance is irrational use of antimicrobial drugs. The goal of the article is to analyze data on outpatient antibiotic consumption in the Republic of Srpska during the period from 2009 to 2014, to present outpatient consumption of the fi rst six antibiotics (the fi fth level of the ATC classifi cation), and, fi nally, to compare the antibiotic use in Republic of Srpska in relation to the antibiotic use in European Union countries. Materials and Methods. Collection and analysis of data on drug consumption is done using ATC/DDD methodology, established by the WHO. Data are taken from the report on drug consumption and distribution in the Republic of Srpska during the period from 2009 to 2014, which are based on submitted data from pharmacies registered in the Republic of Srpska. Results. Results on outpatient antibiotic consumption in the Republic of Srpska, during the period from 2009 to 2014, indicate a total decrease by 3.41 DDDs/1000 inhabitants/day, with the exception of 2010 and 2013, when the antibiotic consumption was increased. In 2014, total outpatient antibiotic consumption in the Republic of Srpska was 31.2% lower than the average consumption in the European Union. During the period from 2010 to 2014, the European Union had a signifi cant increase in antibiotic consumption, while antibiotic consumption in the Republic of Srpska had a tendency of falling. Conclusion. Presented results show a quantitative analysis of antibiotic consumption and provide insight into the outpatient use of antibiotics in the Republic of Srpska, and can be used for further pharmacoepidemiological analysis of antibiotic consumption, which would give a better insight into the therapeutic practice, with the aim of improving the rational pharmacotherapy in the Republic of Srpska. Keywords: antibiotics, outpatient consumption, DDD/1000 inhabitants/day (Scr Med 2016:47:47-52) + , . / 0 1 / 2 3 4 3 5 6 7 8 7 1 7 6 1 9 1 / : 1 0 2 ; < = . : / 3 1 > > ? @ A 4 1 : B 9 C D E F G H I 8 C 7 G H J K L 0 0 A 5 L 0 / @ M = N O P I I 1 B I Q P < . 3 4 0 > 4 0 @ 48 Scripta Medica Vol. 47 • No 1 • April 2016. • www.scriptamedica.com Introduction R S T U V W X Y Z T [ \ Y ] ^ _ ` V a V Y ` V X W V W Y _ T Y ] ` S T b [ T ^ ` T W ` U V W X Y Z c T [ V T W V _ ` S T S V W ` Y [ \ Y ] d S ^ [ e ^ X Y ` S T [ ^ d \ f g _ ] ^ X ` h ` S ^ _ i W ` Y ` S ^ ` U V W X Y Z T [ \ h ` S T T T X ` V Z T X Y _ ` [ Y j Y ] V _ ] T X ` V Y k W U V W T ^ W T W h l S V X S l ^ W ` S T j T ^ U V _ b X ^ k W T Y ] e Y [ a V U V ` \ ^ _ U e Y [ ` ^ j V ` \ V _ S k e ^ _ S V W ` Y [ \ h l ^ W T _ ^ a j T U f m Y l T Z T [ h ` S T U V W X Y Z T [ \ Y ] ^ _ ` V a V Y ` V X W l ^ W Z T [ \ W Y Y _ ] Y j j Y l T U a \ ` S T Y X X k [ [ T _ X T Y ] [ T W V W ` ^ _ X T Y ] W Y e T a ^ X ` T [ V ^ W ` [ ^ V _ W f n _ ] Y [ ` k _ ^ ` T j \ h U k T ` Y ` S T b T _ T ` V X e k ` ^ ` V Y _ W Y ] a ^ X ` T [ V ^ ^ _ U [ T W V W ` ^ _ ` b T _ T W ` [ ^ _ W ] T [ W h ` S T T e T [ b T _ X T ^ _ U W d [ T ^ U Y ] ^ _ ` V a V Y ` V X [ T W V W ` ^ _ X T V W [ V W V _ b T Z T _ ` Y U ^ \ f o p q r _ ` V e V X [ Y a V ^ j [ T W V W ` c ^ _ X T ` Y U ^ \ V W ^ Z T [ \ W T [ V Y k W b j Y a ^ j d k a j V X S T ^ j ` S ` S [ T ^ ` ` Y T T X ` V Z T ` [ T ^ ` e T _ ` Y ] V _ ] T X ` V Y k W U V W T ^ W T W h ^ W l T j j ^ W X Y _ U k X ` V _ b _ k e T [ Y k W Y ] ^ e Y U T [ _ e T U V X ^ j d [ Y X T U k [ T W h ^ _ U V ` [ T s k V [ T W ^ e k j ` V c U V W X V d j V _ ^ [ \ ^ X ` V Y _ V _ Y [ U T [ ` Y W k d d [ T W W Y X X k [ [ T _ X T ^ _ U W d [ T ^ U l Y [ j U l V U T f t r X X Y [ U V _ b ` Y U ^ ` ^ ] [ Y e u v v w h V ` V W T W ` V e ^ ` T U ` S ^ ` ^ a Y k ` u x v v v d T Y c d j T U V T T ^ X S \ T ^ [ V _ ` S T y n ^ W ^ [ T W k j ` Y ] V _ ] T X ` V Y _ W X ^ k W T U a \ [ T W V W ` ^ _ ` a ^ X ` T [ V ^ W ` [ ^ V _ W h l S V X S j T ^ U W ` Y ` S T U V [ T X ` ^ _ U V _ U V [ T X ` X Y W ` W Y ] ^ a Y k ` z f x e V j j V Y _ ^ _ _ k ^ j j \ h V _ X j k U V _ b ` S T X Y W ` Y ] U T X [ T ^ W T U d [ Y U k X ` V Z V ` \ ^ _ U V _ X [ T ^ W T U S T ^ j ` S X ^ [ T X Y W ` W f q { | R S T e ^ V _ X ^ k W T Y ] T e T [ b T _ X T ^ _ U W d [ T ^ U Y ] ^ _ ` V a V Y ` V X [ T W V W ` ^ _ X T V W V [ [ ^ ` V Y _ ^ j ^ _ ` V a V Y ` V X k W T f g ] l T ` ^ i T V _ ` Y X Y _ c W V U T [ ^ ` V Y _ ` S ^ ` Z T [ \ ] T l _ T l ^ _ ` V a V Y ` V X W ^ [ T U T Z T j Y d T U a \ d S ^ [ e ^ X T k ` V X ^ j V _ U k W ` [ \ ` Y U ^ \ h ^ _ U T Z T _ l S T _ l T S ^ Z T ^ _ T l ^ _ ` V a V Y ` V X h a ^ X ` T [ V ^ T Z T _ ` k ^ j j \ S ^ Z T ` S T X ^ d ^ a V j V ` \ ` Y U T Z T j Y d [ T W V W ` ^ _ X T h ` S T [ T ] Y [ T h l T X ^ _ X Y _ X j k U T ` S ^ ` ` S T W V ` k ^ ` V Y _ l T ^ [ T ] ^ X V _ b l V ` S V W Z T [ \ W T [ V Y k W h ^ _ U V ` l V j j a T T Z T _ e Y [ T W T [ V Y k W V ] l T U Y _ Y ` [ ^ ` V Y _ ^ j j \ k W T T } V W ` V _ b ^ _ ` V a V Y ` V X W V _ ` S T ] k ` k [ T f ~ g ` V W T W ` V e ^ ` T U ` S ^ ` a \ u v x v h k _ c j T W W l T S ^ Z T T T X ` V Z T ^ _ ` V a V Y ` V X W h ` S T l Y [ j U d Y d k j ^ ` V Y _ l V j j a T [ T U k X T U a \ z z ` Y    e V j j V Y _ d T Y d j T f € {  r X X Y [ U V _ b ` Y ` S T ‚ Y [ j U m T ^ j ` S ƒ [ b ^ _ V „ ^ ` V Y _ h [ ^ ` V Y _ ^ j k W T Y ] U [ k b W e T ^ _ W ` S ^ ` … d ^ ` V T _ ` W [ T X T V Z T e T U V X ^ ` V Y _ W ^ d d [ Y c d [ V ^ ` T ` Y ` S T V [ X j V _ V X ^ j _ T T U W h V _ U Y W T W ` S ^ ` e T T ` ` S T V [ Y l _ V _ U V Z V U k ^ j [ T s k V [ T e T _ ` W h ] Y [ ^ _ ^ U T s k ^ ` T d T [ V Y U Y ] ` V e T h ^ _ U ^ ` ` S T j Y l T W ` X Y W ` ` Y ` S T e ^ _ U ` S T V [ X Y e e k _ V ` \ † ‡ ‚ m ƒ h z w ˆ x ‰ f Š Objectives: z f R Y ^ _ ^ j \ „ T ` S T U ^ ` ^ Y _ Y k ` d ^ ` V T _ ` X Y _ W k e d ` V Y _ Y ] ^ _ ` V V _ ] T X ` V Z T W ] Y [ W \ W ` T e V X k W T ‡ ‹ b [ Y k d ‰ ^ _ U ^ _ ` V a V c Y ` V X W ‡ ‹ v z b [ Y k d ‰ V _ ` S T Œ T d k a j V X Y ]  [ d W i ^ U k [ V _ b ` S T d T [ V Y U ] [ Y e u v v w ` Y u v z  Ž u f R Y W S Y l Y k ` d ^ ` V T _ ` X Y _ W k e d ` V Y _ Y ] ` S T  [ W ` W V } ^ _ ` V c a V Y ` V X W ^ ` ` S T  ] ` S r R  j T Z T j ‡ g ‘ ‘ ‰ d T [ \ T ^ [ Ž ’ f R Y X Y e d ^ [ T Y k ` d ^ ` V T _ ` ^ _ ` V a V Y ` V X X Y _ W k e d ` V Y _ V _ ` S T Œ T d k a j V X Y ]  [ d W i ^ V _ [ T j ^ ` V Y _ ` Y X Y _ W k e d ` V Y _ V _ ` S T y k [ Y d T ^ _ n _ V Y _ f Materials and Methods  Y j j T X ` V Y _ ^ _ U ^ _ ^ j \ W V W Y ] U ^ ` ^ Y _ U [ k b X Y _ W k e d ` V Y _ V W U Y _ T k W V _ b r R  “ ” ” ” e T ` S Y U Y j Y b \ T W ` ^ a j V W S T U a \ ` S T ‚ m ƒ  Y j j ^ a Y [ ^ ` V _ b  T _ ` [ T ] Y [ ” [ k b  ` ^ ` V W ` V X • T ` S Y U Y j c Y b \ V _ ƒ W j Y f R S T ‚ m ƒ b k V U T j V _ T W ^ [ T k W T U ` Y V e d j T e T _ ` ` S T r R  U [ k b X j ^ W W V  X ^ ` V Y _ ‡ r _ ^ ` Y e V X ^ j R S T [ ^ d T k ` V X  S T e V X ^ j  j ^ W W V  X ^ ` V Y _ ‰ ^ _ U ” ” ” W ‡ ” T  _ T U ” ^ V j \ ” Y W c T W ‰ h ^ _ U ` Y e Y _ V ` Y [ ` S T ^ _ _ k ^ j U [ k b X Y _ W k e d ` V Y _ f w h z v • Y _ V ` Y [ V _ b Y ] ^ _ _ k ^ j X Y _ W k e d ` V Y _ V _ ` S T Œ T d k a j V X Y ]  [ d W i ^ V W [ T b k j ^ ` T U a \ _ ^ ` V Y _ ^ j j T b V W j ^ ` V Y _ f o o p o q r R  “ ” ” ” W \ W ` T e V _ X j k U T W ` S T r _ ^ ` Y e V X ^ j R S T [ ^ d T k ` V X  S T e V X ^ j ‡ r R  ‰  j ^ W W V  X ^ ` V Y _ Y ] U [ k b W ^ _ U U T  _ T U U ^ V j \ U Y W T W ‡ ” ” ” W ‰ ^ W ` S T k _ V ` ] Y [ e Y _ V ` Y [ V _ b ` S T k W T Y ] e T U V c X V _ T W V _ ^ b V Z T _ d Y d k j ^ ` V Y _ Y [ S T ^ j ` S V _ W ` V ` k ` V Y _ f r R  V _ X j k U T W X j ^ W W V  X ^ ` V Y _ Y ] ^ X ` V Z T W k a W ` ^ _ X T W V _ U V T [ c T _ ` b [ Y k d W h U T d T _ U V _ b Y _ ` S T Y [ b ^ _ W Y [ W \ W ` T e W Y ] Y [ c b ^ _ W ` Y l S V X S ` S T \ ^ [ T T T X ` V Z T Y _ h ^ _ U ^ X X Y [ U V _ b ` Y ` S T V [ ` S T [ ^ d T k ` V X ^ j h d S ^ [ e ^ X Y j Y b V X ^ j ^ _ U X S T e V X ^ j d [ Y d T [ ` V T W f ” ” ” V W U T  _ T U ^ W ^ W ` ^ ` V W ` V X ^ j k _ V ` Y ] ^ b [ T T U ^ _ U U T ` T [ c e V _ T U ^ e Y k _ ` Y ] ` S T U [ k b ` S ^ ` V W e Y W ` X Y e e Y _ j \ k W T U ] Y [ ` S T e Y W ` X Y e e Y _ V _ U V X ^ ` V Y _ ] Y [ e T ^ W k [ V _ b T } d Y W k [ T Y ] b V Z T _ d Y d k j ^ ` V Y _ ` Y X T [ ` ^ V _ U [ k b W Y [ b [ Y k d W Y ] U [ k b W f ” ” ” V W e Y W ` j \ T } d [ T W W T U ^ W l T V b S ` k _ V ` W ‡ b h e b h b ‰ h ^ _ U V ] ` S V W V W _ Y ` d Y W W V a j T U k T ` Y Z ^ [ V Y k W [ T ^ W Y _ W ‡ ] Y [ T } ^ e c d j T h ` S T X Y e a V _ T U d [ T d ^ [ ^ ` V Y _ W h Z ^ [ V ^ a j \ U Y W ^ b T h T ` X f ‰ h ` S T k _ V ` U Y W T ‡ n ” ‰ Y ] ^ b V Z T _ d S ^ [ e ^ X T k ` V X ^ j U Y W ^ b T k _ V ` ‡ ` ^ a j T ` h X ^ d W k j T h ^ e d Y k j T ‰ V W k W T U f ” ” ” e T ` S Y U Y j Y b \ V W V _ U T d T _ U T _ ` Y ] d [ V X T ^ _ U _ k e a T [ Y ] V _ S ^ a V ` ^ _ ` W h W Y V ` V W W k V ` ^ a j T ] Y [ U T W X [ V a V _ b ^ _ U X Y e d ^ [ V _ b U [ k b k W T Y Z T [ ` V e T h X Y e d ^ [ V W Y _ Y ] d [ T W X [ V a V _ b a T ` l T T _ U V T [ T _ ` b T Y c b [ ^ d S V X ^ j ^ [ T ^ W ^ _ U U V T [ T _ ` S T ^ j ` S ] ^ X V j V ` V T W f R S T [ T ] Y [ T h ` S V W e T ` S Y U V W W k V ` ^ a j T ] Y [ e Y _ V ` Y [ V _ b d S ^ [ e ^ X Y ` S T [ ^ d \ ^ _ U [ ^ ` V Y _ ^ j V „ ^ ` V Y _ Y ] ` S T [ ^ d \ f ” [ k b X Y _ W k e d ` V Y _ V W T } d [ T W W T U ^ W ` S T _ k e a T [ Y ] ” ” ” W d T [ z v v v V _ S ^ a V ` ^ _ ` W d [ T W X [ V a T U ] Y [ Y _ T U ^ \ ‡ ” ” ” W “ z v v v V _ S ^ a V ` ^ _ ` W “ U ^ \ ‰ h ^ _ U V _ S Y W d V ` ^ j W T ` ` V _ b W ^ W ` S T _ k e a T [ Y ] U T  _ T U U ^ V j \ U Y W T W d T [ z v v d ^ ` V T _ ` c U ^ \ W ‡ ” ” ” W “ z v v d ^ ` V T _ ` c U ^ \ W ‰ f – Results R S T ^ _ ^ j \ W V W Y _ U [ k b X Y _ W k e d ` V Y _ V W a ^ W T U Y _ U ^ ` ^ W k a c e V ` ` T U ] [ Y e w ’ f — ˜ Y ] d S ^ [ e ^ X V T W [ T b V W ` T [ T U V _ ` S T Œ T c d k a j V X Y ]  [ d W