C o l l e c t i o n

o f Research Work

Editorial Board

Editor-in-chief Assoc. Prof. G. Pascalev, MD, PhD

Deputy/ Managing Editor Prof. T. Tsvetkova, MD, PhD, DMSc

Editorial Advisory Board Prof. I. Karnolski, MD, PhD, DMSc Prof. St. Kostianev, MD, PhD, DMSc Prof. Zl. Dimitrova, DChSc

Statistical Advisor Assoc. Prof. N. Mateva, PhD Technical Editors V. Gjulina, N. Atanasova

Members Prof. A. Bakardjiev, MD, PhD Prof. A. Nedeva, MD, PhD, DMSc Prof. B. Chilova, MD, PhD, DMSc Prof. B. Pehlivanov, MD, PhD, DMSc Prof. D. Dimitrakov, MD, PhD, DMSc Prof. D. Getova-Spasova, MD, PhD, DMSc Prof. D. Iluchev, MD, PhD, DMSc Prof. E. Hadjipetrova, MD, PhD, DMSc Prof. F. Mitov, MD, PhD, DMSc Prof. G. Baltadjiev, MD, PhD, DMSc Prof. I. Yovchev, MD, PhD, DMSc Prof. J. Hristov, MD, PhD, DMSc Prof. K. Velkova, MD, PhD, DMSc Prof. M. Kukleva, MD, PhD, DMSc Prof. M. Stoycheva, MD, PhD, DMSc Prof. M. Yaneva, MD, PhD, DMSc

Prof. N. Popivanova, MD, PhD Prof. P. Mandulova, MD, PhD, DMSc Prof. P. Rashkov, PhD, DTSc Prof. P. Solakov, MD, PhD, DMSc Prof. S. Kuzmanova, MD, PhD, DMSc Prof. S. Vladimirov, MD, PhD, DMSc Prof. St. Goranov, MD, PhD, DMSc Prof. T. Mihailov, MD, PhD Prof. V. Anastassov, MD, PhD, DMSc Prof. V. Ishev, MD, PhD, DMSc Prof. V. Sarafian-Ozanian, MD, PhD, DMSc Prof. Z. Zahariev, MD, PhD, DMSc Assoc. Prof. At. Krastev, MD, PhD, DMSc Assoc. Prof. G. Ivanov, MD, PhD, DMSc Assoc. Prof. M. Murdjeva, MD, PhD

International members Prof. Bernard Amor, MD, PhD, DMSc (Paris, France) Prof. George Anastassov, Md, Phd, Ddsc (NYC, USA) Prof. Lidia Benevolenscaya, Md (Moscow, Russia) Prof. John Christakis, Md (Thessaloniki, Greece) Prof. Patrick Dhellemmes, Md, Phd (Lille, France) Prof. Konstantinos Kazakos, MD, PhD (Alexandroupolis, Greece) Prof. Konstantinos Kouskoukis, Md (Alexandroupolis, Greece) Prof. Ingrid Kreissig, MD, PhD, DMSc (Mannheim, Germany) Prof. John Kyriopoulos, Md, Phd, Dmsc (Athens, Greece) Prof. Jukka Meurman, Md, Phd, DOdont (Helsinki, Finland) Prof. Mikhail Mikhailovsky, Md, Phd, Dmsc (Novosibirsk, Russian) Prof. Michael Oellerich, Md, Phd, Dmsc (Göttingen, Germany) Prof. Philippe Pellerin, Md, Phd (Lille, France) Prof. Yves Poumay, Md, Phd, Dmsc (Namur, Belgique) Prof. Constantinos Simopoulos, Md (Alexandroupolis, Greece) Prof. Greg Skalkeas, Md, Academician (Athens, Greece) Prof. Francisco Soriano, Md, Phd (Madrid, Spain) Prof. Eberhard Wieland, Md, PhD, Dmsc (Göttingen, Germany) Language Editor (English): Assoc. Prof. O. Obretenov, DhP Language Editor (Russian): P. Kancheva Typeset by Medical University - Plovdiv, Scientific Department, Dipl. Eng. N. Atanassova Printed in Bulgaria, Plovdiv, Avtoprint LTD ISSN 0204-8043 (print) Number of copies: 1500

Editorial

Correspondence

FOLIA MEDICA, MU, Library and Information Center, V. Gjulina (e-mail: [email protected]) 15 A Vassil Aprilov St., 4002 Plovdiv, Bulgaria

Tel. +359 32/602 588; Fax: +359 32/602 534 http://versita.com/science/medicine/fm/ www.meduniversity-plovdiv.bg, Folia Medica Journal e-mail: [email protected]

52, 3 2010 July - Sept

Contents Reviews Boyan I. Nonchev Cases of Interferon-Alpha and Interferon-Beta-Induced Thyroiditis............................................................................ 5 Ivan P. Novakov, Georgi P. Safev, Stefka E. Peicheva Descending Necrotizing Mediastinitis of Odontogenic Origin – Personal Experience and Literature Review .......................................................................................................................................................... 13

Original Articles Clinical investigations Manabu Abe, Corinne Klett, Eberhard Wieland, Sascha Gille, Olfert Landt Simultaneous Quantification and Genotyping of Hepatitis C Virus RNA by a Two-Step Real-Time PCR Assay on the Lightcycler Instrument................................................................................................................... 21

Experimental investigations George P. Deenichin, Atanas D. Kristev, Vesselin V. Mollov, Valentin I. Turiiski Effect of Elevated Intra-abdominal Pressure on the Contractile Activity and Reactivity of Smooth Muscle Tissue from Rat Gastrointestinal Tract to Galantamine and Drotaverine (No-Spa).................................. 31 Sorana D. Bolboacă, Monica M. Marta, Lorentz Jäntschi Binding Affinity of Triphenyl Acrylonitriles to Estrogen Receptors: Quantitative Structure-Activity Relationships..................................................................................................................................................................... 37

Dental investigations Snejana Ts. Tsanova, Georgi T. Tomov Morphological Changes in Hard Dental Tissues Prepared by Er:YAG Laser (Litetouch, Syneron), Carisolv and Rotary Instruments. A Scanning Electron Microscopy Evaluation..................................................... 46

Short Commmunications Nedialka I. Popivanova, Krassimira N. Chudomirova, Ivan G. Baltadzhiev, Tsvetana I. Abadjieva Hiv/Aids-associated Kaposi’s Sarcoma with Multiple Skin-mucosal Disseminations Following Ultraviolet (РUVA) Photochemotherapy........................................................................................................................ 56

Case Reports Nartsis N. Kaleva, Ivan S. Ivanov, Margarita V. Panova, Tatyana D. Shabanova, Darina S. Delgyanska Hyperinsulinemic Hypoglycemias in Infancy and Childhood – Diagnostic Therapeutic Algorithm with Contribution of Two Cases................................................................................................................................... 62 Maria G. Prancheva, Sasha A. Krasteva, Stoilka G. Tufkova, Teodora P. Karaivanova,Veselina V. Nizamova, Yanko T. Iliev Severe Hypotension and Ischemic Stroke After Disulfiram-Ethanol Reaction......................................................... 70 Yanko T. Iliev, Stoilka G. Tufkova, Maria G. Prancheva A Rare Case of Severe Intoxication from Multiple Bee Stings with a Favorable Outcome................................ 74

С ОД Е РЖ А Н И Е Обзор Б. Нончев

Тиреоидиты, леченные альфа и бета интерферонами..................................................................................5 И. Новаков, Г. Сафев, С. Пейчева

Десцендирующий некротический медиастинит одонтогенного происхождения собственный опыт и литературный обзор...................................................................................................13

Оригинальные статьи Клинические исследования М. Абе, К. Клет, Е. Виланд, С. Гиле, О. Ланд

Одновременноe количественное измерение и генотипизирование рибонуклеиновой кислоты вируса гепатита С посредством применения двушагового PCR теста в реальное время на аппарате Light Cycler.....................................................................................................................21

Экспериментальные исследования Г. Дееничин, А. Крыстев, В. Молов, В. Турийски

Влияние повышенного интраабдоминального давления на сократительную активность и реактивность гладкой мускулатуры гастроинтестинального тракта крысы к Galantamine Drotaverine (No-Spa).......................................................................................................................................31 S. Bolboacă1, M. Marta, L. Jäntschi

Сходство связывания трифенил акрилонитрила с рецепторами эстрогена - зависимость между химической структурой и свойствами.............................................................................................37

Дентальные исследования С. Цанова, Г. Томов

Морфологические изменения в твёрдых зубных тканях, обработанных Er:YAG-лазером (Litetouch, Syneron), Carisolv и ротационными инструментами. Анализ сканирующим электронном микроскопом............................................................................................................................46

Короткие сообщения Н. Попиванова, К. Чудомирова, И. Балтаджиев, Ц. Абаджиева

ХИВ/СПИД-связанная Kaposi's sarcoma с множественной кожно-мукозной диссеминацией после ультрафиолетовой (РUVA) фотохимиотерапии................................................................................56

Казуистика Н. Калева, И. Иванов, М. Панова, Т. Шабанова, Д. Делгянска

Гиперинсулинемические гипогликемии в грудном и детском возрасте - диагностическотерапевтический алгоритм (вклад - два случая).........................................................................................62 М. Пранчева, С. Крыстева, С. Туфкова, Т. Караиванова, В. Низамова, Я. Илиев

Тяжелая гипоксия и ишемический инфаркт мозга при disulfiram-ethanol реакции.................................70 Я. Илиев, С. Туфкова, М. Пранчев

Редкий случай тяжелой интоксикации вследствие многократных ужаливаний пчелами с благоприятным исходом.............................................................................................................................74

Folia Medica 2010; 52(3): 5-12 Copyright © 2010 Medical University Plovdiv doi: 10.2478/v10153-010-0001-6

REVIEWS Cases of interferon-alpha and interferon-beta-induced thyroiditis Boyan I. Nonchev Clinic of Endocrinology and Metabolic Diseases, Medical University, Plovdiv, Bulgaria Abstract Interferons are currently the major treatment modality for several malignant and nonmalignant diseases such as chronic hepatitis C and B, multiple sclerosis, hematological malignancies, malignant melanoma, renal cell carcinoma, etc. Thyroid disorders develop in some of the interferon-treated patients with the incidence ranging from 1% to 35%. These complications may often result in dose reduction or discontinuation of interferon therapy. Interferon induced thyroid disorders can be classified as autoimmune and non-autoimmune thyroiditis. There are many studies on the development of thyroid dysfunction in interferon-alpha treated patients with chronic hepatitis C and in patients with multiple sclerosis treated with interferon-beta. There is a dearth of information about the incidence and characteristics of thyroid abnormalities in patients with hematological malignancies receiving interferon-alpha. A number of genetic determinants are discussed as causes for thyroid impairment (sex, age, ethnic group, genes involved in the thyroid immune regulation), as well as non-genetic factors (related to the underlying disease – hepatitis C virus; multiple sclerosis; therapeutic regimens of interferon administration, iodine concentration in the environment, presence of thyroid autoantibodies at the start of treatment, etc.). In this article we summarize the relevant data about the frequency and characteristics of thyroid disorders in patients treated with interferons, the risk factors and the mechanisms for their development and the peculiarities of the course, detection and treatment of these complications. The review of the literature motivates studying the thyroid function of specific groups of patients receiving interferon in order to clarify the influence of the factors drug and disease on the thyroid gland. Early detection and adequate treatment of thyroid dysfunction in these patients is important to avoid complications that may compromise treatment. Keywords: interferon, thyroiditis, viral hepatitis, multiple sclerosis Introduction

Interferons are natural proteins produced by the cells of the immune system of most vertebrates in response to foreign agents such as viruses, parasites and tumor cells. Discovered in 1950, they were immediately introduced as therapeutic agents able to induce cellular resistance to viral infections. In addition to their antiviral effects, they possess immunomodulator, antiproliferation and anti-tumor properties and act as important factors of growth and differentiation. Currently they are the major therapeutical agents used in the treatment of a considerable number of malignant and benign diseases such as hepatitis C and hepatitis B, hematological malignancies, multiple sclerosis (MS), melanoma,

renal cell carcinoma, etc.������������������������ Despite ����������������������� the good therapeutic response interferons are known to cause a number of side effects – a flu-like syndrome, hematological disorders (myelosuppression), dizziness, neuropsychiatric symptoms, thyroid disorders, that often lead to dose reduction or discontinuation of therapy.1 Adverse side effects can lead cumulatively to a reduction in dose in 40% of the cases and treatment discontinuation in up to 14% of them.1 Many side effects caused by interferons are due to the induction of enhanced immunity with increased cell cytotoxicity or immune dysregulation. In clinical practice different preparations of the interferon group are used: interferon α-2a (Roferon-A), interferon α-2b (Intron A), interferon

Correspondence and reprint request to:B. Nonchev, Clinic of Endocrinology and Metabolic Diseases, Medical University, Plovdiv 15A Vassil Aprilov St, 4002 Plovdiv, Bulgaria Received 8 March 2010; Accepted for publication 28 April 2010

5

B. Nonchev

α-n3 (Alferon N), interferon-α con-1 (Infergen), interferon β-1a (Avonex), interferon β-1b (Betaseron, Betaferon), interferon γ-1b (Actimmune) A relatively new group of drugs are the pegylated interferons - α-2b (PEG-Intron), - α-2a (Pegasys), which have extended half-life and many advantages over the conventional preparations such as a single weekly application, less toxicity, etc. In Bulgaria various regimens involving drugs of the interferon group are very frequently applied. Research now focuses largely on the application of these drugs in treating chronic hepatitis and liver cirrhosis. It is estimated that between 4% and 8% of the population in Bulgaria is infected with HBV and about 1.5% - with HCV. According to data from the National Health Insurance Fund (NHIF), between January 2003 and May 2005, there were a total of 983 patients with chronic hepatitis and liver cirrhosis that were included in the Bulgarian program for antiviral treatment with interferons.2 Interferons are also widely applied in cases of hematological malignancies3 and some neurological diseases such as for instance multiple sclerosis. Interferon therapy and thyroid dysfunctions were found to be associated as early as 1985 in patients with breast cancer and carcinomatoid tumors. There has been quite a lot of research since then and it is now a widely recognised fact that thyroid dysfunction is a common adverse effect associated with the administration of interferon therapy. The incidence of thyroid dysfunction in patients treated with interferon is estimated in several European studies to be relatively high4, and reported in some in the range of 1% to 35%.5 Some prospective studies report that thyroid dysfunction develops in more than 15% of patients with hepatitis C treated with interferon-α6, and that the titers of thyroid autoantibodies (TTA) increase in 40% of the cases.7 High incidence of thyroid dysfunction has also been reported after administration of interferon-β in MS patients - up to 24%.8 The evidence provided by these and some other contemporary analyses show that thyroid disorders are a significant clinical problem for patients receiving interferon.9 The most frequently researched aspect of this problem is development of thyroid dysfunction in hepatitis C patients treated with interferon-α and in multiple sclerosis patients treated with interferon-β.10-13 Types of thyroid dysfunction

The spectrum of possible damage to the thyroid in the course of and after administration of interferon is wide. It can be broadly classified as autoimmune

6

and non-autoimmune interferon-induced thyroiditis (IIT).12 Autoimmune types of thyroiditis can be manifested as positive antithyroid antibodies (ATA) without clinically significant thyroid disease or autoimmune hypothyroidism (with the characteristics of Hashimoto’s thyroiditis), or autoimmune thyrotoxicosis (mimicking Graves’ disease). 12,14 Non-autoimmune forms are presented as destructive thyroiditis or hypothyroidism without ATA. Autoimmune thyroid diseases are complex diseases developing as a result of the impact of certain non-genetic factors on individuals with genetic predisposition. The entire spectrum of autoimmune thyroid diseases (AITD) can be observed in patients treated with interferon - autoimmune Hashimoto’s thyroiditis (HT), Graves disease (GD), and the presence of ATA (thyroglobulin - ТАТ, antithyroid peroxidase ТPO Аb, thyroreceptor ТR Аb) without clinical thyroid dysfunction.5,12,15 Autoimmune interferon induced thyroiditis (IIT)

High titers of ATA is the most common manifestation of an autoimmune process in the thyroid.10 Incidence of “de novo” positive ATA titres varies considerably between studies – from 1.9% to 40%.8,12 The great differences are caused mainly by the design of the studies, the use of different reference limits and also methods of research with different sensitivity and specificity.9 It is assumed that except the de novo formation of ATA, treatment with interferon can lead to intensification of a previous autoimmune conflict.12 Thus, in patients with positive ATA before treatment, several researchers report significant increase in ATA titers after initiation of interferon therapy.6,7,16 It seems that an important factor for antibody formation in both cases is the genetic predisposition to develop thyroid autoimmunity. Established gender and age differences in the prevalence and titers of ATA in patients treated with interferon support this point of view.16,17 For instance, Marazuela et al. demonstrates that ATA emerge significantly more commonly in women (14%) than in men (1%) and correlates directly with age.17 The importance of interferon as trigger-factor for the development of thyroid autoimmunity is supported by the fact that in many cases of positive ATA titers after the initiation of treatment they persist after treatment as well. In a long-term study with an average follow-up of 6.2 years after discontinuation of interferon-α, Carella et al. found that 72.2% of patients with positive ATA during treatment remained with elevated titers Folia Medica 2010; 52(3): 5-12

© 2010 Medical University Plovdiv

Cases of Interferon-Alpha and Interferon-Beta-Induced Thyroiditis

until the end of the study.18 It is assumed that the presence of ATA (especially antithyroid peroxidase) in otherwise healthy persons is a preclinical phase of AITD which combined with an increased thyroid determines an increased risk of developing a clinical disease in up to 5% per year of women.19 Roti et al. indicate that positive TPO Ab before treatment with interferon-α have a high positive prognostic value (67%) for the development of hypothyroidism.6 Data from these and other tests determine the high importance of thyroid immunological tests in patients treated with interferon and the need to perform such tests aimed at prevention, early diagnosis and control over clinical forms of thyroid dysfunction (autoimmune thyroiditis, GD). The latter are a common cause of dose reduction or discontinuation of interferon treatment which compromises the therapeutic effect.12 Based on an in-depth literature analysis Mandac recommends screening of TPO Ab and TAT in all patients with hepatitis C indicated for treatment with interferon-α.12 The most frequent clinical manifestation of interferon-induced thyroiditis (IIT) is the autoimmune Hashimoto’s thyroiditis (HT), with elevated titers of thyroid ATA during the course of the disease (TPO Ab and / or TAT) and hypothyroidism (subclinical or clinical).9 Its prevalence among patients treated with interferon varies significantly (between 2.4% and 19%) assuming that it develops significantly more often in cases with pre-existing autoimmune process in the thyroid.10,15,16 Most authors confirm presence of ATA before starting interferon treatment is a prerequisite and significant risk factor for the development of HT5,7, the occurrence of the disease being accompanied by a significant increase in ATA titers.7,16 Watanabe et al. describe 3 cases of clinical hypothyroidism after initiation of interferon in patients with elevated levels of ATA.16 These results confirm the importance of interferon as a trigger factor for the development of hypothyroidism in susceptible patients.12 In significantly smaller number of cases interferon therapy may lead to the development of GD. In a retrospective study of 321 hepatitis C patients treated with interferon-α, 10 patients developed thyrotoxicosis characterized by completely suppressed TSH, while in 6 cases GD was confirmed by thyroscintigraphy and a positive titer of stimulating ATA.20 Thyrotoxicosis ������������������������������������������ did not fade away spontaneously in any of the patients after discontinuation of interferon treatment. In another large multicenter study, 3 of 237 interferon-treated patients developed GD which required radical treatment.15 In most of Folia Medica 2010; 52(3): 5-12

© 2010 Medical University Plovdiv

the reported cases GD does not go into remission after discontinuation of interferon which requires further treatment.16,20 Duncea et al. report about interferon-induced relapse of GD 10 years after thyroid resection.21 It is known that GD is often accompanied by serious non–thyroid complications - ophthalmopathy, cardiac arrhythmias, etc. Thyroid-associated ophthalmopathy is described in patients with hepatitis C after administration of interferon-α, which highlights the fact that interferon-induced thyroid disorders can lead to severe complications.12 It is these complications that may require dose reduction, in some cases even discontinuation of therapy with interferon thus compromising the therapeutic effect. Non-autoimmune types of IIT

As much as 50% of IIT patients do not develop thyroid autoimmunity, which indicates that the dysfunctions may well be the result of direct effects of interferon on thyrocyte function.6,20,22 A typical manifestation of non-autoimmune IIT is the destructive thyroiditis.9 This is a self-limiting inflammatory disease of the thyroid gland with characteristic phase course - sudden onset of thyrotoxicosis, sometimes accompanied by fever and sensitivity of the gland, followed by a hypothyroid phase and reconstructive phase with normalization of function for several weeks to several months. Sustained hypothyroidism occurs in about 5% of cases. Wong et al. found destructive thyroiditis in four out of ten patients with thyrotoxicosis; it was proven by reduced uptake of technetium in thyroscintigraphy, negative thyrotropin receptor antibodies (ТP Аb) or transient thyrotoxicosis followed by clinical hypothyroidism.20 More than 50% of interferoninduced cases of thyrotoxicosis are believed to be due to destructive thyroiditis while in the rest of the cases GD is implicated.5-7,20,22 Most ������������������� often destructive thyroiditis occurs with minimal or no clinical symptoms which are regarded as side effects of treatment or associated with the underlying disease, which is why its incidence is probably higher than the reported one.12,9 However, according to some authors, patients can develop serious complications that require radical thyroid ablation before treatment is reinitiated.12 Destructive thyroiditis may recur in a second interferon-treatment course, so monitoring is necessary. Hypothyroidism is transient in most cases of destructive thyroiditis. Persistent hypothyroidism usually develops in patients with elevated ATA.6,16 Besides being a result of an underlying autoimmune process, sustained hypothyroidism is reported by

7

B. Nonchev

some authors to be also the result of direct cytotoxic effect of interferon-α on thyroid cells.23 Risk factors for the development of IIT

Determinants of susceptibility to the development of interferon-induced thyroiditis are not fully clarified. A number of genetic factors are discussed (sex, age, ethnicity, genes involved in thyroid immune regulation), as well as many non–genetic factors, related to the underlying disease (hepatitis C virus, MS), therapeutic regimens of interferon administration, content of iodine in the environment, the presence of ATA at the beginning of treatment, etc.12 Genetic factors

There is ample evidence for the importance of the genetic factors for the development of AITD.24 Most likely, these genetic determinants increase the susceptibility of certain individuals to interferoninduced thyroiditis. Data on increased morbidity in women treated with interferon-α support this claim.5,16,17 А pooled analysis of several studies indicates that the risk for the development of thyroid disorders is 4.4 times greater in women than ����������������������������������������� is possible that the increased suscepin men.4 It tibility of the female sex is due to the inheritance of “susceptible” gene determinants associated with the X – chromosome, as well as other non-genetic factors such as estrogenes. Dalgard et a. find that Asian origin is an independent risk factor for the development of thyroid dysfunction in patients treated with interferon-α.25 In recent years several genes have been identified that are associated with thyroid autoimmunity.24 These are genes involved in immune regulation, such as HLA-DR, CTLA-4, PTPN22, and some thyroid specific genes, including the gene for the synthesis of thyroglobulin. They may also contribute to the susceptibility of the organism to IIT.12 So far there have been little data on the relationship between HLA genes and interferon-α-induced thyroid disorders. The issue remains open for future research. Positive titers of ATA at the start of interferon therapy are important risk factors for the development of IIT.5,6,9,16 The presence of ATA is in most cases the preclinical phase of AITDs and is discussed as a marker for the genetic predisposition for their expression.9,19 Summarized data show that the risk of developing thyroid dysfunction in patients with positive ATA at the beginning of treatment is 46.1%

8

compared to 5.4% in patients with negative titers of ATA.5 Importance as risk factor is attributed mostly to thyro-peroxidase antibodies.6,16 Caraccio et al. demonstrates that the only predictive factor for the development of thyroid dysfunction in MS patients treated with interferon-β is the presence of ATA.8 These data are in accordance with the view that interferon-α plays the role of a trigger for the development of AITD in individuals with genetic predisposition, a marker for which is the presence of ATA.9 Non-genetic factors

Factors related to underlying disease Although types of IIT have been detected in patients receiving interferons for various diseases15, the most of the cases are found in patients with type C hepatitis. It is possible that hepatitis C virus infection can play an important role in the etiology of thyroid disorders regardless of the influence of interferon.9 In some earlier studies of patients not treated with interferon such a relationship was not established. For example, Loviselli et al. studied 1310 patients with hepatitis C and found no correlation between the viral infection and the presence of ATA.26 Data from these studies must be viewed with criticism because less sensitive methods for the assessment of ATA were used, and what is more, the impact of other factors of proven effect to AITD was not taken into consideration - mainly iodine intake. There is also lack of standard definition of HCV infection.9 In other, more contemporary analyses the authors establish significant correlation between hepatitis C and thyroid disorders frequency.7,27 The results of a large-scale study of Antonelli et al. show that hypothyroidism and autoimmune deviations are more common in patients with chronic hepatitis C compared with the control group.27 The authors studied four groups of patients: 630 patients with chronic hepatitis C not treated with interferon-α, 389 sex and age-matched healthy subjects from a region with adequate iodine content, 268 patients from a region with iodine deficiency and 86 patients with hepatitis B.������������������������������ Chronic ����������������������������� HCV infection was defined as positive HCV-Ab and high transaminases for more than 6 months. Data analysis found that the incidence of positive ATA (TAT and TPO Ab was significantly greater in patients with hepatitis C than in other groups. In summary the results of numerous studies of Antonelli et al. indicate that the risk for development of AITD is significantly greater in patients with hepatitis С.28

Folia Medica 2010; 52(3): 5-12

© 2010 Medical University Plovdiv

Cases of Interferon-Alpha and Interferon-Beta-Induced Thyroiditis

MS is also associated with higher incidence of AITD according to some researchers.���������� ��������� For example in a study of Munteis et al. on 93 untreated patients with MS incidence of cases with positive thyroid autoantibodies is 5 times higher compared to the control population (P = 0.0001). 13 The authors established hypothyroidism in 6.45% of the patients and in 2.24% of the controls. Similar results are reported by Niederwieser et al. - they report increased incidence of autoimmune types of thyroiditis in men with MS compared with healthy controls.29 In other research papers such correlation is not confirmed.8 Literature review provides scarce data on the incidence of thyroid abnormalities in hematological malignancies. However, in a study of Shinkarkina et al. ATA positive titers are found in 29% of patients with myeloproliferative disorders, which is accepted by the authors as an indication of increased incidence of autoimmune thyroiditis in patients with hematological malignancies.30 Type,

dosage and duration of administration of

interferon preparation

Some research papers indicate that higher doses of interferon and its continued use are risk factors for IIT.5 This view is not supported by other authors.16,25 The combination of interferon-α with ribavirin in modern therapy regimens for the treatment of hepatitis C is also discussed as a possible factor for AITD. It is known that ribavirin induces the Th1 cytokine profile (with an increase of IL-2 and TNFα production) and thus may contribute to susceptibility to IIT.12 In a study of Carella et al. 72 patients with hepatitis C treated with interferon-α and ribavirin and 75 patients with hepatitis C treated only with interferon are analyzed. At the end of treatment period the authors did not observe statistically significant difference in the incidence of autoimmune disorders in both groups of patients (23.6% versus 22.7%).31 However, a significantly higher frequency of autoimmune hypothyroidism in patients on combination therapy is reported.31 Literature data comparing different interferon preparations in terms of their effect on the thyroid are scarce. It should however be noted that in a study of Caraccio et al. the authors do not find statistically significant differences in the incidence of thyroid damage in patients with MS treated with β-1a interferon and patients treated with β-1b interferon.8 In an analysis of all data known so far about the mechanisms of action of interferons Moncani et al. accept the possibility different

Folia Medica 2010; 52(3): 5-12

© 2010 Medical University Plovdiv

preparations to have different potential to induce or reinforce the immune response including at the level of the thyroid gland.22 Iodine intake It is known that iodine intake in population with iodine deficiency may induce thyroid autoimmune response. It is not clear whether replacement therapy with iodine preparations may influence the occurrence of autoimmune thyroid dysfunction during treatment with interferons. There are currently scarce data in this respect - iodine in therapeutic doses in euthyroid patients treated with interferon does not induce a higher incidence of thyroid dysfunction, hypothyroidism in particular.32 Mechanisms for the development of IIT The mechanisms through which interferon therapy causes thyroid disorders are yet unknown.9 The results of numerous studies on this issue suggest immune-mediated effects and also direct toxic effects on the thyroid cells. Immuno-mediated effects of interferon One of the main effects of interferon-α is to increase the antigen expression of the main complex of tissue compatibility on the cell surface. This also applies to the thyroid cells.6 This effect is associated with activation of cytotoxic T cells that may eventually lead to tissue damage and inflammatory response. Another potential mechanism for the development of IIT is associated with the stimulation of Th-1 immune response leading to an increase of interferon gamma and IL-2, which are known to be potent proinflammatory cytokines.33 However, in some patients IIT is presented by the clinical manifestation of GD, which is known to be Th-2 mediated.23 The question that arises is how interferon-α stimulating Th-1 immunity leads to the development of GD. Partial explanation is given by Nagayama et al. based of their research – they are of the opinion that in early stage GD is Th-1 mediated.34 Other possible mechanisms of influence of the interferons are associated with their versatile effects on the immune system – they increase the activity of lymphocytes, macrophages and NK cells, stimulate neutrophilic and lymphocytic activation, increase the production of cytokines such as IL-6, which is known to be involved in the pathogenesis of autoimmune thyroiditis. Direct effects of interferon Given that 50% of IIT are not autoimmune it can be assumed that interferons have direct effects on

9

B. Nonchev

the thyroid.9 This claim is supported by the results of Caraccio et al. who find that that after cultivation of thyroid follicular cells with interferon, the gene expression of thyroglobulin, thyroid peroxidase and sodium iodine symporter are inhibited.8 Other authors report accelerated cell death of thyrocytes after exposure to interferon.35 The results of these and other studies are consistent with the opinion of the presence of direct cytotoxic effects of interferon, which are probably the base of non-autoimmune IIT. Peculiarities in the therapeutic approach and monitoring of patients with IIT Significant clinical importance of IIT motivates some researchers to propose an algorithm for the diagnosis and the treatment of thyroid disorders induced by interferon, emphasizing the need for close cooperation between endocrinologists and hepatologists while solving these problems.12 The authors recommend careful screening for abnormalities in thyroid function before, during and after a course of treatment with interferon. Often in everyday practice symptoms of thyroid dysfunction are attributed to the underlying disease, which is why clinicists should proactively monitor changes in heart rate, excessive sweating, intolerance to cold or heat, changes in body weight, weakness and easy tiredness.9,12 Regardless of the presence or absence of symptoms screening is required for all patients indicated for treatment with interferon. In the published national consensus for diagnosis, treatment and monitoring of patients with chronic viral hepatitis screening of (TSH) is advocated for before and during therapy with interferon-α.36 Moreover, many researchers recommend testing of the values of ATA (TAT, TPO Ab) as an independent prognostic marker for IIT.5,12 In normal TSH and ATA values monitoring of TSH is needed every three months. In higher ATA values monitoring of TSH should be more frequent.12 Upon registration of clinical thyroid dysfunction it is imperative that a full study of thyroid status, which includes further examination of ТR Аb, free thyroid hormone fractions and, if necessary, thyroid scintigraphy. There are some peculiarities in the treatment of IIT unlike treatment of thyroid dysfunction unrelated to interferon. For example, in hepatitis C patients and interferon-induced GD, researchers recommend to proceed directly to radical treatment - radio iodine ablation, because thyreostatics can worsen liver function.12 In patients with evidence of destructive IIT in the phase of thyrotoxicosis and hepatitis C treatment only with β-blockers is

10

recommended as corticosteroids (usually with good effect) are contraindicated in this liver disease. TSH monitoring is recommended for these patients because of frequent progression of destructive thyroiditis towards hypothyroidism. In all cases, the cessation of interferon should be preceded by a consultation with endocrinologist.9 It is important to be aware that reinitiation of interferon can lead to relapse of IIT, which highlights the need for strict monitoring of thyroid function. Upon registration of hypothyroidism patients are usually treated by thyroid hormone replacement, and discontinuation of interferon preparation is not imposed. TSH control every two months is recommended having in mind the possibility of progression of hypothyroidism and the need to increase doses of replacement therapy. Needs for L-thyroxine may vary - they can be increased upon reinitiation of interferon or reduced upon its suspension.17 Conclusions

In conclusion, with the extended use of interferons in various morbid entities (hematological, hepatological, neurological, etc.) many observations have been accumulated and it is a widely accepted fact that the impairment of the thyroid gland is a common side effect of interferon therapy. However, so far no clearly defined guidelines for clinical and therapeutic approach to thyroid complications exist and the behaviour of the medical teams is based primarily on individual assessment.�������������������������� In ������������������������� practice, upon the occurrence of thyroid disorders many clinicists reduce the dose, sometimes even suspend treatment with interferons, thereby compromising their effects on the underlying disease. These data motivate the study of thyroid function in certain groups of patients on interferon treatment in order to clarify the specific influence of the factors – preparation and disease, on thyroid disease. This will help to distinguish the risk of thyroid dysfunction in the studied disease groups, and to establish an algorithm for tracking and behaviour in cases of thyroid abnormalities. Early identification and adequate treatment of thyroid dysfunction in these patients is important to avoid complications compromising treatment. References

1. Russo MW, Fried MW. Side effects of therapy for chronic hepatitis C. Gastroenterology 2003;124(6): 1711-9. 2. Kondeva G, Tarinska N. Program of the NHIF for antiviral treatment of viral hepatitis and cirrhosis – Folia Medica 2010; 52(3): 5-12

© 2010 Medical University Plovdiv

Cases of Interferon-Alpha and Interferon-Beta-Induced Thyroiditis

up to date issues. Bylgarska hepatogastroenterologia, 2006;2: 26-9 (Bulgarian). 3. Raynov Y. New approaches to the treatment of chronic myeloid leukemia. Clinical Hematology and Transfusiology 2004;XL(1-2):22-7 (Bulgarian). 4. Prummel MF, Laurberg P. Interferon-alpha and autoimmune thyroid disease. Thyroid 2003;13:547-51. 5. Koh L, Greenspan F, Yeo P. Interferon-induced thyroid dysfunction: three clinical presentations and review of the literature. Thyroid 1997;7:891-6. 6. Roti E, Minelli R, Giuberti T, et al. Multiple changes in thyroid function in patients with chronic active HCV hepatitis treated with recombinant interferonalpha. Am J Med 1996;101:482-7. 7. Preziati D, La Rosa L, Covini G, et al. Autoimmunity and thyroid function in patients with chronic active hepatitis treated with recombinant interferon alpha2a. Eur J Endocrinol 1995;132:587-93. 8. Caraccio N, Dardano A, Manfredonia F, et. al. Longterm follow-up of 106 multiple sclerosis patients undergoing interferon-beta 1a or 1b therapy: predictive factors of thyroid disease development and duration. J Clin Endocrinol Metab 2005;90(7):4133-7. 9. Tomer Y, Blackard J, Akeno N. Interferon alpha treatment and thyroid dysfunction. Endocrinol Metab Clin N Am 2007;36:1051–66. 10. Carella C, Mazziotti G, Amato G, et al. Interferonα-related thyroid disease: Pathophysiological, epidemiological and clinical aspects. J Clin Endocrinol Metab 2004; 89(8):3656-61. 11. Jamil KM, Leedman PJ, Kontorinis N, et al. Interferon-induced thyroid dysfunction in chronic hepatitis C. J Gastroenterol Hepatol 2009;24(6):1017-23. 12. Mandac J, Chaudhry S, Sherman K, Tomer Y. The clinical and physiological spectrum of interferonalpha induced thyroiditis: toward a new classification. Hepatology 2006;43(4):661-72. 13. Munteis E, Cano JF, Flores JA, et al. Prevalence of autoimmune thyroid disorders in a Spanish multiple sclerosis cohort. Eur J Neurol 2007;14(9):1048‑52. 14. Hristozov K, Кrasnaliev I. Diseases accompanied by hypothyroidism. In: A. Klisarova, ed. Thyroid gland - norm and pathology. Varna 2008: 57-79 (Bulgarian). 15. Oppenheim Y, Ban Y, Tomer Y. Interferon induced autoimmune thyroid disease (AITD): a model for human autoimmunity. Autoimmun Rev 2004; 3:388‑93. 16. Watanabe U, Hashimoto E, Hisamitsu T, et al. The risk factor for development of thyroid disease during interferon alpha therapy for chronic hepatitis C. Am J Gastroenterol 1994;89(3):399-403. 17. Marazuela M, Garcia-Buey L, Gonzalez-Fernandez B, et al. Thyroid autoimmune disorders in patients with chronic hepatitis C before and during interferon-alpha therapy. Clin Endocrinol (Oxf) Folia Medica 2010; 52(3): 5-12

© 2010 Medical University Plovdiv

1996;44:635-42. 18. Carella C, Mazziotti G, Morisco F, et al. Longterm outcome of Interferon-alpha-induced thyroid autoimmunity and prognostic influence of thyroid antibody pattern at the end of the treatment. J Clin Endocrinol Metab 2001;86(5):1925-9. 19. Vanderpump M, Tunbridge W, French J, Appleton D, et al. The incidence of thyroid disorders in the community: a twenty-year follow-up of the Whickham survey. Clin Endocrinol (Oxf) 1995;43:55-68. 20. Wong V, Fu AX, George J, et al. Thyrotoxicosis induced by alpha interferon therapy in chronic viral hepatitis. Clin Endocrinol (Oxf) 2002;56(6):793-8. 21. Duncea I, Pepene C. IFN-alpha-induced recurrence of Graves’ disease ten years after thyroidectomy in chronic viral hepatitis C. Case report. J Gastrointestin Liver Dis 2008;17(4):453-6. 22. Monzani F, Caraccio N, Dardano A, et al. Thyroid autoimmunity and dysfunction associated with type I interferon therapy. Clin Exp Med 2004; 3(4):199‑210. 23. Mazziotti G, Sorvillo F, Stornaiuolo G, et al. Temporal relationship between the appearance of thyroid autoantibodies and development of destructive thyroiditis in patients undergoing treatment with two different type-1 interferons for HCV-related chronic hepatitis: a prospective study. J Endocrinol Invest 2002;25:624-30. 24. Tomer Y, Davies T. Searching for the autoimmune thyroid disease susceptibility genes: from gene mapping to gene function. Endocr Rev 2003;24:694‑717. 25. Dalgard O, Bjoro K, Hellum K, et all. Thyroid dysfunction during treatment of chronic hepatitis C with interferon alpha: no association with either interferon dosage or efficacy of therapy. J Intern Med 2002;251:400-6. 26. Loviselli A, Oppo A, Velluzzi F, et al. Independent expression of serological markers of thyroid autoimmunity and hepatitis virus C infection in the general population: results of a community-based study in northwestern Sardinia. J Endocrinol Invest 1999;22(9):660-5. 27. A ntonelli A, Ferri C, Pampana A, et al. Thyroid disorders in chronic hepatitis C. Am J Med 2004;117(1):10–3. 28. Antonelli A, Ferri C, Ferrari S, et al. Endocrine manifestations of hepatitis C virus infection. Nat Clin Pract Endocrinol Metab 2009;5(1):26-34. 29. Niederwieser G, Buchinger W, Bonelli RM, et al. Prevalence of autoimmune thyroiditis and nonimmune thyroid disease in multiple sclerosis. J Neurol 2003;250(6):672-5. 30. Shinkarkina AP, Vinogradova IuE, Vinogradov DL, et al. Autoantibodies to the thyroid gland in hemoblastoses and cytopenias. Ter Arkh 2003;75(2):62-5 (Russian).

11

B. Nonchev

31. Carella C, Mazziotti G, Morisco F, et al. The addition of ribavirin to interferon-alpha therapy in patients with hepatitis C virus-related chronic hepatitis does not modify the thyroid autoantibody pattern but increases the risk of developing hypothyroidism. Eur J Endocrinol 2002;146:743-9. 32. Minelli R, Braverman LE, Valli MA, et al. Recombinant interferon alpha (rIFN-alpha) does not potentiate the effect of iodine excess on the development of thyroid abnormalities in patients with HCV chronic active hepatitis. Clin Endocrinol (Oxf) 1999;50:95‑100. 33. Mazziotti G, Sorvillo F, Piscopo M, et al. Innate and acquired immune system in patients developing interferon alpha-related autoimmune thyroidi-

tis: a prospective study. J Clin Endocrinol Metab 2005;90(7):4138-44. 34. Nagayama Y, Mizuguchi H, Hayakawa T, et al. Prevention of autoantibody-mediated Graves’- like hyperthyroidism in mice with IL-4, a Th2 cytokine. J Immunol 2003;170:3522-7. 35. Akeno N, Tomer Y. Dissecting the mechanisms of interferon-induced thyroiditis (IIT): direct effects of interferon alpha on thyroid epithelial cells. Presented at the 89th Meeting of the Endocrine Society, Toronto (Canada), June 2, 2007. 36. C onsensus document: Diagnosis, treatment and monitoring of patients with chronic viral hepatitis – diagnostic and therapeutic algorithm. Bulgarska hepatogastroenterologia 2006;1:84-91 (Bulgarian).

Тиреоидиты, леченные альфа и бета интерферонами

возможные причины поражения щитовидной железы указывается на ряд генетических детерминантов (пол, возраст, этническая принадлежность, гены, включенные в тиреоидную иммунную регуляцию) и негетических факторов, связанных с основным заболеванием (гепатит С вирус, множественный склероз, терапевтические режимы применения интерферона, содержание йода в окружающей среде, наличие аутоантител к началу лечения и др.). Обзор обобщает известные к моменту данные о частоте и характеристике тиреоидных нарушений у больных, леченных интерфероном, обобщает факторы риска и механизмы их развития, как и особенности течения, прослеживания и лечения этих осложнений. Имеющиеся в литературе данные мотивируют исследование тиреоидной дисфункции среди определенных групп пациентов, леченных интерфероном, с целью уточнения специфического влияния факторов (препарат и заболевание) на щитовидную железу. Раннее обнаруживание и адекватное лечение тиреоидной дисфункции у этих больных особо важны в целях избежания осложнений, компрометирующих лечение.

Б. Нончев

Резюме В настоящее время интерфероны являются основными лечебными средствами для значительного числа злокачественных и незлокачественных болезней (гепатит С, гепатит В, множественный склероз, злокачественные гемопатии, меланома, почечноклеточная меланома и другие). У некоторых больных, леченных интерфероном, развиваются тиреоидные поражения, распространение которых варьирует в границах 1% - 35%. Эти осложнения являются нередкой причиной редукции доз или прекращения лечения интерфероном. В литературе довольно широко описаны исследования тиреоидной дисфункции у пациентов с гепатитом С, леченных интерфероном альфа и у пациентов с множественным склерозом, леченных интерфероном бета. Данные относительно частоты и характеристики тиреоидных отклонений у больных со злокачественными гемопатиями, леченных интерфероном альфа, недостаточны. Как

12

Folia Medica 2010; 52(3): 5-12

© 2010 Medical University Plovdiv

Folia Medica 2010; 52(3): 13-20 Copyright © 2010 Medical University Plovdiv doi: 10.2478/v10153-010-0002-5

REVIEWS Descending necrotizing mediastinitis of odontogenic origin – personal experience and literature review Ivan P. Novakov, Georgi P. Safev, Stefka E. Peicheva1

Clinic of Thoracic and Abdominal Surgery; Medical University, Plovdiv, 1Clinic of Maxillofacial Surgery, Medical University, Plovdiv, Bulgaria Abstract Descending necrotizing mediastinitis is the most severe form of mediastinal infection. The aim of the study was to present the optimal diagnostic and treatment approach to this severe, life-threatening condition. Patients and methods: Three patients (men, aged 75, 73, and 63) with descending necrotizing mediastinitis hospitalised between April 2007 and February 2009 have been included in the study. The diagnosis of the condition was made based on cervico-thoracic computed tomography and surgical findings. The surgical treatment in each of the cases included bilateral longitudinal cervicotomy, transversal suprasternal cervicotomy and posteriorlateral thoracotomy. Results: The period between the initiation of ambulatory treatment of the dental infection and diagnosing the mediastinitis was 9, 8 and 11 days, respectively. Engagement of all cervical spaces and mediastinal sections with polybacterial (three or more agents) dental infection, originating from third and fourth lower molars was present in each of the patients. Chronic alcoholism and diabetes are factors influencing the course of mediastinitis. The outcome in all the three patients was lethal (within 72 hours). Conclusion: Success in the treatment of descending necrotic mediastinitis of odontogenic origin may be expected only in case of early diagnose and aggressive cervical and mediastinal drainage, performed by bilateral longitudinal cervicotomy and posterior-lateral thoracotomy. Key words: descending mediastinitis, dental infection, cervical cellulitis, cervicotomy, thoracotomy Introduction

The first classification of acute purulent mediastinitis in the antibiotic era dates back to 1937 (H. Neuhof). Depending on the spread of the infection H. Neuhof divided acute mediastinitis into localized (mediastinal abscesses) and diffuse spread (mediastinal phlegmons).1,2 In 1938 E. Pearse described a case of mediastinitis resulting from oropharyngeal infection, and in 1983 A. Estrera et al. formed the group of “descending necrotizing mediastinites”.1-3 These are cases of mediastinitis resulting from bacterial infection, originating from the oropharynx, over 2/3 of which are diagnosed postmortem according to Estrera. It has been found that 60% to 70% of the cases of necrotic mediastinitis are of odontogenic origin – result of dental infection, descending into the mediastinum.1-4

The descending necrotizing mediastinitis is undoubtedly the most severe form of mediastinal infection. The disease has fulminant course with potential occurrence of sepsis and high mortality. Two of the main reasons for the high mortality in patients with descending necrotizing mediastinitis are delay in making the correct diagnosis and delay and/or inappropriate surgical treatment.1,2,5,6 There is still no consensus concerning the optimal surgical approach in patients with descending necrotizing mediastinitis. This is especially applicable to the forms of mediastinal drainage. The opinions vary from drainage of the mediastinum only by transcervical access to routine application of combination of thoracotomy, transcervical mediastinal drainage and tracheostomy.1,6-8 The aim of the current study was to contribute to the search of the optimal approach

Correspondence and reprint request to: Iv. Novakov, Clinic of Thoracic and Abdominal Surgery, Medical University, Plovdiv 15A Vassil Aprilov St, 4002 Plovdiv, Bulgaria Received 16 December 2009; Accepted for publication 31 March 2010

13

Iv. Novakov et al

to the treatment of descending necrotizing mediastinitis by discussing the diagnostic and surgical attitude to this life-threatening condition. Patients and methods

The present study includes three patients (men, aged 75, 73, and 63) diagnosed with descending necrotizing mediastinitis between April 2007 and February 2009. In their cases the condition was caused by dental infection descending into the mediastinum – mediastinitis of odontogenic origin. The source of infection was dental abscess affecting the lower molars (right third, right third, and left fourth, respectively). Chronic alcoholism in two of the patients and diabetes mellitus in the third case were predisposing factors for the progressive course of the dental infection. Appropriate selection of patients is guaranteed by fulfillment of all four criteria suggested by A. Estrera for descending necrotizing mediastinitis3: 1) clinical evidence of severe oropharyngeal infection; 2) characteristic roentgenographic features of mediastinitis; 3) documentation of necrotizing mediastinal infection at operation or necropsy (or both); 4) establishment of the relationship between the oropharyngeal infection and descending necrotizing mediastinitis. The patients had started their treatment in ambulatory settings (treatment of the dental infection). In the three cases presented here the site of admission was a maxillofacial surgery department because of the progression of patient complaints and descent of the inflammatory process to the floor of the oral cavity and the neck. In case of determined descent of the infection into the mediastinum, patients were admitted to a clinic of thoracic surgery. After accomplishment of mediastinal debridement and drainage, post-surgical treatment was performed in an intensive care unit. Diagnostic Methods Conventional roentgenographic examination of the chest and computed tomography of the floor of the oral cavity, the neck and the chest were used in the study. Microbiological investigation of mediastinal exudate was performed to determine the bacteriological agent causing the descending necrotic mediastinitis. Surgical approach The following surgical interventions were performed: 1. incision and drainage of the floor of the oral cavity; 2. bilateral longitudinal cervicotomy with removal of necrotic tissue (debridement) and

14

drainage of cervical fascial spaces; 3. transcervical, suprasternal drainage of the mediastinum; 4. lateral thoracotomy with mediastinal debridement and drainage of all sections of the mediastinum and the respective pleural cavity; 5. application of intrapleural drainage catheter. In each of the patients several drainage procedures, presented in the “Results” section, were performed. Results

The period between initiation of the ambulatory treatment of the dental infection and the admission to the Thoracic Surgery Clinic was established for each of the patients – respectively 9, 8 and 11 days. The infection of the floor of oral cavity presented with identical signs and symptoms in all three patients – pain in the mandible and the throat, dysphagia, trismus, fever (>38.6°С). The findings from the physical examination in all subjects included painful edema of the submandibular area and the neck. Palpable subcutaneous emphysema of the neck was present in two of the cases. Symptoms, that were found in all subjects and presumed descent of the infection into the mediastinum, were dyspnea, chest pain, hyperemia and edema of the upper chest. Widening of the mediastinal shadow, associated with pneumomediastinum was found on conventional chest X-ray in the three study patients. Computed tomography was used to diagnose the infection of the floor of the oral cavity and the neck (Fig. 1). The diagnosis of “descending necrotizing mediastinitis” was confirmed by computed tomography of the chest findings (Fig. 2). The latter investigation allows determination of the progression of the inflammatory process in the neighboring structures – into the pleural space (Figs 2, 3). Spreading of the dental infection (according to the findings from the imagings and the surgery), the isolated bacterial agents, causing mediastinitis, the applied surgical treatment and its outcome are systematized and presented in Table 1 and Figs 4, 5, 6. Discussion

Descending necrotic mediastinitis is defined as the most severe form of acute mediastinal infection. The current study is based entirely on patients with descending necrotic mediastinitis of odontogenic origin. This fact confirms the standpoint that in more than 60% of the cases, the descending necrotic mediastinitis is of odontogenic origin. Several factors contribute to the pathogenesis of this life-threatening Folia Medica 2010; 52(3): 13-20

© 2010 Medical University Plovdiv

Descending Necrotizing Mediastinitis of Odontogenic Origin - Personal Experience and Literature Review

Figure 3. Computed tomography of the chest of a patient with descending necrotizing mediastinitis. Along with engagement of the mediastinum, the inflammatory process affects both pleural cavities (bilateral pleural empyema).

Figure 1. Computed tomography showing descent of the dental infection into the bottom of the oral cavity. Soft-tissue edema (cellulitis) with formation of submandibular abscess on the right (designated with arrows) is present.

Figure 2. Computed tomography of the chest (patient with descending mediastinitis). All sections of the mediastinum are affected. The mediastinum is enlarged, with air collections in it – pneumomediastinum (arrows). The neighboring left pleural cavity is also engaged (left-side empyema).

disease. First, this is the type of the bacterial infection. Descending mediastinitis is typically a result of mixed aerobic-anaerobic bacterial infection with synergy between different agents.9-11 This synergy Folia Medica 2010; 52(3): 13-20 © 2010 Medical University Plovdiv

Figure 4. Picture of a patient with fulminant odontogenic infection, descending into the neck and the mediastinum. The descending infection had caused skin necrosis of a vast area of the neck and the upper part of the chest. Drainage tube, placed in the mediastinum via transcervical access, is marked with arrow.

between bacterial agents is the reason for their high virulence which in turn determines the severe course of the disease. The microbiological examination in our study confirms that the descending mediastinitis is a result of polybacterial infection. The fact that in our results there are no anaerobic bacterial agents isolated from the mediastinum does not mean that they have no participation in the beginning and the

15

Iv. Novakov et al

Figure 5. Right-side thoracotomy in patient with descending necrotic mediastinitis. Debridement of all mediastinal sections was performed. The distal end of a drainage tube placed in the mediastinum via transcervical access is marked with arrow.

Figure 6. Left-side thoracotomy in patient with descending necrotic mediastinitis. Spreading of the inflammatory process causes left-side pleural empyema (purulent exudate in the pleural cavity is marked with arrows).

Table 1. Spreading and type of the dental infection, performed surgical treatment and outcome Patient

1

2

3

Spreading of dental infection

Isolated bacterial infection

Outcome

- submandibular abscess on the right; - bilateral neck cellulitis; - mediastinitis; - bilateral pleural empyema

- Escherichia coli; - Pseudomonas aeruginosa; - Streptococcus salivarius

- right-side submandibular drainage; - bilateral longitudinal cervicotomy; - suprasternal mediastinal drainage; - right-side thoracotomy; - left pleural cavity drainage

- lethal (endotoxic) shock - 48 h after mediastinal drainage

- submandibular abscess on the right; - bilateral neck cellulitis; - mediastinitis; - bilateral pleural empyema

- Staphylococcus aureus; - Escherichia coli; - Pseudomonas aeruginosa; - Streptococcus β-hemoliticus

- right-side submandibular drainage; - bilateral longitudinal cervicotomy; - suprasternal mediastinal drainage; - right-side thoracotomy; - left pleural cavity drainage (tubular drainage)

- lethal (endotoxic) shock - 48 h after mediastinal drainage

- bilateral neck cellulitis; - mediastinitis; - pleural empyema on the left

- Streptococcus β-hemoliticus; - Staphylococcus epidermidis; - Pseudomonas aeruginosa;

- bilateral longitudinal cervicotomy; - suprasternal mediastinal drainage; - left-side thoracotomy;

- lethal (endotoxic) shock - 72 h after mediastinal drainage

development of the disease. In our opinion, the severe changes and the fatal outcome in all three patients are indirectly supporting the presence of anaerobic microorganisms as causing agents of descending necrotic mediastinitis. Other factors, contributing to the pathogenesis of the disease, are the way of descending of the

16

Surgical drainage

infection and the medium for its development. The descent of oral cavity infection into the mediastinum follows the cervical fascial spaces – pretracheal, retropharyngeal (retrovisceral) and perivascular. These are potential spaces, confined of the three layers of the deep cervical fascia. Advancing through the pretracheal space, the infection descends into Folia Medica 2010; 52(3): 13-20

© 2010 Medical University Plovdiv

Descending Necrotizing Mediastinitis of Odontogenic Origin - Personal Experience and Literature Review

the frontal mediastinum, overcoming connective tissue membrane on the level of superior thoracic aperture - suprapleural membrane of Sibson.10,13 The retropharyngeal space begins from the base of the skull passing downwards into the posterior mediastinum, thus enabling direct access of the dental infection to the mediastinum. The descent of the infection via the perivasal space into the mediastinum is along the route of the big cervical blood vessels – carotid artery and internal jugular vein. It has been proven that descending necrotic mediastinitis is most commonly caused by infection of the second and the third lower molars.6,8,9,11 The current study confirms that opinion. The reason behind this fact is that the roots of the second and the third lower molars are situated below m. myohyoideus, which in case of infection of these particular teeth ensures direct access to the submandibular space. Computed tomography results and surgical findings in the patients of the study are representing the easiest and most common way of descent of the dental infection into the mediastinum – from the floor of the oral cavity, through the submandibular space (submandibular process) to the frontal mediastinum. The determination of the way of descent of the infection, as well as which of the mediastinal sections is affected by the descending necrotic mediastinitis is quite conditional. The reason for that standpoint is that synergically acting polybacterial infectious agents find ideal medium for development in the loose connective tissue of the neck and the mediastinum and does not stay isolated in one space.10 The current study confirms the above-mentioned opinion and serves as an ideal example for fulminant development of the dental infection spreading in all cervical spaces and mediastinal sections. It has to be mentioned that numerous predisposing factors are of importance for the severe course of the descending necrotic mediastinitis. Their role is in suppressing organism’s defense, which favors progression of the mediastinal infection.1,2,14 In the current study, three of the most common predisposing factors are present: age over 70 years, alcoholism and diabetes mellitus. Bacterial infection, associated diseases and age affecting the course of the descending mediastinitis, are given objective factors. The outcome of the descending necrotic mediastinitis, however, is determined by other two factors, which are influenceable and are subject of discussion in the present study. These are diagnosing the disease and the methods of its surgical treatment. Folia Medica 2010; 52(3): 13-20 © 2010 Medical University Plovdiv

The overview of a number of publications allows outlining several conclusions, concerning the diagnosing of descending necrotic mediastinitis.4-9,11-15 It has been determined that delayed diagnosing of the disease is the main reason for the high mortality. Some of the reasons for the late diagnosis of mediastinitis are subjective. These are prolonged ambulatory treatment of the dental infection and delayed admission of patients to thoracic surgery clinics. The results of the current study are perfectly representative of an example for a delayed diagnose resulting from subjective factors. Delayed diagnosing of the disease has also its objective explanation. While the diagnosis of cervical infection is easy due to the obvious clinical symptoms and signs, it is not the case with the early engagement of the mediastinum from the inflammatory process, lacking typical clinical symptoms related to it. Because of that reason and taking into account the discussed above fast progression of mediastinitis, we accept the view that in case of clinical presentation of dental infection with trismus, dysphagia and emerging edema of the face and the neck, imaging methods for diagnosing of eventual neck and mediastinal infection must be used immediately. The two imaging methods that are commonly used in diagnosing of mediastinitis are conventional chest X-ray and cervico-thoracic computed tomography. The findings from the conventional X-ray investigation are accepted as late manifestation of the disease. Often, they are non-specific and therefore are of little informative value for determination of the spread of the mediastinal inflammatory process. That is why conventional X-ray is not accepted as a reliable method for early diagnosing of descending necrotic mediastinitis. Cervico-thoracic computed tomography is considered a reliable method for early diagnosing of the disease. Except its diagnostic value, cervico-thoracic tomography also determines spreading of inflammatory-necrotic process into cervical and mediastinal area1,2,16, which allows determination of optimal surgical drainage approach. The high diagnostic value of cervico-thoracic computed tomography has been demonstrated also in this study. We share the opinion for its obligatory application as a diagnostic method in all patients in whom presence of descending necrotic mediastinitis is suspected. Together with formulation of the criteria for descending necrotic mediastinitis in 1983 A. Еstrera also presented the approach to its surgical treatment.3 In case of spreading of the mediastinal infection above the level of the tracheal bifurcation

17

Iv. Novakov et al

(4th thoracic vertebra) Еstrera performed transcervical mediastinal drainage. In case of descent of the mediastinal infection below the level of the carina, he performed transthoracic drainage (thoracotomy). Other authors report successful treatment of descending mediastinitis by combination of transcervical and subxyphoidal mediastinal drainage.1,2,4,17 Some authors report successful mediastinal drainage by means of videothoracoscopic approach.18,19 The presented variants of surgical drainage of the mediastinum may be defined as conservative surgical approach. Data analysis of the these studies shows that conservative surgical treatment is successful only in patients with localized forms of descending mediastinal infection. The origin of infection in these cases is the neck – peritonsilar abscess, lateropharyngeal abscess, esophagus lesion (cervical segment). On the contrary, infections of dental origin are rapidly descending into the cervix and the mediastinum, being prone to diffuse spreading, well-demonstrated here. Therefore, conservative surgical approach is not suitable for the treatment of patients with mediastinitis of odontogenic origin. Success in treatment of acute odontogenic mediastinitis may be achieved only by aggressive surgical drainage of the neck and the mediastinum. Only the transthoracal access – thoracotomy, ensures adequate debridement and drainage of all mediastinal sections. Spreading of the infection from the mediastinum to the pleural cavity (empyema) in most of the patients with odontogenic mediastinitis is a fact that may not be neglected. That is why early thoracotomy is accepted as a standard in the treatment of patients with descending odontogenic mediastinitis, irrespective of the level of mediastinal engagement.5-8,12,14,17,20-22 Among the described in the literature several transthoracic access ways to the mediastinum, we recommend the posterior-lateral thoracotomy. If possible, we perform right-side thoracotomy, since it ensures good and easy access to all mediastinal sections. In case of spreading of the infection into the pleural cavity (empyema), the choice of side for the thoracotomy is consistent with the localization of the empyema. Simultaneously with the mediastinal drainage, it is obligatory to perform also debridement and drainage of the affected cervical fascial spaces. Adequate drainage of the neck is achieved by longitudinal cervicotomy – bilateral, along the frontal edges of mm. sternocleidomastoidei.1,2,5,7,23-25 Except for the longitudinal cervicotomy, we always perform drainage of the submandibular space.

18

The patients in this study underwent adequate in volume, but separated in time drainages of the neck and the mediastinum. In these cases the mediastinitis was diagnosed only after cervical drainage had been performed (treatment of the cervical cellulitis). These are cases of late diagnosing of the odontogenic mediastinitis. Late diagnosing of the disease is the main factor determining the lethal outcome of the disease in all three patients. Prerequisite for avoidance of such fatal, late diagnosing of mediastinitis is determination and accomplishment of the diagnostic treatment plan for patients with dental infection, descending into the bottom of the oral cavity, by a multidisciplinary team, obligatory including thoracic surgeon. Only multidisciplinary surgical team may diagnose at the earliest odontogenic mediastinitis and accomplish its adequate treatment. Conclusions

Analysis of the results of the present study and the overview of the literature allows us to make the following conclusions: The descending necrotic mediastinitis of odontogenic origin presents with the course of a polybacterial infection, usually affecting diffusely the cervical fascial spaces and all mediastinal sections. Success in the treatment of descending necrotic mediastinitis of odontogenic origin may be expected only in case of early diagnosing and aggressive cervical and mediastinal drainage, performed by bilateral longitudinal cervicotomy and posteriorlateral thoracotomy. References

1. Shields TW, Joseph LC, Ponn RB, et al. General thoracic surgery. 6th Edition. Lippincott Williams & Wilkins; 2007. 2. Patterson AG, Pearson GF, Cooper Jd, et al. Pearson’s Thoracic and Esophageal Surgery. 3rd Edition. Elsevier Inc; 2008. 3. Estrera AS, Landau MJ, Grisham JM, et al. Descending necrotizing mediastinitis. Surg Gynecol Obstet 1983;157:545-52. 4. Freeman RK, Villieres E, Verrier ED, et al. Descending necrotizing mediastinitis: an analysis of the effects of surgical debridement on patient mortality. J Throrac Cardiovasc Surg 2000;119:260-7. 5. Corsten MJ, Shamji FM, Odell PF, et al. Optimal treatment of descending necrotizing mediastinitis. Thorax 1997;52:702-8. 6. Sancho LM, Minamoto HA, Senne LU, et al. DeFolia Medica 2010; 52(3): 13-20

© 2010 Medical University Plovdiv

Descending Necrotizing Mediastinitis of Odontogenic Origin - Personal Experience and Literature Review

scending necrotizing mediastinitis: a retrospective surgical experience. Eur Jour of Cardio-thoracic Surg 1999;16:200-5. 7. Brunelli AS, Sabbatini AJ, Catalini GA. Descending necrotizing mediastinitis: surgical drainage and tracheostomy. Arch Otolaryngol Head Neck Surg 1996;122:1326-9. 8. Makeieff MN, Gresillion NP, Berthet JP, et al. Management of descending necrotizing mediastinitis. Laryngoscope 2004;114:772-5. 9. Sakamoto H, Aoki T, Kise Y, et al. Descending necrotizing mediastinitis due to odontogenic infections. Oral Surg Oral Med Oral Pathol 2000;89:412-9. 10. Moncada R, Warpeha R, Pickleman J, et al. Mediastinitis from odontogenic and deep cervical infections - anatomic pathways of propagation. Chest 1978;73:497-502. 11. Tung-Yiu W, Jehn-Shyun H, Ching-Hung C, et al. Cervical necrotizing fasciitis of odontogenic origin: A report of 11 cases. J Oral Maxillofac Surg 2000;58:1347-53. 12. Kinzer SA, Pfeiffer JB, Becker SR, et al. Severe deep neck space infections and mediastinitis of odontogenic origin: clinical relevance and implications for diagnosis and treatment. Acta Oto-laryngologica 2009;129:62-70. 13. Misthos P, Katsaragakis S, Kakaris S, et al. Descending necrotizing anterior mediastinitis: analysis of survival and surgical treatment modalities. J Oral Maxillofac Surg 2007;65:635-9. 14. Endo S, Murayama F, Hasegawa T, et al. Guideline of surgical management based on diffusion of descending necrotizing mediastinitis. Jpn J Thorac Cardiovasc Surg 1999;47:14-19. 15. Findikcioglu AD, Kilic DE, Akin ST, et al. Descend-

Folia Medica 2010; 52(3): 13-20 © 2010 Medical University Plovdiv

ing necrotizing mediastinitis: treatment of a delayed case. Acta Chir Belg 2007;107:462-4. 16. Scaglione M, Pinto A, Giovine S, et al. CT features of descending necrotizing mediastinitis - a pictorial essay. Emerg Radiol Jun 2007;14:77-81. 17. Barbieri M. Descending necrotizing mediastinitis: ten years’ experience. Ear, Nose and Throat Jour 2004;11:707-9. 18. Roberts JR, Smythe RW, Weber RW, et al. Thoracoscopic management of descending necrotizing mediastinitis. Chest 1997;112:850-4. 19. Nakamura Y, Matsura A, Katsura H, et al. Successful video-thoracoscopic drainage for descending necrotizing mediastinitis. Gen Thorac Cardiovasc Surg 2009;57(2):111-5. 20. Papalia E, Rena O, Olario A, et al. Descending necrotizing mediastinitis: surgical management. Eur Jour of Cardio-thoracic Surg 2001;20:739-42. 21. Weatley MJ. Descending necrotizing mediastinitis - transcervical drainage is not enough. Ann Thorac Surg 1990;49:780-6. 22. Roccia F, Pecorari CG, Oltaio AE, et al. Descending necrotizing mediastinitis secondary to retropharyngeal abscess without cervical spread. Gen Thorac Cardiovasc Surg 2008;56:25-7. 23. Henry MC, Alauzen M, Alric P, et al. Descending necrotizing mediastinitis: Advantage of mediastinal drainage with thoracotomy. J Thorac Cardiovasc Surg 1994;107:55-61. 24. Juretic M, Gobic MB, Kukuljia M, et al. Mediastinitis and bilateral pleural empiema caused by odontogenic infection. Radiolo Oncol 2007;41:57-62. 25. Balkan ME, Oktar GL, Oktar MA. Descending necrotizing mediastinitis: a case report and review of the literature. Int Surg 2001;86(1):62-6.

19

Iv. Novakov et al

Десцендирующий некротический медиастинит одонтогенного происхождения – собственный опыт и литературный обзор И. Новаков, Г. Сафев, С. Пейчева

Резюме Десцендирующий некротический медиастинит одонтогенного происхождения представляет собой самую тяжелую форму медиастинальной инфекции. Цель: Работа ставит себе целью представить оптимальный подход при диагностике и лечении пациентов в этом тяжелом жизнеугрожающем состоянии. Пациенты и методы: В исследование включены трое пациентов – мужчин (возраст - 75, 73 и 63 г.) с десцендирующим некротическим медиастинитом, госпитализированных в периоде апрель 2007 – февраль 2009 г. Диагноз заболевания основывается на шейно-торакальной компьютерной томографии и оперативной находке. Оперативное лечение каждого

20

пациента включает двустороннюю лонгитудинальную цервикотомию, трансверсальную супрастернальную цервикотомию и заднебоковую торакотомию. Результаты: Время от амбулаторного лечения дентальной инфекции до диагностицирования медиастинита соответственно 9, 8 и 11 дней. У пациентов установлено поражение всех шейных пространств и отделов медиастинума полибактериальной (три возбудителя и более) дентальной инфекцией, происходящей от нижних третих и четвертых моляров. Налицо и хронический алкоголизм и сахарный диабет как факторы, оказывающие влияние на течение медиастинита. Во всех трех случаях исход летальный (до 72-ого часа). Заключение: Успех при лечении десцендирующего некротического медиастинита одонтогенного происхождения возможен только в случаях ранней диагностики и агрессивного шейного дренажа и медиастинального дренажа, осуществляемых посредством двусторонней лонгитудинальной цервикотомии, трансверсальной супрастернальной цервикотомии и заднебоковой торакотомии.

Folia Medica 2010; 52(3): 13-20

© 2010 Medical University Plovdiv

Folia Medica 2010; 52(3): 21-30 Copyright © 2010 Medical University Plovdiv doi: 10.2478/v10153-010-0003-4

Original Articles

Clinical Investigations Simultaneous quantification and genotyping of hepatitis C virus RNA by a two-step real-time PCR assay on the LightCycler instrument Manabu Abe, Corinne Klett, Eberhard Wieland, Sascha Gille1, Olfert Landt1 Central Institute for Clinical Chemistry and Laboratory Medicine, Klinikum Stuttgart Katharinenhospital, Stuttgart, Germany; 1TIB MOLBIOL GmbH, Berlin, Germany Abstract Background: Clinically, both viral load and genotypes have been found to be major predictors of antiviral therapy outcome regarding chronic hepatitis C and they are, under normal circumstances, performed as separate assays. Design and methods: In order to improve the diagnostic strategy and subsequently reduce the reagent costs we have developed and established the simultaneous quantification and genotyping of hepatitis C virus RNA by a two-step real-time PCR on the LightCycler Instrument (Roche Diagnostics). Results: The quantification assay was calibrated against WHO Standard 96/790. The detection limit was 30 IU/ml, the dynamic range up to 500,000,000 IU/ml. Intra- and interassay imprecisions were 1.2% and 1.9% (n = 10), respectively. The HCV RNA values obtained by real-time PCR assay were highly correlated with those obtained by the Cobas Amplicor HCV monitor test (r = 0.992; p < 0.001). Conclusions: The genotyping was performed by means of the melting temperature analysis. The concordance between our new genotyping method and the Trugene HCV 5’NC Kit was at the level of genotypes 100%. This rapid (3 h) and convenient assay is suitable for HCV genotyping, HCV detection and disease monitoring. Keywords: HCV, real-time PCR, genotyping Introduction

Affecting nearly 300 million people worldwide, hepatitis C virus (HCV) infection is considered to be the most frequent cause of post-transfusion non-A, non-B hepatitis. HCV, a member of the Flaviviridae family, is a single-stranded RNA virus, containing a genome of 9,400 nucleotides. The HCV genome demonstrates considerable diversity of nucleotide sequences.1 Based on the identification of these genomic differences, HCV has been classified into multiple strains.2 It is thought that genetic heterogeneity of HCV may account for some of the differences in disease outcome and response to treatment observed in HCV infected persons.3 HCV genotype 1, which is the most prevalent worldwide, is associated with more severe clinical manifestations, higher levels of HCV viremia and less amenable to treatments such as interferon

alpha or PEG-interferon alpha / ribavirin therapy than HCV genotypes 2 or 3.4,5 Therefore, clinical trials of antiviral therapies require both HCV viral load and genotype information for an appropriate strategy of treatment. Assays for HCV genotyping are generally based on sequencing technology or line-probe test, but these assays are time-consuming, labour intensive, expensive and require significant expertise and additional devices such as an autosequencer.6-8 Recently, several other methods have been published, e.g. heteroduplex mobility analysis using temperature gradient capillary electrophoresis9,10, genotyping with matrix-assisted laser desorption ionizationtime of flight (MALDI-TOF)11, genotyping with genaturing high-performance liquid chromatography12, melting curve analysis with a single set of fluorescence resonance energy transfer probes with

Correspondence and reprint request to:Manabu Abe,Central Institute for Clinical Chemistry and Laboratory Medicine, Klinikum Stuttgart Katharinenhospital, Stuttgart, Germany; E-mail: [email protected] Kriegsbergstrabe 60, D-70174 Stuttgart, Germany Received 25 May 2010; Accepted for publication 07 July 2010

21

Manabu Abe et al

LightCycler13,14. Because we already quantify the HCV RNA viral load by means of the LightCycler, we have tried to combine the quantification and genotyping of HCV on the same instrument using hybridisation probes. After several rounds of optimisation, the simultaneous quantification and genotyping of hepatitis C virus RNA by a two-step real-time PCR on the LightCycler instrument was successfully established. Materials and methods

Clinical specimens EDTA whole blood samples were obtained from 76 patients of the Klinikum Stuttgart, Katharinenhospital, Germany. All samples were sent to the laboratory for routine analysis of HCV viral load. Samples were centrifuged for 2 hours and divided into aliquots, which were stored at – 80°C. All samples were first measured with the Cobas Amplicor HCV Monitor test (Roche Diagnostics, Mannheim, Germany) and then examined with our new method on the LightCycler instrument (Roche Diagnostic, Mannheim, Germany). Sequences of primers and probes Out of different primers tested we have selected the established forward primer NC1 and reverse primer SR215-19 (Fig. 1). With this primer pair the best detection limit was achieved without the need of semi-nested PCR. For genotyping, the hybridization probes published by Bullock et al. were used.13 Internal control Clinical specimens may contain inhibitors of PCR amplification, which can be monitored by using internal controls (IC).20 These controls can be heterologous PCR systems or modified target variants, for example those containing a mutated probe-binding site. DNA based controls monitor the performance of the DNA PCR only, whereas RNA based controls enable to examine in addition the extraction of the sample and the reverse transcription step. We used an internal control containing the target sequence of HCV genotype 1 cloned into a plasmid, replacing the probe-binding site by a fragment from Lambda DNA (GenExpress, Berlin, Germany). Detection of the control derived PCR product was achieved using the hybridization probes HCV-IC-FL (5’- GGT GCC GTT CAC TTC CCG AAT AAC-fluorescein, J02439/2331-2354) and HCV-IC-LC (Red705LC705-CGG ATA TTT TTG ATC TGA CCG AAG CG-phosphate, J02439/2356-2381).

22

In vitro transcription of the internal DNA control into RNA using the T7 promoter The plasmid containing the internal control sequence was linearized prior to in vitro transcription to generate RNA transcripts of defined length. In vitro transcription was performed with T7 RiboMax Express Large Scale RNA Production System (Promega, Mannheim, Germany) according to the instructions of the manufacturer. 500 copies of the RNA transcripts were added to each plasma sample as internal control, enabling to monitor the RNA extraction, reverse transcription, and PCR efficiency. External control As external controls HCV reference standards (#75/98) calibrated against the WHO Standard 96/790 were obtained from the Paul-Ehrlich-Institute, Langen, Germany. Each vial was reconstituted with 0.5 ml of distilled water to yield 25,000 IU/ml of HCV genotype 1a. This concentration is considered equivalent to 75,000 copies/ml according to the data sheet provided by the PEI. Samples RNA from 500 µl EDTA plasma samples was extracted and purified on the QIAamp DSP viral vacuum kit (Qiagen, Hilden, Germany). Reverse transcription reactions (20 µl) were performed in 200 µl-vessels using a block thermocycler (Peqlab, Erlangen, Germany) and contained the following components at the indicated final concentrations or amounts in sterile molecular-grade water: SampleRNA (14 µl), reverse primer SR2 (1.4 µM), dNTPs (each 1.13 mM), superscript II reverse transcriptase (30 U), RNAse Out (10 U), 5x-RT-buffer (Invitrogen). The sample RNA primer dNTP mixture was first denatured by 65°C for 2 min, followed by an annealing step from 65°C to 25°C with 0.3°C/sec. Thereafter, the RT mixture (4.4 µl) was added to the vessels followed by incubation at 40°C for 35 min. The reverse transcriptase was inactivated at 95°C for 1 min. LightCycler Real-Time PCR After the completion of the reverse transcription the LightCycler cDNA PCR was performed with the following components at the indicated final concentrations or amounts in sterile molecular-grade water: PCR reaction mixture (8 µl) contains 1 µM HCV forward primer NC1 (5’-CCC TGT GAG GAA CTA CTG TCT TCA CGC; D10934/ 43-699) 1 µM HCV reverse primer SR2 (5’-GGG CAC TCG CAA GCA CCC TAT; D10934/ 316-296), 0.15 µM HCV

Folia Medica 2010; 52(3): 21-30

© 2010 Medical University Plovdiv

Simultaneous Quantification and Genotyping of Hepatitis C Virus Rna by a Two-Step Real-Time Pcr Assay on the Lightcycler Instrument

Figure 1. Alignment of the HCV genotype sequences showing overlapping and conserved regions of the different HCV genotypes and the locations of the used primers and probes. HCV genotype 1b/2-fw: additional forward primer for the differentiation of genotypes, NC1 common fw primer: forward primer for the quantification, HCV FL anchor: Detection probe with fluorescent colour fluorescein at the 5’end and at the 3’end phosphate, HCV LC sensor: Detection probe with fluorescent colour LC640 at the 3’end, SR2 reverse primer: reverse primer for the quantification and genotyping.

Folia Medica 2010; 52(3): 21-30 © 2010 Medical University Plovdiv

23

Manabu Abe et al

FL anchor (5’-GCCATAGTGGTCTGCGGA ACC GGT-fluorescein; D10934/ 137-160), 0.3 µM HCV LC sensor (5’-LC640-GTA CAC CGG AAT TGC CAG GA-phosphate; D10934/ 163-182), 0.15 µM HCV-IC FL, 0.3 µM HCV-IC LC (all from TIB MOLBIOL), 1 µl Faststart DNA Master Hybridization Probes (Roche Diagnostics), 3 mM MgCl2. The capillaries were loaded with 8 µl PCR reaction mixtures, complemented with 2 µl of sample cDNA and closed. The capillaries were centrifuged for 10 sec at 900 g, transferred into the sample carousel and placed in the LightCycler instrument. The PCR was performed with following cycling program: 95°C for 8 min followed by 40 cycles of 3 sec at 95°C, 5 sec at 50°C and 13 sec at 72°C. Thereafter, the genotyping was performed by means of the melting temperature analysis.13 The melting temperatures were analysed by applying 95°C for 15 sec, 40°C for 15 sec, followed by a stepwise temperature increase of 0.2°C/sec from 40°C to 80°C with the continuous recording of the fluorescence. Data acquisition was performed by applying the following fluorescence channel settings: F2 for the HCV cDNA products and F3 for the IC DNA products. A colour compensation file was generated according to the manufacturer’s instructions and was used in all PCR runs. All PCR was accompanied by a quantification standard, a negative control and a positive control with the known concentration. Performance characteristics The detection limit of the method was established by diluting the HCV RNA reference standard of PaulEhrlich Institute (Langen, Germany) (#75/98) with HCV RNA negative plasma. The copy number which did not lead to a detectable amplification above the crossing point was defined as the detection limit. Linearity was assessed by of a HCV quantification standard (GenExpress, Berlin, Germany) containing 1010 HCV cDNA. For evaluation of the accuracy of the HCVquantification assay a panel of HCV genotypes consisting of types 1a, 1b, 2a, 2b, 2c, 2i, 3a, 4, 5a and 6 obtained from the National Reference Center for hepatitis C (Institute of Virology, Essen, Germany) and 76 clinical samples previously determined on the Cobas Amplicor HCV Monitor Test as the reference method were compared. The intra-assay and inter-assay precision were determined by assessing the crossing point values of an HCV sample containing approximately 20,000 IU/ml 10 times in series on the same day and on 10 consecutive days in single runs.

24

Concerning the evaluation of the accuracy of the HCV genotyping assay a panel of HCV genotypes consisting of types 1a, 1b, 2a, 2b, 2c, 2i, 3a, 4, 5a and 6 obtained from the National Reference Center for hepatitis C (Institute of Virology, Essen, Germany) and 30 clinical samples previously determined on the Trugene HCV 5’NC Kit as the reference method were compared. In order to analyze the impacts of sequence variability we produced the quantification standard for each genotype and created the standard curves. Statistics Method comparison was performed using PassingBablok regression analysis. With a p value below 0.05 the correlation coefficient was considered statistically significant showing excellent agreement between the two data sets investigated. Results

Quantification The detection limit of the HCV RT-LC-PCR assay was, in the first step, determined by diluting the HCV reference standard with HCV negative plasma. It was found to be 30 IU/ml (Table 1). As a second step, the dynamic quantification range of the HCV LC-PCR assay was examined by using a log-fold dilution series of a HCV quantification standard (GenExpress, Berlin, Germany) containing 1010 HCV cDNA. The upper quantification limit extends up to 500,000,000 IU/ml. In a third step, the precision of the HCV LC-PCR assay was determined by assessing the intra- and inter-assay coefficients of variation (CV). For the intra-assay, the crossing-point values of the 10 measurements of an HCV sample with approximately 150,000 copies/ml in a single run were used. The CV of intra-assay was 1.2%. For the interassay CV, the crossing-point values for amplification of HCV from the aforementioned sample of 10 different PCR runs performed on 10 different days were used. The CV of interassay was 1.9%. For evaluation of the accuracy of the HCV quantification assay, a panel of HCV genotypes consisting of types 1a, 1b, 2a, 2b, 2c, 2i, 3a, 4, 5a and 6 and 76 clinical samples previously determined on the Cobas Amplicor HCV Monitor Test as the reference method were compared. It could be shown that results generated with our new protocol were in overall good agreement with the Cobas Amplicor. Using Passing Bablock statistics, correlation coefficients were r = 0.992 (Y = 0.972X – 553.4, p < 0.001, n = 76) between our new assay and target values (Fig. 2). Folia Medica 2010; 52(3): 21-30

© 2010 Medical University Plovdiv

Simultaneous Quantification and Genotyping of Hepatitis C Virus Rna by a Two-Step Real-Time Pcr Assay on the Lightcycler Instrument

Table 1. Percentage of HCV genotype 1a RNA positive replicates in dilutions (HCV negative plasma) of the PEI HCVRNA reference standard (#75/98) HCV RNA (IU/ml)

No. of positive tests/ No. of tests

%

120

10/10

100

60

10/10

100

30

10/10

100

15

7/10

70

Figure 2. Comparison between the new method (Y) and Cobas Amplicor (X). Passing-Bablok Regression analysis: Y = 0.972X + 553.4; r = 0.992 (n = 76).

Concerning the differences of PCR efficiencies there was no significant deviation among the various genotypes. Regarding the influences of the variability of target sequences we performed the multiple measurement series with a panel of HCV genotypes consisting of types 1a, 1b, 2a, 2b, 2c, 2i, 3a, 4, 5a and 6. The coefficient of correlation was r = 0.907 (Y = 1.428X – 148.8, p < 0.001, n = 30) between our new assay and target values (Fig. 3). In order to externally verify our new method we

Folia Medica 2010; 52(3): 21-30 © 2010 Medical University Plovdiv

attend twice a year the external quality assessment scheme for virus genome detection of HCV RNA (Instand e.V., Düsseldorf, Germany) and pass, so far each time, the examination. The ICs were amplified in a competitive fashion together with the samples. The optimal concentration of ICs was 200 IU per capillary. In this concentration there was no inhibition of HCV PCR. When the samples contained more than approximately 2,000 HCV RNA IU/ml, the IC was competitively inhibited.

25

Manabu Abe et al

Figure 3. Comparison between the new method (Y) and target values (X). Passing-Bablok Regression analysis: Y = 1.428X – 148.8; r = 0.907 (n = 30). Table 2. Melting temperature (Tm) of 10 genotypes (± 1°C) 1a

1b

2a

2b

2c

2i

3a

4

5

6

65°C

65°C

60°C

60°C

60°C

60°C

50°C

55°C

60°C

65°C

Genotyping By means of the melting curve analysis we could differentiate four genotypes (Table 2) (Fig. 4). The disadvantage of the melting curve method previously published by Bullock et al.13 is the inability to differentiate between genotypes 2 and 5 (the same melting temperature: 60° ± 1°C), between genotypes 1 and 6 (the same melting temperature: 60° ± 1°C) and also between genotypes 3b and 4 (the same melting temperature: 55° ± 1°C). In order to improve the method we investigated the variations in sequences of the primer target region (Fig. 1). According to the sequence analysis we added the two new forward primers, which made it possible to differentiate the genotypes 1, 6 and genotypes 2, 5. The additional HCV type 1b

26

specific forward primer shows a perfect match with the genotypes 1b, 3a and 3b. This primer has five mismatches with genotypes 4 and 6. The second additional HCV type 2 specific forward primer has 100% concordance with genotype 2 but three mismatches with genotype 5. By means of these additional primers the method can separate the rare genotypes (3b, 5 and 6) from the main genotypes (Table 3). For this improved differentiation only one more PCR is required. In order to validate the accuracy of genotyping we used a panel of various HCV genotypes (1a, 1b, 2a, 2b, 2c, 2i, 3a, 4, 5a and 6 and 30 patient samples, which were at first genotyped with the Trugene HCV 5’NC Kit as the reference method. The results were thereafter compared. The conFolia Medica 2010; 52(3): 21-30

© 2010 Medical University Plovdiv

Simultaneous Quantification and Genotyping of Hepatitis C Virus Rna by a Two-Step Real-Time Pcr Assay on the Lightcycler Instrument

Table 3. Percent amplification efficiency of HCV genotypes Genotype

Slope

Interception

Efficiency

Cp of 105 IU/ml (mean)

1

- 3.493

39.65

93.3%

20.1 (CV: 2.1)

2

- 3.459

40.22

94.6%

20.9 (CV: 2.3)

3a

- 3.610

41.37

89.2%

21.7 (CV: 2.4)

4

- 3.543

40.67

91.5%

21.0 (CV: 3.1)

5

- 3.576

40.57

90.4%

21.6 (CV: 1.6)

6

- 3.519

39.86

92.4%

20.5 (CV: 2.1)

The percent amplification efficiency was calculated as [10(-1/slope) -1] X 100. Cp = crossing point = threshold cycle (CT): The parameter Cp is defined as the fractional cycle number at which the fluorescence passes the fixed threshold (baseline). A plot of the log of initial target copy number for a set of standards versus Cp is a straight line. Quantification of the amount of target in unknown samples is accomplished by measuring Cp and using the standard curve to determine starting copy number. CV: coefficient of variation.

cordance between two methods on the level of genotypes was 100% (data not shown). In order to externally verify our new genotyping method we attend since 2007 once a year the external quality assessment scheme for virus genotyping of HCV RNA (Instand e.V., Düsseldorf, Germany) and pass always the examination.

quences is the most important factor regarding the PCR efficiency. When there are 1-3 mismatches in the primer targeting sequences the PCR efficiency reduces correspondingly. We utilise this fact for further differentiation of genotypes 1, 6 and 2, 5, respectively by means of additional forward primers (Fig. 1).

Table 4. Scheme of interpretations primer: NC1 + SR2

forward primer: HCV type 1b specific

forward primer: HCV type 2 specific

1. PCR (melting temp.)

2. PCR (melting temp.)

2. PCR (melting temp.)

1

positive (65°C)

positive (65°C)

positive (65°C)

2

positive (60°C)

no amplification

positive (60°C)

3a

positive (51°C)

positive (51°C)

positive (51°C)

3b

positive (55°C)

positive (55°C)

positive (55°C)

4

positive (55°C)

no amplification

no amplification

5

positive (60°C)

no amplification

no amplification

6

positive (65°C)

no amplification

no amplification

Genotype

Discussion

Quantification The highly conserved 5’ UTR of HCV is the most suitable target sequence for the quantification of HCV RNA. Concerning the PCR efficiency among the various genotypes (1-3 mismatches in the region for the detection probes according the genotype) there was no significant deviation. We have similar observations with a real-time PCR quantification of all subtypes of the human immunodeficiency virus type 1 using locked nucleic acid-based probes.21 We assume that the perfect match of primer se-

Folia Medica 2010; 52(3): 21-30 © 2010 Medical University Plovdiv

Genotyping Worldwide, 6 major genotypes, and more than one hundred different subtypes of the virus have been identified by nucleotide sequencing.1,2 The genotypic diversity of HCV, due to the mutation rate of the virus, interferes with effective humoral immunity against it. Although neutralizing antibodies to HCV have been detected in the serum of infected patients, these are, at best, short-lived. Moreover, HCV infection has not been shown to induce lasting immunity against re-infection with different virus isolates, or even the same isolate. Thus, neither homologous

27

Manabu Abe et al

Figure 4. Melting curve analysis of a panel of HCV genotypes with 10 reference samples.

nor heterologous immunity appears to develop after acute HCV infection.4 Some HCV genotypes are distributed worldwide, while others are more geographically confined. Genotypes 1a, 1b, 2a, 2b, 2c, and 3a account for more than 90% of the HCV infections in North and South America, Europe, Russia, China, Japan, Australia, and New Zealand. According to data obtained from the Clinic of Gastroenterology, Sofia University Hospital [personal communication Dr. A. Ivanova] genotype 1 dominates in Bulgaria (84.7%) followed by genotype 3a (12.5%). Genotype 3a is more common among younger populations. Other subtypes of genotype 3 are highly prevalent in Nepal, Bangladesh (3b), India, and Pakistan. Most infections in Egypt are genotype 4a, and this and other subtypes of genotype 4 are found in Central Africa. Genotype 5a accounts for about 50% of infections in South Africa. Genotypes 4 and 5 are found only sporadically outside Africa. Genotype 6 isolates are primarily found in Southeast Asia.3,4 It should be noted that the genotype distribution could vary significantly among different population groups in the same geographical area. Associations between genotype and mode of transmission also exist. Genotype 3, for instance, is more prevalent among intravenous drug users.3 The HCV genotype may be an important factor influencing the severity of liver disease. Infection with genotype 1 has been associated with more

28

advanced liver disease and the development of both liver cirrhosis as well as hepatocellular carcinoma.4 Observations have also shown that patients with genotype 1 typically respond weakly to interferon therapy. However, while differences in pathogenicity and responsiveness to antiretroviral therapy have been reported among the genotypes, the biological impact of these differences still remains incompletely defined. Nevertheless, the current limited knowledge base does not undermine the fact that HCV genotyping is an important factor to consider in the management of treatment for HCV infection.3,5 The highly conserved 5’ UTR of HCV can be routinely used to identify HCV RNA in sera and to determine HCV genotypes. Some commercial kits are based on the analysis of the sequence variability of the 5’ UTR of HCV for genotyping (InnoLiPA and TRUGENE). The current limitations of using the 5’ UTR for HCV genotyping are related to the low discriminating power of the 5’ UTR sequence for determination of the particular subtype.22,23 The high level of conservation found in this region does not allow accurate distinction of the HCV genotypes at the subtype level (e.g. subtypes 1a, 1b, 1c). However, these inherent disadvantages of using the 5’ UTR for HCV genotyping are not crucial for actual clinical use. As practice shows, any HCV genotyping system based on the 5’ UTR sequences

Folia Medica 2010; 52(3): 21-30

© 2010 Medical University Plovdiv

Simultaneous Quantification and Genotyping of Hepatitis C Virus Rna by a Two-Step Real-Time Pcr Assay on the Lightcycler Instrument

(including our method) is reliable at the genotype level (e.g., genotypes 1, 2, 3, 4, 5 or 6). The correct differentiation of genotypes is normally sufficient for clinical purpose. At the same time, it should be mentioned that HCV typing systems based on NS5’ or core sequence regions are characterized by sensitivities less than that of the 5’ UTR-based methods.11 ConclusionS

The presented new HCV PCR method proved to be reliable, reproducible and accurate. It combines the adequate lower detection limit of 30 IU/ml plasma with an extended upper detection limit of 500,000,000 IU/ml plasma including the genotyping of HCV within 3 hours and is suitable for both disease detection, HCV therapy monitoring and genotyping. Furthermore our new method reduces the reagent costs immensely because of the simultaneous quantification and genotyping in the single run. References

1. Simmonds P. Genetic diversity and evolution of hepatitis C virus - 15 years on. J Gen Virol 2004; 85:3173‑88. 2. Simmonds P, Bukh J, Combet C, et al. Consensus proposals for a unified system of nomenclature of hepatitis C virus genotypes. Hepatology 2005;42(4):962-73. 3. Zein NN. Clinical significance of hepatitis C virus genotypes. Clin Microbiol Rev 2000;13:223-35. 4. Zeuzem S. Heterogeneous virologic response rates to interferon-based therapy in patients with chronic hepatitis C: Who responds less well? Ann Intern Med 2004;370-81. 5. Chung RT, Andersen J, Volberding P, et al. Alfa2a plus ribavirin versus interferon alpha-2a plus ribavirin for chronic hepatitis C in HIV-coinfected persons. N Engl J Med 2004;351:451-9. 6. Germer JJ, Rys PN, Thorvilson JN, Persing DH. Determination of hepatitis C virus genotype by direct sequence analysis of products generated with the Amplicor HCV test. Clin Microbiol 1999; 37(8):2625-30. 7. Germer JJ, Majewski DW, Rosser M, et al. Evaluation of the TRUGENE HCV 5′NC genotyping kit with the new GeneLibrarian module 3.1.2 for genotyping of hepatitis C virus from clinical specimen. Clin Microbiol 2003;41(10):4855-7. 8. Ross RS, Viazov SO, Holtzer CD, et al. Genotyping of hepatitis C virus isolates using CLIP Sequencing. Clin Microbiol 2000;38(10):3581-4. 9. White PA, Zhai X, Carter I, Zhao Y, Rawlinson Folia Medica 2010; 52(3): 21-30 © 2010 Medical University Plovdiv

WD. Simplified hepatitis C virus genotyping by heteroduplex mobility analysis. Clin Microbiol 2000;38:477-82. 10. Margraf RL, Erali M, Liew M, Wittwer CT. Genotyping hepatitis C virus by heteroduplex mobility analysis using temperature gradient capillary electrophoresis. Clin Microbiol 2004;42(10):4545-51. 11. Ilina EN, Malakhova MV, Generozov EV, Nikolaev EN, Govorun VM. Matrix-assisted laser desorption ionization-time of flight (Mass Spectrometry) for hepatitis C virus genotyping. Clin Microbiol 2005;43(6):2810-5. 12. Liew M, Erali M, Page S, Hillyard D, Wittwer C. Hepatitis C genotyping by denaturing high-performance liquid chromatography. Clin Microbiol 2004;42(1):158-63. 13. Bullock GC, Bruns DE, Haverstick DM. Hepatitis C genotype determination by melting curve analysis with a single set of fluorescence resonance energy transfer probes. Clin Chem 2002;48:2147-54. 14. Haverstick DM, Bullock GC, Bruns DE. Genotyping of hepatitis C virus by melting curve analysis: Analytical characteristics and performance. Clin Chem 2004;12:2405-7. 15. Ratge D, Scheiblhuber B, Nitsche M, Knabbe C. High-speed detection of blood-borne Hepatitis C virus RNA by single-tube real-time fluorescence reverse transcription-PCR with the LightCycler. Clin Chem 2000;46:1987-9. 16. Ratge D, Schreiblhuber B, Landt O, Berg J, Knabbe C. Two-round rapid-cycle RT-PCR in single closed capillaries increases the sensitivity of HCV RNA detection and avoids amplicon carry-over. Clin Virology 2002;24:161-72. 17. H ofgaertner WT, Kant JA, Weck KE. Hepatitis C virus quantisation: optimization of strategies for detecting low-level viremia. Clin Microbiol 2000;38:888-91. 18. M ukaide M, Tanaka Y, Kakuda H, et al. New combination test for hepatitis C virus genotype and viral load determination using Amplocor GT HCV Monitor test v2.0. World J Gastroenterol 2005;11(4):469‑75. 19. Schröter M, Zöllner B, Schäfer P, Laufs R, Feucht HH. Quantitative detection of hepatitis C virus RNA by LightCycler PCR and comparison with two different PCR assays. Clin Microbiol 2001;39:765-8. 20. Leb V, Stöcher M, Valentine-Thon E, et al. Fully automated, internally controlled quantification of Hepatitis B virus DNA by real-time PCR by use of the MagNA Pure LC and LightCycler Instruments. Clin Microbiol 2004;42:585-90. 21. Abe M, Klett C, Wieland E. Gille S, Landt O. Twostep real-time PCR quantification of all subtypes of human immunodeficiency virus type 1 by an

29

Manabu Abe et al

in-house method using locked nucleic acid-based probes. Folia Medica 2008;50(3):5-13. 22. Chen Z, Weck KE. Hepatitis C virus genotyping: interrogation of the 5′ untranslated region cannot accurately distinguish genotypes 1a and 1b. Clin

Одновременноe количественное измерение и генотипизирование рибонуклеиновой кислоты вируса гепатита С посредством применения двушагового PCR теста в реальное время на аппарате Light Cycler М. Абе, К. Клет, Е. Виланд, С. Гиле, О. Ланд

Резюме Введение: Вирусное обременение и вирусный генотип представляют собой самые важные предикторы для исхода антивирусной терапии при хроническом гепатите С. В нормальных условиях их обнаруживают с помощью двух отдельных тестов. Материал и методы: В целях улучшения диагностической стратегии и уменьшения стоимости лабораторных реагентов авторы разработали метод одновременного количественного измерения и генотипизирования РНК гепатитного вируса С

30

Microbiol 2002;40(9):3127-34 23. Nolte FS, Green AM, Fiebelkorn KR, et al. Clinical evaluation of two methods for genotyping hepatitis C virus based on analysis of the 5′ noncoding region. Clin Microbiol 2003;41(4):1558-64.

посредством применения двушагового PCR теста в реальное время на аппарате Light Cycler (Roche Diagnostics). Результаты: Для калибрирования количественного теста применен стандарт ВОЗ 96/790. Верхняя граница теста 30 IU/ml, его динамический охват – до 500,000,000 IU/ml. Внутри- и межтестовые ошибки соответственно 1.2% и 1.9% (n = 10). Стоимости РНК вируса гепатита С, полученные PCR тестом в реальное время сильно коррелируют со стоимостями, полученными тестом Cobas Amplicor HCV Monitor (r = 0.992; p < 0.001). Генетическа типизация проведена с помощью анализа температуры плавления. Согласованность между новым авторским методом генного типизирования и методом Trugene HCV 5’NC - 100%. Новый метод проводится в течение всего трех часов. Он оказывается очень подходящим для генного типизирования, обнаруживания и терапевтического мониторирование вируса гепатита С.

Folia Medica 2010; 52(3): 21-30

© 2010 Medical University Plovdiv

Folia Medica 2010; 52(3): 31-36 Copyright © 2010 Medical University Plovdiv doi: 10.2478/v10153-010-0004-3

Original Articles

Experimental Investigations Effect of elevated intra-abdominal pressure on the contractile activity and reactivity of smooth muscle tissue from rat gastrointestinal tract to galantamine and drotaverine (No-spa) George P. Deenichin, Atanas D. Kristev1, Vesselin V. Mollov, Valentin I. Turiiski1

Department of Special Surgery, 1Department of Medical Physics and Biophysics, Medical University, Plovdiv, Bulgaria Abstract Aim: The aim of the present study was to determine the nature and intensity of changes in the contractile activity and reactivity of gastrointestinal smooth muscle tissue in conditions of increased intra-abdominal pressure. Methods: A method for recording isometric contractions of isolated smooth muscle preparations from gastric corpus, duodenum and sigmoid colon of rats was used. Results: Two groups of rats were used in the study – control animals and animals with elevated abdominal pressure. It was established that pressure of 25 mmHg for 60 min did not cause statistically significant change in the tone and parameters of the spontaneous contractions in all preparation types, as well as in their reactivity to drotaverine (no-spa). Statistically significant increase in the strength of the tonic effects of galantamine (1.10-6 – 1.10-3 mg/ml) was found in all types of smooth muscles preparations isolated from rats with increased abdominal pressure compared with preparations from the control rats. Conclusions: The statistically significant increase in the galantamine-induced effects on smooth muscle preparations is associated with increase in the contractile effectiveness of acetylcholine. M-type cholinergic receptors are predominantly involved in the processes, probably sensibilized from processes activated by the increased intra-abdominal pressure. Key words: abdominal compartment syndrome, increased intra-abdominal pressure, smooth muscles, gastrointestinal tract, cholinergic receptors

Introduction

Compartment syndrome is a condition in which increased pressure within fixed anatomical space impairs the functions of the tissues in this space. Abdominal compartment syndrome is defined as a consequence of intra-abdominal hypertension (IAH) higher than 20 mmHg with clinical evidence of multiorgan failure.1 Intestines are considered as the most susceptible to elevated intra-abdominal pressure.2 Their barrier function is impaired, which causes bacterial translocation from the intestinal lumen to the mesenterial lymph nodes, liver and spleen.3 The edema developing in the intestinal wall and the pathomorphological changes affect the intestinal motor function.5 Gastric and intestinal

motor function is decreased6 and gastrointestinal tract (GI) evacuation is slowed down5, which is associated with higher expression of iNOS7. There is evidence that intra-abdominal hypertension exerts negative effect on the function of the intestinal cells of Cajal, reducing the spontaneous activity of the intestinal musculature.8 Rat models display decreased reactivity of isolated SM from ileum after field electrostimulation, as well as after treatment with acetylcholine.9 Elevated intra-abdominal pressure and abdominal compartment syndrome doubtlessly impair the motility and evacuation function of GI tract. At the same time, there are few studies revealing mechanisms associated with that gastrointestinal

Correspondence and reprint request to: A. Kristev, Department of Medical Physics and Biophysics, Medical University, Plovdiv; E-mail: [email protected] 15A Vassil Aprilov St, 4002 Plovdiv, Bulgaria Received 19 February 2010; Accepted for publication 31 March 2010

31

G. Deenichin et al

dysfunction and its relation with following increase in the intra-abdominal volume and IAH (circulus vitiosus). It is in this respect that the aim of the present study, i.e. to reveal the nature and intensity of the changes in the contractile activity and reactivity of gastrointestinal SM in conditions of increased intra-abdominal pressure, as well as some of the causative mechanisms, makes it up-to-date. Material and methods

Model of abdominal compartment syndrome Adult male rats Wistar (n = 10; 280 – 310 g) were used in the model. A device containing insufflator of atmospheric air and manometer for pressure reading was used to produce the necessary intra-abdominal pressure. The device allowed quick increase in the intra-abdominal pressure without previous laparotomy of the animals, achievement of the required pressure of 25 mmHg, its maintenance for 60 min and easy decompression at the apposite time. The rats were anesthetized with 0.5 ml thiopental applied intraperitoneally and fixed in a supine position. During the experiment the body temperature of the rats was maintained at 36-37°C with a heat lamp. The control group comprised 7 animals weighing from 290 to 310 g, which was not statistically different from the weight of the experimental animals. The rats underwent the above-described procedures except increasing intra-abdominal pressure. All experimental animals were maintained under standard laboratory conditions: temperature 21-23°C, humidity 55 ± 10%, granulated standard food, water available ad libitum and 12/12-hour light-dark cycle. The experiment was made in agreement with the Helsinki Declaration principles in the care and human attitude to experimental animals. The requirements of Order No 25/10. 06. 2003 of the Ministry of Health from 01.01.2004, published in the State Newspaper, issue 59/01.07.2003, amendment in the State newspaper, issue 73/19.08.2003 were followed strictly. Registration

of contractile activity of isolated

smooth muscles

The experiments were performed on smooth muscle (SM) preparations from gastric corpus, duodenum and sigmoid colon dissected from the two groups of rats. The animals were killed previously by decapitation under slight ether anesthesia. Circular (gastric corpus) and longitudinal (sigmoid colon & duodenum) SM preparations 12–13 mm

32

long и 1.0–1.1 mm wide were used. These were fixed stationary – at one end to a glass holder placed in a bath with tempered Krebs solution (37oC) and to a tension transducer Swema (Sweden) at the other. The initial mechanical tension of the SM preparations was 10 mN. The Krebs solution (pH = 7.4) was aerated with a gaseous mixture of 5% СО 2 and 95% О 2. SM tissues were treated with different substances applied by adding aliquots of their concentrated solutions in volume not exceeding 0.5% of the total Krebs solution volume in the tissue bath. Drug-induced effect were assessed according to the basal tone levels and data of spontaneous contractile activity registered after prior 60-minute adaptation of the preparations. During that period the Krebs solution was repeatedly refreshed and the contractile activity of the preparations was tested twice with 1.10-6 mol/l acetylcholine. The change of the spontaneous activity of the SM preparations and their reactivity in treatment with galantamine and drotaverine was studied. The drug choice was determined by their wide use in the clinical practice. The galantamine and drotaverine concentrations used to treat the SM preparations in the tissue bath were comparable with the doses used in the clinical practice. The mechanical activity was registered with Microtechna amplifier (Czech Republic) and recorded with Linsseis recorder (Germany). The solution pH was measured with microcomputer pH-meter 6201 (Jenco Electronics, UK). Drugs, chemicals, solutions

The medical preparations used in the experiments were galantamine (Nivalin) from Sopharma, drotaverine (No-spa) from Chinoin and acetylcholine from Dispersa Baeschlin (Germany). pH of the Krebs solution was 7.40. The solution components included (in mM): NaCl – 120, KCl – 5.9, CaCl2 – 2.5, MgCl2 – 1.2, NaH2PO4 – 1.2, NaHCO3 – 15.4, and glucose – 11.5. The preparation solution contained NaCl : KCl : CaCl2 in a ratio of 27.2 : 1.1 : 1.0.10 All substances used in the preparation of the solutions were products of Merck (Darmstadt, Germany). Statistical analysis Each value was presented as mean ± SЕ. The effects of equimolar drug concentrations on SM preparations from rats with increased intra-abdominal pressure and from control animals were compared for any characteristic of the contractile activity (tone, fre-

Folia Medica 2010; 52(3): 31-36

© 2010 Medical University Plovdiv

Effect of Elevated Intra-abdominal Pressure on the Contractile Activity and Reactivity of Smooth Muscle Tissue from Rat Gastrointestinal Tract to Galantamine and Drotaverine (No-Spa)

quency and amplitude) and for any type of SM tissue alone. The SM preparation tone was measured in mN. The strength of phase contractions (mN) was determined as mean value of at least 8 consecutive spontaneous contractions before and 10 min after treatment with corresponding drug concentration. The frequency (min-1) was determined for a period of 5 min immediately before and 10 min after the drug treatment. The data were analysed with variance analysis (Student’s t-test) using INSTAT software. Differences were accepted as statistically significant at P < 0.05.

galantamine-induced contractions than those of the control preparations (Table 1). 3. Effects of galantamine on the parameters of the spontaneous contractile activity of SM preparations

Increased intra-abdominal pressure to 25 mmHg for 60 minutes does not change statistically significantly (Р > 0.05) the character and strength of the spontaneous phasic contractions of SM preparations from the stomach, duodenum and sigmoid colon of rat.

3.1. On the amplitude of the phasic contractions It is only in a concentration of 1.10-3 mg/ml that galantamine causes statistically significant increase in the strength of the spontaneous contractions of SM preparations from gastric corpus and sigmoid colon. The drug has no effect on the amplitude of the phasic contractions of duodenum. The differences between the effects of equimolar concentrations of galantamine on each type of the SM preparations isolated from IAH and control rats are short of statistical significance. 3.2. On the frequency of phasic contractions Within the examined concentration range galantamine does not influence the spontaneous contractions frequency of SM preparations from different regions of the GI tract of the control and IAH rats.

2. Effects

4. Effects

Results

1. Influence

of the intra-abdominal hypertension

on the contractile activity of isolated

SM

prepa-

rations

of galantamine on the tone of

SM

preparations

Galantamine influences the tone of SM preparations isolated from the GI tracts of both group animals. The effects are contractile in character and show increasing concentration-effect relationship in the concentration zone 1.10-6 – 1.10 -3 mg/ml. The strength of the reactions is different for the different types SM preparations. At most of the concentrations used the preparations from IAH rats show greater strength of

of drotaverine on the tone of

SM

preparations

Drotaverine relaxes SM tissue from the GI tract. The relaxation power is concentration dependent (Table 2). It is greatest in preparations from the stomach and decreases in those from duodenum and sigmoid colon. The differences in the effects of equimolar concentrations from the drug preparations obtained between each of the SM preparations from the control and IAH rats do not reach statistical significance (P > 0.05).

Table 1. Comparison of the strength of tonic contractions (mN) triggered by equimolar concentrations of galantamine on same type SM preparations from control and IAH rats; Smooth muscle preparations Galantamine mg/ml

Control rats

Intra-abdominal hypertension rats Gastric Sigmoid Duodenum corpus colon n = 10 n = 10 n = 10

Gastric corpus n=7

Duodenum n=7

Sigmoid colon n=7

1.10-6

0.14 ± 0.25

0

0

0.25 ± 0.44

0.12 ± 0.46

0.20 ± 0.23

1.10-5

0

0

0

0

0.51 ± 0.63* t = -2.10

0.71 ± 0.36* t = -3.3

1.10-4

1.04 ± 0.50

0

0

1.14 ± 0.42

0.83 ± 0.31* t = -7.03

1.80 ± 1.13* t = -4.05

1.10-3

3.16 ± 0.41

1.89 ± 0.27

3.77 ± 1.10

6.55 ± 1.00* t = -9.11

2.94 ± 0.52* t = -4.92

6.75 ± 2.02* t = -4.31

* P < 0.05. Folia Medica 2010; 52(3): 31-36 © 2010 Medical University Plovdiv

33

G. Deenichin et al

Table 2. Comparison of the strength of reactions (mN) triggered by equimolar concentrations of drotaverine on same type SM preparations from GI tract from control and IAH rats Smooth muscle preparations Drotaverine mg/ml

Control rats

Intra-abdominal hypertension rats Sigmoid Gastric Duodenum corpus colon n = 10 n = 10 n = 10 0.11 ± 0.25 0.11 ± 0.52 -0.22 ± 0.51

Gastric corpus n=7

Duodenum n=7

1.10-6

-0.30 ± 0.06

0

Sigmoid colon n=7 0

1.10-5

-0.34 ± 0.31

0

0

-0.30 ± 0.35

-0.13 ± 0.41

-0.44 ± 1.12

1.10-4

-0.62 ± 0.44

-0.30 ± 0.38

-0.16 ± 0.21

-0.52 ± 0.69

-0.44 ± 0.50

-0.43 ± 1.31

1.10-3

-1.48 ± 0.33

-0.72 ± 0.35

-0.64 ± 0.55

-1.07 ± 0.53

-0.66 ± 0.52

-0.63 ± 1.80

„-“ indicates relaxation; * P < 0.05.

5. Effects

of drotaverine on the parameters of the

SM

spontaneous contractile activity of

prepara-

treatment with 1.10-4 and 1.10-3 mg/ml concentrations of drotaverine is observed.

tions

5.1. On the amplitude of the phasic contractions Drotaverine reduces the spontaneous phasic contractions of SM preparations from all regions of the GI tract (Table 3). No statistically significant differences (P > 0.05) were found in the reaction power between corresponding SM tissues from the control and IAH rats treated with equimolar concentrations of drotaverine.

Discussion

Our research suggests that increase in the intraabdominal pressure within the bounds defined in the experiment (25 mmHg, 60 min) does not cause statistically significant change in the character and power of the spontaneous phasic contractions of SM preparations from stomach, duodenum and sigmoid colon.

Table 3. Comparison of the effects of equimolar concentrations of drotaverine on the strength of spontaneous phasic contractions (mN) of the different types of SM preparations from control and IAH rats Smooth muscle preparations

1.85 ± 0.87

Sigmoid colon n=7 3.19 ± 0.81

Intra-abdominal hypertension rats Sigmoid Gastric Duodenum corpus colon n = 10 n = 10 n = 10 0.66 ± 0.31 1.27 ± 0.25 4.18 ± 1.23

0.76 ± 0.27

1.76 ± 0.87

3.09 ± 0.87

0.56 ± 0.23

1.17 ± 0.25

4.13 ± 1.05

1.10-5

0.71 ± 0.20

1.78 ± 1.02

3.02 ± 0.61

0.63 ± 0.37

1.40 ± 0.35

3.41 ± 0.94

1.10-4

0.67 ± 0.30

0.89 ± 0.62

2.83 ± 0.81

0.57 ± 0.23

1.23 ± 0.25

3.52 ± 1.34

1.10-3

0.14 ± 0.05

0

2.34 ± 1.28

0.33 ± 0.16

0

1.39 ± 0.9

Drotaverine mg/ml

Control rats Gastric corpus n=7

Duodenum n=7

Autocontrols

0.83 ± 0.24

1.10-6

* P < 0.05.

5.2. On the frequency of phasic contractions The drug substance does not influence the frequency of spontaneous contractions of SM preparations from stomach, duodenum and sigmoid colon in the control rats and from stomach and duodenum in the IAH rats. In single preparations4 from sigmoid colon of IAH rats a tendency to increasing frequency of spontaneous contractile activity after

34

There are however changes in the reactivity of the examined SM preparations when treated with galantamine at concentrations ranging from 1.10 -6 mol/l to 1.10 -3 mol/l. Galantamine suppresses acetylcholinesterase activity in SM tissues and the enzyme inhibition is proportional to the increase in the drug concentration.11,12 The elevated concentration of endogenous acetylcholine, as a response to that process, activates the cholinergic Folia Medica 2010; 52(3): 31-36

© 2010 Medical University Plovdiv

Effect of Elevated Intra-abdominal Pressure on the Contractile Activity and Reactivity of Smooth Muscle Tissue from Rat Gastrointestinal Tract to Galantamine and Drotaverine (No-Spa)

receptors, causes elevation of the cytosol level of Са2+ in the SM cells and in turn enhances the contractile activity.13 SM tissues from stomach, duodenum and sigmoid colon react with tonic contractions in both animal groups (control and IAH rats). The contractions are concentration-dependant and are most expressed in the stomach preparations. Although similar in character they differ in strength. Equimolar concentrations of galantamine cause contractions that are statistically significantly more expressed in SM preparations from IAH rats than in SM preparations from control animals. A number of studies show that galantamineinduced tonic effects on the GI tract are associated with influence of the accumulated acetylcholine mainly on the muscarine cholinergic receptors (mAChR) of the SM cells that are effectively blocked by atropine.14 Given this, the difference in the strength of reactions can be accounted for by an IAH-induced change in the behaviour of these receptors. The unidirectional character of changes in all examined SM tissue types indicates most probably an IAH-provoked process causing, in broad terms, sensibilisation of mAChR. The differences in the strength of the effects of galantamine on the amplitude of the phasic contractions of different SM tissue types are result from the effect of the endogenous acetylcholine mainly on the nicotine cholinergic receptors (nAChR) located on the pre- and postganglionic synapses of the neurons in the intramural neuronal structures.15 Their activation stimulates release of neurotransmitters like glutamine, serotonin, dopamine, GAPA and/or VIP etc. with SM effects diverse in character.16 Some of these are proven activators while others are inhibitors of the contractile activity. The absence of statistically significant differences in the galantamine-induced effects on the strength and frequency of the spontaneous contractions of similar SM preparations between the two groups of rats indicates, in broad terms, no effect of IAH on nAChR. Drotaverine inhibits the contractile activity of gastric and intestinal SM preparations by increasing the cytosol concentrations of cAMP and/or cGMP.17 The absence of statistically significant differences in the reactions of same type SM preparations between control and IAH rats shows that within the parameters of the present study the increased intrabdominal pressure does not influence the activity of phosphodiesterase system, which is the target of drotaverine action.18 Folia Medica 2010; 52(3): 31-36 © 2010 Medical University Plovdiv

Conclusions

Intra-abdominal hypertension (25 mmHg, 60 min) does not cause statistically significant changes in the strength and character of spontaneous contractility of SM tissue from stomach, duodenum and sigmoid colon of rats. Intra-abdominal hypertension causes statistically significant increase in the strength of galantamineinduced tonic contractions of the three SM tissue types (gastric corpus, duodenum and sigmoid colon) compared with the same type SM tissues of control animals. The latter is a reason to suggest an increase in the contractile effect of acetylcholine, which is a basic excitatory neurotransmitter in the GI tract. References

1. Sugrue M, Buhkari Y. Intra-abdominal pressure and abdominal compartment syndrome in acute general surgery. World J Surg 2009;33(6):1123-7. 2. Sukhotnik I, Mogilner J, Hayari L, et al. Effect of elevated intra-abdominal pressure and 100% oxygen in superior mesenteric artery blood flow and enterocyte turnover in a rat. Pediatric Surg International 2009;24(12):1347-53. 3. Cheatham ML. Nonoperative management of intraabdominal hypertension and abdominal compartment syndrome. World J Surg 2009;33(6):1116-22. 4. Timoney MF, Zenilman ME. How we manage abdominal compartment syndrome. Contemporary Surgery 2008;64(10):165-70.  5. Madl C, Druml W. Systemic consequences of ileus. Best Practice & Research Clinical Gastroenterology 2009;17(3):445-56. 6. Moore-Olufemi SD, Xue H, Bashir AO, et al. Resuscitation-induced gut edema and intestinal dysfunction. The Journal of Trauma: Injury, Infection and Critical Care 2005; 58(2): 264-70. 7. Moore-Olufemi SD, Xue H, Allen SJ, et al. Inhibition of intestinal transit by resuscitation-induced gut edema is reversed by L-NIL. J Surg Res 2005; 129(1):1-5. 8. Hua Z, Wei C, Wai K, et al. Effects of abdominal infection and intra-abdominal hypertension on intestinal interstitial cells of Cajal. Chinese J Gastrointest Surgery 2008;11(3):256-62. 9. Unsal MA, Imamoglu M, Kadioglu M, et al. The acute alterations in biochemistry, morphology, and contractility of rat-isolated terminal ileum via increased intra-abdominal pressure. Pharmacol Res 2006;53(2):135-41. 10. Blattner R, Classen HG, Dehnert H, Doring HG. Experiments on isolated smooth muscle prepara-

35

G. Deenichin et al

tions. (Eds Barnden IM & Colson R), Hugo Sachs Elektronik KG. Freiburg. 1980, p. 75. 11. Summers W, Kaufman K, Altman F, Fischer J. THA - a review of the literature and its use in treatment of five overdose patients. Clinical Tox 1980;16(3):269-81. 12. Zarotsky V, Sramek JJ, Cutler NR. Galantamine hydrobromide: an agent for Alzheimer’s disease. Am J Health-Syst Pharm 2001;60(5):446-52. 13. James AN, Ryan JP, Parkman HP. Effects of clonidine and tricyclic antidepressants on gastric smooth muscle contractility. Neurogastroenterol Motil

2004;16(2):143-53. 14. Turiiski V, Krustev A, Sirakov V, Getova D. In vivo and in vitro study of the influence of the anticholinesterase drug galantamine on motor and evacuative functions of rat gastrointestinal tract. Eur J Pharmacol 2004;498:233-9.

Вл и я ние п о в ы ш енно го интра а бдо м ина л ь но го да вл ени я на сократительную активность и реактивность гладкой мускулатуры гастроинтестинального тракта крысы к Galantamine Drotaverine (No-Spa) Г. Дееничин, А. Крыстев, В. Молов, В. Турийски

Резюме Ц ель : Установить характер и силу изменений, вызванных повышением интраабдоминального давления на сократительную активность гастроинтестинальной мускулатуры. М етоды : Использован метод регистрации изометрических сокращений изолированных гладкомышечных препаратов, взятых от gastric corpus, duodenum и sigmoid colon крысы.

36

15. Mutafova-Yambolieva V, Yamboliev I, Mihailova D. Comparative effects of the anticholinesterase drug galantamine on the mechanical activity of isolated rat jejunum and ileum. Gen Pharmacol 1993;24(5):1253-6. 16. McConalogue K, Furness JB. Gastrointestinal neurotransmitters. Bailliere’s Clin Endocrinol Metab 1994;8(1);51-76. 17. Shimizu K, Ichikawa T, Urakawa N, Nakajyo S. Inhibitory mechanisms of papaverine on the smooth muscle of guinea pig urinary bladder. Jpn J Pharmacol 2000;83(2):143-9. 18. Kaneda T, Shimizu K, Nakajyo S, Urakawa N. The difference in inhibitory mechanisms of papaverine on vascular and intestinal smooth muscles. Eur J Pharmacol 1998;355(2–3):149–57.

Результаты: Обследовано две группы крыс (контрольная и с повышенным интраабдоминальным давлением). Установлено, что давление 25 mm Hg в течение 60 min значимо не изменяет тонус и параметры спонтанных сокращений всех видов препаратов, как и их реактивность по отношению к drotaverine (no-spa). Наблюдается достоверное нарастание силы тонических эффектов galantamine (1.10-6 - 1.10-3 mg/ml) при различных типах гладкомышечных препаратов, изолированных от крыс с повышенным интраабдоминальным давлением, по отношению к силе контрольных препаратов. Заключение: Достоверно увеличенная сила galantamine-индуцированных эффектов на гладкомышечные препараты связана с нарастанием контрактильной эффективности acetylcholine. В процессы включены в основном М-типа холинергические рецепторы, по всей вероятности сенсибилизированные процессами, спровоцированными повышенным интраабдоминальным давлением.

Folia Medica 2010; 52(3): 31-36

© 2010 Medical University Plovdiv

Folia Medica 2010; 52(3): 37-45 Copyright © 2010 Medical University Plovdiv doi: 10.2478/v10153-010-0005-2

Binding affinity of triphenyl acrylonitriles to estrogen receptors: quantitative structure-activity relationships Sorana D. Bolboacă1, Monica M. Marta1, Lorentz Jäntschi1,2

1”Iuliu

Haţieganu” University of Medicine and Pharmacy Cluj-Napoca, Romania; 2Technical University of Cluj-Napoca, Romania Abstract Aim: The quantitative structure-activity relationship approach was applied to understand the relative binding affinity of triphenyl acrylonitriles to estrogen receptors. Material and methods: A sample of previously studied triphenyl acrylonitriles was divided into training (18 compounds) and test sets (7 compounds) using a stratified random approach. The molecular descriptor family on vertices cutting (MDFV) approach was used in order to translate the structural information into descriptors. The relationship between binding activity and structural descriptors was identified using the multiple linear regression procedure. Results: An optimal three-parameter equation with a determination coefficient of 0.9580 and a cross-validation leave-one-out parameter of 0.9408 was identified. The optimal model was assessed on a test set and a determination coefficient of 0.9004 was obtained. The MDFV model proved not to be significantly different from the previously reported model in terms of goodness-of-fit. In terms of information criteria (Akaike’s, Bayesian, Amemiya, and Hannan-Quinn) and Kubinyi function, the MDFV model proved to perform better than the previously reported model. Conclusion: The optimal MDFV model was able to explain ~96% of the total variance in the estrogenic binding relative affinity of triphenyl acrylonitriles and to have estimation and prediction abilities. Although there were no significant differences in terms of goodness-of-fit, the MDFV model proved to exhibit better information parameters compared to the previously reported model using the same number of molecular descriptors. Keywords: estrogen receptors, relative binding affinity (RBA), triphenyl acrylonitriles (TPT); structure-activity relationship (SAR), molecular descriptors family on vertices (MDFV) Introduction

The interaction of estrogens with tissues is accomplished through estrogen receptors (ER), a group of receptors activated by the hormone 17β-estradiol.1 Two receptors are known to date: ERα (expressed mainly in uterus, stroma of prostate, theca cells of ovary, Leydig cells of testes, epididymus, bone, breast, brain, liver and white adipose2) and ERβ (expressed mainly in colon, epithelium of prostate, testis, granulose cells of ovary, bone marrow, salivary gland, vascular endothelium, brain3,4). A series of hydroxylated and non-hydroxylated triphenyl acrylonitriles (TPEs) proved to have estrogenic activities.5-8 The structure-activity approach has been applied to link topological features of estrogenic drugs and pharmacophoric properties9,10, structural requirements of ER ligand11, or reproductive toxicology.12

A sample of hydroxylated and non-hydroxylated triphenyl acrylonitriles for binding to calf uterus estrogen receptors has been investigated by using quantitative structure-activity relationship approach.1 The best performing model after removing one outlier (the residual value exceeded twice the standard error of estimate) is presented in Eq (1). logRBA = 2.114(±0.243)I12-OH – 0.223(±0.059)S6 – 0.147(± 0.050)S18 – 0.193(± 0.052)Nt n = 24, r = 0.919, r2 = 0.845, EV = 81.415%, F = 27.284, s = 0.595, AVRES = 0.450, PRESS = 10.021, SDEP = 0.646, Presav = 0.515, Q2 = 0.751 (1)

where logRBA = competition for [3H] 17-ß-estradiol binding, S6 and S18 are E-states of C6 and C18 respectively, I12-OH = presence or absence of –OH substitution in C12, Nt = number of free terminal

Correspondence and reprint request to:S. Bolboacă,“Iuliu Haţieganu” University of Medicine and Pharmacy Cluj-Napoca; E-mail: [email protected] 13 Emil Isac, 400023 Cluj-Napoca, Cluj, Romania 37 Received 8 April 2010; Accepted for publication 7 July 2010

S. Bolboacă et al

atoms (excluding H) in C12, n = sample size; r = correlation coefficient, r2 = coefficient of determination, F = F-values (significance level at 1%), EV = explained variance, s = standard error of estimate, AVRES = average of absolute value of residuals, PRESS = predictive residual sum of squares, SDEP = standard deviation of error of predictions, Presav = average of absolute value of predicted residuals, Q2 = cross-validated variance. The study aimed to model the relationship between topological structures of a sample of triphenyl acrylonitriles with binding estrogen receptor using the molecular descriptors family obtained through vertex cutting (MDFV). The best performing model was compared with previously reported model in order to identify the method with highest performances. Material and methods

The sample of triphenyl acrylonitriles previously studied by Mukherjee et al. 13 was included in analysis. The compound abbreviation, 2D structure and estrogenic activity expressed as relative binding affinity to the ER vis-à-vis E2 in logarithmic scale (logRBA) are given in Fig. 1. The 2D structures of the compounds were drawn using HyperChem software (version 8.04). The molecular geometry was constructed using HyperChem, and the molecule was saved as *.mol file. The molecular geometry was optimized using Molecular Modeling Pro Plus: conformational analysis – moly minimizer – makes moderate changes – refine (applied twice). The molecule was then saved as *.hin file (to be introduced in construction of molecular family on vertex cutting), the compounds were validated and the partial changes were computed whenever needed using HyperChem. The sample was split in two sets: training (used to obtain the multiple-linear regression model (MLR)) and test (used to test the MLR equation obtained on training set). A number of 7 molecules were assigned to the test set, using the following approach: • Construct of strata of the sample (25 compounds) based on experimental logRBA values. Seven strata were constructed and the frequency table was obtained. • Extraction randomly a predefined number of molecules from each strata whenever the frequency was higher than 1 (there was one stratum with a single compound; this was assigned to training set). One stratum with observed frequency higher than 4 was randomly to provide 2 com-

38

pound for inclusion in test set. The following compounds were included in test set: triph024, triph016, triph004, triph017, triph006, triph019, and triph009. The logRBA variance of the compounds included in training set proved not to be statistically significant different compared to the compounds included in test set (F-test for variances = 1.28, p-value = 0.4018); the same result was obtained when the means were compared (t-value = -0.0229, p-value = 0.9819). • Normality test on observed logRBA for compounds included in training set (EasyFit v. 5.1.): Kolmogorov-Smirnov statistic14 equal to 0.1243 (p = 0.9118); Anderson-Darling statistic15 equal to 0.5128 (null hypothesis of normality accepted at significance level of 1%); and Chi-Squared statistic16 equal to 1.0122 (p = 0.6028). The molecular descriptors (MDFV)1 were calculated for each compound included in the analysis by applying the following steps (using a series of home-made programs): • Define the investigated set of compounds: logRBA. • Create the following tables: `triph_mdfv` table (contains the named of molecular descriptors), `triph_data` table (contains the *.hin file of molecules prepared for modelling as describe above), and `triph_prop` (contains the experimental logRBA values). • Compute the values of molecular descriptors: candidate fragments obtained by cutting of pairs of vertices after matrix representation of the molecular graph. The MDFV descriptors have a name of 8 letters indicating how they were generated (for details of MDFV see the paper of Bolboacă and Jäntschi.17) • Validate the MDFV descriptors: 2387280 descriptors were calculated for each molecule. 6059 descriptors proved to be valid and were used in MLR analysis after applying three validation criteria (Jarque-Bera value higher than critical value for the observed activity, identity analysis and inter-correlation higher than 0.99). The MLR approach was applied in order to identify the best link between MDFV descriptors and logRBA. Statistica 8.0 software was used in identification of best performing model. The identification of the best performing MDFV - MLR model on training set was performed applying the following criteria: • Highest explanation of the observed logRBA (highest values of significant correlation coefFolia Medica 2010; 52(3): 37-45

© 2010 Medical University Plovdiv

Binding Affinity оf Triphenyl Acrylonitriles тo Estrogen Receptors: Quantitative Structure-Activity Relationships

O O

O N

N

N

N

N

O

triph001 – (-1.046)

O

triph002 – (1.556)

triph003 – (0.342)

O

triph004 – (0.519)

triph005 – (1.792)

O

O O

O

O N

N

N

N

N

O

O

triph006 – (1.869)

triph007 – (0.785)

O

triph008 – (2.220)

triph009 – (1.447)

O

O

O

O

O

N

O

N

N

N

N

O

O

triph011 – (1.968)

triph010 – (0.398)

triph012 – (1.892)

triph013 – (0.959)

O

triph014 – (-0.180)

triph015 – (1.230)

O N

O O

O

N O

N

N

O

N

O

O N

N O

O

triph016 – (-0.444)

triph017 – (0.806)

triph018 – (-2.000)

triph019 – (0.531)

triph020 – (2.033)

N

O

N

H

N

O

N

N

O

O

N

O

N

N

N

N

N

triph021 – (-0.398)

triph022 – (-2.000)

triph023 – (-1.398)

N

O

triph024 – (-2.000)

O

triph025 – (-1.398)

Figure 1. General structure, abbreviation and logLBA (brackets) of triphenyl acrylonitriles.

Folia Medica 2010; 52(3): 37-45 © 2010 Medical University Plovdiv

39

S. Bolboacă et al

ficients between the observed and estimated activity). • Smallest number of descriptors in the model. • Lowest standard error of estimate. • Highest F-value and lowest p-value associated to F-value (significance of MLR model). • Absence of collinearity of descriptors in the model. • Highest value of correlation coefficient in leave-one-out cross-validation. The ability of the best performing MLR model identified in training set was test on test set. A comparison analysis was carried on to compare the best performing MDFV-MLR model with previously reported model with higher correlation coefficient using the following statistics: Akaike’s and related information criteria18 (the smallest the value the better the model was); Kubinyi function (FIT) 19,20 (the highest the value the better the model is) and Z test for comparing two correlation coefficients21. Results

The best performing MDFV model in terms of goodness-of-fit is presented in Eq (3). Ŷ MDFV = 65.11(± 10.35) – TASAAFDL*9.18(± 1.4) + GLCACPDL*(6.69·10 -1 )(± 1.62·10 -1 ) + GMHAAIDR*(7.84·10-5)(± 1.86·10-5) (3)

where ŶMDFV = estrogenic activity estimated by the model, the numbers in round brackets represent the parameter needed to compute the confidence intervals for the slope parameters; TASAAFDL, GLCACPDL, and GMHAAIDR = the MDFV members. Statistical characteristics of the model from Eq(3) are give in Eq(4). ntraining = 18, rtraining = 0.9788 (95%CI [0.9427 – 0.9922]), r2training = 0.9580, r2adj-training = 0.9489, CVtraining = 0.8660 sest = 0.33, F-value (p-value) = 106 (7.16·10-10) tint (p-value) = 13.49 (2.06·10-9), tTASAAFDL (p-value) = -13.72 (1.65·10-9), tGLCACPDL (p-value) = 8.84 (4.18·10-7), tGMHAAIDR (p-value) = 9.02 (3.30·10-7) TASAAFDL: T = 0.941, VIP = 1.063 GLCACPDL: T = 0.926, VIP = 1.080 GMHAAIDR: T = 0.903, VIP = 1.108 r2loo = 0.9408, sloo = 0.39, Floo = 74 (7.88∙10-9) (4)

where training = training set, n = sample size; r = correlation coefficient; r2 = determination coefficient; radj2 = adjusted determination coefficient;

40

CV = coefficient of variation; sest = standard error of estimate; F-value = Fisher statistic of the MLR model; T = tolerance (collinearity diagnostic)); VIF = variance inflation factor (collinearity diagnostic); rloo2 = determination coefficient obtained in leaveone-out analysis; spred = standard error of predicted; Floo = F-value obtained in leave-one-out analysis. The model presented in Eq (3) was validated through its application on test set. Statistical characteristic are presented in Eq (5). ntest = 7, rtest = 0.9489 (95%CI [0.6860 – 0.9926]), r2test = 0.9004, stest = 0.53, Ftest (ptest) = 43 (1.21·10-3) (5)

The values of MDFV descriptors used by model (Eq (3)), estimated and predicted logRBA, and residuals are presented in Table 1. The goodness-of-fit of the model obtained on training set and its application on test set is presented in Fig. 2. The best performing MDFV model (Eq (3)) was compared to the model previously reported (Eq (1), in terms of information criteria (Table 2) and of goodness-of-fit (Z test). The goodness-of-fit of the model from Eq(3) was compared with the goodness-of-fit of the previously reported model (Eq (1)) and a value of 0.795 (p-value = 0.2133) was obtained. Discussion

The linear relation between topological structures of triphenyl acrylonitriles and binding estrogens receptors was successfully modelled. The MDFV approach that implements the fragmentation of vertices in the molecular graph proved able to estimate activities.17,22 Seven information criteria and three weights were computed in order to compare the MDFV model with the model previously reported by Mukherjee et al.17 The best performing model was selected according to Hawkins principles23: highest correlation coefficient, highest Fisher parameter, lowest standard error of the estimate, and smallest possible number of significant parameters (n = 5∙v, where n = sample size, v = number of variables in the model). The model with the highest correlation coefficient, the highest Fisher parameter, the lowest standard error of estimate, and the smallest possible number of significant parameters was considered to be the best performing model (Eq (3)). This model is able to explain ~96% of the total variance in the estrogenic binding relative affinity of triphenyl acrylonitriles. The Fisher-value and its associated significance sustain the significance of the model Folia Medica 2010; 52(3): 37-45

© 2010 Medical University Plovdiv

Binding Affinity оf Triphenyl Acrylonitriles тo Estrogen Receptors: Quantitative Structure-Activity Relationships

Table 1. Values of descriptors, observed, estimated / predicted logRBA and residuals in training and test sets MDFV descriptor

Mol TASAAFDL

GLCACPDL

GMHAAIDR

7.440 7.296 7.408 7.194 7.543 7.304 7.270 7.350 7.286 7.304 7.130 7.130 7.130 7.304 7.130 7.130 7.304 7.130

1.693 -2.002 -1.023 -1.679 0.862 0.783 0.772 -0.836 0.632 0.670 -0.685 0.660 0.728 0.688 0.752 0.777 1.804 -0.852

1148.2 14537.0 23340.0 13358.0 41710.0 22956.0 19946.0 24907.0 22890.0 29383.0 24643.0 22774.0 24238.0 38360.0 20622.0 17342.0 39350.0 21011.0

logRBA

Ŷ-logRBA

Residuals

-2.000 -2.000 -1.398 -1.046 -0.398 -0.180 0.342 -1.398 0.785 0.959 1.230 1.556 1.792 1.892 1.968 2.033 2.220 0.398 0.375† 1.458

-1.9825 -2.0805 -1.7643 -1.0206 -0.3040 0.3677 0.4362 -0.9838 0.4266 0.7962 1.1164 1.8691 2.0290 1.5120 1.7616 1.5209 2.3352 0.7198 0.3753† 1.4269

-0.0175 0.0805 0.3663 -0.0254 -0.0940 -0.5477 -0.0942 -0.4142 0.3584 0.1628 0.1136 -0.3131 -0.2370 0.3800 0.2064 0.5121 -0.1152 -0.3218 0.2422 0.2989

Training set triph018 triph022 triph023 triph001 triph021 triph014 triph003 triph025 triph007 triph013 triph015 triph002 triph005 triph012 triph011 triph020 triph008 triph010

m stdev

KSres (p-value)

0.1070 (0.9721)

ADres (ADcrit 5%)

0.1742 (2.5018)

Test set triph024 triph016 triph004 triph017 triph006 triph019 triph009

7.479 7.332 7.211 7.130 7.231 7.373 7.130

-2.367 -0.649 -0.716 -0.694 -0.858 0.865 0.734

33110.0 25257.0 23290.0 30176.0 39450.0 30626.0 23111.0 m stdev

-2.000 -0.444 0.519 0.806 1.869 0.531 1.447 0.390‡ 1.286

-2.5490 -0.6663 0.2458 1.5440 1.2339 0.3903 1.9446 0.3062‡ 1.5376

0.5490 0.2223 0.2732 -0.7380 0.6351 0.1407 -0.4976 0.4366 0.5151

KSres (p-value)

0.2585 (0.6489)

ADres (ADcrit 5%)

0.4025 (2.5018)

Ŷ-logRBA = logRBA estimated by Eq(3) for training set and predicted logRBA for test set’ m = arithmetic mean; stdev = standard deviation; Comparing means (significance level of 5%): † p = 0.9999; ‡ p = 0.6828; KSres = Kolgomorov-Smirnow statistic applied to residuals (for testing normality); ADres = Anderson-Darling statistics applied to residuals for testing normality; ADcrit 5% = critical value for Anderson-Darling statistics for a significant level of 5%.

Folia Medica 2010; 52(3): 37-45 © 2010 Medical University Plovdiv

41

S. Bolboacă et al

Figure 2. Goodness-of-fit of models: training versus test. Table 2. Validation and comparison of the models Model

Parameter

Eq(3)

Eq(1)

-104.49

-52.16

wi(AICc)

1.00

4.33∙10-12

AICR2 (AIC based on determination coefficient)

1.94

2.96

wi(AICR2)

0.62

0.38

-4.34

-0.86

0.85

0.15

BIC (Bayesian information criterion)

-99.48

-45.18

APC (Amemiya prediction criterion)

0.00

0.10

-107.08

-53.02

8.72

2.59

AICc (corrected Akaike information criterion)

AICu (McQuarrie and Tsai corrected AIC) wi(AICu)

HQC (Hannan-Quinn criterion) FIT (Kubinyi function)

wi = Akaike weights for model i. Parameters: Smallest the better excepting FIT and wi (where largest the better).

from Eq (3) while the t-values and associated significance sustain the significance of the MDFV descriptors used by Eq (3). The characteristics of the descriptor’s contribution to the binding estrogenic activity of triphenyl acrylonitriles revealed the followings: • The interaction between structure and binding activity proved to fulfill via bonds (topology - T) and space (geometry - G). • Dominant atomic properties were represented

42

by electronic affinity (A), melting point (L), and relative atomic mass (M). • The structure on activity scale is logarithm (L) and reciprocal (L). The binding estrogens receptor affinity of TPT proved to be of geometric and topological nature. It depended on compound electronic affinity, melting point and relative atomic mass (Eq (3)). The validity of a linear model is sustained by the absence of collinearity within descriptors used Folia Medica 2010; 52(3): 37-45

© 2010 Medical University Plovdiv

Binding Affinity оf Triphenyl Acrylonitriles тo Estrogen Receptors: Quantitative Structure-Activity Relationships

by the model. The analysis of collinearity was carried out for the model presented in Eq (3) by computing two parameters: tolerance and variance inflation factor. Tolerance, defined as the difference between 1 and determination coefficient, show the degree of instability in the regression coefficients. The variance inflation factor gives the degree to which the standard error of the predictor is increased due to the predictor’s correlation with the other predictors in the model. Values less than 0.10 for tolerance and values greater than 10 for variance inflation factor indicates the presence of multicollinearity.24 The prediction ability of the model in Eq (3) was analyzed in leave-one-out experiment (internal validation) and by applying the model on an external set of compounds (test set, external validation). Internal and external validation analyses must be conducted in SAR analysis since high internal predictivity is not necessary related to high external predictivity25 (effect known as Kibinyi paradox26). The leave-one-out experiment, 17 experiments were conducted with 17 compounds in training set (for identification of MLR model) and 1 compound in test set (the application of the identified MLR model). The values of leave-one-out determination coefficient (0.9408) proved to be close to the determination coefficient of the model from Eq (3) (0.9580) and indicates a good prediction ability (~2% difference between these two determination coefficients). The external validation of the model presented in Eq (3) was carried out and the model was applied on a sample of 7 compounds. The difference between determination coefficient obtained in test set and the determination coefficient of the model in Eq (3) proved to be of 4%. Thus, the predictivity analysis (internal and external) conducted for the model presented in Eq (3), revealed that the model is reliable and could be use to predict the relative binding affinity on estrogen receptors of triphenyl acrylonitriles. Furthermore, the reliability of the model presented in Eq (3) is sustained by the absence of significant difference between the observed and estimated mean of logRBA (training set), respectively observed and predicted mean of logRBA (test set) (p > 0.6, significance level of 5%). A series of parameters were computed in order to compare the best performing MDFV model (Eq (3)) with the model identified by Mukherjee et al. (Eq (1)). The MDFV model (Eq (3)) systematically obtained the best expected values: the smallest Folia Medica 2010; 52(3): 37-45 © 2010 Medical University Plovdiv

values of information criteria (AICc, AICR2, AICu, BIC, APC and HQC), highest values of Akaike’s weights (wi(AICc), wi(AICR2), wi(AICu)) and of the Kubinyi function (FIT). According to the results presented in Table 2, based on AIC values it could be concluded that the model from Eq(3) is more useful compared to model from Eq (1). As far as the goodness of fit of the models on Eq (1) and Eq (3) according to Z test was concerned, these models were not statistically different (p = 0.2133). Even if the models are not statistically different in terms of goodness-of-fit the MDFV model proved to be better in terms of information criteria and Kubinyi function. The present study aimed to model the relative binding affinity to estrogen receptors of TPT by using information extracted from the matrix representation of the compounds. A valid and reliable model with three descriptors was obtained. The model proved its reliability in training and test sets. The analyzed sample size is the main limitation of the present research. Current research in our laboratory aims to characterize activities / properties of other classes of compounds by using the MDFV approach. Conclusions

In the present study an alternative SAR model relating the relative binding affinity on estrogen receptors with the molecular structure of triphenyl acrylonitriles by means of structural descriptors with the multiple linear regression approach. The best performing model proved to be able to explain ~96% of the total variance in the estrogenic binding relative affinity of triphenyl acrylonitriles and exhibits better information parameter compared to previously reported model with the same number of molecular descriptors involved. The assessment in test set of 7 triphenyl acrylonitriles not used in identification of the best MDFV model suggests that it performs predictively. Acknowledgements

The research was supported by CNCSIS-UEFISCSU (project PNII-IDEI458/206/2007). References

1. Dahlman-Wright K, Cavailles V, Fuqua SA, et al. International Union of Pharmacology. LXIV. Estrogen receptors. Pharmacol Rev 2006;58(4):773-81. 2. Jensen EV, Jordan VC. The estrogen receptor: a model for molecular medicine. Clin Cancer Res 2003;9(6):1980-9.

43

S. Bolboacă et al

3. Kuiper GG, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson JA. Cloning of a novel receptor expressed in rat prostate and ovary. Proc Natl Acad Sci USA 1996;93(12):5925-30. 4. Mosselman S, Polman J, Dijkema R. ERβ: identification and characterization of a novel estrogen receptor. FEBS Lett 1996;392:49-53. 5. Harper MJK, Walpole AL. Contrasting endocrine activities of cis and trans isomers in a series of substituted triphenylethylenes. Nature 1966;212(5057):87. 6. Miquel JF, Sekera A, Chaudron T. Synthese de polyphenylethylenes et Synthese de polyphenylethylenes et interferences avec Ie recepteur oestrogene d’uterus de souris. C R Acad Sci (Paris) 1978;286:151‑4. 7. Pons M, Michel A, Crastes de Paulet A, et al. Influence of new hydroxylated Triphenylethylene (TPE) derivatives on estradiol binding to uterine cytosol. J Steroid Biochem 1984;20(1):137-45. 8. Pons M, Bignon E, Chastes De Paulet A, Gilbert J, Ojasoo T, Raynaud JP. Hydroxylated triphenylacrylonitriles adopt a unique orientation within the binding site of the estrogen receptor. J Steroid Biochem 1990;36(5):391-7. 9. Spyrakis F, Cozzini P. How computational methods try to disclose the estrogen receptor secrecy - Modeling the flexibility. Curr Med Chem 2009;16(23):2987‑3027. 10. Mukherjee S, Nagar S, Mullick S, Mukherjee A, Saha A. Pharmacophore mapping of selective binding affinity of estrogen modulators through classical and space modeling approaches: exploration of bridgedcyclic compounds with diarylethylene linkage. J Chem Inf Model 2007;47(2):475-87. 11. Islam MA, Nagar S, Das S, Mukherjee A, Saha A. Molecular design based on receptor-independent pharmacophore: Application to estrogen receptor ligands. Biol Pharm Bull 2008;31(7):1453-60. 12. Cronin MTD, Worth AP. (Q)SARs for predicting effects relating to reproductive toxicity. QSAR and Combinatorial Science 2008;27(1):91-100. 13. Mukherjee S, Mukherjee A, Saha A. QSAR studies with E-state index: predicting pharmacophore signals for estrogen receptor binding affinity of triphenylacrylonitriles. Biol Pharm Bull 2005;28(1):154‑7.

44

14. Kolmogorov A. Confidence limits for an unknown distribution function. The annals of mathematical statistics 1941;12(4):461-3. 15. Anderson TW, Darling DA. Asymptotic theory of certain “goodness-of-fit” criteria based on stochastic processes. Annals of Mathematical Statistics 1952;23(2):193-212. 16. Pearson K. On the criterion that a given system of deviations from the probable in the case of a correlated system of variables is such that it can be reasonably supposed to have arisen from random sampling. Philosophical Magazine 1900;50:157‑75. 17. Bolboacă SD, Jäntschi L. Comparison of quantitative structure-activity relationship model performances on carboquinone derivatives. Scientific World Journal 2009;9:1148-66. 18. Akaike H. Fitting autoregressive models for prediction. Annals of the Institute of Statistical Mathematics 1969;21:243-7. 19. Kubinyi H. Variable selection in QSAR studies. I. An evolutionary algorithm. Quantitative structureactivity relationships 1994;13:285-94. 20. Buckland ST, Burnham KP, Augustin NH. Model selection: An integral part of inference. Biometrics 1997;53(2):603-18. 21. Hinkle DE, Wiersma W, Jurs SG. Applied statistics for the behavioral sciences. 1988, 2nd ed. Boston: Houghton Mifflin Company . 22. Bolboacă SD, Marta MM, Stoenoiu CE, Jäntschi L. Molecular descriptors family on vertex cutting: relationships between acelazolamide structures and their inhibitory activity. Applied Medical Informatics 2009;25(3-4):65-74. 23. Hawkins DM. The problem of overfitting. J Chem Inf Comput Sci 2004;44(1):1-12. 24. Stevens J. Applied multivariate statistics for the social sciences (4th ed.). Mahwah, New Jersey: Lawrence Erlbaum Associates, 2002. 25. Kubinyi H, Hamprecht FA, Mietzner T, Three-dimensional quantitative similarity-activity relationships (3D QSiAR) from SEAL similarity matrices. J Med Chem 1998;41:2553-64. 26. van Drie JH. Pharmacophore discovery - lessons learned. Curr Pharm Des 2003;9:1649-64.

Folia Medica 2010; 52(3): 37-45

© 2010 Medical University Plovdiv

Binding Affinity оf Triphenyl Acrylonitriles тo Estrogen Receptors: Quantitative Structure-Activity Relationships

Сходство связывания трифенил акрилонитрила с рецепторами эстрогена – зависимость между химической структурой и свойствами S. Bolboacă, M. Marta, L. Jäntschi

Резюме Цель: Исследовать относительное сходство связывания трифенил акрилонитрила с рецепторами эстрогена с помощью анализа зависимости между химической структурой и свойствами. Материал и методы: Определенное количество уже исследованного трифенил акрилонитрила разделено на случайном принципе на учебные и тестовые комплекты (соответственно 17 и 18 соединений). С целью привести структурную информацию в структурные дескрипторы авторы использовали анализ молекулярной дескрипторной группы (MDFV). Зависимость связывания рецептора с структурным дескриптором определена с помощью полимерного линейного регрессионного анализа. Р езультаты : Выведено оптимальное трипара-

Folia Medica 2010; 52(3): 37-45 © 2010 Medical University Plovdiv

метрическое уравнение с детерминационным коэффициентом 0.9580 и скрещенным достоверным параметром 0.9408. Оптимальная модель испробована на тестовом комплекте соединений, при чем получена стоимость детерминационного коэффициента 0.9004. Не отмечена сигнификантная разница между MDFV моделью и уже изученной моделью по отношению к статистическому соотношению. В отношении информационных критериев (критерии Akaike, Bayes, Amemiya, and Hannan-Quinn) и функции Kubinyi модель MDFV оказалась лучше, чем изученная раньше модель. Заключение: Оптимальная MDFV модель успела объяснить приблизительно 96% общей дисперсии относительного сходства связывания трифенил акрилонитрила с рецепторами эстрогена, кроме того она демонстрирует оценочные и прогностические свойства. Хотя и не отмечены сигнификантные разницы в статистической адекватности, оказалось, что эта модель имеет более хорошие информационные параметры, чем уже изученная модель, которая использует то же самое число молекулярных дескрипторов.

45

Folia Medica 2010; 52(3): 46-55 Copyright © 2010 Medical University Plovdiv doi: 10.2478/v10153-010-0006-1

Original Articles

Dental Investigations Morphological changes in hard dental tissues prepared by Er:YAG Laser (LiteTouch, Syneron), Carisolv and rotary instruments. A scanning electron microscopy evaluation Snejana Ts. Tsanova, Georgi T. Tomov Department of Operative Dentistry and Endodontics, Faculty of Dental Medicine, Medical University, Plovdiv, Bulgaria Abstract Aim: This in vitro investigation aimed to study by means of scanning electron microscope the morphological changes in hard dental tissues after using several different methods for caries removal and cavity preparation. Materials and methods: Twenty freshly extracted human teeth with carious lesions were used in the study. They were assigned to four groups depending on the method used for preparation: Group 1 – Cavity preparation using Er: YAG laser (LiteTouch, Syneron, Israel). Group 2 – Chemomechanical preparation using colourless Carisolv gel (MediTeam AB, Savedalen, Sweden). Group 3 – Mechanical rotary preparation using diamond burs and air turbine. Group 4 – Mechanical rotary preparation using by steel burs and micromotor. The preparations were performed strictly according to the manufacturer’s instructions for proper use of instruments. The teeth samples were prepared for histological study and investigated by a scanning electron microscope at different magnification; the morphological changes in the tissues were registered and compared. Results: There were considerable differences in the surface characteristics of the dental tissues when we analysed the photomicrographs of the specimens obtained using scanning electron microscopy (SEM). The surface after laser treatment remained highly retentive with no residual smear layer; the second best results in this respect were registered when teeth were chemomechanically excavated with Carisolv gel. The mechanical methods of cavity preparation resulted in surfaces with a smear layer of dentin without any microretentions. Conclusion: The scanning electron microscopy of hard dental tissues prepared using steel and diamond burs showed surfaces covered with a thick smear layer that may be relevant to the subsequent bonding of adhesive restorative materials to the prepared cavity. In preparing the surface using a turbine with diamond burs the smear layer was thinner and part of the dentinal tubules orifices were open in the area of water turbulence. SEM analysis of hard dental tissues prepared with the help of colourless Carisolv gel showed a rough, retentive surface, some of the dentinal tubule lumens obstructed by denaturated collagen and surface contaminants. The teeth surfaces prepared with Er:YAG laser Lite Touch (Syneron) remained without smear layer and clearly exposed dentinal tubules orifices. The surfaces were highly retentive. Key words: scanning electron microscopy, Er:YAG laser, Carisolv Correspondence and reprint request to:S. Tsanova, Department of Operative Dentistry and Endodontics, Faculty of Dental Medicine, Medical University, Plovdiv 3 Hristo Botev blvd., 4002 Plovdiv, Bulgaria Received 21 January 2010; Accepted for publication 31 March 2010 46

Morphological Changes in Hard Dental Tissues Prepared by Er:YAG Laser (LiteTouch, Syneron), Carisolv and Rotary Instruments. A Scanning Electron Microscopy Evaluation

Introduction

In recent years, prevention and early caries detection, as well as changes in the understanding of chemical and biological basis of demineralization process in hard dental tissue and possibilities carious lesion to undergo remineralization supersede the classical operative approach of caries treatment postulated by G. V. Black and promoted minimally invasive preparation (MIP). The main categories of MIP techniques include rotary handpieces and burs, chemo-mechanical cleaning with Carisolv gel, air abrasion and dental lasers.1,2 These trends for the replacement of the conventional method of preparation led to focus the attention of researchers on the impact of alternative techniques for MIP on hard dental tissues and underlying dental pulp. MIP techniques claim to be able to achieve controlled removal of infected and softened dentin while preserving healthy the hard dental tissues and do it with minimal discomfort for the patient. However, currently available data provide contradictory evidence for the impact of alternative techniques of MIP on hard dental tissues compared to conventional preparation. Possible reasons for this are the variety of experimental studies and difficulties to standardize the results of clinical researches. It is worth noting that the researchers giving the most positive evaluation of the alternative methods of preparation (Carisolv, laser) use mainly clinical criteria for evaluation (perception and tolerance of the patient, noise, atraumatic work, color and texture of the dentine when probing, etc) which are all rather subjective. New improved versions of alternative systems for preparation have been made available on the market claiming to be highly clinically efficient, but there is still little information about them (the modified Carisolv colourless gel, multi-frequency high-energy lasers, air-abrasion). This makes it necessary that research in this rapidly developing, promising field of dentistry should be periodically updated.

The objective of the present in vitro study was to evaluate by SEM the ultrastructural changes in hard dental tissues treated with several alternative systems for caries removal and preparation: Er: YAG laser (LiteTouch), chemomechanical preparation with Carisolv gel, conventional preparation with diamond burs/air turbine and steel burs/micromotor. Materials and methods

Experimental design: the study used 20 human teeth freshly extracted because of advanced periodontal disease. The preparations involved natural carious lesions on tooth surface (Fig. 1. a, b, c). According to the preparation technique the teeth were divided into 4 groups of 5 teeth (n = 5): Group 1. Laser preparation by Er: YAG laser (LiteTouch, Syneron, Israel) (Fig. 2 a, b, c) Group 2. Chemomechanical preparation with Carisolv colorless gel (MediTeam AB, Savedalen, Sweden) (Fig. 3 a, b, c) Group 3. Mechanical rotary preparation by diamond burs/air turbine) Group 4. Mechanical rotary preparation by steel burs/micromotor) Preparations are made strictly according to manufacturer’s instructions for service. The removal of caries is proved by clinical methods – observation and probing. After preparation the teeth are immersed for 1 hour in 4% buffered fixative solution of glutaraldehyde (0.075 M, pH 7.3). Then they are rinsed in distilled water and placed for 90 min in cold buffer solution ofsodium cacodylate (0.02M, pH 7.2, 660 mOsm) for fixation of organic matter. Subsequent dehydration is carried out in ethanol in ascending series of 30, 50, 70, 80, 95 and 100% for one hour in each series, the drying of the teeth based on CPD (Critical Point Drier) method in a desiccator. The dried specimens are mounted on a metal stand and gold-coated (200-250 nm) by cathode atomization under vacuum. Scanning microscopy is performed

Figure 1 a, b, c. Extracted teeth with carious lesions. Folia Medica 2010; 52(3): 46-55 © 2010 Medical University Plovdiv

47

S. Tsanova et al

Figure 2 a, b, c. Laser preparation with Er: YAG laser LiteTouch (Syneron, Israel) “Hard tissue mode” (400mJ/20Hz; 8.00W)

Figure 3 a, b, c. Chemomechanical preparation with Carisolv colorless gel and hand excavators.

with the electron microscope of Philips (Holland) 515 model SEM with accelerating voltage of 25 kV in secondary emission mode. For each specimen we made five photographs with the same magnification (x 2000) of randomly chosen areas and different numbers of photos at a different degree of magnification. On the SEM photomicrographs we evaluated, described and compared the morphological findings and differences in the enamel and dentin tissues after treating the teeth using alternative methods for caries removal and cavity preparation. Results

When analyzing the SEM photomicrographs of the specimens examined, it is found that the conventional method of cavity preparation with steel burs and micromotors at low speed without water cooling (group 4) results in contaminated surface covered with smear layer of dentin debris without visible dentinal tubules orifices (Figs 4a, 4b). Thick smear layer covers all treated surfaces. The walls of the

48

cavities are smooth and rounded and the border between enamel and dentin is hardly noticeable. In group 3 (preparation with diamond burs, air turbine and water cooling) a thin, smooth and in some places missing smear layer was observed (Fig. 5a). In the area of water turbulence there were patent dentinal tubules orifices, but without having a clear outline of both tubules lumens and peri- and inter-tubular dentin (Fig. 5b). The boundary between enamel and dentin is unclear and the cavity forms have smooth contours. The dental surface topography after chemomechanical preparation with Carisolv gel (group 2) was clearly rougher compared with that of groups 1 and 2, the dentinal tubules orifices are visible and there is almost no smear layer (Fig. 6a). Preparing the organic matrix using chemomechanical preparation with Carisolv and protecting mineralized dental tissue at the same time result in rough appearance of the treated surfaces and considerable microretention development (Figs 6b, 6c). Denatured collagen fibers and surface contaminations occur in some Folia Medica 2010; 52(3): 46-55

© 2010 Medical University Plovdiv

Morphological Changes in Hard Dental Tissues Prepared by Er:YAG Laser (LiteTouch, Syneron), Carisolv and Rotary Instruments. A Scanning Electron Microscopy Evaluation

Figure 4 a, b. SEM photomicrographs of tooth surfaces prepared with steel burs. The surface is covered with a layer of debris, dentinal tubules orifices are not visible. (x 500, 2000)

Figure 5 a, b. A smooth, thin smear layer covers tooth surfaces prepared with diamond burs and air turbine. In the area of water turbulence partially removed contaminants and single dentinal tubules lumens were observed. (x 500, 2000).

places, blocking the dentinal tubules orifices (Fig. 6d). Cavity forms in Carisolv group follow the initial caries lesions forms without going beyond their boundaries. Folia Medica 2010; 52(3): 46-55 © 2010 Medical University Plovdiv

Cavity forms prepared with Er:YAG laser (Group 1) are characterized by a lack of definite and precise geometric configuration and outlined cavity elements (Fig. 7a). There is rough and irregular

49

S. Tsanova et al

Figure 6 a, b. Dentin surfaces treated with colorless Carisolv gel - clean and highly retentive, with great part of exposed open dentinal tubules (x 500, 2000).

Figure 6 c, d. Dentin surfaces treated with colorless Carisolv gel - there is a rough, granular surface which is highly retentive. In some sections single collagen fibrils are evident (x 3000).

surface without no smear layer (Fig. 7b). Dentinal tubules are clearly exposed. Intertubular dentin is ablated more than peritubular dentin and that made dentinal tubules appearance more prominent

50

(Fig. 7c). In the enamel the typical architectonics of enamel prisms grouped in bundles is observed. Laser ablation of part of the enamel makes the surfaces highly strong retentive (Figs 7d, 7e).

Folia Medica 2010; 52(3): 46-55

© 2010 Medical University Plovdiv

Morphological Changes in Hard Dental Tissues Prepared by Er:YAG Laser (LiteTouch, Syneron), Carisolv and Rotary Instruments. A Scanning Electron Microscopy Evaluation

Figure 7a. A cavity prepared with Er: YAG laser – unclear cavity outlines, craters shading into one another are observed. There are no precise outlined cavity elements. (x 20).

Figure 7 b, c. Laser treated dentin. The surface is clean and free from debris, all dentinal tubules are open. The surface is irregular, rough, which creates high retentivity. At greater magnification the more effective removal of intertubular dentin is seen and that makes dentinal tubules orifices appear convex (x 500, 2000). Discussion

The philosophy of minimally invasive cavity preparation approach is based on several principles – to remove only irreversibly damaged dental tissues and

Folia Medica 2010; 52(3): 46-55 © 2010 Medical University Plovdiv

to avoid macroretention preparation in healthy tissues.1 Additionally, these techniques should protect the underlying pulp and leave the treated surface suitable for adhesive bonding.1 Antibacterial effects

51

S. Tsanova et al

Figure 7 d, e. Enamel surfaces treated with Er: YAG laser revealed characteristic architectonics of bundles of enamel prisms with different orientation. The surface is highly retentive and free from contaminants and smear layer (x 2000, 500).

of the alternative preparation techniques must not be lower than those of standard necrotomy with rotary instruments and even to excel them.1 Nowadays the laser devices available for clinical use are capable of effective, controlled ablation of hard dental tissues.2 Some clinical trials suggested Carisolv gel to be highly efficient in caries removal, leaving clean and retentive dentinal surfaces.2 However, not all researchers agree with these conclusions. Therefore, such studies should be periodically updated due to constant introduction of new technologies. The experimental results of the present study revealed significant differences in the surface morphology of the studied samples which would affect the ability to perform effective adhesive bonding. These morphological differences are highly dependent on the mechanism of action of the specific preparation systems. Laser devices use a variety of physical media as sources for generating different wavelength that is absorbed and interact with specific molecules in human tissues. The explanation for the hard tissue ablation is the water content that evaporates when exposed to laser irradiation creating high internal pressure and subsequent microexplosions. In this interaction of the laser irradiation with tissue if there is inadequate water cooling that will lead

52

to undesirable thermal effects.3 Depending on parameters such as pulse energy and frequency CO2 lasers, Nd: YAG and Er: YAG lasers cause changes in enamel and dentin as roughing, craters, cracking, slicing, carbonification, melting and recrystallisation described in many previous studies.4-6 These changes depend on the laser type, mode of operation, system for water cooling and proper operation. 3 Additionally, the abilities to ablate carious dentin and enamel strongly vary according to different experimental studies.4-6 For argon-fluoride laser and the exciter laser there are data on their ability to remove dental caries which is not of sufficient efficiency.5 Krypton fluoride exciter laser emitting in ultraviolet range has been shown to remove dentin, but enamel resists the attempt for ablation.5 The high-power and high-frequency Er: YAG laser (LiteTouch, Israel), which was used in the present study, has an advanced hydrokinetic system that is said to be capable of effective and safe ablation of hard dental tissues. LiteTouch laser incorporates unique software which allows for the broadest range of energy and frequency settings. The unique LiteTouch hand piece prevents loss of energy and along with the precision control over pulse duration, pulse energy and repetition rate optimize, allows for a wide range of hard tissues procedures. LiteTouch is the first laser in Folia Medica 2010; 52(3): 46-55

© 2010 Medical University Plovdiv

Morphological Changes in Hard Dental Tissues Prepared by Er:YAG Laser (LiteTouch, Syneron), Carisolv and Rotary Instruments. A Scanning Electron Microscopy Evaluation

the world to use a novel mechanism that controls energy output, offering optimal control of treatment parameters. Easy adjustable water spray flow, frequency and energy levels on a touch screen following special software. LiteTouch is also innovative in respect of its optical system incorporated in the ergonomic hand piece working with sapphire tips. The proposed mechanism of action of this system is the photons radiation that laser source delivers in targeted air - water jet, resulting in water droplets microexplosions. It is believed that this process is the mechanism of ablating particles from dental tissues without overheating, and without smear layer formation.7 Another characteristic of this laser is the wavelength (2940 nm) which is absorbed predominantly by water and also the sapphire tips, showing stability in providing focused energy of laser irradiation.8 This combination allows precise microinvasive cavity preparation with minimal heating and optimal rate of radiation absorption by the hydroxylapatite incorporated water.7 The program “hard tissue mode” removes enamel, dentin and dental caries effectively and without visible carbonization or disturbance of the specimen microstructure. Evaluated under electron microscope the dental tissues treated with Er: YAG laser showed rough and irregular surface without presence of a smear layer and with patent dentinal tubules. Intertubular dentin is ablated more than peritubular giving a characteristic appearance of the dentinal surface with mild prominent dentinal tubules. Enamel shows preserved prismatic structure, but also strong retentions due to microexplosions on its surface. Overall, the cavity form is irregular, devoid of strict geometry and dotted with microretentions, but without presence of contaminants or smear layer. The observed changes correspond to changes in hard dental tissues reported by other authors in previous studies on Er: YAG lasers9,10, but without thermally affected surfaces, areas of extensive recrystallisation, melted surfaces or cracks in the dentin, as described in some in vitro studies.3-5 It has also been reported that there are better abilities for adhesive bonding11, faster ablation of enamel and dentin compared with rotating burs12 and an increase in dentinal microhardness after treatment with Er: YAG pulsed lasers13. The latter statement is not confirmed by other studies. The marked surface irregularities and lack of smear layer observed in the recent study, noted also in other studies14,15 provide solid evidence for the physical mechanism of bonding with composite materials after laser treatment11. This fact is not Folia Medica 2010; 52(3): 46-55 © 2010 Medical University Plovdiv

yet fully explored as a possible opportunity to eliminate acid etching of hard dental tissues and its related adverse effects on the underlying dentin and pulp. Carisolv is a chemo-mechanical, minimally invasive method for selective softening of caries in dentin and its subsequent removal with hand excavators.16 The system consists of gel containing three amino acids (glutamine, lysine and leucine) and transparent liquid (0.5% NaOCl), which are mixed immediately before application. The obtained chlorinated amino acids selectively tear the damaged collagen fibers in carious dentin without damaging the underlying demineralized but not denaturated collagen. The macerated infected dentin is removed manually with the help of excavators. Carisolv gel used in this study is colorless and concentration of the amino acids in it is twice as small, while the sodium hypochlorite concentration is increased twofold. The suggested mechanism of action of Carisolv gel is based primarily on the proteolytic effect of NaOCl, which dissolves the denaturated collagen in the caries lesion.16 It is assumed that the three amino acids enhance the effect of NaOCl on the collagen and also reduce the involvement of healthy dental tissues. Carisolv chemical effects on the underlying pulp have been assessed as safe and alkaline pH (~ 11) of the gel neutralizes acids and has a bactericidal effect on cariogenic flora.1,16 The presence of NaOCl in Carisolv is problematic, however, because of the danger of NaOCl inhibiting the bonding agents polymerization. Another clinical problem is the inability of Carisolv to affect the enamel and that requires a combination with rotary instruments in the process of caries excavation.16 Additionally, the results as reported by studies on Carisolv capacity to remove the smear layer vary. According to some authors Carisolv almost completely removes the smear layer remaining visible and patent dentinal tubules.15,17 According to other authors Carisolv is unable to eliminate smear layers and there are no patent dentinal tubules.18 The latter study was conducted on non-carious dentin surface the researchers observing an irregular smear layer over enamel and dentin, and all dentinal tubules orifices filled with debris. A third group of researchers take an intermediate position - according to them Carisolv does not eliminate completely the smear layers, and there are partially patent dentinal tubules and residues of contaminant smear layer covering the dentinal surface.2

53

S. Tsanova et al

The dentin surfaces treated with Carisolv and observed by SEM in the present study are clean, smear layer-free with some remnants of denatured collagen fibers. Conventional rotating burs form smear layer on dental surface while Carisolv increase the surface roughness, leaving a relatively clean area. The dentin topography after Carisolv application is granular and rough compared to preparation with rotating instruments and possesses comparable roughness similar to that observed after laser preparation. The marked structural changes of the dental tissues and surface roughness observed in our study may play a crucial role in adhesion to composite materials, possibly without using etching agents. However, literature data on structural changes after Carisolv preparation vary considerably and we can conclude that this system for chemo - mechanical removal of dental caries is likely sensitive to the technique of application, mineralization and other dentin characteristics.2,19 The results of some contemporary studies showed that despite differences between individual authors, generally the amount of smear layer after treatment with Er: YAG laser and Carisolv in all cases is less than that after preparation with conventional rotating instruments, and surface changes are characterized by markedly rugged topography.2,3,12,15 The morphological features of hard dental tissues observed in our study lead us to the general conclusion that cavity preparation with Er: YAG laser and Carisolv is consistent with the principles of minimally invasive preparation, leaving clean surfaces and strong microretentions suitable for adhesive restorations. These assumptions about the benefits of alternative techniques for minimally invasive preparation of dental tissues for adhesive restorations should be confirmed in future clinical studies. Conclusions

SEM analysis of hard dental tissues treated with steel and diamond burs showed surfaces covered with a thick layer of debris, which could compromise the adhesion of filling materials. Dental tubules orifices are obturated with debris, with exception the areas under water turbulence where the debris is partially removed. Carisolv gel does not affect the enamel or healthy dentine. The surface topography of the dentine remaining after complete caries removal with Carisolv is rougher than that after conventional preparation with rotating burs. No typical smear layer is observed but thin patches of contaminants, much less

54

prominent than after drilling are visible. All laser-treated samples showed no evidence of thermal damage or signs of carbonification and melting. The SEM examination revealed characteristic micro-irregularities of the laser prepared dentin surface without any smear layers, and open dentinal tubules. Intertubular dentin is ablated more than peritubular dentin and that made the dentinal tubules appearance better exposed. Er:YAG laser ablated enamel effectively leaving well exposed enamel prisms without debris. The surfaces are very retentive. References

1. Banerjee A, Watson TF, Kidd EA. Dentine caries excavation: a review of current clinical techniques. Br Dent J 2000;188(9):476-82. 2. Yazici AR, Ozgunaltay G, Dayangac B. A scanning electron microscopic study of different caries removal techniques on human dentin. Oper Dent 2002;27:360-6. 3. Stephanovich M. Scanning electron microscopic study of the effects of Er:YAG lasers and conventional methods of preparation on dentin. SDK and NUS 2005;4(1):153-6 (Bulgarian). 4. McCormack SM, Fried D, Featherstone JD, et al. Scanning electron microscope observations of CO2 laser effects on dental enamel. J Dent Res 1995;74:1702-8. 5. Palamara J, Phakey PP, Orams HJ, et al. The effect on the ultrastructure of dental enamel of excimerdye, argonion and CO2 lasers. Scanning Microsc 1992;6:1061-71. 6. Li ZZ, Code JE, Van De Merwe WP. Er:YAG laser ablation of enamel and dentin of human teeth: determination of ablation rates at various fluences and pulse repetition rates. Lasers Surg Med 1992;12:625‑30. 7. Park NS. Changes in intrapulpal temperature after Er:YAG laser irradiation. Photomed Laser Surg 2007;25:229-32. 8. Eguro T, Aoki A, Maeda T, et al. Energy output reduction and surface alteration of quartz and sapphire tips following Er:YAG laser contact irradiation for tooth enamel ablation. Lasers Surg Med 2009;41:595‑604. 9. Eberhard J, Bode K, Hedderich J, et al. Cavity size difference after caries removal by a fluorescencecontrolled Er:YAG laser and by conventional bur treatment. Clin Oral Investig 2008;12(4):311-8. 10. Matsumoto M, et al. Morphological and compositional changes of human dentin after Er:YAG laser irradiation. J Oral Laser Applications 2003;3:12‑20. 11. Ceballos L, Toledano M, Osorio R, et al. Bonding to Er-YAG-laser-treated dentin. J Dent Res 2002;81(2):119-22. Folia Medica 2010; 52(3): 46-55

© 2010 Medical University Plovdiv

Morphological Changes in Hard Dental Tissues Prepared by Er:YAG Laser (LiteTouch, Syneron), Carisolv and Rotary Instruments. A Scanning Electron Microscopy Evaluation

12. Baraba A, Miletic I, Krmek SJ, Perhavec T, Bozic Z, Anic I. Ablative potential of the erbium-doped yttrium aluminium garnet laser and conventional handpieces: a comparative study. Photomed Laser Surg 2009;11:465-504. 13. C hinelatti MA, et al. Effect of erbium:yttriumaluminum-garnet laser energies on superficial and deep dentin microhardness. Lasers Med Sci 2008;34:135‑40. 14. Raucci-Neto W, Chinelatti MA, Palma-Dibb RG. Ablation rate and morphology of superficial and deep dentin irradiated with different Er:YAG laser energy levels. Photomed Laser Surg 2008;26:523-9. 15. K inoshita J, Kimura Y, Matsumoto K. Comparative study of carious dentin removal by ErCr:YSGG laser and Carisolv. J Clin Laser Med Surg 2003;21:307‑15.

16. Katarski G, Kissov K. Contemporary views of Carisolv treatment of dental caries. Clinical cases. SDK and NUS 2005;4(1):164-72 (Bulgarian). 17. B anerjee A, Kidd EAM, Watson TF. Scanning electron microscopic observations of human dentine after mechanical caries excavation. J Dent 2000;28:179‑86. 18. Cederlund A, Lindskog S, Blomlof J. Effect of a chemomechanical caries removal system (Carisolv) on dentin topography of non-carious dentin. Acta Odontol Scand 1999;57:185-9. 19. Giza S. Comparative studies of carious defects filling using the classical method and dental drill, and using the Carisolv chemomechanical method and the YAG:Er CTL-1601 laser. Ann Acad Med Stetin 2007;53:88-99.

Мор ф ол о г и ч еские и з м енени я в твËрдЫХ ЗубнЫх тканях, обработаннЫх ER:YAG-лазером (LITETOUCH, SYNERON), CARISOLV и ротационнЫми инструментами. Анализ сканирующим Электронном микроскопом

дении указаний производителей относительно эксплуатаций приборов и препаратов. После гистологической обработки зубы-образцы исследованы с помощью сканирующего электронного микроскопа (увеличение х20). Результаты: При анализе сканированных электронномикроскопически (СЭМ) микрофотографий обследованных образцов устанавливаются большие различия в поверхностной анатомии твердых зубных тканей. Лучшее удаление пораженного слоя дентина достигается при лазерном препарировании с помощью Lite Touch, затем при химико-механическом препарировании с помощью Carisolv гелей в отличие от конвенциональных способов зубного препарирования. Выводы: СЭМ анализ ТЗТ, обработанных стальным бором без водяного охлаждения, показывает, что остается толстый загрязненный слой дентина, компрометирующий адгезию восстановляющего материала. СЭМ анализ ТЗТ, обработанных турбиной с водяным охлаждением, показывает наличие тонкого раздавленного слоя дентина. СЭМ анализ ТЗТ, обработанных бесцветными Carisolv гелями, показывает наличие зернистой поверхности, однако часть дентинных канальцев оказываются с закупоренными просветами денатурированным колагеном и поверхностными загрязнениями. СЭМ анализ ТЗТ, обработанных с помощью лазера Lite Touch (Syneron, Israel) демонстрирует эффективное удаление загрязненного слоя дентина и четко экспонированные отверстия дентинных канальцев.

С. Цанова, Г. Томов

Резюме Цель: Работа ставит себе целью в эксперименте in vitro сравнить ультраструктурные изменения в твердых зубных тканях (ТЗТ) после обработки с помощью нескольких альтернативных систем препарирования (Er: YAG – лазер и Carisolv гели) по сравнению с использованием турбинного алмазного бора и стального бора. Материал и методы: В целях исследования использовано 20 только что удаленных человеческих зубов, пораженных кариесом. По системе обработки зубы разделены на 4 группы (одинаковое число зубов в каждой группе). Группа 1 – лазерное препарирование с помощью Er: YAG – лазера (Lite Touch, Syneron, Israel); Группа 2 – химико-механическое препарирование (бесцветные Carisolv гели, MediTeam АВ, Savedalen, Sweden); Группа 3 – механическое ротационное препарирование (турбина с алмазным бором); Група 4 – механическое ротационное препарирование (наконечник со стальным бором). Препарирование произведено при точном соблю-

Folia Medica 2010; 52(3): 46-55 © 2010 Medical University Plovdiv

55

Folia Medica 2010; 52(3): 56-61 Copyright © 2010 Medical University Plovdiv doi: 10.2478/v10153-010-0007-0

SHORT COMMUNICATIONS HIV/AIDS-ASSOCIATED KAPOSI’S SARCOMA WITH MULTIPLE SKIN-MUCOSAL DISSEMINATIONS FOLLOWING ULTRAVIOLET (РUVA) PHOTOCHEMOTHERAPY Nedialka I. Popivanova, Krassimira N. Chudomirova1, Ivan G. Baltadzhiev, Tsvetana I. Abadjieva1, Department of Infectious Diseases, Parasitology and Tropical Medicine, 1Department of Dermatology and Venereology, Medical University, Plovdiv, Bulgaria ABSTRACT HIV/AIDS infection in Bulgaria has spread over about 1200 registered patients and it is supposed that the number of the undetected cases is four times higher. Kaposi’s sarcoma is rarely observed in our country and no cutaneous-mucosal dissemination is reported for the time being. Aim: The aim of the study is to present a case of disseminated Kaposi’s sarcoma in a HIV/ AIDS patient who underwent Psoralen - UVA radiation treatment (Р��������������������� UV������������������� А������������������ ) for total alopecia. Methods: HIV was proved through ELISA and Western blot (InnoLia HIV I/II Score). РСR method (COBAS-Amplicor HIV-1 MT, 1,5) was used to determine viral load (VL). Monitoring was realized by flow-cytometric phenotype analysis of the immune cells. Biopsy of a skin lesion was performed for histomorphological analysis. Computed axial tomography (CAT) of the visceral organs was also applied. Results: The patient’s face, chest, back and upper extremities are covered by more than 50 typical for Kaposi’s sarcoma skin tumors and several isolated lesions are found in the oral cavity mucosa. The histological results show dilated vascular spaces with large endothelial cells������������������������������������������������������������������������������������ ����������������������������������������������������������������������������������� and spindle-like tumor cells in irregularly formed fascicles. Monitoring����������� ���������� of�������� ������� the���� ��� immune���������������������������������������������������������������������������������������� cells���������������������������������������������������������������������������������� ��������������������������������������������������������������������������������������� and������������������������������������������������������������������������������ ��������������������������������������������������������������������������������� the�������������������������������������������������������������������������� ����������������������������������������������������������������������������� viral�������������������������������������������������������������������� ������������������������������������������������������������������������� load��������������������������������������������������������������� ������������������������������������������������������������������� before�������������������������������������������������������� �������������������������������������������������������������� and���������������������������������������������������� ������������������������������������������������������� after���������������������������������������������� ��������������������������������������������������� the������������������������������������������ ��������������������������������������������� application������������������������������ ����������������������������������������� of��������������������������� ����������������������������� highly�������������������� �������������������������� active������������� ������������������� antiretrovi������������ ral therapy (HAART) showed CD4+ T cell number = 0.147x109/l and VL = 216 000 copies HIV-RNA/ml plasma when the disorder was first detected. A very good effect appeared 4 months after the HAART start: the mucous membrane lesions disappeared and the skin tumors decreased by number and dimensions. In the same time the CD4+ Т cell number increased up to 0.255x109/l and VL values decreased < 400 c/ml. Conclusion: ����������������������������������������������������������������������� Disseminated����������������������������������������������������������� ���������������������������������������������������������� form������������������������������������������������������ ����������������������������������������������������� of��������������������������������������������������� Kaposi’s sarcoma can������������������������������ ��������������������������������� ����������������������������� be��������������������������� �������������������������� provoked by additional immunosuppressive factors like the implementation of PUVA therapy. Early initiation of HAART improves the process and prevents visceral dissemination. Key words: Kaposi������������������������������������������������������������������� ’s sarcoma, skin-mucosal ������������������������������������������������������� dissemination����������������������������� , ��������������������������� HIV������������������������ /����������������������� AIDS������������������� , ����������������� PUVA������������� -������������ photo-chemotherapy, HAART Introduction

Kaposi’s sarcoma (�������������������������������� KS������������������������������ ) ���������������������������� is the first reported malignant tumor associated with HIV-infection. Till 1980 when it first appeared in HIV/AIDS-patients it was a very rare type of sarcoma and affected mainly elderly men in Eastern Europe and the Mediterranean region. Nowadays Kaposhi’s sarcoma is most often found in HIV/AIDS and it can affect about 20% of the untreated patients.1 When internal organs are additionally involved the disease may lead to a fatal end. The aim of this study was to present a case of

56

rapid�������������������������������������������� and���������������������������������������� ������������������������������������������� aggressive����������������������������� ��������������������������������������� cutaneous������������������� ���������������������������� -������������������ mucosal����������� dissemina���������� tion of KS in a HIV/AIDS patient who underwent Psoralen-ultraviolet radiation therapy (РUVА) for total alopecia, as well as the effect of highly active antiretroviral therapy (HAART) in such cases. METHODS

HIV���������������������������������������������� ��������������������������������������������� infection was proved at the National confirming laboratory on HIV/AIDS through ELISA and Western blot (InnoLia HIV I/II Score). The immune cells (CD3+,CD4+,CD8+,CD19+,CD56+) were

Correspondence and reprint request to: N. Popivanova, Department of Infectious Diseases, Parasitology and Tropical Medicine, Medical University, Plovdiv 15A Vassil Aprilov St, 4002 Plovdiv, Bulgaria Received 21 May 2010; Accepted for publication 7 July 2010

Hiv/Aids-Associated Kaposi’s Sarcoma with Multiple Skin-mucosal Disseminations Following Ultraviolet (Рuva) Photochemotherapy

phenotype analyzed����������������������������� (��������������������������� Flow-cytometry������������� ) ����������� with determination of the absolute number and percentage of the leukocyte subpopulations. The viral load (VL), expressed by the number of HIV-RNA copies in a ml of plasma (HIV-RNA copies/ml) was determined by a quantitative PCR test (ROCHE; COBASAmplicor HIV-1 Monitor Test, version 1,5). Skin lesion biopsy material was examined histologically. Routine clinical-laboratory and microbiological investigation as well as scanning of the visceral organs were also performed. RESULTS

The patient we present is a 30 years male who was HIV infected in a heterosexual way. He supposes it happened in 2000 when he divorced and indulged in promiscuous sex with different women including country and foreigner sex-workers. At the end of 2006 the patient got total hair fall out. After a two-year unsuccessful treatment with local medical agents the patient underwent over ���������������� 20 intensive ������������� seances with Psoralen-ultraviolet radiation (РUVА). The physician who applied РUVА-course was not aware of the patient’s HIV status. In the course of radiation therapy the patient noticed a small rounded

bluish formation under his eye. At the end of the seances and immediately after them numerous similar new formations appeared on the skin of the body and upper extremities most of them increasing in dimensions. Tumor infiltrates appeared in the oral cavity mucosa as well. The patient pulled through bronchopneumonia. He turned to the Clinic of Infectious Diseases where we observed multiple erythematous to bluish-violaceous, painless, nonbleeding plaques and nodules on the skin as well as several isolated lesions on the oropharingeal mucous membrane. The disorder was diagnosed as Kaposi’s sarcoma. Biopsy of a lesion was performed. No clinical and CAT data of visceral involvement were found. So we initiated HAART. The photographs demonstrate diffuse skin damage of the head, body and the upper extremities with more than 50 tumor formations (Fig. 1, 2 ,3). Histological data show dilated vascular spaces with large endothelial cells, extravasating erythrocytes and dermal hemosiderin deposits. Spindle-like cells in irregularly formed fascicles were also found (Fig. 4). Table 1 presents the results of immune cells and viral load monitoring before and after HAART application.

Figure 1 and Figure 2. Multiple Kaposi's sarcoma tumor formations on the skin of body and extremities. Folia Medica 2010; 52(3): 56-61 © 2010 Medical University Plovdiv

57

N. Popivanova et al

Figure 3. Nodular form of Kaposi's sarcoma. DISCUSSION

Kaposi’s sarcoma is a vascular tumor which affects the skin, mucous membranes, lymph nodes and

the internal organs. It presents proliferation of the endothelial cells which is induced by the human herpes-virus 8 (HHV-8) and is manifested with four epidemiologic forms.2-4 More than a century ago the Hungarian dermatologist Moritz Kaposi described the first form of Kaposi’s sarcoma which was called Classic KS. It affected elderly men from the Mediterranean region and Eastern Europe and caused deforming and often painful but not fatal tumors of the lower extremities. The second form – African endemic KS is more aggressive and rapidly disseminates into the tissues under the skin, the bone and lymphatic systems as well as into the visceral organs. This fatal form of Kaposhi’s sarcoma affects mainly children and young adults from sub-Saharan Africa. The third, iatrogenic form is also aggressive and it is reported to be found in organ-transplanted patients on immunosuppressive therapy. The fourth form, HIV-induced KS, is one of the first sufferings observed in HIV-infected patients and it is an AIDS-defining disease. In contrast to the classic KS form������������������ , ���������������� the tumor formations in HIV-induced KS affect the upper part of the body and they may also appear on the mucous membranes. It is one of the most aggressive KS forms whose lesions can be found in the stomach, intestines, lymph nodes and the lungs. Independent of the clinical-epidemiological variant HHV-8-DNA was present in almost all KS lesions.1,3 Nevertheless it is well known that HHV-8 is a necessary

Figure 4. Histomorphological characteristics of biopsy from Kaposi's sarcoma: Dilated vascular spaces with large endothelial cells, extravasating erythrocytes and hemosiderin deposits. Spindle-like cells in irregularly formed fascicles. Hematoxylin-eosin staining. x 20.

58

Folia Medica 2010; 52(3): 56-61

© 2010 Medical University Plovdiv

Hiv/Aids-Associated Kaposi’s Sarcoma with Multiple Skin-mucosal Disseminations Following Ultraviolet (Рuva) Photochemotherapy

Table 1. Monitoring of immune cells and viral load in a patient with Kaposi's sarcoma Leukocyte subpopulation Flowcytometric detection of the leukocyte subpopulation

Absolute number of the leukocyte subpopulation (Cell number х109/l) At patient’s Four months Six months Reference presentation in after HAART after HAART Limits* the clinic application application

Lymphocytes

1.314

1.131

0.857

1.0 - 2.8

Total Т-cells CD3+

1.064

0.809

0.596

0.7 - 2.5

0.147

0.255

0.230

0.4 - 1.6

0.874

0.520

0,353

0.2 - 1.1

Total В cells CD19+

0.132

0.161

0.170

0.1 - 0.7

NK cells CD3-CD56+

0.111

0.159

0.087

0.1 - 0.7

Т-helper-inducer CD3+CD4+ Т-suppressor/cytotoxic CD3+CD8+

Percentage of the leukocyte subpopulation Total Т-cells CD3+

81

72

70

59 - 85

Т-helper-inducer CD3+CD4+

11

23

27

28 - 60

Т-suppressor/cytotoxic CD3+CD8+

67

46

41

11 - 38

Total В cells CD19+

10

14

20

6 - 23

NK cells CD3-CD56+

8

14

10

6 - 31

0.17

0.49

0.65

0.9 - 3.6

CD4/CD8 index

HIV-RNA copies/ml

Viral loаd (VL) Automatic quantitative РСR test

VL = 216  000

VL < 400

VL < 50

Limit of distinguishable VL 50 copies/ml**

* Laboratory test complex at the National centre of infectious and parasitic diseases (NCIPD). Authorized laboratory of immunology and allergology, Sofia. ** NCIPD, National confirming laboratory of HIV/AIDS, Sofia.

but not independent causative factor for KS. It is quite probable that HHV-8 acts in combination with some proinflammatory and angiogenic factors, such as altered patient’s response to some cytokines and to HIV-1 trans-activator protein Tat which promotes endothelial cell growth. The genes encoded by HHV-8’s DNA have the potential to provoke cell proliferation and prevent apoptosis.3,4 Furthermore, the latency-associated-nuclear-antigen-type1 protein (LANA-1 ��������������������������������������������� protein�������������������������������������� ) is���������������������������������� ������������������������������������ ��������������������������������� strongly������������������������� ������������������������ expressed��������������� �������������� in������������ ����������� the characteristic for KS spindle-like cells which is considered to be important for sustaining certain malignancy associated with the Kaposi’s sarcoma-Herpes virus (KS-HV).1,4,5 Clinically KS in the early stage is manifested by pink patches that turn into reddish or brownish papules and plaques often evolving into nodular forms. It is quite common to involve mucous membrane surfaces in the process.2 Visceral

Folia Medica 2010; 52(3): 56-61 © 2010 Medical University Plovdiv

dissemination of the tumors is characteristic for the aggressive forms. The histological characteristics of KS are identical for the four clinical forms and they include: neoangiogenesis, inflammation and cellular proliferation.5 The inflammatory infiltrate is prominent above the skin and typically precedes spindle tumor cells formation. Spindle-like cells are disorganized and dispersed among the stromal cells. Eosinophilic hyaline globules which are thought to represent remnants of erythrocytes phagocytosed by the tumor cells, are present within the cytoplasm of the spindle cells or extracellularly.4,9 The lesions themselves are vascularised and contain slit-like vascular channels surrounding a pre-existing blood vessel. No atypia and mitotic activity are found in the endothelial cell monolayer. Extravasating erythrocytes and hemosiderin deposits can be seen in the dermis.2 The KS-lesions may shrink upon

59

N. Popivanova et al

first starting HAART.5-7 Such a phenomenon was observed in our patient, whose treatment included a combination of three reverse transcriptase inhibitors - two nucleoside analogs (abacavir, lamivudin) and one non-nucleoside (efavirenz). After four months of treatment the effect was: disappearing of the formations in the oral cavity, skin lesions regress and fading, an increase of CD4+ cells number and decrease of VL values. Six months after HAART initiation the number of CD4+ Т cells kept above the critical threshold for opportunistic infections and their percentage increased more than two times, the CD4/ CD8 index was significantly increased and the viral load�������������������������������������������������� ������������������������������������������������� was���������������������������������������������� ��������������������������������������������� below���������������������������������������� ��������������������������������������� distinguishable������������������������ ����������������������� values����������������� (��������������� Tabl����������� . 1)������� . Various effects of HAART in KS-patients are described in literature and different combinations of medicines are recommend but the common standpoint of all authors is on the immediate start and the favourable effect of applying such a therapy.5,6,7 We did not find out initial or additional manifestations of any KS visceral dissemination. In contrast to the African type, rarely and predominantly isolated KS skin lesions in HIV/AIDS patients have been observed in Bulgaria. Why such a dissemination appeared in a patient whose CD4+ lymphocytes (although strongly decreased before HAART treatment) had not reached the critical values of isolated T-cells in a microliter of plasma, as we observed in a number of HIV/AIDS patients without KS manifestation, is difficult to explain.8,10 The ���������������������������� role of some medical lotions used by the patient for alopecia treatment and especially РUVА may be suspected to be provoking factors for dissemination. Psoralen toxic effects are well known and documented, and laboratory data show activation of the HIV-1 genome at exposure to UV-radiation, including PUVA. It is reported that UV-radiation may help cancerogenesis through either direct induction of DNA-damages or through reduction of the immune system effectiveness in its role to keep certain herpes viruses under control.11 Breuer-McHam et al. found that ultraviolet radiation induced����������������������������������������� HIV ���������������������������������������� activation�������������������������� in����������������������� ������������������������� the ���������������������� skin�������������� and ������������� they supposed the mechanism was associated with Tat and G2M arrest. The authors also supposed that HIV activation might be inhibited by NF-kB blocking agents.12,13 We could find out only one literature report on a KS’s rapid and aggressive onset in a patient with psoriasis, where the authors discussed the preliminarily applied methods of psoriasis medical treatment.14 CONCLUSIONs

Besides HIV-induced immune insufficiency and

60

promiscuity which facilitated meeting and infecting with HHV-8, Kaposi’s sarcoma and especially the rapid disseminated form of the tumor in our patient may be associated with the co-participation of some additional toxic and immunosuppressive factors such as PUVA-photochemotherapy. There are indications that in patients whose blood VL is reduced to undetectable values thanks to HAART, the process of KS dissemination can be stabilized or reversed and in that way involvement of visceral organs can be avoided. For the present the clinical case described by us arouses hopes on a favourite outcome of the process. REFERENCES

1. Hosein Sean. II Cancer. Kaposi’s sarcoma – past and future. CATIE. RonniLyn Pustil –ed. 2008; 20(5):3-9. 2. Dubina M, Goldenberg G. Viral-associated nonmelanoma skin cancers: a review. Am J. Dermatopathol 2009;31(6):561-73. 3. Ganem D. KSHV and the pathogenesis of Kaposi sarcoma: listening to human biology and medicine. J Clin Invest 2010;120(4):939-49. 4. Schwartz RA, Micali G, Nasca MR, Scuderi L. Kaposi sarcoma: a continuing conundrum. J Am Acad Dermatol 2008;59:179-206. 5. Pantanowitz L, Dezube BJ. Advances in the pathobiology and treatment of Kaposi sarcoma. Current opinion in oncology 2004;16(5):443-9. 6. Еl Amari EB, Toutous-Trellu L, Gayet-Ageron A et al. Predicting the evolution of Kaposi sarcoma in the highly active antiretroviral therapy era. AIDS 2008;22(9):1019-28. 7. Dupin N, Del Giudice P. Editorial commentary: treatment of Kaposi sarcoma in the highly active antiretroviral therapy era. Clin Inf Dis 2008;7(3):418-20. 8. Stebbing J, Powels T, Bower M. AIDS-associated Kaposi’s sarcoma associated with a low viral load and a high CD4 cell count. AIDS 2008;22(4):551-2. 9. Eng W, Cockerell CJ. Histological features of Kaposi sarcoma in a patient receiving highly active antiviral therapy. Am J Dermatopathol 2004;26:127-32. 10. Nguyen HQ, Magaret AS, Kitahata MM, et al. Persistent Kaposi sarcoma in the era of highly active antiretroviral therapy: characterizing the predictors of clinical response. AIDS 2008;22(8):937-45. 11. WHO. Does UV interact with the immune system? http://www.who.int/uv/faq/uvhealtfac/en/index4. html 12. Breuer-McHam J, Actor J, Lewis DE, Duvic M.: Ultraviolet Light Induces HIV Activation in Skin and the Mechanism May Be through Tat and G2M Arrest as Found in Jurkat T-cells. J Invest Dermatol 2000;114(4):836, Abstract #522. Folia Medica 2010; 52(3): 56-61

© 2010 Medical University Plovdiv

Hiv/Aids-Associated Kaposi’s Sarcoma with Multiple Skin-mucosal Disseminations Following Ultraviolet (Рuva) Photochemotherapy

13. Breuer-McHam J, Simpson E, Dougherty I, Bonkobara M, Ariizumi K, Lewis DE, Sharp K, Darwson DB, Duvic M, Cruz PD: Activation of HIV in Human Skin by Ultraviolet B Radiation and its Inhibition by NFkB Blocking Agents. Photochemistry and

Photobiology December 2001;74(6),805-10. 14. De Filippi C, Regazzini R, Sacchi S. Reflections on unexpected onset of Kaposi’s sarcoma in a case psoriasis. G Ital Dermatol Venereol. 1990 Dec;125(12):579-81.

ХИВ/СПИД-связанная Kaposi's sarcoma с множественной кожномукозной диссеминацией после ультрафиолетовой (РUVA) фотохимиотерапии

томография (КАТ) висцеральных органов. Результаты: Кожа лица, грудной полости, спины и верхних конечностей покрыта более чем 50 типичными для Kaposi's sarcoma кожными лезиями, а единичные повреждения видны и в полости рта. Гистологические результаты указывают на дилатированные сосудистые пространства с большими эндотелиальными клетками веретеновидной формы (неправильно сформированные). Мониторинг иммунных клеток и VL до и после применения высокоактивной антивирусной терапии (HAART) показывает CD4+ T cells = 0.147x109/l и VL = 216 000 copies HIV-RNA/ml плазмы при обнаружении страдания и очень хорошее воздействие 4 мес после стартирования HAART: исчезновение мукозных и уменьшение числа и размера кожных лезий, повышение CD4+ Т cells до 0.255x109/l, снижение VL < 400 c/ml. З аключение : Диссеминированная форма Kaposi's sarcoma может быть спровоцированной дополнительными иммуносупрессирующими факторами, как PUVA терапия. Раннее стартирование HAART улучшает процесс и предотвращает висцеральную диссеминацию.

Н. Попиванова, К. Чудомирова, И. Балтаджиев, Ц. Абаджиева

Резюме ХИВ/СПИД инфекция в Болгарии охватывает около 1200 регистрированных пациентов, при чем предполагается, что число необнаруженных случаев в 4 раза выше. Kaposi's sarcoma в стране редко встречается и до настоящего времени кожномукозная диссеминация не описана. Цель: Представить диссеминацию Kaposi's sarcoma у ХИВ/СПИД пациента, леченного ультрафиолетовой радиацией (РUVА) по поводу тотальной алопеции. Методы: Доказать наличие ХИВ посредством ELISA и Western blot (InnoLia HIV I/II Score); РСR (COBAS-Amplicor HIV-1 MT, 1.5) для определения VL; мониторинг – флуоцитометрическое фенотизирование иммунных клеток; биопсия кожного повреждения в целях гистоморфологии; компьютерная аксиальная

Folia Medica 2010; 52(3): 56-61 © 2010 Medical University Plovdiv

61

Folia Medica 2010; 52(3): 62-69 Copyright © 2010 Medical University Plovdiv doi: 10.2478/v10153-010-0008-z

CASE REPORTS Hyperinsulinemic hypoglycemias in infancy and childhood diagnostic therapeutic algorithm with contribution of two cases Nartsis N. Kaleva, Ivan S. Ivanov, Margarita V. Panova, Tatyana D. Shabanova, Darina S. Delgyanska Clinic of Pediatric and Genetic Diseases, St George University Hospital, Medical University, Plovdiv, Bulgaria Abstract Hypoglycemia is not an independent diagnosis. It is a pathophysiological syndrome whose cause needs to be identified. Identifying it is just the first step to making the diagnosis as precisely as possible and to preventing brain damage. Timely diagnosis and treatment are factors of paramount importance for the prognosis of affected patients. The aim of this study was to present two of our patients with hyperinsulinemic hypoglycemia because of the rarity of the condition and to propose a diagnostic-therapeutic algorithm of hypoglycemic syndrome in childhood. Identifying the genetic mutations using DNA analysis for both children enabled us to determine the prognosis and to provide genetic counseling about the next pregnancies in the affected families. We make a detailed classification of different types of hypoglycemia and the various therapeutic modalities: dietary, medicinal and surgical depending on the etiology. It is concluded that the highly specialized examinations which ensure the etiological diagnose, treatment, prognosis and genetic consultation demand the participation of a well trained medical team - both in the clinical division and in the laboratory. Key words: hyperinsulinism, hypoglycemia, infancy, genetic analysis, diagnostic algorithm Introduction

Glucose homeostasis is critical for the realization of brain metabolism; insufficient supply of glucose may cause seizures, irreversible brain damage and even death. In infants the percentage of the brain in relation to the other parts of the body is relatively greater than that in adult individuals. That is why glucose requirements of infant’s brain are relatively greater than those of adult’s brain (6-8 mg/kg/min for infants and 2 mg/kg/min for adults). The brain of the newborn and the infant enlarges and develops rapidly which makes it extremely sensitive to hypoglycemia. Lack of good adaptation to starvation is the most common reason for hypoglycemia in children. The adaptation mechanisms of homeostasis during starvation are: 1. glycogenolysis, 2. gluconeosynthesis, 3. lipolysis and 4. fatty acid oxidation and ketogenesis (Fig. 1).1 Each of these mechanisms is strictly regulated by hormones. Insulin suppresses adaptation to hypoglycemic conditions, while counterinsular hormones such as glucagon, growth hormone, cortisol and epinephrine improve it.

Hyperinsulinism is the most frequent cause of persistent hypoglycemia in infancy. An estimated incidence of hyperinsulinemic hypoglycemia (HH) is 1 in 30  000-50  000 live births. It first appears most often in the neonatal period and in infancy. Its clinical presentation is: high weight at birth, plethoric facies, enlarged internal organs, muscular hypotonia, hypothermia, seizures or loss of consciousness. Later in life the symptoms of hyperinsulinemic hypoglycemia vary - hunger, sleepiness, excessive sweating, confusion, behavioural changes, seizures. No matter what the cause is, hypoglycemia should be identified and treated immediately because of the risk of irreparable brain damage if it persists. Further examinations aim to determine the etiology of hyperinsulinemic hypoglycemia which is a guarantee for an adequate approach - concerning treatment (surgical or medicinal), nutrition (the necessity of a specific diet), prognosis (transient or permanent) and heredity with possible risks of involving other children of the family too.1-3 To date there have been only 5 case reported in Bulgaria of patients with hyperinsulinemic hypoglycemia and 4 of them

Correspondence and reprint request to: N. Kaleva, Clinic of Pediatric and Genetic Diseases, St George University Hospital, Medical University, Plovdiv 15A Vassil Aprilov St, 4002 Plovdiv, Bulgaria 62 Received 10 February 2010; Accepted for publication 31 March 2010

Hyperinsulinemic Hypoglycemias in Infancy and Childhood – Diagnostic Therapeutic Algorithm with Contribution of Two Cases

Figure 1. Factors which influence glucose homeostasis (Kelly A, Stanley CA).

proved to have mutations of АВСС8 gene, which codes for SUR 1 subunit of the АТР- depending К- channels. According to Robert S. Gillespie et al. the incidence of hyperinsulinemic hypoglycemia worldwide is 1 in 25 000 newborns.1 The aim of our study was to present two cases with the extremely rare disorder of hyperinsulinemic hypoglycemia in infancy and the difficulties we had in their diagnosing, genetic specification and treatment, as well as to propose a diagnostic and therapeutic algorithm for the treatment of this rare pathological condition.4-8 Case history

Case 1 M.F.Ch. - a female infant admitted to the Clinic of Pediatric and Genetic Diseases for the first time 8 days after birth (disease summary № 3607/09). The baby was born at the Department of Obstetrics and Gynecology of the Smolyan Hospital for Active Treatment after 6th pathological pregnancy at 35 weeks gestation by C-section because of breech presentation and fetal distress. The parents are Bulgarian Mohammedans and they are third cousins. Previously the mother had given birth to a healthy boy who is now 9 years old and she had had 4 spontaneous abortions all of them in the 3rd lunar month. The baby’s weight at birth was 4500 g, her height was 51 cm, she had macrosomia Folia Medica 2010; 52(3): 62-69 © 2010 Medical University Plovdiv

with Cushingoid habitus and was in a condition of perinatal asphyxia. Ductus arteriosus persistent was also found with a small left-to-right shunt. Laboratory test at the Smolyan hospital showed early neonatal recalcitrant hypoglycemia (on from day 2) with glucose levels down to 0.89 mmol/L. For this reason the infant was transferred to the Department of Neonatology at St. George University Hospital in Plovdiv on day 4 after birth. Because of the persistent hypoglycemia concomitant with apneic pauses and cyanosis independent of the intravenous treatment with carbohydrate solutions and for more accurate determination of diagnosis the baby was transferred to the Clinic of Pediatric and Genetic Diseases in Plovdiv 8 days after birth. The neurological status at admission showed muscular hypotonia, hyporeflexia and suppressed suckling and swallowing reflexes. Transfontaneal ultrasound examination showed evidence of grade II hypoxicischemic encephalopathy. The general physical status of the baby was unsatisfactory, her height was 52 cm, the weight was 4260 g, she had 2-3/6 grade systolic murmur at the left sternal border, her liver was palpable 2 cm below the costal arch with softelastic consistency; the spleen was not palpable. Laboratory findings showed hypoproteinemia with total protein levels of 54-49-53 g/L, hypoalbuminemia with albumin levels of 32-29-30 g/L, without other pathological deviations of the biochemical indicators. The baby had anemic syndrome, which

63

N. Kaleva et al

was corrected by blood transfusion. The lactate levels were normal - 1.4 mmol/L (referent values