Estimated cancer incidence in men by site in EMR Estimated cancer incidence in women by site in EMR Lung Bladder Liver Prostate Colorectum Non-Hodgkin lymphoma Stomach Leukaemia Lip, oral cavity Other and unspecified Breast Cervix uteri Colorectum Ovary Non-Hodgkin lymphoma Liver Leukaemia Lip, oral cavity Thyroid Other and unspecified Eastern Mediterranean Health Journal La Revue de Santé de la Méditerranée orientale Volume 20 / No. 6 June/Juin ÁØ{L ëíP_UÐ{dCÐ hix ëÐ}x~A2014 V l m 2 0 N m b r 6 J n 2 0 1 4 So ur ce : G LO BC A N 2 0 12 EASTERN MEDITERRANEAN HEALTH JOURNAL IS the official health journal published by the Eastern Mediterranean Regional Office of the World Health Organization. It is a forum for the presentation and promotion of new policies and initiatives in health services; and for the exchange of ideas, con- cepts, epidemiological data, research findings and other information, with special reference to the Eastern Mediterranean Region. It addresses all members of the health profession, medical and other health educational institutes, interested NGOs, WHO Col- laborating Centres and individuals within and outside the Region. LA REVUE DE SANTÉ DE LA MÉDITERRANÉE ORIENTALE EST une revue de santé officielle publiée par le Bureau régional de l’Organisation mondiale de la Santé pour la Méditerranée orientale. Elle offre une tribune pour la présentation et la promotion de nouvelles politiques et initiatives dans le domaine des ser-vices de santé ainsi qu’à l’échange d’idées, de concepts, de données épidémiologiques, de résultats de recherches et d’autres informations, se rapportant plus particulièrement à la Région de la Méditerranée orientale. Elle s’adresse à tous les professionnels de la santé, aux membres des instituts médicaux et autres instituts de formation médico-sanitaire, aux ONG, Centres collabora- teurs de l’OMS et personnes concernés au sein et hors de la Région. EMHJ is a trilingual, peer reviewed, open access journal and the full contents are freely available at its website: http://www/emro.who.int/emhj.htm EMHJ information for authors is available at its website: http://www.emro.who.int/emh-journal/authors/ EMHJ is abstracted/indexed in the Index Medicus and MEDLINE (Medical Literature Analysis and Retrieval Systems on Line), ISI Web of knowledge, the Cumulative Index to Nursing and Allied Health Literature (CINAHL), CAB International, Lexis Nexis, Scopus and the Index Medicus for the WHO Eastern Mediterranean Region (IMEMR). ©World Health Organization 2014 All rights reserved Disclaimer The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever on the part of the World Health Organization concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted lines on maps represent approximate border lines for which there may not yet be full agreement. The mention of specific companies or of certain manufacturers’ products does not imply that they are endorsed or recommended by the World Health Organization in preference to others of a similar nature that are not mentioned. All reasonable precautions have been taken by the World Health Organization to verify the information contained in this publication. However, the published material is being distributed without warranty of any kind, either express or implied. The responsibility for the interpretation and use of the material lies with the reader. In no event shall the World Health Organization be liable for damages arising from its use. The named authors alone are responsible for the views expressed in this publication. ISSN 1020-3397 Cover designed by Diana Tawadros Internal layout designed by Emad Marji and Diana Tawadros Printed by WHO Regional Office for the Eastern Mediterranean Cover photograph: WHO/J. Nicholson x{bUFfYwí phCn_UÐp[UÐpe^fe=HCÐçPUehdSüÐocCÐLÚ{[>UÐpheH}UÐpdCÐw phýn=UÐ Ónh]_CÐí hwnaCÐí ÊÐÚùÐ éØnUí ºn4 sxíGUÐí ph[UÐ ÓnY{#Ð ;Ò{x{!Ð ÓÐÚØnCÐí ÓnHnhUÐ TOÎpg@Ywí HCÐ ç hdSl= ngfYd_x nY pÉnBíºÓnYd_CÐ YUÙEQíÔn=úÐsýnií ~TÐ}CÐí ºphf_CÐ phYc"Ð EQÓ5^fCÐ Ð|Tíºphehd_UÐ {wn_CÐ }ýnHíph]UÐ ÓnhdcUÐí ºph[UÐ gCÐ Ên\LÌ @ÚnBíhdSüÐ;p[Un=NegCÐØÐ}RúÐíphCn_UÐp[UÐpe^fY Ypiín_CÐ GBÐçOTogZTÐocBÐ Contents La Revue de Santé de la Méditerranée orientale Eastern Mediterranean Health Journal Vol. 20 No. 6 6 ددع نوشرعلا دلجلما• 2014 • From the Editor-in-Chief Filling the gap: restoring EMHJ’s public health identity .......................................................................................................................................................................................... 359 Editorial Cancer control: a reminder of the need for a balanced approach between prevention and treatment Christopher P. Wild .................................................................................................................................................................................................................................................................................... 360 Research articles Pesticide exposure as a risk factor for lymphoproliferative disorders in adults E.A. Salem, M.M. Hegazy and E.A. El Khouley ............................................................................................................................................................................................................................. 363 Detection and genotyping of human papillomavirus in breast cancer tissues from Iraqi patients S.H.M. Ali, N.A.S. Al-Alwan and S.H.M. Al-Alwany ................................................................................................................................................................................................................... 372 Use of human surplus biospecimens in research: a survey from a cancer centre M. Al-Hussaini and A. Abu-Hmaidan ................................................................................................................................................................................................................................................ 378 Smokeless tobacco consumption in a multi-ethnic community in Pakistan: a cross-sectional study S.M. Abbas, A.Y. Alam, M. Usman and K. Siddiqi ......................................................................................................................................................................................................................... 385 Effect of zinc supplementation in children with asthma: a randomized, placebo-controlled trial in northern Islamic Republic of Iran J. Ghaffari, A. Khalilian, E. Salehifar, E. Khorasani and M.S. Rezaii ........................................................................................................................................................................................391 Knowledge and management of fever among Moroccan parents M. Rkain, I. Rkain, M. Safi, M. Kabiri, S. Ahid and B.D.S. Benjelloun .................................................................................................................................................................................... 397 Case report Mosaïcisme 47,XXY/46,XX et anomalie de la différenciation sexuelle : à propos d’un cas O. Lyhyaoui and A. Gaouzi ....................................................................................................................................................................................................................................................................403 Book 20-6.indb 357 6/17/2014 2:41:00 PM Dr Ala Alwan, Editor-in-chief Editorial Board Professor Zulfiqar Bhutta Professor Mahmoud Fahmy Fathalla Professor Rita Giacaman Dr Ziad Memish Dr Sameen Siddiqi Professor Huda Zurayk International Advisory Panel Dr Mansour M. Al-Nozha Professor Fereidoun Azizi Professor Rafik Boukhris Professor Majid Ezzati Dr Zuhair Hallaj Professor Hans V. Hogerzeil Professor Mohamed A. Ghoneim Professor Alan Lopez Dr Hossein Malekafzali Professor El-Sheikh Mahgoub Professor Ahmed Mandil Dr Hooman Momen Dr Sania Nishtar Dr Hikmat Shaarbaf Dr Salman Rawaf Editors Fiona Curlet, Guy Penet (French) Freelance: Alison Bichard, Marie-France Roux Graphics Suhaib Al Asbahi, Diana Tawadros Administration Nadia Abu-Saleh, Yasmeen Sedky Book 20-6.indb 358 6/17/2014 2:41:00 PM طسوتلما قشرل ةيحصلا ةلجلمانوشرعلا دلجلما سداسلا ددعلا 359 From the Editor-in-Chief Filling the gap: restoring EMHJ’s public health identity The Eastern Mediterranean Health Journal, EMHJ, is 20 years old this year. What started with a few submissions and two issues in 1995 has become a monthly publication with over 700 submissions a year. From the outset EMHJ’s stated aims emphasized a public health scope. However, at a time when there were few health/medical journals in the Region and limited publication opportunity for researchers in the Region, EMHJ sought to encourage all health research and provide a venue for researchers to publish their work. This EMHJ successfully did but in the process the Journal’s public health voice was muted with many of the papers pub- lished of limited public health or wide regional relevance. Twenty years later, the landscape of publications in the Region has completely changed, necessitating a refocus of EMHJ on its original mission. From 17 health and biomedi- cal journals indexed in the Index Medicus of the Eastern Mediterranean Region (IMEMR) there are now over 520 published in 20 countries of the Region, offering researchers greater opportunity for publication in many different fields. What is still lacking however is a journal that represents the regional voice for public health and which offers a platform dedicated to raising the profile of public health in the Region. The need for such a platform is evident: public health research and information are vital to provide evidence to inform the development of public health policies and programmes, to keep public health professionals abreast of new policies and initiatives in public health, and to allow the exchange of ideas and concepts in public health. While the Region is quite diverse, there are many commonalities and existing commitments to common action making it expedi- ent to focus on promoting and disseminating public health research and knowledge across the Region, and indeed beyond. EMHJ, as one of the few journals in the EMR with regional and international reach, is in a position to fill the gap, a fact recognized by the new Editorial Board of EMHJ. Thus, at its first meeting in March 2013, a key recommen- dation of the Board was to restore the focus of EMHJ on public health. In the past year the Journal has been more selective in what it considers for publication placing emphasis on arti- cles that focus on public health research and practice. This approach is guided by the decision of the Regional Commit- tee and the regional health priorities it has endorsed, namely health systems’ strengthening, maternal, reproductive and child health and nutrition, noncommunicable diseases, communicable diseases, and emergency preparedness and response. At the same time, for research to provide useful evidence, it must be sound and credible, therefore more rigorous criteria for acceptance have been applied in selec- tion of papers for publication in EMHJ to ensure they meet acceptable standards of scientific rigor and quality. EMHJ, like all research journals, depends on unsolicited submissions, and in order to attract good quality research we recognize that researchers need to be confident that their papers will be efficiently processed and, if accepted, speedily published and widely disseminated. To this end, we introduced an online submission and peer review system last October to streamline the workflow and have succeeded in significantly reducing the lead time from sub- mission to publication. Moreover, the Journal’s visibility is rising. All issues are available and freely accessible on the EMHJ website and visits to the site are climbing. Half of the top 10 countries accessing the EMHJ website in 2013 were from outside the Region, highlighting our international reach. EMHJ’s mailing list continues to expand and email alerts are sent out to inform readers when the latest issue is published. Furthermore, EMHJ is included in numerous international and regional bibliographic databases and indexes, including Medline/PubMed, and is now covered in the Thomson Reuters Web of Science. By 2016, EMHJ will have an impact factor, undoubtedly an important consideration in a researcher’s decision to publish in a given journal. While research papers are our lifeline, equally impor- tant is our role to raise awareness of and keep public health professionals informed about important public health issues and initiatives, especially in relation to the Region. We have therefore extended the content of EMHJ to in- clude invited editorials and reviews, and summary reports of important EMRO meetings with other sections under consideration, such as policy briefs, and perspectives and brief communications for policy-makers and public health practitioners to share their experiences. EMHJ’s focus is firmly back on public health. With a mission to contribute to improving health in the Region through publishing and publicising high quality public health research and information, EMHJ is the public health journal of the Region. Book 20-6.indb 359 6/17/2014 2:41:00 PM EMHJ • Vol. 20 No. 6 • 2014 Eastern Mediterranean Health Journal La Revue de Santé de la Méditerranée orientale 360 Editorial Cancer control: a reminder of the need for a balanced approach between prevention and treatment Christopher P. Wild 1 The recent World Cancer Report 2014 highlighted a number of criti- cal points relevant to cancer control, encompassing occurrence, causes, molecular and cellular mechanisms and early detection, prevention and treatment of the disease (1). There are clear pointers from the body of knowledge in this Report which should contribute to the evidence base for cancer control. Cancer is a growing problem. The GLOBOCAN estimates for 2012 were 14.1 million new cases and 8.2 million deaths worldwide. However, the population demographics of age- ing and growth will result in around 40% more new cancer patients annu- ally by 2025. In the countries of the World Health Organization (WHO) Eastern Mediterranean Region (EMR), the estimated increase is from 555 000 to 819 000 new cases over this period, an increase of 68% in just over a decade (2). In terms of disa- bility-adjusted life years, the low- and middle-income countries are already heavily affected because the common cancers (e.g. liver, stomach, cervix) occur at younger ages and have poorer survival rates (3,4). Indeed, cancer is one of the most common causes of death in the Arab world, ranking third in Oman, for example (5). There is a risk of under-prioritizing cancer, how- ever, because global burden analyses commonly present neoplasms sepa- rately for each organ, and the result is that cancer does not appear in the 20 most common causes of death in EMR (6). Whilst cancer is a universal problem, it is not a uniform one. The patterns and related causes differ markedly by geographical region and level of human development. This het- erogeneity has important implications. For example, chronic infections are linked to 1 in 3 cancers in sub-Saharan Africa but only 1 in 30 in Australia (7). Clearly this must translate into different priorities for national cancer control plans. In the EMR countries the 5 most common cancers among men are lung, bladder, liver, prostate and colorectum, and among women are breast, colorectum, cervix, ovary and non-Hodgkin lymphoma. In con- trast to men, for whom the incidence of these 5 cancers is quite similar, the pattern in women is dominated by breast cancer, with 1 in 3 of all new cancers being breast cancer. In ad- dition, the importance of individual cancer sites varies substantially across EMR countries, e.g. more than 3-fold for cancer of the lung and breast. It is important to note that the above estimates are frequently based on limited national data, circumstanc- es under which it is difficult for coun- tries to make rational cancer control plans. The need to prioritize support to population-based cancer registries has led the International Agency for Research on Cancer and WHO to develop the multi-partner project the Global Initiative for Cancer Registries (8). In the first instance, priority should be given to developing high-quality cancer registries covering populations in defined geographical areas within countries (e.g. a district or the capital region), rather than attempting to implement registration covering the entire country. Initiating national can- cer registration is usually an unrealistic prospect in low- and middle-income countries; either it is technically un- feasible or the cost involved greatly outweighs the additional benefits ob- tained from registration of a sample of the population (9). What priorities should be set to meet this growing cancer challenge? Even in the highest income countries the spiralling costs of cancer treat- ment and care are difficult to sustain. In many countries access to therapy remains limited and personalized medicine is far off. It is implausible for the world to treat its way out of cancer. A high priority must be placed on prevention and early detection of the disease, particularly in low and middle-income countries. Identification of risk factors is the foundation of cancer prevention, and much is already known about this, with typical estimates of 30%–50% of cancers being preventable, based on current knowledge (10). The high rates of smoking in EMR countries only serve to emphasize that the first priority must be tobacco control, no- tably implementation of the WHO Framework Convention on Tobacco 1Director, International Agency for Research on Cancer, Lyon, France. Book 20-6.indb 360 6/17/2014 2:41:00 PM طسوتلما قشرل ةيحصلا ةلجلمانوشرعلا دلجلما سداسلا ددعلا 361 Control. Other priorities on a global scale include vaccinations against human hepatitis B virus and human papilloma viruses, limiting excessive sun exposure, avoiding occupational and environmental carcinogens and measures aimed at tackling alcohol consumption, obesity and physical inactivity (10). Measures to combat some Region-specific risks, including transmission of hepatitis C virus and schistosomiasis, are also important. In many instances preventive ac- tion at the individual level needs to be complemented by legislation and regulation, as has been demonstrated by successes in the case of tobacco control (11). Despite our knowledge about car- cinogenic risks there remain a number of common cancers for which the causes are still poorly understood (e.g. prostate, pancreas, kidney, brain and haematological cancers). Further re- search here is merited and could make use of geographical and temporal vari- ations in cancer patterns or exposures to throw fresh light on risk factors. Examples from the EMR countries include: the causes of the relatively high rates of lymphoma; the impact of air pollution from natural as opposed to man-made sources; the proportion of liver cancer attributable to hepati- tis B and C; the risks associated with waterpipe smoking; and evaluation of exposure to depleted uranium. Re- search studies should seek to utilize the exciting new knowledge and as- sociated laboratory tools developed from studying the molecular and cel- lular underpinnings of cancer towards a better understanding of both causes and prevention (12). Tackling questions of diet, obesity and physical inactivity should be pri- orities for countries of EMR. Certainly the development of adapted, stand- ardized dietary surveillance tools to study nutritional transitions in these countries would provide much-need- ed support to address the epidemic of obesity in both adults and children. In addition, the implementation and evaluation of programmes and poli- cies on diet and obesity, adapted to the Region, need to be prioritized. The etiology of breast cancer, for instance, is dominated by risk factors such as late childbearing and low parity that are related to economic transition and that are less amenable to prevention. Nevertheless, overweight, obesity and lack of physical activity are important and avoidable risk factors in post- menopausal women and should be addressed. The long natural history of cancer and identifiable pre-cancerous lesions in some tumour types offer opportuni- ties for secondary cancer prevention through early detection, including screening. Effective screening depends on having a national, organized pro- gramme. Screening tests, which are just one component of a successful programme, are available for cancers of the cervix, breast, colorectum and oral cavity (in high-risk subjects). There can be no higher priority for sec- ondary cancer prevention in the EMR than identifying effective approaches to the early detection of breast cancer, coupled with adequate treatment and follow-up care. There often remain barriers to implementation of preventive inter- ventions, with a gap between dem- onstration of effectiveness in clinical or community-randomized trials and implementation in everyday health care. Lessons come from cervical and breast cancer screening programmes in Latin America, which have failed to provide tangible benefits despite considerable investment (13,14). In response, there is a clear need for implementation of operational research, making good use of pilot or demonstration studies, to assess the barriers (structural, social, profes- sional) to successful cancer control measures. In addition, interventions are often implemented without ongoing evaluation. This is a missed opportunity. First, because inclusion of a research component into a na- tional programme will provide vital information to improve cancer con- trol programmes; secondly, because the cost of research, where national programmes are already funded, is relatively low; and thirdly, because it provides an opportunity to build national research capacity in areas that are close to policy. In summary, cancer is a complex set of heterogeneous diseases, with remarkable variation in distribu- tion, risk factors, biology, pathology, detection, diagnosis and treatment. Although important cancer control initiatives, particularly in the domain of primary prevention, come through the WHO Global Action Plan for the Prevention and Control of Noncom- municable Diseases 2013–2020, the requirements for comprehensive cancer control in tune with national situations and priorities, especially in the domain of early detection and treatment, demand more focussed in- puts (15). One has only to look at the overwhelming prominence of breast cancer in the EMR countries and at the need for access to specialized tertiary-care centres for cancer therapy to realize that a national cancer control plan must stretch beyond the core elements of the WHO noncommu- nicable diseases agenda. Only in this way will an adequate response emerge to reduce the suffering associated with cancer worldwide. Acknowledgements The author would like to thank Drs Bray, Franceschi and Sankarana- rayanan for their helpful comments and also all colleagues from WHO/ EMRO who provided stimulating discussion at the Regional meeting on Cancer Control and Research Pri- orities in the Eastern Mediterranean Region held in Doha, Qatar, from 20 to 22 October 2013. Book 20-6.indb 361 6/17/2014 2:41:01 PM EMHJ • Vol. 20 No. 6 • 2014 Eastern Mediterranean Health Journal La Revue de Santé de la Méditerranée orientale 362 References 1. Stewart BW, Wild CP. World Cancer Report 2014. Lyon, France: International Agency for Research on Cancer; 2014. 2. GLOBOCAN 2012: estimated cancer incidence, mortality and prevalence worldwide in 2012 [Internet]. Lyon, France: International Agency for Research on Cancer; 2012 (http:// globocan.iarc.fr/Pages/fact_sheets_cancer.aspx, accessed 21 March 2014). 3. Soerjomataram I, Lortet-Tieulent J, Parkin DM, Ferlay J, Mathers C, Forman D, et al. Global burden of cancer in 2008: a system- atic analysis of disability-adjusted life-years in 12 world regions. Lancet. 2012 Nov 24;380(9856):1840–50. PMID:23079588 4. Sankaranarayanan R, Swaminathan R, Brenner H, Chen K, Chia KS, Chen JG, et al. Cancer survival in Africa, Asia, and Cen- tral America: a population-based study. Lancet Oncol. 2010 Feb;11(2):165–73. PMID:20005175 5. Alwan A. Global status report on noncommunicable diseases 2010. Geneva: World Health Organization; 2011. 6. Mokdad AH, Jaber S, Aziz MI, AlBuhairan F, AlGhaithi A, AlHam- ad NM, et al. The state of health in the Arab world, 1990–2010: an analysis of the burden of diseases, injuries, and risk factors. Lancet. 2014 Jan 25;383(9914):309–20. PMID:24452042 7. De Martel C, Ferlay J, Franceschi S, Vignat J, Bray F, Forman D, et al. Global burden of cancers attributable to infections in 2008: a review and synthetic analysis. Lancet Oncol. 2012 Jun;13(6):607–15. PMID:22575588 8. Global Initiative for Cancer Registry Development (GICR) [Internet]. Lyon, France: International Agency for Research on Cancer; 2014 (http://gicr.iarc.fr, accessed 21 March 2014). 9. Bray F, Znaor A, Cueva P, Korir A, Swaminathan R, Ullrich A, et al. Planning and developing population-based cancer registration in low- or middle-income settings. IARC Techni- cal Publication No. 42. Lyon, France: International Agency for Research on Cancer (http://www.iarc.fr/en/publications/ pdfs-online/treport-pub/treport-pub43/index.php accessed 28 May 2014). 10. Vineis P, Wild CP. Global cancer patterns: causes and preven- tion. Lancet. 2014 Feb 8;383(9916):549–57. PMID:24351322 11. Chaloupka FJ, Straif K, Leon ME; Working Group, International Agency for Research on Cancer. Effectiveness of tax and price policies in tobacco control. Tob Control. 2011 May;20(3):235– 8. PMID:21115556 12. Wild CP. The exposome: from concept to utility. Int J Epide- miol. 2012 Feb;41(1):24–32. PMID:22296988 13. Murillo R, Almonte M, Pereira A, Ferrer E, Gamboa OA, Jerón- imo J, et al. Cervical cancer screening programs in Latin Amer- ica and the Caribbean. Vaccine. 2008;26 Suppl 11:L37–L48. PMID:18945401 14. Lee BL, Liedke PE, Barrios CH, Simon SD, Finkelstein DM, Goss PE. Breast cancer in Brazil: present status and future goals. Lan- cet Oncol. 2012;13:e95–e102. PMID:22381937 15. Wild CP. The role of cancer research in noncommunicable disease control. J Natl Cancer Inst. 2012 Jul 18;104(14):1051–8. PMID:22781435 Book 20-6.indb 362 6/17/2014 2:41:01 PM طسوتلما قشرل ةيحصلا ةلجلمانوشرعلا دلجلما سداسلا ددعلا 363 Pesticide exposure as a risk factor for lymphoproliferative disorders in adults E.A. Salem,1 M.M. Hegazy 2 and E.A. El Khouley 3 ABSTRACT In view of the widespread use of pesticides in Egypt and the increasing incidence of leukaemia and lymphoma we aimed to assess pesticide exposure and other selected variables as risk factors for lymphoproliferative disorders (leukaemia and non-Hodgkin lymphoma). In a hospital-based, retrospective, case–control study in 2011– 2012, adult cases of lymphoproliferative disorders (n = 130) were recruited from outpatient clinics in Menoufia, Egypt, while controls (n = 130) were age- and sex-matched fracture patients. Family history of cancer, exposure to X-rays, smoking and use of hair dyes were not risk factors for lymphoproliferative disorders in univariate analysis. History of exposure to pesticides and HCV infection were significant risk factors for lymphoproliferative disorders in multivariate analysis (OR = 2.24; 95% CI: 1.22–4.11 and OR = 2.67; 95% CI: 1.50–4.80 respectively). The risk was significant for cases of non-Hodgkin lymphoma but not chronic lymphocytic leukaemia. 1Department of Public Health and Community Medicine; 2Department of Forensic Medicine and Clinical Toxicology; 3Department of Clinical Oncology; Faculty of Medicine, University of Menoufiya, Menoufiya, Egypt (Correspondence to E.A. Salem: dr_eman53@yahoo.com). Received: 07/03/13; accepted: 23/02/14 ينغلابلا ىدل يوافمللا رثاكتلا تابارطضلا راطتخا لماوع اهرابتعاب ماولها تاديبلم ض ُّرعتلا ليولخا سانيإ ،يزاجح حرفم ،لماس نمايإ نوثحابلا فدهتـسا ،اـموفمللاو مدـلا ضاـضيبا تلادـعم دايدزا عـمو ،رـم في ماوـلها تادـيبلم قاـطنلا عـساولا لماعتـسلاا ءوـض في :ةـصلالخا ضاــ ضيبلاا( يواــ فمللا رــ ثاكتلا تاــ بارطضلا ةاــ قتنم راــ طتخا لــ ماوع اــ هرابتعاب ةــ عونتم تارــ غتم عــ م ماوــ لها تادــ يبلم ضرــ عتلا مــ ييقت ،2012-2011 يــ ماع في دهاوــ شلاو تلااــ حلل ىفــ شتسلما ىــ ع زــ كترت ةيداعتــ سا ةــ سارد نوــ ثحابلا ىرــ جأو ،)ةــ ينيكجدوهلالا اــ موفمللاو 130 عـم ،رـم ،ةـ يفونلما في ةـ يجرالخا تاداـ يعلا نوـعجاري نـمم يواـفمللا رـ ثاكتلا تاـ بارطضاب نـباصلما نـغلابلا نـم ةـ لاح 130 تلمـش ةـيسرلأا قباوـسلا نـم ًلاك نأ نـثحابلل حـضتاو .رـمعلاو سـنلجا ثـيح نـم تلااـلحا نوـلثماُي نـيذلا نـيرخلآا ضىرـلما نـم دهاوـشلا نـم تاـبارطضاب ةـباصلإا راـطتخا لـماوع نـم تـسيل رعـشلا ةـغبصأ مادختـساو ،نـخدتلاو ،ةينيـسلا ةعـشلأل ض ُّرـعتلاو ،ناـطسرلاب ةـباصلإل يدـبكلا باـهتللاا سورـفب ىودـعلاو ماوـلها تادـيبلم ض ُّرـعتلا قباوـس اـمأ .تارـياغتلما دـيحولا لـيلحتلا قـفو كـلذو ،يواـفمللا رـثاكتلا لدـعم( تارـياغتلما ددـعتلما لـيلحتلا قـفو يواـفمللا رـثاكتلا تاـبارطضلا ًاـيئاصحإ اـبه ّدـتعُي يـتلا ةـّمهلما راـطتخلاا لـماوع نـم تـناكف سي ةـلصافب 2.67 ةـيحجرلأا لدـعمو ماوـلها تادـيبلم ضرـعتلل ةبـسنلاب 4.11و 1.22 نـب جـئاتنلا حُوارـت عـم % 95 ةـقث ةـلصافب ،2.24 ةـيحجرلأا اــ به دــ تعي ةــ يهمأ اذ راــ طتخلاا ناكو .)سي يدــ بكلا باــ هتللاا سورــ فب ىودــ علل ةبــ سنلاب 4.80و 1.50 نــ ب جــ ئاتنلا حُوارــ ـت عــ م % 95 ةــ قث .نـمزلما يواـفمللا ضاـضيبلال ةبـسنلاب كـلذك سـيل هـنكلو ،ةـينيكجدوهلالا اـموفملل ةبـسنلاب ًاـيئاصحإ Exposition aux pesticides en tant que facteur de risque des maladies lymphoprolifératives chez l'adulte RÉSUMÉ Étant donné l'utilisation largement répandue des pesticides en Égypte et l'incidence croissante de la leucémie et du lymphome, nous avons tenté d'évaluer l'exposition aux pesticides et d'autres variables sélectionnées en tant que facteurs de risque des maladies lymphoprolifératives (leucémie et lymphome non-hodgkinien). Dans une étude cas-témoins rétrospective menée en milieu hospitalier en 2011–2012, des adultes atteints de maladies lymphoprolifératives (n = 130) ont été recrutés dans des services de consultations externes à Menoufia (Égypte), tandis que les témoins appariés pour l'âge et le sexe (n = 130) recrutés souffraient de fractures. Des antécédents familiaux de cancer, d'exposition aux rayons-X, de consommation de tabac et d'utilisation de teintures capillaires ne constituaient pas des facteurs de risque des maladies lymphoprolifératives selon une analyse univariée. Des antécédents d'exposition aux pesticides et d'infection par le virus de l'hépatite C étaient des facteurs de risque importants de certaines maladies lymphoprolifératives à l'analyse multivariée (OR = 2,24 ; IC à 95 % : 1,22–4,11 et OR = 2,67 ; IC à 95 % : 1,50–4,80 respectivement). Le risque était significatif pour les cas de lymphome non- hodgkinien mais non significatif pour la leucémie lymphocytaire chronique. Book 20-6.indb 363 6/17/2014 2:41:01 PM EMHJ • Vol. 20 No. 6 • 2014 Eastern Mediterranean Health Journal La Revue de Santé de la Méditerranée orientale 364 Introduction Lymphoproliferative disorders are a heterogeneous group of malignant clonal proliferations of lymphocytes. They include disorders of B-, T- and NK-cell lineages all of which are further classified as distinct entities including chronic lymphocytic leukaemia (CLL), non-Hodgkin lymphoma (NHL), Hodgkin lymphoma, Waldenström macroglobulinaemia and multiple my- eloma (1). NHL is a heterogeneous group of over 20 different B- and T- cell neoplasms affecting the immune/ lymphatic system and arising primarily in the lymph nodes (2). Egypt has one of the highest incidence of lymphoma in the world, mainly NHL, which is higher than even the United States of America (USA) (2,3) as well as other developed nations, where Hodgkin lymphoma are more common. In Egypt, NHL is the second most common cancer in adults, and lymphoma is the most common cancer in children (4). The reasons for the increase in NHL are largely unexplained. A known pre- disposing factor that is strongly associ- ated with NHL is immunosuppression (5). Several other risk factors have been identified, including exposure to infec- tious agents and chemical toxins (6,7). Pesticides, which are widely used in agriculture, comprise a wide variety of chemicals mainly used to control dam- age to plants from insects (insecticides), mould (fungicides) and weeds (her- bicides). In Egypt, several pesticides including organophosphate, carbamate and pyrethroid insecticides, fungicides and herbicides are commonly used to increase agricultural productivity (8). Organophosphate pesticides represent more than 80% of the total insecticides used in Egypt (9). While many pesti- cides are carcinogenic in animals and can act through several mechanisms to cause cancer in animals, their role in human carcinogenesis remains has not been fully clarified (10). A signifi- cant association between NHL and agricultural use of organophosphate and carbamate pesticides has been re- ported in studies from the USA (11), Israel (12) and Australia (13). Consid- ering the widespread use of pesticides in Egypt, together with an increasing incidence of leukaemia and lymphoma, it is important to investigate whether there is an association between pesti- cide exposure and lymphoproliferative disorders or not. This study aimed to assess pesti- cide exposure as a risk factor for lym- phoproliferative disorders (leukaemia and NHL) in adults and to study the possible role of other risk factors for leukaemia and NHL in adults, including hepatitis C virus (HCV) infection, fam- ily history of cancer, smoking, exposure to X-rays and use of hair dyes. Methods This was a hospital-based, retrospective, case–control study carried out from 1 April 2011 to 31 October 2012. The study was limited to adults as the epide- miology of childhood lymphoprolifera- tive disorders differs markedly from that of adults regarding specific causes such as post-transplantation conditions or specific infections such as Epstein-Barr virus (14). Subjects Patients Cases of lymphoproliferative disorders (n = 130) were recruited during the pe- riod of the study from patients attending outpatient clinics at the department of oncology, Menoufia University Hospi- tal. The Hospital is a tertiary care centre for Menoufia Governorate, Egypt and these clinics provide outpatient care regarding diagnosis and follow up for patients with a variety of malignan- cies of varying acuity levels. Patients included in this study were over the age of 18 years; 85% of those who were approached consented to participate in the study. They were diagnosed as CLL or NHL after a clear diagnosis which was validated by the clinical oncologist mainly with the help of the surgeons (for biopsy taking), pathologist (for bi- opsy reading and immuophenotyping), clinical pathologist (for laboratory data) and radiodiagnosis physician (for imag- ing). To avoid immunosuppression in these patients all cases were selected before initiation of chemotherapy. The studied cases included 23 cases of CLL and 107 cases of NHL. Diagnostic criteria The diagnosis of CLL was based on the following (15): blood count (for presence of lymphocytosis, i.e. median values 30–50 × 109 of lymphocytes/L) ; bone marrow biopsy (bone marrow is usually hypercellular but can be normocellular; the most characteristic feature is the presence of at least 30% mature-appearing lymphocytes); im- munophenotyping (more than 95% of all cases of CLL have a B-cell pheno- type with monoclonality by light-chain restriction); and cytogenetic and mo- lecular findings (a number of recurrent cytogenetic abnormalities have been identified in CLL). Diagnostic evaluation of patients with NHL included the following (15): history-taking (night sweats, weight loss, fever; neurologic, musculoskeletal or gastrointestinal symptoms); physi- cal examination (lymphadenopathy; pericardial rub, pleural effusion, breast masses; hepatosplenomegaly, bowel obstruction, renal mass, and testicular or ovarian mass; spinal cord compression); biopsy of peripheral lymphadenopa- thy (excisional biopsy); computerized tomography scan of the neck, chest, abdomen and pelvis (to detect enlarged lymph nodes and hepatosplenomegaly and for initial staging and assessment of treatment response); bilateral bone marrow aspirate and biopsy (trephine biopsy should always be carried out if a diagnosis of lymphoma is suspected); complete blood count (peripheral blood lymphocytosis with circulating Book 20-6.indb 364 6/17/2014 2:41:02 PM طسوتلما قشرل ةيحصلا ةلجلمانوشرعلا دلجلما سداسلا ددعلا 365 • History of exposure to various envi- ronmental risk factors, e.g. smoking, X-rays, malaria infection, hair dyes. Serological analysis Blood samples were taken from par- ticipants and serological analysis was carried out for anti-HVC antibodies in serum. Anti-HCV was detected by 3rd-generation enzyme-linked immu- nosorbent assay (ELISA) (Innotest kit, Innogenetics). This qualitatively deter- mines the presence of antibodies to 4 recombinant HCV proteins in serum. The test was done at the laboratory of the National Liver Institute. Statistical analysis The results were collected, tabulated and statistically analysed using SPSS statis- tical package, version 11. The data are presented as descriptive statistics and the chi-squared test was done to study the association between qualitative vari- ables, with Fisher exact test done when more than 25% of the cells contained an expected count < 5. A stepwise logistic re- gression analysis was done to determine the adjusted odds ratio (OR) and 95% confidence interval (CI) for the different risk factors and malignancy. P value < 0.05 was considered statistically significant. Results Background data The mean age of patients with lym- phoproliferative disorders was 46.9 years, and for those with NHL the mean age was 47.5 years. There were more males than females among the cases (59.2% versus 40.8%) and a high proportion lived in rural areas (60.8%). One-third worked as farmers (32.3%). Table 1 shows that there were no statistically significant differences between patients with lymphoprolif- erative disorders and controls regarding age, sex, residence, level of education and occupation (P > 0.05). Most of the females among the cases and controls were housewives. malignant cells is common in low-grade lymphoma); general chemistry panel (including determination of lactate dehydrogenase level, which is manda- tory); HBV and HCV panels (espe- cially in patients anticipated to receive monoclonal antibody therapy and/or chemotherapy); and HIV testing (for association). Controls An equal number of adult controls (n = 130) were recruited, matched to the cases according to age and sex. The cri- teria for selection were being physically and mentally capable of participating and free from cancer and lymphad- enopathy on clinical examination. They were sampled from among all patients of the department of orthopaedics, Me- noufia University Hospital who were arriving with fractures after accidents; 88% of the eligible controls who were contacted agreed to participate in the study. They would be representative of the source population of the cases by region, since both the oncology and or- thopaedic departments of the hospital serve patients from the same area. On the other hand, the risk of exposure to pesticides and to fractures are very likely to be independent. Verbal informed consent was ob- tained from all participants in the study. Ethical approval to perform the study was obtained from the ethics committee in the Faculty of Medicine, University of Menoufia and the management board of the hospital. Data collection All cases and controls were given a ques- tionnaire and serological analysis. Questionnaire Questionnaires have been used success- fully to assess chronic pesticide exposure among farmers, farm workers and the general population, as long-lived bio- logical markers are not available (16). A predesigned questionnaire was pre- pared and validated by the researchers and was pilot tested on 15 patients and 15 controls (out of the participants) at the Hospital to test reliability. Modi- fications were made according to the results obtained. The questionnaire was administered to all participants in a face-to-face interview that lasted 20–30 minutes. The interview was conducted at the hospital by the researchers and 2 well-trained nurses from the hospital who were blind to the study groups. In order to avoid recall bias that occurred after diagnosis of the cases and to allow a minimum latency period, a period of 1 year before the reference date (date of diagnosis) for both cases and controls was used as the duration of recorded exposure. The questionnaire included the follow- ing items: • Sociodemographic data (age, sex, residence, marital status, level of edu- cation, and occupation). Occupation was defined as ever held the occupa- tion if respondents worked for at least 1 year in that occupation. • Family history of malignancies in any first-degree relatives, if it could be ac- curately recalled by the participants. • History of exposure to pesticides (yes/no): types of pesticides (to as- sist participants in recalling the pesti- cides used, a list of the most common brand names was provided); dura- tion of exposure to pesticides; appli- cation method used most often; and use of protective equipment. In this study, the term pesticide referred pri- marily to herbicides, insecticides and fungicides, which are the commonest types used in Egypt. The duration of pesticide use was categorized into 2 groups based on the median years of exposure among subjects who used insecticides (≤ 5 and > 5 years). In- formation on habits such as changing clothes before entering the house, smoking and eating during applica- tion of pesticides and accidents (e.g. accidental poisoning) were recorded. Non-occupational use of pesticides (home, garden) was also included. Book 20-6.indb 365 6/17/2014 2:41:02 PM EMHJ • Vol. 20 No. 6 • 2014 Eastern Mediterranean Health Journal La Revue de Santé de la Méditerranée orientale 366 Risk factors for all lymphoproliferative disorders A history of exposure to pesticides was reported by 38.5% of patients with lym- phoproliferative disorders compared with 26.2% of controls and this was a significant difference (χ2 = 4.50, P < 0.05). Of those exposed to pesticides, significantly more of the cases of lym- phoproliferative disorders had a long duration of exposure (> 5 years) than the controls (86.0% versus 64.7%) (χ2 = 4.10, P < 0.05) (Table 2). No statistically significant differences were found be- tween patients with lymphoproliferative disorders and controls regarding types of pesticide exposure, the main crops sprayed, use of protective measures, and history of regular use of household insecticides (P > 0.05) (Table 2). Regarding other risk factors, infec- tion with HCV was significantly associ- ated with lymphoproliferative disorders (39.2% of cases versus 21.5% of con- trols) (χ2 = 9.62, P < 0.01). None of the other studied variables—family history of cancer, exposure to X-rays, smoking and use of hair dyes—were found to be risk factors for lymphoproliferative disorders (P > 0.05) (Table 3). History of exposure to pesticides and HCV infection were still signifi- cantly associated with lymphoprolifera- tive disorders after adjustment for age, sex and residence (OR = 2.24; 95% CI: 1.22–4.11 and OR = 2.67; 95% CI: 1.50–4.80 respectively) (Table 4). Risk factors for non-Hodgkin lymphoma Similar results were found when analys- ing the cases of NHL separately. A sig- nificant association was shown between NHL and a history of exposure to pesticides (41.1% of cases versus 26.2% of controls) (χ2 = 5.95, P < 0.05) and be- tween NHL and HCV infection (40.2% of cases versus 21.5% of controls) (χ2 = 9.73, P < 0.01) (Table 5). These associa- tions were retained after adjustment for age, sex and residence (OR = 2.46; 95% CI: 1.31–6.63) for exposure to pesticides and (OR = 2.78; 95% CI: 1.51–5.14 for HCV infection) (Table 6). Risk factors for leukaemia No significant association was found with history of exposure to pesticides nor with HCV infection when analysing the cases of CLL separately (P > 0.05) (Table 7). Also, neither family history of cancer nor history of smoking were risk factors for CLL (P > 0.05). Discussion The etiology of NHL and leukaemia is still largely unknown, but exposure to chemicals, in particular pesticides, has been suggested to be a risk factor (17). Table 1 Sociodemographic data of the studied patients with lymphoproliferative disorders (LPD) and controls Sociodemographic data LPD cases (n = 130) Controls (n = 130) Total (n = 260) Test of significance P-value Age (years) Mean (SD) 46.9 (10.9) 48.3 (9.8) 47.6 (10.3) t = 1.11 > 0.05 Range 23–74 26–71 23–74 No. % No. % No. % Sex Male 77 59.2 81 62.3 158 60.8 > 0.05 Female 53 40.8 49 37.7 102 39.2 Residence Urban 51 39.2 46 35.4 97 37.3 χ2 = 0.41 > 0.05 Rural 79 60.8 84 64.6 163 62.7 Marital status Never married 14 10.8 11 8.5 25 9.6 χ2 = 0.39 > 0.05 Ever married 116 89.2 119 91.5 224 86.2 Education level Illiterate 44 33.8 42 32.3 86 33.1 Primary education 43 33.1 45 34.6 88 33.8 χ2 = 1.22 > 0.05 Secondary education 32 24.6 36 27.7 68 26.2 University and above 11 8.5 7 5.4 18 6.9 Occupation Farmer 42 32.3 29 22.3 71 27.3 χ2 = 3.27 > 0.05 Non-farmer 88 67.7 101 77.7 189 72.7 SD = standard deviation. Book 20-6.indb 366 6/17/2014 2:41:02 PM طسوتلما قشرل ةيحصلا ةلجلمانوشرعلا دلجلما سداسلا ددعلا 367 The mean age of patients with lymphoproliferative disorders in the present study was 46.9 years (47.5 years among NHL patients). These results are similar to observations in previous studies of NHL in Egypt (18,19) and in Turkey (20). The male: female ratio in lymphoprolifera- tive disorder cases was 1.45:1 (1.43:1 among NHL patients). This male predominance could be explained by men’s greater exposure to the en- vironmental risk factors at work. The reported male:female ratio among NHL cases varies around the world, but is consistently greater than 1. In the USA, it is approximately (1.6:1) and in some countries it is greater than 2:1 (19). Residence in rural areas was pre- dominant among the cases (60.8%). This could be explained by the fact that the department of oncology at Menoufia University Hospital serves mainly patients coming from the Delta region of Elyot, which has more rural than urban areas. This contrast with a previous study conducted at the National Cancer Institute in Cairo, the capital city of Egypt (18), as cases were mainly coming from Cairo, Giza, and Alexandria Governorates, which have more urban than rural areas. In the present study, farmers showed a higher risk of lymphopro- liferative disorders compared with all other occupations (32.3% of lym- phoproliferative disorder cases were farmers versus 22.3% of controls), although the difference did not reach statistical significance. Attention has focused on farmers in studies of ma- lignant lymphoma because they ex- perience certain exposures, including exposure to zoonoses, pesticides and other chemicals such as fertilizers and solvents (21). Our finding was con- sistent with similar studies in Spain (22), France (23) and Sweden (24). Exposure to pesticides in this study was significantly associated with lymphoproliferative disorders in the multivariate analysis. This risk was increased with duration of exposure to pesticides > 5 years. A significant association was reported for NHL patients in univariate and multivariate analysis (OR = 2.46). The mechanism of pesticide-induced carcinogenicity is still unclear. It has been suggested that alteration of immune function (25) or genetic factors (6,26) may be respon- sible for the carcinogenesis associ- ated with exposure to some pesticides. Also in Egypt, Loffredo et al. found that cases of NHL showed a signifi- cantly higher frequency of exposure to organophosphate insecticides than did controls (OR = 2.5) (27). Similar results were also obtained in a study in the Islamic Republic of Iran in which the authors concluded that persons with exposure to any pesticide had increased risk of NHL than controls (OR = 3.9) (10). However, another large study In the USA of pesticide exposure did not find any association between organophosphate use and NHL (28). It may be that different organophosphate pesticides have dif- ferent effects, but unfortunately we had did not have sufficient numbers of subjects to analyse specific types Table 2 Distribution of patients with lymphoproliferative disorders (LPD) and controls regarding history of exposure to pesticides Risk factor LPD cases (n = 130) Controls (n = 130) χ2 P-value No. % No. % History of exposure to pesticides Yes 50 38.5 34 26.2 4.50 < 0.05 No 80 61.5 96 73.8 If yes: Duration of exposure to pesticides (years) (n = 50) (n = 34) ≤ 5 7 14.0 12 35.3 4.10 < 0.05 > 5 43 86.0 22 64.7 Used protective measures Always 2 4.0 2 5.9 Sometimes 3 6.0 4 11.8 1.09 > 0.05 Never 45 90.0 28 82.4 Type of pesticide Crop insecticide 29 58.0 22 62.9 2.27 > 0.05 Herbicide 10 20.0 6 17.2 Fungicide 7 14.0 2 5.7 Home pesticide 4 8.0 4 14.3 Book 20-6.indb 367 6/17/2014 2:41:03 PM EMHJ • Vol. 20 No. 6 • 2014 Eastern Mediterranean Health Journal La Revue de Santé de la Méditerranée orientale 368 of organophosphates. Another study, in Sweden, found no significant as- sociations between NHL and either organochlorine or organophosphate insecticides (29). No significant association was ob- served in this study between the CLL and history of exposure to pesticides in agriculture. This could be due to the small number of leukaemia patients in the sample (n = 23). A study con- ducted in Greece found an association of pesticide exposure with leukaemia (OR= 2.14) (30). Similar results were obtained from 6 European countries, which found that the risk of CLL was elevated among those ever exposed to inorganic (OR = 1.6) and organic pesticides (OR = 1.5). CLL risk was highest amongst those ever exposed to organophosphates (OR = 2.7) (31). In our study, infection with HCV was associated with an increased risk of developing lymphoproliferative disorders in multivariate analysis, after adjustment for age, sex, residence and history of exposure to pesticides (OR = 2.67). Also, HCV was found to be a risk factor for NHL in the multivariate analysis (OR = 2.78). However, no significant association was found be- tween HCV infection and CLL, which again could be due to the small num- ber of leukaemia patients. A significant association between HCV infection and NHL was pre- viously observed in Egypt (18) and in British Columbia, Canada (32). This association had been hypoth- esized to be due to the persistence of chronic HCV in lymphocytes along with the involvement of genetic and environmental factors (33). Possible mechanisms for malignant transfor- mation include clonal proliferation of B-cells, inhibition of apoptotic cell death or both (34). Polymorphisms in the oxidative stress regulatory enzyme genes GPX1 and MPO may influence NHL risk in HCV-infected Egyptian patients (35). In contrast, other studies, con- ducted in France (36), Canada (37) and India (38,39), found no or weak association between HCV infection and subtypes of NHL. These studies were limited by small sample sizes and Table 3 Distribution of patients with lymphoproliferative disorders (LPD) and controls regarding the studied risk factors Studied risk factor LPD cases (n = 130) Controls (n = 130) χ2 P-value No. % No. % Anti-HCV +ve 51 39.2 28 21.5 9.62 < 0.01 –ve 79 60.8 102 78.5 Family history of cancer (in 1st-degree relatives) Yes 32 24.6 21 16.2 2.87 > 0.05 No 98 75.4 109 83.8 Type of relatives’ cancer Lymphoheamapoietic malignancies 5 3.8 1 0.8 > 0.05 Other 27 20.8 20 15.4 0.60 History of exposure to X-rays Yes 42 32.3 50 38.5 1.07 > 0.05 No 88 67.7 80 61.5 History of smoking Yes 51 39.2 44 33.8 0.81 > 0.05 No 79 60.8 86 66.2 History of using hair dyes Yes 12 9.2 9 6.9 0.47 > 0.05 No 118 90.8 121 93.1 HCV = hepatitis C virus. Table 4 Logistic regression model for the most relevant risk factors for lymphoproliferative disorders Studied risk factor ORa (95% CI) P-value History of exposure to pesticides 2.24 (1.22–4.11) < 0.05 HCV infection 2.67 (1.50–4.80) < 0.01 aAdjusted for age, sex and residence. Variables included in logistic regression were exposure to pesticides and HCV infection. HCV = hepatitis C virus; OR = odds ratio; CI = confidence interval. Book 20-6.indb 368 6/17/2014 2:41:03 PM طسوتلما قشرل ةيحصلا ةلجلمانوشرعلا دلجلما سداسلا ددعلا 369 hence lacked adequate power, or had study populations with a low preva- lence of HCV carriage, and hence have only been able to identify a weak as- sociation. It is also possible that there may be a geographical variations in the association, with co-factors such as geographical differences in viral genotype, population genetics or en- vironmental factors underlying the differences between studies. In our study, we observed that the risk of having lymphoproliferative disorders was greater with HCV infec- tion (OR = 2.67) than with pesticides exposure (OR= 2.24). This might be explained by the high prevalence of HCV infection in Egypt. The Egypt Demographic and Health Survey 2008 concluded that Egypt has the highest prevalence of HCV in the world, esti- mated nationally at 14.7% (40). In this study, family history of can- cer in a first-degree relative and type of relatives' cancer showed no significant association with lymphoproliferative disorders. This could be because the information on family history was self- reported and may therefore be inaccu- rate. This result disagrees with studies in Italy (41) and the USA (42), which reported a significant association be- tween lymphoproliferative disorders and family history of malignancies in first-degree relatives (OR = 1.6 and 2.2 respectively). A history of smoking was not found to be risk factor for lymphoprolifera- tive disorders in our study. Similar re- sults have been reported in the United Kingdom (43) and the USA (44). In contrast to our results, a significant association between tobacco smoke and lymphoid neoplasms has been reported in Italy (45) and the USA (46), but these studies were unable to show a dose–response relationship, leaving limited support for a causal association. In the present study, no signifi- cant association was found between lymphoproliferative disorders and his- tory of using hair dyes. In contrast, a study conducted in Europe found a Table 5 Distribution of patients with non-Hodgkin lymphoma (NHL) and controls regarding history of exposure to pesticides in agriculture and their seroprevalence of anti-HCV Risk factor NHL cases (n = 107) Controls (n = 130) χ2 P-value No. % No. % History of exposure to pesticides in agriculture Yes 44 41.1 34 26.2 5.95 < 0.05 No 63 58.9 96 73.8 Anti-HCV +ve 43 40.2 28 21.5 9.73 < 0.01 ve 64 59.8 102 78.5 HCV = hepatitis C virus. significant association between the use of hair dyes and lymphoid neoplasms (OR = 1.19), as hair dye formulations have long been known to contain mu- tagenic chemicals (47). This difference in the results between the studies could be explained by the fact that 60.8% of our patients were resident in rural areas where the frequent use of hair dyes is not a common practice. This study also showed no signifi- cant association between lymphopro- liferative disorders and history of exposure to X-rays, which could be due to the fact that exposure to X-rays is mainly a risk factor for leukaemia, and cases of CLL accounted only about one-fifth of our total sample (23/130). A similar finding was reported in the USA (48). One of the limitations of the cur- rent study was that pesticide exposure was self-reported by the participants and this might bias the results. Also, the small sample size of CLL cases might lead to inaccurate results regarding risk factors for leukaemia. Case–control studies are prone to bias, especially selection, recall and inter-interviewer bias, even though measures were taken to minimize this, as described earlier. A final limitation of our study was the use of simple regression analysis instead of conditional regression analysis. How- ever, we kept the stratum sizes large to minimize this effect. Table 6 Logistic regression model for the most relevant risk factors for non- Hodgkin lymphoma Risk factor ORa (95% CI) P-value Exposure to pesticides in agriculture 2.46 (1.31–6.63) < 0.01 HCV infection 2.78 (1.51–5.14) < 0.01 aAdjusted for age, sex and residence. Variables included in logistic regression were HCV and history of exposure to pesticides. HCV = hepatitis C virus; OR = odds ratio; CI = confidence interval. Book 20-6.indb 369 6/17/2014 2:41:03 PM EMHJ • Vol. 20 No. 6 • 2014 Eastern Mediterranean Health Journal La Revue de Santé de la Méditerranée orientale 370 Table 7 Distribution of patients with chronic lymphocytic leukaemia (CLL) and controls regarding history of exposure to pesticides in agriculture and their seroprevalence of anti-HCV Risk factor CLL cases (n = 23) Controls (n = 130) χ2 P-value No. % No. % History of exposure to pesticides Yes 6 26.1 34 26.2 0.00 > 0.05 No 17 73.9 96 73.8 Anti-HCV +ve 8 34.8 28 21.5 1.91 > 0.05 –ve 15 65.2 102 78.5 HCV = hepatitis C virus. In conclusion, pesticide exposure and HCV infection were found to be risk factors for lymphoprolifera- tive disorders, including NHL but not leukaemia, in adults. This risk was greatest when the duration of expo- sure to pesticides was greater than 5 years. More research on a large scale is needed, integrating various types of studies, such as surveillance for type pesticide product use, development and application of new biomarkers for pesticide exposure and subtypes of NHL. Competing interests: None declared. References 1. Mahima VG, Patil K, Raina A. Extranodal non-Hodgkin's lym- phoma. An unfamiliar presentation in the oral cavity: a case report. Int J Clin Cases Invest, 2010;1(1):1–6. 2. Soliman AS, Boffetta P. Lymphoma and leukemia. In: Freed- man LS, Edwards BK, Ries LAG, Young JL, editors. Cancer incidence in the four member countries (Cyprus, Egypt, Israel, and Jordan) of the Middle East Cancer Consortium (MECC) compared with US SEER. Bethesda (MD): National Cancer Institute; 2006. pp. 131–40. 3. Curado MP, Edwards B, Shin HR, Storm H, J. Ferlay, Heanue M, et al. Cancer incidence in five continents. Volume 9. Lyon, France: International Agency for Research on Cancer; 2007. p. 101. 4. Wall DA. Lymphoproliferative disorders. 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The relationship be- tween exposure to pesticides and the occurrence of lymphoid neoplasm. Iran Red Crescent Med J. 2012;14(6):337–44. 11. Chiu B, Weisenburger D, Zahm S, Cantor K. Agricultural pesti- cide use, familial cancer, and risk of non-Hodgkin lymphoma. Cancer Epidemiol Biomarkers Prev. 2004;13(4):525–31. 12. Dreiher J, Kordysh E. Non-Hodgkin lymphoma and pesticide exposure: 25 years of research. Acta Haematol. 2006;116:153– 64. 13. Fritschi L, Benke G, Hughes A. Occupational exposure to pes- ticides and risk of non-Hodgkin's lymphoma. Am J Epidemiol. 2005;162(9):849–57. 14. Mueller NE. Hodgkin’s disease. In: Schottenfeld D, Fraumeni JF Jr, editors. Cancer epidemiology and prevention. 2nd ed. New York: Oxford University Press; 1996. p. 893–919. 15. Greer JP, Williams ME. Non-Hodgkin lymphoma in adults. In: Greer JP, Foerster J, Rodgers GM, et al., editors. Wintrobe's Clinical hematology. 12th ed. Philadelphia (PA): Lippincott Williams and Wilkins; 2009. p. 2144–94. 16. Alavanja MC1, Hoppin JA, Kamel F. Health effects of chronic pesticide exposure: cancer and neurotoxicity. Annu Rev Pub- lic Health. 2004;25:155–97. 17. Miligi L, Costantini A, Bolejack V. Non-Hodgkin's lympho- ma, leukemia, and exposures in agriculture: results from the Italian multicenter case–control study. Am J Ind Med. 2003;44(6):627–36. 18. Cowgill K, et al. Case-control study of non-Hodgkin's lym- phoma and hepatitis C virus infection in Egypt. Int J Epidemiol. 2004;33(5):1034–9. 19. Abdel-Fattah MM, Yassine OG. Non-Hodgkin's lymphomas in Alexandria, Egypt; incidence rates and trend study (1995– 2004). Eur J Cancer Prev. 2007 Oct;16(5):479–85. 20. Paydas S, Kilic B, Bugdayci R. Prevalence of hepatitis C virus infection in patients with lymphoproliferative disorders in Southern Turkey. Br J Cancer. 1999;80:1303–5. 21. Mester B, et al. Occupation and malignant lymphoma: a popu- lation based case control study in Germany. BMJ. 2006;63:17– 26. 22. Evan B, et al. 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Epi- demiology. 2003;14(5):43–4. 28. Tatham L, Tolbert P, Kjeldsberg C. Occupational risk factors for subgroups of non-Hodgkin's lymphoma. Epidemiology. 1997;8:551–8. 29. Persson B, Fredrikson M. Some risk factors for non-Hodgkin's lymphoma. Int J Occup Med Environ Health. 1999;12:135–42. 30. Maria K. Pesticide exposure and lymphohaematopoietic cancers: a case-control study in an agricultural region (Larissa, Thessaly, Greece). BMC Public Health. 2011;11:5. 31. Cocco P, Satta G, Dubois S, Pili C, Pilleri M, Zucca M, et al. Lym- phoma risk and occupational exposure to pesticides: results of the Epilymph study. Occup Environ Med .2013;70(2):91–8. 32. Spinelli J, et al. Hepatitis C virus and risk of non-Hodg- kin lymphoma in British Columbia, Canada. Int J Cancer. 2008;122(3):630–3. 33. Gisbert JP, García-Buey L, Arranz R, Blas C, Pinilla I, Khorrami S, et al. The prevalence of hepatitis C virus infection in patients with non-Hodgkin's lymphoma. Eur J Gastroenterol Hepatol. 2004;16(2):135–8. 34. Machida K, et al. Hepatitis C virus triggers mitochondrial permeability transition with production of reactive oxygen species, leading to DNA damage and STAT3 activation. J Virol. 2006;80(14):7199–207. 35. Farawela H, Khorshied M, Shaheen I, Gouda H, Nasef A, Abula- ta N, et al. The association between hepatitis C virus infection, genetic polymorphisms of oxidative stress genes and B-cell non-Hodgkin's lymphoma risk in Egypt. Infect Genet Evol. 2012 Aug;12(6):1189–94. 36. Hausfater P, Cacoub P, Sterkers Y. Hepatitis C virus infection and lymphoproliferative diseases: prospective study on 1,576 patients in France. Am J Hematol. 2001;67:168–71. 37. Collier J, Zanke B, Moore M. No association between hepatitis C and B-cell lymphoma. Hepatology. 1999;29:1259–61. 38. Varma S, Menon MC, Garg A, Malhotra P, Sharma A, Chawla YK, et al. Hepatitis C virus infection among patients with non-Hodgkin’s lymphoma in northern India. Hepatol Int. 2011;5(2):688–92. 39. Dal Maso L, Franceschi S. Hepatitis C virus and risk of lym- phoma and other lymphoid neoplasms: a meta-analysis of epidemiologic studies. Cancer Epidemiol Biomarkers Prev. 2006;15:2078–85. 40. El-Zanaty F, Way A. Egypt Demographic and Health Survey 2008. Cairo, Egypt: Ministry of Health, El-Zanaty and Associ- ates, and Macro International; 2009. 41. Negri E, Talamini R, Montella M, Dal Maso L, Crispo A, Spina M, et al. Family history of hemolymphopoietic and other cancers and risk of non-Hodgkin's lymphoma. Cancer Epidemiol Bio- markers Prev. 2006;15(2):245–50. 42. Zhang Y, Wang R, Holford TR, Leaderer B, Zahm SH, Boyle P, et al. Family history of hematopoietic and non-hematopoietic malignancies and risk of non-Hodgkin lymphoma. Cancer Causes Control. 2007;18(4):351–9. 43. Willett EV, Smith AG, Dovey GJ. Tobacco and alcohol con- sumption and the risk of non-Hodgkin lymphoma. Cancer Causes Control. 2004;15(8):771–80. 44. Jesse D, et al. Associations between anthropometry, cigarette smoking, alcohol consumption, and non-Hodgkin lymphoma in the Prostate, Lung, Colorectal, and Ovarian Cancer Screen- ing Trial. Am J Epidemiol. 2010 June;171(12):1270–81. 45. Talamini L, Polesel J, Montella M. Smoking and non-Hodg- kin lymphoma: case-control study in Italy. Int J Cancer. 2005;115(4):606–10. 46. Diver WR, Patel AV, Thun MJ, Teras LR, Gapstur SM. The associ- ation between cigarette smoking and non-Hodgkin lymphoid neoplasms in a large US cohort study. Cancer Causes Control. 2012 Aug;23(8):1231–40. 47. Sanjose S, Benavente Y, Nieters A. Association between per- sonal use of hair dyes and lymphoid neoplasms in European. Am J Epidemiol. 2006;164:47–55. 48. Boice JD Jr, Morin MM, Glass AG, Friedman GD, Stovall M, Hoover RN, et al. Diagnostic X-ray procedures and risk of leukemia, lymphoma, and multiple myeloma. JAMA. 1991 Mar 13;265(10):1290–4. Book 20-6.indb 371 6/17/2014 2:41:04 PM EMHJ • Vol. 20 No. 6 • 2014 Eastern Mediterranean Health Journal La Revue de Santé de la Méditerranée orientale 372 Detection and genotyping of human papillomavirus in breast cancer tissues from Iraqi patients S.H.M. Ali,1 N.A.S. Al-Alwan 2 and S.H.M. Al-Alwany 1 ABSTRACT Studies have suggested a possible link between breast cancer pathogenesis and human papillomavirus (HPV) infection. This study in Iraq used in situ hybridization to detect the frequency and genotyping of HPV in tissue specimens from 129 patients diagnosed with malignant breast cancer, 24 with benign breast tumours and 20 healthy controls. In the breast cancer group, cocktail HPV genotypes were detected in 60 (46.5%) archived tissue blocks. Of these, genotypes 16 (55.5%), 18 (58.4%), 31 (65.0%) and 33 (26.6%) were detected. Mixed HPV genotypes 16 + 18, 16 + 18 + 31, 16 + 18 + 33, 18 + 33, 16 + 31 and 18 + 31 were found in 5.0%, 25.0%, 8.3%, 7.7%, 10.0% and 13.3% of cancer cases respectively. Only 3 benign breast tumour tissues (12.5%) and none of the healthy breast tissue specimens were HPV-DNA-positive. The detection of high-oncogenic HPV genotypes in patients with breast cancer supports the hypothesis of an etiologic role for the virus in breast cancer development. 1Communicable Disease Research Unit, Baghdad Medical College, Baghdad, Iraq. 2Iraqi National Cancer Research Centre, Baghdad of University, Baghdad, Iraq (Correspondence to N.A.S. Al-Alwan: nadalwan@yahoo.com). Received: 24/12/12; accepted: 26/08/13 تايقارع تاضيرم ىدل يدثلا ناطسر ةجسنأ في ةينيلجا هطمانأ ديدتحو يشربلا يميللحا مرولا سويرف فشك نياولعلا ركاش ،ناولعلا ىدن ،ليع دعس .يشرـبلا يـميللحا مروـلا سورـفب ىودـعلا نـبو يدـثلا ناـطسر تاببـسم نـب طاـبترا دوـجو لماـتحا لىإ تاـساردلا تراـشأ :ةـصلالخا تاـنّيع في يشرـبلا يـميللحا مروـلا سورـفل يـنيلجا طـمنلاو رارـكتلا فـشكل عـضولما في نـجهتلا ةـساردلا هذـه في نوـثحابلا مدختـسا دـقو 20و يدـثلا في ميلـس مرو اـيهدل َص ِّخـُش ةـضيرم 24 بـناج لىإ ،يدـثلا في ثـيبخ ناـطسر اـيهدل َص ِّخـُش ةـضيرم 129 نـم تذـخأ ةيجيـسن يـميللحا مروـلا سورـف نـم ةـيجزم ًاـطمانأ يدـثلا ناـطسرب تاـ باصلما قـيرف ىدـل نوـثحابلا فـشكو .ةـحصلاب نـعتمتي دهاوـشك ةديـس طـمنلاو ،)% 55.5( في 16 يـنيلجا طـمنلا نوـثحابلا فـشك ةيجيـسنلا عـطقلا هذـه نـب نـمو .)% 46.5( ةـظوفمح ةيجيـسن ةـعطق 60 في يشرـبلا ناـطسرلا تلااـح نـب دـجو ماـك .تلااـلحا نـم )% 26.6( في 33 يـنيلجا طـمنلاو ،)% 65.0( في 31 يـنيلجا طـمنلاو ،)% 58.4( في 18 يـنيلجا % 8.3 في 33+18+16و ،تلااـلحا نـم % 25.0 في 31+18+16و ،تلااـلحا نـم 5% في 18+16 يشرـبلا يـميللحا مروـلا سورـفل ةـيجزم ةـينيج طماـنأ نــ كت لمو ناــ طسرلاب ةــ باصلما تلااــ لحا نــ م % 13.3 في 31+18و تلااــ لحا نــ م % 10 في 31+16و تلااــ لحا نــ م % 6.7 في 33+18و ،تلااــ لحا نــ م نـم ةـنيع يأ كاـنه نـكي لمو ،)% 12.5( يشرـبلا يـميللحا مروـلا سورـف اـَنَِدل ةـيبايجإ ميلـس مروـب ةـباصلما يدـثلا جيـسن نـم تاـنيع 3 ىوـس تاـضيرم ىدـل مروـلل لياـعلا دـيلوتلا تاذ يشرـبلا يـميللحا مروـلا سورـفل ةـينيلجا طماـنلأا فـشك نإ .هـل ةـيبايجإ ةميلـسلا يدـثلا جـسن .يدـثلا ناـطسر ثادـحإ في يبــَبـَس رودـل سورـفلا ءادأ ةـيرظن مـعدي يدـثلا ناـطسرب Détection et génotypage du papillomavirus humain dans les tissus mammaires cancéreux de patientes en Iraq RÉSUMÉ Des études ont suggéré qu'un lien était possible entre la pathogénèse du cancer du sein et l'infection à papillomavirus humain. L'étude menée en Iraq a utilisé la méthode d'hybridation in situ pour déterminer la fréquence du papillomavirus humain et pour son génotypage dans les échantillons de tissus prélevés auprès de 129 patientes ayant reçu un diagnostic de cancer du sein malin, de 24 patientes porteuses d'une tumeur du sein bénigne et de 20 femmes témoins en bonne santé. Dans le groupe des patientes atteintes d'un cancer du sein, différents génotypes de papillomavirus humain ont été détectés dans 60 échantillons de tissus conservés (46,5 %). Parmi ceux-ci, on peut citer les génotypes 16 (55,5 %), 18 (58,4 %), 31 (65,0 %) et 33 (26,6 %). Les associations de génotypes de papillomavirus humain 16 + 18, 16 + 18 + 31, 16 + 18 + 33, 18 + 31, 16 + 31 et 18 + 33 ont été observées chez 5,0 %, 25,0 %, 8,3 %, 6,7 %, 10,0 % et 13,3% des cas de cancer respectivement. Seuls trois échantillons de tissu mammaire tumoral bénin (12,5 %) étaient positifs pour l'ADN de papillomavirus humain tandis qu’aucun échantillon de tissu mammaire sain ne l’était. La détection de génotypes du papillomavirus humain hautement oncogènes chez les patientes atteintes de cancer du sein vient appuyer l'hypothèse du rôle étiologique du virus dans l'apparition d'un cancer du sein. Book 20-6.indb 372 6/17/2014 2:41:04 PM طسوتلما قشرل ةيحصلا ةلجلمانوشرعلا دلجلما سداسلا ددعلا 373 Introduction Breast carcinoma is the common- est malignancy among women in developed and developing countries including Iraq (1,2). Local studies in Iraq have demonstrated that most breast cancer patients present in ad- vanced stages with a likely prevalence of more aggressive tumour forms (3,4). Many risk factors have been associated with breast cancer devel- opment and progression including a possible viral etiology (5). Previous studies have demonstrated the pres- ence of high-risk human papillomavi- rus (HPV) genotypes 16, 18 and 33 in breast cancer specimens from diverse populations around the world (6–8). Nevertheless, a definitive relationship between human breast cancer and HPV infection has not yet been deter- mined (9). There is a need to obtain additional evidence in order to assess the possibility of breast cancer preven- tion using HPV vaccines. High-risk HPV encodes a series of proteins, some of which have oncogenic potential (10). The biology of HPV in breast cancer is almost identical to that in cervical cancer, although a signifi- cantly higher viral load is evident in the latter (10). It has been observed that the presence of HPV in breast cancer was associated with the detectable expres- sion of the HPV E6 oncogenic protein and the p16(INKa4) protein, which is an indicator of E7-mediated inacti- vation of the p110(Rb) protein (11). The present study represents the first of its kind in Iraq to explore a possible etiological association between high oncogenic-risk HPV types and breast cancer. Methods Tissue samples This retrospective study used 173 select- ed formalin-fixed, paraffin-embedded blocks from breast tissue samples from 129 patients with breast carcinomas (100 cases of ductal mammary carci- noma not otherwise specified, 17 cases of lobular carcinoma, 5 cases of mixed ductal and lobular carcinoma and 7 cases of medullary carcinoma) and 24 patients with benign epithelial mam- mary lesions. A further 20 blocks from samples from normal breast tissues were labelled as a control group (i.e. normal healthy breast tissues). The age range of the patients was 16–72 years. The specimens were collected dur- ing the period 2008–11 from major hospitals and private laboratories in the mid-Euphrates governorates of Iraq including Babylon, Kerrbla, Al- Najaf, Al-Qadiyisia and Al-Muthana. The diagnoses were based on the re- corded pathological reports of the corresponding patients. Laboratory methods Thick-tissue sections (4 mm) were prepared and stuck onto positively charged slides. An in situ hybridization (ISH) detection system (Zytovision GmbH) was used to target DNA sequences in tissue specimens using digoxigenin-labelled cocktail HPV- DNA probes for a wide range of high-risk HPV genotypes including genotypes 16, 18, 31, 33, 35, 45, 51 and 82. The ISH signals were detected as red discolouration at the site of se- quence complementarity as nuclear signals by proper use of the Zytovision system. Another ISH detection system (Maxim Biotech Inc.) was used to target DNA sequences using a bioti- nylated long DNA probe for HPV genotypes 16, 18, 31 and 33 in tissue specimens. The methods were con- ducted according to the instructions of the manufacturing company. Positive reactions were performed by replacing the probe with a biotinylated house- keeping gene probe. For the negative control, all reagents were added except the diluted probe. Proper use of this ISH detection system gives an intense blue signal at specific sites of the hy- bridization probe in positive test tis- sues (according to the specification of the second differential kit which gave the blue colour). The signal was evalu- ated under light microscopy using × 100 lens for counting the positive cells. The ISH results were given percentage scores based on positive signals and number of cells that gave these signals. Analysis Positive cells were counted in 10 different fields of 100 cells for each sample and the average percentage of positive cells within the 10 fields was determined. Cases were assigned to one of the following percentage score categories: 1%–25% (score 1), 26%–50% (score 2) or > 50% (score 3) (12). The chi-squared test was used to detect the significance between groups. The statistical analysis was done using SPSS program, version 17, and differences were considered significant when P < 0.05 (13). Results Detection and genotyping of HPV Table 1 shows the positive results of the HPV-DNA ISH detection; 60/129 breast cancer cases (46.5%; 95% CI: 37.9%–55.1%) revealed posi- tive red signals. In the benign tumour group 3/24 (12.5%) revealed positive red signals. None of the healthy breast tissues presented positive signals for the HPV ISH test. Of the positive cases, 31/60 (51.7%) breast cancer tis- sues had score 3 (> 50% positive cells), while 2/24 (66.7%) benign tumour tissues had score 3. The microscopic appearance of generic HPV-positive ISH red signals in breast cancer and healthy tissues are illustrated in Figure 1. The signals of ISH for genotyping HPV were detected as blue nuclear signals at the sites of sequence-complementarities (Table Book 20-6.indb 373 6/17/2014 2:41:04 PM EMHJ • Vol. 20 No. 6 • 2014 Eastern Mediterranean Health Journal La Revue de Santé de la Méditerranée orientale 374 with HPV types 18 + 31 (33.3%). A significant difference in the rates of co- infection by high-risk HPV genotypes was observed between the benign and malignant breast tissue groups (P < 0.05). Discussion The majority of molecular events in the genesis of breast cancer are unknown. However, initial studies have reported an association of breast cancers with cervical intraepithelial neoplasia-III-like lesions (14). High oncogenic-risk HPV geno- types such as HPV 16, 18, 31 and 33 were detected in cases of breast cancer in this study. Despite great variability in the HPV detection rates worldwide, the majority of HPV types detected are of the high oncogenic-risk group (HPV genotypes 16 + 18) (15). It has been reported that the association of high oncogenic-risk HPV types is stronger in invasive breast carcino- mas, indicating a possible relationship with the pathology and grade of the disease (16). The transmission route of HPV detected in breast cancer is unclear. 2 and Figure 2). Table 2 shows that in breast cancer cases the frequency of HPV genotype 33 (12.4%) was significantly lower than genotype 31, which was the most frequent genotype (30.2%). The distribution of HPV- DNA genotypes in the malignant tu- mour group were as follows: HPV 16 (25.6%), HPV 18 (27.1%), HPV 31 (30.2%) and HPV 33 (12.4%). These results demonstrate that HPV genotype 31 occurred at a higher rate than other genotypes. The total positive cases in the table refer to positive cases that may have more than one genotype of HPV. Tables 3 and 4 show the rates of co-infection with multiple HPV genotypes in patients with benign and malignant breast tumours. Among the 60 samples in the malignant group positive for HPV, the highest per- centage of co-infection with multiple high-risk of HPV genotypes 16 + 18 + 31 was found in 15 (25.0%) cases. Co- infection with other high-risk HPV genotypes in the malignanat group were as follows: HPV 16 +18 (5.0%), HPV 16 + 18 + 33 (8.3%), HPV 18 + 33 (6.7%), HPV 16 + 31 (10.0%) and HPV 18 + 31 (13.3%). Positive signals indicating high-risk HPV infection were found in 3/24 (12.5%) benign breast tumour tissues, among which 1 case showed mixed HPV infection Table 1 Frequency distribution of human papillomavirus (HPV) DNA signal scoring from samples of malignant breast cancer, benign breast tumour and normal healthy breast tissues HPV signal scoring Malignant breast tumour group Benign breast tumour group Healthy breast group No. % No. % No. % Signal Negative 69 53.5 21 87.5 20 100.0 Positivea 60 46.5 3 12.5 0 0.0 Total 129 100.0 24 100.0 20 100.0 Scoresb 1 9/60 15.0 0/3 0.0 0/0 0.0 2 20/60 33.3 1/3 33.3 0/0 0.0 3 31/60 51.7 2/3 66.7 0/0 0.0 Mean rankc 95.6 67.1 55.5 aMann–Whitney test for difference in mean rank comparing: all 3 study groups (P < 0.001); benign tumour versus healthy groups (P = 0.11); malignant tumour versus healthy groups (P < 0.001); malignant versus benign tumour groups (P = 0.004). Kruskal–Wallis test for difference in signal scoring comparing: malignant versus benign tumour groups for HPV-positive cases (P = 0.52). Healthy controls were not compared because all were HPV negative. bScore 1: 1%–25%; score 2: 26%–50%; score 3: > 50%. cIndicates significant differences in rates of positivity of HPV detection compared with ranks of HPV detection rates in the groups showing benign tumours and healthy breast tissues. Figure 1 Microscopic appearance of generic HPV-positive in situ hybridization red signals in epithelial breast tissues. A = malignant tumour (HPV positive); B = healthy breast (HPV negative) A B Book 20-6.indb 374 6/17/2014 2:41:05 PM طسوتلما قشرل ةيحصلا ةلجلمانوشرعلا دلجلما سداسلا ددعلا 375 Two independent studies suggested a possible haematogenic and/or lymphatic transfer of the virus from one organ to another (17). Investiga- tors revealed the presence of HPV in nipple secretions, suggesting that HPV may transfer in a retrograde fashion from the nipple via the areola, lactif- erous ducts and sinuses (18). In the present study, generic/cocktail high oncogenic-risk types were detected in 46.5% of cases. The reported preva- lence of HPV infection in breast cancer tissues shows a great variation world- wide, ranging from 0% to 86% (19,20). Demographic features and genetic background might contribute to the global geographical differences of HPV prevalence in breast carcinoma tissues. Nevertheless, other reasons for the different results reported in the literature may be attributable to the variation in the numbers of tested samples and sensitivity of the technical methods used in evaluation including the primer sets (21). It has been reported that viri- ons are shed from desquamating keratinocytes (the target cells from HPV infections) and high-risk HPVs can be transmitted by close human non-sexual contact (22). Accord- ingly, we speculate that HPVs may be transmitted by hand from the female perineum to the breast which might occur during showering or bathing. It has been well documented as well that hormonal factors (oestrogen and its derivatives) are known to be involved in breast carcinogenesis (23) and that oestrogen receptor-positive breast carcinomas are more sensitive to ta- moxifen. The presence of oestrogen or progesterone receptors was not related to HPV detection in breast carcinomas (24). The origin of breast cancer remains multifactorial. If HPV is considered to play an etiological role in breast cancer development, as in cervical carcinogenesis, it would be reasonable to expect that it would be possible to detect the virus at an early stage of the disease, and that HPV would be found at least in some normal tis- sues (9). The fact that we did not find viral DNA in healthy breast specimens could support, to a certain extent, the hypothesis that the virus might play a role in the etiology of breast cancer in only a subpopulation of patients. On the other hand, it is logical to believe E D F C B A Table 2 Frequency of positive specific human papillomavirus (HPV) genotypes from samples of malignant breast cancer tissues Genotype Malignant tumour group (n = 129) No. %a 95% CI 31 39 30.2 22.3–38.1 18 35 27.1 19.4–34.8 16 33 25.6 18.1–33.1 33 16 12.4 6.7–18.1 aFriedman test comparing genotypes (P < 0.001). CI = confidence interval for prevalence. Figure 2 Microscopic appearance of human papillomavirus (HPV)-positive in situ hybridization signals of epithelial breast tumour. A = malignant tumour (HPV 16 positive); B = malignant tumour (HPV 18 positive); C = malignant tumour (HPV 31 positive); D = healthy breast (HPV negative); E = malignant tumour (HPV 33 positive); F = healthy breast (HPV negative) Book 20-6.indb 375 6/17/2014 2:41:05 PM EMHJ • Vol. 20 No. 6 • 2014 Eastern Mediterranean Health Journal La Revue de Santé de la Méditerranée orientale 376 that the presence of high-risk types of HPV alone is not sufficient to imple- ment the full process of tumourigen- esis and that further changes would accumulate over time in a stepwise manner to cause the disease. This pos- sible mechanism suggests a need for Table 3 Combined human papillomavirus (HPV) genotypes among positive-HPV cases from samples of malignant breast cancer and benign breast tumour tissues Genotypes Malignant breast tumour group (n = 60) Benign breast tumour group (n = 3) No. % No. % Single genotype 17 28.3 2 66.7 Combined (multi) genotypes 43 71.7 1 33.3a aKruskal Wallis test comparing benign and malignant cancer groups for the presence of co-infection by high-risk HPV genotypes (P < 0.05). further studies with larger cohorts of patients. Adding other investigations and methods to test for E6 and E7 on- cogenes and other transforming genes would be mandatory to determine clearly the exact role of those viruses in the genesis of breast lesions (9,21). The detection of high oncogenic HPV genotypes in patients with breast cancer requires prompt action to con- trol the spread of that sexually transmit- ted infection among the Iraqi general population. Competing interests: None declared. Table 4 Frequency of human papillomavirus (HPV)-genotype combinations among samples of HPV-positive malignant breast cancer and benign breast tumour tissues Genotypes Malignant breast tumour group (n = 43) Benign breast tumour group (n = 1) No. % No. % 16 + 18 3 7.0 0 0.0 16 + 31 6 14.0 0 0.0 16 + 33 2 4.7 0 0.0 18 + 31 8 18.6 1 100.0 18 + 33 4 9.3 0 0.0 16 + 18 + 31 15 34.9 0 0.0 16 + 18 + 33 5 11.6 0 0.0 References 1. GLOBOCAN 2008: cancer incidence and mortality world- wide. Lyon, France: International Agency for Research on Cancer; 2010. 2. Iraqi cancer registry 2008. Baghdad, Iraq: Iraqi Cancer Board, Ministry of Health, 2010. 3. Alwan NA. Breast cancer: demographic characteristics and clinico-pathological presentation of patients in Iraq. East Mediterr Health J. 2010 Nov;16(11):1159–64. PMID:21218740 4. al-Alwan NA. DNA proliferative index as a marker in Iraqi aneuploid mammary carcinoma. East Mediterr Health J. 2000 Sep-Nov;6(5-6):1062–72. PMID:12197329 5. Hsu CR, Lu TM, Chin LW, Yang CC. Possible DNA viral factors of human breast cancer. Cancers (Basel). 2010;2(2):498–512. PMID:24281079 6. Gumus M, Yumuk PF, Salepci T, Aliustaoglu M, Dane F, Ekenel M, et al. HPV DNA frequency and subset analysis in human breast cancer patients’ normal and tumoral tissue samples. J Exp Clin Cancer Res. 2006 Dec;25(4):515–21. PMID:17310842 7. Kulka J, Kovalszky I, Svastics E, Berta M, Füle T. Lymphoe- pithelioma-like carcinoma of the breast: not Epstein-Barr virus-, but human papilloma virus-positive. Hum Pathol. 2008 Feb;39(2):298–301. PMID:18206498 8. Lawson JS, Glenn WK, Salmons B, Ye Y, Heng B, Moody P, et al. Mouse mammary tumor virus-like sequences in hu- man breast cancer. Cancer Res. 2010 May 1;70(9):3576–85. PMID:20388779 9. Heng WK, Glenn YY. Tran W et al. Human papilloma viruses is associated with breast cancer. British Journel of Cancer, 2009, 101:1345–1350. 10. Amarante MK, Watanabe MA. The possible involvement of virus in breast cancer. J Cancer Res Clin Oncol. 2009 Mar;135(3):329–37. PMID:19009309 11. Lawson JS, Glenn WK, Heng B, Ye Y, Tran B, Lutze-Mann L, et al. Koilocytes indicate a role for human papilloma virus in breast cancer. Br J Cancer. 2009 Oct 20;101(8):1351–6. PMID:19773762 12. Zlobec I, Steele R, Michel RP, Compton CC, Lugli A, Jass JR. Scoring of p53, VEGF, Bcl-2 and APAF-1 immunohistochemistry and interobserver reliability in colorectal cancer. Mod Pathol. 2006 Sep;19(9):1236–42. PMID:16741523 Book 20-6.indb 376 6/17/2014 2:41:06 PM طسوتلما قشرل ةيحصلا ةلجلمانوشرعلا دلجلما سداسلا ددعلا 377 13. El-Sisy NA. Immunohistochemical detection of p 53 in amelo- blastoma. J Oral Pathol. 1999;5:478–89. 14. Liu Y, Klimberg VS, Andrews NR, Hicks CR, Peng H, Chiriva- Internati M, et al. Human papillomavirus DNA is present in a subset of unselected breast cancers. J Hum Virol. 2001 Nov- Dec;4(6):329–34. PMID:12082399 15. Kan CY, Iacopetta BJ, Lawson JS, Whitaker NJ. Identification of human papillomavirus DNA gene sequences in human breast cancer. Br J Cancer. 2005 Oct 17;93(8):946–8. PMID:16222323 16. Yasmeen A, Bismar TA, Kandouz M, Foulkes WD, Desprez PY, Al Moustafa AE. E6/E7 of HPV type 16 promotes cell invasion and metastasis of human breast cancer cells. Cell Cycle. 2007 Aug 15;6(16):2038–42. PMID:17721085 17. Widschwendter A, Brunhuber T, Wiedemair A, Mueller- Holzner E, Marth C. Detection of human papillomavirus DNA in breast cancer of patients with cervical cancer history. J Clin Virol. 2004 Dec;31(4):292–7. PMID:15494272 18. de Villiers EM, Sandstrom RE, zur Hausen H, Buck CE. Presence of papillomavirus sequences in condylomatous lesions of the mamillae and in invasive carcinoma of the breast. Breast Can- cer Res. 2005;7(1):R1–11. PMID:15642157 19. Choi YL, Cho EY, Kim JH, Nam SJ, Oh YL, Song SY, et al. Detec- tion of human papillomavirus DNA by DNA chip in breast car- cinomas of Korean women. Tumour Biol. 2007;28(6):327–32. PMID:18391549 20. Lindel K, Forster A, Altermatt HJ, Greiner R, Gruber G. Breast cancer and human papillomavirus (HPV) infection: no evi- dence of a viral etiology in a group of Swiss women. Breast. 2007 Apr;16(2):172–7. PMID:17088061 21. Khan NA, Castillo A, Koriyama C, Kijima Y, Umekita Y, Ohi Y, et al. Human papillomavirus detected in female breast carcinomas in Japan. Br J Cancer. 2008 Aug 5;99(3):408–14. PMID:18648364 22. Rintala MA, Grénman SE, Järvenkylä ME, Syrjänen KJ, Syrjänen SM. High-risk types of human papillomavirus (HPV) DNA in oral and genital mucosa of infants during their first 3 years of life: experience from the Finnish HPV Family Study. Clin Infect Dis. 2005 Dec 15;41(12):1728–33. PMID:16288396 23. Pike MC, Spicer DV, Dahmoush L, Press MF. Estrogens, pro- gestogens, normal breast cell proliferation, and breast cancer risk. Epidemiol Rev. 1993;15(1):17–35. PMID:8405201 24. Chung SH, Wiedmeyer K, Shai A, Korach KS, Lambert PF. Re- quirement for estrogen receptor alpha in a mouse model for human papillomavirus-associated cervical cancer. Cancer Res. 2008 Dec 1;68(23):9928–34. PMID:19047174 Book 20-6.indb 377 6/17/2014 2:41:06 PM EMHJ • Vol. 20 No. 6 • 2014 Eastern Mediterranean Health Journal La Revue de Santé de la Méditerranée orientale 378 Use of human surplus biospecimens in research: a survey from a cancer centre M. Al-Hussaini 1 and A. Abu-Hmaidan 2 ABSTRACT Little is known about the public’s views on the use of human biospecimens for research in the Eastern Mediterranean Region. A study at a cancer centre in Amman, Jordan, assessed patients’ perceptions about the use of blood and tissue samples obtained during clinical care and the use of these in research. A self-administered questionnaire was distributed to a sample of 205 adult cancer patients. Almost all patients (98.0%) accepted the use of their surplus blood samples and archived tissue in research if they consented, with about one-third requesting a specific opt-in consent. Most patients (82.9%) also agreed to donate a blood sample for research purposes only, 84.9% were interested to know the results of that research, but with a specific opt-in consent, and 81.0% accepted sending their samples to research laboratories abroad, even without specific consent. Patients’ views on the potential use of the surplus biospecimens in research were largely concordant with the international literature. 1Department of Pathology and Laboratory Medicine; 2Clinical Research and Cancer Registry Office, King Hussein Cancer Centre, Amman, Jordan (Correspondence to M. Al-Hussaini: mhussaini@khcc.jo). Received: 22/05/13; accepted: 28/10/13 ناطسرلل زكرم نم دمتسم حسم :ثوحبلا في ةجالحا نع ةضئافلا ةيشربلا ةيجولويبلا تانيعلا مادختسا ناديحم وبأ ديمع ،ينيسلحا ءاسيم دــ قو .طــ سوتلما قشر مــ يلقإ في ثوــ حبلا في ةــ يشربلا ةــ يجولويبلا تاــ نيعلا مادختــ سا نــ ع ساــ نلا ءارآ لوــ ح اــ نفراعم يــ ه ٌةــ ليلق :ةــ صلالخا لوـصلحا مـتي يـتلا ةيجيـسنلا تاـنيعلاو مدـلا مادختـسلا ضىرـلما كاردإ مـييقتل ،ندرلأا ،ن َّماـع في ناـطسرلل زـكرم في ةـساردلا هذـه تـيرجأ ضىرـم نـم 205 مـضت ةـنّيع ىـع هـعيزوت مـت تياذ نايبتـسا للاـخ نـم كـلذو ،ثوـحبلا في كـلذ مادختـساو ةـيريسرلا ةـياعرلا للاـخ اـهيلع ةيجـسنلا جذماـنلاو مدـلا تاـنيع نـم ةـجالحا نـع ضـيفي اـم مادختـسا ىـع )مـهنم % 98.0( ًاـبيرقت ضىرـلما عـيجم قـفاو .نـغلابلا ناـطسرلا .ةدـَّيقلما ةـقفاولما نـم ًاـيعون ًاـطمن اوـبلط ثـلثلا نـم برـقي اـم نأ ماـك ،كـلذ ىـع مـهتقفاوم نوـثحابلا بـلط اـم اذإ ثوـحبلا في ،ةـظوفحلما جـئاتن ةـفرعمب نـمتهم مـهنم % 84.9 ناكو ،طـقف ثوـحبلا ضارـغلأ مدـلا تاـنيعب عرـتلا ىـع )مـهنم % 82.9( ضىرـلما مـظعم قـفاو ماـك ىـتح ،دلاـبلا جراـخ تارـتمخ لىإ مـهنم ةذوـخألما تاـنيعلا لاـسرإ ىـع مـهنم % 81.0 قـفاو ماـنيب ،ةدـَّيقم ةـقفاوم عـم نـكلو ،ثوـحبلا كـلت في ةـجالحا نـع ةـضئافلا ةـيجولويبلا تاـنيعلل لـمتحلما مادختـسلاا لوـح ضىرـلما رـظن تاـهجو نأ ّنـبت دـقو .مـهنم ةـصاخ ةـقفاوم نودـب .ليودـلا دـيعصلا ىـع روـشنم وـه اـم عـم قـفاوتت ثوـحبلا Utilisation d'échantillons biologiques humains excédentaires en recherche : une enquête dans un centre anticancer RÉSUMÉ Il existe peu d'informations sur l'opinion du public quant à l'utilisation d'échantillons biologiques humains pour la recherche dans la Région de la Méditerranée orientale. Une étude menée dans un centre anticancer à Amman (Jordanie) a évalué les perceptions des patients concernant l'utilisation des échantillons de sang et de tissu recueillis pendant des soins cliniques et leur utilisation pour la recherche. Un autoquestionnaire a été distribué à un échantillon de 205 patients adultes atteints de cancer. Presque tous les patients (98,0 %) acceptaient l'utilisation des échantillons de sang et de tissus excédentaires conservés pour la recherche, après leur consentement, et environ un tiers exigeait une demande de consentement spécifique. La plupart des patients (82,9 %) consentaient également à faire un don de sang à des fins de recherche uniquement, tandis que 84,9 % souhaitaient connaître les résultats de cette recherche après un consentement spécifique, et 81,0 % acceptaient que leurs échantillons soient envoyés à des laboratoires de recherche à l'étranger, même sans consentement spécifique. Le point de vue des patients sur l'utilisation potentielle des échantillons biologiques excédentaires pour la recherche concordait en grande partie avec les points de vue présentés dans la littérature internationale. Book 20-6.indb 378 6/17/2014 2:41:07 PM طسوتلما قشرل ةيحصلا ةلجلمانوشرعلا دلجلما سداسلا ددعلا 379 Introduction Human biospecimens constitute a valu- able resource for different types of basic and clinical research (1), and surplus blood and archived tissue samples are useful for teaching, audit and quality control in laboratories (2). However, when the primary purpose of biospeci- men collection and tissue archiving is clinical care, patients are generally not informed of the potential use of leftover biospecimens in research, and specific consent addressing this issue may not have been obtained at the time of specimen collection. Although policies, frameworks and legislations regulating the use of biospecimens in research have been introduced in many countries to overcome potential conflicts (3–8), such regulations are generally lacking in developing countries (9). Internation- ally unified guidelines regulating the use of biospecimens in research are urgently needed (10). A search of the medical literature revealed a scarcity of publications in which public or patients’ views on the use of human biospecimens for research have been evaluated in the Re- gion. Most Saudi patients surveyed in one centre agreed to the use of leftover samples in research, without (49%) or with (37%) consent, with only 14% ob- jecting (11). Many Egyptians surveyed did not favour the donation of blood samples for research and were hesitant to have their blood samples donated for genetic research or exported outside the Arab region for research purposes (12). In a study from Morocco, incon- sistencies were found between labora- tories in obtaining informed consent from patients when using their sam- ples in research (13). More recently Ahram et al. explored the attitude of a large cohort (> 3000) of Jordanians towards biobanks, a concept which was positively accepted by a majority of respondents (14). At the King Hussein Cancer Cen- tre in Amman, Jordan, tissue paraffin blocks are archived at the pathology department almost exclusively for clini- cal care, while blood samples and other biological specimens are disposed of once the requested test is completed. Lately an apparent increase in the use of archived tissue and other surplus biospecimens in research has been noted. Although a national clinical re- search law came into effect a few years ago (15), nationwide regulation on the use of surplus biospecimens is still lacking. The local institutional review board/ethics committee tries to oper- ate in accordance with international regulations. It demands anonymiza- tion, or at least coding of specimens, before granting approval for their use in research. For any prospective col- lection of blood or tissue specimens primarily for research purposes, and/ or when sending specimens for exter- nal laboratories for further testing is part of a collaborative national/inter- national research, obtaining specific consent from the concerned patients is mandated. The views of our patients on the potential use of their specimens in re- search have never been studied. Our aim was 3-fold: to assess patients’ awareness about the final destina- tion of blood specimens and tissues obtained during the course of clinical care, once this is completed; to explore patients’ perceptions and any potential obstacles to the use of surplus blood and archived tissue in research; and to compare the results with the interna- tional literature. Methods Sample This was a cross-sectional study over a 2-week period (3 April to 14 April 2010). All patients attending the adult chemotherapy outpatient clinic at King Hussein Cancer Centre were approached and those who agreed to participate in the study were recruited and asked to give informed consent. The questionnaire was anonymous, so that identifiers such as names, hospital numbers or national identity numbers were not collected. Data collection A questionnaire was developed by the investigators in English. It consisted of 31 questions divided into 5 parts: de- mographic data (4 questions); patients’ previous experience of medical research (6 questions); assessment of patients’ awareness of the destination of surplus blood (5 questions) and tissue speci- mens (8 questions) acquired during the course of routine clinical care; and patients’ perceptions on the potential use of biospecimens in research, the ownership, the need and preferred type of consent, and any possible obstacles to the use of those specimens in research (8 questions). Arabic translation was performed by one of the investigators (A.A.H.) and was reviewed by the other investigator (M.A.H.). Research assistants were recruited from the School of Pharmacy at the University of Jordan and were trained in fundamental research eth- ics and on use of the questionnaire. After obtaining the institutional review board approval, a pilot phase was car- ried out in which the questionnaire was distributed to 20 adult patients at- tending the chemotherapy outpatient clinic. Data analysis The participants’ demographic data and their responses to the question- naire are presented as total counts and percentages. The participants’ re- sponses regarding donating and using their biospecimens in research were correlated with their demographic data and any previous participation in research using the chi-squared and Fisher exact tests. A significant P-value was determined at ≤ 0.05. All analyses were performed using SAS, version 9.1. Book 20-6.indb 379 6/17/2014 2:41:07 PM EMHJ • Vol. 20 No. 6 • 2014 Eastern Mediterranean Health Journal La Revue de Santé de la Méditerranée orientale 380 after they had been removed for diag- nostic purposes, half the patients (104, 50.7%) knew that the laboratory doctor (pathologist) was the physician who examined the tissue. Participants listed self-education through active searches in various avail- able media (magazines, Internet, etc.), or through their previous experience, as the main sources of knowledge. Surpris- ingly, only 5.4% of participants consid- ered the medical team as the source of information about blood samples and 7.4% about tissue samples. When several potential uses of surplus blood and archived tissue specimens were listed, comparable results were obtained Results Patients’ background characteristics The questionnaire was distributed to 205 participants (Table 1). There were 55 (26.8%) males and 137 (66.8%) females (missing data on 13 cases). The age ranged between 19 and 80 years old, with a mean of 48.5 years. The majority were of Jordanian nationality and resident in the capital city, Amman (66.3%). Participation in medical research The first question explored any previ- ous participation in medical research. A total of 48 patients (23.4%) had participated in medical research, only 7 of whom (14.6%) had participated previously in studies in which dona- tion of blood samples or examination of tissue biopsies were requested. More than two-thirds of patients (147, 71.7%) had never participated previously in any medical research, since 93 of them (59.2%) had never been approached by researchers. Humanitarian reasons such as helping other cancer patients and/ or benefiting society were listed as the main motivations for participation by 25/48 (52.1%) and 133/147 (90.5%) participants with and without previous research enrolment respectively. Patients’ perceptions about use of biosamples The destination of the surplus blood samples withdrawn during routine clini- cal care was reasonably predicted. Of the participants 83 (40.5%) thought that blood was disposed of immediately once the requested clinical tests were completed. The rest, however, did not have a clear idea about the destination of the surplus blood (Table 2). Compa- rable results were obtained for leftover/ excess tissue samples; only 23.9% were aware that the tissue blocks would rou- tinely be archived in the pathology/ laboratory department at the Centre. Indeed, a similar percentage (23.4%) thought that all tissue samples would be disposed of after the diagnosis was completed (Table 2). The majority of participants (171, 83.3%) declared that the physician or another member of the treating medical team had actually explained the proce- dure involving a tissue biopsy/resection and the indications for performing this procedure. However, 149 (72.7%) con- firmed that the procedural informed consent did not include any informa- tion about what happens to the tissue samples once diagnosis is completed. When they were asked who they thought examined the tissue specimens Table 1 Demographic data of participants and previous experience in research (n = 205) Variable No. % Sex Male 55 26.8 Female 137 66.8 Not stated 13 6.3 Education level Illiterate 10 4.9 High school 91 44.3 College 34 16.6 University 51 24.9 Postgraduate studies 9 4.4 Not stated 10 4.9 Marital status Married 160 78.0 Single 15 7.3 Divorced 9 4.4 Widowed 10 4.9 Not stated 11 5.4 Nationality Jordanian 178 86.8 Non-Jordanian 9 4.4 Not stated 18 8.8 Place of residence in Jordan (n = 178) Amman 118 66.3 Outside Amman 60 33.7 Previous participation in medical research Yes 48 23.4 No 147 71.7 Not stated 10 1.9 Book 20-6.indb 380 6/17/2014 2:41:07 PM طسوتلما قشرل ةيحصلا ةلجلمانوشرعلا دلجلما سداسلا ددعلا 381 in both cases: 139 (67.8%) and 138 (67.3%) patients agreed on using the surplus blood and archived tissue speci- mens respectively in medical research (Table 2). Patients’ perceptions of the ownership of biosamples The last part of the questionnaire fo- cused on assessing patients’ percep- tions of the ownership of their samples and potential obstacles that might face researchers when dealing with bio- specimens in research. The majority of patients believed that ownership lies within the framework of the medical body; 81 (39.5%), 34 (16.6%) and 14 (6.8%) believed that the hospital, labo- ratory or surgeon owned the specimens, respectively. Only 55 (26.8%) believed that the patient owned the specimens and 21 (10.2%) did not know who claimed ownership. Most of the patients (196, 95.6%) showed no interest in retrieving the blood/tissue specimens once clinical care was completed. Interestingly, 170 (82.9%) patients were willing to donate a blood sample primarily for research purposes, even if this was not driven by medical care. An explanatory note in this section of the questionnaire clearly stated that blood and tissue samples were not exhausted completely during the routine clinical work-up, and that tissue specimens were archived in the pathology depart- ment. The overwhelming majority (201, 98.0%) accepted for their surplus/ar- chived samples to be used in research if they consented, and most patients (166, 81.0%) would consent to sending their samples for analysis to external interna- tional laboratories. Many participants (174, 84.9%), however, were interested in knowing the results of the research tests on their specimens. Patients wish to give consent Two-thirds (139, 67.8%) did not re- quest to be consented each time their samples were used in research, i.e. agreed on a general opt-in approach. Patients who wanted to know the results of the research tests were significantly more inclined to require their consent being secured each time prior to using their samples (P = 0.02) (Table 3), whereas those who did not object to their sample being sent to other laboratories were significantly less likely to request their consent being secured prior to any re- search study (P = 0.05) (Table 3). The impact of demographic vari- ables (age, sex and level of education) and prior participation in research were tested against the patients’ willingness to donate blood samples for research (P = 0.90, 0.91, 0.94 and 0.87 respectively) and their attitudes to use of leftover samples in research (P = 0.80, 0.33, 0.59 and 0.27 respectively), the need to be consented each time leftover specimens are used (P = 0.17, 0.33, 0.067 and 0.68 respectively) and sending exporting biospecimens abroad to international laboratories (P = 0.36, 0.59, 0.04 and 0.40 respectively). None of the variables showed statistically significant results, except for education, which tended to affect the decision regarding the need to be consented each time specimens were used for research as well as accepting the export of specimens abroad. Discussion Our study offers an insight into the views of this sample of patients on the use of surplus blood and archived tis- sue specimens in research. The great majority of participants accepted the use of leftover samples in research if they have consented. Patents’ age, sex, Table 2 Patients’ perceptions about the destination and potential use of surplus blood and tissue samples taken in routine clinical care (n = 205) Itema Blood samples Tissue samples No. % No. % What do you think happens to the leftover tissue? Thrown away immediately after the procedure 83 40.5 48 23.4 Used to teach medical students about disease 56 27.3 70 34.1 Used to test new drugs 25 12.2 44 21.5 Preserved and kept (archived) 21 10.2 49 23.9 Don’t know 35 17.1 31 15.1 If you were to decide on what happens to the leftover tissue, which of the following would you agree to? Teaching medical students 141 68.8 129 62.9 Medical research 139 67.8 138 67.3 Diagnosing other diseases 106 51.7 86 42.0 Training doctors 78 38.0 78 38.0 Calibration of medical devices 62 30.2 58 28.3 aPatients could choose more than 1 response per question. Book 20-6.indb 381 6/17/2014 2:41:07 PM EMHJ • Vol. 20 No. 6 • 2014 Eastern Mediterranean Health Journal La Revue de Santé de la Méditerranée orientale 382 educational level and previous partici- pation in research did not significantly affect their acceptance. The findings in our study are in accordance with other studies reported in the international literature (16–18). In other published studies agreement on the use of tissue in research was shown to be unaffected by patients’ demographic characteris- tics or ethnicity and whether they were diseased or not (17,19). Respect for autonomy of research participants is the cornerstone of research ethics. Obtaining informed consent guarantees patients’ auton- omy. Whether an opt-in or opt-out, general or specific, consent is needed in biospecimen research is still being debated. A study on cancer patients in The Netherlands indicated that less than 5% of surveyed colon and breast carcinoma patients declined using their tissue in research. More patients (60%) preferred an opt-out plus approach, in which active verbal information about the possibility to opt-out with all future research on their tissue was preferred to a one time general consent (11%) (20). A focus group survey of the Japa- nese public indicated diverse attitudes towards the use of archived samples in research without consent. Their views ranged from a positive approval of the use of samples without prior consent, to reluctance to accept, to requesting a non-specific prior notice, up to wishing to have the power to opt-out and insist- ence on individual informed consent (21). A majority of Scottish people surveyed were positive about leftover blood being collected and stored for future research use, but they preferred an option for an open-ended consent (22). These same views were shared by a group of Finnish people surveyed (23). On the other hand, a Swedish study investigating public opinion on the use of biospecimens showed that the majority were willing to donate a sample for storage in biobanks for future research under a one-time gen- eral consent, subject to approval by ethics committees (24). Most of our participants indicated that they did not need to consent each time their sam- ples were used in research if an initial consent at the time of collection of the samples was secured, i.e. they were satisfied with a general opt-in consent. Interestingly; these views were similar to what was reported by Ahram et al. in Jordan, in which 90% of the general population preferred an opt-in type of consent, more commonly of a general (75.2%) rather than a specific (16.9%) type (14). This gives credibility to our study since, although our sample was smaller and addressed the views of patients only, the conclusions were similar in both studies. Ownership of the samples removed at the time of routine clinical care is an- other controversial point, with many hot debates between researchers, ethicists and the public, sometimes culminating in lawsuits (25). Ownership implies not only the physical possession of samples, but also the right to any potential ben- efits including financial gains and intel- lectual property. Ownership should be differentiated from custody, whereby specimens are held under the guardian- ship of the custodian, who might not be able to gain the rights to benefits. In a survey of post-surgery patients in the United Kingdom (UK) they listed the hospital (29.1%), the patient (23.2%) or the pathology department (19.7%) as the owner of the samples (26); 15.0% be- lieved that nobody can claim ownership rights, in accordance with the updated version of the Human Tissue Act in UK (27,28). The majority of our surveyed participants thought that the various rep- resentatives of the medical body, i.e. the hospital, the pathology department and/ or the surgeon, had this ownership rights and only 10.2% thought that nobody should claim ownership. Although this is marginally less than the 15% reported by Bryant et al. (26), the difference could be explained on the basis of increased public awareness in the UK following adverse publicity in 2000 about tissue retention, for which public campaigns and legal inquiries were launched, and which has resulted in the amendments made to the Human Tissue Act (29). Some limitations of the current study include the small number of participants recruited from a single institution. In ad- dition, not all questionnaire items were completed by all patients. Despite this, the current study sheds light on the perception of our patients about the potential use of leftover biospecimens in research. Several recommendations emerged, including the need to update the Centre’s procedural consent to in- clude a clear statement on the potential use of archived tissue material in research, where an opt-in or opt-in plus approach Table 3 Patients’ interest in knowing the results of research with biosamples and their willingness for samples to be sent to foreign institutions: correlation with their wish to give informed consent Item Total Wish to give informed consent Yes No No. % % Interested to know results Yes 174 35.5 64.5 No 31 13.3 86.7 χ2 = 5.73, df = 1, P = 0.02 Willing for samples to be sent to foreign institutions Yes 166 29.0 71.0 No 39 45.9 54.1 χ2 = 3.96, df = 1, P = 0.05 Book 20-6.indb 382 6/17/2014 2:41:08 PM طسوتلما قشرل ةيحصلا ةلجلمانوشرعلا دلجلما سداسلا ددعلا 383 References 1. Landon MR. Ethics and policy perspectives on personalized medicine in the post-genomic era. J Biolaw Bus. 2005;8(3):28– 36. PMID:16459425 2. Cohen MC, Blakey S, Donn T, McGovern S, Parry L. An audit of parents’/guardians’ wishes recorded after coronial autopsies in cases of sudden unexpected death in infancy: issues raised and future directions. Med Sci Law. 2009 Jul;49(3):179–84. PMID:19787989 3. Grizzle W, Grody WW, Noll WW, Sobel ME, Stass SA, Trainer T, et al. Recommended policies for uses of human tissue in re- search, education, and quality control. Ad Hoc Committee on Stored Tissue, College of American Pathologists. Arch Pathol Lab Med. 1999 Apr;123(4):296–300. PMID:10320140 4. Ashcroft R. The ethics of reusing archived tissue for research. Neuropathol Appl Neurobiol. 2000 Oct;26(5):408–11. PMID:11054180 5. Upshur RE, Lavery JV, Tindana PO. Taking tissue serious- ly means taking communities seriously. BMC Med Ethics. 2007;8:11. PMID:17963497 6. Bathe OF, McGuire AL. The ethical use of existing samples for genome research. Genet Med. 2009 Oct;11(10):712–5. PMID:19745750 7. Truyers C, Kellen E, Arbyn M, Trommelmans L, Nys H, Hensen K, et al. The use of human tissue in epidemiological re- search; ethical and legal considerations in two biobanks in Belgium. Med Health Care Philos. 2010 May;13(2):169–75. PMID:19936964 8. Schäfer SC, Lehr HA. A case study on the proper use of human tissues for biomedical research at an academic pathology institution in Switzerland. Pathobiology. 2007;74(4):259–63. PMID:17709969 9. Langat SK. Reuse of samples: ethical issues encountered by two institutional ethics review committees in Kenya. Bioethics. 2005 Oct;19(5-6):537–49. PMID:16425489 10. Maschke KJ, Murray TH. Ethical issues in tissue banking for re- search: the prospects and pitfalls of setting international stand- ards. Theor Med Bioeth. 2004;25(2):143–55. PMID:15368751 11. Al-Qadire MM, Hammami MM, Abdulhameed HM, Al Gaai EA. Saudi views on consenting for research on medical records and leftover tissue samples. BMC Med Ethics. 2010;11:18. PMID:20955580 12. Abou-Zeid A, Silverman H, Shehata M, Shams M, Elshabrawy M, Hifnawy T, et al. Collection, storage and use of blood sam- ples for future research: views of Egyptian patients expressed in a cross-sectional survey. J Med Ethics. 2010 Sep;36(9):539– 47. PMID:20663757 13. Tazzite A, Roky R, Avard D; Institut International de Recherche en Ethique Biomédicale. Les implications éthiques de la conservation des échantillons biologiques [The ethical im- plications of conserving biological samples]. J Int Bioethique. 2009 Sep;20(3):87–96, 150–1. PMID:20425942 14. Ahram M, Othman A, Shahrouri M. Public support and consent preference for biomedical research and biobanking in Jordan. Eur J Hum Genet. 2013 May;21(5):567–70. PMID:22968133 15. Ramahi I, Silverman H. Clinical research law in Jordan: an ethical analysis. Dev World Bioeth. 2009 Apr;9(1):26–33. PMID:18302540 16. Jack AL, Womack C. Why surgical patients do not donate tis- sue for commercial research: review of records. BMJ. 2003 Aug 2;327(7409):262. PMID:12896938 17. Chen DT, Rosenstein DL, Muthappan P, Hilsenbeck SG, Miller FG, Emanuel EJ, et al. Research with stored biological samples: what do research participants want? Arch Intern Med. 2005 Mar 28;165(6):652–5. PMID:15795341 18. Hoeyer K, Olofsson BO, Mjörndal T, Lynöe N. The ethics of research using biobanks: reason to question the importance attributed to informed consent. Arch Intern Med. 2005 Jan 10;165(1):97–100. PMID:15642883 19. Goodson ML, Vernon BG. A study of public opinion on the use of tissue samples from living subjects for clinical research. J Clin Pathol. 2004 Feb;57(2):135–8. PMID:14747435 20. Vermeulen E, Schmidt MK, Aaronson NK, Kuenen M, van der Valk P, Sietses C, et al. Opt-out plus, the patients’ choice: preferences of cancer patients concerning information and consent regimen for future research with biological samples archived in the context of treatment. J Clin Pathol. 2009 Mar;62(3):275–8. PMID:19017681 21. Asai A, Ohnishi M, Nishigaki E, Sekimoto M, Fukuhara S, Fukui T. Attitudes of the Japanese public and doctors towards use of archived information and samples without informed consent: preliminary findings based on focus group interviews. BMC Med Ethics. 2002 Jan 9;3:E1. PMID:11825345 22. Treweek S, Doney A, Leiman D. Public attitudes to the storage of blood left over from routine general practice tests and its use in research. J Health Serv Res Policy. 2009 Jan;14(1):13–9. PMID:19103912 23. Tupasela A, Sihvo S, Snell K, Jallinoja P, Aro AR, Hemminki E. Attitudes towards biomedical use of tissue sample collections, consent, and biobanks among Finns. Scand J Public Health. 2010 Feb;38(1):46–52. PMID:19906772 24. Kettis-Lindblad A, Ring L, Viberth E, Hansson MG. Genetic research and donation of tissue samples to biobanks. What do could be adopted. We suggest that the medical team has to pursue a more active role in informing the patients about this vital issue, preferably during the process of consent. On the other hand, increasing public awareness about the value of hu- man biospecimens in research through mass education and public campaigns was previously recommended by Ahram et al. (14). In Jordan, both studies could serve as a baseline and guidance for estab- lishing nationwide guidelines. Acknowledgements The authors would like to thank Dr L. Zaru for her help in the statistical analysis and interpretation as well as valuable comments, Ms D. Al-Remawi for helping in the statistical analysis, Dr F. Attiga for the critical review of the manuscript and Dr J. Ro for final review and help with English correction. The authors would also like to acknowledge the following students from the School of Pharmacy at Jordan University for helping in conducting the study: Z. Abdelmajeed, S. Al Hasan, D. Al Zobi, S. Anshasi, R. Eddeen, A. Fahhad, A. Khamees, H. Khamees and N. Shaheen. We also thank Ms D. Khammash from the Medical School at the Ameri- can University of Beirut for her help in data cleaning. Funding: None. Competing interests: None declared. Book 20-6.indb 383 6/17/2014 2:41:08 PM EMHJ • Vol. 20 No. 6 • 2014 Eastern Mediterranean Health Journal La Revue de Santé de la Méditerranée orientale 384 potential sample donors in the Swedish general public think? Eur J Public Health. 2006 Aug;16(4):433–40. PMID:16207726 25. Charo RA. Body of research–ownership and use of human tis- sue. N Engl J Med. 2006 Oct 12;355(15):1517–9. PMID:17035644 26. Bryant RJ, Harrison RF, Start RD, Chetwood AS, Chesshire AM, Reed MW, et al. Ownership and uses of human tissue: what are the opinions of surgical in-patients? J Clin Pathol. 2008 Mar;61(3):322–6. PMID:18256118 27. Baum M. The use and abuse of human tissue: an analysis of the ethical issues raised by the proposed Human Tissue Act. Med Leg J. 2004;72(Pt 2):67–9. PMID:15239575 28. Angell E, Tarrant C, Dixon-Woods M. Research involving stor- age and use of human tissue: how did the Human Tissue Act 2004 affect decisions by research ethics committees? J Clin Pathol. 2009 Sep;62(9):825–9. PMID:19734481 29. Furness PN. Research using human tissues–a crisis of supply? J Pathol. 2001 Oct;195(3):277–84. PMID:11673823 Book 20-6.indb 384 6/17/2014 2:41:09 PM طسوتلما قشرل ةيحصلا ةلجلمانوشرعلا دلجلما سداسلا ددعلا 385 Smokeless tobacco consumption in a multi-ethnic community in Pakistan: a cross-sectional study S.M. Abbas,1 A.Y. Alam,2 M. Usman 1 and K. Siddiqi 3 ABSTRACT Smokeless tobacco is commonly used in south Asia. In addition to causing oral and pharyngeal cancers, its harmful effects are comparable to smoking tobacco. A cross-sectional survey with systematic sampling was conducted in 2010–2011 to investigate smokeless tobacco use in a multi-ethnic, semi-urban population in Islamabad, Pakistan (n = 2030). The prevalence of smokeless tobacco use was 16.0% (21.6% among males and 8.8% among females); 51.7% of smokeless tobacco users were also cigarette smokers. The rate of smokeless tobacco use was comparatively high among Pakhtun males (38.2%) and Sindhi females (22.4%). The associations between smokeless tobacco use and ethnicity, age group, income level and cigarette smoking were statistically significant among male smokeless tobacco users. Of the sample 41.4% (840/2030) had inadequate knowledge about the health problems associated with smokeless tobacco. Appropriate interventions are needed to raise awareness of the health risks and to prevent smokeless tobacco use. 1Department of Health Sciences, University of York, York, United Kingdom (Correspondence to S.M. Abbas: taureanvibes@hotmail.com). 2King Fahad Hospital, Al Ahsa, Saudi Arabia. 3Department of Health Sciences, Hull York Medical School, University of York, York, United Kingdom. Received: 09/09/13; accepted: 15/01/14 ةضرعتسم ةسارد :ناتسكاب في تاينثلإا ددعتم عمتمج في ينخدت نودب غبتلا كلاهتسا يقيدص نارماك ،نماثع دممح ،ملع رواي ليع ،سابع ملسم ديس ةراـضلا هراـثآ نإـف ،موـعلبلاو مـفلا ناـطسر ببـسي هـنأ لىإ ةـفاضلإاب وـهو .ايـسآ بوـنج في نـخدت نودـب غـبتلا مادختـسا عيـشي :ةـصلالخا ةـفرعلم 2011-2010 نـماعلا في ةـيجهنم تاـنيع ذـخأب ةضرعتـسم ةـسارد نوـثحابلا ىرـجأ دـقو .نـخدتلاب غـبتلا يـطاعت عـم اـهتنراقم نـكمي 2030 تاـنيعلا ددـع غـلبو ،ناتـسكاب ،داـبآ ملاـسإ في تاـينثلإا ةددـعتمو ةـيضرح هبـش ةيناكـس ةـعوممج في نـخدت نودـب غـبتلا مادختـسا ىدـم نأ ماـك ،)ثاـنلإا نـب % 8.8و روـكذلا نـب % 21.6( % 16.0 غـلب دـق نـخدت نودـب غـبتلا مادختـسا راـشتنا لدـعم نأ نـثحابلل حـضتاو .ةـنيع نوـتخاب روـكذ ىدـل ًاـعفترم نـخدت نودـب غـبتلا مادختـسا لدـعم ناك دـقو .رئاجـسلا نوـنخدي اوـناك نـخدت نودـب غـبتلا مدختـسي نـمم % 51.7 نـبو نـخدت نودـب غـبتلا مادختـسا نـب ًاـيئاصحإ هـب ُّدـتعُي طـُبارت كاـنه ناك دـقو .مـهرغ عـم ةـنراقم )% 22.4( دنـسلا ثاـنإ ىدـلو ،)% 38.2( نـم % 41.4 ناكو .روـكذلا نـم نـخدت نودـب غـبتلا مدختـسي نـم نـب كـلذو رئاجـسلا نـخدتو ،لـخدلا ىوتـسمو ،ةـيرمعلا ةـئفلاو ،ةـينثلإا .نـخدت نودـب غـبتلا مادختـسا عـم طبارــتت يـتلا ةـيحصلا تلاكـشلما نـع ةـيفاك فراـعم نوـكلتمي لا )ًادرـف 2030 نـب نـم ًادرـف 840( ةـنيعلا .نـخدت نودـب غـبتلا مادختـسا نـم ةـياقوللو ةـيحصلا رـطاخلما لوـح يـعولا ىوتـسم عـفرل ةـمئلام تلاـخدت لىإ ةـجالحا سـتمو Consommation de tabac sans fumée dans une communauté pluriethnique au Pakistan : une étude transversale RÉSUMÉ La consommation de tabac sans fumée est courante en Asie du Sud. Outre les cancers de la cavité buccale et du pharynx, ses effets nocifs sont comparables à ceux de la consommation de tabac à fumer. Une enquête transversale à partir d'un échantillonnage systématique (n = 2030) a été menée en 2010 et 2011 afin d'évaluer la consommation de tabac sans fumée dans une population semi-urbaine et pluriethnique à Islamabad (Pakistan). La prévalence de la consommation de tabac sans fumée était de 16,0 % (21,6 % chez les hommes et 8,8 % chez les femmes) ; 51,7 % des consommateurs de tabac sans fumée étaient aussi des fumeurs de cigarettes. Le pourcentage de consommation de tabac sans fumée était comparativement élevé chez les hommes pachtounes (38,2 %) et les femmes sindhies (22,4 %). Les associations entre la consommation de tabac sans fumée, le groupe ethnique, la tranche d'âge, le niveau de revenu et la consommation de cigarettes étaient statistiquement significatives chez les consommateurs de tabac sans fumée de sexe masculin. Au sein de l'échantillon, 41,4 % (840/2030) possédaient des connaissances insuffisantes sur les problèmes de santé liés au tabac sans fumée. Des interventions appropriées sont nécessaires pour sensibiliser aux risques sanitaires et prévenir la consommation de tabac sans fumée. Book 20-6.indb 385 6/17/2014 2:41:09 PM EMHJ • Vol. 20 No. 6 • 2014 Eastern Mediterranean Health Journal La Revue de Santé de la Méditerranée orientale 386 Introduction Smokeless tobacco products, which are not burnt but are utilized orally or nasally, have been used in differ- ent countries for centuries and are accessible in many different forms (1,2). The prevalence of smokeless tobacco consumption varies across and within different countries, depend- ing on factors such as socioeconomic status, ethnic origin, sex and age (1,3). These products are most commonly used by south Asian communities, in which around one-third of tobacco is consumed in smokeless form (2). Smokeless tobacco consumption is a threat to public health. It is associ- ated with oropharyngeal, laryngeal, oesophageal and pancreatic cancers, cardiovascular diseases, adverse out- comes of pregnancy and dental disease (4). Furthermore, studies indicate that the use of smokeless tobacco products appears to be increasing, particularly among the younger age groups (5–7). The consumption of smokeless to- bacco products in low- and middle-in- come countries is of particular concern because the products are manufactured and sold without proper regulation and are often consumed with other carcino- genic substances (2). In Pakistan, a low- income country, smokeless tobacco is consumed mainly in the form of naswar (tobacco flavoured with cardamom and menthol) (2), paan or betel quid (containing tobacco, lime, areca nut and other flavourings) (8). Despite its widespread use in Pakistan, little em- pirical research has been carried out on the prevalence and determinants of smokeless tobacco consumption. Studies about the association of use of smokeless tobacco and other substanc- es, such as areca nut, with head and neck and oral cancers have been con- ducted in certain regions of Pakistan, often with a small sample size (8–14). Similarly, a study in Karachi reported a 16% prevalence of smokeless tobacco use among 772 adolescent high-school males (10). The prevalence of smoke- less tobacco use among various eth- nic groups and their knowledge and perceptions concerning to its health implications is not known. We inves- tigated these factors in a semi-urban population in Nurpur Shahan in the outskirts of Islamabad. The population is approximately 14 000, consisting of people who are migrants from all prov- inces and represent different ethnic groups and sociocultural backgrounds Methods A cross-sectional survey with system- atic random sampling was conducted from January 2010 to January 2011. We measured self-reported use of smoke- less tobacco, types of products used and individuals’ perceptions and knowledge of the related health implications. Study setting Nurpur Shahan was selected as the surveillance site. This urban slum was chosen because it has settlements from all provinces and represents a relatively marginalized segment of the society. Sampling The sample was first stratified into males and females. With a precision of + 3%, and an expected prevalence of smoke- less tobacco of 50% in the community and 95% confidence interval (CI), the sample size was estimated as 992 in each group (males and females). To account for possible incomplete data, the sample size was increased to 1015 each, a total of 2030. The sample size was inflated by only 2.3%, because the interviewers were well trained, as pilot-testing further strengthened the rigorous methodol- ogy and because the interviews were conducted face-to-face, ensuring com- pleteness of questionnaires. Males and females aged 5–65 years who were permanent residents of the area and gave informed consent were included in the study. Children aged 5 years and above were included in the study because previous studies con- ducted in South Asian communities has reported smokeless tobacco use in the age range of 5–10 years (15,16). A systematic sampling technique was applied. In each street of the locality, we surveyed the first house followed by the third, fifth and so on. When a particular street was completed, subse- quent streets was approached in a simi- lar fashion. The study was conducted by groups of 4 students, each including at least 1 female, as per the cultural norms of the community. Once a house was selected all members of the family that met the eligibility criteria were con- sented and then interviewed. Data collection Questionnaire The questionnaire components were compiled utilizing previously validated questions. The questionnaire included 3 sections. The first section was to deter- mine the following sociodemographic variables: age (from date of birth or, if unavailable, estimation of age with reference to an index event) (17,18); education (number of years of formal education completed) (17–19); eth- nicity (determined from place of birth and ethnic origin in Pakistan) (20); and income (as rupees per month) (17,18). The second section enquired about respondents’ tobacco consumption: type of smokeless tobacco products used; amount and frequency of use; and cigarette smoking habits. Smokers were define as current and ever-smokers (8–12,15–20). In the third section respondents answered 5 questions designed to assess their knowledge and perceptions about the health implica- tions associated with smokeless tobacco use (8–12,15–20). The questionnaire was first pilot- tested in a few households to address any ambiguity. The questions was translated into Urdu language and then back-translated into English to ensure content validity. Book 20-6.indb 386 6/17/2014 2:41:09 PM طسوتلما قشرل ةيحصلا ةلجلمانوشرعلا دلجلما سداسلا ددعلا 387 The association between smoke- less tobacco use and age group, ethnic- ity, income level and cigarette smoking was statistically significant (P < 0.001) among male smokeless tobacco users (Table 2). Among female smokeless tobacco users there was a significant association between smokeless tobacco use and income level, education and ethnicity. In the total sample educa- tion was the only variable that was not significant (P = 0.06). Nearly half (41.4%) of the study sample had inadequate knowledge about the health problems associated with smokeless tobacco and 34.1% of the sample perceived smokeless to- bacco use as harmless. Only 15 (0.7%) participants suspected an association of cardiovascular disease with smoke- less tobacco use and even fewer (0.4%) reported an association of smokeless tobacco with adverse outcomes of preg- nancy (Table 3). Discussion Smokeless tobacco is an important pub- lic health concern in south Asian coun- tries, as such products are manufactured at a mass scale without any checks on the carcinogenic content of the prod- ucts and information to the public about their harmful health effects (2). The consumption of smokeless tobacco Training of the interviewers The survey was carried out by trained undergraduate medical students as part of their scheduled public health practice field visits. Eight teams, each consisting of 4 students, administered the survey. The students had prior experience of conducting surveys in the same com- munity. The students were formally trained in taking informed consent, administering the questionnaire in ac- cordance with the Strengthening the Reporting of Observational Studies in Epidemiology checklist for conducting cross-sectional studies (21). Interviews The survey questionnaire was admin- istered through face-to-face interviews. A non-coercive approach was adopted, and the participants were not offered any incentives to participate in the sur- vey. Illiterate respondents were given a verbal explanation about the purpose of the research, and informed consent was taken; they were asked to give a thumbprint if they could not sign. A quality assurance framework was used to ensure consistency and quality (22). Moreover, verification checks were done on 5% of the sample (17). The response rate of the survey was 99%. Ethical considerations Ethical clearance for this study was obtained from the ethics committee at Shifa College of Medicine in December 2009. Written informed consent was obtained from all participants in the survey and all data collected were kept confidential. In the case of children (5–13 years old) informed consent was taken from adults in the household. Data analysis The data were entered into SPSS , version 19.0 statistical software. All categorical variables were presented as frequencies and percentages. The rela- tionship between 2 categorical variables was calculated by cross-tabulation and applying chi-squared test and estimat- ing P-values. Results A total of 2030 individuals were sur- veyed including 1132 (55.8%) males and 898 (44.2%) females. The preva- lence of smokeless tobacco use in the total sample was 16.0% (324/2030): 21.6% (245/1132) among males and 8.8% (79/898) among females. The rate of tobacco smoking among male smokeless tobacco users was 188/245 (76.7%) and among female smokeless tobacco users was 49/79 (62.0%). Of the participants 54 (2.6%) reported be- ing daily consumers of paan mixed with tobacco and 84 (4.1%) as daily naswar users (Figure 1). Analysis by ethnic groups showed the highest prevalence of smokeless tobacco use was among Sindhi (23.9%) followed by Pakthtun (22.3%), with Punjabi (12.3%) and Urdu speaking (16.0%) groups reporting the lowest prevalence (Table 1). The prevalence of smokeless tobacco use was highest in the age group 30–39 years (23.6%). Furthermore, smokeless tobacco use was highest among male users in age groups 11–20 years (38.4%) whereas among females it was highest in ages 50+ years. The prevalence of smoke- less tobacco was lowest among the participants earning 9000+ rupees per month and those with the highest level of education. Occasionally 3 times or more a week Daily Never Paan Naswar 4.8%,98 4.3%,88 2.6%,54 4.1%,84 48.6%,989 35.3%,717 Figure 1 Respondents’ self-reported frequency of use of different smokeless tobacco products (n = 2030) Book 20-6.indb 387 6/17/2014 2:41:10 PM EMHJ • Vol. 20 No. 6 • 2014 Eastern Mediterranean Health Journal La Revue de Santé de la Méditerranée orientale 388 among males in the study is comparable to the rates observed in India (26.4%) according to the Global Adult Tobacco Survey (GATS) household survey (23). However, consumption among females was much lower than the rate observed in Indian (18.4%) and Bangladeshi (27.9%) females (23). When compared with the Global Youth Tobacco Survey (GYTS) conducted in Pakistan the prevalence among male smokeless to- bacco users in the study sample exceed- ed the rate (13.8%) among male users surveyed in the GYTS, whereas among female users it was similar (7.4%) (24). Smokeless tobacco use started at an early age in our population, and this is similar to the studies conducted in 3 different rural areas of India and the state of Goa (15,16). Paan mixed with tobacco and nas- war were found to be the 2 commonest types of smokeless tobacco products used, and this agrees with previous Table 1 Prevalence of smokeless tobacco use by demographic characteristics of males and females Variable Both sexes Males Females Total Smokeless tobacco users Total Smokeless tobacco users Total Smokeless tobacco users No. No. % 95% CI No. No. % 95% CI No. No. % 95% CI Overall 2030 324 16.0 14.3–17.5 1132 245 21.6 19.2–24.0 898 79 8.8 7.0–11.0 Age (years) 5–10 269 13 4.8 2.3–7.4 159 10 6.3 2.5–10.0 110 3 2.7 0.3–5.7 11–20 388 81 20.9 16.8–24.8 185 71 38.4 31.3–45.3 203 10 4.9 1.9–7.8 21–29 442 84 19.0 15.3–22.7 267 62 23.2 18.1–28.3 175 22 12.6 7.7–17.5 30–39 246 58 23.6 18.3–28.9 147 48 32.7 25.0–40.2 99 10 10.1 4.2–16.0 40–49 383 42 11.0 7.8–14.0 204 25 12.3 7.8–16.8 179 17 9.5 5.2–13.8 50–59 200 29 14.5 9.6–19.4 115 18 15.7 8.9–22.2 85 11 12.9 5.8–20.0 > 60 102 17 16.7 9.4–23.8 55 11 20.0 9.4–30.6 47 6 12.8 3.2–22.2 Ethnicity Punjabi 1127 139 12.3 10.4–14.2 612 102 16.7 13.6–19.5 515 37 7.2 4.9–9.4 Pakhtun 452 101 22.3 18.5–26.1 228 87 38.2 31.8–44.4 224 14 6.3 3.1–9.5 Sindhi 134 32 23.9 16.6–31.0 85 21 24.7 15.5–33.8 49 11 22.4 10.7–34.1 Balochi 44 9 20.5 8.5–32.3 30 7 23.3 8.2–38.4 14 2 14.3 4.1–32.5 Urdu speaking 162 26 16.0 10.4–21.6 103 16 15.5 8.5–22.5 59 10 16.9 7.3–26.5 Other 111 17 15.3 8.6–22.0 74 12 16.2 7.8–24.6 37 5 13.5 2.5–24.5 Income per month (rupees) < 2000 288 49 17.0 12.1–21.8 90 21 23.3 14.6–32.0 198 28 14.1 9.2–18.9 2000–3499 389 65 16.7 12.9–20.4 96 60 62.5 52.8–72.2 293 5 1.7 0.2–3.2 3500–4999 470 78 16.6 13.2–19.9 184 49 26.6 20.2–32.9 286 29 10.1 6.6–13.6 5000–8999 629 117 18.6 15.6–21.6 525 101 19.2 15.8–22.6 104 16 15.4 8.4–22.2 9000+ 254 15 5.9 3.0–8.8 237 14 5.9 2.9–8.9 17 1 5.9 0.31–16.9 Education None 1048 178 17.0 14.6–19.2 392 129 32.9 28.2–37.5 656 49 7.5 5.4–9.4 Primary 227 39 17.2 12.2–22.0 178 29 16.3 10.8–21.7 49 10 20.4 9.1–31.7 Middle 267 45 16.9 12.3–21.3 159 39 24.5 17.8–31.2 108 6 5.6 1.2–9.8 Matric 185 32 17.3 11.8–22.7 125 26 20.8 13.7–27.9 60 6 10.0 2.4–17.6 Intermediate 135 16 11.9 6.4–17.2 119 9 7.6 2.8–12.4 16 7 43.8 19.4–68.0 Graduate and above 168 14 8.3 4.1–12.5 159 13 8.2 3.9–12.5 9 1 11.1 0.4–31.6 Cigarette smoking Smoker 458 237 51.7 47.1–56.3 395 188 47.6 42.7–52.5 63 49 77.8 67.4–87.9 Non-smoker 1572 87 5.5 4.4–6.6 737 57 7.7 5.8–9.6 835 30 3.6 2.3–4.9 CI = confidence interval. Book 20-6.indb 388 6/17/2014 2:41:10 PM طسوتلما قشرل ةيحصلا ةلجلمانوشرعلا دلجلما سداسلا ددعلا 389 findings from Pakistan (11–13). The consumption of naswar (53.0%) can be explained by the presence of a Pakthun and Balochi ethnic groups in the sample who have a cultural tradition of using this product (12,13). The association between education level and smokeless tobacco use was not significant in the total sample (P = 0.06); however, nearly half of our sample consisted of people with no formal education and the sample size was therefore insufficient to detect associations within other categories. The rate of smokeless tobacco con- sumption fell to 8.3% when education levels increased to graduate and above, a finding similar to a study conducted in Rawalpindi, Pakistan (17). The low level of awareness among our partici- pants about the health implications associated with smokeless tobacco highlights the need for health educa- tion campaigns to raise awareness in the community. This was not a countrywide sur- vey using a sampling strategy that was representative of the whole Pakistan population. Instead, the study was conducted in a diaspora population representing major ethnicities living in the country. Education and income levels in the community differ from those in the urban cities and the as- sociations between smokeless tobacco and socioeconomic factors may vary if cross-sectional studies with a repre- sentative sample size were conducted there. However 66% of the Pakistani population lives in rural areas (25) and the education and income levels in this community study may correspond with the rural population of Pakistan. The awareness levels regarding the health implications associated with smokeless tobacco may also be different in the urban cities where education levels are higher and people have more access to information in the form of social and electronic media. The consumption of smoke- less tobacco at an early age and high prevalence among particular ethnici- ties suggests that there may be a cul- tural acceptance of the habit among this urban slum community. This may also be influenced by level of educa- tion and awareness about the health implications associated with smokeless tobacco. Smokeless tobacco use is cul- turally embedded within South Asian communities (2). Furthermore, it will be worthwhile focusing future research on the ethnic and cultural context. In- formation about the health implications associated with smokeless tobacco use need to be more widely dispersed in the form of mass health education and media campaigns. The different ethnici- ties highlighted in this research reside in other geographical locations within our country and also form ethnic minorities in different countries. The findings of this research can be applied to such communities living abroad. Appropriate Table 2 Relationship of ethnicity, age groups, education, cigarette smoking and income with smokeless tobacco use Variable Significance of association with smokeless tobacco use Total Males Females χ2 P-value χ2 P-value χ2 P-value Age 53 0.001 59.3 0.001 11.5 0.07 Ethnicity 31.7 0.001 34.7 0.001 10.9 0.05 Education 10.5 0.06 9.15 0.103 11.5 0.04 Income 23.0 0.001 31.2 0.001 51.1 0.001 Cigarette smoking 607 0.001 111.3 0.001 5.2 0.02 Table 3 Knowledge and perceptions of the respondents about the health implications associated with smokeless tobacco use Variable Respondents agreeing (n = 2030) No. % 95% CI Smokeless tobacco has carcinogenic tendencies and can cause oral cancers and cancers of other body organs 251 12.3 10.8– 13.7 Smokeless tobacco use can lead to cardiovascular disease 15 0.7 0.3– 1.1 Smokeless tobacco used can lead to adverse outcomes of pregnancy 9 0.4 0.1– 0.7 Smokeless tobacco can lead to dental diseases 223 11.0 9.6– 12.4 Smokeless tobacco has no associated health implications 692 34.1 31.9– 36.1 Don’t know or have no relevant knowledge 840 41.4 38.9– 43.1 CI = confidence interval. Book 20-6.indb 389 6/17/2014 2:41:10 PM EMHJ • Vol. 20 No. 6 • 2014 Eastern Mediterranean Health Journal La Revue de Santé de la Méditerranée orientale 390 References 1. Boffetta P, Hecht S, Gray N, Gupta P, Straif K. Smokeless tobacco and cancer. Lancet Oncol. 2008 Jul;9(7):667–75. PMID:18598931 2. Gupta PC, Ray CS. Smokeless tobacco and health in In- dia and South Asia. Respirology. 2003 Dec;8(4):419–31. PMID:14708551 3. Boffetta P, Aagnes B, Weiderpass E, Andersen A. Smokeless to- bacco use and risk of cancer of the pancreas and other organs. Int J Cancer. 2005 May 10;114(6):992–5. PMID:15645430 4. Critchley JA, Unal B. Health effects associated with smokeless tobacco: a systematic review. Thorax. 2003 May;58(5):435–43. PMID:12728167 5. Connolly, GN, Winn DM, Hecht SS, Henningfeild JE, Walker B Jr & Hoffman D. the re-emergence of smokeless tobacco. N Engl J Med. 1986 Apr 17;314(16):1020–7. 6. Everett SA, Husten CG, Warren CW, Crossett L, Sharp D. Trends in tobacco use among high school students in the United States, 1991–1995. J Sch Health. 1998 Apr;68(4):137–40. PMID:9644605 7. Vainio H, Weiderpass E. Smokeless tobacco: harm reduction or nicotine overload? Eur J Cancer Prev. 2003 Apr;12(2):89–92. PMID:12671531 8. Shah SM, Merchant AT, Luby SP, Chotani RA. Addicted school- children: prevalence and characteristics of areca nut chewers among primary school children in Karachi, Pakistan. J Paediatr Child Health. 2002 Oct;38(5):507–10. PMID:12354270 9. Khawaja MR, Mazahir S, Majeed A, Malik F, Merchant KA, Maqsood M, et al. Chewing of betel, areca and tobacco: per- ceptions and knowledge regarding their role in head and neck cancers in an urban squatter settlement in Pakistan. Asian Pac J Cancer Prev. 2006 Jan-Mar;7(1):95–100. 10. Rozi S, Akhtar S. Prevalence and predictors of smokeless to- bacco use among high-school males in Karachi, Pakistan. East Mediterr Health J. 2007 Jul-Aug;13(4):916–24. PMID:17955775 11. 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Krishnamurthy S, Ramaswamy R, Trivedi U, Zachariah V. Tobacco use in rural Indian children. Indian Pediatr. 1997 Oct;34(10):923–7. PMID:9567556 16. Vaidya SG, Vaidya NS, Naik UD. Epidemiology of tobacco habits in Goa, India. In: Gupta PC, Hamner JE III, Murti PR, edi- tors. Control of tobacco-related cancers and other diseases. Proceedings of an International Symposium, TIFR. Bombay, January 15–19, 1990. Oxford: Oxford University Press; 1992. 17. Alam AY, Iqbal A, Mohamud KB, Laporte RE, Ahmed A, Nishtar S. Investigating socio-economic-demographic determinants of tobacco use in Rawalpindi, Pakistan. BMC Public Health. 2008;8:50. 10.1186/1471-2458-8-50 PMID:18254981 18. Nishtar S, Wierzbicki AS, Lumb PJ, Lambert-Hammill M, Turner CN, Crook MA, et al. Waist-hip ratio and low HDL predict the risk of coronary artery disease in Pakistanis. Curr Med Res Opin. 2004 Jan;20(1):55–62. 19. Khawaja MR, Mazahir S, Majeed A, Malik F, Merchant KA, Maqsood M, et al. Chewing of betel, areca and tobacco: per- ceptions and knowledge regarding their role in head and neck cancers in an urban squatter settlement in Pakistan. Asian Pac J Cancer Prev. 2006 Jan-Mar;7(1):95–100. 20. Rozi S, Akhtar S. Prevalence and predictors of smokeless to- bacco use among high-school males in Karachi, Pakistan. East Mediterr Health J. 2007 Jul-Aug;13(4):916–24. PMID:17955775 21. Strobe checklist for cross sectional studies [Internet]. Bern, Switzerland: STROBE (http://www.strobe-statement.org/ fileadmin/Strobe/uploads/checklists/fileadmin/Strobe/up- loads/checklists/STROBE_checklist_v4_cross-sectional.pdf, accessed 4 April 2014). 22. Lanata CF, Black RE. Lot quality assurance sampling techniques in health surveys in developing countries: advantages and cur- rent constraints. World Health Stat Q. 1991;44(3):133–9. 23. Giovino GA, Mirza SA, Samet JM, Gupta PC, Jarvis MJ, Bhala N, et al.; GATS Collaborative Group. Tobacco use in 3 billion individuals from 16 countries: an analysis of nationally repre- sentative cross-sectional household surveys. Lancet. 2012 Aug 18;380(9842):668–79. PMID:22901888 24. Country fact sheets Pakistan—Karachi (ages 13-15). Global Youth Tobacco Survey (GYTS) [Internet]. Geneva: World Health Organization; 2010 (http://www.emro.who.int/im- ages/stories/tfi/documents/GYTS_FS_PAK_R2.pdf, accessed 4 April 2014). 25. Rural population in Pakistan. Trading Economics [Internet] (http://www.tradingeconomics.com/pakistan/rural-popula- tion-wb-data.html, accessed 4 April 2014). health interventions should be designed to prevent smokeless tobacco use which should be culturally and ethnically adapted. The findings of this research sug- gest that tobacco control policies need to encompass and address smokeless tobacco consumption and not just focus on cigarettes. This could in- volve incorporating questions about smokeless tobacco consumption into national surveys Moreover, research into effective policies and cessation support options should be conducted with prime focus on the provisions of the WHO Framework Convention on Tobacco Control. More research is required to investigate the social de- terminants of smokeless tobacco use among the different ethnic minorities and the constituents of the smokeless tobacco products consumed in the country Competing interests: None declared. Book 20-6.indb 390 6/17/2014 2:41:11 PM طسوتلما قشرل ةيحصلا ةلجلمانوشرعلا دلجلما سداسلا ددعلا 391 Effect of zinc supplementation in children with asthma: a randomized, placebo-controlled trial in northern Islamic Republic of Iran J. Ghaffari,1 A. Khalilian,2 E. Salehifar,3 E. Khorasani 1 and M.S. Rezaii 1 ABSTRACT There are conflicting reports about the benefits of zinc supplements in childhood asthma. This study examined the effect of zinc supplementation in children with asthma attending an outpatient clinic in Sari, Islamic Republic of Iran. In a randomized, double-blind, placebo-controlled clinical trial over 8 weeks, 284 children on inhaled steroids were allocated to receive zinc supplements (50 mg/day) (n = 144) or placebo (n = 140). Cases and controls had low initial serum zinc concentrations [61.8 (SD 7.3) µg/dL and 60.9 (SD 4.3) µg/dL]. After treatment, mean serum zinc level in the case group was significantly higher [129 (SD 20.4) µg/dL] than in the controls [63 (SD 8.6) µg/dL]. There were no significant differences in IgE levels before and after treatment. The case group showed significant improvements in clinical symptoms such as cough, wheezing and dyspnoea and in all spirometry parameters (FVC, FEV1 and FEV1/FVC). 1Department of Paediatrics, Antimicrobial Resistant Nosocomial Infection Research Center; 2Department of Statistics; 3Department of Pharmacology, Mazandaran University of Medical Sciences, Sari, Islamic Republic of Iran (Correspondence to M.S. Rezaii: drmsrezaii@yahoo.com). Received: 01/06/13; accepted: 06/01/14 ةيملاسلإا ناريإ ةيروهجم لماش في دهاوشلاو تلااحلل ةا َّشعم ةسارد :وبرلاب ينباصلما لافطلأا لىع كنزلا نم ةمعادلا ريداقلما رثأ يياضر قداص دممح ،نياسارخ تفع ،رف يلحاص ميهاربإ ،نايليلخ اضرلع ،يرافغ داوج رـثأت ةـساردلا هذـه تـلوانت دـقو .وـبرلاب نـباصلما لاـفطلأا ىـع كـنزلا نـم ةـمعادلا رـيداقلما عـفانم نـع ةـبراضتم رـيراقت كاـنه نإ :ةـصلالخا ةـسارد يـهو ؛ةيملاـسلإا نارـيإ ةـيروهجم في "يراـس" في تاداـيعلا نوـعجاري نـمم وـبرلاب نـباصلما لاـفطلأا ىـع كـنزلا نـم ةـمعادلا رـيداقلما ،ًاقاـشنإ تاديئورتـسلاب نوـلجاعي ًلاـفط 284 تلمـشو عيباـسأ ةـينماث نـم رـثكأ ترمتـسا ةـيمعتلا ةـجودزمو دهاوـشلاو تلااـحلل ةاـ َّشَعُم ةـيريسر نـم لك ىدـل نأ نـثحابلل نـبت دـقو .ًادهاـش ًلاـفط 140 و )موـي/مارغ ليـيم 50( كـنزلا نـم ًماـعاد ًارادـقم اوـقلت ًلاـفط 144 لىإ مـهعيزوت دـعب /مارـغوركم 4.3 ± 60.9و ،تلااـحلل رـل يـيد/مارغوركم 7.3 ± 61.8( ءدـبلا في كـنزلا نـم ةـضفخنم تايوتـسم دهاوـشلاو تلااـلحا لاـفطأ رـل يـيد/مارغ ليـيم 20.4 ± 129 تلااـلحا ةـعوممج في لـصلما في كـنزلا ىوتـسم يطـسو حـبصأ دـقف ةـلجاعلما دـعب اـمأ .)دهاوـشلل رـل يـيد ًاـيئاصحإ اـبه ُّدـتعُي تاـفلاتخا يأ كاـنه نـكي لمو .رـل يـيد/مارغ ليـيم 8.6 ± 63 وـهو دهاوـشلا ىدـل اـمم ًاـيئاصحإ هـب ُّدـتعُي رادـقمب ىـعأ وـهو قـيضو زـيزلأاو لاعـسلا لـثم ةـيريسرلا ضارـعلأا في ًانـستح تلااـلحا ةـعوممج ترـهظأو .ةـلجاعلما دـعبو لـبق E يـعانلما نـلوبولغلا ىوتـسم في يرـفزلا مـجلحا نـب ةبـسنلاو ،لىولأا ةـيناثلا في ّي ِْسرـَقلا يرـفزلا مـجلحاو ،ةـ َّيسرقلا ةـيويلحا ةعـسلا( ةيـسفنتلا تاـسايقلا جـئاتن عـيجمو سـفنلا .)ةـ َّيْسرَقلا ةـيويلحا ةعـسلا لىإ لىولأا ةـيناثلا في ّي ِْسرـَقلا Effet de la supplémentation en zinc chez des enfants asthmatiques : essai randomisé et contrôlé par placebo dans le nord de la République islamique d'Iran RÉSUMÉ Les informations concernant les bénéfices d'une supplémentation en zinc chez l'enfant asthmatique sont contradictoires. L'étude a examiné les effets d'une supplémentation en zinc chez des enfants asthmatiques suivis dans un service de consultations externes à Sari (République islamique d'Iran). Dans un essai clinique randomisé d'une durée de 8 semaines, en double aveugle et contrôlé contre placebo, 284 enfants sous corticostéroïdes inhalés ont été répartis entre un groupe recevant une supplémentation en zinc (50 mg/jour) (n = 144) et un groupe sous placebo (n = 140). Les cas comme les témoins présentaient des concentrations sériques en zinc initiales faibles [61,8 (E.T. 7,3) µg/dL et 60,9 (E.T. 4,3) µg/dL]. Après le traitement, la concentration sérique en zinc moyenne dans le groupe des cas était nettement supérieure [129 (E.T. 20,4) µg/dL] par rapport au groupe des témoins [63 (E.T. 8,6) µg/dL]. Aucune différence significative n'a été observée dans les taux d'IgE avant et après le traitement. Dans le groupe des cas, des améliorations significatives des symptômes cliniques ont été observées, notamment la toux, les sifflements respiratoires, la dyspnée et tous les paramètres spirométriques (CVF, VEMS et VEMS/CVF). Book 20-6.indb 391 6/17/2014 2:41:11 PM EMHJ • Vol. 20 No. 6 • 2014 Eastern Mediterranean Health Journal La Revue de Santé de la Méditerranée orientale 392 Introduction Allergic disorders such as asthma, aller- gic rhinitis, atopic dermatitis and even chronic urticaria have a high prevalence of morbidity, with significant effects on individuals’ quality of life and high economic costs for families and nations (1). Bronchial asthma is a chronic in- flammatory disease of the respiratory tract system and is more common in children than adults (1). The prevalence of asthma varies in different regions of the world. In the Islamic Republic of Iran a meta-analysis has estimated the prevalence of asthma in the country overall to be about 7.6% (2), while in the north of the country, where this study was carried out, a prevalence of 12% was reported (3). The reported prevalence of asthma has been increasing rapidly in recent decades. The etiology of asthma is not clear, and genetic and environmental factors have been implicated in its pathogenesis (4). One hypothesis for the increasing rates of asthma are low consumption of antioxidant foods or increased oxidative stress (1, 5, 6). Different studies suggested that trace elements might be involved in inflam- matory processes such as asthma (1, 4). Zinc levels have been shown to be low in the serum, hair and sputum of patients with asthma (1, 6–8). Trace elements such as selenium and zinc are essential components of antioxidant enzymes and are required to inhibit the production of the free radicals that are thought to aggravate asthma (8, 9). Zinc is involved in cell and tissue growth and plays an important role in DNA and protein synthesis. Furthermore zinc is an important element in oxidant/ antioxidant pathways and is believed to have a pivotal role in bronchial asthma pathways (1, 4, 10). Zinc is a modula- tor of the immune system, decreasing the inflammatory response. It has been suggested that zinc deficiency could de- crease the activity/levels of Th1 helper T-cells and increase the activity/levels of Th2 helper T-cells in asthma (11). Consumption of zinc-containing foods by mothers during pregnancy has been shown to be associated with a lower risk of wheeze and asthma in the child (12). There are conflicting reports about the effect of zinc supplements on childhood asthma. In a study in the Islamic Republic of Iran Pouramjad et al. showed that zinc supplements had no effect on the clinical manifestations of asthma and on pulmonary function tests (13). In contrast Biltagi et el., in Egypt, showed that consumption of omega-3 fatty acid, vitamin C and zinc, singly or in combination, led to significant improvements in pulmonary function tests and sputum inflamma- tory markers in placebo, self-controlled trials of children with asthma (14). The aim of the present study in Sari, Islamic Republic of Iran, was to examine the effect of zinc supplementation on clini- cal symptoms and pulmonary function tests in children with asthma. Methods This study was a double-blind, rand- omized, placebo-controlled clinical trial of the effect of zinc supplementation on clinical symptoms in zinc-deficient children with asthma in Sari, northern Islamic Republic of Iran. Sample The sample was selected from among patients attending the public outpatient allergy clinic at Mazandaran University of Medical Sciences between August 2010 and May 2011. The sample size was calculated based on the results of previous studies (prevalence of asthma 10%–12%). Patients were excluded if they had diabetes mellitus, liver or kid- ney disease, infections or thyroid dys- function, or clear clinical manifestations of zinc deficiency. We also excluded children who had been taking trace ele- ments or vitamin supplements. The diagnosis of moderate and/ or partly controlled bronchial asthma was made based on the child’s history, family history, physical examination and assessment by an allergist and clinical immunologist using Global Initiative for Asthma criteria (15). All patients in this study were using fixed inhaled ster- oids (moderate dose of fluticasone). Of the 345 patients assessed for eligibility, 30 did not meet the inclusion criteria, 10 declined to participate and 5 were excluded for other reasons. The remain- ing 300 patients were enrolled in the clinical trial and randomized to either the case (n = 155) or the control group (n = 145). The study was approved by the ethics committee of Mazandaran Uni- versity of Medical Sciences. Written informed consent was obtained from parents or guardians of the children. Because there were no clinical manifes- tations of zinc deficiency in our patients, there was no ethical requirement to use zinc supplements in the control group. Data collection The patients’ clinical data were assessed by a researcher and a paediatrician. Both were blind to the treatment groups. Clinical symptoms (cough, wheezing, dyspnoea), spirometry indices and se- rum zinc and IgE levels were recorded before and after the intervention. Pulmonary function testing was performed by an expert technician for forced vital capacity (FVC), forced ex- piratory volume in 1 second (FEV1) and FEV1/FVC. The measures were performed without the use of a bron- chodilator. A 5 mL sample of blood was taken from all patients. Blood was centrifuged at 2000 rpm for 15 minutes. Serum zinc level were measured by atomic absorp- tion spectroscopy (Zeeman, Varian) and expressed in μg/dL. Complete blood count, eosinophil count and se- rum total IgE level was performed for all patients. Book 20-6.indb 392 6/17/2014 2:41:11 PM طسوتلما قشرل ةيحصلا ةلجلمانوشرعلا دلجلما سداسلا ددعلا 393 with zinc supplements compared with the control group. Zinc supplementa- tion had a significant effective on all parameters of spirometry including FVC, FEV1 and FEV1/FVC ratio. For example FEV1/FVC ratio before and after the intervention was 73.1 (SD 3.6) and 83.4 (SD 4.7) respectively in the case group (P = 0.007) and 74.7 (SD 3.9) and 77.8 (SD 4.6) respectively in the control group. Discussion Zinc and copper are required for opti- mal activity of the immune system and it has been shown that low levels of these trace elements are important fac- tors in acute and chronic inflammatory states such as bronchial asthma (16). However, the evidence regarding the clinical efficacy of zinc supplementation on children with asthma is conflicting. This study was therefore designed to examine the effects of zinc on clinical symptoms and spirometry parameters in bronchial asthma. We demonstrated that zinc sup- plementation at 50 mg/day for 8 weeks had a significant beneficial effect on both clinical symptoms and lung func- tion in bronchial asthma patients. In other studies, the relationship between serum zinc and bronchial asthma re- mains doubtful (6, 16, 17) or contro- versial (18). In another study in the Intervention After recruitment patients judged to have moderate asthma with associated zinc deficiency (< 70 µg/dL) according to the allergist and clinical immunolo- gist were randomized by a paediatrician to the intervention (zinc supplements, 50 mg/day) or control (placebo) group. Patients received the drug or placebo from the pharmacy and were blind to the treatment. Treatments con- tinue for 8 weeks. Both groups received inhaled fluticasone spray. If a patient had unrelieved symptoms, they were advised to use a short-acting beta-2 agonist (salbutamol). Statistical analysis Analysis was done to determine the clinical improvement and spirometry alteration between the 2 groups. All re- sults are given as the mean and standard deviation (SD) value and data analysis was performed with SPSS software, version 17 statistical program. Data were analysed using paired t-test (for comparing before and after intervention data), independent-test (for compari- son between the 2 groups) and the chi- squared test (for qualitative variables). P-values < 0.05 were assumed to be statistically significant. Results Eligible patients Table 1 shows the age and sex distribu- tion of the 300 enrolled patients (155 cases and 145 controls). There were more boys than girls (59% versus 41%). The mean age was 7.6 (SD 1.6) years and 7.8 (SD 0.5) years respectively in the case and control groups. Of the 155 patients in the case group 11 did not receive the allocated intervention. After the intervention a further 4 children were lost to follow- up and 6 discontinued treatments (of drug and decreased inhaler steroids). Of the 145 in the control group 5 did not receive the allocated intervention. After the intervention 3 were lost to follow-up and 4 discontinued the intervention (of placebo and decreased inhaler steroid). The data were therefore analysed for the 144 cases and 140 controls who com- pleted the study and not for the whole group on the basis of intention to treat. Mean serum zinc levels The mean serum zinc level was 61.8 (SD 7.3) µg/dL and 60.9 (SD 4.3) µg/ dL in the case and control groups re- spectively before treatment (Table 2). After treatment the serum mean levels of zinc increased to 129 (SD 20.4) µg/ dL in the case group compared with 63 (SD 8.6) µg/dL in the controls, and this was a significant difference (P < 0.001). There was no significant difference in IgE levels in both groups before and after treatment (P < 0.05) (Table 2). Clinical symptoms Clinical symptoms evaluated in this study are shown in Table 3. There were significant improvements in the case group compared with the control group in all the clinical symptoms evaluated: cough [relative risk (RR) = 2.3; 95% CI: 1.3–4.1] (P = 0.003), wheezing (RR = 3.6; 95% CI: 1.8–7.4) (P < 0.001) and dyspnoea (RR = 4.2; 95% CI: 1.9–8.7) (P < 0.001). Spirometry parameters Table 4 shows the spirometry param- eters before and after 8 weeks treatment Table 1 Sex and age of the enrolled case and control group patients with asthma Variable Case group Control group Total No. % No. % No. % Total 144 100 140 100 284 100 Sex Female 74 51 67 49 141 41 Male 70 48 73 51 143 59 Age (years) 5–10 88 61 82 59 170 60 11–15 56 49 58 41 114 40 Mean (SD) 7.6 (1.6) 7.8 (0.5) 7.8 (1.2) SD = standard deviation. Book 20-6.indb 393 6/17/2014 2:41:12 PM EMHJ • Vol. 20 No. 6 • 2014 Eastern Mediterranean Health Journal La Revue de Santé de la Méditerranée orientale 394 Islamic Republic of Iran Pouramjad et al. showed that zinc supplementation (50 mg every other day) had no signifi- cant effect on respiratory factors such as spirometry (12). We used a higher dose of zinc sulphate (50 mg every day), however, which might explain the posi- tive effect of zinc supplementation in our study. Furthermore, we evaluated both lung function and clinical symp- toms. Biltagi et al. showed a positive effect not only of zinc supplementation but also supplementation with omega-3 fatty acids and vitamin C in children with moderate asthma (14), and com- bined therapy was more effective than single therapies. Before the intervention all the asth- matic children in our study had low of serum zinc concentrations (< 70 µg/ dL). Studies have suggested that aller- gic diseases such as asthma, leukaemia and coronary artery ectasia are associ- ated with deficiencies of trace elements such as selenium and zinc (14, 19, 20). Furthermore it has been shown that hair zinc levels are lower in children with recurrent wheeze compared with healthy controls and are negatively cor- related with wheezing episodes in the last 6 months (21). Although erythro- cyte zinc levels were not significantly decreased in asthma patients compared with a healthy group of children, there was a significant decrease in patients hospitalized with asthma attacks (22). It has been suggested that zinc deficiency can reduce antioxidant function and lead to exacerbation of bronchial asthma. Soutar et al. reported that limitation of zinc intake was as- sociated with a higher risk of asthma attacks (23). Therefore it seems that zinc could be an effective trace ele- ment in bronchial asthma patients. It has been reported that zinc decreases the incidence and prevalence of acute respiratory tract infection and the sever- ity of symptoms of the common cold (24, 25). El-Kholy et al. reported that adequate dietary intake of zinc and zinc supplementation might decrease the severity of asthmatic attacks (26). Ani- mal studies also demonstrated that zinc supplements reduced inflammatory and airway hyper-responsiveness (27). Prasad et al. showed that zinc decreased oxidative stress and nuclear transcrip- tion factor NF-κB activation in isolated mononuclear cells via induction of zinc finger protein A20 (28). In the present study we were unable to evaluate bronchial epithelial levels of superoxide dismutase, an enzyme which is deficient in asthma patients, but has been hypothesized that zinc might increase the activity of the en- zyme similar to inhaler steroids which have been used in these patients (29). Therefore, it is possible that zinc and inhaler steroids have synergistic effects. Although in this study serum total IgE levels decreased in the case group after zinc treatment, the difference was not significant. It seems that changes in serum total IgE levels may not be an important factor in bronchial asthma patients. Nevertheless Morgan et al. Table 2 Mean serum zinc values and IgE levels before and after treatment for the case and control groups Parameter Case group (n = 144) Control group (n = 140) Before After P-valuea Before After P-valuea Mean (SD) Mean (SD) Mean (SD) Mean (SD) Serum zinc (µg/dL) 61.8 (7.3) 129 (20.4) < 0.001 60.9 (4.3) 63 (8.6) 0.701 Serum IgE (IU/L) 119 (22) 106 (17) 0.423 109 (18) 103 (16) 0.648 aBefore versus after, paired t-test. SD = standard deviation. Table 3 Clinical presentation in the case and control groups before and after treatment Symptom Case group (n = 144) Control group (n = 140) Statistical analysis Before After P-valuea Before After P-valuea RR (95% CI) P-valueb No. % No. % No. % No. % Cough 127 88 101 70 0.004 112 80 103 74 0.427 2.3 (1.3–4.1) 0.003 Wheezing 94 65 68 47 0.002 88 63 81 58 0.638 3.6 (1.8–7.4) < 0.001 Dyspnoea 56 39 35 24 0.003 57 41 49 35 0.537 4.2 (1.9–8.7) < 0.001 Partially controlled asthmac 144 100 73 51 < 0.001 140 100 112 80 0.261 2.0 (1.4–2.8) < 0.001 aBefore versus after, χ2-test; bCase versus control group, independent t-test. cDaytime symptoms more than twice/week; limitations of activities and nocturnal symptoms/activities; need for relief/rescue therapy more than twice/week; lung function (PEF or FEV1) < 80% of predicted or personal best. RR = relative risk; 95% CI = confidence interval. Book 20-6.indb 394 6/17/2014 2:41:12 PM طسوتلما قشرل ةيحصلا ةلجلمانوشرعلا دلجلما سداسلا ددعلا 395 showed zinc supplementation de- creased airways hyper-responsiveness and serum IgE levels in a mouse model of allergic airway inflammation, and suggested that zinc supplements might have an anti-inflammatory effect via inhibition of the NF-κB pathway which leads to a decrease of serum IgE levels (27). Lang et al.’s study showed that zinc supplementation caused decreased eosinophil and lymphocyte levels in the bronchoalveolar lavage of mice (30). There were some limitations to our study. The sample size was small, there was no assessment of patients’ diet and no separate evaluation was made of cases of mild and severe asthma. We did not evaluate other antioxidants such as selenium and magnesium or inflammatory markers in respiratory tract secretions. Conclusions In this study we showed that zinc supplementation at 50 mg/day in children with moderate asthma with zinc deficiency significantly improved both clinical symptoms and lung func- tion. We recommend that any asthma patients with zinc deficiency use zinc supplements. Further studies with larger samples and cross-sectional methods are needed to further understanding the relationship between zinc supplement and bronchial asthma Acknowledgements Funding: this study was a resident thesis (Dr Effat Khorasani) supported by a financial grant from the Mazandaran University of Medical Sciences (thesis no. 1153). RCT registration number: IRCT 201105316660n1 Competing interests: None declared. Table 4 Spirometry parameters in the case and control groups before and after treatment Parameter Case group (n = 144) Control group (n = 140) Before After P-valuea Before After P-valuea Mean (SD) Mean (SD) Mean (SD) Mean (SD) FVC (L) 87.2 (3.4) 96.7 (5.6) < 0.001 86.1 (3.3) 93.4 (4.9) 0.649 FEV1 (L) 71.1 (3.8) 84.7 (3.9) 0.002 72.8 (4.2) 73.1 (4.2) 0.852 FEV1/FVC (FEV1%) 73.1 (3.6) 83.4 (4.7) 0.007 74.7 (3.9) 77.8 (4.6) 0.784 aBefore versus after, paired t-test. SD = standard deviation; FVC = forced vital capacity; FEV1 = forced expiratory volume in 1 second. References 1. Nurmatov U, Devereux G, Sheikh A. Nutrients and foods for the primary prevention of asthma and allergy: system- atic review and meta-analysis. J Allergy Clin Immunol. 2011 Mar;127(3):724–33, e1–30. PMID:21185068 2. Ghaffari J, Aarabi M. The prevalence of pediatric asthma in the Islamic Republic of Iran: a systematic review and meta- analysis. J Pediatr Rev. 2013;1:2–11. 3. Ghaffari J. Prevalence of asthma, allergic rhinitis and eczema in elementary schools in Sari (Iran). Caspian J Intern Med. 2012;3:372–6. 4. Fraenkel DJ, Holgate ST. Etiology of asthma: pathology and mediators. In: Biermann CW, et al., editors. Allergy, asthma and immunology from infancy to adulthood. 3rd ed. Philadel- phia: WB Saunders; 1996. pp. 443–72. 5. Chatzi L, Torrent M, Romieu I, Garcia-Esteban R, Ferrer C, Vioque J, et al. Diet, wheeze, and atopy in school chil- dren in Menorca, Spain. Pediatr Allergy Immunol. 2007 Sep;18(6):480–5. PMID:17680906 6. Kocyigit A, Armutcu F, Gurel A, Ermis B. Alterations in plasma essential trace elements selenium, manganese, zinc, copper, and iron concentrations and the possible role of these ele- ments on oxidative status in patients with childhood asthma. Biol Trace Elem Res. 2004 Jan;97(1):31–41. PMID:14742898 7. Laker M. On determining trace element levels in man: the uses of blood and hair. Lancet. 1982 Jul 31;2(8292):260–2. PMID:6124683 8. Vural H, Uzun K, Uz E, Koçyigit A, Cigli A, Akyol O. Concen- trations of copper, zinc and various elements in serum of patients with bronchial asthma. J Trace Elem Med Biol. 2000 Jun;14(2):88–91. PMID:10941719 9. De Raeve HR, Thunnissen FB, Kaneko FT, Guo FH, Lewis M, Kavuru MS, et al. Decreased Cu,Zn-SOD activity in asthmatic airway epithelium: correction by inhaled corticosteroid in vivo. Am J Physiol. 1997 Jan;272(1 Pt 1):L148–54. PMID:9038914 10. Heo Y, Parsons PJ, Lawrence DA. Lead differentially modifies cytokine production in vitro and in vivo. Toxicol Appl Pharma- col. 1996 May;138(1):149–57. PMID:8658504 11. Miller AL. The etiologies, pathophysiology, and alternative/ complementary treatment of asthma. Altern Med Rev. 2001 Feb;6(1):20–47. PMID:11207455 12. Devereux G, Turner SW, Craig LC, McNeill G, Martindale S, Harbour PJ, et al. Low maternal vitamin E intake during pregnancy is associated with asthma in 5-year-old children. Am J Respir Crit Care Med. 2006 Sep 1;174(5):499–507. PMID:16763215 13. Pouramjad SM, Egtesadi SH, Javad Mousavi SA, Nourmoham- madi I, Yazdani R. Study of zinc serum concentration and effect of zinc supplementation on lung function in asthmatic patients. J Iran Univ Med Sci. 2009;15(60-61):55–61. 14. Biltagi MA, Baset AA, Bassiouny M, Kasrawi MA, Attia M. Omega-3 fatty acids, vitamin C and Zn supplementation in asthmatic children: a randomized self-controlled study. Acta Book 20-6.indb 395 6/17/2014 2:41:12 PM EMHJ • Vol. 20 No. 6 • 2014 Eastern Mediterranean Health Journal La Revue de Santé de la Méditerranée orientale 396 Paediatr. 2009 Apr;98(4):737–42. PMID:19154523 [Article re- tracted in Acta Paediatr. 2012 Aug;101(8):891]. 15. Global strategy for asthma management and prevention. Up- date 2010. Global Initiative for Asthma, 2010 (http://www. ginasthma.org/local/uploads/files/GINA_Report_2010_1.pdf, accessed 22 February). 16. Schwartz J, Weiss ST. Dietary factors and their relation to res- piratory symptoms. The Second National Health and Nutrition Examination Survey. Am J Epidemiol. 1990 Jul;132(1):67–76. PMID:2356815 17. Picado C, Deulofeu R, Lleonart R, Agustí M, Mullol J, Quintó L, et al. Dietary micronutrients/antioxidants and their relation- ship with bronchial asthma severity. Allergy. 2001 Jan;56(1):43– 9. PMID:11167351 18. Anetor JI, Ajose OA, Ige O, Oyeleye AO, Ojo PO. Antioxidant status of adult Nigerian asthmatics: implications for prognosis. Nutr Health. 2003;17(3):221–9. PMID:14703155 19. Kosar F, Taskapan C, Kucukbay Z. Serum levels of selenium, zinc and copper in patients with coronary artery ectasia. Indian Heart J. 2007 Jan-Feb;59(1):38–41. PMID:19098333 20. Zuo XL, Chen JM, Zhou X, Li XZ, Mei GY. Levels of selenium, zinc, copper, and antioxidant enzyme activity in patients with leukemia. Biol Trace Elem Res. 2006 Winter;114(1-3):41–53. PMID:17205986 21. Razi CH, Akelma AZ, Akin O, Kocak M, Ozdemir O, Celik A, et al. Hair zinc and selenium levels in children with recur- rent wheezing. Pediatr Pulmonol. 2012 Dec;47(12):1185–91. PMID:22949381 22. Yilmaz EA, Ozmen S, Bostanci I, Misirlioglu ED, Ertan U. Eryth- rocyte zinc levels in children with bronchial asthma. Pediatr Pulmonol. 2011 Dec;46(12):1189–93. PMID:21815275 23. Soutar A, Seaton A, Brown K. Bronchial reactivity and dietary antioxidants. Thorax. 1997 Feb;52(2):166–70. PMID:9059479 24. Sazawal S, Black RE, Jalla S, Mazumdar S, Sinha A, Bhan MK. Zinc supplementation reduces the incidence of acute lower respiratory infections in infants and preschool children: a double-blind, controlled trial. Pediatrics. 1998 Jul;102(1 Pt 1):1–5. PMID:9651405 25. Prasad AS, Fitzgerald JT, Bao B, Beck FW, Chandrasekar PH. Du- ration of symptoms and plasma cytokine levels in patients with the common cold treated with zinc acetate. A randomized, double-blind, placebo-controlled trial. Ann Intern Med. 2000 Aug 15;133(4):245–52. PMID:10929163 26. el-Kholy MS, Gas Allah MA, el-Shimi S, el-Baz F, el-Tayeb H, Abdel-Hamid MS. Zinc and copper status in children with bronchial asthma and atopic dermatitis. J Egypt Public Health Assoc. 1990;65(5-6):657–68. PMID:2134100 27. Morgan CI, Ledford JR, Zhou P, Page K. Zinc supplementa- tion alters airway inflammation and airway hyperresponsive- ness to a common allergen. J Inflamm (Lond). 2011;8:36. PMID:22151973 28. Prasad AS, Bao B, Beck FW, Kucuk O, Sarkar FH. Antioxidant effect of zinc in humans. Free Radic Biol Med. 2004 Oct 15;37(8):1182–90. PMID:15451058 29. De Raeve HR, Thunnissen FB, Kaneko FT, Guo FH, Lewis M, Kavuru MS, et al. Decreased Cu,Zn-SOD activity in asthmatic airway epithelium: correction by inhaled corticosteroid in vivo. Am J Physiol. 1997 Jan;272(1 Pt 1):L148–54. PMID:9038914 30. Lang C et al. Anti-inflammatory effects of zinc and alterations in zinc transporter mRNA in mouse models of allergic inflam- mation. American Journal of Physiology, 2007, 292:L577– L584. Book 20-6.indb 396 6/17/2014 2:41:13 PM طسوتلما قشرل ةيحصلا ةلجلمانوشرعلا دلجلما سداسلا ددعلا 397 Knowledge and management of fever among Moroccan parents M. Rkain,1 I. Rkain,2 M. Safi,1 M. Kabiri,3 S. Ahid 2,4 and B.D.S. Benjelloun 1 ABSTRACT Parents often have misperceptions about childhood fever, and little information is available about the home management of feverish children in Morocco. In this study of the perceptions, knowledge and practices of families regarding children’s fever, the parents of 264 febrile children aged 0–16 years were interviewed in a paediatric emergency department in Rabat in 2011. Only 3.5% of parents knew the correct temperature definition for fever, 54.4% determined their children’s fever using a thermometer, and the preferred site was rectal. Most of them (96.8%) considered that fever was a very serious condition, which could lead to side-effects such as brain damage (28.9%), seizures (18.8%) paralysis (19.5%), dyspnoea (14.8%) and coma (14.8%). Paracetamol was used by 85.9% and traditional treatments by 45.1%. Knowledge about the correct definition of fever was significantly associated with parents’ profession, educational level and receipt of previous information and advice from health professionals. 1Emergency Medical Department; 3Neonatal Intensive Care Unit, Children’s Hospital, University Hospital Rabat-Salé, Rabat, Morocco. 2Laboratory of Biostatistics, Clinical Research and Epidemiology; 4Pharmacoepidemiology and Pharmacoeconomics Research Team, Faculty of Medicine and Pharmacy, University Mohammed Vth Souissi, Rabat, Morocco (Correspondence to S. Ahid: s.ahid@um5s.net.ma). Received: 03/11/12; accepted: 19/06/13 برغلما في ءابلآا ىدل ىملحا يربدتو فراعلما نوُّلجنب دوعسلا ردب ،ديحأ رمس ،يربك ميرم ،فياصلا ىنم ،نياكر مالهإ ،نياكر ىنم يـجلاعلا رـبدتلا نـع ةـحاتلما تاـمولعلما نأ ماـك ،لاـفطلأا بـيصت يـتلا ىـملحا نـع ةـئطاخ راكـفأ ءاـبلآا ىدـل نوـكي اـم ًارـثك :ةـصلالخا ىـملحا لوـح سرلأا تاـسراممو فراـعمو تاكَردـُم ةـفرعم ةـساردلا هذـه تفدهتـسا دـقو .برـغلما في ةـليلق ىـملحاب باـصلما لـفطلل ليزــنلما ،ًاـماع 16-0 نـب تـحوارت رماـعأ في ىـملحاب اوـبيصأ نـيذلا لاـفطلأا ءاـبآ نـم 264 عـم تلاـباقم نوـثحابلا ىرـجأف ،لاـفطلأا بـيصت يـتلا ةـجردل حـيحصلا فـيرعتلا نوـفرعي ءاـبلآا نـم طـقف % 3.5 نأ نـثحابلل حـضتاو .2011 ماـع في طاـبرلا في لاـفطلأا ئراوـط مـسق في كـلذو ةجرد ساـيقل لـ َّضفلما عـضولما نأو ،ةرارـلحا ساـيقم مادختـساب ىـملحاب لـفطلا ةـباصإ ىـع نوـف َّرعتي مـهنم % 54.4 نأو ،ىـملحا ءاـنثأ ةرارـلحا غاـمدلا ب ُّرـتخ لـثم ةـيبناج تارـثأت لىإ دوـقت دـق ةرـطخ ةـلاح ىـملحا نورـتعي )مـهنم % 96.8( ءاـبلآا مـظعم نأو ،جشرـلا وـه ةرارـلحا في لوماتيـسارابلا مادختـسا مـت دـقو .)% 14.8( تابـسلاو ،)% 14.8( سـفنلا قـيضو ،)% 19.5( للـشلاو ،)% 18.8( تاـجلاتخلااو ،)% 28.9( ُّدـتعُي رادـقمب ىـمحلل حـيحصلا فـيرعتلاب ةـقّلعتلما ةـفرعلما تـَطَباَرَت دـقو .تلااـلحا نـم 45.1% في ةـيديلقتلا تاـلجاعلماو ،تلااـلحا نـم % 85.9 .نـيحصلا نـينهلما نـم تاروـشمو ةقبـسم تاـمولعم مـهيّقَلَت عـمو يـميلعتلا مهاوتـسمو ،ءاـبلآا ةـنهم عـم ًاـيئاصحإ هـب Épisode fébrile chez l’enfant : connaissances des parents marocains et prise en charge par ces derniers RÉSUMÉ Les parents ont souvent des perceptions erronées concernant la fièvre chez l'enfant, et les informations sur la prise en charge des enfants fébriles à domicile sont rares au Maroc. Dans la présente étude sur les perceptions, les connaissances et les pratiques des familles au sujet de la fièvre chez l'enfant, les parents de 264 enfants fébriles âgés de 0 à 16 ans ont été interrogés au sein d'un service d'urgence pédiatrique de la ville de Rabat en 2011. Seuls 3,5 % des parents connaissaient la température exacte définissant un état fébrile et 54,4 % déterminaient la fièvre de leur enfant à l'aide d'un thermomètre, de préférence par voie rectale. La plupart d'entre eux (96,8 %) considéraient que la fièvre était une affection très grave qui pouvait conduire à des effets secondaires tels que des lésions cérébrales (28,9 %), des convulsions (18,8 %), une paralysie (19,5 %), une dyspnée (14,8 %) et un coma (14,8 %). Le paracétamol a été utilisé par 85,9 % des parents et les traitements traditionnels par 45,1 %. La connaissance de la définition exacte de la fièvre était significativement associée à la profession des parents, à leur niveau d'études et à la prise de conseils et d'informations préalable auprès des professionnels de santé. Book 20-6.indb 397 6/17/2014 2:41:13 PM EMHJ • Vol. 20 No. 6 • 2014 Eastern Mediterranean Health Journal La Revue de Santé de la Méditerranée orientale 398 Introduction Fever is one of the most common presenting complaints in paediatrics and general practice and is the cause of nearly 70% of all paediatric visits (1). A number of studies have investigated parents’ knowledge, perceptions, theo- ries and practices of childhood fever (2–5). Parents frequently perceive fever as a disease rather than as a symp- tom or sign of illness (2), as defined by Schmitt in 1980 who introduced the term “fever phobia” to describe parents’ fearful view of fever (3). Insufficient knowledge of parents concerning the cause of fever, and misconceptions about its effects on their children’s health frequently lead to excessive fear and anxiety (4). A frequent finding is that parents are not correctly informed about temperature, defining fever as a medical term (6–8). Impicciatore et al. studying moth- ers’ perceptions and attitudes towards fever and its treatment found that most mothers did not know how to man- age fever (9). Two studies in Greece reached to the same conclusions; Anagnostakis et al. found that parents had incorrect perceptions about fever and worried about temperatures that were considered normal (10), while Mathioudakis et al. found that only 1.4% of parents correctly evaluated and treated fever and 64.6% used the wrong dosages of antipyretics (11). Studies further show that educational level, socioeconomic status and cul- tural background are the main deter- minants of knowledge and judgement of childhood fever (12,13). A higher socioeconomic status and educational level contributed to a more scientifi- cally oriented knowledge of fever. Little information is available about the home management of feverish children in Morocco; in particular, no studies have been published on par- ents’ knowledge, perceptions and atti- tudes regarding fever in their children. The aim of our study was to reveal the perceptions, knowledge and practices of families regarding childhood fever and to discuss the differences between our population and other populations. Methods Study sample This study participants were a con- venience sample of parents of children aged 0–16 years attending the paedi- atric emergency department during 4 months from July to October 2011. Only the parents were asked to par- ticipate in the study. All of the parents who we approached agreed to be in the study and none were excluded. Data collection On arrival at the department, the par- ents of the child were asked to answer to a questionnaire while waiting for examination of the child. The parents were informed about the study, reas- sured about the confidentiality of data and their right to refuse participation without any consequences for the treat- ment of their children and requested to verbally consent to participation. The parents were interviewed using face-to-face interviews in Moroccan Arabic. The interviews, which were conducted in a separate room of the department in order to guarantee pri- vacy, lasted approximately 20 minutes. One physician read questions to the participant from a questionnaire and the parents were given no assistance with answering the questions. The participants were asked open-ended yes/no and multiple-choice questions about sociodemographic data and their knowledge (4 items), beliefs (4 items) and practices (11 items) con- cerning fever. Parents were also asked if they had received advice or general information in the past from physi- cians, pharmacists, nurses or parents regarding the management of fever. A temperature of 38 °C or above was considered to indicate fever (14). Data analysis Statistical analyses were performed using SPSS, version 13.0. Data were presented as percentages and means and standard deviation (SD). Statis- tical significance was determined by the Pearson test. A value of P < 0.05 was considered significant. All variables were coded as dummy variables. In univariate analysis, a logistic regression model was used to search for factors that influenced parents’ knowledge about the definition of fever temperature and their practices regarding fever and to calculate their odds ratios (OR) and 95% confidence intervals (CI). Vari- ables with P < 0.25 were entered into the multivariate analysis Results The total number of parents inter- viewed was 264. The mean age of parents was 31.6 (SD 8.5) years and most of them (81.0%) were mothers. The sociodemographic characteristics of parents are summarized in Table 1. A majority resided in urban areas (88.6%). Only 3.5% of the parents knew the correct temperature definition for fe- ver. Just over half the parents (54.4%) determined their children’s fever using a thermometer, while 44.4% stated that fever could be determined by touching the child’s forehead. Of the parents, 58.9% could read the thermometer. The preferred route of measuring temperature was rectal (Table 2). In our study, 96.8% of parents considered that fever was a very serious condi- tion, which could lead to side-effects such as brain damage (28.9%), seizures (18.8%), paralysis (19.5%), breathing difficulty (14.8%) and coma (14.8%) (Table 2). When fever persisted, 85.7% of par- ents consulted the general practitioner (39.9%) or paediatrician (45.8%). Of all the parents, 72.0% had received no information about fever and 47.3% Book 20-6.indb 398 6/17/2014 2:41:14 PM طسوتلما قشرل ةيحصلا ةلجلمانوشرعلا دلجلما سداسلا ددعلا 399 Discussion The present study is an analysis of Mo- roccan parents’ perceptions, knowledge and interventions about fever. Half of the parents in this study were high- school graduates. Most of the parents were assumed to be socioeconomi- cally relatively advantaged because our emergency department is located in the capital city. The use of a thermometer is the only way to determine whether a child is febrile. All other methods including tac- tile and visual assessment are inaccurate (6); for example, it has been shown that touching the forehead detected only had received advice on management of a feverish state for their children. The source of information was mainly from a paediatrician (23.5%), general practitioner (21.2%) or the experience of grandparents (16.3%). The parents’ interventions for their children’s fever are shown in Table 3. Paracetamol was used by 85.9% of parents and tradi- tional treatments by 45.1%. Table 1 shows the demographic characteristics and receipt of infor- mation/advice of those with correct knowledge about the definition of fever temperature, while Table 4 shows the univariate and multivariate analysis of the data. Variables which did not significantly affect knowledge about their definition of fever were age (P = 0.059), sex (P = 0.241), place of resi- dence (urban/rural) (P = 0.998) and number of children in the family (P = 0.415). Higher socioeconomic level was associated with better knowledge about the definition of fever in the univariate (P < 0.001) but not in the multivariate analysis (P = 0.453). In multivariate analysis, parents’ educa- tional level (P < 0.001), profession (P = 0.016), and previous information (P = 0.007) and advice about fever (P = 0.007) were significantly associated with knowledge regarding the defini- tion of fever (Table 4). Table 1 Parents’ demographic characteristics and receipt of information/advice about childhood fever in relation to correct knowledge about the definition of fever temperature Variable Total (n = 264) Correct knowledge about definition of fever (n = 58) Mean (SD) age (years) 31.6 (8.5) 33.6 (8.1) Mean (SD) no. of children 2.1 (1.4) 1.9 (1.2) No. % No. % Socioeconomic level Low 57 21.6 4 6.9 Middle 192 72.7 44 75.9 High 15 5.7 10 17.2 Educational level Illiterate 92 35.0 9 15.8 Primary school 58 22.1 6 10.5 High school 59 22.4 17 29.8 University 54 20.5 25 43.9 Profession Not working 159 60.9 28 50.0 Retired 1 0.4 0 0.0 Private sector 54 20.7 8 14.3 Public sector 47 18.0 20 35.7 Place of residence Urban 234 88.6 58 100.0 Rural 30 11.4 0 0.0 Received general information about fever Yes 191 72.6 22 38.6 No 72 27.4 35 61.4 Received advice about fever Yes 139 52.7 13 22.4 No 125 47.3 45 77.6 SD = standard deviation. Book 20-6.indb 399 6/17/2014 2:41:14 PM EMHJ • Vol. 20 No. 6 • 2014 Eastern Mediterranean Health Journal La Revue de Santé de la Méditerranée orientale 400 74% of all febrile children (15). In the our study, 54.4% of the parents used a thermometer to detect their children’s fever, but nearly half of them detected fever by touching the children’s fore- head, which is frequently considered by parents to be the most accurate way of measuring an infant’s temperature (16). Axillary temperatures are adequate for clinical screening or fever (15). In our study, three-quarters of parents pre- ferred the rectal route for temperature measurement and only one-quarter preferred to take axillary temperature. This was different to the findings of other studies in the literature (17, 18). In our hospital, we do not have strict policies on paediatric tempera- ture measurement. Rectal or axillary measurement is the preferred route in children younger than 5 years old. Parents may think that measuring the rectal temperature is more appropriate for fever. In the present study, 96.5% of the parents stated the incorrect temperature or did not know the correct temperature for fever. This was similar to the findings of previous reports in culturally diverse populations in different countries (6, 7, 17, 19). The parents with higher educa- tional level demonstrated a significantly higher rate of accuracy in their knowl- edge of fever definition in our study (P = 0.001), in accordance with the findings of previous studies conducted among different populations (20–22). In our study the majority of parents used paracetamol as an antipyretic in treating children’s fever, corroborating the findings of studies in other popula- tions (6, 7, 17). Aspirin and ibuprofen were not preferred in our study sample. In our hospital, we frequently inform parents about the risks of aspirin use in febrile children (such as Reye syndrome and gastrointestinal bleeding). These could be the reasons why the parents in the present study preferred paraceta- mol as an antipyretic. In our Moroccan traditions, iced water is not used as a method of lowering the temperature, while other methods are practised such as towels soaked in cold water. Nearly 60% of the parents in our study believed that fever could have dangerous effects on children, even death. Of all the parents, 28.9% listed brain damage as the most common harmful effect of fever, 18.8% seizures, 19.5% paralysis and 14.8% coma. In the literature, it was reported that many of these beliefs are also shared by pae- diatric health care providers (12) and fever phobia was the message that they conveyed to parents (21). Parental educational status showed a significant effect on the parents’ inter- ventions and knowledge about fever, a finding which corroborates other stud- ies from different populations (6, 17, 20, 23, 24). In our study, parents who had received advice from their doctor and therefore had more information than those who had not had advice, knew bet- ter how to manage their febrile children. Table 2 Parents’ knowledge, practices and beliefs about childhood fever Characteristic No. % (n = 264) Definition of fever temperature (°C) 38–< 38.5 9 3.5 < 38 or > 38.5 162 61.3 Don’t know 93 35.2 Way of measuring child’s temperature Thermometer 143 54.4 Touching the forehead 117 44.5 Chills 5 0.8 If the child drinks a lot 1 0.4 Has a thermometer at home Yes 161 61.2 No 102 38.8 Type of thermometer used to take child’s temperature Rectum 126 73.7 Armpit 43 25.1 Ear 2 1.2 Can read the thermometer Yes 142 58.9 No 99 41.1 Believe fever has a purpose Yes 8 3.1 No 246 96.9 Believe fever is dangerous Yes 153 59.5 No 104 40.5 Dangers specified Neurological 43 28.9 Seizures 28 18.8 Paralysis 29 19.5 Breathing difficulty 22 14.8 Coma 22 14.8 Death 4 2.7 Blindness 1 0.7 Book 20-6.indb 400 6/17/2014 2:41:14 PM طسوتلما قشرل ةيحصلا ةلجلمانوشرعلا دلجلما سداسلا ددعلا 401 Table 3 Therapeutic interventions used by parents to treat childhood fever Variable No. % (n = 264) Physical measures Bath 80 46.8 Undressing 36 21.1 Drinks 27 15.8 Wet towels 28 16.4 Antipyretics Paracetamol 213 85.9 Aspirin 22 8.9 Ibuprofen 13 5.2 Route of administration of antipyretics Rectal 154 64.2 Oral 86 35.8 Traditional treatments Rosewater 65 45.1 Vinegar 23 16.0 Lemon 11 7.6 Anserinea 45 31.3 aChenopodium ambrosioides (wormseed). Table 4 Regression analysis of factors influencing parents’ correct knowledge about the definition of fever temperature Variable Univariate analysis Multivariate analysis OR (95% CI) P-value OR (95% CI) P-value Age 1.03 (1.00–1.07) 0.059 n/a – – Sex 0.67 (0.34–1.32) 0.241 1.01 (0.96–1.06) 0.803 Educational level 2.19 (1.60–2.84) < 0.001 2.36 (1.45–3.85) 0.001 Profession 1.37 (1.09–1.73) 0.007 0.60 (0.40–0.91) 0.016 Socioeconomic level 5.16 (2.41–11.1) < 0.001 1.47 (0.54–4.02) 0.453 Place of residence 0.00 0.00 0.998 n/a – – Received general information 7.27 (3.83–13.8) < 0.001 3.23 (1.39–7.55) 0.007 Received advice 5.45 (2.77–10.7) < 0.001 3.24 (1.38–7.60) 0.007 No. of children 0.90 (0.73–1.14) 0.415 n/a – – OR = odds ratio; CI = confidence interval; n/a = not applicable. One limitation of the present study was that data were collected from parents presenting to one emergency department. Therefore the findings might not be generalizable to the Mo- roccan population. Multi-centre trials may show differences in trends and provide useful comparisons. Competing interests: None declared. References 1. Figueras Nadal C, García de Miguel MJ, Gómez Campderá A, Pou Fernández J, Alvarez Calatayud G, Sánchez Bayle M; Paedi- atric Fever Co-operative Group from the Spanish Paediatric As- sociation. Effectiveness and tolerability of ibuprofen-arginine versus paracetamol in children with fever of likely infectious origin. Acta Paediatr. 2002;91(4):383–90. PMID:12061352 2. What worries parents when their preschool children are acute- ly ill, and why: a qualitative study. BMJ. 1996;313(7063):983–6. PMID:8892420 3. Crocetti M, Moghbeli N, Serwint J. Fever phobia revisited: have parental misconceptions about fever changed in 20 years? Pediatrics. 2001 Jun;107(6):1241–6. PMID:11389237 Book 20-6.indb 401 6/17/2014 2:41:14 PM EMHJ • Vol. 20 No. 6 • 2014 Eastern Mediterranean Health Journal La Revue de Santé de la Méditerranée orientale 402 4. Rideout ME, First LR. Fever: measuring and managing a sizzling symptom. Contemp Pediatr. 2001;18:42–50. 5. Purssell E. Parental fever phobia and its evolutionary correlates. J Clin Nurs. 2009 Jan;18(2):210–8. PMID:18298501 6. Taveras EM, Durousseau S, Flores G. Parents’ beliefs and prac- tices regarding childhood fever: a study of a multiethnic and socioeconomically diverse sample of parents. Pediatr Emerg Care. 2004 Sep;20(9):579–87. PMID:15599258 7. Blumenthal I. What parents think of fever. Fam Pract. 1998 Dec;15(6):513–8. PMID:10078789 8. Krantz C. Childhood fevers: developing an evidence-based anticipatory guidance tool for parents. Pediatr Nurs. 2001 Nov- Dec;27(6):567–71. PMID:12024528 9. Impicciatore P, Violante A, Bonati M. Helping parents to cope when their preschool children are acutely ill. Educational interventions may defuse parents’ concern. BMJ. 1997 Feb 1;314(7077):373. PMID:9040350 10. Kallestrup P, Bro F. Parents’ beliefs and expectations when pre- senting with a febrile child at an out-of-hours general practice clinic. Br J Gen Pract. 2003 Jan;53(486):43–4. PMID:12564276 11. Walsh A, Edwards H, Fraser J. Parents’ childhood fever manage- ment: community survey and instrument development. J Adv Nurs. 2008 Aug;63(4):376–88. PMID:18727765 12. Crocetti M, Moghbeli N, Serwint J. Fever phobia revisited: have parental misconceptions about fever changed in 20 years? Pediatrics. 2001 Jun;107(6):1241–6. PMID:11389237 13. Walsh A, Edwards H. Management of childhood fever by parents: literature review. J Adv Nurs. 2006 Apr;54(2):217–27. PMID:16553708 14. Gal P, Reed MD. Medications. In: Behrman RE, Kleigman RM, Jenson HB, editors. Nelson textbook of pediatrics. 17th ed. Phil- adelphia (Pennsylvania): WB Saunders; 2004. pp. 2432–501. 15. Schmitt BD. Fever in childhood. Pediatrics. 1984 Nov;74(5 Pt 2):929–36. PMID:6333668 16. Fallis WM, Brunsdon-Clark B, Andries A, Gilbert E. A parent’s response prompts a search for current trends in taking the temperature of pediatric ED patients. J Emerg Nurs. 2005 Oct;31(5):462–4. PMID:16198730 17. al-Eissa YA, al-Zamil FA, al-Sanie AM, al-Salloum AA, al-Tuwaijri HM, al-Abdali NM, et al. Home management of fever in chil- dren: rational or ritual? Int J Clin Pract. 2000 Apr;54(3):138–42. PMID:10829354. 18. Kai J. Parents’ perceptions of taking babies’ rectal temperature. BMJ. 1993 Sep 11;307(6905):660–2. PMID:8401054 19. Parmar RC, Sahu DR, Bavdekar SB. Knowledge, attitude and practices of parents of children with febrile convulsion. J Post- grad Med. 2001 Jan-Mar;47(1):19–23. PMID:11590285 20. Al-Nouri L, Basheer K. Mothers’ perceptions of fever in chil- dren. J Trop Pediatr. 2006 Apr;52(2):113–6, discussion 117. PMID:16115838 21. Crocetti M, Sabath B, Cranmer L, Gubser S, Dooley D. Knowl- edge and management of fever among Latino parents. Clin Pediatr (Phila). 2009 Mar;48(2):183–9. PMID:18840888 22. Walsh A, Edwards H, Fraser J. Parents’ childhood fever manage- ment: community survey and instrument development. J Adv Nurs. 2008 Aug;63(4):376–88. PMID:18727765 23. Mackowiak PA. Concepts of fever. Arch Intern Med. 1998 Sep 28;158(17):1870–81. PMID:9759682 24. Poirier MP, Collins EP, McGuire E. Fever phobia: a survey of car- egivers of children seen in a pediatric emergency department. Clin Pediatr (Phila). 2010 Jun;49(6):530–4. PMID:20488812 Book 20-6.indb 402 6/17/2014 2:41:15 PM طسوتلما قشرل ةيحصلا ةلجلمانوشرعلا دلجلما سداسلا ددعلا 403 Rapport de cas Mosaïcisme 47,XXY/46,XX et anomalie de la différenciation sexuelle : à propos d’un cas O. Lyhyaoui 1 et A. Gaouzi 2 Introduction Le mosaïcisme 47,XXY/46,XX est extrêmement rare ; quelques cas ont été rapportés dans la littérature. Il peut être associé à un large spectre de phénotypes tels que le syndrome de Klinefelter, l’ovotestis ou le phénotype féminin. Nous rapportons le cas d’un patient qui présente une anomalie de la différenciation sexuelle à caryotype 47,XXY/46,XX. Observation Nous rapportons l ’observation d’un enfant élevé comme étant un garçon, âgé actuellement de huit ans, présentant une anomalie de la différenciation sexuelle qui a été constatée par les parents dès la naissance. L’enfant a été opéré à l’âge de sept mois pour torsion d’une gonade ectopique droite ; l’aspect macroscopique de cette gonade était en faveur d’un ovaire. L’examen clinique trouve un bourgeon génital médian de 3,7 cm de longueur et de 1,8 cm de largeur, un hypospadias postérieur très sévère, un orifice unique périnéal et des bourrelets génitaux d’aspect vulvaire, sans gonade palpable (Figures 1 et 2). Le caryotype est en faveur d’un mosaïcisme 47,XXY/46,XX avec la présence de 20 % de cellules XXY et 80 % de cellules XX. Le bilan hormonal objective un taux de testostérone totale de base inférieur à 0,05 ng/mL et de 17 OH-progestérone à 0,45 ng/mL. Le cortisol de 8 h et l’ionogramme sanguin sont normaux. Après test à l’hCG - gonadotrophine chorionique humaine - (dose de six injections d’hCG 1500 UI/j, un jour sur deux), la testostérone reste basse, inférieure à 0,01 ng/mL, témoignant de l’absence de tissu testiculaire, avec des taux bas de FSH (hormone folliculostimulante) et de LH (hormone lutéinisante). La génitographie objective la présence d’une cavité vaginale et utérine normale et les deux trompes (Figure 3). La cœlioscopie exploratrice réalisée à l’âge de quatre ans a montré la présence d’un utérus de taille normale avec une trompe droite ligaturée et une trompe gauche de taille normale ; la gonade g a u c h e e s t d ’ a s p e c t o v a r i e n confirmé par la biopsie et l’examen anatomopathologique. Le cas de cet enfant a posé le problème d’orientation du sexe d’élevage sachant que les dérivés mullériens sont de bonne qualité pour une féminisation. L’enfant et sa famille ont bénéficié d’une consultation de psychologie , qui n’était pas concluante pour le choix du sexe. Vu la réticence de la famille pour la féminisation, la décision d’abstention thérapeutique jusqu’à l ’âge de la puberté a été prise par un personnel multidisciplinaire en présence des parents. Discussion Le mosaïcisme 47,XXY/46,XX est extrêmement rare ; il a été rapporté dans cinq cas de personnes ayant des signes évocateurs d’un syndrome de Klinefelter , dans quatre cas d’ovotestis (1), dans un cas de cancer des ovaires hypoplasiques (2) et dans un cas chez un patient avec un phénotype féminin (3). La présence d’une l ignée cel lulaire 46,XX a également été signalée dans cinq autres cas de patients avec un syndrome de Klinefelter qui avaient une mosaïque de plus de deux lignées de cellules différentes. La variabilité phénotypique chez les personnes ayant le même caryotype est souvent rencontrée en mosaïque i m p l i q u a n t l e s c h r o m o s o m e s sexuels (4). Par exemple, le mosaïcisme 45,X/46,XY est associé à un syndrome de Turner, à un phénotype masculin avec dysgénésie gonadique mixte, à une anomalie de la différenciation sexuelle 46,XY et à des hommes apparemment normaux (1) . Le mosaïcisme des chromosomes sexuels pourrait être le résultat du chimérisme, de la perte du chromosome Y à partir de quelques cellules, d’une double fécondation et de la fusion de l’embryon fécondé avec un corps polaire. Le phénotype résultant sera influencé par la distribution d e s d e u x d i ff é r e n t e s l i g n é e s cellulaires dans les différents tissus de l’embryon en développement. 1Service d’Endocrinologie, Diabétologie et Nutrition ; 2Service d’Endocrinologie pédiatrique, Hôpital d’Enfants, CHU Ibn Sina, Rabat (Maroc) (Correspondance à adresser à O. Lyhyaoui : o.lyhyaoui@gmail.com). Reçu : 16/09/12 ; accepté : 08/11/12 Book 20-6.indb 403 6/17/2014 2:41:15 PM EMHJ • Vol. 20 No. 6 • 2014 Eastern Mediterranean Health Journal La Revue de Santé de la Méditerranée orientale 404 Références 1. Velissariou V, Christopoulou S, Karadimas C, Pihos I, Kanaka- Gantenbein C, Kapranos N, et al. Rare XXY/XX mosaicism in a phenotypic male with Klinefelter syndrome: case report. Eur J Med Genet. 2006 Jul-Aug;49(4):331–7. PMID:16829354 2. Gagnon J, Leboeuf G, Ducharme JR. Primary ovarian hypoplasia with XX/XXY blood chromosomes. Union Med Can. 1965 Aug;94:974–84. PMID:14347204 3. Hamlett JD, Timson J, Harris R. XX-XXY mosaicism in a phenotypically normal female. Hum Hered. 1970;20(3):260–4. PMID:5489884 4. Gardner RJM, Sutherland GR. Chromosome abnormalities and genetic counseling. 3rd ed. New York: Oxford University Press; 2004. La présence d’un chromosome Y est un inducteur dominant du phénotype masculin. Il semble qu’une majorité considérable des cas de vrai mosaïcisme du chromosome du sexe sont associés à un phénotype concordant (Y → masculin, pas de Y → féminin). Il est également bien connu que les deux sexes phénotypique et gonadique sont fortement influencés par le pourcentage et la distribution de cellules porteuses de Y dans les gonades, mais pas nécessairement dans le sang. Si la cellule prédominante d a n s l e s g o n a d e s p o r t e u n chromosome Y, le phénotype est celui d’un mâle. Cet effet est basé sur l’expression du gène SRY au-dessus d’un seuil critique dans le développement de la crête urogénitale (1). Cela peut expliquer le cas de notre patient chez lequel le phénotype est principalement celui d’une femme, puisque seulement 20 % des cellules sont porteuses de 47,XXY bien que le mosaïcisme 46,XX eût été observé dans 80 % des cellules. Il est surprenant que si peu de cas de mosaïcisme 47,XXY/46,XX aient été rapportés dans la littérature comparativement à ceux qui ont un mosaïcisme 47,XXY/46,XY (4). Une explication possible pourrait être la sous-estimation de ces patients, en raison de l’échec de détection de la lignée cellulaire 46,XX dans le sang, qui est habituellement utilisée pour l’enquête cytogénétique de routine. Dans le diagnostic prénatal, la détection des cellules 46,XX et des cellules 47,XXY dans la même culture doit être considérée comme contamination maternelle. Conclusion Le mosaïcisme 47,XXY/46,XX est rare et peut être associé à différents phénotypes. Des études cytogénétiques détaillées ainsi que des analyses moléculaires et histologiques devraient être menées, en cas de découverte de mosaïcisme, pour identifier la complexité de la différenciation sexuelle de l’homme et mettre davantage en valeur la diversité de la corrélation phénotype-génotype. Figures 1 & 2 Aspect de l’appareil génital externe du cas en question Figure 3 Image génitographique visualisant la présence d’une cavité vaginale et utérine normale et des deux trompes Book 20-6.indb 404 6/17/2014 2:41:15 PM Subscriptions and Distribution Enquiries regarding subscriptions and distribution of the print edition of EMHJ should be addressed to: Printing and Marketing of Publications at: email: pam@emro.who.int; tel: (+202) 2276 5000; fax: (+202) 2670 2492 or 2670 2494 Permissions Requests for permission to reproduce or translate articles, whether for sale or non-commercial distribution should be addressed to EMHJ at: emhj@emro.who.int Correspondence Editor-in-chief EMHJ WHO Regional Office for the Eastern Mediterranean P.O. 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Yémen Contents V o lu m e 2 0 N u m b er 6 Ju n e 2 0 1 4 From the Editor-in-Chief Filling the gap: restoring EMHJ’s public health identity ...........................................................................................359 Editorial Cancer control: a reminder of the need for a balanced approach between prevention and treatment .............360 Research articles Pesticide exposure as a risk factor for lymphoproliferative disorders in adults .....................................................363 Detection and genotyping of human papillomavirus in breast cancer tissues from Iraqi patients ......................372 Use of human surplus biospecimens in research: a survey from a cancer centre .................................................378 Smokeless tobacco consumption in a multi-ethnic community in Pakistan: a cross-sectional study ..................385 Effect of zinc supplementation in children with asthma: a randomized, placebo-controlled trial in northern Islamic Republic of Iran .............................................................................................................................391 Knowledge and management of fever among Moroccan parents .........................................................................397 Case report Mosaïcisme 47,XXY/46,XX et anomalie de la différenciation sexuelle : à propos d’un cas ..................................403
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Eastern Mediterranean Health Journal [2014; Vol.20, Issue 6]
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