INFECTIOUS DISEASES I DJ JOURNAL

of Pakistan Published by the Infectious Diseases Society of Pakistan

ISSN 1027-0299
Recognised and registered with the Pakistan Medical & Dental Council
NO.PF.11-F-96 (Infectious Diseases) 2560

College of Physicians & Surgeons, Pakistan Higher Education Commission, Pakistan Indexed - WHO EMRO
October - December 2010 Volume 19 Issue 04
CONTENTS
GUEST EDITORIAL

Infectious Diseases Journal of Pakistan

Official Organ of the Infectious Diseases Society of Pakistan President Altaf Ahmed Consultant Microbiology, The Indus Hospital Karachi, Pakistan

PAGE #
224

Gen. Secretary Ejaz A. Khan Department of Pediatrics, Shifa International Hospital, Islamabd, Pakistan Treasurer M. Asim Beg Pathology& Microbiology, Aga Khan University, Karachi, Pakistan Editorial Office Aamer Ikram Editorial Board Naseem Salahuddin: Karachi Naila B Ansari: Karachi Shehla Baqi: Karachi Nurul Iman: Peshawar Ejaz Khan: Islamabad Ayesha Khan: Islamabad Ejaz Vohra: Rumina Hasan: Noaman Siddiqui: Aamir J Khan: D S Akram: Karachi Karachi Abbottabad Karachi Karachi

ORIGINAL ARTICLES General Practitioners Knowledge regarding Tuberculosis: A Survey from Karachi 226 Fauzia Haji Mohammad, Tabinda Ashfaq, Qudsia Anjum,Yaseen Usman Validation of BBL CHROMagar Candida Medium (BD Diagnostics) in Isolating and Differentiating Candida Species in Clinical Specimens 230 Ashraf Hussain, Aamer Ikram, Muhammad Roshan, Luqman Satti Red Cell Distribution Width in the Diagnosis of Iron Deficiency Anemia and Thalassemia Trait Malik Muhammad Adil, Ayesha Junaid, Iffat Zaman, Zeshan Bin Ishtiaque Irrational use of Flagyl (Metronidazole) by Practitioners in Outpatient Clinics Tehmina Munir, Munir Lodhi

Editor:

234

237

Overseas Advisers: Murat Akova: Ankara,Turkey Rayhan Hashmey: UAE Deborah Briggs: U Kansas, USA Peter Chiodini: Royal College Trop Med/Hyg UK Salman Siddiqui: USA Adeel Butt: U of Pittsburgh, USA Farida Jamal: KL, Malyasia Business and Circulation Nasir Hanook
Rights: No part of this issue or associated program may be reproduced, transmitted, transcribed, stored in a retrieval system or translated into language or computer language in any form or means, electronic, mechanical, magnetic, optical, chemical, manual or otherwise without the express permission of the editor/publisher and author(s) of IDJ. Disclaimer: Statements and opinions expressed in the articals, news, letters to the editors and any communications herein are those of the author(s), the editor and the publisher disclaim any responsibility or liability for such material. Neither the editor nor publisher guarantee, warrant, or endorse any product or service advertised in their publication, nor do they guarantee any claim made by the manufacturers of such product or service. Frequency: Infectious Diseases Journal (IDJ) is published quarterly. Designed & Printed by: Mediarc Publications E-259, Ground Floor, E- Market, Block 6, P.E.C.H.S, Karachi. Tel: 34555263, E-mail:guide@super.net.pk Proprietor: Infectious Diseases Society of Pakistan A-53, Block-2, Gulshan-e-Iqbal, Karachi. Ph: 0333-3977011 E-mail: idsp123@yahoo.com

Treatment of Helicobacter pylori Infection; A Controlled Randomized Comparative Clinical Trial 240 Arshad Mehmood, Khan Usmanghani, Abdul Hannan, E. Mohiuddin, Muhammad Akram, Muhammad Asif, Muhammad Riaz ur Rehman Drug Susceptibility Pattern of Typhoidal Salmonellae to the Conventional Anti-Typhoid Drugs; A Current Perspective Anam Imtiaz , Saba Abbasi, Javaid Usman

243

CASE REPORT Central Nervous System ring enhancing lesions in an Immunocompromised Child with Status Epilepticus: A Case Report and Literature Review 246 Amna Batool, Yawar Najam, Ejaz Ahmed Khan, Ismail A Khatri Gelatinous Bone Marrow in AIDS Salman Saleem, Mehreen Ali Khan, Ayesha Hafeez, Aamer Ikram, Usman Rathore NEWS & VIEWS INSTRUCTIONS FOR AUTHORS

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252 254

Price: Rs. 100/-

Colony color and morphology of four most commonly isolated Candida species on CHROMagar plate. Courtesy: Department of Microbiology, Armed Forces Institute of Pathology, Rawalpindi.

Volume 19 Issue 04

Oct-Dec 2010 . 223

GUEST EDITORIAL

Plagiarism in Todays World

Scientific progress has been provided an essential aid with the introduction of the internet. Literature search, correspondence and submission of research articles can all be performed at a fast speed. As in any other field, the use of new inventions can be misused also. This is seen as Plagiarism or intellectual theft, which is an integral component of scientific misconduct. According to the Merriam Webster Online Dictionary plagiarism is defined as, To steal and pass off (the ideas and words of another) as ones own, to use (anothers production) without crediting the source, committing literary theft, to present as new and original an idea or product from an existing source. In other words plagiarism is an act of fraud. It has two components, stealing followed by lying1. Plagiarism has also been stated as one of the most serious crimes in academia2. Authors resort to plagiarism for various reasons, the most important being to increase the number of publications in a short time. As demanded in Pakistan, doctors serving in the government teaching institutions require a fairly large number of research publications in indexed journals for promotion. Being busy practitioners, these professionals at times resort to easy and unfair means for writing articles. Secondly, in this part of the world, most authors do not have a good command over English language and copying verbatim from the net is simple and saves time and energy. At times the author is ignorant about the wrong doing, which is not an acceptable excuse. An important reason is lack of appropriate training. This is because the senior faculty, universities and governing bodies that are responsible for providing the correct guidance, lack expertise, time and funding resources to conduct required training/workshops for the junior doctors. Another reason commonly encountered is the desire to become eminent. Scientists want to have a large number of publications to their credit, so that they can be quoted all over the world. Low moral values are the most important factor, an honest individual would never resort to unfair means. Ethical writing is a reflection of ethical practice3. Whatever the reason, plagiarism is stealing of intellectual property and when detected has to be penalized. It not only brings disgrace to the author besides losing the published material, promotions may be stopped or even services terminated. Some institutions may impose a monitory penalty. Ethics, trust and honesty are the basis of research and publication. Research is essential for the progress of science as the results obtained should be published for the benefit of others. The American College of Physicians in their Ethics Manual have stated that, Dishonesty should not be tolerated - it should be investigated and punished, researchers should be careful, impartial, unbiased and open to investigation and purpose of scientific research should not be self-promotion, personal publicity and financial gain4. Ethics took shape with the Nuremberg Code formed in 1946, The Helsinki Declaration in 1964, and The Belmont Report of 19795. All these have formed a base for important guidelines on Ethics in Research and have been adopted by the World Association of Medical Editors (WAME)6, International Committee of Medical Journal Editors (ICMJE)7 and Committee on Publication Ethics (COPE)8. These guidelines on ethics are followed by most scientific journals. Despite the guidelines from international authorities which have been adopted by most journals and institutions, the act of plagiarism is being detected and reported from all over the world. This dishonesty may start from school and continue to the professional colleges and university. A study on cheating from Croatia which included students in four medical universities, reported more than 99 percent to have admitted to at least one form of educational dishonesty and 78 percent reported to some form of cheating. The study concluded that Academic dishonesty of university students does not begin in higher education; students come in medical schools ready to cheat9. Another questionnaire based study on Plagiarism by Shirazi et al included fourth year medical students and faculty members10. The results revealed that 19% and 22% of students and faculty knew about referencing material from other sources. Surprisingly, 74% students and 69% faculty had observed that colleagues indulge in plagiarizing and were not reported. The study concluded that there was a general lack of information regarding plagiarism among medical students and faculty members. A third cross sectional questionnaire based study conducted by the editorial section of the Journal of Pakistan Medical Association (JPMA) included all authors who submitted their manuscripts for publication in 2010. This study was planned to score the level of perception and practices regarding plagiarism. In this study of JPMA, only 22% of the participants could define plagiarism correctly. The level of perception and practices regarding plagiarism of authors submitting to JPMA was 30% above the 75th Percentile. The study concluded that the authors submitting to JPMA had inadequate knowledge on plagiarism 11 . Plagiarism has been reported earlier from Pakistan. In Pakistan, this problem is not uncommon and many such cases are brought to the notice of editors of medical journals. Surprisingly, the people involved in this matter are usually from a higher academic echelon who had published a similar paper of their own in a local prestigious journal, which was earlier, published in an international journal12. Preventive strategies regarding plagiarism have been advised by Hashim et al, Local literature has advocated using reference managers to prevent plagiarism 13. Detecting plagiarism is not difficult with the availability of the internet and numerous software. Hence, every journal should have a regular screening system. More than this, there is a dire need to root out plagiarism from our teaching institutions. For this, awareness has to be created to consider plagiarism a fraudulent act and which can have drastic and damaging consequences if
224 . Infectious Diseases Journal of Pakistan

detected. Faculty members have to acquaint themselves with the rules and teach their students. Workshops and hands-on training would be an added advantage for the purpose. It is also essential for all institutions, journals and health policy makers to have definite guidelines on plagiarism which will promote ethical research and publication.

Fatema Jawad Editor-in-chief Journal of Pakistan Medical Association

Email: jpma_jpma@hotmail.com

References
1. www.merriam-webster.com/dictionary/plagiarized. Cited 26 December 2010. 2. Pechenik A. A short guide to writing about biology. 4th Edition. New York: Addison Wesley Longman. 2001; p.10. 3. Kolin F C. Successful writing at Work. 6th Edition. Houghton Mifflin. 2002. 4. American College of Physicians Ethics Manual. American College of Physicians. Ann Intern Med 2005; 101: 263-74. 5. Summary from the Nuremberg Code. Trials of War Criminals before the Nuremberg Military Tribunals. Under Control Council Law 10, Volume 2, Nuremberg, October 1946 - April 1949. Washington DC, US Government Printing Office, 1949; pp. 181-2. 6. WAME http://www.wame.org/resources. 7. www.icjme.org. Uniform Requirements for Manuscripts Submitted to Biomedical Journals. 8. Publishing and Editorial issues related to Publication in Biomedical Journals: Overlapping Publications. www.rin.ac.uk/policy/committee-publication ethics-cope-guidel. 9. Taradi SK, Taradi M, Knezevic T, Dogas Z. Students come to medical schools prepared to cheat: a multi-campus investigation. J Med Ethics doi 10.1136/jma.2010.035410. 10. Shirazi B, Jafarey AM, Moazam F. Plagiarism and the medical fraternity: A study of knowledge and attitudes. J Pak Med Assoc 2010; 60:269-73. 11. Jawad F, Ejaz K, Riaz M K, Jafary A, Shirazi B. What is plagiarism and how much authors know about it? Oral presentation at 5th Regional Conference on Medical Journals in the Eastern Mediterranean Region, Karachi-Pakistan, December 2-5, 2010 Abstract Book, page 71. 12. Gadit AA. Plagiarism: how serious is this problem in Pakistan? J Pak Med Assoc 2006; 56: 618. 13. Hashim MJ, Rahim MF, Alam AY. Training in reference management software - a part of new medical informatics workshops in Pakistan. J Ayub Med Coll Abbottabad 2007; 19: 70-1.

Volume 19 Issue 04

Oct-Dec 2010 . 225

ORIGINAL ARTICLE

General Practitioners Knowledge regarding Tuberculosis: A Survey from Karachi

Fauzia Haji Mohammad*, Tabinda Ashfaq*, Qudsia Anjum**,Yaseen Usman* *Department of Family Medicine, Ziauddin University, Karachi **Al Ahli Hospital, Qatar
Abstract Objective To assess the knowledge gaps regarding tuberculosis in general practitioners of Karachi registered for attending the continuous medical education programme. Methods This was a cross sectional survey targeting General Practitioners of Karachi attending the continuous medical education programme organized by the College of Family Medicine, through non-probability purposive sampling. For analysis, they were arbitrarily divided into two groups on the basis of clinical experience; group 1 with less than 5 years and group 2 with more than 5 years of experience. Results A total of 120 general practitioners (GPs) attended the CME programme, out of which 109 completed the questionnaire. 71 (65.13%) were males and 38 (34.86%) were females. Mean age of general practitioners was 37.7 9.9 years and mean duration of their practice was 10.6 8.7 years. The overall knowledge score was found to be slightly higher among general practitioners in group 1. The most common symptom for diagnosis of tuberculosis identified by 38% general practitioners in group 1 was chronic cough, whereas 42% general practitioners in group 2 recognized low grade fever with night sweats. Most general practitioners in both groups, 59% versus 46% identified sputum for acid fast bacillus (AFB) smear as investigation of choice. Only 21% GPs in group 1 versus 37% in group 2 knew about the correct duration of therapy for pulmonary tuberculosis, and 12% group 1 versus 15% group 2 general practitioners knew about the duration of treatment for extra pulmonary tuberculosis. Drugs for initial phase were correctly identified by 55% general practitioners in group 1 and 54% in group 2. The drugs for continuation phase were correctly identified by 10% general practitioners in group 1 and 20% from group 2. Conclusion The study identified gaps in knowledge regarding tuberculosis among general practitioners from Karachi. Their active engagement in educational activities could enhance their Corresponding Author: Fauzia Haji Mohammad, Department of Family Medicine, Ziauddin University, Clifton, Karachi. Email: fauziaakhtar@yahoo.com
226 . Infectious Diseases Journal of Pakistan

knowledge and hence reduce the disease burden and development of multi drug resistant tuberculosis. Key Words CME, General Physicians, Tuberculosis. Introduction Tuberculosis (TB) is an important cause of morbidity and mortality in the developing world. One third of the worlds population, approximately two billion people are infected with Mycobacterium tuberculosis1. In 2006, 1.7 million people died from tuberculosis worldwide, majority from developing countries with more than half of these deaths occurring in Asia. Pakistan, being a third world country ranks eighth in prevalence of tuberculosis2. According to WHO estimated TB burden in 2004, its incidence in Pakistan is 181/100,000 and prevalence is 329/100,000 people3. Tuberculosis has been regarded primarily as a disease of poverty and overcrowding4. Factors contributing to persistent prevalence of this devastating illness in the community include inadequate knowledge of health care professionals, lack of diagnostic tools in health care setup, non-availability of antituberculous drugs and poor patient compliance5. WHO declared tuberculosis as a global emergency in 1993, thus national TB guidelines were launched with a revision in 19986. Although evidence based guideline is available, yet health care professionals lack knowledge for appropriate management of TB. A number of local studies have shown that private practitioners are not compliant with the treatment guidelines7,10. A study done on family physicians in Pakistan targeting knowledge regarding Mantoux test, revealed an overall inadequacy in knowledge; only 18.8% family physicians scored >80% correct responses11. An international study assessed knowledge of health care professionals and community health workers. Although doctors and nurses had better mean scores than non-professionals, yet an overall knowledge gap existed12. A few other international studies also revealed lower levels of knowledge regarding the symptoms and diagnostic procedures for TB among doctors in private practice and primary care physicians13,14. The literature search in the area has suggested updating knowledge of general practitioners (GPs) to improve the scenario for early detection

and treatment of TB. Therefore, this study was aimed to assess the knowledge gaps regarding tuberculosis in general practitioners of Karachi, who were registered for attending the continuous medical education (CME) programme. Material & Methods This was a cross sectional survey targeting the GPs of Karachi registered for attending the CME programme organized in National Institute of Child Health during May-June 2010. This CME programme was organized by the College of Family Medicine for MRCGP (International) exam constituting a few lectures on TB, in order to update GPs knowledge in the light of recent guidelines. The data was collected on a pre-tested self-administered questionnaire before attending the respiratory module. The questionnaire was distributed simultaneously to all of them after verbal informed consent. A total of 120 GPs were surveyed using non-probability purposive sampling method. The sample size was calculated at 95% confidence level and sampling error of 10%, assuming proportion of knowledge among GPs to be 28%. All the results were analyzed using SPSS version 11. A knowledge score of TB was calculated from 18 MCQs (1 point was given for each correct answer). Frequencies were calculated for categorical variables (gender). Mean and standard deviations were calculated for age and year of experience. GPs were divided in two groups on the basis of years of experience for the purposes of analysis, group 1 with less than 5 years and group 2 with more than 5 years of clinical experience. Cross tabulation was done and chi-square test was applied to compare the knowledge between two groups of GPs; p-value of <0.05 was considered significant. Results A total of 109 GPs completed questionnaires which were analyzed. Majority of the respondents were males (65.1%) and 34.9% were females. Mean age of general practitioners was 37.7 9.9 years and mean duration of their practice was 10.6 8.7 years. There were 68 (62.4%) doctors in group 1 and 41(37.6%) in group 2. Table 1 shows the overall knowledge score regarding tuberculosis among GPs. It was found that GPs in group 1 had slightly better knowledge than the doctors in group 2. Only 17.6% GPs in group 1 scored more than 72% as compared to 12.2 % in group 2. 82.4% and 87.8% doctors in two groups respectively scored below 72%. Table 2 summarizes the symptoms, diagnosis, and treatment of TB as recognized by the two groups of GPs. The most common symptom for the diagnosis of TB identified by 38% GPs in group 1 was chronic cough, whereas 42% GPs in group 2 recognized low grade fever with night sweats (p-value 0.437) as the most common presenting complain. Most GPs in the two
Volume 19 Issue 04

Table 1: Knowledge scores versus years of clinical experience Knowledge scores Years of clinical experience < 5 years n % 13 or more <13 12 56 17.66 82.4 > 5 years n % 5 36 12.2 87.8 0.447 p value

groups, 59% and 46% (p-value=0.763) identified sputum for AFB smear as the investigation of choice. Regarding duration of therapy for pulmonary tuberculosis only 21% GPs in group1 knew about the correct duration as compared to 37% GPs in group 2 (p-value 0.095), whereas 12% in group 1 versus 15% in group 2 GPs knew about the correct duration of treatment for extra pulmonary tuberculosis (p-value 0.931). Drugs for initial phase were correctly recognized by 55% GPs in group 1 and 54% GPs in group 2 (p-value 0.206), while the drugs for continuation phase were correctly marked by 10% GPs with <5 years experience and 20% with >5years experience (p-value 0.035). Regarding the side effects of various anti-TB drugs, 41% GPs in group 1 and 39% in group 2 identified peripheral neuropathy as the side effect of isoniazid, 58% from group 1 and 44% from group 2 identified orange color body fluid as the side effect of rifampicin, 59% in group 1 and 44% in group 2 identified vision impairment as the side effect of ethambutol, and 58% from group 1 and 46% from group 2 identified gout as the side effect of pyrazinamide. Discussion GPs are considered as a back bone of health care system and they are the first contact physicians for most of the patients. It has been observed that majority of GPs lack expertise in diagnosing TB, thus leading to increased burden. Several studies have shown poor knowledge regarding TB among GPs probably because in Pakistan after medical graduation most doctors practice independently without adequate clinical experience and supervised training7, 10. In addition, there is also lack of motivation for up gradation of knowledge through CME programmes and workshops. In our study, majority of the respondents were male, the possible reason for this is that majority of female doctors practice as gynecologist and pediatrician. Few local studies have also assessed knowledge of general population about TB, where again there were misconceptions with inadequate knowledge 1 5 - 1 7 . Most common symptom of pulmonary TB is chronic cough, which was identified by few in the current study; this is consistent
Oct-Dec 2010 . 227

Table 2: Knowledge regarding diagnosis of tuberculosis Knowledge of General Practitioners <5 years n=68 >5years n=41 p value %

Most common symptom of tuberculosis High grade fever with chills and rigors Low grade fever with night sweats Chronic cough (> 3 weeks) Weight loss Hemoptysis. Investigation of choice to diagnose pulmonary tuberculosis Complete blood count and ESR Chest X-ray Sputum for AFB smear Tuberculin skin test Blood for AFB smear Three negative sputum samples can exclude the diagnosis Yes No Duration of therapy for pulmonary tuberculosis 6 months 8 months 9 months 12 months Duration of therapy for extra pulmonary tuberculosis 6 months 8 months 9 months 12 months Duration of initial intensive phase and continuation phase 2 months + 7 months 2 months + 6 months 3 months + 6 months 3 months + 5 months Drugs of initial intensive phase HRE HRZE HRSE HRZES Drugs of continuation phase HR RE RZ HE

6 25 26 6 5 7 6 40 7 8 31 37 15 14 33 6 8 8 13 39 17 31 14 6 19 37 5 6 45 7 9 7

9 37 38 9 7 10 9 59 10 12 46 54 22 21 48 9 12 12 19 57 25 45 21 9 28 55 7 10 66 10 14 10

5 17 9 5 5 5 6 19 5 6 23 18 6 15 13 7 5 6 9 21 9 16 7 9 6 22 7 6 17 11 5 8

12 42 22 12 12 12 15 46 12 15 56 44 15 36 32 17 12 15 22 51 22 39 17 22 15 54 17 15 41 27 12 20

0.437

0.763

0.288

0.095

0.931

0.293

0.206

0.035

H=isoniazid, R=rifampicin, Z=pyrazinamide, E=ethambutol, S=streptomycin with findings in a study conducted in Oman18. This may lead to delay in the diagnosis of disease with increasing spread of disease as well as complications. The gold standard test for the diagnosis of pulmonary TB is sputum smear for AFB; correctly
228 . Infectious Diseases Journal of Pakistan

identified by almost half of GPs in both the groups. These figures were almost similar to another study done in Karachi (58.3 %)10. The overall reason for these results is that GPs consider this test to be unreliable and inconvenient in outpatient

Table 3: Knowledge regarding side effects of antituberculous Knowledge of General Practitioners Side effect of Isoniazid 1. Vision impairment 2. Orange colored body fluids 3. Peripheral neuropathy 4. Ototoxicity 5. Gout Side effect of Rifampicin 1. Vision impairment 2. Orange colored body fluids 3. Peripheral neuropathy 4. Ototoxicity 5. Gout Side effect of Ethambutol 1. Vision impairment 2. Orange colored body fluids 3. Peripheral neuropathy 4. Ototoxicity 5. Gout Side effect of Pyrazinamide 1. Vision impairment 2. Orange colored body fluids 3. Peripheral neuropathy 4. Ototoxicity 5. Gout <5 years >5years p-value n=68 % n=41 % 6 23 28 6 5 9 39 7 7 6 40 9 8 6 5 7 6 11 5 39 9 34 41 9 7 13 58 10 10 9 59 13 12 9 7 10 9 16 7 58 5 9 16 5 6 6 18 5 7 5 18 6 5 7 5 6 5 6 5 19 12 22 39 12 15 15 44 12 17 12 44 15 12 17 12 15 12 15 12 46 0.529

disease burden and development of multi-drug resistant tuberculosis. Refrences

1. Tuberculosis fact sheet [Online] 2008 [cited 2008 December 30]. Available from: U R L h t t p : / / w w w. w h o . i n t / m e d i a c e n t r e / f a c t s h e e t s / f s 1 0 4 / en/index.html. WHO Report 2008: Global tuberculosis control-surveillance, planning and financing Geneva: WHO; (WHO/HTM/TB/2008.393). WHO Report 2006.Global tuberculosis control, surveillance, planning and financing. Geneva: WHO; (WHO/HTM/TB/2006.392). S h a b b i r I , M i r z a N , I q b a l R , K h a n S U , Aw a n S R . Clinicoepidemiological profile of one hundred AFB smear positive cases of pulmonary tuberculosis. Pak J Chest Med 2005; 11:29-33. Masroor M, Ahmed I, Qamar R, Imran K, Aurangzeb,Tanveer, Khan MH. Prevalence and pattern of resistance to anti-tuberculosis drugs in our community. Pak J Chest Med 2007;13(1):21-30. Tu b e r c u l o s i s : A G l o b a l E m e rg e n c y. [ o n l i n e ] 1 9 9 9 [ c i t e d 2010 June 11] Available from: URL http://www.nfid.org/ factsheets/tb.shtml. Ahmed M, Fatmi Z, Ahmed J, Ara N. Knowledge, attitude and practice of private practitioners regarding TB-DOTS in a rural district of Sindh, Pakistan. J Ayub Med Coll 2009; 21:28-31. Hussain A, Mirza Z, Qureshi FA, Hafeez A. Adherence of private practitioners with the National Tuberculosis Treatment Guidelines in Pakistan: a survey report. JPMA 2005; 55:17-9. Shehzadi R, Irfan M, Zohra T, Khan JA, Hussain SF. Knowledge regarding management of tuberculosis among general practitioners in northern areas of Pakistan. JPMA 2005; 55:174-6. Khan J, Malik A, Hussain H, Ali NK, Akbani F, Hussain SJ, Kazi GN, Hussain SF. Tuberculosis diagnosis and treatment practices of private physicians in Karachi, Pakistan. East Med Health J 2003; 9:769-75. Ali NS, Jamal K, Khuwaja AK. Family physicians understanding about Mantoux test: A survey from a high endemic country. Asia Pac Fam Med 2010; 9:8. Published online 2010 May 31; DOI: 10.1186/1447-056X-9-8. Keifer EM, Shao T, Carrasquillo O, Nabeta P, Seas C. Knowledge and attitudes of tuberculosis management in San Juan de Lurigancho district of Lima, Peru. J Inf Dev Countries 2009; 3:783-8. Dato MI, Imaz MS. Tuberculosis control and the private sector in a low incidence setting in Argentina. Rev Salud Publica (Boqota) 2009; 11:370-82. Savicevic AJ. Gaps in tuberculosis knowledge among primary health care physician in Croatia: epidemiological study. Coll Antropol 2009; 33:481-6. Mushtaq MU, Majrooh MA, Ahmad W, Rizwan M, Luqman MQ, Aslam MJ, Siddiqui AM, Akram J, Shad MA. Knowledge, attitudes and practices regarding tuberculosis in two districts of Punjab, Pakistan. Int J Tubers Lung Dis 2010; 14:303-10. Khan JA, Irfan M, Zaki A, Beg M, Hussain SF, Rizvi N. Knowledge, attitude and misconceptions regarding tuberculosis in Pakistani patients. JPMA 2006; 56:211-4. Khan SJ, Anjum Q, Khan NU, Nabi FG. Awareness about common diseases in selected female college students of Karachi. JPMA 2005; 55:195-8. Al-Maniari AA, Al-Rawas OA, Al-Ajmi F, De Costa A, Eriksson B, Diwan VK. Tuberculosis suspicion and knowledge among private and public general practitioners: Questionnaire based study in Oman. BMC Public Health 2008; 8:177-183. S. Yadav, A. Patel, S. V. Unadkat, V. V. Bhanushali. Evaluation of management of TB patients by General Practitioners of Jamnagar City. Ind J Com Med 2006; 31:259-60.

2. 3. 4.

0.697
5.

6.

0.521

7.

8.

9.

0.742
10.

11.

setting and also there is poor compliance of patients. Knowledge regarding treatment of pulmonary and extra pulmonary TB was also found to be deficient in both groups, which is consistent with another study done among Pakistani GPs7. Our finding of almost 50% GPs giving treatment for more than recommended duration is similar to a study from Jamnagar India 19. This would result in increased side effects, poor compliance and increased treatment cost. The response for correct drugs for intensive and continuation phase of primary pulmonary TB was less than similar kind of study from Karachi (73.3 %)10. Similarly drugs of initiation and continuation phase were correctly identified by limited number of GPs. The reason is lack of knowledge and familiarization with TB guidelines by GPs. Current situation is expected to result in increased number of multi-drug resistant TB cases. Conclusion The study identified gaps in knowledge regarding TB among GPs from Karachi. Their active engagement in educational activities could enhance their knowledge and hence reduce the

12.

13.

14. 15.

16.

17. 18.

19.

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Oct-Dec 2010 . 229

ORIGINAL ARTICLE

Validation of BBL CHROMagar Candida Medium (BD Diagnostics) in Isolating and Differentiating Candida Species in Clinical Specimens
Ashraf Hussain, Aamer Ikram, Muhammad Roshan, Luqman Satti Department of Microbiology, Armed Forces Institute of Pathology, Rawalpindi
Abstract Objective To determine the diagnostic efficacy of BBL CHROMagar Candida (BD Diagnostics) in isolating and differentiating various Candida species using API 20 C AUX (BioMerieux) as gold standard. Methods One hundred and six isolates of yeasts isolated from various clinical specimens were studied from March 2007 through September 2007. All suspected Candida colonies were presumptively identified on Gram staining and tested up to species level by simultaneous inoculation on CHROMagar Candida medium and API 20 C AUX test strips followed by recommended incubation. Results Out of the total, 52.8% were identified as C. albicans. High sensitivities (98.2%-100%) and specificities (95%-96.8%) were shown by CHROMagar Candida medium for most commonly isolated Candida species of C. albicans, C. krusei, C. tropicalis and C. glabrata. Conclusion CHROMagar Candida medium was easy to use, cost effective and reliable agar medium for isolation and differentiation of most frequently occurring yeast species in the clinical specimens and is recommended for use in peripheral labs. Key words API 20C AUX Medium, Candida Infections, CHROMagar Candida medium, non- albicans Candida species. Introduction The incidence of fungal infections is rising with increasing number of immunocompromised patients, widespread use of broad spectrum antibiotics and invasive procedures1. Candida species are important cause of local and blood stream infections causing significant mortality and morbidity especially in critically ill patients, immunocompromized population and infants. Overall Corresponding Author: Ashraf Hussain, Pathology Department, Combined Military Hospital, Chhor. Email: hussainashraf78@yahoo.com
230 . Infectious Diseases Journal of Pakistan

incidence has risen five fold during this decade and is currently between fourth and sixth most common nosocomial blood isolate in America and Europe2,3. A tilt towards non-albicans Candida has been reported especially in hematological and transplant patients4. Moreover fungemia/colonization ratio of non-albicans Candida has also been found to be more than that of Candida albicans 5. Identification of different Candida species has important therapeutic implication as C. glabrata is less sensitive to ketoconazole and fluconazole than other species and C. krusei displays innate resistance to fluconazole 6 . Presumptive identification of C. albicans is usually done through testing for germ-tube formation7. However, C. tropicalis, C. parapsilosis and Cryptococcus gastricum also have resembling structures8. Therefore it should not be used as a sole criterion for identification of C. albicans. Reference identification procedures using biochemical and morphological studies and conventional methods of yeast identification mainly consisting of assimilation / fermentation characteristics are difficult and require expertise7. Packaged kit and automated systems are expensive and limited by the size of their database 10 . Chromogenic agar media like BBL CHROMagar Candida are easy to use and interpret due to formation of distinct color and morphologies resulting from cleavage of chromogenic substrates by species specific enzymes10. The rationale of the study is to evaluate the diagnostic efficacy of CHROMagar Candida for identification and differentiation of various yeast species in clinical samples as it is now direly needed to precisely identify the pathogen not only at the reference laboratories but also at the peripheral diagnostic facilities. Material and methods This study was conducted at Department of Microbiology, Armed Forces Institute of Pathology, Rawalpindi, from March 2007 through September 2007. One hundred and six yeast isolates yielded from various clinical specimens including blood, high vaginal swabs, urine, sputum, stool and tissues sent for culture and sensitivity to the department of microbiology were included in the study irrespective of age and gender of patients. Upon isolation of a yeast colony, 0.5 MacFarland suspension was prepared in normal saline and 100 uL of the suspension was dispensed on CHROMagar (BD Diagnostics) plate and spread with wire loop. The plates were incubated at 370C for 48 hrs. Identification of Candida species was made according

to the color and morphology of the yeast colonies. Distinct green colored were labeled as Candida albicans, metallic blue color as Candida tropicalis and pinkish colonies with spreading margins and velvety texture were presumptively identified as Candida krusei (Fig 1).

Only four out of these ten yeast species could be identified on CHROMagar Candida medium (Table 2). Distinctive colony morphology is depicted in figure 2. Table 1: Frequency of various yeast species identified on API 20C AUX (n = 106) S. No. Yeast Identified 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Candida albicans Cryptococcus laurentii Candida krusei Candida humicola Candida tropicalis Candida glabrata Candida parapsilosis Rhodotorula rubra Trichosporon cutaneum Trichosporon capitatum Total Number of Isolates 56 2 19 4 11 7 3 1 2 1 106 % 52.8 1.9 17.9 3.8 10.4 6.6 2.8 0.9 1.9 0.9 100

Figure 1: Colony color and morphology of four most commonly isolated Candida species on CHROMagar plate. Clockwise: Pink velvety: C. krusei, green: C. albicans, purple: C. glabrata, blue: C. tropicalis All the yeast isolates were simultaneously inoculated on API 20C AUX (BioMerieux, France) test strips in accordance with the manufacturers instructions. Interpretation was done after 48 and 72 hours of incubation. This method was considered as gold standard in the study and results of CHROMagar Candida medium were compared. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were calculated. Results A total of 106 specimens yielding growth of various yeasts were studied. The mean age for these patients was 42 years (range 1 - 80 years) with greatest number around 30 years of age. 67% (n = 71) specimens were from female patients. The most frequent specimen which yielded Candida spp was urine closely followed by high vaginal swab, 45.3% and 40.6% respectively. Sputum yielded growth of yeast species in 7.5% of the specimens. Other specimens containing yeasts with a lesser frequency included pus and pus swab, blood, throat swab, stool, catheter tip and tissue. Ten different yeast species could be identified using API 20 C AUX medium (Table 1). Candida albicans was found to be the most common yeast present in the clinical specimen (52.8%). This was followed by Candida krusei (17.9%), Candida tropicalis (10.4%) and Candida glabrata (6.6%). Other less frequently isolated yeasts included Candida parapsilosis, Candida humicola, Cryptococcus laurentii, Trichosporon cutaneum, Trichosporon capitatum and Rhodotorula rubra.
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Table 2: Various yeast species identified using CHROMagar Candida (n = 106) S. No 1. 2. 3. 4. Yeast Identified Candida albicans Candida krusei Candida tropicalis Candida glabrata Total Frequency 57 23 14 12 106 Percent 53.8 21.7 13.2 11.3 100

Figure 2: Close view of distinct colony colors and morphology of Candida species on CHROMagar Candida
Oct-Dec 2010 . 231

The sensitivities, specificities negative and positive predictive values of the four Candida species identified on CHROMagar Candida medium are shown in table 3: Table 3: Sensitivities, Specificities, Negative and Positive predictive values for Candida species on CHROMagar Candida Medium Yeast Species Sensitivity % 98.2 100 100 100 Specificity PPV % % 96 95.4 96.8 94.9 96.5 82.6 78.5 58.3 NPV % 97.9 100 100 100

tropicalis. Although CHROMagar candida was able to support growth of all 106 yeast isolates, it placed them in one of the four species of Candida: C. albicans, C. krusei, C. tropicalis or C. glabrata. Generally 10% to 14% of the specimens are found to be containing mixed Candida species, however in our study; we were unable to detect any mixed infections. The reason probably lies in the method of study as the yeast was first isolated on non-differential media like SDA, blood agar and CLED agar etc, and then isolated colonies were tested for species identification on these systems. This might have led to failure to put to test the apparently similar looking yeast colonies of different species. Although detection of mixed Candida infection is also considered to be an advantage with the use of CHROMagar Candida medium, this aspect could not be directly determined during the study. However keeping in view the test results obtained for major Candida species, it can be said with confidence that mixed infections with Candida albicans, C. tropicalis and C. krusei can easily be detected while using this medium for isolation of yeast. It can be appreciated from the results that although C. albicans still remains the major yeast to be isolated from the clinical specimen, non-albicans Candida species now make a very substantial component of the total number. Presuming all the isolates as C. albicans without identifying the actual species can lead to error thus affecting management. In a critical patient, an undesirable outcome due to such an error of presumption is completely unacceptable. Similarly, chronic cases may remain unresponsive to the subsequently used antifungals and their misery may prolong. In this study, the sensitivity and specificity of CHROMagar Candida medium was found to be very high for Candida albicans, C. krusei, C. tropicalis and C. glabrata. This is in accordance with other studies conducted to check these parameters for these species by CHROMagar Candida medium13. Pfaller MA et al, by adhering to the manufecturers guidelines and published criteria of Odds and Bernaerts14, were able to identify correctly 100% of the tested isolates of C. albicans, C. tropicalis and C. krusei and 90% of the isolates of C. glabrata up to the species level14. These four species constituted around 87% of the total isolates in that study; however, despite high sensitivity and specificity obtained for C. glabrata, the PPV for this particular species was only 58.3%. This is due to the fact that some of the relatively infrequently isolated species like C. parapsilosis did give a light purple shade in cream colored colonies, the criteria set for identification of C. glabrata on CHROMagar. The manufacturer doesnt claim the identification of this particular species on this medium, but studies are available in which C. glabrata was successfully identified on this agar medium by its light purplish colony14.

Candida albicans Candida krusei Candida tropicalis Candida glabrata

Discussion With ever increasing number of immunocompromised patients in various medical facilities, isolation of various yeast species is expected to rise. Candida species is the most common yeast causing mortality and morbidity in such patients. Injudicious empirical use of fluconazole without correctly identifying the involved species has resulted not only in treatment failure but also in the development of fluconazole resistant Candida glabrata and Candida krusei sttrains6. While PCR is extremely helpful in definite identification of infection with various microbes, these nucleic acid amplification techniques for Candida are still in the investigatory stage and not available for routine clinical use 11. The classical Wickerhan and Burton method utilizes identification through assessment of assimilation by determining the ability of given yeast isolate to grow in a set of defined minimal liquid media supplemented with different carbohydrates11,12. Though precise, it is laborious and time consuming and therefore not preferable for routine use. Auxanographic technique replaced this for use in clinical laboratory. This is more simple and rapid method and several of its modifications are commercially available such as API 20C, API ID 32C, Vitek, MINITEK etc. These generally are the most frequently employed techniques for the purpose of identification of the yeasts to the species level. However, most of the peripheral laboratories dont have access even to these biochemical identification techniques in developing countries like ours. The main reason is high cost in addition to technical expertise required for performing and interpretation of these tests. Alternative methods are required in routine clinical laboratories which must be cheap and sufficiently reliable. Sabourauds dextrose agar is an excellent medium for primary isolation of yeasts, but it fails to differentiate various species in clinical specimen5. CHROMagar Candida medium by BD Diagnostics is a medium claimed to have high sensitivity and specificity for detection of three of the most commonly isolated yeast species: Candida albicans, Candida krusei and Candida
232 . Infectious Diseases Journal of Pakistan

In this study, all the isolates of C. glabrata were successfully identified as such, but several other isolates like C. parapsilosis were falsely identified as C. glabrata. Interpretation of results when dealing with C. glabrata on CHROMagar has been unreliable in several other studies14, 15. Beighton D et al concluded that colonies identified as C. glabrata varied in color from purple to pale pink that could lead to some degree of confusion with colonies subsequently identified as C. parapsilosis as evident in this study as well15. Although, the PPV in this study for C. glabrata was rather low, the NPV (100%) still highlights its value for this species. This shows that although some of the infrequently isolated Candida species were identified as C. glabrata in this study, none of the C. glabrata present in the specimens were missed. This has a practical significance, since C. glabrata may be involved in several chronic infections like UTI11. Conclusion CHROMagar Candida medium has been found to be easy to use, cost effective and reliable agar medium for isolation and differentiation of most frequently occurring yeast species from the clinical specimen and its usage is recommended for peripheral laboratories. References
1. Moran GP, Sullivan DJ, Coleman DC. Emergence of non-candida albicans species as pathogens. In: Calderone RA Candida and Candidiasis. Washington DC. Am Soc Microbiol 2003; 37-53. Pfaller MA, Diekema DJ, Jones RN, Sader HS, Fluit AC, Hollis RJ. International surveillance of blood stream infections due to candida species: frequency of occurrence and in vitro susceptibilities to fluconazole, ravuconazole, and voriconazole of isolates collected from 1997 through 1999 in the SENTRY Antimicrobial Surveillance Program. J Clin Microbiol 2001; 39:3254-9. Marchetti O, Bille J, Fluckiger U, Eggimann P, Ruef C. Epidemiology of candidemia in Swiss tertiary care hospitals: secular trends 1991-2000.

4. 5.

6.

7. 8.

9.

10.

11.

12. 13.

2.

14.

15.

3.

Clin Infect Dis 2004; 38: 311-20. Schelenz S, Gransden WR. Candidemia in London teaching Hospital: analysis of 128 cases over a 7 year period. Mycoses 2003; 46:390-6. Roilides E, Farmaki E, Evdoridou J, Francesconi A, Kasai M, Filioti J. Candida tropicalis in a neonatal intensive care unit: Epidemiologic and molecular analysis of out break of infection with an uncommon neonatal pathogen. J Clin Microbiol 2003; 41:735-41. Bouchara JP, Declerck P, Cimon B. Planchenault C, De Gentile L, Chabasse D. Routine use of CHROMagar candida medium for presumptive identification of candida yeast species and detection of mixed fungal populations. Clin Microbiol Infect 1996; 2:202-8. Freydiere AM, Guinet R, Bioron P: Yeast identification in the clinical microbiology laboratory: Phenotypical methods. Med Mycol 2001, 39:9-33. Pfaller MA, Messer SA, Hollis RJ, Jones RN, Doem GV, Brandt ME. Trends in species distribution and susceptibility to flunconazole among blood stream isolates of candida species in the United States. Diagn Microbiol Infect Dis 1999; 33:217-22. Koehler AP, Chu KC, Houang ETS, Cheng AF. Simple, reliable and cost effective yeast identification scheme for the clinical laboratory. J Clin Microbiol 1999; 37: 422-6. Bauters TG, Nelis HJ. Comparison of chromogenic and fluorogenic membrane filtration methods for detection of four Candida species. J Clin Microbiol 2002; 40: 1838-9. Hazen KC, Howel SA. Candida, Cryptococcus, and other yeasts of medical importance. In: Murray PR, Baron EJ, Landry ML, Jorgensen JH, Pfaller MA, editors. Manual of Clinical Microbiology. Washington, D.C: ASM Press; 2007. Reiss E, Morrisson CJ. Non culture methods for diagnosis of disseminated candidiasis. Clin Microbiol Rev 1993; 6:311-23. Pfaller MA, Houston A, Coffman S. Application of CHROMagar Candida for rapid screening of clinical specimens for Candida albicans, Candida tropicalis, Candida krusei, and Candida (Turolopsis) glabrata. J Clin Microbiol 1996; 34: 58-61. Odds FC, Bernaerts R. CHROMagar Candida, a new differential isolation medium for presumptive identification of clinically important Candida species. J Clin Microbiol 1994; 32:1923-9. Beighton D, Ludford R, Clark DT, Brailsford SR, Pankhurst CL. Use of CHROMagar Candida medium for isolation of yeasts from dental samples. J Clin Microbiol 1995; 33: 3025-7.

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ORIGINAL ARTICLE

Red Cell Distribution Width in the Diagnosis of Iron Deficiency Anemia and Thalassemia Trait
Malik Muhammad Adil, Ayesha Junaid, Iffat Zaman, Zeshan Bin Ishtiaque Pathology Department, Shifa International Hospital, Islamabad

Abstract Objective To evaluate diagnostic importance of Red Cell Distribution Width (RDW) in differentiating iron deficiency anemia from Thalassemia trait. Patients and methods A total of 100 cases aged 5 months to 50 years of either sex with diagnosed iron deficiency anemia or thalassemia trait were compared with respect to their RDW value. Results RDW value in iron deficiency anemia was between 36.2% to 55.2% (Mean 44.1%). The range of RDW in Thalassemia trait was 14.7% to 24.9% (Mean 19.8%). Conclusions The very high range of RDW in iron deficiency anemia as compared to slight elevation of the value in thalassemia trait in our study suggests that RDW value obtained from simple Complete Blood Counts (CBC) can help in differentiating the two pathologies. Key words Iron deficiency anemia, RDW, Thalassemia trait Introduction Iron deficiency anemia is one of the most common nutritional disorders in the world1. In Pakistan after iron deficiency anemia, beta thalassemia trait is the second most common cause of hypochromic microcytic anemia2. However, in population where thalassemia is also prevalent, it is important to distinguish between these two common causes of microcytic anemia. For the diagnosis of iron deficiency anemia and thalassemia trait, estimation of serum iron, TIBC and level of HbA2 are required3. Red blood cell size variation (anisocytosis), along with poikilocytosis, has been recognized as morphologic hallmarks of some anemias. Traditionally, microscopists subjectively assess anisocytosis as either slight, moderate, or marked. This subjective assessment has limitations, and therefore more Corresponding Author: Malik Muhammad Adil, Department of Medicine, Shifa International Hospital, Islamabad. Email: malikmuhammad.adil@gmail.com
234 . Infectious Diseases Journal of Pakistan

objective quantitative measurements are desirable. It has been suggested that Red Cell Distribution Width (RDW) could fulfill this role4. RDW which is an objective measure of the degree of anisocytosis, has been proposed to be useful in early classification of anemias because it becomes abnormal earlier in nutritional deficiency anemia than any of the other red cell parameters, especially in case of iron deficiency anemia 5,6. Bessman and colleagues have indicated that the use of RDW, made available by new automated blood cell analyzers, has improved the distinction between iron deficiency anemia and thalassemia trait5. However, the reliability of using RDW as a sole method for diagnosis of anemia is uncertain7. The purpose of this study was to determine whether we could reproduce the accuracy of classification in our population using RDW in patients with iron deficiency anemia and thalassemia trait keeping in view the financial constraints in a developing country. If this were so, the time and expense of evaluating iron deficiency anemia and thalassemia trait might be reduced. Material and methods A total of 100 patients (50 with iron deficiency anemia and 50 with thalassemia trait), aged 5 months to 50 years, who reported to Shifa International Hospital, Islamabad, for iron studies and hemoglobin electrophoresis were included in the study. The study was carried out from June 2004 to December 2004. Patients with iron deficiency anemia 5 ml venous blood was collected from each of the subject using aseptic technique. In order to avoid the problem of diurnal variation in iron level, all blood samples were collected between 10 am to 12 noon. The blood was distributed as follows: (a) 3 ml of blood was added to K 2 EDTA at a final concentration of 2.5mg/ml for blood complete examination (b) 1.8 ml was added to plain tube and centrifuged at 1500 rpm for 5 minutes to obtain serum. This serum was analyzed for serum iron and TIBC. Blood complete examination was carried out using SYSMEXKX hematology analyzer. Low, normal & high controls prepared commercially were tested before each batch of samples. Quality control was assured by running normal specimen after every 19 test samples. Serum iron & TIBC were analyzed using ROCHE DIAGNOSTICS reagents on automated clinical

chemistry analyzer HITACHI-911. Commercial controls were run before every batch of samples in order to standardize the sample results. The quantitative determination of both serum iron and TIBC were based upon direct photometric method. The following criteria were used: Anemia was defined as hemoglobin concentration of less than 11.5 gm/dl (WHO criteria). Mean Corpuscular Volume (MCV) <80fl (Normal 9010 fl) RWD <14.6% (Normal 13.41.2%) Serum iron less than 115ug/dl TIBC more than 360 ug/dl Patients with thalassemia trait Hemoglobin electrophoresis was done and quantitative HbA2 levels > 3.5% were taken as thalassemia trait. The proposal was reviewed by internal review board and the study was approved by the Ethics Committee of Shifa College of Medicine. All data was entered in to Statistical package for Social Sciences (SPSS version 10.0). The data was re-validated and later analyzed. Results RDW value in 50 patients with iron deficiency anemia and 50 patients with thalassemia trait are depicted in table 1. Mean RDW in patients with iron deficiency anemia was 44.14.1 % (Range 36.2-55.2%). Elevated RDW was found in all 50 cases none of them were with normal RDW. Mean RDW in patients with thalassemia trait was 181.8 % (Range14.7-24.9 %). Elevated RDW was found in all 50 cases none of them were with normal RDW. Table 2 shows comparison of our results Table 1: RDW values in different types of anemia. Anemia No. of cases RDW % Mean Iron deficiency 50 Thalassemia trait 50 Range Elevated pvalue RDW Cases %

with other studies. Distribution of iron deficiency anemia by RDW is shown in figure 1, 44% of cases had RDW in range of 40.1-45%. Figure 2 shows distribution of Thalassemia trait by RDW, 86% of cases have RDW in range of 14-20%. 50

40

44

Percentage

30

30

20 18 10 8 0 35.1-40 40.1-45 45.1-50 50.1-56 Iron deficiency anemia (RDW)

Fig 1: Distribution of iron deficiency anemia by RDW. 100

80

86

181.8

14.7-24.9

50

100

Percentage

44.14.1 36.2-55.2

50

100

0.001

60

40

Table 2: Comparison with different studies. Studies Elevated RDW Iron deficiency anemia 97% 94% 100% 100% 100% Thalassemia trait 4% 48% 100% 100% 100% 20 14 0 14-20 20.1-25 Thalassemia trait (RDW) Fig 2: Distribution of thalassemia trait by RDW.
Oct-Dec 2010 . 235

Bessman et al 5 Flynn et al 7 Viswanath et al 9 Laso et al 10 Our study

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Discussion The availability of automated blood cell analyzers that provides index of RDW has new approaches to patients with anemia. While the emergency physician is primarily responsible for the detection of patients with anemia, the inclusion of the RDW in the complete blood count has made diagnosing certain anemias easier, especially microcytic8. The measure of elevated RDW was used by Bessman to classify microcytic anemias into two categories5. Anemia with normal RDW (microcytic homogenous) included heterozygous thalassemia and chronic disease, and those with elevated RDW (microcytic heterogeneous) included iron deficiency, S beta thalassemia, hemoglobin H, and RBC fragmentation. In Bessman study, 96% of thalassemia trait cases were with normal RDW (mean RDW 3.71.6%), while 97% of iron deficiency anemia cases were with elevated RDW (mean RDW 16.31.8%). Thus Bessman et al were able to classify 96% of anemias due to thalassemia minor and 97% due to iron deficiency using RDW 5 while Flynn et al 7 results categorized only 55% of thalassemia cases as microcytic homogeneous (normal RDW). In our study RDW was elevated in both cases (iron deficiency anemia and thalassemia trait) but there was great difference between their means i.e. 44.14.1% for iron deficiency and 181.8% for thalassemia trait. In iron deficiency cases the RDW elevated more than double the normal while in thalassemia trait, increase was in fractions, so in general our study did show that it was very unusual for a patient with iron deficiency to have normal RDW. It appears that iron and hemoglobin studies are still required to confirm the diagnoses of iron deficiency and thalassemia in our population. However, cost and time may be saved by following a sequence of investigation in evaluating microcytic RBCs. The CBC with differential and RDW provides the first and most important test with significant cost savings in our population where affordability is main problem in diagnosing these two common conditions. In one study, the result was interesting in a way that they

suggested slight increase in RDW in patients with iron deficiency and moderately elevated RDW in thalassemia trait4. Results of another study from India are in accordance with our study which showed elevated RDW in all cases of iron deficiency anemia 9 . Conclusion We suggest that RDW may be useful in initial differentiation between iron deficient and thalassemia trait patients. In iron deficiency anemia patients, RDW is likely to be moderately to markedly elevated, and thalassemia trait patients show slightly elevated RDW. The cost and time may be saved by following a sequence of steps in evaluating microcytic RBC. References
1. DeMaeyer EM, Dallman P, Gurney JM, Hallberg L, Sood SK, Srikantia SG. Preventing and controlling iron deficiency anemia through primary health care: a guide for health administrators and programme managers 1989:5-58 WHO Geneva, Switzerland. 2. Akhtar F, Malik HS, Anwar M. Prevalence of beta thalassemia trait in patients with hypochromic microcytic anemia. Pak J Pathol 2002; 13(2): 11-3. 3. Weatherall DJ, Clegg JB.Thalassemia syndromes. Oxford 1972; p.113. 4. Roberts GT, El Badawi SB.Red blood cell distribution width index in some hematologic diseases. Am J Clin Pathol 1985; 83(2):222-6. 5. Bessman JD, Gilmer PR Jr, Gardner FH. Improved classification of anemias by MCV and RDW. Am J Clin Pathol 1983; 80(3):322-6. 6. Das Gupta A, Hegde C, Mistri R. Red cell distribution width as a measure of severity of iron deficiency in iron deficiency anemia. Indian J Med Res 1994; 100:177-83. 7. Flynn MM, Reppun TS, Bhagavan NV. Limitations of red blood cell distribution width (RDW) in evaluation of microcytosis. Am J Clin Pathol 1986; 85(4): 445-9. 8. Evans TC, Jehle D. The red blood cell distribution width. J Emerg Med 1991; 9(1):71-4. 9. Viswanath D, Hegde R, Murthy V, Nagashree S, Shah R. Red cell distribution width in the diagnosis of iron deficiency anemia. Indian J Pediatr 2001;68(12):1117-9. 10. Laso FJ, Mateos F, Ramos R, Herrero F, Perez-Arellano JL, Gonzalez Buitrago JM. Amplitude of the distribution of erythrocyte size in the differential diagnosis of microcytic anemia. Med Clin (Barc) 1990; 94(1):1-4.

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ORIGINAL ARTICLE

Irrational Use of Flagyl (Metronidazole) by Practitioners in Outpatient Clinics

Tehmina Munir*, Munir Lodhi** * Department of Pathology, ** Paediatric Department, Combined Military Hospital, Multan.

Abstract Objective To determine the frequency of prescription of flagyl by general practitioners in outpatient clinics in order to limit its use for treatment of acute diarrhoea and other GIT symptoms. Study Design A descriptive study. Place and Duration of Study Combined Military Hospital, Multan between 1st January and 31st May 2010. Methodology Total number of patients who were given flagyl during study period was retrieved from the computerized record of the patients. Clinical diagnosis was not available in most of the cases, so to determine the number of patients with diarrhoea, patients who were advised oral rehydration salts in addition to oral flagyl was determined. A questionnaire about the preference of the physicians for various antibiotics for the treatment of acute diarrhoea was developed and distributed among the doctors working in the outdoor clinics. Results Over a period of 5 months, 4068 patients were prescribed flagyl for their ailment. The age range of the patients was between 9 months to 65 years. Male to female ratio was 3:1. Out of 4068 patients, 1074(26%) were given flagyl along with oral rehydration salts indicating that the antimicrobial was being prescribed for acute diarrhoea. Sixteen doctors working in outdoor/ emergency departments responded to the questionnaire; 14 (87.5%) preferred flagyl, whereas 8 (50%) prescribed oral flagyl for acute as well as chronic gastroenteritis. Out of 14 doctors who said that they prescribed flagyl for acute diarrhoea, 12(75.1%) were highly qualified medical practitioners and only 2 (12.5%) of them were without any postgraduate qualification. Conclusion Our study showed that flagyl was being grossly misused in the Corresponding Author: Tehmina Munir, Department of Pathology, Combined Military Hospital, Multan. Email: tehmunir_doc@yahoo.com
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hospital and being given for the treatment of acute diarrhoea. Appropriate measures need to be taken and importance of better prescribing habits should be highlighted during clinical meetings and discussions. Key Words Diarrhoea, Metronidazole. Introduction Worldwide acute diarrhoea constitutes a major cause of morbidity and mortality, especially in the developing countries1. Most cases of acute diarrhoea are caused by enteric infections. Food and water-borne outbreaks constitute a major portion of diarrhoeas reported in outpatient setup. Significant morbidity and mortality in the developing world is attributable to diarrhoeal diseases2. Childhood diarrhoea is a major cause of morbidity and mortality and causes 3.3 million deaths worldwide. Rotavirus has been reported to be the most common cause of severe childhood diarrhoea in developing as well as developed world3. Other organisms isolated in the stools of patients with diarrhoea are Escherichia coli, Aeromonas spp, Salmonella spp, Campylobacter spp, Entamoeba histolytica, Giardia lamblia, Cryptosporidium etc. Among parasites E. histolytica causes bloody diarrhoea, giardiasis results in chronic diarrhoea and Cryptosporidium causes diarrhoea in immunocompromised individuals4. Most cases of acute diarrhoea are self-limiting or viral and last less than a day. Treatment of diarrhoea primarily consists of rehydration. Bismuth subsalicylate may reduce enterotoxin action and if there is no significant febrile or inflammatory process, low doses of anti-motility agents may offer some relief with minimal risk5,6,7. Appropriate antibiotics may be given for infectious bacterial diarrhoeas8. Most of the doctors performing their duties in our outpatient departments are general duty medical officers who have a tendency to over prescribe medicines and antibiotics. In our setup, metronidazole is being prescribed to patients of any age and sex irrespective of type or cause of diarrhoea. A study was carried out at CMH Multan, 500-bedded hospital, to know about the prescribing habits of the physicians particularly for patients of diarrhoea in the outdoor clinics in order to limit
Oct-Dec 2010 . 237

the use of the antimicrobial for the treatment of acute diarrhoea and other gastrointestinal symptoms. Material & Methods Total number of patients in the hospital, given flagyl (metronidazole) from 1st January to 31st May 2010, was determined from computerized record of the hospital. As most of the doctors do not write the diagnosis on the prescription forms, an indirect attempt was made through number of patients given ORS along with flagyl. A questionnaire about the preference of the physicians for various antibiotics for the treatment of acute diarrhoea was developed and distributed among the doctors working in the outdoor clinics. Fresh fecal specimens were collected in clean container and examined under the microscope to detect Giardia lamblia, Entamoeba histolytica and eggs and ova of other intestinal parasites. An attempt was made to collect the dysenteric and watery specimens and pass them on to laboratory within 15 minutes of collection. Direct and eosin slides were prepared and examined under microscope (10x and 40x). E. histolytica was identified by presence of trophozoites having single nucleus, containing ingested red cells and showing active directional amoeboid movement. Giardia lamblia was identified by the presence of small pear shaped flagellate with a rapid tumbling and spinning motility in fresh diarrhoeal specimens particularly in mucus. Giardia lamblia cysts were looked for in formed specimens. Results During five months, 4068 patients were prescribed flagyl. Mean age was 33 years; range 9 months to 65 years. Male to female ratio was 3:1; 3099 (76%) males and 969 (24%) females. All the outdoor patients were prescribed oral flagyl. Table I shows patients of various age groups who were given flagyl. Out of 4068 patients, 1074 (26.4%) were given flagyl along with oral rehydration salts, indicating that the antimicrobial was being prescribed for acute diarrhoea. Sixteen doctors who were working in outdoor/emergency departments responded to the questionnaire. The preferred Table 1: Age distribution of Patients Age Group Below then 10 years 10 to 29 30 to 49 50 to 69 Above then 70 No. of Patients 77 1363 2120 447 61

antibiotic for acute diarrhoea by 14 doctors was flagyl, whereas 8 prescribed flagyl for acute as well as chronic diarrhoea. Out of 14 doctors prescribing oral flagyl for acute diarrhea, 12 (75%) were highly qualified medical practitioners and only 2 (12.5%) were general duty doctors. During 5 months, 801 stool examinations were performed. Vegetative form of Giardia intestinalis was detected in 17 patients whereas that of E. histolytica was not detected. Discussion The study observed that flagyl was being misused in the hospital, unnecessarily prescribed for 1074 patients of acute diarrhoea. However the extent of the problem may be much bigger as mild cases of diarrhoea and patients with other GIT symptoms might have been treated with flagyl. Our results also showed that most of our qualified practitioners also prefer giving flagyl for acute diarrhoea. Keeping in view the climatic and the sanitary conditions, prevalent diarrhoeal diseases constitute a major proportion of our outpatient workload. A large number of patients were being treated with flagyl reserved for GIT infections like amoebiasis, giardiasis, trichomoniasis, anaerobic infections and Clostridium difficile associated diarrhoea 9. Metronidazole has also been used with other drugs for eradicating H. pylori in patients with duodenal ulcer10. Inappropriate use of antimicrobials with specific reference to flagyl has also been seen in other hospitals of the country as well. In one of the hospitals in Karachi, 39% of general practitioners and 32% of pediatricians prescribed anti-amoebics to more than 30% diarrhoeal patients11. The frequent irrational use of flagyl has been reported in other developing countries like Bangladesh where in one study 17% of the patients were treated with metronidazole in outdoor clinics irrespective of the diagnosis12. A Dutch researcher studied popularity of drugs particularly metronidazole in treating diarrhoea in Philipines13. She attributed this popularity to high frequency of amoebiasis in that country, poor diagnostic methods, unreliable laboratories and aggressive pharmaceutical marketing. Our hospital however had an efficient clinical laboratory and the diagnosis of Giardiasis is rather simple requiring minimal cost and time. Lack of laboratory facilities or the inability of patients to afford microbiological tests were said to be main reason for prescribing antimicrobials in diarrhoeal cases in Pakistan. However, extremely short communication time between doctor and patient was also a major reason for omitting required laboratory tests 14 . Inadequate knowledge might be an important determinant for unrestricted and irrational use of metronidazole. Whereas knowledge may be necessary for good practice, improving knowledge may not improve prescribing practices15. In our study even qualified practitioners prescribed flagyl for cases of

238 . Infectious Diseases Journal of Pakistan

acute diarrhoea. Studies are needed to look in more depth at the reasons for this discordance and the extent to which better knowledge may lead to improvement in prescribing practice for acute diarrhoea. Metronidazole is a potential carcinogen and mutagen in rodents. Acute toxicity causes gastrointestinal tract symptoms whereas chronic toxicity causes neurological damage16. We should be extremely cautious while prescribing metronidazole in cases where its usage is not warranted. Conclusion This comparatively smaller scale study showed that metronidazole is being grossly misused in our hospital. This finding may only be the tip of the iceberg; a larger scale multicentre study may provide the exact extent of inappropriate prescription by the general as well as qualified practitioners. Continued medical education is needed to limit its usage in indicated cases only.

References
1. King CK, Glass R, Bresee JS, Duggan C. Managing Acute Gastroenteritis among Children. Recommendations and Reports- National Center for Infectious Diseases- Nov 21, 2003/52(RR6); 1-16. 2. Sazawal S, Black RE, Bhan MK. Zinc supplementation reduces the incidence of persistent diarrhoea and dysentery among low socioeconomic children in India. J Nutr 1996;126:443-50. 3. Shah M, Yousaf Zai M, Lakhani NB, Chotani RA, Naushad G. Prevalence and correlates of diarrhoea. Ind J Pediatr 2003; 70(3): 207-11. 4. Lawrence S, Friedman Kurt J. Diarrhea and constipation- Isselbacher and Brunwald.eds. In: Harrisons Principles and Practice of Internal Medicine.

Vol 1.13th Ed. McGraw Hill 1994. New York, 213-21. 5. Chaudhury SAR. Prescribing a rational drug. Bangla J Physiol Pharmacol 1991;7:1. 6. Palmar DL, Koster FT, Islam AFMR. A comparison of sucrose and glucose in oral electrolyte therapy of cholera and other severe diarrhoeas. N Eng J Med 1997; 297:1107. 7. King Ck, Glass R, Bresee JS, Duggan C. Managing acute gastroenteritis among children- oral rehydration, maintaining nutritional therapy. MMWR Resource Rep 2003; 52:1-16. 8. Thielman NM, Guerrant RL. Acute infectious diarrhoea. N Eng J Med 2004; 350(1): 38-47. 9. Finegold SM. Metronidazole. In: Mandell, Douglas and Bennetts Principles and Practice of infectious diseases. New York: Churchill Livingstone, 2000; 361- 5. 10. Carpintero P, Blanco M, Pajares JM. Ranitidine versus colloidal bismuth subcitrate in combination with amoxicillin and Metronidazole for eradicating Helicobacter pylori in patients with duodenal ulcer. Clin Infect Dis 1997; 25:1032-7. 11. Murakami K, Okimoto T, Kodama M, Sato R, Wtanabe K, Fujitoka T. Evaluation of three different proton pump inhibitors with amoxicillin and metronidazole in the treatment of Helicobacter pylori infection. J Clin Gastroentrol 2008; 42(2): 139-42. 12. Nizami SQ, Khan IA, Bhutta ZA. Drug Prescribing Practices of general practitioners and pediatricians for childhood diarrhoea in Karachi, Pakistan. Soc Sci Med 1996; 42(8): 1133-9. 13. Gyon AB, Barman A, Ahmed JU, Ahmed AU, Alam MS. A baseline survey on use of drugs at the primary health care level in Bangladesh. Bull WHO 1994; 72(2): 265-71. 14. Van Staa A. Myth and Metronidazole in Manila. The popularity of drugs among prescribers and dispensers in the treatment of diarrhoea. Master thesis in Medicine and cultural Anthropology, University of Amsterdam, 1993. 15. Radyowwijati A, Hilbrand H. Determinants of Antimicrobial use in the developing world. Child Health Research Project Special Report 2002; 4(1): 1-35. 16. Metronidazole (Flagyl) facts, e-MedExpert.com 31 Mar 2008.

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ORIGINAL ARTICLE

Treatment of Helicobacter pylori Infection; A Controlled Randomized Comparative Clinical Trial

Arshad Mehmood*, Khan Usmanghani*, Abdul Hannan*, E. Mohiuddin*, Muhammad Akram*, Muhammad Asif**, Muhammad Riaz ur Rehman** *Department of Clinical Sciences, Faculty of Eastern Medicine, Hamdard University Karachi, Pakistan **College of Conventional Medicine, Faculty of Eastern Medicine, The Islamia University Bahawalpur

Abstract Background Helicobacter pylori induces chronic inflammation of the underlying gastric mucosa and is strongly linked to the development of duodenal and gastric carcinoma. Methods A study was conducted to evaluate the efficacy of Pylorex, a herbal formulation, for treatment of H. pylori infection as compared to triple allopathic therapy (Omeprazole, Amoxicillin, Metronidazole). The therapeutic evaluations of these medicines were conducted on 97 clinically and immunologically diagnosed cases of H. pylori infection. Results H. pylori was eradicated in 16 (32.6%) out of 49 patients by the use of triple allopathic therapy (Control drugs), and in 9 (18.7%) out of 48 patients by the use of Pylorex (Test drug). Conclusion Pylorex possesses a therapeutic value for the treatment of H. pylori associated symptoms but the eradication rate is superior in triple allopathic therapy. Introduction Helicobacter pylori, gram-negative bacterium, is found on the luminal surface of the gastric epithelium. It contains a hydrogenase which produces energy by oxidizing molecular (H2) that is produced by intestinal bacteria. It produces catalase, urease and oxidase. It is capable of forming biofilms and can convert from spiral to a possibly coccoid form. The coccoid form can adhere to gastric epithelial cells in vitro1- 4. Half of the world's population is infected by this bacterium. Actual infection rates vary; people in under developed countries have much higher infection rates than the developed countries where Corresponding Author: Muhammad Akram, Department of Basic Medical Sciences, Faculty of Eastern Medicine, Hamdard University, Madinat-al-Hikmah, Muhammad Bin Qasim Avenue, Karachi, Pakistan. Email: makram0451@hotmail.com
240 . Infectious Diseases Journal of Pakistan

estimated rates are around 25% 5-6. Infections are usually acquired in early childhood. In developed nations it is currently uncommon to find infected children. The percentage of infected people increases with age; about 50% infected over the age of 60 years as compared to around 10% between 18 and 30 years.7-8 Coded herbal formulation Pylorex contains Curcuma longa, Mallotus philippinensis and Glycyrrhiza glabra. These medicinal herbs used in this study were selected on the basis of their traditional use in Greek system of medicine, especially for treatment of H. pylori infection9. This study was conducted to evaluate the efficacy of Pylorex for treatment of H. pylori infection as compared to triple allopathic therapy (Omeprazol, Amoxicillin, Metronidazole) among the population living in Gadap Town. Materials and methods The therapeutic evaluations of these medicines were conducted on clinically and immunologically diagnosed cases of H. pylori infection at Shifa-ul-Mulk Memorial Hospital, for Eastern Medicine, Hamdard University Karachi, from June 2007 to July 2009. All selected patients (n=97) were thoroughly examined. Participants who were willing to undergo treatment and to attend all the follow up visits during the clinical trial were selected. The therapeutic evaluation of the drug was made on the basic improvement in the subjective signs and symptoms, clinical observations and laboratory investigations at periodic intervals during the course of treatment. Patients were randomly assigned to receive triple allopathic therapy (Omeprazole 20 mg twice daily 15 minutes before meal, Amoxicillin 500 mg twice daily and Metronidazole 500 mg twice daily after meal; and 500 mg Pylorex twice daily. The duration of treatment was 15 days with a window for follow up visit of 15-30 days for periodic assessment. Primary analysis was based on antigen test that uses enzyme immunoassay to detect the presence of H. pylori antigen in stool specimens. The samples were tested at Aga Khan Laboratories Karachi. Weekly record of sign and symptoms

was maintained for analyzing the improvement in H. pylori associated symptoms. Disappearance of abdominal pain, heart burning, and regurgitation, fullness of stomach, nausea, vomiting, melena and hematemesis were especially noted. The subjects were randomly divided into two groups; the test and the control groups (Table 1). The data was adjusted based on the number of cases in the light of demographic factor using statistical methods like multinomial logistic regression. P-value less than 0.05 was considered as statistically significant. Results The intent-to-treat population consisted of 97 patients enrolled; 48 were treated with coded herbal formulation Pylorex and 49 with triple allopathic therapy. The mean age of patients prescribed Pylorex was 27 years and 26.1 years for males and females respectively. The mean age of patient prescribed triple allopathic treatment was 26.3 and 28.5 years for males and females respectively.

Table 2: Stool antigen after treatment Treatment group After treatment Test (Pylorex) Control (Triple allopathic therapy) 16 (32.6%) 33 (67.4%) 49 25 72 97 0.359 Total p(n) value

-tive 09 (18.7%) Stool +tive 39 (81.3%) antigen Total 48

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According to the analysis H. pylori was eradicated in 16 patients (32.6%) out of 49 patients by the use of triple allopathic therapy (Control drug), and in 9 patients (18.7%) out of 48 patients by the use of Pylorex (Table 2). All differences that were equal to or more than the set cut-off values were considered clinically significant. Results of stool antigens before and after both the treatments are shown in table 1 and 2. The evaluation of H. pylori eradication was significantly high in the control group as compared with test group. But there was a significant difference in H. pylori associated symptoms as observed between two treated groups at the end of therapy (fig 1).

ea rt Bu rn

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Figure 1: Improvement response in symptoms after treatment than synthetic antibiotics, some patients prefer to use herbal medicines. Thus healthcare professionals should be aware of the available evidence for herbal antibiotics. It has been previously reported that Curcuma longa, Mallotus philippinensis and Glycyrrhiza glabra have anti-H. pylori effects commonly used for the treatment of this infection10-12. In a recent study, anti-H. pylori activity of 50 commonly used Unani (traditional) medicinal plants from Pakistan, extensively utilized for the cure of gastrointestinal disorders, were explored as natural source compounds against H. pylori13. Curcumin is the substance that gives the spice turmeric its yellow color. Dozens of studies have shown that it is chemopreventative, and recently it has been shown to have a strong antibacterial effect against H. pylori. Studies have indicated that curcumin could be considered as a valuable support in the treatment of infections14-15. In a recent study, researchers found that licorice extract produced a potent effect against clarithromycin-resistant H. pylori strains. The authors concluded that licorice extract could form the basis for alternative H. pylori therapeutic agent. Licorice extracts are also effective against H. pylori strains resistant to both amoxicillin and clarithromycin10 . Mallotus philippinensis (Kameela) also has activity against H. Pylori especially against clarithromycin and metronidazole
Oct-Dec 2010 . 241

Discussion Hundreds of plants worldwide are used in traditional medicine as treatment for bacterial infections. Some of these have also been subjected to in vitro screening but the efficacy of such herbal medicines has seldom been rigorously tested in controlled clinical trials. Conventional drugs usually provide effective antibiotic therapy for bacterial infections but there is an increasing problem of antibiotic resistance and a continuing need for new solutions. Although natural products are not necessarily safer Table 1: Baseline stool antigen in patients Treatment group Baseline Test (Pylorex) Control (Triple allopathic therapy) 49 97 Total (n)

Positive stool antigen

48

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resistant strains which could be utilized for the development of antimicrobials against H. pylori related disorders 11 . So taking advantage, the coded herbal formulation Pylorex, contains the three ingredients Curcuma longa, Mallotus philippinensis and Glycyrrhiza glabra for the treatment of H. pylori infection. Triple allopathic therapy is commonly used for the treatment of H. pylori infection but it exerts side effects. In order to overcome this problem, there is a need to find new medicinal agents, which have good efficacy and less adverse effects. The control drugs exhibited side effects like gastrointestinal intolerance nausea and vomiting, whereas Pylorex was well tolerated by the treated patients. More detailed studies are needed to evaluate such herbal medicines. Conclusion The eradication rate of H. pylori is superior in triple allopathic therapy as compared to Pylorex, however Pylorex possesses a therapeutic value for the treatment of associated symptoms. References
1. 2. 3. 4. Brown LM . Helicobacter pylori: epidemiology and routes of transmission. Epidemiol Rev 2000;22 (2): 28397. Olson JW, Maier RJ. Molecular hydrogen as an energy source for Helicobacter pylori . Science 2002;298 (5599): 178890. Stark RM, Gerwig GJ, Pitman RS (1999). Biofilm formation by Helicobacter pylori . Lett Appl Microbiol 1999;28 (2): 1216. Chan WY, Hui PK, Leung KM, Chow J, Kwok F, Ng CS. Coccoid forms

5. 6.

7. 8. 9. 10.

11.

12. 13.

14.

15.

of Helicobacter pylori in the human stomach. Am J Clin Pathol 1994;102 (4): 5037. Pounder RE, Ng D. The prevalence of Helicobacter pylori infection in different countries. Aliment Pharmacol Ther 1995;9(2): 339. Everhart JE, Kruszon-Moran D, Perez-Perez GI, Tralka TS, McQuillan G. Seroprevalence and ethnic differences in Helicobacter pylori infection among adults in the United States. J Infect Dis 2000; 181 (4): 135963. Malaty HM. Epidemiology of Helicobacter pylori infection. Best Pract Res Clin Gastroenterol 2007;21 (2): 20514. Mgraud F. H. pylori antibiotic resistance: prevalence, importance, and advances in testing. Gut 2004;53 (9): 137484. Said HM (1969). Hamdard Pharmacopoeia of Eastern Medicine. Hamdard Foundation Karachi;12: 406. Krausse R, Bielenberg J, Blaschek W, Ullmann U. In vitro anti-Helicobacter pylori activity of extractum liquiritiae, glycyrrhizin and its metabolites. J Antimicrob Chemother 2004;54(1):243-6. Syed Faisal Haider Zaidi, Ikuko Yoshida, Farhana Butt, Muhammed Aasim Yusuf, Khan Usmanghani, Makoto Kadowaki and Toshiro Sugiyama. Potent Bactericidal Constituents from Mallotus philippinensis against Clarithromycin and Metronidazole resistant strains of Japanese and Pakistani Helicobacter pylori . Biol Pharm Bull 2009; 32 : 631-6. Warren JR, Marshall BJ. Unidentified curved bacilli on gastric epithelium in active chronic gastritis. Lancet 1983;1:1273-5. Vaezi MF, Falk GW, Peek RM. CagA-positive strains of Helicobacter pylori may protect against Barretts esophagus. Am J Gastroenterol 2000;95:220611. Mahady GB, Pendland SL, Yun G, Lu ZZ. Turmeric ( Curcuma longa) and curcumin inhibit the growth of Helicobacter pylori, a group 1 carcinogen. Anticancer Res 2002.; 22(6):4179-81. Nostro A, Cellini L, Di Bartolomeo S, Di Campli E, Grande R, Cannatelli MA, Marzio L, Alonzo V. Antibacterial effect of plant extracts against Helicobacter pylori. Phytother Res 2005;3:198-202.

H1N1 INFLUENZA ALERT In recent weeks several young women, mostly peripartum, and a young male have been admitted to three Karachi hospitals with acute lung injury (ARDS) and suspected or confirmed H1N1 influenza pneumonia. They all required ventilatory support. IDSP strongly recommends influenza vaccination, especially in pregnant women, and vigilance for lower respiratory involvement in all patients with influenza-like illness. Early diagnosis and treatment with oseltamivir or zanamivir is known to improve outcome.

242 . Infectious Diseases Journal of Pakistan

ORIGINAL ARTICLE

Drug Susceptibility Pattern of Typhoidal Salmonellae to the Conventional Anti-Typhoid Drugs; A Current Perspective
Anam Imtiaz *, Saba Abbasi*, Javaid Usman** *Student, Army Medical College, National University of Sciences and Technology (NUST), Rawalpindi, Pakistan. **Professor and Head of Department of Microbiology, Army Medical College, National University of Sciences and Technology (NUST), Rawalpindi, Pakistan.

Abstract Introduction Typhoid fever is an important public health issue in developing countries like Pakistan due to overcrowding and poor sanitary conditions. Moreover, development of antibiotic resistance has added to the problems. Objectives To study the current trends of typhoidal salmonellae susceptibility to the conventional anti-typhoid drugs. Material & Methods A descriptive cross-sectional study was carried out at the Microbiology Department of Army Medical College (National University of Sciences and Technology), Rawalpindi from January 2006 to December 2009. All the isolates were dealt with standard microbiological procedures. The antimicrobial sensitivity against the conventional anti-typhoid drugs was determined using Kirby-Bauer disc diffusion method as per the guidelines of Clinical and Laboratory Standards Institute. Results Out of 240 typhoidal Salmonellae isolated, 111 were Salmonella typhi and 129 were Salmonella paratyphi A. An increase in the percentage of multi-drug resistant isolates of typhoidal Salmonellae was found. Conclusion The susceptibility of typhoidal Salmonellae to the conventional anti-typhoid drugs has not improved. Key words Antibiotic Sensitivity, Anti-typhoid Drugs, Typhoid, Typhoidal Salmonellae. Corresponding Author: Anam Imtiaz, Army Medical College, National University of Sciences and Technology (NUST), Rawalpindi, Pakistan. E-mail: anam_ib@hotmail.com
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Introduction Typhoid is one of the major health problems of the developing world where it is responsible for serious morbidity1. Moreover, the widespread acquisition of plasmid mediated resistance against the conventional anti-typhoid drugs has added to the problem2. The resistance to chloramphenicol emerged in 1970s and the multidrug resistant strains came to notice in the late 1980s and early 1990s 3. In Pakistan, the first multi-drug resistant S. typhi was isolated in 1987 and by the end of 1990s isolation rates reached epidemic proportions leaving fluoroquinolones and the 3rd generation cephalosporins as the only treatment options 4, 5. Injudicious use of these drugs has lead to the emergence of resistance against quinolones as well 6-9. In recent years, there have been increasing reports of reversal towards sensitivity to the conventional anti-typhoid drugs from various parts of the world10,11. Such observations prompted us to conduct a study at our institute to determine the trend of susceptibility of typhoidal Salmonellae against the conventional drugs and help local therapeutic recommendations. Methods This study was carried out on typhoidal Salmonellae isolated from blood during January 2006 to December 2009 at the Department of Microbiology of Army Medical College, National University of Sciences and Technology (NUST), Rawalpindi. The blood culture samples were received from the wards of Military Hospital (MH), Rawalpindi; 1100-bedded tertiary care hospital. Five mL of venous blood was collected aseptically using a disposable syringe and added to 50mL of sterile Brain Heart Infusion broth (BHI) (Merck) from adults and 3mL blood in 30mL BHI from children. The top of the culture bottle was cleaned with iodine immediately before the addition of blood. The subcultures were done on MacConkeys agar (Oxoid, Basingstoke, UK) at 24 hr, 48 hr, and 5th and 7th days. Cultures showing no growth till seven days were considered as negative.
Oct-Dec 2010 . 243

The isolates were identified on the basis of morphology and biochemical tests using API 20E (Biomerieux, France) and were confirmed as Salmonella typhi or Salmonella paratyphi A by serological tests using standard antisera (Bio-Rad). Antimicrobial susceptibility testing was done by using chloramphenicol (30g disk), ampicillin (10 g disk) and cotrimoxazole (1.25/23.75 g disk) (Oxoid, UK) on MuellerHinton agar (Oxoid, UK) by the KirbyBauer disk diffusion method according to the guidelines of Clinical and Laboratory Standards Institute (CLSI 2006)12. The plates were incubated at 37C for 24 hours. Escherichia coli ATCC 25922 and Salmonella Typhi AMCOL (local strain) were used as controls. The data was analyzed using SPSS 17.0. Results During the study period, a total of 240 typhoidal Salmonellae were isolated; out of which 111 were Salmonella typhi and 129 were Salmonella paratyphi A. The mean age of the subjects was 33.7 years (range 1 to 90 years) with a male to female ratio of 17:1. There was an increase in the isolation rates of S. paratyphi A as compared to S. typhi from 2007-2009 (Fig 1). The antimicrobial resistance pattern of Salmonella typhi and Salmonella paratyphi A to the conventional anti-typhoid drugs is shown in Table 1 and 2 respectively.

Table 2: Resistance (%) pattern of Salmonella paratyphi A (n=129) to conventional drugs Antibiotic / Year 2006 (%) (n = 39) 5.1 7.7 12.8 2007 (%) (n=30) 16.7 20 20 2008 (%) (n=31) 16.1 12.9 12.9 2009 (%) (n=29 27.6 24.1 20.7

Chloramphenicol Ampicillin Cotrimoxazole

The resistance to the conventional anti-typhoid drugs increased over the four years (2006 to 2009) for Salmonella typhi. Even though a definite increase in the percentage of isolates sensitive to the conventional drugs occurred in 2007 for Salmonella paratyphi A, the percentage of resistant isolates rose subsequently. Discussion Significant variation is found in the resistance patterns of typhoidal Salmonellae in different regions of Pakistan. An interesting trend seen in our study was a decrease in the isolation frequency of typhoidal Salmonellae in the 4 years (from 81 in 2006 to 41 in 2009). The decreased isolation rates may be due to the practice of self medication and empiric antibiotic treatment in cases suspected of typhoid. Moreover, it was observed that the isolation rates of S. paratyphi A exceeded that of S. typhi in 2008 and increased in the subsequent years. This is concurrent to the reports by Anjum et al and Butt et al, of increasing incidence of enteric fever caused by S. paratyphi A in 2002 13,14. For Salmonella paratyphi A, the percentage of resistant isolates increased from 2006 to 2007. This percentage decreased in 2008 and a sudden surge was seen in the year 2009. The development of resistance by the bacteria is related to exposure to drugs. With time, the conventional anti-typhoid drugs were out of use for the treatment of typhoid fever and a reversal towards sensitivity was expected. However, 2009 witnessed a rise in the frequency of resistant isolates of both S. typhi and S. paratyphi A. Hassan et al have reported a steady increase in resistance to cotrimoxazole, ampicillin and chloramphenicol for S. typhi while a fall in resistance for S. paratyphi A 15. In contrast, another regional study has reported an increase in the sensitivity of the typhoidal Salmonellae to chloramphenicol 16. However, similar to our study, resistance to ampicillin and cotrimoxazole was reported to be increasing. In a study conducted in United States in 2009, only 13% of the 2016 isolates studied were found to be resistant to ampicillin, chloramphenicol and cotrimoxazole, which is in sharp contrast to our results 17.

Figure 1 : Frequency of Salmonella typhi and Salmonella paratyphi A from 2006-2009. Table 1: Resistance (%) pattern of Salmonella typhi (n= 111) to conventional drugs Antibiotic / Year 2006 (%) (n = 42) 40.5 42.9 45.2 2007 (%) (n=29) 51.7 51.7 51.7 2008 (%) (n=28) 57.1 46.4 57.1 2009 (%) (n=12) 66.7 75 66.7

Chloramphenicol Ampicillin Cotrimoxazole

244 . Infectious Diseases Journal of Pakistan

Conclusion The susceptibility of typhoidal Salmonellae to the conventional anti typhoid drugs has not improved enough to justify their empirical use. We would like to recommend a judicious use of second line drugs for the treatment of typhoid so that resistance could be contained. References
1. Ochiai RL, Acosta CJ, Danovaro-Holliday MC, Baiqing D, Bhattacharya SK, Agtini MD. A study of typhoid fever in five Asian Countries: disease burden and implication for controls. Bull World Health Organ 2008; 86(4): 260-8. Shanahan PM, Jesudason MV, Thomson CJ, Amyes SG. Molecular analysis of and identification of antibiotic resistance genes in clinical isolates of Salmonella typhi from India. J Clin Microbiol 1998; 36(6):1595-600. Rowe B, Ward LR, Threlfall EJ. Multidrug-Resistant Salmonella typhi: A Worldwide Epidemic. Clin Infect Dis 1997; 24(1): 180-1. Usman J, Karamat K.A, Butt T. Alarming state of emerging resistance in Salmonella Typhi to conventional antityphoid drugs in the Kharian region. JCPSP 1996; 6 (1): 30-2. Thaver D, Zaidi AK, Critchley J, Azmatullah A, Madni SA, Bhutta ZA. A comparison of fluoroquinolones versus other antibiotics for treating enteric fever: meta-analysis. BMJ 2009; 338:1865. Dimitrov T, Udo EE, Albaksami O, Kilani AA, Shehab EM. Ciprofloxacin treatment failure in a case of typhoid fever caused by Salmonella enterica serotype Paratyphi A with reduced susceptibility to ciprofloxacin. J Med Microbiol 2007; 56(2): 277-9 Threlfall EJ, Ward LR. Ward. Decreased susceptibility to Ciprofloxacin in Salmonella enterica serotype Typhi, United Kingdom. Emerg Infect Dis 2001; 7(4): 762-3. Wain J, Hoa NT, Chinh NT, Vinh H, Everett MJ, Diep TS, Day NP, Solomon T, White NJ, Piddock LJ, Parry CM. Quinolone-resistant Salmonella typhi in Vietnam: molecular

9.

10.

11.

2.

12.

3. 4.

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5.

14.

15.

6.

16.

7.

17.

8.

basis of resistance and clinical response to treatment. Clin Infect Dis 1997; 25:1404-10. Nkemngu NJ, Asonganyi ED, Njunda AL. Treatment failure in a typhoid patient infected with nalidixic acid resistant S. enterica serovar Typhi with reduced susceptibility to ciprofloxacin: a case report from Cameroon. BMC Infect Dis 2005; 549. Krishnan P, Stalin M, Balasubramanian S. Changing trends in antimicrobial resistance of Salmonella enterica serovar Typhi and Salmonella enterica serovar Paratyphi A in Chennai. Ind J Pathol Microbiol 2009; 52: 505-8. Dutta S, Sur D, Manna B, Bhattacharya SK, Deen JL, Clemens JD. Rollback of Salmonella enterica serotype Typhi resistance to chloramphenicol and other antimicrobials in Kolkata, India. Antimicrob Agent Chemother 2005; 49:1662-3. Wikler AM, Hindler JF, Patel JB, Bush K, et al. Performance standards for antimicrobial disk susceptibility tests; approved standard-tenth edition; 10th ed. Pennsylvania: CLSI document M02-A10; 2009. Anjum P, Qureshi AH, Parvez MH, Zahoor Ul Haq M, Hamid M. Increasing prevalence of multidrug resistant Salmonella enterica serotype Paratyphi A in patients with enteric fever. Pak J Med Res 2004; 43(2):56-9. Butt T, Ahmad RN, Salman M, Kazmi SY. Changing trends in drug resistance among typhoid Salmonellae in Rawalpindi, Pakistan. East Mediterr Health J 2005; 11: 1038-44. Hasan R, Zafar A, Abbas Z, Mahraj V, Malik F, Zaidi A. Antibiotic resistance among Salmonella enterica serovars Typhi and Paratyphi A in Pakistan (2001-2006). JIDC 2008;2(4): 289-94. Aggarwal A, Vij AS, Oberoi A. A three-year retrospective study on the prevalence, drug susceptibility pattern, and phage types of Salmonella enterica subspecies Typhi and Paratyphi in Christian Medical College and Hospital, Ludhiana, Punjab. J Indian Academy C Med 2007; 8(1):32-5. Lynch MF, Blanton EM, Bulens S, Polyak C, Vojdani J, Stevenson J, Medalla F, Barzilay E, Joyce K, Barrett T, Mintz ED. Typhoid Fever in the United States, 1999-2006. JAMA 2009 26; 302(8): 898-9.

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CASE REPORT

Central Nervous System ring enhancing lesions in an Immunocompromised Child with Status Epilepticus: A Case Report and Literature Review
Amna Batool*, Yawar Najam*, Ejaz Ahmed Khan*, Ismail A Khatri** *Department of Pediatrics and **Neurology, Shifa International Hospital, Islamabad.

Abstract In some developing countries like Pakistan the incidence of Human Immunodeficiency Virus infection in children is unknown and probably underreported . Children usually get HIV infection through vertical transmission and have progressively impaired cellular and humoral immunity. Children can have variable presentation from being asymptomatic to severe symptoms with any organ involvement. Central nervous system involvement is commonly seen in children with HIV infection and has a poor prognosis. Encephalopathy, seizures, status epilepticus, spastic weakness of extremities, microcephaly, developmental delay or regression, can be the presenting CNS features. We present case of a HIV child with status epilepticus history of recurrent infections and had ring enhancing brain lesions. Key words: Brain Lesions, Encephalopathy, HIV Infection, Status Epilepticus Introduction Global burden of HIV infection in children is substantial because of increasing number of infected women1-4. In Pakistan much lower number of cases are reported5-8. Vertical transmission is the most common route of acquiring infection in children. HIV infected children can have a diverse presentation with involvement of any system of the body. CNS involvement is quite common in children. Seizure or status epilepticus can be the presenting feature with or without CNS lesions with diverse etiology. CNS involvement is associated with a poor prognosis. We will review here CNS involvement in a case of pediatric HIV infection focusing on mass lesions. Case report A six-year-old boy presented with generalized tonic-clonic seizures and loss of consciousness for 5 hours. He received blood transfusion prior to referral from a local hospital where he had presented with lethargy, generalized weakness and anemia. Over the past few years, the child had multiple severe bacterial infections including recurrent pneumonias, suppurative Corresponding Author: Amna Batool, Chief Resident, Department of Pediatrics, Shifa International Hospital, Sector H-8/4, Islamabad. Email: dramnabatool@yahoo.com
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otitis media, and skin infections and was treated for pulmonary tuberculosis two years ago. His previous immune workup for recurrent infections had revealed IgG level of greater than 25 g/L on two occasions. There was no known history of previous transfusions or exposures to animals or birds. He was living in Pakistan with his grandparents since age of ten months and his parents were in South Africa. His father, a businessman, was of Pakistani origin and his mother was of African origin. No illnesses were reported in his parents except that his mother was being treated for tuberculosis for last two years in South Africa. On examination, he was a sick looking, frail child. His heart rate was 100/minute, respiratory rate 20/minute, blood pressure 100/60 mmHg and he was afebrile. His weight and height were at the 5th percentile. Neurological examination showed Glasgow Coma Scale of 4/15 and no signs of meningeal irritation. He was having generalized tonic clonic seizures on presentation. He had no lymphadenopathy and had only mild hepatomegaly. The skin showed multiple hyperpigmented macules, few scabbed lesions along with few round blisters and erythematous lesions on face, upper trunk and lower extremities (Fig 1). Rest of the systemic examination was unremarkable. His seizures were controlled using intravenous midazolam and phenytoin. The initial laboratory investigations showed white blood cell count of 12,800/L, hemoglobin 13 g/dL (polymorphs 86%,

Figure 1: Patients skin showing multiple hyperpigmented macules, few scabbed lesions along with few round blisters and erythematous lesions

lymphocytes 12%, monocytes 2%), total protein 8.8 g/dL, sodium 118 mEq/L, potassium 3.7 mEq/L, chloride 87 mEq/L, bicarbonate 21 mEq/L, glucose 100 mg/dL, creatinine 0.14 mg/dL and calcium 8.5 mg/dL. Serum immunoglobulin levels showed IgA 1.7 g/L, IgG 19.6 g/L, IgM 0.74 g/L. Liver function tests and cerebrospinal fluid analysis were normal. EEG showed moderate to severe generalized encephalopathy. Chest X-ray showed bilateral infiltrates and right upper lobe collapse/consolidation. Serum HIV ELISA test was positive which was confirmed by the Western Blot assay. Further evaluation showed negative Rapid Plasma Reagin, toxoplasma IgG and IgM, cytomegalovirus IgM but a positive cytomegalovirus IgG. His CD4+ count was 7 cells/L. Recurrent infections, hypergammaglobulinemia, a very low CD4+ count and a positive HIV antibody led to a diagnosis of AIDS encephalopathy with syndrome of inappropriate antidiuretic hormone (SIADH) secretion. He was managed with intravenous fluids, broad-spectrum antibiotics, anticonvulsants and mechanical ventilation. Axial flair images on MRI brain showed bilateral juxtacortical high signal lesions right >left in frontal and temporo-occipital areas (Fig 2). The multiple enhancing CNS lesions suggested opportunistic infection. Triple antiretroviral therapy (zidovudine, lamivudine and nevirapine) and high dose co-trimoxazole/trimethoprim was commenced immediately. Unfortunately before further diagnostic work-up could be done for the CNS lesions, the childs condition worsened and he died 3 days after initiation of antiretroviral therapy. Discussion

rates of 5-10%, 10-20% and 10-20% respectively3-5. The exact burden of HIV in Pakistan is not known. The USAIDS/WHO and National AIDS Control Program (NACP 2007) estimates that there are 84,000 cases of HIV with 3000-4000 needing treatment1. Pakistan officially has 3073 HIV cases including 332 AIDS cases reported by September 20051. Most reported cases are in the age group of 20-44 years, while males outnumber females by a ratio of 7:1. However, high risk groups exist within the Pakistani population that maintains a 10-20 fold higher prevalence of HIV6, 7. The HIV sero-prevalence of 0.2-11.5% has been reported among various high risk groups in Pakistan8. Under reporting is mainly due to the inadequacy of surveillance and testing systems, social stigmata attached to the disease as well as the lack of awareness among health practitioners and the general population. Once the HIV virus enters in human body it destroys CD4+ lymphocytes thus weakening the immune system and making HIV-infected children vulnerable both to pathogenic and opportunistic microorganisms. So a normal childhood illness will present as a severe, recurrent or persistent infection. Progression of HIV in children depends on age, time of acquisition, CD4+ count and viral load. This process may take several months to years, during which the child may remain asymptomatic. The definitive diagnosis of HIV requires laboratory confirmatory testing. The HIV antibody test is commonly used as a screening test and must be re-confirmed by other tests. Some of the tests used for laboratory diagnosis for HIV in children include: antibody tests like HIV IgG antibody tests, rapid tests; virologic tests like HIV PCR, HIV culture; and P24 antigen assay9-12. Primary CNS infection by HIV is quite common as it is a neurotropic virus. CNS involvement occurs in majority of HIV infected children13-16. CNS involvement is atleast 3 times more common in children than in adults14. Risk factors for involvement of CNS in HIV infected children include: age <2 years, severity of HIV related systemic features and the degree of depression of helper T cells. CNS involvement is mediated mainly by the direct cytotoxic effect of the virus as well as through the infected monocytes and macrophages. Over production of various cytokines including tumor necrosis factor leads to neurological manifestations observed. The neurological manifestations of HIV infection include: progressive encephalopathy, static encephalopathy, seizures, strokes, HIV myopathy and myelopathy, peripheral neuropathy, developmental delay or regression, psychiatric manifestations and sleep problems. Our patient presented with status epilepticus, encephalopathy and CNS enhancing lesions, which probably implied severe
Oct-Dec 2010 . 247

Figure 2: Axial flair images on MRI brain showing bilateral juxtacortical high signal lesions (right >left) in frontal and temporo-occipital areas. Globally the number of children (<15 years) living with HIV was estimated to be more than 2,000,000 in 2007, new infections >350,000, and 270,000 deaths; more than 90% occurring in Sub-Saharan Africa1,2. Importantly, global estimates of HIV in children continues to steadily increase as compared to adults. It is estimated that 90% children acquire the infection during pregnancy period, delivery or breastfeeding with transmission
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HIV CNS involvement with a secondary opportunistic infection. HIV encephalopathy ( HIVE) is the commonest CNS manifestation of HIV infection in children with 50-90% having clinical or radiologic evidence of encephalopathy at some stage14, 15. Presentation is early and approximately 10% children with HIVE present within their first year of life15. Patients can have developmental delay, microcephaly, pyramidal tract signs and spasticity. Risk factors for development of HIVE include high viral load (>5 log10 copies/mL) and lower CD8+ T-lymphocyte percentage (<20% CD8+ T-lymphocytes), presence of hepatomegaly, splenomegaly, or lymphadenopathy in the first 3 months of life17. More than 50% patients die within 3 years of diagnosis with median survival of about 11 months from the time of diagnosis15. Neuro-imaging in HIVE shows white matter disease and cerebral atrophy. White matter lesions are typically seen within the periventricular white matter. On MRI, these lesions appear as low signal on T1 weighted sequences and high signal on T2 weighted sequences18-20. White matter disease may be associated with basal ganglia or white matter calcification. There may be finding of a necrotizing encephalopathy with an associated cardiomyopathy 18 . It is associated with poor prognosis. Seizures are frequently observed as CNS manifestations21, 22. Status epilepticus is also observed quite often and seen in some 8-18% HIV infected children21. It may be caused by direct HIVE, cerebral mass lesions, focal neurological lesions, meningitis, and encephalitis. Electrolyte abnormalities, metabolic disturbances like hyponatremia, hypomagnesaemia and renal failure all lead to increased risk of status epilepticus. It is associated with poor outcome. Our patient presented with status epilepticus, persistent severe hyponatremia and CNS lesions. Generally about 50% of the neurological disorders in HIV infected children are due to cerebral mass lesions. These mass lesions can be of three types i.e. opportunistic infection, CNS neoplasm and cerebrovascular disease. Opportunistic infections include toxoplasmosis, cryptococcosis and cytomegalovirus infection22. The most common is toxoplasmosis, it is seen in up to 50% of patients in Europe and Africa20. Up to 40% of patients with cerebral toxoplasmosis present with seizures. CT scan brain and anti-toxoplasma antibody titer are useful diagnostic aids. CT scan brain shows ring-enhancing lesions that improve on treatment as confirmed by repeat scans. Early and prompt treatment with sulfadiazine and pyrimethamine is associated with rapid recovery. The second most common etiology of cerebral mass lesion is primary CNS lymphoma though generally intracranial neoplasms in HIV infected children are rare23. This mostly presents with seizures and neuroimaging is diagnostic. Involvement of corpus callosum, exclusive white matter involvement, periventricular location and subependymal spread are common distinguishing
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features24. These lesions appear isodense or hyperdense on CT scan and T1 weighted MRI images, whereas they are hypodense on T2 weighted MRI images24, 25. Proton magnetic resonance ( 1 H-MR) spectroscopy and fluorodeoxyglucose positron emission tomography (FDG-PET) are recently used modalities for differentiating focal CNS lesions, in particular lymphoma, from infections such as toxoplasmosis. Both techniques are more sensitive and specific than MRI alone26. Tuberculomas, tubercular abscesses, syphilitic abscesses, nocardia abscesses, cryptococcal abscess and cerebrovascular accidents (CVA) with accompanying edema are some other causes of intracranial mass lesions that present with seizures Our child had multiple ring enhancing lesions which could be of infectious origin such as toxoplasmosis, tuberculosis or fungal. Definitive diagnosis could not be made as the child died within few days of presentation. An HIV infected child who has CNS involvement with ring enhancing lesions requires emergent care. Management of children with neurological involvement needs to be comprehensive; intensive care with ventilatory support, close monitoring of fluids and electrolytes, anticonvulsants and broadspectrum antibiotics should be instituted. Further specific therapy such as antiviral, antituberculous and antiparasitic drugs must be given with additional supporting evidence with low threshold. Antiretroviral therapy must be given accordingly as soon as possible for optimal outcome. Conclusion HIV should be considered in a child with history of repeated infections presenting with status epilepticus or encephalopathy and unexplained MRI abnormalities. Prognosis is generally poor. Reference
1. UNAIDS. 2008 Report on the global AIDS epidemic. Joint United Nations Programme on HIV/AIDS. Available at: h t t p : / / w w w. u n a i d s . o r g / e n / K n o w l e d g e C e n t r e / H I V D a t a / GlobalReport/2008/2008_Global_report.asp. Amy Sarah Ginsburg, Anna Miller, Catherine M. Wilfert. Diagnosis of Pediatric Human Immunodeficiency Virus infection in resource constrained settings. Pediatric Infect Dis J 2006; 25: 105764. Piwoz EG, Ross JS. Use of Population-Specific Infant Mortality Rates to Inform Policy Decisions Regarding HIV and Infant Feeding. J Nutrition 2005; 135: 11139. De Cock KM, Fowler MG, Mercier E, de Vincenzi I, Saba J, Hoff E, Alnwick D, Rogers M, Shaffer N. Prevention of mother-to -child HIV transmission in resource-poor countries: translating research into policy and practice. JAMA 2000; 283: 117582. Hamzullah Khan, Balqis Afridi, Tahniat Ishaq. HIV/AIDS transmission from mother to child with special attention toward transmission through breast-feeding. Rawal Med J 2007; 32(1): 7981. Alia Zaidi, Waheeduz Zaman Tariq, Kashan Abidi Haider, Liaqat Ali, Abdus Sattar, Farah Faqeer, Saeedur Rehman. Seroprevalence of hepatitis B, C and HIV in healthy blood donors in Northwest of Pakistan. Pak J Pathol 2004; 15 (1): 11-6. Yasmin Bhugri. HIV/AIDS in Pakistan. JPMA 2006; 56 (1): 3-4.

2.

3.

4.

5.

6.

7.

8. 9.

10. 11. 12.

13.

14.

15.

16.

Members of the HIV seroprevalence study group of Pakistan. HIV Seroprevalence in Pakistan. AIDS 1996; 10(8): 9267. Ng KP, Saw TL, Baki A, Kamarudin R. Evaluation of three commercial rapid tests for detecting antibodies to human immunodeficiency virus. Med J Malay 2003;58(3):454-60. Hoguin A. Evaluation of three rapid tests for detection of antibodies to HIV-1 non-B subtypes. J Virol Methods 2004; 115(1):105-7. World Health Organization: HIV assays: operational characteristics (phase 1). 2002; Report 13. Wilkinson D, Wilkinson N, Lombard C, Martin D, Smith A, Floyd K, Pallard R. On-site HIV testing in resource-poor settings: is one rapid test enough? AIDS 1997:11(3);377-81. Grubman S, Oleske J. HIV infection in infants, children, and adolescents. In: AIDS and Other Manifestations of HIV Infection. Ed. Wormser GP. 3rd edn. Philadelphia, Lippincott-Raven Publishers, 1998; pp 349 - 71. Tardieu M, Le Chenadec J, Persoz A, Meyer L, Blanche S, Mayaux MJ. HIV-1-related encephalopathy in infants compared with children and adults. French Pediatric HIV Infection Study and the SEROCO Group. Neurology 2000; 54: 1089-95. Angelini L, Zibordi F, Triulzi F, Cinque P, Giudici B, Pinzani R, et al. Age dependant neurologic manifestations of HIV infection in children. Neurol Sci 2000; 21:13542. Vardhaman S. Udgirkar, Milind S. Tullu, Sandeep B. Bavdekar,Vijayalaxmi B. Shaharao, Jaishree R. Kamat, Priya R. Hira. Neurological manifestations of HIV infection. Ind Pediatr 2003; 40:230-4.

17. Gurbindo D, Resino S, Sanchez-Ramon S, Leon JA, Munoz-Fernandez MA. Correlation of viral load and CD8 T-lymphocytes with development of neurological manifestations in vertically HIV-l-infected infants. A prospective longitudinal study. Neuropediatr 1999; 30: 197-204. 18. Haller JO, Cohen HL. Paediatric HIV infection: an imaging update. Pediatr Radiol 1994;24:22430. 19. Jeanes AC, Owens CM. Imaging of HIV disease in children. Br J Radiol 2002 14:8-23. 20. Report of the quality standards subcommittee of the American Academy of Neurology. Evaluation and management of intracranial mass lesions in AIDS. Neurology 1998; 50:21-6. 21. Holtzman DM, Kaku DA, So YT. New-onset seizures associated with human immunodeficiency virus infection: causation and clinical features in 100 cases. Am J Med 1989; 87:1737. 22. Van Paesschen W, Bodian C, Maker H. Metabolic abnormalities and new onset seizures in human immunodeficiency virus-seropositive patients. Epilepsia 1995; 36(2):146150. 23. Owens CM, Allan R, Thomas K, Evans J, Stevens J. The radiological spectrum of vertically-acquired HIV infection. Br J Radiol 1996;69:77782. 24. Provenzale JM, Jinkins JR. Brain and spine imaging findings in AIDS patients. Radiol Clin North Am 1997;35(5):112766. 25. Lee SH, Rao KCVG, Zimmerman RA. Primary tumours in adults. In: Cranial MRI and CT. 3 rd edn. McGraw-Hill, 1992: 295380. 26. Safriel YI, Haller JO, Lefton DR, Obedian R. Imaging of the brain in the HIV-positive child. Pediatr Radiol 2000; 30:72532.

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CASE REPORT

Gelatinous Bone Marrow in AIDS

Salman Saleem*, Mehreen Ali Khan**, Ayesha Hafeez**, Aamer Ikram**, Usman Rathore** *Department of Medicine, Combined Military Hospital, Gujranwala **Department of Pathology, Combined Military Hospital, Quetta
Abstract Bone Marrow aspiration is done in patients of AIDS to assess peripheral dysplasias/pancytopenia and to diagnose bacterial/ fungal infections. Gelatinous transformation of bone marrow is relatively uncommon in HIV infection but if seen in a pancytopenic patient it may be an indication of HIV infection. We report a case of pyrexia of unknown origin whose blood picture revealed pancytopenia. Bone marrow aspiration revealed a gelatinous bone marrow. Based on this finding, anti-HIV antibodies tested by enzyme linked immunosorbant assay turned out to be positive. Keywords Acquired Immunodeficiency Syndrome, Gelatinous Bone Marrow, Pancytopenia. Introduction Gelatinous bone marrow transformation is a rare disorder of unknown pathogenesis having fat cells atrophy, focal loss of haemopoietic tissue and deposition of extracellular gelatinous material. Different diseases are characterized by gelatinous bone marrow transformation and AIDS is among major causes for this change. Case Report A 45-years-old male presented to Department of Medicine, Combined Military Hospital Gujranwala with history of fever and bone pains for three months. The fever was low grade and intermittent in nature. He was having severe bone pains especially limbs. He consulted many doctors in the city but remained undiagnosed. He had been working in UAE in past. On examination, he was febrile and underweight but he denied any weight change during last six months. No unusual lymphadenopathy was noticed. On investigations, his blood complete picture revealed pancytopenia (TLC 3.4x 109 /L; Hb 6.4g/dL; Platelet 48x109/L; Reticulocytes 0.2%). Repeated slides for malarial parasite were negative. His LDH and alkaline phosphatase were raised. Serum bilirubin, ALT, urea, creatinine, electrolytes were within normal limits. No abnormality was detected on X-rays chest. Meanwhile his blood culture revealed Corresponding Author: Mehreen Ali Khan, Department of Pathology, Combined Military Hospital, Quetta. Email: mehreen35@hotmail.com
250 . Infectious Diseases Journal of Pakistan

growth of Acinetobacter baumannii and he was put on antibiotics according to the sensitivity report. However, his fever did not settle even after appropriate antimicrobial therapy for two weeks. Bone marrow aspiration was done from posterior superior iliac spine. The aspirated marrow was jelly like in appearance and difficult to spread over slides. After Giemsa and Leishmans staining, the marrow was reported to be hypocellular with fat spaces and gelatinous transformation. The patient refused to have trephine biopsy due to his severe pain. Review of literature revealed that gelatinous transformation of marrow had been identified in different disorders including AIDS. Patient was reinvestigated and anti-HIV antibodies were positive by ELISA. The diagnosis was confirmed by Western Blot assay. Patient was advised further treatment (HAART) but he preferred to go abroad for further consultation and also he didnt want to disclose his ailment to his family. Discussion Infection with HIV, a retrovirus of lentivirus subgroup, is a frequent cause of immune deficiency giving rise to a wide range of symptoms, opportunistic infections and malignancies1. The virus is transmitted by blood, body fluids, semen and breast milk. The majority of infections occur during heterosexual intercourse. The disease still remains checked in our country due to social and religious obligations. Infection from blood and blood products is possible as screening practices are not strictly adopted in most parts of the country. The official figure was 3073 HIV infected cases in 2005. Around 84,000 cases of HIV were estimated in Pakistan in 2007, according to the UNAIDS and National AIDS Control Program.2 These figures still remain low because of under-diagnosed to undiagnosed cases due to non-availability of expertise or diagnostic modalities. In our country AIDS is a social taboo and patients dont give history suggestive of AIDS. This patient had multiple consultations and investigations for fever but anti-HIV antibodies were not considered previously. Gelatinous bone marrow transformation shows loss of haemopoietic cells, atrophy of fat cells and deposition of gelatinous substance extracellularly3,4. Gelatinous bone marrow is not a specific disease but presence of gelatinous marrow

indicates that patient is having severe generalized illness. AIDS and few other diseases are associated with gelatinous bone marrow transformation like malignancies, alcoholism, malabsorption and anorexia nervosa. Gelatinous bone marrow in seropositive cases of HIV has been studied previously4-6. Mehta et al reported that 29% of the patients with AIDS had gelatinous marrow. Delactretaz observed 38% of HIV infected patients had gelatinous bone marrow6. The most common symptoms of bone marrow necrosis include bone pains (78%), fever (68%), anemia (91%), thrombocytopenia (73%), leucopenia (45%), raised LDH (51%) and raised alkaline phosphatase (40%)7. Our patient had severe bone pains with pancytopenic picture and raised LDH and alkaline phosphatase. However, diagnosis was pointed towards AIDS only after the bone marrow findings. Conclusion Gelatinous bone marrow transformation is an important feature of HIV infection. Sera of all the patients having gelatinous

change in the marrow must be tested for anti-HIV antibodies to establish early diagnosis and prompt treatment. References
1. The White Cells 2: Lymphocytes and their benign disorders. In: Hoffbrandt AV, Pettit JE, Moss PAH, editors. Essential Hematology. 4th ed. Italy: Black Well Science; 2000: p 139. UNAIDS. 2008 Report on the global AIDS epidemic. Joint United Nations Programme on HIV/AIDS. Available at: http://www.unaids.org/en/KnowledgeCentre/HIVData/GlobalReport/ 2008/2008_Global_report.asp Bohm J. Gelatinous transformation of the bone marrow, the spectrum of underlying disease. Am J Surg Pathol 2000;24(1) 56-65. Mehta K, Gascon P, Robby S. The gelatinous bone marrow (serous atrophy) in patient with acquired immunodeficiency syndrome. Evidence of excess sulfates glycosaminoglycan. Arch Pathol Lab Med 1992; 116(5) 504-8. Marche C, Tabbara W, Michon C, Ckair B, Bncaire F Metthiessen L Bone marrow findings in HIV infection a pathological study. Prog AIDS Pathol 1990;2:51-60. Delacretaz F, Perey L, Schnidt PM, Chave JP, Costa J. Histopathology of bone marrow in human immunodeficiency virus infection. Virchows Arch Pathol Anat Histopathol 1987; 411(6):543-51. Janssens AM, Offner FC, Van Hove WZ. Bone marrow necrosis. Cancer 2000;88(8): 1769-80.

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NEWS & VIEWS

Update on Infectious Diseases for the Practicing Clinician October 18th to 23rd 2010 IDSP conducted a course Update on Infectious Diseases for Practicing Clinician, in Karachi. Dr Farheen Ali was Convenor and Dr Fatima Noman Co-Convenor. This is a brief report regarding the feedback that we received from the participants and the issues we came across during the course. Feedback from participants 1. Overall the course was very much appreciated. Most participants agreed that the knowledge they gained was valuable in daily practice. 2. Most requested and stressed that IDSP must continue such courses and make it an annual event. 3. There was a request to include and emphasize emerging infections. 4. There were suggestions to include: a. Nosocomial infections b. TB in pediatric patients c. HIV d. Bedside/hands-on session for certain topics 5. There were requests to distribute Antibiotic Guidelines. 6. The participants appreciated the handouts given; CDs were requested by a few. 7. Most were comfortable with afternoon timings. 8. The evaluation sheet was not properly understood by many. (We should make it simpler in future and in place of 1-5 ranking give actual levels Unsatisfactory to Excellent). Our Feedback and Suggestions The course should definitely be an annual event, as it provides the opportunity to update and educate the practicing physician regarding common and emerging clinical problems. However, the scope should be broadened to include post-graduate residents and fellows as well. It should probably be held around the same time each year, so as to be a marked calendar event for clinicians. We did, however, come across a few issues while putting together this course. We should make a more concerted effort to get handouts prior to the lectures so that participants have them on time. CDs although cost-effective may not be feasible because of the concern that lectures may be re-used without permission. We felt that there was a need for more secretarial help and support. Correspondence and reminders should have been generated timely. Overall the program was well received. We should be able to make this a yearly event. Dr. Farheen Ali
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Emergency Room Conference (ERCON) October 30, 2010: Islamabad Emergency Room Conference (ERCON) was arranged by Organization of ER Physicians. Members of IDSP participated in the conference. Dr. A. Salam Khan, Convenor of the ERCON introduced and welcomed participants. Dr. Naseem Salahuddin explained why she believed that rabies was an orphan disease in Pakistan. Rabies is a common but fatal disease that has never been given its due importance by the health authorities and no attempt has ever been focused on its preventive aspect. The obsolete nerve tissue vaccine continues to be produced and distributed to Government run centers and results in rabies deaths. Dr Seemin Jamali, Director of Emergency Services at JPMC, Karachi, spoke on prevention and treatment of animal bites. Despite JPMC being a Federal Government institute, it has for over a decade obtained modern tissue culture vaccine rather than the NIH provided sheep brain vaccine. Thai Red Cross intradermal regimen is both economical and effective in rabies prophylaxis. Dr Mehmood Javed, ID Consultant at Shifa International Hospital presented a concise lecture on Viral Hemorrhagic Fever and Influenza, and emphasized their preventive aspects. Dr Altaf Ahmad, President IDSP and Director of Lab Services at The Indus Hospital, Karachi, expounded on the importance of Biosafety and Biosecurity measures as being essential procedures in the laboratory for protection of personnel as well as for the environment and community.

8th Annual Conference on Infectious Disease on 11-12th March 2011 The IDSP is organizing the 8th Annual Conference on Infectious Disease on 11-12th March 2011 at Hotel Margalla, in Islamabad. We cordially invite you to participate in the event with your colleagues, trainees and students. We welcome all physicians, surgeons, trainee doctors, nurses and technicians, as they are an essential part of management and prevention of infectious diseases. Ministry of Health and related National Programmes such TB, Malaria and HIV; National Institute of Health; UNICEF; and WHO would also be participating. The theme of the conference is THE AGE OF SUPERBUGS: FACTS AND SOLUTIONS with special emphasis on Antibiotic resistance Viral Hemorrhagic fevers Disaster related infections MDR Tuberculosis HIV/AIDS threats

Zoonosis Nosocomial infections Infections in immunocompromised hosts Biosafety in our Labs Rational use of antibiotics

IDJ over Internet Sincere efforts are being made to ensure easy availability of IDJ over internet in collaboration with Asianet. Hopefully the requirements would be completed in the near future. Editor IDJ

For further information please contact: Dr. Altaf Ahmed Dr. Ejaz A. Khan President IDSP General Secretary IDSP altafvirus@yahoo.com ejazkhan99@hotmail.com

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INSTRUCTIONS FOR AUTHORS

Instructions for Authors

Scope The Infectious Diseases Society of Pakistan sponsors the Infectious Disease Journal of Pakistan (IDJP). The Journal accepts Original Articles, Review Articles, Brief Reports, Case Reports, Short Communications, Letter to the Editor and Notes and News in the fields of Microbiology, Infectious Diseases; with laboratory, clinical or epidemiological aspects. Criteria for publication All articles are peer reviewed by the IDSP panel of reviewers. Authors may also submit the names and contact information of two persons who potentially could serve as unbiased and expert reviewers for their manuscript, but IDSP reserves the right of final selection. Submission of the Manuscript Manuscripts must be formatted according to submission guidelines given below, which are in accordance with the Uniform Requirements for Manuscripts Submitted to Biomedical Journals (originally published in N Engl J Med 1997; 336:309-15). Please submit the manuscript and all enclosures to: The Editor, Infectious Diseases Journal of Pakistan, Department of Pathology, Combined Military Hospital, Quetta, Pakistan. Electronic copy of the manuscript must also be sent to maahin1@yahoo.com. All manuscripts submitted to IDJP must be accompanied by an authorship declaration stating that The authors confirm that the manuscript, the title of which is given, is original and has not been submitted elsewhere. Each author acknowledges that he/she has contributed in a substantial way to the work described in the manuscript and its preparation. Manuscript Categories Original Articles Articles should report original work in the fields of microbiology and infectious diseases. Title page This should list title of the article, full names of each author with highest academic degree(s), institutional addresses and email addresses of all authors. Corresponding author should also be indicated with his/her name, address, telephone, fax number and e-mail address. A short running title of not more than 40 characters (including letters and spaces) be placed at the foot end of the title page. Abstract Abstract should not exceed 200 words and must be structured in to separate sections. Avoid abbreviations or citing references
254 . Infectious Diseases Journal of Pakistan

in the abstract. Key Words Four to five standard key words must be provided. Introduction The section must clearly state the background and importance of the research along with objectives. Materials and Methods Mention the place and duration of study. design of study and details of interventions used must be clearly described. Provide details of subject selection (patients or experimental animals). Details must be sufficient to allow other workers to reproduce the results. Identify precisely all drugs and chemicals used, including generic name(s) and route(s) of administration. Results Present results in logical sequences in the text, tables and illustrations. Articles can have a maximum of five illustrations (in a combination of figures and tables) per article. Discussion Emphasize important aspects of the study and compare with other studies. Do not repeat the details from the results section. Discuss the implications of the findings and their limitations. Link the conclusions with the goals of the study but avoid unqualified statements and conclusion not completely supported by your data. Acknowledgments Acknowledge any sources of support, in the form of grants, equipment or technical assistance. Review Articles Authoritative and state of the art review articles on topical issues are also published, with a word limit of 2000. These should be comprehensive and fully referenced. Articles should contain Abstract, main mext divided into sections, Conclusions and References. Brief Reports Short clinical and laboratory observations are included as Brief Reports. The text should contain no more than 1000 words, 2 illustrations or tables and up to 10 references. Case Reports Instructive cases with a message are published as case reports. Routine syndromes or rare entities without unusual or new features are invariably rejected. The text should contain no more than 1000 words and 2 illustrations or tables. The authorship should not exceed 4 persons. Letter to the Editor These may relate to material published in the IDJP, topic of

interest pertaining to infections diseases, and/or unusual clinical observations. A letter should not be more than 300 words, one figure and 3-5 references. News and Views Pertinent news and updates in infectious diseases from around the world (approximately 200 words). Notices Announcements of conferences, symposia or meetings may be sent for publication at least 12 weeks in advance of the meeting date. Details of programs should not be included. References Number references in the order in which they are first cited in the text. Label references in text, tables and legends by Arabic numerals (in superscript). References cited only in tables or in legends to figures should be numbered in accordance with a sequence established by the first identification of the particular table or illustration. Write all authors, complete title, journal name (standard abbreviation in italics), year, volume, issue, page numbers according to Uniform Requirements of Manuscripts submitted to Biomedical Journals, as cited in N Engl J Med 1997; 336:309-15. Tables and Figures Data reported either in a table or in a figure should be illustrative of information reported in the text, but should not be redundant with the text. Table should be numbered consecutively in Arabic numerals. Tables and Figures legends should be self-explanatory with adequate headings and footnotes.

Illustrations Illustrations should be numbered and given suitable legends. If possible, figures should be submitted in electronic format as either a TIFF (tagged image file format) or JPEG format. Minimum resolution for scanned artwork is: Black & White Illustration Line, e.g. graphs: 600 dpi Halftone, e.g. photographs: 300 dpi Coloured illustrations: 400dpi (images should be split CMYK) Ethical Guidelines The research project must be reviewed by Ethical Committee of the institute. All clinical research articles/studies involving human subjects submitted to the IDJP must adhere to ethical guidelines of their institutions and have informed consent from their patients. All scientific research that uses animals in their study protocols must include a statement on the ethical treatment of animals during the study. Financial support: All authors must disclose any financial support they have received during the course of the study. Conflict of Interest: If there is any conflict of interest then this must be disclosed by the author(s) at the end of the article. Plagiarism Make sure that your article is not copied or reproduced. Plagiarism in research is a crime and the matter will be taken seriously. Instructions updated - December 2010. Editor IDJ

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