JIIMC March 2016.cdr ORIGINAL ARTICLE ABSTRACT Objective: The objective of this study was to study the demographics of intestinal parasitic infections and find out the relative frequency of different intestinal parasites in clinical samples received for routine stool examination from symptomatic patients with diarrhea and/or dysentery. Study Design: A Retrospective cross sectional study. Place and Duration of Study: The study was conducted at Dow Diagnostics Research and Reference Laboratory, Karachi, Pakistan from January 2008 to December 2013 of stool analysis from the patients with symptoms of diarrhea and/or dysentery. Materials and Methods: Microscopic stool examination was performed using direct samples by wet mount under low as well as high power bright field magnification. Tabulations were performed using Microsoft Excel while Chi-square test was applied using Open-epi software. Results: Among the 1815 positive cases infected with single parasites, Entamoebahistolytica was the most commonly found parasite, followed by Giardia labmlia, Blastocystishominis, Ascarislumbricoides and Hymenolepis nana. Among these patients, 1081 (59.6%) were males and 734 (40.4%) were females. Chi-square test was applied using open-episoftware, which showed significant differences among males and females (p=0.03). Conclusion: Protozoal intestinal parasites Entamoebahistolytica and Giardia lambliaare the commonest among all age groups followed by the helminths Ascaris lmbricoides and Hymenolepis nana. Other notable parasites include Entamoeba coli and Taenia species. Key Words: Intestinal Parasites, Parasitic Infections, Entamoeba Histolytica, Giardia Lamblia. 5 in children, physical and mental health problems 6 and diarrheal diseases, which are a major cause of death in Pakistan India, Bangladesh and other 7 developing countries. Previous population based reports from Pakistan show Giardia lamblia to be the most common 8 parasite in Karachi followed by Ascaris lumbricoides 9 and Entamoeba histolytica , though the reports from Bannu and Peshawar (cities situated in Northern Pakistan with a comparatively cold climate) showed Ascaristo be the commonest parasite infecting the school children followed by Enterobiusvermicularis 1 0 , 1 1 a n d H y m e n o l e p i s n a n a . R e p o r t s f r o m Muzaffarabad, a city located further North showed Giardia to be the commonest followed by E. 12 histolytica. Karachi has a semi-arid warm and humid climate and it is home to a number of infectious parasites. Most common parasites reported up to date are Entamoeba species, Ascaris, H . n a n a , G i a r d i a , T. t r i c h u r a a n d Ta e n i a 8,9,13 species. However, the present studies are either 9 old or do not cover the age groups other than 8,13 children, which is also affected by the parasitic infections and add a considerable bulk to the disease Introduction Parasitic infections are a major public health issue in developing world, which affects around 3.5 billion 1 2 peopledirectly and indirectly. They are mostly endemic and specific parasites are prevalent in particular areas, therefore disease pattern is different in different geographical settings. These diseases are associated with climatic conditions, poor sanitation, unsafe drinking water and lack of personal hygiene in tropical and sub-tropical 1,3 countries. They are a major cause of morbidity and mortality and are responsible for chronic diseases 4 such as iron deficiency anemia, growth retardation Demographics of Intestinal Parasitic Infections in Karachi: An Insight from Positive Stool Samples 1 2 3 3 4 5 Muhammad Yahya Noori , Ghazala Khan , Ishrat Abid , Samina Baig , Sambreen Zamir , Shaheen Sharafat Parasitic Intestinal Infections in KarachiJIIMC 2016 Vol. 11, No.1 Correspondence: Dr. Muhammad Yahya Noori Assistant Professor, Pathology Dow International Medical College Dow University of Health Sciences, Karachi E- mail: yahyanoori@gmail.com 1,3,5,6 4 Department of Pathology/Microbiology Dow International Medical College Dow University of Health Sciences, Karachi 2 Faculty of Pharmacy University of Karachi, Karachi Funding Source: NIL ; Conflict of Interest: NIL Received: November 03, 2015; Accepted: February 16, 2016 20 burden. Therefore, a retrospective study was performed to assess the relative disease burden of different parasites across different demographic groups, so that a snapshot can be generated regarding relative frequency of different parasites in symptomatic patients. To study the relative frequency of these parasites in samples from patients coming with diarrhea and/or dysentery results from laboratory records were analyzed for the period of 2008-2013 and were stratified according to age and gender. Materials and Methods The retrospective cross-sectional study based on the laboratory records was carried out in the Dow Diagnostic Research and Reference Laboratory of Health Sciences Karachi from January 2008 to December 2013. All positive stool samples received for routine stool examinations were included in the study, while those which were negative for parasites, ova or cysts were excluded. A total of 1873 positive samples were included in data analysis. Stool samples were collected in sterilized leak proof containers from patients after detailed informed consent. Departmental approval was taken for the study,diagnostic data were de-identified and tabulated without patients' particulars. Each sample was initially examined physically for consistency, color, odor, blood and mucus. Slides were then prepared directly for wet mount by placing a drop of fresh physiological saline at one end of the clean side, while a drop of iodine was placed at the other end. Stool samples were mixed with wooden stick and a small amount was then taken for saline and iodine and covered with cover slip. These were microscopically examined under low (10× = 100 times magnification) and high (40× = 400 times magnification) power bright field for worms, ova, larvae, trophozoites, cyst, erythrocytes, leukocytes and any intestinal mucosal sloughing. Diagnosis was made by observing the differentiating morphological features of protozoal cysts and trophozoites and parasitic ova and larvae. For Entamoeba histolytica, cyts were identified on the basis of presence of four or lesser number of nuclei, while trophozoites were identified as having ingested RBCs and differentiated from Entamoeba coli on the basis of more than five nuclei in cysts and trophozoites without RBCs. Iodamoeba butschlii was identified on the basis of cellular morphology and presence of a single nucleus in Iodine preparation. Blastocystis hominis was identified on the basis of cyst-like forms having a large central body resembling like a large vacuole surrounded by small, multiple nuclei. Ova of Hymenolepis nana were identified on the basis of appearance as they are round to oval with two distinct walls and having 4-8 hair-like processes at each pole on the inner aspect along with three pairs of hookletswithin the inner wall. Ova of Ascaris were identified the basis of yellow brown color with thick shells having an albuminous outer coat and thick transparent wall under the shell. Tabulations were performed using Microsoft 14 Excel while Chi-square test was applied using Open- 15 epi software. Table I: Gender-wise propor�on of infec�ons in pa�ents detected using stool examina�on Table II: Gender-wise propor�on of infec�ons in pa�ents using stool examina�on stra�fied by age Parasitic Intestinal Infections in KarachiJIIMC 2016 Vol. 11, No.1 21 Results Among the 1815 positive cases infected with single parasites, 1081 (59.6%) were males and 734 (40.4%) were females. E. histolytica was the most commonly found parasite in these patients, followed by G. labmlia, B.hominis, A. lumbricoidesand H. nana. A chi-square test was appliedusing Open-episoftware, which showed significant differences among the two groups (p=0.03). Data were stratified according to age groups and gender. It was observed that most commonly affected group was adult males, and altogether 1081 males were found to be mono-infected, most commonly with E. histolytica(n=400) followed by G. lamblia(n=369) and B.hominis (n=120). Similar p atte r n wa s o b s e r ve d i n fe m a l e s a n d E . histolytica(n=321) G. lamblia(n=211) and B. hominis (n=95) were found to be the most common parasites infecting adult females. However in patients, who were three years of age, G. lamblia was found to be the most common parasite, in both males (n=47) and females (n=39). In children, the most common parasites were again Giardia and E. histolytica, though trends were different in males and females as in males Giardia(n=157) infection ra te wa s d e te c te d to b e h i g h e r t h a n E . histolytica(n=114), though in females both infections were found in equal frequency (n=88). Some of the patients were found to be infected with two parasites (n=52), while very few were triply infected (n=6). Organisms found in these cases were mostly G.iardialamblia, E. histolytica, H. nana, E. coli, I. butschlii and A. lumbricoides. Discussion The study found a number of parasitic agents more common in the clinical samples received in Karachi. In our results protozoans like E. histolytica and G. lamblia are among the commonest finding in the local population, with a higher proportion in males as compared to females. Though these protozoal parasites are present in all ages, their high prevalence among children is particularly of concern as amoebiasis can lead to liver abscess, resulting in severe morbidity and mortality. These two parasites have also been reported to be among the commonest in other studies performed in Karachi, which shows that the pattern has not changed over 8,13,16 the years. Though the two studies performed earlier found Giardia to be more common than 8,9 Entamoeba the difference in our findings could simply be explained by the fact that these two studies were population based studies, while our study was mainly performed upon clinical samples. Since both these organisms are spread by contaminated food and water. It is unfortunate that the sanitation and water supply conditions have not improved over the years and is the major contributing factor towards parasitic spread. The lack of hygienic living, inadequate health literacy and p o o r h e a l t h - re l a t e d p ra c t i c e s a m o n g t h e uneducated and low-socioeconomic classes could also be considered important contributing factors towards this situation. An improved water surveillance system and health advocacy activities among the affected population may help in reduction of the burden of these diseases. Giardia infections should be of particular concern in infants and toddlers because they may be related to 17 malnutrition resulting in growth problems. These findings are somewhat slightly different from the Northern Parts of the country as reports published from Bannu and Peshawar showed Ascaristo be the commonest parasite infecting the school children followed by E. vermicularis and H. 10,11 nana which may be attributed to different climatic conditions or other unknown factors. Presence of helminths such as A. lumbricoides and H. nana must be considered important as faecooral transmission of cysts and ova is common and untreated cases in the population may keep on 3 passing the pathogens to those at risk. Since, these parasites along with others have been related to malnutrition in children, their testing and e ra d i c a t i o n s h o u l d b e a p r i o r i t y fo r t h e 18,19 community. Since our study is based on clinical samples, it has its limitations and it may not reflect the exact picture in the community, however it clearly shows the type of intestinal parasites present in the population and highlights the need to develop a strategy to deal with the issues associated with them. A detailed community based survey is necessary to get the exact prevalence measures of intestinal parasitic infection in the local population. Parasitic Intestinal Infections in KarachiJIIMC 2016 Vol. 11, No.1 22 REFERENCES 1. Pullan RL, Smith JL, Jasrasaria R, Brooker SJ. Global numbers of infection and disease burden of soil transmitted helminth infections in 2010. Parasit Vectors. 2014; 7: 37-56. 2. Torgerson PR. One world health: Socioeconomic burden and parasitic disease control priorities. Veterinary parasitology. 2013; 195: 223-32. 3. Hotez PJ, Savioli L, Fenwick A. Neglected tropical diseases of the Middle East and North Africa: review of their prevalence, distribution, and opportunities for control. PLoS Negl Trop Dis. 2012; 6: e1475. 4. Ngui R, Lim YAL, Chong Kin L, Sek Chuen C, Jaffar S. Association between anaemia, iron deficiency anaemia, neglected parasitic infections and socioeconomic factors in rural children of West Malaysia. PLoS Negl Trop Dis. 2012; 6: e1550. 5. DuPont HL. Giardia: both a harmless commensal and a devastating pathogen. The Journal of clinical investigation. 2013; 123: 2352-4. 6. Nazeer JT, Khalifa KES, Von Thien H, El-Sibaei MM, Abdel-Hamid MY, Tawfik RAS, et al. Use of multiplex real-time PCR for detection of common diarrhea causing protozoan parasites in Egypt. Parasitology research. 2013; 112: 595-601. 7. Liu L, Johnson HL, Cousens S, Perin J, Scott S, Lawn JE, et al. Global, regional, and national causes of child mortality: an updated systematic analysis for 2010 with time trends since 2000. The Lancet. 2012; 379: 2151-61. 8. Mehraj V, Hatcher J, Akhtar S, Rafique G, Beg MA. Prevalence and factors associated with intestinal parasitic infection among children in an urban slum of Karachi. PLoS One. 2008; 3: e3680. 9. Siddiqui MI, Bilqees FM, Iliyas M, Perveen S. Prevalence of parasitic infections in a rural area of Karachi, Pakistan. Prevalence. 2002; 52: 315-20. 10. Khan MS, Jehan S, Akram M, Rabnwaz, Zaib M, Lathif Z, et al. Prevalence of intestinal protozoan and worm infestation in primary school going children of 5-10 years of age in district Bannu. Annals of Pakistan Institute of Medical Sciences. 2012; 8: 243-8. 11. Ullah I, Sarwar G, Aziz S, Khan MH. Intestinal worm infestation in primary school children in rural Peshawar. Gomal Journal of Medical Sciences. 2009; 7: 132-6. 12. Chaudhry ZH, Afzal M, Malik MA. Epidemiological factors affecting prevalence of intestinal parasites in children of Muzaffarabad district. Pakistan Journal of Zoology. 2004; 36: 267-71. 13. Mumtaz S, Siddiqui H, Ashfaq T. Frequency and risk factors for intestinal parasitic infection in children under five years age at a tertiary care hospital in Karachi. J Pak Med Assoc.2009; 59: 216-9. 14. Microsoft. Microsoft Excel. Washington: Microsoft; 2010. 15. Dean A, Sullivan K, Soe M. OpenEpi: Open Source Epidemiologic Statistics for Public Health 2015 [updated 2015 5/4/2015; cited 2015]; Available from: www.OpenEpi.com 16. Ahsan ul Wadood AB, ur Rhman A, Qasim KF. Frequency of intestinal parasite infestation in children hospital Quetta. Pakistan J Med Res. 2005; 44. 17. Al-Mekhlafi MSH, Azlin M, Nor Aini U, Shaik A, Saiah A, Fatmah MS, et al. Giardiasis as a predictor of childhood malnutrition in Orang Asli children in Malaysia. Transactions of The Royal Society of Tropical Medicine and Hygiene. 2005; 99: 686-91. 18. Mbuh JV, Nembu NE. Malnutrition and intestinal helminth infections in schoolchildren from Dibanda, Cameroon. Journal of helminthology. 2013; 87: 46- 51. 19. Papier K, Williams GM, Luceres Catubig R, Ahmed F, Olveda RM, McManus DP, et al. Childhood malnutrition and parasitic helminth interactions. Clinical Infectious Diseases. 2014; 59: 234-43. Parasitic Intestinal Infections in KarachiJIIMC 2016 Vol. 11, No.1 23