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PON SCHOLARLY JOURNALS ARCHIVES OF BIOMEDICAL SCIENCES AND HEALTH. 2013. VOL 1 (1): 10-19 2013.

1 PON SCHOLARLY JOURNALS ARCHIVES OF BIOMEDICAL SCIENCES AND HEALTH VOL 1 (1): Vol 1 (1): Original Research Paper PREVALENCE OF ENTAMOEBA HISTOLYTICA/ENT AMOEBA DISPAR AMONG PATIENTS WITH GASTRO-INTESTINAL COMPLAINTS IN LAGOS, NIGERIA. Wellington Aghoghovwia Oyibo, 1 ; Anthonia Azuka Ike 1.; Oladipo Olarinre Oladosu 1 ; Oladele Teslim Ojuromi 2 ; Sanyaolu, Adekunle.O 3 ; Olugbenga Olusanya 1 ; Adetayo Foluso Fagbenro-Beyioku 1 1 Department of Medical Microbiology and Parasitology, College of Medicine, University of Lagos, Idi-Araba, Lagos, Nigeria. 2 Department of Zoology, Lagos State University, Ojo, Lagos. 3 Department of Microbiology, Saint James School of Medicine, Anguilla, BWI. Corresponding Author:Dr. Wellington A. Oyibo ANDI CENTRE OF EXCELLENCE FOR MALARIA DIAGNOSIS International Malaria Microscopy Training and RDT QA Programme WHO/TDR/FIND Malaria Specimen Bank Site Department of Medical Microbiology and Parasitology College of Medicine University of Lagos Idi-Araba Lagos NIGERIA wellao@yahoo.com; waoyibo@gmail.com Tel: ; ABSTRACT Background and Aim: Prevalence study of Entamoeba histolytica / Entamoeba dispar among patients with gastrointestinal presentations in Lagos was carried out in a Secondary and Tertiary Health facility in Lagos State, Nigeria. This study was aimed at updating existing data on intestinal amoebiasis in Lagos. Methods: One hundred and seventy-six (176) stool samples were collected from patients of age ranging from 3 weeks old to 65 years that presented in these hospitals. The collected stool samples were examined by light microscopy after direct wet mount and formol-ether concentration. Results: Intestinal s, including helminthes, were detected in 109 (61.9%) of the 176 stool specimens. Specifically, 43 (24.4%) had Entamoeba histolytica / 10

2 Entamoeba dispar of the total stool samples examined. The prevalence of Entamoeba histolytica/ Entamoeba dispar among the different age groups were as follows: 28.6% (patients <= 20 years); 27.3% (21 30 years); 42.2% (41 50 years) and 53.8% (> 50 years). The overall infection rate of Entamoeba histolytica / Entamoeba dispar by sex was 45% for males and 55% for females. Infection was more prevalent in females than in males (p<0.05) and adults were also more frequently infected than children. Mixed infections between E. histolytica/dispar and Ascaris lumbricoides was the most predominant with an infection rate of 35.0%. Conclusion: Entamoeba histolytica / Entamoeba dispar occurs in patients with gastrointestinal complaints in Lagos. There is need to confirm the species of Entamoeba for effective case reporting. Preventive and control measures such as behavioural change though proper hygienic practices, environmental sanitation, proper disposal of faeces and community education will reduce the transmission of these gastrointestinal s. Keywords: Entamoeba histolytica / Entamoeba dispar; gastro-intestinal parasitoses; s in stool; Prevalence; Entamoeba infection; Introduction Amoebiasis is still one of the major public health problems in tropical and sub-tropical areas, and it is associated with low socioeconomic status and poor hygiene which favours the indirect faecal-oral transmission of causative. Humans are the primary reservoir and infection occurs by ingestion of cysts of Entamoeba histolytica from faecally contaminated material such as water and food (Leber and Novak, 1999). Although about 500 million people each year have amoebiasis, only about 10% experience symptomatic disease (Trol et al., 1997 and Haque et al. 1998). An estimated 100,000 people die yearly from amoebiasis, this disease is the second leading cause of death from parasitic diseases worldwide (Stanley, 2003). The vast majority of these infections are acquired in the developing world as a result of poor hygiene and inadequate source of good drinking water. Classical microscopic examination of the s E. histolytica/dispar in stool cannot differentiate pathogenic from nonpathogenic except through the use of E.histolytica-specific antigen and DNA. Sergeants et al., (1988) have demonstrated that there were differences between isoenzymes of pathogenic and non-pathogenic isolates. Lots of biochemical, immunological and genomic differences between the two species were recognized and led to formal separation of the two species with the name Entamoeba histolytica being retained for the pathogenic species and Brumpt s name Entamoeba dispar being revived for the non-pathogenic species (Ackers, 2002). Not only is microscopy unable to differentiate Entamoeba histolytica from Entamoeba dispar, it can be reported as false-positive results due to misidentification of macrophages and 11

3 non pathogenic species of Entamoeba. After many studies, it is obvious that culture is more sensitive than microscopy, and isoenzyme analysis of cultural amoebae enables differentiation of Entamoeba histolytica from Entamoeba dispar. Amoebic cultures and isoenzyme analysis requires a week to complete and are negative in many microscopy positive samples due to delays in sample processing or due to the probable antiamoebic therapy before stool collection. Detection of Entamoeba histolytica by using ELISA, enzyme-linked immunosorbent assay-based stool antigen detection kits and detection of Entamoeba histolytica specific DNA by polymerase chain reaction (PCR) amplification have been evaluated (Haque et al., 1998; Orlandi and Lampel 2000; Garcia 2000; Gebertsadik, 2004; Roy et al., 2005). These techniques have proven to be more sensitive and specific than microscopy. Nevertheless, studies on the prevalence of E. histolytica / E. dispar and other intestinal had been conducted in different parts of Nigeria at the community setting (Adeyeba and Dipeolu, 1984; Okafor and Azubike 1992; Ogunsanya et al., 1994; Enekwensi and Azubike,1994). This study is aimed at determining the prevalence of E. histolytica / E. dispar in a hospital setting, where there is paucity of data among patients presenting with gastrointestinal symptoms at one Secondary and a Tertiary healthcare setting in Lagos, Nigeria. Materials and Methods Study Area. Lagos is the Nigeria s biggest commercial nerve centre with an estimated population of 13.5 million people (2002 estimate). It lies situate on latitude 6º 30N and 3º 25E. It occupies an area of 79sq km and is by the Gulf of Guinea and habours the boundary of the Atlantic Ocean. The study was conducted in Suru-lere area of Lagos State, Lagos Mainland, and is located between Lat: S and Lon: S. The study sites were General Hospital, Randle Avenue, Surulere and The Lagos University Teaching Hospital (LUTH), Idi- Araba, Lagos. Study design and sample collection: The study design was cross-sectional. Stool specimens were collected from patients that presented with gastrointestinal symptoms such as abdominal pain, flatulence, indigestion and diarrhea for which parasitological stool examination was requested between May, 2006 and September, 2006 at the General Hospital, Randle Avenue, Surulere and The Lagos University Teaching Hospital (LUTH), Idi-Araba, Lagos. Out of the one hundred and seventy six (176) patients from whom stool samples were collected, seventy eight (78) were from General Hospital, Randle Avenue, Surulere, while ninety eight (98) were from The Lagos University Teaching Hospital (LUTH), Idi- Araba, Lagos. Patients clinical presentations were recorded before samples were collected. Each of the 176 patients with 12

4 gastrointestinal presentations that gave written consent was given a sterile, clean, dry, wide-mouth plastic container without preservatives. The patients were instructed on the precautionary measures to adopt so that the specimens will not be contaminated. The specimens were properly labeled with the study identification number, date of collection, age and sex of the patients. The enrolment in this study was voluntary and patients that opted out of the study were not denied access to the appropriate care in the health facility. Permission to conduct this study was granted by the facility. Each of the stool specimens was moved to the Parasitology Laboratory at the College of Medicine, University of Lagos, Idi-Araba, Lagos, and examined within 1 hour of collecting the stool specimen. Macroscopic and microscopic examination was carried out on each sample. Parameters such as stool color and texture as well presence of mucus and blood was recorded. Each sample was subjected to direct microscopical examination with saline and lugol iodine wet mount for the observation of trophozoites as well as the formol-ether con centration technique for the observation of ova and cyst of s. All the slides were examined microscopically using x10 and x40 objective. Data Analyses The results obtained were entered for each patient into a Personal Computer using the 2002 Epi Info Statistical package (Centre for Disease Control, Atlanta, Georgia) package. The proportion of adults and children examined and recorded were analyzed using and compared using chi square. P-values less than 0.05 were considered significant. Results Stool samples collected from one hundred and seventy six (176) patients from Randle General Hospital, Surulere, Lagos and the Lagos University Teaching Hospital (LUTH), Idi-Araba, Lagos were examined. The ages of the patients from whom stool specimens were collected ranged between 3 weeks to 65 years of age. These were made up of: 42 children, between the age of 3 weeks 16 years old and 134 adults, between years old. The consistency of the collected stool samples were: semiformed; 126 (71.6%); formed, 28(15.9%) and watery stools, 22(12.5%). Generally, intestinal s were detected in 109 (61.9%) of the 176 stool specimens. Macroscopic examination of the 109 positive stool samples showed that 15 (13.8%) had blood and mucous while 30 (27.5%) had mucous only. The overall infection rate by sex was 59.8% for male and 63.8% for female. The highest prevalence of parasitic infection in the studied individuals occurred in the years age-group while the least was in the less < 20 years age group (Table 1). Similarly, the highest prevalence in males and females was seen in the 31 13

5 40 years age group as well. Gastroparasitic infections occurred more in females (55%) than in males (45%) (p<0.05). The intestinal parasitoses recorded were: trophozoites of Entamoeba histolytica 1(0.6); Entamoeba histolytica / E. dispar cysts (42 (23.9%); cysts of Endolimax nana 4(2.3%), E. coli cysts 41(23.3%); Trichuris trichiura 3(1.7%) and Ascaris lumbricoides 18(10.2%). The prevalence of Entamoeba histolytica / E. dispar among various age groups and sex showed that the highest infection occurred in individuals > 50 years (53.8%); followed by those of years (42.4%) and years old(41.2%)(table 2). The infectivity of Entamoeba histolytica / E. dispar among males and female occurred in a similar pattern. Forty (22.7%) patients samples had mixed intestinal parasitic infections: Endolimax nana and E. coli 3(7.5%); Trichuris trichiura, Ascaris lumbricoides and E. coli 3(7.5%); E. coli and Ascaris lumbricoides 13(32.5%); E. histolytica/ E. dispar and E. coli,5(12.5%); E. histolytica/ E. dispar, E. coli, and Ascaris lumbricoides, 16(40%). Table 1: Distribution of overall infection rate among age groups and sex Age Range (Years) No. examined MALES FEMALES TOTAL Positive for No. (%) No. examined Positive for No. (%) No. examined Positive for No. (%) <= (30) 15 4 (33.3) 25 7 (28) (53.3) (70) (62.9) (85) (86.4) (85.7) (60) (80) (68.8) > (50) 17 7(41.2) (37.9) TOTAL (59.8) (63.8) (100) 14

6 Table 2: Prevalence of E. histolytica/e. dispar among infected with gastrointestinal s by age groups and sex. MALES FEMALES TOTAL Age Range (years) No. infected with Intestinal Positive for E. histolytica/ E. dispar No. (%) No. Infected with Intestinal Positive for E. histolytica/ E. dispar No. (%) Total Infected with Intestinal Total positive for E. histolytica /E. dispar No. (%) <= (33.3) 4 1 (25) 7 2 (28.6) (25) 14 4 (28.6) 22 6 (27.3) (33.3) 19 9 (47.4) (41.2) (41.2) 16 7 (43.8) (42.4) > (50) 7 4 (57.1) 14 7 (53.8) TOTAL (36.7) (41.7) (39.4) Discussion E. histolytica is the cause of invasive intestinal and extraintestinal infections while E. dispar is nonpathogenic (Haque et al., 1998; Tanyuksel and Petri, 2003). The definitive diagnosis of intestinal amoebiasis depends on the patient s history, presenting symptoms/signs and detection of cysts or trophozoites of the pathogen in the stool. Some researchers suggest that microscopic examination of the stool is sufficient to diagnose E. histolytica in the presence of characteristic microscopic findings (Stanley, 2003). Based on this, the World Health Organization (1997) recommended the following: E. histolytica and E. dispar are morphologically similar and since they cannot be differentiated by light microscopy, they should be reported as E. histolytica /E. dispar; Asymptomatic cases should not be treated unless E. histolytica /E. dispar differentiation is achieved; cases with definitive diagnosis of E. histolytica should receive treatment. In this study, the 24.4% prevalence of E. histolytica/e. dispar infection by microscopy is consistent with a study conducted in South-west Nigeria that reported a 25.5% prevalence rate of E. histolytica/e. dispar (Adeyeba and Dipeolu, 1984) and 20.4 in Ankara, Turkey (Delialioglu et al., 2004). However, the prevalence rate in this study is higher than published earlier reports from the communities in Lagos and other parts of Nigeria: 15

7 0.5% in Lagos (Ogunsanya et al.,1994); 6.8% in Njikoka, Anambra State (Enekwechi and Azuike,1994) and 4.7% in a rural area of Nigeria Okafor and Azubike (1992). These studies were however carried out in the community setting and not in the hospital. However, Gebertsadik et al., (2004) recorded a prevalence rate of 13.2% in outpatients coming to Jimma University Hospital, Ethiopia. The lower rate recorded in these studies may be due to public awareness and improvement in environmental sanitation of the populace. Another reason for the pattern of results in our present study could be because it is hospital based data. Moreover, the prevalence rate of 24.4% recorded in this study is considerable when compared with that from rural area of Ecuador. Gatti et al., (2002) recorded a prevalence rate of 27% (48 of 178 cases). The reason for this disparity may be attributable to differences in study design, patients selection, differing environmental conditions and behavioural pattern of people in those regions. Results from this study also revealed a trend in the occurrence of gastrointestinal s among the patients in Lagos, Nigeria. E. histolytica/e. dispar (24.4%), E. coli (23.3%) and Ascaris lumbricoides (10.2%) were detected more frequently. Cysts of protozoa most frequently detected were that of E. histolytica/e. dispar and E. coli. These findings agree with that of (Adeyeba and Dipeolu, 1984) in survey of gastrointestinal s among humans in South-west Nigeria. Mixed infection of 22.7% was observed in this study and is consistent with earlier report in South-west, Nigeria (Dipeolu, 1984). Furthermore, E. histolytica/e. dispar occurred more in females than in males; and also in adults than in children (Oyerinde et al., 1979). The high prevalence of intestinal parasitic infections 61.9%) could be linked to poor sanitation, nutrition, use of contaminated water and domestic and animal promiscuity (Petri and Singh, 1999; Jackson, 2000). This implied more morbidity of these s on the patients examined. The limitation of this study was the inability to perform an Enzymelinked Immunosorbent Assay (ELISA) to differentiate E. histolytica from E. dispar. This is a gap for future research to address among patients presenting with various gastrointestinal complaints in health facilities. The performance of ELISA will permit the confirmation of E. histolytica as a first step in reporting pathogenic Entamoeba species and the non-pathogenic species in patients presenting with gastrointestinal symptoms. Conclusion In conclusion, this study showed the contribution of E. histolytica / E. dispar among patients with gastrointestinal presentations. It is 16

8 therefore recommended that short and long -term measures would reduce the transmission of the gastro-parasitic disease through simple community improvement in water supply, proper excreta disposal and general environmental and personal hygiene, and by changes in personal behaviour of individuals through advocacy, communication and social mobilization. Competing Interest: None Authors Contribution: WA, AA, OO, OO, AO, OT, and AF conceptualized, analyzed and wrote up the paper. References Ackers, J. P. (2002). The diagnostic implications of the separation of Entamoeba histolytica and E. dispar. J. Biosc. (Supl. 3) 27: Adeyeba, O. A., and Dipeolu, O. O., (1984). A survey of gastrointestinal s in a local government area of south-west Nigeria. Int.J. Zoonoses. 111(1): Brumpt, E., (1925). Étude sommaire de l' "Entamoeba dispar" n. sp. Amibe à kystes quadrinuclées, de l'homme. Bull. Acad. Med. (Paris) 94: Delialioglu, N., Aslan, G., Sozen, M., Babur, C., Kanik, A. and Emekdas, G. (2004). Detection of Entamoeba histolytica/entamoeba dispar in stool specimens by using Enzyme-linked Immunosorbent Assay. Mem Inst Oswald Cruz, Rio De Janeiro. 99(7): Enekwechi, L. C. and Azuike C. N (1994). Survey of the prevalence of intestinal s in children of primary school age. West Afr. J. Med. 13: Garcia, L. S., Shimizu, R. Y. and. Bernard, C. N. (2000). Detection of Giardia lamblia, Entamoeba histolytica/ Entamoeba dispar, and Cryptosporidium parvum antigens in human fecal specimens using the Triage panel enzyme immunoassay. J. Clin. Microbiol. 38: Gatti, S., Swierczynski, G., Robinson, F., Anselmi, M., Corrales, J., Moreira, J., Montalvo, G., Bruno, A., Mascrati, R., Bisoffi, Z. and Scaglia, M. (2002). Amoebic infections due to the Entamoeba histolytica / Entamoeba dispar complex: a study of the incid47-63nce in a remote rural area of Ecuador. Am. J. Trop. Med. Hyg. 67:

9 Gebertsadik, A., Kebede, A., Mezemer, A. and Tasew, G (2004). Detection and differentiation of two morphologically identical species of Entamoeba. Ethiop J. Health Dev. 18: Haque, R., Ali, I. K. M., Akther, S. and. Petri, W. A. Jr. (1998). Comparison of PCR, isoenzyme analysis, and antigen detection for diagnosis of Entamoeba histolytica infection. J. Clin. Microbiol. 36: Jackson,T. F. H. G. (2000). Epidemiology. In J. I. Ravdin(ed.), Amoebiasis. Imperial College Press, London, United Kingdom Leber, A. L., and Novak, S. M. (1999). Intestinal and urogenital amebae, flagellates, and ciliates. In: Manual of Clinical Microbiology, P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken (ed.), 7th ed. ASM Press, Washington, D.C Okafor, C. N. and Azubike C. N. (1992). Studies in intestinal parasitic disease agents in stools of people in rural area in Nigeria, West Afr, J, Med, 11: Oguns anya, T. I., Rotimi, V. O. and Adenuga, A. (1994). A study of the aetiological agents of childhood diarrhea in Lagos, Nigeria. J. Med Microbiol 40: Orlandi, P. A., and Lampel, K. A. (2000). Extraction-free, filterbased template preparation for rapid and sensitive PCR detection of pathogenic parasitic protozoa. J. Clin. Microbiol. 38: Oyerinde, J. P. O., Adegbete-Hochist, A. F and Ogunbi, O. (1979) Prevalence of Intestinal Parasite of man in the metropolitan Lagos. Nig. J. Nat. Sc. 3: Petri,W. A., Jr., and Singh, U. (1999). Diagnosis and management of amoebiasis. Clin. Infect. Dis. 29: Roy, S., Kabir, M., Mondal, D., Ali, M.K., Petri, W.A., and Haque,R., (2005). Real -Time PCR Assay for diagnosis of Entamoeba histolytica infection. J. Clin. Microbiol.43: Sargeaunt, P. G., Williams, J. E. and. Grene, J. D (1978). The differentiation of invasive and non-invasive Entamoeba histolytica by isoenzyme electrophoresis. Trans. R. Soc. Trop. Med. Hyg. 72:

10 Stanley, S. L. (2003). Amoebiasis. The Lancet.361: Tanyuskel,M.,and Petri, W., (2003). Laboratory diagnosis of Amebiasis.Clin Microbiol.Rev. 16: Troll,H., Marti, H. and Weiss, N. (1997). Simple differential detection of Entamoeba histolytica and Entamoeba dispar in fresh stool specimens by sodium acetateacetic acid-formalin concentration and PCR. J. Clin. Microbiol. 35: WHO/ PAHO/UNESCO (1997). A consultation with experts on amoebiasis. Mexico Epidemiol Bull. 18(1):

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INTERNATIONAL JOURNAL OF PHARMACEUTICAL RESEARCH AND BIO-SCIENCE SEROPREVALENCE OF AMOEBIASIS IN A TERTIARY CARE HOSPITAL DEEPINDER CHHINA, VEENU GUPTA, JASDEEP SINGH, POOJA SURI Department of Microbiology,