This study was designed to examine stool specimens of irritable bowel syndrome (IBS) patients for Blastocystis hominis, a common intestinal parasite. One hundred fifty patients were enrolled, 95 IBS cases and 55 controls. These patients provided a medical history, and underwent physical and laboratory evaluations that included stool microscopy and culture for B. hominis and colonoscopy. The 95 cases (51 males and 44 females) had a mean +/- SD age of 37.8 +/- 13.2 years. Stool microscopy was positive for B. hominis in 32% (30 of 95) of the cases and 7% (4 of 55) of the controls (P = 0.001). Stool culture was positive in 46% (44 of 95) of the cases and 7% (4 of 55) of the controls (P \u3c 0.001). Stool culture for B. hominis in IBS was more sensitive than microscopy (P \u3c 0.001). Blastocystis hominis was frequently demonstrated in the stool samples of IBS patients; however, its significance in IBS still needs to be investigated. Stool culture has a higher positive yield for B. hominis than stool microscopy

BEG, M. ASIM

Islam, Muhammad

JAFRI, NADIM

Jafri, Wasim

KHAN, RUSTAM

Yakoob, javed

ZAMAN, VIQAR

English

eCommons@AKU

eCommons@AKUSection of Gastroenterology Department of MedicineApril 2004Irritable bowel syndrome: in search of an etiology:role of Blastocystis hominis.javed YakoobAga Khan University, javed.yakoob@aku.eduWasim JafriAga Khan University, wasim.jafri@aku.eduNADIM JAFRIAga Khan UniversityRUSTAM KHANAga Khan UniversityMuhammad IslamAga Khan University, muhammad.islam@aku.eduSee next page for additional authorsFollow this and additional works at: http://ecommons.aku.edu/pakistan_fhs_mc_med_gastroenterolPart of the Gastroenterology CommonsRecommended CitationYakoob, j., Jafri, W., JAFRI, N., KHAN, R., Islam, M., BEG, M. A., ZAMAN, V. (2004). Irritable bowel syndrome: in search of anetiology: role of Blastocystis hominis.. The American journal of tropical medicine and hygiene., 70(4), 383-385.Available at: http://ecommons.aku.edu/pakistan_fhs_mc_med_gastroenterol/99Authorsjaved Yakoob, Wasim Jafri, NADIM JAFRI, RUSTAM KHAN, Muhammad Islam, M. ASIM BEG, andVIQAR ZAMANThis article is available at eCommons@AKU: http://ecommons.aku.edu/pakistan_fhs_mc_med_gastroenterol/99IRRITABLE BOWEL SYNDROME: IN SEARCH OF AN ETIOLOGY:ROLE OF BLASTOCYSTIS HOMINISJAVED YAKOOB, WASIM JAFRI, NADIM JAFRI, RUSTAM KHAN, MUHAMMAD ISLAM, M. ASIM BEG,* ANDVIQAR ZAMAN*Section of Gastroenterology, Departments of Medicine and Pathology, Aga Khan University Hospital, Karachi, PakistanAbstract. This study was designed to examine stool specimens of irritable bowel syndrome (IBS) patients forBlastocystis hominis, a common intestinal parasite. One hundred fifty patients were enrolled, 95 IBS cases and 55controls. These patients provided a medical history, and underwent physical and laboratory evaluations that includedstool microscopy and culture for B. hominis and colonoscopy. The 95 cases (51 males and 44 females) had a mean ± SDage of 37.8 ± 13.2 years. Stool microscopy was positive for B. hominis in 32% (30 of 95) of the cases and 7% (4 of 55)of the controls (P  0.001). Stool culture was positive in 46% (44 of 95) of the cases and 7% (4 of 55) of the controls(P < 0.001). Stool culture for B. hominis in IBS was more sensitive than microscopy (P < 0.001). Blastocystis hominis wasfrequently demonstrated in the stool samples of IBS patients; however, its significance in IBS still needs to be investi-gated. Stool culture has a higher positive yield for B. hominis than stool microscopy.INTRODUCTIONBlastocystis hominis (B. hominis) is a unicellular protozoanfound in the large intestine of humans. Infection occursworldwide but is commonly found in the tropics and devel-oping countries. The pathogenic potential of B. hominis in thehuman intestine is controversial because the organism hasbeen found in both symptomatic and asymptomatic individu-als. The morphologic forms observed include vacuolar, granu-lar, and amoeboid.1 The morphologic form responsible fortransmitting the disease has not been identified, and spread ispresumed to be via the feco-oral route. The vacuolated formis most commonly found in feces.2 Infection is usually diag-nosed on the basis of direct microscopy of the fecal sampleand observing the vacuolated form of the organism using alight microscope. Cultures of B. hominis, although easy toprepare, are not done routinely, although a previous studydemonstrated that cultures of B. hominis were clearly supe-rior to direct microscopy in terms of sensitivity.3 Superficialinvasion and mucosal inflammation of the intestine with B.hominis have been observed in studies of gnotobiotic guineapigs.4 Although controlled studies of the association betweenB. hominis and diarrhea are lacking, there have been studiesthat have examined the link between B. hominis and irritablebowel syndrome (IBS).5,6 Levels of IgG antibody to B. homi-nis were increased significantly in the patients with IBS com-pared with asymptomatic controls.5 This is suggestive of a linkbetween B. hominis and IBS. The aim of this study was todetermine the prevalence of B. hominis in patients with symp-toms suggestive of IBS.PATIENTS AND METHODSThis prospective study was conducted at the Aga KhanUniversity Hospital in Karachi, Pakistan. Ninety-five patientswith symptoms suggestive of IBS, according to the Rome IIcriteria, who attended the gastroenterology clinic from Janu-ary 2002 to June 2003 were recruited into the study. TheRome II criteria is at least 12 weeks or more, which need notbe consecutive, in the preceding 12 months of abdominal dis-comfort or pain that has two of three features: relieved withdefecation; and/or onset associated with a change in fre-quency of stool; and/or onset associated with a change in formof stool.7 Patients in the IBS group presented with abdominalpain or discomfort associated with altered bowel habits. Incontrol group, there was a recent onset of diarrhea, loss ofappetite, fever, and abdominal discomfort. These patientsprovided a thorough medical history and underwent a physi-cal examination, and the following tests were conducted: com-plete blood count, erythrocyte sedimentation rate, liver func-tion, blood urea nitrogen, creatinine, electrolytes, stool mi-croscopy and culture for B. hominis. Colonoscopy with biopsywas carried out in each of the patients who fulfilled the cri-teria for IBS. Informed consent was obtained for colonoscopyin all the patients as per Aga Khan University Hospital policy.The study was reviewed and approved by ethics review com-mittee of the Department of Medicine of Aga Khan Univer-sity. Technologists were unaware of the classification status ofthe patients. All stool specimens for microscopy and cultureof B. hominis were processed by the same technicians and anote was also made of presence of other parasites such asGiardia lamblia, Entamoeba histolytica, etc. A microbiologicinvestigation was also performed to detect Salmonella spp,Campylobacter jejuni, Clostridium difficile, and Vibrio cho-lera. A viral screen was not performed on stool specimensobtained in view of cost limitations.Microscopy of fecal smears. Fecal sample microscopy wasdone as previously described.3 Briefly, approximately 2 mg offeces was thoroughly emulsified on a glass slide in one drop ofphysiologic saline and covered with a cover slip. A similarpreparation was made on another slide using Lugol’s iodine.These preparations were examined under both the low power(10×) and high dry (40×) objectives.Culture of feces. Cultures were done by inoculating ap-proximately 50 mg of feces into Jones’ medium. For culturingB. hominis, Jones medium without starch was used because itsupports good growth of the parasite as previously described.3The cultures were incubated at 37°C and examined after 24,48, 72, and 96 hours. If no B. hominis were seen up to the endof this period, the cultures were regarded as negative. Thesediment was examined under both the low power (10×) andhigh dry (40×) objectives.Statistical analysis. Results are expressed as the mean ± SDfor continuous variables (e.g, age) and number (percentage)for categorical data (e.g, sex, stool culture, diarrhea, etc.).Univariate analysis was performed using the independentsample t-test. The Pearson chi-square test and Fisher’s exactAm. J. Trop. Med. Hyg., 70(4), 2004, pp. 383–385Copyright © 2004 by The American Society of Tropical Medicine and Hygiene383test were also used whenever appropriate. A P value < 0.05was considered statistically significant. All P values were twosided. Statistical interpretation of data was performed by us-ing the computerized software program SPSS version 10.0(SPSS Inc., Chicago, IL).RESULTSIrritable bowel syndrome group. This group was composedof 51 males and 44 females with a mean ± SD age of 37.7 ± 13years. Symptoms were equally common in males and females.Abdominal pain was seen in 80% (70 of 95) and was de-scribed as cramping in 64% (45 of 70). The bowel habit wasdescribed as diarrhea in 73% (69 of 95) and constipation in13% (12 of 95). Consistency of stool varied from semi-formedin 33% (31 of 95), normal 25% (24 of 95), and loose 42% (40of 95). Clonoscopy showed patchy erythema in the rectumand sigmoid colon in 11% (10 of 95) and 3% (3 of 95), re-spectively. This was later confirmed to be nonspecific inflam-mation.Control group. This group was composed of 19 males and36 females with a mean ± SD age of 44.6 ± 20.5 years. A vagueabdominal pain was present in only 40% (22 of 55). Thebowel habit was described as diarrhea in 87.3% (48 of 55)with loose consistency of stools.Comparison of the IBS and control groups. The IBS symp-toms were equally common in males and females. Seventy-four percent (70 of 95) of the cases and 40% (22 of 55) of thecontrols had abdominal pain (P < 0.001). Seventy-three per-cent (69 of 95) of the cases and 87% (48 of 55) of the controlshad a bowel habit described as diarrhea (P 0.042). In con-trol group, 33% (18 of 55) presented with fever and 42% (23of 55) with loss of appetite, while patients in IBS group didnot demonstrate these features.Stool microscopy. Stool microscopy was positive for B.hominis in 32% (30 of 95) of the IBS patients and in 7.3%(4 of 55) of the control group. Two percent (2 of 95) of theIBS patients were positive for cysts of Entamoeba coli.Stool culture. Stool culture was positive for B. hominis in46% (44 of 95) of the IBS patients and in 7.3% (4 of 55) of thecontrol group. The organisms isolated from stool microscopyand culture in the control group is shown in Table 1.Comparison of stool microscopy and culture. Stool micros-copy in both groups of patients yielded positive results forB. hominis in 23% (34 of 150) compared with 32% (48 of 150)by stool culture. Stool culture for B. hominis was more sen-sitive than microscopy (P < 0.001) (Table 2).DISCUSSIONBlastocystis hominis is one of the most common intestinalprotozoa in humans.6 It appears in both immunocompetentand immunocompromised individuals.8,9 Although several re-ports have suggested that B. hominis could cause gastrointes-tinal disorders, the specific pathogenicity of this organism hasnot yet been defined.10–13 The clinical consequences of B.hominis infection are mainly diarrhea or abdominal pain withnonspecific gastrointestinal symptoms such as nausea, an-orexia, vomiting, weight loss, lassitude, dizziness, and flatu-lence.2 It has been speculated that thick-walled cysts might beresponsible for external transmission, while thin-walled cystsmight reinfect within a host’s intestinal tract.11,12 The trans-missible form of this organism has not yet been defined. It hasbeen suggested that B. hominis could be transmitted via un-treated water.13,14 The various mechanisms suggested for B.hominis-mediated gastrointestinal symptoms include adher-ence of B. hominis to the gut epithelium, triggering a lysismechanism as shown for E. histolytica, G. lamblia, and exis-tence of a diarrheagenic toxin present in culture filtrates andin a B. hominis cell-free fraction.4,15–17In this study, B. hominis was isolated from the feces of 46%of the IBS patients. These patients came from different resi-dential areas of the city and diverse walks of life; however, ahigh prevalence of B. hominis would not reflect their personalhygiene. Although few reports have found that B. hominiswas common in various age groups, our study failed to showa significant association in any age group.10 Several reportshave suggested that the association of persistent bowel dys-function is likely to be associated with deeper penetration ofthe B. hominis and thus more severe mucosal inflamma-tion.18,19 However, evidence of ulceration or invasion of B.hominis into the tissue could not be found by colonoscopyand biopsies in our patients with IBS. This might be consistentwith the results of Phillips and Zierdt, who showed that in-vasion of B. hominis took place only under certain conditionin germ-free guinea pigs.20 Histopathologic examination ofrectal and sigmoid colonic tissues with non-specific inflamma-tion failed to demonstrate B. hominis. This represents a mildincrease in inflammatory cells for which there may not be anecessary and specific etiology, and may be part of the normalvariation in our population.In IBS patients, cysts of Entamoeba coli were also demon-strated on stool microscopy, which is associated with asymp-tomatic carriage. Stool culture appeared to be more sensitivein diagnosing B. hominis infection than microscopy (46% ver-sus 32%), which is consistent with our previous study.3This prevalence study shows an association between B.hominis and IBS, although the study population was small. InTABLE 1Frequency of organisms isolated from the stool of patients in thecontrol group (n 55)BacteriaVibrio cholera 16.3% (9)Campylobacter jejuni 9% (5)Salmonella typhi 5.4% (3)Clostridium difficile 3.6% (2)ParasitesAscaris lumbricoides 9% (5)Blastocystis hominis 7.3% (4)Entamoeba histolytica 5.4% (3)Giardia lamblia 2% (1)Idiopathic 42% (23)TABLE 2Isolation of Blastocystis hominis by stool microscopy and culture inthe irritable bowel syndrome and control groupsCountStool microscopyPNegative PositiveStool culturePositive 16 32 <0.001Negative 100 2Total 116 34YAKOOB AND OTHERS384some IBS patients, the presence of B. hominis may not becasual and responsible for diarrhea. It has been previouslydemonstrated that IBS patients had significantly high-levelsof specific IgG antibodies against B. hominis.5 Also, a sub-group of IBS patients have persistently increased concentra-tions of inflammatory cytokines that include interleukin-1,which by inhibiting absorption of sodium and water, couldcontribute to persistent diarrhea.21 In our study, a significantproportion of IBS patients demonstrated B. hominis in theirfecal samples. However, in view of its pathogenic and non-pathogenic strains, it requires further studies to confirm thepathogenic role of B. hominis in IBS in a larger sample size.Stool culture again has a higher yield for B. hominis than stoolmicroscopy.Received July 2, 2003. Accepted for publication January 13, 2004.Authors’ addresses: Javed Yakoob, Wasim Jafri, Nadim Jafri, Rus-tam Khan, Muhammad Islam Section of Gastroenterology, Depart-ment of Medicine, Aga Khan University Hospital, Karachi, Pakistan.M. Asim Beg and Viqar Zaman, Section of Parasitology, Depart-ments of Medicine and Pathology, Aga Khan University Hospital.Reprint requests: Javed Yakoob, Section of Gastroenterology, De-partment of Medicine, Aga Khan University Hospital, Stadium Road,Karachi-74800, Pakistan, Telephone: 92-21-493-0051 extension 4679,Fax: 92-21-493-4294, E-mail: yakoobjaved@hotmail.com.REFERENCES1. Zierdt CH, 1988. Blastocystis hominis, a long misunderstood in-testinal pathogen. Parasitol Today 4: 15–17.2. Zaman V, 1996. Blastocystis hominis. Weatherall DJ, LedinghamJGG, Warrell D, eds. Oxford Textbook of Medicine. ThirdEdition. Oxford: Oxford University Press, 887.3. Zaman V, Khan K, 1994. A comparison of direct microscopy withculture for the diagnosis of Blastocystis hominis. SoutheastAsian J Trop Med Hyg Public Health 25: 792–793.4. Zierdt CH, 1991. Blastocystis hominis past and future. MicrobiolReview 4: 61–79.5. Hussain R, Jafri W, Zuberi S, Baqai R, Abrar N, Ahmed A,Zaman V, 1997. Significantly increased IgG2 subclass antibodylevels to Blastocystis homonis in patients with Irritable bowelsyndrome. Am J Trop Med Hyg 56: 301–306.6. Ashford RW, Atkinson EA, 1992. Epidemiology of Blastocystishominis in Papua New Guinea: age prevalence and associa-tions with other parasites. Ann Trop Med Parasitol 86: 129–136.7. Thompson WG, Longstreth GF, Drossman DA, Heaton KW,Irvine EJ, Muller-Lissner SA, 1999. Functional bowel disor-ders and functional abdominal pain. Gut 45 (Suppl 2): 1143–1147.8. Icci N, Toma P, Furlani M, Caselli M, Guillini S, 1984. Blasto-cystis hominis: a neglected cause of diarrhea? Lancet i: 966.9. Llibre JM, Tor J, Manterola JM, Carbonell C, Foz M, 1989. Blas-tocystis hominis chronic diarrhea in AIDS patients (letter).Lancet i: 221.10. Taamasri P, Mungthin M, Rangsin R, Tongupprakarn B, AreekulW, Leelayoova S, 2000. Transmission of intestinal blastocysto-sis related to the quality of drinking water. Southeast AsianJ Trop Med Public Health 31: 112–117.11. Singh M, Suresh K, Ho LC, Ng GC, Yap EH, 1995. Elucidationof the life cycle of the intestinal protozoan Blastocystis homi-nis. Parasitol Res 81: 446–450.12. Vdovenko AA, 2000. Blastocystis hominis: origin and significanceof vacuolar and granular forms. Parasitol Res 86: 8–10.13. Nimri L, Batchoun R, 1994. Intestinal colonization of symptom-atic and asymptomatic school children with Blastocystis homi-nis. J Clin Microbiol 32: 2865–2866.14. Kain KC, Noble MA, Freeman HJ, Barteluk RL, 1987. Epide-miology and clinical features associated with Blastocystis homi-nis infection. Diagn Microbiol Infect Dis 8: 235–244.15. Kain KC, Ravdin JI, 1995. Galactose-specific adhesion mecha-nisms of Entamoeba histolytica: model for study of entericpathogens. Methods Enzymol 253: 424–439.16. Thompson RCA, Reynoldson JA, Mendis AHW, 1993. Giardiaand giardiasis. Adv Parasitol 32: 71–160.17. Walderich B, Bernauer S, Renner M, Knobloch J, Burchard GD,1998. Cytopathic effects of Blastocystis hominis on Chinesehamster ovary (CHO) and adenocarcinoma HT 29 cell cul-tures. Trop Med Int Health 3: 385–390.18. Carrascosa M, Martinez J, Perez-Castrillon JL, 1996. Hemor-rhagic proctosigmoiditis and Blastocystis hominis infection.Ann Intern Med 15: 278–279.19. Ghosh K, Ayyaril M, Nirmala V, 1998. Acute GVHD involvingthe gastrointestinal tract and infestation with Blastocystishominis in a patient with chronic myeloid leukaemia followingallogeneic bone marrow transplantation. Bone Marrow Trans-plant 22: 1115–1117.20. Phillips BP, Zierdt CH, 1976. Pathogenic potential in human pa-tients and in gnotobiotes. Exp Parasitol 39: 358–364.21. Collins SM, 1994. Irritable bowel syndrome could be an inflam-matory disorder. Eur J Gastroenterol Hepatol 6: 478–483.B. HOMINIS IN IRRITABLE BOWEL SYNDROME 385

Irritable bowel syndrome: in search of an etiology: role of Blastocystis hominis.

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Irritable bowel syndrome: in search of an etiology: role of Blastocystis hominis.

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