Diagnosis of amoebic colitis by antigen capture ELISA in patients presenting with acute diarrhoea in Cairo, Egypt

Transcription

1 Tropical Medicine and International Health volume 7 no 4 pp april 2002 Diagnosis of amoebic colitis by antigen capture ELISA in patients presenting with acute diarrhoea in Cairo, Egypt Mohamed D. Abd-Alla 1,2 and Jonathan I. Ravdin 2 1 El-Hussein University Hospital, Cairo, Egypt 2 Department of Medicine, University of Minnesota, Minneapolis, MN, USA Summary We studied 84 consecutive patients presenting with acute diarrhoea (less than 1 week in duration) at an outpatient tropical medicine clinic in Cairo, Egypt. The diagnosis of amoebic colitis was established by the presence of Entamoeba histolytica galactose-inhibitable lectin antigen and the presence of occult blood in stool. Controls were 182 healthy regional people and 64 patients complaining of prolonged diarrhoea lasting more than 1 week. Entamoeba histolytica infection was found more frequently in patients with acute diarrhoea (57.1%) than in healthy controls (21.4%) or patients with prolonged diarrhoea (25%) (P < 0.001). There was a higher prevalence of Entamoeba dispar infection in the two control groups (24.2 and 20.3%, respectively, P ¼ and 0.061) compared with those with acute diarrhoea (8.3%). Of the 84 patients with acute diarrhoea 32 had amoebic colitis (38%), and of these, 31 (97%) had at least one positive assay for serum amoebic antibodies (P < compared with control groups). In summary, as determined by antigen-detection enzyme-linked immunosorbent assay, there is an unexpectedly high prevalence of amoebic colitis among patients presenting with acute diarrhoea to a tropical disease clinic in Cairo, Egypt. keywords Entamoeba histolytica, Entamoeba dispar, diarrhoea, enzyme-linked immunosorbent assay, diagnosis, Egypt correspondence Jonathan I. Ravdin, Department of Medicine, University of Minnesota, 420 Delaware Street SE, Minneapolis, MN 55455, USA. Fax: ; ravdi001@tc.umn.edu Introduction Entamoeba histolytica and Entamoeba dispar are morphologically identical but genetically distinct species (Tannich et al. 1989; Tannich & Burchard, 1991): E. histolytica is capable of causing amoebic colitis and liver abscess, and E. dispar is a non-pathogenic commensal (Diamond & Clark 1993). The combined prevalence of both Entamoeba sp. infection varies depending on the region. In Mexico, it has been found to be 25% by Gutierrez et al. (1976) and 83.3% by Romero et al. (1997); on the Indian subcontinent, 38% (Meerovitch et al. 1978; Veerannan 1979; Kulkarni et al. 1987); in sub-saharan and tropical Africa, 25% (Omer et al. 1981); in a Brazilian slum, 40% (Braga et al. 1996); and in an Egyptian village, 45.1% (Abd-Alla et al. 2000a). Intestinal infection with E. histolytica is asymptomatic in up to 90% of those infected (Gathiram & Jackson 1985); but at least people die worldwide each year of amoebic colitis and liver abscess. Misdiagnosis and delayed treatment are common among the estimated 50 million patients suffering from invasive amoebiasis each year; 10% of 469 tourists returning from developing countries with diarrhoea were found to have E. histolytica infection by faecal antigen detection (Jelinek et al. 1996). In another study, late diagnosis and ineffective therapy resulted in 91 patients with intestinal amoebiasis undergoing colonic surgery due to profuse haemorrhage, perforation of amoebic ulcers, gangrene and toxic dilatation, resulting in seven deaths (Dautov 1997). Diagnosis of amoebic infection solely by faecal microscopy is an inaccurate (Krogstad et al. 1978) and timeconsuming procedure. Isolation of amoebic trophozoites by stool culture and differentiation of E. histolytica from E. dispar by zymodeme analysis was considered the gold standard for diagnosing amoebic infection (Ravdin 1988). Recent advances in diagnosis have used antibody recognition of the galactose-inhibitable lectin of E. histolytica in ª 2002 Blackwell Science Ltd 365

2 faeces; the lectin is a highly conserved antigen (Ravdin et al. 1990; Abd-Alla et al. 1993, 1998; Soong et al. 1995; Abo- El-Maged et al. 1996) and a recombinant cysteine-rich section of the lectin produced in E. coli (designated LC3) is also highly antigenic (Song et al. 1995). The recombinant LC3 protein contains all the epitopes recognized by the monoclonal antibodies used in an antigen capture enzymelinked immunosorbent assay (ELISA) (Abo-El-Maged et al. 1996). Detection of amoebic 170 kda lectin antigen in faeces and saliva using epitope-specific monoclonal antibodies provides a quantitative method for differentiating E. histolytica from E. dispar (Abd-Alla et al. 1993, 2000b; Haque et al. 1995). We have repeatedly demonstrated the high sensitivity and specificity of this assay in field conditions in Cairo, Egypt (Abd-Alla et al. 1993; Abo-El-Maged et al. 1996); therefore, we conclude that confirmatory stool culture and zymodeme analysis is not indicated. Entamoeba histolytica lectin antigenemia occurs during invasive amoebiasis (Abd-Alla et al. 1993) and clears within 1 week after treatment of amoebic colitis or liver abscess (Abo-El-Maged et al. 1996). Serum antilectin immunoglobulin G (IgG) antibodies are present within 1 week after onset of symptoms in over 95% of patients with amoebic colitis or liver abscess (Ravdin et al. 1990; Abd-Alla et al. 1992) and may persist for years (Mirelman et al. 1980). In endemic areas, population-wide seropositivity rates of 20 25% limit the use of antiamoebic IgG antibodies as a diagnostic test. The purpose of our study was to determine the incidence of amoebic colitis among 84 consecutive patients presenting to a tropical disease clinic in Cairo, Egypt, with acute diarrhoea, as determined by monoclonal antibody-based ELISA for detection of faecal 170 kda lectin antigen, combined with a stool Hemoccult test. Over 99% of patients with acute amoebic colitis will have occult blood in their stools (Adams & MacLleod 1977). ELISAs to detect a number of different antiamoebic antibodies (serum anti-lc3 IgM, IgG and IgA, and faecal antilectin IgA) were performed to confirm the diagnosis of amoebic colitis and to compare the sensitivity of serology with the faecal antigen detection assay. Materials and methods Human subjects and study samples Sera and stool samples were obtained from 84 consecutive patients presenting over 1-month period with acute diarrhoea of less than 1 week in duration at the outpatient clinic of the Tropical Medicine Department at El-Hussein University Hospital, Cairo, Egypt. There were 30 females and 54 males, with an age range of 9 60 years. Egyptian controls included 182 healthy individuals and 64 patients with prolonged diarrhoea lasting longer than 1 week. Subjects had no history of antiamoebic therapy before entering the study. Control sera were obtained from 56 healthy employees of the University of Virginia who did not have any history of amoebic infection. Control faeces (56) were obtained from the University of Minnesota employees having no history of amoebic infection or recent travel to endemic areas. Egyptian controls infected with E. histolytica or E. dispar were not excluded before entry into the study. Detection of serum and faecal lectin antigen by ELISA The ELISA for detection of 170 kda lectin antigen was performed as described (Abd-Alla et al. 1993). Briefly, 96 flat-bottomed microtiter polystyrene ELISA plates (Costar, Corning, NY, USA) were coated with monoclonal antibody 3F4, which recognizes epitopes present in both E. histolytica and E. dispar lectin, or the 8C12 antibody, which is specific for epitopes present only in E. histolytica lectin epitopes (Petri et al. 1990). Faeces were mixed in an equal volume of phosphate-buffered saline (PBS) containing 2 mm phenylmethylsulphonyl fluoride (PMSF) (USB, Cleveland, OH, USA), serum was diluted 1 : 100 in PBS-Tween with 1% bovine serum albumin (BSA). Serum or faecal samples were added at 100 ll per well and incubated for 2 h at room temperature or overnight at 4 C. Alkaline phosphataseconjugated antilectin monoclonal antibodies 8A3 (recognizing both E. histolytica and E. dispar lectin) or 1G7 (specific for E. histolytica) (Petri et al. 1990) were added at 1 : 1000 dilution and incubated in developing buffer for 2 h at room temperature. Plate readings were corrected for non-specific background by subtracting the optical density (OD) from that of paired wells not coated with monoclonal antibodies but exposed to the identical procedure. The cutoff point was calculated as mean +2 standard deviations (SD) of OD reading of endemic area controls (Ravdin et al. 1990). Detection of faecal antilectin IgA antibodies by ELISA Galactose-inhibitable lectin protein (Petri et al. 1987) was purified as described. ELISA for detection of faecal antilectin IgA antibodies was performed as described (Abo- El-Maged et al. 1996). Briefly, flat-bottomed microtiter ELISA plates were coated with lectin protein (0.1 lg/well) and non-reactive sites were blocked with 1% BSA. Faeces was mixed with equal volumes of PBS-2 mm PMSF, added at 100 ll/well and incubated for 2 h at room temperature or overnight at 4 C. Alkaline phosphatase-conjugated goat antihuman IgA antibodies (Sigma, St Louis, MO, USA) were added (1 : 5000) in PBS Tween containing 1% BSA for incubation over 2 h at room temperature. Correction of 366 ª 2002 Blackwell Science Ltd

3 ELISA OD readings and calculation of the cutoff point were performed as described for lectin antigen detection. Detection of serum anti-lc3 IgG, IgM and IgA antibodies using ELISA This procedure was performed as described previously (Ravdin et al. 1990; Jelinek et al. 1996; Abd-Alla et al. 1998). Recombinant 52 kda LC3 protein (Song et al. 1995) was purified as described. Briefly, 96-well microtiter flat-bottomed polystyrene ELISA plates were coated with LC3 protein and non-reactive sites were blocked with 1% BSA. Serum samples were studied at 1 : 1000 dilution for IgG and at 1 : 500 for IgM or IgA ELISAs, all in PBS Tween (1% BSA) and incubated for 2 h at room temperature. Alkaline phosphatase-conjugated goat antihuman IgG (Sigma, St Louis, MO, USA), IgA or IgM antibodies ICN Biomedicals (Costa Mesa, CA, USA) were diluted (at 1 : 5000 for IgG, 1 : 3000 for IgM, and 1 : 2000 for IgA) in PBS-Tween (1% BSA) for incubation in a 100-ll well for 2 h at room temperature. Correction of non-specific background binding and calculation of the cutoff point were performed as described. Statistics Results were expressed as the mean (+ 3 SD, of percent positive and percent negative). The Z-test (converted to P-value) and unpaired Student s t-test were used to determine the significance of difference (Sox 1986). Results Clinical characteristics Acute diarrhoea was of rapid onset and short duration, an average of 2 days in 95% of cases. Diarrhoea was associated with abdominal pain, distention, dysentery, tenesmus and offensive flatus in most cases. Abdominal examination revealed tenderness mainly along the course of the colon and occasionally centrally. Faeces were loose to fluid in most cases. Prolonged diarrhoea was characterized mainly by increased frequency of defecation, a dull ache along the course of the colon, and semiformed to loose stools for at least 1 week (average 12 days). Prevalence of E. histolytica and E. dispar intestinal infection Table 1 summarizes the results of ELISA for detection of faecal lectin antigen in the two control groups and in Egyptian patients with acute or prolonged diarrhoea. Of 182 Egyptian controls, 39 (21.4%) had E. histolytica antigen and 44 (24.2%) had E. dispar antigen in their faeces (P < compared with patients with acute diarrhoea). Of 84 cases with acute diarrhoea, 48 (57%) had E. histolytica antigen in their faeces and only seven (8.3%) had E. dispar antigen. The low prevalence of E. dispar infection detected in this group may be partly because of the fact that the ELISA method used cannot detect mixed infections if E. histolytica is present (Abd-Alla et al. 1992). None of the subjects infected with E. dispar had occult blood in their stools; in comparison, 32 of 48 patients with acute diarrhoea and E. histolytica antigen in their faeces were Hemoccult positive (P ¼ 0.01). There was no difference in the prevalence of E. histolytica or E. dispar infection between subjects with prolonged diarrhoea and regional controls. Of 56 American controls, two were positive for E. dispar faecal antigen and one for E. histolytica (P < compared with Egyptian patients with acute diarrhoea). Occurrence of amoebic colitis Of 84 patients with acute diarrhoea, 32 had amoebic colitis as defined by a positive ELISA for E. histolytica faecal Table 1 Prevalence of Entamoeba histolytica and E. dispar infection as detected by ELISA for faecal lectin antigen in subjects with acute diarrhoea (< 1 week) compared with prolonged diarrhoea (> 1 week) and healthy controls Positive ELISA results for study subjects Faecal antigendetection ELISA American controls (n ¼ 56) (%) Egyptian controls (n ¼ 182) (%) Patients with acute diarrhoea (n ¼ 84) (%) Patients with prolonged diarrhoea (n ¼ 64) (%) Entamoeba histolytica 1 (1.8) 39 (21.4) 48 (57.1)* 16 (25) (170 kda lectin) E. dispar (170 kda lectin) 2 (3.6) 44 (24.2) 7 (8.3) 13 (20.3) * P < compared with American controls, Egyptian controls, and patients with diarrhoea for > 1 week. P < compared with acute diarrhoea. ª 2002 Blackwell Science Ltd 367

4 Table 2 Prevalence of antilectin antibodies in patients with amoebic colitis compared with subjects with acute diarrhoea due to other causes, prolonged diarrhoea, and healthy Egyptian controls Positive ELISA results for subjects with Acute amoebic colitis (n ¼ 32) (%) Acute diarrhoea with E. histolytica infection (n ¼ 16) (%) Acute diarrhoea without E. Histolytica infection (n ¼ 36) (%) Prolonged diarrhoea (n ¼ 64) Egyptian controls (n ¼ 182) (%) Faecal antilectin IgA 18 (56.3)* 6 (37.5) 7 (19.4) 8 (12.5) 18 (9.9) Serum anti-lc3 IgM 17 (53.1)* 1 (6.3) 8 (22.2) 9 (14.1) 17 (9.3) Serum anti-lc3 IgG 18 (56.3) 6 (37.5) 20 (55.6) 19 (29.7) 18 (9.9) * P < 0.01 compared with acute diarrhoea without E. histolytica infection, prolonged diarrhoea (> 1 week), and Egyptian controls. P < compared with prolonged diarrhoea (> 1 week), and Egyptian controls. antigen and the presence of occult blood in stool. The prevalence of serum anti-lc3 IgM (53.1%) and faecal antilectin IgA (56.3%) antibodies were both significantly higher in patients with acute amoebic colitis than in Egyptian controls (P < 0.001), subjects with prolonged diarrhoea (P < 0.01), and patients with acute diarrhoea without E. histolytica infection (P < 0.01) (Table 2). The prevalence of serum anti-lc3 IgG antibodies (56.3%) was significantly greater in amoebic colitis patients than in Egyptian controls and patients with prolonged diarrhoea (P < and 0.03, respectively). Patients with acute diarrhoea not caused by E. histolytica, those with prolonged diarrhoea, or healthy subjects all had a similar prevalence of serum anti-lc3 IgM or IgA antibodies (P ¼ ). The prevalence of serum anti-lc3 IgG antibodies was higher in subjects with acute diarrhoea without E. histolytica infection, compared with those with prolonged diarrhoea or controls (P < 0.01). Discussion Symptomatic invasive amoebiasis, manifest as amoebic colitis or liver abscess, occurs in approximately 10% of E. histolytica intestinal infections, the remainder are asymptomatic (Gathiram & Jackson 1985). We determined the prevalence of E. histolytica infection among consecutive patients presenting to a Tropical Disease Clinic in Cairo, Egypt with acute diarrhoea and found that 38% had amoebic colitis as defined by occult blood and E. histolytica antigen in faeces. Occult blood is found in the faeces of 99% of patients with acute amoebic colitis (Adams & MacLleod 1977). By contrast, amoebiasis was not found to be a cause of prolonged diarrhoea in this region. Entamoeba histolytica infection was no more common in this group of patients than in regional asymptomatic controls. The results of ELISA for detection or serum antilectin IgM, IgA and IgG antibodies positively correlated with the diagnosis of acute amoebic colitis, as 97% of colitis patients had at least one positive antiamoebic antibody test. However, no individual antibody test was sensitive enough for exclusive use for diagnosis of amoebic colitis in this endemic region. Entamoeba dispar is not known to cause symptoms of diarrhoea (Ravdin 1988; Sather et al. 1990; Abd-Alla et al. 1993; Diamond & Clark 1993), as was the case in this study given its higher prevalence in healthy Egyptian controls. However, the prevalence of asymptomatic E. histolytica and E. dispar infection among healthy controls in Egypt was higher than expected. Acute amoebic colitis is known to be associated with a positive ELISA for serum anti-lc3 IgM antibodies (Petri et al. 1987; Abd-Alla et al. 1998) and intestinal antilectin IgA antibodies (Abo-El-Maged et al. 1996). Serum antilectin or anti-lc3 IgG antibodies (Ravdin et al. 1990; Abd-Alla et al. 1992) persist for years following E. histolytica infection. Therefore, in a highly endemic area it is not surprising that ELISA for serum anti-lc3 IgG antibodies did not correlate well with the occurrence of acute amoebic colitis. The ELISA used in this study detects the same lectin antigen as the Tech lab assay (Haque et al. 1995) (Blacksburg, VA, USA), but our results are not directly applicable to that commercially available test. This study establishes that E. histolytica is one of the most prominent pathogens causing acute diarrhoea in Cairo, Egypt, but does not have a substantial aetiologic role in prolonged diarrhoea. In endemic areas, faecal antigen detection, combined with testing for occult blood in stool, is a more sensitive and effective diagnostic strategy than serology. Acknowledgements This work was supported by grants UO1-AI (ICIDR) and PO1-AI , Project 1, from the National Institute of Health and a grant from the AVIC- ENNE Program, Commission of European communities 368 ª 2002 Blackwell Science Ltd

5 (AVI*-CT ). All studies were approved by the Institutional Review Boards for human subjects research. The authors thank Joan M. Portel and Shana Brooks for excellent secretarial assistance. References Abd-Alla M, El-Hawey A & Ravdin J (1992) Use of an enzyme linked immunosorbent assay to detect anti-adherence protein antibodies in sera from patients with invasive amoebiasis in Cairo, Egypt. American Journal of Tropical Medicine and Hygiene 47, Abd-Alla M, Jackson T, Gathiram V, El-Hawey A & Ravdin J (1993) Differentiation of pathogenic Entamoeba histolytica infection from nonpathogenic infection by detection of galactose-inhibitable adherence protein antigen in sera and feces. Journal of Clinical Microbiology 31, Abd-Alla M, Jackson T & Ravdin J (1998) Serum IgM antibody response to galactose-inhibitable adherence lectin of Entamoeba histolytica. American Journal of Tropical Medicine and Hygiene 59, Abd-Alla M, Jackson T, Reddy S & Ravdin J (2000a) Diagnosis of invasive amoebiasis by enzyme linked immunosorbent assay of saliva to detect amoebic lectin antigen and ati-lectin immunoglobulin G antibodies. Journal of Clinical Microbiology 38, Abd-Alla M, Wahib A & Ravdin J (2000b) Comparison of antigen capture ELISA to stool culture methods for the detection of asymptomatic Entamoeba species infection in Kafer Daod, Egypt. American Journal of Tropical Medicine and Hygiene 62, Abo-El-Maged I, Soong G, El-Hawey A & Ravdin J (1996) Humoral and mucosal IgA antibody response to a recombinant 52-kDa cysteine-rich portion of the Entamoeba histolytica galactose-inhibitable lectin correlates with detection of native 170-kDa antigen in serum patients with amoebic colitis. Journal of Infectious Diseases 174, Adams E & MacLleod I (1977) Invasive amoebiasis. I. Amoebic dysentery and its complications. Medicine 56, Braga L, Lima A, Seara C et al. (1996) Seroepidemiology of Entamoeba histolytica in a slum in northeastern Brazil. American Journal of Tropical Medicine and Hygiene 55, Dautov F (1997) The diagnosis and surgical treatment of intestinal amoebiasis. Meditsinskaia Parazitologiia I Parazitarnye Bolezni 55, Diamond L & Clark C (1993) A redescription of Entamoeba histolytica Schaudinn, 1903 (amended Walker 1911) separating it from Entamoeba dispar, Brumpt, Journal of Eukaryotic Microbiology 40, Gathiram V & Jackson T (1985) Frequency distribution of Entamoeba histolytica zymodemes in a rural South African population. Lancet i, Gutierrez G, Ludlow A, Espinosa G et al. (1976) National serologic survey II. Search for antibodies against Entamoeba histolytica in Mexico. In: Amoebiasis, 1st edn (eds B Sepulveda & L Diamond). Instituto Mexicano del Sgiro Social, Mexico City, pp Haque R, Nevil L, Hahn P & Petri W (1995) Rapid diagnosis of Entamoeba histolytica infection using Entamoeba dispar and Entamoeba histolytica stool antigen kits. Journal of Clinical Microbiology 33, Jelinek T, Peyerl G, Loscher T & Nothdurft H (1996) Evaluation of an antigen-capture enzyme assay for detection of Entamoeba histolytica. stool samples. European Journal of Clinical Microbiology and Infectious Diseases 15, Krogstad D, Spencer H, Healy G et al. (1978) Amoebiasis: Epidemiologic studies in the United States, Annals of Internal Medicine 88, Kulkarni S, Pinker P, Gadkari A & Honda B (1987) Prevalence and patterns of parasitic infections in rural area around Nagpur. Indian Journal of Medical Research 68, Meerovitch E, Healy G & Ambrose-Thomas P (1978) Amoebiasis survey in Calcutta (India), Bangkok (Thailand), Medellin (Columbia), and San Juse (Costa Rico). Canadian Journal of Public Health 69, Mirelman D, Nuchamowitz Y & Stolarsky T (1997) Comparison of use of enzyme-linked immunosorbent assay based kits and PCR amplification of RNA genes for simultaneous detection of Entamoeba histolytica and E. Dispar. Journal of Clinical Microbiology 35, Omer A, McLaren M, Johnson B et al. (1981) A seroepidemiologic survey in the Gezira, Sudan with special reference to arboviruses. American Journal of Tropical Medicine and Hygiene 84, Petri W, Smith R, Schlesinger P & Ravdin J (1987) Isolation of galactose-binding lectin which mediates the in vitro adherence of Entamoeba histolytica. Journal of Clinical Investigation 80, Petri W, Jackson T, Gathiram V, Kress K & Saffer L (1990) Pathogenic and non-pathogenic strains of Entamoeba histolytica can be differentiated by monoclonal antibodies to galactosespecific adherence lectin. Infection and Immunity 581, Ravdin J (1988) Intestinal disease caused by Entamoeba Histolytica. In: Amoebiasis: Human Infection by Entamoeba Histolytica, 1st edn (ed. J Ravdin) Wiley and Sons, New York, pp Ravdin J, Jackson T, Petri J et al. (1990) Association of serum antibodies to adherence lectin with invasive amoebiasis and asymptomatic infection with Entamoeba histolytica. Journal of Infectious Diseases 162, Romero CE, Hernandez JLG, Soto JC, Ramos JMH, Pena JED & Alvarez JO (1997) Detection of anti-entamoeba histolytica salivary antibodies: evaluation of its diagnostic capability in a rural setting. Archives of Medical Research 28, S306 S308. Sather A, Bredenkamp B, Gathiram V, Sinijee A & Jackson T (1990) Detection of Entamoeba histolytica immunoglobulin G and M to plasma membrane antigen by enzyme-linked immunosorbent assay. Journal of Clinical Microbiology 28, Soong G, Kain K, Abd-Alla M, Jackson T & Ravdin J (1995) A recombinant cysteine-rich section of Entamoeba histolytica ª 2002 Blackwell Science Ltd 369

6 galactose-inhibitable lectin is efficacious as a subunit vaccine in the gerbil model of amoebic liver abscess. Journal of Infectious Diseases 171, Sox HC Jr (1986) Probability theory in the use of diagnostic test. Annals of Internal Medicine 104, Tannich E & Burchard G (1991) Differentiation of pathogenic from nonpathogenic Entamoeba histolytica by restriction fragment analysis of a single gene amplified in vitro. Journal of Clinical Microbiology 29, Tannich E, Horsmann R, Knobloch J & Arnold H (1989) Genomic difference between pathogenic and nonpathogenic Entamoeba histolytica. Proceedings of the National Academy of Sciences of the USA 86, Veerannan K (1979) The incidence of intestinal parasites among the patients in a tuberculosis hospital near Madras. Journal of the Indian Medical Association 78, ª 2002 Blackwell Science Ltd