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Diagnostic parameters of serological ELISA for invasive amoebiasis, using antigens preserved without enzymatic inhibitors
Experimental Parasitology 161 (2016) 48e53
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Diagnostic parameters of serological ELISA for invasive amoebiasis, using antigens preserved without enzymatic inhibitors María S. Flores a, *, Perla Carrillo b, Eva Tamez b, Roberto Rangel c, Elba G. Rodríguez b, n a María G. Maldonado a, Armando Isibasi d, Luis Gala gicas, Mexico Instituto de Biotecnología, Facultad de Ciencias Biolo noma de Nuevo Leo n, Monterrey, Mexico Departamento de Microbiología, Facultad de Medicina, Universidad Auto c Houston Methodist Research Institute, Houston, TX, USA d n M Unidad de Investigacio edica en Inmunoquímica, Hospital de Especialidades, Centro M edico Nacional Siglo XXI, Instituto Mexicano del Seguro Social, M exico, DF, Mexico a
b
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
ELISA-IC:MC has important serodiagnostic value in patients with invasive amoebiasis. ELISA-IC:MC is useful in populations from endemic zones carrying antibodies. ELISA-IC:MC presented better diagnostic parameters than IHA. The amoebic antigens are preserved for years without using enzymatic inhibitors. A negative serologic test does not rule out acute invasive amoebiasis.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 June 2015 Received in revised form 23 September 2015 Accepted 6 December 2015 Available online 10 December 2015
Amoebiasis is the third cause of death due to parasites in the world. Although, numerous serodiagnostic and salivary tests have been developed, the majority of these assays lack sensitivity in endemic zones to detect acute amoebic liver abscess. The two main limiting factors to develop reliable assays are the high levels of anti-amoeba antibodies in populations living in endemic zones, and the proteolysis of amoebic extracts even treated with inhibitors. Our group reported a method to preserve amoebic antigens without using enzymatic inhibitors (IC:MC fraction) that shows stability for years. Here we describe the development of a serologic ELISA to diagnose amoebiasis made with IC: MC antigens, and its validation for clinical use in endemic areas. In our study, we included sera from 66 patients diagnosed with acute amoebic liver abscess and 33 volunteers living in an endemic area for amoebiasis. Our assay was compared with an indirect haemaglutination assay (IHA) an ELISA elaborated with antigens derived from untreated trophozoites. The ELISA made with IC: MC antigens presented more reproducibility compared to other assays. Sera from 95% ALA patients showed a positive value. The ELISA (IC: MC) detected 97% of patients with ALA compared to an 81% using IHA. The parameters of ELISA (vs. IHA) were Sensitivity 98% (81%), Specificity 96% (97%), Positive predictive value 98% (96%), Negative predictive value 96% (73%) and Accuracy 98% (87%). A negative serologic test does not rule out the diagnosis of invasive amoebiasis. The ELISA made with antigens preserved without using enzymatic inhibitors has valuable serodiagnostic
Keywords: Amoebiasis ELISA Entamoeba histolytica Diagnosis Antigens Enzymes
* Corresponding author. Instituto de Biotecnología, Facultad de Ciencias Biogicas, Universidad Auto noma de Nuevo Leo n, Avenida Pedro de Alba y Manuel L lo s de los Garza, N.L., Mexico. Barrag an, Ciudad Universitaria, C.P. 66451, San Nicola E-mail addresses: maria.fl[email protected], fl[email protected] (M.S. Flores). http://dx.doi.org/10.1016/j.exppara.2015.12.006 0014-4894/© 2015 Elsevier Inc. All rights reserved.
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value to diagnose acute amoebic liver abscess, even in populations living in endemic zones of amoebiasis carrying antibodies against amoebas. In conclusion, ELISA-IC:MC presented better diagnostic parameters than IHA although a negative serologic test does not rule out acute invasive amoebiasis. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Amoebiasis is the third cause of death due to parasites in the world. It is estimated that one-tenth of the world's population is infected with Entamoeba histolytica and this infection results in up to 100,000 deaths each year. Amoebiasis infections are endemic in most moderate and tropical climates in the world. The amoebiasis is prevalent in South and Central America, Asia and Africa. In some tropical countries, antibody prevalence rates reflecting past or recent infection exceed 50% (Gonzalez et al., 1995; Caballeronez et al., 2009; Salcedo et al., 1994; Haque et al., 2003; Xime Walsh, 1986). Amoebiasis with nonspecific symptoms can mislead the clinical diagnosis. Certain colonoscopic findings predict amoebic colitis while others suggest different diagnoses (Lee et al., 2015). Intestinal amoebiasis is usually diagnosed by the identification of cysts or trophozoites in stools; the invasive pathogenic E. histolytica protozoan must be differentiated from E. dispar and Entamoeba moshkovskii, both are morphologically identical nonpathogenic commensal parasites. E. histolytica stool culture followed by PCR analysis is the gold standard for identification of E. histolytica, however outside the scientific aspect of identification of the Entamoeba strain, in the daily practice, PCR is not used as diagnostic test because costs makes it unfeasible. Mortality from amoebiasis is mainly due to extra intestinal invasion; amoebic liver abscess ALA is the most common. The noninvasive diagnosis of amebic liver abscess is challenging, as most patients at the time of diagnosis do not have a concurrent intestinal infection with E. histolytica. Unfortunately, stools analysis is not useful to diagnose extra intestinal amoebiasis because it is reported that less than 10% of patients suffering from amoebic liver abscess eliminate parasites in feces. Then, fecal testing for E. histolytica parasite antigen or DNA is negative in most patients with ALA (Haque et al., 2010; Haque and Petri, 2006; Katzenstein et al., 1982). Diverse serodiagnostic and salivary tests have been developed to detect extraintestinal amoebiasis, however many of these tests do not have enough sensitivity in acute cases or it decay after a few days of treatment with metronidazole before total resolution of abscess. Some studies have reported the detection of E. histolytica DNA in blood, urine, and saliva simultaneously to improve diagnostic sensitivity for amebic liver abscess, but the cost of these assays is unfeasible for application in poor countries where this parasitosis is endemic (Abd-Alla et al., 2000; Agundis Mata et al., 1996; Fotedar et al., 2007; Merens et al., 2005). The utility of serologic tests is controversial considering the life-long persistence of antibodies following the first infection and the permanent antibodies against amoebas among populations living in endemic zones, even if they have never suffered invasive amoebiasis, nevertheless these populations were exposed to E. histolytica nez et al., 2009). However, (Caballero-Salcedo et al., 1994; Xime serology is the method of choice for diagnosis of extra intestinal amoebiasis in poor countries where amoebiasis is endemic. The indirect haemmaglutination assay (IHA) is yet the assay frequently used by clinical laboratories in endemic countries. In clinical practice, the diagnosis of ALA is based on the positive serological detection of antibodies against E. histolytica and possibly, the
demonstration of a hepatic lesion by imaging techniques. There are limitations to develop good serological assays. One of them is the high serum level of antibodies against amoebas among populations living in endemic zones. These antibodies involve high background noise in serodiagnostic tests, implicating lost of efficacy. Proteolysis of amoebic extracts constitutes another restriction to standardize the assays. Amebas express high levels of enzymes as a molecular mechanism to invade the human host. (SerranoLuna et al., 2013; AraizaeOrozco et al., 1999; Vila et al., 1985; ~ oz et al., 1990, 1984). Worldwide, enzymatic inhibitors are Mun used to reduce the activity of amoebic proteases. Nevertheless, such inhibitors are not completely effective to prevent the amoebic molecules degradation during antigen preparation (Lopez-Revilla et al., 1992; Lopez-Revilla and Baez-Camargo, 1992; PerezMontfort et al., 1987; Zamudio-Prieto et al., 2014). Since proteolysis causes differences in antigenic composition preventing reproducible diagnostic data, our group developed a method to preserve amoebic antigens from trophozoites without using enzymatic inhibitors. The amoebic fraction (IC: MC) obtained by this procedure shows long antigenic stability and the same antigen lot can be used ~ eda, 1995; Flores-de-Castan ~ eda, for several years (Flores-de-Castan ~ eda, 2002a; Flores-de-Castan ~ eda, 2002b; 1999; Flores-de-Castan ~ o et al., 2000). A real challenge Flores et al., 2005; Tamez- Trevin of poor countries remains: to have reliable diagnostic methods, at low cost and applicable in endemic areas for amoebiasis. The aim of this study was to develop an ELISA to diagnose invasive amoebiasis using stable antigens with long shelf-time prepared without enzymatic inhibitors, and validate its clinical use in an area of amoebic endemicity. 2. Material and methods 2.1. Patients and volunteers 2.1.1. Patients with amoebic liver abscess Group of patients with acute amoebic liver abscess Sixty-six patients admitted to the University Hospital “Jose Eleuterio Gonzalez” at Monterrey Nuevo Leon, Mexico. Patients were diagnosed with amoebic liver abscess (ALA) by clinical symptoms, ultrasound image and/or axial tomography (CT) and positive response to antiamoebic drug treatment. Fifty percent of patients required percutaneous drainage. The drained fluid was microbiologic cultured to discard bacterial infection. Stool samples were not examined due to previous reports indicating that only 10% of patients with liver abscess release amebas in stool. (Katzenstein et al., 1982) Serum from each patient was tested by the Cellognost Amoebiasis Indirect Hemagglutination Assay (Behring Diagnostics GmbH, Germany). All patients in this group were positive by Western Blot to diagnose ~ eda, 1999; Flores-deinvasive amoebiasis (Flores-de-Castan ~ eda, 2002b). Castan 2.1.2. Healthy volunteers Group of healthy volunteersThirty-three volunteer students of noma de Nuevo Leo n. Participants in this the Universidad Auto group had neither previous invasive amoebiasis, nor any episode of bloody diarrhea within the previous year. Participants were
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negative at the direct examination for parasites in feces (Faust et al., 1970). They were negative at IHA test (Behring Diagnostics GmbH, Germany) and Western Blot for invasive amoebiasis (Flores-de~ eda, 1999; Flores-de-Castan ~ eda, 2002b). The healthy volunCastan teers lived in an endemic area for amoebiasis located in the northeastern of Mexico. 2.2. E. histolytica 2.2.1. E. histolytica culture HM1-IMSS E. histolytica trophozoites were cultured axenically into borosilicate glass culture tubes, in TYI-S-33 medium supplemented with vitamins and heat inactivated adult bovine serum (Laboratorios Microlab. Mexico) according to Diamond (Diamond, 1968). 2.2.2. Amoebic antigen preserved without enzymatic inhibitors (ELISA-IC: MC) E. histolytica IC: MC fraction was obtained according to Flores ~ eda, 1995; Flores-de-Castan ~ eda, 1999; Flores-de(Flores-de-Castan ~ eda, 2002a; Flores-de-Castan ~ eda, 2002b; Flores et al., Castan 2005). Briefly: the trophozoites were harvested at the end of the logarithmic growth phase and washed with 0.15 M buffered phosphate saline, pH 7.4 (PBS). The pellet was frozen-dried, suspended in chloroform: methanol 20:1 (vol: vol), mixed and centrifuged. The polar solvent insoluble fraction obtained was dried to eliminate residues of solvents and stored at room temperature. Prior to be used, the fraction was resuspended in 0.1 M carbonate buffer pH 9.6 urea 8 M and the mixture boiled in water bath. Supernatant protein concentration was determined according to the Bradford method (Bradford, 1976) The IC: MC fraction might be kept frozen in solution or dried, and stored for up to three years without protein degradation. 2.3. Enzyme-linked immunosorbent assays 2.3.1. Enzyme-linked immunosorbent assay using amoebic antigens preserved without enzymatic inhibitors. (ELISA-IC: MC) The ELISA was performed according to Flores (Flores-de~ eda, 1999; Flores-de-Castan ~ eda, 2002b). Briefly: One hunCastan dred microliters of solution containing 25 mg/mL of the IC: MC fraction supernatant in 0.1 M carbonate buffer pH 9.6 urea 8 M, were incubated overnight at 4 C in order to immobilize the antigen on Corning® polystyrene, flat bottom plates. After washing (0.1 M PBS, 0.05% Tween-20, pH 7.4), the plates were incubated 30 min at 37 C with 300 mL of blocking buffer (2% Bovine serum albumin, 0.1 M PBS, 0.05% Tween-20, pH 7.4, 0.02% thimerosal, pH 7). One hundred microliters per well of each serum were incubated two hours at room temperature and then washed three times. Horseradish peroxidase-conjugated to anti-human immunoglobulin produced in goat was added as secondary antibody. The secondary antibodies tested included anti-human polyvalent immunoglobulins (whole molecule), anti-human IgG (g-chain specific), antihuman IgM (m-chain specific), and anti-human IgA (a-chain specific). The immune reaction was developed using H2O2 as substrate and 3.3-diaminobenzidine tetrahydrochloride as chromogen. The colorimetric reaction absorbance was measured at 490 nm (Microplate reader BIO-RAD. Hercules CA, USA). 2.3.2. Enzyme-linked immunosorbent assay using antigens from untreated trophozoites The ELISA was performed with modifications of above test. Briefly: One hundred microliters of solution containing 25 mg/mL of the freeze dried total trophozoites in 0.1 M carbonate buffer pH 9.6 urea 8 M, were incubated overnight at 4 C to immobilize the
antigen on Corning® polystyrene, flat bottom plates. 2.4. Calculation 2.4.1. Determination of intra-assay and interassay variation ELISA absorbance Index (AI): ALA serum absorbance/control negative serum absorbance. The cut off was calculated with the absorbance mean of negative sera plus one standard deviation. Intra Assay coefficient of variation (intra-assay CV). To determine the deviation within the same plate, ten samples were measured ten times each, on the same plate and assay. Intra assay CV ¼ (The standard deviation of ten measures/AI mean) x 100. Inter-Assay coefficient of variation (inter-assay CV). To determine the deviation within different plates, ten samples were measured in five different plates. Inter-Assay CV ¼ (The standard deviation of the five measurements/AI mean) x 100. 2.4.2. ELISA units (EU) The ELISA units were obtained by the next equation: EU ¼ (100/optical density of control positive sera) x optical density of sample. 2.4.3. Statistics and diagnostic utility parameters Utility parameters were calculated using Friedman Nonparametric Statistics formulated in a 2 2 contingency table. To determine the Diagnostic utility, the parameters of Sensitivity; Specificity; Positive predictive value; Negative predictive value and Accuracy were calculated (Corder and Foreman, 2009; Shankar et al., 2008). 2.5. Indirect haemagglutination assay (IHA) Cellognost Amoebiasis kit was used to test the sera IHA titters. It was performed following the kit instructions (Behring Diagnostics GmbH, Germany). Positive antibody titer 1: 256 sera dilution. 2.6. Stool microscopic identification of parasites Each healthy volunteer provide three fresh stool specimens. Fecal samples emulsified in 10% neutral formalin, were concentrated by flotation (Faust et al., 1970). Each sample was directly examined under the microscope light to search intestinal parasites. 2.7. Chemicals The products, whose origins were not indicated, were reagents from Sigma, St. Louis, MO, USA, or Bio-Rad Laboratories, Richmond, CA, USA. 3. Results 3.1. ELISA assays variability First, assays were run to determine the absorbance index variation in the ELISA tests made with antigens from total trophozoites or ELISA- IC: MC. Checkerboard titration experiments were performed to find the suitable assay conditions and establish the adequate background and signal to noise ratios. As shown in Table 1, absorbance indexes were calculated from the absorbance data of 4 sera samples obtained from ALA patients. Next, each sample was measured in ten wells using the same plate to obtain the intra assay coefficient of CV. As predicted, we observed that AI obtained using sera of patients with ALA, is greater compared to the serum used as negative control. The values represented the arithmetic mean ± standard deviation of absorbance
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Table 1 ELISA assays variability. Sera
ELISA-Total untreated trophozoites Mean AI ± SD (intra assay CV)
1 2 3 4
5.8 3.9 4.8 4.8
± ± ± ±
0.32 0.45 0.65 0.52
(5.5) (11.5) (13.5) (10.8)
ELISA-IC:MC AI variation of the same serum
Mean AI ± SD (intra assay CV)
13.2% 28.5% 41.6% 38.4%
3.5 2.7 3.7 2.8
± ± ± ±
0.18 0.10 0.19 0.25
(5.1) (3.7) (5.1) (8.9)
AI variation of the same serum 10% 9% 11% 13%
Absorbance index: AI, Variation AI: Variation among the highest AI value and the lowest AI value of the same serum. Intra assay coefficient of variation ¼ (The standard deviation of ten measures/AI mean) x 100. Serum from healthy volunteer was used as negative control. The values represented the arithmetic mean ± standard deviation of absorbance obtained for each serum by ten repetitions.
obtained for each serum by ten repetitions. Notably, there is more variation in the ELISA performed with total trophozoites than the ELISA-IC: MC fraction as antigen. Finally, we observed that high and low AI values varied among the same serum, from 13 to 41%, in contrast with variations from nine to 13% in the ELISA - IC: MC as antigen. To obtain the inter-assay coefficient of variation of the ELISAeIC: MC, we tested five different sera samples in five different assays. We observed that the data range between 7 and 13%. 3.2. Antibody isotypes to E. histolytica Next, in order to determine the best option as secondary antibodies in the ELISA test, experiments were performed to identify the immunoglobulin isotypes produced in ALA patients. We evaluated the following antibodies: anti-human polyvalent immunoglobulin (whole molecule), anti-human IgG (g-chain specific), antihuman IgM (m-chain specific), and anti-human IgA (a-chain specific). Fig. 1 shows the ELISA units obtained using sera of patients with ALA and from healthy volunteers. We clearly observed that antibodies binding to isotypes IgA and IgG allowed significant differences between the two groups, nevertheless sera of patients with ALA exhibit a wide dispersion production of IgA antibodies. Taken together, these results show that IgA and IgG are the best immunoglobulins to target amoebic antigens; The total Antihuman polyvalent immunoglobulins allowed detecting differences between the two groups of sera, therefore anti-human polyvalent immunoglobulins will be selected as secondary antibodies for the ELISA test.
3.3. ELISA-IC:MC of the sera from healthy volunteers and patients with ALA Then, collected sera from 66 ALA patients and 33 healthy volunteers were tested in this study. Fig. 2 shows the global reactivity distribution of anti-amoeba antibodies against preserved antigens represented as ELISA-IC:MC units. We estimated an optimal cut off value of 72 ELISA units (EU), with a grey zone ranging from 72 to 80 EU. Consequently, a positive result will contain 80 ELISA units (EU) and a negative result 72 EU. Indeed, sera from 95% ALA patients showed a higher value than the cut off, one serum was within the cut off value and one serum was below, giving a false negative value. As a note, only one of the samples from healthy volunteers was positive, eight were on the gray zone, and the rest of the samples resulted in negative values. The IHA is thus far the assay used by clinical laboratories in poor endemic countries, to diagnosis antibodies to amoebiasis. In addition, we tested the entire ALA patients and healthy volunteer sera by IHA assay, to compare the clinical utility of this test and the ELISA-IC:MC assay developed with preserved E. histolytica antigens without using enzymatic inhibitors. Eighty one % of the sera were positive on IHA and 18.4% showed false negative results, in contrast, the ELISA-IC:MC detected 97% of patients with ALA and it gave 1.5% false negatives. We determine the clinical utility parameters of ELISA-IC:MC and IHA, as shown in Table 2. The IHA values demonstrate that a positive result has 96% probability to be a true positive result. Nevertheless, a negative result has only 73% probability to be a true negative result. Meanwhile ELISA-IC:MC test has 98% probability to be a true positive result, and a negative result has
Fig. 1. Antibody isotypes to Entamoeba histolytica produced by patients with ALA and healthy volunteers living in endemic region.
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Fig. 2. ELISA-IC:MC graphic data distribution of the sera from healthy volunteers and patients with ALA.
Table 2 Clinical parameters obtained by ELISA-IC:MC and IHA tests.
Sensitivity Specificity Positive predictive value Negative predictive value Accuracy
ELISA-IC:MCa
IHA
98% 96% 98% 96% 98%
81% 97% 96% 73% 87%
IHA: Indirect haemagglutination assay (Cellognost Amoebiasis). a ELISA using preserved E. histolytica antigens without enzymatic inhibitors.
96% probability to be a true negative result. ELISA-IC:MC presented more accuracy than IHA. 4. Discussion In areas of high endemicity of amoebiasis, physicians have the training that allows them to efficiently diagnose this parasitosis, however some acute ALA patients present a negative value by IHA test and clinical features suggesting other pathologies. In these clinical cases, it would be desirable to have a quick and inexpensive diagnostic test feasible to be used in non-specialized laboratories. To standardize reliable immune-based tests is important to use inhibitors to prevent the risk of lack of reproducibility of results due to proteolysis (Serrano-Luna et al., 2013; AraizaeOrozco et al., 1999; ~ oz et al., 1990, 1984; Lopez-Revilla et al., 1992; Vila et al., 1985; Mun Lopez-Revilla and Baez-Camargo, 1992; Perez-Montfort et al., 1987). In this study we employed amoebic extracts preserved without the use of enzymatic inhibitors as the antigen in ELISA test. Our group established that this method preserves amoebic antigens and decreases the proteases activity from trophozoites extracts ~ eda, 1995; Flores-de-Castan ~ eda, 1999; Flores-de(Flores-de-Castan ~ eda, 2002a; Flores-de-Castan ~ eda, 2002b; Flores et al., Castan 2005). With stable antigens, it is possible to develop tests with good reproducibility, even using the same antigen lot for long periods of time. In our laboratory we have tested the reproducibility of some commercial ELISA kits and they presented a significant interassay variation (data not shown). For this reason we decided to
compare the ELISA made with antigens preserved without using enzymatic inhibitors versus an ELISA made with antigens from total trophozoites developed by ourselves. The ELISA made with total antigenic extract presented great variation among the repetitions of the same serum, in contrast, the ELISA test using the IC: MC fraction as antigen has the best suitable reproducibility inter and intra assay. During this study, we identified that the immunoglobulin isotypes produced against amoebic antigens are mainly IgG and IgA. The IgA values distribution showed greater scattered than IgG titers. These results agree with previous reports (Carrero et al., 1994; Abioye et al., 1972). Antibodies anti-human polyvalent immunoglobulin (whole molecule) were selected as secondary antibodies as they serve to demonstrate a statistically significant difference response of antibodies to E. histolytica in patients with ALA, regarding healthy volunteers with no history of amoebiasis. Our results add a relevant contribution in the diagnosis of amoebiasis. The ELISA made with IC: MC fraction revealed that the sera from 95% ALA patients had a higher value than the cut off, and 97% of the sera from healthy people had a lower value than the cut off. Healthy individuals living in endemic areas of amoebiasis can contain antibodies to E. histolytica, despite this; the ELISA allows a clear difference between sera of ALA patients from healthy volunteers. Previous reports have shown that IHA is the assay frequently used by clinical laboratories in endemic countries. In the work presented here, we describe the development of an ELISA made with preserved E. histolytica antigens without enzymatic inhibitors, that shows a better clinical diagnostic parameter than the IHA test. Our results explain the seroprevalence differences found in Mexico as reported by Gonzalez et al. (Gonzalez et al., 1995) in the same sampling frame studied using two different tests. The data obtained in different studies will vary considerably according to the type of assay used. In cases of acute ALA, a negative serologic test does not rule out the diagnosis of invasive amoebiasis. Therefore, the parameters of Sensitivity, Specificity, Positive and Negative predictive value and Accuracy indicated that the ELISA made with antigens preserved without enzymatic inhibitors is a reliable
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diagnostic test useful in endemic zones of amoebiasis and affordable to poor countries. Overall, the ELISA for invasive amoebiasis made with antigens preserved without using enzymatic inhibitors has valuable diagnostic use even in populations living in endemic zones of amoebiasis carrying antibodies against amebas. 5. Conclusions The ELISA-IC:MC for invasive amoebiasis made with antigens preserved without using enzymatic inhibitors has valuable diagnostic value even in populations living in endemic zones of amoebiasis carrying antibodies against amebas. ELISA-IC:MC presented better diagnostic parameters than IHA. In cases of acute ALA, a negative serologic test does not rule out the diagnosis of invasive amoebiasis. Conflicts of interest All authors declare that they have no conflict of interest. Ethical approval This research protocol and the consent forms were approved by the Research and Bioethics Committee of the Faculty of Biological Sciences. The research has been performed in accordance with the ethical standards of Declaration of Helsinki. Acknowledgments Eleuterio We acknowledge the Hospital Universitario “Jose n, Me xico for providing support. Gonz alez” at Monterrey Nuevo Leo This project was supported by grants of PAICYTe Universidad noma de Nuevo Leo n and SEP-CONACYT grant 25617. Auto References Abd-Alla, M.D., Jackson, T.F., Reddy, S., Ravdin, J.I., 2000. Diagnosis of invasive amoebiasis by enzyme-linked immunosorbent assay of saliva to detect amoebic lectin antigen and anti-lectin immunoglobulin G antibodies. J. Clin. Microbiol. 38 (6), 2344e2347. Abioye, A.A., Lewis, E.A., McFarlane, H., 1972. Clinical evaluation of serum immunoglobulin in amoebiasis. Immunol 23 (6), 937e946. Agundis Mata, C., Blanco, F., Toledo, J., Reta, F., Kumate, J., Isibasi, A., 1996. Identification of a 35 kDa glycoprotein from Entamoeba histolytica by sera from patients with amoebic liver abscess and with mouse monoclonal antibody. Immunol. Investig. 25 (5e6), 519e529. ~ oz, M., Arias-Negrete, S., 1999. Entamoeba histoAraiza-Orozco, L.M., Avila, E., Mun lytica: surface proteolytic activity and its relationship with in vitro virulence. Folia Parasitol. 46 (3), 161e167. Bradford, M., 1976. A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248e254. Caballero-Salcedo, A., Viveros-Rogel, M., Salvatierra, B., Tapia-Conyer, R., Sepúlvedarrez, G., Ortiz-Ortiz, L., 1994. Seroepidemiology of amoebiasis in Amor, J., Gutie Mexico. Am J. Trop Med. Hyg 50, 412e419. Carrero, J.C., Díaz, M.Y., Viveros, M., Espinoza, B., Acosta, E., Ortiz-Ortiz, L., 1994. Human secretory immunoglobulin A anti-Entamoeba histolytica antibodies inhibit adherence of amebae to MDCK cells. Infect. Immun. 62 (2), 764e767. Corder, G.W., Foreman, D.I., 2009. Nonparametric Statistics for Non-statisticians: a Step-by-step Approach. John Wiley & Sons, Inc., New Jersey. Diamond, L.S., 1968. Techniques of axenic cultivation of Entamoeba histolytica Schaudinn, 1903 and E. histolytica-like ameba. J. Parasitol. 54 (5), 1047e1056. Faust, E.C., Russell, P.F., Jung, R.C., 1970. In: Craig and Faust's Clinical Parasitology, eighth ed. Lea &Febiger, US, pp. 83e101. ~ o, E., Castan ~ eda, F., Tijerina-Menchaca, R., Flores, M.S., Tamez-Trevin
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Entamoeba histolytica: collagenolytic activity and virulence. J. Protozool. 31 (3), 468e470. ~ oz, M., Lamoyi, E., Leo n, G., Tovar, R., Pe rez-García, J., De La Torre, M., Mun Murueta, E., Bernal, R.M., 1990. Antigens in electron-dense granules from Entamoeba histolytica as possible markers for pathogenicity. J. Clin. Microbiol. 28 (11), 2418e2424. Perez-Montfort, R., Ostoa-Saloma, P., Velazquez-Medina, L., Montfort, I., Becker, I., 1987. Catalytic classes of proteinases of Entamoeba histolytica. Mol. Biochem. Parasitol. 26 (1e2), 87e97. ~ a-V pez, M., Ortiz-Estrada, G., de la Serrano-Luna, J., Pin azquez, C., Reyes-Lo Garza, M., 2013. Proteases from Entamoeba spp. and pathogenic free-living amoebae as virulence factors. J. Trop. Med. 2013, 890603. http://dx.doi.org/ 10.1155/2013/890603. Epub 2013 Feb 7. Shankar, G., Devanarayan, V., Amaravadi, L., Barrett, Y.C., Bowsher, R., Finco-Kent, D., Fiscella, M., Gorovits, B., Kirschner, S., Moxness, M., Parish, T., Quarmby, V., Smith, H., Smith, W., Zuckerman, L.A., Koren, E., 2008. Recommendations for the validation of immunoassays used for detection of host antibodies against biotechnology products. J. Pharm. Biomed. Anal. 48 (5), 1267e1281. ~ o, E., Sepúlveda-Saavedra, J., Tijerina-Menchaca, R., FloresTamez-Trevin ~ eda, M.S., 2000. Systemic anti-amoeba antibodies induced orally in mice: Castan detection by an ELISA test using preserved E. histolytica-antigens without enzymatic inhibitors. Arch. Med. Res. 31 (4), 28e29. nchez-Garza, M., Calderon, J., 1985. Entamoeba histolytica and Vila, E.E.A., Sa E. invadens: sulfhydryl-dependent proteolytic activity. J. Protozool. 32 (1), 163e166. Walsh, J.A., 1986. Problems in recognition and diagnosis of amoebiasis: Estimationof the global magnitude of morbidity and mortality. Rev. Infect. 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Diagnostic parameters of serological ELISA for invasive amoebiasis, using antigens preserved without enzymatic inhibitors María S. Flores a, *, Perla Carrillo b, Eva Tamez b, Roberto Rangel c, Elba G. Rodríguez b, n a María G. Maldonado a, Armando Isibasi d, Luis Gala gicas, Mexico Instituto de Biotecnología, Facultad de Ciencias Biolo noma de Nuevo Leo n, Monterrey, Mexico Departamento de Microbiología, Facultad de Medicina, Universidad Auto c Houston Methodist Research Institute, Houston, TX, USA d n M Unidad de Investigacio edica en Inmunoquímica, Hospital de Especialidades, Centro M edico Nacional Siglo XXI, Instituto Mexicano del Seguro Social, M exico, DF, Mexico a
b
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
ELISA-IC:MC has important serodiagnostic value in patients with invasive amoebiasis. ELISA-IC:MC is useful in populations from endemic zones carrying antibodies. ELISA-IC:MC presented better diagnostic parameters than IHA. The amoebic antigens are preserved for years without using enzymatic inhibitors. A negative serologic test does not rule out acute invasive amoebiasis.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 June 2015 Received in revised form 23 September 2015 Accepted 6 December 2015 Available online 10 December 2015
Amoebiasis is the third cause of death due to parasites in the world. Although, numerous serodiagnostic and salivary tests have been developed, the majority of these assays lack sensitivity in endemic zones to detect acute amoebic liver abscess. The two main limiting factors to develop reliable assays are the high levels of anti-amoeba antibodies in populations living in endemic zones, and the proteolysis of amoebic extracts even treated with inhibitors. Our group reported a method to preserve amoebic antigens without using enzymatic inhibitors (IC:MC fraction) that shows stability for years. Here we describe the development of a serologic ELISA to diagnose amoebiasis made with IC: MC antigens, and its validation for clinical use in endemic areas. In our study, we included sera from 66 patients diagnosed with acute amoebic liver abscess and 33 volunteers living in an endemic area for amoebiasis. Our assay was compared with an indirect haemaglutination assay (IHA) an ELISA elaborated with antigens derived from untreated trophozoites. The ELISA made with IC: MC antigens presented more reproducibility compared to other assays. Sera from 95% ALA patients showed a positive value. The ELISA (IC: MC) detected 97% of patients with ALA compared to an 81% using IHA. The parameters of ELISA (vs. IHA) were Sensitivity 98% (81%), Specificity 96% (97%), Positive predictive value 98% (96%), Negative predictive value 96% (73%) and Accuracy 98% (87%). A negative serologic test does not rule out the diagnosis of invasive amoebiasis. The ELISA made with antigens preserved without using enzymatic inhibitors has valuable serodiagnostic
Keywords: Amoebiasis ELISA Entamoeba histolytica Diagnosis Antigens Enzymes
* Corresponding author. Instituto de Biotecnología, Facultad de Ciencias Biogicas, Universidad Auto noma de Nuevo Leo n, Avenida Pedro de Alba y Manuel L lo s de los Garza, N.L., Mexico. Barrag an, Ciudad Universitaria, C.P. 66451, San Nicola E-mail addresses: maria.fl[email protected], fl[email protected] (M.S. Flores). http://dx.doi.org/10.1016/j.exppara.2015.12.006 0014-4894/© 2015 Elsevier Inc. All rights reserved.
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value to diagnose acute amoebic liver abscess, even in populations living in endemic zones of amoebiasis carrying antibodies against amoebas. In conclusion, ELISA-IC:MC presented better diagnostic parameters than IHA although a negative serologic test does not rule out acute invasive amoebiasis. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Amoebiasis is the third cause of death due to parasites in the world. It is estimated that one-tenth of the world's population is infected with Entamoeba histolytica and this infection results in up to 100,000 deaths each year. Amoebiasis infections are endemic in most moderate and tropical climates in the world. The amoebiasis is prevalent in South and Central America, Asia and Africa. In some tropical countries, antibody prevalence rates reflecting past or recent infection exceed 50% (Gonzalez et al., 1995; Caballeronez et al., 2009; Salcedo et al., 1994; Haque et al., 2003; Xime Walsh, 1986). Amoebiasis with nonspecific symptoms can mislead the clinical diagnosis. Certain colonoscopic findings predict amoebic colitis while others suggest different diagnoses (Lee et al., 2015). Intestinal amoebiasis is usually diagnosed by the identification of cysts or trophozoites in stools; the invasive pathogenic E. histolytica protozoan must be differentiated from E. dispar and Entamoeba moshkovskii, both are morphologically identical nonpathogenic commensal parasites. E. histolytica stool culture followed by PCR analysis is the gold standard for identification of E. histolytica, however outside the scientific aspect of identification of the Entamoeba strain, in the daily practice, PCR is not used as diagnostic test because costs makes it unfeasible. Mortality from amoebiasis is mainly due to extra intestinal invasion; amoebic liver abscess ALA is the most common. The noninvasive diagnosis of amebic liver abscess is challenging, as most patients at the time of diagnosis do not have a concurrent intestinal infection with E. histolytica. Unfortunately, stools analysis is not useful to diagnose extra intestinal amoebiasis because it is reported that less than 10% of patients suffering from amoebic liver abscess eliminate parasites in feces. Then, fecal testing for E. histolytica parasite antigen or DNA is negative in most patients with ALA (Haque et al., 2010; Haque and Petri, 2006; Katzenstein et al., 1982). Diverse serodiagnostic and salivary tests have been developed to detect extraintestinal amoebiasis, however many of these tests do not have enough sensitivity in acute cases or it decay after a few days of treatment with metronidazole before total resolution of abscess. Some studies have reported the detection of E. histolytica DNA in blood, urine, and saliva simultaneously to improve diagnostic sensitivity for amebic liver abscess, but the cost of these assays is unfeasible for application in poor countries where this parasitosis is endemic (Abd-Alla et al., 2000; Agundis Mata et al., 1996; Fotedar et al., 2007; Merens et al., 2005). The utility of serologic tests is controversial considering the life-long persistence of antibodies following the first infection and the permanent antibodies against amoebas among populations living in endemic zones, even if they have never suffered invasive amoebiasis, nevertheless these populations were exposed to E. histolytica nez et al., 2009). However, (Caballero-Salcedo et al., 1994; Xime serology is the method of choice for diagnosis of extra intestinal amoebiasis in poor countries where amoebiasis is endemic. The indirect haemmaglutination assay (IHA) is yet the assay frequently used by clinical laboratories in endemic countries. In clinical practice, the diagnosis of ALA is based on the positive serological detection of antibodies against E. histolytica and possibly, the
demonstration of a hepatic lesion by imaging techniques. There are limitations to develop good serological assays. One of them is the high serum level of antibodies against amoebas among populations living in endemic zones. These antibodies involve high background noise in serodiagnostic tests, implicating lost of efficacy. Proteolysis of amoebic extracts constitutes another restriction to standardize the assays. Amebas express high levels of enzymes as a molecular mechanism to invade the human host. (SerranoLuna et al., 2013; AraizaeOrozco et al., 1999; Vila et al., 1985; ~ oz et al., 1990, 1984). Worldwide, enzymatic inhibitors are Mun used to reduce the activity of amoebic proteases. Nevertheless, such inhibitors are not completely effective to prevent the amoebic molecules degradation during antigen preparation (Lopez-Revilla et al., 1992; Lopez-Revilla and Baez-Camargo, 1992; PerezMontfort et al., 1987; Zamudio-Prieto et al., 2014). Since proteolysis causes differences in antigenic composition preventing reproducible diagnostic data, our group developed a method to preserve amoebic antigens from trophozoites without using enzymatic inhibitors. The amoebic fraction (IC: MC) obtained by this procedure shows long antigenic stability and the same antigen lot can be used ~ eda, 1995; Flores-de-Castan ~ eda, for several years (Flores-de-Castan ~ eda, 2002a; Flores-de-Castan ~ eda, 2002b; 1999; Flores-de-Castan ~ o et al., 2000). A real challenge Flores et al., 2005; Tamez- Trevin of poor countries remains: to have reliable diagnostic methods, at low cost and applicable in endemic areas for amoebiasis. The aim of this study was to develop an ELISA to diagnose invasive amoebiasis using stable antigens with long shelf-time prepared without enzymatic inhibitors, and validate its clinical use in an area of amoebic endemicity. 2. Material and methods 2.1. Patients and volunteers 2.1.1. Patients with amoebic liver abscess Group of patients with acute amoebic liver abscess Sixty-six patients admitted to the University Hospital “Jose Eleuterio Gonzalez” at Monterrey Nuevo Leon, Mexico. Patients were diagnosed with amoebic liver abscess (ALA) by clinical symptoms, ultrasound image and/or axial tomography (CT) and positive response to antiamoebic drug treatment. Fifty percent of patients required percutaneous drainage. The drained fluid was microbiologic cultured to discard bacterial infection. Stool samples were not examined due to previous reports indicating that only 10% of patients with liver abscess release amebas in stool. (Katzenstein et al., 1982) Serum from each patient was tested by the Cellognost Amoebiasis Indirect Hemagglutination Assay (Behring Diagnostics GmbH, Germany). All patients in this group were positive by Western Blot to diagnose ~ eda, 1999; Flores-deinvasive amoebiasis (Flores-de-Castan ~ eda, 2002b). Castan 2.1.2. Healthy volunteers Group of healthy volunteersThirty-three volunteer students of noma de Nuevo Leo n. Participants in this the Universidad Auto group had neither previous invasive amoebiasis, nor any episode of bloody diarrhea within the previous year. Participants were
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negative at the direct examination for parasites in feces (Faust et al., 1970). They were negative at IHA test (Behring Diagnostics GmbH, Germany) and Western Blot for invasive amoebiasis (Flores-de~ eda, 1999; Flores-de-Castan ~ eda, 2002b). The healthy volunCastan teers lived in an endemic area for amoebiasis located in the northeastern of Mexico. 2.2. E. histolytica 2.2.1. E. histolytica culture HM1-IMSS E. histolytica trophozoites were cultured axenically into borosilicate glass culture tubes, in TYI-S-33 medium supplemented with vitamins and heat inactivated adult bovine serum (Laboratorios Microlab. Mexico) according to Diamond (Diamond, 1968). 2.2.2. Amoebic antigen preserved without enzymatic inhibitors (ELISA-IC: MC) E. histolytica IC: MC fraction was obtained according to Flores ~ eda, 1995; Flores-de-Castan ~ eda, 1999; Flores-de(Flores-de-Castan ~ eda, 2002a; Flores-de-Castan ~ eda, 2002b; Flores et al., Castan 2005). Briefly: the trophozoites were harvested at the end of the logarithmic growth phase and washed with 0.15 M buffered phosphate saline, pH 7.4 (PBS). The pellet was frozen-dried, suspended in chloroform: methanol 20:1 (vol: vol), mixed and centrifuged. The polar solvent insoluble fraction obtained was dried to eliminate residues of solvents and stored at room temperature. Prior to be used, the fraction was resuspended in 0.1 M carbonate buffer pH 9.6 urea 8 M and the mixture boiled in water bath. Supernatant protein concentration was determined according to the Bradford method (Bradford, 1976) The IC: MC fraction might be kept frozen in solution or dried, and stored for up to three years without protein degradation. 2.3. Enzyme-linked immunosorbent assays 2.3.1. Enzyme-linked immunosorbent assay using amoebic antigens preserved without enzymatic inhibitors. (ELISA-IC: MC) The ELISA was performed according to Flores (Flores-de~ eda, 1999; Flores-de-Castan ~ eda, 2002b). Briefly: One hunCastan dred microliters of solution containing 25 mg/mL of the IC: MC fraction supernatant in 0.1 M carbonate buffer pH 9.6 urea 8 M, were incubated overnight at 4 C in order to immobilize the antigen on Corning® polystyrene, flat bottom plates. After washing (0.1 M PBS, 0.05% Tween-20, pH 7.4), the plates were incubated 30 min at 37 C with 300 mL of blocking buffer (2% Bovine serum albumin, 0.1 M PBS, 0.05% Tween-20, pH 7.4, 0.02% thimerosal, pH 7). One hundred microliters per well of each serum were incubated two hours at room temperature and then washed three times. Horseradish peroxidase-conjugated to anti-human immunoglobulin produced in goat was added as secondary antibody. The secondary antibodies tested included anti-human polyvalent immunoglobulins (whole molecule), anti-human IgG (g-chain specific), antihuman IgM (m-chain specific), and anti-human IgA (a-chain specific). The immune reaction was developed using H2O2 as substrate and 3.3-diaminobenzidine tetrahydrochloride as chromogen. The colorimetric reaction absorbance was measured at 490 nm (Microplate reader BIO-RAD. Hercules CA, USA). 2.3.2. Enzyme-linked immunosorbent assay using antigens from untreated trophozoites The ELISA was performed with modifications of above test. Briefly: One hundred microliters of solution containing 25 mg/mL of the freeze dried total trophozoites in 0.1 M carbonate buffer pH 9.6 urea 8 M, were incubated overnight at 4 C to immobilize the
antigen on Corning® polystyrene, flat bottom plates. 2.4. Calculation 2.4.1. Determination of intra-assay and interassay variation ELISA absorbance Index (AI): ALA serum absorbance/control negative serum absorbance. The cut off was calculated with the absorbance mean of negative sera plus one standard deviation. Intra Assay coefficient of variation (intra-assay CV). To determine the deviation within the same plate, ten samples were measured ten times each, on the same plate and assay. Intra assay CV ¼ (The standard deviation of ten measures/AI mean) x 100. Inter-Assay coefficient of variation (inter-assay CV). To determine the deviation within different plates, ten samples were measured in five different plates. Inter-Assay CV ¼ (The standard deviation of the five measurements/AI mean) x 100. 2.4.2. ELISA units (EU) The ELISA units were obtained by the next equation: EU ¼ (100/optical density of control positive sera) x optical density of sample. 2.4.3. Statistics and diagnostic utility parameters Utility parameters were calculated using Friedman Nonparametric Statistics formulated in a 2 2 contingency table. To determine the Diagnostic utility, the parameters of Sensitivity; Specificity; Positive predictive value; Negative predictive value and Accuracy were calculated (Corder and Foreman, 2009; Shankar et al., 2008). 2.5. Indirect haemagglutination assay (IHA) Cellognost Amoebiasis kit was used to test the sera IHA titters. It was performed following the kit instructions (Behring Diagnostics GmbH, Germany). Positive antibody titer 1: 256 sera dilution. 2.6. Stool microscopic identification of parasites Each healthy volunteer provide three fresh stool specimens. Fecal samples emulsified in 10% neutral formalin, were concentrated by flotation (Faust et al., 1970). Each sample was directly examined under the microscope light to search intestinal parasites. 2.7. Chemicals The products, whose origins were not indicated, were reagents from Sigma, St. Louis, MO, USA, or Bio-Rad Laboratories, Richmond, CA, USA. 3. Results 3.1. ELISA assays variability First, assays were run to determine the absorbance index variation in the ELISA tests made with antigens from total trophozoites or ELISA- IC: MC. Checkerboard titration experiments were performed to find the suitable assay conditions and establish the adequate background and signal to noise ratios. As shown in Table 1, absorbance indexes were calculated from the absorbance data of 4 sera samples obtained from ALA patients. Next, each sample was measured in ten wells using the same plate to obtain the intra assay coefficient of CV. As predicted, we observed that AI obtained using sera of patients with ALA, is greater compared to the serum used as negative control. The values represented the arithmetic mean ± standard deviation of absorbance
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Table 1 ELISA assays variability. Sera
ELISA-Total untreated trophozoites Mean AI ± SD (intra assay CV)
1 2 3 4
5.8 3.9 4.8 4.8
± ± ± ±
0.32 0.45 0.65 0.52
(5.5) (11.5) (13.5) (10.8)
ELISA-IC:MC AI variation of the same serum
Mean AI ± SD (intra assay CV)
13.2% 28.5% 41.6% 38.4%
3.5 2.7 3.7 2.8
± ± ± ±
0.18 0.10 0.19 0.25
(5.1) (3.7) (5.1) (8.9)
AI variation of the same serum 10% 9% 11% 13%
Absorbance index: AI, Variation AI: Variation among the highest AI value and the lowest AI value of the same serum. Intra assay coefficient of variation ¼ (The standard deviation of ten measures/AI mean) x 100. Serum from healthy volunteer was used as negative control. The values represented the arithmetic mean ± standard deviation of absorbance obtained for each serum by ten repetitions.
obtained for each serum by ten repetitions. Notably, there is more variation in the ELISA performed with total trophozoites than the ELISA-IC: MC fraction as antigen. Finally, we observed that high and low AI values varied among the same serum, from 13 to 41%, in contrast with variations from nine to 13% in the ELISA - IC: MC as antigen. To obtain the inter-assay coefficient of variation of the ELISAeIC: MC, we tested five different sera samples in five different assays. We observed that the data range between 7 and 13%. 3.2. Antibody isotypes to E. histolytica Next, in order to determine the best option as secondary antibodies in the ELISA test, experiments were performed to identify the immunoglobulin isotypes produced in ALA patients. We evaluated the following antibodies: anti-human polyvalent immunoglobulin (whole molecule), anti-human IgG (g-chain specific), antihuman IgM (m-chain specific), and anti-human IgA (a-chain specific). Fig. 1 shows the ELISA units obtained using sera of patients with ALA and from healthy volunteers. We clearly observed that antibodies binding to isotypes IgA and IgG allowed significant differences between the two groups, nevertheless sera of patients with ALA exhibit a wide dispersion production of IgA antibodies. Taken together, these results show that IgA and IgG are the best immunoglobulins to target amoebic antigens; The total Antihuman polyvalent immunoglobulins allowed detecting differences between the two groups of sera, therefore anti-human polyvalent immunoglobulins will be selected as secondary antibodies for the ELISA test.
3.3. ELISA-IC:MC of the sera from healthy volunteers and patients with ALA Then, collected sera from 66 ALA patients and 33 healthy volunteers were tested in this study. Fig. 2 shows the global reactivity distribution of anti-amoeba antibodies against preserved antigens represented as ELISA-IC:MC units. We estimated an optimal cut off value of 72 ELISA units (EU), with a grey zone ranging from 72 to 80 EU. Consequently, a positive result will contain 80 ELISA units (EU) and a negative result 72 EU. Indeed, sera from 95% ALA patients showed a higher value than the cut off, one serum was within the cut off value and one serum was below, giving a false negative value. As a note, only one of the samples from healthy volunteers was positive, eight were on the gray zone, and the rest of the samples resulted in negative values. The IHA is thus far the assay used by clinical laboratories in poor endemic countries, to diagnosis antibodies to amoebiasis. In addition, we tested the entire ALA patients and healthy volunteer sera by IHA assay, to compare the clinical utility of this test and the ELISA-IC:MC assay developed with preserved E. histolytica antigens without using enzymatic inhibitors. Eighty one % of the sera were positive on IHA and 18.4% showed false negative results, in contrast, the ELISA-IC:MC detected 97% of patients with ALA and it gave 1.5% false negatives. We determine the clinical utility parameters of ELISA-IC:MC and IHA, as shown in Table 2. The IHA values demonstrate that a positive result has 96% probability to be a true positive result. Nevertheless, a negative result has only 73% probability to be a true negative result. Meanwhile ELISA-IC:MC test has 98% probability to be a true positive result, and a negative result has
Fig. 1. Antibody isotypes to Entamoeba histolytica produced by patients with ALA and healthy volunteers living in endemic region.
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Fig. 2. ELISA-IC:MC graphic data distribution of the sera from healthy volunteers and patients with ALA.
Table 2 Clinical parameters obtained by ELISA-IC:MC and IHA tests.
Sensitivity Specificity Positive predictive value Negative predictive value Accuracy
ELISA-IC:MCa
IHA
98% 96% 98% 96% 98%
81% 97% 96% 73% 87%
IHA: Indirect haemagglutination assay (Cellognost Amoebiasis). a ELISA using preserved E. histolytica antigens without enzymatic inhibitors.
96% probability to be a true negative result. ELISA-IC:MC presented more accuracy than IHA. 4. Discussion In areas of high endemicity of amoebiasis, physicians have the training that allows them to efficiently diagnose this parasitosis, however some acute ALA patients present a negative value by IHA test and clinical features suggesting other pathologies. In these clinical cases, it would be desirable to have a quick and inexpensive diagnostic test feasible to be used in non-specialized laboratories. To standardize reliable immune-based tests is important to use inhibitors to prevent the risk of lack of reproducibility of results due to proteolysis (Serrano-Luna et al., 2013; AraizaeOrozco et al., 1999; ~ oz et al., 1990, 1984; Lopez-Revilla et al., 1992; Vila et al., 1985; Mun Lopez-Revilla and Baez-Camargo, 1992; Perez-Montfort et al., 1987). In this study we employed amoebic extracts preserved without the use of enzymatic inhibitors as the antigen in ELISA test. Our group established that this method preserves amoebic antigens and decreases the proteases activity from trophozoites extracts ~ eda, 1995; Flores-de-Castan ~ eda, 1999; Flores-de(Flores-de-Castan ~ eda, 2002a; Flores-de-Castan ~ eda, 2002b; Flores et al., Castan 2005). With stable antigens, it is possible to develop tests with good reproducibility, even using the same antigen lot for long periods of time. In our laboratory we have tested the reproducibility of some commercial ELISA kits and they presented a significant interassay variation (data not shown). For this reason we decided to
compare the ELISA made with antigens preserved without using enzymatic inhibitors versus an ELISA made with antigens from total trophozoites developed by ourselves. The ELISA made with total antigenic extract presented great variation among the repetitions of the same serum, in contrast, the ELISA test using the IC: MC fraction as antigen has the best suitable reproducibility inter and intra assay. During this study, we identified that the immunoglobulin isotypes produced against amoebic antigens are mainly IgG and IgA. The IgA values distribution showed greater scattered than IgG titers. These results agree with previous reports (Carrero et al., 1994; Abioye et al., 1972). Antibodies anti-human polyvalent immunoglobulin (whole molecule) were selected as secondary antibodies as they serve to demonstrate a statistically significant difference response of antibodies to E. histolytica in patients with ALA, regarding healthy volunteers with no history of amoebiasis. Our results add a relevant contribution in the diagnosis of amoebiasis. The ELISA made with IC: MC fraction revealed that the sera from 95% ALA patients had a higher value than the cut off, and 97% of the sera from healthy people had a lower value than the cut off. Healthy individuals living in endemic areas of amoebiasis can contain antibodies to E. histolytica, despite this; the ELISA allows a clear difference between sera of ALA patients from healthy volunteers. Previous reports have shown that IHA is the assay frequently used by clinical laboratories in endemic countries. In the work presented here, we describe the development of an ELISA made with preserved E. histolytica antigens without enzymatic inhibitors, that shows a better clinical diagnostic parameter than the IHA test. Our results explain the seroprevalence differences found in Mexico as reported by Gonzalez et al. (Gonzalez et al., 1995) in the same sampling frame studied using two different tests. The data obtained in different studies will vary considerably according to the type of assay used. In cases of acute ALA, a negative serologic test does not rule out the diagnosis of invasive amoebiasis. Therefore, the parameters of Sensitivity, Specificity, Positive and Negative predictive value and Accuracy indicated that the ELISA made with antigens preserved without enzymatic inhibitors is a reliable
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diagnostic test useful in endemic zones of amoebiasis and affordable to poor countries. Overall, the ELISA for invasive amoebiasis made with antigens preserved without using enzymatic inhibitors has valuable diagnostic use even in populations living in endemic zones of amoebiasis carrying antibodies against amebas. 5. Conclusions The ELISA-IC:MC for invasive amoebiasis made with antigens preserved without using enzymatic inhibitors has valuable diagnostic value even in populations living in endemic zones of amoebiasis carrying antibodies against amebas. ELISA-IC:MC presented better diagnostic parameters than IHA. In cases of acute ALA, a negative serologic test does not rule out the diagnosis of invasive amoebiasis. Conflicts of interest All authors declare that they have no conflict of interest. Ethical approval This research protocol and the consent forms were approved by the Research and Bioethics Committee of the Faculty of Biological Sciences. The research has been performed in accordance with the ethical standards of Declaration of Helsinki. Acknowledgments Eleuterio We acknowledge the Hospital Universitario “Jose n, Me xico for providing support. Gonz alez” at Monterrey Nuevo Leo This project was supported by grants of PAICYTe Universidad noma de Nuevo Leo n and SEP-CONACYT grant 25617. Auto References Abd-Alla, M.D., Jackson, T.F., Reddy, S., Ravdin, J.I., 2000. Diagnosis of invasive amoebiasis by enzyme-linked immunosorbent assay of saliva to detect amoebic lectin antigen and anti-lectin immunoglobulin G antibodies. J. Clin. Microbiol. 38 (6), 2344e2347. Abioye, A.A., Lewis, E.A., McFarlane, H., 1972. Clinical evaluation of serum immunoglobulin in amoebiasis. Immunol 23 (6), 937e946. Agundis Mata, C., Blanco, F., Toledo, J., Reta, F., Kumate, J., Isibasi, A., 1996. Identification of a 35 kDa glycoprotein from Entamoeba histolytica by sera from patients with amoebic liver abscess and with mouse monoclonal antibody. Immunol. Investig. 25 (5e6), 519e529. ~ oz, M., Arias-Negrete, S., 1999. Entamoeba histoAraiza-Orozco, L.M., Avila, E., Mun lytica: surface proteolytic activity and its relationship with in vitro virulence. Folia Parasitol. 46 (3), 161e167. Bradford, M., 1976. A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248e254. Caballero-Salcedo, A., Viveros-Rogel, M., Salvatierra, B., Tapia-Conyer, R., Sepúlvedarrez, G., Ortiz-Ortiz, L., 1994. Seroepidemiology of amoebiasis in Amor, J., Gutie Mexico. Am J. Trop Med. Hyg 50, 412e419. Carrero, J.C., Díaz, M.Y., Viveros, M., Espinoza, B., Acosta, E., Ortiz-Ortiz, L., 1994. Human secretory immunoglobulin A anti-Entamoeba histolytica antibodies inhibit adherence of amebae to MDCK cells. Infect. Immun. 62 (2), 764e767. Corder, G.W., Foreman, D.I., 2009. Nonparametric Statistics for Non-statisticians: a Step-by-step Approach. John Wiley & Sons, Inc., New Jersey. Diamond, L.S., 1968. Techniques of axenic cultivation of Entamoeba histolytica Schaudinn, 1903 and E. histolytica-like ameba. J. Parasitol. 54 (5), 1047e1056. Faust, E.C., Russell, P.F., Jung, R.C., 1970. In: Craig and Faust's Clinical Parasitology, eighth ed. Lea &Febiger, US, pp. 83e101. ~ o, E., Castan ~ eda, F., Tijerina-Menchaca, R., Flores, M.S., Tamez-Trevin
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