Entamoeba bangladeshi nov. sp., Bangladesh.

: TO THE EDITOR: Diarrheal diseases have a major effect on global health, particularly the health of malnourished children (1). The enteric parasites Entamoeba histolytica and E. moshkovskii are potential causes of diarrheal disease in children (2). For the past 20 years, we have been studying Entamoeba infections in children from the urban slum of Mirpur in Dhaka, Bangladesh (3)

Species-Specific Immunodetection of an Entamoeba histolytica Cyst Wall Protein.

Entamoeba histolytica causes intestinal disease in endemic settings throughout the world. Diagnosis of E. histolytica infection would be improved by the identification of biomarkers that are expressed by cysts of E. histolytica, but not by cysts of closely related commensal species of Entamoeba. Herein, we describe two novel monoclonal antibodies (1A4 and 1D3) produced against a spacer region of the E. histolytica Jacob2 lectin, an outer cyst wall protein. These reagents demonstrated no cross-reaction to E. dispar recombinant antigen and low picomolar molecular detection limits when paired in ELISA sandwich assays. In an immunofluorescence microscopy assay, the α-Jacob2 murine antibodies labeled cysts of three xenically cultured E. histolytica isolates but did not label cysts of three E. bangladeshi isolates. Monoclonal antibody 1A4 did not cross-react with xenic cultures of three E. dispar isolates, demonstrating specificity to E. histolytica, while monoclonal antibody 1D3 cross-reacted with two out of three E. dispar isolates. Both antibodies labeled cysts in formalin-fixed slides, a potential logistical advantage in some settings. The monoclonal antibody 1A4 was also used in an immunofluorescence microscopy assay with formalin-fixed stool specimens. Seven out of ten ELISA-positive stool specimens exhibited 1A4-labeled cyst-like objects, compared to one out of seven ELISA-negative specimens. These results demonstrate that antibodies generated against the flexible spacer of E. histolytica Jacob2 lectin recognize and bind to Jacob2 protein in whole cysts and are capable of differentiating Entamoeba species in fixed specimens. Thus, Jacob2 is a promising biomarker for use in diagnosing E. histolytica infection

ClustalW alignment of the Jacob2 proteins from <i>Entamoeba histolytica</i> HM-1:IMSS (EHI_044500) and <i>Entamoeba dispar</i> SAW760 (EDI_246160).

<p>The ClustalW BLOSUM matrix was used with a gap open cost of 10 and a gap extension cost of 0.1. This figure was generated in Geneious 6.0.3. Cloned residues for “EhJacob” and “EdJacob” recombinant antigens are highlighted in light gray and dark gray respectively.</p

<i>Entamoeba</i> species specificities of a-Jacob antibodies 1A4 (A) and 1D3 (B) in immunofluorescence microscopy.

<p>Smears of xenic <i>Entamoeba</i> isolates were doubly stained with 0.1% Calcofluor White M2R and an anti-Jacob primary antibody with goat anti-mouse Alexa-Fluor 488. Each data point represents the mean Alexa Fluor 488 intensity of a Calcofluor-positive, cyst-like object detected by microscopy. The data points are arranged horizontally by isolate, and black horizontal bars represent the mean Alexa Fluor 488 intensity of each isolate. The overall mean Alexa Fluor 488 intensities of the <i>Entamoeba histolytica</i> isolates (n = 3) were compared to those of <i>E</i>. <i>dispar</i> isolates (n = 3) and of <i>Entamoeba bangladeshi</i> isolates (n = 3) with the Mann-Whitney U test. <i>Eh</i> = <i>Entamoeba histolytica</i>, <i>Ed = Entamoeba dispar</i>, <i>Eb = Entamoeba bangladeshi</i>.</p

Representative photos from an anti-Jacob2 immunofluorescence microscopy assay.

<p>Isolates were doubled stained with 0.1% Calcofluor White M2R and primary anti-Jacob2 monoclonal antibody (1A4 or 1D3) with goat anti-mouse Alexa Fluor 488. The three species examined were pathogen <i>Entamoeba histolytica</i> (1^st column) and commensals <i>Entamoeba dispar</i> and <i>Entamoeba bangladeshi</i> (2^nd and 3^rd columns). Calcofluor was utilized to identify chitinaceous <i>Entamoeba</i> cysts (2^nd and 4^th rows).</p

Summary of screens for α-Jacob murine monoclonal antibodies.

<p>Summary of screens for α-Jacob murine monoclonal antibodies.</p

Monoclonal antibodies 1A4 and 1D3 detecting <i>Entamoeba</i> recombinant Jacob2 antigens in a sandwich ELISA.

<p>Measured antigen concentration ranged from 2.4–2500 pM. Columns and error bars represent mean OD450 ± standard deviation of three replicate assays. Limit of detection (LOD) was calculated as three standard deviations above the mean OD450 of EdJacob at 2500 pM concentration.</p

Species-Specific Immunodetection of an <i>Entamoeba histolytica</i> Cyst Wall Protein

<div><p><i>Entamoeba histolytica</i> causes intestinal disease in endemic settings throughout the world. Diagnosis of <i>E</i>. <i>histolytica</i> infection would be improved by the identification of biomarkers that are expressed by cysts of <i>E</i>. <i>histolytica</i>, but not by cysts of closely related commensal species of <i>Entamoeba</i>. Herein, we describe two novel monoclonal antibodies (1A4 and 1D3) produced against a spacer region of the <i>E</i>. <i>histolytica</i> Jacob2 lectin, an outer cyst wall protein. These reagents demonstrated no cross-reaction to <i>E</i>. <i>dispar</i> recombinant antigen and low picomolar molecular detection limits when paired in ELISA sandwich assays. In an immunofluorescence microscopy assay, the α-Jacob2 murine antibodies labeled cysts of three xenically cultured <i>E</i>. <i>histolytica</i> isolates but did not label cysts of three <i>E</i>. <i>bangladeshi</i> isolates. Monoclonal antibody 1A4 did not cross-react with xenic cultures of three <i>E</i>. <i>dispar</i> isolates, demonstrating specificity to <i>E</i>. <i>histolytica</i>, while monoclonal antibody 1D3 cross-reacted with two out of three <i>E</i>. <i>dispar</i> isolates. Both antibodies labeled cysts in formalin-fixed slides, a potential logistical advantage in some settings. The monoclonal antibody 1A4 was also used in an immunofluorescence microscopy assay with formalin-fixed stool specimens. Seven out of ten ELISA-positive stool specimens exhibited 1A4-labeled cyst-like objects, compared to one out of seven ELISA-negative specimens. These results demonstrate that antibodies generated against the flexible spacer of <i>E</i>. <i>histolytica</i> Jacob2 lectin recognize and bind to Jacob2 protein in whole cysts and are capable of differentiating <i>Entamoeba</i> species in fixed specimens. Thus, Jacob2 is a promising biomarker for use in diagnosing <i>E</i>. <i>histolytica</i> infection.</p></div

Immunofluorescence staining and image analysis of stool specimens stained with antibody 1A4.

<p>ELISA-positive (triangle) and ELISA-negative (square) specimens were stained with DAPI and antibody 1A4 as described in the text. Eight specimens (open symbols) exhibited no detectable cyst-like objects under the DAPI filter. The bottom panels show a typical cyst detectable by (left to right) bright field, DAPI staining, and antibody 1A4 staining microscopy conducted on a fixed stool slide.</p