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Journal of Parasitic Diseases: Official Organ of the Indian Society for Parasitology logoLink to Journal of Parasitic Diseases: Official Organ of the Indian Society for Parasitology
. 2022 Apr 26;46(3):714–721. doi: 10.1007/s12639-022-01490-6

Development of a salivary IgA detection method for accurate diagnosis of amebiasis

Davin Edric V Adao 1, Angeline Odelia C Li 1, Alexander Edward S Dy 1, Windell L Rivera 1,✉
PMCID: PMC9458784  PMID: 36091277

Abstract

An amebiasis detection method was developed based on identifying anti-Entamoeba histolytica IgA in the saliva of infected individuals. The enzyme-linked immunosorbent assay (ELISA)-based detection method was tested along with microscopy and polymerase chain reaction (PCR) on saliva and stool samples from 110 asymptomatic individuals visiting the Manila Health Department − Public Health Laboratory of the City of Manila, Philippines. A receiver operating curve (ROC) was constructed to compare the ELISA results with PCR results. E. histolytica infection was detected in 18 of the 110 individuals. The developed method had an accuracy of 90%, sensitivity of 88.89%, specificity of 90.22%, positive predictive value of 64%, and negative predictive value of 97.65% if a 1:2 dilution of crude saliva sample in phosphate-buffered saline (PBS) was used for diagnosis when compared to PCR. The area under the curve (AUC) of the ROC was 0.9436 if a 1:2 dilution of a crude saliva sample was used. The developed assay presents an easy and accurate method of detecting amebiasis in infected individuals using saliva samples instead of stool or blood samples and has potential applications in both diagnosis and epidemiological studies.

Keywords: Amebiasis, ELISA, Entamoeba histolytica, IgA, Saliva

Introduction

Entamoeba histolytica is a protozoan parasite that causes the intestinal infection known as amebiasis, as well as other extraintestinal infections such as amebic colitis and amebic liver abscess. It is endemic to Central and South America, Asia, and Africa with a prevalence as high as 40% and causes an estimated 55,000 deaths per year (Shirley et al. 2018). Diagnosis of E. histolytica infection is usually through microscopic detection in stool samples. This method cannot distinguish between E. histolytica and its non-invasive relative Entamoeba dispar, or other Entamoeba species (Tanyuksel and Petri 2003). Other methods and kits are also available, most notably enzyme-linked immunosorbent assay (ELISA)-based antigen detection kits such as the TechLab II E. histolytica antigen detection kit (Haque et al. 2000) and the E. histolytica Quik Chek fecal antigen detection kit (Korpe et al. 2012). However, these kits usually require either stool or serum samples for diagnosis (Tanyuksel and Petri 2003; Fotedar et al. 2007a). There have been several studies focusing on the diagnosis of E. histolytica infection through detection of antibodies in saliva samples of patients (del Muro et al. 1990; Ramos et al. 1997, 2005). Specifically, these rely on salivary IgA antibodies, which prevent the adhesion of E. histolytica cells to human cells (Carrero et al. 1994; Haque et al. 2001). The use of saliva samples for diagnosis of E. histolytica infection instead of stool and serum samples is advantageous because it is a less cumbersome, non-invasive method of collecting samples, reducing the risk of contaminating either the patient or the medical technician taking samples.

This study aimed to develop and test an ELISA-based anti-E. histolytica IgA detection method in saliva samples for a safe, rapid, and accurate diagnosis of E. histolytica infection. Salivary anti-E. histolytica IgA is produced during induction of amebic dysentery or amebic liver abscess infection (Valenzuela et al. 2001), possibly as a response to preventing intestinal re-infection (Carrero et al. 2007). Results of the method were compared with polymerase chain reaction (PCR) results from stool DNA extracts since the PCR method has been proven to be a “gold standard” when it comes to E. histolytica diagnosis due to its high sensitivity, specificity, and accuracy compared to other methods (Mirelman et al. 1997; Gonin and Trudel 2003; Tanyuksel and Petri 2003; Fotedar et al. 2007a, b; Stark et al. 2008). Moreover, the developed diagnostic method was compared to PCR instead of other antibody detection kits because the former is better than commercially available antigen and antibody detection kits (Mirelman et al. 1997; Gonin and Trudel 2003; Stark et al. 2008). Also, it is difficult to compare antigen and antibody detection kits with each other due to non-standardized complex antigen and antibody preparations which vary between techniques and kits (Kraoul et al. 1997; Fotedar et al. 2007a).

Materials and methods

Cultivation of E. histolytica

The axenic E. histolytica strain HK9 used in this study was cultured in BI-S-33 medium in 8- or 9-ml screw-capped tubes (Diamond et al. 1978). Cultures were maintained at 35.5 °C.

Sample collection

Stool and saliva samples were collected from 110 individuals who applied for a health permit at the Manila Health Department − Public Health Laboratory of the City of Manila, Philippines. Samples were stored frozen at − 4 °C until use. The protocol for collection of stool and saliva samples was approved by the Philippine Department of Health Research Ethics Committee.

Microscopy of stool samples and DNA extraction

Stool samples were subjected to a formalin-ether concentration technique (FECT) to concentrate E. histolytica cysts. The FECT products were examined under a microscope to identify parasitic worm eggs and protozoan cysts. Approximately 10 µl of FECT products were stained with Lugol’s iodine for microscopic observation.

DNA was extracted from these FECT products using a freeze-thaw organic DNA extraction technique (Rivera et al. 1996). Briefly, FECT products suspended in 100 µL of phosphate-buffered saline (PBS) were exposed to 5 min at 50 °C–65 °C followed by 5 min at − 4 °C. This alternating temperature exposure was repeated 5 times to ensure that E. histolytica cysts were ruptured. FECT products were then incubated with 0.25 mg/ml Proteinase K for 3 h at 55 °C followed by mixing with phenol:chloroform:isoamyl alcohol to separate proteins and other cellular materials and collect the nucleic acids. DNA was then precipitated through overnight incubation with 95% ethanol at − 4 °C and suspended in Tris-EDTA (TE) buffer.

PCR

Presence of E. histolytica and E. dispar in stool samples was determined through PCR of stool DNA extracts using the primer pairs P11/P12 and P13/P14, respectively, using a previously described protocol (Tachibana et al. 1991). PCR products were run through a 2% gel and results visualized under UV light. Detection of E. histolytica using PCR was chosen for comparison due to its high sensitivity and specificity, which makes it suitable as a “gold standard” (Tanyuksel and Petri 2003; Fotedar et al. 2007a, b; Stark et al. 2008).

E. histolytica membrane extraction

Approximately 106 E. histolytica HK9 cells were collected and homogenized in Tris-MgCl2-phenylmethylsulfonyl fluoride (PMSF) buffer using a sterile glass homogenizer. Homogenized membranes were collected through centrifugation at 13,200 rpm for 1.5 h and were later re-suspended and stored in 2X PBS at − 4 °C until use. Protein content was then measured using a Bradford assay. These samples were then diluted to 10 µg/ml with 0.1 M carbonate buffer with NaN3.

ELISA

The presence of anti-E. histolytica IgA in saliva samples was determined using ELISA. Wells of flat-bottomed 96-well plates were coated with 10 µg/ml of crude E. histolytica membrane through overnight 4 °C incubation. These plates were then stored at − 4 °C until use. Only plates frozen for about 1–2 weeks were used to initially determine the presence of anti-E. histolytica antibodies in saliva samples to avoid the effects of prolonged storage on efficacy of the developed assay. Antigen plates were blocked with 3% skim milk in PBS for 1 h and washed. Plates were incubated with saliva samples in 1:2, 1:4, and 1:8 dilutions for 2 h. These were then incubated with anti-human IgA conjugated with alkaline phosphatase (1:5000 dilution) for 1 h. Afterwards, samples were incubated for 30 min with 1 mg/ml of p-nitrophenylphosphate in diethanolamine buffer. Samples were read at 405 and 620 nm. The results used for analysis were 405 nm optical density (OD) readings subtracted with 620 nm OD readings (reference wavelength) to remove background reading.

Determination of expiration date and effect of delay before reading

Plates stored at − 4 °C for one year were used for the expiration date test. Two saliva samples each from PCR and ELISA positive individuals and PCR and ELISA negative individuals were used for testing. ELISA was performed as stated above. The plates were read after 3–10 min, 11–18 min, and 20–27 min after removal from 37 °C incubation with the developing solution to test the effects of delayed readings.

Statistical analysis

A receiver operating characteristic (ROC) curve was constructed based on the data collected from ELISA and PCR. The online program JROCFIT was used to construct the ROC curves as well as compute for sensitivity, specificity, positive predictive value, negative predictive value, and accuracy for 1:2, 1:4, and 1:8 saliva dilutions. A 6-point category was created based on both natural breaks in the data (Sonis 1999) and a positive cut-off point using the method of Frey et al. (1998) to rate the results (Table 1). All readings from PCR negative samples were used to compute the cut-off point using the formula:

Table 1.

Rating categories used on ELISA results with cut-off values computed using the method of Frey et al. (1998) on all the absorbance readings of PCR negative samples. Number of samples (n) = 92, DF = 91, and Student’s t-value at (α = 0.95) = 1.662

Dilution 1:2 1:4 1:8
Mean OD (x̅) 0.0718 0.0704 0.0632
Standard deviation (SD) 0.0303 0.0340 0.0325
Cut-off value 0.1225 0.1273 0.1175
Rating category Diagnosis 1:2 1:4 1:8
1 Definitely negative 0.00–0.052 0.00–0.029 0.00–0.037
2 Probably negative 0.053–0.070 0.030–0.070 0.038–0.067
3 Possibly negative 0.071–0.1225 0.071–0.1272 0.068–0.1175
4 Possibly positive 0.1226–0.170 0.1273–0.156 0.1176–0.134
5 Probably positive 0.171–0.220 0.157–0.170 0.135–0.190
6 Definitely positive 0.221 and above 0.171 and above 0.191 and above
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Cut-off = x̅ + SDf.

where x̅ is the average OD reading of the PCR negative samples, SD is the standard deviation, and f is Inline graphic The value t is the one-tailed Student’s t-test value at n-1 degrees of freedom (DF) where n is the number of samples. Some PCR negative samples with unusually high readings were tested twice and the average readings were used for statistical analyses. Chi-square (χ2) tests were used to find any associations between the prevalences of E. histolytica infection detected using PCR with age and sex of the patients using the program JASP 0.14.1.0.

Results

Collected samples

Stool and saliva samples were taken from 110 individuals aged 16–58 years old with 46 males and 64 females. Most of the subjects were aged 16–25 years old (n = 78). Of the 110 individuals, 18 (16.36%) of them had stool samples that tested positive for E. histolytica in PCR while none tested positive for E. dispar. Fifteen of the 18 individuals with E. histolytica detected using PCR were aged 16–25 years old. Chi-square tests show no association between presence of E. histolytica using PCR detection and sex or age group of individuals (Table 2). Results of microscopy show that 81 out of the 110 individuals had neither worm eggs nor protozoan cysts in their stool samples. In all, there were 8 parasites identified in this study, with Entamoeba sp. found in 8 individuals (Table 3). Only two of these individuals with Entamoeba sp. were positive for E. histolytica in both PCR and ELISA while one was positive only for ELISA and not PCR.

Table 2.

Chi-square test results of association between results of PCR detection of Entamoeba histolytica with sex or age group of patients

PCR PCR
Sex Negative Positive Total Age group Negative Positive Total
Female 54 10 64 16–25 58 15 73
Male 38 8 46 26–35 28 2 30
Total 92 18 110 36–45 3 0 3
46–55 2 1 3
56–65 1 0 1
Total 92 18 110
Value DF p Value DF p
χ2 0.061 1 0.805 χ2 4.409 4 0.353
N 110 N 110
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Table 3.

Microscopy results of stool samples from 110 individuals

Parasite Number of individuals infected Prevalence (%)
No parasites found 81 73.63
Entamoeba sp. 8 9.88
Ascaris lumbricoides 5 4.54
Blastocystis sp. 6 5.45
Dientamoeba fragilis 1 0.91
Iodamoeba butschlii 1 0.91
Endolimax nana 2 1.82
Trichuris trichiura 2 1.82
Hookworm sp. 6 5.45
Unknown worms 4 3.63
Unknown protozoan cysts 2 1.82
Mixed infections 6 5.45
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ELISA and ROC curve results

The cut-off values at each saliva sample dilution were computed using the average and standard deviation of OD readings of all 92 PCR negative samples (Table 1). The t-test value at α = 0.95 and DF = 91 was 1.662 and the computed f was 1.671. The use of 1:2 saliva dilution produced the best results for anti-E. histolytica salivary IgA detection using ELISA. It produced the highest area under curve (0.9436; Fig. 1), sensitivity (88.9%), specificity (90.22%), positive predictive value (64%), and negative predictive value (97.65%) compared to using 1:4 or 1:8 saliva dilutions (Table 4).

Fig. 1.

Fig. 1

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Receiver operating characteristic (ROC) curves constructed using ELISA results from 1:2, 1:4, and 1:8 saliva dilutions and areas under the curve (AUC). The figure was constructed using the online program, JROCFIT

Table 4.

Sensitivity, specificity, positive predictive value, negative predictive values and accuracy of the developed anti-Entamoeba histolytica salivary IgA ELISA detection method using three different saliva sample dilutions (1:2, 1:4, 1:8) when compared to PCR detection in stool samples

Total number of samples 110
Number of PCR negative samples 92 (83.64%)
Number of PCR positive samples 18 (16.36%)
Dilution of saliva sample 1:2 1:4 1:8
Positive in both PCR and ELISA (True positives) 16 12 11
Negative in both PCR and ELISA (True negatives) 83 82 83
PCR positive samples missed by ELISA (False negative) 2 6 7
PCR negative samples missed by ELISA (False positive) 9 10 9
Area under the curve (AUC) 0.9436 0.9150 0.9128
Standard deviation (AUC) 0.0352 0.0348 0.0330
Number of correct cases 99 94 94
Accuracy 90% 85.50% 85.50%
Sensitivity 88.89% 66.67% 61.11%
Specificity 90.22% 89.13% 90.22%
Positive predictive value 64% 54.55% 45%
Negative predictive values 97.65% 93.18% 92.22%
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Expiration date test and effect of delay before reading

The one-year old plate effectively distinguished the E. histolytica positive samples diluted at 1:2 from the negative samples when results were read 3–27 min after incubation with the developing solution (Table 5).

Table 5.

ELISA results of the one-year old antigen plate stored at − 4 °C read at different time periods after adding the developing solution. Values enclosed in parentheses indicate category ratings for the absorbance readings

Saliva sample 1:2 1:4 1:8
Readings within 3–10 min
PCR negative
32,662 0.062(2) 0.062(2) 0.061(2)
32,483 0.088(3) 0.075(3) 0.082(3)
PCR positive
32,535 0.136*(4) 0.143*(4) 0.146*(5)
32,555 0.141*(4) 0.113(3) 0.140*(5)
Readings within 11–18 min
PCR negative
32,662 0.074(3) 0.069(2) 0.068(3)
32,483 0.104(3) 0.090(3) 0.094(3)
PCR positive
32,535 0.159*(4) 0.163*(5) 0.163*(5)
32,555 0.163*(4) 0.128*(4) 0.161*(5)
Readings within 20–27 min
PCR negative
32,662(−) 0.083(3) 0.078(3) 0.077(3)
32,483(−) 0.118(3) 0.103(3) 0.102(3)
PCR positive
32,535(+) 0.179*(5) 0.185*(6) 0.187*(5)
32,555(+) 0.182*(5) 0.145*(4) 0.183*(5)
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*Readings above the cut-off value

Discussion

The developed salivary anti-E. histolytica IgA detection method produced relatively accurate results compared to PCR detection in stool samples for the diagnosis of E. histolytica infection. The developed method had an accuracy of 85.5–90% (Table 3) when compared to PCR, the “gold standard” for E. histolytica detection (Tanyuksel and Petri 2003; Fotedar et al. 2007a, b; Stark et al. 2008) All three dilutions used gave an area under the curve (AUC) higher than 0.90 with the 1:2 saliva dilution giving the highest AUC, sensitivity, specificity, and accuracy as well as positive and negative predictive values (Fig. 1; Table 3). An AUC value close to 1.00 indicates a high accuracy in diagnosis (Sonis 1999; Park et al. 2004). Moreover, ELISA results at the 1:2 saliva dilutions agree well with PCR results compared to the other dilutions. It also performed much better than microscopy, which cannot properly distinguish between E. histolytica and E. dispar due to their identical morphology.

Antigen detection kits – specifically TechLab E. histolytica II – are very accurate and highly specific and sensitive when compared to microscopy and antibody detection (Haque et al. 2001; Abd-Alla et al. 2000a, b; Tanyuksel and Petri 2003; Fotedar et al. 2007a; Chang et al. 2008). A rapid point-of-care antigen detection kit composed of two antibody-lined strips (E. histolytica Quik Chek; TechLab) has also been developed with 100% sensitivity and 100% specificity when compared with the TechLab E. histolytica II kit (Korpe et al. 2012). However, studies have shown that PCR is superior to commercially available antigen detection kits (Mirelman et al. 1997; Gonin and Trudel 2003; Stark et al. 2008). An exception would be the study conducted by Solaymani-Mohammadi et al. (2006) in which there was 100% concordance between the TechLab E. histolytica II kit and PCR, because both techniques found that all symptomatic cases in the study were negative for E. histolytica. Since the present study shows that the developed salivary IgA detection method worked as well as PCR detection, it can be said that the former can perform as well or even better than commercially available E. histolytica antigen diagnostic kits.

Detection of salivary anti-E. histolytica IgA using ELISA has been previously used but both studies were compared with microscopy (del Muro et al. 1990; Punthuprapasa et al. 2001). Low accuracy of the method was attributed to low antibody production and misdiagnosis in microscopy (Punthuprapasa et al. 2001) or differences in detection sensitivity in populations with low prevalence (del Muro et al. 1990). In this study, the salivary anti-E. histolytica IgA detection kit was compared with PCR detection, a method with high sensitivity and specificity for detection of E. histolytica in stool samples (Mirelman 1997; Gonin and Trudel 2003; Tanyuksel and Petri 2003; Fotedar et al. 2007a, b; Stark et al. 2008). The five stool samples positive for Entamoeba cysts but negative in both PCR and ELISA were most likely other species such as Entamoeba coli or E. moshkovskii. Moreover, the salivary assay had high accuracy even when tested in a population with only 16.36% prevalence. The two previous studies tested anti-E. histolytica salivary IgA ELISA detection in populations divided into groups positive or negative for E. histolytica cysts by microscopy (del Muro et al. 1990; Punthuprapasa et al. 2001). This present study better assessed the performance of anti-E. histolytica salivary IgA ELISA detection versus the two other previous studies because it was compared with PCR, which is more sensitive than microscopy and is also able to distinguish between E. histolytica and E. dispar.

Saliva samples are much easier to collect and re-collect from patients compared to stool samples for stool antigen detection kits and PCR. Moreover, the antigen plates for the salivary IgA detection method can be stored for up to a year at − 4 °C without any changes in the method’s results (Table 5). The 96-well format and ease of collecting saliva samples also show the convenience of using the developed method in epidemiological surveys in large sample populations. Thus, it shows potential for use in both field surveys and diagnostics if the components are properly prepared and stored. The E. histolytica TechLab II kit has been proven as a useful diagnostic kit for both amebic liver abscess and amebic dysentery (Haque et al. 2000). The developed method in this study has similar potential if tested with symptomatic patients in future studies. The method has been patented in the Philippines under filing number PH 1/2008/000437.

There were 18 E. histolytica infections and no E. dispar infections identified in this study using PCR. This contrasts with other studies which indicate that E. dispar infection is more prevalent than E. histolytica infection around the world (Rivera et al. 1996, 1998; Abd-Alla et al. 2000b; Abd-Alla and Ravdin 2002; Gonin and Trudel 2003; Nesbitt et al. 2004; Solaymani-Mohammadi et al. 2006; Fotedar et al. 2007a; Stark et al. 2008). Similarly, PCR detection from a previous study showed that E. dispar has higher prevalence in asymptomatic individuals from the Philippines compared to E. histolytica. E. dispar had a prevalence of 7.32% compared to 0.96% for E. histolytica in asymptomatic patients from Baguio City (Rivera et al. 1998). An E. dispar prevalence of 1.03% was also recorded in asymptomatic individuals from a slum community in the City of Manila compared to a 0.36% prevalence of E. histolytica (Rivera et al. 2020).

The use of detection methods with high sensitivity shows that asymptomatic E. histolytica infections in the Philippines may be higher than previously expected. Prevalence was higher in this study (16.36%) compared to previous studies on asymptomatic cases in the Philippines. A 0.36% prevalence of E. histolytica was recorded using PCR detection in asymptomatic individuals in an urban slum community in Manila (Rivera et al. 2020), a 0.96% prevalence in asymptomatic individuals from Baguio City using PCR (Rivera et al. 1998), and a 7.8% prevalence in patients without colitis using ELISA antigen detection (Warren et al. 2012). A 2.9% prevalence of E. histolytica/E. dispar cysts was also recorded in street children from Manila using microscopy (Baldo et al. 2004). Additionally, asymptomatic E. histolytica prevalence may be much higher in groups with high-risk of fecal-oral infection such as patients in mental institutions. A previous study in the Philippines on patients in mental institutions showed a 65.48% prevalence using PCR (Rivera et al. 2006). The prevalence of E. histolytica in this present study using PCR was also much higher compared to other studies in neighboring Southeast Asian countries. PCR detection of E. histolytica showed a prevalence of 11.2% and 9.15% in asymptomatic patients from Vietnam (Blessmann et al. 2003) and Malaysia (Ngui et al. 2012), respectively. A prevalence of 10.8% was also recorded in Orang Asli aborigines from Malaysia using nested PCR (Lau et al. 2013) and a prevalence of 2.5% in schoolchildren from the Thai-Myanmar border (Pattanawong et al. 2021). Further studies using PCR detection may elucidate if a prevalence of E. histolytica around 16% is consistent with asymptomatic patients and if E. histolytica has higher prevalence compared to E. dispar from other parts of the Philippines.

Conclusions

The developed salivary anti-E. histolytica IgA detection method performed well as PCR detection, which is considered the “gold standard” in E. histolytica detection. Moreover, the crude antigen plates used for this assay can be stored at − 4 °C for up to a year before use. Also, saliva samples are easier to collect and re-collect compared to stool or serum samples, which are commonly used in PCR detection and commercially available antigen and antibody detection kits. Thus, the method developed in this study can be a useful and relatively easy-to-use tool in diagnosis or field surveys of E. histolytica infection.

Funding

This study was financially supported by the Office of the Vice President for Academic Affairs of the University of the Philippines System.

Availability of data and material

All relevant data are included in this manuscript.

Declarations

Conflicts of interest/Competing interests

The authors declare that they have no conflicts of interest.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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