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Molecular Identification and Prevalence of Entamoeba histolytica, Entamoeba dispar and Entamoeba moshkovskii in Erbil City, Northern Iraq

SHLER AKRAM FAQE MAHMOOD
SHLER AKRAM FAQE MAHMOOD
 y 
HAWRI MUSTAFA BAKR
HAWRI MUSTAFA BAKR
   | 05 ago 2020
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Article Category: original-paper
Publicado en línea: 05 ago 2020
Páginas: 263 - 272
Recibido: 31 mar 2020
Aceptado: 18 jun 2020
DOI: https://doi.org/10.33073/pjm-2020-028
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© 2020 Shler Akram Faqe Mahmood et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Fig. 1.

Agarose gel electrophoresis analysis for nested PCR products, using primers specific for E. histolytica, positive samples reveal 439 bp bands. Samples 1 to 55, 64,and 65 showed 439 bands; samples 56 to 63 and 66 to 70 tested negative for E. histolytica. Some of these negatives showed 900 bp bands, which are the product of the first PCR and an indicator for the presence of other Entamoeba species. C represented positive control, N represented negative control and M the 100 bp DNA marker.
Agarose gel electrophoresis analysis for nested PCR products, using primers specific for E. histolytica, positive samples reveal 439 bp bands. Samples 1 to 55, 64,and 65 showed 439 bands; samples 56 to 63 and 66 to 70 tested negative for E. histolytica. Some of these negatives showed 900 bp bands, which are the product of the first PCR and an indicator for the presence of other Entamoeba species. C represented positive control, N represented negative control and M the 100 bp DNA marker.

Fig. 2.

Detection of E. dispar after using species specific primers in the second round of the nested PCR; amplification products were analyzed by agarose gel electrophoresis and the stained gels were visualized under UV light. Positive samples exhibited 174 bp bands, which appeared in sample numbers (1, 2, 3, 5, 7, 8, 10, 12, 13, 14, 16, 18, 19,22, 24, 25, 28, 29, 30, 31), (35 to 42), (56 to 63), and (66 to 70). Samples (4, 6, 9, 11, 15, 17, 20, 21, 23, 26, 27, 32, 33, 34, 64, 65) and (43 to 55) tested negative for E. dispar. C represented positive control, N represented negative control and M was the 100 bp DNA marker.
Detection of E. dispar after using species specific primers in the second round of the nested PCR; amplification products were analyzed by agarose gel electrophoresis and the stained gels were visualized under UV light. Positive samples exhibited 174 bp bands, which appeared in sample numbers (1, 2, 3, 5, 7, 8, 10, 12, 13, 14, 16, 18, 19,22, 24, 25, 28, 29, 30, 31), (35 to 42), (56 to 63), and (66 to 70). Samples (4, 6, 9, 11, 15, 17, 20, 21, 23, 26, 27, 32, 33, 34, 64, 65) and (43 to 55) tested negative for E. dispar. C represented positive control, N represented negative control and M was the 100 bp DNA marker.

Fig. 3.

Nested PCR for identification of E. moshkovskii, determined with specific primers for each species, and analyzed by agarose gel electrophoresis. Positive samples amplifying 553 bp amplicon appeared in only three samples (1, 8, and 14); the remaining samples were negative. C represented positive control, N represented negativecontrol, and M was the 100 bp DNA marker.
Nested PCR for identification of E. moshkovskii, determined with specific primers for each species, and analyzed by agarose gel electrophoresis. Positive samples amplifying 553 bp amplicon appeared in only three samples (1, 8, and 14); the remaining samples were negative. C represented positive control, N represented negativecontrol, and M was the 100 bp DNA marker.

Frequency and percentages of positive and negative results for E. histolytica, E. dispar and E. moshkovskii as single and mixed infections.

Entamoeba speciesFrequency & percentage of Positives by PCR per total (microscopic) positivesFrequency & percentage of Negatives by PCR per total (microscopic) positivesFrequency & percentage of positives per populationFrequency and percentage of single infection/positivesFrequency and percentage of mixed infection/positivesFrequency and percentage of single infection/total positivesFrequency and percentage of mixed infection/total positives
E. histolytica57/70 (81.4%)13/70 (18.6%)57/950 (6%)29/57 (50.9%)28/57 (49.1%)29/70 (41.4%)28/70 (40%)
E. dispar41/70 (58.6%)29/70 (41.4%)41/950 (4.3%)13/41 (31.7%)28/41 (68.3%)13/70 (18.6%)28/70 (40%)
E. moshkovskii3/70 (4.3%)67/70 (95.7%)3/950 (0.3%)0/3 (0%)3/3 (100%)0/70 (0%)3/70 (4.3%)

Statistical analysis of the risk factors associated with microscopic positives for Entamoeba species and PCR positives for E. histolytica in asymptomatic subjects.

VariantsTotal frequencyFrequency and percentages of positive by microscopyPercentage within each group95%CIp-value
Lowerupper
Gender
Male524 (55.2%)29 (41.4%)524 (5.5%)  0.036  0.0750.018*
Female426 (44.8%)41 (58.6%)426 (9.6%)  0.069  0.125
Residency
Urban702 (73.9%)48 (68.6%)702 (6.8%)  0.049  0.0900.322
Rural248 (26.1%)22 (31.4%)248 (8.9%)  0.054  0.125
Age group
15–18    87 (9.2%)      4 (5.7%)  87 (4.6%)  0.009  0.0900.76
19–25311 (32.7%)    24 (34.3%)311 (7.7%)  0.048  0.108
26–35328 (34.5%)    26 (37.1%)328 (7.9%)  0.050  0.110
36–45157 (16.5%)    13 (18.6%)157 (8.3%)0.00  0.100
> 45    67 (7.1%)        3 (4.3%)  67 (4.5%)0.00  0.308
Educational level
Primary school298 (31.4%)    25 (35.7%)298 (8.4%)  0.053  0.1150.73
Secondary and high school448 (47.2%)    31 (44.3%)448 (6.9%)  0.044  0.096
Bachelor204 (21.5%)      14 (20%)204 (6.9%)  0.036  0.107
Family size
1–2    92 (9.7%)      5 (7.1%)  92 (5.4%)  0.011  0.1050.563
3–4  228 (24%)    13 (18.6%)228 (5.7%)  0.028  0.086
5–6301 (31.7%)    24 (34.3%)  301 (8%)  0.050  0.112
> 6329 (34.6%)      28 (40%)329 (8.5%)  0.055  0.116
Income status
Poor355 (37.4%)    39 (55.7%)  355 (11%)  0.078  0.1420.004*
Middle class594 (62.5%)    31 (44.3%)594 (5.2%)  0.033  0.069
Wealthy      1 (0.1%)          0 (0%)      1 (0%)0.000.00
Source of water supply
Chlorinated water  646 (68%)    39 (55.7%)  646 (6%)  0.042  0.0790.062
Well water302 (31.8%)    31 (44.3%)302 (10.3%)  0.068  0.138
Others      2 (0.2%)          0 (0%)      2 (0%)0.000.00
Eating out of home
Never259 (27.3%)    17 (24.3%)259 (6.6%)  0.039  0.0960.857
Sometimes310 (32.6%)    24 (34.3%)310 (7.7%)  0.045  0.110
Always381 (40.1%)    29 (41.4%)381 (7.6%)  0.050  0.104
History of taking medications
In the last 2 weeks140 (14.7%)      6 (8.6%)140 (4.3%)  0.013  0.0750.161
More than 2 weeks  810 (85.3)    64 (91.4%)810 (7.9%)  0.059  0.098
Hygiene practice
Washing vegetables and fruits920 (96.8%)    66 (94.3%)920 (7.2%)  0.055  0.0880.204
Eating raw unwashed vegetables and fruits    30 (3.2%)      4 (5.7%)30 (13.3%)  0.029  0.263
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