Diagnosis of intestinal amoebiasis by using nested polymerase chain reaction-restriction fragment length polymorphism assay.
BACKGROUND
Microscopy is unreliable to distinguish the pathogenic Entamoeba histolytica from the nonpathogenic Entamoeba dispar or Entamoeba moshkovskii in stool specimens.


METHODS
Nested polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was carried out to detect E. histolytica, E. dispar, and E. moshkovskii DNA in stool samples of 202 patients positive for E. histolytica, E. dispar, or E. moshkovskii by microscopy or culture and in 35 controls. The TechLab E. histolytica II enzyme-linked immunosorbent assay (ELISA) was performed to detect Gal/GalNAc lectin in 45 stool samples positive for E. histolytica, E. dispar, or E. moshkovskii by microscopy or culture. Rapid-indirect hemagglutination assay (IHA) was performed to detect serum antiamoebic antibodies in the 85 patients positive for E. histolytica, E. dispar, or E. moshkovskii in their stool specimens and in the 35 controls.


RESULTS
Nested PCR-RFLP was positive in 175 of 202 (86.6%) patient stool samples and was negative in all 35 negative control stool samples. ELISA was positive in 29 of 45 (64.4%) patient stool samples. The IHA test was positive in 19 of 85 (22.4%) patient serum samples and in one (2.8%) of the 35 control serum samples. Nested PCR-RFLP detected E. histolytica DNA in stool specimens of 12 (63.2%) of 19 seropositive patients, and in 31 (47%) of 66 seronegative patients. TechLab E. histolytica II ELISA detected E. histolytica antigen in stool specimens of six (54.5%) of 11 seropositive patients, and in 23 (67.6%) of 34 seronegative patients.


CONCLUSIONS
Nested PCR-RFLP was useful for the specific detection of E. histolytica, E. dispar, and E. moshkovskii in stool samples.
Khairnar
Krishna
K
Department of Microbiology, Jawaharlal Institute of Postgraduate Medical Education and Research, Pondicherry 605006, India.
Parija
Subhash Chandra
SC
Palaniappan
Ravisankar
R
D000818:Animals; D000913:Antibodies, Protozoan / Q000032:analysis; D016054:DNA, Protozoan / Q000032:analysis*; D003937:Diagnosis, Differential; D004747:Entamoeba / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D004749:Entamoebiasis / Q000175:diagnosis* / Q000469:parasitology; D004797:Enzyme-Linked Immunosorbent Assay; D005243:Feces / Q000469:parasitology; D006801:Humans; D007411:Intestinal Diseases, Parasitic / Q000175:diagnosis* / Q000469:parasitology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D015203:Reproducibility of Results; D012189:Retrospective Studies
D000913:Antibodies, Protozoan; D016054:DNA, Protozoan
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