The intestinal protozoan parasite E.Histolytica is endemic in large parts of the world and is considered responsible for millions of cases of dysentery and liver abscess each year and responsible for up to 100.000 deaths per year. It is common in developing countries with poor socioeconomic conditions and malnutrition.
Traditionally, the laboratory detection of E.Histolytica in human feces has relied upon the microscopic examination of fresh or fixed stool samples. However, the recent identification of E.dispar as a separate but non pathogenic species which is morphologically indistinguishable from E.histolytica and does not require treatment has indicated the need of alternative methods for detection which are able to differentiate between the two organisms. A number of assays have been developed during recent years such as protein and DNA extraction systems, which are able to distinguish E.histolytica from E.dispar. However most of those assays are not suitable for a rapid diagnosis directly from stool samples, in particular when large numbers of samples have to be processed.
Amplification of amoeba DNA fragments by PCR has been proved to constitute a sensitive and specific method to detect E.histolytica or E.dispar from human feces. The PCR protocols reported so far, however, require further processing of the amplicon, which is time consuming and give false-positive results due to possible cross contamination. Recently developed closed-tube, real time PCR methods can overcome these problems. These methods allow specific detection of the amplicon by binding to one or two fluorescent-labeled probes during PCR. Thus, further downstream analysis is not required, which reduces the time needed to obtain results. In addition, the closed reaction tube minimize the potential for cross-contamination, and the assay output is numerical rather than qualitative, allowing appropriate diagnostic statistics to be applied.
This study was conducted on 40 cases from outpatients and inpatient clinics of Benha University Hospital and from Benha Educational Hospital, in addition to 10 cases of other parasites as G. lamblia and C. parvum
Group I: 20 dysenteric cases (symptomatic).
Group II: 20 non dysenteric cases (symptomatic).
Group III: 10 cases of other parasitic infections as G.lamblia and C.parvum, this group was included in this study to show if cross reaction will occur with the specific primer of E.histolytica during the performance of real time PCR technique.
In addition to control group (+ve control and –ve control)
All studied cases were subjected to:
1- History taking
2-Stool examination by:
Direct smear method
Iodine stained smear
Formol ether concentration technique
Real time PCR testing for parasite DNA in stool samples
All the results obtained were analyzed statistically, and the following results were obtained:
1- Amoebic infection was more prevalent in age group 16-40 years old.
2- Amoebiasis is more prevalent in cases from rural areas than those from urban areas.
3- The percentage of infection was higher in males than females.
4- Abdominal colic &distention are the most common symptoms among dysenteric cases, followed by tenesmus, loss of weight and anorexia& vomiting. Also in non dysenteric cases abdominal colic &distention are the most common symptoms followed by fever and easy fatigue.
5- Direct smear detects 5 cases (10%) with a sensitivity (25%) and specificity (100%), it is a good positive test as +ve predictive value (100%).
6- Iodine stained smear detects 11 cases (22%) with a sensitivity (55%) and specificity (100%), it is a good positive test as +ve predictive value (100%).
7- Formol ether concentration technique revealed the presence of E.histolytica cyst in 20 stool samples (40%).
8- Real time PCR could detect E.histolytica infection in all cases passing E.histolytica in their stool samples and in 6 cases that were negative by microscopy indicating a high sensitivity of real time PCR technique. A total of 26 cases (52%) out of 50 cases under study were confirmed to have E.histolytica infection. |
