Detection Cell-Free DNA (cfDNA) Using Nested-PCR as a Diagnosis Tool for Human Fascioliasis Infection

Abstract

Background: We aimed to detect Fasciola specific deoxyribonucleic acid (DNA) by nested-PCR assay on human stool and urine samples and compare the results with the respective ELISA diagnostic assay.
 Methods: Overall, 206 clinically suspected cases of fascioliasis were enrolled in the study. Blood samples were collected from all the patients, and serum samples were isolated. ELISA assay, using Fasciola somatic antigen (SA), was carried out to detect anti Fasciola antibodies for the collected sera. DNA was randomly extracted from 25 stool and 10 urine samples of seropositive individuals and was evaluated by conventional PCR and nested PCR methods. The nested-PCR results were confirmed by sequencing the 430 bp region of ribosomal ITSI gene. Stool and urine samples from patients with different parasitic diseases and 25 stool samples from healthy individuals served as controls. Urine samples were collected from 10 healthy controls as well.
 Results: Fascioliasis was detected by ELISA in 24.8% of the individuals. Of these, 25 seropositive patients were randomly assigned to the study. Fasciola DNA was identified in the stool samples of 96% of seropositive patients by nested PCR but ova of Fasciola was detected by parasitology methods in only 20% of seropositive cases. Fasciola DNA was identified in 90% of the urine samples by nested PCR. No cross-reactions were observed with other parasites.
 Conclusion: Detection of cfDNA in stool and urine samples has high accuracy and thus can be used for the diagnosis of Fasciola infection in human.

Evaluation of Semi-Nested PCR Compared with Indirect-ELISA to Diagnose Human Fasciolosis

“…Moreover, although there are various methods used to the diagnosis of fasciolosis, each has its limitations and disadvantages. For example, the presence of cross-reactions in serological diagnosis, ineffectiveness of parasitological methods in the diagnosis of acute and chronic stages of fasciolosis are reported ( 7 , 8 – 12 , 13 ), whereas molecular methods are more specific (Real-time PCR or semi-nested PCR) than other methods ( 22 – 25 ). Similarly, comparisons between diagnostic methods (molecular, serological and microscopic) have always been proposed in parasitic diseases to reach a preferred method.…”

“…Preparation and protein concentration of the ES antigen from adult F. gigantica was performed as reported already ( 7 ). All serum samples were examined by indirect ELISA method based on a previous study ( 13 ).…”

“…We extracted DNA from serum using the genomic DNA kit (DNG TM -PLUS) ( 14 ) and AddPrep, both of which were suitable for extraction of genomic DNA from fascioliasis. DNA extraction based on AddPrep Genomic DNA Kit (Product Code: 10023; Korea) ( 13 ).…”

“…The primers were synthesized by Bioneer Company (Korea) ( 13 ) and deposited in GenBank. Table 2 shows the sequence of primers used.…”

“…The problem of serological tests is that it is impossible to detect the disease after treatment and it needs to some molecular tests ( 10 – 12 ). Recent molecular studies on urine and feces using the nested PCR method have shown promising results in this regard ( 13 , 14 ).…”