Development and Evaluation of a Sensitive PCR-ELISA System for Detection of Schistosoma Infection in Feces

Abstract

BackgroundA PCR-enzyme-linked immunosorbent assay (PCR-ELISA) was developed to overcome the need for sensitive techniques for the efficient diagnosis of Schistosoma infection in endemic settings with low parasitic burden.Methodology/Principal FindingsThis system amplifies a 121-base pair tandem repeat DNA sequence, immobilizes the resultant 5′ biotinylated product on streptavidin-coated strip-well microplates and uses anti-fluorescein antibodies conjugated to horseradish peroxidase to detect the hybridized fluorescein-labeled oligonucleotide probe. The detection limit of the Schistosoma PCR-ELISA system was determined to be 1.3 fg of S. mansoni genomic DNA (less than the amount found in a single cell) and estimated to be 0.15 S. mansoni eggs per gram of feces (fractions of an egg). The system showed good precision and genus specificity since the DNA target was found in seven Schistosoma DNA samples: S. mansoni, S. haematobium, S. bovis, S. intercalatum, S. japonicum, S. magrebowiei and S. rhodaini. By evaluating 206 patients living in an endemic area in Brazil, the prevalence of S. mansoni infection was determined to be 18% by examining 12 Kato-Katz slides (41.7 mg/smear, 500 mg total) of a single fecal sample from each person, while the Schistosoma PCR-ELISA identified a 30% rate of infection using 500-mg of the same fecal sample. When considering the Kato-Katz method as the reference test, artificial sensitivity and specificity rates of the PCR-ELISA system were 97.4% and 85.1%, respectively. The potential for estimating parasitic load by DNA detection in feces was assessed by comparing absorbance values and eggs per gram of feces, with a Spearman correlation coefficient of 0.700 (P<0.0001).Conclusions/SignificanceThis study reports the development and field evaluation of a sensitive Schistosoma PCR-ELISA, a system that may serve as an alternative for diagnosing Schistosoma infection.

Comparison of the Kato-Katz and Helmintex methods for the diagnosis of schistosomiasis in a low-intensity transmission focus in Bandeirantes, Paraná, southern Brazil

“…The difficulty of diagnosing schistosomiasis in areas of low-intensity transmission, such as Esteio, has prompted several efforts to improve the sensitivity of egg detection in stool samples, such as using the Percoll and saline density gradient methods (Eberl et al 2002, Coelho et al 2009) or polymerase chain reaction analyses of stool samples (Gomes et al 2010, in addition to the examination of slides using the KK method or combinations of different methods (Siqueira et al 2011).…”

Polymerase chain reaction for the evaluation of Schistosoma mansoni infection in two low endemicity areas of Minas Gerais, Brazil

“…Pontes et al (2003) analysed two slides of three samples using the Kato-Katz method and compared the results to PCR. They demonstrated a Kappa index of 0.8 (excellent) for 194 individuals evaluated in Comercinho, a city endemic for schistosomiasis in MG. Gomes et al (2009) calculated a rate of 0.51 (moderate) between the Kato-Katz and PCR techniques after evaluating 67 residents in the city of Chonin de Cima, another district with endemic schistosomiasis in the same state, by analysing six slides of one faecal sample.…”

“…However, polymerase chain reaction (PCR) has recently been employed in the diagnosis of S. mansoni and has demonstrated high specificity and sensitivity in the detection of DNA in stool samples from patients in endemic areas (Pontes et al 2003, Allan et al 2009, Gomes et al 2009). This method of diagnosis has been shown to be 10-fold more sensitive than the Kato-Katz technique .…”

Two sequential PCR amplifications for detection of Schistosoma mansoni in stool samples with low parasite load

“…OLIVEIRA et al 13 obtained sensitivity using the conventional PCR of an egg in a suspension of 300 µL of feces. Eventually, GOMES et al, 2010 10 , obtained a sensitivity of 0.15 eggs/gram of feces (1.3 fg of DNA), using the PCR-ELISA detection system. Although these studies may report DNA detection values in lower concentrations, it is important to consider that the observation of the bands of DNA concentration may not be suitable for use in routine diagnosis of this infection.…”