Development and evaluation of a sensitive PCR-ELISA system for detection of schistosoma infection in feces

Abstract

Background: A 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 findings: This 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/significance: This 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.

Figure 1. Analytical sensitivity of PCR-ELISA system.

A. Analytical sensitivity of PCR followed by 6% polyacrylamide gel electrophoresis with silver staining and ELISA (optical density, OD) for 10-fold serial dilutions of genomic DNA extracted from a saline solution containing ∼2,000 S. mansoni eggs. The detection limit was 1.3 fg of genomic DNA. A ladder-type banding pattern was exhibited, as expected, due the amplification of the Schistosoma tandem-repeated unit, with the main DNA band of 110 bp present in all samples. B. The Pearson's correlation coefficient between PCR-ELISA OD and log[template DNA]+10 was 0.986 (P<0.0001).

Figure 2. Analytical specificity of PCR followed by 6% polyacrylamide gel electrophoresis with silver staining and ELISA (optical density, OD) for purified DNA from adult S. magrebowiei (lane 2), S. rhodaini (lane 3), S. japonicum (lane 4), S. intercalatum (lane 5), S. haematobium (lane 6), S. bovis (lane 7) and S. mansoni (lane 8) worms.

Lane 1, PCR negative control. The genus specificity of the Schistosoma PCR-ELISA system was demonstrated by the amplification of the 121-bp tandem repeat DNA sequence in all samples with the same set of primers. Absorbance readings were also consistent with the positive control (S. mansoni DNA).

Figure 3. Correlation between log-transformed individual measurements of eggs per gram (epg) of feces and absorbance readings.

A. A Spearman's correlation coefficient of 0.616 (P<0.0001) was found for a comparison considering all 206 samples. B. Considering positive and negative samples for the Kato-Katz technique and the Schistosoma PCR-ELISA system, a Spearman's correlation coefficient of 0.700 (P<0.0001) was observed. The continuous line represents the linear regression, and the hatched line represents the 95% CI.