Comparative evaluation of Sm1-7-qPCR systems incorporated with Maxwell kit-based and NaOH-based DNA extraction methods for the detection of Schistosoma mansoni infection.

BACKGROUND

The World Health Organization's latest roadmap for neglected tropical diseases (NTDs) 2021-2030 has highlighted diagnostics as one of four focus areas to achieve the targets set for 2030. Molecular diagnostic tests, such as quantitative real-time PCR (qPCR), are valuable for diagnosing NTDs, including schistosomiasis. This study aimed to comparatively evaluate two Sm1-7-qPCR systems incorporating different DNA extraction approaches for the detection of Schistosoma mansoni infection.

METHODS

Two copro-DNA-qPCR systems were developed: a Maxwell kit-based copro-DNA-Sm1-7-qPCR system (Sm1-7-qPCR-S1) and a NaOH-based copro-DNA-Sm1-7-qPCR system (Sm1-7-qPCR-S2). Both systems were tested on faecal samples collected from schistosomiasis endemic and non-endemic areas in Uganda (n = 482). A composite reference based on positive results of either the Kato-Katz (KK) or qPCR assays was used for calibration. The S. mansoni positivity rates were analyzed based on the results of different diagnostics. Differences in DNA concentration and cycle threshold (Ct) values from the two copro-DNA-qPCR systems were compared (Wilcoxon signed-rank test).

RESULTS

The overall S. mansoni positivity rate was 52.9% (229/433) according to the composite reference, whereas the KK, Sm1-7-qPCR-S1 and Sm1-7-qPCR-S2 recorded positivity rates of 36.7% (159/433), 43.9% (190/433), and 47.6% (206/433), respectively. Sm1-7-qPCR-S2 demonstrated a sensitivity of 90.0%, outperforming the KK and Sm1-7-qPCR-S1 with sensitivities of 69.4% and 83.0%, respectively (P < 0.0001 and P = 0.015, respectively, χ2m test). For samples positive in both Sm1-7-qPCR systems (n = 179), Ct-values obtained from Sm1-7-qPCR-S2 were significantly lower than those from Sm1-7-qPCR-S1 (P < 0.0001, Wilcoxon signed-rank test). A significant inverse correlation between Ct values and KK faecal egg counts was observed for both systems (Sm1-7-qPCR-S1, r = -0.5637, P < 0.0001; Sm1-7-qPCR-S2, r = -0.5723, P < 0.0001). The KK method showed substantial agreement with the composite reference (ƙ = 0.682), while both Sm1-7-qPCR systems exhibited almost perfect agreement with the reference (ƙ = 0.821 and 0.894, respectively).

CONCLUSIONS

This study highlights that DNA samples extracted from both systems are suitable for robust qPCR amplification. The Sm1-7-qPCR-S2 system offers several advantages over the Sm1-7-qPCR-S1 system, including improved sensitivity, field accessibility, and lower cost. With further refinement, the Sm1-7-qPCR-S2 system could be integrated into the existing schistosomiasis surveillance network to aid the elimination of the disease.