Comparative DNA extraction methods to use for LAMP assay as molecular diagnosis of human schistosomes from urine samples.

BACKGROUND

Schistosomiasis in Africa is an ongoing public health problem that is caused by two major human species, Schistosoma mansoni and S. haematobium, which often cause concurrent infections. Due to the global goal of controlling or eliminating schistosomiasis as a public health problem, the issue of diagnostic sensitivity has become more critical in the assessment of program success. In that regard, the World Health Organization (WHO) has drawn attention to the need for field-applicable tests with high specificity and sensitivity.

METHODS

To address this, we have evaluated the amplification of S. mansoni and S. haematobium cell-free repeat DNA by loop-mediated isothermal amplification (LAMP) from field-collected filtered urine samples from 30 school children in Zambia. We have used four DNA extraction techniques (Qiagen and LAMP-PURE (LP): column-based DNA extraction technique, Chelex, and heating: rapid DNA extraction technique) to determine their impact on LAMP sensitivity and specificity, along with cost analysis and person-time involvement for each approach.

RESULTS

Both Qiagen and LP extraction detected positive infections, but Qiagen extraction is more cost-effective than LP. DNA extraction by LP is the fastest (average 20 min.) compared to the other three methods, although it is the most expensive, including amplification ($9.35 compared to $4.90 for heating extraction and amplification). Chelex extraction is slower and simpler than LP and detects 20% more positive infections than heating. Heating extraction is very fast, inexpensive, and simple to perform. However, LAMP amplification for heating-extracted samples resulted in false negatives, possibly indicating the presence of inhibitor(s).

CONCLUSIONS

We have demonstrated the sensitivity, cost-effectiveness, and time requirement of LAMP with four different DNA extraction approaches for the detection of two schistosome parasite species from a single field-collected urine sample.