Authors

Discussion

Background
Female genital schistosomiasis (FGS) is a neglected gynaecological disease affecting ~56 million women across sub-Saharan Africa. It is caused by eggs of the parasitic trematode Schistosoma haematobium becoming trapped in the female genital organs and surrounding tissues.  Diagnostic advancements for FGS, such as low-resource DNA-based diagnostics, can decentralise screening. However, they can be limited in low-resource settings due to high-resource sample storage and extraction methods used alongside. This study investigated optimal sample storage and DNA extraction methods that can be coupled with low-resource S. haematobium tests for closer-to-the user diagnosis of active FGS.

Methodology
Nylon flocked swabs were spiked with single S. haematobium eggs. and stored in four different storage media at four different temperatures for six different longevities of time (up to 28 days), before being processed with a standard high resource DNA extraction method.
To further investigate the impact of low-resource preservation and DNA extraction methods swabs were stored at 22°C for 24 hours, in a total of five storage conditions- three commercially available storage media, 200µl of distilled water, or completely dry. Swabs were processed using 11  extraction methods that could theoretically be implemented in low-resource laboratories (i.e. needing minimal equipment). All DNA extracts were analysed using the Schistosoma ITS-2-qPCR to measure CT value. Samples with a CT value below a 35 CT cut-off were considered positive. All data was statistically analysed using StataNow.

Results
All conditions including time point (F(5, 284) = 6.80, p< .001), storage media (F(3, 284) = 9.66, p< .001), and temperature (F(3, 284) = 7.4, p= .016) had a statistically significant impact on CT value when analysed using one-way ANOVA tests. Notably, detectable DNA was obtained from swabs exposed to all storage variables. When two-way ANOVA analyses were performed to see if combinations of conditions had compound effects on CT value, only the combination of temperature and timepoint had a significant impact (F(15, 264) = 3.65 p= .032), with a decline in CT value with an increase in temperature and time. 
When comparing storage media and extraction method combinations, both storage and extraction methods had a statistically significant impact on CT value through one-way ANOVA analysis (storage media: (F(4, 115) = 7.4, p< 001, extraction method: (F(10, 154) = 19.99, p<001). When combined, storage media and extraction method had a significant effect on CT value (F(40, 164) = 3.13, p< 001).

Significance
Our experiment shows that DNA can be obtained from S. haematobium eggs/DNA on swabs preserved and stored under very low-resource conditions, including no cold-chain or media, supporting the accessibility of molecular diagnostics for FGS.
High-resource extraction methods produced CT values below cut-off regardless of storage method. However, combinations of dry swab sample storage with low-resource extraction methods produced similar CT values. This suggests a need for further consideration of new low-resource extraction methods.