Evaluation of Two DNA Extraction Methods for Detection of Strongyloides stercoralis Infection

Abstract

is present worldwide, but its prevalence is still uncertain, mainly due to the lack of sensitivity of diagnostic methods. Molecular techniques are under development, but a standardized protocol is still unavailable. We compared the sensitivity of real-time PCR, using two extraction protocols, with that of the Baermann technique. Samples were collected in the framework of the baseline screening of a randomized clinical trial evaluating moxidectin against in Lao People's Democratic Republic. Two stool samples from each participant were processed by the Baermann method, and one subsample was processed by PCR. DNA was extracted using the QIAamp DNA stool minikit based on the standard protocol for the QIAamp DNA minikit (QIA) and using a modification of the QIA procedure (POL). Subsequently, all extracted samples were analyzed by real-time PCR. Overall, 95 samples were analyzed by the three diagnostic methods. Sixty-nine (72.6%) samples were positive according to the Baermann method, 25 (26.3%) by the QIA method, and 62 (65.3%) by the POL method. The sensitivities were 86% (95% confidence interval [CI], 76.7 to 92.9), 31.0% (95% CI, 21.3 to 42.6), and 78.0% (95% CI, 66.8 to 86.1) for the Baermann, QIA, and POL methods, respectively. The sensitivities calculated for each day of the Baermann method separately were 60% (48.4 to 70.8%) and 64% (52.2 to 74.2%) for days 1 and 2, respectively. In conclusion, the POL method revealed a good performance and was comparable to the Baermann test performed on two stool samples and superior to the Baermann method performed on one stool sample. Additional studies are needed to standardize a PCR protocol for diagnosis.

Strongyloides stercoralis prevalence and diagnostics in Vientiane, Lao People’s Democratic Republic

“…RTD-PCR testing was performed on stool samples of 100 mg conserved in 90% ethanol and stored in 1.5 ml cryo tubes at ambient temperature. For isolation of nucleic acids from stool samples the QIAamp DNA mini kit (Qiagen, Germany) was used according to an optimized protocol from Barda and colleagues [ 17 ]. The RTD-PCR targets the generic 28SrRNA gene of Strongyloides spp.…”

“…A plasmid containing the 92 bp amplicon sequence, as well as adjacent base pairs, was used as positive control. The assay was optimized on the plasmid and on characterized DNA from a S. stercoralis drug efficacy trial study [ 17 , 19 ] (data not shown). Analytical sensitivity of the assay was one plasmid copy/μl DNA.…”

Clinical value of serology for the diagnosis of strongyloidiasis in travelers and migrants: A 4-year retrospective study using the Bordier IVD®Strongyloides rattiELISA assay

“…Recently, PCR assays have been developed for S. stercoralis detection in stools, often yielding better performances than microscopy [ 3 , 5 , 26 ]. However, these assays showed heterogeneous performances, greatly depending on the patient population and the reference method used for clinical evaluation [ 2 , 9 ].…”

Strongyloidiasis in Solid Organ Transplantation

“…Multiple molecular methods using PCR are under development but are not yet widely available for clinical use [14,15,67,68]. Strongyloides-specific PCR-based methods can have sensitivity and specificity reaching 100% and can achieve a diagnosis up to one month earlier when compared to conventional microbiological methods [15].…”

“…[35]. Molecular diagnostics such as PCR can be performed if available and it is anticipated that these will likely play a larger role in the future as they become more widely adopted [14,15,20,34,68].…”