Differing Effects of Standard and Harsh Nucleic Acid Extraction Procedures on Diagnostic Helminth Real-Time PCRs Applied to Human Stool Samples

Abstract

This study aimed to assess standard and harsher nucleic acid extraction schemes for diagnostic helminth real-time PCR approaches from stool samples. A standard procedure for nucleic acid extraction from stool and a procedure including bead-beating as well as proteinase K digestion were compared with group-, genus-, and species-specific real-time PCR assays targeting helminths and nonhelminth pathogens in human stool samples. From 25 different in-house and commercial helminth real-time PCR assays applied to 77 stool samples comprising 67 historic samples and 10 external quality assessment scheme samples positively tested for helminths, higher numbers of positive test results were observed after bead-beating-based nucleic acid extraction for 5/25 (20%) real-time PCR assays irrespective of specificity issues. Lower cycle threshold values were observed for one real-time PCR assay after the standard extraction scheme, and for four assays after the bead-beating-based scheme. Agreement between real-time PCR results after both nucleic acid extraction strategies according to Cohen’s kappa ranged from poor to almost perfect for the different assays. Varying agreement was observed in eight nonhelminth real-time PCR assays applied to 67 historic stool samples. The study indicates highly variable effects of harsh nucleic acid extraction approaches depending on the real-time PCR assay used.

Evaluation of the Allplex™ GI-Helminth(I) Assay, the first marketed multiplex PCR for helminth diagnosis

“…The fact that mechanical lysis did not improve the performances suggests that the weakness of the assay is probably due to the amplification step. This is emphasized by a recent study which compared helminth DNA extraction with and without beadbeating, based on the analysis by numerous group-, genus-or species-specific PCRs [11]. This work showed a significant improvement of both in-house and commercial N. americanus PCRs using the bead-beating pretreatment.…”

New Developments in PCR-Based Diagnostics for Bacterial Pathogens Causing Gastrointestinal Infections—A Narrative Mini-Review on Challenges in the Tropics

“…If harsh bead beatingbased nucleic acid extraction as recommended for molecular helminth screenings is applied for PCRs targeting bacterial pathogens, sensitivity is comparable regarding the proportions of positive results compared to column-based standard nucleic acid extraction [62]. However, agreement as indicated by Cohen's kappa [31] leaves room for improvement [62] and it is not always clear whether only sensitivity but also specificity of the PCR approaches is affected by the different extraction approaches [62] in a similar way as discussed above for pre-analytic conditions. Accordingly, there is neither standardization of defined minimum requirements regarding the quality of nucleic acid extraction from stool samples if a broad spectrum of tropical pathogens is desired, nor agreement on which compromises regarding diagnostic sensitivity are considered acceptable for a "one-size-fits-all"-solution for nucleic acid extraction from stool prior to target-specific PCR.…”

“…When stool samples from the tropics are assessed, the diagnostic focus is rarely on bacterial pathogens alone, but parasitic infectious agents are frequently also targeted [ 56 , 57 , 58 ]. In order to release nucleic acids from strong-shelled eggs or cuticle cells of helminthic parasites, harsh nucleic acid extraction methods, i.e., freeze-thawing at the very least [ 59 ] but also more robust, bead beating-based extraction schemes [ 60 , 61 ] have been suggested, although their superiority in terms of sensitivity has not been continuously demonstrated [ 62 ]. For protozoan parasites, similarly harsh nucleic acid extraction procedures have been proposed as well [ 63 , 64 ].…”

“…In any way, such a requirement will not facilitate the implementation of PCR technology at resource-limited sites, so compromises regarding sensitivity are frequently made. If harsh bead beating-based nucleic acid extraction as recommended for molecular helminth screenings is applied for PCRs targeting bacterial pathogens, sensitivity is comparable regarding the proportions of positive results compared to column-based standard nucleic acid extraction [ 62 ]. However, agreement as indicated by Cohen’s kappa [ 31 ] leaves room for improvement [ 62 ] and it is not always clear whether only sensitivity but also specificity of the PCR approaches is affected by the different extraction approaches [ 62 ] in a similar way as discussed above for pre-analytic conditions.…”

“…PCR from stool samples after nucleic acid extraction with commercially available standard nucleic acid extraction kits in line with the manufacturers’ recommendations has been shown to be similarly sensitive as culture for the diagnosis of bacterial pathogens [ 3 ] and even more sensitive than microscopy for the diagnosis of enteric protozoa [ 65 ] in stool samples. Uncertainty, however, remains regarding the reliability of PCR for the diagnosis of enteric helminths in stool samples in comparison to microscopy [ 62 ]. Future studies addressing the definition of optimized nucleic acid extraction conditions for PCRs targeting tropical pathogens from stool are therefore desirable.…”