Optimization of Quantitative PCR Methods for Enteropathogen Detection

Abstract

Detection and quantification of enteropathogens in stool specimens is useful for diagnosing the cause of diarrhea but is technically challenging. Here we evaluate several important determinants of quantification: specimen collection, nucleic acid extraction, and extraction and amplification efficiency. First, we evaluate the molecular detection and quantification of pathogens in rectal swabs versus stool, using paired flocked rectal swabs and whole stool collected from 129 children hospitalized with diarrhea in Tanzania. Swabs generally yielded a higher quantification cycle (Cq) (average 29.7, standard deviation 3.5 vs. 25.3 ± 2.9 from stool, P<0.001) but were still able to detect 80% of pathogens with a Cq < 30 in stool. Second, a simplified total nucleic acid (TNA) extraction procedure was compared to separate DNA and RNA extractions and showed 92% (318/344) sensitivity and 98% (951/968) specificity, with no difference in Cq value for the positive results (ΔCq(DNA+RNA-TNA) = -0.01 ± 1.17, P = 0.972, N = 318). Third, we devised a quantification scheme that adjusts pathogen quantity to the specimen’s extraction and amplification efficiency, and show that this better estimates the quantity of spiked specimens than the raw target Cq. In sum, these methods for enteropathogen quantification, stool sample collection, and nucleic acid extraction will be useful for laboratories studying enteric disease.

Enteropathogen detection in children with diarrhoea, or vomiting, or both, comparing rectal flocked swabs with stool specimens: an outpatient cohort study

“…10 Indeed, when compared head-to-head, stool specimens are superior to swab samples, probably because of the smaller amount of faecal material collected with rectal swabs. The higher rectal swab cycle-threshold values, particularly among discordant samples, 4 probably reflect a smaller amount of faecal material and the dilution with buffers to elute material for nucleic acid extraction. 14,16 This finding is highlighted by the higher cycle threshold values in children with isolated vomiting (appendix p 8).…”

“…4 Moreover, swab specimens are more likely to collect mucosal adherent microorganisms (suggesting a pathogenic role), while stool specimens contain those that exist freely within the lumen. Thus in children with discordant, stool positive-swab negative results and relatively low pathogen abundance (high cycle threshold counts), the detected pathogens might represent non-disease states.…”

“…Additionally, waiting for stool while patients are on site is impractical and post-visit return rates are poor, even in children with diarrhoea, 2 leading to delays or missed diagnostic opportunities that can adversely affect outcomes. 3 Point-of-care acquisition of rectal swabs might overcome these barriers, 4 but there have been few comparative analyses of stool versus swabs, 4–9 and none that have included children with isolated vomiting. Currently, there is a recommendation against the use of rectal swabs as a diagnostic specimen.…”

Use of quantitative molecular diagnostic methods to identify causes of diarrhoea in children: a reanalysis of the GEMS case-control study

“…15 All the assays have been described previously and have been extensively validated (appendix). 15,17,18 Nucleic acid was extracted with the QIAamp Fast DNA Stool mini kit (Qiagen, Hilden, Germany) with pretreatment steps that included bead beating. 18 We added two external controls, bacteriophage MS2 and phocine herpesvirus, to monitor efficiency of nucleic-acid extraction and amplification.…”

“…15,17,18 Nucleic acid was extracted with the QIAamp Fast DNA Stool mini kit (Qiagen, Hilden, Germany) with pretreatment steps that included bead beating. 18 We added two external controls, bacteriophage MS2 and phocine herpesvirus, to monitor efficiency of nucleic-acid extraction and amplification. We included one extraction blank per batch and one no-template amplification control per three cards to exclude laboratory contamination (appendix).…”