Grembi, J. A. | Search bioRxiv

bioRxiv2019

High-throughput multi-parallel enteropathogen quantification via nano-liter qPCR

Quantitative molecular diagnostic methods, such as qPCR, can effectively detect pathogen-specific nucleic acid sequences. However, costs associated with multi-pathogen quantitative molecular diagnostics hinder their widespread use. Nano-liter qPCR (nL-qPCR) is a miniaturized tool for quantification of multiple targets in large numbers of samples based on assay parallelization on a single chip, with potentially significant cost-savings due to rapid throughput and reduced reagent volumes. We evaluated a suite of novel and published assays to detect 17 enteric pathogens using a commercially available nL-qPCR technology. Assay efficiencies ranged from 88-98% (mean 91%) and were reproducible across four operators at two separate facilities. When applied to complex fecal material, assays were sensitive and selective (99.8% of DNA amplified were genes from the target organism). Detection limits were 1-2 orders of magnitude higher for nL-qPCR than an existing enteric TaqMan Array Card (TAC), due to nanofluidic volumes. Compared to the TAC, nL-qPCR displayed 97% (95% CI 0.96, 0.98) negative percent agreement and 63% (95% CI 0.60, 0.66) overall positive percent agreement. Positive percent agreement was 90% for target concentrations above the nL-qPCR detection limits. nL-qPCR assays showed an underestimation bias of 0.34 log10 copies/gram of stool [IQR -0.41, -0.28] compared with the enteric TAC. Higher detection limits, inherent to nL-qPCR, do not hinder detection of clinically relevant pathogen concentrations. With 12 times higher throughput for a sixth of the per-sample cost of the enteric TAC, the nL-qPCR chip described here is a viable alternative for enteropathogen quantification for studies where other technologies are cost-prohibitive.

microbiology↗

bioRxiv2019

Gut microbiota plasticity is correlated with sustained weight loss on a low-carb or low-fat dietary intervention

BackgroundObesity is a complex global health challenge. Although both low-carbohydrate (low-carb) and low-fat diets can lead to weight loss, there is typically substantial variability in weight and related outcomes for both diet approaches among obese but otherwise healthy adults. Elucidating individual characteristics that might contribute to sustained weight loss is critical to developing effective dietary intervention strategies. We hypothesized that structural differences in the gut microbiota explained some portion of the weight loss variability among people randomized to either a low-carb or low-fat diet, possibly related to its effects on dietary compliance.\n\nResultsOur study included two staggered cohorts of obese adults enrolled in the Diet Intervention Examining The Factors Interacting with Treatment Success (DIETFITS) study - a randomized clinical trial of either a low-fat or low-carb diet. In the discovery cohort (n=66), 161 pre-diet fecal samples were sequenced in addition to 157 samples collected after 10-weeks of dietary intervention. In the validation cohort (n = 56), 106 pre-diet fecal samples were sequenced. Pre-diet taxonomic features, such as the Prevotella/Bacteroides ratio, correlated to weight loss in the discovery cohort were not confirmed in the validation cohort. The most robust finding in the discovery cohort indicated that gut microbiota plasticity was linked to 12-month weight loss in a diet-dependent manner; subjects with higher sustained weight loss on a low-fat diet had higher pre-diet daily plasticity, whereas those most successful on the low-carb diet had greater microbiota plasticity over 10 weeks of dietary intervention. Unfortunately, because sample frequency and timing was quite different in the validation cohort, the relationship between plasticity and weight loss could not be studied in this group.\n\nConclusionsThese findings suggest the potential importance of gut microbiota plasticity in sustained weight loss. We highlight the importance of evaluating kinetic trends and in assessing reproducibility in studies of the gut microbiota.

microbiology↗