Polymerase Chain Reaction Assessment Research Articles

Utility of polymerase chain reaction for assessment of onychomycosis during topical therapy.

Increased detection of fungi including non-dermatophyte molds (NDMs) using polymerase chain reaction (PCR) methods is well-established. However, the use of PCR to evaluate ongoing onychomycosis treatment outcome has not been investigated. Nail samples from 28 patients receiving topical efinaconazole were evaluated by both KOH/culture and PCR methods across the study period. Detection of microorganisms by PCR was compared to the culture at baseline and end of study at month 24 (M24). Fungal detection by both methods was evaluated with respect to clinical cure observed as 100% visual clearance of the target toenail. By culture, all 28 subjects were dermatophyte-positive at pre-treatment; only 4/28 also exhibited an NDM microorganism. According to PCR, 24/28 subjects were dermatophyte-positive pre-treatment, with 25/28 also exhibiting NDMs. At M24, 18/28 (64.3%) participants had negative KOH/culture results, in contrast to 4/28 (14.3%) negative PCR results. PCR showed higher rates of NDM detection than the culture at baseline as well as M24. Calculations to compare the diagnostic utility of KOH/culture versus PCR found that positive tests with both methods reliably indicate the presence of onychomycosis, but negative PCR correlated better with onychomycosis cure than did KOH/culture. PCR confirmed a high presence of NDMs pre-treatment, and continued presence of NDMs to M24, with unknown significance requiring further investigation. Though both KOH/culture and PCR have diagnostic limitations, PCR showed better overall utility than culture in predicting onychomycosis topical treatment outcome and should be more strongly considered for evaluation of topical therapies.

Digital Polymerase Chain Reaction for Assessment of Mutant Mitochondrial Carry-over after Nuclear Transfer for In Vitro Fertilization.

The quantification of mitochondrial DNA heteroplasmy for the diagnosis of mitochondrial disease or after mitochondrial donation, is performed mainly using next-generation sequencing strategies (NGS). Digital PCR (dPCR) has the potential to offer an accurate alternative for mutation load quantification. We assessed the mutation load of 23 low-input human samples at the m.11778 locus, which is associated with Leber's hereditary optic neuropathy (LHON) using 2 droplet digital PCR platforms (Stilla Naica and Bio-Rad QX200) and the standard NGS strategy. Assay validation was performed by analyzing a titration series with mutation loads ranging from 50% to 0.01%. A good concordance in mutation rates was observed between both dPCR techniques and NGS. dPCR established a distinctly lower level of background noise compared to NGS. Minor alleles with mutation loads lower than 1% could still be detected, with standard deviations of the technical replicates varying between 0.07% and 0.44% mutation load. Although no significant systematic bias was observed when comparing dPCR and NGS, a minor proportional bias was detected. A slight overestimation of the minor allele was observed for the NGS data, most probably due to amplification and sequencing errors in the NGS workflow. dPCR has proven to be an accurate tool for the quantification of mitochondrial heteroplasmy, even for samples harboring a low mutation load (<1%). In addition, this alternative technique holds multiple benefits compared to NGS (e.g., less hands-on time, more straightforward data-analysis, and a lower up-front capital investment).

Diagnostic efficacy of blastocoel fluid and spent media as sources of DNA for preimplantation genetic testing in standard clinical conditions

To determine whether blastocoel fluid (BF) or spent blastocyst medium (SBM) is a suitable template for genotype and/or karyotype assessment of invitro fertilization-generated embryos. Prospective blinded study. Genetic laboratory. From 26 patients undergoing preimplantation genetic testing (PGT) treatments, 103 trophectoderms (TE), 92 BF samples, and 72 SBM samples. The BF and SBM were retrieved at the time of TE biopsy. Two DNA extraction strategies were evaluated on independent BF and SBM samples. Further enrolled samples were processed using next-generation sequencing and quantitative polymerase chain reaction for assessment of monogenic disorders (PGT-M) or aneuploidy (PGT-A). DNA amplification and concordance rates across BF, SBM, and TE to assess diagnostic efficiency. No differences were detected among the DNA extraction methods tested. In PGT-M tests, for BF and SBM, 2.9% and 20.8% of all samples, respectively, produced a diagnosis concordant with the corresponding TE (n = 2 of 69 and 15 of 72, respectively). The SBM samples were associated with higher discordance rates and higher artifacts/contamination detection compared with BF. In multiple occasions, the maternal mutated variant was detected in the SBM of homozygous wild-type embryos, showing evidence of maternal DNA persistence in culture medium. In PGT-A tests, BF analysis showed high amplification failure rates (65.2%) and an overall concordance rate of 37.5% among amplified samples. Based on current methodologies, BF and SBM genetic analyses do not provide sufficiently reliable results to be employed clinically. Until the risk of maternal contamination can be properly prevented, SBM should not be used for PGT-M purposes.

Resveratrol Inhibits Periodontitis-Related Bone Loss in Rats Subjected to Cigarette Smoke Inhalation.

Alternative therapeutic approaches have been explored to modulate host response to periodontal disease. Knowledge of new strategies to treat periodontitis is particularly relevant in patients presenting augmented risk to periodontitis, such as smokers. The aim of this study is to investigate the impact of resveratrol (RESV) on progression of experimental periodontitis (EP) in the presence of cigarette smoke inhalation (CSI). Rats were assigned to one of three groups: 1) CSI+RESV (n = 20); 2) CSI+placebo (n = 20); and 3) non-CSI (n = 20). CSI was initiated 1 week prior to initiation of RESV or placebo administration (systemically for 30 days) and was continued until the end of the study. EP was induced around the first mandibular and second maxillary molars using ligatures. Specimens from the mandible were processed for morphometric and microcomputed tomography examination of bone volume/levels. Gingival tissues surrounding mandibular molars were collected for quantification of interleukin (IL)-1β, IL-4, IL-6, IL-17, and tumor necrosis factor-α using an assay system. Additional analyses of immunoinflammatory mediator performance (T-helper Type 17 [Th17]/Th2 and Th1/Th2 cell levels) were performed according to Th cell responses in gingival tissues. Gingival tissues of maxillary molars were subjected to real-time polymerase chain reaction for assessment of osteoprotegrin, runt-related transcription factor-2, receptor activator of nuclear factor-kappa B ligand (RANKL), sclerostin, and Dickkopf Wnt signaling pathway inhibitor 1 levels. Higher linear alveolar bone loss (ABL) and lower interradicular bone density were detected in ligated molars in the CSI+placebo group (P <0.05). IL-4 level was the highest, and Th17/Th2 levels were the lowest in RESV-treated rats compared with placebo rats (P <0.05). RESV reduced expression of messenger RNA for RANKL in animals receiving CSI (P <0.05). RESV inhibits EP and CSI-induced supporting ABL and has a beneficial effect on osteo-immunoinflammatory markers.