Development and Validation of Real-Time RT-LAMP Assays for the Specific Detection of Zika Virus.

Two one-step real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays for the detection of Zika virus (ZIKV) were developed, based on two different primer design approaches: (1) open source, based on a combination of sequence diversity clustering (phylogeny and principal component analysis) and LAVA algorithm, using 45 whole genome ZIKV sequences retrieved from the National Center for Biotechnology Information (NCBI) database; (2) standard software for LAMP primer design (Primer Explorer V4), using 59 sequences of the ZIKV 3' UTR. The assays were firstly evaluated with External Quality Assessment panels from INSTAND e.V. (Germany) and EVD-LabNet (The Netherlands) including 4 and 12 unknown samples, respectively, and secondly, with 9 human, mosquito, and monkey ZIKV isolates from Africa (Senegal, Ivory Coast, and Uganda) and America (Brazil). The limit of detection as determined by probit analysis was 181 molecules for both RT-LAMP assays, and 100% reproducibility in the assays was obtained for 103 molecules (4/8 repetitions were positive for 102 molecules). Both assays were specific, amplifying only ZIKV RNA and not cross-detecting other arboviruses included in this study.

Development and validation of four one-step real-time RT-LAMP assays for specific detection of each dengue virus serotype.

BACKGROUND: 4 one-step, real-time, reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays were developed for the detection of dengue virus (DENV) serotypes by considering 2,056 full genome DENV sequences. DENV1 and DENV2 RT-LAMP assays were validated with 31 blood and 11 serum samples from Tanzania, Senegal, Sudan and Mauritania. DENV3 and DENV4 RT-LAMP assays were validated with 25 serum samples from Cambodia. METHODOLOGY/PRINCIPAL FINDINGS: 4 final reaction primer mixes were obtained by using a combination of Principal Component Analysis of the full DENV genome sequences, and LAMP primer design based on sequence alignments using the LAVA software. These mixes contained 14 (DENV1), 12 (DENV2), 8 (DENV3) and 3 (DENV4) LAMP primer sets. The assays were evaluated with an External Quality Assessment panel from Quality Control for Molecular Diagnostics. The assays were serotype-specific and did not cross-detect with other flaviviruses. The limits of detection, with 95% probability, were 22 (DENV1), 542 (DENV2), 197 (DENV3) and 641 (DENV4) RNA molecules, and 100% reproducibility in the assays was obtained with up to 102 (DENV1) and 103 RNA molecules (DENV2, DENV3 and DENV4). Validation of the DENV2 assay with blood samples from Tanzania resulted in 23 samples detected by RT-LAMP, demonstrating that the assay is 100% specific and 95.8% sensitive (positive predictive value of 100% and a negative predictive value of 85.7%). All serum samples from Senegal, Sudan and Mauritania were detected and 3 untyped as DENV1. The sensitivity of RT-LAMP for DENV4 samples from Cambodia did not quite match qRT-PCR. CONCLUSIONS/SIGNIFICANCE: We have shown a novel approach to design LAMP primers that makes use of fast growing sequence databases. The DENV1 and DENV2 assays were validated with viral RNA extracted clinical samples, showing very good performance parameters.

Development of a single-tube one-step RT-LAMP assay to detect the Chikungunya virus genome.

BACKGROUND: A single-tube one-step real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for rapid detection of chikungunya virus (CHIKV) targeting the conserved 6K-E1 target region was developed. The assay was validated with sera collected from a CHIKV outbreak in Senegal in 2015. METHODOLOGY/PRINCIPAL FINDINGS: A novel design approach by combining Principal Component Analysis and phylogenetic analysis of 110 available CHIKV sequences and the LAMP oligonucleotide design software LAVA was used. The assay was evaluated with an External Quality Assessment panel from the European Network for Diagnostics of "Imported" Viral Diseases and was shown to be sensitive and specific and did not cross-detect other arboviruses. The limit of detection as determined by probit analysis, was 163 molecules, and 100% reproducibility in the assays was obtained for 103 molecules (7/8 repetitions were positive for 102 molecules). The assay was validated using 35 RNA samples extracted from sera, and results were compared with those obtained by quantitative RT-PCR carried out at the Institut Pasteur Dakar, demonstrating that the RT-LAMP is 100% sensitive and 80% specific, with a positive predictive value of 97% and negative predictive value of 100%. CONCLUSIONS/SIGNIFICANCE: The RT-LAMP appeared to show superior performance with material stored for months compared to qRT-PCR and can be therefore recommended for use in infrastructures with poor settings.

Development of a recombinase polymerase amplification assay for rapid detection of Francisella noatunensis subsp. orientalis.

Francisella noatunensis subsp. orientalis (Fno) is the causative agent of piscine francisellosis in warm water fish including tilapia. The disease induces chronic granulomatous inflammation with high morbidity and can result in high mortality. Early and accurate detection of Fno is crucial to set appropriate outbreak control measures in tilapia farms. Laboratory detection of Fno mainly depends on bacterial culture and molecular techniques. Recombinase polymerase amplification (RPA) is a novel isothermal technology that has been widely used for the molecular diagnosis of various infectious diseases. In this study, a recombinase polymerase amplification (RPA) assay for rapid detection of Fno was developed and validated. The RPA reaction was performed at a constant temperature of 42°C for 20 min. The RPA assay was performed using a quantitative plasmid standard containing a unique Fno gene sequence. Validation of the assay was performed not only by using DNA from Fno, closely related Francisella species and other common bacterial pathogens in tilapia farms, but also by screening 78 Nile tilapia and 5 water samples. All results were compared with those obtained by previously established real-time qPCR. The developed RPA showed high specificity in detection of Fno with no cross-detection of either the closely related Francisella spp. or the other tested bacteria. The Fno-RPA performance was highly comparable to the published qPCR with detection limits at 15 and 11 DNA molecules detected, respectively. The RPA gave quicker results in approximately 6 min in contrast to the qPCR that needed about 90 min to reach the same detection limit, taking only 2.7-3 min to determine Fno in clinical samples. Moreover, RPA was more tolerant to reaction inhibitors than qPCR when tested with field samples. The fast reaction, simplicity, cost-effectiveness, sensitivity and specificity make the RPA an attractive diagnostic tool that will contribute to controlling the infection through prompt on-site detection of Fno.

Development and validation of a standardized protocol to monitor human dietary exposure by metabolite fingerprinting of urine samples.

Conventional tools for measuring dietary exposure have well recognized limitations. Measurement of food-derived metabolites in biofluids provides an alternative approach and our aim was to develop an experimental protocol which ensures that extraneous variability does not obscure metabolic signals from ingested foods. Healthy adults consumed a standardized meal in the evening before each test day and collected pooled overnight urine. On each test day of three different studies, urine was collected in the fasted state and at different time points after consumption of a standardized breakfast. Metabolite fingerprinting of samples using Flow Infusion Electrospray-Ionization Mass Spectrometry followed by multivariate data analysis showed strong discrimination between overnight, fasting and postprandial samples, in each study separately and when data from the three studies were pooled. Such differences were robust and highly reproducible within individuals on separate occasions. Urine volume was an efficient data normalization factor for metabolite fingerprinting data. Postprandial urines had a stable chemical composition over a period of 2-4 h after eating a standardized breakfast, suggesting that there is a flexible time window for urine collection. Fasting urine samples provided a stable baseline for universal comparisons with postprandial samples. A dietary exposure biomarker discovery protocol was validated by demonstrating that top-ranked signals discriminating between fasting and 2-4 h postprandial urine samples could be linked to metabolites abundant in some components of the standardized breakfast. We conclude that the protocol developed will have value in the search for biomarker leads of dietary exposure. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11306-011-0289-0) contains supplementary material, which is available to authorized users.

Effect of prolonged in vivo administration of progesterone in pregnancy on myometrial gene expression, peripheral blood leukocyte activation, and circulating steroid hormone levels.

OBJECTIVE: We aimed to investigate the effects of progesterone on gene expression and function of both myometrium and circulating leukocytes. METHODS: We recruited women participating in a randomized clinical trial of progesterone to prevent preterm delivery. These participants had a twin pregnancy and were managed in 1 of 2 tertiary referral centers. Participants were treated with progesterone (90 mg vaginally) or placebo from 24 to 34 weeks of pregnancy. The outcome measures were myometrial and leukocyte gene expression and expression of cell surface markers in circulating leukocytes, all quantified ex vivo. RESULTS: Prolonged in vivo administration of progesterone inhibited myometrial expression of connexins 26 and 43, endothelial nitric acid synthase (eNOS), and the prostaglandin receptor EP2 ex vivo. Administration of progesterone also increased numbers of circulating neutrophils while decreasing lymphocyte proportions and decreasing neutrophil CD11b expression. CONCLUSION: The observed effects of prolonged in vivo administration of progesterone will minimize the ability of the uterus to contract as a synctium and the ability of peripheral blood leukocytes to migrate into the myometrium during parturition. We suggest that these are putative mechanisms by which progesterone might prevent preterm birth in women at high risk.

Mitochondrial dysfunction accounts for the stochastic heterogeneity in telomere-dependent senescence.

Aging is an inherently stochastic process, and its hallmark is heterogeneity between organisms, cell types, and clonal populations, even in identical environments. The replicative lifespan of primary human cells is telomere dependent; however, its heterogeneity is not understood. We show that mitochondrial superoxide production increases with replicative age in human fibroblasts despite an adaptive UCP-2-dependent mitochondrial uncoupling. This mitochondrial dysfunction is accompanied by compromised [Ca(2+)]i homeostasis and other indicators of a retrograde response in senescent cells. Replicative senescence of human fibroblasts is delayed by mild mitochondrial uncoupling. Uncoupling reduces mitochondrial superoxide generation, slows down telomere shortening, and delays formation of telomeric gamma-H2A.X foci. This indicates mitochondrial production of reactive oxygen species (ROS) as one of the causes of replicative senescence. By sorting early senescent (SES) cells from young proliferating fibroblast cultures, we show that SES cells have higher ROS levels, dysfunctional mitochondria, shorter telomeres, and telomeric gamma-H2A.X foci. We propose that mitochondrial ROS is a major determinant of telomere-dependent senescence at the single-cell level that is responsible for cell-to-cell variation in replicative lifespan.