The soil transmitted helminths are a group of parasitic worms responsible for extensive morbidity in many of the world's most economically depressed locations. With growing emphasis on disease ...mapping and eradication, the availability of accurate and cost-effective diagnostic measures is of paramount importance to global control and elimination efforts. While real-time PCR-based molecular detection assays have shown great promise, to date, these assays have utilized sub-optimal targets. By performing next-generation sequencing-based repeat analyses, we have identified high copy-number, non-coding DNA sequences from a series of soil transmitted pathogens. We have used these repetitive DNA elements as targets in the development of novel, multi-parallel, PCR-based diagnostic assays.
Utilizing next-generation sequencing and the Galaxy-based RepeatExplorer web server, we performed repeat DNA analysis on five species of soil transmitted helminths (Necator americanus, Ancylostoma duodenale, Trichuris trichiura, Ascaris lumbricoides, and Strongyloides stercoralis). Employing high copy-number, non-coding repeat DNA sequences as targets, novel real-time PCR assays were designed, and assays were tested against established molecular detection methods. Each assay provided consistent detection of genomic DNA at quantities of 2 fg or less, demonstrated species-specificity, and showed an improved limit of detection over the existing, proven PCR-based assay.
The utilization of next-generation sequencing-based repeat DNA analysis methodologies for the identification of molecular diagnostic targets has the ability to improve assay species-specificity and limits of detection. By exploiting such high copy-number repeat sequences, the assays described here will facilitate soil transmitted helminth diagnostic efforts. We recommend similar analyses when designing PCR-based diagnostic tests for the detection of other eukaryotic pathogens.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Elimination and control of Schistosoma japonicum, the most virulent of the schistosomiasis-causing blood flukes, requires the development of sensitive and specific diagnostic tools capable of ...providing an accurate measurement of the infection prevalence in endemic areas. Typically, detection of S. japonicum has occurred using the Kato-Katz technique, but this methodology, which requires skilled microscopists, has been shown to radically underestimate levels of infection. With the ever-improving capabilities of next-generation sequencing and bioinformatic analysis tools, identification of satellite sequences and other highly repetitive genomic elements for use as real-time PCR diagnostic targets is becoming increasingly common. Assays developed using these targets have the ability to improve the sensitivity and specificity of results for epidemiological studies that can in turn be used to inform mass drug administration and programmatic decision making.
Utilizing Tandem Repeat Analyzer (TAREAN) and RepeatExplorer2, a cluster-based analysis of the S. japonicum genome was performed and a tandemly arranged genomic repeat, which we named SjTR1 (Schistosoma japonicum Tandem Repeat 1), was selected as the target for a real-time PCR diagnostic assay. Based on these analyses, a primer/probe set was designed and the assay was optimized. The resulting real-time PCR test was shown to reliably detect as little as 200 ag of S. japonicum genomic DNA and as little as 1 egg per gram of human stool. Based on these results, the index assay reported in this manuscript is more sensitive than previously published real-time PCR assays for the detection of S. japonicum.
The extremely sensitive and specific diagnostic assay described in this manuscript will facilitate the accurate detection of S. japonicum, particularly in regions with low levels of endemicity. This assay will be useful in providing data to inform programmatic decision makers, aiding disease control and elimination efforts.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
There is growing interest in local elimination of soil-transmitted helminth (STH) infection in endemic settings. In such settings, highly sensitive diagnostics are needed to detect STH infection. We ...compared double-slide Kato-Katz, the most commonly used copromicroscopic detection method, to multi-parallel quantitative polymerase chain reaction (qPCR) in 2,799 stool samples from children aged 2-12 years in a setting in rural Bangladesh with predominantly low STH infection intensity. We estimated the sensitivity and specificity of each diagnostic using Bayesian latent class analysis. Compared to double-slide Kato-Katz, STH prevalence using qPCR was almost 3-fold higher for hookworm species and nearly 2-fold higher for Trichuris trichiura. Ascaris lumbricoides prevalence was lower using qPCR, and 26% of samples classified as A. lumbricoides positive by Kato-Katz were negative by qPCR. Amplicon sequencing of the 18S rDNA from 10 samples confirmed that A. lumbricoides was absent in samples classified as positive by Kato-Katz and negative by qPCR. The sensitivity of Kato-Katz was 49% for A. lumbricoides, 32% for hookworm, and 52% for T. trichiura; the sensitivity of qPCR was 79% for A. lumbricoides, 93% for hookworm, and 90% for T. trichiura. Specificity was ≥ 97% for both tests for all STH except for Kato-Katz for A. lumbricoides (specificity = 68%). There were moderate negative, monotonic correlations between qPCR cycle quantification values and eggs per gram quantified by Kato-Katz. While it is widely assumed that double-slide Kato-Katz has few false positives, our results indicate otherwise and highlight inherent limitations of the Kato-Katz technique. qPCR had higher sensitivity than Kato-Katz in this low intensity infection setting.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Most eukaryotic lineages are microbial, and many have only recently been sampled for phylogenetic studies or remain in the "dark area" of the tree of life where there are no molecular data. To assess ...relationships among eukaryotic lineages, we perform a taxon-rich phylogenomic analysis including 232 eukaryotes selected to maximize taxonomic diversity and up to 1554 genes chosen as vertically inherited based on their broad distribution among eukaryotes. We also include sequences from 486 bacteria and 84 archaea to assess the impact of endosymbiotic gene transfer (EGT) from plastids and to detect contamination. Overall, our analyses are consistent with other less taxon-rich estimates of the eukaryotic tree of life, and we recover strong support for five major clades: Amoebozoa, Excavata (without the genus Malawimonas), Opisthokonta, Archaeplastida, and SAR (Stramenopila, Alveolata, and Rhizaria). Our analyses also highlight the existence of "orphan" lineages, lineages that lack robust placement in the eukaryotic tree of life, and indicate the possibility of as yet undiscovered diversity. In analyses including bacteria and archaea, we find that approximately 10% of the 1554 genes, which we choose because they are found in four or five of the five major eukaryotic clades and hence may be more likely to be inherited vertically, appear to have been acquired from cyanobacteria through EGT in photosynthetic lineages. Removing these EGT genes places the green algae as sister to the glaucophytes instead of the red algae, suggesting that unknowingly including genes of plastid origin, and combining them with genes of nuclear origin, may mislead phylogenetic estimates. Finally, the large size of our data set allows comparative analyses of subsets of data; alignments built from randomly sampled sites provide greater support, particularly for deep relationships, than do equivalent-sized data sets built from randomly sampled genes.
Celotno besedilo
Dostopno za:
BFBNIB, DOBA, IZUM, KILJ, NMLJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Molecular-based surveys have indicated that Ancylostoma ceylanicum, a zoonotic hookworm, is likely the second most prevalent hookworm species infecting humans in Asia. Most current PCR-based ...diagnostic options for the detection of Ancylostoma species target the Internal Transcribed Spacer (ITS) regions of the ribosomal gene cluster. These regions possess a considerable degree of conservation among the species of this genus and this conservation can lead to the misidentification of infecting species or require additional labor for accurate species-level determination. We have developed a novel, real-time PCR-based assay for the sensitive and species-specific detection of A. ceylanicum that targets a non-coding, highly repetitive genomic DNA element. Comparative testing of this PCR assay with an assay that targets ITS sequences was conducted on field-collected samples from Argentina and Timor-Leste to provide further evidence of the sensitivity and species-specificity of this assay.
A previously described platform for the design of primers/probe targeting non-coding highly repetitive regions was used for the development of this novel assay. The assay's limits of detection (sensitivity) and cross-reactivity with other soil-transmitted helminth species (specificity) were assessed with real-time PCR experiments. The assay was successfully used to identify infections caused by A. ceylanicum that were previously only identified to the genus level as Ancylostoma spp. when analyzed using other published primer-probe pairings. Further proof of sensitive, species-specific detection was provided using a published, semi-nested restriction fragment length polymorphism-PCR assay that differentiates between Ancylostoma species.
Due to the close proximity of people and domestic/wild animals in many regions of the world, the potential for zoonotic infections is substantial. Sensitive tools enabling the screening for different soil-transmitted helminth infections are essential to the success of mass deworming efforts and facilitate the appropriate interpretation of data. This study describes a novel, species-specific, real-time PCR-based assay for the detection of A. ceylanicum that will help to address the need for such tools in integrated STH deworming programs.
ANZCTR.org.au ACTRN12614000680662.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Optimization of polymerase chain reaction (PCR)-based diagnostics requires the careful selection of molecular targets that are both highly repetitive and pathogen-specific. Advances in both ...next-generation sequencing (NGS) technologies and bioinformatics-based analysis tools are facilitating this selection process, informing target choices and reducing labor. Once developed, such assays provide disease control and elimination programs with an additional set of tools capable of evaluating and monitoring intervention successes. The importance of such tools is heightened as intervention efforts approach their endpoints, as accurate and complete information is an essential component of the informed decision-making process. As global efforts for the control and elimination of both lymphatic filariasis and malaria continue to make significant gains, the benefits of diagnostics with improved analytical and clinical/field-based sensitivities and specificities will become increasingly apparent.
Coupling Illumina-based NGS with informatics approaches, we have successfully identified the tandemly repeated elements in both the Wuchereria bancrofti and Plasmodium falciparum genomes of putatively greatest copy number. Utilizing these sequences as quantitative real-time PCR (qPCR)-based targets, we have developed assays capable of exploiting the most abundant tandem repeats for both organisms. For the detection of P. falciparum, analysis and development resulted in an assay with improved analytical and field-based sensitivity vs. an established ribosomal sequence-targeting assay. Surprisingly, analysis of the W. bancrofti genome predicted a ribosomal sequence to be the genome's most abundant tandem repeat. While resulting cycle quantification values comparing a qPCR assay targeting this ribosomal sequence and a commonly targeted repetitive DNA sequence from the literature supported our finding that this ribosomal sequence was the most prevalent tandemly repeated target in the W. bancrofti genome, the resulting assay did not significantly improve detection sensitivity in conjunction with field sample testing.
Examination of pathogen genomes facilitates the development of PCR-based diagnostics targeting the most abundant and specific genomic elements. While in some instances currently available tools may deliver equal or superior performance, systematic analysis of potential targets provides confidence that the selected assays represent the most advantageous options available and that informed assay selection is occurring in the context of a particular study's objectives.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
Sgr A* is the variable electromagnetic source associated with accretion onto the Galactic center supermassive black hole. While the near-infrared (NIR) variability of Sgr A* was shown to be ...consistent over two decades, unprecedented activity in 2019 challenges existing statistical models. We investigate the origin of this activity by recalibrating and reanalyzing all of our Keck Observatory Sgr A* imaging observations from 2005–2022. We present light curves from 69 observation epochs using the NIRC2 imager at 2.12
μ
m with laser-guide star adaptive optics. These observations reveal that the mean luminosity of Sgr A* increased by a factor of ∼3 in 2019, and the 2019 light curves had higher variance than in all time periods we examined. We find that the 2020–2022 flux distribution is statistically consistent with the historical sample and model predictions, but with fewer bright measurements above 0.6 mJy at the ∼2
σ
level. Since 2019, we have observed a maximum
K
s
(2.2
μ
m) flux of 0.9 mJy, compared to the highest pre-2019 flux of 2.0 mJy and highest 2019 flux of 5.6 mJy. Our results suggest that the 2019 activity was caused by a temporary accretion increase onto Sgr A*, possibly due to delayed accretion of tidally stripped gas from the gaseous object G2 in 2014. We also examine faint Sgr A* fluxes over a long time baseline to search for a quasi-steady quiescent state. We find that Sgr A* displays flux variations over a factor of ∼500, with no evidence for a quiescent state in the NIR.
Robotic assistance with integrated navigation is an area of high interest for improving the accuracy of minimally invasive pedicle screw placement. This study analyzes the accuracy of pedicle screw ...placement between an attending spine surgeon and a resident by comparing the left and right sides of the first 101 consecutive cases using navigated robotic assistance in a private practice clinical setting. A retrospective, Institutional Review Board-exempt review of the first 106 navigated robot-assisted spine surgery cases was performed. One attending spine surgeon and one resident performed pedicle screw placement consistently on either the left or right side (researchers were blinded). A CT-based Gertzbein and Robbins system (GRS) was used to classify pedicle screw accuracy, with grade A or B considered accurate. There were 630 consecutive lumbosacral pedicle screws placed. Thirty screws (5 patients) were placed without the robot due to surgeon discretion. Of the 600 pedicle screws inserted by navigated robotic guidance (101 patients), only 1.5% (9/600) were repositioned intraoperatively. Based on the GRS CT-based grading of pedicle breach, 98.67% (296/300) of left-side screws were graded A or B, 1.3% (4/300) were graded C, and 0% (0/300) were graded D. For the right-side screws, 97.67% (293/300) were graded A or B, 1.67% (5/300) were graded C, and 0.66% (2/300) were graded D. This study demonstrated a high level of accuracy (based on GRS) with no significant differences between the left- and right-side pedicle screw placements (98.67% vs. 97.67%, respectively) in the clinical use of navigated, robot-assisted surgery.
The first analyses of gene sequence data indicated that the eukaryotic tree of life consisted of a long stem of microbial groups "topped" by a crown-containing plants, animals, and fungi and their ...microbial relatives. Although more recent multigene concatenated analyses have refined the relationships among the many branches of eukaryotes, the root of the eukaryotic tree of life has remained elusive. Inferring the root of extant eukaryotes is challenging because of the age of the group (~1.7-2.1 billion years old), tremendous heterogeneity in rates of evolution among lineages, and lack of obvious outgroups for many genes. Here, we reconstruct a rooted phylogeny of extant eukaryotes based on minimizing the number of duplications and losses among a collection of gene trees. This approach does not require outgroup sequences or assumptions of orthology among sequences. We also explore the impact of taxon and gene sampling and assess support for alternative hypotheses for the root. Using 20 gene trees from 84 diverse eukaryotic lineages, this approach recovers robust eukaryotic clades and reveals evidence for a eukaryotic root that lies between the Opisthokonta (animals, fungi and their microbial relatives) and all remaining eukaryotes.
Celotno besedilo
Dostopno za:
BFBNIB, DOBA, IZUM, KILJ, NMLJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Onchocerca volvulus is a filarial parasite that is a major cause of dermatitis and blindness in endemic regions primarily in sub-Saharan Africa. Widespread efforts to control the disease caused by O. ...volvulus infection (onchocerciasis) began in 1974 and in recent years, following successful elimination of transmission in much of the Americas, the focus of efforts in Africa has moved from control to the more challenging goal of elimination of transmission in all endemic countries. Mass drug administration (MDA) with ivermectin has reached more than 150 million people and elimination of transmission has been confirmed in four South American countries, with at least two African countries having now stopped MDA as they approach verification of elimination. It is essential that accurate data for active transmission are used to assist in making the critical decision to stop MDA, since missing low levels of transmission and infection can lead to continued spread or recrudescence of the disease.
Current World Health Organization guidelines for MDA stopping decisions and post-treatment surveillance include screening pools of the Simulium blackfly vector for the presence of O. volvulus larvae using a PCR-ELISA-based molecular technique. In this study, we address the potential of an updated, practical, standardized molecular diagnostic tool with increased sensitivity and species-specificity by comparing several candidate qPCR assays. When paired with heat-stable reagents, a qPCR assay with a mitochondrial DNA target (OvND5) was found to be more sensitive and species-specific than an O150 qPCR, which targets a non-protein coding repetitive DNA sequence. The OvND5 assay detected 19/20 pools of 100 blackfly heads spiked with a single L3, compared to 16/20 for the O150 qPCR assay.
Given the improved sensitivity, species-specificity and resistance to PCR inhibitors, we identified OvND5 as the optimal target for field sample detection. All reagents for this assay can be shipped at room temperature with no loss of activity. The qPCR protocol we propose is also simpler, faster, and more cost-effective than the current end-point molecular assays.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
PDF
