Twenty years ago, the publication of the first bacterial genome sequence, from Haemophilus influenzae, shook the world of bacteriology. In this Timeline, we review the first two decades of bacterial ...genome sequencing, which have been marked by three revolutions: whole-genome shotgun sequencing, high-throughput sequencing and single-molecule long-read sequencing. We summarize the social history of sequencing and its impact on our understanding of the biology, diversity and evolution of bacteria, while also highlighting spin-offs and translational impact in the clinic. We look forward to a 'sequencing singularity', where sequencing becomes the method of choice for as-yet unthinkable applications in bacteriology and beyond.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
Background
Every year, at least one million children become ill with tuberculosis and around 200,000 children die. Xpert MTB/RIF and Xpert Ultra are World Health Organization (WHO)‐recommended rapid ...molecular tests that simultaneously detect tuberculosis and rifampicin resistance in adults and children with signs and symptoms of tuberculosis, at lower health system levels. To inform updated WHO guidelines on molecular assays, we performed a systematic review on the diagnostic accuracy of these tests in children presumed to have active tuberculosis.
Objectives
Primary objectives
• To determine the diagnostic accuracy of Xpert MTB/RIF and Xpert Ultra for (a) pulmonary tuberculosis in children presumed to have tuberculosis; (b) tuberculous meningitis in children presumed to have tuberculosis; (c) lymph node tuberculosis in children presumed to have tuberculosis; and (d) rifampicin resistance in children presumed to have tuberculosis
‐ For tuberculosis detection, index tests were used as the initial test, replacing standard practice (i.e. smear microscopy or culture)
‐ For detection of rifampicin resistance, index tests replaced culture‐based drug susceptibility testing as the initial test
Secondary objectives
• To compare the accuracy of Xpert MTB/RIF and Xpert Ultra for each of the four target conditions
• To investigate potential sources of heterogeneity in accuracy estimates
‐ For tuberculosis detection, we considered age, disease severity, smear‐test status, HIV status, clinical setting, specimen type, high tuberculosis burden, and high tuberculosis/HIV burden
‐ For detection of rifampicin resistance, we considered multi‐drug‐resistant tuberculosis burden
• To compare multiple Xpert MTB/RIF or Xpert Ultra results (repeated testing) with the initial Xpert MTB/RIF or Xpert Ultra result
Search methods
We searched the Cochrane Infectious Diseases Group Specialized Register, MEDLINE, Embase, Science Citation Index, the Cumulative Index to Nursing and Allied Health Literature (CINAHL), Scopus, the WHO International Clinical Trials Registry Platform, ClinicalTrials.gov, and the International Standard Randomized Controlled Trials Number (ISRCTN) Registry up to 29 April 2019, without language restrictions.
Selection criteria
Randomized trials, cross‐sectional trials, and cohort studies evaluating Xpert MTB/RIF or Xpert Ultra in HIV‐positive and HIV‐negative children younger than 15 years. Reference standards comprised culture or a composite reference standard for tuberculosis and drug susceptibility testing or MTBDRplus (molecular assay for detection of Mycobacterium tuberculosis and drug resistance) for rifampicin resistance. We included studies evaluating sputum, gastric aspirate, stool, nasopharyngeal or bronchial lavage specimens (pulmonary tuberculosis), cerebrospinal fluid (tuberculous meningitis), fine needle aspirates, or surgical biopsy tissue (lymph node tuberculosis).
Data collection and analysis
Two review authors independently extracted data and assessed study quality using the Quality Assessment of Studies of Diagnostic Accuracy ‐ Revised (QUADAS‐2). For each target condition, we used the bivariate model to estimate pooled sensitivity and specificity with 95% confidence intervals (CIs). We stratified all analyses by type of reference standard. We assessed certainty of evidence using the GRADE approach.
Main results
For pulmonary tuberculosis, 299 data sets (68,544 participants) were available for analysis; for tuberculous meningitis, 10 data sets (423 participants) were available; for lymph node tuberculosis, 10 data sets (318 participants) were available; and for rifampicin resistance, 14 data sets (326 participants) were available. Thirty‐nine studies (80%) took place in countries with high tuberculosis burden. Risk of bias was low except for the reference standard domain, for which risk of bias was unclear because many studies collected only one specimen for culture.
Detection of pulmonary tuberculosis
For sputum specimens, Xpert MTB/RIF pooled sensitivity (95% CI) and specificity (95% CI) verified by culture were 64.6% (55.3% to 72.9%) (23 studies, 493 participants; moderate‐certainty evidence) and 99.0% (98.1% to 99.5%) (23 studies, 6119 participants; moderate‐certainty evidence). For other specimen types (nasopharyngeal aspirate, 4 studies; gastric aspirate, 14 studies; stool, 11 studies), Xpert MTB/RIF pooled sensitivity ranged between 45.7% and 73.0%, and pooled specificity ranged between 98.1% and 99.6%.
For sputum specimens, Xpert Ultra pooled sensitivity (95% CI) and specificity (95% CI) verified by culture were 72.8% (64.7% to 79.6%) (3 studies, 136 participants; low‐certainty evidence) and 97.5% (95.8% to 98.5%) (3 studies, 551 participants; high‐certainty evidence). For nasopharyngeal specimens, Xpert Ultra sensitivity (95% CI) and specificity (95% CI) were 45.7% (28.9% to 63.3%) and 97.5% (93.7% to 99.3%) (1 study, 195 participants).
For all specimen types, Xpert MTB/RIF and Xpert Ultra sensitivity were lower against a composite reference standard than against culture.
Detection of tuberculous meningitis
For cerebrospinal fluid, Xpert MTB/RIF pooled sensitivity and specificity, verified by culture, were 54.0% (95% CI 27.8% to 78.2%) (6 studies, 28 participants; very low‐certainty evidence) and 93.8% (95% CI 84.5% to 97.6%) (6 studies, 213 participants; low‐certainty evidence).
Detection of lymph node tuberculosis
For lymph node aspirates or biopsies, Xpert MTB/RIF pooled sensitivity and specificity, verified by culture, were 90.4% (95% CI 55.7% to 98.6%) (6 studies, 68 participants; very low‐certainty evidence) and 89.8% (95% CI 71.5% to 96.8%) (6 studies, 142 participants; low‐certainty evidence).
Detection of rifampicin resistance
Xpert MTB/RIF pooled sensitivity and specificity were 90.0% (67.6% to 97.5%) (6 studies, 20 participants; low‐certainty evidence) and 98.3% (87.7% to 99.8%) (6 studies, 203 participants; moderate‐certainty evidence).
Authors' conclusions
We found Xpert MTB/RIF sensitivity to vary by specimen type, with gastric aspirate specimens having the highest sensitivity followed by sputum and stool, and nasopharyngeal specimens the lowest; specificity in all specimens was > 98%. Compared with Xpert MTB/RIF, Xpert Ultra sensitivity in sputum was higher and specificity slightly lower. Xpert MTB/RIF was accurate for detection of rifampicin resistance. Xpert MTB/RIF was sensitive for diagnosing lymph node tuberculosis. For children with presumed tuberculous meningitis, treatment decisions should be based on the entirety of clinical information and treatment should not be withheld based solely on an Xpert MTB/RIF result. The small numbers of studies and participants, particularly for Xpert Ultra, limits our confidence in the precision of these estimates.
Enteroviruses (EV) commonly cause hand, foot and mouth disease (HFMD), and can also cause potentially fatal neurological and systemic complications. In our laboratory, sequencing 5′ untranslated ...region (UTR) of the viral genome has been the routine method of genotyping EVs. During a recent localised outbreak of aseptic meningitis, sequencing the 5′UTR identified the causative virus as EV-A71, which did not fit with the clinical syndrome or illness severity. When genotyped using a different target gene, VP1, the result was different. This led us to evaluate the accuracy of the two different target genome regions and compare them against whole genome sequencing (WGS).
We aimed to optimise the algorithm for detection and characterisation of EVs in the diagnostic laboratory. We hypothesised that VP1 and WGS genotyping would provide different results than 5′UTR in a subset of samples.
Clinical samples from around New South Wales which were positive for EV by commercial polymerase chain reaction (PCR) assays were genotyped by targeting three different viral genome regions: the 5′UTR, VP1 and WGS. Sequencing was performed by Sanger and next generation sequencing. The subtyping results were compared.
Of the 74/118 (63%) samples that were successfully typed using both the 5′UTR and the VP1 method, the EV typing result was identical for 46/74 (62%) samples compared to WGS as the gold standard. The same EV group but different EV types were found in 22/74 (30%) samples, and 6/74 (8%) samples belonged to different EV groups depending on typing method used.
Genotyping with WGS and VP1 is more accurate than 5′UTR. Genotyping by the 5′UTR method is very sensitive, but less specific.
Progress in the diagnosis of leishmaniases depends on the development of effective methods and the discovery of suitable biomarkers. We propose firstly an update classification of Leishmania species ...and their synonymies. We demonstrate a global map highlighting the geography of known endemic Leishmania species pathogenic to humans. We summarize a complete list of techniques currently in use and discuss their advantages and limitations. The available data highlights the benefits of molecular markers in terms of their sensitivity and specificity to quantify variation from the subgeneric level to species complexes, (sub) species within complexes, and individual populations and infection foci. Each DNA-based detection method is supplied with a comprehensive description of markers and primers and proposal for a classification based on the role of each target and primer in the detection, identification and quantification of leishmaniasis infection. We outline a genome-wide map of genes informative for diagnosis that have been used for Leishmania genotyping. Furthermore, we propose a classification method based on the suitability of well-studied molecular markers for typing the 21 known Leishmania species pathogenic to humans. This can be applied to newly discovered species and to hybrid strains originating from inter-species crosses. Developing more effective and sensitive diagnostic methods and biomarkers is vital for enhancing Leishmania infection control programs.
•We describe the current state-of-the-art in plant virus diagnostics.•We compared established technologies including ELISA and real-time PCR with new technologies currently in development. These ...include:.•Field-based testing methods including those utilising LAMP.•Multiplex methods including those based upon Luminex bead arrays.•Methods for virus discovery based upon next-generation sequencing.
Despite the seemingly continuous development of newer and ever more elaborate methods for detecting and identifying viruses, very few of these new methods get adopted for routine use in testing laboratories, often despite the many and varied claimed advantages they possess. To understand why the rate of uptake of new technologies is so low, requires a strong understanding of what makes a good routine diagnostic tool to begin. This can be done by looking at the two most successfully established plant virus detection methods: enzyme-linked immunosorbant assay (ELISA) and more recently introduced real-time polymerase chain reaction (PCR). By examining the characteristics of this pair of technologies, it becomes clear that they share many benefits, such as an industry standard format and high levels of repeatability and reproducibility. These combine to make methods that are accessible to testing labs, which are easy to establish and robust in their use, even with new and inexperienced users. Hence, to ensure the establishment of new techniques it is necessary to not only provide benefits not found with ELISA or real-time PCR, but also to provide a platform that is easy to establish and use. In plant virus diagnostics, recent developments can be clustered into three core areas: (1) techniques that can be performed in the field or resource poor locations (e.g., loop-mediated isothermal amplification LAMP); (2) multiplex methods that are able to detect many viruses in a single test (e.g., Luminex bead arrays); and (3) methods suited to virus discovery (e.g., next generation sequencing, NGS). Field based methods are not new, with Lateral Flow Devices (LFDs) for the detection being available for a number of years now. However, the widespread uptake of this technology remains poor. LAMP does offer significant advantages over LFDs, in terms of sensitivity and generic application, but still faces challenges in terms of establishment. It is likely that the main barrier to the uptake of field-based technologies is behavioural influences, rather than specific concerns about the performance of the technologies themselves. To overcome this, a new relationship will need to develop between centralised testing laboratories offering services and those requiring tests; a relationship which is currently in its infancy. Looking further into the future, virus discovery and multiplex methods seem to converge as NGS becomes ever cheaper, easier to perform and can provide high levels of multiplexing without the use of virus specific reagents. So ultimately the key challenge from a routine testing lab perspective will not be one of investment in platforms–which could even be outsourced to commercial sequencing services–but one of having the skills and expertise to analyse the large datasets generated and their subsequent interpretation. In conclusion, only time will tell which of the next-generation of methods currently in development will become the routine diagnostics of the future. This will be determined through a combination of factors. And while the technology itself will have to offer performance advantages over existing methods in order to supplant them, it is likely to be human factors e.g., the behaviours of end users, laboratories and policy makers, the availability of appropriate expertise, that ultimately determine which ones become established. Hence factors cannot be ignored and early engagement with diagnostic stakeholders is essential.
The lab-on-a-chip concept has led to several point-of-care (POC) diagnostic microfluidic platforms. However, few of these can process raw samples for molecular diagnosis and fewer yet are suited for ...use in a resource-limited setting without permanent electrical infrastructure. We present here a very low cost paper microfluidic device for POC extraction of bacterial DNA from raw viscous samples--a challenge for conventional microfluidic platforms. This is an example of "microfluidic origami" in that the system is activated by folding; demonstrated here is room temperature cell lysis and DNA extraction from pig mucin (simulating sputum) spiked with E. coli without the use of external power. The microfluidic origami device features dry reagent storage and rehydration of the lysis buffer. We demonstrate DNA extraction from samples with a bacterial load as low as 33 CFU ml(-1). Extraction times, starting from the raw sample, have been optimized to about 1.5 h without the use of external power, or to within 1 h using an oven or a heater block. The fabrication of this paper microfluidic device can be translated into high volume production in the developing world without the need for a semiconductor clean room or a microfabrication facility. The sample preparation can be performed with the addition of just the sample, water, ethanol and elute buffer to the device, thus reducing chemical hazards during transport and handling.
Directly labelling locus‐specific primers for microsatellite analysis is expensive and a common limitation to small‐budget molecular ecology projects. More cost‐effective end‐labelling of PCR ...products can be achieved through a three primer PCR approach, involving a fluorescently labelled universal primer in combination with modified locus‐specific primers with 5′ universal primer sequence tails. This technique has been widely used but has been limited largely due to a lack of available universal primers suitable for co‐amplifying large numbers of size overlapping loci and without requiring locus‐specific PCR conditions to be modified. In this study, we report a suite of four high‐performance universal primers that can be employed in a three primer PCR approach for efficient and cost‐effective fluorescent end‐labelling of PCR fragments. Amplification efficiency is maximized owing to high universal primer Tm values (approximately 60+ °C) that enhance primer versatility and enable higher annealing temperatures to be employed compared with commonly used universal primers such as M13. We demonstrate that these universal primers can be combined with multiple fluorophores to co‐amplify multiple loci efficiently via multiplex PCR. This method provides a level of multiplexing and PCR efficiency similar to microsatellite fluorescent detection assays using directly labelled primers while dramatically reducing project costs. Primer performance is tested using several alternative PCR strategies that involve both single and multiple fluorophores in single and multiplex PCR across a wide range of taxa.
During 2015-2016, we evaluated the performance of whole-genome sequencing (WGS) as a routine typing tool. Its added value for microbiological and epidemiologic surveillance of listeriosis was ...compared with that for pulsed-field gel electrophoresis (PFGE), the current standard method. A total of 2,743 Listeria monocytogenes isolates collected as part of routine surveillance were characterized in parallel by PFGE and core genome multilocus sequence typing (cgMLST) extracted from WGS. We investigated PFGE and cgMLST clusters containing human isolates. Discrimination of isolates was significantly higher by cgMLST than by PFGE (p<0.001). cgMLST discriminated unrelated isolates that shared identical PFGE profiles and phylogenetically closely related isolates with distinct PFGE profiles. This procedure also refined epidemiologic investigations to include only phylogenetically closely related isolates, improved source identification, and facilitated epidemiologic investigations, enabling identification of more outbreaks at earlier stages. WGS-based typing should replace PFGE as the primary typing method for L. monocytogenes.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, ODKLJ, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
, a major human pathogen, has a collection of virulence factors and the ability to acquire resistance to most antibiotics. This ability is further augmented by constant emergence of new clones, ...making
a "superbug." Clinical use of methicillin has led to the appearance of methicillin-resistant
(MRSA). The past few decades have witnessed the existence of new MRSA clones. Unlike traditional MRSA residing in hospitals, the new clones can invade community settings and infect people without predisposing risk factors. This evolution continues with the buildup of the MRSA reservoir in companion and food animals. This review focuses on imparting a better understanding of MRSA evolution and its molecular characterization and epidemiology. We first describe the origin of MRSA, with emphasis on the diverse nature of staphylococcal cassette chromosome
(SCC
).
and its new homologues (
,
, and
), SCC
types (13 SCC
types have been discovered to date), and their classification criteria are discussed. The review then describes various typing methods applied to study the molecular epidemiology and evolutionary nature of MRSA. Starting with the historical methods and continuing to the advanced whole-genome approaches, typing of collections of MRSA has shed light on the origin, spread, and evolutionary pathways of MRSA clones.
Objective: To determine whether proactive molecular risk classifier for endometrial cancer (ProMisE) could be used to assess the prognosis of patients with atypical endometrial hyperplasia (AEH) or ...early-stage endometrial cancer (EC) treated with levonorgestrel-releasing intrauterine system (LNG-IUS).
Methods: A retrospective cohort study was conducted among 93 AEH or early-stage EC patients who received LNG-IUS to preserve fertility . By immunohistochemistry and gene sequencing, 4 subtypes of ProMisE were identified (p53 wild type p53 wt, mismatch repair-deficient MMRd, p53-abnormal, and POLE-mutated). The primary outcome was the time to complete response (CR) after LNG-IUS therapy. Secondary outcomes included the recurrence rate after CR and success rate of conception.
Results: Among the 93 patients, 15 (16.1%) were classified as MMRd, 6 (6.5%) as POLE-mutated, 5 (5.4%) as p53-abnormal, and 67 (72.0%) as p53 wt. Comparison of serum cancer antigen 125, family history of tumor, and positive rates of programmed cell death 1 ligand 1 protein and Ki67 protein in 4 groups showed statistically significant differences (p<0.05). Patients with the p53-abnormal subtype had the lowest overall CR rate (40%) and the highest recurrence rate (2/2). Patients with POLE-mutated subtype had the best prognosis, and all 6 patients achieved CR. When patients achieved complete remission, assisted reproductive technology was more likely to help them conceive than natural conception (p<0.05).
Conclusion: Patients with early-stage EC or AEH who are more likely to benefit from fertility-sparing treatment can be identified using ProMisE classifier. Patients with POLE-mutated are suitable for fertility-sparing treatment with LNG-IUS.
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