Performance of a Novel Molecular Method in the Diagnosis of Late-Onset Sepsis in Very Low Birth Weight Infants

OBJECTIVE: To compare the use of a generic molecular assay to 'standard' investigations used to assist the diagnosis of late onset bacterial sepsis in very low birth weight infants (VLBW, <1500g).

METHODS: VLBW infants, greater than 48 hours of age, who were clinically suspected to have sepsis were investigated using standard tests (full blood count, C-reactive protein (at presentation) and blood culture), in addition, blood was taken for a universal molecular assay (16S rRNA reverse transcriptase PCR) for comparison. Clinical data were recorded during the suspected infection episode. A validated sepsis score (NEO-KISS) was used to retrospectively determine the presence of sepsis (independent of blood culture). The performance of each of the tests were compared by sensitivity, specificity, positive/negative likihood ratios (+/-LR) and postive/negative predictive values (PPV/NPV).

RESULTS: Sixty-five babies with suspected clinical sepsis were prospectively included. The performance indicators are presented with 95% confidence limits. For the detection of bacteria, blood culture had sensitivity of 0.57 (0.34-0.78), specificity of 0.45 (0.30-0.61); +LR of 1.05 (0.66-1.66) and-LR of 0.94 (0.52-1.7); PPV of 33.3 (18.56-50.97) and NPV of 68.97 (49.17-87.72). Serum CRP had sensitivity of 0.92 (0.64-1) and specificity of 0.36 (0.17-0.59); +LR of 1.45 (1-2.1) and-LR of 0.21 (0.03-1.5); PPV of 44.46 (26.6-66.6) and NPV of 88.9 (51.8-99.7). The universal molecular assay had sensitivity of 0.76 (0.53-0.92), specificity of 0.95 (0.85-0.99); +LR of 16.8 (4.2-66.3) and-LR of 0.25 (0.1-0.5); PPV of 88.9 (65.3-98.6) and NPV of 89.4 (76.9-96.5).

CONCLUSIONS: In VLBW infants this universal molecular assay performed better in the diagnosis of late onset sepsis (LOS) than blood culture and CRP. Further development is required to explore and improve the performance of the assay in real-time diagnosis.

The BRUTUS Automatic Cryptanalytic Framework: Testing CAESAR Authenticated Encryption Candidates for Weaknesses

This report summarizes our results from security analysis covering all 57 competitions for authenticated encryption: security, applicability, and robustness (CAESAR) first-round candidates and over 210 implementations. We have manually identified security issues with three candidates, two of which are more serious, and these ciphers have been withdrawn from the competition. We have developed a testing framework, BRUTUS, to facilitate automatic detection of simple security lapses and susceptible statistical structures across all ciphers. From this testing, we have security usage notes on four submissions and statistical notes on a further four. We highlight that some of the CAESAR algorithms pose an elevated risk if employed in real-life protocols due to a class of adaptive-chosen-plaintext attacks. Although authenticated encryption with associated data are often defined (and are best used) as discrete primitives that authenticate and transmit only complete messages, in practice, these algorithms are easily implemented in a fashion that outputs observable ciphertext data when the algorithm has not received all of the (attacker-controlled) plaintext. For an implementor, this strategy appears to offer seemingly harmless and compliant storage and latency advantages. If the algorithm uses the same state for secret keying information, encryption, and integrity protection, and the internal mixing permutation is not cryptographically strong, an attacker can exploit the ciphertext–plaintext feedback loop to reveal secret state information or even keying material. We conclude that the main advantages of exhaustive, automated cryptanalysis are that it acts as a very necessary sanity check for implementations and gives the cryptanalyst insights that can be used to focus more specific attack methods on given candidates.

Improving composite damage modelling through automatic placement of cohesive elements

Numerous experimental studies of damage in composite laminates have shown that intralaminar (in-plane) matrix cracks lead to interlaminar delamination (out-of-plane) at ply interfaces. The smearing of in-plane cracks over a volume, as a consequence of the use of continuum damage mechanics, does not always effectively capture the full extent of the interaction between the two failure mechanisms. A more accurate representation is obtained by adopting a discrete crack approach via the use of cohesive elements, for both in-plane and out-of-plane damage. The difficulty with cohesive elements is that their location must be determined a priori in order to generate the model; while ideally the position of the crack migration, and more generally the propagation path, should be obtained as part of the problem’s solution. With the aim of enhancing current modelling capabilities with truly predictive capabilities, a concept of automatic insertion of interface elements is utilized. The consideration of a simple traction criterion in relation to material strength, evaluated at each node of the model (or of the regions of the model where it is estimated cracks might form), allows for the determination of initial crack location and subsequent propagation by the insertion of cohesive elements during the course of the analysis. Several experimental results are modelled using the commercial package ABAQUS/Standard with an automatic insertion subroutine developed in this work, and the results are presented to demonstrate the capabilities of this technique.

Application of the pMHC Array to Characterise Tumour Antigen Specific T Cell Populations in Leukaemia Patients at Disease Diagnosis

Immunotherapy treatments for cancer are becoming increasingly successful, however to further improve our understanding of the T-cell recognition involved in effective responses and to encourage moves towards the development of personalised treatments for leukaemia immunotherapy, precise antigenic targets in individual patients have been identified. Cellular arrays using peptide-MHC (pMHC) tetramers allow the simultaneous detection of different antigen specific T-cell populations naturally circulating in patients and normal donors. We have developed the pMHC array to detect CD8+ T-cell populations in leukaemia patients that recognise epitopes within viral antigens (cytomegalovirus (CMV) and influenza (Flu)) and leukaemia antigens (including Per Arnt Sim domain 1 (PASD1), MelanA, Wilms' Tumour (WT1) and tyrosinase). We show that the pMHC array is at least as sensitive as flow cytometry and has the potential to rapidly identify more than 40 specific T-cell populations in a small sample of T-cells (0.8-1.4 x 106). Fourteen of the twenty-six acute myeloid leukaemia (AML) patients analysed had T cells that recognised tumour antigen epitopes, and eight of these recognised PASD1 epitopes. Other tumour epitopes recognised were MelanA (n = 3), tyrosinase (n = 3) and WT1126-134 (n = 1). One of the seven acute lymphocytic leukaemia (ALL) patients analysed had T cells that recognised the MUC1950-958 epitope. In the future the pMHC array may be used provide point of care T-cell analyses, predict patient response to conventional therapy and direct personalised immunotherapy for patients.

Hydrogel-Forming Microneedle Arrays Allow Detection of Drugs and Glucose In Vivo: Potential for Use in Diagnosis and Therapeutic Drug Monitoring

We describe, for the first time the use of hydrogel-forming microneedle (MN) arrays for minimally-invasive extraction and quantification of drug substances and glucose from skin in vitro and in vivo. MN prepared from aqueous blends of hydrolysed poly(methyl-vinylether-co-maleic anhydride) (11.1% w/w) and poly(ethyleneglycol) 10,000 daltons (5.6% w/w) and crosslinked by esterification swelled upon skin insertion by uptake of fluid. Post-removal, theophylline and caffeine were extracted from MN and determined using HPLC, with glucose quantified using a proprietary kit. In vitro studies using excised neonatal porcine skin bathed on the underside by physiologically-relevant analyte concentrations showed rapid (5 min) analyte uptake. For example, mean concentrations of 0.16 μg/mL and 0.85 μg/mL, respectively, were detected for the lowest (5 μg/mL) and highest (35 μg/mL) Franz cell concentrations of theophylline after 5 min insertion. A mean concentration of 0.10 μg/mL was obtained by extraction of MN inserted for 5 min into skin bathed with 5 μg/mL caffeine, while the mean concentration obtained by extraction of MN inserted into skin bathed with 15 μg/mL caffeine was 0.33 μg/mL. The mean detected glucose concentration after 5 min insertion into skin bathed with 4 mmol/L was 19.46 nmol/L. The highest theophylline concentration detected following extraction from a hydrogel-forming MN inserted for 1 h into the skin of a rat dosed orally with 10 mg/kg was of 0.363 μg/mL, whilst a maximum concentration of 0.063 μg/mL was detected following extraction from a MN inserted for 1 h into the skin of a rat dosed with 5 mg/kg theophylline. In human volunteers, the highest mean concentration of caffeine detected using MN was 91.31 μg/mL over the period from 1 to 2 h post-consumption of 100 mg Proplus® tablets. The highest mean blood glucose level was 7.89 nmol/L detected 1 h following ingestion of 75 g of glucose, while the highest mean glucose concentration extracted from MN was 4.29 nmol/L, detected after 3 hours skin insertion in human volunteers. Whilst not directly correlated, concentrations extracted from MN were clearly indicative of trends in blood in both rats and human volunteers. This work strongly illustrates the potential of hydrogel-forming MN in minimally-invasive patient monitoring and diagnosis. Further studies are now ongoing to reduce clinical insertion times and develop mathematical algorithms enabling determination of blood levels directly from MN measurements.

Assessment of Intervention Fidelity and Recommendations for Researchers Conducting Studies on the Diagnosis and Treatment of Chronic Cough in the Adult: CHEST Guideline and Expert Panel Report

BACKGROUND: Successful management of chronic cough has varied in the primary research studies in the reported literature. One of the potential reasons relates to a lack of intervention fidelity to the core elements of the diagnostic and/or therapeutic interventions that were meant to be used by the investigators.

METHODS: We conducted a systematic review to summarize the evidence supporting intervention fidelity as an important methodologic consideration in assessing the effectiveness of clinical practice guidelines used for the diagnosis and management of chronic cough. We developed and used a tool to assess for five areas of intervention fidelity. Medline (PubMed), Scopus, and the Cochrane Database of Systematic Reviews were searched from January 1998 to May 2014. Guideline recommendations and suggestions for those conducting research using guidelines or protocols to diagnose and manage chronic cough in the adult were developed and voted upon using CHEST Organization methodology.

RESULTS: A total of 23 studies (17 uncontrolled prospective observational, two randomized controlled, and four retrospective observational) met our inclusion criteria. These articles included 3,636 patients. Data could not be pooled for meta-analysis because of heterogeneity. Findings related to the five areas of intervention fidelity included three areas primarily related to the provider and two primarily related to the patients. In the area of study design, 11 of 23 studies appeared to be underpinned by a single guideline/protocol; for training of providers, two of 23 studies reported training, and zero of 23 reported the use of an intervention manual; and for the area of delivery of treatment, when assessing the treatment of gastroesophageal reflux disease, three of 23 studies appeared consistent with the most recent guideline/protocol referenced by the authors. For receipt of treatment, zero of 23 studies mentioned measuring concordance of patient-interventionist understanding of the treatment recommended, and zero of 23 mentioned measuring enactment of treatment, with three of 23 measuring side effects and two of 23 measuring adherence. The overall average intervention fidelity score for all 23 studies was poor (20.74 out of 48).

CONCLUSIONS: Only low-quality evidence supports that intervention fidelity strategies were used when conducting primary research in diagnosing and managing chronic cough in adults. This supports the contention that some of the variability in the reporting of patients with unexplained or unresolved chronic cough may be due to lack of intervention fidelity. By following the recommendations and suggestions in this article, researchers will likely be better able to incorporate strategies to address intervention fidelity, thereby strengthening the validity and generalizability of their results that provide the basis for the development of trustworthy guidelines.

Angle-closure glaucoma: impact, etiology, diagnosis, and treatment.

PURPOSE OF REVIEW:

Recent studies underscore the importance of angle-closure glaucoma (ACG) as a cause of world blindness. A major contribution in assessing the true impact of this disease has been an article estimating the number of persons with occludable angles, angle closure, and blindness from ACG in China as 28.2 million, 9.1 million, and 1.7 million, respectively. Although these numbers are based on data from Singapore and Mongolia, which may be applied to China only with caution, they emphasize the blinding potential of ACG, which is three times as likely to be associated with blindness as open-angle glaucoma (OAG).

RECENT FINDINGS:

Recent reports in the Chinese literature on ACG prevalence suffer from definitional problems that would appear to lead to systematic overestimates of ACG prevalence and underestimates of OAG prevalence. Nonetheless, data from studies by Chinese investigators further emphasize the strong association between ACG and blindness, with fully 16% of subjects with ACG blind in one report-a far higher proportion than for OAG in China and elsewhere. The importance of topiramate as a cause of secondary angle closure has recently been understood, in part, because of a series of 19 such cases reported by investigators at the Food and Drug Administration.

SUMMARY:

Angle closure in this setting appears to be caused by uveal effusion and anterior rotation of the ciliary body with resultant closure of the angle. The condition is not always responsive to laser iridectomy, and elimination of the causative agent appears to be critical. Ultrasonic biomicroscopy is a potential new diagnostic modality for ACG, allowing the measurement of novel parameters, such as the angle opening distance (AOD) at 500 microm (AOD 500). The efficacy of such parameters in improving screening for ACG can only be established by prospective studies of potentially at-risk eyes. A number of novel treatments for AC and angle closure have recently been proposed, including cataract extraction, paracentesis, and argon laser iridoplasty. As with proposed new diagnostic modalities, the efficacy of these treatments remains to be demonstrated with prospective studies, ideally organized in a controlled, randomized fashion.

Corneal structure and biomechanics: impact on the diagnosis and management of glaucoma.

PURPOSE OF REVIEW:

Highlights recent studies relating to the impact of corneal structure and biomechanical properties on glaucoma evaluation and management.

RECENT FINDINGS:

Central corneal thickness has been shown to play a role in the interpretation of intraocular pressure. Central corneal thickness has also been suggested as a glaucoma risk factor. The potential role of other corneal factors, such as stromal makeup, in the accurate measurement of intraocular pressure and the assessment of glaucoma risk remains to be determined.

SUMMARY:

Improved understanding of central corneal thickness and corneal biomechanical properties may someday lead to a better understanding of glaucoma risk and its assessment.

The value of tests in the diagnosis and management of glaucoma.

PURPOSE:

To assess the noneconomic value of tests used in the diagnosis and management of glaucoma, and explore the contexts and factors that determine such value.

DESIGN:

Perspective.

METHODS:

Selected articles from primary and secondary sources were reviewed and interpreted in the context of the authors' clinical and research experience, influenced by our perspectives on the tasks of reducing the global problem of irreversible blindness caused by glaucoma. The value of any test used in glaucoma is addressed by 3 questions regarding: its contexts, its kind of value, and its implicit or explicit benefits.

RESULTS:

Tonometry, slit-lamp gonioscopy, and optic disc evaluation remain the foundation of clinic-based case finding, whether in areas of more or less abundant resources. In resource-poor areas, there is urgency in identifying patients at risk for severe functional loss of vision; screening strategies have proven ineffective, and efforts are hindered by the inadequate allocation of support. In resource-abundant areas, the wider spectrum of glaucoma is addressed, with emphasis on early detection of structural changes of little functional consequence; these are increasingly the focus of new and expensive technologies whose clinical value has not been established in longitudinal and population-based studies. These contrasting realities in part reflect differences among the value ascribed, often implicitly, to the tests used in glaucoma.

CONCLUSIONS:

The value of any test is determined by 3 aspects: its context of usage; its comparative worth and to whom its benefit accrues; and how we define historically what we are testing. These multiple factors

Considerations for optimization of microRNA PCR assays for molecular diagnosis

The remarkable stability of microRNAs in biofluids underlies their potential as biomarkers, but their small size presents challenges for detection by RT-qPCR. The heterogeneity of microRNAs, with each one comprising a series of variants or 'isomiRs', adds additional complexity. Presented here are the key considerations for use of RT-qPCR to measure microRNAs and their isomiRs, with a focus on plasma. Modified nucleotides can be incorporated into primer sequences to enhance affinity and provide increased specificity and sensitivity for RT-qPCR assays. Approaches based upon polyA tailing and use of a common oligo(dT)-based reverse transcription oligonucleotide will detect most isomiRs. Conversely, stem-loop RT oligonucleotides and sequence specific probes can enable detection of specific isomiRs of interest. Next generation sequencing of all the products of a microRNA RT-PCR reaction is a promising new approach for both microRNA quantification and characterization.