Analytical performance of 17 commercially available point-of-care tests for CRP to support patient management at lower levels of the health system.

Accurate and precise point-of-care (POC) testing for C-reactive protein (CRP) can help support healthcare providers in the clinical management of patients. Here, we compared the analytical performance of 17 commercially available POC CRP tests to enable more decentralized use of the tool. The following CRP tests were evaluated. Eight quantitative tests: QuikRead go (Aidian), INCLIX (Sugentech), Spinit (Biosurfit), LS4000 (Lansionbio), GS 1200 (Gensure Biotech), Standard F200 (SD Biosensor), Epithod 616 (DxGen), IFP-3000 (Xincheng Biological); and nine semi-quantitative tests: Actim CRP (ACTIM), NADAL Dipstick (nal von minden), NADAL cassette (nal von minden), ALLTEST Dipstick (Hangzhou Alltest Biotech), ALLTEST Cassette cut-off 10-40-80 (Hangzhou Alltest Biotech), ALLTEST Cassette cut-off 10-30 (Hangzhou Alltest Biotech), Biotest (Hangzhou Biotest Biotech), BTNX Quad Line (BTNX), BTNX Tri Line (BTNX). Stored samples (n = 660) had previously been tested for CRP using Cobas 8000 Modular analyzer (Roche Diagnostics International AG, Rotkreuz, Switzerland (reference standards). CRP values represented the clinically relevant range (10-100 mg/L) and were grouped into four categories (<10 mg/L, 10-40 mg/L or 10-30 mg/L, 40-80 mg/L or 30-80 mg/L, and > 80mg/L) for majority of the semi-quantitative tests. Among the eight quantitative POC tests evaluated, QuikRead go and Spinit exhibited better agreement with the reference method, showing slopes of 0.963 and 0.921, respectively. Semi-quantitative tests with the four categories showed a poor percentage agreement for the intermediate categories and higher percentage agreement for the lower and upper limit categories. Analytical performance varied considerably for the semi-quantitative tests, especially among the different categories of CRP values. Our findings suggest that quantitative tests might represent the best choice for a variety of use cases, as they can be used across a broad range of CRP categories.

Anticipating the future: prognostic tools as a complementary strategy to improve care for patients with febrile illnesses in resource-limited settings.

In low-income and middle-income countries, most patients with febrile illnesses present to peripheral levels of the health system where diagnostic capacity is very limited. In these contexts, accurate risk stratification can be particularly impactful, helping to guide allocation of scarce resources to ensure timely and tailored care. However, reporting of prognostic research is often imprecise and few prognostic tests or algorithms are translated into clinical practice.Here, we review the often-conflated concepts of prognosis and diagnosis, with a focus on patients with febrile illnesses. Drawing on a recent global stakeholder consultation, we apply these concepts to propose three use-cases for prognostic tools in the management of febrile illnesses in resource-limited settings: (1) guiding referrals from the community to higher-level care; (2) informing resource allocation for patients admitted to hospital and (3) identifying patients who may benefit from closer follow-up post-hospital discharge. We explore the practical implications for new technologies and reflect on the challenges and knowledge gaps that must be addressed before this approach could be incorporated into routine care settings.Our intention is that these use-cases, alongside other recent initiatives, will help to promote a harmonised yet contextualised approach for prognostic research in febrile illness. We argue that this is especially important given the heterogeneous settings in which care is often provided for patients with febrile illnesses living in low-income and middle-income countries.

Bacterial versus non-bacterial infections: a methodology to support use-case-driven product development of diagnostics.

Acute febrile illness (AFI) is one of the most common reasons for seeking medical care in low-income and middle-income countries. Bacterial infections account for a relatively small proportion of AFIs; however, in the absence of a simple diagnostic test to guide clinical decisions, healthcare professionals often presume that a non-malarial febrile illness is bacterial in origin, potentially resulting in inappropriate antibiotic use. An accurate differential diagnostic tool for AFIs is thus essential, to both limit antibiotic use to bacterial infections and address the antimicrobial resistance crisis that is emerging globally, without resorting to multiple or complex pathogen-specific assays. The Biomarker for Fever-Diagnostic (BFF-Dx) study is one of the largest fever biomarker studies ever undertaken. We collected samples and classified disease aetiology in more than 1900 individuals, distributed among enrolment centres in three countries on two continents. Identical protocols were followed at each study site, and the same analyses were conducted in each setting, enabling like-with-like comparisons to be made among the large sample set generated. The BFF-Dx methodology can act as a model for other researchers, facilitating wider utility of the work in the future. The established sample collection is now accessible to researchers and companies and will facilitate the development of future fever-related diagnostic tests. Here, we outline the methodology used to determine the sample populations and to differentiate bacterial versus non-bacterial AFIs. Future publications will set out in more detail the study's demographics, the causes of fever identified and the performance of selected biomarkers.

The good and the bad: using C reactive protein to distinguish bacterial from non-bacterial infection among febrile patients in low-resource settings.

C reactive protein (CRP), a marker for the presence of an inflammatory process, is the most extensively studied marker for distinguishing bacterial from non-bacterial infections in febrile patients. A point-of-care test for bacterial infections would be of particular use in low-resource settings where other laboratory diagnostics are not always available, antimicrobial resistance rates are high and bacterial infections such as pneumonia are a leading cause of death. This document summarises evidence on CRP testing for bacterial infections in low-income and middle-income countries (LMICs). With a push for universal health coverage and prevention of antimicrobial resistance, it is important to understand if CRP might be able to do the job. The use of CRP polarised the global health community and the aim of this document is to summarise the 'good and the bad' of CRP in multiple settings in LMICs. In brief, the literature that was reviewed suggests that CRP testing may be beneficial in low-resource settings to improve rational antibiotic use for febrile patients, but the positive predictive value is insufficient to allow it to be used alone as a single tool. CRP testing may be best used as part of a panel of diagnostic tests and algorithms. Further studies in low-resource settings, particularly with regard to impact on antibiotic prescribing and cost-effectiveness of CRP testing, are warranted.

Author Correction: A systematic review of biomarkers to detect active tuberculosis.

In the original version of this Analysis, author Seda Yerlikaya was mistakenly spelled as Seda Yerliyaka. This has now been amended.

A systematic review of biomarkers to detect active tuberculosis.

Millions of cases of tuberculosis (TB) go undiagnosed each year. Better diagnostic tools are urgently needed. Biomarker-based or multiple marker biosignature-based tests, ideally performed on blood or urine, for the detection of active TB might help to meet target product profiles proposed by the World Health Organization for point-of-care testing. We conducted a systematic review to summarize evidence on proposed biomarkers and biosignatures and evaluate their quality and level of evidence. We screened the titles and abstracts of 7,631 citations and included 443 publications that fulfilled the inclusion criteria and were published in 2010-2017. The types of biomarkers identified included antibodies, cytokines, metabolic activity markers, mycobacterial antigens and volatile organic compounds. Only 47% of studies reported a culture-based reference standard and diagnostic sensitivity and specificity. Forty-four biomarkers (4%) were identified in high-quality studies and met the target product profile minimum criteria, of which two have been incorporated into commercial assays. Of the 44 highest-quality biomarkers, 24 (55%) were multiple marker biosignatures. No meta-analyses were performed owing to between-study heterogeneity. In conclusion, TB biomarker discovery studies are often poorly designed and findings are rarely confirmed in independent studies. Few markers progress to a further developmental stage. More validation studies that consider the intended diagnostic use cases and apply rigorous design are needed. The extracted data from this review are currently being used by FIND as the foundation of a dynamic database in which biomarker data and developmental status will be presented.

Toward the Development of a Circulating Free DNA-Based In Vitro Diagnostic Test for Infectious Diseases: a Review of Evidence for Tuberculosis.

The detection of circulating free DNA (cfDNA) has transformed the field of oncology and prenatal diagnostics. Clinical application of cfDNA for disease diagnosis and monitoring, however, is relatively recent in the field of infectious disease. The potential of cfDNA as a noninvasive diagnostic and monitoring tool is especially promising for tuberculosis (TB), as it enables the detection of both pulmonary and extrapulmonary TB from easily accessible urine and/or blood samples from any age group. However, despite the potential of cfDNA detection to identify TB, very few studies are described in the literature to date. A comprehensive search of the literature identified 15 studies that report detecting Mycobacterium tuberculosis DNA in the blood and urine of TB patients with nongenitourinary disease, but in only six of them were the methodological steps considered suitable for cfDNA isolation and detection. The sensitivities and specificities for the diagnosis of pulmonary and extrapulmonary TB cases reported in these six studies are highly variable, falling in the range of 29% to 79% and 67% to 100%, respectively. While most studies could not meet the performance requirements of the high-priority target product profiles (TPP) published by the World Health Organization (WHO), the study results nonetheless show promise for a point-of-care detection assay. Better designed prospective studies, using appropriate samples, will be required to validate cfDNA as a TB biomarker.

Continuous-flow, microfluidic, qRT-PCR system for RNA virus detection.

One of the main challenges in the diagnosis of infectious diseases is the need for rapid and accurate detection of the causative pathogen in any setting. Rapid diagnosis is key to avoiding the spread of the disease, to allow proper clinical decisions to be made in terms of patient treatment, and to mitigate the rise of drug-resistant pathogens. In the last decade, significant interest has been devoted to the development of point-of-care reverse transcription polymerase chain reaction (PCR) platforms for the detection of RNA-based viral pathogens. We present the development of a microfluidic, real-time, fluorescence-based, continuous-flow reverse transcription PCR system. The system incorporates a disposable microfluidic chip designed to be produced industrially with cost-effective roll-to-roll embossing methods. The chip has a long microfluidic channel that directs the PCR solution through areas heated to different temperatures. The solution first travels through a reverse transcription zone where RNA is converted to complementary DNA, which is later amplified and detected in real time as it travels through the thermal cycling area. As a proof of concept, the system was tested for Ebola virus detection. Two different master mixes were tested, and the limit of detection of the system was determined, as was the maximum speed at which amplification occurred. Our results and the versatility of our system suggest its promise for the detection of other RNA-based viruses such as Zika virus or chikungunya virus, which constitute global health threats worldwide. Graphical abstract Photograph of the RT-PCR thermoplastic chip.

Low-cost, real-time, continuous flow PCR system for pathogen detection.

In this paper, we present a portable and low cost point-of-care (POC) PCR system for quantitative detection of pathogens. Our system is based on continuous flow PCR which maintains fixed temperatures zones and pushes the PCR solution between two heated areas allowing for faster heat transfer and as a result, a faster PCR. The PCR system is built around a 46.0 mm × 30.9 mm × 0.4 mm disposable thermoplastic chip. In order to make the single-use chip economically viable, it was manufactured by hot embossing and was designed to be compatible with roll-to-roll embossing for large scale production. The prototype instrumentation surrounding the chip includes two heaters, thermal sensors, and an optical system. The optical system allows for pathogen detection via real time fluorescence measurements. FAM probes were used as fluorescent reporters of the amplicons generated during the PCR. To demonstrate the function of the chip, two infectious bacteria targets were selected: Chlamydia trachomatis and Escherichia coli O157:H7. For both bacteria, the limit of detection of the system was determined, PCR efficiencies were calculated, and different flow velocities were tested. We have demonstrated successful detection for these two bacterial pathogens highlighting the versatility and broad utility of our portable, low-cost, and rapid PCR diagnostic device.