No abstract

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.

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.

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.

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.