Abstract
The clinical signs and symptoms of acute respiratory tract infections (RTIs) are not pathogen specific. Highly sensitive and specific nucleic acid amplification tests have become the ...diagnostic reference standard for viruses, and translation of bacterial assays from basic research to routine clinical practice represents an exciting advance in respiratory medicine. Most recently, molecular diagnostics have played an essential role in the global health response to the novel coronavirus pandemic. How best to use newer molecular tests for RTI in combination with clinical judgment and traditional methods can be bewildering given the plethora of available assays and rapidly evolving technologies. Here, we summarize the current state of the art with respect to the diagnosis of viral and bacterial RTIs, provide a practical framework for diagnostic decision making using selected patient-centered vignettes, and make recommendations for future studies to advance the field.
Molecular assays have revolutionized the diagnosis of acute respiratory tract infections. However, many unanswered questions about the optimal use and cost-effectiveness of these tests remain. Additional prospective diagnostic studies are needed to measure impact on medical decision making and clinical outcomes.
Isothermal molecular diagnostics are bridging the technology gap between traditional diagnostics and polymerase chain reaction-based methods. These new techniques enable timely and accurate testing, ...especially in settings where there is a lack of infrastructure to support polymerase chain reaction facilities. Despite this, there is a significant lack of uptake of these technologies in developing countries where they are highly needed. Among these novel isothermal technologies, recombinase polymerase amplification (RPA) holds particular potential for use in developing countries. This rapid nucleic acid amplification approach is fast, highly sensitive and specific, and amenable to countries with a high burden of infectious diseases. Implementation of RPA technology in developing countries is critically required to assess limitations and potentials of the diagnosis of infectious disease, and may help identify impediments that prevent adoption of new molecular technologies in low resource- and low skill settings. This review focuses on approaching diagnosis of infectious disease with RPA.
The ongoing pandemic has demonstrated the utility of widespread surveillance and diagnostic detection of the novel SARS-CoV-2. Reverse-transcription loop-mediated isothermal amplification (RT-LAMP) ...has enabled broader testing, but current LAMP tests only detect single targets and require separate reactions for controls. With flu season in the Northern Hemisphere, the ability to screen for multiple targets will be increasingly important, and the ability to include internal controls in RT-LAMP allows for improved efficiency. Here we describe multiplexed RT-LAMP with four targets (SARS-CoV-2, influenza A, influenza B, human RNA) in a single reaction using real-time and end point fluorescence detection. Such increased functionality of RT-LAMP will enable even broader adoption of this molecular testing approach and aid in the fight against this public health threat.
This study describes enhancing loop-mediated isothermal amplification through multiplexed real-time and end point detection of SARS-CoV-2 combined with influenza and control targets. By enabling multiple target detection, loop-mediated isothermal amplification can be even more widely used for diagnostics in settings where multiple viral targets are potential infectious agents and where higher-throughput testing is advantageous.
Loop-mediated isothermal amplification (LAMP) is a versatile technique for detection of target DNA and RNA, enabling rapid molecular diagnostic assays with minimal equipment. The global SARS-CoV-2 ...pandemic has presented an urgent need for new and better diagnostic methods, with colorimetric LAMP utilized in numerous studies for SARS-CoV-2 detection. However, the sensitivity of colorimetric LAMP in early reports has been below that of the standard RT-qPCR tests, and we sought to improve performance. Here we report the use of guanidine hydrochloride and combined primer sets to increase speed and sensitivity in colorimetric LAMP, bringing this simple method up to the standards of sophisticated techniques and enabling accurate, high-throughput diagnostics.
This study describes enhancing loop-mediated isothermal amplification through the addition of guanidine chloride and the use of multiple primer sets for different gene targets combined in a single reaction. We demonstrate a five- to tenfold increase in sensitivity for colorimetric SARS-CoV-2 detection, further improved by use of absorbance measurement using a standard plate reader format. These alterations to the simple and rapid colorimetric loop-mediated isothermal amplification method increase performance and can be used as the basis for sensitive isothermal molecular diagnostic tests.
Bloodstream infections are a major cause of death with increasing incidence and severity. Blood cultures are still the reference standard for microbiological diagnosis, but are rather slow. Molecular ...methods can be used as add-on complementary assays. They can be useful to speed up microbial identification and to predict antimicrobial susceptibility, applied to direct blood samples or positive blood cultures.
To review recent developments in molecular-based diagnostic platforms used for the identification of bloodstream infections, with a focus on assays performed directly on blood samples and positive blood cultures.
Peer reviewed articles, conference abstracts, and manufacturers' websites.
We give an update on recent developments of molecular methods in diagnosing BSIs. We first describe the currently available molecular methods to be used for positive blood cultures including: a) in situ hybridization-based methods; b) DNA-microarray-based hybridization technology; c) nucleic acid amplification-based methods; and d) combined methods. Subsequently, molecular methods applied directly to whole blood samples are discussed, including the use of nucleic acid amplification-based methods, T2 magnetic resonance-based methods, and metagenomics for diagnosing BSIs.
Advances in molecular-based methods complementary to conventional blood culture diagnostics and antimicrobial stewardship programmes may optimize infection management by allowing rapid identification of pathogens and relevant antimicrobial resistance genes. Rapid diagnosis of the causing microorganism and relevant resistance determinants is important for early administration and modification of appropriate antimicrobial therapy. Ultimately, this may lead to improved quality and cost-effectiveness of health care, as well as reduced antimicrobial resistance selection.
•A RT-PCR based test and automated testing platform for MTB and drug resistance detection in a single run.•Includes all the reagents required to perform sample to result analysis in 2.5 h.•PCR ...components in lyophilized format.•Inclusion of controls for quality check, ensure result integrity and result reliability.•Analytical sensitivity of 25 CFU/ml for MTB detection, 200 CFU/ml for rpoB, inhA and katG genes.•Compatible with respiratory pulmonary and non- pulmonary specimens.•Storage and transport at ambient temperature (15–30 °C).
A real time-polymerase chain reaction-based test in lyophilized form, was developed to simultaneously identify Mycobacterium tuberculosis complex (MTC) by targeting IS6110, rrs as dual markers, as well as mutations causing rifampicin and isoniazid resistance. The test was evaluated for pulmonary and non-pulmonary specimens from sample isolation to PCR analysis. The test demonstrated limit of detection of 25 CFU/mL for MTB, 200 CFU/mL for rpoB and inhA/katG targets with >95 % CI. The specificity for MTC was supported by a comprehensive clinical validation (n = 100). This load-and-go molecular platform, with features of high throughput, long shelf-life, room temperature storage provides simultaneous detection of MTC and its drug-resistant mutations in minimal time. The test named “PathoDetect TM MTB-RIF and INH resistance detection kit” has been approved by Central Drugs Standard Control Organisation, Indian Council of Medical Research and would have implications for tuberculosis elimination programs.
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