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.
Background:Human papillomavirus (HPV) infection is a powerful prognostic biomarker in a subset of head and neck squamous cell carcinomas, specifically oropharyngeal cancers. However, the role of HPV ...in non-oropharyngeal sites, such as the larynx, remains unconfirmed.Methods:We evaluated a cohort of 324 laryngeal squamous cell carcinoma (LSCC) patients for the expression of p16INK4A (p16) protein by immunohistochemistry (IHC) and for high-risk HPV E6 and E7 mRNA transcripts by RNA in situ hybridisation (ISH). p16 expression and HPV status were correlated with clinicopathological features and outcomes.Results:Of 307 patients assessable for p16 IHC, 20 (6.5%) were p16 positive. Females and node-positive patients were more likely to be p16 positive (P<0.05). There were no other significant clinical or demographic differences between p16-positive and -negative cases. There was no difference in overall survival (OS) between p16-positive and -negative patients with 2-year survival of 79% in each group (HR=0.83, 95% CI 0.36-1.89, P=0.65). There was no statistically significant difference in failure-free survival (FFS) with 2-year FFS of 79% and 66% for p16-positive and -negative patients, respectively (HR=0.60, 95% CI 0.26-1.36, P=0.22). Only seven cases were found to be HPV RNA ISH positive, all of which were p16 IHC positive. There was no statistically significant difference in OS between patients with HPV RNA ISH-positive tumours compared with -negative tumours with 2-year survival of 86% and 71%, respectively (HR=0.76, 95% CI 0.23-2.5, P=0.65). The 2-year FFS was 86% and 59%, respectively (HR=0.62, 95% CI 0.19-2.03, P=0.43).Conclusions:p16 overexpression is infrequent in LSCC and the proportion of cases with high-risk HPV transcripts is even lower. There are no statistically significant correlations between p16 IHC or HPV RNA ISH status and OS or disease outcomes.
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.
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.
•BioFire FilmArray BCID2 panel enable earlier agent identification and detection of resistance genes compared to conventional culture methods.•BioFire FilmArray BCID2 panel appropriate antibiotherapy ...modifications and infection control measures could be taken earlier compared to traditional culture methods.•The use of BioFire FilmArray BCID2 panel is promising in critical bacteremia patient.
The BioFire FilmArray Blood Culture Identification panel (BCID2), a rapid molecular blood culture identification test based on multiplex nested polymerase chain reaction. The aim of this study was to evaluate clinical outcomes between the period before (pre-BCID2 group) and after (post-BCID2 group) the introduction of the BCID2 panel into our routine practice.
The primary endpoint was time to optimal antibiotherapy, and the secondary endpoints were duration of hospital and intensive care unit stay, 7-day, 14-day and 28-day mortality rates after bacteremia.
The median time from empirical antibiotherapy to optimal antimicrobial therapy was 4560 (IQR;3060-7140) minutes in the pre-BCID2 group and 1715 (IQR;1362- 2776.25) minutes (in the post-BCID2 group (p<0.05).
Adding the BCID2 panel may improve antibiotic management in critically ill bacteremia patients
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