Nontuberculous mycobacteria (NTM) infection is similar to Mycobacterium tuberculosis (MTB) infection. Early clinical identification and differentiation of NTM and MTB infections continues to be a major challenge. Nucleic acid amplification tests (NAATs) have the ability to efficiently and rapidly detect pathogens and are widely used in mycobacterial infections. The objective of this study is to estimate the diagnostic accuracy of NAATs for NTM.

The conventional PCR remains a valuable method to detect the newly emergent coronavirus rapidly and accurately. Our investigation aimed to establish the standard materials of SARS-CoV-2 for NAAT detection. We provided formalin-inactivated SARS-CoV-2 and confirmed RNA copy numbers. In addition, the virus genome was confirmed with whole-genome sequencing and identified as Wuhan/WI04/2019. Seven laboratories were invited for this collaborative study, according to the reporting data, we determined the SARS-CoV-2 with the unit of 6.35 Log10 copies/mL as the national standard. The availability of the national standard (NS) of SARS-CoV-2 will facilitate the standardization and harmonization of SARS-CoV-2 NAAT assays.

Introduction: Regular and rapid large-scale screening for pathogens is crucial for controlling pandemics like Coronavirus Disease 2019 (COVID-19). In this study, we present the development of a digital point-of-care testing (POCT) system utilizing microfluidic paper-based analytical devices (μPADs) for the detection of SARS-CoV-2 gene fragments. The system incorporates temperature tuning and fluorescent detection components, along with intelligent and autonomous image acquisition and self-recognition programs. Methods: The developed POCT system is based on the nucleic acid amplification test (NAAT), a well-established molecular biology technique for detecting and amplifying nucleic acids. We successfully detected artificially synthesized SARS-CoV-2 gene fragments, namely ORF1ab gene, N gene, and E gene, with minimal reagent consumption of only 2.2 μL per readout, representing a mere 11% of the requirements of conventional in-tube methods. The power dissipation of the system was low, at 6.4 W. Results: Our testing results demonstrated that the proposed approach achieved a limit of detection of 1000 copies/mL, which is equivalent to detecting 1 copy or a single RNA template per reaction. By employing standard curve analysis, the quantity of the target templates can be accurately determined. Conclusion: The developed digital POCT system shows great promise for rapid and reliable detection of SARS-CoV-2 gene fragments, offering a cost-effective and efficient solution for controlling pandemics. Its compatibility with other diagnostic techniques and low reagent consumption make it a viable option to enhance healthcare in resource-limited areas.

Herein, we describe a phosphorothioated hairpin-assisted isothermal amplification (PHAmp) method for detection of a target nucleic acid. The hairpin probe (HP) is designed to contain a 5' phosphorothioate (PS)-modified overhang, a target recognition site, and a 3' self-priming (SP) region. Upon binding to the target nucleic acid, the HP opens and the SP region is rearranged to serve as a primer. The subsequent process of strand displacement DNA synthesis recycles the bound target to open another HP and produces an extended HP (EP) with a PS-DNA/DNA duplex at the end, which would be readily denatured due to its reduced thermal stability. The trigger then binds to the denatured 3' end of the EP and is extended, producing an intermediate double-stranded (ds) DNA product (IP). The trigger also binds to the denatured 3' end of the IP, and its extension produces the final dsDNA product along with concomitant displacement and recycling of EP. By monitoring the dsDNA products, the target nucleic acid can be identified down to 0.29 fM with a wide dynamic range from 1 nM to 1 fM yielding an excellent specificity to discriminate even a single base-mismatched target. The unique design principle could provide new insights into the development of novel isothermal amplification methods for nucleic acid detection.

DNA amplification is one of the most valuable tools for the clinical diagnosis of nucleic acid-related diseases, but current techniques for DNA amplification are based on intermolecular polymerization reactions, resulting in the risk of errors in the intermolecular reaction pattern. In this article, we introduce the concept of intramolecular polymerization and isomerization cyclic amplification (PICA), which extends a short DNA strand to a long strand containing periodic repeats of a sequence through cyclic alternating polymerization and isomerization. To the best of our knowledge, this is the first time that a real ssDNA self-extension method without any additional auxiliary oligonucleotides has been reported. By interfacing PICA with external molecular elements, it can be programmed to respond to different targets. Herein, we designed two distinct types of amplified nucleic acid detection platforms that can be implemented with PICA, including cyclic reverse transcription (CRT) and cyclic replication (CR). We experimentally demonstrate the mechanisms of CRT-PICA and CR-PICA using mammalian miRNA and virus DNA. The results showed that this proposed detection platform has excellent sensitivity, selectivity, and reliability. The detection level could reach the aM level, that is, several copies of target molecules can be detected if a small volume is taken into account.

Nucleic acid amplification technology (NAAT) has assumed a critical position in disease diagnosis in recent times and contributed significantly to healthcare. Application of these methods has resulted in a more sensitive, accurate and rapid diagnosis of infectious diseases than older traditional methods like culture-based identification. NAAT such as the polymerase chain reaction (PCR) is widely applied but seldom available to resource-limited settings. Isothermal amplification (IA) methods provide a rapid, sensitive, specific, simpler and less expensive procedure for detecting nucleic acid from samples. However, not all of these IA techniques find regular applications in infectious diseases diagnosis. Disease diagnosis and treatment could be improved, and the rapidly increasing problem of antimicrobial resistance reduced, with improvement, adaptation, and application of isothermal amplification methods in clinical settings, especially in developing countries. This review centres on some isothermal techniques that have found documented applications in infectious diseases diagnosis, highlighting their principles, development, strengths, setbacks and imminent potentials for use at points of care.

Ribonucleic acid (RNA) viruses are one of the major classes of pathogens that cause human diseases. The conventional method to detect RNA viruses is real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), but it has some limitations. It is expensive and time-consuming, with infrastructure and trained personnel requirements. Its high throughput requires sophisticated automation and large-scale infrastructure. Isothermal amplification methods have been explored as an alternative to address these challenges. These methods are rapid, user-friendly, low-cost, can be performed in less specialized settings, and are highly accurate for detecting RNA viruses. Microfluidic technology provides an ideal platform for performing virus diagnostic tests, including sample preparation, immunoassays, and nucleic acid-based assays. Among these techniques, nucleic acid isothermal amplification methods have been widely integrated with microfluidic platforms for RNA virus detection owing to their simplicity, sensitivity, selectivity, and short analysis time. This review summarizes some common isothermal amplification methods for RNA viruses. It also describes commercialized devices and kits that use isothermal amplification techniques for SARS-CoV-2 detection. Furthermore, the most recent applications of isothermal amplification-based microfluidic platforms for RNA virus detection are discussed in this article.

The COVID-19 pandemic has highlighted a significant research gap in the field of molecular diagnostics. This has brought forth the need for AI-based edge solutions that can provide quick diagnostic results whilst maintaining data privacy, security and high standards of sensitivity and specificity. This paper presents a novel proof-of-concept method to detect nucleic acid amplification using ISFET sensors and deep learning. This enables the detection of DNA and RNA on a low-cost and portable lab-on-chip platform for identifying infectious diseases and cancer biomarkers. We show that by using spectrograms to transform the signal to the time-frequency domain, image processing techniques can be applied to achieve the reliable classification of the detected chemical signals. Transformation to spectrograms is beneficial as it makes the data compatible with 2D convolutional neural networks and helps gain significant performance improvement over neural networks trained on the time domain data. The trained network achieves an accuracy of 84% with a size of 30kB making it suitable for deployment on edge devices. This facilitates a new wave of intelligent lab-on-chip platforms that combine microfluidics, CMOS-based chemical sensing arrays and AI-based edge solutions for more intelligent and rapid molecular diagnostics.

Battling infection is a major healthcare objective. Untreated infections can rapidly evolve toward the condition of sepsis in which the body begins to fail and resuscitation becomes critical and tenuous. Identification of infection followed by rapid antimicrobial treatment are primary goals of medical care, but precise identification of offending organisms by current methods is slow and broad spectrum empirical therapy is employed to cover most potential pathogens. Current methods for identification of bacterial pathogens in a clinical setting typically require days of time, or a 4- to 8-h growth phase followed by DNA extraction, purification and PCR-based amplification. We demonstrate rapid (70-120 min) genetic diagnostics methods utilizing loop-mediated isothermal amplification (LAMP) to test for 15 common infection pathogen targets, called the Infection Diagnosis Panel (In-Dx). The method utilizes filtration to rapidly concentrate bacteria in sample matrices with lower bacterial loads and direct LAMP amplification without DNA purification from clinical blood, urine, wound, sputum and stool samples. The In-Dx panel was tested using two methods of detection: (1) real-time thermocycler fluorescent detection of LAMP amplification and (2) visual discrimination of color change in the presence of Eriochrome Black T (EBT) dye following amplification. In total, 239 duplicate samples were collected (31 blood, 122 urine, 73 mucocutaneous wound/swab, 11 sputum and two stool) from 229 prospectively enrolled hospital patients with suspected clinical infection and analyzed both at the hospital and by In-Dx. Sensitivity (Se) of the In-Dx panel targets pathogens from urine samples by In-Dx was 91.1% and specificity (Sp) was 97.3%, with a positive predictive value (PPV) of 53.7% and a negative predictive value (NPV) of 99.7% as compared to clinical microbial detection methods. Sensitivity of detection of the In-Dx panel from mucocutaneous swab samples was 65.5% with a Sp of 99.3%, and a PPV of 84% and NPV of 98% as compared to clinical microbial detection methods. Results indicate the LAMP-based In-Dx panel allows rapid and precise diagnosis of clinical infections by targeted pathogens across multiple culture types for point-of-care utilization.

Bacterial infection is a widespread problem in humans that can potentially lead to hospitalization and morbidity. The largest obstacle for physicians/clinicians is the time delay in accurately identifying infectious bacteria, especially their sub-species, in order to adequately treat and diagnose such infected patients. Loop-mediated amplification (LAMP) is a nucleic acid amplification method that has been widely used in diagnostic applications due to its simplicity of constant temperature, use of up to 4 to 6 primers (rendering it highly specific), and capability of amplifying low copies of target sequences. Use of interfacial effect-based monitoring is expected to dramatically shorten the time-to-results of nucleic acid amplification techniques. In this work, we developed a LAMP-based point-of-care platform for detection of bacterial infection, utilizing smartphone measurement of contact angle from oil-immersed droplet LAMP reactions. Whole bacteria (Escherichia coli O157:H7) were assayed in buffer as well as 5% diluted human whole blood. Monitoring of droplet LAMP reactions was demonstrated in a three-compartment, isothermal proportional-integrated-derived (PID)-controlled chip. Smartphone-captured images of droplet LAMP reactions, and their contact angles, were evaluated. Contact angle decreased substantially upon target amplification in both buffer and whole blood samples. In comparison, no-target control (NTC) droplets remained stable throughout the 30 min isothermal reactions. These results were explained by the pre-adsorption of plasma proteins to an oil-water interface (lowering contact angle), followed by time-dependent amplicon formation and their preferential adsorption to the plasma protein-occupied oil-water interface. Time-to-results was as fast as 5 min, allowing physicians to quickly make their decision for infected patients. The developed assay demonstrated quantification of bacteria concentration, with a limit-of-detection at 102 CFU/μL for buffer samples, and binary target or no-target identification with a limit-of-detection at 10 CFU/μL for 5% diluted whole blood samples.

Miniaturized bio-diagnostic devices have the potential to allow for rapid pathogen screening in clinical patient samples, as a low cost and portable alternative to conventional bench-top equipment. Miniaturization of key bio-diagnostic techniques, such as: nucleic acid detection and quantification, polymerase chain reaction (PCR), DNA fingerprinting, enzyme linked immunosorbent assay (ELISA), results in substantial reduction of reaction volumes (expensive samples/reagents) and shorter reaction times. Droplet microfluidics (DMF) is one of several miniaturized bio-sample handling techniques available for manipulating clinical samples and reagents in microliter (10-6 L) to picoliter (10-12 L) volume regime. Electro-actuation of sample and reagent in the form of droplets in the aforementioned volume regime, using dielectrophoresis (DEP) and/or Electrowetting (EW) are achieved by means of patterned, insulated metal electrodes on one or more substrates. In this work, we have utilized electro-actuation based DMF technology, integrated with suitably tailored resistive micro-heaters and temperature sensors, to achieve chip based real-time, quantitative PCR (qRT-PCR). This qRT-PCR micro-device was utilized to detect and quantify the presence of influenza A and C virus nucleic acids, using in-vitro synthesized viral RNA segments. The experimental analysis of the DMF micro-device confirms its capabilities in qRT-PCR based detection and quantification of pathogen samples, with accuracy levels comparable to established commercial bench-top equipment (PCR efficiency ∼95%). The limit of detection (LOD) of the chip based qRT-PCR technique was estimated to be ∼5 copies of template RNA per PCR reaction.

Recent advances in nucleic acid based testing using bio-optical sensor approaches have been introduced but most are based on hybridization between the optical sensor and the bio-molecule and not on an amplification mechanism. Direct nucleic acid amplification on an optical sensor has several technical limitations, such as the sensitivity of the temperature sensor, instrument complexity, and high background signal. We here describe a novel nucleic acid amplification method based on a whispering gallery mode active resonator and discuss its potential molecular diagnostic application. By implanting nanoclusters as active compounds, this active resonator operates without tapered fiber coupling and emits a strong photoluminescence signal with low background in the wavelength of low absorption in an aqueous environment that is typical of biosensors. Our method also offers an extremely low detection threshold down to a single copy within 10 min due to the strong light-matter interaction in a nano-gap structure. We envision that this active resonator provides a high refractive index contrast for tight mode confinement with simple alignment as well as the possibility of reducing the device size so that a point-of-care system with low-cost, high-sensitivity and simplicity.

Abdominal tuberculosis is a severe extrapulmonary tuberculosis, which can lead to serious complications. Early diagnosis and treatment are very important for the prognosis and the diagnosis of abdominal tuberculosis is still difficult. This study aims to evaluate the diagnostic accuracy of nucleic acid amplification tests (NAATs) for abdominal tuberculosis using meta-analysis method.

Mycoplasma species (spp.) bacteria can infect cell cultures, posing a potential threat to recipients of cell therapy products. Conventional Mycoplasma testing methods are highly sensitive but typically require a minimum of 28 days to produce results. This delay is problematic if rapid results are needed to inform treatment decisions. Nucleic acid amplification technique (NAT) methods have been gaining favor for Mycoplasma testing due to their speed and specificity; however, they must first be qualified as meeting or exceeding the sensitivity of the compendial method. We present herein a NAT method for the detection of Mycoplasma that circumvents the need for live Mycoplasma spp. in the test procedure by instead being qualified using Mycoplasma spp. genomic DNA. We have demonstrated a lower limit of detection that exceeds the regulatory requirements set by Health Canada. This assay is now being used to screen clinical cell therapy products manufactured at our center.

Hundreds of studies have evaluated the diagnostic accuracy of nucleic-acid amplification tests (NAATs) for tuberculosis (TB). Commercial tests have been shown to give more consistent results than in-house assays. Previous meta-analyses have found high specificity but low and highly variable estimates of sensitivity. However, reasons for variability in study results have not been adequately explored. We performed a meta-analysis on the accuracy of commercial NAATs to diagnose pulmonary TB and meta-regression to identify factors that are associated with higher accuracy.

Recent advances in electronics and microfluidics have enabled several research groups to develop fully integrated, sample-to-result isothermal nucleic acid amplification test (NAAT) platforms for the point of care. However, high component counts and costs have limited translation of these platforms beyond the clinic to low-resource settings-including homes. Many NAATs include complex, multi-component heater electronics based on flex circuits or multiple printed circuit boards (PCBs) to support essential NAAT steps such as lysis, sample deactivation, and nucleic acid amplification. In contrast, current commercial assays for home use, such as those for pregnancy or ovulation that include electronics, typically have just one onboard PCB. This work describes a generalizable strategy to integrate all heaters and the electronics needed to control them onto a single low-cost, USB-powered PCB. We built a multiplexable disposable NAAT ("MD NAAT") platform that applies these principles, integrating small-area heaters that heat small regions to near-boiling (for pathogen lysis and deactivation) and large-area heaters (for amplification) on the same PCB. We show that both classes of heaters have high intra-board and inter-device reproducibility despite only heating a NAAT cartridge from below. We validated the small-area heaters by lysing methicillin-resistant Staphylococcus aureus (MRSA) cells and the large-area heaters by performing two types of isothermal NAATs (isothermal strand displacement amplification (iSDA) and loop-mediated isothermal amplification (LAMP)). These results demonstrate the merit of integrating NAAT heaters and control electronics onto a single printed circuit board and are a step toward translating NAATs to the home.

Staphylococcus epidermidis is a critical pathogen of nosocomial blood infections, resulting in significant morbidity and mortality. A piezoelectric quartz crystal microbalance (QCM) nucleic acid biosensor array using Au nanoparticle signal amplification was developed to rapidly detect S. epidermidis in clinical samples. The synthesized thiolated probes specific targeting S. epidermidis 16S rRNA gene were immobilized on the surface of QCM nucleic acid biosensor arrays. Hybridization was induced by exposing the immobilized probes to the PCR amplified fragments of S. epidermidis, resulting in a mass change and a consequent frequency shift of the QCM biosensor. To further enhance frequency shift results from above described hybridizations, streptavidin coated Au nanoparticles were conjugated to the PCR amplified fragments. The results showed that the lowest detection limit of current QCM system was 1.3×10³ CFU/mL. A linear correlation was found when the concentration of S. epidermidis varied from 1.3×10³ to 1.3×107 CFU/mL. In addition, 55 clinical samples were detected with both current QCM biosensor system and conventional clinical microbiological method, and the sensitivity and specificity of current QCM biosensor system were 97.14% and 100%, respectively. In conclusion, the current QCM system is a rapid, low-cost and sensitive method that can be used to identify infection of S. epidermidis in clinical samples.

Laboratory based diagnosis of infectious diseases usually relies on culture of the disease causing micro-organism, followed by identification and susceptibility testing. Since Borrelia burgdorferi sensu lato, the etiologic agent of Lyme disease or Lyme borreliosis, requires very specific culture conditions (e.g. specific liquid media, long term cul-ture) traditional bacteriology is often not done on a routine basis. Instead, confirmation of the clinical diagnosis needs ei-ther indirect techniques (like serology or measurement of cellular activity in the presence of antigens) or direct but culture independent techniques, like microscopy or nucleic acid amplification techniques (NAT), with polymerase chain reaction (PCR) being the most frequently applied NAT method in routine laboratories. NAT uses nucleic acids of the disease causing micro-organism as template for amplification, isolated from various sources of clinical specimens. Although the underlying principle, adoption of the enzymatic process running during DNA duplication prior to prokaryotic cell division, is comparatively easy, a couple of 'pitfalls' is associated with the technique itself as well as with interpretation of the results. At present, no commercial, CE-marked and sufficiently validated PCR assay is available. A number of homebrew assays have been published, which are different in terms of target (i.e. the gene targeted by the amplification primers), method (nested PCR, PCR followed by hybridization, real-time PCR) and validation criteria. Inhibitory compounds may lead to false negative results, if no appropriate internal control is included. Carry-over of amplicons, insufficient handling and workflow and/or insufficiently validated targets/primers may result in false positive results. Different targets may yield different analytical sensitivity, depending, among other factors, of the redundancy of a target gene in the genome. Per-formance characteristics (e.g. analytical sensitivity and specificity, clinical sensitivity and specificity, reproducibility, etc.) are, if available, only applicable to a specific assay, running in a specific laboratory. Finally, not only the NAT/PCR method itself, but also the process of DNA isolation from the specimen, is highly diverse and may have fundamental im-pact on the (expected) PCR result. Of concern are distribution effects of DNA, in particular, if only low numbers of bacte-ria/genomes are present in a sample, as it is the case for instance in cerebrospinal fluids. For the ordering physician and for the patient requesting PCR analysis, these 'pitfalls' are usually invisible. As a conse-quence, the reported result (i.e. PCR negative or positive for B. burgdorferi) is hard to interpret, especially, if the reported PCR result is contradictory to the clinical diagnosis or other laboratory findings. Moreover, due to the high number of dif-ferent assays in use, two laboratories, testing the same specimen, might come to different PCR results. The current paper wants to summarize the available PCR/NAT assays for the detection of B. burgdorferi DNA in clinical specimens, with special attention to neurologic disorders, and to discuss the difficulties in PCR analysis and result inter-pretation, associated thereof. In view of growing numbers of patients who are diagnosed of having Lyme disease, and ac-knowledging a substantial growth in knowledge regarding other tick- or vector-borne pathogens, which might be able to induce symptoms comparable to Lyme (neuro-)borreliosis, efforts are urgently needed to standardize and harmonize methods for B. burgdorferi nucleic acid amplification.

Cryptococcosis is a major opportunistic invasive mycosis in immunocompromised patients, but it is also increasingly seen in immunocompetent patients. In the early stages of cryptococcosis, limitations of the detection method may hinder the diagnosis. A molecular diagnostic technique based on nucleic acid sequence-based amplification (NASBA) method was developed to fulfil the need for efficient diagnosis of cryptococcosis.

The number of SARS-CoV-2 detection tests requested to the laboratories has dramatically increased together with an urgent need to release reliable responses in a very short time. The two options taken into consideration and analyzed in the current study were the point-of-care test (POCT) based on the nucleic acid amplification test (NAAT) and the Antigen (Ag) rapid test. The POCT-NAAT-based assay was compared with a rapid antigen test of nasopharyngeal swab samples. If the specimen tested positive, it was followed by viral load quantification and by the functional assessment of the residual infectivity. When the initial cycle threshold (Ct) was below 20 (100%), and in the range of 20-25 (92%) and of 25-30 (88%), a great concordance between the POCT-NAAT and the Ag test was observed. Moreover, the positivity of the antigen test was well correlated to a successful infection in vitro (78%), with greater concordance when the initial Ct below 20 or above 35 (100%) and in the range 20-25 (83%). Our findings showed that most of the swabs which tested positive using the antigen test were able to infect the cells in vitro, suggesting that probably only these samples hold residual infectivity and therefore an increased risk of virus transmission at the moment of being tested.