Rapid, Portable, and Electricity-free Sample Extraction Method for Enhanced Molecular Diagnostics in Resource-Limited Settings.

The COVID-19 pandemic has highlighted the need for rapid and reliable diagnostics that are accessible in resource-limited settings. To address this pressing issue, we have developed a rapid, portable, and electricity-free method for extracting nucleic acids from respiratory swabs (i.e. nasal, nasopharyngeal and buccal swabs), successfully demonstrating its effectiveness for the detection of SARS-CoV-2 in residual clinical specimens. Unlike traditional approaches, our solution eliminates the need for micropipettes or electrical equipment, making it user-friendly and requiring little to no training. Our method builds upon the principles of magnetic bead extraction and revolves around a low-cost plastic magnetic lid, called SmartLid, in combination with a simple disposable kit containing all required reagents conveniently prealiquoted. Here, we clinically validated the SmartLid sample preparation method in comparison to the gold standard QIAamp Viral RNA Mini Kit from QIAGEN, using 406 clinical isolates, including 161 SARS-CoV-2 positives, using the SARS-CoV-2 RT-qPCR assays developed by the US Centers for Disease Control and Prevention (CDC). The SmartLid method showed an overall sensitivity of 95.03% (95% CI: 90.44-97.83%) and a specificity of 99.59% (95% CI: 97.76-99.99%), with a positive agreement of 97.79% (95% CI: 95.84-98.98%) when compared to QIAGEN's column-based extraction method. There are clear benefits to using the SmartLid sample preparation kit: it enables swift extraction of viral nucleic acids, taking less than 5 min, without sacrificing significant accuracy when compared to more expensive and time-consuming alternatives currently available on the market. Moreover, its simplicity makes it particularly well-suited for the point-of-care where rapid results and portability are crucial. By providing an efficient and accessible means of nucleic acid extraction, our approach aims to introduce a step-change in diagnostic capabilities for resource-limited settings.

Amplification Curve Analysis: Data-Driven Multiplexing Using Real-Time Digital PCR.

Information about the kinetics of PCR reactions is encoded in the amplification curve. However, in digital PCR (dPCR), this information is typically neglected by collapsing each amplification curve into a binary output (positive/negative). Here, we demonstrate that the large volume of raw data obtained from real-time dPCR instruments can be exploited to perform data-driven multiplexing in a single fluorescent channel using machine learning methods, by virtue of the information in the amplification curve. This new approach, referred to as amplification curve analysis (ACA), was shown using an intercalating dye (EvaGreen), reducing the cost and complexity of the assay and enabling the use of melting curve analysis for validation. As a case study, we multiplexed 3 carbapenem-resistant genes to show the impact of this approach on global challenges such as antimicrobial resistance. In the presence of single targets, we report a classification accuracy of 99.1% (N = 16188), which represents a 19.7% increase compared to multiplexing based on the final fluorescent intensity. Considering all combinations of amplification events (including coamplifications), the accuracy was shown to be 92.9% (N = 10383). To support the analysis, we derived a formula to estimate the occurrence of coamplification in dPCR based on multivariate Poisson statistics and suggest reducing the digital occupancy in the case of multiple targets in the same digital panel. The ACA approach takes a step toward maximizing the capabilities of existing real-time dPCR instruments and chemistries, by extracting more information from data to enable data-driven multiplexing with high accuracy. Furthermore, we expect that combining this method with existing probe-based assays will increase multiplexing capabilities significantly. We envision that once emerging point-of-care technologies can reliably capture real-time data from isothermal chemistries, the ACA method will facilitate the implementation of dPCR outside of the lab.

Rapid detection and discrimination of chromosome- and MCR-plasmid-mediated resistance to polymyxins by MALDI-TOF MS in Escherichia coli: the MALDIxin test.

Background: Polymyxins are currently considered a last-resort treatment for infections caused by MDR Gram-negative bacteria. Recently, the emergence of carbapenemase-producing Enterobacteriaceae has accelerated the use of polymyxins in the clinic, resulting in an increase in polymyxin-resistant bacteria. Polymyxin resistance arises through modification of lipid A, such as the addition of phosphoethanolamine (pETN). The underlying mechanisms involve numerous chromosome-encoded genes or, more worryingly, a plasmid-encoded pETN transferase named MCR. Currently, detection of polymyxin resistance is difficult and time consuming. Objectives: To develop a rapid diagnostic test that can identify polymyxin resistance and at the same time differentiate between chromosome- and plasmid-encoded resistances. Methods: We developed a MALDI-TOF MS-based method, named the MALDIxin test, which allows the detection of polymyxin resistance-related modifications to lipid A (i.e. pETN addition), on intact bacteria, in <15 min. Results: Using a characterized collection of polymyxin-susceptible and -resistant Escherichia coli, we demonstrated that our method is able to identify polymyxin-resistant isolates in 15 min whilst simultaneously discriminating between chromosome- and plasmid-encoded resistance. We validated the MALDIxin test on different media, using fresh and aged colonies and show that it successfully detects all MCR-1 producers in a blindly analysed set of carbapenemase-producing E. coli strains. Conclusions: The MALDIxin test is an accurate, rapid, cost-effective and scalable method that represents a major advance in the diagnosis of polymyxin resistance by directly assessing lipid A modifications in intact bacteria.

Intact Cell Lipidomics Reveal Changes to the Ratio of Cardiolipins to Phosphatidylinositols in Response to Kanamycin in HeLa and Primary Cells.

Antimicrobial resistance is a major threat the world is currently facing. Development of new antibiotics and the assessment of their toxicity represent important challenges. Current methods for addressing antibiotic toxicity rely on measuring mitochondrial damage using ATP and/or membrane potential as a readout. In this study, we propose an alternative readout looking at changes in the lipidome on intact and unprocessed cells by matrix-assisted laser desorption ionization mass spectrometry. As a proof of principle, we evaluated the impact of known antibiotics (levofloxacin, ethambutol, and kanamycin) on the lipidome of HeLa cells and mouse bone marrow-derived macrophages. Our methodology revealed that clinically relevant concentrations of kanamycin alter the ratio of cardiolipins to phosphatidylinositols. Unexpectedly, only kanamycin had this effect even though all antibiotics used in this study led to a decrease in the maximal mitochondrial respiratory capacity. Altogether, we report that intact cell-targeted lipidomics can be used as a qualitative method to rapidly assess the toxicity of aminoglycosides in HeLa and primary cells. Moreover, these results demonstrate there is no direct correlation between the ratio of cardiolipins to phosphatidylinositols and the maximal mitochondrial respiratory capacity.

Sensitive Detection of Asymptomatic and Symptomatic Malaria with Seven Novel Parasite-Specific LAMP Assays and Translation for Use at Point-of-Care.

Human malaria is a life-threatening parasitic disease with high impact in the sub-Saharan Africa region, where 95% of global cases occurred in 2021. While most malaria diagnostic tools are focused on Plasmodium falciparum, there is a current lack of testing non-P. falciparum cases, which may be underreported and, if undiagnosed or untreated, may lead to severe consequences. In this work, seven species-specific loop-mediated isothermal amplification (LAMP) assays were designed and evaluated against TaqMan quantitative PCR (qPCR), microscopy, and enzyme-linked immunosorbent assays (ELISAs). Their clinical performance was assessed with a cohort of 164 samples of symptomatic and asymptomatic patients from Ghana. All asymptomatic samples with a parasite load above 80 genomic DNA (gDNA) copies per µL of extracted sample were detected with the Plasmodium falciparum LAMP assay, reporting 95.6% (95% confidence interval [95% CI] of 89.9 to 98.5) sensitivity and 100% (95% CI of 87.2 to 100) specificity. This assay showed higher sensitivity than microscopy and ELISA, which were 52.7% (95% CI of 39.7 to 67%) and 67.3% (95% CI of 53.3 to 79.3%), respectively. Nine samples were positive for P. malariae, indicating coinfections with P. falciparum, which represented 5.5% of the tested population. No samples were detected as positive for P. vivax, P. ovale, P. knowlesi, or P. cynomolgi by any method. Furthermore, translation to the point-of-care was demonstrated with a subcohort of 18 samples tested locally in Ghana using our handheld lab-on-chip platform, Lacewing, showing comparable results to a conventional fluorescence-based instrument. The developed molecular diagnostic test could detect asymptomatic malaria cases, including submicroscopic parasitemia, and it has the potential to be used for point-of-care applications. IMPORTANCE The spread of Plasmodium falciparum parasites with Pfhrp2/3 gene deletions presents a major threat to reliable point-of-care diagnosis with current rapid diagnostic tests (RDTs). Novel molecular diagnostics based on nucleic acid amplification are needed to address this liability. In this work, we overcome this challenge by developing sensitive tools for the detection of Plasmodium falciparum and non-P. falciparum species. Furthermore, we evaluate these tools with a cohort of symptomatic and asymptomatic malaria patients and test a subcohort locally in Ghana. The findings of this work could lead to the implementation of DNA-based diagnostics to fight against the spread of malaria and provide reliable, sensitive, and specific diagnostics at the point of care.

Lab-on-chip assay of tumour markers and human papilloma virus for cervical cancer detection at the point-of-care.

Cervical cancer affects over half a million people worldwide each year, the majority of whom are in resource-limited settings where cytology screening is not available. As persistent human papilloma virus (HPV) infections are a key causative factor, detection of HPV strains now complements cytology where screening services exist. This work demonstrates the efficacy of a handheld Lab-on-Chip (LoC) device, with an external sample extraction process, in detecting cervical cancer from biopsy samples. The device is based on Ion-Sensitive Field-Effect Transistor (ISFET) sensors used in combination with loop-mediated isothermal amplification (LAMP) assays, to amplify HPV DNA and human telomerase reverse transcriptase (hTERT) mRNA. These markers were selected because of their high levels of expression in cervical cancer cells, but low to nil expression in normal cervical tissue. The achieved analytical sensitivity for the molecular targets resolved down to a single copy per reaction for the mRNA markers, achieving a limit of detection of 102 for hTERT. In the tissue samples, HPV-16 DNA was present in 4/5 malignant and 2/5 benign tissues, with HPV-18 DNA being present in 1/5 malignant and 1/5 benign tissues. hTERT mRNA was detected in all malignant and no benign tissues, with the demonstrated pilot data to indicate the potential for using the LoC in cervical cancer screening in resource-limited settings on a large scale.

Discrimination of bacterial and viral infection using host-RNA signatures integrated in a lab-on-chip platform.

The unmet clinical need for accurate point-of-care (POC) diagnostic tests able to discriminate bacterial from viral infection demands a solution that can be used both within healthcare settings and in the field, and that can also stem the tide of antimicrobial resistance. Our approach to solve this problem combine the use of host gene signatures with our Lab-on-a-Chip (LoC) technology enabling low-cost POC expression analysis to detect Infectious Disease. Transcriptomics have been extensively investigated as a potential tool to be implemented in the diagnosis of infectious disease. On the other hand, LoC technologies using ion-sensitive field-effect transistor (ISFET), in conjunction with isothermal chemistries, are offering a promising alternative to conventional amplification instruments, owing to their portable and affordable nature. Currently, the data analysis of ISFET arrays are restricted to established methods by averaging the output of every sensor to give a single time-series. This simple approach makes unrealistic assumptions, leading to insufficient performance for applications that require accurate quantification such as Host-Transcriptomics. In order to reliably quantify transcripts on our LoC platform enabling the classification of infectious disease on-chip, we propose a novel data-driven algorithm for extracting time-to-positive values from ISFET arrays. The algorithm proposed correctly outputs a time-to-positive for all the reactions, with a high correlation to RT-qLAMP (0.85, R2 = 0.98, p < 0.01), resulting in a classification accuracy of 100% (CI, 95-100%). This work aims to bridge the gap between translating assays from microarray analysis to ISFET arrays providing benefits on tackling infectious disease and diagnostic testing in hard-to-reach areas of the world.

Loop-Mediated Isothermal Amplification Assay for Detecting Tumor Markers and Human Papillomavirus: Accuracy and Supplemental Diagnostic Value to Endovaginal MRI in Cervical Cancer.

OBJECTIVE: To establish the sensitivity and specificity of a human papillomavirus (HPV) and tumor marker DNA/mRNA assay for detecting cervical cancer that is transferrable to a Lab-on-a-chip platform and determine its diagnostic benefit in early stage disease when used in conjunction with high-resolution endovaginal magnetic resonance imaging (MRI). METHODS: Forty-one patients (27 with Stage1 cervical cancer [Group1] and 14 non-cancer HPV negative controls [Group2]) had DNA and RNA extracted from cervical cytology swab samples. HPV16, HPV18, hTERT, TERC/GAPDH and MYC/GAPDH concentration was established using a loop mediated isothermal amplification (LAMP) assay. Thresholds for tumor marker detection for Group1 were set from Group2 analysis (any hTERT, TERC/GAPDH 3.12, MYC/GAPDH 0.155). Group 1 participants underwent endovaginal MRI. Sensitivity and specificity for cancer detection by LAMP and MRI individually and combined was documented by comparison to pathology. RESULTS: Sensitivity and specificity for cancer detection was 68.8% and 77.8% if any tumor marker was positive regardless of HPV status (scenario1), and 93.8% and 55.8% if tumor marker or HPV were positive (scenario 2). Adding endovaginal MRI improved specificity to 88.9% in scenario 1 (sensitivity 68.8%) and to 77.8%% in scenario2 (sensitivity 93.8%). CONCLUSION: Specificity for cervical cancer detection using a LAMP assay is superior with tumor markers; low sensitivity is improved by HPV detection. Accuracy for early stage cervical cancer detection is optimal using a spatially multiplexed tumor marker/HPV LAMP assay together with endovaginal MRI.

Coupling Machine Learning and High Throughput Multiplex Digital PCR Enables Accurate Detection of Carbapenem-Resistant Genes in Clinical Isolates.

Rapid and accurate identification of patients colonised with carbapenemase-producing organisms (CPOs) is essential to adopt prompt prevention measures to reduce the risk of transmission. Recent studies have demonstrated the ability to combine machine learning (ML) algorithms with real-time digital PCR (dPCR) instruments to increase classification accuracy of multiplex PCR assays when using synthetic DNA templates. We sought to determine if this novel methodology could be applied to improve identification of the five major carbapenem-resistant genes in clinical CPO-isolates, which would represent a leap forward in the use of PCR-based data-driven diagnostics for clinical applications. We collected 253 clinical isolates (including 221 CPO-positive samples) and developed a novel 5-plex PCR assay for detection of blaIMP, blaKPC, blaNDM, blaOXA-48, and blaVIM. Combining the recently reported ML method "Amplification and Melting Curve Analysis" (AMCA) with the abovementioned multiplex assay, we assessed the performance of the AMCA methodology in detecting these genes. The improved classification accuracy of AMCA relies on the usage of real-time data from a single-fluorescent channel and benefits from the kinetic/thermodynamic information encoded in the thousands of amplification events produced by high throughput real-time dPCR. The 5-plex showed a lower limit of detection of 10 DNA copies per reaction for each primer set and no cross-reactivity with other carbapenemase genes. The AMCA classifier demonstrated excellent predictive performance with 99.6% (CI 97.8-99.9%) accuracy (only one misclassified sample out of the 253, with a total of 160,041 positive amplification events), which represents a 7.9% increase (p-value <0.05) compared to conventional melting curve analysis. This work demonstrates the use of the AMCA method to increase the throughput and performance of state-of-the-art molecular diagnostic platforms, without hardware modifications and additional costs, thus potentially providing substantial clinical utility on screening patients for CPO carriage.

Handheld Point-of-Care System for Rapid Detection of SARS-CoV-2 Extracted RNA in under 20 min.

The COVID-19 pandemic is a global health emergency characterized by the high rate of transmission and ongoing increase of cases globally. Rapid point-of-care (PoC) diagnostics to detect the causative virus, SARS-CoV-2, are urgently needed to identify and isolate patients, contain its spread and guide clinical management. In this work, we report the development of a rapid PoC diagnostic test (<20 min) based on reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) and semiconductor technology for the detection of SARS-CoV-2 from extracted RNA samples. The developed LAMP assay was tested on a real-time benchtop instrument (RT-qLAMP) showing a lower limit of detection of 10 RNA copies per reaction. It was validated against extracted RNA from 183 clinical samples including 127 positive samples (screened by the CDC RT-qPCR assay). Results showed 91% sensitivity and 100% specificity when compared to RT-qPCR and average positive detection times of 15.45 ± 4.43 min. For validating the incorporation of the RT-LAMP assay onto our PoC platform (RT-eLAMP), a subset of samples was tested (n = 52), showing average detection times of 12.68 ± 2.56 min for positive samples (n = 34), demonstrating a comparable performance to a benchtop commercial instrument. Paired with a smartphone for results visualization and geolocalization, this portable diagnostic platform with secure cloud connectivity will enable real-time case identification and epidemiological surveillance.

Discovery and validation of a three-gene signature to distinguish COVID-19 and other viral infections in emergency infectious disease presentations: a case-control and observational cohort study.

BACKGROUND: Emergency admissions for infection often lack initial diagnostic certainty. COVID-19 has highlighted a need for novel diagnostic approaches to indicate likelihood of viral infection in a pandemic setting. We aimed to derive and validate a blood transcriptional signature to detect viral infections, including COVID-19, among adults with suspected infection who presented to the emergency department. METHODS: Individuals (aged ≥18 years) presenting with suspected infection to an emergency department at a major teaching hospital in the UK were prospectively recruited as part of the Bioresource in Adult Infectious Diseases (BioAID) discovery cohort. Whole-blood RNA sequencing was done on samples from participants with subsequently confirmed viral, bacterial, or no infection diagnoses. Differentially expressed host genes that met additional filtering criteria were subjected to feature selection to derive the most parsimonious discriminating signature. We validated the signature via RT-qPCR in a prospective validation cohort of participants who presented to an emergency department with undifferentiated fever, and a second case-control validation cohort of emergency department participants with PCR-positive COVID-19 or bacterial infection. We assessed signature performance by calculating the area under receiver operating characteristic curves (AUROCs), sensitivities, and specificities. FINDINGS: A three-gene transcript signature, comprising HERC6, IGF1R, and NAGK, was derived from the discovery cohort of 56 participants with bacterial infections and 27 with viral infections. In the validation cohort of 200 participants, the signature differentiated bacterial from viral infections with an AUROC of 0·976 (95% CI 0·919-1·000), sensitivity of 97·3% (85·8-99·9), and specificity of 100% (63·1-100). The AUROC for C-reactive protein (CRP) was 0·833 (0·694-0·944) and for leukocyte count was 0·938 (0·840-0·986). The signature achieved higher net benefit in decision curve analysis than either CRP or leukocyte count for discriminating viral infections from all other infections. In the second validation analysis, which included SARS-CoV-2-positive participants, the signature discriminated 35 bacterial infections from 34 SARS-CoV-2-positive COVID-19 infections with AUROC of 0·953 (0·893-0·992), sensitivity 88·6%, and specificity of 94·1%. INTERPRETATION: This novel three-gene signature discriminates viral infections, including COVID-19, from other emergency infection presentations in adults, outperforming both leukocyte count and CRP, thus potentially providing substantial clinical utility in managing acute presentations with infection. FUNDING: National Institute for Health Research, Medical Research Council, Wellcome Trust, and EU-FP7.

Rapid Detection of Mobilized Colistin Resistance using a Nucleic Acid Based Lab-on-a-Chip Diagnostic System.

The increasing prevalence of antimicrobial resistance is a serious threat to global public health. One of the most concerning trends is the rapid spread of Carbapenemase-Producing Organisms (CPO), where colistin has become the last-resort antibiotic treatment. The emergence of colistin resistance, including the spread of mobilized colistin resistance (mcr) genes, raises the possibility of untreatable bacterial infections and motivates the development of improved diagnostics for the detection of colistin-resistant organisms. This work demonstrates a rapid response for detecting the most recently reported mcr gene, mcr-9, using a portable and affordable lab-on-a-chip (LoC) platform, offering a promising alternative to conventional laboratory-based instruments such as real-time PCR (qPCR). The platform combines semiconductor technology, for non-optical real-time DNA sensing, with a smartphone application for data acquisition, visualization and cloud connectivity. This technology is enabled by using loop-mediated isothermal amplification (LAMP) as the chemistry for targeted DNA detection, by virtue of its high sensitivity, specificity, yield, and manageable temperature requirements. Here, we have developed the first LAMP assay for mcr-9 - showing high sensitivity (down to 100 genomic copies/reaction) and high specificity (no cross-reactivity with other mcr variants). This assay is demonstrated through supporting a hospital investigation where we analyzed nucleic acids extracted from 128 carbapenemase-producing bacteria isolated from clinical and screening samples and found that 41 carried mcr-9 (validated using whole genome sequencing). Average positive detection times were 6.58 ± 0.42 min when performing the experiments on a conventional qPCR instrument (n = 41). For validating the translation of the LAMP assay onto a LoC platform, a subset of the samples were tested (n = 20), showing average detection times of 6.83 ± 0.92 min for positive isolates (n = 14). All experiments detected mcr-9 in under 10 min, and both platforms showed no statistically significant difference (p-value > 0.05). When sample preparation and throughput capabilities are integrated within this LoC platform, the adoption of this technology for the rapid detection and surveillance of antimicrobial resistance genes will decrease the turnaround time for DNA detection and resistotyping, improving diagnostic capabilities, patient outcomes, and the management of infectious diseases.