Lateral Flow Assay For Detection Research Articles

An isothermal CRISPR- based lateral flow assay for detection of Neisseria meningitidis

BackgroundNeisseria meningitidis can cause life-threatening meningococcal meningitis and meningococcemia. Old standard microbiological results from CSF/blood cultures are time consuming. This study aimed to combine the sensitivity of loop-mediated isothermal nucleic acid amplification (LAMP) with the specificity of CRISPR/Cas12a cleavage to demonstrate a reliable diagnostic assay for rapid detection of N. meningitidis.MethodsA total of n = 139 samples were collected from patients with suspected meningococcal disease and were used for evaluation. The extracted DNA was subjected to qualitative real-time PCR, targeting capsular transporter gene (ctrA) of N. meningitidis. LAMP-specific primer pairs, also targeting the ctrA, were designed and the LAMP products were subjected to CRISPR/Cas12 cleavage reaction. the readout was on a lateral flow strip. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of LAMP-CRISPR/Cas was compared with real-time PCR assays. The limit of detection (LOD) was established with serial dilutions of the target N. meningitidis DNA and calculated by Probit regression analysis.ResultsSix LAMP assay-specific primers were developed targeting the ctrA gene of N. meningitidis, which is conserved in all meningococcal serogroups. The LAMP primers did not amplify DNA from other bacterial DNA tested, showing 100% specificity. The use of 0.4 M betaine increased the sensitivity and stability of the reaction. LAMP-CRISPR/Cas detected meningococcal serogroups (B, C, W). The assay showed no cross-reactivity and was specific for N. meningitidis. The LOD was 74 (95% CI: 47–311) N. meningitidis copies. The LAMP-CRISPR/Cas performed well compared to the gold standard. In the 139 samples from suspected patients, the sensitivity and specificity of the test were 91% and 99% respectively.ConclusionThis developed and optimized method can complement for the available gold standard for the timely diagnosis of meningococcal meningitis and meningococcemia.

Two lateral flow assays for detection of anti-coccidioidal antibodies show similar performance to immunodiffusion in dogs with coccidioidomycosis.

To compare 2 point-of-care lateral flow assays (LFAs) with immunodiffusion (ID) IgG results for anti-coccidioidal antibody detection in dogs with coccidioidomycosis. A further aim was to compare the quantifiable output of 1 of the LFAs to ID antibody titers. Serum banked from 73 client-owned dogs diagnosed with pulmonary or disseminated coccidioidomycosis. ID was used to determine antibody presence and titer against a coccidioidal antigen preparation. All sera were subsequently tested on an LFA based on recombinant chitinase 1 (CTS1) and the commercially available sōna LFA. LFA results were analyzed and compared to ID IgG results and clinical diagnosis. All assays showed similar sensitivities in detecting anti-coccidioidal antibodies (83.6% to 89.0%). When compared with ID IgG, the CTS1 LFA had a positive percent agreement of 100%, while the sōna LFA had a positive percent agreement of 91.4%. Since the CTS1 LFA is semiquantitative, we were able to compare test line densities with ID titers and found a strong correlation between the 2 assays (Spearman ρ = 0.82). This is the first side-by-side evaluation of a commercially available LFA (sōna) and a newer more rapid anti-CTS1 antibody LFA using serum from dogs with coccidioidomycosis. Both LFAs tested have similar sensitivity to ID IgG results. The CTS1 LFA can be read after 10 minutes and is semiquantitative, while the sōna LFA is read after 30 minutes, and the results are subject to interpretation. Accurate and fast detection of anti-coccidioidal antibodies allows clinicians to initiate appropriate treatment without diagnostic delay.

Pen direct writing of SERRS-based lateral flow assays for detection of penicillin G in milk.

Direct pen writing offers versatile opportunities for development of low-cost tests for point-of-care applications. In this work a lateral flow immunoassay (LFIA) test was fabricated by hand "writing" immunoprobes onto hand-cut nitrocellulose strips with a commercial fountain pen. The qualitative capabilities of the test were extended by addition of a Raman reporter and consequent design and fabrication of a Surface Enhanced Resonant Raman Scattering (SERRS)-LFIA test. As proof-of-concept, dual detection of penicillin G was achieved in milk with a visual LOD of 20 ppm and a dynamic range of 0.03-97.5 ppm. Evaluation against equivalent tests performed with conventionally prepared LFIA strips showed comparable results, thus demonstrating the validity of the test. These results demonstrate the potential for further decrease in cost and consequent broader use of LFIA tests in remote regions and resource-limited environments.

Development of a rapid lateral flow assay for detection of anti-coccidioidal antibodies.

Coccidioides spp. are dimorphic fungi that are capable of infecting human and non-human mammals and can cause diverse manifestations of coccidioidomycosis or Valley fever (VF). In combination with clinical symptoms and radiographic findings, antibody-based diagnostic tests are often used to diagnose and monitor patients with VF. Chitinase 1 (CTS1) has previously been identified as the seroreactive antigen used in these diagnostic assays to detect anticoccidial IgG. Here, an indirect enzyme-linked immunosorbent assay to detect IgG to CTS1 demonstrated 165 of 178 (92.7%) patients with a positive result by immunodiffusion (ID) and/or complement fixation (CF) had antibodies to the single antigen CTS1. We then developed a rapid antibody lateral flow assay (LFA) to detect anti-CTS1 antibodies. Out of 143 samples tested, the LFA showed 92.9% positive percent agreement [95% confidence interval (CI), 84.3%-96.9%] and 97.7% negative percent agreement (95% CI, 87.9%-99.6%) with ID and CF assays. Serum or plasma from canines, macaques, and dolphins was also tested by the CTS1 LFA. Test line densities of the CTS1 LFA correlated in a linear manner with the reported CF and ID titers for human and non-human samples, respectively. This 10-min point-of-care test for the rapid detection of anti-coccidioidal antibodies could help to inform healthcare providers in real-time, potentially improving the efficiency of healthcare delivery.

Employment of Aptamer Recognition Elements in Paper Lateral Flow Assays for Detection of a Traumatic Brain Injury Biomarker

Traumatic brain injury (TBI) is one of consequences of outside violence against head, triggering brain function degradation and loss. As compared to conventional enzyme-linked immunosorbent assays (ELISA), paper lateral flow assays (PLFA) can rapidly detect TBI biomarkers in field or at emergency departments. This works shows molecular recognition elements (MRE) play a critical role in PLFAs. Our results show that aptamers have significant advantages over antibodies as MRE. Aptamer shows higher binding affinity, leading to higher detection sensitivity. Besides, aptamers are more stable, allowing for longer shelf life. In short, our work indicates that aptamers have a great promise in point-of-care testing tools due to their reliability, robustness, reproducibility, and massive production capability.

Visualized Lateral Flow Assay for Dual Viral RNA Fragment Detection.

In this technical note, we report an easy-to-produce, reverse-transcription-free, and protein-enzyme-free lateral flow assay for detection of viral RNA fragments by taking SARS-CoV-2 ORF1ab and N as target models. Catalytic hairpin assembly is utilized for dual RNA fragment orthogonal reaction to generate copious amounts of opened hairpin duplexes, which bridge DNA-modified gold nanoparticles and capture strands on the strip to induce coloration. The dual RNA fragments are simultaneously visualized during one time of sample flow, and single-base-mismatched nontarget sequences can be differentiated. The test strip can be flexibly adapted to detect evolutional SARS-CoV-2 variants such as Delta and Omicron. It also shows potential in visually detecting long-sequence virus simulants and achieves a sensitivity comparable to that of RT-qPCR by incorporation with upstream sample amplification. The lateral flow assay should offer a convenient and reliable technique for viral nucleic acid detection.

A portable 3D-printed pretreatment device combined with graded lateral flow assay for detection of S. aureus

There is a need for facile yet reliable bacterial pretreatment and signal-graded strategies for current pathogens' point-of-care testing (POCT). Herein, we present a multifunction and portable 3D-printed magnetic separation device with graded lateral flow assay (LFA) for semi-automated pretreatment and rapid detection of Staphylococcus aureus (S. aureus) in food. The 3D-printed pretreatment device allows purification and enrichment of the palladium@platinum nanoparticles (Pd@Pt NPs)-antibody-S. aureus complex. The recovery rate was 81.7–96.1% in actual food samples. Pd@Pt NPs-mediated signal amplification immunoassay achieved graded detection of S. aureus, reducing the detection limit from 103 CFU/mL to 102 CFU/mL, corresponding to the highest safety limit value (M) and acceptable level limit value (m) of S. aureus in many countries and organizations, respectively. This study paves the way for cost-effective and sensitive POCT with multiplexing potential in applications ranging from pretreatment of samples in the complex matrix as well as foodborne pathogens graded and rapid analysis.

Analytical Validation of Surface-Enhanced Raman Scattering Paper Lateral Flow Assays for Detection of Traumatic Brain Injury Biomarker

Traumatic brain injury (TBI) can cause brain function loss injury-related deaths. Early TBI management is significant, but requires a timely, accurate, and sensitive assessment solution. However, current radiation- or imaging-based TBI diagnostic technologies are either less effective or time-consuming and expensive. Here, a surface-enhanced Raman scattering paper-based lateral flow strip (SERS-PLFS) is developed for rapid detection of neuron-specific enolase (NSE), a TBI biomarker in clinical blood plasma samples. The SERS-PLFS device is validated analytically with clinical human blood plasma samples. The performance metrics including accuracy, precision, reportable range, lower limit of quantification and upper limit of quantification, recovery, uncertainty, selectivity, robustness, are assessed using statistical methods. The validation results show that the SERS-PLFS is reliable in measuring the NSE levels in TBI patients.

A Review of the Nucleic Acid-Based Lateral Flow Assay for Detection of Breast Cancer from Circulating Biomarkers at a Point-of-Care in Low Income Countries.

The current levels of breast cancer in African women have contributed to the high mortality rates among them. In South Africa, the incidence of breast cancer is also on the rise due to changes in behavioural and biological risk factors. Such low survival rates can be attributed to the late diagnosis of the disease due to a lack of access and the high costs of the current diagnostic tools. Breast cancer is asymptomatic at early stages, which is the best time to detect it and intervene to prevent high mortality rates. Proper risk assessment, campaigns, and access to adequate healthcare need to be prioritised among patients at an early stage. Early detection of breast cancer can significantly improve the survival rate of breast cancer patients, since therapeutic strategies are more effective at this stage. Early detection of breast cancer can be achieved by developing devices that are simple, sensitive, low-cost, and employed at point-of-care (POC), especially in low-income countries (LICs). Nucleic-acid-based lateral flow assays (NABLFAs) that combine molecular detection with the immunochemical visualisation principles, have recently emerged as tools for disease diagnosis, even for low biomarker concentrations. Detection of circulating genetic biomarkers in non-invasively collected biological fluids with NABLFAs presents an appealing and suitable method for POC testing in resource-limited regions and/or LICs. Diagnosis of breast cancer at an early stage will improve the survival rates of the patients. This review covers the analysis of the current state of NABLFA technologies used in developing countries to reduce the scourge of breast cancer.

Gold Nanoparticle-Mediated Lateral Flow Assays for Detection of Host Antibodies and COVID-19 Proteins.

Coronaviruses, that are now well-known to the public, include a family of viruses that can cause severe acute respiratory syndrome (SARS) and other respiratory diseases, such as Middle East respiratory syndrome (MERS). Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the seventh member of this coronavirus family, was detected in 2019 and can cause a number of respiratory symptoms, from dry cough and fever to fatal viral pneumonia. Various diagnostic assays ranging from real-time polymerase chain reaction (RT-PCR) to point-of-care medical diagnostic systems have been developed for detection of viral components or antibodies targeting the virus. Point-of-care assays allow rapid diagnostic assessment of infectious patients. Such assays are ideally simple, low-cost, portable tests with the possibility for on-site field detection that do not require skilled staff, sophisticated equipment, or sample pretreatment, as compared to RT-PCR. Since early 2021 when new SARS-CoV-2 variants of concern increased, rapid tests became more crucial in the disease management cycle. Among rapid tests, gold nanoparticle (GNP)-based lateral flow assays (LFAs) have high capacity for performing at the bedside, paving the way to easy access to diagnosis results. In this review, GNP-based LFAs used for either COVID-19 proteins or human response antibodies are summarized and recommendations for their improvement have been suggested.

Development and Evaluation of a Duplex Lateral Flow Assay for the Detection and Differentiation between Rabbit Haemorrhagic Disease Virus Lagovirus europaeus/GI.1 and /GI.2.

Simple SummaryRabbit Haemorrhagic Disease is caused by a virus that affects the liver, the spleen and the lungs of rabbits, causing hepatitis, splenomegaly and haemorrhages. A new genotype of the virus was first reported in France in 2010 and has spread globally since then, replacing most of the circulating former viruses in many countries. The detection of the virus and the differentiation of both genotypes is of crucial importance for disease surveillance. In this article, a rapid test for antigen detection is described and evaluated, providing the first description of a quick and easy-to-use test that allows for the simultaneous detection and differentiation of the genotypes. A total of 136 samples, rabbit liver samples and liver exudates (liquid collected after freeze–thawing) classified as infected and non-infected, were analysed, with good results. These data confirm that the developed rapid test can be used as a reliable diagnostic test for disease surveillance, especially in farms and the field.Rabbit Haemorrhagic Disease Virus 2 (RHDV2, recently named Lagovirus europaeus/GI.2) was first reported in France in 2010 and has spread globally since then, replacing most of the circulating former RHDV (genotype GI.1) in many countries. The detection and differentiation of both genotypes is of crucial importance for the surveillance of the disease. In this article, a duplex lateral flow assay (LFA) for antigen detection is described and evaluated, providing the first description of a quick and easy-to-use test that allows for the simultaneous detection and differentiation of RHDV genotypes GI.1 and GI.2. A panel of GI.1- or GI.2-infected and non-infected rabbit liver samples and liver exudates (136 samples) was analysed, obtaining a total sensitivity of 94.4% and specificity of 100%. These data confirm that the developed duplex LFA can be used as a reliable diagnostic test for RHD surveillance, especially in farms and the field.

An Antibody-Aptamer-Hybrid Lateral Flow Assay for Detection of CXCL9 in Antibody-Mediated Rejection after Kidney Transplantation.

Chronic antibody-mediated rejection (AMR) is a key limiting factor for the clinical outcome of a kidney transplantation (Ktx), where early diagnosis and therapeutic intervention is needed. This study describes the identification of the biomarker CXC-motif chemokine ligand (CXCL) 9 as an indicator for AMR and presents a new aptamer-antibody-hybrid lateral flow assay (hybrid-LFA) for detection in urine. Biomarker evaluation included two independent cohorts of kidney transplant recipients (KTRs) from a protocol biopsy program and used subgroup comparisons according to BANFF-classifications. Plasma, urine and biopsy lysate samples were analyzed with a Luminex-based multiplex assay. The CXCL9-specific hybrid-LFA was developed based upon a specific rat antibody immobilized on a nitrocellulose-membrane and the coupling of a CXCL9-binding aptamer to gold nanoparticles. LFA performance was assessed according to receiver operating characteristic (ROC) analysis. Among 15 high-scored biomarkers according to a neural network analysis, significantly higher levels of CXCL9 were found in plasma and urine and biopsy lysates of KTRs with biopsy-proven AMR. The newly developed hybrid-LFA reached a sensitivity and specificity of 71% and an AUC of 0.79 for CXCL9. This point-of-care-test (POCT) improves early diagnosis-making in AMR after Ktx, especially in KTRs with undetermined status of donor-specific HLA-antibodies.

A recombinase polymerase amplification lateral flow assay for rapid detection of Burkholderia pseudomallei, the causative agent of melioidosis.

Burkholderia pseudomallei causes a fatal and infectious disease, melioidosis or Whitmore's disease in humans and animals. Melioidosis is present in different parts of the world and is endemic in Southeast Asia and Northern Australia. Accurate diagnosis of melioidosis is difficult due to its common flu-like symptoms, potentially long incubation period and erroneous identification as culture contaminant. Early diagnosis of the disease is essentially required for administration of suitable antibiotics and disease containment. The present study reports a rapid, specific and sensitive recombinase polymerase amplification lateral flow assay for detection of B. pseudomallei. Specific primers and probe were designed and the assay was performed at 41°C for 20min in a portable incubator. End products were detected using ready-to-use lateral flow strips. RPA lateral flow assay could detect ≥ 250fg genomic DNA of B. pseudomallei and ≥ 50 copies of recombinant plasmid harbouring the target DNA sequence. The assay was found to be highly specific and did not cross-react with other bacterial strains. In artificially spiked human blood and urine samples, the detection limit of the assay was 4.8 × 104 and 4.95 × 104CFU/mL of B. pseudomallei, respectively. The detection limit of assay after 6h of enrichment of artificially spiked urine samples was found to be 4.95 × 103CFU/mL of B. pseudomallei. Detection limit in artificially spiked tap water and soil samples was determined to be 7.5 × 102CFU/mL and 3.3 × 104CFU per 5g of B. pseudomallei, respectively.

Lateral flow assay for detection and recovery of live cell Neisseria gonorrhoeae

Gonorrhea, caused by Neisseria gonorrhoeae, is a global disease that disproportionately impacts low- and middle-income countries. Current testing methods have failed to significantly reduce disease burden, thereby allowing untreated gonorrhea infections to cause infertility and contribute to the spread of antibiotic resistance. Therefore, low-cost testing is desperately needed to combat this obligate human pathogen. Here, we developed a lateral flow assay (LFA) that identifies N. gonorrhoeae infection at the point of care (POC). The LFA utilizes whole cell bacteria without pretreatment, thus allowing for viable cell recovery after testing. While other LFAs require preheating or multiple lysing steps, our assay displays high accuracy with a single sample application step, thus making it easier for an untrained user and enabling true at-home diagnosis of gonorrhea. Furthermore, we were able to recover viable N. gonorrhoeae cells from the positive test line, thus enabling downstream antibiotic susceptibility testing. This colorimetric, paper-based, reader-free system costs $0.38 USD per test and can improve upon syndromic management currently employed in low-resource and POC settings.

Validation and Concordance Analysis of a New Lateral Flow Assay for Detection of Histoplasma Antigen in Urine.

Histoplasmosis is a major cause of mortality in people living with HIV (PLHIV). Rapid methods to diagnose Histoplasma capsulatum disease could dramatically decrease the time to initiate treatment, resulting in reduced mortality. The aim of this study was to validate a MiraVista® Diagnostics (MVD) Histoplasma urine antigen lateral flow assay (MVD LFA) for the detection of H. capsulatum antigen (Ag) in urine and compare this LFA against the MVista® Histoplasma Ag quantitative enzyme immunoassays (MVD EIA). We assessed the MVD LFA using a standardized reference panel of urine specimens from Colombia. We tested 100 urine specimens, 26 from PLHIV diagnosed with histoplasmosis, 42 from PLHIV with other infectious diseases, and 32 from non-HIV infected persons without histoplasmosis. Sensitivity and specificity of the MVD LFA was 96%, compared with 96% sensitivity and 77% specificity of the MVD EIA. Concordance analysis between MVD LFA and the MVD EIA displayed an 84% agreement, and a Kappa of 0.656. The MVD LFA evaluated in this study has several advantages, including a turnaround time for results of approximately 40 min, no need for complex laboratory infrastructure or highly trained laboratory personnel, use of urine specimens, and ease of performing.

Paper-based loop-mediated isothermal amplification and lateral flow (LAMP-LF) assay for identification of tissues of cattle origin

The present study was made with the objectives of development and standardization of cattle specific paper-based loop-mediated isothermal amplification cum lateral flow assay (LAMP-LFA), as a Point-of-care test (POCT) for identification of tissue of cattle origin. The components of standardized LAMP reaction utilizing cattle specific primer sets were lyophilized over paper buttons, identified best as the carrier of LAMP reagents. Based on probable LAMP amplicon, a pair of probes was designed, tagged and its hybridization with the amplified product of paper LAMP reaction was optimized. The components of lateral flow assay for detection of probe hybridized LAMP products were standardized. Analysis of successful amplification was made by using HNB dye, LAMP-LFA strip, and also by the typical ladder-like pattern on gel electrophoresis. The assay was found highly specific for cattle with an analytical sensitivity of 0.1 pg of absolute DNA. Laboratory validation carried out on samples from different individuals of cattle, coded samples, binary meat admixture, and heat-processed cattle tissues substantiated the accuracy of the assay. Comparison with pre-standardized species-specific PCR assay taken as gold standards revealed 100% conformity. The field utility of the developed assay was further established by its compatibility with the commercial kit eliminating the lengthy DNA extraction step and storage stability of LAMP reagent carrier buttons for 4 months under refrigeration. Thus, the developed assay capable of the result within 3 h in resource-limited settings can be used as POCT for identification of tissue of cattle origin.

Sensitive and rapid lateral-flow assay for early detection of subclinical mammary infection in dairy cows

Detection of subclinical mastitis (SCM) in its initial stage can save great economic losses, improve milk quality and animal welfare. We have developed a semiquantitative lateral flow assay for the detection of SCM in dairy cows targeting myeloperoxidase (MPO) enzyme of milk neutrophils. A competitive immunoassay format was used, and colloidal gold nanoparticles (GNP) were prepared and used as a labelling agent. Monoclonal anti-MPO antibodies were used and assessed for its quality by enzyme-linked immunosorbent assay and dot blot. Conjugation method for GNP and anti-MPO antibodies was standardised, and the conjugate was placed over the conjugate pad. MPO coupled with a carrier protein (OVA) and the species-specific secondary antibodies were placed on test and control lines, respectively. The developed assay was verified with 75 milk samples collected from healthy, SCM and clinical mastitis cows. It displayed a high sensitivity as it could detect MPO as low as 1.5 ng/ml, an accuracy greater than 97% and showed no crossreactivity when crosschecked with other milk proteins. The developed assay can be used as an alternative for SCM diagnostic tests where lab structure are available for obtaining the lysate of milk SCC.

CRISPR-Cas12-based detection of SARS-CoV-2.

An outbreak of betacoronavirus SARS-CoV-2 began in Wuhan, China in December 2019. COVID-19, the disease associated with infection, rapidly spread to produce a global pandemic. We report development of a rapid (<40 min), easy-to-implement and accurate CRISPR-Cas12-based lateral flow assay for detection of SARS-CoV-2 from respiratory swab RNA extracts. We validated our method using contrived reference samples and clinical samples from US patients, including 36 patients with COVID-19 infection and 42 patients with other viral respiratory infections. Our CRISPR-based DETECTR assay provides a visual and faster alternative to the US CDC SARS-CoV-2 real-time RT-PCR assay, with 95% positive predictive agreement and 100% negative predictive agreement..

Photothermal enhancement in sensitivity of lateral flow assays for detection of E-coli O157:H7

Lateral flow immunoassay (LFA) is a well-known point-of-care technology for the detection of various analytes. However, low sensitivity and lack of quantitative results are some of its critical drawbacks. Here we report a photothermal enhanced lateral flow sensor on the basis of the photothermal properties of reduced graphene oxide (rGO) for the detection of E-coli O157:H7 as a model pathogen. The calibration curve of the photothermal method exhibited a linear range from 5 × 10⁵ to 5 × 10⁷ CFU/ml with a correlation coefficient of R2 = 0.96 and a regression equation of y = 8.1x-43 for standard bacteria solutions in phosphate buffer. The limit of detection was ∼5 × 10⁵ CFU/ml for standard bacteria solutions, which was a 10-fold enhancement in sensitivity compared to the qualitative results. Specificity experiments showed that the photothermal method can only detect the target bacteria among 6 types of bacteria strains. It was confirmed that the developed technique could be a highly potential method for the rapid detection field because it can provide fast quantitative results with improved sensitivity.

Evaluation of the RESIST-4 O.K.N.V immunochromatographic lateral flow assay for the rapid detection of OXA-48, KPC, NDM and VIM carbapenemases from cultured isolates.

Purpose This study aimed to evaluate the performance of the RESIST-4 O.K.N.V immunochromatographic lateral flow assay for the detection of OXA-48, KPC, NDM and VIM carbapenemases in 100 clinical Enterobacteriaceae isolates using solid culture media.Methodology In total, 100 clinical Enterobacteriaceae isolates with characterized β-lactamase enzymes (OXA-48 n=46, KPC n =4, NDM n =43 and VIM n =10) were evaluated using the RESIST-4 O.K.N.V assay. The assay was also evaluated using carbapenem-sensitive control strains and confirmed non-carbapenemase-producing Enterobacteriaceae clinical isolates resistant to carbapenems. Inter-rater agreement of the test was evaluated by four different users who tested 11 randomly selected isolates daily over 3 days.Results Overall accuracy of the assay was 99.5 %. For the detection of KPC, OXA-48 and its variants and VIM the assay correctly identified 100 % of the isolates when compared to PCR. Initial performance for NDM detection was sensitivity=95.3 %, specificity=100 %. Two PCR positive Providencia rettgeri isolates rendered false negative results on the assay. Retesting from a carbapenem zone of inhibition rendered a positive result for both isolates increasing the sensitivity to 100 %. No false positive results or cross reactions were detected.Conclusion The RESIST-4 O.K.N.V is reliable, sensitive and specific for the detection of OXA-48, KPC, NDM and VIM carbapenemases. Further evaluation on improving NDM detection in organisms from the Proteeae tribe is warranted to determine optimal test conditions.