Lateral Flow Immunoassay Test Research Articles

Six decades of lateral flow immunoassay: from determining metabolic markers to diagnosing COVID-19.

Technologies based on lateral flow immunoassay (LFIA), known in some countries of the world as immunochromatographic tests, have been successfully used for the last six decades in diagnostics of many diseases and conditions as they allow rapid detection of molecular ligands in biosubstrates. The popularity of these diagnostic platforms is constantly increasing in healthcare facilities, particularly those facing limited budgets and time, as well as in household use for individual health monitoring. The advantages of these low-cost devices over modern laboratory-based analyzers come from their availability, opportunity of rapid detection, and ease of use. The attractiveness of these portable diagnostic tools is associated primarily with their high analytical sensitivity and specificity, as well as with the easy visual readout of results. These qualities explain the growing popularity of LFIA in developing countries, when applied at small hospitals, in emergency situations where screening and monitoring health condition is crucially important, and as well as for self-testing of patients. These tools have passed the test of time, and now LFIA test systems are fully consistent with the world's modern concept of ‘point-of-care testing’, finding a wide range of applications not only in human medicine, but also in ecology, veterinary medicine, and agriculture. The extensive opportunities provided by LFIA contribute to the continuous development and improvement of this technology and to the creation of new-generation formats. This review will highlight the modern principles of design of the most widely used formats of test-systems for clinical laboratory diagnostics, summarize the main advantages and disadvantages of the method, as well as the current achievements and prospects of the LFIA technology. The latest innovations are aimed at improving the analytical performance of LFIA platforms for the diagnosis of bacterial and viral infections, including COVID-19.

Comparative Study Of Blood Culture with Rapid Diagnostic Tests for Diagnosis Of Enteric Fever In A Tertiary Centre Of NE-India.

Introduction: Enteric fever is a global health problem and is also endemic in India. The laboratory diagnosis of enteric fever is very important mainly because in post-antibiotic era most cases are treated empirically and investigated for various causes of PUO including enteric fever. A fast, reliable, and easy to perform serodiagnostic test with a higher sensitivity and specificity is required for rapid diagnosis and management of typhoid cases, thereby enabling clinicians to initiate an early therapy, reducing morbidity and its complications.
 Aim : The study was performed to evaluate the performance of Widal test and Typhidot, a lateral flow immunoassay test.
 Materials and Methods: This study was carried out in the Department of Microbiology in a tertiary care center for 1 year (August 2016– July2017). A total of 110 blood samples were collected from patients (cases) clinically suspected of enteric fever. Culture and Serological tests were done in all the 110 cases. Salmonella isolation was done by conventional methods of culture. Demonstration of presence of anitibodies were done by Widal tests using commercially available kits. Typhidot, a lateral flow immunoassay, was used to test and detect IgM and IgG antibodies against the outer membrane protein (OMP) of the Salmonella Typhi. The performances of the tests were compared, and diagnostic accuracy determined.
 Results : Out of 110 cases, highest percentage of positive cases were found by Typhidot test (29.09%) followed by Widal test (23.63%) . 18.18% cases were confirmed by blood culture. Taking blood culture as gold standard , it was found that Widal test had a sensitivity of 30% and specificity of 78% and Typhidot had a sensitivity of 75% and specificity of 81%.
 Conclusion : Though definitive diagnosis of enteric fever is isolation of the causative organism from the patient, Widal test and Typhidot test are important diagnostic tools in the medical set up. Typhidot is fast becoming a practical alternative to Widal test. Typhidot test had a fairly good sensitivity and specificity, whereas Widal test had good specificity but a lower sensitivity.

The Effect of Sedation, Oral Examination, and Odontoplasty on Systemic Inflammation as Measured by Serum Amyloid A in the Adult Performance Horse.

Serum amyloid A (SAA) is one of the major acute phase proteins in horses. It serves as a marker for systemic inflammation and infection, as the concentration can increase 100- to even 1000-fold during systemic disease processes. The objective of this study was to evaluate the effect of sedation, oral examination, and odontoplasty on systemic inflammation as measured by SAA in the adult performance horse. This study included 32 clinically healthy adult performance horses. Blood samples were collected immediately prior to sedation, oral examination, and odontoplasty and 48 and 72 hours afterward. Serum amyloid A levels were measured directly after venipuncture using a commercially available stall-side lateral flow immunoassay test developed and validated for equine SAA levels. Serum amyloid A values were within normal limits for each of the time periods and there were no significant differences in SAA values between the time periods. The results of this study suggest that sedation, oral examination, and odontoplasty have no systemic inflammatory effects as measured by SAA.

Process parameter optimization for lateral flow immunosensing

Lateral flow immunoassay (LFIA) is a widely used technique in the field of point-of-care diagnostics. Assembly of components such as sample pad, conjugate pad, nitrocellulose membrane (NCM), absorbent pad on the plastic backing laminate to prepare the LFIA strip for analysis is an important factor. Processing of the strip assembly and heat or buffer treatment of strip affects the surface morphology, structure and porosity of the NCM. Flow time and pattern of the fluid flow through the LFIA strip surface affects the sensitivity of the LFIA. In this work, we have studied various combinations of strip component assemblies, the morphology of NCM, and flow time and flow pattern of fluid through the strip surface. The LFIA is developed using the optimized strip assembly for the detection of rabbit IgG, which is commonly used as the substrate for control line formation in most of the LFIA tests. Assembly of the sample pad overlapping on the conjugate pad that overlaps on the NCM at one end of the strip and absorbent pad overlapping on the NCM from other end was found to be optimal. Uniform, leveled, slower fluid flow was observed with this assembly when dried at 55 °C for 20 min. Overlap of the membranes is critical for wicking as the fluid flow through membrane because of capillary force. An optimal concentration of the rabbit IgG required for the control line was found to be 1.2 µg/mL. The reported methodology may be advantageous for the development of LFIA for detection of various biomarkers with improved sensitivity.

Development of a Lateral Flow Immunoassay using recombinant GRA7 antigen to detect Toxoplasma gondii IgG antibodies

Toxoplasma gondii is an intracellular protozoan parasite that causes toxoplasmosis and is of medical importance in pregnant women and immunosuppressed patients. In recent years, many methods have been developed for the detection of infection caused by this parasite; however, most of the developed methods are not adequately sensitive. The dense granular antigen (GRA) 7 is a highly immunogenic protein that is used as a specific antigen for the diagnosis of toxoplasmosis. This study was designed to produce recombinant GRA7 (rGRA7) antigen in bacterial system in order to be applied as an antigen for developing a simple and rapid lateral flow immunoassay test strip using a gold nanoparticle-pAb conjugate probe to detect Toxoplasma IgG-specific antibodies in human sera. After the extraction of genomic DNA from RH strain tachyzoites, polymerase chain reaction (PCR) was performed using specific primers considering restriction sites and BglII and XhoI enzymes. Subsequently, the GRA7 gene was cloned in pET-32a (+) expression vector, and then pET-32a(+)-GRA7 was transformed into E. coli Rosetta (DE3). The induction of protein production was accomplished by IPTG, and the product was finally purified by Ni-NTA affinity chromatography. In order to make the strip test, the anti-human gold nanoparticle conjugate was applied on conjugate pad, rGRA7 antigen was immobilized to a nitrocellulose membrane as the capture agent, and sample and absorbance pads were assembled on a backing card. For the analysis of the sensitivity and specificity of the assay, the selected patients’ sera samples were tested by standard chemiluminescence immunoassay (CLIA) method, and then compared with TOXO-IgG strip results. The findings showed that the use of rGRA7 is an accurate, sensitive, and inexpensive technique for the rapid detection of anti-Toxo­plasma IgG in human sera. Therefore, rGRA7 can be applied as a diagnostic agent in laboratories.

A saliva-based rapid test to quantify the infectious subclinical malaria parasite reservoir.

A large proportion of ongoing malaria parasite transmission is attributed to low-density subclinical infections not readily detected by available rapid diagnostic tests (RDTs) or microscopy. Plasmodium falciparum gametocyte carriage is subclinical, but gametocytemic individuals comprise the parasite reservoir that leads to infection of mosquitoes and local transmission. Effective detection and quantification of these carriers can help advance malaria elimination strategies. However, no point-of-need (PON) RDTs for gametocyte detection exist, much less one that can perform noninvasive sampling of saliva outside a clinical setting. Here, we report on the discovery of 35 parasite markers from which we selected a single candidate for use in a PON RDT. We performed a cross-sectional, multi-omics study of saliva from 364 children with subclinical infection in Cameroon and Zambia and produced a prototype saliva-based PON lateral flow immunoassay test for P. falciparum gametocyte carriers. The test is capable of identifying submicroscopic carriage in both clinical and nonclinical settings and is compatible with archived saliva samples.

SERS-based lateral flow immunoassay of troponin I by using gap-enhanced Raman tags

The lateral flow immunoassay (LFIA) has emerged as a powerful tool for rapid screening owing to its simplicity and flexibility for detection of various biomarkers. However, conventional LFIA strips have several disadvantages, including limits in quantitative analysis and low sensitivity. Here we developed a novel surface-enhanced Raman scattering LFIA based on nonspherical gap-enhanced Raman tags (GERTs), with Raman molecules (RMs) embedded in a 1-nm gap between Au nanorod core and Au shell. Such tags have a strong and uniform SERS response, an order of magnitude higher than that of other common SERS tags such as Au nanorods, nanostars, Au nanoshells with surface-adsorbed RMs, or spherical GERTs with embedded RMs. The feasibility of the tags was demonstrated by the semiquantitative and sensitive detection of the heart disease biomarker cardiac troponin I (cTnI). GERTs were conjugated with monoclonal antibodies and used for LFIA in the same way as ordinary functionalized colloidal gold. The presence of the target antigen, cTnI, was identified by Raman microscopy mapping of the test zone. With the SERS-based LFIA, the limit of cTnI detection was about 0.1 ng/mL. This value is within the diagnostic range of cTnI in the blood serum of patients with heart infarction and is 30 times lower than that of the colorimetric LFIA test using the same antibodies and either GERTs or colloidal gold as labels.

Tobacco Use Classification by Inexpensive Urinary Cotinine Immunoassay Test Strips.

Urinary cotinine is one of the most commonly measured biomarkers reflecting recent exposure to nicotine. In some cases a simple qualitative dichotomization of smokers and non-smokers is all that is required. NicAlert® test strips have been evaluated for this purpose, but other recently introduced, inexpensive single-line test strips have not. In this study we evaluated two such strips with nominal cutoffs of 200 and 10 ng/mL. A total of 800 urine samples with known cotinine concentrations determined by an LC-MS-MS method were examined, including 400 urine samples ranging from 0.23 to more than 24,000 ng/mL by the 200 ng/mL strip, and 400 samples with concentrations <200 ng/mL by the 10 ng/mL cutoff strip. Both test strips performed well in these evaluations. Classification relative to LC-MS-MS by the 200 ng/mL strips had a sensitivity of 99.5% and specificity of 92%, with 95.8% accuracy. The 10 ng/mL strips had a sensitivity of 98.7% and specificity of 90.1%, with 93.3% accuracy. The positive predictive value for the 200 ng/mL strips was 92.6% and the negative predictive value was 99.5%. For the 10 ng/mL strips, the corresponding values were 85.4 and 99.2%, respectively. The prevalence of positive samples was 50% in the 200 ng/mL group, and 37% in the 10 ng/mL set. Each strip was read by two readers with an overall agreement of >98%. Our results suggest that these simple and inexpensive lateral flow immunoassay test strips can provide useful qualitative estimates of nicotine exposures for appropriate applications within the inherent limitations of sensitivity and precision of the immunoassay test strip format.

EFFICIENT CONJUGATION OF AFLATOXIN B1 WITH BOVINE SERUM ALBUMIN APPLYING FOR DEVELOPMENT OF AFLATOXIN B1 QUICK TEST

Aflatoxins are secondary metabolites mostly produced by Aspergillus flavus and Aspergillus parasiticus and found in agricultural foodstuff such as maize grains, peanuts, animal feeds,... These are toxic and cancerous agents for humans and animals. Among them, aflatoxin B1 is the most consideration due to its highest toxicity and presence in samples. Several methods were developed to detect aflatoxin in food, feed and other foodstuffs such as chromatographic methods (TLC, HPTLC, HPLC), LC-MS/MS, FTIR, RIA, ELISA, SPR, electrochemical, and immunodipstick. Among them only immunodipstick method is compatible for field usage. Therefore, aim of the present study is to determine optimal condition for conjugation of aflatoxin B1 (AFB1) with bovine serum albumin (BSA) to develop a lateral flow immunoassay test strip for detection of aflatoxin B1. Optimal conditions for generating mediate compound of aflatoxin B1 – CMO was investigated: AFB1:CMO ratio of 1:2, reflux temperature of 100oC for 2 hours. Concentration of aflatoxin B1 in reaction was 0.27 µg/µl. Conjugation of AFB1 with CMO was confirmed by thin layer chromatography, HPLC and FT-IR methods. Furthermore, optimal conditions for conjugation of mediate AFB1-CMO derivative with BSA were also investigated. Ratio of AFB1-CMO with BSA was determined as 40:1 and equal to predicted theoretical ratio. Efficient conjugation conditions were 25oC for 2 hours in bicarbonate buffer pH 9.5. Generated conjugate of AFB1-BSA was successfully applied to construct a lateral flow immunoassay test strip for detection of aflatoxin B1

Sensitivity and colour intensity enhancement in lateral flow immunoassay tests by adjustment of test line position

In this article, we have enhanced the sensitivity and limit of detection of lateral flow immunoassay tests, exemplified for the detection of human chorionic gonadotropin (pregnancy) through determination of proper test line position. Test line position affects the flow velocity and concentration profile of analytes at the test line, which itself impacts the reaction rate and thus the colour intensity. The flow velocity was precisely modelled, both analytically and numerically, and the predicted analytes' moving front (lateral) velocity was experimentally verified. Evolution of the velocity magnitude was determined to locate points with proper capillary velocity and accumulated bed-volume. This provides insights into the essence of LFIAs performance for simple and easy to practice improvements. The colour intensity was measured by quantifying the RGB of the resulting test line colours. The response colour intensity was enhanced for at least 20%, up to 40% by proper displacement of the test line position, and the limit of detection was cut by half to 10 IU/L.

Lateral flow immunoassay integrated with competitive and sandwich models for the detection of aflatoxin M1 and Escherichia coli O157:H7 in milk

Pathogens, mycotoxins, or antibiotics may exist in a food sample. Micro- and macromolecular substances must be detected quickly. A rapid and convenient lateral flow immunoassay (LFI) integrated with competitive and sandwich models was developed to detect micro- and macromolecular substances. In this study, aflatoxin M1 (AFM1) and Escherichia coli O157:H7 were selected as the micro- and macromolecular substances, respectively. Two test lines in the LFI test strip were evaluated to detect AFM1 and E. coli O157:H7 by competitive and sandwich models. Results showed that the limits of detection for detecting AFM1 and E. coli O157:H7 were 50 pg·mL-1 and 1.58 × 104 cfu·mL-1, respectively. The whole assay time was 30 min. The recoveries of gold nanoparticle-LFI ranged from 78.0 to 111.6% with coefficients of variation in the range of 3.9 to 8.5% for the detection of AFM1. For the detection of E. coli O157:H7, the range of recoveries was from 70.1 to 89.6% with coefficients of variation ranging from 4.9 to 13.0%. This study not only tested sensitivity and specificity, but also was a systematic study of location of 2 test lines of the LFI test strip integrated with competitive and sandwich models.

Rapid design and fielding of four diagnostic technologies in Sierra Leone, Thailand, Peru, and Australia: Successes and challenges faced introducing these biosensors

Febrile illnesses are among the most common reasons for visits to hospitals and clinics worldwide. Since fevers can arise from a wide range of diseases, identifying the causative pathogen is essential not only for effective personal treatment but also for early detection of outbreaks. The Defense Threat Reduction Agency (DTRA) tasked a coalition of commercial, academic, and government researchers with moving diagnostic technology concepts from ideation to field use as rapidly as possible using scientifically sound evaluations. DTRA's 24 Month Challenge program examined >30 technologies before fielding four technologies on four continents. >10,000 in field test results were recorded. Here we discuss our tiered evaluation system to assess candidate technologies developed by commercial partners and the process of field testing those technologies at various front-line clinics in Sierra Leone, Thailand, Peru, and Australia. We discuss successes and challenges for introducing two multiplexed lateral flow immunoassay (LFI) tests that detect malaria, dengue fever, melioidosis, and the plague. Additionally we discuss the use of a LFI reader that assisted the interpretation of the assay, communicated results to a data cloud, and greatly facilitated reach-back support. Lastly, we discuss the concurrent field testing of a multiplexed PCR assay on the FilmArray platform, which had an assay pouch specially designed for the 24 Month Challenge. Either standard-of-care or gold-standard testing were run alongside our fielded technologies to benchmark their performance.

A quantum dot-based lateral flow immunoassay for the sensitive detection of human heart fatty acid binding protein (hFABP) in human serum

We describe the preparation and validation of a novel lateral flow immunoassay test for the detection of human heart fatty acid binding protein (hFABP). Water-soluble CdTe quantum dots (QDs) were selected as the fluorescent label and were linked covalently to anti-hFABP antibodies. Upon conjugation, the secondary structure of the anti-hFABP was preserved and the fluorescence quantum yield of the CdTe QDs increased. The labelled antibodies were transferred to the immunoassay test strip and the antigen–antibody reaction was successfully performed. This evidenced the preserved antibody activity of QD-labelled anti-hFABP towards hFABP, and provided a rapid means for the quantitation of hFABP in human serum within the range of 0–160ng ∙ ml−1, with a much lower detection limit of 221pg.∙ ml−1 compared with other rapid tests based on lateral flow immunoassays. This new immunoassay test has been successfully used for the early detection of acute myocardial infarction.

Comparative Evaluation of diagnostic performance of Blood culture, Lateral flow immunoassay, Tube Widal and Slide Widal Test in diagnosis of Enteric fever using a Composite Reference Standard

Comparative Evaluation of diagnostic performance of Blood culture, Lateral flow immunoassay, Tube Widal and Slide Widal Test in diagnosis of Enteric fever using a Composite Reference Standard

Evaluation of a lateral flow immunoassay for field identification of Solenopsis invicta (Hymenoptera: Formicidae) in Australia

AbstractIn an effort to improve surveillance capacity for the exotic red imported fire ant, Solenopsis invicta, a lateral flow immunoassay (LFA) was recently evaluated by Biosecurity Queensland staff in Australia. The purpose of the research was to assess the ability of the fire ant LFA to discriminate S. invicta from ants found in Australia and to conduct the first field evaluation of the test. In addition to S. invicta, 36 species of ants, collected mainly from Queensland, were evaluated by the LFA, including species from the Dolichoderinae (n = 7), Formicinae (n = 13), Myrmeciinae (n = 1), Myrmicinae (n = 11), Ponerinae (n = 3) and Pseudomyrmicinae (n = 1) subfamilies. The fire ant LFA test correctly identified S. invicta in every instance. No cross reactivity was observed in the other ant species. Field tests by staff previously unfamiliar with the test resulted in suggestions for improving ant collection and manipulation. The fire ant LFA appears to be suitable for use in Australia for rapid confirmation of potential new detections of S. invicta.

IronPhone: Mobile device-coupled point-of-care diagnostics for assessment of iron status by quantification of serum ferritin

Iron deficiency (ID) is an urgent public health problem that has devastating effects on maternal and child health. However, due to poor access and affordability, screening and diagnosis for ID is often limited to proxy hemoglobin measurements alone. Here, we report the development and validation of ironPhone, a mobile-device coupled portable diagnostics for quantification of serum ferritin concentrations, an iron status biomarker, within a few minutes, from a drop of fingerprick blood. The ironPhone diagnostic platform comprises of a smartphone accessory, an app, and a disposable lateral flow immunoassay test strip to quantify serum ferritin. For initial validation in the lab, we optimized and evaluated the performance of ironPhone with known ferritin concentrations in spiked buffer and serum samples. Following lab validation, we performed a human validation by collecting fingerprick whole blood samples from 20 participants to assess iron status using ironPhone and compared the results with the laboratory standard IMMULITE 2000 analyzer. Findings from the ironPhone for the buffer and spiked serum samples provided a calibration curve with R2 values of 0.97 (n=27) and 0.93 (n=12), respectively. On comparison with the laboratory standard IMMULITE analyzer in whole blood samples, a correlation of 0.92 (P<0.0001) was observed with a sensitivity of over 90% for predicting ID (ferritin<15.0µg/L) via the ironPhone, demonstrating its promise for iron status assessment at the point-of-care.

On-site detection of Citrus tristeza virus (CTV) by lateral flow immunoassay using polyclonal antisera derived from virions produced by a recombinant CTV

Maintenance of virus-free citrus in nurseries and orchards is essential to control the spread of aphid-transmitted Citrus tristeza virus (CTV). To this end, a lateral flow immunoassay (LFIA) was developed to detect CTV that was user-friendly and field-deployable. Polyclonal antisera was generated in rabbit against purified virions from Nicotiana benthamiana inoculated with an infectious recombinant cDNA clone of CTV (rCTV). Affinity purified rCTV IgG (1 mg/ml) and Mouse IgG (0.5 mg/ml) were conjugated with gold nanoparticles and coated onto a glass fiber membrane conjugate pad. Affinity purified rCTV IgG and anti-mouse IgG were used as test line and control line, respectively on the test strip. The LFIA detected CTV within 10 min and was sensitive up to a 1:80 dilution of crude plant sap extract. The LFIA was validated using CTV strains T30, T36, VT and RB from greenhouse and field sources and results were 100% in agreement with Enzyme-Linked Immunosorbent Assay and Reverse Transcription quantitative PCR. The immunostrip readily detected CTV after six months in storage in a sealed bag with silica gel at room temperature indicating good stability. The LFIA test strips offered a cost-effective tool to detect CTV in the field or nursery by non-skilled personnel without laboratory equipment.

Magnetic lateral flow immunoassay test strip development - Considerations for proof of concept evaluation.

Lateral flow immunoassays (LFIA) have grown to become the predominant test device format for the diagnostics and point-of-care industries. The demand for robust and reproducible LFIAs has been facilitated through scale-up production methods using specialized and automated instruments. However, the feasibility of a LFIA device can still be evaluated in a small-scale laboratory setting through controlled manual preparation methods. The advent of super-paramagnetic (SPMP) labels for use in lateral flow has heralded the possibility of highly sensitive and stable LFIAs. The methods used for the preparation of a magnetic LFIA prototype device using a reserved suite of laboratory equipment are described.

First Report of Tomato chlorotic spot virus in Sweet Basil (Ocimum basilicum) and Purslane (Portulaca oleracea) in Florida

Typical tosposvirus symptoms, lateral flow immunoassay testing, and sequence data confirm this identification, which is therefore the first report of Tomato chlorotic spot virus infection of sweet basil and purslane in Florida and of the specific plant species from any location.

Utility of a Lateral Flow Immunoassay (LFI) to Detect Burkholderia pseudomallei in Soil Samples.

BackgroundCulture is the gold standard for the detection of environmental B. pseudomallei. In general, soil specimens are cultured in enrichment broth for 2 days, and then the culture broth is streaked on an agar plate and incubated further for 7 days. However, identifying B. pseudomallei on the agar plates among other soil microbes requires expertise and experience. Here, we evaluate a lateral flow immunoassay (LFI) developed to detect B. pseudomallei capsular polysaccharide (CPS) in clinical samples as a tool to detect B. pseudomallei in environmental samples.Methodology/Principal FindingsFirst, we determined the limit of detection (LOD) of LFI for enrichment broth of the soil specimens. Soil specimens (10 grams/specimen) culture negative for B. pseudomallei were spiked with B. pseudomallei ranging from 10 to 105 CFU, and incubated in 10 ml of enrichment broth in air at 40°C. Then, on day 2, 4 and 7 of incubation, 50 μL of the upper layer of the broth were tested on the LFI, and colony counts to determine quantity of B. pseudomallei in the broth were performed. We found that all five soil specimens inoculated at 10 CFU were negative by LFI on day 2, but four of those five specimens were LFI positive on day 7. The LOD of the LFI was estimated to be roughly 3.8x106 CFU/ml, and culture broth on day 7 was selected as the optimal sample for LFI testing. Second, we evaluated the utility of the LFI by testing 105 soil samples from Northeast Thailand. All samples were also tested by standard culture and quantitative PCR (qPCR) targeting orf2. Of 105 soil samples, 35 (33%) were LFI positive, 25 (24%) were culture positive for B. pseudomallei, and 79 (75%) were qPCR positive. Of 11 LFI positive but standard culture negative specimens, six were confirmed by having the enrichment broth on day 7 culture positive for B. pseudomallei, and an additional three by qPCR. The LFI had 97% (30/31) sensitivity to detect soil specimens culture positive for B. pseudomallei.Conclusions/SignificanceThe LFI can be used to detect B. pseudomallei in soil samples, and to select which samples should be sent to reference laboratories or proceed further for bacterial isolation and confirmation. This could considerably decrease laboratory workload and assist the development of a risk map for melioidosis in resource-limited settings.