Overlaid Lateral Flow Immunoassay for the Simultaneous Detection of Two Variant-Specific SARS-CoV-2 Neutralizing Antibodies.

COVID-19 vaccines have been provided to the general public to build immunity since the 2019 coronavirus pandemic. Once vaccinated, SARS-CoV-2 neutralizing antibodies (NAbs-COVID-19) are needed for excellent protection against COVID-19. However, monitoring NAbs-COVID-19 is complicated and requires hospital visits. Moreover, the resulting NAbs-COVID-19 are effective against different strains of COVID-19 depending on the type of vaccine received. Here, an overlaid lateral flow immunoassay (O-LFIA) was developed for the simultaneous detection of two NAbs-COVID-19 against different virus strains, Delta and Omicron. The O-LFIA was visualized with two T-lines with a single device using competition between the free antigen and the antigen-binding antibody. Angiotensin-converting enzyme 2 (ACE2) immobilized on the T-line binds to the antigen remaining after antibody binding. Under the optimum conditions, the proposed device exhibited 50% inhibition concentrations (IC50 values) of 45.1 and 53.6 ng/mL for the Delta and Omicron variants, respectively. Additionally, the proposed platform was applied to real-world samples of animal and human serum, and the developed immunoassay provided results that were in good agreement with those obtained with the standard method. In conclusion, this developed O-LFIA can be used as an alternative method to detect NAbs-COVID-19 and can be enabled for future advancements toward commercialization.

Self-enhancement lateral flow immunoassay for COVID-19 diagnosis.

Equipment-free colorimetric-based lateral flow immunoassay (LFIA) is the most convenient and popular tool for various applications, including diagnostic tools requiring high sensitivity for the detection of pathogens. Thus, improvements and developments of LFIA are constantly being reported. Herein, we enriched the sensitivity of LFIA using the gold enhancement principle, emphasizing needlessly complicated apparatus, only one step for the strip test operation, and typical time incubation (15 min) process. Self-enhanced LFIA was then executed for subsequent flows by overlapping the additionally enhanced pad composed of gold ions and reducing agent on the conjugate pad and the sample pad. Self-enhanced LFIA was performed to detect SARS-CoV-2 antigens in saliva. The obtained result depicted that the achieved sensitivity was up to tenfold compared with that of conventional LFIA by visual measurements. The detection limits of self-enhanced LFIA detecting nucleocapsid protein antigens in the saliva sample was 0.50 and 0.10 ng/mL employed by naked eye detection and calibration curve-based calculation, respectively. When the proposed device was applied to 207 human saliva samples, the diagnostic performance presented a 96.10 % sensitivity and 99.23 % specificity. This self-enhanced LFIA could be implemented in large-scale production and demonstrates higher sensitivity with effortless use, which meets the requirements for point-of-care testing and on-field mass screening.

A novel delayed lateral flow immunoassay for enhanced detection of SARS-CoV-2 spike antigen.

A new detection strategy was developed to improve the sensitivity of a lateral flow immunoassay platform utilizing a delayed hydrophobic barrier fabricated with trimethylsilyl cellulose (TMSC). The SARS-CoV-2 spike receptor-binding domain (SARS-CoV-2 SP RBD) antigen was chosen as a model analyte to demonstrate the superior detectability of this scheme. The novel device consists of 2 separate layers, so-called delayed lateral flow immunoassay (d-LFIA). The upper layer is intended for the analyte or sample flow path, where the test solution flows freely straight to the detection zone to bind with the primary antibody. The lower layer, located just underneath, is designed for the SARS-CoV-2 spike receptor-binding domain-conjugated gold nanoparticles (SARS-CoV-2 SP RBD-AuNPs) used for producing a colorimetric signal. This layer is fabricated with a TMSC barrier to time-delay the movement of SARS-CoV-2 SP RBD-AuNPs, thus allowing the antigen to bind with the primary antibody more efficiently. This platform exhibited a 2.6-fold enhancement in the sensitivity and 9.1-fold improvement in the limit of detection (LOD) as compared with the conventional LFIA. In addition, this d-LFIA device was satisfactorily applied to accurate screening of COVID-19 patients.

Cost-effective paper-based electrochemical immunosensor using a label-free assay for sensitive detection of ferritin.

Ferritin, a blood cell protein containing iron, is a crucial biomarker that is used to estimate the risk assessment of iron deficiency anemia. For point-of-care analysis, a reliable, cost-effective, selective, sensitive, and portable tool is extremely necessary. In this study, a label-free electrochemical immunosensor for detecting ferritin using a paper-based analytical device (ePAD) was created. The device pattern was custom designed onto filter paper to successfully fabricate a deliverable immunosensor. Graphene oxide was first modified onto the working electrode using an inkjet printing technique. An activation step of the electrode surface was then performed using standard 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)/N-hydroxysulfosuccinimide (sulfo-NHS) chemistry. Anti-ferritin antibodies were covalently immobilized onto the amine-reactive ester surface. The amount of ferritin was monitored by observing the electrochemical signal of the selected redox couple by differential pulse voltammetry (DPV). In the presence of ferritin, the sensor showed a considerable decrease in electrochemical response in a concentration-dependent manner. In contrast, there was no observable change in current response detected in the absence of ferritin. The current response provided a good correlation with ferritin concentrations in the range of 1 to 1000 ng mL-1, and the limit of detection (3SD/slope) was found to be 0.19 ng mL-1. This fabricated immunosensor offered good selectivity, reproducibility, and long-term storage stability. In addition, this proposed immunosensor was successfully applied to detect ferritin in human serum with satisfactory results. The promising results suggested that this handmade paper-based immunosensor may be an alternative device for the diagnosis of iron deficiency anemia.

An origami paper-based electrochemical immunoassay for the C-reactive protein using a screen-printed carbon electrode modified with graphene and gold nanoparticles.

An origami paper-based electrochemical immunoassay for C-reactive protein (CRP) detection is described. The assay integrates multiple steps of electrode modification into a single device. A graphene-modified screen-printed carbon electrode (G/SPCE) was employed to enhance sensitivity. Gold nanoparticles were first electrodeposited onto the G/SPCE, followed by a self-assembled monolayer of L-cysteine. The capture anti-CRP was then covalently immobilized on the modified electrode. CRP was quantified by measuring the changes in the charge-transfer resistance of the electrode by using hexacyanoferrate as the redox probe. Cyclic voltammetry and scanning electron microscopy were also applied to verify the successful modification of the electrode. Under optimal conditions, impedance increase in the 0.05-100 µg mL-1 CRP concentration range, and the limit of detection is 15 ng mL-1 (at S/N = 3). The immunoassay was successfully applied to the determination of CRP in a certified human serum sample. This method is simple, low-cost, portable and disposable. Graphical abstract An origami paper-based analytical device (oPAD) is described that integrates the multistep of electrode modification, immobilization and detection into a single device. The direct conjugation between the capture antibody and target molecule was allowed to use in this system. The C-reactive protein (CRP) concentration in serum samples was determined using electrochemical impedance spectroscopy.

Electrochemical lateral-flow device for rapid COVID-19 antigen-diagnostic testing.

Antigen test kits (ATK) are extensively utilized for screening and diagnosing COVID-19 because they are easy to operate. However, ATKs exhibit poor sensitivity and cannot detect low concentrations of SARS-CoV-2. Herein, we present a new, highly sensitive, and selective device obtained by combining the principle of ATKs with electrochemical detection for COVID-19 diagnosis, which can be quantitatively assessed using a smartphone. An electrochemical test strip (E-test strip) was constructed by attaching a screen-printed electrode inside a lateral-flow device to exploit the remarkable binding affinity of SARS-CoV-2 antigen to ACE2. The ferrocene carboxylic acid attached to SARS-CoV-2 antibody acts as an electroactive species when it binds to SARS-CoV-2 antigen in the sample before it flows continuously to the ACE2-immobilization region on the electrode. Electrochemical-assay signal intensity on smartphones increased proportionally to the concentration of SARS-CoV-2 antigen (LOD = 2.98 pg/mL, under 12 min). Additionally, the application of the single-step E-test strip for COVID-19 screening was demonstrated using nasopharyngeal samples, and the results were consistent with those obtained using the gold standard (RT-PCR). Therefore, the sensor demonstrated excellent performance in assessing and screening COVID-19, and it can be used professionally to accurately verify diagnostic data while remaining rapid, simple, and inexpensive.

Label free electrochemical DNA biosensor for COVID-19 diagnosis.

The COVID-19 pandemic has significantly increased the development of the development of point-of-care (POC) diagnostic tools because they can serve as useful tools for detecting and controlling spread of the disease. Most current methods require sophisticated laboratory instruments and specialists to provide reliable, cost-effective, specific, and sensitive POC testing for COVID-19 diagnosis. Here, a smartphone-assisted Sensit Smart potentiostat (PalmSens) was integrated with a paper-based electrochemical sensor to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A disposable paper-based device was fabricated, and the working electrode directly modified with a pyrrolidinyl peptide nucleic acid (acpcPNA) as the biological recognition element to capture the target complementary DNA (cDNA). In the presence of the target cDNA, hybridization with acpcPNA probe blocks the redox conversion of a redox reporter, leading to a decrease in electrochemical response correlating to SARS-CoV-2 concentration. Under optimal conditions, a linear range from 0.1 to 200 nM and a detection limit of 1.0 pM were obtained. The PNA-based electrochemical paper-based analytical device (PNA-based ePAD) offers high specificity toward SARS-CoV-2 N gene because of the highly selective PNA-DNA binding. The developed sensor was used for amplification-free SARS-CoV-2 detection in 10 nasopharyngeal swab samples (7 SARS-CoV-2 positive and 3 SARS-CoV-2 negative), giving a 100% agreement result with RT-PCR.

Detection by using monoclonal antibodies of Yersinia enterocolitica in artificially-contaminated pork.

Monoclonal antibodies against Yersinia enterocolitica were produced by fusion of NS-1 mouse myeloma cells with spleen cells of ICR mice immunized with heat-killed and heat-killed plus SDS-mercaptoethanol treated forms of Y. enterocolitica ATCC 27729 alone or mixed with Y. enterocolitica MU. The twenty-five MAbs obtained from five fusions were divided into nine groups according to their specificities to different bacterial strains and species, as determined by dot blotting. The first five groups of MAbs were specific only to Y. enterocolitica, but did not recognize all of the isolates tested. MAbs in groups 6 and 7 reacted with all isolates of Y. enterocolitica tested but showed cross-reaction with some Yersinia spp. and Edwardsiella tarda, especially in the case of group 7. MAbs in groups 8 and 9 reacted with all isolates of Y. enterocolitica and Yersinia spp., as well as other Gram-negative bacteria that belong to the family Enterobacteriaceae. These MAbs recognized Y. enterocolitica antigens with apparent molecular weights ranging from 10-43 kDa by Western blotting, and could detect Y. enterocolitica from ∼10³-105 colony forming units (CFUs) by dot blotting. The hybridoma clone YE38 was selected for detection of Y. enterocolitica in pork samples which had been artificially-contaminated by inoculation with Y. enterocolitica ATCC 27729 at concentrations of ∼104-106 CFUs/g and incubation in peptone sorbitol bile broth at 4°C. Samples were collected and applied on a nitrocellulose membrane for dot blotting with trypticase soy and cefsulodin-Irgasan-novobiocin agars. After 48 hr of incubation, the detection limit was ∼10²-10³ CFU/g by dot blotting.

Paper-based electrochemical biosensor for diagnosing COVID-19: Detection of SARS-CoV-2 antibodies and antigen.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is emerging as a global pandemic outbreak. To date, approximately one million deaths and over 32 million cases have been reported. This ongoing pandemic urgently requires an accurate testing device that can be used in the field in a fast manner. Serological assays to detect antibodies have been proven to be a great complement to the standard method of reverse transcription-polymerase chain reaction (RT-PCR), particularly after the second week of infection. We have developed a specific and sensitive immunosensor for immunoglobulin detection produced against SARS-CoV-2. Unlike other lateral flow-based assays (LFAs) involving the utilization of multiple antibodies, we have reported a label-free paper-based electrochemical platform targeting SARS-CoV-2 antibodies without the specific requirement of an antibody. The presence of SARS-CoV-2 antibodies will interrupt the redox conversion of the redox indicator, resulting in a decreased current response. This electrochemical sensor was proven effective in real clinical sera from patients with satisfactory results. In addition, the proposed format was also extended to antigen detection (the spike protein of SARS-CoV-2), which presents new possibilities for diagnosing COVID-19.

An automated fast-flow/delayed paper-based platform for the simultaneous electrochemical detection of hepatitis B virus and hepatitis C virus core antigen.

Electrochemical paper-based analytical devices (ePADs) are useful analytical devices that serve as point-of-care testing (POCT) devices for various clinical biomarkers in view of their simplicity, portability, and low-cost format. However, multistep reagent manipulation usually restricts the performance of the device for end users. Herein, we developed a sequential ePAD for sequential immunosensing fluid delivery by integrating dual flow behaviors (fast-flow/delayed) within a single paper platform for the simultaneous detection of hepatitis B surface antigen (HBsAg) and hepatitis C core antigen (HCVcAg). In the present work, a fast-flow channel was used for the automated washing of unbound antigens, while a delayed channel was created to store a redox reagent for further electrochemical analysis with a single buffer loading (the analysis time can be completed within 500 s). Hence, the undesirable complex procedure of multi-step reagent manipulation is scarcely needed by the user. The detection limit of the proposed ePAD was as low as 18.2 pg mL-1 for HBsAg and 1.19 pg mL-1 for HCVcAg. In addition, this proposed ePAD was also proven to be effective in real clinical sera from patients to verify its biological applicability. The ePAD sensor shows high promise as an easy-to-use, portable, and extendable sensor for other multiplex biological assays.

Ultrasensitive electrochemiluminescence sensor based on nitrogen-decorated carbon dots for Listeria monocytogenes determination using a screen-printed carbon electrode.

Current method for identification of foodborne pathogens suffers from its relatively poor performance, consequently limiting its use. Herein, we first describe an ultrasensitive electrochemiluminescence (ECL) sensor based on nitrogen-decorated carbon dots (NCDs) for Listeria monocytogenes (L. monocytogenes) determination using a screen-printed carbon electrode (SPCE). Citric acid serves as carbon source, and ethylenediamine, a molecule containing nitrogen atom, is employed to synthesize CDs. Approximately 4 nm NCD with homogenous size distribution can be produced via a single step green microwave-assisted methodology. The construction of ECL sensor is initiated by the immobilization of capture antibody (Ab1) onto the carboxyl graphene (GOOH)-modified SPCE, where immunocomplexes (antigen and the NCD-labelled secondary antibody (Ab2-NCD)) are formed, resulting in a substantial increment in the ECL signal response in the presence of K2S2O8. The GOOH allows direct formation of the capture antibodies and enhances the electrochemical properties. Under optimal parameters, this sensor exhibits wide linearity (2 to 1.0 × 106 CFU mL-1), high sensitivity (0.104 or 1.0 × 10-1 CFU mL-1) and specificity over the nontargeting studied pathogens and is successfully applied to determine L. monocytogenes in food products. These promising results together with its performance suggest that this proposed platform may serve as an alternative device to effectively control the spread of foodborne diseases.

Chitosan derivative nanocarrier: safety evaluation,antibacterial property and ascorbyl palmitate encapsulation.

A chitosan derivative, methyl ether-terminated poly(ethylene oxide)-4-methoxycinnamolyphthaloylchitosan (PCPLC) was prepared, characterized and self-assembled into nanoparticles. Encapsulation of ascorbyl palmitate (AP) into PCPLC gave 6890.98 nm particles with encapsulation efficiency of 84% at 56% drug loading. The encapsulated AP showed significant improved stability as examined by 1H NMR spectroscopy.The obtained particles displayed no short-term cytotoxicity against the human skin melanoma A-375 cell line using the MTT assay and no short-term skin irritation on human volunteers using a single topical application as patch and photopatch tests. In addition, aqueous suspension of PCPLC nanoparticles successfully inhibited the growth of Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923.

Paper-based immunosensor with competitive assay for cortisol detection.

The measurement of cortisol (stress hormone) is important for diagnosis and monitoring of stress-related diseases. A paper-based immunosensor with a competitive assay was developed for quick and easy detection of cortisol levels in serum. The paper-based sensor was fabricated using a simple and cheap wax printing method. Cortisol conjugated-BSA was immobilized on the paper's surface in the detection zone for the competitive immunoassay. Anti-cortisol mAb-conjugated gold nanoparticles, as signal indicator, were used to detect cortisol in the sample. A 2-step procedure included applying the sample and washing for cortisol determination. The results showed that the device had the ability to differentiate cortisol levels into three ranges: < 25 µg/dL, 25-50 µg/dL and > 50 µg/dL by visual detection. The limit of detection determined by an image processing program was 21.5 µg/dL. This assay was successfully developed to detect cortisol in serum, and showed good recovery and precision. Our results correlated well with those obtained using an electrochemiluminescence method. This paper-based immunosensor provided a rapid and simple screening test for serum cortisol detection.

Morphology, release characteristics, and antimicrobial effect of nisin-loaded electrospun gelatin fiber mat.

Gelatin electrospun (e-spun) fiber mats containing nisin were produced by electrostatic spinning of gelatin-nisin in 70% (vol/vol) acetic acid aqueous solutions. Varying nisin loading concentration (0 to 3% [wt/wt]) did not affect the fiber average diameter, whereas increasing gelatin concentration from 20 to 24% (wt/vol) caused an increase in the average diameter. All nisin-loaded gelatin e-spun fiber mats demonstrated inhibition against Lactobacillus plantarum TISTR 850. However, all fiber mats were fragile and easily dissolved in water. Cross-linking by saturated glutaraldehyde vapor at 37 degrees C for 5 min was done to strengthen the mat. Tensile strength, Young's modulus, and elongation of the cross-linked gelatin-nisin e-spun fiber mats varied in the range of 2.6 to 20.3 MPa, 163 to 966 MPa, and 1.7 to 5.9% , respectively. Cross-linking did not affect the mat's inhibition activity against L. plantarum TISTR 850. Nisin retention in cross-linked antimicrobial gelatin e-spun fiber mats was in the range of 1.0 to 1.22% . Increasing temperature caused an increase in nisin release, but increasing water activity did not cause a significant difference in nisin release over 50 h. After storage at 25 degrees C for 5 months, the antimicrobial gelatin e-spun fiber mat still showed inhibition against L. plantarum TISTR 850. The mats also inhibited the growth of Staphylococcus aureus and Listeria monocytogenes but not Salmonella Typhimurium.

Differentiating between isolates of Vibrio vulnificus with monoclonal antibodies.

Monoclonal antibodies (MAbs) against Vibrio vulnificus (isolate I, VVC and isolate II, VVB) were raised using heat-killed and heat-killed plus SDS-mercaptoethanol treated forms of VVC and VVB for immunizing Swiss mice. Twenty three hybridomas producing MAbs against V. vulnificus were selected and divided into five groups according to their specificities to different V. vulnificus isolates and apparent protein antigens which ranged from approximately 3-50 kDa. Four groups were specific to V. vulnificus without cross reactivity to either other Vibrio spp. or other bacterial species. In dot blot based assays, one group of MAbs were specific to VVC, with a sensitivity of approximately 1.6 x 10(7) CFU ml(-1) (approximately 1.6 x 10(4) cells spot(-1)), and bound to proteins of approximately 50 and approximately 39 kDa. Other MAbs, binding to proteins ranging from approximately 3-14 and approximately 40 kDa, detected VVB (but not VVC) with high sensitivity at approximately 1.6 x 10(5) and 4 x 10(6) CFU ml(-1) (approximately 1.6 x 10(2) and 4 x 10(3) cells spot(-1)), respectively. In addition, certain MAbs were able to recognize V. vulnificus in tissues by means of immunohistochemistry. The remaining groups demonstrated cross reactivity to Vibrio fluvialis. MAbs from this study can, therefore, detect the difference between some isolates of V. vulnificus and in addition to pathogen detection may, with further antibodies, form the basis of serovar typing isolates in the future.

Development of competitive lateral flow immunoassay coupled with silver enhancement for simple and sensitive salivary cortisol detection.

Cortisol is known as a stress biomarker. The measurement of cortisol levels is an early warning indicator for health conditions and diagnosis of stress-related diseases. Herein, a lateral flow immunoassay using a gold nanoparticle label with a silver enhancement system was developed for the simple, sensitive and rapid detection of cortisol. The developed assay was based on a competitive platform of which cortisol-BSA conjugate was immobilized at the test zone to compete with an analyte. The quantitative analysis was performed using gold nanoparticles (AuNPs) as signal labeling. Sequentially, the silver enhancement solution was applied in order to enhance the sensitivity of the assay with the results easily seen by the naked eye. Using this system, the limit of detection (LOD) was found to be 0.5 ng/mL with a 3.6 fold more sensitive detection than without the enhancement system (LOD = 1.8 ng/mL). The salivary cortisol analysis was in the range of 0.5-150 ng/mL (R2 = 0.9984), which is in the clinical acceptable range. For the semi-quantitative analysis, the intensity color of the results was analyzed using an image processing program. The proposed method was successfully applied to detect cortisol in saliva. In addition, the results from our method also complied with the ones of those obtained by using the commercial enzyme-linked immunosorbent assay (ELISA). This developed assay offers great promise for a non-invasive screening test of salivary cortisol.

Burkholderia Hep_Hag autotransporter (BuHA) proteins elicit a strong antibody response during experimental glanders but not human melioidosis.

BACKGROUND: The bacterial biothreat agents Burkholderia mallei and Burkholderia pseudomallei are the cause of glanders and melioidosis, respectively. Genomic and epidemiological studies have shown that B. mallei is a recently emerged, host restricted clone of B. pseudomallei. RESULTS: Using bacteriophage-mediated immunoscreening we identified genes expressed in vivo during experimental equine glanders infection. A family of immunodominant antigens were identified that share protein domain architectures with hemagglutinins and invasins. These have been designated Burkholderia Hep_Hag autotransporter (BuHA) proteins. A total of 110/207 positive clones (53%) of a B. mallei expression library screened with sera from two infected horses belonged to this family. This contrasted with 6/189 positive clones (3%) of a B. pseudomallei expression library screened with serum from 21 patients with culture-proven melioidosis. CONCLUSION: Members of the BuHA proteins are found in other Gram-negative bacteria and have been shown to have important roles related to virulence. Compared with other bacterial species, the genomes of both B. mallei and B. pseudomallei contain a relative abundance of this family of proteins. The domain structures of these proteins suggest that they function as multimeric surface proteins that modulate interactions of the cell with the host and environment. Their effect on the cellular immune response to B. mallei and their potential as diagnostics for glanders requires further study.

In vitro motility of a population of clinical Burkholderia pseudomallei isolates.

Melioidosis, a serious infection caused by Burkholderia pseudomallei, is a leading cause of community-acquired sepsis in Northeast Thailand, and the commonest cause of death from community-acquired pneumonia in the Top End of Northern Australia. The causative organism is a Gram-negative, motile bacillus that is a facultative intracellular pathogen. B. pseudomallei flagella have been proposed as a possible vaccine candidate and putative virulence determinant. Flagella expression was highly conserved for 205 clinical B. pseudomallei isolates, as defined by in vitro swim and swarm motility assays. No association was found between motility and clinical factors including bacteremia and death.

Production of monoclonal antibodies for detection of Vibrio harveyi.

Monoclonal antibodies (MAbs) against Vibrio harveyi were produced from mice immunized with heat-killed and SDS-mercaptoethanol-treated highly virulent V. harveyi 639. Fifteen MAbs were selected and sorted into 6 groups according to their specificity to various proteins of apparent molecular weight ranging from 8 to 49 kDa. Some antibodies were used for detection of V. harveyi at concentrations as low as 10(4) CFU ml(-1) using immunodot blots. Most of the selected MAbs did not show cross-reactivity to other Vibrio species and other gram-negative bacteria tested. Only 1 MAb (VH39-4E) showed slight cross-reactivity to Aeromonas hydrophila. Another MAb (VH24-8H) bound lightly to V. harveyi 1526 but strongly to V. harveyi 639, allowing rapid differentiation. Two of the MAb groups were used to localize V. harveyi in tissues of infected black tiger shrimp Penaeus monodon by immunohistochemistry. This study demonstrates the versatility of a highly specific immunological tool for the detection of V. harveyi in aquaculture and opens the way for further development of convenient test kits.

Enumeration of coliforms and Escherichia coli in frozen black tiger shrimp Penaeus monodon by conventional and rapid methods.

Conventional (most probable number, MPN) and rapid methods-including Chromocult coliform agar (CCA), Fluorocult(R) LMX broth (LMX), and Petrifilm Escherichia coli count plates (PEC) for enumeration of coliforms and E. coli in frozen black tiger shrimp from Thailand were compared in order to assess the possibility of using one of the rapid methods for routine analysis. Enumeration of coliforms and E. coli from 18 samples of regular frozen black tiger shrimp and 156 samples of frozen black tiger shrimp experimentally contaminated with coliforms or E. coli at concentrations of approximately 10, approximately 10(2), and approximately 10(3) CFU g(-1) revealed that at the level of approximately 10 CFU g(-1), coliform numbers ranked as LMX>CCA>MPN=PEC and E. coli as MPN=LMX=PEC=CCA. At the level of approximately 10(2) CFU g(-1), coliform numbers ranked as LMX>MPN=PEC=CCA and E. coli as MPN=LMX>PEC=CCA. At the level of 10(3) CFU g(-1), coliforms ranked as LMX>MPN=CCA>PEC and E. coli as MPN>LMX>CCA>PEC. Agreements with the conventional MPN method for coliforms were LMX 108%, PEC 87.2%, and CCA 91.2% and agreements for E. coli were LMX 101%, PEC 95.7%, and CCA 96.3%. Sensitivities (%) ranked LMX>MPN>CCA=PEC for coliforms and E. coli, whereas equal specificities (100%) of all methods for coliforms and E. coli were demonstrated. Rankings for the other parameters compared were: convenience, PEC>CCA=LMX>MPN; time to detection, MPN>LMX=PEC=CCA; expense, MPN=PEC>CCA>LMX; labor, MPN>LMX=CCA>PEC; accuracy for coliforms, PEC>CCA>MPN>LMX; and accuracy for E. coli, PEC=CCA>LMX>MPN.