A novel peptide pair-based rapid fluorescent diagnostic system for malaria Plasmodium falciparum detection.

Advanced diagnostic materials, such as aptamers, are required due to the scarcity of efficient diagnostic antibodies and the low sensitivity of rapid diagnostic kits at detecting the malaria parasite, Plasmodium falciparum. METHODS: Two peptides M2.9 [(KPTAEQTESPELQSAPEN) and M2.17 (KILFNVYSPLGCTCECWV)] were designed using simple epitope prediction tools and modified against the merozoite surface antigen 2 of P. falciparum (Pf.MSP2) by 3-dimensional modeling based on binding affinity. Based on five prediction tools for hydropathy, M2.17 was selected as an appropriate capture peptide. A peptide-based fluorescence-linked immunosorbent assay (FLISA) and a peptide pair-based fluorescent immunochromatographic test strip (FICT) were developed to detect P. falciparum 3D7 (drug-sensitive) and P. falciparum K1 (multi drugs-resistant) strains. RESULTS: Bioinformatic analysis of two peptides demonstrated the potential binding affinity with the merozoite surface protein 2 of P. falciparum (Pf.MSP2) with a positive hydropathy value. The limit of detection (LOD) of FLISA was 10 parasites/µL and of a peptide pair-linked rapid FICT system was 5 and 200 parasites/µL for P. falciparum 3D7 and K1, respectively. Compared to commercial rapid detection systems (RDTs), a peptide pair-linked FICT system exhibited a 20-fold greater efficiency in detecting P. falciparum 3D7 and specifically discriminated another protozoan spp. CONCLUSION: A peptide pair-linked rapid diagnostic strip could be an alternative to conventional RDTs for monitoring wild-type and drug-resistant malaria parasites.

Rapid immunochromatographic detection of carbapenemases directly from positive blood cultures in patients colonized by carbapenemase-producing bacteria.

OBJECTIVES: Carbapenem resistance is increasing worldwide. Earlier detection of this resistance, combined with appropriate treatment, could improve the prognosis of bloodstream infection. This study aims to evaluate the detection of carbapenemase producing gram-negative bacteria directly from positive blood cultures to quickly adapt antibiotic therapy before the results of antibiotic susceptibility testing are available. METHOD: A prospective single-center study was conducted over a five-month period at Lille University Hospital. Carbapenemase detection by immunochromatographic testing was performed directly from positive blood cultures with gram-negative rods of thirty-five patients previously colonized with carbapenemase-producing bacteria. RESULTS: Among these thirty-five positive blood cultures, 15 carbapenemase-producing strains were directly detected, mainly OXA-48 and NDM. This rapid procedure provided results in less than one hour, compared to several hours for conventional methods. Of the patients with infections caused by carbapenemase-producing isolates, 67% (10 patients) received inappropriate empiric treatment, highlighting the potential of the rapid test to adjust antibiotic therapy sooner. CONCLUSIONS: Carbapenemase detection by immunochromatographic testing directly on blood culture pellets is reliable and can lead to early adaptation of antibiotic therapy in these severe infections.

An immunochromatographic test using whole blood for rapid diagnosis of human paragonimiasis and its diagnostic usefulness.

Paragonimiasis is a harmful food-borne zoonosis caused by lung flukes of the genus Paragonimus. The disease is found on most continents, several million people are at risk of infection, and it is a re-emerging disease in developing countries. The gold standard for diagnosis of pulmonary paragonimiasis requires the finding of eggs in sputa and/or fecal samples. In ectopic paragonimiasis cases, eggs are typically not seen, and supportive information is required such as a history of eating freshwater crabs or crayfishes, radiographic findings and immunological tests. Here, we developed a proof of concept based on lateral flow assay, an immunochromatographic test kit, named the paragonimiasis whole-blood test kit, for detection of specific IgG antibody in simulated whole-blood samples (WBSs) using worm excretory-secretory antigens to diagnose human paragonimiasis. The laboratory diagnostic values of this kit were compared with the detected IgG in serum samples. In simulated WBSs, the diagnostic sensitivity and specificity were 97.8 % and 96.1 %, respectively, while for serum samples, these values were 100.0 % and 94.8 %, respectively. The comparative IgG antibody detections whether a result was positive or negative between simulated WBSs and serum samples did not differ significantly with a concordance of 97.8 % in laboratory conditions using a circumscribed set of samples. The tool is fast and easy to use. The next step involves observing and evaluating native whole blood samples and using specific recombinant antigens need to be evaluated for support diagnosis of paragonimiasis caused by P. heterotremus, P. westermani and P. miyazakii at the bedside or at local and remote hospitals with limited facilities. It will also be valuable for epidemiological surveys in Asia where paragonimiasis is endemic.

A highly sensitive Photothermal Immunochromatographic test strip for detection of aflatoxin B1 based on CoS@TA Nanospheres.

A novel photothermal immunochromatographic test strip (PITS) with tannic acid (TA) modified cobalt sulfide (CoS) nanospheres (CoS@TA) as immuno-probe element was developed for the detection of aflatoxin B1 (AFB1). CoS nanospheres (CoS NPs) with excellent photothermal conversion efficiency (η = 47.5 %) was synthesized and skillfully chelated with TA to improve its dispersion, biocompatibility, and chromatographic properties. After modification, the CoS@TA coupled with monoclonal antibody (mAb) against AFB1 (CoS@TA-mAb) by simple physical adsorption. The CoS@TA based PITS achieved highly sensitive detection of AFB1 with the limit of detection in photothermal signal (photothermal-LOD) of 0.00503 µg/L, which was 19.88-fold higher than the LOD in visual signal (visual-LOD, 0.1 µg/L). The application of TA in the modification of CoS provided ideas to improve the properties of functional nanomaterials such as dispersion and biocompatibility, and the application of CoS@TA in PITS construction laid a methodological foundation for further improving the detection sensitivity of trace targets.

Multiplex immunochromatographic test strip based on multicolor gold nanoparticles for the simultaneous detection of neomycin, penicillin, and chloramphenicol in food samples.

In this study, we demonstrated an approach for the development of multiplex immunochromatographic test strip (MICS) for neomycin (NEO), penicillin (PEN), and chloramphenicol (CAP) detection using three different-colored gold nanoparticles. The gold nanoparticles: gold nanospheres (red), gold nanostars (blue), and gold nanoflowers (black) were applied as labels for MICS fabrication. The proposed MICS achieved a clearly visible limit of detection with cut-off values of 500, 5, and 0.5 µg L-1 for NEO, PEN, and CAP, respectively within only 10 min. Such smartphone-based detection provided a limit of detection of 3.70 µg L-1 for NEO, 0.10 µg L-1 for PEN, and 0.008 µg L-1 for CAP. The analysis of real samples by the developed MICS was well correlated with the enzyme-linked immunosorbent assay and liquid chromatography-mass spectrometry. The proposed MICS has the potential to be used for the simultaneous detection of NEO, PEN, and CAP with rapid, precise, and sensitive results.

Accuracy evaluation of diagnostic methods for visceral leishmaniasis in adult patients with and without HIV infection: clinical management implications.

Visceral leishmaniasis (VL) poses a serious health threat, particularly when untreated, necessitating accurate diagnosis. While the gold-standard method involves identifying amastigotes in bone marrow aspirate (BMA), this procedure is invasive and occasionally contraindicated. Additionally, when VL is associated with HIV infection the serologies accuracies could be affected. This study aims to evaluate and compare diagnostic methods for VL in patients with and without HIV coinfection. We enrolled prospectively 127 consecutive adult VL patients, 48 (37.8%) of whom had HIV coinfection, in Brazil's Midwestern region, where VL is endemic. Parasitological examination served as the reference standard for accuracy analysis, with index tests including immunofluorescent antibody test (IFAT), immunochromatographic test with rK39 protein (rK39-ICT), and blood polymerase chain reaction (PCR). Specificity assessment involved 430 healthy blood donors from the same endemic area. Ninety-two patients had parasitologically confirmed VL. Among HIV-uninfected patients, rK39-ICT exhibited sensitivity comparable to PCR (93.6%; 95% CI: 83.6-100 vs. 97.8%; 95% CI: 93.6-99.2, respectively) and superior to IFAT (71.1%; 95% CI: 57.9-84.3). However, in HIV-infected patients, rK39-ICT sensitivity was notably lower than PCR (40.0%; 95% CI: 22.5-57.5 vs. 97.4%; 95% CI: 92.5-98.9) and similar to IFAT (67.5%; 95% CI: 52.9-82.0). Combining two serological tests in parallel identified 82.1% of parasitologically confirmed VL cases, with a negative likelihood ratio significantly lower than either test alone. No test achieved a specificity of 90%, and there were no significant differences in specificity observed among the index tests. The positivity rate of parasitological examination in the 127 VL patients was higher in HIV-infected compared to HIV-uninfected patients, 91.3% (95% CI: 83.2-99.4) versus 67.6% (95% CI: 56.9-78.3), respectively. These findings underscore the necessity of accounting for HIV infection when choosing VL diagnostic methods. Although rK39-ICT provides reliable results in HIV-uninfected patients, BMA examination remains crucial for accurate diagnosis in individuals with HIV/AIDS. In cases where bone marrow aspiration is contraindicated, employing IFAT and rK39-ICT in parallel could be considered, as the occurrence of both positive results is uncommon in healthy individuals from endemic areas.

Dual-color immunochromatographic test strip for simultaneous sensitive detection of malachite green and leucomalachite green residues in fish and shrimp meat samples.

A sensitive dual immunochromatographic test strip (dual-ICTS) was developed to detect malachite green (MG) and its metabolite, leucomalachite green (LMG), using two types of gold nanoparticles: round-shaped (red) and star-shaped (blue). The detection limits were determined to be 0.221 µg L-1 for MG and 0.214 µg L-1 for LMG, respectively. The dual-ICTS provided a cut-off value of 1.8 µg L-1 for MG and LMG detection. The dual-ICTS successfully detected MG and LMG in food samples, with recovery rates ranging from 86 % to 116 %. The dual-ICTS was evaluated by correlation analysis between the proposed assay and the well-established enzyme-linked immunosorbent assay in the MG and LMG detection. This is the first report on the development of the ICTS that can detect both MG and LMG at the same time within only 5 min, making it a sensitive and rapid tool for on-site detection.

Development and Validation of a Novel Multiplex Real-Time PCR Assay for Rapid Detection of Carbapenemase Genes in Carbapenem-Resistant Enterobacterales Isolates and Clinical Samples.

Purpose: Carbapenem-resistant Enterobacterales (CRE) infection is an urgent threat to human health. This study aimed to develop and validate a novel multiplex real-time PCR (multi-qPCR) assay for the detection of the blaKPC, blaNDM, blaIMP, blaOXA-48-like, and blaVIM genes in CRE isolates and clinical samples, as well as to compare it with three phenotypic methods. Methods: The reliability and limit of detection (LOD) of the multi-qPCR assay were evaluated. PCR and DNA sequencing were used as the reference methods to identify carbapenemase genes in CRE isolates and clinical samples. The accuracy of the multi-qPCR assay, modified carbapenem inactivation and EDTA-modified carbapenem inactivation method (mCIMandeCIM), carbapenemase inhibitor-based combined disk test (CDT), and colloidal gold-based immunochromatographic test was compared with the reference methods with 182 isolates of CRE. Furthermore, 112 clinical samples were collected to validate the efficacy of this multi-qPCR assay. Results: The standard deviations (CVs) of intra-assay and inter-assay of the multi-qPCR assay were ≤ 0.53% and ≤ 2.04% for detecting the five major carbapenemase genes, respectively; while the LOD ranged from 2×102 copies/mL to 8×102 copies/mL. PCR and DNA sequencing confirmed 168 out of 182 CRE isolates producing carbapenemase(s): KPC (n = 93), NDM (n = 46), IMP (n = 8), OXA-48-like (n = 14), VIM (n = 1), KPC&NDM (n = 5), and KPC&NDM&IMP (n = 1). The accuracy of mCIMandeCIM, CDT, Colloidal Gold, and the multi-qPCR assay was 96.2%, 89.6%, 100%, and 100% respectively for detecting carbapenemase(s) producers. Moreover, the sensitivity and specificity of the multi-qPCR assay were all 100% for the detection of each carbapenemase gene in clinical samples, compared with PCR and sequencing. Conclusion: For clinical isolate detection, the multi-qPCR assay is comparable to Colloidal Gold, and superior to mCIMandeCIM and CDT; while for clinical samples detection, it also shows excellent performance. Therefore, the multi-qPCR assay has great potential for clinical diagnosis.

Multifunctional Fe3O4@CuS nanoparticle-driven colorimetric and photothermal immunochromatographic test strip for the sensitive detection of Salmonella typhimurium in milk.

Magnetic nanoparticles are widely employed as signal labeling reporters in immunochromatographic test strips (ICTS) for detecting foodborne pathogens due to their outstanding anti-interference and magnetic enrichment performance. However, the insufficient colorimetric signal brightness of magnetic nanoparticles results in poor sensitivity, hindering their ability to meet the growing demand for advanced ICTS. Herein, we synthesized Fe3O4@CuS core-shell structure nanoparticles using a facile in-situ growth method. These Fe3O4@CuS nanoparticles exhibit a superior photothermal conversion efficiency of 42.12 % and a magnetization strength of 35 emu/g. We developed a dual-readout format ICTS based on Fe3O4@CuS, incorporating both colorimetric and photothermal formats to enhance sensitivity for Salmonella typhimurium detection. The limit of detection for Fe3O4@CuS-ICTS in the colorimetric and photothermal format was 5 × 104 CFU/mL and 7.7 × 10³ CFU/mL, respectively. Additionally, the average recoveries ranged from 91.25 % to 103.39 %, with variations from 2.2 % to 11.1 %, demonstrating good accuracy and precision. Therefore, this work suggests that Fe3O4@CuS nanoparticles, with their superior magnetic, optical, and photothermal properties, can serve as promising signal labeling reporters to improve the detection performance of ICTS and hold potential for constructing more accurate and sensitive point-of-care testing platforms.

Visual Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid On-Site Detection of Escherichia coli O157: H7 in Milk Products.

(1) Background: Rapid on-site testing is an effective method for the detection of Escherichia coli O157: H7(E. coli O157: H7) in food ingredients and the environment. (2) Methods: In this study, we developed colorimetric loop-mediated isothermal amplification (LAMP) and immunochromatographic test strips (ICTs) for the rapid and visual detection of E. coli O157: H7. This study designed new specific LAMP primers for E. coli O157: H7 virulence island genes. After the LAMP amplification, the double-stranded DNA target sequence labeled with digoxin and fluorescein isothiocyanate (FITC) at both ends was bound to the anti-digoxin antibody on the gold nanoparticles. Subsequently, it was further bound to the anti-FITC antibody at the T line of the ICTs, forming a positive test result. Hydroxynaphthyl blue dye was directly added to the LAMP amplification product. A blue color indicated positive results, while a purple color indicated negative results. (3) Results: Two visualization methods showed high specificity for the target strains. The visualization tests had sensitivities of 5.7 CFU mL-1, and the detection limit of the Escherichia coli O157: H7 in artificially contaminated milk samples was 5.7 × 102 CFU mL-1, which was consistent with the results of the standard method (LAMP-electrophoresis method) used in commercial inspection. (4) Conclusions: Both methods could be useful in remote and under-resourced areas.

An Immunochromatographic Test Strip for Rapid Quantitative Control of Monoclonal Antibodies against Programmed Cell Death Protein 1.

Cancer is one of the major public health challenges in the world, which is characterized by rapid progression and high mortality. Immunotherapy, represented by PD-1 monoclonal antibody, has significantly improved the efficacy of malignant tumors and has become one of the most popular immunotherapy methods at present. Therefore, there is an increasing demand for novel detection methods for PD-1 monoclonal antibodies. The aim of this work was to establish a rapid, simple, and sensitive immunochromatographic test strip (ICTS) based on the AuNPs enlargement for both visual and instrumental detection of the PD-1 monoclonal antibody concentration. The mixed solution of NH2OH·HCl and HAuCl4 was used as an enhancement solution to lower the detection limit and achieve higher sensitivity. A test strip reader was used to construct a visualized quantitative detection standard curve for the PD-1 monoclonal antibody concentration. The LOD was 1.58 ng/mL through a triple signal-to-noise ratio. The detection time was within 10 min. The constructed test strips can rapidly, accurately, and efficiently detect the concentration of PD-1 monoclonal antibody in real samples.

Identification of novel biomarkers for anti-Toxoplasma gondii IgM detection and the potential application in rapid diagnostic fluorescent tests.

Toxoplasmosis, while often asymptomatic and prevalent as a foodborne disease, poses a considerable mortality risk for immunocompromised individuals during pregnancy. Point-of-care serological tests that detect specific IgG and IgM in patient sera are critical for disease management under limited resources. Despite many efforts to replace the T. gondii total lysate antigens (TLAs) by recombinant antigens (rAgs) in commercial kits, while IgG detection provides significant specificity and sensitivity, IgM detection remains comparatively low in sensitivity. In this study, we attempted to identify novel antigens targeting IgM in early infection, thereby establishing an IgM on-site detection kit. Using two-dimensional gel electrophoresis (2DE) and mouse serum immunoblotting, three novel antigens, including EF1γ, PGKI, and GAP50, were indicated to target T. gondii IgM. However, rAg EF1γ was undetectable by IgM of mice sera in Western blotting verification experiments, and ELISA coated with PGKI did not eliminate cross-reactivity, in contrast to GAP50. Subsequently, the lateral flow reaction employing a strip coated with 0.3 mg/mL purified rAg GAP50 and exhibited remarkable sensitivity compared with the conventional ELISA based on tachyzoite TLA, which successfully identified IgM in mouse sera infected with tachyzoites, ranging from 103 to 104 at 5 dpi and 104 at 7 dpi, respectively. Furthermore, by using standard T. gondii-infected human sera from WHO, the limit of detection (LOD) for the rapid fluorescence immunochromatographic test (FICT) using GAP50 was observed at 0.65 IU (international unit). These findings underline the particular immunoreactivity of GAP50, suggesting its potential as a specific biomarker for increasing the sensitivity of the FICT in IgM detection.

Evaluation of the diagnostic performance of an immunochromatographic test for Chlamydia trachomatis.

Objectives: To evaluate the diagnostic performance of different brands of immunochromatographic test (ICT) reagents for Chlamydia trachomatis using homogenized samples to provide a reference for reagent quality control. Methods: Eight commercially available ICT reagents were evaluated, of which three used the latex method and five used the colloidal gold method. Analytical performance evaluation using a pure culture broth of C. trachomatis, as well as clinical application validation using cervical epithelial cell samples acquired from the research subjects, were conducted. The concentration of C. trachomatis was quantified using a nucleic acid amplification test. Results: The limit of detection (LOD) of different ICT reagents in the analytical performance evaluation varied from 9.5 × 103 to 1 × 105 IFU/mL, and only one reagent met the LOD specified in the manufacturer's instructions. Likewise, only one reagent in the clinical application validation achieved the analytical LOD, four reagents were 2.1-4.2-fold of the analytical LODs, and three reagents failed to detect positive results in clinical samples. Conclusions: The diagnostic performance of different methods and different brands of ICT reagents in clinical practice was different from the manufacturer's instructions and the results of laboratory evaluation. The diagnostic performance of reagents should be evaluated before they are actually used in clinical practice.

Preparation of Hybrid Magnetic Nanoparticles for Sensitive and Rapid Detection of Phorate Residue in Celery Using SERS Immunochromatography Assay.

Extensive use of pesticides in agricultural production has been causing serious health threats to humans and animals. Among them, phorate is a highly toxic organophosphorus insecticide that has been widely used in planting. Due to its harmful effects on human and animal health, it has been restricted for use in many countries. Analytical methods for the rapid and sensitive detection of phorate residues in agricultural products are urgently needed. In this study, a new method was developed by combining surface-enhanced Raman spectroscopy (SERS) and immunochromatography assay (ICA). Hybrid magnetic Fe3O4@Au@DTNB-Ab nanoprobes were prepared by modifying and growing Au nanoseeds on an Fe3O4 core. SERS activity of the nanoprobe was optimized by adjusting the concentration of the Au precursor. A rapid and sensitive assay was established by replacing the traditional colloidal gold-based ICA with hybrid SERS nanoprobes for SERS-ICA. After optimizing parameters including coating antibody concentrations and the composition and pH of the buffer solution, the limit of detection (LOD) for phorate could reach 1 ng/mL, with a linear range of 5~100 ng/mL. This LOD is remarkably lower than the maximum residue limit in vegetables and fruits set by the Chinese government. The feasibility of this method was further examined by conducting a spiking test with celery as the real sample. The result demonstrated that this method could serve as a promising platform for rapid and sensitive detection of phorate in agricultural products.

High prevalence of anti-Strongyloides antibody in SARS-CoV-2-infected human sera in a Thai hospital: Rapid serological screening.

COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), can stimulate a systemic inflammatory response with severe lung involvement, multisystem dysfunction, and death in some cases. Immunosuppressive treatments have been proposed for management of COVID-19 patients, but these bring the risk of flare-up of pre-existing infections. Strongyloidiasis can become severe or fatal in immunocompromised individuals. This cross-sectional study determined the prevalence of anti-Strongyloides IgG antibody in sera collected from SARS-CoV-2 infected persons in a tertiary-care Thai hospital from January 2021 to January 2022. The survey was conducted using a rapid immunochromatographic test (ICT) kit based on a recombinant antigen of Strongyloides stercoralis known to be IgG-immunoreactive. High prevalence of anti-Strongyloides IgG antibody was found. Out of 297 SARS-CoV-2-infected patients 117 (39.4 %, 95 % CI 33.8-45.2 %) were positive for S. stercoralis according to the ICT kit. In areas where strongyloidiasis is endemic, we suggest using this point-of-care ICT kit for routine rapid screening in seriously ill COVID-19 patients who will be subjected to immunosuppressive treatment. Prompt anthelminthic treatment should be administered to prevent serious systemic strongyloidiasis in at-risk patients.

Computerized analysis of haptens for the ultrasensitive and specific detection of Pyriftalid.

Pyriftalid (Pyr) is one of the most commonly used herbicides and due to its widespread and improper use, it has led to serious pollution of groundwater, soil and other ecosystems, threatening human health. A rapid method to detect Pyr was urgently needed. A high specific monoclonal antibody (mAb) against Pyr with IC50 values of 4.7 ng/mL was obtained by mAb screening technique and method with enhanced matrix effect. The study firstly proposed colloidal gold immunochromatographic test strips (CGIA) for Pyr, which enables rapid qualitative and quantitative determination of a large number of samples anytime and anywhere, so as to effectively monitor Pyr in environment and grain samples. Based on the properties of the desired Pyr antibody, the hapten Pyr-hapten-4 with high structural similarity to Pyr molecule, similar electrostatic potential distribution, and the ability to expose Pyr functional groups was screened out from five different Pyr haptens, which was consistent with mouse antiserum test. The CGIA quickly analyze the Pyr content in positive samples such as water samples, soil samples, paddy samples, brown rice samples within 10 min, the LOD for Pyr by CGIA as low as 1.84 ng/g, the v LOD value as low as 6 ng/g, and the extinction value as low as 25 ng/g. The content of positive samples detected by CGIA was consistent with the quantitative results of LC-MS/MS, the relative accuracy was within the range of 97-103 %. The recovery rate range for Pyr by CGIA was 92.0-99.7 %, and the coefficient of variation was between 1.30-8.56 %. It indicated Pyr-targeted CGIA test strip was an efficient and fast detection method to detect real environment and food samples.

A rapid simultaneous antigen detection of Haemophilus influenzae and Streptococcus pneumoniae for predicting the prognosis of acute otitis media.

BACKGROUND: Rapid identification of causative bacteria in treatment of acute otitis media (AOM) is of paramount importance for appropriate antibiotic use. MATERIALS AND METHODS: This prospective observational study was conducted in 15 hospitals and clinics in Japan between 2018 and 2020. A new rapid antigen test kit (AOS-116), which simultaneously detects antigens for Streptococcus pneumoniae (Sp) and Haemophilus influenzae (Hi), was applied for middle ear fluids (MEFs) and nasopharyngeal secretions (NPSs) in patients with moderate to severe AOM. We investigated relationship between the results of rapid test, severity at initial visit, and clinical course. RESULTS: Regarding performance accuracy based on culture results, AOS-116 showed 1) high (>80%) sensitivity, specificity, and negative predictive value (NPV) in MEFs for both antigens, 2) high sensitivity, specificity, and positive predictive value (PPV) in NPSs for Hi antigen, and 3) high specificity, and PPV in NPSs for Sp antigen. Regarding predictive value of nasopharyngeal culture and antigen detection for causative middle ear pathogens, similar results were observed between AOS-116 and culture, which was characterized with high sensitivity and NPV for both pathogens. MEFs/NPSs positive for Hi antigen were significantly associated with eardrum findings, and severity. MEFs/NPSs positive for pneumococcal antigen were significantly associated with severity of otalgia, fever, and otorrhea. Among patients with prior antimicrobial treatment, improvement tended to be slower in cases positive for Hi than in cases negative. CONCLUSION: The rapid antigen detection test is useful as a decision-making tool for prescribing antimicrobial agents and may play an important role in promoting appropriate antimicrobial use.

Development of a dual immunochromatographic test strip to detect E2 and Erns antibodies against classical swine fever.

Background: It is essential to consider a practical antibody test to successfully implement marker vaccines and validate vaccination efficacy against classical swine fever virus (CSFV). The test should include a serological antibody assay, combined with a tool for differentiating infected from vaccinated animals (DIVA). The immunochromatographic test strip (ICS) has been exclusively designed for detecting CSFV E2 antibodies while lacking in detecting Erns antibodies, which can be employed and satisfy DIVA strategy. This study developed a novel ICS for detecting CSFV E2/Erns dual-antibody. The effectiveness of ICS in evaluating the DIVA capability of two novel chimeric pestivirus vaccine candidates was assessed. Methods: Recombinant E2 or Erns protein was transiently expressed in the plant benthamiana using Agrobacterium tumefaciens. ICS was subsequently assembled, and goat anti-rabbit IgG and recombinant CSFV E2 or Erns protein were plated onto the nitrocellulose membrane as control and test lines, respectively. The sensitivity and specificity of ICS were evaluated using sera with different neutralizing antibody titers or positive for antibodies against CSFV and other pestiviruses. The coincidence rates for detecting E2 and Erns antibodies between ICS and commercial enzyme-linked immunosorbent assay (ELISA) kits were also computed. ICS performance for DIVA capability was evaluated using sera from pigs vaccinated with conventional vaccine or chimeric vaccine candidates. Results: E2 and Erns proteins were successfully expressed in N. benthamiana-produced recombinant proteins. ICS demonstrated high sensitivity in identifying CSFV E2 and Erns antibodies, even at the low neutralizing antibody titers. No cross-reactivity with antibodies from other pestiviruses was confirmed using ICS. There were high agreement rates of 93.0 and 96.5% between ICS and two commercial ELISA kits for E2 antibody testing. ICS also achieved strong coincidence rates of 92.9 and 89.3% with two ELISA kits for Erns antibody detection. ICS confirmed the absence of CSFV Erns-specific antibodies in sera from pigs vaccinated with chimeric vaccine candidates. Conclusion: E2 and Erns proteins derived from the plant showed great potential and can be used to engineer a CSFV E2/Erns dual-antibody ICS. The ICS was also highly sensitive and specific for detecting CSFV E2 and Erns antibodies. Significantly, ICS can fulfill the DIVA concept by incorporating chimeric vaccine candidates.

Assessment of Blood and Semen Detection and DNA Collection from Swabs up to Three Months after Deposition on Five Different Cloth Materials.

Body fluid identification plays a crucial role in criminal investigations. Because of their presence in many cases, blood and semen are the most relevant body fluids in forensic sciences. Based on antigen-antibody reactions binding unique proteins for each body fluid, serological assays represent one of the most rapid and highly specific tests for blood and semen. Currently, few studies have assessed the factors affecting body fluid identification by applying these assays. This work aimed to study the effect of different fabrics from clothes and time since deposition on identification through immunochromatographic tests for blood and semen, DNA isolation, and STR profiling from these samples. Body fluids were deposited on black- and white-dyed denim and cotton fabrics, and on leather. Afterward, blood and semen were sampled at 1 day, 30 days, and 90 days after deposition and identified by using the SERATEC® HemDirect Hemoglobin Test and the PSA Semiquant and SERATEC® BLOOD CS and SEMEN CS tests, respectively. Laboratory and crime scene tests presented similar performances for the detection of blood and semen stains on every tested fabric. No differences were found on band intensities between timepoints for all fabrics. It was possible to recover and identify blood and semen samples up to three months after deposition and to obtain full STR profiles from all the tested fabrics. Both body fluid STR profiles showed differences in their quality between 1 and 90 days after deposition for all fabrics except for black cotton for semen samples. Future research will expand the results, assessing body fluid identification on other substrates and under different environmental conditions.