Bacterial DNA and serum IgG antibody titer assays for assessing infection of human-pathogenic and dog-pathogenic Porphyromonas species in dogs.

Periodontal disease is highly prevalent in both humans and dogs. Although there have been reports of cross-infection of periodontopathic bacteria, methods for assessing it have yet to be established. The actual status of cross-infection remains to be seen. The purpose of this study was to evaluate the utility of bacterial DNA and serum immunoglobulin G (IgG) antibody titer assays to assess infection of human-pathogenic and dog-pathogenic Porphyromonas species in dogs. Four experimental beagles were used for establishing methods. Sixty-six companion dogs at veterinary clinics visiting for treatment and prophylaxis of periodontal disease were used and divided into healthy, gingivitis, and periodontitis groups. Periodontal pathogens such as Porphyromonas gingivalis and Porphyromonas gulae were investigated as target bacteria. DNA levels of both bacteria were measured using species-specific primers designed for real-time polymerase chain reaction (PCR). Serum IgG titers of both bacteria were measured by enzyme-linked immunosorbent assay (ELISA). PCR primers were confirmed to have high sensitivity and specificity. However, there was no relationship between the amount of bacterial DNA and the severity of the periodontal disease. In addition, dogs with periodontitis had higher IgG titers against both bacteria compared to dogs in the healthy and gingivitis groups; there was cross-reactivity between the two bacteria. Receiver operating characteristic (ROC) analysis of IgG titers against both bacteria showed high sensitivity (>90 %) and specificity (>75 %). Since both bacteria were distinguished by DNA assays, the combination of these assays may be useful in the evaluation of cross-infection.

Evaluation of seegene anyplex MTB/NTM real-time detection assay for diagnosis of tuberculous meningitis.

BACKGROUND: Tuberculous meningitis (TBM) is a common central nervous system infectious disease. Polymerase chain reaction (PCR) assay is a useful method for the rapid diagnosis of TBM. The Seegene Anyplex MTB/NTM real-time detection assay has good sensitivity and specificity for detection of tuberculosis in respiratory specimens, though, data regarding other specimens are lacking. This study aims to define the diagnostic role of Seegene Anyplex MTB/NTM real-time detection assay in TBM in adults. METHODS: This was a retrospective study of 367 adults with symptomatic community acquired meningitis between December 2013 and December 2019. Cerebrospinal fluid (CSF) had been sent for conventional diagnosis, including culture to identify Mycobacterium tuberculosis, and Seegene Anyplex MTB/NTM real-time detection assay. Other diagnostic examinations were performed as necessary. RESULTS: Of the 367 patients in the study, 37 were diagnosed with TBM (14 with definite TBM and 23 with probable TBM). Between the total TBM cases (n = 37) and non-TBM cases (n = 330), clinical sensitivity was 32.4% and specificity was 100%, the positive predictive value was 100%, and the negative predictive value was 93.0%. Between the definite TBM cases (n = 14) and non-TBM cases (n = 330), clinical sensitivity was 50.0% and specificity was 100%, the positive predictive value was 100%, and the negative predictive value was 97.9%. CONCLUSION: Due to lack of sensitivity, we suggest Seegeen Anyplex MTB/NTM real-time detection assay should not be used to rule out TBM but is useful for definite diagnosis.

Assessing type I collagen expression and quality in cellular models of osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a group of genetic disorders of bone formation characterized by soft and shorter brittle bones in affected individuals. OI is generally considered a collagenopathy resulting from abnormal expression of type I collagen. As assay system to detect the cellular level and quality of type I collagen would help in rapid and correct detection of OI from the diagnostic perspectives. Here, we report an immunofluorescence assay for detection of type I collagen in fibroblast models of OI and represented them into two broad categories based on the expression level and aggregation characteristics of pro-α1(I). Cell phenotypic assays of pro-α1(I) in OI-related gene knocked down fibroblasts revealed aggregates of pro-α1(I) in conditions with knockdown of SERPINF1, CRTAP, P3H1, PPIB, SERPINH1, FKBP10, TMEM38B, MESD, and KDELR2, whereas pro-α1(I) expression was very low in fibroblasts which had knockdown of IFITM5, SP7, BMP1, WNT1, CREB3L1, MBTPS2, and CCDC134. The expression of pro-α1(I) showed abundant and non-aggregated distribution in the fibroblasts with knockdown of non-OI skeletal disorder-related genes (RAB33B and IFT52). The in vitro assay accurately detected abnormally expressed pro-α1(I) levels in cellular models of various types of OI. Thus, this procedure represents a promising point-of-detection assay for potential diagnosis and therapeutic decisions in OI.

A rapid and high-throughput multiplex genetic detection assay for detection, semi-quantification and virulence genotyping of Helicobacter pylori in non-invasive oral samples.

Aim: This study established a high-throughput multiplex genetic detection assay (HMGA) for rapid identification, semi-quantification and virulence analysis of Helicobacter pylori directly from the clinical non-invasive oral samples. Methods: The gastric mucosa and oral samples were collected from 242 patients in Shanghai from 2021 to 2022. All the samples were detected by routine clinical tests for H. pylori and Sanger sequenced for inconsistent results. A new multiplex PCR assay providing results within 4 hours was designed and optimized involving fluorescent dye-labeled specific primers targeted 16S rRNA gene, semi-quantitative gene ureC and 10 virulence genes of H. pylori. Semi-quantification was carried out by simulating the serial 10-fold dilutions of positive oral samples, and the H. pylori loads in different clinical samples were further compared. The mixed plasmids of virulence genes vacA s1, vacA m1 and vacA m2 were used to evaluate the performance on different genotypes. The consistency of 10 virulence genes in gastric mucosa, saliva, mouthwash and dental plaque of H. pylori-positive patients was compared. Results: The non-invasive HMGA was highly specific for detection of all 12 targets of H. pylori and human internal reference gene ß-globin, and the sensitivity to all target genes could reach 10 copies/µL. Compared with routine clinical tests and sequencing, non-invasive HMGA has a high level (>0.98) of sensitivity, specificity, accuracy, PPV, NPV and kappa coefficient for direct detection of H. pylori in oral samples. Moreover, by detecting peak area levels of ureC, it was confirmed that the H. pylori loads in gastric mucosa were significantly higher than those of the three kinds of oral samples (p<0.05). We also found that 45.0% (91/202) of patients had different H. pylori virulence genes in different oral samples. The concordance of positive detection rates of each virulence gene between saliva and gastric mucosa was more than 78% (p<0.05). Conclusion: The non-invasive HMGA proved to be a reliable method for the rapid H. pylori identification, semi-quantification and detection of 10 virulence genes directly in oral samples, providing a new idea for non-invasive detection of H. pylori.

Revealing viral and cellular dynamics of HIV-1 at the single-cell level during early treatment periods.

While combination therapy completely suppresses HIV-1 replication in blood, functional virus persists in CD4+ T cell subsets in non-peripheral compartments that are not easily accessible. To fill this gap, we investigated tissue-homing properties of cells that transiently appear in the circulating blood. Through cell separation and in vitro stimulation, the HIV-1 "Gag and Envelope reactivation co-detection assay" (GERDA) enables sensitive detection of Gag+/Env+ protein-expressing cells down to about one cell per million using flow cytometry. By associating GERDA with proviral DNA and polyA-RNA transcripts, we corroborate the presence and functionality of HIV-1 in critical body compartments utilizing t-distributed stochastic neighbor embedding (tSNE) and density-based spatial clustering of applications with noise (DBSCAN) clustering with low viral activity in circulating cells early after diagnosis. We demonstrate transcriptional HIV-1 reactivation at any time, potentially giving rise to intact, infectious particles. With single-cell level resolution, GERDA attributes virus production to lymph-node-homing cells with central memory T cells (TCMs) as main players, critical for HIV-1 reservoir eradication.

The Optimal Management of Neisseria gonorrhoeae Infections.

Neisseria gonorrhoeae is one of the most frequent etiologic agents of STDs (sexually transmitted diseases). Untreated asymptomatic gonococcal infection in women can lead to spreading of the infection in the sexually active population and could lead to late consequences, such as sterility or ectopic pregnancies. One important issue about N. gonorrhoeae is its increasing resistance to antibiotics. This paper summarized the newest molecular antimicrobial resistance (AMR) detection assays for Neisseria gonorrhoeae connected with the latest therapeutic antimicrobials and gonococcal vaccine candidates. The assays used to detect AMR varied from the classical minimal inhibitory concentration (MIC) detection to whole-genome sequencing. New drugs against multi drug resistant (MDR) N. gonorrhoeae have been proposed and were evaluated in vivo and in vitro as being efficient in decreasing the N. gonorrhoeae burden. In addition, anti-N. gonorrhoeae vaccine candidates are being researched, which have been assessed by multiple techniques. With the efforts of many researchers who are studying the detection of antimicrobial resistance in this bacterium and identifying new drugs and new vaccine candidates against it, there is hope in reducing the gonorrhea burden worldwide.

Microfluidic Devices for HIV Diagnosis and Monitoring at Point-of-Care (POC) Settings.

Human immunodeficiency virus (HIV) is a global epidemic; however, many individuals are able to obtain treatment and manage their condition. Progression to acquired immunodeficiency syndrome (AIDS) occurs during late-stage HIV infection, which compromises the immune system, making it susceptible to infections. While there is no cure, antiretroviral therapy can be used provided that detection occurs, preferably during the early phase. However, the detection of HIV is expensive and resource-intensive when tested with conventional methods, such as flow cytometry, polymerase chain reaction (PCR), or enzyme-linked immunosorbent assays (ELISA). Improving disease detection in resource-constrained areas requires equipment that is affordable, portable, and can deliver rapid results. Microfluidic devices have transformed many benchtop techniques to on-chip detection for portable and rapid point-of-care (POC) testing. These devices are cost-effective, sensitive, and rapid and can be used in areas lacking resources. Moreover, their functionality can rival their benchtop counterparts, making them efficient for disease detection. In this review, we discuss the limitations of currently used conventional HIV diagnostic assays and provide an overview of potential microfluidic technologies that can improve HIV testing in POC settings.

Establishment of a reverse transcription recombinase-aided amplification detection method for porcine group a rotavirus.

Porcine rotavirus type A (PoRVA) is the main cause of dehydration and diarrhea in piglets, which has a great impact on the development of the pig industry worldwide. A rapid, accurate and sensitive detection method is conducive to the monitoring, control, and removal of PoRVA. In this study, a PoRVA real-time fluorescent reverse transcription recombinase-aided amplification (RT-RAA) assay was developed. Based on the PoRVA VP6 gene, specific primers and probes were designed and synthesized. The sensitivity of RT-RAA and TaqMan probe-based RT-qPCR was 7 copies per reaction and 5 copies per reaction, respectively. The sensitivity of the RT-RAA method was close to TaqMan probe-based RT-qPCR. The detection results of RT-RAA and TaqMan probe-based quantitative real-time RT-PCR methods were completely consistent in 241 clinical samples. Therefore, we successfully established a rapid and specific RT-RAA diagnostic method for PoRVA.

Designing Biological Microsensors with Chiral Nematic Liquid Crystal Droplets.

Biosensing using liquid crystals has a tremendous potential by coupling the high degree of sensitivity of their alignment to their surroundings with clear optical feedback. Many existing set-ups use birefringence of nematic liquid crystals, which severely limits straightforward and frugal implementation into a sensing platform due to the sophisticated optical set-ups required. In this work, we instead utilize chiral nematic liquid crystal microdroplets, which show strongly reflected structural color, as sensing platforms for surface active agents. We systematically quantify the optical response of closely related biological amphiphiles and find unique optical signatures for each species. We detect signatures across a wide range of concentrations (from micromolar to millimolar), with fast response times (from seconds to minutes). The striking optical response is a function of the adsorption of surfactants in a nonhomogeneous manner and the topology of the chiral nematic liquid crystal orientation at the interface requiring a scattering, multidomain structure. We show that the surface interactions, in particular, the surface packing density, to be a function of both headgroup and tail and thus unique to each surfactant species. We show lab-on-a-chip capability of our method by drying droplets in high-density two-dimensional arrays and simply hydrating the chip to detect dissolved analytes. Finally, we show proof-of-principle in vivo biosensing in the healthy as well as inflamed intestinal tracts of live zebrafish larvae, demonstrating CLC droplets show a clear optical response specifically when exposed to the gut environment rich in amphiphiles. Our unique approach shows clear potential in developing on-site detection platforms and detecting biological amphiphiles in living organisms.

Semi-Quantitative Assay to Measure Urease Activity by Urinary Catheter-Associated Uropathogens.

Catheter-associated urinary tract infections (CAUTIs) are one of the most common healthcare-associated infections in the US, accounting for over 1 million cases annually and totaling 450 million USD. CAUTIs have high morbidity and mortality rates and can be caused by a wide range of pathogens, making empiric treatment difficult. Furthermore, when urease-producing uropathogens cause symptomatic CAUTI or asymptomatic catheter colonization, the risk of catheter failure due to blockage increases. The enzyme urease promotes catheter blockage by hydrolyzing urea in urine into ammonia and carbon dioxide, which results in the formation of crystals that coat the catheter surface. If CAUTI is left untreated, the crystals can grow until they block the urinary catheter. Catheter blockage and subsequent failure reduces the quality of life for the chronically catheterized, as it requires frequent catheter exchanges and can promote more severe disease, including dissemination of the infection to the kidneys or bloodstream. Thus, understanding how urease contributes to catheter blockages and/or more severe disease among the broad range of urease-producing microbes may provide insights into better prevention or treatment strategies. However, clinical assays that detect urease production among clinical isolates are qualitative and prioritize the detection of urease from Proteus mirabilis, the most well-studied uropathogenic urease producer. While urease from other known urease producers, such as Morganella morganii, can also be detected with these methods, other uropathogens, including Staphylococcus aureus and Klebsiella pneumonia, are harder to detect. In this study, we developed a high throughput, semiquantitative assay capable of testing multiple uropathogens in a rapid and efficient way. We validated the assay using Jack Bean urease, the urease producing species: Proteus spp., M. morganii, K. pneumonia, and S. aureus strains, and the non-urease producer: Escherichia coli. This modified assay more rapidly detected urease-producing strains compared to the current clinical test, Christensen Urea Agar, and provided semiquantitative values that may be used to further investigate different aspects of urease regulation, production, or activity in these diverse species. Furthermore, this assay can be easily adapted to account for different environmental stimuli affecting urease production, including bacterial concentration, aeration, or addition of anti-urease compounds.

Reoccurring Bovine Anthrax in Germany on the Same Pasture after 12 Years.

The zoonotic disease anthrax, caused by the endospore-forming bacterium Bacillus anthracis, is very rare in Germany. In the state of Bavaria, the last case occurred in July of 2009, resulting in four dead cows. In August of 2021, the disease reemerged after heavy rains, killing one gestating cow. Notably, both outbreaks affected the same pasture, suggesting a close epidemiological connection. B. anthracis could be grown from blood culture, and the presence of both virulence plasmids (pXO1 and pXO2) was confirmed by PCR. Also, recently developed diagnostic tools enabled rapid detection of B. anthracis cells and nucleic acids directly in clinical samples. The complete genome of the strain isolated from blood, designated BF-5, was DNA sequenced and phylogenetically grouped within the B.Br.CNEVA clade, which is typical for European B. anthracis strains. The genome was almost identical to BF-1, the isolate from 2009, separated only by three single nucleotide polymorphisms (SNPs) on the chromosome, one on plasmid pXO2 and three indel regions. Further, B. anthracis DNA was detected by PCR from soil samples taken from spots in the pasture where the cow had fallen. New tools based on phage receptor-binding proteins enabled the microscopic detection and isolation of B. anthracis directly from soil samples. These environmental isolates were genotyped and found to be identical to BF-5 in terms of SNPs. Therefore, it seems that the BF-5 genotype is currently the prevalent one at the affected premises. The area contaminated by the cadaver was subsequently disinfected with formaldehyde.

Current state of technologies and recognition of anti-SSA/Ro antibodies in China: A multi-center study.

BACKGROUND: Previous studies have demonstrated that Ro60 and Ro52 have different clinical implications, and anti-Ro52 antibodies are an independent serum marker of systemic autoimmune diseases, including Sjögren's syndrome. Many different assays have been adopted to detect anti-Sjögren's syndrome antigen A (SSA)/Ro antibodies, while to date no specific approach has been recommended as optimal for anti-SSA/Ro antibody testing. Herein, we performed a multi-center study to explore the current clinical utility of different strategies for anti-SSA/Ro antibody testing in China. METHODS: Twenty-one tertiary care centers were included in this questionnaire-based study. The self-administered questionnaire mainly includes testing methods for anti-SSA/Ro antibodies, reporting system of results, and interpretation of results by clinicians. RESULTS: Six different methods were applied to detect anti-SSA/Ro antibodies in the 21 centers. Line immunoassay (eight different commercial kits) was the most frequently adopted method (21/21, 100%), with different cutoff values and strategies for intensity stratification. There were two reporting systems: One was reported as "anti-SSA antibodies" and "anti-Ro52 antibodies" (12/21, 57%), while the other was "anti-SSA/Ro60 antibodies" and "anti-SSA/Ro52 antibodies" (9/21, 43%). Notably, six centers (29%) considered either positive anti-Ro60 or anti-Ro52 antibodies as positive anti-SSA antibodies, all of which adopted the latter reporting system. CONCLUSION: Significant variabilities existed among anti-SSA/Ro assays. Nearly 30% of centers misinterpreted the definition of positive anti-SSA antibodies, which may be attributed to the confusing reporting systems of line immunoassay. Therefore, we advocate standardization of the nomenclature of anti-SSA/Ro antibodies, changing the "anti-SSA/Ro52" label in favor of the "anti-Ro52" antibodies for a clear designation.

New technologies in molecular allergodiagnostics.

The article presents the characteristics of the ALEX2 (MacroArrayDX, Wien, Austria). It is designed for simultaneous detection of IgE total and specific IgE-aB to 120 extracts and 180 molecules by solid-phase enzyme immunoassay. Extracts and allergen molecules combined with nano-particles are sorbed on a solid-phase substrate, forming a macroscopic multiplex matrix - the immune allergy chip. The Institute of Clinical and Laboratory Standards (CLSI) conducted research on the verification and validation of the ALEX2 in relation to the ImmunoCAP macroarray test system (ThermoFisher Scientific, Uppsala, Sweden), which is often used in allergodiagnostics. The results obtained on the two test systems were comparable. One of the most important features of the ALEX2 test system is that unique allergen molecules and allergenic extracts are included in its composition, and a method has been found to inhibit cross-reactive hydrocarbon determinants (CCDs), which cause frequent non-specific binding of IgE-aT. The use of this test system makes it possible to carry out component allergy diagnostics with the determine of the dominant sensitizing factor in cases of mono- and polyvalent sensitization. The test results affect the determination of indications and the effectiveness of ASIT, allow assessing the risk of anaphylaxis and predicting further treatment tactics for the patient.

Performance of the Diasorin SARS-CoV-2 antigen detection assay on the LIAISON XL.

BACKGROUND: The current reference standard to diagnose a SARS-CoV-2 infection is real-time reverse transcriptase polymerase chain reaction (RT-PCR). This test poses substantial challenges for large-scale community testing, especially with respect to the long turnaround times. SARS-CoV-2 antigen tests are an alternative, but typically use a lateral flow assay format rendering them less suitable for analysis of large numbers of samples. METHODS: We conducted an evaluation of the Diasorin SARS-CoV-2 antigen detection assay (DAA) compared to real-time RT-PCR (Abbott). The study was performed on 248 (74 qRT-PCR positive, 174 qRT-PCR negative) clinical combined oro-nasopharyngeal samples of individuals with COVID-19-like symptoms obtained at a Municipal Health Service test centre. In addition, we evaluated the analytical performance of DAA with a 10-fold dilution series of SARS-CoV-2 containing culture supernatant and compared it with the lateral flow assay SARS-CoV-2 Roche/SD Biosensor Rapid Antigen test (RRA). RESULTS: The DAA had an overall specificity of 100% (95%CI 97.9%-100%) and sensitivity of 73% (95%CI 61.3%-82.7%) for the clinical samples. Sensitivity was 86% (CI95% 74.6%-93.3%) for samples with Ct-value below 30. Both the DAA and RRA detected SARS-CoV-2 up to a dilution containing 5.2 × 102 fifty-percent-tissue-culture-infective-dose (TCID50)/ml. DISCUSSION: The DAA performed adequately for clinical samples with a Ct-value below 30. Test performance may be further optimised by lowering the relative light unit (RLU) threshold for positivity assuming the in this study used pre-analytical protocol . The test has potential for use as a diagnostic assay for symptomatic community-dwelling individuals early after disease onset in the context of disease control.

Selection and characterization of two monoclonal antibodies specific for the Aspergillus flavus major antigenic cell wall protein Aflmp1.

Aspergillus flavus is a major fungal pathogen of plants and an opportunistic pathogen of humans. In addition to the direct impact of infection, it produces immunosuppressive and carcinogenic aflatoxins. The early detection of A. flavus is therefore necessary to diagnose and monitor fungal infection, to prevent aflatoxin contamination of food and feed, and for effective antifungal therapy. Aspergillus-specific monoclonal antibodies (mAbs) are promising as diagnostic and therapeutic reagents for the tracking and treatment of Aspergillus infections, respectively. However, A. flavus has a complex cell wall composition and dynamic morphology, hindering the discovery of mAbs with well-characterized targets. Here we describe the generation and detailed characterization of mAb5.52 (IgG2aκ) and mAb17.15 (IgG1κ), which bind specifically to the highly immunogenic cell wall antigen A. flavus mannoprotein 1 (Aflmp1). Both mAbs were generated using hybridoma technology following the immunization of mice with a recombinant truncated version of Aflmp1 (ExD, including the homologous CR4 domain) produced in bacteria. We show that mAb5.52 and mAb17.15 bind specifically to A. flavus and A. parasiticus cell wall fragments (CWFs), with no cross-reaction to CWFs from other fungal pathogens. Immunofluorescence microscopy revealed that both mAbs bind to the surface of Aspergillus hyphae and that mAb17.15 also binds to spores. The epitope for both mAbs is localized within the CR4 region of the Aflmp1 protein. These Aspergillus-specific mAbs may be useful for the early detection of fungal infection in food/feed crops, for serodiagnosis in patients with invasive aspergillosis caused by A. flavus infection and for the development of antibody-expressing disease-resistant crops.

Multiplex Real-Time PCR Assay for Six Major Carbapenemase Genes.

The global dissemination of carbapenemase-producing Enterobacteriaceae (CPE) is a major concern in public health. Due to the existence of the diversity of carbapenemases, development of an easily available, cost-effective multiplex detection assay for CPE is required worldwide. Using clinically available and reliable equipment, COBAS® z480 (Roche Diagnostics K.K., Tokyo, Japan), we developed a multiplex real-time PCR assay for the detection of two combinations of carbapenemases; first, blaNDM, blaKPC, and blaIMP (Set 1), and second, blaGES, blaOXA-48, and blaVIM (Set 2). We constructed standard curves for each carbapenemase gene using serial dilutions of DNA standards, then applied reference or clinical isolates with each carbapenemase gene to this assay. The multiplex assay showed satisfactory accuracy to detect CPE genes, with the correlation coefficients of greater than 0.99 for all genotypes. The assay appropriately differentiated the reference or clinical strains harboring each carbapenemase gene without cross reactivity. Lastly, the assay successfully detected multiple genes without false-positive reactions by applying six clinical isolates carrying both NDM and OXA-48-like carbapenemase genes. Major advantages of our assay include multiplicity, simple operation, robustness, and speed (1 h). We believe that the multiplex assay potentially contributes to early diagnosis of CPE with a diverse genetic background.

Rapid estimation of Salmonella enterica contamination level in ground beef - Application of the time-to-positivity method using a combination of molecular detection and direct plating.

Little progress has been made in decreasing the incidence rate of salmonellosis in the US over the past decade. Mitigating the contribution of contaminated raw meat to the salmonellosis incidence rate requires rapid methods for quantifying Salmonella, so that highly contaminated products can be removed before entering the food chain. Here we evaluated the use of Time-to-Positivity (TTP) as a rapid, semi-quantitative approach for estimating Salmonella contamination levels in ground beef. Growth rates of 14 Salmonella strains (inoculated at log 1 to -2 CFU/g) were characterized in lean ground beef mTSB enrichments and time-to-detection was determined using culture and molecular detection methods. Enrichments were sampled at five timepoints and results were used to construct a prediction model of estimated contamination level by TTP (superscript indicates time in hours) defined as TTP4: ≥5 CFU/g; TTP6: ≤5, ≥1 CFU/g; TTP8: ≤1, ≥0.01 CFU/g; with samples negative at 8 h estimated ≤0.01 CFU/g. Model performance measures showed high sensitivity (100%) and specificity (83% and 93% for two detection methods) for samples with a TTP4, with false negative rates of 0%.

Two Monoclonal Antibodies That Specifically Recognize Aspergillus Cell Wall Antigens and Can Detect Circulating Antigens in Infected Mice.

Invasive aspergillosis (IA) is a life-threatening disease mainly caused by Aspergillus fumigatus and Aspergillus flavus. Early diagnosis of this condition is crucial for patient treatment and survival. As current diagnostic techniques for IA lack sufficient accuracy, we have raised two monoclonal antibodies (1D2 and 4E4) against A. fumigatus cell wall fragments that may provide a platform for a new diagnostic approach. The immunoreactivity of these antibodies was tested by immunofluorescence and ELISA against various Aspergillus and Candida species in vitro and by immunohistochemistry in A. fumigatus infected mouse tissues. Both monoclonal antibodies (mAbs) showed intensive fluorescence with the hyphae wall of A. fumigatus and A. flavus, but there was no staining with other Aspergillus species or Candida species. Both mAbs also showed strong immunoreactivity to the cell wall of A. fumigatus hyphae in the infected liver, spleen and kidney of mice with IA. The antigens identified by 1D2 and 4E4 might be glycoproteins and the epitopes are most likely a protein or peptide rather than a carbohydrate. An antibody-based antigen capture ELISA detected the extracellular antigens released by A. fumigatus, A. flavus, A. niger and A. terreus, but not in Candida species. The antigen could be detected in the plasma of mice after 48 h of infection by double-sandwich ELISA. In conclusion, both 1D2 and 4E4 mAbs are potentially promising diagnostic tools to investigate invasive aspergillosis.

Rapid detection method of carbapenemase-producing Enterobacteriaceae by MALDI-TOF MS with imipenem/cilastatin (KB) disc and zinc sulfate solution.

INTRODUCTION: Carbapenemase-producing Enterobacteriaceae (CPE) is a major global health threat, and development of rapid detection methods is desired. Here, we established a cost-effective and relatively rapid CPE detection method using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). METHODS: We examined 134 CPE strains (IMP-type, NDM-type, VIM-type, KPC-type, OXA-48-like-type, and GES-type) and 107 non-CPE strains, previously confirmed by genetic tests. The proposed MALDI-TOF MS method involves mixing of a carbapenem drug [here, the commercially available imipenem (IPM) KB disc] and the bacterial strains to be tested, and the consequent drug hydrolysis owing to bacterial carbapenemase activity is confirmed by a waveform spectrum before and after 2 h of the mixing. As metallo-beta-lactamases require zinc in their active site, the false-negatives obtained from our method were cultured in presence of zinc sulfate solution and tested again. RESULTS: Based on the presence or absence of the IPM (+cyano-4-hydroxy-cinnamic acid)-specific waveform peak near 489.45 m/z (±500 ppm), the detection sensitivity and specificity of our method for CPE were determined to be 94.8% and 91.6%, respectively. Seven false-negatives of IMP-type (4), VIM-type (2), and GES-type (1) were found, of which the IMP- and VIM-types tested positive as CPE after culture with zinc sulfate solution. Thus, the overall detection sensitivity improved to 99.3%. CONCLUSION: Our study proposes a new approach for CPE detection using MALDI-TOF MS. Moreover, we propose cultivation of test strains with zinc sulfate solution for efficient detection of IMP-type CPE, not only for MALDI-TOF MS, but also for other detection methods.

Insights into the Formation of DNA-Magnetic Nanoparticle Hybrid Structures: Correlations between Morphological Characterization and Output from Magnetic Biosensor Measurements.

Understanding the binding mechanism between probe-functionalized magnetic nanoparticles (MNPs) and DNA targets or amplification products thereof is essential in the optimization of magnetic biosensors for the detection of DNA. Herein, the molecular interaction forming hybrid structures upon hybridization between DNA-functionalized magnetic nanoparticles, exhibiting Brownian relaxation, and rolling circle amplification products (DNA-coils) is investigated by the use of atomic force microscopy in a liquid environment and magnetic biosensors measuring the frequency-dependent magnetic response and the frequency-dependent modulation of light transmission. This approach reveals the qualitative and quantitative correlations between the morphological features of the hybrid structures with their magnetic response. The suppression of the high-frequency peak in the magnetic response and the appearance of a new peak at lower frequencies match the formation of larger sized assemblies upon increasing the concentration of DNA-coils. Furthermore, an increase of the DNA-coil concentration induces an increase in the number of MNPs per hybrid structure. This study provides new insights into the DNA-MNP binding mechanism, and its versatility is of considerable importance for the mechanistic characterization of other DNA-nanoparticle biosensor systems.