Infecciones por Clostridioides difficile en pediatría. La visión del laboratorio a partir del diagnóstico microbiológico / Clostridioides difficile infections in pediatrics. The view of the laboratory based on microbiological diagnosis

La infección por Clostridioides difficile (ICD) es la principal responsable de diarreas nosocomiales en adultos. En los últimos años se registró un aumento en la incidencia de la ICD en la población adulta que, en cambio, no fue bien caracterizado en pediatría. El objetivo de este trabajo es analizar los datos resultantes del diagnóstico microbiológico de ICD en el Hospital de Pediatría "Prof. Dr. Juan P. Garrahan". Materiales y métodos: se realizó un estudio retrospectivo observacional descriptivo que abarcó desde el 01/01/2018 hasta el 31/12/2021. El diagnóstico se realizó mediante enzimoinmunoensayo para glutamato deshidrogenasa (GDH) y toxinas en materia fecal (MF). Cuando sólo se detectó GDH, se realizó un cultivo toxigénico (CT) de la MF para la detección de toxinas in vitro. Se registraron: edad, sexo y procedencia de los pacientes y recurrencias de las ICD. Se efectuaron estudios de sensibilidad de 387 cepas de C. difficile a metronidazol (MTZ) y vancomicina (VAN). Resultados: en 6632 muestras (1764 pacientes) se registraron 649 estudios positivos (9,8%) (139 pacientes), la mayoría correspondieron a pacientes internados en áreas no críticas. Edad promedio: 7 años (7 ± 4,7). Sexo: 55% masculino. Recurrencias: 62 (45%). Positivos detectados mediante CT: 43%. Sensibilidad antibiótica: 100% a MTZ y 99,7% a VAN. Conclusión: Nuestra población presenta un bajo porcentaje de positividad. Se destaca el rendimiento del CT que permitió el diagnóstico de más de un tercio de los casos. MTZ y VANCO tuvieron excelente actividad in vitro frente a C. difficile (AU)

Clostridioides difficile infection (CDI) is the main cause of nosocomial diarrhea in adults. In recent years there has been an increase in the incidence of CDI in the adult population; however, CDI has not been well characterized in pediatrics. The aim of this study was to analyze the data resulting from the microbiological diagnosis of CDI at Hospital de Pediatría Prof. Dr. Juan P. Garrahan. Materials and methods: a retrospective, observational and descriptive study was conducted from 01/01/2018 to 12/31/2021. Diagnosis was made using enzyme immunoassay for glutamate dehydrogenase (GDH) and toxins in stools. When only GDH was detected, toxigenic culture (TC) of stools was performed for in vitro toxin detection. The age, sex and origin of patients and CDI recurrences were recorded. Sensitivity studies of 387 strains of C. difficile to metronidazole (MTZ) and vancomycin (VAN) were performed. Results: In 6,632 samples (1,764 patients), 649 positive results (9.8%) were recorded (139 patients), most of which corresponded to patients hospitalized in noncritical areas. Mean age: 7 years (7 ± 4.7). Sex: 55% male. Recurrences: 62 (45%). TC-positive results: 43%. Antibiotic sensitivity: 100% to MTZ and 99.7% to VAN. Conclusion: A low percentage of positivity was found in our population. The performance of TC was outstanding, allowing for the diagnosis of more than one third of the cases. MTZ and VANCO had excellent in vitro activity against C. difficile (AU)

A performance evaluation of chemiluminescence enzyme immunoassays on the Sysmex CN-6500 haemostasis analyser.

BACKGROUND: The Sysmex CN-6500 is a new haemostasis analyser with an integrated immunoassay module that performs chemiluminescence enzyme assay (CLEIA) in addition to coagulation, turbidimetric, chromogenic and platelet aggregation tests. AIMS: To evaluate the analytical performance of the CN-6500 against the predicate device (Sysmex HISCL-800) for soluble thrombomodulin (TM), thrombin-antithrombin (TAT), tissue plasminogen activator/plasminogen activator inhibitor 1 complex (tPAI-C) and plasmin α2 plasmin inhibitor complex (PIC) assays. METHODS: Imprecision was assessed by testing two levels of quality control plasmas 10 times on 5 separate days. Comparability was studied in 230 plasmas from normal donors (n = 30), patients with suspected disseminated intravascular coagulation (DIC, n = 100), sepsis (n = 20) or liver disease (n = 20), lipaemic (n = 20), haemolysed (n = 20) and icteric samples (n = 20). Limit of detection, limit of quantitation and linearity were determined by testing serial dilutions of normal plasma. Sample carryover was assessed by testing samples with high and low normal levels of the analytes concerned. RESULTS: The CN-6500 performed 21 CLEIA tests per hour, while simultaneously performing coagulation tests. Acceptable between-run imprecision was obtained using commercial controls with normal and high activity for each analyte (%CV <4%), for all four assays. Excellent linearity was observed (slope 0.89-1.03; r2 >0.99) across the measurement range. The lower limits of detection and quantitation were as follows: TM <0.3/0.6 TU/ml, TAT >0.1/<0.2 ng/ml, PIC <0.004/<0.008 µg/ml and tPAI-C < 0.01/<0.1 ng/ml, respectively. All four assays showed excellent correlation between analysers and were unaffected by haemolysis, icterus or lipaemia. No carryover was observed. CONCLUSIONS: Our data demonstrate that the performance of the CLEIA assays on the CN-6500 is comparable to that of a stand-alone immunoassay analyser.

Detecting ß-Galactosidase-Labeled Cells.

ß-Galactosidase has been used extensively both as a label in enzyme immunoassays and for immunocytochemistry. One good substrate is 5-bromo-4-chloro-3-indolyl-ß-d-galactopyranoside (X-gal), which gives an intense blue product. The product is stable and insoluble in alcohol as well as H2O.

Inkjet-printed electrochemically reduced graphene oxide microelectrode as a platform for HT-2 mycotoxin immunoenzymatic biosensing.

The design and application of an inkjet-printed electrochemically reduced graphene oxide microelectrode for HT-2 mycotoxin immunoenzymatic biosensing is reported. A water-based graphene oxide ink was first formulated and single-drop line working microelectrodes were inkjet-printed onto poly(ethylene 2,6-naphthalate) substrates, with dimensions of 78 µm in width and 30 nm in height after solvent evaporation. The printed graphene oxide microelectrodes were electrochemically reduced and characterized by Raman and X-ray photoelectron spectroscopies in addition to microscopies. Through optimization of the electrochemical reduction parameters, differential pulse voltammetry were performed to examine the sensing of 1-naphthol (1-N), where it was revealed that reduction times had significant effects on electrode performance. The developed microelectrodes were then used as an immunoenzymatic biosensor for the detection of HT-2 mycotoxin based on carbodiimide linking of the microelectrode surface and HT-2 toxin antigen binding fragment of antibody (anti-HT2 (10) Fab). The HT-2 toxin and anti-HT2 (10) Fab reaction was reported by anti-HT2 immune complex single-chain variable fragment of antibody fused with alkaline phosphatase (anti-IC-HT2 scFv-ALP) which is able to produce an electroactive reporter - 1-N. The biosensor showed detection limit of 1.6 ng ∙ mL-1 and a linear dynamic range of 6.3 - 100.0 ng ∙ mL-1 within a 5 min incubation with 1-naphthyl phosphate (1-NP) substrate.

Multifunctional motion-to-color janus transducers for the rapid detection of sepsis biomarkers in whole blood.

Self-propelled particles are revolutionizing sensing applications thanks to a unique motion-based signal generation mechanism in which biorecognition reactions are detected as changes in the velocity of the colloids. Here a new family of self-propelled multifunctional Janus particles is introduced that enables detecting changes in particle motion colorimetrically. The particles consist of an iron oxide core that provides color and magnetism, and a Janus coating that provides biospecific recognition and locomotive properties. In this approach, biomolecular interactions trigger changes in particle motion that are detected as variations in color when spotted on a piece of paper. These variations in color are then read and quantified with a custom-made smartphone app. The high surface area and magnetism of the particles makes them ideal building blocks for developing biosensors because they allow for the rapid capture of a target molecule and the removal of non-specific interactions. Biosensors engineered with the proposed multifunctional particles were able to detect the sepsis biomarker procalcitonin at clinically relevant concentrations within 13 min in whole blood, which is faster than other approaches requiring hour-long incubation steps under controlled conditions to detect the same biomarker in purified serum. The short assay time along with the point-of-need design makes these biosensors suitable for stratifying patients according to their sepsis risk level during triage independently of resource constraints.

Hapten Synthesis, Antibody Development, and a Highly Sensitive Indirect Competitive Chemiluminescent Enzyme Immunoassay for Detection of Dicamba.

Although dicamba has long been one of the most widely used selective herbicides, some U.S. states have banned the sale and use of dicamba because of farmers complaints of drift and damage to nonresistant crops. To prevent illegal use of dicamba and allow monitoring of nonresistant crops, a rapid and sensitive method for detection of dicamba is critical. In this paper, three novel dicamba haptens with an aldehyde group were synthesized, conjugated to the carrier protein via a reductive-amination procedure and an indirect competitive chemiluminescent enzyme immunoassay (CLEIA) for dicamba was developed. The assay showed an IC50 of 0.874 ng/mL which was over 15 times lower than that of the conventional enzyme immunoassay. The immunoassay was used to quantify dicamba concentrations in field samples of soil and soybean obtained from fields sprayed with dicamba. The developed CLEIA showed an excellent correlation with LC-MS analysis in spike-and-recovery studies, as well as in real samples. The recovery of dicamba ranged from 86 to 108% in plant samples and from 105 to 107% in soil samples. Thus, this assay is a rapid and simple analytical tool for detecting and quantifying dicamba levels in environmental samples and potentially a great tool for on-site crop and field monitoring.

Backfilling rolling cycle amplification with enzyme-DNA conjugates on antibody for portable electrochemical immunoassay with glucometer readout.

A simple and feasible electrochemical immunosensing protocol with glucometer readout was designed for the detection of low-abundance disease-related biomarker (alpha-fetoprotein; AFP) on the basis of backfilling rolling cycle amplification (RCA) with invertase-DNA2 conjugates on the detection antibody. The assay consisted of the immunoreaction, RCA reaction, DNA2-invertase hybridization and glucose measurement. Initially, a sandwiched immunoreaction was carried out between anti-AFP capture antibody-coated microplate between nanogold-labeled pAb2 detection antibody conjugated with DNA1 primer (DNA1-AuNP-pAb2) in the presence of target ATP. Thereafter, the carried primers triggered the RCA reaction in the presence of circular DNA template, polymerase and dNTP, to produce numerous repeated oligonucleotide sequences for hybridization with many invertase-DNA2 conjugates. The carried invertase molecules accompanying the hybridization reaction hydrolyzed sucrose into glucose, thereby resulting in the amplification of the detectable signal on a handheld personal glucometer (PGM). Under optimum conditions, the developed immunoassay exhibited high sensitivity for the quantitative screening of AFP within a dynamic range of 0.1-100 ng mL-1 at a low detection limit of 0.087 ng mL-1. Other biomarkers and proteins did not interfere the signals of this system. In addition, this method was utilized to determine human serum samples containing target AFP, and received well-matched results with the referenced enzyme-linked immunosorbent assay (ELISA) method.

One-Step Ultrasensitive Bioluminescent Enzyme Immunoassay Based on Nanobody/Nanoluciferase Fusion for Detection of Aflatoxin B1 in Cereal.

Nanoluciferase (Nluc), the smallest luciferase known, was used as the fusion partner with a nanobody against aflatoxin B1 to develop a bioluminescent enzyme immunoassay (BLEIA) for detection of the aflatoxin B1 in cereal. Nanobody (clone G8) against aflatoxin B1 was fused with nanoluciferase and cloned into a pET22b expression vector, and then transformed into Escherichia coli. The nanobody fusion gene contained a hexahistidine tag for purification by immobilized metal affinity chromatography, yielding a biologically active fusion protein. The fusion protein G8-Nluc retained binding properties of the original nanobody. Concentration of the coelenterazine substrate and buffer composition were also optimized to provide high intensity and long half-life of the luminescent signal. The G8-Nluc was used as a detection antibody to establish a competitive bioluminescent ELISA for the detection of aflatoxin B1 in cereals successfully. Compared to classical ELISA, this novel assay showed more than 20-fold improvement in detection sensitivity, with an IC50 value of 0.41 ng/mL and linear range from 0.10 to 1.64 ng/mL. In addition, the entire BLEIA detection procedure can be completed in one step within 2 h, from sample preparation to data analysis. These results suggest that nanobody fragments fused with nanoluciferase might serve as useful and highly sensitive dual functional reagents for the development of rapid and highly sensitive immunoanalytical methods.

Development of a microdevice for facile analysis of theophylline in whole blood by a cloned enzyme donor immunoassay.

We have developed a microdevice for therapeutic drug monitoring. In this device, dispensing of sample and reagent was accomplished by simple manual operation of a syringe. Moreover, for a simple and rapid measurement, we used cloned enzyme donor immunoassay as a detection principle. These features and the reagent that is enclosed in microdevice beforehand make it possible to complete the facile analysis. In this paper, our model analyte was 1,3-dimethylxanthine (theophylline), a kind of bronchodilator. The fluorescence measurement of theophylline in whole blood was achieved with the limit of detection of 0.73 µg mL-1. This microdevice provides rapid analysis (4 min), requires only a small volume of sample (2 µL) and features simple operation; hence, it is readily applicable to point of care testing.

Chemiluminescence-based biosensor for monitoring astronauts' health status during space missions: Results from the International Space Station.

During space missions, real-time monitoring of astronauts' health status is of crucial importance and therefore there is a strong demand for simple analytical devices that astronauts can use to perform clinical chemistry analyses directly onboard. As part of the "IN SITU Bioanalysis" project, we designed a biosensor for analysing salivary levels of cortisol in astronauts, a marker of chronic stress. The biosensor is based on the Lateral Flow Immunoassay (LFIA) approach coupled with chemiluminescence (CL) detection and comprises a 3D-printed plastic cartridge containing a sealed fluidic element with the LFIA strip, in which the flow of sample and reagents is activated by pressing buttons on the cartridge and sustained by exploiting capillary forces. For measurement, the photon emission is imaged employing a CL reader based on an ultrasensitive cooled charge-coupled device (CCD) camera. The payload was designed to operate in microgravity and to withstand mechanical stress, such as take-off vibrations, and onboard depressurization events, while the microfluidics was developed considering alterations of physical phenomena occurring in microgravity, such as bubble formation, surface wettability and liquid evaporation. The biosensor, which was successfully used by the Italian astronaut Paolo Nespoli during the VITA mission (July-December 2017), demonstrated the feasibility of performing sensitive LFIA analysis of salivary cortisol down to 0.4 ng/mL directly onboard the International Space Station. It could be easily adapted for the analysis of other clinical biomarkers, thus enabling the early diagnosis of diseases and the timely activation of appropriate countermeasures.

Calorimetric sandwich-type immunosensor for quantification of TNF-α.

We report a lab-on-a-chip immunosesnor for quantification of the inflammatory cytokine TNF-α with picomolar sensitivity. The feasibility of the technology was demonstrated via accurate measurement of the concentration of TNF-α in astrocytes cell culture media. The immunoassay was performed in a microfluidic device with an integrated antimony/bismuth thermopile sensor and had a limit of detection of 14 pg mL-1. The device was fabricated using rapid prototyping xurography technique and consisted of two inlets and single outlet. Anti-TNF-α monoclonal antibody was used to capture the analyte while the detection was performed using glucose oxidase-conjugated secondary antibody. Glucose (55 mM) was injected through a sample loop into the fluid flowing within the microfluidic device. A nanovolt meter connected to the thermoelectric sensor recorded the voltage change caused by the enzymatic reaction. Computer simulations using COMSOL Multiphysics were performed to analyze the effect of fluid velocity on the concentration of glucose within the reaction zone. A standard calibration curve was created using serial dilutions of synthetic TNF-α (0-2000 pg mL-1) by plotting the area under the curve of the signal versus the concentration of the analyte. The efficacy of the device was evaluated by quantifying TNF-α in the cell culture medium of lipopolysaccharide stimulated and non-stimulated astrocytes. The results demonstrated high accuracy of the calorimetric immunoassay when compared with gold standard commercial ELISA microplate reader. The immunosensor offers excellent reproducibility, accuracy, and versatility in the choice of the detection enzyme.

Vertical flow-based paper immunosensor for rapid electrochemical and colorimetric detection of influenza virus using a different pore size sample pad.

We present a novel vertical flow-based paper immunosensor for the rapid and sensitive electrochemical and colorimetric detection of influenza H1N1 viruses using a different pore size (DP) sample pad. The DP sample pad consisted of two different pore size papers: larger pores (diameter: 11 µm) facing the inlet, and smaller pores (diameter: 0.45 µm) facing the conjugate pad. This sample pad offered moderate and uniform flows, and hence concentrated horseradish peroxidase tagged antibodies (HRP-Abs)-H1N1 complexes from 40 µL of sample volumes on a conjugate pad for 2 min after sample injection, thereby providing fast detection (6 min for both detection methods) with 100 µL of flushing afterwards, high sensitivity, and the simplicity of the sensor. The filtration characteristics of the DP sample pad were evaluated using fluorescent beads, indicating that only small-sized bio-particles such as viruses can pass through the sample pad. Sandwich immunoreactions of HRP-Ab-H1N1-Ab were performed on the gold paper electrode of the immunoStrip, which was determined by electrochemical impedance spectroscopy (EIS) measurements. Simultaneously, the color signal of free HRP-Ab captured on the colorimetric zone was obtained using a scanner, and the intensity was analyzed using ImageJ. This immunosensor detected H1N1 virus concentration as low as 3.3 plaque forming units (PFU)/mL (phosphate buffer saline; PBS) and 4.7 PFU/mL (saliva) by EIS, and 1.34 PFU/mL (PBS) and 2.27 PFU/mL (saliva) by the colorimetric method. Furthermore, integrating these two detection methods can reduce false results with double assurance, and this device can provide a simple and economical on-site detection platform.

Washing-Free Displacement Immunosensor for Cortisol in Human Serum Containing Numerous Interfering Species.

Simple and sensitive competitive immunosensors for small molecules are difficult to obtain, especially in serum containing numerous interfering species (ISs) with different concentrations. Herein, we report a washing-free and sensitive (competitive) displacement immunosensor for cortisol in human serum, based on electron mediation of Os(bpy)2Cl2 between an electrode and a redox label [oxygen-insensitive diaphorase (DI)] (i.e., electrochemical-enzymatic redox cycling). The anticortisol IgG-DI conjugate bound to a cortisol-immobilized electrode is displaced by competitive binding of cortisol in serum and diffuses away from the electrode during incubation; therefore, the concentration of the displaced conjugate near the electrode becomes very low, even without washing. Electrochemically interfering ascorbic acid is converted to a redox-inactive species by ascorbate oxidase during incubation. The remaining bound conjugate mainly contributes to electrochemical currents. Compared with ferrocene methanol, Fe(CN)64-, and Ru(NH3)63+, the electrochemical and redox cycling behaviors of Os(bpy)2Cl2 are influenced significantly less by ISs in serum. Comparative studies reveal that washing-free displacement assay shows better cortisol-induced signal change than three other assays. The surface concentration of cortisol immobilized on the electrode is optimized, because the electrochemical signal is highly dependent on the surface concentration. When the washing-free displacement immunosensor is applied for the detection of cortisol in artificial serum, cortisol is measured with a detection limit of ∼30 pM within 12 min. The cortisol concentrations measured in clinical serum samples agree well with those obtained using a commercial instrument. The new immunosensor is highly promising for the simple, sensitive, and rapid point-of-care detection of small molecules.

Validating the use of a commercial enzyme immunoassay to measure oxytocin in unextracted urine and saliva of the western lowland gorilla (Gorilla gorilla gorilla).

The neuroendocrine hormone oxytocin, which is an important physiological driver of social behavior and bonding, is increasingly being measured in conjunction with behavior to better understand primate sociality. To date no data are available on oxytocin concentrations within the genus Gorilla; however, as a result of their close genetic relatedness to humans, and tolerance-based social system, Gorilla represents an important group of study. The purpose of this study was to validate the measurement of urinary and salivary oxytocin in western lowland gorillas (Gorilla gorilla gorilla) to help facilitate future study of the interaction between oxytocin and behavior within the subspecies. The primary validation procedure was an intranasal challenge. Elevated oxytocin concentrations were observed in saliva samples taken 15-120 min post challenge. Urine levels remained within baseline range approximately 30 and 90 min following the challenge; however, elevated levels were observed 24 h post challenge. No diurnal variation was found in salivary samples taken at regular intervals throughout the day; however, morning urine samples had higher concentrations than afternoon samples. In addition, samples were collected opportunistically following three social events: play, breeding, and the death of a conspecific. Following the play bouts, salivary oxytocin was almost three times greater than baseline. Salivary oxytocin was also significantly higher 15 min post breeding compared to match-control samples. Following the death of a conspecific, the group mate's urinary oxytocin concentrations decreased by half compared to a baseline period when the group was intact. This study provides a biological validation of the measurement of urinary and salivary oxytocin in western lowland gorillas. These results suggest that urinary oxytocin measurements are suitable for establishing baseline levels, as they represent the build up of the previous day's concentrations, and salivary oxytocin measurements are suitable for assessing changes following specific events.

A new enzyme immunoassay for alpha-fetoprotein in a separate setup coupling an aluminium/Prussian blue-based self-powered electrochromic display with a digital multimeter readout.

A new enzyme immunosensing platform was designed for the simple, rapid and sensitive detection of disease-related biomarkers (alpha-fetoprotein, AFP, was used as a model in this case), coupling an aluminium (Al)/Prussian blue-based self-powered electrochromic display with a digital multimeter readout.

Magnetic multiwalled carbon nanotubes as nanocarrier tags for sensitive determination of fetuin in saliva.

This paper reports the development and performance of an electrochemical immunosensor using magnetic multiwalled carbon nanotubes (m-MWCNTs) as nanocarrier tags for the determination of human fetuin A (HFA), a relevant biomarker of obesity, insulin resistance, and type-2 diabetes as well as for pancreatic and liver cancers and inflammatory processes. Screen-printed carbon electrodes were grafted with p-aminobezoic acid and streptavidin was covalently immobilized on the electrode surface. A biotinylated capture antibody was immobilized through streptavidin-biotin interaction and a sandwich assay configuration was implemented using m-MWCNTs conjugated with HRP and anti-HFA antibodies as the detection label. The determination of HFA was accomplished by measuring the current produced by the electrochemical reduction of benzoquinone at -200 mV upon addition of H2O2 as HRP substrate. The prepared m-MWCNTs were characterized by SEM, TEM, XRD and EDS. All the steps involved in the immunosensor preparation were monitored by electrochemical impedance spectroscopy and cyclic voltammetry. A linear calibration plot for HFA was found between 20 and 2000 pg/mL with a LOD value of 16 pg/mL. This performance is notably better than that reported for an ELISA kit and a chronoimpedimetric immunosensor. The favorable contribution of m-MWCNTs in comparison with MWCNTs without incorporated magnetic particles to this excellent analytical performance is also highlighted. The immunosensor selectivity against other proteins and potentially interfering compounds was excellent. In addition, the usefulness of the immunosensor was demonstrated by the analysis of HFA in saliva with minimal sample treatment.

A Study on Micropipetting Detection Technology of Automatic Enzyme Immunoassay Analyzer.

In order to improve the accuracy and reliability of micropipetting, a method of micro-pipette detection and calibration combining the dynamic pressure monitoring in pipetting process and quantitative identification of pipette volume in image processing was proposed. Firstly, the normalized pressure model for the pipetting process was established with the kinematic model of the pipetting operation, and the pressure model is corrected by the experimental method. Through the pipetting process pressure and pressure of the first derivative of real-time monitoring, the use of segmentation of the double threshold method as pipetting fault evaluation criteria, and the pressure sensor data are processed by Kalman filtering, the accuracy of fault diagnosis is improved. When there is a fault, the pipette tip image is collected through the camera, extract the boundary of the liquid region by the background contrast method, and obtain the liquid volume in the tip according to the geometric characteristics of the pipette tip. The pipette deviation feedback to the automatic pipetting module and deviation correction is carried out. The titration test results show that the combination of the segmented pipetting kinematic model of the double threshold method of pressure monitoring, can effectively real-time judgment and classification of the pipette fault. The method of closed-loop adjustment of pipetting volume can effectively improve the accuracy and reliability of the pipetting system.

Development of an integrated method of concentration and immunodetection of bacteria.

The microbial quality of water is a key aspect to avoid environmental and public health problems. The low pathogen concentration needed to produce a disease outbreak makes it essential to process large water volumes and use sensitive and specific methods such as immunoassays for its detection. In the present work, we describe the development of a device based on microfiltration membranes to integrate the concentration and the immunodetection of waterborne bacteria. A microfiltration membrane treatment protocol was designed to reduce the non-specific binding of antibodies, for which different blocking agents were tested. Thus, the proof of concept of the microbial detection system was also carried out using Escherichia coli as the bacterial pathogen model. E. coli suspensions were filtered through the membranes at 0.5 mL s-1, and the E. coli concentration measurements were made by absorbance, at 620 nm, of the resultant product of the enzymatic reaction among the horseradish peroxidase (HRP) bonded to the antibody, and the substrate 3,3',5,5'-tetramethylbenzidine (TMB). The results showed that the homemade concentration system together with the developed membrane treatment protocol is able to detect E. coli cells with a limit of detection (LoD) of about 100 CFU in 100 mL. Graphical abstract Scheme of the integrated method of concentration and immunodetection of bacteria.

Testing the performance of a prototype lateral flow device using bronchoalveolar lavage fluid for the diagnosis of invasive pulmonary aspergillosis in high-risk patients.

The diagnosis of invasive pulmonary aspergillosis (IPA) increasingly relies on non-culture-based biomarkers in bronchoalveolar lavage (BAL) fluid. The Aspergillus lateral flow device (LFD) is a rapid immunoassay that uses a novel Aspergillus monoclonal antibody to gain specificity. The objective of the study is to compare specificity and sensitivity of the prototype LFD and the galactomannan (GM) enzyme immunoassay in BAL fluid in high-risk patients. A total of 114 BAL samples from 106 patients at high risk for IPA were studied: 8 patients had proven/probable IPA, 16 had possible IPA and 82 did not have IPA. In patients with proven/probable IPA, specificity of LFD was 94% and GM was 89%; sensitivity of LFD was 38% and GM was 75%. Negative predictive value (NPV) for LFD was 94% and for GM was 98%; positive predictive value (PPV) was 38% for both tests. The use of anti-mould prophylaxis did not affect specificity but resulted in decreased NPV of both LFD and GM. Union and intersection analysis showed no improvement in the performance by using both tests. Among patients at risk for IPA, the diagnostic performance of LFD and GM in BAL fluid appears comparable; specificity is high, but sensitivity of both LFD and GM is poor.