Direct Antigen Detection Research Articles

SERS-Temperature Dual-Mode T-type Lateral Flow Strip for Accurate Detection of Free and Total Prostate-Specific Antigens in Blood.

Accurate point-of-care (POC) analysis of cancer markers is the essence in the comprehensive early screening and treatment of cancer. Dual-mode synchronous detection is one of the effective approaches to reduce the probability of false negatives or false positives. As a result, this can greatly improve the accuracy of diagnosis. In this work, a surface-enhanced Raman scattering (SERS)-temperature dual-mode T-type lateral flow strip was fabricated to direct and simultaneous POC detection of total and free prostate-specific antigens (t-PSA and f-PSA) in blood. With the advantage of high stability of T-type lateral flow strip and simultaneous acquirement of assay results for t-PSA and f:t PSA ratio, the proposed method has high accuracy in the diagnosis of prostate cancer, especially in the diagnostic gray zone between 4.0 and 10.0 ng/mL. The SERS-temperature dual-signal has a good linear correlation with either f-PSA or t-PSA. To evaluate the clinical diagnostic performance of the proposed method, spiked human serum samples and the whole blood sample were analyzed. The assay results showed good recovery, and compared with traditional electrochemiluminescence immunoassay (ECLIA) method (t-PSA: 43.151; f/t ratio: 0.08), the results obtained by the proposed method were similar (t-PSA: 40.15 (SERS), 36.21 (temperature); f/t ratio: 0.08 (SERS), 0.08 (temperature), but the detection time (15 min) and cost ($0.05) had been greatly reduced. Therefore, the proposed SERS-temperature synchronous dual-mode T-type lateral flow strip has a strong application potential in the field of accurate large-scale diagnostics of prostate cancer on-site by simultaneous POC detection of t-PSA and f-PSA in blood.

Copper-free click chemistry assisted antibodies for immunodetection of interleukin-10 in saliva

Interleukin-10 (IL-10) is an anti-inflammatory cytokine that is secreted in response to an acute phase inflammation in patients who are suffering from heart failure (HF). The aim of this work was to develop an electrochemical biosensor for determining salivary IL-10 levels. Biofunctionalization strategy was improved through the use of copper-free click chemistry for the developed sensor due to its advantages, leading to high quantitative yields of stable triazoles, rapid reaction, no cytotoxic Cu(I) catalyst requirement, and high specificity of cyclooctynes toward azides. The approach involved in binding of dibenzocyclooctyne acid (DBCO-COOH) to thiol-azide assembled gold microelectrodes, later capturing the monoclonal IL-10 antibody (IL-10 mAb), and ultimately allowing direct detection of IL-10 antigen. Fourier transform infrared spectroscopy (FTIR) and nanoplotter associated with fluorescence microscopy methods have been employed to analyze and prove the biofunctionalization of the gold microelectrodes. Moreover, the electrochemical impedance spectroscopy (EIS) technique was used for detecting IL-10 antigen. The developed immunosensor showed a semi-logarithmic linear range, from 0.1 pg/mL to 5 pg/mL with R2 = 0.9815 and a limit of quantitation (LOQ) of 0.1 pg/mL with relative standard deviation (RSD) of 10.67%. The specificity of the immunosensor was evaluated using an inflammatory cytokine, and none of it generated detectable EIS signals. Finally, the successful analysis of saliva samples from a healthy volunteer without Coronavirus (COVID-19) infection demonstrated the usefulness of the developed immunosensor.

From the lab to the field: handheld surface enhanced Raman spectroscopy (SERS) detection of viral proteins.

Translating sensors from the lab benchtop to a readily available point-of-need setting is desirable for many fields, including medicine, agriculture, and industry. However, this transition generally suffers from loss of sensitivity, high background signals, and other issues which can impair reproducibility. Here we adapt a label-free surface-enhanced Raman spectroscopy (SERS) sensor for SARS-CoV-2 antigens from a lab-based assay to a handheld device. Utilizing a peptide capture molecule, which we previously employed for a surface-based assay, we optimize a simpler and more cost-efficient nanoparticle-based assay. This new assay allows for the direct detection of these viral antigens by SERS, now with the advantages of robustness and portability. We highlight considerations for nanoparticle modification conditions and warn against methods which can interfere with accurate detection. The comparison of these two assays will help guide further development of SERS-based sensors into devices that can be easily used in point-of-care settings, such as by emergency room nurses, farmers, or quality control technicians.

An ultrasensitive fluorescence aptasensor for SARS-CoV-2 antigen based on hyperbranched rolling circle amplification

Sensitive and accurate diagnosis of SARS-CoV-2 infection at early stages can help to attenuate the effects of the COVID-19. Compared to RNA and antibodies detection, direct detection of viral antigens could reflect infectivity more appropriately. However, it is still a great challenge to construct a convenient, accurate and sensitive biosensor with a suitable molecular recognition element for SARS-CoV-2 antigens. Herein, we report a HRCA-based aptasensor for simple, ultrasensitive and quantitative detection of SARS-CoV-2 S1 protein and pseudovirus. The aptamer sequence used here is selected from several published aptamers by enzyme-linked oligonucleotide assay and molecular docking simulation. The sensor forms an antibody-target-aptamer sandwich complex on the surface of microplates and elicits HRCA for fluorescent detection. Without complicated operations or special instruments and reagents, the aptasensor can detect S1 protein with a LOD of 89.7 fg/mL in the linear range of 100 fg/mL to 1 μg/mL. And it can also detect SARS-CoV-2 spike pseudovirus in artificial saliva with a LOD of 51 TU/μL. Therefore, this simple and ultrasensitive aptasensor has the potential to detect SARS-CoV-2 infection at early stages. It may improve the timeliness and accuracy of SARS-CoV-2 diagnosis and demonstrate a strategy to conduct aptasensors for other targets.

Opinion among veterinarians on equine Lyme disease - results of a questionnaire study

Recording equine veterinarians opinion on equine Lyme disease. A survey was conducted to get a general impression of the opinion of German equine vets on the topic of equine Lyme Borreliosis. The main focus of this questionnaire study was set on the diagnostic and therapeutic approach of the horse vets. Overall almost half of the veterinarians (48 %) detected only rarely or never serological antibodies against Bb , 47 %found occasionally and 5 % frequently Borrelia antibodies within their clientel.Concerning the existence of clinical LB in horses, 51 % of the veterinarians reported clinical cases, 43 % thought it might be possible but have not seen cases themselves and 6 % categorically ruled out the existence.On average, further diagnostic measures for LB diseases were initiated 15 ± 9 times per year and 1.4 ± 1.0 cases of equine LB diseases were diagnosed per year. In the majority of 68 % of the cases, the veterinarians chose serological tests as diagnostic measures, followed by a combination of serological tests and Antigen detection tests (27 %) and only 5 % of the veterinarians were using Antigen detection tests exclusively. Blood and skin biopsies were submitted most frequently for direct Antigendetection. The treatment of horses with positive laboratory results indicating Lyme disease and clinical symptoms assessed as suitable was carried out by 93 % of the veterinarians with antibiotics and 7% with homeopathy. Doxycycline (68 %) was used most frequently. The antibiotic treatment was applied over a period of 3 (31 %) to 4 (63 %) weeks.Regarding the question of a possible vaccination, 39 % of veterinarians do not offer vaccination against Lyme borreliosis, 20 % have not yet used it, but do not categorically reject it, and 41 % regularly carry out vaccinations against Lyme borreliosis. The opinion on the clinical relevance of Equine Lyme Borreliosis among german equine veterinarians vary considerably. Uniform diagnostic guidelines and, possibly, recommendations for therapy would serve to counteract the existing uncertainty concerning this disease within the equine veterinary community.

Risk of Developing Asthma After Lower Respiratory Tract Infections with Respiratory Syncytial Virus During Childhood

Background: Recent studies indicate causal relationship between infection by respiratory syncytial virus (RSV) and bronchial asthma. We evaluated the incidence of bronchial asthma in children with RSV positive infection early in their childhood in a nation-wide cohort study. Methods: Children (aged between one month and 15 years) were evaluated for the presence of RSV infection when they presented with one or more acute respiratory tract infection symptoms (fever, cough, cold and wheezing) in a major tertiary care hospital in the Kingdom of Bahrain during a period of seven years. RSV detection was done using nasopharyngeal secretion (NPS) samples by direct antigen detection immunofluorescence technique. Number of children who were later diagnosed with asthma was recorded. Serum IgE levels were estimated. Risk of developing bronchial asthma is represented using relative risk (RR) [95% CI]. Children with asthma without prior RSV infection from the same population formed the historical control. Results: A total of 3782 children diagnosed with respiratory tract infection were recruited. We observed that RSV infection at younger age (during infancy) and severe infection were significantly associated with asthmatic episodes RR [95% CI]: 7 [5.5, 8.2]. Additionally, asthmatics with prior RSV infection had significantly higher total IgE levels (167 ± 37 IU/ml) compared to those without RSV infection (92 ±17 IU/ml). Mean (SD) age of children developing asthma with prior RSV infection was 0.7 (0.42) years compared to the historical control [6.8 (3.8) years] and was statistically significant. Conclusion: Infants with RSV infection have an increased risk of developing bronchial asthma later in the childhood. The more severe the RSV infection, the greater is the severity of bronchial asthma as indicted by serum IgE levels. Asthma in children with RSV infection occurs at much younger age compared to those without RSV infection.

Capacitive Aptasensor Coupled with Microfluidic Enrichment for Real-Time Detection of Trace SARS-CoV-2 Nucleocapsid Protein.

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has lasted for almost 2 years. Stemming its spread has posed severe challenges for clinical virus detection. A long turnaround time, complicated operation, and low accuracy have become bottlenecks in developing detection techniques. Adopting a direct antigen detection strategy, we developed a fast-responding and quantitative capacitive aptasensor for ultratrace nucleocapsid protein detection based on a low-cost microelectrode array (MEA) chip. Employing the solid–liquid interface capacitance with a sensitivity of picofarad level, the tiny change on the MEA surface can be definitively detected. As a result, the limit of detection reaches an ultralow level of femtogram per milliliter in different matrices. Integrated with efficient microfluidic enrichment, the response time of this sensor from the sample to the result is shortened to 15 s, completely meeting the real-time detection demand. Moreover, the wide linear range of the sensor is from 10–5 to 10–2 ng/mL, and a high selectivity of 6369:1 is achieved. After application and evaluation in different environmental and body fluid matrices, this sensor and the detection method have proved to be a label-free, real-time, easy-to-operate, and specific strategy for SARS-CoV-2 screening and diagnosis.

Nonmetallic SERS-based immunosensor byintegrating MoS2 nanoflower and nanosheet towards the direct serum detection of carbohydrate antigen 19-9

Recently, nonmetallic substrates have stimulated great interest in surface-enhanced Raman scattering (SERS)-based immunoassay owing to their good uniformity, stability, and biocompatibility. In this context, a novel nonmetallic SERS-based immunoassay mediated by two-dimensional molybdenum disulfide (MoS2) was delivered for the sensitive and specific monitoring of carbohydrate antigen 19–9 (CA19-9). The effective enrichment of molecules on the large active surfaces of MoS2 as well as potential 532-nm laser-induced charge transfer resonances between them engendered desirable enhancement factor values at the level of 105. Intriguingly, a sandwich immunocomplex combined MoS2 nanoflower and nanosheet exhibited not only a wide linear range from 5 × 10−4 to 1 × 102 IU·mL−1 but also a limit of detection as low as 3.43 × 10−4 IU·mL−1 towards CA19-9. More meaningful, the analytical result for clinical patient serum sample was basically compared with the conventional chemiluminescent immunoassay. Such a novel nonmetallic SERS-based immunosensor with desirable biocompatibility and sensitivity is promising for clinical diagnosis.

A new antigen detection ELISA for the diagnosis of Strongyloides infection

Serodiagnosis is an essential component of the laboratory diagnosis of Strongyloides infection and is usually performed using an indirect IgG antibody test. A direct antigen detection method can complement the IgG assay, particularly for detecting early infection and post-treatment follow-up. In the present study, a recombinant scFv monoclonal antibody against NIE recombinant protein (rMAb23) that we had previously produced was used to develop a Strongyloides antigen detection ELISA (SsAg-ELISA). The assay is based on detecting immune complexes of circulating NIE antigens bound to Strongyloides-specific IgG antibodies. The optimized ELISA parameters were 10 µg/mL of rMAb23 coated on microtitre plate wells, 2% skim milk as blocking reagent, 1:100 serum dilution, and 1:1000 goat anti-human IgG F(ab’)2 conjugated to horseradish peroxidase. Four groups of serum samples were used, i.e., Strongyloides-positive serum samples categorized into Groups IA and IB; the former were from probable chronic infections and the latter from probable early/acute infections. Strongyloides-negative samples comprising Groups II (healthy samples) and III (other infections); the latter were from eleven different types of other parasitic infections. The receiver operating characteristic (ROC) curve showed an area under the curve (AUC) of 1.00, cut-off optical density (OD405) of 0.5002, and 100% diagnostic sensitivity and specificity. The results of the commercial IgG-ELISA and SsAg-ELISA from Group IA were found to be moderately correlated (r = 0.416; p < 0.05). Notably, ANOVA showed that the average ODs405 of Group 1B were significantly higher (p < 0.05) than Group 1A, indicating that the assay may be useful to differentiate early and chronic infection. In conclusion, the developed SsAg-ELISA showed good diagnostic potential, and it merits further evaluation.

Detection of Mycobacterium tuberculosis serum biomarkers and the relation with previous BCG vaccination

Objectives: Accurate diagnosis of tuberculosis (TB) disease is a cornerstone in the management of disease spread and initiation of proper treatment. Detection of Mycobacterium tuberculosis (MTB) antigens in biological fluids might be a reliable way to directly diagnose TB disease. The aim of the current study is to investigate selected TB antigens as potential diagnostic serum biomarkers. Methods: We have explored the detection of three MTB antigens, namely Rv2324, Rv2435c and Rv1284, in serum specimens from confirmed pulmonary TB patients. In addition, serum specimens from Bacillus Calmette-Guerin (BCG)-vaccinated healthy adults and BCG-unvaccinated infants were also tested as negative controls. The three antigens were recombinantly expressed in Escherichia coli and purified using affinity chromatography. Murine antibodies against the three antigens were raised by mice immunization with corresponding recombinant antigens. TB antigen detection in the serum specimens was carried out by the dot blot test. Results: Serum samples from TB patients showed significant reactivity (94.3-100%) with the antigen-specific antibodies. Surprisingly, the three antigens were also detectable in serum specimens (93.1-100%) from healthy adults. On the contrary, the three antigens were barely detectable in sera from BCG-unvaccinated infants (0-14%). Conclusion: These finding, supported by recent literature, highlight the significant interference of BCG vaccination with the diagnosis of TB disease by direct antigen detection. Nevertheless, the three tested antigens could represent useful TB serum biomarkers in countries where BCG vaccination is not routinely performed.

Immunodominant regions prediction of nucleocapsid protein for SARS-CoV-2 early diagnosis: a bioinformatics and immunoinformatics study

ABSTRACT COVID-19 caused by SARS-CoV-2 is sweeping the world and posing serious health problems. Rapid and accurate detection along with timely isolation is the key to control the epidemic. Nucleic acid test and antibody-detection have been applied in the diagnosis of COVID-19, while both have their limitations. Comparatively, direct detection of viral antigens in clinical specimens is highly valuable for the early diagnosis of SARS-CoV-2. The nucleocapsid (N) protein is one of the predominantly expressed proteins with high immunogenicity during the early stages of infection. Here, we applied multiple bioinformatics servers to forecast the potential immunodominant regions derived from the N protein of SARS-CoV-2. Since the high homology of N protein between SARS-CoV-2 and SARS-CoV, we attempted to leverage existing SARS-CoV immunological studies to develop SARS-CoV-2 diagnostic antibodies. Finally, N229-269, N349-399, and N405-419 were predicted to be the potential immunodominant regions, which contain both predicted linear B-cell epitopes and murine MHC class II binding epitopes. These three regions exhibited good surface accessibility and hydrophilicity. All were forecasted to be non-allergen and non-toxic. The final construct was built based on the bioinformatics analysis, which could help to develop an antigen-capture system for the early diagnosis of SARS-CoV-2.

Tailored Biofunctionalized Biosensor for the Label-Free Sensing of Prostate-Specific Antigen.

The increase in the demand and popularity of smart biosensors has brought a novel and innovative concept to develop a diverse range of semen mutual biomarker (i.e., prostate-specific antigen, PSA)-based biodevices for our daily life applications. Using a versatile strategy, here we have developed a next-generation miniaturized capacitive biomarker-based sensor, which facilitates a direct, rapid quantitation and ultrafast detection of prostate-specific antigen (PSA) selectively. To fabricate an affordable PSA biosensor, an interdigitated capacitor (IDC) was functionalized and to detect PSA at concentrations varying from 0.1 to 10 μL/mL, with a response time of 3 s. Moreover, the PSA biosensor showed a high level of selectivity due to the successful probing of the capacitive response-generated biomolecular interactions using external stimuli at the bioelectrode. The resulting IDC-based PSA biosensors are capable of excellent reproducibility and reusability, which are required for real-time biosensing of any targeted biomolecules where low-concentration detection is a key for point-of-care, on-site sensing applications. We anticipate that this research could open exciting opportunities for PSA detection at a low concentration level.

Study of the diagnostic value of interleukin-6 and interleukin-8 in children with acute gastroenteritis.

Gastroenteritis in children is responsible for high morbidity and mortality. Our aim was to determine the serum and fecal levels of interleukin-6 (IL-6) and interleukin-8 (IL-8) in children with acute gastroenteritis of viral and bacterial etiology to assess their utility as diagnostic biomarkers for these infections. In this case-control study, the children were classified according to the pathogen recovered from the stool by bacterial culture or by direct viral antigen detection by enzyme immunoassay (EIA) into 50 children with acute bacterial gastroenteritis and 50 children with acute viral gastroenteritis. In addition, 50 apparently healthy children were included as a control group. Blood and stool samples were subjected to detection of IL-6 and IL-8. There were statistically significant elevations of total leucocytes counts, absolute neutrophils count, C-reactive protein, serum IL-6 and serum IL-8 in children with gastroenteritis compared to healthy children (p<0.001). CRP, serum IL-6 and IL-8 had significantly elevated levels in children with bacterial gastroenteritis compared to viral gastroenteritis. Fecal IL-6 and IL-8 had significantly elevated levels in children with acute gastroenteritis than in healthy control (p<0.001). The area under the curve (AUC) showed that CRP and serum IL-6 could be used as discriminative markers for acute bacterial gastroenteritis in children, in comparison to serum IL-8. Elevated serum IL-6 and CRP can aid in differentiation between viral and bacterial gastroenteritis. Serum IL-8 had limited discrimination ability between viral and bacterial gastroenteritis. Stool levels of IL-6 and IL-8 were elevated in children with viral and bacterial gastroenteritis, however, their assessment by enzyme linked immunosorbent assay had technical limitations to be used as differentiation biomarkers.

Molecular Study of Norovirus in Pediatric Patients with Gastroenteritis

Aim:The aim of the present study was to detect the prevalence of norovirus and genotypes determination by real-time PCR among children below 18 years as an etiology of acute gastroenteritis and to compare rapid detection of norovirus by Enzyme-Linked Immunoassay (ELISA) to virus detection by real-time PCR.Methods:The research was a cross-sectional study conducted on children below 18 years complaining of community-acquired acute gastroenteritis. A stool sample was subjected to direct-antigen detection by ELISA for norovirus and molecular study by real-time polymerase chain reaction.Results:The study included 200 children with acute gastroenteritis with a mean age of 6.7±3.8 years. Norovirus antigen was detected by EIA in 34.5% and by real-time PCR in 30.5% of studied children with genotype GII, the predominant detected genotype (80.97%). Both real-time PCR and antigen detection of norovirus were positive in 43 (70.5%) of the children and negative in 113(81.3%) of the studied children. The sensitivity, specificity, positive predictive value, negative predictive value and accuracy for antigen detection by ELISA were 70.5%, 81.3%, 62.3%, 86.3% and 78%, respectively. Comparison between patients positive for norovirus and those negative for norovirus by real-time PCR revealed non-significant difference as regards age, sex, the season of occurrence and residence.Conclusion:The present study highlights that norovirus prevalence is common among pediatric patients with gastroenteritis above 5 years with GII genotype as the prevalent genotype. There was a significant correlation between positive and negative results of antigen detection of norovirus by ELISA and detection of RNA of norovirus by real-time PCR in stool samples. However, the screening for norovirus by ELISA has limited sensitivity and needs to be associated with a molecular method for accurate diagnosis of sporadic cases of gastroenteritis.

The Evolving Field of Cytopathology and Its Expanding Role in Pathologic Practice.

Cytopathology is one of the most rapidly developing fields in pathology, with significant impact on the diagnostic practice of all organ systems. Its impressive progress not only relies on its minimally invasive nature and cost-effectiveness, but also stems from the clear relationship of its diagnostic categories with risk of malignancy and clinical management, as well as the successful integration of molecular/genetic tests into daily practice. With the increasing clinical demand for less tissue and more specific diagnoses, cytopathology will lead future pathologic development in years to come. In this special section, we capture selected topics in recent cytopathologic progress, including both original studies and timely reviews.One of the most important breakthroughs in cytopathology in 2018 was the establishment and publication of the Milan System for Reporting Salivary Gland Cytopathology.1 The Milan system is based on the studies of many institutions around the globe. In this series, we publish the original article from Temple University (Philadelphia, Pennsylvania) by Luo et al describing their logistics and efforts to build an institutional 6-tier classification system to report salivary gland cytopathology. Slides of 107 salivary gland fine-needle aspirations (FNAs) with subsequent histology slides were reviewed and reclassified using the 6-tier system. The FNA diagnoses were generally consistent with histologic diagnoses for the disease spectrum. The sensitivity, specificity, positive predictive value, and negative predictive value for diagnosing malignancies with this system were 86% (12 of 14), 93% (40 of 43), 80% (12 of 15), and 95% (40 of 42), respectively. The risk of malignancy increased from 0% (0 of 13) for negative for neoplasm to 7% (2 of 29) for benign neoplasm, 67% (2 of 3) for suspicious for malignancy, and 83% (10 of 12) for positive for malignancy. This study points out the rationale and helps to pave the way for the adoption of the Milan System for Reporting Salivary Gland Cytopathology.Cytopathologic materials are a great source for molecular tests critical to guide therapeutic decision-making and accurate diagnoses.2 Doxtader et al describe their experience at the Cleveland Clinic with epidermal growth factor receptor (EGFR) mutation testing by next-generation sequencing and anaplastic lymphoma kinase (ALK) gene rearrangement testing by fluorescence in situ hybridization (FISH) in non–small cell lung carcinomas. At their institution, liquid-based cytology specimens are the primary resource used for molecular testing. In most instances, adequate DNA could be extracted from the residual cell pellets for next-generation sequencing, and ThinPrep slides could be used reliably for FISH testing for ALK gene rearrangements. In occasional cases where the cell pellet material was not adequate for molecular testing, cell blocks and/or surgical pathology specimens were secondary options. In a different original study using an immunocytologic approach, Ren and Klump showed that 87.6% of carcinomas of Müllerian origin in ascites and pleural fluids were positive for PAX8; 19.2%, 18.1%, 23.4%, and 8.5% of high-grade serous carcinomas were positive for GATA3, SOX2, uroplakin II, and SALL4, respectively. Increasing acceptance of molecular tests into cytopathologic practice is also highlighted by a recent recommendation from the International Mesothelioma Interest Group to conduct p16 (by FISH) and BAP1 (by immunohistochemistry) staining in cytologic specimens suspicious for mesothelioma.3The diagnostic power of cytopathology for infectious disease in the lower respiratory tract is highlighted by Baldassarri et al in this series. Cytomorphologic analysis helps to triage and plays a critical supporting role to the microbiology laboratory in prompt and accurate identification of causative organisms, particularly in immunosuppressed patients. Again, cytopathologic materials are perfect for molecular tests including polymerase chain reaction, immunochromatographic assay, and direct antigen detection in identification of bacteria, viruses, and fungal elements.Tremendous progress has been made in the past few years in cytopathology, from the standardized reporting system to FNA sample techniques to molecular tests of tumor-specific markers and specific infectious agents. The continued improvement of cytopathologic practice will undoubtably expand its role in the clinical management of patients.

Respiratory syncytial and influenza virus detecting rapid tests in children younger than 5 years of age in Armenia.

Acute respiratory infections (ARIs) are major causes of morbidity in early childhood. They are mainly caused by viruses, including influenza (INF) and respiratory syncytial viruses (RSV). We aimed to investigate the role of RSV and INF in children hospitalized for ARIs and to show the impact of RSV/INF rapid testing on management of patients. Cross-sectional study using data of inpatient care of children younger than five years hospitalized in Arabkir Medical Center due to ARI from November 1, 2013 to April 1, 2014. Nasopharyngeal swabs were tested for RSV and INF types A and B by direct antigen detection tests. A total of 915 patients, 583 (63.7%) boys and 332 (36.3%) girls were included in the study with the mean age of 18.8 ± 16.3 months. Among them, 390 (42.6%) were tested positive, 3 (0.3%) subjects tested positive both for RSV and INF: 269 (29.4%) for RSV and 124 (13.6%) for INF (A - 121, B - 3). Out of 915 children, 209 (23%) were pretreated with antibiotics, most often with oral amoxicillin/clavulanic acid (n = 54, 25.8%), sulfamethoxazole/trimethoprim (n = 46, 22%), and amoxicillin (n = 38, 18.2%), followed by intramuscular ceftriaxone (n = 37, 17.7%). The usage of antigen tests for detection of respiratory viruses allowed to document high rates of RSV and INF in children admitted to the hospital. In settings where polymerase chain reaction method is not readily available, implementation of rapid tests for detection of respiratory viruses is important in the management of pediatric patients including cohorting and more targeted use of antibiotics.

Experimental study of an evanescent-field biosensor based on 1D photonic bandgap structures.

A photonic bandgap (PBG) biosensor has been developed for the label-free detection of proteins. As the sensing in this type of structures is governed by the interaction between the evanescent field going into the cladding and the target analytes, scanning near-field optical microscopy has been used to characterize the profile of that evanescent field. The study confirms the strong exponential decrease of the signal as it goes into the cladding. This means that biorecognition events must occur as close to the PBG structure surface as possible in order to obtain the maximum sensing response. Within this context, the PBG biosensor has been biofunctionalized with half-antibodies specific to bovine serum albumin (BSA) using a UV-induced immobilization procedure. The use of half-antibodies allows one to reduce the thickness of the biorecognition volume down to ca. 2.5 nm, thus leading to a higher interaction with the evanescent field, as well as a proper orientation of their binding sites towards the target sample. Then, the biofunctionalized PBG biosensor has been used to perform a direct and real-time detection of the target BSA antigen.

Optimization and evaluation of a non-invasive tool for peste des petits ruminants surveillance and control

Peste des petits ruminants (PPR) is a highly contagious and devastating viral disease affecting mainly sheep and goats, but also a large number of wild species within the order Artiodactyla. A better understanding of PPR transmission dynamics in multi-host systems is necessary to efficiently control the disease, in particular where wildlife and livestock co-occur. Notably, the role of wildlife in PPR epidemiology is still not clearly understood. Non-invasive strategies to detect PPR infection without the need for animal handling could greatly facilitate research on PPR epidemiology and management of the disease in atypical hosts and in complex field situations. Here, we describe optimized methods for the direct detection of PPR virus genetic material and antigen in fecal samples. We use these methods to determine the detection window of PPR in fecal samples, and compare the sensitivity of these methods to standard invasive sampling and PPR diagnostic methods using field samples collected at a wildlife-livestock interface in Africa. Our results show that quantitative reverse transcription PCR (RT-QPCR) amplification of PPRV from fecal swabs has good sensitivity in comparison to ocular swabs. Animals infected by PPRV could be identified relatively early on and during the whole course of infection based on fecal samples using RT-QPCR. Partial gene sequences could also be retrieved in some cases, from both fecal and ocular samples, providing important information about virus origin and relatedness to other PPRV strains. Non-invasive strategies for PPRV surveillance could provide important data to fill major gaps in our knowledge of the multi-host PPR epidemiology.

Clinical Evaluation of a Blood Assay to Diagnose Paucibacillary Tuberculosis via Bacterial Antigens.

The diagnosis of active tuberculosis (TB) cases primarily relies on methods that detect Mycobacterium tuberculosis (Mtb) bacilli or their DNA in patient samples (e.g., mycobacterial culture and Xpert MTB/RIF assays), but these tests have low clinical sensitivity for patients with paucibacillary TB disease. Our goal was to evaluate the clinical performance of a newly developed assay that can rapidly diagnose active TB cases by direct detection of Mtb-derived antigens in patients' blood samples. Nanoparticle (NanoDisk)-enriched peptides derived from the Mtb virulence factors CFP-10 (10-kDa culture factor protein) and ESAT-6 (6-kDa early secretory antigenic target) were analyzed by high-throughput mass spectrometry (MS). Serum from 294 prospectively enrolled Chinese adults were analyzed with this NanoDisk-MS method to evaluate the performance of direct serum Mtb antigen measurement as a means for rapid diagnosis of active TB cases. NanoDisk-MS diagnosed 174 (88.3%) of the study's TB cases, with 95.8% clinical specificity, and with 91.6% and 85.3% clinical sensitivity for culture-positive and culture-negative TB cases, respectively. NanoDisk-MS also exhibited 88% clinical sensitivity for pulmonary and 90% for extrapulmonary TB, exceeding the diagnostic performance of mycobacterial culture for these cases. Direct detection and quantification of serum Mtb antigens by NanoDisk-MS can rapidly and accurately diagnose active TB in adults, independent of disease site or culture status, and outperform Mycobacterium-based TB diagnostics.

Preparation of a Latex Agglutination Test (LAT) as A Rapid Direct and Indirect Diagnostic Tool for Detecting Bovine Viral Diarrhea Virus (BVDV) in Cattle

Bovine viral diarrhea virus is a worldwide disease in cattle populations. Carboxylated polystyrene microspheres (0.8 nm) beads (latex) were coated using a passive adsorption method with BVDV (the Nadel strain) for the rapid indirect detection of antibodies (Abs) and with reference anti-BVDV for rapid direct detection of antigen (Ag) in cattle with reproductive disorders. Latex agglutination test (LAT) was compared with indirect fluorescent antibody technique (IFAT) and indirect enzyme linked immunosorbent assay (IELISA) by using 202 serum samples from cows suffering from reproductive disorders, apparently healthy in- contact cows and two healthy cows previously examined as negative controls and 21 liver tissue biopsies from aborted foetuses. The results show that the antibody-coated beads (Ab.c.b.) detected BVDV to 10 TCID50% and that the antigen-coated beads (Ag.c.b.) detected with antibodies to 1/128. In addition, the specificity and sensitivity were 94% and 97.97%, respectively, for Ag.c.b. and 90.9% and 90% ,respectively, for Ab.c.b. Our study confirm that the LAT is a simple, safe, specific, sensitive test and it remains an economic (low cost), accurate and rapid field test for the diagnosis of bovine viral diarrhea virus in cows as other routine serological tests IFAT and IELISA.