Rapid Identification Of Infectious Agents Research Articles

An Overview of Epidemiology and Diagnostic Techniques for Emerging and Re-emerging Viral Infections

In the past decades, the prevalence of emerging viral infections has escalated and is envisaged to continue to grow in the foreseeable future. More than 17 million people die every year from infectious diseases. The most deadly diseases known to humans so far are caused by emerging and re-emerging viruses such as Influenza, Chikungunya, Ebola, HIV, and the Coronavirus disease 19 outbreak, being the most recent. The clinical prognosis of serious illnesses depends on identification of the infectious agent at the onset. This review outlines the epidemiology and diagnostic techniques used to identify viral pathogens that received particular attention in the recent years. The modern diagnostic tools that are used for identification and confirmation of these disease causing agents such as viral antigen identification, viral culture, nucleic acid analysis, and serology are discussed. While rapid identification of infectious agents, quick diagnosis, and the production of vaccines against a specific virus is possible with advanced laboratory techniques, the limited resources delay the implementation of these techniques. The present need is to understand the importance of early and proper implementation of technological advancements for mitigation of damage caused by infective agents, and the introduction of some novel and appropriate approaches on priority basis in endemic and emerging areas.

Pulse-Controlled Amplification-A new powerful tool for on-site diagnostics under resource limited conditions.

BackgroundMolecular diagnostics has become essential in the identification of many infectious and neglected diseases, and the detection of nucleic acids often serves as the gold standard technique for most infectious agents. However, established techniques like polymerase chain reaction (PCR) are time-consuming laboratory-bound techniques while rapid tests such as Lateral Flow Immunochromatographic tests often lack the required sensitivity and/or specificity.Methods/Principle findingsHere we present an affordable, highly mobile alternative method for the rapid identification of infectious agents using pulse-controlled amplification (PCA). PCA is a next generation nucleic acid amplification technology that uses rapid energy pulses to heat microcyclers (micro-scale metal heating elements embedded directly in the amplification reaction) for a few microseconds, thus only heating a small fraction of the reaction volume. The heated microcyclers cool off nearly instantaneously, resulting in ultra-fast heating and cooling cycles during which classic amplification of a target sequence takes place. This reduces the overall amplification time by a factor of up to 10, enabling a sample-to-result workflow in just 15 minutes, while running on a small and portable prototype device. In this proof of principle study, we designed a PCA-assay for the detection of Yersinia pestis to demonstrate the efficacy of this technology. The observed detection limits were 434 copies per reaction (purified DNA) and 35 cells per reaction (crude sample) respectively of Yersinia pestis.Conclusions/SignificancePCA offers fast and decentralized molecular diagnostics and is applicable whenever rapid, on-site detection of infectious agents is needed, even under resource limited conditions. It combines the sensitivity and specificity of PCR with the rapidness and simplicity of hitherto existing rapid tests.

662. Identification of Clinically Relevant Microbes with the MasSpec Pen

BackgroundIn the age of antimicrobial resistance, rapid identification of infectious agents is critical for antimicrobial stewardship and effective therapy. To this end, ambient ionization mass spectrometry techniques have been applied for rapid identification of microbes directly from culture isolates. We have developed a handheld, mass spectrometry-based device, the MasSpec Pen, that permits direct molecular analysis of a biological sample in seconds (Scheme 1). Here, we employ the MasSpec Pen to identify clinically relevant microbes directly from culture isolates.Methods Staphylococcus aureus, Staphylococcus epidermidis, Group A and B Streptococcus, Kingella kingae (K.k), and Pseudomonas aeruginosa (P.a) were cultured on 5% sheep’s blood nutrient agar at 37 °C overnight. Colonies were transferred to a glass slide where they were analyzed directly with the MasSpec Pen coupled to a Q Exactive mass spectrometer (Thermo Scientific) in negative ion mode. For MasSpec Pen analysis, a 10 µL droplet of water was held in contact with the sample surface for 3 seconds and then aspirated to the mass spectrometer for analysis.Data was normalized and the molecular features resulting from the analysis solvent and nutrient medium were removed. The least absolute shrinkage and selection operator (lasso) statistical method was used to build classification models for prediction of bacterial identity. Model performance was evaluated by leave-one-out cross-validation and a validation set of samples.Scheme 1: MasSpec Pen workflow ResultsVarious small molecules were detected including metabolites and glycerophospholipid species. The mass spectral profiles for each species exhibited qualitative differences among them (Figure 1). Additionally, several quorum-sensing molecules were observed in P.a. including hydroxy-heptyl-quinoline (m/z 242.155).Lasso statistical classifiers were created to differentiate organisms at the level of Gram type, genus, and species with each model comprised of a sparse set of molecular features. Accuracies of 90% or greater were achieved for all lasso models and as high as 98% for the differentiation of Staphylococcus (Staph.) and Streptococcus (Strep.).Figure 1: Molecular profiles of species analyzed Figure 2: Statistical classification results ConclusionThese results demonstrate the potential of the MasSpec Pen as a tool for clinical analysis of infected biospecimens.DisclosuresSydney C. Povilaitis, BA, MS Pen Technologies, Inc. (Other Financial or Material Support, Patent) Livia Eberlin, PhD, MS Pen Technolpogies, Inc. (Board Member, Shareholder)

The use of laser-based diagnostics for the rapid identification of infectious agents in human blood.

To investigate the use of a laser-based method of detection as a potential diagnostic test for the rapid identification of infectious agents in human blood. In this study, the successful differentiation of blood spiked with viruses, bacteria or protozoan parasites to clinically relevant levels is demonstrated using six blood types (O+, O-, AB+, A+, A-, B+) using blood from different individuals with blood samples prepared in two different laboratories. Experiments were performed using various compositions of filters, experimental set-ups and experimental parameters for spectral capture. The potential for developing a laser-based diagnostic instrument to detect the presence of parasites, bacteria and viruses in human blood capable of providing analysis results within minutes was demonstrated. There is an ongoing need for clinical diagnostics to adapt to newly emerging agents and to screen simultaneously for multiple infectious agents. A laser-based approach can achieve sensitive, multiplex detection with minimal sample preparation and provide rapid results (within minutes). These properties along with the flexibility to add new agent detection by simply adjusting the detection programming make it a promising tool for clinical diagnosis.

A multiplex loop-mediated isothermal amplification assay for rapid screening of Acinetobacter baumannii and D carbapenemase OXA-23 gene.

Background: Acinetobacter baumannii is a health burden responsible for various nosocomial infections, and bacteremia in particular. The resistance of A. baumannii to most antibiotics including carbapenem has increased. OXA-23-producing A. baumannii is the chief source of nosocomial outbreaks with carbapenem-resistant A. baumannii. Successful antibiotic treatment relies on the accurate and rapid identification of infectious agents and drug resistance. Here, we describe a multiplex loop-mediated isothermal amplification (LAMP) assay for simultaneous and homogeneous identification for A. baumannii infection screening and drug-resistance gene detection. Methods: Four primer pairs were designed to amplify fragments of the recA gene of A. baumannii and the oxa-23 gene. The reaction with a 25 μl of final volume was performed at 63°C for 60 min. For comparative purposes, we used a traditional method of bacterial identification to evaluate assay efficacy. Results: The multiplex LAMP assay enables simultaneous and homogeneous detection of the recA gene of A. baumannii and the oxa-23 gene and requires less than 21 min with no pre-requisite for DNA purification prior to the amplification reaction. The detection is specific to A. baumannii, and the coincidence rate of the multiplex LAMP and the traditional method was 100%. Conclusions: Our data indicate that the multiplex LAMP assay is a rapid, sensitive, simultaneous and homogeneous method for screening of A. baumannii and its drug-resistance gene.

Biosafety and biosecurity in veterinary laboratories

With recent outbreaks of Middle East respiratory syndrome coronavirus (MERS-CoV), anthrax, Nipah and the highly pathogenic avian influenza virus, much emphasis has been placed on the rapid identification of infectious agents globally. As a result, laboratories are building capacity, conducting more advanced and sophisticated research, increasing their staff, and establishing reference collections of dangerous pathogens in an attempt to reduce the impact of infectious disease outbreaks and to characterise disease-causing agents. With this expansion, the global laboratory community has started to focus on laboratory biosafety and biosecurity in order to prevent the accidental and/or intentional release of these agents. Laboratory biosafety and biosecurity systems are used around the world to help to mitigate the risks posed by dangerous pathogens in the laboratory. Veterinary laboratories carry unique responsibilities with regard to workers and communities to handle disease-causing microorganisms safely and securely. Many microorganisms studied in veterinary laboratories not only infect animals, but also have the potential to infect humans. This paper will discuss the fundamentals of laboratory biosafety and biosecurity.

Polymerase-chain reaction/electrospray ionization-mass spectrometry for the detection of bacteria and fungi in bronchoalveolar lavage fluids: a prospective observational study

PLEX-ID uses polymerase chain reaction-electrospray ionization/mass spectrometry for rapid identification of infectious agents in clinical samples. We evaluated its concordance with our centre's standard methods (SM) for bacterial and fungal detection in bronchoalveolar lavage (BAL) fluid in a prospective observational cohort study. The primary outcome was concordance (%) between SM and PLEX-ID. Secondary outcomes included concordance when excluding commensal oral flora, detection of resistance genes, and PLEX-ID's potential impact on clinical management, as determined by two independent reviewers. Included were 101 specimens from 94 patients. BALs were performed primarily for suspected pneumonia (76/101, 75%) and lung transplant work-ups (12/101, 12%). Most specimens yielded at least one organism by either method (92/101, 91%). Among all microorganisms detected (n = 218), 83% and 17% were bacterial and fungal, respectively. Overall concordance between SM and PLEX-ID was 45% (45/101). Concordance increased to 66% (67/101) when discordance for commensal flora was excluded. PLEX-ID failed to detect 21% of all 183 SM-identified organisms, while SM did not identify 28% of the 191 PLEX-ID-identified organisms (p <0.001). There was low concordance for mecA detection. Two infectious-disease specialists' analyses concluded that in most of the 31 discordant, non-commensal cases, PLEX-ID results would have had little or no impact on patient management; in eight cases, however, PLEX-ID would have led to ‘wrong decision-making’. The tested version of PLEX-ID concurred weakly with standard methods in the detection of bacteria and fungi in BAL specimens, and is not likely to be useful as a standalone tool for microbiological diagnosis in suspected respiratory infections.

A rapid method for breath analysis in cystic fibrosis patients.

For easy handling and speed of lung diseases diagnostics, approaches based on volatile organic compounds (VOCs), including those emitted by pathogenic microorganisms, are considered but currently require considerable sampling efforts. We tested whether easy-to-handle and fast detection of lung infections is possible using solid-phase microextraction (SPME) of 100ml of exhaled breath. An analytical procedure for the detection of VOCs from the headspace of epithelial lung cells infected with four human pathogens was developed. The feasibility of this method was tested in a cystic fibrosis (CF) outpatient clinic in vivo. Exhaled breath was extracted by SPME and analyzed by gas chromatography-mass spectrometry (GC-MS). The compositions of VOCs released in the infection model were characteristic for all individual pathogens tested. Exhaled breath of CF patients allowed clear distinction of CF patients and controls by their VOC compositions using multivariate analyses. Interestingly, the major specific VOCs detected in the exhaled breath of infected CF patients in vivo differed from those monitored during bacterial in vitro growth. SPME extraction of VOCs from 100ml of human breath allowed the distinction between CF patients and healthy probands. Our results highlight the importance of assessing the entire pattern of VOCs instead of selected biomarkers for diagnostic purposes, as well as the need to use clinical samples to identify reliable biomarkers. This study provides the proof-of-concept for the approach using the composition of exhaled VOCs in human breath for the rapid identification of infectious agents in patients with lower respiratory tract infections.

Ambient temperature detection of PCR amplicons with a novel sequence-specific nucleic acid lateral flow biosensor

In the field of diagnostics, molecular amplification targeting unique genetic signature sequences has been widely used for rapid identification of infectious agents, which significantly aids physicians in determining the choice of treatment as well as providing important epidemiological data for surveillance and disease control assessment. We report the development of a rapid nucleic acid lateral flow biosensor (NALFB) in a dry-reagent strip format for the sequence-specific detection of single-stranded polymerase chain reaction (PCR) amplicons at ambient temperature (22–25°C). The NALFB was developed in combination with a linear-after-the-exponential PCR assay and the applicability of this biosensor was demonstrated through detection of the cholera toxin gene from diarrheal-causing toxigenic Vibrio cholerae. Amplification using the advanced asymmetric PCR boosts the production of fluorescein-labeled single-stranded amplicons, allowing capture probes immobilized on the NALFB to hybridize specifically with complementary targets in situ on the strip. Subsequent visual formation of red lines is achieved through the binding of conjugated gold nanoparticles to the fluorescein label of the captured amplicons. The visual detection limit observed with synthetic target DNA was 0.3ng and 1pg with pure genomic DNA. Evaluation of the NALFB with 164 strains of V. cholerae and non-V. cholerae bacteria recorded 100% for both sensitivity and specificity. The whole procedure of the low-cost NALFB, which is performed at ambient temperature, eliminates the need for preheated buffers or additional equipment, greatly simplifying the protocol for sequence-specific PCR amplicon analysis.

Mycobacteria of the tuberculosis complex: Genomics, molecular epidemiology, and evolution trends

Data accumulated in the last three decades on the molecular epidemiology of mycobacteria making up the tuberculosis complex (Mycobacterium tuberculosis, M. bovis, M. africanum, M. caprae, M. pinnipedi, M. microti, and M. canettii) are compiled and analyzed. This is the first attempt to classify isolates obtained from tuberculosis patients in various regions of the world on the basis of differences in the mycobacterial genome structure. This classification allows tracing of epidemic chains in tuberculosis outbreaks. A technique for microbiological diagnostics of tuberculosis is elaborated by using molecular epidemiology methods; this provides rapid identification of infectious agents in clinical samples and assessment of their drug resistance. Molecular epidemiology data also form the grounds for our concept of the evolution of mycobacteria belonging to the tuberculosis complex.

Fluorescent DNA chemosensors: identification of bacterial species by their volatile metabolites

Polyfluorophores built on a DNA scaffold (ODFs) were synthesized and tested for fluorescence responses to the volatiles from M. tuberculosis, E. coli and P. putida in closed Petri dishes. Two sensors in a pattern-based response could distinguish the bacterial strains accurately, suggesting the use of ODFs in rapid identification of infectious agents.

Isopycnometric serology: a new technique based on buoyant density changes in latex beads.

WE wish to describe new serological techniques based on changes in buoyant density occurring when antibodies, or antigens, or both, are attached to latex beads. For the density change to be observed, the mass of the attached particles must be an appreciable fraction of the total mass. Using very small carrier beads less than one hundred antibody molecules or one virus particle per bead can be detected. The speed and sensitivity of the method make it directly applicable to the clinically important problem of rapid identification of infectious agents.