Methods For Detection Of Pathogens Research Articles

Clinical and Laboratory Diagnosis of Cryptosporidiosis among Children with Acute Gastroenteritis at a Tertiary Hospital, Cairo, Egypt

Most studies on gastroenteritis have focused on viral and bacterial infections, while gastroenteritis where intestinal protozoan parasites may have played a role has not been well studied. This study was therefore, designed to assess the frequency and several potential risk factors for Cryptosporidium infection among children suffering from acute gastroenteritis and presented to a tertiary hospital in Cairo, Egypt. Effectiveness of modified Ziehl-Neelsen (MZN) and nested polymerase chain reaction (nPCR) for Cryptosporidium detection were evaluated as well. A cross-sectional study was performed during the period from July 2018 to December 2018, where 100 human diarrheic stool samples were collected from children aged 3 months up to 12 years old presented to Ain Shams University Pediatrics Hospital, Cairo, Egypt with acute gastroenteritis. Demographic and clinical data were obtained from the participants. Initial parasite screening was done using the MZN staining method, and microscopically examined for Cryptosporidium infection, while genotyping was based on molecular diagnostic assays using nPCR and sequencing for selected samples. The overall frequency of Cryptosporidium infection was 5% using light microscopy, while 19% of samples were positive by nPCR. Cryptosporidium hominis was the only detected genotype. Clinical picture among cases were not significant in comparison to patients with other causes of gastroenteritis. Cryptosporidium infection is more common below 5 years of age; however, clinical data are not enough for suspicion of infection. Nucleic acid-based methods are more sensitive and specific despite the high cost in developing countries. However, real estimation of Cryptosporidium disease burden is of an outmost importance to achieve prevention and detection of the Cryptosporidium species genetic diversity. Lay summaryCryptosporidium is a protozoan, which causes gastroenteritis in humans. It is most common below 5 years of age; however, diarrhea and vomiting characteristics are not different from other causes of gastroenteritis. General diagnostic methods are inadequate for detection of these infections. Nested polymerase chain reaction (nPCR) and sequencing are accurate methods for pathogen detection and species verification. Our study included 100 Egyptian children with acute gastroenteritis. The overall frequency of Cryptosporidium infection was 5% using light microscopy, while 19% of samples were positive by nPCR. The clinical picture of the children presenting with this disease was not significantly different from those presenting with gastroenteritis due to other causes. This emphasizes the importance of proper diagnosis to know the true burden of the disease.

Biosensors for European Zoonotic Agents: A Current Portuguese Perspective.

Emerging and recurrent outbreaks caused by zoonotic agents pose a public health risk. They result in morbidity and mortality in humans and significant losses in the livestock and food industries. This highlights the need for rapid surveillance methods. Despite the high reliability of conventional pathogen detection methods, they have high detection limits and are time-consuming and not suitable for on-site analysis. Furthermore, the unpredictable spread of zoonotic infections due to a complex combination of risk factors urges the development of innovative technologies to overcome current limitations in early warning and detection. Biosensing, in particular, is highlighted here, as it offers rapid and cost-effective devices for use at the site of infection while increasing the sensitivity of detection. Portuguese research in biosensors for zoonotic pathogens is the focus of this review. This branch of research produces exciting and innovative devices for the study of the most widespread pathogenic bacteria. The studies presented here relate to the different classes of pathogens whose characteristics and routes of infection are also described. Many advances have been made in recent years, and Portuguese research teams have increased publications in this field. However, biosensing still needs to be extended to other pathogens, including potentially pandemic viruses. In addition, the use of biosensors as part of routine diagnostics in hospitals for humans, in animal infections for veterinary medicine, and food control has not yet been achieved. Therefore, a convergence of Portuguese efforts with global studies on biosensors to control emerging zoonotic diseases is foreseen for the future.

New Microbiological Techniques for the Diagnosis of Bacterial Infections and Sepsis in ICU Including Point of Care.

Purpose of ReviewThe aim of this article is to review current and emerging microbiological techniques that support the rapid diagnosis of bacterial infections in critically ill patients, including their performance, strengths and pitfalls, as well as available data evaluating their clinical impact.Recent FindingsBacterial infections and sepsis are responsible for significant morbidity and mortality in patients admitted to the intensive care unit and their management is further complicated by the increase in the global burden of antimicrobial resistance. In this setting, new diagnostic methods able to overcome the limits of traditional microbiology in terms of turn-around time and accuracy are highly warranted. We discuss the following broad themes: optimisation of existing culture-based methodologies, rapid antigen detection, nucleic acid detection (including multiplex PCR assays and microarrays), sepsis biomarkers, novel methods of pathogen detection (e.g. T2 magnetic resonance) and susceptibility testing (e.g. morphokinetic cellular analysis) and the application of direct metagenomics on clinical samples. The assessment of the host response through new “omics” technologies might also aid in early diagnosis of infections, as well as define non-infectious inflammatory states.SummaryDespite being a promising field, there is still scarce evidence about the real-life impact of these assays on patient management. A common finding of available studies is that the performance of rapid diagnostic strategies highly depends on whether they are integrated within active antimicrobial stewardship programs. Assessing the impact of these emerging diagnostic methods through patient-centred clinical outcomes is a complex challenge for which large and well-designed studies are awaited.

Isothermal amplification assay for visual on-site detection of Staphylococcus aureus in Chevon

ABSTRACT The ability to rapidly detect pathogens in food is important from public health and food safety reasons. Traditional culture-based detection methods tend to be laborious, time consuming, technically demanding and may be limited due to their low sensitivity. Rapid detection methods for foodborne pathogens should be specific and sensitive to detect pathogens in low numbers. Sensitivity is important because a single pathogen present in food has the potential risk to cause infection. PCR and Real-time PCR are rapid, sensitive, and specific but require specific instruments and laboratory set-up. In the present study, a simple loop-mediated isothermal amplification (LAMP) assay was developed for rapid detection of Staphylococcus aureus. The LAMP assay was found to be ten times more sensitive than traditional end-point PCR with analytical sensitivity of 0.56 pg/μl and 5.6 pg/μl of DNA, respectively. In spiked chevon samples inoculated with S. aureus, the detection limit of LAMP and PCR assay was 3.3 × 105 CFU/g and 3.3 × 106 CFU/g, respectively, without enrichment. After enriching the samples for 6 h, the detection limit improved to 3.3 × 102 CFU/g and 3.3 × 104 CFU/g, respectively, indicating LAMP to be 100-fold more sensitive than PCR. The detection limit further improved to 3.3 CFU/g of meat after enrichment of 12 h. The developed LAMP was also found to be suitable when evaluated on field samples. The present study reports a simple LAMP assay for rapid visual detection of S. aureus which has potential for use in resource limited settings.

Inertial focusing of microparticles, bacteria, and blood in serpentine glass channels.

Early detection of pathogenic microorganisms is pivotal to diagnosis and prevention of health and safety crises. Standard methods for pathogen detection often rely on lengthy culturing procedures, confirmed by biochemical assays, leading to >24 h for a diagnosis. The main challenge for pathogen detection is their low concentration within complex matrices. Detection of blood-borne pathogens via techniques such as PCR requires an initial positive blood culture and removal of inhibitory blood components, reducing its potential as a diagnostic tool. Among different label-free microfluidic techniques, inertial focusing on microscale channels holds great promise for automation, parallelization, and passive continuous separation of particles and cells. This work presents inertial microfluidic manipulation of small particles and cells (1-10 μm) in curved serpentine glass channels etched at different depths (deep and shallow designs) that can be exploited for (1) bacteria preconcentration from biological samples and (2) bacteria-blood cell separation. In our shallow device, the ability to focus Escherichia coli into the channel side streams with high recovery (89% at 2.2× preconcentration factor) could be applied for bacteria preconcentration in urine for diagnosis of urinary tract infections. Relying on differential equilibrium positions of red blood cells and E. coli inside the deep device, 97% red blood cells were depleted from 1:50 diluted blood with 54% E. coli recovered at a throughput of 0.7 mL/min. Parallelization of such devices could process relevant volumes of 7 mL whole blood in 10 min, allowing faster sample preparation for downstream molecular diagnostics of bacteria present in bloodstream.

Atypical Pathogen Distribution in Chinese Hospitalized AECOPD Patients: A Multicenter Cross-Sectional Study.

PurposeThe proportion of atypical pathogens in patient with AECOPD within mainland China is unknown. The objectives of this study were to determine the distribution of atypical pathogens among Chinese patients with AECOPD, to evaluate the clinical characteristics of different atypical pathogen infections, and to compare different detection methods for atypical pathogens.Patients and MethodsSpecimens were collected from patients with AECOPD from March 2016 to November 2018 at eleven medical institutions in eight cities in China. Double serum, sputum, and urine samples were obtained from 145 patients. Serological and nucleic acid tests were used to assess for Mycoplasma pneumonia and Chlamydia pneumoniae; serological, urinary antigen, and nucleic acid tests were applied to detect Legionella pneumophila. The clinical characteristics of atypical pathogen-positive and -negative groups were also compared.ResultsThe overall positivity rate for Mycoplasma pneumoniae was 20.69% (30/145), with the highest rate being 20.00% (29/145) when determined by passive agglutination.The overall positive rates for Chlamydia pneumoniae and Legionella pneumophila were 29.66% (43/145) and 10.34% (15/145), respectively. The most common serotype of Legionella pneumophila was type 6. The maximum hospitalized body temperature, ratio of eosinophils, C-reactive protein (CRP) level, and procalcitonin (PCT) level of the Mycoplasma pneumoniae-positive group were significantly higher than those of the Mycoplasma pneumoniae-negative group. Patients in the Chlamydia pneumoniae-positive group smoked more, had higher proportions of comorbidities and frequent aggravations in the previous two years than those in the Chlamydia pneumoniae-negative group. Furthermore, the forced expiratory volume in one second to forced vital capacity (FEV1/FVC) ratio assessment of lung function was higher, and the concentration of arterial blood bicarbonate (HCO3−) was lower in the Legionella pneumophila-positive group than in the Legionella pneumophila-negative group.ConclusionOverall, atypical pathogens play an important role in AECOPD. Regarding the testing method, serological testing is a superior method to nucleic acid testing.

Health Monitoring of Laboratory Rodent Colonies-Talking about (R)evolution.

Simple SummaryWhen working with laboratory animals, scientists need to ensure that animals are not suffering from natural infections. Therefore, hygienic standards have been developed over the last 100+ years. The key element of hygienic standardization is the monitoring of infectious agents that can compromise the animal’s health, are dangerous for the personnel or interfere with the research. However, scientists became aware that by eliminating such unwanted infectious agents the overall diversity of all microbes in research animals, the so-called microbiome, has also been reduced. Moreover, it became clear that the microbiome composition has an enormous impact on how research models react, e.g., to treatments. This might hinder the translation of findings in preclinical research to the clinical situation. Therefore, new concepts in hygienic standardization need to be developed that take animal welfare as well as scientific value into account. In this review, we will give an overview of how classical monitoring has been evolved and demonstrate concepts regarding how to handle the microbiome as a yet unknown variable in animal-based research in order to enhance the validity of research findings.The health monitoring of laboratory rodents is essential for ensuring animal health and standardization in biomedical research. Progress in housing, gnotobiotic derivation, and hygienic monitoring programs led to enormous improvement of the microbiological quality of laboratory animals. While traditional health monitoring and pathogen detection methods still serve as powerful tools for the diagnostics of common animal diseases, molecular methods develop rapidly and not only improve test sensitivities but also allow high throughput analyses of various sample types. Concurrently, to the progress in pathogen detection and elimination, the research community becomes increasingly aware of the striking influence of microbiome compositions in laboratory animals, affecting disease phenotypes and the scientific value of research data. As repeated re-derivation cycles and strict barrier husbandry of laboratory rodents resulted in a limited diversity of the animals’ gut microbiome, future monitoring approaches will have to reform—aiming at enhancing the validity of animal experiments. This review will recapitulate common health monitoring concepts and, moreover, outline strategies and measures on coping with microbiome variation in order to increase reproducibility, replicability and generalizability.

Agricultural sustainability can be ensured by adopting dynamic plant pathology, pedagogy

Plant diseases cause serious economic losses (5–15%/up to 25%) in all sorts of economic crop plants irrespective of cash, industrial, horticultural, ornamental, food, fodder, fibre, spices, plantation, aromatic and medicinal plants and forest trees especially in the tropics and subtropics. Farmers, due to lack of expertise, can neither identify the pathogens nor manage them. They are ignorant of varied symptoms produced by the diseases and the mode of field spread of these diseases. Knowledge of the disease cycle and use of resistant varieties are essential for developing effective management strategy against the diseases. Rigorous field training of students/village workers/farmers is the most important pedagogical aspect to ensure sustainability of Indian agriculture. In order to put a long term check on yield/quality losses by overcoming farmer’s ignorance, there is a need to ensure availability of resistant varieties and effective but simple methods of vector control. Intensive training of the farmers through frequent field visits enables them to recognize incidence of the disease and to understand the epidemiological facet of the disease too. Bio agents occurring naturally within the host's environment and crop rotation practices are also helpful. Emphasis is needed to train students in various methods of pathogen detection and to coach a large number of farmer’s team to help them recognize and manage the disease in field. Generally limited number of important diseases and pests affect any crop plant which does not exceed 4–6 and 15–20 respectively in all regions.

Antibiotic-Based Magnetic Nanoprobes Combined with mPCR for Simultaneous Detection of Staphylococcus aureus and Bacillus cereus

Sensitive, simple, and rapid pathogen separation and detection methods are essential for the early diagnosis and immediate medical treatment of bacterial infections. In this study, a novel strategy based on vancomycin (Van)-modified ovalbumin (OVA) mediated magnetic nanoprobes (MBs-OVA-Van) combined with multiplex polymerase chain reaction (mPCR) was developed for efficient isolation and detection of Staphylococcus aureus (S. aureus) and Bacillus cereus (B. cereus). Van conjugated to ovalbumin coated magnetic microcarrier via strong covalent interaction. In addition, Van binds through hydrogen bonds with d-alanyl-d-alanine moieties on the surface of gram-positive bacteria cell wall. Under optimum conditions, the MBs-OVA-Van showed that the capture efficiency (CE) for S. aureus and B. cereus in PBS and juice samples were around 91%, 92% and 89%, 85%, respectively. In addition, with a mixture containing both target bacteria in PBS and juice samples, the CE for S. aureus and B. cereus were recorded at about 90%, 91% and 80%, 84%, respectively. The MBs-OVA-Van plus mPCR method applied in PBS and juice samples for both bacteria showed limits of detection at 102 CFU mL-1 in a < 3 h total assay time. The results indicated that the method holds promise for isolation and enrichment of gram-positive bacteria with S. aureus and B. cereus as models.

Validation of Additional Approaches and Applications for Using the Continuous and Manual Sampling Devices for Raw Beef Trim

In this work, the goal was to determine the efficacy of MicroTally-based sampling in scenarios commonly encountered in the commercial beef processing industry, but outside of the parameters evaluated during the initial proof-of-concept work. The data were derived from 1,650 matched samples collected from 540 individual combo bins at six commercial beef processing plants, comparing MicroTally-based sampling (continuous and manual sampling devices [CSD and MSD]) to N60 Excision and/or N60 Plus methods. Mounting a 61-cm CSD cartridge to a 30-cm-wide conveyor provided sampling that is equivalent to N60 Excision and N60 Plus methods. Mounting a CSD to a chute instead of a conveyor was equivalent to the N60 Plus sampling method. The CSD was shown to be effective for sampling when used in conjunction with a "swinging arm trim diverter" and receiving product in batch mode as opposed to continuous flow. MSD sampling of oval combo bins with trim surface area (≈0.93 m2 [≈1,439 in2]) less than 1 m2 (1,600 in2) was shown to be equivalent to the N60 Plus sample collection method. Peracetic acid applied at the end of the trim conveyor did not negatively impact pathogen index target detection of the CSD even if the samples were shipped overnight before analysis. Pathogen index targets were demonstrated to be useful tools for validating methods designed to measure pathogen prevalence. The data presented herein support equivalency criteria of within 0.5 log CFU per sample for indicator organism counts. These data collectively support various alternative applications of MicroTally-based trim sampling and the application and interpretation of alternative methods for pathogen detection.

Comparative Analysis of Detection Methods for Food-borne Pathogens in Fresh-cut Agricultural Materials

Comparative Analysis of Detection Methods for Food-borne Pathogens in Fresh-cut Agricultural Materials

Application of Metagenomic Next-Generation Sequencing in the Diagnosis of Pulmonary Infectious Pathogens From Bronchoalveolar Lavage Samples.

BackgroundMetagenomic next-generation sequencing (mNGS) is a powerful method for pathogen detection. In this study, we assessed the value of mNGS for bronchoalveolar lavage (BAL) samples in the diagnosis of pulmonary infections.MethodsFrom February 2018 to April 2019, BAL samples were collected from 235 patients with suspected pulmonary infections. mNGS and microbial culture were performed to evaluate the effectiveness of mNGS in pulmonary infection diagnosis.ResultsWe employed mNGS to evaluate the alpha diversity, results suggesting that patients with confirmed pathogens had a lower microbial diversity index compared to that of patients with uncertain pathogens. For the patients admitted to the respiratory intensive care unit (RICU) or on a ventilator, they experienced a lower diversity index than that of the patients in the general ward or not on a ventilator. In addition, mNGS of BAL had a diagnostic sensitivity of 88.89% and a specificity of 14.86% in pulmonary infection, with 21.16% positive predictive value (PPV) and 83.87% negative predictive value (NPV). When rare pathogens were excluded, the sensitivity of mNGS decreased to 73.33%, and the specificity increased to 41.71%. For patients in the simple pulmonary infection group and the immunocompromised group, the main infection types were bacterial infection (58.33%) and mixed-infection (43.18%). Furthermore, mNGS had an advantage over culture in describing polymicrobial ecosystem, demonstrating the microbial distribution and the dominant strains of the respiratory tract in patients with different underlying diseases.ConclusionsThe study indicated that mNGS of BAL samples could provide more accurate diagnostic information in pulmonary infections and demonstrate the changes of respiratory microbiome in different underlying diseases. This method might play an important role in the clinical use of antimicrobial agents in the future.

Rapid Detection of Enterocytozoon hepatopenaei Infection in Shrimp With a Real-Time Isothermal Recombinase Polymerase Amplification Assay.

Enterocytozoon hepatopenaei (EHP) infection has become a significant threat in shrimp farming industry in recent years, causing major economic losses in Asian countries. As there are a lack of effective therapeutics, prevention of the infection with rapid and reliable pathogen detection methods is fundamental. Molecular detection methods based on polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) have been developed, but improvements on detection speed and convenience are still in demand. The isothermal recombinase polymerase amplification (RPA) assay derived from the recombination-dependent DNA replication (RDR) mechanism of bacteriophage T4 is promising, but the previously developed RPA assay for EHP detection read the signal by gel electrophoresis, which restricted this application to laboratory conditions and hampered the sensitivity. The present study combined fluorescence analysis with the RPA system and developed a real-time RPA assay for the detection of EHP. The detection procedure was completed in 3–7 min at 39°C and showed good specificity. The sensitivity of 13 gene copies per reaction was comparable to the current PCR- and LAMP-based methods, and was much improved than the RPA assay analyzed by gel electrophoresis. For real clinical samples, detection results of the real-time RPA assay were 100% consistent with the industrial standard nested PCR assay. Because of the rapid detection speed and the simple procedure, the real-time RPA assay developed in this study can be easily assembled as an efficient and reliable on-site detection tool to help control EHP infection in shrimp farms.

SHERLOCK and DETECTR: CRISPR-Cas Systems as Potential Rapid Diagnostic Tools for Emerging Infectious Diseases.

Infectious diseases are one of the most intimidating threats to human race, responsible for an immense burden of disabilities and deaths. Rapid diagnosis and treatment of infectious diseases offers a better understanding of their pathogenesis. According to the World Health Organization, the ideal approach for detecting foreign pathogens should be rapid, specific, sensitive, instrument-free, and cost-effective. Nucleic acid pathogen detection methods, typically PCR, have numerous limitations, such as highly sophisticated equipment requirements, reagents, and trained personnel relying on well-established laboratories, besides being time-consuming. Thus, there is a crucial need to develop novel nucleic acid detection tools that are rapid, specific, sensitive, and cost-effective, particularly ones that can be used for versatile point-of-care diagnostic applications. Two new methods exploit unpredicted in vitro properties of CRISPR-Cas effectors, turning activated nucleases into basic amplifiers of a specific nucleic acid binding event. These effectors can be attached to a diversity of reporters and utilized in tandem with isothermal amplification approaches to create sensitive identification in multiple deployable field formats. Although still in their beginning, SHERLOCK and DETECTR technologies are potential methods for rapid detection and identification of infectious diseases, with ultrasensitive tests that do not require complicated processing. This review describes SHERLOCK and DETECTR technologies and assesses their properties, functions, and prospective to become the ultimate diagnostic tools for diagnosing infectious diseases and curbing disease outbreaks.

Toward an Integrated Genome-Based Surveillance of Salmonella enterica in Germany.

Despite extensive monitoring programs and preventative measures, Salmonella spp. continue to cause tens of thousands human infections per year, as well as many regional and international food-borne outbreaks, that are of great importance for public health and cause significant socio-economic costs. In Germany, salmonellosis is the second most common cause of bacterial diarrhea in humans and is associated with high hospitalization rates. Whole-genome sequencing (WGS) combined with data analysis is a high throughput technology with an unprecedented discriminatory power, which is particularly well suited for targeted pathogen monitoring, rapid cluster detection and assignment of possible infection sources. However, an effective implementation of WGS methods for large-scale microbial pathogen detection and surveillance has been hampered by the lack of standardized methods, uniform quality criteria and strategies for data sharing, all of which are essential for a successful interpretation of sequencing data from different sources. To overcome these challenges, the national GenoSalmSurv project aims to establish a working model for an integrated genome-based surveillance system of Salmonella spp. in Germany, based on a decentralized data analysis. Backbone of the model is the harmonization of laboratory procedures and sequencing protocols, the implementation of open-source bioinformatics tools for data analysis at each institution and the establishment of routine practices for cross-sectoral data sharing for a uniform result interpretation. With this model, we present a working solution for cross-sector interpretation of sequencing data from different sources (such as human, veterinarian, food, feed and environmental) and outline how a decentralized data analysis can contribute to a uniform cluster detection and facilitate outbreak investigations.

Improving Pulmonary Infection Diagnosis with Metagenomic Next Generation Sequencing.

Pulmonary infections are among the most common and important infectious diseases due to their high morbidity and mortality, especially in older and immunocompromised individuals. However, due to the limitations in sensitivity and the long turn-around time (TAT) of conventional diagnostic methods, pathogen detection and identification methods for pulmonary infection with greater diagnostic efficiency are urgently needed. In recent years, unbiased metagenomic next generation sequencing (mNGS) has been widely used to detect different types of infectious pathogens, and is especially useful for the detection of rare and newly emergent pathogens, showing better diagnostic performance than traditional methods. There has been limited research exploring the application of mNGS for the diagnosis of pulmonary infections. In this study we evaluated the diagnostic efficiency and clinical impact of mNGS on pulmonary infections. A total of 100 respiratory samples were collected from patients diagnosed with pulmonary infection in Shanghai, China. Conventional methods, including culture and standard polymerase chain reaction (PCR) panel analysis for respiratory tract viruses, and mNGS were used for the pathogen detection in respiratory samples. The difference in the diagnostic yield between conventional methods and mNGS demonstrated that mNGS had higher sensitivity than traditional culture for the detection of pathogenic bacteria and fungi (95% vs 54%; p<0.001). Although mNGS had lower sensitivity than PCR for diagnosing viral infections, it identified 14 viral species that were not detected using conventional methods, including multiple subtypes of human herpesvirus. mNGS detected viruses with a genome coverage >95% and a sequencing depth >100× and provided reliable phylogenetic and epidemiological information. mNGS offered extra benefits, including a shorter TAT. As a complementary approach to conventional methods, mNGS could help improving the identification of respiratory infection agents. We recommend the timely use of mNGS when infection of mixed or rare pathogens is suspected, especially in immunocompromised individuals and or individuals with severe conditions that require urgent treatment.

Blood Culture Contamination: Still Hazy After All These Years

Despite the availability of a few methods for direct pathogen detection from whole blood, a blood culture is still the main method for diagnosing bloodstream infections. Detection of organisms in a patient’s blood culture has both diagnostic and prognostic value and is one of the most important functions of the diagnostic microbiology laboratory. Blood cultures support growth of most bacteria and fungi in specialized nutrient broth, pathogen and non-pathogen alike. Thus, growth of microbes in a blood culture can signal the presence of a true bloodstream infection or skin commensal contaminant introduced during the collection of blood. Monitoring blood culture contamination rates is a critical process for microbiology laboratories. Contamination of blood cultures can lead to negative patient outcomes. Blood culture contamination leads to unnecessary antimicrobial therapy, prolonged hospital stays, additional laboratory tests, and additional costs. The National Health Safety Network recently issued a master organism list that takes effect beginning January 2021. This review focuses on discussion of blood culture contamination, the history of benchmarking of the quality metrics related to blood culture contamination, and the need to reduce the impacts of blood culture contamination with standardized benchmarks and best practices to prevent contamination.

Research advances and applications of biosensing technology for the diagnosis of pathogens in sustainable agriculture.

Plant diseases significantly impact the global economy, and plant pathogenic microorganisms such as nematodes, viruses, bacteria, fungi, and viroids may be the etiology for most infectious diseases. In agriculture, the development of disease-free plants is an important strategy for the determination of the survival and productivity of plants in the field. This article reviews biosensor methods of disease detection that have been used effectively in other fields, and these methods could possibly transform the production methods of the agricultural industry. The precise identification of plant pathogens assists in the assessment of effective management steps for minimization of production loss. The new plant pathogen detection methods include evaluation of signs of disease, detection of cultured organisms, or direct examination of contaminated tissues through molecular and serological techniques. Laboratory-based approaches are costly and time-consuming and require specialized skills. The conclusions of this review also indicate that there is an urgent need for the establishment of a reliable, fast, accurate, responsive, and cost-effective testing method for the detection of field plants at early stages of growth. We also summarized new emerging biosensor technologies, including isothermal amplification, detection of nanomaterials, paper-based techniques, robotics, and lab-on-a-chip analytical devices. However, these constitute novelty in the research and development of approaches for the early diagnosis of pathogens in sustainable agriculture.

Proximity ligation assay: an ultrasensitive method for protein quantification and its applications in pathogen detection.

It is of great significance to establish sensitive and accurate pathogen detection methods, considering the continuous emergence or re-emergence of infectious diseases seriously influences the safety of human and animals. Proximity ligation assay (PLA) is developed for the sensitive protein detection and also can be used for the detection of pathogens. PLA employs aptamer or monoclonal/polyclonal antibody-nucleic acid complexes as proximity probes. When the paired proximity probes bind to the same target protein or protein complex, they will be adjacent to each other and form an amplifiable DNA sequence through ligation. Combining the specificity of enzyme-linked immunosorbent assay (ELISA) and sensitivity of polymerase chain reaction (PCR), PLA transforms the detection of protein into the detection of DNA nucleic acid sequence. Therefore, as an ultrasensitive protein assay, PLA has great potential for quantification, localization of protein, and clinical diagnostics. In this review, we summarize the basic principles of PLA and its applications in pathogen detection. KEY POINTS: • Different forms of proximity ligation assay are introduced. • Applications of proximity ligation assay in pathogen detection are summarized. • Proximity ligation assay is an ultrasensitive method to quantify protein and pathogen.

Aptamer-based approaches for the detection of waterborne pathogens.

Waterborne ailments pose a serious threat to public health and are a huge economic burden. Lack of hygiene in drinking and recreational water is the chief source of microbial pathogens in developing countries. Poor water quality and sanitation account for more than 3.4 million deaths a year worldwide. This has urged authorities and researchers to explore different avenues of pathogen detection. There is a growing demand for rapid and reliable sensor technologies, in particular those that can detect in situ and perform in harsh conditions. Some of the major waterborne pathogens include Vibrio cholerae, Leptospira interrogans, Campylobacter jejuni, Shigella spp., enterotoxigenic Escherichia coli, Clostridium difficile, Cryptosporidium parvum, Entamoeba histolytica, and Hepatitis A virus. While conventional methods of pathogen detection like serodiagnosis and microbiological methods have been superseded by nucleic acid amplification methods, there is still potential for improvement. This review provides an insight into aptamers and their utility in the form of aptasensors. It discusses how aptamer-based approaches have emerged as a novel strategy and its advantages over more resource-intensive and complex biochemical approaches.