Infectious Disease Diagnostics Research Articles

Recombinase polymerase amplification: method’s characteristics and applications in diagnostics of infectious diseases

Isothermal amplification techniques have been actively developed in recent years and are gradually introduced into the range of methods for infectious disease diagnostics. One of the fastest isothermal methods is recombinase polymerase amplification (RPA). This review contains information about the principle of RPA, the role of individual reaction components and primer design considerations. It provides information on characteristics of various methods of RPA results detection, effects of inhibitors, temperature and agitation on the efficiency of reaction. Approaches to quantitative and multiplex RPA are described, as well as some variants of portable devices designed to identify infectious agents. The conclusion summarizes advantages and disadvantages of RPA in comparison with other amplification methods.

Metagenomics to improve infectious disease diagnostics in low- and middle-income countries.

Metagenomics to improve infectious disease diagnostics in low- and middle-income countries.

Palm-Sized Lab-In-A-Magnetofluidic Tube Platform for Rapid and Sensitive Virus Detection.

Simple, sensitive, and accurate molecular diagnostics are critical for preventing rapid spread of infection and initiating early treatment of diseases. However, current molecular detection methods typically rely on extensive nucleic acid sample preparation and expensive instrumentation. Here, a simple, fully integrated, lab-in-a-magnetofluidic tube (LIAMT) platform is presented for "sample-to-result" molecular detection of virus. By leveraging magnetofluidic transport of micro/nano magnetic beads, the LIAMT device integrates viral lysis, nucleic acid extraction, isothermal amplification, and CRISPR detection within a single engineered microcentrifuge tube. To enable point-of-care molecular diagnostics, a palm-sized processor is developed for magnetofluidic separation, nucleic acid amplification, and visual fluorescence detection. The LIAMT platform is applied to detect SARS-CoV-2 and HIV viruses, achieving a detection sensitivity of 73.4 and 63.9 copiesµL-1, respectively. Its clinical utility is further demonstrated by detecting SARS-CoV-2 and HIV in clinical samples. This simple, affordable, and portable LIAMT platform holds promise for rapid and sensitive molecular diagnostics of infectious diseases at the point-of-care.

Breaking Boundaries in Pneumonia Diagnostics: Transitioning from Tradition to Molecular Frontiers with Multiplex PCR.

The advent of rapid molecular microbiology testing has revolutionized infectious disease diagnostics and is now impacting pneumonia diagnosis and management. Molecular platforms offer highly multiplexed assays for diverse viral and bacterial detection, alongside antimicrobial resistance markers, providing the potential to significantly shape patient care. Despite the superiority in sensitivity and speed, debates continue regarding the clinical role of multiplex molecular testing, notably in comparison to standard methods and distinguishing colonization from infection. Recent guidelines endorse molecular pneumonia panels for enhanced sensitivity and rapidity, but implementation requires addressing methodological differences and ensuring clinical relevance. Diagnostic stewardship should be leveraged to optimize pneumonia testing, emphasizing pre- and post-analytical strategies. Collaboration between clinical microbiologists and bedside providers is essential in developing implementation strategies to maximize the clinical utility of multiplex molecular diagnostics in pneumonia. This narrative review explores these multifaceted issues, examining the current evidence on the clinical performance of multiplex molecular assays in pneumonia, and reflects on lessons learned from previous microbiological advances. Additionally, given the complexity of pneumonia and the sensitivity of molecular diagnostics, diagnostic stewardship is discussed within the context of current literature, including implementation strategies that consider pre-analytical and post-analytical modifications to optimize the clinical utility of advanced technologies like multiplex PCR.

Large-volume RT-LAMP enables extraction-free amplification of HIV RNA from fingerstick plasma

BackgroundPoint-of-care (POC) nucleic acid amplification tests (NAAT) can significantly expand testing coverage, which is critical for infectious disease diagnostics and monitoring. The development of various isothermal amplification techniques greatly simplifies NAATs, but the cumbersome nucleic acid extraction step remains a bottleneck for the POC. Alternatively, extraction-free amplification, where crude samples are directly added into the assay, substantially simplifies the workflow. However, sample dilution is often needed in extraction-free amplification to reduce assay inhibition from sample matrices. Since NAATs are typically run at small volumes around 20 μL, the input sample quantity is therefore limited, resulting in an inevitable sensitivity loss. ResultsHere we explore the potential to perform isothermal amplification in larger reaction volumes to accommodate larger sample quantities, thereby improving sensitivity in extraction-free amplification. We demonstrated the approach by developing large-volume reverse transcription loop-mediated isothermal amplification (RT-LAMP) for HIV RNA detection from fingerstick plasma. We found that LAMP at reaction volumes up to 1 mL maintained the same performance. We then identified plasma dilution conditions needed to maintain the limit of detection in RT-LAMP. Subsequently, using inactivated HIV virus, we showed the successful detection of 24 HIV RNA copies in a 500 μL RT-LAMP reaction in the presence of 20 μL plasma (fingerstick volumes), translating to a viral load of 1200 copies per mL. To reduce the increased reagent cost with expanded reaction volumes, we further identified lower-cost reagents with maintained assay performance. Moreover, we showed that large-volume LAMP, compared to 20 μL reactions, could tolerate higher concentrations of various inhibitors in the sample, such as albumin and GuSCN. Significance and noveltyNAATs are conventionally conducted at small reaction volumes. Here we demonstrated that LAMP can be run at large reaction volumes (over 100 μL) with maintained assay performance, allowing sample inhibition to be mitigated while accommodating larger sample quantities. The same strategy of expanding reaction volumes could be applied to other isothermal amplification methods and various POC applications, to streamline test workflows and/or improve assay sensitivity.

Recombinase Polymerase and Loop-Mediated Isothermal Amplification in the DNA Diagnostics of Infectious Diseases

Recombinase Polymerase and Loop-Mediated Isothermal Amplification in the DNA Diagnostics of Infectious Diseases

Status check: next-generation sequencing for infectious-disease diagnostics.

Next-generation sequencing (NGS) applications for the diagnostics of infectious diseases has demonstrated great potential with three distinct approaches: whole-genome sequencing (WGS), targeted NGS (tNGS), and metagenomic NGS (mNGS, also known as clinical metagenomics). These approaches provide several advantages over traditional microbiologic methods, though challenges still exist.

Progress in Treatment and Diagnostics of Infectious Disease with Polymers.

In this era of advanced technology and innovation, infectious diseases still cause significant morbidity and mortality, which need to be addressed. Despite overwhelming success in the development of vaccines, transmittable diseases such as tuberculosis and AIDS remain unprotected, and the treatment is challenging due to frequent mutations of the pathogens. Formulations of new or existing drugs with polymeric materials have been explored as a promising new approach. Variations in shape, size, surface charge, internal morphology, and functionalization position polymer particles as a revolutionary material in healthcare. Here, an overview is provided of major diseases along with statistics on infection and death rates, focusing on polymer-based treatments and modes of action. Key issues are discussed in this review pertaining to current challenges and future perspectives.

Clinical and laboratory aspects of infectious mononucleosis in different age groups

Infectious mononucleosis is an acute anthroponotic disease that develops as a result of human infection with viruses of the Herpesviridae family: Epstein – Barr virus (EBV), cytomegalovirus (CMV), herpes viruses of the 6th (HHV-6) and 7th types.The aim of our work was to study the clinical features and laboratory parameters of the course of infectious mononucleosis in patients of three age groups.Material and methods. A retrospective analysis of 156 medical records of patients who were on outpatient treatment by an infectious disease specialist at Clinical Medicine Center MedMix was carried out. Patients were divided into three categories according to the age criterion: the first group from 0 to 5 years consisted of 58 (37.2 %) children, the second group was represented by persons from 5 to 18 years old – 58 (37.2 %), the third group was formed by patients from 18 to 53 years old – 40 (25.6 %) people.Results and its discussion. Among children aged 0 to 5 years, boys (67.2 %) more frequently fell sick, and in the group from 18 to 53 years, females (70 %) were more likely to have the disease. Clinical symptoms such as generalized lymphadenopathy (82.7 %), nasopharyngeal lesions (79.3 %) and changes in the general blood test (86.2 %) were most frequently detected in children aged up to 5 years, while fever (81 %) and oropharyngeal lesions (74.1 %) were most frequently detected in patients aged from 5 to 18 years. Liver damage with the development of acute induced viral hepatitis was recorded in all age groups (24.1, 27.6 and 30 %). EBV infection was most frequently diagnosed among children aged from 5 to 18 years and persons aged from 18 to 53 years (in 62.1 and 70 % of cases, respectively, p < 0.05). Mixed infection with the combination of VEB + CMV + HHV-6 was significantly more frequent in children under 5 years of age (34.5 %; p < 0.05).Conclusions. In childhood, the clinical manifestations of infectious mononucleosis were more pronounced. Such intense course of the disease caused frequent visits to an infectious disease specialist and diagnostics with the establishment of an etiological agent. The dominance of EBV infection in the development of infectious mononucleosis in all groups was established. However, at the age up to 5 years, the most common mixed infections were the following combinations: EBV + CMV, EBV + HHV-6, EBV + CMV + HHV-6 and CMV + HHV-6.

FDA's proposed rule for the regulation of laboratory-developed tests.

In October 2023, the Food and Drug Administration (FDA) released a proposed rule that ends enforcement discretion for laboratory-developed tests (LDTs). The FDA's proposal outlines a five-stage implementation to begin regulating LDTs as they do for commercial in vitro diagnostics (IVDs), including modified FDA-approved/cleared tests. We outline here concerns from the clinical and public health microbiology laboratory perspective. It is our opinion that LDTs performed by individual Clinical Laboratory Improvement Amendments-certified diagnostic laboratories should not be regulated in the same way as commercial IVDs. This rule, if finalized, will negatively impact the diagnostic services currently offered by clinical and public health laboratories and, therefore, patients and the providers who care for them. Ending enforcement discretion will likely stifle diagnostic innovation and decrease access to diagnostic testing and health equity. Furthermore, the lack of infrastructure, including personnel and funding, at the FDA and diagnostic laboratories to support the required submissions for review is an obstacle. Like the FDA, diagnostic laboratories prioritize patient safety, accurate clinical diagnostics, and health equity. Since the scope of the LDT landscape is currently unknown, we are supportive of a registration process, along with non-burdensome adverse event reporting, to first understand the scope of clinical use of LDTs and any associated safety concerns. Any regulatory rule should be based on data that have been gathered systematically, not anecdotes or case reports. A rule must also balance the potential negative impact to patient care with realistic safety risks for infectious disease diagnostics.

Microfluidic systems for infectious disease diagnostics.

Microorganisms, encompassing both uni- and multicellular entities, exhibit remarkable diversity as omnipresent life forms in nature. They play a pivotal role by supplying essential components for sustaining biological processes across diverse ecosystems, including higher host organisms. The complex interactions within the human gut microbiota are crucial for metabolic functions, immune responses, and biochemical signalling, particularly through the gut-brain axis. Viruses also play important roles in biological processes, for example by increasing genetic diversity through horizontal gene transfer when replicating inside living cells. On the other hand, infection of the human body by microbiological agents may lead to severe physiological disorders and diseases. Infectious diseases pose a significant burden on global healthcare systems, characterized by substantial variations in the epidemiological landscape. Fast spreading antibiotic resistance or uncontrolled outbreaks of communicable diseases are major challenges at present. Furthermore, delivering field-proven point-of-care diagnostic tools to the most severely affected populations in low-resource settings is particularly important and challenging. New paradigms and technological approaches enabling rapid and informed disease management need to be implemented. In this respect, infectious disease diagnostics taking advantage of microfluidic systems combined with integrated biosensor-based pathogen detection offers a host of innovative and promising solutions. In this review, we aim to outline recent activities and progress in the development of microfluidic diagnostic tools. Our literature research mainly covers the last 5 years. We will follow a classification scheme based on the human body systems primarily involved at the clinical level or on specific pathogen transmission modes. Important diseases, such as tuberculosis and malaria, will be addressed more extensively.

Application of quantum computing in image processing for recognition of infectious diseases of wheat

This study is devoted to the development and application of quantum methods in the field of diagnostics of infectious diseases of wheat. Taking into account the relevance of the problem of agriculture and the need to improve the efficiency of plant disease control, the work proposes a new approach based on the combined use of quantum computing, image processing and machine learning. Quantum image processing techniques have been applied to improve contrast, filter noise, and analyze key features of infectious diseases in the early stages of their development. The developed quantum machine learning models demonstrate high ac-curacy in image classification, which contributes to earlier and more accurate detection of diseases. The study results highlight the effectiveness of quantum methods in agriculture and provide new tools for more accurate diagnosis of infectious plant diseases. The prospects for introducing this approach into agriculture mean the possibility of improving yields, reducing the use of chemicals and ensuring food security.

An Immiscible-Phase Filtration Device for Isolation, Amplification, and Detection of Nucleic Acids for Clinical Diagnostics.

Clinical diagnostics of infectious diseases via nucleic acid amplification tests (NAATs) depend on a separate step of isolation of nucleic acids from cells/viruses embedded in complex biological matrices. The most recent example has been reverse transcription polymerase chain reaction (RT-PCR) for amplification and detection of SARS-CoV-2 RNA for COVID-19 diagnostics. Kits for RNA extraction and purification are commercially available; however, their integration with amplification systems is generally lacking, resulting in two separate steps, i.e., sample preparation and amplification. This makes NAATs more time-consuming, requiring skilled personnel, and can increase the likelihood of contamination. Here, we describe a setup and methodology to perform the quick extraction and detection of nucleic acids in an integrated manner. In particular, we focus on the use of an immiscible filtration device for capture, isolation,concentration, amplification, and colorimetric detection of SARS-CoV-2 RNA.

Use of Specific Borrelia Phages as a New Strategy for Improved Diagnostic Tests.

The high failure rate of tick-borne infection (TBI)-related testing underscores the need for novel approaches that do not rely on serology and two-tier testing. Delayed diagnosis of TBIs, especially Borrelia infections, results in high healthcare costs and great suffering. There is a significant need for a reliable blood test that can aid in the diagnosis of Lyme disease, particularly when the current FDA-approved serological test is not sensitive enough to detect early Lyme patients who have not yet produced antibodies against Borrelia. Bacteriophages are viruses that specifically associate with their bacterial hosts, particularly prophages, bacteriophages residing in bacteria, and have proven to be tightly correlated with their bacterial hosts. They are poised to have wider applications as markers to detect bacteria, particularly in infectious disease. The gene of choice depends on the prevalence of phages within a particular group of bacteria. Phage genes that have been used as molecular markers to examine phage diversity include structural genes encoding the major capsid protein, the portal protein, the DNA polymerase, and the terminase. Borrelia species carry specific phage sequences that can be used as a proxy to identify the bacteria. Using phages as a proxy for bacteria is beneficial, as phages can be detected more easily than bacteria and can be used to bypass the cryptic and tissue-bound feature that typifies human Borrelia infections.We explored a completely new way of detecting Borrelia using Borrelia-specific bacteriophages as a diagnostic tool. Our detection method, patented by Phelix R&D and Leicester University (WO2018083491A1), could potentially transform infectious disease diagnostics through the innovative use of real-time PCR to target circulating bacteriophage DNA in blood from patients with Lyme disease. Firstly, this bacteriophage-based approach offers increased sensitivity since bacteriophages are typically present in five- to tenfold excess over bacterial cells, making it more accurate and sensitive than conventional bacteria-targeting PCR tests. One of the reasons bacteria-based PCR tests are frequently negative is due to the low bacterial concentration in the blood. Bacteriophage-based PCR surpasses this barrier and offers a direct test, as phages are part of bacteria's own genetic material, in contrast to all existing indirect tests (ELISA, Western BLOT, LTT/ELISPOT test). Secondly, a phage-based test can differentiate between different Lyme disease-causing and relapsing fever-causing Borrelia subtypes (B. burgdorferi s.l., B. miyamotoi, etc.), given that bacteriophages are indicators of bacterial identity. Finally, this test can detect Lyme disease in both early and late stages.

Experimental Identification of Cross-Reacting IgG Hotspots to Predict Existing Immunity Evasion of SARS-CoV-2 Variants by a New Biotechnological Application of Phage Display.

Multiple pathogens are competing against the human immune response, leading to outbreaks that are increasingly difficult to control. For example, the SARS-CoV-2 virus continually evolves, giving rise to new variants. The ability to evade the immune system is a crucial factor contributing to the spread of these variants within the human population. With the continuous emergence of new variants, it is challenging to comprehend all the possible combinations of previous infections, various vaccination types, and potential exposure to new variants in an individual patient. Rather than conducting variant-to-variant comparisons, an efficient approach could involve identifying key protein regions associated with the immune evasion of existing immunity against the virus. In this study, we propose a new biotechnological application of bacteriophages, the phage display platform for experimental identification of regions (linear epitopes) that may function as cross-reacting IgG hotspots in SARS-CoV-2 structural proteins. A total of 34,949 epitopes derived from genomes of all SARS-CoV-2 variants deposited prior to our library design were tested in a single assay. Cross-reacting IgG hotspots are protein regions frequently recognized by cross-reacting antibodies in many variants. The assay facilitated the one-step identification of immunogenic regions of proteins that effectively induced specific IgG in SARS-CoV-2-infected patients. We identified four regions demonstrating both significant immunogenicity and the activity of a cross-reacting IgG hotspot in protein S (located at NTD, RBD, HR1, and HR2/TM domains) and two such regions in protein N (at 197-280 and 358-419 aa positions). This novel method for identifying cross-reacting IgG hotspots holds promise for informing vaccine design and serological diagnostics for COVID-19 and other infectious diseases.

Label-free analysis of bacterial growth and lysis at the single-cell level using droplet microfluidics and object detection-oriented deep learning

Bacteria identification and counting at the small population scale is important to many applications in the food safety industry, the diagnostics of infectious diseases and the study and discovery of novel antimicrobial compounds. There is still a lack of easy to implement, fast and accurate methods to count populations of motile cells at the single-cell level. Here, we report a label-free method to count and localize bacterial cells freely swimming in microfluidic anchored picolitre droplets. We used the object detection oriented YOLOv4 deep learning framework for cell detection from bright-field images obtained with an automated Z-stack setup. The neural network was trained to recognize Escherichia coli cell morphology with an average precision of approximately 84%. This allowed us to accurately identify individual cell division events, enabling the study of stochastic bacterial growth starting from initial populations as low as one cell. This work also demonstrates the ability to study single cell lysis in the presence of T7 lytic bacterial viruses (phages). The high precision in cell numbers facilitated the visualization of bacteria-phage interactions over timescale of hours, paving the way towards deciphering phage life cycles in confined environments.

Application of CRISPR/Cas Technology in Biological Nano‐Analysis

AbstractCurrently, with the growing global demand for infectious disease diagnostics, environmental testing and food safety, CRISPR‐Cas (Clustered Regularly Interspaced Short Palindromic Repeats‐CRISPR associated proteins) system has unlimited potential as one of the emerging technologies in the field of biotechnology. Recent studies have shown that CRISPR‐Cas system has become a powerful weapon for gene editing, disease treatment, molecular diagnosis and imaging. As important biomarkers, various nucleic acid and non‐nucleic acid targets need to be detected from various samples before the situation deteriorates. In addition, there are still some limitations and challenges in the current biosensor platforms based on the CRISPR‐Cas system. Therefore, summarising the strategies, strengths and weaknesses, and development prospects of the CRISPR‐Cas system in nucleic acid and non‐nucleic acid target identification is beneficial to fully explore the potential development directions of CRISPR‐Cas‐based biosensor systems. This review briefly introduces and summarises the classification, mechanisms and applications of the system (diagnostics when combined with some signal detection means). In addition, some of the current limitations, challenges and future prospects for optimisation of biosensor platforms based on this system are discussed.

Determination of Seasonal Influenza Intrahost Viral Diversity as a Function of Host Immune Status

Abstract Background Seasonal influenza viruses evolve predominately via antigenic drift, the slow accumulations of genetic mutations leading to changes in key antigenic sites. Due to the accumulation of antigenic changes, influenza vaccines must be updated each year and, even then, can suffer from limited efficacy due to strain mismatch. Despite high levels of genetic diversity seen across the population, most studies show that intrahost viral diversity in humans following natural infection is limited. It is unclear where the population-level diversity in influenza virus originates. Previous studies have shown that host characteristics, such as pre-existing immunity, may affect the intrahost viral evolution during a single infection. As very young children have limited immunologic memory, we hypothesize these infections may serve as a source of viral diversity. Methods In this study, we characterize influenza genetic diversity in the pediatric population as a function of age and vaccine status. We use next generation sequencing to assess intrahost influenza virus diversity in 200 clinical isolates generated during the 2017-2018 flu season from children between 6 months and 18 years of age. We quantify intrahost diversity as the number and frequency of mutations of influenza virus present within each clinical isolate. The clinical samples were obtained from left over influenza-positive nasopharyngeal swabs from the Children's Hospital of Philadelphia Infectious Disease Diagnostics Laboratory. We stratify these isolates by host subject age and vaccine status (with respect to that season's influenza vaccine). Results The isolates sequenced thus far fall within the 6B.1A, 6B.1A.1, and 6B.1A.5b clades for H1N1 and 3C.2a.1, 3C.2a2, and 3C.3a1 clades for H3N2, as expected. Thus far, there does not appear to be a significant relationship between the number of variants detected and either the age or vaccine status of the subject for either H3N2 or H1N1, though this may change as additional isolates are sequenced. Conclusions Through ongoing studies, we evaluate how host age and vaccination status influences intrahost viral diversity. Using clinical metadata, we investigate the association between viral diversity and the duration of symptoms and disease severity. By identifying a reservoir of influenza diversity, we will be better able to evaluate influenza strains for inclusion in the seasonal vaccine.

Sequence optimized diagnostic assay for Ebola virus detection

Rapid pathogen identification is a critical first step in patient isolation, treatment, and controlling an outbreak. Real-time PCR is a highly sensitive and specific approach commonly used for infectious disease diagnostics. However, mismatches in the primer or probe sequence and the target organism can cause decreased sensitivity, assay failure, and false negative results. Limited genomic sequences for rare pathogens such as Ebola virus (EBOV) can negatively impact assay performance due to undiscovered genetic diversity. We previously developed and validated several EBOV assays prior to the 2013–2016 EBOV outbreak in West Africa, and sequencing EBOV Makona identified sequence variants that could impact assay performance. Here, we assessed the impact sequence mismatches have on EBOV assay performance, finding one or two primer or probe mismatches resulted in a range of impact from minimal to almost two log sensitivity reduction. Redesigning this assay improved detection of all EBOV variants tested. Comparing the performance of the new assay with the previous assays across a panel of human EBOV samples confirmed increased assay sensitivity as reflected in decreased Cq values with detection of three positive that tested negative with the original assay.

Disciples of Pasteur from Russia — 2

In 2022, a global scientific community noted the 200th anniversary of the outstanding scientist, one of founders of a science of microbiology Louis Pasteur. Pasteurs works promoted formation of the microbic theory and gave a momentum to a search of ways for prevention of infections, including hospital ones. Among Pasteurs disciples and colleagues are E. Ru, I.I. Mechnikov as well as many Russian scientists. Not all family names of Pasteurs disciples were revealed to a wide range of readers. Much of them returned home and to bring a marked contribution to development of microbiology and immunology in Russia. Here, we continue seek out and return biographies of the Russian scientists Louis Pasteurs disciples. Among Russian scientists who passed school of Pasteur Institute there may be mentioned many graduates of one of the oldest medical institutions of Russia Military Medical academy as well as associates of I.I. Mechnikov and N.F. Gamalei J.J. Bardach, S.I. Zlatogorov, N.I. Chistovich, L.A. Tarasevich; renowned surgeons V.A. Ratimov and V.A. Oppel, one of founders of Pasteur Institute in Saint Petersburg P.P. Maslakovets. Many Pasteurs followers took part in creating the institute resembling Pasteur Institute nowadays known as the Institute of Experimental Medicine. In the latter regard, we mention E.F. Shperk, V.I. Nedrigajlov, etc. Moreover, it is also necessary to note the first woman in Russia Doctor P.V. Tsiklinskuju who was awarded a title Professor of Bacteriology. Pasteurs disciples received theoretical and practical knowledge from their teacher and returned to own Fatherlands to fully serve with full dedication. They brought with new methods of scientific research and diagnostics of infectious diseases, used already known immune serums and vaccine preparations and developed their own similar agents to combat infections that were relevant in those years: rabies, plague, cholera, typhoid fever, tuberculosis. All Russian scientists, disciples of Louis Pasteur as well as its friend and colleague I.I. Mechnikov continued to serve to a society owing to great achievements in the field of microbiology by transferring own knowledge and experience to numerous disciples continuers of work of the great scientist Louis Pasteur. Many of them were immortalized in the names of research institutes, departments and laboratories.