Comparing variability in diagnosis of upper respiratory tract infections in patients using syndromic, next generation sequencing, and PCR-based methods.

Early and accurate diagnosis of respiratory pathogens and associated outbreaks can allow for the control of spread, epidemiological modeling, targeted treatment, and decision making-as is evident with the current COVID-19 pandemic. Many respiratory infections share common symptoms, making them difficult to diagnose using only syndromic presentation. Yet, with delays in getting reference laboratory tests and limited availability and poor sensitivity of point-of-care tests, syndromic diagnosis is the most-relied upon method in clinical practice today. Here, we examine the variability in diagnostic identification of respiratory infections during the annual infection cycle in northern New Mexico, by comparing syndromic diagnostics with polymerase chain reaction (PCR) and sequencing-based methods, with the goal of assessing gaps in our current ability to identify respiratory pathogens. Of 97 individuals that presented with symptoms of respiratory infection, only 23 were positive for at least one RNA virus, as confirmed by sequencing. Whereas influenza virus (n = 7) was expected during this infection cycle, we also observed coronavirus (n = 7), respiratory syncytial virus (n = 8), parainfluenza virus (n = 4), and human metapneumovirus (n = 1) in individuals with respiratory infection symptoms. Four patients were coinfected with two viruses. In 21 individuals that tested positive using PCR, RNA sequencing completely matched in only 12 (57%) of these individuals. Few individuals (37.1%) were diagnosed to have an upper respiratory tract infection or viral syndrome by syndromic diagnostics, and the type of virus could only be distinguished in one patient. Thus, current syndromic diagnostic approaches fail to accurately identify respiratory pathogens associated with infection and are not suited to capture emerging threats in an accurate fashion. We conclude there is a critical and urgent need for layered agnostic diagnostics to track known and unknown pathogens at the point of care to control future outbreaks.

Interaction of amphiphilic lipoarabinomannan with host carrier lipoproteins in tuberculosis patients: Implications for blood-based diagnostics.

Lipoarabinomannan (LAM), an amphiphilic lipoglycan of the Mycobacterium tuberculosis cell wall, is a diagnostic target for tuberculosis. Previous work from our laboratory and others suggests that LAM is associated with host serum lipoproteins, which may in turn have implications for diagnostic assays. Our team has developed two serum assays for amphiphile detection: lipoprotein capture and membrane insertion. The lipoprotein capture assay relies on capture of the host lipoproteins, exploiting the biological association of host lipoprotein with microbial amphiphilic biomarkers to "concentrate" LAM. In contrast, the membrane insertion assay is independent of the association between pathogen amphiphiles and host lipoprotein association, and directly captures LAM based on its thermodynamic propensity for association with a supported lipid membrane, which forms the functional surface of an optical biosensor. In this manuscript, we explored the use of these assays for the detection of LAM in sera from adults whose tuberculosis status had been well-characterized using conventional microbiological tests, and endemic controls. Using the lipoprotein capture assay, LAM signal/noise ratios were >1.0 in 29/35 (83%) individuals with culture-confirmed active tuberculosis, 8/13 (62%) individuals with tuberculosis symptoms, but no positive culture for M. tuberculosis, and 0/6 (0%) symptom-free endemic controls. To evaluate serum LAM levels without bias associated with potential differences in circulating host lipoprotein concentrations between individuals, we subsequently processed available samples to liberate LAM from associated host lipoprotein assemblies followed by direct detection of the pathogen biomarker using the membrane insertion approach. Using the membrane insertion assay, signal/noise for detection of serum LAM was greater than that observed using the lipoprotein capture method for culture-confirmed TB patients (6/6), yet remained negative for controls (2/2). Taken together, these results suggest that detection of serum LAM is a promising TB diagnostic approach, but that further work is required to optimize assay performance and to decipher the implications of LAM/host lipoprotein associations for diagnostic assay performance and TB pathogenesis.

A centrifugal microfluidic cross-flow filtration platform to separate serum from whole blood for the detection of amphiphilic biomarkers.

The separation of biomarkers from blood is straightforward in most molecular biology laboratories. However, separation in resource-limited settings, allowing for the successful removal of biomarkers for diagnostic applications, is not always possible. The situation is further complicated by the need to separate hydrophobic signatures such as lipids from blood. Herein, we present a microfluidic device capable of centrifugal separation of serum from blood at the point of need with a system that is compatible with biomarkers that are both hydrophilic and hydrophobic. The cross-flow filtration device separates serum from blood as efficiently as traditional methods and retains amphiphilic biomarkers in serum for detection.

Pediatric Tuberculosis: The Impact of "Omics" on Diagnostics Development.

Tuberculosis (TB) is a major public health concern for all ages. However, the disease presents a larger challenge in pediatric populations, partially owing to the lack of reliable diagnostic standards for the early identification of infection. Currently, there are no biomarkers that have been clinically validated for use in pediatric TB diagnosis. Identification and validation of biomarkers could provide critical information on prognosis of disease, and response to treatment. In this review, we discuss how the "omics" approach has influenced biomarker discovery and the advancement of a next generation rapid point-of-care diagnostic for TB, with special emphasis on pediatric disease. Limitations of current published studies and the barriers to their implementation into the field will be thoroughly reviewed within this article in hopes of highlighting future avenues and needs for combating the problem of pediatric tuberculosis.

Modeling and Cost Benefit Analysis to Guide Deployment of POC Diagnostics for Non-typhoidal Salmonella Infections with Antimicrobial Resistance.

Invasive non-typhoidal Salmonella (NTS) is among the leading causes of blood stream infections in sub-Saharan Africa and other developing regions, especially among pediatric populations. Invasive NTS can be difficult to treat and have high case-fatality rates, in part due to emergence of strains resistant to broad-spectrum antibiotics. Furthermore, improper treatment contributes to increased antibiotic resistance and death. Point of care (POC) diagnostic tests that rapidly identify invasive NTS infection, and differentiate between resistant and non-resistant strains, may greatly improve patient outcomes and decrease resistance at the community level. Here we present for the first time a model for NTS dynamics in high risk populations that can analyze the potential advantages and disadvantages of four strategies involving POC diagnostic deployment, and the resulting impact on antimicrobial treatment for patients. Our analysis strongly supports the use of POC diagnostics coupled with targeted antibiotic use for patients upon arrival in the clinic for optimal patient and public health outcomes. We show that even the use of imperfect POC diagnostics can significantly reduce total costs and number of deaths, provided that the diagnostic gives results quickly enough that patients are likely to return or stay to receive targeted treatment.

Detection of Lipid and Amphiphilic Biomarkers for Disease Diagnostics.

Rapid diagnosis is crucial to effectively treating any disease. Biological markers, or biomarkers, have been widely used to diagnose a variety of infectious and non-infectious diseases. The detection of biomarkers in patient samples can also provide valuable information regarding progression and prognosis. Interestingly, many such biomarkers are composed of lipids, and are amphiphilic in biochemistry, which leads them to be often sequestered by host carriers. Such sequestration enhances the difficulty of developing sensitive and accurate sensors for these targets. Many of the physiologically relevant molecules involved in pathogenesis and disease are indeed amphiphilic. This chemical property is likely essential for their biological function, but also makes them challenging to detect and quantify in vitro. In order to understand pathogenesis and disease progression while developing effective diagnostics, it is important to account for the biochemistry of lipid and amphiphilic biomarkers when creating novel techniques for the quantitative measurement of these targets. Here, we review techniques and methods used to detect lipid and amphiphilic biomarkers associated with disease, as well as their feasibility for use as diagnostic targets, highlighting the significance of their biochemical properties in the design and execution of laboratory and diagnostic strategies. The biochemistry of biological molecules is clearly relevant to their physiological function, and calling out the need for consideration of this feature in their study, and use as vaccine, diagnostic and therapeutic targets is the overarching motivation for this review.