Metagenomic next generation sequencing of plasma RNA for diagnosis of unexplained, acute febrile illness in Uganda.

Metagenomic next generation metagenomic sequencing (mNGS) has proven to be a useful tool in the diagnosis and identification of novel human pathogens and pathogens not identified on routine clinical microbiologic tests. In this study, we applied mNGS to characterize plasma RNA isolated from 42 study participants with unexplained acute febrile illness (AFI) admitted to tertiary referral hospitals in Mubende and Arua, Uganda. Study participants were selected based on clinical criteria suggestive of viral infection (i.e., thrombocytopenia, leukopenia). The study population had a median age of 28 years (IQR:24 to 38.5) and median platelet count of 114 x103 cells/mm3 (IQR:66,500 to 189,800). An average of 25 million 100 bp reads were generated per sample. We identified strong signals from diverse virus, bacteria, fungi, or parasites in 10 (23.8%) of the study participants. These included well recognized pathogens like Helicobacter pylori, human herpes virus-8, Plasmodium falciparum, Neisseria gonorrhoeae, and Rickettsia conorii. We further confirmed Rickettsia conorii infection, the cause of Mediterranean Spotted Fever (MSF), using PCR assays and Sanger sequencing. mNGS was a useful addition for detection of otherwise undetected pathogens and well-recognized non-pathogens. This is the first report to describe the molecular confirmation of a hospitalized case of MSF in sub-Saharan Africa (SSA). Further studies are needed to determine the utility of mNGS for disease surveillance in similar settings.

Plasma cell-free RNA signatures of inflammatory syndromes in children.

Inflammatory syndromes, including those caused by infection, are a major cause of hospital admissions among children and are often misdiagnosed because of a lack of advanced molecular diagnostic tools. In this study, we explored the utility of circulating cell-free RNA (cfRNA) in plasma as an analyte for the differential diagnosis and characterization of pediatric inflammatory syndromes. We profiled cfRNA in 370 plasma samples from pediatric patients with a range of inflammatory conditions, including Kawasaki disease (KD), multisystem inflammatory syndrome in children (MIS-C), viral infections, and bacterial infections. We developed machine learning models based on these cfRNA profiles, which effectively differentiated KD from MIS-C-two conditions presenting with overlapping symptoms-with high performance [test area under the curve = 0.98]. We further extended this methodology into a multiclass machine learning framework that achieved 80% accuracy in distinguishing among KD, MIS-C, viral, and bacterial infections. We further demonstrated that cfRNA profiles can be used to quantify injury to specific tissues and organs, including the liver, heart, endothelium, nervous system, and the upper respiratory tract. Overall, this study identified cfRNA as a versatile analyte for the differential diagnosis and characterization of a wide range of pediatric inflammatory syndromes.

Serologic screening for viral infections among blood donors: a study in a blood bank in southern Brazil.

BACKGROUND: Routine screening for viral infections at blood donation is important to avoid transfusion-transmitted infections. It also offers an opportunity to detect an asymptomatic infection. OBJECTIVE: To study changes in serology positivity for viral infections (B and C hepatitis, HTLV-1/2, and HIV) at blood donation in a blood bank from Southern Brazil, comparing two periods of 5 years: the period from 2013 to 2017 with the period from 2018 to 2022. In addition, data on the donor fidelity rate during the studied period were sought. METHODS: Retrospective study using data from 2013 to 2022 from a single blood center electronic database from Curitiba, Southern Brazil. RESULTS: A significant drop in positive serology for all studied viruses was observed: highest in HIV (OR=0.39; 95% CI=0.27-0.57) and lowest in total anti HBc (0.56; 95 CI=0.50-0.63). Anti HBc serology became more commonly seen in women in the period of 2018-2022 when compared to men. No changes in the distribution of positive serology according to donors' ages were observed. Loyalty rates had a median of 70%, with the lowest being 60% in 2013, while the highest was 73% in 2018 and 2022. CONCLUSION: A significant reduction in discarded blood bags due to viral serology was observed when the period of 2013-2017 was compared to 2018-2022 on this blood bank; the highest reduction was observed in HIV serology and the lowest in HBc serology, which became more common in women in the second period. High rates of donor fidelity were observed during the period studied.

Interleukins in the pathogenesis of influenza and other acute respiratory viral infections.

Influenza and other acute respiratory viral infections (ARVIs) are among the most common human diseases. In recent decades, the discovery of cytokines and their significance in the pathogenesis of diseases has led to extensive research on these compounds in various pathologies including ARVIs. The aim of the research was to study the cytokine profile in patients with ARVIs. The cases of 30 patients were investigated. Etiological diagnosis was performed by polymerase chain reaction. Different classes of cytokines in the serum were defined by the enzyme-linked immunosorbent assay (ELISA). The level of cytokines depended on the number of pathogens. The highest levels of pro-inflammatory interleukins and the lowest levels of anti-inflammatory IL-4 were observed in patients with a combination of five or more viruses compared to those with a monoinfection. Analysis of the data showed that in the acute phase, the levels of all studied pro-inflammatory cytokines - IL-2, IL-6, and TNF-α - increased by 8, 39, and 9 times, respectively, compared to those in healthy individuals. In the acute phase of ARVI, the levels of pro-inflammatory cytokines were significantly higher and depended on the severity of the disease. The imbalance of cytokines in the serum has been established in cases of ARVIs, depending on the severity of the disease.

The plasma viral communities associate with clinical profiles in a large-scale haematological patients cohort.

BACKGROUND: Haematological patients exhibit immune system abnormalities that make them susceptible to viral infections. Understanding the relationship between the virome in the blood plasma of haematological patients and their clinical characteristic is crucial for disease management. We aimed to explore the presence of viral pathogens and identify close associations between viral infections and various clinical features. RESULTS: A total of 21 DNA viruses and 6 RNA viruses from 12 virus families were identified from 1383 patients. Patients with haematological diseases exhibited significantly higher diversity, prevalence, and co-detection rates of viral pathogens. During fever episodes, pathogen detection was notably higher, with Epstein-Barr virus (EBV) and Mucorales infections being the most probable culprits for fever symptoms in non-haematological patients. The detection rate of torque teno virus (TTV) significantly increases in haematological patients after transplantation and during primary lung infections. Additionally, TTV-positive patients demonstrate significantly higher absolute neutrophil counts, while C-reactive protein and procalcitonin levels are notably lower. Furthermore, TTV, cytomegalovirus, and parvovirus B19 (B19V) were found to be more prevalent in non-neutropenic patients, while non-viral pathogenic infections, such as Gram-negative bacteria and Mucorales, were more common in neutropenic patients. Pegivirus C (HPgV-C) infection often occurred post-transplantation, regardless of neutropenia. Additionally, some viruses such as TTV, B19V, EBV, and HPgV-C showed preferences for age and seasonal infections. CONCLUSIONS: Analysis of the plasma virome revealed the susceptibility of haematological patients to plasma viral infections at specific disease stages, along with the occurrence of mixed infections with non-viral pathogens. Close associations were observed between the plasma virome and various clinical characteristics, as well as clinical detection parameters. Understanding plasma virome aids in auxiliary clinical diagnosis and treatment, enabling early prevention to reduce infection rates in patients and improve their quality of life. Video Abstract.

Using blood TNF-related apoptosis-inducing ligand levels to discriminate between viral and bacterial infections: A prospective observational study.

OBJECTIVE: The aim of the investigation was to evaluate variations in blood TNF-related apoptosis-inducing ligand (TRAIL) levels between patients with viral and bacterial infections and the diagnostic performance of TRAIL for identifying viral and bacterial infections. METHODS: The investigation included 169 adult (>18 years) patients presenting with medical signs of acute infections (inclusion criteria included a body temperature over 37.5 °C, an onset of symptoms no more than 12 days). Reference standard was based on a rigorous expert panel and the majority of the panel determined the infectious etiology. Finally, 104 patients with 78 bacterial and 26 viral reference standard outcomes were enrolled in this investigation (24 were eliminated depending on the exclusion criteria; 41 had indeterminate reference standard diagnosis). ELISA was employed to measure TRAIL levels in the group of 78 subjects with bacterial infections and 26 individuals with viral infections, and the diagnostic performance of TRAIL was identified by receiver operating characteristic (ROC) analysis. RESULTS: The TRAIL level in individuals with bacterial infections was significantly lower than that in subjects with viral infections (16.59 (2.61-32.6) pg/mL vs. 97.39 (36.18-127.74) pg/mL, P < 0.05). The area under the ROC curve (AUC) of TRAIL was 0.86 (95 %CI:0.79 to 0.94) for identifying bacterial and viral infections. Combining TRAIL with C-reactive protein (CRP), the AUC was 0.94 (95 %CI:0.89 to 1.00). CONCLUSIONS: TRAIL is diagnostic for discriminating between viral and bacterial infections. Combining TRAIL with CRP increases the AUC.

The Blood Virome: A new frontier in biomedical science.

Recent advances in metagenomic testing opened a new window into the mammalian blood virome. Comprised of well-known viruses like human immunodeficiency virus, hepatitis C virus, and hepatitis B virus, the virome also includes many other eukaryotic viruses and phages whose medical significance, lifecycle, epidemiology, and impact on human health are less well known and thus regarded as commensals. This review synthesizes available information for the so-called commensal virome members that circulate in the blood of humans considering their restriction to and interaction with the human host, their natural history, and their impact on human health and physiology.

Rio Negro Virus Infection, Bolivia, 2021.

In May 2021, an agricultural worker originally from Trementinal, Argentina, sought treatment for febrile illness in Tarija, Bolivia, where he resided at the time of illness onset. The patient tested negative for hantavirus RNA, but next-generation sequencing of a serum sample yielded a complete genome for Rio Negro virus.

Red blood cell distribution width as a prognostic biomarker for viral infections: prospects and challenges.

Viral diseases remain a significant global health threat, and therefore prioritization of limited healthcare resources is required to effectively manage dangerous viral disease outbreaks. In a pandemic of a newly emerged virus that is yet to be well understood, a noninvasive host-derived prognostic biomarker is invaluable for risk prediction. Red blood cell distribution width (RDW), an index of red blood cell size disorder (anisocytosis), is a potential predictive biomarker for severity of many diseases. In view of the need to prioritize resources during response to outbreaks, this review highlights the prospects and challenges of RDW as a prognostic biomarker for viral infections, with a focus on hepatitis and COVID-19, and provides an outlook to improve the prognostic performance of RDW for risk prediction in viral diseases.

Ratio of serum amyloid A to C-reactive protein is constant in the same patients but differs greatly between patients with inflammatory diseases.

C-reactive protein (CRP) is commonly monitored to track the activity of inflammation and has become the gold standard in the management of all inflammatory diseases. Indeed, serum amyloid A (SAA) have seemed to correlate moderately with CRP, but the discrepancy of CRP and SAA levels has often been reported, especially in rheumatoid arthritis. Then, we examined CRP reflects a real magnitude of inflammation in patients with rheumatic and infectious inflammatory diseases. A total of 414 patients with infectious and non-infectious inflammatory diseases were enrolled. At initial visit, each patient underwent a clinical assessment and had also laboratory tests such as SAA and CRP. In each patient, we carried out a longitudinal analysis of CRP and SAA levels. We determined the inter-individual correlation between SAA and CRP and also clarified intra-individual changes of SAA/CRP ratio. SAA and CRP levels changed approximately linearly over time within individuals irrespective of rheumatic and infectious inflammatory diseases. However, SAA/CRP ratios differed dramatically between patients (from 0.117 to 50.8, median 5.71). In patients with high SAA/CRP ratio (>8.44), SAA is a better predictor of inflammation than CRP. In contrast, CRP is a better predictor in patients with low ratio (<3.52). Our results suggest that the SAA/CRP ratio differed greatly between individuals but was constant in intra-individuals. Low CRP levels could be accompanied by SAA levels predicting any degree of inflammation, implying that CRP is not reflecting a real magnitude of inflammation. To evaluate the real magnitude of inflammation, to access the SAA/CRP ratio in advance is essential.

Platelet-leukocyte interactions in the pathogenesis of viral infections.

Evolving evidence demonstrates that platelets have major roles in viral syndromes through previously unrecognized viral sensing and effector functions. Activated platelets and increased platelet-leukocyte aggregates are observed in clinical and experimental viral infections. The mechanisms and outcomes of platelet-leukocyte interactions depend on the interacting leukocyte as well as on the pathogen and pathological conditions. In this review, we discuss the mechanisms involved in platelet interactions with leukocytes and its functions during viral infections. We focus on the contributions of human platelet-leukocyte interactions to pathophysiological and protective responses during viral infections of major global health relevance, including acquired immunodeficiency syndrome (AIDS), dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), influenza pneumonia, and COVID-19.

Rapid detection of bacterial infection using a novel single-tube, four-colour flow cytometric method: Comparison with PCT and CRP.

BACKGROUND: A key factor behind the unnecessary use of antibiotics is the lack of rapid and accurate diagnostic tests. In this study, we developed a novel and fast flow cytometric single-tube method to detect bacterial infections within 30 minutes. METHODS: Quantitative flow cytometric four-colour analysis of host biomarkers CD35, CD64, CD329, and MHC class I expression on neutrophils and lymphocytes was performed on samples taken from 841 febrile patients with suspected infection. Obtained data was incorporated into the four-colour bacterial infection (FCBI)-index, using the developed bacterial infection algorithm. FINDINGS: In distinguishing between microbiologically confirmed bacterial (n = 193) and viral (n = 291) infections, the FCBI-index method was superior to serum C-reactive protein (CRP) and procalcitonin (PCT). In 269 confirmed viral respiratory tract infections, 43% (95% CI: 37-49%) of the patients had an increased FCBI-index, suggesting probable bacterial coinfection. INTERPRETATION: The proposed FCBI-index test might be a potent additional tool when assessing appropriateness of empiric antibiotic treatment. FUNDING: This study has been financially supported by Turku University Hospital (Turku, Finland) and The Finnish Medical Foundation.

Genetic Analyses of Common Infections in the Avon Longitudinal Study of Parents and Children Cohort.

The burden of infections on an individual and public health is profound. Many observational studies have shown a link between infections and the pathogenesis of disease; however a greater understanding of the role of host genetics is essential. Children from the longitudinal birth cohort, the Avon Longitudinal Study of Parents and Children, had 14 antibodies measured in plasma at age 7: Alpha-casein protein, beta-casein protein, cytomegalovirus, Epstein-Barr virus, feline herpes virus, Helicobacter pylori, herpes simplex virus 1, influenza virus subtype H1N1, influenza virus subtype H3N2, measles virus, Saccharomyces cerevisiae, Theiler's virus, Toxoplasma gondii, and SAG1 protein domain, a surface antigen of Toxoplasma gondii measured for greater precision. We performed genome-wide association analyses of antibody levels against these 14 infections (N = 357 - 5010) and identified three genome-wide signals (P < 5×10-8), two associated with measles virus antibodies and one with Toxoplasma gondii antibodies. In an association analysis focused on the human leukocyte antigen (HLA) region of the genome, we further detected 15 HLA alleles at a two-digit resolution and 23 HLA alleles at a four-digit resolution associated with five antibodies, with eight HLA alleles associated with Epstein-Barr virus antibodies showing strong evidence of replication in UK Biobank. We discuss how our findings from antibody levels complement other studies using self-reported phenotypes in understanding the architecture of host genetics related to infections.

C-reactive protein or procalcitonin combined with rhinorrhea for discrimination of viral from bacterial infections in hospitalized adults in non-intensive care units with lower respiratory tract infections.

BACKGROUND: Whether procalcitonin (PCT) or C-reactive protein (CRP) combined with certain clinical characteristics can better distinguish viral from bacterial infections remains unclear. The aim of the study was to assess the ability of PCT or CRP combined with clinical characteristics to distinguish between viral and bacterial infections in hospitalized non-intensive care unit (ICU) adults with lower respiratory tract infection (LRTI). METHODS: This was a post-hoc analysis of a randomized clinical trial previously conducted among LRTI patients. The ability of PCT, CRP and PCT or CRP combined with clinical symptoms to discriminate between viral and bacterial infection were assessed by portraying receiver operating characteristic (ROC) curves among patients with only a viral or a typical bacterial infection. RESULTS: In total, 209 infected patients (viral 69%, bacterial 31%) were included in the study. When using CRP or PCT to discriminate between viral and bacterial LRTI, the optimal cut-off points were 22 mg/L and 0.18 ng/mL, respectively. When the optimal cut-off for CRP (≤ 22 mg/L) or PCT (≤ 0.18 ng/mL) combined with rhinorrhea was used to discriminate viral from bacterial LRTI, the AUCs were 0.81 (95% CI: 0.75-0.87) and 0.80 (95% CI: 0.74-0.86), which was statistically significantly better than when CRP or PCT used alone (p < 0.001). When CRP ≤ 22 mg/L, PCT ≤ 0.18 ng/mL and rhinorrhea were combined, the AUC was 0.86 (95% CI: 0.80-0.91), which was statistically significantly higher than when CRP (≤ 22 mg/L) or PCT (≤ 0.18 ng/mL) was combined with rhinorrhea (p = 0.011 and p = 0.021). CONCLUSIONS: Either CRP ≤ 22 mg/L or PCT ≤ 0.18 ng/mL combined with rhinorrhea could help distinguish viral from bacterial infections in hospitalized non-ICU adults with LRTI. When rhinorrhea was combined together, discrimination ability was further improved.

A Novel 29-Messenger RNA Host-Response Assay From Whole Blood Accurately Identifies Bacterial and Viral Infections in Patients Presenting to the Emergency Department With Suspected Infections: A Prospective Observational Study.

OBJECTIVES: The rapid diagnosis of acute infections and sepsis remains a serious challenge. As a result of limitations in current diagnostics, guidelines recommend early antimicrobials for suspected sepsis patients to improve outcomes at a cost to antimicrobial stewardship. We aimed to develop and prospectively validate a new, 29-messenger RNA blood-based host-response classifier Inflammatix Bacterial Viral Non-Infected version 2 (IMX-BVN-2) to determine the likelihood of bacterial and viral infections. DESIGN: Prospective observational study. SETTING: Emergency Department, Campus Benjamin Franklin, Charité-Universitätsmedizin Berlin, Germany. PATIENTS: Three hundred twelve adult patients presenting to the emergency department with suspected acute infections or sepsis with at least one vital sign change. INTERVENTIONS: None (observational study only). MEASUREMENTS AND MAIN RESULTS: Gene expression levels from extracted whole blood RNA was quantified on a NanoString nCounter SPRINT (NanoString Technologies, Seattle, WA). Two predicted probability scores for the presence of bacterial and viral infection were calculated using the IMX-BVN-2 neural network classifier, which was trained on an independent development set. The IMX-BVN-2 bacterial score showed an area under the receiver operating curve for adjudicated bacterial versus ruled out bacterial infection of 0.90 (95% CI, 0.85-0.95) compared with 0.89 (95% CI, 0.84-0.94) for procalcitonin with procalcitonin being used in the adjudication. The IMX-BVN-2 viral score area under the receiver operating curve for adjudicated versus ruled out viral infection was 0.83 (95% CI, 0.77-0.89). CONCLUSIONS: IMX-BVN-2 demonstrated accuracy for detecting both viral infections and bacterial infections. This shows the potential of host-response tests as a novel and practical approach for determining the causes of infections, which could improve patient outcomes while upholding antimicrobial stewardship.

Selenium.

Selenium (Se) is an element commonly found in the environment at different levels. Its compounds are found in soil, water, and air. This element is also present in raw materials of plant and animal origin, so it can be introduced into human organisms through food. Selenium is a cofactor of enzymes responsible for the antioxidant protection of the body and plays an important role in regulating inflammatory processes in the body. A deficiency in selenium is associated with a number of viral diseases, including COVID-19. This element, taken in excess, may have a toxic effect in the form of joint diseases and diseases of the blood system. Persistent selenium deficiency in the body may also impact infertility, and in such cases supplementation is needed.

Future Biomarkers for Infection and Inflammation in Febrile Children.

Febrile patients, suffering from an infection, inflammatory disease or autoimmunity may present with similar or overlapping clinical symptoms, which makes early diagnosis difficult. Therefore, biomarkers are needed to help physicians form a correct diagnosis and initiate the right treatment to improve patient outcomes following first presentation or admittance to hospital. Here, we review the landscape of novel biomarkers and approaches of biomarker discovery. We first discuss the use of current plasma parameters and whole blood biomarkers, including results obtained by RNA profiling and mass spectrometry, to discriminate between bacterial and viral infections. Next we expand upon the use of biomarkers to distinguish between infectious and non-infectious disease. Finally, we discuss the strengths as well as the potential pitfalls of current developments. We conclude that the use of combination tests, using either protein markers or transcriptomic analysis, have advanced considerably and should be further explored to improve current diagnostics regarding febrile infections and inflammation. If proven effective when combined, these biomarker signatures will greatly accelerate early and tailored treatment decisions.

Quantification of Cytokine Storms During Virus Infections.

Highly pathogenic virus infections usually trigger cytokine storms, which may have adverse effects on vital organs and result in high mortalities. The two cytokines interleukin (IL)-4 and interferon (IFN)-γ play key roles in the generation and regulation of cytokine storms. However, it is still unclear whether the cytokine with the largest induction amplitude is the same under different virus infections. It is unknown which is the most critical and whether there are any mathematical formulas that can fit the changing rules of cytokines. Three coronaviruses (SARS-CoV, MERS-CoV, and SARS-CoV-2), three influenza viruses (2009H1N1, H5N1 and H7N9), Ebola virus, human immunodeficiency virus, dengue virus, Zika virus, West Nile virus, hepatitis B virus, hepatitis C virus, and enterovirus 71 were included in this analysis. We retrieved the cytokine fold change (FC), viral load, and clearance rate data from these highly pathogenic virus infections in humans and analyzed the correlations among them. Our analysis showed that interferon-inducible protein (IP)-10, IL-6, IL-8 and IL-17 are the most common cytokines with the largest induction amplitudes. Equations were obtained: the maximum induced cytokine (max) FC = IFN-γ FC × (IFN-γ FC/IL-4 FC) (if IFN-γ FC/IL-4 FC > 1); max FC = IL-4 FC (if IFN-γ FC/IL-4 FC < 1). For IFN-γ-inducible infections, 1.30 × log2 (IFN-γ FC) = log10 (viral load) - 2.48 - 2.83 × (clearance rate). The clinical relevance of cytokines and their antagonists is also discussed.

A host transcriptomic signature for identification of respiratory viral infections in the community.

BACKGROUND: Fever-7 is a test evaluating host mRNA expression levels of IFI27, JUP, LAX, HK3, TNIP1, GPAA1 and CTSB in blood able to detect viral infections. This test has been validated mostly in hospital settings. Here we have evaluated Fever-7 to identify the presence of respiratory viral infections in a Community Health Center. METHODS: A prospective study was conducted in the "Servicio de Urgencias de Atención Primaria" in Salamanca, Spain. Patients with clinical signs of respiratory infection and at least one point in the National Early Warning Score were recruited. Fever-7 mRNAs were profiled on a Nanostring nCounter® SPRINT instrument from blood collected upon patient enrolment. Viral diagnosis was performed on nasopharyngeal aspirates (NPAs) using the Biofire-RP2 panel. RESULTS: A respiratory virus was detected in the NPAs of 66 of the 100 patients enrolled. Median National Early Warning Score was 7 in the group with no virus detected and 6.5 in the group with a respiratory viral infection (P > .05). The Fever-7 score yielded an overall AUC of 0.81 to predict a positive viral syndromic test. The optimal operating point for the Fever-7 score yielded a sensitivity of 82% with a specificity of 71%. Multivariate analysis showed that Fever-7 was a robust marker of viral infection independently of age, sex, major comorbidities and disease severity at presentation (OR [CI95%], 3.73 [2.14-6.51], P < .001). CONCLUSIONS: Fever-7 is a promising host immune mRNA signature for the early identification of a respiratory viral infection in the community.

Interpretation of T cell states from single-cell transcriptomics data using reference atlases.

Single-cell RNA sequencing (scRNA-seq) has revealed an unprecedented degree of immune cell diversity. However, consistent definition of cell subtypes and cell states across studies and diseases remains a major challenge. Here we generate reference T cell atlases for cancer and viral infection by multi-study integration, and develop ProjecTILs, an algorithm for reference atlas projection. In contrast to other methods, ProjecTILs allows not only accurate embedding of new scRNA-seq data into a reference without altering its structure, but also characterizing previously unknown cell states that "deviate" from the reference. ProjecTILs accurately predicts the effects of cell perturbations and identifies gene programs that are altered in different conditions and tissues. A meta-analysis of tumor-infiltrating T cells from several cohorts reveals a strong conservation of T cell subtypes between human and mouse, providing a consistent basis to describe T cell heterogeneity across studies, diseases, and species.