A cell-free DNA metagenomic sequencing assay that integrates the host injury response to infection.

High-throughput metagenomic sequencing offers an unbiased approach to identify pathogens in clinical samples. Conventional metagenomic sequencing, however, does not integrate information about the host, which is often critical to distinguish infection from infectious disease, and to assess the severity of disease. Here, we explore the utility of high-throughput sequencing of cell-free DNA (cfDNA) after bisulfite conversion to map the tissue and cell types of origin of host-derived cfDNA, and to profile the bacterial and viral metagenome. We applied this assay to 51 urinary cfDNA isolates collected from a cohort of kidney transplant recipients with and without bacterial and viral infection of the urinary tract. We find that the cell and tissue types of origin of urinary cfDNA can be derived from its genome-wide profile of methylation marks, and strongly depend on infection status. We find evidence of kidney and bladder tissue damage due to viral and bacterial infection, respectively, and of the recruitment of neutrophils to the urinary tract during infection. Through direct comparison to conventional metagenomic sequencing as well as clinical tests of infection, we find this assay accurately captures the bacterial and viral composition of the sample. The assay presented here is straightforward to implement, offers a systems view into bacterial and viral infections of the urinary tract, and can find future use as a tool for the differential diagnosis of infection.

Diversity and Evolution of Viral Pathogen Community in Cave Nectar Bats (Eonycteris spelaea).

Bats are unique mammals, exhibit distinctive life history traits and have unique immunological approaches to suppression of viral diseases upon infection. High-throughput next-generation sequencing has been used in characterizing the virome of different bat species. The cave nectar bat, Eonycteris spelaea, has a broad geographical range across Southeast Asia, India and southern China, however, little is known about their involvement in virus transmission. Here we investigate the diversity and abundance of viral communities from a colony of Eonycteris spelaea residing in Singapore. Our results detected 47 and 22 different virus families from bat fecal and urine samples, respectively. Among these, we identify a large number of virus families including Adenoviridae, Flaviviridae, Reoviridae, Papillomaviridae, Paramyxoviridae, Parvoviridae, Picornaviridae, and Polyomaviridae. In most cases, viral sequences from Eonycteris spelaea are genetically related to a group of bat viruses from other bat genera (e.g., Eidolon, Miniopterus, Rhinolophus and Rousettus). The results of this study improve our knowledge of the host range, spread and evolution of several important viral pathogens. More significantly, our findings provide a baseline to study the temporal patterns of virus shedding and how they correlate with bat phenological trends.

Urinary leukotriene E4 levels in atopic and non-atopic preschool children with recurrent episodic (viral) wheezing: a potential marker?

UNLABELLED: Backround: Reliable biological markers for the differentiation of asthma phenotypes in preschool children with wheezing are lacking. The purpose of the study is to assess the relationship of urinary Leukotriene E4 (U-LTE4) to particular asthma phenotypes in preschool children with recurrent episodic (viral) wheezing following upper respiratory tract infections with or without atopic predisposition. METHODS: Ninety-six preschool patients with recurrent episodic wheezing participated, 52 atopic and 44 non-atopic, during exacerbation and in remission. Exacerbation was defined on clinical basis (wheeze in the presence of coryzal symptoms). Atopy was determined by specific serum IgE measurement and skin-prick testing. U-LTE4 was determined by enzyme immunoassay. Thirty-six age-matched, non-asthmatic, non-atopic children served as controls. RESULTS: During exacerbation, U-LTE4 was significantly higher in all children with recurrent episodic wheezing in comparison to A: Remission: 642.20 ± 268 versus 399.45 ± 204, p value <0.001 and B: CONTROLS: 642.20 ± 268 versus 271.39 ± 83, p value <0.001. Atopic patients demonstrated significantly higher levels of U-LTE4 compared to non-atopic, both during exacerbation 872.13 ± 246 versus 613.15 ± 150, p value = 0.0013 and during remission 507.59 ± 182 versus 283.59 ± 160, p value <0.001. During remission, a highly significant difference of U-LTE4 was found when controls were compared to atopic patients: 271.39 ± 83 versus 507.59 ± 182, p value = 0.002 but not when compared to non-atopic ones: 271.39 ± 83 versus 283.59 ± 160, p value = 0.432. CONCLUSION: U-LTE4 is strongly associated with the acute wheeze episode in preschool children, more so in atopics. Increased basal levels of U-LTE4 occur only in atopics. This suggests a potential role of U-LTE4 as a marker of atopic, virus-induced asthma in preschool children.

Quantitative nucleic acid amplification methods for viral infections.

BACKGROUND: Over the past 2 decades there have been substantial improvements in the methods used to quantify viral nucleic acid in body fluids and in our understanding of how to use viral load measurements in the diagnosis and management of patients with a number of viral infections. These methods are now integrated into a wide range of diagnostic and treatment guidelines and commonly deployed in a variety of clinical settings. CONTENT: Quantitative nucleic acid amplification methods that are used to measure viral load are described along with key issues and important variables that affect their performance. Particular emphasis is placed on those methods used in clinical laboratories as US Food and Drug Administration-cleared or laboratory-developed tests. We discuss the clinical applications of these methods in patients with HIV-1, hepatitis C virus, hepatitis B virus, cytomegalovirus, Epstein-Barr virus, and BK polyomavirus infections. Finally, the current challenges and future directions of viral load testing are examined. SUMMARY: Quantitative nucleic acid amplification tests provide important information that can be used to predict disease progression, distinguish symptomatic from asymptomatic infection, and assess the efficacy of antiviral therapy. Despite the advances in technology, large challenges remain for viral testing related to accuracy, precision, and standardization. Digital PCR, a direct method of quantification of nucleic acids that does not rely on rate-based measurements or calibration curves, may address many of the current challenges.

Evaluation of urinary prostaglandin E2 metabolite as a biomarker in infants with fever due to viral infection.

We have investigated the clinical feasibility of the major urinary metabolite of prostaglandin (PG) E2, tetranor-PGEM, as a biomarker of inflammation in infants with fever. We tested two different and clinically relevant sampling methods, using self-adhesive urinary bags or gauze pads, with respect to stability of tetranor-PGEM and ease of sampling from infants. Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis was used to quantify tetranor-PGEM in urine, and different normalization parameters, i.e., urinary creatinine and body surface area, were investigated. To study inflammation, infants (1 month-1 year) that were hospitalized with fever of unknown origin at admittance (n=14) were compared to age-matched healthy controls (n=14). Levels of urinary tetranor-PGEM in infants with viral induced fever were increased compared to controls (102.4±56.2 vs. 37.0±21.6pmol/ml/m(2) body surface area, p<0.001). We conclude that urinary tetranor-PGEM is a potential non-invasive biomarker of inflammation in infants.

Hendra virus infection dynamics in Australian fruit bats.

Hendra virus is a recently emerged zoonotic agent in Australia. Since first described in 1994, the virus has spilled from its wildlife reservoir (pteropid fruit bats, or 'flying foxes') on multiple occasions causing equine and human fatalities. We undertook a three-year longitudinal study to detect virus in the urine of free-living flying foxes (a putative route of excretion) to investigate Hendra virus infection dynamics. Pooled urine samples collected off plastic sheets placed beneath roosting flying foxes were screened for Hendra virus genome by quantitative RT-PCR, using a set of primers and probe derived from the matrix protein gene. A total of 1672 pooled urine samples from 67 sampling events was collected and tested between 1 July 2008 and 30 June 2011, with 25% of sampling events and 2.5% of urine samples yielding detections. The proportion of positive samples was statistically associated with year and location. The findings indicate that Hendra virus excretion occurs periodically rather than continuously, and in geographically disparate flying fox populations in the state of Queensland. The lack of any detection in the Northern Territory suggests prevalence may vary across the range of flying foxes in Australia. Finally, our findings suggest that flying foxes can excrete virus at any time of year, and that the apparent seasonal clustering of Hendra virus incidents in horses and associated humans (70% have occurred June to October) reflects factors other than the presence of virus. Identification of these factors will strengthen risk minimization strategies for horses and ultimately humans.

Mass spectrometry-coupled techniques for viral-related disease biomarker identification.

The advent of high-resolution mass spectrometers coupled with proteomic techniques has facilitated the discovery and characterization of novel viral proteins and the detection of virus-induced changes in the cellular proteome. These advances have enabled a more comprehensive characterization of viral interactions involved in infection and pathogenesis, and allowed the discovery of viral biomarkers. This article focuses on the role of mass spectrometry proteomic techniques to identify and characterize both prospective and verified viral biomarkers, and their implications on the diagnosis of disease.

Improved silica-guanidiniumthiocyanate DNA isolation procedure based on selective binding of bovine alpha-casein to silica particles.

DNA purified from clinical cerebrospinal fluid and urine specimens by a silica-guanidiniumthiocyanate procedure frequently contained an inhibitor(s) of DNA-processing enzymes which may have been introduced by the purification procedure itself. Inhibition could be relieved by the use of a novel lysis buffer containing alpha-casein. When the novel lysis buffer was used, alpha-casein was bound by the silica particles in the first step of the procedure and eluted together with DNA in the last step, after which it exerted its beneficial effects for DNA-processing enzymes. In the present study we have compared the novel lysis buffer with the previously described lysis buffer with respect to double-stranded DNA yield (which was nearly 100%) and the performance of DNA-processing enzymes.

[Renal manifestations of viral diseases]. / Manifestations rénales des affections virales.

Like bacterial diseases viral diseases may also be accompanied by functional renal disorders or abnormalities of the urinary sediment. Thus, to find a hematuria or an isolated proteinuria in the context of influenza or hepatitis is not rare at all. In certain cases viral affections may even be accompanied by a nephrotic syndrome. This article aims at the discussion of multiple renal disorders appearing in the context of hepatitis B and C and AIDS.

[Neopterin as marker of immunologic activation in viral and bacterial infections]. / Neopterina como marcador de la activación inmune en infecciones víricas y bacterianas.

OBJECTIVE: Neopterin is released mainly by macrophages during immune system activation. Therefore, the production of neopterin should be increased by infections and its levels in blood and urine are perhaps in relation to the type of infection. PATIENTS AND METHODS: Neopterin levels in urine were determined in 194 healthy children to obtain normal values. Levels of neopterin were also studied in 91 children with viral and 41 with bacterial infections by using high performance liquid chromatography (HPLC). The results are expressed by means of the neopterin/creatinine quotient (neopterin factor = NF). RESULTS: NF significantly increased in 95.6% of the viral infections (sensitivity of 95.6% and specificity of 92.7%) and in 40.4% of the bacterial infections (sensitivity of 40.4% and specificity of 92.7%). CONCLUSIONS: NF rises more frequently in viral than in bacterial infections, reaching higher levels earlier in the former.

Urinary free cortisol values in children under stress.

Children with adrenocortical insufficiency are commonly instructed to increase their baseline glucocorticoid replacement doses by three to five times during periods of stress such as surgery or febrille illness. We conducted this to determine whether these recommendations reflect the actual change in urinary free cortisol (UFC) output during stress. The 24-hour UFC excretion was determined in 78 children who were admitted to a general pediatric department or intensive care unit with temperature > 38.7 degrees C, after major surgery, or during status epilepticus; we reevaluated 43 of the patients 2 weeks after recovery. In addition, the 24-hour UFC levels were determined in 127 healthy children aged 1.8 to 17 years. The UFC level positively correlated with age (r = 0.254; p < 0.001). The amount of UFC per gram of creatinine was inversely correlated with age (r = 0.255; p < 0.001). The amount of UFC per surface area was independent of age. The mean change in the level of UFC per square meter surface area was highest among children who had cardiothoracic surgery and those with multiple trauma. The increase in UFC level during bacterial infection was significantly greater than that during viral infection. The current recommendation to increase the dose to three to five times the baseline glucocorticoid dose during times of stress may underestimate the changes in UFC found in some patients with major surgery, trauma, or certain serious bacterial infections. Production rate studies are needed to prove this point.

The fractional excretion of urea: a new diagnostic test for acute renal allograft rejection.

Fractional excretion of sodium (FENa) has been used in the diagnosis of acute renal allograft failure on the assumption that poor allograft perfusion should result in a low FENa. However, many patients receive medications which affect the active transport of Na+ and thus FENa. In contrast, the fractional excretion of urea (FEurea) is mostly dependent on passive forces and is therefore less influenced by drug therapy. To test the hypothesis that FEurea might be more useful than FENa in evaluating graft failure, we compared FEurea with FENa during 79 episodes of acute renal allograft dysfunction due to acute rejection (AR), cyclosporine nephrotoxicity (CsA-Nx), viral infection, or bacterial infection in 32 children and young adults with renal transplants. There was no significant difference between groups in FENa. However, FEurea was significantly lower (P < 0.05) in patients with CsA-Nx (32.6 +/- 1.9%) and viral infection (32.9 +/- 3.2%) than those with AR (45.1 +/- 1.7%) or bacterial infection (38.9 +/- 2.5%). FEurea was < 35% in 20 of 28 (71.4%) episodes of CsA-Nx and 8 of 11 (72.2%) of viral infection, but only 5 of 36 (13.9%) of AR (P < 0.05). FEurea was also measured during stable graft function, 7-14 days prior to allograft dysfunction. CsA-Nx was associated with a 30.5 +/- 8.3% decrease in FEurea. FEurea did not change in patients with AR. Based on these findings, we present an algorithm to aid in the differential diagnosis of acute renal allograft failure.

Value of urinary neopterin in the differential diagnosis of bacterial and viral infections.

Neopterin is released by stimulated macrophages. In this study we analyzed the diagnostic potential of urinary neopterin concentrations in patients with bacterial and viral infection. All but one of 17 patients with viral infection had increased urinary neopterin concentrations. Patients with bacterial urinary tract infection also showed increased neopterin concentrations, whereas patients with bacterial pneumonia had significantly lower neopterin levels. In addition, patients with acute bacterial pneumonia had lower neopterin levels than patients with protracted infection. A significant inverse correlation between urinary neopterin and hemoglobin concentrations was found. Neopterin concentrations could serve as a helpful additional marker of infectious diseases. Combined with other clinical and laboratory parameters it is a useful parameter for distinguishing between viral and bacterial origins of infection, as was shown by multivariate stepwise linear discriminant analysis.

Temperature-sensitive gel for virus concentration from urine.

Cross-linked, partially hydrolyzed polyacrylamide gels (temperature-sensitive gels) with the property to swell at 4 degrees C and collapse at higher temperatures (greater than 19 degrees C) were used to concentrate bacteriophage T-2 from urine. Samples of urine, 50 ml, seeded with bacteriophage T-2 were reduced to approximately 5 ml, with an average virus recovery of 53%. Subsequent experiments with feline cell-associated herpes virus resulted in a 6-fold decrease in volume with 54% virus recovery. The gel could be used repeatedly without any loss in efficiency.

Episodes of severe metabolic acidosis in a patient with 3-methylglutaconic aciduria.

Persistent excretion of 3-methylglutaconic acid was found in a 6-month-old infant with multiple minor physical malformations and delayed development. During two episodes of intercurrent viral illness, the patient developed severe metabolic acidosis and excreted large amounts of lactate, 3-hydroxybutyrate and acetoacetate. The excretion of 3-methylglutaconic acid did not change during these episodes, nor did it increase following leucine loading. In vitro studies suggest that in this patient, as in the majority of other patients with 3-methylglutaconic aciduria, a primary defect in leucine metabolism is not responsible for the biochemical abnormality.

Neopterin in AIDs, other immunodeficiencies, and bacterial and viral infections.

An increase in total urinary neopterin was observed in 12 of 13 patients with acquired immunodeficiency syndrome (AIDS), seven of 13 patients with lymphadenopathy, one of six healthy homosexual males, seven of ten adult patients with staphylococcal pneumonia, 11 of 12 children with viral infections, four of seven children with bacterial infections, and 12 of 13 children with various immune defects. Extremely high values of total urinary neopterin and monapterin were observed in severely ill patients with AIDS and those with familial hemophagocytic lymphohistiocytosis. Neopterin excretion was normal in two AIDS patients with Kaposi's sarcoma, but without opportunistic infections at that time. On reexamination of one of these patients later on, elevated neopterin values were noted. Parallel increases in neopterin and monapterin were found, whereas biopterin was usually normal. The increase in total neopterin was mainly due to 7,8-dihydroneopterin and was accompanied by an increase in 3'-hydroxysepiapterin. Increased neopterin in urine is assumed to reflect the increase in GTP pool and GTP cyclohydrolase I activity as observed in stimulated monocytes. Thus, neopterin, as a measure of the activation of the nonspecific cellular immune system, may be used diagnostically to detect allograft rejection after transplantations and to follow-up HTLV-III positive patients.

Cytomegalovirus infections in homosexual men. An epidemiological study.

Levels of cytomegalovirus antibody (IgG and IgM) were measured and urine viral cultures were done in 237 homosexual men over a mean period of 14.1 months. The initial prevalence of cytomegalovirus IgG antibody was 86.9%. By the 9th month of follow-up, 71% of serosusceptible men had become infected with cytomegalovirus. During the study period cytomegaloviruria was noted in 32% of seropositive men. Cytomegalovirus IgM antibody was intermittently present in the serum of 95% of IgG-seropositive men, suggesting that frequent reactivation of latent infection or reexposure to exogenous virus had occurred. Of seven sexual practices investigated, only passive anal-genital intercourse correlated with the acquisition of cytomegalovirus infection (p = 0.008).