Next-generation 16S ribosomal RNA gene sequencing is widely used to determine the relative composition of the mammalian gut microbiomes. However, in the absence of a reference, this does not reveal alterations in absolute abundance of specific operational taxonomic units if microbial loads vary across specimens. Here we suggest the spiking of exogenous bacteria into crude specimens to quantify ratios of absolute bacterial abundances. We use the 16S rDNA read counts of the spike-in bacteria to adjust the read counts of endogenous bacteria for changes in total microbial loads. Using a series of dilutions of pooled faecal samples from mice containing defined amounts of the spike-in bacteria Salinibacter ruber, Rhizobium radiobacter and Alicyclobacillus acidiphilus, we demonstrate that spike-in-based calibration to microbial loads allows accurate estimation of ratios of absolute endogenous bacteria abundances. Applied to stool specimens of patients undergoing allogeneic stem cell transplantation, we were able to determine changes in both relative and absolute abundances of various phyla, especially the genus Enterococcus, in response to antibiotic treatment and radio-chemotherapeutic conditioning. Exogenous spike-in bacteria in gut microbiome studies enable estimation of ratios of absolute OTU abundances, providing novel insights into the structure and the dynamics of intestinal microbiomes.

Indole, which is produced from l-tryptophan by commensal bacteria expressing tryptophanase, not only is an important intercellular signal in microbial communities, but also modulates mucosal barrier function and expression of pro- and anti-inflammatory genes by intestinal epithelial cells. Here, we hypothesized that decreased urinary excretion of 3-indoxyl sulfate (3-IS), the major conjugate of indole found in humans, may be a marker of gut microbiota disruption and increased risk of developing gastrointestinal (GI) graft-versus-host-disease. Using liquid chromatography/tandem mass spectrometry, 3-IS was determined in urine specimens collected weekly within the first 28 days after allogeneic stem cell transplantation (ASCT) in 131 patients. Low 3-IS levels within the first 10 days after ASCT were associated with significantly higher transplant-related mortality (P = .017) and worse overall survival (P = .05) 1 year after ASCT. Least absolute shrinkage and selection operator regression models trained on log-normalized counts of 763 operational taxonomic units derived from next-generation sequencing of the hypervariable V3 region of the 16S ribosomal RNA gene showed members of the families of Lachnospiraceae and Ruminococcaceae of the class of Clostridia to be associated with high urinary 3-IS levels, whereas members of the class of Bacilli were associated with low 3-IS levels. Risk factors of early suppression of 3-IS levels were the type of GI decontamination (P = .01), early onset of antibiotic treatment (P = .001), and recipient NOD2/CARD15 genotype (P = .04). In conclusion, our findings underscore the relevance of microbiota-derived indole and metabolites thereof in mucosal integrity and protection from inflammation. Publisher9s Note: There is an Inside Blood Commentary on this article in this issue. Key Points Urinary 3-IS levels predict outcome after ASCT and are associated with antibiotics and NOD2/CARD15 variants.

Motivation: In biomedicine, every molecular measurement is relative to a reference point, like a fixed aliquot of RNA extracted from a tissue, a defined number of blood cells, or a defined volume of biofluid. Reference points are often chosen for practical reasons. For example, we might want to assess the metabolome of a diseased organ but can only measure metabolites in blood or urine. In this case the observable data only indirectly reflects the disease state. The statistical implications of these discrepancies in reference points have not yet been discussed.

Grundproblematik und Fragestellung: In neueren Untersuchungen wurden charakteristische sonographische Zeichen bei Patienten mit klinisch manifester Arteriitis temporalis (AT) beschrieben. Unklar ist, ob die Sonographie auch Patienten mit Polymyalgia rheumatica (PMR) ohne klinische Zeichen einer AT identifizieren kann, bei denen jedoch bioptisch eine Riesenzellarteriitis nachgewiesen wurde. Patienten und Methodik: Wir untersuchten prospektiv 22 Patienten (14 Frauen, 8 Männer, durchschnittliches Alter 67,4 ± 9,1 Jahre) mit PMR ohne klinische Symptome einer AT mittels Duplexsonographie (ATL HDI 3000, Linear 12-5 MHZ). Bei allen Patienten wurde eine farbkodierte Duplexsonographie der Temporalarterien durchgeführt. Die Sonographie erfolgte vor der Biopsie. Bei klinischem Verdacht auf extratemporalen Befall wurden andere Gefäßregionen gezielt mituntersucht. Zur exakten Probeentnahme wurde dem Chirurgen die genaue Lokalisation eines pathologischen Sonographiebefundes mitgeteilt. Die endgültige Diagnose einer AT wurde bei histologischer Sicherung gestellt. Ergebnisse: Bei sieben von 22 (32 %) Patienten mit PMR ohne klinische Zeichen einer AT ließ sich sonographisch ein echoarmer Halo um das Lumen der Temporalarterien darstellen. Bei fünf der sieben Patienten mit sonographisch vermuteter AT zeigten sich histologische Zeichen einer Riesenzellarteriitis. Umgekehrt hatten alle Patienten mit histologisch gesicherter AT (5 von 22, = 23 %) duplexsonographisch pathologische Befunde: In allen Fällen lag ein ausgeprägter Halo mit einer minimalen Dicke von 0,7 mm vor, bei zwei der fünf Patienten darüber hinaus Stenosen der Temporalarterien. Die Sensitivität des sonographischen Halo-Zeichens zur Bestätigung des histologischen Befundes lag damit bei 100 %, die Spezifität bei 88 %. Bei den zwei Patienten mit sonographischen Zeichen einer Arteriitis und unauffälligem histologischem Befund lag die Dicke des Halo-Saumes unter 0,7 mm. Bei zwei Patienten mit AT waren sonographisch Stenosen der Schultergürtel- und Armarterien, bei einem Patienten Stenosen der A. renalis, der A. mesenterica sup. sowie des Truncus coeliacus nachweisbar. Folgerungen: Die farbkodierte Duplexsonographie mit hochfrequenten Schallköpfen scheint eine sichere Diagnose der Arteriitis temporalis bei Patienten mit Polymyalgia rheumatica zu ermöglichen, auch wenn klinische Symptome einer Gefäßentzündung fehlen. Es ist zu prüfen, ob die Diagnose und Behandlung einer bislang asymptomatischen Arteriitis temporalis auf der Grundlage des Ultraschallbefundes ohne Durchführung einer Temporalarterienbiopsie erfolgen kann. Background and objective: Recent studies have described characteristic sonographic signs in patients with manifest temporal arteritis (TA). It was the aim of this study to determine whether sonography can identify TA without clinical signs but biopsy evidence of giant-cell arteritis in patients with rheumatic polymyalgia (RP). Patients and methods: 22 patients (14 women, 8 men; average age 67.4 9.1 years) with RP but no clinical signs of TA were prospectively examined for TA by colour-coded duplex sonography (ATL HDI 3000, linear 12-5 Mhz) before temporal artery biopsy was taken. If there was clinical suspicion of extratemporal involvement, other vessels were also examined selectively. The biopsy was taken from a site identified by the sonography. A definitive diagnosis of TA was made only if there was a positive biopsy. Results: In seven of the 22 patients (32%) sonography showed an echo-poor halo around the lumen of the temporal artery. Five of these seven patients also had histological evidence of giant-cell arteritis. Conversely, all of the five patients had abnormal sonographic findings, namely a marked halo with a minimal thickness of 0.7 mm. Two of the five patients also had temporal artery stenosis, i.e. there was a 100% sensitivity and 80% specificity with respect to the halo sign in conformance with the histology. Two patients with TA were also shown sonographically to have stenosis in arteries of the shoulder girdle and arm. Stenoses in the renal and mesenteric arteries as well of the coeliac trunk were demonstrated in one patient. Conclusions: Colour-coded sonography with a high-frequency transducer head probably provides reliable diagnosis of TA in patients with RP, even in the absence of clinical signs of vascular inflammation. It remains to be proven whether sonography without biopsy is reliable enough for the diagnosis and treatment of asymptomatic TA.

Assessment of renal function relies on the estimation of the glomerular filtration rate (eGFR). Existing eGFR equations, usually based on serum levels of creatinine and/or cystatin C, are not uniformly accurate across patient populations. In the present study, we expanded a recent proof-of-concept approach to optimize an eGFR equation targeting the adult population with and without chronic kidney disease (CKD), based on a nuclear magnetic resonance spectroscopy (NMR) derived ‘metabolite constellation’ (GFR_NMR). A total of 1855 serum samples were partitioned into development, internal validation and external validation datasets. The new GFR_NMR equation used serum myo-inositol, valine, creatinine and cystatin C plus age and sex. GFR_NMR had a lower bias to tracer measured GFR (mGFR) than existing eGFR equations, with a median bias (95% confidence interval [CI]) of 0.0 (−1.0; 1.0) mL/min/1.73 m² for GFR_NMR vs. −6.0 (−7.0; −5.0) mL/min/1.73 m² for the Chronic Kidney Disease Epidemiology Collaboration equation that combines creatinine and cystatin C (CKD-EPI₂₀₁₂) (p < 0.0001). Accuracy (95% CI) within 15% of mGFR (1-P15) was 38.8% (34.3; 42.5) for GFR_NMR vs. 47.3% (43.2; 51.5) for CKD-EPI₂₀₁₂ (p < 0.010). Thus, GFR_NMR holds promise as an alternative way to assess eGFR with superior accuracy in adult patients with and without CKD.