A Dual Marker for Monitoring MDR-TB Treatment: Host-Derived miRNAs and M. tuberculosis-Derived RNA Sequences in Serum.

Background: In the absence of a late marker of treatment failure or relapse in MDR-TB patients, biomarkers based on host-miRNAs coupled with M. tuberculosis-RNAs evaluated in extracellular vesicles (EVs) are an alternative follow-up for MDR-TB disease. Characterization of EVs cargo to identify differentially expressed miRNAs before and after treatment, and to identify M. tuberculosis-derived RNA in serum EVs from resistant TB patients. Methods: EVs were isolated from serum of 26 drug-resistant TB (DR-TB) patients and 16 healthy subjects. Differential expression of miRNAs in pooled exosomes from both untreated and treated patients was assessed and individually validated at different time points during treatment. In addition, M. tuberculosis RNA was amplified in the same samples by qPCR. Results: A multivariate analysis using miR-let-7e-5p, -197-3p and -223-3p were found to be a more sensitive discriminator between healthy individuals and those with TB for both DR-TB (AUC= 0.96, 95%, CI=0.907-1) and MDR-TB groups (AUC= 0.95, 95%, CI= 0.89-1). Upregulation of miR-let-7e-5p were observed at the time of M. tuberculosis negative culture T(3-5) for MDR-TB group or for long-term T(9-15) for MDR-TB group without diabetes (T2DM). A second pathogen-based marker based on 30kDa and 5KST sequences was detected in 33% of the MDR-TB patients after the intensive phase of treatment. The miR-let7e-5p is a candidate biomarker for long-term monitoring of treatment for the group of MDR-TB without T2DM. A dual marker of host-derived miR-let7e-5p and M. tuberculosis-derived RNA for monitoring-TB treatment based in serum EVs. Conclusion: A dual marker consisting of host-derived miR-let7e-5p and M. tuberculosis-derived RNA, could be an indicator of treatment failure or relapse time after treatment was completed.

Screening of 20 Mycobacterium tuberculosis sRNAs in plasma for detection of active pulmonary tuberculosis.

BACKGROUND: Mycobacterium tuberculosis (MTB) sRNAs are abundant. However, the level of MTB sRNA in peripheral blood remains elusive. METHODS: Twenty MTB sRNAs annotated in the reference genome of H37Rv were detected in the plasma of 170 active pulmonary tuberculosis patients and 124 healthy people by qRT-PCR detection system. The differential expression of sRNAs were analyzed in two groups. The value of sRNAs for diagnosis of active tuberculosis were evaluated by ROC curve analysis. RESULTS: Eight of the 20 sRNAs (MTS2823, MTS0997, MTS1338, ASdes, G2, C8, mcr15 and MTS1082) were found in at least 50% of the samples detected. The relative expression levels of MTS2823, MTS0997, MTS1338 and ASdes in plasma of tuberculosis patients were statistically higher than those in healthy controls. ROC curve analysis showed that the AUC of MTS0997, MTS1338, MTS2823 and ASdes were 0.8935 (95% CI 0.8109-0.9760), 0.8722 (95% CI 0.7862-0.9581), 0.8208 (95% CI 0.7246-0.9170) and 0.5792 (95% CI 0.4240-0.7344), respectively. The AUC value of combination of MTS0997, MTS1338 and MTS2823 was 0.914 (95% CI 0.8281-0.9926). CONCLUSIONS: MTB sRNAs MTS2823, MTS0997 and MTS1338 have the potential to be plasma biomarkers for active pulmonary tuberculosis.

Staphylococcus epidermidis undergoes global changes in gene expression during biofilm maturation in platelet concentrates.

BACKGROUND: Staphylococcus epidermidis forms surface-attached aggregates (biofilms) when grown in platelet concentrates (PCs). Comparative transcriptome analyses were undertaken to investigate differential gene expression of S. epidermidis biofilms grown in PCs. STUDY DESIGN AND METHODS: Two S. epidermidis strains isolated from human skin (AZ22 and AZ39) and one strain isolated from contaminated PCs (ST02) were grown in glucose-supplemented Trypticase Soy Broth (TSBg) and PCs. RNA was extracted and sequenced using Illumina HiSeq. Differential expression analysis was done using DESeq, and significantly differentially expressed genes (DEGs) were selected. DEGs were subjected to Kyoto encyclopedia of genes and genomes and Gene Ontology analyses. Differential gene expression was validated with quantitative reverse transcription-PCR. RESULTS: A total of 436, 442, and 384 genes were expressed in AZ22, AZ39, and ST02, respectively. DEG analysis showed that 170, 172, and 117 genes were upregulated in PCs in comparison to TSBg, whereas 120, 135, and 89 genes were downregulated (p < .05) in mature biofilms of AZ22, AZ39, and ST02, respectively. Twenty-seven DEGs were shared by all three strains. While 76 DEGs were shared by AZ22 and AZ39, only 34 and 21 DEGs were common between ST02, and AZ22 and AZ39, respectively. Significant transcriptional expression changes were observed in genes involved in platelet-bacteria interaction, biofilm formation, production of virulence factors, and resistance to antimicrobial peptides and antibiotics. CONCLUSION: Differential gene expression in S. epidermidis is triggered by the stressful PC storage environment. Upregulation of virulence and antimicrobial resistance genes could have clinical implications for transfusion patients.

Blood RNA signatures predict recent tuberculosis exposure in mice, macaques and humans.

Tuberculosis (TB) is the leading cause of death due to a single infectious disease. Knowing when a person was infected with Mycobacterium tuberculosis (M.tb) is critical as recent infection is the strongest clinical risk factor for progression to TB disease in immunocompetent individuals. However, time since M.tb infection is challenging to determine in routine clinical practice. To define a biomarker for recent TB exposure, we determined whether gene expression patterns in blood RNA correlated with time since M.tb infection or exposure. First, we found RNA signatures that accurately discriminated early and late time periods after experimental infection in mice and cynomolgus macaques. Next, we found a 6-gene blood RNA signature that identified recently exposed individuals in two independent human cohorts, including adult household contacts of TB cases and adolescents who recently acquired M.tb infection. Our work supports the need for future longitudinal studies of recent TB contacts to determine whether biomarkers of recent infection can provide prognostic information of TB disease risk in individuals and help map recent transmission in communities.

Elevated plasma phage load as a marker for intestinal permeability in leukemic patients.

Microbial translocation (MT) and altered gut microbiota have been described in acute leukemic patients and contribute to immune activation and inflammation. However, phage translocation has not been investigated in leukemia patients yet. We recruited 44 leukemic patients and 52 healthy adults and quantified the levels of 3 phages in peripheral blood, which were the most positive phages screened from fecal samples. The content of 16S rRNA in plasma was detected by qPCR to assess the intestinal mucosa of these patients. Spearman's rank correlation was used to analyze the relationship between phage load and the relevant clinical data. We found the most prevalent phages in fecal samples were λ phage, Wphi phage, and P22 phage, and λ phage had the highest detection rate in plasma (68%). Phage content was affected by chemotherapy and course of disease and correlated with the levels of CRP (r = 0.43, p = 0.003), sCD14 (r = 0.37, p = 0.014), and sCD163 (r = 0.44, p = 0.003). Our data indicate that plasma phage load is a promising marker for gut barrier damage and that gut phage translocation correlates with monocyte/macrophage activation and systemic inflammatory response in leukemic patients.

Circulating microbial small RNAs are altered in patients with rheumatoid arthritis.

OBJECTIVES: To determine if plasma microbial small RNAs (sRNAs) are altered in patients with rheumatoid arthritis (RA) compared with control subjects, associated with RA disease-related features, and altered by disease-modifying antirheumatic drugs (DMARDs). METHODS: sRNA sequencing was performed on plasma from 165 patients with RA and 90 matched controls and a separate cohort of 70 patients with RA before and after starting a DMARD. Genome alignments for RA-associated bacteria, representative bacterial and fungal human microbiome genomes and environmental bacteria were performed. Microbial genome counts and individual sRNAs were compared across groups and correlated with disease features. False discovery rate was set at 0.05. RESULTS: Genome counts of Lactobacillus salivarius, Anaerobaculum hydrogeniformans, Staphylococcus epidermidis, Staphylococcus aureus, Paenisporosarcina spp, Facklamia hominis, Sphingobacterium spiritivorum, Lentibacillus amyloliquefaciens, Geobacillus spp, and Pseudomonas fluorescens were significantly decreased in the plasma of RA compared with control subjects. Three microbial transfer RNA-derived sRNAs were increased in RA versus controls and inversely associated with disease activity. Higher total microbial sRNA reads were associated with lower disease activity in RA. Baseline total microbial sRNAs were threefold higher among patients who improved with DMARD versus those who did not but did not change significantly after 6 months of treatment. CONCLUSION: Plasma microbial sRNA composition is altered in RA versus control subjects and associated with some measures of RA disease activity. DMARD treatment does not alter microbial sRNA abundance or composition, but increased abundance of microbial sRNAs at baseline was associated with disease activity improvement at 6 months.

Blood transcriptional biomarkers for active pulmonary tuberculosis in a high-burden setting: a prospective, observational, diagnostic accuracy study.

BACKGROUND: Blood transcriptional signatures are candidates for non-sputum triage or confirmatory tests of tuberculosis. Prospective head-to-head comparisons of their diagnostic accuracy in real-world settings are necessary to assess their clinical use. We aimed to compare the diagnostic accuracy of candidate transcriptional signatures identified by systematic review, in a setting with a high burden of tuberculosis and HIV. METHODS: We did a prospective observational study nested within a diagnostic accuracy study of sputum Xpert MTB/RIF (Xpert) and Xpert MTB/RIF Ultra (Ultra) tests for pulmonary tuberculosis. We recruited consecutive symptomatic adults aged 18 years or older self-presenting to a tuberculosis clinic in Cape Town, South Africa. Participants provided blood for RNA sequencing, and sputum samples for liquid culture and molecular testing using Xpert and Ultra. We assessed the diagnostic accuracy of candidate blood transcriptional signatures for active tuberculosis (including those intended to distinguish active tuberculosis from other diseases) identified by systematic review, compared with culture or Xpert MTB/RIF positivity as the standard reference. In our primary analysis, patients with tuberculosis were defined as those with either a positive liquid culture or Xpert result. Patients with missing blood RNA or sputum results were excluded. Our primary objective was to benchmark the diagnostic accuracy of candidate transcriptional signatures against the WHO target product profile (TPP) for a tuberculosis triage test. FINDINGS: Between Feb 12, 2016, and July 18, 2017, we obtained paired sputum and RNA sequencing data from 181 participants, 54 (30%) of whom had confirmed pulmonary tuberculosis. Of 27 eligible signatures identified by systematic review, four achieved the highest diagnostic accuracy with similar area under the receiver operating characteristic curves (Sweeney3: 90·6% [95% CI 85·6-95·6]; Kaforou25: 86·9% [80·9-92·9]; Roe3: 86·9% [80·3-93·5]; and BATF2: 86·8% [80·6-93·1]), independent of age, sex, HIV status, previous tuberculosis, or sputum smear result. At test thresholds that gave 70% specificity (the minimum WHO TPP specificity for a triage test), these four signatures achieved sensitivities between 83·3% (95% CI 71·3-91·0) and 90·7% (80·1-96·0). No signature met the optimum criteria, of 95% sensitivity and 80% specificity proposed by WHO for a triage test, or the minimum criteria (of 65% sensitivity and 98% specificity) for a confirmatory test, but all four correctly identified Ultra-positive, culture-negative patients. INTERPRETATION: Selected blood transcriptional signatures met the minimum WHO benchmarks for a tuberculosis triage test but not for a confirmatory test. Further development of the signatures is warranted to investigate their possible effects on clinical and health economic outcomes as part of a triage strategy, or when used as add-on confirmatory test in conjunction with the highly sensitive Ultra test for Mycobacterium tuberculosis DNA. FUNDING: Royal Society Newton Advanced Fellowship, Wellcome Trust, National Institute of Health Research, and UK Medical Research Council.

Concise whole blood transcriptional signatures for incipient tuberculosis: a systematic review and patient-level pooled meta-analysis.

BACKGROUND: Multiple blood transcriptional signatures have been proposed for identification of active and incipient tuberculosis. We aimed to compare the performance of systematically identified candidate signatures for incipient tuberculosis and to benchmark these against WHO targets. METHODS: We did a systematic review and individual participant data meta-analysis. We searched Medline and Embase for candidate whole blood mRNA signatures discovered with the primary objective of diagnosis of active or incipient tuberculosis, compared with controls who were healthy or had latent tuberculosis infection. We tested the performance of eligible signatures in whole blood transcriptomic datasets, in which sampling before tuberculosis diagnosis was done and time to disease was available. Culture-confirmed and clinically or radiologically diagnosed pulmonary or extrapulmonary tuberculosis cases were included. Non-progressor (individuals who remained tuberculosis-free during follow-up) samples with less than 6 months of follow-up from the date of sample collection were excluded, as were participants with prevalent tuberculosis and those who received preventive therapy. Scores were calculated for candidate signatures for each participant in the pooled dataset. Receiver operating characteristic curves, sensitivities, and specificities were examined using prespecified intervals to tuberculosis (<3 months, <6 months, <1 year, and <2 years) from sample collection. This study is registered with PROSPERO, number CRD42019135618. RESULTS: We tested 17 candidate mRNA signatures in a pooled dataset from four eligible studies comprising 1126 samples. This dataset included 183 samples from 127 incipient tuberculosis cases in South Africa, Ethiopia, The Gambia, and the UK. Eight signatures (comprising 1-25 transcripts) that predominantly reflect interferon and tumour necrosis factor-inducible gene expression, had equivalent diagnostic accuracy for incipient tuberculosis over a 2-year period with areas under the receiver operating characteristic curves ranging from 0·70 (95% CI 0·64-0·76) to 0·77 (0·71-0·82). The sensitivity of all eight signatures declined with increasing disease-free time interval. Using a threshold derived from two SDs above the mean of uninfected controls to prioritise specificity and positive-predictive value, the eight signatures achieved sensitivities of 24·7-39·9% over 24 months and of 47·1-81·0% over 3 months, with corresponding specificities of more than 90%. Based on pre-test probability of 2%, the eight signatures achieved positive-predictive values ranging from 6·8-9·4% over 24 months and 11·2-14·4% over 3 months. When using biomarker thresholds maximising sensitivity and specificity with equal weighting to both, no signature met the minimum WHO target product profile parameters for incipient tuberculosis biomarkers over a 2-year period. INTERPRETATION: Blood transcriptional biomarkers reflect short-term risk of tuberculosis and only exceed WHO benchmarks if applied to 3-6-month intervals. Serial testing among carefully selected target groups might be required for optimal implementation of these biomarkers. FUNDING: Wellcome Trust and National Institute for Health Research.

Host and MTB genome encoded miRNA markers for diagnosis of tuberculosis.

MicroRNAs (miRNAs) are a class of noncoding RNA molecules which are involved in various cellular and physiological processes. Previously, studies have identified several miRNAs that are potential diagnostic biomarkers for various infectious diseases including tuberculosis. We have performed small RNA sequencing using the Ion Torrent PGM platform in extra pulmonary tuberculosis (EPTB) subject's serum samples to identify circulating miRNAs during mycobacterium tuberculosis (MTB) infection. Our analysis identified 20 circulating miRNAs upregulated and 5 miRNAs downregulated during MTB infection in patient's serum. In addition, we have identified 6 MTB genome encoded miRNAs upregulated in EPTB patient's serum samples. Taqman based qRT-PCR analysis of host-genome encoded (hsa-miR-146a-5p and hsa-miR-125b-5p) and MTB genome encoded (MTB-miR5) miRNAs showed increased expression in a cohort of 52 healthy, pulmonary tuberculosis (PTB) and extra pulmonary tuberculosis (EPTB) patients serum samples. Our study identified for the first time a panel of host and MTB genome specific differentially expressed circulating miRNAs in serum samples of an Indian patient cohort with tuberculosis infection with a potential as a non-invasive diagnostic biomarker for tuberculosis infection.

Uncloaking an ancient adversary: Can pathogen biomarker elicitors play a role in confirming extrapulmonary TB and latent TB infection?

Latent tuberculosis infection (LTBI) is diagnosed immunologically using the Mantoux tuberculin skin test (TST) or interferon-gamma release assays (IGRAs). While widely used, immunodiagnostics can produce false negative or false positive results. Pathogen biomarkers provide an alternative, but direct detection in LTBI and extrapulmonary TB cases is challenging. Mycobacterium tuberculosis grows slowly, has limited hematogenous movement, is protected by a lipid rich cell wall, and produces low levels of secreted factors. Here we discuss the potential of elicitors by first considering pathogen markers that may be released following the administration of isoniazid. Isoniazid targets the cell wall of mycobacteria found in extracellular compartments and within monocytes, macrophages, dendritic cells, and lymphatic endothelial cells. Isoniazid's dual-purpose potential as an antibiotic and elicitor is supported by knowledge of latent infection dynamics, time-kill kinetics, and new detection techniques. Within hours, the bactericidal action of isoniazid likely enriches plasma with M. tuberculosis DNA, RNA, proteins/peptides, and lipids. Undoubtedly a portion of these biomarkers are eliminated as some bacilli undergo phagocytosis and lysosomal destruction. However, advances in immunoprecipitation and nucleic acid amplification, combined with the use of larger blood volumes during assay development, may overcome these losses. Other anticipated challenges include determining optimal sample collection times and designing diagnostic workflows that minimize processing-associated marker loss and degradation. Conventional, commercial, and emerging technologies that address these variables are discussed. If realized, isoniazid associated markers could provide proof of concept for novel elicitor-based diagnostic approaches capable of confirming LTBI and empirically treated extrapulmonary TB.

Dynamics of Spirochetemia and Early PCR Detection of Borrelia miyamotoi.

We investigated whether Borrelia miyamotoi disease can be detected in its early stage by using PCR for borrelial 16S rRNA, which molecule (DNA or RNA) is the best choice for this test, and whether spirochetes are present in blood during the acute phase of B. miyamotoi disease. A total of 473 patients with a suspected tickborne infection in Yekaterinburg, Russia, in 2009, 2010, and 2015 were enrolled in this study. Blood samples were analyzed by using quantitative PCR or ELISA, and a diagnosis of borreliosis was confirmed for 310 patients. For patients with erythema migrans, 5 (3%) of 167 were positive for B. miyamotoi by PCR; for patients without erythema migrans, 65 (45%) of 143 were positive for B. miyamotoi by PCR. The median concentration for RNA was 3.8 times that for DNA. Median time for detection of B. miyamotoi in blood was 4 days.

Detection of mycobacterial small RNA in the bacterial culture supernatant and plasma of patients with active tuberculosis.

Bacterial small RNA (sRNA) has been shown to play an important role in control of bacteria virulence, stress response and physiological metabolism by post-transcriptional regulation of gene expression. However, there were few reports about bacterial sRNA as a biomarker of infection. To test the potential role of sRNA in indicating infection of Mycobacterium tuberculosis, total RNA were extracted from the filtrated bacterial cultural supernatant. After synthesis of cDNA by reverse transcription, four Mycobacterial sRNAs including ASdes, ASpks, AS1726, and AS1890, which have been experimentally confirmed by Kristine B in the year of 2009, were detected by real time PCR. The specificity was verified by sequencing of the amplified products. Moreover, we demonstrate that the presence of sRNA Asdes in plasma of 55.56% (15/27) TB patients and 25.00% (6/24) normal controls with BCG vaccination (P < 0.05). Our results suggest that bacterial non-coding sRNA can be detected from either bacterial culture supernatants or patient's plasma. Detecting of Mycobacterial sRNA provides a rapid and relatively noninvasive approach for diagnosing disease and could be developed as a biomarker to identify patients with active tuberculosis to help make informed decisions about proper therapies.1.

Rifaximin Reduces Markers of Inflammation and Bacterial 16S rRNA in Zambian Adults with Hepatosplenic Schistosomiasis: A Randomized Control Trial.

Cirrhosis is the dominant cause of portal hypertension globally but may be overshadowed by hepatosplenic schistosomiasis (HSS) in the tropics. In Zambia, schistosomiasis seroprevalence can reach 88% in endemic areas. Bacterial translocation (BT) drives portal hypertension in cirrhosis contributing to mortality but remains unexplored in HSS. Rifaximin, a non-absorbable antibiotic may reduce BT. We aimed to explore the influence of rifaximin on BT, inflammation, and fibrosis in HSS. In this phase II open-label trial (ISRCTN67590499), 186 patients with HSS in Zambia were evaluated and 85 were randomized to standard care with or without rifaximin for 42 days. Changes in markers of inflammation, BT, and fibrosis were the primary outcomes. BT was measured using plasma 16S rRNA, lipopolysaccharide-binding protein, and lipopolysaccharide, whereas hyaluronan was used to measure fibrosis. Tumor necrosis factor receptor 1 (TNFR1) and soluble cluster of differentiation 14 (sCD14) assessed inflammation. 16S rRNA reduced from baseline (median 146 copies/µL, interquartile range [IQR] 9, 537) to day 42 in the rifaximin group (median 63 copies/µL, IQR 12, 196), P < 0.01. The rise in sCD14 was lower (P < 0.01) in the rifaximin group (median rise 122 ng/mL, IQR-184, 783) than in the non-rifaximin group (median rise 832 ng/mL, IQR 530, 967). TNFR1 decreased (P < 0.01) in the rifaximin group (median -39 ng/mL IQR-306, 563) but increased in the non-rifaximin group (median 166 ng/mL, IQR 3, 337). Other markers remained unaffected. Rifaximin led to a reduction of inflammatory markers and bacterial 16S rRNA which may implicate BT in the inflammation in HSS.

Prospective Discrimination of Controllers From Progressors Early After Low-Dose Mycobacterium tuberculosis Infection of Cynomolgus Macaques using Blood RNA Signatures.

The cynomolgus macaque model of low-dose Mycobacterium tuberculosis infection recapitulates clinical aspects of human tuberculosis pathology, but it is unknown whether the 2 systems are sufficiently similar that host-based signatures of tuberculosis will be predictive across species. By blind prediction, we demonstrate that a subset of genes comprising a human signature for tuberculosis risk is simultaneously predictive in humans and macaques and prospectively discriminates progressor from controller animals 3-6 weeks after infection. Further analysis yielded a 3-gene signature involving PRDX2 that predicts tuberculosis progression in macaques 10 days after challenge, suggesting novel pathways that define protective responses to M. tuberculosis.

Assessment of Validity of a Blood-Based 3-Gene Signature Score for Progression and Diagnosis of Tuberculosis, Disease Severity, and Treatment Response.

Importance: The World Health Organization identified the need for a non-sputum-based triage test to identify those in need of further tuberculosis (TB) testing. Objective: To determine whether the 3-gene TB score can be a diagnostic tool throughout the course of TB disease, from latency to diagnosis to treatment response, and posttreatment residual inflammation. Design, Setting, and Participants: This nested case-control study analyzed the 3-gene TB score in 3 cohorts, each focusing on a different stage of TB disease: (1) the Adolescent Cohort Study profiled whole-blood samples from adolescents with latent Mycobacterium tuberculosis infection, some of which progressed to active TB (ATB), using RNA sequencing; (2) the Brazil Active Screen Study collected whole blood from an actively screened case-control cohort of adult inmates from 2 prisons in Mato Grosso do Sul, Brazil, for ATB from January 2016 to February 2016; and (3) the Catalysis Treatment Response Cohort (CTRC) identified culture-positive adults in primary health care clinics in Cape Town, South Africa, from 2005 to 2007 and collected whole blood for RNA sequencing from patients with ATB at diagnosis and weeks 1, 4, and 24. The CTRC patients also had positron emission tomography-computed tomography scans at diagnosis, week 4, and week 24. Analyses were performed from September 2017 to June 2018. Main Outcomes and Measures: A 3-gene messenger RNA expression score, measured by quantitative polymerase chain reaction or RNA sequencing, was evaluated for distinguishing the following: individuals who progressed to ATB from those who did not, individuals with ATB from those without, and individuals with slower treatment response during TB therapy. Results: Patients evaluated in this study included 144 adolescents from the Adolescent Cohort Study (aged 12-18 years; 96 female and 48 male), 81 adult prison inmates from the Brazil Active Screen Study (aged 20-72 years; 81 male), and 138 adult community members from the CTRC (aged 17-64 years; 81 female and 57 male). The 3-gene TB score identified progression from latent M tuberculosis infection to ATB 6 months prior to sputum conversion with 86% sensitivity and 84% specificity (area under the curve [AUC], 0.86; 95% CI, 0.77-0.96) and patients with ATB in the Brazil Active Screen Study cohort (AUC, 0.87; 95% CI, 0.78-0.95) and CTRC (AUC, 0.94; 95% CI, 0.88-0.99). It also identified CTRC patients with failed treatment at the end of treatment (AUC, 0.93; 95% CI, 0.83-1.00). Collectively, across all cohorts, the 3-gene TB score identified patients with ATB with 90% sensitivity, 70% specificity, and 99.3% negative predictive value at 4% prevalence. Conclusions and Relevance: Across 3 independent prospective cohorts, the 3-gene TB score approaches the World Health Organization target product profile benchmarks for non-sputum-based triage test with high negative predictive value. This gene expression diagnostic approach should be considered for further validation and future implementation.

Metagenomic Investigation of Plasma in Individuals with ME/CFS Highlights the Importance of Technical Controls to Elucidate Contamination and Batch Effects.

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a debilitating disease causing indefinite fatigue. ME/CFS has long been hypothesised to have an infectious cause; however, no specific infectious agent has been identified. We used metagenomics to analyse the RNA from plasma samples from 25 individuals with ME/CFS and compare their microbial content to technical controls as well as three control groups: individuals with alternatively diagnosed chronic Lyme syndrome (N = 13), systemic lupus erythematosus (N = 11), and healthy controls (N = 25). We found that the majority of sequencing reads were removed during host subtraction, thus there was very low microbial RNA content in the plasma. The effects of sample batching and contamination during sample processing proved to outweigh the effects of study group on microbial RNA content, as the few differences in bacterial or viral RNA abundance we did observe between study groups were most likely caused by contamination and batch effects. Our results highlight the importance of including negative controls in all metagenomic analyses, since there was considerable overlap between bacterial content identified in study samples and control samples. For example, Proteobacteria, Firmicutes, Actinobacteria, and Bacteriodes were found in both study samples and plasma-free negative controls. Many of the taxonomic groups we saw in our plasma-free negative control samples have previously been associated with diseases, including ME/CFS, demonstrating how incorrect conclusions may arise if controls are not used and batch effects not accounted for.

Sources and Functions of Extracellular Small RNAs in Human Circulation.

Various biotypes of endogenous small RNAs (sRNAs) have been detected in human circulation, including microRNAs, transfer RNAs, ribosomal RNA, and yRNA fragments. These extracellular sRNAs (ex-sRNAs) are packaged and secreted by many different cell types. Ex-sRNAs exhibit differences in abundance in several disease states and have, therefore, been proposed for use as effective biomarkers. Furthermore, exosome-borne ex-sRNAs have been reported to elicit physiological responses in acceptor cells. Exogenous ex-sRNAs derived from diet (most prominently from plants) and microorganisms have also been reported in human blood. Essential issues that remain to be conclusively addressed concern the (a) presence and sources of exogenous ex-sRNAs in human bodily fluids, (b) detection and measurement of ex-sRNAs in human circulation, (c) selectivity of ex-sRNA export and import, (d) sensitivity and specificity of ex-sRNA delivery to cellular targets, and (e) cell-, tissue-, organ-, and organism-wide impacts of ex-sRNA-mediated cell-to-cell communication. We survey the present state of knowledge of most of these issues in this review.

Diagnosis of childhood tuberculosis and host RNA expression in Africa.

BACKGROUND: Improved diagnostic tests for tuberculosis in children are needed. We hypothesized that transcriptional signatures of host blood could be used to distinguish tuberculosis from other diseases in African children who either were or were not infected with the human immunodeficiency virus (HIV). METHODS: The study population comprised prospective cohorts of children who were undergoing evaluation for suspected tuberculosis in South Africa (655 children), Malawi (701 children), and Kenya (1599 children). Patients were assigned to groups according to whether the diagnosis was culture-confirmed tuberculosis, culture-negative tuberculosis, diseases other than tuberculosis, or latent tuberculosis infection. Diagnostic signatures distinguishing tuberculosis from other diseases and from latent tuberculosis infection were identified from genomewide analysis of RNA expression in host blood. RESULTS: We identified a 51-transcript signature distinguishing tuberculosis from other diseases in the South African and Malawian children (the discovery cohort). In the Kenyan children (the validation cohort), a risk score based on the signature for tuberculosis and for diseases other than tuberculosis showed a sensitivity of 82.9% (95% confidence interval [CI], 68.6 to 94.3) and a specificity of 83.6% (95% CI, 74.6 to 92.7) for the diagnosis of culture-confirmed tuberculosis. Among patients with cultures negative for Mycobacterium tuberculosis who were treated for tuberculosis (those with highly probable, probable, or possible cases of tuberculosis), the estimated sensitivity was 62.5 to 82.3%, 42.1 to 80.8%, and 35.3 to 79.6%, respectively, for different estimates of actual tuberculosis in the groups. In comparison, the sensitivity of the Xpert MTB/RIF assay for molecular detection of M. tuberculosis DNA in cases of culture-confirmed tuberculosis was 54.3% (95% CI, 37.1 to 68.6), and the sensitivity in highly probable, probable, or possible cases was an estimated 25.0 to 35.7%, 5.3 to 13.3%, and 0%, respectively; the specificity of the assay was 100%. CONCLUSIONS: RNA expression signatures provided data that helped distinguish tuberculosis from other diseases in African children with and those without HIV infection. (Funded by the European Union Action for Diseases of Poverty Program and others).

Repeated maternal intramuscular or intraamniotic erythromycin incompletely resolves intrauterine Ureaplasma parvum infection in a sheep model of pregnancy.

OBJECTIVE: Ureaplasma spp are the most commonly isolated microorganisms in association with preterm birth. Maternal erythromycin administration is a standard treatment for preterm prelabor rupture of membranes. There is little evidence of its effectiveness in eradicating Ureaplasma spp from the intrauterine cavity and fetus. We used a sheep model of intrauterine Ureaplasma spp infection to investigate the efficacy of repeated maternal intramuscular and intraamniotic erythromycin treatment to eradicate such an infection. STUDY DESIGN: Thirty ewes with singleton pregnancies received an intraamniotic injection of 10(7) color change units of erythromycin-sensitive Ureaplasma parvum serovar 3 at 55 days' gestation. At 116 days' gestation, 28 ewes with viable fetuses were randomized to receive (1) intraamniotic and maternal intramuscular saline solution treatment (n = 8), (2) single intraamniotic and repeated maternal intramuscular erythromycin treatment (n = 10), or (3) single maternal intramuscular and repeated intraamniotic erythromycin treatment (n = 10). Fetuses were surgically delivered at 125 days' gestation. Treatment efficacy was assessed by culture, quantitative polymerase chain reaction, and histopathologic evaluation. RESULTS: Animals treated with intraamniotic erythromycin had significantly less viable U parvum serovar 3 in the amniotic fluid at delivery. However, neither combination of maternal intramuscular and intraamniotic erythromycin treatment successfully cleared U parvum serovar 3 from the amniotic fluid or fetal tissues. Three de novo erythromycin-resistant U parvum isolates were identified in erythromycin-treated animals. CONCLUSION: Erythromycin treatment, given both to the ewe and into the amniotic cavity, fails to eradicate intrauterine and fetal U parvum serovar 3 infection and may lead to development of erythromycin resistant U parvum.