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.

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.

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.

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.

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.

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).

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.

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.

Detection of tuberculosis in HIV-infected and -uninfected African adults using whole blood RNA expression signatures: a case-control study.

BACKGROUND: A major impediment to tuberculosis control in Africa is the difficulty in diagnosing active tuberculosis (TB), particularly in the context of HIV infection. We hypothesized that a unique host blood RNA transcriptional signature would distinguish TB from other diseases (OD) in HIV-infected and -uninfected patients, and that this could be the basis of a simple diagnostic test. METHODS AND FINDINGS: Adult case-control cohorts were established in South Africa and Malawi of HIV-infected or -uninfected individuals consisting of 584 patients with either TB (confirmed by culture of Mycobacterium tuberculosis [M.TB] from sputum or tissue sample in a patient under investigation for TB), OD (i.e., TB was considered in the differential diagnosis but then excluded), or healthy individuals with latent TB infection (LTBI). Individuals were randomized into training (80%) and test (20%) cohorts. Blood transcriptional profiles were assessed and minimal sets of significantly differentially expressed transcripts distinguishing TB from LTBI and OD were identified in the training cohort. A 27 transcript signature distinguished TB from LTBI and a 44 transcript signature distinguished TB from OD. To evaluate our signatures, we used a novel computational method to calculate a disease risk score (DRS) for each patient. The classification based on this score was first evaluated in the test cohort, and then validated in an independent publically available dataset (GSE19491). In our test cohort, the DRS classified TB from LTBI (sensitivity 95%, 95% CI [87-100]; specificity 90%, 95% CI [80-97]) and TB from OD (sensitivity 93%, 95% CI [83-100]; specificity 88%, 95% CI [74-97]). In the independent validation cohort, TB patients were distinguished both from LTBI individuals (sensitivity 95%, 95% CI [85-100]; specificity 94%, 95% CI [84-100]) and OD patients (sensitivity 100%, 95% CI [100-100]; specificity 96%, 95% CI [93-100]). Limitations of our study include the use of only culture confirmed TB patients, and the potential that TB may have been misdiagnosed in a small proportion of OD patients despite the extensive clinical investigation used to assign each patient to their diagnostic group. CONCLUSIONS: In our study, blood transcriptional signatures distinguished TB from other conditions prevalent in HIV-infected and -uninfected African adults. Our DRS, based on these signatures, could be developed as a test for TB suitable for use in HIV endemic countries. Further evaluation of the performance of the signatures and DRS in prospective populations of patients with symptoms consistent with TB will be needed to define their clinical value under operational conditions. Please see later in the article for the Editors' Summary.

Bacterial RNA extraction and purification from whole human blood using isotachophoresis.

We demonstrate a novel assay for physicochemical extraction and isotachophoresis-based purification of 16S rRNA from whole human blood infected with Pseudomonas putida . This on-chip assay is unique in that the extraction can be automated using isotachophoresis in a simple device with no moving parts, it protects RNA from degradation when isolating from ribonuclease-rich matrices (such as blood), and produces a purified total nucleic acid sample that is compatible with enzymatic amplification assays. We show that the purified RNA is compatible with reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and demonstrate a clinically relevant sensitivity of 0.03 bacteria per nanoliter using RT-qPCR.

[The atypical forms of mycoplasmas persisting in the organism of mycoplasma's infected persons].

The antigens, DNA and RNA of mycoplasmas are preset in the blood serum of persons infected with urogenital mycoplasmas. The planting of patients' tests of blood serum containing antigen M. hominis on the artificial growth mediums resulted in the growth of mini-colonies of mini-cells (20-50 nm). The colonies subcultured hardly but sometimes formed solid bacterial lawn though never acquired "fried-egg" classical mycoplasma form. The proofs of identity of these colonies to M. hominis are presented. The mini-cells possessed infectiousness and ability to persist on a long-run in the internal organs of experimentally infected mice. Apparently, mini-cells are formed under impact of stress factors of the host immune defense and they are one of forms of mycoplasma's persistence in human organism.

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.

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.

[Effects of Gutuo Qingfu Decoction via gastro-enteric perfusion on 16SrRNA in blood of severe multitraumatic patients].

OBJECTIVE: To observe the effects of Gutuo Qingfu Decoction (GQD) via gastro-enteric perfusion on blood level of bacterial 16S rRNA gene in severe multi-traumatic (SMT) patients at early stage. METHODS: Sixty SMT patients were assigned to two groups, the 33 in the treated group and the 27 in the control group. They were treated with the same conventional treatment, but different in the gastro-enteric infusion with GOD for the former and saline for the latter. Blood 16SrRNA gene, body temperature, leukocyte count, C-reactive protein (CRP), and blood bacterial culture positive rate on the 3, 6, 9 post-trauma days were detected, and incidences of infective complication and mortality were observed. RESULTS: Body temperature on day 9 in the treated group was significantly lower than in the control group (37.6 +/- 0.12 degrees C vs 38.1 +/- 0.15 degrees C, P < 0.05); so did the CRP level on day 6 (52.4 +/- 6.3 mg/L vs 104.3 +/- 20.1 mg/L, P < 0.05) and day 9 (42.9 + 7.5 mg/L vs 92.5 +/- 17.1 mg/L, P < 0.05), as well as the positive rates of blood 16SrRNA gene on day 6 and 9 (33.3% vs 59.3% and 30.3% vs 77.8%, P < 0.05 and P < 0.01, respectively). However, the positive rates of blood culture were insignificantly different between the two groups ( P > 0.05). Besides, incidence of infective complication in the treated group was significantly lower than in the control group (30.3% vs 59.3%, P < 0.05). CONCLUSION: Early stage gastrointestinal administration of GQD is likely to have benefits for the improvement of intestinal mucosa barrier and reduction of enteric bacterial translocation in SMT patients, and it may also reduce the incidence of infective complication in these 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.

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.

Use of dried blood smears for detection of feline hemoplasmas using real-time polymerase chain reaction.

The objective of the current study was to determine the sensitivity and specificity of real-time polymerase chain reaction (real-time PCR) for feline hemoplasmas when applied to DNA extracted from dried whole-blood smears in comparison to that for DNA extracted from liquid whole blood. Blood samples were collected into ethylenediamine tetra-acetic acid tubes from 305 cats with possible or suspected hemoplasmosis, and dried blood smears from each sample were prepared. DNA was extracted from blood smears and a 160-microl aliquot of each liquid blood sample by using a robotic extractor and was subjected to real-time PCR for feline glyceraldehyde-3-phosphate dehydrogenase (liquid blood), 18S ribosomal RNA (dried blood), and "Candidatus Mycoplasma haemominutum", Mycoplasma haemofelis, and "Candidatus Mycoplasma turicensis" DNA. When using the results for liquid whole blood as the gold standard, the sensitivity of each assay for "Ca. M. haemominutum", M. haemofelis, and "Ca. M. turicensis" was 49 of 66 (74%), 11 of 13 (85%), and 11 of 20 (55%), respectively. The specificity of each assay was 224 of 234 (96%), 287 of 287 (100%), and 280 of 280 (100%), respectively. When possible, liquid blood samples should be submitted for detection of feline hemoplasmas by using real-time PCR. The improved sensitivity of real-time PCR on blood smears for M. haemofelis compared with that of the other hemoplasma species may reflect the higher organism burdens associated with infection with this species.