Xpert MTB/XDR: a 10-Color Reflex Assay Suitable for Point-of-Care Settings To Detect Isoniazid, Fluoroquinolone, and Second-Line-Injectable-Drug Resistance Directly from Mycobacterium tuberculosis-Positive Sputum.

We describe the design, development, analytical performance, and a limited clinical evaluation of the 10-color Xpert MTB/XDR assay (CE-IVD only, not for sale in the United States). This assay is intended as a reflex test to detect resistance to isoniazid (INH), fluoroquinolones (FLQ), ethionamide (ETH), and second-line injectable drugs (SLIDs) in unprocessed sputum samples and concentrated sputum sediments which are positive for Mycobacterium tuberculosis The Xpert MTB/XDR assay simultaneously amplifies eight genes and promoter regions in M. tuberculosis and analyzes melting temperatures (Tm s) using sloppy molecular beacon (SMB) probes to identify mutations associated with INH, FLQ, ETH, and SLID resistance. Results can be obtained in under 90 min using 10-color GeneXpert modules. The assay can differentiate low- versus high-level resistance to INH and FLQ as well as cross-resistance versus individual resistance to SLIDs by identifying mutation-specific Tm s or Tm patterns generated by the SMB probes. The assay has a limit of detection comparable to that of the Xpert MTB/RIF assay and successfully detected 16 clinically significant mutations in a challenge set of clinical isolate DNA. In a clinical study performed at two sites with 100 sputum and 214 clinical isolates, the assay showed a sensitivity of 94% to 100% and a specificity of 100% for all drugs except for ETH compared to that of sequencing. The sensitivity and specificity were in the same ranges as those of phenotypic drug-susceptibility testing. Used in combination with a primary tuberculosis diagnostic test, this assay should expand the capacity for detection of drug-resistant tuberculosis near the point of care.

CD44 mediates stem cell mobilization to damaged lung via its novel transcriptional targets, Cortactin and Survivin.

Beyond their role in bone and lung homeostasis, mesenchymal stem cells (MSCs) are becoming popular in cell therapy. Various insults may disrupt the repair mechanisms involving MSCs. One such insult is smoking, which is a major risk factor for osteoporosis and respiratory diseases. Upon cigarette smoke-induced damage, a series of reparatory mechanisms ensue; one such mechanism involves Glycosaminoglycans (GAG). One of these GAGs, namely hyaluronic acid (HA), serves as a potential therapeutic target in lung injury. However, much of its mechanisms of action through its major receptor CD44 remains unexplored. Our previous studies have identified and functionally validated that both cortactin (CTTN: marker of motility) and Survivin (BIRC5: required for cell survival) act as novel HA/CD44-downstream transcriptional targets underpinning cell motility. Here, human MSCs were treated with "Water-pipe" smoke to investigate the effects of cigarette smoke condensate (CSC) on these HA-CD44 novel signaling pathways. Our results show that CSC decreased the expression of both CD44 and its downstream targets CTTN and BIRC5 in MSCs, and that HA reversed these effects. Interestingly, CSC inhibited migration and invasion of MSCs upon CD44-targeted RNAi treatment. This shows the importance of CD44-HA/CTTN and CD44-HA/BIRC5 signaling pathways in MSC motility, and further suggests that these signaling pathways may provide a novel mechanism implicated in migration of MSCs during repair of lung tissue injury. These findings suggest that one should use caution before utilizing MSC from donors with history of smoking, and further pave the way towards the development of targeted therapeutic approaches against CD44-associated diseases.

Evaluation of QuantiFERON-TB Gold-Plus in Health Care Workers in a Low-Incidence Setting.

Although launched in 2015, little is known about the accuracy of QuantiFERON-TB Gold-Plus (QFT-Plus) for diagnosis of latent M. tuberculosis infection (LTBI). Unlike its predecessor, QFT-Plus utilizes two antigen tubes to elicit an immune response from CD4+ and CD8+ T lymphocytes. We conducted a cross-sectional study in low-risk health care workers (HCWs) at a single U.S. center to compare QFT-Plus to QuantiFERON-TB Gold in-tube (QFT). A total of 989 HCWs were tested with both QFT and QFT-Plus. Risk factors for LTBI were obtained from a questionnaire. QFT-Plus was considered positive if either antigen tube 1 (TB1) or TB2 tested positive, per the manufacturer's recommendations, or if both TB1 and TB2 tested positive, using a conservative definition. Results were compared using Cohen's kappa and linear regression, respectively. Agreement of QFT with QFT-Plus was high, at 95.6% (95% confidence interval [CI], 94.3 to 96.9; kappa, 0.57). The majority of discordant results between QFT and QFT-Plus TB1 (84.8%) and QFT and QFT-Plus TB2 (88.6%) fell within the range of 0.2 to 0.7 IU/ml. The positivity rate in 626 HCWs with no identifiable risk factors and no self-reported history of positive LTBI tests was 2.1% (CI, 1.0 to 3.2) and 3.0% (CI, 1.7 to 4.3) with QFT and QFT-Plus, respectively. A conservative definition of a QFT-Plus-positive result yielded a positivity rate of 1.0% (CI, 0.2 to 1.7; P value of 0.0002 versus QFT-Plus and 0.07 versus QFT). On follow-up testing, of 11 HCWs with discordant QFT-Plus results, 90.9% (10/11) had a negative QFT result. The QFT-Plus assay showed a high degree of agreement with QFT in U.S. HCWs. A conservative interpretation of QFT-Plus eliminated nearly all nonreproducible positive results in low-risk HCWs. Larger studies are needed to validate the latter finding and to more clearly define conditions under which a conservative interpretation can be used to minimize nonreproducible positive results in low-risk populations.

Clostridium difficile PCR Cycle Threshold Predicts Free Toxin.

There is no stand-alone Clostridium difficile diagnostic that can sensitively and rapidly detect fecal free toxins. We investigated the performance of the C. difficile PCR cycle threshold (CT ) for predicting free toxin status. Consecutive stool samples (n = 312) positive for toxigenic C. difficile by the GeneXpert C. difficile/Epi tcdB PCR assay were tested with the rapid membrane C. Diff Quik Chek Complete immunoassay (RMEIA). RMEIA toxin-negative samples were tested with the cell cytotoxicity neutralization assay (CCNA) and tgcBIOMICS enzyme-linked immunosorbent assay (ELISA). Using RMEIA alone or in combination with CCNA and/or ELISA as the reference method, the accuracy of CT was measured at different CT cutoffs. Using RMEIA as the reference method, a CT cutoff of 26.35 detected toxin-positive samples with a sensitivity, specificity, positive predictive value, and negative predictive value of 96.0% (95% confidence interval [CI], 90.2% to 98.9%), 65.9% (95% CI, 59.0% to 72.2%), 57.4% (95% CI, 52.7% to 62%), and 97.1% (95% CI, 92.8% to 98.9), respectively. Inclusion of CCNA in the reference method improved CT specificity to 78.0% (95% CI, 70.7% to 84.2%). Intercartridge lot CT variability measured as the average coefficient of variation was 2.8% (95% CI, 1.2% to 3.2%). Standardizing the input stool volume did not improve CT toxin specificity. The median CT values were not significantly different between stool samples with Bristol scores of 5, 6, and 7, between pediatric and adult samples, or between presumptive 027 and non-027 strains. In addition to sensitively detecting toxigenic C. difficile in stool, on-demand PCR may also be used to accurately predict toxin-negative stool samples, thus providing additional results in PCR-positive stool samples to guide therapy.

Correction: LprG-Mediated Surface Expression of Lipoarabinomannan Is Essential for Virulence of Mycobacterium tuberculosis.

[This corrects the article DOI: 10.1371/journal.ppat.1004376.].

Interferon Gamma Release Assays for Latent Tuberculosis: What Are the Sources of Variability?

Interferon gamma release assays (IGRAs) are blood-based tests intended for diagnosis of latent tuberculosis infection (LTBI). IGRAs offer logistical advantages and are supposed to offer improved specificity over the tuberculin skin test (TST). However, recent serial testing studies of low-risk individuals have revealed higher false conversion rates with IGRAs than with TST. Reproducibility studies have identified various sources of variability that contribute to nonreproducible results. Sources of variability can be broadly classified as preanalytical, analytical, postanalytical, manufacturing, and immunological. In this minireview, we summarize known sources of variability and their impact on IGRA results. We also provide recommendations on how to minimize sources of IGRA variability.

LprG-mediated surface expression of lipoarabinomannan is essential for virulence of Mycobacterium tuberculosis.

Mycobacterium tuberculosis employs various virulence strategies to subvert host immune responses in order to persist and cause disease. Interaction of M. tuberculosis with mannose receptor on macrophages via surface-exposed lipoarabinomannan (LAM) is believed to be critical for cell entry, inhibition of phagosome-lysosome fusion, and intracellular survival, but in vivo evidence is lacking. LprG, a cell envelope lipoprotein that is essential for virulence of M. tuberculosis, has been shown to bind to the acyl groups of lipoglycans but the role of LprG in LAM biosynthesis and localization remains unknown. Using an M. tuberculosis lprG mutant, we show that LprG is essential for normal surface expression of LAM and virulence of M. tuberculosis attributed to LAM. The lprG mutant had a normal quantity of LAM in the cell envelope, but its surface was altered and showed reduced expression of surface-exposed LAM. Functionally, the lprG mutant was defective for macrophage entry and inhibition of phagosome-lysosome fusion, was attenuated in macrophages, and was killed in the mouse lung with the onset of adaptive immunity. This study identifies the role of LprG in surface-exposed LAM expression and provides in vivo evidence for the essential role surface LAM plays in M. tuberculosis virulence. Findings have translational implications for therapy and vaccine development.

Chemoprevention of rat mammary carcinogenesis by spirulina.

Spirulina (SP) (Arthrospira platensis; previously Spirulina platensis) is a filamentous blue-green microalga (cyanobacterium) with potent dietary phytoantioxidant and anticancerous properties. We investigated the chemopreventive effect of SP against 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat breast carcinogenesis, and further studied its underlying mechanisms of action in vitro. Remarkably, SP cleared DMBA-induced rat mammary tumors, which was clearly confirmed by morphological and histological methods. SP supplementation reduced the incidence of breast tumors from 87% to 13%. At the molecular level, immunohistochemical analysis revealed that SP supplementation reduced expression of both Ki-67 and estrogen α. More interestingly, molecular analysis in the in vitro experiments indicated that SP treatment inhibited cell proliferation by 24 hours, which was accompanied by increased p53 expression, followed by increased expression of its downstream target gene, Cdkn1a (alias p21 or p21(Waf1/Cip1)). In addition, SP increased Bax and decreased Bcl-2 expression, indicating induction of apoptosis by 48 hours after SP treatment. To our knowledge, this is the first report of in vivo chemopreventive effect of SP against DMBA-induced breast carcinogenesis in rat, supporting its potential use in chemoprevention of cancer.

Impact of blood volume, tube shaking, and incubation time on reproducibility of QuantiFERON-TB gold in-tube assay.

Gamma interferon (IFN-γ) release assays (IGRAs) are functional assays used serially to measure the efficacy of novel tuberculosis (TB) vaccines and to screen health care workers for latent tuberculosis infection (LTBI). However, studies have shown nonreproducible IGRA results. In this study, we investigated the effects of blood volume (0.8, 1.0, and 1.2 ml), tube shaking (gentle versus vigorous), and incubation duration (16, 20, and 24 h) on the reproducibility of QuantiFERON-TB Gold In-Tube (QFT-GIT) results for 50 subjects (33 uninfected and 17 infected). The median IFN-γ TB response (TB antigen [Ag] minus nil value) was significantly higher with 0.8 ml blood (1.04 IU/ml) than with 1.0 ml (0.85 IU/ml; P = 0.002) or 1.2 ml (0.49 IU/ml; P < 0.001) for subjects with LTBI. Compared with 0.8 ml (11.8%), there were larger proportions of false-negative results with 1.0 ml (29.4%; P = 0.2) and 1.2 ml (41.2%; P = 0.05) of blood for infected subjects. Blood volume did not significantly change the proportions of positive results in uninfected controls. Compared with gentle shaking, vigorous shaking increased the median IFN-γ response in nil (0.04 versus 0.06 IU/ml; P < 0.001) and TB Ag (0.12 versus 0.24 IU/ml; P = 0.004) tubes and increased TB responses (TB Agvigorous minus nilgentle) (0.02 versus 0.08 IU/ml; P = 0.004). The duration of incubation did not have a significant impact on the proportion of positive results in uninfected or infected subjects. This study identified blood volume and tube shaking as novel preanalytical sources of variability which require further standardization in order to improve the quality and reproducibility of QFT-GIT results.

Can a simple flotation method lower the limit of detection of Mycobacterium tuberculosis in extrapulmonary samples analyzed by the GeneXpert MTB/RIF assay?

The rapid and accurate diagnosis of tuberculosis (TB) in children and extrapulmonary TB in adults continues to be a challenge. In this study, we determined the lower limit of detection (LOD) of the GeneXpert MTB/RIF assay with nonrespiratory specimens and investigated the utility of flotation procedures for concentrating the bacilli. Clinical specimens (9 cerebrospinal fluid [CSF], 13 gastric aspirate, 8 tissue, and 17 stool) were spiked with single-celled Mycobacterium tuberculosis, and the LOD of the GeneXpert assay was determined. Flotation studies were conducted with sucrose and NaCl, and the cycle thresholds of the MTB/RIF assay were compared between treated and untreated samples. There was no significant difference between the LODs of the GeneXpert assay with saline solution (median, 33 CFU/ml) and CSF (median, 25 CFU/ml) (P > 0.05) or gastric aspirate samples (median, 58 CFU/ml) (P > 0.05). The LOD with spiked tissue (median, 1,525 CFU/ml) and stool samples (median, 6,800 CFU/ml) was significantly elevated compared to that determined with saline solution (P ≤ 0.05 and ≤ 0.0005, respectively). Flotation studies with sucrose or NaCl did not consistently result in lowered cycle thresholds in stool or gastric aspirates, but a cycle reduction of >10 was achieved in two of the three pooled CSF samples. Unlike the results seen with tissue and stool samples, there was no significant PCR inhibition in the MTB/RIF assay with CSF and gastric aspirates. Although preconcentration of CSF samples with sucrose and NaCl may enhance detection of M. tuberculosis by PCR, further advances are needed to concentrate the bacilli and eliminate PCR inhibitors in paucibacillary nonrespiratory samples.

Survivin is a novel target of CD44-promoted breast tumor invasion.

The hyaluronan (HA) receptor CD44 plays an essential role in cell-cell or cell-extracellular matrix communications and is a bioactive signal transmitter. Although a number of studies have described the function of CD44 in breast cancer (BC) metastasis, the underlying mechanisms have yet to be determined. By using a validated tetracycline-off-regulated CD44 expression system in the MCF-7 cell line combined with microarray analysis, we identified survivin (SVV) as a potential downstream transcriptional target of CD44. To test the hypothesis that SVV underpins CD44-promoted BC cell invasion, we combined molecular and pharmacologic approaches and showed that CD44 induction increased SVV expression levels, which in turn promotes BC cell invasion. Further, clinical analysis of breast tissue samples showed that SVV expression patterns paralleled those of the standard form of CD44 during breast tumor progression. More interestingly, we identified the PI3K/E2F1 pathway as a potential molecular link between HA/CD44 activation and SVV transcription. In addition to identifying SVV as a target for HA/CD44 signaling, this investigation provides a better understanding of the molecular mechanisms that underpin the novel function of SVV in breast cancer metastasis.

Preanalytical delay reduces sensitivity of QuantiFERON-TB gold in-tube assay for detection of latent tuberculosis infection.

The effects of incubation delays on the accuracy of the QuantiFERON-TB gold in-tube assay (QFT-GIT) were measured. Compared to immediate incubation, 6- and 12-hour delays resulted in positive-to-negative reversion rates of 19% (5/26) and 22% (5/23), respectively. These findings underscore the need for standardizing QFT-GIT preanalytical practices.

Towards understanding the mode of action of the multifaceted cell adhesion receptor CD146.

CD146, also known as melanoma cell adhesion molecule or MCAM, is a key cell adhesion protein in vascular endothelial cell activity and angiogenesis. CD146 promotes tumor progression of many cancers including melanoma and prostate. Strikingly, its expression is frequently lost in breast carcinoma cells, and it may act as a suppressor of breast cancer progression. While upstream mechanisms regulating CD146 are well documented, our understanding of the downstream molecular events underlying its mode of action remains to be elucidated. This review aims to focus on the progress in understanding the signaling mechanisms and the functional relevance of CD146, a multifaceted molecule, in cancer with particular emphasis on its role in inhibiting breast cancer progression.

Deregulation of cell growth and apoptosis in UV-induced melanomagenesis.

We have previously characterized the role of p16/Rb in coordinating the early events in UVB-irradiated skin. As an extension to this work, normal melanocytes and mutant p16-inducible melanoma cell models were employed to elucidate further the coordinated molecular mechanisms occurring during early UVB exposure. Our results showed that melanocytes expressed p16 only at a high UVB dose, with undetectable p53. The Bax/Bcl2 ratio increased at higher dose, indicating that the cells had selected apoptosis program. In the wt-p16 melanoma cells, while low UVB dose upregulated p16, the high dose suppressed it, and further abrogated Cdk6 but not Cdk4. Interestingly, while induction of mutant-p16 increased Cdk4, cdk6 and pRb proteins, UVB exposure did not affect this increase. More interestingly, p16 mutant cells increased their resistance to apoptosis at high UVB-dose, associated with decreased Bax and increased Bcl2 expression. Thus, mutant-p16 appears to dictate a deregulation of cell cycle and increased resistance to apoptosis in melanoma cells. Together, the data indicate a deregulation of p16INK4/Rb pathway as an early event in UVB-induced melanomagenesis.

Diversity of resistance mechanisms in carbapenem-resistant Enterobacteriaceae at a health care system in Northern California, from 2013 to 2016.

The mechanism of resistance in carbapenem-resistant Enterobacteriaceae (CRE) has therapeutic implications. We comprehensively characterized emerging mechanisms of resistance in CRE between 2013 and 2016 at a health system in Northern California. A total of 38.7% (24/62) of CRE isolates were carbapenemase gene-positive, comprising 25.0% (6/24) blaOXA-48 like, 20.8% (5/24) blaKPC, 20.8% (5/24) blaNDM, 20.8% (5/24) blaSME, 8.3% (2/24) blaIMP, and 4.2% (1/24) blaVIM. Between carbapenemases and porin loss, the resistance mechanism was identified in 95.2% (59/62) of CRE isolates. Isolates expressing blaKPC were 100% susceptible to ceftazidime-avibactam, meropenem-vaborbactam, and imipenem-relebactam; blaOXA-48 like-positive isolates were 100% susceptible to ceftazidime-avibactam; and metallo ß-lactamase-positive isolates were nearly all nonsusceptible to above antibiotics. Carbapenemase gene-negative CRE were 100% (38/38), 92.1% (35/38), 89.5% (34/38), and 31.6% (12/38) susceptible to ceftazidime-avibactam, meropenem-vaborbactam, imipenem-relebactam, and ceftolozane-tazobactam, respectively. None of the CRE strains were identical by whole genome sequencing. At this health system, CRE were mediated by diverse mechanisms with predictable susceptibility to newer ß-lactamase inhibitors.

Rapid and specific labeling of single live Mycobacterium tuberculosis with a dual-targeting fluorogenic probe.

Tuberculosis (TB) remains a public health crisis and a leading cause of infection-related death globally. Although in high demand, imaging technologies that enable rapid, specific, and nongenetic labeling of live Mycobacterium tuberculosis (Mtb) remain underdeveloped. We report a dual-targeting strategy to develop a small molecular probe (CDG-DNB3) that can fluorescently label single bacilli within 1 hour. CDG-DNB3 fluoresces upon activation of the ß-lactamase BlaC, a hydrolase naturally expressed in Mtb, and the fluorescent product is retained through covalent modification of the Mtb essential enzyme decaprenylphosphoryl-ß-d-ribose 2'-epimerase (DprE1). This dual-targeting probe not only discriminates live from dead Bacillus Calmette-Guérin (BCG) but also shows specificity for Mtb over other bacterial species including 43 nontuberculosis mycobacteria (NTM). In addition, CDG-DNB3 can image BCG phagocytosis in real time, as well as Mtb in patients' sputum. Together with a low-cost, self-driven microfluidic chip, we have achieved rapid labeling and automated quantification of live BCG. This labeling approach should find many potential applications for research toward TB pathogenesis, treatment efficacy assessment, and diagnosis.

In vitro immunomodulation for enhancing T cell-based diagnosis of Mycobacterium tuberculosis infection.

Interferon-gamma release assays have limited sensitivity for detecting latent tuberculosis infection. In this study, we determine if the addition of immunomodulators to the QuantiFERON-TB Gold In-Tube (QFT-GIT) increased test sensitivity without compromising specificity. We prospectively compared QFT-GIT results with and without incubation with 2 immunomodulators (lipopolysaccharide [LPS] and polyinosine-polycytidylic acid [PolyIC]) in 2 cohorts-113 culture-confirmed tuberculosis (TB) subjects in Hanoi, Vietnam, and 226 documented QFT-GIT-negative, low TB risk health care workers undergoing annual TB screening at a US academic institution. Sensitivity of the tests in TB subjects was 84.1% with the standard QFT-GIT and 85.8% and 74.3% after incubation with LPS and PolyIC, respectively. Specificity in low TB risk health care workers was 100% with the standard QFT-GIT by design and 86.7% with LPS and 63.3% with PolyIC. In conclusion, use of the 2 immunomodulators did not improve sensitivity of the QFT-GIT in TB patients and reduced specificity in low-risk health care workers.

In vitro immunomodulation of a whole blood IFN-γ release assay enhances T cell responses in subjects with latent tuberculosis infection.

BACKGROUND: Activation of innate immunity via pathogen recognition receptors (PRR) modulates adaptive immune responses. PRR ligands are being exploited as vaccine adjuvants and as therapeutics, but their utility in diagnostics has not been explored. Interferon-gamma (IFN-γ) release assays (IGRAs) are functional T cell assays used to diagnose latent tuberculosis infection (LTBI); however, novel approaches are needed to improve their sensitivity. METHODS: In vitro immunomodulation of a whole blood IGRA (QuantiFERON®-TB GOLD In-Tube) with Toll-like receptor agonists poly(I:C), LPS, and imiquimod was performed on blood from subjects with LTBI and negative controls. RESULTS: In vitro immunomodulation significantly enhanced the response of T cells stimulated with M. tuberculosis antigens from subjects with LTBI but not from uninfected controls. Immunomodulation of IGRA revealed T cell responses in subjects with LTBI whose T cells otherwise do not respond to in vitro stimulation with antigens alone. Similar to their in vivo functions, addition of poly(I:C) and LPS to whole blood induced secretion of inflammatory cytokines and IFN-α and enhanced the surface expression of antigen presenting and costimulatory molecules on antigen presenting cells. CONCLUSIONS: In vitro immunomodulation of whole blood IGRA may be an effective strategy for enhancing the sensitivity of T cells for diagnosis of LTBI.