Rapid detection of Mycobacterium tuberculosis by recombinase polymerase amplification.

Improved access to effective tests for diagnosing tuberculosis (TB) has been designated a public health priority by the World Health Organisation. In high burden TB countries nucleic acid based TB tests have been restricted to centralised laboratories and specialised research settings. Requirements such as a constant electrical supply, air conditioning and skilled, computer literate operators prevent implementation of such tests in many settings. Isothermal DNA amplification technologies permit the use of simpler, less energy intensive detection platforms more suited to low resource settings that allow the accurate diagnosis of a disease within a short timeframe. Recombinase Polymerase Amplification (RPA) is a rapid, low temperature isothermal DNA amplification reaction. We report here RPA-based detection of Mycobacterium tuberculosis complex (MTC) DNA in <20 minutes at 39 °C. Assays for two MTC specific targets were investigated, IS6110 and IS1081. When testing purified MTC genomic DNA, limits of detection of 6.25 fg (IS6110) and 20 fg (IS1081)were consistently achieved. When testing a convenience sample of pulmonary specimens from suspected TB patients, RPA demonstrated superior accuracy to indirect fluorescence microscopy. Compared to culture, sensitivities for the IS1081 RPA and microscopy were 91.4% (95%CI: 85, 97.9) and 86.1% (95%CI: 78.1, 94.1) respectively (n = 71). Specificities were 100% and 88.6% (95% CI: 80.8, 96.1) respectively. For the IS6110 RPA and microscopy sensitivities of 87.5% (95%CI: 81.7, 93.2) and 70.8% (95%CI: 62.9, 78.7) were obtained (n = 90). Specificities were 95.4 (95% CI: 92.3,98.1) and 88% (95% CI: 83.6, 92.4) respectively. The superior specificity of RPA for detecting tuberculosis was due to the reduced ability of fluorescence microscopy to distinguish Mtb complex from other acid fast bacteria. The rapid nature of the RPA assay and its low energy requirement compared to other amplification technologies suggest RPA-based TB assays could be of use for integration into a point-of-care test for use in resource constrained settings

Heterogeneous rpoS and rhlR mRNA Levels and 16S rRNA/rDNA (rRNA Gene) Ratios within Pseudomonas aeruginosa Biofilms, Sampled by Laser Capture Microdissection▿

The local environmental conditions in biofilms are dependent on the impinging aqueous solution, chemical diffusion, and the metabolic activities of cells within the biofilms. Chemical gradients established in biofilms lead to physiological heterogeneities in bacterial gene expression. Previously, we used laser capture microdissection (LCM) and quantitative reverse transcription (RT)-PCR to target defined biofilm subpopulations for gene expression studies. Here, we combined this approach with quantitative PCR of bacterial DNA to normalize the amount of gene expression per cell. By comparing the ratio of 16S rRNA to 16S rDNA (rRNA gene), we demonstrated that cells at the top of thick Pseudomonas aeruginosa biofilms have 16S rRNA/genome ratios similar to those of cells in a transition from the exponential phase to the stationary phase. Cells in the middle and bottom layers of these biofilms have ratios that are not significantly different from those of stationary-phase planktonic cultures. Since much of each biofilm appeared to be in a stationary-phase-like state, we analyzed the local amounts of the stationary-phase sigma factor rpoS gene and the quorum-sensing regulator rhlR gene per cell. Surprisingly, the amount of rpoS mRNA was largest at the top of the biofilms at the air-biofilm interface. Less than one rpoS mRNA transcript per cell was observed in the middle or base of the biofilms. The rhlR mRNA content was also greatest at the top of the biofilms, and there was little detectable rhlR expression at the middle or bottom of the biofilms. While the cell density was slightly greater at the bottom of the biofilms, expression of the quorum-sensing regulator occurred primarily at the top of the biofilms, where the cell metabolic activity was greatest, as indicated by local expression of the housekeeping gene acpP and by expression from a constitutive Ptrc promoter. The results indicate that in thick P. aeruginosa biofilms, cells in the 30 μm adjacent to the air-biofilm interface actively express genes associated with stationary phase, while cells in the interior portions do not express these genes and therefore are in a late-stationary-phase-like state and may be dormant

Oligonucleotide primers and probes.

<p>The oligonucleotides chosen for amplification and detection of IS<i>6110</i> and IS<i>1081</i> are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103091#pone-0103091-t002" target="_blank">Table 2</a>. F = dT-FAM, H = tetra hydrofuran and Q = dT-Black Hole Quencher 1.</p

Limit of detection of IS<i>6110</i> RPA and IS<i>1081</i> RPA.

<p>Results shown are for purified DNA extracted from <i>M. bovis</i> BCG. DNA concentrations were measured prior to the preparation of serial dilutions for testing by RPA. The numbers of positive test results and the total number of tests run at each concentration of DNA are recorded.</p

Tuberculosis detection by indirect smear microscopy vs RPA IS<i>6110</i>.

<p>Testing pulmonary specimens (n = 90) by indirect smear microscopy and RPA IS<i>6110</i> to detect tuberculosis, with comparison to liquid culture based test data. RPA <i>IS6110</i> was more sensitive than indirect smear microscopy (87.5% (95% CI: 81.7, 93.2) vs 70.8% (95% CI: 62.91, 78.75)) and also more specific (95.4 (95% CI: 92.3, 98.1) vs 88% (95% CI: 83.6, 92.4)).</p

Real-time RPA amplification of IS<i>1081</i> and IS<i>6110</i>.

<p>Figure <b>2a</b> shows the real time detection of IS<i>1081</i> amplification from a dilution series of quantitated <i>M. bovis</i> BCG DNA with a sensitivity as low as 0.04 pg of DNA per reaction. Figure <b>2b</b> shows the real time detection of IS<i>6110</i> amplification from a dilution series of quantitated <i>M. bovis</i> BCG DNA BCG DNA with a sensitivity as low as 5.0 fg of DNA per reaction.</p

DNA amplification by Recombinase Polymerase Amplification.

<p>The three core proteins, recombinase, single-strand DNA binding protein (SSB) and strand-displacing polymerase enable PCR-like DNA amplification without the need for thermal cycling or an initial chemical or thermal melting step. This diagram was created by TwistDx Ltd (<a href="http://www.twistdx.co.uk/our_technology/" target="_blank">http://www.twistdx.co.uk/our_technology/</a>) and is licensed under a Creative Commons Attribution 3.0 United States License.</p

Strains used for specificity testing.

<p>Non-tuberculous mycobacterial (NTM) strains and other bacterial species used in the determination of the specificity of the IS<i>6110</i> and IS<i>1081</i> RPA assays. All tested negative by both RPA assays. Isolates marked * were supplied by the Mycobacteriology Unit, Institute of Tropical Medicine, Antwerp, Belgium. All other isolates were from the Washington State Public Health Laboratory strain collection.</p

Human herpesvirus 6 reactivation and disease are infrequent in chimeric antigen receptor T-cell therapy recipients

•HHV-6 reactivation in plasma occurred in 6% and possible HHV-6 encephalitis in 0.2% of patients within 12 weeks after CARTx.•HHV-6 reactivation and disease are infrequent after CARTx, and routine HHV-6 monitoring is not warranted.[Display omitted]Human herpesvirus 6B (HHV-6B) reactivation and disease are increasingly reported after chimeric antigen receptor (CAR) T-cell therapy (CARTx). HHV-6 reactivation in the CAR T-cell product was recently reported, raising questions about product and patient management. Because of overlapping manifestations with immune effector cell–associated neurotoxicity syndrome, diagnosing HHV-6B encephalitis is challenging. We provide 2 lines of evidence assessing the incidence and outcomes of HHV-6B after CARTx. First, in a prospective study with weekly HHV-6B testing for up to 12 weeks after infusion, HHV-6B reactivation occurred in 8 of 89 participants; 3 had chromosomally integrated HHV-6 and were excluded, resulting in a cumulative incidence of HHV-6B reactivation of 6% (95% confidence interval [CI], 2.2-12.5). HHV-6B detection was low level (median peak, 435 copies per mL; interquartile range, 164-979) and did not require therapy. Second, we retrospectively analyzed HHV-6B detection in the blood and/or cerebrospinal fluid (CSF) within 12 weeks after infusion in CARTx recipients. Of 626 patients, 24 had symptom-driven plasma testing, with detection in 1. Among 34 patients with CSF HHV-6 testing, 1 patient had possible HHV-6 encephalitis for a cumulative incidence of 0.17% (95% CI, 0.02-0.94), although symptoms improved without treatment. Our data demonstrate that HHV-6B reactivation and disease are infrequent after CARTx. Routine HHV-6 monitoring is not warranted.Kampouri and colleagues report on human herpesvirus 6B (HHV-6B) reactivation and disease following chimeric antigen receptor (CAR) T-cell therapy. A prospective study of 89 patients tested weekly revealed reactivation in 6%, with low-level detection requiring no therapy. In a retrospective study of 626 patients, 24 had symptom-driven plasma testing, with 1 patient testing positive; in 34 patients receiving symptom-driven cerebrospinal fluid testing, 1 patient had HHV-6 that resolved without therapy, with a cumulative incidence of 0.17%. HHV-6B reactivation is infrequent following CAR T-cell therapy, suggesting routine testing is not indicated