A non-sputum triage test to rule out TB disease is a WHO high-priority diagnostic and a combinatory score based on a 3-gene host-signature has shown promise in discriminating TB from other illnesses. We evaluated the accuracy of an early-prototype cartridge-assay (“Xpert MTB Host Response”, or Xpert-MTB-HR-Prototype) of this 3-gene signature on bio-banked blood-samples from PLHIV against a comprehensive microbiological reference standard (CMRS) and against Xpert® MTB/RIF on first sputum alone. We depict results based on performance targets set by WHO in comparison with a laboratory-based CRP assay. Of 201 patients included, 67 were culture-positive for Mycobacterium tuberculosis. AUC for the Xpert-MTB-HR-Prototype was 0·89 (CI 0·83-0·94) against the CMRS and 0·94 (CI 0·89-0·98) against Xpert MTB/RIF. Considering Xpert-MTB-HR-Prototype as a triage test (at nearest upper value of sensitivity to 90%), specificity was 55·8% (CI 47·2-64·1) compared to the CMRS and 85·9% (CI 79·3-90·7) compared to Xpert MTB/RIF as confirmatory tests. Considering Xpert-MTB-HR-Prototype as a stand-alone diagnostic test, at a specificity near 95%, the test achieved a sensitivity of 65·7% (CI 53·7-75·9) while CRP achieved a sensitivity of only 13·6% (CI 7·3-23·4). In this first accuracy study of a prototype blood-based host-marker assay, we show the possible value of the assay for triage and diagnosis in PLHIV.

'MegaPlex PCR' is a robust technology for highly multiplexed amplification of specific DNA sequences. It uses target-specific pairs of PCR primers that are physically separated by surface immobilization. Initial surface-based amplification cycles are then coupled to efficient solution-phase PCR using one common primer pair. We demonstrate this method by coamplifying and genotyping 75 unselected human single-nucleotide polymorphism (SNP) loci.The key challenge in developing a high-multiplex amplification procedure is preventing excessive off-target priming by the many primers in the reaction [1][2][3] . Several previously developed methods use surface immobilization of primer pairs as a way to separate reactions and thus limit primerdimer formation [4][5][6] , but drawbacks of these methods include inefficient amplification, loss of reactants from the surface and considerable primer-dimer formation within pairs of primers.MegaPlex PCR likewise uses physical separation of primer pairs to prevent their interaction, but it additionally includes 'common sequences' at the 5′ ends of the surface primers so that early-stage amplicons can be moved into the solution phase for co-amplification by highly efficient and unbiased PCR using a single common primer pair [6][7][8] (Fig. 1). To reduce primer-dimer formation, MegaPlex PCR uses surface primers that are made partially double-stranded by base-pairing their common 5′ sequences with complementary 'barrier oligonucleotides'. A detailed methods description is available in Supplementary Methods online.During the development of MegaPlex PCR we evaluated a range of targets, input DNAs and reaction conditions. We used membrane arrays and microbeads as binding surfaces, with the beads now routinely attached to microtiter plate wells to provide a platform compatible with automation.Using the microbead support we found MegaPlex PCR to be effective with as little as 200 ng of human genomic DNA, and in various 15-plex reactions it recovered many different target sequences of up to at least 500 bp in size, with little bias against larger amplicons ( Supplementary Fig. 1 online). As occasional targets (~1/10 in preliminary studies) generated severe primer-dimer artifacts, regardless of the multiplex level of the experiment, optimization of MegaPlex PCR was required. One option was the exclusion of these targets by empirical or computational prefiltering, but we selected a modification that avoided the need for target preselection.This modification involves enriching the input genomic DNA for sequences of interest via a crude solution-phase multiplex PCR using specific primers for all the amplification targets. The primer sequences for this can match the specific portions of those on the solid surface, or they can be designed to prime slightly outside these sites in the genomic DNA. This strategy is used routinely in digital molecular counting and genome mapping studies that use limiting-dilution samples, with multiplexing up to at least 1,200 (ref. 9). Although this is less specific ...

A non-sputum triage test to rule out TB disease is a WHO high-priority diagnostic and a combinatory score based on a 3-gene host-signature has shown promise in discriminating TB from other illnesses. We evaluated the accuracy of an early-prototype cartridge-assay (Xpert MTB Host Response, or Xpert-MTB-HR-Prototype) of this 3-gene signature on bio-banked blood-samples from PLHIV against a comprehensive microbiological reference standard (CMRS) and against Xpert MTB/RIF on first sputum alone. We depict results based on performance targets set by WHO in comparison with a laboratory-based CRP assay. Of 201 patients included, 67 were culture-positive for Mycobacterium tuberculosis. AUC for the Xpert-MTB-HR-Prototype was 0.89 (CI 0.83-0.94) against the CMRS and 0.94 (CI 0.89-0.98) against Xpert MTB/RIF. Considering Xpert-MTB-HR-Prototype as a triage test (at nearest upper value of sensitivity to 90%), specificity was 55.8% (CI 47.2-64.1) compared to the CMRS and 85.9% (CI 79.3-90.7) compared to Xpert MTB/RIF as confirmatory tests. Considering Xpert-MTB-HR-Prototype as a stand-alone diagnostic test, at a specificity near 95%, the test achieved a sensitivity of 65.7% (CI 53.7-75.9) while CRP achieved a sensitivity of only 13.6% (CI 7.3-23.4). In this first accuracy study of a prototype blood-based host-marker assay, we show the possible value of the assay for triage and diagnosis in PLHIV.

Objectives A novel 3-gene host transcriptional signature (GBP5, DUSP3 and KLF2) has been validated for tuberculosis (TB) treatment monitoring using laboratory-based RNA sequencing platforms. The signature was recently translated by Cepheid into a prototype cartridge-based test that can be run on the GeneXpert instrument. In this study, we prospectively evaluated the change in the expression of the cartridge-based 3-gene signature following treatment initiation among pulmonary TB patients who were microbiologically cured at the end of treatment. Results The 3-gene signature expression level (TB score) changed significantly over time with respect to baseline among 31 pulmonary TB patients. The greatest increase in TB score occurred within the first month of treatment (median fold-increase in TB score: 1.08 [IQR 0.54–1.52]) and plateaued after 4 months of treatment (median TB score: 1.97 [IQR: 1.03–2.33]). The rapid and substantial increase of the TB score in the first month of treatment holds promise for the early identification of patients that respond to TB treatment. The plateau in TB score at 4 months may indicate early clearance of disease and could direct treatment to be shortened. These hypotheses need to be further explored with larger prospective treatment monitoring studies.

Melting-curve procedures track DNA denaturation in real time and so provide an effective way of assessing sequence variants. Dynamic allele-specific hybridization (DASH) is one such method, based on fluorescence, which uses heat to denature a single allelespecific probe away from one strand of a polymerase chain reaction product attached to a solid support. DASH is a proven system for research genotyping, but here we evaluate it for DNA diagnostics under two scenarios. First , for mutation scanning (resequencing) , a human genomic sequence of 97 bp was interrogated with 15 probes tiled with 12-base overlaps , providing up to fourfold redundancy per base. This test sequence spanned three high-frequency single nucleotide polymorphisms , all of which were correctly revealed in 16 individuals. Second , to score multiple different mutations in parallel , 18 alterations in the gyrA gene of Salmonella were assessed in 62 strains by using wild-type-and mutation-specific probes. Both experiments were performed in a blinded manner , and all results were confirmed by sequencing. All DNA variants were unambiguously resolved in every sample , with no false-positive or false-negative signals across all of the investigations. In conclusion , DASH performs accurately and robustly when applied to DNA diagnostic challenges , including mutation scoring and mutation scanning. (J Mol Diagn 2007, 9