Case-control, diagnostic accuracy study of a non-sputum CD38-based TAM-TB test from a single milliliter of blood

ABSTRACTBackgroundCD4 T cell phenotyping-based blood assays have the potential to meet WHO target product profiles (TPP) of non-sputum-biomarker-based tests to diagnose tuberculosis (TB). Yet, substantial refinements are required to allow their implementation in clinical settings. This study assessed the real time performance of a simplified T cell activation marker (TAM)-TB assay to detect TB in adults from one millilitre of blood with a 24h turnaround time.MethodsWe recruited 479 GeneXpert® positive cases and 108 symptomatic but GeneXpert® negative controls from presumptive adult TB patients in the Temeke District of Dar-es-Salaam, Tanzania. TAM-TB assay accuracy was assessed by comparison with a composite reference standard comprising GeneXpert® and solid culture. A single millilitre of fresh blood was processed to measure expression of CD38 or CD27 by CD4 T cells producing INF-γ and/or TNF-α in response to a synthetic peptide pool covering the sequences of Mycobacterium tuberculosis (Mtb) ESAT-6, CFP-10 and TB10.4 antigens on a 4-color FACSCalibur apparatus.ResultsSignificantly superior to CD27 in accurately diagnosing TB, the CD38-based TAM-TB assay specificity reached 93.4% for a sensitivity of 82.2% with an area under the receiver operating characteristics curve of 0.87 (95% CI 0.84-0.91). The assay performance was not significantly affected by HIV status.ConclusionsWe successfully implemented TAM-TB immunoassay routine testing with a 24h turnaround time at district level in a resource limited setting. Starting from one millilitre of fresh blood and being not influenced by HIV status, TAM-TB assay format and performance appears closely compatible with the optimal TPP accuracy criteria defined by WHO for a non-sputum confirmatory TB test.

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Case–control diagnostic accuracy study of a non-sputum CD38-based TAM-TB test from a single milliliter of blood

Scientific Reports ◽

10.1038/s41598-021-92596-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Hellen Hiza ◽

Jerry Hella ◽

Ainhoa Arbués ◽

Beatrice Magani ◽

Mohamed Sasamalo ◽

...

Keyword(s):

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Peptide Pool ◽

Turnaround Time ◽

Hiv Status ◽

Operating Characteristics ◽

Fresh Blood ◽

Resource Limited ◽

And Performance

AbstractCD4 T cell phenotyping-based blood assays have the potential to meet WHO target product profiles (TPP) of non-sputum-biomarker-based tests to diagnose tuberculosis (TB). Yet, substantial refinements are required to allow their implementation in clinical settings. This study assessed the real time performance of a simplified T cell activation marker (TAM)-TB assay to detect TB in adults from one millilitre of blood with a 24 h turnaround time. We recruited 479 GeneXpert positive cases and 108 symptomatic but GeneXpert negative controls from presumptive adult TB patients in the Temeke District of Dar-es-Salaam, Tanzania. TAM-TB assay accuracy was assessed by comparison with a composite reference standard comprising GeneXpert and solid culture. A single millilitre of fresh blood was processed to measure expression of CD38 or CD27 by CD4 T cells producing IFN-γ and/or TNF-α in response to a synthetic peptide pool covering the sequences of Mycobacterium tuberculosis (Mtb) ESAT-6, CFP-10 and TB10.4 antigens on a 4-color FACSCalibur apparatus. Significantly superior to CD27 in accurately diagnosing TB, the CD38-based TAM-TB assay specificity reached 93.4% for a sensitivity of 82.2% with an area under the receiver operating characteristics curve of 0.87 (95% CI 0.84–0.91). The assay performance was not significantly affected by HIV status. To conclude, we successfully implemented TAM-TB immunoassay routine testing with a 24 h turnaround time at district level in a resource limited setting. Starting from one millilitre of fresh blood and being not influenced by HIV status, TAM-TB assay format and performance appears closely compatible with the optimal TPP accuracy criteria defined by WHO for a non-sputum confirmatory TB test.

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CMV-Reactive T Cells Can Be Isolated From G-CSF Mobilised Peripheral Blood Apheresates.

Blood ◽

10.1182/blood.v116.21.1276.1276 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1276-1276 ◽

Cited By ~ 1

Author(s):

Edward R Samuel ◽

Katy Newton ◽

Stephen Mackinnon ◽

Mark W Lowdell

Keyword(s):

T Cells ◽

Stem Cell ◽

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Peptide Pool ◽

Clinical Use ◽

Cell Responses ◽

Selection Of

Abstract Abstract 1276 Reactivation of cytomegalovirus (CMV) post allogeneic stem cell transplant (SCT) has shown to be controlled by the adoptive transfer of donor derived CMV-reactive T cells (CMV-T). A current method for the generation of CMV-T cells is CMV antigenic stimulation of donor peripheral blood mononuclear cells (PBMC), followed by isolation of interferon-gamma (IFN-g) secreting cells, by magnetic enrichment. The feasibility of generating a CMV-T cell product for clinical use, using PBMC from a G-CSF mobilised donor stem cell apheresis has yet to be fully determined. Small aliquots (<1ml) were taken from the mobilised apheresis of CMV positive donors (n=5), the PBMC separated using density gradient centrifugation and then stimulated in a 16 hour culture using a commercially available CMVpp65 peptide pool (Peptivator – Miltenyi Biotec). CMV-T cells were then isolated using both IFN-g secretion and expression of the activation marker CD25. Results were then compared directly against the same methods in non-mobilised PBMC.Figure 1Figure 1. The proportion of CMVpp65 stimulated CD25+ T cells was equivalent between mobilised (53.15%) and non-mobilised (63.28%) PBMC. The CMV-T response was also predominantly CD4+ in both mobilised (98%) and non-mobilised (88%) PBMC. In contrast IFN-g secretion was shown to identify CMV-T in mobilised PBMC (41.77%), but this was consistently 50–60% lower in frequency compared to non-mobilised PBMC (95.59%). This 1–2 fold reduction in IFN-g+ T cell activation does not appear to effect the ratio of CD4+ to CD8+ T cells, which was comparable between mobilised (1:1.1) and non-mobilised (1:1.5) PBMC (Figure 1). Furthermore a reduction was observed in both yield (6.62% vs. 58.93%) and purity (35.68 vs. 76.01%) post enrichment of IFN-g+ CMV-T cells from mobilised PBMC. Of the IFN-g+ cells isolated from non-mobilised PBMC ∼30% were also positive for CD25 whereas this number was undetectable (<1%) in the mobilised setting) (Figure 2a and b).Figure 2aFigure 2a. Figure 2bFigure 2b. Enrichment of CD25+ CMV-T cells showed parity between mobilised and non-mobilised PBMC in terms of both yield (26.12% vs. 34.75%) and purity (89.92% vs. 93.39%). We have also observed that enrichment of CD25+ CMV-T cells depleted the naturally occurring CD25+ T cells seen in the unstimulated PBMC. Background expression of CD25 has been thought previously to be a barrier to selection of activated cells by CD25. However, CD25+ selections from non-stimulated cultures resulted in a yield of <1%. These results indicate that G-CSF can suppress the Ag-specific T cell responses following in vitro stimulation with a CMVpp65 peptide pool but CMV-T can still be detected and isolated. We hypothesise that the reduced IFN-g+ T cell activation in mobilised PBMC could be as a result of the suppressive nature of an increased frequency of FoxP3+ IL-10 secreting T cells compared to that in non-mobilised PBMC. We have demonstrated G-CSF suppression of CMVpp65 stimulated T cell responses with reference to a type 1 cytokine profile. Both CD25+ and IFN-g+ CMV-T from mobilised and non-mobilised PBMC are being tested for secretion of a profile of type 1 and type 2 cytokines to include TNF-α, IL-2, IL-4 and IL-10. Direct selection of CMV-T by CD25 expression represents a potential approach for isolating these cells for clinical use. ES, KN and this work were supported by a grant from the Technology Strategy Board (TSB) of the UK Government Department of Business, Innovation & Skills Disclosures: No relevant conflicts of interest to declare.

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Severe T cell hyporeactivity in ventilated COVID-19 patients correlates with prolonged virus persistence and poor outcomes

Nature Communications ◽

10.1038/s41467-021-23334-2 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Kerstin Renner ◽

Tobias Schwittay ◽

Sophia Chaabane ◽

Johanna Gottschling ◽

Christine Müller ◽

...

Keyword(s):

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Fresh Blood ◽

Virus Persistence ◽

Clinical Recovery ◽

Immunosuppressive Medication ◽

Downstream Effects ◽

Poor Outcomes ◽

Ventilated Patients

AbstractCoronavirus disease 2019 (COVID-19) can lead to pneumonia and hyperinflammation. Here we show a sensitive method to measure polyclonal T cell activation by downstream effects on responder cells like basophils, plasmacytoid dendritic cells, monocytes and neutrophils in whole blood. We report a clear T cell hyporeactivity in hospitalized COVID-19 patients that is pronounced in ventilated patients, associated with prolonged virus persistence and reversible with clinical recovery. COVID-19-induced T cell hyporeactivity is T cell extrinsic and caused by plasma components, independent of occasional immunosuppressive medication of the patients. Monocytes respond stronger in males than females and IL-2 partially restores T cell activation. Downstream markers of T cell hyporeactivity are also visible in fresh blood samples of ventilated patients. Based on our data we developed a score to predict fatal outcomes and identify patients that may benefit from strategies to overcome T cell hyporeactivity.

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