Identification of Mycobacterium tuberculosis Infection in Infants and Children With Partial Discrimination Between Active Disease and Asymptomatic Infection

Abstract

Background: Improved diagnostic tests are needed for the early identification of Mycobacterium tuberculosis- infected young children exposed to an active TB (aTB) index case. We aimed to compare the diagnostic accuracy of new blood-based tests to that of the tuberculin skin test (TST) for the identification of all infected children and for a potential differentiation between aTB and latent TB infection (LTBI). Methods: 144 children exposed to a patient with aTB were included, and those who met all inclusion criteria (130/144) were classified in three groups based on results from classical investigations: non-infected (NI: n = 69, 53%, median age 10 months), LTBI ( n = 28, 22%, median age 96 months), aTB disease ( n = 33, 25%, median age 24 months). The first whole blood assay consisted of a 7-days in vitro stimulation of blood with four different mycobacterial antigens (40 μl/condition), followed by flow cytometric measurement of the proportions of blast cells appearing among lymphocytes as a result of their specific activation. Thresholds of positivity were determined by Receiver Operating Characteristic (ROC) curve analysis (results of NI children vs. children with LTBI/aTB) in order to identify infected children in a first stage. Other cut-offs were determined to discriminate subgroups of infected children in a second step (results from children with aTB/LTBI). Analysis of blood monocytes and dendritic cell subsets was performed on 100 μl of blood for 25 of these children as a second test in a pilot study. Results: Combining the results of the blast-induced CD3 + T lymphocytes by Heparin-Binding Haemagglutinin and by Culture Filtrate Protein-10 identified all but one infected children (sensitivity 98.2% and specificity 86.9%, compared to 93.4 and 100% for the TST). Further identification among infected children of those with aTB was best achieved by the results of blast-induced CD8 + T lymphocytes by purified protein derivative (sensitivity for localized aTB: 61.9%, specificity 96.3%), whereas high proportions of blood type 2 myeloid dendritic cells (mDC) were a hallmark of LTBI. Conclusions: New blood-based tests requiring a very small volume allow the accurate identification of M. tuberculosis -infected young children among exposed children and are promising to guide the clinical classification of children with aTB or LTBI.

Specific Host Signatures for the Detection of Tuberculosis Infection in Children in a Low TB Incidence Country

“…Samples from a second, independent cohort of children were tested afterwards, representing a validation cohort. This cohort comprised 60 children included in a study evaluating the ability of antigen-induced blast cells as determined by flow cytometry, to identify and correctly classify M. tuberculosis -infected children ( 13 ). This was a whole blood assay, and an IGRA in response to mycobacterial antigens was performed in parallel on PBMC ( 14 , 15 ).…”

Impact of Maternal Pertussis Antibodies on the Infants’ Cellular Immune Responses

“…Flowcytometric acquisition of PT-specific lymphocytes PT-specific lymphocytes (CD3 + , CD3 + CD4 + and CD3 + CD8 + blasts) were measured on heparin blood samples using a specialized Flowcytometric Assay that detects the Specific Cellmediated Immune response in Activated whole blood (FASCIA). Based on previous protocols [16][17][18] whole blood was diluted 1/10 in RPMI 1640 (Life technologies) supplemented with 50 μg/mL gentamycine, 2 mM L-glutamine, MEM non-essential AA, Na pyruvate, 2Bmercapthoethanol and Fetal Bovine Serum. Diluted blood was either left unstimulated (negative control) or stimulated with 5 μg/mL PT antigen (heat-inactivated PT, Bordetella pertussis strain 165, List Biological) or 1 μg/mL Staphylococcal enterotoxin B (SEB, positive control).…”

Electrochemical Detection of Single-Nucleotide Polymorphism Associated with Rifampicin Resistance in Mycobacterium tuberculosis Using Solid-Phase Primer Elongation with Ferrocene-Linked Redox-Labeled Nucleotides

“…TB can be asymptomatic or latent, resulting in a delay in diagnosis, and, in 2019, an estimated 10 million people fell ill with the disease. This delay in diagnosis is compounded by the increasing prevalence of variants of Mycobacterium tuberculosis resistant to rifampicin, the first-line drug of choice for TB treatment. , The sequencing of the whole genome of Mycobacterium tuberculosis revealed that 95–98% of all rifampicin-resistant strains is related to single-nucleotide polymorphisms (SNPs), located in the 81 bp RIF-resistance determining region (RRDR) of the beta subunit of the RNA polymerase (rpoB) gene. − The World Health Organization reported that around half a million people developed rifampicin-resistant TB in 2019, and in 2010, recommended the use of the Xpert MTB/RIF assay (Cepheid, Sunnyvale, CA) for the detection of TB and rifampicin resistance, and while it is widely used, its implementation in developing countries is hampered by many issues, including cost, maintenance, and power requirements. − …”