Sputum lipoarabinomannan (LAM) as a biomarker to determine sputum mycobacterial load: exploratory and model-based analyses of integrated data from four cohorts.

BACKGROUND: Despite the high global disease burden of tuberculosis (TB), the disease caused by Mycobacterium tuberculosis (Mtb) infection, novel treatments remain an urgent medical need. Development efforts continue to be hampered by the reliance on culture-based methods, which often take weeks to obtain due to the slow growth rate of Mtb. The availability of a "real-time" measure of treatment efficacy could accelerate TB drug development. Sputum lipoarabinomannan (LAM; an Mtb cell wall glycolipid) has promise as a pharmacodynamic biomarker of mycobacterial sputum load. METHODS: The present analysis evaluates LAM as a surrogate for Mtb burden in the sputum samples from 4 cohorts of a total of 776 participants. These include those from 2 cohorts of 558 non-TB and TB participants prior to the initiation of treatment (558 sputum samples), 1 cohort of 178 TB patients under a 14-day bactericidal activity trial with various mono- or multi-TB drug therapies, and 1 cohort of 40 TB patients with data from the first 56-day treatment of a standard 4-drug regimen. RESULTS: Regression analysis demonstrated that LAM was a predictor of colony-forming unit (CFU)/mL values obtained from the 14-day treatment cohort, with well-estimated model parameters (relative standard error ≤ 22.2%). Moreover, no changes in the relationship between LAM and CFU/mL were observed across the different treatments, suggesting that sputum LAM can be used to reasonably estimate the CFU/mL in the presence of treatment. The integrated analysis showed that sputum LAM also appears to be as good a predictor of time to Mycobacteria Growth Incubator Tube (MGIT) positivity as CFU/mL. As a binary readout, sputum LAM positivity is a strong predictor of solid media or MGIT culture positivity with an area-under-the-curve value of 0.979 and 0.976, respectively, from receiver-operator curve analysis. CONCLUSIONS: Our results indicate that sputum LAM performs as a pharmacodynamic biomarker for rapid measurement of Mtb burden in sputum, and thereby may enable more efficient early phase clinical trial designs (e.g., adaptive designs) to compare candidate anti-TB regimens and streamline dose selection for use in pivotal trials. Trial registration NexGen EBA study (NCT02371681).

Application of Cerebrospinal Fluid Host Protein Biosignatures in the Diagnosis of Tuberculous Meningitis in Children from a High Burden Setting.

BACKGROUND: The diagnosis of tuberculous meningitis (TBM) especially in children is challenging. New tests are urgently needed for the diagnosis of the disease, especially in resource-limited settings. METHODS: We collected cerebrospinal fluid (CSF) samples from children presenting with symptoms requiring investigation for meningitis at a tertiary hospital in Cape Town, South Africa. Children were later classified as TBM or no TBM using published case definitions. Using a multiplex platform, we investigated the concentrations of biomarkers comprising a previously established 3-marker biosignature (VEGF, IL-13, and LL-37) and other potentially useful host biomarkers as diagnostic candidates for TBM. FINDINGS: Out of 47 children, age, 3 months to 13 years, 23 were diagnosed with TBM and six (16%) were HIV-infected. We validated the previously identified CSF biosignature (sensitivity of 95.7% (95% CI, 79.0-99.2%) and specificity of 37.5% (95% CI, 21.2-57.3%)). However, substitution of IL-13 and LL-37 with IFN-γ and MPO, respectively, resulted in improved accuracy (area under the ROC curve (AUC) = 0.97, 95% CI, 0.92-1.00, up to 91.3% (21/23) sensitivity and up to 100% (24/24) specificity). An alternative four-marker biosignature (sICAM-1, MPO, CXCL8, and IFN-γ) also showed potential, with an AUC of 0.97. CONCLUSION: We validated a previously identified CSF biosignature and showed that refinement of this biosignature by incorporation of other biomarkers diagnosed TBM with high accuracy. Incorporation of these biomarkers into a point-of-care or bedside diagnostic test platform may result in the improved management of TBM in children.

S100A8/A9 regulates CD11b expression and neutrophil recruitment during chronic tuberculosis.

Neutrophil accumulation is associated with lung pathology during active tuberculosis (ATB). However, the molecular mechanism or mechanisms by which neutrophils accumulate in the lung and contribute to TB immunopathology are not fully delineated. Using the well-established mouse model of TB, our new data provide evidence that the alarmin S100A8/A9 mediates neutrophil accumulation during progression to chronic TB. Depletion of neutrophils or S100A8/A9 deficiency resulted in improved Mycobacterium tuberculosis (Mtb) control during chronic but not acute TB. Mechanistically, we demonstrate that, following Mtb infection, S100A8/A9 expression is required for upregulation of the integrin molecule CD11b specifically on neutrophils, mediating their accumulation during chronic TB disease. These findings are further substantiated by increased expression of S100A8 and S100A9 mRNA in whole blood in human TB progressors when compared with nonprogressors and rapidly decreased S100A8/A9 protein levels in the serum upon TB treatment. Furthermore, we demonstrate that S100A8/A9 serum levels along with chemokines are useful in distinguishing between ATB and asymptomatic Mtb-infected latent individuals. Thus, our results support targeting S100A8/A9 pathways as host-directed therapy for TB.

Africa-wide evaluation of host biomarkers in QuantiFERON supernatants for the diagnosis of pulmonary tuberculosis.

We investigated host-derived biomarkers that were previously identified in QuantiFERON supernatants, in a large pan-African study. We recruited individuals presenting with symptoms of pulmonary TB at seven peripheral healthcare facilities in six African countries, prior to assessment for TB disease. We then evaluated the concentrations of 12 biomarkers in stored QuantiFERON supernatants using the Luminex platform. Based on laboratory, clinical and radiological findings and a pre-established algorithm, participants were classified as TB disease or other respiratory diseases(ORD). Of the 514 individuals included in the study, 179(34.8%) had TB disease, 274(51.5%) had ORD and 61(11.5%) had an uncertain diagnosis. A biosignature comprising unstimulated IFN-γ, MIP-1ß, TGF-α and antigen-specific levels of TGF-α and VEGF, identified on a training sample set (n = 311), validated by diagnosing TB disease in the test set (n = 134) with an AUC of 0.81(95% CI, 0.76-0.86), corresponding to a sensitivity of 64.2%(95% CI, 49.7-76.5%) and specificity of 82.7%(95% CI, 72.4-89.9%). Host biomarkers detected in QuantiFERON supernatants can contribute to the diagnosis of active TB disease amongst people presenting with symptoms requiring investigation for TB disease, regardless of HIV status or ethnicity in Africa.

Detection of a combination of serum IgG and IgA antibodies against selected mycobacterial targets provides promising diagnostic signatures for active TB.

Immunoglobulin G (IgG) based tests for the diagnosis of active tuberculosis (TB) disease often show a lack of specificity in TB endemic regions, which is mainly due to a high background prevalence of LTBI. Here, we investigated the combined performance of the responses of different Ig classes to selected mycobacterial antigens in primary healthcare clinic attendees with signs and symptoms suggestive of TB. The sensitivity and specificity of IgA, IgG and/or IgM to LAM and 7 mycobacterial protein antigens (ESAT-6, Tpx, PstS1, AlaDH, MPT64, 16kDa and 19kDa) and 2 antigen combinations (TUB, TB-LTBI) in the plasma of 63 individuals who underwent diagnostic work-up for TB after presenting with symptoms and signs compatible with possible active TB were evaluated. Active TB was excluded in 42 individuals of whom 21 has LTBI whereas active TB was confirmed in 21 patients of whom 19 had a follow-up blood draw at the end of 6-month anti-TB treatment. The leading single serodiagnostic markers to differentiate between the presence or absence of active TB were anti-16 kDa IgA, anti-MPT64 IgA with sensitivity and specificity of 90%/90% and 95%/90%, respectively. The combined use of 3 or 4 antibodies further improved this performance to accuracies above 95%. After successful completion of anti-TB treatment at month 6, the levels of 16 kDa IgA and 16 kDa IgM dropped significantly whereas LAM IgG and TB-LTBI IgG increased. These results show the potential of extending investigation of anti-tuberculous IgG responses to include IgM and IgA responses against selected protein and non-protein antigens in differentiating active TB from other respiratory diseases in TB endemic settings.

Detecting tuberculosis infection in HIV-infected children: a study of diagnostic accuracy, confounding and interaction.

BACKGROUND: Accurate identification of Mycobacterium tuberculosis infection in young and HIV-infected children could guide delivery of preventive therapy, improve resource utilization and help prevent tuberculosis. METHODS: We assessed the performance of the tuberculin skin test (TST) and interferon-γ release assays (IGRAs) for identifying M. tuberculosis infection in South African children presenting for outpatient care. Tuberculosis contact was quantified using a standardized measure of M. tuberculosis exposure. Logistic regression assessed the association among test positivity, age, nutritional and HIV status, while controlling for M. tuberculosis exposure, bacille Calmette-Guérin vaccination and prior tuberculosis treatment. RESULTS: Among 250 (130 HIV infected) children (age 0.25-14.6 years, median 39 months), the proportion positive for each test varied: 34% (TST), 21% (T-SPOT.TB) and 25% (QuantiFERON-TB Gold In-Tube). IGRAs were more likely to be positive in HIV-uninfected compared with HIV-infected children; TST positivity did not differ between these groups. Agreement between tests was good-to-excellent in HIV-uninfected children and poor-to-good in HIV-infected children. In adjusted models, TST and T-SPOT.TB were positively associated with age; this effect varied by HIV status. The QuantiFERON-TB Gold In-Tube was negatively associated with chronic malnutrition; this effect varied by HIV status. Because 93% of children had received bacille Calmette-Guérin, we could not assess the contribution of bacille Calmette-Guérin to false-positive TST results. CONCLUSIONS: Our findings indicate that the TST and IGRAs perform similarly for the detection of M. tuberculosis infection in well-nourished HIV-uninfected children, but test performance is differentially affected by chronic malnutrition, HIV infection and age. Similar to TST interpretation, clinicians and researchers should interpret IGRAs in children with caution taking age, nutritional and HIV status into consideration.