The biochemical and molecular characterisation of respiratory mucins in TB

Includes bibliographical references (leaves 96-112).The role of the dominant respiratory mucins (MUC5AC and MUC5B) and MUC2 has been investigated in chronic airway diseases as it is the mucin glycoprotein that confers upon mucus its biological, rheological and physicochemical properties. Within South Africa, specifically the Western Cape, TB has wreaked havoc especially amongst those of the lower socioeconomic groups. However, despite the prevalence of the disease in South Africa and the known morbidity and mortality associated with mucus and mucin hypersecretion in respiratory diseases, little is known of the association between respiratory mucins and TB. This is a novel study that investigated the association between respiratory mucins and TB at a biochemical and molecular level

The diagnostic accuracy of urine-based Xpert MTB/RIF in HIV-infected hospitalized patients who are smear-negative or sputum scarce

BACKGROUND: Hospitals in sub-Saharan Africa are inundated with HIV-infected patients and tuberculosis (TB) is the commonest opportunistic infection in this sub-group. Up to one third of TB-HIV co-infected patients fail to produce a sputum sample (sputum scarce) and diagnosis is thus often delayed or missed. We investigated the sensitivity of urine-based methods (Xpert MTB/RIF, LAM strip test and LAM ELISA) in such patients. METHODOLOGY/PRINCIPAL FINDINGS: 281 HIV-infected hospitalised patients with clinically suspected TB provided a spot urine sample. The reference standard was culture positivity for Mycobacterium tuberculosis on ≥1 sputum or extra-pulmonary sample. MTB/RIF was performed using 1 ml of both unprocessed and, when possible, concentrated urine. Each unconcentrated urine sample was also tested using the Clearview LAM ELISA and Alere LAM strip test. 42% (116/242) of patients had culture-proven TB. 18% (20/54) were sputum scarce. In sputum-scarce patients, the sensitivity of urine MTB/RIF and LAM ELISA was 40% (95%CI: 22-61) and 60% (95%CI: 39-78), respectively. Urine MTB/RIF specificity was 98% (95%CI: 95-100). Combined sensitivity of urine LAM ELISA and MTB/RIF was better than MTB/RIF alone [MTB/RIF and LAM: 70% (95%CI: 48-85) vs. MTB/RIF: 40% (95%CI: 22-61), p = 0.03]. Significant predictors of urine MTB/RIF positivity were CD4<50 cells/ml (p = 0.001), elevated protein-to-creatinine ratio (p<0.001) and LAM ELISA positivity (p<0.001). Urine centrifugation and pelleting significantly increased the sensitivity of MTB/RIF over unprocessed urine in paired samples [42% (95%CI: 26-58) vs. 8% (95%CI: 0-16), p<0.001]. Urine MTB/RIF-generated C T values correlated poorly with markers of bacillary burden (smear grade and time-to-positivity). Conclusions/Significance This preliminary study indicates that urine-based MTB/RIF, alone or in combination with LAM antigen detection, may potentially aid the diagnosis of TB in HIV-infected patients with advanced immunosuppression when sputum-based diagnosis is not possible. Concentration of urine prior to MTB/RIF-testing significantly improves sensitivity

The diagnostic accuracy of pericardial and urinary lipoarabinomannan (LAM) assays in patients with suspected tuberculous pericarditis.

We evaluated the diagnostic accuracy of urinary and pericardial fluid (PF) lipoarabinomannan (LAM) assays in tuberculous pericarditis (TBP). From October 2009 through September 2012, 151 patients with TBP were enrolled. Mycobacterium tuberculosis culture and/or pericardial histology were the reference standard for definite TBP. 49% (74/151), 33.1% (50/151) and 17.9% (27/151) of patients had definite-, probable-, and non-TB respectively; 69.5% (105/151) were HIV positive. LAM ELISA had the following sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, positive predictive value and negative predictive values (95% confidence interval): urinary - 17.4% (9.1-30.7), 93.8% (71.7-98.9), 2.8 (0.1-63.3), 0.9 (0.8-0.9), 88.9% (56.5-98.0), and 28.3% (17.9-41.6); PF - 11.6% (6.0-21.3), 88% (70.0-95.8), 0.9 (0.08-12.0), 1.0 (0.9-1.1), 72.7% (43.4-90.1), and 26.6% (18.2-36.9). Sensitivity increased with a CD4 ≤ 100 cells/mm(3) from 3.5% to 50% (p < 0.001) for urinary LAM ELISA; for urinary LAM strip test, grade 1 and 2 cut-points performed similarly, irrespective of HIV status or CD4 count. For PF LAM strip tests, switching cut-points from grade 1 to 2 significantly reduced test sensitivity (54.5% versus 19.7%; p < 0.001). Urinary and PF LAM assays have low sensitivity but high specificity for diagnosis of TBP. The sensitivity of urinary LAM is increased in HIV-infected patients with a CD4 ≤ 100 cells/mm(3)

Diagnostic accuracy of quantitative PCR (Xpert MTB/RIF) for tuberculous pericarditis compared to adenosine deaminase and unstimulated interferon-γ in a high burden setting: a prospective study

Background: Tuberculous pericarditis (TBP) is associated with high morbidity and mortality, and is an important treatable cause of heart failure in developing countries. Tuberculous aetiology of pericarditis is difficult to diagnose promptly. The utility of the new quantitative PCR test (Xpert MTB/RIF) for the diagnosis of TBP is unknown. This study sought to evaluate the diagnostic accuracy of the Xpert MTB/RIF test compared to pericardial adenosine deaminase (ADA) and unstimulated interferon-gamma (uIFNγ) in suspected TBP. Methods: From October 2009 through September 2012, 151 consecutive patients with suspected TBP were enrolled at a single centre in Cape Town, South Africa. Mycobacterium tuberculosis culture and/or pericardial histology served as the reference standard for definite TBP. Receiver-operating-characteristic curve analysis was used for selection of ADA and uIFNγ cut-points. Results: Of the participants, 49% (74/151) were classified as definite TBP, 33% (50/151) as probable TBP and 18% (27/151) as non TBP. A total of 105 (74%) participants were human immunodeficiency virus (HIV) positive. Xpert-MTB/RIF had a sensitivity and specificity (95% confidence interval (CI)) of 63.8% (52.4% to 75.1%) and 100% (85.6% to 100%), respectively. Concentration of pericardial fluid by centrifugation and using standard sample processing did not improve Xpert MTB/RIF accuracy. ADA (≥35 IU/L) and uIFNγ (≥44 pg/ml) both had a sensitivity of 95.7% (88.1% to 98.5%) and a negative likelihood ratio of 0.05 (0.02 to 0.10). However, the specificity and positive likelihood ratio of uIFNγ was higher than ADA (96.3% (81.7% to 99.3%) and 25.8 (3.6 to 183.4) versus 84% (65.4% to 93.6%) and 6.0 (3.7 to 9.8); P = 0.03) at an estimated background prevalence of TB of 30%. The sensitivity and negative predictive value of both uIFNγ and ADA were higher than Xpert-MT/RIF (P < 0.001). Conclusions: uIFNγ offers superior accuracy for the diagnosis of microbiologically confirmed TBP compared to the ADA assay and the Xpert MTB/RIF test

Comparison of same day diagnostic tools including Gene Xpert and unstimulated IFN-γ for the evaluation of pleural tuberculosis: a prospective cohort study

Background: The accuracy of currently available same-day diagnostic tools (smear microscopy and conventional nucleic acid amplification tests) for pleural tuberculosis (TB) is sub-optimal. Newer technologies may offer improved detection. Methods: Smear-microscopy, adenosine deaminase (ADA), interferon gamma (IFN-γ), and Xpert MTB/RIF [using an unprocessed (1 ml) and centrifuged (~20 ml) sample] test accuracy was evaluated in pleural fluid from 103 consecutive patients with suspected pleural TB. Culture for M.tuberculosis and/or histopathology (pleural biopsy) served as the reference standard. Patients were followed prospectively to determine their diagnostic categorisation. Results: Of 93 evaluable participants, 40 had definite-TB (reference positive), 5 probable-TB (not definite but treated for TB) and 48 non-TB (culture and histology negative, and not treated for TB). Xpert MTB/RIF sensitivity and specificity (95% CI) was 22.5% (12.4 - 37.6) and 98% (89.2 - 99.7), respectively, and centrifugation did not improve sensitivity (23.7%). The Xpert MTB/RIF internal positive control showed no evidence of inhibition. Biomarker specific sensitivity, specificity, PPV, and NPVs were: ADA (48.85 IU/L; rule-in cut-point) 55.3% (39.8 - 69.9), 95.2% (83.9 - 98.7), 91.4 (73.4 - 95.4), 69.7% (56.7 - 80.1); ADA (30 IU/L; clinically used cut-point) 79% (63.7 - 89), 92.7% (80.6 - 97.5), 91.0 (73.4 - 95.4), 82.7% (69.3 - 90.1); and IFN-γ (107.7 pg/ml; rule-in cut-point) 92.5% (80.2 - 97.5), 95.9% (86.1 - 98.9), 94.9% (83.2 - 98.6), 93.9% (83.5 - 97.9), respectively (IFN-γ sensitivity and NPV better than Xpert [p < 0.05] and rule-in ADA [p < 0.05]). Conclusion: The usefulness of Xpert MTB/RIF to diagnose pleural TB is limited by its poor sensitivity. IFN-γ is an excellent rule-in test and, compared to ADA, has significantly better sensitivity and rule-out value in a TB-endemic setting

Correlation of Mycobacterium Tuberculosis Specific and Non-Specific Quantitative Th1 T-Cell Responses with Bacillary Load in a High Burden Setting

Measures of bacillary load in patients with tuberculosis (TB) may be useful for predicting and monitoring response to treatment. The relationship between quantitative T-cell responses and mycobacterial load remains unclear. We hypothesised that, in a HIV-prevalent high burden setting, the magnitude of mycobacterial antigen-specific and non-specific T-cell IFN-γ responses would correlate with (a) bacterial load and (b) culture conversion in patients undergoing treatment.We compared baseline (n = 147), 2 (n = 35) and 6 month (n = 13) purified-protein-derivative (PPD) and RD1-specific (TSPOT.TB and QFT-GIT) blood RD1-specific (TSPOT.TB; QFT-GIT) responses with associates of sputum bacillary load in patients with culture-confirmed TB in Cape Town, South Africa.IFN-γ responses were not associated with liquid culture time-to-positivity, smear-grade, Xpert MTB/RIF-generated cycle threshold values or the presence of cavities on the chest radiograph in patients with culture-confirmed TB and irrespective of HIV-status. 2-month IGRA conversion rates (positive-to-negative) were negligible [<11% for TSPOT.TB (3/28) and QFT-GIT (1/29)] and lower compared to culture [60% (21/35); p<0.01].In a high burden HIV-prevalent setting T-cell IFN-γ responses to M. tuberculosis-specific and non-specific antigens do not correlate with bacillary load, including Xpert MTB/RIF-generated C(T) values, and are therefore poorly suited for monitoring treatment and prognostication

Point of Care Diagnostics for HIV in Resource Limited Settings: An Overview

Human immunodeficiency virus (HIV) is a global health problem. Early diagnosis, rapid antiretroviral therapy (ART) initiation and monitoring of viral load are the key strategies for effective HIV management. Many people in resource limited settings where timely access to medical care is a challenge and healthcare infrastructure is poor have no access to laboratory facilities and diagnosis is dependent on the presence of point of care (POC) devices. POC instruments have shown to be easy to operate, maintain and transport and can easily be operated by less skilled health workers. Additionally, POC tests do not require laboratory technicians to operate. POC devices have resulted in a growing number of people testing for HIV and thereby receiving treatment early. In recent years, there has been great improvement in the development of POC technologies for early HIV diagnosis, HIV viral load and cluster of differentiation 4 (CD4) measurement. This review discusses POC technologies that are currently available and in the pipeline for diagnosing and monitoring HIV. We also give an overview of the technical and commercialization challenges in POC diagnostics for HIV

Urine-based MTB/RIF-generated C_T values correlates with other markers of bacillary burden.

<p>In <i>m.tb</i> culture and urine MTB/RIF positive patients, MTB/RIF-generated C_T values were correlated with (A) urine LAM ELISA concentration, (B) urine LAM strip grading, (C) either sputum or non-sputum liquid culture time-to-positivity (TTP), (D) only sputum liquid culture time-to-positivity, and (E) smear-grade in smear positive patients.</p

The Deubiquitinating Enzyme USP17 Blocks N-Ras Membrane Trafficking and Activation but Leaves K-Ras Unaffected

The proto-oncogenic Ras isoforms (H, N, and K) have a C-terminal CAAX motif and undergo the same post-translational processing steps, although they traffic to the plasma membrane through different routes. Previously, we have shown that overexpression of the deubiquitinating enzyme USP17 inhibits H-Ras localization to the plasma membrane. Now we report that whereas H-Ras and N-Ras were unable to localize to the plasma membrane in the presence of USP17, K-Ras4b localization was unaffected. EGF stimulation was unable to induce N-Ras membrane localization in USP17-expressing cells. In addition, N-Ras activity and downstream signaling through the MAPK MEK/ERK and PI3K/JNK pathways were blunted. However, we still detected abundant N-Ras localization at the ER and Golgi in USP17-expressing cells. Collectively, our data showed that the deubiquitinating enzyme USP17 blocks EGF-induced N-Ras membrane trafficking and activation, but left K-Ras unaffected