Anke Binder

Diagnostic accuracy of a urine lipoarabinomannan strip-test for TB detection in HIV-infected hospitalised patients

Lack of point-of-care tests for tuberculosis (TB) result in diagnostic delay, and increased mortality and healthcare-related costs. The urine DetermineTM TB-LAM point-of-care strip-test was evaluated in 335 prospectively-recruited hospitalised patients with suspected TB-HIV co-infection (group 1) and from 88 HIV-infected hospitalised patients with non-TB diagnoses (group 2). Cut-off point-specific analyses were performed using: 1) a microbiological reference standard (culture positive versus negative); and 2) a composite reference standard (exclusion of patients with clinical-TB from the culture-negative group). Using the microbiological reference and the manufacturer-recommended grade-1 cut-off point, LAM sensitivity and specificity was 66% (95% CI 57–74%). By contrast, using the composite reference sensitivity was 60% (95% CI 53–67%) and specificity improved to 96% (95% CI 89–100%) (p=0.001). The same pattern was seen when the grade-2 cut-off point was used (specificity 75% versus 96%; p=0.01). In group two patients specificity was poor using the grade-1 cut-off point, but improved significantly when the grade-2 cut-off point was used (90% versus 99%; p=0.009). The grade-2 cut-off point also offered superior inter-reader reliability (p=0.002). Sensitivity was highest in those with a CD4 <200 cells per mL. LAM combined with smear-microscopy was able to rule-in TB in 71% of Mycobacterium tuberculosis culture-positive patients. This preliminary study indicates that the LAM strip-test may be a potentially useful rapid rule-in test for TB in hospitalised patients with advanced immunosuppression. The grade 2, but not the manufacturer-recommended grade 1 cut-off point, offered superior rule-in utility and inter-reader reliability. Larger studies to evaluate cut-off points and diagnostic accuracy are urgently required.

Comparison of Quantitative Techniques including Xpert MTB/RIF to Evaluate Mycobacterial Burden

Introduction Accurate quantification of mycobacterial load is important for the evaluation of patient infectiousness, disease severity and monitoring treatment response in human and in-vitro laboratory models of disease. We hypothesized that newer techniques would perform as well as solid media culture to quantify mycobacterial burden in laboratory specimens. Methods We compared the turn-around-time, detection-threshold, dynamic range, reproducibility, relative discriminative ability, of 4 mycobacterial load determination techniques: automated liquid culture (BACTEC-MGIT-960), [3H]-uracil incorporation assays, luciferase-reporter construct bioluminescence, and quantitative PCR(Xpert -MTB/RIF) using serial dilutions of Mycobacterium bovis and Mycobacterium tuberculosis H37RV. Mycobacterial colony-forming-units(CFU) using 7H10-Middlebrook solid media served as the reference standard. Results All 4 assays correlated well with the reference standard, however, bioluminescence and uracil assays had a detection threshold ≥1×103 organisms. By contrast, BACTEC-MGIT-960 liquid culture, although only providing results in days, was user-friendly, had the lowest detection threshold (<10 organisms), the greatest discriminative ability (1 vs. 10 organisms; p = 0.02), and the best reproducibility (coefficient of variance of 2% vs. 38% compared to uracil incorporation; p = 0.02). Xpert-MTB/RIF correlated well with mycobacterial load, had a rapid turn-around-time (<2 hours), was user friendly, but had a detection limit of ∼100 organisms. Conclusions Choosing a technique to quantify mycobacterial burden for laboratory or clinical research depends on availability of resources and the question being addressed. Automated liquid culture has good discriminative ability and low detection threshold but results are only obtained in days. Xpert MTB/RIF provides rapid quantification of mycobacterial burden, but has a poorer discrimination and detection threshold.

Regulatory T Cells Attenuate Mycobacterial Stasis in Alveolar and Blood-derived Macrophages from Patients with Tuberculosis

RATIONALE There are hardly any data about the frequency of CD4(+)CD25(+)Foxp3(+) regulatory T cells (T-Regs) in the lungs of patients with active tuberculosis (TB). OBJECTIVES To obtain data about the frequency of CD4(+)CD25(+)Foxp3(+) T-Regs, and their impact on mycobacterial containment, in the lungs of patients with active TB. METHODS Patients with pulmonary TB (n = 49) and healthy volunteers with presumed latent TB infection (LTBI; n = 38) donated blood and/or bronchoalveolar lavage (BAL) cells obtained by bronchoscopy. T-cell phenotype (Th1/Th2/Th17/T-Reg) and functional status was evaluated using flow-cytometry and (3)H-thymidine proliferation assays, respectively. H37Rv-infected alveolar and monocyte-derived macrophages were cocultured with autologous T-Regs and purified protein derivative (PPD) preprimed T-Reg-depleted effector cells. Mycobacterial containment was evaluated by counting CFUs. MEASUREMENTS AND MAIN RESULTS In blood and BAL T-Reg levels were higher in TB versus LTBI (P < 0.04), and in TB the frequency of T-Regs was significantly higher in BAL versus blood (P < 0.001). T-Reg-mediated suppression of T-cell proliferation in blood and BAL was concentration-dependent. Restriction of mycobacterial growth in infected alveolar and monocyte-derived macrophages was significantly diminished, and by up to 50%, when T-Regs were cocultured with PPD-primed CD4(+) effector T cells. The levels of CD8(+) T-Regs (CD8(+)CD25(+)Foxp3(+)), IL-17-producing T-Regs (IL-17(+)CD4(+)CD25(+)Foxp3(+)), and IL-17-producing T cells were similar in BAL-TB versus BAL-LTBI. Within the TB group compartmentalization of responses was prominent (T-Reg, IFN-γ, tumor necrosis factor-α, IL-17, and IL-22 significantly higher in BAL vs. blood). CONCLUSIONS In patients with TB the alveolar compartment is enriched for CD4(+) T-Regs. Peripheral blood-derived T-Regs decrease the ability of alveolar and monocyte-derived macrophages to restrict the growth of Mycobacterium tuberculosis in the presence of effector cells. Collectively, these data suggest that CD4(+)CD25(+)FoxP3(+) T-Regs subvert antimycobacterial immunity in human TB.

Will an unsupervised self-testing strategy for HIV work in health care workers of South Africa? A cross sectional pilot feasibility study.

Background In South Africa, stigma, discrimination, social visibility and fear of loss of confidentiality impede health facility-based HIV testing. With 50% of adults having ever tested for HIV in their lifetime, private, alternative testing options are urgently needed. Non-invasive, oral self-tests offer a potential for a confidential, unsupervised HIV self-testing option, but global data are limited. Methods A pilot cross-sectional study was conducted from January to June 2012 in health care workers based at the University of Cape Town, South Africa. An innovative, unsupervised, self-testing strategy was evaluated for feasibility; defined as completion of self-testing process (i.e., self test conduct, interpretation and linkage). An oral point-of-care HIV test, an Internet and paper-based self-test HIV applications, and mobile phones were synergized to create an unsupervised strategy. Self-tests were additionally confirmed with rapid tests on site and laboratory tests. Of 270 health care workers (18 years and above, of unknown HIV status approached), 251 consented for participation. Findings Overall, about 91% participants rated a positive experience with the strategy. Of 251 participants, 126 evaluated the Internet and 125 the paper-based application successfully; completion rate of 99.2%. All sero-positives were linked to treatment (completion rate:100% (95% CI, 66.0–100). About half of sero-negatives were offered counselling on mobile phones; completion rate: 44.6% (95% CI, 38.0–51.0). A majority of participants (78.1%) were females, aged 18–24 years (61.4%). Nine participants were found sero-positive after confirmatory tests (prevalence 3.6% 95% CI, 1.8–6.9). Six of nine positive self-tests were accurately interpreted; sensitivity: 66.7% (95% CI, 30.9–91.0); specificity:100% (95% CI, 98.1–100). Interpretation Our unsupervised self-testing strategy was feasible to operationalize in health care workers in South Africa. Linkages were successfully operationalized with mobile phones in all sero-positives and about half of the sero-negatives sought post-test counselling. Controlled trials and implementation research studies are needed before a scale-up is considered.

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

BackgroundThe 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.MethodsSmear-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.ResultsOf 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]).ConclusionThe 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.

Cigarette smoke impairs cytokine responses and BCG containment in alveolar macrophages

Background There is a strong epidemiological link between smoking and tuberculosis (TB), but the association is confounded by socioeconomic and other factors. A direct relationship between cigarette smoke and poor treatment-related outcomes in patients with TB is therefore questionable. We investigated whether constituents of tobacco smoke impair mycobacterial host immune responses in vitro. Methodology Preparation of a cigarette smoke extract (CSE) from Marlboro Red cigarettes was standardised and reproducibility verified by mass spectroscopy. Macrophages were derived from peripheral blood monocytes (MDM) and alveolar macrophages from bronchoalveolar lavage fluid from healthy non-smoking volunteers. Mycobacterial uptake (flow cytometric detection of fluorescence using green fluorescent protein-labelled BCG), cytokine responses (ELISA) and mycobacterial containment (colony forming units) was evaluated in both macrophage populations with and without co-culture with CSE, nicotine and a nicotine receptor blocker. Results Cigarette smoke failed to impair the uptake of mycobacteria by monocyte-derived or alveolar macrophages. CSE (vs no CSE) reduced the mean (SD) BCG-driven macrophage (MDM) interferon γ (IFN-γ), tumour necrosis factor α (TNF-α) and interleukin 10 (IL-10) responses by 56.4 (18.6)%, 67.0 (33.4)% and 77.7 (27.7)%, respectively (p<0.001). Nicotine alone impaired IL-10 and TNF-α production by 48.8 (37)% and 49 (50)%, respectively (p<0.05) through an α-7 nicotine receptor-independent mechanism. In 5-day cultures, CSE impaired mycobacterial (BCG) containment in both monocyte-derived and alveolar macrophages. Conclusions Cigarette smoke attenuates effector cytokine responses and impairs mycobacterial containment within infected human macrophages derived from the peripheral blood and alveolar compartments, thus supporting the hypothesis that cigarette smoke subverts mycobacteria-related immunity.

Regulatory T Cells Subvert Mycobacterial Containment in Patients Failing Extensively Drug-Resistant Tuberculosis Treatment

&NA; Rationale: The advent of extensively drug‐resistant (XDR) tuberculosis (TB) and totally drug‐resistant TB, with limited or no treatment options, has facilitated renewed interest in host‐directed immunotherapy, particularly for therapeutically destitute patients. However, the selection and utility of such approaches depend on understanding the host immune response in XDR‐TB, which hitherto remains unexplored. Objectives: To determine the host immunological profile in patients with XDR‐TB, compared with drug‐sensitive TB (DS‐TB), using peripheral blood and explanted lung tissue. Methods: Blood and explanted lung tissue were obtained from patients with XDR‐TB (n = 31), DS‐TB (n = 20), and presumed latent TB infection (n = 20). T‐cell phenotype (T‐helper cell type 1 [Th1]/Th2/Th17/regulatory T cells [Tregs]) was evaluated in all patient groups, and Treg function assessed in XDR‐TB nonresponders by coculturing PPD‐preprimed effector T cells with H37Rv‐infected monocyte‐derived macrophages, with or without autologous Tregs. Mycobacterial containment was evaluated by counting colony‐forming units. Measurements and Main Results: Patients failing XDR‐TB treatment had an altered immunophenotype characterized by a substantial increase in the frequency (median; interquartile range) of CD4+CD25+FoxP3+ Tregs (11.5%; 5.9‐15.2%) compared with DS‐TB (3.4%; 1.6‐5.73%; P < 0.001) and presumed latent TB infection (1.8%; 1.2‐2.3%; P < 0.001), which was unrelated to disease duration. Tregs isolated from patients with XDR‐TB suppressed T‐cell proliferation (up to 90%) and subverted containment of H37Rv‐infected monocyte‐derived macrophages (by 30%; P = 0.03) by impairing effector T‐cell function through a mechanism independent of direct cell‐to‐cell contact, IL‐10, TGF (transforming growth factor)‐&bgr;, and CTLA‐4 (cytotoxic T‐lymphocyte‐associated protein 4). Conclusions: Collectively, these data suggest that Tregs may be contributing to immune dysfunction, and bacterial persistence, in patients with XDR‐TB. The relevant cellular pathways may serve as potential targets for immunotherapeutic intervention.

P5.025 Development and Comparative Evaluation of an Innovative HIV Self-Testing Smartphone Application, an Internet-Based and a Paper-Based Instructional Programme in South Africa

Background South Africa has about 11% of the total population living with HIV, the largest to date for any country. Facility-based HIV testing has reached only 50% South Africans because of fear of visibility leading to stigma, embarrassment and discrimination. Alternative strategies like self-testing for HIV may improve engagement, but evidence is limited. For self-testing to be successful, knowledge regarding the process, clear instructions about how to conduct, interpret and seek linkages to counselling and staging is essential. Methods We created an internet-based HIV self-testing programme with a popular oral HIV test. The programme had built-in content for counselling, personal risk staging, instructions to self-test, and to seek counselling and referral. We also created an equivalent paper version and evaluated both programmes in 251 health care professionals working at University of Cape Town, South Africa. The tested internet programme was converted into an interactive, engaging smartphone HIV self-test application. The application was piloted for design, content and comprehension in 12 young adults (aged 18–25 years). Results Internet and paper-based self-testing programmes were well received (91.3%) by participants with overall preference for self-testing reported at 100%. User feedback on the smartphone application was incorporated after pilot evaluation and the following were improved: (a) a user centred design and layout, (b) colourful interface with clear instructions, (c) clarity of content for comprehension, (d) built-in features for expanded access, and (e) overall presentation. After six iterations, a prototype Android application was developed. Conclusion High preference to self-test facilitated the use of the internet and paper-based programmes. This indicates that if validated self-tests are presented with clear instructions to self-test and built-in confidential linkages to counselling and treatment are provided, many more individuals will opt for HIV self-testing. These programmes and the smartphone application will be useful for the scale-up of unsupervised self-testing initiatives in literate populations worldwide.