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Non-sputum tests are needed to improve tuberculosis (TB) diagnosis and close the diagnostic gap. The World Health Organization's target product profile (TPP) for point-of-care (POC) screening tests requires a minimum sensitivity of 90% and a specificity of 70%. Our objective was to identify host blood protein biomarkers meeting TPP criteria. A systematic review was conducted and reported following PRISMA guidelines. Data extraction and quality assessment with Quality Assessment of Diagnostic Accuracy Studies-2 (QUADAS-2) were completed for the included studies. Heterogeneity was assessed. For biomarkers reporting sensitivity and specificity in at least four studies, a random-effects meta-analysis was performed for biomarkers with similar cut-offs. We screened 4,651 citations and included 65 studies that enrolled 16,010 participants and evaluated 156 host proteins. Most (47/65) studies enrolled adult pulmonary TB (PTB), with 15 studies in adult extra-pulmonary TB and 5 in children. Small early-stage discovery studies with case-control design were common (24/65) and had a high risk of bias. For adult PTB, CRP, IP-10, NCAM-1, and SAA met TPP criteria in high-quality studies. There was a high degree of heterogeneity in biomarker cut-offs and study design. CRP at 10 mg/L cut-off was meta-analyzed from 10 studies; pooled sensitivity 86% [95% confidence interval (CI): 80-95] and pooled specificity 67% (95% CI: 54-79). In people living with HIV (six studies), CRP pooled sensitivity was 93% (95% CI: 90-95), and pooled specificity was 59% (95% CI: 40-78). We identified promising biomarkers that performed well in high-quality studies. Data overall are limited and highly heterogenous. Further standardized validation across subgroups in prospective studies is needed before translating into POC assays., Importance: To our knowledge, this is the first comprehensive systematic review of host blood protein biomarkers for tuberculosis (TB), and we identified promising biomarkers for a TB screening test., Competing Interests: The authors declare no conflict of interest.

Rapid and accurate point-of-care (POC) tuberculosis (TB) diagnostics are crucial to bridge the TB diagnostic gap. Leveraging recent advancements in COVID-19 diagnostics, we explored adapting commercially available POC SARS-CoV-2 tests for TB diagnosis in line with the World Health Organization (WHO) target product profiles (TPPs). A scoping review was conducted following PRISMA-ScR guidelines to systematically map POC antigen and molecular SARS-CoV-2 diagnostic tests potentially meeting the TPPs for TB diagnostic tests for peripheral settings. Data were gathered from PubMed/MEDLINE, bioRxiv, medRxiv, publicly accessible in vitro diagnostic test databases, and developer websites up to 23 November 2022. Data on developer attributes, operational characteristics, pricing, clinical performance, and regulatory status were charted using standardized data extraction forms and evaluated with a standardized scorecard. A narrative synthesis of the data is presented. Our search yielded 2003 reports, with 408 meeting eligibility criteria. Among these, we identified 66 commercialized devices: 22 near-POC antigen tests, 1 POC molecular test, 31 near-POC molecular tests, and 12 low-complexity molecular tests potentially adaptable for TB. The highest-scoring SARS-CoV-2 diagnostic tests were the near-POC antigen platform LumiraDx (Roche, Basel, Switzerland), the POC molecular test Lucira Check-It (Pfizer, New York, NY, USA), the near-POC molecular test Visby (Visby, San Jose, CA, USA), and the low-complexity molecular platform Idylla (Biocartis, Lausanne, Switzerland). We highlight a diverse landscape of commercially available diagnostic tests suitable for potential adaptation to peripheral TB testing. This work aims to bolster global TB initiatives by fostering stakeholder collaboration, leveraging SARS-CoV-2 diagnostic technologies for TB, and uncovering new commercial avenues to tackle longstanding challenges in TB diagnosis.

Better access to tuberculosis testing is a key priority for fighting tuberculosis, the leading cause of infectious disease deaths in people. Despite the roll-out of molecular WHO-recommended rapid diagnostics to replace sputum smear microscopy over the past decade, a large diagnostic gap remains. Of the estimated 10·6 million people who developed tuberculosis globally in 2022, more than 3·1 million were not diagnosed. An exclusive focus on improving tuberculosis test accuracy alone will not be sufficient to close the diagnostic gap for tuberculosis. Diagnostic yield, which we define as the proportion of people in whom a diagnostic test identifies tuberculosis among all people we attempt to test for tuberculosis, is an important metric not adequately explored. Diagnostic yield is particularly relevant for subpopulations unable to produce sputum such as young children, people living with HIV, and people with subclinical tuberculosis. As more accessible non-sputum specimens (eg, urine, oral swabs, saliva, capillary blood, and breath) are being explored for point-of-care tuberculosis testing, the concept of yield will be of growing importance. Using the example of urine lipoarabinomannan testing, we illustrate how even tests with limited sensitivity can diagnose more people with tuberculosis if they enable increased diagnostic yield. Using tongue swab-based molecular tuberculosis testing as another example, we provide definitions and guidance for the design and conduct of pragmatic studies that assess diagnostic yield. Lastly, we show how diagnostic yield and other important test characteristics, such as cost and implementation feasibility, are essential for increased effective population coverage, which is required for optimal clinical care and transmission impact. We are calling for diagnostic yield to be incorporated into tuberculosis test evaluation processes, including the WHO Grading of Recommendations, Assessment, Development, and Evaluations process, providing a crucial real-life implementation metric that complements traditional accuracy measures., Competing Interests: Declaration of interests TB reports patent applications in the field of tuberculosis detection and is a shareholder of Avelo. MP serves as an adviser for non-profits such as the Bill & Melinda Gates Foundation, FIND, WHO, and the Stop TB Partnership. All other authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Introduction: Rapid and accurate point-of-care (POC) tuberculosis (TB) diagnostics are a key priority to close the TB diagnostic gap of 3.1 million people without a diagnosis. Leveraging the recent surge in COVID-19 diagnostic innovation, we explored the potential adaptation of commercially available SARS-CoV-2 tests for TB diagnosis, aligning with World Health Organization (WHO) target product profiles (TPPs)., Methods: A scoping review was conducted following PRISMA-ScR guidelines to systematically map commercially available POC molecular and antigen SARS-CoV-2 diagnostic tests potentially meeting the TPPs for TB diagnostic tests for peripheral settings. Data were gathered from PubMed/MEDLINE, bioRxiv, and medRxiv, along with publicly accessible in vitro diagnostic test databases, and developer websites, up to November 23, 2022. Data on developer and test attributes, operational characteristics, pricing, and clinical performance were charted using standardized data extraction forms. Each identified test was evaluated using a standardized scorecard. A narrative synthesis of the charted data is presented., Results: Our database search yielded 2,003 studies, from which 408 were considered eligible. Among these, we identified 58 commercialized diagnostic devices, including 17 near-POC antigen tests, one POC molecular test, 29 near-POC molecular tests, and 11 low-complexity molecular tests. We summarized the detailed characteristics, regulatory status, and clinical performance data of these tests. The LumiraDx (Roche, Switzerland) emerged as the highest- scoring near-POC antigen platform, while Visby (Visby, USA) was the highest-performing near-POC molecular platform. The Lucira Check-It (Pfizer, USA) was noted as the sole POC molecular test. The Idylla TM (Biocartis, Switzerland) was identified as the leading low- complexity molecular test., Discussion: We highlight a diverse landscape of commercially available diagnostic tests suitable for potential adaptation to TB POC testing. This work aims to bolster global TB initiatives by fostering stakeholder collaboration, leveraging COVID-19 diagnostic technologies for TB diagnosis, and uncovering new commercial avenues to tackle longstanding challenges in TB diagnosis.

The COVID-19 pandemic brought diagnostics into the spotlight in an unprecedented way not only for case management but also for population health, surveillance, and monitoring. The industry saw notable levels of investment and accelerated research which sparked a wave of innovation. Simple non-invasive sampling methods such as nasal swabs have become widely used in settings ranging from tertiary hospitals to the community. Self-testing has also been adopted as standard practice using not only conventional lateral flow tests but novel and affordable point-of-care molecular diagnostics. The use of new technologies, including artificial intelligence-based diagnostics, have rapidly expanded in the clinical setting. The capacity for next-generation sequencing and acceptance of digital health has significantly increased. However, 4 years after the pandemic started, the market for SARS-CoV-2 tests is saturated, and developers may benefit from leveraging their innovations for other diseases; tuberculosis (TB) is a worthwhile portfolio expansion for diagnostics developers given the extremely high disease burden, supportive environment from not-for-profit initiatives and governments, and the urgent need to overcome the long-standing dearth of innovation in the TB diagnostics field. In exchange, the current challenges in TB detection may be resolved by adopting enhanced swab-based molecular methods, instrument-based, higher sensitivity antigen detection technologies, and/or artificial intelligence-based digital health technologies developed for COVID-19. The aim of this article is to review how such innovative approaches for COVID-19 diagnosis can be applied to TB to have a comparable impact., (© 2023. The Author(s).)

The current four-symptom screen recommended by the World Health Organization (WHO) is widely used as screen to initiate diagnostic testing for active pulmonary tuberculosis (TB), yet the performance is poor especially when TB prevalence is low. In contrast, more sensitive molecular tests are less suitable for placement at primary care level in low-resource settings. In order to meet the WHO End TB targets, new diagnostic approaches are urgently needed to find the missing undiagnosed cases. Proteomics-derived blood host biomarkers have been explored because protein detection technologies are suitable for the point-of-care setting and could meet cost targets. This study aimed to find a biomarker signature that fulfills WHO's target product profile (TPP) for a TB screening. Twelve blood-based protein biomarkers from three sample populations (Vietnam, Peru, and South Africa) were analyzed individually and in combinations via advanced statistical methods and machine learning algorithms. The combination of I-309, SYWC and kallistatin showed the most promising results to discern active TB throughout the data sets meeting the TPP for a triage test in adults from two countries (Peru and South Africa). The top-performing individual markers identified at the global level (I-309 and SYWC) were also among the best-performing markers at country level in South Africa and Vietnam. This analysis clearly shows that a host protein biomarker assay is feasible in adults for certain geographical regions based on one or two biomarkers with a performance that meets minimal WHO TPP criteria., Competing Interests: The authors declare no conflict of interest.

Background: Sputum is the most widely used sample to diagnose active tuberculosis, but many people living with HIV are unable to produce sputum. Urine, in contrast, is readily available. We hypothesised that sample availability influences the diagnostic yield of various tuberculosis tests., Methods: In this systematic review and meta-analysis of individual participant data, we compared the diagnostic yield of point-of-care urine-based lipoarabinomannan tests with that of sputum-based nucleic acid amplification tests (NAATs) and sputum smear microscopy (SSM). We used microbiologically confirmed tuberculosis based on positive culture or NAAT from any body site as the denominator and accounted for sample provision. We searched PubMed, Web of Science, Embase, African Journals Online, and clinicaltrials.gov from database inception to Feb 24, 2022 for randomised controlled trials, cross-sectional studies, and cohort studies that assessed urine lipoarabinomannan point-of-care tests and sputum NAATs for active tuberculosis detection in participants irrespective of tuberculosis symptoms, HIV status, CD4 cell count, or study setting. We excluded studies in which recruitment was not consecutive, systematic, or random; provision of sputum or urine was an inclusion criterion; less than 30 participants were diagnosed with tuberculosis; early research assays without clearly defined cutoffs were tested; and humans were not studied. We extracted study-level data, and authors of eligible studies were invited to contribute deidentified individual participant data. The main outcomes were the tuberculosis diagnostic yields of urine lipoarabinomannan tests, sputum NAATs, and SSM. Diagnostic yields were predicted using Bayesian random-effects and mixed-effects meta-analyses. This study is registered with PROSPERO, CRD42021230337., Findings: We identified 844 records, from which 20 datasets and 10 202 participants (4561 [45%] male participants and 5641 [55%] female participants) were included in the meta-analysis. All studies assessed sputum Xpert (MTB/RIF or Ultra, Cepheid, Sunnyvale, CA, USA) and urine Alere Determine TB LAM (AlereLAM, Abbott, Chicago, IL, USA) in people living with HIV aged 15 years or older. Nearly all (9957 [98%] of 10 202) participants provided urine, and 82% (8360 of 10 202) provided sputum within 2 days. In studies that enrolled unselected inpatients irrespective of tuberculosis symptoms, only 54% (1084 of 1993) of participants provided sputum, whereas 99% (1966 of 1993) provided urine. Diagnostic yield was 41% (95% credible interval [CrI] 15-66) for AlereLAM, 61% (95% Crl 25-88) for Xpert, and 32% (95% Crl 10-55) for SSM. Heterogeneity existed across studies in the diagnostic yield, influenced by CD4 cell count, tuberculosis symptoms, and clinical setting. In predefined subgroup analyses, all tests had higher yields in symptomatic participants, and AlereLAM yield was higher in those with low CD4 counts and inpatients. AlereLAM and Xpert yields were similar among inpatients in studies enrolling unselected participants who were not assessed for tuberculosis symptoms (51% vs 47%). AlereLAM and Xpert together had a yield of 71% in unselected inpatients, supporting the implementation of combined testing strategies., Interpretation: AlereLAM, with its rapid turnaround time and simplicity, should be prioritised to inform tuberculosis therapy among inpatients who are HIV-positive, regardless of symptoms or CD4 cell count. The yield of sputum-based tuberculosis tests is undermined by people living with HIV who cannot produce sputum, whereas nearly all participants are able to provide urine. The strengths of this meta-analysis are its large size, the carefully harmonised denominator, and the use of Bayesian random-effects and mixed-effects models to predict yields; however, data were geographically restricted, clinically diagnosed tuberculosis was not considered in the denominator, and little information exists on strategies for obtaining sputum samples., Funding: FIND, the Global Alliance for Diagnostics., Competing Interests: Declaration of interests TB reports patent applications in the field of tuberculosis detection, reports consulting fees from the FINDdx, and is a shareholder of Avelo. GMe was supported by the Wellcome Trust (214321/Z/18/Z and 203135/Z/16/Z) and the South African Research Chairs Initiative of the Department of Science and Technology and National Research Foundation of South Africa (grant number 64787). The opinions, findings, and conclusions expressed in this manuscript reflect those of the authors alone. All other authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Introduction: In 2014, the WHO published high-priority target product profiles (TPPs) for new tuberculosis (TB) diagnostics to align end-user needs with test targets and specifications; nevertheless, no TB test meets these targets to date. The COVID-19-driven momentum in the diagnostics world offers an opportunity to address the long-standing lack of innovation in the field of TB diagnostics. This scoping review aims to summarise point-of-care (POC) molecular and antigen tests for COVID-19 diagnosis that, when applied to TB, potentially meet WHO TPPs. This summary of currently available innovative diagnostic tools will guide the development of novel TB diagnostics toward the WHO-set targets., Methods and Analysis: We will follow the Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension Scoping Reviews recommendations. MEDLINE (via PubMed), bioRxiv, MedRxiv and other publicly available in vitro diagnostic test databases were searched on 23 November 2022. POC antigen or molecular tests developed for SARS-CoV-2 detection that meet the eligibility criteria will be included in the review. Developer description, test description, operation characteristics, pricing information, performance and commercialisation status of diagnostic tests identified will be extracted using a predefined standardised data extraction form. Two reviewers will independently perform the screening and data extraction. A narrative synthesis of the final data will be provided., Ethics and Dissemination: No ethical approval is required because individual patient data will not be included. The findings will be published in open-access scientific journals., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Widely available tuberculosis (TB) diagnostics use sputum samples. However, many patients, particularly children and patients living with HIV (PLHIV), struggle to provide sputum. Urine diagnostics are a promising approach to circumvent this challenge while delivering reliable and timely diagnosis. This qualitative study in two high TB/HIV burden countries assesses values and preferences of end-users, along with potential barriers for the implementation of the novel Fujifilm SILVAMP TB-LAM (FujiLAM, Fujifilm, Japan) urine test. Between September 2020 and March 2021, we conducted 42 semi-structured interviews with patients, health care providers (HCPs) and decision makers (DMs) (e.g., in national TB programs) in Malawi and Zambia. Interviews were transcribed verbatim and analyzed using a framework approach supported by NVIVO. Findings aligned with the pre-existing Health Equity Implementation Framework, which guided the presentation of results. The ease and convenience of urine-based testing was described as empowering among patients and HCPs who lamented the difficulty of sputum collection, however HCPs expressed concerns that a shift in agency to the patient may affect clinic workflows (e.g., due to less control over collection). Implementation facilitators, such as shorter turnaround times, were welcomed by operators and patients alike. The decentralization of diagnostics was considered possible with FujiLAM by HCPs and DMs due to low infrastructure requirements. Finally, our findings support efforts for eliminating the CD4 count as an eligibility criterion for LAM testing, to facilitate implementation and benefit a wider range of patients. Our study identified barriers and facilitators relevant to scale-up of urine LAM tests in Malawi and Zambia. FujiLAM could positively impact health equity, as it would particularly benefit patient groups currently underserved by existing TB diagnostics. Participants view the approach as a viable, acceptable, and likely sustainable option in low- and middle-income countries, though adaptations may be required to current health care processes for deployment. Trial registration: German Clinical Trials Register, DRKS00021003. URL: https://www.drks.de/drks_web/setLocale_EN.do., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: CMD was working for FIND until April 2019. FIND at the time partnered with Fujifilm to develop the the SILVAMP TB Assay., (Copyright: © 2022 Herrmann et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: WHO recommends urine lateral-flow lipoarabinomannan (LF-LAM) testing with AlereLAM in HIV-positive inpatients only if screening criteria are met. We assessed the performance of WHO screening criteria and alternative screening tests/strategies to guide LF-LAM testing and compared diagnostic accuracy of the WHO AlereLAM algorithm (WHO screening criteria followed by AlereLAM if screen positive) with AlereLAM and FujiLAM (a novel LF-LAM test) testing in all HIV-positive inpatients., Methods: We searched MEDLINE, Embase, and Cochrane Library from Jan 1, 2011 to March 1, 2020 for studies among adult/adolescent HIV-positive inpatients regardless of tuberculosis signs and symptoms. The reference standards were (1) AlereLAM or FujiLAM for screening tests/strategies and (2) culture or Xpert for AlereLAM/FujiLAM. We determined proportion of inpatients eligible for AlereLAM using WHO screening criteria; assessed accuracy of WHO criteria and alternative screening tests/strategies to guide LF-LAM testing; compared accuracy of WHO AlereLAM algorithm with AlereLAM/FujiLAM testing in all; and determined diagnostic yield of AlereLAM, FujiLAM, and Xpert MTB/RIF (Xpert). We estimated pooled proportions with a random-effects model, assessed diagnostic accuracy using random-effects bivariate models, and assessed diagnostic yield descriptively., Findings: We obtained data from all 5 identified studies (n = 3,504). The pooled proportion of inpatients eligible for AlereLAM using WHO criteria was 93% (95%CI 91, 95). Among screening tests/strategies to guide LF-LAM testing, WHO criteria, C-reactive protein (≥5 mg/L), and CD4 count (<200 cells/μL) had high sensitivities but low specificities; cough (≥2 weeks), hemoglobin (<8 g/dL), body mass index (<18.5 kg/m 2 ), lymphadenopathy, and WHO-defined danger signs had higher specificities but suboptimal sensitivities. AlereLAM in all had the same sensitivity (62%) and specificity (88%) as WHO AlereLAM algorithm. Sensitivity of FujiLAM and AlereLAM was 69% and 48%, while specificity was 88% and 96%, respectively. In 2 studies that collected sputum and non-sputum samples for Xpert and/or culture, diagnostic yield of sputum Xpert was 40-41%, AlereLAM was 39-76%, and urine Xpert was 35-62%. In one study, FujiLAM diagnosed 80% of tuberculosis cases (vs 39% for AlereLAM), and sputum Xpert combined with AlereLAM, urine Xpert, or FujiLAM diagnosed 61%, 81%, and 92% of all cases, respectively., Interpretation: WHO criteria and alternative screening tests/strategies have limited utility in guiding LF-LAM testing, suggesting that AlereLAM testing in all HIV-positive medical inpatients be implemented. Routine FujiLAM may improve tuberculosis diagnosis., Funding: None., Competing Interests: Declaration of Competing Interest TB reports a patent-pending (WO/2019/186,486) in the field of lipoarabinomannan detection, as an inventor, but without ownership/commercial rights., (Copyright © 2022 The British Infection Association. Published by Elsevier Ltd. All rights reserved.)

The Fujifilm SILVAMP TB LAM (FujiLAM) assay offers improved sensitivity compared to Determine TB LAM Ag (AlereLAM) for detecting tuberculosis (TB) among people with HIV. Here, we examined the diagnostic value of FujiLAM testing on early morning urine versus spot urine and the added value of a two-sample strategy. We assessed the diagnostic accuracy of FujiLAM on cryopreserved urine samples collected and stored as part of a prospective cohort of adults with HIV presenting for antiretroviral treatment in Ghana. We compared FujiLAM sensitivity and specificity in spontaneously voided urine samples collected at inclusion (spot urine) versus in the first voided early morning urine (morning urine) and for a one (spot urine) versus two samples (spot and morning urine) strategy. Diagnostic accuracy was determined against both microbiological (using sputum culture and Xpert MTB/RIF testing of sputum and urine to confirm TB) and composite reference standards (including microbiologically confirmed and probable TB cases). Paired urine samples of spot and morning urine were available for 389 patients. Patients had a median CD4 cell count of 176 cells/μL (interquartile range [IQR], 52 to 361). Forty-three (11.0%) had confirmed TB, and 19 (4.9%) had probable TB. Overall agreement for spot versus morning urine test results was 94.6% (kappa, 0.81). Compared to a microbiological reference standard, the FujiLAM sensitivity (95% confidence interval [CI]) was 67.4% (51.5 to 80.9) for spot and 69.8% (53.9 to 82.8) for morning urine, an absolute difference (95% CI) of 2.4% (-10.2 to 14.8). Specificity was 90.2% (86.5 to 93.1) versus 89.0% (85.2 to 92.1) for spot and morning urine, respectively, a difference of 1.2% (-3.7 to 1.4). A two-sample strategy increased FujiLAM sensitivity from 67.4% (51.5 to 80.9) to 74.4% (58.8 to 86.5), a difference of 7.0% (-3.0 to 16.9), while specificity decreased from 90.2% (86.5 to 93.1) to 87.3% (83.3 to 90.6), a difference of -2.9% (-4.9 to -0.8). This study indicates that FujiLAM testing performs equivalently on spot and early morning urine samples. Sensitivity could be increased with a two-sample strategy but at the risk of lower specificity. These data can inform future guidelines and clinical practice. IMPORTANCE This study indicates that FujiLAM testing performs equivalently on spot and early morning urine samples for detecting tuberculosis among people with HIV. Sensitivity could be increased with a two-sample strategy but at the risk of lower specificity. These data can inform future guidelines and clinical practice around FujiLAM.

Background: A novel urine lipoarabinomannan assay (FujiLAM) has higher sensitivity and higher cost than the first-generation AlereLAM assay. We evaluated the cost-effectiveness of FujiLAM for tuberculosis testing among hospitalized people with human immunodeficiency virus (HIV), irrespective of symptoms., Methods: We used a microsimulation model to project clinical and economic outcomes of 3 testing strategies: (1) sputum Xpert MTB/RIF (Xpert), (2) sputum Xpert plus urine AlereLAM (Xpert+AlereLAM), (3) sputum Xpert plus urine FujiLAM (Xpert+FujiLAM). The modeled cohort matched that of a 2-country clinical trial. We applied diagnostic yields from a retrospective study (yields for Xpert/Xpert+AlereLAM/Xpert+FujiLAM among those with CD4 <200 cells/µL: 33%/62%/70%; among those with CD4 ≥200 cells/µL: 33%/35%/47%). Costs of Xpert/AlereLAM/FujiLAM were US$15/3/6 (South Africa) and $25/3/6 (Malawi). Xpert+FujiLAM was considered cost-effective if its incremental cost-effectiveness ratio (US$/year-of-life saved) was <$940 (South Africa) and <$750 (Malawi). We varied key parameters in sensitivity analysis and performed a budget impact analysis of implementing FujiLAM countrywide., Results: Compared with Xpert+AlereLAM, Xpert+FujiLAM increased life expectancy by 0.2 years for those tested in South Africa and Malawi. Xpert+FujiLAM was cost-effective in both countries. Xpert+FujiLAM for all patients remained cost-effective compared with sequential testing and CD4-stratified testing strategies. FujiLAM use added 3.5% (South Africa) and 4.7% (Malawi) to 5-year healthcare costs of tested patients, primarily reflecting ongoing HIV treatment costs among survivors., Conclusions: FujiLAM with Xpert for tuberculosis testing in hospitalized people with HIV is likely to increase life expectancy and be cost-effective at the currently anticipated price in South Africa and Malawi. Additional studies should evaluate FujiLAM in clinical practice settings., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America.)

Background: An accurate point-of-care test for tuberculosis (TB) in children remains an elusive goal. Recent evaluation of a novel point-of-care urinary lipoarabinomannan test, Fujifilm SILVAMP Tuberculosis Lipoarabinomannan (FujiLAM), in adults living with human immunodeficiency virus (HIV) showed significantly superior sensitivity than the current Alere Determine Tuberculosis Lipoarabinomannan test (AlereLAM). We therefore compared the accuracy of FujiLAM and AlereLAM in children with suspected TB., Methods: Children hospitalized with suspected TB in Cape Town, South Africa, were enrolled (consecutive admissions plus enrichment for a group of children living with HIV and with TB), their urine was collected and biobanked, and their sputum was tested with mycobacterial culture and Xpert MTB/RIF or Xpert MTB/RIF Ultra. Biobanked urine was subsequently batch tested with FujiLAM and AlereLAM. Children were categorized as having microbiologically confirmed TB, unconfirmed TB (clinically diagnosed), or unlikely TB., Results: A total of 204 children were enrolled and had valid results from both index tests, as well as sputum microbiological testing. Compared to a microbiological reference standard, the sensitivity of FujiLAM and AlereLAM was similar (42% and 50%, respectively), but lower than that of Xpert MTB/RIF of sputum (74%). The sensitivity of FujiLAM was higher in children living with HIV (60%) and malnourished children (62%). The specificity of FujiLAM was substantially higher than that of AlereLAM (92% vs 66%, respectively). The specificity of both tests was higher in children 2 years or older (FujiLAM, 96%; AlereLAM, 72%)., Conclusions: The high specificity of FujiLAM suggests utility as a "rule-in" test for children with a high pretest probability of TB, including hospitalized children living with HIV or with malnutrition., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Reducing diagnostic delay is key toward decreasing tuberculosis-associated deaths in people living with human immunodeficiency virus. In tuberculosis patients with retrospective urine testing, the point-of-care Fujifilm SILVAMP TB LAM (FujiLAM) could have rapidly diagnosed tuberculosis in up to 89% who died. In FujiLAM negative patients, the probability of 12-week survival was 86-97%., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America.)

BACKGROUNDInadequate tuberculosis (TB) diagnostics are a major hurdle in the reduction of disease burden, and accurate point-of-care tests (POCTs) are urgently needed. We assessed the diagnostic accuracy of Fujifilm SILVAMP TB lipoarabinomannan (FujiLAM) POCT for TB diagnosis in HIV-negative outpatients and compared it with Alere Determine TB LAM Ag (AlereLAM) POCT and a laboratory-based ultrasensitive electrochemiluminescence LAM research assay (EclLAM).METHODSIn this multicenter diagnostic test accuracy study, we recruited HIV-negative adults with symptoms suggestive of pulmonary TB presenting to outpatient health care centers in Peru and South Africa. Urine samples were tested using FujiLAM, AlereLAM, and EclLAM, and the diagnostic accuracy was assessed against a microbiological reference standard (MRS) and a composite reference standard.RESULTSThree hundred seventy-two HIV-negative participants were included and the prevalence of microbiologically confirmed TB was 30%. Compared with the MRS, the sensitivities of AlereLAM, FujiLAM, and EclLAM were 10.8% (95% confidence interval [CI] 6.3%-18.0%), 53.2% (95% CI 43.9%-62.1%), and 66.7% (95% CI 57.5%-74.7%), respectively. The specificities of AlereLAM, FujiLAM, and EclLAM were 92.3% (95% CI 88.5%-95.0%), 98.9% (95% CI 96.7%-99.6%), and 98.1% (95% CI 95.6%-99.2%), respectively. Positive likelihood ratios of AlereLAM, FujiLAM, and EclLAM were 1.4, 46.2, and 34.8, respectively, and positive predictive values were 37.5%, 95.2%, and 93.7%, respectively.CONCLUSIONCompared with AlereLAM, FujiLAM detected 5 times more patients with TB in HIV-negative participants, had a high positive predictive value, and has the potential to improve rapid diagnosis of TB at the point-of-care. EclLAM demonstrated that additional sensitivity gains are possible, which highlights LAM's potential as a biomarker. Additional research is required to assess FujiLAM's performance in prospective cohorts, its cost-effectiveness, and its impact in real-world clinical settings.FUNDINGGlobal Health Innovative Technology Fund, the UK Department for International Development, the Dutch Ministry of Foreign Affairs, the Bill and Melinda Gates Foundation, the Australian Department of Foreign Affairs and Trade, the German Federal Ministry of Education and Research through Kreditanstalt für Wiederaufbau, and the NIH and National Institute of Allergy and Infectious Diseases.

Background: Tuberculosis (TB) is the most common cause of death in people living with HIV (PLHIV), yet TB often goes undiagnosed since many patients are not able to produce a sputum specimen, and traditional diagnostics are costly or unavailable. A novel, rapid lateral flow assay, Fujifilm SILVAMP TB LAM (SILVAMP-LAM), detects the presence of TB lipoarabinomannan (LAM) in urine, and is substantially more sensitive for diagnosing TB in PLHIV than an earlier LAM assay (Alere Determine TB LAM lateral flow assay [LF-LAM]). Here, we present an individual participant data meta-analysis of the diagnostic accuracy of SILVAMP-LAM in adult PLHIV, including both published and unpublished data., Methods and Findings: Adult PLHIV (≥18 years) were assessed in 5 prospective cohort studies in South Africa (3 cohorts), Vietnam, and Ghana, carried out during 2012 to 2017. Of the 1,595 PLHIV who met eligibility criteria, the majority (61%) were inpatients, median age was 37 years (IQR 30-43), 43% had a CD4 count ≤ 100 cells/μl, and 35% were receiving antiretroviral therapy. Most participants (94%) had a positive WHO symptom screen for TB on enrollment, and 45% were diagnosed with microbiologically confirmed TB, using mycobacterial culture or Xpert MTB/RIF testing of sputum, urine, or blood. Previously published data from inpatients were combined with unpublished data from outpatients. Biobanked urine samples were tested, using blinded double reading, with SILVAMP-LAM and LF-LAM. Applying a microbiological reference standard for assessment of sensitivity, the overall sensitivity for TB detection was 70.7% (95% CI 59.0%-80.8%) for SILVAMP-LAM compared to 34.9% (95% CI 19.5%-50.9%) for LF-LAM. Using a composite reference standard (which included patients with both microbiologically confirmed as well as clinically diagnosed TB), SILVAMP-LAM sensitivity was 65.8% (95% CI 55.9%-74.6%), and that of LF-LAM 31.4% (95% CI 19.1%-43.7%). In patients with CD4 count ≤ 100 cells/μl, SILVAMP-LAM sensitivity was 87.1% (95% CI 79.3%-93.6%), compared to 56.0% (95% CI 43.9%-64.9%) for LF-LAM. In patients with CD4 count 101-200 cells/μl, SILVAMP-LAM sensitivity was 62.7% (95% CI 52.4%-71.9%), compared to 25.3% (95% CI 15.8%-34.9%) for LF-LAM. In those with CD4 count > 200 cells/μl, SILVAMP-LAM sensitivity was 43.9% (95% CI 34.3%-53.9%), compared to 10.9% (95% CI 5.2%-18.4%) for LF-LAM. Using a microbiological reference standard, the specificity of SILVAMP-LAM was 90.9% (95% CI 87.2%-93.7%), and that of LF-LAM 95.3% (95% CI 92.2%-97.7%). Limitations of this study include the use of biobanked, rather than fresh urine samples, and testing by skilled laboratory technicians in research laboratories, rather than at the point of care., Conclusions: In this study, we found that SILVAMP-LAM identified a substantially higher proportion of TB patients in PLHIV than LF-LAM. The sensitivity of SILVAMP-LAM was highest in patients with CD4 count ≤ 100 cells/μl. Further work is needed to demonstrate accuracy when implemented as a point-of-care test., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: TB, SGS, AM, SO, RS and CMD were previously or are currently employed by FIND. TB reports a patent in the field of lipoarabinomannan detection. CMD is a member of PLOS Medicine's Editorial Board. The rest of the authors declare no competing interests associated with this manuscript. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication.

Most diagnostic tests for tuberculosis (TB) rely on sputum samples, which are difficult to obtain and have low sensitivity in immunocompromised patients, patients with disseminated TB, and children, delaying treatment initiation. The World Health Organization (WHO) calls for the development of a rapid, biomarker-based, non-sputum test capable of detecting all forms of TB at the point-of-care to enable immediate treatment initiation. Lipoarabinomannan (LAM) is the only WHO-endorsed TB biomarker that can be detected in urine, an easily collected sample. This status update discusses the characteristics of LAM as a biomarker, describes the performance of first-generation urine LAM tests and reasons for slow uptake, and presents considerations for developing the next generation of more sensitive and impactful tests. Next-generation urine LAM tests have the potential to reach adult and pediatric patients regardless of HIV status or site of infection and facilitate global TB control. Implementation and scale-up of existing LAM tests and development of next-generation assays should be prioritized., Competing Interests: T.B. and C.M.D. were previously employed by FIND, and E.M., A.S. and M.R. are currently employed by FIND. T.B. reports a patent application in the field of LAM detection. All other authors declare no conflict of interest.

Background: The novel Fujifilm SILVAMP TB-LAM (FujiLAM) assay detects mycobacterial lipoarabinomannan in urine and has demonstrated superior sensitivity to the Alere Determine TB-LAM Ag (AlereLAM) assay for detection of tuberculosis among hospitalized people with human immunodeficiency virus (PWH). This is the first study to evaluate the assay among a broad population referred for antiretroviral therapy including both outpatients (mainly) and inpatients., Methods: We assessed diagnostic accuracy of FujiLAM and AlereLAM assays in biobanked urine samples from a cohort of adults referred for antiretroviral therapy in Ghana against a microbiological and a composite (including clinical judgement) reference standard, and we assessed the association of FujiLAM test positivity with mortality., Results: We evaluated urine samples from 532 PWH (462 outpatients, 70 inpatients). Against a microbiological reference standard, the sensitivity of FujiLAM was 74.2% (95% confidence interval [CI], 62.0-84.2) compared to 53.0% (95% CI, 40.3-65.4) for AlereLAM, a difference of 21.2% (CI, 13.1-32.5). Specificity was 89.3% (95% CI, 85.8-92.2) versus 95.6% (95% CI, 93.0-97.4) for FujiLAM and AlereLAM, a difference of -6.3% (95% CI -9.6 to -3.3). Specificity estimates for FujiLAM increased markedly to 98.8% (95% CI, 96.6-99.8) in patients with CD4 >100 cells/µL and when using a composite reference standard. FujiLAM test positivity was associated with increased cumulative risk of mortality at 6 months (hazard ratio, 4.80; 95% CI, 3.01-7.64)., Conclusions: FujiLAM offers significantly increased diagnostic sensitivity in comparison to AlereLAM. Specificity estimates for FujiLAM were lower than for AlereLAM but were affected by the limited ability of the reference standard to correctly diagnose tuberculosis in individuals with low CD4 counts., (© The Author(s) 2019. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Improved tuberculosis (TB) prevention and control depend critically on the development of a simple, readily accessible rapid triage test to stratify TB risk. We hypothesized that a blood protein-based host response signature for active TB (ATB) could distinguish it from other TB-like disease (OTD) in adult patients with persistent cough, thereby providing a foundation for a point-of-care (POC) triage test for ATB. Three adult cohorts consisting of ATB suspects were recruited. A bead-based immunoassay and machine learning algorithms identified a panel of four host blood proteins, interleukin-6 (IL-6), IL-8, IL-18, and vascular endothelial growth factor (VEGF), that distinguished ATB from OTD. An ultrasensitive POC-amenable single-molecule array (Simoa) panel was configured, and the ATB diagnostic algorithm underwent blind validation in an independent, multinational cohort in which ATB was distinguished from OTD with receiver operator characteristic-area under the curve (ROC-AUC) of 0.80 [95% confidence interval (CI), 0.75 to 0.85], 80% sensitivity (95% CI, 73 to 85%), and 65% specificity (95% CI, 57 to 71%). When host antibodies against TB antigen Ag85B were added to the panel, performance improved to 86% sensitivity and 69% specificity. A blood-based host response panel consisting of four proteins and antibodies to one TB antigen can help to differentiate ATB from other causes of persistent cough in patients with and without HIV infection from Africa, Asia, and South America. Performance characteristics approach World Health Organization (WHO) target product profile accuracy requirements and may provide the foundation for an urgently needed blood-based POC TB triage test., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The World Health Organization's (WHO) "End TB" strategy calls for development and implementation of novel tuberculosis (TB) diagnostics. Sputum-based diagnostics are challenging to implement and often less sensitive in high-priority populations. Nonsputum, biomarker-based tests may facilitate TB testing at lower levels of the healthcare system, accelerate treatment initiation, and improve outcomes. We provide guidance on the design of diagnostic accuracy studies evaluating nonsputum, biomarker-based tests within the context of WHO's target product profile for such tests. Study designs should account for the intended use when choosing the study population, setting, and reference standards. Although adults with respiratory symptoms may be an initial target population, other high-priority populations regardless of symptoms-including people living with human immunodeficiency virus, those unable to produce sputum samples or with extrapulmonary TB, household contacts, and children-should be considered. Studies beyond diagnostic accuracy that evaluate feasibility and population-level impacts are also needed. A biomarker-based diagnostic may be critical to ending the TB epidemic, but requires appropriate validation before implementation., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Approximately 3.6 million cases of active tuberculosis (TB) go potentially undiagnosed annually, partly due to limited access to confirmatory diagnostic tests, such as molecular assays or mycobacterial culture, in community and primary healthcare settings. This article provides guidance for TB triage test evaluations. A TB triage test is designed for use in people with TB symptoms and/or significant risk factors for TB. Triage tests are simple and low-cost tests aiming to improve ease of access and implementation (compared with confirmatory tests) and decrease the proportion of patients requiring more expensive confirmatory testing. Evaluation of triage tests should occur in settings of intended use, such as community and primary healthcare centers. Important considerations for triage test evaluation include study design, population, sample type, test throughput, use of thresholds, reference standard (ideally culture), and specimen flow. The impact of a triage test will depend heavily on issues beyond accuracy, primarily centered on implementation., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Background: Most tuberculosis-related deaths in people with HIV could be prevented with earlier diagnosis and treatment. The only commercially available tuberculosis point-of-care test (Alere Determine TB LAM Ag [AlereLAM]) has suboptimal sensitivity, which restricts its use in clinical practice. The novel Fujifilm SILVAMP TB LAM (FujiLAM) assay has been developed to improve the sensitivity of AlereLAM. We assessed the diagnostic accuracy of the FujiLAM assay for the detection of tuberculosis in hospital inpatients with HIV compared with the AlereLAM assay., Methods: For this diagnostic accuracy study, we assessed biobanked urine samples obtained from the FIND Specimen Bank and the University of Cape Town Biobank, which had been collected from hospital inpatients (aged ≥18 years) with HIV during three independent prospective cohort studies done at two South African hospitals. Urine samples were tested using FujiLAM and AlereLAM assays. The conduct and reporting of each test was done blind to other test results. The primary objective was to assess the diagnostic accuracy of FujiLAM compared with AlereLAM, against microbiological and composite reference standards (including clinical diagnoses)., Findings: Between April 18, 2018, and May 3, 2018, urine samples from 968 hospital inpatients with HIV were evaluated. The prevalence of microbiologically-confirmed tuberculosis was 62% and the median CD4 count was 86 cells per μL. Using the microbiological reference standard, the estimated sensitivity of FujiLAM was 70·4% (95% CI 53·0 to 83·1) compared with 42·3% (31·7 to 51·8) for AlereLAM (difference 28·1%) and the estimated specificity of FujiLAM was 90·8% (86·0 to 94·4) and 95·0% (87·7-98·8) for AlereLAM (difference -4·2%). Against the composite reference standard, the specificity of both assays was higher (95·7% [92·0 to 98·0] for FujiLAM vs 98·2% [95·7 to 99·6] for AlereLAM; difference -2·5%), but the sensitivity of both assays was lower (64·9% [50·1 to 76·7] for FujiLAM vs 38·2% [28·1 to 47·3] for AlereLAM; difference 26·7%)., Interpretation: In comparison to AlereLAM, FujiLAM offers superior diagnostic sensitivity, while maintaining specificity, and could transform rapid point-of-care tuberculosis diagnosis for hospital inpatients with HIV. The applicability of FujiLAM for settings of intended use requires prospective assessment., Funding: Global Health Innovative Technology Fund, UK Department for International Development, Dutch Ministry of Foreign Affairs, Bill & Melinda Gates Foundation, German Federal Ministry of Education and Research, Australian Department of Foreign Affairs and Trade, Wellcome Trust, Department of Science and Technology and National Research Foundation of South Africa, and South African Medical Research Council., (Copyright © 2019 Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Millions of cases of tuberculosis (TB) go undiagnosed each year. Better diagnostic tools are urgently needed. Biomarker-based or multiple marker biosignature-based tests, ideally performed on blood or urine, for the detection of active TB might help to meet target product profiles proposed by the World Health Organization for point-of-care testing. We conducted a systematic review to summarize evidence on proposed biomarkers and biosignatures and evaluate their quality and level of evidence. We screened the titles and abstracts of 7,631 citations and included 443 publications that fulfilled the inclusion criteria and were published in 2010-2017. The types of biomarkers identified included antibodies, cytokines, metabolic activity markers, mycobacterial antigens and volatile organic compounds. Only 47% of studies reported a culture-based reference standard and diagnostic sensitivity and specificity. Forty-four biomarkers (4%) were identified in high-quality studies and met the target product profile minimum criteria, of which two have been incorporated into commercial assays. Of the 44 highest-quality biomarkers, 24 (55%) were multiple marker biosignatures. No meta-analyses were performed owing to between-study heterogeneity. In conclusion, TB biomarker discovery studies are often poorly designed and findings are rarely confirmed in independent studies. Few markers progress to a further developmental stage. More validation studies that consider the intended diagnostic use cases and apply rigorous design are needed. The extracted data from this review are currently being used by FIND as the foundation of a dynamic database in which biomarker data and developmental status will be presented.

Background: Tuberculosis (TB) infection was responsible for an estimated 1.3 million deaths in 2017. Better diagnostic tools are urgently needed. We sought to determine whether accurate TB antigen detection in blood or urine has the potential to meet the WHO target product profiles for detection of active TB., Materials and Methods: We developed Electrochemiluminescence (ECL) immunoassays for Lipoarabinomannan (LAM) and ESAT-6 detection with detection limits in the pg/ml range and used them to compare the concentrations of the two antigens in the urine and serum of 81 HIV-negative and -positive individuals with presumptive TB enrolled across diverse geographic sites., Results: LAM and ESAT-6 overall sensitivities in urine were 93% and 65% respectively. LAM and ESAT-6 overall sensitivities in serum were 55% and 46% respectively. Overall specificity was ≥97% in all assays. Sensitivities were higher in HIV-positive compared to HIV-negative patients for both antigens and both sample types, with signals roughly 10-fold higher on average in urine than in serum. The two antigens showed similar concentration ranges within the same sample type and correlated., Conclusions: LAM and ESAT-6 can be detected in the urine and serum of TB patients, regardless of the HIV status and further gains in clinical sensitivity may be achievable through assay and reagent optimization. Accuracy in urine was higher with current methods and has the potential to meet the WHO accuracy target if the findings can be transferred to a point-of-care TB test., Competing Interests: TB, CMD and EM are employed by FIND (Geneva, Switzerland), a nonprofit organization that collaborates with industry partners. GBS, MT, AM, TP, DB, and SB are employed by Meso Scale Diagnostics LLC (Rockville, USA) and received funding from FIND and NIH. MK and KK are employed by Otsuka Pharmaceutical Co., Ltd (Tokyo, Japan). FIND, MSD, and Otsuka provided support in the form of salaries for authors [TB, CMD, EM, GBS, MT, AM, TP, DB, SB, MK and KK], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section. TB and AP report patents in the field of LAM detection. The interests of authors do not alter the adherence to PLOS ONE policies on sharing data and materials. (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests) All other authors declare no competing interests.

The detection of circulating free DNA (cfDNA) has transformed the field of oncology and prenatal diagnostics. Clinical application of cfDNA for disease diagnosis and monitoring, however, is relatively recent in the field of infectious disease. The potential of cfDNA as a noninvasive diagnostic and monitoring tool is especially promising for tuberculosis (TB), as it enables the detection of both pulmonary and extrapulmonary TB from easily accessible urine and/or blood samples from any age group. However, despite the potential of cfDNA detection to identify TB, very few studies are described in the literature to date. A comprehensive search of the literature identified 15 studies that report detecting Mycobacterium tuberculosis DNA in the blood and urine of TB patients with nongenitourinary disease, but in only six of them were the methodological steps considered suitable for cfDNA isolation and detection. The sensitivities and specificities for the diagnosis of pulmonary and extrapulmonary TB cases reported in these six studies are highly variable, falling in the range of 29% to 79% and 67% to 100%, respectively. While most studies could not meet the performance requirements of the high-priority target product profiles (TPP) published by the World Health Organization (WHO), the study results nonetheless show promise for a point-of-care detection assay. Better designed prospective studies, using appropriate samples, will be required to validate cfDNA as a TB biomarker., (Copyright © 2019 Fernández-Carballo et al.)

The only currently commercialized point-of-care assay for tuberculosis (TB) that measures lipoarabinomannan (LAM) in urine (Alere LF-LAM) has insufficient sensitivity. We evaluated the potential of 100 novel monoclonal antibody pairs targeting a variety of LAM epitopes on a sensitive electrochemiluminescence platform to improve the diagnostic accuracy. In the screening, many antibody pairs showed high reactivity to purified LAM but performed poorly at detecting urinary LAM in clinical samples, suggesting differences in antigen structure and immunoreactivity of the different LAM sources. The 12 best antibody pairs from the screening were tested in a retrospective case-control study with urine samples from 75 adults with presumptive TB. The best antibody pair reached femtomolar analytical sensitivity for LAM detection and an overall clinical sensitivity of 93% (confidence interval [CI], 80% to 97%) and specificity of 97% (CI, 85% to 100%). Importantly, in HIV-negative subjects positive for TB by sputum smear microscopy, the test achieved a sensitivity of 80% (CI, 55% to 93%). This compares to an overall sensitivity of 33% (CI, 20% to 48%) of the Alere LF-LAM and a sensitivity of 13% (CI, 4% to 38%) in HIV-negative subjects in the same sample set. The capture antibody targets a unique 5-methylthio-d-xylofuranose (MTX)-dependent epitope in LAM that is specific to the Mycobacterium tuberculosis complex and shows no cross-reactivity with fast-growing mycobacteria or other bacteria. The present study provides evidence that improved assay methods and reagents lead to increased diagnostic accuracy. The results of this work have informed the development of a sensitive and specific novel LAM point-of-care assay with the aim to meet the WHO's performance target for TB diagnosis., (Copyright © 2018 Sigal et al.)

Lipoarabinomannan (LAM), the major antigenic glycolipid of Mycobacterium tuberculosis , is an important immunodiagnostic target for detecting tuberculosis (TB) infection in HIV-1-coinfected patients, and is believed to mediate a number of functions that promote infection and disease development. To probe the human humoral response against LAM during TB infection, several novel LAM-specific human mAbs were molecularly cloned from memory B cells isolated from infected patients and grown in vitro. The fine epitope specificities of these Abs, along with those of a panel of previously described murine and phage-derived LAM-specific mAbs, were mapped using binding assays against LAM Ags from several mycobacterial species and a panel of synthetic glycans and glycoconjugates that represented diverse carbohydrate structures present in LAM. Multiple reactivity patterns were seen that differed in their specificity for LAM from different species, as well as in their dependence on arabinofuranoside branching and nature of capping at the nonreducing termini. Competition studies with mAbs and soluble glycans further defined these epitope specificities and guided the design of highly sensitive immunodetection assays capable of detecting LAM in urine of TB patients, even in the absence of HIV-1 coinfection. These results highlighted the complexity of the antigenic structure of LAM and the diversity of the natural Ab response against this target. The information and novel reagents described in this study will allow further optimization of diagnostic assays for LAM and may facilitate the development of potential immunotherapeutic approaches to inhibit the functional activities of specific structural motifs in LAM., (Copyright © 2018 The Authors.)

Background: Nucleic acid amplification tests (NAATs) have proven to be advantageous in the diagnosis of leishmaniases, allowing sensitive diagnosis of: (i) cutaneous leishmaniasis in long duration lesions and (ii) visceral leishmaniasis using a less-invasive sample like peripheral blood, in opposition to tissue aspiration required for parasite demonstration by microscopy. Despite their benefits, the implementation of NAATs for leishmaniasis diagnosis at the point-of-care has not been achieved yet, mostly due to the complexity and logistical issues associated with PCR-based methods., Methods: In this work, we have evaluated the performance of a ready-to-use loop-mediated isothermal amplification (LAMP) kit using two real time fluorimeters to amplify leishmanial DNA obtained by silica column-based and Boil & Spin protocols., Results: The different approaches used to run and interpret the LAMP reactions showed a performance equivalent to PCR and real-time PCR, using spiked and clinical samples. The time to positivity obtained with real-time fluorimetry showed an excellent correlation with both Ct values and parasite load from real-time quantitative PCR., Conclusions: The results obtained open the possibility of using a highly stable, ready-to-use LAMP kit for the accurate diagnosis of leishmaniasis at the point-of-care. Furthermore, the feasibility of relating time to positivity, determined with a portable real-time fluorimeter, with the parasite burden could have a wider application in the management of leishmaniasis, such as in treatment efficacy monitoring or as a pharmacodynamics tool in clinical trials.

Background: Identification of the Mycobacterium tuberculosis immunoproteome and antigens associated with serologic responses in adults has renewed interest in developing a serologic test for childhood tuberculosis (TB). We investigated IgG antibody responses against M. tuberculosis antigens in children with well-characterized TB., Methods: We studied archived sera obtained from hospitalized children with suspected pulmonary TB, and classified as having confirmed TB (culture-confirmed), unlikely TB (clinical improvement without TB treatment), or unconfirmed TB (all others). A multiplexed bead-based assay for IgG antibodies against 119 M. tuberculosis antigens was developed, validated and used to test sera. The area under the curves (AUCs) of the empiric receiver-operator characteristic curves were generated as measures of predictive ability. A cross-validated generalized linear model was used to select the most predictive combinations of antigens., Results: For the confirmed TB versus unlikely TB comparison, the maximal single antigen AUC was 0.63, corresponding to sensitivity 0.60 and specificity 0.60. Older (age: 60+ months old) children's responses were better predictive of TB status than younger (age: 12-59 months old) children's, with a maximal single antigen AUC of -0.76. For the confirmed TB versus unlikely TB groups, the most predictive combinations of antigens assigned TB risk probabilities of 0.33 and 0.33, respectively, when all ages were considered, and 0.57 (interquartile range: 0.48-0.64) and 0.35 (interquartile range: 0.32-0.40) when only older children were considered., Conclusion: An antigen-based IgG test is unlikely to meet the performance characteristics required of a TB detection test applicable to all age groups.

Background: Development of rapid diagnostic tests for tuberculosis is a global priority. A whole proteome screen identified Mycobacterium tuberculosis antigens associated with serological responses in tuberculosis patients. We used World Health Organization (WHO) target product profile (TPP) criteria for a detection test and triage test to evaluate these antigens., Methods: Consecutive patients presenting to microscopy centers and district hospitals in Peru and to outpatient clinics at a tuberculosis reference center in Vietnam were recruited. We tested blood samples from 755 HIV-uninfected adults with presumptive pulmonary tuberculosis to measure IgG antibody responses to 57 M. tuberculosis antigens using a field-based multiplexed serological assay and a 132-antigen bead-based reference assay. We evaluated single antigen performance and models of all possible 3-antigen combinations and multiantigen combinations., Results: Three-antigen and multiantigen models performed similarly and were superior to single antigens. With specificity set at 90% for a detection test, the best sensitivity of a 3-antigen model was 35% (95% confidence interval [CI], 31-40). With sensitivity set at 85% for a triage test, the specificity of the best 3-antigen model was 34% (95% CI, 29-40). The reference assay also did not meet study targets. Antigen performance differed significantly between the study sites for 7/22 of the best-performing antigens., Conclusions: Although M. tuberculosis antigens were recognized by the IgG response during tuberculosis, no single antigen or multiantigen set performance approached WHO TPP criteria for clinical utility among HIV-uninfected adults with presumed tuberculosis in high-volume, urban settings in tuberculosis-endemic countries., (© The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America.)

New diagnostic innovations for tuberculosis (TB), including point-of-care solutions, are critical to reach the goals of the End TB Strategy. However, despite decades of research, numerous reports on new biomarker candidates, and significant investment, no well-performing, simple and rapid TB diagnostic test is yet available on the market, and the search for accurate, non-DNA biomarkers remains a priority. To help overcome this 'biomarker pipeline problem', FIND and partners are working on the development of a well-curated and user-friendly TB biomarker database. The web-based database will enable the dynamic tracking of evidence surrounding biomarker candidates in relation to target product profiles (TPPs) for needed TB diagnostics. It will be able to accommodate raw datasets and facilitate the verification of promising biomarker candidates and the identification of novel biomarker combinations. As such, the database will simplify data and knowledge sharing, empower collaboration, help in the coordination of efforts and allocation of resources, streamline the verification and validation of biomarker candidates, and ultimately lead to an accelerated translation into clinically useful tools., (Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Purpose: Tuberculosis is still a major threat to global health. New tools and strategies to produce disease-related proteins are quintessential for the development of novel vaccines and diagnostic markers., Experimental Design: To obtain recombinant proteins from Mycobacterium tuberculosis (Mtb) for use in clinical applications, a standardized procedure was developed that includes subcloning, protein expression in Mycobacterium smegmatis and protein purification using chromatography. The potential for the different protein targets to serve as diagnostic markers for tuberculosis was established using multiplex immunoassays., Results: Twelve soluble proteins from Mtb, including one protein complex, were purified to near-homogeneity following recombinant expression in M. smegmatis. Protein purity was assessed both by size exclusion chromatography and MS. Multiplex serological testing of the final protein preparations showed that all but one protein displayed a clear antibody response in serum samples from 278 tuberculosis patients., Conclusion and Clinical Relevance: The established workflow comprises a simple, cost-effective, and scalable pipeline for production of soluble proteins from Mtb and can be used to prioritize immunogenic proteins suitable for use as diagnostic markers., (© 2016 The Authors. PROTEOMICS - Clinical Applications Published by WILEY-VCH Verlag GmbH & Co. KGaA.)

In 2014, the World Health Organization (WHO) developed the End TB Strategy in response to a World Health Assembly Resolution requesting Member States to end the worldwide epidemic of tuberculosis (TB) by 2035. For the strategy's objectives to be realised, the next 20 years will need novel solutions to address the challenges posed by TB to health professionals, and to affected people and communities. Information and communication technology presents opportunities for innovative approaches to support TB efforts in patient care, surveillance, programme management and electronic learning. The effective application of digital health products at a large scale and their continued development need the engagement of TB patients and their caregivers, innovators, funders, policy-makers, advocacy groups, and affected communities.In April 2015, WHO established its Global Task Force on Digital Health for TB to advocate and support the development of digital health innovations in global efforts to improve TB care and prevention. We outline the group's approach to stewarding this process in alignment with the three pillars of the End TB Strategy. The supplementary material of this article includes target product profiles, as developed by early 2016, defining nine priority digital health concepts and products that are strategically positioned to enhance TB action at the country level., (The content of this work is ©the authors or their employers. Design and branding are ©ERS 2016.)

Accurate and sensitive online detection tools would benefit both fundamental research and practical applications in aquatic microbiology. Here, we describe the development and testing of an online flow cytometer (FCM), with a specific use foreseen in the field of drinking water microbiology. The system incorporated fully automated sampling and fluorescent labeling of bacterial nucleic acids with analysis at 5-min intervals for periods in excess of 24 h. The laboratory scale testing showed sensitive detection (< 5% error) of bacteria over a broad concentration range (1 × 10(3) -1 × 10(6) cells mL(-1) ) and particularly the ability to track both gradual changes and dramatic events in water samples. The system was tested with bacterial pure cultures as well as indigenous microbial communities from natural water samples. Moreover, we demonstrated the possibility of using either a single fluorescent dye (e.g., SYBR Green I) or a combination of two dyes (SYBR Green I and Propidium Iodide), thus broadening the application possibilities of the system. The online FCM approach described herein has considerable potential for routine and continuous monitoring of drinking water, optimization of specific drinking water processes such as biofiltration or disinfection, as well as aquatic microbiology research in general., (Copyright © 2012 International Society for Advancement of Cytometry.)

As the understanding of variation is the key to a good process and product quality one should pay attention to dynamics on the single-cell level. The basic idea of this approach was to qualify and quantify variations on the single-cell level during bioreactor cultivations by monitoring the expression of an eGFP tagged target protein (human membrane protein) using fully automated real-time, flow injection flow cytometry (FI-FCM). The FI-FCM system consists of a sampling- and defoaming- as well as of a dilution-section. It allows a very short monitoring interval (5 min) and is able to dilute the reactor sample by a factor ranging up to more than 10,000. In bioreactor cultivations of recombinant Pichia pastoris expressing the eGFP tagged target protein, high correlations (R(2)≥ 0.97) between the FI-FCM fluorescent signal and other, however, population-averaged fluorescence signals (off-line fluorescence, in situ fluorescence probe) were obtained. FI-FCM is the only method able to distinguish between few cells with high fluorescence and many cells with low fluorescence intensity and proved that cells differ significantly from each other within the population during bioreactor cultivations. Single-cell fluorescence was distributed over a broad range within the cell population. These distributions strongly suggest that (a) the AOX-I promoter is leaky and (b) a fraction of the population is able to express more protein of interest within shorter time and (c) a fraction of the population does not express the fusion protein at all. These findings can help in the selection of high producing, stable strains. To show the platform-independency of the system, it has successfully been tested during bioreactor cultivations of three different strains (P. pastoris, Saccharomyces cerevisiae, Escherichia coli). Along with its applications in PAT, the FI-FCM could be used as a platform-independent (prokaryotes and eukaryotes) method in various other applications; for example in the closed-loop-control of bioprocesses using different kinds of fluorescent reporters, (waste- and drinking-) water analysis, clone selection in combination with FACS or even for surgery applications., (Copyright © 2011 Elsevier B.V. All rights reserved.)