Detection Of Lipoarabinomannan Research Articles

Role of detection of lipoarabinomannan (LAM) in urine for diagnosis of pulmonary tuberculosis in HIV patients: Egyptian experience

BackgroundTuberculosis remains a worldwide problem fueled by the HIV epidemic. TB infection impacts HIV progression and mortality even with treatment. Egypt has increasing HIV prevalence, although still in low prevalent areas.ResultsUrinary LAM was positive in 22 (95.7%) of TB patients and 1 (1.9%) of non TB group. Sensitivity was 95.7%, specificity 98.1%, positive and negative predictive values were 95.7% and 98.1% respectively, with accuracy 97.4%. Urinary LAM ELISA assay has the highest sensitivity (95.7%) in relation to other tests used for TB detection in HIV patients and its concentration was highly correlated to CD4 cell count and the extent of radiological changes.ConclusionThe use of urinary LAM in HIV patients is rapid, safe, available, and helpful tool for ruling in TB especially for those who cannot expectorate, critically ill, with low CD4, or presented by multiple system affection.

Urine lipoarabinomannan in HIV uninfected, smear negative, symptomatic TB patients: effective sample pretreatment for a sensitive immunoassay and mass spectrometry

Our study sought to determine whether urine lipoarabinomannan (LAM) could be validated in a sample cohort that consisted mainly of HIV uninfected individuals that presented with tuberculosis symptoms. We evaluated two tests developed in our laboratory, and used them on clinical samples from Lima, Peru where incidence of HIV is low. ELISA analysis was performed on 160 samples (from 140 adult culture-confirmed TB cases and 20 symptomatic TB-negative child controls) using 100 μL of urine after pretreatment with Proteinase K. Two different mouse monoclonal antibodies-CS35 and CHCS9-08 were used individually for capture of urine LAM. Among cases, optical density (OD450) values had a positive association with higher bacillary loads. The 20 controls had negative values (below the limit of detection). The assay correctly identified all samples (97–100% accuracy confidence interval). For an alternate validation of the ELISA results, we analyzed all 160 urine samples using an antibody independent chemoanalytical approach. Samples were called positive only when LAM surrogates—tuberculostearic acid (TBSA) and d-arabinose (d-ara)—were found to be present in similar amounts. All TB cases, including the 40 with a negative sputum smear had LAM in detectable quantities in urine. None of the controls had detectable amounts of LAM. Our study shows that urinary LAM detection is feasible in HIV uninfected, smear negative TB patients.

Mycobacterial antigens in pleural fluid mononuclear cells to diagnose pleural tuberculosis in HIV co-infected patients

BackgroundExtra pulmonary manifestation of tuberculosis (TB) accounts for approximately one-half of TB cases in HIV-infected individuals with pleural TB as the second most common location. Even though mycobacteria are cleared, mycobacterial antigens may persist in infected tissues, causing sustained inflammation and chronicity of the disease. The aim of this study was to explore various mycobacterial antigens in pleural effusions, the impact of HIV infection and CD4+ T-cell depletion on the presence of antigens, and the diagnostic potential of antigens for improved and rapid diagnosis of pleural TB.MethodsPleural fluid specimens were collected from patients presenting with clinically suspected pleural TB, and processed routinely for culture, cytology, and adenosine deaminase activity analysis. HIV status and CD4+ T-cell counts were recorded. Pleural fluid mononuclear cells (PFMC) were isolated, and cell smears were stained with acid-fast staining and immunocytochemistry for various mycobacterial antigens. Real-time and nested-PCR were performed. Patients were categorized as pleural TB or non-TB cases using a composite reference standard. Performance of the mycobacterial antigens as diagnostic test was assessed.ResultsA total of 41 patients were enrolled, of which 32 were classified as pleural TB and 9 as non-TB. Thirteen patients had culture confirmed pleural TB, 26 (81%) were HIV-TB co-infected, and 64% had < 100 CD4+ T-cells/microL. Both secreted and cell-wall mycobacterial antigens were detected in PFMC. Lipoarabinomannan (LAM) was the most frequently detected antigen. There was no direct correlation between positive culture and antigens. Cases with low CD4+ T-cell counts had higher bacterial and antigen burden. By combining detection of secreted antigen or LAM, the sensitivity and specificity to diagnose pleural TB was 56 and 78%, respectively, as compared to 41 and 100% for culture, 53 and 89% for nested PCR, and 6 and 100% for real-time PCR.ConclusionMycobacterial antigens were detectable in PFMC from tuberculous pleural effusions, even in cases where viable mycobacteria or bacterial DNA were not always detected. Thus, a combination of secreted antigen and LAM detection by immunocytochemistry may be a complement to acid-fast staining and contribute to rapid and accurate diagnosis of pleural TB.

Detection of mycobacterial lipoarabinomannan in serum for diagnosis of active tuberculosis

Urinary detection of Mycobacterium tuberculosis lipoarabinomannan (LAM) for tuberculosis (TB) diagnosis is well characterized, but the utility of serum LAM detection remains unclear. We developed an assay for serum LAM detection using single-molecule array (Simoa), purified M. tuberculosis LAM, and anti-LAM monoclonal antibodies and evaluated performance on diluted/heat-treated serum samples from patients with and without active TB and/or HIV. The Simoa assay had a limit of detection of 0.35 pg/mL and lower limit of quantification of 0.942 pg/mL. Corrected serum LAM concentrations ranged from 0 to 132.0 pg/mL [median 1.71, interquartile range (IQR) 0.94–6.80] in 90 TB+ patients and from 0 to 2.29 pg/mL (median 1.03, IQR 0.47–1.69) in 55 TB− patients. Using a cutoff of 2.3 pg/mL for 100% specificity, assay sensitivity was 37% in all TB+ subjects (33/90; 95% CI 0.27–0.48), 47% in TB+/HIV+ subjects (26/55; 0.34–0.61), and 60% in TB+/HIV+/smear+ subjects (21/35; 0.42–0.76). Mycobacterial LAM is detectable in serum with high specificity and reasonable sensitivity using Simoa.

Evaluation of Mycobacterium tuberculosis lipoarabinomannan antigen assay and rapid serology blood test for the diagnosis of bovine tuberculosis in Ethiopia

BackgroundBovine tuberculosis (bTB) is prevalent in dairy cattle in Ethiopia. Currently used diagnostic tools such as the single intradermal comparative tuberculin test (SICTT) are time consuming and labor intensive. A rapid, easy-to-use and cost-effective diagnostic test would greatly contribute to the control of bTB in developing countries like Ethiopia. In the present study, two point-of-care diagnostic tests were evaluated for the detection of bTB: LIONEX® Animal TB Rapid test, a membrane-based test for the detection of antibodies to Mycobacterium bovis in blood and ALERE® Determine TB Lipoarabinomannan (LAM) Ag, an immunoassay for the detection of lipoarabinomannan (LAM) antigen (Ag) of mycobacteria in urine. A combination of the SICTT and gamma interferon (IFN-γ) test was used as the gold standard for the validation of these point-of-care tests, as it was not feasible to slaughter the study animals to carry out the historical gold standard of mycobacterial culture. A total of 175 heads of cattle having three different bTB infection categories (positive SICTT, negative SICTT, and unknown SICTT status) were used for this study.ResultThe sensitivity and specificity of TB LAM Ag were 72.2% (95% CI = 62.2, 80.4) and 98.8% (95% CI = 93.6, 99.7), respectively, while the sensitivity and specificity of the LIONEX Animal TB rapid test assay were 54% (95% CI = 44.1 64.3) and 98.8% (95% CI = 93.6, 99.7) respectively. The agreement between TB LAM Ag and SICTT was higher (κ = 0.85; 95% CI = 0.65–0.94) than between TB LAM Ag and IFN-γ (κ = 0.67; 95% CI = 0.52–0.81). The agreement between LIONEX Animals TB Rapid blood test and SICTT was substantial, (κ = 0.63; 95% CI = 0.49–0.77) while the agreement between LIONEX Animal TB rapid blood test and IFN-γ test was moderate (κ = 0.53; 95% CI = 0.40–0.67). Analysis of receiver operating curve (ROC) indicated that the area under the ROC curve (AUC) for TB LAM Ag was 0.85 (95% CI = 0.79–0.91) while it was 0.76 (95% CI; =0.69–0.83) for LIONEX Animal TB rapid test assay.ConclusionThis study showed that TB LAM Ag had a better diagnostic performance and could potentially be used as ancillary either to SICTT or IFN-γ test for diagnosis of bTB.

Detection of Mycobacterium tuberculosis lipoarabinomannan and CFP-10 (Rv3874) from urinary extracellular vesicles of tuberculosis patients by immuno-PCR.

Extracellular vesicles (EVs), the small circulating vesicles released from urine samples of tuberculosis (TB) patients, contain a pool of biomarkers. We recently detected Mycobacterium tuberculosis lipoarabinomannan (LAM) and CFP-10 (Rv3874) biomarkers from the urinary EVs of pulmonary TB (PTB) and extrapulmonary TB (EPTB) patients by immuno-polymerase chain reaction (I-PCR) assay and the results were compared with the analogous enzyme-linked immunosorbent assay (ELISA). The detection limits of both purified LAM and CFP-10 were determined to be 1 fg/mL with I-PCR, which was 106 times lower than ELISA. Detection of LAM and CFP-10 biomarkers in urinary EVs of TB patients by I-PCR showed superiority over ELISA. Notably, LAM I-PCR revealed sensitivities of 74.3 and 67.9% in PTB (n=74) and EPTB (n=53) patients, respectively, with specificities of 91.5-92.8% (n=116). Moreover, the sensitivities attained with LAM I-PCR were significantly higher (P<0.01) than with CFP-10 I-PCR. After further improving the sensitivity and specificity of the assay, our I-PCR based on LAM detection in urinary EVs may be used as an adjunct test for rapid diagnosis of TB.

Risk score for predicting mortality including urine lipoarabinomannan detection in hospital inpatients with HIV-associated tuberculosis in sub-Saharan Africa: Derivation and external validation cohort study.

BackgroundThe prevalence of and mortality from HIV-associated tuberculosis (HIV/TB) in hospital inpatients in Africa remains unacceptably high. Currently, there is a lack of tools to identify those at high risk of early mortality who may benefit from adjunctive interventions. We therefore aimed to develop and validate a simple clinical risk score to predict mortality in high-burden, low-resource settings.Methods and findingsA cohort of HIV-positive adults with laboratory-confirmed TB from the STAMP TB screening trial (Malawi and South Africa) was used to derive a clinical risk score using multivariable predictive modelling, considering factors at hospital admission (including urine lipoarabinomannan [LAM] detection) thought to be associated with 2-month mortality. Performance was evaluated internally and then externally validated using independent cohorts from 2 other studies (LAM-RCT and a Médecins Sans Frontières [MSF] cohort) from South Africa, Zambia, Zimbabwe, Tanzania, and Kenya. The derivation cohort included 315 patients enrolled from October 2015 and September 2017. Their median age was 36 years (IQR 30–43), 45.4% were female, median CD4 cell count at admission was 76 cells/μl (IQR 23–206), and 80.2% (210/262) of those who knew they were HIV-positive at hospital admission were taking antiretroviral therapy (ART). Two-month mortality was 30% (94/315), and mortality was associated with the following factors included in the score: age 55 years or older, male sex, being ART experienced, having severe anaemia (haemoglobin < 80 g/l), being unable to walk unaided, and having a positive urinary Determine TB LAM Ag test (Alere). The score identified patients with a 46.4% (95% CI 37.8%–55.2%) mortality risk in the high-risk group compared to 12.5% (95% CI 5.7%–25.4%) in the low-risk group (p < 0.001). The odds ratio (OR) for mortality was 6.1 (95% CI 2.4–15.2) in high-risk patients compared to low-risk patients (p < 0.001). Discrimination (c-statistic 0.70, 95% CI 0.63–0.76) and calibration (Hosmer-Lemeshow statistic, p = 0.78) were good in the derivation cohort, and similar in the external validation cohort (complete cases n = 372, c-statistic 0.68 [95% CI 0.61–0.74]). The validation cohort included 644 patients between January 2013 and August 2015. Median age was 36 years, 48.9% were female, and median CD4 count at admission was 61 (IQR 21–145). OR for mortality was 5.3 (95% CI 2.2–9.5) for high compared to low-risk patients (complete cases n = 372, p < 0.001). The score also predicted patients at higher risk of death both pre- and post-discharge. A simplified score (any 3 or more of the predictors) performed equally well. The main limitations of the scores were their imperfect accuracy, the need for access to urine LAM testing, modest study size, and not measuring all potential predictors of mortality (e.g., tuberculosis drug resistance).ConclusionsThis risk score is capable of identifying patients who could benefit from enhanced clinical care, follow-up, and/or adjunctive interventions, although further prospective validation studies are necessary. Given the scale of HIV/TB morbidity and mortality in African hospitals, better prognostic tools along with interventions could contribute towards global targets to reduce tuberculosis mortality.

Diagnostic utility of urinary lipoarabinomannan in pulmonary tuberculosis

Background Urinary lipoarabinomannan (LAM) detection is a promising approach for the rapid diagnosis of active tuberculosis (TB). Objective to assess the diagnostic accuracy of quantitative urine Lipoarabinomannan (LAM) test in the diagnosis of pulmonary tuberculosis and evaluate its efficacy as a follow up test after 3 months of the first line of antituberculous therapy. Methods This study was carried out on 40 patients (32 male and 8 female) with active tubercular infection LAM level was measured in urine by enzyme-linked immunosorbant assay (ELISA). Results Patients with smear positive specimens had higher urine LAM level: grade 1+ (mean ± SD of 1.47 ± 0.85 ng/ml), grade 2+ (1.63 ± 1.05 ng/ml), grade 3+ (4.32 ± 2.56 ng/ml) than that of smear negative (1.42 ± 0.50 ng/ml) (P Conclusion Our finding showed that the urine LAM test is simple, rapid, and reliable diagnostic modality for active pulmonary tuberculosis. Quantitative LAM detection results increased progressively with bacillary burden. Urine LAM testing may be an accessible method to monitor response to anti-TB therapy among active pulmonary tuberculosis patients.

Label-Free and Real-Time Detection of Tuberculosis in Human Urine Samples Using a Nanophotonic Point-of-Care Platform.

Tuberculosis (TB) is the leading global cause of death from a single infectious agent. Registered incidence rates are low, especially in low-resource countries with weak health systems, due to the disadvantages of current diagnostic techniques. A major effort is directed to develop a point-of-care (POC) platform to reduce TB deaths with a prompt and reliable low-cost technique. In the frame of the European POCKET Project, a novel POC platform for the direct and noninvasive detection of TB in human urine was developed. The photonic sensor chipis integrated in a disposable cartridge and is based on a highly sensitive Mach-Zehnder Interferometer (MZI) transducer combined with an on-chip spectral filter. The required elements for the readout are integrated in an instrument prototype, which allows real-time monitoring and data processing. In this work, the novel POC platform has been employed for the direct detection of lipoarabinomannan (LAM), a lipopolysaccharide found in the mycobacterium cell wall. After the optimization of several parameters, a limit of detection of 475 pg/mL (27.14 pM) was achieved using a direct immunoassay in undiluted human urine in less than 15 min. A final validation of the technique was performed using 20 clinical samples from TB patients and healthy donors, allowing the detection of TB in people regardless of HIV coinfection. The results show excellent correlation to those obtained with standard techniques. These promising results demonstrate the high sensitivity, specificity and applicability of our novel POC platform, which could be used during routine check-ups in developing countries.

Novel High Sensitivity Tuberculosis Point-of-Care Test for People Living with HIV

Background: Most tuberculosis-related deaths in people living with HIV (PLHIV) could be prevented with earlier diagnosis and treatment. The only commercially available tuberculosis point-of-care test (Alere DetermineTM TB LAM Ag (AlereLAM)) has suboptimal sensitivity which restricts its use in clinical practice. The novel Fujifilm SILVAMP TB LAM (FujiLAM) test was designed to achieve superior diagnostic accuracy. Methods: We performed a blinded assessment of FujiLAM on biobanked urine samples from three independent prospective cohort studies of hospitalized PLHIV at two South African district hospitals. Diagnostic accuracy was determined against both microbiological (MRS) and composite reference standards (CRS, including clinical diagnoses), in comparison with AlereLAM. Findings: A total of 968 newly admitted PLHIV were included. The prevalence of microbiologically-confirmed TB was 62% and the median CD4 count 86 cells/µl. In a meta-analysis, the estimated sensitivity of the FujiLAM was superior at 70·4% (95%CI: 53·0-83·1) over that of AlereLAM at 42·3% (31·7-51·8), against the MRS, with a difference of 28.1% (21.5-34.4). Specificities were 90·8% (86·0-94·4) and 95·0% (87·7-98·8) for the FujiLAM and AlereLAM against the MRS, respectively, with a non-significant difference of -4.2% (-12.7-4.4). Against the CRS, specificities of both assays were higher (FujiLAM 95·7% and AlereLAM 98·2%) but sensitivities were lower (FujiLAM 64·9% and AlereLAM 38·2%). In the subgroup of patients with CD4 ≤100 cells/µl, the sensitivity of FujiLAM was 84·2% (71·4-91·4) versus 57·3% (42·2-69·6) for AlereLAM. Interpretation: In comparison to AlereLAM, FujiLAM offers superior diagnostic sensitivity, while maintaining specificity, and has the potential to transform rapid point-of-care tuberculosis diagnosis for hospitalized PLHIV. FujiLAM's suitability for settings of intended use needs to be assessed in a prospective evaluation. Funding: GHIT Fund, UK DFID, Dutch MoFA, BMGF, Australian DFAT, German BMBF through KfW, Wellcome Trust, DST/ NRF of South Africa, and South African MRC. Declaration of Interest: TB, EI, AM, SO, CCB, and CMD are 384 employed by FIND. FIND is a not for-profit foundation that supports the evaluation of publicly prioritized tuberculosis assays and the implementation of WHO-approved (guidance and prequalification) assays using donor grants. FIND has product evaluation agreements with several private sector companies that design diagnostics for tuberculosis and other diseases. These agreements strictly define FIND’s independence and neutrality vis-a-vis the companies whose products get evaluated and describe roles and responsibilities. AP and TB report patents in the field of lipoarabinomannan detection. All other authors declare no competing interests. Ethical Approval: All study-related activities were approved by the Human Research Ethics Committee (HREC) of the University of Cape Town (UCT). Written informed consent was obtained from patients, as per study protocols. Study participation did not affect standard of care. Reporting followed STARD guidelines.12 167 Retrospective urine LAM testing was supervised by the sponsor, FIND, and was performed at UCT in April 2018.

Detection and Diagnostic Accuracy of Rapid Urine Lipoarabinomannan Lateral-Flow Assay in Pulmonary Tuberculosis patients in Nigeria

Background: Tuberculosis (TB) is a public health challenge in both developed and developing countries. Early diagnosis is essential in preventing the further spread of the disease, but the control programs are currently facing a number of constraints and fewer than 25% of all tuberculosis cases especially childhood cases are detected. We aimed to evaluate diagnostic accuracy of a commercially available qualitative immunoassay for the detection of lipoarabinomannan (LAM) antigen of Mycobacteria in human urine by comparing its sensitivity and specificity in TB patients with the AFB and GeneXpert in individuals with presumptive tuberculosis cases. Methods: A cross-sectional study that consecutively enrolled 53 eligible TB adults’ patients attending TB Centre, Mangu, Plateau State from February to March 2017. We applied the LAM test on urine collected as a spot and early morning sample. Diagnostic accuracy was analyzed for a microbiological TB reference standard based on Gene Xpert MTB/RIF results and for a composite reference standard including clinical data. Performance of sputum smear microscopy (AFB) was included for comparison. Results: The mean age of the respondents was 41.0±17.0 years.) The male proportion was 36(68.0%) and female was 17(32.0%). The patients with HIV-1 Co-infection were 9(23.8%). Of the 53 patients, the positive testing rate of TB using LAM test was 11 (20.8 %). The proportion of those who tested positive using Gene Xpert was 9(17.0%) and AFB was 33(62.2%), and the sensitivity and specificity were 33.3% and 93.2%, respectively. Negative and positive predictive values were 87.23% and 50.0%, diagnostic accuracy was 83.02%. Conclusion: The study showed great sensitivity of urine LAM test suggesting it could be useful as point of care diagnostic test for presumptive TB cases. Its high negative predictive value suggests a role in screening out uninfected patients; though GeneXpert had superior sensitivity, but the ease of the LAM test holds operational advantage as a screening method, however larger studies are needed to further determine diagnostic accuracy.

Detection of lipoarabinomannan in urine and serum of HIV-positive and HIV-negative TB suspects using an improved capture-enzyme linked immuno absorbent assay and gas chromatography/mass spectrometry

TB diagnosis and treatment monitoring in resource limited regions rely heavily on serial sputum smear microscopy and bacterial culture. These microbiological methods are time-consuming, expensive and lack adequate sensitivity. The WHO states that improved TB diagnosis and treatment is imperative to achieve an end to the TB epidemic by 2030. Commercially available lipoarabinomannan (LAM) detection tools perform at low sensitivity that are highly dependent on the underlying immunological status of the patient; those with advanced HIV infection perform well. In this study, we have applied two novel strategies towards the sensitive diagnosis of TB infection based on LAM: Capture ELISA to detect LAM in paired urine and serum samples using murine and human monoclonal antibodies, essentially relying on LAM as an ‘immuno-marker’; and, secondly, detection of α-d-arabinofuranose and tuberculostearic acid (TBSA)- ‘chemical-markers’ unique to mycobacterial cell wall polysaccharides/lipoglycans by our recently developed gas chromatography/mass spectrometry (GC/MS) method. Blinded urine specimens, with microbiologically confirmed active pulmonary TB or non TB (HIV+/HIV–) were tested by the aforementioned assays. LAM in patient urine was detected in a concentration range of 3–28 ng/mL based on GC/MS detection of the two LAM-surrogates, d-arabinose and tuberculostearic acid (TBSA) correctly classifying TB status with sensitivity > 99% and specificity = 84%. The ELISA assay had high sensitivity (98%) and specificity (92%) and the results were in agreement with GC/MS analysis. Both tests performed well in their present form particularly for HIV-negative/TB-positive urine samples. Among the HIV+/TB+ samples, 52% were found to have >10 ng/mL urinary LAM. The detected amounts of LAM present in the urine samples also appears to be associated with the gradation of the sputum smear, linking elevated LAM levels with higher mycobacterial burden (odds ratio = 1.08–1.43; p = 0.002). In this small set, ELISA was also applied to parallel serum samples confirming that serum could be an additional reservoir for developing a LAM-based immunoassay for diagnosis of TB.

Developing and Deploying Universal Diagnostic Platforms for One-Health Biosurveillance

ObjectiveOur goal is to develop deployable strategies for infectious disease diagnosis at the point-of-care that are applicable to multiple hosts of infection - conforming to the global One Health Strategy for diagnostics. We aim to develop methods that do not require prior knowledge of the pathogen in question, and can facilitate rapid and effective decision-making and situational awareness.IntroductionThere is an urgent need for diagnostic strategies for infections which are host-independent, so as to effectively track zoonotic spread, monitor animal carriers of pathogens, and evaluate transmission dynamics. Infection of a host - pathogen or human- by an animal results in recognition by the immune response, which consequently causes release of inflammatory mediators. Many scientists have explored the use of cytokines as diagnostic indicators of disease, but the conserved nature of the immune response in humans and animals results in cross-reactivity among many pathogens, making evaluation of the results difficult, especially in high disease burden populations. Measuring the pathogen-specific signature, however, is advantageous - as it offers discrete identification of active infection, and discrimination from exposure. It also offers a universal strategy that can be applied to human and animal hosts of infection - allowing for One Health Biosurveillance. Achieving this, however, requires the development of a) tailored strategies for the measurement of biochemically disparate pathogen signatures in clinical samples and b) ultra-sensitive detection of such signatures in the host. The sensor team at Los Alamos National Laboratory is working on both of these aspects, and the development of One Health Diagnostic platforms, the focus of the work presented here.MethodsMany of the biomarkers secreted by bacterial pathogens and recognized by innate immune receptors elicit host cytokine responses that are amphiphilic (largely glycolipids, lipoproteins or lipoglycans). Based on such, our team has developed tailored methods – membrane insertion and lipoprotein capture for the capture and detection of these greasy and stealthy molecules in infected blood. Sensitivity and specificity, assay optimization, alinearity associated with lipidic molecules and assay parameter development for biomarkers associated with bacterial pathogens will be presented. Considerations for clinical sampling for amphiphile detection in blood, and clinical study design will be demonstrated. Our team has developed an ultra-sensitive biosensor for detection of biomarkers in complex matrices based on the evanescent field properties of single mode planar optical waveguides. Detection of amphiphilic biomarkers in clinical samples, using the waveguide-based biosensor and tailored methods for the detection of such molecules has allowed for the diagnosis of infection in both human and animals hosts. Herein, we present two examples of the same; 1) diagnosis of tuberculosis in humans, cattle and badgers using pathogen-based diagnostic strategies; and 2) detection of Gram-negative Lipopolysacharides in beef and in patients with Salmonella-induced sepsis.ResultsWe have developed tailored ultra-sensitive waveguide-biosensor assays for the detection of Lipoarabinomannan (and other biomarkers) from Mycobacteria and demonstrated feasibility in blinded clinical studies in humans and cattle, demonstrating one -health compatibility.We have also demonstrated detection of lipopolysacharides from eight different serotypes of Shiga-toxin carrying E. coli in beef, and Salmonella Typhimurium in pediatric patients using the same approach.ConclusionsWe have demonstrated clinical feasibility of a One Health Strategy for point-of-care diagnostics of bacterial infections in blood for tuberculosis and Gram-negative pathogens in clinical samples. The results will be demonstrated with several discussion points on the consideration of unconventional biochemistry of pathogens for diagnostics, factors influencing point-of-care deployment and integration of diagnostic platforms for biosurveillance. One Health Considerations and challenges therein will also merit discussion.ReferencesSakamuri, R; et al. Accurate Tracking of Bovine Tuberculosis Biomarkers in Infected Cattle using a Novel Biomarker Capture Strategy, Analytical Sciences, 33(4): 457-460, 2017.Noormohammed A et al. Detection of Salmonella LPS in Patient blood by Lipoprotein Capture. Proc. SPIE 10072, Optical Diagnostics and Sensing XVII: Toward Point-of-Care Diagnostics, 100720A , 2017. doi:10.1117/12.2253506Stromberg L et al. Membrane Insertion for the Detection of Lipopolysaccharides: Exploring the Dynamics of Amphiphile-in-Lipid Assays, PloS One, April 2016.Sakamuri RM et al. Association of lipoarabinomannan with high density lipoprotein in blood: implications for diagnostics.Tuberculosis (Edinb). 2013 May;93(3):301-7. doi: 10.1016/j.tube.2013.02.015. Epub 2013 Mar 16Mukundan H et al. Understanding the interaction of Lipoarabinomannan with membrane mimetic architectures. Tuberculosis, 92(1) 32-47, 2012.Mukundan H et al., Waveguide-based Sensors for Pathogen Detection, Invited Review, Sensors, 9(7), 5783-5809.

Evaluation of lipoarabinomannan in the diagnosis of tuberculosis

The detection of lipoarabinomannan (LAM) antigens in body fluids has several potential advantages compared with the diagnostic methods used currently. The aim of this study was to evaluate the possible role of the detection of LAM in the serum and the urine as a diagnostic aid in the diagnosis of different forms of tuberculosis (TB). This study included 62 newly confirmed tuberculosis cases classified into two groups: group A included patients with pulmonary TB (n=36), and was further divided into two groups: group A1 [the smear-positive pulmonary TB group (n=24)] and group A2 [the smearnegative pulmonary TB group (n=12)]; group B included the extrapulmonary TB group (n=26); and 10 apparently healthy individuals served as the control group. The LAM level was measured in the serum and the urine by an enzyme-linked immunosorbant assay. The mean level of quantitative serum LAM was higher in group A1 (0.55±0.20?ng/ml) compared with group A2 (0.44±0.30?ng/ml) or group B (0.41±0.27?ng/ml). The mean level of quantitative urine LAM was higher in group A1 (0.81±0.24?ng/ml) compared with group B (0.72±0.35?ng/ml) and group A2 (0.65±0.37?ng/ml; P<0.001). The quantitative urine LAM test correlated positively with the degree of bacillary burden (P<0.05). Quantitative serum LAM had a sensitivity of 88.7%, specificity 90%, accuracy 88.9%, positive predictive value 98.2%, and negative predictive value 56.3%. Quantitative urine LAM had a sensitivity of 85.5%, specificity 90%, accuracy 86.1%, positive predictive value 98.1%, and negative predictive value 50%. A combination of serum and urine LAM tests identified that 98.4% of the cases with a positive TB culture correlated with higher serum LAM levels. Advanced chest radiography involvement and TB culture correlated with higher urine LAM levels (P<0.05). The LAM test is a valuable addition in the diagnosis of TB and its different forms. A combination of quantitative serum and urine LAM increased the sensitivity of the test. The quantitative urine LAM test offers additional clinical insight into the degree of TB disease severity and has more applicability.

Capillary electrophoresis as a method to determine underivatized urinary lipoarabinomannans, a biomarker of active tuberculosis caused by Mycobacterium tuberculosis.

Tuberculosis is a devastating contagious disease caused by Mycobacterium tuberculosis. This is the first report describing the development of novel capillary electrophoresis methods to detect lipoarabinomannans shed into the blood circulation by replicating bacteria. The novelty of the methods is the detection without derivatization. The lipoarabinomannan is detected owing to the ionization of the diverse functional groups of the structure, such as the multibranched mannan domain or the phosphatidyl group. Four alkaline solutions were used; normal polarity in three of them and reversed polarity in one. Urinary lipoarabinomannans by saccharide domains were identified with direct absorbance detection. The accuracy and the analytical sensitivity were then validated with cello-, manno- and xylooligosaccharides. Lipoarabinomannan detection was feasible within 20 min (RSD 2.1%). This method worked at the dynamic range of 0.1-10 μg/mL. With reversed polarity, indirect absorbance detection, and pH 9.0 electrolyte were used, the analytes migrated already within 5 min (RSD 0.01%). Inorganic nonabsorbing ions were used for this method optimization. This improvement resulted in the detection limit of 1 pg/mL in water and in the linear dynamic range of 1 pg/mL to 10 ng/mL. In conclusion, the described method has great potential as a point-of-care assay for clinical use.

Detection of Mycobacterial Lipoarabinomannan with A Monoclonal Antibody Qualitative ELISA in Urine of Tuberculous Meningitis Patients

BACKGROUND: Tuberculous meningitis is the most severe manifestation of tuberculosis. The diagnostic approach of tuberculous meningitis is difficult. Combination of clinical, laboratory and radiological criteria were used in diagnostic approach of tuberculous meningitis. Urinary mycobacterial lipoarabinomannan (LAM) antigen detection is a promising diagnostic tool. Detection of mycobacterial antigen in concentrated urine sample is predicted to improve the positivity rate of the qualitative enzyme-linked immunosorbent assay (ELISA) diagnostic tool. The purpose of this study is to examine the detection ability of a monoclonal antibody qualitative ELISA in concentrated and unconcentrated urine of tuberculous meningitis patients.METHODS: This research is a descriptive, crosssectionally designed. The study was conducted in the Clinical Pathology Department laboratory of Dr. Hasan Sadikin Hospital, in July-October 2014. A total of 27 patients diagnosed as tuberculous meningitis patients were included and the subjects were classified into possible and probable criteria according to consensus criteria. The subjects were classified as definite if the cerebrospinal fluid culture was positive for Mycobacterial tuberculosis growth. The subjects were examined for the presence of LAM in unconcentrated and concentrated urine with a monoclonal antibody qualitative ELISA method.RESULTS: Unconcentrated urinary LAM examination positivity was 0% while in concentrated urine was 14.8%. The positivity of concentrated urinary LAM were higher among the definite criteria group.CONCLUSION: Concentrating urine sample increase the positivity rate of urinary LAM detection with ELISA method as high as 14.8%. The urinary antigen detection is higher among the definite tuberculous meningitis patients.KEYWORDS: LAM, concentrated urine, tuberculous meningitis, qualitative ELISA

Detection of lipoarabinomannan (LAM) in urine is an independent predictor of mortality risk in patients receiving treatment for HIV-associated tuberculosis in sub-Saharan Africa: a systematic review and meta-analysis.

BackgroundSimple immune capture assays that detect mycobacterial lipoarabinomannan (LAM) antigen in urine are promising new tools for the diagnosis of HIV-associated tuberculosis (HIV-TB). In addition, however, recent prospective cohort studies of patients with HIV-TB have demonstrated associations between LAM in the urine and increased mortality risk during TB treatment, indicating an additional utility of urinary LAM as a prognostic marker. We conducted a systematic review and meta-analysis to summarise the evidence concerning the strength of this relationship in adults with HIV-TB in sub-Saharan Africa, thereby quantifying the assay’s prognostic value.MethodsWe searched MEDLINE and Embase databases using comprehensive search terms for ‘HIV’, ‘TB’, ‘LAM’ and ‘sub-Saharan Africa’. Identified studies were reviewed and selected according to predefined criteria.ResultsWe identified 10 studies eligible for inclusion in this systematic review, reporting on a total of 1172 HIV-TB cases. Of these, 512 patients (44 %) tested positive for urinary LAM. After a variable duration of follow-up of between 2 and 6 months, overall case fatality rates among HIV-TB cases varied between 7 % and 53 %. Pooled summary estimates generated by random-effects meta-analysis showed a two-fold increased risk of mortality for urinary LAM-positive HIV-TB cases compared to urinary LAM-negative HIV-TB cases (relative risk 2.3, 95 % confidence interval 1.6–3.1). Some heterogeneity was explained by study setting and patient population in sub-group analyses. Five studies also reported multivariable analyses of risk factors for mortality, and pooled summary estimates demonstrated over two-fold increased mortality risk (odds ratio 2.5, 95 % confidence interval 1.4–4.5) among urinary LAM-positive HIV-TB cases, even after adjustment for other risk factors for mortality, including CD4 cell count.ConclusionsWe have demonstrated that detectable LAM in urine is associated with increased risk of mortality during TB treatment, and that this relationship remains after adjusting for other risk factors for mortality. This may simply be due to a positive test for urinary LAM serving as a marker of higher mycobacterial load and greater disease dissemination and severity. Alternatively, LAM antigen may directly compromise host immune responses through its known immunomodulatory effects. Detectable LAM in the urine is an independent risk factor for mortality among patients receiving treatment for HIV-TB. Further research is warranted to elucidate the underlying mechanisms and to determine whether this vulnerable patient population may benefit from adjunctive interventions.Electronic supplementary materialThe online version of this article (doi:10.1186/s12916-016-0603-9) contains supplementary material, which is available to authorized users.

Detection of lipoarabinomannan (LAM) in urine is indicative of disseminated TB with renal involvement in patients living with HIV and advanced immunodeficiency: evidence and implications.

TB is the leading cause of HIV/AIDS-related deaths globally. New diagnostic tools are urgently needed to avert deaths from undiagnosed HIV-associated TB. Although simple assays that detect lipoarabinomannan (LAM) in urine have been commercially available for years, their specific role and utility were initially misunderstood, such that they have been slower to emerge from the diagnostics pipeline than otherwise might have been expected. In this article, we review and explain how urine-LAM assays should be understood as diagnostics for disseminated TB in HIV-positive patients with advanced immunodeficiency, in whom haematogenous TB dissemination to the kidneys serves as the primary mechanism by which LAM enters the urine. These insights are critical for the appropriate design of studies to evaluate these assays and to understand how they might be most usefully implemented. This understanding also supports the 2015 WHO recommendations on the restricted use of these assays in sick HIV-positive patients with advanced immunodeficiency.

A sensitive urinary lipoarabinomannan test for tuberculosis.

We have previously developed a diagnostic test for tuberculosis based on detection of mycobacterial lipoarabinomannan (LAM) in urine. The method depended on a laborious concentration step. We have now developed an easy to perform test based on a magnetic immunoassay platform, utilizing high avidity monoclonal antibodies for the detection of LAM in urine. With this method the analytical sensitivity of the assay was increased 50-100-fold compared to conventional ELISA. In a pilot study of HIV-negative patients with microbiologically verified TB (n=17) and healthy controls (n=22) the sensitivity of the test was 82% and the specificity 100%. This is in stark positive contrast to a range of studies using available commercial tests with polyclonal anti-LAM Abs where the sensitivity of the tests in HIV-negative TB patients was very low.

Detection of lipoarabinomannan in urine for identification of active tuberculosis among HIV‐positive adults in Ethiopian health centres

To assess the diagnostic performance of urine lipoarabinomannan (LAM) detection for TB screening in HIV-positive adults in Ethiopia. Testing for LAM was performed using the Determine TB-LAM lateral flow assay on urine samples from participants in a prospective cohort with baseline bacteriological categorisation for active TB in sputum. Characteristics of TB patients with regard to LAM status were determined. Participants were followed for 6months to evaluate survival, retention in care and incident TB. Positive LAM results were found in 78/757 participants. Among 128 subjects with definite (confirmed by culture and/or Xpert MTB/RIF) TB, 33 were LAM-positive (25.8%); the respective figure for clinically diagnosed cases was 2/20 (10%). Five of the remaining 43 LAM-positive individuals had died during the 6-month follow-up period, whereas 38 remained in care without clinical signs of TB. The overall sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were 25.8%, 92.9%, 42.3% and 86.0%, respectively. Among TB patients, LAM positivity was associated with higher WHO clinical stage, lower body mass index (BMI), CD4 cell and haemoglobin levels, and with increased mortality. A combination algorithm of urine LAM testing and sputum smear microscopy detected 49 (38.2%) of definite TB cases; among those with CD4 count ≤100cells/mm3 , this proportion was 66.7%. The performance of urine LAM testing for TB detection was poor in this population. However, this was improved among subjects with CD4 count ≤100cells/mm3 . In combination with sputum microscopy urine, LAM could be considered for targeted TB screening in this subgroup.