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

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Abstract

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 (OD ₄₅₀) 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.

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Limit of blank, limit of detection and limit of quantitation.

David Armbruster, Terry Pry (2008)

* Limit of Blank (LoB), Limit of Detection (LoD), and Limit of Quantitation (LoQ) are terms used to describe the smallest concentration of a measurand that can be reliably measured by an analytical procedure. * LoB is the highest apparent analyte concentration expected to be found when replicates of a blank sample containing no analyte are tested. LoB = mean(blank) + 1.645(SD(blank)). * LoD is the lowest analyte concentration likely to be reliably distinguished from the LoB and at which detection is feasible. LoD is determined by utilising both the measured LoB and test replicates of a sample known to contain a low concentration of analyte. * LoD = LoB + 1.645(SD (low concentration sample)). * LoQ is the lowest concentration at which the analyte can not only be reliably detected but at which some predefined goals for bias and imprecision are met. The LoQ may be equivalent to the LoD or it could be at a much higher concentration.

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Diagnostic accuracy of a low-cost, urine antigen, point-of-care screening assay for HIV-associated pulmonary tuberculosis before antiretroviral therapy: a descriptive study

Stephen D Lawn, Andrew D Kerkhoff, Monica Vogt … (2012)

Summary Background The diagnostic accuracy of sputum smear microscopy and routine chest radiology for HIV-associated tuberculosis is poor, and culture-based diagnosis is slow, expensive, and is unavailable in most resource-limited settings. We assessed the diagnostic accuracy of a urine antigen test Determine TB-LAM Ag (Determine TB-LAM; Alere, Waltham, MA, USA) for screening for HIV-associated pulmonary tuberculosis before antiretroviral therapy (ART). Methods In this descriptive study, consecutive adults referred to a community-based ART clinic in Gugulethu township, South Africa, were all screened for tuberculosis by obtaining sputum samples for fluorescence microscopy, automated liquid culture (gold-standard test), and Xpert MTB/RIF assays (Cepheid, Sunnyvale, CA, USA) and urine samples for the Clearview TB-ELISA (TB-ELISA; Alere, Waltham, MA, USA) and Determine TB-LAM test. Patients with Mycobacterium tuberculosis cultured from one or more sputum samples were defined as cases of tuberculosis. The diagnostic accuracy of Determine TB-LAM used alone or combined with sputum smear microscopy was compared with that of sputum culture and the Xpert MTB/RIF assay for all patients and subgroups of patients stratified by CD4 cell count. Findings Patients were recruited between March 12, 2010, and April 20, 2011. Of 602 patients enrolled, 542 were able to provide one or more sputum samples, and 94 had culture-positive tuberculosis (prevalence 17·4%, 95% CI 14·2–20·8). Complete results from all tests were available for 516 patients (median CD4 count, 169·5 cells per μL; IQR 100–233), including 85 culture-positive tuberculosis, 24 of whom (28·2%, 95% CI 19·0–39·0) had sputum smear-positive disease. Determine TB-LAM test strips provided results within 30 min. Agreement was very high between two independent readers of the test strips (κ=0·97) and between the test strips and TB-ELISA (κ=0·84). Determine TB-LAM had highest sensitivity at low CD4 cell counts: 66·7% (95% CI 41·0–86·7) at <50 cells per μL, 51·7% (32·5–70·6) at <100 cells per μL, and 39·0% (26·5–52·6) at <200 cells per μL; specificity was greater than 98% for all strata. When combined with smear microscopy (either test positive), sensitivity was 72·2% (95% CI 46·5–90·3) at CD4 counts less than 50 cells per μL, 65·5% (45·7–82·1) at less than 100 cells per μL, and 52·5% (39·1–65·7) at less than 200 cells per μL, which did not differ statistically from the sensitivities obtained by testing a single sputum sample with the Xpert MTB/RIF assay. Interpretation Determine TB-LAM is a simple, low-cost, alternative to existing diagnostic assays for tuberculosis screening in HIV-infected patients with very low CD4 cell counts and provides important incremental yield when combined with sputum smear microscopy. Funding Wellcome Trust.

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Tuberculosis transmission by patients with smear-negative pulmonary tuberculosis in a large cohort in the Netherlands.

Alma Tostmann, Sandra Kik, Nico A. Kalisvaart … (2008)

Sputum smear microscopy is commonly used for diagnosing tuberculosis (TB). Although patients with sputum smear-negative TB are less infectious than patients with smear-positive TB, they also contribute to TB transmission. The objective of this study was to determine the proportion of TB transmission events caused by patients with smear-negative pulmonary TB in The Netherlands. All patients in The Netherlands with culture-confirmed TB during the period 1996-2004 were included in this study. Patients with identical DNA fingerprints in Mycobacterium tuberculosis isolates from sputum samples were clustered. The first patients in a cluster were considered to be the index patients; all other patients were considered to have secondary cases. In addition, we examined transmission from sources by conventional contact tracing. We analyzed 394 clusters with a total of 1285 patients. On the basis of molecular linkage only, 12.6% of the secondary cases were attributable to transmission from a patient with smear-negative TB. The relative transmission rate among patients with smear-negative TB, compared with patients with smear-positive TB, was 0.24 (95% confidence interval, 0.20-0.30). Secondary cases in clusters with an index patient with smear-negative TB more frequently had smear-negative status (odds ratio, 1.86; 95% confidence interval, 1.18-2.93), compared with secondary cases in clusters with an index patient with smear-positive TB. Conventional contact tracing revealed that 26 (6.2%) of the 417 sources, as identified by the Municipal Health Services, had smear-negative TB. In The Netherlands, patients with smear-negative, culture-positive TB are responsible for 13% of TB transmission. Countries that have ample resources should expand their TB-control efforts to include prevention of transmission from patients with smear-negative, culture-positive pulmonary TB.

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Author and article information

Contributors

Delphi Chatterjee: delphi.chatterjee@colostate.edu

Journal

Journal ID (nlm-ta): Sci Rep

Journal ID (iso-abbrev): Sci Rep

Title: Scientific Reports

Publisher: Nature Publishing Group UK (London )

ISSN (Electronic): 2045-2322

Publication date (Electronic): 3 February 2021

Publication date PMC-release: 3 February 2021

Publication date Collection: 2021

Volume: 11

Electronic Location Identifier: 2922

Affiliations

[1 ]GRID grid.47894.36, ISNI 0000 0004 1936 8083, Mycobacteria Research Laboratory, Department of Microbiology, Immunology and Pathology, , Colorado State University, ; Fort Collins, CO 80523 USA

[2 ]Socios en Salud Sucursal Peru, Lima, 15001 Peru

[3 ]GRID grid.8536.8, ISNI 0000 0001 2294 473X, Programa Academico de Tuberculose, Faculdade de Medicina, , Universidade Federal do Rio de Janeiro, ; Rio de Janeiro, 21941-590 Brazil

[4 ]GRID grid.38142.3c, ISNI 000000041936754X, Department of Global Health and Social Medicine, , Harvard Medical School, ; Boston, MA 02115 USA

Article

Publisher ID: 82445

DOI: 10.1038/s41598-021-82445-4

PMC ID: 7859189

PubMed ID: 33536495

SO-VID: 75788c6f-9703-4a01-9e20-f25469dc6236

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Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

History

Date received : 17 September 2020

Date accepted : 20 January 2021

Funding

Funded by: FundRef http://dx.doi.org/10.13039/100000002, National Institutes of Health;

Award ID: RO1 AI132680

Funded by: NIH/NIAID

Award ID: U19 AI109755

Award Recipient : Molly F. Franke

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ScienceOpen disciplines: Uncategorized

Keywords: microbiology,biomarkers,medical research,immunological techniques,mass spectrometry,diseases,infectious diseases,tuberculosis

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ScienceOpen disciplines: Uncategorized

Keywords: microbiology, biomarkers, medical research, immunological techniques, mass spectrometry, diseases, infectious diseases, tuberculosis

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

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Point-of-Care Testing for Infectious Diseases Super Collection

Most cited references 42

Limit of blank, limit of detection and limit of quantitation.

Diagnostic accuracy of a low-cost, urine antigen, point-of-care screening assay for HIV-associated pulmonary tuberculosis before antiretroviral therapy: a descriptive study

Tuberculosis transmission by patients with smear-negative pulmonary tuberculosis in a large cohort in the Netherlands.

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