Elsevier

Tuberculosis

Volume 96, January 2016, Pages 31-36
Tuberculosis

Diagnostics
Use of lateral flow assays to determine IP-10 and CCL4 levels in pleural effusions and whole blood for TB diagnosis

https://doi.org/10.1016/j.tube.2015.10.011Get rights and content

Summary

One of the key problems in combating TB is the lack of fast and accurate diagnostic tests that are affordable and easy to use in resource-limited settings. We have used a field-friendly up-converting phosphor (UCP) reporter technology in a lateral flow (LF) based test for the diagnosis of respiratory infections. In this study we analysed samples obtained from patients presenting with symptoms suggestive of TB but prior to confirmation by microbiology in The Gambia. Following clinical and microbiological evaluation they were classified as either having TB or other respiratory disorder (ORD). Analysis of blood was performed for those with pulmonary TB and pleural fluid for those with pleural TB. UCP-LF test for detection and quantitation of IP-10 and CCL4 were used being the two chemokine markers that have been shown to increase in active TB disease. UCP-LF test accurately determined concentrations of both markers as compared to ELISA and multiplex cytokine array. However, only IP-10 could discriminate between TB and ORD, and this was significantly enhanced by analysing the site of infection (pleural fluid), which showed 92% correct classification. Future work will assess the use of multiple markers to increase diagnostic accuracy.

Introduction

Tuberculosis (TB) is caused by infection with Mycobacterium tuberculosis (Mtb), which is transmitted via aerosolized droplets from index cases with active TB disease [1]. In 2013, 8.6 million people were diagnosed with TB and 1.5 million people died [2]. One of the major challenges in combating active TB is the lack of fast and accurate diagnostic tests. Current tests such as Acid Fast Bacilli (AFB) smear microscopy, Mtb culture and molecular based diagnostic tests (i.e. GeneXpert MTB-RIF) all have limitations, most notably the requirement for infrastructure, thus limiting roll-out to peripheral clinics, where the majority of patients are seen [3]. In addition, analysis of the pathogen in TB is challenging in paucibacillary patients including children [4]. Current blood-based tests utilising host-derived production of interferon gamma (IFN-γ) following stimulation with Mtb antigens (IFN-γ release assays (IGRA)) also require infrastructure and cannot differentiate active and latent forms of TB [5]. However, recent work has shown increased diagnostic accuracy when a combination of analytes other than IFN-γ [6], [7] or samples from the site of infection [8], [9] were analysed. Progression to the development of user-friendly rapid tests for TB, based on host-derived markers would be of major global public health importance.

Diagnostics employing the rapid lateral flow (LF) assay format require little infrastructure and are routinely used for home testing of pregnancy, monitoring of diabetes and screening for infectious diseases such as HIV and malaria [10], [11], [12]. Previous studies have described the application of sensitive fluorescent up-converting phosphor (UCP) reporter technology combined with the LF assay format to diagnose bacterial and viral infections [13]. User-friendly dry reagent formats for storage at ambient temperature combined with lightweight portable strip readers have enabled implementation as a field friendly device in different sites in Africa [14], [15], [16]. Similar types of UCP-LF assays have also been developed for the quantitative analysis of blood samples to determine levels of IFN-γ [17], IL-10 [18] and chemokine IP-10 [16], [17] and CCL4 [19].

While there are many analytes of interest in regards to TB diagnostics, two candidates that hold great promise are IP-10 and CCL4 (MIP-1β). IP-10 is a chemokine produced by monocytes and other immune cells. It is highly elevated in both pleural effusions and whole blood supernatants of TB patients and has been suggested as a potential biomarker for treatment monitoring and disease progression [20], [21]. Moreover, we have shown that levels in pleural effusions are not affected by the HIV status of the patient [8], [22]. CCL4 is a chemoattractant for natural killer cells, monocytes and other immune cells to the site of infection. It induces the synthesis and release of other pro-inflammatory cytokines such as IL-1, IL-6 and TNF-α from fibroblasts and macrophages [23], [24]. In addition a human CD8+ regulatory T cell subset has been identified that mediates suppression of Th1 cell immunity to mycobacteria through CCL4 [25]. CCL4 has previously been shown to discriminate between TB and ORD in saliva and serum [26] as well as between tuberculoid and borderline tuberculoid (TT/BT) leprosy and exposed healthy individuals [27].

The study described here is the first to apply UCP-LF assays using pleural fluid as a potential tool for the diagnosis of pleural TB. We analysed IP-10 and CCL4 levels in pleural effusions or Mtb antigen-stimulated whole blood supernatants from subjects with pleural or pulmonary TB respectively, and compared these to subjects with other respiratory disorders (ORDs). Our findings hold promise for future development of an IP-10 based rapid diagnostic test for TB in resource-poor settings.

Section snippets

Study participants and samples

Adult human immune-deficiency virus (HIV) uninfected participants were recruited following written informed consent. Participants presented at the MRC outpatients department with symptoms suggestive of TB (i.e. cough > 2 weeks plus one other system such as weight loss, night sweats) but prior to microbiological confirmation. Patients were classified as having TB or ORD based on bacteriological, clinical observations and chest X-ray (CXR) as described previously [9]. Patients with evidence of

Participant characteristics

Only HIV-seronegative subjects were included in this study. For WBA supernatant detection, 10 TB and 10 non-TB (ORD) subjects were analysed and for pleural fluid analysis, 19 TB and 11 non-TB (ORD) subjects were analysed (Table 1). There was no difference in the median age between the TB and non-TB subjects, but the TB group had a significantly higher proportion of males compared to the non-TB group (81% compared to 49%). The degree of Mtb sensitisation was similar for both groups of WBA

Discussion

This is the first study to show the clinical utility of UCP-LF assays for diagnosis of pleural TB. UCP-LF strips were used to detect IP-10 and CCL4 levels in Mtb antigen-stimulated blood or pleural fluid (from different subjects) and correlated with findings by ELISA and multiplex cytokine array. We found good correlation between LF and ELISA for both analytes but only found significant diagnostic accuracy for IP-10. In addition, analysis at the site of infection (i.e. pleural fluid) resulted

Acknowledgements

We thank all study participants and the TB clinic and ward at MRC, Fajara. We also thank Claudia Schacht and Julia Buech (EURICE GmbH, Saarbrücken, Germany) for project management.

References (31)

  • J.S. Sutherland et al.

    Highly accurate diagnosis of pleural tuberculosis by immunological analysis of the pleural effusion

    PLoS ONE

    (2012)
  • M.O.C. Ota et al.

    Rapid diagnosis of tuberculosis using ex vivo host biomarkers in sputum

    Eur Respir J

    (2014)
  • N.E. Rosenberg et al.

    Detection of acute HIV infection: a field evaluation of the determine® HIV-1/2 Ag/Ab combo test

    J Infect Dis

    (2012)
  • D. Eibach et al.

    Evaluation of the malaria rapid diagnostic test VIKIA malaria Ag Pf/Pan™ in endemic and non-endemic settings

    Malar J

    (2013)
  • P.L. Corstjens et al.

    Tools for diagnosis, monitoring and screening of Schistosoma infections utilizing lateral-flow based assays and upconverting phosphor labels

    Parasitology

    (2014)
  • Cited by (30)

    • Caloric Restriction Promotes Immunometabolic Reprogramming Leading to Protection from Tuberculosis

      2021, Cell Metabolism
      Citation Excerpt :

      The balance between activation and control of non-protective inflammation is critical for MTB outcomes. Our cellular, histological, and omics analyses identified a reduced amount of proinflammatory cytokines in CR conditions, suggesting a balanced immune response capable of fighting MTB infection without causing major collateral lung damage (Lyadova et al., 2010; Mishra et al., 2013; Petruccioli et al., 2016; Cafaro et al., 2016; Sutherland et al., 2016; Ribeiro-Rodrigues et al., 2002). The better control of MTB outgrowth and the protection phenotype induced by CR may also be dependent on higher induction of trained immunity (Joosten et al., 2018).

    • Illuminating Host-Mycobacterial Interactions with Genome-wide CRISPR Knockout and CRISPRi Screens

      2020, Cell Systems
      Citation Excerpt :

      M. bovis BCG infection induced the production of many cytokines and chemokines in THP-1 cells (Figures S6A and S6B). IFNAR1 and TYK2 gene knockdown in THP-1 cells abolished the production of infection-induced cytokines and chemokines, including IL-1Ra (interleukin-1 receptor antagonist), which inhibits the pro-inflammatory function of IL-1α and IL-1β by binding to their receptors, as well as CXCL10 (C-X-C motif chemokine ligand 10) and CCL4 (C-C motif chemokine ligand 4), important biomarkers for monitoring TB treatment and disease progression (Sutherland et al., 2016) (Figure S6C). Inhibiting these hits might represent potential HDTs for TB.

    • Fingerstick test quantifying humoral and cellular biomarkers indicative for M. leprae infection

      2019, Clinical Biochemistry
      Citation Excerpt :

      Although leprosy is one of the oldest recorded debilitating diseases it has remained a health problem in several countries, predominantly in poor-resourced regions, and imposes a significant social and financial burden on society in these economically underprivileged areas. New tools to break the ongoing transmission are required, in particular low-complexity diagnostic tests for detection of M. leprae infection and early diagnosis of leprosy could definitely be game-changers [24,50–52]. Characteristic for leprosy is its unique disease spectrum [41], in susceptible individuals, with on one hand paucibacillary disease (tuberculoid leprosy) accompanied with enhanced serum levels of pro-inflammatory cytokines and chemokines (IFN-γ, IP-10) and on the other hand multibacillary disease (lepromatous leprosy) with high M. leprae-specific antibody titres and increased levels of regulatory cytokines like IL-10 [41,53,54].

    • Measuring bovine γδ T cell function at the site of Mycobacterium bovis infection

      2017, Veterinary Immunology and Immunopathology
      Citation Excerpt :

      Our sequencing and co-culture system allowed us to identify several novel chemokines that were upregulated by γδ T cells responding to M. bovis antigen, including CCL4, CCL8 and CXCL10. CCL4 production in the context of TB has been previously described (Sutherland et al., 2016), however not in the bovine model and not by γδ T cells. We observed increased CCL4 expression in the co-cultures from both M. bovis-infected and naïve animals, suggesting a potential role for this chemokine throughout BTB disease progression.

    View all citing articles on Scopus
    1

    Current address: WHO Regional Office for Africa, Brazzaville, Congo.

    View full text