Abstract
Mortality from human immunodeficiency virus (HIV)–associated tuberculosis (TB) is high, particularly among hospitalized patients. In 433 people with HIV hospitalized with symptoms of TB, we investigated plasma matrix metalloproteinases (MMP) and matrix-derived biomarkers in relation to TB diagnosis, mortality, and Mycobacterium tuberculosis (Mtb) bloodstream infection (BSI). Compared to other diagnoses, MMP-8 was elevated in confirmed TB and in Mtb-BSI, positively correlating with extracellular matrix breakdown products. Baseline MMP-3, -7, -8, -10, and PIIINP were associated with Mtb-BSI and 12-week mortality. These findings implicate MMP dysregulation in pathophysiology of advanced HIV-TB and support MMP inhibition as a host-directed therapeutic strategy for HIV-TB.
Keywords: tuberculosis, HIV, mortality, matrix metalloproteinase, procollagen III N-terminal propeptide, matrix degradation product, biomarker
In HIV-associated TB, plasma matrix metalloproteinases (MMP), including MMP-8, and procollagen III N-terminal propeptide are associated with Mycobacterium tuberculosis bloodstream infection and 12-week mortality. This implicates MMP dysregulation in pathophysiology of advanced HIV-TB and supports MMP inhibition as a host-directed therapeutic strategy.
Tuberculosis (TB) is a leading infectious cause of death worldwide, resulting in an estimated 1.3 million deaths annually [1]. The End TB Strategy aims to reduce TB deaths by 95% between 2015 and 2035 [2]. In people with human immunodeficiency virus (HIV), TB is the leading cause of death [3]. To meet End TB targets, identification of and interventions for those at highest risk of poor outcomes are needed, particularly for use in low-resource settings where the burden of TB falls most heavily. Further understanding of the causes of mortality and pathophysiology of TB disease is required to prevent TB deaths in people with HIV.
We have previously reported that plasma matrix metalloproteinase (MMP)-1, MMP-8 (neutrophil collagenase), and procollagen III N-terminal propeptide (PIIINP), a matrix degradation product released during collagen turnover, are elevated in patients with active TB compared to patients without TB, highlighting collagen turnover as a feature of TB disease, in both HIV-positive and -negative cohorts [4, 5]. Plasma PIIINP was significantly higher in HIV-positive patients with newly diagnosed active TB compared to HIV-negative patients, positively correlated with HIV viral load, and was elevated during TB immune reconstitution inflammatory syndrome [4].
Here, we evaluated plasma MMP, PIIINP, and the extracellular matrix components collagen type IV alpha 1 chain (Col4α1) and hyaluronic acid (HA) in a cohort of patients hospitalized with advanced HIV and TB symptoms, thereby focusing on the population most requiring interventions to reduce mortality. We aimed to evaluate potential as diagnostic biomarkers and hypothesized that disseminated Mycobacterium tuberculosis (Mtb) in HIV-TB may drive systemic MMP upregulation and consequently tissue damage. We report a novel association of elevated plasma MMP with Mtb-bloodstream infection (BSI) and mortality in advanced HIV-TB, providing pathophysiological insights.
METHODS
The study was approved by the University of Cape Town Human Research Ethics Committee (REC, reference 057/2013) and London School of Hygiene and Tropical Medicine REC (reference 11710). Full methods have been reported elsewhere [6]; see also the Supplementary Methods. Eligible patients were adults with HIV infection and a CD4 count ≤350 cells/μL, admitted to Khayelitsha Hospital, Cape Town, with a probable new diagnosis of TB. Exclusion criteria were pregnancy, TB treatment within 1 month prior to admission or >3 doses of TB treatment prior to enrollment, or unknown HIV status (declined testing). Participants provided written informed consent. Participants were investigated by TB culture (sputum, blood), Xpert (sputum, urine), and urine lipoarabinomannan (LAM; Alere Determine TB LAM assay) prior to initiation of TB treatment. Vital status was determined at 12 weeks.
Participants were classified retrospectively as having microbiologically confirmed TB if Mtb was identified in clinical samples; clinical TB if TB was likely and treatment for TB was given following World Health Organization guidelines but no microbiological confirmation was obtained; no TB if TB was excluded on clinical and microbiological grounds; or LAM TB if criteria for no TB was met but urine LAM was positive ≥2 by 2 independent readers [6].
Inclusion required the availability of ethylenediaminetetraacetic acid plasma at enrollment. Plasma MMP were quantified by Luminex array (Bio-Rad Bio-Plex 200 system; R&D Systems, United Kingdom). PIIINP, Col4α1, and HA were quantified by enzyme-linked immunosorbent assays (Cloud Clone Corp, China). HA and Col4α1 measurement were limited to a subset of 73 randomly selected participants. Statistical analysis was performed in Prism 9 and R Studio (2023.03.1). Comparisons of analytes by TB diagnosis was between no TB and either confirmed TB or clinical TB and a Bonferroni correction was performed as 2 groups were compared for 1 analyte. Statistical significance was inferred by a P value <.05. Comparisons between 2 groups were by Mann-Whitney U test unless otherwise stated.
The relationship between MMP/PIIINP concentrations and Mtb-BSI or mortality was depicted using a Loess nonparametric smoother and assessed using a mixed-effects logistic regression model, including a random effect on intercept for plate, to account for technical variation between plates, as analytes were measured across multiple plates. Hierarchical clustering analysis was performed by Ward method based on Euclidean distance, to assess the association between analytes, including neutrophil count, neutrophil percentage, and procalcitonin, but excluding Col4α1 and HA, as they had only been measured in a subset of patients. This was on scaled data, excluding extreme outliers (observations with a value >4 median absolute deviations from the variable median applied after transformation). Pearson correlation to assess the association between analytes was performed on log-transformed values, excluding extreme outliers.
RESULTS
Plasma samples were available for 437 participants (Supplementary Table 1). TB diagnosis was confirmed in 313 (71.6%) and clinical TB in 48 (11.0%). Only 4 participants met the criteria for LAM TB, so these were excluded from subsequent analyses. In the no TB group (n = 72 [16.5%]), community-acquired pneumonia (n = 34 [47.2%]) was the most frequent diagnosis, followed by other blood pathogen (n = 8 [11.1%]), Pneumocystis jirovecii pneumonia (n = 6 [8.33%]), and cryptococcal disease (n = 6 [8.33%]). The median CD4 count was 56 cells/μL (interquartile range [IQR], 18.0–112 cells/μL) in confirmed TB, 93 (IQR, 53.8–182 cells/μL) in clinical TB, and 89.5 (IQR, 29.0–224 cells/μL) in no TB.
Vital status at 12 weeks was known for 427 of 433 (98.6%) participants. Death occurred in 82 of 433 (18.9%) participants at a median of 16.0 days (IQR, 2.75–44.3 days). Mortality at 12 weeks in confirmed TB was 19.8% (62/313). Mtb-BSI was present in 133 of 313 (42.5%) participants with confirmed TB and was associated with increased mortality: 37 of 133 (27.8%) participants with Mtb-BSI died compared to 23 of 173 (13.3%) without Mtb-BSI (P = .002 by Fisher exact test). Participant characteristics are reported by vital status and Mtb-BSI in Supplementary Table 2. More Mtb-BSI occurred in men than women, but there was no difference in mortality. Older age was associated with mortality, but not Mtb-BSI. Being antiretroviral therapy (ART) naive or having defaulted was associated with increased Mtb-BSI, compared to being ART treated. Mtb-BSI and mortality were associated with lower CD4 counts.
Plasma MMP-8 Is Elevated in Patients Hospitalized With HIV-TB
We first examined plasma MMP and matrix-derived biomarker concentrations in no TB in comparison to confirmed TB or clinical TB. Plasma MMP-8 was significantly elevated in confirmed TB compared to no TB (median, 23 712 pg/mL [IQR, 7688–47 571 pg/mL] vs 10 602 pg/mL [IQR, 2019–32 205 pg/mL]; P = .003; Figure 1A and Supplementary Table 3), as was plasma Col4α1 (Figure 1B). Plasma MMP-3 and -10 were lower in participants with confirmed TB compared to no TB and clinical TB, while MMP-1, -7, and -9, PIIINP, and HA did not differ between groups. These findings were similar in male and female participants (Supplementary Figure 1). MMP-8 did not differ significantly by sex (Supplementary Table 4 and Supplementary Figure 2). Analyte associations with age and CD4 count for confirmed TB are shown in Supplementary Figure 2.
Figure 1.
Elevated matrix metalloproteinase-8 in human immunodeficiency virus (HIV)-associated tuberculosis (TB), Mycobacterium tuberculosis bloodstream infection (Mtb-BSI), and TB mortality. Plasma matrix metalloproteinase (MMP)-8 (A) and Col4α1 (B) were elevated in hospitalized participants with HIV infection and microbiologically confirmed TB, compared to hospitalized HIV-positive participants with symptoms due to other diagnoses (no TB). Differences between confirmed TB and TB diagnosed clinically were not statistically significant. Plasma MMP-8 was increased in participants with confirmed TB and Mtb-BSI compared to those without (C) and in those who had died compared to those who had survived at 12 weeks (D). Col4α1 was measured in a subset of 73 participants. *P < .05, **P < .01, ***P < .001.
Plasma MMP-8 Is Associated With Mtb-BSI and Mortality in HIV-TB
MMP are primarily transcriptionally regulated; however, MMP-8 may be stored in neutrophil granules. Exploring the hypothesis that disseminated Mtb drives MMP-8 upregulation and release from neutrophils and that this is associated with poor outcomes in HIV-TB, we next examined MMP-8 in the presence or absence of Mtb-BSI and by vital status at 12 weeks. We found that in confirmed TB, those with Mtb-BSI had elevated plasma MMP-8 (Figure 1C) compared to those without (median, 40 003 pg/mL [IQR, 20 006–70 583 pg/mL] vs 11 451 pg/mL [IQR, 4697–30 789 pg/mL]; P < .001). Plasma MMP-8 was elevated in participants with confirmed TB who died compared to those who survived (median, 32 811 pg/mL [IQR, 12 060–66 934 pg/mL] vs 20 201 pg/mL [IQR, 6050–40 561 pg/mL]; P = .002; Figure 1D). Col4α1 and HA did not differ by vital status (data not shown), although Col4α1 was elevated in confirmed TB with Mtb-BSI compared to without Mtb-BSI (median, 11.2 ng/mL [IQR, 7.76–9.92 ng/mL] vs 8.75 ng/mL [IQR, 9.76–13.5 ng/mL]; P < .001). Plasma MMP-8 positively correlated with Col4α1 (r = 0.535, P < .001) and PIIINP (r = 0.404, P < .001).
Multiple MMP and PIIINP Is Associated With HIV-TB Severity
We next examined the association of other plasma MMP and PIIINP with Mtb-BSI and mortality in confirmed TB. We found a positive association between plasma MMP-3, -7, -8, and -10 and PIIINP for Mtb-BSI and mortality (Figure 2A and 2B). We performed hierarchical clustering analysis including neutrophil count, neutrophil percentage, and procalcitonin as biomarkers of acute inflammation (Supplementary Figure 2). MMP-8 most closely clustered with PIIINP and procalcitonin. Procalcitonin, but not neutrophil count, positively associated with Mtb-BSI and mortality (Figure 2C and 2D).
Figure 2.
Plasma matrix metalloproteinase (MMP) and procollagen III N-terminal propeptide (PIIINP) are associated with Mycobacterium tuberculosis bloodstream infection (Mtb-BSI) and mortality in human immunodeficiency virus–associated tuberculosis. Probability (dependent variable, y-axis) of Mtb-BSI (A and C) and mortality (B and D) by analyte log concentration (predictor variable, x-axis). Loess fit to data (colored lines with shaded 95% confidence intervals [CI]) demonstrates the functional relationship, with measured concentrations shown as a jittered scatterplot at 0 (survived/no Mtb-BSI) or 1 (died/Mtb-BSI) on the y-axis. Odds ratios (OR) and 95% CIs are from logistic regression models, with adjustment for random effects by plate. In A and B, log MMP or PIIINP concentrations are in pg/mL. In C and D, log neutrophil count is ×109/L and log procalcitonin concentration is in μg/L.
DISCUSSION
Patients with advanced HIV-1 hospitalized with symptoms of TB are at high risk of mortality. We found that baseline plasma MMP-8 and Col4α1 were increased in hospitalized patients with HIV infection who were confirmed to have TB compared to those who eventually received an alternative diagnosis. Contrary to our previous finding in outpatients, in this hospitalized cohort, plasma PIIINP was not elevated in patients with TB compared to symptomatic patients with alternative diagnoses.
Elevated plasma MMP-8, together with MMP-3, -7, and -10 and PIIINP, were associated with Mtb-BSI and mortality at 12 weeks, suggesting that MMP upregulation and matrix turnover are features of TB disease severity. This is in keeping with our recently reported finding that elevated plasma MMP-8 concentrations at the end of TB treatment are associated with persistent Mtb culture positivity [7]. MMP-8 was most closely associated with procalcitonin (an acute phase reactant) and the matrix degradation product PIIINP (released during type III collagen turnover), suggesting that MMP-8 activity, collagen turnover, and acute inflammation are closely related processes in HIV-TB [5, 8]. HA, a polysaccharide component of the extracellular matrix that is released during extracellular matrix turnover, has previously been found to predict AIDS events or death in people with HIV commencing ART [9]. Plasma HA was not associated with mortality in this study.
While we have demonstrated an association between plasma MMP and mortality in HIV-TB, our study does not prove a causal relationship. Elevated MMP may be a function of Mtb bacterial load or Mtb dissemination, which is itself associated with mortality. In vitro, virulent Mtb is able to induce neutrophil-derived MMP-8 secretion, via an NF-kB–dependent mechanism, so it is possible that bloodstream Mtb directly stimulates neutrophil MMP-8 release [10]. However, elevated MMP-8 may also occur via upregulation of proinflammatory cellular networks, and the finding that MMP-8 is associated more closely with procalcitonin than neutrophil count or percentage is consistent with networks upregulating MMP-8 [11]. Increased MMP-8 in TB was positively associated with PIIINP, a matrix degradation product released during type III collagen turnover, and Col4α1, a component of type IV collagen. MMP inhibition with doxycycline has been shown to be safe and effective for TB in HIV-negative patients [12]. Our results support the case for clinical trials of MMP inhibition with doxycycline as a host-directed therapy for HIV-TB.
Prior studies have examined MMP-8 in patients with TB, predominantly in those who are HIV negative [11]. Our finding of elevated plasma MMP-8 in participants with confirmed TB compared to those symptomatic with other illnesses is consistent with previous reports in outpatients, although this is the first report in people with HIV who are hospitalized [4, 5, 13, 14]. MMP-8 is emerging as a biomarker that may help identify patients with active TB in some settings, if used in combination with other screening tools. Here, we also report elevated plasma Col4α1 in HIV-TB. Elevated Col4α1 was also associated with Mtb-BSI. Col4α1 is a subunit of type IV collagen, a key component of basement membranes that has not previously been studied in human TB, to our knowledge.
A strength of this study is the combination of rigorous clinical and mycobacterial analyses that was employed to determine TB status. A limitation pertains to the use of a single blood culture to determine the presence or absence of Mtb-BSI. It is likely that sequential blood cultures would have a higher diagnostic accuracy for Mtb-BSI [15]. We have not adjusted for known predictors of mortality in our analysis, as causal pathways are not well understood.
In summary, in people with HIV hospitalized with a clinical syndrome compatible with TB, plasma MMP-8 was elevated in confirmed TB, likely due to Mtb-driven tissue damage, compared to other diagnoses. MMP-3, -7, -8, and -10 and PIIINP were associated with Mtb-BSI and mortality at 12 weeks, implicating MMP dysregulation in HIV-TB morbidity. MMP inhibition is a potential therapeutic target for people with HIV-TB, who urgently need improved treatment outcomes.
Supplementary Data
Supplementary materials are available at The Journal of Infectious Diseases online (http://jid.oxfordjournals.org/). Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.
Supplementary Material
Contributor Information
Naomi F Walker, Centre for Infectious Diseases Research in Africa, Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Observatory, South Africa; TB Centre and Department of Clinical Research, London School of Hygiene and Tropical Medicine, United Kingdom; Department of Clinical Sciences and Centre for Tuberculosis Research, Liverpool School of Tropical Medicine, United Kingdom.
Charlotte Schutz, Centre for Infectious Diseases Research in Africa, Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Observatory, South Africa; Department of Medicine, University of Cape Town, Observatory, South Africa.
Amy Ward, Department of Medicine, University of Cape Town, Observatory, South Africa.
David Barr, Centre for Infectious Diseases Research in Africa, Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Observatory, South Africa; Wellcome-Liverpool-Glasgow Centre for Global Health Research, University of Liverpool; Department of Infectious Diseases, Queen Elizabeth University Hospital, Glasgow.
Charles Opondo, Department of Medical Statistics, London School of Hygiene and Tropical Medicine.
Muki Shey, Centre for Infectious Diseases Research in Africa, Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Observatory, South Africa; Department of Medicine, University of Cape Town, Observatory, South Africa.
Paul T Elkington, National Institute for Health and Care Research Biomedical Research Centre, School of Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton.
Katalin A Wilkinson, Centre for Infectious Diseases Research in Africa, Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Observatory, South Africa; Department of Medicine, University of Cape Town, Observatory, South Africa; The Francis Crick Institute, London.
Robert J Wilkinson, Centre for Infectious Diseases Research in Africa, Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Observatory, South Africa; Department of Medicine, University of Cape Town, Observatory, South Africa; The Francis Crick Institute, London; Department of Infectious Diseases, Imperial College London, United Kingdom.
Graeme Meintjes, Centre for Infectious Diseases Research in Africa, Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Observatory, South Africa; Department of Medicine, University of Cape Town, Observatory, South Africa.
Notes
Acknowledgments. We thank the Western Cape Provincial Government and staff at Khayelitsha Hospital for their support of the study.
Author contributions. G. M., C. S., A. W., and D. B. conceived the clinical study and recruited the clinical cohort. N. F. W., G. M., P. T. E., and D. B. conceived the laboratory study. N. F. W., K. A. W., M. S., and D. B. conducted laboratory analyses. N. F. W., C. S., C. O., and D. B. performed data analysis. N. F. W. wrote the first draft of the manuscript. All authors contributed to the manuscript and approved the final submitted report.
Disclaimer. The funders had no role in the study design, data collection, data analysis, data interpretation, or writing of this report. The opinions, findings and conclusions expressed in this manuscript reflect those of the authors alone.
Financial support. This research was funded in whole, or in part, by Wellcome (098316, 214321/Z/18/Z, 203135/Z/16/Z). For the purpose of open access, the author has applied a CC-BY public copyright licence to any Author Accepted Manuscript version arising from this submission. N. F. W. was supported by a National Institute for Health and Care Research (NIHR) Academic Clinical Lectureship and funding from the Academy of Medical Sciences UK, Medical Research Council (MRC) UK, British Heart Foundation, Arthritis Research UK, Royal College of Physicians and Diabetes UK (Starter Grant for Clinical Lecturers), and the British Infection Association (Project Grant). M. S. is supported by Wellcome (211360/Z/18/Z) and the National Research Foundation of South Africa (NRF, #UID127558). P. T. E. was supported by MRC MR/P023754/1 and MR/W025728/ 1. R. J. W. is supported by the Francis Crick Institute, which is funded by Wellcome (CC2112), Cancer Research UK (CC2112), and UK Research and Innovation (CC2112). R. J. W. also receives support from Wellcome (203135), the European and Developing Countries Clinical Trials Partnership (SRIA 2015–1065), and the National Institutes of Health (grant number U01AI115940). R. J. W. is supported in part by the NIHR Biomedical Research Centre of Imperial College National Health Service Trust. C. S. was funded by the South African MRC under the National Health Scholars Programme. G. M. was supported by Wellcome (098316, 214321/Z/18/Z, and 203135/Z/16/Z) and the South African Research Chairs Initiative of the Department of Science and Technology and NRF (grant number 64787).
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