1. Immunology and Inflammation

Tryptophan metabolism determines outcome in tuberculous meningitis: a targeted metabolomic analysis

  1. Edwin Ardiansyah
  2. Julian Avila-Pacheco
  3. Le Thanh Hoang Nhat
  4. Sofiati Dian
  5. Dao Nguyen Vinh
  6. Hoang Thanh Hai
  7. Kevin Bullock
  8. Bachti Alisjahbana
  9. Mihai G Netea
  10. Riwanti Estiasari
  11. Trinh Thi Bich Tram
  12. Joseph Donovan
  13. Dorothee Heemskerk
  14. Tran Thi Hong Chau
  15. Nguyen Duc Bang
  16. Ahmad Rizal Ganiem
  17. Rovina Ruslami
  18. Valerie ACM Koeken
  19. Raph L Hamers
  20. Darma Imran
  21. Kartika Maharani
  22. Vinod Kumar
  23. Clary B Clish
  24. Reinout van Crevel
  25. Guy Thwaites
  26. Arjan van Laarhoven  Is a corresponding author
  27. Nguyen Thuy Thuong Thuong  Is a corresponding author
  1. Research Center for Care and Control of Infectious Diseases, Universitas Padjadjaran, Indonesia
  2. Department of Internal Medicine and Radboud Center of Infectious Diseases (RCI), Radboud University Medical Center, Netherlands
  3. The Broad Institute of MIT and Harvard, United States
  4. Oxford University Clinical Research Unit, Viet Nam
  5. Department of Neurology, Hasan Sadikin Hospital, Faculty of Medicine, Universitas Padjadjaran, Indonesia
  6. Department of Internal Medicine, Hasan Sadikin Hospital, Faculty of Medicine, Universitas Padjadjaran, Indonesia
  7. Department of Neurology, Cipto Mangunkusumo Hospital, Faculty of Medicine Universitas Indonesia, Indonesia
  8. Centre for Tropical Medicine and Global Health, Nuffield Department of Medicine, University of Oxford, United Kingdom
  9. London School of Hygiene and Tropical Medicine, United Kingdom
  10. Department of Medical Microbiology and Infection Prevention, Amsterdam University Medical Centre, Netherlands
  11. Hospital for Tropical Diseases, Viet Nam
  12. Pham Ngoc Thach Hospital for Tuberculosis and Lung Disease, Viet Nam
  13. Department of Biomedical Science, Faculty of Medicine, Universitas Padjadjaran, Indonesia
  14. Department of Computational Biology for Individualised Infection Medicine, Centre for Individualised Infection Medicine (CiiM) & TWINCORE, joint ventures between the Helmholtz-Centre for Infection Research (HZI) and the Hannover Medical School (MHH), Germany
  15. Oxford University Clinical Research Unit Indonesia, Faculty of Medicine Universitas Indonesia, Indonesia
https://doi.org/10.7554/eLife.85307
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  1. Edwin Ardiansyah
  2. Julian Avila-Pacheco
  3. Le Thanh Hoang Nhat
  4. Sofiati Dian
  5. Dao Nguyen Vinh
  6. Hoang Thanh Hai
  7. Kevin Bullock
  8. Bachti Alisjahbana
  9. Mihai G Netea
  10. Riwanti Estiasari
  11. Trinh Thi Bich Tram
  12. Joseph Donovan
  13. Dorothee Heemskerk
  14. Tran Thi Hong Chau
  15. Nguyen Duc Bang
  16. Ahmad Rizal Ganiem
  17. Rovina Ruslami
  18. Valerie ACM Koeken
  19. Raph L Hamers
  20. Darma Imran
  21. Kartika Maharani
  22. Vinod Kumar
  23. Clary B Clish
  24. Reinout van Crevel
  25. Guy Thwaites
  26. Arjan van Laarhoven
  27. Nguyen Thuy Thuong Thuong
(2023)
Tryptophan metabolism determines outcome in tuberculous meningitis: a targeted metabolomic analysis
eLife 12:e85307.
https://doi.org/10.7554/eLife.85307
  2. Download BibTeX
  3. Download .RIS
5 figures, 4 tables and 3 additional files

Figures

Figure 1
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Tryptophan metabolism pathway.

Tryptophan is metabolized mainly through the kynurenine pathway through indoleamine 2,3-dioxygenase 1 (IDO1), generating kynurenine and its downstream metabolites (blue box). IDO1 is partly stimulated by M. tuberculosis-induced interferon gamma (IFN-γ) production by T helper 1cells.

Figure 2 with 5 supplements
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Six-month survival curve of tuberculous meningitis (TBM) patients.

Patients were stratified by cerebrospinal fluid (CSF) tryptophan concentrations tertiles.

Figure 2—figure supplement 1
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Cerebrospinal fluid (CSF) parameters of tuberculous meningitis (TBM) patients in Indonesia and Vietnam.

Distributions of leukocytes, polymorphonuclear cells, mononuclear cells, protein, and the ratio of CSF/blood glucose in Indonesian (purple) and Vietnamese (yellow) patients are depicted by violin plots.

Figure 2—figure supplement 2
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Stability of metabolites over-time.

The concentrations of tryptophan metabolites (in log2 scale) were measured in cerebrospinal fluid (CSF) samples from Indonesian (purple) and Vietnamese (yellow) tuberculous meningitis (TBM) patients were recruited between 2007 and 2018.

Figure 2—figure supplement 3
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Six-month survival curve of tuberculous meningitis (TBM) patients stratified by HIV status.

Patients were stratified by tertiles based on cerebrospinal fluid (CSF) tryptophan concentrations (red = high tryptophan, gray = intermediate tryptophan, blue = low tryptophan).

Figure 2—figure supplement 4
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In-hospital mortality for 17 HIV-positive patients with cryptococcal meningitis.

Holm-adjusted Wilcoxon rank-sum test p-values are presented :*p<0.05, **p<0.01.

Figure 2—figure supplement 5
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Cerebrospinal fluid (CSF) tryptophan distributions according to mycobacterial load.

(A) Comparing CSF culture-negative versus -positive patients and (B) among patients with a positive CSF Xpert, in culture-positive and -negative tuberculous meningitis (TBM) patients, CSF tryptophan was associated with CSF Xpert Ct-values from a low (high Ct-value) to low (low Ct-value) load.

Figure 3 with 1 supplement
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Cerebrospinal fluid and plasma metabolites concentrations in tuberculous meningitis (TBM) and all controls for the tryptophan metabolites associated with outcome: tryptophan and kynurenine.

(A) Relative concentrations based on peak ion intensity and (B) absolute concentrations in μM. Boxplots are shown with Holm-adjusted Wilcoxon rank-sum test p-values are presented: (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). No measurements were excluded for these graphs. Note: plasma measurements were available for a subset of 300 TBM patients and all controls.

Figure 3—figure supplement 1
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Boxplots of cerebrospinal fluid (CSF) and plasma metabolites concentrations in tuberculous meningitis (TBM) and controls.

Relative concentrations based on peak ion intensities are shown. CSF and plasma concentrations are not directly comparable. Boxplots are shown with Holm-adjusted Wilcoxon rank-sum test p-values are presented: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Note: plasma measurements were available for a subset of 300 TBM patients and all controls.

Figure 4 with 1 supplement
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Correlation between tryptophan metabolites and with clinical and cerebrospinal fluid (CSF) parameters.

Significant Spearman’s correlation coefficients are presented in the correlation matrix, while the ones with not significant correlations were blank. Red indicates positive correlations, and blue indicates negative ones. The color gradient shows the strength of the associations.

Figure 4—figure supplement 1
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Associations between cerebrospinal fluid (CSF)/plasma metabolite ratios (y-axis) and CSF protein levels (as a proxy of CSF barrier leakage, x-axis).

Of note, 71 patients had undetectable plasma levels of 3-indolacetic acid and were removed from this graph.

Figure 5 with 1 supplement
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Associations of cerebrospinal fluid (CSF) tryptophan with IFN-γ (A) and with TNF-α (B) in 176 Indonesian (left) and 304 (Vietnamese) tuberculous meningitis (TBM) patients.

The boxplots on the left of each plot show the association of metabolites with cytokines below the detection limit. Abbreviations: IFN-γ: interferon gamma, TNF-α: tumor necrosis factor alpha, LOD: lower limit of detection.

Figure 5—figure supplement 1
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Correlation between cerebrospinal fluid (CSF) tryptophan metabolites and inflammatory markers measured with O-link in Indonesian (A) and Vietnamese (B) tuberculous meningitis patients.

Inflammatory markers were clustered based on their correlation coefficients using hierarchical clustering. Red indicates positive correlation, and blue indicated negative correlation.

Tables

Table 1
Patient baseline characteristics.
Tuberculous meningitisNon-infectious controlBacterial meningitis*Cryptococcal meningitis*
(n = 1069)(n = 54)(n = 50)(n = 60)
Clinical features
Age, years34 (27–44)35 (25–44)46 (34–57)33 (27–37)
Sex, % male700 (65.5%)30 (55.6%)12 (60.0%)26 (78.8%)
Glasgow Coma Scale14 (12–15)15 (12–15)13 (9–14)15 (13–15)
HIV, % positive284 (26.6%)11 (20.4%)0 (0%)60 (100%)
Tuberculous meningitis grade (%)
Grade I287 (27.3%)---
Grade II584 (55.6%)---
Grade III179 (17.0%)---
Cerebrospinal fluid features
Leukocytes, cells/μl150 (49–336)2 (1–3)1900 (739–5460)86 (24–192)
Neutrophils, cells/μl22 (3–99)1 (0–1)1527 (538–4986)17 (6–109)
Mononuclear cells, cells/μl98 (38–207)2 (1–3)307 (134–646)31 (6–89)
Protein, g/l1.46 (0.90–2.40)0.40 (0.26–0.59)1.90 (1.10–3.80)0.76 (0.58–1.60)
CSF to blood glucose ratio0.28 (0.17–0.40)0.60 (0.56–0.70)0.46 (0.17–1.00)0.50 (0.30–1.00)
M. tuberculosis culture or ZN staining or GeneXpert positive686 (64.17%)---
Outcomes
Outcome at day 60
Alive825 (77.2%)---
Deceased231 (21.6%)---
Lost to follow-up13 (1.2%)---
Outcome at day 180
Alive731 (68.4%)---
Deceased304 (28.4%)---
Lost to follow-up34 (3.2%)---

  1. Categorical variables are presented in N (%); continuous variables are presented in median (IQR). Abbreviation: CSF = cerebrospinal fluid.

  2. *

    Clinical metadata available for 40% of bacterial meningitis and 56% of cryptococcal meningitis patients.

Table 2
Univariate Cox regression for influence of cerebrospinal fluid (CSF) metabolites on 60-day mortality.
MetabolitesHR*95% CI*p-valueFDR†
Tryptophan1.161.10, 1.24<0.001<0.001
Kynurenine1.000.93, 1.07>0.9>0.9
Kynurenic acid1.000.93, 1.070.9>0.9
3-Hydroxyanthranilic acid1.010.97, 1.050.60.9
Xanthurenic acid0.950.90, 1.000.050.2
Quinolinic acid0.920.85, 1.000.0380.2
Niacinamide1.030.95, 1.110.50.8
3-Indoleacetic acid1.110.96, 1.290.20.4
N-Acetylserotonin1.010.94, 1.090.70.9
5-Methoxytryptophol1.110.93, 1.320.30.5

  1. Baseline cerebrospinal fluid (CSF) metabolites were measured in 1069 TBM patients. Cox regression models were stratified by cohort and adjusted by sex, age, and HIV status. Hazard ratio (HR) was calculated per twofold increase in metabolite concentration. Bold: False Discovery Rate (FDR) < 0.05.

  2. *

    HR = hazard ratio, CI = confidence interval.

  3. †

    FDR = false discovery rate; Benjamini and Hochberg correction for multiple testing.

Table 3
Univariate Cox regression for influencen of baseline cerebrospinal fluid (CSF) metabolites on early and late mortality.
MetaboliteEarly mortality (days 0–14)Late mortality (days 14–180)
HR*95% CI*p-valueFDR†HR*95% CI*p-valueFDR†
Tryptophan1.141.06, 1.23<0.0010.0051.171.08, 1.26<0.001<0.001
Kynurenine1.030.95, 1.130.40.610.91, 1.10>0.9>0.9
Kynurenic acid1.050.96, 1.140.30.50.950.86, 1.040.30.5
3-Hydroxyanthranilic acid1.020.97, 1.060.50.61.010.96, 1.060.60.7
Xanthurenic acid0.960.90, 1.030.20.50.960.89, 1.040.30.5
Quinolinic acid0.890.81, 0.980.020.10.90.81, 1.000.0520.2
Niacinamide1.020.92, 1.120.70.71.050.95, 1.160.30.5
3-Indoleacetic acid1.10.92, 1.320.30.51.180.97, 1.430.0930.2
N-Acetylserotonin1.050.96, 1.140.30.50.960.88, 1.060.40.5
5-Methoxytryptophol1.090.87, 1.350.50.61.291.04, 1.590.020.1

  1. Baseline cerebrospinal fluid (CSF) metabolites were measured in 1069 TBM patients. Cox regression models were stratified by sites and adjusted by age, sex, and GCS. Bold: False Discovery Rate (FDR) < 0.05.

  2. *

    HR = hazard ratio, CI = confidence interval.

  3. †

    Benjamini and Hochberg correction for multiple testing.

Table 4
Univariate Cox regression for influence of baseline plasma metabolites on 60-day mortality.
MetaboliteHR*95% CI*p-valueFDR†
Tryptophan0.80.56, 1.160.20.4
Kynurenine1.541.22, 1.93<0.0010.002
Kynurenic acid1.21.01, 1.430.0360.2
3-Hydroxyanthranilic acid1.131.00, 1.280.0450.2
Xanthurenic acid1.110.98, 1.250.10.3
Quinolinic acid0.990.88, 1.120.90.9
Niacinamide0.920.79, 1.070.30.4
3-Indoleacetic acid1.040.97, 1.120.30.4
N-Acetylserotonin1.050.91, 1.220.50.6
5-Methoxytryptophol1.040.85, 1.280.70.8

  1. Baseline plasma tryptophan metabolites were measured in a subset 300 patients. Cox regression models were stratified by sites and adjusted by age, sex, and GCS. Bold: False Discovery Rate (FDR) < 0.05.

  2. *

    HR = hazard ratio, CI = confidence interval.

  3. †

    Benjamini and Hochberg correction for multiple testing.

Additional files

MDAR checklist
https://cdn.elifesciences.org/articles/85307/elife-85307-mdarchecklist1-v2.docx
Source data 1

The source data includes clinical metadata and LC-MS data before pre-processing.

https://cdn.elifesciences.org/articles/85307/elife-85307-data1-v2.xlsx
Reporting standard 1
https://cdn.elifesciences.org/articles/85307/elife-85307-repstand1-v2.doc

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  1. Edwin Ardiansyah
  2. Julian Avila-Pacheco
  3. Le Thanh Hoang Nhat
  4. Sofiati Dian
  5. Dao Nguyen Vinh
  6. Hoang Thanh Hai
  7. Kevin Bullock
  8. Bachti Alisjahbana
  9. Mihai G Netea
  10. Riwanti Estiasari
  11. Trinh Thi Bich Tram
  12. Joseph Donovan
  13. Dorothee Heemskerk
  14. Tran Thi Hong Chau
  15. Nguyen Duc Bang
  16. Ahmad Rizal Ganiem
  17. Rovina Ruslami
  18. Valerie ACM Koeken
  19. Raph L Hamers
  20. Darma Imran
  21. Kartika Maharani
  22. Vinod Kumar
  23. Clary B Clish
  24. Reinout van Crevel
  25. Guy Thwaites
  26. Arjan van Laarhoven
  27. Nguyen Thuy Thuong Thuong
(2023)
Tryptophan metabolism determines outcome in tuberculous meningitis: a targeted metabolomic analysis
eLife 12:e85307.
https://doi.org/10.7554/eLife.85307