Akkerman, Onno W

Alffenaar, Jan-Willem C

Bolhuis, Mathieu S

Ghimire, Samiksha

Jongedijk, Erwin M

Touw, Daan J

van den Elsen, Simone H J

van der Werf, Tjip S

Wessels, Mireille

University of Groningen

   University of GroningenDose optimisation of first-line tuberculosis drugs using therapeutic drug monitoring in salivavan den Elsen, Simone H J; Akkerman, Onno W; Wessels, Mireille; Jongedijk, Erwin M;Ghimire, Samiksha; van der Werf, Tjip S; Bolhuis, Mathieu S; Touw, Daan J; Alffenaar, Jan-Willem CPublished in:European Respiratory JournalDOI:10.1183/13993003.00803-2020IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionFinal author's version (accepted by publisher, after peer review)Publication date:2020Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):van den Elsen, S. H. J., Akkerman, O. W., Wessels, M., Jongedijk, E. M., Ghimire, S., van der Werf, T. S.,Bolhuis, M. S., Touw, D. J., & Alffenaar, J-W. C. (2020). Dose optimisation of first-line tuberculosis drugsusing therapeutic drug monitoring in saliva: feasible for rifampicin, not for isoniazid. European RespiratoryJournal, 56(4), [2000803]. https://doi.org/10.1183/13993003.00803-2020CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.      Early View    Research letter    Dose optimisation of first-line tuberculosis drugs using therapeutic drug monitoring in saliva: feasible for rifampicin, not for isoniazid   Simone H.J. van den Elsen, Onno W. Akkerman, Mireille Wessels, Erwin M. Jongedijk, Samiksha Ghimire, Tjip S. van der Werf, Mathieu S. Bolhuis, Daan J. Touw, Jan-Willem C. Alffenaar    Please cite this article as: van den Elsen SHJ, Akkerman OW, Wessels M, et al. Dose optimisation of first-line tuberculosis drugs using therapeutic drug monitoring in saliva: feasible for rifampicin, not for isoniazid. Eur Respir J 2020; in press (https://doi.org/10.1183/13993003.00803-2020).       This manuscript has recently been accepted for publication in the European Respiratory Journal. It is published here in its accepted form prior to copyediting and typesetting by our production team. After these production processes are complete and the authors have approved the resulting proofs, the article will move to the latest issue of the ERJ online.    Copyright ©ERS 2020  Dose optimisation of first-line tuberculosis drugs using therapeutic drug monitoring in saliva: feasible for rifampicin, not for isoniazid. Simone HJ van den Elsena,b, Onno W Akkermanc,d, Mireille Wesselsa, Erwin M Jongedijka, Samiksha Ghimirea, Tjip S van der Werfd,e, Mathieu S Bolhuisa, Daan J Touwa, and Jan-Willem C Alffenaara,f,g,h#. a University of Groningen, University Medical Center Groningen, Department of Clinical Pharmacy and Pharmacology, Groningen, The Netherlands. b Hospital Group Twente, Department of Clinical Pharmacy, Almelo and Hengelo, the Netherlands c University of Groningen, University Medical Center Groningen, Tuberculosis Center Beatrixoord, Haren, The Netherlands. d University of Groningen, University Medical Center Groningen, Department of Pulmonary Diseases and Tuberculosis, Groningen, The Netherlands. e University of Groningen, University Medical Center Groningen, Department of Internal Medicine, Groningen, The Netherlands. f Sydney Pharmacy School, Faculty of Medicine and Health, The University of Sydney, Sydney, Australia. g Westmead hospital, Sydney, Australia. h Marie Bashir Institute of Infectious Diseases and Biosecurity, Sydney, Australia. # corresponding author: J.W.C. Alffenaar, University of Sydney, Faculty of Medicine and Health, School of Pharmacy, Pharmacy Building A15, NSW 2006, Australia. Email: johannes.alffenaar@sydney.edu.au Take home message: Therapeutic drug monitoring using saliva samples is feasible for rifampicin despite low penetration, but is not feasible for isoniazid which showed inexplicable highly variable saliva-serum concentration ratios.   To the editor: The persisting worldwide burden of tuberculosis (TB) is worrisome. In 2018, an estimated 10 million individuals developed TB and 1.45 million deceased.[1] The increase in drug resistance is an important point of concern. Resistance can be acquired by inappropriate drug management, non-compliance, and insufficient drug exposure.[2, 3] The last is frequently described for the first-line TB drugs rifampicin and isoniazid  due to large inter-individual pharmacokinetic variability.[3] Therapeutic drug monitoring (TDM) can be used to verify drug exposure and adjust individual drug dosages if needed.[4] The efficacy of rifampicin and isoniazid is associated with the ratio of the steady-state area under the concentration-time curve from 0-24 h to minimal inhibitory concentration (AUC0-24/MIC) with a target value of >271 for rifampicin and >567 for isoniazid.[5, 6] Traditional TDM uses plasma or serum samples, whereas other matrices like dried blood spot and saliva have been recommended as alternatives suitable for programmatic use.[4, 7] Collecting saliva samples is non-invasive and simple with the perspective of home-based self-sampling.[8] Salivary concentrations of rifampicin and isoniazid have been studied before, but highly variable saliva-serum concentration ratios across studies were observed.[8] Moreover, none of these studies assessed the feasibility of TDM using saliva samples. Therefore, the aim of this prospective study was to evaluate the feasibility of saliva instead of serum samples for TDM of rifampicin and isoniazid in patients with TB. Adult patients with TB admitted at the Tuberculosis Center Beatrixoord in Haren, the Netherlands who were treated with rifampicin and/or isoniazid and had routine TDM for rifampicin or isoniazid were eligible for inclusion. All patients provided informed consent. This study was approved by the ethical review board of the University Medical Center Groningen (IRB 2016/069) and registered at Clinicaltrials.gov (NCT03080012). All samples were taken after >14 days of treatment (steady state) and stored at -80 °C pending analysis. Saliva and serum samples were collected simultaneously according to the routine TDM schedule which usually included samples drawn before, and 0.5, 1, 2, 3, 4, and 6 hours after drug intake. Two different methods of saliva collection were used. The Salivette (Sarstedt, Nümbrecht, Germany) was utilized for sputum culture negative patients. Membrane filtration was applied to the samples of sputum culture positive patients to minimize infection hazard.[9, 10] The recovery of both sampling methods was determined for rifampicin and isoniazid at concentrations of 1 and 7 mg/L as described.[11] Rifampicin recovery at 1 mg/L was 64% (coefficient of variation [CV] 9%) using the Salivette and 67% (5%) using membrane filtration, while at 7 mg/L recovery was 102% (2%) and 99% (8%), respectively. For isoniazid, recovery (CV) at 1 mg/L was 77% (8%) using the Salivette and 68% (4%) using membrane filtration, whereas at 7 mg/L recovery was 91% (1%) and 88% (3%). After analysis, the salivary drug concentrations were corrected for the recovery of the applied sampling method. The pH of each saliva sample was determined by two independent researchers using pH indicator strips (range 4.0-7.0 and 2.0-9.0, Merck KGaA, Darmstadt, Germany). Saliva and serum samples were analysed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods.[12, 13] The method for rifampicin was recently updated and validated using the more suitable internal standard [2H8]-rifampicin. Cross-validation in saliva was successfully performed for both drugs. Bias and precision of spiked pooled saliva met the pre-set criteria of <20% for lower limit of quantification (LLOQ; rifampicin 0.1 mg/L, isoniazid 0.2 mg/L) as well as <15% for low (rifampicin 0.5 mg/L, isoniazid 0.4 mg/L), medium (rifampicin 5.0 mg/L, isoniazid 4.0 mg/L), and high (rifampicin 8.0 mg/L, isoniazid 6.4 mg/L) concentrations. Saliva-serum ratios were calculated using the paired drug concentrations for each time point as well as the non-compartmental AUC0-24 (MWPharm version 3.82, Mediware, Groningen, The Netherlands) in both matrices. The saliva-serum concentration ratios were evaluated using Passing Bablok regression and Bland-Altman plots (Analyze-it 4.81; Analyze-it Software Ltd., Leeds, United Kingdom). Cmax was defined as highest observed drug concentration and Tmax as time of Cmax. Intra-individual variation was assessed as CV (%) of the saliva-serum ratios within one pharmacokinetic curve, while inter-individual variation was calculated as CV of the mean saliva-serum ratios of all curves. Characteristics of the study population, pharmacokinetic parameters (Cmax, Tmax, AUC0-24) in both matrices, and saliva-serum ratios are shown in Table 1.  Penetration of rifampicin into saliva was low and slightly delayed. This resulted in undetectable salivary concentrations when collected before drug intake, 0.5 h, or 1 h after drug intake. Saliva and serum concentrations (>1 h after drug administration) correlated well with a regression line of saliva concentration=0.074+0.112*serum concentration (95% confidence interval [CI] of intercept -0.0311 to 0.161; 95% CI slope 0.087-0.138, r=0.803). Bland-Altman analysis led to a mean (95% CI) saliva-serum concentration ratio of 0.13 (0.12-0.14) with SD of 0.04. The AUC0-24 saliva-serum ratio was slightly higher, but comparable (Table 1). An AUC0-24 conversion factor was calculated as serum-saliva AUC0-24 ratio and resulted in a median (IQR) of  6.5 (6.2-7.9). Inter- and intra-individual variation were both approximately 20%. Isoniazid saliva-serum ratios were much higher than for rifampicin and can be explained by the difference in protein binding (10% versus 90%). Passing-Bablok regression resulted in a regression line of saliva concentration=-0.055+0.812*serum concentration (95% CI intercept -0.556 to 0.460; 95% CI slope 0.185-1.244, r=0.889). The Bland-Altman analysis showed a mean (95% CI) saliva-serum concentration ratio of 0.80 (0.65-0.95) with SD of 0.46. Intra-individual variation was 22.3%, while inter-individual variation was relatively large (48.3%) which could suggest that isoniazid penetration into saliva is influenced by other factors. Salivary pH was not related to the saliva-serum ratio of isoniazid and rifampicin. A limitation of this study is the lack of data on salivary flow and protein binding. Both could introduce variation in the saliva-serum ratios.[8] However, we aimed to evaluate the feasibility of salivary TDM and consider it unfeasible if protein binding and salivary flow have to be determined in each patient. Moreover, no influence of salivary pH on saliva-serum ratios was detected, whereas salivary pH is related to salivary flow.[8] Despite this limitation, we propose that rifampicin AUC0-24 in serum can be satisfactorily estimated using the AUC0-24 in saliva applying a conversion factor of 6.5 and used for AUC0-24 guided dose optimization in TB patients. The sampling burden can be reduced by collecting samples only at 2, 3, 4, and 6 hours after drug intake, since the other salivary rifampicin concentrations  (0, 0.5, 1 h) were undetectable . Simple HPLC-UV methods [14] are available in TB endemic areas, but usually not LC-MS/MS. Additional testing is recommended to determine if these analytical techniques are also able to assess low rifampicin concentrations in saliva. The results of isoniazid are less encouraging. Based on the findings in this study, we would not recommend TDM of isoniazid in saliva. The major cause of the large variation of isoniazid saliva-serum ratios remains unclear, as is the case with moxifloxacin [10]. A future study could focus on the identification of acetylator phenotype using saliva samples. Unfortunately, our sample size was too small to distinguish three groups with different drug clearance rates and we did not perform NAT2 genotyping. In general, we conclude that TDM for isoniazid using saliva samples will not be an equivalent alternative to traditional TDM as already shown for moxifloxacin [10] and amikacin [15], but it can be useful in home screening of rifampicin drug exposure in patients with TB as has been established for linezolid [10] and levofloxacin [11].    Table 1. Patient characteristics, non-compartmental pharmacokinetic (PK) parameters (Cmax, Tmax, AUC0-24) in serum and saliva, salivary pH, as well as saliva-serum ratios. Presented as median (interquartile range), unless stated otherwise.  Rifampicin (n=11) Isoniazid (n=8) Study population Male [n(%)] 9 (82%) 6 (75%) Age (years) 34 (25-54) 54 (49-58) Bodyweight (kg) 69 (58-71) 68 (57-72) Creatinine concentration (µmol/L) 62 (51-72) 65 (49-75) Dose (mg/kg) 10.2 (8.5-12.3) 5.4 (4.2-6.5) Serum PK Cmax (mg/L) 8.70 (5.99-12.12) 3.50 (1.65-4.75) Tmax (h) 2 (2-3) 2 (1-2) AUC0-24 (mg*h/L) 38.01 (34.44-76.50) 17.83 (7.80-20.74)  Saliva PK Cmax (mg/L) 1.21 (1.08-1.35) 1.57 (0.93-2.75) Tmax (h) 3 (2-4) 1 (1-2) AUC0-24 (mg*h/L) 5.88 (5.08-7.94) 7.62 (7.28-11.73) Salivary pH 6.1 (5.5-7.0) 6.1 (5.8-6.8) Saliva-serum ratio Paired concentration ratio Inter-individual variation (%CV) Intra-individual variation [mean (range) of %CV] 0.126 (0.109-0.154) 21.5% 17.2% (7.4%-24.0%) 0.763 (0.413-1.158) 48.3% 22.3% (9.2%-36.5%)  AUC0-24 ratio 0.154 (0.127-0.162) 0.824 (0.492-1.200)    References: 1.  World Health Organization. Global tuberculosis report 2019. 2019. 2.  Pasipanodya JG, McIlleron H, Burger A, Wash PA, Smith P, Gumbo T. Serum drug concentrations predictive of pulmonary tuberculosis outcomes. J. Infect. Dis. 2013; 208: 1464–1473. 3.  Srivastava S, Pasipanodya JG, Meek C, Leff R, Gumbo T. Multidrug-resistant tuberculosis not due to noncompliance but to between-patient pharmacokinetic variability. J. Infect. Dis. 2011; 204: 1951–1959. 4.  Alffenaar J-WC, Gumbo T, Dooley KE, Peloquin CA, McIlleron H, Zagorski A, Cirillo DM, Heysell SK, Silva DR, Migliori GB. Integrating pharmacokinetics and pharmacodynamics in operational research to End TB. Clin. Infect. Dis. 2019; ciz942. 5.  Jayaram R, Gaonkar S, Kaur P, Suresh BL, Mahesh BN, Jayashree R, Nandi V, Bharat S, Shandil RK, Kantharaj E, Balasubramanian V. Pharmacokinetics-pharmacodynamics of rifampin in an aerosol infection model of tuberculosis. Antimicrob. Agents Chemother. 2003; 47: 2118–2124. 6.  Gumbo T, Louie A, Liu W, Brown D, Ambrose PG, Bhavnani SM, Drusano GL. Isoniazid bactericidal activity and resistance emergence: integrating  pharmacodynamics and pharmacogenomics to predict efficacy in different ethnic populations. Antimicrob. Agents Chemother. 2007; 51: 2329–2336. 7.  Ghimire S, Bolhuis MS, Sturkenboom MGG, Akkerman OW, de Lange WCM, van der Werf TS, Alffenaar J-WC. Incorporating therapeutic drug monitoring into the World Health Organization hierarchy of tuberculosis diagnostics. Eur. Respir. J. 2016; 47: 1867–1869. 8.  van den Elsen SHJ, Oostenbrink LM, Heysell SK, Hira D, Touw DJ, Akkerman OW, Bolhuis MS, Alffenaar J-WC. Systematic Review of Salivary Versus Blood Concentrations of Antituberculosis Drugs and Their Potential for Salivary Therapeutic Drug Monitoring. Ther. Drug Monit. 2018; 40: 17–37. 9.  van den Elsen SHJ, van der Laan T, Akkerman OW, van der Zanden AGM, Alffenaar J-WC, van Soolingen D. Membrane Filtration Is Suitable for Reliable Elimination of Mycobacterium tuberculosis from Saliva for Therapeutic Drug Monitoring. J. Clin. Microbiol. 2017. p. 3292–3293. 10.  van den Elsen SHJ, Akkerman OW, Jongedijk EM, Wessels M, Ghimire S, van der Werf TS, Touw DJ, Bolhuis MS, Alffenaar J-WC. Therapeutic drug monitoring using saliva as matrix: an opportunity for linezolid, but challenge for moxifloxacin. Eur Respir J. 2020; 1901903. 11.  Ghimire S, Maharjan B, Jongedijk EM, Kosterink JGW, Ghimire GR, Touw DJ, van der Werf TS, Shrestha B, Alffenaar J-WC. Evaluation of Saliva as a Potential Alternative Sampling Matrix for Therapeutic Drug Monitoring of Levofloxacin in Patients with Multidrug-Resistant Tuberculosis. Antimicrob. Agents Chemother. 2019; 63: e02379-18. 12.  Sturkenboom MGG, van der Lijke H, Jongedijk EM, Kok WT, Greijdanus B, Uges DRA, Alffenaar J-WC. Quantification of isoniazid, pyrazinamide and ethambutol in serum using liquid chromatography-tandem mass spectrometry. J. Appl. Bioanal. 2015; 1: 89–98. 13.  de Velde F, Alffenaar JWC, Wessels AMA, Greijdanus B, Uges DRA. Simultaneous determination of clarithromycin, rifampicin and their main metabolites in human plasma by liquid chromatography-tandem mass spectrometry. J. Chromatogr. B 2009; 877: 1771–1777. 14.  Goutal S, Auvity S, Legrand T, Hauquier F, Cisternino S, Chapy H, Saba W, Tournier N. Validation of a simple HPLC-UV method for rifampicin determination in plasma: Application to the study of rifampicin arteriovenous concentration gradient. J. Pharm. Biomed. Anal. 2016; 123: 173–178. 15.  van den Elsen SHJ, Akkerman OW, Huisman JR, Touw DJ, van der Werf TS, Bolhuis MS, Alffenaar J-WC. Lack of penetration of amikacin into saliva of tuberculosis patients. Eur. Respir. J. 2018; 51: 1702024.   

English

Dose optimisation of first-line tuberculosis drugs using therapeutic drug monitoring in saliva:feasible for rifampicin, not for isoniazid

Proceedings - University of Groningen

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Dose optimisation of first-line tuberculosis drugs using therapeutic drug monitoring in saliva: feasible for rifampicin, not for isoniazid

ARTS repository - University of Groningen

   University of GroningenDose optimisation of first-line tuberculosis drugs using therapeutic drug monitoring in salivavan den Elsen, Simone H J; Akkerman, Onno W; Wessels, Mireille; Jongedijk, Erwin M;Ghimire, Samiksha; van der Werf, Tjip S; Bolhuis, Mathieu S; Touw, Daan J; Alffenaar, Jan-Willem CPublished in:European Respiratory JournalDOI:10.1183/13993003.00803-2020IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionFinal author's version (accepted by publisher, after peer review)Publication date:2020Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):van den Elsen, S. H. J., Akkerman, O. W., Wessels, M., Jongedijk, E. M., Ghimire, S., van der Werf, T. S.,Bolhuis, M. S., Touw, D. J., & Alffenaar, J-W. C. (2020). Dose optimisation of first-line tuberculosis drugsusing therapeutic drug monitoring in saliva: feasible for rifampicin, not for isoniazid. European RespiratoryJournal, 56(4), Article 2000803. Advance online publication. https://doi.org/10.1183/13993003.00803-2020CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.      Early View    Research letter    Dose optimisation of first-line tuberculosis drugs using therapeutic drug monitoring in saliva: feasible for rifampicin, not for isoniazid   Simone H.J. van den Elsen, Onno W. Akkerman, Mireille Wessels, Erwin M. Jongedijk, Samiksha Ghimire, Tjip S. van der Werf, Mathieu S. Bolhuis, Daan J. Touw, Jan-Willem C. Alffenaar    Please cite this article as: van den Elsen SHJ, Akkerman OW, Wessels M, et al. Dose optimisation of first-line tuberculosis drugs using therapeutic drug monitoring in saliva: feasible for rifampicin, not for isoniazid. Eur Respir J 2020; in press (https://doi.org/10.1183/13993003.00803-2020).       This manuscript has recently been accepted for publication in the European Respiratory Journal. It is published here in its accepted form prior to copyediting and typesetting by our production team. After these production processes are complete and the authors have approved the resulting proofs, the article will move to the latest issue of the ERJ online.    Copyright ©ERS 2020  Dose optimisation of first-line tuberculosis drugs using therapeutic drug monitoring in saliva: feasible for rifampicin, not for isoniazid. Simone HJ van den Elsena,b, Onno W Akkermanc,d, Mireille Wesselsa, Erwin M Jongedijka, Samiksha Ghimirea, Tjip S van der Werfd,e, Mathieu S Bolhuisa, Daan J Touwa, and Jan-Willem C Alffenaara,f,g,h#. a University of Groningen, University Medical Center Groningen, Department of Clinical Pharmacy and Pharmacology, Groningen, The Netherlands. b Hospital Group Twente, Department of Clinical Pharmacy, Almelo and Hengelo, the Netherlands c University of Groningen, University Medical Center Groningen, Tuberculosis Center Beatrixoord, Haren, The Netherlands. d University of Groningen, University Medical Center Groningen, Department of Pulmonary Diseases and Tuberculosis, Groningen, The Netherlands. e University of Groningen, University Medical Center Groningen, Department of Internal Medicine, Groningen, The Netherlands. f Sydney Pharmacy School, Faculty of Medicine and Health, The University of Sydney, Sydney, Australia. g Westmead hospital, Sydney, Australia. h Marie Bashir Institute of Infectious Diseases and Biosecurity, Sydney, Australia. # corresponding author: J.W.C. Alffenaar, University of Sydney, Faculty of Medicine and Health, School of Pharmacy, Pharmacy Building A15, NSW 2006, Australia. Email: johannes.alffenaar@sydney.edu.au Take home message: Therapeutic drug monitoring using saliva samples is feasible for rifampicin despite low penetration, but is not feasible for isoniazid which showed inexplicable highly variable saliva-serum concentration ratios.   To the editor: The persisting worldwide burden of tuberculosis (TB) is worrisome. In 2018, an estimated 10 million individuals developed TB and 1.45 million deceased.[1] The increase in drug resistance is an important point of concern. Resistance can be acquired by inappropriate drug management, non-compliance, and insufficient drug exposure.[2, 3] The last is frequently described for the first-line TB drugs rifampicin and isoniazid  due to large inter-individual pharmacokinetic variability.[3] Therapeutic drug monitoring (TDM) can be used to verify drug exposure and adjust individual drug dosages if needed.[4] The efficacy of rifampicin and isoniazid is associated with the ratio of the steady-state area under the concentration-time curve from 0-24 h to minimal inhibitory concentration (AUC0-24/MIC) with a target value of >271 for rifampicin and >567 for isoniazid.[5, 6] Traditional TDM uses plasma or serum samples, whereas other matrices like dried blood spot and saliva have been recommended as alternatives suitable for programmatic use.[4, 7] Collecting saliva samples is non-invasive and simple with the perspective of home-based self-sampling.[8] Salivary concentrations of rifampicin and isoniazid have been studied before, but highly variable saliva-serum concentration ratios across studies were observed.[8] Moreover, none of these studies assessed the feasibility of TDM using saliva samples. Therefore, the aim of this prospective study was to evaluate the feasibility of saliva instead of serum samples for TDM of rifampicin and isoniazid in patients with TB. Adult patients with TB admitted at the Tuberculosis Center Beatrixoord in Haren, the Netherlands who were treated with rifampicin and/or isoniazid and had routine TDM for rifampicin or isoniazid were eligible for inclusion. All patients provided informed consent. This study was approved by the ethical review board of the University Medical Center Groningen (IRB 2016/069) and registered at Clinicaltrials.gov (NCT03080012). All samples were taken after >14 days of treatment (steady state) and stored at -80 °C pending analysis. Saliva and serum samples were collected simultaneously according to the routine TDM schedule which usually included samples drawn before, and 0.5, 1, 2, 3, 4, and 6 hours after drug intake. Two different methods of saliva collection were used. The Salivette (Sarstedt, Nümbrecht, Germany) was utilized for sputum culture negative patients. Membrane filtration was applied to the samples of sputum culture positive patients to minimize infection hazard.[9, 10] The recovery of both sampling methods was determined for rifampicin and isoniazid at concentrations of 1 and 7 mg/L as described.[11] Rifampicin recovery at 1 mg/L was 64% (coefficient of variation [CV] 9%) using the Salivette and 67% (5%) using membrane filtration, while at 7 mg/L recovery was 102% (2%) and 99% (8%), respectively. For isoniazid, recovery (CV) at 1 mg/L was 77% (8%) using the Salivette and 68% (4%) using membrane filtration, whereas at 7 mg/L recovery was 91% (1%) and 88% (3%). After analysis, the salivary drug concentrations were corrected for the recovery of the applied sampling method. The pH of each saliva sample was determined by two independent researchers using pH indicator strips (range 4.0-7.0 and 2.0-9.0, Merck KGaA, Darmstadt, Germany). Saliva and serum samples were analysed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods.[12, 13] The method for rifampicin was recently updated and validated using the more suitable internal standard [2H8]-rifampicin. Cross-validation in saliva was successfully performed for both drugs. Bias and precision of spiked pooled saliva met the pre-set criteria of <20% for lower limit of quantification (LLOQ; rifampicin 0.1 mg/L, isoniazid 0.2 mg/L) as well as <15% for low (rifampicin 0.5 mg/L, isoniazid 0.4 mg/L), medium (rifampicin 5.0 mg/L, isoniazid 4.0 mg/L), and high (rifampicin 8.0 mg/L, isoniazid 6.4 mg/L) concentrations. Saliva-serum ratios were calculated using the paired drug concentrations for each time point as well as the non-compartmental AUC0-24 (MWPharm version 3.82, Mediware, Groningen, The Netherlands) in both matrices. The saliva-serum concentration ratios were evaluated using Passing Bablok regression and Bland-Altman plots (Analyze-it 4.81; Analyze-it Software Ltd., Leeds, United Kingdom). Cmax was defined as highest observed drug concentration and Tmax as time of Cmax. Intra-individual variation was assessed as CV (%) of the saliva-serum ratios within one pharmacokinetic curve, while inter-individual variation was calculated as CV of the mean saliva-serum ratios of all curves. Characteristics of the study population, pharmacokinetic parameters (Cmax, Tmax, AUC0-24) in both matrices, and saliva-serum ratios are shown in Table 1.  Penetration of rifampicin into saliva was low and slightly delayed. This resulted in undetectable salivary concentrations when collected before drug intake, 0.5 h, or 1 h after drug intake. Saliva and serum concentrations (>1 h after drug administration) correlated well with a regression line of saliva concentration=0.074+0.112*serum concentration (95% confidence interval [CI] of intercept -0.0311 to 0.161; 95% CI slope 0.087-0.138, r=0.803). Bland-Altman analysis led to a mean (95% CI) saliva-serum concentration ratio of 0.13 (0.12-0.14) with SD of 0.04. The AUC0-24 saliva-serum ratio was slightly higher, but comparable (Table 1). An AUC0-24 conversion factor was calculated as serum-saliva AUC0-24 ratio and resulted in a median (IQR) of  6.5 (6.2-7.9). Inter- and intra-individual variation were both approximately 20%. Isoniazid saliva-serum ratios were much higher than for rifampicin and can be explained by the difference in protein binding (10% versus 90%). Passing-Bablok regression resulted in a regression line of saliva concentration=-0.055+0.812*serum concentration (95% CI intercept -0.556 to 0.460; 95% CI slope 0.185-1.244, r=0.889). The Bland-Altman analysis showed a mean (95% CI) saliva-serum concentration ratio of 0.80 (0.65-0.95) with SD of 0.46. Intra-individual variation was 22.3%, while inter-individual variation was relatively large (48.3%) which could suggest that isoniazid penetration into saliva is influenced by other factors. Salivary pH was not related to the saliva-serum ratio of isoniazid and rifampicin. A limitation of this study is the lack of data on salivary flow and protein binding. Both could introduce variation in the saliva-serum ratios.[8] However, we aimed to evaluate the feasibility of salivary TDM and consider it unfeasible if protein binding and salivary flow have to be determined in each patient. Moreover, no influence of salivary pH on saliva-serum ratios was detected, whereas salivary pH is related to salivary flow.[8] Despite this limitation, we propose that rifampicin AUC0-24 in serum can be satisfactorily estimated using the AUC0-24 in saliva applying a conversion factor of 6.5 and used for AUC0-24 guided dose optimization in TB patients. The sampling burden can be reduced by collecting samples only at 2, 3, 4, and 6 hours after drug intake, since the other salivary rifampicin concentrations  (0, 0.5, 1 h) were undetectable . Simple HPLC-UV methods [14] are available in TB endemic areas, but usually not LC-MS/MS. Additional testing is recommended to determine if these analytical techniques are also able to assess low rifampicin concentrations in saliva. The results of isoniazid are less encouraging. Based on the findings in this study, we would not recommend TDM of isoniazid in saliva. The major cause of the large variation of isoniazid saliva-serum ratios remains unclear, as is the case with moxifloxacin [10]. A future study could focus on the identification of acetylator phenotype using saliva samples. Unfortunately, our sample size was too small to distinguish three groups with different drug clearance rates and we did not perform NAT2 genotyping. In general, we conclude that TDM for isoniazid using saliva samples will not be an equivalent alternative to traditional TDM as already shown for moxifloxacin [10] and amikacin [15], but it can be useful in home screening of rifampicin drug exposure in patients with TB as has been established for linezolid [10] and levofloxacin [11].    Table 1. Patient characteristics, non-compartmental pharmacokinetic (PK) parameters (Cmax, Tmax, AUC0-24) in serum and saliva, salivary pH, as well as saliva-serum ratios. Presented as median (interquartile range), unless stated otherwise.  Rifampicin (n=11) Isoniazid (n=8) Study population Male [n(%)] 9 (82%) 6 (75%) Age (years) 34 (25-54) 54 (49-58) Bodyweight (kg) 69 (58-71) 68 (57-72) Creatinine concentration (µmol/L) 62 (51-72) 65 (49-75) Dose (mg/kg) 10.2 (8.5-12.3) 5.4 (4.2-6.5) Serum PK Cmax (mg/L) 8.70 (5.99-12.12) 3.50 (1.65-4.75) Tmax (h) 2 (2-3) 2 (1-2) AUC0-24 (mg*h/L) 38.01 (34.44-76.50) 17.83 (7.80-20.74)  Saliva PK Cmax (mg/L) 1.21 (1.08-1.35) 1.57 (0.93-2.75) Tmax (h) 3 (2-4) 1 (1-2) AUC0-24 (mg*h/L) 5.88 (5.08-7.94) 7.62 (7.28-11.73) Salivary pH 6.1 (5.5-7.0) 6.1 (5.8-6.8) Saliva-serum ratio Paired concentration ratio Inter-individual variation (%CV) Intra-individual variation [mean (range) of %CV] 0.126 (0.109-0.154) 21.5% 17.2% (7.4%-24.0%) 0.763 (0.413-1.158) 48.3% 22.3% (9.2%-36.5%)  AUC0-24 ratio 0.154 (0.127-0.162) 0.824 (0.492-1.200)    References: 1.  World Health Organization. Global tuberculosis report 2019. 2019. 2.  Pasipanodya JG, McIlleron H, Burger A, Wash PA, Smith P, Gumbo T. Serum drug concentrations predictive of pulmonary tuberculosis outcomes. J. Infect. Dis. 2013; 208: 1464–1473. 3.  Srivastava S, Pasipanodya JG, Meek C, Leff R, Gumbo T. Multidrug-resistant tuberculosis not due to noncompliance but to between-patient pharmacokinetic variability. J. Infect. Dis. 2011; 204: 1951–1959. 4.  Alffenaar J-WC, Gumbo T, Dooley KE, Peloquin CA, McIlleron H, Zagorski A, Cirillo DM, Heysell SK, Silva DR, Migliori GB. Integrating pharmacokinetics and pharmacodynamics in operational research to End TB. Clin. Infect. Dis. 2019; ciz942. 5.  Jayaram R, Gaonkar S, Kaur P, Suresh BL, Mahesh BN, Jayashree R, Nandi V, Bharat S, Shandil RK, Kantharaj E, Balasubramanian V. Pharmacokinetics-pharmacodynamics of rifampin in an aerosol infection model of tuberculosis. Antimicrob. Agents Chemother. 2003; 47: 2118–2124. 6.  Gumbo T, Louie A, Liu W, Brown D, Ambrose PG, Bhavnani SM, Drusano GL. Isoniazid bactericidal activity and resistance emergence: integrating  pharmacodynamics and pharmacogenomics to predict efficacy in different ethnic populations. Antimicrob. Agents Chemother. 2007; 51: 2329–2336. 7.  Ghimire S, Bolhuis MS, Sturkenboom MGG, Akkerman OW, de Lange WCM, van der Werf TS, Alffenaar J-WC. Incorporating therapeutic drug monitoring into the World Health Organization hierarchy of tuberculosis diagnostics. Eur. Respir. J. 2016; 47: 1867–1869. 8.  van den Elsen SHJ, Oostenbrink LM, Heysell SK, Hira D, Touw DJ, Akkerman OW, Bolhuis MS, Alffenaar J-WC. Systematic Review of Salivary Versus Blood Concentrations of Antituberculosis Drugs and Their Potential for Salivary Therapeutic Drug Monitoring. Ther. Drug Monit. 2018; 40: 17–37. 9.  van den Elsen SHJ, van der Laan T, Akkerman OW, van der Zanden AGM, Alffenaar J-WC, van Soolingen D. Membrane Filtration Is Suitable for Reliable Elimination of Mycobacterium tuberculosis from Saliva for Therapeutic Drug Monitoring. J. Clin. Microbiol. 2017. p. 3292–3293. 10.  van den Elsen SHJ, Akkerman OW, Jongedijk EM, Wessels M, Ghimire S, van der Werf TS, Touw DJ, Bolhuis MS, Alffenaar J-WC. Therapeutic drug monitoring using saliva as matrix: an opportunity for linezolid, but challenge for moxifloxacin. Eur Respir J. 2020; 1901903. 11.  Ghimire S, Maharjan B, Jongedijk EM, Kosterink JGW, Ghimire GR, Touw DJ, van der Werf TS, Shrestha B, Alffenaar J-WC. Evaluation of Saliva as a Potential Alternative Sampling Matrix for Therapeutic Drug Monitoring of Levofloxacin in Patients with Multidrug-Resistant Tuberculosis. Antimicrob. Agents Chemother. 2019; 63: e02379-18. 12.  Sturkenboom MGG, van der Lijke H, Jongedijk EM, Kok WT, Greijdanus B, Uges DRA, Alffenaar J-WC. Quantification of isoniazid, pyrazinamide and ethambutol in serum using liquid chromatography-tandem mass spectrometry. J. Appl. Bioanal. 2015; 1: 89–98. 13.  de Velde F, Alffenaar JWC, Wessels AMA, Greijdanus B, Uges DRA. Simultaneous determination of clarithromycin, rifampicin and their main metabolites in human plasma by liquid chromatography-tandem mass spectrometry. J. Chromatogr. B 2009; 877: 1771–1777. 14.  Goutal S, Auvity S, Legrand T, Hauquier F, Cisternino S, Chapy H, Saba W, Tournier N. Validation of a simple HPLC-UV method for rifampicin determination in plasma: Application to the study of rifampicin arteriovenous concentration gradient. J. Pharm. Biomed. Anal. 2016; 123: 173–178. 15.  van den Elsen SHJ, Akkerman OW, Huisman JR, Touw DJ, van der Werf TS, Bolhuis MS, Alffenaar J-WC. Lack of penetration of amikacin into saliva of tuberculosis patients. Eur. Respir. J. 2018; 51: 1702024.   

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Dose optimisation of first-line tuberculosis drugs using therapeutic drug monitoring in saliva:feasible for rifampicin, not for isoniazid

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