Dotting the I's and crossing the T's: A South Australian perspective on variability in troponin thresholds for myocarditis risk in clozapine treatment

a University of Adelaide, Discipline of Psychiatry, Adelaide, South Australia, Australia b Basil Hetzel Institute, Woodville, South Australia, Australia c Central Adelaide Local Health Network, Adelaide, South Australia, Australia d Office of the Chief Psychiatrist, Adelaide, South Australia, Australia e Centre for Medicine Use and Safety, Faculty of Pharmacy and Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia f SA Pharmacy, Southern Adelaide Local Health Network, Bedford Park, South Australia, Australia g Department of Chemistry, School of Physical Sciences, University of Adelaide, Adelaide, South Australia, Australia h SA Heart, Adelaide, South Australia, Australia i SA Pathology, Adelaide, South Australia, Australia

Clozapine is the most effective treatment for schizophrenia resistant to other antipsychotic medications but is a third line due to a range of serious side effects (Clark et al., 2014).Internationally, Australia reports the highest rates of clozapine induced myocarditis (CIM), up to 8 % in the first months of treatment (Dawson et al., 2018;Nachmani Major et al., 2020).High rates in Australia have been attributed to greater clinical awareness and screening programs, or alternatively to more rapid titration (Dawson et al., 2018;de Leon et al., 2022).In contrast, rates of CIM in countries such as Denmark may be very low (0.03 %), in the context of slower outpatient titration (de Leon et al., 2023;Rohde et al., 2018).Early identification and intervention for CIM is vital.Data from the World Health Organization's pharmacovigilance database (Vigibase), suggest 58.1 % of cases are serious, including 5 % being fatal (De Las Cuevas et al., 2022).However, there is also high variability in severity of CIM reported internationally, ranging from 96.6 % severe in the United States (17.3 % mortality) to 34 % (2.9 % mortality) in Australia.Given that clozapine is usually ceased as part of the management of CIM with associated risk of relapse and chronic symptom burden, there is a need to balance the sensitivity of monitoring and to work with Cardiology to better understand the absolute risk associated with intermediate changes in markers of cardiac pathology, particularly in Australia where monitoring is more wide-spread.
Risk factors for CIM include age, ethnicity, obesity, dose titration rate, concurrent inflammation and sodium valproate treatment, all of which may interact to determine clozapine plasma levels (de Leon et al., 2022).Clozapine itself causes an initial inflammatory response that inhibits its own metabolism via Cytochrome P450 enzyme CYP 1A2 and hence requires slow titration (Clark et al., 2018;de Leon et al., 2022).One exploratory study has identified genetic associations between CIM and SNPs from HLA regions associated with risk of inflammation, as well as the GNA15 gene associated with heart failure, however results need further replication (Lacaze et al., 2020).
CIM typically presents with mild tachycardia followed by a flu-like illness around days 10-19 of treatment.C-reactive protein (CRP) can be elevated, and tachycardia may worsen.Over the next 1-5 days plasma CRP and troponin levels rise associated with electrocardiogram (ECG) changes and reduction of left ejection fraction.If clozapine is ceased at this time, cardiac function can return to normal over the next 5 days (Ronaldson et al., 2011).ECG changes including PR segment depression in precordial and limb leads and PR segment depression in leads with ST segment elevation may be present but have low specificity, approximated at 47 % (Butta et al., 2020).Echocardiography during CIM usually shows at least mild ventricular dilatation and mild global ventricular dysfunction (predominantly left ventricle).However, some cases show normal heart function.There are reports of associated pericardial effusion, apical hypokinesis and thrombus formation (Bellissima et al., 2018).Cardiac magnetic resonance imaging (CMR) can confirm these findings.Other investigations such as B-type natriuretic peptide (BNP), elevated in heart failure, and creatine kinase (CKmB) elevated with myocardial damage, can be used to refine risk assessment (Bellissima et al., 2018).
Troponin proteins, central to normal cardiac function, are released into blood plasma with cardiac ischaemia, stress and in the context of CIM.Cardiac contractility or excitation-contraction coupling is driven by the cyclic depolarisation of cardiac muscle, subsequent increase in intracellular calcium levels and resultant changes in the cross-bridging of thin filaments (actin) and thick filaments (myosin) (Park et al., 2017).Actin filaments are formed from a helix of actin strands with tropomyosin protein located at regular intervals within the helix groove.Actin/myosin bridging is regulated by the three-protein troponin complex: troponin C (calcium-binding), I (inhibitory) and T (tropomyosin binding).At a molecular level contraction is triggered by calcium binding to troponin C, which then releases inhibition by Troponin I enabling actin-myosin cross-bridging and contraction.Troponin T both anchors the troponin complex and modulates calcium sensitivity (Rasmussen and Jin, 2021).Current troponin tests are highly sensitive assays of the striated muscle specific troponin I or T and although both occur in cardiac and skeletal muscles, they are encoded by different genes yielding specific isoforms.
Monitoring for CIM in Australia is recommended by manufacturers and is relatively widespread (eCPMS, 2019).These expert consensusbased protocols appear effective for the identification of myocarditis, although subsequent management shows substantial variability and prospective studies are required for full validation (Nachmani Major et al., 2020).Australian protocols support weekly testing for the first month of treatment and any time if a fever of 38 • C or more is present.These protocols use thresholds first established by Ronaldson et al., CRP > 100 mg/L and high sensitivity (hs) troponin of twice normal level, to indicate risk of myocarditis and trigger clozapine cessation (Ronaldson et al., 2011).On average CRP is elevated 5 days prior to troponin, providing an early indication of evolving risk (Mcneil et al., 2013).For troponin values between 1 and 2 times normal, clozapine can continue but daily repeat testing is required.In a recent large UK-based retrospective cohort study, the accuracy of these thresholds was confirmed (Area Under the Receiver Operating Curve for troponin = 0.975; CRP = 0.896) (Segev et al., 2021).CRP was more specific (96.7 % versus 91.4 %), while troponin was more sensitive (83.3 % versus 33.3 %) and showed superior positive predictive value (50 % versus 29.9 %).Combined tachycardia, raised CRP and troponin show 100 % sensitivity.Given the range of differential causes for CIM-like presentations including acute coronary syndrome or respiratory infection and associated viral myocarditis, accurate diagnosis is complex (Goodison et al., 2015).Up to 80 % of cases where risk is identified may be false positive and can result in inappropriate cessation of clozapine and relapse (Segev et al., 2021).Given substantially higher rates of severe CIM have been reported for the UK in Vigibase (34 vs 87.5 %) (De Las Cuevas et al., 2022), Australian physicians may have a lower threshold for CIM diagnosis and these accuracy metrics need further local prospective study.
The South Australian (SA) Public Mental Health Service manages over 1000 clozapine treated patients.We implemented weekly CRP and troponin screening for CIM over the first month of treatment in 2011 (Clark et al., 2014;Health, 2022).A state-wide monitoring system has identified potential CIM cases with elevated CRP and troponin in almost 8 % of patients within first few months of commencement (Nachmani Major et al., 2020).The assessment and treatment processes for patients with troponin values 1 to 2 times normal are variable, suggesting need for a more standardised approach.In some cases clozapine is ceased early, while in others monitoring is stopped prematurely after an early negative echocardiogram (Nachmani Major et al., 2020).Consistent with these findings, one large international study reported inappropriate cessation as high as 18.3 % in patients where myocarditis was ruled out (Segev et al., 2021).
We report here an interesting case of clozapine initiation complicated by elevated troponin T but normal troponin I in a 22-year-old nonsmoking male of Sri Lankan origins, with treatment resistant schizophrenia.The management of this case led to a review of variation in hs troponin laboratory protocols and reference ranges across SA and subsequent adjustment of local guidelines.In this paper, we also consider the value of personalised titration to prevent such adverse events.

A complex case of intermediate troponin elevation during clozapine titration
Over the year prior to clozapine initiation the patient experienced auditory hallucinations, ideas of reference, paranoid delusions and occasional aggressive behaviour.Initially, he was treated with aripiprazole to 30 mg and then paliperidone long-acting injection to 150 mg monthly, both ceased due to inefficacy and akasthisia.Clozapine was commenced during inpatient admission according to manufacturer protocol (for detail see Supplementary Table 1).Dose was titrated to 300 mg nocte, tolerated well other than tachycardia to 130 bpm and mild constipation managed with coloxyl and senna.The patient was also treated with rosuvstatin 10 mg and colecalciferol 1000 Units daily for hyperlipidaemia and vitamin D deficiency respectively.Troponin T (SA Pathology (SAP), threshold 17 ng/L for males) and CRP testing was within normal range during inpatient stay.Clozapine plasma level was 518 μg/L at discharge (day 31) when blood testing moved to a private pathology lab using troponin I (threshold 54 ng/L, Australian Clinical Labs (ACL)).Psychotic symptoms improved leaving some residual intermittent referential ideas and auditory hallucinations, most prominent in music.During the initial weeks of outpatient follow up atenolol was titrated to 100 mg under cardiology guidance for persistent tachycardia.There were no abnormal findings on repeat ECG or transthoracic echocardiography and heart rate was stabilised at 100 bpm.Given persistent, at times distressing positive symptoms the patient requested a higher clozapine dose, which was slowly increased over 3 months in 25 mg increments to 450 mg (peak clozapine level 885 μg/L, day 134) with limited benefit.Amisulpride augmentation was trialled from day 134.Given high clozapine levels and tachycardia, troponin I (ACL) and CRP levels were monitored monthly.On day 160 the patient changed laboratories back to SAP and Troponin T testing returned an intermediate elevated result of 24 ng/L (threshold 17 ng/L) with normal CRP.Daily, repeat troponin T testing (SAP) according to protocol over the next week confirmed intermediate elevation above 20 ng/L.Surprisingly on day 172, troponin I (ACL) was normal (<3) in the context of ongoing elevated troponin T (SAP).Elevated troponin levels (SAP) persisted following the cessation of amisulpride, addition of Ivabradine for better rate control (HR 80 bpm) and reduction of clozapine in increments to 175 mg (day 228, level 216 μg/L).Over the next 3 months troponin slowly decreased and finally dropped below threshold on day 331.Over the following 12 months, monthly troponin T (SA pathology) gradually dropped to negligible levels of 4-6 ng/L (SAP).Repeat ECG and Echocardiography remained normal.Maintenance of clozapine treatment during this period was critical for the exceptional functional improvement of the patient, who moved from part-time to full time work followed by enrolment in a university degree.Progress was supported by case-management and cognitive therapy for resistant positive symptoms, which were no worse at lower clozapine dose and became less prominent over time.Low mood and anxiety associated with positive symptoms also improved with the addition of sertraline titrated to 50 mg.

Variability in troponin sensitivity and thresholds between commercially available tests
In light of the apparent differences in local thresholding for troponin tests and increasing anecdotal reports of equivocal results between 1 and 2 times normal, we contacted Clinpath, Australian Clinical Labs and SA Pathology, the main local pathology providers to survey this variation.We identified wide analytical variability in the type and sensitivity of troponin immunoassays used across SA pathology providers.One provider used only troponin I, 1 only troponin T and the third used both types of assay with 2 versions of troponin I depending on laboratory.Thresholds for troponin t-tests range from 13 to 14 ng/L in females and 14-17 in males.While those for troponin I range from 16 to 48 ng/L in females and 26-72 in males (See Supplementary Table 2).Commercially available tests are all of high sensitivity and are gold standard for the assessment of myocardial damage.Over time the sensitivity of these tests at the lower end of the range has increased substantially.Troponin T assays are known to be more sensitive and less specific than Troponin I with increased likelihood of false positive results (Body and Carlton, 2018).A single patient may use one or all of these providers across their treatment course and as such there is no absolute standard range for 1 to 2 times normal values.Variability in troponin assay thresholding is likely an international dilemma given the range of immunoassays commercially available (Chaulin, 2022).Consequently, in discussion with local laboratories we have adjusted the generic 1 to 2 times normal threshold in our local SA clozapine cardiac guidelines (Health, 2022) to fixed values for consistency across all CIM monitoring (see Table 1).

Interpretation and management of intermediate troponin level elevation
For correct interpretation of troponin values, common non-cardiac causes of elevated levels such as fibrin clots, sepsis or renal failure, existing heart failure and specifically for troponin T, skeletal muscle damage associated with trauma or myopathy including that associated with statin treatment, should also be considered (Chaulin, 2022;Mair et al., 2018).Other factors include elevated heterophilic antibodies, rheumatoid factor, alkaline phosphatase, and cross-reactions of diagnostic antibodies (anti-cTn) with skeletal troponin molecules (Chaulin, 2022).While this patient was treated with a statin, there was no report of myalgia.The median time to statin related myopathy for Rosuvastatin is 30 days, 3rd quartile 92 days.Although statin related myopathy remains a potential confounder (Akimoto et al., 2018), the absence of myalgia and normal liver enzymes make this unlikely.
There is emerging evidence for successful clozapine rechallenge and also for continuation of clozapine during mild episodes meeting CIM criteria (Richardson et al., 2021;Ronaldson et al., 2012).Griffin et al. have proposed a rechallenge protocol using prophylactic β-blockers in patients that maintain at least 50 % left ventricular ejection fraction (LVEF).More complex therapy according to American College of Cardiology/American Heart Association Heart Failure guidelines, is recommended if LVEF drops to between 49 and 30 % (Griffin et al., 2021).Evidence for rechallenge remains at the level of isolated case reports and such protocols need further study.Clearly in this case continuation of clozapine during intermediate troponin elevation was critical to psychosocial recovery.
Our case clearly does not meet the accepted criteria for CIM and highlights that CRP/troponin testing needs to be interpreted carefully within the clinical presentation as a marker of evolving myocarditis risk rather than an absolute indication to cease clozapine.Risk adverse responses by clinicians may contribute to reported inappropriate clozapine cessation (Segev et al., 2021).Whilst we cannot predict that the patient would have developed frank myocarditis in this case, the panel provided information that was used to rationally modify treatment via close collaboration between psychiatry and cardiology, all be it with additional patient and health service burden of repeat testing.Further multilevel analysis of the personal and economic impact of the protocol is required.Education programs and the development of close collaborations between local psychiatry and cardiology teams are imperative.

The case for personalised titration
East Asian ethnicity is associated with reduced CYP1A2 activity and effective clozapine dose may be as low as 175 mg to 300 mg for this group (de Leon et al., 2022).The use of personalised titration can be considered to minimise adverse events such as myocarditis that are potentially related to clozapine levels (de Leon et al., 2022).Decision making in the case presented in this paper was complicated by the balance between treatment resistance, patient preference and variability in troponin assays across laboratories.However, increasing dose beyond 300 mg even in the context of clozapine resistance did not carry a favourable risk/benefit ratio.
In conclusion, our case highlights that the interpretation of intermediate troponin results is uncertain and that clozapine may be continued safely, while treatment is optimised to reduce risk of emergent myocarditis.Based on our experiences, in an Australian context, we have developed and implemented a standardised set of thresholds for hs troponin monitoring that considers both the wide variability in thresholds and sensitivity of commercially available hs troponin immunoassays and manufacturer recommendations for repeated daily testing with intermediate elevation.Potentially, unique features of Australian prescribing, such as more rapid titration rates, predispose patients to higher rates of myocarditis.Hence the impact of monitoring maybe negligible in countries such as Denmark where slow outpatient titrations predominate.Further prospective study of the accuracy of monitoring thresholds, along with the individual and economic impacts of repeated CRP/Troponin testing and the integration of personalised approaches to clozapine titration are all required.Where CRP/Troponin monitoring is implemented, guidelines should be updated to alert prescribers to the potential variability between laboratories and for false positive troponin results associated with comorbidities and concomitant treatment.Close partnerships between Cardiology, Clinical Pathology and Psychiatry are critical, not only for optimisation local management but to develop a better shared understanding of the optimal approach to assessment and

Table 1
SA Health guideline recommendations for interpretation of troponin results used in CIM screening.
(SA Health, 2022)ECG and Echocardiography or CMR are part of assessment(SA Health, 2022).**In the absence of chest pain and changes on ECG suggestive of ischemia.