Isoniazid and rifampicin exposure during treatment in drug-susceptible TB.

BACKGROUND: Observational real-world studies on therapeutic drug monitoring (TDM) in relation to pharmacokinetic (PK) target values are lacking. This study aims to describe the PK of rifampicin (RIF) and isoniazid (INH) in a real-world setting of patients with drug-susceptible TB in relation to frequently used threshold values.METHODS: A total of 116 patients with TB using standard doses of RIF and INH and who had TDM as part of clinical care were included. Maximum plasma concentration (Cmax) and 24 h area under the concentration time curve (AUC24) at standard and revised doses were described in relation to the threshold values (Cmax ≥8 mg/L for RIF and ≥3 mg/L for INH).RESULTS: For RIF (100 patients), median Cmax and median AUC24 were respectively 7.9 mg/L (IQR 6.0-11.0) and 35.8 mg*h/L (IQR 27.4-57.3) at the first TDM measurement after a standard dose of 600 mg. For INH (90 patients), median Cmax and median AUC24 were respectively 2.9 mg/L (IQR 1.3-2.5) and 12.5 mg*h/L (IQR 8.7-18.9) at the first TDM after a standard dose 300 mg. Overall, more than 50% of study participants had drug exposure below threshold values at the first TDM.CONCLUSION: Our study shows that the measured Cmax values for both RIF and INH were frequently below the pre-specified targets, emphasising the need for better justification of drug exposure targets. These TDM results highlight the need for validating PK targets of anti-TB drugs associated with clinically relevant outcomes.

Tolerability and pharmacokinetic evaluation of inhaled dry powder hydroxychloroquine in healthy volunteers.

RATIONALE: Inhaled antimicrobials enable high local concentrations where needed and, compared to orally administration, greatly reduce the potential for systemic side effects. In SARS-CoV-2 infections, hydroxychloroquine sulphate (HCQ) administered as dry powder via inhalation could be safer than oral HCQ allowing higher and therefore more effective pulmonary concentrations without dose limiting toxic effects. OBJECTIVES: To assess the local tolerability, safety and pharmacokinetic parameters of HCQ inhalations in single ascending doses of 5, 10 and 20 mg using the Cyclops dry powder inhaler. METHODS: Twelve healthy volunteers were included in the study. Local tolerability and safety were assessed by pulmonary function tests, electrocardiogram and recording adverse events. To estimate systemic exposure, serum samples were collected before and 0.5, 2 and 3.5 h after inhalation. RESULTS AND DISCUSSION: Dry powder HCQ inhalations were well tolerated by the participants, except for transient bitter taste in all participants and minor coughing irritation. There was no significant change in QTc-interval or drop in FEV1 post inhalation. The serum HCQ concentration remained below 10 µg/L in all samples. CONCLUSION: Single doses of inhaled dry powder HCQ up to 20 mg are safe and well tolerated. Our data support that further studies with inhaled HCQ dry powder to evaluate pulmonary pharmacokinetics and efficacy are warranted.

Clinical standards for drug-susceptible pulmonary TB.

BACKGROUND: The aim of these clinical standards is to provide guidance on 'best practice´ for diagnosis, treatment and management of drug-susceptible pulmonary TB (PTB).METHODS: A panel of 54 global experts in the field of TB care, public health, microbiology, and pharmacology were identified; 46 participated in a Delphi process. A 5-point Likert scale was used to score draft standards. The final document represents the broad consensus and was approved by all 46 participants.RESULTS: Seven clinical standards were defined: Standard 1, all patients (adult or child) who have symptoms and signs compatible with PTB should undergo investigations to reach a diagnosis; Standard 2, adequate bacteriological tests should be conducted to exclude drug-resistant TB; Standard 3, an appropriate regimen recommended by WHO and national guidelines for the treatment of PTB should be identified; Standard 4, health education and counselling should be provided for each patient starting treatment; Standard 5, treatment monitoring should be conducted to assess adherence, follow patient progress, identify and manage adverse events, and detect development of resistance; Standard 6, a recommended series of patient examinations should be performed at the end of treatment; Standard 7, necessary public health actions should be conducted for each patient. We also identified priorities for future research into PTB.CONCLUSION: These consensus-based clinical standards will help to improve patient care by guiding clinicians and programme managers in planning and implementation of locally appropriate measures for optimal person-centred treatment for PTB.

Comparative cardiovascular safety of strontium ranelate and bisphosphonates: a multi-database study in 5 EU countries by the EU-ADR Alliance.

Strontium ranelate use, compared with oral bisphosphonates, is not associated with increased risk of AMI in patients with no contraindications for SR use. However, current strontium ranelate (compared with current bisphosphonate) appears associated with 25-30% excess risk of VTE and 35% excess risk of CVDeath. INTRODUCTION: Evaluate the risk of cardiac and thromboembolic events among new users of SR and oral BPs without contraindications for SR. METHODS: We conducted three multi-national, multi-database (Aarhus-Denmark, HSD-Italy, IPCI-Netherlands, SIDIAP-Spain, THIN-UK) case-control studies nested within a cohort of new users of SR/BP. We matched cases of acute myocardial infarction (AMI), venous thromboembolism (VTE), and cardiovascular death (CVDeath), up to 10 controls on gender, year of birth, index date, and country. Conditional logistic regression was used to estimate odds ratios (OR) and 95% confidence intervals (CIs) according to current SR vs current BP use and current vs past SR use, adjusting for potential confounders. Data were pooled using random effects meta-analysis. RESULTS: No excess risk of AMI (5477 cases/54,674 controls) was found with current SR vs current BP (OR 0.89 (95%CI 0.70, 1.12)) nor with current vs past SR use (0.71(0.56, 0.91)). For VTE (5614 cases/6036 controls), an excess risk was found with current SR compared with current BP use, 1.24 (0.96, 1.61), and current vs past SR use, 1.30 (1.04, 1.62). For CVDeath (3019 cases/29,871 controls), an increased risk was seen with current SR vs current BP use, 1.35 (1.02, 1.80), but not with current vs past SR use (0.68 (0.48, 0.96)). CONCLUSION: In patients without contraindications for SR, we found no evidence of an increased risk of AMI but a 25-30% excess risk of VTE and a 35% excess risk of CVDeath with current SR vs current BP users. This is despite a reduction in risk in CVDeath with current vs past SR users. The latter disparity could still be partially explained by cessation of preventative therapies in end-of-life or residual confounding by indication.

How to design a study to evaluate therapeutic drug monitoring in infectious diseases?

BACKGROUND: Therapeutic drug monitoring (TDM) is a tool to personalize and optimize dosing by measuring the drug concentration and subsequently adjusting the dose to reach a target concentration or exposure. The evidence to support TDM is however often ranked as expert opinion. Limitations in study design and sample size have hampered definitive conclusions of the potential added value of TDM. OBJECTIVES: We aim to give expert opinion and discuss the main points and limitations of available data from antibiotic TDM trials and emphasize key elements for consideration in design of future clinical studies to quantify the benefits of TDM. SOURCES: The sources were peer-reviewed publications, guidelines and expert opinions from the field of TDM. CONTENT: This review focuses on key aspects of antimicrobial TDM study design: describing the rationale for a TDM study, assessing the exposure of a drug, assessing susceptibility of pathogens and selecting appropriate clinical endpoints. Moreover we provide guidance on appropriate study design. IMPLICATIONS: This is an overview of different aspects relevant for the conduct of a TDM study. We believe that this paper will help researchers and clinicians to design and conduct high-quality TDM studies.

Correction to: Impact of risk minimisation measures on the use of strontium ranelate in Europe: a multi-national cohort study in 5 EU countries by the EU-ADR Alliance.

The original version of this article, published on 26 November 2019 contained a mistake.

Impact of risk minimisation measures on the use of strontium ranelate in Europe: a multi-national cohort study in 5 EU countries by the EU-ADR Alliance.

INTRODUCTION: In May 2013 and March 2014, the European Medicines Agency (EMA) issued two decisions restricting the use of strontium ranelate (SR). These risk minimisation measures (RMM) introduced new contraindications and limited the indications of SR therapy. The EMA required an assessment of the impact of RMMs on the use of SR in Europe. Methods design: multi-national, multi-database cohort Setting: electronic medical record databases based on hospital (Denmark) and primary care provenance (Italy, Spain, the Netherlands, UK). PARTICIPANTS: the database source populations were included for population-based analyses, and SR users for patient-level analyses. INTERVENTION: New RMMs included contraindications (ischaemic heart disease, peripheral arterial disease, cerebrovascular disease, uncontrolled hypertension) and restricted SR indication to severe osteoporosis with initiation by experienced physician and not as first line anti-osteoporosis therapy. METHODS: Prevalence and incidence rates of SR use in the population; prevalence of contraindications and restricted indications in SR users, plus 1-year therapy persistence. Drug use measures were calculated in three periods for comparison: reference (2004 to May 2013), transition (June 2013 to March 2014) and assessment (from April 2014 to end 2016). RESULTS: The study population included 143 million person-years(PY) of follow-up and 76,141 incident episodes of SR treatment. Average monthly prevalence rates of SR use dropped by 86.4% from 62.6/10,000 PY (95 CI 62.4-62.9) in the reference to 8.5 (8.5-8.6) in the assessment period. Similarly, the incidence rate of SR use fell by 97.3% from 7.4/10,000 PY (7.4-7.4) to 0.2 (0.2-0.2) between the reference and assessment period. The prevalence of any contraindication decreased, whilst the prevalence of restricted indications increased in these periods. One-year persistence decreased in the assessment compared with reference period. CONCLUSIONS: Our study demonstrates a substantial impact of the regulatory action to restrict use of SR in Europe: SR utilisation overall decreased strongly. The proportion of patients fulfilling the restricted indications, without contraindications, increased after the proposed RMMs.

No evidence found for an increased risk of long-term fatigue following human papillomavirus vaccination of adolescent girls.

INTRODUCTION: In 2013, the Netherlands Pharmacovigilance Center Lareb published an overview of reports of long-lasting fatigue following bivalent HPV-vaccination (2vHPV). After an update of this overview in 2015, concerns regarding the safety of 2vHPV was picked up by the media, which led to further reports of long-lasting fatigue. Therefore, the Dutch National Institute for Public Health and the Environment (RIVM) investigated a possible association between HPV-vaccination and long-term fatigue. METHODS: In this retrospective cohort study conducted in the Integrated Primary Care Information database, we investigated the occurrence of chronic fatigue syndrome (CFS), fatigue ≥6 months and 3-6 months in all girls born in 1991-2000 during the follow-up period January 1st 2007-December 31st 2014 (2007-2008 pre-vaccination and 2009-2014 post-vaccination). Patients with certain fatigue ≥6 m were asked for consent to link their primary care information with vaccination data. Incidence rates per 10,000 person years (PY) for 12-16-year-old girls were compared between pre- and post-HPV-vaccine era. A self-controlled case series (SCCS) analysis was performed using consenting vaccinated cases. A primary high-risk period of 12 months after each dose was defined. RESULTS: The cohort consisted of 69,429 12-16-year-old girls accounting for 2758 PY pre-vaccination and 57,214 PY post-vaccination. Differences between pre- and post-vaccination incidences (CFS: 3.6 (95% CI 0.5-25.7)/10,000 PY and 0.9 (0.4-2.1); certain fatigue ≥6 m: 7.3 (1.8-29.0) and 19.4 (16.1-23.4); certain fatigue 3-6 m: 0.0 and 16.6 (13.6-20.3), respectively) were not statistically significant. SCCS analyses in 16 consenting vaccinated cases resulted in an age-adjusted RR of 0.62 (95%CI 0.07-5.49). CONCLUSIONS: Fatigue ≥6 m and 3-6 m was frequently found among adolescent girls, but CFS was rarely diagnosed. No statistically significant increased incidence rates were found post-vaccination compared to similar age groups of girls pre-vaccination. The SCCS analysis included a low number of cases but revealed no elevated risk of certain fatigue ≥6 m in the high-risk period.

Pharmacokinetics of rifampicin in adult TB patients and healthy volunteers: a systematic review and meta-analysis.

Objectives: The objectives of this study were to explore inter-study heterogeneity in the pharmacokinetics (PK) of orally administered rifampicin, to derive summary estimates of rifampicin PK parameters at standard dosages and to compare these with summary estimates for higher dosages. Methods: A systematic search was performed for studies of rifampicin PK published in the English language up to May 2017. Data describing the Cmax and AUC were extracted. Meta-analysis provided summary estimates for PK parameter estimates at standard rifampicin dosages. Heterogeneity was assessed by estimation of the I2 statistic and visual inspection of forest plots. Summary AUC estimates at standard and higher dosages were compared graphically and contextualized using preclinical pharmacodynamic (PD) data. Results: Substantial heterogeneity in PK parameters was evident and upheld in meta-regression. Treatment duration had a significant impact on the summary estimates for rifampicin PK parameters, with Cmax 8.98 mg/L (SEM 2.19) after a single dose and 5.79 mg/L (SEM 2.14) at steady-state dosing, and AUC 72.56 mg·h/L (SEM 2.60) and 38.73 mg·h/L (SEM 4.33) after single and steady-state dosing, respectively. Rifampicin dosages of at least 25 mg/kg are required to achieve plasma PK/PD targets defined in preclinical studies. Conclusions: Vast inter-study heterogeneity exists in rifampicin PK parameter estimates. This is not explained by the available modifying variables. The recommended dosage of rifampicin should be increased to improve efficacy. This study provides an important point of reference for understanding rifampicin PK at standard dosages as efforts to explore higher dosing strategies continue in this field.

Use of antihistamines and risk of ventricular tachyarrhythmia: a nested case-control study in five European countries from the ARITMO project.

PURPOSE: After regulatory restrictions for terfenadine and astemizole in '90s, only scarce evidence on proarrhythmic potential of antihistamines has been published. We evaluate the risk of ventricular tachyarrhythmia (VA) related to the use of individual antihistamines. METHODS: A matched case-control study nested in a cohort of new users of antihistamines was conducted within the EU-funded ARITMO project. Data on 1997-2010 were retrieved from seven healthcare databases: AARHUS (Denmark), GEPARD (Germany), HSD and ERD (Italy), PHARMO and IPCI (Netherlands) and THIN (UK). Cases of VA were selected and up to 100 controls were matched to each case. The odds ratio (OR) of current use for individual antihistamines (AHs) was estimated using conditional logistic regression. RESULTS: For agents largely used to prevent allergic symptoms, such as cetirizine, levocetirizine, loratadine, desloratadine and fexofenadine, we found no VA risk. A statistically significant, increased risk of VA was found only for current use of cyclizine in the pooled analysis (ORadj, 5.3; 3.6-7.6) and in THIN (ORadj, 5.3; 95% CI, 3.7-7.6), for dimetindene in GEPARD (ORadj, 3.9; 1.1-14.7) and for ebastine in GEPARD (ORadj, 3.3; 1.1-10.8) and PHARMO (ORadj, 4.6; 1.3-16.2). CONCLUSIONS: The risk of VA associated with a few specific antihistamines could be ascribable to heterogeneity in pattern of use or in receptor binding profile.

Real life data on incidence and risk factors of severe asthma exacerbations in children in primary care.

Real-life data on the incidence rates (IR) and risk factors of severe asthma exacerbations in children are sparse. We aimed to assess IR and risk factors of severe asthma exacerbations in children in real life. We conducted a population-based cohort study using a Dutch GP database containing complete medical records of >1 million patients. All records of children with physician-diagnosed asthma aged 5-18 years between 2000 and 2012 were examined for exacerbations, defined as either hospitalization, emergency department visit or need of systemic steroids for asthma. IR was expressed as number of exacerbations per person year (PY). We identified 14,303 asthmatic children with 35,118 PY of follow-up and 732 exacerbations. The overall IR was 2.1/100PY (95% CI 1.9-2.2), 4.1/100PY (3.8-4.4) for children on asthma treatment. Re-exacerbation occurred in 2% (1.3-4.3) of patients within 1 month, in 25% (20.6-28.8) within 1 year. Predictors for (frequent) exacerbations were age, medication use and prior exacerbations (all p < 0.001). The overall IR of severe asthma exacerbations was 4/100PY in children on asthma treatment, highest in spring and fall. 25% of the patients with an exacerbation will experience a next exacerbation within 1 year. More severe asthma is a predictor of subsequent and future exacerbations.

Emerging drugs and alternative possibilities in the treatment of tuberculosis.

INTRODUCTION: Tuberculosis (TB) remains a global health problem. Drug resistance, treatment duration, complexity, and adverse drug reactions associated with anti-TB regimens are associated with treatment failure, prolonged infectiousness and relapse. With the current set of anti-TB drugs the goal to end TB has not been met. New drugs and new treatment regimens are needed to eradicate TB. AREAS COVERED: Literature was explored to select publications on drugs currently in phase II and phase III trials. These include new chemical entities, immunotherapy, established drugs in new treatment regimens and vaccines for the prophylaxis of TB. EXPERT OPINION: Well designed trials, with detailed pharmacokinetic/pharmacodynamic analysis, in which information on drug exposure and drug susceptibility of the entire anti-TB regimen is included, in combination with long-term follow-up will provide relevant data to optimize TB treatment. The new multi arm multistage trial design could be used to test new combinations of compounds, immunotherapy and therapeutic vaccines. This new approach will both reduce the number of patients exposed to inferior treatment and the financial burden. Moreover, it will speed up drug evaluation. Considering the investments involved in development of new drugs it is worthwhile to thoroughly investigate existing, non-TB drugs in new regimens.

Risk of cardiac valvulopathy with use of bisphosphonates: a population-based, multi-country case-control study.

UNLABELLED: Analyses of healthcare data from 30 million individuals in three countries showed that current use of bisphosphonates may be associated with a small increased risk of cardiac valvulopathy (vs. those not exposed within the previous year), although confounding cannot be entirely ruled out. The observed tendency for decreased valvulopathy risk with cumulative duration of bisphosphonate use >6 months may even indicate a protective effect with prolonged use. Further studies are still needed to evaluate whether bisphosphonates increase or decrease the risk of valvulopathy. INTRODUCTION: A signal of cardiac valve disorders with use of bisphosphonates was identified in the literature and EudraVigilance database, which contains reports of suspected adverse drug reactions from worldwide sources. The aim of this study was to evaluate the association using population-based healthcare data. METHODS: This was a case-control study among users of bisphosphonates and other drugs for osteoporosis in six healthcare databases covering over 30 million individuals in Italy, Netherlands and the UK from 1996 to 2012. Prescriptions/dispensations were used to assess drug exposure. Newly diagnosed cases of cardiac valvulopathy were identified via disease codes/free-text search. Controls were matched to each case by age, sex, database and index date. Adjusted odds ratios (ORs) were estimated using conditional logistic regression for the pooled data and meta-analysis of individual database risk estimates. RESULTS: A small but statistically significant association was found between exposure to bisphosphonates as a class and risk of valvulopathy. Overall risk was 18 % higher (95 % CI 12-23 %) in those currently exposed to any bisphosphonate (mainly alendronate and risedronate) vs. those not exposed within the previous year. Risk of valve regurgitation was 14 % higher (95 % CI 7-22 %). Decreased valvulopathy risk was observed with longer cumulative duration of bisphosphonate use, compared to use of less than 6 months. Meta-analyses of database-specific estimates confirmed results from pooled analyses. CONCLUSIONS: The observed increased risks of cardiac valvulopathy with bisphosphonate use, although statistically significant, were quite small and unlikely to be clinically significant. Further studies are still needed to evaluate whether bisphosphonates increase or decrease the risk of valvulopathy and to investigate possible mechanisms for the association.

Sudden cardiac and sudden unexpected death related to antipsychotics: A meta-analysis of observational studies.

To estimate the risk of sudden cardiac death (SCD) or sudden unexpected death (SUD) related to individual antipsychotics, a meta-analysis of observational studies was performed. Adjusted odds ratio (OR) of SCD/SUD with 95% confidence intervals (CI) were extracted and pooled; heterogeneity was studied using Q statistic and I(2) index, and its potential causes (e.g., hERG blockade potency) explored using meta-regression. Two cohort (740,306 person-years) and four case-control (2,557 cases; 17,670 controls) studies, investigating nine antipsychotics, were included. Compared with nonusers, the risk was increased for quetiapine (OR = 1.72, 95% CI: 1.33-2.23), olanzapine (OR = 2.04, 1.52-2.74), risperidone (OR = 3.04, 2.39-3.86), haloperidol (OR = 2.97, 1.59-5.54), clozapine (OR = 3.67, 1.94-6.94), and thioridazine (OR = 4.58, 2.09-10.05). Heterogeneity was found (Q = 20.0, P = 0.01; I(2) = 60.0%), and the increasing mean hERG blockade potency (P = 0.01) accounted for 43% of this. The SCD/SUD risk differed between individual antipsychotics, and mean hERG blockade potency could be an explanatory factor. This should be considered when initiating antipsychotic treatment.

Application of a Systems Pharmacology-Based Placebo Population Model to Analyze Long-Term Data of Postmenopausal Osteoporosis.

Osteoporosis is a progressive bone disease characterized by decreased bone mass resulting in increased fracture risk. The objective of this investigation was to test whether a recently developed disease systems analysis model for osteoporosis could describe disease progression in a placebo-treated population from the Early Postmenopausal Intervention Cohort (EPIC) study. First, we qualified the model using a subset from the placebo arm of the EPIC study of 222 women who had similar demographic characteristics as the 149 women from the placebo arm of the original population. Second, we applied the model to all 470 women. Bone mineral density (BMD) dynamics were changed to an indirect response model to describe lumbar spine and total hip BMD in this second population. This updated disease systems analysis placebo model describes the dynamics of all biomarkers in the corresponding datasets to a very good approximation; a good description of an individual placebo response will be valuable for evaluating treatments for osteoporosis.

Risk of atrial fibrillation among bisphosphonate users: a multicenter, population-based, Italian study.

UNLABELLED: Bisphosphonate treatment is used to prevent bone fractures. A controversial association of bisphosphonate use and risk of atrial fibrillation has been reported. In our study, current alendronate users were associated with a higher risk of atrial fibrillation as compared with those who had stopped bisphosphonate (BP) therapy for more than 1 year. INTRODUCTION: Bisphosphonates are widely used to prevent bone fractures. Controversial findings regarding the association between bisphosphonate use and the risk of atrial fibrillation (AF) have been reported. The aim of this study was to evaluate the risk of AF in association with BP exposure. METHODS: We performed a nested case-control study using the databases of drug-dispensing and hospital discharge diagnoses from five Italian regions. The data cover a period ranging from July 1, 2003 to December 31, 2006. The study population comprised new users of bisphosphonates aged 55 years and older. Patients were followed from the first BP prescription until an occurrence of an AF diagnosis (index date, i.e., ID), cancer, death, or the end of the study period, whichever came first. For the risk estimation, any AF case was matched by age and sex to up to 10 controls from the same source population. A conditional logistic regression was performed to obtain the odds ratio with 95% confidence intervals (CI). The BP exposure was classified into current (<90 days prior to ID), recent (91-180), past (181-364), and distant past (≥365) use, with the latter category being used as a reference point. A subgroup analysis by individual BP was then carried out. RESULTS: In comparison with distant past users of BP, current users of BP showed an almost twofold increased risk of AF: odds ratio (OR) = 1.78 and 95% CI = 1.46-2.16. Specifically, alendronate users were mostly associated with AF as compared with distant past use of BP (OR, 1.97; 95% CI, 1.59-2.43). CONCLUSION: In our nested case-control study, current users of BP are associated with a higher risk of atrial fibrillation as compared with those who had stopped BP treatment for more than 1 year.

Examining a possible association between human papilloma virus (HPV) vaccination and migraine: results of a cohort study in the Netherlands.

UNLABELLED: Since the introduction of the bivalent human papilloma virus (HPV) vaccine in the Netherlands, migraine has been reported as a notable event in the passive safety surveillance system. Research on the association between HPV vaccination and migraine is needed. Therefore, potential migraine cases in 2008-2010 were selected from a group of general practitioners and linked to the vaccination registry. Data were analysed in three ways: (i) incidences of migraine postvaccination (2009/2010) were compared to pre-vaccination incidences (2008); (ii) in a cohort, incidence rates of migraine in vaccinated and unvaccinated girls were compared and (iii) in a self-controlled case series analysis, the relative incidence of migraine in potentially high-risk periods was compared to non-high-risk periods. Incidence rates of migraine for 12- to 16-year-old girls and boys postvaccination were slightly higher than pre-vaccination incidence rates. Incidence rate ratios (IRRs) for vaccinated compared to unvaccinated girls were not statistically significantly higher. Furthermore, the RR for migraine in the high-risk period of 6 weeks following each dose versus non-high-risk period was 4.3 (95% confidence interval (CI) 0.69-26.6) for certain migraine. CONCLUSION: Using different methods, no statistically significant association between HPV vaccination and incident migraine was found. However, the number of cases was low; to definitively exclude the risk, an increased sample size is needed.