Comparison of Methods to Estimate Low-Density Lipoprotein Cholesterol in Patients With High Triglyceride Levels.

Importance: Low-density lipoprotein cholesterol (LDL-C) is typically estimated with the Friedewald or Martin/Hopkins equation; however, if triglyceride levels are 400 mg/dL or greater, laboratories reflexively perform direct LDL-C (dLDL-C) measurement. The use of direct chemical LDL-C assays and estimation of LDL-C via the National Institutes of Health Sampson equation are not well validated, and data on the accuracy of LDL-C estimation at higher triglyceride levels are limited. Objective: To compare an extended Martin/Hopkins equation for triglyceride values of 400 to 799 mg/dL with the Friedewald and Sampson equations. Design, Setting, and Participants: This cross-sectional study evaluated consecutive patients at clinical sites across the US with patient lipid distributions representative of the US population in the Very Large Database of Lipids from January 1, 2006, to December 31, 2015, with triglyceride levels of 400 to 799 mg/dL. Data analysis was performed from November 9, 2020, to March 23, 2021. Main Outcomes and Measures: Accuracy in LDL-C classification according to guideline-based categories and absolute errors between estimated LDL-C and dLDL-C levels. Patients were randomly assigned 2:1 to derivation and validation data sets. Levels of dLDL-C were measured by vertical spin-density gradient ultracentrifugation. The LDL-C levels were estimated using the Friedewald method, with a fixed ratio of triglycerides to very low-density lipoprotein cholesterol (VLDL-C ratio of 5:1), extended Martin/Hopkins equation with a flexible ratio, and Sampson equation with VLDL-C estimation by multiple least-squares regression. Results: A total of 111 939 patients (mean [SD] age, 52 [13] years; 65.0% male) with triglyceride levels of 400 to 799 mg/dL were included, representing 2.2% of 5 081 680 patients in the database. Across all individual guideline LDL-C classes (<40, 40-69, 70-99, 100-129, 130-159, 160-189, and ≥190), estimation of LDL-C by the extended Martin/Hopkins equation was most accurate (62.1%) compared with the Friedewald (19.3%) and Sampson (40.4%) equations. In classifying LDL-C levels less than 70 mg/dL across all triglyceride strata, the extended Martin/Hopkins equation was most accurate (67.3%) compared with Friedewald (5.1%) and Sampson (26.4%) equations. In addition, for classifying LDL-C levels less than 40 mg/dL across all triglyceride strata, the extended Martin/Hopkins equation was most accurate (57.2%) compared with the Friedewald (4.3%) and Sampson (14.4%) equations. However, considerable underclassification of LDL-C occurred. The magnitude of error between the Martin/Hopkins equation estimation and dLDL-C was also smaller: at LDL-C levels less than 40 mg/dL, 2.7% of patients had 30 mg/dL or greater differences between dLDL-C and estimated LDL-C using the Martin/Hopkins equation compared with the Friedewald (92.5%) and Sampson (38.7%) equations. Conclusions and Relevance: In this cross-sectional study, the extended Martin/Hopkins equation offered greater LDL-C accuracy compared with the Friedewald and Sampson equations in patients with triglyceride levels of 400 to 799 mg/dL. However, regardless of method used, caution is advised with LDL-C estimation in this triglyceride range.

Assessment of Coronary Artery Calcium Scoring to Guide Statin Therapy Allocation According to Risk-Enhancing Factors: The Multi-Ethnic Study of Atherosclerosis.

Importance: The 2018 American Heart Association/American College of Cardiology Guideline on the Management of Blood Cholesterol recommends the use of risk-enhancing factor assessment and the selective use of coronary artery calcium (CAC) scoring to guide the allocation of statin therapy among individuals with an intermediate risk of atherosclerotic cardiovascular disease (ASCVD). Objective: To examine the association between risk-enhancing factors and incident ASCVD by CAC burden among those at intermediate risk of ASCVD. Design, Setting, and Participants: The Multi-Ethnic Study of Atherosclerosis is a multicenter population-based prospective cross-sectional study conducted in the US. Baseline data for the present study were collected between July 15, 2000, and July 14, 2002, and follow-up for incident ASCVD events was ascertained through August 20, 2015. Participants were aged 45 to 75 years with no clinical ASCVD or diabetes at baseline, were at intermediate risk of ASCVD (≥7.5% to <20.0%), and had a low-density lipoprotein cholesterol level of 70 to 189 mg/dL. Exposures: Family history of premature ASCVD, premature menopause, metabolic syndrome, chronic kidney disease, lipid and inflammatory biomarkers, and low ankle-brachial index. Main Outcomes and Measures: Incident ASCVD over a median follow-up of 12.0 years. Results: A total of 1688 participants (mean [SD] age, 65 [6] years; 976 men [57.8%]). Of those, 648 individuals (38.4%) were White, 562 (33.3%) were Black, 305 (18.1%) were Hispanic, and 173 (10.2%) were Chinese American. A total of 722 participants (42.8%) had a CAC score of 0. Among those with 1 to 2 risk-enhancing factors vs those with 3 or more risk-enhancing factors, the prevalence of a CAC score of 0 was 45.7% vs 40.3%, respectively. Over a median follow-up of 12.0 years (interquartile range [IQR], 11.5-12.6 years), the unadjusted incidence rate of ASCVD among those with a CAC score of 0 was less than 7.5 events per 1000 person-years for all individual risk-enhancing factors (with the exception of ankle-brachial index, for which the incidence rate was 10.4 events per 1000 person-years [95% CI, 1.5-73.5]) and combinations of risk-enhancing factors, including participants with 3 or more risk-enhancing factors. Although the individual and composite addition of risk-enhancing factors to the traditional risk factors was associated with improvement in the area under the receiver operating curve, the use of CAC scoring was associated with the greatest improvement in the C statistic (0.633 vs 0.678) for ASCVD events. For incident ASCVD, the net reclassification improvement for CAC was 0.067. Conclusions and Relevance: In this cross-sectional study, among participants with CAC scores of 0, the presence of risk-enhancing factors was generally not associated with an overall ASCVD risk that was higher than the recommended treatment threshold for the initiation of statin therapy. The use of CAC scoring was associated with significant improvements in the reclassification and discrimination of incident ASCVD. The results of this study support the utility of CAC scoring as an adjunct to risk-enhancing factor assessment to more accurately classify individuals with an intermediate risk of ASCVD who might benefit from statin therapy.

Anomalous Origin of the Right Coronary Artery Causing Myocardial Ischemia: A Case for a Multimodality Imaging Approach.

A 46-year-old man was admitted with non-ST elevation myocardial infarction and newly diagnosed acutely decompensated heart failure. Echocardiogram demonstrated left ventricular ejection fraction of 30% with basal inferior and inferolateral akinesis. Coronary angiography showed mild diffuse coronary artery disease and an anomalous right coronary artery arising from the left coronary cusp. Further imaging was consistent with ischemia in the right coronary distribution. Etiology of ischemia was thought to be the anomalous right coronary artery, and surgical unroofing of the right coronary ostium was performed. Here, we report a multimodality imaging approach, including cardiac magnetic resonance, cardiac computed tomographic angiography, and single-photon emission computed tomography, to support the diagnosis and management of a patient with anomalous right coronary artery arising from the left coronary cusp.

Importance of the triglyceride level in identifying patients with a Type III Hyperlipoproteinemia phenotype using the ApoB algorithm.

BACKGROUND: Hyperlipoproteinemia Type III (HLP3), also known as dysbetalipoproteinemia, is defined by cholesterol and triglyceride (TG) enriched remnant lipoprotein particles (RLP). The gold standard for diagnosis requires demonstration of high remnant lipoprotein particle cholesterol (RLP-C) by serum ultracentrifugation (UC), which is not readily available in daily practice. The apoB algorithm can identify HLP3 using total cholesterol (TC), plasma triglyceride (TG), and apoB. However, the optimal TG cutoff is unknown. OBJECTIVE: We analyzed apoB algorithm defined HLP3 at different TG levels to optimize the TG cutoff for the algorithm. METHODS: 128,485 UC lipid profiles in the Very Large Database of Lipids (VLDbL) were analyzed. RLP-C was assessed at TG ≥ 133 mg/dL, ≥175 mg/dL, ≥200 mg/dL, and ≥ 250 mg/dL. Sensitivity (Sn), specificity (Sp), positive predictive value (PPV), negative predictive value (NPV), and prevalence adjusted and bias-adjusted kappa (PABAK) were calculated against UC Criterion (VLDL-C/TG ≥ 0.25) for HLP3. RESULTS: The median age (IQR) was 57 years (46-68). 45% were men, 20.1% had diabetes, and 25.5% had hypertension. The median RLP-C level for the TG cutoffs (mg/dL) of ≥ 133, ≥ 175, ≥ 200, and ≥ 250 were 34, 43, 50, and 62 mg/dL, respectively, compared to 67 mg/dL in UC defined HLP3. TG ≥ 133 mg/dL yielded optimal results (Sn 29.5%, Sp 98.5%, PABAK 0.96, PPV 13.6%, NPV 99.4%). CONCLUSION: TG ≥ 133 mg/dL allows for high sensitivity in screening for HLP3. Higher TG cutoffs may identify more severe HLP3 phenotypes, but with a large loss in sensitivity for HLP3.

Modern prevalence of the Fredrickson-Levy-Lees dyslipidemias: findings from the Very Large Database of Lipids and National Health and Nutrition Examination Survey.

INTRODUCTION: Five decades ago, Fredrickson, Levy, and Lees (FLL) qualitatively characterized clinical dyslipidemias with specific implications for cardiovascular and non-cardiovascular morbidity and mortality. They separated disorders of elevated cholesterol and triglycerides into five phenotypes (types I-V) based on their lipoprotein profile. Although clinicians generally consider them rare entities, modern FLL prevalence may be greater than previously reported. MATERIAL AND METHODS: We performed a cross-sectional analysis in 5,272 participants from the 2011-2014 National Health and Nutrition Examination Survey and 128,506 participants from the Very Large Database of Lipids study with complete, fasting lipid profiles. We used a validated algorithm to define FLL phenotypes employing apolipoprotein B, total cholesterol, and triglycerides. RESULTS: Overall prevalence of FLL phenotypes was 33.9%. FLL prevalence in the general population versus clinical lipid database was: type I (0.05 vs. 0.02%), type IIa (3.2 vs. 3.9%), type IIb (8.0 vs. 10.3%), type III (2.0 vs. 1.7%), type IV (20.5 vs. 24.1%), and type V (0.15 vs. 0.13%). Those aged 40-74 years had a higher overall prevalence compared to other age groups (p < 0.001) and men had overall higher prevalence than women (p < 0.001). Those with diabetes (51.6%) or obese BMI (49.0%) had higher prevalence of FLL phenotypes compared to those without diabetes (31.3%; p < 0.001) and normal BMI (18.3%; p < 0.001). CONCLUSIONS: FLL phenotypes are likely far more prevalent than appreciated in clinical practice, in part due to diabetes and obesity epidemics. Given the prognostic and therapeutic importance of these phenotypes, their identification becomes increasingly important in the era of precision medicine.

Modern prevalence of dysbetalipoproteinemia (Fredrickson-Levy-Lees type III hyperlipoproteinemia).

INTRODUCTION: Dysbetalipoproteinaemia (HLP3) is a disorder characterized by excess cholesterol-enriched, triglyceride-rich lipoprotein remnants in genetically predisposed individuals that powerfully promote premature cardiovascular disease if untreated. The current prevalence of HLP3 is largely unknown. MATERIAL AND METHODS: We performed cross-sectional analysis of 128,485 U.S. adults from the Very Large Database of Lipids (VLDbL), using four algorithms to diagnose HLP3 employing three Vertical Auto Profile ultracentrifugation (UC) criteria and a previously described apolipoprotein B (apoB) method. We evaluated 4,926 participants from the 2011-2014 National Health and Nutrition Examination Survey (NHANES) with the apoB method. We examined demographic and lipid characteristics stratified by presence of HLP3 and evaluated lipid characteristics in those with HLP3 phenotype discordance and concordance as determined by apoB and originally defined UC criteria 1. RESULTS: In U.S. adults in VLDbL and NHANES, a 1.7-2.0% prevalence is observed for HLP3 with the novel apoB method as compared to 0.2-0.8% prevalence in VLDbL via UC criteria 1-3. Participants who were both apoB and UC criteria HLP3 positive had higher remnant particles as well as more elevated triglyceride/apoB and total cholesterol/apoB ratios (all p < 0.001) than those who were apoB method positive and UC criteria 1 negative. CONCLUSIONS: HLP3 may be more prevalent than historically and clinically appreciated. The apoB method increases HLP3 identification via inclusion of milder phenotypes. Further work should evaluate the clinical implications of HLP3 diagnosis at various lipid algorithm cut-points to evaluate the ideal standard in the modern era.

The evolution of the heart-healthy diet for vascular health: A walk through time.

The rate of cardiovascular disease (CVD) mortality reduction in the United States has plateaued recently, despite the development of novel preventive pharmacotherapies, increased access to care, and healthcare spending. This is largely due to American's poor dietary patterns and practices causing increasing trends in the prevalence of obesity and type 2 diabetes mellitus. For decades, dietary guidelines on 'healthy diets' to reduce CVD risk, grounded in epidemiological research, have been nationally distributed to Americans. In this review, we highlight landmark events in modern nutrition science and how these have framed past and current understandings of diet and health. We also follow the evolution of dietary recommendations for Americans throughout the years, with an emphasis on recommendations aimed to reduce risk for CVD and mortality. Secondly, we examine how the low-fat ideology came to dominate America in the last decades of the 20th century and subsequently contributed to an excess intake of refined carbohydrates which, in the context of an increasingly sedentary lifestyle, may have fueled the obesity epidemic. We then examine the current major evidence-based dietary patterns and specific dietary approaches to reduce CVD risk, reviewing the literature surrounding nutritional components of the heart-healthy diet and discussing the dietary patterns proven most effective for CVD prevention: the Dietary Approaches to Stop Hypertension (DASH) diet, the Mediterranean diet, and the healthy vegetarian diet. Finally, we discuss emerging dietary trends, considerations for nutrition counseling, and future directions within the important field of nutrition, with the ultimate goal of improving vascular health.

A Clinician's Guide to Healthy Eating for Cardiovascular Disease Prevention.

Despite continued advances in health care, the cardiovascular disease (CVD) mortality rate has plateaued in recent years and appears to be trending upward. Poor diet is a leading cause of obesity and type 2 diabetes mellitus, which are leading contributors to CVD morbidity and mortality. Although dietary modification is a cornerstone of CVD prevention, implementation in clinical practice is limited by inadequate formal training in nutrition science. In this report, we review the individual components of a heart-healthy diet, evidence-based dietary recommendations, and the impact of diet on CVD risk factor prevention and management. Furthermore, we examine the unique difficulties of dietary counseling in low-socioeconomic-status environments and provide an evidence-based approach to better serve these populations. We utilized PubMed searches in adults with no date restriction with the following search terms: "carbohydrate," "fat," protein," "DASH," "Mediterranean," "plant-based," "vegetarian," "cardiovascular disease," "obesity," "weight loss," "diabetes," "socioeconomic status," and "race." In this review, we demonstrate that patients should focus on implementing a general diet plan that is high in fruits, whole grains, legumes, and nonstarchy vegetables while low in trans-fats, saturated fats, sodium, red meat, refined carbohydrates, and sugar-sweetened beverages. The Dietary Approaches to Stop Hypertension, Mediterranean, and vegetarian diets have the most evidence for CVD prevention. Clinicians should understand the barriers that patients may face in terms of access to healthy dietary choices. Further research is needed to determine the dietary changes that are most economically, socioculturally, and logistically feasible to reduce these barriers. Improvement in diet is a public health priority that can lead to a significant population-level reduction in CVD morbidity and mortality. It is imperative that clinicians understand current dietary practice guidelines and implement evidence-based dietary counseling in those at high risk for CVD.

An Elusive Case of Mycosis Fungoides: Case Report and Review of the Literature.

Erythroderma refers to a spectrum of skin diseases resulting in diffuse erythema and scaling encompassing ≥ 90% of the body surface area. The differential diagnosis ranges from primary dermatologic diseases such as atopic dermatitis and psoriasis to potentially deadly causes such as staphylococcal toxic shock syndrome, toxic epidermal necrolysis, and malignancy. Cutaneous T cell lymphoma (CTCL) is an uncommon but highly morbid cause of erythroderma. This non-Hodgkin lymphoma remains a diagnostic challenge due to its variable clinical presentation and varied histologic features. Mycosis fungoides (MF) is the most common form of CTCL. Making a timely diagnosis is challenging as it may mimic inflammatory diseases of the skin including eczema, psoriasis, lichen planus, and cutaneous lupus. We present a case of a 58-year-old man who presented with 5 years of cutaneous symptoms and several months of fevers and night sweats, ultimately diagnosed as MF. Owing to diffuse CD30 positivity, he was a candidate for brentuximab vedotin, an antibody-drug conjugate medication that selectively targets the CD30 antigen. This resulted in an excellent therapeutic response.

Impact of improved low-density lipoprotein cholesterol assessment on guideline classification in the modern treatment era-Results from a racially diverse Brazilian cross-sectional study.

BACKGROUND: The Martin/Hopkins low-density lipoprotein cholesterol equation (LDL-CN) was previously demonstrated as more accurate than Friedewald LDL-C estimation (LDL-CF) in a North American database not able to take race into account. OBJECTIVES: We hypothesized that LDL-CN would be more accurate than LDL-CF and correlate better with LDL particle number (LDL-P) in a racially diverse Brazilian cohort. METHODS: We performed a cross-sectional analysis of 4897 participants in the Brazilian Longitudinal Study of Adult Health, assessing LDL-CF and LDL-CN accuracy via overlap with ultracentrifugation-based measurement among clinical guideline LDL-C categories as well as mg/dL and percent error differences. We analyzed by triglyceride categories and correlated LDL-C estimation with LDL-P. RESULTS: LDL-CN demonstrated improved accuracy at 70 to <100 and <70 mg/dL (P < .001), with large errors ≥20 mg/dL about 9 times more frequent in LDL-CF at LDL-C <70 mg/dL, mainly due to underestimation. Among individuals with LDL-C <70 mg/dL and triglycerides ≥150 mg/dL, 65% vs 100% of ultracentrifugation-based low-density lipoprotein cholesterol calculation fell within appropriate categories of estimated LDL-CF and LDL-CN, respectively (P < .001). Similar results were observed when analyzed for age, sex, and race. Participants at LDL-C <70 and 70 to <100 mg/dL with discordantly elevated LDL-CN vs LDL-CF had a 58.5% and 41.5% higher LDL-P than those with concordance (P < .0001), respectively. CONCLUSIONS: In a diverse Brazilian cohort, LDL-CN was more accurate than LDL-CF at low LDL-C and high triglycerides. LDL-CN may avoid underestimation of LDL-C and better reflect atherogenic lipid burden in low particle size, high particle count states.

Anticoagulation risk assessment for patients with non-valvular atrial fibrillation and venous thromboembolism: A clinical review.

Non-valvular atrial fibrillation and venous thromboembolism anticoagulation risk assessment tools have been increasingly utilized to guide implementation and duration of anticoagulant therapy. Anticoagulation significantly reduces stroke and recurrent venous thromboembolism risk, but comes at the cost of increased risk of major and clinically relevant non-major bleeding. The decision for anticoagulation in high-risk patients is complicated by the fact that many risk factors associated with increased thromboembolic risk are simultaneously associated with increased bleeding risk. Traditional risk assessment tools rely heavily on age, sex, and presence of cardiovascular comorbidities, with newer tools additionally taking into account changes in risk factors over time and novel biomarkers to facilitate more personalized risk assessment. These tools may help counsel and inform patients about the risks and benefits of starting or continuing anticoagulant therapy and can identify patients who may benefit from more careful management. Although the ability to predict anticoagulant-associated hemorrhagic risk is modest, ischemic and bleeding risk scores have been shown to add significant value to therapeutic management decisions. Ultimately, further work is needed to optimally implement accurate and actionable risk stratification into clinical practice.

Clinician's Guide to the Updated ABCs of Cardiovascular Disease Prevention: A Review Part 1.

Efforts to better control risk factors for cardiovascular disease and prevent the development of subsequent events are crucial to maintaining healthy populations. In today's busy practice environment and with the overwhelming pace of new research findings, ensuring appropriate emphasis and implementation of evidence-based preventive cardiovascular care can be challenging. The ABCDEF approach to cardiovascular disease prevention is intended to improve dissemination of contemporary best practices and facilitate the implementation of comprehensive preventive strategies for clinicians. This review serves as a succinct yet authoritative overview for internists and subspecialty cardiologists not otherwise focused on cardiovascular prevention. The goal of this 2-part series is to compile a state-of-the-art list of elements central to both primary and secondary prevention of cardiovascular disease, using an ABCDEF checklist, with particular focus on recent society guideline updates. In Part 1 we highlight developments in cardiovascular risk assessment tools, summarize important recent aspirin trials, discuss prevention considerations in atrial fibrillation, and review guidelines for blood pressure categorization, goals, and therapy.

Clinician's Guide to the Updated ABCs of Cardiovascular Disease Prevention: A Review Part 2.

Efforts to better control risk factors for cardiovascular disease and prevent the development of subsequent cardiovascular events are crucial to maintaining healthy populations. In today's busy practice environment and with the overwhelming pace of new research findings, ensuring appropriate emphasis and implementation of evidence-based preventive cardiovascular care can be challenging. The ABCDEF approach to cardiovascular disease prevention is intended to improve dissemination of contemporary best practices and ease the implementation of comprehensive preventive strategies for clinicians. This review serves as a succinct yet authoritative overview for interested internists as well as for cardiologists not otherwise focused on cardiovascular disease prevention. The goal of this 2-part series is to compile a state-of-the-art list of elements central to primary and secondary prevention of cardiovascular disease, using an ABCDEF checklist. In Part 2, we review new recommendations about lipid-modifying strategies, contemporary best practice for tobacco cessation, new evidence related to cardiovascular risk reduction in diabetes using novel therapies, ways to implement a heart-healthy diet, modern interventions to improve physical exercise, and how best to prevent the onset of heart failure.

Discordant Dry Eye Disease (An American Ophthalmological Society Thesis).

PURPOSE: To improve understanding of dry eye disease and highlight a subgroup of patients who have a component of central sensitization and neuropathic pain contributing to their condition. METHODS: Prospective, cross-sectional, IRB-approved study comparing isolated dry eye disease (n=48) to fibromyalgia (positive control; n=23) and healthy (negative control; n=26) individuals with ocular surface examination, corneal confocal microscopy, quantitative sensory testing, and self-reported ocular symptoms and systemic associations. A subset of patients also underwent skin biopsy and/or brain neuroimaging. Dry eye patients were split into concordant (ie, those with dry eyes on examination) and discordant (ie, those with dry eye symptoms but normal examination) subgroups for further analysis. We hypothesized that on the systemic measures included, concordant patients would resemble healthy controls, whereas discordant patients would show evidence of centralized mechanisms similar to fibromyalgia. RESULTS: Schirmer test and Ocular Surface Disease Index (OSDI) scores indicated significant decreases in tear production (Schirmer: healthy, 18.5±8.2 mm; dry, 11.2±5.4 mm; fibromyalgia, 14.4±7.5; P<.001) and increases in self-reported dry eye symptoms (OSDI: healthy, 1.9±3.0; dry, 20.3±17.7; fibromyalgia, 20.3±17.1; P<.001) in the dry eye and fibromyalgia patients, compared to controls. The discordant subgroup had decreased corneal nerve density and decreased visual quality-of-life scores, similar to patients with fibromyalgia. Concordant patients were more similar to healthy controls on these measures. CONCLUSIONS: Individuals with discordant dry eye may have a central pathophysiologic mechanism leading to their eye pain symptoms, which could be an important factor to consider in treatment of chronic idiopathic dry eye.

Sperm morphology in two house mouse subspecies: do wild-derived strains and wild mice tell the same story?

Being subject to intense post-copulatory selection, sperm size is a principal determining component of male fitness. Although previous studies have presented comparative sperm size data at higher taxonomic levels, information on the evolution of sperm size within species is generally lacking. Here, we studied two house mouse subspecies, Mus musculus musculus and Mus musculus domesticus, which undergo incipient speciation. We measured four sperm dimensions from cauda epididymis smears of 28 wild-caught mice of both subspecies. As inbred mouse strains are frequently used as proxies for exploring evolutionary processes, we further studied four wild-derived inbred strains from each subspecies. The subspecies differed significantly in terms of sperm head length and midpiece length, and these differences were consistent for wild mice and wild-derived strains pooled over genomes. When the inbred strains were analyzed individually, however, their strain-specific values were in some cases significantly shifted from subspecies-specific values derived from wild mice. We conclude that: (1) the size of sperm components differ in the two house mouse subspecies studied, and that (2) wild-derived strains reflect this natural polymorphism, serving as a potential tool to identify the genetic variation driving these evolutionary processes. Nevertheless, we suggest that more strains should be used in future experiments to account for natural variation and to avoid confounding results due to reduced variability and/or founder effect in the individual strains.