Impact of COVID-19 on Healthcare Resource Utilisation Among Patients with Inflammatory Bowel Disease in the USA.

BACKGROUND AND AIMS: The impact of the COVID-19 pandemic on patients with inflammatory bowel disease [IBD] is largely unknown. We characterised the impact of COVID-19 on IBD care by conducting an analysis of US health care claims data. METHODS: We obtained de-identified, open-source, health insurance claims data, from January 2019 to December 2020, from the Symphony Health Integrated Dataverse for US adults with IBD, and measured the rates per 1000 patients of five outcomes: colonoscopies; new biologic or small molecule treatment initiations or treatment switches; new biologic or small molecule treatment initiations or treatment switches in patients who had a colonoscopy within the previous 60 days; IBD-related surgeries; and telehealth consultations. RESULTS: For 2019 and 2020, 1.32 million and 1.29 million patients with IBD, respectively, were included in the analysis. In March-April 2020, the rates of colonoscopies [17.39 vs 34.44], new biologic or small molecule treatment initiations or switches in patients who had a colonoscopy within the previous 60 days [0.76 vs 1.18], and IBD-related surgeries [2.33 vs 2.99] per 1000 patients were significantly decreased versus January-February 2020; significant year on year decreases versus 2019 were also observed. Telehealth utilisation increased in March 2020 and remained higher than in 2019 up to December 2020. CONCLUSIONS: Reduction in colonoscopies and subsequent initiation/switching of treatments during the COVID-19 pandemic suggest lost opportunities for therapy optimisation which may have an impact on longer-term patient outcomes. Increased utilisation of telehealth services may have helped address gaps in routine clinical care.

Potential benefits of immunomodulator use with vedolizumab for maintenance of remission in ulcerative colitis.

BACKGROUND AND AIM: This study aimed to determine the efficacy and safety of vedolizumab treatment with or without concomitant immunomodulator use in Japanese patients with moderate-to-severe ulcerative colitis. METHODS: Among enrolled patients in a phase 3 study conducted in Japan (clinicaltrials.gov, NCT02039505), data from patients allocated to 300-mg intravenous vedolizumab for induction and maintenance phases were used for this exploratory analysis. Efficacy endpoints were clinical response, clinical remission, and mucosal healing at week 10 and clinical remission and mucosal healing at week 60, and disease worsening and treatment failure during the maintenance phase. RESULTS: At week 10, the differences in clinical response, clinical remission, and mucosal healing rates between the subgroups (those with concomitant immunomodulator use minus those without) were 0.7 (95% confidence interval: -14.3, 15.7), 3.3 (95% confidence interval: -8.5, 15.2), and 1.8 (95% confidence interval: -13.0, 16.5), respectively. At week 60, the differences in clinical remission and mucosal healing between the subgroups with and without concomitant immunomodulator use were 26.1 (95% confidence interval: -3.5, 55.6) and 29.9 (95% confidence interval: 1.4, 58.4), respectively. The proportions of patients without treatment failure at day 330 of the maintenance phase were 90.7% with concomitant immunomodulator use and 73.7% without. No marked differences in incidence of infections were observed between subgroups. CONCLUSIONS: This study suggested the possibility that concomitant immunomodulator use may be beneficial to maintain the clinical efficacy of vedolizumab.

Real-World Effectiveness of Vedolizumab Dose Escalation in Patients With Inflammatory Bowel Disease: A Systematic Literature Review.

Background: Vedolizumab is a gut-selective anti-lymphocyte trafficking agent approved for the treatment of moderate to severely active inflammatory bowel disease (IBD: ulcerative colitis [UC] and Crohn's disease [CD]). Methods: A systematic literature review (SLR) of real-world studies was conducted to assess the effectiveness of dose escalation of vedolizumab every 8 weeks (Q8W) during maintenance treatment to achieve a response in patients who were either vedolizumab responders experiencing secondary loss of response (SLOR) or non-responders. MEDLINE and EMBASE databases were searched from January 2014 to August 2021. Results: Screening of SLR outputs identified 72 relevant real-world study publications featuring dose escalation of vedolizumab maintenance therapy. After qualitative review, ten eligible studies (9 articles, 1 abstract) were identified as reporting clinical response and/or clinical remission rates following escalation of intravenous vedolizumab 300 mg Q8W maintenance dosing to every 4 weeks (Q4W) maintenance dosing in adult patients with UC/CD (≥10 patients per study). Overall, 196/395 (49.6%) patients with IBD had a response within 54 weeks of vedolizumab maintenance dose escalation. Although definitions for clinical response/remission varied across the 10 studies, clinical response rates after escalated vedolizumab Q8W maintenance dosing ranged from 40.0% to 73.3% (9 studies) and from 30.0% to 55.8% for remission (4 studies) over a range of 8 to <58 weeks' follow-up. Conclusions: This synthesis of real-world effectiveness data in vedolizumab-treated patients with IBD indicates that approximately half were able to achieve or recapture clinical response after escalating vedolizumab maintenance dosing.

Week 2 Symptomatic Response with Vedolizumab as a Predictive Factor in Japanese Anti-TNFα-Naive Patients with Ulcerative Colitis: A post hoc Analysis of a Randomized, Placebo-Controlled Phase 3 Trial.

BACKGROUND AND AIM: To evaluate the onset of symptomatic response with vedolizumab in patients with moderate-to-severe ulcerative colitis in Japan. METHODS: Patients were randomized to receive vedolizumab 300 mg or placebo at Weeks 0, 2, and 6. Mayo subscores were analyzed in patients with baseline stool frequency (SF) ≥1 and rectal bleeding (RB) ≥1. In patients with baseline SF ≥2 and RB ≥1, the proportion who achieved SF ≤1 and RB = 0 was determined. RESULTS: Patients were randomized to vedolizumab (n = 164) or placebo (n = 82). Decrease from baseline in mean SF subscore was greater with vedolizumab versus placebo from Week 2 (-6.6%; 95% confidence interval [CI], -16.2, 3.0), with a greater difference in anti-tumor necrosis factor (TNF)α-naive patients (vedolizumab vs. placebo, -13.2%; 95% CI, -29.7, 3.3). Mean percentage decrease from baseline RB subscore was numerically greater with vedolizumab versus placebo from Week 6 in anti-TNFα-naive patients (-10.7%; 95% CI, -33.0, 11.5). More patients in the anti-TNFα-naive subgroup achieved SF ≤1 and RB = 0 with vedolizumab versus placebo at Week 2 (14.8%; 95% CI, 2.5, 27.0) and Week 6 (20.3%; 95% CI, 4.4, 36.2). Patients with SF ≤1 and RB = 0 at Week 2 had higher clinical response, clinical remission, and mucosal healing rates at Week 10 than those without. CONCLUSIONS: Our results indicate that vedolizumab induces a rapid symptomatic response, particularly in anti-TNFα-naive patients, and suggest that early symptomatic improvement predicts treatment response at Week 10 (NCT02039505).

Efficacy of biologic therapies for biologic-naïve Japanese patients with moderately to severely active ulcerative colitis: a network meta-analysis.

BACKGROUND/AIMS: Several biologic therapies are approved in Japan to treat moderately to severely active ulcerative colitis (UC), but there are no published comparative efficacy studies in a Japanese population. We compared the efficacy of biologics approved in Japan (adalimumab, infliximab, golimumab, and vedolizumab) for treating biologic-naïve patients with UC at their approved doses. METHODS: A targeted literature review identified 4 randomized controlled trials of biologics for UC in biologicnaïve Japanese patients. For each study, efficacy outcome data from induction (weeks 6-12) and maintenance (weeks 30-60) treatment were extracted for analysis. Treatment effects on clinical response, clinical remission, and mucosal healing relative to the average placebo results across all trials were estimated using network meta-analyses followed by transformation into probabilities and odds ratios (OR). RESULTS: At the end of induction, the likelihood of clinical response and clinical remission was highest with infliximab (OR: 2.12 and 2.35, respectively) and vedolizumab (OR: 2.10 and 2.32, respectively); the likelihood of mucosal healing was highest with infliximab (OR: 2.24) and adalimumab (OR: 1.86). During maintenance, the likelihood of clinical response and clinical remission was highest with vedolizumab (OR: 6.44 and 4.68, respectively) and golimumab (OR: 5.13 and 3.84, respectively); the likelihood of mucosal healing was significantly higher than placebo with all biologics. CONCLUSIONS: All active treatments were efficacious compared with placebo. Infliximab and vedolizumab had the highest odds for induction of clinical response, remission, and mucosal healing. Golimumab and vedolizumab had numerically higher odds of achieving efficacy outcomes in the maintenance phase.

Optimizing Selection of Biologics in Inflammatory Bowel Disease: Development of an Online Patient Decision Aid Using Conjoint Analysis.

OBJECTIVES: Recent drug approvals have increased the availability of biologic therapies for inflammatory bowel disease (IBD), making it difficult for patients with ulcerative colitis (UC) and Crohn's disease (CD) to navigate treatment options. Here we developed a conjoint analysis to examine patient decision-making surrounding biologic medicines for IBD. We used the results to create an online patient decision aid that generates a unique "preferences report" for each patient to assist with shared decision-making with their provider. METHODS: We administered an adaptive choice-based conjoint survey to IBD patients that quantifies the relative importance of biologic attributes (e.g., efficacy, side effect profile, mode of administration, and mechanism of action) in decision making. The conjoint software determined individual patient preferences by calculating part-worth utilities for each attribute. We conducted regression analyses to determine if demographic and disease characteristics (e.g., type of IBD and severity) predicted how patients made decisions. RESULTS: 640 patients completed the survey (UC=304; CD=336). In regression analyses, demographics and IBD characteristics did not predict individual patient preferences; the main exception was IBD type. When compared to UC, CD patients were more likely to report side effect profile as most important (odds ratio (OR) 1.63, 95% confidence interval (CI) 1.16-2.30). Conversely, those with UC were more likely to value therapeutic efficacy (OR 1.41, 95% CI 1.01-2.00). CONCLUSIONS: Biologic decision-making is highly personalized; demographic and disease characteristics poorly predict individual preferences, indicating that IBD patients are unique and difficult to statistically categorize. The online decision tool resulting from this study (www.ibdandme.org) may be used by patients to support shared decision-making and optimize personalized biologic selection with their provider.

Patient Understanding of the Risks and Benefits of Biologic Therapies in Inflammatory Bowel Disease: Insights from a Large-scale Analysis of Social Media Platforms.

BACKGROUND: Few studies have examined inflammatory bowel disease (IBD) patients' knowledge and understanding of biologic therapies outside traditional surveys. Here, we used social media data to examine IBD patients' understanding of the risks and benefits associated with biologic therapies and how this affects decision-making. METHODS: We collected posts from Twitter and e-forum discussions from >3000 social media sites posted between June 27, 2012 and June 27, 2015. Guided by natural language processing, we identified posts with specific IBD keywords that discussed the risks and/or benefits of biologics. We then manually coded the resulting posts and performed qualitative analysis using ATLAS.ti software. A hierarchical coding structure was developed based on the keyword list and relevant themes were identified through manual coding. RESULTS: We examined 1598 IBD-related posts, of which 452 (28.3%) centered on the risks and/or benefits of biologics. There were 5 main themes: negative experiences and concerns with biologics (n = 247; 54.6%), decision-making surrounding biologic use (n = 169; 37.4%), positive experiences with biologics (n = 168; 37.2%), information seeking from peers (n = 125; 27.7%), and cost (n = 38; 8.4%). Posts describing negative experiences primarily commented on side effects from biologics, concerns about potential side effects and increased cancer risk, and pregnancy safety concerns. Posts on decision-making focused on nonbiologic treatment options, hesitation to initiate biologics, and concerns about changing or discontinuing regimens. CONCLUSIONS: Social media reveals a wide range of themes governing patients' experience and choice with IBD biologics. The complexity of navigating their risk-benefit profiles suggests merit in creating online tailored decision tools to support IBD patients' decision-making with biologic therapies.

Gastroenterologists' Perceptions Regarding Ulcerative Colitis and Its Management: Results from a Large-Scale Survey.

INTRODUCTION: Misperceptions about ulcerative colitis (UC) may influence management strategies and limit opportunities for improving patient outcomes. This study assessed physicians' perceptions of UC, concepts of disease severity and remission, and treatment goals. METHODS: Gastroenterologists who typically treated ≥10 adults with UC per month were recruited for a large-scale, web-based survey. Participants were asked about their perceptions of UC (often vs. Crohn's disease [CD]), treatment goals, and medication use. Response data were evaluated via descriptive statistics and univariate and multivariable analyses. RESULTS: Gastroenterologists (N = 500) with a mean of 16.5 years (standard deviation, 8.7 years) in practice participated. In comparison to CD, survey respondents perceived UC as being easier to diagnose, having better treatment outcomes, and being associated with later prescribing of biologics. Treatment goals commonly considered to have the greatest importance included quality of life improvement (31.2% of respondents), maintenance of clinical remission (17.4%), and mucosal healing (17.4%). When respondents evaluated the performance of medication classes in achieving these goals, biologics were rated significantly higher than all other classes (P < 0.05). However, the most common drivers for the initiation of biologic therapy were the development of steroid refractoriness (66.8%) and steroid dependency (65.8%). Medication class use by UC severity was generally consistent with the traditional step-up approach to UC therapy, with biologics being used most commonly for severe UC. CONCLUSION: These results suggest a possible disparity between treatment goals and therapeutic management in UC. An increased awareness of general UC perceptions is an important step toward a better overall understanding of the disease and, ultimately, toward improved management aligned with treatment goals. FUNDING: This study was sponsored by the American Gastroenterological Association (AGA), and the design and conduct of the study as well as article processing charges and the open access fee for this publication were funded by Takeda Pharmaceuticals International, Inc. (TPI).

Balancing opioid-induced gastrointestinal side effects with pain management: Insights from the online community.

Opioids cause gastrointestinal (GI) symptoms such as nausea, vomiting, pain, and (in 40 percent) constipation that diminish patients' quality of life. Outside traditional surveys, little is known about the opioid-induced constipation (OIC) patient experience and its impact on pain management. The purpose of this study was to use data from social media platforms to qualitatively examine patient beliefs about OIC and other prominent GI side effects, their impact on effective pain management and doctor-patient interaction. The authors collected Tweets from March 25 to July 31, 2014, and e-forum posts from health-related social networking sites regardless of timestamp. The authors identified specific keywords related to opioids and GI side effects to locate relevant content in the dataset, which was then manually coded using ATLAS.ti software. The authors examined 2,519,868 Tweets and more than 1.8 billion e-forum posts, of which, 88,586 Tweets and 9,767 posts satisfied the search criteria. Three thousand three individuals experienced opioidinduced GI side effects, mostly related to phenanthrenes (n = 1,589), and 1,274 (42.4 percent) individuals described constipation. Over-the-counter medications and nonevidence-based natural approaches were most commonly used to alleviate constipation. Many individuals questioned, rotated, reduced, or stopped their opioid treatments as a result of their GI side effects. Investigation of social media reveals a struggle to balance pain management with opioid-induced GI side effects, especially constipation. Individuals are often unprepared to treat OIC, to modify opioid regiments without medical advice, and to resort to using natural remedies and treatments lacking scientific evidence of effectiveness. These results identify opportunities to improve physician-patient communication and explore effective treatment alternatives.

X-ray fluorescence microscopy reveals large-scale relocalization and extracellular translocation of cellular copper during angiogenesis.

Although copper has been reported to influence numerous proteins known to be important for angiogenesis, the enhanced sensitivity of this developmental process to copper bioavailability has remained an enigma, because copper metalloproteins are prevalent and essential throughout all cells. Recent developments in x-ray optics at third-generation synchrotron sources have provided a resource for highly sensitive visualization and quantitation of metalloproteins in biological samples. Here, we report the application of x-ray fluorescence microscopy (XFM) toin vitro models of angiogenesis and neurogenesis, revealing a surprisingly dramatic spatial relocalization specific to capillary formation of 80-90% of endogenous cellular copper stores from intracellular compartments to the tips of nascent endothelial cell filopodia and across the cell membrane. Although copper chelation had no effect on process formation, an almost complete ablation of network formation was observed. XFM of highly vascularized ductal carcinomas showed copper clustering in putative neoangiogenic areas. This use of XFM for the study of a dynamic developmental process not only sheds light on the copper requirement for endothelial tube formation but highlights the value of synchrotron-based facilities in biological research.

Subtractive transcriptomics: establishing polarity drives in vitro human endothelial morphogenesis.

Although investigations of mature normal and tumor-derived capillaries have resulted in characterization of these structures at the phenotypic level, less is known regarding the initial molecular cues for cellular assembly of endothelial cells into human capillaries. Here, we employ a novel combination of microenvironmental manipulation and microarray data filtration over narrowly delineated temporal data series to identify the morphogenesis component apart from the proliferation component, as pooled human microvascular-derived endothelial cells are induced to form capillary-like structures in vitro in a murine tumor-derived matrix. The 217 morphogenesis-specific genes identified using this subtractive transcriptomics approach are mostly independent of the angiogenic proteins currently used as therapeutic targets for aberrant angiogenesis. Quantitative real-time PCR was used to validate 20% of these transcripts. Immunofluorescent analysis of proliferating and tube-forming cells validates at the protein level the morphogenesis-specific expression pattern of 16 of the 217 gene products identified. The transcripts that are selectively up-regulated in tube-forming endothelial cells reveal a temporal expression pattern of genes primarily associated with intracellular trafficking, guided migration, cytoskeletal reorganization, cellular adhesion, and proliferation inhibition. These data show that a sequential up-regulation of genes that establish and maintain polarity occurs during migration and morphogenesis of in vitro human endothelial cells undergoing tubulogenesis; some of which may well be effective as novel antiangiogenic drug targets.

Drug resistance-associated pfCRT mutations confer decreased Plasmodium falciparum digestive vacuolar pH.

Elucidating the altered physiology of various chloroquine resistant (CQR) strains of Plasmodium falciparum is essential for understanding the molecular basis of CQR. In this study, we have devised several new methods for analyzing digestive vacuolar (DV) pH for individual intraerythrocytic parasites under continuous perfusion. These use controlled illumination power and novel data acquisition software, and are based on either acridine orange (AO) emission spectra or ratiometric 5-(and 6-)carboxy-2',7'-dimethyl-3'-hydroxy-6'-N-ethylaminospiro [isobenzofuran-1(3H),9'-(9H)xanthen]-3-one (DM NERF) excitation. Results show that DV pH is more acidic for laboratory strains of CQR parasites relative to chloroquine sensitive (CQS). Using mutant pfcrt allelic exchange clones not previously exposed to chloroquine (CQ), we now show a direct association between acid DV pH, CQ resistance and mutation of pfcrt to either South American (7G8) or South East Asian (Dd2) CQR-associated alleles. Surprisingly, these alleles confer a similar degree of DV acidification. Verapamil (VPL) reversed acid DV pH for the Dd2 mutant C3(Dd2) clone, in a surprisingly rapid fashion, but did not reverse acid DV pH for the 7G8 mutant C6(7G8) clone. Thus, there is a direct link between expression of two major CQR-associated pfcrt alleles and altered parasite DV physiology. The data also support models that envision direct but allele-specific interaction between PfCRT and VPL.

Chloroquine resistance modulated in vitro by expression levels of the Plasmodium falciparum chloroquine resistance transporter.

Plasmodium falciparum malaria is increasingly difficult to treat and control due to the emergence of parasite resistance to the major antimalarials, notably chloroquine. Recent work has shown that the chloroquine resistance phenotype can be conferred by multiple amino acid mutations in the parasite digestive vacuole transmembrane protein PfCRT. Here, we have addressed whether chloroquine resistance can also be affected by changes in expression levels of this protein. Transient transfection reporter assays revealed that truncation of the pfcrt 3'-untranslated region just prior to putative polyadenylation sites resulted in a 10-fold decrease in luciferase expression levels. Using allelic exchange on a chloroquine-resistant line (7G8 from Brazil), this truncated 3'-untranslated region was inserted downstream of the pfcrt coding sequence, in the place of the endogenous 3'-untranslated region. The resulting pfcrt-modified "knockdown" clones displayed a marked decrease in pfcrt transcription and an estimated 30-40% decrease in PfCRT protein expression levels. [3H]hypoxanthine incorporation assays demonstrated up to a 40% decrease in chloroquine with or without verapamil IC50 levels of pfcrt knockdown clones, relative to the 7G8 parent. Single-cell photometric analyses were consistent with an altered intracellular pH in the knockdown clones, providing further evidence for a relationship between PfCRT, pH regulation, and chloroquine resistance. Genetic truncation of 3'-untranslated regions provides a useful approach for assessing the impact of candidate genes on drug resistance or other quantifiable phenotypes in P. falciparum.

Chloroquine resistance in the malarial parasite, Plasmodium falciparum.

Malarial parasites remain a health problem of staggering proportions. Worldwide, they infect about 500 million, incapacitate tens of millions, and kill approximately 2.5 million (mostly children) annually. Four species infect humans, but most deaths are caused by one particular species, Plasmodium falciparum. The rising number of malarial deaths is due in part to increased drug resistance in P. falciparum. There are many varieties of antimalarial drug resistance, and there may very well be several molecular level contributions to each variety. This is because there are a number of different drugs with different mechanisms of action in use, and more than one molecular event may sometimes be relevant for resistance to any one class of drugs. Thus, "multidrug" resistance in a clinical setting likely entails complex combinations of overlapping resistance pathways, each specific for one class of drug, that then add together to confer the particular multidrug resistance phenotype. Nonetheless, rapid progress has been made in recent years in elucidating mechanisms of resistance to specific classes of antimalarial drugs. As one example, resistance to the antimalarial drug chloroquine, which has been the mainstay therapy for decades, is becoming well understood. This article focuses on recent advances in determining the molecular mechanism of chloroquine resistance, with particular attention to the biochemistry and biophysics of the P. falciparum digestive vacuole, wherein changes in pH have recently been found to be associated with chloroquine resistance.

Solution structures of antimalarial drug-heme complexes.

Paramagnetic metal centers [such as Fe(III) found within ferriprotoporphyrin IX heme (FPIX)] exert through space effects on the relaxation rate of nearby proton spins that depend critically on the metal-proton distance. We have measured these effects for all protons of several antimalarial drugs that bind to FPIX by systematically varying the drug:heme molar ratio in high field NMR experiments. These measurements allow us to determine precise FPIX Fe-drug H distances for the solution structures of noncovalent complexes formed between FPIX mu-oxo dimers and the antimalarial drugs chloroquine (CQ), quinine (QN), and quinidine (QD). Using these distances, we then performed distance restraint calculations to determine the lowest-energy solution structures of these complexes. Structures were solved for neutral, monoprotic (+1), and diprotic (+2) forms of the drugs. Analysis of these structures allows us to visualize for the first time the stereospecific differences between QN and QD binding to FPIX and the differences in populations of QN and QD solution structures upon changes in digestive vacuolar pH for drug resistant malarial parasites [Dzekunov, S. M., et al. (2000) Mol. Biochem. Parasitol. 110, 107-124]. The data indicate a previously unrecognized key role for the CQ aliphatic chain in stabilizing FPIX-CQ complexes, and suggest how lengthening or shortening the chain might perturb stability. We also define FPIX:drug stoichiometries of 2:1 for the complexes formed at physiological FPIX concentrations, in contrast to the 4:1 and 5:1 stoichiometries previously determined at higher FPIX concentrations [Dorn, A., et al. (1998) Biochem. Pharmacol. 55, 727-736]. These atomic resolution antimalarial drug-heme structures should help elucidate how these drugs inhibit formation of hemozoin during metabolism of heme within the malarial parasite Plasmodium falciparum and assist ongoing development of strategies for circumventing antimalarial drug resistance.

Alternative mutations at position 76 of the vacuolar transmembrane protein PfCRT are associated with chloroquine resistance and unique stereospecific quinine and quinidine responses in Plasmodium falciparum.

Chloroquine resistance (CQR) in Plasmodium falciparum is associated with multiple mutations in the digestive vacuole membrane protein PfCRT. The chloroquine-sensitive (CQS) 106/1 line of P. falciparum has six of seven PfCRT mutations consistently found in CQR parasites from Asia and Africa. The missing mutation at position 76 (K76T in reported population surveys) may therefore be critical to CQR. To test this hypothesis, we exposed 106/1 populations (10(9)-10(10) parasites) to a chloroquine (CQ) concentration lethal to CQS parasites. In multiple independent experiments, surviving CQR parasites were detected in the cultures after 28 to 42 days. These parasites showed novel K76N or K76I PfCRT mutations and corresponding CQ IC(50) values that were approximately 8- and 12-fold higher than that of the original 106/1 IC(50). A distinctive feature of the K76I line relative to 106/1 parasites was their greatly increased sensitivity to quinine (QN) but reduced sensitivity to its enantiomer quinidine (QD), indicative of a unique stereospecific response not observed in other CQR lines. Furthermore, verapamil had the remarkable effect of antagonizing the QN response while potentiating the QD response of K76I parasites. In our single-step drug selection protocol, the probability of the simultaneous selection of two specific mutations required for CQR is extremely small. We conclude that the K76N or K76I change added to the other pre-existing mutations in the 106/1 PfCRT protein was responsible for CQR. The various mutations that have now been documented at PfCRT position 76 (K76T, K76N, K76I) suggest that the loss of lysine is central to the CQR mechanism.