Na+ Influx Induced by New Antimalarials Causes Rapid Alterations in the Cholesterol Content and Morphology of Plasmodium falciparum.

Among the several new antimalarials discovered over the past decade are at least three clinical candidate drugs, each with a distinct chemical structure, that disrupt Na+ homeostasis resulting in a rapid increase in intracellular Na+ concentration ([Na+]i) within the erythrocytic stages of Plasmodium falciparum. At present, events triggered by Na+ influx that result in parasite demise are not well-understood. Here we report effects of two such drugs, a pyrazoleamide and a spiroindolone, on intraerythrocytic P. falciparum. Within minutes following the exposure to these drugs, the trophozoite stage parasite, which normally contains little cholesterol, was made permeant by cholesterol-dependent detergents, suggesting it acquired a substantial amount of the lipid. Consistently, the merozoite surface protein 1 and 2 (MSP1 and MSP2), glycosylphosphotidylinositol (GPI)-anchored proteins normally uniformly distributed in the parasite plasma membrane, coalesced into clusters. These alterations were not observed following drug treatment of P. falciparum parasites adapted to grow in a low [Na+] growth medium. Both cholesterol acquisition and MSP1 coalescence were reversible upon the removal of the drugs, implicating an active process of cholesterol exclusion from trophozoites that we hypothesize is inhibited by high [Na+]i. Electron microscopy of drug-treated trophozoites revealed substantial morphological changes normally seen at the later schizont stage including the appearance of partial inner membrane complexes, dense organelles that resemble "rhoptries" and apparent nuclear division. Together these results suggest that [Na+]i disruptor drugs by altering levels of cholesterol in the parasite, dysregulate trophozoite to schizont development and cause parasite demise.

Neer Award 2015: Analysis of cytokine profiles in the diagnosis of periprosthetic joint infections of the shoulder.

BACKGROUND: Periprosthetic joint infection (PJI) after shoulder arthroplasty can present a diagnostic and therapeutic challenge. This study evaluated the diagnostic utility of broader synovial fluid cytokine analysis for identifying PJI in patients undergoing revision shoulder arthroplasty. METHODS: Synovial fluid levels of 9 cytokines (interleukin [IL] 6, granulocyte-macrophage colony-stimulating factor, IL-1ß, IL-12, IL-2, IL-8, interferon-γ, IL-10, and tumor necrosis factor-α) were measured in 75 cases of revision shoulder arthroplasty with a multiplex immunoassay. Cases were classified into infection categories and groups based on objective perioperative findings. Differences in cytokine levels among infection groups were evaluated. Receiver operating characteristic curves were used to assess the diagnostic utility of the individual synovial fluid cytokines and combinations of cytokines in determining infection status. RESULTS: Synovial IL-6, granulocyte-macrophage colony-stimulating factor, interferon-γ, IL-1ß, IL-2, IL-8, and IL-10 were significantly elevated in cases of revision shoulder arthroplasty classified as infected. Individually, IL-6, IL-1ß, IL-8, and IL-10 showed the best combination of sensitivity and specificity for predicting infection, and a combined cytokine model consisting of IL-6, tumor necrosis factor-α, and IL-2 showed better diagnostic test characteristics than any cytokine alone, with sensitivity of 0.80, specificity of 0.93,, positive and negative predictive values of 0.87 and 0.89, and positive and negative likelihood ratios of 12.0 and 0.21. CONCLUSIONS: Individual and combined synovial fluid cytokine analysis were both more effective than routine perioperative testing, such as serum erythrocyte sedimentation rate and C-reactive protein, in the diagnosis of PJI of the shoulder. Once validated, combined synovial fluid cytokine analysis could be used as a predictive tool to determine the probability of PJI in patients undergoing revision shoulder arthroplasty and better guide treatment.

Evaluation of commercially available reagents for diagnosis of histoplasmosis infection in immunocompromised patients.

Urinary histoplasma antigen measurement can be useful for diagnosing systemic histoplasmosis and for monitoring treatment response, especially in immunocompromised patients. However, testing has traditionally been limited to specialized reference laboratories, as immunoassay reagents for the antigen were not widely available. Recently, a polyclonal-antibody-based in vitro diagnostic (IVD) kit for histoplasma antigen detection was released, as well as monoclonal-antibody reagents against the target. We evaluated the analytical and clinical performance of the two reagents. Both assays were capable of detecting histoplasma antigen in urine samples over a wide dynamic range, although the monoclonal assay showed improved precision and analytical sensitivity relative to the polyclonal IVD. In a test set of clinically characterized patient samples, the monoclonal laboratory-developed test (LDT) demonstrated 90.5% sensitivity and 96.3% specificity versus 61.9% sensitivity and 79.3% specificity for the polyclonal IVD, with areas under the curve (AUCs) of 0.987 and 0.754, respectively. The major differences between the two assays were higher background reactivity in healthy donors with the polyclonal assay and an increased signal response in positive samples for the monoclonal assay. The impact of these differences on monitoring treatment response was evaluated in a series of patients undergoing treatment for histoplasmosis. While all the assays gave similar qualitative estimates of treatment response, responses were more evident using the monoclonal assay. In summary, we conclude that while multiple assays are available for measuring histoplasma antigen in urine, a monoclonal-antibody-based assay appears to provide improved analytical performance for management of immunocompromised histoplasmosis patients.

Limited utilization of serologic testing in patients undergoing duodenal biopsy for celiac disease.

BACKGROUND: Clinical algorithms for the workup of celiac disease often recommend the use of serologic assays for initial screening, followed by duodenal biopsy for histologic confirmation. However, the majority of duodenal biopsies submitted to pathology for "rule out celiac" are negative. The objective of this study was to determine the underlying causes for this low diagnostic yield. METHODS: We performed a retrospective review of pathology reports from 1432 consecutive duodenal biopsies submitted for pathologic assessment to "rule out celiac" and correlated biopsy results with results for concurrent serologic testing for celiac autoantibodies. RESULTS: The majority of patients had no record of serologic testing prior to biopsy, and evidence of positive serology results was found in only 5% of patients. Most duodenal biopsies were submitted as part of a multi-site GI sampling strategy that included biopsies from other locations. In this context, serologic results correlated with the likelihood of significant duodenal and non-duodenal findings, and were also helpful in evaluating patients with indeterminate duodenal histology. CONCLUSIONS: The presence of a positive screening test for celiac autoantibodies does not appear to be a major driver in the decision to submit duodenal biopsies for evaluation of celiac disease, which accounts for the low incidence of findings in these samples. In patients where celiac serology testing was performed, the results were a good predictor of the likelihood of findings on biopsy.

Remarkable stability in patterns of blood-stage gene expression during episodes of non-lethal Plasmodium yoelii malaria.

BACKGROUND: Microarray studies using in vitro cultures of synchronized, blood-stage Plasmodium falciparum malaria parasites have revealed a 'just-in-time' cascade of gene expression with some indication that these transcriptional patterns remain stable even in the presence of external stressors. However, direct analysis of transcription in P. falciparum blood-stage parasites obtained from the blood of infected patients suggests that parasite gene expression may be modulated by factors present in the in vivo environment of the host. The aim of this study was to examine changes in gene expression of the rodent malaria parasite, Plasmodium yoelii 17X, while varying the in vivo setting of replication. METHODS: Using P. yoelii 17X parasites replicating in vivo, differential gene expression in parasites isolated from individual mice, from independent infections, during ascending, peak and descending parasitaemia and in the presence and absence of host antibody responses was examined using P. yoelii DNA microarrays. A genome-wide analysis to identify coordinated changes in groups of genes associated with specific biological pathways was a primary focus, although an analysis of the expression patterns of two multi-gene families in P. yoelii, the yir and pyst-a families, was also completed. RESULTS: Across experimental conditions, transcription was surprisingly stable with little evidence for distinct transcriptional states or for consistent changes in specific pathways. Differential gene expression was greatest when comparing differences due to parasite load and/or host cell availability. However, the number of differentially expressed genes was generally low. Of genes that were differentially expressed, many involved biologically diverse pathways. There was little to no differential expression of members of the yir and pyst-a multigene families that encode polymorphic proteins associated with the membrane of infected erythrocytes. However, a relatively large number of these genes were expressed during blood-stage infection regardless of experimental condition. CONCLUSIONS: Taken together, these results indicate that 1) P. yoelii gene expression remains stable in the presence of a changing host environment, and 2) concurrent expression of a large number of the polymorphic yir and pyst-a genes, rather than differential expression in response to specific host factors, may in itself limit the effectiveness of host immune responses.

A combined transcriptome and proteome survey of malaria parasite liver stages.

For 50 years since their discovery, the malaria parasite liver stages (LS) have been difficult to analyze, impeding their utilization as a critical target for antiinfection vaccines and drugs. We have undertaken a comprehensive transcriptome analysis in combination with a proteomic survey of LS. Green fluorescent protein-tagged Plasmodium yoelii (PyGFP) was used to efficiently isolate LS-infected hepatocytes from the rodent host. Genome-wide LS gene expression was profiled and compared with other parasite life cycle stages. The analysis revealed approximately 2,000 genes active during LS development, and proteomic analysis identified 816 proteins. A subset of proteins appeared to be expressed in LS only. The data revealed exported parasite proteins and LS metabolic pathways including expression of FASII pathway enzymes. The FASII inhibitor hexachlorophene and the antibiotics, tetracycline and rifampicin, that target the apicoplast inhibited LS development, identifying FASII and other pathways localized in the apicoplast as potential drug targets to prevent malaria infection.

Evaluating the frequency of positive paraneoplastic antibodies and associated malignancy risk.

OBJECTIVE: To evaluate the association between malignancy and frequently positive paraneoplastic antibodies. METHODS: A retrospective cohort study was carried out for all patients who received paraneoplastic antibody testing in 2013-2014 at a tertiary referral center. Available medical records on included patients were reviewed through July 2020. Patients were divided into antibody positive and negative subgroups. Focused analysis was performed on the subgroup of patients who received testing via a commonly used antibody panel. RESULTS: A total of 1860 patients (the full cohort) received 19,323 antibody testing via panel or individual antibody testing, and were followed-up for a mean period of 36.2 months (range 0-83 months). Altogether 229 antibodies in 196 patients were positive, and 9 (3.9%) in 7 patients were against onconeuronal antigens. The remaining 220 (96.1%) were positive for mostly antibodies against cell surface or synaptic antigens. A total of 1161 patients received Mayo Clinic paraneoplastic antibody panel tests (the panel cohort), and 14.9% (173) of these patients possessed one or more positive antibodies. For the panel cohort, no difference was found between antibody positive and negative groups with respect to the prevalence of previously existing malignancy (15.6% versus 16.6%, p = 0.745) or incidence of new malignancy (4.0% vs. 3.7%, p = 0.848) during the follow-up period. No difference was observed in the incidence of new malignancy during follow-up between the antibody positive and negative groups for the 7 most frequently positive antibodies. CONCLUSIONS: The presence of frequently positive antibodies, mostly to cell surface or synaptic antigens, is not clearly associated with the development of malignancy in the subsequent three years.

Elevated levels of the Plasmodium yoelii homologue of macrophage migration inhibitory factor attenuate blood-stage malaria.

The excessive production of proinflammatory cytokines plays a significant role in the pathogenesis of severe malaria. Mammalian macrophage migration inhibitory factor (MIF) (mMIF) is an immune mediator that promotes a sustained proinflammatory response by inhibiting the glucocorticoid-mediated downregulation of inflammation. In addition, Plasmodium parasites also encode a homologue of mammalian MIF that is expressed in asexual-stage parasites. We used the Plasmodium yoelii murine model to study the potential role of parasite-encoded MIF in the pathogenesis of malaria. Antibodies raised against purified, non-epitope-tagged P. yoelii MIF (PyMIF) were used to localize expression in trophozoite- and schizont-stage parasites and demonstrate extracellular release. In vitro, recombinant PyMIF was shown to actively induce the chemotaxis of macrophages but did not induce or enhance tumor necrosis factor alpha (TNF-α) production from peritoneal macrophages. To examine the role of parasite-derived PyMIF in vivo, two transgenic parasite lines that constitutively overexpress PyMIF were generated, one in a nonlethal P. yoelii 17X background [Py17X-MIF(+)] and the other in a lethal P. yoelii 17XL background [Py17XL-MIF(+)]. Challenge studies with transgenic parasites in mice showed that the increased expression of PyMIF resulted in a reduction in disease severity. Mice infected with Py17X-MIF(+) developed lower peak parasitemia levels than controls, while malaria-associated anemia was unaltered. Infection with Py17XL-MIF(+) resulted in a prolonged course of infection and a reduction in the overall mortality rate. Combined, the data indicate that parasite-derived MIF does not contribute significantly to immunopathology but, through its chemotactic ability toward macrophages, may attenuate disease and prolong infection of highly virulent parasite isolates.

A one-step NIST traceable HPLC method for quantitation of vitamin B6 and 4-pyridoxic acid in human plasma.

OBJECTIVES: Vitamin B6 deficiency is associated with a wide spectrum of clinical syndromes. While vitamin B6 status is primarily assessed by measuring the biologically active form of the vitamin, pyridoxal 5-phosphate (PLP), concurrent measurement of the final metabolite 4-pyridoxic acid (PA) can provide additional information regarding supplement intake and hypophosphatasia. The aim of this study is to develop a simple method traceable to the NIST standard reference material 3950 for simultaneous detection of PLP and PA. DESIGN & METHODS: A one-step reverse phase HPLC method with fluorescence detection was developed by evaluating different derivatization conditions, the use of an internal standard and different calibration strategies. The assay analytical performance was evaluated. RESULTS: Pre-column derivatization with semicarbazide showed the best overall performance in terms of signal to noise ratio, retention time and peak shape when compared to pre- or post-column derivatization with chlorite, pre-column or in-mobile phase derivatization using sodium bisulfite. The final method provided an analytical measurement range from 7.8 to 350 â€‹nmol/L for PLP and 3.3-339 â€‹nmol/L for PA, total imprecision <15% and <5% for PLP and PA respectively. Calibration against the NIST standard produced measured values within 3% of NIST assigned PLP values. The use of 4-deoxypyridoxine as internal standard did not improve precision or accuracy when compared to calibration using 5-level external standards. CONCLUSIONS: This method combines derivatization and protein precipitation in one step and is traceable to NIST standard reference material 3950. It is simple and reliable for routine evaluation of vitamin B6 nutrition status.

Functional characterization of a redundant Plasmodium TRAP family invasin, TRAP-like protein, by aldolase binding and a genetic complementation test.

Efficient and specific host cell entry is of exquisite importance for intracellular pathogens. Parasites of the phylum Apicomplexa are highly motile and actively enter host cells. These functions are mediated by type I transmembrane invasins of the TRAP family that link an extracellular recognition event to the parasite actin-myosin motor machinery. We systematically tested potential parasite invasins for binding to the actin bridging molecule aldolase and complementation of the vital cytoplasmic domain of the sporozoite invasin TRAP. We show that the ookinete invasin CTRP and a novel, structurally related protein, termed TRAP-like protein (TLP), are functional members of the TRAP family. Although TLP is expressed in invasive stages, targeted gene disruption revealed a nonvital role during life cycle progression. This is the first genetic analysis of TLP, encoding a redundant TRAP family invasin, in the malaria parasite.

The closed MTIP-myosin A-tail complex from the malaria parasite invasion machinery.

The Myosin A-tail interacting protein (MTIP) of the malaria parasite links the actomyosin motor of the host cell invasion machinery to its inner membrane complex. We report here that at neutral pH Plasmodium falciparum MTIP in complex with Myosin A adopts a compact conformation, with its two domains completely surrounding the Myosin A-tail helix, dramatically different from previously observed extended MTIP structures. Crystallographic and mutagenesis studies show that H810 and K813 of Myosin A are key players in the formation of the compact MTIP:Myosin A complex. Only the unprotonated state of Myosin A-H810 is compatible with the compact complex. Most surprisingly, every side-chain atom of Myosin A-K813 is engaged in contacts with MTIP. While this side-chain was previously considered to prevent a compact conformation of MTIP with Myosin A, it actually appears to be essential for the formation of the compact complex. The hydrophobic pockets and adaptability seen in the available series of MTIP structures bodes well for the discovery of inhibitors of cell invasion by malaria parasites.

Medication Exposure in Highly Adherent Psychiatry Patients.

Medication exposure is dependent upon many factors, the single most important being if the patient took the prescribed medication as indicated. To assess medication exposure for psychotropic and other medication classes, we enrolled 115 highly adherent psychiatry patients prescribed five or more medications. In these patients, we measured 21 psychotropic and 38 nonpsychotropic medications comprising a 59 medication multiplex assay panel. Strict enrollment criteria and reconciliation of the electronic health record medication list prior to study initiation produced a patient cohort that was adherent with 91% of their prescribed medications as determined by comparing medications detected empirically in blood to the electronic health record medication list. In addition, 13% of detected medications were not in the electronic health record medication list. We found that only 53% of detected medications were within the literature-derived reference range with 41% below and 6% above the reference range specific to each medication. When psychotropic medications were analyzed near trough-level, only sertraline was found to be within the literature-derived reference range for all patients tested. Concentrations of the remaining medications indicated extensive exposure below the reference range. This is the first study to empirically and comprehensively assess medication exposure obtained in comorbid polypharmacy patients, minimizing the important behavioral factor of adherence in the study of medication exposure. These data indicate that low medication exposure is extensive and must be considered when therapeutic issues arise, including the lack of response to medication therapy.

Assessment of Patient Medication Adherence, Medical Record Accuracy, and Medication Blood Concentrations for Prescription and Over-the-Counter Medications.

Importance: Inaccurate medication records and poor medication adherence result in incomplete knowledge of therapy for patients. Objective: To study accuracy of medical records and patient adherence by measuring blood concentrations of medications. Design, Setting, and Participants: This cross-sectional study validated a serum-based liquid chromatography-tandem mass spectrometry assay to simultaneously quantify 263 medications used for acute and chronic conditions. The assay panel was applied to 3 clinical patient cohorts: residual serum from 1000 randomly selected samples sent for routine clinical chemistry testing between April 8 and October 6, 2015 (residuals cohort), 50 prospectively enrolled patients in a gastroenterology clinic between March 1 and March 15, 2016, who were prescribed more than 5 medications (gastroenterology care cohort), and a convenience cohort of 296 patients with hypertension who sought care in an emergency department (ED care cohort) between July 1, 2012, and April 25, 2013. Integrated data analysis of the cohorts was performed from August 22 to November 29, 2017. Main Outcomes and Measures: Medication serum concentrations, electronic health record medication lists, and predicted drug interactions. Results: Of the 1346 total samples, 1000 came from the residuals cohort (640 women and 360 men; median age, 60 years [interquartile range (IQR), 44-71 years]), 50 from the gastroenterology care cohort (30 women and 20 men; median age, 66 years [IQR, 62-70 years]), and 296 from the ED care cohort (160 women and 136 men; median age, 59 years [IQR, 52-66 years]). Median medication adherence, defined as the subset of detected medications from the prescription record, was 83% (IQR, 50%-100%) in the residuals cohort, 100% (IQR, 84%-100%) in the gastroenterology care cohort, and 78% (IQR, 57%-100%) in the ED care cohort. Patients adherent to 1 medication were more often adherent to other medications. Among patients prescribed 3 medications or more, there were no significant associations between medication adherence and sex or number of prescribed medications, and there was a modest association between adherence and age. By comparing detected vs prescribed medications, we detected a median of 0 (IQR, 0-2) medications per patient that were not listed in the electronic health record in the residuals cohort, 1 (IQR, 0-2) medication per patient that was not listed in the electronic health record in the gastroenterology care cohort, and 1 (IQR, 0-2) medication per patient that was not listed in the electronic health record in the ED care cohort. A total of 435 patients (43.5%) in the residuals cohort had no discrepancy between the electronic health record and detected medication lists, 22 patients (44.0%) in the gastroenterology care cohort had no discrepancy between the electronic health record and detected medication lists, and 41 patients (13.9%) in the ED care cohort had no discrepancy between the electronic health record and detected medication lists. Half of adverse drug reaction alerts occurred among medications detected without prescription. Conclusions and Relevance: Comprehensive medication monitoring offers promise to improve adherence, the accuracy of medical records, and the safety for patients with polypharmacy.

Simple HPLC Method with Internal Standard for Evaluation of Vitamin B1 Status By Use of Whole Blood.

BACKGROUND: Two primary assays are routinely used for evaluating a patient's vitamin B1 status: plasma free thiamine and whole blood thiamine diphosphate (TDP). TDP is the bioactive form of vitamin B1 and best reflects body stores. Plasma free thiamine levels are driven by recent dietary intake. The objective of this study was to develop a simple HPLC method with an internal standard (IS) that simultaneously measures TDP and thiamine in whole blood, and to assess the use of this single-tube assay to provide comprehensive evaluation of vitamin B1 status. METHODS: The final assay used amprolium thiochrome as an IS, and the sample preparation procedure takes approximately 1 h. Whole blood thiamine and plasma thiamine were concurrently measured for 126 subjects. RESULTS: The analytical measurement range was 1.7 to 442.3 nmol/L (TDP) and 1.7 to 375.4 nmol/L (thiamine), with interassay precisions of 4.0% to 4.8% (TDP) and 2.9% to 8.0% (thiamine), respectively. Method comparison with a reference laboratory HPLC method showed r = 0.9625, slope = 1.021, and intercept = 0.982 (n = 53) for TDP quantification. Whole blood thiamine correlated closely with plasma thiamine levels but were slightly higher with a mean difference of 1.0 nmol/L (range: -3.0 to 5.0 nmol/L). The reference interval for whole blood TDP and thiamine was 84.3 to 213.3 nmol/L and 1.7 to 21.9 nmol/L, respectively. CONCLUSIONS: This assay provides a simple and reliable HPLC method with a suitable IS for quantification of both TDP and thiamine from whole blood. It also eliminates the need for separate samples for TDP and thiamine measurement, which will allow both short-term and long-term vitamin B1 status to be assessed from a single sample.

Medication adherence, medical record accuracy, and medication exposure in real-world patients using comprehensive medication monitoring.

BACKGROUND: Poor adherence to medication regimens and medical record inconsistencies result in incomplete knowledge of medication therapy in polypharmacy patients. By quantitatively identifying medications in the blood of patients and reconciling detected medications with the medical record, we have defined the severity of this knowledge gap and created a path toward optimizing medication therapy. METHODS AND FINDINGS: We validated a liquid chromatography-tandem mass spectrometry assay to detect and/or quantify 38 medications across a broad range of chronic diseases to obtain a comprehensive survey of patient adherence, medical record accuracy, and exposure variability in two patient populations. In a retrospectively tested 821-patient cohort representing U.S. adults, we found that 46% of medications assessed were detected in patients as prescribed in the medical record. Of the remaining medications, 23% were detected, but not listed in the medical record while 30% were prescribed to patients, but not detected in blood. To determine how often each detected medication fell within literature-derived reference ranges when taken as prescribed, we prospectively enrolled a cohort of 151 treatment-regimen adherent patients. In this cohort, we found that 53% of medications that were taken as prescribed, as determined using patient self-reporting, were not within the blood reference range. Of the medications not in range, 83% were below and 17% above the lower and upper range limits, respectively. Only 32% of out-of-range medications could be attributed to short oral half-lives, leaving extensive exposure variability to result from patient behavior, undefined drug interactions, genetics, and other characteristics that can affect medication exposure. CONCLUSIONS: This is the first study to assess compliance, medical record accuracy, and exposure as determinants of real-world treatment and response. Variation in medication detection and exposure is greater than previously demonstrated, illustrating the scope of current therapy issues and opening avenues that warrant further investigation to optimize medication therapy.

Characterization of a Plasmodium falciparum Orthologue of the Yeast Ubiquinone-Binding Protein, Coq10p.

Coenzyme Q (CoQ, ubiquinone) is a central electron carrier in mitochondrial respiration. CoQ is synthesized through multiple steps involving a number of different enzymes. The prevailing view that the CoQ used in respiration exists as a free pool that diffuses throughout the mitochondrial inner membrane bilayer has recently been challenged. In the yeast Saccharomyces cerevisiae, deletion of the gene encoding Coq10p results in respiration deficiency without inhibiting the synthesis of CoQ, suggesting that the Coq10 protein is critical for the delivery of CoQ to the site(s) of respiration. The precise mechanism by which this is achieved remains unknown at present. We have identified a Plasmodium orthologue of Coq10 (PfCoq10), which is predominantly expressed in trophozoite-stage parasites, and localizes to the parasite mitochondrion. Expression of PfCoq10 in the S. cerevisiae coq10 deletion strain restored the capability of the yeast to grow on respiratory substrates, suggesting a remarkable functional conservation of this protein over a vast evolutionary distance, and despite a relatively low level of amino acid sequence identity. As the antimalarial drug atovaquone acts as a competitive inhibitor of CoQ, we assessed whether over-expression of PfCoq10 altered the atovaquone sensitivity in parasites and in yeast mitochondria, but found no alteration of its activity.

Precision profiling and components of variability analysis for Affymetrix microarray assays run in a clinical context.

Although gene expression profiling using microarray technology is widely used in research environments, adoption of microarray testing in clinical laboratories is currently limited. In an attempt to determine how such assays would perform in a clinical laboratory, we evaluated the analytical variability of Affymetrix microarray probesets using two generations of human Affymetrix chips (U95Av2 and U133A). The study was designed to mimic potential clinical applications by using multiple operators, machines, and reagent lots, and by performing analyses throughout a period of several months. A mixed model analysis was used to evaluate the relative contributions of multiple factors to overall variability, including operator, instrument, run, cRNA/cDNA synthesis, and changes in reagent lots. Under these conditions, the average probeset coefficient of variation (CV) was relatively low for present probesets on both generations of chips (mean coefficient of variation, 21.9% and 27.2% for U95Av2 and U133A chips, respectively). The largest contribution to overall variation was chip-to-chip (residual) variability, which was responsible for between 40 to 60% of the total variability observed. Changes in individual reagent lots and instrumentation contributed very little to the overall variability. We conclude that the approach demonstrated here could be applied to clinical validation of Affymetrix-based assays and that the analytical precision of this technique is sufficient to answer many biological questions.

Use and clinical interpretation of pneumococcal antibody measurements in the evaluation of humoral immune function.

Pneumococcal vaccination is a commonly used technique for assessing the humoral immune status of a patient suspected of having immunodeficiency. Interpretation of what constitutes an adequate response, however, can be challenging. This is due to the complexity of the data generated from serotype-specific assays, historical variations in the assays used to measure pneumococcal antibodies, and varying recommendations on the relevant cut points that define response. In this review, we summarize the historical evolution of assays used for this purpose and discuss the analytical considerations that have influenced published data. We also examine current clinical recommendations for defining an adequate response to vaccination, with a particular focus on the interpretation of serotype-specific data generated by multiplex assays.

Co-prescription trends in a large cohort of subjects predict substantial drug-drug interactions.

Pharmaceutical prescribing and drug-drug interaction data underlie recommendations on drug combinations that should be avoided or closely monitored by prescribers. Because the number of patients taking multiple medications is increasing, a comprehensive view of prescribing patterns in patients is important to better assess real world pharmaceutical response and evaluate the potential for multi-drug interactions. We obtained self-reported prescription data from NHANES surveys between 1999 and 2010, and confirm the previously reported finding of increasing drug use in the elderly. We studied co-prescription drug trends by focusing on the 2009-2010 survey, which contains prescription data on 690 drugs used by 10,537 subjects. We found that medication profiles were unique for individuals aged 65 years or more, with ≥98 unique drug regimens encountered per 100 subjects taking 3 or more medications. When drugs were viewed by therapeutic class, it was found that the most commonly prescribed drugs were not the most commonly co-prescribed drugs for any of the 16 drug classes investigated. We cross-referenced these medication lists with drug interaction data from Drugs.com to evaluate the potential for drug interactions. The number of drug alerts rose proportionally with the number of co-prescribed medications, rising from 3.3 alerts for individuals prescribed 5 medications to 11.7 alerts for individuals prescribed 10 medications. We found 22% of elderly subjects taking both a substrate and inhibitor of a given cytochrome P450 enzyme, and 4% taking multiple inhibitors of the same enzyme simultaneously. By examining drug pairs prescribed in 0.1% of the population or more, we found low agreement between co-prescription rate and co-discussion in the literature. These data show that prescribing trends in treatment could drive a large extent of individual variability in drug response, and that current pairwise approaches to assessing drug-drug interactions may be inadequate for predicting real world outcomes.