Occurrence of Colletotrichum musicola causing anthracnose on Manihot esculenta.

Cassava (Manihot esculenta) is one of the main food sources of energy in developing countries owing to its starch-rich roots (Pinweha et al., 2015). Anthracnose is considered the most destructive disease of the aerial part of this crop (Bragança et al., 2016; Liu et al., 2019), and it is caused by species such as Colletotrichum plurivorum, C. karstii, C. fructicola, C. siamense (Liu et al., 2019), and C. theobromicola (Oliveira et al, 2016). In 2019, leaves with irregular necrotic spots, typical symptoms of anthracnose, were collected in Pará, Brazil. Commercial sampled fields showed 20% of incidence of anthracnose. Colletotrichum strains were isolated and cultured on potato dextrose agar at 25 ºC with a 12-h light photoperiod from surface-disinfected (70% alcohol and 1% sodium hypochlorite) lesion transition area. Five of the obtained isolates exhibited brown colonies on the upper and lower surfaces. Conidia were hyaline, cylindrical and aseptate, 12.82-15.23 µm × 3.52-5.25 µm in size. These phenotypic characters were similar to those belonging to C. orchidearum sensu lato (Damm et al. 2019). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), ß-tubulin (TUB2), chitin synthase 1(CHS-1), and histone HIS3 partial gene were amplified and sequenced for one representative isolate (UFT/Coll89). Sequences were deposited in GenBank [Accession numbers: MT396235 (GAPDH), MT800856 (TUB2), MT800870 (CHS-1), and MT856672 (HIS3)]. BLASTn searches of CHS-1 and HIS3 sequences showed 100% identity to C. musicola. Maximum Likelihood Phylogenetic analysis, including previously published sequences of closely related species, placed the isolate from Cassava in the C. musicola clade with 100% support, and confidently it assigned to this species. Pathogenicity was proven with inoculations by spraying a conidial suspension (106 conida mL-1) on 3-month-old cassava plants (three unwounded leaves per plant). The plants were placed in a humid chamber at 25 °C for 48h, and a 12-h photoperiod. The negative control was represented by plants inoculated with sterile distilled water. The experiment was repeated twice. The same symptoms observed in the field were reproduced only in inoculated leaves, from which the pathogen was reisolated lesions fulfilling Koch's postulates. No symptoms were observed on the negative control. To our knowledge, this is the first report of C. musicola joining a group of new and emergent species of Colletotrichum causing anthracnose in cassava producing regions around the world. The identification of this species causing cassava anthracnose is crucial to improve the disease control strategies and resistance breeding programs.

First report of Colletotrichum truncatum causing anthracnose in cassava in Brazil.

Cassava (Manihot esculenta Crantz) presents significant economic importance in Brazil and other developing countries due to its use in human and animal feeding. In 2019, cassava plants sampled in Pará state (Brazil) presented necrotic and irregular leaf spots, characteristic symptoms of cassava anthracnose. About 90% of the plants were symptomatic, and disease severity was higher during months with high temperature and humidity. Fragments of symptomatic tissues were removed from the lesion transition area, surface disinfested (45 s in 70% ethanol, 1 min in 1% NaOCl, and rinsed twice in sterile water), and plated on potato dextrose agar. Cultures were incubated at 25 °C under continuous light for 7 days. Among the obtained isolates, seven presented grey felt-like mycelium with white sectors, reverse greyish, and hyaline, aseptate, smooth-walled, falcate conidia with average size 20.7-30.7 (26.1 ± 2.1) × 2.4-4.8 (3.5 ± 0.5) µm. Phenotypical features were similar to C. truncatum (Damm et al. 2019). The representative isolate UFT/Coll87 was chosen for further assays. The identity of the isolate was determined by maximum likelihood analysis using sequences of actin (ACT, GenBank accession number MT321653), ß-tubulin (TUB2, MT856673) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, MT800857) partial regions. Colletotrichum isolate from cassava nested with C. truncatum isolates in a clade with 100% support, being confidently assigned to this species. Koch's postulates were fulfilled to confirm the pathogenicity of UFT/Coll87. Inoculation was carried out in three cassava plants by spraying a conidial suspension (106 conida mL-1) on unwounded leaves (three leaves per plant). Plants sprayed with sterile water represented negative control. Inoculated plants were kept in a humid chamber for 48 h, 25 °C, and a 12-h photoperiod. The experiment was repeated 2 times. Typical cassava anthracnose symptoms were observed 10 days after inoculation. No symptoms were observed in negative control. The pathogen was reisolated from symptomatic leaves and was phenotypically identical to the original isolate UFT/Coll87, fulfilling Koch's postulates. Colletotrichum fructicola, C. karstii, C. plurivorum, and C. siamense were reported causing cassava anthracnose in China (Liu et al. 2019). In Brazil, C. chrysophilum, C. fructicola, C. siamense and C. theobromicola were reported in association with cassava (Bragança et al. 2016; Oliveira et al. 2018; Machado et al. 2020). To our knowledge, this is the first report of C. truncatum causing cassava anthracnose worldwide. Our finding is important for disease management due to the high host range of C. truncatum. The pathogen can reduce the cassava yield, and the crop may serve as a potential inoculum source since it is commonly cultivated near to other crops that are also infected by C. truncatum.

Dietary habits in a group of patients with multiple sclerosis are similar to those of healthy control subjects.

BACKGROUND: Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system. Over time, patients with MS accumulate neurological disabilities. MS typically affects young adults and is associated with an inflammatory profile of cytokines and lymphocytes. If a patient were to consume a potentially inflammatory diet, it is possible that the evolution of MS in that individual would be more aggressive. OBJECTIVE: To investigate whether patients with MS living in and around the city of Santos, São Paulo, Brazil, had a profile of inflammatory diet. METHODS: Patients with MS and healthy control subjects were individually interviewed, and the 24-hour Diet Recall and the Bristol Stool Form Scale were applied. Salt intake was calculated using the WebDiet 2.0 software. RESULTS: There were no remarkable differences in dietary habits between healthy control subjects (n=34) and patients with MS (n=66), except for higher consumption of carbohydrates by patients. Both patients with MS and control subjects had higher protein and lower carbohydrate intake than the World Health Organization's recommended daily amounts. There was no correlation between food intake and neurological disability in patients with MS. CONCLUSION: The dietary patterns of patients with MS and healthy controls were similar in the city of Santos, São Paulo, Brazil, and surrounding towns, except for higher intake of carbohydrates by patients. No profile of pro-inflammatory diets was identified among the patients with MS enrolled in this study.

First report of Colletotrichum chrysophillum causing cassava anthracnose in Brazil.

Cassava (Manihot esculenta Crantz) has significant socioeconomic relevance in Brazil and other developing countries, as one of the main sources of carbohydrates for human and animal consumption (De Oliviera et al., 2011). Among the cassava crop diseases, anthracnose is one of the main limiting factors for production and may be caused by species like Colletotrichum plurivorum, C. karstii, C. fructicola, and C. siamense (Bragança et al., 2016; Liu et al., 2019; Oliveira et al., 2016; Sangpueak; Phansak; Buensanteai, 2018). Severity in the field is variable, depending on the resistance of the variety used and is also highly influenced by the climate, being the most severe disease under high humidity and high temperature. Under these conditions, it can cause losses of up to 100%. In 2019, cassava leaves presenting dark brown necrotic injuries of different sizes and irregular borders-typical anthracnose symptoms- were collected from commercial plantations in the states of Pará and Tocantins, Brazil. Symptomatic tissue fragments were superficially disinfected, placed in plates with potato dextrose agar (PDA), and incubated under 25 ± 2 °C for seven days. In the 56 isolates used in the morphological identification, the colonies were white and gray at the top and dark gray in the bottom with sector formation. The conidia were hyaline, cylindrical, and aseptic, 10.04 to 17.83 µm long × 3.29 to 5.75 µm wide. These phenotypical characteristics were similar to those of C. gloeosporioides lato sensu species (Weir et al., 2012). Genomic DNA was extracted from two representative isolates (UFT/Coll69, collected in the municipality of Casa de Tábua-PA; UFT/Coll82, collected in Pau Darco-PA) and the APN2 / MAT-IGS, DNA lyase (Apn2), and glyceraldehyde-3-phosphate dehydrogenase-IGS (GAP2-IG) intergenic spacers were amplified and sequenced. The nucleotide sequences were deposited in the GenBank (accession numbers: MT409462, MT396231, MT759633, MT396239, MT396232, MT800846). The BLASTn (Basic Local Alignment Search Tool) showed a 99 to 100% similarity with Colletotrichum chrysophillum. The maximum likelihood phylogenetic analysis grouped the isolates in the C. chrysophillum clade, with a high bootstrap value (98%). Based on morphocultural characteristics and the phylogenetic analysis, the isolates associated with M. esculenta anthracnose were identified as C. chrysophillum, with a frequency of 6.67% among Colletotrichum colonies isolated from cassava leaves. The inoculation of three isolates was carried out in three plants, three leaves for each plant, by spraying spore solution with a concentration of 1×106 conidia / ml, without wounding the leaves and placed in a humid chamber at 25 ° C for ten days. Control plants were inoculated with sterile distilled water. From the 2nd day after inoculation, small irregular necrotic lesions appeared that increased in size over time, while control plants remained asymptomatic. Both were pathogenic and the symptoms caused after inoculation were similar to each other and to those observed in the field. In Brazil, anthracnose by C. chrysophillum was reported in cashew (Veloso et al., 2018) and banana trees (Vieira et al., 2017). To our knowledge, this is the first report of cassava anthracnose disease by C. chrysophillum.

Dietary habits in a group of patients with multiple sclerosis are similar to those of healthy control subjects / Hábitos alimentares de pacientes com esclerose múltipla são semelhantes àqueles de controles saudáveis

ABSTRACT Background: Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system. Over time, patients with MS accumulate neurological disabilities. MS typically affects young adults and is associated with an inflammatory profile of cytokines and lymphocytes. If a patient were to consume a potentially inflammatory diet, it is possible that the evolution of MS in that individual would be more aggressive. Objective: To investigate whether patients with MS living in and around the city of Santos, São Paulo, Brazil, had a profile of inflammatory diet. Methods: Patients with MS and healthy control subjects were individually interviewed, and the 24-hour Diet Recall and the Bristol Stool Form Scale were applied. Salt intake was calculated using the WebDiet 2.0 software. Results: There were no remarkable differences in dietary habits between healthy control subjects (n=34) and patients with MS (n=66), except for higher consumption of carbohydrates by patients. Both patients with MS and control subjects had higher protein and lower carbohydrate intake than the World Health Organization's recommended daily amounts. There was no correlation between food intake and neurological disability in patients with MS. Conclusion: The dietary patterns of patients with MS and healthy controls were similar in the city of Santos, São Paulo, Brazil, and surrounding towns, except for higher intake of carbohydrates by patients. No profile of pro-inflammatory diets was identified among the patients with MS enrolled in this study.

RESUMO Introdução: Esclerose múltipla (EM) é uma doença crônica e autoimune do sistema nervoso central. Ao longo do tempo, pacientes com EM acumulam incapacidades neurológicas. A EM tipicamente afeta adultos jovens e se associa a um perfil inflamatório de citocinas e linfócitos. Se um paciente tiver uma dieta potencialmente inflamatória, é possível que a evolução da EM seja mais agressiva neste indivíduo. Objetivo: Investigar se pacientes com EM residentes na cidade de Santos e região, São Paulo, Brasil, apresentam um perfil de dieta inflamatória. Métodos: Pacientes com EM e controles saudáveis foram individualmente entrevistados e um Recordatório da Dieta de 24 horas e a Escala de Bristol de Tipo de Fezes foram aplicadas. A ingesta de sal foi calculada usando o WebDiet 2.0 software. Resultados: Não houve diferença marcante nos hábitos alimentares de controles saudáveis (n=34) e pacientes com EM (n=66), exceto pelo maior consumo de carboidratos pelos pacientes. Tanto os pacientes com esclerose múltipla quanto controles tinham maior consumo de proteína e menor consumo de carboidratos do que o recomendado pela Organização Mundial de Saúde. Não houve correlação entre o consumo de alimentos e a incapacidade neurológica dos pacientes com EM. Conclusão: O padrão de dieta de pacientes com EM e controles saudáveis é semelhante na cidade de Santos, SP, Brasil e região circunvizinha, exceto pelo maior consumo de carboidratos pelos pacientes. Não foi identificado um perfil de dieta pró-inflamatória entre os pacientes incluídos neste estudo.

Bioequivalence evaluation of sparse sampling pharmacokinetics data using bootstrap resampling method.

Bioequivalence studies are an essential part of the evaluation of generic drugs. The most common in vivo bioequivalence study design is the two-period two-treatment crossover design. The observed drug concentration-time profile for each subject from each treatment under each sequence can be obtained. AUC (the area under the concentration-time curve) and Cmax (the maximum concentration) are obtained from the observed drug concentration-time profiles for each subject from each treatment under each sequence. However, such a drug concentration-time profile for each subject from each treatment under each sequence cannot possibly be available during the development of generic ophthalmic products since there is only one-time point measured drug concentration of aqueous humor for each eye. Instead, many subjects will be assigned to each of several prespecified sampling times. Then, the mean concentration at each sampling time can be obtained by the simple average of these subjects' observed concentration. One profile of the mean concentration vs. time can be obtained for one product (either the test or the reference product). One AUC value for one product can be calculated from the mean concentration-time profile using trapezoidal rules. This article develops a novel nonparametric method for obtaining the 90% confidence interval for the ratio of AUCT and AUCR (or CT,max/CR,max) in crossover studies by bootstrapping subjects at each time point with replacement or bootstrapping subjects at all sampling time points with replacement. Here T represents the test product, and R represents the reference product. It also develops a novel nonparametric method for estimating the standard errors (SEs) of AUCh and Ch,max in parallel studies by bootstrapping subjects treated by the hth product at each time point with replacement or bootstrapping subjects treated by the hth product at all sampling time points with replacement, h = T, R. Then, 90% confidence intervals for AUCT/AUCR and CT,max/CR,max are obtained from the nonparametric bootstrap resampling samples and are used for the evaluation of bioequivalence study for one-time sparse sampling data.

Evaluating elevated release liner adhesion of a transdermal drug delivery system (TDDS): a study of Daytrana™ methylphenidate transdermal system.

BACKGROUND: Complaints from healthcare providers that the adhesive on the Daytrana™ methylphenidate transdermal drug delivery system (TDDS) adhered to the release liner to such an extent that the release liner could not be removed prompted this study. Daytrana™ has a packaging system consisting of a moisture-permeable pouch contained within a sealed tray containing a desiccant; the tray is impermeable to ambient moisture. The objective of this project was to determine if the Daytrana™ packaging system influenced the difficulty in removing the release liner. METHOD: Both a sealed tray and an open tray containing sealed pouches were placed into an environmental chamber at 25°C and 60% relative humidity for 30 days; afterwards, release liner removal testing using a peel angle of 90° and a peel speed of 300 mm/min was performed. RESULTS: TDDS from open chamber trays required less force to remove the release liner than did TDDS from closed chamber trays. For the 10 mg/9 h TDDS and the 15 mg/9 h TDDS (the dosages examined), there were substantial differences in release liner removal force between an old lot and a new lot for closed chamber trays but not for open chamber trays. CONCLUSION: The results demonstrate that for this particular TDDS, storage conditions such as humidity influence release liner adhesion. This project also demonstrates that, to ensure adequate product quality, adhesion needs to become an important design parameter, and the design of a TDDS should consider the ability to remove the release liner under anticipated storage conditions.

Release liner removal method for transdermal drug delivery systems (TDDS).

A release liner removal test is a valuable test for assessing the quality of a transdermal drug delivery system (i.e., TDDS, patch). This test measures the force required to remove the release liner from a patch. The objective of the present study was to establish sample preparation and instrument parameters for measuring release liner removal adhesion for TDDS. Ten TDDS were evaluated (six drugs for a total of 29 lots). Patches which had a rate-controlling membrane were run as-is, since they could not be cut to a precise width without sacrificing their structural integrity. Patches that were square or rectangular in shape were run as-is, and the width of these patches was determined using a digital caliper. Patches which were not square or rectangular in shape and did not have a rate-controlling membrane were cut to a precise width using a specimen cutter. Double-sided tape was used to adhere the liner side of the transdermal system to a clean stainless steel test panel. A release liner peel adhesion method for TDDS is proposed using a dwell time of approximately 3 min, a peel angle of 90 degrees , and a peel speed of 300 mm/min.

Evaluation of substrates for 90 degrees peel adhesion--a collaborative study. II. Transdermal drug delivery systems.

In a previous study on peel adhesion for medical tapes, it was shown that a stainless steel (SS) substrate better discriminated among medical tapes than a high-density polyethylene (HDPE) substrate. The objective of this study was to determine if a SS substrate would also better distinguish among transdermal drug delivery systems (TDDSs). Five TDDSs (Vivelle Dot, Climara, Catapres-TTS, Duragesic, and Mylan Fentanyl) were evaluated on three different substrates (SS, HDPE, and human cadaver skin). All measurements were made using a dwell time of approximately 3 min, a peel angle of 90 degrees, and a peel speed of 300 mm/min. Differences among TDDSs were greater for SS than for HDPE, using the F statistic for testing for differences among TDDSs means as a measure of heterogeneity, thereby indicating greater discrimination by SS.

Statistical issues including design and sample size calculation in thorough QT/QTc studies.

After several drugs were removed from the market in recent years because of death due to ventricular tachycardia resulting from drug-induced QT prolongation (Khongphatthanayothin et al., 1998; Lasser et al., 2002; Pratt et al., 1994; Wysowski et al., 2001), the ICH Regulatory agencies requested all sponsors of new drugs to conduct a clinical study, named a Thorough QT/QTc (TQT) study, to assess any possible QT prolongation due to the study drug. The final version of the ICH E14 guidance (ICH, 2005) for "The Clinical Evaluation of QT/QTc Interval Prolongation and Proarrhythmic Potential for Nonantiarrhythmic Drugs" was released in May 2005. The purpose of the ICH E14 guidance (ICH, 2005) is to provide recommendations to sponsors concerning the design, conduct, analysis, and interpretation of clinical studies to assess the potential of a drug to delay cardiac repolarization. The guideline, however, is not specific on several issues. In this paper, we try to address some statistical issues, including study design, primary statistical analysis, assay sensitivity analysis, and the calculation of the sample size for a TQT study.

Evaluation of substrates for 90 degrees peel adhesion--a collaborative study. I. Medical tapes.

As part of a method development for peel testing, an interlaboratory comparison among Food and Drug Administration-Center for Drug Evaluation and Research, Food and Drug Administration-Center for Devices and Radiological Health and Southwest Research Institute was conducted using medical tapes. The aim was to determine which readily available substrate [stainless steel (SS), high density polyethylene (HDPE) or Vitro-Skin(R)] would best distinguish among various medical tapes. Five medical tapes (3M 1523, 3M 1525L, 3M 1776, Mepiform(R) and Mediderm(R) 3505) were evaluated on four different substrates (SS, HDPE, Vitro-Skin, and human cadaver skin) using the following peel parameters: approximately 3 min dwell time, 90 degrees peel angle, and 300 mm/min peel rate. No substrate mimics cadaver skin for all five tapes. SS had the best ability to distinguish among the medical tapes. Overall, for quality control purposes (yielding good discrimination and precision), SS would be the optimal substrate.

Stability, dose uniformity, and palatability of three counterterrorism drugs-human subject and electronic tongue studies.

PURPOSE: These studies evaluated the ability of common household food and drink products to mask the bitter taste of three selected anti-terrorism drugs. METHODS: Three anti-terrorism drugs (doxycycline, ciprofloxacin hydrochloride, and potassium iodide) were mixed with a variety of common household food and drinks, and healthy adult volunteers evaluated the resulting taste and aftertaste. In parallel, the ASTREE Electronic Tongue was used to evaluate taste combinations. Stability of the mixtures over time was monitored, as was the dosage uniformity across preparations. RESULTS: Foods and drinks were identified that satisfactorily masked the bitter flavor of each drug. Dose uniformity and stability were also acceptable over the range studied, although some combinations were significantly less stable than others. The electronic tongue was able to differentiate between tastes, but ranked masking agents in a different order than human volunteers. CONCLUSIONS: Doxycycline, potassium iodide, and ciprofloxacin, which are stockpiled in solid tablet form, can conveniently be prepared into more palatable formulations, using common household foods and drinks. The electronic tongue can be used to perform an initial screening for palatability.

Use of screening algorithms and computer systems to efficiently signal higher-than-expected combinations of drugs and events in the US FDA's spontaneous reports database.

Since 1998, the US Food and Drug Administration (FDA) has been exploring new automated and rapid Bayesian data mining techniques. These techniques have been used to systematically screen the FDA's huge MedWatch database of voluntary reports of adverse drug events for possible events of concern. The data mining method currently being used is the Multi-Item Gamma Poisson Shrinker (MGPS) program that replaced the Gamma Poisson Shrinker (GPS) program we originally used with the legacy database. The MGPS algorithm, the technical aspects of which are summarised in this paper, computes signal scores for pairs, and for higher-order (e.g. triplet, quadruplet) combinations of drugs and events that are significantly more frequent than their pair-wise associations would predict. MGPS generates consistent, redundant, and replicable signals while minimising random patterns. Signals are generated without using external exposure data, adverse event background information, or medical information on adverse drug reactions. The MGPS interface streamlines multiple input-output processes that previously had been manually integrated. The system, however, cannot distinguish between already-known associations and new associations, so the reviewers must filter these events. In addition to detecting possible serious single-drug adverse event problems, MGPS is currently being evaluated to detect possible synergistic interactions between drugs (drug interactions) and adverse events (syndromes), and to detect differences among subgroups defined by gender and by age, such as paediatrics and geriatrics. In the current data, only 3.4% of all 1.2 million drug-event pairs ever reported (with frequencies > or = 1) generate signals [lower 95% confidence interval limit of the adjusted ratios of the observed counts over expected (O/E) counts (denoted EB05) of > or = 2]. The total frequency count that contributed to signals comprised 23% (2.4 million) of the total number, 10.4 million of drug-event pairs reported, greatly facilitating a more focused follow-up and evaluation. The algorithm provides an objective, systematic view of the data alerting reviewers to critically important, new safety signals. The study of signals detected by current methods, signals stored in the Center for Drug Evaluation and Research's Monitoring Adverse Reports Tracking System, and the signals regarding cerivastatin, a cholesterol-lowering drug voluntarily withdrawn from the market in August 2001, exemplify the potential of data mining to improve early signal detection. The operating characteristics of data mining in detecting early safety signals, exemplified by studying a drug recently well characterised by large clinical trials confirms our experience that the signals generated by data mining have high enough specificity to deserve further investigation. The application of these tools may ultimately improve usage recommendations.