Generic lamotrigine extended-release tablets are bioequivalent to innovator drug in fully replicated crossover bioequivalence study.

OBJECTIVE: Lamotrigine is a commonly prescribed antiepileptic drug. U.S. Food and Drug Administration (FDA)-funded clinical studies have demonstrated bioequivalence (BE) for generic lamotrigine immediate-release (IR) products in epilepsy patients with generic substitution. To address the potential concerns about the risk of generic-brand substitution of lamotrigine extended-release (ER) products, considering the complexity of controlled release systems and pharmacokinetic variations associated with possible within-subject variability (WSV), this prospective study assessed (1) BE of generic and brand lamotrigine ER products in a fully replicated BE study design in healthy subjects and (2) whether such fully replicated study design and WSV data can better support the approval of generic lamotrigine ER products. METHODS: This open-label, single-dose, two-treatment, four-period, two-sequence, fully replicated crossover BE study compared generic lamotrigine ER tablet to brand Lamictal XR (200 mg) in 30 healthy subjects under fed conditions. Pharmacokinetics (PK) profiles were generated based on intensive blood sampling up to 144 h. RESULTS: The two products showed comparable peak plasma concentration (Cmax ), area under the concentration-time curve (AUC) from time zero to the last measurable time point (AUC0-t ) and AUC extrapolated to infinity (AUC0-inf ), whereas median time to Cmax (Tmax ) values differed, that is, 10 h for generic and 22 h for brand products, respectively. WSVs for PK metrics were small (~8% of Cmax and ~6% of AUC) and similar between these two products. PK simulation predicted equivalent PK measurements of both products at steady state and after brand-to-generic switch, except the first day upon switching. No serious adverse events were reported. SIGNIFICANCE: The generic lamotrigine ER tablet product demonstrates BE to the brand product in a fully replicated BE study design with healthy subjects, supporting the adequacy of the two-way crossover study design to demonstrate BE and generic-brand substitution of lamotrigine ER products.

Attribution of Listeria monocytogenes human infections to food and animal sources in Northern Italy.

Listeriosis is a foodborne illness characterized by a relatively low morbidity, but a large disease burden due to the severity of clinical manifestations and the high case fatality rate. Increased listeriosis notifications have been observed in Europe since the 2000s. However, the reasons for this increase are largely unknown, with the sources of sporadic human listerioris often remaining elusive. Here we inferred the relative contributions of several putative sources of Listeria monocytogenes strains from listerioris patients in Northern Italy (Piedmont and Lombardy regions), using two established source attribution models (i.e. 'Dutch' and 'STRUCTURE') in comparative fashion. We compared the Multi-Locus Sequence Typing and Multi-Virulence-Locus Sequence Typing profiles of strains collected from beef, dairy, fish, game, mixed foods, mixed meat, pork, and poultry. Overall, 634 L. monocytogenes isolates were collected from 2005 to 2016. In total, 40 clonal complexes and 51 virulence types were identified, with 36% of the isolates belonging to possible epidemic clones (i.e. genetically related strains from unrelated outbreaks). Source attribution analysis showed that 50% of human listerioris cases (95% Confidence Interval 44-55%) could be attributed to dairy products, followed by poultry and pork (15% each), and mixed foods (15%). Since the contamination of dairy, poultry and pork products are closely linked to primary production, expanding actions currently limited to ready-to-eat products to the reservoir level may help reducing the risk of cross-contamination at the consumer level.

Characterization of Salmonella enterica Isolates from Selected U.S. Swine Feed Mills by Whole-Genome Sequencing.

Every year salmonellosis is responsible for $2.3 billion in costs to the U.S. food industry, with nearly 6% of the reported cases associated with pork and/or pork products. Several studies have demonstrated the role of pigs as Salmonella reservoirs. Furthermore, this pathogen has been identified as a potential biological hazard in many livestock feeds. The overall objective of this research was to characterize Salmonella enterica isolates in selected U.S. swine feed mills by whole-genome sequencing (WGS) and evaluate isolates in association with the season and feed production stages. Salmonella isolates were collected from 11 facilities during a previous study. Samples were analyzed for Salmonella prevalence following the U.S. Department of Agriculture guidelines and confirmed by PCR. WGS was carried out on either the MiSeq or NextSeq sequencer. De novo genome assemblies were obtained with the Shovill pipeline, version 0.9. ResFinder and SPIFinder were used to identify antibiotic resistance genes and pathogenicity islands. Finally, their phylogenetic relationship and diversity were determined by core genome multilocus sequence typing. Overall, our analysis showed the presence of S. enterica in the feed mill environment. Isolates belonged to 16 different serotypes. Salmonella Agona, Salmonella Mbandaka, Salmonella Senfenberg, and Salmonella Scharzengrund were the most frequently found, and 18 single-nucleotide polymorphism clusters were identified. In silico analysis showed that 40% of the strains carried at least one antimicrobial resistance gene. All isolates in this study could be considered of public health concern and pathogenic potential. Our findings underscore the potential role of the feed mill environment as the pathogen entry route into the human food value chain.

Identification of a major Listeria monocytogenes outbreak clone linked to soft cheese in Northern Italy - 2009-2011.

BACKGROUND: Molecular subtyping and enhanced surveillance in Lombardy region identified a cluster of possibly related listeriosis cases from 2006 to 2010. This cluster grouped 31 isolates that belonged to serotype 1/2a and Sequence Type 38 (ST38) as defined by Multilocus Sequence Typing (MLST). METHODS: Our study expanded the previous investigation to include cases from 2011 to 2014 and used Multi-Virulence-Locus Sequence Typing (MVLST) on all ST38 isolates to better understand their epidemiology and possibly identify a common source outbreak. RESULTS: Out of 306 L. monocytogenes clinical isolates collected, 43 (14.1%) belonged to ST38 with cases occurring in nine out of twelve Lombardy provinces. The ST38 isolates were split by MVLST into two Virulence Types (VTs): VT80 (n = 12) and VT104 (n = 31). VT104 cases were concentrated between 2009 and 2011 in two provinces, Bergamo and Milan. An epidemiologic investigation was performed and in one case, a matching VT104 isolate was retrieved from a soft cheese sample from a patient's refrigerator. CONCLUSIONS: Our findings revealed a major listeriosis outbreak in Northern Italy linked to soft cheese in 2009-2011, which went undetected by local health authorities. Our study shows that integrating subtyping methods with conventional epidemiology can help identify the source of L. monocytogenes outbreak clones.

Epidemiology and Molecular Typing of Pregnancy-Associated Listeriosis Cases in Lombardy, Italy, over a 10-Year Period (2005-2014).

In developed countries, pregnancy-related listeriosis accounts for 20-43% of total invasive listeriosis. This work describes the first pregnancy-related listeriosis survey in Italy based on two data sources, that is, mandatory notification system and regional laboratory-based network. Out of 610 listeriosis cases reported over a 10-year period, 40 were pregnancy-related (6.6%). Among these, 29 pregnancy-related isolates were available and have been analysed with serotyping, Pulsed-Field Gel Electrophoresis, and Multi-Virulence-Locus Sequence Typing. No maternal fatality was recorded, but 11 (29.7%) pregnancies resulted in a foetal death, a miscarriage, or a birth of a foetus dying immediately after birth. The average incidence of pregnancy-related listeriosis was 4.3 cases per 100000 births, and the proportion of pregnancy-associated listeriosis among ethnic minorities was significantly higher compared to the general population (30.0% versus 3.5%, P < 0.01). L. monocytogenes isolates belonged to serotypes 1/2a, 1/2b, and 4b, with the latter significantly more prevalent among pregnancy-related isolates. Twenty different pulsotypes were distinguished and 16 out of the 29 isolates were classified into seven clusters. A total of 16 virulence types (VTs) were identified. Five VTs accounted for 45% of the total cases and coincided with those of previously described Epidemic Clones (ECs) of L. monocytogenes.

Development of a rapid SNP-typing assay to differentiate Bifidobacterium animalis ssp. lactis strains used in probiotic-supplemented dairy products.

Identification at the genus, species, and strain levels is desirable when a probiotic microorganism is added to foods. Strains of Bifidobacterium animalis ssp. lactis (BAL) are commonly used worldwide in dairy products supplemented with probiotic strains. However, strain discrimination is difficult because of the high degree of genome identity (99.975%) between different genomes of this subspecies. Typing of monomorphic species can be carried out efficiently by targeting informative single nucleotide polymorphisms (SNP). Findings from a previous study analyzing both reference and commercial strains of BAL identified SNP that could be used to discriminate common strains into 8 groups. This paper describes development of a minisequencing assay based on the primer extension reaction (PER) targeting multiple SNP that can allow strain differentiation of BAL. Based on previous data, 6 informative SNP were selected for further testing, and a multiplex preliminary PCR was optimized to amplify the DNA regions containing the selected SNP. Extension primers (EP) annealing immediately adjacent to the selected SNP were developed and tested in simplex and multiplex PER to evaluate their performance. Twenty-five strains belonging to 9 distinct genomic clusters of B. animalis ssp. lactis were selected and analyzed using the developed minisequencing assay, simultaneously targeting the 6 selected SNP. Fragment analysis was subsequently carried out in duplicate and demonstrated that the assay yielded 8 specific profiles separating the most commonly used commercial strains. This novel multiplex PER approach provides a simple, rapid, flexible SNP-based subtyping method for proper characterization and identification of commercial probiotic strains of BAL from fermented dairy products. To assess the usefulness of this method, DNA was extracted from yogurt manufactured with and without the addition of B. animalis ssp. lactis BB-12. Extracted DNA was then subjected to the minisequencing protocol, resulting in a SNP profile matching the profile for the strain BB-12.

Multi-virulence-locus sequence typing of 4b Listeria monocytogenes isolates obtained from different sources in India over a 10-year period.

Listeria monocytogenes is an emerging foodborne pathogen responsible for listeriosis. The incidence of listeriosis has increased during the last 2 decades due to the increase in consumption of ready-to-eat foods and change in food consumption habits. Outbreaks and sporadic cases of listeriosis have been reported in developed countries. These reports have helped determine the safety practices needed to control listeriosis. Although L. monocytogenes has been reported from humans, animals, and a variety of foods in India, limited data exist with respect to prevalence and distribution of L. monocytogenes in the Indian subcontinent. The Indian Listeria Culture Collection Centre in Goa maintains all of the isolates received for subtyping and molecular characterization. Of the listerial isolate collection maintained by this center, three fourths of the isolates are of 4b serotype, while the number of other serotypes is very low. Therefore, we screened L. monocytogenes serotype 4b isolates to determine their relevance to previously defined epidemics and/or outbreaks using multi-virulence-locus sequence typing (MVLST). A total of 25 isolates in serogroup 4b of L. monocytogenes were randomly selected from a repository of 156 L. monocytogenes 4b isolates obtained from different sources in India over a period of 10 years. MVLST sequence types (virulence types, VTs) were compared to known epidemic clones and other known isolates in the L. monocytogenes MVLST database. The 25 isolates were grouped into three clusters. Cluster I comprised 21 isolates including animal (n=9), human (n=4), and food (n=8), which matched Epidemic Clone I (ECI, VT20). Three isolates-two from animal and one from food-formed a cluster while a single animal isolate was placed into two novel VTs (VT98 and VT99), respectively. Based on these findings, it can be inferred that ECI has been isolated from a variety of sources and places and has persisted in India for at least 10 years.

Novel epidemic clones of Listeria monocytogenes, United States, 2011.

We identified a novel serotype 1/2a outbreak strain and 2 novel epidemic clones of Listeria monocytogenes while investigating a foodborne outbreak of listeriosis associated with consumption of cantaloupe during 2011 in the United States. Comparative analyses of strains worldwide are essential to identification of novel outbreak strains and epidemic clones.

Ruminant rhombencephalitis-associated Listeria monocytogenes strains constitute a genetically homogeneous group related to human outbreak strains.

Listeriosis is a disease that causes significant economic losses at the farm level because of high morbidity and mortality in ruminants. This study was performed to investigate the role of ruminants in the epidemiology of listeriosis in northern Italy and the possible association of animal-adapted strains of Listeria monocytogenes with strains associated with human disease. Twenty ruminant rhombencephalitis isolates previously confirmed as L. monocytogenes by bacteriology and PCR were characterized by serotyping, pulsed-field gel electrophoresis, multi-virulence-locus sequence typing (MVLST), and multiplex single nucleotide polymorphism (mSNP) typing for the detection of epidemic clones. Subtyping results were subsequently compared with those obtained from human, food, and environmental isolates of L. monocytogenes, including 311 isolates from the University of Turin, Grugliasco, Italy, and 165 isolates representing major human listeriosis outbreaks worldwide, in addition to other unrelated isolates. Both mSNP typing and MVLST showed that 60% of the isolates analyzed belonged to epidemic clone I (ECI), which has been epidemiologically linked to several human outbreaks of listeriosis. In particular, the 1981 Canada outbreak was linked to the use of sheep manure and the 1985 California outbreak was linked to the use of raw cow's milk. In our study, ECI isolates were collected from different ruminant species on geographically and temporally distinct occasions for the last 13 years. Our results support the hypothesis that ruminants represent possible natural reservoirs of L. monocytogenes strains capable of causing epidemics of listeriosis in humans.

Enterobacterales draft genome sequences: 15 historical (1998-2004) and 30 contemporary (2015-2016) clinical isolates from Pakistan.

The continued emergence and spread of antimicrobial resistance among pathogenic bacteria are ever-growing threats to health and economy. Here, we report the draft genomes for 45 Enterobacterales clinical isolates, including historical and contemporary drug-resistant organisms, obtained in Pakistan between 1998 and 2016: 5 Serratia, 3 Salmonella, 3 Enterobacter, and 34 Klebsiella.

Usage of Compartmental Models in Predicting COVID-19 Outbreaks.

Accurately predicting the spread of the SARS-CoV-2, the cause of the COVID-19 pandemic, is of great value for global regulatory authorities to overcome a number of challenges including medication shortage, outcome of vaccination, and control strategies planning. Modeling methods that are used to simulate and predict the spread of COVID-19 include compartmental model, structured metapopulations, agent-based networks, deep learning, and complex network, with compartmental modeling as one of the most widely used methods. Compartmental model has two noteworthy features, a flexible framework that allows users to easily customize the model structure and its high adaptivity that allows well-matured approaches (e.g., Bayesian inference and mixed-effects modeling) to improve parameter estimation. We retrospectively evaluated the prediction performances of the compartmental models on the CDC COVID-19 Mathematical Modeling webpage based on data collected between August 2020 and February 2021, and subsequently discussed in detail their corresponding model enhancement. Finally, we presented examples using the compartmental models to assist policymaking. By evaluating all models in parallel, we systemically evaluated the performance and evolution of using compartmental models for COVID-19 pandemic prediction. In summary, as a 100-year-old epidemic approach, the compartmental model presents a powerful tool that is extremely adaptive and can be readily customized and implemented to address new data or emerging needs during a pandemic.

Biofilm formation and genomic features of Listeria monocytogenes strains isolated from meat and dairy industries located in Piedmont (Italy).

Listeria monocytogenes is considered a major challenge for the food industry as it can persist for long periods in food processing plants by forming biofilms. The aims of this study were: i) to assess the biofilm producing ability of 57 Listeria monocytogenes isolates previously subjected to whole-genome sequencing (WGS); ii) to compare the levels of biofilm formation with the presence or absence of biofilm associated genes. To determine the presence or absence of a known set of biofilm associated genes, a comparative genomic analysis was performed on each strain. Among Listeria monocytogenes isolates, 58 %, 38.5 % and 3.5 % exhibited weak, moderate or strong biofilm production, respectively. No difference in biofilm production was observed between food and environmental isolates. The percentage of Listeria monocytogenes strains isolated from meat products (57 %) classified as moderate or strong biofilm producers was higher than the percentage obtained for strains isolated from dairy products (28 %). The presence of the Stress Survival Islet 1, the arsD stress gene and the truncated inlA protein was significantly associated with increased levels of biofilm. Combining biofilm phenotype with molecular and genotyping data may provide the opportunity to better understand the relationship between genes linked to biofilm formation in Listeria monocytogenes.

Novel multiplex single nucleotide polymorphism-based method for identifying epidemic clones of Listeria monocytogenes.

A novel primer extension-based, multiplex minisequencing assay targeting six highly informative single nucleotide polymorphisms (SNPs) in four virulence genes correctly identified and differentiated all four epidemic clones (ECs) of Listeria monocytogenes and 9 other strains initially misclassified as non-ECs. This assay allows rapid, accurate, and high-throughput screening for all known ECs of L. monocytogenes.

Genotypic and Phenotypic Characterization of Antimicrobial Resistance Profiles in Non-typhoidal Salmonella enterica Strains Isolated From Cambodian Informal Markets.

Non-typhoidal Salmonella enterica is a pathogen of global importance, particularly in low and middle-income countries (LMICs). The presence of antimicrobial resistant (AMR) strains in market environments poses a serious health threat to consumers. In this study we identified and characterized the genotypic and phenotypic AMR profiles of 81 environmental S. enterica strains isolated from samples from informal markets in Cambodia in 2018-2019. AMR genotypes were retrieved from the NCBI Pathogen Detection website (https://www.ncbi.nlm.nih.gov/pathogens/) and using ResFinder (https://cge.cbs.dtu.dk/services/) Salmonella pathogenicity islands (SPIs) were identified with SPIFinder (https://cge.cbs.dtu.dk/services/). Susceptibility testing was performed by broth microdilution according to the Clinical and Laboratory Standards Institute (CLSI) standard guidelines M100-S22 using the National Antimicrobial Resistance Monitoring System (NARMS) Sensititre Gram Negative plate. A total of 17 unique AMR genes were detected in 53% (43/81) of the isolates, including those encoding tetracycline, beta-lactam, sulfonamide, quinolone, aminoglycoside, phenicol, and trimethoprim resistance. A total of 10 SPIs (SPI-1, 3-5, 8, 9, 12-14, and centisome 63 [C63PI]) were detected in 59 isolates. C63PI, an iron transport system in SPI-1, was observed in 56% of the isolates (n = 46). SPI-1, SPI-4, and SPI-9 were present in 13, 2, and 5% of the isolates, respectively. The most common phenotypic resistances were observed to tetracycline (47%; n = 38), ampicillin (37%; n = 30), streptomycin (20%; n = 16), chloramphenicol (17%; n = 14), and trimethoprim-sulfamethoxazole (16%; n = 13). This study contributes to understanding the AMR genes present in S. enterica isolates from informal markets in Cambodia, as well as support domestic epidemiological investigations of multidrug resistance (MDR) profiles.

Draft Genome Sequences of Two Extensively Drug-Resistant Strains of Acinetobacter baumannii Isolated from Clinical Samples in Pakistan.

Infections in immunocompromised patients that are caused by extensively drug-resistant (XDR) Acinetobacter baumannii strains have been increasingly reported worldwide. In particular, carbapenem-resistant A. baumannii strains are a prominent cause of health care-associated infections. Here, we report draft genome assemblies for two clinical XDR A. baumannii isolates obtained from hospitalized patients in Pakistan.

Draft Genome Sequences of 81 Salmonella enterica Strains from Informal Markets in Cambodia.

Salmonella enterica is an important global pathogen due to its contribution to human morbidity and death. The presence of S. enterica in Southeast Asian informal markets is amplified by cross-contamination between market surfaces and food products. Here, we describe the draft genome sequences of 81 Salmonella enterica isolates from informal markets in Cambodia.

Listeria monocytogenes in Gorgonzola: subtypes, diversity and persistence over time.

L. monocytogenes represents a primary concern in the production of Gorgonzola, a Protected Designation of Origin (PDO) Italian blue-veined cheese produced only in the Piedmont and Lombardy regions. L. monocytogenes isolates (N=95) obtained from Gorgonzola rinds, paste, and production/ripening environments were serotyped and then genotyped using Pulsed Field Gel Electrophoresis (PFGE). The goal of this study was to investigate the variability of L. monocytogenes PFGE-types across different PDO Gorgonzola manufacturers (N=22). The majority of the strains (88%) were serotyped as 1/2a. PFGE identified 2 major pulse-types grouping 62 strains, detected from different plants and years, suggesting the presence of persistent and niche-adapted L. monocytogenes. In 9 plants, environmental strains shared the same pulse-types with strains from rinds or paste, suggesting a possible transmission pathway. Encouragingly, L. monocytogenes was retrieved from only 1 paste, indicating that production processes were under control in 21 plants. In the remaining plant, un-effective pasteurization or cross-contamination during production processes could be the cause of the contamination. Consequently, it is imperative that producers operate under the total respect of the Good Manufacturing Practices and following the principles of the Hazard Analysis Critical Control Point plans, in order to contain contamination throughout the whole processing.

In-depth comparative analysis of Illumina® MiSeq run metrics: Development of a wet-lab quality assessment tool.

Whole genome sequencing of bacterial isolates has become a daily task in many laboratories, generating incredible amounts of data. However, data acquisition is not an end in itself; the goal is to acquire high-quality data useful for understanding genetic relationships. Having a method that could rapidly determine which of the many available run metrics are the most important indicators of overall run quality and having a way to monitor these during a given sequencing run would be extremely helpful to this effect. Therefore, we compared various run metrics across 486 MiSeq runs, from five different machines. By performing a statistical analysis using principal components analysis and a K-means clustering algorithm of the metrics, we were able to validate metric comparisons among instruments, allowing for the development of a predictive algorithm, which permits one to observe whether a given MiSeq run has performed adequately. This algorithm is available in an Excel spreadsheet: that is, MiSeq Instrument & Run (In-Run) Forecast. Our tool can help verify that the quantity/quality of the generated sequencing data consistently meets or exceeds recommended manufacturer expectations. Patterns of deviation from those expectations can be used to assess potential run problems and plan preventative maintenance, which can save valuable time and funding resources.

Whole genome sequencing uses for foodborne contamination and compliance: Discovery of an emerging contamination event in an ice cream facility using whole genome sequencing.

We review how FDA surveillance identifies several ways that whole genome sequencing (WGS) improves actionable outcomes for public health and compliance in a case involving Listeria monocytogenes contamination in an ice cream facility. In late August 2017 FDA conducted environmental sampling inside an ice cream facility. These isolates were sequenced and deposited into the GenomeTrakr databases. In September 2018 the Centers for Disease Control and Prevention contacted the Florida Department of Health after finding that the pathogen analyses of three clinical cases of listeriosis (two in 2013, one in 2018) were highly related to the aforementioned L. monocytogenes isolates collected from the ice cream facility. in 2017. FDA returned to the ice cream facility in late September 2018 and conducted further environmental sampling and again recovered L. monocytogenes from environmental subsamples that were genetically related to the clinical cases. A voluntary recall was issued to include all ice cream manufactured from August 2017 to October 2018. Subsequently, FDA suspended this food facility's registration. WGS results for L. monocytogenes found in the facility and from clinical samples clustered together by 0-31 single nucleotide polymorphisms (SNPs). The FDA worked together with the Centers for Disease Control and Prevention, as well as the Florida Department of Health, and the Florida Department of Agriculture and Consumer Services to recall all ice cream products produced by this facility. Our data suggests that when available isolates from food facility inspections are subject to whole genome sequencing and the subsequent sequence data point to linkages between these strains and recent clinical isolates (i.e., <20 nucleotide differences), compliance officials should take regulatory actions early to prevent further potential illness. The utility of WGS for applications related to enforcement of FDA compliance programs in the context of foodborne pathogens is reviewed.

Draft Genome Sequences of Antimicrobial-Resistant Shigella Clinical Isolates from Pakistan.

Shigella spp. are the most common cause of dysentery in developing countries and the second leading cause of diarrheal deaths worldwide. Multidrug-resistant (MDR) Shigella spp. are a serious threat to global health. Herein, we report draft genome sequences for three MDR Shigella isolates from Pakistan, two Shigella flexneri isolates and one Shigella sonnei isolate.