Taxon Appearance From Extraction and Amplification Steps Demonstrates the Value of Multiple Controls in Tick Microbiota Analysis

Background: The development of high-throughput sequencing technologies has substantially improved analysis of bacterial community diversity, composition, and functions. Over the last decade, high-throughput sequencing has been used extensively to identify the diversity and composition of tick microbial communities. However, a growing number of studies are warning about the impact of contamination brought along the different steps of the analytical process, from DNA extraction to amplification. In low biomass samples, e.g., individual tick samples, these contaminants may represent a large part of the obtained sequences, and thus generate considerable errors in downstream analyses and in the interpretation of results. Most studies of tick microbiota either do not mention the inclusion of controls during the DNA extraction or amplification steps, or consider the lack of an electrophoresis signal as an absence of contamination. In this context, we aimed to assess the proportion of contaminant sequences resulting from these steps. We analyzed the microbiota of individual Ixodes ricinus ticks by including several categories of controls throughout the analytical process: homogenization, DNA extraction, and DNA amplification. Results: Controls yielded a significant number of sequences (1, 126–13, 198 mean sequences, depending on the control category). Some operational taxonomic units (OTUs) detected in these controls belong to genera reported in previous tick microbiota studies. In this study, these OTUs accounted for 50.9% of the total number of sequences in our samples, and were considered contaminants. Contamination levels (i.e., the percentage of sequences belonging to OTUs identified as contaminants) varied with tick instar and sex: 76.3% of nymphs and 75% of males demonstrated contamination over 50%, while most females (65.7%) had rates lower than 20%. Contamination mainly corresponded to OTUs detected in homogenization and extraction reagent controls, highlighting the importance of carefully controlling these steps. Conclusion: Here, we showed that contaminant OTUs from sample laboratory processing steps can represent more than half the total sequence yield in sequencing runs, and lead to unreliable results when characterizing tick microbial communities. We thus strongly advise the routine use of negative controls in tick microbiota studies, and more generally in studies involving low biomass samples

Temporal patterns in Ixodes ricinus microbial communities: an insight into tick-borne microbe interactions

Background: Ticks transmit pathogens of medical and veterinary importance and are an increasing threat to human and animal health. Assessing disease risk and developing new control strategies requires identifying members of the tick-borne microbiota as well as their temporal dynamics and interactions. Methods: Using high-throughput sequencing, we studied the Ixodes ricinus microbiota and its temporal dynamics. 371 nymphs were monthly collected during three consecutive years in a peri-urban forest. After a Poisson lognormal model was adjusted to our data set, a principal component analysis, sparse network reconstruction, and differential analysis allowed us to assess seasonal and monthly variability of I. ricinus microbiota and interactions within this community. Results: Around 75% of the detected sequences belonged to five genera known to be maternally inherited bacteria in arthropods and to potentially circulate in ticks: Candidatus Midichloria, Rickettsia, Spiroplasma, Arsenophonus and Wolbachia. The structure of the I. ricinus microbiota varied over time with interannual recurrence and seemed to be mainly driven by OTUs commonly found in the environment. Total network analysis revealed a majority of positive partial correlations. We identified strong relationships between OTUs belonging to Wolbachia and Arsenophonus, evidence for the presence of the parasitoid wasp Ixodiphagus hookeri in ticks. Other associations were observed between the tick symbiont Candidatus Midichloria and pathogens belonging to Rickettsia. Finally, more specific network analyses were performed on TBP-infected samples and suggested that the presence of pathogens belonging to the genera Borrelia, Anaplasma and Rickettsia may disrupt microbial interactions in I. ricinus. Conclusions: We identified the I. ricinus microbiota and documented marked shifts in tick microbiota dynamics over time. Statistically, we showed strong relationships between the presence of specific pathogens and the structure of the I. ricinus microbiota. We detected close links between some tick symbionts and the potential presence of either pathogenic Rickettsia or a parasitoid in ticks. These new findings pave the way for the development of new strategies for the control of ticks and tick-borne diseases. [MediaObject not available: see fulltext.] © 2021, The Author(s)

Risk profiles and one-year outcomes of patients with newly diagnosed atrial fibrillation in India: Insights from the GARFIELD-AF Registry.

BACKGROUND: The Global Anticoagulant Registry in the FIELD-Atrial Fibrillation (GARFIELD-AF) is an ongoing prospective noninterventional registry, which is providing important information on the baseline characteristics, treatment patterns, and 1-year outcomes in patients with newly diagnosed non-valvular atrial fibrillation (NVAF). This report describes data from Indian patients recruited in this registry. METHODS AND RESULTS: A total of 52,014 patients with newly diagnosed AF were enrolled globally; of these, 1388 patients were recruited from 26 sites within India (2012-2016). In India, the mean age was 65.8 years at diagnosis of NVAF. Hypertension was the most prevalent risk factor for AF, present in 68.5% of patients from India and in 76.3% of patients globally (P < 0.001). Diabetes and coronary artery disease (CAD) were prevalent in 36.2% and 28.1% of patients as compared with global prevalence of 22.2% and 21.6%, respectively (P < 0.001 for both). Antiplatelet therapy was the most common antithrombotic treatment in India. With increasing stroke risk, however, patients were more likely to receive oral anticoagulant therapy [mainly vitamin K antagonist (VKA)], but average international normalized ratio (INR) was lower among Indian patients [median INR value 1.6 (interquartile range {IQR}: 1.3-2.3) versus 2.3 (IQR 1.8-2.8) (P < 0.001)]. Compared with other countries, patients from India had markedly higher rates of all-cause mortality [7.68 per 100 person-years (95% confidence interval 6.32-9.35) vs 4.34 (4.16-4.53), P < 0.0001], while rates of stroke/systemic embolism and major bleeding were lower after 1 year of follow-up. CONCLUSION: Compared to previously published registries from India, the GARFIELD-AF registry describes clinical profiles and outcomes in Indian patients with AF of a different etiology. The registry data show that compared to the rest of the world, Indian AF patients are younger in age and have more diabetes and CAD. Patients with a higher stroke risk are more likely to receive anticoagulation therapy with VKA but are underdosed compared with the global average in the GARFIELD-AF. CLINICAL TRIAL REGISTRATION-URL: http://www.clinicaltrials.gov. Unique identifier: NCT01090362

Expression and localization of cystic fibrosis transmembrane conductance regulator in the rat endocrine pancreas.

Impaired glucose tolerance and overt diabetes mellitus are becoming increasingly common complications of cystic fibrosis (CF), most probably merely as a result of increased life expectancy. In order to understand the pathophysiology of cystic fibrosis-related diabetes (CFRD), knowledge on the possible expression and cell distribution of the cystic fibrosis transmembrane conductance regulator (CFTR) protein within the endocrine pancreas is required. In this report, we establish the first evidence for expression of CFTR protein in rat pancreatic islets by using independent techniques. First reverse transcriptase-polymerase chain reaction (RT-PCR) amplification showed that CFTR mRNA is present in isolated islets of Langerhans. Furthermore, the analysis of flow cytometry-separated islet cells indicated that the level of CFTR transcripts is significantly higher in the non-beta than in beta-cell populations. The expression of CFTR protein in rat islet cells was also demonstrated by Western blotting and the level of expression was also found significantly higher in the non-beta than in beta-cell populations. Last, in situ immunocytochemistry studies with two monoclonal antibodies recognizing different CFTR epitopes indicated that CFTR expression occurs mainly in glucagon-secreting alpha-cells.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe