Mycobacterium tuberculosis has been ravaging humans by inflicting respiratory tuberculosis since centuries. Bacillus Calmette Guerine (BCG) is the only vaccine available for tuberculosis, and it is known to be poorly effective against adult tuberculosis. Proteins belonging to the ESAT-6 family and PE/PPE family show immune responses and are included in different vaccine trials. Herein, we study the functional and structural characterization of a 248 amino acid long putative protein novel hypothetical protein 1 (NHP1) present in the RD7 region of Mycobacterium tuberculosis  using bioinformatics tools.Mycobacterium smegmatis protein as the putative protein with homology. Structural evaluations were done with Ramachandran plot analysis, ProSA-web, and VERIFY3D, and with GalaxyWEB server, a more stable structure was validated with good stereo chemical properties.Physicochemical properties were studied using Expasy ProtParam and SMS software. We predicted different B-cell and T-cell epitopes by using the immune epitope database (IEDB) and also tested antigenicity, immunogenicity, and allergenicity. Secondary structure of the protein predicted 30% alpha helices, 20% beta strands, and 48% random coils. Tertiary structure of the protein was predicted using the Robetta server using the The present study of a subtracted genomic locus using various bioinformatics tools indicated good immunological properties of the putative mycobacterial protein, NHP1. Evidence obtained from the analyses of NHP1 using structure prediction tools strongly point to the fact that NHP1 is an ancient protein having flavodoxin folding structure with ATP binding sites. Positive scores were obtained for antigenicity, immunogenicity, and virulence too, implying the possibility of NHP1 to be a potential vaccine candidate. Such computational studies might give clues for developing newer vaccines for tuberculosis, which is the need of the hour.The online version contains supplementary material available at 10.1186/s43141-022-00340-5. Mycobacterium tuberculosis shows resistance to commonly used drugs, which makes the disease more difficult to treat [M. tuberculosis was done in 1998, which showed that more than half of the genome encodes hypothetical proteins [M. tuberculosis complex share 99% of genome sequence similarity but exhibit different pathogenic outcomes due to the coevolution of the species with animals and humans [M. tuberculosis and M. leprae showed 219 genes unique for Mycobacterium species of which a few codes for highly conserved ESAT-6 family protein [M. tuberculosis are considered as immunodominant regions. Genes specific to human isolates were identified in the RD7 region of H37Rv strain, for example, the mce3 gene  located on the cell surface which plays an important role in the survival of the pathogen in the host [The World Tuberculosis Report, 2020, estimated ten million newly infected tuberculosis (TB) cases in 2019 of which 1.2 million people were HIV-negative and 2,08,000 people were HIV positive . Furtherto treat . The firproteins . Membersd humans . A larged humans . Biologi protein . RD1, RDthe host . RD7 regthe host .M. tuberculosis [M. tuberculosis were identified using computational tools of which only seven secretory proteins were previously reported [M. tuberculosis proteins were done using in silico methods of which few were predicted to have vaccine potential, ribosome binding regions, GTP binding domains, etc. [Computer-aided drug discovery studies in the field of tuberculosis research are a good model to find new drug targets against rculosis . SMRT sereported . Hypothens, etc. . Reversens, etc. .M. tuberculosis H37Rv and Indian clinical isolates. Using the initial 384 bp subtracted product as a probe for RFLP with different restriction enzymes followed by sequencing and assembly of the subcloned fragments, a 4.5 kb subtractive fragment was obtained by genome walking. Three potential ORFs were found in the 4.5 loci, NHP1 being one among them [Mycobacterium canetti and predicted 5 potential ORFs. The present study is an in silico approach of the functional and structural studies of one of the novel hypothetical proteins (NHP1), present in the N4.5 locus of clinical isolates. Being a primitive protein and also being located in the RD7 region make NHP1 an ideal vaccine candidate to be explored. In order to check the probable use of NHP1 protein as a potential vaccine candidate against tuberculosis, various computational tools including Expasy ProtParam, SMS suite, NCBI CDD blast, PFAM, SMART, TB Pred, TMHMM, InterPro, VaxiJen, AllerTop, AllergenFP, VirulentPred, GOR4, SOPMA, PSIPRED, Robetta, PROCHECK, and GalaxyWEB server were used. Three positive and negative controls each were used for the comparison of results of the in silico study. The 3D structural studies of NHP1 were done using homology protein modeling. Proteins with high sequence similarity were selected as templates using the MODELLER server. Along with the homology template model, a position-specific scoring matrix (PSSM) and hidden Markov model (HMM)-based server Robetta were considered to develop the 3D structure of the query protein. The developed structures were refined and validated using different quality assessment tools including PROCKECK. The refinement of the developed 3D structure of the protein was also done using the GalaxyWEB server which is based on molecular dynamic simulation. The present study aimed at exploring the possibility of NHP1 protein as a potential vaccine candidate against tuberculosis, a disease for which there are no many vaccines in the market.Our present focus, the NHP1 locus, was obtained by subtractive hybridization in an earlier study using ong them . NCBI BLThe nucleotide sequence of N4.5 deposited earlier by our research group was retrieved from the National Center for Biotechnology Information (NCBI) GenBank (accession number — GU994138.2). NHP1 locus had 747 bp encoding a protein of 248 amino acids. The nucleotides encoding esxA , esxB , and fbpB  used as positive controls were retrieved from NCBI GenBank. whiB2 , tuf elongation factor , and cyp144  used as negative control were also retrieved from NCBI GenBank. The physical and chemical characters, immunological properties, and the secondary and tertiary structures of the protein were studied using different bioinformatics tools.https://web.expasy.org/protparam/ server was used for physical and chemical characterization of the proteins. The prediction included molecular weight, isoelectric point, extinction coefficient, and instability index. SMS (Sequence Manipulation Suite) v2.0 [https://www.bioinformatics.org/sms2/ was also used to predict the physical properties using the sequence of 248 amino acids along with the positive and negative controls as input.Expasy’s ProtParam  https://te) v2.0  https://https://www.ncbi.nlm.nih.gov/Structure/cdd/ wrpsb.cgi, PFAM [http://pfam.xfam.org/ and SMART [http://smart.embl-heidelberg.de/smart/set_mode.cgi were used to search the conserved domains in the target protein and control samples.Bioinformatics tools including CDD-BLAST  https://gi, PFAM  http://pnd SMART  http://shttps://webs.iiitd.edu.in/tbpred/submission.html), CELLO  [http://cello.life.nctu.edu.tw/), TMHMM [http://www.cbs.dtu.dk/services/TMHMM/), SignalP  and SOSUI [https://harrier.nagahama-i-bio.ac.jp/sosui/) were used to study whether the protein is localized in the cytoplasm or on the cell surface. InterPro [https://www.ebi.ac.uk/interpro/) tool which predicts functional analysis of proteins by comparing the referral sequences with predicted databases provided by different softwares was used to study the protein. Proteins are further classified into different families after domain prediction and site analysis. Furthermore, PFP-FunDSeqE  [http://www.csbio.sjtu.edu.cn/bioinf/PFP-FunDSeqE/) server was used to find protein folding patterns based on functional domain and its evolutionary information. NHP1 protein along with positive and negative controls was studied using these servers.Subcellular localization prediction tools like TBpred  (https:/diction)  (http://), TMHMM  (http://nd SOSUI  (https:/InterPro  (https:/rmation)  (http://http://www.ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.html) server was used to predict the antigenicity of the query proteins. Antigenicity of the protein was studied using the server ANTIGENpro (http://scratch.proteomics.ics.uci.edu/explanation.html). Allergenicity is very crucial in the development of vaccines. AllerTOP v.2.0 [https://www.ddg-pharmfac.net/AllerTOP/) and AllergenFP [http://ddg-pharmfac.net/AllergenFP/data.html) servers were used to predict the allergenicity of the protein. ToxinPred [https://webs.iiitd.edu.in/raghava/toxinpred/algo.php) is a free server which helps in predicting the toxic regions of a protein sequence if any. This server can also be used to identify a new toxic peptide present in organisms.VaxiJen v2.0  (http://OP v.2.0  (https:/lergenFP  (http://oxinPred  (https:/http://203.92.44.117/virulent/) was used to predict the virulence factors. VirulentPred is based on the SVM method with 81.8% accuracy.VirulentPred  (http://http://crdd.osdd.net/raghava/hlapred/) and NetCTL [http://www.cbs.dtu.dk/services/NetCTL/) servers were used to predict the T-cell epitopes of the proteins. The NetCTL server was used to predict cytotoxic T-lymphocyte (CTL) epitopes of the query proteins. NetCTL prediction depends on MHCI binding affinity, C-terminal cleavage function, and transporter function associated with antigen processing. IEDB MHCII [http://tools.iedb.org/mhcii/) server was used for identifying helper T-lymphocyte (HTL) epitopes. Human/HLA-DR were chosen as the species/locus with 7 allele human leukocyte antigen (HLA), and 15-m-long epitopes were retrieved. The percentile rank was compared with the Swiss-Prot database showing high MHCII affinity. T-cell epitopes immunogenicity prediction tool was used to study if the predicted T-cell epitopes elicit an immune response. Immunogenicity of class-I peptide MHC complex (pMHC) (http://tools.iedb.org/immuno genicity/) is based on the positions and the properties of the amino acids.HLAPred (d NetCTL  (http://DB MHCII  (http://https://webs.iiitd.edu.in/raghava/bcepred/bcepred_submission.html) and ABCpred [https://webs.iiitd.edu.in/raghava/abcpred/ABC_method.html) were used for the prediction of linear B-cell epitopes. BepiPred by IEDB [http://tools.iedb.org/bcell/) resources was also used to predict linear B epitopes from the protein structure. In this server, amino acid characteristic scales and hidden Markov Model were used to predict linear B-cell epitopes.BcePred  (https:/ ABCpred  (https:/ by IEDB  (http://http://bioinf.cs.ucl.ac.uk/psipred/) and self-optimized prediction method with alignment (SOPMA) [https://npsa-prabi.ibcp.fr/NPSA/npsa_sopma.html) and GOR IV [https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_gor4.html) servers were used to predict the secondary structure of NHP1 protein.Online PSI-blast-based secondary structure prediction (PSIPRED)  (http:// (SOPMA)   and Robetta [https://robetta.bakerlab.org/) servers. The model protein structures obtained were validated using PROCHECK [http://www.csb.yale.edu/Excite/AT-CSBquery.html) and VERIFY3D [https://saves.mbi.ucla.edu/). PROCHECK was used to check the stereochemical parameters along with the bond angles and bond lengths of the protein structure. ProSA-web [https://prosa.services.came.sbg.ac.at/prosa.php) server was used for Z-score validation. Galaxy Refine — GalaxyWEB server [http://galaxy.seoklab.org/cgi-bin/help.cgi?key=METHOD&type= REFINE) was used to refine the predicted homology model.The protein tertiary structure was modelled using MODELLER  , with a theoretical pI 5.09, and estimated half-life of 30 h. The hypothetical protein had an average GRAVY of 0.035, making the protein hydrophobic. The aliphatic index of the protein is 86.65, as the volume occupied by alanine, valine, leucine, and isoleucine is defined. Various physicochemical parameters of NHP1 are depicted in Table In silico studies of the putative 747 bp long gene in the N4.5 region of M. tuberculosis and 99% identity with many isolates of M. canetti. CDD-BLAST searches conserved domains by scanning the position-specific scoring matrices (PSSM) with all the conserved domains in the database with a protein query. CDD-BLAST, PFAM, and SMART did not show the presence of any conserved domain in the query protein.Conserved domains are sequences or structural units reoccurring in an evolutionary perspective. NHP1 nucleotide sequence subjected to NCBI BLAST showed 100% identity with many isolates of The 248 amino acid long sequence was used as the input in SOSUI, TMHMM, and CELLO to study the subcellular localization of the protein. These servers categorize the protein into transmembrane or cytoplasmic ones Figs.  and 2. TVaxiJen servers predicted the protein as antigenic based on the physical and chemical properties, where the default threshold of 0.4 was chosen as the antigenicity measure. The query protein had an antigenicity score of 0.4509. The positive control proteins gave a VaxiJen threshold of esxA-0.557, esxB-0.782, and fbp-0.584 and negative control proteins whiB2-0.356, tuf ef-0.398, and cyp144-0.37, respectively. ANTIGENPro server predicted the antigenicity of the query protein as 0.764607. AllerTOP v.2.0 and AllergenFP predicted the query protein to be a probable non-allergen. Among the positive controls used, esxA protein was predicted as a probable allergen and toxic in nature, whereas esxB and fbpB proteins were nontoxic and non-allergen in nature. According to ToxinPred, whiB2 protein was studied as toxin, and tuf ef protein according to AllergenFP was studied as a probable allergen candidate. ToxinPred data predicted NHP1 protein to be nontoxic. VirulentPred data has shown NHP1 to be a virulent protein. Table HLAPred predicted 22 T-cell epitopes for the NHP1 protein. Predicted binders were scanned for genomic molecular mimicry against different species where no identical sequences were seen which can be used as a vaccine candidate. NetCTL server predicted the presence of seven MHC ligands with a threshold of 0.75 as shown in Table ABCpred was used to predict the B-cell epitope of the NHP1 protein using artificial neural networks. A threshold of 0.5 along with an epitope length of 16 amino acids was used in the ABCpred server. Eighteen epitopes were predicted by the ABCpred server above (0.92–0.56) the threshold level, where the default threshold value was 0.51. BcePred predicted the presence of six B-cell epitopes in the query protein with an accuracy of 58.7% at 2.3 threshold. Physicochemical characters like hydrophobicity, polarity, and flexibility were combined together at a threshold of 2.38 for linear B-cell epitope prediction by BcePred. BepiPred predicted the presence of B epitopes in the amino acid sequence of NHP1 with a threshold ranging from 0.2 to 0.722 Fig. . BepiPreThe secondary structure of NHP1 was predicted using different computational tools Table . SOPMA pThe conventional method of determining the three-dimensional protein structure is by NMR spectroscopy and X-ray diffraction. 3D homology models of NHP1 protein were predicted using the homology protein structure modeling method. Homology modeling has led to a new breakthrough in the field of computational biology. This modeling is very effective in studying molecular evolution since evolutionarily-related proteins share a common structure. MODELLER server is based on homology and comparative modeling of the protein sequence. MODELLER selected the most appropriate structure similar to NHP1 protein in the PDB database. The template 4TMD_A showed a probability of 99.56% with an E-value 1.8e-13 with a target length of 195. The predicted model was saved as PDB format. 3D structure of the protein was predicted using the Robetta server. Robetta is an automated tool for protein structure prediction using homology modeling and de novo structure prediction method.The predicted structures were analyzed using PROCHECK, VERIFY3D, and ProSA. Ramachandran plot was analyzed for the predicted model. The Ramachandran plot of the Robetta structure indicated 92.7% of residues with 188 residues in favorable regions and 20 residues in allowed regions. The number of non-glycine and non-proline residues was 212. ProSA web server was used for Z-score validation. The Z score of the 3-D sequence was estimated as −6.06. Analysis of the predicted structure using the VERIFY3D server showed that 80.24% of the residues have an average 3D-1D confirmation score. The refined image of the protein was predicted by the GalaxyWEB Refine server. GalaxyWEB Refine is used to improve the accuracy of the predicted model using side-chain perturbation. Refined 3D homology model of NHP1 had 95.1% residues as favored in the Ramachandran plot, with an RMSD value of 0.362 , esxB , and fbpB  which were extensively studied for immunological properties both in silico and in vivo and further subjected to vaccine studies were selected as positive control. whiB2 (Rv3260c), tuf elongation factor (Rv0686), and cyp144 (Rv1777) proteins that are important for the biochemical functions of the bacteria were used as negative control.M. tuberculosis using different computational tools like CELLO, TMHMM, and HMMTOP predicted these proteins as cytoplasmic transmembrane proteins with no signal peptides. Homology modeling of the proteins was done using RaptorX, SWISS-MODEL server, and Phyre 2. Molecular docking of the proteins was done with azole drugs using AutoDock Vina and revealed that the P450 enzymes were found to act as a target site for novel drugs. Different azole drugs docked against P450 enzymes were predicted to be good drug candidates against M. tuberculosis [M. tuberculosis membrane proteins are known to be immunogenic. Surface membrane proteins are considered as potential vaccine targets, while cytoplasmic proteins are possible drug targets [M. tuberculosis are generally potent activators of human T-cell responses [Subcellular localization studies play an important role in identifying proteins as drug targets, and most of the prediction tools are validated against mycobacterial proteins . NHP1 pr targets . Since mesponses , NHP1 alM. tuberculosis H37Rv was published in 2016 which can be used for comparative studies. These 999 hypothetical proteins were further studied for conserved domains using CDD Blast and ScanProsite. Ninety-eight proteins were selected from this set and further studied for structural and functional characterization. Proteins with plasma membrane localization and transmembrane topology prediction were studied for antigenicity using VaxiJen server. Proteins having an antigenic score of > 5 were considered and selected for epitope prediction of which two proteins were shown to be having 15 linear epitopes and 21 conformational epitopes. This study has shown these epitopes can be candidates for developing new drugs or vaccines against tuberculosis [A study of 999 hypothetical proteins of https://webs.iiitd.edu.in/cgibin/atpint/chkres?8477). PFP-FunDSeqE server results depict NHP1 protein to have a flavodoxin-like folding. Proteins with flavodoxin-like folding patterns are considered as ancient origin structures. Flavodoxin family proteins of M. bovis BCG, M. canetti, M. pinnipedii, and few other species of Mycobacterium were found to be deposited in EMBL UniProt (https://www.uniprot.org/uniref/UniRef100_ A0A0K2HZ71). Flavin cofactors were present in flavodoxin folding proteins in the electron transport chain, and in few bacteria, these proteins were induced in low iron conditions [ATPint: tool predicted the ATP binding residues of the NHP1 protein  showed 6 MHC ligands. Only 2 MHC ligands above the threshold value were found in all three negative controls under study. IEDB MHC II predicted the presence of T-helper cell epitopes of which fbpB and cyp144 were having T-helper cell epitopes. BepiPred predicted the presence of B epitope alleles for positive and negative controls. In comparison with all positive and negative controls, NHP1 protein showed immunological properties which tend to make it a vaccine candidate.M. tuberculosis. The 3D structure of NCGM946K2_146 gene of M. tuberculosis was studied using tools like SWISS-MODEL server, Phyre2, and MODELLER, of which MODELLER was found to be the most efficient tool for homology protein structure prediction. The structural and functional studies of NCGM946K2_146 pointed to those of a potential therapeutic drug candidate [M. tuberculosis H37Rv [M. tuberculosis were carried out using GOR-IV server. SOPMA and GOR IV predicted 28–33% of alpha helical structure, 18–22% extended strand structure and 44–49% random coil structure, and 3% beta turn structure. PSIPRED predicted the secondary structure similar to the prediction of GOR-IV and SOPMA with high confidence of prediction. The tertiary structure of the NHP1 protein was studied using homology modeling. Membrane proteins including NP_216679, NP_218309, and NP_218312 of M. tuberculosis were studied for physical and chemical characterization, secondary structure prediction, and functional characterization using different computer tools. These proteins were found to be membrane proteins with transmembrane helices.The secondary structure of NHP1 was predicted using SOPMA, PSIPRED, and GOR-IV. SOPMA and PSIPRED were earlier used to predict the structure of the hypothetical protein CGM946K2_146 of andidate . PSIPREDis H37Rv . Structu3D structural studies of NHP1 were done using homology protein modeling. Proteins with high sequence similarity were selected as templates using the MODELLER server. Along with the homology template model, a position-specific scoring matrix (PSSM) and hidden Markov model (HMM)-based server Robetta were considered to develop the 3D structure of the query protein. The developed structures were refined and validated using different quality assessment tools including PROCHECK. The refinement of the developed 3D structure of the protein was also done using the Galaxy web server which is based on molecular dynamic simulation.M. smegmatis, showed 99.56% sequence similarity with 195 amino acids among the 248 long amino acid NHP1 sequence. Robetta server was used to predict the structure of NHP1 using 4TMD_A as model protein structure. 3D structure of a multiepitope vaccine using Ag85A (Rv3804c), Mtb32A (Rv0125), Rv2684, and Rv2608 developed previously using ITASSER showed 85.9% favored region, 8.9% allowed region, and 5.2% disallowed region in Ramachandran plot [The physical and chemical parameters of a multiepitope vaccine developed using reverse vaccine technology were studied using Expasy ProtParam which predicted the vaccine developed as acidic, hydrophilic, and stable in nature . Anotherran plot . Ramacharan plot . Since tran plot .Homology protein modeling has led to a new breakthrough in the field of computational biology. In homology modeling, new protein structures are designed using a known protein structure. This method is very effective in studying evolutionarily related proteins since they share common structural domains. Crystallographic studies of membrane proteins are challenging, since they can be affected by detergents and other physical and chemical properties of the protein. Total number of protein structures deposited in Protein Data Bank (PDB) until 2021 is 183,584 of which 2037 are membrane proteins. Generally, a sequence identity more than 50% can give reliable models with only slight errors in the loops and side-chain positions. Homology protein modeling is a time-saving method when compared to X-ray crystallography technique.M. canetti, which is an ancestral smooth tubercle bacillus. Studies on the nature of protein folds have given us insights on the presence of a flavodoxin-like fold in NHP1 pointing to the ancient nature of this mycobacterial protein, a corroboration of our earlier research on the N4.5 region’s close similarity with the corresponding genomic locus in the smooth tubercle bacillus, M. canettii. Thus, the evidence collected through various in silico approaches to better understand NHP1 has led us to the conclusion that it is a transmembrane, signal peptide of M. tuberculosis with sufficient antigenicity and immunogenicity as depicted by prediction servers like VaxiJen, AntigenPro, AllerTOP, and HLAPred. An ideal vaccine to put a curb on the spread of tuberculosis should target multiple immune system components. As it is very difficult to create a vaccine with all these attributes, multiple vaccines or combination approaches would be the ideal strategy to look forward to. In this context, the current study exploring the possibility of NHP1 as a potential vaccine candidate assumes importance as it has immunogenic features comparable with those of esxA, esxB, Ag85B, etc. which are currently explored vaccine candidates.Cell-mediated response is significant in the progress of the disease in a patient. Bacterial membrane proteins play a significant role in the host-pathogen interaction. Hence, the protein under study offers a promise of immunogenicity and hence important as a candidate vaccine. The 3D structure of NHP1 was predicted with 99% confidence using the Robetta server. NHP1 protein has also shown 99% sequence similarity with M. canetti and showed the presence of flavodoxin-like folding patterns with ATP binding sites stressing the evolutionarily ancient nature of the protein. Evidence from our earlier studies of the sequences upstream and downstream of NHP1 loci has also revealed almost 100% similarity with the corresponding regions of M. canetti, the ancient smooth tubercle bacillus. These two facts drive home the ancient nature of this locus. This revelation is particularly important as India has a lot of clinical isolates of the pathogen falling under the category of ancestral strains. NHP1 protein showed high antigenic threshold along with epitopes of T cells and B cells making the protein a vaccine candidate similar to esxB and fbpB proteins and other proteins in the RD7 region. The comparative analysis of NHP1 protein with positive and negative controls showed that NHP1 protein has comparable immunological properties and hence is likely to have vaccine potential. Data from these computational studies have revealed NHP1 to be a membrane protein with good antigenicity and non-allergenicity. Thus, the current approach has helped in identifying a multiepitope protein present in the RD7 region of M. tuberculosis clinical strains as a novel vaccine candidate using immunoinformatics tools which can be further analyzed using animal models for its efficacy.The structural and functional properties of a putative protein, NHP1, in the RD7 region of the genome were studied using in silico approaches. This was found to be an ORF in a subtracted genomic region N4.5 in clinical isolates in India. The initial studies on this protein depicted it to be a novel multiepitope possible subunit vaccine candidate. NHP1, predicted to be a membrane protein with signal sequence, showed 99% similarity with the counterpart in Additional file 1.


Tamarix taklamakanensis M. T. Liu, belonging to the genus Tamarix (family Tamaricaceae), is an endangered shrub endemic to arid basins in northwestern China. Most of species in this genus have high medicinal value. The complete chloroplast genome was reported in this study. The chloroplast genome with a total size of 156,177 bp consists of two inverted repeats  separated by a large single-copy region  and a small single-copy region . Further annotation revealed the chloroplast genome contains 106 genes, including 73 protein coding genes, 29 tRNA genes, and 4 rRNA genes. A total of 64 simple sequence repeats (SSRs) were identified in the chloroplast genome. This information will be useful for study on the evolution and genetic diversity of T. taklamakanensis in the future. Tamarix taklamakanensis M. T. Liu is an Endangered desert shrub belonging to the genus Tamarix, Tamaricaceae, and it often grows in arid desert areas of northwestern China. Since this species shows a strong drought resistant and heat tolerance, it plays an important role in sand fixation and reforestation on mobile sand dunes as a pioneer tree species of Western China . The voucher specimen  was stored in the herbarium of Tarim University. The leaves total genomic DNA was extracted using CTAB method , yielding approximately 50 Gb of high-quality sequences. First, the clean data was quality-controlled by using FastQC v0.11.9 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Then, the whole chloroplast genome was assembled using GetOrganelle v1.7.3   as the reference genome and used PGA (https://github.com/quxiaojian/PGA)  and Bayesian inference (BI)  . The ML nce (BI)  based onnce (BI) . This re


Anticoagulation was associated with improved survival of hospitalized coronavirus disease 2019 (COVID-19) patients in large-scale studies. Yet, the development of COVID-19-associated coagulopathy (CAC) and the mechanism responsible for improved survival of anticoagulated patients with COVID-19 remain largely elusive. This investigation aimed to explore the effects of anticoagulation and low-molecular-weight heparin (LMWH) in particular on patient outcome, CAC development, thromboinflammation, cell death, and viral persistence.P = 0.009].Data of 586 hospitalized COVID-19 patients from three different regions of Austria were evaluated retrospectively. Of these, 419 (71.5%) patients received LMWH and 62 (10.5%) received non-vitamin-K oral anticoagulants (NOACs) during hospitalization. Plasma was collected at different time points in a subset of 106 patients in order to evaluate markers of thromboinflammation (H3Cit-DNA) and the cell death marker cell-free DNA (cfDNA). Use of LMWH was associated with improved survival upon multivariable Cox regression . Interestingly, neither LMWH nor NOAC was associated with attenuation of D-dimer increase over time, or thromboinflammation. In contrast, anticoagulation was associated with a decrease in cfDNA during hospitalization, and curtailed viral persistence was observed in patients using LMWH leading to a 4-day reduction of virus positivity upon quantitative polymerase chain reaction [13 (interquartile range: 6–24) vs. 9 (interquartile range: 5–16) days, Time courses of haemostatic and thromboinflammatory biomarkers were similar in patients with and without LMWH, indicating either no effects of LMWH on haemostasis or that LMWH reduced hypercoagulability to levels of patients without LMWH. Nonetheless, anticoagulation with LMWH was associated with reduced mortality, improved markers of cell death, and curtailed viral persistence, indicating potential beneficial effects of LMWH beyond haemostasis, which encourages use of LMWH in COVID-19 patients without contraindications.  While the rapid development of effective vaccines provides hope for the management of the pandemic, availability as well as the mutational burden require further research on patient care in COVID-19.Neutrophil extracellular traps (NETs), caused by decondensation of nuclear DNA from neutrophils, are associated with disease progression in COVID-19.Within this observational study, we report on a multicentre cohort of hospitalized COVID-19 patients in Austria. We evaluated the predictive potential of previously reported haemostatic biomarkers for mortality and investigated markers of thromboinflammation and cell death in these patients. Further, we explored the relationship between LMWH intake and potential underlying mechanisms responsible for the observed improved outcome of COVID-19 patients receiving anticoagulation by analysing their impact on thromboinflammation, cell death, and duration of viral persistence.n = 586) admitted to three different large-scale treatment centres in Austria  between 14 February 2020 and 18 September 2020 were analysed. Patient demographics including comorbidities, use of anticoagulation, and additional medication relevant for COVID-19 treatment were recorded. Routine laboratory analysis was performed every second day. Nasopharyngeal swabs and quantitative polymerase chain reaction (qPCR) for SARS-CoV-2 were performed according to the Charité protocolMethods and www.who.int; Clinical management of COVID-19 Interim guidance 27 May 2020). Accordingly, patients were classified as mild, moderate, severe, or critical based on clinical evaluation and disease dynamic within the first week after admission to hospital. Patients without COVID-19 symptoms at admission were added to the cohort of mild patients. Outcome data were available for all patients at the time of analysis. The study was conducted in accordance with the Declaration of Helsinki. The recovery of data at Kaiser Franz Josef Hospital in Vienna was part of the randomized controlled ACOVACT study  approved by the local ethics committee (EK1315/2020), which aims to compare the effect of different antiviral agents and adjunctive treatments on outcome of hospitalized COVID-19 patients. Data gathered at the remaining two centres were prospectively evaluated and retrospectively analysed in this investigation. Accordingly, the study was approved by the ethics committee of the Medical University of Innsbruck (EK1167/2020) and the Kepler University Hospital Linz (EK1085/2020). Patients in the ACOVACT study gave written informed consent prior to study inclusion, while patient consent was waved for all remaining participants.In this retrospective multicentre study, clinical data from COVID-19 patients . Patient demographics are given inTables Figure ). Disease severity at admission was indicative for outcome . Intensive care unit (ICU) treatment was required in 111 patients (18.9%) and 66 patients (11.3%) required invasive ventilation. Overall, 88 patients (15.0%) died during their hospital stay. The main cause of death in this cohort was respiratory failure in 49 patients (55.7%), followed by multiorgan failure , cardiac decompensation , sepsis , and thromboembolic complications . Cause of death is unknown in three patients (3.4%). Missing data are presented in In total, 586 consecutive patients were retrospectively analysed in this study , PT , international normalized ratio , and activated partial thrombin time , showing an increased risk for mortality for patients with any deranged haemostatic biomarker. The determined predictive effects of each biomarker remained significant after addition of age to the logistic regression model . However, D-dimer and INR were not associated with mortality in the multivariable logistic regression model including age after multiple imputation , while PT  and aPTT  remained significant in the final model. Ultimately, none of the evaluated biomarkers improved the prediction of fatal outcome based on age .Age predicted fatal outcome with an odds ratio (OR) of 1.109 [95% confidence interval (95% CI): 1.082–1.137, P = 0.046, P < 0.001, P = 0.041, respectively; P = 0.014, uncomplicated vs. non-survivors: P < 0.001, ICU vs. non-survivors: P = 0.058; P = 0.001, uncomplicated vs. non-survivors: P = 0.013, ICU vs. non-survivors: P = 0.467; P = 0.018, uncomplicated vs. non-survivors: P = 0.151, ICU vs. non-survivors: P = 0.897; r = −0.069, P = 0.531; r = 0.280, P = 0.010; In this cohort, D-dimer levels, H3Cit–DNA complexes, and cfDNA measured upon admission were associated with higher COVID-19 disease severity at admission . There was no difference in H3Cit-DNA dynamics over time between patients with different outcomes (P = 0.123), while patients with ICU treatment and non-survivors displayed higher levels of H3Cit-DNA throughout the entire observational period when compared to patients with uncomplicated hospitalization (P < 0.001). In contrast, cfDNA dynamics varied according to outcome . In particular, patients with uncomplicated hospitalization and patients requiring ICU treatment showed a decrease over time, while non-survivors displayed a steady increase in cfDNA throughout hospitalization.H3Cit–DNA complexes in circulation decreased during hospitalization (Table 3). Of note, the reported multivariable Cox regression model was carried out on the data set obtained after multiple imputations to account for missing data. Age, hypertension, cardiovascular diseases, chronic renal insufficiency, history of malignancy, requirement of oxygen at admission, oxygen saturation at admission, COVID-19 disease severity at admission, and use of LMWH were significantly associated with survival in univariable Cox regression. The multivariable model included all parameters being significant upon univariable Cox regression. Ultimately, older age, history of malignancy, and increasing COVID-19 disease severity increased the risk for mortality. In addition, LMWH remained significantly associated with reduced risk for mortality, as depicted by a hazard ratio (HR) of 0.561 (95% CI: 0.348–0.906). Additionally, a multivariable Cox regression model for adjustment to potential confounders and disparities in baseline demographics between survivors and non-survivors was fit. Here, we also included parameters that were not significant upon univariable Cox regression . Of note, we observed an increase in the proportion of LMWH treatment throughout the observed study period (P = 0.001). Importantly, the association of LMWH with improved hospital survival was not affected by the time point of study inclusion, as the interaction term of LMWH × time of inclusion was not significantly associated with survival , while both the main effect of LMWH use and time of inclusion remained significant in the Cox regression analysis. The association of LMWH with survival further remained significant after including presence of cardiovascular diseases  or chronic renal insufficiency  as confounders for LMWH use. Additionally, as patients on anticoagulants frequently used further drugs , as well as in patients with severe and critical COVID-19 at admission . Respective data for use of NOAC can be found in After observing an association of LMWH with survival in a data set including multiple imputations, we aimed for description of these data on the original data set. Kaplan–Meier analysis according to status of LMWH intake is shown in y period Figure S5er drugs Table S4,er drugs Figure S6Figure ). In particular, the effect of clinical course on D-dimer slopes was similar between patients treated with LMWH and patients who were not anticoagulated (P = 0.890). Likewise, use of NOAC was not associated with D-dimer increase in ICU patients and non-survivors . There was no difference in H3Cit-DNA dynamics over time between LMWH users and patients without LMWH use . Results obtained in patients using NOAC showed a similar pattern (P = 0.009) irrespective of anticoagulation. Interestingly, cfDNA dynamics differed according to LMWH use , which was comparable between patients using NOAC , leading to a reduction in median time of viral persistence . Of note, the association of LMWH with curtailed viral persistence remained significant after additional adjustment for corticosteroids, antiviral agents, such as remdesivir/favipiravir or lopinavir/ritonavir, and camostat  and after correction for chronic renal insufficiency . Similarly, correction for these parameters did not alter the results obtained for NOAC which still did not associate with shortened viral persistence  not receiving anticoagulants, 337 (75.7%) receiving LMWH, and 48 patients (10.8%) receiving NOAC. Patients with LMWH use displayed a significantly increased probability for earlier negativity of SARS-CoV-2 qPCR upon age-adjusted Cox regression  received therapeutic doses of LMWH and the statistical power for the respective analyses was not sufficient. Of note, previous reports were not able to assess a difference between prophylactic and therapeutic uses of LMWH in more than 4,000 patients.Taken together, the present investigation confirms an association of anticoagulants with improved survival of COVID-19 patients in a large Central European Multicentre Cohort and suggests a beneficial effect of LMWH use on SARS-CoV-2 viral persistence. While the exact pathomechanisms underlying these observations cannot be investigated due to the retrospective observational study design, the present study encourages the evaluation of viral persistence in randomized controlled trials assessing the effect of LMWH in COVID-19 patients in order to establish a causal relation of the presented findings. Limiting viral persistence, thereby shortening hospitalization and contagiousness is a relevant aspect during this pandemic.Cardiovascular Research online.D.P. collected and analysed the data, prepared the figures, and wrote the manuscript; S.H. performed statistical analyses, prepared the figures, and wrote the manuscript; W.C.S., J.S., A.P., A.S., K.K., D.A., T.S., F.F., H.H., E.P., P.K., B.R., M.T.T., P.G., C.F., C.S., T.S., and M.K. collected and analysed the data; I.P., C.B., P.S., G.W., R.B.W., H.J.F.S., B.J., and A.Z. provided resources and interpreted the data; A.A. conceived the study, analysed and interpreted the data, and wrote the manuscript.Conflict of interest: none declared.This work is part of the ACOVACT study of the Medical University of Vienna and is financially supported by the Austrian Federal Ministry of Education, Science and Research, the Medical-Scientific Fund of the Mayor of Vienna (COVID024), and the Austrian Science Fund .The data presented in this article are represented in the main text and in the article’s cvab308_Supplementary_DataClick here for additional data file.


Mansonella perstans and Loa loa in children, adolescents and adults living across Bioko Island, Equatorial Guinea are lacking. We aimed to demonstrate that blood retained on malaria rapid diagnostic tests, commonly deployed for malaria surveys, could be used as a source of nucleic acids for molecular based detection of M. perstans and L. loa. We wanted to determine the positivity rate and distribution of filarial nematodes across different age groups and geographical areas as well as to understand level of co-infections with malaria in an asymptomatic population.Regular and comprehensive epidemiological surveys of the filarial nematodes M. perstans, L. loa and Plasmodium spp. parasites were monitored by qPCR in a cross-sectional study using DNA extracted from a subset malaria rapid diagnostic tests (mRDTs) collected during the annual malaria indicator survey conducted on Bioko Island in 2018.M. perstans and L. loa were 6.6% and 1.5%, respectively. M. perstans infection were more prominent in male (10.5%) compared to female (3.9%) survey participants. M. perstans parasite density and positivity rate was higher among older people and the population living in rural areas. The socio-economic status of participants strongly influenced the infection rate with people belonging to the lowest socio-economic quintile more than 3 and 5 times more likely to be L. loa and M. perstans infected, respectively. No increased risk of being co-infected with Plasmodium spp. parasites was observed among the different age groups.We identified DNA specific for the two filarial nematodes investigated among 8.2% (263) of the 3214 RDTs screened. Positivity rates of M. perstans and L. loa. Our study demonstrates that employing mRDTs probed with blood for malaria testing represents a promising, future tool to preserve and ship NAs at room temperature to laboratories for molecular, high-throughput diagnosis and genotyping of blood-dwelling nematode filarial infections. Using this approach, asymptomatic populations can be reached and surveyed for infectious diseases beyond malaria.We found otherwise asymptomatic individuals were infected with  Mansonella perstans and Loa loa are filarial nematodes that infect millions of people living in less developed areas, predominantly in sub-Saharan Africa. Both parasites are neglected among other filarial nematodes because both are regarded as causing mainly asymptomatic infections. The aim of this study was to explore the feasibility of using malaria rapid diagnostic tests (mRDTs) deployed during malaria surveys as a convenient sampling strategy for molecular surveillance of blood-dwelling filarial nematode infections. Our findings demonstrate the potential of mRDTs as a source of parasite DNA beyond malaria, providing an opportunity to expand current knowledge on the distribution and populations mostly affected by M. perstans and L. loa infections to Equatorial Guinea, located in Central-West Africa. M. perstans, M. streptocerca and M. ozzardi [Mansonella sp "DEUX" [Mansonella spp. generally alternate between an insect vector and humans who are the final hosts. The insect vectors transmitting M. perstans belong to the genus Culicoides [M. perstans is transferred to humans during the next feeding round [M. perstans are detected by microscopic examination in thick or thin blood films stained with Giemsa in blood samples taken at any time of the day [M. perstans infections in endemic populations, and as for other filarial infections most infections seem to be clinically silent [M. perstans infections include eosinophilia, angioedema, arthralgia, fever, headache, pruritus, skin eruption, serositis, neurologic manifestations, ocular or palpebral pruritus, visual impairment and chest pain [Human filariases are vector-borne infectious diseases that encompass Mansonellosis and loiasis . Mansone ozzardi . Recentlp "DEUX" . Mansonep "DEUX" ,4. The llicoides . When feng round . The thing round . Adult m the day ,6. Littly silent ,5. Cliniest pain .Loa loa in endemic countries of Central, and Western Sub-Saharan Africa [L. loa larvae are transmitted to humans during blood feeding of an infected vector fly belonging to the genus Chrysops spp. [L. loa can be challenging since adult worms can be present without detectable mf in blood [At least 10 million people are infected with n Africa . L. loa ops spp. . Diagnosin blood .In areas where different filarial nematodes are co-endemic, misclassification of mf by microscopy can be problematic ,9. ThereM. perstans and L. loa in otherwise asymptomatic individuals. In doing so, we described the association of filarial infections with demographic and geographic factors and assessed the level of multi-parasitism of these nematodes with the highly endemic malaria parasites on Bioko Island. We also used Cq values as a proxy of filarial parasite density and compared this measurement against demographic and geographic characteristics of the investigated population as well as the time of the day of RDT sample collection.The aim of this work was to demonstrate that blood retained on malaria rapid diagnostic tests (mRDTs) is a source of nucleic acids for molecular based detection of The MIS in 2018 was approved by the Ministry of Health and Social Welfare of Equatorial Guinea and the Ethics Committee of the London School of Hygiene & Tropical Medicine (Ref. No. LSHTM: 5556). Consent was sought from eligible respondents before the administration of the questionnaire. A signed authorization was requested from the parents or legal guardians of children, and adult participants to use their samples for further analyses. Laboratory experiments were performed in accordance with relevant guidelines and regulations.2, Bioko forms part of the nation of Equatorial Guinea and is administratively divided into four districts: Malabo, the capital of Equatorial Guinea, and Baney both located in the northern part of the Island; and Luba and Riaba both located in the Southern part. The Island has an estimated population size of 270´000 people, with the majority (85%) living in Malabo [Bioko Island is located on the West African continent shelf, precisely in the Gulf of Guinea and separated from Cameroon by no more than 32 kilometres of shallow ocean. With its total land surface of approximately 2000 kmn Malabo . Bioko hn Malabo .Malaria has historically been hyper-endemic in Bioko Island with a prevalence of 45% among 2–14 year old children before the launch of the Malaria control program . The Bio2 grid. Coinfections were estimated at the same spatial resolution, for comparison.The 2018, the MIS on Bioko Island was conducted on a representative sample using the Malaria Pf/PAN (HRP2/pLDH) Ag Combo rapid diagnostic test . Consent to store used mRDTs for further molecular analyses was obtained from 13,505 survey participants and unique barcode labels were affixed to the used mRDTs that were shipped to the Swiss Tropical and Public Health Institute for further analyses. Each mRDT barcode was linked to a household unique identification code [A subset of the mRDT were selected for molecular analysis of the blood retained on the mRDT based on the mRDT test outcome for malaria. Out of the 1376 malaria positive mRDTs identified during the 2018 MIS, we analysed the nucleic acids from 1065 mRDTs (77.4%). In addition, other malaria negative mRDT were selected for the molecular analysis.Plasmodium spp. parasites [M. perstans and L. loa, by qPCR using well described marker genes [We reused the extracted nucleic acids (NAs) from a study which was published recently by our group . Brieflyarasites . For theer genes ,21. We hL. loa and M. perstans DNA. In a multiplex qPCR reaction, the L. loa specific qPCR assay amplifies a 62 base pair (bp) fragment from the hypothetical protein LLMF72 [M. perstans specific qPCR assay is based on amplification of a 187 bp fragment of the 18S ribosomal RNA gene [2O was run as a non-template control, and a mix of M. perstans and L. loa DNA served as a positive control for each run. Each reaction contained 2 μL of template DNA and 8 μL master mix consisting of 1 x Luna Universal Probe qPCR Master Mix , 0.4 μM of M. perstans forward primer, 0.4 μM of M. perstans reverse primer, 0.25 μM of the Yakima Yellow-labelled M. perstans probe, 0.8 μM of L. loa forward primer, 0.8 μM of L. loa reverse primer, 0.4 μM of FAM-labelled L. loa-specific minor groove binder (MGB) probe, 0.4 μM HsRNaseP forward primer, 0.4 μM of HsRNaseP-reverse primer and 0.4 μM of Cy5-labelled HsRNaseP probe. Using the Bio-Rad CFX96 Real-Time PCR System , amplification program was 1min at 95°C, followed by 50 cycles of 15s at 95°C and 45s at 55°C. Samples were considered positive if the quantification cycle (Cq) value was <50.A multiplex qPCR assay, herein referred to as llmp-qPCR, was developed and performed to detect n LLMF72  and the RNA gene . The humRNA gene . All reaPlasmodium spp. and P. falciparum detection and quantification that has been developed by our group and described previously [Plasmodium 18S rDNA sequence (Pspp18S) and the P. falciparum-specific acidic terminal sequence of the var genes (PfvarATS). The human RNaseP (HsRNaseP) gene served as an internal control to assess the quality of DNA extraction and qPCR amplification. The PlasQ was performed on NA extracted from mRDT and samples with Cq value <45 of either of the two targets, PfvarATS or Pspp18S, were considered positive for Plasmodium spp. Then, results obtained were linked to llmp-qPCR results obtained from the same aliquot of NA extracted from identical mRDT to assess co-infection status between Plasmodium spp., L. loa and M. perstans. Coinfections were estimated at the same 1x1 km2 grid, for comparison.The PlasQ is a multiplex RT-qPCR assay for eviously ,22. ThisM. perstans and 457 bp for L. loa. PCR products of 10 and 23 samples tested positive by llmp-qPCR for L. loa and M. perstans, respectively, were sequenced from both ends by Sanger Sequencing  to confirm specificity of the qPCR assays. Samples covering a large and representative range of different Cq values were selected for sanger sequencing of the ribosomal internal transcribed spacer 1 region (https://www.geneious.com). A consensus sequence of all 23 M. perstans and 10 L. loa sequences of 417 bp and 390 bp length, respectively, served as a query sequence to identify all GenBank entries with >90% identity and >95% coverage using BLAST. Additionally, representative sequences for M. streptocerca (KR868771) and M. ozzardi (EU272180) deposited to GenBank were included. Geneious Prime software (version 2021.0.3) was used for the multiple sequence alignment and to generate the phylogenetic analysis using its in-build neighbour-joining (NJ) clustering method [Onchocerca volvulus (EU272179) as an outgroup. The resulting newick file was imported into R for final phylogeny and visualization using the ggplot2, ggtree, and treeio packages.The ribosomal internal transcribed spacer 1 region was amplified with a set of primers that bind universally to all filarial species and are designed to highlight interspecific differences . The PCR1 region . Sequencg method . Branch g method  with OncPlasmodium spp. and M. perstans or L. loa were expressed as the product and 95% confidence interval (95% CI) of Plasmodium spp., M. perstans and L. loa the prevalence stratified by age group. The ELIMU-MDx platform was used for quality control, management and analysis of qPCR data [-value < 0.05 was considered statistically significant.Households were assigned scores based on the type of assets and amenities they own  to derive a surrogate of Socio-Economic Status (SES), using Principal Component Analysis (PCA). Households were ranked based on their score and the distribution was further divided into five equal categories (quintiles), each with approximately 20% of the households. The first quintile corresponded to the lowest wealth index (WI) and the fifth to the highest WI. The household WI categories were also assigned to permanent (de jure) household members. Predicted co-infection rates for PCR data . StatistPlasmodium spp. co-infections, we over-sampled mRDTs from two specific sub-populations. Firstly, malaria positive mRDTs were preferentially selected and processed and secondly, for filarial nematodes infections, mRDTs from adults living in rural districts were enriched for selection and analysis. A graphical representation of the over-sampling is shown in M. perstans and L. loa infections stratified by geographical location, age and socio-economic status is shown in A total of 4774 households, including 20’012 individuals, from across Bioko Island participated in the MIS 2018. 13’505 participants provided an additional consent for molecular analysis of the mRDT collected. To increase the probability to identify individuals with filarial nematodes and M. perstans and L. loa in a single qPCR reaction (M. perstans and/or L. loa. The proportion of mRDTs positive for M. perstans was 6.6% (213) compared to 1.5% (50) for L. loa. M. perstans and L. loa stratified by age (A), socio-economic status (B) and gender (C). People living in rural districts have significantly higher positivity rates for M. perstans than people living in the urban areas. Positivity rates in rural districts ranged from 17.1% (Luba) to 13.2% (Baney) compared to 2.1% in the urban district of Malabo. On the contrary, no significant differences in L. loa positivity rates were observed between rural and urban districts. L. loa was more prevalent in the two Southern districts, Riaba (3.9%) and Luba (2.7%), compared to the Northern districts of Malabo (1.5%) and Baney (0.7%). M. perstans infection rates in high-endemic rural districts increased significantly with age and the highest positivity rate was observed in participants older than 60 years (p< 0.00001). L. loa was found at higher rates in participants older than 20 years of age in urban as well as in rural areas (p = 0.0001)  . Among cividuals . People L. loa infections was comparable between male and female individuals combined for rural and urban districts (p = 0.4), M. perstans was significantly higher among male (21.1%) compared to the female (9.0%) inhabitants of rural districts (p< 0.00001) (While the proportion of 0.00001) .M. perstans and ten samples positive for L. loa identified by the llmp-qPCR assay were all confirmed by sequence analysis. The M. perstans sequences clustered closely with each other and other M. perstans sequences, but are distinct from Mansonella spp. DEUX sequences. The L. loa sequences generated in this study are closely related to sequences from Central- and West-Africa deposited in GenBank.Specificity of the qPCR-based species identification was confirmed by sequence analysis of the conserved ribosomal internal transcribed spacer 1 region that encompassed 390 to 450 bp depending on the filarial species . To our P. falciparum for all samples collected during the MIS based on mRDT positivity in Plasmodium spp. when analysed by RT-qPCR    . The dif40 years . Combinifrom age , no signfrom age , while t= 0.018) . Interes= 0.018) .M. perstans and L. loa in Equatorial Guinea that also includes the evaluation of geographical distribution and association with socio-economic status.We conducted a larger scale, cross-sectional study of samples including paediatric, adolescent, adult and elderly populations residing in urban and rural regions of Bioko Island. To the best of our knowledge, this report represents the first molecular epidemiological study of L. loa and M. perstans infections among Equato-Guineans living in Spain [L. loa and M. perstans on Bioko Island using microscopy and qPCR-based detection [Between 1978 and 2020, a total of 20 publications described Mansonellosis, Loasis, Onchocerciasis, and lymphatic filariasis in the context of Equatorial Guinea . Five ofin Spain –31 or Siin Spain , while tin Spain –35. One etection .M. perstans and L. loa. The widespread availability and use of mRDTs in malaria endemic regions that are also endemic to M. perstans and L. loa, the simplicity of mRDT collection and storage, would make this approach convenient for large-scale molecular epidemiological studies covering Plasmodium spp., M. perstans and L. loa. Using our extraction protocol based on mRDTs, high quality and sufficient quantities of M. perstans and L. loa specific DNA fragments were obtained as demonstrated by successful Sanger sequencing of the ribosomal internal transcribed spacer 1 region. Therefore, apart from amplifying short DNA fragments usually used for qPCR, our NA extraction method also allows to amplify larger fragments suitable for genotyping of the pathogens of interest. In future, switching to more polymorphic markers for genotyping in combination with next generation sequencing technologies might improve tracking of infections and importantly help to understand if there are multiple strain infections that possibly accumulate over time in the elderly population showing the highest parasite density of M. perstans infections by qPCR.We have extended our mRDT-based molecular surveillance tool, originally developed for malaria, to the filarial nematodes M. perstans and L. loa test system was estimated to be $4 per sample, from which $3 were spent for NA extraction and $1 on the llmp-qPCR assay. Noteworthy, the same aliquot of extracted NA was used for genomic characterization and quantification of malaria parasites as described [The cost of our mRDT-based escribed  making tM. perstans (6.6%) are more prevalent than L. loa (1.5%) and that M. perstans infections can be mostly found in the older, male, population living in rural parts of Bioko Island. This finding reconfirms previous reports [M. perstans and L. loa compared to a qPCR-based cross-sectional study conducted on Bioko Island in 2018. Ta and colleagues found that 8.8% and 0.7% of persons tested were infected with M. perstans and L. loa, respectively [L. loa found in both studies could be underestimated and partially explained by the fact that 70% of infected individuals do not show mf circulation in peripheral blood, with occult infection or occasional presence of adult worms under conjunctive tissue [Here, we found that infections with  reports –39. We fectively . The sime tissue .M. perstans infection found in rural areas on the East coast of Bioko Island might reflect the distribution or abundance of the vector and its active transmission in those areas. The vector Culicoides presence is more likely associated to aquatic environments, banana and plantain stems [M. perstans parasite densities compared to younger people might be due to the cumulative effect of reinfection during their lifespan [The higher parasite density, as expressed by the qPCR’s Cq values, of in stems  that miglifespan .M. perstans and L. loa cause chronic infections that are associated with strong immune modulation in the human host [M. perstans might negatively impact on mf clearance [M. perstans infections might impair vaccine induced immune responses and protection by exerting strong immune modulatory effects as described for other helminth infections [It has been shown that filarial worms including man host ,42. Theslearance  as well learance . In addifections ,45. Therfections ,47.Quantitative measurements of filarial nematodes might become important in the context of development of novel drug interventions against Mansonellosis, loiasis, lymphatic filariasis and onchocerciasis in areas with high co-infections between these parasites . The lacL. loa and M. perstans, both blood-borne pathogens. Onchocercha volvulus and M. stretocerca were described on Bioko Island [Wuchereria bancrofti. Also, we have used primers/probe combinations in our Llmp-qPCR assay that could most likely not amplify the newly identified Mansonella sp “DEUX, thereby potentially omitting this novel Mansonella species described recently [Our study presented had some limitations. We restricted our analysis to o Island  but theirecently . We condIn summary, our approach of repurposing used mRDT as source of NA provides a promising, future tool that enables a cost-effective approach to monitor the prevalence, genotypes, parasite densities and co-infections of filarial nematodes and potentially other blood borne infectious diseases. Also, PCR amplification and sequencing of DNA fragments allowing for genotyping extends the range of possible applications of using NA stored on mRDTs. This method might be of particular interest in settings with limited access to cool chains, laboratory infrastructure and in populations not necessarily served by clinics and health posts in rural areas.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 FigSelection of mRDTs used for NA extraction and molecular analysis stratified by mRDT result (A), age groups (B) and district (C).(TIFF)Click here for additional data file.S2 FigM. perstans and L. loa. (B) Curves in purple show amplification of the RNaseP gene used as an internal extraction and qPCR amplification control. (C) Curves in yellow show the amplification and detection of M. perstans-specific 18S target by qPCR. (D) Curves in blue show the amplification and detection of the L. loa-specific LLMF72 target by qPCR.(A) Multiplex qPCR amplification of the human RNase P gene, (TIF)Click here for additional data file.S3 FigL. loa (white) and M. perstans (grey). Grouped by age (A), gender (B), urban or rural residence (C) and day time of blood collection (D).Variation of Llmp-qPCR Cq values for (TIFF)Click here for additional data file.S4 FigM. perstans or L. loa positive samples, sorted by their Cq values are shown. Samples selected for Sanger Sequencing are highlighted in red.All (TIFF)Click here for additional data file.


Salvadora persica (Miswak), Commiphora molmol (myrrh) and Azadirachta indica (neem) in combination with commercially available antimicrobial agents: penicillin, tetracycline, ofloxacin and fluconazole on endodontic pathogens such as Enterococcus faecalis, Streptococcus mitis,Actinomyces naeslundii and Candida albicans. Materials and Methods: Microbiological samples from the root canals of the teeth undergoing retreatment were taken using sterile paper points kept at full length in the canal for 30 s. The disc diffusion method was used to check the susceptibility of microbes to the plant extracts and antimicrobials by measuring the diameter of the inhibition zones. Against the microbes, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)/minimum fungicidal concentration (MFC) of the plant extracts were assessed. The fractional inhibitory concentration index (FICI) was used to estimate the synergistic effect of plant extracts combined with antimicrobials against the resistant endodontic microbes. Results: The findings clearly indicate the effectiveness of all the three plant extracts, Commiphora molmol, Azadirachta indica, Salvadora persica, against all the experimental pathogenic microorganisms except for the ineffectiveness of Azadirachta indica, Salvadora persica against Candidaalbicans. Maximum antimicrobial activity was displayed by Azadirachta indica against Enterococcusfaecalis  and the minimum antimicrobial activity was displayed by Commiphora molmol against Actinomyces naeslundii . The best synergy was displayed by Commiphora molmol with fluconazole against Candidaalbicans (FICI = 0.45). Conclusions: The current study delineates the variable antimicrobial activity of plant extracts against the experimental endodontic pathogenic microorganisms. Plant extracts in conjunction with various antimicrobials can be valuable aids in combating relatively resistant endodontic microorganisms that have been the cause of worry in recent years, leading to failure even in treatment procedures following all required protocols.Background and objectives: This study aimed to evaluate the synergistic antimicrobial activity of extracts obtained from  Enterococci are the most common bacteria found in the oral cavity, gastrointestinal tract, and vagina of humans and animals. Previously, Enterococci were regarded as non-virulent, but now they are recognized as one of the major causes of nosocomial infections worldwide [Enterococci are Gram-positive cocci that can occur singly, in pairs, or as short chains. They are facultative anaerobes, possessing the ability to grow in the presence or absence of oxygen. In endodontics, Enterococcus species, especially Enterococcus faecalis , are associated with chronic periodontitis and failed root canal treatments involving chronic apical periodontitis [orldwide . Enterocdontitis ,3.Enterococcus pose a great challenge for dentists owing to their complex nature, which helps them to survive the harsh environments, including extreme alkaline pH, salt concentrations and high temperatures of 60 °C [E. faecalis to be associated with endodontic failure is its ability to invade dentinal tubules and strong adhesion to collagen [E. faecalis ranges from 100 to 400 μm into dentinal tubules.of 60 °C ,5,6. Thecollagen , which iE. faecalis are cell surface-associated protein, namely, enterococcal surface protein (ESP), secreted toxins, such as cytolysin, hemolysin, gelatinase, aggregation substance (AS), serine protease, and cell wall polysaccharide. These virulence traits are attributed to the pathogenicity islands, which are virulence-coding genes present on the genome. These genes encode for transposases, transcriptional regulators, and proteins are known to have potential roles in enhancing virulence [E. faecalis has begun to pose a therapeutic challenge to physicians due to its ease of acquiring and transferring antimicrobial drug resistance. Management of such cases can sometimes be more demanding with the complex internal root canal anatomy of some teeth. However, this aspect has been greatly solved with the advent of cone-beam computed tomography (CBCT) in the field of endodontics [The virulence traits of irulence . E. faecodontics ,10,11.Candida albicans  is a fungus usually seen in 21% of primary infections and 18% of retreatment cases [Candida can survive in extreme environments by biofilm formation and using its physiochemical properties to suit the local conditions. This is the prime reason that it can persist in highly alkaline environments created by calcium hydroxide medications [Actinomyces naeslundii (A. naeslundii) and other actinomyces species cause extra radicular infections [Actinomycosis, caused by Actinomyces species, is an example of extra-radicular infection independent of the intra-radicular infections [Streptococcus mitis (S. mitis) is a Gram-positive coccus normally found in the oral cavity and can attach tooth surfaces by expressing specific proteins known as adhesins. Its cell wall contains peptidoglycans and lipoteichoic acids, which can influence inflammatory reactions and enhance pain modulation [Streptococcus can invade dentinal tubules and, by releasing different extracellular proteins, can adapt and survive in adverse environmental conditions [nt cases ,13. Candications . Actinomfections . This midulation . Streptonditions .Salvadora persica (S. persica), also known as miswak, has antimicrobial properties and prevents E. faecalis bacteria from attaching the tooth surface [Streptococcus mutans (S. mutans) count as fluoride interacts with bacterial glycolytic enzymes [Commiphora molmol (C. molmol). Myrrh belongs to the family Burseraceae [C. molmol consists of 57–61% water-soluble gum, 25–40% alcohol-soluble resin, 7–17% volatile oil, and 3–4% impurities. In ancient times, it was used in inflammatory and infectious diseases. Azadirachta indica (A. indica), commonly known as neem, is a tree, which belongs to the Meliaceae family. It has many biological activities, such as antimicrobial, antiviral, antifungal, anti-inflammatory, antimalarial, antipyretic, antioxidant, analgesic, immune-stimulant, anti-fertility, anti-acne, antihypoglycemic, anti-cancer and nematicidal properties [ surface . It has  enzymes . Because enzymes  and low seraceae , which aoperties ,23.S. persica, C. molmol and A. indica combined with commercially available antimicrobial agents: penicillin, tetracycline, ofloxacin and fluconazole on endodontic pathogens, such as E. faecalis, S. mitis, A. naeslundii and C. albicans.This study was aimed to evaluate the synergistic antimicrobial activity of extracts obtained from An in vitro experimental study design was used for the current study. Patients visiting the Department of Endodontics, College of Dentistry, King Khalid University, Saudi Arabia for retreatment of failed root canals from May 2018 to May 2019 were included in the study. The patients having radiological proof depicting apical periodontitis following diagnostic directions accepted by AAE  were incS. persica, gum resin of C. molmol and bark of A. indica) were taken from the traditional market of Abha, Saudi Arabia. The reliability of the specimens was checked by the plant taxonomist. Various antimicrobials, such as penicillin, tetracycline, ofloxacin and fluconazole  in blend with various plant extracts were used to assess the presence of the collaborative effect. The detailed protocol of the study is depicted in the flowchart  was used to vaporize the filtrate underneath decreased pressure and temperature. In ethanol (0.2 g/mL), dried plant extracts were dissolved again to be used in antimicrobial susceptibility assay. Preparation of stock solutions was done, and two-fold dilution of the stock ranging from 50 μg/mL to 0.2g/mL was used to achieve a final working volume to be later used in determining MIC and MBC/MFC.The dried herbs of The teeth undergoing retreatment were assessed for their location, number of canals, and presenting signs and symptoms . Intracoronal and extracoronal restorations were considered unacceptable if they had a marginal defect, fracture, secondary caries, or a dislodged restoration. A periapical radiograph was used to evaluate the condition of obturation . An obtuMicrobial growth and isolation were done in the specific culture media presented in E. faecalis colony was isolated for identification by biochemical tests, such as Voges–Prosjauer, indole, citrate, urease, hydrogen sulfide (H2S) production and L-pyrrolidinyl arylamidase (PYR) test. A. neslundii and S. mitis were tested by API rapid ID32A kit (bioMérieux) as described by the manufacturer. They were also tested for esculin hydrolysis. Acid production from arabinose, fructose, cellobiose, inositol, glycogen, mannitol, lactose, ribose, and trehalose (at 1% w/v) and salicin and starch (at 0.5% w/v) in peptone-yeast extract broth was observed. C. albicans colonies showed a rough, filamentous border, composed of hyphae and pseudohyphae. C. albicans was identified by a germ tube test.An Disc diffusion method was used to examine antimicrobial sensitivity assay. Lysogeny broth (LB) media were used to inoculate cultures of microbes at 37 °C for 3 h. Phosphate-buffered saline was used to adjust turbidity to 0.5 McFarland’s index. Mueller–Hinton agar (MHA) plates were used for microbial lawn culture. MHA plates were used to place the selected antimicrobial plates  and incubated in suitable conditions at 37 °C for 24 h. The microbial growth inhibition zone was measured around each antimicrobial agent in millimeters as described .The agar well diffusion method was used to examine the antimicrobial activity of the extracts. LB broth media was inoculated with cultures of microbes for 3 h at 37 °C, and 0.5 McFarland’s index in phosphate-buffered saline was used to adjust the turbidity. The cap of the sterile syringe was used to prepare the wells of 6 mm diameter in LB agar, and a lawn culture was formed from a diluted culture with the help of a sterile cotton swab. The wells were filled with 20 μL of extract from various plants (0.2 g/mL) with incubation of the plates at 37 °C for 24 h. The diameter of the inhibition zone was measured around each well in millimeters as described . The wel5 CFU/mL. MIC was considered as the lowest concentration of extracts that showed no visible microbial growth and turbidity after 24 h of incubation.Micro-broth dilution assays using Mueller–Hinton broth were used to determine MICs of the plant extracts against microbial strains. The range of the extract’s concentration was from 50 μg/mL to 0.2 g/mL. The wells of polystyrene sterile flat-bottomed 96-well plates were filled with 180 μL culture of all microbial strains. The triplicate wells for each strain were loaded with 20 μL from the 2-fold dilution of the plant extract. In the control group, the triplicate wells were loaded with 20 μL of ethanol (5%). Each strain had a starting inoculum of 1.5 × 10Subculturing of 100 μL of the culture from each well of the micro-broth assay was done on MHA plates at 37 °C for 24 h to obtain the MBC/MFC of the extracts. MBC/MFC was considered as the lowest concentration of the extracts at which there was no microbial growth.The synergistic antimicrobial activity of the plant extracts was determined in combination with various antimicrobial agents by checkboard methods.The antimicrobial activity of the plant extract and antimicrobial agent combination was interpreted as one of the following categories: synergy; additive effect, indifferent; or antagonism. The fractional inhibitory concentration (FIC) of each agent was calculated as the MIC of the agent in combination, divided by the MIC of the agent alone.The sum of the FIC or FIC index (FICI) is therefore, analyzed as:The FICI results were interpreted as follows: <0.5 synergy; 0.5 to 1 additive effect; 1–2 indifferent or no effect; and >2 antagonism .t-test.The experiments were conducted in triplicates. For the results, data were outlined as mean ± standard deviation (SD). SPSS  was used for statistical analysis. The significance of the difference between the mean expression of the experimental and the control samples was calculated using a one-tail Student’s S. mitis was resistant to all the antimicrobials.In this study, the screening activity of the antimicrobials against different strains was performed before evaluating the antimicrobial activity of the extracts from various plants. C. albicans (p < 0.05).To assess antimicrobial activity, the various plant extracts were used to treat all four microbes. A zone of inhibition was viewed as significant for susceptibility of the microbe to the plant extract if the zone size was more than 8 mm. albicans  (p < 0.0p < 0.05), while C. albicans was not susceptible to neem and miswak extract (p = 0.179). The highest MIC and MBC value was displayed by C. molmol against A. neslundii, whereas the lowest MIC and MBC value was displayed by A. indica against E.faecalis  [A. indica on E. faecalis [A. indica against C. albicans followed sodium hypochlorite and propolis [A. indica demonstrated antimicrobial property against all endodontic pathogenic microbes (p < 0.05) except the C. albicans (p = 0.179). The antibacterial activity of neem was the most against E. faecalis  followed by A. neslundii  and S. mitis .Ethanol was used as a solvent to withdraw the vital compounds from the plant products following the previous studies ,41,42. Ta (neem) . Severalfaecalis ,45,46. Apropolis . On the E. faecalis, S. mutans, L. acidophilus and P. aeruginosa [C. albicans. The highest antibacterial activity of miswak was displayed against S. mitis  followed by E. faecalis  and A. neslundii .Miswak in the form of chewing sticks, due to their antibacterial, antifungal, antiviral, and antiplaque properties, have a widespread application in and around Middle Eastern countries . Againstruginosa ,49. In tE. faecalis, F. nucleatum [C. albicans  followed by S. mitis , E. faecalis  and A. neslundii .Myrrh has been investigated for its antimicrobial properties against oral pathogens and found to have promising results against ucleatum  and helpE. faecalis, while for A. neslundii it was indifferent with penicillin (FICI = 1.23), tetracycline (FICI = 1.18) and ofloxacin (FICI = 1.005). The best effect of combining various plant extracts with different antimicrobials against E. faecalis was an additive effect of combining C. molmol with penicillin (FICI = 0.51), which was followed by C. molmol with tetracycline (FICI = 0.57) and C. molmol with ofloxacin (FICI = 0.71). Miswak was effective against S. mitis in synergy with ofloxacin (FICI = 0.49). Against A. neslundii, it had an additive effect with tetracycline (FICI = 0.64) and ofloxacin (FICI = 0.91), and against E. faecalis, it had an additive effect with penicillin (FICI = 0.79). The best effect of combining various plant extracts with different antimicrobials against A. neslundii was an additive effect of combining C. molmol with tetracycline (FICI =0.54 ), which was followed by S. persica with tetracycline (FICI = 0.64) and S. persica with ofloxacin (FICI = 0.91). Myrrh was in synergy with ofloxacin and fluconazole against both S. mitis (FICI = 0.49) and C. albicans (FICI = 0.45), respectively. It exhibited an additive effect against E. faecalis with all the three antimicrobials: penicillin (FICI = 0.51), tetracycline (FICI = 0.57), ofloxacin (FICI = 0.71), while an indifferent effect was observed against A. neslundii with penicillin (FICI = 1.25) and ofloxacin (FICI = 1.40). The best effects of combining various plant extracts with different antimicrobials against S. mitis were the synergistic effect of C. molmol with ofloxacin (FICI = 0.49) and S. persica with ofloxacin (FICI = 0.49). Against C. albicans, the synergistic effect of C. molmol with fluconazole, was the best synergistic effect (FICI = 0.45) against the different microbes tested with various combinations.The effect of combining two drugs can be antagonistic, indifferent, additive, or synergistic. Few previous in vitro studies have evaluated the effects of combining various antibiotics with different plant extracts that have reduced MIC for antibiotics ,52. One C. albicans [Tetracyclines, being broad-spectrum antibiotics, are used as intracanal antibiotics against polymicrobial endodontic microorganisms, along with others like metronidazole and newer BioPure MTAD . However, intracanal microorganisms are resistant to these antibiotics ,54,55, aalbicans . This prC. molmol, A. indica, S. persica, against all the experimental endodontic pathogenic microorganisms. A. indica and S. persica were ineffective against C. albicans. Synergies of plant extracts with antimicrobials helped enhance overall antimicrobial effects. The best synergy was displayed by C. molmol with fluconazole against C.albicans. In addition, C. molmol and S. persica both exhibited synergy with ofloxacin against S. mitis. The effects of synergies may help in improving the treatment modalities against endodontic pathogens. However, further studies are needed to evaluate their biocompatibility, effect on biofilms and their mechanisms for synergy.The results from the study indicate that plants in the form of their extracts are good sources of biologically active antimicrobial agents. Plant extracts in conjunction with various antimicrobial agents can be a valuable aid in combating the relatively resistant endodontic microorganisms that have been the cause of worry in recent years leading to failure even in the treatment procedures following all the required protocols. The current study clearly indicates the effectiveness of all the three plant extracts,


Staphylococcus aureus is the major cause of endocarditis, and its mortality has remained high despite therapeutic procedures over time. A case of left-sided native valve endocarditis caused by methicillin-sensitive Staphylococcus aureus which responded well to moxifloxacin monotherapy is described. An 83-year-old woman with a history of current hospitalization presented with fatigue and fever. Transthoracic echocardiography depicted vegetation, and blood cultures were positive for Staphylococcus aureus. After a 14-day intravenous administration of moxifloxacin, a good clinical response was achieved, and antibiotic regimen transitioned to oral moxifloxacin for an additional four-week therapy. Staphylococcus aureus (S. Aureus), being the most common cause of infective endocarditis, (IE) is an intractable microorganism [organism , 2. Infeorganism . Treatmeorganism , 5.A 83-year-old woman with a history of chronic atrial fibrillation presented to an outpatient clinic with fatigue and fever. She has been hospitalized a week before her admission due to decompensated cardiac failure and given intravenous diuretics. On physical examination, her body temperature was 38.7°C, pulse rate was 110 beats/minute, blood pressure was 140/80 mmHg, and respiratory rate was 18 breaths/minute with an oxygen saturation of 96% on ambient air. Overall, the patient appeared weak. Auscultatory exam revealed irregularly irregular heart beats and a 2/6 holosystolic murmur at the apex and a 1/6 diastolic murmur at the left lower sternal border. She had minimal left pulmonary crackles on inspiration. There were no digital clubbing, Osler's nodes, or Roth's spots. Jugular venous pressure was normal, and there was no peripheral edema. Because of her previous stay, a hospital-acquired infection was suspected along with COVID-19. Hence, empirical parenteral antibiotics moxifloxacin (MOX) (400 mg once daily) and cefoperazone-sulbactam combination (1.0 g + 1.0 g twice daily) were administrated immediately. The decision was awaited until the laboratory test results.3 with 94.6% of segment form, a platelet count of 307.000/uL, and C-reactive protein of 56.6 mg/dL. A real-time polymerase chain reaction assay performed on a nasopharyngeal swab specimen was negative for COVID-19. Prior to initiation of empirical regimen, blood samples were taken for culture from different venipuncture sites. On the second day of moxifloxacin therapy, the patient's fever returned to normal. Subsequently, test results for blood cultures came positive for methicillin-sensitive S. aureus that was sensitive to quinolones (Electrocardiogram was compatible with atrial fibrillation. No obvious infiltrates on chest X-ray were seen. Her laboratory data included hemoglobin of 10.7 g/dL, white blood cell count of 21,060 cells/mminolones  (Table 1In addition to resolution of fever, improvement in general condition of the patient and a decrease in the number of leukocytes motivated the decision to continue solely with parenteral MOX. Meanwhile, Janeway lesions appeared on both legs . On day Initial antibiotic treatment in infectious diseases is generally empirical because the determination of a pathogen may take days from time to time . Both ESS. Aureus shows that, with standard oral doses, MOX is both inhibitory and bactericidal against strains of S. Aureus [S. aureus [S. aureus were not resistant to MOX. Unless a high ciprofloxacin resistance existed, MOX treatment did not fail when compared to ciprofloxacin and vancomycin [Moxifloxacin has a prolonged serum half-life, broad spectrum of antimicrobial activity, and good safety features such as excellent absorption, single daily dosing, and good tolerability. MOX belongs to the fourth generation of quinolone antibiotics. In selected patients, thanks to its high activity against Gram-positive bacteria, MOX may be preferred as first-line strategy . In vivo. Aureus . In anim. aureus . In vitr. aureus , and all. aureus . Strainsncomycin .S. aureus IE due to well-established high bacterial densities within vegetation. Depending on the bactericidal activity of the selected regimen, a shorter duration of antimicrobial therapy only in unique instances with uncomplicated IE may be suggested [S. aureus IE. Although these regimens are not allowed to be utilized in left-sided IE [Duration of therapy should be up to six weeks in uggested . Accordisided IE , recent sided IE , 5.One cohort study  and a raS. aureus IE with a 4th generation quinolone antibiotic with simplicity is the key message in this report. This case is the 1st reported case of sequential intravenous and oral MOX monotherapy as first-line treatment of left-sided native valve IE caused by methicillin-sensitive S. Aureus.Treatment of