Plasmodium spp. parasites that must infect and survive within mosquito salivary glands (SGs) prior to host transmission. Recent advances in transcriptomics and the complete genome sequencing of mosquito vectors have increased our knowledge of the SG genes and proteins involved in pathogen infection and transmission. Membrane solute carriers are key proteins involved in drug transport and are useful in the development of new interventions for transmission blocking. Herein, we applied transcriptomics analysis to compare SGs mRNA levels in Anopheles stephensi fed on non-infected and P. berghei-infected mice. The A. stephensi solute carriers prestinA and NDAE1 were up-regulated in response to infection. These molecules are predicted to interact with each other, and are reportedly involved in the maintenance of cell homeostasis. To further evaluate their functions in mosquito survival and parasite infection, these genes were knocked down by RNA interference. Knockdown of prestinA and NDAE1 resulted in reduction of the number of sporozoites in mosquito SGs. Moreover, NDAE1 knockdown strongly impacted mosquito survival, resulting in the death of half of the treated mosquitoes. Overall, our findings indicate the importance of prestinA and NDAE1 in interactions between mosquito SGs and Plasmodium, and suggest the need for further research.Malaria is caused by mosquito-borne  Malaria is caused by mosquito-borne Plasmodium parasites. In particular, adult female mosquitoes of the genus Anopheles are efficient Plasmodium vectors in many diverse ecosystems3. Anopheles stephensi is a competent vector for both Plasmodium falciparum and P. vivax, the most virulent malaria-associated species3, as well as for Plasmodium species that infect rodents and, thus, this mosquito species is widely used as a laboratory model. A. stephensi is found throughout the Indian subcontinent—with its territory extending from the Arabian Peninsula, Iran, and Iraq, to Bangladesh, southern China, Myanmar, and Thailand4.Over the last 15 years, massive prevention measures and new treatment tools have greatly decreased the global malaria burden. However, despite these advances, about 214 million new malaria cases and 438,000 associated deaths were registered in 2015 alone9. Sporozoite invasion of mosquito SGs is receptor-mediated, and involves several parasite and SG proteins11. Characterization of the salivary components involved in parasite infection and transmission would greatly enhance our understanding of the mosquito–host–pathogen interface, and potentially reveal candidate targets for malaria prevention and control.Malaria transmission from mosquitoes to vertebrate hosts occurs through the mosquito’s salivary glands (SGs), and for this reason many studies have focused on this organ to develop new malaria control measuresAnopheles spp. after feeding or infection have created transcriptome catalogues that reveals the influence of Plasmodium infection process in certain genes12. Recently, a role of a membrane glucose transporter gene in Anopheles gambiae was reported in malaria transmission using RNA interference studies5. Membrane proteins are responsible for the transport of a high diversity of substrates through the SGs, maintaining basic cell physiological functions and preserving optimal conditions for completion of the parasite life cycle14.Sialotranscriptomic and functional genomic studies of 15. Over 300 human SLCs have been identified and studied with regards to their pharmacological applications as key proteins for drug transport16. Thus, research focusing the ion transporters in mosquito SGs will be useful not only for increasing our knowledge of these biological processes, but also for the development of new interventions for transmission blocking17.Solute carrier (SLC) families include membrane proteins that transport solutes, such as ions and metabolites maintain ionic homeostasis using ion or electrochemical gradientsP. berghei-infected and on non-infected mice, and RNA sequencing (RNA-seq) was performed. Gene expression levels were determined and compared between the infected and non-infected sialomes. The membrane ion transporters prestinA and NDAE1 were found to be up-regulated in response to infection. Based on the gene expression during Plasmodium infection, putative co-interaction and transmembrane function to maintain cell homeostasis, these genes were selected to perform silencing experiments. Further gene silencing of prestinA and NDAE1 resulted in significantly reduced vector survival and decreased sporozoite numbers in the SG. This work completes the analysis of the first Illumina-derived mRNAs assembly from P. berghei-infected A. stephensi SGs, constituting a valuable tool for future research18. Our present results suggest that membrane ion transporters in A. stephensi SGs affect Plasmodium infection, potentially representing new targets for development of measures to block malaria transmission.In the present study, SGs were isolated from mosquitoes fed on 20. Thus, the analysis of the transcriptomic response of A. stephensi SG to P. berghei infection can predict, at some extend, proteome regulation. Previous studies have investigated the Anopheles sp. sialotranscriptome24, but concerning A. stephensi, there is only a previous work reporting an immune related response to P. berghei infection18. Herein, RNA-seq analysis enabled the production of two catalogs of transcripts corresponding to the P. berghei infected and non-infected SGs, which constitute a body of fundamental information to help with the selection of potential key targets for malaria control.Recently‚ it was demonstrated that, according to the system under study, transcriptomic changes are highly correlated with protein levelsP. berghei infection , of which 1996 (79%) were upregulated and 540 (21%) were downregulated. These findings suggest a vast network of interactions between the vector and parasite, with certain molecules likely being essential for parasite maintenance in the mosquito and/or for part of the mosquito’s response to infection. Other studies have shown comparable results26.From the RNA sequencing data, we obtained a total of 26,357 and 36,393 transcripts for control and infected samples, respectively. Among these transcripts, 2536 showed differential expression in response to A. stephensi transcripts were functionally annotated using Gene Ontology (GO) terms, and were grouped into 14 classes: cell function, chemosensory, detoxification, diverse, immunity, metabolism, proteolysis, recognition, regulatory, replication-transcription-translation (RTT), signaling, structural, transport, and unknown function. Figure Plasmodium infection, the data concerning immunity class was discussed elsewhere18.The differentially expressed A. stephensi SG transcripts revealed that the more commonly represented functional classes in this data set included metabolism, RTT, cell function, and transport , while less common functional classes included recognition, detoxification, and chemosensory   Fig. . The mosPlasmodium infection, including 8 that were upregulated (fold-change of 5.67 to 1.49) and 6 that were downregulated (fold-change of −3.16 to −1.39), were analyzed (see Supplementary Table P = 0.009)  activity mediating this metabolic pathway28. Another transcript, AGAP003184 (fold-change of 2.67), reportedly plays a role in the heme-biosynthetic pathway favoring Plasmodium survival30.Changes in metabolism-related transcripts may be associated with increases of metabolites favoring sporozoite development within SGs before transmission to a vertebrate hostA. gambiae enables parasite adhesion32, suggesting its involvement in the metabolic process. Herein, the chondroitin 4-sulfotransferase represented by AGAP005721 was upregulated in P. berghei-infected SGs, supporting the possibility that this molecule may be related to parasite adhesion to SGs.Previous studies show that chondroitin glycosaminoglycan presence in the apical midgut microvilli of P. falciparum-infected A. gambiae33 and in the SGs of P. berghei-infected A. coluzzii5. Moreover, differential transcription has been detected in hemocytes of P. berghei-infected A. coluzzii34, and a high number of upregulated genes related to DNA replication were found in dengue virus-infected A. aegypti35. It is possible that the Plasmodium parasite may interfere with or manipulate the expressions of genes required for invasion and development within the SGs.Translational control of gene expression may result from cell stress and could allow cells to regulate protein levels in relation to infection processes. A total of 363 transcripts from the RTT functional class were differentially expressed, including 15.28% of upregulated and 10.74% of downregulated transcripts. Increased translational regulation has also been observed in 18. Since the innate immune response is activated when pathogens are recognized, it is expected that this class will be strongly represented in studies focusing on infection.The results concerning the immune response were discussed in a previous study conducted by the authors36. This activity also ensures that SGs meet the requirements for Plasmodium survival37.Membrane transport proteins mediate the passage of specific molecules and/or ions across the membrane bilayer of the SGs, controlling nutrient uptake, removal of metabolic wastes and xenobiotics, and the maintenance of transmembrane electrochemical gradientsP. berghei infection impacts the expression of transport-related transcripts, suggesting that the parasite may exploit A. gambiae cellular mechanisms. Moreover, a microarray-based experiment revealed the upregulation of 326 transport-related genes among a total of 4978 transcripts from A. gambiae SGs after P. berghei infection23.A SAGE analysis revealed that A. coluzzii5. Within this category, 50 transcripts were identified as ion transmembrane transporters. These proteins mediate the passage of specific molecules and/or ions through SGs membrane, and are associated with a diverse range of physiological roles, including the maintenance of transmembrane electrochemical gradients36. In particular, we identified transcripts related to the solute carrier family (SLC), including AGAP007054 (SLC13), AGAP007743 (SLC16), AGAP005405 (SLC39), AGAP000097 (SLC25), AGAP005537 (SLC35), AGAP010725 (SLC26), and NDAE1 (SLC4). Based on the influence and shared function of transporters in mosquito SGs during infection, and the high expression levels of such genes in our data, we selected the solute carriers SLC26 and NDAE1 as targets for further studies. These molecules are described below in greater detail.Our present results showed that transport-related transcripts were among the most differentially expressed , with 9.52% upregulated and 8.89% downregulated. This is in accordance with a previous report in Plasmodium infection. The transcript AGAP010035 from the cell function class was upregulated (fold-change of 2.34), and is related to the TOR nutritional hormone signaling pathway, which is affected by parasite infection in the midgut38. Additionally, we detected downregulation of two salivary gland surface (SGS) proteins, SGS4 (fold-change of −1.51) and SGS5 (fold-change of and −2.75), which are considered highly immunogenic and related to both blood feeding and parasite infection40, suggesting parasite expression regulation.Some genes that were identified in this study belonged to functional classes other than those mentioned above, and have been previously reported as associated with 41. The proteolysis gene AGAP009212 (fold-change of 1.49)—known as SRPN6, a malaria parasite invasion marker in mosquitoes—suggests that serpins are associated with the immune response against Plasmodium infection in A. stephensi. In fact, mosquito serpins control melanization45 and host homeostasis during blood feeding44. Previous reports showed that serine proteases are also related to dengue virus transmission in Aedes mosquitoes46.Proteins from the proteolysis class are involved in several proteolytic events during blood feeding, including digestion of blood, sugar, and extracellular matrix components47. Studies using P. berghei reveal the induction of cell death and apoptosis during infection49. Our present data showed upregulation of the initiator caspase CASPL2 (fold-change of 0.95), which is reportedly related to apoptosis during parasite infection. Ramphul et al.and collaborators (2015) silenced initiator caspases, such as CASP-L1, CASP-L2 and IAP1, and reported no significant changes in Plasmodium infection intensity or infection prevalence among mosquitoes50. Downstream substrates of caspases, such as Bcl proteins, have been found in the A. stephensi sialome. Here we detected upregulation of Anob-1 (AGAP011552) (fold-change of 1.44), which is a protein from the Bcl-2 family (DEBCL) that acts as an apoptosis regulator and is related to autophagy51.Apoptosis, i.e., programmed cell death, is a crucial cellular mechanism for tissue homeostasis and normal immune system functioning, which is highly regulated by a variety of caspases, Bcl-2 proteins, and inhibitors of apoptosis (IAPs)prestinA and NDAE1 genes, respectively, were annotated as encoding a protein with sulfate transmembrane transporter activity (Gene Ontology annotation: http://www.ebi.ac.uk/QuickGO/GTerm?id = GO:0015116) and a protein with anion transmembrane transporter activity (Gene Ontology annotation: http://www.ebi.ac.uk/QuickGO/GTerm?id = GO:0008509). PrestinA is an anion exchanger belonging to the SLC26 family, while NDAE1 participates in anion transport and belongs to SLC4 family15. The transmembrane localizations of prestinA and NDAE1 were confirmed using location predictor software.The Drosophila melanogaster (dPrestin) and A. gambiae (AgPrestinA and AgPrestinB) as homologues of mammalian SLC26a5. AgPrestinA and AgPrestinB are highly expressed in the SGs and midgut, respectively52. In our data, the mosquito orthologue was found over-expressed  in Plasmodium-infected SGs. PrestinB is a transmembrane protein that supports the exchange of ions HCO3–, oxalate2−, SO42−, and formate across the midgut membrane15. Uptake of SO42− by prestinB is essential for biosynthesis of chondroitin sulfate (ChSO4), an epithelial ligand that allows midgut invasion31. PrestinB also mediates the uptake of bicarbonate produced by carbonic anhydrase or supplied by Na+-driven anion exchanger (NDAE1)53 that was also found to be over-expressed  as revealed in our studies. NDAE1 is a solute carrier that plays a central role in bicarbonate, sulfate, and oxalate metabolism, contributing to maintaining pH homeostasis in the cell lumen. Disruption of this gene is reportedly lethal to Drosophila larva54. As expected, both these SLC transcripts were found over represented in the previously reported sialotranscriptome of A. coluzzii5. Moreover, both SLC are presented in both A. gambiae (NCBI (https://www.ncbi.nlm.nih.gov/) with the Accession no. UP_000007062) and A. stephensi (VectorBase (https://www.vectorbase.org/) with the Accession no. AsteI2.3.) proteomes, which indicates the possibility of these transporters being expressed in the mosquito. We further analyzed the potential interaction between these two solute carriers using STRING software for prediction of protein–protein interaction  may interact with at least two possible network partners: the proteins NDAE1 and K13811. NDAE1 (AGAP009736) showed the closest relationship, with a high confidence score of 0.729. The protein K13811, a 3′-phosphoadenosine 5′-phosphosulfate synthase (666 aa) encoded by the AGAP001256 gene, showed an interaction score of 0.731 with prestinA. In sulfation reactions, K13811 is considered an obligate cosubstrate for active sulfate55. Remarkably, our data showed that the AGAP001256 transcript was also upregulated (fold-change of 1.86). NDAE1 network predictions indicated the existence of two proteins: prestinA (confidence score of 0.729) and FKBP12 , which was not present in the transcriptomic data.A. stephensi protein is closely related to the majority known proteins from other malaria vectors.Furthermore, the evolutionary history of the prestin Fig.  indicateA. stephensi and A. maculatus NDAE1 proteins are in the same monophyletic group, being closely related  and the NDAE1 sequence (99% reverse and forward primer). Synthetized dsRNA was checked for integrity, and ~276 ng of dsRNA was injected into each mosquito. The dsRNA was injected fourteen days post-infection because at this time point the ruptured oocyts release sporozoites which can then migrate to the salivary glands. After silencing assays, we evaluated the gene expression levels  from VectorBase. The products showed high similarity to the els Fig. , percentels Fig. , survivaels Fig. , and numels Fig. .Figure 5prestinA and NDAE1 genes is represented in the prestinA . NDAE1 was knocked down by 92.98%, significantly reducing the gene expression level . PrestinA and NDAE1 expression levels were also evaluated under the effect of knockdown of the other gene. PrestinA silencing was associated with a decline of NDAE1 mRNA levels , and NDAE1 silencing was associated with reduced prestinA expression , suggesting interaction between these two proteins. Double knockdown (silencing 62.02% of prestinA and 55.25% of NDAE1) resulted reduced mRNA expression levels for both genes  compared with the control group. The lower silencing efficiency in this case can be explained by the amount of specific dsRNA used, since in the double knockdown, we inoculated mosquitoes with ~138 ng/injection of each dsRNA.In Fig. inA Fig. , NDAE1   and fondue (a component of the hemolymph clot), show that manipulation of their gene expressions in A. gambiae G3 strain mosquitoes resulted in survival patterns similar to that of the control57. However fondue-silenced mosquitoes reportedly influence fungal infections57. Our results showed that double knockdown profoundly affected mosquito mortality, with a 23% population reduction. Moreover, knockdown of NDAE1 alone led to a 51% population reduction (P < 0.05) . On the other hand, prestinA is SG exclusive, and thus the disruption of mRNA is tissue specific.Under the conditions of the present study, our results showed that 05) Fig. . Since a05) Fig. . This inprestinA reduced infection by 42.2% (P = 0.003), the NDAE1 silenced group showed an infection reduction of 73.5%, (P = 0.006), and double knockdown resulted in a reduction of 53.2% (P = 0.001)  Fig. .31, support that reducing transporter expression in A. stephensi SGs, and thus lowering sulfate uptake and leading to deficient chondroitin sulfate biosynthesis, could prevent Plasmodium invasion by reducing the ability for parasite adhesion.These results together with previous findingsPlasmodium invasion/multiplication in mosquito tissues60. Manipulation of NDAE1 expression in the whole vector may produce a synergetic effect, resulting in an acute reduction of sporozoites in SGs due to pH alteration. Silencing of both genes may have altered system equilibrium, exposing P. berghei to different/adverse environmental parameters. Figure A. stephensi-SGs cells and their influence on Plasmodium infection.Sustained pH homeostasis is essential for viable A. gambiae37 and A. coluzzii, as well as P. berghei maintenance in SGs. Our present findings provide crucial information for the selection of target genes for preventing malaria transmission, including our report of the A. stephensi sialotranscriptome during P. berghei infection and our functional characterization of prestinA and NDAE1. These ion and solute membrane transporters are tightly connected, and were demonstrated to be upregulated after infection. Moreover, RNAi-mediated gene knockdown results suggested that the proteins encoded by these genes may affect Plasmodium viability in the SGs. Further investigation is needed to improve our understanding of basic transport, transport regulation, and how these processes regulate vector–parasite interaction. To eradicate and control malaria disease, it is crucial that we explore new targets for developing control measures and transmission-blocking vaccines.Suppression of ion and solute exchangers can disrupt different metabolic pathways; therefore, these transmembrane proteins may be useful in new transmission-blocking approaches. The impact of trehalose and glucose transporters has already been demonstrated in terms of survival of rd October (permit number n° 023357) and the Council of Ethics of the IHMT. All experiments were performed in accordance with relevant guidelines and regulations.Animals were maintained at the Institute of Hygiene and Tropical Medicine (IHMT), in accordance to Europe Directive 86/609/ EEC and Portuguese law (Decreto-Lei No. 129/92). Animal experiments were conducted with the approval of the Divisão Geral de Alimentação e Veterinária (DGAV), Portugal, under Art° 8, Portaria n°1005/92 from 23A. stephensi mosquitoes were obtained from the IHMT insectary and reared at 20 °C in 70% humidity, under a 12 h light/dark photoperiod. Adult mosquitoes were fed ad libitum on a 10% glucose solution. For experiments, only mosquitoes aged between 3–5 days were used.The P. berghei -infected mice were obtained. Female CD1 mice were intraperitoneally inoculated with 107 P. berghei ANKA parasitized red blood cells. The levels of parasitaemia were measured from blood smears of the mouse tail using light microscopy after staining with Hemacolor® kit . Mice were used when the parasitaemia reached about 10% and exflagellation was observed (4–6 exflagellations/field).To validate gene expression and gene knockdown efficiency, mosquitoes fed on uninfected and Three hundred female mosquitoes per group were allowed to feed on anesthetized mice, and only fully engorged females were kept at 19–21 °C and 80% humidity.5. In order to check Plasmodium infection, midguts were dissected at day 8–9 post-blood-meal (PBM) confirming the presence of oocysts; at day 18 PBM, SGs were extracted and kept either in ice cold PBS for sporozoites quantification or in ice-cold RNA later  for RNA extraction.Midguts and SGs were dissected from cold anaesthetized female mosquitoes, under a stereoscopic microscope at 4X magnification . Detailed methodology of tissue and RNA isolation is described elsewhereRNA was promptly extracted using RiboZol™  and stored at −80 °C. The quantification and integrity of the RNA was assessed using a ND-1000 Spectrophotometer  and a Agilent 2100 Bioanalyser® system , respectively.P. berghei-infected mice. Individual SGs of from each condition were grouped to obtain the quantity needed to perform high throughput sequencing. Sequencing was performed at Parque Científico de Madrid . From 1 µg of total-RNA, enrich poly (A) mRNA were used to bead with magnetic beads with Oligo (dT) to construct the cDNA library. Next, from purified mRNAs, two different fragmentation conditions were applied so that a “shorter” and a “longer” preparation was made for both control and infected RNA samples. Further, double-stranded cDNAs were synthesized and subjected to end-repair, adenylation, ligation with sequencing adapters and PCR amplification. Proper fragments were purified by size using AMPure XP beads  and size and quality confirmed using an Agilent High Sensitivity DNA kit . The size range of fragments was around 310 bp for the short insert and 465 bp for the longer insert preparations. Next, the libraries were pooled and titrated using qPCR to get an accurate estimation of concentration. In a Cluster Station , clusters were generated and using a GAIIx equipment , the libraries were sequenced using a 2 × 100 cycle sequencing run program separated by a paired-end turnaround. TruSeq RNA kit  was used to prepare cDNA library as per manufacturer instructions. Based on the results provided by FPCM, Era7 Company performed the transcriptomic analysis. Trimmed sequences were used for de novo assembly using Oases program and functional information obtained from Uniprot  number, Interpro motifs). For each condition, Bowtie and Samtools were used to map and determine the RPKM values of reads. After clustering transcripts, eXpress program was used to obtain the quantification per protein, and each set of transcripts annotated by the same protein was considered as a Unigene provided with functional annotation. Differential expression analysis of Unigenes in infected versus uninfected mosquitoes was carried out using EdgeR62. For this data, each transcript compiles information of UniProt, RefSeq, GO, VectorBase and NCBI Taxonomy Enzyme databases. After the initial analysis, transcripts were functionally annotated into 14 classes: cell function, chemosensory, detoxification, diverse, immunity, metabolism, proteolysis, recognition, regulatory, RTT, signaling, structural, transport and unknown. Previous studies have investigated the sialotranscriptome of A. stephensi regarding exclusively the immunity class18. The remaining data is analyzed in the present study.RNA-seq was conducted in SGs of mosquitoes fed on uninfected and Up or down gene regulation was also considered in this analysis. RNA-seq data can be found in the database: ArrayExpress (www.ebi.ac.uk/arrayexpress) with the Accession no. E-MTAB-3964.A. stephensi SGs with differential regulation and belonging to different functional classes with a potential interference in the parasite development, were chosen for RNA-seq validation through relative qPCR. Using three biological replicates of each condition (infected and non-infected), total RNA (100 ng/µL of each sample) were used to synthesize cDNA using the iScript™ cDNA Synthesis Kit . qPCR reactions of 20 μl were performed in triplicate using IQ™ SYBR® Green Supermix kit  in a CFX96 Touch Real-time PCR  thermocycler. The following conditions were used: an initial cycle of denaturation at 95 °C for 10 min; followed by 45 cycles of 95°C for 15 seconds and temperature of each primer set for 45 seconds. Fluorescence readings were taken at 62 °C after each cycle and a melting curve (60–95 °C) was performed. The sequences of the primers were design using Primer3 platform (http://bioinfo.ut.ee/primer3–0.4.0/) and their conditions are listed in Supplementary Table Fourteen transcripts identified by RNA-seq of ribosomal protein S7 (RPS7) (Vectorbase: ASTE004816) as reference gene . Pearson’s correlation was used to compare the expression values between RNA-seq and qPCR methods for the 14 selected genes  in order to identify known/novel protein-protein interactions. Since STRING software until date only accepts A. gambiae data, orthologues from A. stephensi obtained sequences were used64. Briefly, the program by single protein name or amino acid sequence generates the network images based on a spring model. To predict transmembrane domains/protein localization, TMHMM, based on a hidden Markov model66, WoLF PSORT67 and MultiLoc268 were applied to amino acid sequences.Genes selected for functional analysis were further characterized using the software STRING 10.0  (Katoh & Standley 2016) and all positions containing gaps were exclude with GUIDANCE2 . Molecular Evolutionary Genetics Analysis  software was used to obtain the best model to build69 a Maximum Likelihood tree based on a bootstrapping method with 1000 replicates. Generated trees were visualized and edited using FigTree v1.4.370.From VectorBase database (https://www.vectorbase.org/), the amino acid sequence of the selected SLC proteins were used to search for the orthologues (https://www.vectorbase.org/blast). The sequences of the orthologues and main vectors of malaria were obtained. prestinA (ASTE009391) and NDAE1 (ASTE003102) on P. berghei-infected A. stephensi mosquitoes. Previously obtained mosquito RNA was used to synthesize cDNA using the iScript™ cDNA Synthesis Kit  and cDNA was used to amplify fragments of interest, using iProof™ High-Fidelity DNA Polymerase  and specific primers containing T7 promoter sequences at the 5′-end  (GenBank: NM_009735). Fourteen days PBM, cold anesthetized female mosquitoes were injected intrathoraxically with 69 nl (4 mg/ml) of each dsRNA using a nano-injector . For double knockdown a mixture of equal parts of dsRNA was used. At this time point, the majority of oocysts ruptured and the sporozoites start to invade salivary glands.For dsRNA injection three independent assays per group (target gene) were conducted one week apart. For each group, 100 72. Four days post injection, three biological replicas containing 15 to 30 SGs per treatment were used to perform sporozoite quantification, by light microscopy using a haemocytometer5. Infection levels were compared between control and treated groups using the non-parametric Mann-Whitney test (SPSS v24.0)73. To assess gene knockdown efficiency three biological replicas of 20–40 SGs per group were used to extract total RNA and synthetize cDNA as described previously. qPCR assays were performed under the conditions aforementioned. Gene expression was analysed by the CFX Manager™ Software  using Pfaff method63.After dsRNA injection, the number of viable mosquitoes was evaluated using Log Rank, Breslow and Tarone-Ware testssupplementary information


At a median follow-up of 17 months, patients with higher NLR at baseline had poorer progression-free survival (PFS) and overall survival (OS). On multivariate analysis, elevated NLR at baseline was independently associated with PFS and OS . In addition, patients with high pre- and post-treatment NLR demonstrated worse clinical outcomes than other groups. Our results suggest that NLR is an independent prognostic indicator for patients with ESCC undergoing dCRT and changes in NLR level with treatment may indicate therapeutic benefit.The present study evaluated the clinical and prognostic value of neutrophil-to-lymphocyte ratio (NLR) in patients with locally advanced esophageal squamous cell carcinoma (ESCC) treated with definitive chemoradiotherapy (dCRT). A total of 517 patients with ESCC were enrolled and analysed retrospectively. The NLR was calculated at three time points: baseline, post-treatment, and at the time of tumor progression. Elevated NLR was defined as a ratio ≥5. High NLR at baseline was present in 204 (39%) patients and was significantly correlated with larger tumour size, advanced TNM stage, worse ECOG performance status, and dCRT response ( The prognosis of esophageal squamous cell carcinoma (ESCC) remains extremely poor in spite of improvements in surgical techniques, with a 5-year survival rate ranging from 15% to 25%2et al.Recently, several studies have revealed that the presence of an ongoing systemic inflammatory response is associated with adverse outcomes in variety of solid organ malignancies, including ESCC71011131415Therefore, the aim of the present study was to examine the association of pretreatment NLR with treatment response rate, progression-free survival (PFS), and overall survival (OS) in patients with locally advanced ESCC treated with dCRT. We also analysed the impact of change of NLR with treatment to investigate its role as a response indicator.Criteria for reporting recommendations for tumor markers in prognosis study (REMARK) were followed wherever possible.The study protocol was approved by the Institutional Review Board for human studies of Huai’an First People’s Hospital, Huai’an, China; and informed written consent was obtained from all subjects. The study was performed in accordance with the approved guidelines. Patients with locally advanced ESCC treated with dCRT at the Nanjing Medical University Huai’an First Hospital between January 2006 and May 2010 were identified and retrospectively analysed. The inclusion criteria were as follows: (a) histologically confirmed primary ESCC by available biopsy specimens; (b) previously untreated; (c) Karnofsky score ≥70; (d) age ≤75 years; (e) no other significant medical disease. Patients with any evidence of active infection or presence of a chronic inflammatory condition were ineligible. Patients with hematology disease were excluded. Tumor were staged according to the conventional tumor-node-metastasis (TNM) classification for esophageal carcinoma , and the pretreatment clinical staging was based on the results of barium swallow, esophagogastroduodenoscopy (EGD), neck, chest, or abdominal CT examination, and bone scan.Clinicopathologic characteristics including age, gender, Eastern Cooperative Oncology Group performance status (ECOG PS), tumor length, tumor differentiation, smoking status, and TNM stage were extracted from patients medial records. The laboratory data collected included hemoglobin concentration, absolute WBC count, absolute neutrophil count, and absolute lymphocyte count. At diagnosis, data on serum carcinoembryonic antigen (CEA) and squamous cell carcinoma antigen (SCCA) levels were also collected. The NLR was defined by dividing the number of absolute neutrophil count by the number of absolute lymphocyte count. A NLR of 5 or greater was considered elevated in accordance with previous studies1819For patients with adequate bone marrow, renal, and hepatic function, chemotherapy was performed with a PF-based regimen (cisplatin/fluorouracil). Chemotherapy started on day 1, concurrent with initiation of radiotherapy. Cisplatin (80 mg per square metre of body surface area) was administered intravenously on Day 1, and fluorouracil (1 g per square metre of body surface area) was administered as a continuous infusion from Day 1 to Day 4. Two courses of chemotherapy were administered at a 4-week interval during radiotherapy.All patients were treated with external-beam radiation using 6 or 15 MV LINAC (Siemens ONCOR). The radiation treatment was delivered as three-dimensional conformal radiation therapy or intensity-modulated radiation treatment to ensure tumour coverage and spare adjacent normal organs. Information from EGD examination and CT scan was studied in detail before delineation of target tumour volume. A total radiation dose of 50–60 Gy  was given to the target of the gross oesophageal mass and enlarged lymph nodes.To evaluate the treatment response, esophagogram, EGD and CT scan were performed 4 weeks after completion of dCRT. The response to treatment was assessed basically according to the following criteria. CR was defined as complete regression of all assessable lesions; partial response (PR) was defined as more than 50% reduction in primary tumor size or more of the sum of the lesions and no progression of assessable lesions; stable disease (SD) was defined as a reduction of <50% or increase <25% in tumor size; progressive disease (PD) was defined as an increase ≥25% in primary tumor volume or appearance of new lesions. We divided these categories into two groups: the effective group consisted o CR and PR, the resistant group consisted of SD and PD.Follow-up evaluation was performed every 3 months for the first year, every 6 months for an additional 2 years, and then at the end of each year to study end or until death. During the follow-up period, diagnostic examinations were performed when recurrence and/or metastasis was suspected. Follow-up data were obtained from patients’ medical records and/or telephone interviews.The primary endpoints were OS and PFS. OS was defined as the time from diagnosis to death (event), or last follow-up (censored), and PFS was calculated from the date of therapy initiation to the time of disease progression (event), or last date of follow up (censored).U test or Kruskal-Wallis test. Categorical variables were compared using Fisher’s exact test or chi-square test. Survival curves were plotted with Kaplan–Meier method, and the differences were compared using a log-rank test. Univariate and multivariate Cox regression analyses were performed to evaluate potential prognostic factors for survival, and only variables that showed statistical significance in univariate analysis were subsequently entered into multivariate analysis. All statistical analyses were conducted using SPSS Statistics version 20.0 . A two-sided p value less than 0.05 was considered statistically significant.Continuous data were expressed as the median (range), and categorical variables were reported as frequencies and percentages. Continuous data were analysed using Mann-Whitney A total of 517 patients met the inclusion criteria and were selected for this study. Most of the patients were male , and the median age at diagnosis was 65 years . There were 83 (16%) cases with stage II disease, 377 (73%) cases with stage III disease, and 57 (11%) cases with stage IV disease. Median tumour length was 4 cm  and 224 primary tumours (43%) were longer than 5 cm. Detailed patient characteristics at baseline are shown in All 517 patients underwent concurrent dCRT with two cycles of PF. After treatment, CR, PR, SD, and PD were observed in 88 (17%), 203 (39%), 211 (41%), and 15 patients (3%), respectively. After dCRT, 17 patients (3%) underwent esophagectomy and 160 patients (31%) received adjuvant chemotherapy. With a median follow-up of 17 months , 431 (83%) of the 517 patients died. Of these, the cause of death was progression of recurrent disease in 396 (92%) patients, treatment-related esophagoaortic fistula in 3 (1%) patients, and other causes in the remaining 32 (7%) patients. The median PFS and OS for the whole cohort of patients were 12 months and17 months, respectively.9/L , 4.06 × 109/L , 1.12 × 109/L , and 3.24 , respectively.For all patients, the median values for baseline serum WBC count, neutrophil count, lymphocyte count, and NLR were 5.86 × 10At baseline, 204 (39%) patients had a high baseline NLR ≥ 5 and 313 (61%) patients had NLR < 5. The relationships between clinicopathologic variables and pretreatment NLR are shown in p = 0.033), larger tumor size (p < 0.001), distant lymph node metastasis (p < 0.001), advanced TNM stage (p < 0.001), and low response rate to dCRT .Tumour responses to dCRT for the 517 patients are shown in vs. NLR < 5, median PFS 9 vs. 15 months, p < 0.001, vs. NLR < 5, median OS 12 vs. 20 months, p < 0.001, To further examine whether pretreatment NLR was associated with outcomes of ESCC patients after dCRT, Kaplan–Meier survival analysis was used to compare the low (n = 313) and high (n = 204) NLR subgroups p < 0.05, Results of univariate analysis indicated that ECOG PS (≥2), tumor length (≥5), lymph node metastasis (positive), tumor stage (III/IV), dCRT response (SD + PD), SCCA level (≥1.5), and baseline NLR radio (≥5) were significantly correlated with decreased PFS or OS  and OS  and OS  had a worse dCRT response rate and poorer PFS and OS. Although several studies have shown an association between NLR and prognosis of patients with ESCC, they mainly reported results for patients treated with surgery21et al. examined a cohort of 295 esophageal cancer patients treated with esophagectomy and found no association between pretreatment NLR and tumor stageAs a biomarker of inflammation and immunology, increased NLR was previously correlated with advanced stage in endometrial cancer, small-cell lung cancer, and colorectal cancer2324Currently, definitive chemoradiotherapy with a PF regimen is an important component of the treatment of locally advanced ESCC, and the clinical CR to dCRT is widely accepted as the most important predictor of patient outcome26293031Recently, mounting evidence indicates that the existence of systemic inflammation response, as evidenced by NLR, mGPS, CRP, and PLR, is correlated with a worse prognosis in patients with cancers3334353825A previous study demonstrated the prognostic significance of pre- and post-treatment NLR in prostate cancer patients who received second-line chemotherapyThe present study provides the first clinical evidence supporting NLR as a biomarker for prognosis of patients with locally advanced ESCC. However, research limitations exist in our study. First, the retrospective design of the study may lead to bias, and the results must be validated in prospective study. Second, the total number of patients included is relatively small. In addition, unknown physiological factors that potential affecting the NLR might influence our results.In conclusion, our results demonstrated that pretreatment NLR ≥ 5 was an independent prognostic factor for poor survival in patients with locally advanced ESCC treated with dCRT. Moreover, changes in NLR level with treatment may indicate therapeutic benefit.How to cite this article: Zhou, X.-L. et al. Neutrophil-to-lymphocyte ratio as a prognostic biomarker for patients with locally advanced esophageal squamous cell carcinoma treated with definitive chemoradiotherapy. Sci. Rep.7, 42581; doi: 10.1038/srep42581 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


To evaluate prevalence and patterns of drug resistance among pulmonary tuberculosis (TB) patients in Hangzhou City, China.Sputum samples of smear positive TB patients enrolled in 2011 and 2015 were collected and tested for drug susceptibility, and demographic and medical record data were extracted from the electronic database of China Information System for Disease Control and Prevention. Chi-square test was used to compare drug resistance prevalence between new and treated patients and between male and female patients, and Chi-square test for trend was used to compare the prevalence over calendar years 2011 and 2015.Of 1326 patients enrolled in 2015, 22.3% had resistance to any first-line anti-TB drugs and 8.0% had multi-drug resistance (MDR); drug resistance rates among previously treated cases were significantly higher than among new cases. Significant declines of resistance to isoniazid, rifampin, ethambutol and streptomycin, and MDR from 2011 to 2015 were observed among previously treated patients, while a significant decline of resistance to rifampin was observed among new cases.While the prevalence of acquired drug resistance decreased due to due to implementation of DOTS-Plus program, the prevalence of primary drug resistance due to transmission remained high. Greater efforts should be made to screen drug resistance for case finding and to reduce transmission through improving the treatment and management of drug-resistant patients. China is one of the countries with the highest burden of tuberculosis (TB) disease in the world. Although its ranking in total TB cases dropped in 2015 from second to third behind India and Indonesia , 4. StudHangzhou City is located in eastern China, about a hundred miles away from Shanghai. It comprises 13 districts, one county-level city, and two counties, and has 7.2 million local residents and over 2 million migrant populations.All smear-positive pulmonary TB patients who lived in Hangzhou in years 2011 and 2015 were included in this study. The highest value was used for analysis if patients had multiple drug susceptibility testing (DST) results in the study years.TB is a notifiable disease in China. Over 68,000 health facilities report notifiable diseases to the national, real-time, internet-based disease reporting system, known as the China Information System for Disease Control and Prevention (CIS-DCP). Hangzhou City Center for Disease Control and Prevention (CDC) is authorized for access to the sociodemographic information and medical records of TB patients who live in Hangzhou in this system.TB cases were diagnosed following Chinese clinical guideline for TB diagnosis and treatment. Three sputum samples were collected from each participant at different time points  prior to initiation of treatment, and were examined for acid-fast bacilli (AFB). Two specimens with the highest bacterial counts were used for culture. TB culture was performed as follows: First, decontaminating and digesting the sputum with equal volume of 4% sodium hydroxide for 15 min; Then, inoculating 0.1 ml specimen into the Lowenstein–Jensen medium, and culturing it in incubator at 37 °C; After that, observing the colony growth, which was confirmed by microscopic examination for AFB through Ziehl-Neelsen staining.Species identification of mycobacteria was performed by conventional biochemical tests. Drug sensitivity test was performed using the proportion method on Löwenstein-Jensen medium, with the following concentrations: 0.2 micrograms per milliliter (μg/ml) for isoniazid, 2.0 μg/ml for ethambutol, 2.0 μg/ml for ofloxacin, 4.0 μg/ml for streptomycin, 30 μg/ml for kanamycin, and 40 μg/ml for rifampin. The critical growth proportion for drug resistance was 1% for all drugs. All drugs were obtained from Sigma Life Science Company (USA). The standard sensitive strain H37Rv was tested in each set of the tests and again within each set if the batch of medium was changed. The drug sensitivity test result or the H37Rv should be sensitive. All drug sensitivity tests in years 2011 and 2015 were performed by the same staff in the TB reference laboratory at Hangzhou CDC, a part of World Health Organization/International Union against Tuberculosis and Lung Disease Global Project on Anti-Tuberculosis Drug Resistance Surveillance.P value < 0.05 was considered statistically significant.Statistical analysis was conducted with SPSS 12.0 software. Chi-square tests or Fisher’s exact tests were used comparing drug resistance rates between new and treated patients and between male and female patients. Chi-square tests for trend were used for comparing the difference of drug resistance rates from 2011 to 2015. M. tuberculosis. Six patients were excluded due to lack of drug sensitivity test results for first-line anti-TB drugs; therefore, 1326 (70.2%) patients were included in the analysis.The general characteristics and drug resistance rates of 1184 participants in 2011 were reported elsewhere ; of thesOf 1326 patients, 961 (72.5%) were male and 365 (27.5%) were female; 1305 (98.42%) were Han Chinese and 21 (1.58%) were other ethnic minorities; age ranged from 12 to 94 years (mean 54); 289 (21.8%) were migrants; 1020 (76.9%) were new cases and 306 (23.1%) were previously treated cases; 874 (65.9%) had a drug sensitivity test result for ofloxacin and 875 (66.0%) for kanamycin.In 2015, about 18% (184/1020) new TB patients were resistant to at least one first-line drug, while the prevalence of drug resistance among previously treated patients was double  . There were significant declines in resistance to isoniazid, rifampin, ethambutol, streptomycin, and multi-drugs among previously treated patients, while among new patients there was a significant decline for rifampin only  than among treated cases (22.2%). The MDR prevalence is comparable to the global average, e.g., 3.3% among new cases and 20% among previously treated cases , whereasStudies have shown that there is increasing or persistently high prevalence of drug resistance among TB patients in Mainland China , 6, 14, Second, the widely used regimens for both new and treated TB patients in China are two months of isoniazid, rifampicin, pyrazinamide and ethambutol followed by four months of isoniazid and rifampicin (2HRZE/4HR) or two months of isoniazid, rifampicin, pyrazinamide, ethambutol and streptomycin followed by six months of isoniazid and rifampicin (2HRZES/6HR) .PreviousOur study has limitations. First, our study sample only included sputum smear-positive TB patients, but other study showed 17% of drug-resistant and 20% of MDR cases were linked to sputum smear-negative sources . TherefoIn summary, our study found higher prevalence of drug resistance and MDR among treated TB patients than among new patients in Hangzhou City, and showed a decreasing trend from years 2011–2015. DOTS-Plus program should be expanded, and greater efforts should be made to screen drug resistance for case finding and to reduce transmission through improving the treatment and management of drug-resistant patients.


Syphilis is a global health concern disproportionately affecting HIV-infected populations. In Alberta, Canada, the incidence of syphilis in the general population has recently doubled with 25% of these infections occurring in HIV-infected patients. The Southern Alberta HIV Clinic (SAC) and Calgary STI Program (CSTI) analyzed the epidemiologic characteristics of incident syphilis infections in our well-defined, HIV-infected population over 11 years.Since 2006, as routine practice of both the Southern Alberta Clinic (SAC) and Calgary STI Programs (CSTI), syphilis screening has accompanied HIV viral load measures every four months. All records of patients who, while in HIV care, either converted from being syphilis seronegative to a confirmed seropositive or were re-infected as evidenced by a four-fold increase in rapid plasma reagin (RPR) after past successful treatment, were reviewed.P < 0.001), males  and men who have sex with men (MSM) as their HIV risk activity .Incident syphilis was identified 249 times in 194 HIV-infected individuals. There were 36 individuals with repeated infections (28.5% of episodes). Following a prior decline in annual incident syphilis rates, the rates have tripled from 8.08/1000 patient-years : 4.14–14.75) in 2011, to 27.04 per 1000 person-years (95% CI: 19.45–36.76) in 2016. Half of the syphilis episodes were asymptomatic. Patients diagnosed with syphilis were twice as likely not to be taking ART and had a higher likelihood of having plasma HIV RNA viral loads > 1000 copies/mL (19%). Incident syphilis was seen predominantly in Caucasians  contains supplementary material, which is available to authorized users. The global reemergence of syphilis as a common and clinically significant sexually transmitted infection (STI) is forcing healthcare providers to search for the new elements driving this epidemic. It is estimated that worldwide, there are nearly twelve million new infections with syphilis annually . SyphiliSyphilis, similar to other STI’s, acts synergistically with HIV, both increasing susceptibility to infection and leading to worse health outcomes –6. AsympThis increase in episodes of syphilis being seen in southern Alberta prompted the Calgary STI Program (CSTI) and the Southern Alberta Clinic (SAC), to coordinate their initiatives to improve syphilis screening in those engaged in HIV care. In this retrospective cohort study, we aimed to characterize incident syphilis episodes in HIV-infected individuals, assessing patient demographics and markers of HIV control. Such knowledge may be essential for developing improved and targeted preventative strategies especially for patients engaged in ongoing HIV care.SAC and CSTI are the sole referral agencies for both syphilis and HIV within the region, facilitating inclusivity of this retrospective cohort study. SAC provides HIV care to all HIV-infected individuals living within southern Alberta, Canada and CSTI provides all health care services for STI’s from testing to treatment.In a quality assurance project  at both SAC and CSTI programs, routine serologic screening for syphilis regardless of risk accompanied regular four-month HIV viral load measurements between January 1, 2006 and December 31, 2016. The records of all episodes of incident syphilis infection occurring in HIV-infected patients were reviewed. Every indeterminate or positive syphilis serology for a SAC patient was discussed with or referred to CSTI at the time of testing.N = 2448). Patients were followed until December 31, 2016 or until they either moved, died or were lost-to-follow-up. All patients, who while in HIV care, converted from being seronegative for syphilis to a confirmed positive status or were re-infected with syphilis were reviewed through the SAC database and a CSTI chart review. A control population was identified through the SAC database to be used for comparison of demographic data and included all HIV-infected patients followed at SAC with at least one regular visit between January 1, 2006 and December 31, 2016 whose treponemal or non treponemal syphilis test remained negative.All individuals followed at SAC with at least one regular visit between January 1, 2006 and December 31, 2016 were included in this study , however in 2008 the screening test was changed to an enzyme immunoassay (EIA), a treponemal test. The RPR continued to be used as a confirmatory test as well as for monitoring response to therapy. In Calgary, the secondary confirmatory test was either the fluorescent treponemal antibody absorption test (FTA-ABS) or the line immunoassay (INNO-LIA). Repeat syphilis episodes were identified by a four-fold increase in RPR after a prior documented successful treatment course for syphilis and were evaluated and staged by an STI specialist (RR).Clinical and sociodemographic data was obtained from a review of both SAC and CSTI databases and charts for all patients included in the study. These data included: age at HIV diagnosis; gender , self-reported ethnicity , Other); most likely HIV exposure risk , and year of HIV diagnosis. Other self-reported data fields included cigarette smoking (current or past), alcohol abuse (> 14 drinks/week or self-described binge drinking), recreational drug use (i.e. intravenous drug use), intimate partner violence exposure , and ever unstable housing .Neisseria gonorrhoeae, Chlamydia trachomatis and Hepatitis C virus (HCV) were self-reported and documented in CSTI charts at the time of syphilis diagnosis.For patients with an incident syphilis infection, detailed standardized information was collected by one physician (RL), through a comprehensive review of both SAC and CSTI charts and databases. Multiple sources including nursing interviews, social work reports, self-administered questionnaires, laboratory reports and routine STI and HIV care notes from charts and databases were used. These data included self-described sexual practices, ART use and HIV viral loads collected both prior to and after each positive syphilis test. Prior history of comorbid infections including During routine HIV appointments, all patients were asked if they were sexually active and if the activities would be considered “unsafe” (condomless sex). For those engaging in unsafe sexual practices, counselling on both HIV and STI transmission risks was provided. The number of self-reported sexual partners in the period six months prior to testing positive for syphilis was documented. All data was anonymized prior to analysis.Demographic and risk factors of patients in control group were compared with those with syphilis infection, those with repeat episodes of syphilis, those not on ART, and those not virally suppressed  using chi-squared testing. Age and sex adjusted incident rate (IR) of syphilis infection were calculated from 2006 to 2016 using the 2006 demographic as reference. Incidence rates adjusted for HIV risk exposure in addition to age and sex were also analyzed. Incidence rates were calculated using patients “active in care,” meaning they had attended at least one SAC visit each year to contribute to a patient-year of follow up. All statistical analysis was performed using Stata, . All charts were created with Microsoft Excel.Between 1/1/2006 and 12/31/2016, the mean number of patients “active in care” at SAC was 1517 per year (range: 1086 in 2006 to 1889 in 2016). During this time, there were 20,203 syphilis tests done on a total of 2448 patients who attended at least one regular SAC visit. On average, there were 180 days between each syphilis test per patient. The average number of syphilis screening tests that were done per patient each year over the 11-year period was 2.1. In 2006 the average number of tests per year was 1.3, whereas in 2016 this was 2.8. For high risk patients (MSM) screening rates were more frequent with the average testing over 11 years being 2.4 tests per year. The average number of viral load measurements per patient per year was 3.03 (range 2.58–3.37).Of the 2448 HIV-infected patients followed at SAC and CSTI during the study period, encompassing 15,175 person-years, we identified 360 potential episodes of syphilis in 305 different patients that met the broad study criteria for incident syphilis. Of the 360 potential episodes, 38 were excluded after being classified as false positive syphilis tests. Inadequate availability of comprehensive data led to exclusion of 32 episodes along with 41 additional episodes, who although tested in Alberta, had since moved out of province. These episodes were not included in incident rate calculations as validity of the tests ensuring incident infection as well as characteristics of the diagnosis were not available. We therefore analyzed 249 episodes in 194 individuals. Over half (50.8%) of incident syphilis cases were asymptomatic and were only identified by routine screening. In 71/249 (28.5%) of the infections, syphilis occurred in individuals who had previously been successfully treated for syphilis on one or more past occasions and were identified by a four-fold increase in RPR titer.The average IR between 2006 and 2016 was 12.88 per 1000 person-years of follow up : 9.64–16.11). When adjusted for HIV exposure category in addition to age and sex, the average IR between 2006 and 2016 was 13.22 per 1000 person-years of follow up (95% CI: 8.20–21.34). Incident rates decreased from 15.50 in 2006 (95% CI: 9.04–26.35) to 5.87 in 2008 (95% CI: 2.68–12.77). The rise in syphilis rates in 2009 , with MSM being the leading HIV exposure risk group  and in Caucasians (72% vs. 56%), P = < 0.001). Compared to HIV-infected but with no evidence of past or current syphilis infection, those with syphilis were more likely to have a history of alcohol abuse  and a history of recreational drug use   at time of syphilis infection were very common with 42% of the population studied having had at least one other STI prior to incident syphilis infection other than HIV. STI co-infections included; 32% of cases having a prior history of N. gonorrhoeae, 24% having had documented C. trachomatis and 1.5% were also co-infected with HCV.The incident syphilis infections were seen mainly in males (94%) compared to the control group ; the mean length of time between the HIV positive test and a positive syphilis test was 7.7 years (range 0.1–25.6) with 31% having been HIV positive for greater than 10 years . Self-reported alcohol abuse was also more common in those not on ART   of patients were not on ART in contrast to 11% of other HIV-infected patients in care in the same time period. Those not on ART with incident syphilis were more likely to be PWID (23%), compared to those on ART , Caucasian  and MSM  and a greater proportion were PWID   of patients had detectable HIV viremia  (Table P = 0.007), Caucasian  and MSM  compared to those in the control population of HIV-infected, syphilis negative individuals. A trend towards current or past recreational drug use  was noted but not reaching significance in those with repeat infections  and C. trachomatis (39%).Over one-quarter (28%) of syphilis infections occurred in individuals previously successfully treated for a past episode and were identified by a four-fold increase in RPR. Over two-thirds 47/71) were second episodes of syphilis, with 21% (15/71) experiencing syphilis for the third time, 9% (6/71) for the fourth time and 4%  for the fifth time. Those experiencing repeat episodes of syphilis were more likely male , repeat syphilis positive, syphilis positive not on ART, syphilis positive not HIV Virologically suppressed (VL > 40 copies/mL). 1Indigenous includes Aboriginal, Metis and Inuit; ACB includes African, Caribbean, Black; Other includes IndoAsian, Hispanic, East Asian, and other. 2MSM = self-reported men who have sex with men identification; HET = self-reported heterosexual identification; PWID = self-reported intravenous drug use identification; Other HIV Risk factor behavior includes: blood transfusions, hemophiliac, neonatal, postnatal infection, unknown or not reported. 3History of Cigarette Smoking-Current or Past; History of Alcohol Abuse- > 14 drinks/week or binge drinking; History of Recreational Drug Use –Current or Past; History of Intimate Partner Violence-Current or Past. (DOCX 24 kb)


Carica papaya is used traditionally and showed in vitro antimalarial activity. This study attempted to evaluate in vivo antimalarial activity of C. papaya in mice. Currently, antimalarial drug resistance poses a serious challenge. This stresses the need for newer antimalarial compounds. In vivo antimalarial activity of solvent fractions of the plant was carried out against early P. berghei infection in mice. Parasitemia, temperature, PCV, and body weight of mice were recorded. Windows SPSS version 16 (one-way ANOVA followed by Tukey's post hoc test) was used for data analysis. C. papaya fruit rind and root produced a significant (p < 0.001) chemosuppressive effect. A maximum parasite suppression of 61.78% was produced by pet ether fraction of C. papaya fruit rind in the highest dose (400 mg/kg/day). Only 400 mg/kg/day dose of chloroform fraction of C. papaya root exhibited a parasite suppression effect (48.11%). But, methanol fraction of the plant parts produced less chemosuppressive effect. The pet ether and chloroform fractions of C. papaya fruit rind had the highest antimalarial activity and could be a potential source of lead compound. Further study should be done to show the chemical and metabolomic profile of active ingredients. Pet ether fraction of Even though a remarkable progress has been made, malaria remains a major health problem in Sub-Saharan Africa. It is endemic in 91 countries. The number of people infected with malaria in Sub-Saharan Africa is estimated to be 114 million in 2015. Children are especially vulnerable, accounting for more than two-thirds of global malaria deaths [However, safe and effective mode of treatment is needed to control malaria and its complications. The increasing antimalarial drug resistance, insecticide resistance, and behavioral changes in Anopheles vectors threaten effective antimalarial drug therapy and malaria control and elimination . Artemis Cinchona species) and artemisinin (obtained from Artemisia annua) are derived from plants [The famous and potent antimalarial compounds quinine  bred and maintained at Ethiopian Public Health Institute were used. The animals were kept in cages and housed in a standard animal house under natural 12/12 h light dark cycle at room temperature, the animal house of School of Veterinary Medicine, Jimma University. They were maintained on standard pelleted diet and water ad libitum. All mice were acclimatized for one week before the study. This study was approved by the ethical review board of college of health science of Jimma University with a reference number HRPGC/578/2015. All experiments were conducted in accordance with the internationally accepted guidelines on laboratory animal use, care, and handling .The acute toxicity studies were conducted as per the OECD guidelines 425. Acute oral toxicity of each of the solvent fractions was evaluated in healthy female mice aged of 6–8 weeks. Five female mice were fasted for three hours and orally given a dose of 2000 mg/kg of the solvent fractions. The mice were observed for lacrimation, hair erection, behavioral change, reduction in their motor, feeding activities, and mortality for three hours and followed for 24 hours and/or 14 days . P. berghei ANKA strain obtained from Ethiopian Public Health Institute and maintained at animal house facility was used. For the parasite maintenance serial passage of blood from infected mice to noninfected ones was made. A donor mouse with a parasitemia of approximately 30% was sacrificed and blood collected in a Petri-dish containing 2% trisodium citrate as anticoagulant. The blood was then diluted with 0.9% normal saline. Each mouse used in the experiment was inoculated intraperitoneally with 0.2 ml of 1 × 107  P. Berghei infected red blood cells [Chloroquine sensitiveod cells .In vivo antiplasmodial activity of the plant extract against early P. berghei infection was carried out according to the method described by Peter et al. (1975). Based on acute toxicity test, three doses  were selected for the in vivo antimalarial study of the solvent fractions [ractions .After parasite inoculation, 30 mice were randomly assigned into five groups (three treatment groups and two controls), 6 mice per group. The negative control group was treated with the vehicle 2% Tween 80. Likewise, positive control group was treated with standard drug chloroquine 25 mg/kg/day. The remaining three groups received three different doses  of the plant extracts. The doses were administered orally at a volume of 10 ml/kg. The mice were treated after three hours of infection and continued for three days.Weight, rectal temperature, and packed cell volume (PCV) were recorded just before infection and on day four postinfection. On day four, a thin blood film was prepared from the tail blood of each mouse. The blood films were fixed with methanol and stained with 10% Giemsa for 10 min. Blood films were examined microscopically to determine parasitemia and parasite suppression.The mean parasitemia and % parasitemia suppression were calculated and expressed as follows :(1)%  PaFor packed cell volume (PCV) determination, blood was drawn from the tail of the different group of mice using heparinized capillary tubes before infection and on day 4 after infection.The tubes were filled with blood up to (3/4)th of their volume and sealed at the dry end with sealing clay. The tubes were then placed in hematocrit centrifuge with the sealed end outwards and centrifuged for 5 min at 5,000 rpm .(3)Packe Carica papaya were qualitatively screened for the presence of secondary metabolites. Thus, tests for alkaloids, flavonoids, terpenoids, phenolic compounds, tannins, saponins, anthraquinones, and cardiac glycosides were performed using standard test procedures [The solvent fractions of root and rind ofocedures , 27.p values less than 0.05 were considered to be statistically significant.The data was analyzed using windows software SPSS version 16 and expressed as mean ± standard error of mean (M ± SEM). Statistical significance was determined by one-way analysis of variance (ANOVA), followed by Tukey post hoc test to compare the measured parameters  within and between groups. The analysis was performed with 95% confidence interval and  Carica papaya fruit rind has a considerable antiplasmodial activity in vivo against P. berghei on early infections. In this study, the petroleum ether fraction of C. papaya fruit rind produced a dose dependent chemosuppressive effect at three doses evaluated , with a chemosuppression of 23.03%, 34.38%, and 61.78%, respectively (p < 0.001) parasite suppression as compared to negative control. The highest chemosuppressive effect (61.78%) was exhibited by petroleum ether fraction of C. papaya fruit rind at 400 mg/kg/day dose.The findings from the four-day suppressive test showed that petroleum ether ofectively . At all  C. papaya fruit rind. On the other hand, the standard drug, chloroquine, caused chemosuppression of 100%. Compared to negative control, only the highest dose (400 mg/kg/day) of petroleum ether (p < 0.001) and chloroform (p < 0.05) fractions caused a statistically significant prolongation of survival time. However, the methanol fraction was not associated with significant prolongation of survival time when compared with the negative control.However, a mild chemosuppression was produced by the methanol fraction of C. papaya root fractions produced a dose dependent and statistically significant (p < 0.001) chemosuppressive effect at the three doses evaluated . The parasite suppression by petroleum ether fraction was 21.85%, 31.53%, and 43.77% for 100, 200, and 400 mg/kg/day doses, respectively. Likewise, the parasite suppression by chloroform fraction was 9.77%, 25.25%, and 48.11% for 100, 200, and 400 mg/kg/day doses. The 400 mg/kg/day dose of the ether and chloroform fractions produced the highest parasite suppression relative to the other doses.As shown in p < 0.05) survival time prolongation effect, with the mean survival time of 9.83 and 10.17 days, respectively. Similarly, 400 mg/kg/day dose of the chloroform fraction exhibited statistically significant (p < 0.01) survival time (10.17 ± 0.75 days) prolongation effect compared to the negative control group. This effect was still by much lower (p < 0.001) than that attained by chloroquine (30 ± 0.00).Both 200 and 400 mg/kg/day doses of the ether fraction caused statistically significant ( C. papaya fruit rind significantly (p < 0.01) prevented reduction in PCV as compared to the negative control. Likewise, only the highest administered dose (400 mg/kg/day) of chloroform fraction of C. papaya fruit rind significantly (p < 0.01) prevented reduction in PCV  shows that the higher two doses (200 and 400 mg/kg/day) of petroleum ether fraction ofn in PCV . C. papaya fruit rind exhibited a significant protection against body temperature reduction on day 4. Analysis of percent of body temperature change, between days 0 and 4, indicated that P. berghei infected mice treated with the three doses of the pet ether and chloroform fractions showed a statistically significant (p < 0.01) difference when compared to negative control  ofp < 0.05, 200 mg/kg with p < 0.01, and 400 mg/kg with p < 0.001) of petroleum ether fraction of C. papaya root significantly attenuated the PCV reduction compared with the negative control. Similarly, the 200 mg/kg (p < 0.05) and 400 mg/kg (p < 0.001) doses of chloroform fraction showed a statistically significant PCV protection effect unlike methanol fraction  difference when compared to negative control. This difference was not significant when compared with chloroquine treated group. On the other hand, the effect of petroleum ether and chloroform fractions on body weight was not dose dependent and consistent. As indicated in  C. papaya root solvent fractions produced a significant (p < 0.05) parasite induced body weight reduction.Analysis of percent of body temperature change, between days 0 and 4, revealed that at the evaluated doses the three fractions Carica papaya showed the presence of secondary metabolites such as alkaloids, flavonoids, polyphenols, tannins, and terpenoids as indicated in In the qualitative phytochemical analysis, the solvent fractions of fruit rind and root of Artemisia annua and quinine from Cinchona bark [ In vivo evaluations of antimalarial activity begin with the use of the rodent malaria parasite. In addition, in vivo studies take into account any prodrug effect and the role of immune system in controlling malaria infection unlike in vitro ones [ in vivo antimalarial activity of solvent fractions of Carica papaya root and fruit rind using the 4-day suppressive test, which mainly evaluates the antimalarial activity of candidates on early infections, against P. berghei in mice. In acute toxicity studies, the observation of no death or sign of toxicity with an oral dose of 2000 mg/kg of the fractions indicated the solvent fractions are safe for use.Antimalarial drug resistance remains a major challenge and continued to emerge creating an obstacle in malaria control and elimination , 28. At ona bark . In vivotro ones . Accordi of Carica papaya root and fruit rind exhibited a statistically significant (p < 0.001) and dose dependent parasite suppression effect on early infections (Tables  Carica papaya fruit rind in the highest dose (400 mg/kg/day), with the longest survival time compared to other fractions treated mice and negative control. Likewise, chloroform fraction of Carica papaya root exhibited a higher chemosuppression effect of 48.11% at 400 mg/kg/day dose. The parasite suppression exhibited by these solvent fractions was comparable with similar studies done on D. angustifolia [ Lophira alata [ Parkia biglobosa [In this study, all the solvent fractions p < 0.00 and doses Tables . A maximstifolia , Lophirara alata , and Pariglobosa . But, a Malaria infected mice suffer from anemia because of erythrocyte destruction, either by malaria multiplication or by spleen reticuloendothelial cell action . An ideaIn vivo antiplasmodial activity can be classified as moderate, good, and very good if an extract displayed percent of parasite suppression equal to or greater than 50% at a dose of 500 mg, 250 mg, and 100 mg/kg body weight per day, respectively [ Carica papaya fruit rind and chloroform fraction of Carica papaya root exhibited a moderate antiplasmodial activity.ectively , 35. Bas Carica papaya fruit rind produced a promising in vivo antiplasmodial activity. This result was in agreement with in vitro antiplasmodial activity of pet ether (IC50 = 15.19 μg/mL) and methanol (IC50 > 100 μg/mL) fractions of C. papaya fruit rind in previous study [ C. papaya produced antiplasmodial activity with IC50 of 46.23 μg/mL [ C. papaya fruit rind had the highest antimalarial activity relative to the other fractions and the plant parts. This is attributed to the possible presence of the active metabolites. On phytochemical screening, terpenoids, flavonoids, alkaloids, and phenols are present in both petroleum ether and chloroform fractions that might be responsible for the observed antimalarial activity. These phytochemicals might also exert a synergistic antiplasmodial effect. But, weak antimalarial activity by the methanol fraction may be due to the presence of trace active constituents in the administered dose.In this study, pet ether fraction of23 μg/mL . This si C. papaya fruit rind. From this study, the active antimalarial compound found in C. papaya fruit rind is possibly nonpolar or semipolar in nature. Moreover, there may be a commercial potential in extracting the active compound from this plant, which grows abundantly throughout the tropics, the rind of which is discarded as waste.This finding may be an indicator for the presence of potential compounds with higher antimalarial activity in the petroleum ether fraction of C. papaya fruit rind and root exhibited a moderate antiplasmodial effect with the longest survival time compared to negative control. From this finding, the petroleum ether fraction of C. papaya fruit rind had the highest antimalarial activity and could be targeted as potential source of lead compound in the development of new antimalarial agent. Therefore, further study should be done on pet ether fraction of C. papaya fruit rind to show the chemical and metabolomic profile of active ingredients from this plant.The higher dose of pet ether and chloroform fraction of


Wolbachia infection in insects, the knowledge about its relations with beetle species, populations and individuals, and the effects of bacteria on these hosts, is still unsatisfactory. In this review we summarize the current state of knowledge about Wolbachia occurrence and interactions with Coleopteran hosts.Despite great progress in studies on Wolbachia presence among beetles. These publications were then examined with respect to the distribution and taxonomy of infected hosts and diversity of Wolbachia found in beetles. Sequences of Wolbachia genes  were used for the phylogenetic analyses.An intensive search of the available literature resulted in the selection of 86 publications that describe the relevant details about Wolbachia has been confirmed in 204 beetle species and that the estimated average prevalence of this bacteria across beetle species is 38.3% and varies greatly across families and genera (0–88% infected members) and is much lower (c. 13%) in geographic studies. The majority of the examined and infected beetles were from Europe and East Asia. The most intensively studied have been two groups of herbivorous beetles: Curculionidae and Chrysomelidae. Coleoptera harbor Wolbachia belonging to three supergroups: F found in only three species, and A and B found in similar numbers of beetles (including some doubly infected); however the latter two were most prevalent in different families. A total of 59% of species with precise data were found to be totally infected. Single infections were found in 69% of species and others were doubly- or multiply-infected. Wolbachia caused numerous effects on its beetle hosts, including selective sweep with host mtDNA (found in 3% of species), cytoplasmic incompatibility (detected in c. 6% of beetles) and other effects related to reproduction or development . Phylogenetic reconstructions for Wolbachia genes rejected cospeciation between these bacteria and Coleoptera, with minor exceptions found in some Hydraenidae, Curculionidae and Chrysomelidae. In contrast, horizontal transmission of bacteria has been suspected or proven in numerous cases .The collected publications revealed that Wolbachia infection across beetle species and populations is very uneven. Even the basic data about infection status in species and frequency of infected species across genera and families is very superficial, as only c. 0.15% of all beetle species have been tested so far. Future studies on Wolbachia diversity in Coleoptera should still be based on the Multi-locus Sequence Typing system, and next-generation sequencing technologies will be important for uncovering Wolbachia relations with host evolution and ecology, as well as with other, co-occurring endosymbiotic bacteria.The present knowledge about  Recentl species ; however species . Due to Wolbachia has been detected in 10–70% of examined hosts   . Furthernfection . WolbachWolbachia infection among various taxonomic groups of nematodes and arthropods. Over the last years, such reviews have been prepared for the following groups: filarial nematodes (Filarioidea)  , crustacrioidea) , spidersrioidea) , mites (rioidea) , springtrioidea) , Heteroprioidea) , ants (Frioidea) , wasps (rioidea)  and buttrioidea) . SurprisWolbachia by referring to all the abovementioned aspects of research. Moreover, we have highlighted future research directions concerning Wolbachia relationships with their diverse Coleopteran hosts.In this review we have summarized the current state of knowledge on the relations between beetles and Wolbachia” AND “Coleoptera” and “Wolbachia” AND “beetles”. Our final literature search for this analysis was conducted on December 22, 2017. This produced 322 results. Each result was inspected to determine whether or not it contained information on the subject matter. Articles that had no relevance  were excluded. After the removal of duplicates, 65 were excluded from the remaining articles (n = 239) for not being direct reports about Wolbachia-Coleoptera relations, 44 were excluded because they examined other hosts and only referred to publications on Coleoptera, and 44 others were excluded because they referred to data already presented in previous publications on Coleoptera. Each document was read critically for the information that it contained on Wolbachia-Coleoptera relations, with special reference to answering the study questions listed below. Wolbachia” AND “Coleoptera” or “beetle” were used) led to either studies not related with Wolbachia infection in beetles (which only included references to some other studies on either bacteria or beetles), or to Wolbachia sequences submitted to GenBank but without any references to published (and reviewed) articles. Searches in NCBI (GenBank) resulted only in the finding of some beetle hosts, which have been already described in papers found via Web of Science searches.We searched the scientific literature with Web of Knowledge databases, using the following combination of keywords linked by AND (the Boolean search term to stipulate that the record should contain this AND the next term): “Wolbachia infection in Coleoptera with respect to the following: the (i) characteristics of the publications (to determine the scope and progress of studies on Wolbachia) (n = 86), (ii) geographic distribution of infected beetle species and populations (n = 84), (iii) sampling design  (n = 63), (iv) characteristics of the markers (genes) used for genotyping the bacteria (n = 82) and their hosts (n = 34), (v) numbers and frequencies of species found to be infected in particular beetle families and genera (n = 58), (vi) supergroup prevalence in examined taxonomic groups (n = 43), (vii) strain distribution and diversity in populations and individuals (n = 30), (vii) effects of Wolbachia on its beetle hosts (n = 39). Statistical analyses  were done in Statistica 11 (Statsoft).We examined the collected data on various aspects of https://www.ncbi.nlm.nih.gov/genbank/) and the Wolbachia MLST database (https://pubmlst.org/wolbachia/) all available sequences of Wolbachia genes found in any species of beetle. We restricted further analyses to the most widely used bacteria genes, i.e., 16S rDNA and cell division protein gene ftsZ. Because of the different lengths and spans of available sequences, the long parts of the 3′ and 5′ ends of each gene were trimmed, which resulted in alignments of length 663 bp for 16S rDNA and 241 bp for ftsZ. The length of the ftsZ alignment was particularly short as two different sets of primers have been used for its amplification, and its amplicons only overlapped across a relatively short part of the gene. Phylogenetic trees were only reconstructed for unique gene variants found in particular host taxa. Trees were inferred using Maximum Likelihood (ML) implemented in the IQ-TREE web server (http://www.iqtree.org/)   under thee.org/)  with 10,ee.org/)  and otheThe nomenclature of host taxa and their systematic positions throughout the paper follow the articles from which the data was derived.Wolbachia infection in Coleoptera comprised 86 papers  and Chrysomelidae (34), following Coccinellidae (10), Tenebrionidae (9), and Sylvanidae (3) . The memWolbachia infection were from Europe, and a relatively high number of species were from Asia and both Americas, whereas only ten infected species were from Africa, and three from Australia-Oceania . Fewer studies (23.1%) analyzed rDNA , EF1α (14.0%), Wingless (2.2%), Histone H3 (2.2%) and microsatellites (6.1%). In Wolbachia-related studies, host genes have been used for several purposes like (i) using host DNA as a control for genetic material quality, (ii) barcoding for host species identification, (iii) phylogenetics, phylogeography and population genetics, (iv) estimating co-evolutionary relations between the bacteria and host, and (v) detecting some of the effects of Wolbachia on its hosts .The most often used 16S rDNA . Most stWolbachia infection belong to 23 families . In total 204 beetle species were found to harbor Wolbachia infection; however, the distribution of infected species among families varied markedly. The highest numbers of infected beetle species were found for the Curculionidae (81 species), Chrysomelidae (49 species), Hydraenidae (14 species), Buprestidae (13 species), Coccinellidae (12 species) and Dytiscidae (8 species) (Wolbachia (The beetles examined with respect to species) . In all olbachia . HoweverAltica , Naupactus , Hydraena  and Agrilus  . If considering only the most widely examined families, Chrysomelidae and Curculionidae, the difference in infection frequency between these two groups was insignificant . Geographic studies on Wolbachia prevalence in insects have found much lower frequencies of infection in Coleoptera species: the bacterium was found in only 10.5% of beetles from Panama and 13.5% of beetles from North America  . In totaspecies) , Table 1species) ; howeverspecies) , infecti America .Wolbachia strains belonged to three supergroups . However, they occurred at very different proportions in different groups of beetles, and these differences were significant . Overall, the proportion of beetle species found to be infected with Wolbachia strains belonging to supergroups A or B was similar, with approximately 12% of all species harboring either supergroup  , 63 species were reported to be totally infected , whereas 43 species had this bacterium in only some individuals  . A single Wolbachia strain was observed in 43 species (species with available data n = 62), whereas two strains were reported in 10 species  these numbers were 12, 5 and five, respectively and in Curculionidae (n = 37), 30, 3 and four, respectively .Among the various beetle species, viduals) , whereasctively) . The samctively) . These dectively . The numWolbachia affected beetle hosts in several ways. Linkage disequilibrium and/or selective sweep between bacteria and host genomes  were detected in six species (3% or 9% if excluding Chrysomelidae and Curculionidae): two (4%) Chrysomelidae —16 (33%) species of Chrysomelidae  transfer of bacteria genes to the autosomes of the host  (16S sequences from Xylosandrus spp. and Curculio spp. (Curculionidae), or Oreina cacaliae and Galeruca tanaceti (Chrysomelidae) clustered as a sister lineage to all other A and B sequences (16S sequences assigned to supergroup B was much greater than those assigned to supergroup A (The tree reconstructed for ergroups . F superionidae) . Sequencequences . Overallrgroup A .ftsZ included 131 sequences found in 114 host beetle species. The ftsZ phylogenetic tree resulted in a topology similar to that of 16S rDNA—it included groups of sequences belonging to A, B and F supergroups (Agrilus araxenus and Sphaerobothris aghababiani (both Buprestidae). Moreover, the supergroup B clade was divided into two clusters, among which one included a small group of sequences found in four beetle hosts: Chelymorpha alternans (Chrysomelidae), Eurymetopus fallax, Sitophilus oryzae and Conotrachelus nenuphar   (The tree reconstructed for ergroups . Supergrionidae) . Also inionidae) . There wionidae) .Wolbachia strains identified on the basis of polymorphism of two genes show that there is no strict correlation between host phylogeny and bacterial strain relationships. Even in studies that covered multiple related species , evidence for direct inheritance of Wolbachia strains from common ancestors is restricted to Hydraenidae . Also, knowledge about infection at the geographic scale is very uneven, and only Europe and Asia  have been relatively well investigated. There is a huge gap in the knowledge for African, Australian and Oceanian beetles, where a high diversity of beetles exists and probably a similar diversity of Wolbachia could be expected , between Wolbachia and its hosts and especially between Wolbachia and other microorganisms, amplicon-sequencing  or genome-sequencing are needed. This could be achieved thanks to the development of next-generation sequencing technologies (NGS). Surprisingly, despite fast development of NGS in the last years, very few studies have used this technology for studying Wolbachia in beetle populations. For example, five studies sequenced 16S amplicons generated from microbiota and detected Wolbachia  in Coleoptera and their relations, both with the host and Wolbachia. So far, only seven studies have found Wolbachia with Rickettsia and/or Spiroplasma together in beetle hosts  hosts that could not also be examined in beetle hosts. Also, the extraordinary diversity of beetles  makes this group an excellent target for Wolbachia studies. The presented summary about Wolbachia infection in beetles shows that despite numerous studies, there are still many issues that need to be investigated. We hope that this systematic review will facilitate various future studies on Wolbachia infection among beetles.In our opinion, beetles are still an insufficiently examined group of 10.7717/peerj.4471/supp-1Table S1Click here for additional data file.10.7717/peerj.4471/supp-2Figure S1Wolbachia 16S rDNA gene sequences obtained from hosts belonging to various species of Coleoptera (data downloaded from NCBI GenBank https://www.ncbi.nlm.nih.gov/genbank/). Statistical supports  are presented above the branches (shown only if >0.50).Maximum likelihood phylogenetic tree reconstructed based on Click here for additional data file.10.7717/peerj.4471/supp-3Figure S2Wolbachia ftsZ gene sequences obtained from hosts belonging to various species of Coleoptera (data downloaded from NCBI GenBank https://www.ncbi.nlm.nih.gov/genbank/). Statistical supports  are presented above the branches (shown only if >0.50).Maximum likelihood phylogenetic tree reconstructed based on Click here for additional data file.10.7717/peerj.4471/supp-4Supplemental Information 1Click here for additional data file.


We performed a MeSH term-based bibliometric analysis aiming to assess the publication trends of EAACI journals, namely Allergy, Pediatric Allergy and Immunology (PAI) (from 1990 to 2015) and Clinical and Translational Allergy (CTA) (from its inception in 2011 to 2015). We also aimed to discuss the impact of the creation of CTA in the publication topics of Allergy and PAI. We analysed a total of 1973 articles and 23,660 MeSH terms. Most MeSH terms in the three journals fell in the category of “basic immunology and molecular biology” (BIMB). During the studied period, we observed an increase in the proportion of MeSH terms on BIMB, and a decreasing proportion of terms on allergic rhinitis and aeroallergens. The observed changes in Allergy and PAI publication topics hint at a possible impact from CTA creation.The online version of this article (10.1186/s13601-018-0191-1) contains supplementary material, which is available to authorized users. To the editorBibliometric methods are used to quantitatively analyse published scientific literature for potential identification of relevant patterns , 2. We p® terminology, while titles and abstracts were text-mined to obtain further MeSH terms. This was achieved using the online tool Syn4Data (http://www.syn4data.med.up.pt/). Two independent researchers verified the obtained results and excluded inadequate/wrongly attributed or generalist/uninformative MeSH terms  and 2015, we randomly selected and analysed 20% of Allergy publications and 40% of PAI publications. Additionally, we assessed all CTA publications until 2015. We retrieved the title, abstract and keywords of selected publications—keywords were converted into MeSH terms via Unified Medical Language SystemFor each journal, we obtained the frequency of publications to which each MeSH term was attributed. MeSH terms were categorised into nine “thematic” categories—(1) asthma and lower airways diseases, (2) allergic rhinitis and allergy to aeroallergens, (3) food allergy and nutrition, (4) drug allergy, (5) anaphylaxis and insect venom allergy, (6) skin and eye diseases, (7) diagnosis methods, (8) asthma and allergy therapy, and (9) basic immunology and molecular biology (BIMB). For each journal, we assessed the time trends of each category, performing a univariable logistic regression with time (in years) as independent variable.We then compared the proportions of each category terms between the three journals. To address whether CTA had any influence in the topics published by Allergy and PAI, we compared MeSH terms proportions in these latter two journals prior and after 2011. Multivariable logistic regressions were performed for each category, adjusting for changes of the Editors-in-chief. Similar comparisons were performed concerning the proportions of publications assessing animal models.n = 1001) and MeSH terms  were from Allergy, followed by PAI  and CTA . Allergy and CTA had significantly more publications with animal models  than PAI (5.6%) . More post-2011 than pre-2011 Allergy publications assessed animal models .We assessed 23,660 MeSH terms from 1973 articles. Most publications ; p < 0.001], and “BIMB”   and 29.5% of CTA (p < 0.001). In Allergy, more terms belonged to BIMB category after than before 2011 , even after adjustment for Editorial changes . In PAI, the opposite trend was observed , even after adjusting for the Editors-in-chief  ; p < 0.001), “asthma and lower airways diseases” , and “allergic rhinitis and allergy to aeroallergens” ; this latter decrease was significant even after adjusting for Editorial changes . The proportion of “allergic rhinitis and allergy to aeroallergens” terms was also lower in post- than pre-2011 PAI articles . Consistently, within the three journals altogether, there were significant decreases over time in the percentage of MeSH terms on “diagnosis methods”  and “allergic rhinitis and allergy to aeroallergens” .On the contrary, post-2011 Allergy publications had lower proportion of terms on “diagnosis methods”  (A), and absolute and relative frequencies of MeSH terms of each thematic category (B) among Allergy, Pediatric Allergy and Immunology (PAI), and Clinical and Translational Allergy (CTA) journals.