Bartonella spp. are Gram-negative, facultative, intracellular bacteria transmitted by hematophagous insects. Several species cause zoonotic diseases such as cat-scratch disease. Bartonella henselae and Bartonella clarridgeiae are the main species found in Thailand, however, there have been few studies on Bartonella spp. In addition, the hematological evaluation of Bartonella-infected animals is limited in Thailand. The aims of this study were prevalence investigation and hematological evaluation of Bartonella-infected dogs and cats residing in Bangkok, Thailand. (2) Methods: In total, 295 dogs and 513 cats were molecularly evaluated to detect Bartonella spp. using PCR with primers targeting the partial gltA, rpoB, ftsZ, ribC, and groEL genes. In total, 651 domestic animals were evaluated for hematological parameters compared between Bartonella-positive and Bartonella-negative animals. (3) Results: Overall, the prevalence of Bartonella spp. was 1.61% which was found only in free-ranging cats (2.83%). Bartonella henselae and B. clarridgeiae were confirmed from a concatenated phylogenetic tree of partial gltA and ribC genes, with 100% bootstrapping replication. For other housekeeping gene sequences, mixed infection was expected from the amplicons of rpoB, ftsZ, and groEL. Importantly, the mean corpuscular volume (MCV) was significantly increased in Bartonella-positive cats. (4) Conclusions: We suggest that B. henselae and B. clarridgeiae are important species and are still circulating in domestic animals, especially cats. The evaluation of blood parameters, especially a raised MCV, should be of concern in Bartonella infection in asymptomatic cats. Additionally, the knowledge of how to prevent Bartonella-related diseases should be promoted with people in at-risk situations.(1) Background:  Bartonella spp. are Gram-negative, facultative, fastidious intracellular bacteria causing various pathological changes and bacteremia in their natural and accidental hosts. Several bloodsucking arthropods have been confirmed as vectors transmitting these pathogens including cat fleas  [Pulex irritans) [Pediculus humanus) [Luztomyia verrucarum) [Bartonella spp. such as the brown dog tick  [Ixodes spp. [Dermacentor spp. [Bartonella and candidatus Bartonella have been described and more than 35 species have revealed their whole genome.s felis) , human frritans) , human lhumanus) , and sanrucarum) . Additioguineus)  and the des spp.  and Dermtor spp. ). Up to B. henselae (cat-scratch disease), B. bacilliformis (Carrion’s disease and verruga peruana), and B. quintana (trench fever) [Bartonella spp. from various animal hosts, both companion and wild animals [B. henselae, which can cause an asymptomatic infection among the hosts, such as carnivores, rodents, and ruminants [Human bartonelloses are caused by h fever) . Humans  animals . Bartone animals . At leasuminants ,11,12.Bartonella spp. [Bartonella-infected animals, particularly cats and dogs, is still unclear. Several studies in other countries have evaluated the blood parameters in Bartonella-infected animals. Importantly, blood parameters are frequently used to diagnose and monitor animal health. Even if domestic cats and dogs are popular in Thailand, there has been little hematological study comparing uninfected- and infected-Bartonella animals.Dogs and cats are defined as family members sharing household environments with humans in both urban and rural areas, and they are considered as a source of zoonotic infection, especially of bartonellosis . In addilla spp.  and are lla spp. , but doglla spp. . The clilla spp. . The evaBartonella-contaminated vector feces, and blood-sucking arthropod bite [Bartonella in domestic animals in Thailand. The objectives of this study were to investigate the prevalence of Bartonella spp. in domestic dogs and cats and to evaluate the hematological parameters of Bartonella-infected dogs and cats in Bangkok, Thailand.In Thailand, dogs and cats that are both free-ranging and owned cohabit human communities. Bangkok is a province where the density of temples and household are considered high. Free-ranging animals were frequently found in temples which were a place for cultural activities and festivals, and owned animals were also found in the household. Significantly, the pattern of familiarity between humans and their belonging animals might induce zoonotic infection, particularly bartonellosis. The main proven modes of transmission in the human are animal scratch or bite with pod bite . Howevern = 513) and dogs (n = 295) residing in Bangkok, Thailand, consisting of 634 free-ranging (from temples) and 174 owned animals (from KUVTH). The ratio of males to females in the animals was 1.26:1 in the owned and 0.83:1 in the free-ranging animals. The mean ± standard deviation age of studied animals was 3.03 ± 2.01 years in the free-ranging animals (2.83 ± 1.71 years in cats and 3.29 ± 2.33 years in dogs), and in the owned animals was 3.47 ± 1.75 years (3.22 ± 1.71 years in cats and 3.58 ± 1.77 years in dogs). The numbers for related factors  are presented in In total, 808 blood samples were collected from domestic cats (Bartonella spp. in the domestic dogs and cats was 1.61% (13/808). Only domestic cats presented Bartonella DNA in extracted blood samples, however, there was an evidence only in free-ranging cats (2.83%) residing in temples  in the current study. Bartonella spp. were detected in animals residing in 10/54 (18.52%) temple clusters that were located in three zones of Bangkok (p = 0.35). Additionally, there were no significant differences for gender (p = 0.34), age group (p = 0.51), and ectoparasite infestation (p = 0.22) in the examined animals.The overall prevalence of  temples . Notably Bangkok . The posgltA-positive amplicons showed a similar direction with matched species of ribC gene results. However, rpoB, ftsZ, and groEL revealed different species (Bartonella. Amplicons of gltA and ribC matched with two species of Bartonella consisting of B. henselae (%identity = 97.01–100% of gltA and 86.62–100% of ribC) and B. clarridgeiae (%identity = 94.59–100% of gltA and 99.54–100% of ribC). Additionally, the similarity was up to 100% for rpoB, ftsZ, and groEL in all sequences, and details of sequencing data are revealed in gltA and ribC sequences showed two lineages of Bartonella that were identical to B. henselae and B. clarridgeiae, with 100% bootstrapping replication  in Bartonella-infected free-ranging cats (p < 0.05). Moreover, more than 36% of positive cats had low PCV, HGB, and MCHC. The reference of blood parameters was included in In total, 650 examined animals (80.45%) were evaluated for blood parameters. The hematological results of the examined animal population were presented in Bartonella spp. in domestic dogs and cats in Bangkok, Thailand. The results indicated that the prevalence of Bartonella in the total cat population evaluated was 2.53%, however, Bartonella spp. was observed only in free-ranging cats. The current study found two species of Bartonella (B. henselae and B. clarridgeiae) which was similar with another study in Thailand that also reported that B. henselae and B. clarridgeiae as the main species in this region [Bartonella bacteremia was found in 13 cats, however, all dogs were negative to Bartonella in this study. In cats, this positive proportion contrasted with other Bartonella prevalence reported in Brazil [Bartonella spp. in dogs was not significantly different from studies in China [Bartonella spp. were detected in cardiac tissues [Bartonella was positive only in cardiac tissue but no evidence in blood [Bartonella DNA in their blood samples. Comparisons of prevalence differences between recent and previous studies are presented in Bartonella [Bartonella than owned animals [Bartonella was the main factor causing a false negative. For the detection method, sensitivity was the impacted factor that caused the different prevalence. Moreover, the concentration of Bartonella DNA might not match with the sensitivity of the test [Bartonella detection [This study detected evidence of s region . Evidencn Brazil ,19, Green Brazil , Italy [n Brazil ,22, Germn Brazil , New Caln Brazil , USA [25n Brazil ,27,28,29n Brazil , Israel n Brazil , Portugan Brazil , France n Brazil ,34, and n Brazil , but wasn Brazil ,36, Spain Brazil , Scotlann Brazil , Irelandn Brazil , and Chiin China , Spain [in China , Cape Vein China , and Grein China , but con tissues ,43 and b tissues ,44,45,46in blood . Conversin blood  and catsin blood . This exrtonella . Strayed animals  and thesthe test  and causBartonella spp. in the current study is quite low. The low prevalence in this study was suspected to be caused by direct pathogen detection from the bacteremia level in blood samples [Bartonella spp. [Bartonella spp. detection in clinical samples, e.g., blood, other body fluids, and tissues [Bartonella diagnosis in humans and experimental cats [Bartonella spp. were quite more abundant. Due to the phylogenetic study, the longest sequence product of cPCR was more useful than the nested PCR for analysis. Additionally, the Bartonella Alphaproteobacteria growth medium (BAPGM) pre-enrichment was suggested for the specimen culture in Bartonella spp. to increase the sensitivity rate of PCR detection compared with the direct DNA extraction from pure samples [Bartonella spp. had been also co-isolated with Bartonella spp. especially environmental bacteria, non-pathogenic commensal organism, and skin normal flora [The prevalence of  samples ,50,51 anlla spp.  at the s tissues . Comparetal cats . Althoug samples . Even ifal flora ,56, and Bartonella DNA. Additionally, some studies concluded that the prevalence of Bartonella infection in stray animals was higher than in pet animals [Bartonella-positive animals. This was consistent with other studies that found no difference in prevalence based on gender, residing location, living pattern, age, and ectoparasitic appearance [Bartonella spp [Only free-ranging cats were found with  animals ,49. Howepearance . A contrella spp . We founBartonella henselae and B. clarridgeiae were the major species detected in cats [B. henselae [B. vinsonii subsp. arupensis [Bartonella infection in Thailand. Bartonella henselae was reported as the main species causing zoonotic infection, especially via cat-scratch disease. In Southeast Asia, it has been suggested that B. henselae and B. clarridgeiae are the species affecting human health and of veterinary importance [B. henselae and B. clarridgeiae are transmitted to humans via cat fleas (C. felis) [C. felis is the major vector as the pathogen-transmissible insect for feline bartonellosis [ in cats . These s in cats ,58,59,60henselae  and B. vrupensis . Of thesportance . In the . felis)  which ar. felis)  and C. fnellosis .gltA gene of Bartonella spp. for screening. Using gltA detection, this gene had a high specificity for Bartonella detection in extracted DNA samples from blood [gltA and ribC-amplified amplicons revealed a similar species of Bartonella. Additionally, other studies mentioned that positive Bartonella samples targeting the ribC gene were also positive to gltA in all samples due to the higher detection power of gltA [rpoB, ftsZ, and groEL) in the current study. This might have been due to the low discriminatory power of some genes, except for rpoB, for species differentiation in the Bartonella genus [Bartonella spp. in individual cats. From the results, it was difficult to make any conclusions regarding the mixed infection at this time. Nevertheless, co-infection should be investigated using other more sensitive techniques.The current study used primers targeting the om blood . The cur of gltA ,69,70. Hla genus . On the Bartonella-positive free-ranging cats had raised values for MCV. Over 36% of bacteremic cats had low values for HGB and MCHC. Another study in dogs mentioned that Bartonella-infected blood was significantly low in hemoglobin (HGB), erythrocytic mass (RBC), and hematocrit (HCT) [Bartonella-positive animals were significantly low in HGB, MCH, and MCHC [B. bacilliformis and were described in various ways [Bartonella-positive animals frequently showed anemia, eosinophilia, neutrophilia, and thrombocytopenia in [Bartonella-infected cats mostly showed mild or no abnormality of blood panels [Bartonella spp. were rarely described in naturally infected cats presented as healthy carriers [Bartonella in red blood cells in the replication stage [B. henselae growth and hemoglobin was a potential source of heme in vivo [Bartonella-positive cats in this study. Nevertheless, there are some limitations including the criteria for owned animals’ inclusion. A significant inclusion criterion for blood donors of studied samples that might affect Bartonella detection in owned animals required an ectoparasite control. Flea- and tick-controlled animals were possibly a chance of negative Bartonella detection. Due to the ectoparasite infestation, there was a risk factor for Bartonella infection in dogs and cats [Bartonella detection in ectoparasite was not included in this study. For further study, Bartonella spp. in animal-infesting ectoparasites should be examined to describe the dynamics of pathogen transmission.it (HCT) . The samand MCHC . The mecous ways . Bartonepenia in ,74. Addid panels . Howeverd panels . Interescarriers ,76. For on stage , and (2)on stage . In addi in vivo  which exand cats . HoweverBlood samples were collected by veterinarians and involved the gentle restraint technique. This animal research study was approved by the Kasetsart University Institutional Animal Care and Use Committee under the Ethical Review Board of the Office of National Research Council of Thailand (NRCT). All the laboratories used in this study met the standards followed by verification of the Institutional Biosafety Committee (IBC), Faculty of Veterinary Medicine, Kasetsart University .In this study, we surveyed both free-ranging and owned animals. Free-ranging animals were dogs and cats residing in temples of Bangkok. These animals were fed by animal care takers of each temple. Moreover, the ectoparasite control history of free-ranging animals was unknown. Owned animals were dogs and cats registered at the Blood Bank (BB) unit of Kasetsart University Veterinary Teaching Hospital (KUVTH) for blood donors. Owned animals lived only in the household and they roamed only in the area around their houses. The criteria for blood donors at BB of KUVTH included a limited age (1–7 years), flea and tick continue control, no blood receive history, no drug usage, and required body weight (dogs ≥ 17 kg and cats ≥ 4 kg).Bartonella spp. and the remaining volume was used for hematological evaluation.This cross-sectional study was conducted in the Bangkok metropolis of Thailand. The animal caretakers and owners of animals that resided in temples and registered with the BB at KUVTH, respectively were invited to participate in this study. Animal that had resided in temples were defined as free-ranging animals and animals that had donated blood at the BB of KUVTH were defined as owned animals. The temples that consented in this study were in three zones of Bangkok . Thirty-General data were collected for both free-ranging and owned animals including gender, living area, breed, age, and ectoparasite presentation at the surveyed time. Data of free-ranging animals were interviewed from the animal caretaker, however, data of owned animals were brought from the KUVTH record database and BB unit logbook. For ectoparasite presentation, they were examined at the time of blood collection and type of ectoparasite was recorded.TM Blood DNA Extraction Mini Kit, Favorgen Biotech Corporation, Pingtung, Taiwan) following the manufacturer’s instructions. The final volume of eluted solution (100 µL) was stored at −20 °C until the PCR-based detection.The separated blood samples were extracted for genomic DNA using commercial kits  gene . The 25 µL of PCR mixture contained 0.5 µL of dNTPs solution (0.2 mM each), 1X of Taq Reaction buffer (mixed with MgSO4), 4 pmol/µL of each primer, 0.04 U/µL of Taq DNA polymerase  Inc., Richmond, BC, Canada), 0.8% of dimethyl sulfoxide, and 3 µL of DNA template. The amplified products were kept at 4 °C until electrophoresis. Electrophoresis was conducted using agarose gel under a 0.5X TAE buffer. The process of electrophoresis was run at 100 V for 40–60 min depending on the expected amplicon size. The sample bands which were suspected as being positive for the Bartonella amplicon size were purified using a DNA purification kit , according to the manufacturer’s instructions.Conventional PCR was performed to detect tA) gene . The ampBartonella-suspected samples were amplified for other housekeeping gene fragments (rpoB), cell division protein (ftsZ), 60 kDa chaperonin (groEL), and riboflavin synthase (ribC). The amplification cycles for each housekeeping gene are shown in gltA.The ragments  consisti®, Seoul, Korea). The obtained DNA sequences were edited using the SnapGene ® Viewer software version 5.2.4  and the phylogenetic relationship was analyzed using the MEGA-X software . Concatenated sequences were used to establish a phylogenetic tree under the fitted parameter model of the maximum likelihood method with 500 bootstrapping replications. All amplicon sequences were submitted to GenBank (accession numbers MW575344–MW575394).The purified amplicons were analyzed using Sanger’s sequencing technology by a commercial company . Additionally, a manual differential count from the Diff-Quick stained blood smear was also performed in all of the samples. Common blood panels were analyzed based on the leukocyte count (WBC) and the proportions of neutrophils (NEU), lymphocytes (LYM), eosinophils (EOS), monocytes (MON), and by the numbers of basophils (BAS), the erythrocytic count (RBC), hemoglobin concentration (HGB), packed cell volume (PCV), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and platelet count (PLT).The remaining blood samples were sent to the Hematological Unit of KUVTH and to a private hematological company. All of the blood samples were analyzed using the laser flow cytometry technique by an automated hematology analyzer . There was no evidence of Bartonella in domestic dogs. Two zoonotic species were identified in this study (B. henselae and B. clarridgeiae). There was a high proportion detected in temple clusters located in the inner zone of Bangkok, however, there were no significant differences for other associated factors. The 100% bootstrapping replication of the concatenated phylogenetic tree based on the gltA and ribC genes confirmed the two species of Bartonella spp. in this study. Nevertheless, other genes  revealed different species from the same individual sample and mixed infection was expected. Bartonella-positive, free-ranging cats had raised MCV values compared to negative cats, additionally, there were low values of HGB and MCHC in over 36% of the Bartonella-positive cats. Based on these results, we suggest that zoonotic Bartonella spp., especially B. henselae and B. clarridgeiae, are important in Thailand and a prevention program, such as routine health checks of animals and humans, should be implemented. Importantly, abnormal red blood cell parameters such as a high corpuscular volume or low hemoglobin-related parameters in healthy cats should also be considered as indicators of feline subclinical bartonellosis in Thailand. Moreover, the knowledge of Bartonella-related diseases protection should be intervened in the risk person.Overall, the prevalence of


Trypanosoma brucei is a parasitic protist that causes African sleeping sickness. The establishment of T. brucei cell lines has provided a significant advantage for the majority of T. brucei research. However, these cell lines were isolated and maintained in culture for decades, occasionally accumulating changes in gene expression. Since trypanosome strains have been maintained in culture for decades, it is possible that difference may have accumulated in fast-evolving non-coding RNAs between trypanosomes from the wild and those maintained extensively in cultures. To address this, we compared the lncRNA expression profile of trypanosomes maintained as cultured cell lines (CL) to those extracted from human patients, wildtype (WT). We identified lncRNAs from CL and WT from available transcriptomic data and demonstrate that CL and WT have unique sets of lncRNAs expressed. We further demonstrate that the unique and shared lncRNAs are differentially expressed between CL and WT parasites, and that these lncRNAs are more evenly up-regulated and down-regulated than protein-coding genes. We validated the expression of these lncRNAs using qPCR. Taken together, this study demonstrates that lncRNAs are differentially expressed between cell lines and wildtype T. brucei and provides evidence for potential evolution of lncRNAs, specifically in T. brucei maintained in culture. Trypanosoma brucei is the causative agent for African trypanosomiasis, a neglected tropical disease that leads to sleep disturbance and is almost always fatal if untreated [T. brucei invades the mammalian host and enters the bloodstream. Mammalian host specificity depends on the subspecies of the parasite: T. brucei rhodesiense (Tbr) and T. brucei gambiense (Tbg) infect human hosts, while T. brucei brucei (Tbb) primarily infects cattle. Tbb is unable to survive in humans due to its susceptibility to trypanolytic factors (TLF) present in human sera [ntreated . The disman sera . Tbr oveman sera ,4 while man sera ,6,7,8,9.man sera .T. brucei has a distinctly unique evolutionary lineage and is an interesting research organism [T. brucei studies are sourced from isolates collected decades ago [T. brucei cell lines vary in their expression of RNA editing pathways [In addition to causing debilitating disease, organism . The estorganism ,13. Howeades ago . In factades ago ,15. Furtades ago ,17, and pathways . Additiopathways . While tT. brucei, one lncRNA has been shown to have a functional role in parasite differentiation during the bloodstream stage [T. brucei. In our study, we pooled T. brucei RNA-seq datasets from both cell line (CL) and wildtype (WT) sources and used two lncRNA identification tools to identify a high-confidence set of lncRNAs. We then show that the CL and WT strains each has a unique set of lncRNAs and also share common lncRNAs that have different expression patterns compared to protein-coding genes. We further verify the expression of newly identified lncRNA expression using quantitative RT-PCR.Long non-coding RNA (lncRNA) is a class of non-coding RNA that is over 200 nucleotides in length with little to no protein-coding potential ,25,26,27am stage . LncRNAsam stage . While lam stage . Althougam stage ,32. ThusT. brucei cell lines were isolated decades ago [T. brucei research, including transcriptomic analysis. While RNA-seq has been performed several times in CL T. brucei [T. brucei has rarely been investigated using transcriptomic analysis. Despite recent interest in the roles of lncRNAs, a thorough comparison of the differences in these RNAs has not been performed. To compare lncRNAs from CL and WT T. brucei, we used thirty-three RNA-seq datasets from three published transcriptomic studies for lncRNA identification [T. brucei cell line in culture or in mice. On the other hand, an RNA-seq dataset was considered WT if the source parasites were recently collected from human blood or cerebrospinal fluid (CSF). For the CL studies, any datasets from the procyclic form of T. brucei was discarded, as no WT dataset is available for this life stage. For the WT datasets, any dataset that had lower than 1% alignment rate to the T. brucei TREU927 genome was discarded. This criterion was chosen as the WT samples collected directly from patients were contaminated with host tissue, and the published work showed that 1–10% of the reads were trypanosome-specific [A vast majority of the available ades ago , and hav. brucei ,34,35,36fication ,33,34. Aspecific . OverallT. brucei TREU927 genome and de novo assembled into transcripts. These assemblies were merged, and rRNAs and transcripts matching spliced-leader RNA (SL-RNA) were removed from the assembly. The resulting assembly then underwent the lncRNA identification process using two different tools  or expression site-associated gene (ESAG) as most of these transcripts are annotated as pseudogenes, degenerates, or frameshifted. With these criteria, we removed 43 transcripts previously classified as mRNAs from the lncRNA list. In the past, pseudogenes have been regarded as “junk genes” that were considered defunct pieces of protein-coding genes. Nevertheless, some pseudogenes have now been shown to produce lncRNAs that can have further regulatory functions [T. brucei.LncRNAs identified through this process included novel transcripts and transcripts that have already been annotated in the unctions ,48. WithT. brucei being isolated decades ago, most CL T. brucei were collected from ungulates [In addition to CL ngulates . We specp < 0.01)  were up-regulated and 2855 (20.3%) were down-regulated in the WT when compared to the CL (p < 0.01)  of the WT to the CL. Among the 978 identified lncRNAs, 139 (14.2%) transcripts were up-regulated in WT while 133 (13.6%) transcripts were down-regulated in WT when compared to the CL ( < 0.01) A. We the < 0.01) B. The brTo validate the lncRNA identification pipeline, we performed qPCR analysis on several identified lncRNAs. However, after the data were assembled using Stringtie, we were quite surprised. The data predicted that a significant number of lncRNA were potentially spliced. We questioned the validity of the output, and consequently performed a de novo assembly of Tb protein coding genes where only two spliced genes are already known. Using the same analysis, our data were consistent and supported the currently established two spliced protein coding genes. We selected five lncRNAs with unique sequences for RT-qPCR analysis. Of the five lncRNAs, three were mono-exonic lncRNAs and we identified two as potentially multi-exonic lncRNAs. Tb927.11.10190 (telomerase reverse transcriptase) was used as the reference gene . ExpressT. brucei. We identified 978 lncRNAs comprised of 536 novel transcripts and 445 transcripts that have been annotated as mRNA, pseudogenic transcript, or ncRNA in the genome. We then provided data that both CL and WT each have a unique set of lncRNAs that are expressed exclusively in each strain, and that lncRNAs are differentially expressed between CL and WT. Further, we compared lncRNA differential expression to protein-coding genes to show that lncRNAs are proportionally more evenly up-regulated and down-regulated while protein-coding genes are largely down-regulated in WT when compared to CL. Finally, we verified the expression of these lncRNAs using RT-qPCR. Our findings support that cell lines have a distinct class of differentially genes when compared to non-cultured samples [In this study, we pooled 33 RNA-seq datasets and used two lncRNA identification tools to identify potential lncRNAs in CL and WT  samples ,17. The T. brucei TREU927 reference genome actually annotates many multi-exonic transcripts. Out of 9881 transcripts labeled as protein-coding genes, four transcripts have multiple exons. Furthermore, of the 1383 transcripts labeled as pseudogenes, 26 transcripts are annotated with multiple exons. The presence of a predicted higher number of multi-exonic transcripts in the pseudogenic transcripts, which can potentially lead to pseudogene-derived lncRNAs [T. brucei. Furthermore, the RT-qPCR for multi-exonic lncRNAs confirmed their expression despite the fact that the primers were designed to target exon junction that are thousands of base pairs apart. Therefore, the data suggest the existence of multi-exonic lncRNAs in T. brucei. Further investigation is in underway.A strange finding in the identified lncRNAs is that our data suggest that many of these transcripts are spliced. Trypanosomatids are known to have very few verified cis-spliced transcripts ,51,52. T lncRNAs ,48, suggPrevious transcriptomic analysis in cultured and bloodstream/CSF suggested that cultured trypanosomes potentially have faster division times than human-sourced trypanosomes due to higher levels of RNA-binding proteins, cytoskeletal proteins, mitochondrial proteins, translation initiation factors, and proteasome subunits . Our anaT. brucei. Furthermore, lncRNA conservation has multiple levels: sequence, structure, function, and syntenic context of neighboring genes [T. brucei. The lncRNA, grumpy, controls differentiation from proliferating slender form to non-proliferating stumpy form in bloodstream T. brucei [The existence of unique lncRNAs does not necessarily require that they have unique functions in CL or WT. LncRNAs are more conserved in terms of tissue-specificity , and botng genes . Therefong genes . Likewis. brucei , signify. brucei . In termT. brucei TREU927 genome sequence and annotation were downloaded from TriTrypDB [The riTrypDB . The mosT. brucei RNA-seq datasets (PRJEB19907) [T. brucei grown in mice and collected from blood and fat tissue (PRJEB11801) [T. brucei collected from human patients  [T. brucei brucei EATRO 1125, clone AnTat1.1 strain while the five CL parasites were T. brucei brucei EATRO 1125 Antat1.1E 90–13 strain. The WT parasites were T. brucei rhodesiense collected from patients from Kaberamaido district of North-Eastern Uganda. FastQC was used on all datasets to check for the dataset quality [https://www.bioinformatics.babraham.ac.uk/projects/fastqc/, accessed on 8 January 2020), and Trimmomatics was used to trim any adapters present in the datasets [T. brucei TREU927 genome using STAR [https://github.com/alexdobin/STAR, accessed on 2 October 2020) and de novo assembled into transcripts using Stringtie [Thirty-three RNA-seq datasets from three studies were used for this study: (1) four runs from cultured bloodstream form EB19907) , (2) fivEB11801) , and (3) quality  . The following databases were used for riboPicker: SILVA rRNA database for large subunit (version 132) and small subunit (version 138) rRNA sequences [https://www.arb-silva.de/, accessed on 5 June 2020) and Rfam database for 5S and 5.8S subunit rRNA sequences (release 14.2) [https://rfam.org/, accessed on 5 June 2020). Since the SILVA small subunit data file was too large to index with riboPicker, it was broken into six smaller sized files for indexing.The merged assembly was processed through riboPicker to identify and remove any rRNAs  (http://equences  (https:/se 14.2)  . To decrease the false positive rate due to the optimized sensitivity, the specificity thresholds for mRNA and lncRNA were each increased to 0.99. CPC2 was run on the default setting [http://cpc2.gao-lab.org/, accessed on 10 September 2020). The transcript ID was used to find the lncRNA transcripts that were shared by the results from both lncRNA identification tools.After the rRNA filtering, the assembly was processed with two different lncRNA identification tools: FEELnc and CPC2. The FEELnc coding potential calculation module was performed on shuffle mode with relaxed ORF definition and multi k-mer frequencies to increase sensitivity of the module   with DEv. 4.0.5 , readr [v. 4.0.5 , and txiv. 4.0.5  librariev. 4.0.5  package.T. brucei brucei Lister 427 were cultured in T75 vented cap culture flasks  in complete HMI-9 media  containing 10% FBS , 10% Serum Plus medium supplement , and 1X penicillin-streptomycin . The trypanosomes were maintained in a humidified incubator at 37 °C and 5% CO2, and they were sub-cultured every 48 h. After checking for parasite density for log growth phase, the trypanosomes were collected, pelleted, and flash frozen.Bloodstream form T. brucei using the Direct-zol RNA Miniprep Kit  with a minor modification. To decrease the genomic DNA contamination, on-column DNase I treatment was performed twice. RNA concentration and quality were assessed on a Nanodrop 8000 Spectrophotometer . The cDNA generation was performed with Superscript II Reverse Transcriptase kit  with oligo dT primer following the manufacturer’s protocol.RNA was extracted from frozen pellets of T 1-Step Kit . using two Q-qPCR machines . The no-RT reactions were set up to determine DNA contamination in the RNA samples. For reference gene, Tb927.11.10190 was chosen as it was determined to be an ideal reference gene for T. brucei qPCR studies [The qRT-PCR reactions were performed with Power SYBR Green RNA-to-C studies . The exp


Helicobacter pylori infection is a chronic infection that is closely related to the occurrence of gastric tumorigenesis. Non-coding RNA has been demonstrated to play a very important role in the organism, exerting a prominent role in the carcinogenesis, proliferation, apoptosis, invasion, metastasis, and chemoresistance of tumor progression. H. pylori infection affects the expression of non-coding RNA at multiple levels such as genetic polymorphisms and signaling pathways, thereby promoting or inhibiting tumor progression or chemoresistance. This paper mainly introduces the relationship between H. pylori-infected gastric cancer and non-coding RNA, providing a new perspective for gastric cancer treatment.Gastric cancer is a common malignant tumor of the digestive system. Its occurrence and development are the result of a combination of genetic, environmental, and microbial factors.  Helicobacter pylori infection  lymphoma  are increasing annually . The assnfection . H. pyloong time . H. pylolymphoma . Variousevent GC . The Kyo treated .H. pylori-induced GC. LncRNA is an ncRNA that is greater than 200 nucleotides in length. It has many known functions, including transcriptional interference, regulation of alternative splicing, generation of endogenous siRNA, regulation of protein activity, and alteration of protein positioning (Non-coding RNA (ncRNA) refers to RNA that does not encode protein, which has been divided into long ncRNAs (lncRNAs) and short ncRNAs including microRNAs (miRNAs), PiWi-interacting RNAs (piRNAs), small nucleolar RNAs (snoRNAs), small interfering RNAs (siRNAs), tRNA-derived small RNAs (tsRNAs), circular RNAs (circRNAs), and heterochromatin-derived 24nt small RNA in plants according to their length. These RNAs are derived from genomic transcription, but they are not translated into proteins; and they play their respective biological roles at the RNA level. Among these RNAs, lncRNA, miRNA, and some special small ncRNAs (sncRNAs) are mainly involved in the progress of itioning . In addiitioning . MiRNA iitioning .H. pylori-infected GC. H. pylori infection modulates expression of ncRNA and changes the expression of related target genes. Their impact on tumor progression and drug resistance treatment has been categorized and summarized, and a new perspective for clinical treatment is provided.This review mainly summarizes the mechanism of ncRNA in Helicobacter pylori presents large regional differences worldwide, which is related to factors such as geography and basic health conditions. H. pylori survival is facilitated in an acidic environment, and it colonizes in the gastric mucosa by virtue of its spiral shape, exercise ability, adhesion factors, and urease and ammonia production, subsequently producing a complex inflammatory response, damaging the gastric mucosa, and subsequently producing digestive diseases via the expression of various pathogenic markers such as cytotoxin-related gene A (CagA), BabA adhesin, and empty vesicular toxin (VacA)  . Flagellal cells . The viral cells . All thein vitro and in vivo  in  stomach , 2005. HH values . A novelon assay . Howeveron assay .H. pylori, interacts with the fliK mRNA and downregulates bacterial motility and adhesion ability, significantly impairing bacterial adhesion to host gastric cell lines   of ll lines . The sRNs (SSRs) . Phase vHelicobacter pylori not only plays a role via its own nosotoxin but also participates in the homeostasis regulation of intestinal flora, and H. pylori eradication treatment has a significant effect on the change in intestinal flora , a competing endogenous RNA (ceRNA) network was constructed, and four lncRNAs  showed higher expression in R_026827 ] in H. pd by PCR . Among ted genes . Seven ued genes . LINC001nfection .H. pylori-infected GES-1 cells, for which a concrete example is the downregulation of MUC2 by lncRNA AF147447 to suppress cell proliferation and invasion in the H. pylori-infection background  was found to be induced by H. pylori infection and to promote the migration and proliferation of GC cells  and increased expression (p = 0.03) of miR-133a (H. pylori-positive gastritis (p < 0.004)  . Other eH. pylori-produced virulence factors. MiR-34a was found to be significantly reduced in the H. pylori + GC group by rTip-α (a toxin secreted by H. pylori), while its overexpression decreased the level of TLP4, TNF-α, and IL-6. Viability was enhanced by rTip-α but decreased by miR-34a, which induces cell proliferation  mutation-positive] infection suppresses miR-134 expression when compared with H. pyloriCagA+/P– tissues  expression to promote EMT and tumor growth  expression, which is an important coreceptor in the WNT signaling pathway, promoting cell proliferation, while miR-27b targets the 3′UTR of FZD7 to suppress FZD7 expression in GC  and thus promoting proliferation and invasion and inhibiting apoptosis  upregulation, and this upregulation can enhance reactive oxygen species resistance to prevent cell death (H. pylori infection in patients with GC (p < 0.05) (H. pylori-induced gastric carcinogenesis by inhibiting the expression of lncRNA SOX2OT (SOX2 overlapping transcript) and SOX2, a master regulator of the pluripotency of cancer stem cells   . MiR-375em cells . MiR-375em cells . A specid BECN1)  (summarid BECN1) .H. pylori eradication and aspirin use have been suggested to prevent development of the intestinal metaplasia in GC (H. pylori infection to spasmolytic polypeptide-expressing metaplasia (SPEM)  inhibits this upregulation. COE upregulates PDCD4 expression by decreasing the methylation of its promoter and inhibits H. pylori-induced inflammation and EMT  downregulation in GC cells  properties like self-renewal and tumor-initiating capacity, while miR-320a and miR-4496 target β-catenin and ABCG2  at the transcriptional and posttranscriptional levels to attenuate CagA induction. Furthermore, the combination treatment of miR-320a/-4496 with 5-fluorouracil in an orthotopic mouse model has been shown to attenuate gastric tumorigenesis and metastatic potential  . H. pylo and EMT . H. pylosistance . H. pylosistance . GKN1 suGC cells . H. pyloof FBXW7 . Accompaotential . Rebamipic drugs . It was ic drugs . And miRic drugs . In addiic drugs , miR-181ic drugs , miR-22 ic drugs , and so ic drugs   in the detection of GC  has been regarded as a modular scaffold of WDR45 and KAT2A histone modifiers. It can regulate the localization and histone modification of SOD2. GClnc1 shows a strong correlation with the carcinogenesis, invasion, growth, and prognosis of GC. Another is clinical analysis showed that 78% of GC patients with higher GClnc1 expression are H. pylori-infected. The mechanism underlying the interaction between H. pylori and GClnc1 merits further exploration  of ncRNA in r for GC . For exar for GC . And Cirr for GC . At presCW and YH wrote the manuscript. SW, BZ, and YB revised the manuscript. YH and QL was responsible for searching the references. CY projected and edited the manuscript. XX and SY reviewed the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Pseudomonas aeruginosa is often co-isolated with other microbes, likely engaging in inter-species interactions. In the case of chronic co-infections, this cohabitation can last for a long time and evolve over time, potentially contributing to the clinical outcome. Interactions involving the emerging pathogens Achromobacter spp. have only rarely been studied, reporting inhibition of P. aeruginosa biofilm formation. To evaluate the possible evolution of such interplay, we assessed the ability of Achromobacter spp. isolates to affect the biofilm formation of co-isolated P. aeruginosa strains during long-term chronic co-infections. We observed both competition and cohabitation. An Achromobacter sp. isolate secreted exoproducts interfering with the adhesion ability of a co-isolated P. aeruginosa strain and affected its biofilm formation. Conversely, a clonal Achromobacter sp. strain later isolated from the same patient, as well as two longitudinal strains from another patient, did not show similar competitive behavior against its P. aeruginosa co-isolates. Genetic variants supporting the higher virulence of the competitive Achromobacter sp. isolate were found in its genome. Our results confirm that both inter-species competition and cohabitation are represented during chronic co-infections in CF airways, and evolution of these interplays can happen even at the late stages of chronic infection.In the lungs of patients with cystic fibrosis (CF), the main pathogen  Pseudomonas aeruginosa, the most common pathogen isolated from CF airways, is particularly difficult to eradicate and is associated with an accelerated decline in lung function, with a poor prognosis [Staphylococcus aureus and Haemophilus influenzae are the main pediatric pathogens, while Burkholderia cepacia complex, Achromobacter spp., Stenotrophomonas maltophilia and nontuberculous mycobacteria are mainly found in adults [Achromobacter spp. gained attention as important emerging pathogens that can cause severe chronic infections in CF patients, associated with lung inflammation and decline in respiratory function [Achromobacter genus comprises 22 species [Achromobacter xylosoxidans is the most often isolated species among CF patients, followed by Achromobacter ruhlandii, Achromobacter insuavis, Achromobacter insolitus, Achromobacter dolens, Achromobacter agrifaciens and Achromobacter spanius [Development of chronic lung infections and progressive inflammation is the major cause of morbidity and ultimate mortality for patients with cystic fibrosis (CF) . Colonizrognosis . Other rn adults . In partfunction ,6,7,8,9 function ,11. The  species ; Achromo spanius ,15,16,17Due to the polymicrobial nature of CF lung infection, it is likely that microbes could engage in inter-species interactions, acting competitively or synergistically with each other to gain an adaptive advantage, thereby influencing the community composition, resistance to antibiotics and the course of airway disease ,19. In tP. aeruginosa is often co-isolated with other microbial species sharing the same environment. While its interactions—including both cooperation and competition—with classical pathogens Burkholderia spp. and S. aureus have been extensively studied [Achromobacter spp. and S. maltophilia is still limited. Despite the reported co-isolation of P. aeruginosa and Achromobacter spp. from sputum samples and the increasing number of patients becoming chronically infected with the latter [P. aeruginosa biofilm formation can be affected by A. xylosoxidans [Achromobacter spp. isolates to affect the biofilm of P. aeruginosa strains sharing the same lung environment during long-term chronic co-infections and searched for genetic features of virulence possibly associated with the competition ability. studied ,30,31,32e latter ,9,33,34,soxidans . To evalP. aeruginosa and Achromobacter spp. clinical isolates were longitudinally collected from two CF patients chronically co-infected for over 9 years: patient A since 1996, and patient B since 1999. For each patient, the two species were isolated from the same sputum sample twice: in 2005 and 2008 from patient A, and in 2008 and 2014 from patient B. The general information on each isolate is presented in P. aeruginosa isolates from patients A and B showed 82% and 83% similarity, respectively, while Achromobacter spp. isolates from both patients showed 87% similarity. While all Achromobacter spp. isolates had been initially identified as A. xylosoxidans [A. insuavis [A. insuavis strain collected from this patient, named isolate A1, was previously classified as a hypermutator [P. aeruginosa isolates from patient A belong to the DK08 clone type, sampled from multiple patients at the Copenhagen CF Center [soxidans , a receninsuavis . The firrmutator . P. aeruF Center .Achromobacter spp. isolates from the two patients [P. aeruginosa isolates underwent the phenotypic evolution known to occur during CF chronic infection: the growth rate significantly diminished over time in both patients, while the adhesion ability increased significantly in patient A , and the fluorescence emission was checked  via long, peritrichous flagella but lack twitching motility [Achromobacter spp.’s ability to adhere on hydrogel contact lenses [Achromobacter spp. on polystyrene and glass, and the formation of loosely attached aggregates characterized by the scattering and dispersal of planktonic cells. Interestingly, when cultured with P. aeruginosa, the two microbes could form mixed biofilms, suggesting that Achromobacter spp.’s adhesion might be enhanced on biotic surfaces.As regards the biofilm mode of growth, motility ,45, whicmotility . Indeed,motility . Howevert lenses ,49 and tt lenses . Our curt lenses  results P. aeruginosa strain, interfering with its adhesion ability and affecting its biofilm formation capability. Conversely, the clonal strain A2 later isolated from the same patient, as well as two Achromobacter sp. strains longitudinally collected from another patient, did not show similar competitive behavior against their P. aeruginosa co-isolates. Thus far, only one recent study evaluated the occurrence of inter-species interactions between Achromobacter sp. and P. aeruginosa CF isolates, reporting that P. aeruginosa biofilm formation can be affected by A. xylosoxidans [A. insuavis, suggesting that this behavior might be common to various species of the genus. Interestingly, this isolate was previously classified as a hypermutator [P. aeruginosa biofilm were morphologically different clones collected from the same sputum sample of a chronically infected patient [Achromobacter spp.’s competitive behavior should be further verified.Concerning inter-species interactions, we observed that only the A1 isolate has inhibitory effects against the co-isolated soxidans . The isormutator , and the patient , a situa patient . AlthougVgrG paralogues have also been reported to regulate bacterial motility, biofilm formation and protease production in Aeromonas sp. [HlyD gene, while a deleterious variant was present in the genome of its clonal late isolate. HlyD is essential for the secretion of the RTX hemolytic toxin HlyA from Escherichia coli [P. aeruginosa. Additionally, the genes related to the quorum sensing system were checked in all the P. aeruginosa isolates for the presence of variants, but no mutations were detected that could indicate down-regulation of this system.Investigating the genomic features that could be implicated in the observed competition, in the genome of the A1 isolate, we identified genetic variants supporting its higher virulence. Only in this isolate, we detected a type VI secretion system tip protein, the VgrG gene, whose product is known to be involved in inter-species competition between Gram-negative bacteria . Additioonas sp. . Moreovehia coli  and seemhia coli . We prevhia coli , furtherAchromobacter spp. are usually rich in mobile genetic elements carrying antibiotic resistance [P. aeruginosa might influence the course of infection and the outcome of antibiotic therapies.Interestingly, 3 years after the A1 and P1 strains’ isolation, their competitive interplay evolved towards a more indolent cohabitation or even cooperation—similar to the situation observed in patient B—which might represent a survival advantage. Although major adaptations of bacteria causing CF chronic infections are likely to happen during the early stage of chronic infection, while in the late stage, the situation is supposedly more stable, we observed variations in the interplay between two microorganisms in the late stage of a chronic co-infection, suggesting that adaptive mechanisms are still ongoing. In the later stage, isolates A2 and P2 could also grow together in mixed biofilm communities, supporting the possibility that close microbial interactions might occur between them. Indeed, bacterial proximity within biofilm communities can favor social exchanges of signal molecules and genetic elements, influencing many aspects of the community itself such as the microbial composition, nutrient availability, and antibiotic resistance . Althougsistance ,55, whosAchromobacter spp.’s competitive abilities. Future studies on a larger scale, involving more strains from more patients, are needed to better understand the interplay between competition and adaptation in the lungs of CF patients. Further mechanisms involved in inter-species interactions should also be explored, such as regulation of quorum sensing and secretion of specific virulence factors or metabolic by-products. In addition, evaluating interactions involving other microbial species would increase insights into the extent and complexity of such interplays and their contribution to the clinical outcome. This highlights the importance and necessity of further studies with a larger number of isolates, encouraging further research on this subject.In conclusion, despite our observations being limited to restricted mechanisms on a small number of selected strains, our results show that both inter-species competition and cohabitation are represented during chronic co-infections in CF airways, and evolution of these interplays can happen at the late stages of chronic infection. Furthermore, we provided insights on virulence mechanisms that could be involved in Achromobacter spp. and P. aeruginosa were collected from two CF patients followed in the CF clinic at Rigshospitalet in Copenhagen, Denmark. The use of the stored bacterial isolates was approved by the local ethics committee at the Capital Region of Denmark (Region Hovedstaden) with the registration number H-4-2015-FSP. P. aeruginosa and Achromobacter spp. were isolated from the same sputum sample twice from each patient: in 2005 and 2008 from patient A, and in 2008 and 2014 from patient B.Four clinical isolates of 600) was measured using a spectrophotometer, cultures were diluted to 0.05 OD/mL in LB medium and 150 µL/well was incubated in a 96-well plate for 20–24 h, with shaking at 37 °C. Using an automated plate reader, OD600 was measured every 20 min. Growth rate was calculated using GraphPad Prism 7.0.Bacterial strains were plated on LB agar and incubated at 37 °C for 24–48 h. One colony was picked from the plate and inoculated in 10 mL LB medium, with shaking at 37 °C overnight. Optical density at 600 nm  was normalized on growth (absorbance at 600 nm). In competition assays, P. aeruginosa adhesion was measured in the presence/absence of 10% Achromobacter spp. culture supernatant.Bacterial strains were plated on LB agar and incubated at 37 °C for 24–48 h. One colony was picked from the plate and inoculated in 10 mL LB medium, with shaking at 37 °C overnight. ODP. aeruginosa strains were tagged with a mini-Tn7 construct carrying gentamycin resistance and GFPmut3b genes under the control of the growth-dependent E. coli ribosomal promoter rrnB P1 [P. aeruginosa by conjugative transfer as described by Choi and Schweizer [P. aeruginosa strains were mixed with E. coli donor and helper strains (pRK2013 and pNTS2), and a drop of bacterial suspension was placed in the center of an LB agar plate and incubated at 37 °C overnight. Transconjugants were selected by plating bacteria on LB agar containing gentamycin and trimethoprim. Mini-Tn7 insertion was checked by colony PCR using PTn7R and PglmSF primers [P. aeruginosa GFP-tagged strains were cultured in a 96-well plate for 12 h, with shaking at 37 °C, while fluorescence  and OD600 were measured every 20 min using an automated plate reader. rrnB P1 . The conchweizer . Briefly primers . To asse2, CaCl2 and trace metals). Biofilm formation was observed by confocal laser scanning microscopy for GFP-tagged cells and Syto62 staining of total cells. For statistical analysis, at least 7 pictures/channel were taken, homogeneously distributed along the channel. Pictures were visualized and elaborated using Imaris 7.4 software. Biomass was calculated using Comstat2 software [A flow chamber system was assembled and sterilized following the protocol from Tolker-Nielsen and Sternberg . Brieflysoftware .Achromobacter spp. sequences) and PRJEB40978 (P. aeruginosa sequences). Sequence data can be found with the experiment accession numbers ERX3614542 (strain A1), ERX3614543 (strain A2), ERX3614548 , ERX3614549 , ERS5248144 (strain P1), ERS5248145 (strain P2), ERS5248146 (strain P3) and ERS5248147 (strain P4). Genotypic relatedness among longitudinal isolates was verified by checking core genome similarities obtained using the Harvest-OSX64-v1.1.2 suite [A. xylosoxidans NH44784-1996 and P.aeruginosa PAO1 (RefSeq accessions: GCF_000967095.2 and GCF_000006765.1) were used as reference genomes. Bowtie 2 v2.3.4.1 [Whole genome sequencing and assembly were performed as previously described . Sequenc.2 suite . Variantv2.3.4.1  was used


Klebsiella pneumoniae (K. pneumoniae), was very high in Iran. This study is aimed at assessing the genetic diversity, antimicrobial resistance pattern, and biofilm formation in K. pneumoniae isolates obtained from patients with COVID-19 and ventilator-associated pneumonia (VAP) hospitalized in an intensive care unit (ICU) in Iran.  Patients with acute respiratory distress syndrome caused by coronavirus disease 2019 (COVID-19) are at risk for superadded infections, especially infections caused by multidrug resistant (MDR) pathogens. Before the COVID-19 pandemic, the prevalence of MDR infections, including infections caused by MDR K. pneumoniae isolates were obtained from seventy patients with COVID-19 hospitalized in the ICU of Shahid Beheshti hospital, Kashan, Iran, from May to September, 2020. K. pneumoniae was detected through the ureD gene. Antimicrobial susceptibility testing was done using the Kirby-Bauer disc diffusion method, and biofilm was detected using the microtiter plate assay method. Genetic diversity was also analyzed through polymerase chain reaction based on enterobacterial repetitive intergenic consensus (ERIC-PCR). The BioNumerics software  was used for analyzing the data and drawing dendrogram and minimum spanning tree. Findings. K. pneumoniae isolates had varying levels of resistance to antibiotics meropenem (80.4%), cefepime-aztreonam-piperacillin/tazobactam (70%), tobramycin (61.4%), ciprofloxacin (57.7%), gentamicin (55.7%), and imipenem (50%). Around 77.14% of isolates were MDR, and 42.8% of them formed biofilm. Genetic diversity analysis revealed 28 genotypes (E1–E28) and 74.28% of isolates were grouped into ten clusters . Clusters were further categorized into three major clusters, i.e., clusters E, H, and J. Antimicrobial resistance to meropenem, tobramycin, gentamicin, and ciprofloxacin in cluster J was significantly higher than cluster H, denoting significant relationship between ERIC clusters and antimicrobial resistance. However, there was no significant difference among major clusters E, H, and J respecting biofilm formation.  In this cross-sectional study, seventy K. pneumoniae isolates obtained from patients with COVID-19 have high antimicrobial resistance, and 44.2% of them have genetic similarity and can be clustered in three major clusters. There is a significant difference among clusters respecting antimicrobial resistance. Coronavirus disease 2019 (COVID-19) is a new emerging disease in human population. The World Health Organization introduced COVID-19 as a pandemic in March 11, 2020. The disease is caused by a virus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [The SARS-CoV-2 virus often affects ciliated cells in the alveolar epithelium and reduces their normal activities such as airway clearance. Subsequent gradual accumulation of fluid and residuals in the lung results in acute respiratory distress syndrome. Some afflicted patients need hospitalization in intensive care unit (ICU) to receive intensive care and mechanical ventilation .coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii [Pulmonary infiltration and mechanical ventilation predispose patients with COVID-19 in ICU to secondary bacterial infections. Previous studies reported that 40%–86% of patients with COVID-19 receiving mechanical ventilation developed ventilator-associated pneumonia (VAP). The prevalence of VAP among patients in ICU varies according to their clinical conditions, ICU admission policies, and types of treatment [aumannii . Howeveraumannii .Klebsiella pneumoniae (K. pneumoniae) is one of the most common pathogens contributing to VAP in ICU in the United States and Middle East countries like Iran [K. pneumoniae (MDRKp) have been a major public health concern. Several studies reported the high prevalence of nosocomial infections caused by K. pneumoniae strains which were resistant to third generation cephalosporins, aminoglycosides, and quinolones. Some MDRKp strains have changed to extensive drug resistance (XDR) strains. These strains are usually a major threat to patients with serious health conditions such as COVID-19 due to the ineffectiveness of treatments against them and their high mortality rate [ike Iran . It is aike Iran . In receity rate –10.K. pneumoniae can form biofilm which is an extracellular matrix consisted of proteins, exopolysaccharides, deoxyribonucleic acid (DNA), and lipopeptides and protects bacteria against antibiotics [ibiotics , 12. Bioibiotics . Thick bibiotics .K. pneumoniae has some virulence factors, such as capsular polysaccharides, lipopolysaccharides, types 1 and 3 fimbriae, outer membrane proteins, and factors determining iron acquisition and nitrogen source, and uses them for survival and evasion from the immune system of the body [Moreover, the body , 16. Adethe body .Bacteria have different genetic characteristics which determine their virulence and behaviors. Determining the genetic diversity of bacteria helps determine the most prevalent bacterial strains in a given setting, determine the sources of infections, and determine the best preventive measures and infection control policies for that setting , 19. TypEnterobacteriaceae family [Enterobacterial repetitive intergenic consensus (ERIC) is one of the repetitive elements which vary in different bacterial genomes in terms of pattern and number. Polymerase chain reaction (PCR) based on ERIC (ERIC-PCR) has successfully been used for the genotyping, strain diversity assessment, population analysis, and epidemiological assessments of different microbial pathogens and determining their taxonomy and phylogenetic relatedness . Comparee family .K. pneumoniae among patients with COVID-19 in the ICU of this hospital. The present study was conducted to narrow this gap. The study is aimed at assessing the genetic diversity, antimicrobial resistance pattern, and biofilm formation in K. pneumoniae isolates obtained from patients with COVID-19 and VAP in ICU.Kashan is a city in the center of Iran. Before the onset of the COVID-19 pandemic, high prevalence of MDRKp infections had been reported in the leading hospital of the city . HoweverThis descriptive study was conducted during the second wave of COVID-19 in Iran, i.e., from May to September, 2020.K. pneumoniae isolates were obtained from seventy patients with COVID-19 hospitalized in the ICU of Shahid Beheshti hospital, Kashan, Iran. This hospital is the main COVID-19 care center in Kashan city in the center of Iran. All K. pneumoniae isolates were obtained through sampling from patients' tracheal secretions and then were immediately cultured in an appropriate culture medium in the laboratory of the Microbiology Department of Kashan University of Medical Sciences, Kashan, Iran. Data on participants' demographic characteristics were collected from their medical records.Seventy The Ethics Committee of Kashan University of Medical Sciences, Kashan, Iran, approved this study (code: IR.KAUMS.MEDNT.REC.1399.034). Sampling and data collection were performed under the supervision of this committee. All participants provided written informed consent.K. pneumoniae to the species level was performed based on biochemical reactions, including reaction on SH2/indole/motility (SIM) medium, triple sugar iron (TSI) agar, urease production on urea agar, growth on Simmons' citrate agar medium, methyl red/Vogues-Proskauer (MR/VP), and ornithine decarboxylase (OD) test [K. pneumoniae isolates through detecting the ureD gene. This gene is responsible for urea hydrolysis. The forward primer 5′-CCCGTTTTACCCGGAAGAAG-3′ and the reverse primer 5′-GGAAAGAAGATGGCATCCTGC-3′ were employed for the amplification of 243 base pairs (bp) of the ureD gene [μL as follows: initial denaturation at 95°C for three minutes, denaturation at 95°C for thirty thirty-second cycles, annealing at 95°C for 45 seconds, elongation at 72°C for sixty seconds, and final extension at 72°C for sixty seconds. The final products of PCR were electrophoresed on agarose gel.Phenotypic detection of OD) test . Before K. pneumoniae isolates using the Kirby-Bauer disc diffusion method based on the guidelines of the Clinical and Laboratory Standards Institute (CLSI) [K. pneumoniae isolates was identified with respect to the following ten antibiotics: piperacillin/tazobactam , cefepime , imipenem , meropenem , gentamicin , tobramycin , aztreonam , ciprofloxacin , polymyxin B (300 μg), and colistin . K. pneumoniae isolates which were resistant to three or more antimicrobial categories were considered as MDR [coli ATCC 25922 was used as control.Antimicrobial susceptibility testing was performed for all detected e (CLSI) . Accordid as MDR . Escheri8 cfu/ml). This suspension was tenfold diluted to the concentration of 5 × 105 (cfu/ml), and then, 200 μL bacterial suspension was inoculated into 96-well flat-bottomed sterile microplate. Negative control wells containing 200 μL TSB were also included in each test. Incubation at a temperature of 37°C was performed for 24 hours. After that, wells were gently washed three times using 200 μL of distilled water, dried, and fixed using 99% methanol during twenty minutes. Thereafter, biofilm mass was stained for fifteen minutes using 200 μL of 0.1% crystal violet dye. After air drying the wells, the dye of the biofilm which had lined the walls of the microplate was resolubilized using 5% isopropanol acid. Finally, the microplate was spectrophotometrically measured using a microplate reader at a wave length of 570 nm [Biofilm was detected through the MTP method. MTP is a quantitative method to detect biofilm using a microplate reader. An overnight culture in the TSB culture medium was performed for each isolate at a temperature of 37°C. Then, a bacterial suspension in TSB was prepared and adjusted to 0.5 McFarland  was used for band profile analysis. This version of the software has the options for analyzing and interpreting phenotypic and genotypic data such as the data obtained in gel- and sequence-based typing methods. For dendrogram construction, genetic similarity analysis was performed using Unweighted Pair Group Mean Method with Arithmetic mean (UPGMA), Dice similarity coefficient, and 1% band position tolerance. Only bands which sized 100–3000 bp according to the ladder were considered for analysis. The BioNumerics software creates groups with specific similarity range. The difference among band patterns was depicted using numbers over lines in dendrogram  or numbeP values less than 0.05 were considered statistically significant. Data analysis was performed using the SPSS software (v. 16.0).Data were analyzed using the Fisher's exact and the Chi-square tests. K. pneumoniae isolates were obtained from patients with COVID-19 and VAP in ICU. Thirty-six isolates were from male patients, and 34 were from female patients. The mean of patients' age was 65.7 ± 10.5 years , CPM-AZT-PTZ (70%), TOB (61.4%), CIP (57.7%), GM (55.7%), and IMI (50%). K. pneumoniae for ten tested antibiotics. All K. pneumoniae isolates were sensitive to polymyxin B and colistin, while 77.14% of isolates were MDRKp.Antimicrobial susceptibility testing showed that K. pneumoniae isolates formed biofilm. Biofilm analysis showed that 2.8% of isolates were strong biofilm producer (n = 2), 31.4% of them were moderate biofilm producer (n = 22), 8.5% of them were weak biofilm producer (n = 6), and the remaining 57.1% of them did not form biofilm (n = 40) (Findings showed that 42.8% of (n = 40) .K. pneumoniae isolates (E1–E28). These ERIC types consisted of ten genotypes with a genetic similarity of more than 80% and eighteen isolates with a genetic similarity of less than 80%. Each of these eighteen isolates was placed in a separate ERIC type (n = 52) were placed in ten clusters . The clusters were further categorized into three major clusters, i.e., clusters E, H, and J, with ten, ten, and eleven isolates, respectively. Moreover, 21 isolates were placed in seven minor clusters, namely, clusters A, B, C, D, F, G, and I, each with 2–4 isolates . Moreover, antimicrobial resistance to TOB in cluster H was significantly greater than cluster E. However, there was no significant difference between clusters J and H respecting antimicrobial resistance  and were resistant to routinely used antibiotics for treating K. pneumoniae infections such as beta-lactams, aminoglycosides, and quinolones. Study results also revealed the high prevalence of antimicrobial resistance among K. pneumoniae isolates. The high prevalence of MDRKp infection in the present study was in line with the findings of previous studies [K. pneumoniae among patients with VAP in ICU [This study assessed the genetic diversity, antimicrobial resistance pattern, and biofilm formation in  studies , 31. ForP in ICU , 32. MDRP in ICU . These fP in ICU , around P in ICU .K. pneumoniae infections. As K. pneumoniae is a major cause of VAP in ICU, its resistance to these antibiotics makes treatment difficult and causes challenges for the management of VAP among patients with COVID-19 in ICU. The mechanisms of resistance to carbapenems such as Mer among K. pneumoniae include changes in membrane permeability, production of wide-spectrum beta-lactamases, changes in porins, and expression of the efflux system genes [K. pneumoniae resistance to fluoroquinolones such as CIP is plasmid-mediated horizontal gene transfer [K. pneumoniae isolates. Previous studies also reported acquired resistance to aminoglycosides such as GM among both gram-negative and gram-positive bacteria [The high prevalence of antimicrobial resistance to antibiotics Mer and CIP is an important finding because these were the most commonly used antibiotics in the study setting for the management of em genes . Moreovetransfer , 38. Ourbacteria , 40. Thebacteria .K. pneumoniae isolates were able to form biofilm, and 34.2% of those with this ability were moderate to strong biofilm producers. Biofilm formation is an important step in the development and stabilization of opportunistic nosocomial VAP because it protects bacteria against phagocytosis. Colonization of VAP-induced bacteria in endotracheal tube and ventilator circuit is very common, and there is a well-known significant relationship between such colonization and nosocomial pneumonia [K. pneumoniae is a major cause of VAP, and biofilm formation is an important pathogenic factor among patients with VAP induced by K. pneumoniae. Biofilm formation ability seems to have direct relationship with environmental resistance among K. pneumoniae strains [K. pneumoniae reported a biofilm formation rate of 62.5% [K. pneumoniae strains formed biofilm [K. pneumoniae is attributable to the differences in the geographical area, setting, and sample size of the studies. Effective management of infections caused by biofilm-producing microorganisms using available antibiotics is essential in healthcare settings. Recently, some novel biofilm-eliminating strategies have been developed. Examples of these strategies are use of bacteriophage, weak organic acids, and photo irradiation. Nonetheless, further studies are still needed to produce firmer evidence in this area [Study findings also showed that 42.8% of  strains . A formeof 62.5% . Another biofilm . This dihis area .K. pneumoniae isolates obtained from the study setting. Several previous studies in Iran and other countries also reported genetic diversity among K. pneumoniae isolates. For instance, a study on CST-resistant K. pneumoniae isolates in the southwest of Iran reported 23 ERIC types among 26 isolates [. pneumoniae isolates. Moreover, a study into the genetic relatedness of MDRKp isolates in hospitals in Egypt reported 21 ERIC types and great genetic diversity [K. pneumoniae due to differences in its nucleotide sequences [MST and dendrogram analysis in this study revealed 28 different ERIC types, namely, ten prevalent types and eighteen unique types. This finding denotes the great diversity of the isolates . Anotheriversity . These cequences .K. pneumoniae isolates in the largest cluster, i.e., cluster J, had high antimicrobial resistance to antibiotics Mer, CPM, TOB, AZT, GM, CIP, and PTZ, and the most prevalent antimicrobial resistance pattern among them was Mer+CPM+TOB+AZT+IMI+GM+CIP+PTZ. Antimicrobial resistance to Mer, TOB, GM, and CIP in this cluster was significantly higher than cluster H. Moreover, antimicrobial resistance to TOB in cluster J was significantly higher than cluster E. These findings denote significant relationship between ERIC clusters and antimicrobial resistance. However, there was no significant difference among the three major clusters E, H, and J respecting biofilm formation. The high diversity of K. pneumoniae isolates in the present study is an important finding because more than 70% of these isolates are MDR and can use mechanisms such as horizontal gene transfer to transfer this resistance to the bacteria which induce healthcare-associated infections. Different clones may have different antimicrobial resistance patterns and thereby can cause more difficulties in the treatment of their associated infections [Study findings also showed that fections . Given tThe rates of VAP between centers managing COVID-19 are likely to vary depending on the clinical characteristics of the patients managed, differential ICU admission policies, and clinical factors such as use of immunosuppressive therapies. However, it is thought that increasing the number of nurses in the ICU, educating nurses about oral hygiene, and head of bed elevation in these centers can help reduce VAP cases.K. pneumoniae isolates obtained from patients with COVID-19 have high antimicrobial resistance, and the prevalence of MDR strains among them is very high (77%). Moreover, 42.8% of these isolates are biofilm producer, and 44.2% of them have genetic similarity and are clustered in three main clusters with significant differences among clusters respecting genetic similarity and antimicrobial resistance of the isolates. Determining the characteristics of these isolates helps medical specialists more effectively manage their associated infections and use more effective infection management policies for each setting. Further studies are recommended for assessing genetic diversity, ERIC types, and antimicrobial resistance pattern among different bacterial species which cause VAP among patients with COVID-19 in order to facilitate patient recovery. Future studies are needed to assess bacterial colonization among patients with COVID-19 at the time of their hospital admission.This study suggests that


Ranunculus yunnanensis Franch is endemic in Yunnan and Sichuan Provinces, southwestern China. Here, we report the complete chloroplast (cp) genome of R. yunnanensis. The chloroplast genome is 156,050 bp in length, with 111 encoded genes, including 78 protein-coding genes, 29 tRNA genes, and four rRNA genes. Maximum-likelihood phylogenetic reconstruction using the existing data of Ranunculus shows that R. yunnanensis is revealed at the basal position of the marsh buttercup clade. This result has improved a better understanding of the internal relationship of the Ranunculus. Ranunculus yunnanensis Franch is a perennial herb of Ranunculus only distributed in north, northwest Yunnan, and southwest Sichuan . The voucher specimen (20190716-24) is deposited in the Museum of Beijing Forestry University . A sequencing library was sequenced using Illumina nova-seq 6000 platform. The cp genome was assembled from the de novo datasets using the GetOrganelle pipeline  as the reference sequence. The cp genome of R. yunnanensis was annotated with Plastid Genome Annotator (PGA)  region and a small single copy  region are separated by two reverse repeats  regions. The genome contains 111 encoded genes, including 78 protein-coding genes, 29 tRNA genes, and four rRNA genes. The total GC content was 37.9%, while the GC contents in LSC, SSC, and IR regions were 36.1%, 31.5%, and 43.5%, respectively. Annotated cp genome sequence was submitted to GenBank with an accession number MZ703201.The complete cp genome of Ranunculus shows that R. yunnanensis is at the basal position within the marsh buttercup species (Ranunculus.Phylogenetic analysis was carried out in the software toolkit phylosuite (Zhang et al.  species . Our res


Helicobacter pylori (H. pylori) prevalence is lower in patients with inflammatory bowel disease (IBD) than in those without IBD, suggesting that H. pylori plays a protective role in IBD. It has been reported that IBD may occur due to H. pylori eradication; however, it is unclear whether H. pylori eradication increases the incidence of IBD. Moreover, the effect of H. pylori eradication on IBD activity is unclear.H. pylori duodenal ulcer. H. pylori eradication (clarithromycin and amoxicillin for 7 days and a proton-pump inhibitor) led to symptom improvement the day after treatment initiation. However, he developed diarrhea and his FC level remained high despite improvement in duodenal ulcer symptoms and endoscopic findings of H. pylori eradication. Colonoscopy results indicated UC relapse.An 11-year-old boy diagnosed with ulcerative colitis (UC) was in clinical remission, with treatment involving 5-aminosalicylic acid. Fecal calprotectin (FC) level had decreased to 33.2 mg/kg, indicating mucosal healing. At age 12, he experienced epigastric pain on an empty stomach, which was relieved with dietary intake. His FC level was elevated without UC symptoms, such as diarrhea and bloody stools. He was diagnosed with H. pylori eradication may worsen UC activity. However, further studies are required as this case report involved only one pediatric patient with increased UC activity after H. pylori eradication. Helicobacter pylori (H. pylori). The prevalence of H. pylori is lower in patients with IBD than in those without IBD, which suggests H. pylori plays a protective role in the development of IBD. [H. pylori eradication [H. pylori eradication increases the incidence of IBD as well as the effect of H. pylori eradication on IBD activity remains unclear. We describe a pediatric case of UC, worsened through H. pylori eradication for the treatment of duodenal ulcer.The prevalence of inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn’s disease, has increased worldwide.  Its path of IBD. , 4 It hadication –7; howevAn 11-year-old boy consulted a family doctor for bloody diarrhea. The patient had no specific medical or family history. A colonoscopy from the rectum to the sigmoid colon led to the diagnosis of pediatric UC with a pediatric ulcerative colitis activity index (PUCAI) of 30. He was administered 5-aminosalicylic acid (5-ASA) suppositories (1 g/day) and was referred to our hospital for subsequent treatment. His symptoms promptly improved to a PUCAI of 5. His blood examination results were unremarkable but the fecal calprotectin (FC) level was elevated to 3,190 mg/kg. A complete colonoscopy was performed, which revealed inflammatory findings from the rectum to the transverse colon. Moreover, the cecum and ascending colon showed loss of vascular permeability and adherent purulent mucus Fig. .Fig. 1CoH. pylori infection. The H. pylori infection was treated using clarithromycin and amoxicillin for 7 days and a proton-pump inhibitor. The patient’s symptoms improved the day after treatment initiation. In week 33, an EGD was performed to evaluate the therapeutic effect. The duodenal ulcer had healed, and scarring was observed . He reported no UC symptoms, including diarrhea and bloody stools ; however, an elevated FC level was noted (week 20). Esophagogastroduodenoscopy (EGD) findings indicated an A1 ulcer on the lower wall of the duodenal bulb Fig. A. A rapiH. pylori eradication. The patient subsequently developed diarrhea  in week 35 and a UC relapse was considered. Cytomegalovirus antibody, antigenemia, and tuberculosis (T-SPOT assay) tests were negative. Colonoscopy showed small aphthae and edematous mucosa throughout the colon in week 36 -10 and suppressing IL-17 producing effector T-helper cells. [H. pylori-infected IBD patients, and this report showed NLRP12 has a negative correlation with the disease activity in pediatric IBD patients. [H. pylori in IBD patients is required.The mechanism by which r cells.  Gravina r cells.  Chen et atients.  Further H. pylori eradication, the patient had an increased FC level, and he gradually developed diarrhea. Therefore, the elevated FC level was considered to be due to UC activity. The FC level is the most helpful biomarker of endoscopic disease activity. [H. pylori eradication may help to detect endoscopic and histological relapse prior to clinical relapse. Endoscopic and histological remission in addition to clinical remission should be confirmed in patients with H. pylori-induced peptic ulcer cases.18.In our patient, endoscopic mucosal healing was not confirmed after the initial 5-ASA treatment. However, an FC level < 250 mg/kg is indicative of endoscopic and histological mucosal healing.  Based onctivity.  No otherctivity.  FC monitH. pylori eradication. Measuring the FC level is non-invasive and allows early detection of relapse during the asymptomatic phase. Further studies are required to determine the effect of H. pylori eradication in relation to pediatric IBD activity.To date, no studies concerning pediatric IBD have evaluated the FC level prior to and post


Helicobacter pylori infection and portal hypertension (PH)-associated gastrointestinal (GI) diseases, such as esophageal varices and portal hypertensive gastropathy (PHG), the impact of H. pylori infection on PH-related GI complications has not yet been elucidated. This meta-analysis investigated the association between H. pylori infection and the risk of PH-related GI complications. An electronic search for original articles published before May 2020 was performed using PubMed, EMBASE, and the Cochrane Library. Independent reviewers conducted the article screening and data extraction. We used the generic inverse variance method for the meta-analysis, and Begg’s rank correlation test and Egger’s regression test to assess publication bias. A total of 1,148 cases of H. pylori infection and 1,231 uninfected controls were included from 13 studies. H. pylori infection had no significant association with esophageal varices . Although H. pylori infection was significantly associated with PHG in case-control studies , no significant differences were found in the cohort studies  or all studies combined . In conclusion, H. pylori infection was not associated with the risk of PH-related GI complications. Clinicians should carefully treat cirrhotic patients with PH-related GI complications, regardless of H. pylori infection.Despite the importance of  Liver cirrhosis is a disease characterized by chronic fibrosis and dysfunction of the liver. In cirrhotic patients, structural abnormalities, including hepatic vascular resistance, and functional defects, including endothelial dysfunction and increased hepatic vascular tone, increase hepatic resistance to portal blood flow and elevate portal pressure. Alterations in intrahepatic hemodynamics provoke splanchnic vasodilation as an adaptive response. As cirrhosis progresses, splanchnic vasodilation becomes too intense to have serious effects on systemic circulation. Varices and portal hypertensive gastropathy (PHG) can develop as complications of portal hypertension (PH) in patients with cirrhosis. Varices and PHG are observed in approximately 50% and 3–14% of cirrhotic patients, respectively, and can be the main causes of massive bleeding, leading to hemodynamic instability and a life-threatening disease course [Helicobacter pylori can colonize the acidic gastric mucosal surface. Gastric colonization by H. pylori can induce peptic ulcer disease, atrophic gastritis, gastric cancer, and mucosa-associated lymphoid tissue lymphoma. H. pylori also contributes to non-GI disorders, such as iron-deficient anemia, vitamin B12 deficiency, and idiopathic thrombocytopenic purpura. Extragastric diseases that were previously considered to be independent of H. pylori have recently been found to be related to H. pylori infection [H. pylori infection [H. pylori infection and cirrhosis, regardless of PH-related GI complications [H. pylori infection [H. pylori eradication therapy can also improve hyperammonemia in patients with cirrhosis [H. pylori infection in cirrhotic patients with varices or PHG, these show controversial results concerning the relationship between H. pylori infection and PH-related GI complications [H. pylori infection and PH-related complications in patients with cirrhosis.nfection . Multiplnfection –6. Many ications . Moreovenfection . H. pyloirrhosis . Althougications –20. TherHelicobacter,” “pylori,” “pyloridis,” “Helicobacter pylori,” or “Campylobacter pylori” and “liver cirrhosis,” “liver fibrosis,” “hepatic fibrosis,” “liver failure,” “hepatic failure,” “esophageal varix,” “varix,” “variceal,” or “portal hypertensive gastropathy” which are described in S1 Text. We manually searched major international gastroenterology and hepatology conference abstracts and the references of the selected articles. Additionally, we found articles through internet searches to identify further relevant studies. The current study was conducted following the Preferred Reporting Items for Systematic review and Meta-Analysis protocols (PRISMA) guidelines [Two authors (Y.K.J. and D.H.L.) independently carried out a comprehensive systematic search for published articles from inception to May 2020 using PubMed, EMBASE, and the Cochrane Library. The search was limited to human studies without language restrictions. Search terms included “idelines .H. pylori infection and PH-related GI complications; 3) appropriate diagnostic tests for H. pylori infection conducted prior to the evaluation of PH-related GI complications; and 4) available raw data reported in the comparison arms.Studies that met the following eligibility criteria were included: 1) observational studies, including cohort and case-control studies; 2) studies that evaluated the relationship between Studies that did not include original articles were excluded. Also, studies were excluded if they were case studies, had no control group, or were not performed on human subjects. We excluded articles that did not explicitly state institutes or hospitals where the research was performed. When duplicated publications were identified, we included the most recent and thorough articles.H. pylori detection, and outcome results in patients with or without H. pylori infection.Data extraction was conducted independently by two authors (Y.K.J. and D.H.L.). The following data were collected from the included studies: first author, year of publication, study design, demographic and clinical information of the study patients , methods of The retrieved articles were independently reviewed by fully qualified investigators , and disagreements were resolved through discussions among investigators. The Newcastle-Ottawa quality assessment Scale (NOS), which was developed as a judgment tool for the quality of nonrandomized studies such as case-control or cohort studies in meta-analyses, was used to evaluate the quality of the selected studies . The queH. pylori-infected patients, we assumed that the ORs were similar to RRs. The generic inverse variance method was used to combine the results across studies. Heterogeneity among studies was assessed using the I2 statistics test considering low heterogeneity (<25%), moderate heterogeneity (25–75%), and high heterogeneity (>75%). Subgroup analyses based on NOS information were also performed. The probability of publication bias was assessed using the Begg’s rank correlation test and Egger’s regression test. Statistical significance was set at P <0.05.All statistical analyses were performed using Stata software  and R . The odds ratio (OR), relative risk (RR), and 95% confidence interval (CI) were considered to be the effect sizes. We used the unadjusted OR and RR values calculated from the raw data. Considering the low incidence of PH-related GI complications in Fig 1.Through the database search, 4,429 potentially eligible studies were identified . We excluded 498 duplicated studies. After evaluating study titles and abstracts, 3,788 studies and 121 studies were excluded. After a full-length article review, 11 studies were excluded, and other 11 studies were included. Among these excluded studies, 2 studies were not accessible, 2 studies did not have relevant content, and 7 studies did not report the complete outcomes of interest. Two studies were identified after additional searches. Finally, 13 studies (8 cohort studies and 5 case-control studies) were included in the meta-analysis –19,24,25Table 1. A total of 2,379 patients were included in the meta-analysis. H. pylori-specific serum immunoglobulin G was most frequently used for the diagnosis of H. pylori infection [H. pylori-positive patients and H. pylori-negative patients, respectively [H. pylori-positive patients was 54.1 ± 10.8 years and that of H. pylori-negative patients was 54.8 ± 9.4 years. There was no significant age difference according to H. pylori infection. The median NOS score of the trials that were included was 7 (range 5–8), and we considered 5 of 13 trials to be of high methodological quality .The baseline characteristics and quality assessments of the 13 trials are summarized in nfection ,17,19,25nfection ,26 did nectively ,19,20. MH. pylori infection and PH-related GI complications are shown in Table 2. H. pylori infection was not significantly associated with esophageal varices . Similarly, there was no association between H. pylori infection and PHG . Subgroup analyses based on information on the study design and NOS were conducted. Figs 2 and 3 show the unadjusted RR/OR and 95% CI of PH-related GI complications by H. pylori infection in cohort studies and case-control studies, respectively. There was no meaningful relationship between esophageal varices and H. pylori in either cohort  or case-control studies . H. pylori infection was shown to be unrelated to PHG in cohort studies , but in case-control studies of the relationship between H. pylori and PHG, it was noted that there was some relationship between H. pylori infection and PHG .The meta-analysis results of the association between Fig 4. Five cohort studies were included in the subgroup analysis, and all case-control studies were excluded. There was no significant correlation between H. pylori infection and PH-related GI complications in patients with cirrhosis .For subgroup analysis, we selected highly qualified articles with NOS>7 and analyzed them in HP>0.10) in the pooled estimates of cirrhotic patients with varices. The corresponding pooled ORs were not significantly influenced by omitting any single study, as shown in S3 Table. Publication bias in this study was unremarkable. All P-values obtained from I2 (HP), Begg’s (BeggP) and Egger’s methods (EggP) are shown in Tables 2 and 3.There was no heterogeneity found  gene in liver tissue is associated with the severity of hepatic fibrosis. Moreover, H. pylori infection can lead to and aggravate NAFLD [H. pylori [H. pylori eradication greatly improves cirrhosis and cirrhotic complications [Many studies have suggested a strong relationship between irrhosis ,28–31. Tte NAFLD ,32. The . pylori . H. pyloications ,33,34.H. pylori infection in cirrhotic patients is different, although many published studies have demonstrated a positive relationship between liver cirrhosis and H. pylori infection. Vasodilation mainly contributes to the development of PH by increasing resistance to portal flow and expansion of collateral circulation [H. pylori spiral form into an inviable coccid form [H. pylori can survive in acidic environment, and its growth was limited in neutral pH [H. pylori colonization. The severity of gastric vascular congestion is not associated with the possibility of H. pylori infection [The relationship between PH-related GI complications and culation . Nitric cid form ,37. H. putral pH ,39. The utral pH . Howeverutral pH . Gastricnfection –44.H. pylori infection can be suppressed in patients with PH, including varix and PHG, although H. pylori infection can increase in patients with cirrhosis and result in poor prognosis. Our recent study revealed an insignificant relationship between PH-related GI complications and H. pylori infection as in the previous studies. Analysis using three case-control studies showed the possibility of a positive relationship between PH-related GI complications and H. pylori infection. However, case-control studies are less reliable than cohort studies in terms of evidence-based medicine. Moreover, the selected case-control studies had a lower quality than cohort studies. Subgroup analysis, including only articles with NOS>7, failed to show a meaningful relationship between H. pylori infection and PH-related GI complications. However, in a cohort study of patients with cirrhosis, patients with H. pylori infection had a poor prognosis, including PH-related GI complications, as compared with those without [ without .H. pylori infection and PH-related complications, this meta-analysis may be the first to establish a comprehensive and reliable analysis. Prior studies covered only one or a few etiologies of chronic liver disease, but this study handles multiple etiologies of cirrhosis, including viral hepatitis, alcoholic hepatitis, autoimmune hepatitis, and even PBC. This meta-analysis deals with all H. pylori diagnostic techniques used in actual clinical practice. In addition, the studies included in this meta-analysis have similarities in the composition of sex and gender of the participants. There are low to moderate grades of heterogeneity and inconsistency across studies on PHG. Also, there was no detectable publication bias according to Begg’s rank correlation test and Egger’s regression test.Although there have been previous studies on the relationship between H. pylori infection and PH-related GI complications are reported, more influential and reliable meta-analyses will be available. A meta-analysis that deals with the relationship between H. pylori infection and the risk of bleeding due to PH-related GI complications is also required because there are conflicting reports about the relationship between variceal bleeding and H. pylori infection [However, this study has some limitations and requires careful consideration when interpreting the results of the investigation. The total number of articles included in the meta-analysis was relatively small (13 articles). In addition, 5 out of 13 articles were case-control studies; therefore, there are only 8 cohort studies with high reliability. Because meta-analyses comprise already published articles, their conclusions are affected by the number and quality of the chosen studies. If more highly qualified original articles on the subject of the connection between nfection ,46.pylori patients with cirrhosis is unlikely to be attributed to the development of PH-associated GI complications. Knowing that H. pylori infection is not associated with PH-related GI complications in cirrhosis patients will help clinicians treat PH-related GI complications and H. pylori-induced gastritis in cirrhosis patients. Clinicians can treat PH-related GI complications in patients with cirrhosis, regardless of H. pylori infection.In summary, our study demonstrated that gastric colonization of H. S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S1 TextSearch strategies for PubMed, EMBASE and the Cochrane Library.(DOCX)Click here for additional data file.