Dermacentor and Haemaphysalis species. Individual ticks from pools with pathogen detection were then further screened by targeted amplification and then NS, which provided extensive genome data and revealed probable pathogen Haseki Tick Virus . Two distinct TTV2 clades were observed in phylogenetic analysis, one of which included closely related Dermacentor reticulatus Uukuviruses. JMTV detection indicated integrated virus sequences. Overall, we observed an expansion of newly documented pathogenic tick-borne viruses into Europe, with TTV1 being identified on the continent for the first time. These viruses should be included in the diagnostic assessment of symptomatic cases associated with tick bites and vector surveillance efforts. NS is shown as a useful tool for monitoring tick-associated pathogens in pooled or individual samples.We analysed both pooled and individual tick samples collected from four countries in Eastern Europe and the Black Sea region, using metagenome-based nanopore sequencing (NS) and targeted amplification. Initially, 1337 ticks, belonging to 11 species, were screened in 217 pools. Viruses (21 taxa) and human pathogens were detected in 46.5% and 7.3%, respectively. Tick-borne viral pathogens comprised Tacheng Tick Virus 2 , Jingmen Tick Virus  and Tacheng Tick Virus 1 . An association of tick species with individual virus taxa was observed, with the exception of TTV2, which was observed in both  Accounting for a major portion of vector-borne diseases in many countries, tick-borne infections may produce a significant disease burden on local healthcare providers and constitute an eminent public health threat3. With ticks acting as biological vectors, a wide palette of pathogenic microorganisms including viruses, protozoans and bacteria can be transmitted to humans and other susceptible vertebrates3.The overall prevalence of tick-borne infections is increasing globally, due to the expansion of tick populations into new geographic regions and increased human exposure as a result of environmental and climatic changes4. Those considered significant human or animal pathogens due to disease severity, distribution, potential to emerge and notable impact include Crimean-Congo hemorrhagic fever virus (CCHFV) and Nairobi sheep disease virus (NSDV) (family Nairoviridae genus Orthonairovirus); Tick-borne encephalitis virus (TBEV), Omsk hemorrhagic fever virus (OHFV), Kyasanur forest disease virus (KFDV), Powassan virus (POWV) and Alkhurma hemorrhagic fever virus (AHFV) (family Flaviviridae genus Flavivirus); Severe fever with thrombocytopenia syndrome virus (SFTSV) and Heartland virus (HRTV) (family Phenuiviridae genus Banyangvirus); Colorado tick fever virus (CTFV) , Bourbon virus (BRBV)  and African swine fever virus (ASFV) 5. Expanding activity zones and changing epidemiology have been documented for some of these viruses, such as for CCHFV around the Mediterranean Sea region, POWV in North America and AHFV in Africa5. In addition to these established tick-borne pathogens, a number of newly characterized viruses have been associated with human infections, including Jingmen Tick Virus (JMTV) and Alongshan virus (ALSV), (family Flaviviridae), Tacheng Tick Virus 1 (TTV1) and Songling virus (SGLV) , Tacheng Tick Virus 2 (TTV2) (family Phenuiviridae genus Uukuvirus) and Haseki Tick Virus (HTV) (family Flaviviridae)11. Although cases have been reported from China , Kosovo (JMTV) and the Russian Federation (HTV), information on pathogenesis, distribution and public health impact of these viruses are currently scarce.Viruses transmitted by ticks are taxonomically heterogenous and exhibit diverse characteristics4. Metagenome-based screening, enabling analysis of the nucleic acid content of any sample without prior information facilitates bio- or xeno-surveillance, where ticks can be utilized as “blood bags” to screen pathogens over multiple hosts12. Metagenome-based screening by Nanopore Sequencing (NS) has been documented as a powerful tool to detect known and novel tick-borne viruses13. In this study, we analyzed pooled and subsequently individual ticks collected from several locations in Eastern Europe and the Black Sea region using NS and targeted amplification for several recently described tick-borne viruses.Expansion and spillover of tick-borne viruses may be easily overlooked until the emergence of symptomatic case clusters or local epidemics involving humans or domestic animals. Timely identification of the circulating pathogens and assessment of public health threats rely heavily on surveillanceIn total, 1337 ticks, belonging to 11 species, were included in the study and screened in 217 pools. Virus sequences were detected in 101 pools (46.5%) and viruses documented as tick-borne human pathogens were observed in 16 (7.3%). In pools with detectable viruses, probable coinfections, evidenced by multiple virus sequences from various families/genera, were present in 31 (30.6%). Co-infections of human pathogens were not detected in pools. Tick species and virus detection prevalences according to the country of collection are provided in Table Dermacentor marginatus and Dermacentor reticulatus ticks, and also in a Haemaphysalis punctata pool from Georgia. Ixodes ricinus harbored the broadest virus diversity (10 virus taxa) and all JMTV detections. Information on individual pools with detectable virus sequences are provided in Supplementary Tables Viruses belonging to 21 separate taxa were documented in the tick pools . A pool with TTV2 detection from Ukraine and JMTV detection from Poland could not be included in PCR screening due to the lack of sufficient material for testing.We performed targeted PCR and additional NS on individual ticks from pools with detectable viral pathogens. Fifty-nine individual samples from 15 pools were amplified and subsequently sequenced, including samples with expected band sizes for TTV2 and JMTV observed in electrophoresis (n = 31), selected samples negative in targeted amplification (n = 6) and controls comprising a female and a male Dermacentor reticulatus uukuvirus (DRUV) (n = 1). Subsequently, we sequenced all PCR positive (n = 29) and selected PCR negative (n = 3) individuals. Expected viruses, based on NS sequencing, were observed in 19 individual samples—18 PCR (+) and 1 PCR (−). The largest contigs were characterized as TTV2 (n = 15) and DRUV (n = 4) by BLAST, with mapped read counts of 3–86 and sizes between 332 and 5659 bp on the L and S genome segments. Breakup of pooled and single samples and virus detections are provided in Supplementary Table Using targeted PCR, we screened all individuals from 10 pools from Georgia and 3 pools from Poland, which were NS positive for TTV2 (n = 12) and closely-related We further analyzed 6 larger TTV2 L segment contigs (> 2330 bp) from different individuals and collection sites. Pairwise comparisons revealed up to 9.9% and 2.5% diversity on nucleotide and amino acid alignments, respectively (Supplementary Table A large portion (1885 of 2189 amino acids) of the TTV2 L segment, encoding for the RNA-dependent RNA polymerase , was available in two individual samples from Georgia . The sequences demonstrated 6.6% and 1.7% divergence based on nucleotide and putative amino acid alignments, respectively, which increased to 32.1% nucleotide and 5.7% amino acid divergence in pairwise comparisons with the closest relative (TTV2 strain Iasi20)  was detected in 11.5% of the pools, which were exclusively D. reticulatus ticks. Sequences with highest identities to members of the Norwavirus genus (family Nairoviridae) were also common, detected in 10.1% of the pools and in I. ricinus ticks. Our phylogenetic analyses based on Bunyavirus polymerase and nucleocapsid amino acid alignments revealed diverse virus sequences related to Grotenhout virus and other proposed members, distinct from the recently described congeneric pathogen Beiji nairovirus  , present in 6.4% (14/217) of pooled and 23.1% (9/39) of single D. reticulatus ticks. This virus is related to HTV (unclassified Riboviria), with 91.8% and 94.4% nucleotide and putative amino acid identities, respectively. Despite being absent in pooled ticks, sequences with highest BLAST identities to HTV were observed in four individual D. reticulatus samples from Georgia and Poland, with a prevalence of 10.2% (4/39) (Supplementary Table Another frequently observed virus was Riboviria), Tacheng Tick Virus 3 , Dermacentor reticulatus Rhabdovirus 1  and Betaricinrhavirus chimay (Rhabdoviridae) were identified during analyses of individual ticks, with < 10 mapped reads  including Tacheng Tick Virus 5  and HTV  in the sample cohort and produced abundant virus genome data for further downstream analysis.Uukuvirus tachengense species, TTV2 was initially described during a metagenomic investigation of arthropods from China and has since been detected in D. marginatus, Dermacentor nuttalli, Dermacentor silvarum, and Hyalomma asiaticum ticks17. Subsequently, it has been documented as the causative agent in a patient with tick-associated febrile disease from China, with implications of person-to-person transmission through droplets or direct contact with body fluids9. Further detections of TTV2 in Hyalomma / Dermacentor spp. from Kazakhstan (according to GenBank records) and D. reticulatus from Romania are reported18. TTV2 is also documented in D. marginatus and the prominent CCHFV vector, H. marginatum, from ecologically diverse locations in Asia Minor, with evidence for trans-stadial or horizontal virus transmission among ticks20. Interestingly, we observed two distinct TTV2 clades in our phylogenetic analysis, where one of the groups also included DRUV, an unclassified Ribovirus separately detected in 4.1% of the tick pools in the study. DRUV was recently described from Croatia in D. reticulatus pools21, but to date there is scant information. Genomes of both TTV2 and DRUV consist of L and S segments, while seemingly lacking the glycoprotein encoding M segment, even in cell culture grown isolates21. The presence of TTV2 and DRUV divergent clades and human exposure in Europe or around the Black Sea region require further investigation.Among the tick-borne viral pathogens identified, TTV2 was the most frequent, detected in 5.9% of the tick pools (13/217) from several sites in three of the four countries of collection, including Eastern Europe (Poland and Ukraine) and the Asian Black Sea region (Georgia). Detections were further confirmed in individual targeted PCR and NS 15. Initially described during virus discovery efforts in arthropods from China, TTV1 was also detected in febrile diseases associated with tick bites, and as a co-infecting agent in a case with Rickettsial fever and meningitis22. Virus excretion patterns in infected individuals suggested possible transmission by direct contact with body fluids or droplets, similar to CCHFV, as noted for TTV29. Virus exposure was further documented in local sheep, cattle and human populations in China, with Dermacentor spp. suggested as probable vectors9. Virus genomes were detected in D. marginatus, D. silvarum, D. nuttalli, and H. asiaticum ticks in China, and also identified from spleen tissues of great gerbils (Rhombomys opimus)23. Recently, the virus has been documented in pools of Hyalomma aegyptium from southern Turkey24. It remains to be determined whether the lack of detection in targeted amplification in our tick cohort reflects genome diversity or low sensitivity of the PCR.We further detected TTV1 in 0.4% of the pools, comprising nus Fig. . This isI. ricinus samples collected from Poland and Georgia  appear to be widely distributed, having been identified in a diverse spectrum of tick species as well as in bats, cattle and rodents from Asia, Africa, Europe and the Americas25. JMTV and Alongshan virus (another Jingmenvirus) have been documented to cause human infections and subsequent seroconversion, with further evidence for exposure in domestic animals and humans7. Co-infections with JMTV were reported among individuals with CCHFV in Kosovo, with possible impacts on disease outcome26. In Europe, JMTV-infected ticks were reported from Romania, Serbia and the Thrace region of Turkey, while Alonghsan virus detection is more pronounced in northern Europe28. Recently, the integration of the JMTV polymerase gene into I. ricinus genome was documented, in ticks of both sexes and I. ricinus embryo-derived tick cell line IRE/CTVM1929. The integrated fragment of the viral genome was reported to be variable in size and forming several genovariants, with an average of 1.5 copies per cell. Several tick species other than I. ricinus were further suggested to contain the integrated segments. In field collected I. ricinus samples, the integration event was observed with a prevalence of up to 34.3% in various regions of Russia29. The JMTV findings in our study strongly suggest integrated virus sequences being detected, rather than replicating viruses. We also have previously reported NS as capable of detecting all JMTV genome segments, surpassing the sensitivity of screening by nested PCR13. Documentation of virus integration in tick vectors also has ramifications for PCR-based screening, which may require additional genome targets or steps to exclude amplification of integrated fragments.JMTV is the best studied emerging tick-borne pathogen observed in our cohort. It was identified in 0.9% of the samples, comprising ia Table . ClassifD. reticulatus samples, respectively. This virus was first described in D. reticulatus from Croatia, sharing significant NS3–NS5 protein homologies with members of the Pestivirus genus21. It is closely related to other recently documented pestivirus-like viruses, Bole Tick Virus 4 (BTV4), Trinbago Virus (TBOV), as well as HTV21. HTV genomes were recently documented in retrospectively screened sera from individuals with febrile disease associated with tick bites, with prevalences of 0.9–9.3% in different regions of the Russian Federation, with and without TBEV or Borrelia spp.11. We detected HTV in 10.2% of the single D. reticulatus samples from Georgia and Poland and observed clustering of the sequences with HTVs as well as DRPV1 in phylogenetic analyses. Given these findings and limited sequence variation between complete HTV and DRPV1 polyprotein coding sequences, these viruses represent sub-lineages of the same taxon, observed in geographically segregated regions. The capacity of either virus to produce human symptomatic infections requires further investigation.Our findings on HTV and DRPV1 demand further discussion. We detected DRPV1 in 6.4% and 23.1% of the pooled and individual I. ricinus ticks from Bulgaria and Poland. Norwavirus is a recently described genus in the family Nairoviridae, which currently includes Grotenhout Virus (GRHV) as the sole member of the Grotenhout norwavirus species15. Closely related viruses have been documented in northern Europe and central Asia, such as Pustyn Virus (PTV) and Norway Nairovirus 1 (NWNV1), but these strains currently remain taxonomically unclassified15. Similar to TTV2, norwaviruses lack the genomic segment M, which encodes for nairovirus glycoproteins. Beiji nairovirus (BJNV), a norwavirus-related virus, was documented as a potential agent causing tick-borne febrile diseases in China, supported by experimental inoculations7. The public health implications of norwaviruses currently remain underexplored.Finally, we detected Norwaviruses, distinct from Beiji nairovirus, in 10.1% of the pools, comprising In conclusion, our findings reveal several tick-borne viral pathogens, recently reported from various regions from Asia, to be present in various locations in Eastern Europe and Asian Black Sea region. In any of the sampling regions, actual prevalences of individual viruses is hard to assess, due to our cross-sectional collection and non-targeted screening strategy employed in the study. Moreover, local virus clades or integrated virus genomes might undermine targeted screening due to sequence variation (TTV1) or amplification of the integrated fragments (JMTV). Currently, the lack of standardized antibody testing further hinders investigation of previous exposures and serological diagnosis. Nevertheless, these viruses should be considered in the diagnostic assessment of symptomatic cases associated with tick bites and tested when possible. NS proves to be a useful tool for monitoring tick-associated pathogens in pooled and individual samples.30 and stored at − 80 °C. Subsequently, samples from Bulgaria, Poland and Georgia were shipped to Walter Reed Biosystematics Unit (WRBU), Museum Support Center, Smithsonian Institution, MD, USA, in dry ice. Samples from Ukraine were pooled according to species and collection site, processed locally and nucleic acids were shipped to WRBU for further processing. The dorsal and ventral sides of intact specimens were imaged using IDX machine .Questing adult ticks were collected via drag/flagging at 75 ad hoc sites in Bulgaria (2021–2022), Poland (2021), Georgia (2021) and Ukraine 2012–2013) Nucleic acid extraction was carried out individually following homogenization in sterile 1 × PBS  and ATL Lysis Buffer with regent DX  with 5 mm stainless steel grinding balls  using TissueLyser . Following centrifugation, the supernatant was purified using the IndiMag Pathogen Kit  on BioSprint 96 DNA/RNA Purification System , according to the manufacturer recommendations.32. Subsequently, 5 µl of the individual tick nucleic acids were pooled according to species, sex and locality of collection (up to a maximum of 9), and stored in − 80 °C.Morphological identification of the individual ticks was confirmed using DNA barcoding, by amplification of the 658 bp region of the mitochondrial cytochrome c oxidase subunit I (COI) gene, as described previouslyPooled nucleic acids were subjected to cDNA synthesis using NEBNext Ultra II RNA First Strand and Non-Directional RNA Second Strand Synthesis modules, utilizing a random primer mix . Double stranded cDNA was cleaned up using Agencourt AMPure XP reagent  and quantitated by Qubit dsDNA HS Assay Kit . The NEBNext Ultra II End repair/dA-tailing and Quick Ligation modules , and Ligation Sequencing Kit SQK-LSK109  were used to prepare libraries, which were further quantitated by Qubit  using 1 × dsDNA HS Assay Kit. Each sample was separately barcoded with the Native Barcoding Expansion 96-EXP-NBD 196 . An epMotion 5075 workstation  was used for automated liquid handling and library preparation, carried out according to the manufacturer recommendations. Sequencing libraries containing 20–24 barcoded pools were loaded on a single ONT R9.4.1 flow cell and run on the GridION sequencing device  for 48 h.33. The amplicons of the nested amplification  were visualized by electrophoresis on 1.5% agarose gels using GelGreen Nucleic Acid Gel Stain .Nested polymerase chain reaction (PCR) was used to amplify JMTV, TTV1 and TTV2 in single tick samples with the corresponding virus sequences observed in pooled NS. We employed previously published primer sets and conditions for each assay, in a 30 µl total volume per sample. The assays targeted JMTV NS5-like protein on segment 1 and the nucleocapsid protein on segment S for TTV1 and TTV234. Raw reads were trimmed with Porechop to remove adapter sequences and then filtered with NanoFilt to remove reads with q-scores ≤ 9 and read lengths ≤ 100 bp35. Tick genome data were then removed as hosts using Minimap2 v2.24 and Samtools v1.937. Subsequently, the data was aligned to the National Center for Biotechnology Information (NCBI) non-redundant (NR) database using DIAMOND v2.0.1438 and visualized using MEGAN6 (v6.23.2)39.Following NS, base-calling and demultiplexing was carried out on the GridION with the MinKNOW operating software v21.11.7 (Oxford Nanopore Technologies) and Guppy v5.1.1340. Read mapping was carried out using Minimap2 plug-in for Geneious Prime, with default settings optimized for nanopore data. Longest reads were used for similarity assessment when contigs were not available. Alignment and pairwise sequence comparisons were carried out using CLUSTAL W41. Potential genetic exchange and recombination were assessed using the RDP4 software in the default settings42. Protein domain and motif searches were performed using the NCBI conserved domain search tool and MOTIF Search in the PFAM database44. Phylogenetic analysis was performed on sequences using IQ-TREE 245. Optimal evolutionary models and partitioning schemes were determined for nucleotide and amino acid sequence alignment using the automatic model selection tools (-m MFP + MERGE). The maximum likelihood extended majority-rule consensus trees were constructed using the ultrafast bootstrap approximation approach (UFBoot) for 1000 replicates46.Sequences were handled using Geneious Prime (v2022.2.1) . BLASTn and BLASTp algorithms were used for similarity searches in the NCBI databaseSupplementary Figure 1.Supplementary Figure 2.Supplementary Figure 3.Supplementary Tables.


Amblyomma, Ixodes, Dermacentor, and Hyalomma genera may transmit certain viruses such as Bourbon virus (BRBV), Dhori virus (DHOV), Powassan virus (POWV), Omsk hemorrhagic fever virus (OHFV), Colorado tick fever virus (CTFV), Crimean-Congo hemorrhagic fever virus (CCHFV), Heartland virus (HRTV), Kyasanur forest disease virus (KFDV), etc. that affect humans and certain wildlife. The tick vectors may become infected through feeding on viraemic hosts before transmitting the pathogen to humans and animals. Therefore, it is vital to understand the eco-epidemiology of tick-borne viruses and their pathogenesis to optimize preventive measures. Thus this review summarizes knowledge on some medically important ticks and tick-borne viruses, including BRBV, POWV, OHFV, CTFV, CCHFV, HRTV, and KFDV. Further, we discuss these viruses’ epidemiology, pathogenesis, and disease manifestations during infection.Ticks are obligatory hematophagous arthropods that harbor and transmit infectious pathogens to humans and animals. Tick species belonging to  Ticks are responsible for more than 95% of vector-borne diseases in the United States . Tick-boAmblyomma, Haemaphysalis, Ixodes, Rhipicephalus, etc., and over 700 species worldwide , soft ticks (Argasidae), and Nuttalliellidae, Ixodidae is the most prominent family, including genera like orldwide . Ixodes sulcatus  distribusulcatus ; I. rici ricinus  in Europiculatus  in some apularis ; D. andendersoni ; and Ambericanum  in Northapularis , A. amerericanum , D. andendersoni , and Haepinigera  go throupinigera . Among tpinigera .TBVs use a variety of mechanisms to evade host immune responses for successful tissue colonization in humans and animals . DespiteThis review thus emphasizes the epidemiological characteristics of some medically important tick vectors and TBVs, including Bourbon virus (BRBV), POWV, Omsk hemorrhagic fever virus (OHFV), Colorado tick fever virus (CTFV), Crimean-Congo hemorrhagic fever virus (CCHFV), Heartland virus (HRTV), and Kyasanur forest disease virus (KFDV) . Details2.Orthomyxoviridae family, genus Thogotovirus, infecting humans and animals via tick bites in most parts of Africa, the Middle East, and Europe , proper diagnosis is required to understand preventive strategies. Future research into the transmission and underlying pathogenic mechanisms of BRBV would aid in our understanding of their ecology.BRBV is a member of the d Europe . Virusesd Europe . The inc animals . In Ehim animals . The firArkansas . AdvanceArkansas  and micrArkansas , are rec2.1.Oz-virus isolated in Japan , frog (XLK-WG), hamster (BHK21Cl-15), duck embryo (DE), human (HeLa and HUH-7), Aedes, Anopheles , Dermacentor ANE58, DAE15, Hyalomma, and Amblyomma RAE/CTVM1, HAE/CTVM9 AVL/CTVM17 , first isolated from a human who died from encephalitis in Powassan, Ontario, Canada, in 1958 (deer tick virus) (flavivirus family has an ssRNA genome (about 11 kb) with a 5′ UTR (loops A and B) and a 3′ UTR that becomes infectious when it enters the host cell  . The flaost cell . Flaviviartments . Except 3.1.Ixodes scapularis ticks and white-footed mice in northern and north-central America  after feeding on infected rodent blood  transmit OHFV to humans and animals after feeding on infected hosts  after an extraneural or intracerebral injection (Macaca radiata) showed no obvious pathological or histological symptoms, and no virus was isolated from their blood. Furthermore, high aminotransferase levels were observed, indicating viral replication in the liver  and vascular tissues (vascular tropism). However, isorders , similarisorders . The effisorders . The OHFisorders . Generalisorders  in the snjection . Other wnjection . Some innjection . The virnjection . Furthernjection , which mnjection . In a stnjection , meningonjection . In conthe liver . Direct he liver . There hhe liver . Currenthe liver . Based ohe liver . Generalhe liver .5.via H. spinigera bites. The KFDV transmission cycle involves multiple Haemaphysalis species, notably H. spinigera, some Ixodes species, and hosts, such as bats, birds, monkeys, rodents, and shrews . Humans are merely incidental hosts for the KFDV and play no role in disease transmission , bats, black-faced langurs, bonnet macaques, grey langurs, ground-nesting birds, Indian crested porcupines, cattle, and gerbils are all susceptible to the KFDV infection . Infecte6.Reovirales order, Spinareoviridae family, and Coltivirus genus comprises a 12-segmented dsRNA genome (D. andersoni) bites (D. andersoni (The CTFV member of the A genome . The CTFA genome . CTF is i) bites . Ticks bndersoni .6.1.D. albopictus, D. arumapertus, D. andersoni, D. occidentalis, Haemaphysalis leporispalustris, Otobius lagophilus, I. sculptus, and I. spinipalpis may also transmit the CTFV to animals   confirmed cases, with an overall of 27 deaths (14 suspected and 13 PCR-confirmed cases). These reported cases are significantly higher than the 33 PCR-confirmed reported cases in 2021. Cases have been reported in several areas of Iraq, and the outbreak may put additional strain on an already overburdened healthcare system. Most confirmed cases had a history of direct contact with animals, including butchers and livestock breeders. Over half of the confirmed cases were among males aged 15–44 years (n = 176) and found that CCHFV-specific antibodies were detected in 17 sera, indicating CCHFV circulation in the westernmost region of the Balkans, Bosnia and Herzegovina, which raises concerns about CCHFV transmission out of this region (CCHF transmitted  Arabia) , Africa  Arabia) , and Eur Arabia) . In huma Arabia) . Later,  Arabia) , raising Arabia) . These f Arabia)  and Maur Arabia) , suggest Arabia) . Later,  Arabia) . Most of Arabia) . Apart f44 years . Travele44 years , France 44 years , Germany44 years , and a t44 years . In Iran44 years . In 199844 years . Regardi44 years . ELISA a44 years . In addi44 years . A recens region . A crosss region .7.2.via which pathogens mediate vertebrate host inflammatory and immune responses , etc., poses threats to public health. The genus Bandavirus contains viruses that comprise three segments, i.e., small (S ~ 1.7 kb in size), medium (M ~ 3.2 kb in size), and large (L ~ 6.4 kb in size) (via the plasma membrane (The genus in size) . The M sin size) . The S smembrane .8.1.A. americanum) was first reported in northwestern Missouri in 2009  on their bodies 2 weeks before becoming ill . For example, two HRTV-infected farmers in northwestern Missouri developed ehrlichiosis-like mild fever, leukopenia, and thrombocytopenia  to track the emergence of TBVs. Surveillance programs should also identify relevant animals or mammals that help the tick vectors complete their life cycle. For example, birds and bats have gained attention due to their role in spreading ticks and their associated viruses during seasonal migration . Humans TS and XX conceptualized the study. QL conducted the database search. TS, XX, BW, and ZB narratively screened the databases, then extracted their data and prepared the initial draft of the manuscript. TS prepared the final draft of the manuscript. XX supervised the project and critically assessed the manuscript. All authors contributed to the article and approved the submitted version.This study was supported by the Yunnan Key R&D Program (No. 202103AQ100001) and Yunnan Major Scientific and Technological Project (No. 202202AG050013).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


We describe a 4-year-old male patient in Ohio, USA, who had encephalitis caused by Powassan virus lineage 2. Virus was detected by using metagenomic next-generation sequencing and confirmed with IgM and plaque reduction neutralization assays. Clinicians should recognize changing epidemiology of tickborne viruses to enhance encephalitis diagnosis and management. Powassan virus (POWV) is a tickborne flavivirus that causes encephalitis with severe neurologic sequelae  was negative  was collected by lumbar puncture, revealing a leukocyte count of 44 cells/μL (reference range <10 cells/μL) of which 85% were lymphocytes; glucose and protein levels were normal. The patient’s BIOFIRE FILMARRAY Meningitis/Encephalitis PCR panel . Leading diagnoses were autoimmune encephalitis and acute necrotizing encephalopathy of childhood (ANEC). The patient exhibited severe encephalopathy, nystagmus, right hemiparesis, and diffuse hypertonia. He was treated with high dose methylprednisolone, plasmapheresis, and intravenous immunoglobulins. NS3 gene (https://www.cdc.gov/ncezid/dvbd/specimensub), which showed IgM against POWV (capture ELISA), negative POWV-specific PCR, and a positive plaque reduction neutralization test at 1:8 dilution (reference range <1:2), confirming the final diagnosis was ANEC caused by POWV. The patient continued having severe neurologic sequalae requiring a tracheostomy, gastrostomy tube, and inpatient rehabilitation. At follow-up 1 year after admission, he had been decannulated, was able to orally ingest liquids and solids, and ambulated independently, but he had substantial language and cognitive deficits. Genetic testing for familial ANEC type 1 was negative. We sent CSF obtained on hospital day 5 to the University of California San Francisco for metagenomic next-generation sequencing (mNGS), which detected a single 140-nt POWV sequence . We perfNS3 gene , panel BNS3 gene , panel C>3 reads spanning >3 regions of the viral genome , and other viral agents were negative, excluding alternative diagnoses. Our case highlights the ability of mNGS to identify rare or unexpected pathogens that cause encephalitis. Providers should recognize changing epidemiology of tickborne viruses, such as POWV, to enhance encephalitis diagnosis and management. When cases are identified, local public health departments should complete comprehensive entomological and epidemiologic studies to determine virus prevalence.Additional information for Powassan virus infection detected by metagenomic next-generation sequencing, Ohio, USA.


Orthopoxvirus, producing a febrile skin rash disease in humans. Monkeypox is zoonotic transmitted and transmitted from human to human in several ways. Even though this disease is self-limited, it creates important community health worries due to its inconvenience and widespread complications. Herein, we discussed the up-to-date current situation of monkeypox regarding its epidemiology, clinical manifestations, current in-use therapeutics, necessary protective measures, and response to potential occurrences considering the recent pandemic. Also, in this review, a comparative genomic analysis of the recent circulating strains that have been recovered from various countries including, Egypt, USA, Spain, Japan and South Africa has been investigated.The 2022 monkeypox outbreak has created a new global health threat and pandemic. Monkeypox virus is a descendant of the genus  The recent monkeypox outbreaks in 2022 have created a new global health threat because of their high number of cases and the speed of infection. Infection of monkeypox is characterized by a febrile rash disease in humans and characteristic skin lesions. Monkeypox is transmitted to humans via various routes including, direct contact with infected humans or animals, or vial droplet means. Although, the disease is self-limited; however, it can cause important public health consequences, particularly in pediatric, immunocompromised individuals and during pregnancy. This review introduces the monkeypox illness epidemiology, clinical manifestations, genomic mutation of the circulating viral strains, and management. Viruses are still responsible for large numbers of new cases of medically significant recurrent infections and widespread sets of various and serious pathological conditions affecting both humans and animals . VirusesOrthopoxvirus, where four viruses, have been reported to cause human infections and these including the monkeypox virus, vaccinia virus, cowpox virus, and the eradicated variola virus [Orthopoxvirus genus is a member of the family; Poxviridae, the large, enveloped viruses, and their genome comprises linear, double-stranded DNA [Orthopoxviruses have plastic genomes characterized by a central region occupied by core genes and peripheral regions with variable gene content and carrying “accessory” genes that mainly encode proteins involved in host–virus interactions [Monkeypox is a descendant of the genus la virus . Orthoponded DNA . The genractions . Analysiractions . Around ractions . Monkeypractions . Monkeypractions . Clade 1ractions . Moreoveractions  and genoractions . The Wesractions . Unlike ractions .www.who.int/emergencies/disease-outbreak-news (accessed on 06 June 2023). On the other hand, the WHO unveiled a new episode in May 2022, leading to a pattern shift with over 780 confirmed cases from 27 nonendemic areas of the monkeypox virus and no history of travel to endemic regions [Although smallpox which is caused by the variola virus, is the only known human viral disease without an animal reservoir, the monkeypox virus has a broad variety of authorized animal reservoirs . Human m regions .www.ncbi.nlm.nih.gov/nuccore/OP597769.1/ (accessed March 2023) was compared with homologous complete genomic DNA sequences of the USA “NCBI accession code, OP752103.1” www.ncbi.nlm.nih.gov/nuccore/OP752103.1/ (accessed March 2023), Japan “NCBI accession code, LC722946.1” www.ncbi.nlm.nih.gov/nuccore/LC722946.1 (accessed March 2023), Spain “NCBI accession code, ON838939.1” www.ncbi.nlm.nih.gov/nuccore/ON838939.1 (accessed March 2023) and South Africa “NCBI accession code, ON918611.1” www.ncbi.nlm.nih.gov/nuccore/ON918611.1 (accessed March 2023) using Multiple Alignment using Fast Fourier Transform . The Egyptian monkeypox isolate was coded hMpxV/Egypt/MOH-NRC-0002/2022, and was collected by the Egyptian Ministry of Health on 26 September 2022 from infected humans (Homo Sapiens) and the complete genome (197201 bp) was deposited and released to the public on the NCBI GenBank database on 7 October 2022. The respective multiple sequence alignments of the respective genomes were carried out to highlight the nonsynonymous single nucleotide polymorphisms (SNPs) and detect the reflected missense or non-sense SNPs of the coded proteins. We have aligned several sequences of several circulating strains for each country  and we found that almost all the deposited sequences in the NCBI GenBank database were almost identical on the nucleotide levels (98–100% identity) with no missense or nonsense mutations could be detected. The result of this analysis and the complete list of the accompanied changes is displayed in The monkeypox complete genome DNA sequence of the Egypt strain “NCBI accession code, OP597769.1”  is applied to treat serious side effects of resulted from vaccinia vaccination, it is recommended for the therapy of monkeypox during recent epidemics . VIGIV iOrthopoxvirus are characterized by their genomic stability, however, based on the complete genome sequences of the currently circulating monkeypox strain there is evidence of 34 nonsynonymous missense SNPs of the monkeypox strain isolated in 2022 as compared with the USA, Spain, Japan and South Africa strains. Whether these SNPs are associated with the magnitude of the viral pathogenicity, the spread of infection as well as in protection efficiency of the commercially available vaccines still has to be clinically investigated.The monkeypox disease rises in non-endemic locations indicating that the pathogens are not limited to geographical borders. The current monkeypox outbreak besides the COVID-19 pandemic has become another danger and increased fear among the population worldwide. Therefore, a global health framework should develop compelling strategies as well as strict protective and control measures should be undertaken to alleviate the spread of viral infection. The monkeypox uprising has been a focal point of regard for researchers, clinicians, epidemiologists, and policy pioneers. Nonetheless, priority ought to be given to control efforts that should depend on upgraded case finding, contact tracing, isolation, good hygiene practices, and immunization. Although the viral members of genus Several necessary protective measures and responses to potential occurrences in light of the recent monkeypox pandemic should be strictly implemented to contain this newly emerged vial pandemic. Although this virus is characterized by a genetically stable genomic DNA however, there is evidence of newly emerged mutation resulting in nonsynonymous missense single nucleotide polymorphism (SNP) in 34 ORFs (19.1%) out of the 178 ORFs of the complete genome in the genomic DNA of the monkeypox strain that was recently isolated in Egypt. the complete genome of the USA, Spain, Japan, and South Africa strains showed no SNPs when they were compared with each other. The SNPs detected in each ORF were either one, two, three, or four missense SNPs, with a percentage of, 79.4%, 11.8%, 8.8%, and 2.9%, respectively. Whether these SNPs will be associated with the pathogenicity and spread of infection as well as in the protection efficiency of the commercially available vaccines still has to be investigated and biochemically identified in the future.Viruses are still emerging and responsible for many new cases of medically significant recurrent infections and widespread sets of human and animal infectious diseases worldwide.Orthopoxvirus, producing a febrile rash disease.The 2022 monkeypox outbreak has created a new global health threat and pandemic. Monkeypox virus is a descendant of the genus Monkeypox is zoonotic transmitted and transmitted from human to human in several ways.Comparative genomic analysis of the monkeypox strains that have been recently isolated in the USA, Spain, Japan, and South Africa showed no detected nonsynonymous SNPs when they were compared with each other. However, the SNPs were determined in 34 open reading frames (ORFs) of the complete genome of the Egyptian strain out of the 178 ORFs (19.1%) of the complete genome when compared with the recently isolated USA strain in 2022.The SNPs detected in each ORF were either one, two, three, or four missense SNPs, with a percentage of, 79.4%, 11.8%, 8.8%, and 2.9%, respectively.Whether these SNPs are associated with the pathogenicity, spread of infection as well as in protection efficiency of the commercially available vaccines still has to be investigated and biochemically identified.Diagnostic tests are critical in confirming monkeypox infection and must be correlated with clinical and epidemiological information.Both conventional PCR and RT-PCR tests can be used to confirm the suspected case of monkeypox and to evaluate the viral burden in blood, respectively.Morphological identification of the poxviruses can be done using the electron microscope from a tissue biopsy or vesicular exudate.Immunochemical examinations are performed for the existence of specific antigens of the Orthopoxvirus.The monkeypox immunoglobulins IgG and IgM serology tests on a blood sample to identify recent or past exposure.Western blot analysis of certain viral proteins can be used to confirm the diagnosis.The smallpox vaccine is predicted to provide partial immunity against monkeypox infection due to their genetic similarities and had shown up to 85% cross-protection.The live attenuated vaccines including, ACAM2000, Modified Vaccinia Ankara, and LC16m8 are new-generation smallpox vaccines with improved safety profiles compared with first and second-generation vaccines and arouse satisfactory antibody production.To control the current outbreak of the monkeypox virus, health sectors have appointed policies to dispense smallpox vaccines for healthcare practitioners.Modified vaccinia Ankara-Bavarian Nordic strain (MVA-BN), a third-generation smallpox vaccine was permitted in 2019 against monkeypox.On May 24, 2022, the US CDC decided to commute some of their MVA-BN, for persons at high risk of coming in contact with monkeypox virus.A non-replicating (MVN-BN) and minimally replicating (LC16) vaccines are recommended for children, and pregnant and breastfeeding women.There is currently no particular treatment for monkeypox virus infections however CDC advised the use of anti-smallpox drugs to be used to treat monkeypox disease.Tecovirimat (ST-246) is available orally (200 mg capsule) and was authorized by the Food and drug administration (FDA) to cure human smallpox and monkeypox in adults and pediatric patients.Both cidofovir and brincidofovir act by inhibiting the viral DNA polymerase and are also evolved to treat smallpox and monkeypox.Serious side effects due to vaccinia vaccination, such as eczema vaccinium, severe generalized vaccinia, and vaccinia virus-induced infections can be managed by using Vaccinia Immune Globulin Intravenous (VIGIV).


Ixodes persulcatus ticks were positive for viral RNA. Clinicians should be aware of this potential health threat and include this emerging virus in the differential diagnosis for tick-bitten patients in this region.We identified Yezo virus infection in a febrile patient who had a tick bite in northeastern China, where 0.5% of  Tickborne orthonairoviruses have been considered a major public health threat worldwide (Yezo virus (YEZV), a new tickborne orthonairovirus discovered in Japan in 2021, can cause acute febrile illness in humans, whose clinical symptoms include thrombocytopenia and leukopenia  for viral detection by using reverse transcription PCR  Table 1.Ixodes persulcatus. At that time, no obvious clinical symptoms, such as rash, itching, and discomfort, occurred. However, fever developed, followed by light headache, dizziness, blurred vision, chest distress, shortness of breath, fatigue, and arthralgia in 1 week  and platelet counts  were within reference ranges. Serum levels of liver aminotransferases were slightly increased   Table 2.The patient was hospitalized for 8 days. Headache, dizziness, and arthralgia continued throughout the hospitalization, whereas the clinical signs of fever, blurred vision, chest distress, shortness of breath, and fatigue were relieved or disappeared . LymphopA blood sample was collected at admission and was negative for the tick-borne pathogens that have been identified in northeastern China . The patHaemaphysalis japonica, 431 H. concinna, 1,110 Dermacentor silvarum, and 1,075 I. persulcatus)   . YEZV RN2%–1.0%) . The preI. persulcatus ticks) by using specific primers  in tick-bitten patients in northeastern China, compared with YEZV patients in Japan (5/248). To date, no severe YEZV-infected patient has been reported, and the patients in the 2 countries recovered completely. Thus, active surveillance should be performed on the tick-bitten populations to evaluate the prevalence and clinical characteristics of YEZV infection in the studied regions.H. megaspinosa, I. ovatus, and I. persulcatus ticks in Hokkaido, Japan, showing a prevalence of 0.0%–5.7% , and other tick species, such as H. japonica, H. concinna, and D. silvarum, were negative for YEZV RNA. Those results indicate that I. persulcatus ticks might serve as a potential vector for YEZV in northeastern China.YEZV RNA has been detected in I. persulcatus tick bites in northeastern China. Phylogenetic analysis confirmed the association between febrile illness and the virus. To date, there are >8 pathogenetic tickborne viruses in humans and animals found in northeastern China: tickborne encephalitis virus (YZEV was identified in a tick-bitten patient who had febrile illness and Additional information on Yezo virus infection in tick-bitten patient and ticks, northeastern China.


Phytophthora sojae, an oomycete pathogen that causes root and stem rot in soybean. Meanwhile, PsRLK6 is recognized as a novel type of MAMP by an unknown plant LRR receptor-like protein and triggers immune responses in soybean, tomato, and Nicotiana benthamiana. The findings reveal dual roles of a pathogen LRR-RLK in determining both life through sexual reproduction and death through triggering plant immunity.Leucine-rich repeat receptor-like kinases (LRR-RLKs) are well known to act in plant growth, development, and defense responses. Plant LRR-RLKs locate on cell surface to sense and initiate responsive signals to a variety of extracellular stimuli, such as microbe-associated molecular patterns (MAMPs) released from microorganisms. LRR-RLKs are also present in microbes and function in microbial growth and development, but their roles in communicating with hosts are largely unknown. A recent study published in Nature Communications uncovered that a microbial LRR-RLK, PsRLK6, is required for oospore development in the sexual reproduction of  Phytophthora sojae, is detected as a MAMP by a Nicotiana LRR-RLP RXEG1  are recognized by pattern recognition receptors (PRRs) in plants, such as leucine-rich repeat receptor-like kinases (LRR-RLKs) and receptor-like proteins (LRR-RLPs), triggering plant immunity. MAMPs are known to be a variety of molecules secreted or released from bacteria, fungi, and oomycetes, including oligosaccharides, peptides, peptidoglycans, oligogalacturonides, and lipids . Therefore, PsRLK6ECD was selected for further investigation. Purified PsRLK6ECD protein triggered other PTI (pattern-triggered immunity) responses in N. benthamiana, including phosphorylation of MAPK and upregulation of PR1a and CYP71D20, and consistently improved resistance to P. capsici. PsRLK6ECD-induced immune responses and P. capsici resistance were significantly compromised in BAK1-silenced or sobir1 knockout N. benthamiana plants, indicating that BAK1 and SOBIR1 are required for PsRLK6ECD-triggered plant immunity. PsRLK6ECD also induced ROS production, PR1a expression, and disease resistance in soybean and tomato, but not in Arabidopsis. To identify the critical region of PsRLK6 for eliciting plant immunity, Pei et al. used AlphaFold 2 to predict the 3D structure of PsRLK6ECD. Their data indicate that PsRLK6ECD has a signal peptide domain, an LRR capping domain, seven atypical LRR domains, and an LRR C-terminal domain. By constructing a series of truncated mutant variants, Pei et al. identified that LRR5-6 is sufficient for inducing ROS accumulation and P. capsici resistance in N. benthamiana. Purified LRR5-6 peptide also induced ROS burst in soybean and tomato. Therefore, two LRR domains of PsRLK6ECD are critical for its elicitor activity. Phylogenetic analysis showed that 53 PsRLK6 homologs were found in different oomycete species, but not in tested fungi, bacteria, and plants. The ECDs of PsRLK6 homologs from Phytophthora infestans, Phytophthora capsici, Phytophthora parasitica, Phytophthora nicotianae, Phytophthora cactorum, and Pythium oligandrum also induced H2O2 accumulation and P. capsici resistance in N. benthamiana leaves, suggesting that the elicitor activity of RLK6 is conserved in oomycetes.Pei and colleagues expressed the extracellular domains (ECDs) of all 24 PsRLKs from P. sojae-soybean interaction, Pei et al. monitored the immune responses of soybean challenged with knockout mutant, overexpression, or wild-type P. sojae strains. Their data revealed that the overexpression strain induced higher ROS burst, PR1a expression, and MAPK activation than the wild-type and knockout mutant. In consistence, the virulence of the overexpression strain was severely compromised. However, the virulence of the knockout mutant was not affected compared to the wild-type. Then, what is the biological significance of PsRLK6 for P. sojae? As Pei et al. observed no obvious phenotypes of the knockout mutant ΔPsRLK6 in filamentous growth and zoospore development, they further checked the role of PsRLK6 during the sexual reproduction of P. sojae. Pei et al. found that the production of oospores, which act as an important inoculum for the next growing season (Tyler ΔPsRLK6 mutant compared to the wild-type when cultured on lima bean agar medium or during infection with soybean. By contrast, oospore production was increased in the overexpression strains. Consistent with these results, the expression level of PsRLK6 was significantly upregulated during oospore maturation on lima bean agar medium and in the late stages of infection on soybean roots. Altogether, PsRLK6 is required for oospore development in P. sojae.To determine the role of PsRLK6 during on Tyler , was conP. sojae by enhancing the production of oospores, which is indicative of a reproductive or 'life' strategy as employed by plant RLKs during plant fertilization (Berger P. sojae. The findings of this study are novel in that a receptor in pathogen is recognized by another receptor in plant, establishing a new type of MAMP that mediates the cell-to-cell communication between the pathogen and the plant. Unlike other MAMPs, which typically consist of secreted virulence factors or cell wall components (Yu et al. P. sojae. PsRLK6 is a transmembrane protein and its ECD may be cleaved and released into the plant apoplast where pathogen-plant communications might occur. The PsRLK6 ECD may be released by a controlled proteolysis process and sensed by a plant LRR-RLP, which deserve investigation in future studies. It would also be intriguing to investigate the mechanisms by which P. sojae coordinates PsRLK6-mediated sexual development and evasion of host immunity triggered by PsRLK6.In summary Fig. , Pei et n Berger . Additio


Hemophilia A (HA) is a rare, inherited, serious bleeding disorder characterized by a deficiency of blood clotting factor VIII (FVIII). HA is associated with considerable economic burden.To identify, review, and summarize published studies on the health care resource use and costs of managing HA in the United States using a targeted literature review.A comprehensive and targeted literature search was conducted in Embase, MEDLINE, and Cochrane Database of Systematic Reviews covering the period 2010 to 2022. We supplemented the search by searching gray literature . Eligibility criteria were developed based on the population, interventions, comparators, and outcomes framework. For comparability, costs were adjusted to 2021 US dollars.A total of 40 publications, including 17 full-text papers, 21 abstracts, and 2 Institute for Clinical and Economic Review reports, met eligibility criteria. Total annual health care costs per patient ranged from $213,874 to $869,940 and are mainly driven by the cost and intensity of prophylaxis with FVIII replacement concentrates, bypassing agents, and, most recently, emicizumab. Generally, we observed substantial heterogeneity in estimated treatment costs for HA, which varied depending on HA severity, treatment type and intensity, age, weight, and inhibitor status. Patients with inhibitors incurred much higher costs, but annual FVIII treatment costs are increasing over time among a subset of adult patients without inhibitors. Only 2 studies reported indirect costs; these were $13,220 and $27,978 annually among patients without and with inhibitors, respectively. Parents of children with HA spent $8,252 on non–mental health services and $258 on mental health services annually.The annual health care costs of managing HA are substantial and vary widely, depending on the study population definitions and intensity of prophylaxis. Total health care costs are dominated by the cost of prophylaxis. Indirect costs are also important. More robust studies in various settings, subpopulations, and assessing the impact of emerging therapies are required to fully elucidate the changing societal and economic impact, particularly regarding indirect costs and productivity loss for individuals living with HA. Hemophilia A (HA) is a rare genetic condition affecting 32,000 male patients in the United States. Plasma-derived and recombinant clotting factors reflect standard of care. Extended half-life products, newly approved nonfactor agents , and gene therapies in development are contributing to changes in care management. With new therapies, the impact on costs needs to be better understood. We have summarized the published literature on the substantial costs and resource use of managing HA in the United States. Implications for managed care pharmacyThe cost of managing HA is substantial and varies by disease severity, treatment type and intensity, age, weight, and inhibitor status. This study provides an in-depth analysis of current health care costs and resource utilization, providing a useful benchmark to understand the comparative cost-effectiveness of future therapies that may alter the costs of managing HA . This will have implications for clinical and coverage policies that guide managed care pharmacy benefits.1Congenital hemophilia A (HA) is a rare, inherited, X-linked coagulation disorder characterized by a deficiency of blood clotting factor (F)VIII. People living with HA demonstrate different health care resource use (HCRU) than those with hemophilia B (HB), requiring separate evaluations. Genetic assessment, coagulation tests, and factor assessments now make it possible to quickly and efficiently diagnose hemophilia, differentiate genotype, and predict the risk of inhibitor development.2 Patients with HA may experience chronic joint diseases from repeated bleeding into the joints, neurological damage, and damage to other organ systems.Approximately 85% of people diagnosed with hemophilia have type A and most are male. Roughly 400 new cases of HA are diagnosed annually in the United States. Approximately 50%-60% of all patients with HA have severe disease , are diagnosed within the first year of life, and present with clinically significant bleeding-related complications.World Federation of Hemophilia Guidelines for the Management of Hemophilia, the use of prophylaxis, or regular administration of therapeutic products aimed at maintaining hemostasis to prevent bleeding, is always recommended for people with severe disease (and for some with moderate disease) over episodic replacement therapy at the time of a bleed, although “on-demand” treatment is additionally recommended to treat a breakthrough hemorrhage.3 Access to innovative therapies allows patients with HA to live as long as individuals without HA. A new generation of extended half-life (EHL) clotting factor concentrates and hemostasis rebalancing agents  have shown benefit in preventing bleeding episodes and maintaining hemostasis with longer treatment intervals than standard half-life (SHL) products, without loss of efficacy, reducing the need for on-demand blood products.5 FDA approval of emicizumab in 2017 has provided an additional treatment option demonstrating lower annualized bleeding rates in patients with HA, thus substantially reducing the disease burden for patients with and without inhibitors and impacting the utilization and costs of other agents and health care resources.8 On the near horizon are gene therapies that may provide a functional cure for HA.10The goal of treatment is to prevent bleeding and joint damage and empower patients to practice self-management. Per the 15 Peyvandi et al reviewed earlier studies on quality of life and cost outcomes for patients with mild HA, which excluded patients with moderate and severe HA.16 Patients with more severe disease often require more intense therapy and medical attention. Because of the wide array of available treatment strategies and data sources, these costs can vary. The objective of this targeted literature review was to identify, review, and summarize the published literature on the costs of managing congenital HA and to show the variation in resource use and costs.The medical and pharmaceutical costs of treating and preventing bleeding episodes in patients with HA have previously been evaluated in various data sources  to be substantial.Supplementary Tables 1 and 2, available in online article).A targeted literature review was performed to identify published evidence on medical resource use and health care costs (direct and indirect costs) for the treatment and management of patients with HA. We defined the search strategy, study inclusion and exclusion criteria, and outcomes of interest in a prespecified protocol (Supplementary Tables 3-5). We included any retrospective or prospective studies conducted on patients with HA in the United States that reported costs and/or health care resource use in a real-world setting. The population of interest was all patients with congenital HA in the United States, including all ages and severities, regardless of inhibitor status or therapy type. Studies were excluded if patients with congenital HA were mixed with other patients with hemophilia . Clinical trials and economic models were excluded if they were not the original source of the resource use and cost data.Eligibility criteria were developed based on the population, interventions, comparators, and outcomes framework  to ensure that any new publications in the intervening months were included. We searched relevant abstracts, posters, and presentations of relevant scientific conferences from the past 6 years (2016 to 2022). A manual search of reference lists and the Institute for Clinical and Economic Review website was also completed to identify additional studies. Abstracts were excluded as duplicates if the full-text paper was also identified in the search.A single reviewer completed study screening through title and abstract review based on the predefined inclusion and exclusion criteria. Full-text review and data extraction on 20% of the included studies was conducted by 2 of the authors (Ms Chen and Dr Cheng) for standardization and calibration using an extraction form with predefined outcomes of interest. The remaining studies were reviewed and extracted by a single author. The second reviewer checked for accuracy and completeness. Discrepancies were resolved through discussion.17 If no currency year was reported in the study, the latest year of the study period was assumed for the base year . Cost outcomes were summarized into different categories: aggregated health care costs, drug costs, outpatient and emergency department (ED) costs, inpatient costs, other costs , and indirect costs. Resource use outcomes included drug utilization, inpatient visit, ED visit, outpatient visit, indirect resource use (work or school absenteeism), other resource use , and bleeding rate.Costs were adjusted to 2021 US dollars using the 2021 Consumer Price Index in Medical Care.The initial electronic database search conducted on November 22, 2021, identified a total of 1,967 records across all sources, and another 30 records were identified through the manual search in other sources and repeated search in May 2022. After duplicate removal of 751 reports, 1,246 titles and abstracts were screened, and 82 studies were eligible for full-text review. A total of 42 records were excluded based on population, interventions, comparators, and outcomes criteria, which resulted in a total of 40 studies being included in the final analysis. The final list of publications comprised 17 full-text papers, 21 conference abstracts, and 2 Institute for Clinical and Economic Review reports and is reported in the PRISMA Diagram format .A total of 17 full-text publications were included in the review, with 11 studies published between 2017 and 2022 and 6 studies published earlier between 2012 and 2016. Separation of these publishing dates was done, as FDA approval of emicizumab in 2017 may have triggered more studies looking at costs and resource use after 2017. Most of the full-text publications (n = 15) included patients with HA across severities. The remaining 2 studies focused on adult patients with severe and moderate to severe HA, respectively. The real-world studies adopted a variety of data sources, including MarketScan research database (n = 6), Optum research database (n = 3), chart review (n = 2), American Thrombosis and Hemostasis Network (ATHN) Registry (n = 2), Humana research database (n = 1), US Department of Defense database (n = 1), Premier inpatient database (n = 1), and survey questionnaire administered to patients and caregivers (n = 1). Of the 17 included full-text publications, the majority (n = 12) reported costs and HCRU data. Two studies only reported costs data and 2 studies only reported resource use data. Indirect costs and HCRU were assessed in 2 publications .Supplementary Table 6). The highest total annual health care costs were incurred by patients with HA and inhibitors who were treated with bypassing agents.18Among studies reporting total annual health care expenditures of managing HA, the total health care costs per patient averaged from $213,874 to $869,940 , EHL products , plasma-derived products , and emicizumab . In addition, 2 studies conducted prior to the approval of emicizumab reported average annual treatment costs using bypassing agents stratified by age groups, which were estimated at $439,536 and $1,051,567 among pediatric patients covered by Medicaid and employer-sponsored insurance, respectively.21 Furthermore, there appears to be an increasing trend in total annual factor costs over time, although study populations across studies may still be heterogeneous regarding geographical distribution, payer types, and demographic characteristics because of different data sources.22After restricting the population to adult male patients with HA without inhibitors, we observed the highest annual FVIII treatment costs among patients who used EHL products in 2 studies  compared with other FVIII therapies such as SHL and plasma-derived products .12,21 Fu23 The annual outpatient costs averaged from $4,772 to $109,776 and from $828 to $3,929 for ED visits. The highest annual outpatient cost was incurred by patients who received bypassing therapy and used ambulatory services, which was 4 times higher than those who received factor replacement therapy .18 In contrast, the median annual outpatient costs were similar . Six studies reported inpatient costs, which averaged from $12,336 to $237,648.24Supplementary Table 6 summarizes the details of costs associated with treating bleeds, home health visits, and other medical costs in each study.Five studies recorded costs associated with outpatient and ED visits.25 Guh et al found 4.8 times higher mean annual health care expenditures comparing those with and without inhibitors in a population with employer-sponsored insurance  and a 3.6 times higher expenditure in the Medicaid-insured population .20 Only one study in pediatric patients reported on the use of emicizumab, which is now the recommended standard of care for people with HA with an inhibitor.3 This study showed the total annual cost of prophylaxis among pediatric patients with inhibitors was $1,269,500 prior to using emicizumab and $337,910 after using emicizumab, a significant reduction in costs (P < 0.001). Among pediatric patients without inhibitors, the annual cost of prophylaxis was reduced from $466,880 to $363,362 following initiation of emicizumab (P = 0.009).A total of 11 studies reported total health care and drug costs for patients with HA by inhibitor status or conducted subgroup analyses among patients with or without inhibitors . All stu26 The average annual total indirect costs per patient were estimated at $13,220 and $27,978 among patients without and with inhibitors, respectively. Subgroup analysis results by disease severity and age groups are summarized in Supplementary Table 7. Kim et al estimated the impact of caring for children with HA on parents’ utilization of nonmental and mental health services using MarketScan commercial medical and pharmacy claims. They found higher mean cost for nonmental health services for parents of children with HA compared with control parents  (Supplementary Table 7).Only 2 publications assessed indirect HCRU and its associated costs. Zhou et al reported information on absenteeism and imputed indirect costs using the human capital approach, which used wages as a proxy measure of work time output.Supplementary Table 8), most studies reported more than 1 type (n = 10), which included utilization of FVIII concentrate (n = 6),27 emicizumab (n = 1),13 and bypassing agents (n = 3),24 admission to hospitals (n = 10),28 outpatient/physician office visits (n = 5),28 ED visits (n = 8),28 hemophilia treatment center (HTC) visits (n = 2),27 and home health visits (n = 2).22 Several studies reported clinical outcomes associated with hemophilia-related bleeding events, whereas 2 studies reported joint procedures and surgeries.29We found a range of results with substantial variability. Of the 14 studies with HCRU , compared with those with mild, moderate, or severe disease on prophylaxis.Health care costs for patients with HA are substantial and vary widely, depending on the study population definitions and the intensity of prophylaxis. Total health care costs are dominantly driven by the cost of prophylaxis , especially for those with severe disease on continuous prophylaxis. Studies conducted consistently demonstrate much higher costs for patients presenting with inhibitors compared with those without inhibitors. Overall, 74%-96% of total health care costs were attributed to the cost of treatment, which varied depending on the presence of inhibitors, disease severity, and treatment agents. Studies of adult patients with HA consistently showed higher product costs because currently used treatments employ weight-based dosing. Because different studies used various definitions of disease severity, it is difficult to compare cost estimates across studies. However, Zhou et al reported total direct medical costs, clotting factor costs, and indirect costs among patients without inhibitors stratified by disease severity, with the highest total direct costs incurred by those with severe HA and on prophylaxis treatment .13 Two studies examined the use of different prophylaxis products and compared prophylaxis treatment costs for moderate to severe and severe patients at HTCs using the ATHN dataset.21 Both studies found the highest costs from those being treated with EHL products. The longitudinal study by Croteau et al also found that EHL prescribing continues to rise nationally,11 which might explain our finding of potentially rising FVIII treatment costs over time. EHL products with longer half-lives require less frequent injections compared with SHL treatments and would consequently result in reduced prophylactic treatment burden for patients and caregivers. Nevertheless, how further growth in use of EHLs will be impacted by emerging non-factor replacements and gene therapies is unclear.Among patients without inhibitors, total costs and treatment costs varied by the type of treatment. One study found lower costs of prophylaxis and the total cost of bleeding events after initiating emicizumab.20 In addition, people with HA who develop an inhibitor are twice as likely to be hospitalized for a bleeding complication, which resulted in significantly higher inpatient costs than those without inhibitors.23As expected, the additional health care costs associated with inhibitors can be substantial for those who have them. Clotting factor costs, especially agents that bypass inhibitors (bypassing agents), accounted for the majority of the total costs because of the amount of treatment required to stop bleeding among those with high titer inhibitors. The average total annual health care expenditures varied across 3 claims-based retrospective studies  because of different insurance mix of the populations, with the lowest costs from those with Medicaid insurance and the highest costs from those with employer-sponsored insurance.30 They found that HA is also associated with considerable indirect costs for both pediatric and adult patients, mainly due to school absenteeism among children, functional disability of adult patients, and underemployment of parents of affected children. HA is a costly disorder, not only because of its high medical expenses but also because of the high indirect costs incurred. Differential data in terms of indirect costs and productivity loss among various key subpopulations may be beneficial to fully determine the burden of illness associated with HA.Two full-text publications and one conference abstract reported indirect costs among patients with HA.25The most frequently examined resource use outcomes include the following: (1) annual counts of hospital stays and office, ED, and home health visits, (2) proportion of patients with at least 1 of these visits, (3) length of inpatient stays, and (4) proportion of patients on each type of treatment. Less focus is on treatment duration and dosing patterns, as administrative claims data lack details that would provide information on timing of actual administration of FVIII concentrate, the prescribed dose, or the use of FVIII for continuous or intermittent prophylaxis. Although the presence of inhibitors is relatively uncommon (~25%-30% of patients with severe HA), studies have shown consistently higher rates of hospitalizations and ED visits.Data on patients with HA are commonly aggregated across disease severity and/or mixed with a hemophilia B population. Furthermore, most studies used a variety of outcome measures, scales, and time points, with very few studies using consistent formats. Because of the diversity in reporting, direct comparisons and meta-analyses were not possible, despite our efforts to extract outcomes data for comparable populations. However, this restriction to select only relevant data for populations of patients with hemophilia A facilitated the clean extraction and comparison of costs across published literature.Supplementary Table 9.Risk of bias was not formally assessed in the included studies given that the goal of the targeted literature review was to consolidate the existing evidence and not use it to conduct a meta-analysis. Additionally, results of the conference abstracts were not discussed given the scope of the study; however, data were extracted, and the results are included in 31 Disease severity and inhibitor status is not specified in the coding. Therefore, most studies reported outcomes in populations with various levels of disease severity, with inhibitor status assumed if bypassing agents were observed in the patients’ data. Thus, it is difficult to determine the costs of illness by hemophilia severity level and inhibitor status in studies using claims data. On the contrary, observational studies using data obtained from HTCs or electronic medical records have the advantage of containing richer clinical information.Conducting administrative claims data analyses in a population of patients with hemophilia is challenging because of the relatively small patient population and limitations of diagnostic coding. Using validated algorithms can improve specificity of patient identification.10 Using validated algorithms can improve patient identification.31Importantly, the definitions of prophylaxis vary across studies. For instance, one study defined 3 prophylaxis cohorts over a 12-month period (1) based on quantity of claims, requiring patients with 6 or more non-inpatient, non-ED FVIII claims with 15 or more days supply; (2) based on frequency of claims, requiring no supply gaps more than 60 days between dispensings; (3) based on days of coverage, requiring patients having at least 273 days supply. This study found that the highest costs come from the subgroup with the third definition of prophylaxis use, highlighting how different methods of identifying patients managed with FVIII prophylaxis influence the cost estimates observed.32 Furthermore, a few studies did not report currency year and/or adjust for medical inflation. In cases where a currency year was not reported, we adjusted for inflation using the last year of the study’s follow-up period. Together, these factors complicate the understanding of the economic burden of managing HA with emerging treatment options.Cost categories associated with managing HA were defined differently in some studies, thus requiring attention when interpreting and comparing study findings. For instance, some studies included medical and pharmacy costs in the calculation of FVIII prophylaxis treatment costs, whereas others only examined aggregate drug spending. Use of FVIII concentrate can be reflected on either medical claims or pharmacy claims, depending on each patient’s specific benefit design. Therefore, careful understanding of the data is needed to avoid double-counting or omitting certain treatment costs. Differences in cost estimates highlighted the importance of knowing the data granularity and cost benchmarks used. For example, the ATHN dataset lacks specific cost data . Therefore, such studies estimated annual costs of FVIII prophylaxis based on use and median wholesale acquisition cost, which excludes the confidential discount rates and rebates agreed between drug manufacturers and payers. However, pharmacy claims are often able to tease apart the average wholesale price, actual payment to the pharmacy , and the patient’s out-of-pocket cost for the medication.The economic burden of HA is substantial to the US payers and caregivers. Cost estimates vary widely, depending on the study population definitions, the intensity of prophylaxis, and the presence or absence of inhibitors. In all studies, total health care costs are dominantly driven by the cost of prophylaxis  especially for patients with inhibitors and noninhibitor patients with severe disease on continuous prophylaxis. Various subgroups add to the complexity of understanding the cost burden. More robust studies in various settings and subpopulations are crucial to fully elucidate the economic burden, particularly regarding indirect costs and productivity loss for patients with HA. Cost and resource studies should also incorporate the patient/caregiver/societal perspectives. As new treatments for HA emerge, future research examining resource use and costs is required to inform subsequent economic studies.


Acid tropical soils may become more productive when treated with agricultural lime, but optimal lime rates have yet to be determined in many tropical regions. In these regions, lime rates can be estimated with lime requirement models based on widely available soil data. We reviewed seven of these models and introduced a new model (LiTAS). We evaluated the models’ ability to predict the amount of lime needed to reach a target change in soil chemical properties with data from four soil incubation studies covering 31 soil types. Two foundational models, one targeting acidity saturation and the other targeting base saturation, were more accurate than the five models that were derived from them, while the LiTAS model was the most accurate. The models were used to estimate lime requirements for 303 African soil samples. We found large differences in the estimated lime rates depending on the target soil chemical property of the model. Therefore, an important first step in formulating liming recommendations is to clearly identify the soil property of interest and the target value that needs to be reached. While the LiTAS model can be useful for strategic research, more information on acidity-related problems other than aluminum toxicity is needed to comprehensively assess the benefits of liming. Soil acThe amount of lime required to adjust soil acidity depends on the soil, the target crop(s), and the liming materials used. In temperate regions, lime requirements are commonly estimated with locally-calibrated quick tests using buffer solutions . These tThis situation differs for many smallholder farmers in sub-Saharan Africa  and otheHere, we provide a comprehensive review of lime requirement models for tropical acid soils that can be used with readily available soil data. The remainder of the paper is organized into seven sections. In Section 2, we introduce key concepts related to estimating lime requirements that have been a source of confusion and inconsistency. Section 3 presents the materials and methods used. In Section 4, we describe and discuss seven published lime requirement models for tropical soils and introduce a new model named LiTAS. The models are grouped by their target soil properties, and their accuracy is evaluated with experimental data from soil incubation studies. Section 5 presents a case study in which we run all models for a dataset of 303 African soil samples. We show substantial differences between models in the estimated lime requirement for acid tropical soils. In Section 6, we discuss the implications of our findings, and we conclude the paper in Section 7.2(H2O) of 5.5 or less for most of the year (2 or 1 M KCl. Soil pH(CaCl2) is more stable against the seasonal changes in the electrolyte concentration of the soil solution that can affect pH(H2O) measurements ((KCl) is used to measure the pH while accounting for the exchangeable acidity of the soil ((H2O) is generally higher than pH(CaCl2) and pH(KCl) the soil . In acid pH(KCl) .2+, Mg2+, K+, and Na+) and the accumulation of exchangeable acidity (Al3+ and H+). The main problem with soil acidity in the tropics is not the low pH as such, but rather the associated aluminum (Al) toxicity that constrains crop growth  regions are inherently acid because intense weathering processes have resulted in the displacement and leaching of basic  exchangeable cations  of the soil occupied by exchangeable acid cations . In tropical soils (except in histosols), nearly all exchangeable acidity is exchangeable Al3+; thus, Al saturation approximates acidity saturation  but derived from exchangeable acidity measurements.The terms exchangeable acidity and exchangeable Alterature . Indeed,terature . Consequ2.1.22+ and Mg2+ deficiencies coexist with Al toxicity problems in many acidic soils . Moreover, some highly weathered acid soils can have very low ECEC and, thus, low exchangeable Ca2+ and Mg2+ but low acidity saturation, resulting in Ca and Mg deficiencies without Al toxicity problems (Caproblems . Therefoproblems . Organic2.1.3(V) is the sum of all exchangeable bases  divided by the Cation Exchange Capacity at pH 7 (CEC7). CEC7 differs from ECEC, especially in acid soils, where CEC7 ≫ ECEC. For ECEC, exchangeable acid cations (Al3+ and H+) are extracted with a neutral unbuffered salt solution. In contrast, a pH 7 buffer solution is used for CEC7, which extracts both exchangeable and non-exchangeable acidity , comprising the potential acidity. The magnitude of the potential acidity of the soil depends on the type and amount of clay and organic matter. Although there is some inverse parallelism between acidity saturation and base saturation, these terms are not complementary because they have different denominators .A higher “base saturation” is an alternative to a lower acidity saturation in setting a target for alleviating soil acidity problems . Base saContrary to Al toxicity and acidity saturation, there is no direct relation between base saturation and crop yields. Instead, a minimum base saturation threshold is defined such that, above it, no soil acidity problems are detected . Therefo2.1.4Most lime requirement methods used in temperate regions target soil pH by estimating the lime rate required to raise the pH to a specific level (6 to 6.5 for most crops and soils) with locally-calibrated models . In acidExchangeable acidity has a negative exponential association with soil pH . Very hi2.1.5Acid tropical soils usually have very low plant-available phosphorus because of the high P fixation capacity of Fe and Al oxides often found in these soils. Liming has the associated benefit of increasing P availability, which might result in significant yield responses, particularly when P fertilization is low . However2.2LR) are commonly expressed in charges per soil mass  or in the equivalent mass in tons  of pure calcium carbonate (CaCO3) per unit area in hectares (ha). To transform lime rates between charges per soil mass and calcium carbonate mass per area, soil bulk density (sbd) and liming depth (ld) are needed. Lime rates in t ha−1 and cmolc kg−1 are the same when sbd = 1 g cm−3 and ld = 20 cm. Thus, LR can be converted from charges per soil mass to calcium carbonate mass per area with Eq. sbd is expressed in g cm−3 and ld in cm.Lime rates  of the liming material to be applied. In addition, other models  to facilitate their use and evaluation. The R package, data, and scripts used for analysis in this paper are available on GitHub (https://github.com/cropmodels/limer).A literature review was conducted to identify lime requirement models that only require soil properties available in soil databases to estimate lime rates for acid tropical soils. The terms “acid*” AND “soil*” AND (“lim* requirement” OR “lim* recommendation” OR “lim* rate”) were used in the Web of Science and Google Scholar databases to screen and retrieve relevant literature and references therein. Methods that required additional soil tests to measure the soil’s buffering capacity e.g.,  and methECEC or acidity saturation . Therefore, these values might not reflect all the variability in the original data.The compiled data include strongly acidic to moderately acidic soils, four soil orders, and different tropical regions . We incly) and predicted (ŷ) lime rates was assessed with the root mean squared error (cr) agreement between observed (ent (rc) . We repeent (rc) . For theECEC, and 7CEC, in which exchangeable acidity was extracted with 1 M KCl and 7CEC measured in 1 M NH4OAc buffered at pH 7, ending with a total of 303 African soils. The origin of the selected soil samples is shown on a map in Lastly, we compared lime rate estimates from different models using soil data from the Africa Soil Profile Database AfSP, . The AfS44.1This section describes five published lime requirement models based on acidity saturation  and introduces a new model (LiTAS), which were evaluated with data from four soil incubation studies .4.1.1c kg−1) of 1.5 times the initial exchangeable acidity (cmolc kg−1) was enough to reduce the acidity saturation to at most 15%, which was considered to be a threshold below which most crops are not affected by acidity  and 2 for beans and other crops sensitive to acidity, including many vegetable and fruit crops  that received a lime rate of exactly 1.5 times the initial exchangeable acidity, only one ended with an acidity saturation >15%, but it was very close to that value (18%). Furthermore, all soil samples with a lime rate of at least twice the initial exchangeable acidity had a final acidity saturation of 6% or less. Hence, when the goal of liming is to reduce the acidity saturation to a level that does not affect crop growth, liming is only needed when the acidity saturation is above 15% (or 5% for sensitive crops). In such cases, lime rates of 1.5 (or two for sensitive crops) times the initial exchangeable acidity would suffice for most tropical soils.This simple model worked well for almost all the experimental data available from the four studies . Out of lf of 2 was recommended for most soil types, except for sandy soils (lf = 1) and clay soils  to estimate lime rates  is divided by 100 to change it to a fraction, and the subscript i indicates the initial and f the final values. The unit of LR, exch. acid, and ECEC is cmolc kg−1.To derive their formula, + and Al3+), Ca2+, and Mg2+ instead of ECEC because these were the cations measured by + and Na+, was considered negligible, as these are normally very low in acid soils. Thus, the sum of exchangeable acidity, Ca2+, and Mg2+ was considered equivalent to the ECEC. We present the formula using ECEC, noting that ECEC might not always include all cations but should always include the exchangeable Al3+, Ca2+, and Mg2+, as these are the most abundant cations in acid soils. If data on exchangeable K+ and Na+ are available, they might be included depending on which exchangeable cations were considered for the TAS.The original formula uses the sum of exchangeable acidity (Hlf) that equals 1.5 or 2 depending on the relation between initial exchangeable acidity, TAS, and ECEC. The authors defined the following rule: “factor 1.5 is replaced by 2 when the estimated liming requirement using the factor 1.5 is greater than the chemical lime equivalent of the exchangeable Al (acidity).” Thus:Since not all the applied lime reacts with the exchangeable acidity, the formula is multiplied by a lime factor  of 1.2 g cm−3 and a lime incorporation depth (ld) of 20 cm  and a unit conversion from cmolc kg−1 to t ha−1. Based on preliminary data from Sitiung, Indonesia, c of exchangeable acidity was neutralized per cmolc of CaCO3 and computed the lf as the inverse of that fraction (1/0.53 = 1.9). The accuracy of their model was slightly lower than that of the c kg−1 to tons of CaCO3 per ha, lf to 1.4, assuming sbd = 1 g cm−3 and ld = 15 cm  was developed to provide fertilizer (N and P) and liming recommendations for acid soils with nutrient problems . In NuMa, low ECEC per unit of clay) require almost twice the lime amount of soils with high clay activity  to neutralize the same amount of exchangeable acidity charges. In addition, they considered that reducing the acidity saturation below 19% requires an additional amount of lime equivalent to 10% of the ECEC per percentage point. The NuMaSS model predicts lime rates in tons per hectare by assuming ld = 15 cm and sbd = 1 g cm−3.This model uses different lime factors depending on the soil’s clay activity (effective cation exchange capacity of the soil’s clay fraction). According to its authors, soils with low clay activity (i.e.LR was transformed from t ha−1 to cmolc kg−1 (Eq. lf and lower LR) was assumed when data on soil clay content were not available , indicating that the second term of the formula for TAS < 19% should be revised or omitted. Unfortunately, the software is no longer available, and the data used to derive the formula are unavailable, so the model cannot be further scrutinized.The NuMaSS formula adds much complexity to the formula of 4.1.5X is the sum of the minimum quantity of exchangeable Ca and Mg required by the crop  model developed for the state of Minas Gerais, Brazil  also haslf , the Ca2+ stays in the cation exchange complex and becomes available for the crop . However, while more complicated, the derived models did not improve the prediction accuracy ASf, and ΔECEC is the change in the effective cation exchange capacity, which equals the difference between the increase in exchangeable bases (Δexch. bases) minus the neutralized exchangeable acidity (Δexch. acid). ΔECEC is usually positive (Δexch. bases > Δexch. acid) because ECEC increases at higher pH; thus, it increases with liming (Δh liming .(7)ASf(%fAS = TAS), fAS can be replaced with TAS in Eq. TAS becomes a function of the initial soil properties (iECEC and iexch. acid), the increase in exchangeable bases (Δexch. bases), and the exchangeable acidity neutralized (Δexch. acid). Therefore, to estimate the required LR to reach a given TAS, we need to find the association of Δexch. acid and Δexch. bases with LR so that the two former variables can be replaced for some function of LR in Eq. fAS ≥ 5%, these two associations can be modeled with a linear regression without intercept (P < 0.001).Considering that our goal is to make the final acidity saturation equal to the target acidity saturation , which is an inverse function of TAS  is the sum of exchangeable bases over 7CEC, expressed as a percentage , 7CEC can be distributed to tV and iV in Eq. Since 2+ (and Mg2+) positive charges from the lime become part of the exchangeable complex . It excludes soil samples with LR > ipot. acid because the increase in exchangeable bases cannot be greater than what the cation exchange complex can take. When LR ≤ 50% ipot. acid, there is almost a one-to-one association between the lime rate and the increase in exchangeable bases charges (Δexch. bases = LR × 0.95(±0.05) ∀ LR < 0.5 × ipot. acid, exch. bases = LR × 0.8(±0.03) ∀ 0.5 × ipot acid < LR < ipot acid, tV ≤ 50%, but it does not perform well at higher base saturation targets (lf) that depends on Vt could be considered for the model. For example, the lf could be 1.05 when Vt ≤ 50%  and then slightly increase as tV approaches 100%, with a maximum Vt of 1.25 .The base saturation model implicitly assumes that all Ca complex . Fig. 3B targets . Consequ4.34.3.1OM, g kg−1) or potential acidity (Eq. X) and lower than the potential acidity of the soil (ipot. acid). Thus, the estimated lime requirement results from a series of rules such that it selects the lowest LR from the four nonlinear models that is higher than X and lower than ipot. acid. When no model returns a lime rate higher than X, the estimated LR is X. If the selected LR (either from the models or X) is greater than the initial potential acidity of the soil, the estimated LR equals ipot. acid. This model always recommends liming because the Ca and Mg available in the soil are ignored when computing crop requirements, and it thus assumes that all Ca and Mg must be provided by liming. Therefore, the minimum lime rate is X (Ca and Mg crop requirements), except when X is higher than pot. acid, in which case LR = pot. acid.dity Eq. . It alsoLR from the nonlinear models instead of the average, it often underpredicts LR.The model parameters were calibrated with the same soil incubation study data from The 4.3.2One of the most straightforward and oldest methods to determine the lime required to raise the pH to a specific level is to take a soil sample and measure the quantity of base required to produce that pH change by a slow titration in the lab . The amo5TAS) and a 50% target base saturation (Vt) were defined for maize, and a 5% TAS and 70% Vt for groundnut. Although we refer to two specific crops, these are common critical acidity values of many other cereal and legume crops. Models targeting pH were not included because these models are location-specific and have low accuracy when extrapolated to other regions  and second two models with different targets .We computed lime requirements for 303 African soils of pH between 3.5 and 6.5  with the regions . Lime rac kg−1. In contrast, the LiTAS model estimated lower lime rates than other models on average and for most soils, with larger differences for the more sensitive crop  but comparable in magnitude (mean difference = 0.47 cmolc kg−1). The acidity saturation model predicted higher lime rates in soils with very low pH for the more tolerant crop  with greater accuracy than the original LiTAS was the only model based on a target acidity saturation  or, more likely, because of the incubation study per se . Experimental results have an error component, including systematic errors that are consistent within one experiment but differ between experiments, introducing statistical bias. This bias can be reduced with standardized procedures. However, lime incubation studies are not fully standardized and differ in the incubation time and temperature, liming materials, and water additions, among other variables . For inscubation . A more lf) is a continuous function of TAS. The lf depending on TAS and the initial acidity saturation, using a discontinuous rule with two fixed levels of lf. However, the proposed rule did not always improve accuracy, not even for Cochrane et al.’s own data. In the MG5 and NuMaSS methods, the lf depends on clay content or activity. Our review does not show evidence for a need to adjust the lf as a function of clay, despite the wide range of clay content and soils included in the four soil incubation studies used here. Adjusting the lf and lime rates by clay content might be a workaround to account for differences in soil bulk density when the method returns lime rates in tons per ha without directly including the soil bulk density in the formulas. Nevertheless, clay type and content could be considered in future corrections of the TAS method, particularly if there are high deviations in the association between lime rate and Δexch. acid and Δexch. bases.A novel feature of the LiTAS model is that the lime factor (It seems counterintuitive that while both the acidity saturation and base saturation models were highly accurate for their target, the lime requirements they predicted were sharply different. These differences highlight the importance of identifying the soil chemical property most associated with the crop yield response to liming. Tropical soils can have several acidity problems affecting crop growth . It migh6.2Lime requirement models can be useful for strategic research on potential lime use in tropical regions where liming is still rare and experimental evidence is scarce . These mECEC, could help the development of site-specific lime recommendations. In the meantime, farm-level lime requirement estimates need to be informed by locally measured soil properties and could also consider additional local soil-quality indicators, such as soil color, soil texture, or the presence of specific plant species (However, this does not mean that the quality of the readily available soil data used by the models reviewed here is sufficient for farm-level recommendations . Soil pH species . The soi7Liming can increase crop productivity in acid soils, but the lime rate required to achieve this is unknown for many tropical regions. While lime requirement models could be very useful, the proliferation of models introduces uncertainty about which model to use. We discussed the strengths and weaknesses of various lime requirement models that can be used with data readily available in spatial soil databases. We showed important differences in the amount of lime required according to these models, especially when considering different target soil chemical properties. LiTAS, the new acidity saturation model introduced here, is more accurate than all prior models across many acid tropical soils from different regions and can effectively estimate the lime rate required to lower the acidity saturation to a specific target. This model could be incorporated into more comprehensive models once lime rates needed for other acidity problems are well established.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.