Dipeptidyl peptidase III (DPPIII) member of M49 peptidase family is a zinc-dependent metallopeptidase that cleaves dipeptides sequentially from the N-terminus of its substrates. In Leishmania, DPPIII, was reported with other peptidases to play a significant role in parasites’ growth and survival. In a previous study, we used a coding sequence annotated as DPPIII to develop and evaluate a PCR assay that is specific to dermotropic Old World (OW) Leishmania species. Thus, our objective was to further assess use of this gene for Leishmania species identification and for phylogeny, and thus for diagnostic and molecular epidemiology studies of Old World Leishmania species.DDPIII genes were searched in all Leishmania genomes and aligned to design PCR primers and identify relevant restriction enzymes. A PCR assays was developed and seventy-two Leishmania fragment sequences were analyzed using MEGA X genetics software to infer evolution and phylogenetic relationships of studied species and strains. A PCR-RFLP scheme was also designed and tested on 58 OW Leishmania strains belonging to 8 Leishmania species and evaluated on 75 human clinical skin samples.Orthologous DPPIII importance for parasite survival. Inter- and intra-specific genetic diversity was used to develop universal amplification of a 662bp fragment. Sequence analyses and phylogenies confirmed occurrence of 6 clusters congruent to L. major, L. tropica, L. aethiopica, L. arabica, L. turanica, L. tarentolae species, and one to the L. infantum and L. donovani species complex.Sequence analysis showed 478 variable sites (302 being parsimony informative). Test of natural selection (dN-dS)  inferred a negative selection, characteristic of essential genes, corroborating the Leishmania species identification was designed using double digestions with HaeIII and KpnI and with SacI and PvuII endonucleases. Overall, this PCR-RFLP yielded distinct profiles for each of the species L. major, L. tropica, L. aethiopica, L. arabica and L. turanica and the L. (Sauroleishmania) L. tarentolae. The species L. donovani, and L. infantum shared the same profile except for strains of Indian origin. When tested on clinical samples, the DPPIII PCR showed sensitivities of 82.22% when compared to direct examination and was able to identify 84.78% of the positive samples.A PCR-RFLP algorithm for DPPIII gene is suitable to detect and identify Leishmania species and to complement other molecular methods for leishmaniases diagnosis and epidemiology. Thus, it can contribute to evidence-based disease control and surveillance.The study demonstrates that  Leishmania species. Leishmaniases are epidemiologically complex diseases. Their complexity relates to the diversity of species and transmission cycle components, often poorly elucidated. Risk factors for changing eco-epidemiological profiles include environmental and climate changes, migrations and conflicts, contributing to leishmaniases emergence in disease-free areas, leading to co-sympatry of the major pathogens in the same transmission areas and even foci. Taxonomical identification of the parasites is a central issue for patient management, molecular epidemiology and thus disease control and surveillance. Thus, development of adequate specific and sensitive DNA tools to identify and classify Leishmania parasites for surveillance and control remains a research priority. The DPPIII gene is an essential gene conserved in all Leishmania species. By comparative sequence analyses of the DPPIII gene fragments and the development of DPPIII-based PCR RFLP assays, we showed its potential for molecular diagnosis, epidemiology and taxonomy. We conclude that this molecular marker can be used to complement other molecular methods for OW Leishmania species identification and discrimination and can contribute to evidence- based disease control and surveillance.Leishmaniases correspond to a group of neglected tropical diseases caused by protozoan parasites members of more than 20  Leishmania species parasites. They are transmitted to humans by the bites of infected female phlebotomine sandflies. Three main disease forms are encountered: cutaneous, visceral and mucocutaneous leishmaniases. An estimated 1 to 1.5 million cutaneous leishmaniasis (CL) and 500000 visceral leishmaniasis (VL) cases are annually reported worldwide [Leishmania is crucial for selecting the most appropriate therapy to be administrated to patients [Leishmaniases correspond to a group of diseases caused by more than 20 protozoan patients –5. Paraspatients –9. But npatients . Leishmapatients ,12, the patients ,14, migrpatients , all conpatients  and potepatients ,18.Leishmania parasites. Advances in systematics and taxonomy methods have improved knowledge about parasite evolution, phylogeny, and molecular species identification. Leishmania species discrimination and evolutionary relationships have been inferred using a range of assays targeting molecular markers including coding regions such as for heat shock proteins (hsp 20 [hsp70 [glycoprotein 63 (gp63) [cytochrome oxidase II [cysteine protease B [Tryparedoxine peroxidase [In Tunisia and worldwide, these diseases show emerging trends in their epidemiology resulting in changes in their distribution illustrated by the spread of the species from their classical foci to neighboring areas –24. Iden (hsp 20  or hsp700 [hsp70 ), glycop3 (gp63) –29, cytoidase II ,31, cystotease B ,33, Tryproxidase , and nonroxidase –37 and sroxidase . Some asroxidase , others roxidase  or speciroxidase , or are roxidase ,40–42. Iroxidase ,44 but aroxidase ,45–47 anDipeptidyl peptidase III (DPPIII) belongs to the M49 peptidase family of zinc-dependent enzymes that sequentially cleave dipeptides from the N-terminus of its substrates [DPPIII protein in protein turn over, oxidative stress and pain modulation and inflammation [Leishmania, the protein was characterized in L. braziliensis demonstrating its role in parasite survival in the vector, or host environments through peptide degradation and thus use of amino acids for energy production in stressful environment [L. donovani, which classified it as a candidate virulence factor that might be part of a stress response of the parasite [DPPIII gene is present in all Leishmania and other trypanosomatids sequenced genomes [Trypanosoma genus including T. brucei and T. cruzi species [L. braziliensis DPPIII translated sequence has a 65–80% similarity rate to other microorganisms belonging to Endotrypanum, Blechomonas, Crithidia and Leptomonas genera [Leishmania, the gene is conserved in species infecting humans and other mammals, with predicted 88–89% amino acids sequence identity to the L. braziliensis enzyme. In a previous study, primers targeting one part of the gene were used to amplify L. major, L. tropica, L. arabica and L. aethiopica DNAs but the reaction did not yield any product with the viscerotropic L. infantum and L. donovani species DNAs nor with L. tarentolae [Leishmania species including in countries which are also endemic for viscerotropic species. Therefore, we further investigated genetic diversity of the DPPIII gene and products of a new PCR in a panel of strains belonging to a range of pathogenic and non- pathogenic endemic species in the Old World to assess its relevance as a molecular marker for phylogeny and species identification, and thus to complement others for molecular epidemiology studies of Old World Leishmania species and disease surveillance.Metallopeptidases constitute a diverse group of distinct evolutionary families. bstrates . Studiesironment . Presencparasite . A 2040b genomes . However species ,52. The s genera . Within rentolae . In thisThis work complies with the ethical standards as required by the ethical committee of Institut Pasteur de Tunis, lead institution in the studies .The samples correspond to scrapings done in the frame of routine diagnosis of suspected CL patients with consent of patients in prospective studies (samples 1–13). In case of samples 14 to 75, they were also collected in the frame of routine diagnosis of suspected patients, the preserved samples’ remains were used retrospectively with the consent of the ethics committee of IPT with respect of patient anonymity.Leishmania DNA belonging to 8 Leishmania species isolated from a range of hosts: human cases, reservoirs or vectors, in various geographical origins, to cover a maximum of Old-World parasite species, available in our local DNA bank as detailed in In total, we analyzed 58 Seventy-five DNAs extracted from clinical samples using DNA purification kits as recommended by the supplier (Qiagen QIAamp DNA Mini Kit) were used . The samHaeIII RFLP was used as an identification technique as previously described [Leishmania DNAs inputs that were at the limit of detection.CL was confirmed for 45 patients by direct examination (DE) under microscope (x1000) of Giemsa- stained smears and subsequent observation of amastigotes. Twenty- six were DE negative, and in case of 4 CL patients, the direct examination was not done or not reported on the data collection file. For molecular identification purposes, ITS1-PCR followed by escribed . No- temDPPIII PCR assays were computed using DE as gold standard. Besides sensitivity and specificity, we generated the receiver operating curves (ROC) using the scikit-learn library under Python3. The area under curve (AUC) was calculated to estimate the ability of a given diagnosis test to correctly differentiate positive versus negative cases.Performances of ITS1 PCR and dipeptidyl peptidase III gene sequences of different Leishmania species (http://rebase.neb.com/). Two generic primers were manually designed in a gene region selected upon this multiple alignment analysis in conserved parts of the gene (http://www.premierbiosoft.com/netprimer/). NCBI primer-blast analysis did not find a target template in Homo sapiens genome database. Finally, the retained primers cover the region of the gene defined by the positions 832 to 1494 and have the following sequences: DPPIII-F 5’-AGGAGTGGGTGAAGGATGTG-3’ and DPPIII-R 5’-CAGCAAGCAGAGGTACAGC-3’.Available  species  were retthe gene  flanking2, 200 μM of each 2’- deoxy-nucleoside 5’-triphosphate, 1U Taq Platinium DNA polymerase (Invitrogen), 0.3 μM of each primer. No template reactions were the negative PCR controls. The thermal cycling parameters of the assay were as follows: initial denaturation at 94°C for 3 min followed by 35 cycles consisting of 94°C for 1 min, 62°C for 2 min, and 72°C for 2 min, and a final extension step of 5 min at 72°C. Amplicons were visualized on a 1.5% agarose gel under UV light in presence of ethidium bromide.The reaction was set up to amplify 0.2 ng of DNA in 25μL using a mix that contained 1x standard PCR buffer, 1.5 mM MgClAnalytical detection limit of the assay was assessed testing serial ten-fold DNA dilutions, ranging from 20ng/μl to 0.002fg/μl, whereby 1μl was used for PCR.DPPIII F/R PCR products were also sequenced or digested using selected enzymes.The DPPIII-F and DPPIII-R oligonucleotides, the BigDye Terminator v3.1 Cycle sequencing Kit (Applied Biosystem), and an ABI 3500 sequencer. The chromatograms were visualized and manually adjusted using DNA Baser sequence assembler v4 program (2013) .Direct sequencing of PCR products was performed on both strands using the Leishmania species with a minimum number of cuts and without generating too small fragments (<50bp) using predicted RFLP patterns visualized with the SnapGene 2.8.3 program from GSL Biotech, available at snapgene.com. Double digestions were done in 20μl reactions using 1 unit of each restriction enzyme, HaeIII and KpnI or SacI and PvuII, as recommended by the manufacturer (New England Biolabs Inc.) using 1x CutSmart buffer, and 10μl of reaction product. Incubations were done at 37°C for 2h. Then the restriction fragments were visualized on a 3% agarose gel.In case of RFLP analysis, the enzymes were first selected for their ability to differentiate OW Leishmania (representing 13 Leishmania species), one L. (Sauroleishmania) and two Leptomonas species (Leptomonas pyrrhocoris and Leptomonas seymouri) were aligned using ClustalW program, and MEGAX software was used to build phylogenetic trees. Then, sequenced DPPIII fragments of a selection of DNAs  and indels. We did multiple sequence alignments using the entire DPPIII gene (2040bp) of 19 published database sequences, covering 13 Leishmania, 1 L. (Sauroleishmania) and 2 Leptomonas species  and the number of non-synonymous substitutions per non-synonymous sites . The deduced difference between the non-synonymous and synonymous distances per site from averaging over all sequence pairs dN-dS is -0.164 (SE = 0.013). The probability of rejection of the null hypothesis of strict neutrality (dN = dS) using the codon- based Z test of neutrality was in favor of the deviation of the DPPIII gene from neutrality and tendency for a negative selection .We used phylogenetic analysis to assess taxonomical potential of the  species . Sequenc species . To deteDPPIII gene  including variable restriction sites, that also encodes for the active site motives, HELLGH and EECRAE, which are involved in zinc binding. This region was flanked by interspecies conserved sequences that we used to design a novel primers pair, the gene . It is wLeishmania and L. (Sauroleishmania) species, having different geographical origins and hosts  DPPIII sequences were retrieved from the TritrypDB database and included in the analysis , C (28%), A (20.7%), G (33.2%); indels (0.5%); GC content (61.3%) and the average pairwise similarity (96.3%). The selective evolutionary pressure on the 662 bp DPPIII fragment was determined, and it showed that as for the entire gene, the studied fragment is under negative selection .To further validate taxonomical potential of the selected 662bp  origins . In addianalysis . This amL. tropica and L. donovani, some SNPs appeared as specific to some of their strains, may be linked to the geographical origins  and the Old- World species . Among the Old- World species, 6 clusters were observed that are congruent to L. major, L. tropica, L. aethiopica, L. arabica, L. turanica, L. tarentolae species, and another one to the L. infantum and L. donovani species complex. This is in accordance with the phylogeny constructed using the entire DPPIII gene.The Maximum Likelihood based on the amplified lignment  showed aAll the groups mentioned above were also observed using UPGMA, NJ and ME phylogenies –S5 Figs L. tropica showed two groups according to their geographical origins. The first group gathers the Mediterranean strains LA28, AM, CJ and Leep0920, and the second, more heterogeneous, includes strains from the Middle East and India. The SNPs that differentiate these subgroups are situated at the positions 1131 and 1350 of the DPPIII gene  and L. (Sauroleishmania) tarentolae to identify within the 662bp fragment, the variable restriction sites, which are specific to species- or group of species. We selected four restriction enzymes (RE)  that distinguish between the different Leishmania species/group of species according to predicted double- digestion restriction fragments length polymorphisms, as detailed in Leishmania species identification is possible using the scheme on Leishmania species.In parallel, we took profit of the variable restriction sites to develop PCR-RFLP based identification as an alternative to the sequence analysis. Indeed, to develop the species identification scheme, we used published sequences of Old World HaeIII and KpnI RE would identify L. major, L. tropica, L. aethiopica, L. tarentolae, L. donovani but would not distinguish within the group made of L. infantum, L. turanica and L. arabica , L. arabica and L. turanica species DNA products were used in a PvuII and SacI double digestion. We obtained a different and unique restriction pattern for each of L. arabica, L. turanica and L. infantum species DNA as predicted by the in silico analysis; the African and Middle Eastern L. donovani and L. infantum DNAs shared the same profile  as gold standard. The + and 46 DPPIII PCR+. The distribution of positive results across the 3 tests on all the samples is shown on - were DPPIII PCR+. However, 8 ITS1 PCR+ samples were DPPIII PCR-. Two DE+ samples were negative by both PCR assays. Measured sensitivity using DE as gold standard was 82.22% for DPPIII and 88.8% for ITS1. Specificity was estimated to be 76.92% for both methods. Computed areas under the curve (AUC_ROC) were 0.80 and 0.83 respectively  subgenus represented by L. tarentolae species and annotated as a putative DPPIII protein, the CDS is initiated much downstream from the ATG site in other Leishmania genes, resulting in a shorter coding region (470bp vs 2040bp).Dipeptidyl Peptidase III protein coding sequence has been identified as a potential target for DNA parasite identification. The gene is present in the genome of chomonas  but is lathogens . InteresDPPIII gene as a target to amplify a 664bp fragment from L. major, L. tropica, L. arabica and L. aethiopica, while the PCR was negative for strains of L. infantum, L. donovani, and L. tarentolae species [DPPIII gene as a molecular marker for Old World Leishmania taxonomy and molecular epidemiology. For this purpose, we used sequences or strains belonging to a range of pathogenic, non- pathogenic  Leishmania species to humans, and monoxenous kinetoplastids  that also could be opportunistic to Leishmania species in humans [Leishmania taxa, geographical origins, hosts and diseases resulting in a representative set of DNAs of pathogenic Leishmania species in the OW; L. aethiopica and non-pathogenic Leishmania were represented each by a unique DNA.We previously used  species . Thus, tn humans –78. We aLeishmania genus evolution is mainly marked by the development of a digenetic life cycle and the introduction of the intracellular amastigote stage in the development cycle [Leishmania MLSA groups [DPPIII gene that contains the zinc binding motifs of the active site, in 69 Leishmania DNAs and 19 Database sequences, also showed a clear separation between the New World  and the Old World species . L. tarentolae was grouped with Old World species which is in accordance with the most recent classification [nt cycle . In thisnt cycle , or LeisA groups  and the fication .Interestingly, the analysis also suggested that the gene is under negative selection, a known feature of essential genes  that corL. major were different from those of the closely related, supposedly non- pathogenic to human, L. arabica, L. turanica and L. gerbilli. These species, known to be endemic in Middle East and Central Asia, share the same transmission areas and even cycle components as L. major; mixed infections were even described [The two sequence types that were observed for escribed –86. The L. infantum, and L. donovani which is a closely related species. These parasites have a still debated taxonomy. While some studies described them as two distinct groups [L. donovani complex) due to the low genetic diversity between them [L. donovani complex based on SNP variations clearly separated these parasites into five major groups that coincided with their geographic origin [L. donovani as a distinct genetic sub-group from East African strains [cpB sequence analysis, Sudanese and Ethiopian L. donovani strains constituted a distinct subgroup from other L. donovani complex strains that included L. infantum from Tunisia and L. donovani from Kenya, India, and Iran [L. donovani strains from the North (L. infantum) and East African (L. donovani) ones, corroborating close relatedness of the African parasites of this species complex, not allowing to identify an L. infantum sequence type.Two sequences types were also observed for t groups ,88, otheeen them ,47. In ac origin . Microsa strains ,90. In aand Iran . In thisDPPIII fragment sequence analyses also showed that, among the Old World studied species, L. tropica was the most polymorphic. Indeed, we noticed the presence of 5 haplotypes corresponding to 3 different geographical origins. The first haplotype was observed for the Mediterranean strains from Tunisia and Greece; three other haplotypes corresponded to Middle Eastern parasites, and the last one was shared between the unique Indian strain and a Middle Eastern one. Geographical subdivision within L. tropica was also observed in other studies using more sophisticated methods such as microsatellites [The tellites  and genotellites .L. aethiopica and non- pathogenic Leishmania species. Our findings would need to be validated using additional strains. However, we have observed that each of their sequences were sufficiently different from the other studied ones to be distinguished. L. (Sauroleishmania), including Leishmania tarentolae species, are reptile parasites, described in different evolution studies as deriving from the mammalian forms of Leishmania [Leishmania genus [L. tarentolae was detected and identified in hard and soft tissues of a 300 years old Brazilian mummy, emphasizing the fact that these parasites might infect and visceralize in humans [We tested only one DNA in case of ishmania ,90,93, aia genus . L. taren humans . This spLeishmania taxonomy marker by developing a new DPPIII PCR-RFLP based parasite identification where one or two steps would be needed for taxonomical assignments of the parasites in the Old World. Thereby, HaeIII/KpnI double digestion allows successful identification of L. major, L. tropica, L. aethiopica, the group representing L. infantum and L. donovani species, and even the species L. tarentolae of the subgenus L. (Sauroleishmania). Depending on the studied geographical region, this first analysis step could be sufficient to identify the endemic species making the assays cheaper and quicker. However, exceptions will be if imported species are observed and studied, or, in case of Asian and Middle Eastern regions, where L. turanica and L. arabica are respectively co-sympatric to L. major, and co-endemic with L. infantum or L. donovani. A second SacI/PvuII digestion step will precise the species identity that could not be ascertained during the first step. Although the L. gerbilli and L. turanica sequences are different, the selected enzymes did not generate discriminating RFLP patterns. Therefore, for these parasite species the sequence analysis is more appropriate for their identification.In this study, we further validated DPPIII gene as a L. tropica and L. aethiopica species. However it was possible to differentiate them using species- specific PCR and MLMT assays [HaeIII & KpnI) of a single maker, and we recommend the validation of its use for the identification of L. aethiopica. Using ITS1 PCR-HaeIII RFLP method [L. aethiopica digestion profile looked remarkably like the L. infantum/L.donovani species profile. It needed the use of metaphor agarose gel electrophoresis or a second digestion step (using CfoI) to differentiate these species. In case of DPPIII-RFLP assay, we recommend use of 2% - 3% agarose gel electrophoresis to achieve optimal resolution of the L. aethiopica RFLP pattern so it can be clearly distinguished from the L. donovani one  presented distinct profiles. But Southern blotting and radioactive hybridization were required to allow full discriminative resolution of the digestion profiles, notably to detect the small sized bands that had faint intensity. An alternative for detection of these bands was the use of specific conditions for the electrophoresis  [In addition, as expected from the sequence analyses,  species ,98. Howeerformed . Differee brand) .DPPIII PCR on clinical samples demonstrated its ability to detect Leishmania parasites in DE+ human samples without reacting with human DNA as shown in Leishmania negative samples. The performances of this method were shown close to those of ITS1 PCR using direct examination diagnosis as gold standard, although sensitivity of ITS1 was higher. Their AUC ROC also pointed to the ability of these tests to distinguish True Positive from False Positive cases. The performance difference could be due to copy number of the targets, here a single copy DPPIII gene versus multi-copy ITS1, or also by the fact that our target sequence (662bp) is longer than the ITS1 one (300-350bp). In a similar context, reducing the target length was shown to greatly improve the sensitivity of PCR assays [DPPIII PCR was able to detect 3 DE+ samples that were not detected by ITS1 PCR despite the repeated nature of this target. Multicopy or repetitive sequences, notably intergenic and spacer regions, could be prone to sequence variations and more rapid accumulation of mutations than in coding regions [Cpb) could also be difficult to analyze due to complexity created by isogene variations [DPPIII and ITS1 amplified products were relatively easy to analyze, although in other ongoing studies it was not uncommon for us to observe difficult to interpret ITS1 RFLP profiles hampering Leishmania identification. We could identify the Leishmania species using one RFLP step in case of 39 DPPIII+ samples, while in case of 7 the amplicons were in too low amount to be further processed.The application of R assays . Interes regions . Interes regions . Heterog regions . RFLP pariations . In caseDPPIII gene is a suitable marker for taxonomy and demonstrated ability of the developed DPPIII PCR to detect and identify parasites within human samples. So it could be used for diagnosis and molecular epidemiology studies to complement other methods including the ITS1 PCR assay. Indeed, it is recommended to use a combination of markers and techniques to provide an accurate diagnosis [In the present study, we demonstrated by a phylogenetic approach that the iagnosis  and to aiagnosis ,102 in cLeishmania taxonomical identification constitutes a key step to conduct molecular epidemiology investigations, to monitor disease outbreak and spread, and to implement rapid and adequate preventive measures. Molecular analyses of the DPPIII gene demonstrate it to be a plausible complement / alternative for such purposes in North Africa. Further investigations need be done to assess gene diversity in the under-represented species of this study, especially L. donovani in the Indian subcontinent, L. aethiopica in Africa, and L. turanica, L. gerbilli and L. arabica in Asian and Middle Eastern foci. This marker may contribute to advance in the fight against leishmaniases.Application of molecular identification methods for robust https://tritrypdb.org/tritrypdb/)TritrypDB ((http://rebase.neb.com/)The Restriction Enzyme Database (REBASE) www.geneious.com)Geneious v3.6.2 (http://www.premierbiosoft.com/netprimer/)NetPrimer (https://www.megasoftware.net/)MEGA 6 (www.snapgene.com)SnapGene (www.DnaBaser.com)DNA Baser sequence assembler v4 program ; L.d: L. donovani; **East African L. donovani strain ; L.tu: L. turanica; L.ar: L. arabica; L.tar: L. tarentolae.MW: 100bp Molecular Weight. (TIF)Click here for additional data file.S2 FigLeishmania parasites was inferred by using the Maximum Likelihood method and Tamura 3-parameter model. The bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history of the taxa analyzed. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Tamura 3 parameter model, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites ). This analysis involved 72 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. There were a total of 662 positions in the final dataset. Evolutionary analyses were conducted in MEGA X.The evolutionary history of (TIF)Click here for additional data file.S3 FigThe bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history of the taxa analyzed. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The evolutionary distances were computed using the Tamura 3-parameter method and are in the units of the number of base substitutions per site. The rate variation among sites was modeled with a gamma distribution (shape parameter = 0.65). This analysis involved 72 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All ambiguous positions were removed for each sequence pair (pairwise deletion option). There were a total of 662 positions in the final dataset. Evolutionary analyses were conducted in MEGA X.(TIF)Click here for additional data file.S4 FigThe bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history of the taxa analyzed. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The evolutionary distances were computed using the Tamura 3-parameter method and are in the units of the number of base substitutions per site. The rate variation among sites was modeled with a gamma distribution (shape parameter = 0.65). The ME tree was searched using the Close-Neighbor-Interchange (CNI) algorithm at a search level of 1. The Neighbor-joining algorithm was used to generate the initial tree. This analysis involved 72 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All ambiguous positions were removed for each sequence pair (pairwise deletion option). There were a total of 662 positions in the final dataset. Evolutionary analyses were conducted in MEGA X.(TIF)Click here for additional data file.S5 FigThe bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history of the taxa analyzed. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The evolutionary distances were computed using the Tamura 3-parameter method and are in the units of the number of base substitutions per site. The rate variation among sites was modeled with a gamma distribution (shape parameter = 0.65). This analysis involved 72 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All ambiguous positions were removed for each sequence pair (pairwise deletion option). There were a total of 662 positions in the final dataset. Evolutionary analyses were conducted in MEGA X.(TIF)Click here for additional data file.S6 FigL.m: L. major, L.i: L. infantum, L.t: L. tropica, L.d: L. donovani, L.ae: L. aethiopica, L.ar: L. arabica, L.tu: L. turanica, L.ta: L. tarentolae.A. After double digestion with HaeIII&KpnI endonucleases. B. After double digestion with SacI and PvuII of the PCR products. The parasites that could not be distinguished by HaeIII/KpnI digestion are differentiated by the SacI/PvuII restriction. MW: 100 bp Molecular Weight. (TIF)Click here for additional data file.S7 FigHaeIII-KpnI. MW: 50bp Molecular Weight. L. m: L. major (IL24), L. i: L. infantum (Drep 5) and L. t: L. tropica (LA28). Lanes 1–13: Clinical samples.DNA was isolated directly from clinical samples, amplified by DPPIII-PCR and double digested with (TIF)Click here for additional data file.S8 Fig(A) Second round digestion of L. aethiopica strain. MW: 50bp Molecular weight; L. i: L. infantum (LV08); L. d: L. donovani (L1005); L. tu: L. turanica (95A); L. ara: L. arabica (J238); L. aeth: L. aethiopica (L100). (B) Two round digestion of L. aethiopica strain visualized on a 2% agarose gel. Lane 1: HaeII-KpnI double digestion, Lane 2: SacI-PvuII double digestion.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.


F8 gene from probands and age, ABO blood type, FVIII:C, VWF:Ag, and the result of targeted gene analysis in females. We diagnosed 110 females as carriers, and their FVIII:C and FVIII:C/VWF:Ag ratio were lower than those in 69 non-carriers . In receiver operating characteristic analysis, the areas under the curve (AUC) of the FVIII:C/VWF:Ag ratio and FVIII:C were 0.936 and 0.876, respectively. The cut-off value of FVIII:C/VWF:Ag ratio (0.81) at the maximum Youden J index provided a sensitivity of 82.8% and specificity of 96.6%. The cut-off value of FVIII:C (83.8 IU/dL) showed a sensitivity of 81.8% and specificity of 79.7%. Considering the AUC, the FVIII:C/VWF:Ag ratio is a good screening test to detect haemophilia A carriers, as evidenced by its specificity of 96.6%; however, it may also induce false-negative results.Analyses of factor VIII procoagulant activity (FVIII:C) and the FVIII:C to VWF:Ag ratio (FVIII:C/VWF:Ag ratio) have been investigated as screening bioassays to detect haemophilia carriers. This study aimed to determine the validity of the FVIII:C/VWF:Ag ratio and FVIII:C analyses as screening tests. We reviewed the medical records of 137 genetically confirmed, proband haemophilia A patients and 179 of their familial females who had undergone carrier testing. The collected data included the severity and mutation type of  F8 gene is deficient in haemophilia A. In X-linked inheritance, a female heterozygote with a mutation on only one of the two X-chromosomes, the so-called carrier, often has FVIII:C of more than 50 IU/dL of normal activity. Approximately two-thirds of carriers have FVIII:C > 60 IU/dL [Congenital haemophilia A is one of the most common congenital bleeding disorders. Almost all of the patients are male because X-linked recessive traits transmit hereditary haemophilia A. However, haemophilia A may also develop sporadically. Mild to moderate haemophilia A is familial in 70% of patients, whereas severe haemophilia A is familial in only 45% of patients . In Kore60 IU/dL . BecauseF8 gene. Linkage analysis using various restriction fragment length polymorphisms (RFLPs) or variable numbers of tandem repeats is representative indirect mutation analysis. Although linkage analysis is a technically simple and cost-saving method, the misdiagnosis rate attributable to DNA recombination error ranges from 1% to 5% depending on restriction enzyme markers [To diagnose a carrier, researchers have applied direct and indirect mutation analysis of the  markers . The intFor these reasons, direct mutation analysis, such as intron 22 inversion or DNA sequencing, is preferred even though it is labour-intensive and costly. Pathogenic mutations can be identified in 98% of patients by direct mutation analysis . On the We retrospectively reviewed the medical records of 137 patients genetically confirmed with haemophilia A and 179 of their familial females who had undergone carrier testing between April 2007 and May 2021 at the Korea Hemophilia Foundation (KHF) clinic. For the avoidance of bias which could be caused by very low FVIII:C, female haemophilia A patients having less than 5 IU/dL of FVIII:C and typical bleeding diathesis such as haemarthrosis were excluded. We collected information on the disease severity and mutation type of F8 gene from probands. From the subjected females, age, family relationship with probands, bleeding diathesis, ABO blood type, FVIII:C, VWF:Ag, and the results of targeted gene analysis were derived. We collected the information regarding bleeding diathesis from verbal questions and answers.® reagent or CS-2500 automated coagulation analyser  using the Dade Actin FS  reagent. VWF:Ag was measured with an ACL 9000TM automated coagulation analyser using HemosILTM VWF antigen .FVIII:C was measured using the one-stage assay with ACL 9000TM  using Synthasil®  was performed for DNA sequencing. Multiple ligation-dependent probe amplification was applied for the gene dosage assay. As for females who had wanted to undergo carrier testing, we carried out targeted gene mutation analysis for the same pathogenic mutation as their own probands. We classified the pathogenic mutations into null  and non-null mutations (missense and splicing).The gold standard for the identification of a female as a haemophilia carrier was genetic testing. Inversion testing, DNA sequencing and the gene dosage assay were subsequently carried out until the detection of the pathogenic mutations. Long-distance PCR or inverse-shifting PCR according to the research of Rossetti et al. was utilized for inversion tests ,13. Sangp-value < 0.05. We performed ROC analysis using MEDCALC® ) software. The target sample size was calculated as 143 using G*power 3.1.9.7, with an effect size of 0.3, a power of 0.8, and a significance level of 0.05 [Statistical analysis was performed using SPSS Statistics version 20 . Data that satisfied the normality requirement were expressed as the mean ± SD. Otherwise, the data are described as median (range). Differences between carrier subjects and non-carrier subjects were analysed using the chi-square test and Mann–Whitney U test depending on the variable scales. Linear correlation analysis was used to determine the correlation between FVIII:C and VWF:Ag. In addition, binomial logistic regression was used for correlation analysis between FVIII:C and bleeding diathesis. Statistical significance was defined as a  of 0.05 .A total of 316 subjects were included in this study . A singlp = 0.052), but carriers reported bleeding diathesis more commonly  than non-carriers , especially subcutaneous haematoma, epistaxis and traumatic bleeding.Bleeding diathesis was investigated in the medical records of 74.9% (134/179) of the females. While 68 females reported not increased bleeding tendency, 66 females complained of increased bleeding manifestations. Multiple bleeding diathesis was identified in 12 out of 45 symptomatic carriers and 2 out of 21 symptomatic non-carriers. The most frequent bleeding diathesis was hypermenorrhoea, followed by subcutaneous haematoma and epistaxis. The difference was statistically insignificant (p = 0.000). These findings were similar when the data of carriers and non-carriers defined as O or non-O blood type subjects were compared. FVIII:C and VWF:Ag were lower in the O blood type than in the lower non-O blood type (p = 0.000). However, the FVIII:C/VWF:Ag ratio was not significantly different  (p = 0.976), VWF:Ag , and FVIII:C/VWF:Ag ratio  were observed. There was a strong positive correlation between FVIII:C and VWF:Ag , and the correlation was stronger in non-carriers  than in carriers . In terms of mutation types, there was no statistical difference in the FVIII:C, VWF:Ag, and FVIII:C/VWF:Ag ratio. There was no significant association between FVIII:C and bleeding diathesis . However, females with FVIII:C < 40 IU/dL manifested an increased bleeding tendency (p = 0.045) compared to females with FVIII:C > 40 IU/dL. When it was defined to carriers, bleeding diathesis became more remarkable in carriers with FVIII:C of less than 40 IU/dL (p = 0.004). On the other hand, 22 carriers and none of the non-carriers had FVIII:C less than 40 IU/dL.FVIII:C was measured in all females, but VWF:Ag was tested in 160 females. The median FVIII:C, VWF:Ag, and FVIII:C/VWF:Ag ratio of the total females were 74.5 IU/dL, 103.0 IU/dL, and 0.77 respectively . In carr= 0.770) . Between®, the sensitivity and specificity of FVIII:C/VWF:Ag ratio at criterion value of 0.73 were estimated at 75.8% and 96.6%, respectively. Given that the prevalence of haemophilia A carriage was 0.02%, the positive and negative predictive values were 0.86% and 99.99%, respectively. We performed ROC analysis to determine the optimal cut-off value , sensitivity (81.8%), and specificity (79.7%) were lower than those of FVIII:C/VWF:Ag.Authors should discuss the results and how they can be interpreted from the perspective of previous studies and of the working hypotheses. The findings and their implications should be discussed in the broadest context possible. Future research directions may also be highlighted.F8 mutation analysis is the gold standard for diagnosing haemophilia A carriers. Nevertheless, several studies regarding FVIII:C/VWF:Ag ratio and FVIII:C were conducted to determine the possibility of screening tests for haemophilia A carriership [riership ,10,11. IThe proportion of severe probands in this study was approximately 10% higher than that of the KHF registrants . It is aBleeding diathesis in carriers has been reported in a variable range. While Paroskie et al. reported that 72% of obligate haemophilia A carriers had a high frequency of bleeding symptoms , Seck etp = 0.001) [p < 0.001) between FVIII:C and bleeding diathesis [The correlation between bleeding diathesis and FVIII:C is also controversial. Seck et al. reported the FVIII:C level was significantly different between carriers with and without bleeding diathesis  . Howeveriathesis . This fip = 0.62). This result indicated that the significant impact of ABO type on FVIII:C level was mediated through an effect on VWF:Ag. In our study, 31.1% of VWF:Ag and 33.2% of FVIII:C were reduced in O blood type subjects compared to those of non-O blood type subjects. A stronger correlation of FVIII:C and VWF:Ag in non-carriers (r = 0.781) than in carriers (r = 0.556) suggests that FVIII:C levels are more dependent on VWF:Ag levels in non-carriers.VWF represents a high-molecular weight adhesive glycoprotein that plays an essential role in primary haemostasis by promoting platelet adhesion to the subendothelium and platelet plug formation at the sites of vascular injury. VWF has a second role in haemostasis which is to bind FVIII and protect it from premature clearance and degradation ,21,22. IFor the screening of diseases with a heavy burden, a test with high sensitivity is usually preferred, and it is known that a measure with high specificity is more eligible for the screening of curable or treatable diseases. Shetty et al. indicated that the lowest misclassification rate of 7% among the carriers was seen when a cut-off value of 0.7 was chosen . The YouEven though the AUC of FVIII:C showed very good results, the AUC of the FVIII:C/VWF:Ag ratio was excellent; thus, the FVIII:C/VWF:Ag ratio rather than FVIII:C should be considered for the screening of haemophilia A carriers .This study has some limitations. First, data on bleeding diathesis were collected using simple verbal questions and answers. Bleeding assessment tools, such as quantitative bleeding score, might be essential to conclude bleeding diathesis in carriers. Second, this study did not consider the menstrual cycle and oral contraceptives which may affect the FVIII:C and VWF:Ag levels.In conclusion, the gold standard for the diagnosis of haemophilia A carriership is mutation analysis. However, in case genetic analysis of the pathogenic mutation is unavailable, the FVIII:C/VWF:Ag ratio can be considered as at least a screening test.


Candida. The recent emergence of antifungal drug resistance and increased incidences of infections caused by non-albicans Candida species merit the need for developing immune therapies against Candida infections. Although the role of cellular immune responses in anti-Candida immunity is well established, less is known about the role of humoral immunity against systemic candidiasis. This review summarizes currently available information on humoral immune responses induced by several promising Candida vaccine candidates, which have been identified in the past few decades. The protective antibody and B-cell responses generated by polysaccharide antigens such as mannan, β-glucan, and laminarin, as well as protein antigens like agglutinin-like sequence gene (Als3), secreted aspartyl proteinase (Sap2), heat shock protein (Hsp90), hyphally-regulated protein (Hyr1), hyphal wall protein (Hwp1), enolase (Eno), phospholipase (PLB), pyruvate kinase (Pk), fructose bisphosphate aldolase (Fba1), superoxide dismutase gene (Sod5) and malate dehydrogenase (Mdh1), are outlined. As per studies reviewed, antibodies induced in response to leading Candida vaccine candidates contribute to protection against systemic candidiasis by utilizing a variety of mechanisms such as opsonization, complement fixation, neutralization, biofilm inhibition, direct candidacidal activity, etc. The contributions of B-cells in controlling fungal infections are also discussed. Promising results using anti-Candida monoclonal antibodies for passive antibody therapy reinforces the need for developing antibody-based therapeutics including anti-idiotypic antibodies, single-chain variable fragments, peptide mimotopes, and antibody-derived peptides. Future research involving combinatorial immunotherapies using humanized monoclonal antibodies along with antifungal drugs/cytokines may prove beneficial for treating invasive fungal infections.Systemic candidiasis is an invasive fungal infection caused by members of the genus  Candida. Candida species can exist inside human hosts as commensals and have emerged as important agents of opportunistic infections. Superficial infections are frequently observed in response to overgrowth or disruption of microbial flora, and/or environmental changes in individuals. However, in the event of breakdown of tissue barriers and during immune-compromising conditions, superficial infections lead to dissemination of Candida in the bloodstream, which is then referred to as invasive or systemic candidiasis [Candida species, which include C. albicans, C. tropicalis, C. glabrata,C. parapsilosis, and C. krusei. More recently, a multidrug-resistant Candida species, C. auris has been linked to major outbreaks of invasive infections in healthcare facilities around the globe [Candida species has been increasing over the past decade and multidrug-resistant mechanisms to Candida species pose a serious threat to public health worldwide. According to the CDC’s report regarding the antibiotic resistance threat in 2017, more than 34,000 cases and 1700 deaths annually were due to drug-resistant Candida species [Candida strains is a major cause of concern. Apart from antifungal drug resistance, longitudinal studies have detected a shift towards non-albicans Candida (NAC) species for the past few decades [C. albicans to NAC species varies by region [Candidiasis broadly refers to fungal infections caused by members of the genus didiasis . Systemididiasis . Accordididiasis . Globalldidiasis . Every ydidiasis . More thhe globe . Currenthe globe . Despite species . Additio decades . Populaty region .Candida infection is crucial for high-risk individuals such as immunocompromised patients, premature infants, cancer patients, and those with invasive treatments for long periods in hospital settings. To resolve the burden and challenges posed by Candida mediated systemic candidiasis, there is a strong specific medical need for vaccine/s or immunotherapies that target Candida species. Therefore, new alternative immunotherapeutic approaches are urgently needed to treat systemic candidiasis caused by Candida species. For a long time, the role of cellular immunity has overshadowed the contribution of humoral immunity in host defense against invasive candidiasis. The main aim of this review is to disseminate currently available information and experimental evidence regarding the role of B-cells and antibody-mediated immune responses against Candida vaccine antigens from studies done mostly in C. albicans. In this review article, we have summarized the lead anti-Candida vaccine candidates and humoral immune responses induced by them for conferring protection. Identification of fungal antigens, which elicit protective antibodies can initiate the design of multi-valent or multi-epitope Candida vaccine/s. Recent advances regarding monoclonal antibodies and their mechanisms of protection, anti-idiotypic antibodies, single-chain variable fragments, and peptide mimotopes are discussed, which may be useful for the development of direct antibody-based, as well as combination immunotherapies against invasive candidiasis.Increased interest in the development of new vaccines against Candida, and serve as efficient receptors for phagocytosis of opsonised Candida yeasts and induction of proinflammatory cytokines for recruiting and activating phagocytes [Candida, as demonstrated by various studies using complement protein C3 depleted or C3 deficient animals [Candida yeast cells, thereby limiting the fungal burden early upon infection [C. albicans more slowly, showed significantly impaired survival and had significantly increased Candida CFUs (colony forming units) during disseminated candidiasis [Candida early during infection and were found more effective in killing C. albicans than DCs or macrophages [C. albicans [C. albicans produce tumor necrosis factor-α (TNF-α), which is required for surviving systemic candidiasis [Candida, they were reported to be less efficient than macrophages at fungal killing [Candida infections, since neutrophil activation is essential for clearance of Candida, with neutropenia being a major risk factor for invasive fungal infections [Candida killing has been established and they are the only immune cells capable of successfully inhibiting the conversion of Candida blastopores into hyphae, a key fungal virulence trait [C. albicans by neutrophils [C. tropicalis in disseminated candidiasis [C. albicans infection [C. albicans infections in T/B/NK-cell-deficient mice, compared with T/B-cell-deficient SCID (severe combined immunodeficiency) mice [Candida infection has been reported using human NK cells, which could mediate direct cytotoxic effects on the fungus [Candida directly via cell surface receptors or indirectly via cytokines and interferons [The physical barriers, like skin and the mucosal epithelial surfaces existing in the mouth, upper airways, the gastrointestinal and genitourinary tracts, are mainly considered the first line of defense against fungal pathogens . Epithelagocytes . The com animals ,16. In a animals . The rolnfection . Macrophdidiasis ,19. Monorophages . Monocytalbicans , and mondidiasis . Furtherdidiasis . DCs pladidiasis ,25. DCs didiasis . Althoug killing . Neutropfections . The rolce trait ,26. Neutce trait . Uppulurtrophils . A recendidiasis . Naturalnfection . It has cy) mice . The role fungus . It has erferons .+ T helper cells and CD8+ cytotoxic T-cells are the predominant players involved in controlling Candida infection [In addition to innate immunity, adaptive immunity is essential for a successful elimination and development of optimal protective immunity against invasive candidiasis. Cell-mediated adaptive immune responses constitute the mainstay of host defense mechanisms against systemic candidiasis, wherein both CD4nfection .+ T-cell immune responses have been believed to be protective against Candida infection [+ T-cells are known to contribute towards Th1, Th2, and Th17 immunity during antifungal immune response [+ T-cells do not have direct cytolytic activity, they still play a critical role against Candida infection. The significance of the CD4+ T-cell response in inducing protective immunity against C. albicans is indicated by the prevalence of oropharyngeal candidiasis in AIDS (acquired immunodeficiency syndrome) patients whose CD4+ T-cell count is depleted [CD4 T-cells: Historically, CD4nfection . CD4+ T-response . Althougdepleted . It has depleted ,37,38,39depleted ,41.+ T-cells are known to mediate resistance to systemic fungal infections, primarily through their direct cytotoxic activity and cytokine secretion [+ T-cells in the direct killing of fungi has been established [+ T-cells can restrict fungal infection [CD8 T-cells: CD8ecretion ,43. The ablished  and cytonfection .Candida infection. Studies have shown that certain cytokines enhance phagocyte killing of Candida species and specific cytokines expressed by antigen presenting cells like DCs and macrophages are crucial for the differentiation of T-helper (Th) cells [Candida. Protection against fungal infections has been extensively correlated with Th1/Th17 mediated cellular immunity [C. albicans and a decrease in mortality was observed in mice treated with recombinant GM-CSF during disseminated candidiasis [Candida strains [Candida-associated abscesses in a mucosal model [Candida infection in mice, neutralization of endogenously synthesized IFN-γ by a specific antibody has been shown to prevent the development of a protective Th1 response [Candida host defense by inducing nitric oxide production by macrophages, as well as Candida-specific immunoglobulin production [Candida infection [C. tropicalis infection compared to controls [C. albicans infection [C. albicans hyphae [−/− deficient mice [C. albicans infection [C. albicans infection [Candida immunity at mucosal surfaces. It has been shown that IL-17 secreted by Th17 cells keeps fungal infection in check by recruiting neutrophils and inducing defensin secretion by epithelial cells at mucosal sites [C. albicans infections at mucosal sites [Candida infection [C. albicans mediated systemic infection.Cytokines: Multiple different cytokines and chemokines are associated with protection against h) cells ,47. Th1 immunity ,49. The didiasis ,51. IFN- strains ,52, and al model . While aresponse . Furtherresponse . In addiresponse ,56. IFN-oduction ,57. The nfection , and depcontrols . TNF-α acontrols . The prenfection  and Benos hyphae . Circulaent mice ,64. It hent mice . The rolent mice  and IL-1ent mice . Netea enfection . A previnfection . Recentlnfection . Th17 ceal sites . In a real sites , and IL-al sites . Additioal sites . Unlike al sites ,74. Howenfection  and a prnfection . FurtherAlthough cellular immunity has been considered to be the central component during systemic candidiasis, there is an important role of humoral immunity in controlling invasive fungal infections. Although the complement system, collectins, and antimicrobial peptides are part of humoral immunity, the primary focus of this section is to evaluate the humoral responses mediated by B-cells and antibodies that confer protection during systemic candidiasis.C. albicans mediated candidiasis [Candida infection in mice [C. albicans strain [C. albicans infection [C. albicans in the absence of B-cells, contributing to protective immunity to systemic candidiasis [−CD5+ B-cells transferred from immune rats had lesser Candida CFUs when compared to controls, but showed a significant delay in fungal clearance when compared to animals administered with immune T-cells [Candida-immunized rats in naïve animals resulted in protection against vaginal candidiasis [in vitro addition of rituximab in PBMCs  to deplete B-cells, led to reduced anti-Candida responses [C. dubliniensis/S. aureus challenge and C. albicans/S. aureus rechallenge, and protection against lethal C. albicans/S. aureus was not mediated by adaptive immunity [C. albicans was cleared more efficiently in TgVH3B4 mice after fungal inoculation than control mice [C. albicans infection led to enhanced proliferation of B-1 cells, which resulted in increased numbers of B-1a B-cells and C. albicans-specific B-cells in TgVH3B4 mice, which may have a role in fungal clearance [C. albicans, the generated antibodies were capable of stimulating opsonophagocytic macrophage activity in vitro and provided protection against disseminated candidiasis in vivo [In a previously published study, Sinha et al. showed that B-cell deficiency did not increase the susceptibility of the animals to didiasis . Further in mice . In addis strain . Previounfection ,81. Howenfection . Jones-Cdidiasis . It has  T-cells . Anotherdidiasis . Furtheresponses . Lilly eimmunity . Severalimmunity ,88. A strol mice . Notablylearance . A recen in vivo . Notably in vivo .Antibodies are the effector molecules of the adaptive immune responses, which restrict the fungal burden and aid in its clearance . EffectoC. albicans to human oral epithelial cells [Candida antibody response, while controls with functional B-cells were able to generate protective antibody response against Candida infection. The B-cell deficient mice were more prone to death, had reduced body weight, and were also found to be more susceptible to Candida infection. This finding suggested that B-cells and antibodies are responsible for the protection against Candida infection [Candida antibodies in passive immunization and protection of the host against candidiasis has been studied by Cassone et al. [Candida IgA antibody was not protective against recurrent vaginal C. albicans infection, and the vaginal anti-Candida IgA and IgG antibodies levels were similar in women with or without vaginal candidiasis [Candida IgA, IgE, IgG, and subclass  antibody levels in serum and vaginal washes from women with or without vulvovaginal candidiasis, another study demonstrated significantly higher levels of IgA in vaginal washes and lower in serum, present in patients. Additionally, similar levels of serum anti-Candida IgA and IgG antibodies were observed in women with or without a history of Candida vaginitis [Candida human domain antibodies inhibited attachment of fungus to the vaginal epithelium, which protected rats from experimental vaginitis [C. albicans protected mice against disseminated candidiasis [C. albicans cell wall surface proteins demonstrated significantly higher serum levels of C. albicans-specific antibodies [Antibody isotypes IgM, IgG, and IgA are the main antibodies produced against fungal pathogens. IgA, which is a predominant antibody involved in conferring immunity at mucosal surfaces, is known to prevent the binding or attachment of al cells . Maiti enfection . The role et al. . Howevere et al. . A prevididiasis . On charaginitis . A studyaginitis . An earldidiasis . Productdidiasis . Vaccinatibodies . Moreovetibodies .Candida vaccine/s. Although several different anti-Candida vaccine candidates have been identified, only a few of them have progressed to clinical trial evaluation to date [Candida vaccine/s that can efficiently immunize subjects at risk of developing invasive fungal infections [Candida vaccines [C. albicans have been explored as vaccine candidates  glucan without any β- branching from Alcaligenes faecalis), zymosan  glucan from Saccharomyces cerevisiae) and pustulan  linked β-glucan from lichen Lasallia pustulata), are experimental alternatives for β-glucan that are used for studying antifungal immune responses.The β-1,3-glucans are structurally complex homopolymers of glucose, present on the C. albicans cells showed protective immune response by producing anti-β-glucan antibodies, which were protective in a mice model of disseminated candidiasis [Candida growth [Cryptococcus neoformans, by exerting anticryptococcal activities in vitro and in vivo through its binding to C. neoformans cell wall β-glucan [C. albicans infection [C. albicans, compared to non-protective anti-β--glucan antibodies [Bromuro et al. reported that mice immunized with didiasis . When mia growth . In a seβ-glucan . Furtherβ-glucan ,149. Recβ-glucan . The β-gβ-glucan . Furthernfection . On evaltibodies .in vitro and/or in vivo microbicidal activity against C. albicans [C. albicans cell wall β-glucan were identified in various animal serum samples and were seen to participate in immune response to pathogenic fungi [Candida infections [in vitro and since its activity could be neutralized by laminarin  and not pustulan , it was speculated that it interacted with β-glucan containing yeast killer toxin receptor present on C. albicans [Candida species [C. albicans β-glucan, and are responsible for exerting an inhibitory effect on the growth of C. albicans [Anti-β-glucan antibodies are detectable in normal animal and human sera, and when elicited by glucan-based vaccines they can exert a fungicidal protective activity . Studiesalbicans . Furtheric fungi . Chiani ic fungi . On analic fungi . Recentlfections . Of notealbicans ,159. Add species . Furtheralbicans .Galleria mellonella as a model for Candida infection, a dose-dependent protection was observed when β-glucan was injected in high amount in the hemocoel of insects [C. albicans B-cell β-1,2-mannotriose epitope attached to the Fba peptide T-cell epitope, the resulting three component synthetic vaccine could induce protective antibodies to all three epitopes of the fully synthetic construct [in vivo against systemic C. albicans infection via protective antibody generation [Candida β--glucan), was observed to induce an effective humoral immune response , and post-vaccination serum exhibited anti-Candida growth activity in vitro in a mucocutaneous infection model [Candida and demonstrated synergy with the antifungal fluconazole in killing C. albicans in vitro. Further, they also showed protective activity in vivo, suggesting their use in combinatorial antifungal therapy [C. albicans mutant with β--glucan exposed on its cell-surface was observed to induce the generation of protective antibodies against invasive candidiasis in mice, which was primed by IL-18 secretion [Candida innate and humoral adaptive immune responses [Candida cell wall. Seo et al. demonstrated that during fungal infection, β-glucan-stimulated Dectin-1 may cooperate with TLR4 to specifically enhance IgG1 production by mouse B-cells, suggesting a role of β-glucan in the B-cell mediated antibody response [Huang et al. found that immunization with β-glucan particles complexed with ovalbumin, not only induced robust Th1- and Th17-based CD4 T-cell responses, but also induced strong humoral antibody responses (IgG2c) in mice . Using l insects . Inocula insects . By inco insects . Furtheronstruct . Synthesonstruct . Furtherneration . Active on model . Mouse m therapy . More reecretion . On evalesponses . It has response . Hoogeboresponse .Laminaria digitata, consisting of β- glucan repeating units, with sporadic β- branches [in vitro and was neutralized by laminarin, suggesting that candidacidal activity was mediated by the interactions between killer peptides and β-glucan moiety present on Candida cell surface [C. albicans and could inhibit their growth in vitro in the absence of host immune cells. Their results showed that protection mechanisms of the glyco-conjugate vaccine included direct antifungal properties of anti-β-glucan antibodies [C. albicans β-mannan antigen. The laminarin vaccine could bind to the Dectin-1 receptor, which resulted in amplification and immunomodulation of the immune response, and also altered the distribution of IgG subclasses [Laminarin is a β-glucan entity isolated from brown algae, branches . It is a surface . Torosantibodies . Torosantibodies . It has tibodies . Adamo etibodies . Laminarbclasses . When labclasses .Candida species. Hsps can protect humans from systemic candidiasis and are a major target for the immune system in invasive fungal infections. Particularly, Hsp90 confers protection against C. albicans and can bridge the gap between innate and specific humoral immunity. Matthews et al. demonstrated that sera from patients recovering from C. albicans mediated systemic infection produced antibodies specific to both Candida and heat-shock protein Hsp90 [C. albicans in invasive candidiasis [C. albicans infection and improvement of survival rates in mice suggested that antibody to Hsp90 epitopes could be protective in murine invasive candidiasis [C. albicans, C. glabrata, and C. krusei infections [in vitro as well as in vivo efficacy in a murine candidiasis model [et al., wherein intradermal administration of Hsp90 protein and DNA vaccine resulted in improved survival rates in BALB/c mice during systemic candidiasis, which correlated with increased levels of anti-Candida Hsp90 serum IgG antibodies [C. albicans Hsp90 protein elicited protective immune responses against systemic candidiasis through both antibody-mediated and cell-mediated immune responses [C. albicans infection in C57BL/6J mice, confirming that Hsp90 can serve as a potential vaccine candidate for systemic candidiasis by inducing both protective humoral and cellular immunity [Candida Hsp90 could elicit increased levels of both serum and vaginal Hsp90-specific IgG and IgA antibodies, resulting in enhanced humoral response during experimental vaginal candidiasis [Astragalus membranaceus, the efficacy of recombinant Hsp90 protein vaccine was enhanced against systemic candidiasis by significant enhancement of IgG antibody titers and various interleukins in the serum of Hsp90-immunized mice [Heat shock proteins (Hsps) control basic physiological activities or virulence via interaction with a variety of diverse regulators of cellular signaling pathways. They are expressed in response to thermal stability, morphogenesis, cell cycle regulation, apoptosis, and drug resistance in in Hsp90 . Moreovein Hsp90 . Burnie didiasis . A humandidiasis ,180. Prefections . Using afections . Mycografections . Interesfections . The synis model ,186. Thetibodies . Additioesponses . Furtherimmunity . Raska edidiasis . On incodidiasis . Knotigodidiasis . Yang etzed mice . Recentlzed mice .Candida albicans strain [Candida infection, Als3 protein contributes to in vitro fungal killing by oral and vaginal epithelial cells [The Als3 encoded protein, which is a member of the agglutinin-like sequence family, is essentially an agglutinin, playing a crucial role in both fungal adhesion  and invas strain . A separal cells . C. albicans infection. Moreover, Als1 vaccinated B-cell-deficient mice were not susceptible to intravenous C. albicans infection [C. albicans and non-albicans Candida species by reducing kidney fungal burden [C. albicans infection [Candida protection, Spellberg et al. reported that Als3 vaccine-induced Th1 immunity was able to generate cross-vaccine protection against Staphylococcus aureus mediated bacterial systemic infection [Candida and Staphylococcus aureus infection [C. albicans and S. aureus infection by inducing Th1/Th17 immune responses and by enhancing neutrophil phagocytic killing of both organisms [Candida infection was mainly accomplished by the generation of cell-mediated immunity; indicating that antibody response can be used as surrogate markers of vaccine-mediated protection [C. albicans like adherence, filamentation, and biofilm formation [C. albicans germ tubes to human epithelial and vascular endothelial cells [In a seminal study, Spellberg et al. reported that the murine systemic candidiasis model can be used in the evaluation of pathogenesis and antifungal drug efficacy . Using tnfection ,203. Ibrl burden . Spellbel burden . Howeverl burden ,204. Micnfection . Apart fnfection ,207. Neinfection . Lin et rganisms . Spellbeotection . On analotection . Uppulurormation . A monocormation . Furtheral cells . Candida infection [in vivo [Candida and methicillin-resistant S. aureus infections by eliciting a strong B and T-cell response [C. albicans infections by inducing higher anti-Als3 antibody titers, which interfered with C. albicans ability to adhere and invade endothelial cells and form biofilms in vitro [+ T-cells against multidrug resistant C. auris infection in mice [In a Phase I clinical trial, Schmidt et al. were the first to report that NDV-3 vaccine formulation (using Als3) induced a safe and robust immune response in healthy humans, characterized by significantly high titers of anti-Als3 IgG and IgA1 antibodies . Unlike nfection . Regardl[in vivo . In addiresponse . Furtherresponse . NDV-3A response . It has in vitro . Recentl in mice .C. albicans, and has a well-established role in fungal virulence. The Sap2 genes of Candida contributes immensely to fungal pathogenesis by degrading most host proteins at epithelial sites, and can also hydrolyze complement [C. albicans, wherein Sap mutation resulted in attenuation of virulence during disseminated infections [Candida mutant strains lacking Sap2 exhibited attenuated virulence in a rat model of vaginal candidiasis [Sap2 gene belongs to the secreted aspartyl proteinase (Sap) family, which includes 10 members (Sap1-Sap10) . Sap2 ismplement . Gene difections . Using tfections . De Berndidiasis .C. albicans proteinases induced antibody responses in humans in response to Candida infection [C. albicans vaginal infection in a rat model [Candida-mediated vaginitis [C. albicans mediated vulvovaginal candidiasis [Previous studies have demonstrated that the nfection ,223. Casat model . De Bernaginitis . It has aginitis ,224. In aginitis ,226. Totaginitis . In a lodidiasis . Ghadjardidiasis .C. albicans had increased levels of anti-mannan and anti-Sap2 antibodies in vaginal fluid, which mediated protection against vaginal C. albicans infection [C. albicans mediated vaginitis in rats [C. albicans from the gastrointestinal tract of dermatitis patients [C. albicans infection, establishing the protective effect of anti-Sap2 antibodies against C. albicans mediated disseminated Candida infection [C. albicans [Animals infected with nfection . Both in in rats . Furtherpatients . Vilanovnfection . In a mualbicans .Candida infections can be attenuated by Sap2 inhibition has been supported to some extent in animal models by treatment with aspartic protease inhibitor pepstatin in mucosal and peritoneal Candida infections [C. albicans vaginitis, through induction of increased anti-Sap2 IgG and IgA antibodies titers in Sap2 immunized rats [The antibodies generated against Sap2 immunization have a role in enzyme neutralization, and the hypothesis that fections . Using azed rats . Furtherzed rats .C. albicans vaginitis [C. albicans infection [C. albicans infection in mice, immunization with murine DCs sensitized by pulsing with Sap2 protein was more immunoprotective [Candida vaginitis by inhibiting the inflammatory response during vaginitis caused by C. albicans in mice [C. albicans infection [C. albicans mediated systemic infection [Candida biofilm inhibition ability in vitro, enhanced neutrophil-mediated fungal killing, and protected naïve mice against systemic infection on passive transfer. Additionally, the findings of this study suggested a role of Candida-specific B1 and B2 B-cells during early stages of Sap2-mediated immune response [Bernardis et al. demonstrated that intramuscular immunization with a virosomal formulation of the Sap2 vaccine (PEV-7) induces protective antibody response in mouse and rat models . Immunizaginitis . The PEVaginitis  and PEV7aginitis . A studynfection . Notablyotective . Pericol in mice . Further in mice . More renfection . Additionfection . A recennfection . Anti-Saresponse .C. albicans hyphae, and has no effect on the fungus germination [C. albicans virulence by resisting phagocyte killing, a major host defense mechanism against candidiasis. Luo G et al. reported that rHyr1 protein plus alum adjuvant vaccination enhanced survival and reduced fungal burden in both immunocompetent and immunocompromised mice against disseminated candidiasis [C. albicans infection by directly neutralizing Hyr1 protein in vitro, resulting in enhanced mouse neutrophil-killing activity [C. albicans Hyr1 protein), has been shown to protect mice against lethal candidemia [Acinetobacterbaumannii mediated bacteremia and pneumonia [C. albicans and A. baumannii mixed biofilm formation in vitro [C. albicans Hyr1 protein provided cross-kingdom protection against gram-negative bacteria [Candida IgG, which were differentiated in vitro and screened against recombinant Hyr1 cell wall protein and whole fungal cell wall preparations [Candida antibodies enhanced phagocytosis and protected against disseminated candidiasis [HYR1 is a hyphae-expressed gene, required for hyphal growth and virulence. Bailey et al. showed that Hyr1 protein was strictly expressed on mination . Hyr1 isdidiasis . Additioactivity . Both acndidemia . It has ndidemia . Uppulurneumonia . Polycloin vitro , and monbacteria . Rudkin arations . The Hyrdidiasis . These sC. albicans hyphal cell wall protein, which is a substrate for mammalian transglutaminases. Hwp1 is an adhesion protein, expressed on the hyphal cell wall of C. albicans, which promotes the cross-linking of fungus with epithelial cells. It is a member of the GPI (glycosylphosphatidylinositol) anchor-dependent family of cell wall proteins, which is known to participate in covalent bonds formation with primary amines and buccal epithelial cells. Hwp1 protein has been shown to be important for both in vivo hyphal development and pathogenicity of C. albicans [in vitro and in vivo studies, making it a potential therapeutic target [C. albicans infection and celiac disease, wherein patients with celiac disease had high levels of anti-Hwp1 antibodies [Candida Hwp1-specific monoclonal antibodies could protect mice against invasive C. auris infection, by significantly enhancing survival and reducing fungal burdens [The Hwp1 gene encodes a albicans . Expressc target . Studiesc target . Naglik c target . Interestibodies . Vaccinatibodies . Recentl burdens .C. albicans adhesion with epithelial cells [C. albicans in mice. In humans, antibody responses to enolase are low or absent during colonization but increase after disseminated infection in immunocompetent hosts. In a study by Sundstorm et al., enolase was found to be an immunodominant humoral immunogen, which induced lymphocyte activation and stimulated both cell-mediated and humoral immune responses in mice [C. albicans infection. Passive transfer of enolase-specific antiserum reduced fungal burden in the target organs, and enolase-specific IgG1 and IgG2a antibodies could enhance neutrophil-mediated killing of C. albicans strains by opsonization [Candida enolase and fructose-bisphosphate aldolase can be used in the diagnosis of systemic candidiasis [Saccharomyces cerevisiae cells displaying enolase 1 antigen on their surfaces could elicit an immune response and aid the survival of mice challenged with C. albicans [C. albicans enolase by phage display technology and evaluated its role during in vitro and in vivo conditions. This monoclonal antibody inhibited the growth as well as plasminogen binding activity of C. albicans. Further, CaS1 administration enhanced survival time, reduced fungal burdens and also reduced the levels of inflammatory cytokines in a mice model of in vivo candidiasis [Enolase is an abundantly expressed cytosolic enzyme involved in the glycolysis pathway. It is secreted in the extracellular medium and is also found on the surface of fungal cell wall . It medial cells . The pre in mice . Passive in mice . In a se in mice . Since i in mice . Anothernization . It has didiasis ,261. Shialbicans . Anotheralbicans . Recentldidiasis .C. albicans. Phospholipase B (PLB) enzyme has both hydrolase and acyltransferase activities, and can be found in secreted or intracellular forms [C. albicans reduced the activity of phospholipase A2 enzyme and also attenuated fungal virulence [Candida strain than the parent Candida strain to both HUVEC and HT-29 epithelial cells in vitro [Candida PLB may play a crucial role in the dissemination of C. albicans via gastrointestinal and hematogenous routes [C. albicans could restore fungal virulence in vivo. The study used anti-PLB1 antibody to show that PLB1 is secreted during the invasion of the gastric mucosa by both parental and revertant strains, suggesting for the use of phospholipases as a vaccine candidate [Candida PLB, making it an attractive candidate for diagnostic use as well. Not much is reported in literature regarding B-cell or antibody mediated responses against PLB, except that it can be used as a potential vaccine candidate due to its hyphal-specific nature [Phospholipases are a heterogeneous group of enzymes that hydrolyze one or more ester linkages in glycerophospholipids, and have been proposed to contribute to the virulence of ar forms . It playirulence . The conirulence . Using sin vitro . These ss routes ,268. Mukandidate . Studiesc nature ,270.Candida cells from the host’s immune system [Candida and is considered to be one of the main immunodominant proteins [C. glabrata infection and has an immunodominant role [C. glabrata, for designing an effective epitope-based peptide vaccine [Fructose-bisphosphate aldolase (Fba1) is a multifunctional protein and an important enzyme of glycolytic pathway. It can facilitate fungal attachment to human cells or abiotic surfaces, and protects e system . In addiproteins . When β-proteins . Passiveproteins . Using aproteins . Not onlproteins . Phage vproteins . A studyant role . Furtherant role . Adams eant role . Using a vaccine .C. albicans cell wall protein Pyruvate kinase (Pk), which is a glycolytic enzyme, is of special interest, due to its involvement in oxidative stress as well as in the formation of biofilms. On screening for C. albicans sequences that encode proteins that are immunogenic during infections in humans, Swoboda et al. identified few glycolytic enzymes, including pyruvate kinase and alcohol dehydrogenase as non-ubiquitous immunogens during C. albicans infections [C. albicans infected mice immune sera obtained on different days post-infection using two-dimensional polyacrylamide gel electrophoresis, Pitarch et al. demonstrated the presence of pyruvate kinase as an immunoreactive antigen [C. albicans infection in mice and displayed the highest immunogenicity [The fections . Using afections . On assa antigen . More regenicity .C. albicans, six members of the SOD gene family have been identified, amongst which, Sod5 is exclusively extracellular, and is attached to the fungal cell surface via GPI anchors [C. albicans in invasive mouse models [C. albicans mediated disseminated candidiasis, it has been seen that the loss of the Sod5 gene resulted in attenuation of fungal virulence [C. albicans mutants lacking Sod5 were more susceptible to killing by macrophages and neutrophils and were associated with increased reactive oxygen species production by macrophages and neutrophils [Candida biofilm formation on venous catheters in a rodent model [C. albicans may be a novel therapeutic approach to combat systemic fungal disease [Superoxide dismutase (Sod) are antioxidant enzymes that convert superoxide radicals into less damaging hydrogen peroxide. In  anchors . Sod5 is anchors  and is re models . Since Se models , it is ce models ,270. Usiirulence . C. albitrophils . Additiont model . It has  disease .C. albicans malate dehydrogenase (Mdh1) has been screened by few proteome studies as a candidate for a vaccine against candidiasis [C. albicans mediated systemic infection in mice [didiasis ,286. Thedidiasis . On eval in mice .Candida infections. The fungal pathogen contains certain virulence factors on its cell wall, which contributes to immune evasion. Generation of innate and adaptive immune responses are essential for fungal clearance and the major players of innate and adaptive immunity include phagocytes, cytokines, complement proteins, T-cells, B-cells, and antibodies. Although cellular immune responses are critical for conferring protection against systemic fungal infection, the potential of humoral immunity in contributing to protective responses is still debated. In various studies, several Candida virulence antigens, like cell wall polysaccharides, surface proteins, and enzymes have been investigated as vaccine candidates to assess the protective immune responses against fungal challenge in animal models of systemic candidiasis. Many different subunit and conjugate vaccines have been prepared by using Sap2, Als3, Hyr1, enolase, mannan, and glucan, out of which only Sap2 and Als3 vaccines have successfully completed phase-1 trials while the rest of the vaccines are under preclinical trials. Most antifungal vaccines exert protection by inducing either (or both) B-cell and T-cell responses. In this review, we have consolidated the currently-available information on Candida vaccine antigens, induced B-cells, antibody immune responses and latest findings on the role of different types of antibodies in the antifungal defense against Candida infections.In the past few decades, significant progress has been made in understanding the protective host immune responses generated against invasive C. albicans by opsonization. Antibodies against Hsp90 exhibit direct antifungal activity by inhibiting Candida Hsp90 antigen, which is indispensable for fungus viability. Anti-mannan antibodies can activate both classical and alternate pathway of complement system and enhance opsonophagocytic killing of C. albicans. Direct antifungal activity is also reported for β-glucan and laminarin antibodies, which are known to inhibit fungal growth and adherence. Comparatively fewer reports are available for identifying mechanisms of protection for antibodies generated against Hwp1, PLB, Pk, Fba, Sod5, and Mdh1 vaccine antigens. In addition to functioning as antigen-presenting cells that contribute to the activation and differentiation of T-cells, B-cells can directly bind to fungal antigens and differentiate into antibody producing plasma cells which are the key players of antifungal humoral immune response. B-cells also produce memory B-cells that prevent secondary infections. B-cell-deficient mice fail to develop Th2-dependent immunity and have decreased levels of IL-4, IL-10, and TGF-β cytokines. Further, an antibody-independent role of B-cells during fungal infection has been seen on depleting B- cells in vitro. In addition, B1 B-cells have also been implicated in contributing to innate immunity against Candida species.Data from multiple literature reports summarized in this review clearly show that antibodies induced in response to leading vaccine candidates do contribute to protection against systemic candidiasis by utilizing a variety of mechanisms . Anti-SaCandida monoclonal antibodies and human-recombinant antibodies are considered promising immunotherapeutics against systemic candidiasis. Monoclonal antibodies generated against leading vaccine antigens have been shown to elicit various anti-Candida immune responses such as opsonophagocytosis, neutralization, fungal growth inhibition, biofilm inhibition, and direct candidacidal activity  can cross-react and confer protection against different fungal pathogens, makes a strong case for utilizing antibodies in adjunctive therapy. Synergistic combinations of monoclonal antibodies and antifungal drugs would not only provide a more potent and effective solution for combating emerging drug resistance in Candida species, but the enhanced specificity is also expected to improve clinical outcomes. Developing novel immunomodulatory approaches using monoclonal antibodies combined with administration of recombinant cytokines is another promising way for enhancing their therapeutic effect. In addition, new and upcoming technologies, such as multivalent antibodies, antibody-drug conjugates or antibody-antibiotic conjugates can be utilized for treatment of life-threatening invasive fungal infections.Likewise, anti-idiotypic antibodies and mimotopes which recognize yeast killer toxins are known to exhibit direct candidacidal activity. Antibody-derived peptides and single chain variable fragments have been used in many studies for active or passive vaccination in mice/rat model of experimental disseminated candidiasis. The finding that anti-Candida species merits the need for developing alternate immunotherapies against invasive candidiasis.The serious threat posed by invasive fungal infections is a persisting problem and new therapeutic options are urgently required including vaccine/s and/or antibody-based therapies. Challenges associated with the extensive use of antifungal drugs like antifungal drug-associated toxicity and emergence of antifungal drug resistance, along with increased incidences of infections due to multi-drug resistant non-albicans Candida vaccine/s.Due to the promising potential of humoral immunity in conferring antifungal protection, identification of fungal antigens which elicit protective antibodies will be crucial for designing effective multi-valent and/or multi-epitope-based anti-There is a need to synthesize novel peptides and oligosaccharides using a combination of protective antigens that could be used to make multi-epitope conjugate vaccines including B-cell and T-cell epitopes for generating memory. Although few experimental vaccines have been designed, extensive animal-based studies are lacking.Candida monoclonal antibodies along with elucidation of their protection mechanisms, will be beneficial in generating more precise antibody-based therapies. More clinical trials are needed to validate passive antibody therapy against invasive candidiasis.Additionally, identification, isolation and characterization of protective anti-Novel cytokine-based adjunctive immunotherapies and immunomodulators should be explored for the treatment of invasive candidiasis. More studies are needed to demonstrate the feasibility of such immunotherapies for improving the prognosis of invasive candidiasis.Candida. More studies are needed to assess the safety and efficacy of combination therapies.Synergistic use of monoclonal antibodies in combination with anti-fungal drugs has been seen to impart cooperative protection against Susceptibility of immunocompromised individuals having deficiencies in an immune cell repertoire to invasive candidiasis justifies future research for characterization of the protective B-cell and T-cell repertoires, aiding the development of novel vaccination strategies.


Hemiculter leucisculus  is a small cyprinid fish that has a wide distribution in East Asia. In this study, we characterized the complete mitochondrial genome of H. leucisculus in Hainan Island using Illumina MiSeq platform. The mitogenome contained 16,621 bp with AT content of 56.2%. The mitogenome of H. leucisculus comprised 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and one control region (D-loop). Phylogenetic analyses indicated that H. leucisculus in the Hainan Island formed independent lineage and the species of H. leucisculus might be a paraphyletic taxon.The sharpbelly,  Hemiculter leucisculus , is a small cyprinid fish that has a wide distribution in the drainage basins of China, Korea island, Japan, Mongolia, and Russia (www.fishbase.org). In China, H. leucisculus widely occupies various freshwater habitats, such as rivers, lakes, reservoirs, and even pools  on 27 October 2019 and was carefully distinguished this species according to the ichthyography by Luo and Chen  under the voucher numbers HL2019001. We sequenced the complete mitochondrial genome using the Illumina MiSeq platform  and assembled the raw sequence reads into contigs using SPAdes 3.9.0. The complete mitochondrial genomes were finally obtained utilizing the contigs in SOAPdenovo . This mitogenome showed similar AT contents with that in previous reported H. leucisculus populations in the Hainan Island, we downloaded published Hemiculter mitogenomes and built Bayesian inference (BI) and maximum-likelihood (ML) trees using the combined DNA sequences of 13 PCGs and corresponding amino acids in RAXML-VI-HPC (Stamatakis Xenocypris davidi (Genbank nos: KF039718) from Xenocyprinae was selected as an outgroup. Optimal nucleotide substitution model of GTR + I + G for phylogenetic analyses were selected using the Akaike information criterion in MRMODELTEST version 2.3 (Nylander H. bleekeri (H. leucisculus in the Hainan Island formed an independent lineage and H. leucisculus was a paraphyletic taxon (To infer the phylogenetic status of Nylander . For theNylander , while BNylander . The disbleekeri . All treic taxon . However