Bartonella spp. comprise a genus of Gram-negative alphaproteobacteria that are slow growing, fastidious, and facultative intracellular pathogens with zoonotic potential. Immunofluorescent antibody assays (IFAs), Western blot (WB), and enzyme-linked immunosorbent assays (ELISAs), the frequently used modalities for the serological diagnosis of canine and human Bartonelloses, generate numerous false negative results. Therefore, the development of a reliable serodiagnostic assay for Bartonelloses is of clinical and epidemiological importance. Pap31, a heme binding surface protein of B. henselae, is associated with bacterial adhesion and related to bacterial colonization. To our knowledge, B. henselae Pap31 and its fragments , middle (MD), and C-terminal (CTD) domains) have not been investigated for the serodiagnosis of canine and human Bartonelloses. In this study, we evaluate the diagnostic utility of B. henselae recombinant whole Pap31 (rPap31) and Pap31 fragments by ELISA using sera from 70 dogs , and 34 Bartonella spp. IFA- and PCR-negative (control dogs)) and 36 humans  and 18 controls)). In the dogs, the area under the curve (AUC) score of recombinant whole Pap31 was 0.714 with a sensitivity of 42% and specificity of 94% at an OD cutoff value of 0.8955. Among the evaluated recombinant Pap31 proteins for the diagnosis of canine Bartonelloses, rPap31-NTD yielded the highest AUC score of 0.792 (95% CI 0.688–0.895) with a sensitivity of 44% and specificity of 100% at a cutoff value of 1.198. In concordance with this finding, rPap31-NTD also had the highest AUC score of 0.747 (95% CI 0.581–0.913) among the Pap31 recombinant proteins for the diagnosis of human Bartonelloses, with 39% sensitivity and 94% specificity at a cutoff value of 1.366. Recombinant whole Pap31 (rPap31) resulted in 72% sensitivity and 61% specificity at a cutoff value of 0.215 for human Bartonelloses. Due to either low sensitivity or questionable specificity, our findings indicate that recombinant Pap31 and the selected fragments may not be appropriate diagnostic targets in detecting anti-Bartonella antibodies in Bartonella-infected dogs and humans. The findings from this study can be used to further assess the antigenicity and immunogenicity of B. henselae Pap31 as a diagnostic target. Bartonella spp. Comprise a genus of Gram-negative alphaproteobacteria that are slow growing, fastidious, and facultative intracellular pathogens with zoonotic potential [Bartonella spp. are transmitted by arthropod vectors, including fleas, lice, sand flies, and ticks, by animal bites and scratches, or via direct contact with Bartonella-infected clinical specimens [. Bartonella spp. are associated with a broad spectrum of clinical signs and pathological abnormalities in dogs [Bartonella spp. develop clinical manifestations that are similar or identical to diseases observed in human patients infected with the same Bartonella spp., including bacteremia, encephalitis, endocarditis, fever of an unknown origin, lymphadenomegaly, myocarditis, ocular disease (uveitis), peliosis hepatis, and vasculitis [Bartonella spp. have been reported to infect dogs, with B. henselae, B. koehlerae, and B. vinsonii subsp. berkhoffii being the species or subspecies most frequently identified in sick dogs in North America [Bartonella infection in dogs have not yet been critically investigated.otential ,2,3,4. Bpecimens ,6. Barto in dogs ,2,3,4. Dsculitis ,8,9,10. Bartonella by culture, the amplification of Bartonella DNA by PCR, and the detection of Bartonella antibodies by serological assays. Although serology can only confirm exposure, immunofluorescent antibody assays (IFAs), Western Blot (WB), and enzyme-linked immunosorbent assays (ELISAs) are most frequently used for the diagnosis of canine and human Bartonelloses [Bartonella IFA antigens, each tested as an independent serological assay [Bartonella antibodies were not detected in 38% of the Bartonella spp. bacteremic dogs, which indicated that IFA substantially underestimates the true serological prevalence of Bartonella spp. infections in dogs. In concordance with our initial findings of poor IFA sensitivity, using a panel of 3 Bartonella IFA antigens, we subsequently confirmed even lower IFA sensitivity (6%) in dogs with hemangiosarcoma, despite the amplification of Bartonella spp. DNA from 73% (80/110) of the dogs [Bartonella spp. infection in most (71%) PCR-positive dogs by qPCR and ddPCR amplification, B. henselae WB also had a low sensitivity (33%) in dogs with hemangiosarcoma [Currently, the diagnosis of canine and human Bartonelloses is performed by the isolation of nelloses ,18,19,20al assay . In thatthe dogs . Similarosarcoma . Due to Bartonella henselae Pap31, an outer membrane protein, appears to be an important virulence factor for bacterial attachment and the colonization of mammalian cells, thus promoting the establishment of B. henselae infection in the host [Bartonella henselae Pap31, homologous to the hemin binding protein family of B. quintana, is also involved in heme acquisition [Bartonella henselae Pap31 shares 58.4% identity with the heme binding protein A (HbpA) of B. quintana and 31.7% identity with OMP31 porin protein of Brucella melitensis. Pap31 proteins act as adhesins for fibronectin, heparin, and human umbilical endothelial cells (HUVECs), thereby mediating host–pathogen interactions [B. bacilliformis, the etiological agent of Carrion’s disease, has been previously evaluated as a candidate antigen for the development of a reliable serological assay for human Bartonelloses [B. henselae infective endocarditis patients also identified Pap31 as an immunoreactive candidate protein for the serodiagnosis of Bartonelloses [the host ,25,26. Buisition ,25. Bartractions . The Papnelloses . Anothernelloses .B. henselae recombinant Pap31 and selected fragments have not been investigated for the serodiagnosis of canine and human Bartonelloses. Therefore, we evaluated the diagnostic utility of B. henselae full-length recombinant Pap31 (rPap31) and recombinant Pap31 fragments  by ELISA. Sera from dogs and humans exposed to or infected with Bartonella spp. and control groups  were used to screen recombinant Pap31 proteins and peptides. We hypothesized that B. henselae Pap31 protein elicits a sensitive and specific humoral immune response in dogs and humans exposed to Bartonella spp. The aims of this study are: (1) to evaluate the sensitivity and specificity of the entire B. henselae rPap31 protein; (2) to compare the sensitivity and specificity of the recombinant Pap31 N-terminal domain (rPap31-NTD), middle domain (rPap31-MD), and C-terminal domain (rPap31-CTD); and (3) to evaluate the diagnostic utility of B. henselae Pap31 linear B-cell epitopes for the serodiagnoses of canine and human Bartonelloses.To our knowledge, B. henselae Pap31 with a likelihood probability of 0.9986. Lipoprotein signal peptide (Sec/SPII), Tat signal peptides (Tat/SPI), and other signal peptides were absence in B. henselae Pap31 with a likelihood probability of <0.0007. The signal peptide (Sec/SPI) cleavage site was predicted between amino acid positions 22 and 23 with a probability of 0.9902. No transmembrane helices were predicted using TMHMM-2.0, as described in the methods section. Bartonella henselae Pap31 protein was predicted to be an outer membrane protein as determined by PSORTb with a localization score of 9.93 and by Protter, Based on signal peptide analysis, a standard secretory signal peptide (Sec/SPI) was found in B. henselae Pap31 protein using the IEDB Analysis Resource software (BepiPred 2.0) revealed five linear B-cell epitope regions, as represented by the yellow areas above the threshold score of 0.5, In silico analysis of E. coli BL21 (DE3) clones confirmed the insertion of the entire pap31 gene, as well as the three pap31 gene fragments into the pET200D/TOPO expression system in the correct reading frame and current orientation. BLAST searches indicated that the in-frame translated amino acid pap31 gene insert in the recombinant plasmid had 100% homology with B. henselae Pap31 (GenBank: AAC39274.1). With the exception of rPap31-MD, purified rPap31 and two rPap31 fragments (rPap31-NTD and rPap31-CTD) yielded a single band, as confirmed by Coomassie stained SDS-PAGE and Western blot analysis, Sanger sequencing of plasmids isolated from the respective recombinant Bartonella spp.; n = 36) and Group II (n = 34 control dogs) for canine Bartonelloses. When testing sera from Group I and II dogs, the sensitivity and specificity of rPap31 was 42% and 94%, respectively, at an OD450 nm cutoff value of 0.8955, with a maximum value of the Youden index as determined by ROC analysis, In this study, the entire length recombinant Pap31 and three domains of rPap31 proteins were tested in an ELISA format using Group I (infected with p < 0.05) for Bartonella antibodies in naturally infected dogs when compared to the controls, Recombinant Pap31-NTD yielded the highest AUC of 0.792 (95% CI 0.688–0.895), Bartonella spp; n = 18) and Group IV  human serum samples were tested to evaluate the diagnostic utility of the rPap31 and rPap31 domains for the diagnosis of human Bartonelloses. Recombinant Pap31 resulted in 72% sensitivity and 61% specificity at a cutoff value of 0.215, Group III (infected with p = 0.01189) in rPap31-NTD reactivity among human Group III compared to Group IV (control group), p > 0.05) of Bartonella spp. antibodies when testing naturally infected human sera (Group III) compared to control sera, There was a significant difference , Bartonella IFA and rPap31-NTD ELISA (kappa = 0.48) and between Bartonella IFA and rPap31-MD ELISA (kappa = 0.46), when the IFA and ELISA assays were performed on the dogs’ sera samples, Bartonella IFA and rPap31 ELISA (kappa = 0.352) and between Bartonella IFA- and rPap31-CTD-based ELISA (kappa = 0.337) for the canine Bartonelloses, Bartonella IFA results with ELISA results for human Bartonelloses, there was a fair agreement between Bartonella IFA and rPap31 ELISA (kappa = 0.333) and a moderate agreement between Bartonella IFA and rPap31-NTD ELISA (kappa = 0.444), Bartonella IFA and rPap31-MD or rPap31-CTD ELISA was slight  for human Bartonelloses.When comparing Bartonella IFA-positive dogs were also positive on rPap31-NTD ELISA, while 19 (56%) of the 34 Group II control dogs tested negative by rPap31-NTD ELISA, Bartonella IFA-positive and 8 (44%) Group IV controls tested ELISA-positive by rPap31-NTD at a cutoff value of 0.795, At an OD cutoff value of 0.646, 33 of the 36 (92%) Group I Bartonella henselae IFA-positive dogs (n = 8) or IFA-positive humans (n = 8) naturally infected with Bartonella spp. In addition, using dot blot analysis, these peptides were also not reactive with the same 16 B. henselae IFA-positive dog and human sera.Despite the coating of ELISA plates with high concentrations of B-cell peptides  and the use of dog or human sera at 1:50 and 1:100 dilution, none of these peptides were reactive with the sera from Bartonella infection in dogs is questionable due to low sensitivity. Among the rPap31 protein fragments, rPap31-NTD yielded the highest AUC score of 0.792 (95% CI 0.688–0.895) with a sensitivity of 44% and specificity of 100% at a cutoff value of 1.198 when testing dog sera, indicating the relatively poor diagnostic sensitivity of rPap31 and the selected fragments for the diagnosis of canine Bartonelloses. In concordance with the findings from dogs, rPap31-NTD also had the highest AUC score of 0.747 (95% CI 0.581–0.913) among the rPap31 protein fragments for the diagnosis of human Bartonelloses, with 39% sensitivity and 94% specificity at a cutoff value of 1.366. Of the rPap31 protein fragments, the ELISA seroreactivity of rPap31-NTD was significantly different when comparing human Group III  and Group IV (control) individuals. However, there was no difference in the reactivity for the two other rPap31 protein fragments when human IFA-reactive sera were compared to control sera. Due to low sensitivity and questionable specificity, our findings indicate that recombinant Pap31 and none of the selected Pap31 fragments are appropriate as diagnostic targets for detecting anti-Bartonella antibodies in Bartonella-infected dogs or humans.In this study, we evaluated the diagnostic utility of rPap31 and three Pap31 fragments  by ELISA for canine and human Bartonelloses. When testing sera from the dogs and humans that were IFA-positive and/or PCR-positive, the rPap31-NTD and rPap31-MD were the most strongly reactive among the selected recombinant proteins. In dogs, the AUC score of recombinant whole Pap31 (rPap31) was 0.714, with a sensitivity of 42% and specificity of 94% at the OD cutoff value of 0.8955 with a maximum Youden index value, suggesting the diagnostic utility of rPap31 for the diagnosis of Bartonella antibodies present in the control group sera. Alternatively, some IFA seronegative humans (control group) may have been exposed to a Bartonella spp., but immunofluorescence was not visualized using cell cultured Bartonella spp. antigens, as was previously reported (16). In the context of specificity, B. henselae Pap31 shares homology with Neisseria opacity proteins (Opa), Brucella OMP31 (a putative porin), and Agrobacterium tumefaciens OMP25 (an immunogenic surface protein) [B. henselae Pap31 protein with proteins of other microorganisms most likely contributed to the low specificity found in this study. These findings indicate that recombinant Pap31 proteins may generate false positive results due to the cross-reactivity with antibodies induced against antigenically similar proteins found in other microorganisms.Nonetheless, rPap31 appears to be highly specific (94%) for the diagnosis of canine Bartonelloses, the specificity of rPap31 was only 61% for the diagnosis of human Bartonelloses. In contrast to the significant differentiation of the infected and control dog groups, there was no significant difference in rPap31 seroreactivity between the infected and control humans. For humans, rPap31 ELISA resulted in 72% sensitivity and 61% specificity, at a cutoff value of 0.215, indicating the potential cross-reactivity of rPap31 with non-protein) ,30. WithA number of factors, such as the selection of cutoff values, antigen preparation, sample population, and standard methods used for the calculation of cutoff values, may influence the assessment of the diagnostic accuracy of an ELISA . The diaBartonella spp. proteins that are associated with survival, multiplication, or bacterial adaptation in accidental and reservoir hosts should be useful targets for the diagnosis of Bartonelloses. Additionally, certain proteins that are important for the pathogen’s survival can be useful for vaccine development but not for serological diagnosis, because of their low antigenicity and high immunogenicity. As a hallmark of these intra-erythrocytic bacteria, the penetration of erythrocytes is an invasion strategy used to obtain heme, which is essential for the growth of Bartonella spp. in vivo and in vitro [Bartonella spp., including the heme binding protein A (HbpA) of B. quintana and Pap31 of B. henselae, play an active role in hemin acquisition, survival, and disease pathogenesis [B. bacilliformis recombinant Pap31 for the diagnosis of Oroya fever, the authors demonstrated that Pap31 antigens were highly induced in growing cultures of B. bacilliformis and were immunologically recognized dominant proteins in infected humans, supporting the application of Pap31 for the ELISA and WB diagnosis of human Bartonelloses [B. bacilliformis [B. bacilliformis recombinant pap31 was not reactive with sera from patients with Coxiella burnetti, Brucella spp., or B. henselae infection, supporting a lack of recombinant B. bacilliformis Pap31 cross-reactivity with these microorganisms [B. bacilliformis findings, B. henselae recombinant Pap31 proteins were reactive with sera from a number of dog and human controls in this study. Our findings indicate that the optimization of a recombinant Pap31 protein-based serological assay, perhaps by combining rPap31 fragments in a chimeritope may be needed to enhance sensitivity, specificity, and diagnostic accuracy for the diagnosis of human Bartonelloses.Logically, in vitro ,26,30,32ogenesis ,26,30. Togenesis ,33,34. Pnelloses . Dichterliformis . In anotB. henselae IFA-positive dogs or humans. Post-inoculation sera from dogs that were experimentally infected with either B. henselae San Antonio 2 or B. henselae CSU 1 strains, and dogs experimentally infected with B. vinsonii subsp. berkhoffii genotype III, were also not reactive to these synthetic peptides, suggesting that three-dimensional epitope conformation is likely of critical importance for the documentation of Pap31 seroreactivity. These findings are consistent with the poor seroreactivity results of B. bacilliformis Pap31 linear epitopes reported in a previous study [B. bacilliformis infection were reactive to B. bacilliformis Pap31 linear epitopes in line blots. It is also possible that B. henselae Pap31 peptide epitopes represent a minimally immunogenic region of the B. henselae Pap31 protein, and hence were not reactive with sera from B. henselae IFA-positive dogs and humans in this current study.Despite the predicted high antigenicity scores of the four selected antigenic Pap31 B-cell epitopes , none of these epitope peptides were reactive with sera from us study , where oBartonella spp. PCR- and IFA-negative) samples are critical for assessing the specificity of serological assays for the diagnosis of Bartonelloses. A limitation of this study is the potential inclusion of serum samples from sick dogs and humans into the respective Bartonella spp. PCR- and IFA-negative control groups, which would negatively impact our assessment of ELISA specificity for the diagnosis of canine and human Bartonelloses. Dog sera were submitted to the NCSU-CVM-VBDDL by veterinarians to test for evidence of exposure to or infection with canine vector-borne disease (CVBD) organisms; therefore, despite negative IFA (with a documented poor sensitivity) and PCR results (similarly poor sensitivity in association with chronic infection), these dogs could have been previously exposed to 1 or more of the 40 plus Bartonella species [Rickettsia rickettsii appeared to reactivate occult and previously undetected Bartonella spp. infections [Bartonella spp. environmental exposures [Bartonella infection and misdiagnosed as unexposed to or uninfected by Bartonella PCR, BAPGM, and IFA testing due to the less than perfect sensitivity of each of these diagnostic assays [Bartonella infection in healthy and sick dogs [The selection of true negative control  for human sera. Therefore, the potential cross-reactivity of recombinant Pap31 proteins with sera from humans infected with phylogenetically related pathogens was not addressed in our study. In addition, the information on infecting Bartonella spp., genotype, or strain was lacking for 33 Group I (IFA-positive) dogs and 9 Group III (IFA-positive) humans. NCBI Blast searches indicate that B. henselae Pap31 shares 50% to 100% identity with Pap31 sequences of other Bartonella spp., including medically important animal and human pathogens: B. henselae Pap31 (92 to 100%), B. koehlerae Pap31 (83%), B. quintana Pap31 (55%), and 50% with B. bacilliformis Pap31 (50%). Based upon IFA testing, the sera used in this study may or may not have detected antibodies to all known Bartonella spp., genotypes, or strains, which further complicates the evaluation of Pap31-based ELISA sensitivity and specificity.Another limitation of this study is the lack of historical and clinical information for the diagnostic specimens submitted for NCU-CVM-VBDDL testing. Although all Group III human patients were tested because of a self-reported history of chronic illness, these sera represented a very heterogenous sample set in the context of symptomatology and duration of illness. This information would be of importance to compare Pap31 seroreactivity versus disease status and duration. Additionally, only B. henselae Pap31 as a diagnostic target. Although our results are potentially promising, further studies are needed to optimize a B. henselae Pap31-based ELISA for the diagnosis of Bartonella infection in dogs and humans.In conclusion, with additional assessment and optimization, the recombinant Pap31 and rPap31-NTD fragment may be appropriate for canine Bartonelloses diagnostic applications, whereas rPap31-NTD may have application for the diagnosis of human Bartonelloses. The findings from this study can be used to further assess the antigenicity and immunogenicity of Bartonella PCR, Bartonella droplet digital PCR (ddPCR), and Bartonella IFA testing results for the dog and human clinical samples that were used for comparative ELISA in this study are summarized in Bartonella exposure. In this study, the serum samples from 70 dogs  and Group II: 34 Bartonella spp. IFA- and PCR-negative (control dogs)) and 36 humans  and Group IV: 18 Bartonella spp. IFA- and PCR-negative (control humans)) were tested by rPap31, rPap31-NTD, rPap31-MD, and rPap31-CTD-based ELISA. All 70 dogs’ sera were submitted to NCSU-VBDDL for diagnostic testing between 2016 and 2019. All 36 humans’ sera were obtained from the repository maintained by the NCSU-CVM-IPRL  between 2009 to 2020.The Bartonella spp. naturally infected dogs (Bartonella IFA-positive). A cutoff titer of ≥1:64 was used to define an IFA-positive titer. Of the 36 dogs, 1 dog had a B. henselae IFA IgG titer of 1:64, whereas the remaining 35 dogs had B. henselae IFA IgG titers of ≥1:128. Of these 36 dogs, 23 were concurrently B. vinsonii subsp. berkhoffii genotype I seropositive (IFA titer range ≥ 1:64 to 1:4096) and 32 dogs were B. koehlerae seropositive (IFA titer range ≥ 1:64 to 1:4096). A total of 21 of the 36 dogs were IFA-positive (titers ≥ 1:64) to all 3 Bartonella spp. . Of the 36 Group I dogs, Bartonella DNA was amplified from the blood of 3 dogs: 2 dogs were PCR-positive for B. vinsonii and 1 dog was PCR-positive for B. henselae,Seventy archived sera from dogs previously tested at the North Carolina State University, College of Veterinary Medicine, Vector Borne Diseases Diagnostic Laboratory (NCSU-CVM-VBDDL) or the Intracellular Pathogens Research Laboratory, NCSU-CVM (NCSU-CVM-IPRL) were selected for comparative ELISA testing utilizing each of the purified recombinant Pap31 proteins, Bartonella PCR, BAPGM enrichment blood culture, and the IFA serological panel used to test these study participants were previously published [Rickettsia rickettsii, Ehrlichia canis, Babesia canis, and Babesia gibsoni, and were seronegative to Anaplasma phagocytophilum, Anaplasma platys, Borrelia burgdorferi, Ehrlichia canis, and Ehrlichia ewingii by ELISA . The blood from these 24 dogs was PCR-negative after whole blood DNA purification for Babesia, Ehrlichia, Anaplasma, Rickettsia, hemotropic Mycoplasma, and Leishmania spp.Based on NCSU-CVM-VBDDL serological and PCR testing, 24 of the 36 Group I dogs were seronegative for evidence of exposure and PCR-negative for evidence of infection with other canine vector-borne disease (CVBD) organisms that are routinely tested for in the NCSU-CVM-VBDDL. Detailed methods for the ublished ,21. SpecR. rickettsii (IFA titer 1:128), Babesia canis (IFA titer 1:2048), and Babesia gibsoni (IFA titer 1:2048); 1 dog was seroractive to R. rickettsii (IFA titer 1:512) and Babesia canis (IFA titer 1:64); 1 dog was seropositive to Babesia canis (IFA titer 1:1024) and Babesia gibsoni (IFA titer 1:4096); and 1 dog was seropositive to Ehrlichia canis (IFA titer 1:2048). The CVBD PCR and serological testing results were not available for the remaining eight Bartonella spp. IFA seropositive dogs.Of the remaining 12 dogs, 4 were seropositive as follows: 1 dog was seroractive to Bartonella spp., Bartonella spp. . All 34 Group II dogs were PCR-negative after whole blood DNA extraction for Bartonella spp. Whole blood EDTA from 21 of 34 Group II dogs was processed in Bartonella Alpha Proteobacteria Growth Medium (BAPGM) in the NCSU-CVM-IPRL, as described previously [Bartonella DNA was not amplified from any extracted blood sample before and after the BAPGM enrichment blood culture. Bartonella DNA was also not amplified from reagent controls or BAPGM-negative (un-inoculated) culture controls. Bacterial growth was not visualized following the direct culture of blood or subculture from BAPGM onto Trypticase soy agar II (TSA) supplemented with 5% sheep blood plates. Due to the inadequate blood volumes for BAPGM enrichment blood culture, testing results were not available for the remaining 13 dogs.Group II consisted of 34 dogs for which diagnostic testing in the NCSU-CVM-VBDDL and NCSU-CVM-IRPL did not provide evidence of the exposure to or infection with a eviously . BartoneBabesia, Ehrlichia, Anaplasma, Rickettsia, hemotropic Mycoplasma, and Leishmania spp., and were seronegative to Anaplasma phagocytophilum, Anaplasma platys, Borrelia burgdorferi, Ehrlichia canis, and Ehrlichia ewingii by ELISA  or in-house IFA assays. A total of 10 of these 16 dogs were R. rickettsii seropositive (IFA titer of ≥1:64). Rickettsia rickettsii is phylogenetically related to the genus Bartonella, belonging to the class alphaproteobacteria [R. rickettsii IFA-positive dogs were included in the Group II dogs. Additional serological testing for the exposure to CVBD organisms was not available for the remaining for 18 dogs.Sixteen Group II dogs were PCR-negative after whole blood DNA extraction for bacteria . TherefoB. henselae rPap31 and its recombinant fragments  for the serodiagnosis of human Bartonelloses, a set of 36 diagnostic serum samples obtained from the repository maintained by the NCSU-CVM-IPRL  were tested by ELISA. Detailed methods for the Bartonella PCR, BAPGM enrichment blood culture, and the IFA serological panel used to test these study participants were previously published [Bartonella spp. All 18 individuals were B. henselae, B. koehlerae, or B. vinsonii subsp. berkhoffii genotype III seropositive. Fifteen Group III humans were B. henselae seropositive (IFA titer of ≥1:64), B. koehlerae and B. vinsonii subsp. berkhoffii genotype III IFA seropositive, but were B. henselae seronegative. Of the 18 people, 8 were IFA seropositive to B. vinsonii subsp. berkhoffii genotype I, 10 to B. vinsonii subsp. berkhoffii genotype II, 14 to B. vinsonii subsp. berkhoffii genotype III, and 11 to B. koehlerae. A total of 5 of the 18 people were seropositive to all 5 IFA antigens B. henselae, B. koehlerae, and B. vinsonii subsp. Berkhoffii genotypes I, II, and III.To evaluate the diagnostic utility of ublished . Group IBartonella blood qPCR, or droplet digital PCR (ddPCR) [Bartonella PCR-positive and B. henselae IFA-positive  and B. henselae IFA-positive (IFA titer of ≥1:64); 6 were Bartonella ddPCR-positive  and B. henselae IFA seropositive; and 1 B. henselae seropositive individual was infected with B. vinsonii subsp. berkhoffii genotype I. The three remaining people were Bartonella ddPCR-positive, B. henselae IFA-negative, but were seropositive to both B. koehlerae and B. vinsonii subsp. berkhoffii genotype III.Based on  (ddPCR)  and IFA Bartonella spp. IFA-negative and were Bartonella PCR-negative from blood and BAPGM enrichment blood culture, B. henselae, B. vinsonii subsp. berkhoffii genotypes I, II, and III, and B. koehlerae. Based on the Bartonella PCR and BAPGM enrichment culture testing, Bartonella DNA was not amplified from any blood or BAPGM enrichment blood culture.To evaluate ELISA rPap31 proteins for specificity, Group IV sera (n = 18) were selected from the study participants that were B. henselae Pap31 protein (1 to 279 amino acids) was derived from the NCBI database. To determine the signal peptide and its cleavage site, signal peptides of the Pap31 protein were determined using the Signal P-5.0 Server . The transmembrane topology of B. henselae was examined by TMHMM.v2.0 (http://www.cbs.dtu.dk/services/TMHMM/ accessed on 8 June 2020). PSORTb v3.0.3 was used to predict the Pap31 subcellular localization site of Pap31 (https://www.psort.org/psortb/ accessed on 8 June 2020). The integration and visualization of annotated and predicted protein sequence features of B. henselae Pap31 were predicted using Protter (https://wlab.ethz.ch/protter/start/ accessed on 8 June 2020).The amino acid sequence of the B. henselae San Antonio (Bh SA2) Pap31 (rPap31) protein and each of the three Pap31 fragments  were cloned and expressed using the Escherichia coli expression system. Four Pap31 primer sets were used for conventional PCR amplification, as shown in Escherichia coli were used as negative controls. Genomic DNA from B. henselae Houston-1 was used as a positive control. For all 4 PCR assays, conventional PCR was performed in an Eppendorf Mastercycler EP gradient under the following conditions: a single hot-start cycle at 95 °C for 2 min followed by 30 cycles of denaturing at 95 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 30 s. Amplification was completed by an additional cycle at 72 °C for 2 min. The PCR products were analyzed by 2% agarose gel electrophoresis with detection using gel red . Prior to the ligation reaction for cloning, PCR products were purified by gel extraction using PureLink quick gel extraction and a PCR purification combo kit .The entire pap31 gene was cloned and expressed using a ChampionTM pET200 Directional TOPO® Expression kit . Whole pap31 (encoding 25 to 279 amino acids (aa)) and 3 fragments of the pap31 gene, C-terminal (25 to 94 aa), middle (95 to 187 aa), and N-terminal (188 to 279 aa) domains, were cloned according to the manufacturer’s instructions , The E. coli BL21 (DE3) Star™ chemically competent cells and plated on LB plates containing 50 µg/mL of kanamycin. Positive clones were confirmed by plasmid extraction followed by sequencing. Amplicon products were sequenced using T7 and T7 reverse primers by Sanger sequencing . Chromatogram evaluation and sequence alignment was performed using SnapGene software  to confirm the in-frame cloning of pap31 and pap31 gene fragments. The nucleotide sequence homology of the PCR amplicon was performed with available nucleotide sequences at the NCBI databases using the NCBI BLAST program (v2.0).Recombinant plasmids were subsequently transformed into E. coli BL21 (DE3) clone containing pap31 inserts was grown in 25 mL of LB broth at 37 °C containing 50 µg/mL of kanamycin. When the optical density (OD600 nm) of the culture bacteria reached 0.6 to 0.8, 1mM IPTG was added to induce protein expression. Cultures were examined for protein expression at 0, 3, 6, and 8 h post-IPTG induction at 30 °C by immunoblotting of whole cell lysates using a Pierce™ 6X-His Epitope-Tag mouse monoclonal antibody  and an anti-mouse IgG secondary antibody .To confirm and monitor the expression of recombinant proteins, each TM Cobalt spin columns, according to the manufacturer’s instructions . For purification, each recombinant clone was grown at 37 °C overnight in a 25 mL LB broth containing 50 µg/mL of kanamycin in a shaker at 200 rpm. After overnight incubation, 5ml of the overnight culture was transferred to fresh 1000 mL LB broth containing 50 µg/mL of kanamycin and was incubated at 37 °C for 2 h in a shaking platform at 200 rpm. After 2 h, IPTG was added to a final concentration of 1 mM and incubated at 30 °C for 8 h. The cultures were then centrifuged at 5000× g to obtain the pellets. The pellets were processed using BugBuster Master Mix  to purify inclusion bodies according to the manufacturer’s instructions. After the inclusion body purification, the pellets were then subsequently solubilized in 1× PBS containing 6 M of urea. His-tagged proteins were extracted from inclusion body suspensions using HisPur™ Cobalt spin columns . Wash buffer (1× PBS with 6 M urea and 5 mM imidazole) and Elution buffer (1× PBS with 6 M urea and 100 mM imidazole) were used during the His spin column purification steps. Eluted proteins were analyzed by SDS-PAGE in Criterion™ using 4–15% gradient polyacrylamide tris-glycine precast midi gels  at a constant current (100 V) for 1 h 50 min in a 1× running buffer . A pre stained broad-range (10–250 kDa) molecular weight protein marker  was used as a standard. Fractionated proteins were visualized by staining the gel overnight with Bio-safe™ Coomassie brilliant blue . Purified recombinant proteins were verified by WB using Pierce™ 6X-His Epitope-Tag mouse monoclonal antibody  and anti-mouse IgG secondary antibody . Proteins were dialyzed into phosphate-buffered saline  using Slide-A-Lyzer™ Dialysis Cassettes, .Recombinant rPap31s  were purified by column-chromatography using HisPurBartonella spp. were used to screen the purified recombinant proteins. In brief, ELISA plate wells were coated with 100 µL of 10 µg/mL recombinant purified proteins using a carbonate buffer, pH 9.6. Plates were incubated at 4 °C overnight. After overnight coating, plates were washed with 1× PBS 4 times. Plates were then incubated with a blocking solution (1× PBS containing 3% milk) at room temperature (RT) for 2 h, followed by washing with 1× PBS containing 0.05% Tween-20  4 times. Plates were incubated with 100 µL of dog or human sera at 1:100 dilution in 1× PBS containing 3% milk at RT for 1 h. Plates were washed with 1× PBS containing 0.05% Tween-20 4 times, followed by incubation with 100 µL of secondary antibody for 1 h at RT. HRP-conjugated goat anti-dog IgG  and HRP-conjugated goat anti-human IgG  in 1× PBS containing 3% milk were used as secondary antibodies for dog and human ELISA testing, respectively. After secondary antibody incubation, the plates were washed 5 times with 1× PBS containing 0.05% Tween. A total of 50 µL of 1-Step™ Ultra TMB-ELISA substrate solution  was then added to the plate wells and incubated at RT for 15 min, followed by the addition of 2M H2SO4 to stop the reaction. The absorbance was measured at 450 nm of the wavelength using a Tecan plate reader. Sera from dogs naturally infected with B. henselae (B. henselae IFA titer = 1: 512) and Bartonella PCR-negative and IFA-negative dogs were used as the positive and negative controls, respectively. The plate wells coated with only coating buffer (without protein) were used as blanks to determine the subtract background noise. Based on the results from the negative controls, a baseline was established for scoring individual samples as positive or negative for infection with Bartonella. The average absorbance value was calculated for each set of duplicate samples.The sensitivity and specificity of purified recombinant Pap31 proteins were evaluated by ELISA using sera from Group I (n = 36) and II  dogs, and from Group III (n = 18) and IV  humans, as described in the Methods Sections for canine and human Bartonelloses, respectively. Each protein was immobilized in duplicate in ELISA plate wells (500 ng/well), using carbonate buffer and standard methods that have been well described . Sera frB. henselae Pap31 protein sequence to predict the linear B-cell epitopes [B. henselae Pap31, https://web.expasy.org/protparam/ accessed on 20 January 2020). The protein secondary structure of B. henselae Pap31 was determined using PSIPRED (http://bioinf.cs.ucl.ac.uk/psipred/ accessed on 22 January 2020). The antigenicity of the predicted linear B-cell epitopes was evaluated by VaxiJen 2.0. A total of 53 linear B-cell epitopes were predicted by the above-mentioned 6 algorithms, Several B-cell epitope prediction tool methods have been developed over the years, based on a Hidden Markov Model (HMM) with an amino acid propensity scale, Neural Networks, Support Vector Machines, and SVM models trained on the frequency of amino acid pairs (AAPs) ,46,47. Bepitopes ,48. Subsepitopes , ABCPredepitopes , BCPredsepitopes , FBCPredepitopes , and SVMepitopes , were alB. henselae IFA-positive Group I dogs (IFA titer of ≥1:256), and four Group II dogs (Bartonella PCR-negative and IFA-negative). Twenty-eight-day post-inoculation sera from two dogs that were infected subcutaneously with B. henselae SA2 and one dog infected with B. vinsonii subsp. berkhoffii genotype III [B. henselae CSU 1 strain was kindly provided Dr. Michael Lappin, the Department of Clinical Sciences, Colorado State University. For human testing, sera from eight Group III B. henselae IFA-positive (IFA titer of ≥1:128) and four Group IV (control) humans were used. Of the eight B. henselae IFA-positive human samples, two serum samples were from B. henselae PCR-positive humans.To assess the antigenicity and seroreactivity of four Pap31 B-cell epitopes for canine Bartonelloses, Pap31 B-cells epitopes were tested by ELISA and dot blot. Dog ELISA was performed using three serum samples from three experimentally inoculated dogs, five type III , were obBartonella IFA-positive dogs; n = 8) and humans (Bartonella IFA-positive n = 8) with a B. henselae IgG titer of ≥1:128) in 1× TBST containing 3% nonfat dry milk for 1 h. A negative-control dog or negative human serum sample  were chosen from the VBDDL or IPRL archives, respectively. After washing with 1× TBST, a secondary antibody -conjugated goat anti-dog whole IgG or AP-conjugated anti-human) was added at serial dilutions  in 1× TBS-T containing 3% nonfat dry milk, and then incubated at room temperature for an hour. After washing, the membrane strips were developed using a commercially available substrate solution containing nitroblue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3′-indolyphosphate p-toluidine salt . Image acquisition was performed using a ChemiDoc imaging system . The blots were analyzed with Image Lab software .ELISA was performed, as described above in the Methods Section, with minor modifications. ELISA plate wells were coated with 100 µL of 10 and 20 µg/mL peptides using a carbonate buffer. Sera from dogs and humans were used at two dilutions (1:50 and 1:100 dilution). Secondary antibodies were used as described above in the Methods Section. Dot blot was performed as follows: peptides at a concentration of 15 μg and 20 μg were spotted onto PVDF membranes and air dried. The strips were blocked overnight with 5% nonfat dry milk in 1× TBS containing 0.05% Tween-20 (TBST). After overnight blocking, each strip containing the peptides was incubated with 2 dilutions (1:50 and 1:100) of primary antibody  cutoff values were determined to maximize the Youden index, as previously described (53). The Youden index is the metric for assessing the performance of a diagnostic test. The index is defined by Equation (1), and a, b, c, and d in (1), respectively, denote the numbers of true positives, false negatives, false positives, and true negatives. J = 0 represents a diagnostic test, which gives the same proportion of positives for both the control and infected groups, and J = 1 represents a diagnostic test with no false positive and false negative detected. The optimal cutoff value is the one that maximizes the J.To determine the sensitivity, specificity, and cutoff values for ELISA, the receiver operating characteristic (ROC) analysis was performed with 95% Cis, as previously described ,55. SincBartonella IFA testing results, the positive, negative, and overall percent agreement between ELISA and IFA assays were calculated as previously described [Bartonella IFA and ELISA tests, the kappa statistic was calculated as previously described [p-values of less than 0.05 were considered statistically significant.To compare the agreement between the ELISA and escribed . To measescribed . Differe


A statically significant decrease in weight SDS (p < 0.05) was noted during treatment, but the effect size was slight (r = 0.09). The atomoxetine treatment had no significant impact on height SDS, BMI SDS, and serum growth parameters, such as the levels of IGF-1, IGFBP-3, and thyroid function. The SNAP-IV results showed a significant improvement in the core symptoms of ADHD, while the CPRS results indicated a significant improvement in controlling ADHD symptoms across two different domains, learning problems (r = 0.81) and hyperactivity (r = 0.86). No severe adverse reactions were observed in the course of treatment, and the most common adverse reactions were gastrointestinal symptoms. (4) Conclusions: Atomoxetine is an effective and safe treatment for primary school children with ADHD. In China, it may be an excellent choice to alleviate parenting stress and improve the condition of primary school children with ADHD. Moreover, our study indicated that the serum levels of IGF-1 and IGFBP-3 were within the normal range in newly diagnosed ADHD children, and atomoxetine will not affect the serum concentration of growth parameters, such as IGF-1, IGFBP-3, and thyroid function, in the short term. However, the treatment may reduce appetite, resulting in a reduction in the Children’s weight for a short period. Further observational studies to monitor the long-term effects of atomoxetine on primary school children are recommended.(1) Objective: Atomoxetine is a selective norepinephrine reuptake inhibitor used to treat attention-deficit/hyperactivity disorder (ADHD) in children over six years old. Although it is common knowledge that primary school children with ADHD often present with difficulties in the morning prior to school and in the evening, these two periods, and the family interactions they involve, are often neglected in studies of ADHD. Questionnaire–Children with Difficulties (QCD) has been widely used in China to evaluate parents’ perceptions of ADHD and patients’ daily behaviors during different times. In the long term, the efficacy and safety of atomoxetine have been well established in previous studies. Still, the short-term effects of atomoxetine treatment on serum growth parameters, such as IGF-1, IGFBP-3, and thyroid function, are not well documented. Therefore, this study was the first one using the QCD to quantify the efficacy of atomoxetine treatment in the morning prior to school and in the evening, and has investigated the possible influence on the growth parameters of Chinese primary school children with ADHD. (2) Method: This prospective study was conducted at the Department of Pediatrics at the Affiliated Hospital of Jiangnan University from August 2019 to February 2021. Changes in the children’s behavior and core ADHD symptoms following treatment were assessed using three parent-reported questionnaires, including Children with Difficulties (QCD), the Swanson, Nolan, and Pelham IV scale (SNAP-IV), and the Conners’ parents rating scales (CPRS). The height, weight, and body mass index (BMI) were measured and corrected to reflect the standard deviations (SDS) in Chinese children based on age and gender. Serum growth parameters, such as insulin-like growth factor 1 (IGF-1), insulin-like growth factor-binding protein 3 (IGFBP-3), and thyroid function, were also measured to assess the children’s growth development. Any adverse drug reactions were assessed every three weeks. (3) Result: Finally, 149 children were enrolled in this study, and they completed 12 weeks of atomoxetine treatment. The QCD results indicated that the atomoxetine treatment could significantly alleviate behavioral difficulties in primary children with ADHD, especially in the morning prior to school ( Attention-deficit/hyperactivity disorder (ADHD) is the most common childhood neurodevelopmental disorder, with a prevalence of approximately 5% worldwide . It is dBehavioral and psychosocial treatments (BPT) are the recommended nonpharmacological treatments to alleviate core the symptoms of ADHD, and they are the first-line treatments for very young children (<5 years old) ,5. HowevStudies performed during the last decade indicate that the development of ADHD involves a complex interaction of the neurobiological and neurochemical systems. However, the precise etiology and pathophysiology behind this disease are still not fully understood ,13,14. NAlthough it is well known that primary school children with ADHD often present with difficulties in the morning prior to school and in the evening, these two periods, and the family interactions that they entail, are often neglected in studies of ADHD ,29. A noDiagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-V); (4) received a 12-week treatment with atomoxetine for the first time. Patients with developmentally induced growth suppression disorders, congenital malformations, or chronic somatic diseases, such as asthma, arthritis, and chronic back/neck pain, were excluded from the study. Patients suffering from neural disorders  and acute diseases at the time of admission (including upper respiratory tract infection and enteritis) were also excluded. Patients who received previous pharmacological treatment with either atomoxetine or methylphenidate, or others, were not included. The workflow used to screen for eligible participants is illustrated in This prospective study was conducted at the Department of Pediatrics at the Affiliated Hospital of Jiangnan University from August 2019 to February 2021. The criteria for study inclusion were: (1) aged between 6 and 12 years; (2) newly diagnosed ADHD patients; (3) met the ADHD diagnostic criteria via clinical assessment according to the The guardians or parents of the patients participating in the study provided written informed consent for us to obtain anonymous data from the children’s medical records at the very beginning. They were also fully informed that thyroid function, serum IGF-1, and IGFBP-3 levels were measured for the clinical assessment of growth development and for the purpose of clinical research. The Research Ethics Committees of the Affiliated Hospital of Jiangnan University  approved the study (No.LS2019047).The children received pharmacologic therapy as indicated by the second edition of the Chinese ADHD guidelines, released in 2015 . AtomoxeTo quantify the effects of atomoxetine at different periods of a day, and its efficacy at alleviating the core symptoms of ADHD, an inventory of the improvement in the children’s behavior and core symptoms of ADHD was kept by using three parent-reported questionnaires, completed electronically, including the Chinese version of the Questionnaire–Children with Difficulties (QCD), the Chinese version of the Swanson, Nolan, and Pelham IV scale (SNAP-IV), and the Conners’ Parents Rating Scales (CPRS). The QCD scale consists of 20 questions that focus on the difficulties faced by children with ADHD before going to school (Q 1~4), during school (Q 5~7), after school (Q 8~10), in the evening (Q 11~14), and at night (Q 15~18), and on their overall behavior (Q 19~20). Four different level scores were used: 0 means disagree, 1 means partially agree, 2 means mostly agree, and 3 means complete agree. This questionnaire is a reliable and valid tool with which to assess daily ADHD symptoms in various Chinese studies ,37. SNAPTo investigate the possible adverse reactions to atomoxetine, an adapted semi-structured interview was recorded every three weeks by the guardians or parents electronically  based ont-test. For all tests, a p-value below 0.05 was considered to be statistically significant.Statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS) software version 23.0 . The results were presented as means ± standard deviations. The pre-treatment and post-treatment variables were compared using a paired A total of 155 patients chose atomoxetine treatment via a shared decision-making process, with input from the physician and the guardians or parents. The parents/guardians of six children refused to provide their medical records regarding atomoxetine treatment. Finally, 149 children were enrolled in this study and completed 12 weeks of atomoxetine treatment, including 56 girls and 93 boys. The demographics and clinical characteristics are summarized in p < 0.001). The effect sizes of the atomoxetine treatment, assessed by measuring the difference in the QCD scores between baseline and during the six different treatment time points, are also provided . Our results indicate that the atomoxetine treatment could significantly alleviate behavioral difficulties in primary children with ADHD, especially in the morning prior to school and in the evening.The results of the fully completed patient-reported QCDs at baseline and during the six different treatment time points are summarized in p < 0.05). The effect sizes of the atomoxetine treatment measured the difference in the SNAP-IV scores between baseline and after treatment .The SNAP-IV scores are summarized in p < 0.05), while the effect sizes of the atomoxetine treatment were r = 0.06, r = 0.81, r = 0.35, and r = 0.81, respectively.The CPRS scores are summarized in p < 0.05), but the effect size is slight (r = 0.09). However, the height SDS, BMI SDS, and the levels of serum growth parameters did not differ significantly from the baseline after 12 weeks of treatment (p > 0.05).The serum IGF-1 and IGFBP-3 levels were normal for their sex and age . The heiOver the course of the 12 weeks, no severe adverse reactions, such as suicide attempt or suicidal ideation, were reported. Gastrointestinal adverse reactions were reported by 56.38% of participants during the first three weeks of treatment. The most common gastrointestinal symptom was the loss of appetite or anorexia (35.57%), followed by constipation (12.08%) and nausea and vomiting (8.72%). The incidence of neurological side effects was 24.16%, with lethargy (13.42%) being the most common symptom reported, followed by dizziness (6.71%) and emotional instability (4.03%). These adverse reactions were effectively alleviated by changing the work and rest time, the medicine administration time, the prandial time, and the diet. Furthermore, the incidence of adverse reactions decreased significantly in the later stage of the treatment, as shown in https://www.nmpa.gov.cn, accessed on 16 October 2021). At the same time, clonidine and bupropion were recommended as the second-line medications [The current prospective study investigated the improvement of behavioral difficulties at different times after a 12-week atomoxetine treatment for primary children diagnosed with ADHD aged between 6 and 12 years, and the possible influence on the growth parameters of this treatment. Although behavioral and psychosocial treatments were recommended before pharmacological therapy by the United Kingdom and Germany ,51, effeications . As a noications ,53. Shorications ,55. Howeications ,57. Hencications ,59. Our p < 0.001, r = 0.66) and in the evening  was assessed by comparing QCD scores at different times. Our QCD results indicated that atomoxetine could significantly reduce the functional difficulties experienced by ADHD children in these two periods. Family interactions were another neglected point in previous studies of ADHD [To the best of our knowledge, this is the first report using the QCD to examine the efficiency of atomoxetine for children aged 6–12 years old with ADHD, in terms of reducing the functional difficulties at different times. The QCD was created by Yamashita, and is widely used in Asian countries, such as China and Japan . The QCD of ADHD . Althoug of ADHD , previou of ADHD ,63. Henc of ADHD ,65,66. C of ADHD ,68,69, oIn this study, the levels of serum growth parameters were closely monitored throughout treatment. These markers have essential roles in promoting children’s growth and development ,71,72. Ip < 0.05), the effect size was slight (r = 0.09). A survey by Germinario revealed that the height and weight gradually returned to normal after two years of ADHD treatment. This improvement was more evident for atomoxetine when compared with treatment using methylphenidate [Our study reported no severe adverse reactions, such as suicide attempt or suicidal ideation, throughout treatment. The most significant initial adverse reactions were gastrointestinal symptoms, including the loss of appetite or anorexia, constipation, nausea, and vomiting. These symptoms might be caused by temporary disorders of the central norepinephrine system associated with hunger or satiety . Intereshenidate . Consisthenidate .Our study has some limitations that have to be acknowledged. First of all, all enrolled patients were from a single center, so a selection bias could not be entirely ruled out. Secondly, although all questionnaires were completed by parents/guardians electronically at the clinical department and there were no missing data, the expectations of parents/guardians and their insufficient training by the clinicians might have introduced a positive bias into our findings. Furthermore, although all enrolled patients were newly diagnosed with ADHD, this study did not compare the use of atomoxetine with a placebo. Therefore, the symptom improvement noted in ADHD children could have resulted from natural growth and development. Finally, the small number of patients enrolled in this study may affect the generalizability of the research findings.In summary, this study determined that atomoxetine positively affects behavioral difficulties in primary school children with ADHD and confirmed that atomoxetine would not affect growth development in the short term. In addition, our study was the first one using the QCD to prove that atomoxetine treatment can significantly reduce the functional difficulties experienced by ADHD children in the morning prior to school and in the evening. Atomoxetine may be a good choice in China to alleviate the stress of parents/guardians, and to improve family interactions with primary school children with ADHD. Moreover, our study indicated that the serum levels of IGF-1 and IGFBP-3 were within the normal range in newly diagnosed ADHD patients, and that atomoxetine will not affect the serum concentration of growth parameters in the short-term.


Introduction: Suicide is one of the leading causes of death among adolescents. Although it is known that both perfectionism and attention-deficit/hyperactivity disorder (‘ADHD’) are important risk factors for suicide, there are no studies that have investigated the relationship between suicidal behavior and perfectionism among people with ADHD.Aim: The current study investigates the association between perfectionism and suicide in adolescents with ADHD.Method: Subjects included 88 adolescents with ADHD and 96 non-clinical control adolescents. We used the Multidimensional Perfectionism Scale to evaluate perfectionism as well as its separate traits, and the Mini International Neuropsychologic Interview Kid to evaluate psychiatric disorders and suicidal behavior. Differences between the groups were statistically evaluated using t-tests, a Poisson regression analysis with suicide as a discrete variable, and a logistic regression analysis with suicide as a binary variable.Results: Compared to the control group, the ADHD group showed a significantly lower level on the adaptive ‘Organization’ trait of perfectionism, but not on any other trait, and a significantly higher level of suicidal behavior.p = 0.003), and for the ‘Personal Standards’ trait .Logistic regression results indicated a significant association for perfectionism in general  and with the ‘Personal standards’ trait model .Poisson regression analysis also showed a significant association with perfectionism in general (IRR = 0.90; Discussion: Our results indicate that a low level of perfectionism, in particular ‘Personal standards’, may be a risk factor for suicidal behavior in adolescents with ADHD. We recommend that psychoeducation and therapy of adolescents with ADHD should consider focusing on adaptive perfection as a possible risk factor for suicide as well. Attention-Deficit/Hyperactivity Disorder (‘ADHD’) is one of the most diagnosed psychiatric disorder among children and adolescents , with a Both impulsivity and perfectionism  are tranImportantly, there is also a growing body of research evidence that both ADHD is associated with an increased risk of suicide, which is a leading cause of death among adolescents e.g., , 16]. Ac. Ac16)].In addition to this, several studies have also indicated a positive relationship between perfectionism and suicidal behavior , 19–21 sBased on these recent new insights, the main aim of the present study was to explore if perfectionism may be a risk factor for suicidal behavior in adolescents with ADHD. We believe that increased knowledge in this area may help to improve suicide prevention in patients with ADHD.The study was conducted with the approval of the Ethical Committee of the Medical Research Council, Hungary . Subjects were provided with written informed consents after being verbally informed on the nature of the study. After signing the informed consent, a diagnostic interview was recorded by a psychologist (see below). Afterwards, self-rated questionnaires were completed by all subjects. No compensation was provided to the participants.Adolescents with ADHD were recruited from the Vadaskert Child Psychiatric Hospital and Outpatient Clinic, Budapest, Hungary, in Szent Rokus Hospital and Outpatient Clinic, Baja and Tolna County's Balassa Janos Hospital and Outpatient Clinic, Szekszard. Control group participants were recruited from several high schools in Hungary. Both groups included subjects between 13 and 18 years. Further inclusion criteria for the clinical group included an ADHD diagnosis from a psychiatrist which needed to be confirmed by a structured psychiatric diagnostic interview (see below).Exclusion criterion for both the ADHD and control groups included a medical history of intellectual disability. Further exclusion criteria for the control group included: current or previous psychiatric diagnoses or psychological treatment (see below).Psychiatric symptoms and disorders were diagnosed using the modified version of the Mini International Neuropsychologic Interview for Children and Adolescents (MINI Kid). The modified version of the MINI Kid evaluates the presence of twenty-four psychiatric diagnoses according to the Diagnostic and Statistical Manual of Mental Disorders fifth edition (DSM-5) , including ADHD and their subthreshold forms and suicidal behavior , 23. TheCurrent suicidal behavior was assessed via the following questions of the MINI Kid: “In the past month did you” “Wish you were dead?”, “Want to hurt yourself?”, “Think about killing yourself?”, “Think of a way to kill yourself?”, “Attempt suicide?” Have a method in mind to kill yourself?” Participants met the criterion for suicidal behavior after one “yes” answer and according to the points, suicidal behavior level was either “low” or “moderate” or “high” , 23. TheThe level of perfectionism was evaluated with the ‘Multidimensional Perfectionism Scale’ (‘FMPS’), a self-report scale developed by Frost et al. : It contMaladaptive traits cause a negative reaction if standards are not reached and include: ‘Concern Over Mistakes’ (reflecting negative reactions to errors), ‘Parental Criticism’ , ‘Parental Expectation’ , ‘Doubt About Actions’ (the tendency to doubt abilities in actions). Adaptive traits do not evoke negative reactions if the goal is not reached and include: ‘Personal Standards’ (setting high standards for assessments), ‘Organization’ (the ability and importance placed on orderliness).Response method used a 5-point liker scale (1 = strongly disagree and 5 = strongly agree). The internal consistency of the subscales ranged from 0.77 to 0.93 . The totA structured parent-rated questionnaire was developed specifically for this study to gather information about the demographic data of the participants, including the combined education level of the parents, family history of psychological or psychiatric treatment, economic activity of the father/mother , as well as the structure of the family. The questionnaire consisted of closed questions and each answer belonged to a point.t-tests. The distribution of the suicide scale of the MINI Kid significantly violated normality assumptions . Effect sizes were calculated with Cohen's d for the parametric test (t-test), a rank correlation r- value for the non-parametric Mann-Whitney test  as a relative measure of association. The OR is the ratio of the odds of an event occurring in one group to the odds of it occurring in another group.For the second approach, suicidal behavior was evaluated as a continuous discrete variable and evaluated using a Poisson regression. For this approach, the Incidence Rate Ratio (‘IRR’) was calculated as a measure of association.Within this analysis, over dispersion was evaluated using the alpha statistic. If standard deviations were not equal , a negative binomial regression was used instead of a Poisson regression .For both approaches, we evaluated perfectionism either as a unidimensional or multidimensional concept. The unidimensional approach includes only one total score of perfection, based on all traits except ‘Organization’. For the multidimensional approach we included all traits of perfection (except ‘Organization’) as separate variables.(182) = 0.69, p < 0.001].The other explanatory variable included in each regression analyses was a combined ADHD diagnosis. The two ADHD variables  were combined into one variable because of their high dependence .In the present study the ADHD group included 88 adolescents  and the control group 96 adolescents . There was no significant difference in age between the groups. In regard to gender, there was a significant difference between the clinical and control group: in the control group 46% of the participants were girls whereas in the clinical group only 8% of the participants were girls [χ2(183) = 3.153 p = 0.002]; effect size medium The comparison of the ADHD and control group regarding each perfectionism scale can be found in (182) = −2.975, p = 0.003, r = 0.22].Negative binominal regression analysis used suicidal behavior as a discrete variable. According to the AIC index, the model without demographic variables showed the best fit.p – 0.012), indicating that if the combined ADHD scale increased by one unit, the incident rate for suicidal behavior increases with 57 %. The IRR for ‘Total Perfectionism’ was 0.90, (p < 0.001), which indicates that if the perfectionism scale increases by one point, it would reduce the incident rate of suicide behavior scale with 10%. The detailed results of the simple model regression can be found in For the simple model, the IRR for the variable ADHD was 1.57 (p = 0.019), indicating that for each unit increase in the Likert-scale for ‘Personal Standards’, the percent change in the incident rate of suicidal behavior was 19%.The extended model revealed a significant association for ‘Personal standards’ , indicating that an increase in one unit on the perfectionism score reduced the chance of suicidal behavior by 7%. The presence of ADHD increased the chance of suicidal behavior by 48% . The simple model indicated a significant association for ‘total perfectionism’ ]. This result indicates that an increase of one unit in ‘Personal Standards’ reduced the chance of suicidal behavior by approximately 8%.With the extended model including five perfectionism traits, the factor ‘Personal Standards’ was significantly associated with suicide behavior [OR: 0.82 . Each subject as well as their parents provided written informed consents after being verbally informed on the nature of the study.LK-P organized and conducted the data collection and wrote the first drafts of the article. JB was the supervisor of the research and the manuscript process. BF contributed to the data collection, BV conducted the statistical analyses, and JJ made the proofreading of the manuscript and helped in the finalization process. All funders should be credited and all grant numbers should be correctly included in this section. All authors contributed to the article and approved the submitted version.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.


Overlap in symptom domains particularly in the field of impulsivity and emotional dysregulation in attention deficit hyperactivity disorder (ADHD) and borderline personality disorder (BPD) have stimulated further research activities since our last review from 2014.Disentangling features of impulsivity in ADHD and BPD revealed that impulsivity is a feature of both disorders with patients suffering from both ADHD and BPD having highest impulsivity ratings. BPD individuals have more problems using context cues for inhibiting responses and their impulsivity is stress-dependent, whereas ADHD patients have more motor impulsivity and therefore difficulties interrupting ongoing responses. For emotion regulation difficulties the ranking order ranges from ADHD to BPD to the comorbid condition, again with the patients suffering from both, ADHD and BPD, having the most pronounced emotion regulation problems.Environmental influences namely adverse childhood events were shown to be linked to both ADHD and BPD. Traumatic experiences seem independently linked to impulsivity features. Thus, some authors point to the risk of misdiagnosis during childhood and the necessity to screen for traumatic experiences in both patient groups.Genetic research confirmed genetic overlap of BPD with bipolar disorder (BD) and schizophrenic disorders, as well as genetic overlap of BD and ADHD. A population-based study confirmed the high co-occurrence and familial co-aggregation of ADHD and BPD. Interesting questions in the field of gene-environment-interactions are currently dealt with by genetic and epigenetic research.Few studies have investigated treatment strategies for the comorbid condition, though the issue is highly important for the management of patients suffering from both disorders and presenting with the highest symptom scores.Research on the different impulsivity features might point to a necessity of disorder-specific treatment strategies in the field of impulse control. Future research is needed to base treatment decisions for the comorbid condition on an evidence basis. Attention deficit hyperactivity disorder (ADHD) and borderline personality disorder (BPD) are common psychiatric disorders with prevalences of about 5% for ADHD)  and abouADHD and BPD share important symptom domains, namely impulsivity and emotion dysregulation. Impulsivity as a feature of actions out of spontaneous impulses without thinking about consequences is a core symptom in both disorders, although the psychopathological descriptions of impulsivity in ADHD and BPD in the DSM-criteria are not the same: the type of impulsivity used to define BPD refers to impulsive self-harm and can occur as an associated feature in ADHD, whereas core impulsivity in ADHD is defined as impatience when waiting, talking over people, and interrupting others . EmotionParticularly comorbid patients, suffering from ADHD and BPD and their characteristics have been focus of new studies published since 2015 –14. The This article gives an overview on the state of knowledge on ADHD and BPD and the interrelation between the two disorders updating our review from 2014 on the subject .The aim of this update is to improve and broaden knowledge on the relationship of ADHD and BPD. Information for clinicians will be extracted and a scientifically oriented summary of new findings will be presented to identify research directions.A search for studies published since 2014 and dealing with ADHD and BPD in adults was conducted for the following bibliographic databases: Pubmed, Embase, Medline, PsychInfo, Central . The following terms were used: (ADHD OR (attention deficit) OR (attention-deficit) OR hyperactivity*) AND  OR borderline*). The search included all fields in Pubmed, Embase, Medline and PsychInfo databases and abstract, title and keywords in the Central bibliography  37).We searched relevant registers for studies on ADHD and BPD. Furthermore, we screened the reference lists of relevant articles manually. Studies of any design focusing on ADHD and BPD were screened and categorized. We used the categories genetic, epigenetic and environmental risk factors, shared or overlapping symptoms and treatment to group the findings. The systematic search was conducted in April 2020. Originally, as this narrative review is an update to our article from 2014 , only stThe main symptomatic overlap between ADHD and BPD is noted in the domains of impulsivity and emotional dysregulation. But on closer examination, these domains are not the same in ADHD and BPD.Impulsivity is a core symptom of both ADHD and BPD. Impulsivity as a construct remains poorly defined. In a broader sense, „to act without reflecting the consequences" has been considered an acceptable clinical definition of impulsivity. As mentioned above, the descriptive definitions of impulsive behavior used to establish the diagnoses ADHD or BPD are not the same according to the DSM-criteria: the type of impulsivity used to define BPD refers to impulsive self-harm, whereas core impulsivity in ADHD is defined as impatience when waiting, talking over people, and interrupting others from what they are doing . MethodsResearch suggests that impulsivity is a multifaceted construct that via different traits can lead to maladaptive behavior . In bothA questionnaire study  using seO’Malley et al.  comparedA classical test for cognitive control, ADHD related attention problems and response inhibition deficits is the Continuous Performance Test (CPT) . In the However, directly linking cognitive tests to behavior is not always possible since associations between neuropsychological test results and symptoms or behavior are often weak and a causal link has not been clearly demonstrated. It is nevertheless interesting that impaired response inhibition in the narrow sense (interruption of ongoing actions) is reported for ADHD and not for BPD – in line with the idea that impulsivity in ADHD reflects a more neurocognitive disorder whereas impulsivity in BPD has a more “behavioral” and composite etiology.In the framework of hypotheses generated based on scientific work related to the theories and assumptions of Marsha Linehan’s Dialectical Behavioral Therapy (DBT) stress has been assumed to be a powerful factor influencing levels of impulsivity especially in BPD patients. To understand the differences and commonalities between ADHD and BPD in the realm of impulsivity better it has been hypothesized that ADHD is characterized by high levels of trait impulsivity whereas BPD is characterized by state impulsivity or, in other words, stress-induced high impulsivity levels.To elucidate these assumptions, Cackowski et al.  investigWith the aim to further investigate the impact of acute stress on self-reported and neuropsychological measures of impulsivity in ADHD and BPD the study from Krause-Utz et al.  followedImpulsivity in different psychiatric disorders and its association with early trauma experiences is subject of a study in 744 participants published by Richard-Lepouriel and colleagues . Six cliAnother study compared 165 BPD patients and 165 healthy controls in terms of impulsivity, traumatic experiences, ADHD symptoms and dissociative symptom load . High scIn BPD, difficulties of emotion regulation are considered the core pathology . In ADHDRüfenacht et al.  comparedSubstance use disorders (SUD) are important comorbidities of ADHD and BPD patients. A mediation analysis in 305 male SUD patients confirmed that difficulties in emotion regulation did partially mediate the relationship between ADHD symptoms and symptoms of BPD .In another attempt to contribute some more explanations on the relationship between retrospectively reported symptoms of ADHD in childhood and BPD, Fossati et al.  assessedLittle is known about shared risk factors for ADHD and BPD. Overlapping genetic risks raise interesting questions about the distinctiveness of the categorial diagnoses.Heritability estimates of ADHD are ranging between 70 and 80%  while faThe most robust findings in the field of genetic risk arise from estimating genetic overlap using genome wide association studies (GWAS). Polygenic risk scores (PRS) are calculated as an estimate of an individual’s genetic liability to a trait or disease, according to their genotype profile and relevant GWAS data .g = 0.28 [P = 2.99 × 10− 3]), schizophrenia (rg = 0.34 [P = 4.37 × 10− 5]) and major depression (rg = 0.57 [P = 1.04 × 10− 3]) was found. We have not found a GWAS that directly examined the genetic overlap between ADHD and BPD.A GWAS of BPD indicated a genetic overlap with bipolar disorder (BD), major depression and schizophrenia . Geneticp = 0.860) whereas a strong association between BD-PRS and increased odds of ADHD-diagnosis was found . Again, a direct association between ADHD and BPD has not been examined in this study.In a prospective population-based study using a PRS-approach, Mistry et al.  examinedKuja-Halkola et al.  investigA review and meta-analysis of genetic factors in BPD did not identify a susceptibility gene for the development of BPD . The aut3AR) and the severity of ADHD, BPD and BD [3AR methylation status will mediate the effect of childhood maltreatment on psychopathology in adulthood. For the methylation analysis, blood samples were taken from the three clinical groups . The results revealed an association between maltreatment and severity of the disorder. There was a significant association of childhood maltreatment, especially physical abuse, with 5-HT3AR methylation levels.Amad et al.  pronouncD and BD . FollowiThe interrelation between ADHD, traumatic experiences and BPD is still subject of debate. GxE-interactions are thought to be important factors in the development of symptom-expression in both BPD and ADHD. In general, individuals with a particular genotype are at risk to develop more severe symptoms in the presence of environmental adversity of any sort , 42.Brown et al.  investigDalbudak and Evren  investigFerrer et al.  analyzedFor total trauma-scores, there was a highly significant gradual increase: “No BPD-no ADHD” (48.45) < ADHD (53.64) < BPD (54.99) < BPD + ADHD (61.16). The same ranking applied to total abuse, emotional abuse and sexual abuse. According to the total trauma-scores, comorbid patients seem to suffer more childhood trauma than non-comorbid patients do. The ADHD-group has slightly lower scores compared with the BPD-group.The recent investigations on ACE and severity of ADHD add further support to the evidence already discussed in our preceding review: Not only patients with BPD but also patients with ADHD - and especially comorbid ADHD+BPD-patients - have increased rates of childhood trauma and adverse events. Having ADHD could go along with one of the genetic backgrounds on which environmental risks play - leading to development of BPD. Thus, children with ADHD might be more likely to develop BPD when exposed to trauma.Treatment strategies for BPD are mostly psychotherapeutic , 48. Trealways be considered when treating comorbid personality disorders, with the aim to reduce ADHD-dependent distress, improve functioning in the daily lives and enhance control over the behavior. According to Asherson and colleagues, patients are more likely to engage and benefit from psychological treatment programs for BPD when comorbid ADHD is treated.Asherson et al.  stated aBeneficial but small to moderate effects of methylphenidate (MPH) on emotional dysregulation in adult ADHD (without taking into account comorbidity) have been shown in the past  and confGvirts et al.  administThe evidence presented in this review illustrates that research efforts since 2014 have produced some inconsistent results, some progress in differentiation of seemingly overlapping ADHD/BPD-symptomatology and some interesting ideas for future research.As ADHD frequently co-occurs with a wide range of other neurodevelopmental and psychiatric disorders the specificity of the clinically observed close link between ADHD and BPD has to be further defined and sharpened. The aim of such better understanding of this co-occurrence can be a basis for a better understanding of systematic nosologic and etiopathogenetic connections and might have the potential to contribute to better treatment decisions and rational preventive measures. It has been suggested that the co-occurrence of ADHD and BPD is partly due to shared genetic factors. Estimated genetic correlation may be the best way to indicate genetic overlap between disorders. This was estimated at 0.59 in an older study discussed in our previous review . In our It has been proposed to look further into plausible targets of GxE interactions and epigenetics. In general, research on candidate genes, single genes and GxE findings have to be seen with caution because results have often not been replicated. As stated in our genetics-section, the most robust findings arise from GWAS-data.We found only one study on epigenetic alterations and their association with traumatic experiences in BPD, ADHD and BD . BecauseResearch on environmental risk factors revealed a correlation between ADHD and adverse childhood events. Retrospectively, patients with BPD + ADHD report more childhood maltreatment. Risky, impulsive and novelty seeking behaviors in children with ADHD might elevate the risk for exposure to traumatic situations. Some authors point to the risk of misdiagnosing ADHD in children that suffer mainly from emotional neglect and abuse and therefore recommend general screening for adverse events and traumatic experiences in all children presenting for ADHD diagnosis. In doing so, impulsivity features that can be correlated to traumatic experiences might be clearly distinguishable from ADHD, leading to fewer misdiagnoses. Conversely, it could be argued, that experiencing a trauma may lead to more severe ADHD symptoms. Thus, the precise nature of the interrelation between ADHD, traumatic experiences, impulsivity and BPD is still subject of debate.In order to understand the discussion about the genetic background of ADHD, BPD and the comorbid ADHD+BPD-patients, it is important to be familiar with the concept of pleiotropy. Genetic pleiotropy means that the same genetic background can lead to different phenotypes. In a recent review on this topic, Lee et al.  summarizDifferent potential models can be derived from the genetic, epigenetic and environmental risk factor research at this point: 1) Same genes lead to two distinct disorders (genetic pleiotropy); (2) BPD and ADHD are overlapping, e.g. they are different presentations of the same underlying disorder; 3) ADHD is leading to higher risk for developing BPD. Model  ADHD is  shall be Same genIn our previous review we postulated that ADHD children are at elevated risk for adversities and traumatic experiences in childhood and that this contributes to the development of BPD in adolescence and adulthood. Recent studies in the field of epigenetics and genetics of vulnerability-genes add interesting aspects to our previous discussion about a “sensitive” genotype in combination with a non-fitting environment in childhood. The vulnerability might not (exclusively) be dependent on disorder-specific susceptibility-genes (e.g. ADHD-related susceptibility genes) but also on epigenetic changes caused by interpersonal stress Fig. . Due to To decide whether BPD and ADHD are distinct or overlapping disorders, it would be helpful to know whether symptoms like impulsivity and emotional dysregulation are the same in both disorders or if there are differences in etiology and thus different treatment options. There have been several efforts to disentangle “ADHD-typical” and “BPD-typical” impulsivity and features of impulsivity and emotional dysregulation in ADHD and BPD. This line of research has seen the most differentiated progress in recent years, although most reviewed studies have small sample sizes and need confirmation through reproduction. Summarized findings of the reviewed studies on impulsivity and impulsivity under stress indicate that – quantitatively – impulsivity is a feature of both, ADHD and BPD. In addition, impulsivity level seems highest in patients with comorbid ADHD and BPD Tables  and 2. TEmotion regulation is a symptom of both disorders and patients with ADHD, patients with BPD and patients with both, ADHD+BPD, seem to have – in this order – growing intensity of emotion regulation problems with the comorbid patients having the most severe emotion regulation difficulties and most dysfunctional emotion regulation habits. Emotion regulation difficulties seem to function as a mediator between ADHD symptoms and BPD features. In analogy to the discussion about impulsivity and the distinguishing features of impulsivity in ADHD and BPD, it would be interesting to further clarify the qualitative nature of emotional dysregulation in ADHD and BPD.Treatment decisions for comorbid patients with ADHD and BPD today are based on expert advice and not on systematic scientific evidence. Only one naturalistic study reported on methylphenidate treatment in patients with BPD and ADHD passing through a DBT-treatment program reported promising results.The above-mentioned conceptualization of the relationship between ADHD and BPD might also have implications for future ideas about treatment options. Currently there are no known predictors of good or poor response to ADHD drug treatments. Trauma has not been identified as a predictor of poor response to drug treatments in ADHD - this might be an important area for future enquiry. A background of trauma may or may not be associated with a good response to medications. To date, this discussion remains speculative, as causal directions are not well investigated.Based on the presented evidence and models it is conceivable that BPD may be a heterogeneous entity that develops based on unfavorable learning histories or traumatic experiences in the context of early otherness  and in the absence of a fitting environment or in the context of adverse events or childhood trauma. Some authors have discussed subtypes of BPD and have considered that severe ADHD with associated behavioral and emotional symptoms might reflect a subtype of BPD. Other factors that have important implications for the treatment decisions are other developmental disorders, organic disorders with psychiatric symptoms and complex PTSD that lead to similar behavioral and emotional symptoms. Another possibility is that ADHD and BPD are different entities and co-occur in individuals.Knowledge about the relationship between ADHD and BPD has grown since 2014. Results of genetic studies remain unspecific and inconsistent. Epigenetic research and research focussing on hypothesized vulnerability genes or sites seem a promising avenue in the future (see Fig.


Photons above 700 nm have minimal photosynthetic activity when applied alone  was first observed by Emerson and Lewis  in greenThis 400–700 nm range was recommended by McCree  from amoRecent advances in light-emitting diode (LED) technology have enabled researchers to re-visit the Emerson Enhancement Effect and study not only the short-term photosynthetic responses of single leaves under low light but also the long-term responses of plant canopies under higher photon flux densities. Zhen and van Iersel  found thZhen et al.  studied In a subsequent study, Zhen and Bugbee  measured2 fixed per mole of absorbed photons). CQY was equal among treatments from planting to harvest, confirming the important role of far-red photons for photosynthesis.Zhen and Bugbee  followedWe have studied the effects of far-red photons at the photosystem, leaf, and canopy level, with consistent results at all three scales. Collectively, these findings provide compelling evidence for the photosynthetic value of far-red photons when combined with shorter-wavelength photons, as long as the far-red flux does not exceed about 30% of the total photon flux. This is equal to or higher than the fraction of far-red photons in sunlight under which plants have evolved.Unfortunately, the Design Lights Consortium , followipercent increase in net photosynthetic rate that diminished at higher background light intensities, which is expected.Design Lights Consortium also argued that the data presented in Zhen and van Iersel  suggesteIn addition, the Design Lights Consortium  also misLastly, the Design Lights Consortium  raised cSince the efficacy of horticultural fixtures (μmoles of photons per joule) is calculated as photosynthetic photons/energy use, the efficacy of fixtures that include far-red photons will be counted as lower than fixtures that do not emit far-red photons. This occurs with lower color temperature white LEDs and especially fixtures with far-red LEDs. Fixture efficacy is used by energy efficiency programs to determine fixture eligibility for rebate or incentive programs. Until the definition of PAR is expanded to include far-red photons , lighting manufacturers will be discouraged from including far-red LEDs in fixtures. Changing the definition from PAR to ePAR and including photons from 400 to 750 nm will facilitate the development of fixtures with higher efficacy and encourage innovation /(400–750 nm)].This opinion piece originated from discussions among all authors. SZ wrote the first draft with input from MvI and BB. All authors revised the manuscript and have approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.