Giardia duodenalis assemblages A and B. Differentiation between human infections with these assemblages, as well as between single-assemblage (A or B) and mixed-assemblage (A and B) infections, is therefore needed to better understand the pathological impact of infection with either, or both, assemblages. We assessed the prevalence of G. duodenalis assemblages A and B using 305 fecal samples provided by school-age children situated along the southern shoreline of Lake Malawi. Concurrently, intestinal pathology data were also collected to test for association(s) between assemblage infection status and intestinal health. Prevalence of G. duodenalis infection was 39.3% by real-time polymerase chain reaction. Of all identified infections, 32% were single G. duodenalis assemblage A and 32% were single G. duodenalis assemblage B, whereas 33% were mixed-assemblage infections. Fifteen unique G. duodenalis assemblage A and 13 unique G. duodenalis assemblage B β-giardin haplotypes were identified. There was a positive association between single infection with G. duodenalis assemblage B and both self-reporting of abdominal pain  and self-reporting of diarrhea . No association between single infection with assemblage A and any form of intestinal pathology was found. Additionally, there was a positive association between mixed-assemblage infections and self-reporting of abdominal pain . Our study highlights the importance G. duodenalis assemblage typing and reaffirms the need for improved access to water, sanitation and hygiene infrastructure in rural areas of low- and middle-income countries.Almost all human giardiasis infections are caused by  Giardia duodenalis .–Human giardiasis is a globally important intestinal parasitic disease caused by infection with the eukaryotic protozoan diplomonad –,,G. duodenalis taxon assemblages A through H, almost all human infections are caused by assemblages A and B,–Although cosmopolitan in distribution, prevalence of human giardiasis can be particularly high in rural areas of low- and middle-income countries (LMICs) where adequate water, sanitation, and hygiene (WASH) infrastructure is lacking, including those in sub-Saharan Africa.G. duodenalis assemblages A and B, as well as between those burdened with single-assemblage (A or B) or mixed-assemblage (A and B) infections.,–G. duodenalis assemblages A and B as well as between single- and mixed-assemblage infections using molecular characterization and taxon assemblage typing is needed to better understand the pathological impact of infection with either, or both, assemblages and thus also for improved disease surveillance and control.,,G. duodenalis A and B assemblages is often high and where other parasitic and neglected tropical diseases (NTDs) that cause similar intestinal pathologies, such as intestinal schistosomiasis, can be coendemic.,,Recent evidence suggests that there may be marked differences in the types of and degree of pathology experienced by those infected with either –Lake Malawi is one of seven African Great lakes. With an approximate water surface area of 29,600 km,,G. duodenalis assemblages A and B in this area are currently available. We therefore evaluated the prevalence of human giardiasis infection in school-age children attending four primary schools situated in close proximity to Lake Malawi’s southern shoreline using both point-of-contact rapid diagnostic tests (RDTs) as well as a diagnostic 18S real-time polymerase chain reaction (PCR).G. duodenalis infections to assemblage level to identify the prevalence of differing infection statuses . This was done using nested PCR with subsequent Sanger sequencing together with a confirmatory G. duodenalis assemblage A- and B-specific duplex real-time PCR assay.,Only limited data on the prevalence of human giardiasis in Mangochi district have been reported.G. duodenalis infection by QUIK CHEK® RDTs  were provided with a single dose of tinidazole under the supervision of the study clinician (S. A. K.). For anonymity, all study participants were assigned an individual study-specific identification code and all laboratory analysis was carried out blinded from any other data.Ethical approval and research authorizations were approved in the United Kingdom by the Liverpool School of Tropical Medicine (LSTM) Research Ethics Committee (application 17-018) and in Malawi by the National Health Sciences Research Committee (1805). Informed, written assent was obtained from the guardians of all child participants enrolled in the study before any data or clinical sample collection. Study participants who tested positive for ,,N = 121; Mchoka school, N = 121; Sungusya school, N = 60 and Malinde [St. Martin’s] school, N = 61). The number of study participants was weighted more toward schools situated on the western side of the lake based on previous parasitological surveys and anticipated prevalence’s of G. duodenalis infection.,,Questionnaire responses and fecal material were provided by school-age children situated along the southern shoreline of Lake Malawi, Mangochi district, Malawi, in November of 2021. Four primary schools were selected for parasitological surveillance (two on the western side of the lake and two on the eastern side of the lake) based on their close proximity to the lake’s shoreline and previously identified prevalence’s of urogenital and intestinal schistosomiasis; suggesting frequent community lake water contact.supplemental material). Yes–no responses were given to the following questions: Do you drink the lake [Lake Malawi] water? Do you have regular contact with livestock cattle (cows)? Do you currently have abdominal (stomach) pain? and Do you currently have loose stool (diarrhea)?Questionnaire responses were provided by all 362 children across all four primary schools to infer lake water consumption and cattle (cow) contact behaviors, as well as current intestinal pathology , . A subset of fecal material from each stool sample was used for RDT testing as required. In addition, approximately 2 g of fecal material from each stool sample was filtered through a standard 212-µM gauge filter and stored within a labeled 2-mL screwcap tube containing 1 mL 100% ethanol for preservation and transportation. All RDT testing and fecal preservation in ethanol was carried out no more than 9 hours after stool sample provision. All ethanol-preserved fecal material was transported to the LSTM under ambient conditions for DNA extraction and genetic analyses.One stool sample was provided by 313 (86.2%) study participants across all four primary schools . QUIK CHEK RDTs that did not present a visible test band were deemed negative and scored as 0, whereas QUIK CHEK RDTs that presented a visible test band were deemed positive and scored according to band strength: light infection (faint band), 1; moderate infection (moderate band), 2; heavy infection (strong band), 3.N = 105; Mchoka school, N = 94). FOB-RDTs that did not present a visible test band were deemed negative and scored as 0, whereas FOB-RDTs that presented a visible test band were deemed positive and scored according to band strength: light outcome (faint band), 1; moderate outcome (moderate band), 2; heavy outcome (strong band), 3.Fecal occult blood (FOB) GP/Pro lateral-flow RDTs  were used to screen for any overt presence of blood within the feces of study participants , according to manufacturer’s instructions. The FOB-RDT was carried out using fecal material from 199 stool samples across two schools  GP/Pro lateral-flow RDTs  were used to screen for any overt presence of calprotectin within the feces of study participants , according to manufacturer’s instructions. The FC-RDT was carried out using fecal material from 98 stool samples provided by study participants at Samama school. FC-RDTs that did not present a visible test band were deemed negative and scored as 0, whereas FC-RDTs that presented a visible test band were deemed positive and scored according to band strength: light outcome (faint band), 1; moderate outcome (moderate band), 2; heavy outcome (strong band), 3.G. duodenalis assemblage types are found within the 18S small subunit ribosomal ribonucleic acid (SSU rRNA) and within genes coding for triosephosphate isomerase (tpi) enzymes, glutamate dehydrogenase (gdh) enzymes, and β-giardin (bg) proteins.,,,,–G. duodenalis–positive samples,G. duodenalis infections to assemblage level using both nested PCR targeting the bg locusG. duodenalis assemblage A- and B-specific duplex real-time PCR assay targeting the tpi locus.The genetic loci used most extensively to characterize ,G. duodenalis and PhHV-1 DNA during an initial diagnostic 18S real-time PCR, as detailed subsequently.DNA was isolated from each ethanol-preserved fecal sample using the QIAamp DNA mini kit  according to manufacturer’s instructions with minor revisions including a bead-beating step using a MagNA Lyser cell disrupter  as detailed previously.G. duodenalis–specific nuclear 18S rRNA region within the small subunit ribosomal ribonucleic acid (SSU RNA),Supplemental Table 1A–C. This real-time PCR assay included one G. duodenalis positive control using 5 μL of G. duodenalis template DNA  PCR thermocycler . All real-time PCR assay plates were prepared using a Myra liquid handling system . A cycle threshold (Ct) value of ≤ 39 was deemed positive for infection with G. duodenalis infection during the initial diagnostic 18S real-time PCR screen described earlier, a nested PCR was carried out to detect and amplify a single-copy 511-bp region of the G. duodenalis bg locus. This was then used for genotyping and phylogenetic analysis according to a previously published methodology,bg locus being only single copy. Details of all primer sequences, both reaction mixes used, and both initial and nested PCR conditions can be found in Supplemental Table 2A–C, respectively. The initial PCR included a positive control using 2 μL of reference G. duodenalis template DNA .Using all fecal DNA extract isolates deemed positive for Nested PCR products were purified using the ExoSAP-IT PCR Product Clean-Up Reagent (ThermoFisher Scientific) according to manufacturer’s instructions and were then sequenced in both forward and reverse directions using Sanger sequencing.G. duodenalis assemblages were identified by carrying out nucleotide Basic Local Alignment Search Tool (BLAST) searches within the NCBI databaseG. duodenalis assemblages A and B in tandem. To check whether any sequences were more closely related to G. duodenalis assemblages C through H, additional nucleotide BLAST searches were carried out using each cleaned sequence in turn against the G. duodenalis (non–assemblage-specific) database. Nucleotide BLAST hits with the greatest match and query cover scores as well as lowest E-values were considered the most closely related assemblage and the sequence of each most closely related BLAST hit was downloaded from GenBank in FASTA format to serve as an assemblage-specific reference sequence. Five randomly selected G. duodenalis assemblage A forward sequences and five randomly selected G. duodenalis assemblage B forward sequences were uploaded to the GenBank repositoryG. duodenalis assemblages A and B, a multiple alignment with fast Fourier transform (MAFFT) alignment was carried out using all obtained assemblage A and B reference sequences  within Geneious Prime . This MAFFT alignment was then visualized within Genious Prime to identify which nucleotide positions showed single nucleotide polymorphism (SNP) variations between both assemblages. Chromatograms of all study sample sequences (forward direction and grouped according to school site) were then MAFFT aligned to this existing reference alignment using Geneious Prime and visualized at SNP positions to check for double-peaking with both assemblage-specific nucleotides, suggesting co-infection with both A and B assemblages.To identify any samples co-infected with both To infer evolutionary relationships between all study sequences identified as single-assemblage infections, all eight reference sequences and all sequences identified as mixed-assemblage infections were removed from this MAFFT alignment, which was then exported from Geneious Prime and imported into PopART version 1.7.G. duodenalis infection during the initial diagnostic 18S real-time PCR screen described earlier, a duplex real-time PCR was carried out to detect and amplify G. duodenalis assemblage A- and B-specific fragments within the tpi locus (both single-copy targets 77-bp in length) according to previous work,bg and tpi loci. Details of primer/probe sequences, reaction mix used, and PCR conditions can be found in Supplemental Table 3A–C. This real-time PCR assay included three positive controls: one using 3 μL of reference G. duodenalis assemblage A template DNA, one using 3 μL of reference G. duodenalis assemblage B template DNA, and one using 3 μL of reference G. duodenalis mixed-assemblage A and B template DNA . All real-time PCR assay plates were prepared using a Myra liquid handling system (Bio Molecular Systems). A Ct value ≤ 45 was deemed positive for infection with either All statistical analyses and generation of figures were caried out within R Studio version 2021.09.0, build 351 ,Any association between diagnostic 18S real-time PCR Ct values and participant age was measured using a Spearman’s rank coefficient test. This was calculated using the cor.test function as part of the R stats package.Diagnostic outcomes (positive/negative) of QUIK CHEK, FOB- and FC-RDTs were compared with that of the diagnostic 18S real-time PCR. Sensitivity, specificity, positive predictive, and negative predictive values were calculated using the epi.tests function as part of the epiR package version 2.0.39.G. duodenalis . Odds ratios were also calculated to measure any association between self-reported regular contact with livestock cattle , and infection with G. duodenalis . In addition, ORs were calculated to measure any association between infection with G. duodenalis  and presence of FOB (according to FOB-RDT outcomes), self-reporting of abdominal (stomach) pain, and self-reporting of loose stool (diarrhea).Odds ratios (OR) were calculated to measure any association between self-reported consumption of lake water (as ascertained by yes–no questionnaire responses) and infection with G. duodenalis assemblage A (either single assemblage A infection or mixed assemblage A and B infection versus no infection and single assemblage B infection) and self-reported consumption of lake water, as well as self-reported regular contact with livestock cattle. Likewise, ORs were calculated in this manner to measure any association between infection with G. duodenalis assemblage B and self-reported consumption of lake water, as well as self-reported regular contact with livestock cattle.Odds ratios were also calculated to measure any association between infection with G. duodenalis assemblage A (versus no infection), and presence of FOB (according to FOB-RDT outcomes), self-reporting of abdominal (stomach) pain, and self-reporting of loose stool (diarrhea). Likewise, ORs were calculated in this manner to measure any association between single infection with G. duodenalis assemblage B and presence of FOB (according to FOB-RDT outcomes), self-reporting of abdominal (stomach) pain, and self-reporting of loose stool (diarrhea). These analyses were restricted only to single-assemblage infections versus no infection only (and not to the mixed-assemblage infections) because mixed-assemblage infections may confound pathological outcomes. Finally, ORs were also calculated to measure any association between mixed infection with both G. duodenalis assemblages A and B and presence of FOB (according to FOB-RDT outcomes), self-reporting of abdominal (stomach) pain, and self-reporting of loose stool (diarrhea). Odds ratios were calculated using the oddsratio function as part of the epitools package version 0.5.10.1.Furthermore, ORs were calculated to measure any association between single infection with G. duodenalis infection using the diagnostic 18S real-time PCR across all four schools was 305. Of these participants, responses to all questions are provided in The total number of questionnaire respondents also assessed for G. duodenalis assemblage A, single infection with G. duodenalis assemblage B, or mixed infection with both G. duodenalis assemblages A and B) identified was 289. Of these participants, responses to the questions “do you currently have abdominal (stomach) pain?” and “do you currently have loose stool (diarrhea)?” can are provided in The total number of questionnaire respondents that were either deemed negative for infection using the diagnostic 18S real-time PCR or that had positive infection status . Of these, 16  were deemed light infections; 50  were deemed moderate infections; and 42  were deemed heavy infections.A total of 109 (34.9% of 312) participants were deemed positive for A total of 56 (27% of 207) participants were deemed positive for overt FOB using the FOB-RDT. Of these, one  was deemed light outcome; 47  were deemed moderate outcome; and eight  were deemed heavy outcome.A total of 12 (12% of 101 participants) were deemed positive for overt FC using the FC-RDT. Of these, none were deemed light outcome; eight  were deemed moderate outcome; and four  were deemed heavy outcome.DNA extraction was performed using 310 of all 313 (99%) ethanol-preserved fecal samples because three fecal samples were deemed too low in volume to carry out DNA extraction.G. duodenalis infection using the diagnostic 18S real-time PCR . Of all fecal DNA isolates deemed positive for G. duodenalis infection, 54  were deemed light infections; 54  were deemed moderate infections; and 12  were deemed heavy infections, as ascertained by diagnostic 18S real-time PCR Ct values. The diagnostic outcome of all assay control samples was as expected. All samples retested for quality assurance purposes  gave the same diagnostic outcome as the initial screen with G. duodenalis–positive samples giving a Ct value within ± Ct 5 given during the initial screen.Of the 310 samples screened, five failed to amplify the PhHV-1 internal DNA extraction control in both initial and repeat screens and were therefore omitted from further analysis. Of the remaining 305 samples, 120 (39.3%) were deemed positive for G. duodenalis bg locus was successfully amplified in 108 samples , all of which were then purified and sequenced in both forward and reverse directions using Sanger sequencing. One sample, provided by a study participant attending Mchoka school, was omitted from any further analysis because this was deemed a poor-quality sequence. As such, a total of 107 samples , school N = 14) were used for genotyping and phylogenetic analysis. The diagnostic outcome of all assay control samples was as expected.The 511-bp region of the G. duodenalis bg locus, 38 samples  were identified as single G. duodenalis assemblage A infections , whereas 34 samples  were identified as single G. duodenalis assemblage B infections . A total of 35 samples  were identified as mixed G. duodenalis assemblage A and B infections . No samples were identified as G. duodenalis assemblages C-H. A TCS haplotype network constructed using all MAFFT aligned study sequences  identified as single-assemblage infections can be seen in When targeting the G. duodenalis bg loci haplotypes were identified within this study group, 15 of which were associated with G. duodenalis assemblage A and 13 with G. duodenalis assemblage B. There is clear and distinct divergence between G. duodenalis assemblages A and B, haplotypes of which are separated by 14 SNPs. Twenty-one of 38 (55%) sequences identified as G. duodenalis assemblage A shared a common haplotype and two of 38 (5%) also shared a common haplotype. The remaining 15 G. duodenalis assemblage A sequence haplotypes were unique and not shared. However, one sequence (SUN07) was more closely related to G. duodenalis assemblage B than the remaining 37 G. duodenalis assemblage A sequences, and two sequences (MCH80 and MCH91) were far more genetically distinct than the remaining 36 G. duodenalis assemblage A sequences . Likewise, 11 of 34 (32%) sequences identified as G. duodenalis assemblage B shared one common haplotype, and six of 34 (18%) also shared a common haplotype. Five sequences each shared a common haplotype with one other sequence, and six G. duodenalis assemblage B sequence haplotypes were unique and not shared. One sequence (MCH34) was more closely related to G. duodenalis assemblage A than the remaining 33 G. duodenalis assemblage B sequences, albeit by only two SNPs. All four schools were well represented within haplotypes across both G. duodenalis assemblages, and so no evidence of G. duodenalis divergence by locality (school site) was suggested.Twenty-eight unique G. duodenalis tpi locus was successfully amplified in 78 samples , representing 65% of all samples deemed positive for G. duodenalis infection by diagnostic 18S real-time PCR and 55% of samples with infection status identified using nested PCR/Sanger sequencing (targeting the bg locus). When targeting the G. duodenalis tpi locus, 20 samples  were identified as single G. duodenalis assemblage A infections , whereas 45 samples  were identified as single G. duodenalis assemblage B infections . A total of 13  samples were identified as mixed G. duodenalis assemblage A and B infections . The diagnostic outcome of all assay control samples was as expected. All samples retested for quality assurance purposes  gave the same diagnostic outcome as the initial screen with G. duodenalis–positive samples giving a Ct value within ± Ct 5 given during the initial screen.The 77-bp region of the 18 G. duodenalis infections identified as single assemblage A when targeting the bg locus were also identified as single assemblage A when targeting the tpi locus. One infection identified as single assemblage A when targeting the bg locus was identified as mixed-assemblage A and B when targeting the tpi locus, and two infections identified as mixed-assemblage A and B when targeting the bg locus were identified as single assemblage A when targeting the tpi locus. The remaining 19 infections identified as single assemblage A when targeting the bg locus could not be identified when targeting the tpi locus due to failure in amplifying the G. duodenalis assemblage A-specific tpi fragment using the duplex G. duodenalis assemblage A- and B-specific real-time PCR.In total, G. duodenalis infections identified as single assemblage B when targeting the bg locus were also identified as single assemblage B when targeting the tpi locus. No infections identified as single assemblage B when targeting the bg locus identified as mixed-assemblage A and B when targeting the tpi locus, and 16 infections identified as mixed-assemblage A and B when targeting the bg locus were identified as single assemblage B when targeting the tpi locus. The remaining six infections identified as single assemblage B when targeting the bg locus could not be identified when targeting the tpi locus due to failure in amplifying the G. duodenalis assemblage B-specific tpi fragment using the duplex G. duodenalis assemblage A- and B-specific real-time PCR. No discordance between assemblage-type was found between bg and tpi loci across all single-assemblage infections successfully identified using both targets.Likewise, 28 of G. duodenalis infections identified as mixed assemblage A and B infections when targeting the bg locus were also identified as mixed assemblage A and B infections when targeting the tpi locus. The remaining 23 of these infections could not be identified as mixed-assemblage infections due to failure in amplifying either or both G. duodenalis assemblage A- or assemblage B-specific tpi fragments using the duplex G. duodenalis assemblage A- and B-specific real-time PCR. Of these 23 infections that could not be identified as mixed infections using the G. duodenalis assemblage A- and B-specific real-time PCR, two were identified as single-infection assemblage A (as assemblage B DNA was not detected), and 16 were identified as single-infection assemblage B (as assemblage A DNA was not detected). Furthermore, neither G. duodenalis assemblage A or B DNA was detected in five samples deemed mixed-assemblage infections (by nested PCR/Sanger sequencing targeting the bg locus) when using the G. duodenalis assemblage A- and B-specific real-time PCR (targeting tpi loci).In addition, 12 of G. duodenalis assemblage-specific real-time PCR assay (targeting tpi loci) compared with nested PCR/Sanger sequencing (targeting the bg locus) and relative to diagnostic 18S real-time PCR Ct values, sensitivity of the assay appears to decrease as diagnostic 18S real-time PCR Ct values increase. This suggests that G. duodenalis assemblages in samples with a lower concentration of DNA , may be less likely to be correctly identified when targeting the tpi region using this real-time PCR assay , appears reliable when screening single-assemblage (A or B) infections between diagnostic 18S real-time PCR Ct values 14 to ∼30. However, this assay appears unreliable when screening single-assemblage infections when diagnostic 18S real-time PCR Ct values are > 30, particularly when screening samples identified as single G. duodenalis assemblage A infections by nested PCR/Sanger sequencing (targeting the bg locus). In addition, although performance is slightly improved with lower diagnostic 18S real-time PCR Ct values, when screening mixed-assemblage (A and B) infections, the assay appears unreliable regardless of diagnostic 18S real-time PCR Ct value. This is most likely because of the assay’s poor performance in detecting and amplifying G. duodenalis assemblage A DNA .No association between participant age and diagnostic 18S real-time PCR Ct values was found (P = 2.2 e-16) , a specificity of 96% (95% CI: 92–98), a positive predictive value of 93% (95% CI: 87–97), and a negative predictive value of 90% (95% CI: 85–94). In addition, there was a strong negative and significant correlation between QUIK CHEK RDT band strength and diagnostic 18S real-time PCR Ct values  . There wWhen comparing diagnostic outcomes of the FOB-RDT against the diagnostic 18S real-time PCR, FOB-RDT had a sensitivity of 28% (95% CI: 18–39), a specificity of 77% (95% CI: 68–84), a positive predictive value of 46% (95% CI: 32–61), and a negative predictive value of 60% (95% CI: 51–68). When comparing diagnostic outcomes of the FC-RDT against the diagnostic 18S real-time PCR, FC-RDT had a sensitivity of 6% (95% CI: 1–17), a specificity of 82% (95% CI: 69–91), a positive predictive value of 25% (95% CI: 5–57), and a negative predictive value of 48% (95% CI: 37–59).G. duodenalis . In addition, there was a statistically insignificant and weak negative association between self-reported regular contact with livestock cattle, and infection with G. duodenalis . There was a statistically insignificant and weak positive association between infection with G. duodenalis and presence of FOB . However, there was a statistically significant and strong positive association between infection with G. duodenalis and both self-reporting of abdominal pain , and self-reporting of loose stool (diarrhea) .There was a statistically insignificant and weak positive association between self-reported consumption of lake water and infection with G. duodenalis assemblage A . There was also a statistically insignificant and weak positive association between self-reported consumption of lake water and infection with G. duodenalis assemblage B . In addition, there was a statistically insignificant and weak positive association between self-reported regular contact with livestock cattle and infection with G. duodenalis assemblage A . There was also a statistically insignificant and weak negative association between self-reported regular contact with livestock cattle and infection with G. duodenalis assemblage B .There was a statistically insignificant and weak positive association between self-reported consumption of lake water and infection with G. duodenalis assemblage A and presence of FOB  and self-reporting of abdominal pain . Additionally, there was no association between single infection with G. duodenalis assemblage A and self-reporting of loose stool/diarrhea . There was a statistically insignificant positive association between single infection with G. duodenalis assemblage B and presence of FOB ; however, there was a statistically significant and strong positive association between single infection with G. duodenalis assemblage B and both self-reporting of abdominal pain , and self-reporting of loose stool/diarrhea .There was a statistically insignificant and weak negative association between single infection with G. duodenalis assemblages A and B and presence of FOB  and statistically insignificant positive association between mixed infection with both G. duodenalis assemblages A and B and self-reporting of loose stool/diarrhea, . There was, however, a statistically significant and strong positive association between mixed infection with both G. duodenalis assemblages A and B and self-reporting of abdominal (stomach) pain . On the basis of assay sensitivity and specificity compared with diagnostic 18S real-time PCR, the overt presence of FC as measured by RDT was deemed a poor marker for G. duodenalis infection and so was omitted from OR analyses.There was a statistically insignificant negative association between mixed infection with both G. duodenalis assemblages A and B as well as between single- and mixed-assemblage infections is essential to better understand the pathological impact of infection with either, or both, assemblages and thus also for improved disease surveillance and control.G. duodenalis infection statuses  and test for any association(s) between infection status and intestinal pathology.Differentiation between human infections with G. duodenalis infection status was identified in 107 study participants when targeting the bg locus. Although mixed-assemblage (A and B) G. duodenalis infections often appear to comprise approximately one-third of all infections,,–G. duodenalis assemblage A or assemblage B.,–G. duodenalis assemblages A and B haplotypes  and when attempting to confirm mixed-assemblage infections, regardless of diagnostic 18S Ct value . Although real-time PCR offers a more rapid and less expensive means of assemblage typing G. duodenalis infections , further work is needed to optimize and improve this real-time PCR assay, especially when attempting to genotype low-intensity single-assemblage infections or mixed infections where the intensity of infection of each individual assemblage may also be low.tpi locus being only single-copy, such as nested PCR . Whilst further and more thorough assessment of the assay is needed to confirm diagnostic performance , the assay showed excellent promise as a rapid and reliable diagnostic tool capable of accurately diagnosing G. duodenalis infections in resource-limited settings. Both FOB and FC RDTs were deemed unreliable as a means of measuring intestinal pathology caused by G. duodenalis infection because no association between the presence of FOB or FC and G. duodenalis infection was found when using these assays.When targeting G. duodenalis infection. There was also no association found between self-reporting of these behaviors and infection with either G. duodenalis assemblage A or B. It may therefore be alternative recognized routes of giardiasis transmission, such as through contamination of food or eating utensils , during food preparation and/or consumption, through direct contact with infected individuals, or through unintentional consumption of lake water , that is the primary mode of G. duodenalis infection within this study group.,Surprisingly, no association was found between self-reported consumption of lake water, as well as self-reported regular contact with livestock cattle, and G. duodenalis assemblage B and both self-reporting of abdominal (stomach) pain and self-reporting of loose stool (diarrhea), but no identified association between single infection with G. duodenalis assemblage A and any form of intestinal pathology. With regard to self-reported diarrhea specifically, these data are in agreement with some previous studies that have also found an association between single infection with G. duodenalis assemblage B and symptomatic diarrhea ,,,–,,,,,G. duodenalis assemblage B and stomach or abdominal pain ,,,,,Interestingly, however, within this study group, there was a statistically significant positive association between single infection with G. duodenalis infection type and intestinal pathology.,G. duodenalis assemblage A and B were similar. Alternatively, other studies have suggested that it is perhaps host–parasite factors, such as host age, nutritional status and immunological status, that are likely to play a major role in G. duodenalis assemblage-specific pathologies.G. duodenalis assemblage-specific pathologies is warranted.Currently, there is no clear correlation between P = 0.06), there was a positive association between mixed infection with both G. duodenalis assemblages A and B and self-reporting of loose stool/diarrhea, as well as a statistically significant positive association between mixed infection with both assemblages and self-reporting of abdominal pain. Although an interesting observation, conclusions as to whether these associations are a result of individuals being infected with G. duodenalis assemblage B , or whether there is some potential interplay between mixed infection with both assemblages and intestinal pathology, cannot be drawn using these data; thus, additional work is needed to scrutinize further whether infection with human-infecting G. duodenalis assemblages A and B has a more detrimental impact on intestinal health than single infection with just one assemblage.Additionally, although statistically insignificant  or mixed-assemblage (A and B) infections. Furthermore, these data also suggest that asymptomatic G. duodenalis infections may be linked to G. duodenalis infection type , as also suggested elsewhere.Nevertheless, in keeping with previous studies, these data also suggest that there may be marked differences in the degree of pathology experienced by those infected with either G. duodenalis subassemblages AI–AIII, BIII, and BIV, which may also potentially differ in their pathogenic impact, could not be identified. To investigate any differences in pathological impact within G. duodenalis assemblage types, more thorough genetic analyses are needed.G. duodenalis infection status and intestinal pathology, additional and more extensive pathology testing, such as through measuring participant height and weight,,An immediate limitation of this study is that gdh locus, to confirm identified infection types—particularly in light of the fairly poor performance of the G. duodenalis assemblage-specific real-time PCR assay, targeting tpi loci, used here. Further development and optimization of reliable markers and assays for multi-locus G. duodenalis assemblage typing should continue to be carried out,,,Another limitation included the absence of additional genetic targets, such as the G. duodenalis assemblage A and mild/intermittent diarrhea, whereas infection with G. duodenalis assemblage B was associated with severe/persistent diarrhea. Although severity of pathological symptoms such as diarrhea was not investigated here, future studies should seek to investigate further any associations between G. duodenalis infection type(s) and the severity of pathological symptoms, rather than just presence and absence of symptoms.It should also be highlighted that previous studies have also found statistically significant differences in the severity of pathological symptoms, such as diarrhea, between those infected with either assemblage. As an interesting example, Homan and MankG. duodenalis  or through intestinal pathology being exacerbated by coinfection with G. duodenalis and other intestinal pathogens.G. duodenalis assemblage types and infection status.Furthermore, the pathogenic impact of other waterborne pathogens prevalent in this area known to cause similar intestinal pathologies, such as intestinal schistosomiasis and cryptosporidiosis, were not considered within any analyses and so may be confounding results, either through intestinal pathology being present within individuals not infected with G. duodenalis assemblage A and B single and mixed infections are common in school-age children situated along the southern shoreline of Lake Malawi, Mangochi district, Malawi, Within this study group, it was found that there may be marked differences in the degree of intestinal pathology experienced by those infected with G. duodenalis assemblage A compared with those infected with G. duodenalis assemblage B, as well as between those burdened with single- and mixed-assemblage infections. This study therefore highlights the importance of molecular methods that can be used to identify G. duodenalis assemblage types as well as the importance of investigating their impact on human pathology.Both G. duodenalis but also of poor access to clean and safe drinking water and sanitation facilities. In doing so, not only can the pathological impact of human giardiasis be better understood, but, through improved disease surveillance and control and improved access to WASH infrastructure, a reduction in disease-associated pathologies experienced by the world’s most disadvantaged communities can also be achieved.Molecular surveillance methods such as these should continue to be used, particularly in areas lacking adequate access to WASH infrastructure, to better understand the detrimental health impacts not only of infection with Supplemental materialsSupplemental materials


Molecular circuits and devices with temporal signal processing capability are of great significance for the analysis of complex biological processes. Mapping temporal inputs to binary messages is a process of history-dependent signal responses, which can help understand the signal-processing behavior of organisms. Here, we propose a DNA temporal logic circuit based on DNA strand displacement reactions, which can map temporally ordered inputs to corresponding binary message outputs. The presence or absence of the output signal is determined by the type of substrate reaction with the input so that different orders of inputs correspond to different binary outputs. We demonstrate that a circuit can be generalized to more complex temporal logic circuits by increasing or decreasing the number of substrates or inputs. We also show that our circuit had excellent responsiveness to temporally ordered inputs, flexibility, and expansibility in the case of symmetrically encrypted communications. We envision that our scheme can provide some new ideas for future molecular encryption, information processing, and neural networks. In nature, how living systems process internal and external signals is complicated and diverse and depends on the complexity, type, and temporal order of the signals. Living organisms respond to variable identities and orders of signals with their internal temporal regulatory networks and thus exhibit diverse behaviors and phenotypes . For exaIn prior studies, several schemes have been proposed to respond to temporally ordered inputs ,7 and siInspired by this idea, we propose a scheme for DNA temporal logic circuits based on DNA strand displacement reactions. According to our design rules, our circuits can be generalized to larger-scale temporal logic circuits by varying the number of substrates or inputs. Specifically, we studied the mapping relationship between the temporally ordered inputs and the binary message when the number of inputs and substrates is different. Here, we present the corresponding simulation model. The circuit was applied to the symmetric encryption of binary messages to demonstrate the effectiveness of mapping temporally ordered inputs into binary messages. We constructed a five-input, five-substrate DNA temporal logic circuit using a simulation model and used the circuit as the key for encryption and decryption. Finally, we used this circuit to demonstrate a case of symmetrically encrypted communication of binary messages to show its responsiveness, flexibility, and expansibility.All DNA samples used in this paper were purchased from Sangon Biotech. Co., Ltd. . The unmodified DNA strands were purified by polyacrylamide gel electrophoresis. DNA strands modified with a fluorophore or quencher were purified by high-performance liquid chromatography. The fluorophores of the three modifications were FAM, ROX, and CY5.5, respectively, and the corresponding quenching agents of the three modifications were BHQ1, BHQ2, and BHQ3, respectively. The concentration of DNA molecules was measured using the NanoDrop 2000 spectrophotometer  at an absorbance of λ = 260 nm.2+ buffer  and placed inside a polymerase chain reaction (PCR) thermocycler to anneal. The temperature was set to be heated at 95 °C for 10 min, cooled to 25 °C at the rate of 1 °C/min, and maintained at 25 °C for 10 min.All substrates were dissolved as single strands of DNA at the same concentration (3 µM) in 50 µL of 1 × TAE/Mg2+ buffer and reacted for 2 h at 37 °C. The samples to be tested were mixed with 3 µL of 50% glycerol solution and analyzed in a 12% native polyacrylamide gel after running at a constant pressure of 85 V for 120 min.All samples were dissolved in 30 µL of 1 × TAE/Mg2+ buffer at 37 °C for fluorescence analysis. All fluorescence experiments were repeated more than three times to ensure reproducibility.All fluorescences were measured using a real-time PCR system . All samples were added to 50 µL of 1 × TAE/MgA DNA temporal logic circuit is defined in To implement the above principle of mapping temporally ordered inputs to binary messages, we designed the circuit shown in All the release and inhibition reactions are similar DNA strand displacement reactions; so, we illustrate only the release and inhibition reactions of S2 in The results of circuit mapping the temporally ordered inputs to binary messages are indicated by the fluorescence experiments shown in n inputs and n substrates can be designed by increasing or decreasing the number of inputs and substrates. Therefore, we studied the circuit when the number of inputs and the number of substrates changed.The DNA temporal logic circuit constructed above is a reaction system with three inputs and three substrates. The design rules and responsiveness to mapping temporally ordered inputs to binary messages have been described. Notably, a larger-scale temporal circuit with more inputs and substrates can be easily extended according to our design rules. Theoretically, a DNA circuit with any arbitrary number of When the number of substrates was changed, we first explored the temporal logic circuit of three inputs and two substrates. The design of the specific circuit is shown in We explored the temporal logic circuit with four inputs and three substrates. The binary messages of the temporally ordered inputs map are also different from all previous circuits. The design of the specific circuit is shown in DNA temporal logic circuits are essentially the mapping of different orders of inputs to specific binary messages. According to our design rules, more inputs can be mapped by varying the number of inputs and substrates. Theoretically, the expansibility allows the number of binary bits output to increase arbitrarily. The n + 1 inputs n substrates circuit can ensure the integrity of n bits of binary messages when mapping. This means that any binary message we want to express can be included. Therefore, we propose to encrypt and decrypt binary messages through our scheme. According to the explicit mapping relationship between the input order and the output, the timing input is equivalent to a binary message when transmitted. Specifically, we created a five-input, five-substrate temporal logic circuit using a simulation model and used the model to demonstrate the case of encrypted communication with binary messages.The information communication process of the sender, Alice, and the receiver, Bob, is shown in In this study, we proposed a general design rule for DNA temporal logic circuits, which could map temporally ordered inputs to different binary messages. We applied this scheme to symmetric encryption to protect binary information during communication. Accordingly, we demonstrated that our circuit had excellent responsiveness to temporally ordered inputs, flexibility, and expansibility, which could be applied to the symmetrically encrypted communication of binary messages.It should be noted that the release and inhibition reactions were key in achieving the responses to different input orders, and the substrate could lead to different binary number outputs according to the different reactions that occurred. In the experiments, we set the temperature condition of these reactions to 37 °C; thus, our framework could adapt to the biological environment. Individual output signals could also be designed to participate in downstream reactions. All these features provided a modular interface for subsequent extensions and facilitate the potential implementation of more complex and larger-scale synthetic circuits. DNA circuits with a temporal memory function are considered to have the potential to provide more opportunities for the future intelligent behavior of artificial molecular machines . We hope


Fertility preservation in pregnant women recently diagnosed with cancer can be a challenge. Raised levels of human chorionic gonadotropin (Beta-hCG) and progesterone in this population of patients may pose a problem for the prompt initiation of controlled ovarian stimulation (COS) due to a potential negative feedback of these hormones on folliculogenesis; however, it is not feasible to wait for negativization of serum beta-hCG levels before starting controlled ovarian stimulation. In literature, very few cases have been reported regarding the preservation of fertility in pregnant women recently diagnosed with cancer. We performed an extended revision of the literature to evaluate the current knowledge of the management of fertility preservation in women with cancer and we examined two cases closely. The first case study involved a cancer patient who underwent surgical abortion at 6.5 weeks of gestation followed by administration of mifepristone to detach any minimal residual trophoblast and consequently to decrease serum beta-hCG and progesterone levels before starting COS. In the second case study, the cancer patient underwent surgical abortion at 7.1 weeks of gestation and simultaneous unilateral oophorectomy for ovarian tissue cryopreservation due to a limited time for COS. By analyzing the results of these studies, it could be hypothesized that mifepristone administration may favor the decrease of serum beta-hCG and progesterone levels in order to permit rapid initiation of COS. In cases where COS is not feasible, ovarian tissue cryopreservation should be considered as an alternative fertility preservation technique. Hematological cancers and breast cancers are among the most common oncological diseases diagnosed in adolescents and young adults and their associated treatments can negatively impact fertility. Due to the diagnosis of such tumors at younger ages and the trend of delaying motherhood until older ages , a risinA review of studies was performed evaluating the management of fertility preservation in pregnant women recently diagnosed with cancer. A comprehensive review of the literature was performed using PubMed, Scopus, Embase, ScienceDirect and Google Scholar up to March 2023. Key search terms included the following: “fertility preservation” AND “hCG” OR “abortion” OR “pregnancy termination” and “ovarian tissue cryopreservation” AND “hCG” OR “abortion” OR “pregnancy termination.” Inclusion criteria consisted of studies reported in English regarding women undergoing fertility preservation for oncological reasons after first-trimester abortion. To avoid confounding mechanisms due to different hormonal levels, reports about fertility preservation beyond the first trimester or in cancer patients who recently gave birth, were excluded. In addition, we reported two unpublished cases of fertility preservation in oncological patients who presented in early pregnancy.The electronic searches provided a total of 877 citations, after the removal of 60 duplicate records, 817 citations remained. Of these, 811 records were excluded after title/abstract screening (not relevant to the review) and 6 manuscripts were included in the review. These manuscripts report six cases of COS for fertility preservation in cancer patients after first-trimester abortion with varying outcomes . The serWe chose to include two unpublished cases in our report. To our knowledge, this is the first reported case regarding fertility preservation after a combined medical and surgical abortion, as well as the first reported case of fertility preservation with cryopreservation of ovarian tissue alone in a pregnant woman with cancer Table . The firA 28-year-old woman presented to our hospital with night sweats and itching for a period of two months. No other symptoms were reported. Prior to this occasion her personal medical history had been unremarkable. Fine-needle aspiration of a two centimeter-large cervical lymph node was performed and ultimately a diagnosis of Classic Hodgkin Lymphoma, stage IIA was determined. The diagnostic pathway was then completed with an excisional lymph node biopsy, a total-body computerized tomography and a positron emission tomography. Hematologists indicated initiation of a chemotherapy protocol with doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD) within a month from the first fertility preservation consultation. While performing a complete lab assessment before beginning COS, high levels of serum beta-hCG were detected (5959 U/L). A transvaginal ultrasound was then performed, which confirmed a pregnancy dated at 6.1-weeks. After a multidisciplinary discussion with hematologists and gynecologists, the patient was informed about the risks of continuing the pregnancy and the existing fertility preservation techniques were evaluated. The woman opted for pregnancy termination, and a surgical abortion procedure was successfully performed at 6 weeks 5 days of gestation. On the day of the D&C procedure, beta-hCG levels were 19064 U/L. The patient received Mifepristone 600 mg (RU486) as an off-label treatment after providing informed consent. The day after RU486 administration, serum beta-hCG levels were 6802 U/L, while 2 days after serum levels were 2258 U/L and 6 days after drug administration, beta-hCG levels further dropped to 308.9 U/L , followed by adjuvant chemotherapy with Epirubicin, Cyclophosphamide and Paclitaxel. Her gynecological history included two vaginal deliveries in 2007 and 2021. During fertility preservation workup, the patient stated that her last menstrual period had occurred eleven days before the consultation. Five antral follicles were detected during ultrasound examination. The woman underwent blood sampling to measure serum beta-hCG levels and AMH levels. Beta-hCG levels were 2390 U/L and AMH levels were 1.05 ng/ml. The patient was informed in a multidisciplinary discussion with oncologists and gynecologists about the risks of continuing the pregnancy. Two weeks after diagnosis of the pregnancy, the woman opted for pregnancy termination. The possible abortion procedures and fertility preservation techniques were evaluated. The woman successfully underwent a surgical abortion procedure at 7 weeks and 1 day and laparoscopic right ovariectomy for ovarian tissue cryopreservation contextually. Ovarian tissue was cryopreserved with the slow freezing method  , during In the case of the removal of ovarian tissue for cryopreservation, it is ideal to avoid the couple having to relocate to a center specialized in fertility preservation. If the reference center for the cryopreservation of ovarian tissue does not offer the possibility of abortion, or vice versa, in order to reduce patient stress and avoid multiple surgeries, it is possible to perform retrieval of ovarian tissue (biopsy or ovariectomy) in any center and send the specimens to the referral fertility center within several hours.LH-like action [In conclusion, the need to start fertility preservation therapy immediately after an interruption of pregnancy is challenging. From available data in the literature and the two cases described, hCG levels may affect stimulation outcomes. In fact, just one case report in the literature hypothesized that beta-hCG can improve the outcome of COS through a e action , and othe action , 7, 10.


Migraine is a neurological disorder with recurrent headaches accompanied by burdens in social life. Primary palmar hyperhidrosis is a chronic condition with excessive sweating of the palms that can significantly impair quality of life. Primary hyperhidrosis can cause anxiety, and stress, including anxiety, is the most common inducer of migraine headaches. Recently, oxybutynin has been used for primary palmar hyperhidrosis. We herein describe a 26-year-old female migraine patient with primary palmar hyperhidrosis whose migraine attacks and burdens were attenuated after the prescription of an oxybutynin lotion formula. The patient’s monthly headache days (MHD) and monthly acute medication intake days (AMD) at the first visit were 10 and 9. Headache Impact Score 6 (HIT-6) at the initial visit was 63. After the prescription of Japanese herbal kampo medicine Goreisan (TJ-17), Goshuyuto (TJ-31), and 200 mg of valproic acid, MHD, AMD, and HIT-6 decreased gradually. However, these parameters could not improve sufficiently at nine months: MHD 4, AMD 4, and HIT-6 52. We first prescribed a lotion formulation of 20% oxybutynin hydrochloride at nine months. After this, migraine was further attenuated, and stress related to primary palmar hyperhidrosis was reduced; at 12 months, the patient had achieved MHD 2, AMD 2, and HIT-6 48. She will continue receiving primary palmar hyperhidrosis treatment while tapering off migraine prophylaxis. While the exact mechanisms connecting migraine and primary hyperhidrosis remain uncertain, this case raises important questions about the potential interplay between stress, sweating, and migraine triggers. Migraine is a neurological disorder characterized by recurrent episodes of severe headache, often accompanied by other symptoms such as nausea, vomiting, and sensitivity to light and sound. The epidemiological prevalence in Japan is 8.4%, and those in other countries are higher . MigrainPrimary palmar hyperhidrosis is a chronic condition characterized by excessive sweating of the palms that can significantly impair daily activities and quality of life. The epidemiological survey revealed the prevalence of primary palmer hyperhidrosis as 5.22% among the 5- to 64-year-old population . TherefoWe herein describe a migraine patient with primary palmar hyperhidrosis whose migraine attacks and burdens were attenuated after the prescription of a lotion formulation of 20% oxybutynin hydrochloride.A 26-year-old female presented with a chronic headache on a numeric rating scale of 2/10 to 8/10 as an outpatient, with a long-standing history of primary palmar hyperhidrosis and recurrent migraine attacks. The headache was not pulsatile but unilateral. She had experienced headache aggravation and avoided physical activity due to the headache, and she also felt nausea. The attacks last for about six hours. She had suffered from migraines since age 16, and the frequency and intensity of migraine attacks had worsened over the last three years. She had never consulted a neurologist about her headache or a dermatologist about her palmar hyperhidrosis. She used over-the-counter analgesics as acute medication, and no prophylactic medications had been tried. We diagnosed migraine without aura , and we let her keep a headache diary. The monthly headache days (MHD) and monthly acute medication intake days (AMD) at the first visit were 10 and 9. Headache Impact Score 6 (HIT-6) at the initial visit was 63. The HIT-6 score is obtained from a simple summation of the six items and ranges between 36 and 78, with larger scores reflecting greater impact. Headache impact severity levels can be categorized using score ranges based on the HIT-6 interpretation guide. The four headache impact severity categories are little or no impact (49 or less), some impact (50-55), substantial impact (56-59), and severe impact (60-78) [According to the guideline , describAfter starting prophylactic medications, those parameters gradually decreased. However, at nine months, the patient’s MHD, AMD, and HIT-6 were 4, 4, and 52, respectively. Because of concerns about drowsiness, there was no desire to further increase the dose of valproic acid or add other medications. It was only then, for the first time, that she also asked about palmar hyperhidrosis. She has experienced excessive sweating of her palms since adolescence, which significantly affected her social interactions and self-confidence. We prescribed a lotion formulation of 20% oxybutynin hydrochloride. At 12 months, the patient had achieved MHD 2, AMD 2, and HIT-6 48. MHD and AMD decreased, and HIT-6 was within the normal range as it was below 50. She will continue receiving primary palmar hyperhidrosis treatment while tapering off valproic acid and Japanese herbal kampo medicines. Also, she reported that her palmar hyperhidrosis-induced stress was significantly reduced after the initiation of oxybutynin treatment.The presented case report describes a patient with primary palmar hyperhidrosis and migraine attacks whose migraine frequency and burdens were attenuated after the prescription of a lotion formulation of 20% oxybutynin hydrochloride. This case raises several interesting points regarding the potential relationship between primary palmar hyperhidrosis, migraine, and the therapeutic use of oxybutynin.The potential link between hyperhidrosis and migraineThe patient had been experiencing excessive sweating of her palms since adolescence, which might have led to social distress and impaired self-confidence. The relationship between primary palmar hyperhidrosis and migraines is intriguing, especially considering that stress ,7, incluA recent investigation has revealed that individuals suffering from migraines exhibit heightened sensitivity to wetness, particularly when exposed to cold and wet stimuli on their foreheads, as opposed to healthy individuals. This sensitivity to cold and wet conditions could potentially be attributed to the involvement of the transient receptor potential cation channel subfamily M (melastatin) member 8 (TRPM8) . This chOxybutynin's potential mechanism of actionOxybutynin is an anticholinergic medication  that hasIn addition to CGRP, novel therapeutic targets have been found for migraine . AcetylcIt has also been shown that migraine patients have reduced sympathetic activity during the interictal phase of attacks. Therefore, it is possible that oxybutynin may have normalized autonomic nervous system activity by inhibiting acetylcholine and suppressing the parasympathetic nervous system , therebyLimitations and considerationsAs a limitation, we did not examine how much stress was directly due to hyperhidrosis, such as with a stress check test. The patient initially received prophylactic medications for her migraines, which led to a gradual reduction in migraine frequency and burden. However, a complete resolution was not achieved, and concerns about drowsiness limited the ability to further increase the dose of the prophylactic medication. It was only after addressing the primary palmar hyperhidrosis with oxybutynin that the patient achieved a substantial improvement in both her migraine symptoms and her palmar hyperhidrosis. This suggests the possibility of a synergistic effect when treating both conditions simultaneously. While adequate prophylactic medications have not yet been developed for migraine, it may be a good idea to do what may improve the situation, even if only slightly.The presented case highlights a unique observation of a patient with primary palmar hyperhidrosis and migraines experiencing significant improvements in migraine frequency and burdens following the initiation of oxybutynin treatment for primary palmar hyperhidrosis. While the exact mechanisms connecting these conditions remain uncertain, this case raises important questions about the potential interplay between stress, sweating, and migraine triggers. Further research is needed to elucidate the underlying mechanisms and explore the potential benefits of combined therapeutic strategies for patients with coexisting primary palmar hyperhidrosis and migraines.


Hyperuricemia and right ventricular dysfunction (RVD) are both widespread in heart failure with preserved ejection fraction (HFpEF) patients. RVD is associated with a poor prognosis in HFpEF. The correlation between serum uric acid (UA) levels and right ventricular function is unclear. The prognostic performance of UA in patients with HFpEF needs further validation.p < 0.001) was independently associated with RVD, and UA >7 mg/dl  was associated with heart failure readmission in patients with HFpEF.A total of 210 patients with HFpEF were included in the study and divided into two groups according to UA level: the normal UA group (≤7 mg/dl) and the high UA group (>7 mg/dl). The variables examined included clinical characteristics, echocardiography, and serum biochemical parameters. Right ventricular function was assessed by tricuspid annular plane systolic excursion (TAPSE) and tricuspid annular peak systolic velocity (TAPSV). Baseline characteristics were compared between the two groups, and the correlation between baseline UA and RVD was assessed using multifactorial binary logistic regression. Kaplan–Meier curves were used to describe all-cause mortality and heart failure readmission. Results showed that right ventricular function parameters were worse in the high UA group. After adjusting for UA, left ventricular posterior wall thickness (LVPWT), N-terminal B-type natriuretic peptide (NT-proBNP), atrial fibrillation (AF), and low-density lipoprotein cholesterol (LDL-C), UA (odds ratio = 2.028; Elevated serum UA is closely associated with RVD and significantly associated with the heart failure readmission rate in patients with HFpEF. Heart failure with preserved ejection fraction (HFpEF) is generally considered a syndrome with pathophysiological heterogeneity, whose prevalence has increased rapidly over the past two decades , 2. FactAs the end product of purine metabolism in the human body, uric acid (UA) is commonly associated with the development and progression of cardiovascular diseases such as peripheral artery disease, coronary artery disease (CAD), hypertension, and atrial fibrillation (AF) –7. The pRight ventricular dysfunction (RVD) is one of the common manifestations in the HFpEF population . Former 2, based on CKD-EPI formula), (3) urate-lowering therapy, (4) malignant tumor, (5) severe hepatic impairment (elevated liver enzymes: three times over upper reference limit or liver cirrhosis), and (6) infections. According to Chinese guidelines for the diagnosis and management of hyperuricemia and gout in 2019, hyperuricemia is defined as above 7.0 mg/dl  acute coronary syndrome or right myocardial infarction history, (2) severe renal impairment , blood urea nitrogen (BUR), creatinine (Cr), direct bilirubin (DB), hemoglobin (Hb), glycosylated hemoglobin (HbAlc), high-sensitivity C-reactive protein (hs-CRP), LDL-C, N-terminal B-type natriuretic peptide (NT-proBNP), and UA.Blood samples were taken on the second morning after admission. UA was performed through the central laboratory using ABBOTT. Conversion of each UA measurement from micromoles per liter to milligrams per deciliter was conducted by dividing it by 60.2 + right atrial pressure, estimated based on the diameter and collapse of the inferior vena cava.All the echocardiographic examinations were conducted by trained echocardiographers according to the guidelines of the American Society of Echocardiography (ASE) . The staEndpoints examined include readmission for heart failure and all-cause mortality. Heart failure readmission was determined by two senior doctors in the heart failure ward. Deaths were confirmed by population management consultations and hospital death certificates. Enrolled patients were followed up by telephone or WeChat every 4 months until the end of August 2022 or death. Postcharge clinical events were obtained through telephone follow-up and medical records from other hospitals.t-test, rank sum test, or Chi-square test were selected based on data characteristics. Pearson’s or Spearman’s tests were used to assess the association of variables with UA, TAPSE, and TAPSV. Based on published data and clinical relevance, we performed a univariate analysis of UA, gender, CAD, diabetes, AF, body mass index (BMI), systolic blood pressure (SBP), heart rate, left ventricular posterior wall thickness (LVPWT), and interventricular septal thickness (IVST). Based on the results of univariate binary logistic analysis, different models were developed to determine the odds ratio (OR) between UA and RVD. The long-term cumulative incidence of all-cause mortality and heart failure readmission was estimated using Kaplan–Meier curves. The predictive value of variables for heart failure readmission was tested by Cox’s univariate proportional hazards regression analysis. Variables in univariate Cox regression were included in multivariate Cox regression, and subgroup analysis was performed to demonstrate the potential effects of UA >7 mg/dl. Statistical significance was p < 0.05. Results were expressed as a hazard ratio (HR) with 95% confidence intervals (95% CI). All statistical analyses were performed using SPSS version 24.0 .We used percentages for qualitative data. Normally distributed quantitative data were presented as mean ± standard deviation (SD), and abnormally distributed quantitative data were presented as median (interquartile range (IQR)). The receiver operating characteristic (ROC) curve was used to determine the predictive value of UA for RVD. When comparing baseline data for HFpEF patients with UA > 7 mg/dl and UA ≤ 7 mg/dl, independent sample p < 0.05; In total, 210 eligible HFpEF patients were recruited to the study (59% women), with 80 patients assigned to the high UA group. Compared to the normal UA group, patients in the high UA group were more prevalent with CAD, AF, and higher New York Heart Association (NYHA) class heart failure, higher SBP, diastolic blood pressure (DBP), heart rate, BUR, Cr, NT-proBNP, DB, and hs-CRP, but lower Alb and LDL-C at baseline  but negatively correlated with TAPSE, TAPSV, LDL-C, and Alb (p < 0.05). In addition, TAPSE and TAPSV had positive associations with IVST, LVPWT, LDL-C, and Alb, but were negatively associated with PASP, heart rate, Cr, NT-proBNP, hs-CRP, and DB . The best cutoff value of UA for predicting RVD was 7.15 mg/dl, yielding sensitivity and specificity of 69.4% and 85.5%, respectively , AF , IVST , LVPWT , and other biochemical indexes including NT-proBNP, Cr, DB, and Alb  compared to the normal UA group, and all-cause mortality also trended to be higher without statistical significance (p = 0.062). The rate of heart failure readmission was higher in both male (p = 0.002) and female (p = 0.018) patients in the high UA group (p < 0.05). Indicators in univariate Cox regression were taken into multivariate Cox regression, and high UA  and NT-proBNP  were independently related to heart failure readmission rate after adjusting of high UA, gender, NYHA class, CAD, AF, RVD, NT-proBNP, and Cr (During a median follow-up period of 278 (190–443) days, 52 (24.8%) patients were readmitted for heart failure, and 11 (5.2%) patients died. Kaplan–Meier curves for heart failure readmission and all-cause mortality are displayed in The results of this prospective cohort analysis showed that UA levels were associated with the adverse change in right ventricular function in HFpEF patients, with RVD measured by TAPSE and TAPSV. Patients in the high UA group had a higher rate of heart failure readmission, but no statistical difference in all-cause mortality between the two groups was detected.The European Society of Cardiology (ESC) guidelines highlight UA measurement as an additional marker for the stratification of cardiovascular risk , althougVariables frequently used to assess RV function in patients with heart failure include RV ejection fraction, the longitudinal strain of the RV, TAPSE, and TAPSV. In the current study, TAPSE and TAPSV were used to assess right ventricular function, which is recommended by ASE to improve the accuracy of RVD . They arThe mechanisms to account for elevated UA and RVD in HFpEF patients appear to be unclear and remain to be elucidated. The possible explanations for the findings are as follows: Firstly, increased UA production due to the upregulation of xanthine oxidase and decreased UA excretion owing to lactic acid accumulation and reduced renal perfusion resulted in a high prevalence of hyperuricemia in heart failure patients , 31. SecPrevious studies in China did not report gender differences in UA levels in HFpEF patients, so we grouped patients according to Chinese hyperuricemia guidelines: the normal UA group (UA ≤ 7.0 mg/dl) and the high UA group (UA > 7.0 mg/dl). However, the enrolled population showed that gender differences in UA levels did exist. In addition, several studies have shown that the effect of gender on the prognosis of HFpEF is still controversial –39. In op = 0.07) or all-cause mortality ; p = 0.25) in patients with HFrEF  for the publication of any potentially identifiable images or data included in this article.X-LD contributed to the study design, data analysis, and manuscript preparation. H-WY, JX, X-FZ, JZ, MS, X-SW, Z-QL, LG, Z-YL, and PG were involved in the acquisition of data. D-YZ and QY worked on the study concept, design, and final proof. All authors read and approved the final manuscript.


Plants close plasmodesmata to restrict cell-to-cell communication after pathogen perception and in response to a range of stresses. All these stimuli trigger callose deposition at the plasmodesmal neck, suggesting a convergence of signaling. We have defined the hierarchy of molecular components and signals required to mediate plasmodesmal closure in immune responses, identifying a PDLP–NHL3 complex as a critical node that integrates multiple signaling cascades to regulate plasmodesmata. The plant immune system relies on the perception of molecules that signal the presence of a microbe threat. This triggers signal transduction that mediates a range of cellular responses via a collection of molecular machinery including receptors, small molecules, and enzymes. One response to pathogen perception is the restriction of cell-to-cell communication by plasmodesmal closure. We previously found that while chitin and flg22 trigger specialized immune signaling cascades in the plasmodesmal plasma membrane, both execute plasmodesmal closure via callose synthesis at the plasmodesmata. Therefore, the signaling pathways ultimately converge at or upstream of callose synthesis. To establish the hierarchy of signaling at plasmodesmata and characterize points of convergence in microbe elicitor-triggered signaling, we profiled the dependence of plasmodesmal responses triggered by different elicitors on a range of plasmodesmal signaling machinery. We identified that, like chitin, flg22 signals via RESPIRATORY BURST OXIDASE HOMOLOGUE D (RBOHD) to induce plasmodesmal closure. Further, we found that PLASMODESMATA-LOCATED PROTEIN 1 (PDLP1), PDLP5, and CALLOSE SYNTHASE 1 (CALS1) are common to microbe- and salicylic acid (SA)-triggered responses, identifying PDLPs as a candidate signaling nexus. To understand how PDLPs relay a signal to CALS1, we screened for PDLP5 interactors and found NON-RACE SPECIFIC DISEASE RESISTANCE/HIN1 HAIRPIN-INDUCED-LIKE protein 3 (NHL3), which is also required for chitin-, flg22- and SA-triggered plasmodesmal responses and PDLP-mediated activation of callose synthesis. We conclude that a PDLP–NHL3 complex acts as an integrating node of plasmodesmal signaling cascades, transmitting multiple immune signals to activate CALS1 and plasmodesmata closure. The innate immune system of plants relies in part on the perception of invading pathogens at the cell surface. Cell-surface perception initiates signal transduction to induce cellular responses via molecular machinery that includes receptor kinase complexes, secondary messengers, and enzymes. This machinery can function across different subcellular structures, cells, and tissues, and be co-opted to mediate diverse defense responses. A single stimulus can initiate multiple signaling cascades: For example, receptor activation can trigger a range of responses that include a burst in production of reactive oxygen species (ROS) and changes to gene expression. Further, different stimuli can independently initiate cascades that converge on common responses such as the activation of callose synthesis in different cellular locations triggered by a range of microbial elicitors. By understanding the hierarchy and relationship of different signaling cascades, we can reveal the immune signaling network and identify critical nodes.The microbe-associated molecular pattern (MAMP) elicitors chitin and flg22 induce plasmodesmata closure –3 via thPlasmodesmal closure in response to a range of pathogen and stress elicitors is ultimately executed by callose deposition at the plasmodesmata neck, produced by callose synthases . PLASMODWe sought to leverage our knowledge of plasmodesmal machinery that functions in specific responses to determine the relationships between signaling cascades triggered by different immune elicitors. We found that flg22, like chitin, elicits a cascade that signals via RBOHD, and that PDLP1, PDPL5, and CALS1 are common to microbe elicitor- and SA-triggered responses. To dissect the possibility that PDLPs act as a signaling nexus and understand how they activate callose synthesis, we identified NDR1 (NON-RACE SPECIFIC DISEASE RESISTANCE)/HIN1 (HAIRPIN-INDUCED)-LIKE protein 3 (NHL3) as a PDLP5 interactor. Specifically, NHL3 is a key plasmodesmal component required for PDLP-mediated activation of callose synthesis. The data support a model in which NHL3 interacts with PDLPs to form a central integrator of plasmodesmal immune signaling, transmitting information to activate CALS1 and plasmodesmata closure.rbohD mutants using microprojectile bombardment assays and found that rbohD cannot close plasmodesmata in response to flg22 (ADataset S1), demonstrating that flg22-triggered plasmodesmal closure signals via RBOHD. For chitin, RBOHD dependence is linked to phosphorylation of S133 and S347  and that S133 and S347 of RBOHD are required for both flg22- and chitin-triggered plasmodesmal response.To investigate whether and where plasmodesmal signaling pathways converge upstream of callose deposition, we surveyed known plasmodesmal signaling components for their role in response to specific elicitors. Focusing initially on chitin- and flg22-triggered plasmodesmal responses, we hypothesized that as chitin-triggered plasmodesmal closure is mediated by activation of RBOHD , and thaSI Appendix, Fig. S2). Therefore, SA responses have a similar temporal profile to chitin and flg22 responses, allowing for the possibility that SA, chitin, and flg22 induce plasmodesmal closure via a similar mechanism.SA-triggered plasmodesmata closure is independent of RBOHD  but, likpdlp5 (CDataset S1) nor cals1 (DDataset S1) mutants closed their plasmodesmata in response to flg22 or chitin. As CALS1 produces callose and must lie at the endpoint of the signaling pathways, we conclude that flg22, chitin, and SA signaling at plasmodesmata converge downstream of RBOHD, and at, or upstream of, PDLP5.To explore whether flg22, chitin, and SA signaling converge on the same pathway upstream of callose deposition, we tested the requirement for known mediators of SA-triggered plasmodesmata closure, PDLP5 and CALS1, in chitin and flg22 responses. Using microprojectile bombardment assays, we found that neither rbohD exhibits normal plasmodesmal closure response to SA  by CRISPR/Cas9 gene editing that is perturbed in flg22-induced plasmodesmal closure  and found that this mutant can close its plasmodesmata in response to SA (FDataset S1). Likewise, lym2 (GDataset S1) shows wild-type-like SA-induced plasmodesmata closure.As SA is known to be synthesized in response to flg22 treatment , 9, we ase to SA , and thaics1) mutant, in which pathogen-induced SA synthesis and accumulation is significantly reduced . Collectively, these data suggest SA itself does not act as a secondary messenger within microbe-triggered plasmodesmal cascades, but rather initiates a signaling pathway that converges with other responses.To assay for a direct role of SA in chitin- and flg22-triggered plasmodesmal closure, we asked whether these elicitors trigger plasmodesmal closure if SA synthesis is perturbed. Thus, we assayed for plasmodesmal responses in the ISOCHORISMATE SYNTHASE 1 . Therefore, PDLP1 is also required for elicitor-triggered signaling cascades in plasmodesmata, supporting a model in which PDLPs are common to multiple signaling pathways and function downstream of, or at, the point at which these signals converge.PDLP1 positively regulates callose deposition at plasmodesmata and at the downy mildew haustorial membrane  but has Arabidopsis thaliana (Arabidopsis) cDNA library with a PDLP5 bait . The screen identified 65 prey hits, and we refined this list based on their presence in plasmodesmal proteomes of Arabidopsis suspension culture cells . NHL3 (AT5G06320) is present in both proteomes and is a membrane-associated protein like PDLP5. Thus, we chose NHL3 for further characterization as a likely PDLP5 interactor.Having established that PDLP5 functions in MAMP- and SA-triggered plasmodesmal signaling, and considering it is a positive regulator of callose deposition , we hypore cells , 15, proWe first examined the subcellular localization of NHL3 to determine whether a bona fide interaction with PDLP5 is plausible. We C-terminally tagged NHL3 with mCherry and observed that NHL3-mCherry is located in the plasma membrane, as previously reported , 17, FigA. HoweveNicotiana benthamiana leaves. Immunoprecipitation of mCherry-tagged proteins showed that PDLP5-eGFP coimmunoprecipitated with NHL3-mCherry, but not with LTI6b-mCherry . NHL3-mCherry, or the negative control LTI6b (LOW TEMPERATURE INDUCED PROTEIN 6b)-mCherry, was transiently coexpressed with PDLP5-eGFP in -mCherry B, supporN. benthamiana. NHL3 was either N- or C-terminally tagged with the N terminus of yellow fluorescent protein (YFPN) and coexpressed with a cytosolic (YFPC)  or a secreted (SP-YFPC) C terminus of YFP . YFP fluorescence was visible in the positive control combinations: cytosolic YFPC coexpressed with cytosolic YFPN , and SP-YFPC with SP-YFPN, . YFP fluorescence was similarly detected when NHL3-YFPN or YFPN-NHL3 was coexpressed with cytosolic YFPC , but not with SP-YFPC . Therefore, both termini of NHL3 are cytosolic facing.To further test this association, we used Förster resonance energy transfer–fluorescence lifetime imaging (FRET-FLIM) analysis. As NHL3 has been differentially predicted to carry one or two transmembrane domains , and a sACT2 promoter to drive expression (pACT2::LTI6b-mCherry) and found that, unlike previous reports . This allowed us to resolve our FRET-FLIM analysis to plasmodesmata within the plasma membrane. We found that the fluorescence lifetime of PDLP5-eGFP was reduced at plasmodesmata in the presence of NHL3-mCherry, but not LTI6b-mCherry (Dataset S2), indicating that FRET occurs between PDLP5 and NHL3. Alongside co-IP data and the initial split ubiquitin screen, this supports a model in which PDLP5 and NHL3 form a complex for plasmodesmal signaling.We assayed for interaction between NHL3-mCherry and PDLP5-eGFP fusion proteins using FRET-FLIM. As a negative control, we fused LTI6b to mCherry and an  reports , LTI6b lNHL gene family has homology to NDR1 and the TOBACCOHIN1 genes . Furthermore, while nhl3 mutants were able to close their plasmodesmata in response to H2O2, they could not in response to SA (BDataset S3), phenocopying the pdlp5 and cals1 mutants . When NHL3 and PDLP5 were coexpressed, we did not detect a further increase in callose per plasmodesmal deposit, but we did detect an increase in the number of deposits detected per field of view compared to when both proteins were individually expressed . This might arise from more plasmodesmata producing callose above the detection threshold and indicate increased callose synthesis. These data support a function of NHL3 in cooperating with PDLP5 to increase the activity of callose synthases, thereby positively regulating callose deposition at plasmodesmata.Reasoning that NHL3 likely cooperates with PDLP5 to positively regulate callose deposition at plasmodesmata, we assayed for the impact of NHL3 overexpression on callose deposition at plasmodesmata using quantitative imaging of aniline blue–stained callose. When  control A, indepePDLP5  and PDL[PDLP1OE ], which desmata . TherefPlants deploy a suite of signaling cascades with specific molecular components to initiate and execute defense responses against invading pathogens. These cascades, and the machinery they require, can be specific to subcellular structures such as is observed in plasmodesmata, where microbe elicitors trigger discrete responses. Despite the specificity in signaling observed in plasmodesmata, pathways initiated by different elicitors converge on the common response of callose synthesis to mediate plasmodesmal closure. Thus, plasmodesmal responses likely converge at a node that integrates multiple independent inputs.SI Appendix, Fig. S1]. As an NADPH oxidase, RBOHD produces ROS that act as small molecule transmitters of plasmodesmal responses. How ROS are perceived and transmit information within this cascade remains an open question and the observation that hydrogen peroxide can induce plasmodesmata closure via an independent pathway , as well as its dependence on PDLP1 . Irrespective of the working model, PDLPs are only found in seed plants  protein LEA_2 domain found close to the C terminus, which has no known function. LEA_2 domain proteins are produced during responses to dehydration or hyperosmotic stress, and have been postulated to stabilize macromolecules against freezing, dehydration, ionic or osmotic stress . They ha37PDLP1 and PDLP5 overexpression phenotypes are reverted to wild-type levels in the absence of NHL3 (PDLPs have two extracellular DOMAIN OF UNKNOWN FUNCTION (DUF) 26 domains. Structural analysis of the PDLP5 DUF26 domains suggests these are lectin dimers, which could form an extended binding cleft for a carbohydrate polymer . While t of NHL3 B–F sugge of NHL3 ; resolviHow plasmodesmata specifically respond to a range of immunity- and stress-associated elicitors is a critical question that underpins how organism-level responses are regulated in a multicellular context. Further, how immune signals are localized, specialized, and integrated is key to mapping the molecular signaling networks that orchestrate cellular responses to pathogens. Characterization of plasmodesmal signaling has revealed that a PDLP–NHL3 complex integrates different signaling cascades to activate callose synthesis and close plasmodesmata during immune responses. Thus, we have uncovered a mechanism by which diverse signals are integrated to produce a single response, further characterizing the signaling cascades that regulate cell-to-cell connectivity during immune responses. How NHL3 functions and the full range of signals it can integrate, and resolving how small molecules such as ROS and SA are received in plasmodesmal responses, remain questions to be addressed to understand the molecular dynamics of plasmodesmal closure and callose synthesis across plant responses.SI Appendix.Extended Methods and Materials are available in A. thaliana wild-type used in this study is Col-0. Mutants and transgenic lines are in the Col-0 background. Lines used in this study are lym2 ; pdlp5 _46_E06, ]; cals1 atrbohD, ]; RBOHDS_035427, ]; nhl3-2_150318, ]; PDLP5OBombardment assays were performed as described in ref. Arabidopsis leaf or N. benthamiana leaf was infiltrated with 1% or 0.1% aniline blue in PBS buffer (pH 7.4), respectively. Plasmodesmata callose deposits were imaged from the abaxial side by a confocal microscope (LSM Zeiss 800) using a 63× water immersion objective (Plan-Apochromat 63×/1.0 M27). Aniline blue was excited with a 405-nm UV laser and collected at 430 to 550 nm. Z-stacks from multiple areas (specified in figure legends) per leaf were collected. We used an automated image analysis pipeline to quantify aniline blue–stained plasmodesmata, available at ref. The 7th or 8th The split-ubiquitin screen was performed by DualSystems Biotech, Switzerland .SI Appendix). Immunoblotting was carried out with anti-GFP  or anti-RFP-biotin  primary antibodies, and anti-mouse IgG  or anti-rabbit-HRP  secondary antibodies.Protein extraction and co-IP was performed as described  with sliTransiently expressed constructs for colocalization and split-YFP analysis were imaged by confocal microscopy (LSM Zeiss 800) 2 d postinfiltration using a water immersion 63× objective (C-Apocromat 63×/1.20WKorr UV VIS IR-water). GFP/YFP was excited with a 488-nm argon laser and collected at 505 to 530 nm while mCherry was excited with a 561-nm DPSS laser and collected at 600 to 640 nm.N. benthamiana transiently expressing constructs of interest were imaged using a STELLARIS 8 FALCON microscope (Leica Microsystems). The abaxial side of the leaf sample was imaged using a 63× objective . GFP was excited with a 488-nm WLL laser and collected at 503 to 530 nm. Acquisition was set at a mode of xyz, format size of 512 × 512, scan speed of 40 Hz, single-line repetitions, and a pixel dwell time of 3.16 µs. To reduce the risk of bleaching the sample, laser power was adjusted to ensure no more than 1.0 photon per pulse occurred. Datasets were acquired by scanning each image until a suitable number of photon counts per pixel were reached in the brightest channel, up to a maximum of 1,000 counts per pixel. Data were analyzed using the LAS X software. Plasmodesmal regions of interest were specified using the intensity threshold and manual selection. Using a two-exponential decay for GFP, a fit was generated and the mean τ amplitude-weighted GFP lifetime (in ns) for all plasmodesmata per image was calculated.Leaves of Arabidopsis seeds were surface sterilized with 70% ethanol and 2% bleach with 0.02% Triton X-100, then washed with sterile dH2O and sown onto Murashige and Skoog medium 1% sucrose 0.8% agar plates 16-h light at 22 °C. Photos were taken 21 d postgermination for determination of rosette area. Individual plants were traced by hand, and area of the rosette was measured in FIJI.For rosette area phenotyping and subsequent callose staining, B. cinerea spores were harvested in 0.25× potato dextrose broth and adjusted to a concentration of 2.5 × 105 spores/mL. The spore suspension was incubated at room temperature for 2 h with continual shaking to germinate the spores. Expanded leaves of 5-wk-old Arabidopsis plants adhered in a 1.5% agar H2O plate, were inoculated with 2 µL of the spore suspension, with a droplet placed in between the mid vein and the leaf edge of each leaf side . Plates were sealed and left in a cabinet at 22 °C, with 10-h light. Developing disease lesions were photographed 2 d postinoculation and measured using FIJI. Each prepared plate had three to four leaves per genotype, and the average lesion area was taken for each genotype as a single biological replicate.Data presented as boxplots have the middle line marking the median, the box indicating the upper and lower quartiles, and the whiskers showing the minimum and maximum values within 1.5× interquartile range. Details of the statistical analysis are given in the figure legends. Data were analyzed using RStudio 2021.09.01 Build 351/R version 4.0.3.Appendix 01 (PDF)Click here for additional data file.Dataset S01 (XLSX)Click here for additional data file.Dataset S02 (XLSX)Click here for additional data file.Dataset S03 (XLSX)Click here for additional data file.Dataset S04 (XLSX)Click here for additional data file.