FOXO inhibition rescues α-defensin expression in human intestinal organoids.

To mediate critical host-microbe interactions in the human small intestine, Paneth cells constitutively produce abundant levels of α-defensins and other antimicrobials. We report that the expression profile of these antimicrobials is dramatically askew in human small intestinal organoids (enteroids) as compared to that in paired tissue from which they are derived, with a reduction of α-defensins to nearly undetectable levels. Murine enteroids, however, recapitulate the expression profile of Paneth cell α-defensins seen in tissue. WNT/TCF signaling has been found to be instrumental in the regulation of α-defensins, yet in human enteroids exogenous stimulation of WNT signaling appears insufficient to rescue α-defensin expression. By stark contrast, forkhead box O (FOXO) inhibitor AS1842856 induced the expression of α-defensin mRNA in enteroids by >100,000-fold, restoring DEFA5 and DEFA6 to levels comparable to those found in primary human tissue. These results newly identify FOXO signaling as a pathway of biological and potentially therapeutic relevance for the regulation of human Paneth cell α-defensins in health and disease.

Are Synovial Inflammatory Markers Increased in Patients Who Have Aseptic Total Hip Arthroplasty Dislocation Indicated for Revision?

BACKGROUND: Previous studies have speculated on elevated synovial inflammatory markers in patients undergoing surgical revision for total hip arthroplasty (THA) dislocation. However, this assumption is based on small patient series and a full investigation according to International Consensus Meeting (ICM) criteria has not yet been performed. METHODS: Patients who had aseptic THA dislocation indicated for revision surgery were identified retrospectively. Only patients who had available diagnostic workup according to ICM 2018 criteria, including preoperative and intraoperative parameters, were included. For comparison, we analyzed a matched cohort of patients indicated for aseptic THA revision for other conditions. The 2 cohorts each consisted of 55 patients and were not different regarding age, sex, BMI, or implant fixation. RESULTS: There was no difference in synovial white blood cell count (2,238 ± 2,544 versus 2,533 ± 3,448 c/µL; P = .601), alpha-defensin quotient (0.14 ± 0.11 versus 0.19 ± 0.28; P = .207), or polymorphonuclear neutrophil percentage (% PMN) (36.7 ± 22.6 versus 31.3 ± 24.5%; P = .312) between the groups. In the dislocation cohort, 20% of patients had a synovial white blood cell count of 3,000 c/µL or higher, compared with 18% in the control cohort. However, all patients in the dislocation cohort were below the cutoff for alpha-defensin or % PMN. CONCLUSION: In patients who have aseptic THA dislocation, synovial inflammatory markers are not elevated compared with patients undergoing aseptic revision for other complications. A detailed preoperative analysis of synovial inflammatory markers using ICM criteria appears critical in patients who have a THA dislocation to exclude periprosthetic joint infection. LEVEL OF EVIDENCE: Level III, retrospective, comparative study.

Human intelectin-2 (ITLN2) is selectively expressed by secretory Paneth cells.

Intelectins (intestinal lectins) are highly conserved across chordate evolution and have been implicated in various human diseases, including Crohn's disease (CD). The human genome encodes two intelectin genes, intelectin-1 (ITLN1) and intelectin-2 (ITLN2). Other than its high sequence similarity with ITLN1, little is known about ITLN2. To address this void in knowledge, we report that ITLN2 exhibits discrete, yet notable differences from ITLN1 in primary structure, including a unique amino terminus, as well as changes in amino acid residues associated with the glycan-binding activity of ITLN1. We identified that ITLN2 is a highly abundant Paneth cell-specific product, which localizes to secretory granules, and is expressed as a multimeric protein in the small intestine. In surgical specimens of ileal CD, ITLN2 mRNA levels were reduced approximately five-fold compared to control specimens. The ileal expression of ITLN2 was unaffected by previously reported disease-associated variants in ITLN2 and CD-associated variants in neighboring ITLN1 as well as NOD2 and ATG16L1. ITLN2 mRNA expression was undetectable in control colon tissue; however, in both ulcerative colitis (UC) and colonic CD, metaplastic Paneth cells were found to express ITLN2. Together, the data reported establish the groundwork for understanding ITLN2 function(s) in the intestine, including its possible role in CD.

It can be unnecessary to combine common synovial fluid analysis and alpha-defensin tests for periprosthetic joint infection diagnosis.

BACKGROUND: Periprosthetic joint infection is a serious complication after total joint arthroplasty. Despite that alpha-defensin was used as diagnostic test in the 2018 ICM (international consensus meeting) criteria, its position in the PJI diagnostic pipeline was controversial. Therefore, we performed a retrospective pilot study to identify whether synovial fluid alpha-defensin test was necessary when corresponding synovial fluid analysis (WBC count, PMN% and LE tests) was performed. METHODS: Between May 2015 and October 2018, a total of 90 suspected PJI patients who underwent revisions after TJA were included in this study. Based on the 2018 ICM criteria, the interobserver agreements between preoperative diagnostic results and postoperative diagnostic results and the interobserver reliability between preoperative diagnostic results and postoperative diagnostic results with or without synovial fluid alpha-defensin tests were calculated. After that, the ROC analysis, and the direct cost-effectiveness of adding alpha-defensin was performed. RESULTS: There were 48,16 and 26 patients in the PJI group, inconclusive group and non-PJI group, respectively. Adding the alpha-defensin tests into 2018 ICM criteria can't change the preoperative diagnostic results, postoperative diagnostic results, and the concordance between preoperative and postoperative diagnostic results. Moreover, the Risk-benefit Ratio is over 90 per changed decision and the direct cost-effectiveness of alpha-defensin was more than $8370($93*90) per case. CONCLUSIONS: Alpha-defensin assay exhibit high sensitivity and specificity for PJI detection as a standalone test based on the 2018 ICM criteria. However, the additional order of Alpha-defensin can't offer additional evidence for PJI diagnosis when corresponding synovial fluid analysis was performed (synovial fluid WBC count, PMN% and LE strip tests). EVIDENCE LEVEL: Level II, Diagnostic study.

Alpha-defensin as a diagnostic tool in revision total knee arthroplasties with unexpected positive intraoperative cultures and unexpected culture negative intraoperative cultures.

PURPOSE: Unexpected-positive-intraoperative-cultures (UPIC) are common in presumed aseptic revision-total-knee-arthroplasties (rTKA). However, the clinical significance is not entirely clear. In contrast, in some presumably septic rTKA, identification of an underlying pathogen was not possible, so-called unexpected-negative-intraoperative-cultures (UNIC). The purpose of this study was to evaluate the potential use of synovial alpha-defensin (AD) levels in these patients. METHODS: Synovial AD levels from 143 rTKAs were evaluated retrospectively from our prospectively maintained institutional periprostetic joint infection (PJI) biobank and database. The 2018-International Consensus Meeting (ICM) criteria was used to define the study groups. Samples from UPICs with a minimum of one positive intraoperative culture (ICM 2- ≥ 6) (n = 20) and UNIC's (ICM ≥ 6) (n = 14) were compared to 34 septic culture-positive samples (ICM ≥ 6) and 75 aseptic culture-negative (ICM 0-1). Moreover, AD-lateral-flow-assay (ADLF) and an enzyme-linked-immunosorbent-assay (ELISA) in detecting the presence of AD in native and centrifuged synovial fluid specimens was performed. Concentration of AD determined by ELISA and ADLF methods, as well as microbiological, and histopathological results, serum and synovial parameters along with demographic factors were analysed. RESULTS: AD was positive in 31/34 (91.2%) samples from the septic culture-positive group and in 14/14 (100%) samples in the UNIC group. All UPIC samples showed a negative AD result. Positive AD samples were highly associated with culture positive and histopathological results (p < 0.001). No high-virulent microorganisms (0/20) were present in the UPIC group, compared to infected-group (19/34; 55.9%). High virulent microorganisms showed a positive AD result in 89.5% (17/19) of the cases. Methicillin resistant Staphylococcus epidermis (MRSE) infections had significantly higher AD levels than with methicillin susceptible S. epidermdis (MSSE) (p = 0.003). ELISA and ADLF tests were positive with centrifuged (8/8) and native (8/8) synovial fluid. CONCLUSION: AD showed a solid diagnostic performance in infected and non-infected revisions, and it provided an additional value in the diagnosis of UPIC and UNIC associated to rTKAs. Pathogen virulence as well as antibiotic resistance pattern may have an effect on AD levels. Centrifugation of synovial fluid had no influence on ADLF results.

Refining the Role of Routine Synovial Alpha-Defensin in Periprosthetic Joint Infection Following Total Knee Arthroplasty: An Analysis of Limitations.

BACKGROUND: The utility of the synovial alpha-defensin test in diagnosing periprosthetic joint infections (PJIs) remains controversial. This study aimed to examine the diagnostic utility of this test. METHODS: A retrospective review was conducted to identify adults evaluated for PJI following total knee arthroplasty at a single institution. Patient demographics, laboratory results, and operative details were recorded. Using the 2018 Musculoskeletal Infection Society (MSIS) criteria, cases were categorized as definitive, inconclusive, or negative for PJI. The sensitivity, specificity, positive predictive value, and negative predictive value of each MSIS criterion was determined. The number of patients whose PJI diagnosis was contingent on alpha-defensin positivity was calculated. RESULTS: Overall, 172 total knee arthroplasty patients were included, who had an average age of 70.4 years (range, 39 to 95). Of the 21 patients who met major criteria, 20 (95.2%) were alpha-defensin positive. Of the remaining 151 patients, 85 did not meet minor criteria, all of whom were alpha-defensin negative. Among the 30 patients who met minor criteria, 28 (93.3%) were alpha-defensin positive and 2 (6.7%) were negative. The remaining 36 patients were deemed inconclusive preoperatively. In total, alpha-defensin testing changed the diagnosis in only 9 of 172 patients (5.2%). The sensitivity, specificity, positive predictive value, and negative predictive value of alpha-defensin in this cohort were 94.1, 100, 100, and 97.6, respectively. CONCLUSION: Alpha-defensin may assist in the diagnosis of PJI when a preoperative workup is inconclusive. However, this test is often unnecessary when the diagnosis of PJI can be made using the 2018 MSIS criteria.

Leukocyte esterase and alpha-defensin in periprosthetic joint infection: predictive quality and correlation in a prospective study.

PURPOSE: Periprosthetic joint infection (PJI) is a rare but serious complication of total joint arthroplasty (TJA). An accurate diagnosis of PJI preoperatively does not exist. Alpha-defensin (AD) is a proven and common indicator. The diagnostic marker of leukocyte esterase (LE) promises some advantages: feasibility, availability, and fast result reporting. The aim of this study was the evaluation of the predictive quality and correlation between both diagnostic tools in the diagnosis of PJI. METHODS: A prospective study was conducted between April 2018 and August 2022. All patients with suspicion of PJI on hip and knee joint were included and underwent a routine and standardized joint punction. For laboratory diagnostics of AD, the synovial liquid was analyzed by ELISA. The sample was additionally applied to a LE test strip (Combur 10 Test, Roche Diagnostics, Mannheim, Germany). RESULTS: A total of 249 patients were examined (mean age 67.12 ± 11.89; gender distribution man/woman 139 (55.8%)/110(44.2%), hip/knee 71(28.5%)/178 (71.5%). According to EBJIS criteria, PJI was diagnosed in 54 (21.7%) patients. AD showed excellent results with an AUC of 0.930 (sensitivity/specificity 0.870/0.990). LE yielded very good results with an AUC of 0.820 (sensitivity/specificity 0.722/0.918). Both parameters showed a strong positive correlation. CONCLUSION: LE is a rapidly available alternative in PJI diagnostics. The simultaneous determination of both markers may enhance diagnostic reliability. A routine usage may shorten the time from diagnosis to treatment of PJI.

The optimal diagnostic cut-off of WBC and PMN counts for joint aspiration in periprosthetic joint infection is 2479/µL and 67%, respectively: ICM criteria thresholds are too high.

BACKGROUND: Various organizations have published definitions for periprosthetic joint infection (PJI) with significant differences in the cut-offs of white blood cell (WBC) count and polymorphonuclear (PMN) leukocyte cells. Herein, we aim to analyze optimal cut-offs in patients which are planned to undergo a prosthesis revision and compare them with the actual published thresholds of the International Consensus Meeting (ICM) and European Bone and Joint Infection Society (EBJIS). METHODS: A test kit was compiled in a monocentric prospective study, according to the ICM criteria (2018) and 2021 EBJIS criteria. The kit was implemented using: blood samples (including leukocyte count and C-reactive protein); samples for examining the synovial fluid (WBC count, PMN cell differentiation, microbiological culture for incubation over 14 days, alpha-defensin ELISA laboratory test, and leukocyte-esterase test). The cut-offs for WBC and PMN counts were investigated using ROC analyses and Youden index. The ICM 2018 criteria were applied, using alpha-defensin in all cases. Patients which have to undergo a prosthesis revision were included, a pre-operative joint aspiration had been performed, and the patients had been followed up prospectively. RESULTS: 405 patients were examined with the compiled test kit; 100% had a complete dataset with respect to alpha-defensin; 383 patients, according to WBC count; and 256, according to PMN cell differentiation The cut-off of 2478.89 cells/µl in the WBC count (sensitivity: 87.70%; specificity: 88.10%) and the cut-off of 66.99% in PMN differentiation showed the best accuracy (sensitivity: 86.00%; specificity: 88.80%). Other published cut-offs for WBC were tested in this cohort and showed the following accuracy: 3000/µl (EBJIS/ICM; sensitivity: 82.10%; specificity: 91.00%), 2000/µl (sensitivity: 89.60%; specificity: 83.40%), and 1500/µl (sensitivity: 91.50%; specificity: 75.00%). The published cut-offs for PMN had the following accuracy in this cohort: 80% (ICM; sensitivity: 66.3%; specificity: 96.50%), 70% (sensitivity: 82.6%; specificity: 90%), and 65% (EBJIS, sensitivity: 86%; specificity: 88.8%). CONCLUSIONS: This study aims to improve current cut-offs for PMN- and WB-Count, even though PJI diagnosis is based on the combination of all defined tests. The optimal diagnostic cut-off of WBC and PMN counts was found to be 2479/µL and 67%, respectively, whereas ICM cut-offs in this cohort seem too high, as they provide high specificity but very low sensitivity. On the other hand, a cut-off for WBC count of 1500/µl alone would be very low, leading to low specificity and very high suspicion of PJI. The current consensus guidelines could be actualized considering these results to significantly improve the diagnostic quality. LEVEL OF EVIDENCE: II.

Alleviation of Hepatic Steatosis by Alpha-Defensin Is Associated with Enhanced Lipolysis.

Background and Objectives: The neutrophilic peptide, alpha-defensin, is considered an evolving risk factor intimately linked with lipid mobilization. It was previously linked to augmented liver fibrosis. Here, we assess a potential association between alpha-defensin and fatty liver. Materials and Methods: A cohort of transgenic C57BL/6JDef+/+ male mice that overexpress the human neutrophil-derived alpha-defensin in their polymorphonuclear neutrophils (PMNs) were assessed for liver steatosis and fibrosis development. Wild type (C57BL/6JDef.Wt) and transgenic (C57BL/6JDef+/+) mice were maintained on a standard rodent chow diet for 8.5 months. At the termination of the experiment, systemic metabolic indices and hepatic immunological cell profiling were assessed. Results: The Def+/+ transgenic mice exhibited lower body and liver weights, lower serum fasting glucose and cholesterol, and significantly lower liver fat content. These results were associated with impaired liver lymphocytes count and function (lower CD8, NK cells, and killing marker CD107a). The metabolic cage demonstrated dominant fat utilization with a comparable food intake in the Def+/+ mice. Conclusions: Chronic physiological expression of alpha-defensin induces favorable blood metabolic profile, increased systemic lipolysis, and decreased hepatic fat accumulation. Further studies are needed to characterize the defensin net liver effect.

Human Neutrophil α-Defensins 1-3 Are Upregulated in the Microenvironment of Fibrotic Liver.

Background and Objectives: Neutrophil infiltration is an established signature of Non-Alcoholic Fatty Liver Disease (NAFLD) and Steatohepatitis (NASH). The most abundant neutrophilic peptide, alpha-defensin, is considered a new evolving risk factor in the inflammatory milieu, intimately involved in lipid mobilization. Our objective is to assess for potential association between alpha-defensin immunostains and NAFLD severity. Materials and Methods: We retrospectively investigated the liver biopsies of NAFLD/NASH patients, obtained at Hillel Yaffe Medical center between the years 2012 and 2016. Patients' characteristics were recorded, including relevant blood tests at the time of biopsy. Each biopsy was semi-quantitatively scored using NAFLD Activity Score (NAS) and NASH fibrosis stage. The biopsies were immunostained for alpha-defensin. The precipitation of alpha-defensin was correlated to NAS and fibrosis. Results: A total of 80 biopsies were evaluated: male ratio 53.2%, mean age 44.9 ± 13.2 years, 54 had fibrosis grades 0-2, and 26 were grade 3-4. Conventional metabolic risk factors were more frequent in the high-grade fibrosis group. Immunostaining for alpha-defensin disclosed higher intensity (a.u.) in grade 3-4 fibrosis relative to grades 0-2, 25% vs. 6.5%, p < 0.05, respectively. Moreover, alpha-defensin staining was nicely co-localized with fibrosis. Conclusions: In our group of NASH/NAFLD patients, higher metabolic risk profile was associated with higher fibrosis grade. Immunostaining for alpha-defensin showed patchy intense staining concordant with high fibrosis, nicely co-localized with histological fibrosis. Whether alpha-defensin is a profibrotic risk factor or merely risk marker for fibrosis must be clarified in future studies.

Which Minor Criteria is the Most Accurate Predictor for the Diagnosis of Hip and Knee Periprosthetic Joint Infection in the Asian Population?

BACKGROUND: The aim of this study was to evaluate the diagnostic performance of minor criteria from the 2018 International Consensus Meeting (ICM) for the diagnosis of chronic periprosthetic joint infection (PJI) in an Asian population. METHODS: We retrospectively reviewed 76 patients who underwent a revision knee or hip arthroplasty at an academic institution between September 2018 and December 2019. All major and minor 2018 ICM criteria were available for all patients included. Cases with at least 1 major criterion or score ≥6 in minor criteria were considered as infected. The diagnostic performance was evaluated by a receiver operative characteristic curve analysis and area under the curve (AUC) for each minor criterion. An AUC value of more than 0.9 was considered outstanding and 0.8-0.9 as excellent. RESULTS: When using 2018 ICM threshold, the diagnostic performance ranked based on AUC was the following: alpha defensin (0.92), positive histology (0.83), leukocyte esterase (0.82), synovial white blood cell (0.81), serum erythrocyte sedimentation rate (0.78), synovial polymorphonuclear neutrophils (0.77), serum C-reactive protein (0.74), D-dimer (0.59), single positive culture (0.53), and positive intraoperative purulence (0.51). Alpha defensin was considered as an outstanding test among the 2018 ICM minor criteria. Positive histology, leukocyte esterase, and synovial white blood cell were considered as excellent tests. CONCLUSION: Based on our findings, alpha-defensin has the best diagnostic performance in Asian population among the minor criteria of 2018 ICM.

Alpha-Defensin Offers Limited Utility in Work-Up Prior to Reimplantation in Chronic Periprosthetic Joint Infection in Total Joint Arthroplasty Patients.

BACKGROUND: Alpha-defensin (AD) is a synovial biomarker included in the 2018 consensus criteria for diagnosing periprosthetic joint infection (PJI). Its value in assessing eradication of infection prior to second stage reimplantation is unclear. The purpose of this study was to evaluate the impact of AD on eligibility for reimplantation following resection for chronic PJI. METHODS: This study included patients who previously underwent resection arthroplasty for PJI. Synovial fluid aspirated from 87 patients was retrospectively reviewed. All patients completed a 6-week course of intravenous antibiotics and an appropriate drug holiday. Synovial white blood cell count, percentage neutrophils, and culture from the AD immunoassay laboratory were reviewed with serum erythrocyte sedimentation rate and C-reactive protein values from our institution. A modified version of the 2018 consensus criteria was used, including white blood cell count, percentage neutrophils, erythrocyte sedimentation rate, and C-reactive protein. AD was then added to determine if it changed diagnosis or clinical management. RESULTS: Four patients were categorized as "infected" (score >6), none exhibited a positive AD or positive culture. Sixty eight patients were diagnosed as "possibly infected" (score 2 to 5), none had a positive AD, and one had a positive culture (Cutibacterium acnes). AD did not change the diagnosis from "possibly infected" to "infected" in any case or alter treatment plans. Fifteen patients had a score of <2 (not infected) and none had a positive AD. CONCLUSION: The routine use of AD in the work-up prior to a second-stage arthroplasty procedure for PJI may not be warranted.

[Implementation of a standardized clinical test kit for diagnostics of periprosthetic infections in the clinical routine. German version]. / Implementierung eines standardisierten Test-Kits zur Diagnostik von periprothetischen Infektionen in der klinischen Routine.

BACKGROUND: The number of primary arthroplasties is increasing and the proportion of revision arthroplasties is becoming increasingly more important. The need for standardized and guideline-based diagnostics for the safe detection of a periprosthetic joint infection (PJI) is becoming apparent. In the past 10 years various organizations have published definitions and diagnostic guidelines. The implementation of an inhouse standard test kit could help to simplify the process and could improve the diagnostic quality. METHOD: In 2016 a test kit was compiled in a monocentric prospective study, taking the International Consensus Meeting (ICM) criteria 2014 and the Infectious Diseases Society of America (IDSA) criteria into account, which also fulfils the definitions of the ICM criteria 2018 and criteria of the European Bone and Joint Infection Society 2021. The test kit was implemented in the clinical setting of a special department for aseptic and septic revision arthroplasty. The usability and accuracy of the test kit were examined. RESULTS: The test kit was implemented using blood samples (leukocyte count; C­reactive protein, CRP), samples for examining the synovial fluid (white blood cell count, PMN cell differentiation, microbiological culture for incubation over 14 days, alpha-defensin enzyme-linked immunosorbent assay, ELISA, leukocyte esterase test strips) together with information and request forms. Between April 2016 and February 2020 a total of 405 patients were investigated. Within 3 calendar years, the use of the test kit increased from 59% initially to 86%, and finally to 96% of cases in the third calendar year. The leukocyte esterase test strip was reliable in only 72%, due to undifferentiated readability or blood contamination. The costs increased by the only commercially available alpha-defensin ELISA test by approx. 52€ per puncture. The best individual test showed a sensitivity/specificity of 92.8%/95.2% with alpha-defensin. It was calculated which combinations showed a similar test quality and different combinations, such as CRP+ cell count+ microbiology showed a sensitivity/specificity both of around 90%. Metallosis is a challenge for preoperative PJI diagnostics. DISCUSSION: In a prospective study it was shown, that the implementation of the standardized test kit lead to a guideline based PJI diagnostic in all cases and thus to a significantly increase of the diagnostic quality. There is currently no single test that reliably excludes or proves an infection. The alpha-defensin laboratory ELISA test showed the best test accuracy, whereby the consideration of test combinations is obligatory and at the same time safe.

Evaluation of alpha defensin, IL-1 receptor antagonist, and IL-18 levels in COVID-19 patients with macrophage activation syndrome and acute respiratory distress syndrome.

BACKGROUND: Many laboratory parameters have been associated with morbidity and mortality in SARS-CoV-2 (COVID-19), which emerged in an animal market in Wuhan, China in December 2019 and has infected over 20 million people. This study investigated the relationship between serum interleukin (IL)-18, IL-1 receptor antagonist (IL-1Ra), and alpha defensin levels and the clinical course and prognosis of COVID-19. MATERIALS AND METHODS: This study included 100 patients who were admitted to the chest diseases department and intensive care unit of our hospital and diagnosed with COVID-19 by real-time polymerase chain reaction (PCR) of nasopharyngeal swab samples between March 24 and May 31, 2020. The control group consisted of 50 nonsymptomatic health workers with negative real-time PCR results in routine COVID-19 screening in our hospital. RESULTS: Serum alpha defensin, IL-1Ra, and IL-18 levels were significantly higher in patients who developed macrophage activation syndrome (MAS) and acute respiratory distress syndrome (ARDS) compared to patients who did not (p < .001 for all). Alpha defensin, IL-1Ra, and IL-18 levels were significantly higher in COVID-19 patients with and without MAS or ARDS when compared to the control group (p < .001 for all). When the 9 patients who died were compared with the 91 surviving patients, IL-1Ra and IL-18 levels were found to be significantly higher in the nonsurvivors (p < .001). CONCLUSION: Our findings of correlations between alpha defensin and levels of IL-1Ra and IL-18, which were previously shown to be useful in COVID-19 treatment and follow-up, indicates that it may also be promising in treatment.

Whole blood RNA sequencing reveals a differential transcriptomic profile associated with cervical insufficiency: a pilot study.

BACKGROUND: The uterine cervix is a mechanical and immunological barrier against ascending infection during pregnancy. Cervical insufficiency (CI), a painless cervical dilation that occurs in the mid-trimester, is an important cause of extremely preterm birth. We hypothesized that women with CI have a differential transcriptomic profile. Therefore, we compared the transcriptomic profile of peripheral blood in women with CI and that of controls. METHODS: RNA sequencing was used to generate the global gene expression profiles of 11 women with CI and 4 controls, and differential expression analysis was performed to identify genes showing significant expression changes between the CI (n = 11) and control (n = 4) groups as well as between the CI-preterm (n = 7) and CI-term (n = 4) groups. Gene set enrichment was assessed in terms of Gene Ontology processes, and a subset of differentially expressed genes in CI was validated in a different sample-set by qRT-PCR and ELISA. RESULTS: Thirty genes were differentially expressed between the CI and control groups. Differentially upregulated genes in the CI group included neutrophil-mediated immunity-associated (DEFA3 and ELANE) and bicarbonate transport-related genes. The serum concentration of alpha defensin 3 was significantly higher in women with CI than in controls (P = 0.014). Analysis of differential gene expression according to pregnancy outcomes revealed 338 differentially expressed genes between the CI-term and CI-preterm groups. Immune and defense response to organism-associated genes and influenza A and NOD-like receptor signaling pathways were upregulated in the CI-term group. CONCLUSIONS: Our results revealed significant differences in the whole blood transcriptomic profiles of women with CI compared to those of controls. Different immune responses in women with CI may affect pregnancy outcomes.

Alpha-Defensin Offers Limited Utility in Routine Workup of Periprosthetic Joint Infection.

BACKGROUND: Alpha-defensin (AD) is a synovial biomarker included as a minor criterion in the scoring system for diagnosing periprosthetic joint infection (PJI). The purpose of this study is to study the impact of AD on diagnosis and management of PJI. METHODS: Synovial fluid from 522 patients after total knee and hip arthroplasty was retrospective reviewed. Synovial white blood cell count, percentage of neutrophils, and culture from the AD immunoassay laboratory were reviewed with serum erythrocyte sedimentation rate and C-reactive protein values from our institution. A modified version of the 2018 scoring system for diagnosis of PJI was used, only scoring white blood cell count, percentage of neutrophils, erythrocyte sedimentation rate, and C-reactive protein. AD was then analyzed with these scores to determine if AD changed diagnostic findings or clinical management. RESULTS: Eight-two patients were categorized as "infected" (score ≥6), of which 76 patients had positive AD. Of the 6 "infected" patients with negative AD, 2 had positive cultures (Staphylococcus epidermidis). Two-hundred thirteen patients were diagnosed as "possibly infected" (score 2-5). Fourteen of these patients had positive AD, of which 5 had positive cultures assisting with the diagnosis. The AD test changed the diagnosis from "possibly infected" to "infected" in 8 patients (1.5%) but only altered treatment plan in 6 patients (1.1%). A score <2 (not infected) was calculated in 227 patients with no patients having positive AD. CONCLUSION: AD may be beneficial in some cases where laboratory values are otherwise equivocal; however, its routine use for the diagnosis of PJI may not be warranted.

Alpha-Defensin Is Not Superior to Traditional Diagnostic Methods for Detection of Periprosthetic Joint Infection in Total Hip Arthroplasty and Total Knee Arthroplasty Patients.

BACKGROUND: Synovial fluid alpha-defensin (AD) may improve diagnostic accuracy of periprosthetic joint infection (PJI) following total knee (TKA) and hip (THA) arthroplasty but is only available as send-out test. This study evaluated laboratory result accuracy between send-out test vs hospital labs and if AD made a difference in treatment plan. METHODS: A retrospective review was performed of 152 consecutive patients with a TKA or THA joint aspiration for painful or clinically concerning joint. Synovial fluid was sent to our institution (hospital-based labs, HBL) and send-out immunoassay laboratory (Synovasure). Patients were scored with specific criteria from validated scoring system for PJI using HBL and Synovasure results. The score with and without AD test was compared to determine if AD impacted patient management. RESULTS: Overall, there was strong agreement between institutions for PJI diagnosis (Cohen's kappa score 0.96). Twenty-nine patients had PJI diagnosis (score ≥6), of which 28 (97%) had positive AD with 1 false-negative result. Sixty-three patients had inconclusive score (between 2 and 5) and 60 patients had negative PJI diagnosis (score ≤1). Of these patients, 5 underwent surgery for infection. Two patients had surgery for positive AD, 2 for positive culture, and 1 because of elevated HBL results. The AD test changed the PJI diagnosis and influenced decision for surgery in only 1.3% (2/152) of patients. CONCLUSION: Minimal differences were found in laboratory values between institutions. The addition of AD may be useful in cases of equivocal laboratory results but does not appear to be necessary for routine diagnosis of PJI after TKA/THA. LEVEL EVIDENCE: Level III.

The Alpha-Defensin Prosthetic Joint Infection Test Has Poor Validity for Native Knee Joint Infection.

BACKGROUND: The alpha-defensin test known as Synovaure has been very effective in diagnosis of prosthetic joint infections (PJIs). Being able to easily and accurately differentiate septic and inflammatory arthropathies in native joints would improve diagnostic workup and management. We tested the ability of an alpha-defensin test to distinguish septic from inflammatory or crystalline arthropathy in the native knee. METHODS: 40 native knee joint fluid specimens were tested with cell count, fluid analysis, and culture and alpha-defensin testing. We determined the sensitivity and specificity of the alpha-defensin test using culture-positive fluid as the gold standard for septic arthropathy and positive crystals as the gold standard for crystalline arthropathy. RESULTS: The Synovasure PJI test had 100% specificity for septic arthritis coupled with a 28% false-positive rate when applied to native knee aspirations. False-positive rate was 5.3 times higher in patients with crystals found in the joint fluid. CONCLUSION: Alpha-defensin testing, in the form of the Synovasure PJI test, has a high-false-positive rate when used to distinguish septic and inflammatory arthritis in the native knee joint. Future work will need to determine the sensitivity and specificity of the newer native joint panel. Clinicians should be cognizant of the specific alpha-defensin test used when sampling native knee synovial fluid.

What is the Clinical Utility of Synovial Alpha Defensin Testing of Antibiotic Spacers Before Reimplantation?

BACKGROUND: The purpose of this study was to evaluate the diagnostic performance of standalone alpha defensin (AD) testing of antibiotic spacers during two stage exchange and to determine if the addition of AD testing to other commonly used laboratory tests improves the ability to detect persistent infection in an antibiotic spacer. METHODS: Cases of two-stage exchange for periprosthetic joint infection from 2016 to 2019 at a single institution were retrospectively reviewed. Cases were classified as persistently infected or not infected in accordance with 2014 and 2018 Musculoskeletal Infection Society criteria to determine if AD provided any clinical utility beyond the other commonly used tests that make up both criteria. Delphi Consensus criteria at 1 year were used as the gold standard for determining recurrent periprosthetic joint infection. RESULTS: Fifty-two spacers (25 hips and 27 knees) in 51 patients were included for analysis. Five spacers were persistently infected based on Musculoskeletal Infection Society criteria. One spacer underwent reresection and the remaining 4 underwent reimplantation with no subsequent infectious complications. All 48 patients who were categorized as not infected underwent reimplantation; at 1 year postoperatively, 7 (13%) had failed due to infection. Three spacers (6%) had a positive AD test. Two spacers with positive AD tests underwent reimplantation, neither had failed at 1 year postoperatively. Sensitivity of standalone AD testing was 0%, and specificity was 96%. CONCLUSION: Standalone AD testing for the purpose of predicting repeat infection after two-stage exchange arthroplasty exhibits sensitivity of 0% and low predictive value. Addition of synovial AD testing did not increase the diagnostic performance of commonly used synovial and serologic markers of infection. LEVEL OF EVIDENCE: IV-retrospective cohort study.

Leukocyte esterase test and alpha-defensin test have similar accuracy for the diagnosis of periprosthetic joint infection.

BACKGROUND: The diagnosis of periprosthetic joint infection (PJI) after total joint replacement remains challenging. Synovial biomarkers are recommended as the major diagnostic criteria for PJI. The purpose of this study was to test the accuracy of the alpha-defensin test and compare it with that of the leukocyte esterase (LE) test for the diagnosis of PJI. METHODS: We obtained 130 hip or knee aspirates from May 2015 to September 2018. PJI was defined according to the European Bone and Joint Infection Society (EBJIS) criteria. Synovial fluid samples were tested with the alpha-defensin ELISA and a LE strip. For the LE strip test, besides using the traditional threshold 500 (equal to ++), we are also using an improved one, with 500 (equal to ++) serving as the threshold for the tests before centrifugation and both 250 and 500 indicating positive results after centrifugation. The receiver operating characteristic (ROC) curves, sensitivity, specificity, positive predictive value, negative predictive value, and Cohen's Kappa value were calculated for the LE and alpha-defensin tests. RESULTS: The area under the curve (AUC) of alpha-defensin, LE strip test with traditional, and improved interpretation strategy were 0.875, 0.854, and 0.877, respectively. The Cohen's Kappa value for the alpha-defensin tests was 0.826 with the traditional LE interpretation strategy and 0.875 with the improved strategy. CONCLUSION: The present study shows that the use of the LE strip to test synovial fluid yielded an accuracy similar to that of the alpha-defensin immunoassay for the diagnosis of PJI; this finding supports the 2018 ICM PJI definition, which considers positive alpha-defensin and LE test results to be equivalent.