Investigation of the pathogen-specific antibody response in periprosthetic joint infection.

PURPOSE: Periprosthetic joint infections (PJIs) are a very demanding complication of arthroplasty. Diagnosis of PJI and pathogen identification pose considerable challenges in clinical practice. We hypothesized that the pathogen-specific immune response to PJI reflects the infection process, provides clinically relevant information on disease course, and has the potential to further optimize antimicrobial therapy. METHODS: We conducted a prospective matched cohort pilot study with 13 patients undergoing two-stage septic revision arthroplasty (PJI patients) between 06/2020 and 06/2021, as well as 11 control patients undergoing one-stage aseptic revision arthroplasty (Non-PJI patients). Pre-, intra- and postoperative serum samples were collected at standardized time points. We developed a custom Luminex®-based quantitative bead-based suspension array (Infection Array; IA), and used it for simultaneous measurement of antibody specificities against 32 pathogens commonly associated with PJI in 267 serum samples. RESULTS: The IA was able to trace the dynamics of the pathogen-specific humoral immune response in all patients against PJI-related pathogens, prominently coagulase-negative staphylococci and streptococci. Pathogen-specific serum antibody titers declined in 62% of PJI patients over the course of treatment, while no changes in antibody titers were observed in 82% of Non-PJI patients during this study. Our serological data strongly suggested that antibody signatures reflect an immune response to microbial invasion. CONCLUSION: Our results provide insights into the pathophysiology of PJI and information on the individual disease courses. The IA is therefore a promising and novel serological tool of high resolution for monitoring the immunoproteomic footprints of infectious pathogens in the course of PJI.

Acquired hypogammaglobulinemia and pathogen-specific antibody depletion after solid organ transplantation in human immunodeficiency virus infection: A brief report.

Hypogammaglobulinemia (HGG) frequently occurs in recipients after types of (SOT). The incidence and significance of HGG in HIV+ recipients of SOT are just being explored. We reported that 12% of the recipients in the SOT in multi-center HIV-TR (HIV-TR) Study developed moderate or severe HGG at 1 year. In LT recipients, this was associated with serious infections and death. We have now further characterized the decreased antibodies in HIV+ SOT recipients who developed HGG. We measured the levels of pathogen-specific antibodies and poly-specific self-reactive antibodies (PSA) in relation to total IgG levels from serial serum samples for 20 HIV+ SOT recipients who developed moderate to severe HGG following SOT. Serum antibody levels to measles, tetanus toxoid, and HIV-1 were determined by EIA. Levels of PSAs were determined by incubating control lymphocytes with patient serum, staining with anti-human IgG Fab-FITC, and analysis by flow cytometry. Levels of PSA were higher compared to healthy, HIV-uninfected controls at pre-transplant baseline and increased by weeks 12 and 26, but the changes were not significant. Likewise, anti-HIV antibody levels remained unchanged over time. In contrast, antibody levels against measles and tetanus were significantly reduced from baseline by week 12, and did not return to baseline, even after 2 years. For HIV patients who develop moderate to severe HGG after transplant, the reduction in IgG levels is associated with a significant decrease in pathogen-specific antibody titers, while PSA levels and anti-HIV antibodies are unchanged. This may contribute to infectious complications and other clinical endpoints.

Card9 mediates susceptibility to intestinal pathogens through microbiota modulation and control of bacterial virulence.

OBJECTIVE: In association with innate and adaptive immunity, the microbiota controls the colonisation resistance against intestinal pathogens. Caspase recruitment domain 9 (CARD9), a key innate immunity gene, is required to shape a normal gut microbiota. Card9-/- mice are more susceptible to the enteric mouse pathogen Citrobacter rodentium that mimics human infections with enteropathogenic and enterohaemorrhagic Escherichia coli. Here, we examined how CARD9 controls C. rodentium infection susceptibility through microbiota-dependent and microbiota-independent mechanisms. DESIGN: C. rodentium infection was assessed in conventional and germ-free (GF) wild-type (WT) and Card9-/- mice. To explore the impact of Card9-/-microbiota in infection susceptibility, GF WT mice were colonised with WT (WT→GF) or Card9-/- (Card9-/- →GF) microbiota before C. rodentium infection. Microbiota composition was determined by 16S rDNA gene sequencing. Inflammation severity was determined by histology score and lipocalin level. Microbiota-host immune system interactions were assessed by quantitative PCR analysis. RESULTS: CARD9 controls pathogen virulence in a microbiota-independent manner by supporting a specific humoral response. Higher susceptibility to C. rodentium-induced colitis was observed in Card9-/- →GF mice. The microbiota of Card9-/- mice failed to outcompete the monosaccharide-consuming C. rodentium, worsening the infection severity. A polysaccharide-enriched diet counteracted the ecological advantage of C. rodentium and the defective pathogen-specific antibody response in Card9-/- mice. CONCLUSIONS: CARD9 modulates the susceptibility to intestinal infection by controlling the pathogen virulence in a microbiota-dependent and microbiota-independent manner. Genetic susceptibility to intestinal pathogens can be overridden by diet intervention that restores humoural immunity and a competing microbiota.

Binding and Neutralizing Capacity of Respiratory Syncytial Virus (RSV)-Specific Recombinant IgG Against RSV in Human Milk, Gastric and Intestinal Fluids from Infants.

Oral administration of pathogen-specific recombinant antibodies may help to prevent infant gastrointestinal (GI) pathogen infection; however, to neutralize an infectious agent, these antibodies must resist degradation in the GI tract. Palivizumab, a recombinant antibody specific for the respiratory syncytial virus (RSV), was used as a model for pathogen-specific IgG in human milk. The aim was to compare the remaining binding capacity of palivizumab in milk between three mothers after exposure to an in vitro model of infant gastrointestinal digestion (gastric and duodenal fluids) using ELISA. The neutralizing capacity of palivizumab in pooled human milk, gastric contents, and stools from preterm infants was also evaluated for blocking RSV with green fluorescent protein (RSV-GFP) infection in Hep-2 cells using confocal and inverted microscopy and flow cytometry. The reduction of palivizumab binding capacity in human milk and digested samples was slightly different between mothers. Overall, palivizumab decreased 50% after simulated gastric digestion with pepsin and 62% after simulated intestinal digestion with pancreatin. Palivizumab (2-8 µg/mL) in human milk or stool samples blocked RSV (3.4 × 104 FFU/mL) infection (no syncytia formation on Hep-2 cells) by microscopy. Syncytia formation was detected on Hep-2 cells when RSV was incubated in gastric contents or virus medium with 2-4 µg/mL of palivizumab, but no infection was observed at 8 µg/mL. No fluorescence (absence of infected cells) was detected when palivizumab (100 µg/mL) was incubated in human milk or medium with RSV-GFP (1.1 × 105 FFU/mL), whereas fluorescence increased with the reduced concentration of palivizumab using flow cytometry. These results suggest that undigested and digested matrices could change the binding and neutralizing capacity of viral pathogen-specific antibodies.

Multiple-Point Evaluation Algorithms for Enhanced Precision of Pathogen-Specific Cerebrospinal Fluid/Serum Antibody Index Calculation.

The determination of pathogen-specific antibody indices (AIs) of CSF and serum is an essential cornerstone in assessing neurological diseases and demands reliable high precision. Various companies provide ELISA kits for the detection of respective antibody concentrations and base AI calculation on a single CSF/serum pair of optical densities (ODs), combined with selection rules. The remainder of OD measurements is not used. There is no averaging of measurement errors and result stabilization. OD data from Siemens Enzygnost ELISA measurements of 2012-2016 proficiency survey samples for measles/rubella/varicella zoster/herpes simplex virus (MRZH) reaction (INSTAND e.V.) were reanalyzed. Several reference methods for calculating Q values from ODs using multiple-point evaluation are described. The methods are based on the α method and the four-parameter logistic (4PL) equation. Statistical analysis shows standard deviations of relative AI differences from AI target values to be significantly lower if derived from multiple-point evaluation instead of single-pair evaluation. Thus, the virus-averaged hit rate of a 10% target AI environment can be improved from 49% up to 69%. Waiving the usage of a standard curve in favor of parameter fitting significantly improves calculational precision for Siemens Enzygnost assays. Patient safety, diagnostic assay costs, and laboratory effectiveness might be improved for other test distributors as well.

Circulating pathogen-specific plasmablasts in female patients with upper genital tract infection.

Mucosal antibodies constitute the first line of adaptive immune defence against invaders in the female genital tract (FGT), yet the sequence of events leading to their production is surprisingly poorly characterized. We explored the induction of pathogen-specific antibody-secreting cells (ASC) as a response to an acute infection in the upper FGT. We recruited 12 patients undergoing surgery due to an upper FGT infection (7/12 blood culture positive, 5/12 negative) and six healthy controls. Pathogens were sampled during surgery and PBMC collected in the acute phase of the disease (days 7-10). We searched by ELISPOT circulating pathogen-specific ASC and explored their frequency, immunoglobulin isotype distribution, and expressions of homing receptors (α4ß7, L-selectin, and CLA). All patients had circulating ASC specific to the infective bacteria; the geometric mean was 434 (95%CI 155-1234) ASC (IgA + IgG + IgM)/106 PBMC. IgA ASC predominated in 7/12, IgG ASC in 3/12, and IgM ASC in 2/12 cases. Of all the pathogen-specific ASC, 60% expressed α4ß7, 67% L-selectin, and 9% CLA. This study is the first to show induction of pathogen-specific ASC in the peripheral blood in bacterial infection in the human FGT. Our findings reveal that such FGT-originating pathogen-specific ASC are predominated by IgA ASC and exhibit a homing receptor profile resembling that of ASC in acute urinary tract infection. The data thus suggest a characteristic profile shared by the urogenital tract.