Multicentric evaluation of a specific intrathecal anti-Treponema pallidum IgG index as a diagnostic biomarker of neurosyphilis: results from a retro-prospective case-control study.

BACKGROUND AND OBJECTIVES: The diagnosis of neurosyphilis (NS) lacks a true 'gold standard', making the diagnosis challenging while consequences of a misdiagnosis are potentially severe. The aim of this study was to evaluate the diagnostic performance of measuring an antibody index (AI) for the intrathecal synthesis of specific anti-Treponema pallidum (T. pallidum) IgG for the diagnosis of NS. METHODS: Specific anti-T. pallidum IgG were measured simultaneously in paired cerebrospinal fluid (CSF)-serum samples collected retrospectively and prospectively between 2007 and 2022, from patients suspected of NS, in Switzerland. An AI was calculated to account for blood-brain barrier integrity. Area under the receiver operating characteristic curve, sensitivity/specificity and positive/negative predictive values of AI test were estimated. Two NS definitions were used: NS1 included patients with NS suspicion presenting with neurological symptoms and/or acute neurosensory signs, and positive T. Pallidum Hemagglutinations Assay (TPHA)/T. pallidum particle agglutination assay (TPPA) serology and CSF-TPHA/TPPA ≥320, and either CSF-leucocytes >5 cells/mm3 and/or CSF-protein >0.45 g/L and/or a reactive CSF-venereal disease research laboratory (VDRL)/rapid plasma reagin (RPR) test. NS2 included patients with suspected NS presenting with acute ocular and/or otologic symptoms, and positive TPHA/TPPA serology, and a favourable response to NS treatment. Controls were patients diagnosed with any other central nervous system (CNS) pathologies and with positive TPHA/TPPA serology. RESULTS: The study included 71 NS (43 NS1 and 28 NS2) and 110 controls. With a threshold of ≥1.7, sensitivity and specificity of the specific AI test were 90.7% (CI 77.7 to 97.4) and 100% (CI 96.7 to 100.0), respectively, for NS1 and 14.3% (CI 4 to 32.7) and 100% (CI 96.7 to 100.0) for NS2. In patients suspected of NS with a CNS involvement (NS1 group), NS could be confirmed by the positivity of this specific AI. CONCLUSIONS: Measurement of an intrathecal synthesis index of specific anti-T. pallidum IgG in patients with CSF inflammatory signs appears to be a valuable diagnostic test. However, in otic or ocular syphilis, presenting few CSF abnormalities, AI is not sufficient alone to confirm NS diagnosis. TRIAL REGISTRATION: Swiss Association of Research Ethics Committees number 2019-00232.

Towards an Early Clinical and Biological Resistance Detection in Dermatophytosis: About 2 Cases of Trichophyton indotineae.

Trichophyton indotineae causes resistant dermatophytosis to terbinafine. The global spread of terbinafine-resistant Trichophyton indotineae strains with mutations in the squalene epoxidase gene is a major issue. This emerging species is now more frequently isolated in Europe and we report here two cases of T. indotineae tinea corporis in Switzerland, one with in vitro resistance to terbinafine and a second with in vitro susceptibility but a clinical resistance. Mycology isolation from cultures and sequencing ITS gene were used to confirm T. indotineae infection. In vitro antifungal susceptibility was tested in a microplate with a colorimetric detection of fungal viability for the determination of the minimal inhibitory concentration (MIC). Facing these emerging resistances and since there are a limited number of antifungal agents available to treat dermatophytosis, the early detection of terbinafine resistance should be a prerequisite in the management of T. indotineae infections.

Intrathecal Synthesis Index of Specific Anti-Treponema IgG: a New Tool for the Diagnosis of Neurosyphilis.

Neurosyphilis (NS) diagnosis is challenging because clinical signs are diverse and unspecific, and a sensitive and specific laboratory test is lacking. We tested the performance of an antibody index (AI) for intrathecal synthesis of specific anti-Treponema IgG by enzyme-linked immunosorbent assay (ELISA) for NS diagnosis. We conducted a retroprospective monocentric study including adults with neurological symptoms who had serum and cerebral spinal fluid (CSF) samples collected between 2006 and 2021. Two NS definitions were used. NS1 included patients with neurological symptoms, positive Treponema pallidum particle agglutination (TPPA) serology, and CSF-TPPA of ≥320, as well as CSF-leukocytes of >5 cells/mm3 and/or CSF-protein of >0.45 g/L and/or a reactive CSF-VDRL/RPR test. NS2 included patients with acute ocular and/or otologic symptoms, positive TPPA serology, and a response to NS treatment. Controls were patients with central nervous system disorders other than neurosyphilis. Anti-Treponema pallidum IgG were measured simultaneously in serum and CSF, and AI was calculated according to Reiber diagram. We assessed the AI test area under the curve (AUC), sensitivity/specificity, and estimated positive and negative predictive values. In total, 16 NS1 patients, 11 NS2 patients, and 71 controls were included. With an AI of ≥1.7 as a positive test for NS diagnostic, specificity was 98.6% (95% confidence interval [CI 95%] of 92.4 to 100.0) and sensitivity was 81.3% (CI 95% of 54.4 to 96.0) for NS1 and 98.6% (CI 95% 92.4 to 100.0) and 27.3% (CI 95% 6.0 to 61.0), respectively, for NS2. Positive and negative predictive values were >95% for NS1 and >85% for NS2, for prevalence above and below 20%. Measuring an AI for intrathecal synthesis of specific anti-Treponema pallidum IgG is a new promising tool highly specific for NS diagnosis. IMPORTANCE In the context of a lack of a gold standard for the diagnosis of neurosyphilis due to either nonspecific or nonsensitive tests, we present in this article a new promising tool highly specific for NS diagnosis. This new test involves measuring an intrathecal synthesis index of specific anti-Treponema IgG by ELISA.

Head-to-Head Accuracy Comparison of Three Commercial COVID-19 IgM/IgG Serology Rapid Tests.

BACKGROUND: Comparative data of SARS-CoV-2 IgM/IgG serology rapid diagnostic tests (RDTs) is scarce. We thus performed a head-to-head comparison of three RDTs. METHODS: In this unmatched case-control study, blood samples from 41 RT-PCR-confirmed COVID-19 cases and 50 negative controls were studied. The diagnostic accuracy of three commercially available COVID-19 RDTs: NTBIO (RDT-A), Orient-Gene (RDT-B), and MEDsan (RDT-C), against both a recombinant spike-expressing immunofluorescence assay (rIFA) and Euroimmun IgG ELISA, was assessed. RDT results concordant with the reference methods, and between whole blood and plasma, were established by the Kendall coefficient. RESULTS: COVID-19 cases' median time from RT-PCR to serology was 22 days (interquartile range (IQR) 13-31 days). Whole-blood IgG detection with RDT-A, -B, and -C showed 0.93, 0.83, and 0.98 concordance with rIFA. Against rIFA, RDT-A sensitivity (SN) was 92% (95% CI: 78-98) and specificity (SP) 100% (95% CI: 91-100), RDT-B showed 87% SN (95% CI: 72-95) and 98% SP (95% CI: 88-100), and RDT-C 100% SN (95% CI: 88-100) and 98% SP (95% CI: 88-100). Against ELISA, SN and SP were above 90% for all three RDTs. CONCLUSIONS: RDT-A and RDT-C displayed IgG detection SN and SP above 90% in whole blood. These RDTs could be considered in the absence of routine diagnostic serology facilities.

Diagnostic accuracy of Augurix COVID-19 IgG serology rapid test.

AIMS: To validate the diagnostic accuracy of the Augurix SARS-CoV-2 IgM/IgG rapid immunoassay diagnostic test (RDT) for COVID-19. METHODS: In this unmatched 1:1 case-control study, blood samples from 46 real-time RT-PCR-confirmed SARS-CoV-2 hospitalized cases and 45 healthy donors (negative controls) were studied. Diagnostic accuracy of the IgG RDT was assessed against both an in-house recombinant spike-expressing immunofluorescence assay (rIFA), as an established reference method (primary endpoint), and the Euroimmun SARS-CoV-2 IgG enzyme-linked immunosorbent assays (ELISA) (secondary endpoint). RESULTS: COVID-19 patients were more likely to be male (61% vs 20%; P = .0001) and older (median 66 vs 47 years old; P < .001) than controls. Whole blood IgG-RDT results showed 86% and 93% overall Kendall concordance with rIFA and IgG ELISA, respectively. IgG RDT performances were similar between plasma and whole blood. Overall, RDT sensitivity was 88% (95% confidence interval [95%CI]: 70-96), specificity 98% (95%CI: 90-100), PPV 97% (95%CI: 80-100) and NPV 94% (95%CI: 84-98). The IgG-RDT carried out from 0 to 6 days, 7 to 14 days and > 14 days after the SARS-CoV-2 RT-PCR test displayed 30%, 73% and 100% positivity rates in the COVID-19 group, respectively. When considering samples taken >14 days after RT-PCR diagnosis, NPV was 100% (95%CI:90-100), and PPV was 100% (95%CI:72-100). CONCLUSIONS: The Augurix IgG-RDT done in whole blood displays a high diagnostic accuracy for SARS-CoV-2 IgG in high COVID-19 prevalence settings, where its use could be considered in the absence of routine diagnostic serology facilities.

Desmoplakin interacts with the coil 1 of different types of intermediate filament proteins and displays high affinity for assembled intermediate filaments.

The interaction of intermediate filaments (IFs) with the cell-cell adhesion complexes desmosomes is crucial for cytoskeletal organization and cell resilience in the epidermis and heart. The intracellular desmosomal protein desmoplakin anchors IFs to the cell adhesion complexes predominantly via its four last carboxy-terminal domains (C-terminus). However, it remains unclear why the C-terminus of desmoplakin interacts with different IF types or if there are different binding affinities for each type of IFs that may influence the stability of cell-specific adhesion complexes. By yeast three-hybrid and fluorescence binding assays, we found that the coiled-coil 1 of the conserved central rod domain of the heterodimeric cytokeratins (Ks) 5 and 14 (K5/K14) was required for their interaction with the C-terminus of desmoplakin, while their unique amino head- and C-tail domains were dispensable. Similar findings were obtained in vitro with K1/K10, and the type III IF proteins desmin and vimentin. Binding assays testing the C-terminus of desmoplakin with assembled K5/K14 and desmin IFs yielded an apparent affinity in the nM range. Our findings reveal that the same conserved domain of IF proteins binds to the C-terminus of desmoplakin, which may help explain the previously reported broad binding IF-specificity to desmoplakin. Our data suggest that desmoplakin high-affinity binding to diverse IF proteins ensures robust linkages of IF cytoskeleton and desmosomes that maintain the structural integrity of cellular adhesion complexes. In summary, our results give new insights into the molecular basis of the IF-desmosome association.

Fast identification of dermatophytes by MALDI-TOF/MS using direct transfer of fungal cells on ground steel target plates.

Dermatophytes cause human infections limited to keratinised tissues. We showed that the direct transfer method allows reliable identification of non-dermatophytes mould and yeast by MALDI-TOF/MS. We aimed at assessing whether the direct transfer method can be used for dermatophytes and whether an own mass spectra library would be superior to the Bruker library. We used the Bruker Biotyper to build a dermatophyte mass spectra library and assessed its performance by 1/testing a panel of mass spectrum produced with strains genotypically identified and, 2/comparing MALDI-TOF/MS identification to morphology-based methods. Identification of dermatophytes using the Bruker library is poor. Our library provided 97% concordance between ITS sequencing and MALDI-TOF/MS analysis with a panel of 1104 spectra corresponding to 276 strains. Direct transfer method using unpolished target plates allowed proper identification of 85% of dermatophytes clinical isolates most of which were common dermatophytes. A homemade dermatophyte MSP library is a prerequisite for accurate identification of species absent in the Bruker library but it also improves identification of species already listed in the database. The direct deposit method can be used to identify the most commonly found dermatophytes such as T. rubrum and T. interdigitale/mentagrophytes by MALDI-TOF/MS.

Hu antigen R (HuR) heterogeneous expression quantification as a prognostic marker of melanoma.

BACKGROUND: Prognostic markers for melanoma, particularly for stage II disease, are needed for the risk-benefit evaluation of future adjuvant therapies. The mainly nuclear RNA-binding protein human antigen R (HuR) regulates the protein expression of thousands of mRNAs, its own heterogeneous expression could therefore reflect tumor heterogeneity and plasticity. Here, we evaluate its quantification in primary melanoma as a marker of metastatic outcome. METHODS: We conducted an immunohistochemistry-based automated quantification of HuR nuclear expression heterogeneity in primary melanomas, most with Breslow thickness ≥ 1 mm and calculated the dimensionless fourth moment, that is, the kurtosis of HuR (HuR K) expression distribution. Twelve tumors from patients with no metastatic disease were compared to a similar number of tumors from patients who had metastatic disease at 2 years follow up. RESULTS: HuR K value appeared significantly higher in the non-metastatic group comparatively to the metastatic group (P = 2.84 × 10-3 , 1-tailed Wilcoxon rank-sum test). Moreover, compared to the Breslow thickness, HuR K value appeared as a more robust marker of metastatic outcome (respective areas under receiver operating characteristic curves 0.84 and 0.87). CONCLUSION: Our data need confirmation on a large cohort, however strongly suggest that HuR expression heterogeneity quantification using kurtosis, could be used as a prognostic marker in melanoma.

IL-22 capacitates dermal fibroblast responses to TNF in scleroderma.

OBJECTIVES: Interleukin (IL) 22 mRNA in systemic sclerosis (SSc) skin and Th22 cells in SSc peripheral blood are increased, but the role of IL-22 in fibrosis development remains poorly understood. METHODS: Biopsies were obtained from the involved skin of 15 SSc, 4 morphea and 8 healthy donors (HD). The presence of IL-22+ cells in the skin was determined by immunostaining. The in vitro response of HD and SSc fibroblasts to IL-22, IL-22 in conjunction with tumour necrosis factor (TNF) or keratinocyte conditioned medium was assessed by ELISA, radioimmunoassay (RIA), real-time PCR and western blot. The in vivo response in mice was assessed by histomorphometry. RESULTS: IL-22+ cells were over-represented in the dermis and epidermis of morphea and in the epidermis of SSc compared with HD. The majority of dermal IL-22+ cells were T cells. Dermal fibroblasts expressed both IL-22 receptor subunits IL-10RB and IL-22RA, expression of which was enhanced by TNF and reduced by transforming growth factor (TGF)-ß. IL-22 induced rapid phosphorylation of p38 and ERK1/2 in fibroblasts, but failed to induce the synthesis of chemokines and extracellular matrix components. However, IL-22 enhanced the production of monocyte chemotactic protein 1, IL-8 and matrix metalloproteinase 1 induced by TNF. Fibroblast responses were maximal in the presence of conditioned medium from keratinocytes activated by IL-22 in conjunction with TNF. Dermal thickness was maximal in mice injected simultaneously with IL-22 and TNF. CONCLUSIONS: IL-22 capacitates fibroblast responses to TNF and promotes a proinflammatory fibroblast phenotype by favouring TNF-induced keratinocyte activation. These results define a novel role for keratinocyte-fibroblast interactions in the context of skin fibrosis.

L-Tryptophan as a Novel Potential Pharmacological Treatment for Wound Healing via Aryl Hydrocarbon Receptor Activation.

BACKGROUND: The aryl hydrocarbon receptor has been shown to be involved in wound healing. OBJECTIVE: The aim of this study was to assess the effect of tryptophan on wound healing in vitro and in a clinical trial. METHODS: The ability of tryptophan and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to increase wound healing was assessed in an in vitro scratch wound model in human keratinocytes. Topical tryptophan and vehicle were assessed for 12 weeks in 51 patients with lower limb ulcers that were resistant to conventional therapies. RESULTS: TCDD 0.1 nM and tryptophan 1 µM increased the rate of scratch recovery in a culture model. Topical tryptophan induced stronger pain relief and faster re-epithelialization than its vehicle in patients with lower limb ulcers. CONCLUSION: Tryptophan shows promising potential as a novel topical treatment for wound healing.

High IL-17E and low IL-17C dermal expression identifies a fibrosis-specific motif common to morphea and systemic sclerosis.

BACKGROUND: High interleukin (IL)-17A levels are characteristically found in the skin of systemic sclerosis (SSc) individuals. Our aim was to investigate whether the dermal expression of IL-17A and related IL-17 family members (i.e. IL-17C, IL-17E and IL-17F) could distinguish fibrotic from healthy skin and could show similarities in SSc and morphea, two disorders with presumed distinct pathogenesis, but characterized by skin fibrosis. METHODS: Biopsies were obtained from the involved skin of 14 SSc, 5 morphea and 8 healthy donors (HD) undergoing plastic surgery. Immunohistochemistry/immunofluorescence techniques were coupled to a semi-automated imaging quantification approach to determine the presence of the IL-17 family members in the skin. The in vitro effects induced by the IL-17 family members on fibroblasts from normal and SSc individuals were assessed by ELISA and RIA. RESULTS: Positive cells for each of the IL-17 isoforms investigated were present in the dermis of all the individuals tested, though with variable frequencies. SSc individuals had increased frequency of IL-17A+ (p = 0.0237) and decreased frequency of IL-17F+ (p = 0.0127) and IL-17C+ cells (p = 0.0008) when compared to HD. Similarly, morphea individuals had less frequent IL-17C+ cells (p = 0.0186) in their skin but showed similar number of IL-17A+ and IL-17F+ cells when compared to HD. Finally, IL-17E+ cells were more numerous in morphea (p = 0.0109) and tended to be more frequent in SSc than in HD. Fibroblast production of IL-6, MMP-1 and MCP-1 was enhanced in a dose-dependent manner in the presence of IL-17E and IL-17F, but not in the presence of IL-17C. None of the cytokine tested had significant effect on type I collagen production. Of interest, in SSc the frequency of both IL-17A and IL-17F positive cells increased with disease duration. CONCLUSIONS: The frequency of IL-17A and IL-17F distinguish SSc to morphea individuals while dermal expression of IL-17C (low) and IL-17E (high) identifies a fibrosis-specific motif. The specific IL-17C/IL-17E cytokine combination may thus play a role in the development of fibrosis.

Hyalurosome gene regulation and dose-dependent restoration of skin atrophy by retinaldehyde and defined-size hyaluronate fragments in dermatoporosis.

BACKGROUND: Dermatoporosis is an emerging clinical condition caused by chronological skin aging, long-term sun exposure and chronic use of corticosteroids; however, genomic expression in dermatoporosis and the efficacy of different therapeutic approaches to prevent and treat dermatoporosis have not been investigated so far. OBJECTIVE: We examined the possible effect of topical retinaldehyde (RAL) and defined-size hyaluronate fragments (HAFi) on the expression of hyalurosome genes potentially involved in the pathogenesis of dermatoporosis. We also explored the effect of different concentrations of HAFi on skin thickness. METHODS: 13 persons were separated into a young control group (n = 8) and a dermatoporosis group (n = 5). Topical treatment of both groups with a combination of 0.05% RAL and 1 or 0.2% HAFi was applied on the forearm twice daily for 30 days. Forearm skin biopsies of both groups were performed before and after application. Hyalurosome genes CD44, heparin-binding epidermal growth factor (HB-EGF), ErbB1, hyaluronate synthase 3 (HAS3) and Hyal2 were chosen as potential markers of dermatoporosis. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed for quantification of mRNA expression of the target hyalurosome genes. Measurement of forearm skin thickness before and after treatment was performed by ultrasonography. Analysis of the results was done by Student's t test. A p value <0.05 was considered statistically significant. RESULTS: In qRT-PCR analysis the relative expression of hyalurosome (CD44, HAS3, HB-EGF) genes was found to be reduced in patients prior to topical treatment and to be notably increased following treatment. The reduced expression of CD44 and HAS3 in patients was specifically restored in dermatoporotic patients after treatment. No difference in skin thickness was observed in controls after treatment. The treatment caused a significant increase in skin thickness in dermatoporotic patients. This increase was more significant with 1% HAFi when compared to 0.2% HAFi. RAL and HAFi also caused a significant reduction in purpuric lesions in patients with dermatoporosis. CONCLUSION: Our results indicate that topically applied RAL and HAFi regulate hyalurosome gene expression in dermatoporosis and that they show a dose-dependent effect on the correction of skin atrophy in dermatoporotic patients.

Interaction of plectin with keratins 5 and 14: dependence on several plectin domains and keratin quaternary structure.

Plectin, a cytolinker of the plakin family, anchors the intermediate filament (IF) network formed by keratins 5 and 14 (K5/K14) to hemidesmosomes, junctional adhesion complexes in basal keratinocytes. Genetic alterations of these proteins cause epidermolysis bullosa simplex (EBS) characterized by disturbed cytoarchitecture and cell fragility. The mechanisms through which mutations located after the documented plectin IF-binding site, composed of the plakin-repeat domain (PRD) B5 and the linker, as well as mutations in K5 or K14, lead to EBS remain unclear. We investigated the interaction of plectin C terminus, encompassing four domains, the PRD B5, the linker, the PRD C, and the C extremity, with K5/K14 using different approaches, including a rapid and sensitive fluorescent protein-binding assay, based on enhanced green fluorescent protein-tagged proteins (FluoBACE). Our results demonstrate that all four plectin C-terminal domains contribute to its association with K5/K14 and act synergistically to ensure efficient IF binding. The plectin C terminus predominantly interacted with the K5/K14 coil 1 domain and bound more extensively to K5/K14 filaments compared with monomeric keratins or IF assembly intermediates. These findings indicate a multimodular association of plectin with K5/K14 filaments and give insights into the molecular basis of EBS associated with pathogenic mutations in plectin, K5, or K14 genes.

Michel's Transport Medium as an Alternative to Liquid Nitrogen for PCR Analysis of Skin Biopsy Specimens.

Formalin fixation and paraffin embedding are standard procedures for histopathological diagnosis and allow long-term archiving of tissue specimens. The cross-linking properties of formalin cause fragmentation of nucleic acids and reduce the sensitivity of PCR analysis. Michel's medium is a well-established transport medium used by dermatologists for biopsy transport to maintain tissue-fixed immunoreactants prior to direct immunofluorescence and immunoelectron microscopy. Here we report that Michel's medium also allows short-term preservation of DNA for PCR analysis and permits amplification of amplicons larger than 1 kb. Therefore, Michel's medium appears to be a reserve medium for performing PCR when no other samples are available.

Phosphorylation of serine 4,642 in the C-terminus of plectin by MNK2 and PKA modulates its interaction with intermediate filaments.

Plectin is a versatile cytolinker of the plakin family conferring cell resilience to mechanical stress in stratified epithelia and muscles. It acts as a critical organizer of the cytoskeletal system by tethering various intermediate filament (IF) networks through its C-terminal IF-binding domain (IFBD). Mutations affecting the IFBD cause devastating human diseases. Here, we show that serine 4642, which is located in the extreme C-terminus of plectin, is phosphorylated in different cell lines. Phosphorylation of S4642 decreased the ability of plectin IFBD to associate with various IFs, as assessed by immunofluorescence microscopy and cell fractionation studies, as well as in yeast two-hybrid assays. Plectin phosphorylated at S4642 was reduced at sites of IF network anchorage along cell-substrate contacts in both skin and cultured keratinocytes. Treatment of SK-MEL-2 and HeLa cells with okadaic acid increased plectin S4642 phosphorylation, suggesting that protein phosphatase 2A dephosphorylates this residue. Moreover, plectin S4642 phosphorylation was enhanced after cell treatment with EGF, phorbol ester, sorbitol and 8-bromo-cyclic AMP, as well as during wound healing and protease-mediated cell detachment. Using selective protein kinase inhibitors, we identified two different kinases that modulate the phosphorylation of plectin S4642 in HeLa cells: MNK2, which is downstream of the ERK1/2-dependent MAPK cascade, and PKA. Our study indicates that phosphorylation of S4642 has an important regulatory role in the interaction of plectin with IFs and identifies a novel link between MNK2 and the cytoskeleton.