Evaluation of Sézary cell marker expression and cell death behaviour upon in vitro treatment by flow cytometry in Sézary syndrome patients.

The diagnosis of Sézary syndrome (SS) relies on the identification of blood Sézary cells (SC) by different markers via flow cytometry. Treatment of SS is challenging since its pathogenesis is characterized by cell death resistance rather than hyperproliferation. In this study, we establish an integrated approach that considers both the expression of SC markers and sensitivity to cell death both spontaneously and upon in vitro treatment. Peripheral blood mononuclear cells were isolated from 20 SS patients and analysed for the SC markers CD7 and CD26 loss as well as CD158k and PD1 gain. The cells were then treated with different established and experimental therapies in vitro and cell death was measured. Spontaneous and therapeutically induced cell death were measured and correlated to cellular marker profiles. In the marker-positive cells, spontaneous cell death sensitivity was reduced. Different treatments in vitro managed to specifically induce cell death in the putative CTCL cell populations. Interestingly, a repeated analysis after 3 months of treatment revealed the CTCL cell death sensitivity to be restored by therapy. We propose this novel integrated approach comprising the evaluation of SC marker expression and analysis of cell death sensitivity upon treatment that can also enable a better therapy stratification.

Health-related quality of life and its influencing factors in patients with primary cutaneous B-cell lymphomas: A multicentric study in 100 patients.

BACKGROUND: Primary cutaneous B-cell lymphomas (CBCL) are a group of rare malignant skin diseases that represent approximately 20%-30% of all primary cutaneous lymphomas (PCL). Previous studies revealed impaired health-related quality of life (HRQoL) in patients diagnosed with primary cutaneous T-cell lymphoma (CTCL). Currently, only small-sized studies investigated HRQoL in CBCL patients and lacked detailed analysis of respective subtypes. OBJECTIVES: This study aims to investigate HRQoL in CBCL patients to identify independent factors of HRQoL impairment in CBCL patients. METHODS: One hundred CBCL patients were recruited from eight German PCL centres in this multicentric, cross-sectional study from 2021 to 2022. The patients completed the dermatologic HRQoL questionnaire Skindex-29 and an investigator-designed 'CBCL-Questionnaire' with additional questions on HRQoL and clinical characteristics. RESULTS: The Skindex-29 revealed that HRQoL in CBCL patients is impaired on a mild to moderate level. The multiple regression analysis identified parameters like worries about dying, feeling prejudiced/discriminated and impairment of daily activities to be independently associated with impairment of HRQoL. Highest scores for HRQoL impairment were found in patients with primary cutaneous follicle centre lymphoma while on rituximab treatment and in patients with primary cutaneous marginal zone lymphoma while on watchful waiting. CONCLUSIONS: HRQoL is impaired in CBCL patients, even though, in the face of indolent disease course and favourable prognosis in the majority of cases. Of note, our investigator-designed tool identified worries about dying, feeling prejudiced/discriminated, and the type of treatment to have a negative impact on patients' HRQoL. Our study highlights the importance of a thorough patient-doctor communication to capture overall disease burden because generic HRQoL tools might lack of disease-specific items.

[Calprotectin-a key for diagnosing chronic inflammatory bowel disease in inflammatory dermatoses? : Three case reports]. / Calprotectin ­ ein Schlüssel zur Diagnose einer chronisch entzündlichen Darmerkrankung bei entzündlichen Dermatosen? : Drei Kasuistiken.

We report on three patients with inflammatory dermatoses as a result of a chronic inflammatory bowel disease (IBD). The diagnosis of IBD was based on the initial determination of the fecal calprotectin value. Although the patients did not report any intestinal complaints, the increased calprotectin value in the stool was the reason for further gastroenterological evaluation. This article highlights the importance of determining the fecal calprotectin value as another parameter in the diagnosis of inflammatory dermatoses.

Treatment of early-stage mycosis fungoides: results from the PROspective Cutaneous Lymphoma International Prognostic Index (PROCLIPI) study.

BACKGROUND: The PROspective Cutaneous Lymphoma International Prognostic Index (PROCLIPI) study is a prospective analysis of an international database. Here we examine front-line treatments and quality of life (QoL) in patients with newly diagnosed mycosis fungoides (MF). OBJECTIVES: To identify (i) differences in first-line approaches according to tumour-nodes-metastasis-blood (TNMB) staging; (ii) parameters related to a first-line systemic approach and (iii) response rates and QoL measures. METHODS: In total, 395 newly diagnosed patients with early-stage MF (stage IA-IIA) were recruited from 41 centres in 17 countries between 1 January 2015 and 31 December 2018 following central clinicopathological review. RESULTS: The most common first-line therapy was skin-directed therapy (SDT) (322 cases, 81·5%), while a smaller percentage (44 cases, 11·1%) received systemic therapy. Expectant observation was used in 7·3%. In univariate analysis, the use of systemic therapy was significantly associated with higher clinical stage (IA, 6%; IB, 14%; IIA, 20%; IA-IB vs. IIA, P < 0·001), presence of plaques (T1a/T2a, 5%; T1b/T2b, 17%; P < 0·001), higher modified Severity Weighted Assessment Tool (> 10, 15%; ≤ 10, 7%; P = 0·01) and folliculotropic MF (FMF) (24% vs. 12%, P = 0·001). Multivariate analysis demonstrated significant associations with the presence of plaques (T1b/T2b vs. T1a/T2a, odds ratio 3·07) and FMF (odds ratio 2·83). The overall response rate (ORR) to first-line SDT was 73%, while the ORR to first-line systemic treatments was lower (57%) (P = 0·027). Health-related QoL improved significantly both in patients with responsive disease and in those with stable disease. CONCLUSIONS: Disease characteristics such as presence of plaques and FMF influence physician treatment choices, and SDT was superior to systemic therapy even in patients with such disease characteristics. Consequently, future treatment guidelines for early-stage MF need to address these issues.

Should we be imaging lymph nodes at initial diagnosis of early-stage mycosis fungoides? Results from the PROspective Cutaneous Lymphoma International Prognostic Index (PROCLIPI) international study.

BACKGROUND: Early-stage mycosis fungoides (MF) includes involvement of dermatopathic lymph nodes (LNs) or early lymphomatous LNs. There is a lack of unanimity among current guidelines regarding the indications for initial staging imaging in early-stage presentation of MF in the absence of enlarged palpable LNs. OBJECTIVES: To investigate how often imaging is performed in patients with early-stage presentation of MF, to assess the yield of LN imaging, and to determine what disease characteristics promoted imaging. METHODS: A review of clinicopathologically confirmed newly diagnosed patients with cutaneous patch/plaque (T1/T2) MF from PROspective Cutaneous Lymphoma International Prognostic Index (PROCLIPI) data. RESULTS: PROCLIPI enrolled 375 patients with stage T1/T2 MF: 304 with classical MF and 71 with folliculotropic MF. Imaging was performed in 169 patients (45%): 83 with computed tomography, 18 with positron emission tomography-computed tomography and 68 with ultrasound. Only nine of these (5%) had palpable enlarged (≥ 15 mm) LNs, with an over-representation of plaques, irrespectively of the 10% body surface area cutoff that distinguishes T1 from T2. Folliculotropic MF was not more frequently imaged than classical MF. Radiologically enlarged LNs (≥ 15 mm) were detected in 30 patients (18%); only seven had clinical lymphadenopathy. On multivariate analysis, plaque presentation was the sole parameter significantly associated with radiologically enlarged LNs. Imaging of only clinically enlarged LNs upstaged 4% of patients (seven of 169) to at least IIA, whereas nonselective imaging upstaged another 14% (24 of 169). LN biopsy, performed in eight of 30 patients, identified N3 (extensive lymphomatous involvement) in two and N1 (dermatopathic changes) in six. CONCLUSIONS: Physical examination was a poor determinant of LN enlargement or involvement. Presence of plaques was associated with a significant increase in identification of enlarged or involved LNs in patients with early-stage presentation of MF, which may be important when deciding who to image. Imaging increases the detection rate of stage IIA MF, and identifies rare cases of extensive lymphomatous nodes, upstaging them to advanced-stage IVA2.

European dermatology forum: Updated guidelines on the use of extracorporeal photopheresis 2020 - Part 2.

BACKGROUND: Following the first investigational study on the use of extracorporeal photopheresis for the treatment of cutaneous T-cell lymphoma published in 1983, this technology has received continued use and further recognition for additional earlier as well as refractory forms. After the publication of the first guidelines for this technology in the JEADV in 2014, this technology has maintained additional promise in the treatment of other severe and refractory conditions in a multidisciplinary setting. It has confirmed recognition in well-known documented conditions such as graft-vs.-host disease after allogeneic bone marrow transplantation, systemic sclerosis, solid organ transplant rejection including lung, heart and liver and to a lesser extent inflammatory bowel disease. MATERIALS AND METHODS: In order to further provide recognized expert practical guidelines for the use of this technology for all indications, the European Dermatology Forum (EDF) again proceeded to address these questions in the hands of the recognized experts within and outside the field of dermatology. This was done using the recognized and approved guidelines of EDF for this task. All authors had the opportunity to review each contribution as it was added. RESULTS AND CONCLUSION: These updated 2020 guidelines provide at present the most comprehensive available expert recommendations for the use of extracorporeal photopheresis based on the available published literature and expert consensus opinion. The guidelines were divided into two parts: PART I covers Cutaneous T-cell lymphoma, chronic graft-vs.-host disease and acute graft-vs.-host disease, while PART II will cover scleroderma, solid organ transplantation, Crohn's disease, use of ECP in paediatric patients, atopic dermatitis, type 1 diabetes, pemphigus, epidermolysis bullosa acquisita and erosive oral lichen planus.

Smart local orbitals for efficient calculations within density functional theory and beyond.

Localized basis sets in the projector augmented wave formalism allow for computationally efficient calculations within density functional theory (DFT). However, achieving high numerical accuracy requires an extensive basis set, which also poses a fundamental problem for the interpretation of the results. We present a way to obtain a reduced basis set of atomic orbitals through the subdiagonalization of each atomic block of the Hamiltonian. The resulting local orbitals (LOs) inherit the information of the local crystal field. In the LO basis, it becomes apparent that the Hamiltonian is nearly block-diagonal, and we demonstrate that it is possible to keep only a subset of relevant LOs that provide an accurate description of the physics around the Fermi level. This reduces to some extent the redundancy of the original basis set, and at the same time, it allows one to perform post-processing of DFT calculations, ranging from the interpretation of electron transport to extracting effective tight-binding Hamiltonians, very efficiently and without sacrificing the accuracy of the results.

Clinical diversity and treatment approaches to blastic plasmacytoid dendritic cell neoplasm: a retrospective multicentre study.

BACKGROUND: Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare, aggressive type of haematologic precursor malignancy primarily often manifesting in the skin. We sought to provide a thorough clinical characterization and report our experience on therapeutic approaches to BPDCN. METHODS: In the present multicentric retrospective study, we collected all BPDCN cases occurring between 05/1999 and 03/2018 in 10 secondary care centres of the German-Swiss-Austrian cutaneous lymphoma working group. RESULTS: A total of 37 BPDCN cases were identified and included. Almost 90% of the patients had systemic manifestations (bone marrow, lymph nodes, peripheral blood) in addition to skin involvement. The latter presented with various types of cutaneous lesions: nodular (in more than 2/3) and bruise-like (in 1/3) skin lesions, but also maculopapular exanthema (in circa 1/6). Therapeutically, 22 patients received diverse combinations of chemotherapeutic regimens and/or radiotherapy. Despite initial responses, all of them ultimately relapsed and died from progressive disease. Eleven patients underwent haematopoietic stem cell transplantation (HSCT; autologous HSCT n = 3, allo-HSCT n = 8). The mortality rate among HSCT patients was only 33.33% with a median survival time of 60.5 months. CONCLUSION: Our study demonstrates the clinical diversity of cutaneous BPDCN manifestations and the positive development observed after the introduction of HSCT.

European dermatology forum - updated guidelines on the use of extracorporeal photopheresis 2020 - part 1.

BACKGROUND: Following the first investigational study on the use of extracorporeal photopheresis for the treatment of cutaneous T-cell lymphoma published in 1983, this technology has received continued use and further recognition for additional earlier as well as refractory forms. After the publication of the first guidelines for this technology in the JEADV in 2014, this technology has maintained additional promise in the treatment of other severe and refractory conditions in a multi-disciplinary setting. It has confirmed recognition in well-known documented conditions such as graft-versus-host disease after allogeneic bone marrow transplantation, systemic sclerosis, solid organ transplant rejection including lung, heart and liver and to a lesser extent inflammatory bowel disease. MATERIALS AND METHODS: In order to further provide recognized expert practical guidelines for the use of this technology for all indications, the European Dermatology Forum (EDF) again proceeded to address these questions in the hands of the recognized experts within and outside the field of dermatology. This was done using the recognized and approved guidelines of EDF for this task. All authors had the opportunity to review each contribution as it was added. RESULTS AND CONCLUSION: These updated 2020 guidelines provide at present the most comprehensive available expert recommendations for the use of extracorporeal photopheresis based on the available published literature and expert consensus opinion. The guidelines are divided in two parts: PART I covers cutaneous T-cell lymphoma, chronic graft-versus-host disease and acute graft-versus-host disease while PART II will cover scleroderma, solid organ transplantation, Crohn's disease, use of ECP in paediatrics practice, atopic dermatitis, type 1 diabetes, pemphigus, epidermolysis bullosa acquisita and erosive oral lichen planus.

The PROCLIPI international registry of early-stage mycosis fungoides identifies substantial diagnostic delay in most patients.

BACKGROUND: Survival in mycosis fungoides (MF) is varied and may be poor. The PROCLIPI (PROspective Cutaneous Lymphoma International Prognostic Index) study is a web-based data collection system for early-stage MF with legal data-sharing agreements permitting international collaboration in a rare cancer with complex pathology. Clinicopathological data must be 100% complete and in-built intelligence in the database system ensures accurate staging. OBJECTIVES: To develop a prognostic index for MF. METHODS: Predefined datasets for clinical, haematological, radiological, immunohistochemical, genotypic, treatment and quality of life are collected at first diagnosis of MF and annually to test against survival. Biobanked tissue samples are recorded within a Federated Biobank for translational studies. RESULTS: In total, 430 patients were enrolled from 29 centres in 15 countries spanning five continents. Altogether, 348 were confirmed as having early-stage MF at central review. The majority had classical MF (81·6%) with a CD4 phenotype (88·2%). Folliculotropic MF was diagnosed in 17·8%. Most presented with stage I (IA: 49·4%; IB: 42·8%), but 7·8% presented with enlarged lymph nodes (stage IIA). A diagnostic delay between first symptom development and initial diagnosis was frequent [85·6%; median delay 36 months (interquartile range 12-90)]. This highlights the difficulties in accurate diagnosis, which includes lack of a singular diagnostic test for MF. CONCLUSIONS: This confirmed early-stage MF cohort is being followed-up to identify prognostic factors, which may allow better management and improve survival by identifying patients at risk of disease progression. This study design is a useful model for collaboration in other rare diseases, especially where pathological diagnosis can be complex.

Occurrence of occult CSF leaks during standard FESS procedures.

OBJECTIVE: To determine the incidence of occult cerebrospinal fluid leaks (CSF) after functional endoscopic sinus surgery (FESS) and to evaluate the diagnostic performance of beta2-transferrin in blood-contaminated conditions. STUDY DESIGN: Prospective cohort study. METHODS: An analysis of 57 intraoperative samples using hydrogel 6 beta2-transferrin assay after FESS was undertaken. In case of CSF positive samples and continuing rhinorrhea, reanalysis after more than 1 year was conducted. In-vivo analysis of a primary spontaneous CSF leak sample took place to verify difficulties in detecting beta2-transferrin in blood-contaminated settings. Own titrations were performed to evaluate detection limits of CSF by beta2-transferrin and beta-trace protein assays in these settings. RESULTS: An incidence of 13% for occult CSF leaks after FESS was found. In blood-contaminated conditions, routine beta2-transferrin assays showed low sensitivity. In over 1 year follow-up, all samples were negative for CSF and none of them developed clinical relevant CSF leaks or meningitis. CONCLUSION: Occult and clinically irrelevant CSF leaks do occur in a significant proportion of patients during and shortly after FESS. Intra- and postoperatively, routine beta2-transferrin assays show low sensitivity. They should not be used in these settings. The clinical course of patients with occult CSF leaks indicated possibility of an uneventful follow-up.

Global patterns of care in advanced stage mycosis fungoides/Sezary syndrome: a multicenter retrospective follow-up study from the Cutaneous Lymphoma International Consortium.

BACKGROUND: Advanced-stage mycosis fungoides (MF)/Sézary syndrome (SS) patients are weighted by an unfavorable prognosis and share an unmet clinical need of effective treatments. International guidelines are available detailing treatment options for the different stages but without recommending treatments in any particular order due to lack of comparative trials. The aims of this second CLIC study were to retrospectively analyze the pattern of care worldwide for advanced-stage MF/SS patients, the distribution of treatments according to geographical areas (USA versus non-USA), and whether the heterogeneity of approaches has potential impact on survival. PATIENTS AND METHODS: This study included 853 patients from 21 specialist centers (14 European, 4 USA, 1 each Australian, Brazilian, and Japanese). RESULTS: Heterogeneity of treatment approaches was found, with up to 24 different modalities or combinations used as first-line and 36% of patients receiving four or more treatments. Stage IIB disease was most frequently treated by total-skin-electron-beam radiotherapy, bexarotene and gemcitabine; erythrodermic and SS patients by extracorporeal photochemotherapy, and stage IVA2 by polychemotherapy. Significant differences were found between USA and non-USA centers, with bexarotene, photopheresis and histone deacetylase inhibitors most frequently prescribed for first-line treatment in USA while phototherapy, interferon, chlorambucil and gemcitabine in non-USA centers. These differences did not significantly impact on survival. However, when considering death and therapy change as competing risk events and the impact of first treatment line on both events, both monochemotherapy (SHR = 2.07) and polychemotherapy (SHR = 1.69) showed elevated relative risks. CONCLUSION: This large multicenter retrospective study shows that there exist a large treatment heterogeneity in advanced MF/SS and differences between USA and non-USA centers but these were not related to survival, while our data reveal that chemotherapy as first treatment is associated with a higher risk of death and/or change of therapy and thus other therapeutic options should be preferable as first treatment approach.

Brentuximab vedotin in CD30+ cutaneous lymphoma: How do we treat, how shall we treat? A review of the literature.

Brentuximab vedotin is an antibody-drug conjugate that brings the antimicrotubule agent monomethyl auristatin E into CD30-expressing cells. Some prior studies demonstrated good efficacy in cutaneous lymphomas. The standard therapeutic scheme is 1·8 mg kg-1 every 3 weeks. The background of this work is the fact that cutaneous lymphoma has a different pathophysiology and a dynamic other than systemic lymphomas. The objectives of this review were to get an overview of the currently used therapeutic regimen, and to check whether dose reduction or modified time intervals could be of benefit in a similar way with less toxicity. Therefore, we conducted a systematic review of the literature indexed in PubMed and the Cochrane Central Register of Controlled Trials up to April 2016. The procedure was based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) criteria. The review showed that the currently used therapeutic regimen is 1·8 mg kg-1 every 3 weeks. No publications of dose-finding studies in CD30+ cutaneous T-cell lymphoma (CTCL) were found. Two cases of patients, treated with a dose < 1·8 mg kg-1 , have been published. Brentuximab vedotin seems to be a powerful treatment option in refractory CD30+ CTCL, and there is a trend that dose reductions, as well as prolonged treatment intervals, work without any loss of response and with fewer side-effects.

Dose-escalation study evaluating pegylated interferon alpha-2a in patients with cutaneous T-cell lymphoma.

BACKGROUND: This open-label, multicenter, dose-escalation study evaluated the safety, tolerability, and efficacy of subcutaneous pegylated (40 kD) interferon α-2a (PEG-IFN α-2a) in patients with cutaneous T-cell lymphoma (CTCL). PATIENTS AND METHODS: PEG-IFN α-2a was administered subcutaneously at 180 (n = 4), 270 (n = 6), or 360 µg (n = 3) once weekly for 12 weeks. Efficacy was assessed by the proportion of patients with complete response (CR) or partial response (PR). RESULTS: PEG-IFN α-2a was generally well tolerated, with a moderate number of reductions or withholding of doses because of adverse events (AEs) (25% (n = 1), 66% (n = 4), and 0% (n = 0) in the 180-, 270-, and 360-µg/week groups, respectively). The only dose-limiting toxicity was a grade 3 elevation of liver enzymes in the 270-µg dose group. The most common AEs were fatigue, acute flu-like symptoms, and hepatic toxicity. The major response rate (CR or PR) was 50% in the 180-µg group (CR, 50%; PR, 0%), 83% in the 270-µg group (CR, 67%; PR, 17%), and 66% in the 360-µg group (CR, 33%; PR, 33%). CONCLUSION: PEG-IFN α-2a at doses up to 360 µg once weekly was well tolerated in patients with CTCL up to the highest dose group and showed good response rates. Due to their good tolerance even in high doses, they might be an option for patients not tolerating standard IFN-α preparations. However, for this purpose and to evaluate comparability between standard and PEG-IFN larger clinical trials are needed, alone and in combination with oral photochemotherapy (PUVA).

[WHO classification and clinical spectrum of cutaneous lymphomas]. / WHO-Klassifikation und klinisches Spektrum der kutanen Lymphome.

BACKGROUND: Cutaneous lymphomas are rare skin cancers with a wide clinical spectrum. OBJECTIVE: To give an overview of the current classification and the clinical spectrum of cutaneous lymphomas. MATERIAL AND METHODS: Analysis and summary of the current literature concerning the different entities of cutaneous lymphomas. RESULTS: A few modifications in the nomenclature of cutaneous lymphoma have been introduced in the revised version of the WHO classification 2016. In the last years new types of lymphomatoid papulosis were described. Moreover, two subgroups of folliculotropic mycosis fungoides are now mentioned. The CD4+ small/medium T­cell lymphoproliferative disorder is no longer classified as an overt lymphoma. As new entities the EBV+ mucocutaneous ulcer, an EBV-associated diffuse large B­cell lymphoma (not otherwise specified) and a primary cutaneous acral CD8+ T­cell (provisional) lymphoma are being considered. CONCLUSION: The classification of cutaneous lymphomas is based on the revised version of the WHO classification (2016). The clinical pathological correlation is an elementary component for correct diagnosis.

Phase II multicentre trial of oral quisinostat, a histone deacetylase inhibitor, in patients with previously treated stage IB-IVA mycosis fungoides/Sézary syndrome.

BACKGROUND: Quisinostat is a hydroxamate, second-generation, orally available pan-histone deacetylase inhibitor. OBJECTIVES: To evaluate the efficacy and safety of oral quisinostat in patients with previously treated cutaneous T-cell lymphoma (CTCL). METHODS: Patients received quisinostat 8 mg or 12 mg on days 1, 3 and 5 of each week in 21-day treatment cycles. Primary efficacy end point was cutaneous response rate (RR) based on the modified Severity Weighted Assessment Tool (mSWAT). Secondary end points included global RR, duration of response (DOR) in skin, progression-free survival (PFS), pruritus relief, safety and pharmacodynamic markers. RESULTS: Eight of 26 (25 evaluable) patients achieved ≥ 50% reduction in mSWAT score at least once, with confirmed cutaneous response in six (RR 24%). There was a low global RR of 8%. DOR in skin ranged from 2·8 to 6·9 months. Median PFS was 5·1 months. Pruritus relief was more frequent in cutaneous responders (67%) than nonresponders (32%). Serial tumour biopsies revealed an increase in acetylated tubulin, indicating a target effect of histone deacetylase 6. Twenty-one of 26 (81%) patients were withdrawn from the study before or at clinical cut-off; five (19%) continued to receive treatment with quisinostat. The most common drug-related adverse events were nausea, diarrhoea, asthenia, hypertension, thrombocytopenia and vomiting. Grade 3 drug-related adverse events included hypertension, lethargy, pruritus, chills, hyperkalaemia and pyrexia. CONCLUSIONS: Quisinostat 12 mg three times weekly is active in the treatment of patients with relapsed or refractory CTCL, with an acceptable safety profile. Combination therapy with other drugs active in CTCL may be appropriate.

[HIV window period. Acute retroviral syndrome]. / Diagnostische Lücke. Akutes retrovirales Syndrom.

A 24-year-old man with no significant medical history was admitted to this hospital because of an acute exanthema, myalgia and malaise. A fourth-generation screening test for HIV was reactive but the following confirmation test was not. Although the patient did not belong to a high risk group, we decided to perform a HIV-RNA-PCR. The result showed a high virus load so that we finally diagnosed a primary HIV infection.