Clinical and diagnostic findings in COVID-19 patients: an original research from SG Moscati Hospital in Taranto Italy.

The coronavirus disease 2019 (COVID-19) pandemic is a worldwide medical challenge due to the scarcity of proper information and remedial resources. The ability to efficiently avoid a further SARS-CoV-2 pandemic will, therefore, depend on understanding several factors which include host immunity, virus behavior, prevention measures, and new therapies. This is a multi-phase observatory study conducted in the SG Moscati Hospital of Taranto in Italy that was converted into COVID-19 Special Care Unit for SARS-Co-V2 risk management. Patients were admitted to the 118 Emergency Pre-Hospital and Emergency Department based on two diagnostic criteria, the nasopharyngeal swab assessed by reverse-transcriptase-polymerase-chain-reaction (RT-PCR) and CT-scan image characterized by ground glass opacity. Patients were divided into four groups, positive-positive (ER-PP), negative-positive (ER-NP), negative-negative (ER-NN) and a group admitted to the ICU (ER-IC). A further control group was added when the T and B lymphocyte subsets were analyzed. Data included gender, age, vital signs, arterial blood gas analysis (ABG), extensive laboratory results with microbiology and bronchoalveolar lavage fluid (BALF) which were analyzed and compared. Fundamental differences were reported among the groups. Males were significantly higher in PP, ICU, and NP groups, from 2 to 4-fold higher than females, while in the NN group, the number of females was mildly higher than males; the PP patients showed a marked alkalotic, hypoxic, hypocapnia ABG profile with hyperventilation at the time of admission; finally, the laboratory and microbiology results showed lymphopenia, fibrinogen, ESR, CRP, and eGFR were markedly anomalous. The total number of CD4+ and CD8+ T cells was dramatically reduced in COVID-19 patients with levels lower than the normal range delimited by 400/µL and 800/µL, respectively, and were negatively correlated with blood inflammatory responses.

Combining immunofluorescence with immunoblot assay improves the specificity of autoantibody testing for myositis.

OBJECTIVE: Immunoblot (IB) methods are widely used to detect myositis-specific autoantibodies (MSAs); however, false-positive results are common. In this study, we aimed to determine whether associating the anti-nuclear antibody (ANA) IIF pattern may help to improve the specificity of MSA detection by IB in patients with idiopathic inflammatory myositis (IIM). METHODS: Serum samples from 104 patients presenting with muscle weakness/myalgia and positive to at least one MSA by IB (MYOS12 Diver and MIOS7 Diver, D-tek) were tested for ANAs on HEp-2000 cells (Immuno Concepts). The chi-square test was used to analyse the concordance of the MSA result and its corresponding pattern by ANA testing between patients with and without IIM. RESULTS: Eighty-three of the 104 patients had a diagnosis of definite IIM, while in 21 cases, patients were affected by other autoimmune diseases or various non-systemic diseases. Forty nine of 83 (59%) patients in the IIM group and 4/21 (19%) in the non-IIM group showed a concordance between ANA pattern and MSAs by IB (P < 0.001). MSA monopositivity was significantly associated with IIM (91.6%) compared with 61.9% in the non-IIM group (P = 0.0005). CONCLUSIONS: Considering both the MSA result and its corresponding pattern by ANA testing may help to improve the specificity of MSA detection by IB and to confirm the diagnosis of MSA-associated IIM. The monopositivity of MSAs is an important additional tool to validate IB results.

An effective algorithm for the serological diagnosis of idiopathic inflammatory myopathies: The key role of anti-Ro52 antibodies.

BACKGROUND: Patients with suspected idiopathic inflammatory myopathies (IIM) are commonly tested for the presence of anti-nuclear antibodies (ANA) by indirect immunofluorescence (IIF) on HEp-2 cell substrates. However, ANA-IIF false negative tests may occur in IIM because some antigens, such as Jo1 and Ro52, may be scarcely expressed on HEp-2 cells. In addition, cytoplasmic staining is often not appropriately investigated by a specific antibody assay, leading to decreased clinical sensitivity of the ANA test. We evaluated the diagnostic impact of different strategies using different combination of myositis-related autoantibody tests. METHODS: Sera from 51 patients with an established diagnosis of IIM were tested for ANA by IIF on HEp-2 cells and for myositis-specific antibodies (MSA) and myositis-associated antibodies (MAA) by lineblot methods. RESULTS: Forty-four/51 (86.3%) samples tested positive with at least one of the three methods and seven were negative with all methods. Of the 44 positive samples, 9 (20.5%) tested negative for the ANA-IIF test and positive for MAA/MSA. Anti-Ro52 were the most prevalent autoantibodies in IIM patients (21/51; 41%), frequently associated with anti-Jo1 antibodies (13/21; 62%). 13 (16%) anti-Ro52 and anti-Jo1 negative samples were reactive to MSA. CONCLUSIONS: Our findings suggest that when IIM is clinically suspected, the optimal diagnostic algorithm is to associate the ANA-IIF screening test with a specific test for anti-Ro52 and anti-Jo1 antibodies. Should all these tests be negative, serological tests for MSA are recommended.

Improvement of renal function in epidermolysis bullosa patients after gluten free diet: two cases.

Epidermolysis bullosa (EB) is a rare inherited genetic disease characterized by an abnormal response of the skin and mucosa to mechanical trauma. Dystrophic EB (DEB) is very often associated with many extra cutaneous complications. Those complications involve either epithelial associated tissues or other organs. In particular, several renal complications have been described for DEB in the recessive form, such as amyloidosis, post-infection glomerulonephritis, upper and lower urinary tract obstruction and IgA-Nephropathy (IgAN). In the cases reported below we have two patients diagnosed with DEB that showed compromised renal function and proteinuria. The switch of the normal diet toward a gluten free diet resulted beneficial for both patients, since renal function was rescued and proteinuria cured. Moreover, a general health status improvement was recognised, given that nutritional condition was ameliorated and bone growing enhanced. Furthermore, in both patients the presence of autoantibodies anti-COL7 indicating an autoimmune form of the disease. Therefore, patients received low doses of betametasone useful to reduce inflammatory state and to control immune system function. In conclusion, our results prompt us to hypothesized that in these patients, due to the fragility of the intestinal mucosa, the absence in the diet of gluten may be beneficial.

Multicentric evaluation of a second generation assay to detect antiviral citrullinated peptide antibodies: a collaborative study by the Forum Interdisciplinare per la Ricerca nelle Malattie Autoimmuni.

AIMS: A novel immunoenzymatic assay using viral citrullinated peptides derived from Epstein-Barr virus-encoded proteins (viral citrullinated peptide 2 (VCP2)) has been developed and evaluated by means of a multicentre collaborative study. METHODS: Three hundred nine sera from patients with established rheumatoid arthritis (RA), 36 with early arthritis, 12 with juvenile arthritis and 453 controls were tested for VCP2 and cyclic citrullinated peptide (CCP) antibodies. RESULTS: The VCP2 assay showed 78.3% sensitivity and 97.1% specificity. VCP2 and CCP had a high concordance rate in patients with RA (88%) and controls (97%). However, 36 RA sera were positive in the CCP assay but negative on VCP2, and two RA sera reacted only on VCP2. CONCLUSIONS: The new VCP2 assay is endowed with high sensitivity and specificity. VCP2-positive RA sera are mostly but not completely contained in the CCP-positive population. Studies are in progress to establish whether the VCP2 assay can detect clinically distinct subsets of patients with RA.

Diagnostic accuracy of different immunological methods for the detection of antineuronal antibodies in paraneoplastic neurological syndromes.

The aim of this work was to evaluate the diagnostic accuracy of three different analytical methods for the detection of antineuronal antibodies and outline how they might be used to diagnose Paraneoplastic Neurological Syndromes (PNS) in a more effectively and rationally way. One hundred and four patients with neurological diseases were studied: 38 with paraneoplastic neurological disorder, 44 with other neurological diseases, and 22 with systemic autoimmune diseases and neurological disorders. 20 healthy subjects and 18 subjects with tumour without neurological disorders were also studied. Antineuronal antibodies were tested using three methods: Western blot (WB); Line-blot (LB); and indirect immunofluorescence (IIF) on primate cerebellum. The diagnostic sensitivity of the IIF, WB and LB methods was 28.9%, 26.3% and 36.8%, respectively, and their specificity was 95.2%, 97.1% and 98.1% respectively. The combined use of the three methods brought the sensitivity to 39.4%. The results of this study show that the methods used in clinical laboratories for the detection of antineuronal antibodies have good specificity. Among the three methods assessed, LB showed the highest diagnostic accuracy and also allowed for recognition of fine antibody specificities. According to these results we can suggest that LB should be used as the method of choice to search for paraneoplastic antibodies.

[Accuracy and standardization of diagnostic methods for the detection of antibodies to citrullinated peptides]. / Accuratezza diagnostica e standardizzazione dei metodi per la determinazione degli anticorpi anti-peptidi citrullinati.

Anti-citrullinated peptide antibodies (ACPA) have a very high specificity for rheumatoid arthritis, much more than that of the rheumatoid factor. In addition, ACPA can be found in sera in the pre-clinical phase, are associated with more severe joint destruction and with higher disease activity. In recent years, keeping pace with new knowledge and with progress made in the antigenic composition of tests and in the characterization of immunogenic epitopes, many immunoenzymatic (ELISA) methods of second and third generation have been produced and marketed commercially, and their use has spread among clinical laboratories. Today, completely automated methods are also available, which are easy to use and with a higher throughput, rendering the diagnostic utility of testing ever faster and more effective. This review takes into consideration the more important characteristics of the new ACPA-ELISA tests now commercially available, and also considers recent progress in standardizing test results.

Pemphigus vulgaris with circulating anti-desmoglein 3 and anti-BP180 antibodies: a case report and brief review of cases with coexistence of pemphigus vulgaris and bullous pemphigoid.

Pemphigus vulgaris (PV) and bullous pemphigoid (BP) are two autoimmune blistering diseases involving the skin and the mucous membranes characterized by circulating autoantibodies directed against desmosomal cadherins or antigens expressed in the basement membrane zone, respectively. The simultaneous presence of clinical and/or immunopathological features of PV and BP in the same patient has been reported in very few cases in the literature to date. Most of these cases had exclusive cutaneous involvement, while a minority showed concomitant oral lesions. We describe the case of a 59-year-old female patient with a 10-year history of refractory PV lesions limited to mucous membranes (conjunctiva, oral cavity and genital mucosa), which were controlled by the addition of mycophenolate sodium to oral prednisone. Immunofluorescence studies revealed findings consistent with PV, whereas enzyme-linked immunosorbent assay revealed circulating anti-BP180 antibodies in association with anti-desmoglein 3 antibodies. The significance and relevance of this finding are briefly discussed, in light of the literature data.

Accuracy of the first fully automated method for anti-cardiolipin and anti-beta2 glycoprotein I antibody detection for the diagnosis of antiphospholipid syndrome.

In this study we evaluated the diagnostic accuracy for antiphospholipid syndrome (APS) of new fully automated immunoassays for anticardiolipin (aCL) and anti-beta2 glycoprotein I (anti-beta2-GPI) auto-antibody detection (EliA-Phadia), and compared the results with those obtained with Orgentec and Inova ELISA methods. Sixty-two APS patients and 123 controls (20 syphilis, 33 Lyme disease, 30 HCV infection and cryoglobulinemia, 40 healthy subjects) were studied. Using the 99(th) percentile cutoff, the sensitivity and specificity of EliA aCL IgG, aCL IgM, anti-beta2-GPI IgG, and anti-beta2-GPI IgM were 69.4% and 81.9%, 64.5% and 86.7%, 64.5% and 98.8%, and 53.2% and 92.8%, respectively. Using the Sydney criteria cutoff (>40 GPL/MPL units), sensitivity and specificity of EliA aCL IgG and aCL IgM were 45.2% and 98.8%, and 35.5% and 97.5%, respectively. The best diagnostic efficiency was obtained combining the aCL tests (>40 GPL/MPL units) with the anti-beta2-GPI tests (sensitivity 85.7%, specificity 90.4%). The area under the ROC curves for EliA, Orgentec, and Inova methods were 0.870, 0.940, and 0.850 for aCL IgG; 0.820, 0.820, and 0.820 for aCL IgM; 0.910, 0.960, and 0.920 for anti-beta2-GPI IgG; 0.840, 0.840, and 0.820 for anti-beta2-GPI IgM, respectively. Finally, the overall agreement between EliA assays and the other two ELISA methods ranged from moderate (anti-beta2-GPI IgG EliA versus Orgentec: Cohen's k = 0.426) to good (anti-beta2-GPI IgM EliA vs. Inova: k = 0.841). In conclusion, newly developed EliA methods for antiphospholipid antibody detection perform similarly to other ELISA assays and represent a useful tool for APS laboratory diagnosis in daily practice.

Diagnostic accuracy of IgA anti-tissue transglutaminase antibody assays in celiac disease patients with selective IgA deficiency.

Clinical studies have estimated a 10- to 20-fold increased risk for celiac disease (CD) in patients with selective IgA deficiency (SIgAD). For this reason, screening for CD is mandatory in SIgAD patients, but it represents a special challenge since the specific IgA class antibodies against gliadin (AGA), endomysium (EMA), and tissue-transglutaminase (tTG) are not produced in patients with CD. IgG class counterparts of these antibodies may be informative; in particular IgG EMA has been demonstrated to be a valid marker for diagnosing CD in SIgAD cases, but it is not used much in clinical laboratories, because it is cumbersome and involves some technical difficulties. Even if it was widely used in clinical laboratories, the measuring of IgG AGA has shown a less-than-optimum diagnostic accuracy, so that now it tends to be substituted by tests for anti-tTG IgG, for which the few available studies have shown diagnostic performances superior to AGA. Since it is not known whether various available methods for measuring IgG anti-tTG antibodies offer similar diagnostic performances, we have compared the results obtained from nine second-generation commercial methods (D-tek, Phadia, Immco, Orgentec, Radim, Euroimmun, Inova, Aesku, Generic Assays), measuring IgG anti-tTG antibodies in 20 patients with CD and SIgAD and in 113 controls (9 patients with SIgAD without CD, 54 patients with chronic liver disease, and 50 healthy individuals). Diagnostic sensitivity, calculated by means of ROC plot analysis, ranged between 75% and 95%, and specificity ranged from 94% to 100%. In the same population, the diagnostic sensitivity and specificity of AGA IgG were 40% and 87%, respectively. Even though they perform differently, all IgG anti-tTG methods evaluated are reliable serological assays for the diagnosis of CD in SIgAD patients, with diagnostic accuracy superior to the AGA IgG method. The methods that use a mix of tTG and gliadin peptides as the antigenic preparation have a specificity slightly lower than that of the methods that use only tTG.

[Prevalence of anti-CCP antibodies in systemic sclerosis]. / Prevalenza degli anticorpi anti-peptide ciclico citrullinato (anti-CCP) nella sclerosi sistemica.

Joint involvement in systemic sclerosis (SSc) commonly occurs as arthralgias, while a true arthritis is less frequent. The most common arthritis developing in SSc is rheumatoid arthritis (RA) and its diagnosis may be misled by concomitant joint contracture or tendon sheath involvement due to SSc. Anti-citrullinated cyclic peptide (CCP) antibodies are an emerging tool to diagnose RA and have shown to be more specific than rheumatoid factor. We assessed the prevalence of anti-CCP antibodies in SSc patients and evaluated their sensitivity and specificity for associated RA. Searching for RF and anti-CCP antibodies and joint examination were carried out in sixty consecutive SSc patients. Hands and feet standard x-rays were performed in patients complaining with arthralgia and/or arthritis. Six out of sixty (10%) SSc patients had RA according to 1987 ARA revised criteria. Anti-CCP were detected in 5 patients (sensitivity 83%) and RF was present in all RA patients (sensitivity 100%). However, anti-CCP antibodies had a much higher specificity (94%) than RF (41%) for RA. Our study suggests that anti-CCP antibodies are a useful test to identify patients with SSc having also RA. This is crucial in the management of SSc because may allow an adequate therapy of RA and prevent further joint damage in patients who already have a poor quality of life.

Low specificity of anti-tissue transglutaminase antibodies in patients with primary biliary cirrhosis.

The association between celiac disease (CD) and primary biliary cirrhosis (PBC) is well documented in medical literature; however, a high frequency of false positive results of the anti-transglutaminase (anti-tTG) test has been reported in patients with PBC. To verify if the positive results for anti-tTG autoantibody are false positives due to cross reactivity with mitochondrial antigens, we studied 105 adult patients affected with PBC, positive for anti-mitochondrial M2 antibodies. Anti-tTG IgA antibodies were studied by using six different immunoenzymatic assays that employ the tTG antigen obtained from different sources (human recombinant, placenta, red blood cells, and guinea pig liver). On the whole, 28 out of 105 PBC subjects tested positive for anti-tTG IgA antibodies, but only two were eventually found to be affected by CD; the other 26 were shown to be false positive. The specificity of the various antigenic substrates ranged from 88.5% of the human erythrocytes tTG to 97.1% of the human recombinant tTG. The results of this study showed that a true association between PBC and CD was present in only 2% of the patients and that, in most cases, the false positive results were attributable to the type of substrate utilized in the assay.

Accuracy of semiquantitative immunoenzymatic methods in quantitation of anti-topoisomerase I (Scl-70) antibodies.

Reports of a possible correlation between anti-Scl-70 antibody concentration and clinical manifestations in systemic sclerosis patients have recently appeared in the scientific literature. The goal of our study was to evaluate, by means of a multicenter study, the analytical reliability of immunoassay systems in the quantitative measurement of Scl-70 antibodies. Three blind samples (H, M, L) at different anti-Scl-70 antibody concentrations, and a low concentration antibody serum (LPC) used as a common calibrator, were sent three times in a 6-month time span to 39 Italian clinical laboratories. Each laboratory was asked to calculate dosages following the enzyme-linked immunosorbent assay (ELISA) method they used and report the optical density values of each sample (ODs), of the cutoff serum provided by the manufacturer of the kit used (ODco) and of LPC (ODLPC). The overall analytical imprecision (between methods and between laboratories) of the three different determinations of the values respectively expressed in ODs, ODs/ODco and ODs/ODLPCratio was 47.1, 52.8 and 34.0% for sample H, 56.2, 47.4% and 34% for sample M and 84.6, 86.0 and 86.6% for sample L. The average intra-method analytical imprecision was, respectively, 20.7, 29.8 and 18.6% for sample H, 24.6, 26.5 and 19.3% for sample M, and 30.6, 28.1 and 20.2% for sample L. The commercial ELISA methods currently used to determine the presence of anti-Scl-70 autoantibodies show considerable differences in the quantitative determination. The best results for reproducibility analyses have been obtained when the values were expressed as a ratio between the ODs of the sample and of the common calibrator (ODs/ODLPC). Forward-looking clinical studies that can clarify the usefulness of quantitative determination of anti-Scl-70 antibodies in the monitoring of diffuse scleroderma patients can be performed only when standard serum with a known antibody concentration and calibration curves for quantitative ELISA measurements are made available.

Proteomic: new advances in the diagnosis of rheumatoid arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic autoimmune disease affecting the joints. A number of novel treatment modalities have been introduced over the past years, and rheumatologists are now attempting to institute optimal treatment in recent-onset arthritis. To facilitate diagnosis during the early stages of disease, when often not all clinical symptoms are manifest, a good serological marker is needed. METHODS: Antibodies directed to citrullinated proteins provide this ability. The most sensitive assay for detecting these antibodies is the so-called anti-cyclic citrullinated peptide, second generation (CCP II) enzyme-linked immunosorbent assay (ELISA). RESULTS: The diagnostic and prognostic potential of anti-CCP antibodies and the availability of a fully automated assay method lead us to conclude that the test is satisfactory for routine use as a serological marker of RA. In addition, we consider the potential of multiplex autoantibody assays, including miniaturized, high-throughput microarray technology, to improve diagnosis and prognostication in early onset arthritis patients.

Total testing process: appropriateness in laboratory medicine.

BACKGROUND: The need to reduce costs in Laboratory Medicine is often related to the possibility of reducing test requests without taking into account patients' outcomes. Therefore, the term "appropriateness" in Laboratory Medicine as referred to the specific steps (pre-analytical, analytical, post-analytical) and related to the clinical process could allow the improvement of clinical effectiveness and economic efficiency. METHODS: Our experience has shown an improvement in analytical appropriateness (reorganization and re-engineering by Laboratory automation) and pre-analytical appropriateness (critical revision of the panel for cardiac markers) by evaluating the workload and errors rate in the pre-analytical phase. RESULTS: We obtained an economic saving (119,580 euro/year) in cardiac markers request (analytical appropriateness: 60%, pre-analytical appropriateness: 40%) and also an improvement in clinical appropriateness (diagnosis and therapy). CONCLUSIONS: Our data confirm the need to improve communications between physicians and Laboratory Medicine as regards the pre-analytical step and to implement educational programs for defining criteria and procedures. Appropriateness in analytical and post-analytical steps contribute to achieve economic saving (Core lab, POCT) and improvement of the turn-around time (TAT).

Application of a diagnostic algorithm in autoantibody testing: assessment of clinical effectiveness and economic efficiency.

BACKGROUND: Guidelines aim to assist physicians about appropriate health care for specific clinical circumstances. Therefore, they must be continuously updated, integrated and tailored to local situations. METHODS: We applied recently developed guidelines for autoantibody testing by assessing their economic (efficiency) and clinical (effectiveness) impact. Since June 2002, a test order algorithm has been adopted for autoantibody testing requests (3258). In particular, the guidelines were modified taking into account the needs of different departments and the results were compared to those (2762) of the previous period (January-May 2002) that had not been integrated with any diagnostic algorithm. RESULTS: A significant reduction in the number of anti-double stranded DNA (anti-dsDNA) (21.4%) and anti-Extractable Nuclear Antigens (anti-ENA) (19%) was found (p<0.0001), while the number of anti-nuclear antibody (ANA) test was unchanged (p=n.s.); further reduction in clinically inappropriate test request rates (23%) was observed. CONCLUSIONS: The application of guidelines allowed the improvement of diagnostic tests' efficiency and clinical effectiveness (patient's outcomes), thus confirming the need to apply eventual modifications to the diagnostic process taking into consideration different clinical needs.

IgA and IgG tissue transglutaminase antibody prevalence and clinical significance in connective tissue diseases, inflammatory bowel disease, and primary biliary cirrhosis.

An association between celiac disease (CD) and other autoimmune diseases such as connective tissue diseases (CTD), inflammatory bowel diseases (IBD), and primary biliary cirrhosis (PBC) has been reported in several studies. However, a high rate of false positives in autoantibody testing was noted, especially when tissue transglutaminase (tTG) from guinea pig liver was used. Thus, the real prevalence of CD in CTD, IBD, and PBC is unclear. In a case-control study, 400 patients with CTD, 170 with IBD, 48 with PBC, and 120 healthy subjects were investigated for CD by the analysis of IgA and IgG tTG antibodies using the more specific human recombinant tTG immunoenzymatic assay. Patients and controls with positive findings were further tested for antiendomysial antibodies by indirect immunofluorescence and HLA typing, and those found positive by either of these tests underwent duodenal biopsy to confirm a possible diagnosis of CD. Twelve patients were positive for IgA or IgG tTG antibodies, showing an overall prevalence of 1.9%. Only 1 healthy subject (0.8%) had a low level positive reaction for IgA anti-tTG. Among the 12 patients and the healthy subject, only 2 (1 SLE and 1 ulcerative colitis patient) were subsequently confirmed to be affected with CD by positive EMA, HLA, and small bowel biopsy findings. The highest rate of false positives was found in PBC patients (10.4%). For these reasons, serological screening testing for CD is not recommended in CTD patients or in subjects affected with IBD or PBC, unless there is a relevant clinical suspicion of CD.

Maxillary brown tumor in secondary hyperparathyroidism requiring urgent parathyroidectomy.

Brown tumors are unusual but serious complications of renal osteodystrophy, and can be successfully treated by parathyroidectomy or by pharmacological treatment of hyperparathyroidism. Brown tumors in patients with severe hyperparathyroidism (HPT) secondary to renal failure have been increasingly reported. We describe an unusual case of brown tumors at the maxillary bone and the seventh right rib, in a 57-year old man with a long history of hemodialysis. The maxillary lesion caused serious local discomfort due to its rapid growth. In this setting, surgical total parathyroidectomy was chosen as the most adequate therapeutic approach, given the previous unsatisfactory response to calcitriol. After successful parathyroidectomy, rapid healing was achieved with sclerosis of both brown tumors, as documented by serial computerized tomograms. In conclusion, although vitamin D therapy has been beneficial in several cases of secondary hyperparathyroidism complicated by brown tumors, we propose that whenever regression of the tumor bulk is urgently needed, as in our case, parathyroidectomy should be the first treatment choice.