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Serum aminoterminal type III procollagen peptide (sPIIIP) has been proposed as an index of hepatic fibroplasia. sPIIIP was retrospectively evaluated in 34 treated and five untreated patients affected by chronic active hepatitis with or without cirrhosis by an RIA test. Serum samples taken before and after 6 months of treatment were tested in all cases. In 15 of the treated and all untreated patients, 6-20 (median 13) sera, corresponding to a median follow-up of 43 months were studied. Before treatment, the sPIIIP median value was 18.6 ng/ml; after 6 months of treatment, it decreased to 13.6 ng/ml (p less than 0.005). Follow-up sPIIIP levels were significantly lower in treated than in untreated patients (p less than 0.05), at each interval considered, except for the last control (39 months). In seven patients, treatment was discontinued: sPIIIP rose rapidly in six; four of them were retreated and this was followed by a new decrease. In four patients, sPIIIP was tested weekly from the onset of the treatment: it reverted to normal values within the first week in all cases, while GOT decreased later. sPIIIP is significantly and rapidly reduced by steroids. Steroid withdrawal is generally followed by a rebound, with a new decrease when treatment is restarted. Since sPIIIP is more rapidly lowered than GOT levels, the above data support the hypothesis that steroids can directly affect collagen metabolism.

Summary Background Serum antinuclear antibodies giving the ‘multiple nuclear dots’ or the ‘rim-like/membranous’ patterns are frequently detected by indirect immunofluorescence on HEp-2 cells in patients with primary biliary cirrhosis. Aim To assess the accuracy of multiple nuclear dot and rim-like/membranous antinuclear antibodies for the diagnosis of primary biliary cirrhosis. Methods Sera from 4371 consecutive patients referred to our laboratory were analysed under code for antinuclear antibodies testing by indirect immunofluorescence on HEp-2 cells. Results Review of the clinical records of the 4371 patients allowed identification of 101 patients with antimitochondrial antibody-positive primary biliary cirrhosis and 22 with antimitochondrial antibody-negative variant. Multiple nuclear dot and/or rim-like/membranous patterns were found in 59 (1.3%) of the 4371 patients: 31 antimitochondrial antibody-positive primary biliary cirrhosis, 17 antimitochondrial antibody-negative primary biliary cirrhosis and 11 non-primary biliary cirrhosis. The specificity for primary biliary cirrhosis of both the antinuclear antibodies pattern was 99%. Positive predictive value and likelihood ratio for a positive test were 86% (95% CI: 72.7–94) and 221 (95% CI: 91.7–544) for multiple nuclear dot, 79% (95% CI: 62.2–90.1) and 132 (95% CI: 56.8–312.7) for rim-like/membranous, respectively. Conclusions Multiple nuclear dot and rim-like/membranous antinuclear antibodies are rare findings. Their positivity strongly suggests the diagnosis of primary biliary cirrhosis, irrespective of antimitochondrial antibody status. The high specificity for primary biliary cirrhosis makes them a useful diagnostic tool especially in antimitochondrial antibody-negative patients.

Background. The frequency of apoptosis in bile duct cells of primary biliary cirrhosis is still unclear, spanning from rare to 50% in the various reports. Aim. To study bile duct cell apoptosis in stage I primary biliary cirrhosis lesions. Patients. Nine stage I-II biopsies with a total number of 26 bile ducts of different sizes, selected from a larger series on the basis of the expression on serial frozen sections of HLA-DR and Fas antigens. Methods. Apoptosis was evaluated by a DNA fragmentation assay on frozen sections, according to the manufacturer's protocol and by expression of apoptosis related cytokeratin neoepitopes. Bile duct cell proliferation was assessed by MIB1 [Ki-67] expression. Results. Apoptosis was frequently found in inflammatory cells of portal tracts and sinusoids. Apoptosis of hepatocytes was also systematically observed. Only 4 positive bile duct cells were found in 3 bile ducts from 3 biopsies. Quantitative evaluation was not attempted. Cholangiocyte proliferation was observed in the same ducts and occasionally in other biopsies. Conclusions. These data suggest that cholangiocyte death by apoptosis at the level of typical primary biliary cirrhosis lesions is a rare event, at least in early stages of the disease. The observed rate of proliferation was consistent with the rate of apoptosis.

The process of immunosenescence does not seem to involve the allergic (IgE-mediated) reaction, as allergic sensititation not only is maintained during aging, but can also become clinically evident at 60 years and older.

The administration of ursodeoxycholic acid, a hydrophilic bile acid not hepatotoxic to humans, has been suggested for treatment of primary biliary cirrhosis to improve cholestasis and reduce hepatocellular damage. Efficacy of treatment has been studied mainly in patients with asymptomatic or early-stage disease. In January 1988, to establish the efficacy and safety of ursodeoxycholic acid in a population with more severe disease, we started a multicenter, double-blind, placebo-controlled trial in patients with symptomatic disease, that is, with pruritus or serum bilirubin exceeding 2 mg/dl. Forty-four patients were assigned to ursodeoxycholic acid, 500 mg daily (corresponding to about 8.7 mg/kg body weight in these patients), and 44 to a placebo. As planned at the beginning of the study, a preliminary analysis was performed when all patients had been followed for at least 6 months (33 patients up to 12 months). Pruritus, self-evaluated by the patients, and cholestyramine consumption, as recorded in a diary, decreased significantly ( p p p p p p

Serum IgA antiendomysial antibodies (EmA) were found in 61 (87%) of 70 adults and children with untreated celiac disease, whereas IgA antigliadin antibodies (AGA) and IgA R1-antireticulin antibodies (R1-ARA) were positive in 71% and 47%, respectively, of the same patients. Two of the nine untreated celiacs negative for IgA EmA showed positivity for IgA AGA. While IgA AGA and R1-ARA disappeared in all the celiacs, tested one year after gluten-free diet, IgA EmA persisted at low titer in seven (18%) of these 38 subjects, although the jejunal biopsy showed a complete regrowth of jejunal villi. All the disease control patients as well as the blood donors tested were always negative for the three IgA antibodies. Our results state that the search for both IgA EmA and AGA gives the best results in the screening of celiac disease, since the positivity for at least one of these two antibodies allows identification with a 100% specificity of the 90% of untreated celiac patients.

Liver fibrosis was induced in rats after administration of dimethylnitrosamine (DMN) intraperitoneally three times a week for 3 weeks. Incomplete septa appeared after 7 days and evidence of nodulation of the parenchyma was observed after 21 days. Both distribution of extracellular matrix components (collagen type I, type III and type IV, laminin, fibronectin, heparan sulphate proteoglycan) and the distribution of desmin as a marker of lipocytes (Ito cells) and of iso-α-smooth muscle actin were studied with immunoperoxidase. Changes in the distribution of extracellular matrix components outlined both the formation of septa and the development of nodules with changes in the sinusoidal pattern evoking aspects of capillarization. The number of desmin-positive cells increased in DMN-treated animals, showing a prominent reaction in the fibrous septa. In the normal liver, lipocytes were positive for laminin and negative for actin, but septal and juxta-septal lipocytes were positive for both antigens, suggesting the presence of transitional cells with mixed immunoreactivity. This was confirmed by ultrastructural studies which showed typical intraseptal myofibroblasts and other elements exhibiting the structural features of both myofibroblasts and lipocytes.

SUMMARY Serum IgA anti-gliadin antibodies (AGA) were positive in 25 (68%) of 37 untreated adults with coeliac disease belonging mostly to IgAl subclass (88%) and only in a few cases to IgA2(12%). Anti-secretory component IgA AGA were present in serum of seven patients (28%) by immunofluores-cence and in nine (36%) by ELISA. The search for IgA AGA in jejunal juice of eight untreated children with coeliac disease was positive in seven cases (88%), with consistent finding of anti-secretory component IgA AGA. These antibodies belonged with equal proportion to IgAl and IgA2 subclasses. This study shows that in intestinal secretions of untreated coeliac disease cases the IgA immune response to gliadin is confined to polymeric anti-secretory component IgA with the same prevalence of IgAl and IgA2 subclasses, while in serum IgA AGA are largely monomeric, more frequently of IgAl than of IgA2 subclass, and with a lower proportion of polymeric anti-secretory component IgA (20–36%). The finding of secretory IgA AGA in serum of patients with coeliac disease could result from a spill-over from the intestinal mucosal synthesis into the circulation.

In 46 adult patients with coeliac disease, 41 (89%) of whom were positive for IgA and/or IgG antigliadin antibodies (AGA) when untreated, we investigated after a gluten-free diet the relationship between the persistence of AGA, the persistence of jejunal lesions, and the duration and compliance with the diet. IgG AGA were positive in 21 coeliac patients (46%) after variable periods of gluten-free diet and were associated with IgA positivity only in 4 cases (9%). Both IgA and IgG AGA positivity appeared to be more related to the lack of improvement of the jejunal lesions than to the strictness and duration of gluten withdrawal. Nine coeliacs showed no improvement of jejunal lesions after the gluten-free diet. Of these 9, 4 showed persistent IgA AGA, while the remaining 5 resulted IgAAGA-negative as before when untreated. Though intestinal biopsy remains the best means of determining the positive effect of gluten withdrawal, the persistence of IgA AGA in treated coeliacs is always predictive of the persistence of severe jejunal lesions. The persistence of IgG AGA, on the contrary, should be regarded as an immunological memory.

Background Anti-ganglioside antibodies have been described in sera of coeliac patients with peripheral neuropathy and cerebellar ataxia. Aims To investigate the correlation between anti-ganglioside antibodies and neurological involvement in coeliac disease before and after gluten-free diet. Patients and methods Twenty-two untreated coeliac patients with neurological dysfunction and 30 untreated coeliacs without neurological dysfunction, 20 patients with neurological disorders, 50 autoimmune disease and 20 blood donors were tested for anti-GM1, anti-GD1b and anti-GQ1b IgG and IgM antibodies by enzyme-linked immunosorbent assay. Results IgG antibodies to at least one of the three antigens tested were positive in 64% of coeliac patients with neurological symptoms compared to 30% of coeliacs without neurological dysfunction ( P = 0.02), 50% of patients with neurological disorders ( P = ns), 20% with autoimmune diseases ( P = 0.003) and none of blood donors ( P = 0.0001). A strict gluten-free diet determined anti-ganglioside antibody disappearance in about half of coeliacs. Conclusions A significant correlation between anti-ganglioside antibodies and neurological disorders in patients with an underlying coeliac disease has been found. Anti-ganglioside antibodies may represent a new immunological marker to identify neurological impairment in patients with coeliac disease.

Summary Background : Besides the autoantibodies included in the diagnostic criteria of type 1 autoimmune hepatitis, many other autoantibodies have been described in this condition. Recently, antibodies against cyclic citrullinated peptide have been validated as specific diagnostic and prognostic markers of rheumatoid arthritis. Aim : To assess whether these antibodies are part of the autoantibody repertoire of type 1 autoimmune hepatitis and correlate with rheumatological manifestations. Methods : Antibodies against cyclic citrullinated peptide were tested by a commercially available enzyme-linked immunosorbent assay. Results : The antibodies were found in 12 of 133 (9%) type 1 autoimmune hepatitis, two of 49 (4%) with primary biliary cirrhosis, one of 80 (1%) with hepatitis C virus-related chronic liver disease and 53 of 89 (60%) with rheumatoid arthritis serum samples. High titres were found only in rheumatoid arthritis and type 1 autoimmune hepatitis. No clinical (in particular rheumatological manifestations), biochemical or immunoserological differences were detectable between antibodies against cyclic citrullinated peptide positive and negative type 1 autoimmune hepatitis sera, with the exception of rheumatoid factor, always negative in the positive ones. Conclusions : Antibodies against cyclic citrullinated peptide can be detected in a subgroup of patients with type 1 autoimmune hepatitis. They might be part of the wide range of autoantibody production characteristic of this condition and/or, less probably, be predictive of future rheumatoid arthritis development.

Background, The pathogenesis of non-alcoholic steatohepatitis remains unclear from several points of view. Minimal diagnostic criteria are still not defined. Aim. To gather information useful for diagnosis and to improve the understanding of pathogenic mechanisms. Patients. A series of 14 patients with non-alcoholic steatohepatitis, identified among liver outpatients, were paired for age, sex and alanine amino transferase values with 14 patients with hepatitis C virus infection without steatosis. Methods. Clinical, biochemical and immunohistological examination, including characterisation of inflammatory cell population, evaluation of type 111 collagen and tenascin deposition, activation of stellate cells, hepatocellular apoptosis and proliferation. Results. Patients with non-alcoholic steatohepatitis were more frequently obese, had higher triglyceride concentrations and lower gamma-globulins. T lymphocytes outnumbered polymorphonuclear cells, both in hepatitis C and in steatohepatitis, with a larger number of CD8 lymphocytes in patients with viral hepatitis but a comparable number of granulocytes. This resulted in a higher granulocytes to T cells ratio in steatohepatitis, possibly making these cells more easily detectable in spite of similar absolute numbers. Portal fibrosis and piecemeal necrosis were prevalent in hepatitis C virus infection, pericentral fibrosis was similar. Hepatocellular, apoptosis and proliferation as well as stellate cell activation were less relevant in steatohepatitis than in hepatitis C virus infection in spite of similar alanine amino transferase levels. Conclusions. These data provide a possible explanation for the relatively low tendency to progression of non-alcoholic steatohepatitis in most patients despite increased alanine amino transferase and suggest that non-death-related release of alanine amino transferase might occur in non-alcoholic steatohepatitis. This makes liver biopsy an essential part of the clinical setting supporting diagnosis, evaluation of severity and possibly definition of the evolutionary trend.

Editor,—Recent studies have demonstrated that coeliac disease can develop months or years after the clinical onset of insulin dependent diabetes mellitus (IDDM).1 Over the last 10 years, 200 consecutive children with IDDM at the onset have been tested for IgG and IgA antigliadin antibodies (AGA) by indirect immunofluorescence2 and for IgA antiendomysial antibodies (EmA) using monkey oesophagus and, more recently, human umbilical cord as substrate.3 An antibody follow up was also performed in 151 of these patients every 3–6 months. At initial testing six diabetic children (four girls and two boys, median age 9 years, range 2–13) were positive for both IgA EmA and IgA AGA (associated with IgG …

Sera from 800 patients referred to our laboratory for celiac sprue screening were tested for IgA antiendomysial antibodies on human umbilical cord (HUC-EmA) and for IgA and IgG antigliadin antibodies (AGA) on rodent tissue by indirect immunofluorescence. Thirty-three patients (4.1%) were positive for at least one of the two antibodies ; IgA HUC-EmA were found in 25 patients, whereas IgA and IgG AGA were detected in 19 and 24 subjects, respectively. Twenty-seven (82% ) of the 33 patients with immunological markers showed subtotal or severe partial villous atrophy consistent with celiac sprue, whereas the remaining six cases had normal histological findings. None of 30 antibody-negative biopsied patients showed abnormalities of duodenal mucosa. Of the 27 newly diagnosed celiacs, only 21 (78% ) were positive for IgA and/or IgG AGA, whereas IgA HUC-EmA were found in all but two cases (93%). In these two cases, positive only for IgG AGA, celiac sprue was associated with IgA deficiency. Our study proves that the routine use of IgA HUC-EmA increases celiac sprue finding by up to 15% when compared with AGA.

Since celiac disease screening by traditional IgA anti-endomysial antibody test is limited by high costs of monkey esophagus commercial kits as well as by rising ethical problems related to the endangered species, the identification of an inexpensive and commonly available substrate for this antibody determination is urgently required. To achieve this goal, we compared the prevalence of IgA anti-endomysial antibodies detected on monkey esophagus with that on human umbilical cord. Fifty-seven (95%) of 60 untreated adult celiacs were positive for these antibodies on monkey esophagus as well as on human umbilical cord. IgA anti-endomysial antibodies, detected on both tissues, were negative in all 200 disease and healthy controls tested, displaying a 100% specificity for gluten-sensitive enteropathy. These data suggest that human umbilical cord can replace monkey esophagus for IgA anti-endomysial antibodies test. Human umbilical cord allows unlimited testing for celiac disease screening on wide series of high-risk subjects, permitting identification of greater numbers of asymptomatic celiac patients with a remarkable saving of money and bypassing the ethical problems related to killing monkeys.

To obtain information on the mechanisms of hepatocellular damage and the determinants of response to interferon, hepatitis C virus (HCV) genotype, tissue HCV antigens, hepatocellular expression of HLA-A,B,C and intercellular adhesion-1 molecules, and the number of lobular T lymphocytes were studied in 38 anti-HCV-positive patients. 14 patients did not show a primary response to interferon treatment. HCV genotype 1b was detected in 11 of them. They displayed higher scores of HCV-positive hepatocytes, HLA-A,B,C, and ICAM-1 molecules expression than with the responders. HCV-infected hepatocytes maintained the capacity to express HLA-A,B,C and ICAM-1 molecules. CD8-positive T cells in contact with infected hepatocytes and Councilman-like bodies were observed. A significant correlation was found between the number of lobular CD8-positive T cells and alanine amino transferase levels. No differences were observed in clinical, biochemical, and histological features between patients with high and low number of hepatocytes containing HCV antigens. These data suggest a prominent role of T cell-mediated cytotoxicity in the genesis of hepatocellular damage. The high expression of interferon-inducible antigens like HLA-A,B,C molecules suggests the presence of strong activation of the interferon system possibly related to high HCV replication in nonresponder patients infected with genotype 1b.

Study of the reaction of celiac disease-related antibodies with human substrates has been hampered by the immunological cross-reactivity between fluorescein isothiocyanate-conjugated anti-human immunoglobulins and immunoglobulins normally present in human tissues. In order to overcome this problem we extracted IgG from 2 celiac disease sera (positive for specific IgG and IgA antibodies) using a genetically engineered recombinant form of streptococcal protein G covalently immobilized on 4% agarose beads. The separated IgG and IgA was conjugated with fluorescein isothiocyanate and used in direct immunofluorescence on 0 blood group human duodenum, liver, and myocardium. Staining of the lamina propria beneath the villous epithelium, the endomysium of smooth muscle layers in the duodenum, and the liver sinusoidal and pericellular myocardial matrix was observed. The reactivity of celiac disease-related antibodies with various human tissues indicates that these antibodies are the result of a systemic immune response directed against all tissues containing matrix proteins.

By using abdominal ultrasonography (UlS), deep nodes were detected in 41 of 85 (48%) HIV-1 positive subjects, most of them heroin addicts, but in none of 85 healthy HIV-negative controls. Computerized tomography, performed in 10 cases of lymphadenopathy, invariably confirmed the UlS findings. Prevalence [asymptomatic carriers: 8/15 (53%); PGL patients: 8/18 (44%); ARC: 13/27 (48%); AIDS: 12/25 (48%)], number, size, and site of deep nodes were comparable among the different CDC groups. No correlation was found between abdominal and superficial lymphadenopathy. Median serum concentrations of gammaglobulins (g/dl) and IgG (mg/dl) were higher in patients with than without deep nodes (2.25 vs 1.87 and 2540 vs 1900, respectively) (p0.01) as well as in cases with than without superficial nodes (2.15 vs 1.80 and 2340 vs 1941, respectively) (p0.05). Abdominal lymphadenopathy occurred during all stages of HIV infection even in asymptomatic carriers: this should be considered in the differential diagnosis of UlS-detected deep nodes. Enlargement of either deep or superficial nodes seems to reflect a state of polyclonal B cell activation.

To resolve conflicting reports about the occurrence of antibodies against hepatitis C virus (HCV) in patients with autoimmune chronic active hepatitis (AI-CAH), sera from UK and Italian patients were tested with the original anti-HCV assay (Ortho) and a novel anti-HCV assay (UBI) based entirely on synthetic HCV peptides. 28 (60%) of 47 Italian patients with type-1 AI-CAH were anti-HCV-positive by Ortho ELISA, 25 of whom were also strongly positive by the UBI assay. 15 (60%) of 25 UK patients with type-1 AI-CAH were HCV-positive by Ortho ELISA but only 2 were positive by the UBI assay. Similarly, 29 (88%) of 33 Italian patients with type-2 AI-CAH, but 0 of 10 UK patients, were very strongly anti-HCV-positive with the UBI assay. Italian patients with AI-CAH appear to have a high frequency of genuine exposure to HCV, whereas seropositivity by the Ortho HCV ELISA in UK patients is likely to represent a false-positive result. These findings indicate important geographical and/or genetic influences in autoimmune liver disease among different populations.

SUMMARY The specificities of anti-nuclear antibodies (ANA) reacting with multiple nuclear dots (MND-ANA) present in about 15% primary biliary cirrhosis sera were studied by Western blot analysis with nuclear fractions from a human cell line. Reactivity with two broad bands of 78–92 kD and 96–00 kD of the insoluble fraction was present exclusively in MND-ANA-positive sera. Antibodies eluted from these proteins specifically retained the immunofluorescence reactivity of MND-ANA. Immunomorphological analysis by a pre-embedding technique revealed that the antibody specifically binds to nuclear regions resembling in size and number nuclear bodies. Since these structures are absent in immature rate endometrial cell and can be induced by diethylstilbestrol. we tested MND-ANA by immunofluorescence on cryostat sections of uteri from hormone-treated and untreated immature rats. A strong reaction of nuclear dots was observed predominantly in endometrial cells of hormone treated rats. We thus conclude that MND-ANA present in primary biliary cirrhosis sera are directed against 78–92-kD and 96–100-kD nuclear proteins located in nuclear bodies.

Serum samples from 66 seropositive subjects (56 with a history of intravenous drug abuse), including asymptomatic carriers and patients with persistent generalised lymphadenopathy (PGL), AIDS related complex (ARC), and AIDS, were tested by indirect immunofluorescence on rat tissue sections and HEp-2 cells for the presence of antibodies to nuclei, smooth muscle, intermediate filaments (anti-IMF) and microfilaments (anti-MF). Counterimmunoelectrophoresis was also used to detect antibodies to extractable nuclear antigens. Smooth muscle antibodies with the V pattern or antinuclear antibodies, mainly of the speckled type, or anti-IMF, occurred in 35 cases, being widely distributed in all groups. Such an autoantibody response resembles the "viral" autoimmunity described in various infectious diseases and in particular that of non-A, non-B post-transfusion hepatitis. Autoantibodies may be of some prognostic relevance, as the prevalence of smooth muscle antibodies V increased as the disease progressed (asymptomatic carriers 20%, those with PGL 29%, those with ARC 47%, and those with AIDS 63%. In the PGL group autoantibody positivity correlated with the presence of skin anergy. The fact that autoantibodies were more frequently detected in patients with circulating immune complexes suggests that these can contain autoantibodies and the corresponding autoantigens.

Smooth muscle antibodies with anti-actin specificity are commonly regarded as markers of autoimmune liver disease. However, there are interpretational problems because different techniques have been used for their identification and therefore the results are difficult to compare. The present paper reports the results of a new method for the identification of anti-actin antibodies (indirect immunofluorescence on cryostat sections of liver from rats chronically injected with phalloidin). The results have been compared with those obtained by four other techniques: demonstration by immunofluorescence of kidney peritubular reactivity (SMAT), of anti-microfilament antibodies (on HEp-2 cells and vinblastine-treated peripheral blood mononuclear cells) and counterimmunoelectrophoresis with purified muscle actin as antigen. The new method proved to be the most sensitive and specific. Furthermore, its reproducibility was found to be high, the interpretation easy and the cost low. The clinical significance of anti-actin antibodies in patients with chronic liver disease is also discussed.

Background It is believed that a subset of CU is immune-mediated and, indeed, autoimmunity to thyroid specific antigens and high affinity IgE receptor has been described in a proportion of such cases. Aim To verify the occurrence of other autoimmune phenomena and to establish whether screening for a wide spectrum of autoantibodies is cost-effective in the diagnostic evaluation of this condition. Methods Sera from 184 patients with CU were tested by indirect immunofluorescence for antinuclear (ANA), anti smooth muscle (SMA), antimitochondrial (AMA) and anti liver and kidney microsomal (LKM-1) antibodies. A large subset of the same sera were tested by the same technique also for anti neutrophil cytoplasmic (ANCA) and antiendomysial (EmA) antibodies. Results LKM-1 was never detected and AMA was found in 1 case (0.5%). ANA (8.7%) and SMA (11.4%) were more frequently found than in healthy controls, though the difference was not statistically significant. Furthermore, titers were generally low: less than 1:80 in over 90% cases. Similar results were obtained with ANCA, which were found in 8% cases. None of the autoantibody positive patients has developed any autoimmune disorder so far. IgA EmA was found in 1 case (0.8%), who was histologically diagnosed as celiac and put on a gluten-free diet. Conclusions Wide spectrum autoantibody testing is not cost-effective in CU and is advisable only when there is a high clinical probability of underlying autoimmune disease or low cost laboratory facilities are available. In this latter case, we suggest to cut the screening dilution to 1:160 in order to increase the specificity.