Anti-MOG autoantibodies in Italian multiple sclerosis patients: specificity, sensitivity and clinical association

There is considerable evidence that multiple sclerosis (MS) is an immune-mediated disease characterized by infiltration of inflammatory cells into the CNS and demyelination. Several myelin proteins may be encephalitogenic, including myelin basic protein, proteolipid protein and myelin oligodendrocyte glycoprotein (MOG), the latter being expressed on the external layer of myelin sheaths and hence accessible to antibody attack. We investigated MOG autoreactivity in serum and cerebrospinal fluid (CSF) by ELISA, employing the recombinant extracellular domain of MOG as antigen. We tested serum samples from 262 MS patients (175 relapsing-remitting, 43 primary progressive and 44 secondary progressive), 131 patients with other neurological diseases (OND) and 307 healthy controls. No patients or controls were receiving immunomodulating treatments. We found anti-MOG antibodies in the serum of 13.7% MS patients, mainly in those with secondary progressive MS (25%), in 13.7% of OND patients and in 6.2% of controls. We found a direct correlation (R(2) = 0.6, P = 0.002) between disease severity and anti-MOG titer only in patients with primary and secondary progressive MS. Anti-MOG antibodies were present in the CSF of 11.4% MS patients and 18.9% OND patients. Intrathecal synthesis of anti-MOG antibodies was demonstrated in four (4.5%) of MS patients and no OND patients. Anti-MOG antibodies are not specific for MS; however, they may characterize a subset of MS patients and this may be revealed by serial assays in relation to changing disease phase.     

Demyelination in in vitro culture in multiple sclerosis: facts and controversies

The myelinotoxicity of sera from multiple sclerosis (MS) patients was determined by the assessment of visible myelin damage in guinea-pig spinal cord-spinal ganglia and in rat cerebella cultivated on collagen-coated coverslips in Leighton tubes. No change was seen in 22 cases. These data show that serum myelinotoxicity is low in MS - as compared with that of Experimental Allergic Encephalomyelitis. It appears a little specific phenomenon, the mechanism of which remains unclear. It can be easily assessed only in very sensitive culture techniques and is best measured by biochemical methods. This does not preclude the pathophysiological significance of the serum myelinotoxicity in MS. Supernatants of cerebro-spinal fluid (CSF) cell cultures in 10 MS patients caused no demyelination. Pooled concentrated supernatants of CSF cell cultures from 13 to 20 MS patients--containing from 7.3 to 11.7 micrograms IgG/ml gave no patent in vitro demyelination in 3 different experiments. The released products of CSF cell cultures in MS are not very toxic for myelin. However these experiments have to be repeated with more sensitive culture techniques, biochemical assay of myelinotoxicity and more concentrated CSF cultures supernatants.     

Autoreactive T and B cells responding to myelin proteolipid protein in multiple sclerosis and controls.

The pathogenesis of multiple sclerosis (MS) could involve an autoimmune response to proteolipid protein (PLP). Immunization of experimental animals with this major myelin protein can lead to experimental allergic encephalomyelitis. To identify a possible role of PLP as target antigen in MS, we evaluated T cell immunity to PLP in blood and cerebrospinal fluid (CSF) from patients with MS and controls by counting cells which in response to PLP in short-term cultures secreted interferon-gamma. The PLP-specific B cell response was analyzed by counting cells secreting anti-PLP antibodies. PLP-reactive T cells were detected in blood of most MS patients (mean value 1 per 20,408 mononuclear cells), and at 41-fold higher numbers in CSF (mean 1 per 500 CSF cells). Anti-PLP IgG antibody-secreting cells were detected in blood from most MS patients (mean 1 per 30,303 cells), but such cells were 49-fold more frequent in CSF (mean 1 per 625 cells). PLP-reactive T and B cells were also detected in blood and CSF from control patients, but at much lower numbers. A strong and persistent autoimmune response to PLP as well as to other myelin proteins, enriched in CSF, is proposed to be pathogenetically important in MS.     

The Pathology of an Autoimmune Astrocytopathy: Lessons Learned from Neuromyelitis Optica

Neuromyelitis optica (NMO) is a disabling autoimmune astrocytopathy characterized by typically severe and recurrent attacks of optic neuritis and longitudinally extensive myelitis. Until recently, NMO was considered an acute aggressive variant of multiple sclerosis (MS), despite the fact that early studies postulated that NMO and MS may be two distinct diseases with a common clinical picture. With the discovery of a highly specific serum autoantibody (NMO‐IgG), Lennon and colleagues provided the first unequivocal evidence distinguishing NMO from MS and other central nervous system (CNS) inflammatory demyelinating disorders. The target antigen of NMO‐IgG was confirmed to be aquaporin‐4 (AQP4), the most abundant water channel protein in the CNS, mainly expressed on astrocytic foot processes at the blood–brain barrier, subpial and subependymal regions. Pathological studies demonstrated that astrocytes were selectively targeted in NMO as evidenced by the extensive loss of immunoreactivities for the astrocytic proteins, AQP4 and glial fibrillary acidic protein (GFAP), as well as perivascular deposition of immunoglobulins and activation of complement even within lesions with a relative preservation of myelin. In support of these pathological findings, GFAP levels in the cerebrospinal fluid (CSF) during acute NMO exacerbations were found to be remarkably elevated in contrast to MS where CSF‐GFAP levels did not substantially differ from controls. Additionally, recent experimental studies showed that AQP4 antibody is pathogenic, resulting in selective astrocyte destruction and dysfunction in vitro, ex vivo and in vivo. These findings strongly suggest that NMO is an autoimmune astrocytopathy where damage to astrocytes exceeds both myelin and neuronal damage. This chapter will review recent neuropathological studies that have provided novel insights into the pathogenic mechanisms, cellular targets, as well as the spectrum of tissue damage in NMO.     

Lymphocytes from multiple sclerosis patients produce elevated levels of gamma interferonin vitro

The production of gamma interferon (IFN) by mononuclear cells (MNC) from patients with exacerbating/remitting multiple sclerosis (MS) and controls was evaluated. After 3 days of culture with concanavalin A, the amount of gamma IFN in supernatant fluids was determined by radioimmunoassay. MNC from MS patients produced significantly (P<0.001) more gamma IFN than MNC from either normal controls or patients with other neurologic diseases. Levels of gamma IFN in the serum and CSF were also measured. Despite the relative absence of gamma IFN in serum (4 positive of 30), all CSF samples tested had low, but detectable, levels of gamma IFN (0.3 to 1.4 U/ml). These studies suggest that some of the autoimmune features and immunologic abnormalities in MS may be related to elevated gamma IFN production.     

Intrathecal CD4+ and CD8+ T‐cell responses to endogenously synthesized candidate disease‐associated human autoantigens in multiple sclerosis patients

MS pathology is potentially orchestrated by autoreactive T cells, but the antigens recognized remain unknown. A novel APC/T‐cell platform was developed to determine intrathecal CD4⁺ and CD8⁺ T‐cell responses to candidate MS‐associated autoantigens (cMSAg) in clinically isolated syndrome (CIS, n = 7) and MS (n = 6) patients. Human cMSAg encoding open reading frames (n = 8) were cloned into an Epstein–Barr virus (EBV)‐based vector to express cMSAg at high levels in EBV‐transformed B‐cells (BLCLs). Human cMSAg cloned were myelin‐associated and ‐oligodendrocyte glycoprotein, myelin basic protein, proteolipid protein, ATP‐dependent potassium channel ATP‐dependent inwards rectifying potassium channel 4.1, S100 calcium‐binding protein B, contactin‐2, and neurofascin. Transduced BLCLs were used as autologous APC in functional T‐cell assays to determine cMSAg‐specific T‐cell frequencies in cerebrospinal fluid derived T‐cell lines (CSF‐TCLs) by intracellular IFN‐γ flow cytometry. Whereas all CSF‐TCL responded strongly to mitogenic stimulation, no substantial T‐cell reactivity to cMSAg was observed. Contrastingly, measles virus fusion protein‐specific CD4⁺ and CD8⁺ T‐cell clones, used as control of the APC/T‐cell platform, efficiently recognized transduced BLCL expressing their cognate antigen. The inability to detect substantial T‐cell reactivity to eight human endogenously synthesized cMSAg in autologous APC do not support their role as prominent intrathecal T‐cell target antigens in CIS and MS patients early after onset of disease.     

Interleukin-12 and Perforin mRNA Expression is Augmented in Blood Mononuclear Cells in Multiple Sclerosis

Cytokines are suggested to orchestrate an abnormal immune response in multiple sclerosis (MS). The regulatory cytokine interleukin (IL)-12 induces T-helper (Th) cell switch to the Th1 type and the production by cytotoxic T cells of perforin, a cell lysis-inducing factor. It has been suggested that Th1-like cytokines may promote the development of MS, and the production of perforin to induce oligodendrocyte damage. In-situ hybridization with radiolabelled synthetic oligonucleotide probes was used to detect and enumerate mononuclear cells (MNC) expressing IL-12 and perforin mRNA in blood and cerebrospinal fluid (CSF) from patients with MS and controls. Plasma and CSF levels of IL-12 (p70) were evaluated by ELISA. Higher numbers of IL-12 and perforin mRNA-expressing MNC were registered in blood in MS and also in controls with aseptic meningoencephalitis (AM) compared to healthy subjects. There were a few patients with other non-inflammatory neurological diseases who also had high levels of IL-12 or perforin mRNA expressing blood MNC. A parallel elevation was observed for IL-12 (p70) concentrations in plasma. In the MS patients' CSF, there was a further augmentation of IL-12 mRNA expressing MNC. To evaluate autoantigen-induced IL-12 and perforin mRNA expression, blood MNC were cultivated ± myelin basic protein (MBP), a candidate autoantigen in MS. Higher numbers of MBP-reactive IL-12 and perforin mRNA expressing blood MNC were detected in MS than controls. The augmentation of both IL-12 and perforin in MS might reflect ongoing inflammatory processes in MS and could represent targets for future treatments.     

MBP、IL-16在Guillain—Barré综合征发病中的机制

目的探讨髓鞘碱性蛋白（MBP）和白细胞介素16（IL-16）在Guillain-Barré综合征（GBS）发病过程中的变化。方法用ELISA法检测GBS组（36例）中MBP和IL-16的水平，并与多发性硬化（MS）组（45例）及对照组（33例）相比较。结果GBS、MS和对照组3组中CSFMBP的水平均明显高于Serum MBP的水平（P〈0．01），而CSF IL-16的水平均明显低于Serum IL-16的水平（P〈0．01）。GBS组CSF中MBP的水平明显高于对照组（P〈0．01）但低于MS组（P〈0．01），Serum中MBP水平和对照组相比无明显差异但低于MS组（P〈0．01）。CSF中的IL-16水平明显高于对照组（P〈0．05），但与MS组相比无明显差异，Serum中IL-16的水平低于对照组（P〈0．01）但与MS组相比无明显差异。结论GBS发病早期神经根髓鞘的脱失可能更严重，中枢神经系统局部产生的IL-16参与了GBS神经根脱髓鞘的炎症过程。     

Immunodetection of a Hepatitis C Virus (HCV) Antigen and Th1/Th2 Cytokines in Cerebrospinal Fluid of Meningitis Patients

Abstract

Infection with hepatitis C virus (HCV) has become the most important public health problem in Egypt. HCV infection has been implicated in diseases of the central nervous system. Cerebrospinal fluid (CSF) and serum samples from 91 patients with meningitis (62 males and 29 females, mean age of 37 years) were investigated. Anti‐HCV antibodies and HCV antigen were evaluated in patients CSF and serum using enzyme linked immunosorbent assay. The levels (mean?±?SD?pg/ml) of Th1 cytokines (IFN‐γ and TNF‐α) and Th2 interleukines (IL‐10 and IL‐4) were also determined. The anti‐HCV antibodies were detected in high percentages both in CSF samples (71%) and in sera (90%). Also, the HCV antigen was detected in about 60% of tested CSF and serum samples. The levels of IFN‐γ and IL‐10 cytokines were significantly higher (P?<?0.05) in both serum and CSF of patients positive for HCV antigen than those negative. HCV antigen was detected in the CSF of meningitis patients with a significant upregulation of Th1 and Th2 responses. The high incidence of HCV infection may draw light on the etiological role of HCV in the pathogensis of meningitis diseases in our study group.     

Fibroblast growth factor-2 levels are elevated in the cerebrospinal fluid of multiple sclerosis patients

In recent years a role has been recognized for fibroblast growth factor (FGF)-2 in the pathogenesis of demyelination and the failure of remyelination in experimental models of multiple sclerosis (MS). FGF-2 levels were determined using a sensitive immunoassay in the cerebrospinal fluid (CSF) of 20 patients with clinically isolated syndrome (CIS), 40 patients with relapsing-remitting (R-R) MS, and 30 patients with secondary progressive (SP) MS, correlated with MRI measures. Control CSF samples were obtained from 20 subjects who underwent lumbar puncture for diagnostic purposes and for whom all instrumental and laboratory analyses excluded systemic and nervous system diseases. FGF-2 levels in the CSF of MS and CIS patients were significantly higher than controls (P<0.001 and P<0.05, respectively). The highest levels were detected in R-R MS patients during relapse and in SP MS patients with an increase of 1 point in EDSS scores in the last 6 months. A significant correlation was found in SP MS patients with lesional load (R=0.43, P<0.01) but not with parenchymal fractions as measures of brain atrophy. A slight increase in serum FGF-2 levels was also found in R-R MS patients during relapse with gadolinium enhancing lesions and in SP patients with disability progression. These findings support the implication of FGF-2 in the pathogenesis of MS and concur with recent reports of the involvement of FGF receptor signalling in the disruption of myelin production in differentiated oligodendrocytes and in the loss of adult oligodendrocytes and myelin in vivo due to FGF-2.     

Soluble HLA class I and class II antigens in patients with multiple sclerosis

Abstract: Soluble HLA class I (sHLA-I) and soluble HLA class II (sHLA-II) antigen levels during different stages of disease were investigated in paired serum and cerebrospinal fluid (CSF) samples from 37 patients with multiple sclerosis (MS) using ELISA and Western blot analysis. Soluble HLA-II antigens in the serum of untreated patients with the relapsing-remitting type of MS (RRMS) were found to be significantly elevated in acute relapse as compared to values obtained from patients under steroid treatment, in remission or healthy controls. No significant differences in circulating sHLA-I levels could be detected. In contrast, a trend towards increased intrathecal production of sHLA-I molecules in the CSF was observed in untreated RRMS patients in acute relapse, whereas the levels of soluble HLA-II antigens in the CSF were below the detection limit of the ELISA method. Our observations underline the presence of systemic immune activation in MS patients, as reflected in elevated serum sHLA-II antigen levels, and reveal a dichotomy between sHLA class I and II antigen production in the peripheral blood versus CSF in acute MS. Serial measurements of sHLA-II antigen levels might represent a non-invasive method to assess disease activity in MS patients.     

Multiple Sclerosis: Cells Secreting Antibodies Against Myelin-Associated Glycoprotein are Present in Cerebrospinal Fluid

We evaluated the B-cell response in cerebrospinal fluid (CSF) and blood by enumerating cells secreting antibodies to myelin-associated glycoprotein (MAG) and, for reference, to myelin basic protein (MBP), two myelin components which may constitute targets for autoimmune attack in multiple sclerosis (MS). Among 25 untreated MS patients, 12 had cells in CSF secreting anti-MAG IgG antibodies (mean value 1 per 1429 CSF cells) and three also had cells secreting anti-MAG antibodies of the IgM isotype but at lower levels. In CSF from 2 out of 10 MS patients examined, anti-MAG and anti-MBP IgG antibody-secreting cells were present concurrently. Antibody-secreting cells were less frequent in blood and bone marrow, reflecting compartmentalization to CSF. Anti-MAG antibody-secreting cells were found in CSF from only 1 out of 27 control patients. The intrathecal production of anti-MAG and anti-MBP antibodies may be important in the pathogenesis of MS.     

Myelin antigen reactive T cells in cerebrovascular diseases.

T cell reactivities to the putative autoantigens myelin basic protein (MBP), MBP peptides with amino acid residues 110-128 and 148-165, and myelin proteolipid protein (PLP) were examined in patients with acute ischaemic cerebrovascular disease (CVD) and, for comparison, in patients with inflammatory neurological diseases and other neurological diseases. A quantitative measure of these T cell reactivities was obtained by assessing numbers of T cells among blood and cerebrospinal fluid (CSF) mononuclear cells that secreted IFN-gamma in response to antigen in vitro. Higher numbers of T cells reactive with each of these four antigens were detected in peripheral blood from patients with CVD compared with patients of the two control groups. Among blood cells from the CVD patients, their average number was 2.3-4.2/10(5) mononuclear cells. MBP reactive T cells were several-fold enriched in the CSF of CVD patients. The findings strongly suggest that brain damage in context with acute CVD leads to an in vivo expansion of myelin reactive T cells.     

LATENT IMMUNOGLOBULIN G (Gm) ALLOTYPES: OCCURRENCE IN THE CEREBROSPINAL FLUID IN SOME NEUROPATHOLOGICAL STATES

Gm allotypes were detected and quantitated by radioimmunoassay (RIA) in paired serum and CSF samples from patients suffering from various neurological diseases. Of 115 patients with neurological disorders (65 MS and 50 others), seven subjects displayed one or two allotypes in their CSF which were absent in serum. The Gm phenotype in the patient's serum allowed us to infer the genotype without the need of familial data. A comparison of the regression curves obtained in RIA from the unexpected allotype in CSF and the counterpart in a normal serum pool argued for an identity of the Gm antigen carried by both inhibitory molecules. The unexpected allotype(s) in CSF can be considered as the product of a latent Gm gene which may be activated by either immune perturbations due to the disease per se or some particular immune regulations in the central nervous system.     

Cells of Cerebrospinal Fluid of Multiple Sclerosis Patients Secrete Antibodies to Myelin Basic Protein In Vitro

To characterize the role of B lymphocytes in the pathogenesis of Multiple Sclerosis (MS), we have isolated mononuclear cells from cerebrospinal fluid (CSF) and stimulated them with a polyclonal B-cell mitogen (pokeweed mitogen). This study has been done with MS patients selected for the occurrence of an acute attack in the course of the disease and with patients hospitalized for other neurological diseases. Five of the 11 MS patients had B lymphocytes producing in vitro antibodies (Abs) directed against purified human myelin basic protein (hMBP), as revealed by Western blot analysis. None of the 20 patients with other neurological diseases showed such a reactivity. The produced Abs recognized only 1 or 2 hMBP peptides without dominance for a certain peptide. This result emphasizes the presence of B cells producing Abs against MBP in CSF of MS patients and shows the interest of studying mononuclear cells of CSF as a good marker of the pathogenesis.     

Cells producing antibodies specific for myelin basic protein region 70-89 are predominant in cerebrospinal fluid from patients with multiple sclerosis.

Cells secreting antibodies against guinea pig myelin and synthetic myelin basic protein (MBP) peptides were evaluated in cerebrospinal fluid (CSF) and blood from patients with multiple sclerosis (MS) and a variety of other neurological diseases (OND). The peptides used, reproducing amino acid sequences 1-20, 70-89, 108-126, or 157-166 of MBP, were selected on the basis of their hydrophilic and encephalitogenic properties. Low numbers of cells secreting IgG antibodies against myelin or each of the MBP peptides (about 1 per 50,000) were detected in peripheral blood, with no difference between MS and OND. In CSF, cells secreting IgG antibodies to MBP 70-89 were more frequently (p = 0.007) detected in patients with MS (1/380 IgG-secreting cells on average) than in patients with OND (1/2083 IgG-secreting cells on average). The frequencies of cells secreting antibodies against myelin or the three other MBP peptides were similar in MS and OND. Thus, evaluation of B cell immunity at the cellular level indicates that MBP 70-89 is an immunodominant B cell epitope in MS. It is not clear whether this intrathecal anti-MBP 70-89 IgG antibody response has any pathogenetic relevance in MS or is the result of myelin breakdown.     

Multiple sclerosis: effect of myelin basic protein on interleukin 1, interleukin 2 production and interleukin 2 receptor expression in vitro.

The production of interleukin 1 (IL-1) and interleukin 2 (IL-2) by peripheral blood mononuclear cells (PBMC) stimulated with human myelin basic protein (MBP) was assessed in vitro in multiple sclerosis (MS) patients in relapse, patients with other neurological diseases (OND) and healthy subjects. Myelin basic protein significantly increased both IL-1 and IL-2 production by PBMC from MS patients during relapse when compared to OND patients or healthy controls. The most efficient concentration of MBP for the induction of IL-1 and IL-2 was 50 micrograms/ml. The optimal IL-1 production occurred after 48 h of PBMC culture and optimal IL-2 production after 72 h of PBMC culture. Anti-Tac monoclonal antibody (MoAb) was used to study IL-2 receptor expression on the same sample of PBM used for IL-2 study in MS patients in relapse. In addition IL-2 receptor expression was studied in PBMC from chronic progressive MS patients. In both MS groups IL-2 receptor expression on PBMC stimulated with MBP appeared higher than in control groups, but these differences were not statistically significant. IL-2 receptor expression on cerebrospinal fluid lymphocytes (CSF-L) either unstimulated or MBP-stimulated was, however, significantly higher in both MS groups when compared to OND patients. These results confirm the presence of activated lymphocytes in the CSF of MS patients during active stages of disease and suggest that this activation may be related to expansion of MBP specific cells.     

Enolase and Arrestin are Novel Nonmyelin Autoantigens in Multiple Sclerosis

Introduction: Although myelin autoimmunity is known to be a major factor in the pathogenesis of multiple sclerosis (MS), the role of nonmyelin antigens is less clear. Given the complexity of this disease, it is possible that autoimmunity against nonmyelin antigens also has a pathogenic role. Autoantibodies against enolase and arrestin have previously been reported in MS patients. The T-cell response to these antigens, however, has not been established. Methods: Thirty-five patients with MS were recruited, along with thirty-five healthy controls. T-cell proliferative responses against non-neuronal enolase, neuron-specific enolase (NSE), retinal arrestin, β-arrestin, and myelin basic protein were determined. Results: MS patients had a greater prevalence of positive T-cell proliferative responses to NSE, retinal arrestin, and β-arrestin than healthy controls (p<0.0001). The proliferative response against NSE, retinal arrestin, and β-arrestin correlated with the response against myelin basic protein (p≤0.004). Furthermore, the proliferative response against retinal arrestin was correlated to β-arrestin (p<0.0001), whereas there was no such correlation between non-neuronal enolase and NSE (p = 0.23). Discussion: There is accumulating evidence to suggest that the pathogenesis of MS involves more than just myelin autoimmunity/destruction. Autoimmunity against nonmyelin antigens may be a component of this myriad of immunopathological events. NSE, retinal arrestin, and β-arrestin are novel nonmyelin autoantigens that deserve further investigation in this respect. Autoimmunity against these antigens may be linked to neurodegeneration, defective remyelination, and predisposition to uveitis in multiple sclerosis. Further investigation of the role of these antigens in MS is warranted.     

Immune studies in multiple sclerosis

The serum and cerebrospinal fluid of patients with multiple sclerosis, in presence of human complement, caused destruction of glial cells and myelin in cultured nervous tissue, particularly in the acute stage of the disease. Cytotoxicity of the cerebrospinal fluid (CSF) was not wholly specific for multiple sclerosis, being observed also in cases of encephalitis and certain viral diseases. The degree of cytotoxicity of the CSF correlated with the increase in γ-globulin (IgG) in the cerebrospinal fluid. Immunoelectrophoresis studies of the CSF showed the presence in 50% of cases of an abnormal fast γ-globulin component.

These findings have two important implications. Firstly they support the concept that demyelination in multiple sclerosis and related diseases is the result of an autoimmune process. Secondly they are useful for the laboratory diagnosis of multiple sclerosis and its differentiation from non-demyelinating disorders of the nervous system.

     

Evidence of Local Synthesis of Smooth-Muscle Antibodies in the Central Nervous System in Isolated Cases of Multiple Sclerosis and Chronic Lymphocytic Meningoencephalitis

Paired samples of serum and concentrated cerebrospinal fluid (CSF) from 53 patients with multiple sclerosis (MS) and from 36 patients with other neurological diseases were adjusted to an equal concentration of IgG and examined for occurrence of auto-antibodies. Anti-nuclear antibodies, anti-mitochondrial antibodies, and rheumatoid factor were not detected with increased frequency in the materials studied. Smooth-muscle antibody (SMA) was, however, detected in CSF and serum from three patients with MS and one patient with chronic lymphocytic meningoencephalitis, and in the CSF only from one additional patient with MS. The SMA activities of the CSF were 4 to 16 times higher than in the matching sera, suggesting a local SMA synthesis within the central nervous system. The SMA was associated with electrophoretically restricted fractions of IgG, but an association between SMA and oligoclonal IgG of the CSF could not be demonstrated in the SMA-positive patients by the methods used.