The 1952 outbreak of encephalitis in California; laboratory methods for etiologic diagnosis

The general procedures used in the diagnosis of neurotropic viral diseases are outlined and are discussed with specific reference to western equine encephalitis. Cerebrospinal fluid is considered practically worthless as a starting material, in attempts to isolate the causal agent. The material of choice in attempting to recover the virus is central nervous system tissue, available only in instances of fatal infection. In the usual case, the diagnosis depends upon serologic or immunologic methods. These methods are aimed at detecting the presence of specific antibodies and of increases in the content of antibodies in the blood during the course of the illness. The in vitro complement fixation test is considered a better diagnostic tool than the in vivo neutralization test, since rises in titer are more readily detectable by the former technique than by the latter.     

Immunoenzyme method in the diagnosis of human brucellosis

The optimum conditions for the determination of specific antibodies in the sera of brucellosis patients by means of enzyme immunoassay (EIA) have been selected. The comparative study of the specificity and sensitivity of EIA and other serological tests has demonstrated that EIA has high diagnostic effectiveness in the diagnosis of acute and chronic brucellosis. The presence of direct correlation between the results of EIA and Coombs' test is observed, which is indicative of the capacity of EIA for detecting both complete and incomplete specific antibodies. It should be pointed out that in all cases the titer of specific antibodies in EIA has been found to be 5-16 times higher than in Coombs' test, the passive hemagglutination test, and agglutination test.     

False-Positive Anti-Toxoplasma Fluorescent-Antibody Tests in Patients with Antinuclear Antibodies

The indirect fluorescent-antibody (IFA) method for diagnosis of toxoplasmosis is widely used and is considered to be as specific as the Sabin-Feldman dye test. After observing a patient with systemic lupus erythematosus (SLE) who had a positive toxoplasma IFA test but a negative dye test, we studied sera with high titers of antinuclear antibodies from 16 SLE patients and from 2 with rheumatoid arthritis for Toxoplasma antibodies in the immunoglobulin G and M (IgG and IgM) IFA tests and the dye test. Results of these tests were compared with titers of antinuclear antibodies, precipitating antibodies to single-strand deoxyribonucleic acid (DNA), and binding antibodies by use of DNA labeled with (3)H-actinomycin D. Of 18 patients, 11 had IgG and 4 had IgM IFA Toxoplasma antibodies; only 2 had antibodies detectable in the dye test. The immunofluorescence patterns in the Toxoplasma IFA test were indistinguishable from those obtained in patients with toxoplasmosis without antinuclear antibodies. Absorption of SLE sera with DNA did not result in a decrease in Toxoplasma IFA titers. When SLE sera were absorbed with live T. gondii, a marked drop in IgG IFA titer was observed as well as a decrease in titers of antinuclear antibodies and (3)H-DNA binding. Treatment of Toxoplasma cells with deoxyribonuclease and ribonuclease did not decrease their fluorescence. These results suggest that T. gondii nuclear antigens can absorb antinuclear antibodies but do not have exposed substrates for deoxyribonuclease. Tests in which organisms containing "nuclear" antigens for IFA detection of antibodies to these organisms are used may result in "false-positives" with sera containing antinuclear antibodies.     

Detection of Brucella antigens by dot immunoassay using specific antibodies labelled with colloid gold particles]

Dot immunoassay was developed to improve the quality of laboratory diagnosis of brucellosis. Particles of colloid gold were used as a marker of specific antibodies. The method was used for detecting Brucella antigens in artificially contaminated environmental objects (soil and water) and in biological material (milk, blood serum, and visceral homogenates of animals). The sensitivity of the test system was 19.5.10(3)-62.5.10(4) CFU/ml. Specificity of the assay was tested with 10 heterologous antigenically closely related bacterial species. The proposed test system is simple, economic, highly sensitive and specific, and requires no expensive equipment and reagents.     

FACTS AND FALLACIES ABOUT GONORRHEA AND SYPHILIS

The limitations and special usefulness of clinical and laboratory diagnostic techniques in the diagnosis of gonorrhea are poorly understood and utilized by the average practitioner today. Most physicians and clinics, lulled by complacency or lack of ancillary aid in the area of diagnosis, proceed by measures based in many instances upon past fallacy rather than upon the facts recently developed by research in this disease. The same circumstances apply concerning treatment and management of this disease, particularly in females.All physicians are potentially capable of giving excellent treatment for syphilis today. The problem is to properly diagnose the disease, manage the patient and deal with the source. Looming large in the area of diagnosis is the interpretation of serologic tests for syphilis. No serologic test diagnoses syphilis, but rather gives information as to the immunologic status of the the patient in relation to reagin and treponemal antibodies. None of the antibodies measured in these tests are absolutely specific for syphilis alone.There is no substitute for a well-informed physician, who knows his patient, to relate and interpret even the best of treponemal serologic tests.     

Dot immunodetection for sphingomyelinase with monoclonal antibody

An assay for the detection of sphingomyelinase with monoclonal antibodies is described. The assay takes advantage of nitrocellulose membranes as antigen adsorbent on which a dilute sample can be concentrated as a spot, using a specially designed 96-well filtration device which is commercially available. The method requires only 6 micrograms of the extracts from leukocytes and liver, and it is 10 times more sensitive than the colorimetric assay. This reduced amount of sample material also has the merit of requiring only a 0.5-ml blood sample from patients. The combination of this dot immunoassay with the monoclonal antibody allows a sensitive and a specific assay, and is also applicable as a screening test on a large number of samples. Furthermore, the possibility of differential diagnosis of Niemann-Pick disease types by detecting isoenzymes by this method was examined after isoelectric focusing of placental sphingomyelinase.     

Etiology of intestinal Enterobacteriaceae infections in young children

Enterobacteria, potentially capable of causing infections, were isolated from the feces of 88.7% of young children in the intestinal department of an infectious disease hospital. Opportunistic bacteria were considered to be the causative agents of infections only in cases of their high concentration in the material under test. In about a half of the cases the etiological role of the suspected microorganisms was confirmed by the detection of antibodies in low titers. The presence of maternal antibodies did not interfere with diagnostic procedures. The detection of antibodies to autocultures, even in a single case, is of diagnostic importance in the examination of young children. Autoserologically confirmed mixed infection was found to take a more prolonged course than autoserologically confirmed monoinfection.     

Evaluation of the usefulness of the ELISA method for determining the level of enterovirus antibodies in serum and cerebrospinal fluid

The usefulness of the methods was compared: complement fixation test (CFT), neutralization test (NT) and ELISA IgG and IgM against enteroviruses for the evaluation of specific immune reaction in sera and cerebrospinal fluid (CSF) samples of patients with confirmed enterovirus infections. The criteria were established for the assessment of ELISA results in rapid diagnosis of enterovirus neuroinfections. The criteria accepted by the producer lowered the sensitivity of the method and the possibility of recognition of local synthesis of antibodies in the CNS. The use of serum negative in CFT and negative CSF as reference for the determination made possible using of that kit for rapid diagnosis of neuroinfections. The modified ELISA IgG test makes possible determination of antibodies in CSF and serum, and accepting the generally recognized criteria for local production of antibodies in the CNS the ELISA test makes possible rapid diagnosis of neuroinfections which is not possible by other methods.     

Specific antibodies and mycobacterial antigens in pulmonary tuberculosis patients sera. Note I

206 sera collected from different groups of subjects were analyzed by immunoenzymatic methods regarding the content of Mycobacterium tuberculosis specific antibodies and mycobacterial antigens. The results underlined that two assays offered improved serologic diagnosis of tuberculosis over a single antibody test. BCG vaccination interferes with serologic tests for tuberculosis when polyspecific antibodies or mixture of common and specific antigens are used as immunologic reagents. A mycobacterial antigens circadian variation in correlation with vesperal fever in tuberculous patients was not revealed. The mycobacterial antigens seem to become undetectable, in sera, after 6 months of efficient treatment.     

Use of microcapsular leptospiral antigens for detecting specific antibodies

The sensitivity of microcapsular leptospiral antigens, produced by Japan Lyophilization Laboratory and intended for use in tests for the detection of antibodies to leptospires in the sera of experimentally immunized laboratory animals, were studied. The comparative study of the microcapsular agglutination (MCA) test and other serological tests, such as the microagglutination (MA) test and the indirect enzyme immunoassay (EIA), was made. The leptospiral antigens under study were found to actively react with serospecific and group-specific antibodies. In infected guinea pigs and rabbits specific antibodies could be detected from days 3-4 in the MCA test and only from days 5-7 in the MA test. The average antibody level determined by titration in the MCA test was 3.3 times higher and in indirect EIA, 4.3 times higher than that determined by titration in the MA test. These data make it possible to recommend the use of microcapsular leptospiral antigens for the early diagnosis of leptospirosis.     

Limitations of immunofluorescence tests in the diagnosis of infectious mononucleosis

The relative value of heterophil agglutinins (HA) and of specific EBV antibodies in the diagnosis of infectious mononucleosis (IM) was assessed in 108 cases of the disease and in 280 controls. Among the 108 cases 93 were HA-positive by sheep cells in at least one of their sera, while 15 were HA-negative by the same test. Among the 280 controls false-positive HA tests were not encountered except in eight cases with the horse cell microtitre tests. With one of the two slide tests at least two false-positive tests and 12 false-negative tests were also found but these sera had low titres in microtitre tests. The HA life-span was found to be unexpectedly long in a few cases, sheep cell HA lasting up to 8 to 10 months and horse cell HA up to 21 to 23 months.Many false-positive tests may therefore not be true false-positives and may result from the persistence of HA following unrecognized mononucleosis months before. Virtually all cases of IM had (or developed) antibodies to Epstein-Barr virus, viral capsid antigen (EBV-VCA), whereas only half of the controls were EBV-VCA-positive. The comparative analysis of nonspecific and specific test results in mononucleosis allows the following conclusions: (1) horse cell microtitre tests and the monospot test are more sensitive than sheep cell microtitre tests and the monotest; (2) false-negative results are occasionally seen with the latter tests but not with the former; (3) more false-positive results, however, are probably seen with the former tests; and (4) specific EBV-IgM and EBV-EA antibody tests are useful in the diagnosis of selected borderline cases of mononucleosis.     

Vimentin-typing in diagnostic surgical pathology: a comparative study using four antibodies after different fixations

Vimentin-typing was carried out on various normal and neoplastic tissues using four anti-vimentin antibodies in order to evaluate the effect of different fixation treatments on tissue reactivity in comparison to the results obtained on frozen sections. All antisera were reactive on frozen material; on paraffin embedded material staining of tissues depended on the type of fixation method applied (formalin, methacarn or absolute alcohol) and each antibody behaved differently in relation to the fixative used. Only mesenchymal normal structures were revealed on frozen material whilst on paraffin embedded material three of the four antibodies reacted also with non-mesenchymal normal structures (epithelia, central and peripheral nervous system cells). All four antibodies decorated, regardless of treatment, neoplastic cells of mesenchymal and non-mesenchymal derivation, but not germ cells or germ cell tumors. The reactivity of vimentin to its specific antibodies depends on the fixative used: therefore, in routine pathology more than one antiserum should be available for testing. Furthermore, given the variety of non-mesenchymal structures stained by the anti-vimentin antibodies, the differential diagnosis of undifferentiated tumors must not be based on vimentin positivity alone. The expression of vimentin by non-mesenchymal neoplastic cells seems to parallel that of normal tissues during embryogenesis; therefore, this intermediate filament appears to be not only a marker of mesenchymal cells but also of many immature elements.     

Detection of antibodies to DNA: a technical assessment

Antibodies to DNA can be found in the circulation of the majority of patients with Systemic Lupus Erythematosus (SLE). They are quite specific for this disease, which makes their detection an important diagnostic aid to the clinician. Fluctuations in the level of anti-dsDNA in an individual patient generally parallel the clinical state of that patient. Furthermore, the presence of anti-dsDNA may precede the diagnosis of SLE by more than a year. Four methods relevant for the measurement of anti-dsDNA antibodies are discussed in this paper: the ELISA, the indirect immunofluorescence test onCrithidia luciliae, the PEG assay, and the Farr assay. Each of these methods detects a part of the spectrum of anti-dsDNA antibodies present in the circulation of an individual patient. The ELISA is the most sensitive method, whereas the Farr assay is the most specific for SLE. However, with the latter method only antibodies of a relative high avidity for DNA are detected. Mild forms of SLE, where patients only have anti-dsDNA of a low avidity in their circulation, may easily be missed by this technique. Clinically, high avidity anti-dsDNA is related with the more frequent occurrence of nephritis, whereas low avidity anti-dsDNA antibodies are more often found in patients with central nervous system involvement.     

The use of trypan blue for counterstaining in the Sepharose bead immunofluorescence test. The application of the test for the demonstration of primate retrovirus-specific antibodies and antigens.

The Sepharose bead immunoflurorescence test was performed by counterstaining the beads with trypan blue. This results in a red staining of negative beads which allows an easy distinction from positive green-fluorescent beads. Sepharose beads conjugated with viral proteins or antiviral antibodies were used to demonstrate Mason-Pfizer monkey virus (MPMW)- and simian sarcoma virus (SSV) - specific antigens or antibodies. The test shows a high sensitivity and specificity and needs a small amount of material.     

The kinetics of specific serum antibodies in sheep infested with sheep bot larvae]

The kinetics of specific antibodies of the blood serum of sheep experimentally infested with 80, 160 and 1000 specimens of Oestrus ovis larvae was examined. The affinity pure serum IgG and the immunoferment analysis (ELISA) were used for qualitative estimation of specific antibodies. It has been shown that the level of specific antibodies correlates with the larval biomass and is connected with ontogenesis of this parasite. The younger animals, which were infested for the first time, are characterized by more intensive production of specific IgG than adult reinfested ones. The ways of immunity response formation in animals infested with Oestrus ovis larvae are considered.     

Detection of antibodies to DNA: a technical assessment.

Antibodies to DNA can be found in the circulation of the majority of patients with Systemic Lupus Erythematosus (SLE). They are quite specific for this disease, which makes their detection an important diagnostic aid to the clinician. Fluctuations in the level of anti-dsDNA in an individual patient generally parallel the clinical state of that patient. Furthermore, the presence of anti-dsDNA may precede the diagnosis of SLE by more than a year. Four methods relevant for the measurement of anti-dsDNA antibodies are discussed in this paper: the ELISA, the indirect immunofluorescence test on Crithidia luciliae, the PEG assay, and the Farr assay. Each of these methods detects a part of the spectrum of anti-dsDNA antibodies present in the circulation of an individual patient. The ELISA is the most sensitive method, whereas the Farr assay is the most specific for SLE. However, with the latter method only antibodies of a relative high avidity for DNA are detected. Mild forms of SLE, where patients only have anti-dsDNA of a low avidity in their circulation, may easily be missed by this technique. Clinically, high avidity anti-dsDNA is related with the more frequent occurrence of nephritis, whereas low avidity anti-dsDNA antibodies are more often found in patients with central nervous system involvement.     

Results of serological reactions in patients with presumptive diagnosis of toxoplasmosis]

Even though Toxoplasma gondii is an ubiquitous parasite that can effect most of human structures and organs, not all clinical manifestations suggestive of being produced by it are caused by this protozoon. For these reasons sera samples of patients suspected of having toxoplasmosis are sent to the laboratory for detecting specific antibodies which would facilitate the differential diagnosis. Thus, 716 sera from suspected patients, mainly from the Metropolitan Region of Chile, were sent to the Parasitology Laboratory of Chile University in order to carry out in them, specific serological tests for toxoplasmosis: indirect hemagglutination test (IHAT), Sabin Feldman reaction (SFT) and complement fixation test (CFT). Were considered positive: IHAT and/or SFT with titers > or = 1:16 and CFT with titer > or = 1:5. The pathologies for demanding these serological tests were obstetrical problems 210 (29.3%), congenital problems 193 (27.0%), ophthalmopathies 81 (11.3%), adenopathies 77 (10.8%), AIDS 67 (9.4%), myocardiopathies 46 (6.4%) and miscellaneous 42 (5.9%). The positivity found in these sera was higher in ophthalmopathies (61.7%), followed by obstetrical problems, miscellaneous problems, myocardiopathies and AIDS (50.7-52.4%), less frequent was the positivity in adenopathies (35.1%) and congenital problems (23.1%). In general, the 43.7% of positivity for toxoplasmosis found in these patients is higher than the 37.0% found in the general population. High titers of IHAT and SFT plus positive CFT was found in 13-fold higher proportion than in the general population.     

Diagnosis of ovine enzootic abortion using an indirect ELISA (rOMP91B iELISA) based on a recombinant protein fragment of the polymorphic outer membrane protein POMP91B of Chlamydophila abortus

Chlamydophila abortus is of major economic importance worldwide as one of the principal causes of abortion in sheep. Serological diagnosis of infection by the complement fixation test (CFT) is complicated by false positive reactions resulting from cross-reactive antibodies to Chlamydophila pecorum. To improve diagnosis an indirect enzyme-linked immunosorbent assay (iELISA) based on a recombinant protein fragment of the C. abortus polymorphic outer membrane protein POMP91B (rOMP91B iELISA) was assessed using a panel of 281 sera from experimentally and naturally infected sheep. The iELISA performed well, being more sensitive (84.2%) and specific (98.5%) than the CFT. Furthermore, the iELISA was better at differentiating C. abortus- from C. pecorum-infected animals. The new rOMP91B iELISA test will prove a valuable tool for the routine serodiagnosis of C. abortus infection.     

A new type of anti-ganglioside antibodies present in neurological patients

High titers of anti-GA1 antibodies have been associated with neurological syndromes. In most cases, these antibodies cross-react with the structurally related glycolipids GM1 and GD1b, although specific anti-GA1 antibodies have also been reported. The role of specific anti-GA1 antibodies is uncertain since the presence of GA1 in the human nervous system has not been clarified. A rabbit was immunized with GD1a and its sera were screened for antibody reactivity by standard immunoassay methods (HPTLC-immunostaining and ELISA). Anti-GD1a antibodies were not detected but, unexpectedly, anti-GA1 IgG-antibodies were found. Antibody binding to GA1 was inhibited by soluble GA1 but also by GD1a. These results indicate that the rabbit produced antibodies that recognize epitopes present on the glycolipids, that are absent or not exposed on solid phase adsorbed GD1a. We investigated the presence of these unusual anti-ganglioside antibodies in normal and neurological patient sera. Approximately, 10% of normal human sera contained low titer of specific anti-GA1 IgG-antibodies but none of them recognized soluble GD1a. High titers of IgG-antibodies reacting only with GA1 were detected in 12 patient sera out of 325 analyzed. Of these, 6 sera showed binding that was inhibited by soluble GD1a and four of them also by GM1. This new type of anti-ganglioside antibodies should be considered important elements for understanding of the pathogenesis of these diseases as well as their diagnosis.     

Reversal of snake neurotoxin binding to mammalian acetylcholine receptor by specific antiserum

Snake curaremimetic toxins are known to bind to the nicotinic acetylcholine receptor (AcChoR) [Changeux et al. (1970) Proc. Natl Acad. Sci. USA, 67, 1241-1247], thus blocking neuromuscular transmission, and producing respiratory failure in mammals. In the present paper we show that the toxic effects of Naja nigricollis toxin alpha to mammals can be efficiently reversed by toxin-alpha-specific antibodies. In vivo we observed that return to normal breathing in toxin-alpha-intoxicated and ventilated rats was 12 times faster after injection of specific antiserum or monoclonal antibody (M-alpha 1) as compared with control animals. Ex vivo we observed that return to normal contraction of a toxin-alpha-blocked phrenic nerve-hemidiaphragm preparation was 14 times more rapid after treatment with specific antiserum than after washings. In vitro we observed that antibodies accelerated the reversal of binding of [3H]toxin alpha to AcChoR prepared from rat diaphragm. The observation made in vitro furthermore indicates that antibodies are capable of destabilizing the [3H]toxin-AcChoR complex. A similar destabilization phenomenon occurs also in vivo, as inferred from measurements of receptor occupancy by [3H]toxin alpha in diaphragm of anaesthetized rats in the presence or absence of antibodies. The property of antibodies to reverse neurotoxin binding to AcChoR may be considered as a critical test for evaluation of the quality of a neurotoxin-specific antisera.