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External quality assessment in serological diagnostic virology in Norway 1982-84.

Serum specimens for external quality assessment in virology were sent as an open distribution to 10-13 clinical microbiological laboratories during the period 1982-84. Antibodies to rubella were tested by hemagglutination-inhibition (HI); other viral antibodies were tested by complement fixation (CF). The median of the antibody-titres for each annual batch of serum specimens is used to describe and evaluate the laboratory performance. Generally speaking the antibody titres in both tests were found to cover a wide range.

Antibodies, Viral↗

[Assay of specific anti-Chlamydia pneumoniae antibodies by ELISA method. 2. studies on clinical usefulness and serological diagnostic standards].

We measured anti-Chlamydia pneumoniae (C. pneumoniae) specific antibody titers by means of a newly-developed enzyme-linked immunosorbent assay (ELISA) method using an anti-C. pneumoniae specific antibody detection reagent. The clinical usefulness of this method was hereby evaluated. The IgG, IgA and IgM titers in 418 serum specimens obtained from patients with respiratory tract infections were measured by this new ELISA method, and the results were compared with the titers determined for the same specimens with the micro immunofluorescence (Micro-IF) method. The results showed good correlation coefficients for IgG, IgA and IgM. The two assay methods showed high agreement rates for positivity and for negativity. Specimens which did not yield the same results with the ELISA method and the Micro-IF method were subjected to analysis by the Western blot method, and the rates of agreement with the ELISA results were high. In addition, the child (0 approximately 15 yrs old; n = 122) and adult (16 approximately 90 yrs old; n = 133) cases were classified on the basis of being antigen-positive or antigen-negative at the initial examination, and their antibody-positive rates were determined. The adults showed no statistically significant differences in the antibody-positive rates for either IgG or IgA antibodies as a function of the pretreatment antigen status. However, the children showed statistically significant (p < 0.001) differences in the antibody-positive rates for both IgG and IgA antibodies as a function of the antigen status in the antigen-positive group compared with the rates in the antigen-negative group. Furthermore, the IgM-positive rates for the children were high in the antigen-positive group compared with the rates in the antigen-negative group, and the difference was statistically significant (p < 0.001). The IgM-positive rates in the adults were also significantly (p < 0.05) different between the antigen-positive group and the antigen-negative group. The Micro-IF method was applied to 34 specimens from antigen-positive patients, and 22 specimens were found to show an IgG titer of > or = 512 or an IgM titer of > or = 16. The diagnoses of these patients were acute respiratory disease in sixteen, pneumonia in four. Application of the ELISA-method to those 22 specimens showed all of them to exhibit IgG absorbance of > or = 0.6 and IgA absorbance of 0.2. The results described above indicate the clinical usefulness of our new ELISA method for the detection of antibodies specific for C. pneumoniae. The significance of this ELISA method for serological diagnosis of C. pneumoniae infections and the criteria for diagnosis of acute infections were also discussed.

Adolescent↗

[Hepatitis A foci in children's collectives studied by using clinico-biochemical, virological and serological diagnostic methods].

In 4 foci of hepatitis A (HA) at children's institutions in Moscow 218 children and 30 staff members were examined. Simultaneously with clinico-biochemical studies, specific IgM and IgG in the blood and hepatitis A virus (HAV) antigen in feces were determined by radioimmunoassay. Different forms of HA were detected in 29.1% of children in kindergartens, 19.4% of school children and 3.3% of adults, which was due to great differences in the size of the immune stratum among them (IgG to HAV was detected in 8-10% of children aged 3-6 years and in 83% of adults). In 2/3 of children with HA in the foci the disease was not accompanied by jaundice, 1/5 of them having the inapparent form of this infection. In 45% of the patients HAV antigen was detected in feces, irrespective of the form of HA, 5-14 days before the appearance of hyperfermentemia, and in 2/3 of them this antigen was also detected during the first 6 days after that. Simultaneously with an increase in aminotransferase activity, all of the HA patients showed the presence of specific IgM in their blood.

Adult↗

Viral RNA in the resolution of human immunodeficiency virus type 1 diagnostic serology.

BACKGROUND: Assays for human immunodeficiency virus (HIV) antibody testing are characterized by very high levels of sensitivity and specificity. Nonetheless, serologic testing of HIV-infected individuals before they produce multispecific HIV antibodies will not detect and confirm those individuals as positive for HIV. This study was carried out to determine whether viral RNA in such specimens is detectable in a quantitative RNA polymerase chain reaction assay. STUDY DESIGN AND METHODS: Study specimens were identified through the United States military HIV testing program. Thirty-five individuals were studied whose HIV type 1 (HIV-1) infection status was serologically inconclusive at initial testing (i.e., reactive on enzyme immunoassay, nondiagnostic or nonreactive on Western blot), but who were documented to be infected (n = 15) or uninfected (n = 20) through the testing of subsequently collected (follow-up) sera. HIV-1 RNA was detected in sera by the use of a commercially available, quantitative, reverse transcriptase-polymerase chain reaction assay. RESULTS: Specimens from 15 individuals subsequently confirmed to be positive for HIV antibody were all positive for HIV-1 RNA. Specimens from 19 of 20 individuals subsequently confirmed to be negative for HIV antibody were negative for HIV-1 RNA. In the one HIV-1 RNA-positive serum from this group, the RNA copy number was low; specimen contamination during prior laboratory testing was suspected. CONCLUSION: Viral RNA was detected in specimens collected for serologic diagnosis of HIV infection in which no special precautions had been taken to preserve the nucleic acid. Such molecular methods potentially add a much needed tool to current HIV testing algorithms.

AIDS Serodiagnosis↗

Stability of freeze dried horseradish peroxidase conjugated monoclonal antibodies used in diagnostic serology.

Two murine monoclonal antibodies, an IgG1 isotype specific for the heavy chain of bovine IgG1, the second an IgG3 isotype and specific for an epitope of the O-polysaccharide of Brucella abortus were conjugated with horseradish peroxidase. The conjugated antibodies were freeze dried in the presence of a number of additives to preserve activity and tested for stability over an 18 month period. Addition of 0.3M trehalose or 0.8% lactalbumin and 3.2% sucrose resulted in the lowest loss of activity if the conjugated antibodies were freeze dried in glass vials. Freeze drying in polypropylene vials resulted in a more rapid rate of deterioration with some additives or an accelerated rate of decline after an initial plateau of lesser loss of activity. The use of polypropylene vials and lactalbumin and sucrose were included in this study because of their low cost compared to glass vials and trehalose. While each antibody behaved differently, addition of trehalose or lactalbumin and sucrose to the conjugated antibodies aided in the preservation of enzyme activity. Both additives provide a suitable method for long term storage of freeze dried or freeze dried and reconstituted monoclonal antibody-enzyme conjugates.

Animals↗

Inducible proteinase of Candida albicans in diagnostic serology and in the pathogenesis of systemic candidosis.

The extracellular acid proteinase of Candida albicans was purified from culture filtrates by a single-column chromatographic step. The purity of the enzyme and its unique antigenic properties were confirmed by polyacrylamide-gel electrophoresis and by reaction with homologous and heterologous anti-sera. The purified enzyme (PP), which was a carboxyl proteinase, contained mannan as an integral part of the molecule. C. albicans proteinase was detected in experimental candida kidney lesions by indirect immunoflourescence. Precipitating antibodies to PP and to cytoplasmic extract (CE) were detected in sera from rabbits with chronic, experimental, systemic candidosis; however precipitins to PP were not found in sera from infected rabbits in which tissue invasion was prevented by antifungal treatment. In retrospective tests with sera from healthy subjects and from patients with and without proven systemic candidosis a qualitative distinction between true and false-positive precipitins to PP was not found; however, whereas 72% of sera from proven cases of deep-seated candida infection had anti-PP titres greater than 4 and greater than or equal to anti-CE titres, these same quantitative criteria were met by only 15% of sera from patients for whom information of a diagnosis of candidosis was not available. The purified proteinase was therefore a more specific antigen than the widely used cytoplasmic extract for detection of antibodies in cases of candidosis.

Animals↗

Simplified radioimmunoassay for diagnostic serology.

A simplified, indirect radioimmunoassay is described for Escherichia coli, vaccinia virus, and herpesvirus. The antigens were affixed to glass cover slips; thus both the primary and secondary reactions take place on the cover slips, and the unbound antiserum is easily separated from the bound antiserum by rinsing. Rabbit or human immune sera were reacted with the antigens, and the primary immune complex was quantitated by a secondary reaction with (125)I-indicator globulin (anti-rabbit or anti-human). A direct relationship between the antiserum concentration and the (125)I absorption was established. Variations in titers were detectable, and the titers were comparable to complement fixation titers. Homologous and heterologous reactions were distinguishable. The method affords an objective, quantitative, and qualitative evaluation of antibody, and results are reproducible.

Absorption↗

Chlamydia trachomatis serology: diagnostic value of outer membrane protein 2 compared with that of other antigens.

Different immunoassays using recombinant antigens or synthetic peptides were evaluated for the serodiagnosis of Chlamydia trachomatis infections. Antigens used included cysteine-rich outer membrane protein 2 (OMP2), heat shock protein 60, the polypeptide encoded by open reading frame 3 of the plasmid (pgp3), synthetic peptides derived from species-specific epitopes in variable domain IV of the major OMP (MOMP) (Labsystems, Helsinki, Finland), and a fragment of the total lipopolysaccharide (Medac, Hamburg, Germany). Because cross-reactions between chlamydial species have been reported, Chlamydia pneumoniae-specific antibodies were also determined by immunoassays (Labsystems). Responses obtained with serum samples from patients with well-defined diseases (i.e., urethral or endocervical samples from which C. trachomatis DNA was amplified) were compared to those obtained with samples from healthy blood donors. The best sensitivity (79%) associated with the best specificity (82%) was obtained when immunoglobulin G (IgG) responses to both MOMP and pgp3 were considered. The highest sensitivity (89%) was obtained with anti-OMP2 IgG, but the lowest specificity (57%) was obtained with this antibody, due to probable cross-reactivity with C. pneumoniae OMP2.

Adult↗

Diagnostic serologic testing of cage and aviary birds for chlamydiosis and suggested confirmatory testing.

A 2 x 2 contingency table was constructed to demonstrate the relationships between detectable chlamydial antibody activity and clinical health status of tested birds. The table revealed that 65.5% of clinically ill birds were antibody positive by elementary body agglutination (EBA) (> or = 10 titers) and 59.0% were antibody positive by latex agglutination (LA). Thus, EBA was slightly more sensitive than LA in detecting antibody activity. Of the clinically normal birds, 96.7% were antibody negative (< 10 titers) by EBA and 98.3% were antibody negative by LA. Individual serum or plasma samples from a group of mixed types of psittacine birds and cockatiels were tested as a separate group, and relationships between EBA-detectable antibody activity and health status were obtained from a 2 x 2 contingency table. Sixty-six percent of birds clinically ill with signs of chlamydiosis in the mixed-type group were antibody positive, whereas only 32.3% of clinically ill cockatiels were antibody positive. Statistical analysis of the contingency table using a chi-square test demonstrated that the EBA test differentiates between individual birds on the basis of health status (P < 0.001). When testing paired serum or plasma samples by EBA, LA, and direct complement fixation (DCF), the highest percentage of significant (> or =4-fold change) titer decreases was detected by LA, and the highest percentage of significant titer increases was detected by DCF. Examples of EBA, LA, and DCF titers in paired and multiple serum or plasma samples are presented to show the variety of responses that can occur. Results reflected variations seen in individual testing of birds with titer variability seen in the first sample tested. Additional types of testing believed necessary for confirming or ruling out an infectious process in birds are outlined. The current interpretations of serologic results are given.

Agglutination Tests↗

[Congenital infection: diagnostic serology of the mother not always definitive].

In 2 infants, a girl and a boy, congenital viral infection was diagnosed in the neonatal period. The prenatal examination (serologic investigation for Toxoplasma gondii, rubella virus, cytomegalovirus, herpes simplex virus and syphilis (TORCHES)) was negative. In both cases prenatal ultrasonography was abnormal and suggested intrauterine infection. The infants were born with typical symptoms of multisystem disease, known as symptomatic congenital cytomegalovirus infection (jaundice, petechiae, hepatosplenomegaly, intrauterine growth retardation, microcephaly and cerebral calcifications) and congenital rubella syndrome (intrauterine growth retardation, congenital heart disease, cataract, hepatosplenomegaly and cerebral calcifications), respectively. Both had severe cerebral damage. To diagnose severe congenital infection in the first trimester of pregnancy in presence of congenital anomalies in utero there are other possible methods than TORCHES investigation, such as polymerase chain reaction and virus culture in amniotic fluid or in foetal blood obtained by cord puncture.

Adult↗

Detection of IgE antibodies specific for 1-phenyl-2,3-dimethyl-3-pyrazoline-5-one by RAST: a serological diagnostic method for sensitivity to pyrazoline drugs.

Certain adverse reactions to pyrazoline drugs resemble IgE-mediated hypersensitivity. However, convincing evidence of antigen-antibody interactions is not fully demonstrated. In this study, IgE antibodies specific for 1-phenyl-2,3-dimethyl-3-pyrazoline-5-one have been found in 17 out of 19 serum samples from individuals sensitive to pyrazoline drugs with 4-aminoantipyrine discs by Radio Allergo Sorbent Test (RAST). In contrast, we have not found any positive results from 10 normal donors without sensitivity to pyrazoline drugs after ingestion of metamizol 500 mg/day for 14 days. Therefore, our results provide further evidence in favor of an IgE-dependent mechanism in patients suffering from sensitivity to pyrazoline drugs. The determination of specific IgE antibodies could be used as a serodiagnostics method.

Adult↗