A one-tube quantitative HIV-1 RNA NASBA nucleic acid amplification assay using electrochemiluminescent (ECL) labelled probes.

Quantification of HIV-1 viral RNA based on co-amplification of an internal standard Q-RNA dilution series requires a number of NASBA nucleic acid amplification reactions. For each internal standard Q-RNA concentration a separate NASBA amplification has to be performed. The development of an electrochemiluminescent (ECL) detection system with a dynamic signal detection range over 5 orders of magnitude enabled simplification of the Q-NASBA protocol. Three distinguishable Q-RNAs (QA, QB and QC) are mixed with the wild-type sample at different amounts (i.e. 10(4) QA, 10(3) QB and 10(2) QC molecules) and co-amplified with the wild-type RNA in one tube. Using ECL-labelled oligonucleotides the wild-type, QA, QB and QC amplificates are separately detected with a semi-automated ECL detection instrument and the ratio of the signals determined. The amount of initial wild-type RNA can be calculated from the ratio of wild-type signal to QA, QB and QC signals. This one-tube Q-NASBA protocol was compared to the earlier described protocol with six amplifications per quantification using model systems and HIV-1 RNA isolated from plasma of HIV-1-infected individuals. In all cases the quantification results of HIV-1 RNA were comparable between the two methods tested. Due to the use of only one amplification per quantification in the one-tube Q-NASBA protocol the QA, QB and QC internal standard RNA molecules can be added to the sample before nucleic acid isolation. The ratio of QA:QB:QC:WT RNAs, from which the initial input of WT-RNA is calculated, will remain constant independent of any loss that might occur during the nucleic acid isolation.

Identification and molecular characterization of two diagnostically relevant marker proteins of the Epstein-Barr virus capsid antigen complex.

The molecular specificity of the IgG response against Epstein-Barr virus (EBV) was studied in 345 randomly collected sera of normal healthy individuals. The sera were tested on immunoblots containing antigens of the cell line HH514.c16 (a superinducible derivate of P3HR1), noninduced or induced for the expression of early antigens (EA) or viral capsid antigens (VCA), and from the EBV-negative cell line Ramos-Nut. This study reveals a remarkable similar antigen recognition pattern of IgG class antibodies in sera of healthy EBV carriers. The protein bands recognized predominantly have molecular weights of 18 kD, 36/38 kD, 40 kD, 72 kD, and 160 kD. The 72 kD and 36/38 kD bands were identified as EBNA1 and "Zebra," respectively, using reading frame-specific antisera. The bands at 160 kD (major capsid protein), 40 kD, and 18 kD were identified as VCA-class proteins. Of all EBV-seropositive sera tested, 98% reacted with either p18 or p40 or both. The synthesis of the antigens p18 and p40 was inhibited by phosphonoacetic acid, indicating that these were true late proteins. The detection of p18 and p40 in purified virion and capsid preparations confirms that these proteins are structural components of viral capsid antigen complex.

Raised serum IgA to common cell envelope antigens supports enterobacterial inductive contribution to pathogenesis of secondary ankylosing spondylitis.

Ankylosing spondylitis (AS) is closely associated with the histocompatibility antigen HLA-B27. Pathogenesis of AS is thought to involve interactions between B27 and certain enterobacterial antigens. However, enterobacterial involvement is uncertain and contested by some. The present paper demonstrates raised serum IgA to a common enterobacterial heat modifiable major outer membrane protein (h-momp; Mr 35,000) in active AS (N = 25; IgA = 1485 +/- 20) compared with controls, who were hospital patients without known arthropathies or gastro-intestinal disease (N = 12; IgA = 548 +/- 59). Serum IgG and IgM did not differ statistically. Raised serum IgA to h-momp might indicate enterobacterial antigenic stimulation from the gastro-intestinal tract and thus support an inductive contribution of enterobacterial antigens to the pathogenesis of secondary AS. It does not necessarily imply direct involvement in the pathogenesis of primary AS. H-momp appears to be a convenient tool for serological studies of AS and at present is likely to be more suitable than other bacterial antigens.

Lack of serologically defined arthritogenic Shigella flexneri cell envelope antigens in post-dysenteric arthritis.

Post-dysenteric or reactive arthritis (ReA) is closely associated with HLA-B27. This histocompatibility antigen is heterogeneous and consists of 2 serologically defined variants: B27M1+M2+ and B27M1+M2-. This paper gives a qualitative evaluation of the antibodies present in the sera of 62 patients with dysentery due to Shigella flexneri 2a, a known arthritogenic bacterium. The patients were classified in 4 groups: B27M1+M2+ReA+ (n = 5), B27M1+M2+ReA- (n = 7); B27M1+M2-ReA- (n = 1); B27-ReA- (n = 49). The isolated infectant possessed cell envelope antigens with B27M2-like epitopes (Mr 20,000). Analysis of the spectrum of antibodies directed against the separated cell envelope antigens of S. flexneri in the sera of these patients revealed 7 main patterns of reactivity. The detectable immunogens encompassed protein stainable antigens (Mr 98, 78, 68, 54, 50, 44, 41, 35, 14 and 13 kDa), lipopolysaccharides and peptidoglycan. None of the sera possessed detectable antibodies to the B27M2-like antigen. Consequently, this antigen is unlikely to be associated with ReA, and this applies equally to other antigens or patterns of antigens. The arthritogenicity of S. flexneri may therefore not be determined by the presence or absence of detectable antibody titers to certain cell envelope antigens. We hypothesize that other properties of these antigens could be of significance.

HLA-B27M1M2 and high immune responsiveness to Shigella flexneri in post-dysenteric arthritis.

The heterogeneous HLA-B27 antigen is closely associated with post-infectious or reactive arthritis (ReA) and is comprised of two serologically defined variants: B27M1+M2+ and B27M1+M2-. An outbreak of dysentery (n = 120) caused by a Shigella flexneri 2a strain, which possessed cell envelope antigens with epitopes resembling B27M2, resulted in five B27M1+M2+ patients with ReA. The remaining seven B27M1+M2+, one B27M1+M2- and all but three B27-negative patients remained free of joint symptoms; the latter three displayed arthralgia. IgM, IgG and IgA serum titers were statistically raised in all patient groups, but were exceptionally and persistently high in the B27M1+M2+ patients with ReA, especially IgA, as determined in acute-phase sera and sera sampled 1 year after dysentery. B27M1+M2+ thus appears to be a marker for a subset of disease, characterized by a high immune response. It is concluded that the B27M2 epitope is not unequivocally disease-related to Shigella ReA, that B27M1+M2+ is not likely to be the only immune-response-regulating gene involved in this form of ReA and that cross-reactivity between bacterial antigenic epitopes and B27 can only be part of a multifactorial process leading to ReA and in itself not sufficient to produce ReA. The intensity of the immune response appears to be another important factor.

Serum IgA to enterobacteria in ankylosing spondylitis.

The aetiology of ankylosing spondylitis (AS) may involve certain enterobacteria. It is therefore interesting that serum polymeric IgA, a precursor of secretory IgA, was statistically elevated in active AS (n = 35) and that levels were comparable to those found in yersiniosis (n = 12); this might indicate antigenic stimulation by bacteria which are present in the intestines of AS patients. However, specific serum IgA to the incriminated enterobacteria Klebsiella, Shigella and Yersinia, as determined by ELISA, was not raised in the above AS patients. Nor were these titres raised in patients with idiopathic reactive arthritis (n = 21). In contrast, yersiniosis (n = 12) and shigellosis (n = 96) patients displayed marked increases in specific serum IgA titres to the respective infectants. It is proposed that AS may involve a set of enterobacteria rather than a few suspected species. Thus, despite the lack of raised group averages, screening of individual patients for specific IgA to several indicated bacteria might disclose whether or not raised serum IgA is related to enterobacterial activity. Apart from this, the above supports other reports indicating that serum IgA may be a useful parameter to assist in monitoring of disease activity in AS. Finally, it is suggested that study of a homogeneous group of reactive arthritis patients might facilitate aetiological research of seronegative arthropathies such as AS.

Antibodies to Enterobacteriaceae in ankylosing spondylitis.

Serological studies on ankylosing spondylitis (AS; N = 82) show that although statistically more AS patients than controls (N = 24) may possess elevated serum titres to enterobacteria such as Salmonella, Shigella and Yersinia, this does not necessarily imply enterobacterial involvement in AS, as other groups without enteritis or arthropathies that frequent health care facilities (N = 72) may also display this phenomenon, presumably due to increased exposure. Moreover, an inventory of all detectable antibody reactivities to the separated cell envelope antigens of five enterobacterial species suspected of involvement in AS (notably Enterobacter, Klebsiella, Salmonella, Shigella and Yersinia) failed to reveal statistical associations with AS. This might be explained, assuming that the aetiology of AS entails a set of enterobacteria rather than a few individual species. It is proposed that serological studies on AS should be supported by additional information, e.g. that of the faecal carriage, and that these combined studies encompassing other enterobacteria, in addition to Klebsiella, might be fruitful.

Susceptibility and HLA-B27 in post-dysenteric arthropathies.

A recent outbreak of bacillary dysentery in The Netherlands revealed that, despite the close association of HLA-B27 with post-dysenteric or reactive arthritis (ReA), not even in one family did all HLA-B27 positive patients infected by an arthritogenic bacterium, develop ReA. This dissociation shows that additional factors beside B27 may determine susceptibility to ReA.

Susceptibility of 126 isolates of Escherichia coli from the cerebrospinal fluid of neonates to five antibiotics.

One hundred and twenty-six clinical isolates of Escherichia coli from the CSF of neonates, obtained in the Netherlands during 1976 to 1982, were tested for sensitivity to five antibiotics. The usefulness of the generally recommended initial therapy, a combination of ampicillin and gentamicin, is supported in the majority of cases. On the basis of the in-vitro results, cefotaxime would have been effective as a therapy for all cases. Ampicillin and cefuroxime resistance occurred mostly in neonates who had received antibiotics prophylactically, neonates whose mothers had fever during labour or in neonates who had been nursed in incubators for more than one week.

Susceptibility to six antibiotics of group B streptococci isolated from cerebrospinal fluid.

64 clinical isolates of group B streptococci from cerebrospinal fluids of neonates were tested for susceptibility to 6 antibiotics. The strains were obtained in The Netherlands during 7 years. The usefulness of the generally recommended initial therapy, a combination of ampicillin and gentamicin, is supported.