Relapsing sepsis episodes of Escherichia coli with CTX-M ESBL or derepressed ampC genes in a patient with chronic autoimmune pancreatitis complicated by IgG4 hypergammaglobulinaemia.

Bloodstream recurrent infections have been reported for a variety of opportunistic bacteria. These are often either catheter related or are caused by indwelling devices. A case of relapsing sepsis with two Escherichia coli strains carrying extended-spectrum ß-lactamase and derepressed ampC genes is reported. The patient had seven episodes of bloodstream infections within 1 year and was diagnosed with chronic autoimmune pancreatitis and IgG4 hypergammaglobulinaemia. Abscesses were found in his spleen and pancreas cauda, which was finally resected. Relapses of bacteraemia with resistant enterobacteria should be considered during perioperative protection. Surgical removal of the infective focus could be curative.

Actinobaculum schaalii: identification with MALDI-TOF.

Actinobaculum schaalii is an emerging uropathogen. So far, its identification has been performed with 16S rRNA gene sequencing or PCR. The diagnosis has often been delayed due to fastidious growth and identification problems. Eleven clinical isolates of A. schaalii from bloodstream infections that were initially identified with 16S rRNA sequencing analysis were recovered and later identified with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF). We present a review of bacteriological data of these patients, an algorithm for fast laboratory work-up and advocate the use of sensitized culture of urine to allow better recovery of A. schaalii in susceptible patients.

Effector memory T-cells dominate immune responses in tuberculosis treatment: antigen or bacteria persistence?

OBJECTIVE: To compare ex vivo immunological responses upon stimulation of lymphocytes with Mycobacterium tuberculosis-specific antigens in three groups: 1) subjects diagnosed with tuberculosis (TB) in the early 1940s and 1950s but who did not receive anti-tuberculosis chemotherapy (n = 5), 2) subjects treated with anti-tuberculosis agents prior to the rifampicin (RMP) era (n = 26) and 3) subjects who received RMP as a part of modern combination therapy (n = 7). DESIGN: A total of 38 healthy subjects, mean age 70 +/- 13 years, with a history of previously treated TB were recruited. Peripheral blood mononuclear cells were collected and analysed as a batch by ELISpot. Representative samples with high reactivities were further immunophenotypically characterised. RESULTS: No differences between the studied groups were detected with regard to the frequencies of reactive lymphocytes. The dominant immunophenotypic profile of the representative responders, irrespective of the treatment schemes, was CD4+CD45RO+CD45RA-CD27-CD28-CCR7-, compatible with the fast reacting effector memory T-cell lineage (T(EM)). CONCLUSION: Specific T(EM) cells persist even in subjects treated for TB decades ago with modern anti-tuberculosis chemotherapy. Additional studies are needed to address the question of what drives the survival of T(EM) after adequate treatment: persistence of antigens or bacteria.

Feasibility of commercial interferon-gamma-based methods for the diagnosis of latent Mycobacterium tuberculosis infection in Finland, a country of low incidence and high bacille Calmette-Guérin vaccination coverage.

The performances of the QuantiFERON-TB Gold in Tubes (QFGT), T SPOT-TB (ELISPOT) and the Mantoux test were compared for the diagnosis of latent tuberculosis infection in Finland, a country of low tuberculosis incidence. In Cohort A (16 students), freshly isolated peripheral blood mononuclear cells (PBMCs), and in Cohort B (21 school children), cryopreserved PBMCs, were used for the ELISPOT assay. Cryopreservation of cells in fetal calf serum, but not in serum-free medium, produced false-positive results. Discrepancies between the results of the assays were observed. It was concluded that the accuracy of these ex-vivo methods needs additional evaluation.

Use of quantitative and objective enzyme immunoassays to investigate the possible association between Chlamydia pneumoniae and Mycoplasma pneumoniae antibodies and asthma.

Sera from 150 consecutive patients with established asthma and 150 matched controls were examined for Chlamydia pneumoniae IgG and IgA with commercially available enzyme immunoassays (EIAs) detecting immune response solely to surface proteins of elementary bodies. The assays were also modified to measure combined immune response to surface proteins and family-specific lipopolysaccharide antigen. Mycoplasma pneumoniae IgG and IgA were measured with new commercial EIAs utilising P1-enriched protein fraction as an antigen. No statistically significant differences between the patient groups in terms of prevalence or levels of antibodies to either organism were found with these methods.

Development of enzyme immunoassays to detect salivary sIgA to Chlamydia pneumoniae and Mycoplasma pneumoniae.

Enzyme immunoassays (EIAs) for the detection of secretory IgA antibody (sIgA) to Chlamydia pneumoniae and Mycoplasma pneumoniae from saliva are described. The presence of salivary sIgA in healthy laboratory personnel (mean age 40, range 25-62 years) was detected using conjugates of antibodies directed against secretory and alpha-chain domains. The EIA results for the detection of C pneumoniae sIgA antibodies were confirmed by a sensitive microimmunofluorescence method used as a reference. Circulating IgA antibody levels in sera were also determined using commercial EIAs. Secretory IgA antibodies to both C pneumoniae and M. pneumoniae were detectable only from persons with positive or borderline circulating IgA antibodies. Moreover, C. pneumoniae sIgA was found in the saliva of a clinically healthy person whose serum IgA antibody levels had been constantly elevated during the past 7 years. In conclusion, because of their specificity the described methods could be used in further delineation of the role of anti-C pneumoniae and M. pneumoniae sIgA antibodies. However, owing to the unexpected high frequency of these antibodies in saliva of clinically healthy persons, it seems unlikely that a single sIgA measurement from saliva is diagnostically more powerful than a single IgA measurement from serum to study and interpret the involvement of these pathogens in chronic respiratory diseases.

Multicenter evaluation of the novel enzyme immunoassay based on P1-enriched protein for the detection of Mycoplasma pneumoniae infection.

The aim of the study was to evaluate new Mycoplasma pneumoniae IgG, IgA and IgM EIA methods based on the enrichment of P1-protein (ThermoLabsystems, Helsinki, Finland) (L) for the detection of acute infection. This evaluation was performed in two independent routine clinical microbiology laboratories. The first laboratory used samples preselected by IgG and IgM Platelia enzyme immunoassay (P) and the second used samples preseleced by Serion ELISA Classic M. pneumoniae IgG, IgM (V). The L method was also compared to the FDA approved method of ImmunoWell M. pneumoniae IgG and IgM (G). When the agreement of two methods was applied as a serologic criteria for an acute infection, the following ratios of acute to nonacute infection were calculated 32/86 (totally 118) in the first and 20/72 (totally 92) in the second laboratory. In the first laboratory, the corresponding ratios by methods were 35/83 (sensitivity 100%, specificity 96.5%), 31/87 (sensitivity 97%, specificity 100%), and 55/63 (sensitivity 100%, specificity 79%) for the L, P and G methods, respectively. In the second laboratory, the ratios were 21/71 (sensitivity 100%, specificity 99%), 16/76 (sensitivity 83%, specificity 100%), and 53/39 (sensitivity 100, specificity 69%) for the L, V and G methods, respectively. Taking into account that the tested sample material was preselected by the P and V methods, which may have introduced some bias in their favor, the newly developed L method utilizing P1-enriched protein was found reliable for serodiagnosis of acute M. pneumoniae infection. The method G was the least specific in detection of acute infection.

Improved sensitivity and specificity of enzyme immunoassays with P1-adhesin enriched antigen to detect acute Mycoplasma pneumoniae infection.

An in-house P1-enriched (168-kDA protein) Mycoplasma pneumoniae antigen preparation was compared in IgG, IgA and IgM enzyme immunoassays (EIAs) to the respective EIAs employing crude antigen lysate, antigen prepared by Triton X-114 partition and two commercial antigens, one of which was an ether-extracted antigen and the other a P1-enriched antigen. In addition, three commercial kits from Sanofi Pasteur, Novum Diagnostica and Savyon Diagnostics were also assessed for comparison. Diagnostic sensitivity was studied with paired samples from adults (n=37) with acute respiratory illness interpreted as acute, recent or past infection to M. pneumoniae on the basis of the results of complement fixation test (CFT). If the consensus of at least two methods is taken as the true positive for acute infection, the diagnostic sensitivities of combined IgG and IgM EIAs were 100% for the Platelia(R), Sero MP and in-house EIAs whereas for the Novum EIAs and CFT- 97% and 74%, respectively. Moreover, the sensitivity of the P1-enriched antigen was proven superior on the basis of systematically highest OD(405 nm) ratios between convalescent and acute serum samples. Analytical specificity was studied by screening serum samples from 92 Finnish blood donors and 111 serum samples from cord blood. Diagnostic specificity was studied in a blind testing of 30 paired serum samples from infants with pneumonia of variable etiology. No single misinterpretation of acute infection from the group of samples with other respiratory diseases did occur. The present study confirmed and extended the earlier observations of the usefulness of P1-enriched antigen for reliable serologic diagnosis of acute M. pneumoniae infection.

A preliminary evaluation of a new enzyme immunoassay to detect Chlamydia pneumoniae-specific antibodies.

New enzyme immunoassays (EIAs) for determination of specific IgG, IgA, and IgM antibody titers to Chlamydia pneumoniae were evaluated independently in three research laboratories. Specificity of the EIAs was enhanced by removing LPS from the chlamydial antigen. The performance of these EIAs was evaluated in comparison with the microimmunofluorescence (MIF) test using specimens from: (i) a group of adult patients with community-acquired pneumonia (CAP) previously diagnosed as having an acute chlamydial infection by the complement fixation test or the whole inclusion fluorescence test; (ii) from a group of adult patients with acute respiratory tract infections; and (iii) from a group of young children consecutively presenting with acute respiratory tract infections. The MIF test and the EIAs detected acute infections in paired serum specimens from 12 of 14 patients from the first group. Eleven of these 12 patients were positive in both tests. The MIF test detected seven acute infections in single convalescence serum specimens from eight patients. Two of these were also positive in the EIAs. Paired serum specimens from the second group of adult patients (n=12) were collected during an epidemic of C. pneumoniae. The EIAs detected six acute infections. The MIF test detected two additional patients with acute infections. From the group of young children (n=30), the EIAs detected two patients with acute infections. Our conclusion from this preliminary evaluation is that these EIAs could be useful for laboratory diagnosis of acute C. pneumoniae infections. Comprehensive prospective studies should provide suitable data to calculate the sensitivity, specificity, and predictive values.

The use of serologic tests for the diagnosis of chlamydial infections.

Serology is commonly used for the diagnosis of acute Chlamydia pneumoniae infections and also for the diagnosis of complicated Chlamydia trachomatis infections. Furthermore, recent sero-epidemiological studies have linked C. pneumoniae infection with several diseases traditionally considered non-infectious. The objectives of this mini-review are to critically review and discuss some selected analytical and methodological aspects, controversies and current problems in chlamydial serodiagnosis. To illustrate our views we present some original data of the comparison of current technologies. The review of the literature revealed high variability in methodologies applied to different studies. This observation was supported by our own data, which explains occasional conflicting clinical interpretation. Although the microimmunofluorescence (MIF) technique is generally considered as the gold standard for serodiagnosis of chlamydial infections, assay conditions are highly variable and hence pose a major problem in the interpretation of the results. For instance, many recent studies linking C. pneumoniae and atherosclerosis have utilized MIF techniques with variable threshold criteria for the positivity, in combination with selection bias of cases and controls possibly leading to conflicting results. Variability of assay conditions is also a common problem with Western blots, and interpretation is problematic when both anti-C. pneumoniae and anti-C. trachomatis antibodies are present. Furthermore, there is a lot of disagreement in serological criteria applied to recently emerged enzyme immunoassay (EIA) techniques when these assays are used for acute and non-acute clinical conditions and their association with Chlamydiae. In conclusion, standardization of serological techniques and the development of uniform criteria for interpretation of serologic findings is necessary to increase our knowledge of the biology of Chlamydiae, pathogenesis of any chlamydial infection and chronic infections in particular.

High prevalence of congenital toxoplasmosis in Brazil estimated in a 3-year prospective neonatal screening study.

BACKGROUND: A pilot neonatal screening programme revealed a high (approximately 1 per 4800 live births) prevalence of congenital toxoplasmosis (CT) in the State of Rio Grande do Sul, Brazil. The purpose of this paper was to estimate in a larger prospective study the prevalence of CT in the country. METHODS: At the beginning of the study, an in-house indirect enzyme immunoassay (EIA) was used, to be later replaced with a commercial capture IgM fluorometric enzyme immunoassay (FEIA). Both methods detect specific anti-Toxoplasma gondii IgM-class antibodies eluted from dried blood spots. RESULTS: Of the total of 140,914 samples received from all over the country, 47 cases were identified and confirmed as CT. This finding suggests a prevalence of 1 per 3000 live births. Of the 47 patients, only eight (17%) had clinical manifestations: two had intracranial calcifications, four had retinal scars, one had an intracranial calcification and retinal scars, and one had hepatosplenomegaly with lymphoadenopathy. The testing was paid for by the patients' families who volunteered for the study and gave their informed consent. CONCLUSION: The 3-year prospective study using sensitive detection methods, reliable confirmation, and feedback from clinicians showed that CT has an extraordinarily high prevalence in Brazil, in fact the highest ever reported in the world. Although the long-term efficacy of treatment of CT has not been well documented, in view of the availability of reliable diagnostics, confirmation and monitoring, functional logistics, and networking for screening, the insidious nature of the sequelae and the very high prevalence of the disease, neonatal screening for CT should be considered an alternative to no screening at all.

Prevalence of Chlamydia pneumoniae and Mycoplasma pneumoniae immunoglobulin G and A antibodies in a healthy Finnish population as analyzed by quantitative enzyme immunoassays.

Chlamydia pneumoniae and Mycoplasma pneumoniae immunoglobulin G (IgG) and IgA antibody seroprevalence rates and antibody levels related to age and gender were studied. The samples (n = 742) were collected during a nonepidemic period and analyzed by quantitative enzyme immunoassays (EIAs). Seroprevalence to C. pneumoniae was found to increase sharply in young children, and in the 15- to 19-year-old group it reached levels as high as 70 and 60% for IgG and IgA, respectively. After adolescence, seroprevalence showed a transient decrease and then continued to increase, although less dramatically than in early childhood. In the elderly the seroprevalence of IgG antibodies reached 75 and 100% in women and men, respectively. The corresponding rates of IgA antibodies were 73 and 100%. When a randomly selected subgroup of samples (n = 66) was analyzed in parallel by a microimmunofluorescence test and an EIA for C. pneumoniae IgA antibodies, similar seroprevalence rates were obtained (36 versus 35%). Seroprevalence to M. pneumoniae was already found to increase very sharply in 2- to 4-year-old children, reaching 16% for IgG and 8% for IgA. Seroprevalence to M. pneumoniae also continued to increase in adolescence, but in contrast to that to C. pneumoniae, the increase leveled off at about 40 to 50% in adulthood. In subjects aged over 65 years, prevalence did not exceed 60% for IgG or 35% for IgA. The seroprevalence patterns as well as the medians and variations of levels of C. pneumoniae and M. pneumoniae IgG antibodies were similar to those of corresponding IgA antibodies. Compared to IgG antibodies, IgA antibodies do not seem to be of additional value in the diagnosis of infections caused by these pathogens when single serum specimens are studied.

Improvement of immunoglobulin M capture immunoassay specificity: toxoplasma antibody detection method as a model.

In the Toxoplasma gondii immunoglobulin M (IgM) capture fluorometric enzyme immunoassay used as a model, nonspecific responses due to the binding of human IgM to horseradish peroxidase (HRP) conjugates were observed despite the removal of the Fc portion of the immunoglobulin. This interaction may be mediated through the binding of human IgM to the HRP moiety of the conjugate. Addition of polymerized HRP into the reaction mixture reduced nonspecific signals in the majority of low false-positive serum reactions. Other plausible sites of interaction are conserved epitopes of mouse immunoglobulins presenting antigenic similarities with the allotopes of other species. Fragmentation of mouse antimicrobial IgG to Fab' and selection of proper conjugation procedure improved assay specificity.

Multivariant confirmation of sickle cell disease using a non-radioactive minisequencing reaction.

A non-radioactive solid-phase minisequencing method for confirmation of abnormal hemoglobin variants causing sickle cell disease has been developed. In this method amplified 5'-biotinylated target sequences containing normal and mutation sites are immobilized onto streptavidin-coated microplates. Detection primers corresponding to target sequences are annealed immediately adjacent to the mutation site and single-step, hapten-labeled nucleotide primer extension reactions are performed. The incorporation of the labeled nucleotide is detected through immunological reaction with an enzyme-labeled anti-hapten conjugate and a substrate. The method enables confirmation of mutations of the beta-globin gene variants (Hbs S, C, E D-Punjab, O-Arab) and the alpha-globin gene variant (Hb G-Philadelphia). The test was evaluated using characterized dried blood spot specimens (n = 100) The advantages of the procedure are easy performance and objectiveness. The non-radioactive minisequencing assay will prove helpful for genotyping in neonatal screening for hemoglobinopathies and in prenatal and pre-implantational diagnostics.

Multicenter evaluation of a fluorometric enzyme immunocapture assay to detect toxoplasma-specific immunoglobulin M in dried blood filter paper specimens from newborns.

An easy-to-perform fluorometric enzyme immunocapture assay (FEIA) was developed by Labsystems, Helsinki, Finland, to detect toxoplasma-specific immunoglobulin M (IgM) in dried blood spots. Assay materials were distributed to two sites that have programs in place designed to identify infants born with congenital toxoplasma infection: the Statens Serum Institut, Copenhagen, Denmark, and the New England Regional Newborn Screening Program, Boston, Mass. Each site tested over 700 dried blood samples from healthy newborns to define a cutoff at the 99.5 percentile (5 enzyme immunounits for Copenhagen and 4 enzyme immunounits for Boston). Each site then applied its own cutoff of interpret results for dried blood spots prepared from either adults with serology suggestive of acute infection (Copenhagen) or infants determined to be congenitally infected on the basis of serological criteria (Boston). In Copenhagen, 35 of 38 adult samples were either positive to a small degree or borderline positive for IgA. These samples thus may not represent acute infection. In Boston, of 26 congenitally infected infants, 22 were positive by FEIA. The four infant specimens not positive by FEIA were either negative or borderline positive by the standard Boston assay. These results demonstrate that the IgM FEIA is a potential alternative to other filter paper assay for toxoplasma-specific IgM currently in use for newborns.

Population-based differences in thyrotropin and thyroxine distributions in healthy newborns revealing results from independent reagent evaluation.

An independent evaluation of reagents for the determination of thyroxine and thyrotropin from dried blood spot samples taken from newborns between the third and fifth day of life revealed striking differences in the thyrotropin distribution among newborns from Byelorussia. An analysis of the thyrotropin distribution from Byelorussian newborns showed that 40% of samples had over 5 mIU/l blood. In other European populations comparable in respect to timing of blood collection, this fraction varied from only 1% (Stockholm, Sweden) to 3.7% (Lille, France). The reason for the "shift right" in Byelorussian newborns remains to be further investigated. This shift can be attributed to the synergetic effects of mild-moderate iodine deficiency and/or as yet unidentified environmental factors. The differences observed in cumulative distribution patterns obtained by two commercial methods question the use of absolute figures (such as the proportion of samples over 5 mIU/l) for the purpose of inter-population comparisons.

Analytical quality control in neonatal screening.

This critical review questions the present understanding in monitoring of analytical quality control in neonatal screening. Current status and historical background of the analytical quality control, particularly of the tests intended for the screening of congenital hypothyroidism and some inborn errors of metabolism, is reviewed. The reasons why attempts to standardize immunoassays through the preparation of a so-called "gold standard" (e.g. for thyrotropin) will not resolve noncomparability of results are discussed. The review presents arguments for the necessity of elaboration of international guidelines for methods assessment and comparison with an emphasis on their clinical relevance.

New neonatal thyrotropin enzyme immunoassay with fluorimetric detection: comparison with time-resolved fluoroimmunoassay.

This short communication compares a novel fluorimetric microplate enzyme immunoassay (FEIA) with a commercial time-resolved fluoroimmunoassay for the determination of thyrotropin in dried blood spots. The evaluation was performed using a retrospective study design with newborn blood samples from three screening centres. Non-parametric Spearman rank correlation analysis revealed highly significant positive correlation between methods: rs = 0.465, p < 0.0001 (Hannover), rs = 0.659, p < 0.0001 (Minsk), rs = 0.755, p < 0.0001 (Helsinki). Wilcoxon signed rank test performed for paired FEIA and time-resolved fluoroimmunoassay showed that the results obtained by both tests represented the same distribution (p < 0.0001). The new method, using fluorimetric detection, can be performed with the instrumentation commonly used for the screening of congenital hypothyroidism and phenylketonuria. Results are obtained within three to four hours after arrival of the sample in the laboratory. Preliminary evaluation indicates the method to be a suitable alternative to time-resolved fluoroimmunoassay for neonatal thyroid function screening.