Chlamydophila pneumoniae diagnostics: importance of methodology in relation to timing of sampling.

The diagnostic impact of PCR-based detection was compared to single-serum IgM antibody measurement and IgG antibody seroconversion during an outbreak of Chlamydophila pneumoniae in a military community. Nasopharyngeal swabs for PCR-based detection, and serum, were obtained from 127 conscripts during the outbreak. Serum, drawn many months before the outbreak, provided the baseline antibody status. C. pneumoniae IgM and IgG antibodies were assayed using microimmunofluorescence (MIF), enzyme immunoassay (EIA) and recombinant ELISA (rELISA). Two reference standard tests were applied: (i) C. pneumoniae PCR; and (ii) assay of C. pneumoniae IgM antibodies, defined as positive if >or=2 IgM antibody assays (i.e. rELISA with MIF and/or EIA) were positive. In 33 subjects, of whom two tested negative according to IgM antibody assays and IgG seroconversion, C. pneumoniae DNA was detected by PCR. The sensitivities were 79%, 85%, 88% and 68%, respectively, and the specificities were 86%, 84%, 78% and 93%, respectively, for MIF IgM, EIA IgM, rELISA IgM and PCR. In two subjects, acute infection was diagnosed on the basis of IgG antibody seroconversion alone. The sensitivity of PCR detection was lower than that of any IgM antibody assay. This may be explained by the late sampling, or clearance of the organism following antibiotic treatment. The results of assay evaluation studies are affected not only by the choice of reference standard tests, but also by the timing of sampling for the different test principles used. On the basis of these findings, a combination of nasopharyngeal swabbing for PCR detection and specific single-serum IgM measurement is recommended in cases of acute respiratory C. pneumoniae infection.

Prevention of antibiotic-associated diarrhoea by a fermented probiotic milk drink.

OBJECTIVE: To study the preventive effect of a milk drink fermented with multistrain probiotics on antibiotic associated diarrhoea (AAD). DESIGN: Double-blind placebo controlled study. SETTING: University Hospital of North Norway. SUBJECTS AND METHODS: Of 853 patients treated with antibiotics, 87 met the inclusion criteria, and were randomized to ingestion of a fermented milk drink containing LGG, La-5 and Bb-12 (n=46) or placebo with heat-killed bacteria (n=41), during a period of 14 days. A diary was recorded, and stool samples were collected for microbiological analyses. RESULTS: Sixty-three patients completed the study according to the protocol; two patients (5.9%) in the treatment group and eight (27.6%) in the placebo group developed AAD (P=0.035). The relative risk of developing AAD was 0.21 (95% confidence interval: 0.05-0.93) when given probiotic milk drink. CONCLUSION: A fermented multistrain probiotic milk drink may prevent four of five cases of AAD in adult hospitalized patients. SPONSORSHIP: TINE BA, Oslo, Norway.

Clostridium difficile infections related to antibiotic use and infection control facilities in two university hospitals.

We investigated whether a reduction in antibiotic use at the Aker University Hospital (Aker) led to a reduction in Clostridium difficile-associated diarrhoea (CDAD). We compared the incidence of CDAD in Aker and Tromsoe University Hospitals (Tromsoe) and related it to antibiotic use and facilities for infection control between 1993-2001. For this purpose we also performed point prevalence studies. Total antibiotic use was the same in the two hospitals. In spite of a reduction in the use of broad-spectrum antibiotics in Aker the incidence of CDAD increased during 1993-1999. In Tromsoe the use of broad-spectrum antibiotics and clindamycin was two to three times higher than in Aker, but until 1999 the incidence of CDAD remained constant and only half that of Aker. After 1999 the incidence of CDAD was halved in Aker, and increased three-fold in Tromsoe. Point prevalence studies in 2001 revealed an equal prevalence of antibiotic-associated diarrhoea. The facilities for infection control were better in Tromsoe. The percentage of single rooms were 8% in Aker and 14% in Tromsoe, and the percentage of single rooms with a WC was 6% in Aker and 12% in Tromsoe. The bed occupancy was much higher in Aker than in Tromsoe. Lack of facilities for infection control and higher bed occupancy could have contributed to the higher incidence of CDAD in Aker in spite of decreased use of broad-spectrum antibiotics and clindamycin. To limit CDAD in hospitals the focus must be on both rational antibiotic use and infection control.

Chlamydia pneumoniae IgA- and IgG antibodies in young survivors of myocardial infarction. A comparison of antibody detection by a microimmunofluorescence test and an enzyme immunoassay.

OBJECTIVES: Chronic Chlamydia pneumoniae infection is considered as a cardiovascular risk factor and antibodies are commonly analysed by the subjective microimmunofluorescence (MIF) test. We wanted to investigate the C. pneumoniae IgA- and IgG seroprevalence in young survivors of myocardial infarction and matched controls, and to compare the agreement of detecting antibodies between a MIF test and an enzyme immunoassay (EIA). DESIGN: A total of 61 patients hospitalized as a result of myocardial infarction, 51 patients hospitalized with chest pain and negative exercise-ECG and 61 age and sex matched controls (mean age 53.3 years, range 40-60 years) were included in this case-control study. Serological comparisons were expressed as sensitivity, specificity and interrater agreement (K or Kw) of the EIA test related to the MIF test. RESULTS: Presence of IgA (cut off = 16) antibodies was significantly higher in coronary heart patients compared with controls for both assays (P = 0.02 by the MIF and P = 0.05 by the EIA test). The presence of IgG (cut off = 32) antibodies was significantly higher amongst patients (P = 0.05) when analysed by the MIF-test, but not with the EIA-test (P = 0.16). The strength of agreement between the assays was good for both IgA- (Kw = 0.67) and IgG (Kw = 0.79) analyses. However, only 52.8% of the IgA samples classified as strong positive (cut-off = 32) by the MIF test were strong positive by the EIA test (K = 0.56). Only 73.2% of the negative IgG samples (<32) by the MIF-test turned out negative by the EIA-test (K = 0.73). CONCLUSIONS: Dependent on assay and cut-off level, there is an increased C. pneumoniae IgA- and IgG seroprevalence in young survivors of myocardial infarction compared with controls. Despite the subjective interpretation of MIF-titres, the strength of agreement between the EIA and MIF tests was good for both antibody classes. However, misclassification of highly positive IgA samples and negative IgG samples by the MIF test may influence study conclusions. We conclude that the choice of serological method is of major importance when evaluating a possible relationship between C. pneumoniae and coronary heart disease.

Immunodeficiency in alpha-mannosidosis: a matched case-control study on immunoglobulins, complement factors, receptor density, phagocytosis and intracellular killing in leucocytes.

UNLABELLED: Patients with the autosomal recessive lysosomal storage disease alpha-mannosidosis suffer from recurrent infections. To study the mechanisms of this immunodeficiency, six patients were matched against six healthy controls and their humoral and cellular immunocompetence investigated. No differences in the number of circulating leucocytes including B-cells, levels of immunoglobulin main classes, nor IgG subclasses were observed. However, post-immunisation serum levels of specific antibodies against poliovirus, diphtheria toxin and tetanus toxin were significantly reduced. In patients, the density of the complement-binding receptor CD11b and the Fc-receptor CD16 was significantly enhanced on monocytes and polymorphonuclear neutrophils (PMN) and the number of phagocytosing PMN was significantly increased in the presence of pooled human serum. This was not observed in the presence of autologous serum, indicating altered opsonic properties. Also in normal PMN, phagocytosis was inhibited by a factor in the serum from the patients. Despite maintained oxidative burst, patient PMN demonstrated insufficient intracellular bacterial killing. CONCLUSION: Our data indicate that patients with alpha-mannosidosis have an immunodeficiency at both the humoral and cellular level.

Immunity to diphtheria among children in Northern Norway and North-Western Russia.

In 1990, diphtheria re-emerged in Russia and during the following four-year period the resulting epidemic reached all the Newly Independent States (NIS) of the former Soviet Union. Several neighbouring countries of NIS, Norway included, have experienced sporadic imported cases. A sero-epidemiological study among children in Northern Norway and North-Western Russia was performed in order to evaluate protection against diphtheria and how differences in vaccination programmes affect immunity. A total of 664 sera, 400 from Norwegian and 264 from Russian children, were examined for antibodies against diphtheria, using an in vitro toxin neutralisation method. The Russian children studied had satisfactory protection in all age groups examined. The Norwegian children had poor protection against diphtheria from the age of 7 years until they received the booster dose at the age of 11. Therefore, a revision of the Norwegian vaccination programme, including a booster dose at school-entry age, seems warranted.

Detection of diphtheria antitoxin by four different methods.

OBJECTIVE: To investigate the reliability of the different methods used in Norway and Russia for detection of diphtheria antitoxin. METHODS: One hundred and twenty-two sera were selected among Russian serum samples previously collected for seroepidemiologic studies of diphtheria antitoxin. The sera were selected to cover the total antitoxin range and were analyzed by four different antidiphtheria toxin assays: an in vitro toxin neutralization test using Vero cells (in vitro NT), an in vivo neutralization test using rabbit skin inoculation (in vivo NT), an indirect enzyme immunoassay (EIA) and a passive hemagglutination assay (PHA). The results were expressed according to the international standard as: not protected (<0.01 IU/mL), relatively protected (0.01-0.1 IU/mL) or protected (≥0.1 IU/mL). The sensitivity, specificity and inter-rater agreement (K or Kw) of each method were related to the in vitro NT selected as the reference method. RESULTS: The in vivo NT test corresponded very well with the in vitro NT in its ability to differentiate between protection/relative protection and no protection (sensitivity 97%, specificity 87% and K=0.84). The EIA test showed a high sensitivity (96%), but since many sera were categorized as protected rather than not protected, the specificity (30%) and inter-rater agreement (K=0.29) were low. The PHA test had a very high specificity (100%) but a low sensitivity (86%). CONCLUSIONS: The agreement between the two neutralization tests was high. If none of the neutralization assays is routinely available, the PHA test can be used to predict the need for vaccination on an individual basis but should not be used for seroepidemiologic studies, since the protection rate for diphtheria would be falsely too low, due to the lower sensitivity. The indirect EIA test used in this study should not be used routinely.

The effect of highly purified eicosapentaenoic and docosahexaenoic acids on monocyte phagocytosis in man.

The n-3 fatty acids (FA) from marine sources are known to exert antiinflammatory effects on monocyte function. There is still controversy whether n-3 FA may increase the susceptibility to infections. The present study was designed to assess the effect of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on monocyte phagocytosis and respiratory burst activity. Fifty-eight healthy men were randomized to take a daily supplement of 3.8 g highly purified EPA (n = 20), 3.6 g DHA (n = 19), or corn oil (n = 19) for 7 wk. Mononuclear leukocytes were collected, isolated, and cryopreserved prior to and after dietary supplementation. Paired samples were analyzed in the presence of autologous serum in a crossover design. Monocyte phagocytosis and respiratory burst activity were measured by flow cytometry after ingestion of Escherichia coli. Monocytes retained their phagocytic ability and respiratory burst activity after supplementation. No reduction in internalization of bacteria was registered. Dietary n-3 FA and particularly EPA improved bacterial adherence to the monocyte surface. In the crossover experiments, there was an adverse effect of serum enriched with n-3 FA on bacterial adherence. We conclude that monocytes retain their phagocytic potential after supplementation with purified EPA and DHA.

Retention of phagocytic functions in cryopreserved human monocytes.

Phagocytosis and respiratory burst activity were measured by flow cytometry in fresh and cryopreserved human monocytes, after ingestion of Escherichia coli and Staphylococcus aureus. Mononuclear leukocytes, isolated from 15 healthy donors, were divided into two portions, of which one was examined immediately and the other was cryopreserved for 3 weeks. Morphological characteristics and expression of receptors involved in phagocytosis were similar in fresh and cryopreserved monocytes. Furthermore, both internalization of bacteria and respiratory burst activity remained unchanged after cryopreservation. Transmission electron microscopy confirmed actual internalization of bacteria and not merely bacterial attachment to monocytes. Monocytes were demonstrated to retain integral cellular functions during cryopreservation. This may imply that the method has potential for use in basal and clinical trials.