Multicenter analytical evaluation of a high-sensitivity troponin T assay.

BACKGROUND: High-sensitivity cardiac troponin assays are being introduced clinically for earlier diagnosis of acute myocardial infarction (AMI). We evaluated the analytical performance of a high-sensitivity cardiac troponin T assay (hscTnT, Roche Diagnostics) in a multicenter, international trial. METHODS: Three US and 5 European sites evaluated hscTnT on the Modular® Analytics E170, cobas® 6000, Elecsys 2010, and cobas® e 411. Precision, accuracy, reportable range, an inter-laboratory comparison trial, and the 99th percentile of a reference population were assessed. RESULTS: Total imprecision (CVs) were 4.6-36.8% between 3.4 and 10.3 ng/L hscTnT. Assay linearity was up to 10,000 ng/L and the limit of blank and detection were 3 and 5 ng/L, respectively. The 99th percentile reference limit was 14.2 ng/L (n=533). No significant differences between specimen types, assay incubation time, or reagent lots existed. A substantial positive bias (76%) exists between the 4th generation and hscTnT assays at the low end of the measuring range (<50 ng/L). hscTnT serum pool concentrations were within 2SD limits of the mean of means in the comparison trial, indicating comparable results across multiple platforms and laboratories. CONCLUSION: The Roche hscTnT assay conforms to guideline precision requirements and will likely identify additional patients with myocardial injury suspicious for AMI.

Comparison of midregional pro-atrial natriuretic peptide with N-terminal pro-B-type natriuretic peptide in the diagnosis of heart failure.

OBJECTIVES: The concentration of atrial natriuretic peptide (ANP) in the circulation is approximately 10- to 50- fold higher than B-type natriuretic peptide (BNP). We sought to compare the accuracy of midregional pro-atrial natriuretic peptide (MRproANP) measured with a novel sandwich immunoassay with N-terminal pro-B-type natriuretic peptide (NTproBNP) in the diagnosis of heart failure. DESIGN: The diagnosis of heart failure was adjudicated by two independent cardiologists using all available clinical data (including BNP levels) in 287 consecutive patients presenting with dyspnoea to the emergency department (ED). MRproANP and NTproBNP levels were determined at presentation in a blinded fashion. RESULTS: Heart failure was the adjudicated final diagnosis in 154 patients (54%). Median MRproANP was significantly higher in patients with heart failure as compared to patients with other causes of dyspnoea (400 vs. 92 pmol L(-1), P < 0.001). The diagnostic accuracy of MRproANP was very high with an area under the receiver operating characteristic curve of 0.92 and was comparable with that of NTproBNP (0.92, P = 0.791). Moreover, MRproANP provided incremental diagnostic information to BNP and NTproBNP in patients presenting with BNP levels in the grey zone between 100 and 500 pg mL(-1). CONCLUSION: Midregional pro-atrial natriuretic peptide is as accurate in the diagnosis of heart failure as NTproBNP. MRproANP seems to provide incremental information on top of BNP or NT-proBNP in some subgroups and should be further investigated in other studies.

Chlamydia pneumoniae DNA in peripheral venous blood samples from patients with carotid artery stenosis.

In the study presented here, peripheral blood specimens obtained from patients with atherosclerosis were examined for the presence of Chlamydia pneumoniae to determine whether these specimens can be used for routine testing. Chlamydia pneumoniae DNA was detected in 7 of 56 patients with carotid stenosis and in three of four patients with other atherosclerotic diseases, but it was not detected in any of 50 healthy controls or in any of 59 age- and gender-matched patients suffering from other nonatherosclerotic diseases. IgG antibodies indicative of an active Chlamydia pneumoniae infection were detected by microimmunofluorescence in two of nine PCR-positive patients but in none of 41 PCR-negative patients. Four of nine serum samples obtained from PCR-positive patients contained IgA antibodies compared to 5 of 41 samples obtained from PCR-negative patients.

Chlamydia pneumoniae serology: interlaboratory variation in microimmunofluorescence assay results.

The lack of standardization in chlamydia serology has made interpretation of published data difficult. This study was initiated to determine the extent of interlaboratory variation of microimmunofluorescence (MIF) test results for the serodiagnosis of Chlamydia pneumoniae infections. Identical panels of 22 sera were sent to 14 laboratories in eight countries for the determination of IgG and IgM antibodies by MIF. Although there was extensive variation in the numeric titer values, the overall percentage agreement with the reference standard titers from the University of Washington was 80%. For results by serodiagnostic category, the best agreement was for four-fold rise in IgG titers, while the lowest agreement was for negative or low IgG titers. Agreement for IgM titers was 50%-95%. Four laboratories failed to discern false-positive IgM titers possibly because of the presence of rheumatoid factor. Further studies are underway to determine the source of interlaboratory variation for the MIF test.

Detection of seroconversion and persistence of Chlamydia trachomatis antibodies in five different serological tests.

Microimmunofluorescence (MIF), a Chlamydia trachomatis species-specific enzyme immunoassay incorporating lipopolysaccharide-extracted Chlamydia trachomatis L2 elementary bodies, two different synthetic peptide-based species-specific tests, and a recombinant lipopolysaccharide genus-specific test were performed on multiple follow-up sera (n = 104 total) from 16 women with Chlamydia trachomatis-positive cervical swabs. These women included five with IgG seroconversions, five with Chlamydia trachomatis reinfections after initial therapy, and six with serologic follow-up of more than 6 years after antibiotic therapy. Of all the tests employed in this study, MIF IgG reverted earliest to negative titers, while MIF IgA was the least sensitive. The lipopolysaccharide-extracted elementary body enzyme immunoassay exhibited the closest correlation with the MIF test. The highest test sensitivity was observed in one of the synthetic peptide-based tests, which detected earliest seroconversions and longest IgG persistence. The other synthetic peptide-based test gave false-negative results in 2 of 16 women and did not detect seroconversion earlier than the MIF test. Seroconversion and persistence of genus-specific IgG--cross-reactivity with Chlamydia pneumoniae--against lipopolysaccharide were similar to species-specific IgG. A significant serologic response to reinfection was observed only in women with signs of pelvic inflammatory disease. Species-specific tests of high sensitivity and reproducibility are best suited for gynecological diagnostic purposes.

In vitro susceptibilities of Chlamydia pneumoniae isolates from German patients and synergistic activity of antibiotic combinations.

The susceptibilities of six Chlamydia pneumoniae type strains and of six German patient isolates to erythromycin, azithromycin, roxithromycin, clarithromycin, doxycycline, ofloxacin, and rifampin were investigated. MICs and minimal chlamydicidal concentrations were all within the ranges reported previously. Combinations of azithromycin with either ofloxacin, doxycycline, or rifampin, as well as combinations of three antibiotics (rifampin, azithromycin, and ofloxacin or doxycycline), showed synergistic activity against C. pneumoniae.

[Aspergillus antigen detection: comparison of a new sandwich ELISA with the latex agglutination test in patients with histologically proven invasive aspergillosis]. / Aspergillus-Antigen-Nachweis: Vergleich eines neuen Sandwich-ELISA mit dem Latex-Agglutinationstest bei Patienten mit histologisch gesicherter invasiver Aspergillose.

A new sandwich antigen enzyme-linked immunosorbent assay (ELISA) for the detection of Aspergillus galactomannan in blood plasma was compared with the latex-agglutination test by investigation of six patients with histologically proven invasive aspergillosis. The ELISA gave positive results in all six patients while the latex test recognized 5/6 as positive. On an average the antigen ELISA gave positive results 19 days earlier than the latex test.

Infection of an intravenous port system with Metschnikowia pulcherrima Pitt et Miller.

A patient with short bowel syndrome as a consequence of multiple intestinal resections for Crohn's disease, had a port system implanted to improve her nutritional status. One year later she presented with fever, weakness and nighttime sweating. Metschnikowia pulcherrima Pitt et Miller was grown in blood cultures from the port system. After antifungal chemotherapy using fluconazole and removal of the implant, the patient's condition improved markedly and her fever and sweating disappeared. We conclude that Metschnikowia pulcherrima can turn into a human pathogen in patients with indwelling catheters for parenteral nutrition. Chemotherapy with fluconazole and, whenever possible, removal of the implant, appear to be adequate treatment.

[Chlamydia pneumoniae infection in a family]. / Chlamydia-pneumoniae-Infektion in einer Familie.

UNLABELLED: HISTORY AND SYMPTOMS: For 11 weeks a 38-year-old woman had suffered from a respiratory infection with peribronchitis, nocturnal coughing fits and earache. INVESTIGATIONS, TREATMENT AND COURSE: The Chlamydia-CFR titre was raised. Subsequent throat swabs of her husband and two daughters grew Chlamydia pneumoniae (C.p.), but not in her case; 5 days earlier she had been started on roxithromycin. 3 weeks before the patient fell ill her two daughters had a flu-like illness with cough and subfebrile temperature and her husband also had flu-like symptoms, which regressed after few days. Antibiotic treatment with roxithromycin improved the symptoms in the mother and older daughter, but the younger daughter was not given treatment because she had no symptoms at the time the microorganism had been isolated. She developed joint symptoms, like those of reactive arthritis, 12 weeks after the mother's illness had begun, and conjunctivitis 5 weeks later. CONCLUSIONS: It is likely that the daughters had the C.p. infection first and then infected their parents. While the father's and older daughter's illness quickly regressed, the mother became quite ill. Her serology was positive for a primary infection in adulthood, but in the daughters the serology was negative and, despite demonstration of the organism, the diagnosis of acute C.p. infection could not be made.

Evaluation of a new commercial microimmunofluorescence test for detection of antibodies to Chlamydia pneumoniae, Chlamydia trachomatis, and Chlamydia psittaci.

A new commercial test for chlamydial serology, the MRL-Micro-Immunofluorescent Test (MRL; MRL Diagnostics, USA) was compared with the standard microimmunofluorescence test (MIF) using sera from 246 patients. Chlamydia pneumoniae immunoglobulin G (IgG) antibodies were detected in 46.3% (MIF) and 64.2% (MRL) of sera and Chlamydia trachomatis IgG in 23.2% (MIF) and 25.2% (MRL); Chlamydia psittaci IgG antibodies were found with the MRL in 1% of the sera from a general population and in 17.3% of preselected sera with elevated complement fixation titers. Titers were usually higher with the MRL. IgG titers of > or = 1:512 were detected in only 2% of sera using the standard MIF but in 30% using the MRL. In 16 sera from three Chlamydia pneumoniae culture-positive patients, the diagnosis of acute infection could be confirmed serologically in one with the MRL test but in none with the MIF test, indicating a higher sensitivity of the MRL.

Reactions of polyclonal and neutralizing anti-p54 monoclonal antibodies with an isolated, species-specific 54-kilodalton protein of Chlamydia pneumoniae.

A recently described 54-kDa protein has been detected in six type strains and three patient isolates of Chlamydia pneumoniae by immunoblotting with sera from patients positive for antibodies to C. pneumoniae by the microimmunofluorescence test. This protein was not found in either C. trachomatis E or C. psittaci Z 432 as an antigen, confirming its species specificity. The 54-kDa protein was isolated by continuous-elution electrophoresis and immunoglobulin G monoclonal antibodies (MAbs) against the isolated antigen were produced. MAb 8B11E6 reacted only with the 54-kDa band of C. pneumoniae and not with C. trachomatis E or C. psittaci in a Western immunoblot assay. This antibody was purified and tested for neutralizing activity together with three additional anti-p54-active MAbs (8B11E6, 8B11B4, and 10F1C1). In Buffalo green monkey cells, all of the MAbs significantly reduced the infectivity of C. pneumoniae elementary bodies, whereas no neutralizing activity could be observed with C. trachomatis E or C. psittaci Z 432. These results not only confirm the species specificity of the 54-kDa protein but also indicate that this protein might play an important role in the pathogenesis of C. pneumoniae infection. Furthermore, the results suggest a possible protective role of anti-p54 antibodies in an adaptive immune response.

Evidence of labile inhibitors in the detection of Chlamydia trachomatis in cervical specimens by polymerase chain reaction.

A total of 276 cervical swabs (241 from first visits and 35 from follow-up visits) from 241 women were tested for Chlamydia trachomatis by polymerase chain reaction (PCR) and enzyme immunoassay (EIA). Sixty-one smears (53 from first visits and 8 from follow-up visits) from 53 women were stained by direct fluorescent antibody (DFA). Twenty-one (8.7%) women had positive swabs in at least two different tests. All follow-up swabs (collected between 3 days and 3 weeks after the first clinical visit) were positive in at least one test when the woman had been positive at the first visit and no antibiotic treatment had been initiated. Including swabs from follow-up visits and DFA results, the respective sensitivities and specificities of the assays were as follows: PCR, 75.9% and 100%; EIA, 69% and 98.4%. The seven swabs that were false negative by PCR (tested initially after thawing from -20 degrees C) were mailed nonrefrigerated to the assay manufacturer, where they tested true positive. These data point to labile inhibitors of the PCR, predominantly cervical mucus.

Immune response to Chlamydia trachomatis heat-shock protein in infertile female patients and influence of Chlamydia pneumoniae antibodies.

A total of 446 sera from 245 patients with primary or secondary infertility, all of whom were examined laparoscopically, 117 patients with Chlamydia trachomatis-positive cervical swabs, and 84 control persons (50 obstetric patients and 34 female blood donors) were tested for antibodies to Chlamydia trachomatis and to Chlamydia pneumoniae with the microimmunofluorescence (MIF) test. MIF test antibody rates were highest in patients with complete tubal occlusion (73%) and in patients with proven Chlamydia trachomatis infection (74%), whereas only 9 to 10% of the control group showed Chlamydia trachomatis antibodies. Reaction to the 60 kDa antigen of Chlamydia trachomatis, a heat-shock protein (hsp) analogue, has been suggested as a possible marker for the development of chronic sequelae after Chlamydia trachomatis infection. Immunoblot analysis of 222 sera (169 infertility patients, 20 antigen-positive patients, and 33 mothers) showed a significantly higher anti-hsp antibody rate in patients with complete tubal occlusion than in infertility patients with normal fallopian tubes (76% vs. 19%, p < 0.001). The presence of antibodies not only to Chlamydia trachomatis but also to Chlamydia pneumoniae in the MIF test was associated with a significantly higher rate of anti-hsp antibodies and with complete tubal occlusion. This association did not appear to be due to cross-reactivity between Chlamydia pneumoniae and Chlamydia trachomatis antibodies in the MIF test.

Hyalohyphomycoses due to Fusarium spp.--two case reports and review of the literature.

We report a case of a severe Fusarium solani keratitis in a 82-year-old patient with a history of surgical trauma. Antimycotic therapy and keratoplasty led to markedly improved vision. Identification of the fungus was complicated by the fact that the isolate did not produce the typical macroconidia. The second case was a fatal disseminated Fusarium verticillioides infection in a 69-year-old patient during neutropenia after chemotherapy of acute myelogenous leukemia. The patient developed pneumonia, fever, skin lesions, myalgia, and fungaemia. The clinical signs, diagnosis and therapy of localized and disseminated Fusarium infections are outlined and discussed in view of the literature.

Chlamydia trachomatis species specific serology: ImmunoComb Chlamydia bivalent versus microimmunofluorescence (MIF).

The ImmunoComb Chlamydia Bivalent IgG/IgA (Orgenics, Israel) is a new quantitative serologic test that employs LPS extracted Chlamydia trachomatis L2 and LPS extracted Chlamydia pneumoniae elementary bodies on two separate antigenic spots. The Bivalent C. trachomatis specific test results were compared with microimmunofluorescence (MIF), the gold standard of chlamydial species specific serology. For C. trachomatis IgG the Bivalent was highly concordant with the MIF: the rate of positive titres (IgG > or = 1:8) was 10% vs. 11% in 100 blood donors, 18% vs. 16% in 111 obstetric patients (6% antigen prevalence), 26% vs. 22% in sterile women with open (n = 54) and 86% vs. 84% with occluded (n = 51) tubes, and 88% vs. 85% in 103 women with C. trachomatis positive cervical smears. Surprisingly, the Bivalent differed considerably from the MIF in IgA prevalence: in obstetric patients (8% vs. 4%), sterile women with open (13% vs. 6%) and occluded (71% vs. 20%) tubes, and women with positive cervical smears (78% vs. 24%). Bivalent IgA appeared to be more sensitive than MIF IgA and showed a stronger correlation with positive cervical smears in obstetric patients (sensitivity 67% vs. 0%, specificity 95% vs. 96%, positive prediction 44% vs. 0%, negative prediction 98% vs. 94%) and with tubal occlusion in sterile women (sensitivity 71% vs. 20%, specificity 87% vs. 94%, positive prediction 84% vs. 77%, negative prediction 76% vs. 55%). MIF IgM was of little diagnostic help. Supplemental to the often difficult C. trachomatis antigen detection, the easily performed Bivalent IgG/IgA appears to be of great value in routine diagnosis of genital chlamydial infections.

[Comparison of immunodiffusion and counterimmunoelectrophoresis for the detection of precipitating antibodies against Candida and Aspergillus antigens]. / Vergleich von Immundiffusion und Uberwanderungs-Elektrophorese zum Nachweis präzipierender Antikörper gegen Candida- und Aspergillus-Antigene.

Early diagnosis of disseminated fungal infections is a major problem in patients at risk, e.g. patients with malignancies or other severe illnesses. A number of serological tests for the detection of fungal antigens or antibodies can be performed in addition to culture methods. Valuable serological tests exist for the detection of precipitating antibodies to Candida sp. or Aspergillus sp. Precipitating antibodies against intracellular fungal antibodies can be detected by the immunodiffusion test (ID) or by counterimmunoelectrophoresis (CIE). In our study 103 patients' sera were examined in parallel with these two methods for antibodies against Candida sp. and 100 sera for antibodies against Aspergillus sp. The results indicate that counterimmunoelectrophoresis is more sensitive than immunodiffusion, and that the results of CIE also correlate better with elevated titers in other serological tests, e.g. the hemagglutination test or the immunofluorescence test. One of the limitations is that precipitating antibodies cannot be detected until relatively late in the course of infection. This disadvantage is further intensified by the long duration of performance of the immunodiffusion test in the laboratory. In comparison with the ID test, the detection of precipitating antibodies by counterimmunoelectrophoresis shortened the duration of performance in our laboratory by up to 5 days.

Identification of Chlamydia pneumoniae-specific protein antigens in immunoblots.

The immunoblot patterns of 248 sera, all examined previously by the microimmunofluorescence test (MIF) for species-specific Chlamydia antibodies, were analyzed. Predominant specific antibody activity was directed to the 54 kDa protein of Chlamydia pneumoniae, which was recognized by 93% of sera positive for Chlamydia pneumoniae by MIF but by only 2% of sera positive for Chlamydia trachomatis and negative for Chlamydia pneumoniae and by 3% of sera negative for both Chlamydia pneumoniae and Chlamydia trachomatis. This antigen appears to be specific for Chlamydia pneumoniae. Other Chlamydia pneumoniae-specific protein antigens were recognized far less frequently. Absorption analysis indicated that the 54 kDa protein is located on the surface of the Chlamydia pneumoniae elementary bodies.

Comparison of serological tests for the detection of antibodies against Chlamydia trachomatis and Chlamydia pneumoniae in rheumatological patients.

In cases of reactive arthritis, a suspected Chlamydia trachomatis infection is often detected by serological methods. However, mostly tests with genus-specific antigens are used, neglecting the fact that antibodies against Chlamydia pneumoniae are highly prevalent in the adult population. Therefore we tested sera of 129 patients with various rheumatological disorders and of 18 healthy persons in parallel with a genus-specific test (IPAZYME) and with the species-specific microimmunofluorescence test for C. trachomatis and C. pneumoniae antibodies. The data showed that 55% of the 64 IPA-positive results were caused by antibodies (IgG) against Chlamydia pneumoniae, only 6% by anti-Chlamydia trachomatis IgG and 20% by both specificities. For IgA antibodies, the percentages were 44%, 12.5% and 12.5% respectively. In 12 IPA-positive cases, the MIF showed no reaction. 58% of all 147 sera tested with MIF had IgG antibodies against C. pneumoniae, 5% had anti-C. trachomatis IgG and 8% IgG against both species. The percentages for IgA were 29%, 2% and 2%, respectively. IgM positivity in MIF disappeared after absorption with rheumatoid factor absorbent. No significant differences were found between the various groups of patients. The data suggest that due to the high prevalence of anti-C. pneumoniae antibody, genus-species tests cannot be used as screening tests for the serological diagnosis of C. trachomatis infections.

Prevalence of antibodies to Chlamydia pneumoniae TWAR in a group of German medical students.

Prevalence of antibodies to Chlamydia pneumoniae TWAR in Germany has not been previously evaluated. Therefore a healthy adult population of 353 German medical students (mean age 24 years) was examined with a species-specific microimmunofluorescence test for IgG, IgM and IgA antibodies to C. pneumoniae and in parallel to Chlamydia trachomatis. Altogether, 229 persons had IgG antibodies to C. pneumoniae (64.9%), 136 had IgA antibodies (38.5%), while the serum of only one contained specific IgM. Prevalence rates were higher in males (69.4%) than in females (57.3%). The total prevalence for antibodies to C. trachomatis was 5.9%. The results indicate that C. pneumoniae infections are highly endemic in Germany, and that primary infection probably takes place in children or young adults. Prevalence of antibodies to C. pneumoniae in this group of healthy young adults was about 10-fold higher than that to C. trachomatis. This finding needs to be taken into account when genus-specific tests are used for studying Chlamydial antibodies.