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Despite major improvements in the diagnosis of pathogenic organisms causing acute respiratory infections (ARIs), details of infections caused by atypical pathogens are not well understood, particularly in developing countries. This clinical and epidemiological research was conducted in Bangladesh to explore the prevalence of atypical pathogens in causing childhood pneumonia. Sixty-four children with ARI were studied at the Pediatric Outpatient Department of Dhaka Medical College Hospital, Bangladesh, during September through December 2000. In addition to clinical examination, hematological, radiological, and bacteriological examinations were performed. Antibody titers from paired sera against Mycoplasma pneumoniae and Legionella spp. in the acute and convalescent phases revealed that none of these children were infected with M. pneumoniae, while only one serum sample was positive for L. pneumophila serogroup 4. Antibody titers against Chlamydophila (Chlamydia) pneumoniae, determined by an indirect microimmunofluorescence method, and by an enzyme-linked immunosorbent assay (ELISA) kit (HITAZYME C. pneumoniae kit) indicated that 13 children (20.3%) were infected with C. pneumoniae. Our results indicate a high prevalence rate of C. pneumoniae, suggesting it is as an important causative pathogen of childhood pneumonia in Bangladesh.

BACKGROUND Traditional risk factors of cardiovascular disease do not fully explain the accelerated atherosclerosis present in patients with end-stage renal disease (ESRD). The goal of this study was to identify the association of clinical and laboratory factors including seropositivity for Chlamydia pneumoniae determined by a specific enzyme-linked immunosorbent assay (ELISA) with the presence of coronary artery disease identified by coronary angiography in ESRD patients. METHODS We prospectively enrolled 161 consecutive ESRD patients undergoing haemodialysis for >6 months (106 men, 55 women; mean age 63.1+/-10.2 years; mean dialysis duration 91.3+/-90.1 months). All patients underwent coronary angiography within 1 week after blood sampling. The associations of coronary artery disease with clinical parameters including C. pneumoniae IgA and IgG seropositivity were analysed using multiple logistic regression models. RESULTS Coronary stenosis >50% was found in 102 of 161 haemodialysis patients (63.4%). Of the 102 patients, 75.5% were asymptomatic. Seropositivity for C. pneumoniae IgA was found in patients with coronary stenosis (77 out of 102, 75.5%) more frequently (P

"HITAZYME C. pneumoniae" (or "HITAZYME CPN", for short) is a diagnostic reagent that has been recently developed by adopting an ELISA method for detection of anti-Chlamydia pneumoniae (C. pneumoniae) antibodies. When this reagent is used under a current diagnostic standard that has been set as a provisional standard, however, high antibody positive rates are often produced for both IgG and IgA even using the specimens of healthy persons. So, it is difficult to distinguish C. pneumoniae-infected patients from healthy persons. Therefore, this time, we tried to establish a new diagnostic standard by setting up of special cut-off values for a single serum and rise rates of antibody titers for paired sera to improve the accuracy for diagnosis of C. pneumoniae infection. For a single serum testing, we set a special cut-off value at ID 3.00 for both IgG and IgA, so that most healthy persons fall within the range of the "negative" zone. This value was based on the calculation of "Mean+2SD" using measurement results (or IDs) of healthy persons. When this cut-off value was applied, the rate of > or = ID 3.00 for either IgG or IgA was 7.6% for healthy persons, and 64.9% for infected patients. (The rate reached 76.4% when the highest IDs of multiple specimens taken from each patient for this test were used in calculation) As a diagnostic standard for a single serum, therefore, it was defined that: "If ID is 3.00 or greater for IgG and/or IgA, it is highly likely that the case has an acute or a present infection." Using paired sera, we could confirm almost a linear relationship between the results by HITAZYME CPN and those by micro-IF method. Under micro-If method, if the antibody titer increases four times or greater using paired sera, acute infection is diagnosed. As it was found that the four-fold increase in antibody titer corresponds to the increase of 1.35 in ID for IgG and 1.00 for IgA, we defined a diagnostic standard for paired sera as follows: "If ID increases by 1.35 or greater for IgG, and/or if ID increases by 1.00 or greater for IgA, the case may be diagnosed as acute infection."

Studies were conducted with the goal of developing a kit for assaying anti-Chlamydia pneumoniae antibodies in human serum which would enable judging positive cases with high specificity by means of an objective numerical index. Thus, an enzyme-linked immunosorbent assay (ELISA) method employing a C. pneumoniae outer membrane complex protein was established. Elementary bodies (EB) were purified from the YK-41 strain of C. pneumoniae, and subsequent treatment with Sarkosyl, DNase and RNase yielded chlamydial outer membrane complex (COMC). COMC was employed as the antigen and immobilized on 96-well microplates for ELISA method. This ELISA method was used to test 51 serum specimens from patients who had been demonstrated to be positive for C. pneumoniae antigen (throat swab: PCR positive), and the levels of IgG, IgA and IgM antibodies were assayed. For each specimen, comparison was made with the antibody titers determined by the micro immunofluorescence test (Micro-IF method). The results showed good correlation coefficients of 0.950 for IgG, 0.852 for IgA and 0.866 for IgM. In addition, the two assay methods showed the following high agreement rates: 90.2% for IgG, 84.3% for IgA and 82.4% for IgM. Specimens which did not yield the same results with the ELISA method and Micro-IF method were subjected to analysis by Western blot method, and the rates of agreement with the ELISA results were 80% for IgG, 87.5% for IgA and 88.9% for IgM. These data indicate the efficacy of this new ELISA method. Moreover, COMC was reacted with mouse antisera to three Chlamydia species, and the mouse IgG antibody was assayed. Anti-C. pneumoniae antiserum showed the strongest reactivity, whereas weaker reactivity was shown by anti-C. trachomatis antiserum (1/32nd of the reactivity of the anti-C. pneumoniae antiserum) and anti-C. psittaci antiserum (1/4th). In addition, sera from patients infected with C. trachomatis or C. psittaci (Psittacosis) were subjected to the ELISA method using COMC from C. pneumoniae. It was found that the correlation between the ELISA and Micro-IF methods was higher in relation to the anti-C. pneumoniae antibody titer than either the anti-C. trachomatis antibody titer or anti-C. psittaci antibody titer. These findings indicate this new assay kit based on the ELISA method has high specificity for C. pneumoniae.

Simkania negevensis has been associated with bronchiolitis in infants and community-acquired pneumonia in adults. Reports of exposure to this microorganism are only available from Israel, North America and Western Europe. Currently, no standard method for diagnosis of S. negevensis infection has been established nor have prevalence rates been shown in Japan. For the first time we demonstrated the ability of the microimmunofluorescence (MIF) test to detect S. negevensis-specific immunoglobulin G and exposure to S. negevensis in Japan. The positive rate in healthy volunteers was 4.3% (25/588), with rates increasing with age. Results indicate the usefulness of the MIF test as a serological method for detecting S. negevensis-specific antibodies. A standard serological test for infection with S. negevensis is needed.

To determine whether Chlamydia pneumoniae (C. pneumoniae) infection is associated with hypertension in Japanese adults, we measured serum levels of IgA (a marker of reinfection) and of IgG (a marker of previous infection) antibodies to C. pneumoniae by enzyme-linked immunosorbent assay in 112 adults including normotensive and untreated hypertensive subjects and in 117 hypertensive subjects who had been receiving treatment for more than 3 years. In 112 adults, positivity rate for IgA was lower (P < .01) in hypertensive than in normotensive or borderline hypertensive subjects. Positivity rates for IgA and IgG together, which indicate persistent infection of C. pneumoniae, were lower (P < .01) in hypertensive than in normotensive subjects. IgA levels were inversely correlated with systolic blood pressure (SBP) (r = 0.530, P = .0001) and with diastolic blood pressure (DBP) (r = 0.398, P = .0001). In the 117 hypertensive subjects treated with medication, positivity rate for IgA was lower (P < .01) in subjects with poor control than in those with good control. Positivity rates for IgA and IgG together were lower (P < 0.01) in the poor control group than in the good or fair control groups. IgA levels were correlated inversely with SBP and DBP. In both 112 adults and 117 hypertensive patients, levels of SBP or DBP were inversely associated with positivity rates for IgA and IgG together in multiple logistic regression analysis. The results suggest an inverse relationship between high blood pressure and C. pneumoniae infection in Japanese adults.

To develop EIA kit with low cross-reactivity for the quantitative detection of anti-chlamydial antibodies, we examined the preparation of trachomatis antigens and its specificity to mouse antisera and human sera. The chlamydial elementary body (EB) purified from C. trachomatis L2/434/Bu strain was treated by Sarkosyl, dithiothreitol and SDS by turns to obtain the soluble EB outer membrane (COMC). SDS-PAGE showed that the major components of the COMC were 96K, 60K and 39.5 KDa peptides. The reactivity of the COMC immobilized to 96 wells microtiter plate to mouse anti-serum to C. trachomatis was higher than the other two mouse anti-sera to C. psittaci and pneumoniae. In human sera, the cut off values were calculated from an average optical density plus its two-fold standard deviation obtained by the testing of 100 samples of healthy human sera. We evaluated the specificity of the kit to 17 anti-C. pneumoniae, 9 C. trachomatis and 4 C. psittaci antibodies positive patients' sera judged by the MFA method respectively. The results showed that the concordance ratio of IgG and IgA were 88%, 100% in anti-C. pneumoniae, 89%, 78% in anti-C. trachomatis and 50%, 50% in anti-C. psittaci respectively. From the results obtained in this study, we concluded that the HITAZYME method which had a very low cross-reactivity to C. pneumoniae is clinically useful in the serodiagnosis of C. trachomatis infections, even if it has a little common antigenicity with C. psittaci antigen.