Prevalence of Chlamydia pneumoniae antibodies in patients with acute respiratory infections in Israel.

AIMS--To evaluate the prevalence of antibodies to Chlamydia pneumoniae (TWAR) in relation to other aetiological agents of acute respiratory infections in Israeli patients. METHOD--Serum samples from 604 patients (183 children and 421 adults) were collected over three years. Antibodies to C pneumoniae, C trachomatis, and Legionella sp were evaluated using the microimmunofluorescence (MIF) assay. Antibodies to Mycoplasma pneumoniae were detected using the Serodia Myco II test. RESULTS--Antibodies to TWAR were detected in 319 (51.3%) sera. Twenty one patients had MIF results indicative of recent infection. TWAR prevalence and antibody titres in children (aged 1-10 years) were low, gradually increased in teenagers (11-18 years), and were highest in adults and elderly patients. In contrast to the consistently noted TWAR antibody prevalence and serological evidence of recent infection during the study period, a significant decrease in those variables was recorded for C trachomatis. Six patients had serological evidence of recent infection with both C pneumoniae and C trachomatis. The presence of antibodies to Mycoplasma pneumoniae and Legionella sp was tested in 473 of the patients; 29 had antibodies to M pneumoniae and 23 to Legionella sp. Six patients (including five children) had serological evidence of recent infection with M pneumoniae and four with Legionella sp. CONCLUSION--C pneumoniae should be considered in patients with acute respiratory diseases. MIF is the preferred method for monitoring the presence of antibodies to this organism.     

Detection of Chlamydia pneumoniae and Chlamydia psittaci in sputum samples by PCR.

AIMS--To use the polymerase chain reaction (PCR) to detect Chlamydia pneumoniae and Chlamydia psittaci in sputum samples. METHODS--A nested PCR was developed, the first stage of which amplified DNA from both C pneumoniae and C psittaci while the second stage targeted specifically at C pneumoniae, allowing the two species to be differentiated. The primers were designed not to amplify sequences from C trachomatis. A panel of 26 sputum samples from patients with community acquired pneumonia evaluated previously by enzyme linked immunosorbent assay (ELISA), direct immunofluorescence (DIF), and culture was tested blind by PCR. Most of these specimens also had accompanying serial serum samples which were tested for species specific antibodies using microimmunofluorescence (micro-IF). RESULTS--PCR detected C pneumoniae DNA in 10 of the 26 samples and C psittaci DNA in four. There was good concordance between ELISA, DIF, micro-IF and PCR in the C pneumoniae group. Two of the C psittaci identified by PCR were labelled C pneumoniae by DIF but the PCR results were supported by serology or a history of bird contact. Of the PCR negative group: six were true negative results; two contained C trachomatis. There were four discrepant results. CONCLUSIONS--The data suggest that PCR is effective in the detection of C pneumoniae. The sensitivity for C psittaci is inevitably lower due to the strategy taken but specificity seemed to be good.     

Chlamydia pneumoniae respiratory infections among patients infected with the human immunodeficiency virus

Thirteen cases ofChlamydia pneumoniae infection in patients seropositive for the human immunodeficiency virus (HIV) are described. The occurrence, the clinical spectrum, and the significance of the infection during HIV disease are compared with data reported in the literature.Chlamydia pneumoniae infection was established by a serologic micro-immunofluorescence test using standard diagnostic criteria. In four cases the results of serological tests were confirmed by direct immunofluorescence on respiratory specimens. Five patients developed focal pneumonia but recovered completely after specific antibiotic treatment. Three patients developed severe and diffuse interstitial pulmonary involvement, two of whom died of acute respiratory failure. Five patients developed upper respiratory tract infection. Using 39 pair-matched HIV-seropositive subjects as controls, the cases of infection were found to be significantly associated with a previously diagnosed pulmonary disease. Upon retrospective analysis of 319 consecutive cases of pneumonia among HIV-infected patients,Chlamydia pneumoniae was the sole agent detected in eight (2.5%) cases, andChlamydia pneumoniae together with other infectious agents was detected in seven (2.2%) cases.Chlamydia pneumoniae is a possible cause of severe respiratory infection in Italian HIV-infected immunocompromised patients, and its presence must be suspected when patients do not respond to therapy with beta-lactam agents or to anti-Pneumocystiscarinii treatment.     

Comparison of five serologic tests for diagnosis of acute infections by Chlamydia pneumoniae

Serology is often used to diagnose acute infections by Chlamydia pneumoniae. In this study paired sera from patients with acute respiratory tract infection during an epidemic of C. pneumoniae infections were examined by five different antibody tests. These tests were the complement fixation (CF) test, the microimmunofluorescence (MIF) test, a recombinant enzyme immunoassay (rEIA) (Medac) based on a recombinant lipopolysaccharide of chlamydia and measuring antibodies to a common chlamydial antigen, and two tests that utilize preparations of C. pneumoniae organisms, the SeroCp-EIA (Savyon) (with preserved lipopolysaccharide) and the LOY-EIA (Labsystems) (without this antigen). Both of the last two tests should measure specific antibodies to C. pneumoniae, although cross-reacting antibodies may also be detected by the SeroCp-EIA. Acute infection of C. pneumoniae was serologically confirmed in 44% of the cases by at least two different tests. Using an expanded "gold standard," i.e., the presence of significant reactions in at least two tests, the sensitivity of the CF test was 69%, that of the MIF test was 88%, that of the rEIA was 89%, that of the LOY-EIA was 96%, and that of the SeroCp-EIA was 92%. Specificity was high for all methods, but adjustments of diagnostic criteria were made to several of the tests. The basis for these adjustments and supportive data are presented. Infections of C. pneumoniae were detected in patients from 8 to 83 years of age. Two peaks in the incidence of such infections were observed: one among young teenagers and a second in adults 30 to 45 years of age, corresponding to parents of young teen-agers. The tests were equally sensitive in different age groups. Reinfections seemed to be rare.     

Clinical features of Chlamydia pneumoniae acute respiratory infection

Chlamydia pneumoniae is a worldwide respiratory pathogen involved in 6–20% of community-acquired pneumonias and in about 5% of acute exacerbations of chronic bronchitis. Preliminary data also indicate a possible association between Chlamydia pneumoniae infection and asthma. Further studies are needed to elucidate whether Chlamydia pneumoniae is merely a precipitant of asthma symptoms or is actually one of the causes of asthma.     

Asymptomatic respiratory tract infection with Chlamydia pneumoniae TWAR.

Chlamydia pneumoniae is a newly recognized organism associated with respiratory tract infections. Asymptomatic infection with C. pneumoniae, although it has been suggested to occur, has not been previously documented. We describe two asymptomatic individuals infected with this organism; these infections demonstrate that C. pneumoniae is able to establish a subclinical infection.     

Usefulness of enzyme linked immunosorbent assays species specific in the detection of Chlamydia trachomatis and Chlamydophila pneumoniae IgG antibodies in patients with genital infections or respiratory tract infections

Chlamydia trachomatis (Ct) and Chlamydophila pneumoniae (Cpn) are obligate intracellular bacteria causing genital tract infections (GTI) and respiratory tract infections (RTI), respectively. Antigenic cross-reactivity between the two species may complicate serologic diagnosis. In this study, we compared the performance of two ELISA tests in relation to microimmunofluorescence (MIF) for the detection of Ct and Cpn IgG antibodies. We also explored the degree of cross-reactivity by ELISA and MIF. Among 278 positive sera for Cpn and/or Ct IgG antibodies in the MIF, 153 were from patients with GTI and 125 were from patients with RTI. These sera were tested by our in house MIF test and by two commercial ELISA: SeroCP and SeroCT for the detection of anti-Cpn IgG antibodies and anti-Ct IgG antibodies, respectively. In sera from patients with RTI, correlation between MIF and SeroCP was 92%. The specificity of this test was 38.5%. In fact, among the 140 sera from patients with GTI and that cross-reacted in MIF, only six were confirmed by the two ELISA tests as having IgG antibodies to Ct. The correlation between MIF and SeroCT was 80%. The specificity of this test was 100%. Indeed, among the 65 sera from patients with RTI with cross-reactions in MIF, 30 sera showed a negative SeroCT test. SeroCT was highly specific and could diminish considerably the extent of cross-reactions. Whilst, SeroCP test was not specific enough to distinguish between the presence of IgG antibodies and Cpn or Ct.     

Use of Counter and rocket immunoelectrophoresis in acute respiratory infections due to Streptococcus pneumoniae.

The use of Counter immunoelectrophoresis (CIE) for the detection of pneumococcal capsular antigen in the sputum and serum of patients suffering from acute respiratory infections is described. The CIE of sputum gave positive results in 224 (99%) out of 225 samples in which Streptococcus pneumoniae was isolated by cultural techniques, and in 23 (9%) out of 262 samples in which no or other potential pathogens had been isolated. In the detection of capsular antigen in serum, CIE was positive in 32 (35%) out of 92 pneumonia cases and was associated with an increase in mortality.     

Ultrastructural defects of respiratory tract cilia associated with chronic infections

Ultrastructural defects were demonstrated in nasal and bronchial cilia from a 12-year-old boy with repeated upper and lower respiratory tract infections. Numerous abnormalities were found, including single axonemes surrounded by excess cytoplasmic matrix, compound cilia, intracytoplasmic microtubular doublets, and cilia contained within a periciliary sheath. Dynein arms were missing from the majority of the peripheral microtubular doublets. The most striking abnormality, however, was a disorientation of cilia as judged by the increased variation in the orientation of central microtubules. Because of these ultrastructural abnormalities, it is highly likely that ciliary motility was markedly decreased and that defective mucociliary transport was responsible for chronic and repeated upper respiratory tract infections.     

Multiplex polymerase chain reaction for the simultaneous detection of Mycoplasma pneumoniae, Chlamydia pneumoniae, and Chlamydia psittaci in respiratory samples.

AIMS: To develop a multiplex polymerase chain reaction (PCR) for the simultaneous detection of Mycoplasma pneumoniae, Chlamydia pneumoniae, and Chlamydia psittaci in respiratory samples. METHODS: Oligonucleotide primers for the amplification of the DNA of these three organisms were optimised for use in combination in the same reaction. PCR products were detected by hybridisation with pooled internal probes using an enzyme linked immunosorbent assay. Those with positive signals were further differentiated using species specific probes. Quality of DNA extraction and PCR inhibition were controlled by amplification of a human mitochondrial gene. A panel of 53 respiratory samples with known results was evaluated blindly. This was followed by a retrospective study on sputa collected from 244 patients with suspected community acquired pneumonia. RESULTS: The multiplex assay had a lower sensitivity than PCR with individual primers by about one log. The resultant sensitivity was considered acceptable for diagnostic use. Of the panel of 53 samples, nine of 11 M pneumoniae, 11 of 11 C pneumoniae, six of seven C psittaci, and 24 of 24 negative samples were correctly identified. Of the 244 patients with pneumonia, seven (2.9%) had detectable M pneumoniae, six (2.5%) had C pneumoniae, and one (0.4%) had C psittaci. The case notes from 11 patients were studied. The PCR finding was of possible significance in at least eight of these patients. CONCLUSIONS: This multiplex PCR assay has the potential to be used as a diagnostic and epidemiological tool. Further prospective studies are needed to establish its clinical value.     

Detection of viral, Chlamydia pneumoniae and Mycoplasma pneumoniae infections in exacerbations of asthma in children.

BACKGROUND: A high frequency of virus infections has been recently pointed out in the exacerbations of asthma in children. OBJECTIVES: To confirm this, using conventional and molecular detection methods, and expanding the study to younger children. STUDY DESIGN: One hundred and thirty-two nasal aspirates from 75 children hospitalized for a severe attack of asthma were studied (32 infants, mean age 9.1 months; and 43 children, mean age 5.6 years). According to the virus, a viral isolation technique, immunofluorescence assays (IFA) or both were used for the detection of rhinovirus, enterovirus, respiratory syncytial (RS) virus, adenovirus, coronavirus 229E, influenza and parainfluenza virus. Polymerase chain reaction (PCR) assays were used for the detection of rhinovirus, enterovirus, RS virus, adenovirus, coronavirus 229E and OC43, Chlamydia pneumoniae and Mycoplasma pneumoniae. RESULTS: Using IFA and viral isolation techniques, viruses were detected in 33.3% of cases, and by PCR techniques, nucleic acid sequences of virus, Chlamydia pneumoniae and Mycoplasma pneumoniae were obtained in 71.9% of cases. The combination of conventional and molecular techniques detects 81.8% of positive samples. Two organisms were identified in the same nasal sample in 20.4% of the cases. The percentage of detections was higher (85.9%) in the younger group than in the other (77%). The most frequently detected agents were rhinovirus (46.9%) and RS virus (21.2%). Using PCR rather than conventional techniques, the detection rates were increased 5.8- and 1.6-fold in rhinovirus and RS virus infections, respectively. The detection levels of the other organisms are as follows: 9.8, 5.1, 4.5, 4.5, 4.5, 3.7, and 2.2% for enterovirus, influenza virus, Chlamydia pneumoniae, adenovirus, coronavirus, parainfluenza virus, and Mycoplasma pneumoniae, respectively. CONCLUSION: These results confirm the previously reported high frequency of rhinovirus detection in asthmatic exacerbations in children. They also point out the frequency of RS virus detection, and emphasize the fact that PCR assays may be necessary to diagnose respiratory infections in asthma.     

Evaluation of a commercial test for antibodies to the chlamydial lipopolysaccharide (Medac) for serodiagnosis of acute infections by Chlamydia pneumoniae (TWAR) and Chlamydia psittaci

A commercial test (rELISA) based on a recombinant chlamydial lipopolysaccharide (LPS) antigen has been evaluated for the diagnosis of acute infections caused by Chlamydia pneumoniae (TWAR) and Chlamydia psittaci. This test and a microimmunofluorescence test (MIF) were compared in 160 patients with community-acquired pneumonia. Seventeen of nineteen cases with significant titre changes detected by rELISA were confirmed by MIF. The two remaining cases not confirmed by MIF were considered false-positive reactions. One case positive by MIF only was judged not to be a true-positive reaction. All three cases occurred in patients with Mycoplasma pneumoniae infection and may be the result of a mitogenic effect. High antibody titres have been used to indicate acute C. pneumoniae infection. We found high MIF or rELISA titres to be equally common in patients and controls; no association between the two tests was detected. An unexpected cross-reactivity between the rELISA antigen and parvovirus was observed, which might have diagnostic implications. Both MIF and rELISA detected acute C. pneumoniae and C. psittaci infection, and there was good agreement between the tests. Single serum diagnosis was generally not feasible with either MIF or rELISA.     

Asthma, atopy and Chlamydia pneumoniae antibodies in adults

BACKGROUND: Factors involved in the development of inflammation and asthma in nonatopic subjects have remained largely obscure, although there is some evidence to suggest that certain infections may play a role. OBJECTIVE: We investigated the association between serological evidence of Chlamydia pneumoniae infection and asthma in adults, and the possible modifying effect of the patients' atopic status on this association. METHODS: Four hundred and thirty consecutive patients who attended the hospital between 1992 and 1993 with symptoms suggestive of asthma, rhinitis or allergy were enrolled. Diagnostic procedures including lung function measurements and skin-prick tests were performed in all patients. The patients with established asthma (n = 332) were divided into those with recent asthma (n = 224, onset 1985 onward) and longstanding asthma (n = 108, onset before 1985). The controls (n = 98) comprised all subjects who did not meet the criteria of asthma. Serum immunoglobulin (Ig)G, IgA and IgM antibodies to C. pneumoniae were measured by the microimmunofluorescence test. RESULTS: In women, the prevalences of elevated IgG (a titre of >/= 128) and IgA (>/= 32) antibody levels and the age-adjusted geometric mean titres (GMT) of IgG and IgA antibodies were invariably highest among subjects with nonatopic longstanding asthma. Elevated IgG titres in women occurred in 11% of controls, in 28% of nonatopic recent onset asthmatics, and in 43% of asthmatics with nonatopic longstanding disease; for men the respective figures were 33, 50 and 64%. Logistic regression analysis controlling for age, sex and smoking showed that asthma was significantly associated with elevated IgG antibody levels to C. pneumoniae (odds ratio 3.3, 1.6-6.8 for longstanding asthma, 2.3, 1. 2-4.4 for recent asthma, and among women only 4.2, 1.6-10.9 for longstanding asthma, and 3.0, 1.3-7.2 for recent asthma). When the atopics and nonatopics were analysed separately, an even stronger relationship in the nonatopics was obtained for longstanding asthma (6.0,2.1-17.1). In contrast, the relationship between atopic asthma, either recent or longstanding, and elevated IgG titres was not significant, indicating that asthma per se does not predispose to C. pneumoniae infection. CONCLUSIONS: Asthma was significantly associated with elevated IgG antibody levels to C. pneumoniae, and this association was strongest for nonatopic longstanding asthma.     

Prevalence of antibodies to Chlamydia pneumoniae in an Israeli population without clinical evidence of respiratory infection

AIMS: To estimate the occurrence of recent, past, and "persistent" infections with Chlamydia pneumoniae--as indicated by serology--in an Israeli population without clinical evidence of respiratory infection. METHODS: Serum samples from 402 subjects (172 children and 230 adults), without known respiratory symptoms, were collected. Antibodies to C pneumoniae (IgG, IgA, and IgM) were evaluated using the microimmunofluorescence (MIF) assay. Antibody prevalence and indication of recent, past, and persistent infections were calculated and their distribution determined according to age, sex, and season. RESULTS: Antibodies to C pneumoniae were detected in 53 children (31%) and 171 adults (74%). Recent infection was indicated in only one of 50 children under 5 years of age, in nine of 122 older children, and in 19 of 230 adults. IgM antibodies were detected in nine children, but only in three adults. Past infection was indicated in six of 96 young children (aged 1-10 years), in 28 of 76 teenagers, and in 128 of 230 adults. Persistent infection was indicated in three young children, in six teenagers, and in 24 adults, with a significantly higher frequency (p = 0.012) in men (18 of 117) than in women (six of 113). No seasonal differences could be detected. CONCLUSIONS: Infection with C pneumoniae was detected serologically in children and adults without clinical signs of respiratory disease. These results should serve as a basis for studies on the role of C pneumoniae infections and their sequelae in Israel and contribute to the general understanding of asymptomatic infection with C pneumoniae.     

Comparison of throat swabs with sputum specimens for the detection of Chlamydia pneumoniae antigen by direct immunofluorescence.

AIM: To compare throat swabs with sputum specimens for Chlamydia pneumoniae antigen detection. METHODS: During a one year period, sputum and throat swabs from 50 patients over 15 years of age with acute or persisting lower respiratory tract infection were examined for C pneumoniae antigen by direct immunofluorescence. RESULTS: C pneumoniae antigen was detected in 18/50 patients (36.0%) from sputum, throat swab, or both. Paired sputum and throat swabs were received from 35/50 patients (70.0%). C pneumoniae antigen was detected in either or both specimens from 14/35 patients (40.0%). Of the 14 positive patients, both specimens were positive in nine (64.3%), throat swab only in four (28.6%), and sputum only in one (7.1%). Of the remaining 15 patients from whom only a single specimen was sent, a further three of eight throat swabs and one of seven sputum specimens were positive. There was no statistically significant difference between the results obtained from the two types of specimen. CONCLUSIONS: Throat swabs may be as good as sputum for the detection of C pneumoniae antigen.     

Chlamydia pneumoniae and multiple infections in the aorta contribute to atherosclerosis

Our previous study on herpesvirus infection including Herpes simplex virus type 1 (HSV-1), Epstein-Barr virus (EBV), cytomegalovirus (CMV) and atherosclerosis revealed that the prevalence of herpesvirus is higher in atherosclerotic aorta than in non-atherosclerotic aorta. Infections with two or three forms of the virus have been found only in atherosclerotic aorta. In our current study, we examined both Chlamydia pneumoniae and Chlamydia trachomatis in herpesvirus-infected aortic tissues, by means of immunohistochemistry, polymerase chain reaction, Southern hybridization, in situ hybridization, electron microscopy and electron-microscopic immunohistochemistry. In particular, the bacteria were found in atherosclerotic lesions. In atherosclerotic aorta, 40% of tissues examined were positive for C. pneumoniae in contrast to absence of this bacteria in non-atherosclerotic aorta. Elementary bodies of C. pneumoniae were found in macrophage-like cells in the intima of atherosclerotic aorta by electron microscopy. Chlamydia trachomatis was not found in both atherosclerotic and non-atherosclerotic aorta. Our findings suggest that multiple infections in aortic tissue contribute to the development of atherosclerosis. Furthermore, the absence of C. pneumoniae compared to herpesviruses in normal arterial tissue suggests that C. pneumoniae is specific for atherosclerotic lesions. In contrast to 'abortive infection' of viruses in arteries, C. pneumoniae infection was demonstrated in macrophages by electron microscopy and electron-microscopic immunohistochemistry in atherosclerotic lesion.Chlamydia pneumoniae may be the most important pathogen related to the development of atherosclerosis.     

Nonvalue of sputum culture in the management of lower respiratory tract infections.

Establishment of the microbiological etiology of bacterial pneumonia by sputum culture is confounded by both lack of recovery of fastidious pathogens and contamination of specimens with oropharyngeal flora. We reviewed the clinical records from 249 patients over a 3-month period for evidence of pneumonia. Gram staining and cultures were performed on 381 specimens isolated from this population of patients. Recovery of respiratory tract pathogens was accomplished with 354 specimens from 226 patients; 27 specimens yielded normal flora in culture but were smear positive. An additional 256 specimens submitted to our microbiology laboratory did not meet smear criteria for purulence nor did they yield respiratory tract pathogens in culture. A total of 637 specimens submitted to the microbiology laboratory were evaluated for sputum purulence by the criteria of Bartlett. Of the total 354 specimens which were positive in culture for a pathogen, 182 (52%) were submitted from 150 patients with no objective evidence of pneumonia. The majority of specimens obtained from patients without pneumonia were nonpurulent. However, 71 of 182 culture-positive specimens obtained from 50 patients without pneumonia were purulent. Approximately half of these patients (31 of 50) had other pulmonary or upper respiratory tract pathology which could account for the sputum purulence. Among the 172 culture-positive specimens from 76 patients with pneumonia, only 100 (58%) were acceptable by smear criteria. An additional 23 patients provided expectorated purulent sputum from which no respiratory tract pathogen could be isolated. Of these 23, 7 had pneumonia. We conclude that sputum culture and Gram staining are neither specific nor sensitive as diagnostic tools. Objective criteria for purulence of Gram-stained specimens must be applied before their inoculation into culture media. Specimens should be sought only from patients with objective evidence of pneumonia.