Ultrastructural study of Chlamydia pneumoniae in a continuous-infection model.

We have established an in vitro model of long-term continuous Chlamydia pneumoniae infection in HEp-2 cells. Using transmission electron microscopy, we demonstrated the presence of spontaneous abnormal chlamydial inclusions similar in appearance to the persistent chlamydial forms induced in vitro by treatment with cytokines or antibiotics or by nutrient deprivation.

Is there an association between Kawasaki disease and Chlamydia pneumoniae?

The clinical and epidemiological features of Kawasaki disease (KD) are consistent with an infectious cause. Because chronic infection with Chlamydia pneumoniae has been implicated in the pathogenesis of atherosclerosis, it has been suggested that it may also be involved in the pathogenesis of KD. Paired sera (baseline pretreatment and 1 year after treatment with intravenous immunoglobulin [IVIG]) from 26 children with KD and 29 age-matched controls were examined by microimmunofluorescence (MIF) serology and immunoblotting. There were no significant differences in the prevalence of anti-C. pneumoniae IgG, IgA, or IgM between cases and controls; however, 73%-85% of sera from cases and controls reacted with C. pneumoniae proteins by immunoblotting. There was significantly more reactivity in the pre-IVIG, but not post-IVIG, KD sera compared with sera from controls to proteins at 72-74 kDa and 74-76 kDa. They may be heat shock proteins. The results of this study do not support an association between KD and C. pneumoniae on the basis of MIF and immunoblot analysis.

In vitro activities of gemifloxacin (SB 265805, LB20304) against recent clinical isolates of Chlamydia pneumoniae.

We compared the in vitro activity of gemifloxacin, a new quinolone antibiotic, to the activities of levofloxacin, moxifloxacin, trovafloxacin, erythromycin, and doxycycline against 20 isolates of Chlamydia pneumoniae. Gemifloxacin was the most active quinolone tested, with a MIC at which 90% of the isolates are inhibited and a minimal bactericidal concentration at which 90% of strains tested are killed of 0.25 microg/ml, but this activity was less than those of doxycycline and erythromycin.

In vitro activities of azithromycin and ofloxacin against Chlamydia pneumoniae in a continuous-infection model.

Chlamydia pneumoniae is a well-established cause of community-acquired pneumonia and bronchitis in adults and children. Chronic infections with C. pneumoniae have been implicated in the development of atherosclerosis and other diseases in humans. Methods currently used for the culture and propagation of C. pneumoniae are not analogous to the infection as it occurs in vivo. We have established a model of continuous C. pneumoniae infection in vitro. HEp-2 cells inoculated with CM-1 and TW-183 strains have been persistently infected for periods of over 1.5 and 2 years, respectively. The cultures were maintained without centrifugation or the addition of cycloheximide, fresh host cells, or chlamydia. We observed cycles of host cell lysis, detachment, and regrowth with both strains of C. pneumoniae. Continuous C. pneumoniae infections may more closely resemble the actual events as they occur in vivo and, therefore, may be a better model for the in vitro study of C. pneumoniae infection. When we used continuously infected cells to determine the effects of azithromycin and ofloxacin on C. pneumoniae propagation in vitro, we found that both drugs reduced but did not completely eliminate the organism. This may be an important observation, as the failure of antibiotic therapy against C. pneumoniae infection in humans has been described.

Activity of grepafloxacin and other fluoroquinones and newer macrolides against recent clinical isolates of Chlamydia pneumoniae.

Chlamydia pneumoniae is a frequent cause of community-acquired respiratory tract infection including pneumonia and bronchitis. Quinolones have attracted interest as potential therapy for community-acquired respiratory tract infections because they are active against a wide range of pathogens including C. pneumoniae and Mycoplasma pneumoniae. The in vitro susceptibilities of C. pneumoniae were determined for grepafloxacin, levofloxacin, moxifloxacin, trovafloxacin, clarithromycin and azithromycin. Isolates of C. pneumoniae tested included two reference strains, TW-183 and CM-1, and 12 recent clinical isolates from adults with community-acquired pneumonia. Susceptibility testing was performed in HEp-2 cells grown in 96-well microtiter plates. The MIC was the lowest antibiotic concentration at which no inclusions were seen. The MBC was the lowest concentration which resulted in no inclusions after passage in antibiotic-free medium. Grepafloxacin was the most active quinolone tested with an MIC50 of 0.125 mg/l, MIC90 and MBC90 of 0.5 mg/l. Grepafloxacin may have a role in the treatment of C. pneumoniae infections, but prospective clinical studies utilizing culture are lacking.

Antibody response to Chlamydia pneumoniae infection in children with respiratory illness.

Serologic diagnosis of Chlamydia pneumoniae infection has been based on the microimmunofluorescence test (MIF). However, recent prospective studies in children have found that >50% infected with C. pneumoniae failed to develop any antibodies detectable by MIF. In this study, single sera from 46 culture-positive and 42 culture-negative children with respiratory infection and known MIF status were examined by immunoblotting. Forty-one (89.1%) of the single sera from culture-positive and 27 (64.3%) from culture-negative children reacted to C. pneumoniae antigens in immunoblot. C. pneumoniae proteins most frequently recognized by sera from culture-positive patients were at 101-102, 72-76, 50-52, 48-49, 43-44, 41-42, and 30-31 kDa. However, there did not appear to be a correlation of specific band patterns and culture status.

In vitro activity of trovafloxacin against Chlamydia pneumoniae.

The in vitro susceptibilities of 12 strains of Chlamydia pneumoniae to a new quinolone, trovafloxacin, and ofloxacin, doxycycline, erythromycin, and azithromycin were determined. The activity of trovafloxacin was similar to that of ofloxacin, with a MIC at which 90% of the isolates are inhibited and a minimal concentration at which 90% of the isolates are killed of 1.0 microg/ml, but trovafloxacin was less active than doxycycline, erythromycin, and azithromycin.

In-vitro activity of dirithromycin against Chlamydia pneumoniae.

The in-vitro susceptibilities of 12 strains of Chlamydia pneumoniae were determined for dirithromycin, a new macrolide antibiotic, erythromycyclamine, its active metabolite, and erythromycin. Both dirithromycin and erythromycyclamine had an MIC90 of 2 mg/L, as compared with 0.062 mg/L for erythromycin. The combination of dirithromycin and erythromycyclamine appeared to be additive. Determination of the role of dirithromycin for the treatment of C. pneumoniae infection will depend on the results of prospective, controlled studies utilizing culture.

Evaluation of Chlamydia immunoglobulin M (IgM), IgG, and IgA rELISAs Medac for diagnosis of Chlamydia pneumoniae infection.

Chlamydia pneumoniae is an important pathogen responsible for a variety of respiratory diseases in humans. Cell culture remains the most specific method for C. pneumoniae diagnosis, but it is labor-intensive and time-consuming. Thus, serology, particularly microimmunofluorescence (MIF) testing, is frequently utilized. However, the MIF test has a significant subjective component. We evaluated a new serological test: Chlamydia Immunoglobulin M (IgG, IgA, and IgM rELISAs Medac, based on a recombinant Chlamydia-specific lipopolysaccharide (LPS) fragment, for the diagnosis of C. pneumoniae infection. The results of this study demonstrated that the use of rELISAs Medac with single sera does not appear to be sensitive or specific for diagnosis of C. pneumoniae infection compared to culture. In children, sensitivities of the rELISAs compared to culture did not exceed 34.2%, and the specificities ranged from 68.4% (IgG) to 91.2% (IgA). In adults, the sensitivities of the rELISAs were slightly higher, up to 77.8% (IgA or IgG), but the specificities ranged from a very low 20.8% for IgA or IgG to 81.1% for IgM. When multiple sera were tested, the results of the rELISAs Medac correlated with culture results in five of eight (62.5%) patients. However, this offers only a retrospective diagnosis, which makes it difficult to manage these patients prospectively.

Use of polymerase chain reaction for the detection of Chlamydia trachomatis in ocular and nasopharyngeal specimens from infants with conjunctivitis.

BACKGROUND: Chlamydia trachomatis is the most common identifiable infectious cause of neonatal conjunctivitis. Nonculture tests including enzyme immunoassays and direct fluorescent antibody tests have been shown to perform well for the diagnosis of chlamydial conjunctivitis with sensitivities and specificities > or = 90%. However, the performance with respiratory specimens has been less than satisfactory. METHODS: We compared a new, commercially available polymerase chain reaction (PCR) assay, Roche AMPLICOR (Roche Diagnostic Systems, Branchburg, NJ) with culture for the detection of C. trachomatis in conjunctival and nasopharyngeal specimens from infants with conjunctivitis. We also evaluated AMPLICOR for the detection of C. trachomatis in the urine of mothers of positive infants. RESULTS: Ocular and nasopharyngeal specimens from 75 infants with conjunctivitis were obtained for culture and PCR. AMPLICOR was equivalent to culture for eye specimens and more sensitive than culture for nasopharyngeal specimens. The sensitivity, specificity and positive and negative predictive values of PCR compared with culture for conjunctival specimens were 92.3, 100, 100 and 98.4%, respectively. The sensitivity, specificity and positive and negative predictive values for nasopharyngeal specimens were 100, 97.2, 60 and 100%, respectively. We also detected C. trachomatis by PCR in the urine of 12 mothers of culture positive infants. CONCLUSIONS: PCR performed comparably to culture for detection of C. trachomatis in conjunctival and nasopharyngeal specimens from infants with conjunctivitis.

Evaluation of a new optical immunoassay for diagnosis of neonatal chlamydial conjunctivitis.

The BioStar OIA Chlamydia test (BioStar, Inc., Boulder, Colo.) is a novel immunoassay system that uses changes in reflection of light to directly detect chlamydial antigen in clinical specimens. We compared the optical immunoassay (OIA) with culture for detecting Chlamydia trachomatis in ocular specimens from infants with suspected chlamydial conjunctivitis. We initially performed a retrospective evaluation, testing 152 ocular specimens previously collected for culture with the OIA. The sensitivity and specificity were 94.2 and 97%, respectively. A subsequent prospective study evaluating 37 ocular specimens from infants with suspected C. trachomatis conjunctivitis revealed a sensitivity and specificity of 100 and 92.6%, respectively.

[Immunodiagnostic preparations based on monoclonal antibodies to Chlamydia trachomatis]. / Immunodiagnosticheskie preparaty na osnove monoklonal'nykh antitel k Chlamydia trachomatis.

Highly sensitive diagnostic preparations for the detection of C. trachomatis by direct immunofluorescent and enzyme immunoassay techniques were obtained with the use monoclonal antibodies to C. trachomatis genus-specific polysaccharide antigen. The enzyme immunoassay diagnostic preparation permitted the detection of C. trachomatis in experimental specimens in a dose of 4-14 ng protein. The results of the primary clinical trial of the immunofluorescent preparation revealed that its effectiveness was not lower than that of similar foreign commercial preparations. The use of this diagnosticum for the study of clinical material obtained from 1,603 patients with different venereal diseases showed the presence of chlamydial contamination in 40.2% of the examined patients. The data thus obtained made it possible to recommend the newly developed immunofluorescent preparation for diagnosing infections caused by C. trachomatis.

[The immunochemical and biological properties of monoclonal antibodies to Chlamydia trachomatis]. / Immunokhimicheskie i biologicheskie svoistva monoklonal'nykh antitel k Chlamydia trachomatis.

In this work some properties of 11 monoclonal antibodies to C.trachomatis, obtained in our earlier investigations, were studied and the antigens recognized by these McAb were characterized. Some McAb reacted with a genus-specific thermostable antigen, sensitive to sodium metaperiiodate and having a mol. wt. of 10 - 12 kD. Other McAb interacted with C.trachomatis species- and subspecies-specific thermostable proteins with a mol. wt. of 40 kD. Two McAb reacted with thermolabile protein with a mol. wt. of 32 kD and some C.trachomatis unidentified protein. The in vitro study of two McAb, active against subspecies-specific proteins, revealed the neutralizing activity of these McAb. Thus McAb obtained in this investigation may be for the differential identification of different determinants and for further analysis of the antigenic structure of C.trachomatis, as well as for the development of modern immunodiagnostic preparations.