Studies of persistent infection by Chlamydia trachomatis serovar K in TPA-differentiated U937 cells and the role of IFN-gamma.

Inoculation of phorbol ester-differentiated U937 cells as a model for human macrophages with Chlamydia trachomatis of the urogenital serovar K resulted in a persistent infection, with maximal growth at day 7, until day 10 post-infection. At these times inclusion bodies were present in 0.5-2% of the cells. Typical inclusion bodies containing elementary bodies and reticulate bodies were observed by electron microscopy. Furthermore, single chlamydial particles resembling atypical elementary or intermediate bodies were identified in the cytoplasm in > 80% of the host cells. IFN-gamma exerts antichlamydial activity in epithelial and fibroblastoid cells, but the infection of U937 cells by C. trachomatis was not affected by IFN-gamma. The activity of the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO) was not detected in untreated or in IFN-gamma-treated or chlamydiae-infected or mock-infected U937 cells. The presence of atypical persisting chlamydiae and the lack of IDO expression in U937 cells indicates that the development of these atypical bacteria is independent from IFN-gamma-mediated tryptophan deprivation and other IFN-gamma-mediated effects. Evaluation of persistently infected cells revealed that the expression of the chlamydial major outer-membrane protein, heat-shock protein (hsp60) and lipopolysaccharide (LPS) antigens was not significantly altered in the course of the culture. An intense staining of the LPS on the surface of the host cells was demonstrated by immunofluorescence. The data show that phorbol ester-differentiated U937 cells restrict chlamydial growth strongly but not completely through a mechanism distinct from IDO-mediated tryptophan deprivation. The mechanisms of persistence of chlamydiae in monocytes, which differ considerably from those described for other cells, require further investigation.

Evaluation of ELISA to detect Chlamydia trachomatis antigen in urine samples from arthritis patients.

OBJECTIVE: To determine whether examination of urine samples using ELISA allows the detection of asymptomatic C. trachomatis infection in arthritis patients. METHODS: The in vitro sensitivity of IDEIA Chlamydia ELISA to detect C. trachomatis in urine samples was determined by the investigation of serial dilutions of chlamydial elementary bodies. In a clinical study, urine samples from 402 consecutive arthritis patients (182 men and 220 women) in a tertiary care rheumatology clinic were examined for asymptomatic chlamydial infection by ELISA and the results were compared to culture and direct immunofluorescence assay (DFA, MicroTrak) of urogenital swabs. RESULTS: The in vitro sensitivity of ELISA for detecting purified elementary bodies of C. trachomatis serovar K in urine was 60 infection forming units. Twenty-three of 402 arthritis patients (6%) had asymptomatic chlamydial infection as shown by DFA and culture from urogenital smears. The ELISA method identified only 3 of 17 swab-positive patients among 271 patients when urine specimens were collected during the clinical visit, while the assay detected all 6 swab-positive patients among 131 patients when first-voided early morning urine specimens were used (p < 0.001). CONCLUSION: It is mandatory to examine only first voided early morning urine samples if ELISA is used instead of DFA or culture from urogenital swabs to detect asymptomatic chlamydial infection in arthritis patients.

Ultrastructural and molecular analyses of the persistence of Chlamydia trachomatis (serovar K) in human monocytes.

Previous studies have suggested that monocytes may play a role in the dissemination of Chlamydia trachomatis, and in establishment of persistent infection with this bacterium. Infection of cultured human peripheral blood monocytes with C. trachomatis serovar K produced persistent, nonproductive infection. Transmission electron microscopy of such infected cultures revealed single or multiple Chlamydia in monocyte inclusions over a culture period of 10 days. Those inclusions were aberrant, and normal reticulate bodies within the inclusions were not observed. Immunoelectron microscopy showed the chlamydial major outer membrane protein and lipopolysaccharide to be associated with the bacterial plasma membrane. Lipopolysaccharide was also identified in the monocyte cytoplasm. Molecular analyses of primary chlamydial rRNA transcripts demonstrated that the organism is viable and metabolically active within monocyte inclusions. However, attempts to overcome chlamydial growth arrest by incubation of Chlamydia-infected monocytes with tryptophan, and antibodies against alpha interferon, gamma interferon, or tumor necrosis factor, were all ineffective, suggesting that known mechanisms of growth inhibition do not hold in human monocytes. These observations indicate that infection of human peripheral blood monocytes with C. trachomatis may be involved in the genesis/maintenance of extra-urogenital inflammation, since non-culturable, metabolically active bacteria persist in those cells.

Sensitivities of PCR, MicroTrak, ChlamydiaEIA, IDEIA, and PACE 2 for purified Chlamydia trachomatis elementary bodies in urine, peripheral blood, peripheral blood leukocytes, and synovial fluid.

Routine microbiological diagnosis of Chlamydia-induced reactive arthritis is based mainly on the detection of Chlamydia trachomatis with urogenital swabs or in urine. Because chlamydial antigen, rRNA, and DNA are present in low quantities in the inflamed joint, highly sensitive assays are needed to detect C. trachomatis not only at the primary site of infection but also in peripheral blood and peripheral blood leukocytes, which are suspected carriers for dissemination, and in synovial fluid. To evaluate possible tools for this purpose, the sensitivities of PCR, MicroTrak, Chlamydia EIA, IDEIA, and PACE 2 for the detection of defined numbers of purified C. trachomatis elementary bodies (EB) in urine, peripheral blood, peripheral blood leukocytes, and synovial fluid were determined. In urine, PCR detected 2, MicroTrak and ChlamydiaEIA detected 2 x 10(3), and PACE 2 and IDEIA detected 2 x 10(4) EB per ml. In peripheral blood, only PCR and MicroTrak detected C. trachomatis, with detection limits of 100 and 2 x 10(7) EB per ml, respectively. For peripheral blood leukocytes, the detection limits were 2 EB per ml for PCR and 2 x 10(4) EB per ml for MicroTrak, ChlamydiaEIA, IDEIA, and PACE 2. In synovial fluid, PCR detected 200, MicroTrak and IDEIA detected 2 x 10(5), and PACE 2 detected 10(6) EB per ml. ChlamydiaEIA was unable to detect 2 x 10(6) EB per ml in synovial fluid. In summary, PCR was found to be the most sensitive method. The sensitivities of the other methods tested were at least 1,000 times lower than that of PCR. PCR should therefore be considered a most promising tool for routine diagnosis of Chlamydia-induced arthritis.

[Is there a correlation between tubal occlusions in chronic salpingitis and urogenital chlamydia infections?]. / Gibt es einen Zusammenhang zwischen Tubenverschlüssen bei chronischer Salpingitis und urogenitalen Chlamydieninfektionen?

A prospective study was performed to analyse the relationship between urogenital infections caused by Chlamydia trachomatis and occlusions of the fallopian tubes with histologically confirmed chronic salpingitis and salpingitis isthmica nodosa. 110 infertile patients were tested for C. trachomatis infection. 23 patients with tubal occlusions and histologically confirmed chronic salpingitis (group 1) and eight patients with salpingitis isthmica nodosa (group 2) were compared to 13 patients with tubal occlusions after tuboligation (group 3), and to 66 patients with patent fallopian tubes as demonstrated by laparoscopy or hysterosalpingography (group 4). The prevalence of infections of the endocervix or urethra and the presence of Chlamydia in urine was low in all four groups. However, in groups 1 and 2, the median Chlamydia IgG and IgA serum antibody titres were significantly higher (p < or = 0.0002) than in groups 3 and 4. This result illustrates the association between urogenital infections with Chlamydia and tubal occlusions with histologically documented chronic salpingitis and salpingitis isthmica nodosa.

Intracellular persistence of chlamydial major outer-membrane protein, lipopolysaccharide and ribosomal RNA after non-productive infection of human monocytes with Chlamydia trachomatis serovar K.

The replication of Chlamydia trachomatis serovar K was studied in human peripheral blood monocytes (PBMo). The intracellular fate of the bacteria was examined by determining the presence of chlamydial major outer-membrane protein (MOMP), lipopolysaccharide (LPS) and ribosomal RNA (rRNA). In-vitro infection of PBMo with C. trachomatis serovar K was not productive. However, chlamydial MOMP antigen, demonstrated by immunofluorescence, was present in PBMo for up to 14 days. Infected monocytes also contained chlamydial rRNA, measured by in-vitro hybridisation, and LPS, measured by enzyme immunoassay, for up to 14 days. These data are compatible with the hypothesis that the infection of PBMo with C. trachomatis may play a role in the systemic distribution of chlamydial antigens, leading to systemic manifestations of urogenital chlamydial infection.

Chlamydial rRNA in the joints of patients with Chlamydia-induced arthritis and undifferentiated arthritis.

Synovial fluid and synovial membrane specimens of 11 patients with Chlamydia-induced arthritis (CIA), 24 patients with undifferentiated arthritis (UndA), 4 patients with post-enteritic reactive arthritis, 3 patients with Lyme arthritis and 9 patients with rheumatoid arthritis were investigated for the presence of Chlamydia trachomatis (C. trachomatis). A single stranded DNA-probe was used for nucleic acid hybridization with ribosomal RNA (rRNA) from C. trachomatis. In 4 patients (CIA = 1, UndA = 3) chlamydial rRNA was found in the synovial fluid. In one additional patient (CIA) the specimen of a synovial membrane biopsy was positive for chlamydial rRNA. The detection of intra-articular chlamydial rRNA is discussed as an indicator for the presence of viable Chlamydiae in inflamed joints.