OBJECTIVETo establish a gap ligase chain reaction (G-LCR) assay for the detection of Chlamydia trachomatis (Ct) in neonates with pneumonia.

METHODSA G-LCR DNA amplification assay that targeted the outer major membrane protein gene (omp1) of Ct was developed to detect Ct. The sensitivity and specificity of the G-LCR test was examined by the use of highly purified elementary bodies (EBs). Nasopharyngeal swabs taken from 328 neonates with pneumonia were analyzed by Gap-LCR and cell culture.

RESULTSThe detection limit of G-LCR was 2 EBs. G-LCR could detect five species of Ct and was not cross-reacted with C psittaci and other bacteria. The prevalence of Ct in 328 neonates with pneumonia, using an expanded gold standard of a positive cell culture or two confirmed positive non-culture tests, was 21% (69/328). After analysis of discrepant results, the sensitivity, specificity, and positive and negative predictive values for the G-LCR were 98.6%, 100%, 100% and 99.6%, respectively; whereas those for culture were 86.9%, 100%, 100% and 96.6%, respectively.

CONCLUSIONThis study demonstrated that the G-LCR was a highly sensitive nonculture technique and good alternative test for the detection of chlamydial infections.

Chlamydia Infections ; diagnosis ; microbiology ; Chlamydia trachomatis ; genetics ; Female ; Humans ; Infant, Newborn ; Ligase Chain Reaction ; methods ; Male ; Sensitivity and Specificity

OBJECTIVETo establish a methed of cleavage fragment length polymorphism (CFLP) analysis with a primer labeled at the 5'-end with digoxigenin for genotyping of Chlamydia trachomatis (Ct). The methods for detection of Ct by major outer membrane protein (MOMP) gene (ompl) with nested polymerase chain reaction (ompl-nPCR) were studied. The incidence of Ct infection in pregnant women, the common genotypes and vertical transmission rate of Ct in Chongqing area during the past one year was also investigated.

METHODSThe samples were taken from cervical scrapes of parturient women and nasopharygeal swabs of their neonates from April 2003 to Feb. 2004 in Chongqing Women and Children's Health Care Institute. Totally 300 pairs (605 specimens) were detected by using ompl-nPCR, ompl-PCR (inside pair of primers was used directly) and plasmid-PCR. The results were judged by the modified gold standard (MGS). The ompl-nPCR amplified DNA was purified by recovery of DNA from agarose gel electroelution into dialysis bags. The DNA amplified from ompl-nPCR was sequenced by ABI PRISM 377 DNA sequencer. CFLP assay with a primer labeled at the 5'-end with digoxigenin was created for genotyping of Ct, and was primarily applied.

RESULTSThe minimum detectable levels of ompl-nPCR and ompl-PCR corresponded to 2.5 elementary body (EB) and 25 EB, respectively. The sensitivity of ompl-nPCR was 10 times that of ompl-PCR. The positive rate of Ct in the samples from the pregnant women was 11% (33/300). The vertical transmission rate of Ct from mothers to their infants was 24.2% (8/33). The rate of Ct isolated from nasopharyngeal swabs 5 - 10 days after birth was 38.9% (7/18), which was significantly greater than that [3.0% (1/33)] detected within 24 hours after birth (chi(c)(2) = 8.79, P < 0.01). Of the 33 Ct-positive samples from pregnant women, 9 had vaginal delivery and 24 had caesarean section. The vertical transmission rates in vaginal delivery group and caesarean section group were 66.7% (6/9) and 8.3% (2/24), respectively (chi(c)(2) = 9.16, P < 0.01). Incidence of premature rupture of membrane in Ct-positive group was 30.3% (10/33), which was greater than that of Ct-negative groups (13.5%, 36/267, chi(2) = 6.40, P < 0.05). Four different patterns were observed in the 16 Ct-positive samples from 8 pregnant women and 8 matched maternal-infants by using CFLP, which were confirmed by DNA sequencing later. They were type E (3 pairs), type F (2 pairs), type H (2 pairs) and type D (1 pair). Each pair of matched maternal-infantile samples presented identical CFLP pattern.

CONCLUSIONSThis study revealed the infection rate of Ct in pregnant women, vertical transmission rate of Ct and the common genotypes of Ct in Chongqing Women and Children's Health Care Institute. The CFLP assay by using a primer labeled at the 5'-end with digoxigenin was first used for genotyping of Ct. The assay showed a good sensitivity and reproducibility, no radioactive contamination, and is simple. Therefore the assay is a potential new method for Ct genotyping.

Cervix Uteri ; microbiology ; Chlamydia Infections ; diagnosis ; Chlamydia trachomatis ; genetics ; DNA Primers ; Female ; Genes, Bacterial ; genetics ; Genotype ; Humans ; Polymerase Chain Reaction ; Polymorphism, Genetic ; Pregnancy ; Pregnancy Complications, Infectious ; diagnosis

OBJECTIVETo prepare antibodies against pORF5 plasmid protein of Chlamydia trachomatis and develop double-antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISAs) for the detection of genital C. trachomatis infections.

METHODSThe pORF5 protein was expressed in Escherichia coli and used to immunize BALB/c mice and New Zealand rabbits to produce monoclonal antibodies (mAbs) and polyclonal antibody (pAb) for DAS-ELISAs. Clinical samples from 186 urogenital infection patients (groups I) and 62 healthy donors (groups II) were detected in parallel by the DAS-ELISAs developed in this study and by IDEIA PCE commercial ELISA.

RESULTSTwo hybridoma cell lines, named 2H4 and 4E6, stably secreting specific mAbs against pORF5 were obtained. The mAb 2H4 was recognized by 32 (17.20%, positive recognition rate) and 25 (13.44%), mAb 2H4 by 0 (0%) and 2 (3.22%) samples from groups I and II, respectively. The sensitivities of mAbs 2H4 and 4E6 were 92.11% and 77.78% and the specificities were 100% and 96.88%, respectively in relation to the IDEIA PCE commercial ELISA. The sensitivities of detection for the DAS-ELISAs were 10 ng/mL (based on 2H4) and 18 ng/mL (based on 4E6).

CONCLUSIONTwo DAS-ELISAs were developed in this study that provided a feasible and effective assay that could be considered alternative tools for the serodiagnosis of C. trachomatis infection.

Adolescent ; Adult ; Chlamydia Infections ; diagnosis ; Chlamydia trachomatis ; pathogenicity ; Enzyme-Linked Immunosorbent Assay ; methods ; Female ; Humans ; Male ; Middle Aged ; Urogenital System ; microbiology ; Young Adult

No abstract available.
Adolescent ; Adult ; Antigens, Bacterial/analysis ; Chlamydia Infections/*diagnosis/epidemiology/microbiology ; Chlamydia trachomatis/immunology ; DNA, Viral/analysis ; Female ; Humans ; Mass Screening/*methods ; Papillomavirus Infections/*diagnosis/epidemiology/virology ; Papillomavirus, Human/genetics ; Poland/epidemiology ; Prevalence ; *Sexual Behavior ; Urban Population

The purified elementary bodies of C. pneumoniae TW-183 were used for immunization of male BALB/c mice, the spleen cells of these mice were fused with SP2/0 cells and the hybrid cells were cloned by limiting dilution. One clone that secreted the C. pneumoniae monoclonal antibody (Cpn-McAb) stably was obtained finally. The Cpn-McAb belonged to IgG2b class and anti-Cpn-MOMP; the outcome of micro-immunofluorescence showed its weak cross reaction with the C. psittaci elementary body but it has no cross reaction with C. trachoma elementary body. It has the same speciality of the imported Cpn-McAb. For the evaluation of Cpn-McAb, the peripheral blood mononuclear cell specimens of 454 patients were detected by self-made Cpn-McAb and imported Cpn-McAb at the same time. The positive rates of Cpn-antigen were 53.3% for self-made Cpn-McAb and 52.6% for imported Cpn-McAb,showing high concordance between them (Kappa=0.714). The results showed that self-made Cpn-McAb has almost the same high specificity and sensitivity as imported Cpn-McAb, so the self-made Cpn-McAb may replace imported Cpn-McAb to detect Cpn specific antigen and be helpful to diagnosing and treating the clinical diseases associated with Cpn infection.
Animals ; Antibodies, Bacterial ; immunology ; Antibodies, Monoclonal ; biosynthesis ; genetics ; Antibody Specificity ; Chlamydia Infections ; diagnosis ; microbiology ; Chlamydophila pneumoniae ; immunology ; Hybridomas ; secretion ; Male ; Mice ; Mice, Inbred BALB C

BACKGROUND/AIMS: Fitz-Hugh-Curtis syndrome is defined as perihepatitis associated with pelvic inflammatory disease (PID). We retrospectively analyzed clinical and laboratory manifestations as well as the therapeutic response in patients with clinically diagnosed Fitz-Hugh-Curtis syndrome. METHODS: A cohort of 25 patients with PID and perihepatitis (as diagnosed by dynamic abdominal computed tomography (CT)) was enrolled. The prognosis, clinical manifestations, and physical examination, laboratory, and CT findings were analyzed. RESULTS: The mean (+/-SD) age of the patients was 32(+/-8) years, and all of them were sexually active, premenopausal women, and presented with abdominal pain. Of these, 52% complained of vaginal discharge. On physical examination, right upper-quadrant tenderness was the most common finding (84%), with lower-abdominal tenderness being present in 20% of patients. On laboratory examination, erythrocyte sedimentation rate and C-reactive protein were increased in 76% and 92% of the patients, respectively. The white blood cell count was increased in 60% of them. Most patients had a normal liver function test. Using a specimen of the cervical discharge, the polymerase chain reaction to test for Chlamydia trachomatis were positive in 87% (13/15) of the patients, and Chlamydia antigen was found in 75% (9/12) of them. Dynamic abdominal CT revealed subcapsular enhancement of the liver in the arterial phase. All of the patients improved with antibiotic therapy. CONCLUSIONS: Symptoms and physical findings suggestive of PID are not present in many patients with Fitz-Hugh-Curtis syndrome. When a premenopausal woman complains of upper abdominal pain and shows CT findings compatible with perihepatitis, examination of cervical discharge would be recommended to assess the possibility of Fitz-Hugh-Curtis syndrome.
Adult ; Anti-Bacterial Agents/therapeutic use ; Chlamydia Infections/diagnosis/microbiology ; Chlamydia trachomatis/isolation & purification ; Cohort Studies ; Diagnosis, Differential ; Drug Therapy, Combination ; Female ; Gonorrhea/complications/*diagnosis/radiography ; Hepatitis/complications/*diagnosis/radiography ; Humans ; Pelvic Inflammatory Disease/complications/*diagnosis/radiography ; Retrospective Studies ; Syndrome ; Tomography, X-Ray Computed

BACKGROUND: Fitz-Hugh-Curtis (FHC) syndrome is inflammation of the liver capsule associated with pelvic inflammatory disease. We measured Chlamydia trachomatis antibodies in 30 female patients with acute abdominal pain for diagnosis of FHC-syndrome, and the results were compared with other tests. METHODS: A dual-polymerase chain reaction was used for the detection of C. trachomatis in the cervix, and a micro-immunofluorescence test was performed to measure the antibody to C. trachomatis in serum. Cervical specimens were stained with Gram stain and cultured on chocolate agar for detection of Neisseria gonorrhoeae, and abdominal computed tomography (CT) and pelvic examinations were performed. RESULTS: Of the 30 patients examined, 19 were diagnosed as having FHC-syndromes and 11 abdominal pains without FHC-syndrome. C. trachomatis was detected from one of the five patients studied, and no N. gonorrhoeae was isolated from the patients with FHC-syndrome. High titers of IgG antibody (1:512-1:1,024) to C. trachomatis were demonstrated in all patients with FHC-syndrome. The CT scan revealed perihepatitis in 14 patients with FHC-syndrome. CONCLUSIONS: All patients with FHC-syndrome are associated with C. trachomatis infections, and a high titer of C. trachomatis antibody (IgG) is a very useful marker for FHC-syndrome.
Adolescent ; Adult ; Aged ; Antibodies, Bacterial/*analysis ; Cervix Uteri/chemistry ; Chlamydia Infections/*diagnosis/microbiology ; Chlamydia trachomatis/*immunology/isolation & purification ; Female ; Hepatitis/diagnosis ; Humans ; Immunoglobulin G/analysis ; Immunoglobulin M/analysis ; Middle Aged ; Pelvic Inflammatory Disease/complications/*diagnosis ; Syndrome ; Tomography Scanners, X-Ray Computed ; Young Adult