High treatment failure rate is better explained by resistance gene detection than by minimum inhibitory concentration in patients with urogenital Chlamydia trachomatis infection.

OBJECTIVE: The aim of this study was to investigate the relationships between treatment outcomes of patients with urogenital Chlamydia trachomatis infections and minimum inhibitory concentrations (MICs) and drug resistance genes. METHODS: The clinical data of 92 patients diagnosed with Chlamydia trachomatis (C. trachomatis) infections were collected. Of these patients, 28 received regular treatment with azithromycin and 64 received minocycline. All patients underwent three monthly follow-ups after the completion of treatment. The microdilution method was used for the in vitro susceptibility tests. The acquisition of 23S rRNA mutations and presence of the tet(M) gene were detected by gene amplification and sequencing. RESULTS: The MICs of azithromycin, clarithromycin, erythromycin, tetracycline, doxycycline, and minocycline were comparable for isolates from the treatment failure and treatment success groups. Higher detection rates of 23S rRNA gene mutations and tet(M) were found in the treatment failure group (57.14% and 71.43%, respectively) than in the treatment success group (14.29% and 30.23%, respectively) (p < 0.05). The A2057G, C2452A, and T2611C gene mutations of 23S rRNA were detected in eight clinical isolates from the azithromycin treatment failure group, while the T2611C gene mutation was detected in one clinical strain from the treatment success group. CONCLUSIONS: The detection of resistance genes could better explain the high treatment failure rate than the MIC results in patients with urogenital C. trachomatis infections, highlighting the need for genetic antimicrobial resistance testing in infected patients.

Ironing Out the Unconventional Mechanisms of Iron Acquisition and Gene Regulation in Chlamydia.

The obligate intracellular pathogen Chlamydia trachomatis, along with its close species relatives, is known to be strictly dependent upon the availability of iron. Deprivation of iron in vitro induces an aberrant morphological phenotype termed "persistence." This persistent phenotype develops in response to various immunological and nutritional insults and may contribute to the development of sub-acute Chlamydia-associated chronic diseases in susceptible populations. Given the importance of iron to Chlamydia, relatively little is understood about its acquisition and its role in gene regulation in comparison to other iron-dependent bacteria. Analysis of the genome sequences of a variety of chlamydial species hinted at the involvement of unconventional mechanisms, being that Chlamydia lack many conventional systems of iron homeostasis that are highly conserved in other bacteria. Herein we detail past and current research regarding chlamydial iron biology in an attempt to provide context to the rapid progress of the field in recent years. We aim to highlight recent discoveries and innovations that illuminate the strategies involved in chlamydial iron homeostasis, including the vesicular mode of acquiring iron from the intracellular environment, and the identification of a putative iron-dependent transcriptional regulator that is synthesized as a fusion with a ABC-type transporter subunit. These recent findings, along with the noted absence of iron-related homologs, indicate that Chlamydia have evolved atypical approaches to the problem of iron homeostasis, reinvigorating research into the iron biology of this pathogen.

Optimized testing for C. trachomatis DNA in synovial fluid samples in clinical practice.

AIM: No standardized polymerase chain reaction (PCR) assay is available for detection of Chlamydia trachomatis (C. tr.) in synovial fluid (SF) for diagnostic use in clinical practice. This study tested the performance of two optimized molecular biology methods, to determine which is best suited for detecting C. tr. in SF clinical samples from patients with various rheumatologic diseases. METHODS: Two DNA extraction methods, i.e., (1) alkaline lysis and (2) QIAEX II Gel Extraction Kit® + cetyltrimethylammonium bromide (CTAB; Qiagen, Hilden, Germany), and C. tr.-omp1-152 bp PCR were tested in SF samples from a total of 329 patients with the following diagnoses: reactive arthritis (ReA; n = 10, 4 patients had posturethritic ReA), undifferentiated arthritis (UA; n = 66), rheumatoid arthritis (RA; n = 169), psoriatic arthritis (PSA; n = 12), and osteoarthritis (OA) n = 72. RESULTS: In SF samples, C. tr.-omp1-152 bp PCR in combination with alkaline lysis DNA extraction allowed detection of more C. tr.-positive samples: 3/10 (30%) ReA patients (all with posturethritic ReA) and 20/66 (38%) UA patients were positive, compared to the 0/10 (0%) patients with ReA and 1/66 (2%) with UA detected using the QIAEX II Gel Extraction Kit® + CTAB. Moreover, 2/12 (17%) SF samples from PSA patients tested positive with alkaline lysis. All samples from patients with OA and RA tested negative. CONCLUSION: Alkaline lysis in combination with C. tr.-omp1-152 bp PCR emerged as the most sensitive method for identification of C. tr. in clinical SF samples.

Bubonic lymphogranuloma venereum with multidrug treatment failure.

A patient with proctitis and inguinal buboes diagnosed with lymphogranuloma venereum (LGV) was treated with doxycycline 21 days, azithromycin 20 days and moxifloxacin for a further 12 days because of progressive worsening of inguinal symptoms. Despite extensive antibiotic treatment, the inguinal LGV lesions persisted; however, the patient recovered spontaneously after three months.

A cohort study of Chlamydia trachomatis treatment failure in women: a study protocol.

BACKGROUND: Chlamydia trachomatis is the most commonly diagnosed bacterial sexually transmitted infection in the developed world and diagnosis rates have increased dramatically over the last decade. Repeat infections of chlamydia are very common and may represent re-infection from an untreated partner or treatment failure. The aim of this cohort study is to estimate the proportion of women infected with chlamydia who experience treatment failure after treatment with 1 gram azithromycin. METHODS/DESIGN: This cohort study will follow women diagnosed with chlamydia for up to 56 days post treatment. Women will provide weekly genital specimens for further assay. The primary outcome is the proportion of women who are classified as having treatment failure 28, 42 or 56 days after recruitment. Comprehensive sexual behavior data collection and the detection of Y chromosome DNA and high discriminatory chlamydial genotyping will be used to differentiate between chlamydia re-infection and treatment failure. Azithromycin levels in high-vaginal specimens will be measured using a validated liquid chromatography-tandem mass spectrometry method to assess whether poor azithromycin absorption could be a cause of treatment failure. Chlamydia culture and minimal inhibitory concentrations will be performed to further characterize the chlamydia infections. DISCUSSION: Distinguishing between treatment failure and re-infection is important in order to refine treatment recommendations and focus infection control mechanisms. If a large proportion of repeat chlamydia infections are due to antibiotic treatment failure, then international recommendations on chlamydia treatment may need to be re-evaluated. If most are re-infections, then strategies to expedite partner treatment are necessary.

Effects of transcutaneous electrical stimulation (1 pulse/sec) through custom-made disposable surface electrodes covering Omura's ST36 area of both legs on normal cell telomeres, oncogen C-fosAb2, integrin alpha5beta1, chlamydia trachomatis, etc. in breast cancer & alzheimer patients.

Our previous study indicated that when extremely reduced normal cell (NC) telomeres in various cancer patients are increased over 500 ng BDORT units, abnormally high cancer cell telomeres and cancer-related markers such as Oncogen C-fosAb2 (Onco.)& Integrin alpha5beta1 (Integ.), & 8-OH-dG as well as bacterial & viral infections, mercury, asbestos, chromium, & beta-amyloid (1-42) markedly reduced due to improved circulation & excretion of these substances in urine. Since 1995, we have been using press-needle stimulation of Omura's ST36 with 200x press-release procedure 4x a day, with significant improvements in various cancer patients. In this study, Transcutaneous Electrical Stimulation (TES) at 60 pulses/min, which is close to patient's heart rate, was given between Omura's ST36 of both legs of the breast cancer & Alzheimer's patients. After about 10 minutes of TES, NC telomeres increased from 1 yg (= 10-24 g) to 500-525 ng; Integ. reduced from 85-75 ng to 0.5 ng & Chlamydia trachomatis (CT) reduced from 4500-3500 ng to 0.5 ng. An additional 10 minutes TES increased NC telomeres to 800-875 ng, while Integ. reduced to 0.5 yg & CT became less than 0.1 yg. After a total 30 minutes of TES, NC telomeres increased to 1000-1200ng BDORT units, with decreases in Integ. and Onco. to less than 0.1 yg. CT reduced to << 0.1 yg. About 24 hours later, NC telomeres were still 300 ng & both Integ. and Onco. were 2.5 ng. CT was approximately 20 ng. In Alzheimer patient, abnormally high beta-Amyloid (1-42) of 7-12 ng markedly reduced to within normal value of less than 1.5 ng by 20-30 min TES. Stimulation beyond 30 minutes gradually reduced NC telomeres. TES pulse rate of 4 pulses/sec for the same patient initially increased NC telomere up to 750-950 ng BDORT units within 20 minutes, but when stimulation continued more than 20 min, NC telomeres rapidly reduced to -150 ng in less than 10 min of TES with reduced beneficial effects.

Baicalin suppresses expression of Chlamydia protease-like activity factor in Hep-2 cells infected by Chlamydia trachomatis.

In this study, we tested the ability of Baicalin to block Chlamydia trachomatis infection and found that the Baicalin blocked infection of Hep-2 cells. Then, we looked into the expression of RFX5 and CPAF gene in Chlamydia-infected cells. We found that RFX5 and CPAF were up-regulated and down-regulated respectively by Baicalin. Since CPAF is responsible for degrading RFX5, we suggest that CPAF was a primary target of Baicalin and played an important role in regulating RFX5. Our findings demonstrate that Baicalin can inhibit C. trachomatis effectively and therefore, can be considered as potential agents for therapy of Chlamydia infectious diseases.

Visual DNA microarrays for simultaneous detection of Ureaplasma urealyticum and Chlamydia trachomatis coupled with multiplex asymmetrical PCR.

Visual DNA microarrays, based on gold label silver stain (GLSS) and coupled with multiplex asymmetrical PCR, were developed for simultaneous, sensitive and specific detection of Ureaplasma urealyticum and Chlamydia trachomatis. 5'-end-amino-modified oligonucleotides, which were immobilized on glass surface, acted as capturing probes that were designed to bind complementary biotinylated targets DNA. The gold-conjugated streptavidins were introduced to the microarray for specific binding to biotin. The black image of microarray spots, resulting from the precipitation of silver onto nanogold particles bound to streptavidins, were used to detect biotinylated targets DNA visually or with a visible light scanner. Multiplex asymmetrical PCR of U. urealyticum, C. trachomatis and Bacillus subtilis (used as positive control) was performed to prepare abundant biotinylated single-stranded targets DNA, which affected detection efficiency and sensitivity of hybridization on microarray. Plenty of clinical samples of U. urealyticum and C. trachomatis from infected patients were tested using home-made DNA microarrays. For its high sensitivity, good specificity, simplicity, cheapness and speed, the present visual gene-detecting technique has potential applications in clinical fields.

Clinical efficacy of clarithromycin against uterine cervical and pharyngeal Chlamydia trachomatis and the sensitivity of polymerase chain reaction to detect C. trachomatis at various time points after treatment.

The detection and eradication of pharyngeal Chlamydia trachomatis in patients with chlamydial uterine cervicitis (commercial sex workers and others) were investigated. Pharyngeal C. trachomatis was detected in 75.0% of the commercial sex workers and in 21.9% of the other subjects. All the pharyngeal C. trachomatis-positive patients had a past history of orogenital contact. Chlamydial infection was treated with clarithromycin for 7 or 14 days. The presence of C. trachomatis was determined by polymerase chain reaction (PCR) on days 8, 15, and 22 after completion of the treatment. In the 7-day treatment group, the eradication rate of pharyngeal C. trachomatis was 53.3%, 56.7%, and 60.0% on days 8, 15, and 22, respectively, after completion of the treatment, while the eradication rate of cervical C. trachomatis was 83.3%, 96.7%, and 100% on days 8, 15, and 22, respectively. The eradication rate of pharyngeal C. trachomatis in the 7-day treatment was significantly lower than that of cervical C. trachomatis, while there was no significant difference in the 14-day treatment. The eradication rate of pharyngeal C. trachomatis in the 14-day treatment was significantly higher than that in the 7-day treatment. Since the DNA of dead organisms may be detected because of high PCR sensitivity, appropriate therapeutic judgment by PCR could be done around day 22 after completion of the treatment.

Chlamydia trachomatis genes whose products are related to energy metabolism are expressed differentially in active vs. persistent infection.

The Chlamydia trachomatis genome encodes glycolysis and pentose phosphate pathway enzymes, two ATP/ADP exchange proteins, and other energy transduction-related components. We asked if and when chlamydial genes specifying products related to energy transduction are expressed during active vs. persistent infection in in vitro models and in synovia from Chlamydia-associated arthritis patients. Hep-2 cells infected with K serovar were harvested from 0-48 h post-infection (active infection). Human monocytes identically infected were harvested at 1, 2, 3, 5 days post-infection (persistent). RNA from each preparation and from synovial samples PCR-positive/-negative for Chlamydia DNA was subjected to RT-PCR targeting (a) chlamydial primary rRNA transcripts and adt1 mRNA, (b) chlamydial mRNA encoding enzymes of the glycolysis (pyk, gap, pgk) and pentose phosphate (gnd, tal) pathways, the TCA cycle (mdhC, fumC), electron transport system (cydA, cydB), and sigma factors (rpoD, rpsD, rpoN). Primary rRNA transcripts and adt1 mRNA were present in each infected preparation and patient sample; controls were negative for chlamydial RNA. In infected Hep-2 cells, all energy transduction-related genes were expressed by approximately 11 h post-infection. In monocytes, pyk, gap, pgk, gnd, tal, cydA mRNA were present in 1-2-day-infected cells but absent at 3 days and after; cydB, mdhC, fumC were expressed through 5 days post-infection. RT-PCR targeting mRNA from sigma factor genes indicated that lack of these gene products cannot explain selective transcriptional down-regulation during persistence. Analyses of RNA from synovial tissues mirrored those from the monocyte system. These data suggest that in the first phase of active chlamydial infection, ADP/ATP exchange provides energy required for metabolism; in active growth, glycolysis supplements host ATP. In persistence host, rather than bacterially produced, ATP is the primary energy source. Metabolic rate in persistent C. trachomatis is lower than in actively growing cells, as judged from assays for relative chlamydial primary rRNA transcript levels in persistent vs. actively growing cells.

In vitro analysis of the change in resistance of Chlamydia trachomatis under exposure to sub-MIC levofloxacin for a therapeutic term.

BACKGROUND: While fluoroquinolone-resistant Chlamydia trachomatis strains have not been clinically isolated, they were isolated in an in vitro study recently. METHODS: To determine whether C. trachomatis strains develop resistance under sub-MIC antibacterial exposure in a clinical therapeutic term, C. trachomatis strains were exposed to sub-MIC levofloxacin (LVFX) for about 2 weeks. The MIC of LVFX was measured and DNA fingerprinting was performed every 72 h by PCR using random primers. RESULTS: There was almost no change in the MIC under exposure to 0.125 microg/ml LVFX. However, some mutational changes in DNA fingerprints developed. CONCLUSIONS: In clinical therapeutic terms, resistant strains of C. trachomatis will probably not develop, even if sub-MIC LVFX is employed.

Multiple drug-resistant Chlamydia trachomatis associated with clinical treatment failure.

In vitro susceptibility testing and genotyping were done on urogenital isolates of Chlamydia trachomatis from 3 patients, 2 of whom showed evidence of clinical treatment failure with azithromycin and one of whom was the wife of a patient. All 3 isolates demonstrated multidrug resistance to doxycycline, azithromycin, and ofloxacin at concentrations >4.0 microg/mL. Recurrent disease due to relapsing infection with the same resistant isolate was documented on the basis of identical genotypes of both organisms. This first report of clinically significant multidrug-resistant C. trachomatis causing relapsing or persistent infection may portend an emerging problem to clinicians and public health officials.

Frequency of apolipoprotein E (APOE) allele types in patients with Chlamydia-associated arthritis and other arthritides.

Genetic background is important in determining whether certain infecting bacteria disseminate to the joint and cause arthritis. We assessed whether APOE genotype is associated with the presence of DNA from Chlamydia or other bacteria in synovial tissues of patients with various arthritides. Nucleic acids from synovial tissues of 135 patients were screened by PCR for DNA from Chlamydia trachomatis, C. pneumoniae and other bacteria (pan-bacteria). APOE genotype was determined by a PCR-based method for all patients in each of four resulting groups comprised of about 35 individuals each, positive for C. trachomatis only, C. pneumoniae only, other bacteria, or no bacteria. RT-PCR was used to assess synovial APOE expression. The latter assays confirmed that APOE mRNA is present in synovial tissue. Determination of APOE genotype showed that patients PCR-negative in all assays, and those positive in the C. trachomatis - and pan-bacteria- (excluding Chlamydia) directed assays, had distributions of the APOE epsilon2, epsilon3 and epsilon4 alleles mirroring those of the general population (i.e. about 8%, 79% and 13%, respectively). In contrast, 68% of patients with C. pneumoniae DNA in synovium possessed a copy of the epsilon4 allele. These results indicate that no association exists between APOE genotype and synovial presence of C. trachomatis or other bacteria. However, individuals bearing at least one copy of the APOE epsilon4 allele may be at increased risk for synovial infection by C. pneumoniae.