Comparison of plasmid- and chromosome-based polymerase chain reaction assays for detecting Chlamydia trachomatis nucleic acids

Several laboratories have demonstrated that the polymerase chain reaction (PCR) is more sensitive than culture or enzyme immunoassay (EIA) for detecting Chlamydia trachomatis in genitourinary tract specimens when various DNA targets are used for amplification, including the cryptic plasmid, major outer membrane protein (MOMP), or rRNA genes. We compared the performances of five different PCR assays, including assays with two plasmid, two MOMP, and one rRNA targets, by amplifying serial dilutions of C. trachomatis DNA and testing genitourinary tract specimens. By using published procedures, two different plasmid primers had sensitivities of 0.1 fg for C. trachomatis plasmid DNA and 10 fg for total cellular DNA. The sensitivities of the assays with the two MOMP primers were 0.1 and 10 pg, and the sensitivity for the assay with the rRNA primers was 1 pg for cellular DNA. Both plasmid-based assays detected 38 of 38 confirmed Chlamydiazyme-positive specimens, whereas the assays with the MOMP and rRNA primers detected 36 of 38 and 29 of 38 confirmed Chlamydiazyme-positive specimens, respectively. Six of 18 Chlamydiazyme-negative specimens collected from individuals whose specimens were positive by culture or immunofluorescence were positive by both plasmid-based PCRs; 4 of these were positive by PCR with the MOMP primers and 3 were positive by PCR with the rRNA primers. The results obtained with both purified DNA and genitourinary tract specimens indicated that the plasmid-based PCRs are more sensitive than bacterial chromosome-based PCRs for detecting C. trachomatis.     

Community-based urine screening for Chlamydia trachomatis with a ligase chain reaction assay

BACKGROUND: Urine tests for Chlamydia trachomatis permit expansion of screening beyond traditional clinic environments. Prevention of infection in teenagers is a high priority. OBJECTIVE: To define the prevalence of C. trachomatis among teenagers by using the ligase chain reaction assay on urine specimens and to evaluate leukocyte esterase testing of urine specimens as an indicator of infection. DESIGN: Cross-sectional study. SETTING: An adolescent clinic, a juvenile detention facility, seven school-based clinics, and three community-based youth organizations in Seattle, Washington. PARTICIPANTS: 10,118 sexually active teenagers and young adults. MEASUREMENTS: Chlamydia trachomatis infection detected in urine specimens by ligase chain reaction assay and leukocyturia detected by leukocyte esterase testing. RESULTS: The prevalence of chlamydial infection among female participants was 8.6% and declined with increasing age; among male participants, it was 5.4% and increased with increasing age. In female participants, independent predictors of infection were being 17 years of age or younger (odds ratio [OR], 1.49), having had two or more sex partners in the previous 2 months (OR, 1.61), and having genitourinary symptoms (OR, 1.46). In male participants, independent predictors were being of nonwhite race or ethnicity (OR, 2.00 to 3.08), having had two or more sex partners in the previous 2 months (OR, 1.57), and having used a condom during the most recent sexual encounter (OR, 0.67). For identifying infection in male participants, the sensitivity of leukocyte esterase testing was 58.9%, the specificity was 94.9%, the positive predictive value was 38.4%, and the negative predictive value was 97.7%. CONCLUSIONS: Chlamydial infection is common in teenagers and young adults in community settings. The urine ligase chain reaction assay will permit widespread screening for C. trachomatis, but leukocyte esterase testing had low sensitivity for selecting persons for screening with this assay. Indicators of chlamydial infection differed substantially in male and female participants.     

Use of DNA purification kits for polymerase chain reaction testing of Gen-Probe Chlamydia trachomatis PACE 2 specimens

Psychological stress and chronic anxious behavior have a tremendous impact on our heart and biological rhythm of the body. Both are responsible for new development or promotion of coronary heart disease and may be associated with unpredictable adverse coronary events.     

Selective screening for Chlamydia trachomatis infection in a primary care population of women

The potency of food chemicals to induce cell aging was evaluated in human diploid fibroblast cells HAIN-55 having a finite replicative potential by using in vitro aging markers, i.e., decreases of maximum proliferative potential (lifespan) of cells, saturation density in monolayer culture (SD), plating efficiency (PE) and mitotic index (MI), and an increase of cells with polyploid karyotypes. By treatment twice with low concentration of genotoxic chemicals aflatoxin B1, allylisothiocyanate or trans-cinnamaldehyde (severe clastogenic flavoring agent; Kasamaki et al., 1982), lifespan (expressed by the number of cumulative cell population doubling (CPD)) of the treated cells was reduced by 8-12 CPDs accompanied by change of the other aging markers. By successive treatment (29 or 25 times) with non-genotoxic chemical aspartame (N-L-aspartyl-L-phenylalanine) or L-canavanine (structural analogue of L-arginine), lifespan of the treated cells was also slightly shortened (by 2-6 CPDs) compared with the untreated control cells. In the process of cell aging, Mitochondrial activity (MTT activity) decreased almost in parallel with the decrease of SD and MI. On the basis of these results, a variety of genotoxic and non-genotoxic chemicals were examined by using MTT activity as the aging marker for their effects on the aging of HAIN-55 cells and bovine artery endothelial cells which also had a finite replicative potential. The results showed that seven genotoxic and nine non-genotoxic chemicals promoted cell aging.     

Detection of Chlamydia trachomatis in semen by the polymerase chain reaction in male members of infertile couples

We found that on pulmonary auscultation, fine crackles could be induced by changing the posture from sitting to supine and/or from supine to supine with passive leg elevation in patients without obvious congestive heart failure. We named these crackles "posturally induced crackles (PIC)." To investigate the relationship between PIC and long-term prognosis after myocardial infarction, we followed up 262 patients who recovered from acute myocardial infarction for a mean period of six years. Cardiac death occurred in three of 78 PIC-negative patients and in 28 of 143 PIC-positive patients. PIC-negative patients had a significantly better long-term prognosis than PIC-positive patients according to the Kaplan-Meier survival curves for cardiac death (p < 0.01). In a multilogistic model based on 70 appropriate cases, PIC was the third most important prognosticator after recovery from myocardial infarction and the number of diseased coronary vessels and the pulmonary capillary wedge pressure ranked first and second, respectively.     

The in situ polymerase chain reaction for detection of chlamydia trachomatis

The in situ polymerase chain reaction (PCR) is a technique that has important applications in the diagnosis of viral and bacterial diseases. This study investigated an in situ PCR assay established to detect the presence of Chlamydia trachomatis in endocervical swabs. In addition, histological sections of endocervical squamous cell carcinoma were analyzed because previous studies had revealed a significant association with C. trachomatis. A total of 20 cervical neoplasms (squamous cell carcinoma in situ; n = 10; invasive squamous cell carcinoma; n = 10) and endocervical smears taken from five patients with and without inflammatory changes were analyzed by conventional PCR. Chlamydial DNA was found in 10 histological samples (six carcinomas in situ, four invasive carcinomas) and in one endocervical swab from a patient with known C. trachomatis infection. Positive specimens were used for establishing an in situ PCR assay (IS-PCR). After IS-PCR, these samples showed dense cytoplasmic staining of endocervical cells (smears) and non-neoplastic epithelial cells (cervical neoplasms). The other tumor samples and smears did not demonstrate positive PCR reaction. The results indicate that in situ PCR is an effective technique for localizing C. trachomatis in target cells because IS-PCR detection of chlamydial DNA correlated with histological and cytological features.     

Detection of Chlamydia trachomatis and Ureaplasma urealyticum from aborted tissues by polymerase chain reaction technique]

Helicobacter pylori (Hp) eradication in peptic ulcer disease is associated with a greatly reduced recurrence rate. The optimal drug regimen for HP eradication remains uncertain. It is also unclear if eradication of Hp in duodenitis and antral gastritis improves symptoms. The aims of this study were to compare the efficacy of three drug regimens in the eradication of Hp and to assess if Hp eradication improved symptoms in patients with duodenitis and antral gastritis. Patients (n = 79) found to have duodenal ulcer, duodenitis and/or antral gastritis with a positive urease test (CLO) at endoscopy were allocated to one of the three regimens: A. omeprazole 20 mg b.d. and clarithromycin 500 mg t.d.s. for two weeks (n = 27), B. De-Nol 240 mg b.d. for four weeks, metronidazole 400 mg t.d.s. and amoxicillin 500 mg t.d.s. for one week (n = 26), and C. omeprazole 20 mg b.d. and amoxicillin 500 mg t.d.s. for two weeks (n = 26). In conclusion, traditional 'triple' therapy with bismuth and two antibiotics achieved the highest Hp eradication rate and was best tolerated. Recolonisation with Hp was uncommon after eradication. Dyspeptic symptoms improved with Hp eradication in duodenitis and antral gastritis.     

Comparison of performances of two commercially available tests, a PCR assay and a ligase chain reaction test, in detection of urogenital Chlamydia trachomatis infection

The diagnostic performance of a PCR test (Roche Cobas Amplicor CT/NG Test) and that of a ligase chain reaction (LCR) test (Abbott LCx Chlamydia trachomatis assay) were compared by using endocervical and urethral swab specimen culture as a reference test. First-void urine (FVU) and endocervical and urethral swab specimens were collected from 1,015 unselected patients attending a sexually transmitted disease clinic and a clinic for adolescents in Helsinki, Finland. Chlamydia trachomatis was cultured from samples from the endocervix or urethra. PCR was performed with fresh and frozen urine and the culture transport medium. LCR was performed with fresh and frozen urine and LCx swab transport medium. Diagnostic consistency and diagnostic accuracy were statistically tested. The test results were identical for 984 patients (97%). Discrepant results were observed for 31 patients. Overall, LCR and PCR showed excellent kappa coefficients of consistency for both swab and FVU specimens (0.93 and 0.95, respectively). Sixty-one patients (6%) were culture positive. Testing of FVU by LCR or PCR increased the overall positivity rates to 7.0 and 7.7%, respectively. While PCR of FVU detected the greatest number of C. trachomatis infections (sensitivity, 96.1%), for some PCR-positive FVU specimens the results could not be confirmed (specificity, 99.6%). PCR and LCR were more sensitive than culture (sensitivities, 92 and 93% versus 79% for culture) in the diagnosis of genital C. trachomatis infection. In conclusion, both tests can be recommended for use in the clinical laboratory and for the screening of asymptomatic C. trachomatis infections.     

Prevalence of cervical Chlamydia trachomatis infection in a female population seeking contraception counseling

To analyze the outcome of systemic lupus erythematosus (SLE) associated with acute disseminated intravascular coagulation (DIC) and also to clarify the clinical factor(s) contributing to the outcome, we retrospectively investigated 120 SLE patients treated between 1981 and 1991. Eight of these patients (6.7%) developed acute DIC; four recovered and the other four died within 2 weeks of onset. Infection preceded acute DIC in all these patients. Acute DIC associated with atypical pneumonia was always fatal, while the patients with pharyngitis or urinary tract infection survived when they were treated adequately. Comparison of the dead and surviving groups revealed that the activity of SLE before the onset of DIC, the severity of DIC, and the treatment given for DIC and the coexistent infection were not significantly related to a fatal outcome. However, severe infection such as atypical pneumonia in patients with secondary immunodeficiency was likely to be fatal irrespective of the presence of DIC.     

Diagnosis of extrapulmonary tuberculosis by a commercial polymerase chain reaction kit

The Roche Amplicor Mycobacterium tuberculosis PCR test was compared with mycobacterial culture for direct detection of M. tuberculosis in extrapulmonary specimens. From January 1995 to October 1996, 124 clinical specimens from 112 patients were assessed, including 47 body fluids, 61 tissue specimens and 16 abscesses. The sensitivity and specificity of Amplicor PCR compared to culture were 63.6% and 93.1% respectively. Analysis of 7 PCR-positive, culture-negative specimens confirmed that all were from patients with recently diagnosed tuberculosis under treatment. Eight specimens were PCR negative-culture positive, including a pleural fluid containing inhibitory substances. On acid-fast bacilli (AFB) smear-negative specimens, sensitivity and specificity were 53 and 100% respectively. The best results for Amplicor PCR were obtained with abscesses and biopsies. It is concluded that this test, highly specific for the diagnosis of tuberculosis, is at least as sensitive on extrapulmonary specimens as on smear-negative respiratory specimens.     

Urinary inhibitors of polymerase chain reaction and ligase chain reaction and testing of multiple specimens may contribute to lower assay sensitivities for diagnosing Chlamydia trachomatis infected women

Clinical and morphologic features of a progressive polyneuropathy in young mature Alaskan Malamutes are described. Clinical signs included progressive paraparesis, synchronous pelvic limb gait, exercise intolerance, hyperesthesia, hyporeflexia, muscle atrophy, and tetraplegia. Electromyographic testing revealed diffuse fibrillation potentials and positive sharp waves in limb muscles, especially in muscles below the elbow and stifle. Pathologic findings in skeletal muscles and peripheral nerves included neurogenic muscle atrophy, focal or diffuse loss of myelinated nerve fibers, myelinoaxonal necrosis, and variable demyelination or remyelination. Ultrastructural changes included axonal degeneration, presence of numerous Büngner bands, and denervated Schwann cell subunits. The nature and distribution of abnormal electrophysiologic and pathologic findings were suggestive of a distal sensorimotor polyneuropathy, which we have termed idiopathic polyneuropathy of Alaskan Malamutes to distinguish this condition from hereditary polyneuropathy of Norwegian Alaskan Malamutes, last described in 1982.     

Demonstration of Chlamydia trachomatis in inguinal lymphadenitis of lymphogranuloma venereum: a light microscopy, electron microscopy and polymerase chain reaction study

Intravacuolar organisms in vacuolated macrophages were associated with areas of necrosis and suppuration in 12 patients with suppurative inguinal lymphadenitis. The intravacuolar organisms measured 0.2 to 2.0 micrometers in diameter, stained Gram negative with the Brown-Hopp's tissue Gram stain, faintly blue with hematoxylin and eosin stain, and black with the Warthin-Starry silver impregnation stain. The organisms lined vacuolar membranes and/or clumped in centers of vacuoles. Electron microscopy revealed elementary and reticulate bodies and intermediate forms characteristic of the genus Chlamydia. Cultures of three lymph nodes in McCoy cells grew Chlamydia trachomatis, lymphogranuloma venereum (LGV) serovars. Polymerase chain reaction using primers for chlamydial 16S ribosomal DNA confirmed the organisms as Chlamydia in lymph nodes from nine patients. Recognition of chlamydial organisms by light microscopy in tissue sections of lymph nodes allows a definitive diagnosis of lymphogranuloma venereum.     

Performance and cost-effectiveness of selective screening criteria for Chlamydia trachomatis infection in women. Implications for a national Chlamydia control strategy

At 9 mM glucose, experimental results show that mitochondrial phosphate depletion (induced by glucose phosphorylation, catalyzed by mitochondrial hexokinase) reduces the activities of the respiratory chain, oxidative phosphorylation, and glutaminase. Consequently, the 14C-lactate oxidation to 14CO2 is lowered in the presence of glucose. The fall of ATP level triggers a high aerobic glycolysis by deinhibiting fructose-6-P kinase. NADH, generated by enhanced glyceraldehyde-3-P dehydrogenase activity, increases the reducing power. Moreover, the lactate dehydrogenase (LDH) system is shifted toward lactate formation, while NAD+ is regenerated and the oligomycin-inhibited ATP production is replaced by the iodoacetate-inhibited ATP production. From 14CO2 production and lactate accumulation it is calculated that about 60% of 14C-glucose which disappears is channelled into extraglycolytic reactions. On the contrary, 82% of glucose below l mM is metabolized through non-glycolytic reactions. The pyruvate kinase-M2 (PK-M2) inhibition does not limit the glycolytic flow from 9 mM glucose, but it may cause sustained gluconeogenesis.     

Detection of chlamydia trachomatis by polymerase chain reaction assay in nonbacterial prostatitis

BACKGROUND: Melasma is a common disorder of facial hyperpigmentation among Asian women. Many modalities of treatment are available but none is satisfactory. OBJECTIVE: This study was undertaken to see if glycolic acid peels are effective and safe in the treatment of melasma and fine facial wrinkling. METHODS: Ten Asian women with moderate to severe melasma were recruited into the study. The women had twice daily applications of a cream containing 10% glycolic acid and 2% hydroquinone (Neostrata AHA Age Spot and Skin Lightening Gel) to both sides of the face, and glycolic acid peels every 3 weeks (20-70%) to one-half of the face using Neostrata Skin Rejuvenation System. All patients had to use a sunblock (SPF 15%). At regular intervals and at the end of 26 weeks (or after eight peels) the degree of improvement of pigmentation and fine facial wrinkling on each side of the face were assessed. Any skin irritation or side effects were also noted. Assessment was by an independent dermatologist, the patients themselves, and the use of the Munsell color chart and photographs. The nonparametric Wilcoxon Rank-Sum test was used for statistical analysis. RESULTS: The melasma and fine facial wrinkling improved on both sides of the face. The side that received glycolic acid peels did better but the results were not statistically significant (P > 0.059). CONCLUSION: A cream containing 10% glycolic acid and 2% hydroquinone (Neostrata AHA Age Spot and Skin Lightening Gel) improved melasma and fine facial wrinkling in Asian women. In combination with glycolic acid peels at 3-week intervals the lightening of melasma is subjectively much better. This improvement does not reach statistical significance and the sample size is small (n = 10).     

Vaginal douching as a risk factor for cervical Chlamydia trachomatis infection

Females with Turner syndrome (TS) are alleged to have increased numbers of melanocytic naevi. Although a high count of acquired melanocytic naevi (AMN) is one of the major risk factors for melanoma, this malignancy has been reported only rarely in patients with TS. The purpose of this study was to explore the effects of environmental and genetic factors on AMN count and density in TS. AMN count and density in 24 patients with TS treated with growth hormone (GH). 24 GH-treated females with GH deficiency (GHD) and 24 normal females were compared in a cross-sectional study. The average AMN density in TS was 50 naevi/m2 as compared with 18 naevi/m2 in the GHD group and 24 naevi/m2 in normal controls (P = 0.001 and P = 0.004, respectively). Duration of GH therapy did not correlate with AMN count (P = 0.44) or AMN density (P = 0.81). The pattern of distribution of naevi between constantly exposed, intermittently exposed and unexposed skin was similar in all groups. Sun exposure was the major factor that affected the regional AMN densities in the control groups, but not in the TS group. The findings of our study indicate that the effects of environmental factors on AMN count and density may vary among genetically different populations. A review of the literature suggested that melanoma is no more prevalent in TS than in the general population.     

Evaluation of polymerase chain reaction for detection of Mycoplasma meleagridis infection in turkeys

Different methods were compared for the detection of the turkey pathogen Mycoplasma meleagridis in a turkey field flock before and after antibiotic (oxytetracycline) treatment. They included culture, serology (detection of antibodies by ELISA) and a polymerase chain reaction (PCR) based assay developed by Boyle et al. [Boyle, J.S., Good, R., Morrow, C.J., 1995. Detection of the turkey pathogens Mycoplasma meleagridis and M. iowae by amplification of genes coding for rRNA. J. Clin. Microbiol. 33, 1335-1338]. Culture and PCR assay with tracheal swab samples were much more sensitive than ELISA with serum samples. Percentages of infected birds detected by culture or PCR for samples collected prior to antibiotic treatment were almost identical but the percentage of positive samples detected after antibiotic treatment was much higher with the PCR test.     

Is urine leukocyte esterase test a useful screening method to predict Chlamydia trachomatis infection in women?

We evaluated the use of the leukocyte esterase test (LET) on first-catch urine specimens from women as a screening test to predict infection with Chlamydia trachomatis. For diagnosis, we used Abbott's ligase chain reaction (LCR) on urine specimens and isolation by tissue culture (TC) on cervical brushes. Of 4,053 women attending sexually transmitted disease and family planning clinics, 4.3% (n = 174) were positive by TC and 5.9% (n = 239) were positive by LCR. When LET was compared to TC, the sensitivity, specificity, positive predictive value, and negative predictive value were 54.0, 67.0, 6.8, and 97.0%, respectively. The corresponding performance of LET versus LCR was 53.1, 67.3, 10.1, and 95.8%. Almost half of the laboratory-confirmed chlamydial infections were negative by LET. The low specificity probably reflects multiple causes of pyuria in women and results in a low positive predictive value. LET is neither sensitive nor specific as a predictor of chlamydial infection and cannot be recommended for use as a screening test for C. trachomatis with first-catch urine samples from females from low- or moderate-prevalence populations.     

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.