Urine Specimens from Pregnant and Nonpregnant Women Inhibitory to Amplification of \u3cem\u3eChlamydia trachomatis\u3c/em\u3e Nucleic Acid by PCR, Ligase Chain Reaction, and Transcription-Mediated Amplification: Identification of Urinary Substances Associated with Inhibition and Removal of Inhibitory Activity

The presence of endogenous amplification inhibitors in urine may produce false-negative results for the detection of Chlamydia trachomatis nucleic acids by tests such as PCR, ligase chain reaction (LCR), and transcription-mediated amplification (TMA). Consecutive urine specimens from 101 pregnant women and 287 nonpregnant women submitted for urinalysis were processed for C. trachomatis detection. Aliquots were spiked with the equivalent of one C. trachomatis elementary body and were tested by three commercial assays: AMPLICOR CT/NG, Chlamydia LCX, and Chlamydia TMA. The prevalence of inhibitors resulting in complete inhibition of amplification was 4.9% for PCR, 2.6% for LCR, and 7.5% for TMA. In addition, all three assays were partially inhibited by additional urine specimens. Only PCR was more often inhibited by urine from pregnant women than by urine from nonpregnant women (9.9 versus 3.1%; P = 0.011). A complete urinalysis including dipstick and a microscopic examination was performed. Logistic regression analysis revealed that the following substances were associated with amplification inhibition: beta-human chorionic gonadotropin (odd ratio [OR], 3.3) and crystals (OR, 3.3) for PCR, nitrites for LCR (OR, 14.4), and hemoglobin (OR, 3.3), nitrites (OR, 3.3), and crystals (OR, 3.3) for TMA. Aliquots of each inhibitory urine specimen were stored at 4 and -70°C and a dilution of 1:10 (84% for PCR, 100% for LCR, and 92% for TMA). Five urine specimens (three for PCR and two for TMA) required phenol-chloroform extraction to remove inhibitors. The results indicate that the prevalence of nucleic acid amplification inhibitors in female urine is different for each technology, that this prevalence may be predicted by the presence of urinary factors, and that storage and dilution remove most of the inhibitors

Rapid Diagnosis of Trichomonas vaginalis by Testing Vaginal Swabs in an Isothermal Helicase-Dependent AmpliVue Assay

The AmpliVue™ Trichomonas Assay (Quidel) is a new FDA cleared rapid test for qualitative detection of Trichomonas vaginalis (TV) DNA in female vaginal specimens. The assay is based on BioHelix’s Helicase-Dependent Amplification (HDA) isothermal technology in conjunction with a disposable lateral-flow detection device, with a total turn-around time of approximately 45 minutes

Smoking, season, and detection of chlamydia pneumoniae DNA in clinically stable COPD patients

BACKGROUND: The prevalence and role of Chlamydia pneumoniae in chronic obstructive pulmonary disease (COPD) remain unclear. METHODS: Peripheral blood mononuclear cells were obtained from 100 outpatients with smoking-related, clinically stable COPD, and induced sputum was obtained in 62 patients. RESULTS: Patients had mean age (standard deviation) of 65.8 (10.7) years, mean forced expiratory volume in one second of 1.34 (0.61) L, and 61 (61.0%) were male. C. pneumoniae nucleic acids were detected by nested polymerase chain reaction in 27 (27.0%). Current smoking (odds ratio {OR} = 2.6, 95% confidence interval {CI}: 1.1, 6.6, P = 0.04), season (November to April) (OR = 3.6, 95% CI: 1.4, 9.2, P = 0.007), and chronic sputum production (OR = 6.4, 95% CI: 1.8, 23.2, P = 0.005) were associated with detection of C. pneumoniae DNA. CONCLUSIONS: Prospective studies are needed to examine the role of C. pneumoniae nucleic acid detection in COPD disease symptoms and progression

Association of circulating Chlamydia pneumoniae DNA with cardiovascular disease: a systematic review

BACKGROUND: Chlamydia pneumoniae antigens, nucleic acids, or intact organisms have been detected in human atheroma. However, the presence of antibody does not predict subsequent cardiovascular (CV) events. We performed a systematic review to determine whether the detection of C. pneumoniae DNA in peripheral blood mononuclear cells (PBMC) was associated with CV disease. METHODS: We sought studies of C. pneumoniae DNA detection in PBMC by polymerase chain reaction (PCR) among patients with CV disease or other clinical conditions. We pooled studies in which CV patients were compared with non-diseased controls. We analyzed differences between studies by meta-regression, to determine which epidemiological and technical characteristics were associated with higher prevalence. RESULTS: Eighteen relevant studies were identified. In nine CV studies with control subjects, the prevalence of circulating C. pneumoniae DNA was 252 of 1763 (14.3%) CV patients and 74 of 874 (8.5%) controls, for a pooled odds ratio of 2.03 (95% CI: 1.34, 3.08, P < 0.001). Prevalence was not adjusted for CV risk factors. Current smoking status, season, and age were associated with C. pneumoniae DNA detection. High prevalence (>40%) was found in patients with cardiac, vascular, chronic respiratory, or renal disease, and in blood donors. Substantial differences between studies were identified in methods of sampling, extraction, and PCR targets. CONCLUSIONS: C. pneumoniae DNA detection was associated with CV disease in unadjusted case-control studies. However, adjustment for potentially confounding measures such as smoking or season, and standardization of laboratory methods, are needed to confirm this association

The evaluation of diagnostic tests for sexually transmitted infections

Diagnostic tests should receive method- and use-effectiveness evaluations. Method-effectiveness evaluations determine sensitivity, specificity and predictive values for new tests. Use-effectiveness evaluations determine how practical or convenient a new test will be in a specific setting and may not be performed in a formal way in North American laboratories. To perform a clinical method evaluation of diagnostic tests, a good relationship between laboratory and clinical personnel is essential. Studies are usually conducted separately on populations of men and women, and should include sampling from different prevalence groups. Test performance comparisons may be made on a single specimen type or on more than one specimen from the same patient, which allows for the expansion of a reference standard and includes the ability of a particular assay, performed on a specimen type to diagnose an infected individual. The following components of the evaluation should be standardized and carefully followed: specimen identification; collection; transportation; processing; quality control; reading; proficiency testing; confirmatory testing; discordant analysis - sensitivity, specificity and predictive value calculations; and record keeping. Methods are available to determine whether sample results are true or false positives or negatives. Use-effectiveness evaluations might determine the stability or durability of supplies and equipment; the logistics of shipping, receiving and storing supplies; the clarity and completeness of test instructions; the time and effort required to process and read results; the subjectivity factors in interpretation and reporting; and the costs. These determinations are usually more apparent for commercial assays than for homemade tests

Powassan virus multiplication in Dermacentor andersoni ticks

All stages of Dermacentor andersoni (Stiles) ticks were infected by feeding on rabbits injected intravenously with large doses of Powassan virus 48 to 72 hours after the ticks became attached. Viremia titers at least 10²˙⁵ mouse LD₅₀ per ml were required to establish infection in 1 to 5% of ticks ingesting viremic blood. The "infection-threshold" of D. andersoni adults for Powassan virus was elevated when viremia was induced towards the end of engorgement. When each stage in the tick's life cycle was infected a viral-eclipse phase was demonstrated in gut cells. Other organs such as salivary glands, Gene's organ glands and accessory glands supported virus multiplication. This was demonstrated by infectivity titrations and observation of fluorescent foci when the tissues were stained with Powassan virus antiserum conjugated with fluorescein isothiocyanate (FITC). Transstadial transfer of Powassan virus was observed through ecdysis of larvae to nymphs and from nymphs to adults. This occurred both after feeding on hamsters (which produced a substantial viremia) and guineapigs (which did not become viremic). Virus-was detected only in larval gut and invaded nymphal salivary glands during the molt. Infection localized in gut and salivary glands of engorged nymphs and flat adults, and was not detected in adult female accessory and Gene's glands until repletion. Adult males maintained high virus titers in salivary glands only. Transovarial transfer was not detected although infectivity was present on the surface of eggs laid by the mediation of an infected Gene's organ gland. Powassan virus was detected in salivary gland secretions but not in rectal excretions in adults or nymphs. Transmission of Powassan virus to hamsters, guinea-pigs, and rabbits was demonstrated by the bite of D. andersoni nymphs and adults which were infected by ingestion of blood by their antecedent larvae.Science, Faculty ofMicrobiology and Immunology, Department ofGraduat

The laboratory diagnosis of Chlamydia trachomatis infections

Lower genital tract infections with Chlamydia trachomatis are predominantly asymptomatic in men and women. Diagnostic technology has provided several approaches to the diagnosis of C trachomatis. Outside of cells, Chlamydia can die or degrade without optimal storage and transportation. Because some of the other assays perform better on certain specimen types, it is important for laboratories to recognize these differences and provide advice to physicians and nurses collecting patient specimens, with the objective of diagnosing lower genital tract infections to prevent transmission and upper tract damage. Most invasive specimens, such as cervical or urethral swabs, may be collected for culture, antigen or nucleic acid detection. Noninvasive samples such as first-void urine and vaginal swabs can be easily collected by the patient; these samples must be tested by more sensitive nucleic acid amplification tests. These newer investigative strategies should enable implementation of screening programs to identify and treat partners. Serology has not been particularly useful for the diagnosis of acute C trachomatis infections in adults. Presently, it appears that antibiotic-resistant C trachomatis is not a clinical problem. Laboratories providing C trachomatis diagnosis require participation in continuous quality improvement programs

The laboratory diagnosis of sexually transmitted infections in cases of sexual assault and abuse

Laboratory staff dealing with samples from victims must be aware that such patients have been psychologically traumatized and deserve special care. The help of a sexual assault care team should be sought if available, and appropriate specimens should be collected two to 10 days after an incident, preferably in a single visit. Specimens should be clearly labelled, and the laboratory should be informed. In the laboratory, all procedures need to be clearly documented. There are special requirements for the collection of forensic specimens and associated records, which may later be required for legal proceedings. The laboratory must know what the current legal status is for any test being used in that community. The present article serves as a guideline to more detailed practice standards for the investigation of individual sexually transmitted infections in assault and abuse situations

Canadian Laboratory Standards for Sexually Transmitted Infections: Best Practice Guidelines

Sexually transmitted infections (STI) continue to spread, and show no international boundaries. Diseases such as gonorrhea and syphilis, which we thought were under control in Canadian populations, have increased in incidence. Sexually transmitted or associated syndromes such as cervicitis, enteric infections, epididymitis, genital ulcers, sexually related hepatitis, ophthalmia neonatorum, pelvic inflammatory disease, prostatitis and vulvovaginitis present a challenge for the physician to identify the microbial cause, treat the patient and manage contacts. During the past 10 years, new technologies developed for the diagnosis of STIs have provided a clearer understanding of the real accuracy of traditional tests for the diagnosis of infections caused by Chlamydia trachomatis, Neisseria gonorrhoeae, Treponema pallidum, herpes simplex viruses, hepatitis B virus, human papillomaviruses, HIV, Haemophilus ducreyi, Trichomonas vaginalis and mycoplasmas. This has presented a major challenge to the diagnostic laboratory, namely, selecting the most sensitive and specific test matched with the most appropriate specimens to provide meaningful and timely results to facilitate optimal patient care