Monitoring of Chlamydia trachomatis infections after antibiotic treatment using RNA detection by nucleic acid sequence based amplification.

AIM: To investigate the value of RNA detection by nucleic acid sequence based amplification (NASBA) for the monitoring of Chlamydia trachomatis infections after antibiotic treatment. METHODS: Cervical smears (n = 97) and urine specimens (n = 61) from 25 C trachomatis positive female patients were analysed for the presence of C trachomatis 16S ribosomal RNA (rRNA) by NASBA and C trachomatis plasmid DNA by the polymerase chain reaction (PCR) before and up to five weeks after antibiotic treatment. RESULTS: Chlamydia trachomatis RNA was found in all cervical smears taken before antibiotic treatment (n = 24) and in two smears taken one week after antibiotic treatment; no C trachomatis RNA was detected after two weeks or more. In contrast, C trachomatis DNA was found in all such specimens before treatment, and 21 of 25, six of 21, and five of 20 smears were found to be positive at one, two, and three weeks after treatment, respectively. After four weeks, only one of six smears was positive, and this smear had been negative in the two preceding weeks. Of the 61 urine samples investigated, C trachomatis DNA and C trachomatis RNA were found in all before treatment (n = 15), whereas one week after treatment four of 15 were C trachomatis DNA positive and C trachomatis RNA was detected in one sample only. CONCLUSIONS: These data show that RNA detection by NASBA can be used successfully to monitor C trachomatis infections after antibiotic treatment. Furthermore, it might be possible to use urine specimens as a test of cure because neither C. trachomatis DNA or RNA could be detected two weeks or more after treatment.     

From CBCL to DSM: a comparison of two methods to screen for DSM-IV diagnoses using CBCL data.

The screening efficiency of 2 methods to convert Child Behavior Checklist (CBCL) assessment data into Diagnostic and Statistical Manual of Mental Disorders (4th ed. [DSM-IV]; American Psychiatric Association, 1994) diagnoses was compared. The Machine-Aided Diagnosis (MAD) method converts CBCL input data directly into DSM-IV symptom criteria. The Achenbach System of Empirically Based Assessment (ASEBA) proceeds more indirectly and uses DSM-oriented scales. The power of the 2 methods to predict DSM-IV diagnoses obtained via administration of the structured Diagnostic Interview Schedule for Children (DISC-IV) interview in a clinical sample was examined. DISC-IV interviews and CBCL reports from parents of 44 children, 25 boys, and 19 girls, ages 6 to 17 were used. The results showed comparable levels of predictive power for the 2 methods. Both methods were able to predict DSM-IV diagnoses and therefore can be used for screening DSM-IV diagnoses.     

Adequacy of family history taking in ovarian cancer patients: a population-based study

The aim of this study was to evaluate the adequacy of family history taking in epithelial ovarian cancer (EOC) patients and to identify factors that determine adequacy. Furthermore, the validity of family history taking was assessed by comparison with self-administered questionnaires. Medical records of all 1,112 EOC patients registered by the nation-wide cancer registry and diagnosed in eleven Dutch hospitals between 1996 and 2006 were reviewed. Adequate family history taking was defined as a written notification of the presence or absence of relatives with breast or ovarian cancer. Factors that were correlated with family history taking were identified using univariable and multivariable logistic regression.?147 patients filled in a postal questionnaire. An adequate family history was taken in 41% of all cases. Younger age, an academic hospital and having undergone surgery and/or chemotherapy were associated with adequate family history taking. The comparison with self-administered questionnaires showed a disagreement in 64% mainly due to missing data in medical records. Documentation on family history is either absent or inadequate in the medical records in the majority of EOC patients. These data urge for better uptake of hereditary cancer risk assessment. Different strategies for this assessment like improved family history taking and genetic testing in EOC patients should be explored.     

Detection of Mycoplasma pneumoniae in spiked clinical samples by nucleic acid sequence-based amplification

Isothermal nucleic acid sequence-based amplification (NASBA) was applied to the detection of Mycoplasma pneumoniae. M. pneumoniae RNA prepared from a plasmid construct was used to assess the sensitivity of the assay, and an internal control for the detection of inhibitors was constructed. The sensitivity of the NASBA assay was 10 molecules of wild-type M. pneumoniae RNA generated in vitro and 5 color-changing units (CCU) of M. pneumoniae. An appropriate specimen preparation procedure was developed: after protease treatment of the respiratory specimens, guanidine thiocyanate lysis solution (4.7 M guanidine thiocyanate [Sigma-Aldrich NV], 46 mM Tris-HCl [Merck, Darmstadt, Germany], 20 mM EDTA [Sigma-Aldrich NV], 1.2% [wt/vol] Triton X-100 [Sigma-Aldrich NV], pH 6.2.) was added. With spiked throats, nasopharyngeal aspirates, bronchoalveolar lavage specimens, and sputum specimens, the sensitivity of the NASBA assay in the presence of the internal control was 2 x 10(4) molecules of in vitro-generated RNA or 5 CCU of M. pneumoniae. The sensitivity of the NASBA assay was comparable to that of a PCR targeted to the P1 adhesin gene. Fifteen clinical specimens positive for M. pneumoniae by PCR were also positive by NASBA. These results indicate that the sensitivity of detection of M. pneumoniae in spiked respiratory samples by NASBA is high. Together with the use of the internal control, the assay merits evaluation as a diagnostic tool.     

Performance of different mono- and multiplex nucleic acid amplification tests on a multipathogen external quality assessment panel

An external quality assessment (EQA) panel consisting of a total of 48 samples in bronchoalveolar lavage (BAL) fluid or transport medium was prepared in collaboration with Quality Control for Molecular Diagnostics (QCMD) (www.qcmd.org). The panel was used to assess the proficiency of the three laboratories that would be responsible for examining the 6,000 samples to be collected in the GRACE Network of Excellence (www.grace-lrti.org). The main objective was to decide on the best-performing testing approach for the detection of influenza viruses A and B, parainfluenza virus types 1 to 3, respiratory syncytial virus (RSV), human metapneumovirus, coronavirus, rhinovirus, adenovirus, Chlamydophila pneumoniae, Mycoplasma pneumoniae, and Legionella pneumophila by nucleic acid amplification techniques (NAATs). Two approaches were chosen: (i) laboratories testing samples using their in-house procedures for extraction and amplification and (ii) laboratories using their in-house amplification procedures on centrally extracted samples. Furthermore, three commercially available multiplex NAAT tests-the ResPlex (Qiagen GmbH, Hilden, Germany), RespiFinder plus (PathoFinder, Maastricht, The Netherlands), and RespiFinder Smart 21 (PathoFinder) tests-were evaluated by examination of the same EQA panel by the manufacturer. No large differences among the 3 laboratories were noticed when the performances of the assays developed in-house in combination with the in-house extraction procedures were compared. Also, the extraction procedure (central versus local) had little effect on performance. However, large differences in amplification efficacy were found between the commercially available tests; acceptable results were obtained by using the PathoFinder assays.     

Relation between injected volume and optical parameters in refilled isolated porcine lenses

PURPOSE: This study was performed to elucidate the correlation between added lens refill material and enhanced lens power as well as the correlation between lens refilling volume and accommodative amplitude as determined by equatorial stretching of ex vivo refilled pigs' lenses. METHODS: Nine porcine lenses were refilled with increasing amounts of silicone oil. After each refill step, the lens power, the lens power change, and the lens thickness were measured both in the relaxed state and with a 3-mm larger ciliary body diameter. In addition, the spherical aberration of the refilled lenses was also quantified. RESULTS: Injection of 0.04 mL silicone material into the relaxed lens enhanced the lens power by 1 D. A 0.54-mm increase of the lens thickness in relaxed lenses added 1 D to the lens power. Increasing the lens refilling volume decreased the lens power changes measured at 3-mm ciliary body stretch. Spherical aberration was positive in the refilled lenses and increased with increasing lens refilling volume. CONCLUSION: The correlation found between the refilling volume and the lens power (0.04 mL D(-1)), as well as the correlation between the lens thickness and the lens power (0.54 mm D(-1)), might be important factors to be controlled in conjunction with surgery, as these also determine the lens power in the presence of this refill material. An increasing lens filling volume is associated with decreasing accommodative amplitude. The positive spherical aberration of refilled porcine lenses presents a sharp contrast to the negative aberration of natural pigs' lenses. Different lens contours and the transition from a gradient to a homogeneous refractive index might be responsible for this change in spherical aberration.     

RNA amplification by nucleic acid sequence-based amplification with an internal standard enables reliable detection of Chlamydia trachomatis in cervical scrapings and urine samples.

In the present study, the suitability of RNA amplification by nucleic acid sequence-based amplification (NASBA) for the detection of Chlamydia trachomatis infection was investigated. When comparing different primer sets for their sensitivities in NASBA, use of both the plasmid and omp1 targets resulted in a detection limit of 1 inclusion-forming unit (IFU), while the 16S rRNA appeared to be the most sensitive RNA target for amplification (10(-3) IFU). In contrast, for DNA amplification by PCR, the plasmid target was optimal (10(-2) IFU), which is 10 times less sensitive than rRNA NASBA. To exclude false negativity in NASBA detection because of inhibition of amplification and/or inefficient sample preparation, an internal standard was developed. The internal control was added prior to sample preparation. This 16S rRNA NASBA with an internal control was compared with a plasmid DNA PCR by using a group of C. trachomatis-negative (n = 41) and -positive (n = 37) cervical scrapings, as determined by enzyme immunoassay (EIA). In addition, urine samples from the EIA-positive women were tested (n = 17). Both NASBA and PCR assays were able to detect C. trachomatis in all EIA-positive cervical scrapings, the corresponding urine samples, and two samples from the EIA-negative group. The internal NASBA standard was found clearly in all EIA-negative samples. In conclusion, these results indicate that detection of C. trachomatis by RNA amplification by NASBA with an internal standard is a suitable and highly sensitive detection method, with potential use in the diagnosis of urogenital C. trachomatis infections with cervical scrapings as well as urine specimens.