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The commercial polymerase chain reaction (PCR) test, SeptiFast, is designed to identify the DNA of individual bacterial and fungal pathogens in whole blood. We aimed to evaluate the usefulness of the test for the detection of community-onset bloodstream infections. We prospectively included adult patients who were subjected to blood culture (BC) at an infectious diseases department. For the evaluation, one BC/PCR set (two BC bottles and one PCR tube) per patient was used. When several sets were obtained and analyzed, the first set with any positive result was evaluated. Among 1,093 consecutively included patients, BC was positive in 138 and PCR was positive in 107. Fifty positive PCR results were supported by BC in the same BC/PCR set, ten were supported by other cultures, and, additionally, ten were supported by the clinical presentation. Compared with BC, PCR showed specificities and negative predictive values of >97% for all detectable pathogens. The following sensitivities and positive predictive values (PPVs) were noted: Staphylococcus aureus, 67% and 43%; Streptococcus pneumoniae, 12% and 67%; other Streptococcus species, 43% and 77%; Escherichia coli, 53% and 56%; and Klebsiella species, 43% and 23%. If support from other cultures and the clinical presentation were included in the reference standard, the PPVs for the detection of these bacteria were 57%, 100%, 92%, 75%, and 69%, respectively. Although the specificities were high, the low sensitivities and suboptimal PPVs noted in the present study discourage routine use of the test in its present form for the detection of community-onset bloodstream infections.

Background: The new variant of Chlamydia trachomatis (nvCT), discovered in Sweden in 2006, contains a 377-bp cryptic plasmid deletion, which includes the targets for the COBAS Amplicor/TaqMan C trachomatis/Neisseria gonorrhoea and Abbott m2000rt C trachomatis/N gonorrhoea tests., Objectives: To evaluate the new real-time COBAS TaqMan CT test v2.0 (CTM CT v2.0) for C trachomatis diagnostics and to investigate whether the proportion of nvCT was affected by the introduction of genetic diagnostics detecting nvCT (LightMix 480HT) in Orebro county, Sweden., Methods: CTM CT v2.0 compared with LightMix 480 HT PCR for the diagnosis of C trachomatis was evaluated. Discrepant samples were analysed using BD ProbeTec ET and Abbott m2000rt RealTime CT II. All previously LightMix and cell culture-positive samples were analysed using an nvCT-specific PCR., Results: The sensitivity, specificity, negative predictive value and positive predictive value of CTM CT v2.0 for examined samples (n = 1058) was 100%, 99.8%, 100% and 98.2%, respectively. Of 11,577 consecutive PCR samples, 9.4% (n = 1084) were positive and 34.3% (n = 372) of these were nvCT. Of 2306 consecutive culture samples, 5.0% (n = 116) were C trachomatis positive and 38.8% (n = 45) of these were nvCT., Conclusions: CTM CT v2.0 is a sensitive and specific method for C trachomatis detection. Studies including larger numbers of symptomatic and asymptomatic patients as well as genital and extragenital samples, and in comparison with other internationally validated and, ideally, US Food and Drug Administration-approved C trachomatis nucleic acid amplification tests are imperative. The proportion of nvCT remains high in Orebro county, Sweden, despite the introduction of genetic diagnostics to detect the mutant.

The aim of this study was to determine whether a patient's endocervical swab specimen can be transported in first void urine (FVU) as combined specimens for the detection of Mycoplasma genitalium by real-time PCR. The study also compared two different DNA extraction methods for observation of possible PCR inhibition. Three specimens, one endocervical swab specimen transported in 2-SP medium, one endocervical swab specimen transported in FVU and a FVU specimen, were collected from 329 women. All sample types underwent manual DNA extraction whereas in the DNA extraction study, 329 endocervical swab specimens transported in FVU were subjected to both manual Chelex and automated BioRobot M48 DNA extraction. A total of 100 endocervical swab specimens transported in FVU from patients PCR-negative for M. genitalium in the study were used in the PCR inhibition analysis. M. genitalium was detected in 25/329 (7.6 %) women. The endocervical swab specimens transported in 2-SP medium and transported in FVU were positive for M. genitalium in 17/25 (68 %) and 24/25 (96 %) women, respectively. The FVU specimens alone were positive for M. genitalium in 22/25 (88 %) women. In the DNA extraction study, M. genitalium DNA was detected in 24/329 (7.3 %) and 28/329 (8.5 %) of endocervical swab specimens transported in FVU subjected to manual Chelex extraction and automated BioRobot M48 extraction, respectively. Partial PCR inhibition was detected in 6 % of samples subjected to manual Chelex extraction whereas no inhibition was detected with the automated BioRobot M48 extraction. Thus endocervical swab specimens transported in FVU demonstrate higher sensitivity than FVU specimens only and have considerably increased sensitivity compared with endocervical swab specimens transported in 2-SP medium for detection of M. genitalium DNA. Moreover, automated BioRobot M48 extraction was shown to be superior to a crude manual Chelex extraction, leaving no PCR inhibition and giving a slightly higher DNA yield and/or better sensitivity.

The aim of this study was to compare conventional 16S rRNA gene PCR, real-time 16S rRNA gene PCR and real-time Mycoplasma genitalium adhesin protein (MgPa) gene PCR as detection methods for M. genitalium infection. The study also determined the prevalence of M. genitalium in male and female patients attending a sexually transmitted infections clinic in a rural area in the west of Sweden. First void urine (FVU) and/or urethral swabs were collected from 381 men, and FVU and/or cervical swabs and/or urethral swabs were collected from 298 women. A total of 213 specimens were used in the PCR comparative study: 98 consecutively sampled specimens from patients enrolled in the prevalence study, 36 consecutively sampled specimens from patients with symptoms of urethritis and 79 specimens from patients positive for M. genitalium by real-time MgPa gene PCR in the prevalence study. A true-positive M. genitalium DNA specimen was defined as either a specimen positive in any two PCR assays or a specimen whose PCR product was verified by DNA sequencing. The prevalence of M. genitalium infection in men and women was 27/381 (7.1 %) and 23/298 (7.7 %), respectively. In the PCR comparative study, M. genitalium DNA was detected in 61/76 (80.3 %) of true-positive specimens by conventional 16S rRNA gene PCR, in 52/76 (68.4 %) by real-time 16S rRNA gene PCR and in 74/76 (97.4 %) by real-time MgPa gene PCR. Real-time MgPa gene PCR thus had higher sensitivity compared with conventional 16S rRNA gene PCR and had considerably increased sensitivity compared with real-time 16S rRNA gene PCR for detection of M. genitalium DNA. Real-time MgPa gene PCR is well suited for the clinical diagnosis of M. genitalium.

A multiplex PCR (mPCR) was developed for simultaneous detection of specific genes for Streptococcus pneumoniae (lytA), Mycoplasma pneumoniae (P1), Chlamydophila pneumoniae (ompA), and Haemophilus influenzae (16S rRNA, with verification PCR for P6). When the protocol was tested on 257 bacterial strains belonging to 37 different species, no false negatives and only one false positive were noted. One Streptococcus mitis out of thirty was positive for lytA. In a pilot application study of 81 sputum samples from different patients with suspected lower respiratory tract infection (LRTI), mPCR identified S. pneumoniae in 25 samples, H. influenzae in 29, M. pneumoniae in 3, and C. pneumoniae in 1. All samples culture positive for S. pneumoniae (n=15) and H. influenzae (n=15) were mPCR positive for the same bacteria. In a pilot control study with nasopharyngeal swabs and aspirates from 10 healthy adults, both culture and mPCR were negative. No PCR inhibition was found in any of the mPCR-negative sputum or nasopharyngeal samples. Whether all samples identified as positive by mPCR are truly positive in an aetiological perspective regarding LRTI remains to be evaluated in a well-defined patient material. In conclusion, the mPCR appears to be a promising tool in the aetiological diagnostics of LRTI.

A real-time LightCycler PCR (LC-PCR) with hybridization probes for detection of Mycoplasma genitalium in endocervical and first void urine specimens was developed and compared to a conventional PCR. The primers for both assays were identical and designed to amplify a 427 bp fragment of the 16S rRNA gene of M. genitalium. The LC-PCR assay had a detection limit of < 5 bacterial genomes per reaction when dilutions of genomic DNA from a type strain of M. genitalium were tested. First void urine from 398 men and first void urine and endocervical specimens from 301 women attending an STD clinic were analysed by LC-PCR and by the conventional PCR. Using the conventional PCR as reference, the LC-PCR had a specificity of 99.7 % and a sensitivity of 72.2 % for the detection of M. genitalium in first void urine samples from men. There was no significant difference in the performance of the LC-PCR assay compared to the conventional PCR when endocervical swabs were considered (58 and 65 %, respectively) or with a set of endocervical swab/urine specimens for which the LC-PCR assay detected 73 % of the infections (specificity = 98.6 % and sensitivity = 68.2 %) while the conventional PCR detected 85 % of the infections. With female urine specimens there was a significant difference between the two assays (38 and 73 %, respectively; P = 0.01 McNemar's test). This illustrates the need to analyse both endocervical and urine specimens, because M. genitalium DNA was detected in only one of the two specimens in a great number of the M. genitalium-infected women. The lower sensitivity of the LC-PCR assay was probably caused by a combination of inhibition and limitations regarding the amount of template DNA. The LC-PCR assay was easy to perform and the simultaneous amplification and detection eliminated the need for further handling of PCR products. With improvement in sample preparation methods and increased volumes of the template DNA, the LC-PCR assay could be a useful routine diagnostic method.

The aim of this study was to increase the sensitivity of an earlier version of an HIV-2 nested PCR assay based on primers in the gag, pol, LTR, and env regions. The assay was first optimized with regard to concentrations of dNTP, MgCl2, and primers, using a method that allowed optimization of all three parameters in only two test runs. We then designed and optimized new primer sets in the LTR, gag, and gag/pol regions that were based on more isolates than were the former (old) primer sets. Samples from 57 HIV-2 antibody-positive individuals were tested with the four old primer sets as well as with the three new primer sets. Five primer sets from this run (new gag, new gag/pol, old LTR, old env, and new LTR) were then tested with 35 more samples, giving a total number of 92 tested samples from HIV-2-infected individuals. At initial testing of the 92 samples a combination of primer sets from two different regions yielded a sensitivity ranging from 93.5 to 98.9%. After repeated testing the sensitivity ranged from 96.7 to 100% for the different primer combinations. The specificity was 100% for all primer sets except old LTR, which had a specificity of 97%. In conclusion, it is possible to create a more sensitive PCR assay by optimizing the different PCR parameters as well as by including primer sets based on more isolates.

The aim of the study was to evaluate the sensitivity of patients' self-sampled vaginal specimens, first-catch urine (FCU), combined vaginal/FCU specimens and endocervical specimens for detecting chlamydial infection in women. Women attending sexually transmitted disease clinics, youth clinics and a women's health clinic were enrolled. They self-collected a vaginal specimen with two swabs, which were placed into a sterile tube and into a tube containing a buffer medium, respectively. An FCU sample was collected and aliquoted into both an empty tube and the tube containing the vaginal swab. A clinician collected an endocervical swab. The samples were sent to laboratories for analysis using polymerase chain reaction testing and strand displacement amplification testing, respectively. The sensitivities calculated in all 171 Chlamydia trachomatis-infected women were equal for endocervical specimens (97.1%), vaginal specimens (96.5%) and combined vaginal/FCU specimens (95.3%), whereas the sensitivity for FCU was significantly lower (87.7%). The sensitivity of vaginal specimens for the detection of C. trachomatis is as high as that of combined vaginal/FCU specimens. [ABSTRACT FROM AUTHOR]

Objectives: To study the treatment efficacy of tetracyclines and azithromycin in Mycoplasma genitalium positive patients attending an STD clinic.Methods: All M genitalium positive patients (34 men and 26 women) attending an STD clinic during a 6 month period were treated with antibiotics. All patients known to be partners of M genitalium positive patients and those who were M genitalium positive, but not initially treated, were treated with azithromycin. Patients with urethritis and/or cervicitis were treated with tetracyclines before their M genitalium status was known.Results: 10 of 14 women (71%) and 10 of 16 men (63%) treated with tetracyclines were M genitalium positive at follow up, whereas all patients treated with azithromycin (16 men and 20 women) were M genitalium negative, at the 4 week follow up visit.Conclusions: These results suggest that tetracyclines are not sufficient to eradicate M genitalium. Randomised controlled treatment trials are urgently needed. [ABSTRACT FROM AUTHOR]