bluuebunny/crossref_metadata_2025_split huggingface dataset:
Performance of the Gen-Probe Transmission-Mediated Amplification Research Assay Compared to That of a Multitarget Real-Time PCR for
Mycoplasma genitalium
Detection
Authors: Hardick Justin , Giles Julie , Hardick Andrew , Hsieh Yu-Hsiang , Quinn Thomas , Gaydos Charlotte 
Published: April 2006

ABSTRACT

Mycoplasma genitalium
(MG) can cause nongonococcal urethritis and is potentially associated with urethritis, endometritis, and cervicitis. Several assays have been developed to detect MG. Molecular amplification assays for organism detection can be problematic due to the potential for false-positive and false-negative results. Confirmatory testing is often required in these situations, requiring additional time and resources. Use of multigene targets could integrate both detection and verification at lower cost. Utilizing two targets, the MgPa adhesion gene and the 16S rRNA gene, a multitarget real-time (MTRT) PCR for the detection of MG was developed. Samples from patients attending sexually transmitted disease clinics were collected in duplicate. Urine samples from males (
n
= 286) and self-collected vaginal swabs from females (
n
= 321) were analyzed by MTRT PCR for MG and the Gen-Probe transmission-mediated amplification (TMA) assay, which targets MG rRNA for detection (TMA-MG research use only). Utilizing the criteria of any two targets being positively amplified, the MTRT PCR had a sensitivity and specificity of 91.8% (101 positive samples/110 samples tested) and 99.5% (495/497), respectively, with a positive predictive value (PPV) of 98.1% (101/103) and a negative predictive value (NPV) of 98.2% (495/504). The Gen-Probe TMA-MG assay had a sensitivity, specificity, PPV, and NPV of 98.1% (108/110), 98.1% (488/497), 92.3% (108/117), and 99.5% (488/490), respectively. Comparison between the MTRT PCR and TMA-MG assay by kappa statistic analysis indicated that an overall kappa value was 0.941 (95% confidence interval, 0.907 and 0.976). Both assays demonstrated accuracy in the detection of MG from urine samples from male patients and self-collected vaginal swabs from female patients.
Development of Conventional and Real-Time Nucleic Acid Sequence-Based Amplification Assays for Detection of
Chlamydophila pneumoniae
in Respiratory Specimens
Authors: Loens K. , Beck T. , Goossens H. , Ursi D. , Overdijk M. , Sillekens P. , Ieven M. 
Published: April 2006

ABSTRACT

Isothermal nucleic acid sequence-based amplification (NASBA) was applied to the detection of
Chlamydophila pneumoniae
16S rRNA by using the NucliSens basic kit (bioMérieux, Boxtel, The Netherlands). The assay was originally developed as a conventional NASBA assay with electrochemiluminescence detection and was subsequently adapted to a real-time NASBA format by using a molecular beacon.
C. pneumoniae
RNA prepared from a plasmid construct was used to assess the analytical sensitivity of the assay. The sensitivity of the NASBA assay was 10 molecules of in vitro wild-type
C. pneumoniae
RNA and 0.1 inclusion-forming unit (IFU) of
C. pneumoniae
. In spiked respiratory specimens, the sensitivity of the
C. pneumoniae
NASBA assay varied between 0.1 and 1 IFU/100 μl sample, depending on the type of specimen. Finally, conventional and real-time NASBA were applied to respiratory specimens previously tested by PCR. A 100% concordance between the test results was obtained.
Effects of Delayed-Entry Conditions on the Recovery and Detection of Microorganisms from BacT/ALERT and BACTEC Blood Culture Bottles
Authors: Sautter R. L. , Bills A. R. , Lang D. L. , Ruschell G. , Heiter B. J. , Bourbeau P. P. 
Published: April 2006

ABSTRACT

Manufacturers generally recommend that blood culture bottles be loaded into instruments within a short time of collection. However, in our experience, delays often occur prior to loading the bottles. We examined the effect of holding bottles under various temperatures (T)—room temperature (RT), 4°C, 37°C, and RT for 2 h following incubation at 37°C (to simulate transit [TR])—and for various holding times of 4, 12, and 24 h. We utilized the BacT/ALERT system with FA and FN bottles and the BACTEC system with Plus (PL) and Lytic 10 (LY) bottles. Standardized inocula and 5 ml of blood were added to each bottle. Fifteen organisms were evaluated based upon expected performance: aerobic (FA and PL), anaerobic (FN and LY 10), and facultative (all bottles). Based upon expected performance, the FA and FN bottles recovered 458 of 468 organisms and 282 of 288 organisms, respectively, whereas the PL and LY bottles recovered 453 of 468 organisms and 257 of 288 organisms, respectively (
P
= <0.001, FN versus LY). There were 3, 11, 21, and 27 false-negative results for bottles held at 4°C, RT, 37°C, and TR, respectively. There were 4, 8, and 50 false-negative results for bottles held for 4, 12, and 24 h, respectively. Our results support holding these four bottle types at 4°C or at RT for up to 24 h and at 37°C for up to 12 h. We propose that manufacturers only need to make claims for “delayed entry” when these bottles are held for more than 24 h at 4°C or at RT or for more than 12 h at 37°C.
Characteristic Signatures of the
lytA
Gene Provide a Basis for Rapid and Reliable Diagnosis of
Streptococcus pneumoniae
Infections
Authors: Llull Daniel , López Rubens , García Ernesto 
Published: April 2006

ABSTRACT

The nucleotide sequences of the
lytA
gene from 29 pneumococcal isolates of various serotypes and 22 additional streptococci of the mitis group (including two
Streptococcus pseudopneumoniae
strains) have been compared and found to correspond to 19 typical (927-bp-long) and 20 atypical (921-bp-long) alleles. All the
Streptococcus pneumoniae
strains harbored typical
lytA
alleles, whereas nonpneumococcal isolates belonging to the mitis group always carried atypical alleles. A sequence alignment showed that the main difference between typical and atypical
lytA
alleles resided in 102 nucleotide positions (including the 6 bp absent from atypical alleles). These nucleotides were perfectly conserved in all the typical alleles studied, and the corresponding nucleotides of the atypical alleles were also perfectly conserved. The presence in these signatures of distinctive restriction sites (namely, SnaBI, XmnI, and BsaAI) allowed the development of a simple, reliable, and fast method that combines PCR amplification of the
lytA
gene, digestion with BsaAI, and separation of the products by agarose gel electrophoresis. This assay allows the rapid and consistent identification of true
S. pneumoniae
strains and represents an improved diagnostic tool for the study of pneumococcal carriage.
Follow-Up on Diagnostic Proficiency of Laboratories Equipped To Perform Orthopoxvirus Detection and Quantification by PCR: the Second International External Quality Assurance Study
Authors: Niedrig Matthias , Meyer Hermann , Panning Marcus , Drosten Christian 
Published: April 2006

ABSTRACT
Two years after the first external quality assurance study on bioterrorism-relevant viruses, we have conducted a follow-up study on orthopoxvirus detection by PCR. Thirty-three laboratories (27 European, 4 Austral-Asian, and 2 American) participated. Samples contained 0 to 40,000,000 DNA copies of lyophilized monkeypox, cowpox, and vaccinia virus per ml. Laboratories achieved a >80% detection chance above 56,234 copies per ml. Global sensitivity was not significantly improved over that of the first study. Twenty-seven and 9 participants, respectively, were able to genotype and quantify virus. Four of 27 genotyping results were incorrect. Quantification accuracy was significantly better for vaccinia virus than for the other viruses. False-positive results occurred in 22 (11.8%) of all 186 tests on negative samples, but 18 of these were contributed by only five laboratories. Fifty-five percent of laboratories could appropriately detect PCR inhibition. The use of either real-time PCR or commercial diagnostic kits had significant positive influence on laboratory performance.
Molecular Epidemiology and Evolution in an Outbreak of Fulminant Hepatitis B Virus
Authors: Bracho Maria Alma , Gosalbes María José , González Francisco , Moya Andrés , González-Candelas Fernando 
Published: April 2006

ABSTRACT
In order to establish the transmission pathway for two outbreak patients affected by fulminant hepatitis B (FHB) following a shared period of hospitalization, we sequenced the complete genomes of the hepatitis B viruses (HBV) isolated from them as well as from the suspected common source and 11 additional controls. Phylogenetic and statistical analyses of these sequences revealed that the two FHB patients were indeed infected by a common source and that the fatal development of the disease did not appear to be associated with any mutation previously reported to be related to FHB. These data have also allowed us to estimate the extent and distribution of genetic variability along the genomes of HBV genotype D samples from the same source population. As a result of these analyses, we provide an improved statistical method to individualize the assignment of each suspected patient and the source of an outbreak and information on which genome region to analyze in the molecular epidemiological assessment of hepatitis B virus transmission cases.
Development of Real-Time Reverse Transcriptase PCR Assays To Detect and Serotype Dengue Viruses
Authors: Chien Li-Jung , Liao Tsai-Ling , Shu Pei-Yun , Huang Jyh-Hsiung , Gubler Duane J. , Chang Gwong-Jen J. 
Published: April 2006

ABSTRACT
Serotyping dengue virus (DENV) from suspect human specimens is crucial for developing sound epidemiological control measurements early in the transmission season and for effective patient management. We modified DENV consensus D1 (mD1) and serotype-specific TS2 (mTS2) and redesigned serotype-specific TS1 (rTS1) and TS4 (rTS4) as described previously in the conventional capsid and premembrane gene (C-prM) protocol (R. S. Lanciotti, C. H. Calisher, D. J. Gubler, G.-J. Chang, A. V. Vorndam, J. Clin. Microbiol. 30:545-551, 1992). In addition, we designed two new sets of amplimers and probes, located at nonstructural protein 5 (NS5) and the 3′ noncoding region (3′NC) of DENV. The NS5 protocol utilizes two flaviviral consensus outer amplimers (mFU1 and CFD2) and four dengue virus serotype-specific TaqMan fluorogenic probes. The 3′NC protocol uses two DENV consensus amplimers, DC10418 and CDC10564. The conventional gel-based, heminested detection method was adapted for the C-prM protocol for detecting and serotyping dengue viruses. In addition, we developed the real-time SYBR green I and postamplification melting temperature curve analysis for the mD1/TS and 3′NC protocols using identical amplification conditions. The NS5 amplimer/probe set was formulated as a one-tube, multiplex, real-time reverse transcriptase PCR for serotype identification. Three sets of amplimers and probes were verified for their specificity in tests with yellow fever, Japanese encephalitis, St. Louis encephalitis, and West Nile viruses; optimized against 109 DENV strains; and validated for detection of the virus in sera from two different panels of acute-phase human dengue serum specimens and one panel of virus isolates from dengue patients' serum specimens. Clinical evaluation by two separate laboratories indicated that the C-prM was more sensitive (100%) than the NS5 (91%) or the 3′NC (91%) protocol.
New Commercially Available PCR and Microplate Hybridization Assay for Detection and Differentiation of Human Polyomaviruses JC and BK in Cerebrospinal Fluid, Serum, and Urine Samples
Authors: Moret Hélène , Brodard Véronique , Barranger Côme , Jovenin Nicolas , Joannes Martine , Andréoletti Laurent 
Published: April 2006

ABSTRACT

JC and BK human polyomaviruses (family
Polyomaviridae
) may cause severe neurological or urinary tract pathologies in immunocompromised hosts. In the present study, we evaluated a new commercially available PCR and microplate colorimetric hybridization assay for the standardized differential detection of JC virus (JCV) and BK virus (BKV) genomes in clinical samples. This JC/BK Consensus test was first evaluated by testing serial dilutions of JCV or BKV plasmid DNA standards and was then compared with an in-house reference PCR assay for the detection of JC and BK virus genomes in 70 cerebrospinal fluid (CSF) samples of patients with neurological disorders and in 75 serum or plasma samples and 125 urine samples of renal graft recipients. This new test allowed a limit of detection of 10 copies and 1 copy of JC and BK virus genomes, respectively, and was able to differentiate various levels of JCV, BKV, and mixed JCV and BKV DNA genomes in a single reaction tube. Our results showed 100% specificity and sensitivity for the JC/BK Consensus test with CSF samples. With serum or plasma samples, this test had a sensitivity and a specificity of 100% for both JCV and mixed JCV and BKV DNA detection and a sensitivity and a specificity of 100 and 97.8% for BKV DNA detection, respectively. With urine samples, the sensitivity and specificity were 100 and 96.6%, respectively, for JCV DNA detection; 100 and 89.4%, respectively, for BKV DNA detection; and 44.4 and 100%, respectively, for mixed JCV and BKV DNA detection. In conclusion, our data indicate that this new test, the JC/BK Consensus test, is valuable for the sensitive and specific differential detection of single JCV and BKV infections in CSF, serum or plasma, and urine samples. The use of this reliable PCR assay would improve the routine virological diagnosis as well as the clinical care of immunocompromised patients with polyomavirus-related pathologies.
Incidence and Time Course of
Leishmania infantum
Infections Examined by Parasitological, Serologic, and Nested-PCR Techniques in a Cohort of Naïve Dogs Exposed to Three Consecutive Transmission Seasons
Authors: Oliva Gaetano , Scalone Aldo , Foglia Manzillo Valentina , Gramiccia Marina , Pagano Annalisa , Di Muccio Trentina , Gradoni Luigi 
Published: April 2006

ABSTRACT

Most experience in the comparison of diagnostic tools for canine leishmaniasis comes from cross-sectional surveys of dogs of different ages and breeds and in cases with unknown onset and duration of leishmaniasis. A longitudinal study was performed on 43 beagle dogs exposed to three transmission seasons (2002 to 2004) of Mediterranean leishmaniasis and examined periodically over 32 months through bone marrow microscopy and nested PCR (n-PCR), lymph node culture, serology (immunofluorescent-antibody test), and evaluation of clinical parameters. Starting from January 2003, the highest rate of positives was detected by n-PCR at all assessments (from 23.3% to 97.3%). Sensitivities of serologic and parasitological techniques were lower but increased with time, from 15.8% to 75.0 to 77.8%. Some dogs that tested positive by n-PCR but negative by other tests (“subpatent infection”) remained so until the end of the study or converted to negative in subsequent assessments, whereas all dogs with positive serology and/or microscopy/culture (“asymptomatic patent infection”) exhibited progressive leishmaniasis; 68% of them developed clinical disease (“symptomatic patent infection”) during the study, at 7 (range, 3 to 14) months after being positive to all tests. Postexposure infection incidences were high and were significantly different between 2002 and 2003 exposures (39.5% and 91.7%, respectively). The time course of infection was highly variable in each dog, with three patterns being identified: (i) rapid establishment of a patent condition (0 to 2 months from detection of infection); (ii) a prolonged subpatent condition (4 to 22 months) before progression; and (iii) a transient subpatent condition followed by 10 to 21 months of apparent
Leishmania-
negative status before progression.
Use of a Variable Amplicon Typing Scheme Reveals Considerable Variation in the Accessory Genomes of Isolates of
Burkholderia pseudomallei
Authors: Duangsonk Kwanjit , Gal Daniel , Mayo Mark , Hart C. Anthony , Currie Bart J. , Winstanley Craig 
Published: April 2006

ABSTRACT

Melioidosis, a disease caused by the bacterium
Burkholderia pseudomallei
, is endemic in southeast Asia and northern Australia. We used suppression subtractive hybridization (SSH) to identify sequences that varied between two
B. pseudomallei
isolates from Australia and determined the distribution of 45 SSH-derived sequences among a panel of
B. pseudomallei
and
B. thailandensis
isolates. Sequences exhibiting variable prevalence were included in a variable amplicon typing (VAT) scheme designed to score the presence or absence of 14 PCR amplicons. VAT analysis was carried out with 48 isolates from Thailand, which were typed by multilocus sequence typing (MLST), and 44 isolates from Australia of known MLST type. The VAT scheme could be used to divide the 48 isolates from Thailand into 23 VAT types and the 44 isolates from Australia into 36 VAT types. Some of the sequences included in the VAT scheme were more commonly PCR positive among isolates from Australia than among isolates from Thailand, and vice versa. No isolate from Australia was PCR positive for genomic island 11 or a putative transposase sequence, whereas four SSH-derived sequences were far more prevalent among the Australian isolates. Analysis based on the VAT scheme indicated that the isolates clustered into groups, some of which were mainly or exclusively from one geographical origin. One cluster included Australian isolates that were mostly associated with severe disease, including rare neurological melioidosis, suggesting that the content of the accessory genome may play an important role in determining the clinical manifestation of the disease.
Is Serological Testing a Reliable Tool in Laboratory Diagnosis of Syphilis? Meta-Analysis of Eight External Quality Control Surveys Performed by the German Infection Serology Proficiency Testing Program
Authors: Müller Iris , Brade Volker , Hagedorn Hans-Jochen , Straube Erich , Schörner Christoph , Frosch Matthias , Hlobil Harald , Stanek Gerold , Hunfeld Klaus-Peter 
Published: April 2006

ABSTRACT

The accuracy of diagnostic tests is critical for successful control of epidemic outbreaks of syphilis. The reliability of syphilis serology in the nonspecialist laboratory has always been questioned, but actual data dealing with this issue are sparse. Here, the results of eight proficiency testing sentinel surveys for diagnostic laboratories in Germany between 2000 and 2003 were analyzed. Screening tests such as
Treponema pallidum
hemagglutination assay (mean accuracy, 91.4% [qualitative], 75.4% [quantitative]),
Treponema pallidum
particle agglutination assay (mean accuracy, 98.1% [qualitative], 82.9% [quantitative]), and enzyme-linked immunosorbent assays (ELISAs) (mean qualitative accuracy, 95%) were more reliable than Venereal Disease Research Laboratory (VDRL) testing (mean accuracy, 89.6% [qualitative], 71.1% [quantitative]), the fluorescent treponemal antibody absorption test (FTA-ABS) (mean accuracy, 88% [qualitative], 65.8% [quantitative]), and immunoblot assays (mean qualitative accuracy, 87.3%). Clearly, immunoglobulin M (IgM) tests were more difficult to manage than IgG tests. False-negative results for samples that have been unambiguously determined to be IgM and anti-lipoid antibody positive accounted for 4.7% of results in the IgM ELISA, 6.9% in the VDRL test, 18.5% in the IgM FTA-ABS, and 23.0% in the IgM immunoblot assay. For negative samples, the mean percentage of false-positive results was 4.1% in the VDRL test, 5.4% in the IgM ELISA, 0.7% in the IgM FTA-ABS, and 1.4% in the IgM immunoblot assay. On average, 18.3% of participants misclassified samples from patients with active syphilis as past infection without indicating the need for further treatment. Moreover, 10.2% of laboratories wrongly reported serological evidence for active infection in samples from patients with past syphilis or in sera from seronegative blood donors. Consequently, the continuous participation of laboratories in proficiency testing and further standardization of tests is strongly recommended to achieve better quality of syphilis serology.
Time to Blood Culture Positivity as a Predictor of Clinical Outcome of
Staphylococcus aureus
Bloodstream Infection
Authors: Marra Alexandre R. , Edmond Michael B. , Forbes Betty A. , Wenzel Richard P. , Bearman Gonzalo M. L. 
Published: April 2006

ABSTRACT

Few studies have assessed the time to blood culture positivity as a predictor of clinical outcome in bloodstream infections (BSIs). The purpose of this study was to evaluate the time to positivity (TTP) of blood cultures in patients with
Staphylococcus aureus
BSIs and to assess its impact on clinical outcome. We performed a historical cohort study with 91 adult patients with
S. aureus
BSIs. TTP was defined as the time between the start of incubation and the time that the automated alert signal indicating growth in the culture bottle sounded. Patients with BSIs and TTPs of culture of ≤12 h (
n
= 44) and >12 h (
n
= 47) were compared. Septic shock occurred in 13.6% of patients with TTPs of ≤12 h and in 8.5% of patients with TTP of >12 h (
P
= 0.51). A central venous catheter source was more common with a BSI TTP of ≤12 h (
P
= 0.010). Univariate analysis revealed that a Charlson score of ≥3, the failure of at least one organ (respiratory, cardiovascular, renal, hematologic, or hepatic), infection with methicillin-resistant
S. aureus
, and TTPs of ≤12 h were associated with death. Age, gender, an APACHE II score of ≥20 at BSI onset, inadequate empirical antibiotic therapy, hospital-acquired bacteremia, and endocarditis were not associated with mortality. Multivariate analysis revealed that independent predictors of hospital mortality were a Charlson score of ≥3 (odds ratio [OR], 14.4; 95% confidence interval [CI], 2.24 to 92.55), infection with methicillin-resistant
S. aureus
(OR, 9.3; 95% CI, 1.45 to 59.23), and TTPs of ≤12 h (OR, 6.9; 95% CI, 1.07 to 44.66). In this historical cohort study of BSIs due to
S. aureus
, a TTP of ≤12 h was a predictor of the clinical outcome.
Origin of Class 1 and 2 Integrons and Gene Cassettes in a Population-Based Sample of Uropathogenic
Escherichia coli
Authors: Solberg Owen D. , Ajiboye Remi M. , Riley Lee W. 
Published: April 2006

ABSTRACT

The prevalence of urinary tract infections (UTI) caused by trimethoprim-sulfamethoxazole (TMP-SMX)-resistant
Escherichia coli
is increasing and varies geographically in the United States. Recent community-based UTI studies have demonstrated geographic clustering of an
Escherichia coli
clonal group, suggesting occurrence of a community outbreak of UTI. A large proportion of this clonal group (designated CgA) isolated from women in a California college community was found to be resistant to TMP-SMX. We wished to determine if the acquisition of TMP-SMX resistance by CgA occurred before or after the CgA strains were introduced into this community. Between October 1999 and January 2000 and between October 2000 and January 2001, 482
E. coli
isolates were consecutively collected from the urine samples of women with UTI at a student health clinic and analyzed for determinants of TMP-SMX resistance. In particular, the distribution of integrons harboring resistance cassettes for TMP-SMX (
dfr
) was examined. Among 95 TMP-SMX-resistant isolates, 68 and 27 isolates carried class 1 and class 2 integrons, respectively. A class 1 integron was found in 25 (93%) of 27 TMP-SMX-resistant CgA isolates but in only 43 (63%) of 68 TMP-SMX-resistant non-CgA isolates (
P
< 0.001) and in none of 44 TMP-SMX-susceptible
E. coli
isolates (
P
< 0.0001). CgA strains carried only a single arrangement of class 1 gene cassettes (
dfrA17-aadA5
), while the non-clonal group strains carried nine different cassette arrangements. These results support the idea that CgA strains acquired their resistance at a common site prior to their spread to the college community.