bluuebunny/crossref_metadata_2025_split huggingface dataset:
16S rRNA Gene Sequencing versus the API 20 NE System and the VITEK 2 ID-GNB Card for Identification of Nonfermenting Gram-Negative Bacteria in the Clinical Laboratory
Authors: Bosshard P. P. , Zbinden R. , Abels S. , Böddinghaus B. , Altwegg M. , Böttger E. C. 
Published: April 2006

ABSTRACT

Over a period of 26 months, we have evaluated in a prospective fashion the use of 16S rRNA gene sequencing as a means of identifying clinically relevant isolates of nonfermenting gram-negative bacilli (non-
Pseudomonas aeruginosa
) in the microbiology laboratory. The study was designed to compare phenotypic with molecular identification. Results of molecular analyses were compared with two commercially available identification systems (API 20 NE, VITEK 2 fluorescent card; bioMérieux, Marcy l'Etoile, France). By 16S rRNA gene sequence analyses, 92% of the isolates were assigned to species level and 8% to genus level. Using API 20 NE, 54% of the isolates were assigned to species and 7% to genus level, and 39% of the isolates could not be discriminated at any taxonomic level. The respective numbers for VITEK 2 were 53%, 1%, and 46%, respectively. Fifteen percent and 43% of the isolates corresponded to species not included in the API 20 NE and VITEK 2 databases, respectively. We conclude that 16S rRNA gene sequencing is an effective means for the identification of clinically relevant nonfermenting gram-negative bacilli. Based on our experience, we propose an algorithm for proper identification of nonfermenting gram-negative bacilli in the diagnostic laboratory.
Reactivity of Genotype-Specific Recombinant Proteins of Human Erythrovirus B19 with Plasmas from Areas Where Genotype 1 or 3 Is Endemic
Authors: Parsyan Armen , Kerr Shane , Owusu-Ofori Shirley , Elliott Gordon , Allain Jean-Pierre 
Published: April 2006

ABSTRACT
Human erythrovirus (parvovirus) B19 (B19) is a common human pathogen. The recent discovery of three genotypes, 1 to 3, raised issues related to the ability of genotype-specific antigens to cross-react with antibodies elicited by other genotypes. This study assessed antibody capture and immunoglobulin G (IgG) cross-reactivity between genotype 1 and genotype 3 recombinant antigens and analyzed the potential gain of adding VP1 protein to VP2 capsid antigen. Plasma samples from genotype 1- or genotype 3-infected populations were blindly tested with blindly prepared reagents. The IgG reactivity was assessed with baculovirus-expressed VP2 or VP1 and VP2 recombinant genotype 1 or genotype 3 proteins in a standardized enzyme immunoassay (EIA). A high degree of agreement (>95%) between EIA results was observed, with Spearman correlation coefficient and kappa reliability coefficient results of ≥0.95 for samples from the United Kingdom and ≥0.77 for samples from Ghanaian children, respectively. Most discrepant results were related to equivocal reactivity. The addition of VP1 to VP2 capsids did not significantly impact antibody detection. These data suggest that the currently available genotype-1-based IgG EIA is suitable to detect antibody to B19 genotype 3 in Ghanaian children. However, samples from the Ghanaian adult population exhibited atypical results in the assay, possibly due to the high levels of nonspecific IgG antibodies found in adults living in this region. Within these limitations, the study demonstrates that genotype 1 and genotype 3 antigens are equally effective in detecting either antibody species.
Dynamics of Appearance and Expansion of a Prolyliminopeptidase- Negative Subtype among
Neisseria gonorrhoeae
Isolates Collected in Sydney, Australia, from 2002 to 2005
Authors: Limnios E. Athena , Nguyen Nhu-Lan , Ray Sanghamitra , McIver Christopher J. , Tapsall John W. 
Published: April 2006

ABSTRACT

Recent studies have demonstrated a wide geographic circulation of isolates of
Neisseria gonorrhoeae
that fail to produce prolyliminopeptidase (PIP). Tests based on the production of this enzyme are important elements of a number of identification systems for gonococci. We documented the appearance, expansion, and contraction of subtypes of 165 PIP-negative gonococci detected in an extended and systematic sample of the 3,926
N. gonorrhoeae
isolates collected in Sydney, Australia, from July 2002 to September 2005. Their appearance, peak, and decline followed an “epidemic” curve. At the peak of their prevalence in 2003, PIP-negative gonococci comprised 22% of all isolates. Closely related phenotypes accounted for 162/165 of the PIP-negative gonococci. Algorithms for confirmation of
N. gonorrhoeae
should take account of the temporal and geographic variability of gonococci by utilizing two or more distinct confirmatory methods.
Use of TaqMan Real-Time Reverse Transcription-PCR for Rapid Detection, Quantification, and Typing of Norovirus
Authors: Trujillo A. Angelica , McCaustland Karen A. , Zheng Du-Ping , Hadley Leslie A. , Vaughn George , Adams Susan M. , Ando Tamie , Glass Roger I. , Monroe Stephan S. 
Published: April 2006

ABSTRACT
Noroviruses (NoVs) are the most commonly identified cause of outbreaks and sporadic cases of acute gastroenteritis. We evaluated and optimized NoV-specific TaqMan real-time reverse transcription (RT)-PCR assays for the rapid detection and typing of NoV strains belonging to genogroups GI and GII and adapted them to the LightCycler platform. We expanded the detection ability of the assays by developing an assay that detects the GIV NoV strain. The assays were validated with 92 clinical samples and 33 water samples from confirmed NoV outbreaks and suspected NoV contamination cases. The assays detected NoV RNA in all of the clinical specimens previously confirmed positive by conventional RT-PCR and sequencing. Additionally, the TaqMan assays successfully detected NoV RNA in water samples containing low viral concentrations and inhibitors of RT and/or PCR, whereas the conventional method with region B primers required dilution of the inhibitors. By means of serially diluted NoV T7 RNA transcripts, a potential detection limit of <10 transcript copies per reaction mixture was observed with the GII assay and a potential detection limit of <100 transcript copies per reaction mixture was observed with the GI assay. These results and the ability to detect virus in water that was negative by RT-PCR demonstrate the higher sensitivity of the TaqMan assay compared with that of a conventional RT-PCR assay. The TaqMan methods dramatically decrease the turnaround time by eliminating post-PCR processing. These assays have proven useful in assisting scientists in public health and diagnostic laboratories report findings quickly to outbreak management teams.
Forty-Eight-Hour Diagnosis of Onychomycosis with Subtyping ofTrichophyton rubrumStrains
Authors: Kardjeva V. , Summerbell R. , Kantardjiev T. , Devliotou-Panagiotidou D. , Sotiriou E. , Gräser Y. 
Published: April 2006

ABSTRACTA novel strategy for the molecular identification of fungal agents of onychomycosis (includingTrichophyton rubrum) has been designed based on the use of species-specific and universal primers in conjunction with a commercial kit that allows the extraction of DNA directly from the nail specimens. The microsatellite marker T1, which is based on a (GT)n repeat, was applied for the species-specific identification ofTrichophyton rubrum. To evaluate how oftenScopulariopsisspp. are detected in nail specimens, a second primer pair was designed to amplify specifically a 336-bp DNA fragment of the 28S region of the nuclear rRNA gene ofS. brevicaulisand closely related species. Other fungal species were identified using amplification of the internal transcribed spacer (ITS) region of the rRNA gene, followed by restriction fragment length polymorphism analysis or sequencing. In addition, polyacrylamide gel separation of the T1-PCR product allowed subtyping ofT. rubrumstrains. We studied 195 nail specimens (the “nail sample”) and 66 previously collected etiologic strains (the “strain sample”) from 261 onychomycosis patients from Bulgaria and Greece. Of the etiologic agents obtained from both samples,T. rubrumwas the most common organism, confirmed to be present in 76% of all cases and serving as the sole or (rarely) mixed etiologic agent in 199 of 218 cases (91%) where the identity of the causal organism(s) was confirmed. Other agents seen included molds (6% of cases with identified etiologic agents; mainlyS. brevicaulis) and other dermatophyte species (4%; most frequentlyTrichophyton interdigitale). Simultaneous infections with two fungal species were confirmed in a small percentage of cases (below 1%). The proportion of morphologically identified cultures revealed by molecular study to have been misidentified was 6%. Subtyping revealed that all but fiveT. rubrumisolates were of the common type B that is prevalent in Europe. In comparison to microscopy and culture, the molecular approach was superior. The PCR was more sensitive (84%) than culture (22%) in the nail sample and was more frequently correct in specifically identifying etiologic agents (100%) than microscopy plus routine culture in either the nail or the strain samples (correct culture identifications in 96% and 94% of cases, respectively). Using the molecular approach, the time for diagnosing the identity of fungi causing onychomycosis could be reduced to 48 h, whereas culture techniques generally require 2 to 4 weeks. The early detection and identification of the infecting species in nails will facilitate prompt and appropriate treatment and may be an aid for the development of new antifungal agents.
Oncogenic Human Papillomavirus (HPV) Type Distribution and HPV Type 16 E6 Variants in Two Spanish Population Groups with Different Levels of HPV Infection Risk
Authors: Ortiz M. , Torres M. , Muñoz L. , Fernández-García E. , Canals J. , Cabornero A. I. , Aguilar E. , Ballesteros J. , del Amo J. , García-Sáiz A. 
Published: April 2006

ABSTRACTThe aim of this study is to determine oncogenic human papillomavirus (HPV) types and HPV type 16 (HPV16) variant distribution in two Spanish population groups, commercial sex workers and imprisoned women (CSW/IPW) and the general population. A multicenter cross-sectional study of 1,889 women from five clinical settings in two Spanish cities was conducted from May to November 2004. Oncogenic HPV infection was tested by an Hybrid Capture II (HC2) test, and positive samples were genotyped by direct sequencing using three different primer sets in L1 (MY09/11 and GP5+/GP6+) and E6/E7. HPV16 variants were identified by sequencing the E6, E2, and L1 regions. Four hundred twenty-five samples were positive for the HC2 test, 31.5% from CSW/IPW and 10.7% from the general population. HPV16 was the most frequent type. Distinct profiles of oncogenic HPV type prevalence were observed across the two populations. In order of decreasing frequency, HPV types 16, 31, 58, 66, 56, and 18 were most frequent in CSW/IPW women, and types 16, 31, 52, 68, 51, and 53 were most frequent in the general population. We analyzed HPV16 intratype variants, and a large majority (78.7%) belonged to the European lineage. AA variants were detected in 16.0% of cases. African variants belonging to classes Af1 (4.0%) and Af2 (1.3%) were detected. Different HPV types and HPV16 intratype variants are involved in oncogenic HPV infections in our population. These results suggest that HPV type distribution differs in CSW/IPW women and in the general population, although further analysis is necessary.
Comparison of PCR Assays for Diagnosis of Cutaneous Leishmaniasis
Authors: Bensoussan Esther , Nasereddin Abedelmajeed , Jonas Flory , Schnur Lionel F. , Jaffe Charles L. 
Published: April 2006

ABSTRACT

Three PCR assays for diagnosing leishmaniasis were compared and validated against parasite cultures and microscopic evaluation of stained tissue smears using 92 specimens from suspected cases of cutaneous leishmaniasis (CL) in Israel and the West Bank. Samples from imported and locally acquired disease were examined. The kinetoplast DNA (kDNA) PCR showed the highest sensitivity (98.7%) of any assay, correctly diagnosing 77/78 of the confirmed positive samples, followed by the rRNA gene internal transcribed spacer 1 (ITS1) PCR (71/78 positive, 91.0% sensitivity) and then the spliced leader mini-exon PCR (42/78 positive, 53.8% sensitivity). Either parasite culture or microscopy alone detected 62.8% (49/78) or 74.4% (58/78) of the positive specimens, respectively, while culture and microscopy together improved overall sensitivity to 83.3% (65/78). Except for the kDNA PCR that had six false positives, all other assays were 100% specific. Further, restriction enzyme analysis of the ITS1 PCR product enabled identification of 74.6% of the positive samples, which included strains of
Leishmania major
(50.9%),
Leishmania tropica
(47.2%), and the
Leishmania braziliensis
complex (1.9%). This suggests that a PCR using kDNA should be used for the diagnosis of CL and that an ITS1 PCR can be reliably used for the diagnosis of CL when rapid species identification is needed.
Simultaneous Detection of Pathogens in Clinical Samples from Patients with Community-Acquired Pneumonia by Real-Time PCR with Pathogen-Specific Molecular Beacon Probes
Authors: Morozumi Miyuki , Nakayama Eiichi , Iwata Satoshi , Aoki Yasuko , Hasegawa Keiko , Kobayashi Reiko , Chiba Naoko , Tajima Takeshi , Ubukata Kimiko 
Published: April 2006

ABSTRACT

In this study, real-time PCR with pathogen-specific molecular beacons (MB) and primers was evaluated for prediction of community-acquired pneumonia (CAP) causative agents, detecting six main CAP agents,
Streptococcus pneumoniae
,
Haemophilus influenzae
,
Mycoplasma pneumoniae
,
Chlamydophila pneumoniae
,
Legionella pneumophila
, and
Streptococcus pyogenes
, simultaneously. The PCR assay was evaluated for fresh clinical specimens from infants and children (
n
= 389) and from adults (
n
= 40). The MB probes and primers are both pathogen specific, namely, the
lytA
gene for
S. pneumoniae
, the
mip
gene for
L. pneumophila
, and 16S rRNA genes for the remaining four organisms. DNA extraction of clinical specimens was performed with a commercially available EXTRAGEN II kit, and amplification was performed with Stratagene Mx3000P. The limit of detection for these pathogens ranged from 2 copies to 18 copies. The whole process from DNA extraction to the analysis was finished in less than 2 h. The obtained sensitivity and specificity of this real-time PCR study relative to those of conventional cultures were as follows: 96.2% and 93.2% for
S. pneumoniae
, 95.8% and 95.4% for
H. influenzae
, 100% and 100% for
S. pyogenes
, and 100% and 95.4% for
M. pneumoniae
, respectively. The sensitivity and specificity for
M. pneumoniae
relative to those of a serologic assay were 90.2% and 97.9%, respectively. In six clinical samples of
C. pneumoniae
, the real-time PCR gave positive predictable values, and in those cases, elevation of the titer value was also observed. In conclusion, we demonstrated that a real-time PCR assay with pathogen-specific MB is useful in identifying CAP causative agents rapidly and in examining the clinical course of empirical chemotherapy in a timely manner, supporting conventional culture methods.
Single-Assay Combination of Epstein-Barr Virus (EBV) EBNA1- and Viral Capsid Antigen-p18-Derived Synthetic Peptides for Measuring Anti-EBV Immunoglobulin G (IgG) and IgA Antibody Levels in Sera from Nasopharyngeal Carcinoma Patients: Options for Field Screening
Authors: Fachiroh J. , Paramita D. K. , Hariwiyanto B. , Harijadi A. , Dahlia H. L. , Indrasari S. R. , Kusumo H. , Zeng Y. S. , Schouten T. , Mubarika S. , Middeldorp J. M. 
Published: April 2006

ABSTRACT

Assessment of immunoglobulin A (IgA) antibody responses to various Epstein-Barr virus (EBV) antigen complexes, usually involving multiple serological assays, is important for the early diagnosis of nasopharyngeal carcinoma (NPC). Through combination of two synthetic peptides representing immunodominant epitopes of EBNA1 and viral capsid antigen (VCA)-p18 we developed a one-step sandwich enzyme-linked immunosorbent assay (ELISA) for the specific detection of EBV reactive IgG and IgA antibodies in NPC patients (EBV IgG/IgA ELISA). Sera were obtained from healthy donors (
n
= 367), non-NPC head and neck cancer patients (
n
= 43), and biopsy-proven NPC patients (
n
= 296) of Indonesian and Chinese origin. Higher values of optical density at 450 nm for EBV IgG were observed in NPC patients compared to the healthy EBV carriers, but the large overlap limits its use for NPC diagnosis. Using either EBNA1 or VCA-p18 peptides alone IgA ELISA correctly identified 88.5% and 79.8% of Indonesian NPC patients, with specificities of 80.1% and 70.9%, whereas combined single-well coating with both peptides yielded sensitivity and specificity values of 90.1 and 85.4%, respectively. The positive and negative predictive values (PPV and NPV, respectively) for the combined EBNA1 plus VCA EBV IgA ELISA were 78.7% and 93.9%, respectively. In the Indonesia panel, the level of EBV IgA reactivity was not associated with NPC tumor size, lymph node involvement, and metastasis stage, sex, and age group. In the China panel the sensitivity/specificity values were 86.2/92.0% (EBNA1 IgA) and 84.1/90.3% (VCA-p18 IgA) for single-peptide assays and 95.1/90.6% for the combined VCA plus EBNA1 IgA ELISA, with a PPV and an NPV for the combined EBV IgA ELISA of 95.6 and 89.3%, respectively. Virtually all NPC patients had abnormal anti-EBV IgG diversity patterns as determined by immunoblot analysis. On the other hand, healthy EBV carriers with positive EBV IgA ELISA result showed normal IgG diversity patterns. By using EBV IgG immunoblot diversity as confirmation assay for EBV IgA ELISA-positive samples, the sensitivity and specificity for NPC diagnosis increased to 98% and 99.2%, respectively, in the Indonesian NPC samples. The use of these combined methods for seroepidemiological screening studies is proposed.
Relationship of Phylogenetic Background, Biofilm Production, and Time to Detection of Growth in Blood Culture Vials with Clinical Variables and Prognosis Associated with
Escherichia coli
Bacteremia
Authors: Martínez Jose A. , Soto Sara , Fabrega Anna , Almela Manel , Mensa Josep , Soriano Alex , Marco Francesc , Jimenez de Anta María T. , Vila Jordi 
Published: April 2006

ABSTRACT

In patients with
Escherichia coli
bacteremia, data on the relationship of phylogenetic background, biofilm production, and degree of bacteremia with clinical variables and prognosis are scarce. During a 1-year period, all adults with bacteremia due to
Escherichia coli
diagnosed at a university center were enrolled. Determination of phylogenetic background, biofilm production, and genotyping was performed with all strains, and the time to positivity of blood culture vials was recorded. A total of 185 episodes of diverse-source
E. coli
bacteremia was analyzed. Strains of phylogroup D were predominant (52%). Phylogroup A isolates were associated with pneumonia and prior antibiotic intake, B1 with an abdominal source of infection, B2 with the absence of urological abnormalities, and D with urological abnormalities and age below 65 years. Resistance to antibiotics and no biofilm production were concentrated in phylogroup A strains. Biofilm production was not associated with any clinical variable. An immunocompromising condition (odds ratio [OR] = 5.01, 95% confidence interval [CI] = 1.4 to 17.9), peritonitis (OR = 17, 95% CI = 3.32 to 87), pneumonia (OR = 9.97, 95% CI = 1.96 to 50.6), and ≤7 h to bacteremia detection (OR = 4.37, 95% CI = 1.38 to 13.8) were the best predictors of a fatal outcome. Results from this study suggest that the distribution of phylogenetic backgrounds among
E. coli
strains involved in diverse-source bacteremia may be subject to geographical variation and that, in afflicted individuals, some high-risk sources, the patient's underlying condition, and the degree of bacteremia are more important than microbial factors in determining the outcome. Time to positivity of blood culture vials may be a variable of potential clinical impact.
Transmission of an Uncultivated
Bergeyella
Strain from the Oral Cavity to Amniotic Fluid in a Case of Preterm Birth
Authors: Han Yiping W. , Ikegami Akihiko , Bissada Nabil F. , Herbst Melissa , Redline Raymond W. , Ashmead Graham G. 
Published: April 2006

ABSTRACT

Intrauterine infection is a recognized cause of preterm birth. The infectious organisms are believed to originate primarily from the vaginal tract and secondarily from other parts of the body. It is plausible that microbes in the oral cavity can be transmitted to the pregnant uterus. However, direct evidence supporting such a transmission is lacking. In this study, amniotic fluids of 34 pregnant women were examined by PCR using 16S and 23S rRNA universally conserved primers. Bacterial DNA was amplified from the only patient with clinical intrauterine infection and histologic necrotizing acute and chronic chorioamnionitis. One strain,
Bergeyella
sp. clone AF14, was detected and was 99.7% identical to a previously reported uncultivated oral
Bergeyella
strain, clone AK152, at the 16S rRNA level. The same strain was detected in the subgingival plaque of the patient but not in her vaginal tract. The 16S-23S rRNA sequence of clone AF14 matched exactly with the sequences amplified from the patient's subgingival plaque. These observations suggest that the
Bergeyella
strain identified in the patient's intrauterine infection originated from the oral cavity. This is the first direct evidence of oral-utero microbial transmission. The patient's periodontal health during pregnancy was unclear. She did not have detectable periodontal disease during postpartum examination.
Bergeyella
spp. had not been previously associated with preterm birth and were detected in subgingival plaque of women without clinical levels of intrauterine infection. Uncultivated species may be overlooked opportunistic pathogens in preterm birth. This study sheds new light on the implication of oral bacteria in preterm birth.
Use of a Short Fragment of the C-Terminal E Gene for Detection and Characterization of Two New Lineages of Dengue Virus 1 in India
Authors: Domingo C. , Palacios G. , Jabado O. , Reyes N. , Niedrig M. , Gascón J. , Cabrerizo M. , Lipkin W. I. , Tenorio A. 
Published: April 2006

ABSTRACT
Here we propose the use of a 216-nucleotide fragment located in the carboxyl terminus of the E gene (E-COOH) and a pairwise-based comparison method for genotyping of dengue virus 1 (DENV-1) strains. We have applied this method to the detection and characterization of DENV-1 in serum samples from travelers returning from the tropics. The results obtained with the typing system correlate with the results obtained by comparison of the sequences of the entire E gene of the strains. The approach demonstrates utility in plotting the distribution and circulation of different genotypes of DENV-1 and also suggests the presence of two new clades of Indian strains. The integration of the method with an online database and a typing characterization tool enhances its strength. Additionally, the analysis of the complete E gene of DENV-1 strains suggested the occurrence of a nondescribed recombination event in the China GD23-95 strain. We propose the use of this methodology as a tool for real-time epidemiological surveillance of dengue virus infections and their pathogenesis.