bluuebunny/crossref_metadata_2025 huggingface dataset:
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

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

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

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

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

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

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

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

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

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.
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

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

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

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

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

A 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

The 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

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

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

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

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

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

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.
Phenotypic Switching in
Candida lusitaniae
on Copper Sulfate Indicator Agar: Association with Amphotericin B Resistance and Filamentation
Authors: Miller Nancy S. , Dick James D. , Merz William G.
Published: April 2006

Candida lusitaniae
is an opportunistic yeast pathogen that has the ability to develop resistance to amphotericin B (AmB). The mechanism(s) for this resistance is not well understood, although there are data supporting mutations in sterol pathways and other data supporting phenotypic switching (PS). The goal of this study was to determine whether
C. lusitaniae
has a PS system and to characterize any phenotypes, including any changes in AmB MICs. When 10
4
CFU of an AmB-resistant (MIC of 16 to 32 μg/ml) clinical strain was plated on yeast-peptone-dextrose (YPD) agar with 1 mM CuSO
4
, three colony colors were observed: light brown (LB) ≫ dark brown (DB) > white (W), similar to the result for
Candida glabrata
. Switching did occur with high AmB resistance (MIC of 256 μg/ml) being associated with W, whereas LB and DB colonies had MICs of 2 to 8 μg/ml and 2 to 16 μg/ml, respectively. Filamentation (pseudohyphae) was associated with DB colonies. All phenotypes occurred spontaneously with greater frequency (∼10
−2
to 10
−4
) than spontaneous mutations, and all phenotypes were reversible, fulfilling the two PS criteria. High AmB MICs were always associated with W colonies but not with all W colonies. Detection of PS on YPD-CuSO
4
is also similar to that in
Candida glabrata
, and we hypothesize that this is due to similarities in metallothionein gene expression. Phenotypic switching represents a key strategy in
C. lusitaniae
that confers a selective advantage during environmental challenges, including the ability to switch to AmB resistance.
Seroprevalence and Molecular Epidemiology of Human T-Cell Leukemia Virus Type 1 (HTLV-1) and HTLV-2 in Blood Donors from Dakar, Senegal
Authors: Diop Saliou , Calattini Sara , Abah-Dakou Julienne , Thiam Doudou , Diakhaté Lamine , Gessain Antoine
Published: April 2006

In 2002, human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2 seroprevalence was 0.16% (8/4,900) in blood donors from Dakar, Senegal. Most of the positive donors originated from the country’s southern region. Seven donors were infected by HTLV-1 (of cosmopolitan subtype), and one was infected by HTLV-2. These data highlight the problem of transfusion safety in this area where HTLV-1-associated lymphoproliferative and neurological diseases are endemic.
Microevolution of the Direct Repeat Locus of
Mycobacterium tuberculosis
in a Strain Prevalent in San Francisco
Authors: Aga Roxanne S. , Fair Elizabeth , Abernethy Neil F. , DeRiemer Kathryn , Paz E. Antonio , Kawamura L. Masae , Small Peter M. , Kato-Maeda Midori
Published: April 2006

We describe a microevolutionary event of a prevalent strain of
Mycobacterium tuberculosis
that caused two outbreaks in San Francisco. During the second outbreak, a direct variable repeat was lost. We discuss the mechanisms of this change and the implications of analyzing multiple genetic loci in this context.
Discovery of the Neutralizing Epitope Common to Influenza B Virus Victoria Group Isolates in Japan
Authors: Nakagawa Naoko , Suzuoki Jun , Kubota Ritsuko , Kobatake Shinzo , Okuno Yoshinobu
Published: April 2006

Monoclonal antibody 9B2 possesses hemagglutination inhibition activity against all the 2002/2003 influenza B virus Victoria group isolates in Kobe, Japan, as well as representative strains isolated between 1987 and 1997. The 9B2 epitope localizes three-dimensionally in the vicinity of antigenic site A of the hemagglutinin molecule, and amino acid substitutions in this region affected the binding of 9B2.
Dismembering the ethical physician
Authors: Genuis S J
Published: April 2006

Physicians may experience ethical distress when they are caught in difficult clinical situations that demand ethical decision making, particularly when their preferred action may contravene the expectations of patients and established authorities. When principled and competent doctors succumb to patient wishes or establishment guidelines and participate in actions they perceive to be ethically inappropriate, or agree to refrain from interventions they believe to be in the best interests of patients, individual professional integrity may be diminished, and ethical reliability is potentially compromised. In a climate of ever-proliferating ethical quandaries, it is essential for the medical community, health institutions, and governing bodies to pursue a judicious tension between the indispensable regulation of physicians necessary to maintain professional standards and preserve public safety, and the support for “freedom of conscience” that principled physicians require to practise medicine in keeping with their personal ethical orientation.
Diffusion weighted magnetic resonance imaging for acute stroke: practical and popular
Authors: Tan P L , King D , Durkin C J , Meagher T M , Briley D
Published: April 2006

Aim

To evaluate the feasibility and impact of diffusion weighted magnetic resonance imaging (DW MRI) as the first line neuroimaging of stroke at a district general hospital.

Methods

Prospective audit of all in-patients admitted with clinically suspected acute stroke and referred for imaging over a consecutive 17 week period. The data collected included scan type, time from cerebral event to imaging request, and time from formal radiological request to neuroimaging. Clinicians' (general physicians, neurologists, and radiologists) perceptions were assessed by a questionnaire.

Results

148 patients had neuroimaging for clinically suspected stroke during this period. Eighty one per cent of patients (120 of 148) had DW MRI as first line. Ninety two per cent of these patients had DW MRI within 24 hours of the formal radiological request. Twenty eight patients did not undergo DW MRI because lack of MRI safety, clinical state, unavailability because of maintenance service or lack of trained staff. Clinicians found the introduction of the DW MRI based service a significant improvement on computed tomography, especially for equivocal cases.

Conclusion

DW based MRI service is both feasible and sustainable in the setting of a district general hospital and most clinicians feel that this is a significant improvement to stroke services.
Effects of ageing on smell and taste
Authors: Boyce J M , Shone G R
Published: April 2006

Disorders of taste and smell commonly present diagnostic dilemmas to the medical profession. This may be secondary to the lack of knowledge and understanding of these conditions. There seems to be a low level of interest in the disorders, when compared with disruption of the other senses such as sight and hearing. Nevertheless, impairment of these senses are common and may be life threatening, especially when they involve the elderly patient. The aetiology of the conditions is widespread, and extend beyond the content of this article. This article will relate only to how the ageing process may contribute to sensory dysfunction. It will focus on how the ageing process changes the normal anatomy and physiology of the senses, how this effects the person's quality of life, and the current management of these conditions.
Aetiology, diagnosis, and management of hypopituitarism in adult life
Authors: Prabhakar V K B , Shalet S M
Published: April 2006

Hypopituitarism is a complex medical condition associated with increased morbidity and mortality, requires complicated treatment regimens, and necessitates lifelong follow up by the endocrinologist. The causes, clinical features, and the management of hypopituitarism including endocrine replacement therapy are considered in this review article.