bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation of anAnaplasmasp. Organism from White-Tailed Deer by Tick Cell Culture
Authors: Munderloh Ulrike G. , Tate Cynthia M. , Lynch Meghan J. , Howerth Elizabeth W. , Kurtti Timothy J. , Davidson William R.
Published: September 2003

We used tick cell culture to isolate a bacterium previously referred to as the “white-tailed deer (WTD) agent” from two captive fawns inoculated with blood from wild WTD (Odocoileus virginianus). Buffy coat cells were added to ISE6 tick cell cultures and incubated at 34°C, and 8 days later,Anaplasma-like inclusions were demonstrated in Giemsa-stained culture samples. The microbes became established and could be continuously passaged in tick cells. The identity of a culture isolate designated WTD76 was verified as the WTD agent by using specific PCR primers and by DNA sequencing. Comparison with sequences available in GenBank indicated that the isolate was most closely related first toAnaplasma platysand second toAnaplasma phagocytophilum, supporting its placement in the genusAnaplasma. Transmission electron microscopy of thisAnaplasmasp. organism in tick cell cultures revealed large inclusions filled with pleomorphic and rod-shaped bacteria. Tick cells infected with theAnaplasmasp. organism were used to successfully infect a naive deer, thereby proving the infectivity of the isolate for deer.
Diagnosis of Tick-Borne Encephalitis by a μ-Capture Immunoglobulin M-Enzyme Immunoassay Based on Secreted Recombinant Antigen Produced in Insect Cells
Authors: Jääskeläinen Anu , Han Xiuqi , Niedrig Matthias , Vaheri Antti , Vapalahti Olli
Published: September 2003

Acute tick-borne encephalitis is diagnosed by detection of IgM antibodies to tick-borne encephalitis virus (TBEV) (genus
Flavivirus
) in patient serum. TBEV membrane (M) and envelope (E) proteins have previously been shown to form virus-like particles when expressed in mammalian cells. We expressed the prM/M and E proteins in insect cells with a recombinant baculovirus system and obtained antigenic protein secreted into the cell culture medium, as evidenced by detection by a panel of five monoclonal antibodies to TBEV E protein. According to the sedimentation pattern in sucrose gradient centrifugation, the proteins were most likely secreted as virus-like particles. A μ-capture immunoglobulin M-enzyme immunoassay (IgM-EIA) test was developed and compared to a commercially available TBEV-IgM test (Progen) based on inactivated purified TBEVs. With a panel of 100 TBEV-IgM-negative, 50 TBEV-IgM-positive, and seven dengue virus-IgM-positive serum samples from our diagnostic laboratory, a sensitivity of 100% and specificity of 99% were obtained, and the correlation coefficient of EIA absorbances with the reference test was 0.93. The antigen was also suitable for IgG antibody detection in an immunofluorescent assay format. This is the first time that secreted, fully antigenic E protein has been produced in insect cells for this arthropod-borne flavivirus.
Use of Discriminatory Probes for Strain Typing of Formalin-Fixed, Rabies Virus-Infected Tissues by In Situ Hybridization
Authors: Nadin-Davis Susan A. , Sheen Mary , Wandeler Alexander I.
Published: September 2003

An in situ hybridization (ISH) method has been developed to overcome difficulties encountered in the viral typing of formalin-fixed rabies virus-infected brain tissue. Rabies viruses representative of all strains normally encountered in diagnostic submissions throughout Canada, including 3 strains of terrestrial hosts (arctic fox, western skunk, mid-Atlantic raccoon), 10 strains circulating in several bat reservoirs (BBCAN1 to BBCAN7, LACAN, SHCAN, and MYCAN), and the Evelyn-Rokitniki-Abelseth (ERA) strain, used as an oral vaccine for fox rabies control in Ontario, were targeted. Partial phosphoprotein gene fragments generated from reverse transcription (RT)-PCR products of specimens of each viral type were molecularly cloned and used to produce negative-sense digoxigenin-labeled RNA transcripts. Conditions permitting the use of these transcripts as strain-specific probes were optimized by blotting analyses with RT-PCR amplicons generated with representative rabies viruses and by ISH applied to mouse brains inoculated with these strains. The successful application of this methodology to two rabies virus-positive specimens that were also identified by traditional methods and the retrospective typing of two archival rabies virus-positive equine specimens is described. This technique provides a typing regimen for rabies virus isolates submitted in a form that is normally recalcitrant to alternate typing strategies.
Corynebacterium nigricans
sp. nov.: Proposed Name for a Black-Pigmented
Corynebacterium
Species Recovered from the Human Female Urogenital Tract
Authors: Shukla Sanjay K. , Bernard Kathryn A. , Harney Mary , Frank Daniel N. , Reed Kurt D.
Published: September 2003

Six independent isolates of an unusual black-pigmented
Corynebacterium
species (strains CN-1, CN-2, CN-3415, W70124, 91-0032, and 92-0360) were recovered from the human female urogenital tract. Four of the six source patients had complications of pregnancy, including spontaneous abortion, preterm labor, and low amniotic fluid volume at the time of the pathogen isolation. One isolate was recovered from a vaginal ulcer. All six strains yielded black-pigmented colonies on sheep blood agar, chocolate agar, and colistin-nalidixic acid agar after 24 to 48 h of incubation at 35°C. The dry, adherent colonies pitted the agar surface. The cells were coccobacillary to rod-shaped, catalase positive, nonmotile, and nonlipophilic. Only five of six isolates were available for characterization. Biochemical and chemotaxonomic studies revealed that the strains belong to the genus
Corynebacterium
but differ from known corynebacterial species. Comparative 16S rRNA gene sequence analysis showed that the strains are closely related and form a new subline within the genus
Corynebacterium
. We propose the name
Corynebacterium nigricans
sp. nov. for this group of coryneforms. The type strain of
Corynebacterium nigricans
is CN-1. It is deposited in the American Type Culture Collection (assigned strain number ATCC 700975) and in the Institute Pasteur collection (assigned strain number CIP 107346).
Use of Loop-Mediated Isothermal Amplification of the IS900Sequence for Rapid Detection of CulturedMycobacterium aviumsubsp.paratuberculosis
Authors: Enosawa M. , Kageyama S. , Sawai K. , Watanabe K. , Notomi T. , Onoe S. , Mori Y. , Yokomizo Y.
Published: September 2003

We evaluated the usefulness of loop-mediated isothermal amplification (LAMP) in detecting specific gene sequences ofMycobacterium aviumsubsp.paratuberculosis(MAP). A total of 102 primer sets for LAMP was designed to amplify the IS900, HspX, and F57 gene sequences of MAP. Using each of two primer sets (P-1 and P-2) derived from the IS900fragment, it was possible to detect MAP in a manner similar to that used with nested PCR. The sensitivity of LAMP with P-1 was 0.5 pg/tube, which was more sensitive than nested PCR. When P-2 was used, 5 pg/tube could be detected, which was the same level of sensitivity as that for nested PCR. LAMP with P-1 was specific. Although only 2Mycobacterium scrofulaceumstrains out of 43 non-MAP mycobacterial strains were amplified, the amplification reaction for these strains was less efficient than for MAP strains, and their products could be distinguished from MAP products by restriction digestion. LAMP with P-2 resulted in very specific amplification only from MAP, the same result obtained with nested PCR. Our LAMP method was highly specific, and the white turbidity of magnesium pyrophosphate, a by-product of the LAMP reaction, allowed simple visual detection. Our method is rapid, taking only 2 h, compared with 4 h for nested PCR. In addition, the LAMP method is performed under isothermal conditions and no special apparatus is needed, which makes it more economical and practical than nested PCR or real-time PCR. These results indicate that LAMP can provide a rapid yet simple test for the detection of MAP.
Comparison and Evaluation of Real-Time PCR, Real-Time Nucleic Acid Sequence-Based Amplification, Conventional PCR, and Serology for Diagnosis of
Mycoplasma pneumoniae
Authors: Templeton Kate E. , Scheltinga Sitha A. , Graffelman A. Willy , van Schie Jolanda M. , Crielaard Jantine W. , Sillekens Peter , van den Broek Peterhans J. , Goossens Herman , Beersma Matthias F. C. , Claas Eric C. J.
Published: September 2003

Mycoplasma pneumoniae
is a common cause of community-acquired pneumonia and lower-respiratory-tract infections. Diagnosis has traditionally been obtained by serological diagnosis, but increasingly, molecular techniques have been applied. However, the number of studies actually comparing these assays is limited. The development of a novel duplex real-time PCR assay for detection of
M. pneumoniae
in the presence of an internal control real-time PCR is described. In addition, real-time nucleic acid sequence-based amplification (NASBA) on an iCycler apparatus is evaluated. These assays were compared to serology and a conventional PCR assay for 106 clinical samples from patients with lower-respiratory-tract infection. Of the 106 samples, 12 (11.3%) were positive by all the molecular methods whereas serology with acute sample and convalescent samples detected 6 (5.6%) and 9 (8.5%), respectively. Clinical symptoms of the patients with
Mycoplasma
-positive results were compared to those of the other patients with lower-respiratory-tract infections, and it was found that the results for mean lower age numbers as well as the presence of chills, increased erythrocyte sedimentation rate, and raised C-reactive protein levels showed significant differences. Molecular methods are superior for diagnosis of
M. pneumoniae
, providing more timely diagnosis. In addition, using real-time methods involves less hands-on time and affords the ability to monitor the reaction in the same tube.
Molecular Epidemiology of Pulmonary Tuberculosis in Belgrade, Central Serbia
Authors: Vuković Dragana , Rüsch-Gerdes Sabine , Savić Branislava , Niemann Stefan
Published: September 2003

In order to gain precise data on the actual epidemiology of tuberculosis (TB) in Belgrade, central Serbia, we conducted the molecular epidemiological investigation described herein. IS
6110
restriction fragment length polymorphism (RFLP) typing of 176
Mycobacterium tuberculosis
isolates was performed. These strains were obtained from 48.4% of all patients diagnosed with culture-proven pulmonary TB from April through September 1998 and from May through October 1999. Clusters containing strains with identical RFLP IS
6110
patterns were assumed to have arisen from recent transmission. Of the 176 cases, 55 (31.2%) were grouped into 23 clusters ranging in size from two to six patients. Nearly 80% of clustered patients were directly interviewed, and transmission between family-unrelated contacts was found to be predominant in the study population. Classical contact investigation identified only 2 (3.6%) of the 55 clustered patients. The clustering of TB patients was not associated with any demographic or clinical characteristic other than infection with multidrug-resistant (MDR)
M. tuberculosis
strains. Nearly 70% of MDR strains were clustered, which indicates active transmission of MDR TB in Belgrade. However, this was not observed by conventional epidemiologic surveillance. In conclusion, the first molecular epidemiologic analysis of TB in the region revealed frequent recent transmission of TB and pointed out significant shortcomings of the current concept for conventional contact tracing. The results presented also demonstrate that transmission of MDR TB in Belgrade is not optimally controlled, and they provide support for the development of improved control strategies, including application of molecular methods.
Applicability of a Real-Time Quantitative PCR Assay for Diagnosis of Respiratory Syncytial Virus Infection in Immunocompromised Adults
Authors: van Elden L. J. R. , van Loon A. M. , van der Beek A. , Hendriksen K. A. W. , Hoepelman A. I. M. , van Kraaij M. G. J. , Schipper P. , Nijhuis M.
Published: September 2003

Respiratory syncytial virus (RSV) accounts for the majority of respiratory virus infections, producing high mortality rates in immunocompromised patients with hematologic malignancies. The available methods for the rapid detection of RSV by antigen detection or PCR either lack sensitivity, require complex laboratory manipulation, or have not been evaluated in this patient population. To assess the applicability of a TaqMan-based real-time PCR technique for the detection of RSV A and B in immunocompromised adults, we developed a rapid, sensitive detection method that simultaneously detects RSV A and B and can be applied in routine diagnostics. The specificity of the assay was assessed using a panel of reference strains of other respiratory viruses and RSV. Electron microscopy-counted stocks of RSV A and B were used to develop a quantitative PCR format. Eleven copies of viral RNA could be detected for RSV A strain Long, and 14 copies could be detected for RSV B strain 9320, corresponding to 50% tissue culture infective doses of 0.86 and 0.34, respectively. The assay was evaluated on 411 combined nose and throat swabs derived from immunocompromised adults with or without signs of respiratory tract infection. The diagnostic efficacy of the TaqMan PCR determined on the clinical samples showed that this real-time PCR technique was substantially more sensitive than the combination of conventional viral culture and shell vial culture. None of the clinical specimens derived from patients without signs of respiratory illness were found to be positive for RSV by real-time TaqMan PCR.
Real-Time PCR Assay Compared to Nested PCR and Antigenemia Assays for Detecting Cytomegalovirus Reactivation in Adult T-Cell Leukemia-Lymphoma Patients
Authors: Ikewaki Junji , Ohtsuka Eiichi , Kawano Rie , Ogata Masao , Kikuchi Hiroshi , Nasu Masaru
Published: September 2003

We analyzed the efficiency of the quantitative real-time PCR assay for cytomegalovirus (CMV) reactivation in adult T-cell leukemia-lymphoma (ATL) patients and compared the results with those obtained with qualitative nested PCR and antigenemia assays. The viral load obtained by the real-time PCR assay closely paralleled the number of antigen-positive cells obtained with the antigenemia assay. Real-time PCR revealed that a large number of DNA copies could be present even in samples assessed as negative or low in antigen-positive cells (0 to 10 antigen-positive cells/50,000 cells) by antigenemia assay. CMV copy numbers did not differ between the negative and low-antigen-positive groups. When the input concentration for real-time PCR assay was 2,500 to 5,000 copies/ml, the positivity rate for the nested PCR assay was 47.3%, while the positivity rate was more than 90% at an input concentration of ≥50,000 copies/ml. Real-time PCR is more sensitive than the antigenemia and nested PCR assays. Moreover, real-time PCR was able to detect CMV reactivation earlier than the antigenemia and nested PCR assays through the use of longitudinal analysis in four ATL patients with CMV pneumonia. In longitudinal assessments, analysis of the results suggested that a cutoff level of 5,000 copies/ml might be used to initiate treatment. Real-time PCR is more suitable for monitoring CMV reactivation in ATL patients than the antigenemia and nested PCR assays. CMV viral loads of 5,000 copies/ml are proposed as the cutoff for initiating antiviral therapy in ATL patients.
Molecular Typing of
Salmonella enterica
Serovar Typhi Isolates from Various Countries in Asia by a Multiplex PCR Assay on Variable-Number Tandem Repeats
Authors: Liu Yichun , Lee May-Ann , Ooi Eng-Eong , Mavis Yeo , Tan Ai-Ling , Quek Hung-Hiang
Published: September 2003

A multiplex PCR method incorporating primers flanking three variable-number tandem repeat (VNTR) loci (arbitrarily labeled TR1, TR2, and TR3) in the CT18 strain of
Salmonella enterica
serovar Typhi has been developed for molecular typing of
S. enterica
serovar Typhi clinical isolates from several Asian countries, including Singapore, Indonesia, India, Bangladesh, Malaysia, and Nepal. We have demonstrated that the multiplex PCR could be performed on crude cell lysates and that the VNTR banding profiles produced could be easily analyzed by visual inspection after conventional agarose gel electrophoresis. The assay was highly discriminative in identifying 49 distinct VNTR profiles among 59 individual isolates. A high level of VNTR profile heterogeneity was observed in isolates from within the same country and among countries. These VNTR profiles remained stable after the strains were passaged extensively under routine laboratory culture conditions. In contrast to the
S. enterica
serovar Typhi isolates, an absence of TR3 amplicons and a lack of length polymorphisms in TR1 and TR2 amplicons were observed for other
S. enterica
serovars, such as
Salmonella enterica
serovar Typhimurium,
Salmonella enterica
serovar Enteritidis, and
Salmonella enterica
serovar Paratyphi A, B, and C. DNA sequencing of the amplified VNTR regions substantiated these results, suggesting the high stability of the multiplex PCR assay. The multiplex-PCR-based VNTR profiling developed in this study provides a simple, rapid, reproducible, and high-resolution molecular tool for the epidemiological analysis of
S. enterica
serovar Typhi strains.
Comparing First-Void Urine Specimens, Self-Collected Vaginal Swabs, and Endocervical Specimens To DetectChlamydia trachomatisandNeisseria gonorrhoeaeby a Nucleic Acid Amplification Test
Authors: Shafer Mary-Ann , Moncada Jeanne , Boyer Cherrie B. , Betsinger Kelli , Flinn Scott D. , Schachter Julius
Published: September 2003

We set out to determine the prevalences ofChlamydia trachomatisandNeisseria gonorrhoeaeby ligase chain reaction as well as to determine the prevalence ofTrichomonas vaginalisby culture in a large and diverse national sample of non-health-care-seeking young women entering the military; we also sought to compare the abilities of three different techniques of collecting specimens (first-void urine, self-collected vaginal swab, and clinician-collected endocervical swab) to identify a positive specimen. A cross-sectional sample of young women was voluntarily recruited; as a part of their routine entry pelvic examination visit, they completed a self-administered reproductive health questionnaire and provided first-void urine (used to detectC. trachomatisandN. gonorrhoeae) and self-collected vaginal swabs (used to detectC. trachomatis,N. gonorrhoeae, andT. vaginalis). The number of positive tests divided by the number of sexually active women screened by each sampling method determined the rates of prevalence. The rate of infection with any of the three sexually transmitted diseases (STDs) tested was 14.1%. The total positive rates for each STD (identified by ≥1 specimen) were the following: forC. trachomatis, 11.6%;N. gonorrhoeae, 2.4%; andT. vaginalis, 1.7%. The proportions of positives identified by specimen type were, forC. trachomatisandN. gonorrhoeae, respectively, endocervix, 65 and 40%; urine, 72 and 24%; and vagina, 81 and 72%. The proportions of positives when specimen results were combined were, forC. trachomatisandN. gonorrhoeae, respectively, cervix plus urine, 86 and 49%; cervix plus vagina, 91 and 93%; and vagina plus urine, 94 and 79%. We concluded that STDs were epidemic in this population. Self-collected vaginal swabs identified the highest number of positive test results among single specimens, with the combined cervix-vagina results identifying the highest number of positive results. Self-collected vaginal swab collections are a feasible alternative to cervical specimen collections in this population, and the use of multiple types of specimens increases the positive yield markedly.
Unexpected Detection of Animal VP7 Genes among Common Rotavirus Strains Isolated from Children in Mexico
Authors: Laird A. R. , Ibarra V. , Ruiz-Palacios G. , Guerrero M. L. , Glass R. I. , Gentsch J. R.
Published: September 2003

In the course of characterizing 103 rotaviruses from children in Mexico, we found that the majority of strains were globally common types (55.4% of total), while uncommon types represented 5.7%, mixed infections with common types represented 14.8%, and partially or fully nontypeable isolates represented about 24%. Serotype G9 was detected for the first time in Mexico. We sequenced a subset of strains that were G nontypeable by reverse transcriptase PCR and found surprisingly that two strains having common human rotavirus P genotypes (8 and 6) had serotype G3 and G4 VP7 gene sequences that shared closer homology with canine and porcine strains, respectively, than with human strains, suggesting that these isolates represented reassortants between human and animal rotaviruses.
Screening of Volunteer Students in Yaounde (Cameroon, Central Africa) forChlamydia trachomatisInfection and Genotyping of IsolatedC. trachomatisStrains
Authors: Ngandjio Antoinette , Clerc Maithe , Fonkoua Marie Christine , Thonnon Jocelyn , Njock Friede , Pouillot Regis , Lunel Françoise , Bebear Christiane , de Barbeyrac Bertille , Bianchi Anne
Published: September 2003

The prevalence ofChlamydia trachomatisinfection was 3.78% out of 1,277 volunteer students screened by direct fluorescence assay and Cobas Amplicor PCR. The infection was associated with the nonuse or inconsistent use of condoms in women (P= 0.026) and a previous sexually transmitted infection in men (P= 0.023). The most frequent genotypes determined by sequencing theomp1genes of 25 clinical isolates were E (44%) and F (20%), and some strains harbored mutations, but E genotype strains did not.
Chemically Defined Media for Growth of
Haemophilus influenzae
Strains
Authors: Coleman Hannah N. , Daines Dayle A. , Jarisch Justin , Smith Arnold L.
Published: September 2003

A chemically defined medium that supports the growth of both encapsulated and nontypeable
Haemophilus influenzae
strains in broth to densities that are ≥ 10
9
CFU/ml or on agar plates is described. The mean generation time of a panel of clinical isolates was comparable to that in rich, chemically undefined media (brain-heart infusion broth supplemented with heme and β-NAD).
Outbreak of Serogroup C Meningococcal Disease Caused by a Variant of
Neisseria meningitidis
Serotype 2a ET-15 in a Community of Men Who Have Sex with Men
Authors: Tsang Raymond S.W. , Kiefer Lori , Law Dennis K. S. , Stoltz Jan , Shahin Rita , Brown Shirley , Jamieson Frances
Published: September 2003

We describe an outbreak, in a community of men who have sex with men, of serogroup C meningococcal disease caused by a genetic variant of the serotype 2a ET-15
Neisseria meningitidis
characterized by a point mutation in the gene coding for the serotype 2a antigen. A microbiological characterization of the outbreak strain is presented in this report.
Confirmation of Nontypeable
Streptococcus pneumoniae-
Like Organisms Isolated from Outbreaks of Epidemic Conjunctivitis as
Streptococcus pneumoniae
Authors: Carvalho Maria Gloria S. , Steigerwalt Arnold G. , Thompson Terry , Jackson Delois , Facklam Richard R.
Published: September 2003

Eleven isolates representing five distinct outbreaks of pneumococcal conjunctivitis were examined for phenotypic and genetic characteristics. None of the strains possessed capsules, and all strains were susceptible to optochin, bile soluble, and Gen-Probe AccuProbe test positive. All 11 isolates were confirmed as
Streptococcus pneumoniae
by DNA-DNA reassociation experiments.
Evaluation of INNO-LiPA MYCOBACTERIA v2: Improved Reverse Hybridization Multiple DNA Probe Assay for Mycobacterial Identification
Authors: Tortoli Enrico , Mariottini Alessandro , Mazzarelli Gianna
Published: September 2003

INNO-LiPA MYCOBACTERIA (Innogenetics, Ghent, Belgium) is a reverse hybridization DNA probe assay that has been recently improved by increasing the number of identifiable mycobacterial species to 16. Our assessment, performed with 197 mycobacteria belonging to 81 taxa, revealed 100% specificity and sensitivity for 20 out of 23 probes. The probes specific forMycobacterium fortuitumcomplex, for theMycobacterium avium-intracellulare-scrofulaceumgroup, and forMycobacterium intracellularetype 2 cross-reacted with several mycobacteria rarely isolated from clinical specimens. The overall sensitivity was 100%, and the overall specificity was 94.4%.
Role of Baseline Human Immunodeficiency Virus Genotype as a Predictor of Viral Response to Tenofovir in Heavily Pretreated Patients
Authors: Barrios Ana , de Mendoza Carmen , Martín-Carbonero Luz , Ribera Esteve , Domingo Pere , Galindo María José , Gálvez Juan , Estrada Vicente , Dalmau David , Asensi Víctor , Soriano Vincent
Published: September 2003

Human immunodeficiency virus (HIV)-infected patients (
n
= 153) failing antiretroviral therapy after exposure to compounds from all three drug families were monitored for 6 months after beginning a rescue intervention program including tenofovir (TDF). At 3 months, levels of HIV RNA in plasma dropped by a mean of 0.9 log
10
and the mean CD4 count increased by 52 cells/μl. At 6 months, HIV RNA levels had dropped by a mean of 1.06 log
10
and the mean CD4 count had increased by 49 cells/μl. Only five (3.7%) patients discontinued TDF use due to adverse events. In the multivariate analysis, the presence of M41L and/or L210W at baseline was the only viral determinant of a lower response to TDF.
Population Dynamics of Persistent
Staphylococcus aureus
Isolated from the Airways of Cystic Fibrosis Patients during a 6-Year Prospective Study
Authors: Kahl Barbara C. , Duebbers Angelika , Lubritz Gabriele , Haeberle Johannes , Koch Hans G. , Ritzerfeld Barbara , Reilly Marion , Harms Erik , Proctor Richard A. , Herrmann Mathias , Peters Georg
Published: September 2003

Molecular typing of normal (
n
= 456) and small-colony-variant (SCV;
n
= 239)
Staphylococcus aureus
isolates cultured from the airways of 52 of 72 cystic fibrosis (CF) patients (72.2%) during a 6-year prospective study revealed a median long-term persistence of 37 months (range, 6 to 70). SCV persisted longer in the airways than the normal
S. aureus
(statistically not significant). Pulsed-field gel electrophoresis identified six prevalent clonal lineages, which were cultured from more than one patient (3 to 12 patients), and 39 individual clones, which were isolated only from single patients. The SCV phenotype was not restricted to a distinct clonal lineage but occurred in many different clones. Most patients (33 of 52, 63.46%) harbored single clones. This study provides a basis for improved understanding of
S. aureus
colonization and infection dynamics in CF patients.
Prevalence of Fragilysin Gene in
Bacteroides fragilis
Isolates from Blood and Other Extraintestinal Samples
Authors: Foulon Ina , Piérard Denis , Muyldermans Gaëtan , Vandoorslaer Kristof , Soetens Oriane , Rosseel Paul , Lauwers Sabine
Published: September 2003

Of 166
Bacteroides fragilis
isolates, 26.2% of 103 isolates from blood and 20.6% of 63 extraintestinal isolates harbored the fragilysin gene (difference not statistically significant). Clinical characteristics and evolution were comparable in patients with
B. fragilis
bacteremia with or without this enterotoxin. Fragilysin seems not to be an important virulence factor in
B. fragilis
disease.
Highly Sensitive PCR Assay for Routine Diagnosis of African Swine Fever Virus in Clinical Samples
Authors: Agüero M. , Fernández J. , Romero L. , Sánchez Mascaraque C. , Arias M. , Sánchez-Vizcaíno J. M.
Published: September 2003

This work provides a novel, highly sensitive, hot start PCR method for rapid and specific detection of African swine fever virus (ASFV) that can be used as a routine diagnostic test for ASFV in surveillance, control, and eradication programs. A confirmatory test of the specificity of this method based on restriction endonuclease analysis was also developed.
Streptococcus pneumoniae
Endocarditis: Persistence of DNA on Heart Valve Material 7 Years after Infectious Episode
Authors: Branger S. , Casalta J. P. , Habib G. , Collard F. , Raoult D.
Published: September 2003

We amplified by PCR and sequenced
Streptococcus pneumoniae rpoB
from DNA of the cardiac valve of a man who had presented with pneumococcal endocarditis 7 years earlier. Histopathologically, the valve did not show evidence of endocarditis. This case raises the question of persistence of DNA without any evidence of infection.
Development of a 5′ Nuclease-Based Real-Time PCR Assay for Quantitative Detection of Cariogenic Dental Pathogens
Streptococcus mutans
and
Streptococcus sobrinus
Authors: Yoshida Akihiro , Suzuki Nao , Nakano Yoshio , Kawada Miki , Oho Takahiko , Koga Toshihiko
Published: September 2003

A 5′ nuclease TaqMan PCR assay was developed for the quantitative detection of the major cariogenic bacteria
Streptococcus mutans
and
Streptococcus sobrinus
. The absolute and relative numbers of bacteria were measured by this method. This assay will be useful for quantifying these organisms in oral specimens and for analyzing biofilm formation.
Emergence of Invasive Serotype VIII Group B Streptococcal Infections in Denmark
Authors: Ekelund Kim , Slotved Hans-Christian , Nielsen Hans Ulrik , Kaltoft Margit S. , Konradsen Helle B.
Published: September 2003

Serotype VIII group B streptococcus has only rarely been described outside Japan. The Streptococcus Unit, Statens Serum Institut, performed national surveillance of invasive group B streptococcal (GBS) diseases in Denmark in 1999 to 2002 and identified seven clinical GBS isolates of serotype VIII in blood from seven patients admitted to different hospitals.
Latex Assay for Serotyping of Group B
Streptococcus
Isolates
Authors: Slotved H.-C. , Elliott J. , Thompson T. , Konradsen H. B.
Published: September 2003

We developed a group B streptococcus (GBS) latex serotyping kit that reduces the numbers of GBS nontypeable isolates by nearly 50%. A total of 232 isolates were tested, and 203 isolates were serotyped by the GBS latex test, while the capillary precipitation test serotyped 184 isolates.
Detection of
Mycoplasma pneumoniae
by Real-Time Nucleic Acid Sequence-Based Amplification
Authors: Loens K. , Ieven M. , Ursi D. , Beck T. , Overdijk M. , Sillekens P. , Goossens H.
Published: September 2003

Real-time isothermal nucleic acid sequence-based amplification (RT-NASBA) was applied to the detection of
Mycoplasma pneumoniae
. In vitro-generated
M. pneumoniae
RNA was used to assess the sensitivity of the assay. The 95% hit rate was 148 molecules of
M. pneumoniae
RNA in the amplification and 10
4
molecules of in vitro-generated RNA after nucleic acid extraction. The sensitivity of the RT-NASBA and the conventional NASBA assays corresponded to 5 color-changing units (CCU) of
M. pneumoniae
. In spiked throat swabs, nasopharyngeal aspirates, bronchoalveolar lavages, and sputum, the sensitivity of both NASBA assays corresponded to 5 to 50 CCU of
M. pneumoniae
. A total of 17 clinical specimens positive for
M. pneumoniae
by PCR were also positive by conventional NASBA, but one specimen was negative by RT-NASBA. These results indicate that the sensitivity of detection of
M. pneumoniae
by RT-NASBA in respiratory samples might be slightly reduced compared to that by conventional NASBA. However, the real-time assay is superior in speed and ease of handling.
Genital Human Papillomavirus Genotypes in Northwestern Tanzania
Authors: Mayaud Philippe , Weiss Helen A. , Lacey Charles J. N. , Gill Dilbinder K. , Mabey David C. W.
Published: September 2003

Using MY09-MY11 PCR and human papillomavirus (HPV) typing by reverse blot hybridization, we found a 34% cervical HPV prevalence among 561 pregnant women in Tanzania. One hundred three of 123 women (84%) with typeable samples harbored high-risk oncogenic strains. HPV type 16 (HPV-16) was the most prevalent subtype (18%) among HPV-infected women and among women with cervical neoplasia (3 of 19). A multivalent vaccine for HPV-16, -18, -31, -33, and -35 would be necessary to prevent 50% of the neoplasia in this population.
Direct Detection of Multidrug-Resistant
Mycobacterium tuberculosis
in Clinical Specimens in Low- and High-Incidence Countries by Line Probe Assay
Authors: Johansen Isik Somuncu , Lundgren Bettina , Sosnovskaja Anaida , Thomsen Vibeke Østergaard
Published: September 2003

The INNO-LiPA Rif.TB assay is designed for the detection of
rpoΒ
gene mutations causing rifampin resistance in isolates. We applied the method directly to 60 Lithuanian and Danish clinical specimens to detect rifampin resistance rapidly. Results were obtained in 78.3% of clinical specimens, and all were concordant with those obtained by BACTEC 460. The assay could have major impact on the management of multidrug-resistant tuberculosis.
Difference in
FKS1
Gene Sequences between Serotypes A and D of
Cryptococcus neoformans
Authors: Tanaka Reiko , Imanishi Yumi , Nishimura Kazuko
Published: September 2003

We compared sequences of the glucan synthase (
FKS1
) gene in serotypes A and D of
Cryptococcus neoformans
. Four introns were present in serotype D but not serotype A. PCR with primers that flank these introns permits simple differentiation of serotypes A and D.
Shell Vial Culture as a Tool for Isolation of
Brucella melitensis
in Chronic Hepatic Abscess
Authors: Rovery Clarisse , Rolain Jean Marc , Raoult Didier , Brouqui Philippe
Published: September 2003

A 50-year-old man was admitted with a liver abscess and positive serology for
Brucella
spp. Liver pus and blood cultures remained sterile on conventional culture. Inoculation of liver pus onto eukaryotic cells by the centrifugation-shell vial technique yielded
Brucella melitensis
, identified by 16S rRNA gene amplification and sequencing.
In Vivo Development of Quinolone Resistance in
Salmonella enterica
Serotype Typhimurium DT104
Authors: Kristiansen Malthe A. Melau , Sandvang Dorthe , Rasmussen Thorkild Bremholm
Published: September 2003

Salmonella enterica
serotype Typhimurium definitive phage type 104 was isolated several times from the same patient over a period of 2 years. The strain developed reduced sensitivity to fluoroquinolones, and a mutation in the
gyrA
gene that is associated with reduced sensitivity to quinolones was identified.
Use of Multiplex PCR To Identify
Staphylococcus aureus
Adhesins Involved in Human Hematogenous Infections
Authors: Tristan Anne , Ying Liu , Bes Michele , Etienne Jerome , Vandenesch Francois , Lina Gerard
Published: September 2003

We have developed a multiplex PCR procedure to determine the distribution of nine adhesin genes in
Staphylococcus aureus
isolates. Only genes encoding bone sialoprotein binding protein and fibronectin binding protein B were significantly associated with hematogenous osteomyelitis/arthritis and native-valve endocarditis, respectively, suggesting their involvement in hematogenous tissue infections.
Virulence of
Rhodococcus equi
Isolated from Cats and Dogs
Authors: Takai Shinji , Martens Ronald J. , Julian Alan , Garcia Ribeiro Márcio , Rodrigues de Farias Marconi , Sasaki Yukako , Inuzuka Kazuho , Kakuda Tsutomu , Tsubaki Shiro , Prescott John F.
Published: September 2003

Nine cat isolates and nine dog isolates of
Rhodococcus equi
from clinical material were investigated for the presence of the virulence-associated antigens (VapA and VapB) and virulence plasmids. Five of the cat isolates and one dog isolate were VapA positive and contained an 85-kb type I or an 87-kb type I plasmid. The remaining 12 isolates were avirulent
R. equi
strains and contained no virulence plasmids.
Mutations in
katG
,
inhA
, and
ahpC
Genes of Brazilian Isoniazid-Resistant Isolates of
Mycobacterium tuberculosis
Authors: Silva Márcia Susana N. , Senna Simone G. , Ribeiro Marta O. , Valim Andréia R. M. , Telles Maria Alice , Kritski Afrânio , Morlock Glenn P. , Cooksey Robert C. , Zaha Arnaldo , Rossetti Maria Lucia R.
Published: September 2003

The presence of mutations in specific regions of the
katG
,
inhA
, and
ahpC
genes was analyzed with 69
Mycobacterium tuberculosis
isoniazid-resistant isolates from three Brazilian states. Point mutations in codon 315 of the
katG
gene were observed in 87.1, 60.9, and 60% of the isolates from Rio Grande do Sul, Rio de Janeiro, and São Paulo, respectively. Mutations in the
inhA
gene were identified only in one isolate from RJ State, and the
ahpC
promoter region revealed mutations in distinct positions in 12.9, 21.7, and 6.7% of the isolates from RS, RJ and SP, respectively.
Identification of Oral
Peptostreptococcus
Isolates by PCR-Restriction Fragment Length Polymorphism Analysis of 16S rRNA Genes
Authors: Riggio Marcello P. , Lennon Alan
Published: September 2003

Oral
Peptostreptococcus
isolates tentatively identified by conventional microbiological culture methods were identified to the species level by a combination of PCR amplification of 16S rRNA genes and restriction enzyme analysis of the amplified products. This method is a reliable and rapid alternative to conventional methods for identification of these bacterial species.
Multiplex PCRs for Identification of Necrotoxigenic
Escherichia coli
Authors: Van Bost Sigrid , Jacquemin Etienne , Oswald Eric , Mainil Jacques
Published: September 2003

Two multiplex PCRs were developed for the detection of necrotoxigenic
Escherichia coli
virulence genes. M1 contained the primers for the toxins and the aerobactin, and M2 contained the primers for the adhesins. They were validated by single PCRs performed with reference
E. coli
strains and by multiplex PCRs with necrotoxigenic
E. coli
strains isolated from different animal species.
Disseminated Nosocomial Fungal Infection by
Aureobasidium pullulans
var.
melanigenum
: a Case Report
Authors: Bolignano Giuseppe , Criseo Giuseppe
Published: September 2003

We report on a rare case of disseminated nosocomial fungal infection due to
Aureobasidium pullulans
var.
melanigenum
in a severely traumatized patient. Repeated blood and urine cultures yielded multicellular filamentous hyphal structures of varying size accompanied by budding yeast-like-cells of ellipsoidal morphology. The patient became asymptomatic after fluconazole therapy.
Survey of Human Genes of Retroviral Origin: Identification and Transcriptome of the Genes with Coding Capacity for Complete Envelope Proteins
Authors: de Parseval Nathalie , Lazar Vladimir , Casella Jean-François , Benit Laurence , Heidmann Thierry
Published: September 2003

Sequences of retroviral origin occupy approximately 8% of the human genome. Most of these “retroviral” genes have lost their coding capacities since their entry into our ancestral genome millions of years ago, but some reading frames have remained open, suggesting positive selection. The complete sequencing of the human genome allowed a systematic search for retroviral envelope genes containing an open reading frame and resulted in the identification of 16 genes that we have characterized. We further showed, by quantitative reverse transcriptase PCR using specifically devised primers which discriminate between coding and noncoding elements, that all 16 genes are expressed in at least some healthy human tissues, albeit at highly different levels. All envelope genes disclose significant expression in the testis, three of them have a very high level of expression in the placenta, and a fourth is expressed in the thyroid. Besides their primary role as key molecules for viral entry, the envelope genes of retroviruses can induce cell-cell fusion, elicit immunosuppressive effects, and even protect against infection, and as such, endogenous retroviral envelope proteins have been tentatively identified in several reports as being involved in both normal and pathological processes. The present study provides a comprehensive survey of candidate genes and tools for a precise evaluation of their involvement in these processes.
Antiviral Treatment Down-Regulates Peripheral B-Cell CD81 Expression and CD5 Expansion in Chronic Hepatitis C Virus Infection
Authors: Zuckerman Eli , Kessel Aharon , Slobodin Gleb , Sabo Edmond , Yeshurun Daniel , Toubi Elias
Published: September 2003

Hepatitis C virus (HCV) infection is associated with immune-mediated abnormalities and B-cell lymphoproliferation. Recently, CD81 was identified as an HCV receptor on B lymphocytes, providing a mechanism by which B cells are infected and activated by the virus. It has recently been shown that peripheral B-cell CD81 overexpression and CD5+subpopulation expansion correlate with HCV viral load and are associated with the development of HCV-related autoimmunity. In the present study, we assessed the effects of combination antiviral therapy (alfa interferon and ribavirin) on peripheral B-cell CD81 expression and CD5 expansion and the presence of autoimmune markers. Peripheral B-cell CD5 expression and the mean fluorescence intensity of CD81 were assessed by flow cytometry before and after treatment in 15 HCV-infected patients, in 10 untreated patients, and in 25 healthy controls. A significant posttreatment decrease in peripheral B-cell CD81 expression and disappearance of CD5+B-cell expansion were observed in all nine patients in whom a complete and sustained virological response was achieved (P< 0.01) (comparable to those for healthy controls). The decrease in CD81 overexpression and CD5 expansion in these patients was associated with a decrease and/or disappearance of autoimmune markers. In contrast, in nonresponders overexpression of CD81 and expansion of the CD5+B-cell subpopulation were not significantly changed and were comparable to those for untreated patients. In conclusion, antiviral therapy down-regulates peripheral B-cell CD81 expression and the CD5+population, either directly or by its effect on HCV RNA load. The overexpression of CD81 and the expansion of the population of CD5+peripheral B cells in HCV-infected patients may possibly play a role in the development of HCV-associated autoimmunity and lymphoproliferation.
Down-Modulation of Mature Major Histocompatibility Complex Class II and Up-Regulation of Invariant Chain Cell Surface Expression Are Well-Conserved Functions of Human and Simian Immunodeficiency Virus
nef
Alleles
Authors: Schindler Michael , Würfl Stephanie , Benaroch Philippe , Greenough Thomas C. , Daniels Rod , Easterbrook Philippa , Brenner Matthias , Münch Jan , Kirchhoff Frank
Published: September 2003

Recently, it has been demonstrated that the human immunodeficiency virus type 1 (HIV-1) Nef from laboratory strains down-modulates cell surface expression of mature major histocompatibility complex class II (MHC-II) molecules, while up-regulating surface expression of the invariant chain (Ii) associated with immature MHC-II (P. Stumptner-Cuvelette, S. Morchoisne, M. Dugast, S. Le Gall, G. Raposo, O. Schwartz, and P. Benaroch, Proc. Natl. Acad. Sci. USA
98:
12144-12149, 2001). These Nef functions could contribute to impaired CD4
+
-T-helper-cell responses found in HIV-1-infected patients with progressive disease. However, it is currently unknown whether
nef
alleles derived from HIV-1-infected individuals or from other primate lentiviruses also modulate MHC-II and Ii. In the present study, we demonstrate that both activities are conserved among primary HIV-1
nef
alleles, as well as among HIV-2 and simian immunodeficiency virus (SIV)
nef
alleles. Down-modulation of mature MHC-II required high levels of Nef expression. In contrast, surface expression of Ii was already strongly increased at low to medium levels of Nef expression. Notably,
nef
genes derived from two of four HIV-1-infected long-term nonprogressors did not up-regulate Ii, whereas
nef
alleles derived from 10 individuals with progressive disease were active in this assay. Unlike other in vitro Nef functions, the average activity of Nef in modulating MHC-II and Ii surface expression did not change significantly during the course of infection. Mutational analysis confirmed that MHC-II down- and Ii up-regulation are functionally separable from each other and from other Nef functions and identified acidic residues, located at the base of the flexible C-proximal loop of Nef, that are critical for increased Ii expression. Overall, our results suggest that the ability of Nef to interfere with MHC-II antigen presentation might play a role in AIDS pathogenesis.
Kids campaign against tobacco
Authors: Davis R M
Published: September 2003

Counter-advertising contests, in which children are encouraged to submit artwork promoting awareness about the harmful effects of smoking, provide an effective form of education
Students’ opinion of tobacco control policies recommended for US colleges: a national survey
Authors: Rigotti N A , Regan S , Moran S E , Wechsler H
Published: September 2003

Objective:Comprehensive tobacco control policies for US colleges and universities have been proposed by several groups in order to counter the rising use of tobacco by students enrolled in these institutions. Student opinion of these policies is not known, and concern about student opposition is one barrier that deters administrators from adopting the policies. This study measured student support for recommended college tobacco control policies.

Design:Mailed survey of US college students (2001 Harvard School of Public Health College Alcohol Study).

Setting:119 nationally representative, four-year colleges and universities in the USA.

Participants:10 904 randomly selected undergraduate students enrolled at participating schools.

Main outcome measures:Students’ opinion of 7 proposed tobacco control policies.

Results:A majority of students supported each policy. Over three quarters of students favoured smoke-free policies for all college buildings, residences, and dining areas, while 71% supported prohibiting tobacco advertising and sponsorship of campus social events, 59% favoured prohibiting tobacco sales on campus, and 51% supported smoke-free campus bars. All policies had more support among non-smokers than smokers (p < 0.001). Among smokers, support for policies was inversely related to intention to quit and intensity of tobacco consumption. Because college students’ tobacco consumption is low, a majority of smokers favoured banning smoking in college buildings and dining areas and prohibiting tobacco marketing on campus.

Conclusions:Student support for proposed campus tobacco control policies is strong, even among smokers, and broadly based across demographic subgroups. These findings should provide reassurance to college administrators who are considering adopting these policies.
Long term compliance with California’s Smoke-Free Workplace Law among bars and restaurants in Los Angeles County
Authors: Weber M D , Bagwell D A S , Fielding J E , Glantz S A
Published: September 2003

Objective:To assess long term compliance with the California Smoke-Free Workplace Law in Los Angeles County freestanding bars and bar/restaurants.

Design:Population based annual site inspection survey of a random sample of Los Angeles County freestanding bars and bar/restaurants was conducted from 1998 to 2002.

Main outcome measures:The primary outcomes of interest were patron and employee smoking. The secondary outcomes of interest were the presence of ashtrays and designated outdoor smoking areas.

Results:Significant increases in patron non-smoking compliance were found for freestanding bars (45.7% to 75.8%, p < 0.0001) and bar/restaurants (92.2% to 98.5%, p < 0.0001) between 1998 and 2002. Increases in employee non-smoking compliance were found for freestanding bars (86.2% to 94.7%, p < 0.0003) and bar/restaurants (96.5% to 99.2%, p < 0.005).

Conclusions:This study provides clear evidence that the California Smoke-Free Workplace Law has been effective at reducing patron and employee smoking in Los Angeles County bars and restaurants. Recommendations include educational campaigns targeted to freestanding bar owners and staff to counter perceptions of lost revenue, more rigorous enforcement, and more severe penalties for repeat violators such as alcohol licence revocation. Policymakers can enact smoke-free restaurant and bar policies to protect employees and patrons from secondhand smoke, confident that these laws can be successfully implemented.