bluuebunny/crossref_metadata_2025 huggingface dataset:
Prevalence of Anti-Human Papillomavirus Type 16, 18, 31, and 58 Virus-Like Particles in Women in the General Population and in Prostitutes
Authors: Touzé Antoine , de Sanjosé Silvia , Coursaget Pierre , Almirall M. Rosa , Palacio Virgilio , Meijer Chris J. L. M. , Kornegay Janet , Bosch F. Xavier
Published: July 2002

Genital human papillomavirus (HPV) infection is sexually transmitted. The aim of the study was to characterize serological responses to HPV types 16, 18, 31, and 58 by exploring type-specific virus-like particles (VLPs) in two groups of women with very distinct sexual behaviors. Anti-VLP antibodies for types 16, 18, 31, and 58 and HPV DNA in cervical cells were investigated with 177 prostitutes and 283 age-matched controls from the female general population in Spain. Anti-VLP positivity increased with number of lifetime sexual partners in women from the general population, and no seroresponse was found in virgins. However, in prostitutes HPV infection was characterized by higher multireactivity to three or four VLPs (25%) than the general population (3%) and by a more frequent antibody response to HPV-58 than in the general population. About 75% of the women seropositive for type 58 had been born in a Latin American country. Seroprevalence of HPV and cervical HPV DNA in prostitutes were 14 and 10 times higher than observed in women in the general population (prevalence odds ratio [POR] of HPV seropositivity, 14.04 [95%; CI = 8.4 to 23.6] and POR for HPV DNA, 10.4 [95% CI = 3.9 to 27.6). Our results indicate that prostitutes are at an increased risk of oncogenic HPV infections, and they confirm the validity of anti-VLPs as markers of present or past HPV infection, that the number of sexual partners is the major determinant in acquisition of oncogenic HPV, and that anti-VLPs could be used as a marker of repeated infection in prostitutes.
Rapid Typing of Human Adenoviruses by a General PCR Combined with Restriction Endonuclease Analysis
Authors: Allard Annika , Albinsson Bo , Wadell Göran
Published: July 2002

We have developed a system for rapid typing of adenoviruses (Ads) based on a combination of PCR and restriction endonuclease (RE) digestion (PCR-RE digestion). Degenerated consensus primers were designed, allowing amplification of DNA from all 51 human Ad prototype strains and altogether 44 different genome variants of Ad serotypes 1, 3, 4, 5, 7, 11, 19, 40, and 41. The 301-bp amplimer of 22 prototype strains representing all six subgenera and the genome variant was selected as a target for sequencing to look for subgenus and genome type variabilities. The sequences obtained were used to facilitate the selection of specific REs for discrimination purposes in a diagnostic assay by following the concept of cleavage or noncleavage of the 301-bp amplimer. On the basis of these results, a flowchart was constructed, allowing identification of subgenus B:2 and D serotypes and almost complete distinction of subgenus A, B:1, C, E, and F serotypes. Application of the PCR-RE digestion system to clinical samples allowed typing of 34 of 40 clinical samples positive for Ad. The genome type determined by this method was identical to that obtained by traditional RE typing of full-length Ad DNA. The remaining six samples were positive only after a nested PCR. Therefore, to reduce the risk of false-negative results, samples scored negative by the PCR-RE digestion system should be evaluated by the described nested PCR. Used in combination, the PCR-RE digestion method and the nested PCR provide a reliable and sensitive system that can easily be applied to all kinds of clinical samples when rapid identification of adenoviruses is needed.
Improved, Low-Cost Selective Culture Medium for
Actinobacillus actinomycetemcomitans
Authors: Alsina M. , Olle E. , Frias J.
Published: July 2002

Actinobacillus actinomycetemcomitans
is considered to be one of the major oral putative pathogens, especially in cases of juvenile periodontitis. This microorganism requires nutritionally complex media for growth, and therefore the media for its primary isolation usually include blood agar or serum in their base. In this study we present a new medium, Dentaid-1, which improves the detection of
A. actinomycetemcomitans
in periodontal samples. In its composition, blood and serum have been omitted, hence reducing its cost and making it a more restrictive medium against the growth of other microorganisms with high nutritional requirements. The growth yields of pure cultures of the bacteria on Dentaid-1 were comparable to those on nonselective blood agar. Moreover, clinical efficacy was evaluated in subgingival samples from 77 subjects with adult periodontitis. Dentaid-1 detected
A. actinomycetemcomitans
in 24 subjects, while a previously described tryptic soy-serum-bacitracin-vancomycin agar detected the microorganism in only 19 subjects (79.1%). Dentaid-1 is a low-cost, noninhibitory formula for the improved diagnosis and monitoring of patients subgingivally infected by this important oral putative pathogen.
Evaluation of a Medium (STGG) for Transport and Optimal Recovery of
Streptococcus pneumoniae
from Nasopharyngeal Secretions Collected during Field Studies
Authors: O'Brien Katherine L. , Bronsdon Melinda A. , Dagan Ron , Yagupsky Pablo , Janco Jacob , Elliott John , Whitney Cynthia G. , Yang Yong-Hong , Robinson Lisa-Gaye E. , Schwartz Benjamin , Carlone George M.
Published: July 2002

Field studies of
Streptococcus pneumoniae
(pneumococci) nasopharyngeal (NP) colonization are hampered by the need to directly plate specimens in order to ensure isolate viability. A medium containing skim milk, tryptone, glucose, and glycerin (STGG) has been used to transport and store NP material, but its ability to preserve pneumococci has not been evaluated. Our objective was to qualitatively and semiquantitatively evaluate the ability of STGG to preserve pneumococci in NP secretions. Entwined duplicate calcium alginate NP swab samples were obtained from children. One swab was plated directly onto a gentamicin blood agar plate; the other was placed in STGG. Growth from the directly plated specimen was compared with growth from an STGG aliquot immediately cultured or stored at −70°C for 9 weeks, −20°C for 9 weeks, or 4°C for 5 days. Of 186 specimens, 96 (52%) were positive for pneumococci from the direct plating; 94 (98%) of these were positive from the fresh STGG specimen. Pneumococci were recovered from all 38 positive specimens frozen at −70°C, all 18 positive specimens frozen at −20°C, and 18 of 20 positive specimens stored at 4°C. Recovery of pneumococci after storage of NP material in STGG medium at −70°C is at least as good as that from direct plating. Storage at −20°C is also acceptable. Storage at 4°C for 5 days is not ideal.
Performance Assessment of New Multiplex Probe Assay for Identification of Mycobacteria
Authors: Tortoli Enrico , Nanetti Anna , Piersimoni Claudio , Cichero Paola , Farina Claudio , Mucignat Giorgio , Scarparo Claudio , Bartolini Laura , Valentini Roberta , Nista Domenico , Gesu Giampietro , Tosi Cristiana Passerini , Crovatto Marina , Brusarosco Giuliana
Published: July 2002

A new DNA probe assay (INNO LiPA Mycobacteria; Innogenetics, Ghent, Belgium) for the simultaneous identification, by means of reverse hybridization and line-probe technology, of
Mycobacterium tuberculosis
complex,
Mycobacterium kansasii
,
Mycobacterium xenopi
,
Mycobacterium gordonae
, the species of the
Mycobacterium avium
complex (MAC),
Mycobacterium scrofulaceum
, and
Mycobacterium chelonae
was evaluated on a panel of 238 strains including, besides representatives of all the taxa identifiable by the system, a number of other mycobacteria, some of which are known to be problematic with the only other commercial DNA probe system (AccuProbe; Gen-Probe, San Diego, Calif.), and two nocardiae. The new kit, which includes a control probe reacting with the whole genus
Mycobacterium
, correctly identified 99.6% of the strains tested; the one discrepancy, which remained unresolved, concerned an isolate identified as MAC intermediate by INNO LiPA Mycobacteria and as
Mycobacterium intracellulare
by AccuProbe. In five cases, because of an imperfect checking of hybridization temperature, a very slight, nonspecific, line was visible which was no longer evident when the test was repeated. Two strains whose DNA failed amplification at the first attempt were regularly identified when the test was repeated. Interestingly, the novel kit dodged all the pitfalls presented by the strains giving anomalous reactions with AccuProbe. A unique feature of INNO LiPA Mycobacteria is its ability to recognize different subgroups within the species
M. kansasii
and
M. chelonae
, while the declared overlapping reactivity of probe 4 with some
M. kansasii
and
Mycobacterium gastri
organisms and of probe 9 with MAC,
Mycobacterium haemophilum
, and
Mycobacterium malmoense
, may furnish a useful aid for their identification. The turnaround time of the method is approximately 6 h, including a preliminary PCR amplification.
Single Rapid Real-Time Monitored Isothermal RNA Amplification Assay for Quantification of Human Immunodeficiency Virus Type 1 Isolates from Groups M, N, and O
Authors: de Baar Michel P. , van Dooren Maaike W. , de Rooij Esther , Bakker Margreet , van Gemen Bob , Goudsmit Jaap , de Ronde Anthony
Published: July 2002

Because human immunodeficiency virus type 1 (HIV-1) subtypes and circulating recombinant forms (CRFs) are spreading rapidly worldwide and are becoming less confined to a geographical area, RNA assays that can detect and quantify all HIV-1 isolates reliably are in demand. We have developed a fast, real-time monitored RNA assay based on an isothermal nucleic acid sequence-based amplification technology that amplifies a part of the long terminal repeat region of the HIV-1 genome. Real-time detection was possible due to the addition of molecular beacons to the amplification reaction that was monitored in a fluorimeter with a thermostat. The lower level of detection of the assay was 10 HIV-1 RNA molecules per reaction, and the lower level of quantification was 100 copies of HIV-1 RNA with a dynamic range of linear quantification between 10
2
and 10
7
RNA molecules. All HIV-1 groups, subtypes, and CRFs could be detected and quantified with equal efficiency, including the group N isolate YBF30 and the group O isolate ANT70. To test the clinical utility of the assay, a series of 62 serum samples containing viruses that encompassed subtypes A through G and CRFs AE and AG of HIV-1 group M were analyzed, and these results were compared to the results of a commercially available assay. This comparison showed that the quantification results correlated highly (
R
2
= 0.735) for those subtypes that could be well quantified by both assays (subtypes B, C, D, and F), whereas improved quantification was obtained for subtypes A and G and CRFs AE and AG. A retrospective study with six individuals infected with either a subtype A, B, C, or D or an AG isolate of HIV-1 group M, who were treated with highly active antiretroviral therapy, revealed that the assay was well suited to the monitoring of therapy effects. In conclusion, the newly developed real-time monitored HIV-1 assay is a fast and sensitive assay with a large dynamic range of quantification and is suitable for quantification of most if not all subtypes and groups of HIV-1.
Genotypic Characterization of
Toxoplasma gondii
Strains Associated with Human Toxoplasmosis in Spain: Direct Analysis from Clinical Samples
Authors: Fuentes Isabel , Rubio Jose M. , Ramı́rez Carmen , Alvar Jorge
Published: July 2002

Genetic analysis of the
SAG2
locus was performed to determine the prevalence of the different genotypes of
Toxoplasma gondii
(strain types I, II, and III) associated with human toxoplasmosis in Spain. This determination was made directly from primary clinical samples, obviating the previous process of isolation in mice or cell culture. A total of 34 isolates of
T. gondii
, collected from immunocompromised patients and congenital infection cases, were analyzed. Restriction fragment length polymorphism in PCR-amplified
SAG2
products was used to group strains into one of the three genotypes of
T. gondii
. Complete characterization of the
SAG2
gene was successful in 76.5% of the cases, demonstrating the feasibility of direct genotype analysis from clinical samples of different origins. Strains of
T. gondii
type II were the most prevalent in immunocompromised patients, with 52% of cases, while strains of type I were present in 75% of the congenital infection cases. These data differ from previous reports that show type II strains to be mostly associated with all kinds of human toxoplasmosis. These differences might be an effect of selection in the process of culture and isolation of the samples performed by other researchers prior to strain characterization.
Relevance of Reactivity in Commercially Available Hepatitis C Virus Antibody Assays
Authors: Polywka Susanne , Schröter Matthias , Feucht Heinz-Hubert , Zöllner Bernhard , Laufs Rainer
Published: July 2002

Sera from 2,148 patients were tested with a third-generation microparticle enzyme immunoassay (MEIA), a confirmatory assay, and a reverse transcription-PCR. Overall, 85.6% of reactivities were confirmed, 13.2% were shown to be unspecifically reactive, and 1.2% were indeterminate. The rate of confirmed MEIA reactivities clearly depended on the strength of the reactivity.
Use of Recombinant Mitogillin for Improved Serodiagnosis ofAspergillus fumigatus-Associated Diseases
Authors: Weig Michael , Frosch Matthias , Tintelnot Kathrin , Haas Andrea , Groß Uwe , Linsmeier Bernd , Heesemann Jürgen
Published: July 2002

During human infection,Aspergillus fumigatussecretes a 18-kDa protein that can be detected as an immunodominant antigen in the urine of infected patients. Recently, this protein was shown to be mitogillin, a ribotoxin that cleaves a single phosphodiester bond of the 29S rRNA of eukaryotic ribosomes. We proved the immunogenic capacity of mitogillin in a rabbit animal model, indicating its usefulness as an antigen for serological diagnosis of invasive aspergillosis. The mitogillin gene fromA. fumigatuswas transferred from plasmid pMIT+ to expression vector pQE30 and expressed inEscherichia colias a fusion protein. Purified recombinant mitogillin was recognized by serum immunoglobulin G (IgG) of polyclonal rabbit sera that were obtained by immunization with purified native mitogillin. Consequently, we developed an enzyme-linked immunosorbent assay for detection of IgG, IgM, and IgA antibodies to recombinant mitogillin. In serum samples of patients suffering from aspergilloma (AO;n= 32), invasive pulmonary aspergillosis (IPA;n= 42), or invasive disseminated aspergillosis (IDA;n= 40), a good correlation of production of IgG antibody against mitogillin and clinical disease was observed (for patients with AO, 100% [32 of 32] were positive; for patients with IPA, 64% [31 of 42] were positive; for patients with IDA, 60% [24 of 40] were positive). In contrast, positive titers for serum IgG and IgM antibodies against mitogillin were found in only 1.3% of the serum samples of healthy volunteers and positive titers for IgA antibody were found in only 1.0% of the serum samples of healthy volunteers (n= 307; specificity = 95.4%). These results indicate that recombinant mitogillin expressed inE. colican be used for improvement of the serodiagnosis ofA. fumigatus-associated diseases.
iceA
Genotypes of
Helicobacter pylori
Strains Isolated from Brazilian Children and Adults
Authors: Ashour Abdussalam Ali Ramadan , Birchal Collares Guilherme , Nogueira Mendes Edilberto , Ribeiro de Gusmão Valquı́ria , Queiroz Dulciene Maria de Magalhães , Prazeres Magalhães Paula , Teles de Carvalho Anfrisina Sales , de Oliveira Celso Affonso , Ferreira Nogueira Ana Margarida Miguel , Aguiar Rocha Gifone , Rocha Andreia Maria Camargos
Published: July 2002

Data concerning the geographic distribution of
iceA
alleles are scarce, and information on the association of the gene with the disease is rare and still controversial. Furthermore, no such study has been developed in Brazil, where duodenal ulcer and gastric adenocarcinoma are very common. We investigated, by PCR, the frequency of
iceA
alleles and
cagA
status in
Helicobacter pylori
strains isolated from 142 patients (62 children and 80 adults; 66 female; mean age, 30.0 years; age range, 3 to 78 years) with gastritis, duodenal ulcer, or gastric adenocarcinoma.
iceA
was identified in bacterium samples obtained from all patients. Eleven (7.7%) of them were infected with multiple strains. Among the patients with nonmixed infection,
iceA2
allele was detected in 118 (90.1%).
iceA2
allele was associated with ulcer (
P
= 0.02) and with carcinoma (
P
= 0.001).
iceA2
amplicons of 229, 334, or 549 bp were detected, but none of them was associated with the patient's disorder.
iceA2
strains were more frequent in patients older than 7 years (
P
= 0.001). The gene was also more frequent in strains obtained from males (
P
= 0.02).
cagA
was more common in strains obtained from carcinoma (
P
= 0.0008) and ulcer patients (
P
< 0.006).
cagA
-positive strains were more frequent in children older than 7 years (
P
< 0.003). No association between
cagA
status and sex was found (
P
= 0.28). In conclusion, we think
iceA
should not be used as a reliable marker for predicting the clinical outcome of
H. pylori
infection.
High Prevalence of VanB2 Vancomycin-Resistant
Enterococcus faecium
in Taiwan
Authors: Lu Jang-Jih , Perng Cherng-Lih , Ho Ming-Fa , Chiueh Tzong-Shi , Lee Wei-Hwa
Published: July 2002

Thirty-six VanB glycopeptide-resistant
Enterococcus faecium
isolates were collected from patients in five different hospitals in Taiwan. The vancomycin resistance genes were amplified by the long
vanB
PCR, which amplifies the 6,373-bp
vanB
gene cluster including the

vanR
B2
, vanS
B2
, vanY
B2
, vanW
B2
, vanH
B2
, vanB2

, and

vanX
B2

genes. The deduced amino acid sequences were found to be 95 to 98% homologous to those of the
vanB1
gene cluster: VanR
B1
, 97%; VanS
B1
, 97%; VanY
B1
, 96%; VanH
B1
, 95%; VanB1, 96%; and VanX
B1
, 98%. Restriction enzyme analysis of the long
vanB
PCR products revealed that all 36 isolates had the same
vanB2
-specific pattern. DNA sequence analysis of the
vanB2
gene, which is a
d
-Ala–
d
-Lac ligase gene, revealed that none of the 36 sequences were identical to the previously published
vanB2
sequence. Thirty-one isolates had 1 nucleotide different from the published
vanB2
sequence. The sequences of the other five isolates differed from the published
vanB2
sequence by 2 or 3 nucleotides. Four isolates with a low or moderate resistance to vancomycin (MIC = 4 to 32 μg/ml) were found to have the same leucine-to-methionine change at amino acid position 308 of the
vanB2
gene. The genomic DNAs of all 36 isolates were digested with
Sma
I and then typed by pulsed-field gel electrophoresis (PFGE). Eight different PFGE types (I to VIII) were observed, and type I was found to be prevalent in all hospitals examined in this study. This result suggests that intra- and interhospital dissemination of this
E. faecium
strain has occurred in Taiwan.
Frequent Detection of the Human Herpesvirus 6-Specific Genomes in the Livers of Children with Various Liver Diseases
Authors: Ozaki Yoshikazu , Tajiri Hitoshi , Tanaka-Taya Keiko , Mushiake Sotaro , Kimoto Akihito , Yamanishi Koichi , Okada Shintaro
Published: July 2002

This study was performed to investigate the frequency of human herpesvirus 6 (HHV-6) infection of the liver in children with a variety of liver diseases and to evaluate the role of HHV-6 infection in pediatric patients with prolonged non-B non-C hepatitis. Detection of the HHV-6 genomes in liver, in peripheral blood mononuclear cells (PBMC), and in plasma was performed by PCR or by in situ hybridization. Liver biopsy materials from 48 patients, in whom HHV-6 infection was serologically confirmed, were available for PCR analysis. Sequences of the HHV-6B genome were detectable in the livers of 36 of 48 patients (75%). The presence of the genome was not associated with serum transaminase activities. The genome was detectable in PBMC of 22 of 31 (71%) patients tested. In these 31 patients HHV-6 was detected in both the livers and PBMC of 20, was detected in PBMC but not in the livers of 2, was detected in the livers but not in PBMC of 3, and was detected in neither of samples of 6. In situ hybridization of the livers of six patients showed the presence of the HHV-6B genome in the nuclei of hepatocytes. The anti-HHV-6 immunoglobulin M antibody was detectable in 2 of 9 of the non-B non-C hepatitis patients, whereas none of the 22 patients with etiology-defined liver diseases tested positive. Cell-free viral DNA was not detectable in either group of patients. Our results showed that HHV-6B is frequently present in the livers of children with a variety of liver diseases but do not support the assumption that HHV-6B infection of the liver is associated with prolonged non-B non-C hepatitis.
Isolation and Characterization of
Borrelia burgdorferi
Sensu Lato Strains in an Area of Italy Where Lyme Borreliosis Is Endemic
Authors: Ciceroni Lorenzo , Ciarrochi Simonetta , Ciervo Alessandra , Mondarini Valeria , Guzzo Francesco , Caruso Giuseppe , Murgia Rossella , Cinco Marina
Published: July 2002

Between 1993 and 1998, we isolated
Borrelia burgdorferi
sensu lato from 55 of the 119 patients with clinically diagnosed Lyme borreliosis who were admitted to “San Martino” Hospital in Belluno, Veneto, an Adriatic region in northeastern Italy where Lyme borreliosis is endemic. Upon hospitalization, all patients presented erythema migrans. Isolates were typed using ribosomal DNA PCR-restriction fragment length polymorphism (RFLP) analysis of the
rrfA-rrlB
intergenic spacer. Of the 41 isolates typed, 37 belonged to
Borrelia afzelii
, 2 to
Borrelia garinii
, and 2 to
B. burgdorferi
sensu stricto. Pulsed-field gel electrophoresis, performed on 21 strains (13 new isolates and 8 controls), revealed different RFLP patterns within the
B. garinii
and
B. afzelii
strains; among the five
B. garinii
strains and the 12
B. afzelii
strains, three or two different RFLP patterns were identified, according to the restriction enzyme used. The protein patterns of the new isolates confirmed their genotypic classification and revealed the level of expression of some immunodominant proteins like OspA and other characteristic Osps. These findings constitute the first report of such a high recovery rate of
B. burgdorferi
from patients in a very restricted area in Italy; they also indicate the predominance of the genospecies
B. afzelii
in the study area and the heterogeneity of the circulating strains.
Nomenclature of Major Antimicrobial-Resistant Clones of
Streptococcus pneumoniae
Defined by the Pneumococcal Molecular Epidemiology Network
Authors: McGee L. , McDougal L. , Zhou J. , Spratt B. G. , Tenover F. C. , George R. , Hakenbeck R. , Hryniewicz W. , Lefévre J. C. , Tomasz A. , Klugman K. P.
Published: July 2002

The emergence of disease caused by penicillin-resistant and multidrug-resistant pneumococci has become a global concern, necessitating the identification of the epidemiological spread of such strains. The Pneumococcal Molecular Epidemiology Network was established in 1997 under the auspices of the International Union of Microbiological Societies with the aim of characterizing, standardizing, naming, and classifying antimicrobial agent-resistant pneumococcal clones. Here we describe the nomenclature for 16 pneumococcal clones that have contributed to the increase in antimicrobial resistance worldwide. Guidelines for the recognition of these clones using molecular typing procedures (pulsed-field gel electrophoresis, BOX-PCR, and multilocus sequence typing) are presented, as are the penicillin-binding profiles and macrolide resistance determinants for the 16 clones. This network can serve as a prototype for the collaboration of scientists in identifying clones of important human pathogens and as a model for the development of other networks.
Direct Detection of
Legionella
Species from Bronchoalveolar Lavage and Open Lung Biopsy Specimens: Comparison of LightCycler PCR, In Situ Hybridization, Direct Fluorescence Antigen Detection, and Culture
Authors: Hayden R. T. , Uhl J. R. , Qian X. , Hopkins M. K. , Aubry M. C. , Limper A. H. , Lloyd R. V. , Cockerill F. R.
Published: July 2002

We developed a rapid thermocycling, real-time detection (also known as real-time PCR) method for the detection of
Legionella
species directly from clinical specimens. This method uses the LightCycler (Roche Molecular Biochemicals, Indianapolis, Ind.) and requires approximately 1 to 2 h to perform. Both a
Legionella
genus PCR assay and
Legionella pneumophila
species-specific PCR assay were designed. A total of 43 archived specimens from 35 patients were evaluated, including 19 bronchoalveolar lavage (BAL) specimens and 24 formalin-fixed, paraffin-embedded open lung biopsy specimens. Twenty-five of the specimens were culture-positive for Legionella (9 BAL specimens and 16 tissue specimens). BAL specimens were tested by LightCycler PCR (LC-PCR) methods and by a direct fluorescent antibody (DFA) assay, which detects
L. pneumophila
serogroups 1 to 6 and several other
Legionella
species. Tissue sections were tested by the two LC-PCR methods, by DFA, by an in situ hybridization (ISH) assay, specifically designed to detect
L. pneumophila
, and by Warthin-Starry (WS) staining. The results were compared to the “gold standard” method of bacterial culture. With BAL specimens the following assays yielded the indicated sensitivities and specificities, respectively:
Legionella
genus detection by
Legionella
genus LC-PCR, 100 and 100%;
Legionella
genus detection by DFA assay, 33 and 100%; and
L. pneumophila
detection by
L. pneumophila
species-specific LC-PCR, 100 and 100%. With open lung biopsy specimens the following assays yielded the indicated sensitivities and specificities, respectively:
Legionella
genus detection by LC-PCR 68.8 and 100%;
Legionella
genus detection by DFA assay, 44 and 100%;
Legionella
genus detection by WS staining, 63 and 100%;
L. pneumophila
species-specific detection by LC-PCR, 17 and 100%; and
L. pneumophila
species-specific detection by ISH, 100 and 100%. The analytical sensitivity of both LC-PCR assays was <10 CFU/reaction. LC-PCR is a reliable method for the direct detection of
Legionella
species from BAL specimens. The
Legionella
genus LC-PCR assay could be performed initially; if positive,
L. pneumophila
species-specific LC-PCR could then be performed (if species differentiation is desired). The speed with which the LC-PCR procedure can be performed offers significant advantages over both culture-based methods and conventional PCR techniques. In contrast, for the methods evaluated, culture was the best for detecting multiple
Legionella
species in lung tissue. WS staining,
Legionella
genus LC-PCR, and
L. pneumophila
species-specific ISH were useful as rapid tests with lung tissue.
Malaria Rapid Diagnostic Devices: Performance Characteristics of the
Para
Sight F Device Determined in a Multisite Field Study
Authors: Forney J. Russ , Magill Alan J. , Wongsrichanalai Chansuda , Sirichaisinthop Jeeraphat , Bautista Christian T. , Heppner D. Gray , Miller R. Scott , Ockenhouse Christian F. , Gubanov Alex , Shafer Robyn , DeWitt Caroline Cady , Quino-Ascurra Higinio A. , Kester Kent E. , Kain Kevin C. , Walsh Douglas S. , Ballou W. Ripley , Gasser Robert A.
Published: July 2002

Microscopic detection of parasites has been the reference standard for malaria diagnosis for decades. However, difficulty in maintaining required technical skills and infrastructure has spurred the development of several nonmicroscopic malaria rapid diagnostic devices based on the detection of malaria parasite antigen in whole blood. The
Para
Sight F test is one such device. It detects the presence of
Plasmodium falciparum
-specific histidine-rich protein 2 by using an antigen-capture immunochromatographic strip format. The present study was conducted at outpatient malaria clinics in Iquitos, Peru, and Maesod, Thailand. Duplicate, blinded, expert microscopy was employed as the reference standard for evaluating device performance. Of 2,988 eligible patients, microscopy showed that 547 (18%) had
P. falciparum
, 658 (22%) had
P. vivax
, 2 (0.07%) had
P. malariae
, and 1,750 (59%) were negative for
Plasmodium
. Mixed infections (
P. falciparum
and
P. vivax
) were identified in 31 patients (1%). The overall sensitivity of
Para
Sight F for
P. falciparum
was 95%. When stratified by magnitude of parasitemia (no. of asexual parasites per microliter of whole blood), sensitivities were 83% (>0 to 500 parasites/μl), 87% (501 to 1,000/μl), 98% (1,001 to 5,000/μl), and 98% (>5,000/μl). Device specificity was 86%.
Cariogenic Actinomyces Identified with a β-Glucosidase-Dependent Green Color Reaction toGardenia jasminoidesExtract
Authors: Chen Li , Ma Lili , Park No-Hee , Shi Wenyuan
Published: July 2002

The oral bacteriaActinomyces naeslundiiandActinomyces viscosusare known to contribute to the initiation and progression of human dental caries, especially root caries. We report that bothA. naeslundiiandA. viscosusreact with a component in theGardenia jasminoidesextract to produce a distinct green product. This green color reaction was found to be dependent on the bacterial β-glucosidase. The reaction is specific for cariogenic actinomyces, and it can detect as few as 104cells ofA. naeslundiiandA. viscosusper ml.
Enzyme-Linked Immunosorbent Assay Specific to Dengue Virus Type 1 Nonstructural Protein NS1 Reveals Circulation of the Antigen in the Blood during the Acute Phase of Disease in Patients Experiencing Primary or Secondary Infections
Authors: Alcon Sophie , Talarmin Antoine , Debruyne Monique , Falconar Andrew , Deubel Vincent , Flamand Marie
Published: July 2002

During flavivirus infection in vitro, nonstructural protein NS1 is released in a host-restricted fashion from infected mammalian cells but not vector-derived insect cells. In order to analyze the biological relevance of NS1 secretion in vivo, we developed a sensitive enzyme-linked immunosorbent assay (ELISA) to detect the protein in the sera of dengue virus-infected patients. The assay was based on serotype 1 NS1-specific mouse and rabbit polyclonal antibody preparations for antigen immunocapture and detection, respectively. With purified dengue virus type 1 NS1 as a protein standard, the sensitivity of our capture ELISA was less than 1 ng/ml. When a panel of patient sera was analyzed, the NS1 antigen was found circulating from the first day after the onset of fever up to day 9, once the clinical phase of the disease is over. The NS1 protein could be detected even when viral RNA was negative in reverse transcriptase-PCR or in the presence of immunoglobulin M antibodies. NS1 circulation levels varied among individuals during the course of the disease, ranging from several nanograms per milliliter to several micrograms per milliliter, and peaked in one case at 50 μg/ml of serum. Interestingly, NS1 concentrations did not differ significantly in serum specimens obtained from patients experiencing primary or secondary dengue virus infections. These findings indicate that NS1 protein detection may allow early diagnosis of infection. Furthermore, NS1 circulation in the bloodstream of patients during the clinical phase of the disease suggests a contribution of the nonstructural protein to dengue virus pathogenesis.
Catheter-Related Bacteremia Due to
Kocuria kristinae
in a Patient with Ovarian Cancer
Authors: Basaglia G. , Carretto E. , Barbarini D. , Moras L. , Scalone S. , Marone P. , De Paoli P.
Published: July 2002

We report on the first case of a catheter-related recurrent bacteremia caused by
Kocuria kristinae
, a gram-positive microorganism belonging to the family
Micrococcaceae
, in a 51-year-old woman with ovarian cancer. This unusual pathogen may cause opportunistic infections in patients with severe underlying diseases.
Improving Sensitivity of Direct Microscopy for Detection of Acid-Fast Bacilli in Sputum: Use of Chitin in Mucus Digestion
Authors: Farnia P. , Mohammadi F. , Zarifi Z. , Tabatabee D. J. , Ganavi J. , Ghazisaeedi K. , Farnia P. K. , Gheydi M. , Bahadori M. , Masjedi M. R. , Velayati A. A.
Published: July 2002

In order to try to improve the results of direct smear microscopy, we used the mucus-digesting quality of chitin in tuberculosis (TB) laboratories. For this purpose, a total of 430 sputum specimens were processed by the
N
-acetyl-
l
-cysteine concentration, sodium hypochlorite (NaOCl) liquefaction, chitin sedimentation, and direct microscopy methods. Then, the smear sensitivity for acid-fast bacillus detection by chitin-treated sputum was compared with the sensitivity of smears prepared by other methods. Our results showed that the chitin solution took less time to completely homogenize the mucoid sputum than did the
N
-acetyl-
l
-cysteine and NaOCl methods. The
N
-acetyl-
l
-cysteine concentration method demonstrated sensitivity and specificity levels of 83 and 97%, respectively. In comparison, the sensitivity of chitin sedimentation was 80%, with a specificity of 96.7%. The NaOCl liquefaction method showed a sensitivity of 78%, with a specificity of 96%. Finally, the sensitivity of direct microscopy was lower than those of the other tested methods and was only 46%, with a specificity of 90%. The chitin and NaOCl liquefaction methods are both easy to perform, and they do not require additional equipment (centrifuges). Also, our results demonstrated that the chitin method is less time-consuming than the NaOCl method, since only 30 min of incubation is required to bring complete sedimentation of bacilli in chitin-treated sputum whereas the NaOCl method needs 10 to 12 h to give the same results in the same sputum specimens. Therefore, the chitin liquefaction and sedimentation method may provide better results in TB laboratories of developing countries than the
N
-acetyl-
l
-cysteine concentration, NaOCl overnight sedimentation, and direct smear microscopy methods.
Specific Identification of
Listeria welshimeri
and
Listeria monocytogenes
by PCR Assays Targeting a Gene Encoding a Fibronectin-Binding Protein
Authors: Gilot Philippe , Content Jean
Published: July 2002

We have cloned and sequenced a
Listeria welshimeri
DNA fragment homologous to the previously described fibronectin-binding protein-encoding gene (
fbp
) of
Listeria monocytogenes
(P. Gilot, Y. Jossin, and J. Content, J. Med. Microbiol., 49:887-896, 2000). This
L. welshimeri
DNA fragment expresses a 24.8-kDa protein that binds to human fibronectin. Based on the
fbp
sequences, we developed novel PCR assays for the identification of
L. welshimeri
and
L. monocytogenes
.
Periodontal Bacteria in Rabbit Mandibular and Maxillary Abscesses
Authors: Tyrrell Kerin L. , Citron Diane M. , Jenkins Jeffrey R. , Goldstein Ellie J. C.
Published: July 2002

Despite the high incidence of odontogenic abscesses in pet rabbits, published data on the bacteriology of these infections are lacking, and clinical cultures are often ambiguous, making antibiotic choices difficult. In order to define the bacteriology of these infections, 12 rabbit mandibular and maxillary abscesses were cultured aerobically and anaerobically. All specimens yielded pathogenic bacteria, including
Fusobacterium nucleatum
,
Prevotella heparinolytica
,
Prevotella
spp.,
Peptostreptococcus micros
,
Streptococcus milleri
group,
Actinomyces israelii
, and
Arcanobacterium haemolyticum
. These organisms are consistent with the characterized bacteriology of periodontal disease in human and other mammalian studies. The isolates were tested against 10 antimicrobial agents commonly used to treat rabbits; 100% of the strains tested were susceptible to clindamycin, 96% were susceptible to penicillin and ceftriaxone, 54% were susceptible to ciprofloxacin, and only 7% were susceptible to trimethoprim-sulfamethoxazole.
Molecular Typing of Selected
Enterococcus faecalis
Isolates: Pilot Study Using Multilocus Sequence Typing and Pulsed-Field Gel Electrophoresis
Authors: Nallapareddy Sreedhar R. , Duh Ruay-Wang , Singh Kavindra V. , Murray Barbara E.
Published: July 2002

The present study compared the recently developed multilocus sequence typing (MLST) approach with a well-established molecular typing technique, pulsed-field gel electrophoresis (PFGE), for subspecies differentiation of
Enterococcus faecalis
isolates. We sequenced intragenic regions of three
E. faecalis
antigen-encoding genes (
ace
, encoding a collagen and laminin adhesin;
efaA
, encoding an endocarditis antigen; and
salA
, encoding a cell wall associated antigen) and one housekeeping gene (
pyrC
) of 22
E. faecalis
isolates chosen largely for their temporal and geographical diversity, but also including some outbreak isolates. MLST analysis of polymorphic regions of these four genes identified 13 distinct sequence types (STs) with different allelic profiles; the composite sequences generated from the four sequenced gene fragments of individual isolates showed 98.3 to 100% identity among the 22 isolates. We also found that the allelic profiles from two sequences,
ace
and
salA
, were sufficient to distinguish all 13 STs of this study. The 13 STs corresponded to 12 different PFGE types, with one previously designated PFGE clone (a widespread U.S. clone of β-lactamase-producing isolates) being classified into two highly related STs which differed at 2 of 2,894 bases, both in the same allele. MLST also confirmed the clonal relationships among the isolates of two other PFGE clonal groups, including vancomycin resistant isolates. Thus, this pilot study with representative
E. faecalis
isolates suggests that, similar to PFGE, the sequence-based typing method may be useful for differentiating isolates of
E. faecalis
to the subspecies level in addition to identifying outbreak isolates.
Identification, Characterization, and Distribution of a Shiga Toxin 1 Gene Variant (
stx
1c
) in
Escherichia coli
Strains Isolated from Humans
Authors: Zhang Wenlan , Bielaszewska Martina , Kuczius Thorsten , Karch Helge
Published: July 2002

By using sequence analysis of Shiga toxin 1 (Stx 1) genes from human and ovine Stx-producing
Escherichia coli
(STEC) strains, we identified an Stx1 variant in STEC of human origin that was identical to the Stx1 variant from ovine STEC, but demonstrated only 97.1 and 96.6% amino acid sequence identity in its A and B subunits, respectively, to the Stx1 encoded by bacteriophage 933J. We designated this variant “Stx1c” and developed
stxB
1
restriction fragment length polymorphism and
stx
1c
-specific PCR strategies to determine the frequency and distribution of
stx
1c
among 212 STEC strains isolated from humans.
stx
1c
was identified in 36 (17.0%) of 212 STEC strains, 19 of which originated from asymptomatic subjects and 16 of which were from patients with uncomplicated diarrhea.
stx
1c
was most frequently (in 23 STEC strains [63.9%]) associated with
stx
2d
, but 12 (33.3%) of the 36 STEC strains possessed
stx
1c
only. A single STEC strain possessed
stx
1c
together with
stx
2
and was isolated from a patient with hemolytic-uremic syndrome. All 36
stx
1c
-positive STEC strains were
eae
negative and belonged to 10 different serogroups, none of which was O157, O26, O103, O111, or O145. Stx1c was produced by all
stx
1c
-containing STEC strains, but reacted weakly with a commercial immunoassay. We conclude that STEC strains harboring the
stx
1c
variant account for a significant proportion of human STEC isolates. The procedures developed in this study now allow the determination of the frequency of STEC strains harboring
stx
1c
among clinical STEC isolates and their association with human disease in prospective studies.
Evaluation of an Automated Sample Preparation Protocol for Quantitative Detection of Hepatitis C Virus RNA
Authors: Stelzl Evelyn , Kormann-Klement Andrea , Haas Josef , Daghofer Elisabeth , Santner Brigitte I. , Marth Egon , Kessler Harald H.
Published: July 2002

The COBAS AMPLIPREP instrument for automated sample preparation has recently been introduced. In this study, the COBAS AMPLIPREP/COBAS AMPLICOR HCV MONITOR test, which includes this new molecular device, was evaluated and compared to the COBAS AMPLICOR HCV MONITOR test, which includes a manual extraction protocol. Interassay and intra-assay variation, precision, and linearity were determined, and a total of 130 clinical specimens were investigated. For determination of interassay variation, coefficients of variation were found to be between 9 and 59% for the COBAS AMPLIPREP/COBAS AMPLICOR HCV MONITOR test and between 13 and 69% for the COBAS AMPLICOR HCV MONITOR test. For determination of intra-assay variation, coefficients of variation were found to be between 7 and 13% for the COBAS AMPLIPREP/COBAS AMPLICOR HCV MONITOR test and between 8 and 16% for the COBAS AMPLICOR HCV MONITOR test. When precision of the COBAS AMPLIPREP/COBAS AMPLICOR HCV MONITOR test was tested, all results were found to be within ±0.5 log of the expected results. Determination of linearity resulted in a quasilinear curve over 3 logs. When clinical samples were tested with the COBAS AMPLIPREP/COBAS AMPLICOR HCV MONITOR test and compared with the COBAS AMPLICOR HCV MONITOR test, all results were found within ±0.5 log. In conclusion, the assay, which included the new molecular device, proved to be suitable for the routine molecular laboratory. It was found to be laborsaving and easy to handle.
Suppression of Acute Viremia by Short-Term Postexposure Prophylaxis of Simian/Human Immunodeficiency Virus SHIV-RT-Infected Monkeys with a Novel Reverse Transcriptase Inhibitor (GW420867) Allows for Development of Potent Antiviral Immune Responses Resulting in Efficient Containment of Infection
Authors: Mori Kazuyasu , Yasutomi Yasuhiro , Sawada Shuzo , Villinger Francois , Sugama Kazushige , Rosenwith Brigitte , Heeney Jonathan L. , Überla Klaus , Yamazaki Shudo , Ansari Aftab A. , Rübsamen-Waigmann Helga
Published: July 2002

A nonnucleoside reverse transcriptase (RT) inhibitor, GW420867, was tested for postexposure prophylaxis (PEP) in rhesus macaques experimentally infected with 100 50% tissue culture infective doses of a chimeric simian/human immunodeficiency virus (SHIV) containing the RT gene of HIV-1 (SHIV-RT). Animals were either mock treated, or treated for 4 weeks starting at 8 or 24 h postinfection (p.i.) with GW420867. While such therapy led to undetectable plasma viremia in three of six monkeys, a transient plasma viremia was noted in the other three treated animals at 2 to 4 weeks following cessation of therapy. Following this transient viremia all drug-treated animals showed low or undetectable levels of plasma viremia up to the last sample examined at 90 weeks p.i. Despite low and/or undetectable viremia, virus-specific cytotoxic T lymphocyte and viral Env-specific proliferative responses were seen in the peripheral blood mononuclear cells of both mock- and drug-treated animals as early as 3 weeks p.i. Such virus-specific cellular responses, however, were better maintained in the drug-treated animals than the mock-treated animals. In contrast to the virus-specific cellular response, the magnitude and kinetics of virus specific humoral responses appeared to correlate with the detection of viremia. These data support the view that a short-term PEP with GW420867 permits the generation and maintenance of long-lasting virus-specific cell-mediated immune responses while markedly reducing viral loads to undetectable levels for a prolonged period of time (90 weeks) and leads to long-term disease protection. This model provides a unique means to define mechanisms and correlates of disease protection.
Depletion of Lymphocytes and Diminished Cytokine Production in Mice Infected with a Highly Virulent Influenza A (H5N1) Virus Isolated from Humans
Authors: Tumpey Terrence M. , Lu Xiuhua , Morken Timothy , Zaki Sherif R. , Katz Jacqueline M.
Published: July 2002

Previously, we observed that several virulent influenza A (H5N1) viruses which caused severe or fatal disease in humans were also lethal in BALB/c mice following dissemination of the virus to solid organs, including the brain. In contrast, one particular human H5N1 virus was nonlethal in mice and showed no evidence of systemic spread. To compare H5N1 viruses of varying pathogenicity for their ability to alter the mammalian immune system, mice were infected with either influenza A/Hong Kong/483/97 (HK/483) (lethal) or A/Hong Kong/486/97 (HK/486) (nonlethal) virus and monitored for lymphocyte depletion in the blood, lungs, and lymphoid tissue. Intranasal infection with HK/483 resulted in a significant decrease in the total number of circulating leukocytes evident as early as day 2 postinfection. Differential blood counts demonstrated up to an 80% drop in lymphocytes by day 4 postinfection. In contrast, nonlethal HK/486-infected mice displayed only a transient drop of lymphocytes during the infectious period. Analysis of lung and lymphoid tissue from HK/483-infected mice demonstrated a reduction in the number of CD4+and CD8+T cells and reduced synthesis of the cytokines interleukin-1β and gamma interferon and the chemokine macrophage inflammatory protein compared with HK/486-infected mice. In contrast, the cytokine and chemokine levels were increased in the brains of mice infected with HK/483 but not HK/486. Evidence of apoptosis in the spleen and lung of HK/483-infected mice was detected in situ, suggesting a mechanism for lymphocyte destruction. These results suggest that destructive effects on the immune system may be one factor that contributes to the pathogenesis of H5N1 viruses in mammalian hosts.
High-Efficiency Utilization of the Bovine Integrin α
v
β
3
as a Receptor for Foot-and-Mouth Disease Virus Is Dependent on the Bovine β
3
Subunit
Authors: Neff Sherry , Mason Peter W. , Baxt Barry
Published: July 2002

We have previously reported that
Foot-and-mouth disease virus
(FMDV), which is virulent for cattle and swine, can utilize the integrin α
v
β
3
as a receptor on cultured cells. Since those studies were performed with the human integrin, we have molecularly cloned the bovine homolog of the integrin α
v
β
3
and have compared the two receptors for utilization by FMDV. Both the α
v
and β
3
subunits of the bovine integrin have high degrees of amino acid sequence similarity to their corresponding human subunits in the ectodomains (96%) and essentially identical transmembrane and cytoplasmic domains. Within the putative ligand-binding domains, the bovine and human α
v
subunits have a 98.8% amino acid sequence similarity while there is only a 93% similarity between the β
3
subunits of these two species. COS cell cultures, which are not susceptible to FMDV infection, become susceptible if cotransfected with α
v
and β
3
subunit cDNAs from a bovine or human source. Cultures cotransfected with the bovine α
v
β
3
subunit cDNAs and infected with FMDV synthesize greater amounts of viral proteins than do infected cultures cotransfected with the human integrin subunits. Cells cotransfected with a bovine α
v
subunit and a human β
3
subunit synthesize viral proteins at levels equivalent to those in cells expressing both human subunits. However, cells cotransfected with the human α
v
and the bovine β
3
subunits synthesize amounts of viral proteins equivalent to those in cells expressing both bovine subunits, indicating that the bovine β
3
subunit is responsible for the increased effectiveness of this receptor. By engineering chimeric bovine-human β
3
subunits, we have shown that this increase in receptor efficiency is due to sequences encoding the C-terminal one-third of the subunit ectodomain, which contains a highly structured cysteine-rich repeat region. We postulate that amino acid sequence differences within this region may be responsible for structural differences between the human and bovine β
3
subunit, leading to more efficient utilization of the bovine receptor by this bovine pathogen.