bluuebunny/crossref_metadata_2025 huggingface dataset:
Performance of the ImmunoCard STAT! E. coli O157:H7 Test for Detection of
Escherichia coli
O157:H7 in Stools
Authors: Mackenzie Andrew , Orrbine Elaine , Hyde Lucie , Benoit Michelle , Chan Frank , Park Choong , Alverson Jessica , Lembke Amy , Hoban Daryl , Kennedy William
Published: December 2019

ImmunoCard STAT! E. coli O157:H7 (Meridian Diagnostics, Inc., Cincinnati, Ohio) is a novel rapid (10-min) test for the presence of
Escherichia coli
O157:H7 in stools. The test may be performed either directly on stool specimens or on an overnight broth culture of stool. In a multicenter prospective study, 14 of 14 specimens positive by culture for
E. coli
O157:H7 were positive by the ImmunoCard STAT! O157:H7 test, and there were no false positives from 263 culture-negative specimens. In a retrospective study, the test was positive in 339 (81%) of 417 stored culture-positive specimens and the specificity was 95% (98 of 103 specimens). No false positives were associated with alternate stool pathogens. The ImmunoCard STAT! O157:H7 test has high sensitivity and specificity.
Detection of Human Cytomegalovirus pp67 Late Gene Transcripts in Cerebrospinal Fluid of Human Immunodeficiency Virus Type 1-Infected Patients by Nucleic Acid Sequence-Based Amplification
Authors: Zhang Fan , Tetali Surya , Wang Xue Ping , Kaplan Mark H. , Cromme Frans V. , Ginocchio Christine C.
Published: December 2019

This study examined the clinical correlation between the presence of human cytomegalovirus (HCMV) pp67 mRNA in cerebrospinal fluid (CSF) and active HCMV central nervous system (CNS) disease in patients with human immunodeficiency virus type 1 (HIV-1). In total, 76 CSF specimens collected from 65 HIV-1-positive patients diagnosed with HCMV CNS disease, other non-HCMV-related CNS diseases, or no CNS disease were tested for the presence of HCMV pp67 mRNA using the NucliSens cytomegalovirus (CMV) pp67 assay (Organon Teknika, Durham, N.C.). The results were compared to those of a nested PCR for the detection of HCMV glycoprotein B DNA and to those obtained by viral culture (54 samples). CSF specimens collected from patients without HCMV CNS disease yielded the following results: pp67 assay negative, 62 of 62 specimens; culture negative, 41 of 41 specimens; and PCR negative, 56 of 62 specimens (6 specimens were positive). CSF specimens collected from patients with HCMV CNS disease yielded the following results: pp67 assay positive, 9 of 13 specimens; PCR positive, 13 of 13 specimens; and culture positive, 2 of 13 specimens. After resolution of the discordant results, the following positive and negative predictive values (PPV and NPV, respectively) for the diagnosis of HCMV CNS disease were determined. The PPV for PCR, pp67 assay, and culture were 68.4, 100, and 100%, respectively, and the NPV for PCR, pp67 assay, and culture were 100, 97.0, and 82.7%, respectively. The sensitivities for DNA PCR, pp67 assay, and culture for the detection of HCMV were 100, 84.6, and 18%, respectively, and the clinical specificities were 90.5, 100, and 100%, respectively. This study indicates that the detection of HCMV pp67 mRNA in CSF has good correlation with active HCMV CNS disease, whereas CSF culture is insensitive and qualitative DNA PCR may detect latent nonreplicating virus in CSF from patients without HCMV CNS disease.
Quantitation of Cell-Free and Cell-Associated Kaposi's Sarcoma-Associated Herpesvirus DNA by Real-Time PCR
Authors: White Irene E. , Campbell Thomas B.
Published: December 2019

A real-time PCR assay for quantitation of Kaposi's sarcoma-associated herpes virus (KSHV or human herpesvirus 8) DNA was evaluated. The linear dynamic range was 10 to 10
5
copies of KSHV DNA (
r
2
> 0.99). The accuracy of DNA purification and quantitation was less than ±0.4 log
10
copies for samples that contained from 10 to 10
5
copies of KSHV DNA. Cell-associated KSHV DNA was quantitated over a range of infected cell frequencies from 0.1 to 10
−5
, and cell-free KSHV DNA in plasma was quantitated over a range of 100 to 10
6
copies/ml. Real-time PCR provides a convenient method for quantitation of cell-free and cell-associated KSHV DNA in laboratory and clinical specimens.
Direct PCR of
Cryptococcus neoformans MAT
α and
MAT
a
Pheromones To Determine Mating Type, Ploidy, and Variety: a Tool for Epidemiological and Molecular Pathogenesis Studies
Authors: Chaturvedi Sudha , Rodeghier Birgit , Fan Jinjiang , McClelland Carol M. , Wickes Brian L. , Chaturvedi Vishnu
Published: December 2019

Cryptococcus neoformans MAT
α and
MAT
a pheromones were amplified by direct PCR. Nucleotide sequence analyses revealed unique restriction enzyme sites. Sixty strains were used to devise a restriction fragment length polymorphism typing scheme that yielded three variety-specific patterns. Additionally, pheromone-specific PCR allowed easier identification of diploid
C. neoformans
strains than flow cytometry.
Recombinant Chimeric Borrelia Proteins for Diagnosis of Lyme Disease
Authors: Gomes-Solecki Maria J. C. , Dunn John J. , Luft Benjamin J. , Castillo Jonathan , Dykhuizen Daniel E. , Yang Xiaohua , Glass John D. , Dattwyler Raymond J.
Published: December 2019

Current serologic Lyme disease tests use whole borrelia cells as the source of antigen. These assays are difficult to standardize and to optimize for sensitivity and specificity. To help solve these problems, we constructed a library of recombinant chimeric proteins composed of portions of key antigens ofBorrelia burgdorferi. These proteins were then used to develop an enzyme-linked immunosorbent assay. We compared our assay with the most sensitive of three whole-cell borrelia assays. We found that the recombinant assay could detect antibodies significantly better from early Lyme disease sera (P< 0.05), and had the same sensitivity for late Lyme disease sera, as the most sensitive whole-cell borrelia assay. On potentially cross-reactive sera, the recombinant assay was more specific, but not significantly so, than the best whole-cell borrelia assay. Optimization of the recombinant assay offers the potential for a significant improvement in both sensitivity and specificity.
A Simple Restriction Fragment Length Polymorphism-Based Strategy That Can Distinguish the Internal Genes of Human H1N1, H3N2, and H5N1 Influenza A Viruses
Authors: Cooper Lynn A. , Subbarao Kanta
Published: December 2019

A simple molecular technique for rapid genotyping was developed to monitor the internal gene composition of currently circulating influenza A viruses. Sequence information from recent H1N1, H3N2, and H5N1 human virus isolates was used to identify conserved regions within each internal gene, and gene-specific PCR primers capable of amplifying all three virus subtypes were designed. Subtyping was based on subtype-specific restriction fragment length polymorphism (RFLP) patterns within the amplified regions. The strategy was tested in a blinded fashion using 10 control viruses of each subtype (total, 30) and was found to be very effective. Once standardized, the genotyping method was used to identify the origin of the internal genes of 51 influenza A viruses isolated from humans in Hong Kong during and immediately following the 1997–1998 H5N1 outbreak. No avian-human or H1-H3 reassortants were detected. Less than 2% (6 of 486) of the RFLP analyses were inconclusive; all were due to point mutations within a restriction site. The technique was also used to characterize the internal genes of two avian H9N2 viruses isolated from children in Hong Kong during 1999.
Characterization of a Toxin A-Negative, Toxin B-Positive Strain of
Clostridium difficile
Responsible for a Nosocomial Outbreak of
Clostridium difficile
-Associated Diarrhea
Authors: Alfa Michelle J. , Kabani Amin , Lyerly David , Moncrief Scott , Neville Laurie M. , Al-Barrak Ali , Harding Godfrey K. H. , Dyck Brenda , Olekson Karen , Embil John M.
Published: December 2019

Clostridium difficile
-associated diarrhea (CAD) is a very common nosocomial infection that contributes significantly to patient morbidity and mortality as well as to the cost of hospitalization. Previously, strains of toxin A-negative, toxin B-positive
C. difficile
were not thought to be associated with clinically significant disease. This study reports the characterization of a toxin A-negative, toxin B-positive strain of
C. difficile
that was responsible for a recently described nosocomial outbreak of CAD. Analysis of the seven patient isolates from the outbreak by pulsed-field gel electrophoresis indicated that this outbreak was due to transmission of a single strain of
C. difficile
. Our characterization of this strain (HSC98) has demonstrated that the toxin A gene lacks 1.8 kb from the carboxy repetitive oligopeptide (CROP) region but apparently has no other major deletions from other regions of the toxin A or toxin B gene. The remaining 1.3-kb fragment of the toxin A CROP region from strain HSC98 showed 98% sequence homology with strain 1470, previously reported by M. Weidmann in 1997 (GenBank accession number
Y12616
), suggesting that HSC98 is toxinotype VIII. The HSC98 strain infecting patients involved in this outbreak produced the full spectrum of clinical illness usually associated with
C. difficile
-associated disease. This pathogenic spectrum was manifest despite the inability of this strain to alter tight junctions as determined by using in vitro tissue culture testing, which suggested that no functional toxin A was produced by this strain.
Rapid Identification of
Candida dubliniensis
Using a Species-Specific Molecular Beacon
Authors: Park Steven , Wong May , Marras Salvatore A. E. , Cross Emily W. , Kiehn Timothy E. , Chaturvedi Vishnu , Tyagi Sanjay , Perlin David S.
Published: December 2019

Candida dubliniensis
is an opportunistic fungal pathogen that has been linked to oral candidiasis in AIDS patients, although it has recently been isolated from other body sites. DNA sequence analysis of the internal transcribed spacer 2 (ITS2) region of rRNA genes from reference
Candida
strains was used to develop molecular beacon probes for rapid, high-fidelity identification of
C. dubliniensis
as well as
C. albicans
. Molecular beacons are small nucleic acid hairpin probes that brightly fluoresce when they are bound to their targets and have a significant advantage over conventional nucleic acid probes because they exhibit a higher degree of specificity with better signal-to-noise ratios. When applied to an unknown collection of 23 strains that largely contained
C. albicans
and a smaller amount of
C. dubliniensis
, the species-specific probes were 100% accurate in identifying both species following PCR amplification of the ITS2 region. The results obtained with the molecular beacons were independently verified by random amplified polymorphic DNA analysis-based genotyping and by restriction enzyme analysis with enzymes
Bsm
AI and
Nsp
BII, which cleave recognition sequences within the ITS2 regions of
C. dubliniensis
and
C. albicans
, respectively. Molecular beacons are promising new probes for the rapid detection of
Candida
species.
Detection of Complement-Mediated Antibody-Dependent Bactericidal Activity in a Fluorescence-Based Serum Bactericidal Assay for Group BNeisseria meningitidis
Authors: Mountzouros Kenneth T. , Howell Alan P.
Published: December 2019

Serum bactericidal assays (SBAs) for Group B meningococci are considered the methods of choice for the evaluation of functional antimeningococcal antibodies. Many investigators regard SBAs as time- and labor-intensive. Variations in SBA protocols among different laboratories make interpretation of results difficult. Here we describe a fluorescence-based serum bactericidal assay (fSBA) and compare the results obtained with the fSBA to the results obtained with a more conventional SBA. The results generated by both assays were dependent upon the surviving bacteria after incubation, and the assay mixtures contained identical components. Differences between assays lie in how the surviving bacteria are quantified. The fSBA described in the paper uses the fluorescent dye alamarBlue (M. V. Lancaster and R. D. Fields, U.S. patent 5501959, March 1996). The fluorescent signals generated in the fSBA correlate to the oxidative respiration of surviving bacteria. Viable bacteria were detected between 6 and 8 h directly from reaction mixtures in 96-well plates by the fSBA, whereas colonies isolated on semisolid media could be counted after 24 h of incubation. The bactericidal titers generated by both assays were nearly identical. The fSBA described here can be used as an assay for the screening of large quantities of individual sera as complement sources or as a method for the detection of functional antibodies directed against group BNeisseria meningitidisin both human and mouse antisera.
Application of a Reverse Transcription-PCR for Identification and Differentiation of Aichi Virus, a New Member of the Picornavirus Family Associated with Gastroenteritis in Humans
Authors: Yamashita T. , Sugiyama M. , Tsuzuki H. , Sakae K. , Suzuki Y. , Miyazaki Y.
Published: December 2019

Aichi viruses isolated in Vero cells from seven patients in five gastroenteritis outbreaks in Japan, five Japanese returning from Southeast Asian countries, and five local children in Pakistan with gastroenteritis were examined for differentiation based on their reactivities with a monoclonal antibody to a standard strain (A846/88) and a reverse transcription-PCR (RT-PCR) of three genomic regions. The RNA sequences were determined for 519 bases of these 17 isolates at the putative junction between the C terminus of 3C and the N terminus of 3D. The analyses revealed an approximately 90% homology between these isolates, which were then divided into two groups: group 1 (genotype A) included six isolates from four outbreaks and one isolate from a traveler and group 2 (genotype B) included one isolate from the other outbreak, four isolates from returning travelers, and all of the isolates from the Pakistani children. Based on the isolate sequences, a primer pair and a biotin-labeled probe were designed for amplification and detection of 223 bases at the 3C-3D junction of Aichi virus RNA in fecal specimens. The Aichi virus RNA was detected in 54 (55%) of 99 fecal specimens from the patients in 12 (32%) of 37 outbreaks of gastroenteritis in Japan. Of the 12 outbreaks, 11 were suspected to be due to genotype A. These results indicated that RT-PCR can be a useful tool to detect Aichi virus in stool samples and that a sequence analysis of PCR products can be employed to identify the prevalent strain in each incident.
Identification of Members of the
Burkholderia cepacia
Complex by Species-Specific PCR
Authors: Whitby Paul W. , Carter Karen B. , Hatter Kenneth L. , LiPuma John J. , Stull Terrence L.
Published: December 2019

Definitive identification of the species in the
Burkholderia cepacia
complex by routine clinical microbiology methods is difficult. Phenotypic tests to identify
B. multivorans
and
B. vietnamiensis
have been established; more recent work indicates
B. stabilis
may also be identified by growth characteristics and biochemical tests. However, attempts to identify genomovars I and III have, thus far, proved unsuccessful. Previously, we demonstrated the utility of two primer pairs, directed to the rRNA operon, to specifically identify the
B. cepacia
complex in a PCR. One of these primer pairs, G1-G2, only amplified a DNA fragment from genomovars I and III and
B. stabilis
in a PCR with genomic DNA isolated from prototypical strains representing the five genomovars. Sequence analysis of the rRNA operon for all the genomovars indicated that this primer pair targeted a region shared by these isolates. Further analysis revealed a region of heterogeneity between genomovar III and
B. stabilis
internal to the amplified product of G1-G2. Primers designed to target this region were tested with prototypical strains following an initial amplification with the G1-G2 primer pair. New primers specific for the prototypical genomovar III and
B. stabilis
were designated SPR3 and SPR4, respectively. Analysis of 93 isolates representing 18 genomovar I, 13
B. multivorans
, 36 genomovar III, 11
B. stabilis
, and 15
B. vietnamiensis
isolates was performed. DNA from all isolates of genomovars I and III and
B. stabilis
was amplified by G1-G2. Genomovar III isolates yielded a product with SPR3/G1 while
B. stabilis
amplified with SPR4-G1. Genomovar I isolates were amplified by either SPR3-G1 or SPR4-G1, but not both.
B. multivorans
yielded a product with SPR3-G1 but not G1-G2, and
B. vietnamiensis
isolates were negative in all PCRs. Thus using an algorithm with G1-G2, SPR3-G1, and SPR4-G1 primers in a PCR analysis, genomovar III isolates can be separated from
B. stabilis
and the identity of
B. multivorans
and
B. vietnamiensis
can be confirmed.
Molecular Epidemiology of an Outbreak of Fulminant Hepatitis B
Authors: Petrosillo Nicola , Ippolito Giuseppe , Solforosi Laura , Varaldo Pietro E. , Clementi Massimo , Manzin Aldo
Published: December 2019

A nosocomial outbreak of hepatitis B occurred among the inpatients of a hematology unit. Nine of the 11 infected patients died from fulminant hepatitis. An investigation was conducted to identify the source of infection and the route of transmission. Two clusters of nosocomial hepatitis B were identified. The hepatitis B virus (HBV) genome from serum samples of all case patients, of one HBsAg-positive patient with acute reactivation of the infection, and of eight acutely infected, unrelated cases was identified by PCR amplification of viral DNA and was entirely sequenced. Transmission was probably associated with breaks in infection control practices, which occurred as single events from common sources or through a patient-to-patient route, likely the result of shared medications or supplies. Sequence analysis evidenced close homology among the strains from the case patients and that from the patient with reactivation, who was the likely source of infection. Molecular analysis of viral isolates evidenced an accumulation of mutations in the core promoter/precore region, as well as several nucleotide substitutions throughout the genome. The sequences of all patients were compared with published sequences from fulminant and nonfulminant HBV infections.
Genetic and Serologic Evaluation of Capsule Production by Bovine Mammary Isolates ofStaphylococcus aureusand OtherStaphylococcusspp. from Europe and the United States
Authors: Tollersrud T. , Kenny K. , Reitz A. J. , Lee J. C.
Published: December 2019

Bovine mastitis caused byStaphylococcus aureusis responsible for major economic losses to the dairy industry, and more-effective therapeutic or preventive approaches are sorely needed. The predominance of staphylococcal capsular polysaccharide types 5 and 8 among human isolates from many sources is well documented, but there seems to be a greater variation in the distribution of capsular serotypes among isolates from cows. A total of 636 isolates ofS. aureusfrom cases of bovine mastitis in Sweden, Denmark, Finland, Iceland, Ireland, and the United States were investigated for production of capsular polysaccharide types 5 and 8. Approximately half of all the European isolates tested were of serotype 8, although variation among countries and among isolates of clinical and subclinical origin was observed. Sweden had the highest frequency (87%) of serotypeable isolates, and Finland had the lowest (48%). Capsule types 5 and 8 accounted for only 42% of the U.S. isolates tested. A few isolates showed weak reactivity with CP5 antiserum in a colony blot assay, and an enzyme-linked immunosorbent assay inhibition method confirmed that the levels of capsule produced by these strains were <10% of those produced by control strains. Fifty isolates that failed to react with capsular antisera all possessed the genes for production of capsular polysaccharide type 5 or 8. These results underscore the variability in capsule production by bovine isolates ofS. aureusfrom different geographic regions. This information is important for the rational design of a capsule-based vaccine to preventS. aureusbovine mastitis.
Genetic Diversity, Distribution, and Serological Features of Hantavirus Infection in Five Countries in South America
Authors: Padula P. J. , Colavecchia S. B. , Martínez V. P. , Gonzalez Della Valle M. O. , Edelstein A. , Miguel S. D. L. , Russi J. , Riquelme J. Mora , Colucci N. , Almirón M. , Rabinovich R. D.
Published: December 2019

Since 1995 when the first case of hantavirus pulmonary syndrome (HPS) was reported in Patagonia, there have been more than 400 cases of HPS reported in five countries in South America. The first case of HPS was associated with Andes (AND) virus. In this study, we report on the genetic diversity, geographical distribution, and serological features of hantavirus infection in six countries in South America based on 87 HPS cases from Argentina, Bolivia, Chile, Paraguay, and Uruguay. An early immunoglobulin M (IgM), IgA, and IgG humoral response was observed in almost all HPS cases. The IgM response appears to peak 1 or 2 days after the onset of symptoms. Peak IgG antibody titers occur mostly after the first week. Low IgG titers or the absence of IgG was associated with higher mortality rates. The IgA response peaks around day 15 and then rapidly decreases. The results of phylogenetic analysis based on partial M-fragment G1- and G2-encoding sequences showed that HPS cases from the five countries were infected with viruses related to AND or Laguna Negra (LN) virus. Within AND virus-infected persons, at least five major genetic lineages were found; one lineage was detected in Uruguayan and Argentinean cases from both sides of the Rio de la Plata river. Two Paraguayan patients were infected with a virus different from LN virus. According to the results of phylogenetic analyses, this virus probably belongs to a distinct lineage related more closely to the AND virus than to the LN virus, suggesting that there is probably an
Oligoryzomys
-borne viral variant circulating in Paraguay. These studies may contribute to a better understanding of hantavirus human infection in South America.
Borna Disease in a Free-Ranging Lynx (
Lynx lynx
)
Authors: Degiorgis M.-P. , Berg A.-L. , Hård af Segerstad C. , Mörner T. , Johansson M. , Berg M.
Published: December 2019

A free-ranging lynx (
Lynx lynx
) was shot because of its abnormal behavior. Histopathological examination revealed a nonsuppurative meningoencephalitis. In situ hybridization, immunohistochemistry, and reverse transcriptase PCR analysis showed the presence of Borna disease virus infection in the brain. To our knowledge, this is the first confirmed case of Borna disease in a large felid.
Detection of Rabies Virus Antigen in Dog Saliva Using a Latex Agglutination Test
Authors: Kasempimolporn S. , Saengseesom W. , Lumlertdacha B. , Sitprija V.
Published: December 2019

Dog bites are responsible for more than 90% of human rabies deaths in Asia. We developed a simple and inexpensive test based on latex agglutination (LA) for rabies virus antigen detection in dog saliva. Rabies virus antigen could be detected by agglutination on a glass slide using latex particles coated with gamma globulin. By evaluation of paired saliva-brain specimens from 238 dogs, the LA test using saliva was 99% specific and 95% sensitive compared to the fluorescent antibody test (FAT) on brain smears. The advantages of the LA test over the standard FAT are that it is comparatively simple and there is no need to kill the animal before examination.
Molecular Identification ofParacoccidioides brasiliensisby PCR Amplification of Ribosomal DNA
Authors: Motoyama Andrea B. , Venancio Emerson J. , Brandão Gilberto O. , Petrofeza-Silva Silvana , Pereira Ildinete S. , Soares Celia M. A. , Felipe Maria Sueli S.
Published: December 2019

We have amplified and sequenced the 5.8S and 28S ribosomal DNA genes and intergenic regions ofParacoccidioides brasiliensis, strain Pb01. Using primers specifically designed for both ribosomal DNA regions, we were able to discriminate betweenP. brasiliensisand other human pathogenic fungi by PCR. The use of this molecular marker could be important for paracoccidiodomycosis diagnosis and ecological and molecular epidemiological studies ofP. brasiliensisin Latin America.
Human Infection with
Halomonas venusta
following Fish Bite
Authors: von Graevenitz Alexander , Bowman John , Del Notaro Carlo , Ritzler Michael
Published: December 2019

Halomonas venusta
, a moderately halophilic, nonfermentative gram-negative rod, is reported for the first time as a human pathogen in a wound that originated from a fish bite.
Role of IS6110-Targeted PCR, Culture, Biochemical, Clinical, and Immunological Criteria for Diagnosis of Tuberculous Meningitis
Authors: Caws M. , Wilson S. M. , Clough C. , Drobniewski F.
Published: December 2019

An open prospective clinical, microbiological, and molecular analysis of a national molecular diagnostic service for tuberculous meningitis (TBM) using an in-house IS6110-targeted PCR for molecular “Fastrack” diagnosis was carried out. Between April 1997 and June 1998. Consecutive cerebrospinal fluid (CSF) samples from 131 patients were assessed. Against a culture on the same sample, PCR had a sensitivity of 75% and a specificity of 94%. Of samples from patients classified as definite or probable TBM cases based on clinical criteria, 81% had raised CSF protein levels and 73% had a lymphocytosis, although 57% of all submitted samples showed a raised lymphocyte count. While only 46% had a CSF glucose level below the normal range, the CSF glucose level was significantly lower (P = 0.0281) than in cases of meningitis of other etiologies. Levels of tumor necrosis factor alpha were also found to be significantly raised in definite or probable TBM cases (P = 0.028), while adenosine deaminase levels were not. The study showed IS6110-targeted PCR to be a rapid, sensitive, and specific test in routine use for the diagnosis of TBM.
Comparative Fingerprinting Analysis ofCampylobacter jejuni subsp. jejuni Strains by Amplified-Fragment Length Polymorphism Genotyping
Authors: Lindstedt Bjørn-Arne , Heir Even , Vardund Traute , Melby Kjetil K. , Kapperud Georg
Published: December 2019

Amplified-fragment length polymorphism (AFLP) analysis with the endonucleases BglII and MfeI was used to genotype 91 Campylobacter jejuni subsp. jejunistrains from outbreaks and sporadic cases. AFLP-generated fragments were labeled with fluorescent dye and separated by capillary electrophoresis. The software packages GeneScan and GelCompar II were used to calculate AFLP pattern similarities and to investigate phylogenetic relationships among the genotyped strains. The AFLP method was compared with two additional DNA-based typing methods, pulsed-field gel electrophoresis (PFGE) using SmaI and restriction fragment length polymorphism analysis on PCR products (PCR-RFLP) of theflaA and flaB genes. We found that AFLP analysis of C. jejuni strains is a rapid method that offers better discriminatory power than do both PFGE and PCR-RFLP. AFLP and, to a lesser extent, PCR-RFLP could differentiate strains within the same PFGE profiles, which also makes PCR-RFLP an alternative to PFGE. We were able to clearly distinguish 9 of 10 recognized outbreaks by AFLP and to identify similarities among outbreak and sporadic strains. Therefore, AFLP is suitable for epidemiological surveillance ofC. jejuni and will be an excellent tool for source identification in outbreak situations.
Differences in Surface-Exposed Antigen Expression between Helicobacter pylori Strains Isolated from Duodenal Ulcer Patients and from Asymptomatic Subjects
Authors: Thoreson Ann-Catrin E. , Hamlet Annika , Çelik Janet , Byström Mona , Nyström Susanne , Olbe Lars , Svennerholm Ann-Mari
Published: December 2019

We have analyzed possible qualitative and quantitative differences in antigen expression between Helicobacter pylori strains isolated from the antrum and different locations in the duodenum of 21 duodenal ulcer (DU) patients and 20 asymptomatic subjects (AS) by enzyme-linked immunosorbent assay (ELISA) and inhibition ELISA. Almost all antral and duodenal strains grown in vitro expressed theN-acetyl-neuroaminyllactose-binding hemagglutinin, flagellins (subunits FlaA and FlaB), urease, a 26-kDa protein, and a neutrophil-activating protein. In 75% of both the DU patients and the AS, antral H. pylori strains expressed either the blood group antigen Lewis y (Ley) alone or together with the Lex antigen. However, duodenal H. pyloristrains of DU patients expressed Ley antigen more frequently than corresponding strains of AS (P < 0.05). Presence of Ley on H. pylori was related to the degree of active duodenitis (P < 0.05). Duodenal H. pylori strains isolated from AS were significantly more often Lewis nontypeable than duodenal strains of DU patients (P < 0.01). Presence of H. pylori blood group antigen-binding adhesin (BabA) was significantly higher on both antral and duodenal strains isolated from DU patients than on corresponding strains isolated from AS (P < 0.05). BabA-positive duodenal H. pylori strains isolated from DU patients were associated with active duodenitis more frequently than corresponding strains isolated from AS (P < 0.01). Infection with H. pylori strains positive for Ley and BabA in the duodenum is associated with development of duodenal ulcer formation.
Seroepidemiological Survey of Infection by Feline Leukemia Virus and Immunodeficiency Virus in Madrid and Correlation with Some Clinical Aspects
Authors: Arjona Alvaro , Escolar Elena , Soto Iria , Barquero Nuria , Martin Daniel , Gomez-Lucia Esperanza
Published: December 2019

A study of 180 healthy cats found that 15.6% were feline leukemia virus (FeLV) positive, 8.3% were feline immunodeficiency virus (FIV) positive, and 1.1% were FIV and FeLV positive, which corresponded to 30.4, 13.8, and 2.6, of 115 cats with FIV- and FeLV-related symptoms, respectively. Differences were seen in the sexes and ages of the populations studied. Anemia, leukopenia, and lymphopenia were the most frequent hematological abnormalities in infected cats.
Quality Control Limits for Broth Microdilution Susceptibility Tests of Ten Antifungal Agents
Authors: Barry A. L. , Pfaller M. A. , Brown S. D. , Espinel-Ingroff A. , Ghannoum M. A. , Knapp C. , Rennie R. P. , Rex J. H. , Rinaldi M. G.
Published: December 2019

Broth microdilution susceptibility tests of Candidaspecies have now been standardized by the National Committee for Clinical Laboratory Standards (NCCLS). An eight-laboratory collaborative study was carried out in order to document reproducibility of tests of Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258 by the NCCLS method. Replicate broth microdilution tests were used to define control limits for 24- and 48-h MICs of amphotericin B, flucytosine, fluconazole, voriconazole, ketoconazole, itraconazole, caspofungin (MK 0991), ravuconazole (BMS 207147), posaconazole (SCH 56592), and LY 303366.
Fecal Excretion of Salmonella entericaSerovar Typhimurium Following a Food-Borne Outbreak
Authors: Murase Toshiyuki , Yamada Mikiko , Muto Tetsunori , Matsushima Akiyoshi , Yamai Shiro
Published: December 2019

Fecal excretion of Salmonella enterica serovar Typhimurium organisms was observed in patients and in people not showing symptoms who were involved in an outbreak of food-borne infection with this organism. Excretion of organisms was prolonged in the patients who were given antimicrobial drugs compared with those who were not. The isolates were indistinguishable by their pulsed-field gel electrophoresis patterns and biotyping from the strain recovered from the roast pork that had been consumed by all of the people. This indicates that these isolates obtained from the infected people had originated in the contaminated pork.
Genetic Classification of “Rickettsia heilongjiangii” and “Rickettsia hulinii,” Two Chinese Spotted Fever Group Rickettsiae
Authors: Zhang J. Z. , Fan M. Y. , Wu Y. M. , Fournier P. E. , Roux V. , Raoult D.
Published: December 2019

To determine the phylogenetic position of two new rickettsial strains isolated from ticks in China, 16S ribosomal DNA,gltA, and ompA (apart from the tandem repeat units) genes were amplified by PCR and sequenced. The phylogenetic relationships between these strains and other rickettsiae were inferred from the comparison of sequences of the three genes by the parsimony, neighbor-joining, and maximum-likelihood methods. The results demonstrated that the 054 strain, a rickettsia pathogenic in humans, and the HL-93 strain were related and clustered together withRickettsia japonica. Significant statistical bootstrap values (100 and 92%) supported the nodes in this cluster. Based on previous genotypic and antigenic data and the phylogenetic analysis presented here, the 054 and HL-93 strains should be considered as new species, and we formally propose that they be named “Rickettsia heilongjiangii” and “Rickettsia hulinii,” respectively.
Comparison of Rapid Centrifugation Assay with Conventional Tissue Culture Method for Isolation of Dengue 2 Virus in C6/36-HT Cells
Authors: Roche Rosmari Rodríguez , Alvarez Mayling , Guzmán María G. , Morier Luis , Kourí Gustavo
Published: December 2019

A rapid centrifugation assay was compared with conventional tube cell culture for dengue virus isolation in both sera and autopsy samples from dengue and dengue hemorrhagic fever/dengue shock syndrome fatal cases. The rapid centrifugation assay allowed isolation of virus from 16.6% more samples than the conventional method, and it shortened the time for dengue virus detection. Finally, it allowed the isolation of dengue 2 virus in 42.8% of tissue samples from five fatal cases. Our results suggest that the rapid centrifugation assay may be useful for detection of dengue virus in clinical specimens.
A Functional Antigenomic Promoter for the Paramyxovirus Simian Virus 5 Requires Proper Spacing between an Essential Internal Segment and the 3′ Terminus
Authors: Murphy Susan K. , Ito Yasuhiko , Parks Griffith D.
Published: December 2019

A previous analysis of naturally occurring defective interfering (DI) RNA genomes of the prototypic paramyxovirus simian virus 5 (SV5) indicated that 113 bases at the 3′ terminus of the antigenome were sufficient to direct RNA encapsidation and replication. A nucleotide sequence alignment of the antigenomic 3′-terminal 113 bases of members of the
Rubulavirus
genus of the
Paramyxoviridae
family identified two regions of sequence identity: bases 1 to 19 at the 3′ terminus (conserved region I [CRI]) and a more distal region consisting of antigenome bases 73 to 90 (CRII) that was contained within the 3′ coding region of the L protein gene. To determine whether these regions of the antigenome were essential for SV5 RNA replication, a reverse genetics system was used to analyze the replication of copyback DI RNA analogs that contained a foreign gene (GL, encoding green fluorescence protein) flanked by 113 5′-terminal bases and various amounts of SV5 3′-terminal antigenomic sequences. Results from a deletion analysis showed that efficient encapsidation and replication of SV5-GL DI RNA analogs occurred when the 90 3′-terminal bases of the SV5 antigenomic RNA were retained, but replication was reduced ∼5- to 14-fold in the case of truncated antigenomes that lacked the 3′-end CRII sequences. A chimeric copyback DI RNA containing the 3′-terminal 98 bases including the CRI and CRII sequences from the human parainfluenza virus type 2 (HPIV2) antigenome in place of the corresponding SV5 sequences was efficiently replicated by SV5 cDNA-derived components. However, replication was reduced ∼20-fold for a truncated SV5-HPIV2 chimeric RNA that lacked the HPIV2 CRII sequences between antigenome bases 72 and 90. Progressive deletions of 6 to 18 bases in the region located between the SV5 antigenomic CRI and CRII segments (3′-end nucleotides 21 to 38) resulted in a ∼25-fold decrease in SV5-GL RNA synthesis. Surprisingly, replication was restored to wild-type levels when these length alterations between CRI and CRII were corrected by replacing the deleted bases with nonviral sequences. Together, these data suggest that a functional SV5 antigenomic promoter requires proper spacing between an essential internal region and the 3′ terminus. A model is presented for the structure of the 3′ end of the SV5 antigenome which proposes that positioning of CRI and CRII along the same face of the helical nucleocapsid is an essential feature of a functional antigenomic promoter.
Reactivation of the Previously Silenced Cytomegalovirus Major Immediate-Early Promoter in the Mouse Liver: Involvement of NFκB
Authors: Löser Peter , Jennings Gary S. , Strauss Michael , Sandig Volker
Published: December 2019

The cytomegalovirus (CMV) major immediate-early promoter/enhancer is active in many cell culture systems and is considered to be one of the strongest promoters in vitro. However, when this promoter was used in in vivo approaches to gene therapy, it was silenced within a few weeks in several organs including the liver. In this study, we demonstrated transcriptional inactivation of the CMV promoter in mouse liver. In contrast to the CMV promoter, a hybrid promoter consisting of a minimal CMV promoter and the enhancer II of hepatitis B virus was active for at least 11 weeks in mouse liver. While investigating the reason for the shutdown of the CMV promoter, we did not find evidence for methylation of adenovirus DNA in the region of transgene insertion, but we could show that the silenced CMV promoter was reactivated after lipopolysaccharide treatment of mice or partial hepatectomy. Both stimuli are known to activate the transcription factor NFκB, which binds to four sites in the CMV promoter/enhancer. We show that expression from the CMV promoter in hepatocyte-derived cell lines in vitro depends on NFκB. In vivo experiments demonstrate that NFκB, which is not present in mouse hepatocytes in vivo, is activated after infection with recombinant adenoviruses and that the time course of NFκB activation parallels that of CMV promoter-dependent expression. Moreover, adenovirus infection of transgenic mice carrying a CMV promoter-drivenlacZgene leads to strong activation of the expression of this gene in the liver. Thus, NFκB is involved in the activation of the CMV promoter in the liver.
DNA Immunization with Japanese Encephalitis Virus Nonstructural Protein NS1 Elicits Protective Immunity in Mice
Authors: Lin Yi-Ling , Chen Li-Kuang , Liao Ching-Len , Yeh Chia-Tsui , Ma Shiou-Hwa , Chen Jin-Ling , Huang Yue-Ling , Chen Shih-Shun , Chiang Hsien-Yuan
Published: December 2019

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, is a zoonotic pathogen that is prevalent in some Southeast Asian countries and causes acute encephalitis in humans. To evaluate the potential application of gene immunization to JEV infection, we characterized the immune responses from mice intramuscularly injected with plasmid DNA encoding JEV glycoproteins, including the precursor membrane (prM) plus envelope (E) proteins and the nonstructural protein NS1. When injected with the plasmid expressing prM plus E, 70% of the immunized mice survived after a lethal JEV challenge, whereas when immunized with the plasmid expressing NS1, 90% of the mice survived after a lethal challenge. As a control, the mice immunized with the DNA vector pcDNA3 showed a low level (40%) of protection, suggesting a nonspecific adjuvant effect of the plasmid DNA. Despite having no detectable neutralizing activity, the NS1 immunization elicited a strong antibody response exhibiting cytolytic activity against JEV-infected cells in a complement-dependent manner. By contrast, immunization with a construct expressing a longer NS1 protein (NS1′), containing an extra 60-amino-acid portion from the N terminus of NS2A, failed to protect mice against a lethal challenge. Biochemical analyses revealed that when individually expressed, NS1 but not NS1′ could be readily secreted as a homodimer in large quantity and could also be efficiently expressed on the cell surface. Interestingly, when NS1 and NS1′ coexisted in cells, the level of NS1 cell surface expression was much lower than that in cells expressing NS1 alone. These data imply that the presence of partial NS2A might have a negative influence on an NS1-based DNA vaccine. The results herein clearly illustrate that immunization with DNA expressing NS1 alone is sufficient to protect mice against a lethal JEV challenge.
Infectious Cellular Load in Human Immunodeficiency Virus Type 1 (HIV-1)-Infected Individuals and Susceptibility of Peripheral Blood Mononuclear Cells from Their Exposed Partners to Non-Syncytium-Inducing HIV-1 as Major Determinants for HIV-1 Transmission in Homosexual Couples
Authors: Blaak Hetty , van’t Wout Angélique B. , Brouwer Margreet , Cornelissen Marion , Kootstra Neeltje A. , Albrecht-van Lent Nel , Keet René P. M. , Goudsmit Jaap , Coutinho Roel A. , Schuitemaker Hanneke
Published: December 2019

To study risk factors for homosexual transmission of human immunodeficiency virus type 1 (HIV-1), we compared 10 monogamous homosexual couples between whom transmission of HIV-1 had occurred with 10 monogamous homosexual couples between whom HIV-1 transmission had not occurred despite high-risk sexual behavior. In the group of individuals who did not transmit virus, peripheral cellular infectious load was lower and the CD4
+
T-cell counts were higher than in the group of transmitters. HIV-1 RNA levels in serum did not differ between transmitters and nontransmitters. Compared with peripheral blood mononuclear cells (PBMC) from normal healthy blood donors, 8 of 10 nonrecipients and only 3 of 8 recipients had PBMC with reduced susceptibility to in vitro infection with non-syncytium-inducing (NSI) HIV-1 variants isolated from either their respective partners or an unrelated individual. No difference in susceptibility was observed for infection with a syncytium-inducing variant. Among the individuals who had PBMC with reduced susceptibility, five nonrecipients and one recipient had PBMC that were equally or even less susceptible to NSI variants than PBMC that had low susceptibility and that were derived from healthy blood donors that were heterozygous for a 32-bp deletion in the CCR5 gene (CCR5 Δ32). Three of these individuals (all nonrecipients) had a CCR5 Δ32 heterozygous genotype themselves, confirming an association between low susceptibility to NSI variants and CCR5 Δ32 heterozygosity. All three recipients with less susceptible PBMC had partners with a high infectious cellular load; inversely, both nonrecipients with normally susceptible PBMC had partners with a very low infectious cellular load. These results suggest that a combination of susceptibility of target cells and inoculum size upon homosexual exposure largely determines whether HIV-1 infection is established.
Antibody-Secreting Cell Responses and Protective Immunity Assessed in Gnotobiotic Pigs Inoculated Orally or Intramuscularly with Inactivated Human Rotavirus
Authors: Yuan Lijuan , Kang S.-Y. , Ward Lucy A. , To Thanh L. , Saif Linda J.
Published: December 2019

Newborn gnotobiotic pigs were inoculated twice perorally (p.o.) (group 1) or intramuscularly (i.m.) (group 2) or three times i.m. (group 3) with inactivated Wa strain human rotavirus and challenged with virulent Wa human rotavirus 20 to 24 days later. To assess correlates of protection, antibody-secreting cells (ASC) were enumerated in intestinal and systemic lymphoid tissues from pigs in each group at selected postinoculation days (PID) or postchallenge days. Few virus-specific ASC were detected in any tissues of group 1 pigs prior to challenge. By comparison, groups 2 and 3 had significantly greater numbers of virus-specific immunoglobulin M (IgM) ASC in intestinal and splenic tissues at PID 8 and significantly greater numbers of virus-specific IgG ASC and IgG memory B cells in spleen and blood at challenge. However, as for group 1, few virus-specific IgA ASC or IgA memory B cells were detected in any tissues of group 2 and 3 pigs. Neither p.o. nor i.m. inoculation conferred significant protection against virulent Wa rotavirus challenge (0 to 6% protection rate), and all groups showed significant anamnestic virus-specific IgG and IgA ASC responses. Hence, high numbers of IgG ASC or memory IgG ASC in the systemic lymphoid tissues at the time of challenge did not correlate with protection. Further, our findings suggest that inactivated Wa human rotavirus administered either p.o. or parenterally is significantly less effective in inducing intestinal IgA ASC responses and conferring protective immunity than live Wa human rotavirus inoculated orally, as reported earlier (L. Yuan, L. A. Ward, B. I. Rosen, T. L. To, and L. J. Saif, J. Virol. 70:3075–3083, 1996). Thus, more efficient mucosal delivery systems and rotavirus vaccination strategies are needed to induce intestinal IgA ASC responses, identified previously as a correlate of protective immunity to rotavirus.
Mucosal and Parenteral Vaccination against Acute and Latent Murine Cytomegalovirus (MCMV) Infection by Using an Attenuated MCMV Mutant
Authors: MacDonald Margaret R. , Li Xi-Yang , Stenberg Richard M. , Campbell Ann E. , Virgin Herbert W.
Published: December 2019

We used a live attenuated murine cytomegalovirus (MCMV) mutant to analyze mechanisms of vaccination against acute and latent CMV infection. We selected MCMV mutant RV7 as a vaccine candidate since this virus grows well in tissue culture but is profoundly attenuated for growth in normal and severe combined immunodeficient (SCID) mice (V. J. Cavanaugh et al., J. Virol. 70:1365–1374, 1996). BALB/c mice were immunized twice (0 and 14 days) subcutaneously (s.c.) with tissue culture-passaged RV7 and then challenged with salivary gland-passaged wild-type MCMV (sgMCMV) intraperitoneally (i.p.) on day 28. RV7 vaccination protected mice against challenge with 105PFU of sgMCMV, a dose that killed 100% of mock-vaccinated mice. RV7 vaccination reduced MCMV replication 100- to 500-fold in the spleen between 1 and 8 days after challenge. We used the capacity to control replication of MCMV in the spleen 4 days after challenge as a surrogate for protection. Protection was antigen specific and required both live RV7 and antigen-specific lymphocytes. Interestingly, RV7 was effective when administered s.c., i.p., perorally, intranasally, and intragastrically, demonstrating that attenuated CMV applied to mucosal surfaces can elicit protection against parenteral virus challenge. B cells and immunoglobulin G were not essential for RV7-induced immunity since B-cell-deficient mice were effectively vaccinated by RV7. CD8 T cells, but not CD4 T cells, were critical for RV7-induced protection. Depletion of CD8 T cells by passive transfer of monoclonal anti-CD8 (but not anti-CD4) antibody abrogated RV7-mediated protection, and RV7 vaccination was less efficient in CD8 T-cell-deficient mice with a targeted mutation in the β2-microglobulin gene. Although gamma interferon is important for innate resistance to MCMV, it was not essential for RV7 vaccination since gamma interferon receptor-deficient mice were protected by RV7 vaccination. Establishment of and/or reactivation from latency by sgMCMV was decreased by RV7 vaccination, as measured by diminished reactivation of MCMV from splenic explants. We found no evidence for establishment of splenic latency by RV7 after s.c. vaccination. We conclude that RV7 administered through both systemic and mucosal routes is an effective vaccine against MCMV infection. It may be possible to design human CMV vaccines with similar properties.