bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

ABSTRACT
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

ABSTRACT

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

ABSTRACT

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

ABSTRACTCurrent 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

ABSTRACT
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

ABSTRACT

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

ABSTRACT

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

ABSTRACTSerum 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

ABSTRACT
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

ABSTRACT

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

ABSTRACT
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

ABSTRACTBovine 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

ABSTRACT

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

ABSTRACT

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

ABSTRACT
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

ABSTRACTWe 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

ABSTRACT

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.