bluuebunny/crossref_metadata_2025_split huggingface dataset:
Rapid Identification of Nine Microorganisms Causing Acute Respiratory Tract Infections by Single-Tube Multiplex Reverse Transcription-PCR: Feasibility Study
Authors: Gröndahl Britta , Puppe Wolfram , Hoppe Andrea , Kühne Inka , Weigl Josef A. I. , Schmitt Heinz-Josef 
Published: December 2019

ABSTRACT

Acute respiratory tract infections (ARIs) are leading causes of morbidity and, in developing countries, mortality in children. A multiplex reverse transcription-PCR (RT-PCR) assay was developed to allow in one test the detection of nine different microorganisms (enterovirus, influenza A and B viruses, respiratory syncytial virus [RSV], parainfluenzaviruses type 1 and type 3, adenovirus,
Mycoplasma pneumoniae
, and
Chlamydia pneumoniae
) that do not usually colonize the respiratory tracts of humans but, if present, must be assumed to be the cause of respiratory disease. Clinical samples from 1,118 children admitted to the Department of Pediatrics because of an ARI between November 1995 and April 1998 were used for a first clinical evaluation. Detection of one of the microorganisms included in the assay was achieved for 395 of 1,118 (35%) clinical samples, of which 37.5% were RSV, 20% were influenza A virus, 12.9% were adenovirus, 10.6% were enterovirus, 8.1% were
M. pneumoniae
, 4.3% were parainfluenzavirus type 3, 3.5% were parainfluenzavirus type 1, 2.8% were influenza B virus, and 0.2% were
C. pneumoniae
. Seasonal variations in the rates of detection of the different organisms were observed, as was expected from the literature. The levels of concordance with the data obtained by commercially available enzyme immunoassays were 95% for RSV and 98% for influenza A. The results show that the multiplex RT-PCR–enzyme-linked immunosorbent assay is a useful and rapid diagnostic tool for the management of children with ARI. Studies of the overall benefit of this method with regard to the use of antibiotics, the use of diagnostic procedures including additional microbiological tests, and hospitalization rate and duration are warranted.
Comparison of PCR, Culture, and Serological Tests for Diagnosis of
Mycoplasma pneumoniae
Respiratory Tract Infection in Children
Authors: Dorigo-Zetsma J. W. , Zaat S. A. J. , Wertheim-van Dillen P. M. E. , Spanjaard L. , Rijntjes J. , van Waveren G. , Jensen J. S. , Angulo A. F. , Dankert J. 
Published: December 2019

ABSTRACT

For diagnosis of
Mycoplasma pneumoniae
infection we compared two rapid tests, PCR and the immunoglobulin M immunofluorescence assay (IgM IFA), with culture and the complement fixation test (CFT), in a prospective study among 92 children with respiratory tract infection and 74 controls. Based on positivity of culture and/or CFT as the diagnostic criterion, nine patients (10%) were diagnosed with
M. pneumoniae
infection. All patients positive by culture were also positive by PCR. In all controls cultures, PCRs, and serological assays were negative, except in one with a positive IgM IFA. The IgM IFA had a low positive predictive value of 50%. Only a combination of PCR (seven patients) and CFT (seven patients) allowed diagnosis of all cases.
Detection of Penicillin Susceptibility in
Streptococcus pneumoniae
by
pbp2b
PCR-Restriction Fragment Length Polymorphism Analysis
Authors: O’Neill A. M. , Gillespie S. H. , Whiting G. C. 
Published: December 2019

ABSTRACT

A PCR-restriction fragment length polymorphism strategy directed against the
pbp2b
gene was evaluated for identification of penicillin susceptibility. A total of 106 United Kingdom (U.K.), 30 Danish, and 11 Papua New Guinean strains were tested. Of the U.K. strains, all the susceptible and all but one of the resistant isolates were correctly assigned. By using conventional definitions of “not resistant” and “not susceptible,” the sensitivities were 97.5 and 94.4%, the specificities were 100 and 98.9%, the positive predictive values were 100 and 94.4%, and the negative predictive values were 93.1 and 98.9%, respectively. This technique may allow susceptible (MIC, <0.1 mg/liter) and resistant (MIC, >1 mg/liter) isolates to be distinguished in a single PCR.
Proficiencies of Three Anaerobic Culture Systems for Recovering Periodontal Pathogenic Bacteria
Authors: Doan Nguyen , Contreras Adolfo , Flynn Jane , Morrison John , Slots Jørgen 
Published: December 2019

ABSTRACT

Anaerobic culture is employed routinely in the primary isolation of periodontal pathogenic bacteria. However, little or no data exist on the relative abilities of the Coy anaerobic chamber (Coy Laboratory Products, Grass Lake, Mich.), the GasPak (Becton Dickinson Microbiology Systems, Cockeysville, Md.), and the AnaeroPack (Mitsubishi Gas Chemical America, Inc., New York, N.Y.) systems to grow important periodontal species, including
Porphyromonas gingivalis
,
Prevotella intermedia/nigrescens
,
Bacteroides forsythus
,
Eubacterium
species,
Campylobacter
species,
Fusobacterium
species, and
Peptostreptococcus micros
. A total of 78 specimens from advanced periodontitis lesions were collected anaerobically, plated on enriched blood agar medium, and incubated at 35°C for 5 to 7 days in each anaerobic culture system. The three culture systems were equally efficient in isolating
Porphyromonas gingivalis
and
Prevotella intermedia/nigrescens
. The Coy anaerobic chamber yielded the highest proportional recoveries of
Campylobacter
(
P
= 0.0001; nonparametric analysis of variance) and
Eubacterium
(
P
= 0.009). The Coy anaerobic chamber and the GasPak system demonstrated higher proportional recoveries of
Bacteroides forsythus
(
P
= 0.0006) and
Peptostreptococcus micros
(
P
= 0.0001) than the AnaeroPack system. The AnaeroPack system was most efficient in growing
Fusobacterium
species (
P
= 0.0001). Overall, the Coy anaerobic chamber and the GasPak system showed the highest proportional recoveries of putative periodontal pathogens, but the recoveries by the various anaerobic test systems varied considerably from sample to sample.
Molecular Fine-Specificity Analysis of Antibody Responses to Human Cytomegalovirus and Design of Novel Synthetic-Peptide-Based Serodiagnostic Assays
Authors: Greijer Astrid E. , van de Crommert Jos M. G. , Stevens Servi J. C. , Middeldorp Jaap M. 
Published: December 2019

ABSTRACT
To identify single immunodominant marker proteins which can replace complex virion antigen in serodiagnostic assays, we investigated in detail the molecular fine specificity of antibody responses in different individuals with latent or active human cytomegalovirus (HCMV) infection. An overview of the HCMV proteins recognized by human antibodies was obtained by immunoblotting. For selected immunodominant proteins the epitope fine specificity of the antibody response was determined by a peptide-scanning enzyme-linked immunosorbent assay (ELISA). Epitope clusters were synthesized as combination peptides and were used for further serologic analysis of immunoglobulin M (IgM) and IgG reactivities with panels of sera from different groups of patients in comparison to those with cytomegalovirus (CMV) virion antigen. Several serum samples had significantly higher reactivities with peptides than with the CMV virion antigen. However, individual serum samples occasionally recognized diverse peptide epitopes, stressing the importance of using combinations of peptides in serologic assays. From these studies we were able to define a specific combination of peptides derived from pp52 (UL44) and pp150 (UL32) for the specific and highly sensitive early detection of HCMV IgM, whereas a combination of peptides from pp150 (UL32), gB (UL55), and pp28 (UL99) was selected to give optimal and specific reactivity with HCMV IgG. On the basis of the results obtained with these peptide combinations, new, highly specific serodiagnostic assays were constructed. These assays had sensitivities of 98.9 and 96.4% for IgG and IgM, respectively, in comparison with the results obtained with the “gold standard,” the virion antigen-based ELISA. From the results of this study we conclude that specific combinations of highly defined synthetic peptides can replace complex HCMV virion extracts used in current serodiagnostics and may add to further standardization of HCMV serology.
Identity of Ehrlichial DNA Sequences Derived from
Ixodes ricinus
Ticks with Those Obtained from Patients with Human Granulocytic Ehrlichiosis in Slovenia
Authors: Petrovec M. , Sumner J. W. , Nicholson W. L. , Childs J. E. , Strle F. , Barlič J. , Lotrič-Furlan S. , AvšičŽupanc T. 
Published: December 2019

ABSTRACT

Adult
Ixodes ricinus
(Acari: Ixodidae) ticks collected near Ljubljana, Slovenia, were tested for the agent of human granulocytic ehrlichiosis (HGE) by using PCR assays based on the 16S rRNA gene. Three (3.2%) of 93 ticks were found to contain granulocytic ehrlichiae. Nucleotide sequences of portions of the bacterial
groESL
heat shock operon amplified from these ticks were identical or nearly (99.8%) identical to those previously determined for human patients with HGE from Slovenia, providing additional evidence that the ticks were infected with the HGE agent. This study identified
I. ricinus
as the likely vector for these ehrlichial pathogens of humans in this part of Europe.
Improvement of Immunoglobulin M Capture Immunoassay Specificity: Toxoplasma Antibody Detection Method as a Model
Authors: Tuuminen Tamara , Seppänen Helena , Pitkänen Eeva-Marja , Palomäki Pekka , Käpyaho Kirsti 
Published: December 2019

ABSTRACT

In the
Toxoplasma gondii
immunoglobulin M (IgM) capture fluorometric enzyme immunoassay used as a model, nonspecific responses due to the binding of human IgM to horseradish peroxidase (HRP) conjugates were observed despite the removal of the Fc portion of the immunoglobulin. This interaction may be mediated through the binding of human IgM to the HRP moiety of the conjugate. Addition of polymerized HRP into the reaction mixture reduced nonspecific signals in the majority of low false-positive serum reactions. Other plausible sites of interaction are conserved epitopes of mouse immunoglobulins presenting antigenic similarities with the allotopes of other species. Fragmentation of mouse antimicrobial IgG to Fab′ and selection of proper conjugation procedure improved assay specificity.
Multicenter Evaluation of the Abbott LCx
Mycobacterium tuberculosis
Ligase Chain Reaction Assay
Authors: Lumb Richard , Davies Kirsten , Dawson David , Gibb Robert , Gottlieb Thomas , Kershaw Clair , Kociuba Katherine , Nimmo Graeme , Sangster Norma , Worthington Michele , Bastian Ivan 
Published: December 2019

ABSTRACT

Four Australian hospital laboratories evaluated the performance of the Abbott LCx
Mycobacterium tuberculosis
assay with 2,347 specimens (2,083 respiratory and 264 nonrespiratory specimens) obtained from 1,411 patients. A total of 152 specimens (6.5%) were culture positive for
Mycobacterium tuberculosis
complex (MTBC); of these, 79 (52%) were smear positive. After resolution of discrepant data, the overall sensitivity, specificity, and positive and negative predictive values for the LCx assay were 69.7, 99.9, 99.1, and 97.7% respectively. For smear-positive respiratory specimens that were culture positive for MTBC, the values were 98.5, 100, 100, and 98.4%, respectively, while the values for smear-negative respiratory specimens were 41.5, 99.9, 96.4, and 98%, respectively. Relative operating characteristic curves were constructed to demonstrate the relationship between sensitivity and specificity for a range of possible cutoff values in the LCx assay. These graphs suggested that the assay sensitivity for respiratory samples could be increased from 70.2 to 78.6%, while the specificity would be reduced from 99.9 to 99.4% by inclusion of a grey zone (i.e., LCx assay values of between 0.2 and 0.99). An algorithm is presented for the handling of specimens with LCx assay values within this grey zone.
Application of Infrequent-Restriction-Site PCR to Clinical Isolates of
Acinetobacter baumannii
and
Serratia marcescens
Authors: Yoo Jin-Hong , Choi Jung-Hyun , Shin Wan-Shik , Huh Dong-Ho , Cho Yoon-Kyung , Kim Kyung-Mi , Kim Mi-Young , Kang Moon-Won 
Published: December 2019

ABSTRACT

We applied infrequent-restriction-site PCR (IRS-PCR) to the investigation of an outbreak caused by 23 isolates of
Acinetobacter baumannii
in an intensive care unit from November 1996 to May 1997 and a pseudoepidemic caused by 16 isolates of
Serratia marcescens
in a delivery room from May to September 1996. In the epidemiologic investigation of the outbreak caused by
A. baumannii
, environmental sampling and screening of all health care workers revealed the same species from the Y piece of a mechanical ventilator and the hands of two health care personnel. IRS-PCR showed that all outbreak-related strains were genotypically identical and that three strains from surveillance cultures were also identical to the outbreak-related strains. In a pseudoepidemic caused by
S. marcescens
, IRS-PCR identified two different genotypes, and among them one genotype was predominant (15 of 16 [93.8%] isolates). Extensive surveillance failed to find any source of
S. marcescens
. Validation of the result of IRS-PCR by comparison with that of field inversion gel electrophoresis (FIGE) showed that they were completely concordant. These results suggest that IRS-PCR is comparable to FIGE for molecular epidemiologic studies. In addition, IRS-PCR was less laborious and less time-consuming than FIGE. To our knowledge, this is the first report of the application of IRS-PCR to
A. baumannii
and
S. marcescens
.
Genetic Characterization of
Cryptosporidium
Strains from 218 Patients with Diarrhea Diagnosed as Having Sporadic Cryptosporidiosis
Authors: McLauchlin J. , Pedraza-Díaz S. , Amar-Hoetzeneder C. , Nichols G. L. 
Published: December 2019

ABSTRACT

Samples of whole feces in which
Cryptosporidium
oocysts were recognized by hospital laboratories were collected from 218 patients with diarrhea. All samples were reexamined by light microscopy, and oocysts were detected in 211 samples. A simple and rapid procedure for the extraction of DNA from whole feces was developed, and this was used to amplify fragments of the
Cryptosporidium
outer wall protein (COWP), the thrombospondin-related adhesive protein C1 (TRAP-C1), and the 18S rRNA genes by PCR. For seven samples oocysts were not detected by microscopy and DNA failed to be amplified by the three PCR procedures. Among the 211 samples “positive” by microscopy, the sensitivities of PCRs for the 18S rRNA, COWP, and TRAP-C1 gene fragments were 97, 91, and 66%, respectively. The sensitivities of all three PCR procedures increased with increasing numbers of oocysts as observed by microscopy. Two genotypes of the COWP and TRAP-C1 genes can be detected by PCR-restriction fragment length polymorphism analysis. With this series of samples, the same genotypes of the COWP and TRAP-C1 genes always segregated together. A combined genotyping data set was produced for isolates from 194 samples: 74 (38%) were genotype 1 and 120 (62%) were genotype 2. Genotype 2 was detected in a significantly greater proportion of the samples with small numbers of oocysts, and genotype 1 was detected in a significantly greater proportion of the samples with larger numbers of oocysts. There were no significant differences in the distribution of the genotypes by patient sex and age. The distribution of the genotypes was significantly different both in patients with a history of foreign travel and in those from different regions in England.
Multicenter Laboratory Validation of Susceptibility Testing of
Mycobacterium tuberculosis
against Classical Second-Line and Newer Antimicrobial Drugs by Using the Radiometric BACTEC 460 Technique and the Proportion Method with Solid Media
Authors: Pfyffer Gaby E. , Bonato Donald A. , Ebrahimzadeh Adeleh , Gross Wendy , Hotaling Jacqueline , Kornblum John , Laszlo Adalbert , Roberts Glenn , Salfinger Max , Wittwer Franziska , Siddiqi Salman 
Published: December 2019

ABSTRACT

In a large multicenter study involving six major study sites in the United States, Canada, and Europe, the susceptibilities of 272
Mycobacterium tuberculosis
strains to classical second-line antituberculosis (anti-TB) drugs (capreomycin, cycloserine, ethionamide, and kanamycin) and newer compounds (amikacin, clofazimine, ofloxacin, and rifabutin) were determined by the radiometric BACTEC 460 procedure and the conventional proportion method on Middlebrook 7H10 agar. Previously established critical concentrations for classical second-line anti-TB drugs were compared with several concentrations in liquid medium to establish equivalence. MICs of newer compounds determined in liquid medium were either the same or up to four times lower than those determined in agar medium. After establishing critical concentrations (breakpoints) in the extended testing of clinical isolates, we obtained an excellent overall correlation between the two systems, with no errors with amikacin, kanamycin, and ofloxacin and very few major or very major errors with the other drugs; however, for cycloserine, no breakpoint concentration could be recommended due to repeatedly inconsistent results by both methods. Based on these data we conclude that the BACTEC 460 procedure is a simple and rapid method requiring 4 to 8 days on average to generate accurate antimicrobial susceptibility testing (AST) results for eight anti-TB drugs other than those considered primary ones. These data not only fill a major gap of knowledge regarding the critical test concentrations of secondary anti-TB drugs but also provide a baseline for future evaluations of
M. tuberculosis
AST with the more recently developed, nonradiometric broth-based culture systems.
Comparison of Enzyme Immunoassay and Reverse Transcriptase PCR for Identification of Serotype G9 Rotaviruses
Authors: Coulson Barbara S. , Gentsch Jon R. , Das Bimal K. , Bhan M. K. , Glass Roger I. 
Published: December 2019

ABSTRACT

While only four globally important rotavirus G serotypes (1 to 4) have been documented, many studies suggest that serotype G9 viruses may be widely distributed and more important than previously recognized. We have evaluated 10 serotype G9 rotavirus-neutralizing monoclonal antibodies (MAbs) directed to VP7, which bound by direct enzyme immunoassay (EIA) to P1A[8], G9 rotaviruses F45, WI61, and AU32, for their ability to recognize the New Delhi G9 rotavirus 116E. Only one MAb (MAb F45:1) bound to P[11], G9 virus 116E to a high titer by EIA. This MAb was incorporated into an indirect EIA for G serotyping, which was validated with prototype cultivable human rotaviruses of G types 1 to 4 and 9. The EIA was compared with genotyping by reverse transcriptase PCR (RT-PCR) under code for the determination of the G types of rotaviruses obtained from neonates in New Delhi, India. The sensitivities of RT-PCR and EIA (after two additional freeze-thaw cycles) for the typing of G9 rotaviruses were 91 and 86%, respectively, for 24 culture-adapted rotavirus strains. The untypeable culture-adapted rotavirus samples also were unreactive with VP7 group antigen-reactive MAb 60. After two additional freeze-thaw cycles, only 26 of 42 (62%) of stools containing rotavirus typed as G9 by RT-PCR were positive for G9 rotavirus by EIA. Stools containing rotavirus untypeable by EIA contained significantly less MAb 60-reactive VP7 antigen (
P
= 0.0001) than the stools containing typeable rotavirus. Thus, RT-PCR genotyping was the more sensitive method for determination of G9 type, but a serotype was readily determined in rotavirus samples containing MAb 60-reactive VP7 antigen by an EIA that incorporates MAb F45:1.
The Three Major Spanish Clones of Penicillin-Resistant
Streptococcus pneumoniae
Are the Most Common Clones Recovered in Recent Cases of Meningitis in Spain
Authors: Enright Mark C. , Fenoll Asunción , Griffiths David , Spratt Brian G. 
Published: December 2019

ABSTRACT

One hundred six isolates of
Streptococcus pneumoniae
recovered in Spain from patients with meningitis in 1997 and 1998 were characterized by multilocus sequence typing. A heterogeneous collection of genotypes was associated with meningitis in Spain: 65 different sequence types were resolved and, even at a genetic distance of 0.43, there were 37 distinct lineages. Thirty-eight percent of the isolates, including all isolates of serotypes 6B, 9V, 14, and 23F, were resistant to penicillin, and 24% of the isolates were members of the three major Spanish penicillin-resistant or multidrug-resistant clones of serotypes 6B, 9V, and 23F or serotype variants of these clones. These three clones (MICs, 1 to 2 μg of penicillin/ml) were the most common clones associated with pneumococcal meningitis in Spain during 1997 and 1998. Only two of the other clones associated with meningitis were penicillin resistant (MICs, 0.12 to 0.5 μg/ml). One of the two most prevalent penicillin-susceptible clones causing meningitis (serotype 3) has not been detected outside of Spain, whereas the other (serotype 18C) has been recovered from patients with meningitis in the United Kingdom, The Netherlands, and Denmark. The prevalence of meningitis caused by isolates of the three major Spanish penicillin-resistant or multiply antibiotic-resistant clones, which are now globally distributed, is disturbing and clearly establishes their ability to cause life-threatening disease.
Diphtheria in the Republic of Georgia: Use of Molecular Typing Techniques for Characterization of
Corynebacterium diphtheriae
Strains
Authors: Sulakvelidze Alexander , Kekelidze Merab , Gomelauri Tsaro , Deng Yingkang , Khetsuriani Nino , Kobaidze Ketino , De Zoysa Aruni , Efstratiou Androulla , Morris J. Glenn , Imnadze Paata 
Published: December 2019

ABSTRACT

Sixty-six
Corynebacterium diphtheriae
strains (62 of the gravis biotype and 4 of the mitis biotype) isolated during the Georgian diphtheria epidemic of 1993 to 1998 and 13 non-Georgian
C. diphtheriae
strains (10 Russian and 3 reference isolates) were characterized by (i) biotyping, (ii) toxigenicity testing with the Elek assay and PCR, (iii) the randomly amplified polymorphic DNA (RAPD) technique, and (iv) pulsed-field gel electrophoresis (PFGE). Fifteen selected strains were ribotyped. Six RAPD types and 15 PFGE patterns were identified among all strains examined, and 12 ribotypes were found among the 15 strains that were ribotyped. The Georgian epidemic apparently was caused by one major clonal group of
C. diphtheriae
(PFGE type A, ribotype R1), which was identical to the predominant epidemic strain(s) isolated during the concurrent diphtheria epidemic in Russia. A dendrogram based on the PFGE patterns revealed profound differences between the minor (nonpredominant) epidemic strains found in Georgia and Russia. The methodologies for RAPD typing, ribotyping, and PFGE typing of
C. diphtheriae
strains were improved to enable rapid and convenient molecular typing of the strains. The RAPD technique was adequate for biotype differentiation; however, PFGE and ribotyping were better (and equal to each other) at discriminating between epidemiologically related and unrelated isolates.
Quantification of Human Cytomegalovirus DNA in Amniotic Fluid of Mothers of Congenitally Infected Fetuses
Authors: Revello Maria Grazia , Zavattoni Maurizio , Furione Milena , Baldanti Fausto , Gerna Giuseppe 
Published: December 2019

ABSTRACT

A quantitative PCR assay was used to quantitate human cytomegalovirus DNA in amniotic fluid of mothers of 21 fetuses with congenital infection. Seven fetuses presented ultrasound abnormalities or were born with symptoms, whereas 14 fetuses were subclinically infected. Although the median DNA level was higher in symptomatic fetuses, the difference was not statistically significant (
P
= 0.09).