bluuebunny/crossref_metadata_2025_split huggingface dataset:
Application of Competitive PCR to Cerebrospinal Fluid Samples from Patients with Herpes Simplex Encephalitis
Authors: Domingues R. B. , Lakeman F. D. , Mayo M. S. , Whitley R. J. 
Published: December 2019

ABSTRACT
The purpose of the present study was to determine if the quantity of herpes simplex virus (HSV) DNA in the cerebrospinal fluid (CSF) of patients with herpes encephalitis would be useful in establishing the prognosis of the disease and to determine the effect of antiviral therapy on the clearance of viral DNA from the CSF. Quantitation of HSV DNA was done by constructing an internal standard (IS) from the glycoprotein B amplicon which had a 25-bp deletion between primer annealing sites. Each CSF specimen was coamplified with the IS and the ratio of the amount of HSV/amount of IS was compared to the ratios on a standard curve constructed with the same IS plus known amounts of HSV DNA. CSF specimens were available from 16 patients who were treated with intravenous acyclovir, and the amount of HSV DNA ranged from <25 to 18,000 copies per μl in CSF obtained before or within 4 days of the initiation of acyclovir therapy. Patients with >100 copies of HSV DNA per μl were older, were found by computed tomography to have lesions, and had poorer outcomes than patients with <100 copies. Follow-up CSF specimens were available from seven patients. In six of these seven patients, the HSV DNA levels decreased during therapy. One patient had a twofold increase in HSV DNA levels after 1 week of therapy and died on day 8. The application of this assay may be helpful in establishing the prognosis and in the monitoring of patients with herpes simplex encephalitis.
5′ Nuclease PCR Assay To Detect
Yersinia pestis
Authors: Higgins James A. , Ezzell John , Hinnebusch B. Joseph , Shipley Michelle , Henchal Erik A. , Ibrahim M. Sofi 
Published: December 2019

ABSTRACT

The 5′ nuclease PCR assay uses a fluorescently labeled oligonucleotide probe (TaqMan) to rapidly detect and quantitate DNA templates in clinical samples. We developed a 5′ nuclease PCR assay targeting the plasminogen activator gene (
pla
) of
Yersinia pestis
. The assay is species specific, with a detection threshold of 2.1 × 10
5
copies of the
pla
target or 1.6 pg of total cell DNA. The assay detected
Y. pestis
in experimentally infected
Xenopsylla cheopis
fleas and in experimentally infected monkey blood and oropharyngeal swabs. The TaqMan assay is simple to perform and rapid and shows promise as a future field-adaptable technique.
Comparison of NNA Agar Culture and Selective Broth Culture for Detection of Group B Streptococcal Colonization in Women
Authors: Dunne W. Michael , Holland-Staley Carol A. 
Published: December 2019

ABSTRACT

In 1996, the Centers for Disease Control and Prevention recommended the use of a selective broth culture for the improved detection of genital tract or anorectal carriage of group B streptococci (GBS) in pregnant women. In order to verify this recommendation in our laboratory, we compared the sensitivity of Todd-Hewitt medium with gentamicin and nalidixic acid (SBM) with our current method of direct plating on blood agar medium containing neomycin and nalidixic acid (NNA). Five hundred consecutive cervicovaginal and anorectal specimens submitted for GBS culture were included in the study. Swabs were plated onto NNA and the swabs were immersed in SBM, followed by overnight incubation at 35°C. On the following day, the NNA plates were examined for colonies typical of GBS and the organisms were identified by the CAMP test or by latex agglutination. SBM cultures were subcultured onto blood agar and CNA agar plates, and the plates were reincubated for 24 h. Negative specimens from either medium were incubated for an additional 24 h and were examined again before finalization of the results. GBS were recovered from 78 specimens by both methods; from SBM only for 17 specimens (sensitivity, 86%) and from NNA only for 16 specimens (sensitivity, 85%). A moderate to heavy growth of
Enterococcus faecalis
was observed on plates containing NNA-positive, SBM-negative specimens. Competitive growth studies suggested that
E. faecalis
suppressed the growth potential of GBS in SBM. Our study suggests that direct plating on NNA, as a single method, is equivalent in sensitivity to SBM for the recovery of GBS, and the results are often available 24 h sooner. However, it appears that both direct plating and selective broth amplification techniques are required for the maximum level of identification of colonization with GBS in pregnant women.
Evaluation of PCR, Culture, and Serology for Diagnosis of
Chlamydia pneumoniae
Respiratory Infections
Authors: Verkooyen R. P. , Willemse D. , Hiep-van Casteren S. C. A. M. , Mousavi Joulandan S. A. , Snijder R. J. , van den Bosch J. M. M. , van Helden H. P. T. , Peeters M. F. , Verbrugh H. A. 
Published: December 2019

ABSTRACT

We prospectively studied 156 patients with a diagnosis of community-acquired pneumonia requiring admission. Several respiratory specimens were obtained for the detection of
Chlamydia pneumoniae
by cell culture and PCR. Three serum samples were obtained from each patient. Serological diagnosis of a
C. pneumoniae
infection was determined by the microimmunofluorescence (MIF) test, the complement fixation (CF) test, and recombinant lipopolysaccharide (LPS) enzyme-linked immunosorbent assay (ELISA; referred to as the rDNA LPS ELISA). Twenty-three patients (15%) had serological results compatible with acute
C. pneumoniae
infection; nine (39%) of these subjects were
C. pneumoniae
PCR positive. Twenty-two patients (14%) had positive PCR results without serological evidence of an acute
C. pneumoniae
infection. An attempt was made to calculate the sensitivities and specificities of the MIF test, rDNA LPS ELISA, and PCR for the diagnosis of chlamydial community-acquired pneumonia. Several “gold standards” were defined. Generally, the sensitivities of the rDNA LPS ELISA and MIF were comparable, while the sensitivity of the CF test was shown to be very low. Independent of the gold standard used, the best PCR results were obtained with nasopharyngeal specimens. However, the predictive value of a positive
C. pneumoniae
PCR result for patients with community-acquired pneumonia remains unknown and may be low. Although a widely accepted gold standard is still lacking, the rDNA LPS ELISA may currently be the preferred tool for diagnosing acute respiratory
Chlamydia
infections in routine clinical practice. However, the MIF test remains the method of choice for determining the prevalence of
C. pneumoniae
infections in a given community.
Random Amplification of Polymorphic DNA and Microsatellite Genotyping of Pre- and Posttreatment Isolates of
Candida
spp. from Human Immunodeficiency Virus-Infected Patients on Different Fluconazole Regimens
Authors: Metzgar David , van Belkum Alex , Field Dawn , Haubrich Richard , Wills Christopher 
Published: December 2019

ABSTRACT

Twelve patients infected with the human immunodeficiency virus (HIV) and with CD4 cell counts below 100 cells/μl received fluconazole daily (200 mg; five patients) or weekly (400 mg; seven patients) for fungal prophylaxis during a 6-month period. Oropharyngeal swabs were taken at regular intervals in order to detect colonization with
Candida
spp. All yeast isolates were examined with respect to the development over time of fluconazole resistance. Genetic diversity among the strains was assessed in order to discriminate between selection of a resistant subclone and patient recolonization. Genotyping was performed through random amplification of polymorphic DNA (RAPD) analysis. Specific site polymorphisms were assayed by tracking length variability in several microsatellite loci. Finally, to maximize resolution, one of these loci (
ERK1
) was analyzed by nucleotide sequencing. Although the number of strains analyzed was too small to allow statistical verification, it appeared that when fluconazole was given weekly, a smaller fraction of the strains showed diminished sensitivity than when it was given daily. Genetic analyses allowed three different scenarios to be discerned. Resistance development in an otherwise apparently unchanged strain was seen for 1 of the 12 patients. Clear strain replacement was observed for 3 of the remaining 11 patients. For all other patients minor differences were seen in either the RAPD genotype or the microsatellite allele composition during the course of treatment. In general, microsatellite sequence data is in agreement with data obtained by other methods, but occasionally within-patient heterogeneity is indicated. The present results show that during fluconazole treatment colonizing strains can remain identical, be replaced by clearly different strains, or undergo small changes. Within a patient there may be different levels of intrastrain variation.
Enzyme Immunoassay Detecting Teichoic and Lipoteichoic Acids versus Cerebrospinal Fluid Culture and Latex Agglutination for Diagnosis of
Streptococcus pneumoniae
Meningitis
Authors: Stuertz Kristin , Merx Imke , Eiffert Helmut , Schmutzhard Erich , Mäder Michael , Nau Roland 
Published: December 2019

ABSTRACT

A newly developed enzyme immunoassay (EIA) was used to detect the presence of pneumococcal teichoic and lipoteichoic acids in cerebrospinal fluid (CSF) from patients with
Streptococcus pneumoniae
meningitis who were being treated with antibiotics. All initial CSF samples, which on culture grew
S. pneumoniae
, were positive in the EIA. A total of 14 subsequent culture-negative samples gave clear signals in the EIA up to day 15 after the onset of antibiotic treatment. For 11 CSF specimens, culture, microscopy, and latex agglutination were negative while the EIA detected pneumococcal antigens. The EIA did not react either with CSF of patients with meningitis caused by bacteria other than
S. pneumoniae
or by viral pathogens. In conclusion, this EIA can be a valuable tool for the diagnosis of
S. pneumoniae
meningitis from CSF samples in cases in which prior antimicrobial therapy minimizes the usefulness of culture or other antigen detection tests.
Specific Serological Diagnosis of Leprosy with a Recombinant
Mycobacterium leprae
Protein Purified from a Rapidly Growing Mycobacterial Host
Authors: Triccas James A. , Roche Paul W. , Britton Warwick J. 
Published: December 2019

ABSTRACT

In this report we demonstrate the utility of an monoclonal antibody inhibition enzyme-linked immunosorbent assay based on the
Mycobacterium leprae
35-kDa protein, purified from the rapidly growing host
Mycobacterium smegmatis
, for the serodiagnosis of multibacillary leprosy. The assay proved highly specific (97.5%) and sensitive (90%) and compared favorably with two other established methods routinely utilized for leprosy serodiagnosis.
Strategy for Optimizing DNA Amplification in a Peripheral Blood PCR Assay Used for Diagnosis of Human Brucellosis
Authors: Morata Pilar , Queipo-Ortuño Maria Isabel , de Dios Colmenero Juan 
Published: December 2019

ABSTRACT
We studied two of the possible factors which can interfere with specific DNA amplification in a peripheral-blood PCR assay used for the diagnosis of human brucellosis. We found that high concentrations of leukocyte DNA and heme compounds inhibit PCR. These inhibitors can be efficiently suppressed by increasing the number of washings to four or five and decreasing the amount of total DNA to 2 to 4 μg, thereby avoiding false-negative results.
Evidence of Nosocomial Infection in Japan Caused by High-Level Gentamicin-Resistant
Enterococcus faecalis
and Identification of the Pheromone-Responsive Conjugative Plasmid Encoding Gentamicin Resistance
Authors: Ma Xinghua , Kudo Michiaki , Takahashi Ayako , Tanimoto Koichi , Ike Yasuyoshi 
Published: December 2019

ABSTRACT

A total of 1,799
Enterococcus faecalis
isolates were isolated from inpatients of Gunma University Hospital, Gunma, Japan, between 1992 and 1996. Four hundred thirty-two (22.3%) of the 1,799 isolates had high-level gentamicin resistance. Eighty-one of the 432 isolates were classified and were placed into four groups (group A through group D) with respect to the
Eco
RI restriction endonuclease profiles of the plasmid DNAs isolated from these strains. The 81 isolates were isolated from 36 patients. For 35 of the 36 patients, the same gentamicin-resistant isolates were isolated from the same or different specimens isolated from the same patient at different times during the hospitalization. For one other patient, two different groups of the isolates were isolated from the same specimen. Groups A, B, C, and D were isolated from 5, 14, 12, and 6 patients, respectively. The strains had multiple-drug resistance. The restriction endonuclease digestion patterns of the
E. faecalis
chromosomal DNAs isolated from isolates in the same group were also identical. The patients who had been infected with the gentamicin-resistant isolates from each group were geographically clustered on a ward(s). These results suggest that the isolates in each group were derived from a common source and had spread in the ward. The gentamicin-resistant isolates exhibited a clumping response upon exposure to pheromone (
E. faecalis
FA2-2 culture filtrate). The gentamicin resistance transferred at a high frequency to the recipient
E. faecalis
isolates by broth mating, and the pheromone-responsive plasmids encoding the gentamicin resistance were identified in these isolates.
Specific Differentiation between
Mycobacterium bovis
BCG and Virulent Strains of the
Mycobacterium tuberculosis
Complex
Authors: Magdalena Juana , Supply Philip , Locht Camille 
Published: December 2019

ABSTRACT

A PCR procedure based on the intergenic region (IR) separating two genes encoding a recently identified mycobacterial two-component system, named SenX3-RegX3, was developed and was shown to be suitable for identifying
Mycobacterium bovis
BCG. The
senX3-regX3
IR contains a novel type of repetitive sequence, called mycobacterial interspersed repetitive units (MIRUs). All tested BCG strains exclusively contained 77-bp MIRUs within the
senX3-regX3
IR, whereas all non-BCG
M. tuberculosis
complex strains contained a 53-bp MIRU, in addition to the 77-bp MIRUs. All 148 strains analyzed so far could be divided into eight different groups according to the copy numbers of the 77-bp MIRU and to the presence or absence of the 53-bp MIRU. BCG strains contained either one, two, or three 77-bp MIRUs. The other strains contained one to five 77-bp MIRUs invariably followed by a 53-bp MIRU. The consistent absence of the 53-bp MIRU in BCG strains and its presence in virulent strains allowed us to develop an enzyme-linked immunosorbent assay using specific capture oligonucleotide probes to distinguish between BCG and other
M. tuberculosis
complex strains.
Improved Identification of Mycobacteria by Using the Microbial Identification System in Combination with Additional Trimethylsulfonium Hydroxide Pyrolysis
Authors: Müller K.-Dieter , Schmid Ernst N. , Kroppenstedt Reiner M. 
Published: December 2019

ABSTRACT

The MIDI automated Microbial Identification System (MIS) uses gas chromatography (GC) analysis of whole-cell fatty acid methyl esters (FAMEs) between 9 and 20 carbons in length to characterize a wide range of bacterial genera and species, including mycobacteria. Mycolic acid cleavage products (MACPs) with chain lengths of C
22
to C
26
are not released by MIDI sample preparation of mycobacteria. Therefore, the MIS library search report often matches several mycobacterial species without any significant difference in the similarity indices. The problem is solved by adding trimethylsulfonium hydroxide (TMSH) instead of sodium sulfate in the last step of sample preparation, thus allowing the identification of MACPs in addition to FAMEs. Only one GC run parameter has to be changed: the temperature program must be extended from 260 to 310°C. The MIS library search report for the identification of bacteria is not disturbed by TMSH. The combination of conventional library search report with the information of typical MACP patterns yields significantly better discrimination of mycobacterial species than the MIDI method allows.
Persistence of Clones of Coagulase-Negative Staphylococci among Premature Neonates in Neonatal Intensive Care Units: Two-Center Study of Bacterial Genotyping and Patient Risk Factors
Authors: Vermont Clementien L. , Hartwig Nico G. , Fleer André , de Man Peter , Verbrugh Henri , van den Anker John , de Groot Ronald , van Belkum Alex 
Published: December 2019

ABSTRACT

From 1 January 1995 until 1 January 1996, we studied the molecular epidemiology of blood isolates of coagulase-negative staphylococci (CoNS) in the Neonatal Intensive Care Units (NICUs) of the Sophia Children’s Hospital (SCH; Rotterdam, The Netherlands) and the Wilhelmina Children’s Hospital (WCH; Utrecht, The Netherlands). The main goal of the present study was to detect putatively endemic clones of CoNS persisting in these NICUs. Pulsed-field gel electrophoresis was used to detect the possible presence of endemic clones of clinical significance. In addition, clinical data of patients in the SCH were analyzed retrospectively to identify risk factors for the acquisition of positive blood cultures. In both centers, endemic CoNS clones were persistently present. Thirty-three percent of the bacterial isolates derived from blood cultures in the SCH belonged to a single genotype. In the WCH, 45% of all bacterial strains belonged to a single clone. These clones were clearly different from each other, which implies that site specificity is involved. Interestingly, we observe that the clonal type in the SCH differed significantly from the incidentally occurring strains with respect to both the average pH and partial CO
2
pressure of the patient’s blood at the time of bacterial culture. We found that the use of intravascular catheters, low gestational age, and a long hospital stay were important risk factors for the development of a putative CoNS infection. When the antibiotic susceptibility of the bacterial isolates was assessed, a clear correlation between the nature of the antibiotics most frequently used as a first line of defense versus the resistance profile was observed. We conclude that the intensive use of antibiotics in an NICU setting with highly susceptible patients causes selection of multiresistant clones of CoNS which subsequently become endemic.
Detection of a Novel Strain of Porcine Circovirus in Pigs with Postweaning Multisystemic Wasting Syndrome
Authors: Morozov Igor , Sirinarumitr Theerapol , Sorden Steven D. , Halbur Patrick G. , Morgan Marsha K. , Yoon Kyoung-Jin , Paul Prem S. 
Published: December 2019

ABSTRACT
Swine infectious agents, especially viruses, are potential public health risks associated with the use of pig organs for xenotransplantation in humans. Therefore, there is a need for better characterization of swine viruses and for the development of diagnostic tests for their detection. We report here isolation of a novel strain of porcine circovirus (PCV) from pigs with postweaning multisystemic wasting syndrome (PMWS). Affected pigs exhibited severe interstitial pneumonia and lymphoid depletion. The complete nucleotide sequence (1,768 nucleotides) of the genome of the PCV isolate was determined and compared with the sequence of the PCV strain isolated from PK-15 cells. Sequence comparison revealed significant differences between the two PCV strains, with an overall DNA homology of 76%. Two major open reading frames (ORFs) were identified. ORF1 was more conserved between the two strains, with 83% nucleotide homology and 86% amino acid homology. ORF2 was more variable, with nucleotide homology of 67% and amino acid homology of 65%. PCR and in situ hybridization demonstrated abundant viral DNA in various organs of pigs with PMWS. In situ hybridization demonstrated that this strain of PCV targets multiple organs and infects macrophages, lymphocytes, endothelial cells, and epithelial cells.
Rapid and Specific Detection of ToxigenicStaphylococcus aureus: Use of Two Multiplex PCR Enzyme Immunoassays for Amplification and Hybridization of Staphylococcal Enterotoxin Genes, Exfoliative Toxin Genes, and Toxic Shock Syndrome Toxin 1 Gene
Authors: Becker Karsten , Roth Ricarda , Peters Georg 
Published: December 2019

ABSTRACTTwo multiplex PCR enzyme immunoassays (PCR-EIAs) were developed forStaphylococcus aureusexotoxin gene screening as an alternative to the conventional biological assays, which depend on detectable amounts of toxins produced. One set of oligonucleotide primers and probes was designed to search for enterotoxin A to E genes (entA,entB,entC,entD, andentE), and the other one was designed to detect the staphylococcal exfoliative toxin genes (etaandetb) and the toxic shock syndrome toxin 1 gene (tst). Oligonucleotide primers were used as published previously, modified or newly developed to meet the requirements of both good size-distinguishable amplification bands of multiplex PCR and the temperature limit of the uracil DNA glycosylase system for carryover protection. Amplification products were visualized by agarose gel electrophoresis, and specificity was controlled with the aid of a DNA EIA system using oligonucleotide probes derived from the sequences of theS. aureustoxin genes. PCR procedures were performed by using template nucleic acids extracted from a panel ofS. aureusreference strains and from a collection of 50 clinical strains. The PCR results were compared with those of immunological toxin production assays. This multiplex PCR-EIA system offers an alternative method for the rapid, sensitive, specific, and simultaneous detection of the clinically important exotoxin potency of isolatedS. aureusstrains for diagnostic purposes as well as research studies.
PCR Detection of North American and Central African Isolates of Epizootic Hemorrhagic Disease Virus (EHDV) Based on Genome Segment 10 of EHDV Serotype 1
Authors: Aradaib Imadeldin E. , Wilson William C. , Schore Caroline E. , Mohammed Mohammed E. H. , Yilma Tilahun D. , Cullor James S. , Osburn Bennie I. 
Published: December 2019

ABSTRACTPCR amplification technology for the detection of epizootic hemorrhagic disease virus (EHDV) ribonucleic acid in cell culture and clinical specimens was developed. With oligoribonucleotide primers selected from genome segment 10 of EHDV serotype 1 (EHDV-1), which codes for two nonstructural proteins (NS3 and NS3a), the PCR-based assay resulted in a 535-bp PCR product. RNAs from North American EHDV-1 prototype, EHDV-2 prototype, and a number of EHDV field isolates, including the Central African isolates of EHDV-5 and EHDV-318 propagated in cell cultures, were detected by this PCR-based assay. The specific 535-bp PCR products were visualized onto agarose gels, and the identity of the PCR products was confirmed by chemiluminescent hybridization with a 352-bp internal probe. The sensitivity of the EHDV PCR assay was increased by chemiluminescent hybridization; by this EHDV-NS3 PCR, 10 fg of EHDV RNA was detected (equivalent to 600 viral particles). Amplification product was not detected when the PCR-based assay was applied to RNAs from North American bluetongue virus prototype serotypes 2, 10, 11, 13, and 17; total nucleic acid extracts from uninfected BHK-21 cells; or unfractionated blood from calves and deer that were EHDV seronegative and virus isolation negative. The described EHDV PCR-based assay with primers derived from segment 10 of EHDV-1 resulted in detection of EHDV RNA from blood and tissues collected from calves and deer with natural and experimental EHDV infections and provides a valuable tool to study the epidemiology of EHDV infection in susceptible ruminants.