bluuebunny/crossref_metadata_2025 huggingface dataset:
Enzyme-Linked Immunosorbent Assay Using Recombinant OspC and the Internal 14-kDa Flagellin Fragment for Serodiagnosis of Early Lyme Disease
Authors: Rauer Sebastian , Spohn Nicole , Rasiah Christiane , Neubert Uwe , Vogt Arnold
Published: December 2019

The outer surface protein C (OspC) and the internal 14-kDa flagellin fragment of strain GeHo ofBorrelia burgdorferisensu stricto were expressed as recombinant proteins inEscherichia coliand were purified for use in an immunoglobulin M (IgM) enzyme-linked immunosorbent assay (OspC–14-kDa antigen ELISA). No hint at disturbing protein-protein interferences, which might influence the availability of immunoreactive epitopes, was found when the recombinant antigens were combined in the ELISA. The recombinant OspC–14-kDa antigen ELISA was compared to a commercial IgM ELISA that used a detergent cell extract fromBorrelia afzeliiPKo as the antigen. According to the manufacturer’s information, the cell extract contains, in addition to other antigens, the following diagnostically relevant antigens: the 100-kDa (synonyms, 93- and 83-kDa antigens), 41-kDa, OspA, OspC, and 17-kDa antigens. The specificity was adjusted to 95% on the basis of data for 154 healthy controls. On testing of 104 serum samples from patients with erythema migrans (EM), the sensitivity of the recombinant ELISA (46%) for IgM antibodies was similar to that of the commercial ELISA (45%). However, when 42 serum samples from patients with polyclonal B-cell stimulation due to an Epstein-Barr virus infection were tested, false-positive reactions were significantly less frequent in the recombinant ELISA (10%) than in the whole-cell-extract ELISA (23%). OspC displays sequence heterogeneity of up to 40% according to the genomospecies. However, when the reactions of serum specimens from controls and EM patients with OspC from representative strains ofB. burgdorferisensu stricto (strain GeHo) andB. afzelii(strain PKo) were compared in an ELISA, almost no differences in specificity and sensitivity were seen. This demonstrates that the sera predominantly recognize the common epitopes of OspC tested in this study. In conclusion, we suggest that the OspC–14-kDa antigens ELISA is a suitable test for the detection of an IgM response in early Lyme disease.
Comparison of RapID Yeast Plus System with API 20C System for Identification of Common, New, and Emerging Yeast Pathogens
Authors: Espinel-Ingroff A. , Stockman L. , Roberts G. , Pincus D. , Pollack J. , Marler J.
Published: December 2019

The ability to identify yeast isolates by the new enzymatic RapID Yeast Plus System was compared to the ability to identify yeast isolates by the API 20C system. A total of 447 yeast isolates representing
Blastoschizomyces capitatus
, 17
Candida
spp., 5
Cryptococcus
spp.,
Geotrichum
spp., 2
Hanseniaspora
spp.,
Hansenula anomala
,
Hansenula wingei
, 3
Rhodotorula
spp.,
Saccharomyces cerevisiae
,
Sporobolomyces salmonicolor
,
Trichosporon beigelii
, and 2
Prototheca
spp. were evaluated. Also, five quality control strains (
Candida
spp. and
Cryptococcus laurentii
) with well-documented reactivities by the RapID Yeast Plus System were used. Each isolate was evaluated by both methods with a 48-h culture grown at 30°C on Sabouraud dextrose agar (Emmons modification) by following the recommendations of the manufacturers. The RapID Yeast Plus System enzymatic reactions were read after 4 h of incubation, and the API 20C carbohydrate assimilation identification profiles were obtained after 72 h of incubation. There was good (95.7%) agreement between the identifications obtained by the two methods with the eight common
Candida
spp. and with
Cryptococcus neoformans
. The agreement was lower when the emerging
Candida
spp. and other yeast-like pathogens were tested (79.1 and 75.2%, respectively). These preliminary data suggest the potential utility of the RapID Yeast Plus System for use in the clinical laboratory for the rapid identification of common yeast pathogens as well as certain new and emerging species.
Identification of a Novel Enteric
Helicobacter
Species in a Kitten with Severe Diarrhea
Authors: Foley Janet E. , Solnick Jay V. , Lapointe Jean-Martin , Jang Spencer , Pedersen Niels C.
Published: December 2019

A previously undescribed
Helicobacter
sp. was recovered from a cat with severe diarrhea. Based upon the absence of any other identifiable cause of diarrhea, this helicobacter may be involved in the development of the disease signs. The organism could not be cultured but was described on the basis of 16S rRNA gene sequence analysis and morphology and appeared to be a new species, with
Helicobacter canis
being the most genetically similar species. The presence of a diarrhea-inducing helicobacter in a companion animal may pose a risk of zoonosis.
Soft Salt-Mannitol Agar–Cloxacillin Test: a Highly Specific Bedside Screening Test for Detection of Colonization with Methicillin-Resistant
Staphylococcus aureus
Authors: Mir Nuria , Sánchez Miguel , Baquero Fernando , López Blanca , Calderón Celia , Cantón Rafael
Published: December 2019

The early detection of colonization with methicillin-resistant
Staphylococcus aureus
(MRSA) of patients in intensive-care units is an essential step in the strategy for preventing MRSA epidemics. In this study, tubes containing soft salt-mannitol agar with cloxacillin (6 μg/ml) (SSMAC) were prepared for inoculation of clinical samples at patients’ bedsides by personnel of an intensive-care unit. A total of 1,914 swabs from different sample sites of 81 patients were dipped into SSMAC tubes, and after 24 h of incubation (in an incubator located near the intensive-care unit), an evident color change was considered by the intensive-care-unit personnel to be an MRSA alarm. Sixty-three (3.3%) SSMAC tubes were considered positive for MRSA, 1,827 (95.4%) were considered negative, and 24 (1.2%) were considered intermediate. Compared with values for parallel conventional surveillance cultures for MRSA, excluding tubes with intermediate results, the SSMAC test had a sensitivity of 72.7%, a specificity of 99.2%, a positive predictive value of 76.2%, and a negative predictive value of 99.0%. When intermediate tubes were considered positive, the corresponding values were 75.3, 98.2, 63.2, and 99.0%, respectively. The sensitivity and specificity values of the test to identify MRSA-colonized patients were 89.4 and 100%, respectively. Oropharyngeal and naris specimens were the most reliable samples for MRSA detection. False-negative results were frequent in bronchial aspirates with low (<10
3
to 10
6
CFU/ml) MRSA counts. False-positive results were mainly due to methicillin-resistant
Staphylococcus haemolyticus
. The SSMAC tube is a useful, rapid, and inexpensive tool for the early identification of MRSA-colonized patients and, consequently, for the implementation of measures to prevent the spread of MRSA.
Evaluation of Use of a New Chromogenic Agar in Detection of Urinary Tract Pathogens
Authors: Samra Z. , Heifetz M. , Talmor J. , Bain E. , Bahar J.
Published: December 2019

CHROMagar Orientation, a new chromogenic medium, was evaluated for the detection and differentiation of gram-positive and gram-negative pathogenic microorganisms in 900 urine samples from hospitalized patients. Performance characteristics of the medium were evaluated in comparison to those of 5% sheep blood and MacConkey agars by direct inoculation of the urine samples on the three media. Four gram-negative and two gram-positive strains as well as one yeast control strain from the American Type Culture Collection were used to ensure quality control. CHROMagar Orientation succeeded in detecting all the urine pathogens that were detected by the reference media, including gram-negative bacilli, staphylococci, streptococci, and yeasts. Colony color and morphology on CHROMagar Orientation accurately differentiated
Escherichia coli
,
Proteus mirabilis
,
Proteus vulgaris
,
Pseudomonas aeruginosa
, and
Acinetobacter
spp. Owing to the similarity in the pigmentation produced by
Klebsiella
,
Enterobacter
, and
Citrobacter
isolates, the medium failed to distinguish among them; however, these isolates were easily recognized as coliforms because of their metallic blue coloration. Staphylococci were clearly perceptible:
S. aureus
and
S. epidermidis
grow in regular-size colonies that range from opaque white to yellowish, and
S. saprophyticus
produces opaque pink colonies. All streptococcus strains, including those from groups B and C, were detected. They grow as undifferentiated flat dry diffused colonies, and additional tests were required for identification. Enterococci were easily discriminated by their strong turquoise pigmentation and their typical growth on the agar’s surface. Yeast grow in typical creamy wet convex colonies. The accuracy of antibiotic susceptibility determinations according to standard methods was also tested by picking isolates directly from CHROMagar Orientation. The results showed excellent correlation with those obtained with microorganisms picked from reference media. Owing to the ease in differentiating mixed flora on CHROMagar Orientation, antimicrobic susceptibility tests were performed directly from primary isolates in all cases without the need for subcultures.
Short-Term Follow-Up by Serology of Patients Given Antibiotic Treatment for
Helicobacter pylori
Infection
Authors: Laheij R. J. F. , Witteman E. M. , Bloembergen P. , de Koning R. W. , Jansen J. B. M. J. , Verbeek A. L. M.
Published: December 2019

Helicobacter pylori
serology and in particular enzyme-linked immunosorbent assays for the measurement of immunoglobulin G (IgG) antibody titers form an accurate means of diagnosing
H. pylori
infection in patients before treatment.
H. pylori
serology is of limited value in monitoring treatment because of the slow decline in antibody titers. In the present study we aimed to measure the most suitable moment after antibiotic treatment at which serology should be used to monitor treatment. Sixty-four patients who had nonulcer dyspepsia and
H. pylori
infection and who underwent upper gastrointestinal endoscopy because of persistent dyspeptic symptoms were included in the study.
H. pylori
cure was confirmed by histology and culture 5 weeks after the completion of the antibiotic treatment. Serological examination was performed before therapy and at 5 weeks, 10 weeks, and 1 year after the completion of antibiotic treatment. Diagnostic performance was assessed by receiver-operating characteristic analysis. The areas under the receiver-operating characteristic curves of the
H. pylori
antibody titers at 5 weeks, 10 weeks, and 1 year after the completion of treatment were 0.53 (95% confidence interval [CI], 0.36 to 0.69), 0.60 (95% CI, 0.43 to 0.76), and 0.78 (95% CI, 0.63 to 0.93), respectively. The areas under the receiver-operating characteristic curves of the changes in
H. pylori
IgG antibody titers at 5 weeks, 10 weeks, and 1 year after the completion of treatment in comparison with the pretreatment titers were 0.85 (95% CI, 0.72 to 0.97), 0.96 (95% CI, 0.89 to 1.0), and 1.0 (95% CI, not estimable), respectively. We conclude that serology forms a useful means of monitoring treatment in patients with nonulcer dyspepsia and
H. pylori
infection as early as 10 weeks and maybe even sooner after the completion of treatment for the infection.
Use of Roche AMPLICOR
Mycobacterium tuberculosis
PCR in Early Diagnosis of Tuberculous Meningitis
Authors: Bonington Alec , Strang J. I. George , Klapper Paul E. , Hood Steven V. , Rubombora William , Penny Miranda , Willers Rose , Wilkins Edmund G. L.
Published: December 2019

Several nucleic acid-based amplification tests are available for the detection of
Mycobacterium tuberculosis
, but few data are available on their use in the diagnosis of tuberculous meningitis (TBM). We performed a prospective study to assess the Roche AMPLICOR
Mycobacterium tuberculosis
PCR test (TB AMPLICOR) for use in the diagnosis of TBM and compared it with direct Ziehl-Neelsen staining of smears, radiometric culture for
M. tuberculosis
, and clinical and cerebrospinal fluid (CSF) findings. Eighty-three CSF specimens collected from 69 patients with suspected meningitis in South Africa were tested by TB AMPLICOR. On the basis of clinical and laboratory findings, 40 of these patients were treated for TBM and 29 patients were not treated for TBM. Ten CSF samples from 10 patients were positive by TB AMPLICOR. Seven of these 10 patients were classified as having definite TBM, 2 were classified as having probable TBM, and 1 was classified as having possible TBM. The sensitivity of TB AMPLICOR for detecting cases of definite and probable TBM in patients from whom CSF specimens had been collected less than 10 days into antituberculosis treatment was 60.0%. Specimens from all 29 patients not treated for TBM were negative by the TB AMPLICOR, giving a 100% specificity. TB AMPLICOR is therefore more sensitive than the combination of Ziehl-Neelsen staining of smears and radiometric culture for
M. tuberculosis
and is a rapid and highly specific diagnostic test for TBM.
Use of Ligase Chain Reaction with Urine versus Cervical Culture for Detection of
Chlamydia trachomatis
in an Asymptomatic Military Population of Pregnant and Nonpregnant Females Attending Papanicolaou Smear Clinics
Authors: Gaydos Charlotte A. , Howell M. Rene , Quinn Thomas C. , Gaydos Joel C. , McKee Kelly T.
Published: December 2019

Ligase chain reaction (LCR) (Abbott Laboratories, Abbott Park, Ill.) with first-catch urine specimens was used to detect
Chlamydia trachomatis
infections in 465 asymptomatic military women attending clinics for routine Papanicolaou smear tests. Results were compared to results of cervical culture to determine the sensitivity of the urine LCR and the possible presence of inhibitors of amplification in pregnant and nonpregnant women. Discrepant results for LCR and culture were resolved by direct fluorescent antibody staining of culture sediments, two different PCR assays, and LCR for the outer membrane protein 1 gene. The prevalence of
Chlamydia
in specimens by urine LCR was 7.3% compared to 5% by culture. For 434 women with matching specimens, there were 11 more specimens positive by LCR than were positive by culture, of which all but one were determined to be true positives. There were four culture-positive, LCR-negative specimens, all from nonpregnant women. The sensitivity, specificity, and positive and negative predictive values of urine LCR after discrepant results were resolved were 88.6, 99.7, 96.9, and 99.0%, respectively. The sensitivity of culture was 71.4%. From the 148 pregnant women (prevalence by LCR, 6.8%), there were no patients who were cervical culture positive and urine LCR negative to indicate the presence in pregnant women of inhibitors of LCR. Additionally, a subset of 55 of the LCR-negative frozen urine specimens from pregnant women that had been previously processed in LCR buffer were inoculated with 5 cell culture inclusion forming units of
C. trachomatis
each and retested by LCR; all tested positive, indicating the absence of inhibitors of LCR in urine from these pregnant women. The use of LCR testing of urine specimens from asymptomatic women, whether pregnant or not, offers a sensitive and easy method to detect
C. trachomatis
infection in women.
Comparative Evaluation of National Committee for Clinical Laboratory Standards Broth Macrodilution and Agar Dilution Screening Methods for Testing Fluconazole Susceptibility of
Cryptococcus neoformans
Authors: Kirkpatrick William R. , McAtee Robert K. , Revankar Sanjay G. , Fothergill Annette W. , McCarthy Dora I. , Rinaldi Michael G. , Patterson Thomas F.
Published: December 2019

A simple screening method for fluconazole susceptibility of
Cryptococcus neoformans
using 2% dextrose Sabouraud dextrose agar (SabDex) with fluconazole was compared to the National Committee for Clinical Laboratory Standards (NCCLS) broth macrodilution method. By this method, fluconazole-susceptible
C. neoformans
isolates are significantly smaller on medium with fluconazole than on fluconazole-free medium. Isolates with decreased susceptibility have normal-size colonies on medium containing fluconazole. The 48-h NCCLS broth macrodilution MICs (NCCLS MICs) for isolates with normal-size colonies on 8- or 16-μg/ml fluconazole plates were predicted to be ≥8 or ≥16 μg/ml, respectively. On medium with 16 μg of fluconazole per ml, all strains (84 of 84) for which the NCCLS MICs were <16 μg/ml were correctly predicted, as were all isolates (7 of 7) for which the MICs were ≥16 μg/ml. Agar dilution appears to be an effective screening method for fluconazole resistance in
C. neoformans.
Evaluation of Murex CMV DNA Hybrid Capture Assay for Detection and Quantitation of Cytomegalovirus Infection in Patients following Allogeneic Stem Cell Transplantation
Authors: Hebart Holger , Gamer Daphne , Loeffler Juergen , Mueller Claudia , Sinzger Christian , Jahn Gerhard , Bader Peter , Klingebiel Thomas , Kanz Lothar , Einsele Hermann
Published: December 2019

Murex hybrid capture DNA assay (HCS) is a solution hybridization antibody capture assay for detection and quantitation of cytomegalovirus (CMV) DNA in leukocytes. To determine whether CMV HCS is sensitive enough to initiate and monitor antiviral therapy after allogeneic stem cell transplantation (SCT), 51 consecutive SCT recipients were prospectively screened for the appearance of CMV infection by HCS, PCR, and culture assays from blood samples. Preemptive antiviral therapy was initiated after the second positive PCR result in all patients, as previously reported, and HCS was not considered for clinical decision making. A total of 417 samples were analyzed. Of these, 21 samples were found to be positive by PCR and HCS, 88 samples were PCR positive but HCS negative, and 308 were negative by both assays. Concordance of results between PCR and HCS and between HCS and blood culture was observed in 78.9 and 95.9% of the samples assayed, respectively. PCR was found to be more sensitive than HCS, and HCS was more sensitive than the blood culture assay (
P
< 0.0001). Four patients with symptomatic CMV infection were PCR positive prior to the onset of CMV-related symptoms, whereas HCS detected CMV DNA in three patients prior to and one at onset of CMV disease. The numbers of genomes per milliliter of blood were higher in patients with symptomatic CMV infection than in those with asymptomatic CMV infection (
P
= 0.06). None of the HCS-negative patients developed CMV disease. Thus, all patients with CMV disease were correctly identified by HCS; however, the lower sensitivity limit of the HCS assay may still be insufficient to allow diagnosis of CMV infection early enough to prevent CMV disease in patients following allogeneic SCT.
Simultaneous Detection and Identification of Human Parainfluenza Viruses 1, 2, and 3 from Clinical Samples by Multiplex PCR
Authors: Echevarría Juan E. , Erdman Dean D. , Swierkosz Ella M. , Holloway Brian P. , Anderson Larry J.
Published: December 2019

Reverse transcription (RT)-PCR assays have been widely described for use in the diagnosis of human parainfluenza viruses (HPIVs) and other respiratory virus pathogens. However, these assays are mostly monospecific, requiring separate amplifications for each HPIV type. In the present work, we describe multiplex RT-PCR assays that detect and differentiate HPIV serotypes 1, 2, and 3 in a combined reaction. Specifically, a mixture of three pairs of primers to conserved regions of the hemagglutinin-neuraminidase gene of each HPIV serotype was used for primary amplification, yielding amplicons with similar sizes. For typing, a second amplification was performed with a mixture of nested primers, yielding amplicons with sizes easily differentiated by agarose gel electrophoresis. A modified single-amplification RT-PCR assay with fluorescence-labeled nested primers, followed by analysis of the labeled products on an automated sequencing gel, was also evaluated. Fifteen temporally and geographically diverse HPIV isolates from the Centers for Disease Control and Prevention archives and 26 of 30 (87%) previously positive nasopharyngeal specimens (8 of 10 positive for HPIV serotype 1 [HPIV1], 9 of 10 positive for HPIV2, and 9 of 10 positive for HPIV3) were positive and were correctly typed by both assays. Negative results were obtained with naso- or oropharyngeal specimens and/or culture isolates of 33 unrelated respiratory tract pathogens, including HPIV4, enterovirus, rhinovirus, respiratory syncytial virus, adenovirus, influenza virus, and
Streptococcus pneumoniae
. Our multiplex RT-PCR assays provide sensitive, specific, and simplified tools for the rapid diagnosis of HPIV infections.
Simplified Quantitative Assay System for Measuring Activities of Drugs against IntracellularLegionella pneumophila
Authors: Higa Futoshi , Kusano Nobuchika , Tateyama Masao , Shinzato Takashi , Arakaki Noriko , Kawakami Kazuyoshi , Saito Atsushi
Published: December 2019

We developed a new simple assay for the quantitation of the activities of drugs against intracellularLegionella pneumophila. The cells of a murine macrophage-like cell line (J774.1 cells) allowed the intracellular growth and replication of the bacteria, which ultimately resulted in cell death. The infected J774.1 cell monolayers in 96-well microplates were first treated with antibiotics and were further cultured for 72 h. The number of viable J774.1 cells in each well was quantified by a colorimetric assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and an enzyme-linked immunosorbent assay reader. The number of growing bacteria in each well was also determined by counting the numbers of CFU on buffered charcoal yeast extract-α agar plates. Viable J774.1 cell counts, determined by the colorimetric assay, were inversely proportional to the number of intracellular replicating bacteria. The minimum extracellular concentrations (MIECs) of 24 antibiotics causing inhibition of intracellular growth ofL. pneumophilawere determined by the colorimetric assay system. The MIECs of beta-lactams and aminoglycosides were markedly higher than the MICs in buffered yeast extract-α broth. The MIECs of macrolides, fluoroquinolones, rifampin, and minocycline were similar to the respective MICs. According to their intracellular activities, clarithromycin and sparfloxacin were the most potent among the macrolides or fluoroquinolones tested in this study. Our results indicated that the MTT assay system allows comparative and quantitative evaluations of the intracellular activities of antibiotics and efficient processing of a large number of samples.
Apparent Increased Prevalence of High-Level Aminoglycoside-Resistant
Enterococcus durans
Resulting from False Identification by a Semiautomated Software System
Authors: Tsakris Athanassios , Woodford Neil , Pournaras Spyros , Kaufmann Mary , Douboyas John
Published: December 2019

Identification of enterococci by using a semiautomated system (PASCO; Difco Laboratories, Detroit, Mich.) in the AHEPA University Hospital, Thessaloniki, Greece, revealed a high proportion of
Enterococcus durans
, particularly among isolates highly resistant to gentamicin and streptomycin. When 14 isolates were further tested by a conventional biochemical scheme and by PCR, all were reidentified as
Enterococcus faecalis
. Antibiotic resistance and pulsed-field gel electrophoresis patterns showed that unrelated strains were misidentified.
Comparison of the Rapid Yeast Plus Panel with the API20C Yeast System for Identification of Clinically Significant Isolates of
Candida
Species
Authors: Heelan Judith S. , Sotomayor Edgar , Coon Kimberly , D’Arezzo Julia B.
Published: December 2019

The RapID Yeast Plus system (Innovative Diagnostic Systems, Norcross, Ga.) is a qualitative micromethod employing conventional tests and single-substrate chromogenic tests and having a 4-h incubation period. This system was compared with the API20C (bioMerieux Vitek, Hazelwood, Mo.) system, a 24- to 72-h carbohydrate assimilation method. One hundred thirty-three clinical yeast isolates, including 57 of
Candida albicans
, 26 of
Candida tropicalis
, 23 of
Candida glabrata
, and 27 of other yeasts, were tested by both methods. When discrepancies occurred, isolates were further tested by the Automated Yeast Biochemical Card (bioMerieux Vitek). Germ tube production and microscopic morphology were used as needed to definitively identify yeast isolates. The RapID Yeast Plus system correctly identified 125 yeast isolates, with an overall accuracy of 94% (125 of 133). Excellent correlation was found in the recognition of the three yeasts most commonly isolated from human sources. The test was 99% (105 of 106 isolates) accurate with
C. albicans
,
C. tropicalis
, and
C. glabrata
. The RapID Yeast Plus system compares favorably with the API20C system and provides a simple, accurate alternative to conventional assimilation methods for the rapid identification of the most commonly encountered isolates of
Candida
species.
Survey of
Klebsiella pneumoniae
Strains Producing Extended-Spectrum β-Lactamases: Prevalence of SHV-12 and SHV-2a in Korea
Authors: Kim Jungmin , Kwon Youngmi , Pai Hyunjoo , Kim Jong-Won , Cho Dong-Taek
Published: December 2019

Fifty-three clinical isolates of extended-spectrum β-lactamase (ESBL)-producing
Klebsiella pneumoniae
collected from three hospitals in Korea were investigated for phenotypical and genotypical characterizations. Among these, 39 strains (74%) were shown by isoelectric focusing to carry SHV-type β-lactamases: 27 strains showed the pI 8.2 β-lactamase, and another 12 strains showed the pI 7.6 β-lactamase. The SHV gene of each of these strains was amplified by PCR, followed by nucleotide sequencing analysis. The gene of the pI 8.2 β-lactamase was found to be identical to the sequences encoding SHV-12, and the gene of the pI 7.6 β-lactamase was identical to the sequences encoding SHV-2a. A total of eight cefoxitin-resistant strains were found to have the plasmid-mediated AmpC-type β-lactamase, with a pI of 8.0, and this was confirmed to be CMY-1 β-lactamase by PCR and hybridization analysis. Noteworthy in this study is the fact that SHV-12 and SHV-2a have been the most commonly identified SHV-type ESBLs in Korea.
Detection of
Leptospira
DNA in Patients with Aseptic Meningitis by PCR
Authors: Romero Eliete C. , Billerbeck Ana E. C. , Lando Valéria S. , Camargo Eide D. , Souza Candida C. , Yasuda Paulo H.
Published: December 2019

Samples of cerebrospinal fluid from 103 patients with aseptic meningitis were tested by PCR for detection of leptospires, and the results were compared with those of the microscopic agglutination test (MAT) and an enzyme-linked immunosorbent assay for detection of immunoglobulin M (ELISA-IgM). Of these samples, 39.80% were positive by PCR and 8.74 and 3.88% were positive by MAT and ELISA-IgM, respectively.
Comparison of DNA Enzyme Immunoassay and Line Probe Assays (Inno-LiPA HCV I and II) for Hepatitis C Virus Genotyping
Authors: Le Pogam S. , Dubois F. , Christen R. , Raby C. , Cavicchini A. , Goudeau A.
Published: December 2019

Two methods for genotyping hepatitis C virus (DNA enzyme immunoassay [DEIA] and line probe assay [Inno-LiPA HCV I and II]) were compared on 120 samples and of these 87% were assigned to the same subtype by both assays. There were 15 subtyping discrepancies which involved 5% of type 1 isolates and 90% of type 2 isolates. Amplified products from the core and 5′ untranslated regions (UTR) were sequenced to resolve conflicts. Type 1 discordant samples had a guanosine at position −99 in the 5′ UTR, a characteristic of genotype 1b, and a core region typical of subtype 1a. The eight isolates classified as 2a/2c by LiPA and as subtype 2c by DEIA belonged to type 2.
Sensitivities and Specificities of Premier
E. coli
O157 and Premier EHEC Enzyme Immunoassays for Diagnosis of Infection with Verotoxin (Shiga-Like Toxin)-Producing
Escherichia coli
Authors: Mackenzie A. M. R. , Lebel P. , Orrbine E. , Rowe P. C. , Hyde L. , Chan F. , Johnson W. , McLaine P. N. ,   The Synsorb Pk Study Investigators
Published: December 2019

This study describes the performance of two rapid enzyme immunoassays, Premier
E. coli
O157 and Premier EHEC (Meridian Diagnostics Inc., Cincinnati, Ohio) for the detection in stools of
Escherichia coli
O157 and verotoxins (Shiga-like toxins), respectively. Both tests were performed on stools from 876 children presenting to eight emergency departments with diarrhea. Standard culture, including
E. coli
O157:H7 isolation, was performed, and paired sera were taken for anti-O157-lipopolysaccharide antibody determination. Stools from patients enrolled in the study, and those yielding discordant results, were sent to a reference laboratory for repeat testing and further investigation, including cytotoxicity and non-O157 verotoxin-producing
E. coli
culture. Results were classified as field results (obtained in the eight site laboratories) and resolved results (obtained after repeat testing in the central laboratory). The “gold standard” for sensitivity of both tests and for specificity of Premier
E. coli
O157 was isolation of
E. coli
O157:H7 or a fourfold anti-O157 antibody rise. Specimens positive by the Premier EHEC test and negative for
E. coli
O157 culture were examined for non-O157 verotoxin-producing
E. coli
. The field sensitivity of Premier
E. coli
O157 was 86%, that of Premier EHEC was 89%, and the specificity of Premier
E. coli
O157 was 98%. Ten of 13 discordant Premier
E. coli
O157 results were reassigned as true results after repeat testing. Ten non-O157 verotoxin-producing
E. coli
isolates were recovered from Premier EHEC-positive,
E. coli
O157 culture-negative stools. Only one specimen gave an unequivocally false-positive Premier EHEC result. Both tests are highly sensitive and are specific if correctly performed. The Premier EHEC test will be particularly valuable as a practical routine test for the detection of non-O157 verotoxin-producing
E. coli.
Assessment of Resolution and Intercenter Reproducibility of Results of Genotyping
Staphylococcus aureus
by Pulsed-Field Gel Electrophoresis of
Sma
I Macrorestriction Fragments: a Multicenter Study
Authors: van Belkum Alex , van Leeuwen Willem , Kaufmann Mary Elizabeth , Cookson Barry , Forey Françoise , Etienne Jerome , Goering Richard , Tenover Fred , Steward Christine , O’Brien Frances , Grubb Warren , Tassios Panayotis , Legakis Nicholas , Morvan Anne , El Solh Névine , de Ryck Raf , Struelens Marc , Salmenlinna Saara , Vuopio-Varkila Jaana , Kooistra Mirjam , Talens Adriaan , Witte Wolfgang , Verbrugh Henri
Published: December 2019

Twenty well-characterized isolates of methicillin-resistant
Staphylococcus aureus
were used to study the optimal resolution and interlaboratory reproducibility of pulsed-field gel electrophoresis (PFGE) of DNA macrorestriction fragments. Five identical isolates (one PFGE type), 5 isolates that produced related PFGE subtypes, and 10 isolates with unique PFGE patterns were analyzed blindly in 12 different laboratories by in-house protocols. In several laboratories a standardized PFGE protocol with a commercial kit was applied successfully as well. Eight of the centers correctly identified the genetic homogeneity of the identical isolates by both the in-house and standard protocols. Four of 12 laboratories failed to produce interpretable data by the standardized protocol, due to technical problems (primarily plug preparation). With the five related isolates, five of eight participants identified the same subtype interrelationships with both in-house and standard protocols. However, two participants identified multiple strain types in this group or classified some of the isolates as unrelated isolates rather than as subtypes. The remaining laboratory failed to distinguish differences between some of the related isolates by utilizing both the in-house and standardized protocols. There were large differences in the relative genome lengths of the isolates as calculated on the basis of the gel pictures. By visual inspection, the numbers of restriction fragments and overall banding pattern similarity in the three groups of isolates showed interlaboratory concordance, but centralized computer analysis of data from four laboratories yielded percent similarity values of only 85% for the group of identical isolates. The differences between the data sets obtained with in-house and standardized protocols could be the experimental parameters which differed with respect to the brand of equipment used, imaging software, running time (20 to 48 h), and pulsing conditions. In conclusion, it appears that the standardization of PFGE depends on controlling a variety of experimental intricacies, as is the case with other bacterial typing procedures.
Evaluation of a Commercially Available Reverse Transcription-PCR Assay for Diagnosis of Enteroviral Infection in Archival and Prospectively Collected Cerebrospinal Fluid Specimens
Authors: Pozo Francisco , Casas Inmaculada , Tenorio Antonio , Trallero Gloria , Echevarria Jose M.
Published: December 2019

A commercially available reverse transcription (RT)-PCR method (AMPLICOR EV; Roche Diagnostic Systems, Inc., Branchburg, N.J.) was evaluated for detection of enteroviruses in cerebrospinal fluid from patients with neurological disease. This assay was compared with virus isolation in cell culture and an in-house RT-PCR method designed with a nonoverlapping region of the enteroviral genome. A panel of 200 cerebrospinal fluid specimens prospectively collected from patients with a wide variety of neurological symptoms, including 50 patients involved in three different outbreaks of acute aseptic meningitis, was assayed. A second panel of 97 archived cerebrospinal fluid specimens, stored for 2 to 5 years, from patients with aseptic meningitis associated with several enterovirus outbreaks was also studied. From the first panel, enteroviruses were detected in 13 of 50 specimens by cell culture (26%), in 43 of 50 specimens by AMPLICOR EV (86%), and in 46 of 50 specimens by the in-house assay (92%) from patients with aseptic meningitis associated with outbreak and 1 of 29, 3 of 29, and 4 of 29 specimens, respectively, from sporadic cases of aseptic meningitis. The remaining 121 cerebrospinal fluid specimens from patients with other neurological syndromes were negative by all tests. From the second panel, enteroviral RNA was detected by the AMPLICOR test (31 of 97 specimens, 32%) and the in-house assay (39 of 97 specimens, 40%). According to our results, patients with aseptic meningitis should be analyzed for enteroviral infection in cerebrospinal fluid by RT-PCR methods, and the AMPLICOR EV test is a suitable tool for performing such studies. Archival cerebrospinal fluid specimens are less suitable for evaluation of the performance of RT-PCR methods designed for enterovirus detection.
16S rRNA Sequence Diversity in
Mycobacterium celatum
Strains Caused by Presence of Two Different Copies of 16S rRNA Gene
Authors: Reischl U. , Feldmann K. , Naumann L. , Gaugler B. J. M. , Ninet B. , Hirschel B. , Emler S.
Published: December 2019

Direct sequencing of the 16S rRNA gene (16S rDNA) of
Mycobacterium celatum
isolates showed ambiguities, suggesting heterogeneity. Cloned 16S rDNA yielded two copies of the gene, which differed by insertion of a thymine at position 214 and by additional mismatches. Restriction fragment length polymorphism analysis confirmed the presence of two copies of 16S rDNA within the bacterial chromosome.
Diagnosis of Group A Streptococcal Necrotizing Fasciitis by Using PCR To Amplify the Streptococcal Pyrogenic Exotoxin B Gene
Authors: Louie Lisa , Simor Andrew E. , Louie Marie , McGeer Allison , Low Donald E.
Published: December 2019

This study evaluated a PCR assay for detection of the streptococcal pyrogenic exotoxin B (
speB
) gene from tissue biopsy specimens of patients with necrotizing fasciitis.
speB
was detected in specimens from all 10 patients with necrotizing fasciitis due to group A streptococcus. The assay was negative for all 11 patients without culture or serologic evidence of streptococcal infection. These results suggest that the detection of
speB
by PCR may be useful for confirming group A streptococcal infection when cultures are negative or not available.
The L1 Major Capsid Protein of Human Papillomavirus Type 16 Variants Affects Yield of Virus-Like Particles Produced in an Insect Cell Expression System
Authors: Touze Antoine , El Mehdaoui Slimane , Sizaret Pierre-Yves , Mougin Christine , Muñoz Nubia , Coursaget Pierre
Published: December 2019

The L1 major capsid proteins of six human papillomavirus type 16 (HPV-16) strains were expressed in insect cells by using recombinant baculoviruses. Virus-like particles (VLPs) which appeared similar to empty virions were identified by electron microscopy for all HPV strains investigated. However, the yield of VLPs produced varied in a range from 1 to 79 depending on the HPV-16 strain. The L1 proteins of these strains differed by up to 15 amino acids from the L1 protein of the prototype HPV-16 strain. Mutations in the amino acid region from residues 83 to 97 seemed to affect the level of expression of the L1 protein. These results are important when considering the development of HPV vaccines and serological tests. They indicate that strains inducing high levels of VLP production must be selected for the development of vaccines. Moreover, the L1 proteins of all strains investigated were able to bind with DNA. We also investigated the seroreactivities of VLPs derived from three different HPV-16 strains from Algeria, Senegal, and the Philippines by testing sera from women from 11 countries in immunoglobulin G-specific enzyme-linked immunosorbent assays. We observed a strong correlation between the reactivities of the three different VLP variants, independent of the geographical origin of the sera investigated. These results indicate that the three strains investigated are serologically cross-reactive despite the fact that their L1 proteins differ in 14 amino acids and suggest that VLPs derived from only one HPV-16 strain could be sufficient for the development of an HPV-16 vaccine and anti-HPV-16 tests.
Sudden Increase in Isolation of Group B Streptococci, Serotype V, Is Not Due to Emergence of a New Pulsed-Field Gel Electrophoresis Type
Authors: Elliott J. A. , Farmer K. D. , Facklam R. R.
Published: December 2019

Until recently, group B streptococcus, serotype V (GBS-V), was an infrequent cause of disease. It is now recognized as a significant cause of infections in both children and adults. To determine if this increase was due to the recent introduction and spread of a single clone of GBS-V, we analyzed, by pulsed-field gel electrophoresis (PFGE), the
Sma
I chromosomal DNA digests of 45 bacteria: 41 isolated from human infections between 1986 and 1996 in the United States, 2 from human infections in Argentina, and 2 from naturally infected mice. Seventeen patterns were found and arbitrarily designated patterns A to Q. Pattern N constituted 24 (53%) of the isolates and was found in all of the years tested and from all surveillance areas, as well as in both isolates from Argentina, and was very similar to the GBS-V isolated from a mouse. Pattern P was found in three isolates, pattern F was found in two, and the remaining patterns were found in one isolate each. We concluded that the majority of isolates of GBS-V are of one PFGE subtype and that this subtype was predominate before the increase in disease caused by GBS-V and that GBS-V disease is caused by several different subtypes.
Differentiation of
Rhizomucor
Species by Carbon Source Utilization and Isoenzyme Analysis
Authors: Vastag Mária , Papp Tamás , Kasza Zsolt , Vágvölgyi Csaba
Published: December 2019

Nineteen
Rhizomucor miehei
and
Rhizomucor pusillus
isolates were assayed for their ability to utilize 87 various substrates as a single carbon source. Besides a difference in sucrose utilization, distinctive differences were found in the utilization of glycine, phenylalanine, and β-alanine. Five isoenzyme systems also proved useful for the determination of markers of distinctive value at a species level. Data were used to obtain information about the genetic polymorphism of these species: a high degree of variability was found among the
R. pusillus
isolates, whereas the group of
R. miehei
isolates was more homogeneous genetically.
Associations between Heat-Stable (O) and Heat-Labile (HL) Serogroup Antigens of
Campylobacter jejuni
: Evidence for Interstrain Relationships within Three O/HL Serovars
Authors: Jackson C. J. , Fox A. J. , Jones D. M. , Wareing D. R. A. , Hutchinson D. N.
Published: December 2019

A comparative examination of the heat-stable (O) and heat-labile (HL) serogrouping results for 9,024 sporadic human isolates of
Campylobacter jejuni
revealed conserved associations between specific O and HL antigens (O/HL serovars). Forty-nine percent of the isolates which grouped for both O and HL antigens belonged to one of three serovars: O 4 complex/HL 1 (17.9%), O 1/HL 2 (16.8%), or O 50/HL 7 (14.5%). Other common serovars were O 2/HL 4 (8.3%), O 6/HL 6 (8.1%), O 53/HL 11 (4.5%), O 19/HL 17 (3.3%), O 5/HL 9 (3.3%), O 9/HL 9 (3.2%), and O 23/HL 5 (3.1%). These 10 serovars accounted for 83.1% of the serogroupable isolates. A large number of strains (41.3%) could be typed by only one of the two methods or could not be serogrouped (11%). Strains belonging to three serovars, O 2/HL 4, O 50/HL 7, and O 23/HL 5, were further characterized by combining data from expressed features (O/HL serogroups, phage groups, and biotypes) with restriction fragment length polymorphism genotypes. These polyphasic data demonstrated that within each serovar, individual isolates showed substantial conservation of both genomic and phenotypic characteristics. The essentially clonal nature of the three serovars confirmed the potential of combined O and HL serogrouping as a practical and phylogenetically valid method for investigating the epidemiology of sporadic
C. jejuni
infection.
Reduction of Diagnostic Window by New Fourth-Generation Human Immunodeficiency Virus Screening Assays
Authors: Weber Bernard , Mbargane Fall El Hadji , Berger Annemarie , Doerr Hans Wilhelm
Published: December 2019

In order to reduce the diagnostic window between the time of human immunodeficiency virus (HIV) infection and laboratory diagnosis, new screening enzyme-linked immunosorbent assays (ELISAs) which permit the simultaneous detection of HIV antigen and antibody have been developed. Two fourth-generation assays, HIV DUO (Biomérieux) and HIV Combi (Boehringer Mannheim), for the combined detection of HIV antigen and antibody, were compared with a third-generation assay (HIV-1/HIV-2 3rd Generation Plus enzyme immunoassay [EIA]; Abbott) and a p24 antigen test (HIV-1 Ag monoclonal; Abbott). A total of 17 seroconversion panels, 15 cell culture supernatants infected with different HIV type 1 (HIV-1) subtypes, and 255 potentially cross-reactive serum samples were tested. Ten seroconversions were detected an average of 8.1 days earlier with HIV DUO and 7.5 days earlier with HIV Combi than with the third-generation ELISA. Overall, in the 17 seroconversion panels tested, HIV DUO detected HIV-1 infection an average of 4.8 days and HIV Combi detected infection an average of 4.4 days earlier than HIV-1/HIV-2 3rd Generation Plus EIA. HIV antigen was detected with HIV DUO and HIV Combi in all of the 15 cell culture supernatants infected with different HIV-1 subtypes, including subtype O. With fourth-generation assays, considerably fewer false-positive results (
n
= 4 to 6) were obtained, in comparison with the third-generation EIA (
n
= 18). Fourth-generation assays permit an earlier diagnosis of HIV infection than third-generation antibody screening assays through the detection of p24 antigen, which may be present in serum samples from individuals with recent HIV infection prior to seroconversion.
Molecular Characterization of Pneumococcal Nasopharynx Isolates Collected from Children during Their First 2 Years of Life
Authors: Sluijter Marcel , Faden Howard , de Groot Ronald , Lemmens Nicole , Goessens Wil H. F. , van Belkum Alex , Hermans Peter W. M.
Published: December 2019

Pneumococcal colonization was studied in 19 children monitored from birth through the age of 2 years. For this purpose, pneumococcal isolates were characterized by capsular typing, restriction fragment end labeling (RFEL), and penicillin-binding protein (PBP) genotyping. Fifty-eight isolates were collected and were found to belong to 10 capsular types, 31 RFEL types, and 7 PBP genotypes. Thirty-nine percent of the isolates had reduced susceptibility to penicillin. All seven highly resistant strains (MICs, >1 μg/ml) were identical to the pandemic clone 23F. Children were culture positive between one and eight times at 13 scheduled visits. Although the infants were frequently recolonized with different strains, colonization with one particular strain often persisted for several months. Isolation of a previously detected capsular type was common, and the chromosomal homogeneity tended to be high when it occurred. Horizontal transfer of capsular genes between strains of different RFEL types was demonstrated in one child. The ecological advantage of transfer of capsular genes is unclear unless survival of the organism on a mucosal surface may be linked to immunoprotective pressure against particular capsular types.
Relevant Criteria for Detecting Microsporidia in Stool Specimens
Authors: Chioralia Gabriela , Trammer Thomas , Kampen Helge , Seitz Hanns M.
Published: December 2019

By using different staining techniques, 479 stool specimens from 212 diarrheic patients with AIDS were examined for microsporidian spores. Calcofluor fluorescence staining of 119 specimens revealed fluorescent ovoid structures of microsporidian size. Staining of these samples according to the method of Weber et al. (R. Weber, R. T. Bryan, R. L. Owen, C. M. Wilcox, L. Gorelkin, and G. S. Visvesvara, N. Engl. J. Med. 326:161–166, 1992) with trichrome produced six specimens with pinkish spores containing the characteristic microsporidian belt-like structure. The 6 specimens were processed for transmission electron microscopy, as were another 21 specimens which did not present the belt-like structure after trichrome staining but which looked highly suspicious after fluorescence staining. In these 21 samples, only fungal spores and, particularly, bacterial
Clostridium
spores were demonstrated, whereas in the 6 samples diagnosed positive after trichrome staining, the existence of microsporidia could be verified by electron microscopy. Based on our observations, we propose that the belt-like structure seen with the Weber stains in microsporidian spores corresponds to structures existing in priming-stage spores. The results suggest that routine microscopical fecal diagnosis for microsporidian infection should include a screening by fluorescence staining and, subsequently, a confirmatory viewing of fluorescence-positive samples after trichrome staining.
Genotypic Characterization of
Salmonella enteritidis
Phage Types by Plasmid Analysis, Ribotyping, and Pulsed-Field Gel Electrophoresis
Authors: Ridley A. M. , Threlfall E. J. , Rowe B.
Published: December 2019

Pulsed-field gel electrophoresis (PFGE) was used to resolve
Xba
I and
Spe
I macrorestriction fragments from 60 defined phage type (PT) reference strains of
Salmonella enteritidis
. The level of discrimination was compared to that afforded by plasmid profile analysis and ribotyping. Twenty-eight distinct
Xba
I pulsed-field profiles (PFPs) were observed, although a single type, PFP X1, predominated. Absence of the 57-kb
spv
-associated fragment was observed for three PT reference strains, and the profile was designated PFP X1A. The
Xba
I macrorestriction profiles of a further four PT reference strains were altered by the presence of plasmid-associated bands. Twenty-six
Spe
I-generated PFPs (plus one subtype) were observed for the same strains. No
Spe
I fragment corresponding to the 38-MDa serovar-specific plasmid was detected. The distribution of
Xba
I and
Spe
I profiles did not always correspond, producing a total of 32 combined PFPs for the 60 PT reference strains. This compared with a total of 18 different plasmid profiles and three
Pvu
II ribotypes generated by the same strains. The results of this study indicate that PFGE may offer an improved level of discrimination over other genotypic typing methods for the epidemiological typing of
S. enteritidis
.