bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiplex PCR for Typing and Subtyping Influenza and Respiratory Syncytial Viruses
Authors: Stockton J. , Ellis J. S. , Saville M. , Clewley J. P. , Zambon M. C.
Published: December 2019

A multiplex reverse transcription (RT)-PCR method that has been developed is capable of detecting and subtyping influenza A (H1N1 and H3N2) and B viruses as well as respiratory syncytial virus (RSV) types A and B in respiratory clinical samples taken as part of a national community-based surveillance program of influenza-like illness in England and Wales. The detection of each different pathogen depended on distinguishing five amplification products of different sizes on agarose gels following RT-PCR with multiple primer sets. The multiplex RT-PCR was tested with 65 nasopharyngeal apirates from which RSV had been isolated and 237 combined nose and throat swabs from which influenza A (H1N1 and H3N2) or B virus had been detected by virus isolation, as well as 40 respiratory samples from which other viruses including cytomegalovirus, herpes simplex virus, enteroviruses, and parainfluenza viruses had been grown. For the typing and subtyping of influenza A and B viruses and RSV types A and B, the multiplex RT-PCR gave an excellent (100%) correlation with the results of conventional typing and subtyping with specific antisera. Multiplex RT-PCR can also be used to accurately detect more than one viral template in the same reaction mixture, allowing viral coinfections to be identified with the same respiratory specimen.
Use of Molecular and Reference Susceptibility Testing Methods in a Multicenter Evaluation of MicroScan Dried Overnight Gram-Positive MIC Panels for Detection of Vancomycin and High-Level Aminoglycoside Resistances in Enterococci
Authors: Chen Yao-Shen , Marshall S. A. , Winokur P. L. , Coffman S. L. , Wilke W. W. , Murray P. R. , Spiegel C. A. , Pfaller M. A. , Doern G. V. , Jones R. N.
Published: December 2019

Modified MicroScan gram-positive MIC no. 8 panels (PM-8) were analyzed for their improved ability to detect vancomycin resistance (VR) and high-level aminoglycoside resistance (HLAR) in enterococci. A validation study design that utilized selected challenge strains, recent clinical isolates, and reproducibility experiments in a multicenter format was selected. Three independent medical centers compared the commercial panels to reference broth microdilution panels (RBM) and Synergy Quad Agar (QA). Resistance was verified by demonstration of VR and HLAR genes by PCR tests. The study was conducted in three phases. (i) In the challenge phase (CP), two well-characterized sets of enterococci were obtained from the Centers for Disease Control and Prevention; one set contained 50 isolates for VR testing and one contained 48 isolates for HLAR testing. In addition, a set of 47 well-characterized isolates representing diverse geographic areas, obtained from earlier national surveillance studies, was tested at the University of Iowa College of Medicine (UICM). (ii) In the efficacy phase (EP), each laboratory tested 50 recent, unique clinical isolates by all methods. (iii) In the reproducibility Phase (RP), each laboratory tested the same 10 strains by all methods in triplicate on three separate days. All isolates from the EP were sent to the UICM for molecular characterization of
vanA
, -
B
, -

C
1

, -

C
2–3

, and HLAR genes. In the CP, the ranking of test methods by error rates (in parentheses; very major and major errors combined, versus PCR results) were as follows: for high-level streptomycin resistance (HLSR), QA (12.0%) > PM-8 (5.2%) > RBM (1.6%); for high-level gentamicin resistance (HLGR), RBM (3.7%) > PM-8 (3.1%) > QA (2.6%); and for VR, RBM = QA (3.0%) > PM-8 (1.2%). In the EP, agreement between all methods and the reference PCR result was 98.0% for HLSR, 99.3% for HLGR, and 98.6% for VR. In the RP, the percentages of results ± 1 log
2
dilution of the all-participant mode were as follows: for VR, 100% (PM-8), 98.9% (QA), and 90.0% (RBM); for HLSR, 99.6% (RBM), 98.5% (PM-8), and 82.2% (QA); and for HLGR, 99.6% (RBM), 99.3% (PM-8), and 98.1% (QA). The ability of the PM-8 to detect VR and HLAR in enterococci was comparable to those for reference susceptibility and molecular PCR methods and was considered acceptable for routine clinical laboratory use.
Detection of
Candida dubliniensis
in Oropharyngeal Samples from Human Immunodeficiency Virus-Infected Patients in North America by Primary CHROMagar Candida Screening and Susceptibility Testing of Isolates
Authors: Kirkpatrick William R. , Revankar Sanjay G. , Mcatee Robert K. , Lopez-Ribot Jose L. , Fothergill Annette W. , McCarthy Dora I. , Sanche Stephen E. , Cantu Rebecca A. , Rinaldi Michael G. , Patterson Thomas F.
Published: December 2019

Candida dubliniensis
has been associated with oropharyngeal candidiasis in patients infected with human immunodeficiency virus (HIV).
C. dubliniensis
isolates may have been improperly characterized as atypical
Candida albicans
due to the phenotypic similarity between the two species. Prospective screening of oral rinses from 63 HIV-infected patients detected atypical dark green isolates on CHROMagar Candida compared to typical
C. albicans
isolates, which are light green. Forty-eight atypical isolates and three control strains were characterized by germ tube formation, differential growth at 37, 42, and 45°C, identification by API 20C, fluorescence, chlamydoconidium production, and fingerprinting by Ca3 probe DNA hybridization patterns. All isolates were germ tube positive. Very poor or no growth occurred at 42°C with 22 of 51 isolates. All 22 poorly growing isolates at 42°C and one isolate with growth at 42°C showed weak hybridization of the Ca3 probe with genomic DNA, consistent with
C. dubliniensis
identification. No
C. dubliniensis
isolate but only 18 of 28
C. albicans
isolates grew at 45°C. Other phenotypic or morphologic tests were less reliable in differentiating
C. dubliniensis
from
C. albicans
. Antifungal susceptibility testing showed fluconazole MICs ranging from ≤0.125 to 64 μg/ml. Two isolates were resistant to fluconazole (MIC, 64 μg/ml) and one strain was dose dependent susceptible (MIC, 16 μg/ml). MICs of other azoles, including voriconazole, itraconazole, and SCH 56592, for these isolates were lower.
C. dubliniensis
was identified in 11 of 63 (17%) serially evaluated patients. Variability in phenotypic characteristics dictates the use of molecular and biochemical techniques to identify
C. dubliniensis
. This study identifies
C. dubliniensis
in HIV-infected patients from San Antonio, Tex., and shows that
C. dubliniensis
is frequently detected in those patients by using a primary CHROMagar screen.
Direct Detection of Respiratory Syncytial Virus, Parainfluenza Virus, and Adenovirus in Clinical Respiratory Specimens by a Multiplex Reverse Transcription-PCR Assay
Authors: Osiowy Carla
Published: December 2019

Diagnosis of respiratory virus infections currently involves detection by isolation or antigen detection, which usually identifies only a single suspected agent. To permit identification of more than one respiratory virus in clinical specimens, a rapid detection method involving a single-step, multiplex reverse transcription-PCR (RT-PCR) assay was developed. The assay included five primer sets that amplified the RNA of respiratory syncytial virus subtypes A and B, parainfluenza virus types 1, 2, and 3, and adenovirus types 1 to 7. Initially the assay was tested on tissue culture-grown virus and was found to be specific for all 12 prototype viruses tested, with no interassay cross amplification or amplification of other respiratory viruses. Assay sensitivity allowed a detection range of 0.2 50% tissue culture infectious dose (TCID
50
) for adenovirus to 250 TCID
50
for parainfluenza virus type 1. The multiplex RT-PCR assay was also able to directly detect viruses in respiratory specimens, with virus being detected in 41 of 112 samples as compared to 34 of 112 samples detected by direct immunofluorescence or antigen detection following specimen culture. This suggests that the multiplex RT-PCR assay can be used as a rapid and sensitive diagnostic method for major respiratory viruses.
Comparison of Phenotypic and Genotypic Methods for Detection of Diphtheria Toxin among Isolates of Pathogenic Corynebacteria
Authors: Efstratiou Androulla , Engler Kathryn H. , Dawes Charlotte S. , Sesardic Dorothea
Published: December 2019

We have compared molecular, immunochemical, and cytotoxic assays for the detection of diphtheria toxin from 55 isolates of
Corynebacterium diphtheriae
and
Corynebacterium ulcerans
originally isolated in five different countries. The suitabilities and accuracies of these assays for the laboratory diagnosis of diphtheria were compared and evaluated against the “gold standard” in vivo methods. The in vivo and Vero cell cytotoxicity assays were accurate in their abilities to detect diphtheria toxin but were time-consuming; however, the cytotoxicity assay is a suitable in vitro alternative to the in vivo virulence test. There was complete concordance between all the phenotypic methods. Genotypic tests based upon PCR were rapid; however, PCR must be used with caution because some isolates of
C. diphtheriae
possessed toxin genes but failed to express a biologically active toxin. Therefore, phenotypic confirmation of toxigenicity for the microbiological diagnosis of diphtheria is recommended.
Characterization of a Type-Common Human Recombinant Monoclonal Antibody to Herpes Simplex Virus with High Therapeutic Potential
Authors: De Logu Alessandro , Williamson R. Anthony , Rozenshteyn Roman , Ramiro-Ibañez Fernando , Simpson Cindy D. , Burton Dennis R. , Paolo Sanna Pietro
Published: December 2019

We report the characterization of a type-common human recombinant monoclonal antibody previously isolated by antigen selection from a phage-displayed combinatorial antibody library established from a herpes simplex virus (HSV)-seropositive individual. Competition with well-characterized murine monoclonal antibodies and immunodetection of gD truncations revealed that this antibody recognizes the group Ib antigenic site of glycoprotein D, a highly conserved and protective type-common determinant. To our knowledge, this is the first human group Ib monoclonal antibody ever described. The antibody also displayed first-order neutralization kinetics and a high neutralization rate constant, was capable of completely inhibiting syncytium formation by a fusogenic strain of HSV type 1, and efficiently neutralized low-passage clinical isolates of both HSV serotypes. Taken together with our earlier observations of the in vivo antiviral activities of this human recombinant antibody in animal models of HSV infection, the present results support the high therapeutic potential of this antibody.
Detection of
Ureaplasma urealyticum
by PCR and Biovar Determination by Liquid Hybridization
Authors: Povlsen K. , Jensen J. S. , Lind I.
Published: December 2019

An assay which combines the direct detection of
Ureaplasma urealyticum
with biovar determination was developed and applied to 618 urogenital specimens.
U. urealyticum
was detected by inhibitor-controlled PCR. A 429-bp fragment of the urease gene was amplified. The amplicons were labelled with digoxigenin during PCR. Biovar determination was performed by liquid hybridization with biotin-labelled biovar-specific probes, and the hybrids were detected with peroxidase-conjugated sheep anti-digoxigenin immunoglobulin G Fab fragments. Results of PCR and culture for 453 urogenital specimens from women and 105 urethral specimens from men could be compared. Among the specimens from women, 63% were PCR positive as well as culture positive, 0.9% were positive only by PCR, and 4% were positive only by culture. Among the specimens from men, 15% were PCR positive as well as culture positive, 1% were positive only by PCR, and 9% were positive only by culture. By using culture as the reference method, the PCR had a sensitivity of 94% and a specificity of 98% when applied to specimens from women and a sensitivity of 64% and a specificity of 99% when applied to specimens from men. Overall, 80% of the PCR-positive specimens contained biovar 1,13.5% contained biovar 2, and 6.5% contained both biovars.
Serratia ficaria
: a Misidentified or Unidentified Rare Cause of Human Infections in Fig Tree Culture Zones
Authors: Anahory T. , Darbas H. , Ongaro O. , Jean-Pierre H. , Mion P.
Published: December 2019

Serratia ficaria
, an enterobacterium involved in the fig tree ecosystem, has been isolated from human clinical samples in rare instances, and its role as a pathogen is unclear. In 7 years, we have isolated
S. ficaria
from seven patients; it was the only pathogen in 4 patients, including a patient with septicemia described previously and three patients with gallbladder empyemas described in the present report. From March 1995 to July 1997, the incidence of biliary infections due to
S. ficaria
was 0.7%. We discuss the digestive carriage of this bacterium and its epidemiology with respect to the fig tree life cycle. Since fig trees grow around the Mediterranean as well as in the United States (California, Louisiana, Hawaii),
S. ficaria
should be more frequently isolated. In our experience, various strains have been misidentified or unidentified by commercial systems. Incorrect identification could be an additional explanation for the paucity of reported cases.
S. ficaria
produces nonpigmented, lactose-negative colonies which give off a potatolike odor. This odor is the primary feature of
S. ficaria
and must prompt reexamination of the identifications proposed by commercial systems. We tested 42 novel strains using three commercial systems: Vitek gram-negative identification (GNI) cards and API 20E and ID 32E strips (bioMérieux, Marcy-l’Etoile, France). The percentages of positivity that we have obtained were lower than those published previously for the following characteristics: lipase, gelatinase, DNase, and rhamnose. The best system for the recognition of
S. ficaria
is ID 32E, which correctly identified 27 of 42 strains. The API 20E system gave correct identifications for only two strains.
S. ficaria
was not present in the Vitek GNI card system database.
Comparison of Genomic Methods for Differentiating Strains of
Enterococcus faecium
: Assessment Using Clinical Epidemiologic Data
Authors: Savor Connie , Pfaller Michael A. , Kruszynski Julie A. , Hollis Richard J. , Noskin Gary A. , Peterson Lance R.
Published: December 2019

Genomic DNA extracted from 45 vancomycin-resistant
Enterococcus faecium
(VRE) isolates was cleaved with
Hin
dIII and
Hae
III and subjected to agarose gel electrophoresis. The ability of this method (restriction endonuclease analysis [REA]) to distinguish strains at the subspecies level was compared with results previously determined by pulsed-field gel electrophoresis (PFGE). Chart reviews were performed to provide a clinical correlation of possible epidemiologic relatedness. A likely clinical association was found for 29 patients as part of two outbreaks. REA found 21 of 21 isolates were the same type in the first outbreak, with PFGE calling 19 strains the same type. In the second outbreak with eight patient isolates,
Hin
dIII found six were the same type and two were unique types.
Hae
III found three strains were the same type, two strains were a separate type, and three more strains were unique types, while PFGE found three were the same type and five were unique types. No single “ideal” method can be used without clinical epidemiologic investigation, but any of these techniques is helpful in providing focus to infection control practitioners assessing possible outbreaks of nosocomial infection.
Two-Color Hybridization Assay for Simultaneous Detection of
Bordetella bronchiseptica
and Toxigenic
Pasteurella multocida
from Swine
Authors: Register Karen B. , Lee Ruby M. , Thomson Cindy
Published: December 2019

Bordetella bronchiseptica
and toxigenic
Pasteurella multocida
are the etiologic agents of swine atrophic rhinitis. Methods currently used for their identification are time-consuming and suffer from a lack of sensitivity. We describe a colony lift-hybridization assay for detection of
B. bronchiseptica
and toxigenic
P. multocida
that can be performed with a single colony lift derived from a primary isolation plate without the need for pure subcultures of suspect bacteria. Membranes are hybridized simultaneously to probes derived from the
B. bronchiseptica alcA
gene and the
P. multocida toxA
gene. A multicolor development procedure permits sequential detection of bound probes. The assay was tested with 84 primary isolation plates generated from nasal swabs from swine with clinical signs of atrophic rhinitis. Comparison of the results from the colony lift-hybridization assay with those from conventional testing, based on a combination of colony morphology, biochemical reactions, mouse lethality, and enzyme-linked immunosorbent assay, indicated that the colony lift assay has superior sensitivity and comparable specificity. This technique has wide application for diagnostic and experimental studies.
Evaluation of Strategies for Molecular Fingerprinting for Use in the Routine Work of a Mycobacterium Reference Unit
Authors: Wilson Stuart M. , Goss Stephen , Drobniewski Francis
Published: December 2019

We have investigated the role of two rapid PCR-based typing methods, IS
6110
-based PCR and spacer-oligonucleotide typing, within a national tuberculosis reference service. The validity of clusters with IS
6110
restriction fragment length polymorphism fingerprints with less than 6 bands was also investigated in the context of referred isolates.
First Isolation of
Trichophyton fischeri
in the United States
Authors: Rosenthal S. A. , Scott J. S. , Summerbell R. C. , Kane J.
Published: December 2019

A 49-year-old male with
Pneumocystis carinii
pneumonia was seen at Bellevue Hospital in New York, N.Y. Sputum samples yielded cultures of
Candida lusitaniae
,
Mycobacterium avium
, and a filamentous fungus,
Trichophyton fischeri. T. fischeri
is a nonpathogenic fungus which resembles the dermatophyte
Trichophyton rubrum
. This is the first record of the species from U.S. sources. This case exemplifies the ecological differences between
T. fischeri
and
T. rubrum
and illustrates how correct identification of the former species can minimize diagnostic confusion. The two species are distinguished from each other by the type of growth on Casamino Acids-erythritol-albumin agar and by micromorphological differences.
Enhancement of the AMPLICOR Enterovirus PCR Test with a Coprecipitant
Authors: Taggart E. William , Byington Carrie L. , Hillyard David R. , Robison John E. , Carroll Karen C.
Published: December 2019

The incorporation of a commercially available coprecipitant into the AMPLICOR enterovirus PCR test specimen preparation enhanced the sensitivity and reproducibility of this assay. Fifty-five previously tested archived cerebrospinal fluids (CSF) specimens were tested in a blind study in duplicate with and without Pellet Paint coprecipitant (Novagen, Inc., Madison, Wis.). Of these specimens, 26 had previously been determined to be positive and 29 had previously been determined to be negative. All previously positive CSF specimens were positive when Pellet Paint was used and only 18 were positive without Pellet Paint. No previously negative specimens were positive on repeat testing with or without Pellet Paint. The background signal was not affected by the addition of Pellet Paint. These data support the utility of a coprecipitant in minimizing false-negative results.
Evaluation of a Leukocyte Stabilization Reagent for Use in the Cytomegalovirus pp65 Antigenemia Assay
Authors: Bush Charlene E. , Sluchak-Carlsen Julia A.
Published: December 2019

New erythrocyte lysis and leukocyte stabilization reagents (Streck Laboratories, Inc.) were tested in the cytomegalovirus pp65 antigenemia assay, to determine if whole-blood processing time could be delayed to 24 h postdraw. The combination of these reagents gave results comparable to those for patient samples processed immediately after blood draw.
Correlation between Presence of Viable Bacteria and Presence of Endotoxin in Middle-Ear Effusions
Authors: Dingman Jeffrey R. , Rayner Mark G. , Mishra Suman , Zhang Yingze , Ehrlich Miles D. , Post J. Christopher , Ehrlich Garth D.
Published: December 2019

The presence of endotoxin (detected by the
Limulus
amebocyte lysate assay) was compared to the presence of viable
Haemophilus influenzae
and
Moraxella catarrhalis
(detected by PCR) in 106 middle-ear effusions from pediatric patients with chronic otitis media. Endotoxin was found in 81 of the 106 specimens. Of these 81 specimens, 66 (81.5%) also tested positive for one or both of the gram-negative bacteria
H. influenzae
and
M. catarrhalis
. The data suggest that viable gram-negative bacteria, detectable by PCR but often undetectable by culture, may be the source of endotoxin in middle-ear effusions.
Fluconazole Disk Diffusion Susceptibility Testing of
Candida
Species
Authors: Kirkpatrick William R. , Turner Thomas M. , Fothergill Annette W. , McCarthy Dora I. , Redding Spencer W. , Rinaldi Michael G. , Patterson Thomas F.
Published: December 2019

We describe a simple procedure for detecting fluconazole-resistant yeasts by a disk diffusion method. Forty clinical
Candida
sp. isolates were tested on RPMI-glucose agar with either 25- or 50-μg fluconazole disks. With 25-μg disks, zones of inhibition of ≥20 mm at 24 h accurately identified 29 of 29 isolates for which MICs were ≤8 μg/ml, and with 50-μg disks, zones of ≥27 mm identified 28 of 29 such isolates. All 11 isolates for which MICs were >8 μg/ml were identified by using either disk. Disk diffusion may be a useful screening method for clinical microbiology laboratories.
Constant Low Rate of Fungemia in Norway, 1991 to 1996
Authors: Sandven Per , Bevanger Lars , Digranes Asbjørn , Gaustad Peter , Haukland Hanne H. , Steinbakk Martin ,   The Norwegian Yeast Study Group
Published: December 2019

Since 1991 information on yeast isolates from blood cultures has been recorded prospectively from all microbiological laboratories (5 university and 16 county or local hospital laboratories) in Norway (population, 4.3 million). From 1991 to 1996 a total of 571 episodes of fungemia in 552 patients occurred (1991, 109 episodes; 1992, 81 episodes; 1993, 93 episodes; 1994, 89 episodes; 1995, 98 episodes; and 1996, 101 episodes). The fungemia rates per 10,000 patient days were 0.29 in 1991 and 0.27 in 1996. The average rates for the years 1991 to 1996 were 0.37 for the university laboratories and 0.20 for the other laboratories. These rates are low compared to the rate (0.76) in five Dutch university hospitals in 1995 and the rate (2.0) in Iowa in 1991. The four most frequently isolated species were
Candida albicans
(66%),
Candida glabrata
(12.5%),
Candida parapsilosis
(7.6%), and
Candida tropicalis
(6.4%). The incidences of both
C. albicans
(range, 63 to 73%) and
C. glabrata
(range, 8.4 to 15.7%) varied somewhat throughout this period, but no significant increase or decrease was noted. MICs of amphotericin B, flucytosine, and fluconazole were determined for 89% of the isolates. All were susceptible to amphotericin B, and only 29 (5.6%) strains had decreased susceptibility to flucytosine. All
C. albicans
isolates were susceptible to fluconazole. The percentage of yeast isolates with decreased susceptibility to fluconazole (MICs, ≥16 μg/ml) did increase, from 9.6% in 1991 and 1992 to 12.2% in 1994, 16.1% in 1995, and 18.6% in 1996. This was largely due to increases in the percentages of resistant
C. glabrata
and
Candida krusei
strains in the last 2 years. Compared to the incidence in other countries, it is remarkable that Norway has such a low and constant incidence of fungemia. A possible reason for this difference might be a restricted antibiotic use policy in Norway.
Evaluation of the Abbott LCx Assay for Detection of
Neisseria gonorrhoeae
in Endocervical Swab Specimens from Females
Authors: Kehl Sue C. , Georgakas Kristina , Swain Geoffrey R. , Sedmak Gerald , Gradus Stephen , Singh Ajaib , Foldy Seth
Published: December 2019

The Abbott LCx
Neisseria gonorrhoeae
assay (Abbott Laboratories, Abbott Park, Ill.) uses a ligase chain reaction (LCR) amplification in the LCx probe system for detection of a specific nucleotide sequence in the Opa-encoding gene of
N. gonorrhoeae
. We evaluated the LCx assay in a comparison with conventional culture employing modified Thayer-Martin media for the detection of
N. gonorrhoeae
from female endocervical specimens obtained from patients attending a sexually transmitted disease clinic. Discordantly LCR-positive and culture-negative specimens were further evaluated by testing with another LCR assay which used an
N. gonorrhoeae
-specific pilin probe. Specimens positive by both LCR assays were considered confirmed LCx-positive specimens. A specimen was considered to contain
N. gonorrhoeae
when it was either culture positive or culture negative and confirmed LCx positive. A total of 403 female endocervical specimens were evaluated. The prevalence of
N. gonorrhoeae
in this population was 8.7%. The sensitivity and specificity of the LCx assay were 94.3 and 99.4%, and those of culture were 77.1 and 100%, respectively. The Abbott LCx assay is a rapid, sensitive method for detection of
N. gonorrhoeae
in female endocervical specimens.
In Vitro Comparison of NALC-NaOH, Tween 80, and C
18
-Carboxypropylbetaine for Processing of Specimens for Recovery of Mycobacteria
Authors: Thornton Charles G. , MacLellan Kerry M. , Brink Thomas L. , Passen Selvin
Published: December 2019

A recent article (C. G. Thornton et al., J. Clin. Microbiol. 36:1996–2003, 1998) reported a new specimen-processing method for improved recovery of mycobacteria. This method used C
18
-carboxypropylbetaine (CB-18) and increased both smear and culture sensitivity. The companion article (C. G. Thornton et al., J. Clin. Microbiol. 36:2004–2013, 1998) described initial improvements to this method. Additional significant parameters of the CB-18 processing method are identified herein. First, eliminating the incubation step was shown to further improve culture sensitivity. Subsequently, recovery of several mycobacterial isolates by the CB-18 method was compared to a contemporary processing method that combines NALC and NaOH (NALC-NaOH) and a Tween 80-based method. Recovery of the tuberculous isolates following NALC-NaOH processing averaged 20% and ranged from 1.6 to 45%, whereas recovery of the nontuberculous isolates averaged 11% and ranged from 0.1 to 55%. Recovery of the tuberculous and nontuberculous isolates by the Tween 80-based method ranged from 22 to 92% and 27 to 93%, respectively, with averages of 58 and 65%, respectively. Recovery of the tuberculous and nontuberculous mycobacteria following CB-18 processing averaged 86 and 73%, respectively, with ranges from 61 to over 100% and from 43 to over 100%, respectively. Other parameters of the CB-18 method were also examined, including recovery versus CB-18 concentration and the relationship between CB-18 concentration and the tuberculocidal effect. The tuberculocidal effect was time dependent but independent of concentration, whereas recovery was directly proportional to concentration. Increasing the CB-18 concentration to 4 mM provided quantitative recovery on solid medium; however, higher concentrations of CB-18 were not compatible with liquid culture. Examination of the relationship between increasing CB-18 and lecithin concentrations suggested that lecithin could not overcome the deleterious effects of CB-18 in liquid culture at these higher concentrations.
PCR-Enzyme Immunoassay for Detection of
Streptococcus pneumoniae
DNA in Cerebrospinal Fluid Samples from Patients With Culture-Negative Meningitis
Authors: Cherian Thomas , Lalitha M. K. , Manoharan Anand , Thomas Kurien , Yolken Robert H. , Steinhoff Mark C.
Published: December 2019

A PCR-based assay was developed to amplify a conserved region of the pneumococcal autolysin gene. The amplified product was labelled with digoxigenin-labelled dUTP and was detected with a biotin-labelled probe in an enzyme immunoassay (EIA). The assay was initially tested with suspensions of various serotypes of
Streptococcus pneumoniae
and other gram-positive and gram-negative bacteria and was then applied to cerebrospinal fluid (CSF) specimens from patients with meningitis and those with other neurological disorders. The assay detected all the serotypes of
S. pneumoniae
tested, whereas all the other bacterial strains tested were negative. Seven of the 8 CSF specimens positive for pneumococcus by culture or latex agglutination (LA) were positive by PCR-EIA, whereas all 10 specimens positive for other organisms were negative. Among 11 patients with clinically diagnosed meningitis but with negative culture and LA results, 5 were positive by PCR-EIA. The assay was negative for all but one patient without meningitis; it was positive with the CSF from a child with immunodeficiency and pneumococcal abscesses on the scalp. PCR-EIA is a useful tool for the diagnosis of meningitis, especially when culture and LA are negative because of prior antibiotic treatment.
Nasal Carriage of
Staphylococcus aureus
and Epidemiology of Surgical-Site Infections in a Sudanese University Hospital
Authors: Ahmed Abdalla O. A. , van Belkum Alex , Fahal Ahmed H. , Elnor A. E. Abu , Abougroun El Sir A. M. , VandenBergh Marjolein F. Q. , Zijlstra Ed E. , Verbrugh Henri A.
Published: December 2019

Surgical site infections (SSI) due to
Staphylococcus aureus
among 256 male and 158 female patients (mean age, 28 years) undergoing elective surgery at the Soba University Hospital (Khartoum, Sudan) were studied. During an 11-month study period all patients were analyzed for nasal carriage of
S. aureus
at the time of admission. Follow-up of the development of SSI proceeded until 4 weeks after the operations. In addition, nasal swabs were obtained periodically during the same period from 82 members of the staff. In order to discriminate autoinfection from cross infection, bacterial isolates were typed by random amplification of polymorphic DNA (RAPD), pulsed-field gel electrophoresis (PFGE) of DNA macrorestriction fragments, and restriction fragment length polymorphism analysis of the protein A and coagulase genes. Preoperative cultures revealed the presence of
S. aureus
in the noses of 98 patients (24%). The overall number of postsurgical wound infections in the entire group was 57 (14%), 24 of which were due to
S. aureus
. Only 6 of the 98 nasal
S. aureus
carriers suffered from wound infections by the same species. In these six cases the infecting strain could not be genetically discriminated from the nasal inhabitant, substantiating autoinfection. However, nasal carriage of
S. aureus
is not a significant risk factor for the development of SSI in this setting (6 of 98 patients with autoinfection versus 18 of 316 patients [414 − 98 patients] with cross infection;
P
= 0.81), most probably due to the fact that noncarriers are at a significant and relatively large risk for acquiring an independent
S. aureus
SSI. The other
S. aureus
strains causing SSI showed a high degree of genetic heterogeneity, demonstrating that it is not an epidemic strain that is causing the SSI. Among the staff personnel screened, 47.4% did not carry
S. aureus
in the nose at any time during the study period, whereas 13.2% persistently carried a single strain in the nose. Another 39.5% could be classified as intermittent carriers. When strains derived from staff personnel were genetically typed, it was demonstrated that most of the strains represented genetic variants clearly differing from the isolates causing SSI. On the other hand, possible cross colonization among staff personnel and even cross infection from staff personnel to patients or from patient to patient were demonstrated in some cases, but epidemic spread of a single strain or a few clonally related strains of
S. aureus
could be excluded.
Corynebacterium macginleyi
Has to Date Been Isolated Exclusively from Conjunctival Swabs
Authors: Funke Guido , Pagano-Niederer Maja , Bernauer Wolfgang
Published: December 2019

Fifteen strains of
Corynebacterium macginleyi
were exclusively isolated from conjunctival swabs of patients with either conjunctivitis or corneal ulcers. Up to now, only three
C. macginleyi
strains had been described in the literature. The characteristics of the 15 patients from whom
C. macginleyi
was isolated are outlined, characteristics useful for the identification of
C. macginleyi
are described, and the antimicrobial susceptibility pattern of the species is provided.
C. macginleyi
is uniformly susceptible to penicillins, quinolones, and aminoglycosides. Although considered to be of rather low pathogenicity
C. macginleyi
seems to have the potential to cause superinfections in selected patients with ocular surface problems.
Typing of Human
Mycobacterium avium
Isolates in Italy by IS
1245
-Based Restriction Fragment Length Polymorphism Analysis
Authors: Lari Nicoletta , Cavallini Michela , Rindi Laura , Iona Elisabetta , Fattorini Lanfranco , Garzelli Carlo
Published: December 2019

All but 2 of 63
Mycobacterium avium
isolates from distinct geographic areas of Italy exhibited markedly polymorphic, multibanded IS
1245
restriction fragment length polymorphism (RFLP) patterns; 2 isolates showed the low-number banding pattern typical of bird isolates. By computer analysis, 41 distinct IS
1245
patterns and 10 clusters of essentially identical strains were detected; 40% of the 63 isolates showed genetic relatedness, suggesting the existence of a predominant AIDS-associated IS
1245
RFLP pattern.
Multicenter Evaluation of a Modified Protocol for the RAPIDEC Staph System for Direct Identification of
Staphylococcus aureus
in Blood Cultures
Authors: van Griethuysen Arjanne , Buiting Anton , Goessens Wil , van Keulen Peter , Wintermans Rob , Kluytmans Jan
Published: December 2019

A modified protocol for the RAPIDEC Staph system (bioMérieux, Marcy-l’Etoile, France) for direct identification of
Staphylococcus aureus
in blood cultures was evaluated in a multicenter study. A total of 129 blood cultures (BACTEC 9000 Blood Culture System; Becton Dickinson Diagnostic Instrument Systems, Sparks, Md.) containing gram-positive cocci in clusters were analyzed by conventional methods and by RAPIDEC Staph in accordance with the manufacturer’s protocol and in accordance with a modified protocol. The sensitivity, specificity, and positive and negative predictive values obtained with the manufacturer’s protocol were 90.5, 97.7, 95.0, and 95.5%, respectively, and those obtained with the modified protocol were 100, 96.6, 93.3, and 100%, respectively. The modified protocol for the RAPIDEC Staph is easier to perform than the manufacturer’s protocol and is very reliable.
Development of a Scheme for Genotyping
Helicobacter pylori
Based on Allelic Variation in Urease Subunit Genes
Authors: Owen Robert J. , Slater Eleanor R. , Xerry Jacqueline , Peters Tansy M. , Teare E. Louise , Grant Andrew
Published: December 2019

Helicobacter pylori
urease subunit genes in 383 isolates from 10 countries were investigated by PCR-restriction fragment length polymorphism (
Hae
III) analysis. Eighty-two different
ureAB
profiles were documented by reference to known sequences. Variation among 51% of strains was accounted for by 10 predominant patterns, which provided a unique framework for categorizing isolates with geographically diverse origins.
Rapid Differentiation of Closely Related
Candida
Species and Strains by Pyrolysis-Mass Spectrometry and Fourier Transform-Infrared Spectroscopy
Authors: Timmins Éadaoin M. , Howell Susan A. , Alsberg Bjørn K. , Noble William C. , Goodacre Royston
Published: December 2019

Two rapid spectroscopic approaches for whole-organism fingerprinting of pyrolysis-mass spectrometry (PyMS) and Fourier transform-infrared spectroscopy (FT-IR) were used to analyze a group of 29 clinical and reference
Candida
isolates. These strains had been identified by conventional means as belonging to one of the three species
Candida albicans
,
C. dubliniensis
(previously reported as atypical
C. albicans
), and
C. stellatoidea
(which is also closely related to
C. albicans
). To observe the relationships of the 29 isolates as judged by PyMS and FT-IR, the spectral data were clustered by discriminant analysis. On visual inspection of the cluster analyses from both methods, three distinct clusters, which were discrete for each of the
Candida
species, could be seen. Moreover, these phenetic classifications were found to be very similar to those obtained by genotypic studies which examined the
Hin
fI restriction enzyme digestion patterns of genomic DNA and by use of the 27A
C. albicans
-specific probe. Both spectroscopic techniques are rapid (typically, 2 min for PyMS and 10 s for FT-IR) and were shown to be capable of successfully discriminating between closely related isolates of
C. albicans
,
C. dubliniensis
, and
C. stellatoidea
. We believe that these whole-organism fingerprinting methods could provide opportunities for automation in clinical microbial laboratories, improving turnaround times and the use of resources.
Comparison of PCR, Isoenzyme Analysis, and Antigen Detection for Diagnosis of
Entamoeba histolytica
Infection
Authors: Haque Rashidul , Ali I. K. M. , Akther S. , Petri William A.
Published: December 2019

The diagnosis of amebiasis by microscopic identification of the parasite in stool is insensitive and unable to distinguish the invasive parasite
Entamoeba histolytica
from the commensal parasite
E. dispar
. In this study, we have tested a PCR technique for the detection of
E. histolytica
and compared it with isoenzyme analysis and the TechLab
E. histolytica
-specific antigen detection test. The nested-PCR test we used is based on amplification of the small subunit rRNA gene of
E. histolytica
and
E. dispar
followed by restriction digest analysis of the PCR product. Single stool samples were obtained from 98 patients from Dhaka, Bangladesh, with diarrhea: 88 patients diagnosed by microscopy and/or culture with
E. histolytica
and/or
E. dispar
infection and 10 patients without infection. Isoenzyme analysis identified 53 of the infections as
E. histolytica
and 28 as
E. dispar
. PCR and isoenzyme identification of
E. histolytica
agreed in 96% (51 of 53) of amebic cultures. PCR for
E. histolytica
was negative in all 10 samples that were negative for
E. histolytica
by isoenzyme and antigen detection. PCR and antigen detection had comparable sensitivities when performed directly on fresh stool specimens, identifying 87% (46 of 53) and 85% (45 of 53), respectively, of
E. histolytica
infections identified by isoenzyme analysis. The correlation of results by antigen detection and PCR for identification of
E. histolytica
in stool was 93% (45 of 48 cases). Mixed infections with
E. histolytica
and
E. dispar
were detected by PCR in 14% (12 of 88) of cases. In conclusion, all three techniques for specific identification of
E. histolytica
in fresh stool showed excellent correlation. Only the TechLab
E. histolytica
antigen detection test was both rapid and technically simple.
Antibodies against Early Proteins of Human Papillomaviruses as Diagnostic Markers for Invasive Cervical Cancer
Authors: Meschede Wolfgang , Zumbach Klaus , Braspenning Joris , Scheffner Martin , Benitez-Bribiesca Luis , Luande Jeff , Gissmann Lutz , Pawlita Michael
Published: December 2019

Cervical cancer is the most prevalent tumor in developing countries and the second most frequent cancer among females worldwide. Specific human papillomaviruses (HPVs) and, most notably, HPV types 16 and 18 are recognized as being causally associated with this malignancy. Antibodies against early HPV proteins E6 and E7 have been found more often in patients with tumors than in controls. Existing peptide enzyme-linked immunosorbent assays (ELISAs) for the detection of anti-E6 and anti-E7 antibodies in human sera have low levels of sensitivity and specificity and thus are not suitable for use as diagnostic tools. Based on highly purified recombinant native proteins, we developed four sandwich ELISAs for the detection of antibodies against HPV type 16 and 18 E6 and E7 proteins. We demonstrate their sensitivities and high degrees of specificity for cervical cancer. Among a total of 501 serum specimens from unselected patients with invasive cervical cancer, 52.9% reacted positively in at least one of the four assays. In contrast, among 244 serum specimens from control subjects without cervical cancer, only 2 reactive serum specimens (0.8%) were found. For 19 of 19 antibody-positive patients, the HPV type indicated by seroreactivity was identical to the HPV DNA type found in the tumor, which also indicates a high degree of specificity for antibody detection with respect to HPV type. In a direct comparison of 72 serum specimens from patients with cervical cancer, 56% of the specimens reacted in at least one of the four protein ELISAs, whereas 40% reacted in at least one of seven peptide ELISAs covering the four antigens. These assays could be of value for the detection of invasive cervical cancer in settings in which cytology-based early tumor screening is not available, for the clinical management of patients diagnosed with cervical cancer, and for the immunological monitoring of E6 and E7 vaccination trials.
Molecular Fingerprinting of
Mycobacterium tuberculosis
and Risk Factors for Tuberculosis Transmission in Paris, France, and Surrounding Area
Authors: Gutiérrez M. C. , Vincent V. , Aubert D. , Bizet J. , Gaillot O. , Lebrun L. , Le Pendeven C. , Le Pennec M. P. , Mathieu D. , Offredo C. , Pangon B. , Pierre-Audigier C.
Published: December 2019

Forty-three percent of the tuberculosis cases reported in France are from the Ile de France region. The incidence of tuberculosis in this region is 33 cases per 100,000 inhabitants, twice the national average. A restriction fragment length polymorphism (RFLP) analysis was performed with clinical isolates of
Mycobacterium tuberculosis
isolated during 1995 in 10 hospitals in Paris and surrounding areas to detect tuberculosis transmission and define the factors associated with clustering in this population. The molecular markers used were the insertion sequence IS
6110
and the direct repeat (DR) sequence. Social, demographic, and clinical data were collected from the patients’ medical files. Ten patients with isolates with a single copy of IS
6110
were excluded from further analysis. Twenty-four patients with false-positive cultures due to laboratory contamination (based on RFLP analysis with IS
6110
and examination of patient data) were also excluded. The study was then conducted with 272 strains isolated from 272 patients. Further fingerprinting was performed by using the DR element with strains with patterns by RFLP analysis with IS
6110
that differed by one band only and strains with identical patterns by RFLP analysis with IS
6110
and with low numbers of copies of IS
6110
. The combined use of both markers identified unique patterns for 177 strains and clustered 95 (35.7%) strains in 26 groups, each containing isolates from 2 to 12 patients. The clustering was strongly associated with homelessness and the male sex. It was not associated with age, birth in a foreign country, human immunodeficiency virus positivity, or residence in hostels or prison. Isolates from homeless people were often included in large clusters, and homeless people could be the source of tuberculosis transmission for more than 50% of the clustered patients. These results suggest that homeless people play a key role in the spread of
M. tuberculosis
in the community and that poor socioeconomic conditions are the main risk factors associated with active tuberculosis transmission.
Viruses and Bacteria in the Etiology of the Common Cold
Authors: Mäkelä Mika J. , Puhakka Tuomo , Ruuskanen Olli , Leinonen Maija , Saikku Pekka , Kimpimäki Marko , Blomqvist Soile , Hyypiä Timo , Arstila Pertti
Published: December 2019

Two hundred young adults with common colds were studied during a 10-month period. Virus culture, antigen detection, PCR, and serology with paired samples were used to identify the infection. Viral etiology was established for 138 of the 200 patients (69%). Rhinoviruses were detected in 105 patients, coronavirus OC43 or 229E infection was detected in 17, influenza A or B virus was detected in 12, and single infections with parainfluenza virus, respiratory syncytial virus, adenovirus, and enterovirus were found in 14 patients. Evidence for bacterial infection was found in seven patients. Four patients had a rise in antibodies against
Chlamydia pneumoniae
, one had a rise in antibodies against
Haemophilus influenzae
, one had a rise in antibodies against
Streptococcus pneumoniae
, and one had immunoglobulin M antibodies against
Mycoplasma pneumoniae
. The results show that although approximately 50% of episodes of the common cold were caused by rhinoviruses, the etiology can vary depending on the epidemiological situation with regard to circulating viruses. Bacterial infections were rare, supporting the concept that the common cold is almost exclusively a viral disease.
Use of Gas Chromatographic Fatty Acid and Mycolic Acid Cleavage Product Determination To Differentiate among
Mycobacterium genavense
,
Mycobacterium fortuitum
,
Mycobacterium simiae
, and
Mycobacterium tuberculosis
Authors: Chou S. , Chedore P. , Kasatiya S.
Published: December 2019

Three
Mycobacterium genavense
strains and three American Type Culture Collection reference strains each of
Mycobacterium fortuitum
,
Mycobacterium simiae
, and
Mycobacterium tuberculosis
were subcultured onto Mycobacteria 7H11 agar (Difco Laboratories, Detroit, Mich.) supplemented with mycobactin J (Allied Laboratories, Fayette, Mo.). After 4 weeks of incubation at 37°C in 10% CO
2
, the cultures were analyzed by gas-liquid chromatography (GLC) for their fatty acids and mycolic acid cleavage products.
M. fortuitum
was clearly differentiated from
M. genavense
by the presence of the specific marker 2-methyloctadecenoic acid in
M. fortuitum
and by the ratio of tetracosanoic acid to hexacosanoic acid. This ratio was <1 for
M. genavense
and >3 for
M. fortuitum. M. fortuitum
also contained docosanoic acid, which was not detected in
M. genavense. M. genavense
,
M. simiae
, and
M. tuberculosis
, which have similar GLC profiles, were also differentiated from each other by the presence of either
cis
-10-hexadecenoic acid or
cis
-11-hexadecenoic acid and by tetradecanoic acid content.
Comparison of Use of Cerebrospinal Fluid, Serum, and Throat Swab Specimens in Diagnosis of Enteroviral Acute Neurological Infection by a Rapid RNA Detection PCR Assay
Authors: Andréoletti Laurent , Blassel-Damman Nathalie , Dewilde Anny , Vallée Louis , Cremer Robin , Hober Didier , Wattré Pierre
Published: December 2019

A PCR assay for detection of enterovirus RNA in multiple specimen types from patients with neurological infections was evaluated. Combined PCR assay of cerebrospinal fluid and serum (systemic specimens) was more sensitive than assaying either specimen alone in children but not in adults. Compared with PCR in systemic specimens, detection of enterovirus RNA in throat swabs showed a sensitivity of 62.5% and a specificity of 75.6%.