bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection of Fluconazole-Resistant
Candida
Strains by a Disc Diffusion Screening Test
Authors: Sandven Per
Published: December 2019

A commercial disc diffusion test has been evaluated as a screening method for the detection of
Candida
species with decreased susceptibility to fluconazole. A total of 1,407
Candida
strains of different species were tested, and the results were compared with the MIC results. The recently published National Committee for Clinical Laboratory Standards breakpoint criteria have been used. Isolates were classified as susceptible if the MIC for the isolates was ≤8 μg/ml, susceptible-dose dependent (S-DD) if the MIC was 16 to 32 μg/ml, and resistant if the MIC was ≥64 μg/ml. All 77 resistant strains and 121 of 122 S-DD strains had fluconazole zone diameters of ≤21 mm, and most of the strains (91%) had zone diameters of ≤15 mm. It was not possible to distinguish between resistant and S-DD strains by the disc test. Among a total of 1,208 strains found to be susceptible by the microdilution method, 49 (4.1%) yielded fluconazole zone sizes of ≤21 mm and would have been misclassified as resistant or S-DD strains on the basis of the disc test. For the majority (86%) of these 49 strains the fluconazole MIC was 8 μg/ml. The fluconazole disc test is recommended as a simple and reliable screening test for the detection of
Candida
strains with decreased susceptibility to fluconazole. Fluconazole MICs should be determined for strains found to be resistant by the disc test. The reason for confirmatory testing is twofold: to determine if isolates are resistant or S-DD, since the disc test does not make this distinction, and to identify fluconazole-susceptible strains that are found to be falsely resistant by the fluconazole disc test.
Large-Scale Survey of
Campylobacter
Species in Human Gastroenteritis by PCR and PCR–Enzyme-Linked Immunosorbent Assay
Authors: Lawson A. J. , Logan J. M. J. , O'neill G. L. , Desai M. , Stanley J.
Published: December 2019

A PCR-based study of the incidence of enteropathogenic campylobacter infection in humans was done on the basis of a detection and identification algorithm consisting of screening PCRs and species identification by PCR-enzyme-linked immunosorbent assay. This was applied to DNA extracted from 3,738 fecal samples from patients with sporadic cases of acute gastroenteritis, submitted by seven regional Public Health Laboratories in England and Wales over a 2-year period. The sending laboratories had cultured “
Campylobacter
spp.” from 464 samples. The PCR methodologies detected 492
Campylobacter
-positive samples, and the combination of culture and PCR yielded 543
Campylobacter
-positive samples. There was identity (overlap) for 413 samples, but 79 PCR-positive samples were culture negative, and 51 culture-positive samples were PCR negative. While there was no statistically significant difference between PCR and culture in detection of
C. jejuni-C. coli
(PCR, 478 samples; culture, 461 samples), PCR provided unique data about mixed infections and non-
C. jejuni
and non-
C. coli
campylobacters. Mixed infections with
C. jejuni
and
C. coli
were found in 19 samples, and mixed infection with
C. jejuni
and
C. upsaliensis
was found in one sample; this was not apparent from culture. Eleven cases of gastroenteritis were attributed to
C. upsaliensis
by PCR, three cases were attributed to
C. hyointestinalis
, and one case was attributed to
C. lari
. This represents the highest incidence of
C. hyointestinalis
yet reported from human gastroenteritis, while the low incidence of
C. lari
suggests that it is less important in this context.
Identification of
Bartonella
Species Directly in Clinical Specimens by PCR-Restriction Fragment Length Polymorphism Analysis of a 16S rRNA Gene Fragment
Authors: Matar Ghassan M. , Koehler Jane E. , Malcolm Georgia , Lambert-Fair Mary Ann , Tappero Jordan , Hunter Suzan B. , Swaminathan Bala
Published: December 2019

It is now established that two species of
Bartonella
, namely,
Bartonella henselae
and
B. quintana
, cause bacillary angiomatosis in human immunodeficiency virus-infected patients. In addition,
B. henselae
causes cat scratch disease and
B. quintana
,
B. henselae
, and
B. elizabethae
can cause bacteremia and endocarditis in immunocompetent persons. We have developed a PCR-restriction fragment length polymorphism-based assay for direct detection and identification to species level of
Bartonella
in clinical specimens. This is accomplished by PCR amplification of
Bartonella
DNA using primers derived from conserved regions of the gene carrying the 16S ribosomal DNA, followed by restriction analysis using
Dde
I and
Mse
I restriction endonucleases. We amplified a
Bartonella
genus-specific 296-bp fragment from 25 clinical samples obtained from 25 different individuals. Restriction analysis of amplicons showed that identical patterns were seen from digestion of
B. henselae
and
B. quintana
amplicons with
Dde
I, whereas a different unique pattern was seen by using the same enzyme with
B. vinsonii
and
B. elizabethae
. With
Mse
I digestion,
B. henselae
and
B. vinsonii
gave nearly identical patterns while
B. quintana
and
B. elizabethae
gave a different pattern. By combining the restriction analysis data generated with
Mse
I and
Dde
I, unique “signature” restriction patterns characteristic for each species were obtained. These patterns were useful in identifying the
Bartonella
species associated with each tissue specimen.
Methods for Improved Detection of Oxacillin Resistance in Coagulase-Negative Staphylococci: Results of a Multicenter Study
Authors: Tenover Fred C. , Jones Ronald N. , Swenson Jana M. , Zimmer Barbara , McAllister Sigrid , Jorgensen James H.
Published: December 2019

A multilaboratory study was undertaken to determine the accuracy of the current National Committee for Clinical Laboratory Standards (NCCLS) oxacillin breakpoints for broth microdilution and disk diffusion testing of coagulase-negative staphylococci (CoNS) by using a PCR assay for
mecA
as the reference method. Fifty well-characterized strains of CoNS were tested for oxacillin susceptibility by the NCCLS broth microdilution and disk diffusion procedures in 11 laboratories. In addition, organisms were inoculated onto a pair of commercially prepared oxacillin agar screen plates containing 6 μg of oxacillin per ml and 4% NaCl. The results of this study and of several other published reports suggest that, in order to reliably detect the presence of resistance mediated by
mecA
, the oxacillin MIC breakpoint for defining resistance in CoNS should be lowered from ≥4 to ≥0.5 μg/ml and the breakpoint for susceptibility should be lowered from ≤2 to ≤0.25 μg/ml. In addition, a single disk diffusion breakpoint of ≤17 mm for resistance and ≥18 mm for susceptibility is suggested. Due to the poor sensitivity of the oxacillin agar screen plate for predicting resistance in this study, this test can no longer be recommended for use with CoNS. The proposed interpretive criteria for testing CoNS have been adopted by the NCCLS.
Leuconostoc pseudomesenteroides
as a Cause of Nosocomial Urinary Tract Infections
Authors: Cappelli Elisabete A. , Barros Rosana R. , Camello Thereza Cristina F. , Teixeira Lúcia M. , Merquior Vânia Lúcia C.
Published: December 2019

The phenotypic and genotypic characterization of five clinical isolates of
Leuconostoc pseudomesenteroides
associated with nosocomially acquired urinary tract infections is described. All the strains were susceptible to chloramphenicol, clindamycin, erythromycin, gentamicin, and tetracycline; all were resistant to nalidixic acid, norfloxacin, and vancomycin; and all were intermediately affected by ampicillin and penicillin. Analysis of chromosomal DNA by pulsed-field gel electrophoresis after treatment with
Sma
I indicated a clonal relationship of the isolates. The results provide evidence for the possibility of nosocomial transmission of this unusual opportunistic, vancomycin-resistant pathogen.
Seroepidemiology of Human Group C Rotavirus in South Africa
Authors: Steele A. D. , James V. L. A.
Published: December 2019

Sera from three separate healthy population cohorts were used to determine the incidence of group C rotavirus infections in 1,356 South Africans. Using an enzyme-linked immunosorbent assay based on a recombinant group C rotavirus VP6 protein, the total percent positivity was found to be 34.4% (range, 33 to 38%), with almost half of the population infected after the age of 20 years.
Analysis of GB Virus C Markers in Families Over Three Generations
Authors: Chen Margaret , Fischler Björn , Hultgren Catharina , Halasz Robert , Nemeth Antal , Sällberg Matti
Published: December 2019

GB virus C (GBV-C) markers were analyzed in two to three generations in three families with documented vertical transmission of GBV-C. None of the maternal grandparents had GBV-C markers, whereas the male spouses had GBV-C envelope 2 antibodies. Evidence was found for intrafamilial transmission but not for GBV-C transmission over three generations.
Cord Formation in MB/BacT Medium Is a Reliable Criterion for Presumptive Identification of
Mycobacterium tuberculosis
Complex in Laboratories with High Prevalence of
M. tuberculosis
Authors: Badak F. Zuhre , Goksel Servet , Sertoz Ruchan , Guzelant Asuman , Kizirgil Ahmet , Bilgic Altinay
Published: December 2019

We evaluated cord formation in MB/BacT broth as a rapid method for presumptive identification of the
Mycobacterium tuberculosis
complex. Kinyoun acid-fast-stained smears from 370 positive MB/BacT bottles were examined for the presence of serpentine cording. The smears were examined independently by two observers. Observer 1 (the supervisor of the mycobacteriology laboratory) examined all of the smears while observer 2 (a clinical microbiologist not familiar with acid-fast bacillus [AFB] microscopy) examined 148 randomly chosen smears that were read by observer 1 without knowledge of which smear was which. The sensitivity, specificity, and positive and negative predictive values of cording for the presumptive identification of
M. tuberculosis
read by observer 1 were 88.2, 97.4, 99.2, and 69.7%, respectively. These values were reported at 90.6, 52.3, 82.8, and 69.7%, respectively, by observer 2. Our laboratory prevalence of
M. tuberculosis
among positive cultures was 78% during the time this study was conducted. At the time of positive signal of the MB/BacT bottles, the broth of the bottles had sufficient cell mass to allow for observation of the presence or absence of serpentine cording. The presence of cords in MB/BacT broth is a reliable criterion for rapid, predictive identification of the
M. tuberculosis
complex for laboratories with a high proportion of the
M. tuberculosis
complex when the smears are examined by a microbiologist who has experience with AFB staining.
Identification of
Candida dubliniensis
in a Prospective Study of Patients in the United States
Authors: Jabra-Rizk Mary Ann , Baqui A. A. M. A. , Kelley Jacqueline I. , Falkler William A. , Merz William G. , Meiller Timothy F.
Published: December 2019

Although
Candida albicans
remains the fungal species most frequently isolated as an opportunistic oral pathogen, other yeast species are often identified in human immunodeficiency virus (HIV)-seropositive patients.
Candida dubliniensis
phenotypically resembles
C. albicans
in many respects, yet it can be identified and differentiated as a unique
Candida
species by its phenotypic and genetic profiles. The purpose of the present study was to prospectively test for the presence of
C. dubliniensis
among clinical isolates and to determine the clinical and demographic characteristics of patients harboring
C. dubliniensis
. Over a 90-day period, isolates from 724 patients that were presumptively identified as
C. albicans
were screened for
C. dubliniensis
by use of tests for germ tube and chlamydospore production, by detection of an inability to grow at 45°C, by colony color on CHROMagar Candida medium, and by the results of a sugar assimilation test with the API 20C AUX yeast identification system. Among 699 isolates retrieved from those specimens evaluated, 5 from 25 HIV-seropositive patients and 1 isolate from a patient whose HIV status was unknown were shown to be consistent by phenotyping and by electrophoretic karyotyping with the European reference strain of
C. dubliniensis
. One of the
C. dubliniensis
isolates had dose-dependent susceptibility to fluconazole (MIC, 16 μg/ml). These results confirm the presence of this interesting species in the United States and support the need for further investigations into the prevalence and pathogenesis of
C. dubliniensis.
Limits in Reliability of Glycoprotein G-Based Type-Specific Serologic Assays for Herpes Simplex Virus Types 1 and 2
Authors: Schmid D. Scott , Brown Denise R. , Nisenbaum Rosane , Burke Rae Lyn , Alexander D’Anna , Ashley Rhoda , Pellett Philip E. , Reeves William C.
Published: December 2019

Type-specific serologic assays for herpes simplex virus (HSV) types 1 and 2 based on glycoprotein G-1 (gG-1) (HSV-1) and gG-2 (HSV-2) discriminate between antibodies against HSV-1 and HSV-2. We previously developed a Western blot assay using gG-1 and gG-2 expressed in baculovirus, performed extensive validation studies, and determined that it was both sensitive and specific for type-specific detection of HSV antibody. Here we report that, among a cohort of Thai military recruits, the serostatus of some individuals changed from positive to negative over time (6.6% among those ever positive for HSV-1, and 14.9% among those ever positive for HSV-2). We tested a subset of these specimens in three other gG-based assays: an enzyme-linked immunosorbent assay, an immunoblot strip assay, and a Western blot assay. Positive-to-negative shifts occurred in every assay; the frequency of the shifts ranged from 6.1% to 21.2% of the specimen sets tested. There was only limited agreement among the assays concerning which individuals lost reactivity. This inaccuracy, exhibited by all of the assay protocols, was not predicted by validation studies employing specimens from cross-sectional studies and was most pronounced in HSV-2 testing. This argues for the inclusion of serial blood specimens in serologic assay validation procedures.
Rickettsia helvetica
in
Ixodes ricinus
Ticks in Sweden
Authors: Nilsson Kenneth , Lindquist Olle , Liu Ai Jie , Jaenson Thomas G. T. , Friman Göran , Påhlson Carl
Published: December 2019

In the present study further characterization of the amplified sequence of the citrate synthase gene of the spotted fever group
Rickettsia
isolated from
Ixodes ricinus
ticks in Sweden showed that it has 100% homology with the deposited sequence of the citrate synthase gene of
Rickettsia helvetica
. The restriction fragment length polymorphism (RFLP) pattern of an amplified 382-bp product of the citrate synthase sequence, defined by primers RpCS877 and RpCS1258, yielded fragments for our isolate that could be visualized as a double band that migrated at approximately 44 bp, another double band at 85 bp, and a single band at nearly 120 bp after digestion with the restriction enzyme
Alu
I. When calculating a theoretical PCR-RFLP pattern of the sequence of the citrate synthase gene of
R. helvetica
from the known positions where the
Alu
I enzyme cuts, we arrived at the same pattern that was obtained for our isolate, a pattern distinctly different from the previously published PCR-RFLP pattern for
R. helvetica
. Investigation of 125 living
I. ricinus
ticks showed a higher prevalence of rickettsial DNA in these ticks than we had found in an earlier study. Rickettsial DNA was detected by amplification of the 16S rRNA gene, for which a seminested primer system consisting of two oligonucleotide primer pairs was used. Of the 125 ticks, some were pooled, giving a total of 82 tick samples, of which 20 were found to be positive for the rickettsial DNA gene investigated. When considering the fact that some of the positive samples were pooled, the minimum possible prevalence in these ticks was 20 of 125 (16%) and the maximum possible prevalence was 46 of 125 (36.8%). These prevalence estimates conform to those of other studies of spotted fever group rickettsiae in hard ticks in Europe.
Stability of Insertion Sequence IS
1245
, a Marker for Differentiation of
Mycobacterium avium
Strains
Authors: Bauer Jeanett , Andersen Åse Bengård
Published: December 2019

Recently a novel insertion element, IS
1245
, has been described and suggested for use as a probe in restriction fragment length polymorphism studies of
Mycobacterium avium
strains. An important issue in this context is the stability of the insertion element. We analyzed single colonies of
M. avium
cultures and found frequent small one- to two-band changes. However, following repeated in vitro passages over 1 year, similar one- to two-band changes were observed in the IS
1245
patterns of only six
M. avium
strains investigated.
Hemolysin as a Virulence Factor for Systemic Infection with Isolates of
Mycobacterium avium
Complex
Authors: Maslow Joel N. , Dawson David , Carlin Elizabeth A. , Holland Steven M.
Published: December 2019

Isolates of the
Mycobacterium avium
complex were examined for hemolysin expression. Only invasive isolates of
M. avium
were observed to be hemolytic (
P
< 0.001), with activity the greatest for isolates of serovars 4 and 8. Thus,
M. avium
hemolysin appears to represent a virulence factor necessary for invasive disease.
Detection of Viable
Mycobacterium tuberculosis
by Reverse Transcriptase-Strand Displacement Amplification of mRNA
Authors: Hellyer T. J. , DesJardin L. E. , Teixeira L. , Perkins M. D. , Cave M. D. , Eisenach K. D.
Published: December 2019

Numerous assays have been described for the detection of DNA and rRNA sequences that are specific for the
Mycobacterium tuberculosis
complex. Although beneficial to initial diagnosis, such assays have proven unsuitable for monitoring therapeutic efficacy owing to the persistence of these nucleic acid targets long after conversion of smears and cultures to negative. However, prokaryotic mRNA has a typical half-life of only a few minutes and we have previously shown that the presence of mRNA is a good indicator of bacterial viability. The purpose of the present study was to develop a novel reverse transcriptase-strand displacement amplification system for the detection of
M. tuberculosis
α-antigen (85B protein) mRNA and to demonstrate the use of this assay in assessing chemotherapeutic efficacy in patients with pulmonary tuberculosis. The assay was applied to sequential, noninduced sputum specimens collected from four patients: 10 of 11 samples (91%) collected prior to the start of therapy were positive for alpha-antigen mRNA, compared with 1 of 8 (13%), 2 of 8 (25%), 2 of 8 (25%), and 0 of 8 collected on days 2, 4, 7, and 14 of treatment, respectively. In contrast, 39 of 44 samples (89%) collected on or before day 14 were positive for α-antigen DNA. The loss of detectable mRNA corresponded to a rapid drop over the first 4 days of treatment in the number of viable organisms present in each sputum sample, equivalent to a mean fall of 0.43 log
10
CFU/ml/day. Analysis of mRNA is a potentially useful method for monitoring therapeutic efficacy and for rapid in vitro determination of drug susceptibility.
Serologic Testing for Human Granulocytic Ehrlichiosis at a National Referral Center
Authors: Comer James A. , Nicholson William L. , Olson James G. , Childs James E.
Published: December 2019

An indirect immunofluorescence assay (IFA) was used to identify patients with antibodies reactive to the human granulocytic ehrlichiosis (HGE) agent. Serum samples collected from clinically ill individuals were submitted to the Centers for Disease Control and Prevention by physicians via state health departments from throughout the United States and tested against a panel of ehrlichial and rickettsial pathogens. Antibodies reactive to the HGE agent were detected in 142 (8.9%) of 1,602 individuals tested. There were 19 confirmed and 59 probable (n= 78) cases of HGE as defined by seroconversion or a fourfold or higher titer to the HGE agent than to theEhrlichia chaffeensisantigens. The average age of patients with HGE was 57 years, and males accounted for 53 (68%) of the patients. Cases of HGE occurred in 21 states; 47 (60%) of the cases occurred in Connecticut (n= 14), New York (n= 18), and Wisconsin (n= 15). Onset of HGE was identified from April through December, with cases peaking in June and July. The earliest confirmed cases of HGE occurred in 1987 in Wisconsin and 1988 in Florida. No fatalities were reported among the 78 patients with confirmed or probable HGE. Reactivity to the HGE agent and to eitherCoxiella burnetii,Rickettsia rickettsii, orRickettsia typhiwas infrequent; however, 74 (52%) of the 142 individuals who were positive for HGE had at least one serum sample that also reacted to theE. chaffeensisantigen. Thirty-four persons with confirmed or probable human monocytic ehrlichiosis due toE. chaffeensisalso had antibodies to the HGE agent in at least one serum sample. The specific etiologic agent for 30 patients was not ascribed because of similarity of titers to both ehrlichial antigens. The use of both antigens may be required to correctly diagnose most cases of human ehrlichiosis, especially in geographic regions where both the HGE agent andE. chaffeensisoccur.
Laboratory Diagnosis of Toscana Virus Infection by Enzyme Immunoassay with Recombinant Viral Nucleoprotein
Authors: Soldateschi Dario , Dal Maso Grazia Maria , Valassina Marcello , Santini Laura , Bianchi Silvia , Cusi Maria Grazia
Published: December 2019

A recombinant enzyme immunoassay (rEIA) to detect serum immunoglobulin M (IgM) and IgG to Toscana virus (TOSV) was developed with the aim of establishing a simple and easily available assay for diagnosing acute and/or previous infections. The rEIA, based on the recombinant nucleoprotein of TOSV expressed in
Escherichia coli
, was evaluated with 97 serum samples collected in an area where TOSV is endemic and compared to an analogous assay based on cell-derived TOSV. Discordant results were resolved by immunoblotting (IB). Twenty-two of these samples, obtained from subjects hospitalized during the summer season with meningitis of suspected TOSV etiology, were further characterized by indirect immunofluorescence and IB, and detection of specific TOSV RNA sequences in the cerebrospinal fluid of these patients was attempted by nested PCR. The results indicated that rEIA was able to diagnose acute TOSV infection by detection of specific serum IgM in all of the subjects with TOSV meningitis confirmed by nested PCR or serology. The overall sensitivity and specificity of rEIA were both 100% for IgM detection and 100 and 96.6%, respectively, for IgG detection. Thus, rEIA appears to be a simple and reliable laboratory test for the diagnosis of acute TOSV infection and for the assessment of immune status.
Case Report and Molecular Analysis of Subacute Sclerosing Panencephalitis in a South African Child
Authors: Vardas Eftyhia , Leary P. M. , Yeats Jane , Badrodien Waseila , Kreis Stephanie
Published: December 2019

This is the first case of subacute sclerosing panencephalitis from South Africa in which the molecular characteristics of the causative measles virus were examined. The virus found is classified as genotype D3, which has not previously been found in Africa and was last circulating in the United States before 1992.
Evaluation of the Ultrasensitive Roche Amplicor HIV-1 Monitor Assay for Quantitation of Human Immunodeficiency Virus Type 1 RNA
Authors: Erali Maria , Hillyard David R.
Published: December 2019

The ultrasensitive Amplicor HIV-1 Monitor test (Roche Diagnostic Systems) was evaluated for precision, linearity, and sensitivity and was compared to the standard Amplicor assay. The ultrasensitive assay reliably quantified samples in the range from 50 to 50,000 human immunodeficiency virus type 1 RNA copies/ml with acceptable correlation with the standard Amplicor test.
Reproducibility and Performance of the Second-Generation Branched-DNA Assay in Routine Quantification of Human Immunodeficiency Virus Type 1 RNA in Plasma
Authors: Murphy Donald G. , Gonin Patrick , Fauvel Micheline
Published: December 2019

We examined the reproducibility of a second-generation branched-DNA (bDNA) assay (Quantiplex HIV RNA 2.0) for quantification of human immunodeficiency virus type 1 (HIV-1) RNA in plasma by retesting 325 specimens on separate runs and on different lots. The performance of the bDNA test was also assessed by data analysis obtained during routine testing of 15,365 specimens. Upon retesting, 96 and 86% of specimens displaying RNA levels above 5,000 and between 500 and 5,000 copies/ml, respectively, showed less than a 0.3 log
10
(twofold) difference with their initial values. Assay variability was found to increase as viral load decreased. Overall, the bDNA version 2.0 assay was found to be a reproducible and efficient test for routine quantification of HIV-1 RNA in plasma.
Helicobacter pylori
Can Be Induced To Assume the Morphology of
Helicobacter heilmannii
Authors: Fawcett Paul T. , Gibney Kathleen M. , Vinette Kathleen M. B.
Published: December 2019

Cultures of
Helicobacter pylori
obtained from the American Type Culture Collection (strain 43504) were grown as isolated colonies or lawns on blood agar plates and in broth culture with constant shaking. Examination of bacterial growth with Gram-stained fixed preparation and differential interference contrast microscopy on wet preparations revealed that bacteria grown on blood agar plates had a morphology consistent with that normally reported for
H. pylori
whereas bacteria from broth cultures had the morphologic appearance of
Helicobacter heilmannii
. Bacteria harvested from blood agar plates assumed an
H. heilmannii
-like morphology when transferred to broth cultures, and bacteria from broth cultures grew with morphology typical of
H. pylori
when grown on blood agar plates. Analysis by PCR of bacteria isolated from blood agar plates and broth cultures indicated that a single strain of bacteria (
H. pylori
) was responsible for both morphologies.
Immunohistochemical Detection of JC Virus in Nontumorous Renal Tissue of a Patient with Renal Cancer but without Progressive Multifocal Leukoencephalopathy
Authors: Aoki Naoto , Kitamura Tadaichi , Tominaga Takashi , Fukumori Nobutaka , Sakamoto Yosimitsu , Kato Kenzo , Mori Mayumi
Published: December 2019

We performed immunohistochemical staining on the nontumorous renal tissue of 45 patients with renal cancer but without progressive multifocal encephalopathy using JCV-specific antibody. For one patient we found positive staining of the nuclei of the renal collecting ducts. Immunoelectron microscopic examination of the positive cell nuclei revealed electron-dense polyomavirus-like particles.
Use of an Oxacillin Disk Screening Test for Detection of Penicillin- and Ceftriaxone-Resistant Pneumococci
Authors: Jetté Louise P. , Sinave Christian
Published: December 2019

In a context of worldwide emergence of resistance among
Streptococcus pneumoniae
strains, early detection of strains with decreased susceptibility to β-lactam antibiotics is important for clinicians. If the 1-μg oxacillin disk diffusion test is used as described by the National Committee for Clinical Laboratory Standards, no interpretation is available for strains showing zone sizes of ≤19 mm, and there is presently no disk diffusion test available for screening cephalosporin resistance. The zones obtained by the diffusion method by using the 1-μg oxacillin disk were compared with penicillin MICs for 1,116 clinical strains and with ceftriaxone MICs for 695 of these strains. Among the 342 strains with growth up to the 1-μg oxacillin disk margin, none were susceptible (MIC, ≤0.06 μg/ml), 62 had intermediate resistance (MIC, 0.12 to 1.0 μg/ml), and 280 were resistant (MIC, ≥2.0 μg/ml) to penicillin. For ceftriaxone, among 98 strains with no zone of inhibition in response to oxacillin, 68 had intermediate resistance (MIC, 1.0 μg/ml), and 22 were resistant (MIC, ≥2.0 μg/ml). To optimize the use of the disk diffusion method, we propose that the absence of a zone of inhibition around the 1-μg oxacillin disk be regarded as an indicator of nonsusceptibility to penicillin and ceftriaxone and recommend that such strains be reported as nonsusceptible to these antimicrobial agents, pending the results of a MIC quantitation method.
Contaminations Occurring in Fungal PCR Assays
Authors: Loeffler Juergen , Hebart Holger , Bialek Ralf , Hagmeyer Lars , Schmidt Diethard , Serey Francois-Prâseth , Hartmann Matthias , Eucker Jan , Einsele Hermann
Published: December 2019

Successful in vitro amplification of fungal DNA in clinical specimens has been reported recently. In a collaboration among five European centers, the frequency and risk of contamination due to airborne spore inoculation or carryover contamination in fungal PCR were analyzed. The identities of all contaminants were specified by cycle sequencing and GenBank analysis. Twelve of 150 PCR assays that together included over 2,800 samples were found to be contaminated (3.3% of the negative controls were contaminated during the DNA extraction, and 4.7% of the PCR mixtures were contaminated during the amplification process). Contaminants were specified as
Aspergillus fumigatus
,
Saccharomyces cerevisiae
, and
Acremonium
spp. Further analysis showed that commercially available products like zymolyase powder or 10× PCR buffer may contain fungal DNA. In conclusion, the risk of contamination is not higher in fungal PCR assays than in other diagnostic PCR-based assays if general precautions are taken.
Isolation of Lyme DiseaseBorreliafrom Puffins (Fratercula arctica) and Seabird Ticks (Ixodes uriae) on the Faeroe Islands
Authors: Gylfe Åsa , Olsen Björn , Straševičius Darius , Marti Ras Nuria , Weihe Pál , Noppa Laila , Östberg Yngve , Baranton Guy , Bergström Sven
Published: December 2019

This is the first report on the isolation of Lyme diseaseBorreliafrom seabirds on the Faeroe Islands and the characteristics of its enzootic cycle. The major components of theBorreliacycle include the puffin (Fratercula arctica) as the reservoir andIxodes uriaeas the vector. The importance of this cycle and its impact on the spread of human Lyme borreliosis have not yet been established.Borreliaspirochetes isolated from 2 of 102 sampled puffins were compared to the borreliae previously obtained from seabird ticks,I. uriae. Therrf-rrlintergenic spacer and therrsand theospCgenes were sequenced and a series of phylogenetic trees were constructed. Sequence data and restriction fragment length polymorphism analysis grouped the strains together withBorrelia garinii. In a seroepidemiological survey performed with residents involved in puffin hunting on the Faeroe Islands, 3 of 81 serum samples were found to be positive by two commonly used clinical tests: a flagellin-based enzyme-linked immunosorbent assay (ELISA) and Western blotting. These three positive serum samples also had high optical density values in a whole-cell ELISA. The finding of seropositive Faeroe Islanders who are regularly exposed toI. uriaeindicate that there may be a transfer ofB. gariniiby this tick species to humans.
Predictive Fluorescent Amplified-Fragment Length Polymorphism Analysis of
Escherichia coli
: High-Resolution Typing Method with Phylogenetic Significance
Authors: Arnold Catherine , Metherell Lou , Willshaw Geraldine , Maggs Anthony , Stanley John
Published: December 2019

The fluorescent amplified-fragment length polymorphism (FAFLP) assay potentially amplifies a unique set of genome fragments from each bacterial clone. It uses stringently hybridizing primers which carry a fluorescent label. Precise fragment sizing is achieved by the inclusion of an internal size standard in every lane. Therefore, a unique genotype identifier(s) can be found in the form of fragments of precise size or sizes, and these can be generated reproducibly. In order to evaluate the potential of FAFLP as an epidemiological typing method with a valid phylogenetic basis, we applied it to 87 strains of
Escherichia coli
. These comprised the EcoR collection, which has previously been classified by multilocus enzyme electrophoresis (MLEE) and which represents the genetic diversity of the species
E. coli
, plus 15 strains of the clinically important serogroup O157. FAFLP with an unlabelled nonselective
Eco
RI primer (
Eco
+0) and a labelled selective
Mse
I primer (
Mse
+TA) gave strain-specific profiles. Fragments of identical sizes (in base pairs) were assumed to be identical, and the genetic distances between the strains were calculated. A phylogenetic tree derived from measure of distance correlated closely with the MLEE groupings of the EcoR collection and placed the verocytotoxin-producing O157 strains on an outlier branch. Our data indicate that FAFLP is suitable for epidemiological investigation of
E. coli
infection, providing well-defined and reproducible identifiers of genotype for each strain. Since FAFLP objectively samples the whole genome, each strain or isolate can be assigned a place within the broad context of the whole species and can also be subjected to a high-resolution comparison with closely related strains to investigate epidemiological clonality.
Typing of Human Enteroviruses by Partial Sequencing of VP1
Authors: Oberste M. Steven , Maher Kaija , Kilpatrick David R. , Flemister Mary R. , Brown Betty A. , Pallansch Mark A.
Published: December 2019

Human enteroviruses (family
Picornaviridae
) are the major cause of aseptic meningitis and also cause a wide range of other acute illnesses, including neonatal sepsis-like disease, acute flaccid paralysis, and acute hemorrhagic conjunctivitis. The neutralization assay is usually used for enterovirus typing, but it is labor-intensive and time-consuming and standardized antisera are in limited supply. We have developed a molecular typing system based on reverse transcription-PCR and nucleotide sequencing of the 3′ half of the genomic region encoding VP1. The standard PCR primers amplify approximately 450 bp of VP1 for most known human enterovirus serotypes. The serotype of an “unknown” may be inferred by comparison of the partial VP1 sequence to those in a database containing VP1 sequences for the prototype strains of all 66 human enterovirus serotypes. Fifty-one clinical isolates of known serotypes from the years 1991 to 1998 were amplified and sequenced, and the antigenic and molecular typing results agreed for all isolates. With one exception, the nucleotide sequences of homologous strains were at least 75% identical to one another (>88% amino acid identity). Strains with homologous serotypes were easily discriminated from those with heterologous serotypes by using these criteria for identification. This method can greatly reduce the time required to type an enterovirus isolate and can be used to type isolates that are difficult or impossible to type with standard immunological reagents. The technique may also be useful for the rapid determination of whether viruses isolated during an outbreak are epidemiologically related.
Direct Quantification of the Enteric Bacterium
Oxalobacter formigenes
in Human Fecal Samples by Quantitative Competitive-Template PCR
Authors: Sidhu H. , Holmes R. P. , Allison M. J. , Peck A. B.
Published: December 2019

Homeostasis of oxalic acid appears to be regulated, in part, by the gut-associated bacterium
Oxalobacter formigenes
. The loss of this bacterium from the gut flora is associated with an increased susceptibility to hyperoxaluria, a condition which can lead to the formation of calcium oxalate crystalluria and kidney stones. In order to identify and quantify the presence of
O. formigenes
in clinical specimens, a quantitative-PCR-based assay system utilizing a competitive DNA template as an internal standard was developed. This quantitative competitive-template PCR test allows for the rapid, highly specific, and reproducible quantification of
O. formigenes
in fecal samples and provides a prototype for development of DNA-based quantitative assays for enteric bacteria.
Femur Osteomyelitis Due to a Mixed Fungal Infection in a Previously Healthy Man
Authors: Cimerman M. , Gunde-Cimerman N. , Zalar P. , Perkovic T.
Published: December 2019

We describe a previously healthy, 22-year-old man who, after a closed fracture of the femur and subsequent operation, developed chronic osteomyelitis. Within a few days, infected bone fragments, bone, and wound drainage repeatedly yielded three different filamentous fungi:
Aspergillus fumigatus
,
Aspergillus flavus
, and
Chalara ellisii
. Histologic examination of the bone revealed septate hyphae. After sequential necrotomies of the femur and irrigation-suction drainage with added antimycotic therapy, the infection ceased and the fracture healed. This case is unique in that it is the only known instance in which a long bone was affected in an immunocompetent individual, with no evidence of any systemic infection, by a mixed population of two different
Aspergillus
spp. and the rare filamentous fungus
C. ellisii
. Environmental factors that could potentiate the infection include blood and edema fluid resulting from the surgical procedure and the presence of the osteosynthetic plate.
Isolation of
Malassezia furfur
from a Cat
Authors: Crespo M. J. , Abarca M. L. , Cabañes F. J.
Published: December 2019

During a survey of the occurrence of
Malassezia
species in the external ear canals of cats without otitis externa,
Malassezia furfur
was isolated. This is the first report of the isolation of
M. furfur
from cats.
Evaluation of the Digene Hybrid Capture II CT-ID Test for Detection of
Chlamydia trachomatis
in Endocervical Specimens
Authors: Girdner Jennifer L. , Cullen Allison P. , Salama Teresa G. , He Ling , Lorincz Attila , Quinn Thomas C.
Published: December 2019

The performance characteristics of the new signal amplification-based Hybrid Capture (HC) II CT-ID test system (Digene, Silver Spring, Md.) with endocervical specimens were compared to those of tissue culture and PCR (AMPLICOR CT PCR; Roche Molecular Systems, Branchburg, N.J.) for detection of
Chlamydia trachomatis
in 587 women. HC II CT-ID identified 62 of 65 confirmed
C. trachomatis
-positive patients (sensitivity of 95.4%) and was negative for 517 of 522 patients who were negative by culture and PCR (specificity of 99.0%). Twelve of the 65 confirmed positive patients were negative by culture but were identified by both HC II CT-ID and PCR (sensitivity of culture was 81.5% [
P
< 0.01]). In comparison, PCR detected 59 of 65 positive specimens (sensitivity of 90.8%) and had a specificity of 99.6% (520 of 522). These results demonstrate that the Digene HC II CT-ID test is a highly sensitive and specific assay for the detection of
C. trachomatis
infection in endocervical specimens.
First-Glance Diagnosis of
Strongyloides stercoralis
Autoinfection by Stool Microscopy
Authors: Sing Andreas , Leitritz Lorenz , Bogner Johannes R. , Heesemann Jürgen
Published: December 2019

We report a case of autoinfection due to
Strongyloides stercoralis
in a 27-year-old Ethiopian AIDS patient living in Germany for nearly 3 years. This case was diagnosed on the basis of a single-view field in microscopy of a freshly obtained formalin-fixed stool specimen showing both rhabditiform and filariform larvae. The diagnosis of autoinfection by microscopy is discussed in detail.
Pseudoepidemic of
Aspergillus niger
Infections Traced to Specimen Contamination in the Microbiology Laboratory
Authors: Laurel Valerie L. , Meier Patricia A. , Astorga Alicia , Dolan Donna , Brockett Royce , Rinaldi Michael G.
Published: December 2019

We report a pseudo-outbreak of
Aspergillus niger
that followed building construction in our clinical microbiology laboratory. Because outbreaks of invasive aspergillosis have been linked to hospital construction, strategies to minimize dust in patient care areas are common practice. We illustrate that the impact of false-positive cultures on patient care should compel laboratories to prevent specimen contamination during construction.
Misclassification of Susceptible Strains of
Staphylococcus aureus
as Methicillin-Resistant
S. aureus
by a Rapid Automated Susceptibility Testing System
Authors: Ribeiro Julival , Vieira F. D. , King Tom , D’Arezzo Julia B. , Boyce John M.
Published: December 2019

Eight
Staphylococcus aureus
strains initially identified by Vitek GPS-BS or GPS-SA cards as resistant to oxacillin, but susceptible to most non-beta-lactam antibiotics, were found on further testing to be susceptible to oxacillin and ceftizoxime by disk diffusion tests. For all these strains, the MICs of oxacillin were ≤0.5 μg/ml by agar dilution tests, and the strains were oxacillin susceptible when tested by the BBL Crystal MRSA ID and a Vitek machine with GPS-101 cards. None grew on oxacillin-salt agar screening plates. None were positive for
mecA
gene sequences by PCR. When
S. aureus
strains tested by Vitek GPS-SA or GPS-BS cards appear resistant to only penicillin and oxacillin, a confirmatory test such as the oxacillin-salt agar screening method should be performed.
Identification of Medically Relevant
Trichosporon
Species Based on Sequences of Internal Transcribed Spacer Regions and Construction of a Database for
Trichosporon
Identification
Authors: Sugita Takashi , Nishikawa Akemi , Ikeda Reiko , Shinoda Takako
Published: December 2019

The nucleotide sequences of the internal transcribed spacer (ITS) 1 and 2 regions in the rRNA gene were determined by directly sequencing PCR-amplified fragments for all of the species (17 species and five varieties) in the genus
Trichosporon
. Comparative sequence analysis suggests that six medically relevant species,
T. asahii
,
T. asteroides
,
T. cutaneum
,
T. inkin
,
T. mucoides
, and
T. ovoides
, can be readily identified by their ITS sequences. In addition, the sequence analysis showed that conspecific strains have fewer than 1% nucleotide differences in the ITS 1 and 2 regions overall. Molecular phylogenetic trees are also presented.
Differentiation of
Helicobacter pylori
Isolates Based on Lectin Binding of Cell Extracts in an Agglutination Assay
Authors: Hynes Sean O. , Hirmo Siiri , Wadström Torkel , Moran Anthony P.
Published: December 2019

Plant and animal lectins with various carbohydrate specificities were used to type 35 Irish clinical isolates of
Helicobacter pylori
and the type strain NCTC 11637 in a microtiter plate assay. Initially, a panel of eight lectins with the indicated primary specificities were used:
Anguilla anguilla
(AAA),
Lotus tetragonolobus
(Lotus A), and
Ulex europaeus
I (UEA I), specific for α-
l
-fucose;
Solanum tuberosum
(STA) and
Triticum vulgaris
(WGA), specific for β-
N
-acetylglucosamine;
Glycine max
(SBA), specific for β-
N
-acetylgalactosamine;
Erythrina cristagali
(ECA), specific for β-galactose and β-
N
-acetylgalactosamine; and
Lens culinaris
(LCA), specific for α-mannose and α-glucose. Three of the lectins (SBA, STA, and LCA) were not useful in aiding in strain discrimination. An optimized panel of five lectins (AAA, ECA, Lotus A, UEA I, and WGA) grouped all 36 strains tested into eight lectin reaction patterns. For optimal typing, pretreatment by washing bacteria with a low-pH buffer to allow protein release, followed by proteolytic degradation to eliminate autoagglutination, was used. Lectin types of treated samples were stable and reproducible. No strain proved to be untypeable by this system. Electrophoretic and immunoblotting analyses of lipopolysaccharides (LPSs) indicated that the lectins interact primarily, but not solely, with the O side chain of
H. pylori
LPS.
Epidemiological Analysis of Sequential
Pseudomonas aeruginosa
Isolates from Chronic Bronchiectasis Patients without Cystic Fibrosis
Authors: Pujana I. , Gallego L. , Martín G. , López F. , Canduela J. , Cisterna R.
Published: December 2019

PCR fingerprinting was used for the epidemiological investigation of 64
Pseudomonas aeruginosa
isolates collected from 16 chronic bronchiectasis patients without cystic fibrosis: 56% of the patients harbored one clone, 12.5% carried a single major type with minor variants, and 31.5% carried two clones. Only a minority of the acquisitions of antibiotic resistance was related to the acquisition of exogenous strains. Mucoid and nonmucoid sets of isolates did not display any consistent differences in their patterns. The genetic similarity among the clones ranged from 10 to 69%. Cross-infection or common-source exposure did not appear to have occurred.
Variation in Microbial Identification System Accuracy for Yeast Identification Depending on Commercial Source of Sabouraud Dextrose Agar
Authors: Kellogg James A. , Bankert David A. , Chaturvedi Vishnu
Published: December 2019

The accuracy of the Microbial Identification System (MIS; MIDI, Inc.) for identification of yeasts to the species level was compared by using 438 isolates grown on prepoured BBL Sabouraud dextrose agar (SDA) and prepoured Remel SDA. Correct identification was observed for 326 (74%) of the yeasts cultured on BBL SDA versus only 214 (49%) of yeasts grown on Remel SDA (
P
< 0.001). The commercial source of the SDA used in the MIS procedure significantly influences the system’s accuracy.
Scopulariopsis chartarum
Systemic Mycosis in a Dog
Authors: Welsh Ronald D. , Ely Ray W.
Published: December 2019

Scopulariopsis chartarum
was reported as the agent of a multisystemic infection in a dog. The clinical syndromes in this dog with a fulminating mycotic disease mimicked those observed in dogs infected with canine distemper virus.
Proficiency of Clinical Laboratories in Spain in Detecting Vancomycin-Resistant
Enterococcus
spp
Authors: Alonso-Echanove Juan , Robles Belen , Jarvis William R. ,   The Spanish VRE Study Group
Published: December 2019

Studies in a variety of U.S. clinical laboratories have demonstrated difficulty in detecting intermediate and low-level vancomycin-resistant enterococci (VRE). The misclassification of “at least intermediate resistant isolates” as vancomycin susceptible may have both clinical implications and a negative impact on measures to control the spread of VRE. No published study has assessed the ability of clinical laboratories in Europe to detect VRE. So, the apparent low prevalence of VRE in European hospitals may be, in part, secondary to the inability of these laboratories to detect all VRE. In an effort to assess European laboratories’ proficiency in detecting VRE, we identified 22 laboratories in Spain and asked them to test four VRE strains and one susceptible enterococcal strain from the Centers for Disease Control and Prevention collection. Each organism was tested by the routine antimicrobial susceptibility testing method used by each laboratory. Overall, VRE were correctly identified in 61 of 88 (69.1%) instances. The accuracy of VRE detection varied with the level of resistance and the antimicrobial susceptibility method. The high-level-resistant strain (
Enterococcus faecium
; MIC, 512 μg/ml) was accurately detected in 20 of 22 (91.3%) instances, whereas the intermediate-resistant isolate (
Enterococcus gallinarum
; MIC, 8 μg/ml) was accurately detected in only 11 of 22 (50%) instances. Classification errors occurred in 27 of 88 (30.9%) instances. Misclassification as vancomycin susceptible was the most common error (16 of 27 [59.3%] instances). Our study shows that the participating Spanish laboratories had an overall acceptable proficiency in detecting VRE but that a substantial proportion of VRE isolates with low or intermediate levels of resistance were not detected. We recommend that studies be conducted to validate laboratory proficiency testing as an important step in the prevention and control of the spread of antimicrobial resistance.