bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of CHROMagar Staph. aureus, a New Chromogenic Medium, for Isolation and Presumptive Identification of
Staphylococcus aureus
from Human Clinical Specimens
Authors: Gaillot Olivier , Wetsch Muriel , Fortineau Nicolas , Berche Patrick
Published: December 2019

CHROMagar Staph. aureus (CSA) is a new chromogenic medium for presumptive identification of
Staphylococcus aureus
as mauve colonies after 24 h of incubation. We conducted a preliminary study with 100
S. aureus
and 45 coagulase-negative
Staphylococcus
(CoNS) stock isolates plated on CSA. All
S. aureus
isolates yielded mauve colonies after 24 h of incubation at 37°C, while CoNS isolates grew as blue, white, or beige colonies. Culture on CSA was then prospectively compared to a conventional laboratory method, i.e., culture on 5% horse blood agar (HBA), catalase test, and latex agglutination test (HBA-catalase-latex), for isolation and presumptive identification of
S. aureus
from 2,000 consecutive clinical samples. Among the 310
S. aureus
isolates recovered by at least one of the two methods, 296 grew as typical mauve colonies on CSA, while only 254 yielded catalase-positive, latex-positive colonies on HBA. The sensitivity of CSA was significantly higher than that of the conventional method (95.5 and 81.9%, respectively;
P
< 0.001) and allowed the recovery of important clinical isolates that were undetected on blood agar. The specificities of the two methods were not significantly different, although that of CSA was slightly higher (99.4% versus 98.9% for HBA-catalase-latex;
P
= 0.08). On the basis of its excellent sensitivity and specificity, ease of identification of positive colonies, and absence of complementary testing, CSA can be recommended as a routine plating medium for presumptive identification of
S. aureus
in clinical specimens.
Comparison of Polyvinyl Alcohol Fixative with Three Less Hazardous Fixatives for Detection and Identification of Intestinal Parasites
Authors: Jensen B. , Kepley W. , Guarner J. , Anderson K. , Anderson D. , Clairmont J. , De l'aune William , Austin E. H. , Austin G. E.
Published: December 2019

Polyvinyl alcohol (PVA) containing the fixative mercuric chloride is considered the “gold standard” for the fixation of ova and parasites in the preparation of permanently stained smears of stool specimens. However, mercuric chloride is potentially hazardous to laboratory personnel and presents disposal problems. We compared three new alternative, nontoxic fixatives with PVA, analyzing ease of sample preparation and quality of smears. Sixty-eight fresh stool specimens were divided into aliquots and placed in each of four different fixatives: PARASAFE (PS) (Scientific Devices Laboratory, Inc., Des Plaines, Ill.), ECOFIX (EC) (Meridian Diagnostics, Inc., Cincinnati, Ohio), Proto-Fix (PF) (Alpha-Tec Systems, Inc., Vancouver, Wash.), and low-viscosity PVA fixative (PVA) (Meridian). Specimens were processed and stained according to each manufacturer's directions. Parasites were found in 31 of 68 slide preparations with PVA, 31 with PF, 30 with EC, and 30 with PS.
Blastocystis hominis
and
Iodamoeba bütschlii
were preserved in a readily identifiable state by all methods of fixation. However, some parasites were more easily identified with some of the fixatives because of differences in parasite distortion. For example,
Entamoeba histolytica
(
Entamoeba dispar
) was detected in 13 stools fixed with PF, 7 with PVA, and 6 with EC but none with PS. Likewise,
Chilomastix mesnili
was identified in 13 specimens fixed with PF, 8 with EC, and 5 with PVA but only 1 with PS, while
Entamoeba coli
was seen much less frequently with PS than with the other three fixatives. A dirty background was observed in 41% of specimens prepared with PS, whereas background quality was acceptable with other fixatives. Sample preparation was most rapid with PS, although the EC method involved the fewest steps. In conclusion, PVA and PF produced the least parasite distortion, while PS proved unsatisfactory for the identification of
E. histolytica
,
E. coli
, and
C. mesnili
. Both PF and EC appear to be acceptable, environmentally safe substitutes for PVA.
Comparison of a Commercial Enzyme-Linked Immunosorbent Assay with Immunofluorescence and Complement Fixation Tests for Detection of
Coxiella burnetii
(Q Fever) Immunoglobulin M
Authors: Field Peter R. , Mitchell Jody L. , Santiago Avelina , Dickeson David J. , Chan Sau-Wan , Ho David W. T. , Murphy Alan M. , Cuzzubbo Andrea J. , Devine Peter L.
Published: December 2019

A commercially available enzyme-linked immunosorbent assay (ELISA) for the diagnosis of Q fever (PanBio
Coxiella burnetii
immunoglobulin M [IgM] ELISA, QFM-200) was compared to the indirect fluorescent antibody test (IFAT) for
C. burnetii
IgM and the complement fixation test (CFT). The ELISA demonstrated 92% agreement with the reference method (IFAT), and gave a sensitivity of 99% (69 of 70 samples) and a specificity of 88% (106 of 121). Specificity can be increased with confirmation by IFAT. CFT was found to have a specificity of 90% (107 of 119), although it was lacking in sensitivity (73%; 51 of 70). No cross-reactivity was observed in the ELISA with serum samples from patients with mycoplasma (
n
= 6), chlamydia (
n
= 5), or legionella (
n
= 4) infections, although 2 of 5 patients with leptospirosis and 1 of 4 samples containing rheumatoid factor (RF) demonstrated positive results in the ELISA. Results indicate that the performance of the PanBio
C. burnetii
(Q fever) IgM ELISA (
F
= 187) is superior to that of CFT (
F
= 163), and consequently the ELISA should be a useful aid in the diagnosis of acute Q fever.
Identification of Coryneform Bacterial Isolates by Ribosomal DNA Sequence Analysis
Authors: Tang Yi-Wei , Von Graevenitz Alexander , Waddington Michael G. , Hopkins Marlene K. , Smith Douglas H. , Li Haijing , Kolbert Christopher P. , Montgomery Stacy O. , Persing David H.
Published: December 2019

Identification of coryneform bacteria to the species level is important in certain circumstances for differentiating contamination and/or colonization from infection, which influences decisions regarding clinical intervention. However, methods currently used in clinical microbiology laboratories for the species identification of coryneform bacteria are often inadequate. We evaluated the MicroSeq 500 16S bacterial sequencing kit (Perkin-Elmer Biosystems, Foster City, Calif.), which is designed to sequence the first 527 bp of the 16S rRNA gene for bacterial identification, by using 52 coryneform gram-positive bacilli from clinical specimens isolated from January through June 1993 at the Mayo Clinic. Compared to conventional and supplemented phenotypic methods, MicroSeq provided concordant results for identification to the genus level for all isolates. At the species level, MicroSeq provided concordant results for 27 of 42 (64.3%)
Corynebacterium
isolates and 5 of 6 (83.3%)
Corynebacterium
-related isolates, respectively. Within the
Corynebacterium
genus, MicroSeq gave identical species-level identifications for the clinically significant
Corynebacterium diphtheriae
(4 of 4) and
Corynebacterium jeikeium
(8 of 8), but it identified only 50.0% (15 of 30) of other species (
P
< 0.01). Four isolates from the genera
Arthrobacter
,
Brevibacterium
, and
Microbacterium
, which could not be identified to the species level by conventional methods, were assigned a species-level identification by MicroSeq. The total elapsed time for running a MicroSeq identification was 15.5 to 18.5 h. These data demonstrate that the MicroSeq 500 16S bacterial sequencing kit provides a potentially powerful method for the definitive identification of clinical coryneform bacterium isolates.
Selective Isolation ofeae-Positive Strains of Shiga Toxin-ProducingEscherichia coli
Authors: Fukushima Hiroshi , Hoshina Ken , Gomyoda Manabu
Published: December 2019

Culture on cefixime, tellurite, and sorbitol-MacConkey agar after HCl treatment facilitated the growth of 410 (94%) of 436eae-positive Shiga toxin-producingEscherichia coli(STEC) strains and 17 (16%) of 107eae-negative STEC strains. This selectivity was closely related to acid resistance inE. coliand tellurite resistance ineae-positive STEC strains.
Pseudomembranous Colitis Caused by a Toxin A
−
B
+
Strain of
Clostridium difficile
Authors: Limaye Ajit P. , Turgeon David K. , Cookson Brad T. , Fritsche Thomas R.
Published: December 2019

We report a case of severe pseudomembranous colitis due to a toxin A
−
B
+
strain of
Clostridium difficile
in an immunosuppressed patient and discuss the implications for diagnostic testing in suspected
C. difficile
-associated diarrhea.
Aerococcus urinae
in Urinary Tract Infections
Authors: Zhang Qing , Kwoh Christopher , Attorri Silvia , Clarridge Jill E.
Published: December 2019

Aerococcus urinae
is a rarely reported pathogen, possibly due to difficulties in the identification of the organism.
A. urinae
is a gram-positive coccus that grows in pairs and clusters, produces alpha-hemolysis on blood agar, and is negative for catalase and pyrrolidonyl aminopeptidase. Some of these characteristics and its being absent from the databases of most commercial identification systems could allow
A. urinae
to be misidentified as a streptococcus, enterococcus, or staphylococcus. We report two cases of urinary tract infection (UTI) caused by
A. urinae
and characterize these isolates by morphology, biochemical testing, whole-cell fatty acid analysis, 16S rRNA gene sequencing, and antibiotic susceptibilities. Most patients infected with
A. urinae
are elderly males with predisposing conditions who present initially with UTI. Because
A. urinae
is resistant to sulfonamides, treatment could be inappropriate, with infections resulting in serious complications, including death. It is important for the clinician and the microbiologist to consider
A. urinae
a potential pathogen and proceed with thorough microbiological identification.
Distribution of Rotavirus VP4 Genotypes and VP7 Serotypes among Nonhospitalized and Hospitalized Patients with Gastroenteritis and Patients with Nosocomially Acquired Gastroenteritis in Austria
Authors: Frühwirth M. , Brösl S. , Ellemunter H. , Moll-Schüler I. , Rohwedder A. , Mutz I.
Published: December 2019

To assess the potential benefits of a reassortant tetravalent rotavirus vaccine, we investigated stool specimens from children in three different groups by reverse transcription-PCR (RT-PCR) for rotavirus G and P types: (i) children not hospitalized with community-acquired rotavirus-acute gastroenteritis (RV-AGE), (ii) children hospitalized for RV-AGE, and (iii) children with nosocomially acquired RV-AGE. From a total of 553 samples investigated, 335 were positive by enzyme-linked immunosorbent assay, of which 294 (88%) were positive by RT-PCR. Among the RT-PCR-positive samples, the predominant types were G1P[8] (84%), followed by G4P[8] (9%) and G3P[8] (2%). No differences between the three groups were observed, suggesting that community vaccination will diminish the most cost-relevant cases of hospitalizations and nosocomial infections.
Helicobacter mesocricetorum
sp. nov., a Novel Helicobacter Isolated from the Feces of Syrian Hamsters
Authors: Simmons J. H. , Riley L. K. , Besch-Williford C. L. , Franklin C. L.
Published: December 2019

A spiral-shaped bacterium with bipolar, single, nonsheathed flagella was isolated from the feces of Syrian hamsters. The bacterium grew as a thin spreading film at 37°C under microaerobic conditions, did not hydrolyze urea, was positive for catalase and alkaline phosphatase, reduced nitrate to nitrite, did not hydrolyze hippurate, and was sensitive to nalidixic acid but resistant to cephalothin. Sequence analysis of the 16S rRNA gene and biochemical and phenotypic criteria indicate that the novel bacterium is a helicobacter. The novel bacterium is most closely related to the recently described mouse enteric helicobacter,
Helicobacter rodentium
. This is the first urease-negative
Helicobacter
species with nonsheathed flagella isolated from feces of asymptomatic Syrian hamsters. We propose to name this novel helicobacter
Helicobacter mesocricetorum
. The type strain is MU 97-1514 (GenBank accession number
AF072471
).
Comparison of Agar Diffusion Methodologies for Antimicrobial Susceptibility Testing of
Pseudomonas aeruginosa
Isolates from Cystic Fibrosis Patients
Authors: Burns Jane L. , Saiman Lisa , Whittier Susan , Larone Davise , Krzewinski Jay , Liu Zhenling , Marshall Steven A. , Jones Ronald N.
Published: December 2019

Pseudomonas aeruginosa
is the most common pathogen infecting the lungs of patients with cystic fibrosis (CF). Improved antimicrobial chemotherapy has significantly increased the life expectancy of these patients. However, accurate susceptibility testing of
P. aeruginosa
isolates from CF sputum may be difficult because the organisms are often mucoid and slow growing. This study of 597 CF isolates of
P. aeruginosa
examined the correlation of disk diffusion and Etest (AB BIODISK, Solna, Sweden) results with a reference broth microdilution method. The rates of interpretive errors for 12 commonly used antipseudomonal antimicrobials were determined. The disk diffusion method correlated well (zone diameter versus MIC) for all of the agents tested. However, for mucoid isolates, correlation coefficients (
r
values) for piperacillin, piperacillin-tazobactam, and meropenem were <0.80. The Etest correlation with reference broth microdilution results (MIC versus MIC) was acceptable for all of the agents tested, for both mucoid and nonmucoid isolates. Category interpretation errors were similar for the disk diffusion and Etest methods with 0.4 and 0.1%, respectively, very major errors (false susceptibility) and 1.1 and 2.2% major errors (false resistance). Overall, both agar diffusion methods appear to be broadly acceptable for routine clinical use in susceptibility testing of CF isolates of
P. aeruginosa
.
Standardization of Immunoglobulin M Capture Enzyme-Linked Immunosorbent Assays for Routine Diagnosis of Arboviral Infections
Authors: Martin Denise A. , Muth David A. , Brown Teresa , Johnson Alison J. , Karabatsos Nick , Roehrig John T.
Published: December 2019

Immunoglobulin M antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA) is a rapid and versatile diagnostic method that readily permits the combination of multiple assays. Test consolidation is especially important for arthropod-borne viruses (arboviruses) which belong to at least three virus families: theTogaviridae,Flaviviridae, andBunyaviridae. Using prototype viruses from each of these families and a panel of well-characterized human sera, we have evaluated and standardized a combined MAC-ELISA capable of identifying virus infections caused by members of each virus family. Furthermore, by grouping antigens geographically and utilizing known serological cross-reactivities, we have reduced the number of antigens necessary for testing, while maintaining adequate detection sensitivity. We have determined that a 1:400 serum dilution is most appropriate for screening antiviral antibody, using a positive-to-negative ratio of ≥2.0 as a positive cutoff value. With a blind-coded human serum panel, this combined MAC-ELISA was shown to have test sensitivity and specificity that correlated well with those of other serological techniques.
Distinguishing Species of the
Burkholderia cepacia
Complex and
Burkholderia gladioli
by Automated Ribotyping
Authors: Brisse Sylvain , Verduin Cees M. , Milatovic Dana , Fluit Ad , Verhoef Jan , Laevens Severine , Vandamme Peter , Tümmler Burkhard , Verbrugh Henri A. , van Belkum Alex
Published: December 2019

Several species belonging to the genus
Burkholderia
are clinically relevant, opportunistic pathogens that inhabit major environmental reservoirs. Consequently, the availability of means for adequate identification and epidemiological characterization of individual environmental or clinical isolates is mandatory. In the present communication we describe the use of the Riboprinter microbial characterization system (Qualicon, Warwick, United Kingdom) for automated ribotyping of 104 strains of
Burkholderia
species from diverse sources, including several publicly accessible collections. The main outcome of this analysis was that all strains were typeable and that strains of
Burkholderia gladioli
and of each species of the
B. cepacia
complex, including
B. multivorans
,
B. stabilis
, and
B. vietnamiensis
, were effectively discriminated. Furthermore, different ribotypes were discerned within each species. Ribotyping results were in general agreement with strain classification based on restriction fragment analysis of 16S ribosomal amplicons, but the resolution of ribotyping was much higher. This enabled automated molecular typing below the species level. Cluster analysis of the patterns obtained by ribotyping (riboprints) showed that within
B. gladioli
,
B. multivorans
, and
B. cepacia
genomovar VI, the different riboprints identified always clustered together. Riboprints of
B. cepacia
genomovars I and III,
B. stabilis
, and
B. vietnamiensis
did not show distinct clustering but rather exhibited the formation of loose assemblages within which several smaller, genomovar-specific clusters were delineated. Therefore, ribotyping proved useful for genomovar identification. Analysis of serial isolates from individual patients demonstrated that infection with a single ribotype had occurred, despite minor genetic differences that were detected by pulsed-field gel electrophoresis of DNA macrorestriction fragments. The automated approach allows very rapid and reliable identification and epidemiological characterization of strains and generates an easily manageable database suited for expansion with information on additional bacterial isolates.
Distribution and Molecular Characterization of
Porphyromonas gingivalis
Carrying a New Type of
fimA
Gene
Authors: Nakagawa Ichiro , Amano Atsuo , Kimura Richard K. , Nakamura Takayuki , Kawabata Shigetada , Hamada Shigeyuki
Published: December 2019

Fimbriae of
Porphyromonas gingivalis
are filamentous appendages on the cell surface and are thought to be one of the virulence factors. The
fimA
gene encoding the subunit protein of fimbriae, fimbrillin (FimA), was classified into four typeable variants (types I to IV). We previously examined the distribution of
P. gingivalis
in terms of
fimA
genotypes in periodontitis patients using a
fimA
type-specific PCR assay. However, some patients harbored
P. gingivalis
with untypeable
fimA
. In this study, we have cloned a new type (type V) of
fimA
from dental plaque samples.
P. gingivalis
with type V
fimA
was isolated from dental plaque of a periodontitis patient, and the isolate was named HNA-99. The deduced amino acid sequences were compared with those of type I
P. gingivalis
ATCC 33277, type II strain HW24D1, type III strain 6/26, and type IV strain HG564, and the homologies were found to be 45, 44, 43, and 55%, respectively. Southern blot analysis showed that the clinical isolate HNA-99 possessed
P. gingivalis
-specific genes
sod
and
kgp
. However, in terms of serological specificities, type V FimA showed a difference from other types of FimA. In addition, type V
P. gingivalis
bacteria were detected in 16.4% (12 of 73) of the
P. gingivalis
-positive patients with periodontitis by PCR assay using specific primers. Thus, a new type of
fimA
gene is now established, and the
fimA
genotyping could be useful in determining the disease-associated genotypes of
P. gingivalis
involved in the development of adult periodontitis.
Evaluation of the LiPA MYCOBACTERIA Assay for Identification of Mycobacterial Species from BACTEC 12B Bottles
Authors: Miller Nancimae , Infante Susanna , Cleary Tim
Published: December 2019

The LiPA MYCOBACTERIA (Innogenetics NV, Ghent, Belgium) assay was used to identify mycobacterial isolates using culture fluid from positive BACTEC 12B bottles. The LiPA method involves reverse hybridization of a biotinylated mycobacterial PCR fragment, a 16 to 23S rRNA spacer region, to oligonucleotide probes arranged in lines on a membrane strip, with detection via biotin-streptavidin coupling by a colorimetric system. This system identifies
Mycobacterium
species and differentiates
M. tuberculosis
complex,
M. avium-M. intracellulare
complex, and the following mycobacterial species:
M. avium
,
M. intracellulare
,
M. kansasii
,
M. chelonae
group,
M. gordonae
,
M. xenopi
, and
M. scrofulaceum
. The mycobacteria were identified in the laboratory by a series of tests, including the Roche AMPLICOR
Mycobacterium tuberculosis
(MTB) test, the Gen-Probe ACCUPROBE, and a PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the 65-kDa heat shock protein gene. The LiPA MYCOBACTERIA assay detected 60 mycobacterium isolates from 59 patients. There was complete agreement between LiPA and the laboratory identification tests for 26
M. tuberculosis
complex, 9
M. avium
, 3
M. intracellulare
complex, 3
M. kansasii
, 4
M. gordonae
, and 5
M. chelonae
group (all were
M. abscessus
) isolates. Three patient samples were LiPA positive for
M. avium-M. intracellulare
complex, and all were identified as
M. intracellulare
by the PCR-RFLP analysis. Seven additional mycobacterial species were LiPA positive for
Mycobacterium
spp. (six were
M. fortuitum
, and one was
M. szulgai
). The LiPA MYCOBACTERIA assay was easy to perform, and the interpretation of the positive bands was clear-cut. Following PCR amplification and gel electrophoresis, the LiPA assay was completed within 3 h.
Evaluation of Commercially Available Preservatives for Laboratory Detection of Helminths and Protozoa in Human Fecal Specimens
Authors: Pietrzak-Johnston S. M. , Bishop H. , Wahlquist S. , Moura H. , Da Silva N. De Oliveira , Da Silva S. Pereira , Nguyen-Dinh P.
Published: December 2019

Formalin and mercuric chloride-based low-viscosity polyvinyl alcohol (LV-PVA) are widely used by most diagnostic parasitology laboratories for preservation of helminth eggs and protozoan cysts and trophozoites in fecal specimens. Concerns about the toxicity of formalin and the difficulty of disposal of LV-PVA are powerful incentives to use alternate preservatives. Such alternatives have been marketed by several companies and are often presented as one-vial, non-mercuric chloride fixatives that aim at performing the same role as formalin and PVA combined. We compared five, one-vial commercial preservatives, two from Meridian Diagnostics, Inc. (Ecofix and sodium acetate-acetic acid-formalin), and one each from Scientific Device Laboratories, Inc. (Parasafe), Alpha Tec Systems, Inc. (Proto-fix), and Streck Laboratories, Inc. (STF), with 10% formalin and LV-PVA. Fecal specimens obtained from patients in a Brazilian hospital were aliquoted within 12 h of collection into the seven preservatives mentioned above and were processed after 1 month at the Centers for Disease Control and Prevention. Direct and concentrated permanent smears as well as concentrates for 20 positive specimens (a total of 259 processed samples) were prepared, stained according to the manufacturers' instructions, examined, and graded. Positive specimens contained one or more parasites with stages consisting of eggs, larvae, cysts, and a few trophozoites of
Giardia intestinalis
. Criteria for assessment of the preservatives included the quality of the diagnostic characteristics of helminth eggs, protozoan cysts, and trophozoites, ease of use, and cost. Acceptable alternatives to formalin for wet preparations were found. Ecofix was found to be comparable to the traditional “gold standard” LV-PVA for the visualization of protozoa in permanent stained smears. This study suggests that more acceptable alternatives to the traditional formalin and LV-PVA exist.
Macrorestriction Fragment Profiles Reveal Genetic Variation of
Cowdria ruminantium
Isolates
Authors: de Villiers E. P. , Brayton K. A. , Zweygarth E. , Allsopp B. A.
Published: December 2019

Macrorestriction profile analysis by pulsed-field gel electrophoresis (PFGE) was used to distinguish between seven isolates of
Cowdria ruminantium
from geographically different areas. Characteristic profiles were generated for each isolate by using the restriction endonucleases
Ksp
I,
Sal
I, and
Sma
I with chromosomal sizes ranging between 1,546 and 1,692 kb. Statistical analysis of the macrorestriction profiles indicated that all the isolates were distinct from each other; these data contribute to a better understanding of the epidemiology of this pathogen and may be exploited for the identification of genotype-specific DNA probes.
Evaluation of Quantitative PCR and Branched-Chain DNA Assay for Detection of Hepatitis B Virus DNA in Sera from Hepatocellular Carcinoma and Liver Transplant Patients
Authors: Chen Tao , Luk John M. , Cheung Siu-Tim , Yu Wan-Ching , Fan Sheung-Tat
Published: December 2019

This study evaluated the applicability of quantitative PCR (Q-PCR) and branched-chain DNA assays for detection of hepatitis B virus (HBV) DNA in sera. For 42 samples, the detection rates were 81 and 41%, respectively, with a correlation coefficient of 0.633. The Q-PCR is useful for early monitoring of HBV load in high-risk patients.
Comparison of PCR-Ribotyping, Arbitrarily Primed PCR, and Pulsed-Field Gel Electrophoresis for Typing
Clostridium difficile
Authors: Bidet Philippe , Lalande Valérie , Salauze Béatrice , Burghoffer Béatrice , Avesani Véronique , Delmée Michel , Rossier Anne , Barbut Frédéric , Petit Jean-Claude
Published: December 2019

Clostridium difficile
is now recognized as the major agent responsible for nosocomial diarrhea in adults. Among the genotyping methods available, arbitrarily primed PCR (AP-PCR), PCR-ribotyping, and pulsed-field gel electrophoresis (PFGE) have been widely used for investigating outbreaks of
C. difficile
infections. However, the comparative typing ability, reproducibility, discriminatory power, and efficiency of these methods have not been fully investigated. We compared the results of three methods—AP-PCR with three different primers (AP3, AP4, and AP5), PCR-ribotyping, and PFGE (with
Sma
I endonuclease)—to differentiate 99 strains of
C. difficile
that had been previously serogrouped. Typing abilities were 100% for PCR-ribotyping and AP-PCR with AP3 and 90% for PFGE, due to early DNA degradation in strains from serogroup G. Reproducibilities were 100% for PCR-ribotyping and PFGE but only 88% for AP-PCR with AP3, 67% for AP-PCR with AP4, and 33% for AP-PCR with AP5. Discriminatory power for unrelated strains was >0.95 for all the methods but was lower for PCR-ribotyping among serogroups D and C. PCR-based methods were easier and quicker to perform, but their fingerprints were more difficult to interpret than those of PFGE. We conclude that PCR-ribotyping offers the best combination of advantages as an initial typing tool for
C. difficile
.
Performance of Phenotypic and Genotypic Methods To Determine the Clinical Relevance of Serial Blood Isolates of
Staphylococcus epidermidis
in Patients with Septicemia
Authors: Sloos J. H. , Dijkshoorn L. , Vogel L. , van Boven C. P. A.
Published: December 2019

Five typing methods, including biotyping (API ID32; BioMérieux, Marcy l'Etoile, France), quantitative antibiogram typing based on actual zone sizes, plasmid typing, randomly amplified polymorphic DNA (RAPD) analysis (with primer M13 and primer set ERIC-2–1026), and pulsed-field gel electrophoresis (PFGE), were compared with a previously performed method of DNA fingerprinting by AFLP (amplified fragment length polymorphism analysis) for their performance in the typing of blood isolates of
Staphylococcus epidermidis
. Sixteen epidemiologically unrelated strains and 11 sets of four blood culture isolates from 11 patients with septicemia were used. The stabilities and reproducibilities of the patterns, the discriminatory capacities of the methods, and the ability to apply the methods to blood culture isolates were used as performance criteria. All strains tested were typeable by each method, and the patterns were stable and reproducible. The numbers of different types within the collection of 16 epidemiologically different isolates were 5 by biotyping, 14 by antibiogram typing, 4 by plasmid typing, 9 by the RAPD assay (combination of results with primer M13 and primer set ERIC-2–1026), and 16 by PFGE. Within the 11 sets of four blood culture isolates the types found by quantitative antibiogram typing, plasmid typing, and PFGE were unique for each set, whereas by biotyping and RAPD analysis some types were observed in more than one set. The results of biotyping did not correspond with the results of the other methods or the results of AFLP. For 6 of the 11 sets, the results of all methods except those of biotyping corresponded completely. Quantitative antibiogram typing, PFGE, and AFLP proved to be the most accurate of the six typing methods tested.
Rapid Solid-Phase Immunoassay for Detection of Methicillin-Resistant
Staphylococcus aureus
Using Cycling Probe Technology
Authors: Fong Whalley K. , Modrusan Zora , Mcnevin John P. , Marostenmaki Johanna , Zin Ben , Bekkaoui Faouzi
Published: December 2019

A Cycling Probe Technology (CPT) assay with a lateral-flow device (strip) was developed for the detection of the
mecA
gene from methicillin-resistant
Staphylococcus aureus
(MRSA) cultures. The assay uses a
mecA
probe (DNA-RNA-DNA) labeled with fluorescein at the 5′ terminus and biotin at the 3′ terminus. The CPT reaction occurs at a constant temperature, which allows the probe to anneal to the target DNA. RNase H cuts the RNA portion of the probe, allowing the cleaved fragments to dissociate from the target DNA, making the target available for further cycling. The strip detection step uses a nitrocellulose membrane with streptavidin and immunoglobulin G antibody impregnated on the surface. In the absence of the
mecA
gene, the uncut probe is bound to an antifluorescein-gold conjugate and is then captured by the streptavidin to form a test line. In the presence of the
mecA
gene, the probe is cut and no test line is formed on the strip. A screen of 324
S. aureus
clinical isolates by the CPT-strip assay showed a 99.4% sensitivity and a 100% specificity compared to the results of PCR for the detection of the
mecA
gene. Specificity testing showed that the CPT-strip assay did not exhibit any cross-reactivity with a panel of
mecA
-negative non-
S. aureus
isolates. The CPT-strip assay is simple and does not require sophisticated equipment. Furthermore, the assay takes 1.5 h starting from a primary culture to the time to detection of the
mecA
gene in
S. aureus
isolates.
Use of Pooled Fecal Culture for Sensitive and Economic Detection ofMycobacterium aviumsubsp.paratuberculosisInfection in Flocks of Sheep
Authors: Whittington R. J. , Fell S. , Walker D. , McAllister S. , Marsh I. , Sergeant E. , Taragel C. A. , Marshall D. J. , Links I. J.
Published: December 2019

Ovine Johne's disease, or paratuberculosis, occurs in many countries. In Australia, surveillance using serology is used as part of a control program, but the testing regime is costly relative to its sensitivity. For this reason, culturing ofMycobacterium aviumsubsp.paratuberculosisin fecal samples pooled from a number of sheep was evaluated. Initially, the effect of pooling on the sensitivity of fecal culture was evaluated using samples from 20 sheep with multibacillary paratuberculosis and 20 sheep with paucibacillary paratuberculosis, each confirmed histologically. All multibacillary cases and 50% of paucibacillary cases were detected by culturing of feces at a pooling rate of 1 infected plus 49 uninfected sheep. In a pilot-scale study in 1997,M. aviumsubsp.paratuberculosiswas detected by pooled fecal culture on 93% of 27 infected farms which were identified originally based on history, clinical signs, and one or more rounds of testing using serologic and histopathologic examinations. Pooled fecal culture was compared with serologic examination for submissions from 335 farms where both tests had been conducted on the same sheep and was significantly more sensitive (P< 0.001). Computer simulation of random sampling indicated that the testing of 6 pools of 50 sheep would provide 95% confidence in detecting ≥2% prevalence of infection. The estimated laboratory cost of pooled fecal culture when applied as a flock test is approximately 30% that of serologic examination, and sample collection costs are lower. It is recommended that pooled fecal culture replace serologic examination for detection ofM. aviumsubsp.paratuberculosisinfection at the flock level.
Quantitation of Varicella-Zoster Virus DNA in Whole Blood, Plasma, and Serum by PCR and Electrochemiluminescence
Authors: de Jong Menno D. , Weel Jan F. L. , Schuurman Tim , Wertheim-van Dillen Pauline M. E. , Boom René
Published: December 2019

We describe a highly sensitive assay for quantitation of varicella-zoster virus (VZV) DNA in blood, involving PCR amplification, solution hybridization with Tris-(2,2′-bipyridine)-ruthenium(II) chelate-labeled probes, and measurement by electrochemiluminescence (ECL). Extraction and amplification efficiencies were monitored by the inclusion of internal control (IC) DNA, mimicking the VZV target, in the DNA extraction. Viral DNA load was calculated from the ratio of VZV and IC ECL signals. The lower limit of sensitivity was 20 VZV DNA copies/ml of plasma or serum and 80 copies/ml of whole blood. In reconstruction experiments, expected and calculated VZV DNA loads were in excellent accordance. Blood specimens from 42 VZV-infected patients were tested for the presence of VZV DNA and showed detection rates of 86% in patients with varicella and 81% in patients with herpes zoster. In specimens obtained during the first week after onset of the rash, detection rates were 100 and 89%, respectively. Viral DNA was detected in all immunocompromised patients with herpes zoster, emphasizing the risk of disseminated disease in this patient group. VZV DNA load was similar in patients with varicella and multidermatomal herpes zoster and lower in patients with unidermatomal zoster. Despite the cell-associated nature of the virus, VZV DNA was detected in serum and plasma at high copy numbers, and at similar frequencies compared to whole-blood specimens. Quantitation of VZV DNA in blood is of potential importance for diagnosis and clinical management of VZV-infected patients. Plasma and serum provide convenient matrices for this purpose.
Prevalence of GB Virus C (Also Called Hepatitis G Virus) Markers in Norwegian Blood Donors
Authors: Nordbø Svein Arne , Krokstad Sidsel , Winge Per , Skjeldestad Finn Egil , Dalen Are B.
Published: December 2019

GB virus C (GBV-C), also called hepatitis G virus (HGV), occurs worldwide, but the clinical significance of this virus is still unclear. Plasma samples from 1,001 blood donors were tested by reverse transcription PCR using primers from the NS5 region and by a commercial enzyme-linked immunosorbent assay (ELISA) for the detection of immunoglobulin G antibodies against the putative envelope of HGV (anti-HGV E2). GBV-C/HGV RNA was present in the plasma from 2.5% of the blood donors, and anti-HGV E2 antibodies could be detected in 10.5% of the samples. Only one of the blood donors with viremia had elevated levels of alanine aminotransferase. Among ELISA-positive donors, there was a significantly higher percentage (16.5%) of individuals who had been treated by acupuncture than individuals who had not been given this treatment (9.4%). No other variables showed significant differences. Screening of medical records from 401 recipients of blood from PCR-positive donors revealed no association with liver disease. Four of 12 partners (33%) were HGV RNA positive, and sequence analyses of the strains showed that four of the couples probably were infected with the same strains, while strains from different couples were not identical. Anti-HGV E2 antibodies were detected in serum samples from four other partners. The prevalence of GBV-C/HGV among blood donors in our region is dramatically higher than the prevalence of hepatitis C virus (0.03%).
Analytical Sensitivity, Reproducibility of Results, and Clinical Performance of Five PCR Assays for Detecting
Chlamydia pneumoniae
DNA in Peripheral Blood Mononuclear Cells
Authors: Mahony J. B. , Chong S. , Coombes B. K. , Smieja M. , Petrich A.
Published: December 2019

Chlamydia pneumoniae
has been associated with atherosclerosis and coronary artery disease (CAD), and its DNA has been detected in atheromatous lesions of the aorta, carotid, and coronary arteries by a variety of PCR assays. The objective of this study was to compare the performances of five published PCR assays in the detection of
C. pneumoniae
in peripheral blood mononuclear cells (PBMCs) from patients with coronary artery disease. The assays included two conventional PCRs, one targeting a cloned
Pst
I fragment and one targeting the 16S rRNA gene; two nested PCRs, one targeting the 16S rRNA gene and one targeting
ompA
; and a touchdown enzyme time release (TETR) PCR, targeting the 16S rRNA gene. All PCRs had similar analytical sensitivities and detected a minimum of 0.005 inclusion-forming units (IFU) of
C. pneumoniae
; the
ompA
nested PCR and the TETR PCR were slightly more sensitive and detected 0.001 IFU. Assay reproducibility was examined by testing 10 replicates of
C. pneumoniae
DNA by each assay. All five assays showed excellent reproducibility at high levels of DNA, with scores of 10 out of 10 for 0.01 IFU, but exhibited decreased reproducibility for smaller numbers of
C. pneumoniae
IFU for all tests. Pairwise comparison of test results indicated that there was a significant difference between tests (Cochran Q = 32.0,
P
< 0.001), with the
Pst
I fragment (
P
< 0.001) and 16S rRNA (
P
= 0.002) assays having lower reproducibility than the nested
ompA
and TETR assays. To further analyze assay sensitivity,
C. pneumoniae
-infected U-937 mononuclear cells were added to whole blood, and extracted mononuclear-cell DNA was tested by each assay. All five assays showed similar sensitivities, detecting 15 infected cells; three assays detected 3 infected cells, while all assays were negative at the next dilution (1.5 infected cells). A striking difference in performance of the five assays was seen, however, when PBMCs from CAD patients were tested for
C. pneumoniae
DNA. The
ompA
nested PCR detected
C. pneumoniae
DNA in 11 of 148 (7.4%) specimens, the 16S rRNA nested PCR detected 2 positives among the 148 specimens (1.4%) (
P
< 0.001), and the other 3 assays detected no positive specimens (
P
< 0.001, compared with the
ompA
assay). These results indicate that analytical sensitivity alone does not predict the ability of an assay to detect
C. pneumoniae
in whole-blood-derived PBMCs. Before standardized assays can be used in wide-scale epidemiological studies, further characterization of these assays will be required to improve our understanding of their performance in the detection of
C. pneumoniae
in clinical material.
Characterization of United Kingdom Isolates ofCorynebacterium pseudotuberculosisUsing Pulsed-Field Gel Electrophoresis
Authors: Connor Kathleen M. , Quirie Malcolm M. , Baird Graham , Donachie William
Published: December 2019

Caseous lymphadenitis is a chronic suppurative disease caused byCorynebacterium pseudotuberculosisand is responsible for serious economic losses to the sheep and goat industry. Caseous lymphadenitis was first reported for goats in the United Kingdom in 1990 and for sheep in 1991. Recent evidence suggests that the prevalence of the disease within the national flock is increasing. Fifty isolates ofC. pseudotuberculosisfrom the United Kingdom comprising sheep and horse isolates, the original goat outbreak strain, and the type strain were characterized by biotyping, antimicrobial susceptibility, production of phospholipase D, and genotyping by pulsed-field gel electrophoresis usingSfiI andSmaI. All of the isolates were confirmed asC. pseudotuberculosis, and all produced phospholipase D but none reduced nitrate. Restriction withSfiI generated 16 to 18 bands between 48.5 and 290 kb and differentiated six pulsotypes. We conclude that 80% of the strains tested were epidemiologically related to the outbreak strain and that the equine profile was distinct both phenotypically and genotypically.
Diagnosis and Clinical Virology of Lassa Fever as Evaluated by Enzyme-Linked Immunosorbent Assay, Indirect Fluorescent-Antibody Test, and Virus Isolation
Authors: Bausch D. G. , Rollin P. E. , Demby A. H. , Coulibaly M. , Kanu J. , Conteh A. S. , Wagoner K. D. , McMullan L. K. , Bowen M. D. , Peters C. J. , Ksiazek T. G.
Published: December 2019

The Lassa virus (an arenavirus) is found in West Africa, where it sometimes causes a severe hemorrhagic illness called Lassa fever. Laboratory diagnosis has traditionally been by the indirect fluorescent-antibody (IFA) test. However, enzyme-linked immunosorbent assays (ELISAs) for Lassa virus antigen and immunoglobulin M (IgM) and G (IgG) antibodies have been developed that are thought to be more sensitive and specific. We compared ELISA and IFA testing on sera from 305 suspected cases of Lassa fever by using virus isolation with a positive reverse transcription-PCR (RT-PCR) test as the “gold standard.” Virus isolation and RT-PCR were positive on 50 (16%) of the 305 suspected cases. Taken together, Lassa virus antigen and IgM ELISAs were 88% (95% confidence interval [CI], 77 to 95%) sensitive and 90% (95% CI, 88 to 91%) specific for acute infection. Due to the stringent gold standard used, these likely represent underestimates. Diagnosis could often be made on a single serum specimen. Antigen detection was particularly useful in providing early diagnosis as well as prognostic information. Level of antigenemia varied inversely with survival. Detection by ELISA of IgG antibody early in the course of illness helped rule out acute Lassa virus infection. The presence of IFA during both acute and convalescent stages of infection, as well as significant interobserver variation in reading the slides, made interpretation difficult. However, the assay provided useful prognostic information, the presence of IFA early in the course of illness correlating with death. The high sensitivity and specificity, capability for early diagnosis, and prognostic value of the ELISAs make them the diagnostic tests of choice for the detection of Lassa fever.
18S Ribosomal DNA-Based PCR for Diagnosis of
Trichomonas vaginalis
Authors: Mayta Holger , Gilman Robert H. , Calderon Maritza M. , Gottlieb Aren , Soto Giselle , Tuero Iskra , Sanchez Sixto , Vivar Aldo
Published: December 2019

Trichomonas vaginalis
remains the most common sexually transmitted parasite in the world and is considered a major risk factor in the transmission of the human immunodeficiency virus. A PCR technique using primers targeting a specific region of the 18S rRNA gene of
T. vaginalis
was developed. The PCR test was standardized using 15 reference strains, giving a single product of 312 bp in all strains. No amplification was observed when DNA from related organisms or human DNA was used as a target. The test was evaluated on 372 vaginal swab specimens and 361 urine samples from women attending infertility and obstetric clinics at two separate hospitals in Lima, Peru. Compared to
T. vaginalis
culture, the overall sensitivity and specificity of PCR of vaginal swab samples was 100% and 98%, respectively. The PCR of urine samples was 100% sensitive and 99.7% specific compared to culture of vaginal swab, but the sensitivity drops to 83.3% when compared to PCR of vaginal swabs. All culture-positive samples were found to be positive by PCR in either urine or vaginal secretion. None of the PCR-negative samples were positive by culture. The origin of the amplification was confirmed by digestion of PCR products with
Hae
III. This PCR assay, which is easy to perform and has a high sensitivity and specificity, should be useful for routine diagnosis of
T. vaginalis
infection.
Recognition of Enteropathogenic
Escherichia coli
Virulence Determinants by Human Colostrum and Serum Antibodies
Authors: Parissi-Crivelli Aurora , Parissi-Crivelli Joaquín M. , Girón Jorge A.
Published: December 2019

Human colostra and sera collected from Mexican mothers and their children at birth and 6 months thereafter were studied for the presence of antibodies against the bundle-forming pilus and several chromosomal virulence gene products (intimin and secreted proteins EspA and EspB) of enteropathogenic
Escherichia coli
(EPEC). Among 21 colostrum samples studied, 76, 71.5, 57, and 47% of them contained immunoglobulin A (IgA) antibodies against EspA, intimin, EspB, and BfpA, respectively. Interestingly, there was a difference in IgG response to EPEC antigens between the sera from neonates and sera from the same children 6 months later. While the number of neonates reacting to Esps and intimin diminished when they reached 6 months of age, those reacting with BfpA increased from 9 to 71%. Intimin from an enterohemorrhagic
E. coli
strain was also recognized by most of the samples reacting with EPEC intimin. These data suggest that Bfp and Esps elicit an antibody response during the early days of life of neonates and support the value of breast-feeding in areas of the world where bacterial diarrheal infections are endemic.
Comparison of Sequencing by Hybridization and Cycle Sequencing for Genotyping of Human Immunodeficiency Virus Type 1 Reverse Transcriptase
Authors: Hanna George J. , Johnson Victoria A. , Kuritzkes Daniel R. , Richman Douglas D. , Martinez-Picado Javier , Sutton Lorraine , Hazelwood J. Darren , D'Aquila Richard T.
Published: December 2019

The performances of two methods of nucleotide sequencing were compared for the detection of drug resistance mutations in human immunodeficiency virus type 1 reverse transcriptase (RT) in viruses isolated from highly RT inhibitor-experienced individuals. Of 11,677 amino acids deduced from population PCR products by both cycle sequencing and sequencing by hybridization to high-density arrays of oligonucleotide probes, 97.4% were concordant by both methods, 0.8% were discordant, and 1.7% had an ambiguous determination by at least one method. A higher rate of discordance (3.9%) was observed among RT inhibitor resistance-associated codons. In 45% of the isolates, RT codon 67 was deduced as the wild-type Asp by hybridization sequencing but as the zidovudine resistance-associated Asn by cycle sequencing. In other resistance-associated codon discordances, cycle sequencing also more commonly called a known resistance-associated amino acid than hybridization sequencing did. The nucleotide sequence in the vicinity of several codons with discordant calls influenced population-based hybridization sequencing. For isolates evaluated by additional sequencing of molecular clones of PCR products by both methods, the discordance between methods was less frequent (0.4% of all 5,994 amino acids and 0 of 494 drug resistance-associated codons). At positions which were discordant or ambiguous in the population sequences, the results of sequencing of clones by both methods were usually in agreement with the population cycle sequencing result. In summary, most RT codons were highly concordant by both methods of population-based sequencing, with discordances due in large part to genetic mixtures within or adjacent to discordant codons.
Detection of Human Herpesvirus 6 and Varicella-Zoster Virus in Tear Fluid of Patients with Bell's Palsy by PCR
Authors: Pitkäranta A. , Piiparinen H. , Mannonen L. , Vesaluoma M. , Vaheri A.
Published: December 2019

Human herpesvirus 6 DNA was detected by PCR in the tear fluid of 7 (35%) of 20 patients with Bell's palsy and of 1 (5%) of 20 healthy controls. Varicella-zoster virus was detected by PCR in the tear fluid of 2 of 20 Bell's palsy patients but in none of the tear fluids from 20 healthy controls. These findings suggest an association between human herpesviruses and Bell's palsy.