bluuebunny/crossref_metadata_2025 huggingface dataset:
Insertion Element IS
3
-Based PCR Method for Subtyping
Escherichia coli
O157:H7
Authors: Thompson Curt J. , Daly Claire , Barrett Timothy J. , Getchell Jane P. , Gilchrist Mary J. R. , Loeffelholz Mike J.
Published: December 2019

An
Escherichia coli
O157:H7 subtyping method based on PCR amplification of variable DNA sequences between the repetitive element IS
3
was developed. Template DNA was prepared by boiling cells in Chelex. Two separate IS
3
PCR amplifications were performed for each isolate: one with a single primer (primer IS3A) and one with two primers (primers IS3A and IS3B). The IS
3
PCR subtyping method was applied to 35 epidemiologically related and unrelated
E. coli
O157:H7 isolates that had been previously characterized by pulsed-field gel electrophoresis (PFGE). PFGE identified 25 different subtypes (difference of one or more bands). PCR with single primer IS3A and primer pair IS3A-IS3B identified 6 and 14 different subtypes, respectively. By combining the results of the two PCR amplifications, 15 different IS
3
PCR subtypes were identified. While not as sensitive as PFGE, IS
3
PCR subtyping grouped all outbreak-related isolates. IS
3
PCR banding patterns were reproducible between amplifications and between subcultures. IS
3
PCR could serve as a simple, rapid screening method for the identification of unrelated
E. coli
O157:H7 isolates.
Temporal Correlations between Tick Abundance and Prevalence of Ticks Infected withBorrelia burgdorferiand Increasing Incidence of Lyme Disease
Authors: Stafford Kirby C. , Cartter Matthew L. , Magnarelli Louis A. , Ertel Starr-Hope , Mshar Patricia A.
Published: December 2019

The abundance of host-seekingIxodes scapularisnymphs, the principal vector for the Lyme disease agent,Borrelia burgdorferi, in Old Lyme, Lyme, and East Haddam, Connecticut, was compared with the incidence of reported human Lyme disease in the 12-town area around the Connecticut River and the State of Connecticut for the period 1989 to 1996. Ticks were sampled from lawns and woodlands by dragging flannel over the vegetation and examined for the presence ofB. burgdorferiby indirect fluorescent antibody staining. The infection rate of the nymphal ticks byB. burgdorferiduring the 9-year period was 14.3% (of 3,866), ranging from 8.6% (1993) to 24.4% (1996). The incidence of Lyme disease was positively correlated with tick abundance in the 12 town area (r= 0.828) and the State of Connecticut (r= 0.741). An entomological risk index based upon the number ofI. scapularisticks infected byB. burgdorferiwas highest in 1992, 1994, and 1996 and was highly correlated with the incidence of Lyme disease in Connecticut (r= 0.944). The number of Lyme disease cases has been influenced, in part, by annual changes in population densities ofI. scapularisand, presumably, a corresponding change in the risk of contact with infected ticks. Based upon tick activity and spirochetal infection rates, epidemiologically based Lyme disease case reports on a regional scale appear to reflect real trends in disease.
Detection of Bovine Torovirus in Fecal Specimens of Calves with Diarrhea from Ontario Farms
Authors: Duckmanton Lynn , Carman Susy , Nagy Éva , Petric Martin
Published: December 2019

Breda virus (BRV), a member of the genusTorovirus, is an established etiological agent of disease in cattle. BRV isolates have been detected in the stools of neonatal calves with diarrhea in both Iowa and Ohio and in several areas of Europe. However, this virus has been reported only once in Canada. Therefore, a study was performed to determine the extent to which bovine torovirus is present in calves with diarrhea from farms in southern Ontario. A total of 118 fecal samples from symptomatic calves and 43 control specimens from asymptomatic calves were examined by electron microscopy (EM) and reverse transcription-PCR (RT-PCR) for the presence of torovirus. Torovirus RNA was detected in 43 of the 118 diarrheic samples (36.4%) by RT-PCR with primers designed in the conserved 3′ end of the torovirus genome. By EM, torovirus particles were observed in 37 of the 118 specimens (31.4%). All but one of these samples were also positive by RT-PCR. The incidence of torovirus in the asymptomatic control specimens by RT-PCR was only 11.6%. To establish the identity of the particles observed in the diarrheic specimens, five of the amplicons from samples positive by both RT-PCR and EM were cloned and sequenced. Nucleotide sequence analysis revealed that the bovine torovirus found in southern Ontario manifests between 96 and 97% sequence identity to the BRV type 1 strain found in Iowa. This study shows that bovine torovirus is a common virus in the fecal specimens of calves with diarrhea from farms in southern Ontario and thus may be an important pathogen of cattle.
Rapid Diagnosis of Extrapulmonary Tuberculosis by Ligase Chain Reaction Amplification
Authors: Gamboa Fredy , Dominguez José , Padilla Eduardo , Manterola José M. , Gazapo Elena , Lonca Joan , Matas Lurdes , Hernandez Agueda , Cardona Pere Joan , Ausina Vicente
Published: December 2019

A rapid amplification-based test for the diagnosis of extrapulmonary tuberculosis, the LCx
Mycobacterium tuberculosis
Assay from Abbott Laboratories, was evaluated. Results from the LCx
M. tuberculosis
Assay were compared with those from culture and the final clinical diagnosis for each patient. A total of 526 nonrespiratory specimens from 492 patients were tested. The specimens included urine; feces; lymph node exudates; pleural, cerebrospinal, articular, and ascitic fluids; tissue biopsies; gastric aspirates; purulent exudates; blood; and bone marrow aspirates. After combination of the culture results and the patient’s clinical data, a total of 135 specimens were collected from 122 patients with a diagnosis of extrapulmonary tuberculosis. The sensitivity, specificity, and positive and negative predictive values for the LCx
M. tuberculosis
Assay were 77.7, 98.7, 95.2, and 93.1%, respectively; these values rose in resolved cases of TB to 78.5, 100, 100, and 93.1%, respectively. For 37 (27.4%) specimens from patients smear positive for the disease and 98 (72.6%) specimens from patients smear negative for the disease, the sensitivities of the LCx
M. tuberculosis
Assay were 100 and 71.1%, respectively. Statistically significant differences (
P
< 0.01) in sensitivities were found between culture and the LCx
M. tuberculosis
Assay. These differences were even greater among smear-negative specimens. The results demonstrate that the LCx
M. tuberculosis
Assay will provide rapid and valuable information for the diagnosis of extrapulmonary tuberculosis.
Multicenter Comparison of ESP Culture System II with BACTEC 460TB and with Lowenstein-Jensen Medium for Recovery of Mycobacteria from Different Clinical Specimens, Including Blood
Authors: Tortoli Enrico , Cichero Paola , Chirillo M. Gabriella , Gismondo M. Rita , Bono Letizia , Gesu Giampietro , Simonetti M. Tullia , Volpe Gisella , Nardi Giampietro , Marone Piero
Published: December 2019

The recently developed ESP Culture System II (AccuMed, Chicago, Ill.) was compared with radiometric BACTEC 460TB (Becton Dickinson, Towson, Md.) and with Lowenstein-Jensen medium for recovery of mycobacteria from over 2,500 clinical specimens both of respiratory and nonrespiratory origin, including blood. The majority of the 219 mycobacterial isolates (129) belonged to the
Mycobacterium tuberculosis
complex, followed by 37 isolates of the
Mycobacterium avium
complex (MAC) and 53 isolates of eight other mycobacterial species. Rates of recovery obtained with BACTEC, ESP, and Lowenstein-Jensen medium were 89, 79, and 64%, respectively, with such differences being statistically significant. Different media and systems appeared to behave differently when the more frequently detected organisms were considered:
M. tuberculosis
complex isolates grew better with BACTEC, and MAC isolates grew better with ESP. An analysis of the combinations of Lowenstein-Jensen medium with BACTEC and with ESP did not reveal significant differences in recovery rates. With regard to the times needed for the detection of positive cultures, they were significantly longer on Lowenstein-Jensen medium (average, 28 days) than with the remaining two systems, between which there was no difference (average, 18 days). We conclude, therefore, that the ESP system, when used in combination with a solid medium, performs as well as the thoroughly validated radiometric BACTEC system and offers the advantages of full automation and absence of radioisotopes.
Use of Voriconazole in Treatment of
Scedosporium apiospermum
Infection: Case Report
Authors: Girmenia Corrado , Luzi Giovanni , Monaco Monica , Martino Pietro
Published: December 2019

We report a case of disseminated
Scedosporium apiospermum
infection in a neutropenic patient with acute myeloid leukemia. Due to progression of the mycosis after 7 days of amphotericin B lipid complex therapy and to high susceptibility of the mold to voriconazole in vitro, the patient was treated with intravenous voriconazole. After a few days of therapy, fever disappeared and skin lesions improved. However, the patient died after 1 month due to intestinal bleeding. Despite a negative outcome, this case seems to indicate a promising role for voriconazole in the treatment of
S. apiospermum
infections.
Comparison of a 2,3-Bis(2-Methoxy-4-Nitro-5-Sulfophenyl)-5-[(Phenylamino)Carbonyl]-2
H
-Tetrazolium Hydroxide (XTT) Colorimetric Method with the Standardized National Committee for Clinical Laboratory Standards Method of Testing Clinical Yeast Isolates for Susceptibility to Antifungal Agents
Authors: Hawser S. P. , Norris H. , Jessup C. J. , Ghannoum M. A.
Published: December 2019

MICs for clinical
Candida
and
Cryptococcus
isolates were determined by a method incorporating the colorimetric indicator 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2
H
-tetrazolium hydroxide (XTT), and the results were compared with MICs obtained by the National Committee for Clinical Laboratory Standards approved standard method (M27-A). One hundred percent of all isolates demonstrated agreement within 2 dilutions between the MICs of amphotericin B, fluconazole, itraconazole, ketoconazole, and flucytosine obtained by the two methods. These data suggest that an XTT-based method could provide a useful means for the determination of antifungal susceptibility of yeasts.
Comparison of Three Methods for Testing Azole Susceptibilities of
Candida albicans
Strains Isolated Sequentially from Oral Cavities of AIDS Patients
Authors: Tortorano Anna Maria , Viviani Maria Anna , Barchiesi Francesco , Arzeni Daniela , Rigoni Anna Lisa , Cogliati Massimo , Compagnucci Patrizia , Scalise Giorgio
Published: December 2019

Three susceptibility testing procedures were compared to determine fluconazole, itraconazole, and ketoconazole MICs against 47
Candida albicans
strains isolated sequentially from the oral cavities of five AIDS patients undergoing azole therapy. They included the broth microdilution method (BM), performed according to the National Committee for Clinical Laboratory Standards’ tentative standard, the agar dilution method (AD), and the Etest; the latter two tests were performed both in Casitone agar (AD-Cas and Etest-Cas) and in RPMI (AD-RPMI and Etest-RPMI). Twenty-four- and 48-h MICs obtained by AD and Etest were compared with 48-h MICs obtained by BM. The MICs of all the azoles determined by BM were usually lower than those obtained by the other methods, mainly due to different reading criteria. In order to assess the most appropriate way of evaluating the agreement of MICs obtained by different methods with those produced by the proposed reference method (BM), we used the mean differences calculated according to Bland and Altman’s method. Comparison of fluconazole MICs obtained by BM and AD-Cas yielded a mean difference of 3, and the percentages of agreement within ±2 dilutions were 98 and 100% at 24 and 48 h, respectively. For ketoconazole and itraconazole MICs, lower mean differences were noted, and agreement ranged from 96 to 100%. Agreement between the AD-RPMI and BM results was poor for all azoles, and an increase in MICs was always observed between the 1st- and 2nd-day readings. Similarly, Etest-Cas gave better agreement with BM than did Etest-RPMI for all the azoles. BM, AD-Cas, and Etest-Cas each demonstrated a progressive increase in fluconazole MICs against strains isolated sequentially from a given patient, in accordance with the decreased clinical response to fluconazole.
Molecular Analysis of and Identification of Antibiotic Resistance Genes in Clinical Isolates of
Salmonella typhi
from India
Authors: Shanahan Philippa M. A. , Jesudason Mary V. , Thomson Christopher J. , Amyes Sebastian G. B.
Published: December 2019

A representative sample of 21
Salmonella typhi
strains isolated from cultures of blood from patients at the Christian Medical College and Hospital, Vellore, India, were tested for their susceptibilities to various antimicrobial agents. Eleven of the
S. typhi
strains possessed resistance to chloramphenicol (256 mg/liter), trimethoprim (64 mg/liter), and amoxicillin (>128 mg/liter), while four of the isolates were resistant to each of these agents except for amoxicillin. Six of the isolates were completely sensitive to all of the antimicrobial agents tested. All the
S. typhi
isolates were susceptible to cephalosporin agents, gentamicin, amoxicillin plus clavulanic acid, and imipenem. The antibiotic resistance determinants in each
S. typhi
isolate were encoded by one of four plasmid types. Plasmid-mediated antibiotic resistance genes were identified with specific probes in hybridization experiments; the genes responsible for chloramphenicol, trimethoprim, and ampicillin resistance were chloramphenicol acetyltransferase type I, dihydrofolate reductase type VII, and TEM-1 β-lactamase, respectively. Pulsed-field gel electrophoresis analysis of
Xba
I-generated genomic restriction fragments identified a single distinct profile (18 DNA fragments) for all of the resistant isolates. In comparison, six profiles, different from each other and from the resistance profile, were recognized among the sensitive isolates. It appears that a single strain containing a plasmid conferring multidrug-resistance has emerged within the
S. typhi
bacterial population in Vellore and has been able to adapt to and survive the challenge of antibiotics as they are introduced into clinical medicine.
Rapid Identification of
Candida albicans
and Other Human Pathogenic Yeasts by Using Short Oligonucleotides in a PCR
Authors: Mannarelli B. M. , Kurtzman C. P.
Published: December 2019

A PCR system that can quickly and accurately identify 14 species of human pathogenic yeasts was developed. The procedure distinguished between nine species of a closely related clade,
Lodderomyces elongisporus
,
Candida parapsilosis
, a new
Candida
sp.,
C. sojae
,
C. tropicalis
,
C. maltosa
,
C. viswanathii
,
C. albicans
, and
C. dubliniensis
and between another five more divergent species,
Pichia guilliermondii
,
C. glabrata
,
C. zeylanoides
,
C. haemulonii
, and
C. haemulonii
type II. A rapid DNA extraction procedure that yields purified DNA in about 1 h is also described. The system uses uniform conditions with four primers for each reaction, two 40- to 50-mer universal primers that serve as a positive control and two 23- to 30-mer species-specific primers. Species-specific primers were derived from a 600-nucleotide variable region (D1/D2) at the 5′ end of the large-subunit (26S) ribosomal DNA gene and were generally designed to use mismatches at the 3′ end. Universal primers were developed from conserved nucleotide sequences in the small-subunit (18S) rRNA gene. In this system, a control 1,200- to 1,300-base DNA fragment was produced in all reactions and a smaller 114- to 336-base DNA fragment was produced if the chromosomal DNA from the target species was present. The PCR procedure is rapid and easy to interpret and may be used with mixed cultures.
Tubulointerstitial Nephritis Due to a Mutant Polyomavirus BK Virus Strain, BKV(Cin), Causing End-Stage Renal Disease
Authors: Smith R. D. , Galla J. H. , Skahan K. , Anderson P. , Linnemann C. C. , Ault G. S. , Ryschkewitsch C. F. , Stoner G. L.
Published: December 2019

A renal biopsy from a 36-year-old man with AIDS showed a severe tubulointerstitial nephritis with intranuclear inclusions in epithelial cells. Electron microscopy revealed the characteristic findings of a polyomavirus (PyV) infection, and immunofluorescence indicated the presence of BK virus (BKV) antigen. Inoculation of rhesus monkey kidney cell cultures both with urine and with buffy coat blood cells resulted in a cytopathic response which was subsequently confirmed to be due to BKV. Further characterization of the viral DNA from the kidney by PCR amplification and Southern blot analysis with PyV and strain-specific primers and probes indicated that the virus was closely related to the BK(Dun) strain but different in its apparent sequence arrangement. Subsequent cycle sequencing showed a dinucleotide mutation of TG→AA which substitutes hydrophilic Gln for hydrophobic Leu in a sequence homologous to an origin DNA-binding domain of simian virus 40 T antigen. It is suggested that the mutation and a coding region rearrangement of this strain of BKV designated BKV(Cin) has the potential to alter viral DNA replication and enhance pathogenicity.
Antimicrobial Susceptibilities and Molecular Epidemiology ofSalmonella entericaSerotype Enteritidis Strains Isolated in Hong Kong from 1986 to 1996
Authors: Ling J. M. , Koo I. C. , Kam K. M. , Cheng A. F.
Published: December 2019

The incidence of salmonellosis has been increasing in Hong Kong since 1989. The most commonSalmonella entericaserotype isolated in 1994 wasS. enteritidis. The antimicrobial susceptibilities and molecular epidemiology of 275S. enteritidisstrains isolated in this locality between 1986 and 1996 were studied. Over 99% of the isolates were susceptible to 17 of the 19 antimicrobial agents tested. One isolate harbored an autotransferring plasmid that confers resistance to tetracycline and trimethoprim-sulfamethoxazole. Another isolate harbored a mobilizable plasmid that confers resistance to ampicillin and cephalothin. This isolate was found to produce a β-lactamase with a pI of 5.2. A total of 264 isolates (96%) were found to harbor one to five plasmids, and the majority (254) harbored a 60-kb plasmid. Of these isolates, 94% contained identical 60-kb plasmids. Based on plasmid profiles, plasmid and chromosomal fingerprints, ribotypes, and randomly amplified polymorphic DNA (RAPD) patterns, 170 (62%) isolates were allocated to group 1b. About 90% of isolates had identical or similar DNA fingerprints, ribotypes, and RAPD patterns, suggesting that a predominant clone ofS. enteritidiswas circulating in Hong Kong during the period being studied.
Detection and Identification ofActinobacillus pleuropneumoniaeSerotype 5 by Multiplex PCR
Authors: Lo Terry M. , Ward Christine K. , Inzana Thomas J.
Published: December 2019

Serotyping ofActinobacillus pleuropneumoniaeis based on detection of the serotype-specific capsular antigen. However, not all isolates can be serotyped, and some may cross-react with multiple serotyping reagents. To improve sensitivity and specificity of serotyping and for early detection, a multiplex PCR assay was developed for detection ofA. pleuropneumoniaeand identification of serotype 5 isolates. DNA sequences specific to the conserved export and serotype-specific biosynthesis regions of the capsular polysaccharide ofA. pleuropneumoniaeserotype 5 were used as primers to amplify 0.7- and 1.1-kb DNA fragments, respectively. The 0.7-kb fragment was amplified from all strains ofA. pleuropneumoniaetested with the exception of serotype 4. The 0.7-kb fragment was not amplified from any heterologous species that are also common pathogens or commensals of swine. In contrast, the 1.1-kb fragment was amplified from all serotype 5 strains only. The assay was capable of amplifying DNA from less than 102CFU. TheA. pleuropneumoniaeserotype 5 capsular DNA products were readily amplified from lung tissues obtained from infected swine, although the 1.1-kb product was not amplified from some tissues stored frozen for 6 years. The multiplex PCR assay enabled us to detectA. pleuropneumoniaerapidly and to distinguish serotype 5 strains from other serotypes. The use of primers specific to the biosynthesis regions of otherA. pleuropneumoniaeserotypes would expand the diagnostic and epidemiologic capabilities of this assay.
Detection of
Bacteroides fragilis
Enterotoxin Gene by PCR
Authors: Shetab Razeq , Cohen Stuart H. , Prindiville Thomas , Tang Yajarayma J. , Cantrell Mary , Rahmani Darush , Silva Joseph
Published: December 2019

Bacteroides fragilis
constitutes about 1% of the bacterial flora in intestines of normal humans. Enterotoxigenic strains of
B. fragilis
have been associated with diarrheal diseases in humans and animals. The enterotoxin produced by these isolates induces fluid changes in ligated intestinal loops and an in vitro cytotoxic response in HT-29 cells. We developed a nested PCR to detect the enterotoxin gene of
B. fragilis
in stool specimens. After DNA extraction, a 367-bp fragment was amplified with two outer primers. The amplicon from this reaction was subjected to a second round of amplification with a set of internal primers. With these inner primers, a 290-bp DNA fragment was obtained which was confirmed as part of the
B. fragilis
enterotoxin gene by Southern blotting with a nonradioactive internal probe and a chemiluminescence system. By this approach,
B. fragilis
enterotoxin gene sequences were detected in eight known enterotoxigenic human isolates and nine enterotoxigenic horse isolates. No amplification products were obtained from DNA extracted from 28 nonenterotoxigenic
B. fragilis
isolates or
B. distasonis
,
B. thetaiotaomicron
,
B. uniformis
,
B. ovatus
,
Escherichia coli
, or
Clostridium difficile
. The sensitivity of this assay allowed us to detect as little as 1 pg of enterotoxin DNA sequences or 100 to 1,000 cells of enterotoxigenic
B. fragilis
/g of stool. Enterotoxin production of all isolates was confirmed in vitro in HT-29 cells. A 100% correlation was obtained between enterotoxin detection by cytotoxin assay and the nested PCR assay. This rapid and sensitive assay can be used to identify enterotoxigenic
B. fragilis
and may be used clinically to determine the role of
B. fragilis
in diarrheal diseases.
Stabilized, Freeze-Dried PCR Mix for Detection of Mycobacteria
Authors: Klatser Paul R. , Kuijper Sjoukje , van Ingen Cor W. , Kolk Arend H. J.
Published: December 2019

We report here the development of a freeze-drying procedure allowing stabilization at ambient temperature of preoptimized, premixed, and predispensed PCR mixes aimed at the detection of mycobacteria in clinical materials. The freeze-dried mixes retained activity at 4°C and at 20°C for 1 year and for 3 months at 37°C, as judged by their performance with 50 and 500 fg of purified
Mycobacterium bovis
BCG target DNA.
A PCR Specific for
Escherichia coli
O157 Based on the
rfb
Locus Encoding O157 Lipopolysaccharide
Authors: Desmarchelier Patricia M. , Bilge Sima S. , Fegan Narelle , Mills Leanne , Vary James C. , Tarr Phillip I.
Published: December 2019

A PCR was developed for the detection of
Escherichia coli
O157 based on the
rfbE
O-antigen synthesis genes. A 479-bp PCR product was amplified specifically from
E. coli
O157 in cell lysates containing 200 or 2 CFU following crude DNA extraction. The PCR detected <1 CFU of
E. coli
O157 per ml in raw milk following enrichment.
Conservation of Restriction Sites in Isolates of
Streptococcus pneumoniae
with Diverse Restriction Fragment Patterns
Authors: Hall Lucinda M. C. , Duke Brigid
Published: December 2019

Separation of large restriction fragments by pulsed-field gel electrophoresis is a commonly used method for epidemiological typing of
Streptococcus pneumoniae
and many other bacterial species. Information on the genetic changes underlying the restriction fragment polymorphisms that allow discrimination between isolates is scarce. In this study fragments adjacent to
Apa
I sites in a clinical isolate of
S. pneumoniae
were cloned and used to probe
Hin
dIII and
Hin
dIII-plus-
Apa
I genomic DNA digests from other isolates with very different
Apa
I fragment patterns. If for a given isolate the
Hin
dIII fragment detected by the probe was reduced in size on digestion with
Apa
I, it was deduced that the
Apa
I site was conserved in that isolate. The results demonstrate that of six
Apa
I sites in PN93/908 examined, five were retained in 11 genetically different isolates and one was retained in 2 isolates but lost in 9 others. It was concluded that point mutations at restriction sites are unlikely to account for the restriction fragment length polymorphism observed and that much of the polymorphism may be due to DNA rearrangements, possibly resulting from the insertion or deletion of mobile DNA elements.
Defective Human T-Cell Lymphotropic Virus Type I (HTLV-I) Provirus in 10 Chilean Seronegative Patients with Tropical Spastic Paraparesis or HTLV-I-Associated Myelopathy
Authors: Ramirez Eugenio , Cartier Luis , Rios Maritza , Fernandez Jorge
Published: December 2019

We studied the presence of
tax
and
ltr
genes from human T-cell lymphotropic virus type I (HTLV-I) provirus in the peripheral blood mononuclear cells from 15 seronegative patients with tropical spastic paraparesis or HTLV-I-associated myelopathy by PCR. Only a region of the
tax
gene from 10 patients was amplified. The nucleotide homologies of six Chilean isolates to the ATK-1 clone ranged between 98.7 and 99.4%.
International Surveillance of Bloodstream Infections Due to
Candida
Species: Frequency of Occurrence and Antifungal Susceptibilities of Isolates Collected in 1997 in the United States, Canada, and South America for the SENTRY Program
Authors: Pfaller M. A. , Jones R. N. , Doern G. V. , Sader H. S. , Hollis R. J. , Messer S. A. , Group for The Sentry Participant
Published: December 2019

An international program of surveillance of bloodstream infections (BSIs) in the United States, Canada, and South America between January and December 1997 detected 306 episodes of candidemia in 34 medical centers (22 in the United States, 6 in Canada, and 6 in South America). Eighty percent of the BSIs were nosocomial and 50% occurred in patients hospitalized in an intensive care unit. Overall, 53.3% of the BSIs were due to
Candida albicans
, 15.7% were due to
C. parapsilosis
, 15.0% were due to
C. glabrata
, 7.8% were due to
C. tropicalis
, 2.0% were due to
C. krusei
, 0.7% were due to
C. guilliermondii
, and 5.8% were due to
Candida
spp. However, the distribution of species varied markedly by country. In the United States, 43.8% of BSIs were due to non-
C. albicans
species.
C. glabrata
was the most common non-
C. albicans
species in the United States. The proportion of non-
C. albicans
BSIs was slightly higher in Canada (47.5%), where
C. parapsilosis
, not
C. glabrata
, was the most common non-
C. albicans
species.
C. albicans
accounted for 40.5% of all BSIs in South America, followed by
C. parapsilosis
(38.1%) and
C. tropicalis
(11.9%). Only one BSI due to
C. glabrata
was observed in South American hospitals. Among the different species of
Candida
, resistance to fluconazole (MIC, ≥64 μg/ml) and itraconazole (MIC, ≥1.0 μg/ml) was observed with
C. glabrata
and
C. krusei
and was observed more rarely among other species. Isolates of
C. albicans
,
C. parapsilosis
,
C. tropicalis
, and
C. guilliermondii
were all highly susceptible to both fluconazole (99.4 to 100% susceptibility) and itraconazole (95.8 to 100% susceptibility). In contrast, 8.7% of
C. glabrata
isolates (MIC at which 90% of isolates are inhibited [MIC
90
], 32 μg/ml) and 100% of
C. krusei
isolates were resistant to fluconazole, and 36.9% of
C. glabrata
isolates (MIC
90
, 2.0 μg/ml) and 66.6% of
C. krusei
isolates were resistant to itraconazole. Within each species there were no geographic differences in susceptibility to fluconazole or itraconazole.
Comparison of Roche Cobas Amplicor
Mycobacterium tuberculosis
Assay with In-House PCR and Culture for Detection of
M. tuberculosis
Authors: Eing Bodo R. , Becker Andrea , Sohns Arthur , Ringelmann Ronald
Published: December 2019

The new Roche Cobas Amplicor
Mycobacterium tuberculosis
assay, which is a semiautomated version of the manually performed Roche Amplicor
M. tuberculosis
test, was compared to culture and an IS
6110
-based in-house PCR protocol. A total of 1,681 specimens from 833 patients, including specimen types other than sputum, were tested in parallel by both the in-house PCR and the Cobas Amplicor
M. tuberculosis
assay. After we resolved discrepant PCR results, the sensitivity, specificity, and positive and negative predictive values for the Cobas Amplicor
M. tuberculosis
assay were 66.33, 99.71, 94.36, and 97.66%, respectively. The corresponding values for the in-house PCR were 91.08, 99.85, 97.87, and 99.37%, respectively. For culture- and smear-positive specimens, the sensitivity of the Cobas Amplicor
M. tuberculosis
test was 96.42% (in-house PCR, 100%). If only smear-negative sputum specimens were considered, the Cobas Amplicor
M. tuberculosis
assay exhibited a sensitivity of 45.45% (in-house PCR, 63.63%) relative to that of culture. With a modified protocol for DNA extraction (washing of samples plus ultrasonication), both PCR methods performed better with gastric aspirates than with sputum samples (sensitivity of the Cobas Amplicor
M. tuberculosis
assay with smear-negative gastric aspirates, 70.00%; sensitivity of in-house PCR, 90.00%). With dithiothreitol being used for liquefaction of specimens in this study, the Cobas Amplicor
M. tuberculosis
assay exhibited an inhibition rate of 9.16%. In our view, the new Cobas Amplicor
M. tuberculosis
test (i) is well suited for typing of smear-positive specimens, (ii) may also be applied to gastric aspirates and other types of specimens if DNA extraction methods are modified appropriately, and (iii) exhibits a sensitivity with smear-negative sputum specimens which makes it recommendable that a minimum of three samples from the same patient be tested.
Microtiter Assay for Detecting
Campylobacter
spp. and
Helicobacter pylori
with Surface Gangliosides Which Bind Cholera Toxin
Authors: Sack David A. , Lastovica Albert J. , Chang Sunny H. , Pazzaglia Gary
Published: December 2019

Campylobacter jejuni
with Gm1 ganglioside in the core of its lipopolysaccharide has been associated with Guillain-Barrésyndrome. Since this epitope may be of considerable pathophysiologic importance and since this ganglioside binds cholera toxin, a rapid screening assay to detect bacteria that bind cholera toxin as an indication of Gm1 on their surfaces was developed. In the assay, bacterial lawns were grown on agar plates, harvested with phosphate-buffered saline, boiled, and incubated with a standard concentration of cholera B subunit. Preparations from strains with Gm1 were observed to inhibit the binding of cholera B subunit to Gm1 in a microtiter enzyme-linked immunosorbent assay. By using this assay with two groups of strains, 37 positive strains were detected among the 197 tested. Species with positive isolates included
C. jejuni
,
Campylobacter coli
, and
Helicobacter pylori
. The assay is capable of testing large numbers of isolates and should prove useful in future clinical and epidemiological studies of bacteria with this epitope.
Clinical Importance of Identifying Coagulase-Negative Staphylococci Isolated from Blood Cultures: Evaluation of MicroScan Rapid and Dried Overnight Gram-Positive Panels versus a Conventional Reference Method
Authors: Weinstein Melvin P. , Mirrett Stanley , Van Pelt Linda , McKinnon Mary , Zimmer Barbara L. , Kloos Wesley , Reller L. Barth
Published: December 2019

We evaluated the clinical usefulness of species identification of blood isolates of coagulase-negative staphylococci as a predictor of the clinical significance of the isolates. In addition, we compared results of species identification obtained with MicroScan Rapid Gram-Positive Identification panels and Dried Overnight (Conventional) Gram-Positive Identification panels with those obtained by a tube reference method. Two hundred eighty-five blood isolates were tested, including 92 judged to represent true bacteremia and 193 judged to represent contamination. The most common species detected were
Staphylococcus epidermidis
,
Staphylococcus hominis
, and
Staphylococcus haemolyticus
. These three species accounted for nearly 98% of the clinically significant isolates and 89% of the contaminants. The isolation of other species almost always represented contamination. However, identification of the three most common species did not help distinguish pathogens from contaminants. Both the Rapid and the Dried Overnight Gram-Positive panels identified
S. epidermidis
strains accurately, but the panels performed less well for the other species. Analysis revealed that
S. hominis
was frequently misidentified due to the presence of a previously unknown subspecies. Based on the initial results, revised investigational Dried Overnight Gram-Positive Identification panels (CPID-2) were prepared and tested. The CPID-2 panels identified 85 to 95% of
S. epidermidis
strains, 76 to 86% of
S. hominis
strains, and 88 to 92% of
S. haemolyticus
strains with high probability (>85%) and, overall, represented a significant improvement over the other panels for identification of these staphylococcal species.
Use of Fluorescent-Antibody Staining of Cytocentrifuge-Prepared Smears in Combination with Cell Culture for Direct Detection of Respiratory Viruses
Authors: Doing Kirk M. , Jerkofsky Mary Ann , Dow Elaine G. , Jellison Jo Ann
Published: December 2019

Over a 3-year period, 1,003 respiratory samples were collected and examined for selected respiratory viruses with cytocentrifuged prepared smears stained with fluorescently labeled antibodies (IFA) in conjunction with cell culture. IFA results were compared with results obtained by cell culture. Viruses were isolated or detected by direct means in 401 samples. Agreement between culture and IFA was 90%.
Comparison of Sample Preparation Methods for Detection of
Legionella pneumophila
in Culture-Positive Bronchoalveolar Lavage Fluids by PCR
Authors: Jaulhac B. , Reyrolle M. , Sodahlon Y. K. , Jarraud S. , Kubina M. , Monteil H. , Piémont Y. , Etienne J.
Published: December 2019

A prospective study was conducted on 25
Legionella pneumophila
culture-positive and 98 culture-negative bronchoalveolar lavage fluid samples to compare two DNA preparation methods: a rapid modified Chelex-based protocol and a proteinase K method. PCR was found to be more sensitive with the Chelex-based method (
P
= 0.03). No difference was found concerning the inhibition rate.
Isolation and Characterization of Sorbitol-Fermenting Shiga Toxin (Verocytotoxin)-Producing
Escherichia coli
O157:Hßtrains in the Czech Republic
Authors: Bielaszewska Martina , Schmidt Herbert , Karmali Mohamed A. , Khakhria Rasik , Janda Jan , Bláhová Kvĕta , Karch Helge
Published: December 2019

Two sorbitol-fermenting (SF) Shiga toxin-producing
Escherichia coli
(STEC) O157:H− strains were isolated from patients with hemolytic-uremic syndrome in the Czech Republic in 1995. Their phenotypic and genotypic characteristics and genomic DNA fingerprints were identical or closely related to those of SF STEC O157:H− strains isolated in Germany in 1988 to 1997. This indicates that the Czech isolates belong to the SF STEC O157 clone which is widespread in Germany. It is the first finding of the clone outside Germany.
Molecular Epidemiology of Reemergent
Vibrio cholerae
O139 Bengal in India
Authors: Mukhopadhyay Asish Kumar , Basu Arnab , Garg Pallavi , Bag Prasanta K. , Ghosh Amit , Bhattacharya S. K. , Takeda Yoshifumi , Nair G. Balakrish
Published: December 2019

We report the prevalence of the O139 serogroup in Calcutta, India, after its reemergence in August 1996 and the spread of the reemerged clone to other parts of the country by using previously established molecular markers. Phenotypically, the reemerged
Vibrio cholerae
O139 displayed a difference compared to those that appeared in late 1992 and 1993 in that the current O139 strains are sensitive to co-trimoxazole. Ribotyping with the enzyme
Bgl
I produced two rRNA restriction patterns in the O139 strains isolated after August 1996, and these patterns were identical to those exhibited by strains of O139 isolated in 1992. Three clones of
V. cholerae
O139 are currently prevailing in the country, with strains exhibiting three bands after
Hin
dIII digestion and hybridization with a
ctxA
probe being dominant. The reemergence of
V. cholerae
O139 in Calcutta after a 32-month quiescent period reestablishes the O139 serogroup as an entity which is likely to play a crucial role in the temporal antigenic variations among the serogroups of
V. cholerae
causing cholera.
Fatal
Hormonema dematioides
Peritonitis in a Patient on Continuous Ambulatory Peritoneal Dialysis: Criteria for Organism Identification and Review of Other Known Fungal Etiologic Agents
Authors: Shin Jong Hee , Lee Sang Ku , Suh Soon Pal , Ryang Dong Wook , Kim Nam Ho , Rinaldi Michael G. , Sutton Deanna A.
Published: December 2019

We report a fatal case a fungal peritonitis caused by the yeast-like dematiaceous mould
Hormonema dematioides
in a 45-year-old woman. The woman had a 13-year history of insulin-dependent diabetes mellitus and had been on continuous ambulatory peritoneal dialysis for chronic renal failure.
H. dematioides
was repeatedly isolated from the dialysate culture specimens collected on days 3, 9, 16, and 20 of her hospital stay. Preliminary culture reports on day 7 of the growth of a yeast-like fungus, a probable
Candida
species, prompted the administration of fluconazole (FLU). Intraperitoneal and intravenous FLU failed to eliminate the mould, and the patient expired on day 21 of her hospital stay. We use this case to present what appears to be the first report of fungal peritonitis due to
H. dematioides
, to provide laboratorians with criteria for differentiating this organism from the similar mould
Aureobasidium pullulans
and from various yeast genera, and to provide a review of known fungal taxa inciting peritonitis.
Streptococcus iniae
, a Human and Animal Pathogen: Specific Identification by the Chaperonin 60 Gene Identification Method
Authors: Goh Swee Han , Driedger David , Gillett Sandra , Low Donald E. , Hemmingsen Sean M. , Amos Mayben , Chan David , Lovgren Marguerite , Willey Barbara M. , Shaw Carol , Smith John A.
Published: December 2019

It was recently reported that
Streptococcus iniae
, a bacterial pathogen of aquatic animals, can cause serious disease in humans. Using the chaperonin 60 (Cpn60) gene identification method with reverse checkerboard hybridization and chemiluminescent detection, we identified correctly each of 12
S. iniae
samples among 34 aerobic gram-positive isolates from animal and clinical human sources.
Molecular Epidemiology of Two Consecutive Outbreaks of Parainfluenza 3 in a Bone Marrow Transplant Unit
Authors: Zambon Maria , Bull Tim , Sadler Carol J. , Goldman John M. , Ward Katherine N.
Published: December 2019

Two consecutive nosocomial outbreaks of parainfluenza 3, in which 5 of 15 infected patients died, occurred in an adult bone marrow transplant unit. Parainfluenza 3 strain variation was assessed by reverse transcription-PCR sequencing of part of the parainfluenza 3 F gene, including the noncoding region, directly from clinical samples. Sequence data from the outbreaks were compared with those from 15 other parainfluenza 3 isolates circulating concurrently in the community; altogether, 13 strains which fell into three lineages were identified. Four immunosuppressed patients shed virus persistently for between 1 and 4 months without change in sequence. The first outbreak lasted 4 months and involved three parainfluenza 3 strains, and one persistently infected patient was implicated as the source of infection for three others. The second outbreak lasted for 1 month but involved only one strain. These data indicate that introduction of community parainfluenza 3 strains to the bone marrow transplant unit was followed by person-to-person transmission within the unit rather than reintroduction of virus from the community.
Direct Detection of
eae
-Positive Bacteria in Human and Veterinary Colorectal Specimens by PCR
Authors: Hubbard A. L. , Harrison D. J. , Moyes C. , McOrist S.
Published: December 2019

A PCR test based on the amplification of an
eae
-specific sequence was designed and evaluated for its ability to directly detect homologous sequences in enteropathogenic
Escherichia coli
and
Citrobacter
spp. (amplification of
eae
open reading frame, 178 bp) in sections of the intestines of humans and animals with colonic lesions. Positive PCR results were observed with
eae
-positive reference strains of
E. coli
and
Citrobacter rodentium
(
Citrobacter freundii
biotype 4280). Known
eae
-negative reference strains of
E. coli
and other laboratory strains of enteric bacteria were negative by the amplification test. The sensitivity of the PCR for detection of
eae
-positive
E. coli
and
C. rodentium
was between 1 and 2 CFU. To detect these sequences directly from sections of fixed colon from human and veterinary sources, PCR conditions were modified by the addition of 0.1 mM 8-methoxypsoralen to eliminate extraneous bacterial DNA from the PCR amplification cocktail without added template. Sections of colon from three pigs experimentally affected with colon lesions due to enteropathogenic (attaching and effacing)
E. coli
were PCR positive for bacterial
eae
genome. Sections from control animals were negative. Sections of colon from one of 18 biopsies from confirmed AIDS patients and from 22 of 35 colorectal cancer patients were PCR positive for bacterial
eae
genome. The PCR test was a simple and quick method of detecting bacterial
eae
genome in human and veterinary clinical specimens. This method may remove the need for initial culture and detection of the gene by DNA probing from potential associated lesions. The clear relationship of bacteria containing the
eae
gene with colonic lesions in the pigs and mice indicates that a similar relationship is possible for human patients having similar lesions.
Rapid and Specific Molecular Identification of Methicillin-Resistant
Staphylococcus aureus
in Endotracheal Aspirates from Mechanically Ventilated Patients
Authors: Vannuffel Pascal , Laterre Pierre-François , Bouyer Michele , Gigi Jacques , Vandercam Bernard , Reynaert Marc , Gala Jean-Luc
Published: December 2019

Multiplex amplification of
femA
and
mecA
genetic determinants allowed an early and rapid identification of methicillin-resistant
Staphylococcus aureus
(MRSA) in endotracheal aspirates of mechanically ventilated patients.
femA
and/or
mecA
amplification and bacteriological results were concordant in 57 of 60 samples. In all three discrepant cases, complementary bacteriological tests confirmed the presence of MRSA first identified by molecular analysis. These results underline the value and rapidity of this molecular diagnosis for MRSA infection and control surveillance in intensive care units. Rapid MRSA detection is expected to have a significant clinical impact not only on patient outcome but also on the costs for isolation and treatment.
Prevalence of and Factors Associated with Visceral Leishmaniasis in Human Immunodeficiency Virus Type 1-Infected Patients in Southern Spain
Authors: Pineda Juan A. , Gallardo José A. , Macías Juan , Delgado Juan , Regordán Carmen , Morillas Francisco , Relimpio Federico , Martín-Sánchez Joaquina , Sánchez-Quijano Armando , Leal Manuel , Lissen Eduardo
Published: December 2019

The actual prevalence of visceral leishmaniasis among human immunodeficiency type 1 (HIV-1)-infected patients in the Mediterranean basin remains unknown. There is also controversy about the risk factors for
Leishmania infantum
and HIV-1 coinfection. To appraise the prevalence of visceral leishmaniasis in patients infected with HIV-1 in southern Spain and to identify factors associated with this disease, 291 HIV-1 carriers underwent a bone marrow aspiration, regardless of their symptoms. Giemsa-stained samples were searched for
Leishmania
amastigotes. Thirty-two (11%) patients showed visceral leishmaniasis. Thirteen (41%) patients had subclinical cases of infection. Centers for Disease Control and Prevention (CDC) clinical category C was the factor most strongly associated with this disease (adjusted odds ratio [OR], 1.88 [95% confidence interval, 1.22 to 2.88]), but patients with subclinical cases of infection were found in all CDC categories. Female sex was negatively associated with visceral leishmaniasis (adjusted OR, 0.42 [95% confidence interval, 0.18 to 0.97]). Intravenous drug users showed a higher prevalence than the remaining patients (13.3 versus 4.9%;
P
= 0.04), but such an association was not independent. These results show that visceral leishmaniasis is a very prevalent disease among HIV-1-infected patients in southern Spain, with a high proportion of cases being subclinical. Like other opportunistic infections, subclinical visceral leishmaniasis can be found at any stage of HIV-1 infection, but symptomatic cases of infection appear mainly when a deep immunosuppression is present. There is also an association of this disease with male sex and intravenous drug use.
Evaluation of the AMPLICOR Cytomegalovirus Test with Specimens from Human Immunodeficiency Virus-Infected Subjects
Authors: Boivin Guy , Handfield Julie , Toma Emil , Murray Gilles , Lalonde Richard , Tevere Vincent J. , Sun Rita , Bergeron Michel G.
Published: December 2019

The AMPLICOR cytomegalovirus (CMV) test, a new qualitative assay for the detection of CMV DNA in plasma, was compared to conventional methods and quantitative PCR (Q-PCR) assays by using leukocytes and plasma from 179 blood samples from subjects with AIDS. For the diagnosis of CMV disease, cell-based assays such as a Q-PCR with polymorphonuclear leukocytes (Q-PCR-PMNL) and a pp65 antigenemia assay had the highest sensitivities but suffered from a lack of specificity. The best agreement between the results of the Q-PCR-PMNL assay and those of the AMPLICOR test was found when a threshold diagnostic value of 690 copies per 10
5
cells was selected for the Q-PCR-PMNL assay. In that context, the AMPLICOR CMV test had a sensitivity of 96.4% and a specificity of 95.3% when results were compared to results of the cell-based PCR assay. This threshold was close to the one described as associated with the best sensitivity and specificity for the diagnosis of CMV disease in a recently published study (4). Blood samples that tested positive by the Q-PCR-PMNL assay but negative by the AMPLICOR CMV test were associated with viral loads (mean, 785 copies, median, 96 copies per 10
5
leukocytes) lower than the viral loads of blood samples that tested positive by both assays (mean, 21,452 copies; median, 9,784 copies per 10
5
leukocytes) (
P
= 0.003). The AMPLICOR CMV test gave positive results at least 48 days before the development of symptomatic CMV disease in a longitudinal analysis of a limited subset of patients (
n
= 6) from whom sequential specimens were available for testing. In conclusion, the AMPLICOR CMV test is a very convenient assay combining rapidity, simplicity, and the possibility of batch testing. A positive result by this test seems particularly important since this implies, in most instances, the presence or the imminence of CMV disease, although a negative test result does not rule out disease.