bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of Two New Automated Assays for Hepatitis B Virus Surface Antigen (HBsAg) Detection: IMMULITE HBsAg and IMMULITE 2000 HBsAg
Authors: Weber Bernard , Dengler Thomas , Berger Annemarie , Doerr Hans Wilhelm , Rabenau Holger
Published: January 2003

In recent years the diagnostic industry has developed new automated immunoassays for the qualitative detection of hepatitis B virus (HBV) surface antigen (HBsAg) in serum and plasma samples that are performed on analyzers that permit a high-speed throughput, random access, and primary tube sampling. The aim of the present study was the evaluation of two new automated HBsAg screening assays, IMMULITE HBsAg and IMMULITE 2000 HBsAg, from Diagnostic Products Corporation. The new HBsAg assays were compared to well-established tests (Auszyme Monoclonal [overnight incubation, version B], IMx HBsAg, AxSYM HBsAg, and Prism HBsAg [all from Abbott] and Elecsys HBsAg [Roche Diagnostics]). In the evaluation were included seroconversion panels, sera from the acute and chronic phases of infection, dilution series of various HBsAg standards, HBV subtypes and S gene mutants. To challenge the specificity of the new assays, sera from HBsAg-negative blood donors, pregnant women, and dialysis and hospitalized patients and potentially cross-reactive samples were investigated. IMMULITE HBsAg and IMMULITE 2000 HBsAg, although not as sensitive as the Elecsys HBsAg assay, were equivalent to the AxSYM HBsAg assay and showed a higher sensitivity than the Auszyme Monoclonal B and IMx HBsAg systems for detection of acute infection in seroconversion panels. The specificities (100%) of both IMMULITE assays on unselected blood donors and potentially interfering samples were comparable to those of the alternative assays after repeated testing. In conclusion, the new IMMULITE HBsAg and IMMULITE 2000 HBsAg assays show a good sensitivity for HBsAg detection compared to other well-established tests. The specificity on repeatedly tested samples was equivalent to that of the alternative assays. The rapid turnaround time, primary tube sampling, and on-board dilution make it an interesting assay system for clinical laboratory diagnosis.
Comparative Genotyping of
Campylobacter jejuni
by Amplified Fragment Length Polymorphism, Multilocus Sequence Typing, and Short Repeat Sequencing: Strain Diversity, Host Range, and Recombination
Authors: Schouls Leo M. , Reulen Sanne , Duim Birgitta , Wagenaar Jaap A. , Willems Rob J. L. , Dingle Kate E. , Colles Frances M. , Van Embden Jan D. A.
Published: January 2003

Three molecular typing methods were used to study the relationships among 184
Campylobacter
strains isolated from humans, cattle, and chickens. All strains were genotyped by amplified fragment length polymorphism (AFLP) analysis, multilocus sequence typing (MLST), and sequence analysis of a genomic region with short tandem repeats designated clustered regularly interspaced short palindromic repeats (CRISPRs). MLST and AFLP analysis yielded more than 100 different profiles and patterns, respectively. These multiple-locus typing methods resulted in similar genetic clustering, indicating that both are useful in disclosing genetic relationships between
Campylobacter jejuni
isolates. Group separation analysis of the AFLP analysis and MLST data revealed an unexpected association between cattle and human strains, suggesting a common source of infection. Analysis of the polymorphic CRISPR region carrying short repeats allowed about two-thirds of the typeable strains to be distinguished, similar to AFLP analysis and MLST. The three methods proved to be equally powerful in identifying strains from outbreaks of human campylobacteriosis. Analysis of the MLST data showed that intra- and interspecies recombination occurs frequently and that the role of recombination in sequence variation is 50 times greater than that of mutation. Examination of strains cultured from cecum swabs revealed that individual chickens harbored multiple
Campylobacter
strain types and that some genotypes were found in more than one chicken. We conclude that typing of
Campylobacter
strains is useful for identification of outbreaks but is probably not useful for source tracing and global epidemiology because of carriage of strains of multiple types and an extremely high diversity of strains in animals.
Occurrence and Phenotypic Characteristics of Extended-Spectrum β-Lactamases among Members of the Family
Enterobacteriaceae
at the Tel-Aviv Medical Center (Israel) and Evaluation of Diagnostic Tests
Authors: Navon-Venezia Shiri , Hammer-Munz Orly , Schwartz David , Turner Dan , Kuzmenko Boris , Carmeli Yehuda
Published: January 2003

We assessed the prevalence and phenotypic characteristics of extended-spectrum β-lactamase (ESBL) producers among cefuroxime-resistant (CXM-R) (MIC ≥ 32 μg/ml) members of the family
Enterobacteriaceae
in our institution. The 438 CXM-R clinical isolates obtained from nonurine sources among inpatients were screened. ESBL production was confirmed by disk diffusion assay using cefpodoxime (CPD), cefotaxime (CTX), and ceftazidime (CTZ) with and without clavulanate (CLAV). A difference of ≥5 mm in the size of the zone of inhibition in the presence of CLAV for at least one of the agents was considered representative of the ESBL phenotype: 186 isolates (42.5%) were confirmed as ESBL producers. The isolates tested and the rates of ESBL producers were as follows:
Klebsiella
spp. (
n
= 81), 79%;
Proteus
spp. (
n
= 58), 62%;
Escherichia coli
(
n
= 64), 53%;
Enterobacter
spp. (
n
= 69), 42%;
Serratia
spp. (
n
= 70), 14%;
Citrobacter
spp. (
n
= 25), 24%;
Providencia
spp. (
n
= 21), 24%;
Morganella
spp. (
n
= 41), 5%; and
Kluyvera
(
n
= 3), 0%. The overall sensitivity of isolated ESBL confirmatory tests was 79% for CPD-CLAV, 66% for CTZ-CLAV, and 91% for CTX-CLAV. Sensitivities of CTZ-CLAV confirmatory tests for
Klebsiella
spp.,
Proteus
spp.,
E. coli
, and
Enterobacter
spp. were 84, 22, 76, and 62%, respectively, and those for CTX-CLAV were 95, 97, 94, and 83%, respectively. They were 90% for CPD-CLAV and CTZ-CLAV, 95% for CPD-CLAV and CTX-CLAV, and 100% for CTZ-CLAV and CTX-CLAV. ESBL production was highly prevalent among
Enterobacteriaceae.
Using resistance to CXM as an ESBL screening criterion is a suitable option in high-incidence areas where
Klebsiella
spp. are not the dominant ESBL producers. This screening criterion may simplify the screening test and improve its sensitivity, although at the price of testing more isolates. The CTX-CLAV combination confirmed ESBL producers better than the CTZ-CLAV combination, with sensitivity varying between species. Combined CTZ-CLAV and CTX-CLAV testing detected all these strains; CPD-CLAV provided no additional benefit.
Frequent Recovery of a Single Clonal Type of Multidrug-Resistant
Staphylococcus aureus
from Patients in Two Hospitals in Taiwan and China
Authors: Aires de Sousa M. , Crisóstomo M. I. , Santos Sanches I. , Wu J. S. , Fuzhong J. , Tomasz A. , de Lencastre H.
Published: January 2003

One hundred thirty-two methicillin-resistant
Staphylococcus aureus
(MRSA) isolates recovered from patients with
S. aureus
infections between January 1998 and February 1999 in two hospitals, one located in Taipei, Taiwan, and another in Nanjing, People's Republic of China, were examined for antibiotic susceptibility and for clonal type by a combination of three methods: hybridization of
Cla
I restriction digests with
mecA
- and Tn
554
-specific DNA probes and pulsed-field gel electrophoresis of chromosomal
Sma
I digests. Selected isolates representing each clonal type were also analyzed by
spaA
typing, multilocus sequence typing, and a multiplex PCR method capable of identifying the structural type of the staphylococcal cassette chromosome
mec
(SCC
mec
) carried by the bacteria. The overwhelming majority of isolates (126 of 132 or 95%) belonged to minor variants of a single clonal type resembling the Brazilian and Hungarian epidemic MRSA clones, which showed a common
spaA
type and which were either sequence type 239 (ST239) or ST241 (a single-locus variant of ST239) in association with SCC
mec
type III or IIIA.
Multicenter Evaluation of the Performance Characteristics of the NucliSens HIV-1 QT Assay Used for Quantitation of Human Immunodeficiency Virus Type 1 RNA
Authors: Ginocchio Christine C. , Kemper Marti , Stellrecht Kathleen A. , Witt Donald J.
Published: January 2003

The analytical performance of the NucliSens HIV-1 QT assay, a highly sensitive test based on nucleic acid sequence-based amplification technology, was evaluated in a multicenter trial. Assay specificity was evaluated with 502 plasma (EDTA) specimens from human immunodeficiency virus type 1 (HIV-1)-seronegative volunteer donors. No HIV-1 RNA was reported in any of the donor specimens. Analytical sensitivity and reproducibility were estimated with panels prepared from a high-titer well-characterized HIV-1 RNA stock (5.84 × 10
8
RNA copies/ml). The assay's dynamic range was linear from 10
6
to 10
1
HIV-1 RNA copies, with a lower detectable limit of 25 copies/ml and a 95% detection rate of 176 copies/ml. Sensitivity of the assay to detect HIV-1 RNA in clinical specimens from patients (
n
= 101) and in commercially available or prepared panels (
n
= 24) was compared with NASBA HIV-1 RNA QT (an earlier version of NucliSens HIV-1 QT) and with the Food and Drug Administration-approved standard and ultrasensitive AMPLICOR HIV-1 MONITOR, version 1.0, assays. Detection of HIV-1 RNA was reproducible over a 5-log range (mean standard deviation = 0.15 log). The NucliSens and the standard AMPLICOR assays were equivalent in detection of HIV-1 RNA (concentration, 10
3
to 10
5
copies/ml) in 57 clinical specimens. The NucliSens assay was more sensitive in detecting HIV-1 RNA at lower concentrations (≤10
2
copies/ml) (44 of 44) than either the standard AMPLICOR test (12 of 19) or the NASBA assay (10 of 25). A 25% increase in HIV-1 RNA detection frequency with panels was observed with the NucliSens assay (23 of 24) compared with the standard AMPLICOR test (17 of 24). The new assay was highly specific and demonstrated good sensitivity with a broad linear dynamic range.
Pneumocystis jiroveci
Internal Transcribed Spacer Types in Patients Colonized by the Fungus and in Patients with Pneumocystosis from the Same French Geographic Region
Authors: Nevez Gilles , Totet Anne , Jounieaux Vincent , Schmit Jean-Luc , Dei-Cas Eduardo , Raccurt Christian
Published: January 2003

Pneumocystis jiroveci
(human-derived
Pneumocystis
) infections can display a broad spectrum of clinical presentations, of which pulmonary colonization with the fungus may represent an important part, occurring frequently in patients with various underlying diseases and presenting alternative diagnoses of acute pneumocystosis (
Pneumocystis carinii
pneumonia [PCP]). There are few data concerning the
P. jiroveci
genotypes involved in pulmonary colonization, whereas several genotypes responsible for PCP in immunocompromised patients have been described. In this study,
P. jiroveci
genotypes have retrospectively been investigated and compared in 6 colonized patients and in 11 patients with PCP who were in the same hospital. Seventeen archival bronchoalveolar lavage samples were genotyped at internal-transcribed spacer 1 (ITS1) and ITS2 of the nuclear rRNA operon. Fourteen different genotypes were identified, of which 1 was found only in colonized patients, 10 were found only in patients with PCP, and 3 were found in both patient populations. Mixed infections were diagnosed in 2 of the 6 colonized patients and in 6 of the 11 patients with PCP. The results show that similar genotypes can be responsible for PCP as well as pulmonary colonization. There is a high diversity of genotypes in colonized patients and in patients with PCP. Mixed infections may occur in these two patient populations. These shared features of
P. jiroveci
ITS genotypes in colonized patients and patients with PCP suggest that human populations infected by
P. jiroveci
, whatever the clinical manifestation, may play a role as a common reservoir for the fungus.
Characteristics of
Massilia timonae
and
Massilia timonae
-Like Isolates from Human Patients, with an Emended Description of the Species
Authors: Lindquist David , Murrill Donna , Burran W. Patrick , Winans Gloria , Janda J. Michael , Probert Will
Published: January 2003

The description of
Massilia timonae
, a nonfermentative aerobic gram-negative rod, was based on a single strain. A subsequent report of a second isolate has been recently published. Phenotypic descriptions of these two strains were based primarily on commercial test kit results. We have identified three additional strains as
M. timonae
by 16S rRNA sequence analysis and have characterized them phenotypically in parallel with the type strain of
M. timonae
, CIP 105350, by conventional test methods. A fourth strain, designated
M. timonae
-like, was also characterized. All four strains were isolated from human patients: two were blood isolates, one was isolated from cerebrospinal fluid, and one was isolated from bone. The four strains and the type strain were quite similar phenotypically. However, in contrast to the original description, the strains were found to be oxidase positive and arginine dihydrolase negative and to have lateral flagella as well as a single polar flagellum. Additionally the strains produced acid oxidatively from some carbohydrates. Other phenotypic characteristics, including cellular fatty acids, agreed with the original description. Based on our emended description,
M. timonae
and
M. timonae
-like strains can be differentiated from other aerobic nonfermentative gram-negative rods by conventional biochemical tests combined with cellular fatty acid analysis.
Genetic Diversity of
Neisseria gonorrhoeae
Housekeeping Genes
Authors: Viscidi Raphael P. , Demma James C.
Published: January 2003

Molecular typing of
Neisseria gonorrhoeae
strains is an important tool for epidemiological studies of gonococcal infection and transmission. The recently developed multilocus sequence typing (MLST) method is based on the genetic variation among housekeeping genes. As a preliminary investigation for the development of such a method, we characterized the genetic diversity at 18 gonococcal housekeeping gene loci. Approximately 17,500 nucleotides, spanning 18 loci, were sequenced from 24 isolates. Including strain FA 1090, which has been fully sequenced, and three unique
glnA
sequences from GenBank, the number of alleles identified for the 18 loci ranged from 2 to 18, with a mean of 8.3 alleles per locus. The majority of polymorphic sites were distributed randomly along the genes, consistent with evolution of DNA sequences by point mutation. In addition, several examples of clustered mutations and insertions or deletions were detected, which most likely occurred by recombinational events. While purifying selection is the dominant force driving the evolution of these housekeeping genes, positive selection also appeared to operate on the
abcZ
and
gpdh
loci. The 25 completely characterized strains each had a unique allelic profile with as few as three loci (
pilA
,
abcZ
, and
pip
or
pgi2
). Molecular typing based on the allelic profile of housekeeping genes resolved the isolates better than either
porB
nucleotide sequencing or typing of the
opa
gene. The allelic profiles for the
pilA
,
abcZ
, and
serC
loci of paired strains from 16 sexual contacts were identical. A potential MLST for
N. gonorrhoeae
, based on ∼500- to 600-bp gene fragments of seven housekeeping gene loci, would include the
pilA
,
abcZ
,
serC
,
glnA
,
gdh
,
gnd
, and
pip
loci.
Comparison of Recovery of Blood Culture Isolates from Two BacT/ALERT FAN Aerobic Blood Culture Bottles with Recovery from One FAN Aerobic Bottle and One FAN Anaerobic Bottle
Authors: Riley Julie A. , Heiter Barbara J. , Bourbeau Paul P.
Published: January 2003

Traditionally, a routine blood culture for adult patients consisted of paired aerobic and anaerobic bottles, but the routine use of an anaerobic blood culture bottle has been challenged in recent years. In this study, we compared the recovery of two FAN aerobic bottles with one FAN aerobic and one FAN anaerobic bottle. Each pair of bottles was collected by a separate collection procedure, and each bottle held a recommended 8- to 12-ml draw. A total of 704 clinically significant isolates were recovered from 8,620 sets (17,240 pairs), with 487 (69.2%) isolates recovered from one or both bottles in each pair of bottles, 86 isolates (12.2%) recovered only from the FAN aerobic-FAN aerobic pair, and 131 isolates (18.6%) recovered only from the FAN aerobic-FAN anaerobic pair. Significantly more total organisms (
P
= 0.002), gram-positive cocci (
P
= 0.03),
Staphylococcus aureus
(
P
= 0.05),
Enterobacteriaceae
other than
Escherichia coli
(
P
= 0.02), and anaerobes (
P
= 0.01) were recovered from the FAN aerobic-FAN anaerobic pair than from the FAN aerobic-FAN aerobic pair. A separate analysis was performed on the 618 isolates that were recovered from the FAN aerobic-FAN anaerobic pair to compare recovery by bottle type. Significantly more
S. aureus
(
P
= 0.005) and anaerobes (
P
< 0.001) were recovered from the FAN anaerobic bottle, while significantly more coagulase-negative staphylococci (
P
= 0.01),
Streptococcus pneumoniae
(
P
= 0.03), and other gram-negative bacilli (
P
= 0.004) were recovered from the FAN aerobic bottle. These results support the routine use of a FAN anaerobic bottle for use in the culture of blood with the BacT/ALERT system in our institution. These results also suggest that the decision of whether to routinely utilize an anaerobic blood culture bottle should be influenced by the overall recovery of bacteria and yeast, the recovery of specific types of bacteria or yeast, the medium type, and the blood culture system utilized by the laboratory.
Extended Virulence Genotype of Pathogenic
Escherichia coli
Isolates Carrying the
afa-8
Operon: Evidence of Similarities between Isolates from Humans and Animals with Extraintestinal Infections
Authors: Girardeau Jean Pierre , Lalioui Lila , Said A. Mohamed Ou , De Champs Christophe , Le Bouguénec Chantal
Published: January 2003

The afimbrial AfaE-VIII adhesin is common among
Escherichia coli
isolates from calves with intestinal and/or extraintestinal infections and from humans with sepsis or pyelonephritis. The virulence genotypes of 77
Escherichia coli afa-8
isolates from farm animals and humans were compared to determine whether any trait of commonality exists between isolates of the different host species. Over half of the extraintestinal
afa-8
isolates were associated with
pap
and
f17Ac
adhesin genes and contained virulence genes (
pap
,
hly
, and
cnf1
) which are characteristic of human extraintestinal pathogenic
E. coli
(ExPEC). PapG, which occurs as three known variants (variants I to III), is encoded by the corresponding three alleles of
papG
. Among the
pap
-positive strains, new
papG
variants (
papGrs
) that differed from the isolates with genes for the three adhesin classes predominated over isolates with
papG
allele III, which in turn were more prevalent than those with allele II. The data showed the substantial prevalence of the enteroaggregative
E. coli
heat-stable enterotoxin gene (
east1
) among
afa-8
isolates. Most of the
afa-8
isolates harbored the high-pathogenicity island (HPI) present in pathogenic
Yersinia
; however, two-thirds of the HPI-positive strains shared a truncated HPI integrase gene. The presence of ExPEC-associated virulence factors (VFs) in extraintestinal isolates that carry genes typical of enteric strains and that express O antigens associated with intestinal
E. coli
is consistent with transfer of VFs and O-antigen determinants between ExPEC and enteric strains. The similarities between animal and human ExPEC strains support the hypothesis of overlapping populations, with members of certain clones or clonal groups including animal and human strains. The presence of multiple-antibiotic-resistant bovine
afa-8
strains among such clones may represent a potential public health risk.
Quality Assessment Program for Genotypic Antiretroviral Testing Improves Detection of Drug Resistance Mutations
Authors: Sayer D. C. , Land S. , Gizzarelli L. , French M. , Hales G. , Emery S. , Christiansen F. T. , Dax E. M.
Published: January 2003

Genotypic antiretroviral testing is now widely used for the management of patients who are undergoing antiretroviral therapy for human immunodeficiency virus infection. The assays are complex, and there is considerable potential for variation between laboratories. Informative and ongoing quality assessment programs (QAPs) which address all aspects of testing are required. The panel distribution of clinical material is a critical component of QAPs. We report on the results and data from a recent panel. Four cryopreserved plasma samples from treated donors were distributed to nine laboratories. Three laboratories performed testing by commercial assays, and six laboratories used in-house assays, with one laboratory reporting results from two in-house assays. There was complete concordance between results for 95.9% of the nucleotide sequence and 94.5% of the amino acid sequence. Despite this overall high level of concordance, the degree of concordance at drug resistance mutation (DRM) sites when DRMs were present was considerably less (38% of DRM sites). Consequently, only 3 of the 10 methods reported 100% of DRMs as present. This elevated discrepancy rate is almost certainly a result of variability in the identification of mixtures of nucleotides (mixtures) at any site within the sequence. In addition, laboratories differed in the number of codons in the reverse transcriptase gene that were sequenced and their ability to amplify all samples. This panel distribution demonstrated a requirement for laboratory participation in ongoing QAPs and the optimization of assays with standards that contain mixtures.
Detection and Differentiation of
Entamoeba histolytica
and
Entamoeba dispar
Isolates in Clinical Samples by PCR and Enzyme-Linked Immunosorbent Assay
Authors: Gonin Patrick , Trudel Louise
Published: January 2003

Differential diagnosis of
Entamoeba histolytica
(pathogenic) and
Entamoeba dispar
(nonpathogenic), which are two morphologically identical species of amebae, is essential both for treatment decision and public health knowledge. The study reported here was designed to choose a reference differentiation technique. Stool samples (
n
= 95) were tested by microscopy, TechLab enzyme-linked immunosorbent assays (ELISAs), and an in-house PCR. The target for the PCR amplification was a small region (135 bp) of the SSU rRNA selected to increase the sensitivity of the test. Sixty-eight specimens tested positive by PCR: 2 for
E. histolytica
and 66 for
E. dispar
. For detection of
E. dispar
, ELISA performance was lower than that of microscopy in this reference context, while PCR was much more sensitive than microscopy. Given the low proportion of
E. histolytica
cases, test performance for this species is difficult to assess. However, for differentiation, PCR performed well on simulated samples, while ELISA gave a discordant result for one of the two samples PCR positive for
E. histolytica
during the study. This report also confirms that
E. dispar
infection is significantly higher among travelers and underlines the possibility of acquiring
E. histolytica
infection in regions that are not areas of endemicity. Because of its lower sensitivity, the interest of ELISA for
Entamoeba
detection and differentiation in stools seems questionable in nontropical regions. On the other hand, results suggest that PCR should be useful as a reference test for sensitive differentiation of both species and to contribute to physicians' decision in treatment of
E. histolytica-
or
E. dispar
-infected patients.
Comparison of LightCycler PCR, Rapid Antigen Immunoassay, and Culture for Detection of Group A Streptococci from Throat Swabs
Authors: Uhl J. R. , Adamson S. C. , Vetter E. A. , Schleck C. D. , Harmsen W. S. , Iverson L. K. , Santrach P. J. , Henry N. K. , Cockerill F. R.
Published: January 2003

We compared the performance characteristics of a real-time PCR method, the LightCycler Strep-A assay (Roche Applied Science, Indianapolis, Ind.), to those of a rapid antigen immunoassay, the Directigen 1-2-3 Group A Strep Test kit (BD Diagnostic Systems, Sparks, Md.), and a standard culture method for detection of group A streptococci (GAS) from 384 throat swabs. The LightCycler PCR produced more positive results (
n
= 58) than either culture (
n
= 55) or the Directigen immunoassay (
n
= 31). The results of the LightCycler PCR and the Directigen method were independently compared to the results of the accepted “gold standard,” bacterial culture. The sensitivities, specificities, and positive and negative predictive values for this comparison were as follows: for the Directigen method, 55, 99, 97, and 93%, respectively; for the LightCycler PCR, 93, 98, 88, and 99%, respectively. In no case was a throat swab positive by both the LightCycler PCR and the Directigen method but negative by culture. The medical histories of patients whose throat swabs were negative by culture but positive by either the LightCycler PCR (
n
= 7) or the Directigen method (
n
= 1) were reviewed. All of these patients had signs or symptoms compatible with GAS disease, and therefore, all of these discordant positive results (along with positive results by either the Directigen method or the LightCycler PCR that agreed with the culture results) were counted as true positives for statistical analysis. For this analysis, the LightCycler PCR detected more true-positive results than the culture method (58 versus 55 swabs); however, this difference was not statistically significant (
P
= 0.5465). In contrast, statistically significantly more true-positive results occurred by culture than by the Directigen method (55 versus 31 swabs;
P
< 0.0001) and by the LightCycler PCR than by the Directigen method (58 versus 31 swabs;
P
< 0.0001). The LightCycler PCR is a suitable stand-alone method for the detection of GAS from throat swabs. Additionally, this method requires less than half the personnel time and the procedure can be completed in considerably less time (∼1 h) than our standard approach (up to 2 days) for detection of GAS in throat swabs (i.e., testing by the Directigen method with negative results verified by culture).
Evaluation of a Novel Automated Chemiluminescent Assay System for Antimicrobial Susceptibility Testing
Authors: Manome Isao , Ikedo Masanari , Saito Yoshihiko , Ishii Keiko Kumura , Kaku Mitsuo
Published: January 2003

The newly developed Rapid Lumi Eiken/IS60 (RL/IS60) system automatically determines MICs by detecting chemiluminescence produced in the reaction of a chemiluminescent probe and oxygen metabolites from living microorganisms. The present study evaluated this system for accuracy in antimicrobial susceptibility testing. Chemiluminescence intensities after 4 h of cultivation of clinically important strains were plotted against various concentrations of antimicrobial agents, which resulted in curves reflecting the levels of susceptibility. Sixty-percent inhibitory concentrations based on the susceptibility curves agreed with MICs determined by the reference microdilution method. When the MICs of antimicrobial agents for four quality control (QC) strains (Staphylococcus aureus,Enterococcus faecalis,Escherichia coli, andPseudomonas aeruginosa) were determined by the RL/IS60 system, most (91.1%) of them were within the QC limits proposed by the National Committee for Clinical Laboratory Standards. The system was further assessed for a total of 162 clinical isolates, includingE. coli,Citrobacter freundii,Enterobacter cloacae,Klebsiella pneumoniae,Serratia marcescens,Proteus mirabilis,Morganella morganii,P. aeruginosa,Haemophilus influenzae,S. aureus, coagulase-negative staphylococci,Enterococcus faecalis,Enterococcus faecium, andStreptococcus pneumoniae. Overall, there was 90.6% agreement between the RL/IS60 system and the reference microdilution method. Our results suggest that the RL/IS60 system provides rapid and reliable MICs of a variety of antimicrobial agents for clinical isolates as well as QC strains.
Identification of a Gene Encoding Heat-Resistant Agglutinin in
Escherichia coli
as a Putative Virulence Factor in Urinary Tract Infection
Authors: Srinivasan Usha , Foxman Betsy , Marrs Carl F.
Published: January 2003

Escherichia coli
causes the vast majority of urinary tract infections (UTI) in both ambulatory and hospital patients. Several uropathogenic virulence factors have been identified, but half of all
E. coli
isolates that cause UTI have none or only one of the known virulence factors. Thus, it is reasonable to presume that other bacterial factors may be important in UTI pathogenesis. In order to find additional uropathogenic
E. coli
genes, we used genomic subtraction to identify DNA regions present in a uropathogenic strain of
E. coli
(1128-11). Genomic subtraction yielded 40 tester-specific fragments, including a novel heat-resistant agglutinin (
hra
) gene fragment.
hra
occurred in 55% of 486 UTI strains compared to 28% of 165 rectal strains (
P
= 0.001). The
hra
gene in 1128-11 was cloned, sequenced, and found to have 91% homology to the
hra
gene from
E. coli
meningitis strain RS218. The genetic organization of genes flanking
hra
in 1128-11 is distinct from the
hra
found in
E. coli
strains J96 and RS218. In our UTI and rectal specimen collections,
hra
was positively associated with a number of known virulence genes, including pathogenicity island genes
hly
and
cnf
, which are absent in 1128-11. The presence of
hra
in 1128-11 independent of
hly/cnf
suggests multiple mechanisms by which
hra
can be acquired by pathogenic
E. coli
strains. The flanking genes suggest that in 1128-11,
hra
may be part of a novel variant of a pathogenicity island V.
Evaluation of a Human Group A Rotavirus Assay for On-Site Detection of Bovine Rotavirus
Authors: Maes Roger K. , Grooms Daniel L. , Wise Annabel G. , Han Cunqin , Ciesicki Valerie , Hanson Lora , Vickers Mary Lynne , Kanitz Charles , Holland Robert
Published: January 2003

Neonatal diarrhea induced by bovine group A rotavirus causes significant economic loss in the dairy and beef industry due to increased morbidity and mortality, treatment costs, and reduced growth rates. The objective of this study was to evaluate a human group A rotavirus assay (ImmunoCardSTAT Rotavirus [ICS-RV]) as an on-site diagnostic test for bovine rotavirus. When used with a collection of bovine diarrhea samples submitted to the Virology Section of the Diagnostic Center for Population and Animal Health at Michigan State University and compared to a bovine group A rotavirus-specific reverse transcription-PCR (RT-PCR), the ICS-RV assay had a sensitivity and specificity of 87.0 and 93.6%, respectively. A commercially available group A rotavirus enzyme-linked immunosorbent assay (ELISA) (Pathfinder; Sanofi Diagnostics, Redmond, Wash.), when used with the same fecal sample collection and compared to the same RT-PCR, had a sensitivity and specificity of 78.3 and 67.7%, respectively. Subsequently, the ICS-RV assay, RT-PCR, and a different commercially available group A rotavirus ELISA (Rotaclone; Meridian Diagnostics, Cincinnati, Ohio) were used to evaluate fecal samples collected from neonatal calves experimentally infected with bovine rotavirus. When diarrheic fecal samples that were positive for bovine rotavirus by RT-PCR were evaluated, the ICS-RV assay and the Rotaclone assay detected bovine rotavirus 85 and 95% of the time, respectively. Based on these studies, the ICS-RV assay appears to be an excellent test for detecting group A bovine rotaviruses. This assay may be useful as an on-site diagnostic test for veterinarians as an aid in the management of bovine neonatal diarrhea.
Culture-Independent Molecular Analysis of Microbial Constituents of the Healthy Human Outer Ear
Authors: Frank Daniel N. , Spiegelman George B. , Davis William , Wagner Eileen , Lyons Eric , Pace Norman R.
Published: January 2003

Molecular-phylogenetic sequence analyses have provided a new perspective on microbial communities by allowing the detection and identification of constituent microorganisms in the absence of cultivation. In this study we used broad-specificity amplification of ribosomal DNA (rDNA) genes to survey organisms present in the human outer ear canal. Samples were obtained from 24 individuals, including members of three extended families, in order to survey the resident microbiota and to examine microbial population structures in individuals related by familial or household associations. To examine the stability of the microbial populations, one individual was sampled four times and another twice over a 14-month period. We found that a distinct set of microbial types was present in the majority of the subjects sampled. The two most prevalent rDNA sequence types that were identified in multiple individuals corresponded closely to those of
Alloiococcus otitis
and
Corynebacterium otitidis
, commonly thought to be associated exclusively with infections of the middle ear. Our results suggest, therefore, that the outer ear canal may serve as a reservoir for normally commensal microbes that can contribute to pathogenesis upon introduction into the middle ear. Alternatively, culture analyses of diseases of the middle ear may have been confounded by these contaminating commensal organisms.
Performance of the APTIMA Combo 2 Assay for Detection of
Chlamydia trachomatis
and
Neisseria gonorrhoeae
in Female Urine and Endocervical Swab Specimens
Authors: Gaydos C. A. , Quinn T. C. , Willis D. , Weissfeld A. , Hook E. W. , Martin D. H. , Ferrero D. V. , Schachter J.
Published: January 2003

The greater sensitivity of nucleic acid amplification tests (NAATs) for
Chlamydia trachomatis
and
Neisseria gonorrhoeae
permits the use of urine and other noninvasive specimens, which can increase the reach and decrease the costs of public health screening programs aimed at controlling these infections. This study evaluated the performance of the APTIMA Combo 2 assay, a multiplex assay based on the transcription-mediated amplification reaction, for the simultaneous detection of both pathogens in endocervical swab and urine specimens from females. Combo 2 assay results were compared with patient infected status, which were available by using other commercial NAATs. Sensitivity and specificity for
C. trachomatis
were 94.2 and 97.6%, respectively, in swabs and 94.7 and 98.9%, respectively, in first-catch urine (FCU). Sensitivity and specificity for
N. gonorrhoeae
were 99.2 and 98.7%, respectively, in swabs and 91.3 and 99.3%, respectively, in FCU. The assay reliably detected both infections in coinfected patients. The Combo 2 assay can be recommended for use with endocervical swab and urine specimens from females, especially for screening tests for asymptomatic women in sexually transmitted disease surveillance programs. This Food and Drug Administration-cleared assay can be a useful tool in efforts to reduce the prevalence and incidence of
C. trachomatis
and
N. gonorrhoeae
infections in sexually active women and to prevent their costly and serious sequelae.
Validation of a Urine-Based PCR-Enzyme-Linked Immunosorbent Assay for Use in Clinical Research Settings To Detect
Trichomonas vaginalis
in Men
Authors: Kaydos-Daniels S. Cornelia , Miller William C. , Hoffman Irving , Banda Topia , Dzinyemba Willard , Martinson Francis , Cohen Myron S. , Hobbs Marcia M.
Published: January 2003

Trichomonas vaginalis
infection is highly prevalent worldwide and is associated with urethritis, prostatitis, and urethral strictures in men. However, the natural history and importance of
T. vaginalis
in men are poorly understood, in part because of difficulties in diagnosing infection. Traditional detection methods rely on culture and wet-mount microscopy, which can be insensitive and time consuming. Urethral swabs are commonly used to detect
T. vaginalis
in men, but discomfort from specimen collection is a barrier to large studies. One thousand two hundred twenty-five Malawian men attending sexually transmitted disease and dermatology clinics were enrolled in this cross-sectional study to validate detection by urine-based PCR-enzyme-linked immunosorbent assay (ELISA) with urine and urethral swab culture as the reference standard. This assay for detection of amplified
T. vaginalis
DNA in first-catch urine (≤30 ml) performed with a sensitivity of 92.7%, a specificity of 88.6%, and an adjusted specificity of 95.2% compared to culture of urethral swabs or urine sediment. For clinical research settings in which urethral swabs are not available and culture is not feasible, the urine-based PCR-ELISA may be useful for detection of trichomoniasis in men.
Prospective Study of the Performance of Vibrational Spectroscopies for Rapid Identification of Bacterial and Fungal Pathogens Recovered from Blood Cultures
Authors: Maquelin K. , Kirschner C. , Choo-Smith L.-P. , Ngo-Thi N. A. , van Vreeswijk T. , Stämmler M. , Endtz H. P. , Bruining H. A. , Naumann D. , Puppels G. J.
Published: January 2003

Rapid identification of microbial pathogens reduces infection-related morbidity and mortality of hospitalized patients. Raman spectra and Fourier transform infrared (IR) spectra constitute highly specific spectroscopic fingerprints of microorganisms by which they can be identified. Little biomass is required, so that spectra of microcolonies can be obtained. A prospective clinical study was carried out in which the causative pathogens of bloodstream infections in hospitalized patients were identified. Reference libraries of Raman and IR spectra of bacterial and yeast pathogens highly prevalent in bloodstream infections were created. They were used to develop identification models based on linear discriminant analysis and artificial neural networks. These models were tested by carrying out vibrational spectroscopic identification in parallel with routine diagnostic phenotypic identification. Whereas routine identification has a typical turnaround time of 1 to 2 days, Raman and IR spectra of microcolonies were collected 6 to 8 h after microbial growth was detected by an automated blood culture system. One hundred fifteen samples were analyzed by Raman spectroscopy, of which 109 contained bacteria and 6 contained yeasts. One hundred twenty-one samples were analyzed by IR spectroscopy. Of these, 114 yielded bacteria and 7 were positive for yeasts. High identification accuracy was achieved in both the Raman (92.2%, 106 of 115) and IR (98.3%, 119 of 121) studies. Vibrational spectroscopic techniques enable simple, rapid, and accurate microbial identification. These advantages can be easily transferred to other applications in diagnostic microbiology, e.g., to accelerate identification of fastidious microorganisms.
Evaluation of 11 PCR Assays for Species-Level Identification of
Campylobacter jejuni
and
Campylobacter coli
Authors: On Stephen L. W. , Jordan Penelope J.
Published: January 2003

We examined the sensitivity and specificity of 11 PCR assays described for the species identification of
Campylobacter jejuni
and
Campylobacter coli
by using 111 type, reference, and field strains of
C. jejuni
,
C. coli
, and
Campylobacter lari
. For six assays, an additional 21 type strains representing related
Campylobacter
,
Arcobacter
, and
Helicobacter
species were also included. PCR tests were initially established in the laboratory by optimizing conditions with respect to five type and reference strains of
C. jejuni
,
C. coli
, and
C. lari
. One PCR test for
C. coli
failed to give appropriate results during this initial setup phase and was not evaluated further. The remaining 10 assays were used to examine heated lysate and purified DNA templates as appropriate of well-characterized type, reference, and field strains of
C. jejuni
(
n
= 62),
C. coli
(
n
= 34), and
C. lari
(
n
= 15). The tests varied considerably in their sensitivity and specificity for their respective target species. No assay was found to be 100% sensitive and/or specific for all
C. jejuni
strains tested, but four assays for
C. coli
gave appropriate responses for all strains examined. Between one and six strains of
C. jejuni
gave amplicons in four of seven
C. jejuni
PCR tests only where purified DNA was used as the template; corresponding results were seen with one strain of
C. coli
in each of three assays for the latter species. Our findings indicate that a polyphasic strategy for PCR-based identification should be used to identify
C. jejuni
and
C. coli
strains. The data may assist laboratories in selecting assays suited for their needs and in designing evaluations of future PCR tests aimed to identify these species.
Characterization of Members of the
Legionellaceae
Family by Automated Ribotyping
Authors: Cordevant Christophe , Tang Jane S. , Cleland David , Lange Marc
Published: January 2003

In order to implement a new and reliable method for characterizing different species of
Legionella
, a genetic fingerprinting study with an automated ribotyping system (RiboPrinter) was completed with members of this genus which were deposited at the American Type Culture Collection. The RiboPrinter examined the different patterns of
Eco
RI digestion fragments from the rRNA operons of 110 strains, representing 48 of the 49 described
Legionella
species as well as 70 serogroups of those species. Distinctive and consistent patterns were obtained for the type strains of the 48 species investigated.
Legionella pneumophila
subsp.
fraseri
and
L. pneumophila
subsp.
pascullei
each generated a specific pattern, whereas
L. pneumophila
subsp.
pneumophila
produced six different fingerprint patterns. No correlation seemed to exist between the ribotypes obtained and the 15 serotypes of
L. pneumophila
. For the other species, those with two known serogroups presented two distinctive patterns with the RiboPrinter with the exception of
L. hackeliae
and
L. quinlivanii
, which yielded only one pattern. We also encountered ribotypes for strains which were not identified to the species level. The ribotypes generated for these strains with the RiboPrinter did not match those generated for known type strains, suggesting the putative description of new serogroups or species. Although the automated system did not have sufficient discriminatory ability to serve as an epidemiological tool in a clinical setting, it appeared to be a powerful tool for general genomic analysis of the
Legionella
isolates (e.g., determination of new species) and assessment of the interrelationship among
Legionella
strains through the RiboPrinter database connection.
Antimicrobial Susceptibility Testing of Carbapenems: Multicenter Validity Testing and Accuracy Levels of Five Antimicrobial Test Methods for Detecting Resistance in
Enterobacteriaceae
and
Pseudomonas aeruginosa
Isolates
Authors: Steward Christine D. , Mohammed Jasmine M. , Swenson Jana M. , Stocker Sheila A. , Williams Portia P. , Gaynes Robert P. , McGowan, John E. , Tenover Fred C.
Published: January 2003

From January 1996 to May 1999, Project ICARE (Intensive Care Antimicrobial Resistance Epidemiology) received 448 nonduplicate clinical isolates of
Enterobacteriaceae
and
Pseudomonas aeruginosa
that were reported to be imipenem intermediate or resistant. However, broth microdilution (BMD) confirmatory testing at the Project ICARE central laboratory confirmed this result in only 11 of 123 (8.9%)
Enterobacteriaceae
isolates and 241 of 325 (74.2%)
P. aeruginosa
isolates. To investigate this overdetection of imipenem resistance, we tested 204 selected isolates from the Project ICARE collection plus five imipenem-resistant challenge strains at the Centers for Disease Control and Prevention against imipenem and meropenem by agar dilution, disk diffusion, Etest (AB BIODISK North America, Inc., Piscataway, N.J.), two MicroScan WalkAway conventional panels (Neg MIC Plus 3 and Neg Urine Combo 3) (Dade MicroScan, Inc., West Sacramento, Calif.), and two Vitek cards (GNS-116 containing meropenem and GNS-F7 containing imipenem) (bioMérieux Vitek, Inc., Durham, N.C.). The results of each test method were compared to the results of BMD testing using in-house-prepared panels. Seven imipenem-resistant and five meropenem-resistant isolates of
Enterobacteriaceae
and 43 imipenem-resistant and 21 meropenem-resistant isolates of
P. aeruginosa
were identified by BMD. For
Enterobacteriaceae
, the imipenem and meropenem test methods produced low numbers of very major and major errors. All test systems in the study produced low numbers of very major and major errors when
P. aeruginosa
was tested against imipenem and meropenem, except for Vitek testing (major error rate for imipenem, 20%). Further testing conducted in 11 of the participating ICARE hospital laboratories failed to pinpoint the factors responsible for the initial overdetection of imipenem resistance. However, this study demonstrated that carbapenem testing difficulties do exist and that laboratories should consider using a second, independent antimicrobial susceptibility testing method to validate carbapenem-intermediate and -resistant results.
Attempted Isolation of
Nanobacterium
sp. Microorganisms from Upper Urinary Tract Stones
Authors: Drancourt Michel , Jacomo Véronique , Lépidi Hubert , Lechevallier Eric , Grisoni Vincent , Coulange Christian , Ragni Edith , Alasia Claude , Dussol Bertrand , Berland Yvon , Raoult Didier
Published: January 2003

A single team has reported isolation of nanobacteria in human and bovine blood products, as well as, more recently, kidney stones. This has raised controversy. To confirm the data, we searched for nanobacteria from 10 aseptically removed upper urinary tract (UUT) stones. We used scanning electronic microscopy (SEM) with four stones and culture of stones on either 3T6 fibroblast monolayers or liquid RPMI medium. Detection of nanobacteria was made with a commercially available monoclonal antibody, 16S ribosomal DNA amplification with specific primers, and transmission electronic microscopy (TEM) of inoculated cells. SEM showed nanoparticles in four of four UUT stones similar to those recently described. TEM of inoculated 3T6 cell monolayers has shown transient intracytoplasmic vacuolar formations containing 200- to 500-nm particles in 3 of 10 cell cultures. Gimenez staining, Hoechst staining, and specific monoclonal immunofluorescence failed to reveal nanobacteria. Finally, we could not grow
Nanobacterium
sp. microorganisms by the techniques described. Although with SEM, we observed nanoparticles morphologically similar to nanobacteria, we failed to isolate
Nanobacterium
sp. microorganisms in culture and to prove the bacterial nature of these nanoparticles in stones.
M Protein Gene Type Distribution among Group A Streptococcal Clinical Isolates Recovered in Mexico City, Mexico, from 1991 to 2000, and Durango, Mexico, from 1998 to 1999: Overlap with Type Distribution within the United States
Authors: Espinosa Luz Elena , Li Zhongya , Barreto Demostenes Gomez , Jaimes Ernesto Calderon , Rodriguez Romeo S. , Sakota Varja , Facklam Richard R. , Beall Bernard
Published: January 2003

To examine the type distribution of pathogenic group A streptococcal (GAS) strains in Mexico, we determined the
emm
types of 423 GAS isolates collected from ill patients residing in Mexico (Durango or Mexico City). These included 282 throat isolates and 107 isolates from normally sterile sites. Of the other isolates, 38 were recovered from other miscellaneous infections. A total of 31 different
emm
types were found, revealing a broad overlap between commonly occurring
emm
types in Mexico and the United States. The information obtained in this study is consistent with the possibility that multivalent, M type-specific vaccines prepared for GAS strain distribution within the United States could theoretically protect against the majority of GAS strains causing disease in the two cities surveyed in Mexico.