bluuebunny/crossref_metadata_2025 huggingface dataset:
Development and Evaluation of a Rapid Latex Agglutination Test Using a Monoclonal Antibody To Identify
Candida dubliniensis
Colonies
Authors: Marot-Leblond Agnes , Beucher Bertrand , David Sandrine , Nail-Billaud Sandrine , Robert Raymond
Published: January 2006

Cell components of the dimorphic pathogenic fungus
Candida dubliniensis
were used to prepare monoclonal antibodies (MAbs). One MAb, designated 12F7-F2, was shown by indirect immunofluorescence to be specific for a surface antigen of
Candida dubliniensis
yeast cells. No reactivity was observed with other fungal genera or with other
Candida
species, including
Candida albicans
, that share many phenotypic features with
C. dubliniensis
. The use of different chemical and physical treatments for cell component extraction suggested that the specific epitope probably resides on a protein moiety absent from
C. albicans
. However, we failed to identify the target protein by Western blotting, owing to its sensitivity to heat and sodium dodecyl sulfate. MAb 12F7-F2 was further used to develop a commercial latex agglutination test to identify
C. dubliniensis
colonies (Bichro-dubli Fumouze test; Fumouze Diagnostics). The test was validated on yeast strains previously identified by PCR and on fresh clinical isolates; these included 46
C. dubliniensis
isolates, 45
C. albicans
isolates, and other yeast species. The test had 100% sensitivity and specificity for
C. dubliniensis
isolated on Sabouraud dextrose, CHROMagar Candida, and CandiSelect media and 97.8% sensitivity for
C. dubliniensis
grown on Candida ID medium. The test is rapid (5 min) and easy to use and may be recommended for routine use in clinical microbiology laboratories and for epidemiological investigations.
Use of Quantitative Real-Time PCR To Study the Kinetics of Extracellular DNA Released from
Candida albicans
, with Implications for Diagnosis of Invasive Candidiasis
Authors: Kasai Miki , Francesconi Andrea , Petraitiene Ruta , Petraitis Vidmantas , Kelaher Amy M. , Kim Hee-sup , Meletiadis Joseph , Sein Tin , Bacher John , Walsh Thomas J.
Published: January 2006

Quantitative real-time PCR (qPCR) is considered one of the most sensitive methods to detect low levels of DNA from pathogens in clinical samples. To improve the design of qPCR for the detection of deeply invasive candidiasis, we sought to develop a more comprehensive understanding of the kinetics of DNA released from
Candida albicans
in vitro and in vivo. We developed a
C. albicans
-specific assay targeting the rRNA gene complex and studied the kinetics of DNA released from
C. albicans
alone, in the presence of human blood monocytes (H-MNCs), and in the bloodstream of rabbits with experimental disseminated candidiasis. The analytical qPCR assay was highly specific and sensitive (10 fg). Cells of
C. albicans
incubated in Hanks balanced salt solution (±10% bovine serum albumin [BSA]) or RPMI (±10% BSA) showed a significant release of DNA at
T
equal to 24 h compared to
T
equal to 0 h (
P
≤ 0.01).
C. albicans
incubated with H-MNCs exhibited a greater release of DNA than
C. albicans
cells alone over 24 h (
P
= 0.0001). Rabbits with disseminated candidiasis showed a steady increase of detectable DNA levels in plasma as disease progressed. Plasma cultures showed minimal growth of
C. albicans
, demonstrating that DNA extracted from plasma reflected fungal cell-free DNA. In summary, these studies of the kinetics of DNA release by
C. albicans
collectively demonstrate that cell-free fungal DNA is released into the bloodstream of hosts with disseminated candidiasis, that phagocytic cells may play an active role in increasing this release over time, and that plasma is a suitable blood fraction for the detection of
C. albicans
DNA.
Identification of Invasive Serotype 1 Pneumococcal Isolates That Express Nonhemolytic Pneumolysin
Authors: Kirkham Lea-Ann S. , Jefferies Johanna M. C. , Kerr Alison R. , Jing Yu , Clarke Stuart C. , Smith Andrew , Mitchell Tim J.
Published: January 2006

Recently, there has been an increase in invasive pneumococcal disease (IPD) caused by serotype 1
Streptococcus pneumoniae
throughout Europe. Serotype 1 IPD is associated with bacteremia and pneumonia in Europe and North America, especially in neonates, and is ranked among the top five most prevalent pneumococcal serotypes in at least 10 countries. The currently licensed pediatric pneumococcal vaccine does not afford protection to this serotype. Upon screening of 252 clinical isolates of
S. pneumoniae
, we discovered mutations in the pneumolysin gene of two out of the four serotype 1 strains present in the study group. Analysis of an additional 28 serotype 1 isolates from patients with IPD from various Scottish Health Boards, revealed that >50% had mutations in their pneumolysin genes. This resulted in the expression of nonhemolytic forms of pneumolysin. All of the strains producing nonhemolytic pneumolysin were sequence type 306 (ST306), whereas those producing “wild-type” pneumolysin were ST227. The mutations were in a region of pneumolysin involved in pore formation. These mutations can be made in vitro to give the nonhemolytic phenotype. Pneumolysin is generally conserved throughout all serotypes of
S. pneumoniae
and is essential for full invasive disease; however, it appears that serotype 1 ST306 does not require hemolytically active pneumolysin to cause IPD.
Viridans Streptococci Isolated by Culture from Blood of Cancer Patients: Clinical and Microbiologic Analysis of 50 Cases
Authors: Han Xiang Y. , Kamana Mallika , Rolston Kenneth V. I.
Published: January 2006

Clinical and microbiologic studies of 50 cases of viridans streptococcal bacteremia in cancer patients were performed. The bacteria were identified to species level by sequencing analysis of the 16S rRNA gene. At least nine
Streptococcus
spp. were found, including
S. mitis
(25 strains, 50.0% of 50); currently unnamed
Streptococcus
spp. (11 strains);
S. parasanguis
(five strains);
S. anginosus
(three strains);
S. salivarius
(two strains); and one strain each of
S. gordonii, S. sanguis, S. sobrinus
, and
S. vestibularis
. There were no
S. oralis
strains. Among 11 antibiotics of nine classes tested, no resistance to vancomycin, linezolid, or quinupristin-dalfopristin was seen. Resistance to penicillin (MIC, 4 to 12 μg/ml) was noted only among
S. mitis
strains (28.0%, 7/25) and not non-
S. mitis
strains (0/25) (
P
= 0.004). Significantly more
S. mitis
strains than non-
S. mitis
strains were resistant to fluoroquinolones and to ≥3 classes of antibiotics. Isolation of quinolone-resistant organisms was associated with the prior usage of quinolones (
P
= 0.002). Quantitative blood cultures showed that the strains resistant to levofloxacin or gatifloxacin were associated with higher colony counts than were their corresponding nonresistant strains. The young and elderly patients also had higher levels of bacteremia caused predominantly by
S. mitis
. Septic shock was present in 17 (34.0% of 50) patients, and 13 of those cases were caused by
S. mitis
(
P
= 0.007). These results suggest that
S. mitis
is the most common cause of viridans streptococcal bacteremia in cancer patients and is more resistant to antibiotics than other species.
Detection of Rhinoviruses by Tissue Culture and Two Independent Amplification Techniques, Nucleic Acid Sequence-Based Amplification and Reverse Transcription-PCR, in Children with Acute Respiratory Infections during a Winter Season
Authors: Loens K. , Goossens H. , de Laat C. , Foolen H. , Oudshoorn P. , Pattyn S. , Sillekens P. , Ieven M.
Published: January 2006

Five hundred seventeen consecutive nasopharyngeal aspirates were collected between October 1998 and May 1999 for episodes of acute respiratory tract infections in children presenting at the University Hospital of Antwerp. Culture and nucleic acid amplification techniques—nucleic acid sequence-based amplification (NASBA) and reverse transcription-PCR (RT-PCR)—were applied to detect rhinoviruses (RVs). Other respiratory viruses were detected by immunofluorescence (IF) analysis of the specimens and IF analysis of shell vial cultures. Among the 517 specimens, 219 viral agents were identified. They were, in decreasing order, rhinoviruses (93 [18.0%]), respiratory syncytial virus (76 [14.7%]), adenoviruses (16 [3.1%]), influenza viruses (15 [2.9%]), enteroviruses (15 [2.9%]), and herpes simplex virus (4 [0.8%]). For the evaluation of rhinovirus detection, culture positivity and/or a positive reaction in the two independent amplification methods was used as an expanded “gold standard.” Based on this standard, the sensitivity, specificity, positive predictive value, and negative predictive value of culture were 44.7, 100, 100, and 99.8%, and those of NASBA and RT-PCR were 85.1, 98.3, 83.3, and 98.5% and 82.9, 93.4, 55.7, and 98.2%, respectively. NASBA and RT-PCR produced comparable results and were significantly more sensitive than virus culture. RVs showed the highest incidence in acute respiratory tract infections in children.
Detection of
cfxA
and
cfxA2
, the β-Lactamase Genes of
Prevotella
spp., in Clinical Samples from Dentoalveolar Infection by Real-Time PCR
Authors: Iwahara Kaori , Kuriyama Tomoari , Shimura Satoshi , Williams David W. , Yanagisawa Maki , Nakagawa Kiyomasa , Karasawa Tadahiro
Published: January 2006

While most bacteria involved in dentoalveolar infection are highly susceptible to penicillin, some
Prevotella
strains exhibit resistance to this agent through the production of β-lactamase. The production of β-lactamase by
Prevotella
spp. is in turn associated with the expression of the genes
cfxA
and
cfxA2
. The aim of the present study was to determine the prevalence of
cfxA
and
cfxA2
in
Prevotella
strains by use of real-time PCR and to assess the performance of this molecular method for the direct detection of the genes in 87 clinical samples (pus and root canal exudates) from dentoalveolar infection. Production of β-lactamase by each isolate was determined using a nitrocefin disk. β-Lactamase production was seen in 31% of
Prevotella
isolates, while all isolates of other species were β-lactamase negative. The penicillin resistance of isolates strongly correlated with the production of β-lactamase. Real-time PCR was found to detect the
cfxA
and
cfxA2
genes from at least five cells per reaction mixture (5 × 10
3
CFU/ml of pus). Using real-time PCR, the presence of
cfxA
and
cfxA2
was evident for all 48 β-lactamase-positive
Prevotella
strains. In contrast, neither β-lactamase-negative
Prevotella
(
n
= 91) or non-
Prevotella
(
n
= 31) strains were positive for the genes. In this study, 31 of the 87 samples yielded β-lactamase-positive
Prevotella
results, and
cfxA
and
cfxA2
were detected in all 31 samples. Of the 56 culture-negative samples, 8 (14%) were positive for
cfxA
and
cfxA2
by the real-time PCR. This sensitive and specific molecular method offers a rapid clinical test for aiding in the selection of an appropriate antibiotic for treatment of dentoalveolar infection. Although penicillin remains largely effective in the treatment of dentoalveolar infection, β-lactamase-stable antibiotics should be considered in cases in which β-lactamase-positive
Prevotella
strains are involved.
Virological, Serological, and Clinical Features of an Outbreak of Acute Gastroenteritis Due to Recombinant Genogroup II Norovirus in an Infant Home
Authors: Tsugawa Takeshi , Numata-Kinoshita Kazuko , Honma Shinjiro , Nakata Shuji , Tatsumi Masatoshi , Sakai Yoshiyuki , Natori Katsuro , Takeda Naokazu , Kobayashi Shinichi , Tsutsumi Hiroyuki
Published: January 2006

Norovirus (NV) is an important cause of acute nonbacterial gastroenteritis worldwide. Recently, several sporadic cases due to naturally occurring recombinant NVs have been reported. In January 2000, there was an outbreak of gastroenteritis in an infant home in Sapporo, Japan. Of 34 residents of the home that were less than 2 years old, 23 developed gastrointestinal symptoms and NV infection was confirmed by conventional reverse transcription-PCR to detect the RNA polymerase region of genogroup II NV. In this virus, the RNA polymerase region shared 86% nucleotide identity with Hawaii virus but only 77% with Mexico virus; however, its capsid region shared only 70% identity with Hawaii virus but 90% with Mexico virus. On the other hand, both regions shared a higher 96% nucleotide identity with Arg320 virus, which was found in Mendoza, Argentina, in 1995 and considered to be a recombinant of Hawaii and Mexico viruses. The findings indicate that the virus involved in the outbreak was similar and may have evolved from the Arg320 virus. Clinically the cases were more severe than those of previously reported sporadic or outbreak cases of NV infection.
Long-Term Population-Based Genotyping Study of
Mycobacterium tuberculosis
Complex Isolates in the French Departments of the Americas
Authors: Brudey Karine , Filliol Ingrid , Ferdinand Séverine , Guernier Vanina , Duval Philippe , Maubert Bertrand , Sola Christophe , Rastogi Nalin
Published: January 2006

The three French overseas departments of the Americas are characterized both by insular (Guadeloupe and Martinique) and continental (French Guiana) settings with a tuberculosis case detection rate that varies from less than 10 per 100,000 per year in insular areas to an estimated incidence of more than 55 per 100,000 in French Guiana. Under a long-term genotyping program, more than three-fourths of all the
Mycobacterium tuberculosis
isolates (
n
= 744) received from the three settings were fingerprinted over a 10-year period (1994 to 2003) by spoligotyping and variable number of tandem DNA repeats (VNTRs) in order to understand the current trends in their detection rates, drug resistance, and groups and subpopulations at risk of contracting the disease and to pinpoint the circulating phylogeographical clades of the bacilli. The major difference in the study populations was the nationality of the patients, with a high percentage of immigrants from high-incidence neighboring countries in French Guiana and a low but increasing percentage in the French Caribbean. The rate of recent transmission was calculated to be 49.3% in French Guiana, compared to 27.2% and 16.9% in Guadeloupe and Martinique, respectively. At the phylogeographic level, 77.9% of the isolates studied belonged to four major clades (Haarlem, Latin-American and Mediterranean, T, and X) which are already reported from neighboring Caribbean islands in an international database and may underline potential interregional transmission events.
Use of a Mycobacteriophage-Based Assay for Rapid Assessment of Susceptibilities of
Mycobacterium tuberculosis
Isolates to Isoniazid and Influence of Resistance Level on Assay Performance
Authors: Galí N. , Domínguez J. , Blanco S. , Prat C. , Alcaide F. , Coll P. , Ausina V.
Published: January 2006

We standardized and assessed the performance of an in-house microtiter assay for determining the susceptibilities of
Mycobacterium tuberculosis
clinical isolates to isoniazid based on mycobacteriophage amplification technology. Seventy isolates (43 resistant and 27 sensitive according to the BACTEC 460 radiometric method and MIC determination) were studied. The isoniazid resistance molecular mechanism was previously determined by sequencing the entire
katG
gene and the
mabA-inhA
regulatory region. The sensitivity of the mycobacteriophage-based assay in detecting isoniazid resistance was 86.1%, the specificity achieved was 92.6%, and the overall accuracy was 88.6%. In order to assess the possible influence of resistance levels on the mycobacteriophage-based-assay sensitivity, the results were analyzed according to the isoniazid MICs. All the isolates exhibiting high-level resistance (MIC ≥ 2 μg/ml) were scored as resistant by the mycobacteriophage-based assay (100% concordance), and 95% showed mutations or deletions in the catalytic domain of the
katG
gene. In contrast, 26.1% of the low-level-resistance strains (MICs, 0.25 to 1 μg/ml) were misclassified, and 66.7% had alterations in the
mabA-inhA
regulatory region. The mycobacteriophage-based assay could be used as a rapid method to detect the isoniazid susceptibility pattern, although data from those areas with high rates of low-level-resistance strains should be interpreted with caution. The features of the assay make it suitable for widespread application due to its low technical demand and cost.
Development and Validation of a Rotor-Gene Real-Time PCR Assay for Detection, Identification, and Quantification of
Chlamydia trachomatis
in a Single Reaction
Authors: Jalal Hamid , Stephen Hannah , Curran Martin D. , Burton Janet , Bradley Michelle , Carne Christopher
Published: January 2006

A multitarget real-time PCR (MRT-PCR) for detection of
Chlamydia trachomatis
DNA was developed and validated. There were three targets for amplification in a single reaction: the cryptic plasmid (CP), the major outer membrane protein (MOMP) gene, and an internal control. The assay had the following characteristics: (i) detection and confirmation of the presence of
C. trachomatis
DNA in a single reaction, (ii) detection of all genovars of
C. trachomatis
without any cross-reactivity with pathogenic bacteria or commensal organisms of the oropharynx and genital tract, (iii) a 95% probability of detection with three copies of MOMP and one copy of CP per reaction mixture, (iv) identification of the inhibition of amplification, (v) a quantitative dynamic range of 25 to 250,000 genome copies per reaction mixture, (vi) high intra- and interassay reproducibilities, and (vii) correct identification of all samples in the validation panel. There were 146 COBAS Amplicor PCR (Amplicor PCR)-positive samples and 122 Amplicor PCR-negative samples in the panel. MRT-PCR detected CP DNA alone in 6 (4%) Amplicor PCR-positive samples and both CP and MOMP DNAs in 140 (96%) of 146 Amplicor PCR-positive samples. The quantity of MOMP DNA in 95 (68%) of 140 samples was within the dynamic range of the assay. The median
C. trachomatis
load in these samples was 321 genome copies per reaction mixture (range, 26 to 40,137 genome copies per reaction mixture). Due to the inclusion of two different
C. trachomatis
-specific targets, the assay confirmed 259 (97%) of 268 results in a single reaction. This assay could be used in the qualitative format for the routine detection of
C. trachomatis
and in the quantitative format for study of the pathogenesis of
C. trachomatis
-associated diseases. The assay demonstrated the potential to eliminate the need for confirmatory testing in almost all samples, thus reducing the turnaround time and the workload.
Listeria monocytogenes
Serotype 4b Strains Belonging to Lineages I and III Possess Distinct Molecular Features
Authors: Liu Dongyou , Lawrence Mark L. , Gorski Lisa , Mandrell Robert E. , Ainsworth A. Jerald , Austin Frank W.
Published: January 2006

A collection of
Listeria monocytogenes
serotype 4b strains belonging to lineages I and III were examined by PCR and Southern blot analysis using species-, virulence-, and serotype-specific primers and probes. Whereas four serotype 4b lineage I strains reacted in PCR with the serotype 4b-, 4d-, and 4e-specific ORF2110 and virulence-specific
lmo1134
and
lmo2821
primers, all nine serotype 4b lineage III strains were negative by ORF2110 and
lmo1134
primers. In addition, the nine serotype 4b lineage III strains formed two separate groups through their reactions in PCR with virulence-specific
lmo2821
primers. Southern blot analysis using species-specific
lmo0733
and virulence-specific
lmo2821
gene probes largely confirmed the PCR results. These findings indicate that
L. monocytogenes
serotype 4b strains belonging to lineages I and III possess distinct molecular features.
Four-Year Persistence of a SingleCandida albicansGenotype Causing Bloodstream Infections in a Surgical Ward Proven by Multilocus Sequence Typing
Authors: Viviani Maria Anna , Cogliati Massimo , Esposto Maria Carmela , Prigitano Anna , Tortorano Anna Maria
Published: January 2006

The present study represents the first application of multilocus sequence typing to retrospectively investigate a suspected outbreak ofCandida albicansbloodstream infection cases that occurred in the same hospital ward between July 1987 and October 1991. Results demonstrated that eight bloodstream infections were caused by the same strain, endemic in the ward, over a 4-year period.
Clonal Spread of Serogroup W135 Meningococcal Disease in Turkey
Authors: Kilic Abdullah , Urwin Rachel , Li Haijing , Saracli Mehmet A. , Stratton Charles W. , Tang Yi-Wei
Published: January 2006

Six cases ofNeisseria meningitidisserogroup W135 meningococcal infection have been reported in Turkey since 2003. Seven isolates recovered from four meningococcal meningitis patients and two asymptomatic carriers produced three distinct pulsed-field gel electrophoresis (PFGE) patterns. Multilocus sequence typing and antigen gene sequencing showed that five isolates were indistinguishable from ST-11 (ET-37) serogroup W135 meningococci, which were first isolated in Saudi Arabia and were responsible for the worldwide outbreak among Hajj pilgrims and their contacts in 2000. The remaining two isolates, which had related PFGE patterns, differed from each other at only one of the genetic loci characterized but were not related to the ST-11 clonal complex. None of the six individuals recalled contact with a pilgrim or had traveled on the Hajj. These six individuals exhibited no time or place relationships to each other, except for the two asymptomatic carriers, who were soldiers and served in the same military unit. These data demonstrate that serogroup W135 meningococci with different genotypes, including the Hajj epidemic strain, are endemic in Turkey.
Comparative Study of the New Colorimetric VITEK 2 Yeast Identification Card versus the Older Fluorometric Card and of CHROMagar Candida as a Source Medium with the New Card
Authors: Aubertine C. L. , Rivera M. , Rohan S. M. , Larone D. H.
Published: January 2006

The new VITEK 2 colorimetric card was compared to the previous fluorometric card for identification of yeast. API 20C was considered the “gold standard.” The new card consistently performed better than the older card. Isolates from CHROMagar Candida plates were identified equally as well as those from Sabouraud dextrose agar.
Does Nasal Cocolonization by Methicillin-Resistant Coagulase-Negative Staphylococci and Methicillin-Susceptible
Staphylococcus aureus
Strains Occur Frequently Enough To Represent a Risk of False-Positive Methicillin-Resistant
S. aureus
Determinations by Molecular Methods?
Authors: Becker Karsten , Pagnier Isabelle , Schuhen Brigitte , Wenzelburger Frauke , Friedrich Alexander W. , Kipp Frank , Peters Georg , von Eiff Christof
Published: January 2006

By analyzing the colonization of the anterior nares in cardiothoracic surgery patients on admission, nasal cocolonization by methicillin-susceptible
Staphylococcus aureus
and methicillin-resistant coagulase-negative staphylococci was detected in 8/235 (3.4%) specimens. Consequently, in a low-methicillin-resistant
S. aureus
(MRSA) setting, a molecular MRSA screening test targeting the
mecA
gene and an
S. aureus
-specific gene in parallel and applied directly to clinical specimens would be associated with an unacceptable positive predictive value of about 40%.
Evaluation of Three Diagnostic Methods, Including Real-Time PCR, for Detection of
Dientamoeba fragilis
in Stool Specimens
Authors: Stark D. , Beebe N. , Marriott D. , Ellis J. , Harkness J.
Published: January 2006

Dientamoeba fragilis
is a protozoan parasite of humans that infects the mucosa of the large intestine and is associated with gastrointestinal disease. We developed a 5′ nuclease (TaqMan)-based real-time PCR assay, targeting the small subunit rRNA gene, for the detection of
D. fragilis
in human stool specimens and compared its sensitivity and specificity to conventional PCR and microscopic examination by a traditional modified iron-hematoxylin staining procedure. Real-time PCR exhibited 100% sensitivity and specificity.
Utility of the VITEK 2 Advanced Expert System for Identification of Extended-Spectrum β-Lactamase Production in
Enterobacter
spp
Authors: Schwaber Mitchell J. , Navon-Venezia Shiri , Chmelnitsky Inna , Leavitt Azita , Schwartz David , Carmeli Yehuda
Published: January 2006

Forty clinical isolates of
Enterobacter
spp. were identified as extended-spectrum β-lactamase (ESBL) producers by disk diffusion. The VITEK 2 Advanced Expert System (AES) identified the ESBL phenotype in only 25 isolates (62.5%), and erroneously reported cephalosporin susceptibility in 11 isolates (28%). Refinements in the AES are required in order to improve ESBL detection in
Enterobacter
.
Diagnostic Oligonucleotide Microarray Fingerprinting of
Bacillus
Isolates
Authors: Chandler Darrell P. , Alferov Oleg , Chernov Boris , Daly Don S. , Golova Julia , Perov Alexander , Protic Miroslava , Robison Richard , Schipma Matthew , White Amanda , Willse Alan
Published: January 2006

A genome-independent microarray and new statistical techniques were used to genotype
Bacillus
strains and quantitatively compare DNA fingerprints with the known taxonomy of the genus. A synthetic DNA standard was used to understand process level variability and lead to recommended standard operating procedures for microbial forensics and clinical diagnostics.
Rapid, Noninvasive Diagnosis of Visceral Leishmaniasis in India: Comparison of Two Immunochromatographic Strip Tests for Detection of Anti-K39 Antibody
Authors: Sundar Shyam , Maurya Radheshyam , Singh Rakesh K. , Bharti K. , Chakravarty Jaya , Parekh Ashish , Rai Madhukar , Kumar Kailash , Murray Henry W.
Published: January 2006

Used with blood or serum, a new anti-K39 antibody immunochromatographic strip test (IT-Leish; DiaMed AG) proved sensitive (range, 99 to 100%) and specific (range, 95 to 100%) for the noninvasive serodiagnosis of visceral leishmaniasis in India. Used with serum, the IT-Leish test and the existing Kalazar Detect test (InBios International, Inc.) yielded comparable results for symptomatic infection and identified apparent subclinical infection in 15 to 32% of healthy residents in a region where visceral leishmaniasis is highly endemic.
Safe
Mycobacterium tuberculosis
DNA Extraction Method That Does Not Compromise Integrity
Authors: Warren Robin , de Kock Marianna , Engelke Erica , Myburgh Roseline , Gey van Pittius Nico , Victor Thomas , van Helden Paul
Published: January 2006

Concern has been raised about the efficacy of the heat killing of mycobacteria during the isolation of DNA. We demonstrate a method that allows for the efficient killing of
Mycobacterium tuberculosis
without compromising DNA integrity for subsequent molecular investigation. This method reduces the risk of infection to the research scientist.
Utility of Extended Blood Culture Incubation for Isolation of
Haemophilus, Actinobacillus, Cardiobacterium, Eikenella
, and
Kingella
Organisms: a Retrospective Multicenter Evaluation
Authors: Petti Cathy A. , Bhally Hasan S. , Weinstein Melvin P. , Joho Kim , Wakefield Teresa , Reller L. Barth , Carroll Karen C.
Published: January 2006

The incidence of and average time to detection for
Haemophilus, Actinobacillus, Cardiobacterium, Eikenella
, and
Kingella
(HACEK) bacteria in blood cultures with standard incubation and the utility of extended incubation of blood culture bottles were reviewed at four tertiary care microbiology laboratories. HACEK organisms were isolated from 35 (<0.005%) of 59,203 positive blood cultures. None of 407 blood cultures with extended incubation grew HACEK or other bacteria. Bacteremia from HACEK bacteria is rare, and extended incubation of blood cultures to recover HACEK bacteria is unnecessary.
Multiple-Locus Variable-Number Tandem Repeat Analysis for Typing of
Staphylococcus epidermidis
Authors: Johansson Anders , Koskiniemi Satu , Gottfridsson Per , Wiström Johan , Monsen Tor
Published: January 2006

We applied a high-resolution PCR-based typing method, multiple-locus variable-number tandem repeat analysis (MLVA), for discrimination of 30 multidrug-resistant clinical isolates of
Staphylococcus epidermidis
. The results of MLVA were congruent with results obtained by pulsed-field gel electrophoresis (PFGE). MLVA generated discrete character data, and its discriminatory capacity was comparable to that of PFGE.
Prevalence and Evolution of Methicillin-Resistant
Staphylococcus aureus
in Spanish Hospitals between 1996 and 2002
Authors: Vindel A. , Trincado P. , Gómez E. , Cabrera R. , Boquete T. , Solá C. , Valdezate S. , Saez-Nieto J. A.
Published: January 2006

Pulsed-field gel electrophoretic analysis of 2,144 methicillin-resistant
Staphylococcus aureus
(MRSA) strains isolated from patients in Spanish hospitals over a 7-year period revealed 17 predominant profiles. Typing showed the replacement of Iberian clone E1 (ST247-MRSA-I) by two prevalent clones, E7 and E8, that are closely related to each other and have the same genetic background as ST125-MRSA-IV.
Rapid Identification ofMycobacterium tuberculosisBeijing Genotypes on the Basis of the Mycobacterial Interspersed Repetitive Unit Locus 26 Signature
Authors: Rao K. Rajender , Ahmed Niyaz , Srinivas Sriramula , Sechi Leonardo A. , Hasnain Seyed E.
Published: January 2006

Mycobacterium tuberculosisBeijing strains are prevalent in many parts of the world and often give rise to large institutional outbreaks. Such highly transmissible strains, often associated with multidrug resistance, are likely underrepresented in outbreaks reported from developing countries, mainly due to nonavailability of fast detection methods suitable in epidemiological surveillance studies. We evaluated a PCR assay based on amplification of mycobacterial interspersed repetitive unit locus 26 as a stand-alone method for unambiguous identification of Beijing strains. The method was used on blinded samples from 10 standard strains whose Beijing status was already confirmed by spoligotyping. All 10 strains were accurately identified, and their profiles were corroborated successfully with spoligotypes. The method was also applied to 70 different non-Beijing clinical isolates from different countries to allow discrimination of isolates. Owing to its accuracy, simplicity, and rapidity, the assay can be employed in laboratory-level testing of isolates linked to certain outbreaks. The test can also be adopted for direct application on clinical samples to save time on culturing bacilli for genotyping.
First Isolation of
Bartonella alsatica
from a Valve of a Patient with Endocarditis
Authors: Raoult Didier , Roblot France , Rolain Jean-Marc , Besnier Jean-Marc , Loulergue Joëlle , Bastides Frédéric , Choutet Patrick
Published: January 2006

We report the first documented case of endocarditis in a man infected with
Bartonella alsatica
, which causes bacteremia in healthy wild rabbits.
B. alsatica
was identified by serology and culture and by PCR of an aortic valve specimen.
B. alsatica
should be added to the list of zoonotic agents of blood culture-negative endocarditis.
Nocardia cyriacigeorgica
Septicemia
Authors: Elsayed Sameer , Kealey Angela , Coffin Carla S. , Read Ron , Megran David , Zhang Kunyan
Published: January 2006

We report two cases of
Nocardia cyriacigeorgica
septicemia and disseminated infection in the setting of profound immunodeficiency. In both instances, diagnosis was rapidly facilitated by 16S rRNA gene sequencing of blood culture isolates. These constitute the first confirmed reports of
Nocardia cyriacigeorgica
bloodstream infection in humans.
Specimens from a Vesicular Lesion Caused by Molluscum Contagiosum Virus Produced a Cytopathic Effect in Cell Culture That Mimicked That Produced by Herpes Simplex Virus
Authors: Bell Constance A. , Eberly Allison P. , Takata Geraldine , Combs Raelene K. , Deweese Nathan E. , Whelen A. Christian
Published: January 2006

Infection with molluscum contagiosum virus, a poxvirus, normally has a typical clinical presentation; therefore, laboratory confirmation is infrequently sought and the virus is rarely isolated in culture. As reported herein, viral culture of specimens from atypical lesions may produce an abortive infection in limited cell lines and a cytopathic effect suggestive of herpes simplex virus.
Multilocus Sequence Typing of
Porphyromonas gingivalis
Strains from Different Geographic Origins
Authors: Enersen Morten , Olsen Ingar , van Winkelhoff Arie J. , Caugant Dominique A.
Published: January 2006

Porphyromonas gingivalis
is an important periodontal pathogen that can be isolated from both active and inactive periodontal lesions. Apparently, differences in virulence between
P. gingivalis
strains exist, but the mechanisms underlying these differences are not yet fully understood. To obtain more information about pathogenicity and virulence of
P. gingivalis
, it is relevant to assess the genetic population structure of the species and to examine the occurrence of putative virulence factors against the genetic background. Presently, multilocus sequence typing (MLST) is the best method for analyzing bacterial population structures. Forty
P. gingivalis
strains from worldwide sources were analyzed by MLST. Internal 310- to 420-bp DNA fragments of the eight ubiquitous chromosomal genes,
ftsQ, hagB, gdpxJ, pepO, mcmA, recA, pga
, and
nah
, were amplified by PCR and then sequenced. The number of alleles at individual loci ranged from 2 to 19, and a total of 33 allelic profiles, or sequence types (STs), were identified. Nucleotide variation between alleles was located at one or a few sites. Identical or similar STs were found in isolates from different geographic regions. Our results showed signs of a clonal population structure with a level of recombination not as high as that previously suggested for the species. We also found that
P. gingivalis
isolates from individual patients were genetically heterogeneous.
Effects of Cow Age and Pregnancy on
Bartonella
Infection in a Herd of Dairy Cattle
Authors: Maillard R. , Grimard B. , Chastant-Maillard S. , Chomel B. , Delcroix T. , Gandoin C. , Bouillin C. , Halos L. , Vayssier-Taussat M. , Boulouis H.-J.
Published: January 2006

Bartonella
spp. are small hemotropic bacteria infecting mammals. Four
Bartonella
species have been recently described in cattle and wild ruminants. To date, the biology and possible pathogenic role of
Bartonella
species isolated from ruminants are poorly understood. Therefore, a dairy herd of 448 cows and heifers was surveyed in order to establish the prevalence of
Bartonella bovis
and
B. chomelii
infections, the level of bacteremia, and the relationship between bacteremia and age or pregnancy status. The putative impact of
Bartonella
infection on production performance (individual milk cell count, milk yield) and reproductive status (success of artificial insemination [AI], placental retention, embryonic death, and abortion) was also assessed. The overall mean prevalence of
B. bovis
bacteremia was 59%, with the highest prevalence in heifers (92.5%). No
B. chomelii
was isolated, and 95% (114/120) of the
B. bovis
strains isolated and tested by PCR-restriction fragment length polymorphism belonged to type I. The level of bacteremia was higher in pregnant cows than in nonpregnant cows (
P
= 0.05), and the level of bacteremia rose during the last two-thirds of gestation (
P
< 0.001). There was no correlation between bacteremia and milk yield, individual milk cell count, success of first AI, interval between two calvings, or incidence of abortion and embryonic death. The interval from calving to first AI was shorter and the incidence of placental retention was lower in bacteremic animals than in nonbacteremic ones (
P
= 0.03 and
P
= 0.01, respectively).
Positive Correlation between Epstein-Barr Virus Viral Load and Anti-Viral Capsid Immunoglobulin G Titers Determined for Hodgkin's Lymphoma Patients and Their Relatives
Authors: Besson Caroline , Amiel Corinne , Le-Pendeven Catherine , Brice Pauline , Fermé Christophe , Carde Patrice , Hermine Olivier , Raphael Martine , Abel Laurent , Nicolas Jean-Claude
Published: January 2006

Markers of Epstein-Barr virus (EBV) infection include measures of specific serological titers and of viral load (VLo) in peripheral blood mononuclear cells. Few studies have investigated the correlation between these two phenotypes. Here, we found that there was no correlation between VLo and either anti-EBV nuclear antigen type 1 or anti-early antigen immunoglobulin G (IgG) titer but that anti-viral capsid antigen (VCA) IgG titer increased with VLo in peripheral blood mononuclear cells in patients with Hodgkin's lymphoma (P= 3.10−3). A similar pattern was observed in healthy first-degree relatives (parents and siblings) of patients (P= 6.10−4). Our results indicate that anti-VCA IgG titers and EBV VLo are specifically correlated EBV phenotypes.
Association between Preterm Birth and Vaginal Colonization by Mycoplasmas in Early Pregnancy
Authors: Kataoka Soromon , Yamada Takashi , Chou Kazutoshi , Nishida Ryutaro , Morikawa Mamoru , Minami Mashiho , Yamada Hideto , Sakuragi Noriaki , Minakami Hisanori
Published: January 2006

To examine the association between colonization by two newly classified species of genital ureaplasmas (
Ureaplasma parvum
and
U. urealyticum
) in early pregnancy and subsequent late abortion or preterm birth at <34 weeks of gestation, four species of genital mycoplasmas—
Mycoplasma genitalium, M. hominis, U. parvum
, and
U. urealyticum—
as well as
Chlamydia trachomatis
and
Neisseria gonorrhoeae
were examined by PCR-based methods in a prospective cohort study of 877 women with singleton pregnancies at <11 weeks of gestation. Antibiotics were used only in cases in which
C. trachomatis
and/or
N. gonorrhoeae
was detected. Multivariate logistic-regression analysis was used to assess independent risk factors after taking maternal low body weight and past history of preterm birth into account.
M. genitalium, M. hominis, U. parvum, U. urealyticum, C. trachomatis
, and
N. gonorrhoeae
were detected in 0.8%, 11.2%, 52.0%, 8.7%, 3.2%, and 0.1% of these 877 women, respectively. Twenty-one (2.4%) women experienced late abortion or preterm birth at <34 weeks of gestation. Three factors—detection of
U. parvum
in the vagina (odds ratio [OR], 3.0; 95% confidence interval [CI], 1.1 to 8.5); use of antibiotics, such as penicillin and cefatrizine, for incidental inflammatory complications before 22 weeks of gestation (OR, 4.2; 95% CI, 1.6 to 10.0); and past history of preterm birth (OR, 10.4; 95% CI, 2.7 to 40.5)—were independently associated with late abortion and preterm birth. In conclusion, vaginal colonization with
U. parvum
, but not
U. urealyticum
, is associated with late abortion or early preterm birth.
Legionella anisa
, a Possible Indicator of Water Contamination by
Legionella pneumophila
Authors: van der Mee-Marquet Nathalie , Domelier Anne-Sophie , Arnault Laurence , Bloc Daniel , Laudat Patrice , Hartemann Philippe , Quentin Roland
Published: January 2006

Legionella anisa
is one of the most frequent species of
Legionella
other than
Legionella pneumophila
in the environment and may be hospital acquired in rare cases. We found that
L. anisa
may mask water contamination by
L. pneumophila
, suggesting that there is a risk of
L. pneumophila
infection in immunocompromised patients if water is found to be contaminated with
Legionella
species other than
L. pneumophila
.
Direct Identification of Mycobacteria in Primary Liquid Detection Media by Partial Sequencing of the 65-Kilodalton Heat Shock Protein Gene
Authors: McNabb Alan , Adie Kathy , Rodrigues Mabel , Black William A. , Isaac-Renton Judith
Published: January 2006

We investigated extending the use of direct partial
hsp65
gene sequencing for the identification of mycobacteria to isolates in primary liquid detection media as an economical, feasible, and more rapid means of identification. During the course of the study, the
hsp65
sequence-based identifications for isolates from 670 primary liquid detection media determined to be positive for acid-fast bacilli were compared to the identifications derived from Accuprobes, biochemical test panels, or 16S rRNA gene sequencing. Preliminary analysis indicated a 97.6% concordance, with a final agreement of 99.1% between the identification algorithms.
hsp65
sequencing costs (US$32.84) were greater than the cost of identification with Accuprobe (US$19) but less than the cost of the biochemical test panel identification (average cost, US$98.90) and equivalent to the cost of 16S rRNA sequencing, although there was a referral cost (US$59.85) for the shipping of isolates to another reference laboratory. Analysis indicated that our laboratory would have recognized a cost savings of approximately $12,000 by using
hsp65
sequencing to identify isolates from specimens with a negative fluorescent- smear status and would have achieved further savings by using it as an alternative to biochemical panel testing for fluorescent-smear-positive specimens. The time to identification by
hsp65
gene sequencing was slightly longer than that required by the Accuprobe assay (1 versus 2 days), shorter than that required by the biochemical test panels (2 days versus 26 days on average), and more rapid than referral for 16S rRNA gene sequencing.
Armored RNA as Virus Surrogate in a Real-Time Reverse Transcriptase PCR Assay Proficiency Panel
Authors: Hietala S. K. , Crossley B. M.
Published: January 2006

In recent years testing responsibilities for high-consequence pathogens have been expanded from national reference laboratories into networks of local and regional laboratories in order to support enhanced disease surveillance and to test for surge capacity. This movement of testing of select agents and high-consequence pathogens beyond reference laboratories introduces a critical need for standardized, noninfectious surrogates of disease agents for use as training and proficiency test samples. In this study, reverse transcription-PCR assay RNA targets were developed and packaged as armored RNA for use as a noninfectious, quantifiable synthetic substitute for four high-consequence animal pathogens: classical swine fever virus; foot-and-mouth disease virus; vesicular stomatitis virus, New Jersey serogroup; and vesicular stomatitis virus, Indiana serogroup. Armored RNA spiked into oral swab fluid specimens mimicked virus-positive clinical material through all stages of the reverse transcription-PCR testing process, including RNA recovery by four different commercial extraction procedures, reverse transcription, PCR amplification, and real-time detection at target concentrations consistent with the dynamic ranges of the existing real-time PCR assays. The armored RNA concentrations spiked into the oral swab fluid specimens were stable under storage conditions selected to approximate the extremes of time and temperature expected for shipping and handling of proficiency panel samples, including 24 h at 37°C and 2 weeks at temperatures ranging from ambient room temperature to −70°C. The analytic test performance, including the reproducibility over the dynamic range of the assays, indicates that armored RNA can provide a noninfectious, quantifiable, and stable virus surrogate for specific assay training and proficiency test purposes.
Validation of VITEK 2 Version 4.01 Software for Detection, Identification, and Classification of Glycopeptide-Resistant Enterococci
Authors: Abele-Horn Marianne , Hommers Leif , Trabold René , Frosch Matthias
Published: January 2006

We evaluated the ability of the new VITEK 2 version 4.01 software to identify and detect glycopeptide-resistant enterococci compared to that of the reference broth microdilution method and to classify them into the
vanA, vanB, vanC1
, and
vanC2
genotypes. Moreover, the accuracy of antimicrobial susceptibility testing with agents with improved potencies against glycopeptide-resistant enterococci was determined. A total of 121 enterococci were investigated. The new VITEK 2 software was able to identify 114 (94.2%) enterococcal strains correctly to the species level and to classify 119 (98.3%) enterococci correctly to the glycopeptide resistance genotype level. One
Enterococcus casseliflavus
strain and six
Enterococcus faecium vanA
strains with low-level resistance to vancomycin were identified with low discrimination, requiring additional tests. One of the
vanA
strains was misclassified as the
vanB
type, and one glycopeptide-susceptible
E. facium
wild type was misclassified as the
vanA
type. The overall essential agreements for antimicrobial susceptibility testing results were 94.2% for vancomycin, 95.9% for teicoplanin, 100% for quinupristin-dalfopristin and moxifloxacin, and 97.5% for linezolid. The rates of minor errors were 9% for teicoplanin and 5% for the other antibiotic agents. The identification and susceptibility data were produced within 4 h to 6 h 30 min and 8 h 15 min to 12 h 15 min. In conclusion, use of VITEK 2 version 4.01 software appears to be a reliable method for the identification and detection of glycopeptide-resistant enterococci as well as an improvement over the use of the former VITEK 2 database. However, a significant reduction in the detection time would be desirable.
Internal Transcribed Spacer Sequencing versus Biochemical Profiling for Identification of Medically Important Yeasts
Authors: Ciardo D. E. , Schär G. , Böttger E. C. , Altwegg M. , Bosshard P. P.
Published: January 2006

In this study, we established an in-house database of yeast internal transcribed spacer (ITS) sequences. This database includes medically important as well as colonizing yeasts that frequently occur in the diagnostic laboratory. In a prospective study, we compared molecular identification with phenotypic identification by using the ID32C system (bioMérieux) for yeast strains that could not be identified by a combination of CHROMagar Candida and morphology on rice agar. In total, 113 yeast strains were included in the study. By sequence analysis, 98% of all strains were identified correctly to the species level. With the ID32C, 87% of all strains were identified correctly to the species or genus level, 7% of the isolates could not be identified, and 6% of the isolates were misidentified, most of them as
Candida rugosa
or
Candida utilis
. For a diagnostic algorithm, we suggest a three-step procedure which integrates morphological criteria, biochemical investigation, and sequence analysis of the ITS region.
Absence of
Spiroplasma
or Other Bacterial 16S rRNA Genes in Brain Tissue of Hamsters with Scrapie
Authors: Alexeeva Irina , Elliott Ellen J. , Rollins Sandra , Gasparich Gail E. , Lazar Jozef , Rohwer Robert G.
Published: January 2006

Spiroplasma
spp. have been proposed to be the etiological agents of the transmissible spongiform encephalopathies (TSEs). In a blind study, a panel of 20 DNA samples was prepared from the brains of uninfected hamsters or hamsters infected with the 263K strain of scrapie. The brains of the infected hamsters contained ≥10
10
infectious doses/g. The coded panel was searched for bacterial 16S rRNA gene sequences, using primers selective for spiroplasma sequences, primers selective for mollicutes in general, and universal bacterial primers. After 35 PCR cycles, no samples were positive for spiroplasma or any other bacterial DNA, while control
Spiroplasma mirum
genomic DNA, spiked at 1% of the concentration required to account for the scrapie infectivity present, was readily detected. After 70 PCR cycles, nearly all samples yielded amplified products which were homologous to various bacterial 16S rRNA gene sequences, including those of frequent environmental contaminants. These sequences were seen in uninfected as well as infected samples. Because the concentration of scrapie infectivity was at a known high level, it is very unlikely that a bacterial infection at the same concentration could have escaped detection. We conclude that the infectious agent responsible for TSE disease cannot be a spiroplasma or any other eubacterial species.