bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of PCR with the Routine Procedure for Diagnosis of Tuberculosis in a Population with High Prevalences of Tuberculosis and Human Immunodeficiency Virus
Authors: Kivihya-Ndugga Lydia , van Cleeff Maarten , Juma Ernest , Kimwomi Joseph , Githui Willie , Oskam Linda , Schuitema Anja , van Soolingen Dick , Nganga Lucy , Kibuga Daniel , Odhiambo Joseph , Klatser Paul
Published: March 2004

Direct smear examination with Ziehl-Neelsen (ZN) staining for the diagnosis of tuberculosis (TB) as employed in most low-income countries is cheap and easy to use, but its low sensitivity is a major drawback. The low specificity of chest X-rays, used for the diagnosis of smear-negative TB, risks high levels of overdiagnosis. Major advances in molecular techniques, which rapidly identify mycobacterial DNA in sputa, may overcome these obstacles. In this study, the AMPLICOR PCR system was used to diagnose pulmonary TB in a developing country with high prevalences of both TB and human immunodeficiency virus (HIV). The sensitivity and specificity of this technique were compared to those of the usual diagnostic techniques. Sputum specimens were collected from 1,396 TB suspects attending the Rhodes Chest Clinic, Nairobi, Kenya. The specimens were analyzed for the presence of
Mycobacterium tuberculosis
by PCR; culture on Löwenstein-Jensen medium was used as the “gold standard.” All culture-positive samples were genotyped to identify the mycobacterial species. The sensitivity and specificity of PCR were 93 and 84%, respectively. HIV status did not affect the sensitivity of PCR. A total of 99.7% of the true smear-positive and 82.1% of the true smear-negative TB patients were correctly identified by PCR. PCR detected
M. tuberculosis
in 11.7% of the culture-negative suspects, 60% of which had one or two PCR-positive sputum specimens. Of the 490 positive cultures, 486 were identified as
M. tuberculosis
. The high sensitivity of Amplicor PCR merits usage in a clinical setting with high TB and HIV burdens. Thus, PCR can be considered as an alternative to ZN staining in combination with chest X-ray for diagnosis of TB; however, cost-effectiveness studies and operational studies are required to support an evidence-based decision of introducing PCR for TB control in high-burden environments.
Temperature-Mediated Heteroduplex Analysis for Detection of
pncA
Mutations Associated with Pyrazinamide Resistance and Differentiation between
Mycobacterium tuberculosis
and
Mycobacterium bovis
by Denaturing High- Performance Liquid Chromatography
Authors: Mohamed Amr M. , Bastola Dhundy R. , Morlock Glenn P. , Cooksey Robert C. , Hinrichs Steven H.
Published: March 2004

The goal of this study was to apply temperature-mediated heteroduplex analysis using denaturing high-performance liquid chromatography to identify pyrazinamide (PZA) resistance in
Mycobacterium tuberculosis
isolates and simultaneously differentiate between
M. tuberculosis
and
Mycobacterium bovis
. Features that contributed to an optimal assay included the use of two different reference probes for the
pncA
gene targets from wild-type
M. tuberculosis
and wild-type
M. bovis
, optimization of the column temperature, increasing the starting concentration of the elution buffer, and reducing the rate of elution buffer increase (slope). A total of 69 strains were studied, including 48 wild-type
M. tuberculosis
strains (13 were PZA-resistant strains) and 21
M. bovis
strains (8 were BCG strains). In all isolates tested, wild-type
M. tuberculosis
generated a single-peak pattern when mixed with the
M. tuberculosis
probe and a double-peak pattern with the
M. bovis
probe. In contrast, all
M. bovis
isolates generated a double-peak pattern when mixed with the
M. tuberculosis
probe and a single-peak pattern with the
M. bovis
probe. PZA-resistant mutant
M. tuberculosis
isolates generated characteristic patterns that were easily distinguishable from both wild-type
M. tuberculosis
and
M. bovis
isolates. Chromatographic patterns generated by the two reference probes allowed the rapid detection of PZA resistance with the simultaneous ability to distinguish between
M. tuberculosis
and
M. bovis
. This approach may allow the detection of drug resistance-associated mutations, with potential application to clinical and epidemiological aspects of tuberculosis control.
Human β-Defensins 2 and 3 Demonstrate Strain-Selective Activity against Oral Microorganisms
Authors: Joly Sophie , Maze Connie , McCray Paul B. , Guthmiller Janet M.
Published: March 2004

Human β-defensins 2 and 3 (HBD-2 and HBD-3) are inducible peptides present at sites of infection in the oral cavity. A few studies have reported broad-spectrum antimicrobial activity for both peptides. However, no comprehensive study has thoroughly investigated their potential against oral pathogens. The purpose of this study was to test the effectiveness of HBD-2 and HBD-3 against a collection of oral organisms (
Actinobacillus actinomycetemcomitans
,
Fusobacterium nucleatum
,
Porphyromonas gingivalis
,
Peptostreptococcus micros
,
Actinomyces naeslundii
,
Actinomyces israelii
,
Streptococcus sanguis
,
Streptococcus mutans
,
Candida tropicalis
,
Candida parapsilosis
,
Candida krusei
,
Candida glabrata
, and
Candida albicans
). Radial diffusion assays were used to test HBD-2 and HBD-3 activities against at least three strains of each species. There was significant variability in MICs, which was strain specific rather than species specific. MICs ranged from 3.9 to >250 μg/ml for HBD-2 and from 1.4 to >250 μg/ml for HBD-3. HBD-3 demonstrated greater antimicrobial activity and was effective against a broader array of organisms. Overall, aerobes were 100% susceptible to HBD-2 and HBD-3, whereas only 21.4 and 50% of the anaerobes were susceptible to HBD-2 and HBD-3, respectively. HBD-2 and HBD-3 also demonstrated strain-specific activity against the
Candida
species evaluated. Interestingly, an association between HBD-2 and HBD-3 activities was noted. This suggests that the two peptides may have similar mechanisms yet utilize distinct pathways. The lack of activity against specific anaerobic strains and
Candida
warrants further investigation of the potential resistance mechanisms of these organisms. Finally, the significant variability between strains underlies the importance of testing multiple strains when evaluating activities of antimicrobial peptides.
Multicenter Evaluation of the MB/BACT System for Susceptibility Testing of
Mycobacterium tuberculosis
Authors: Bemer Pascale , Bodmer Thomas , Munzinger Juerg , Perrin Monique , Vincent Véronique , Drugeon Henri
Published: March 2004

The reliability of the MB/BACT system for susceptibility testing of
Mycobacterium tuberculosis
to pyrazinamide, rifampin, isoniazid, streptomycin, and ethambutol was compared to the BACTEC 460TB system. The proportion method was used to resolve discrepant results by an independent arbiter. Two interpretative methods were used, with an undiluted control (direct control) and a diluted control (10
−1
control). As no significant difference was observed between the two controls, the method with the direct control was adopted as the most accurate one. One hundred sixty-six strains were tested, with an overall agreement of 98.3%. After resolution of the 18 discrepant results by the proportion method, the sensitivity and specificity of the MB/BACT system were 100% for rifampin, isoniazid, and pyrazinamide. For ethambutol, sensitivity was 92.3% at the critical concentration and 33% at the high concentration, and specificity was 100% at both concentrations. For streptomycin, sensitivity was 100% at the critical concentration and 80% at the high concentration, and specificity was 98.6% at the critical concentration and 100% at the high concentration. The rifampin, isoniazid, streptomycin, and ethambutol susceptibility test results were obtained in 6.6 days with the MB/BACT versus 5 days with the BACTEC 460TB. The pyrazinamide susceptibility test results were obtained in 7.8 days with the MB/BACT, versus 6.7 days with the BACTEC 460TB. These data demonstrate that the fully automated MB/BACT system is a very reliable method for rapid susceptibility testing of
M. tuberculosis
against rifampin, isoniazid, and pyrazinamide. Sensitivity results have to be improved for ethambutol and streptomycin, especially at the high concentration.
Rapid Quantification ofYersinia enterocoliticain Pork Samples by a Novel Sample Preparation Method, Flotation, Prior to Real-Time PCR
Authors: Wolffs Petra , Knutsson Rickard , Norling Börje , Rådström Peter
Published: March 2004

The development of real-time PCR thermal cycles in the late 1990s has opened up the possibility of accurate quantification of microorganisms in clinical, environmental, and food samples. However, a lack of suitable sample preparation methods that allow rapid quantification of the nucleic acids, remove PCR inhibitors, and prevent false-positive results due to DNA originating from dead cells has limited the use of quantitative PCR. We have used for the first time a new variant of density gradient centrifugation, called flotation, as a user-friendly sample preparation method prior to PCR. This paper describes the use of this sample preparation method, without DNA purification, for direct detection and quantification ofYersinia enterocoliticain PCR-inhibitory meat juice from pork. Flotation combined with qPCR could overcome PCR interference in juice from pork, as was shown by amplification efficiencies of 1.006 ± 0.021 and 1.007 ± 0.025, which are comparable to the amplification efficiency obtained for purified DNA samples (1.005 ± 0.059). Applying flotation to meat juice samples containing natural background flora and spiked with different levels ofY. enterocoliticashowed that direct quantification ofY. enterocoliticawas possible down to a level of at least 4.2 × 103CFU per ml of meat juice, even in the presence of 106CFU of background flora per ml. Finally, the results showed that samples containing large amounts ofY. enterocoliticaDNA did not result in a positive PCR signal. This indicates that the risk of false-positive results due to detection of DNA originating from dead cells can be greatly reduced by using flotation prior to PCR.
Enteroaggregative
Escherichia coli
Virulence Factors Are Found To Be Associated with Infantile Diarrhea in Brazil
Authors: Zamboni Andresa , Fabbricotti Sandra H. , Fagundes-Neto Ulysses , Scaletsky Isabel C.A.
Published: March 2004

We have previously shown that enteroaggregative
Escherichia coli
(EAEC) is an important pathogen among Brazilian infants. Most EAEC strains harbor a plasmid (pAA) from which a DNA fragment has been used as a probe (EAEC probe). To better understand the characteristics of EAEC in Brazil, 109 strains carrying and lacking the EAEC probe sequence were tested for the presence of pAA plasmid-borne and chromosomal factors. Common virulence factors of probe-positive and probe-negative isolates included the presence of the Pet, EAST-1, Shf, Irp2, ShET1/Pic, and Hly virulence markers. The presence of AggR or one other virulence factor (AAF/I, AAF/II, AAF/III, or Aap) was predominantly identified only in probe-positive strains. In EAEC probe-positive strains, the virulence marker Aap was found significantly more frequently (
P
= 0.023) in isolates from children with diarrhea (22%) than in isolates from controls (3%). EAST-1 and Shf were the markers most frequently detected (61%) in EAEC probe-negative strains and were found to be significantly associated with diarrhea (
P
= 0.003 and
P
= 0.020, respectively). Furthermore, our data suggest that AggR can be used as an important genetic marker for EAEC probe-positive strains.
Expanded Geographical Distribution of the N Family of
Mycobacterium tuberculosis
Strains within the United States
Authors: Milan S. Joy , Hauge Kirsten A. , Kurepina Natalia E. , Lofy Kathryn H. , Goldberg Stefan V. , Narita Masahiro , Nolan Charles M. , McElroy Peter D. , Kreiswirth Barry N. , Cangelosi Gerard A.
Published: March 2004

The N and W-Beijing families of
Mycobacterium tuberculosis
are phylogenetically closely related. The ability of the W-Beijing family to rapidly cause widespread disease is well described; however, few outbreaks involving the N family have been reported outside the New York City, N.Y., area. During 2002 to 2003, Seattle, Wash., experienced a rapidly expanding tuberculosis outbreak involving 38 persons in a 23-month period. The outbreak strain, SBRI9, exhibited the genotypic properties of the N family. Its IS
6110
restriction fragment length polymorphism pattern was identical or nearly identical to those of two N family strains that were responsible for clusters of tuberculosis cases, including a large nosocomial outbreak, in New York City and New Jersey from 1989 to 1990. It was also identical to strains involved in late 1990s tuberculosis cases in Michigan, Maryland, and Arkansas. Further monitoring of the N family may show that it shares with the W-Beijing family the propensity to spread rapidly, suggesting that this characteristic evolved prior to the divergence of the two genetic lineages.
Rapid and Sensitive Detection ofMycobacterium aviumsubsp.paratuberculosisin Bovine Milk and Feces by a Combination of Immunomagnetic Bead Separation-Conventional PCR and Real-Time PCR
Authors: Khare Sangeeta , Ficht Thomas A. , Santos Renato L. , Romano Juan , Ficht Allison R. , Zhang Shuping , Grant Irene R. , Libal Melissa , Hunter David , Adams L. Garry
Published: March 2004

Immunomagnetic bead separation coupled with bead beating and real-time PCR was found to be a very effective procedure for the isolation, separation, and detection ofMycobacterium aviumsubsp.paratuberculosisfrom milk and/or fecal samples from cattle and American bison. Samples were spiked withM. aviumsubsp.paratuberculosisorganisms, which bound to immunomagnetic beads and were subsequently lysed by bead beating; then protein and cellular contaminants were removed by phenol-chloroform-isopropanol extraction prior to DNA precipitation. DNA purified by this sequence of procedures was then analyzed by conventional and real-time IS900-based PCR in order to detectM. aviumsubsp.paratuberculosisin feces and milk. By use of this simple and rapid technique, 10 or fewerM. aviumsubsp.paratuberculosisorganisms were consistently detected in milk (2-ml) and fecal (200-mg) samples, making this sensitive procedure very useful and cost-effective for the diagnosis of clinical and subclinical Johne's disease (paratuberculosis) compared to bacteriological culture, which is constrained by time, labor, and expense under diagnostic laboratory conditions.
Development of an Equine Herpesvirus Type 4-Specific Enzyme-Linked Immunosorbent Assay Using a B-Cell Epitope as an Antigen
Authors: Maeda Ken , Mizukoshi Fuminori , Hamano Masataka , Kai Kazushige , Iwata Hiroyuki , Kondo Takashi , Matsumura Tomio
Published: March 2004

The equine herpesvirus type 4 (EHV-4)-specific region of glycoprotein G has served as an antigen for serodiagnosis and seroepizootic studies of EHV-4 infection (B. S. Crabb and M. J. Studdert, J. Virol.
67:
6332-6338, 1993; S. Yasunaga, K. Maeda, T. Matsumura, K. Kai, H. Iwata, and T. Inoue, J. Vet. Med. Sci.
60:
1133-137, 1998; S. Yasunaga, K. Maeda, T. Matsumura, T. Kondo, and K. Kai, J. Vet. Med. Sci.
62:
687-691, 2000). Here we identified a major B-cell epitope in the type-specific region of EHV-4 and applied it as an antigen in enzyme-linked immunosorbent assays (ELISAs). A 24-amino-acid repeat sequence expressed as a glutathione
S
-transferase fusion protein specifically reacted as well as the type-specific region with sera from foals infected with EHV-4. Five synthetic peptides (12-mer peptides) in the repeat sequence were included as ELISA antigens. The results indicated that the 12-mer peptide MKNNPIYSEGSL contained a major B-cell epitope specific for EHV-4 infection. Inclusion of this 12-mer peptide in ELISAs for an epidemiological study specifically detected EHV-4 infection in the field. These results indicated that the 12-mer epitope was responsible for the type-specific antibody response and therefore is useful for seroepizootic studies and serodiagnosis of EHV-4 infection.
Characterization of Shiga Toxin-Producing
Escherichia coli
Strains Isolated from Human Patients in Germany over a 3-Year Period
Authors: Beutin Lothar , Krause Gladys , Zimmermann Sonja , Kaulfuss Stefan , Gleier Kerstin
Published: March 2004

We have investigated 677 Shiga toxin-producing
Escherichia coli
(STEC) strains from humans to determine their serotypes, virulence genes, and clinical signs in patients. Six different Shiga toxin types (1, 1c, 2, 2c, 2d, and 2e) were distributed in the STEC strains. Intimin (
eae
) genes were present in 62.6% of the strains and subtyped into intimins α1, β1, γ1, ε, θ, and η. Shiga toxin types 1c and 2d were present only in
eae
-negative STEC strains, and type 2 was significantly (
P
< 0.001) more frequent in
eae
-positive STEC strains. Enterohemorrhagic
E. coli
hemolysin was associated with 96.2% of the
eae
-positive strains and with 65.2% of the
eae
-negative strains. Clinical signs in the patients were abdominal pain (8.7%), nonbloody diarrhea (59.2%), bloody diarrhea (14.3%), and hemolytic-uremic syndrome (HUS) (3.5%), and 14.3% of the patients had no signs of gastrointestinal disease or HUS. Infections with
eae
-positive STEC were significantly (
P
< 0.001) more frequent in children under 6 years of age than in other age groups, whereas
eae
-negative STEC infections dominated in adults. The STEC strains were grouped into 74 O:H types by serotyping and by PCR typing of the flagellar (
fliC
) genes in 221 nonmotile STEC strains. Eleven serotypes (O157:[H7], O26:[H11], O103:H2, O91:[H14], O111:[H8], O145:[H28], O128:H2, O113:[H4], O146:H21, O118:H16, and O76:[H19]) accounted for 69% of all STEC strains. We identified 41 STEC strains belonging to 31 serotypes which had not previously been described as human STEC. Twenty-six of these were positive for intimins α1 (one serotype), β1 (eight serotypes), ε (two serotypes), and η (three serotypes). Our study indicates that different types of STEC strains predominate in infant and adult patients and that new types of STEC strains are present among human isolates.
Multiplex PCR Assay for Differentiation ofHelicobacter felis,H. bizzozeronii, andH. salomonis
Authors: Baele M. , Van den Bulck K. , Decostere A. , Vandamme P. , Hänninen M.-L. , Ducatelle R. , Haesebrouck F.
Published: March 2004

Helicobacter felis,Helicobacter bizzozeronii, andHelicobacter salomonisare frequently found in the gastric mucous membrane of dogs and cats. These large spiral organisms are phylogenetically highly related to each other. Their fastidious nature makes it difficult to cultivate them in vitro, hampering traditional identification methods. We describe here a multiplex PCR test based on the tRNA intergenic spacers and on the urease gene, combined with capillary electrophoresis, that allows discrimination of these three species. In combination with previously described 16S ribosomal DNA-based primers specific for the nonculturable “CandidatusHelicobacter suis,” our procedure was shown to be very useful in determining the species identity of “Helicobacter heilmannii”-like organisms observed in human stomachs and will facilitate research concerning their possible zoonotic importance.
Isolation and Characterization of
Borrelia hermsii
Associated with Two Foci of Tick-Borne Relapsing Fever in California
Authors: Fritz Curtis L. , Bronson Lawrence R. , Smith Charles R. , Schriefer Martin E. , Tucker James R. , Schwan Tom G.
Published: March 2004

Relapsing fever, caused by the spirochete
Borrelia hermsii
and transmitted by the soft tick
Ornithodoros hermsi
, is endemic in many rural mountainous areas of California. Between 1996 and 1998, 12 cases of relapsing fever associated with two exposure sites in northern California were investigated. Follow-up at exposure sites included collection of soft ticks and serum specimens from sylvatic rodents. Attempts to cultivate spirochetes were made through inoculation of patient blood into mice and by feeding
Ornithodoros
ticks on mice. Three isolates of
B. hermsii
were recovered from two blood specimens and one pool of ticks. The protein and plasmid profiles of the three isolates were comparable to those of previous
B. hermsii
isolates from the western United States. Western immunoblotting of patient sera demonstrated an expanding immunologic response to antigens within four distinct molecular weight regions by 3 to 4 weeks postonset. Antibody to
B. hermsii
was detected in sera from 4 of 11 yellow-pine chipmunks (
Tamias amoenus
); no other rodent species collected were seropositive.
Rapid Serodiagnosis of Active Pulmonary
Mycobacterium tuberculosis
by Analysis of Results from Multiple Antigen-Specific Tests
Authors: Okuda Yoshinari , Maekura Ryoji , Hirotani Atsusi , Kitada Seigo , Yoshimura Kenji , Hiraga Touru , Yamamoto Yuoko , Itou Masami , Ogura Takeshi , Ogihara Toshio
Published: March 2004

We have prospectively analyzed three antigens for serodiagnosis of tuberculosis (TB). These antigens were tuberculous glycolipid antigen, lypoarabinomannan polysaccharide antigen, and antigen 60 (A60), which was derived from purified protein derivatives. Of the 131 patients with active pulmonary TB, 57 were both smear and culture negative and 14 had chronic active pulmonary TB that remained smear positive for >12 months of chemotherapy. One hundred twenty healthy adults were controls. The percentages of patients positive in all three tests were 58.8% for smear-positive active pulmonary TB and 71.4% for chronic active pulmonary TB. When the results of the three serodiagnostic tests were evaluated in combination, the sensitivity increased to 91.5% in patients with active pulmonary TB and to 86.0% in smear- and culture-negative patients. The false-positive rate of the three-test combination was 12.5% in the healthy control groups. In conclusion, it was not possible to detect all of the antibodies against antigenic substances in the cell walls of the tuberculous bacilli in the sera of all TB patients by using available serodiagnostic tests. However, the combined use of tests with three separate antigens maximizes the effectiveness of serodiagnosis.
Optimization of Quantitative Detection of Cytomegalovirus DNA in Plasma by Real-Time PCR
Authors: Boeckh Michael , Huang MeeiLi , Ferrenberg James , Stevens-Ayers Terry , Stensland Laurence , Nichols W. Garrett , Corey Lawrence
Published: March 2004

Previous studies have shown that detection of cytomegalovirus (CMV) DNA in plasma is less sensitive than the antigenemia assay for CMV surveillance in blood. In 1,983 blood samples, plasma PCR assays with three different primer sets (UL125 alone, UL126 alone, and UL55/UL123-exon 4) were compared to the pp65 antigenemia assay and blood cultures. Plasma PCR detected CMV more frequently in blood specimens than either the antigenemia assay or cultures, but of the three PCR assays, the double-primer assay (UL55/UL123-exon 4) performed best with regard to sensitivity, specificity, and predictive values compared to antigenemia: 122 of 151 antigenemia-positive samples were detected (sensitivity, 80.1%), and there were 122 samples that were PCR positive-antigenemia negative (specificity, 93%). Samples with discrepant results had a low viral load (median, 0.5 cells per slide; 1,150 copies per ml) and were often obtained from patients receiving antiviral therapy. CMV could be detected by other methods in 15 of 29 antigenemia positive-PCR negative samples compared to 121 of 122 PCR positive-antigenemia negative samples (
P
< 0.001). On a per-subject basis, 21 of 25 patients (antigenemia positive-PCR negative) and all 57 (PCR positive-antigenemia negative) could be confirmed at different time points during follow-up. The higher sensitivity of the double-primer assay resulted in earlier detection compared to antigenemia in a time-to-event analysis of 42 CMV-seropositive stem cell transplant recipients, and two of three patients with CMV disease who were antigenemia negative were detected by plasma PCR prior to the onset of disease. Interassay variability was low, and the dynamic range was >5 log
10
. Automated DNA extraction resulted in high reproducibility, accurate CMV quantitation (
R
= 0.87,
P
< 0.001), improved sensitivity, and increased speed of sample processing. Thus, primer optimization and improved DNA extraction techniques resulted in a plasma-based PCR assay that is significantly more sensitive than pp65 antigenemia and blood cultures for detection of CMV in blood specimens.
Translocation of
Enterococcus faecalis
Strains across a Monolayer of Polarized Human Enterocyte-Like T84 Cells
Authors: Zeng Jing , Teng Fang , Weinstock George M. , Murray Barbara E.
Published: March 2004

We used a two-chamber system to study transcytosis of
Enterococcus faecalis
across monolayers of human colon carcinoma-derived T84 cells, which show structural resemblance to the native intestine. Among 16
E. faecalis
isolates from different sources, the well-characterized strain OG1RF and 8 other isolates (2 endocarditis isolates, 1 urine isolate, and all 5 fecal isolates) showed translocation in this assay, while 6 clinical isolates (3 endocarditis and 3 urine isolates), the recipient strain JH2-2, and the control,
Escherichia coli
DH5α, had no detectable translocation. Of two OG1RF mutants involving the previously studied
epa
(enterococcal polysaccharide antigen) gene cluster, known to be needed for virulence and resistance to killing by polymorphonuclear leukocytes, one
epa
mutant (TX5179) was unable to translocate, while TX5180, with an
epa
disruption farther downstream, showed a moderate decrease in translocation relative to that of the wild-type strain OG1RF (
P
< 0.01), indicating that the
epa
gene cluster is important for translocation across a T84 monolayer. This observation was confirmed by complementation of the
epa
mutant (TX5179) with
epa
genes and restoration of its translocation ability. In conclusion, we have demonstrated translocation of at least some strains of
E. faecalis
across T84 monolayers, although strains differ considerably in this ability, and we have demonstrated that
epa
mutations can cause marked changes in successful translocation. These results suggest that this model may be a useful in vitro system for studying the process of translocation from the intestinal tract.
Random Amplified Polymorphic DNA and Phenotyping Analysis of
Salmonella enterica
Serovar Enteritidis Isolates Collected from Humans and Poultry in Uruguay from 1995 to 2002
Authors: Betancor Laura , Schelotto Felipe , Martinez Araci , Pereira Marinela , Algorta Gabriela , Rodríguez M. Alejandra , Vignoli Rafael , Chabalgoity José A.
Published: March 2004

Molecular and phenotyping techniques were applied to study
Salmonella enterica
serovar Enteritidis strains both from human cases of infection and of avian origin isolated in Uruguay from 1995 to 2002. A group of 62 isolates was subjected to random amplified polymorphic DNA (RAPD) assay and analysis of antibiotic resistance patterns. Twenty-one of these strains were further characterized by phage typing and analysis of their protein expression profiles. RAPD fingerprinting with five different primers discriminated 10 different genetic profiles. Of the 62 strains tested, 48 had a single major genetic profile, whereas the other nine profiles were evenly distributed among the other strains. The genetic diversity was greater among strains of animal origin than among isolates of human origin. Comparative examination of the results obtained by RAPD analysis and phenotypic analysis and by strain source provided evidence of the reliable discriminatory power of RAPD analysis in our study. Six avian isolates with antibiotic resistance were detected: two were nalidixic acid resistant and four had a particular β-lactam resistance pattern. The last four isolates all had the same unusual phage type (phage type 4b); however, RAPD analysis differentiated them into two groups. Two isolates with unique RAPD profiles were recovered from distinct human cases, suggesting that the technique differentiates unrelated strains. Overall, the results show the existence of a predominant genetic type that is present in poultry and that is transmitted to humans. There are also several other genotypes, but only a few of them could be recovered from human sources, suggesting the existence of different pathogenic traits among strains circulating in the country.
First Culture Isolation of
Borrelia lonestari
, Putative Agent of Southern Tick-Associated Rash Illness
Authors: Varela Andrea S. , Luttrell M. Page , Howerth Elizabeth W. , Moore Victor A. , Davidson William R. , Stallknecht David E. , Little Susan E.
Published: March 2004

Southern tick-associated rash illness (STARI) is a Lyme disease-like infection described in patients in the southeastern and south-central United States, where classic Lyme disease is relatively rare. STARI develops following the bite of a lone star tick (
Amblyomma americanum
) and is thought to be caused by infection with an “uncultivable” spirochete tentatively named
Borrelia lonestari
. In this study, wild lone star ticks collected from an area where
B. lonestari
is endemic were cocultured in an established embryonic tick cell line (ISE6). The cultures were examined by dark-field microscopy for evidence of infection, and spirochete identity and morphology were evaluated by flagellin B and 16S rRNA gene sequence, by reaction to
Borrelia
-wide and
B. burgdorferi
-specific monoclonal antibodies, and by electron microscopy. Live spirochetes were first visualized in primary culture of
A. americanum
ticks by dark-field microscopy 14 days after the cell culture was inoculated. The sequences of the flagellin B and 16S rRNA genes of cultured spirochetes were consistent with previously reported sequences of
B. lonestari
. The cultured spirochetes reacted with a
Borrelia-
wide flagellin antibody, but did not react with an OspA antibody specific to
B. burgdorferi
, by indirect fluorescent antibody testing. Electron microscopy demonstrated organisms that were free and associated with ISE6 cells, with characteristic
Borrelia
sp. morphology. This study describes the first successful isolation of
B. lonestari
in culture, providing a much needed source of organisms for the development of diagnostic assays and forming a basis for future studies investigating the role of the organism as a human disease agent.
Computer-Assisted Surveillance for Detecting Clonal Outbreaks of Nosocomial Infection
Authors: Hacek Donna M. , Cordell Ralph L. , Noskin Gary A. , Peterson Lance R.
Published: March 2004

Whole-house surveillance for healthcare-associated infection is no longer the recommended practice because of the large personnel time investment required. We developed a computer-based tracking system using microbiologic data as an aid in detecting potential outbreaks of healthcare-associated infections on a hospital-wide basis. Monthly total isolates of 25 clinically significant hospital pathogens were tallied from 1991 to 1998 to form a database for future comparison. Two different algorithm tools (based on increases of organism numbers over baseline) were applied to determine alert thresholds for suspected outbreaks using this information. The first algorithm (2SD) defined an alert as two standard deviations above the mean monthly number of isolates. The second (MI) defined an alert as either a 100% increase from the baseline organism number over 2 months or a ≥50% increase (compared to baseline) during a three-consecutive-month period. These two methods were compared to standard infection control professional surveillance (ICP) for the detection of clonal outbreaks over 12 months. Overall, a total of seven clonal outbreaks were detected during the 1-year study. Using standard methods, ICP investigated nine suspected outbreaks, four of which were associated with clonal microbes. The 2SD method signaled a suspected outbreak 15 times, of which three were clonal and ICP had detected one. The MI method signaled a suspected outbreak 30 times; four of these were clonal, and ICP had detected one. The sensitivity and specificity values for ICP, 2SD, and MI for detecting clonal outbreaks were 57, 43, and 57% and 17, 83, and 67%, respectively. Statistical methods applied to clinical microbiology laboratory information system data efficiently supplement infection control efforts for outbreak detection.
Surveillance of Childhood Influenza Virus Infection: What Is the Best Diagnostic Method To Use for Archival Samples?
Authors: Frisbie Brent , Tang Yi-Wei , Griffin Marie , Poehling Katherine , Wright Peter F. , Holland Kathy , Edwards Kathryn M.
Published: March 2004

Despite the clinical importance of influenza virus in pediatric respiratory infections, the optimal set of diagnostic tests to use when conducting studies using archival samples is not clear. In this study, we compared diagnostic tests for influenza virus in 75 children younger than 5 years of age who presented with symptomatic respiratory infection during one of four influenza seasons, had negative viral cultures for other respiratory pathogens, and had both an archival nasal aspirate obtained at the time of illness and serology spanning that influenza season. For all eligible children, we compared the results of viral culture performed at the time of collection with serology and PCR of archival nasal aspirates. Using real-time viral culture as the “gold standard,” the test characteristics of PCR of archival nasal aspirates (sensitivity, 82%; specificity, 100%) and serology (sensitivity, 82%; specificity, 87%) were similar. The relatively low sensitivity of PCR of archival nasal samples in this study compared to that of PCR of fresh samples in a previous study suggests that RNA degradation occurred despite storage of the specimens at −70°C. RNA degradation would also explain why only 11 (52%) of 21 archival nasal samples that had positive influenza virus cultures at the time of collection had positive repeat cultures in the summer of 2000. Thus, in archival specimens stored at −70°C, PCR was more sensitive than viral culture. However, testing of fresh specimens had the highest yield in this study. Studies of optimal methods for specimen storage are needed.
Laboratory Detection of
Haemophilus influenzae
with Decreased Susceptibility to Nalidixic Acid, Ciprofloxacin, Levofloxacin, and Moxifloxacin Due to
gyrA
and
parC
Mutations
Authors: Pérez-Vázquez María , Román Federico , Aracil Belén , Cantón Rafael , Campos José
Published: March 2004

The detection of clinical isolates with decreased fluoroquinolone susceptibilities and a resistance mechanism is of epidemiological and clinical interest. We studied the susceptibilities of 62 clinical isolates and 2 American Type Culture Collection reference strains of
Haemophilus influenzae
to ciprofloxacin, levofloxacin, moxifloxacin, and nalidixic acid by the microdilution and disk diffusion methods. The ciprofloxacin MICs for 34 of the isolates were ≥0.12 μg/ml (range, 0.12 to 32 μg/ml), and the ciprofloxacin MICs for 28 matched control isolates were ≤0.06 μg/ml. In addition, we sequenced the quinolone resistance-determining regions (QRDRs) of
gyrA
and
parC
of all strains. The log
2
MICs of all quinolones were plotted against the inhibition zone diameters. The MICs and inhibition zone diameters selected to screen for the resistance mechanism were based on the susceptibility distribution data and the presence or absence of amino acid changes in the QRDRs of GyrA and ParC. Strains for which ciprofloxacin MICs were ≤0.06 μg/ml, levofloxacin and moxifloxacin MICs were ≤0.03 μg/ml, and nalidixic acid MICs were ≤2.0 μg/ml lacked modifications in the QRDR of GyrA. In contrast, all strains for which ciprofloxacin, levofloxacin, and moxifloxacin MICs were ≥0.5 μg/ml and the vast majority of those for which nalidixic acid MICs were ≥32 μg/ml exhibited amino acid changes in GyrA and ParC. Nalidixic acid and the other three fluoroquinolones studied could be used to screen
H. influenzae
isolates for the detection of decreased susceptibilities to quinolones due to the acquisition of two amino acid changes in the QRDRs of GyrA and ParC (sensitivity, >95%; specificity, >80%).
Characterization of Three New Enterococcal Species,
Enterococcus
sp. nov. CDC PNS-E1,
Enterococcus
sp. nov. CDC PNS-E2, and
Enterococcus
sp. nov. CDC PNS-E3, Isolated from Human Clinical Specimens
Authors: Carvalho Maria da Glória S. , Steigerwalt Arnold G. , Morey Roger E. , Shewmaker Patricia Lynn , Teixeira Lúcia M. , Facklam Richard R.
Published: March 2004

As a reference laboratory, the
Streptococcus
Laboratory at the Centers for Disease Control and Prevention (CDC) is frequently asked to confirm the identity of unusual or difficult-to-identify catalase-negative, gram-positive cocci. In order to accomplish the precise identification of these microorganisms, we have systematically applied analysis of whole-cell protein profiles (WCPP) and DNA-DNA reassociation experiments, in conjunction with conventional physiological tests. Using this approach, we recently focused on the characterization of three strains resembling the physiological groups I (strain SS-1730), II (strain SS-1729), and IV (strain SS-1728) of enterococcal species. Two strains were isolated from human blood, and one was isolated from human brain tissue. The results of physiological testing were not consistent enough to allow confident inclusion of the strains in any of the known enterococcal species. Resistance to vancomycin was detected in one of the strains (SS-1729). Analysis of WCPP showed unique profiles for each strain, which were not similar to the profiles of any previously described
Enterococcus
species. 16S ribosomal DNA (rDNA) sequencing results revealed three new taxa within the genus
Enterococcus
. The results of DNA-DNA relatedness experiments were consistent with the results of WCPP analysis and 16S rDNA sequencing, since the percentages of homology with all 25 known species of
Enterococcus
were lower than 70%. Overall, the results indicate that these three strains constitute three new species of
Enterococcus
identified from human clinical sources, including one that harbors the
vanA
gene. The isolates were provisionally designated
Enterococcus
sp. nov. CDC Proposed New Species of
Enterococcus
1 (CDC PNS-E1), type strain SS-1728
T
(= ATCC BAA-780
T
= CCUG 47860
T
);
Enterococcus
sp. nov. CDC PNS-E2, type strain SS-1729
T
(= ATCC BAA-781
T
= CCUG 47861
T
); and
Enterococcus
sp. nov. CDC PNS-E3, type strain SS-1730
T
(= ATCC BAA-782
T
= CCUG 47862
T
).
Antibiotic-Associated Diarrhea Accompanied by Large-Scale Alterations in the Composition of the Fecal Microbiota
Authors: Young Vincent B. , Schmidt Thomas M.
Published: March 2004

Alterations in the diversity of the gut microbiota are believed to underlie the development of antibiotic-associated diarrhea (AAD). A molecular phylogenetic analysis was performed to document temporal changes in the diversity of fecal bacteria of a patient who developed AAD. Antibiotic administration was associated with distinct changes in the diversity of the gut microbiota, including a marked decrease in the prevalence of butyrate-producing bacteria. Following the discontinuation of the antibiotic, resolution of diarrhea was accompanied by a reversal of these changes, providing the first direct evidence linking changes in the community structure of the gastrointestinal bacteria with the development of AAD.
Simultaneous Detection of
Entamoeba histolytica
,
Giardia lamblia
, and
Cryptosporidium parvum
in Fecal Samples by Using Multiplex Real-Time PCR
Authors: Verweij Jaco J. , Blangé Roy A. , Templeton Kate , Schinkel Janke , Brienen Eric A. T. , van Rooyen Marianne A. A. , van Lieshout Lisette , Polderman Anton M.
Published: March 2004

Entamoeba histolytica
,
Giardia lamblia
, and
Cryptosporidium
are three of the most important diarrhea-causing parasitic protozoa. For many years, microscopic examination of stool samples has been considered to be the “gold standard” for diagnosis of
E. histolytica
,
G. lamblia
,
and C. parvum
infections. Recently, more specific and sensitive alternative methods (PCR, enzyme-linked immunosorbent assay, and direct fluorescent-antibody assay) have been introduced for all three of these parasitic infections. However, the incorporation in a routine diagnostic laboratory of these parasite-specific methods for diagnosis of each of the respective infections is time-consuming and increases the costs of a stool examination. Therefore, a multiplex real-time PCR assay was developed for the simultaneous detection of
E. histolytica
,
G. lamblia
, and
C. parvum
in stool samples. The multiplex PCR also included an internal control to determine efficiency of the PCR and detect inhibition in the sample. The assay was performed on species-specific DNA controls and a range of well-defined stool samples, and it achieved 100 percent specificity and sensitivity. The use of this assay in a diagnostic laboratory would provide sensitive and specific diagnosis of the main parasitic diarrheal infections and could improve patient management and infection control.
Comparison of the Semisolid Agar Antifungal Susceptibility Test with the NCCLS M38-P Broth Microdilution Test for Screening of Filamentous Fungi
Authors: Kuzucu Cigdem , Rapino Barbara , McDermott Laura , Hadley Susan
Published: March 2004

Antifungal susceptibility testing of pathogenic molds is being developed. A simple screening semisolid agar antifungal susceptibility (SAAS) test accurately measures susceptibilities of yeasts. The performance of the SAAS screening test for filamentous fungi was assessed by comparing MICs of four antifungals (amphotericin B [AMB], AMB lipid complex [ABEL], itraconazole [ITZ], and posaconazole [POS]) for 54 clinical mold isolates with the results of the National Committee for Clinical Laboratory Standards (NCCLS) proposed broth microdilution method (M38-P). The SAAS test utilized inocula stabbed into tubes of 0.5% semisolid heart infusion agar. In both tests MICs were read after incubation at 35°C for 48 h. The isolates tested were
Aspergillus fumigatus
,
Aspergillus niger
,
Aspergillus flavus
, other
Aspergillus
spp.,
Fusarium
spp.,
Penicillium
sp.,
Mucor
sp.,
Scedosporium prolificans
,
Trichophyton
sp., and an unidentified dematiaceous mold. Concordance of test results was determined as the percent agreement of MICs ± 1 dilution. The overall agreement between the tests for each drug was as follows: AMB, 94%; ABEL, 83%; ITZ, 94%; POS, 94%. For the
Aspergillus
spp., all but one were susceptible to ITZ by SAAS test; all were susceptible to POS (MIC range, 0.25 to 4 μg/ml). Three of six non-
Aspergillus
molds that were resistant to AMB and ABEL by SAAS (MIC ≥ 2 μg/ml) were also resistant by the NCCLS test. The SAAS test compared favorably to the NCCLS broth microdilution test for molds, and most of the clinical isolates tested were susceptible to all four drugs.
Improved Antigenic Methods for Differential Diagnosis of Bovine, Caprine, and Cervine Alphaherpesviruses Related to Bovine Herpesvirus 1
Authors: Keuser Véronique , Schynts Frédéric , Detry Bruno , Collard Alfred , Robert Béatrice , Vanderplasschen Alain , Pastoret Paul-Pierre , Thiry Etienne
Published: March 2004

The control of infectious bovine rhinotracheitis induced by bovine herpesvirus 1 (BoHV-1) requires sensitive and specific diagnostic assays. As BoHV-1 is antigenically and genetically related to four other alphaherpesviruses of ruminants—namely, BoHV-5, caprine herpesvirus 1 (CpHV-1), cervine herpesvirus 1 (CvHV-1) and CvHV-2—diagnostic tests able to discriminate BoHV-1 from these related viruses are needed to avoid misdiagnosis, especially because some of these viruses are able to cross the species barrier. In this study, murine monoclonal antibodies (MAbs) specific for BoHV-1, BoHV-5, CpHV-1, CvHV-1, and CvHV-2 were produced with the aim of setting up an immunofluorescence assay able to discriminate between these related herpesviruses. Produced MAbs were selected for their viral specificity by enzyme-linked immunosorbent assay and indirect immunofluorescence staining of virus-infected cells. Radioimmunoprecipitation characterization of the selected MAbs revealed that four of them are directed against glycoprotein C (gC) and one of them is directed against gD of these related viruses. The obtained results demonstrate that the antibodies produced allow an unambiguous discrimination of each of the four alphaherpesviruses related to BoHV-1.
Hairpin Primers for Simplified Single-Nucleotide Polymorphism Analysis of
Mycobacterium tuberculosis
and Other Organisms
Authors: Hazbón Manzour Hernando , Alland David
Published: March 2004

We describe a novel, simple, rapid, and highly sensitive method to detect single-nucleotide polymorphisms (SNPs) in
Mycobacterium tuberculosis
and other organisms. Amplification refractory mutation (ARMS) SNP assays were modified by converting the SNP-detecting linear primers in the ARMS assay to hairpin-shaped primers (HPs) through the addition of a 5′ tail complementary to the 3′ end of the linear primer. The improved ability of these primers to detect SNPs in
M. tuberculosis
was compared in a real-time PCR with SYBR-I green dye. Linear primers resulted in incorrect or indeterminate allele designation for 6 of the 13 SNP alleles tested in seven different SNP assays, while HPs determined the correct SNP in all cases. We compared the cycle threshold differences (Δ

C
t

) between the reactions containing primer-template matches and the reactions containing primer-template mismatches (where a larger Δ

C
t

indicates a more robust assay). The use of HPs dramatically improved the mean Δ

C
t

values for the SNP assays (7.6 for linear primers and 11.2 for HPs). We designed 98 different HP assays for SNPs previously associated with resistance to the antibiotic isoniazid to test the large-scale utility of the HP approach. Assay design was successful in 72.4%, 83.7%, 88.8%, and 92.9% of the assays after one to four rounds of assay design, respectively. HP SNP assays are simple, sensitive, robust, and inexpensive. These advantages favor the application of this technique for SNP assays of
M. tuberculosis
and other organisms.
Preferential Presence of Decorin-Binding Protein B (BBA25) and BBA50 Antibodies in Cerebrospinal Fluid of Patients with Neurologic Lyme Disease
Authors: Fikrig Erol , Coyle Patricia K. , Schutzer Steven E. , Chen Manchuan , Deng Zhidian , Flavell Richard A.
Published: March 2004

Borrelia burgdorferi
antibodies preferentially present in cerebrospinal fluid (CSF) were examined by differentially probing a
B. burgdorferi
expression library with CSF and sera from patients with neurologic Lyme disease. Several phage clones selectively reacted with CSF, and these genes were then expressed in recombinant form and used to detect specific antibody in an enzyme-linked immunosorbent assay. Decorin-binding protein B (BBA25) and BBA50 (hypothetical protein) elicited immunoglobulin G (IgG) or IgM detectable in CSF—but not sera—of patients, demonstrating preferential antibody production during neuroborreliosis.
Parasitic Mycelial Forms of
Coccidioides
Species in Mexican Patients
Authors: Muñoz B. , Castañón L. R. , Calderón I. , Vázquez M. E. , Manjarrez M. E.
Published: March 2004

Of 26 cases of coccidioidomycosis reported here, 15 showed hyphae, atypical parasitic structures of
Coccidioides
spp. in fresh cytologic and/or histologic specimen preparations. The finding of this morphology could have implications which should be considered, especially when the disease affects areas of nonendemicity.
Human Monoclonal Antibody Fragment Specific for Glycoprotein G in Herpes Simplex Virus Type 2 with Applications for Serotype-Specific Diagnosis
Authors: Bugli Francesca , Manzara Stefania , Torelli Riccardo , Graffeo Rosalia , Santangelo Rosaria , Cattani Paola , Fadda Giovanni
Published: March 2004

A combinatorial library was used to select a human monoclonal antibody fragment (Fab) with high affinity for G glycoprotein in herpes simplex virus type 2 (HSV-2). Tests with 112 clinical specimens demonstrated successful discrimination between HSV-2 and HSV-1, showing the potential of Fab as a low-cost tool for HSV subtyping in clinical diagnosis.