bluuebunny/crossref_metadata_2025 huggingface dataset:
Surge Capacity for Response to Bioterrorism in Hospital Clinical Microbiology Laboratories
Authors: Shapiro Daniel S.
Published: December 2003

Surge capacity is the ability to rapidly mobilize to meet an increased demand. While large amounts of federal funding have been allocated to public health laboratories, little federal funding has been allocated to hospital microbiology laboratories. There are concerns that hospital laboratories may have inadequate surge capacities to deal with a significant bioterrorism incident. A workflow analysis of a clinical microbiology laboratory that serves an urban medical center was performed to identify barriers to surge capacity in the setting of a bioterrorism event and to identify solutions to these problems. Barriers include a national shortage of trained medical technologists, the inability of clinical laboratories to deal with a dramatic increase in the number of blood cultures, a delay while manufacturers increase production of critical products and then transport and deliver these products to clinical laboratories, and a shortage of class II biological safety cabinets. Federal funding could remedy staffing shortages by making the salaries of medical technologists comparable to those of similarly educated health care professionals and by providing financial incentives for students to enroll in clinical laboratory science programs. Blood culture bottles, and possibly continuous-monitoring blood culture instruments, should be added to the national antibiotic stockpile. Federal support must ensure that companies that manufacture essential laboratory supplies are capable of rapidly scaling up production. Hospitals must provide increased numbers of biological safety cabinets and amounts of space dedicated to clinical microbiology laboratories. Laboratories should undertake limited cross-training of technologists, ensure that adequate packaging supplies are available, and be able to move to a 4-day blood culture protocol.
Acquisition and Duration of Vancomycin-Resistant Enterococcal Carriage in Relation to Strain Type
Authors: Mascini E. M. , Jalink K. P. , Kamp-Hopmans T. E. M. , Blok H. E. M. , Verhoef J. , Bonten M. J. M. , Troelstra A.
Published: December 2003

In May 2000, the first outbreak of vancomycin-resistant
Enterococcus faecium
(VREF) was detected in the University Medical Center Utrecht in the nephrology ward. The question arose why some VREF strains spread among hospitalized patients, whereas other strains do not. Thirty patients who were found to be colonized with VREF between May and November 2000 were included in the study. Molecular typing confirmed that 19 of them carried an identical epidemic strain which harbored the
esp
gene while 11 were colonized by nonepidemic strains that were all
esp
negative. Acquisition of the outbreak strain was significantly associated with diabetes mellitus, renal transplantation, and extensive use of antibiotics, especially cephalosporins, in the 2-month period before the first isolation of VREF. To establish the duration of colonization, prospective surveillance of VREF carriage for a 6-month period starting from the first isolation of VREF was realized for 20 patients. After 6 months, VREF was still recovered from 60% of carriers of the outbreak strain versus 20% of carriers of nonepidemic strains (
P
< 0.01). However, antibiotic use during the follow-up period was significantly higher by carriers of the outbreak strain than by carriers of nonepidemic strains. The fact that the outbreak strain was recovered for a longer period of time than nonepidemic strains may facilitate dissemination of the strain. The results support a careful restrictive antibiotic policy for wards at risk for spread of VREF and implementation of isolation precautions for patients who are colonized with
esp
-positive outbreak strains.
Growth of
Helicobacter pylori
in Medium Supplemented with Cyanobacterial Extract
Authors: Vega Alba E. , Cortiñas Teresa I. , Mattana Claudia M. , Silva Humberto J. , Puig de Centorbi Olga
Published: December 2003

Solid and liquid media supplemented only with a cyanobacterial extract (CE) and free of fetal calf serum (FCS), blood, and its derivatives support the growth of
Helicobacter pylori
. A total of 11 strains of
H. pylori
isolated from gastric biopsy samples were successfully subcultured in Mueller-Hinton agar supplemented with 0.4% CE. When this medium was used for primary isolation of
H. pylori
, a low isolation rate (30%) was observed because of the abundant growth of contaminants. The growth kinetics of eight isolates and
H. pylori
reference strain NCTC 11638 in Mueller-Hinton broth (MHB) supplemented with 0.7% CE were estimated by use of growth parameters, and the results were compared with those obtained with MHB-5% FCS. For four strains the cellular concentrations obtained with CE were statistically higher (
P
< 0.05) than those obtained with FCS, and in some cases these values were similar to the highest values reported in the literature. Depending on the strain, the specific growth rates obtained with CE were similar to or increased compared with those obtained with FCS. The replacement of FCS by CE in
H. pylori
cultures would facilitate the retrieval of cultures with high cellular densities as a source of cellular and extracellular proteins free of serum. Also, CE has advantages over conventional supplements, such as easier conservation and compliance with the pressing tendency at present to avoid the use of products derived from animals.
Multilocus Variable-Number Tandem Repeat AnalysisDistinguishes Outbreak and SporadicEscherichia coliO157:H7Isolates
Authors: Noller Anna C. , McEllistrem M. Catherine , Pacheco Antonio G. F. , Boxrud David J. , Harrison Lee H.
Published: December 2003

Escherichia coliO157:H7 is a major cause of food-borne illness in the United States. Outbreak detection involves traditional epidemiological methods and routine molecular subtyping by pulsed-field gel electrophoresis (PFGE). PFGE is labor-intensive, and the results are difficult to analyze and not easily transferable between laboratories. Multilocus variable-number tandem repeat (VNTR) analysis (MLVA) is a fast, portable method that analyzes multiple VNTR loci, which are areas of the bacterial genome that evolve quickly. Eighty isolates, including 21 isolates from five epidemiologically well-characterized outbreaks from Pennsylvania and Minnesota, were analyzed by PFGE and MLVA. Strains in PFGE clusters were defined as strains that differed by less than or equal to one band by usingXbaI and the confirmatory enzymeSpeI. MLVA was performed by comparing the number of tandem repeats at seven loci. From 6 to 30 alleles were found at the seven loci, resulting in 64 MLVA types among the 80 isolates. MLVA correctly identified the isolates from all five outbreaks if only a single-locus variant was allowed. MLVA differentiated strains with unique PFGE types. Additionally, MLVA discriminated strains within PFGE-defined clusters that were not known to be part of an outbreak. In addition to being a simple and validated method forE. coliO157:H7 outbreak detection, MLVA appears to have a sensitivity equal to that of PFGE and a specificity superior to that of PFGE.
Evolution ofsfbIEncoding Streptococcal Fibronectin-Binding Protein I: Horizontal Genetic Transfer and Gene Mosaic Structure
Authors: Towers Rebecca J. , Fagan Peter K. , Talay Susanne R. , Currie Bart J. , Sriprakash Kadaba S. , Walker Mark J. , Chhatwal Gursharan S.
Published: December 2003

Streptococcal fibronectin-binding protein is an important virulence factor involved in colonization and invasion of epithelial cells and tissues byStreptococcus pyogenes. In order to investigate the mechanisms involved in the evolution ofsfbI, thesfbIgenes from 54 strains were sequenced. Thirty-four distinct alleles were identified. Three principal mechanisms appear to have been involved in the evolution ofsfbI. The amino-terminal aromatic amino acid-rich domain is the most variable region and is apparently generated by intergenic recombination of horizontally acquired DNA cassettes, resulting in a genetic mosaic in this region. Two distinct and divergent sequence types that shared only 61 to 70% identity were identified in the central proline-rich region, while variation at the 3′ end of the gene is due to deletion or duplication of defined repeat units. Potential antigenic and functional variabilities in SfbI imply significant selective pressure in vivo with direct implications for the microbial pathogenesis ofS. pyogenes.
PCR Typing of Genetic Determinants for Metallo-β-Lactamases and Integrases Carried by Gram-Negative Bacteria Isolated in Japan, with Focus on the Class 3 Integron
Authors: Shibata Naohiro , Doi Yohei , Yamane Kunikazu , Yagi Tetsuya , Kurokawa Hiroshi , Shibayama Keigo , Kato Haru , Kai Kumiko , Arakawa Yoshichika
Published: December 2003

From January 2001 to December 2002, 587 strains of gram-negative bacterial isolates demonstrating resistance to ceftazidime and a combination of sulbactam and cefoperazone were subjected to a disk diffusion screening test using sodium mercaptoacetic acid; 431 strains (73.4%) appeared to produce metallo-β-lactamase (MBL). Of these 431 strains, 357 were found by PCR to carry genes for IMP-1 type MBL (
bla
IMP-1
), while only 7 and 67 strains carried the IMP-2 gene (
bla
IMP-2
) and the VIM-2 gene (
bla
VIM-2
), respectively. Neither VIM-1 nor SPM-1 type MBL genes were found among the strains tested. Of 431 strains, 427 carried the
intI1
gene, and 4 strains carrying both the
intI1
and
intI3
genes were reidentified as
Pseudomonas putida
harboring
bla
IMP-1
. Of these four
P. putida
strains, three strains and one strain, respectively, were separately isolated from two hospitals located in the same prefecture, and the three strains showed very similar pulsed-field gel electrophoresis patterns. Of 357
bla
IMP-1
carriers, 116, 53, 51, 47, and 30 strains were identified as
Pseudomonas aeruginosa
,
Alcaligenes xylosoxidans
,
P. putida/fluorescens
,
Serratia marcescens
, and
Acinetobacter baumannii
, respectively. Four strains carrying
bla
IMP-2
were reidentified as
P. putida
. Sixty-three
P. aeruginosa
strains and four
P. putida
strains carried
bla
VIM-2
. Of 427
intI1
-positive strains, 180, 53, 51, 47, and 35 were identified as
P. aeruginosa
,
A. xylosoxidans
,
P. putida/fluorescens
,
S. marcescens
, and
A. baumannii
, respectively. In the present study, it was confirmed that strains carrying
bla
IMP-1
with a class 1 integron are the most prevalent type in Japan, although several
intI3
carriers have also been identified sporadically in this country.
Use of an Immunoglobulin G Avidity Assay Based on Recombinant Antigens for Diagnosis of PrimaryToxoplasma gondiiInfection during Pregnancy
Authors: Beghetto Elisa , Buffolano Wilma , Spadoni Andrea , Del Pezzo Mariassunta , Di Cristina Manlio , Minenkova Olga , Petersen Eskild , Felici Franco , Gargano Nicola
Published: December 2003

The objective of this work was to develop an antibody-specific immunoglobulin G (IgG) avidity assay to discriminate between acute and latent phases ofToxoplasma gondiiinfection by using recombinant antigens. One hundred twenty-one serum samples from women who developed IgG antibodies againstToxoplasmaduring pregnancy were used. The IgG avidities of antibodies directed against epitopes carried by fragments of GRA3, GRA7, MIC3, and SAG1 antigens were measured by performing parallel enzyme immunoassays. The avidity index forToxoplasma-specific antibodies against a homogeneous mixture of recombinant GRA3, GRA7, MIC3, and SAG1 antigens correlated closely with the IgG avidity of antibodies against lysed whole-cellT. gondiiantigen. The avidity assay performed with the recombinant MIC3 antigen highlighted the presence of avidity low-antibodies IgG exclusively in sera collected within 2 months after primary infection. The presence ofT. gondii-specific, low-avidity IgG antibodies against recombinant MIC3 antigen can be used to determine the point of infection withT. gondiiwithin a 2-month time frame after infection.
Molecular Parameters for Precise Diagnosis of Asymptomatic Epstein-Barr Virus Reactivation in Healthy Carriers
Authors: Maurmann Susanne , Fricke Lutz , Wagner Hans-Joachim , Schlenke Peter , Hennig Holger , Steinhoff Jürgen , Jabs Wolfram J.
Published: December 2003

Asymptomatic Epstein-Barr virus (EBV) reactivations periodically occur in oral mucosa-associated lymphoid tissues. Until now, EBV reactivation has been diagnosed by serologic profiles that suggest virus replication. Serologic responses, however, are delayed and do not necessarily indicate ongoing replicative activity. The aim of the present study was to establish in healthy carriers parameters for a molecular diagnosis of reactivated EBV infection. Recent studies emphasized the association of an increase in peripheral-B-cell viral load with replicative activity at remote sites. Therefore, real-time PCR was used to quantitate EBV genomes in the peripheral blood mononuclear cells (PBMC) (viral load) and plasma samples (viremia) of 22 healthy EBV-seropositive blood donors over a period of 15 months. Furthermore, transcription of the immediate-early gene encoding BZLF1 was investigated in the PBMC of all volunteers. Serology suggested reactivation in nine donors, of whom all but one showed at least once a significant increase in viral load. Another five individuals also exhibited significant changes in viral load but no serologic response. Of the 13 volunteers with significant increases in viral load, 6 had a period of viremia accompanying the rise in viral load. A stable viral load without viremia and negative serology was seen in eight adults. BZLF1 mRNA was undetectable throughout. We conclude that for healthy subjects serology underestimates the frequency of asymptomatic EBV reactivations. Prospective examination of peripheral viral load and viremia is suitable for the exact diagnosis of EBV reactivation, which might be of advantage for immunocompromised patients in whom EBV reactivations are considerably harmful.
Changes in Gene Cassettes of Class 1 Integrons among
Escherichia coli
Isolates from Urine Specimens Collected in Korea during the Last Two Decades
Authors: Yu Hak Sun , Lee Je Chul , Kang Hee Young , Ro Dong Woo , Chung Jae Young , Jeong Young Sook , Tae Seong Ho , Choi Chul Hee , Lee Eun Young , Seol Sung Yong , Lee Yoo Chul , Cho Dong Taek
Published: December 2003

Gene cassettes of class 1 integrons in
Escherichia coli
isolates from urine specimens collected in Korea during the last 2 decades were characterized.
intI1
was detected in 54% of the isolates, yet gene cassette regions were amplified in only 43% of the isolates.
intI2
was detected in 29 (5%) isolates, and no
intI3
was detected in this study. Twenty-one different genes, including genes encoding resistance to antibiotics, an alcohol dehydrogenase gene (
adhE
), and unknown genes, were detected. The genes most commonly found in class 1 integrons were those for aminoglycoside and trimethoprim resistance. The occurrence of aminoglycoside resistance genes in class 1 integrons decreased, and the presence of
dfr
genes increased rapidly, during the last 2 decades. Single-gene cassettes were predominant during the 1980s, while multigene cassettes predominated from the 1990s on. The
aadA1
,
aadA2
, and
blaP1-aadA2
gene cassettes were frequently found in isolates from the 1980s but were not detected in isolates recovered since 2000.
dfrA12-aadA2
and
dfrA17-aadA5
were the most prevalent gene cassettes among isolates recovered from the 1990s on. In conclusion, class 1 integrons would appear to be responsible for resistance to antibiotics commonly used to treat urinary tract infections, and selection of a specific gene cassette was found to occur over the course of time.
Culture and Phenotypic Characterization of a
Wolbachia pipientis
Isolate
Authors: Fenollar Florence , La Scola Bernard , Inokuma Hisashi , Dumler J. Stephen , Taylor Mark J. , Raoult Didier
Published: December 2003

The recent isolation of
Wolbachia pipientis
in the continuous cell line Aa23, established from eggs of a strain of the Asian tiger mosquito
Aedes albopictus
, allowed us to perform extensive characterization of the isolate. Bacterial growth could be obtained in C6/36, another
A. albopictus
cell line, at 28°C and in a human embryonic lung fibroblast monolayer at 28 and 37°C, confirming that its host cell range is broader than was initially thought. The bacteria were best visualized by Diff-Quik and May-Grünwald-Giemsa staining. Proteins from 213 to 18 kDa with two major protein bands of 65 and 25 kDa were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By Western blotting with specific polyclonal mouse and rabbit antisera, dominant immunoreactive antigens were found at approximately 100, 80, and 30 kDa. The genome size was calculated to be 1,790 ± 17 kb by pulsed-field gel electrophoresis. The sequence of the citrate synthase gene (
gltA
) of
W. pipientis
was determined by gene walking. Its position in the phylogenetic tree constructed with
gltA
confirmed that found in a phylogenetic tree constructed with 16S rRNA genes and that it belongs in the α subgroup of the class
Proteobacteria
and that it is closely related to but independent from the genera
Ehrlichia
,
Anaplasma
, and
Neorickettsia
.
Typing of Methicillin-Resistant
Staphylococcus aureus
in a University Hospital Setting by Using Novel Software for
spa
Repeat Determination and Database Management
Authors: Harmsen Dag , Claus Heike , Witte Wolfgang , Rothgänger Jörg , Claus Hermann , Turnwald Doris , Vogel Ulrich
Published: December 2003

The
spa
gene of
Staphylococcus aureus
encodes protein A and is used for typing of methicillin-resistant
Staphylococcus aureus
(MRSA). We used sequence typing of the
spa
gene repeat region to study the epidemiology of MRSA at a German university hospital. One hundred seven and 84 strains were studied during two periods of 10 and 4 months, respectively. Repeats and
spa
types were determined by Ridom StaphType, a novel software tool allowing rapid repeat determination, data management and retrieval, and Internet-based assignment of new
spa
types following automatic quality control of DNA sequence chromatograms. Isolates representative of the most abundant
spa
types were subjected to multilocus sequence typing and pulsed-field gel electrophoresis. One of two predominant
spa
types was replaced by a clonally related variant in the second study period. Ten unique
spa
types, which were equally distributed in both study periods, were recovered. The data show a rapid dynamics of clone circulation in a university hospital setting.
spa
typing was valuable for tracking of epidemic isolates. The data show that disproval of epidemiologically suggested transmissions of MRSA is one of the main objectives of
spa
typing in departments with a high incidence of MRSA.
High Prevalence of Hepatitis B Virus Pre-S Mutant in Countries Where It Is Endemic and Its Relationship with Genotype and Chronicity
Authors: Huy Tran Thien-Tuan , Ushijima Hiroshi , Win Khin Maung , Luengrojanakul Pairoj , Shrestha Pradeep Krishna , Zhong Zhao-Hua , Smirnov Andrei V. , Taltavull Teresa Casanovas , Sata Tetsutaro , Abe Kenji
Published: December 2003

It has been reported that hepatitis B virus (HBV) mutants carrying mutations in the pre-S region can be found in infected patients. In this study, we investigated the prevalence of the HBV variant with the pre-S mutant in different geographic regions, including countries with low and high levels of endemic HBV infection, and analyzed the correlation with clinical findings. We examined 387 HBV DNA-positive serum samples from individuals among 12 countries, consisting of Vietnam, Myanmar, Thailand, China, Korea, Nepal, Japan, Russia, Spain, United States, Bolivia, and Ghana. HBV pre-S mutants were detected in 71 (18.3%) of 387 serum samples tested. This mutant was the most prevalent in Vietnam (36%), followed by Nepal (27.3%), Myanmar (23.3%), China (22.4%), Korea (14.3%), Thailand (10.5%), Japan (7.7%), and Ghana (4.3%). In contrast, no case with this mutation was found in Russia, Spain, United States, and Bolivia. Among the HBV deletion mutations, 15.5% (11 of 71) occurred in the pre-S1 and 46.5% (33 of 71) in the pre-S2 regions. Eight (11.3%) cases had a mutation in both the pre-S1 and pre-S2 regions. In addition, a point mutation at the pre-S2 starting codon was observed in 19 (26.7%) cases. The detection rate of the HBV mutant in patients with hepatocellular carcinoma was significantly higher than in other patients (
P
< 0.05). Furthermore, these mutants were found more frequently in genotype B (25%) and genotype C (24.5%) than in the other genotypes (
P
< 0.05). Our results indicated that there was a high prevalence of HBV pre-S mutation in regions of endemic HBV infection in Asia. Furthermore, the pre-S mutation appeared to be correlated with hepatocellular carcinoma and HBV genotypes.
Evaluation of a PCR Assay for Quantitation of
Rickettsia rickettsii
and Closely Related Spotted Fever Group Rickettsiae
Authors: Eremeeva Marina E. , Dasch Gregory A. , Silverman David J.
Published: December 2003

A spotted fever rickettsia quantitative PCR assay (SQ-PCR) was developed for the detection and enumeration of
Rickettsia rickettsii
and other closely related spotted fever group rickettsiae. The assay is based on fluorescence detection of SYBR Green dye intercalation in a 154-bp fragment of the rOmpA gene during amplification by PCR. As few as 5 copies of the rOmpA gene of
R. rickettsii
can be detected. SQ-PCR is suitable for quantitation of
R. rickettsii
and 10 other genotypes of spotted fever group rickettsiae but not for
R. akari
,
R. australis
,
R. bellii
, or typhus group rickettsiae. The sensitivity of SQ-PCR was comparable to that of a plaque assay using centrifugation for inoculation. The SQ-PCR assay was applied successfully to the characterization of rickettsial stock cultures, the replication of rickettsiae in cell culture, the recovery of rickettsial DNA following different methods of extraction, and the quantitation of rickettsial loads in infected animal tissues, clinical samples, and ticks.
Evaluation of the INNO-LiPA HBV Genotyping Assay for Determination of Hepatitis B Virus Genotype
Authors: Osiowy Carla , Giles Elizabeth
Published: December 2003

Specific genotypes of hepatitis B virus (HBV) are increasingly recognized for their clinical significance and association with particular viral mutations. Although many HBV genotyping methods exist, there has been no standardized or commercially available method for direct molecular typing of the HBV genome. A newly available line probe assay (INNO-LiPA HBV Genotyping assay; Innogenetics N.V., Ghent, Belgium) that allows the identification of HBV genotypes A to G was assessed by comparison with pre-S1/pre-S2 sequence analysis of the isolates in 188 serum specimens. All seven genotypes were detected by the line probe assay (LiPA), and complete concordance between LiPA and sequence analysis was observed for 152 specimens (81%). LiPA was able to detect 19 mixed genotype infections not detected by amplicon sequencing, which for the most part were confirmed by cloning and sequencing of the pre-S1/pre-S2 amplicon. Four specimens had discrepant results between the two methods, and five specimens had indeterminate results by LiPA. The HBV DNA in four specimens was unable to be amplified by the nested INNO-LiPA HBV DR amplification primers; however, the HBV DNA in six specimens unable to be genotyped by sequencing was clearly genotyped by LiPA. The INNO-LiPA HBV Genotyping assay appears to be useful for the rapid genotyping of HBV, particularly for the sensitive detection of mixed genotype infections.
Genetic Heterogeneity in the rRNA Gene Locus of
Trichophyton tonsurans
Authors: Gaedigk Andrea , Gaedigk Roger , Abdel-Rahman Susan M.
Published: December 2003

Trichophyton tonsurans
is the major pediatric pathogen in tinea capitis, causing disparate disease presentations. Little is known about genetic variation, which may ultimately be linked to divergent disease status. This investigation was aimed at identifying genetic variants of
T. tonsurans
by methods that can facilitate strain discrimination in population-based studies. Ninety-two isolates were acquired from six U.S. microbiology laboratories, and genomic DNA was isolated from mature colonies. The nontranscribed spacer (NTS) was amplified by PCR, and products from isolates with various amplicon sizes were fully sequenced. Nested amplification, targeting a variable internal repeat (VIR) region, allowed assignment of variant type by fragment size. Subvariant type was assigned by a combination of PCR-restriction fragment length polymorphism-based assays. Five variants differing in size (348 to 700 bp) and sequence were identified within the VIR region comprised of several large repeats (104, 140, and 194 bp) arranged in tandem. Seven single-nucleotide polymorphisms (SNPs) were detected across the NTS, with five occurring in the constant regions flanking the VIR region and two occurring in the VIR region. Additionally, a 10-bp insertion and a 14-bp deletion were identified upstream of the VIR region. The combination of SNPs revealed seven haplotype patterns which were stable upon serial passage over 1 year. No sequence variations were identified within the internal transcribed spacer regions. Unique NTS sequences were utilized to develop a duplex PCR assay that discriminated
T. tonsurans
from other dermatophytes. Of the 92 isolates evaluated, this genotyping scheme distinguished 12 distinct strains, providing evidence of genetic heterogeneity in
T. tonsurans
.
Human Herpesvirus 8 Load in Matched Serum and Plasma Samples of Patients with AIDS-Associated Kaposi's Sarcoma
Authors: Polstra Abeltje M. , van den Burg Remco , Goudsmit Jaap , Cornelissen Marion
Published: December 2003

Human herpesvirus 8 (HHV-8) (or Kaposi's sarcoma [KS]-associated herpesvirus) is associated with all forms of KS. HHV-8 DNA load in peripheral blood mononuclear cells (PBMCs) of KS patients has been shown to correlate with the clinical stage of the disease. Studies have been done to assess the HHV-8 viral load in different sample types from KS patients and its clinical relevance. This paper describes the design and evaluation of a quantitative real-time (TaqMan) PCR assay for routine diagnosis of HHV-8 infection. The linear dynamic range was 5 to 5 × 10
6
copies of HHV-8 DNA (
r
2
> 0.99). The assay is very sensitive, specific, and easily reproducible (less than 2% variability) and can be used for different clinical samples, such as serum, plasma, and PBMCs. The question of which clinical sample, serum or plasma, is preferable for HHV8 DNA testing was addressed, using this newly developed real-time PCR assay. From 85 patients with diagnosed AIDS-KS, matched plasma and serum samples were collected. Of the 85 patients tested, 35 were positive for HHV-8 DNA in both plasma and serum (41%), 8 were positive in serum but not plasma, and 7 had detectable HHV-8 DNA only in plasma. The HHV-8 load was similar in both plasma and serum, and no significant difference was found. However, more inhibition was seen in the plasma samples with the use of a system quality control, seal herpesvirus type 1. Therefore, our results suggest that serum is the preferred material for HHV-8 load testing, since there is less possible hindrance in the amplification than with plasma.
Development of a Multitarget Real-Time TaqMan PCR Assay for Enhanced Detection of
Francisella tularensis
in Complex Specimens
Authors: Versage Jessica L. , Severin Darlena D. M. , Chu May C. , Petersen Jeannine M.
Published: December 2003

Tularemia is the zoonotic disease caused by the gram-negative coccobacillus
Francisella tularensis.
Its wide distribution in the environment poses a challenge for understanding the transmission, ecology, and epidemiology of the disease.
F. tularensis
is also considered a potential biological weapon due to its extreme infectivity. We have developed a multitarget real-time TaqMan PCR assay capable of rapidly and accurately detecting
F. tularensis
in complex specimens. Targeted regions included the IS
Ftu2
element and the
23kDa
,
fopA
, and
tul4
genes. Analysis of the four TaqMan assays demonstrated that three (IS
Ftu2
,
23kDa
, and
tul4
) performed within our established criterion of a detection limit of one organism. The combined use of the three assays was highly specific, displaying no cross-reactivity with the non-
Francisella
bacteria tested and capable of differentially diagnosing both
F. tularensis
and
Francisella philomiragia
. When the multitarget TaqMan assay (IS
Ftu2
,
23kDa
, and
tul4
) was compared to culturing, using environmentally contaminated specimens, the TaqMan PCR assay was significantly more sensitive than culturing (
P
≤ 0.05). The sensitive and specific nature of this rapid multitarget TaqMan assay provides a valuable new tool that with future evaluations can be used for analyzing clinical specimens, field samples during bioterrorism threat assessment, and samples from outbreaks and for improving our understanding of the ecology and environmental prevalence of
F. tularensis
.
Culture-Independent Identification of Pathogenic Bacteria and Polymicrobial Infections in the Genitourinary Tract of Renal Transplant Recipients
Authors: Domann Eugen , Hong George , Imirzalioglu Can , Turschner Simon , Kühle Johannes , Watzel Corinna , Hain Torsten , Hossain Hamid , Chakraborty Trinad
Published: December 2003

Renal transplant recipients are predisposed to urinary tract infections caused by both common uropathogens and opportunistic bacteria resulting frequently in significant polymicrobial infections. In this study, a culture-independent 16S rRNA-based approach was established to identify unusual, fastidious, or anaerobic bacteria and to investigate bacterial diversity in urinary tract specimens. Similarly sized amplicons encompassing the V6 to V8 region of the 16S rRNA were analyzed with denaturing high-performance liquid chromatography (DHPLC) (WAVE System). Artificial mixtures of single amplicons from commonly encountered uropathogenic bacteria produced distinct peak profiles whose identities were confirmed by sequencing individually collected peak products. We evaluated the application of the method on 109 urinary tract specimens from renal transplant recipients; 100% correlation was found for culture-positive specimens, and DHPLC generated peak profiles. However, for culture-negative specimens, DHPLC facilitated the detection of novel peak profiles. DNA sequencing of these individual peaks was used to identify the bacteria involved. Thus, in PCR-positive but culture-negative samples the method allowed detection of previously known uropathogens such asCorynebacterium urealyticumandGardnerella vaginalis, but also unusual agents includingAnaerococcus lactolyticus,Bacteroides vulgatus,Dialister invisus,Fusobacterium nucleatum,Lactobacillus iners,Leptotrichia amnionii,Prevotella buccalis,Prevotella ruminicola,Rahnella aquatilis, andStreptococcus intermediuswere detected as single pathogens or as constituents of polymicrobial infections. The method described is reproducible and rapidly and enables both DHPLC-based profiling and sequence-based investigation of microbial communities and polymicrobial infections. A detailed understanding of infections found in recipients of renal transplants will guide antibiotic therapy regimens and provide new perspectives for decreasing the risk of graft rejection.
Natural Acquisition of
Helicobacter pylori
Infection in Newborn Rhesus Macaques
Authors: Solnick Jay V. , Chang Kikuko , Canfield Don R. , Parsonnet Julie
Published: December 2003

Helicobacter pylori
infection is usually acquired in childhood, but precise estimates of the age of acquisition are difficult to obtain in young children. Since serial endoscopic biopsies are not feasible in human infants, we examined acquisition of
H. pylori
infection that is known to occur in socially housed nonhuman primates. By 12 weeks of age, 8 of 20 newborns (40%) were culture positive for
H. pylori
, and prevalence reached 90% by 1 year of age. Newborns from infected dams were more commonly infected than those from uninfected dams, particularly during the peripartum period, suggesting that close contact during this time may facilitate transmission. Transient infection was uncommon and occurred only after the first positive culture. These results suggest that in a high-prevalence environment, persistent
H. pylori
infection may be acquired at an earlier age than was previously thought. Since clean, potable water was readily available, contamination of water supply is not essential for widespread infection at an early age in areas where hygiene is otherwise poor. Furthermore, breastfeeding seems to offer little protection, since newborn macaques breastfeed during the first year of life and typically are fully weaned only when another newborn arrives the following spring.
Loop-Mediated Isothermal Amplification for Detection of African Trypanosomes
Authors: Kuboki Noritaka , Inoue Noboru , Sakurai Tatsuya , Di Cello Francescopaolo , Grab Dennis J. , Suzuki Hiroshi , Sugimoto Chihiro , Igarashi Ikuo
Published: December 2003

While PCR is a method of choice for the detection of African trypanosomes in both humans and animals, the expense of this method negates its use as a diagnostic method for the detection of endemic trypanosomiasis in African countries. The loop-mediated isothermal amplification (LAMP) reaction is a method that amplifies DNA with high specificity, efficiency, and rapidity under isothermal conditions with only simple incubators. An added advantage of LAMP over PCR-based methods is that DNA amplification can be monitored spectrophotometrically and/or with the naked eye without the use of dyes. Here we report our conditions for a highly sensitive, specific, and easy diagnostic assay based on LAMP technology for the detection of parasites in theTrypanosoma bruceigroup (includingT. brucei brucei,T. brucei gambiense,T. brucei rhodesiense, andT. evansi) andT. congolense. We show that the sensitivity of the LAMP-based method for detection of trypanosomes in vitro is up to 100 times higher than that of PCR-based methods. In vivo studies in mice infected with human-infectiveT. brucei gambiensefurther highlight the potential clinical importance of LAMP as a diagnostic tool for the identification of African trypanosomiasis.
Epidemiology of
Aspergillus terreus
at a University Hospital
Authors: Baddley John W. , Pappas Peter G. , Smith Anita C. , Moser Stephen A.
Published: December 2003

Invasive fungal infections due to
Aspergillus
species have become a major cause of morbidity and mortality among immunocompromised patients.
Aspergillus terreus
, a less common pathogen, appears to be an emerging cause of infection at our institution, the University of Alabama hospital in Birmingham. We therefore investigated the epidemiology of
A. terreus
over the past 6 years by using culture data; antifungal susceptibility testing with amphotericin B, voriconazole, and itraconazole; and molecular typing with random amplification of polymorphic DNA-PCR (RAPD-PCR). During the study period, the percentage of
A. terreus
isolates relative to those of other
Aspergillus
species significantly increased, and
A. terreus
isolates frequently were resistant to amphotericin B. Molecular typing with the RAPD technique was useful in discriminating between patient isolates, which showed much strain diversity. Further surveillance of
A. terreus
may better define epidemiology and determine whether this organism is becoming more frequent in relation to other
Aspergillus
species.
Nosocomial Outbreak of Infections by
Proteus mirabilis
That Produces Extended-Spectrum CTX-M-2 Type β-Lactamase
Authors: Nagano Noriyuki , Shibata Naohiro , Saitou Yuko , Nagano Yukiko , Arakawa Yoshichika
Published: December 2003

Nineteen multidrug-resistant
Proteus mirabilis
strains were isolated from 19 patients suffering from infections probably caused by
P. mirabilis
. These strains were recovered from urine or other urogenital specimens of 16 inpatients and three outpatients with a hospitalization history in a urology ward of Funabashi Medical Center, from July 2001 to August 2002. These strains demonstrated resistance to cefotaxime, ceftriaxone, cefpodoxime, and aztreonam, while they were highly susceptible to ceftazidime (MIC, ≤0.5 μg/ml). The resistance level of these strains to cefotaxime was decreased by the presence of clavulanic acid. Therefore, the strains were speculated to produce extended-spectrum class A β-lactamases. These strains were later found to carry
bla
CTX-M-2
genes by both PCR and sequencing analyses. The profiles of
Sma
I-digested genomic DNA of 19 isolates were distinguished into five different clusters by biased sinusoidal field gel electrophoresis. Four of them, consisting of 18 isolates, were suggested to be a clonal expansion. These findings suggested that a nosocomial outbreak of infections by CTX-M-2-producing
P. mirabilis
had occurred in our medical center. Most patients suffered from urogenital malignancies with long-term catheterization. Cefazolin, cefoperazone-sulbactam, and/or levofloxacin were mostly administered to the patients, but these agents seemed ineffective for eradication of CTX-M-2 producers. Early recognition and rapid identification of colonizing antimicrobial-resistant bacteria, including CTX-M-2-producing
P. mirabilis
, would be the most effective measures to cope with further spread of this kind of hazardous microorganism in clinical environments.
Listeria monocytogenes
Serotype Identification by PCR
Authors: Borucki Monica K. , Call Douglas R.
Published: December 2003

Serotyping is a universally accepted subtyping method for
Listeria monocytogenes
. Identification of the strain serotype permits differentiation between important food-borne strains (1/2a, 1/2b, and 4b) and provides a “gold standard” for comparing isolates analyzed in different labs and with different techniques. Although an efficient enzyme-linked immunosorbent assay serotyping protocol was described recently, identification of PCR serotyping primers would further increase the ease and accessibility of this classification system. Serotyping PCR primers were designed from variable regions of the
L. monocytogenes
genome. Three primer sets were used in conjunction with a previously described Division III primer set in order to classify 122
L. monocytogenes
strains into five serotype groups [1/2a(3a), 1/2b, 1/2c(3c), 4b(d,e), and 4a/c]. Results of the PCR method agreed with those of the conventional slide agglutination method for 97, 100, 94, and 91% of strains belonging to serotypes 1/2a, 1/2b, 1/2c, and 4b, respectively.
Antibiotic-Resistant Invasive Pediatric
Streptococcus pneumoniae
Clones in Israel
Authors: Greenberg David , Dagan Ron , Muallem Marie , Porat Nurith
Published: December 2003

Antibiotic-resistant international clones of
Streptococcus pneumoniae
are increasingly reported in different parts of the world. We investigated the spread of these clones through an active surveillance performed at the Israeli Streptococcal National Center during 1998 and 1999. Isolates were tested for antibiotic susceptibility, serotyped, and genotyped by random amplified polymorphic DNA analysis and pulsed-field gel electrophoresis. Of 437 isolates, 276 (63.4%) were antibiotic resistant and 156 (35%) were penicillin nonsusceptible (PNS). The PNS isolates were less frequently encountered in southern Israel (27 of 136 [20%]) than in other regions (127 of 301 [42%]). Among 276 antibiotic-resistant isolates, 43 fingerprint patterns were observed. The most common clones were 9V/14-a (19.2%), 5-a (17.8%), and 1-a (10%). The 9V/14-a clone was less common, while the 1-a clone was more frequent in the south than in other regions. The 5-a clone was more common in Jerusalem than in other regions. Among the Jewish and Arab populations the most frequent clones were 9V/14-a (20%) and 1-a (25%), respectively. Three international clones, 9V/14-a-Spain
9V
-3, 6B-a-Spain
6B
-2, and 5-a-Colombia
5
-19, comprised 40% of all antibiotic-resistant isolates and 56% of all PNS isolates. The seven-valent conjugate vaccine covers 58% of the most common clones, all highly PNS clones, and 94% of the multidrug-resistant clones in Israel, while the nine-valent vaccine covers all of them. The most common antibiotic-resistant invasive pediatric
S. pneumoniae
clones—mainly the three international ones—contribute significantly to increases in antibiotic resistance. Their geographic distribution varies within the country and between the different populations.
Evaluation of Methodology for Serotyping Invasive and Nasopharyngeal Isolates of
Haemophilus influenzae
in the Ongoing Surveillance in Brazil
Authors: Bokermann Sérgio , Zanella Rosemeire C. , Lemos Ana Paula S. , de Andrade Ana Lúcia S. S. , Brandileone Maria Cristina de C.
Published: December 2003

To assess the magnitude of discrepant results obtained by routine
Haemophilus influenzae
serotyping, 258 isolates, collected by the epidemiological surveillance system in Brazil from individuals with invasive diseases or carriage, were evaluated by two slide agglutination (SlAg) methods: SlAg method 1, by which strains were initially screened with a serotype b-specific antiserum, and SlAg method 2, by which strains were tested against all serotype-specific antisera in parallel. Investigators comparing results of the two SlAg methods with those obtained by capsule type-specific PCR were blinded to the method used. The serotype prevalence rates found by the three methods were significantly different, involving discrepancies mainly between serotype b and noncapsulated (NC) isolates. For invasive isolates (
n
= 131), the overall agreement rate between SlAg method 1 or 2 and PCR was 68.0 or 88.3%, respectively, whereas for colonizing isolates (
n
= 127) the corresponding rate was 46.5 or 94.2%, respectively. SlAg method 2 improved the ascertainment of serotypes over that obtained with SlAg method 1, demonstrating good correlation with PCR. Use of the polyvalent antiserum as a screening reagent for SlAg for invasive and colonizing isolates showed poor discriminatory power, with a sensitivity of 65.8% and a specificity of 91.7%. We stress the importance of using a well-standardized SlAg methodology and suggest that reference laboratories should utilize PCR routinely to confirm SlAg results and to check all nonspecific SlAg reactions and apparent NC isolates by SlAg in order to provide reliable data on the prevalence of
H. influenzae
serotypes in the
H. influenzae
type b vaccine era.
Quantitative Validation of Media for Transportation and Storage of
Streptococcus pneumoniae
Authors: Charalambous B. M. , Batt S. L. , Peek A. C. , Mwerinde H. , Sam N. , Gillespie S. H.
Published: December 2003

The need to design effective
Streptococcus pneumoniae
vaccines and to monitor resistance means that it is essential to have efficient methods to determine carriage rates. Two liquid media, consisting of skim milk, glycerol, glucose, and tryptone soya broth (STGG) or skim milk, glycerol, and glucose (SGG) alone, were evaluated for their ability to maintain pneumococcal viability. Optimal recovery of
S. pneumoniae
was achieved when swabs were transferred to STGG medium prior to plating onto blood agar-gentamicin selective plates (22%) compared to 7% when plated out directly (
P
< 0.0001 by Fisher's exact test). Both STGG and SGG media are appropriate for the long-term storage of pneumococci and primary swab samples at −70°C, with no decrease in viable count observed following repeated freeze-thaw cycles. Samples could be stored refrigerated for up to 3 days in either STGG or SGG medium with no significant loss of viability. Viability decreased progressively in storage at 20 to 30°C, with greater losses of viability occurring at the higher temperatures. There were no significant differences in viability between isolates in the two media. STGG preserved pneumococci significantly better (about twofold) than SGG medium at 21°C (
P
< 0.0001) and 30°C (
P
< 0.0001). Samples can be stored for 4 and 2.5 days at 6 to 8°C, 28 and 17 h at 21°C, and 15 and 7 h at 30°C in STGG and SGG media, respectively. For field studies undertaken in resource-limited environments, SGG medium can be prepared by using locally available materials. The quantitative data reported in this study will enable researchers to plan appropriate transport and storage protocols.
Evidence ofBorrelia lonestariDNA inAmblyomma americanum(Acari: Ixodidae) Removed from Humans
Authors: Stromdahl Ellen Y. , Williamson Phillip C. , Kollars Thomas M. , Evans Sandra R. , Barry Ryan K. , Vince Mary A. , Dobbs Nicole A.
Published: December 2003

We used a nested PCR withBorreliaflagellin gene (flaB) primers and DNA sequencing to determine ifBorrelia lonestariwas present inAmblyomma americanumticks removed from military personnel and sent to the Tick-Borne Disease Laboratory of the U.S. Army Center for Health Promotion and Preventive Medicine. In our preliminary investigation, we detectedBorreliasequences in 19 of 510A. americanumadults and nymphs from Ft. A. P. Hill, Va. During the 2001 tick season, theflaBprimers were used to test allA. americanumsamples as they were received, and 29 of 2,358A. americanumsamples tested individually or in small pools were positive. PCRs with 2,146A. americanumsamples in 2002 yielded 26 moreBorrelia-positive samples. The positive ticks in 2001 and 2002 were from Arkansas, Delaware, Kansas, Kentucky, Maryland, New Jersey, North Carolina, Tennessee, and Virginia. The last positive sample of the 2001 season was a pool of larvae. To further investigate larval infection, we collected and tested questingA. americanumlarvae from Aberdeen Proving Ground, Md.; 4 of 33 pools (40 larvae per pool) were positive. Infection of unfed larvae provides evidence of the maintenance ofB. lonestariby means of transovarial transmission. Sequence analysis revealed that the amplicons were identical to sequences of theB. lonestari flaBgene in GenBank. Despite the low prevalence of infection, the risk ofB. lonestaritransmission may be magnified becauseA. americanumis often abundant and aggressive, and many tick bite victims receive multiple bites.
Denaturing High-Performance Liquid Chromatography for Detecting and Typing Genital Human Papillomavirus
Authors: Li Jianduan , Gerhard Daniela S. , Zhang Zhengyan , Huettner Phyllis C. , Wright Jason , Nguyen Loan , Lu Danielle , Rader Janet S.
Published: December 2003

Human papillomaviruses (HPVs) are important in the development of human cancers, including cervical and oral tumors. However, most existing methods for HPV typing cannot routinely distinguish among the more than 100 distinct types of HPV or the natural HPV intratypic variants that have also been documented. To address this problem, we developed a novel method, general primer-denaturing high-performance liquid chromatography (GP-dHPLC), for the detection and typing of genital HPV using an automated 96-well plate format. GP-dHPLC uses general primer PCR (GP-PCR) to amplify the viral DNA and then analyzes the GP-PCR products by denaturing high-performance liquid chromatography (dHPLC). A number of different primer pairs with homology to most known genital HPV types were tested, and the L1C1-L1C2M pair specific for the L1 region of the viral genome was chosen. A set of HPV standard control patterns, consisting of those for HPV types 16, 18, 31, 33, 39, 45, 51, 52, 56, 58, 59, 6, and 11, was established for genital HPV typing. One hundred eighty-six frozen and formalin-fixed cervical cancer tissue samples were analyzed for the presence of HPV and the HPV type by this method, and 95.8% of them were found to contain HPV DNA. GP-dHPLC accurately discriminated among HPV variants that differed by as little as one nucleotide. Several new variants of HPV types 16, 18, 39, 45, 52, and 59 were identified. Moreover, multiple HPV infections were detected in 26.6% of the samples. Our results indicate that HPV typing by GP-dHPLC permits discrimination of common genital HPV types, detection of multiple HPV infections, and identification of HPV variants in clinical samples.