bluuebunny/crossref_metadata_2025 huggingface dataset:
Sequence Diversity of the Intergenic Spacer Region of the rRNA Gene of
Malassezia globosa
Colonizing the Skin of Patients with Atopic Dermatitis and Healthy Individuals
Authors: Sugita Takashi , Kodama Minako , Saito Masuyoshi , Ito Tomonobu , Kato Yukihiko , Tsuboi Ryoji , Nishikawa Akemi
Published: July 2003

The lipophilic yeast
Malassezia globosa
is one of the major constituents of the mycoflora of the skin of patients with atopic dermatitis (AD). We compared the genotypes of
M. globosa
colonizing the skin surface of 32 AD patients and 20 healthy individuals for polymorphism of the intergenic spacer (IGS) 1 region of the rRNA gene. Sequence analysis demonstrated that
M. globosa
was divided into four major groups, which corresponded to the sources of the samples, on the phylogenetic tree. Of the four groups, two were from AD patients and one was from healthy subjects. The remaining group included samples from both AD patients and healthy subjects. In addition, the IGS 1 region of
M. globosa
contained short sequence repeats: (CT)
n
, and (GT)
n
. The number of sequence repeats also differed between the IGS 1 of
M. globosa
from AD patients and that from healthy subjects. These findings suggest that a specific genotype of
M. globosa
may play a significant role in AD, although
M. globosa
commonly colonizes both AD patients and healthy subjects.
Identification of Enteroviruses by Using Monoclonal Antibodies against a Putative Common Epitope
Authors: Shin Soo-Youn , Kim Ki-Soon , Lee Yoon-Sung , Chung Yoon-Seok , Park Kwi-Sung , Cheon Doo-Sung , Na Byoung-Kuk , Kang Yoonsung , Cheong Hyang-Min , Moon Youngjoon , Choi Jee-Hye , Cho Hang-Eui , Min Na-Young , Son Jin-Sook , Park Young-Hoon , Jee Youngmee , Yoon Jae-Deuk , Song Chul-Yong , Lee Kwang-Ho
Published: July 2003

A common epitope region of enteroviruses was identified by sequence-independent single-primer amplification (SISPA), followed by immunoscreening of 11 cDNA libraries from two Korean enterovirus isolates (echoviruses 7 and 30) and a coxsackievirus B3 (ATCC-VR 30). The putative common epitope region was localized in the N terminus of VP1 when the displayed recombinant proteins from the phages were chased by the convalescent-phase sera. The genomic region encoding the common epitope region was amplified and then expressed by using the vector pGEX-5X-1. The antigenicity of the expressed recombinant protein was identified by Western blotting with guinea pig antisera for six different serotypes of enteroviruses. After successive immunization of mice with the recombinant common epitope protein, splenocytes were extracted and hybridized with P3X63-Ag8-653 cells. A total of 24 hybridomas that produced monoclonal antibodies (MAbs) against the putative common epitope of enteroviruses were selected. Four of these were immunoglobulin G1 isotypes with a kappa light chain. These MAbs recognized 15 Korean endemic serotypes and prototypes of enteroviruses in an indirect immunofluorescence assay. These results suggest that the expressed protein might be a useful antigen for producing group common antibodies and that the use of the MAbs against the putative common epitope of enteroviruses might be a valuable diagnostic tool for rapidly identifying a broad range of enteroviruses.
Optimization of Computer Software Settings Improves Accuracy of Pulsed-Field Gel Electrophoresis Macrorestriction Fragment Pattern Analysis
Authors: Duck William M. , Steward Christine D. , Banerjee Shailen N. , McGowan, John E. , Tenover Fred C.
Published: July 2003

Computer-assisted analysis of pulsed-field gel electrophoresis (PFGE) libraries can facilitate comparisons of fragment patterns present on multiple gels. We evaluated the ability of the Advanced Analysis (version 4.01) and Database (version 1.12) modules of the Phoretix gel analysis software package (Nonlinear USA, Inc., Durham, N.C.) to accurately match DNA fragment patterns. Two gels containing 38 lanes of
Sma
I-digested
Enterococcus faecalis
OG1RF DNA were analyzed to assess the impact of (i) varying the lane position of the standards, (ii) using gel plugs made at different times, and (iii) normalizing the fragment patterns by using molecular weight (MW) algorithms versus retardation factor (
R

f

) algorithms. Two sets of PFGE libraries (one containing
Sma
I restriction patterns from 62
Enterococcus faecium
isolates and the other containing
Sma
I restriction patterns of 89
Staphylococcus aureus
isolates) were analyzed to assess the impact of varying the matching tolerance algorithm (designated as the vector box setting [VBS]) in the Phoretix software. Varying the lane position of standards on a gel and using gel plugs made on different days resulted in different VBSs, although it was not possible to judge whether those differences were statistically significant. Normalization of
E. faecalis
OG1RF fragment patterns by
R

f

and MW methodology yielded no statistically significant differences in variability between the same fragment on different lanes. Suboptimal VBSs decreased the specificity with which related isolates were grouped together in dendrograms. The optimal VBS for analysis of PFGE fragment patterns from
E. faecalis
isolates differed from that for
S. aureus
isolates and sometimes was not that recommended by the manufacturer. Thus, computer-assisted analysis of PFGE patterns seemed to compensate for the intra- and intergel variation evaluated in the present study, and optimizing the software for the species to be tested was a critical preliminary step before further PFGE library analysis.
High Prevalence of Human Metapneumovirus Infection in Young Children and Genetic Heterogeneity of the Viral Isolates
Authors: Viazov S. , Ratjen F. , Scheidhauer R. , Fiedler M. , Roggendorf M.
Published: July 2003

RNA of the newly identified human metapneumovirus (HMPV) was detected in nasopharyngeal aspirates of 11 of 63 (17.5%) young children with respiratory tract disease. Markers of infection caused by another member of the
Pneumovirinae
subfamily of the family
Paramyxoviridae
, respiratory syncytial virus (RSV), were identified in 15 of these patients (23.8%). Three patients were simultaneously infected with HMPV and RSV. Studies of the clinical characteristics of HMPV-infected children did not reveal any difference between HMPV-infected patients and a control population of RSV-infected patients with regard to disease severity, but the duration of symptoms was significantly shorter for HMPV-infected patients. Phylogenetic analysis of the amplified viral genome fragments confirmed the existence and simultaneous circulation within one epidemic season of HMPV isolates belonging to two genetic lineages.
Quantification of Hepatitis C Virus (HCV) in Liver Specimens and Sera from Patients with Human Immunodeficiency Virus Coinfection by Using the Versant HCV RNA 3.0 (Branched DNA-Based) DNA Assay
Authors: Tedeschi Rosamaria , Pivetta Eliana , Zanussi Stefania , Bidoli Ettore , Ros Mirna , di Gennaro Giampiero , Nasti Guglielmo , De Paoli Paolo
Published: July 2003

The new generation assay Versant HCV RNA 3.0v (Bayer Diagnostics) was evaluated to quantify hepatitis C virus (HCV) RNA levels in liver biopsy specimens from patients with HCV and human immunodeficiency virus (HIV) coinfection. A total of 25 liver biopsies and sera collected at the time of liver biopsy were used. The efficiency of HCV RNA recovery from spiked samples was between 38.6 and 50.7%, and reproducible measurements of viral load were observed (the intra- and interrun coefficients of variation were 0.5 to 13% and 3.5 to 24.7%, respectively), with good specificity and sensitivity. Linearity was evaluated in the range of 96,154 to 769 IU/μg by using a serially diluted high-titer sample. Coinfected patients had high HCV RNA viral loads in serum and liver (498,471 IU/ml and 231,495 IU/μg, respectively), and both levels were correlated (
r
= 0.63;
P
< 0.01). The amount of hepatic HCV RNA was significantly higher among patients with genotype 1 than among patients with genotype 3 (
P
< 0.01). The virological end-of-treatment response in the serum was associated with a lower pretreatment intrahepatic HCV viral load (
P
= 0.03). The new version of b-DNA is a sensitive, specific, and reproducible method for quantitating HCV RNA in the liver. Given its positive analytical performance, the assay will be used to evaluate the HCV RNA levels in the serum and liver during follow-up of patients treated with an anti-HCV therapeutic regimen.
Major Outbreak of Toxic Shock-Like Syndrome Caused byStreptococcus mitis
Authors: Lu Hong-Zhou , Weng Xin-Hua , Zhu Bai , Li Haijing , Yin You-Kuan , Zhang Yong-Xin , Haas David W. , Tang Yi-Wei
Published: July 2003

Severe illness caused by viridans streptococci rarely occurs in immunocompetent hosts. Between December 1990 and May 1991, thousands of patients in the YangZi River Delta area of Jiangsu Province, China, suffered from scarlet fever-like pharyngitis. Fewer cases occurred in subsequent years with the same seasonality. Approximately half of the cases developed complications characteristic of streptococcal toxic shock-like syndrome (TSLS). Throat cultures yielded predominant growth of alpha-hemolytic streptococci. All cases admitted to Haian People's Hospital were investigated. Clinical specimens were collected, medical records were reviewed, and bacterial isolates were identified phenotypically and analyzed by 16S rRNA gene sequencing and pulsed-field gel electrophoresis (PFGE). Proteins were purified from culture supernatants by extraction, ammonium sulfate precipitation, and fast-protein liquid chromatography. Biological activities of protein components were determined by subcutaneous inoculation into rabbits. A total of 178 cases of non-beta-hemolytic streptococcal scarlet fever-like pharyngitis were studied. In 88 (79.3%) of 111 patients, oropharyngeal swab cultures grew morphologically identical alpha-hemolytic streptococci. A protein in culture supernatants was pyrogenic in rabbits, was mitogenic for splenocytes, and enhanced rabbit susceptibility to endotoxin challenge. The N-terminal amino acid sequence of this 34-kDa protein showed no homology with knownStreptococcuspyrogenic exotoxins. The organism was identified asStreptococcus mitisbased on biochemical and 16S rRNA sequence analyses. Representative outbreak isolates from 1990 to 1995 displayed identical PFGE patterns. This TSLS outbreak in southeastern China was caused by a toxigenic clone ofS. mitis. An apparently novel toxin may explain the unusual virulence of this organism.
Field Evaluation of the
gag
-Based Heteroduplex Mobility Assay for Genetic Subtyping of Circulating Recombinant Forms of Human Immunodeficiency Virus Type 1 in Abidjan, Côte d'Ivoire
Authors: Sawadogo Souleymane , Adjé-Touré Christiane , Bilé Celestin E. , Ekpini Rene E. A. , Chorba Terence , Nkengasong John N.
Published: July 2003

The
gag
-based heteroduplex mobility assay (
gag
-HMA) was evaluated for its ease and reliability in subtyping circulating recombinant forms (CRFs) of human immunodeficiency virus type 1 (HIV-1) in Côte d'Ivoire. One hundred thirty-two plasma samples were analyzed blindly for HIV-1 subtypes by sequencing the
pol
gene and by
gag
-HMA. DNA sequencing was used as the “gold standard.” Of the 132 samples sequenced, 108 (82%) were CRF02_AG, 14 (11%) were pure subtype A, 5 (4%) were subtype G, 3 (2%) were subtype D, 1 was CRF01_AE, and 1 was subtype H. The
gag
-HMA correctly classified 126 (95.5%) of the samples. Of the 108 samples that were classified as CRF02_AG by DNA sequencing, 107 (99%) were correctly identified by
gag
-HMA, resulting in a positive predictive value of 96.4%. The
gag
-HMA seems to be a valuable tool for understanding the molecular epidemiology of HIV-1 CRF02_AG in Côte d'Ivoire and West Africa, which could be important for developing and evaluating AIDS vaccines, although DNA sequencing remains necessary for accurate molecular epidemiology.
Comparison of Two Commercial Enzyme-Linked Immunosorbent Assays with an Immunofluorescence Assay for Detection ofLegionella pneumophilaTypes 1 to 6
Authors: Malan Annette K. , Martins Thomas B. , Jaskowski Troy D. , Hill Harry R. , Litwin Christine M.
Published: July 2003

Members of the genusLegionellaare characterized as gram-negative, motile, freshwater-dwelling bacteria that were responsible for a pneumonia outbreak among American Legion members in 1976. Because clinicians routinely order serologic testing forLegionella pneumophilaserogroups 1 to 6 as a screen for possibleL. pneumophilainfections, we evaluated the Wampole LaboratoriesL. pneumophilatype 1 to 6 immunoglobulin G (IgG) and IgM combined enzyme-linked immunosorbent assay (ELISA) and the Zeus ScientificL. pneumophilatype 1 to 6 IgG-IgM-IgA multispecific combined ELISA systems and compared them to an IgG-specific immunofluorescence assay (IFA) forL. pneumophilaserogroups 1 to 6. The Centers for Disease Control and Prevention recommends that the positive titer cutoff for an IFA be 1:256. Regardless of where the positive IFA cutoff titer is placed, however, the sensitivity of both commercial assays was below what would be acceptable for a screening assay. With a 1:256 IFA titer as the positive cutoff, the agreement, sensitivity, and specificity of the Wampole ELISA were 74.6, 21.4, and 98.4%, respectively. The agreement, sensitivity, and specificity of the Zeus ELISA were 72.6, 10.5, and 100.0%, respectively. We recommend that any laboratories attempting to replace an IFA type 1 to 6 screen with an alternative ELISA carefully investigate the sensitivity of the replacement assay.
Invasive Disease Due to Nontypeable
Haemophilus influenzae
among Children in Arkansas
Authors: O'Neill Joshua M. , St. Geme Joseph W. , Cutter David , Adderson Elisabeth E. , Anyanwu Juliana , Jacobs Richard F. , Schutze Gordon E.
Published: July 2003

In this study, we reviewed cases of invasive disease due to nontypeable
Haemophilus influenzae
among children hospitalized at Arkansas Children's Hospital from 1993 to 2001. A total of 28 cases were examined, including 21 associated with bacteremia and 4 associated with meningitis. Of the patients examined, 86% were ≤4 years of age, and 68% had underlying medical conditions. Characterization of the bacterial isolates by multilocus sequence type genotyping revealed significant overall genetic diversity, similar to the diversity in the general population structure for nontypeable
H. influenzae
. However, four separate pairs of isolates were closely related genetically, a relationship confirmed by pulsed-field gel electrophoresis and Southern hybridization studies using probes for the major
H. influenzae
adhesin genes. These results suggest that selected strains of nontypeable
H. influenzae
may have more invasive potential, especially in young children and patients with underlying medical conditions. At this point, the specific factors that contribute to enhanced virulence remain unclear.
Mycobacterium bovis
subsp.
caprae
Caused One-Third of Human
M. bovis
-Associated Tuberculosis Cases Reported in Germany between 1999 and 2001
Authors: Kubica Tanja , Rüsch-Gerdes Sabine , Niemann Stefan
Published: July 2003

The prevalence of the
Mycobacterium bovis
subsp.
caprae
and
M. bovis
subsp.
bovis
among German tuberculosis cases caused by the bovine tubercle bacillus from 1999 to 2001 was determined. Isolates from 166 patients living in Germany and 10 animals were analyzed by conventional laboratory procedures, spoligotyping, and partly by PCR-restriction fragment length polymorphism analysis of the
gyrB
gene. By spoligotyping, 55 of 176 isolates (31%) could be identified as
M. bovis
subsp.
caprae
, and 121 (69%) were confirmed as
M. bovis
subsp.
bovis.
In general, a low variability of spoligotypes with 59 distinct patterns and a cluster rate of 77% (136 isolates/19 clusters) was determined. About half of all isolates were grouped in the three main clusters with 29, 30, and 35 isolates, respectively. Differences in age and gender between the patient groups infected with
M. bovis
subsp.
bovis
and
M. bovis
subsp.
caprae
did not reach statistical significance. However, marked differences in the geographical prevalence of
M. bovis
subsp.
caprae
were observed, ranging from fewer than 10% of all
M. bovis
isolates in the north up to more than 80% of isolates in the south of Germany. In conclusion,
M. bovis
subsp.
caprae
accounts for a high ratio of human
M. bovis
-associated tuberculosis cases in Germany and was more frequently found in the southern part.
Molecular Epidemiology of
Shigella
in aTaiwan Township during 1996 to2000
Authors: Chen Jiann-Hwa , Chiou Chien-Shun , Chen Pei-Chun , Liao Tung-Liang , Liao Tsai-Ling , Li Jen-Ming , Hsu Wen-Bin
Published: July 2003

A
previously identified
Shigella flexneri
serotype 2a strain was
responsible for an outbreak of shigellosis in a Taiwan township in
August 1996. In order to find the relationship between this outbreak
strain and subsequent
Shigella
infections in the
area, 59, 47, 35, and 20
Shigella
isolates recovered in 1997,
1998, 1999, and 2000, respectively, were collected and typed by
serological and pulsed-field gel electrophoresis (PFGE) techniques. Of
these 161 isolates, 139 isolates were
S. flexneri
serotype 2a,
and one-third of them (47 isolates) exhibited the outbreak pattern. The
remaining 92
S. flexneri
serotype 2a isolates displayed 49
different
Not
I-PFGE patterns. Forty-five patterns were closely
related to the outbreak pattern, with deletions of three specific
Not
I fragments occurring with high frequency. While the
outbreak strain remained the main cause of shigellosis after the
outbreak, the continuous emergence of closely related though poorly
transmissible strains from the outbreak strain contributed to the
observed annual decrease of shigellosis in the
area.
Reverse Transcription-PCR Assays for Detection of Bovine Enteric Caliciviruses (BEC) and Analysis of the Genetic Relationships among BEC and Human Caliciviruses
Authors: Smiley J. R. , Hoet A. E. , Tråvén M. , Tsunemitsu H. , Saif L. J.
Published: July 2003

Two genetically distinct bovine enteric caliciviruses (BECs) have been identified: the norovirus (NLV) Jena and Newbury Agent-2 (NA-2) BECs, which are genetically related to human noroviruses, and the Nebraska (NB) BECs, which is related to sapoviruses and lagoviruses but may also represent a new calicivirus genus. The prevalence of these two BEC genotypes in cattle is unknown. Although reverse transcription-PCR (RT-PCR) primers for human NLV recognize NLV-BECs, the genetic relationships between NLV from humans and the NLV-BECs commonly circulating in cattle is undefined. In the present study, veal calf fecal samples were assayed for enteric caliciviruses by using six RT-PCR primer sets designed for the detection of human NLVs or BECs. Caliciviruses genetically related to the NLV-BEC Jena and NA-2 strains or to the recently characterized NB BEC strain were identified in three of four and four of four sampled veal herds, respectively. Extended 3′-terminal genome sequences of two NLV-BECs, designated CV95-OH and CV186-OH, encoding the RNA-dependent RNA polymerase (RdRp; open reading frame 1 [ORF-1]), VP1 (ORF-2), and VP2 (ORF-3) genes were determined. Phylogenetic and sequence identity analyses of each genome region demonstrated these viruses to be most closely related to the NLV-BEC Jena and NA-2 strains. In initial testing, the human P289-P290 (P289/290) primer set was found to be the most sensitive for calicivirus detection. However, its failure to identify all positive fecal pools (as determined by other assays) led us to design two new primer sets, CBECU-F/R and NBU-F/R, for the sensitive and specific detection of NLV-BEC (NLV-BEC Jena and NA-2) and BEC-NB-like viruses, respectively. The RT-PCR assays with the new primers were compared against other primer sets, including P289/290. Composite results of the tests completed by using the new assays identified 72% (54 of 75) of veal calf fecal samples as positive, with 21 of 21 sequenced reaction products specific for the target RdRp gene. The same design strategy used for the new BEC assays may also be applicable to the design of similar assays for the detection of human caliciviruses (HuCVs). Our data support the genetic relationship between NLV-BECs and NLV-HuCVs but with the NLV-BECs comprising two clusters within a third NLV genogroup.
Characterization of Serotype G9 Rotavirus Strains Isolated in the United States and India from 1993 to 2001
Authors: Laird A. R. , Gentsch J. R. , Nakagomi T. , Nakagomi O. , Glass R. I.
Published: July 2003

The emergence of rotavirus serotype G9 as a possible fifth globally common serotype in the last decade, together with its increasing detection in association with various genome constellations, raises questions about the origins and epidemiological importance of recent G9 isolates. We examined a collection of 40 G9 strains isolated in the United States from 1996 to 2001 and in India since 1993 to determine their VP7 gene sequences, P types, E types, subgroup specificities, and RNA-RNA hybridization profiles. With the exception of two U.S. strains, all of the study strains shared high VP7 gene sequence homology (<2.5% sequence divergence on both the nucleotide and amino acid levels) and were more closely related to other recent isolates than to the first G9 strains isolated in the 1980s. The VP7 gene sequence and RNA-RNA hybridization profiles of the long-E-type strains showed greater variation than the short-E-type strains, suggesting that the latter strains are the result of a relatively recent reassortment event of the G9 VP7 gene into a short-E-type lineage. No evidence for reassortment of genes other than VP4 and VP7 between major human rotavirus genogroups was observed. Except for Om46 and Om67, which formed a distinct clade, phylogenetic analysis showed that most of the study strains grouped together, with some subgroups forming according to genetic constellation, geographic location, and date of isolation. The high potential of G9 strains to generate different P and G serotype combinations through reassortment suggests that it will be important to determine if current vaccines provide heterotypic protection against these strains and underscores the need for continued surveillance for G9 and other unusual or emerging rotavirus strains.
Identification ofcagATyrosine Phosphorylation DNA Motifs inHelicobacter pyloriIsolates from Peptic Ulcer Patients by Novel PCR-Restriction Fragment Length Polymorphism and Real-Time Fluorescence PCR Assays
Authors: Owen Robert J. , Sharp Sally I. , Chisholm Stephanie A. , Rijpkema Sjoerd
Published: July 2003

Cag pathogenicity island-containingHelicobacter pylori(type I) induces signal transduction pathways resulting in tyrosine phosphorylation of proteins adjacent to the site of bacterial adhesion on host gastric epithelial cells. Conventional block PCR-restriction fragment length polymorphism (RFLP) and real-time LightCycler (LC) PCR hybridization assays, validated by direct sequencing, were designed to test for the presence of three nucleotide sequences corresponding to tyrosine phosphorylation motifs (TPMs) A, B, and C in 84 isolates ofH. pyloritype I from patients in England. Overall, the PCR assays demonstrated that one or more TPMs were present in 62 strains (75%). Motif A was common (71% of strains), whereas motifs B and C were rarer (8% of strains). Strains lacking a TPM were typically vacuolating cytotoxin genotypevacAm2. Motif A was widely distributed in relation to disease severity and was more commonly (but not significantly [P= 0.071]) associated with gastric ulcer than with duodenal ulcer (86 versus 56%). The LC hybridization assay provided a rapid means of detecting all three motifs, but RFLP analysis was more specific for TPM-A. TPMs provide novel additional strain markers for definingcagAvariation, including identification of RFLP types within TPM-A. The presence of a particular TPM was not of direct diagnostic value, either singly or in combination, but the higher proportion of TPM-A strains in gastric ulcer patients merits further investigation.
Effects of Rapid Detection of Bloodstream Infections on Length of Hospitalization and Hospital Charges
Authors: Beekmann S. E. , Diekema D. J. , Chapin K. C. , Doern G. V.
Published: July 2003

Current automated continuous-monitoring blood culture systems afford more rapid detection of bacteremia and fungemia than is possible with non-instrument-based manual methods. Use of these systems has not been studied objectively with respect to impact on patient outcomes, including hospital charges and length of hospitalization. We conducted a prospective, two-center study in which the time from the obtainment of the initial positive blood culture until the Gram stain was called was evaluated for 917 cases of bloodstream infection. Factors showing univariate associations with a shorter time to notification included higher body temperature and respiratory rate and higher percentage of immature neutrophils. Multiple linear regression models determined that the primary predictors of both increased microbiology laboratory and total hospital charges for patients with bloodstream infection were nonmicrobiologic and included length of stay and host factors such as the admitting service and underlying illness score. Significant microbiologic predictors of increased charges included the number of blood cultures obtained, nosocomial acquisition, and polymicrobial bloodstream infections. Accelerated failure time regression analysis demonstrated that microbiologic factors, including time until notification, organism group, and nosocomial acquisition, were independently associated with length of hospitalization after bacteremia, as were the factors of admitting service, gender, and age. Our data suggest that an increased time to notification of bloodstream infection is independently associated with increased length of stay. We conclude that the time to notification is an obvious target for efforts to shorten length of stay. The newest generation of automated continuous-monitoring blood culture systems, which shorten the time required to obtain a positive result, should impact length of hospitalization.
Genetic Polymorphism and Molecular Epidemiology ofLeishmania(Viannia)braziliensisfrom Different Hosts and Geographic Areas in Brazil
Authors: Cupolillo Elisa , Brahim Lúcia Regina , Toaldo Cristiane B. , de Oliveira-Neto Manoel Paes , de Brito Maria Edileuza Felinto , Falqueto Aloisio , de Farias Naiff Maricleide , Grimaldi Gabriel
Published: July 2003

Numerical zymotaxonomy and variability of the internal transcribed spacers (ITS) between the small and large subunits of the rRNA genes were used to examine strain variation and relationships in natural populations ofLeishmania(Viannia)braziliensis. A total of 101 strains from distinct hosts and Brazilian geographic regions were assigned to 15 zymodemes clustered in two major genetic groups. The great number of isolates (48.5%) placed in zymodeme IOC/Z-27 were collected on the Atlantic coast. The high molecular diversity found in populations in the Amazon Basin was related to the great number of sandfly vector(s) in that region. The results of the restriction fragment length polymorphism analysis of the ITS depicted considerable intraspecific variation. Genotypic groups A, B, and C contained 39, 40, and 22 isolates, which were divided into 16, 10, and 15 genotypes, respectively. The genetic polymorphism observed demonstrates the degree of diversity ofL.(V.)braziliensisstrains from different regions where they are endemic. The results reinforce the clonal theory forLeishmaniaparasites showing the genetic diversity of this pathogen and an association ofL.(V.)braziliensisgenotypes with specific transmission cycles, probably reflecting an adaptation of different clones to the vector species involved.
Evaluation of Real-Time PCR versus PCR with Liquid-Phase Hybridization for Detection of Enterovirus RNA in Cerebrospinal Fluid
Authors: Lai K. Kay-Yin , Cook Linda , Wendt Sharon , Corey Lawrence , Jerome Keith R.
Published: July 2003

A LightCycler and two TaqMan real-time PCR assays were evaluated against an older PCR with liquid-phase hybridization method for the detection of enterovirus RNA in 74 patient samples. The two-step LightCycler and the two-step TaqMan formats correlated well with each other (
r
2
= 0.90) and were equally sensitive compared to the liquid-phase hybridization method, whereas the one-step recombinant
Tth
DNA polymerase format was rather insensitive, detecting enterovirus RNA in only about one-half of those patient samples previously positive by liquid-phase hybridization. The two-step TaqMan method was optimized utilizing 10 μl of cDNA and demonstrated the highest degree of analytical sensitivity among the methods evaluated in our study, being able to reproducibly quantify down to 510 copies of enteroviral RNA/ml of cerebrospinal fluid. This new assay can be performed in 4 h, is much less labor intensive, and showed less cross-reactivity with rhinovirus than the liquid-phase hybridization assay. Thus, the two-step TaqMan assay should prove useful in the diagnosis of enteroviral meningitis versus bacterial meningitis, thereby resulting in timely and appropriate clinical management that can amount to significant cost savings to the patient and health care system.
Evaluation of the NCCLS Extended-Spectrum β-Lactamase Confirmation Methods forEscherichia coliwith Isolates Collected during Project ICARE
Authors: Tenover Fred C. , Raney Patti M. , Williams Portia P. , Rasheed J. Kamile , Biddle James W. , Oliver Antonio , Fridkin Scott K. , Jevitt Laura , McGowan, John E.
Published: July 2003

To determine whether confirmatory tests for extended-spectrum β-lactamase (ESBL) production inEscherichia coliare necessary, we selected 131E. coliisolates that met the National Committee for Clinical Laboratory Standards (NCCLS) screening criteria for potential ESBL production from the Project ICARE (Intensive Care Antimicrobial Resistance Epidemiology) strain collection. For all 131 isolates, the broth microdilution (BMD) MIC of at least one extended-spectrum cephalosporin was ≥2 μg/ml. For 21 of 131 (16%) isolates, the ESBL confirmatory test was positive; i.e., the BMD MICs of ceftazidime or cefotaxime decreased by ≥3 doubling dilutions in the presence of clavulanic acid (CA) or the disk diffusion zone diameters increased by ≥5 mm around ceftazidime or cefotaxime disks in the presence of CA. All 21 isolates were shown by PCR to contain at least one of the genesblaTEM,blaSHV, andblaOXA, and in isoelectric focusing (IEF) tests, all isolates demonstrated at least one β-lactamase band consistent with a TEM, SHV, or OXA enzyme. Of the 21 isolates, 3 showed a CA effect for cefotaxime by BMD but not by disk diffusion testing. A total of 59 (45%) of the 131 isolates demonstrated decreased susceptibility to cefpodoxime alone (MIC = 2 to 4 μg/ml), and none had a positive ESBL confirmatory test result. These were classified as false positives according to ESBL screen test results. For the remaining 51 (39%) isolates, the cefpodoxime MICs ranged from 16 to >128 μg/ml and the MICs for the other extended-spectrum cephalosporins were highly variable. All 51 isolates gave negative ESBL confirmatory test results. Most showed IEF profiles consistent with production of both a TEM and an AmpC β-lactamase, and representative isolates of several phenotypic groups showed changes in porin profiles; these 51 isolates were considered true negatives. In all, only 16% of 131E. coliisolates identified as potential ESBL producers by the current NCCLS screening criteria were confirmed as ESBL producers. Thus, changing the interpretation of extended-spectrum cephalosporins and aztreonam results from the susceptible to the resistant category without confirming the presence of an ESBL phenotype would lead to a large percentage of false resistance results and is not recommended. However, by increasing the cefpodoxime MIC screening breakpoint to ≥8 μg/ml, 45% of the false-positive results could be eliminated. NCCLS has incorporated this change in the cefpodoxime screening breakpoint in its recent documents.
Identification and Differentiation ofLeishmaniaSpecies in Clinical Samples by PCR Amplification of the Miniexon Sequence and Subsequent Restriction Fragment Length Polymorphism Analysis
Authors: Marfurt Jutta , Nasereddin Abed , Niederwieser Igor , Jaffe Charles L. , Beck Hans-Peter , Felger Ingrid
Published: July 2003

We recently developed a new PCR-restriction fragment length polymorphism (RFLP)-based assay using the miniexon sequence from the genusLeishmania. Here we report the application of this new genotyping method to naturally infected clinical samples for the differentiation of New and Old WorldLeishmaniaspecies. Of the newly developed assay and four currently applied diagnostic tests (i.e., in vitro cultivation, serology, and two other molecular assays using either the small subunit-internal transcribed spacer sequence or a repetitive genomic sequence), the miniexon assay showed the highest sensitivity, 89.7%, compared to 70.6, 57.1, 51.7, and 79.3%, respectively. Species differentiation was robust and reliable compared with that by two otherLeishmaniagenotyping techniques. The assay provides a valuable tool for the identification ofLeishmaniadirectly from clinical samples and enables determination of the infecting species by a facile technique with high discrimination power. SinceLeishmaniacauses a broad spectrum of diseases distinguished by different parasite and host factors, detection and characterization of the infecting species is crucial for the confirmation of a diagnosis as well as the establishment of the clinical prognosis and the initiation of an adequate therapeutic approach. The miniexon PCR-RFLP assay will facilitate such determination and might improve diagnosis and treatment of leishmaniasis.
Improved Assay To Detect Neutralizing Antibody following Vaccination with Diluted or Undiluted Vaccinia (Dryvax) Vaccine
Authors: Newman Frances K. , Frey Sharon E. , Blevins Tamara P. , Mandava Mahendra , Bonifacio, Andres , Yan Lihan , Belshe Robert B.
Published: July 2003

The assessment of immunogenicity of a diluted vaccinia vaccine for possible widespread use of a diluted vaccine in the event of a bioterrorist attack prompted us to focus on the development of a sensitive and specific plaque reduction neutralization (PRN) assay to assess the antibody response of volunteers to a vaccinia (Dryvax) vaccine. Two incubation times, 1 h or overnight (approximately 15 h), were explored for the neutralization step of the assay. In addition, serum samples were evaluated using both sonicated and nonsonicated virus in PRN assays with 1 and 15 h of incubation. The use of the overnight incubation method resulted in the detection of antibody in two vaccinated individuals who exhibited a take, i.e., a major reaction indicative of successive vaccination as defined by the Centers for Disease Control and Prevention, but did not have a fourfold increase in antibody to vaccinia virus by the 1-h-incubation method and increased the sensitivity from 94 to 100%. In addition to the increased sensitivity of the assay, we noted a significant increase (approximately 40-fold) in the PRN titer of serum samples tested with the 15-h-incubation method. The use of sonicated virus increased the reproducibility of the virus titers and PRN titers. Forty-two percent of the samples tested using sonicated virus had a PRN titer that was fourfold higher or greater than that of nonsonicated virus in the assay. A PRN titer that was threefold higher or greater was observed in more than half (58%) of the samples using sonicated virus. Therefore, the more sensitive, specific, and reproducible plaque neutralization assay for the detection of antibody to vaccinia virus is the method using a 15-h-incubation time and freshly sonicated vaccinia virus.
Rotavirus Diarrhea Severity Is Related to the VP4 Type in Mexican Children
Authors: Mota-Hernández Felipe , José Calva Juan , Gutiérrez-Camacho Claudia , Villa-Contreras Sofía , Arias Carlos F. , Padilla-Noriega Luis , Guiscafré-Gallardo Héctor , Guerrero María de Lourdes , López Susana , Muñoz Onofre , Contreras Juan F. , Cedillo Roberto , Herrera Ismael , Puerto Fernando I.
Published: July 2003

This report is of a community-based case control study to assess whether the severity of acute diarrhea by rotavirus (RV) in young children is associated with a particular VP7 (G) or VP4 (P) RV serotype. Five hundred twenty children younger than 2 years of age with diarrhea lasting less than 3 days were age and gender matched with 520 children with no diarrhea. The G and P serotypes were determined with specific monoclonal antibodies, and the VP4 serotype specificity in a subgroup was confirmed by genotyping. Infection with a G3 serotype led to a higher risk of diarrhea than infection with a G1 serotype. Infection with a G3-nontypeable-P serotype was associated with more severe gastroenteritis than infection with a G3 (or G1) P1A[8] serotype. A child with diarrhea-associated dehydration was almost five times more likely to be infected with a G3-nontypeable-P serotype than a child without dehydration (
P
< 0.001). Moreover, the two predominant monotypes within serotype P1A[8] had significantly different clinical manifestations. In this study, the severity of RV-associated diarrhea was related to different P serotypes rather than to G serotypes. The relationship between serotype and clinical outcomes seems to be complex and to vary among different geographic areas.
Comparison of Culture Screening Methods for Detection of Nasal Carriage of Methicillin-Resistant
Staphylococcus aureus
: a Prospective Study Comparing 32 Methods
Authors: Safdar Nasia , Narans Leah , Gordon Barbara , Maki Dennis G.
Published: July 2003

Screening for carriage of methicillin-resistant
Staphylococcus aureus
(MRSA) is fundamental to modern-day nosocomial infection control, both for epidemiologic investigation and day-to-day decisions on barrier isolation. Numerous microbiologic techniques have been advocated for screening for nasal carriage of MRSA, including the use of charcoal rather than rayon swabs, preincubation of swabs in Stuart's medium, preincubation of swabs in salt-containing trypticase soy broth (TSB), use of mannitol-salt agar (MSA), use of MSA containing oxacillin (MSA
Ox
), use of Mueller-Hinton agar containing oxacillin (MHA
Ox
), and the use of MSA containing lipovitellin with an oxacillin disk (MSAL
Ox
). We report a prospective clinical trial undertaken to test all of these methods concurrently. Patients at high risk for MRSA carriage were screened with eight consecutive nasal swabs (four standard rayon, four charcoal-coated rayon), which were processed by primary plating on MSA, MSA
Ox
, MHA
Ox
, and MSAL
Ox
; Stuart's preincubation for 72 h followed by plating on the solid media; overnight enrichment in salt-containing TSB followed by plating; and Stuart's preincubation for 72 h followed by overnight enrichment in TSB and plating. All of the above methods were repeated with charcoal swabs. Each patient was screened by 32 culture methods. Forty-three (42%) of 102 patients studied were positive for MRSA by one or more methods. Among the four media evaluated with direct plating, MSAL
Ox
was 11 to 25% more sensitive for detecting MRSA (MSAL
Ox
versus MSA
Ox
or MHA
Ox
or MSA, each
P
< 0.01). Preincubation in Stuart's medium for 72 h did not enhance recovery of MRSA. Enrichment in salt-containing TSB further increased yield 9%. MSAL
Ox
also showed the best specificity, 93%. Charcoal swabs showed no advantage over standard rayon swabs. Our results suggest that the highest yield will be achieved by using standard rayon swabs that are enriched overnight in TSB with inoculation onto MSAL
Ox
medium. Direct inoculation of swabs onto MSAL
Ox
allows detection of 90% of MRSA carriers.
Comparison of LightCycler-Based PCR, COBAS Amplicor CMV Monitor, and pp65 Antigenemia Assays for Quantitative Measurement of Cytomegalovirus Viral Load in Peripheral Blood Specimens from Patients after Solid Organ Transplantation
Authors: Pang Xiaoli L. , Chui Linda , Fenton Jayne , LeBlanc Barbara , Preiksaitis Jutta K.
Published: July 2003

In order to evaluate the LightCycler-based PCR (LC-PCR) as a diagnostic assay technique, a classical pp65 antigenemia assay and the commercially available COBAS Amplicor CMV Monitor (CACM) assay were compared to the LC-PCR assay for the detection and quantitation of cytomegalovirus (CMV) load in 404 parallel specimens of peripheral blood from 66 patients after solid organ transplantation. A good correlation existed among these three assays (
r
≅ 0.6,
P
< 0.0001). The LC-PCR assay was the most sensitive (54% of specimens positive) compared to the CACM (48.6%) and the pp65 antigenemia (26%) assays. The LC-PCR assay detected all samples found positive by using both the CMV pp65 antigenemia assay and the CACM assay. The LC-PCR also had the widest dynamic range (from 250 to 10
7
DNA copies/ml of plasma). No cross-reactions were found among CMV and Epstein-Barr virus, varicella-zoster virus, or herpes simplex virus in the LC-PCR by using amplification with specifically designed primer pairs. Precision, expressed as the coefficient of variation, was <3% with standard DNA from cell cultures and between 6.55 and 14.1% with clinical specimens in repeat LC-PCR runs. One run of the LC-PCR took half of the time required for the semiautomated CACM procedure. Because of its sensitivity, specificity, cost-effectiveness, and simplicity, the LC-PCR assay could replace the pp65 antigenemia and the CACM assays as the preferred technique for the surveillance, diagnosis, and monitoring of response of CMV diseases in high-risk populations.
Environmental Isolation of
Balamuthia mandrillaris
Associated with a Case of Amebic Encephalitis
Authors: Schuster Frederick L. , Dunnebacke Thelma H. , Booton Gregory C. , Yagi Shigeo , Kohlmeier Candice K. , Glaser Carol , Vugia Duc , Bakardjiev Anna , Azimi Parvin , Maddux-Gonzalez Mary , Martinez A. Julio , Visvesvara Govinda S.
Published: July 2003

This report describes the first isolation of the ameba
Balamuthia mandrillaris
from an environmental soil sample associated with a fatal case of amebic encephalitis in a northern California child. Isolation of the ameba into culture from autopsied brain tissue confirmed the presence of
Balamuthia
. In trying to locate a possible source of infection, soil and water samples from the child's home and play areas were examined for the presence of
Balamuthia
. The environmental samples (plated onto nonnutrient agar with
Escherichia coli
as a food source) contained, in addition to the ameba, a variety of soil organisms, including other amebas, ciliates, fungi, and nematodes, as contaminants. Presumptive
Balamuthia
amebas were recognized only after cultures had been kept for several weeks, after they had burrowed into the agar. These were transferred through a succession of nonnutrient agar plates to eliminate fungal and other contaminants. In subsequent transfers, axenic
Naegleria
amebas and, later, tissue cultures (monkey kidney cells) served as the food source. Finally, the amebas were transferred to cell-free axenic medium. In vitro, the
Balamuthia
isolate is a slow-growing organism with a generation time of ∼30 h and produces populations of ∼2 × 10
5
amebas per ml. It was confirmed as
Balamuthia
by indirect immunofluorescence staining with rabbit anti-
Balamuthia
serum and human anti-
Balamuthia
antibody-containing serum from the amebic encephalitis patient. The environmental isolate is similar in its antimicrobial sensitivities and identical in its 16S ribosomal DNA sequences to the
Balamuthia
isolate from the deceased patient.
Molecular Typing of Methicillin-Resistant
Staphylococcus aureus
: Can PCR Replace Pulsed-Field Gel Electrophoresis?
Authors: Strandén A. , Frei R. , Widmer A. F.
Published: July 2003

Pulsed-field gel electrophoresis (PFGE) is considered the “gold standard” for molecular typing of methicillin-resistant
Staphylococcus aureus
(MRSA). However, the method is time-consuming and expensive, and its discriminatory power may not be necessary in outbreak situations. We used a rapid multiplex PCR-based method with published primers and compared the results with those obtained by PFGE. A total of 75 clinical isolates were typed: 59 strains originated from our prospectively collected clinical strains and were epidemiologically unrelated; 16 strains came from an outbreak that was epidemiologically well defined in time and space. A primer mix of the
spa
gene, the
coa
gene, and the hypervariable region adjacent to
mec
A gene was used for multiplex PCR. Both PFGE and PCR clustered the 75 strains into 41 different genotypes. Concordance of the results was 100% for strains originating from the outbreak. Overall, both methods produced concordant results in 72% of cases. A total of 16% were clustered together by PFGE, but not by PCR and 12% were clustered together by PCR but not by PFGE, respectively. The turnaround time was only 8 h for PCR but 5 days for PFGE. This PCR-based method is excellent for rapid and inexpensive typing of MRSA in an outbreak setting, but the discriminatory power and reproducibility are still insufficient to replace PFGE in longitudinal studies in the endemic setting.
Evaluation of an Isothermal Signal Amplification Method for Rapid Detection of Methicillin-Resistant
Staphylococcus aureus
from Patient-Screening Swabs
Authors: Levi Katrina , Bailey Claire , Bennett Alexandra , Marsh Peter , Cardy Don L. N. , Towner Kevin J.
Published: July 2003

A new molecular assay (CytAMP) utilizing isothermal signal-mediated amplification of RNA was evaluated for rapid detection of methicillin (oxacillin)-resistant
Staphylococcus aureus
(MRSA). The assay targeted the
coa
(coagulase) and
mecA
genes, thereby simultaneously identifying
S. aureus
and methicillin (oxacillin) resistance. Results were obtained in approximately 3.5 h as a color signal in 96-well microtiter plates. The detection limit was between 2 × 10
5
and 10
6
CFU/assay, equivalent to 4 × 10
6
to 2 × 10
7
CFU/ml in an overnight broth. This level of growth was obtained with an initial inoculum of 10 to 50 CFU. The CytAMP assay and a
mecA
-
femB
PCR assay both detected 113 MRSA strains among 396 clinical isolates of bacteria (CytAMP sensitivity and specificity were both 100% relative to those of PCR). Conventional culture detected 109 MRSA strains, but with 19 false-positive and 23 false-negative results relative to both molecular methods. Discrepancies were also observed for 100 enrichment broths containing MRSA screening swabs, with 11 broths culture negative but PCR positive. CytAMP and PCR were more in agreement, but six broths were CytAMP negative and PCR positive. Five of these contained 10
2
to 10
5
CFU/assay (below the CytAMP detection limit of 2 × 10
5
CFU/assay), and the sixth contained 10
6
CFU/assay. Overall, culture and CytAMP had similar sensitivities and specificities relative to those of PCR, but the CytAMP assay enabled swabs to be analyzed as a batch following overnight incubation in enrichment broth, with results reported before 12 noon the next day.