bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection and Genotyping of Oocysts of
Cryptosporidium parvum
by Real-Time PCR and Melting Curve Analysis
Authors: Tanrıverdi Sultan , Tanyeli Atila , Başlamışlı Fikri , Köksal Fatih , Kılınç Yurdanur , Feng Xiaochuan , Batzer Glenda , Tzipori Saul , Widmer Giovanni
Published: August 2002

Several real-time PCR procedures for the detection and genotyping of oocysts of
Cryptosporidium parvum
were evaluated. A 40-cycle amplification of a 157-bp fragment from the
C. parvum
β-tubulin gene detected individual oocysts which were introduced into the reaction mixture by micromanipulation. SYBR Green I melting curve analysis was used to confirm the specificity of the method when DNA extracted from fecal samples spiked with oocysts was analyzed. Because
C. parvum
isolates infecting humans comprise two distinct genotypes, designated type 1 and type 2, real-time PCR methods for discriminating
C. parvum
genotypes were developed. The first method used the same β-tubulin amplification primers and two fluorescently labeled antisense oligonucleotide probes spanning a 49-bp polymorphic sequence diagnostic for
C. parvum
type 1 and type 2. The second genotyping method used SYBR Green I fluorescence and targeted a polymorphic coding region within the GP900/poly(T) gene. Both methods discriminated between type 1 and type 2
C. parvum
on the basis of melting curve analysis. To our knowledge, this is the first report describing the application of melting curve analysis for genotyping of
C. parvum
oocysts.
Dichotomy of Glycoprotein G Gene in Herpes Simplex Virus Type 1 Isolates
Authors: Rekabdar Elham , Tunbäck Petra , Liljeqvist Jan-Åke , Lindh Magnus , Bergström Tomas
Published: August 2002

Herpes simplex virus type 1 (HSV-1) encodes 11 envelope glycoproteins, of which glycoprotein G-1 (gG-1) induces a type-specific antibody response. Variability of the gG-1 gene among wild-type strains may be a factor of importance for a reliable serodiagnosis and typing of HSV-1 isolates. Here, we used a gG-1 type-specific monoclonal antibody (MAb) to screen for mutations in the immunodominant region of this protein in 108 clinical HSV-1 isolates. Of these, 42 isolates showed no reactivity to the anti-gG-1 MAb. One hundred five strains were further examined by DNA sequencing of the middle part of the gG-1 gene, encompassing 106 amino acids including the immunodominant region and epitope of the anti-gG-1 MAb. By phylogenetic comparisons based on the sequence data, we observed two (main) genetic variants of the gG-1 gene among the clinical isolates corresponding to reactivity or nonreactivity to the anti-gG-1 MAb. Furthermore, four strains appeared to be recombinants of the two gG-1 variants. In addition, one strain displayed a gG-1-negative phenotype due to a frameshift mutation, in the form of insertion of a cytosine nucleotide. When immunoglobulin G reactivity to HSV-1 in sera from patients infected with either of the two variants was investigated, no significant differences were found between the two groups, either in a type-common enzyme-linked immunosorbent assay (ELISA) or in a type-specific gG-1 antigen-based ELISA. Despite the here-documented existence of two variants of the gG-1 gene affecting the immunodominant region of the protein, other circumstances, such as early phase of infection, might be sought for explaining the seronegativity to gG-1 commonly found in a proportion of the HSV-1-infected patients.
Detection and Characterization of Hepatitis C Virus RNA in Seminal Plasma and Spermatozoon Fractions of Semen from Patients Attempting Medically Assisted Conception
Authors: Bourlet Thomas , Levy Rachel , Maertens Anne , Tardy Jean-Claude , Grattard Florence , Cordonier Hélène , Laurent Jean-Louis , Guerin Jean-François , Pozzetto Bruno
Published: August 2002

To investigate the risk of transmission of hepatitis C virus (HCV) via semen in assisted reproduction techniques, semen samples from 32 men chronically infected with HCV attending a center for assisted procreation were tested for HCV RNA by a reverse transcription-PCR protocol by using a modified version of the Cobas AMPLICOR HCV assay (version 2.0; Roche Diagnostics). The sensitivity of the test was 40 copies/ml. Four of 32 seminal plasma samples (12.5%) were found to be positive for the presence of HCV RNA. The median HCV load in blood was significantly higher in patients who were found to be positive for the presence of HCV RNA in semen than in those who tested negative (
P
= 0.02). In one man, seven consecutive seminal plasma samples tested positive for HCV RNA, as did two consecutive motile spermatozoon fractions; the corresponding fractions obtained after migration of the spermatozoa remained negative. Despite the absence of the proven infectivity of virus in semen samples that test positive for HCV RNA, these findings highlight the fact that seminal fluid may exhibit prolonged HCV RNA excretion. The usefulness of HCV RNA detection in both seminal plasma and spermatozoon fractions before the start of a program of medically assisted reproduction in couples in whom the male partner is chronically infected with HCV would need to be evaluated prospectively with a larger population of subjects exhibiting HCV RNA in their semen.
Development of a Real-Time Reverse Transcriptase PCR Assay for Type A Influenza Virus and the Avian H5 and H7 Hemagglutinin Subtypes
Authors: Spackman Erica , Senne Dennis A. , Myers T. J. , Bulaga Leslie L. , Garber Lindsey P. , Perdue Michael L. , Lohman Kenton , Daum Luke T. , Suarez David L.
Published: August 2002

A real-time reverse transcriptase PCR (RRT-PCR) assay based on the avian influenza virus matrix gene was developed for the rapid detection of type A influenza virus. Additionally, H5 and H7 hemagglutinin subtype-specific probe sets were developed based on North American avian influenza virus sequences. The RRT-PCR assay utilizes a one-step RT-PCR protocol and fluorogenic hydrolysis type probes. The matrix gene RRT-PCR assay has a detection limit of 10 fg or approximately 1,000 copies of target RNA and can detect 0.1 50% egg infective dose of virus. The H5- and H7-specific probe sets each have a detection limit of 100 fg of target RNA or approximately 10
3
to 10
4
gene copies. The sensitivity and specificity of the real-time PCR assay were directly compared with those of the current standard for detection of influenza virus: virus isolation (VI) in embryonated chicken eggs and hemagglutinin subtyping by hemagglutination inhibition (HI) assay. The comparison was performed with 1,550 tracheal and cloacal swabs from various avian species and environmental swabs obtained from live-bird markets in New York and New Jersey. Influenza virus-specific RRT-PCR results correlated with VI results for 89% of the samples. The remaining samples were positive with only one detection method. Overall the sensitivity and specificity of the H7- and H5-specific RRT-PCR were similar to those of VI and HI.
Distribution of Environmentally Regulated Genes of
Streptococcus suis
Serotype 2 among
S. suis
Serotypes and Other Organisms
Authors: de Greeff Astrid , Buys Herma , Verhaar Robin , van Alphen Loek , Smith Hilde E.
Published: August 2002

The occurrence of 36 environmentally regulated genes of
Streptococcus suis
strain 10 among all 35
S. suis
serotypes was determined by using hybridization with the amplified genes as probes. In addition, the distribution of these genes among the virulence phenotypes of serotypes 1 and 2 was assessed. Hybridization was also performed with various other streptococcal species and nonstreptococcal bacterial species which may be present in pigs. Interestingly, probe ivs-25/iri-1, similar to
agrA
and
sapR
, hybridized only with
S. suis
serotype 1 and 2 strains with virulent phenotypes and is therefore suitable as a diagnostic parameter. Only one probe was specific for
S. suis
. This probe's sequence was identical to the
epf
gene, a putative virulence factor of
S. suis
. Probe ivs-31 was similar to a virulence factor of
S. suis
, namely, a gene encoding a fibronectin- and fibrinogen-binding protein. This probe hybridized only with oral streptococci. Nearly half of the probes (45%) hybridized with the oral streptococci (
S. oralis
,
S. milleri
,
S. sanguis
,
S. gordonii
, and
S. mitis
) and with
Streptococcus pneumoniae
. This indicates a close relationship between
S. suis
, the oral streptococci, and
S. pneumoniae
with respect to the selected environmentally regulated genes. One probe only hybridized with gram-negative species and therefore seems to be obtained by
S. suis
from a gram-negative organism by horizontal transfer.
Effects of Various Test Media on the Activities of 21 Antimicrobial Agents against
Haemophilus influenzae
Authors: Jacobs Michael R. , Bajaksouzian Saralee , Windau Anne , Appelbaum Peter C. , Lin Gengrong , Felmingham David , Dencer Christine , Koeth Laura , Singer Mendel E. , Good Caryn E.
Published: August 2002

As considerable variation in the antimicrobial susceptibility of
Haemophilus influenzae
has been reported, the effects of various test media on the susceptibility of
H. influenzae
were studied. MICs were determined by three laboratories for 21 antimicrobial agents against a panel of 100 selected isolates. Testing was performed using a reference NCCLS frozen broth microdilution method with
Haemophilus
test medium (HTM) broth and dried commercial MIC trays rehydrated with the following media: in-house and commercially prepared HTM broth, Mueller-Hinton broth with 2% lysed horse blood and NAD, IsoSensitest broth with 2% lysed horse blood and NAD, and IsoSensitest broth-based HTM. Overall, all results were very reproducible, with the MIC at which 50% of the isolates tested are inhibited (MIC
50
), MIC
90
, and geometric mean MIC being within one doubling dilution by all six methods and at all three testing centers for 15 of the 21 agents tested. Interlaboratory differences were more marked than intralaboratory differences or differences among media. Cefprozil, cefaclor, and trimethoprim-sulfamethoxazole results differed the most, while results for ampicillin, amoxicillin-clavulanic acid, cefdinir, cefixime, ceftriaxone, and clarithromycin were the most reproducible. However, these variations in results caused considerable differences in susceptibility rates for agents for which NCCLS susceptible breakpoints were close to the geometric mean MIC, particularly for cefaclor and cefprozil. This was much less of a problem when pharmacokinetic-pharmacodynamic breakpoints were used. Reproducible susceptibility results were obtained for a wide range of agents against
H. influenzae
in three laboratories using a variety of media that support the growth of this fastidious species.
Detection of
Trichomonas vaginalis
on Modified Columbia Agar in the Routine Laboratory
Authors: Stary Angelika , Kuchinka-Koch Angelika , Teodorowicz Lilianna
Published: August 2002

Broth culture of
Trichomonas vaginalis
is considered the “gold standard” for the diagnosis of trichomoniasis. Two studies were carried out to evaluate modified Columbia agar (MCA) for the isolation of
T. vaginalis
from clinical samples. Study I compared isolation on MCA to that on liquid medium with 889 vaginal samples. Out of 63 samples positive for
T. vaginalis
(7.1% of total), MCA identified 62 (98.4%) and broth identified 58 (92.1%). In study II, trichomoniasis was diagnosed within the scope of a screening program for a total of 39,585 men and women by culture on MCA and direct microscopy. Culture on MCA detected 199 (98.5%) and Gram staining detected 163 (80.7%) of 202 positive specimens. Wet-mount preparations used for symptomatic patients identified 103 (92.8%) of 111 cases. Culture of
T. vaginalis
from clinical samples on MCA is highly sensitive and reliable, as well as timesaving, and therefore suitable for screening of symptomatic and asymptomatic individuals.
Mycobacterium microti
Infection (Vole Tuberculosis) in Wild Rodent Populations
Authors: Cavanagh Rachel , Begon Michael , Bennett Malcolm , Ergon Torbjørn , Graham Isla M. , de Haas Petra E. W. , Hart C. A. , Koedam Marianne , Kremer Kristin , Lambin Xavier , Roholl Paul , Soolingen Dick van
Published: August 2002

Mycobacterium microti
(vole tuberculosis) infections in small wild mammals were first described more than 60 years ago in several populations in Great Britain. Few studies of vole tuberculosis have been undertaken since then, and little is known about the relationship between
M. microti
isolates originating from different populations or at different times or of the prevalence of this infection in wild rodent populations, despite human cases of
M. microti
infections being increasingly reported. In this study, field voles (
Microtus agrestis
), bank voles (
Clethrionomys glareolus
), and wood mice (
Apodemus sylvaticus
) were found to be infected, with up to 8% having external tuberculous signs, in wild populations in Northumberland and Cheshire, England. Spoligotyping applied directly to the clinical material simultaneously detected and typed
M. microti
bacteria in skin lesions, lymph glands, and internal abcesses. IS
6110
restriction fragment length polymorphism typing of cultured bacteria was used to compare these isolates with previously isolated strains from both animals and humans. This demonstrated that although the current rodent isolates were distinct from those isolated from voles in the 1930s in Great Britain, they had a high degree of similarity to these strains and were distinct from the
M. microti
isolates from humans, a pig, and a ferret from The Netherlands. Thus,
M. microti
infection seems to be widespread in wild rodent populations, but more studies are needed to understand how
M. microti
might be transmitted from animals to humans and to determine better the zoonotic risk posed.
Simultaneous Detection of
Anaplasma marginale
and a New
Ehrlichia
Species Closely Related to
Ehrlichia chaffeensis
by Sequence Analyses of 16S Ribosomal DNA in
Boophilus microplus
Ticks from Tibet
Authors: Wen Bohai , Jian Rui , Zhang Youzhi , Chen Rong
Published: August 2002

To identify ehrlichial agents in
Boophilus microplus
ticks, DNA samples of
B. microplus
collected from the Tibet Autonomous Region and Sichuan Province of China were screened by a nested PCR. Sixteen of 43 (37%) DNA samples of
B. microplus
from Tibet were positive in nested PCR analysis. All 27 samples from Sichuan were negative. The screen identified two ehrlichial agents based on different 16S rRNA genes that were found after amplifying and sequencing the 5′-end fragments of the 16S rRNA genes. One sequence was identical to that of the gene of
Anaplasma marginale
, an etiological agent of animal anaplasmosis. The other sequence was most similar to that of the gene of
Ehrlichia chaffeensis
, an etiological agent of human monocytic ehrlichiosis. The sequence of 1,501 bases from the novel ehrlichial agent was obtained and showed the greatest levels of sequence similarity (97 to 98%) to 16S rRNA gene sequences of the members of the
E. canis
group of the genus
Ehrlichia
. Sequence comparison of the 16S rRNA gene with the members of the genus
Ehrlichia
reveals that the novel ehrlichial agent detected in
B. microplus
ticks is a new species of the genus
Ehrlichia
and is most closely related to
E. chaffeensis
.
Rapid Detection of Methicillin Resistance in Coagulase-Negative Staphylococci with the VITEK 2 System
Authors: Horstkotte Matthias A. , Knobloch Johannes K.-M. , Rohde Holger , Dobinsky Sabine , Mack Dietrich
Published: August 2002

The aim of the present study was to evaluate the accuracy of the new VITEK 2 system (bioMérieux, Marcy l' Etoile, France) for the detection of methicillin resistance in coagulase-negative staphylococci (CoNS) by using AST-P515 and AST-P523 test cards. Analyses of the VITEK 2 oxacillin MIC determination evaluated according to the actual breakpoint (≥0.5 μg/ml) of the National Committee for Clinical Laboratory Standards resulted in a high sensitivity of 99.2% but a moderate specificity of 80%. The newly included oxacillin resistance (OR) test of the VITEK 2 system displayed a high sensitivity and a high specificity of 97.5 and 98.7%, respectively. Concordance between the results of the
mecA
PCR and the VITEK 2 oxacillin MIC was observed for almost all
Staphylococcus epidermidis
strains, but the reduced specificity was attributable to higher oxacillin MICs for
mecA
-negative non-
S. epidermidis
strains, especially
S. saprophyticus
,
S. lugdunensis
, and
S. cohnii
. Evaluation of alternative oxacillin MIC breakpoints of 1, 2, or 4 μg/ml resulted in improved degrees of specificity of 84, 90.7, and 97.3%, respectively. Only minor changes occurred in the corresponding sensitivity values, which were 98.4, 97.5, and 97.5%, respectively. Methicillin resistance in CoNS was detected after 7 and 8 h in 91.1 and 93.5% of the
mecA
-positive strains, respectively, by the VITEK 2 OR test and in 86.3 and 89.5% of the
mecA
-positive strains, respectively, by VITEK 2 oxacillin MIC determination. After 7 and 8 h the VITEK 2 OR test classified 59.2 and 78.9% of the
mecA
-negative strains, respectively, as susceptible to oxacillin, whereas comparable values were obtained 2 h later by VITEK 2 oxacillin MIC determination. The results of our study encourage the use of the VITEK 2 system, which proved to be a highly reliable and rapid phenotypic method for the detection of methicillin resistance in CoNS.
New Variants of
Vibrio cholerae
O1 Biotype El Tor with Attributes of the Classical Biotype from Hospitalized Patients with Acute Diarrhea in Bangladesh
Authors: Nair G. Balakrish , Faruque Shah M. , Bhuiyan N. A. , Kamruzzaman M. , Siddique A. K. , Sack David A.
Published: August 2002

The sixth pandemic of cholera and, presumably, the earlier pandemics were caused by the classical biotype of
Vibrio cholerae
O1, which was progressively replaced by the El Tor biotype representing the seventh cholera pandemic. Although the classical biotype of
V. cholerae
O1 is extinct, even in southern Bangladesh, the last of the niches where this biotype prevailed, we have identified new varieties of
V. cholerae
O1, of the El Tor biotype with attributes of the classical biotype, from hospitalized patients with acute diarrhea in Bangladesh. Twenty-four strains of
V. cholerae
O1 isolated between 1991 and 1994 from hospitalized patients with acute diarrhea in Matlab, a rural area of Bangladesh, were examined for the phenotypic and genotypic traits that distinguish the two biotypes of
V. cholerae
O1. Standard reference strains of
V. cholerae
O1 belonging to the classical and El Tor biotypes were used as controls in all of the tests. The phenotypic traits commonly used to distinguish between the El Tor and classical biotypes, including polymyxin B sensitivity, chicken cell agglutination, type of
tcpA
and
rstR
genes, and restriction patterns of conserved rRNA genes (ribotypes), differentiated the 24 strains of toxigenic
V. cholerae
O1 into three types designated the Matlab types. Although all of the strains belonged to ribotypes that have been previously found among El Tor vibrios, type I strains had more traits of the classical biotype while type II and III strains appeared to be more like the El Tor biotype but had some classical biotype properties. These results suggest that, although the classical and El Tor biotypes have different lineages, there are possible naturally occurring genetic hybrids between the classical and El Tor biotypes that can cause cholera and thus provide new insight into the epidemiology of cholera in Bangladesh. Furthermore, the existence of such novel strains may have implications for the development of a cholera vaccine.
Comparative Assessment of Genotyping Methods for Epidemiologic Study of
Burkholderia cepacia
Genomovar III
Authors: Coenye Tom , Spilker Theodore , Martin Alissa , LiPuma John J.
Published: August 2002

We analyzed a collection of 97 well-characterized
Burkholderia cepacia
genomovar III isolates to evaluate multiple genomic typing systems, including pulsed-field gel electrophoresis (PFGE), BOX-PCR fingerprinting and random amplified polymorphic DNA (RAPD) typing. The typeability, reproducibility, and discriminatory power of these techniques were evaluated, and the results were compared to each other and to data obtained in previous studies by using multilocus restriction typing (MLRT). All methods showed excellent typeability. PFGE with
Spe
I was more reproducible than RAPD and BOX-PCR fingerprinting. The discriminatory power of the methods was variable, with PFGE and RAPD typing having a higher index of discrimination than BOX-PCR fingerprinting. In general, the results obtained by PFGE, BOX-PCR fingerprinting, and MLRT were in good agreement. Our data indicate that different genomic-based methods can be used to type
B. cepacia
genomovar III isolates depending on the situation and the epidemiologic question being addressed. PFGE and RAPD fingerprinting are best suited to addressing small-scale studies (i.e., local epidemiology), whereas BOX-PCR fingerprinting is more appropriate for large-scale studies (i.e., global epidemiology). In this regard, BOX-PCR fingerprinting can be considered a rapid and easy alternative to MLRT.
Determination of NovelBorreliaGenospecies in SwedishIxodes ricinusTicks
Authors: Fraenkel Carl-Johan , Garpmo Ulf , Berglund Johan
Published: August 2002

A total of 301 adult questingIxodes ricinusticks were collected at 15 different locations along the south and east coasts of Sweden to determine theBorreliagenospecies diversity. Thirty-two ticks (11%) were found to be positive by nested PCR withBorrelia burgdorferisensu lato-specific primers. Species determination was based on partial sequencing of the 16S rRNA gene and the flagellin gene. Five differentBorreliaspecies were found. The nucleotide sequence of theBorreliaDNA found in two ticks differed extensively from the nucleotide sequences of theBorreliaDNA found in the other ticks, and analysis revealed that they were closely related to the relapsing fever borrelia speciesBorrelia miyamotoi. This is the first report of aB. miyamotoi-like borrelia inI. ricinusand in Europe. Moreover, theBorreliaDNA of two ticks (6%) clustered within theB. valaisianacomplex.B. valaisianahas not previously been reported in Sweden.B. afzeliiDNA was found in 14 ticks (44%), andB. gariniiDNA was found in 10 ticks (31%).B. burgdorferisensu stricto DNA was found in four ticks (13%). We conclude that all of the known human-pathogenic species (B. garinii,B. afzelii, andB. burgdorferisensu stricto) andB. valaisianafound elsewhere in Europe are also present in the Swedish host-seeking tick population and that aB. miyamotoi-likeBorreliaspecies seems to be present inI. ricinusticks in Europe.
Molecular Epidemiology of Erythromycin Resistance in
Streptococcus pneumoniae
Isolates from Blood and Noninvasive Sites
Authors: Amezaga Maria Rosario , Carter Philip E. , Cash Phillip , McKenzie Hamish
Published: August 2002

Erythromycin-resistant isolates of
Streptococcus pneumoniae
from blood cultures and noninvasive sites were studied over a 3-year period. The prevalence of erythromycin resistance was 11.9% (19 of 160) in blood culture isolates but 4.2% (60 of 1,435) in noninvasive-site isolates. Sixty-two of the 79 resistant isolates were available for study. The M phenotype was responsible for 76% (47 of 62) of resistance, largely due to a serotype 14 clone, characterized by multilocus sequence typing as ST9, which accounted for 79% (37 of 47) of M phenotype resistance. The ST9 clone was 4.8 times more common in blood than in noninvasive sites. All M phenotype isolates were PCR positive for
mef
(A), but sequencing revealed that the ST9 clone possessed the
mef
(A) sequence commonly associated with
Streptococcus pyogenes
. All M phenotype isolates with this
mef
(A) sequence also had sequences consistent with the presence of the Tn
1207.1
genetic element inserted in the
celB
gene. In contrast, isolates with the
mef
(E) sequence normally associated with
S. pneumoniae
contained sequences consistent with the presence of the mega insertion element. All MLS
B
isolates carried
erm
(B), and two isolates carried both
erm
(B) and
mef
(E). Fourteen of the 15 MLS
B
isolates were tetracycline resistant and contained
tet
(M). However, six M phenotype isolates of serotypes 19 (two isolates) and 23 (four isolates) were also tetracycline resistant and contained
tet
(M). MICs for isolates with the
mef
(A) sequence were significantly higher than MICs for isolates with the
mef
(E) sequence (
P
< 0.001). Thus, the ST9 clone of
S. pneumoniae
is a significant cause of invasive pneumococcal disease in northeast Scotland and is the single most important contributor to M phenotype erythromycin resistance.
Rational Design of DNA Sequence-Based Strategies for Subtyping
Listeria monocytogenes
Authors: Cai Steven , Kabuki Dirce Yorika , Kuaye Arnaldo Yoshiteru , Cargioli Theresa Gina , Chung Michael S. , Nielsen Rasmus , Wiedmann Martin
Published: August 2002

The ability to differentiate bacteria beyond the species level is essential for identifying and tracking infectious disease outbreaks and to improve our knowledge of the population genetics, epidemiology, and ecology of bacterial pathogens. Commonly used subtyping methods, such as serotyping, phage typing, ribotyping, and pulsed-field gel electrophoresis, can yield ambiguous results that are difficult to standardize and share among laboratories. DNA sequence-based subtyping strategies can reduce interpretation ambiguity. We report the development of a rational approach for designing sequence-based subtyping methods.
Listeria monocytogenes
was selected as the model organism for testing the efficacy of this approach. Two housekeeping genes (
recA
and
prs
), one stress response gene (
sigB
), two virulence genes (
actA
and
inlA
), and two intergenic regions (
hly
-
mpl
and
plcA
-
hly
) were sequenced for 15
L. monocytogenes
isolates. Isolates were chosen from a representative collection of more than 1,000
L. monocytogenes
isolates to reflect the genetic diversity of this species. DNA sequences were aligned, and sliding window analyses were performed for each gene to define 600-bp-long regions that were (i) most polymorphic (using ProSeq) or (ii) most discriminatory (using a new algorithm implemented in WINDOWMIN). Complete gene sequences for
actA
(1,929 bp) and
inlA
(2,235 bp) provided the highest discrimination (identifying 15 and 14 allelic types, respectively). WINDOWMIN allowed identification of 600-bp regions within these genes that provided similar discriminatory power (yielding 15 and 13 allelic types, respectively). The most discriminatory 600-bp fragments identified in the housekeeping and stress response genes differentiated the isolates into 8 to 10 subtypes; intergenic region sequences yielded 8 and 12 allelic types based on 335- and 242-bp sequences for
hly
-
mpl
and
plcA
-
hly
, respectively. Regions identified as most polymorphic were not necessarily most discriminatory; therefore, application of the WINDOWMIN algorithm provided a powerful tool for determining the best target regions for DNA sequence-based subtyping. Our specific results also show that inclusion of virulence gene target sequences in a DNA sequence-based subtyping scheme for
L. monocytogenes
is necessary to achieve maximum subtype differentiation.
Possible Connection between a Widely Disseminated Conjugative Gentamicin Resistance (pMG1-Like) Plasmid and the Emergence of Vancomycin Resistance in
Enterococcus faecium
Authors: Tomita Haruyoshi , Pierson Carl , Lim Suk Kyung , Clewell Don B. , Ike Yasuyoshi
Published: August 2002

A total of 640 vancomycin-resistant
Enterococcus faecium
(VRE) isolates, which were obtained between 1994 and 1999 from the Medical School Hospital of the University of Michigan, Ann Arbor, were used in this study. Of the 640 strains, 611 and 29 were VanA and VanB VRE, respectively, based on PCR analysis. Four hundred ninety-two (77%) of the strains exhibited resistance to concentrations of gentamicin from 64 μg/ml (MIC) to more than 1,024 μg/ml (MIC). The gentamicin resistance of each of 261 (53%) of the 492 gentamicin-resistant strains was transferred to
E. faecium
at a frequency of about 10
−5
to 10
−6
per donor cell in broth mating. More than 90% of vancomycin resistances of the 261 strains cotransferred with the gentamicin resistances to
E. faecium
strains by filter mating. The conjugative gentamicin resistance plasmids were identified and were classified into five types (A through E) with respect to their
Eco
RI restriction profiles. The transfer frequencies of each type of plasmid between
E. faecium
strains or
Enterococcus faecalis
strains were around 10
−3
to 10
−5
per donor cell or around 10
−6
to 10
−7
per donor cell, respectively, in broth mating. Type A and type B were the most frequently isolated, at an isolation frequency of about 40% per VRE isolate harboring the gentamicin resistance conjugative plasmid. The plasmids did not show any homology in Southern hybridization with the pheromone-responsive plasmids and broad-host-range plasmids pAMβ1 and pIP501. The
Eco
RI or
Nde
I restriction fragments of each type of plasmids hybridized to the conjugative gentamicin resistance plasmid pMG1 (65.1 kb), which was originally isolated from an
E. faecium
clinical isolate, and transfer efficiently in broth mating.
Detection and Quantification of Oral Treponemes in Subgingival Plaque by Real-Time PCR
Authors: Asai Yasuyuki , Jinno Takayoshi , Igarashi Hajime , Ohyama Yoshinori , Ogawa Tomohiko
Published: August 2002

Oral treponemes have been associated with periodontal diseases. We developed a highly sensitive and specific method to detect and quantify cultivable oral treponemes (
Treponema denticola
,
Treponema vincentii
, and
Treponema medium
) in 50 subgingival plaque samples from 13 healthy subjects as well as 37 patients with periodontal diseases using real-time PCR assays with specific primers and a TaqMan probe for each 16S rRNA sequence. The specificity for each assay was examined by using DNA specimens from various treponemal and other bacterial species. The TaqMan real-time PCR was able to detect from 10
3
to 10
8
cells of the oral treponemes, with correlation coefficients as follows:
T. denticola
, 0.984;
T. vincentii
, 0.991; and
T. medium
, 0.984. The frequencies of occurrence of these three oral treponemes in subgingival plaque samples were as follows:
T. denticola
, 68.0%;
T. vincentii
, 36.0%; and
T. medium
, 48.0%. In addition, the number of
T. denticola
,
T. vincentii
, and
T. medium
cells in plaque samples detected by real-time PCR ranged from 3 to 15,184, 1 to 447, and 1 to 7,301 cells/pg of plaque DNA, respectively. Increased numbers of
T. denticola
cells were detected in plaque samples from deep periodontal pockets, and
T. medium
was also detected in deep pockets. On the other hand,
T. vincentii
was mainly found in shallow pockets. These results suggest that various oral treponemes are associated with the formation of each stage of periodontal disease.
High Prevalence of Human Papillomavirus (HPV) Infections and High Frequency of Multiple HPV Genotypes in Human Immunodeficiency Virus-Infected Women in Brazil
Authors: Levi José E. , Kleter Bernhard , Quint Wim G. V. , Fink Maria C. S. , Canto Cynthia L. M. , Matsubara Regina , Linhares Iara , Segurado Aluísio , Vanderborght Bart , Neto José Eluf , van Doorn Leen-Jan
Published: August 2002

A group of 208 human immunodeficiency virus (HIV)-infected women in Brazil were studied for the presence of human papillomavirus with the general SPF
10
PCR primer set. Virtually all (98%) women were found positive for human papillomavirus (HPV) DNA. Genotyping by the reverse hybridization line probe assay (HPV-LiPA) revealed a high prevalence of multiple genotypes (78.9% of the cases), with an average of 3.1 genotypes per patient (range, 1 to 10 genotypes). HPV 6 was the most prevalent genotype and was observed in 80 (39.2%) patients, followed by types 51 (31.9%), 11 (26.0%), 18 (24.0%), and 16 (22.5%). Of the genotypes detected, 40.9% were low-risk genotypes. Twenty-two (10.5%) patients showed normal (Pap I) cytology, 149 (71.6%) patients had inflammation (Pap II), and 28 patients (13.4%) had a Pap III score. The prevalence of high-risk genotypes increased with the cytological classification. There were no significant associations between the number of HPV genotypes detected and the cytological classification, HIV viral load, and CD4 count in these patients. In conclusion, the highly sensitive SPF
10
LiPA system shows that a very high proportion of HIV-infected women in Brazil are infected with HPV and often carry multiple HPV genotypes.
Leptotrichia amnionii
sp. nov., a Novel Bacterium Isolated from the Amniotic Fluid of a Woman after Intrauterine Fetal Demise
Authors: Shukla Sanjay K. , Meier Paul R. , Mitchell Paul D. , Frank Daniel N. , Reed Kurt D.
Published: August 2002

A novel bacterium was isolated and characterized from the amniotic fluid of a woman who experienced intrauterine fetal demise in the second trimester of pregnancy. The bacterium was a slow-growing, gram-negative anaerobic coccobacillus belonging to the genus
Leptotrichia
. Unlike
Leptotrichia sanguinegens
, the isolate did not grow in chopped-meat glucose broth or on sheep blood agar upon subculturing. The isolate was characterized by sequencing and analyzing its 16S rRNA gene. The 1,493-bp 16S ribosomal DNA sequence had only 96% homology with
L. sanguinegens.
Several phylogenetic analyses indicated that
L. amnionii
is a distinct species and most closely related to
L. sanguiegens
.
Fast, Noninvasive Method for Molecular Detection and Differentiation of
Malassezia
Yeast Species on Human Skin and Application of the Method to Dandruff Microbiology
Authors: Gemmer Christina M. , DeAngelis Yvonne M. , Theelen Bart , Boekhout Teun , Dawson, Thomas L.
Published: August 2002

Malassezia
fungi have been the suspected cause of dandruff for more than a century. Previously referred to as
Pityrosporum ovale
,
Pityrosporum orbiculare
, or
Malassezia
, these fungi are now known to consist of at least seven
Malassezia
species. Each species has a specific ecological niche, as well as specific biochemical and genetic characteristics.
Malassezia
yeasts have fastidious culture conditions and exceedingly different growth rates. Therefore, the results of surveys of
Malassezia
based on culture methods can be difficult to interpret. We developed a molecular technique, terminal fragment length polymorphism analysis, to more accurately survey the ecology of
Malassezia
yeasts without bias from culture. This technique involves fluorescent nested PCR of the intergenic transcribed spacer (ITS) ITS I and ITS II region ribosomal gene clusters. All known
Malassezia
species can be differentiated by unique ITS fragment lengths. We have used this technique to directly analyze scalp samples from subjects enrolled in a demographic scalp health study. Results for subjects assigned composite adherent scalp flaking scores (ASFS) <10 were compared to those for subjects assigned composite ASFS >24.
Malassezia restricta
and
M. globosa
were found to be the predominant
Malassezia
species present in both groups. Importantly, we found no evidence of
M. furfur
in either group, indicating that
M. furfur
can be eliminated as the causal organism for dandruff. Both groups also showed the presence of non-
Malassezia
fungi. This method, particularly when it is used in combination with existing fungal ITS databases, is expected to be useful in the diagnosis of multiple other fungal infections.
Detection ofRickettsia prowazekiiin Body Lice and Their Feces by Using Monoclonal Antibodies
Authors: Fang Rong , Houhamdi Linda , Raoult Didier
Published: August 2002

In order to identifyRickettsia prowazekiiin lice, we developed a panel of 29 representative monoclonal antibodies selected from 187 positive hybridomas made by fusing splenocytes of immunized mice with SP2/0-Ag14 myeloma cells. Immunoblotting revealed that 15 monoclonal antibodies reacted with the lipopolysaccharide-like (LPS-L) antigen and 14 reacted with the epitopes of a 120-kDa protein. Only typhus group rickettsiae reacted with the monoclonal antibodies against LPS-L.R. felis, a recently identified rickettsial species, did not react with these monoclonal antibodies, confirming that it is not antigenically related to the typhus group. Monoclonal antibodies against the 120-kDa protein were highly specific forR. prowazekii.We successfully applied a selected monoclonal antibody against the 120-kDa protein to detect by immunofluorescence assayR. prowazekiiin smears from 56 wild and laboratory lice, as well as in 10 samples of louse feces infected or not infected with the organism. We have developed a simple, practical, and specific diagnostic assay for clinical specimens and large-scale epidemiological surveys with a sensitivity of 91%. These monoclonal antibodies could be added to the rickettsial diagnostic panel and be used to differentiateR. prowazekiifrom other rickettsial species.
Rapid-Cycle PCR and Fluorimetry for Detection of Mycobacteria
Authors: Lachnik Jacqueline , Ackermann Birgit , Bohrssen Antje , Maass Silvia , Diephaus Catharina , Puncken Axel , Stermann Marion , Bange Franz-Christoph
Published: August 2002

In this study we used LightCycler PCR amplification and product detection by fluorescence resonance energy transfer probes to identify mycobacteria and differentiate between
Mycobacterium tuberculosis
complex,
Mycobacterium avium,
and other nontuberculous mycobacteria. Targeting the 16S rRNA gene, three different probes specific for mycobacteria,
M
.
tuberculosis
complex, and
M
.
avium
were constructed. As few as five genome copies of target nucleic acid were detected by the probes, illustrating the high sensitivity of the system. All 33 mycobacterial species tested but none of the closely related actinomycetes and other bacteria produced a specific fluorescence signal. A specificity of 100% was also demonstrated for the
M
.
tuberculosis
complex-specific probe and the
M
.
avium
-specific probe. Within 45 min, the LightCycler method correctly detected mycobacteria and specifically identified
M
.
tuberculosis
complex and
M
.
avium
without any post-PCR sample manipulation. In view of future clinical studies, we also constructed and tested an internal control which could be used to assure successful amplification and detection of mycobacteria. Monitoring of PCR inhibition will be essential for evaluation of this system for direct detection of mycobacteria in clinical specimens. Finally, we tested our system on sputum seeded with mycobacteria and were able to detect as few as 10 organisms. At present, this system is the fastest available method for identification and differentiation of mycobacteria from culture-positive specimens and offers an excellent alternative to previously established nucleic acid amplification-based techniques for the diagnostic mycobacterial laboratory.
Clinical and Epidemiological Correlates of Genotypes within the
Mycobacterium avium
Complex Defined by Restriction and Sequence Analysis of
hsp65
Authors: Smole Sandra C. , McAleese Fionnuala , Ngampasutadol Jutamas , von Reyn C. Fordham , Arbeit Robert D.
Published: August 2002

Species identification of isolates of the
Mycobacterium avium
complex (MAC) remains a difficult task. Although
M
.
avium
and
Mycobacterium intracellulare
can be identified with expensive, commercially available probes, many MAC isolates remain unresolved, including those representing
Mycobacterium lentiflavum
as well as other potentially undefined species. PCR restriction analysis (PRA) of the
hsp65
gene has been proposed as a rapid and inexpensive approach. We applied PRA to 278 MAC isolates, including 126 from blood of human immunodeficiency virus (HIV)-infected patients, 59 from sputum of HIV-negative patients with chronic obstructive pulmonary disease, 88 from environmental sources, and 5 pulmonary isolates from a different study. A total of 15 different PRA patterns were observed. For 27 representative isolates, a 441-bp fragment of the
hsp65
gene was sequenced; based on 54 polymorphic sites, 18 different alleles were defined, including 12 alleles not previously reported. Species and phylogenetic relationships were more accurately defined by sequencing than by PRA or commercial probe. The distribution of PRA types and, by implication, phylogenetic lineages among blood isolates was significantly different from that for pulmonary and environmental isolates, suggesting that particular lineages have appreciably greater virulence and invasive potential.
Malignant Catarrhal Fever-Like Disease in Barbary Red Deer (Cervus elaphus barbarus) Naturally Infected with a Virus Resembling Alcelaphine Herpesvirus 2
Authors: Klieforth Robert , Maalouf Gabriel , Stalis Ilse , Terio Karen , Janssen Donald , Schrenzel Mark
Published: August 2002

Eight Barbary red deer (Cervus elaphus barbarus) developed clinical signs suggestive of malignant catarrhal fever (MCF) over a 28-day period. These animals were housed outdoors with four other species of ruminants. Affected red deer had lethargy, ocular signs, and nasal discharge and were euthanatized within 48 h. Lesions included ulcers of the muzzle, lips, and oral cavity associated with infiltrates of neutrophils and lymphocytes. Serologically, six of seven red deer tested during the outbreak were positive by competitive enzyme-linked immunosorbent assay for antibodies to a shared MCF virus antigen. PCR using oligonucleotide primers designed for a conserved protein of alcelaphine herpesviruses 1 (AlHV-1) and 2 (AlHV-2) and for conserved regions of a herpesvirus DNA polymerase gene was positive for tissues from all eight clinically affected animals and negative for eight out of eight red deer without clinical signs of MCF. DNA sequencing of PCR amplicons from the diseased red deer indicated that they were infected with a novel herpesvirus closely related to AlHV-2; immunohistochemistry using polyclonal anti-AlHV-2 serum and in situ hybridization demonstrated the presence of virus within salivary glands adjacent to oral lesions of affected animals. A survey of other ruminants near the outbreak subsequently showed that normal Jackson's hartebeest (Alcelaphus buselaphus jacksoni) that were cohoused with the diseased red deer were infected with the same virus and were shedding the virus in nasal excretions. These findings suggest that a herpesvirus closely related to AlHV-2 caused the MCF-like disease epizootic in Barbary red deer and that the virus may have originated from Jackson's hartebeest.
Practical Approach for Typing Strains of
Leishmania infantum
by Microsatellite Analysis
Authors: Bulle Béatrice , Millon Laurence , Bart Jean-Mathieu , Gállego Montserrat , Gambarelli Françoise , Portús Montserrat , Schnur Lee , Jaffe Charles L. , Fernandez-Barredo Salceda , Alunda José María , Piarroux Renaud
Published: August 2002

Currently the universally accepted standard procedure for characterizing and identifying strains of
Leishmania
is isoenzyme analysis. However, in the Mediterranean area, despite their very wide geographical distribution, most
Leishmania infantum
strains belong to zymodeme MON-1. In order to increase our understanding of polymorphism in strains of
L. infantum
, we developed PCR assays amplifying 10 microsatellites and sequenced PCR products. The discriminative power of microsatellite analysis was tested by using a panel of 50
L. infantum
strains collected from patients and dogs from Spain, France, and Israel, including 32 strains belonging to zymodeme MON-1, 8 strains belonging to zymodemes MON-24, MON-29, MON-33, MON-34, or MON-80, and 10 untyped strains. Five of the microsatellites were polymorphic, revealing 22 genotypes, whereas the five remaining microsatellites were not variable. In particular, MON-1 strains could be separated into 13 different closely related genotypes. MON-33 and MON-34 strains also gave two additional genotypes closely related to MON-1, while MON-29, MON-24, and MON 80 strains exhibited more divergent genotypes. Among the foci examined, the Catalonian focus displayed a high polymorphism, probably reflecting isoenzyme polymorphism, while the Israeli focus exhibited a low polymorphism that could be consistent with the recent reemergence and rapid spread of canine leishmaniasis in northern and central Israel. The strains originating from the south of France and the Madrid, Spain, area displayed significant microsatellite polymorphism even though they were monomorphic by isoenzyme analysis. In conclusion, microsatellite polymorphism exhibits a high discriminative power and appears to be suitable for characterization of closely related strains of
L. infantum
in epidemiological studies.
Mycobacterium africanum
Subtype II Is Associated with Two Distinct Genotypes and Is a Major Cause of Human Tuberculosis in Kampala, Uganda
Authors: Niemann S. , Rüsch-Gerdes S. , Joloba M. L. , Whalen C. C. , Guwatudde D. , Ellner J. J. , Eisenach K. , Fumokong N. , Johnson J. L. , Aisu T. , Mugerwa R. D. , Okwera A. , Schwander S. K.
Published: August 2002

The population structure of 234
Mycobacterium tuberculosis
complex strains obtained during 1995 and 1997 from tuberculosis patients living in Kampala, Uganda (East Africa), was analyzed by routine laboratory procedures, spoligotyping, and IS
6110
restriction fragment length polymorphism (RFLP) typing. According to biochemical test results, 157 isolates (67%) were classified as
M. africanum
subtype II (resistant to thiophen-2-carboxylic acid hydrazide), 76 isolates (32%) were classified as
M. tuberculosis
, and 1 isolate was classified as classical
M. bovis
. Spoligotyping did not lead to clear differentiation of
M. tuberculosis
and
M. africanum
, but all
M. africanum
subtype II isolates lacked spacers 33 to 36, differentiating them from
M. africanum
subtype I. Moreover, spoligotyping was not sufficient for differentiation of isolates on the strain level, since 193 (82%) were grouped into clusters. In contrast, in the IS
6110
-based dendrogram,
M. africanum
strains were clustered into two closely related strain families (Uganda I and II) and clearly separated from the
M. tuberculosis
isolates. A further characteristic of both
M. africanum
subtype II families was the absence of spoligotype spacer 40. All strains of family I also lacked spacer 43. The clustering rate obtained by the combination of spoligotyping and RFLP IS
6110
analysis was similar for
M. africanum
and
M. tuberculosis
, as 46% and 49% of the respective isolates were grouped into clusters. The results presented demonstrate that
M. africanum
subtype II isolates from Kampala, Uganda, belong to two closely related genotypes, which may represent unique phylogenetic branches within the
M. tuberculosis
complex. We conclude that
M. africanum
subtype II is the main cause of human tuberculosis in Kampala, Uganda.
Fluorescent Amplified Fragment Length Polymorphism Analysis ofSalmonella entericaSerovar Typhimurium Reveals Phage-Type- Specific Markers and Potential for Microarray Typing
Authors: Hu Honghua , Lan Ruiting , Reeves Peter R.
Published: August 2002

Fluorescent amplified fragment length polymorphism (AFLP) was applied to 46Salmonella entericaserovar Typhimurium isolates of Australian origin comprising nine phage types, by using the restriction enzymesMseI andEcoRI and all 16 possibleMseI +1-EcoRI +1 primer pair combinations. AFLP in the present study showed a very good discrimination power with a Simpson index of diversity of 0.98, and 35 different AFLP patterns were observed in the 46 isolates. AFLP grouped most serovar Typhimurium isolates by phage type and enabled differentiation of phage types. Furthermore, 84 phage-type-specific polymorphic AFLP fragments, for which presence or absence correlated with phage type (including 25 with one exception to phage type specificity) were observed in the 46 strains studied. Eighteen phage-type-specific AFLP fragments were cloned and sequenced. Fifteen are of known genes or have a homologue in the databases. Three sequences are plasmid related, eight are phage related, and four relate to chromosomal genes. Twelve of the 18 fragments are polymorphic because the DNA is present or absent as indicated by Southern hybridization, and we see good potential to use sequences of these fragments as the basis for multiplex PCR and development of a microarray-based molecular phage-typing method for serovar Typhimurium.